Design, synthesis, and biological evaluation of triazole-pyrimidine-methylbenzonitrile derivatives as dual A_2A/A_2B adenosine receptor antagonists

Figures & data

Figure 1. Chemical structures of A₂ adenosine receptor antagonists.

Figure 2. Design of dual A_2A/A_2B AR antagonists containing quinoline or its open-ring bioisosteres based on the structure of triazole-pyrimidine-methylbenzonitrile.

Scheme 1. Synthetic route of target compounds 7a–7n. Reagents and conditions: (a) Pd(dppf)Cl₂, (Bpin)₂, potassium acetate, 1,4-dioxane, N₂, reflux, 4 h; (b) 4,6-dichloropyrimidin-2-amine, K₂CO₃, Pd(PPh₃)₄, DMF, N₂, 45 °C, 5 min, then compound 2 was added, 115 °C, 5 h; (c) Et₃N, TMSA, Pd(PPh₃)₂Cl₂, CuI, dry THF, N₂, reflux, 16 h; (d) TBAF(1M in THF), THF, 0 °C to r.t., 20 h; (e) Compound 6, CuSO₄·5H₂O, sodium l-ascorbate, 60 °C, DMF, t-BuOH, 12 h; (f) SnCl₂, 70 °C, 2 h; (g) for 7 h: LiOH, H₂O, t-BuOH, r.t., 8 h; for 7i–7l: The corresponding amine derivatives, MeOH, THF, 45 °C, 18 h; (h) TMSiA, DIPEA, THF, r.t., 24 h; (i) DPPA, DBU, THF, r.t., 10 h.

Scheme 2. Synthetic route of the target compound 17. Reagents and conditions: (a) 4,6-dichloropyrimidin-2-amine, K₂CO₃, Pd(PPh₃)₄, DMF, N₂, 45 °C, 5 min, then compound 11 was added, 115 °C, 5 h; (b) Et₃N, TMSA, Pd(PPh₃)₂Cl₂, CuI, dry THF, N₂, reflux, 16 h; (c) TBAF(1M in THF), THF, 0 °C to r.t., 20 h; (d) MeMgBr, THF, −10 °C to r.t., 12 h; (e) DPPA, DBU, THF, r.t., 10 h; (f) CuSO₄·5H₂O, sodium l-ascorbate, 60 °C, DMF, t-BuOH, 12 h.

Scheme 3. Synthetic route of the target compound 22. Reagents and conditions: (a) MsCl, Et₃N, 0 °C, DCM, 1 h, then TBAF (1 M in THF) was added, 50 °C, 12 h; (b) 4,6-dichloropyrimidin-2-amine, TMSA, Et₃N, Pd(PPh₃)₂Cl₂, CuI, dry THF, N₂, 80 °C, 12 h; (c) Pd(dppf)Cl₂, K₂CO₃, 1,4-dioxane, N₂, 100 °C, 4 h; (d) TBAF (1 M in THF), CuSO₄·5H₂O, sodium l-ascorbate, H₂O, t-BuOH, 60 °C,12 h.

Table 1. Antagonist activity (%inhibition) of compounds 7, 17, and 22 at 10–1000 nM on cAMP levels in CHO cells expressing hA_2A AR or hA_2B AR ^a.

Compound	Ar^1	Ar^2	hA2A AR (%)			hA2B AR (%)
			1000 nM	100 nM	10 nM	100 nM
7a			43.54 ± 0.77	16.52 ± 1.90	28.24 ± 8.79	3.51 ± 3.19
7c			48.45 ± 1.45	38.16 ± 10.85	35.9 ± 5.39	85.43 ± 0.04
7d			91.27 ± 4.28	63.38 ± 1.39	40.83 ± 9.01	80.27 ± 1.84
7e			67.76 ± 4.91	50.68 ± 5.51	44.99 ± 1.25	82.45 ± 7.81
7f			92.41 ± 0.43	84.21 ± 2.94	54.91 ± 1.19	102.64 ± 0.24
7g			107.32 ± 0.89	56.02 ± 2.03	19.61 ± 2.13	52.94 ± 3.96
7h			92.61 ± 1.91	55.18 ± 4.27	37.91 ± 2.32	54.75 ± 2.09
7i			102.58 ± 2.82	95.93 ± 3.36	32.73 ± 1.80	103.33 ± 0.63
7j			100.79 ± 4.62	82.17 ± 1.26	22.45 ± 6.68	82.38 ± 4.52
7k			94.87 ± 2.05	70.52 ± 6.37	21.69 ± 5.43	78.28 ± 2.66
7l			89.66 ± 1.31	52.3 ± 8.46	13.46 ± 3.71	84.34 ± 4.72
7m			69.63 ± 1.01	28.64 ± 4.03	21.51 ± 4.32	69.38 ± 4.01
7n			54.77 ± 2.41	29.78 ± 0.38	18.77 ± 3.88	63.52 ± 4.93
17			92.65 ± 2.87	92.28 ± 0.61	17.63 ± 2.78	64.42 ± 0.89
22			93.61 ± 2.76	94.73 ± 4.43	22.39 ± 11.87	19.88 ± 1.85
AB928^b	AB928	AB928	101.35 ± 5.55	96.8 ± 7.16	20.04 ± 6.46	102.85 ± 1.04

^aData are expressed as means ± SEM.

^bUsed as a positive control.

Table 2. IC₅₀ values of selected compounds on cAMP assays in CHO cells expressing hA_2A or hA_2B AR^a.

Compound	cAMP IC50 (nM)
	hA2A AR	hA2B AR
7f	24.04 ± 0.06	102.64 ± 0.24%^b, 10.79 ± 0.67%^c
7i	6.007 ± 0.05	14.12 ± 2.05
17	320.5 ± 94.04	80.03 ± 13.45
22	67.19 ± 10.92	178.5 ± 9.89
AB928	2.754 ± 0.12	25.48 ± 0.35

^aData are expressed as means ± SEM.

^bPercentage of inhibition (I%) determined at 100 nM concentration of compound 7f.

^cPercentage of inhibition (I%) determined at 10 nM concentration of compound 7f.

Table 3. Radioligand binding affinity data (IC₅₀) for selected compounds against hA_2A AR^a.

Compound	cAMP assays IC50 (nM)	Radioligand binding assays IC50 (nM)
7f	24.04 ± 0.06	158.0 ± 56.42
7i	6.007 ± 0.05	63.55 ± 13.03
AB928	2.754 ± 0.12	25.23 ± 0.74

^aData are expressed as means ± SEM.

Figure 3. The efficiencies of selected compounds for IL-2 production. The efficiencies of selected antagonists on T-cell activation at 10 nM, 100 nM, and 1000 nM were tested in the presence or absence of 1000 nM NECA. The DMSO control was tested in the absence of both antagonists and NECA. The NECA control was tested without antagonists in the presence of 1000 nM NECA.

Figure 4. Binding modes of compound 7i at A_2A AR (a) and hA_2B AR (b) binding cavities have indicated docking orientations and some key receptor residues. Depths of the two binding pockets have been circled with the red dotted line. 2D diagrams of ligand-target interactions for docking compound 7i with the A_2A AR (c) and hA_2B AR (d) predicted by Discovery Studio.

Table 4. Metabolic stability assay in rat and human liver microsomes^a.

Compound	Microsomal metabolic stability
	T1/2 (min)		CLint(liver)(mL/min/g)		Remaining (%, T = 60min)
	Hum	Rat	Hum	Rat	Hum	Rat
7f	n.c.	93.65	n.c.	0.78	104.65	64.04
7i	1155	67.28	0.06	1.08	98.76	50.67
AB928	433.13	33.8	0.17	2.15	94.40	28.10
7-EC	8.12	7.26	9.0	10.0	0.72	0.35

^aT_1/2, half-life, CL_int(liver), intrinsic clearance, n.c., not calculated.

Figure 5. Plasma concentration-time profiles of compounds 7f and 7i following oral administration (a) and intravenous administration (b).

Table 5. Pharmacokinetic parameters of compounds 7f and 7i ^a.

Parameter	7f		7i
	Oral	i.v.	Oral	i.v.
t1/2 (h)	4.12	2.67	4.82	2.19
tmax(h)	2.00	0.25	1.00	0.08
Cmax(µg/L)	7162.80	5010.30	1007.40	7266.60
Cmax/Dose^b	238.76	501.03	33.58	726.66
AUC0→t(µg/L*h)	45,490.39	1728.68	7904.90	4484.58
AUC0→t/Dose^c	1516.35	1728.68	263.50	448.46
AUC0→∞(µg/L*h)	46,232.45	17315.97	8145.18	4484.81
AUC0→∞/Dose^c	1541.08	1731.60	271.51	448.48
F(%)	87.7		58.8

^aPK parameters (mean, n = 3), C_max, maximum concentration; T_max, time to reach C_max; AUC_0→t, Area under the curve from zero to the last measurable plasma concentration; AUC_0→∞, Area under the curve from the last measurable plasma concentration to infinity.

^bUnit is (µg/L)/(mg/kg).

^cUnit is (µg/L*h)/(mg/kg).