The role of 5-arylalkylamino- and 5-piperazino- moieties on the 7-aminopyrazolo[4,3-d]pyrimidine core in affecting adenosine A₁ and A_2A receptor affinity and selectivity profiles

Figures & data

Figure 1. Previously reported pyrazolo[4,3-d]pyrimidines A–C and triazolotriazines ZM-241385 and D.

Figure 2. Herein reported pyrazolo[4,3-d]pyrimidine derivatives 1–24.

Scheme 1. Reagents and conditions: (a) Ph-B(OH)₂, Cu(OAc)₂, pyridine, CH₂Cl₂, 4 Å molecular sieves, room temperature; (b) MeI or PhCH₂Br, NaH, anhydrous THF, room temperature; (c) cyclohexene, Pd/C, 150 °C, mw; (d) R–N=C=S, DMF, room temperature; (e) 0.1 M aqueous NaOH, CH₃I, room temperature; (f) NH₄Cl, formamide, 110–150 °C mw; (g) compounds 2, 11, 12, BBr₃, anhydrous CH₂Cl₂, room temperature or reflux.

Scheme 2. Reagents and conditions: (a) N,N-dimethylaniline, POCl₃, 150 °C, mw; (b) 33% aqueous NH₃, 100 °C, mw; (c) benzylamine, ethyldiisopropylamine, tert-butanol, 200 °C, mw; (d) ethyldiisopropylamine, N-methylpyrrolidone, 130–150 °C, mw; (e) compound 20, LiAlH₄, anhydrous THF, room temperature.

Scheme 3. Reagents and conditions: (a) CF₃COOH, CH₂Cl₂, reflux; (b) RCOCl, NEt₃, anhydrous THF, room temperature.

Table 1. Binding affinity at hA₁, hA_2A and hA₃ ARs and potencies at hA_2B ARs.

			Binding experiments^a Ki (nM) or I%			cAMP assays IC50 (nM) or I%
	R	R2	hA1^b	hA2A^c	hA3^d	hA2B^e
1	Ph	Ph	67 ± 5	412 ± 37	13 ± 2	2%
2	4-OMe-C6H4	Ph	33%	8%	27 ± 3	1%
3	2,4-diClC6H3	Ph	1%	1%	61 ± 8	2%
4	4-OH-C6H4	Ph	481 ± 42	40%	8%	10%
5	Ph-CH2	Ph	25 ± 3	123 ± 11	28 ± 3	2%
6	Ph-CH2CH2	Ph	11.5 ± 1.2	40%	38%	1%
7	Ph-CH2CH2CH2	Ph	785 ± 72	29%	24%	1%
8	Ph	Me	1%	4%	1%	1%
9	Ph-CH2	Me	9%	1%	20%	1%
10	Ph-CH2	CH2Ph	19%	1%	1%	1%
11	4-OMe-C6H4-CH2CH2	Ph	153 ± 11	26%	19%	1%
12	4-OH-C6H4-CH2CH2	Ph	27%	150 ± 14	1%	1%
13	3,4-diOMe-C6H3-(CH2)2	Ph	415 ± 39	189 ± 18	22%	17%
14	3,4-diOH-C6H3-(CH2)2	Ph	71 ± 6	238 ± 24	3%	1%
A^f	Ph	–	150 ± 12	110 ± 10	39%	420 ± 38
B^f	Ph-CH2	–	15%	35%	17%	2%
C^f	Ph-CH2CH2	–	5.31 ± 0.42	55 ± 5	12%	42%
ZM-241385^g		–	714	1.6	743	75^h

^a K_i values are means ± SEM of four separate assays each performed in duplicate. Percentage of inhibition (I%) are determined at 1 μM concentration of the tested compounds.

^b Displacement of specific [³H]DPCPX competition binding assays to hA₁CHO cells.

^c Displacement of specific [³H]ZM241385 competition binding to hA_2ACHO cells.

^d Displacement of specific [¹²⁵I]AB-MECA competition binding to hA₃CHO cells.

^e cAMP experiments in hA_2BCHO cells, stimulated by 200 nM NECA. Percentage of inhibition (I%) are determined at 1 μM concentration of the tested compounds.

^f Ref. 24.

^g Ref. 5.

^h K_i value obtained from binding experiments at recombinant hA_2B.

Table 2. Binding affinity at hA₁, hA_2A and hA₃ ARs and potencies at hA_2B ARs.

		Binding experiments^a Ki (nM) or I%			cAMP assays I%
	R	hA1^b	hA2A^c	hA3^d	hA2B^e
15	Ph	647 ± 53	20%	20%	1%
16	CH2Ph	162 ± 14	1%	1%	1%
17	(CH2)2Ph	518 ± 42	40%	40%	1%
18	CH2C6H2-2,4,6-F	204 ± 18	34%	39%	1%
19	CH2C6H3-2-Cl-4-F	193 ± 17	29%	1%	2%
20	CO-2-furyl	580 ± 47	16%	13%	2%
21	COOtBu	615 ± 49	33%	21%	1%
22	CH2-2-furyl	92 ± 8	38%	5%	16%
23	COCH2tBu	29%	2%	1%	2%
24	COCH2Ph	429 ± 36	3%	1%	3%

^a K_i values are means ± SEM of four separate assays each performed in duplicate. Percentage of inhibition (I%) are determined at 1 μM concentration of the tested compounds.

^b Displacement of specific [³H]DPCPX competition binding assays to hA₁CHO cells.

^c Displacement of specific [³H]ZM241385 competition binding to hA_2ACHO cells.

^d Displacement of specific [¹²⁵I]AB-MECA competition binding to hA₃CHO cells.

^e cAMP experiments in hA_2BCHO cells, stimulated by 200 nM NECA. Percentage of inhibition (I%) are determined at 1 μM concentration of the tested compounds.

Figure 3. Interaction Energy Fingerprints (IEFs) comparison between compound 1 and compound ZM-241385 used as reference. Panels A, B and C report the comparison analysis for hA₁, hA_2A and hA₃ receptor subtypes, respectively. On the left side is shown the electrostatic contribution comparison, while on the right the hydrophobic one. In each subsection, the IEFs of compound 1 are shown above the IEFs of the reference ZM-241385.

Figure 4. Comparison of the proposed binding mode of compound 1 (sky blue), compound A (pink), and reference pose of ZM-241385 (green) on hA₁, hA_2A and hA₃ subtype receptors (panels A, B and C, respectively). Protein residues mainly involved in binding are shown as sticks (tan). The zoom makes TM1 not visible, while TM6 and TM7 are rendered in a transparent manner to give a more clear visualization of the binding site.

Figure 5. Results of the IEFs comparison between all compounds and reference compound ZM-241385. RMSDs and RMSD_trend between electrostatic (panels A and B, respectively) and hydrophobic (panels C and D, respectively) Energy Fingerprints of each compound (y-axis) and reference ZM-241385 are reported for hA₁, hA_2A and hA₃ receptors (x-axis). A colorimetric scale going from blue to red represents favorable to unfavorable values. An exclamation point identifies those poses that have a positive van der Waals and/or electrostatic potential (and for which was not possible to select an alternative pose with negative values).