Synthesis and Anti-tumor Activities of Novel [1,2,4]triazolo[1,5-a]pyrimidines

Abstract

In order to find novel anti-tumor compounds a series of novel [1,2,4]triazolo[1,5-a]pyrimidine derivatives has been designed and synthesized. The structures of all the compounds were confirmed by IR, ¹H-NMR, MS and elemental analysis. Their anti-tumor activities against cancer cell lines (HT-1080 and Bel-7402) were tested in vitro by the MTT method. Among them, compound 19 displayed the best anti-tumor activity, with IC₅₀ values of 12.3 μM and 6.1 μM against Bel-7402 and HT-1080 cell lines, respectively.

Introduction

Purine analogs are widely used in the treatment of various diseases, particularly cancer. The clinical application of 6-mercaptopurine [1] and thioguanine [2] in cancer treatment and the development of potent purine based CDK inhibitors, such as the purvalanols [3,4], olomoucine [3] and roscovitine [5], together with other findings based on the purine scaffold, have greatly inspired and directed parallel developments in the chemistry and anti-tumor research of related heterocyclic analogs, including pyrrolo-pyrimidines [6], pyrazolo-pyrimidines [7,8], imidazolo-pyridines [9], triazolo-pyrimidines [10], pyrazolo-pyridazines [11] and imidazo-pyrazines [12].

[1,2,4]Triazolo[1,5-a]pyrimidines, a subtype of bioisosteric purine analogs, were also reported to possess potential anti-tumor activities, especially those bearing functional groups at the C-5, C-6 or C-7 positions [13,14,15]. However, substitutions at the C-2 position seemed to be less attractive. Recently, the good antiproliferative ability of some [1,2,4]triazolo[1,5-a]pyrimidines bearing functional groups at the C-2 and C-7 positions was disclosed [16,17], which greatly encouraged us to explore more potent analogs of such structures. Consequently, we carried out a series of modifications upon the [1,2,4]triazolo[1,5-a]pyrimidine scaffold by introducing functional groups into the C-2 and C-7 positions. Herein, we report the synthesis and in vitro anti-tumor evaluation of a new series of 7-anilino-5-methyl-2-(3-((5-(substituted-aminomethyl)furan-2-yl)methylthio)propyl)[1,2,4]triazol[1,5-a]- pyrimidines (6-20).

Results and Discussion

Chemistry

Our synthetic route to the target compounds is shown in Scheme 1.

Scheme 1. Synthesis of target compounds.

Scheme 1. Synthesis of target compounds.

Reagents and conditions: a) aminoguanidine, γ-butyrolactone, Py, reflux, 10 h; b) ethyl acetoacetate, acetic acid, reflux, 30 h, c) methanolic ammonia, room temperature, 24 h; d) POCl₃, reflux, 3 h; e) R¹NH₂, ⁱPrOH, 50°C, 3 h; f) furan-2-ylmethanethiol, NaH, DMF, 50°C, 0.5 h; g) HCHO, R²R³NH, AcOH, 50°C, 4 h.

Commercially available γ-butyrolactone was first transformed into the substituted [1,2,4]triazole 1 with aminoguanidine carbonate in the presence of pyridine in 40% yield. Condensation of 1 with ethyl acetoacetate was carried out in acetic acid at reflux following the procedure reported by Okabe [18], to give a mixture of 2 and 2′ (the acetylated product of 2), which further treated with methanolic ammonia to give 2 in 88% yield. Subsequent treatment of 2 with phosphorus oxychloride afforded 3 in 94% yield. Displacement of the chloro group of 3 with various anilines provided the intermediates 4a-4c in 87-93% yields.

Etherification of 4a-4c with (furan-2-yl)methanethiol in the presence of sodium hydride provided intermediates 5a-5c. Subsequent aminomethylation of 5a-5c with various secondary amines and formaldehyde, followed by chromatographic purification, gave the final products 6-20 in 45-80% yields.

Anticancer activities

All target compounds 6-20 were evaluated for their in vitro anti-tumor activity by the MTT method. The IC₅₀ values against Bel-7402 (Human Liver Cancer Cell Lines) and HT-1080 (Human Fibro Sarcoma Cell Lines) cell lines are summarized in Table 1.

Table 1. The anticancer activities of compounds 6-20.

Compd.	IC50 (μM)		Compd.	IC50 (μM)
	Bel-7402	HT-1080		Bel-7402	HT-1080
6	>300	>300	14	54.1	30.8
7	72.5	52.1	15	44.6	36.3
8	67.3	57.4	16	42.1	32.2
9	>300	237	17	13.9	28.4
10	>300	280	18	22.1	29.3
11	45.8	35.1	19	12.3	6.1
12	77.6	41.4	20	22.2	14.8
13	>300	261.9	cisplatin	35.5	22.7

Table 1. The anticancer activities of compounds 6-20.

Compd.	IC50 (μM)		Compd.	IC50 (μM)
	Bel-7402	HT-1080		Bel-7402	HT-1080
6	>300	>300	14	54.1	30.8
7	72.5	52.1	15	44.6	36.3
8	67.3	57.4	16	42.1	32.2
9	>300	237	17	13.9	28.4
10	>300	280	18	22.1	29.3
11	45.8	35.1	19	12.3	6.1
12	77.6	41.4	20	22.2	14.8
13	>300	261.9	cisplatin	35.5	22.7

Table 1 shows that both substituents on the aniline ring and variations on the 5"-furylmethyl moiety of the side-chain have a substantial influence on the anti-tumor activity. The 3-fluorophenylamino substituted series showed comparable or better anti-tumor activities than the phenylamino substituted one (cf. 7 and 11, 8 and 12). Morever, introduction of the 4-fluoro-3-(trifluoromethyl)phenylamino group into the C-7 position was found to be quite favorable for increasing anti-tumor activity (comparison of 8 and 17, 14 and 20). Among the whole series, compound 19 showed the best anti-tumor activity with IC₅₀ values of 12.3 μM and 6.1 μM against the Bel-7402 and HT-1080 cell lines, respectively, which was four times more potent than cisplatin. On the other hand, among the hydrophilic groups introduced into the 5"-furylmethyl of the side-chain at the C-2 position of the scaffold, piperidinyl, pyrrolidinyl, diethylamino and dimethylamino groups rendered better anti-tumor activity, while morpholino, 4-methylpiperizinyl resulted in a drastic decrease in anti-tumor potency (comparison of 6, 9 and 7, 8). However, despite the negative effects of morpholino and 4-methyl-piperizinyl substitutions, anti-tumor potency could be retained in the 4-fluoro-3-(trifluoro-methyl)phenyl amino series, as illustrated by the comparison of 6 and 15, 13 and 18, suggesting that the contribution of 4-fluoro-3-(trifluoromethyl)phenyl amino groups to the potency might partly compensate for the negative effect of morpholino and 4-methylpiperizinyl substitution.

Conclusions

In conclusion, a novel series of [1,2,4]triazolo[1,5-a]pyrimidines were synthesized and found to be active against various tumor cell lines. Compounds with disubstitution on the aniline ring and piperidinyl, pyrrolidinyl, diethylamino or dimethylamino on the 5"-furylmethyl moiety of the side-chain showed better anti-tumor activities than other series. Among this series, compound 19 showed the best anti-tumor activity and will be considered in a further study.

Experimental Section

General

Melting points were determined by the capillary tube method, and the thermometer was uncorrected. Mass spectra were obtained on an Agilent 1100 HPLC-MS instrument. ¹H-NMR spectra were run in DMSO-d₆ or CDCl₃, with TMS at the internal standard, on a Bruker ARX-300 instrument operating at 300 MHz. IR spectra (KBr disks) were recorded on a Bruker IFS 55 instrument. Elemental analysis was performed with a Carlo-Erba 1106 Elemental analysis instrument.

5-Amino-3-(3-hydroxypropyl)-4H-[1,2,4]triazole (1).

A mixture of aminoguanidine (65 g, 0.55 mol), γ-butyrolactone (43 g, 0.50 mol) and pyridine (900 mL) was heated to reflux for 10 h. The resulting mixture was concentrated and filtered to give a pale solid, which was recrystallized from absolute ethanol (150 mL) to give 1 (28 g, 40%) as a white solid, m. p. 146-147 °C, (lit. 149-150 °C [19]); MS [MH⁺] (m/z): 142.1; ¹H-NMR (DMSO-d₆) δ: 1.71 (pent, 2H, CH₂), 2.44 (t, 2H, J = 6.0 Hz, CH₂), 3.41 (t, 2H, J = 6.2 Hz, CH₂), 4.45 (s, 1H, OH), 5.55 (br s, 2H, NH₂), 11.51 (br s, 1H, NH); Anal. calcd. for C₅H₁₀N₄O: C 42.24, H 7.09, N 39.41; Found: C 42.01, H 7.01, N 39.23.

7-Hydroxyl-2-(3-hydroxypropyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine (2).

A mixture of 1 (28 g, 0.20 mol), ethyl acetoacetate (30.8 g, 0.24 mol) and acetic acid (300 mL) was heated to reflux for 30 h and then concentrated and filtered to give a mixture of 2 and 2′ as a pale yellow solid. This solid was added into satd. ammonia/methanol (300 mL) and stirred at room temperature for 24 h. The mixture was evaporated, water was added and then acidified with dilute HCl to pH 5-6, filtered and recrystallized from ethanol to give 2 (37 g, 88%), m. p. 204-206 °C; MS [MH⁺] (m/z): 208.1; ¹H-NMR (DMSO-d₆) δ: 1.85 (pent, 2H, CH₂), 2.30 (s, 1H, CH₃), 2.72 (t, 2H, J = 6.0 Hz, CH₂), 3.45 (t, 2H, J = 6.1 Hz, CH₂), 4.49 (br s, 1H, OH), 5.76 (s, 1H, C₆-H), 12.98 (br s, 1H, C₇-OH); Anal. calcd. for C₉H₁₂N₄O₂: C 51.92, H 5.81, N 26.91; Found: C 51.86, H 5.68, N 26.79.

7-Chloro-2-(3-chloropropyl)-5-methyl-[1, 2, 4]triazolo[1,5-a]pyrimidine (3).

A mixture of 2 (37 g, 0.18 mol) and phosphorus oxychloride (265 g, 1.70 mol) was heated to reflux for 3 h, and then concentrated in vacuo to give a red oil, which was poured into water and extracted with chloroform (250 mL×3). The organic layer was then washed with water three times, dried with anhydrous magnesium sulfate and concentrated to give 3 (41 g, 94%) as a yellow solid (LC purity: 96%, MS [MH⁺] (m/z): 245.2), which was directly used in next step without purification.

General procedure for the synthesis of 7-anilino-5-methyl-2-(3-chloroproyl)-[1,2,4]triazolo[1,5-a]pyrimidines 4a-4c.

A mixture of R¹NH₂ (44 mmol), 3 (10 g, 40 mmol) and isopropanol (100 mL) was heated at 50°C for 3 h. After cooling to room temperature and the following filtration, a yellow solid was obtained. Recrystallization of the solid from methanol gave 4a-4c as light yellow powders (87-93%).

General procedure for the synthesis of 7-anilino-5-methyl-2-(3-(furan-2-ylmethylthio)propyl)- [1,2,4]triazolo [1,5-a]pyrimidines 5a-5c.

Furan-2-ylmethanethiol (8.6 g, 75 mmol) was added dropwise into a suspension of NaH (1.8 g 75 mmol) and dry DMF (80 mL) at room temperature. After the addition, the mixture was stirred for 5 min, and then 4a-4c (25 mmol) was added. The mixture was heated at 50°C for 30 min and then poured into water. The brown oil which separated from the water was triturated with diethyl ether, then filtered and recrystallized from ethyl acetate/cyclohexane to afford 5a-5c as gray solids (80%-85%).

General procedure for the synthesis of 6-20.

Formaldehyde (0.6 g, 8 mmol) was added into the solution of R²R³NH (13 mmol) in acetic acid (20 mL). The mixture was stirred at 30°C for 10 min, and then 5a-5c (5 mmol) was added. After stirring at 50°C for 4 h, the mixture was concentrated in vacuo. The residue was taken up in water (50 mL), basified with concentrated aqueous sodium hydroxide to pH 9-10, extracted with methylene dichloride, and then dried over magnesium sulfate. Evaporation of the solvent provided an oil residue, which was purified by chromatography to give the final products 6-20.

7-Phenylamino-5-methyl-2-(3-((5-(morpholinomethyl)furan-2-yl)methylthio)propyl)-[1,2,4]triazolo [1,5-a]pyrimidine (6).

Yield: 60%; m.p. 167-168 °C; MS [MH⁺] (m/z): 479.2; IR (KBr) cm^-1: 3445.5 (υ_NH), 2963.4 (υ_CH3), 1609.3, 1576.3 (υ_C=C, υ_C=N), 1479.8 (δ_CH2), 1328.5 (υ_C-N); ¹H-NMR (DMSO-d₆)δ: 2.02 (pent, 2H, CH₂), 2.51 (s, 3H, CH₃), 2.59 (m, 6H, 3×CH₂), 2.88 (t, 2H, J = 7.2 Hz, CH₂), 3.61 (br s, 4H, 2×CH₂), 3.73 (s, 2H, CH₂), 3.77 (s, 2H, CH₂), 6.23 (d, 1H, J =2.6 Hz, furyl-H), 6.27 (d, 1H, J =2.7 Hz, furyl-H), 6.34 (s, 1H, C₆-H), 7.29~7.48 (m, 5H, Ph-5×H); Anal. calcd. for C₂₅H₃₀N₆O₂S: C 62.74, H 6.32, N 17.56 Found: C 62.59 H 6.25, N 17.45.

7-Phenylamino-5-methyl-2-(3-((5-(piperidin-1-ylmethyl)furan-2-yl)methylthio)propyl)-[1,2,4]triazolo [1,5-a]pyrimidine (7).

Yield: 64%; m.p. 169-170°C; MS [MH⁺] (m/z): 477.1; IR (KBr) cm^-1: 3447.2 (υ_NH), 2966.5 (υ_CH3), 1609.1, 1575.9 (υ_C=C, υ_C=N), 1478.0 (δ_CH2), 1329.6 (υ_C-N); ¹H-NMR (CDCl₃)δ: 1.42 (m, 2H, CH₂), 1.64 (m, 4H, 2×CH₂), 2.16 (pent, 2H, CH₂), 2.49 (br s, 4H, 2×CH₂), 2.52 (s, 3H, CH₃), 2.64 (t, 2H, J = 7.1 Hz, CH₂), 2.99 (t, 2H, J = 7.3 Hz, CH₂), 3.57 (s, 2H, CH₂), 3.72 (s, 2H, CH₂), 6.11 (d, 1H, J =3.0 Hz, furyl-H), 6.14 (d, 1H, J =3.0 Hz, furyl-H), 6.32 (s, 1H, C₆-H), 7.35~7.50 (m, 5H, Ph-5×H); Anal. calcd. for C₂₆H₃₂N₆OS : C 65.52, H 6.77, N 17.63 Found: C 65.44, H 6.63, N 17.60.

7-Phenylamino-5-methyl-2-(3-((5-(pyrrolidin-1-ylmethyl)furan-2-yl)methylthio)propyl)-[1,2,4]triazolo [1,5-a]pyrimidine (8).

Yield: 64%; m.p. 162-164°C; MS [MH⁺] (m/z): 463.2; IR (KBr) cm^-1: 3440.5 (υ_NH), 2965.2 (υ_CH3), 1611.5, 1576.1 (υ_C=C, υ_C=N), 1478.3 (δ_CH2), 1329.8 (υ_C-N); ¹H-NMR (CDCl₃)δ: 1.82 (m, 4H, 2×CH₂), 2.15 (pent, 2H, CH₂), 2.52 (s, 3H, CH₃), 2.64 (t, 2H, J = 7.2 Hz, CH₂), 2.69 (br s, 4H, 2×CH₂), 2.99 (t, 2H, J = 7.3 Hz, CH₂), 3.71 (s, 2H, CH₂), 3.72 (s, 2H, CH₂), 6.11 (d, 1H, J =3.0 Hz, furyl-H), 6.17 (d, 1H, J =2.9 Hz, furyl-H), 6.32 (s, 1H, C₆-H), 7.35~7.50 (m, 5H, Ph-5×H); Anal. calcd. for C₂₅H₃₀N₆OS: C 64.91, H 6.54, N 18.17 Found: C 64.79, H 6.43, N 18.11.

7-Phenylamino-5-methyl-2-(3-((5-((4-methylpiperazin-1-yl)methyl)furan-2-yl)methylthio)propyl)-[1,2,4] triazolo[1,5-a]pyrimidine (9).

Yield: 73%; m.p. 176-179°C; MS [MH⁺] (m/z): 492.2; IR (KBr) cm^-1: 3442.6 (υ_NH), 2966.8 (υ_CH3), 1609.3, 1576.8 (υ_C=C, υ_C=N), 1479.2 (δ_CH2), 1329.0 (υ_C-N); ¹H-NMR (DMSO-d₆)δ: 2.01 (pent, 2H, CH₂), 2.41 (s, 3H, CH₃), 2.61 (t, 2H, J = 6.9 Hz, CH₂), 2.74 (br s, 7H, CH₃, 2×CH₂), 3.17 (br s, 4H, 2×CH₂), 3.72 (s, 2H, CH₂), 3.77 (s, 2H, CH₂), 6.22 (m, 2H, furyl-2×H), 6.36 (s, 1H, C₆-H), 7.29~7.48 (m, 5H, Ph-5×H); Anal. calcad. for C₂₆H₃₃N₇OS: C 63.52, H 6.77, N 19.94 Found: C 63.47, H 6.85, N 19.82.

7-(3-Fluorophenylamino)-5-methyl-2-(3-((5-(morpholinomethyl)furan-2-yl)methylthio)propyl)-[1,2,4] triazolo[1,5-a]pyrimidine (10).

Yield: 80%; m.p. 173-175°C; MS [MH⁺] (m/z): 497.1; IR (KBr) cm^-1: 3442.8 (υ_NH), 2970.5 (υ_CH3), 1608.0, 1575.2 (υ_C=C, υ_C=N), 1478.5 (δ_CH2), 1329.4 (υ_C-N); ¹H-NMR (DMSO-d₆) δ: 2.03 (pent, 2H, CH₂), 2.44 (s, 3H, CH₃), 2.61 (t, 2H, J = 7.1 Hz, CH₂), 2.70 (br s, 4H, 2×CH₂), 2.90 (t, 2H, J = 7.3 Hz, CH₂), 3.66 (br s, 4H, 2×CH₂), 3.79 (s, 2H, CH₂), 3.83 (s, 2H, CH₂), 6.26 (d, 1H, J =2.9 Hz, furyl-H), 6.33 (d, 1H, J =2.8 Hz, furyl-H), 6.52 (s, 1H, C₆-H), 7.13 (dd, 1H, J_FH=8.4 Hz, J_HH=7.7 Hz, Ph-4H), 7.34 (br d, 1H, J_FH=7.7 Hz, Ph-2H), 7.34 (br d, 1H, J_HH=7.7 Hz, Ph-6H), 7.50 (dd, 1H, J₁=7.8 Hz, J₂=7.7 Hz, Ph-5H); Anal. calcd. for C₂₅H₂₉FN₆O₂S : C 55.28, H 5.89, N 16.92 Found: C 55.21, H 5.78, N 16.79.

7-(3-Fluorophenylamino)-5-methyl-2-(3-((5-(piperidin-1-ylmethyl)furan-2-yl)methylthio)propyl) [1,2,4] triazolo[1,5-a]pyrimidine (11).

Yield: 68%; m.p. 170-172 °C; MS [MH⁺] (m/z): 495.1; IR (KBr) cm^-1: 3444.5 (υ_NH), 2963.5 (υ_CH3), 1609.5, 1576.5 (υ_C=C, υ_C=N), 1479.8 (δ_CH2), 1329.1 (υ_C-N); ¹H-NMR (DMSO-d₆)δ: 1.48 (br s, 2H, CH₂), 1.71 (br s, 2H, 2×CH₂), 2.02 (pent, 2H, CH₂), 2.45 (s, 3H, CH₃), 2.63 (t, 2H, J = 7 Hz, CH₂), 2.91 (t, 2H, J = 7.1 Hz, CH₂), 3.03 (br s, 4H, 2×CH₂), 3.82 (s, 2H, CH₂), 4.26 (s, 2H, CH₂), 6.35 (br s, 1H, furyl-H), 6.53 (br s, 2H, C₆-H, furyl-H), 7.14 (dd, 1H, J_FH=8.5 Hz, J_HH=7.7 Hz, Ph-4H), 7.34 (br d, 1H, J_FH=7.9 Hz, Ph-2H), 7.34 (br d, 1H, J_HH=7.9 Hz, Ph-6H), 7.52 (dd, 1H, J₁=7.7 Hz, J₂=7.7 Hz, Ph-5H); Anal. calcd. for C₂₆H₃₁FN₆OS: C 63.13, H 6.32, N 16.99 Found: C 63.05, H 6.21, N 16.88.

7-(3-Fluorophenylamino)-5-methyl-2-(3-((5-(pyrrolidin-1-ylmethyl)furan-2-yl)methylthio)propyl)-[1,2,4] triazolo[1,5-a]pyrimidine (12).

Yield: 65%; m.p. 166-168 °C; MS [MH⁺] (m/z): 481.1; IR (KBr) cm^-1: 3442.8 (υ_NH), 2968.5 (υ_CH3), 1610.2, 1574.2 (υ_C=C, υ_C=N), 1478.2 (δ_CH2), 1328.9 (υ_C-N); ¹H-NMR (CDCl₃) δ: 1.82 (m, 4H, 2×CH₂), 2.14 (pent, 2H, CH₂), 2.55 (s, 3H, CH₃), 2.64 (t, 2H, J = 7.2 Hz, CH₂), 2.69 (br s, 4H, 2×CH₂), 2.99 (t, 2H, J = 7.2 Hz, CH₂), 3.71 (s, 4H, 2×CH₂), 6.11 (br s, 1H, furyl-H), 6.16 (br s, 1H, furyl-H), 6.38 (s, 1H, C₆-H), 7.03 (dd, 1H, J_FH=8.1 Hz, J_HH=7.0 Hz, Ph-4H), 7.14 (br d, 1H, J_FH=7.1 Hz, Ph-2H), 7.14 (br d, 1H, J_HH=7.1 Hz, Ph-6H), 7.45 (dd, 1H, J₁=7.2 Hz, J₂=7.0 Hz, Ph-5H); Anal. calcd. for C₂₅H₂₉FN₆OS: C 62.48, H 6.08, N 17.49 Found: C 62.38, H 5.99, N 17.41.

7-(3-Fluorophenylamino)-5-methyl-2-(3-((5-((4-methylpiperazin-1-yl)methyl)furan-2-yl)methylthio)pro pyl)-[1,2,4]triazolo[1,5-a]pyrimidine (13).

Yield: 77%; m.p. 179-180 °C; MS [MH⁺] (m/z): 510.2; IR (KBr) cm^-1: 3445.1 (υ_NH), 2967.8 (υ_CH3), 1608.5, 1575.9 (υ_C=C, υ_C=N), 1479.5 (δ_CH2), 1329.1 (υ_C-N); ¹H-NMR (DMSO-d₆) δ: 2.02 (pent, 2H, CH₂), 2.43 (s, 3H, CH₃), 2.57~2.62 (m, 6H, 3×CH₂), 2.73 (s, 3H, CH₃), 2.89 (t, 2H, J = 6.9 Hz, CH₂), 3.16 (br s, 4H, 2×CH₂), 3.56 (s, 2H, CH₂), 3.76 (s, 2H, CH₂), 6.22 (m, 2H, furyl-2×H), 6.51 (s, 1H, C₆-H), 7.12 (dd, 1H, J_FH=8.6 Hz, J_HH=7.5 Hz, Ph-4H), 7.33 (br d, 1H, J_FH=7.8 Hz, Ph-2H), 7.33 (br d, 1H, J_HH=7.8 Hz, Ph-6H), 7.51 (dd, 1H, J₁=7.5 Hz, J₂=7.5 Hz, Ph-5H); Anal. calcd. for C₂₆H₃₂FN₇OS: C 61.27, H 6.33, N 19.24 Found: C 61.15, H 6.25, N 19.12.

7-(3-Fluorophenylamino)-5-methyl-2-(3-((5-((dimethylamino)methyl)furan-2-yl)methylthio)propyl)-[1,2,4]triazolo[1,5-a]pyrimidine (14).

Yield: 52%; m.p. 156-157 °C; MS [MH⁺] (m/z): 455.2; IR (KBr) cm^-1: 3443.5 (υ_NH), 2964.5 (υ_CH3), 1608.2, 1575.4 (υ_C=C, υ_C=N), 1479.8 (δ_CH2), 1329.5 (υ_C-N); ¹H-NMR (CDCl₃) δ: 2.15 (pent, 2H, CH₂), 2.30 (s, 6H, 2×CH₃), 2.55 (s, 3H, CH₃), 2.64 (t, 2H, J = 7.1 Hz, CH₂), 2.99 (t, 2H, J = 7.2 Hz, CH₂), 3.51 (s, 2H, CH₂), 3.72 (s, 2H, CH₂), 6.13 (br s, 2H, furyl-2×H), 6.40 (s, 1H, C₆-H), (dd, 1H, J_FH=8.3 Hz, J_HH=7.1 Hz, Ph-4H), 7.15 (br d, 1H, J_FH=7.3 Hz, Ph-2H), 7.15 (br d, 1H, J_HH=7.3 Hz, Ph-6H), 7.45 (dd, 1H, J₁=7.2 Hz, J₂=7.1 Hz, Ph-5H); Anal. calcd. for C₂₃H₂₇FN₆OS: C 60.77, H 5.99, N 18.49 Found: C 60.69, H 5.78, N 18.38.

7-(4-Fluoro-3-(trifluoromethyl)phenylamino)-5-methyl-2-(3-((5-(morpholinomethyl)furan-2-yl)methyl lthio)propyl)-[1,2,4]triazolo[1,5-a]pyrimidine (15).

Yield: 75%; m.p. 190-192 °C; MS [MH⁺] (m/z): 565.1; IR (KBr) cm^-1: 3446.5 (υ_NH), 2870.3 (υ_CH3), 1622.3, 1581.2, 1505.3 (υ_C=C, υ_C=N), 1478.1 (δ_CH2), 1323.5 (υ_C-N); ¹H-NMR (DMSO-d₆) δ: 2.02 (pent, 2H, CH₂), 2.42 (s, 3H, CH₃), 2.61 (t, 2H, J = 7.2 Hz, CH₂), 2.75 (br s, 4H, 2×CH₂), 2.90 (t, 2H, J = 7.1 Hz, CH₂), 3.11 (br s, 4H, 2×CH₂), 3.65 (br s, 2H, CH₂), 3.79(br s, 2H, CH₂), 6.26 (d, 1H, J =3.0 Hz, furyl-H), 6.33 (d, 1H, J =2.9 Hz, furyl-H), 6.41 (s, 1H, C₆-H), 7.64 (dd, 1H, J_FH=10.0 Hz, J_HH=9.5 Hz, Ph-5H), 7.83 (m, 2H, Ph-2, 6-2×H); Anal. calcd. for C₂₆H₂₈F₄N₆O₂S : C 55.31, H 5.00, N 14.88 Found: C 55.19, H 4.79, N 14.72.

7-(4-Fluoro-3-(trifluoromethyl)phenylamino)-5-methyl-2-(3-((5-(piperidin-1-ylmethyl)furan-2-yl)meth ylthio)propyl)-[1,2,4]triazolo[1,5-a]pyrimidine (16).

Yield: 54%; m.p. 182-183 °C; MS [MH⁺] (m/z): 563.1; IR (KBr) cm^-1: 3443.6 (υ_NH), 2953.7 (υ_CH3), 1620.4, 1575.8, 1505.3 (υ_C=C, υ_C=N), 1476.7 (δ_CH2), 1323.0 (υ_C-N); ¹H-NMR (DMSO-d₆) δ: 1.44 (m, 2H, CH₂), 1.66 (m, 4H, 2×CH₂), 2.02 (pent, 2H, CH₂), 2.41 (s, 3H, CH₃), 2.61 (t, 2H, J = 6.9 Hz, CH₂), 2.89 (t, 2H, J = 7.1 Hz, CH₂), 3.00 (br s, 4H, 2×CH₂), 3.79 (s, 2H, CH₂), 4.09 (s, 2H, CH₂), 6.30 (br s, 1H, furyl-H), 6.40 (s, 1H, C₆-H), 6.47 (br s, 1H, furyl-H), 7.63 (dd, 1H, J_FH=9.9 Hz, J_HH=9.0 Hz, Ph-5H), 7.79~7.83 (m, 2H, Ph-2, 6-2×H); Anal. calcd. for C₂₇H₃₀F₄N₆OS: C 57.64, H 5.37, N 14.94 Found: C 57.49, H 5.15, N 14.87.

7-(4-Fluoro-3-(trifluoromethyl)phenylamino)-5-methyl-2-(3-((5-(pyrrolidin-1-ylmethyl)furan-2-yl) methylthio)propyl)-[1,2,4]triazolo[1,5-a]pyrimidine (17).

Yield: 54%; m.p. 179-180 °C; MS [MH⁺] (m/z): 549.1; IR (KBr) cm^-1: 3445.1 (υ_NH), 2953.7 (υ_CH3), 1620.4, 1505.3 (υ_C=C, υ_C=N), 1479.5 (δ_CH2), 1323.0 (υ_C-N); ¹H-NMR (CDCl₃) δ: 1.81 (m, 4H, 2×CH₂), 2.13 (pent, 2H, CH₂), 2.53 (s, 3H, CH₃), 2.62 (t, 2H, J = 7.2 Hz, CH₂), 2.67 (br s, 4H, 2×CH₂), 2.99 (t, 2H, J = 7.3 Hz, CH₂), 3.71 (s, 2H, CH₂), 3.72 (s, 2H, CH₂), 6.10 (d, 1H, J =3.1 Hz, furyl-H), 6.14 (d, 1H, J =3.0 Hz, furyl-H), 6.16 (s, 1H, C₆-H), 7.36 (dd, 1H, J_FH=9.2 Hz, J_HH=9.0 Hz, Ph-5H), 7.61 (m, 2H, Ph-2, 6-2×H); Anal. calcd. for C₂₆H₂₈F₄N₆OS: C 56.92, H 5.14, N 15.32 Found: C 56.85, H 5.21, N 15.18.

7-(4-Fluoro-3-(trifluoromethyl)phenylamino)-5-methyl-2-(3-((5-((4-methylpiperazin-1-yl)methyl)furan-2-yl)methylthio)propyl)-[1,2,4]triazolo[1,5-a]pyrimidine (18).

Yield: 79%; m.p. 179-180 °C; MS [MH⁺] (m/z): 578.0; IR (KBr) cm^-1: 3444.7 (υ_NH), 2965.7 (υ_CH3), 1610.2, 1575.8 (υ_C=C, υ_C=N), 1478.7 (δ_CH2), 1329.5 (υ_C-N); ¹H-NMR (DMSO-d₆) δ: 2.02 (pent, 2H, CH₂), 2.41 (s, 3H, CH₃), 2.59 (t, 2H, J = 7.1 Hz, CH₂), 2.72 (br s, 7H, CH₃, 2×CH₂), 2.89 (t, 2H, J = 7.1 Hz, CH₂), 3.15 (br s, 4H, 2×CH₂), 3.55 (s, 2H, CH₂), 3.76 (s, 2H, CH₂), 6.21 (m, 2H, furyl-2×H), 6.40 (s, 1H, C₆-H), 7.63 (dd, 1H, J_FH=9.9 Hz, J_HH=9.2 Hz, Ph-5H), 7.83 (m, 2H, Ph-2, 6-2×H); Anal. calcd. for C₂₇H₃₁F₄N₇OS : C 56.14, H 5.41, N 16.97 Found: C 56.21, H 5.29, N 16.86.

7-(4-Fluoro-3-(trifluoromethyl)phenylamino)-5-methyl-2-(3-((5-((diethylamino)methyl)furan-2-yl)-methylthio)propyl)-[1,2,4]triazolo[1,5-a]pyrimidine (19).

Yield: 45%; m.p. 172-174 °C; MS [MH⁺] (m/z): 551.2; IR (KBr) cm^-1: 3445.2 (υ_NH), 2970.1 (υ_CH3), 1575.6, (υ_C=C, υ_C=N), 1479.5 (δ_CH2), 1329.5 (υ_C-N); ¹H-NMR (CDCl₃) δ: 1.11 (t, 6H, J = 6.9 Hz, 2×CH₃), 2.15 (pent, 2H, CH₂), 2.54 (s, 3H, CH₃), 2.61 (m, 6H, 3×CH₂), 2.99 (t, 2H, J = 7.4 Hz, CH₂), 3.72 (s, 4H, 2×CH₂), 6.12 (m, 2H, furyl-2H), 6.18 (s, 1H, C₆-H), 7.36 (dd, 1H, J_FH=9.0 Hz, J_HH=9.0 Hz, Ph-5H), 7.64 (m, 2H, Ph-2, 6-2×H); Anal. calcd. for C₂₆H₃₀F₄N₆OS : C 56.71, H 5.49, N 15.26 Found: C 56.57, H 5.39, N 15.12.

7-(4-Fluoro-3-(trifluoromethyl)phenylamino)-5-methyl-2-(3-((5-((dimethylamino)methyl)furan-2-yl)-methylthio)propyl)-[1,2,4]triazolo[1,5-a]pyrimidine (20).

Yield: 47%; m.p. 166-168 °C; MS [MH⁺] (m/z): 523.1; IR (KBr) cm^-1: 3443.9 (υ_NH), 2966.3 (υ_CH3), 1609.5, 1575.3 (υ_C=C, υ_C=N), 1479.8 (δ_CH2), 1328.2 (υ_C-N); ¹H-NMR (CDCl₃) δ: 2.14 (pent, 2H, CH₂), 2.33 (s, 6H, 2×CH₃), 2.54 (s, 3H, CH₃), 2.63 (t, 2H, J = 7.1 Hz, CH₂), 2.99 (t, 2H, J = 7.2 Hz, CH₂), 3.56 (s, 2H, CH₂), 3.72 (s, 2H, CH₂), 6.13 (br s, 1H, furyl-H), 6.17 (br s, 2H, furyl-H , C₆-H), 7.36 (dd, 1H, J_FH=9.0 Hz, J_HH=8.7 Hz, Ph-5H), 7.64 (m, 2H, Ph-2, 6-2×H); Anal. calcd. for C₂₄H₂₆F₄N₆OS : C 55.16, H 5.01, N 16.08 Found: C 55.03, H 4.92, N 16.15.

Pharmacology

The anticancer activities of compounds 6-20 were evaluated in vitro on Bel-7402 (Human Liver Cancer cell lines) and HT-1080 (Human Fibro Sarcoma cell lines) by measuring cell viability by the MTT method, with cisplatin as the positive control. The cells were seeded in RPM I 1640 medium (100 μL) in a 96-well plate at a concentration of 4,000 cells per well. After culturing for 12 h at 37 °C and 5% CO₂, cells were incubated with various concentrations of the samples for 24 h. MTT was added at a terminal concentration of 5μg/mL and incubated with the cells for 4 h. The formazan crystals were dissolved in DMSO (100 μL) in each well and the optical density was measured at 492 nm (for the absorbance of MTT formazan) and 630 nm (for the reference wavelength). The IC₅₀ was calculated using the Bacus Laboratories Incorporated Slide Scanner (Bliss) software. Each compound was tested in triplicate at every concentration.