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 1,3-benzodizine. 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.

Figure 1

Structures of vatalanib succinate (PTK787) and vandetanib (ZD6474).

Results and Discussion

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.

Table 1

the structures of target compounds and tdeir antiproliferative effects in MVECs.

Compounds	R1	R2	R3	GI50 (μM) a
8a	H	H	4-Cl	>80
8b	CH3CH2O	CH3O	4-Cl	26.54±1.43
8c	CH3CH2O	CH3O	4-CH3	42.56±2.79
8d	CH3CH2O	CH3O	3-Cl, 4-F	38.81±1.86
8e	CH3(CH2)4O	CH3O	4-Cl	25.35±1.02
8f	CH3(CH2)4O	CH3O	3-OCF3	42.64±2.27
8g	CH3(CH2)4O	CH3O	4-OCF3	37.98±1.98
8h	Cl(CH2)3O	CH3O	3,5-di-Cl	28.84±1.40
8i	Cl(CH2)3O	CH3O	3,4-di-Cl	11.02±0.49
8j	Cl(CH2)3O	CH3O	4-Cl	18.59±1.31
8k	Cl(CH2)3O	CH3O	4-CH3	17.08±0.65
8l	Cl(CH2)3O	CH3O	4-F	23.05±0.92
8m	Cl(CH2)3O	CH3O	3-Cl, 4-F	7.98±0.35
8n	Cl(CH2)3O	CH3O	3-OCF3	24.86±0.86
8o	Cl(CH2)3O	CH3O	4-OCF3	23.19±1.12
8p	Cl(CH2)3O	CH3O	3-CF3, 4-F	15.22±0.51
8q	Cl(CH2)3O	CH3O	3-CF3	21.38±1.54
8r	Cl(CH2)3O	CH3O	H	>80
vatalanib succinate				38.15±2.07
(PTK787)

a. GI50 is the concentration that inhibits 50% of cell growth. The cells were treated with various concentrations of the tested compounds for 4 days and cell growth inhibition was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data shown are means ± SD of three independent experiments.

Compounds 8a-r were prepared according to the synthetic route outlined in Scheme 1. 4-Cyano-2-methoxyphenol (1) reacted with various alkyl halides to afford intermediates 2a-c in satisfactory yields. Compounds 2a-c were selectively nitrated at 30 oC 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 oC, 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 6a-r 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.

Scheme 1

Synthetic route to the target compounds.

Reagents and conditions: (a) alkyl halides, DMF, K2CO3, 37 oC; (b) HNO3, 30 oC; (c) Pd/C, cyclohexene, ethanol, reflux; (d) NaNO2/HCl, 0 oC; substituted aniline, 0 oC; (e) 70% ethanol, reflux; (f) AcOH, reflux.

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 (R2) at the C6 position and an alkoxy group (R1) 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 (R1) 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 3-chloropropoxy group at the C7 position, but with a different substituted group (R3) 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 GI50 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 3-chloropropoxy 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.

Table 2

Antiproliferative effects of compound 8m and PTK787 in several tumor cell lines.

Compounds	GI50 (μM) a
	T47D	DU-145	PC-3	LL/2	B16F0
8m	5.04±0.32	4.96±0.43	6.91±0.30	3.79±0.19	5.19±0.22
PTK787	36.32±1.88	41.28±2.47	63.68±3.55	31.15±2.01	18.71±1.67

a. Cells were treated with various concentrations of the tested compounds for 4 days and the cell growth inhibition was determined using the MTT assay. Data shown are means ± SD of three independent experiments.

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 1,2,4-benzotriazine, 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.

Experimental

General

Reagents (analytical grade) were obtained from commercial suppliers and used without further purification unless otherwise noted. 1H- and 13C-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. K2CO3 (1.39 g, 10.1 mmol) was added and the mixture was stirred at 20 oC 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 oC for 6 h and finally poured into a mixture of ice/H2O (100 mL). After stirring for 10 min, a precipitate was formed. It was filtered off, washed with H2O, and air-dried to yield the 4-substituted-3-methoxybenzonitriles 2a-c as white solids.

4-Ethoxy-3-methoxybenzonitrile (2a): Yield: 91.8%; mp: 102-103 oC. 1H-NMR (DMSO-d) δ: 7.36 (2H, m, H-2, H-6), 7.04 (1H, s, H-5), 4.11 (2H, q, J = 6.9 Hz, CH3CH2O-), 3.81 (3H, s, -OCH3), 1.35 (3H, t, J = 6.9 Hz, CH3CH2O-); LC-MS: 178.1 (M+H)+; Anal. Calcd. for C10H11NO2: C 67.78, H 6.26, N 7.90; Found: C 67.79, H 6.24, N 7.91.

3-Methoxy-4-pentyloxybenzonitrile (2b): Yield: 89.0%; mp: 55-56 oC. 1H-NMR (DMSO-d) δ: 7.37 (2H, m, H-2, H-6), 7.09 (1H, d, J = 8.1 Hz, H-5), 4.02 (2H, t, CH3(CH2)3CH2O-), 3.79 (3H, s, -OCH3), 1.71 (2H, m, CH3CH2CH2CH2CH2O-), 1.34 (4H, m, CH3CH2CH2CH2CH2O-), 0.88 (3H, t, J = 6.9 Hz, CH3CH2CH2CH2CH2O-); LC-MS: 220.1 (M+H)+; Anal. Calcd. for C13H17NO2: C 71.21, H 7.81, N 6.39; Found: C 71.20, H 7.80, N 6.39.

4-(3-Chloropropoxy)-3-methoxybenzonitrile (2c): Yield: 87.2%. 1H-NMR (DMSO-d) δ: 7.41 (2H, m, H-2, H-6), 7.15 (1H, d, J = 8.8 Hz, H-5), 4.16 (2H, t, J = 6.0 Hz, ClCH2CH2CH2O-), 3.79 (5H, m, -OCH3, ClCH2CH2CH2O-), 2.20 (2H, m, ClCH2CH2CH2O-); LC-MS: 226.1 (M+H)+; Anal. Calcd. for C11H12ClNO2: C 58.54, H 5.36, N 6.21; Found: C 58.52, H 5.37, N 6.20.

General procedure for the synthesis of 4-substituted-5-methoxy-2-nitrobenzonitriles, 3a-c

A solution of 4-substituted-3-methoxybenzonitriles 2 (9.10 mmol) in nitric acid (10 mL) was heated to 30 oC for 2 h, poured into ice-water (100 mL), filtered, and washed with water to afford compounds 3 as light yellow solids.

4-Ethoxy-5-methoxy-2-nitrobenzonitrile (3a): Yield: 88.1%; mp: 198-199 oC. 1H-NMR (DMSO-d) δ: 7.85 (1H, s, H-3), 7.70 (1H, s, H-6), 4.25 (2H, q, J = 6.9 Hz, CH3CH2O-), 3.97 (3H, s, -OCH3), 1.37 (3H, t, J = 6.9 Hz, CH3CH2O-); LC-MS: 223.1 (M+H)+; Anal. Calcd. for C10H10N2O4: C 54.05, H 4.54, N 12.61; Found: C 54.03, H 4.55, N 12.60.

5-Methoxy-2-nitro-4-pentyloxybenzonitrile (3b): Yield: 92.9%; mp: 136-137 oC. 1H-NMR (DMSO-d) δ: 7.89 (1H, s, H-3), 7.80 (1H, s, H-6), 4.16 (2H, t, J = 6.3 Hz, CH3CH2CH2CH2CH2O-), 3.95 (3H, s, -OCH3), 1.72 (2H, m, CH3CH2CH2CH2CH2O-), 1.35 (4H, m, CH3CH2CH2CH2CH2O-), 0.89 (3H, t, J = 6.9 Hz, CH3CH2CH2CH2CH2O-); LC-MS: 265.1 (M+H)+; Anal. Calcd. for C13H16N2O4: C 59.08, H 6.10, N 10.60; Found: C 59.10, H 6.11, N 10.59.

4-(3-Chloropropoxy)-5-methoxy-2-nitrobenzonitrile (3c): Yield: 95.3%; mp: 133-134 oC. 1H-NMR (DMSO-d) δ: 7.91 (1H, s, H-3), 7.71 (1H, s, H-6), 4.32 (2H, t, J = 6.0 Hz, ClCH2CH2CH2O-), 4.00 (3H, s, -OCH3), 3.78 (2H, t, J = 6.3 Hz, ClCH2CH2CH2O-), 2.23 (2H, m, ClCH2CH2CH2O-); LC-MS: 271.0 (M+H)+; Anal. Calcd. for C11H11ClN2O4: C 48.81, H 4.10, N 10.35; Found: C 48.82, H 4.11, N 10.34.

General procedure for the synthesis of 4-substituted-2-amino-5-methoxybenzonitriles, 4a-c

A mixture of compound 3 (3.69 mmol) and Pd/C (10%, 7.00 g) in anhydrous ethanol (10.0 mL) was stirred and heated under reflux. Cyclohexene (2.30 mL, 22.6 mmol) was added dropwise. The mixture was refluxed overnight, then cooled to 40 °C, filtered, and washed with ethanol. The filtrate was concentrated to yield a solid. The crude product was suspended in ethanol, stirred at 40 °C for 30 min, cooled to room temperature, filtered to produce pure compound 4 as a yellow solid.

2-Amino-4-ethoxy-5-methoxybenzonitrile (4a): Yield: 49.1%; mp: 135-136 °C. 1H-NMR (DMSO-d) δ: 6.87 (1H, s, H-6), 6.39 (1H, s, H-3), 5.58 (2H, s, NH2), 3.96 (2H, q, J = 6.9 Hz, CH3CH2O-), 3.65 (3H, s, -OCH3), 1.33 (3H, t, J = 6.9 Hz, CH3CH2O-); LC-MS: 193.1 (M+H)+; Anal. Calcd. for C10H12N2O2: C 62.49, H 6.29, N 14.57; Found: C 62.50, H 6.30, N 14.58.

2-Amino-5-methoxy-4-pentyloxybenzonitrile (4b): Yield: 50.1%; mp: 136-137 °C. 1H-NMR (DMSO-d) δ: 6.85 (1H, s, H-6), 6.38 (1H, s, H-3), 5.57 (2H, s, NH2), 3.88 (2H, t, J = 6.3 Hz, CH3CH2CH2CH2CH2O-), 3.63 (3H, s, -OCH3), 1.70 (2H, m, CH3CH2CH2CH2CH2O-), 1.34 (4H, m, CH3CH2CH2CH2CH2O-), 0.88 (3H, t, J = 6.9 Hz, CH3CH2CH2CH2CH2O-); LC-MS: 235.1 (M+H)+; Anal. Calcd. for C13H18N2O2: C 66.64, H 7.74, N 11.96; Found: C 66.62, H 7.73, N 11.97.

2-Amino-4-(3-chloropropoxy)-5-methoxybenzonitrile (4c): Yield: 47.2%; mp: 118-119 oC. 1H-NMR (DMSO-d δ: 6.89 (1H, s, H-6), 6.43 (1H, s, H-3), 5.62 (2H, s, NH2), 4.03 (2H, t, J = 6.1 Hz, ClCH2CH2CH2O-), 3.77 (2H, t, J = 6.4 Hz, ClCH2CH2CH2O-), 3.65 (3H, s, -OCH3), 2.18 (2H, m, ClCH2CH2CH2O-); LC-MS: 241.1 (M+H)+; Anal. Calcd. for C11H13ClN2O2: C 54.89, H 5.44, N 11.64; Found: C 54.88, H 5.45, N 11.63.

General procedure for the synthesis of 7-alkoxy-6-methoxy-4-substituted-1,2,3-benzotriazines 8a-r

Compound 4 (or compound 5 when preparing 8a, 10.0 mmol) in 10N-hydrochloric acid (30.0 mL) was cooled to 0 oC and diazotized with sodium nitrite (0.71 g) in water (10.0 mL). The diazonium solution was neutralized with excess of sodium acetate trihydrate and stirred for 2 h at 0 oC with a corresponding substituted aniline (10.0 mmol). The solution was kept overnight at 4 oC, filtered, and washed with water. The crude material was purified on a silica gel column with petroleum ether : ethyl acetate (v/v) = 10:1 to yield a yellow solid 6. Compound 6 was boiled in 70% ethanol (25.0 mL) for 1 h, then the solution was evaporated under reduced pressure to dryness. Acetic acid (10.0 mL) was added and the solution was refluxed for 2 h, cooled, poured into water (100 mL), filtered, and dried to afford 7-alkoxyl-6-methoxy-4-substituted-1,2,3-benzotriazines 8.

4-(4-chloroanilino)-1,2,3-benzotriazine (8a): Yield: 68.9%; mp: 230 oC. IR (KBr): 3266, 3086, 1621, 1559, 1493, 1424, 1147, 766 cm-1; 1H-NMR (DMSO-d) δ: 10.02 (1H, s, NH), 8.62 (1H, d, J = 8.1 Hz), 8.23 (1H, d, J = 8.1 Hz), 8.13 (1H, t, J = 8.2 Hz), 8.05 (1H, m), 7.90 (2H, m), 7.52 (2H, m); LC-MS: 257.1 (M+H)+; 13C-NMR (DMSO-d) δ : 151.02, 143.53, 137.58, 134.73, 132.38, 128.66, 128.06, 127.47, 124.28, 121.87, 109.04; Anal. Calcd. for C13H9ClN4: C 60.83, H 3.53, N 21.83; Found: C 60.79, H 3.51, N 21.85.

4-(4-Chloroanilino)-7-ethoxy-6-methoxy-1,2,3-benzotriazine (8b): Yield: 72.1%; mp: 230 oC. IR (KBr): 3296, 2928, 1612, 1560, 1494, 1423, 1291, 818 cm-1; 1H-NMR (DMSO-d) δ: 9.68 (1H, s, NH), 7.92 (3H, m, H-8, H-2’, H-6’), 7.51 (3H, m, H-8, H-3’, H-5’), 4.29 (2H, q, J = 6.6 Hz, CH3CH2O-), 4.03 (3H, s, -OCH3), 1.44 (3H, t, J = 6.9 Hz, CH3CH2O-); LC-MS: 331.1 (M+H)+; 13C-NMR (DMSO-d) δ: 154.22 (C-7), 153.27 (C-6), 150.12 (C-10), 141.65 (C-9), 137.95 (C-1’), 128.57, 127.48, 123.95, 106.78 (C-5), 103.84 (C-4), 100.09 (C-8), 64.74 (CH3CH2O-), 56.73 (-OCH3), 14.42 (CH3CH2O-); Anal. Calcd. for C16H15ClN4O2: C 58.10, H 4.57, N 16.94; Found: C 58.01, H 4.56, N 16.96.

7-Ethoxy-6-methoxy-4-(4-methylanilino)-1,2,3-benzotriazine (8c): Yield: 73.2%; mp: 249-251 oC. IR (KBr): 33381, 2980, 1614, 1512, 1426, 1283, 1244, 1103, 856, 816 cm-1; 1H-NMR (DMSO-d) δ: 9.50 (s, 1H, NH), 7.90 (s, 1H, H-8), 7.70 (d, 2H, J = 8.2 Hz, ArH-2’H, 6’H), 7.53 (s, 1H, H-5), 7.25 (d, 2H, J = 8.2 Hz, ArH-3’H, 5’H), 4.29 (q, 2H, J = 6.9 Hz, CH3CH2O-), 4.02 (s, 3H, CH3O-), 2.34 (s, 3H, ArH-4’CH3), 1.44 (t, 3H, J = 6.8 Hz, CH3CH2O-); LC-MS: 311.2 (M+H)+; 13C-NMR (DMSO-d) δ: 154.16, 153.19, 150.30, 141.62, 138.86, 128.72, 124.04, 122.70, 106.77, 103.82, 100.12, 64.73, 56.72, 20.89, 14.46; Anal. Calcd. for C17H18N4O2: C 65.79, H 5.85, N 18.05; Found: C 65.68, H 5.81, N 18.07.

4-(3-Chloro-4-fluoroanilino)-7-ethoxy-6-methoxy-1,2,3-benzotriazine (8d): Yield: 64.1%; mp: 248-250 oC. IR (KBr): 3288, 1609, 1554, 1500, 1417, 1292, 1237, 1222, 1106 cm-1; 1H-NMR (DMSO-d) δ: 9.66 (s, 1H, NH), 8.17 (m, 1H, ArH-2’H), 7.87 (s, 1H, H-8), 7.82 (m, 1H, ArH-5’H), 7.58 (s, 1H, H-5), 7.51 (d, 1H, J = 9 Hz, ArH-6’H), 4.31 (q, 2H, J = 6.9 Hz, CH3CH2O-), 4.03 (s, 3H, CH3O-), 1.44 (t, 3H, J = 6.9 Hz, CH3CH2O-); LC-MS: 349.2 (M+H)+; 13C-NMR (DMSO-d) δ: 154.43, 154.29, 153.62, 152.10, 150.35, 141.80, 136.65, 124.10, 123.00, 122.90, 119.55, 117.10, 116.80, 107.15, 104.13, 100.28, 64.69, 56.65, 14.46; Anal. Calcd. for C16H14ClFN4O2: C 55.10, H 4.05, N 16.06; Found: C 55.00, H 4.07, N 16.04.

4-(4-Chloroanilino)-6-methoxy-7-pentyloxy-1,2,3-benzotriazine (8e): Yield: 69.3%; mp: 213 oC. IR (KBr): 3378, 2954, 1612, 1562, 1507, 1426, 1245, 817 cm-1; 1H-NMR (DMSO-d) δ: 9.63 (1H, s, NH), 7.91 (3H, m, H-8, H-2’, H-6’), 7.53 (3H, m, H-8, H-3’, H-5’), 4.24 (2H, t, J = 6.0 Hz, CH3CH2CH2CH2CH2O-), 4.03 (3H, s, -OCH3), 1.82 (2H, m, CH3CH2CH2CH2CH2O-), 1.40 (4H, m, CH3CH2CH2CH2CH2O-), 0.92 (3H, t, J = 6.0 Hz, CH3CH2CH2CH2CH2O); LC-MS: 373.2 (M+H)+; 13C-NMR (DMSO-d) δ: 154.31 (C-7), 153.27 (C-6), 150.30 (C-10), 141.63 (C-9), 138.88 (C-1’), 128.73, 127.40, 123.91, 106.72 (C-5), 103.82 (C-4), 100.05 (C-8), 68.98 (CH3CH2CH2CH2CH2O-), 56.79 (-OCH3), 28.17 (CH3CH2CH2CH2CH2O-), 27.78 (CH3CH2CH2CH2CH2O-), 21.98 (CH3CH2CH2CH2CH2O-), 14.04 (CH3CH2CH2CH2CH2O-); Anal. Calcd. for C19H21ClN4O2: C 61.21, H 5.68, N 15.03; Found: C 61.11, H 5.64, N 14.99.

6-Methoxy-7-pentyloxy-4-(3-trifluoromethoxyanilino)-1,2,3-benzotriazine (8f): Yield: 73.1%; mp: 181-183 oC. IR (KBr): 3421, 2931, 1613, 1508, 1446, 1258, 1216, 1163, 1105, 845, 784 cm-1; 1H-NMR (DMSO-d) δ: 9.72 (s, 1H, NH), 8.04 (s, 1H, ArH-2’H), 7.92 (d, 1H, J = 8.9 Hz, ArH-4’H), 7.91 (s, 1H, H-8), 7.60 (s, 1H, H-5), 7.56 (t, 2H, J = 8.6 Hz, ArH-5’H), 7.14 (d, 1H, J = 8.7 Hz, ArH-6’H), 4.24 (t, 2H, J = 6.3 Hz, CH3CH2CH2CH2CH2O-), 4.05 (s, 3H, CH3O-), 1.83 (m, 2H, CH3CH2CH2CH2CH2O-), 1.42 (m, 4H, CH3CH2CH2CH2CH2O-), 0.92 (t, 3H, J = 6.9 Hz, CH3CH2CH2CH2CH2O-); LC-MS: 423.2 (M+H)+; 13C-NMR (DMSO-d) δ: 154.23, 153.52, 150.28, 148.76, 141.90, 141.06, 130.41, 124.25, 121.80, 118.10, 115.20, 114.04, 106.92, 104.11, 100.19, 68.98, 56.79, 28.17, 27.78, 21.98, 14.04; Anal. Calcd. for C20H21F3N4O3: C 56.87, H 5.01, N 13.26; Found: C 56.81, H 5.00, N 13.23.

6-Methoxy-7-pentyloxy-4-(4-trifluoromethoxyanilino)-1,2,3-benzotriazine (8g): Yield: 70.2%; mp: 230-232 oC. IR (KBr): 3426, 2959, 1615, 1510, 1428, 1247, 1200, 1165, 1102, 850, 786 cm-1; 1H-NMR (DMSO-d) δ: 9.69 (s, 1H, NH), 7.98 (d, 2H, J = 9.0 Hz, ArH-3’H, 5’H), 7.90 (s, 1H, H-8), 7.58 (s, 1H, H-5), 7.46 (d, 2H, J = 8.7 Hz, ArH-2’H, 6’H), 4.24 (t, 2H, J = 6.3 Hz, CH3CH2CH2CH2CH2O-), 4.04 (s, 3H, CH3O-), 1.83 (m, 2H, CH3CH2CH2CH2CH2O-), 1.42 (m, 4H, CH3CH2CH2CH2CH2O-), 0.92 (t, 3H, J = 6.9 Hz, CH3CH2CH2CH2CH2O-); LC-MS: 423.2 (M+H)+; 13C-NMR (DMSO-d) δ: 154.31, 153.27, 150.30, 144.05, 141.54, 137.91, 125.24, 123.93, 122.14, 121.45, 106.86, 103.83, 100.08, 68.97, 56.76, 28.17, 27.76, 21.97, 14.04; Anal. Calcd. for C20H21F3N4O3: C 56.87, H 5.01, N 13.26; Found: C 56.88, H 5.03, N 13.27.

7-(3-Chloropropoxy)-4-(3,5-dichloroanilino)-6-methoxy-1,2,3-benzotriazine (8h): Yield: 72.9%; mp: 110-112 oC. IR (KBr): 3430, 2919, 1620, 1508, 1423, 1289, 849 cm-1; 1H-NMR (DMSO-d) δ: 9.74 (1H, s, NH), 8.10 (2H, d, J = 1.77 Hz, H-2’, H-6’), 7.91 (1H, s, H-8), 7.68 (1H, s, H-5), 7.37 (1H, s, H-4’), 4.39 (2H, t, J = 6.15 Hz, ClCH2CH2CH2O-), 4.06 (3H, s, -OCH3), 3.84 (2H, t, J = 6.5 Hz, ClCH2CH2CH2O-), 2.30 (2H, m, ClCH2CH2CH2O-); LC-MS: 413.0 (M+H)+; 13C-NMR (DMSO-d) δ: 154.57 (C-7), 153.92 (C-6), 150.31 (C-10), 142.11 (C-9), 141.94, 134.28, 122.82, 120.06, 107.51 (C-5), 104.54 (C-4), 100.44 (C-8), 66.29 (ClCH2CH2CH2O-), 57.26 (-OCH3), 42.19 (ClCH2CH2CH2O-), 31.84 (ClCH2CH2CH2O-); Anal. Calcd. for C17H15Cl3N4O2: C 49.36, H 3.65, N 13.54; Found: C 49.28, H 3.66, N 13.56.

7-(3-Chloropropoxy)-4-(3,4-dichlorophenyl)-6-methoxy-1,2,3-benzotriazine (8i): Yield: 72.4%; mp: 120-122 oC. IR (KBr): 3424, 1616, 1510, 1426, 1281, 848 cm-1; 1H-NMR (DMSO-d) δ: 9.74 (1H, s, NH), 8.31 (1H, d, J = 2.31 Hz, H-2’), 7.93 (2H, m, H-8, H-6’), 7.71 (1H, m, H-5’), 7.67 (1H, s, H-5), 4.38 (2H, t, J = 6.0 Hz, ClCH2CH2CH2O-), 4.05 (3H, s, -OCH3), 3.84 (2H, t, J = 6.4 Hz, ClCH2CH2CH2O-), 2.30 (2H, m, ClCH2CH2CH2O-); LC-MS: 413.0 (M+H)+; 13C-NMR (DMSO-d) δ: 154.11 (C-7), 153.43 (C-6), 149.95 (C-10), 141.66 (C-9), 139.23, 130.84, 130.51, 125.02, 123.14, 121.90, 107.10 (C-5), 104.11 (C-4), 100.08 (C-8), 65.86 (ClCH2CH2CH2O-), 56.85 (-OCH3), 41.87 (ClCH2CH2CH2O-), 31.46 (ClCH2CH2CH2O-); Anal. Calcd. for C17H15Cl3N4O2: C 49.36, H 3.65, N 13.54; Found: C 49.31, H 3.66, N 13.56.

4-(4-Chloroanilino)-7-(3-chloropropoxy)-6-methoxy-1,2,3-benzotriazine (8j): Yield: 67.9%; mp: 253 oC. IR (KBr): 3425, 2925, 1616, 1561, 1495, 1424, 1245, 839 cm-1; 1H-NMR (DMSO-d) δ: 9.66 (1H, s, NH), 7.91 (3H, m, H-8, H-2’, H-6’), 7.62 (1H, s, H-5), 7.50 (2H, d, J = 8.8 Hz, H-3’, H-5’), 4.37 (2H, t, J = 6.0 Hz, ClCH2CH2CH2O-), 4.04 (3H, s, -OCH3), 3.84 (2H, t, J = 6.4 Hz, ClCH2CH2CH2O-), 2.29 (2H, m, ClCH2CH2CH2O-); LC-MS: 379.0 (M+H)+; 13C-NMR (DMSO-d) δ: 153.99 (C-7), 153.29 (C-6), 150.12 (C-10), 141.57 (C-9), 137.91 (C-1’), 128.59, 127.54, 123.99, 107.07 (C-5), 104.07 (C-4), 100.23 (C-8), 65.82 (ClCH2CH2CH2O-), 56.82 (-OCH3), 41.87 (ClCH2CH2CH2O-), 31.47 (ClCH2CH2CH2O-); Anal. Calcd. for C17H16Cl2N4O2: C 53.84, H 4.25, N 14.77; Found: C 53.79, H 4.24, N 14.71.

7-(3-Chloropropoxy)-6-methoxy-4-(4-methylanilino)-1,2,3-benzotriazine (8k): Yield: 70.1%; mp: 247 oC. IR (KBr): 3420, 2924, 1615, 1513, 1425, 1282, 1242, 859 cm-1; 1H-NMR (DMSO-d) δ: 9.55 (1H, s, NH), 7.92 (1H, s, H-8), 7.69 (2H, d, J = 8.4 Hz, H-2’, H-6’), 7.59 (1H, s, H-5), 7.25 (2H, d, J = 8.4 Hz, H-3’, H-5’), 4.37 (2H, t, J = 6.0 Hz, ClCH2CH2CH2O-), 4.03 (3H, s, -OCH3), 3.84 (2H, t, J = 6.3 Hz, ClCH2CH2CH2O-), 2.34 (3H, s, -CH3), 2.29 (2H, m, ClCH2CH2CH2O-); LC-MS: 359.1 (M+H)+; 13C-NMR (DMSO-d) δ: 154.23 (C-7), 153.51 (C-6), 150.62 (C-10), 141.59 (C-9), 136.43 (C-1’), 133.64, 129.48, 123.22, 107.28 (C-5), 104.42 (C-4), 100.86 (C-8), 66.14 (ClCH2CH2CH2O-), 57.22 (-OCH3), 42.24 (ClCH2CH2CH2O-), 31.84 (ClCH2CH2CH2O-), 20.99(Ph-CH3); Anal. Calcd. for C18H19ClN4O2: C 60.25, H 5.34, N 15.61; Found: C 60.28, H 5.33, N 15.56.

7-(3-Chloropropoxy)-4-(4-fluoroanilino)-6-methoxy-1,2,3-benzotriazine (8l): Yield: 71.2%; mp: 166 oC. IR (KBr): 3437, 1620, 1510, 1428, 1284, 842 cm-1; 1H-NMR (DMSO-d) δ: 9.63 (1H, s, NH), 7.91 (1H, s, H-8), 7.84 (2H, m, H-2’, H-6’), 7.61 (1H, s, H-5), 7.29 (2H, t, J = 8.7 Hz, H-3’, H-5’), 4.37 (2H, t, J = 6.0 Hz, ClCH2CH2CH2O-), 4.03 (3H, s, -OCH3), 3.84 (2H, t, J = 6.4 Hz, ClCH2CH2CH2O-), 2.29 (2H, m, ClCH2CH2CH2O-); LC-MS: 363.1 (M+H)+; 13C-NMR (DMSO-d) δ: 160.67, 157.48, 154.28 (C-7), 153.56 (C-6), 150.64 (C-10), 141.79 (C-9), 135.46 (C-1’), 125.15, 125.04, 115.79, 115.50, 107.40 (C-5), 104.29 (C-4), 100.67 (C-8), 66.18 (ClCH2CH2CH2O-), 57.17 (-OCH3), 42.22 (ClCH2CH2CH2O-), 31.86 (ClCH2CH2CH2O-); Anal. Calcd. for C17H16ClFN4O2: C 56.28, H 4.45, N 15.44; Found: C 56.20, H 4.43, N 15.56.

4-(3-Chloro-4-fluoroanilino)-7-(3-chloropropoxy)-6-methoxy-1,2,3-benzotriazine (8m): Yield: 77.1%; mp: 138-139 oC. IR (KBr): 3314, 2923, 1616, 1571, 1511, 1427, 1285, 849 cm-1; 1H-NMR (DMSO-d) δ: 9.70 (1H, s, NH), 8.18 (1H, d, J = 4.4 Hz, H-2’), 7.90 (1H, s, H-8), 7.82 (1H, m, H-6’), 7.64 (1H, s, H-5), 7.51 (1H, t, J = 9.1 Hz, H-5’), 4.38 (2H, t, J = 5.9 Hz, ClCH2CH2CH2O-), 4.04 (3H, s, -OCH3), 3.84 (2H, t, J = 6.4 Hz, ClCH2CH2CH2O-), 2.29 (2H, m, ClCH2CH2CH2O-); LC-MS: 397.0 (M+H)+; 13C-NMR (DMSO-d) δ: 155.47 (C-4’), 154.32 (C-7), 153.64 (C-6), 152.25 (C-4’), 150.32 (C-10), 141.81 (C-9), 136.57 (C-1’), 124.15 (C-2’), 122.93 (C-6’), 122.84 (C-6’), 119.47 (C-6’), 117.14 (C-5’), 116.85 (C-5’), 107.33 (C-5), 104.28 (C-4), 100.56 (C-8), 66.19 (ClCH2CH2CH2O-), 57.21 (-OCH3), 42.18 (ClCH2CH2CH2O-), 31.86 (ClCH2CH2CH2O-); Anal. Calcd. for C17H15Cl2FN4O2: C 51.40, H 3.81, N 14.10; Found: C 51.41, H 3.85, N 14.08.

7-(3-Chloropropoxy)-6-methoxy-4-(3-trifluoromethoxyanilino)-1,2,3-benzotriazine (8n): Yield: 65.3%; mp: 154-155 oC. IR (KBr): 3427, 1615, 1572, 1511, 1448, 1256, 854 cm-1; 1H-NMR (DMSO-d) δ: 9.75 (1H, s, NH), 8.05 (1H, s, H-2’), 7.93 (2H, s, H-8, H-6’), 7.65 (1H, s, H-5), 7.59 (1H, t, J = 8.2 Hz, H-5’), 7.15 (1H, d, J = 7.9 Hz, H-4’), 4.38 (2H, t, J = 5.9 Hz, ClCH2CH2CH2O-), 4.06 (3H, s, -OCH3), 3.84 (2H, t, J = 6.4 Hz, ClCH2CH2CH2O-), 2.29 (2H, m, ClCH2CH2CH2O-); LC-MS: 429.1 (M+H)+; 13C-NMR (DMSO-d) δ: 154.34 (C-7), 153.65 (C-6), 150.40 (C-10), 148.82, 141.96 (C-9), 141.12 (C-1’), 130.54, 124.05, 122.30, 118.90, 115.91, 114.64, 107.32 (C-5), 104.41 (C-4),100.46 (C-8), 66.15 (ClCH2CH2CH2O-), 57.17 (-OCH3), 42.17 (ClCH2CH2CH2O-), 31.82 (ClCH2CH2CH2O-); Anal. Calcd. for C18H16ClF3N4O3: C 50.42, H 3.76, N 13.07; Found: C 50.37, H 3.78, N 13.06.

7-(3-Chloropropoxy)-6-methoxy-4-(4-trifluoromethoxyanilino)-1,2,3-benzotriazine (8o): Yield: 63.0%; mp: 150 oC. IR (KBr): 3444, 1618, 1574, 1510, 1429, 1247, 852 cm-1; 1H-NMR (DMSO-d) δ: 9.68 (1H, s, NH), 7.93 (2H, d, J = 9.0 Hz, H-2’, H-6’), 7.88 (1H, s, H-8), 7.59 (1H, s, H-5), 7.40 (2H, d, J = 8.7 Hz, H-3’, H-5’), 4.33 (2H, t, J = 5.9 Hz, ClCH2CH2CH2O-), 4.00 (3H, s, -OCH3), 3.79 (2H, t, J = 6.6 Hz, ClCH2CH2CH2O-), 2.25 (2H, m, ClCH2CH2CH2O-); LC-MS: 429.1 (M+H)+; 13C-NMR (DMSO-d) δ: 154.41 (C-7), 153.70 (C-6), 150.54 (C-10), 144.48, 141.97 (C-9), 138.51 (C-1’), 125.64, 124.23, 122.34, 121.79, 107.46 (C-5), 104.43 (C-4), 100.68 (C-8), 66.23 (ClCH2CH2CH2O-), 57.22 (-OCH3), 42.21 (ClCH2CH2CH2O-), 31.85 (ClCH2CH2CH2O-); Anal. Calcd. for C18H16ClF3N4O3: C 50.42, H 3.76, N 13.07; Found: C 50.39, H 3.75, N 13.04.

7-(3-Chloropropoxy)-4-(4-fluoro-3-trifluoromethylanilino)-6-methoxy-1,2,3-benzotriazine (8p): Yield: 71.1%; mp: 150 oC. IR (KBr): 3452, 2920, 1614, 1508, 1432, 1290, 1129 cm-1; 1H-NMR (DMSO-d) δ: 9.82 (1H, s, NH), 8.18 (2H, m, H-6’, H-2’), 7.91 (1H, s, H-8), 7.65 (1H, s, H-5), 7.60 (1H, m, H-5’), 4.38 (2H, t, J = 6.0 Hz, ClCH2CH2CH2O-), 4.05 (3H, s, -OCH3), 3.84 (2H, t, J = 6.3 Hz, ClCH2CH2CH2O-), 2.30 (2H, m, ClCH2CH2CH2O-); LC-MS: 431.1 (M+H)+; 13C-NMR (DMSO-d) δ: 156.36, 154.04 (C-7), 153.35 (C-6), 150.01 (C-10), 141.53 (C-9), 135.74 (C-1’), 128.37, 128.26, 124.46, 120.85, 120.26, 117.61, 117.33, 107.05 (C-5), 103.94 (C-4), 100.10 (C-8), 65.83 (ClCH2CH2CH2O-), 56.80 (-OCH3), 41.78 (ClCH2CH2CH2O-), 31.44 (ClCH2CH2CH2O-); Anal. Calcd. for C18H15ClF4N4O2: C 50.19, H 3.51, N 13.01; Found: C 50.17, H 3.53, N 13.00.

7-(3-Chloropropoxy)-6-methoxy-4-(3-trifluoromethylanilino)-1,2,3-benzotriazine(8q): Yield: 75.2%; mp: 180-181 oC. IR (KBr): 3438, 2925, 1623, 1576, 1510, 1448, 1244, 799 cm-1; 1H-NMR (DMSO-d) δ: 9.81 (1H, s, NH), 8.30 (1H, s, H-2’), 8.25 (1H, d, J = 8.4 Hz, H-6’), 7.94 (1H, s, H-8), 7.70 (1H, d, J = 7.8 Hz, H-5’), 7.66 (1H, s, H-5), 7.51 (1H, d, J = 8.1 Hz, H-4’), 4.38 (2H, t, J = 6.0 Hz, ClCH2CH2CH2O-), 4.06 (3H, s, -OCH3), 3.84 (2H, t, J = 6.4 Hz, ClCH2CH2CH2O-), 2.29 (2H, m, ClCH2CH2CH2O-); LC-MS: 413.1 (M+H)+; 13C-NMR (DMSO-d) δ: 154.10 (C-7), 153.41 (C-6), 150.14 (C-10), 141.66 (C-9), 139.87 (C-1’), 129.88, 129.53, 129.32, 125.71, 119.93, 118.17, 107.09 (C-5), 104.11 (C-4), 100.14 (C-8), 65.85 (ClCH2CH2CH2O-), 56.83 (-OCH3), 41.84 (ClCH2CH2CH2O), 31.45 (ClCH2CH2CH2O-); Anal. Calcd. for C18H16ClF3N4O2: C 52.37, H 3.91, N 13.57; Found: C 52.32, H 3.94, N 13.56.

4-Anilino-7-(3-chloropropoxy)-6-methoxy-1,2,3-benzotriazine (8r): Yield: 70.1%; mp: 243 oC. IR (KBr): 3435, 2922, 1614, 1566, 1502, 1447, 1421, 1285, 1241, 1108, 850, 744 cm-1; 1H-NMR (DMSO-d) δ: 9.59 (1H, s, NH), 7.94 (1H, s, H-8), 7.84 (2H, d, J = 7.8 Hz, ArH-2’H, 6’H), 7.61 (1H, s, H-5), 7.45 (2H, t, J = 7.8 Hz, ArH-3’H, 5’H), 7.18 (1H, t, J = 7.5 Hz, ArH-4’H), 4.37 (2H, t, J = 6.0 Hz, ClCH2CH2CH2O-), 4.04 (3H, s, -OCH3), 3.84 (2H, t, J = 6.3 Hz, ClCH2CH2CH2O-), 2.29 (2H, m, ClCH2CH2CH2O-); LC-MS: 345.2 (M+H)+; 13C-NMR (DMSO-d) δ: 153.88 (C-7), 153.17 (C-6), 150.28 (C-10), 141.50 (C-9), 138.83 (C-1’), 128.68 (C-3’, C-5’), 124.03 (C-4’), 122.88 (C-2’, C-6’), 107.01 (C-5), 104.02 (C-4), 100.31 (C-8), 65.78 (ClCH2CH2CH2O-), 56.81 (-OCH3), 41.89 (ClCH2CH2CH2O-), 31.48 (ClCH2CH2CH2O-); Anal. Calcd. for C17H17ClN4O2: C 59.22, H 4.97, N 16.25; Found: C 59.28, H 4.96, N 16.17.

Antiproliferative activity assay

Cell lines: The cell lines used were obtained from ATCC (Rockville, MD, USA). T47D human breast cancer cells, DU145 and PC-3 human prostate cancer cells were cultured in RPMI1640 medium (Gibco; New York, NY, USA) supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, 1 mM L-glutamine, 5 µg/mL insulin and 10% (v/v) heat-inactivated fetal bovine serum (FBS). MVECs, LL/2 murine lewis lung carcinoma cells and B16F0 melanoma cells were cultured in DMEM with 4 mM L-glutamine adjusted to contain 4.50 g/L glucose and 10% (v/v) heat-inactivated FBS.

Cell growth inhibition assay: The antiproliferative activities of these compounds were determined by the MTT assay. Cells (2×103 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 oC. 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 GI50 concentration was calculated [18].