Design, Synthesis, and Antitumor Activities of Some Novel Substituted 1,2,3-Benzotriazines

A series of novel substituted 1,2,3-benzotriazines based on the structures of vatalanib succinate (PTK787) and vandetanib (ZD6474) were designed and synthesized. The antiproliferative effects of these compounds were tested on microvascular endothelial cells (MVECs) using the MTT assay. Introduction of a methoxy and a 3-chloropropoxy group into the 1,2,3-benzotriazines increased the antiproliferative effects. 4-(3-Chloro-4-fluoroanilino)-7-(3-chloropropoxy)-6-methoxy-1,2,3-benzotriazine (8m) was the most effective compound. It was 4-10 fold more potent than PTK787 in inhibiting the growth of T47D breast cancer cells, DU145 and PC-3 prostate cancer cells, LL/2 murine Lewis lung cancer cells and B16F0 melanoma cells.


Introduction
Vascular endothelial growth factor (VEGF) plays an important role in both physiological and pathological angiogenesis by binding to its receptor, VEGFR [1]. The expression of VEGFR is increased in the majority of cancers and is associated with survival, migration and invasion of solid tumor cells [1]. Elevated VEGF levels have been found to increase resistance to chemotherapy [2,3] and VEGF signaling has been used as a therapeutic target for the treatment of cancer. VEGFR inhibitors such as vatalanib succinate (PTK787/ZK222584) and vandetanib (ZD6474) have been used in clinical trials in several types of cancer [4][5][6][7]. Although both agents showed antitumor activities alone, combinations with chemotherapeutic agents seem to be required for reaching therapeutic effects [8][9][10][11][12]. To improve the antiproliferative abilities of PTK787 and ZD6474 (Figure 1), we have analyzed the structures of PTK787 and ZD6474. PTK787 contains 2,3-benzodizine and ZD6474 contains 1,3benzodizine. We thought that the replacement of the benzodizine with 1,2,3-benzotriazine might have increased activities compared to PTK787. Thus, we designed and synthesized eighteen novel compounds (8a-m) with a basic 1,2,3-benzotriazine scaffold and the introduction of a methoxy group or a short alkoxy group. The antiproliferative effects of these compounds were determined in microvascular endothelial cells (MVECs) using MTT assay and the antiproliferative effects of compound 8m were found to be the most profound in inhibiting growth of MVECs. Therefore, it was further investigated in T47D breast cancer cells, DU145 and PC-3 prostate cancer cells, LL/2 murine Lewis lung cancer cells and B16F0 melanoma cells.

Design and synthesis of novel 1,2,3-benzotriazines
Recently, it was reported that 1,2,4-benzotriazines were inhibitors of Src and the binding model of 1,2,4-benzotriazines with Src showed the importance of the N1 and N2 atoms [13,14]. We considered that VEGFR and Src might react with substrates in a similar fashion as both of them are tyrosine kinases. Both PTK787 and ZD6474 contain a substituted anilino group. ZD6474 contains a methoxy group at the C6 position and a short side chain at the C7 position. We thought that these substituents are required for the antitumor activity. Thus, we decided using 1,2,3-benzotriazine to replace the phthalazine in PTK787 or the quinazoline in ZD6474 and to add a methoxy group at the C6 position and a short alkoxy group at the C7 position of 1,2,3-benzotriazine. A series of target compounds, 8a-r, were obtained and their structures are listed in Table 1.
Compounds 8a-r were prepared according to the synthetic route outlined in Scheme 1. 4-Cyano-2methoxyphenol (1) reacted with various alkyl halides to afford intermediates 2a-c in satisfactory yields. Compounds 2a-c were selectively nitrated at 30 o C to give nitro compounds 3a-c in 87-95% yields. The reduction of the nitro compounds 3a-c to the corresponding amines 4a-c was catalyzed by Pd/C [15]. Compounds 4a-c were diazotized and then coupled with substituted anilines at 0 o C, followed by chromatographic purification to afford triazenes 6b-r. Compound 6a was prepared by the coupling of diazotized 2-aminobenzonitrile (5) and 4-chloroaniline. The cyclization of compounds 6ar in 70% ethanol formed intermediates 7a-r, which then rearranged to compounds 8a-r after refluxing in acetic acid [16]. As intermediates 7a-r were unstable, they were used directly in the next step without further purification.

Antiproliferative activities in MVECs
The antiproliferative effects of compounds 8a-r in MVECs were determined (Table 1) and the structure-activity relationships were analyzed. The results revealed that compounds with a methoxy group (R 2 ) at the C6 position and an alkoxy group (R 1 ) at the C7 position (8b-r) had an increased activity compared to that of compound 8a which did not have substitutions at the C6 and C7 positions. The compounds with a 3-chloropropoxy group (R 1 ) at the C7 position (8h-r) were more active than compounds with an ethoxy group (8b-d) or a pentyloxy group (8e-g).
By comparing the activities of compounds with a methoxy group at the C6 position and a 3chloropropoxy group at the C7 position, but with a different substituted group (R 3 ) at the C4 anilino group (8h-r), it revealed that a substitution at the C4 anilino increased the antiproliferative activity in MVECs compared to compound 8r, which did not have a substitution at the C4 anilino group. The electronic effect of a substituent on the anilino group did not influence the antiproliferative activities (comparing compounds 8k-o). Compounds with two substituents at the C3' and C4' positions of the C4 anilino group (8i, 8m, and 8p) were more active than compounds with two substituents at the C3' and C5' positions (8h) or compounds with one substituent (8j, 8k, 8l, 8n, 8o, 8q). Compound 8m was the most potent one in inhibiting proliferation of MVECs with a GI 50 value of 7.98 μM. The compounds with substituents at the anilino group (compounds 8h-q) were more effective than PTK787 in inhibiting growth of MVECs after the introduction of a methoxy group at the C6 position and a 3chloropropoxy group at the C7 position. The antiproliferative effects of compound 8m were then tested in tumor cell lines. The antiproliferative effects of compound 8m were determined in human T47 breast cancer cells, DU-145 and PC-3 prostate cancer cells, murine LL/2 Lewis lung cancer cells and B16F0 melanoma cells using MTT assay (Table 2). Compound 8m was more effective than PTK787 to inhibit cell growth in all the tested cell lines.

Conclusions
In summary, our data indicate that 1,2,3-benzotriazines substituted with a methoxy group at the C6 position and a 3-chloropropoxy group at the C7 position exhibit antiproliferative activities. Most of these compounds are more effective than PTK787 in inhibiting proliferation of MVECs. Compound 8m is the most potent and exhibits greater anti-tumor cell growth activity activities than PTK787. Since we did not stimulate the cell growth of MVECs and tumor cells with VEGF, the observed antiproliferative effects of these compounds, including PTK787, may be through a pathway independent of VEGFR inhibition. Because of the high similarity of 1,2,3-benzotriazine and 1,2,4benzotriazine, the increased antiproliferative effects of the designed compounds in tumor cells may be due to the inhibition of Src, or other tyrosine kinases which is not inhibited by PTK787 [17]. The effects of our novel 1,2,3-benzotriazines on the activities of VEGFRs, Src and other kinases are under investigation.

General
Reagents (analytical grade) were obtained from commercial suppliers and used without further purification unless otherwise noted. 1 H-and 13 C-NMR spectra were recorded on a Bruker ARX-300 instrument with tetramethylsilane as the internal standard. IR spectra were recorded on a Bruker IR-27G spectrometer. MS were determined on either Finnigan MAT/USA spectrometer (LC-MS). Elemental analysis was determined on a Carlo-Erba 1106 Elemental analysis instrument (Carlo Erba, Milan, Italy). The melting points were determined on an electrically heated X4 digital visual melting point apparatus and were uncorrected. The structures of the synthesized compounds were characterized by melting point (mp), infrared (IR) spectra, nuclear magnetic resonance (NMR) spectra and mass spectral (MS) data.

General procedure for the synthesis of 4-substituted-3-methoxybenzonitriles 2a-c
A solution of 4-cyano-2-methoxyphenol (1, 1.00 g, 6.70 mmol) in anhydrous DMF (4.00 mL) was stirred and cooled with a water bath. K 2 CO 3 (1.39 g, 10.1 mmol) was added and the mixture was stirred at 20 o C for 1 h. The corresponding alkyl halide (8.13 mmol) was added dropwise, the mixture was stirred at room temperature overnight, then heated at 37 o C for 6 h and finally poured into a mixture of ice/H 2 O (100 mL). After stirring for 10 min, a precipitate was formed. It was filtered off, washed with H 2 O, and air-dried to yield the 4-substituted-3-methoxybenzonitriles 2a-c as white solids.
Cell growth inhibition assay: The antiproliferative activities of these compounds were determined by the MTT assay. Cells (2×10 3 cells/well in 96-well plates) were incubated for 24 h, then various concentrations of each compound were added to each well and the cells were cultured for another 4 days at 37 o C. MTT solution (50 μL of 2 mg/mL) was added per well and the culture continued for an additional 4 h. The medium was removed by aspiration and the cells were dissolved in 200 μL DMSO. The absorbance at 570 nm was measured in the 96-well plate reader. Growth inhibition is reported as compared to untreated cells (%) and GI 50 concentration was calculated [18].