Synthesis and structure–activity relationship studies of tyrosine-based antagonists at the human P2X₇ receptor

Abstract

Analogues of the P2X₇ receptor antagonist KN-62, modified at the piperazine and arylsulfonyl groups, were synthesized and assayed at the human P2X₇ receptor for inhibition of BzATP-induced effects, that is, uptake of a fluorescent dye (ethidium bromide) in stably transfected HEK293 cells and IL-1β release in differentiated THP-1 cells. Substitution of the arylsulfonyl moiety with a nitro group increased antagonistic potency relative to methyl substitution, such that compound 21 was slightly more potent than KN-62. Substitution with D-tyrosine in 36 and sterically bulky tyrosyl 2,6-dimethyl groups in 9 enhanced antagonistic potency.

The P2 purinergic receptor family is divided into P2X and P2Y subfamilies. The P2X receptors are ligandgated cation channels activated by extracellular ATP (adenosine-5-triphosphate). P2X receptors are further classified into seven receptor subtypes, P2X₁–P2X₇.^1,2 In particular, the P2X₇ receptor, formerly designated as the P2Z receptor,^3,4 is highly expressed in cells of the immune system such as macrophages,⁵ lymphocytes,⁶ mast cells,⁷ and microglia.⁸ The P2X₇ receptor, which displays 35–40% structural homology with other subtypes of P2X receptors, consists of two hydrophobic membrane-spanning domains (M1,M2), a large extracellular loop, a short intracellular N-terminus, and a long intracellular C-terminus.⁹ Interestingly, the C terminus of the P2X₇ receptor is 239 amino acids longer (AA 352–595) than that of other P2X receptor subtypes. The long intracellular C-terminal domain of the P2X₇ receptor modulates several functions, including signal transduction events and cytolytic pore formation.^10–12 The activation of P2X₇ receptors results in two distinct responses, depending on the exposure time to agonist. A brief stimulation of the receptor by extracellular ATP opens a membrane channel permeable to small cations (Na⁺, Ca²⁺, K⁺). However, repeated or sustained activation by ATP induces the formation of a pore permeable to large molecular weight molecules/ions up to 900 Da in mass, such as ethidium bromide (314 Da) and YO-Pro-1 (376 Da), leading to cellular death or cell lysis.^13–15

The P2X₇ receptor is implicated in the regulation of the expression and secretion of cytokines and inflammatory mediators including interleukin (IL)-1,¹⁶ IL-2,¹⁷ IL-18,¹⁸ and tumor necrosis factor (TNF)-α.¹⁹ Notably, the P2X₇ receptor plays an important role in the processing and release of the proinflammatory cytokine IL-1β in the immune system by a complex sequence of events: Initially, the activated P2X₇ receptor causes the depletion of K⁺ leading to the stimulation of IL-1β converting enzyme (caspase-1), which converts LPS-activated pro-IL-1β to mature IL-1β.¹⁶ Therefore, development of antagonists for inhibition of the P2X₇ receptor could be a therapeutic strategy to treat inflammatory diseases (Fig. 1).

Figure 1.

P2X₇ receptor antagonists.²¹–²⁷

Among potent and specific non-competitive antagonists at the human (h) P2X₇ receptor developed so far²⁰ is a tyrosyl derivative, the isoquinoline KN-62 (1-(N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl)-4-phe-nylpiperazine) 1, first known as an inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaMK II).²¹ Compound 2 (MRS2306), modified from KN-62, with the general structure R¹-Tyr(OR²)-piperazinyl-R³, exhibited an IC₅₀ value of 40 nM and was more potent than KN-62 in an ATP-induced K⁺ efflux assay in hP2X₇-expressing HEK293 cells.²² Another series of conformationally constrained and N-arylpiperazinemodified analogues was reported.^23,24 An N-p-fluorophenylpiperazine analogue, 3, displayed higher antagonistic potency than KN-62 in an ethidium bromide uptake assay and significantly inhibited ATP-stimulated secretion of IL-1β from human macrophages. Other P2X₇ receptor antagonists, such as the thiazolidine-2,4-dione derivative 4, the 4,5-diarylimidazoline derivative 5, and the 1-benzyl-5-phenyltetrazole derivative 6, have been reported by several industrial pharmaceutical laboratories.^25–27

In the present study, we report the structure–activity relationship (SAR) of a novel series of Cbz-substituted tyrosyl derivatives having substituted piperazine and arylsulfonyl groups. The compounds were evaluated in an assay of the uptake of a fluorescent dye ethidium bromide induced by 2′- and 3′-O-(4-benzoyl-benzoyl)-ATP (BzATP), which activates the hP2X₇ receptor stably expressed in HEK293 cells, and in an ELISA for IL-1β release from differentiated THP-1 cells.

Two variable positions of the skeleton, that is, the arylpiperazine and arylsulfonate moieties, were systematically modified through facile acylation and sulfonylation reactions as shown in Scheme 1 – Scheme 3. The starting materials, the compounds 7, 10, 11, and 12, were first reacted with a variably substituted arylpiperazine or Boc-piperazine to produce an amide in the presence of coupling reagents such as EDC·HCl or Bop-Cl. Then, the phenol was derivatized with various sulfonamide groups by the reaction of a para-substituted arylsulfonyl chloride in the presence of DMAP as catalyst. Compound 21 was used as the starting material in a synthetic route that led to the dimerized compound 40 (Scheme 3). This symmetric dimer was designed for the purpose of probing the proximity of adjacent binding sites on the receptor.²² The nitro group of compound 21 was reduced with NaBH₄-copper (II) bisacetylacetonate to an amine, which was subsequently converted to the isothiocyanate in compound 38 by a reaction with thiophosgene. The isothiocyanate group in 38 was reacted with 1,4-diaminobutane to produce the primary amine congener 39. Finally, to make the dimer 40 a R⁵–R⁵ linker was created by a coupling reaction between 38 and 39.

Scheme 1.

Synthesis of a representative P2X₇ receptor antagonist consisting of a 2,6-dimethyltyrosine derivative. Reagents: (a) benzoyl piperazine, HOBt, EDC.HCl, DMF, rt,8h; (b) p-methylbenzenesulfonyl chloride, DMAP, Et₃N, CH₂Cl₂, rt, 12h

Scheme 3.

Dimerization of P2X₇ receptor antagonists through formation of a thiourea linkage. Reagents and conditions: (a) NaBH₄, Cu(acac)₂, methanol, rt, 80–85%; (b) CSCl₂, chloroform, water, sodium bicarbonate, 70–85%; (c) 1,4-diaminobutane, CH₂Cl₂, rt, 40–45%; (d) dry DMF, at 80 °C, 52%.

The SAR studies of tyrosine-based derivatives 9, 16–36, and 40, which have various substitutions at the toluenesulfonyl, substituted piperazine, and benzyloxycarbonyl (Cbz) groups, were assessed in the ethidium⁺ accumulation assay. Experiments were performed using HEK293 cells stably transfected with cDNA encoding the hP2X₇ receptor. As shown in Table 1, compound 9 with 2,6-dimethyl groups at the phenyl ring of tyrosine displayed a 3-fold increase of the antagonistic activity compared with compound 16. In addition, when the methyl group was adjacent to the benzoyl piperazine group (e.g., 17 and 18), the antagonist effects were also increased by 2-fold compared with compound 16 regardless of the stereochemistry of the chiral carbon of the piperazine group. Thus, the steric effects near the sulfonyl ester of the phenyl group of tyrosine and the benzoyl group of piperazine ring were tolerated in the antagonism of the receptor. However, unlike compound 17 or 18, their regioisomer, 19, contained a methyl group adjacent to the α-carbonyl of the tyrosyl moiety, which reduced the antagonistic effect. In a comparison between compounds 20 and 16, the presence of a methyl group at the tyrosyl α-nitrogen decreased the antagonistic activity by 2-fold at the hP2X₇ receptor, which is consistent with previously reported results.²³ The 4-nitro group at the R⁵ position (compound 21) increased the antagonistic effect by 5-fold compared with the corresponding 4-methyl group of 16; compound 21 was slightly more potent than KN-62.

Table 1.

Activities of synthesized tyrosine-based derivatives on ethidium accumulation in hP2X₇-expressing HEK293 cells²⁸

Compound	R/S	R1	R2	R3	R4	R5	IC50a ± SE (nM)
KN-62 (positive control)							175 ± 35
9b	(S)	H	Ph-CO	H	H	CH3	219 ± 69
16	(S)	H	Ph-CO	H	H	CH3	599 ± 22
17	(S)	H	Ph-CO	(S)CH3	H	CH3	260 ± 77
18	(S)	H	Ph-CO	(R)CH3	H	CH3	256 ± 96
19	(S)	H	Ph-CO	H	(R)CH3	CH3	504 ±136
20	(S)	CH3	Ph-CO	H	H	CH3	1360 ± 580
21	(S)	H	Ph-CO	H	H	NO2	117 ± 44
22	(S)	H	p-(CH3)2N-Ph-CO	(S)CH3	H	CN	539 ± 90
23	(S)	H	p-(CH3)2N-Ph-CO	(S)CH3	H	I	55 ± 5%c
24	(S)	H	p-(CH3)2N-Ph-CO	(S)CH3	H	CH3	54 ± 3%c
25	(S)	H	p-(CH3)2N-Ph-CO	H	H	CH3	2010 ± 340
26	(S)	H	p-CF3-Ph-CO	H	H	CH3	724 ± 171
27	(S)	H	p-F-Ph-CO	H	H	CH3	319 ± 65
28	(S)	H	p-Br-Ph-CO	H	H	CH3	481 ± 41
29	(S)	H	Boc	H	H	F	5880 ± 3280
30	(S)	H	Boc	H	H	Br	517 ± 18
31	(S)	H	Boc	H	H	I	3540 ± 907
32	(S)	H	Boc	H	H	CN	780 ± 107
33	(S)	H	Boc	H	H	OCF3	725 ±118
34	(S)	H	Boc	H	H	CH2Br	2460 ± 470
35	(S)	H	Boc	H	H	CH3	1540 ± 102
36	(R)	H	Boc	H	H	CH3	861 ± 123
40d							28 ± 1%c

^aIC₅₀ = 50% inhibitory concentrations were obtained from concentration–response curves. Data values are expressed as means ± SD. All experiments were repeated at least 3–11 times.

^bThis compound has 2,6-dimethyl groups at the phenyl ring of the tyrosyl moiety (see Scheme 1).

^cThe compounds 23, 24, and 40 represented the % inhibition at 10 µM concentration level.

^dThis compound is a dimer linked through formation of a thiourea linkage (see Scheme 3). 16, MRS2427; 21, MRS2447; 27, MRS2544.

The rank order of potency according to IC₅₀ values of para-substituents at the benzoyl group was F (27) > Br (28) > CF₃ (26) > (CH₃)₂N (25). Comparing compounds 17 versus 24 and 16 versus 25 show that a 4-dimethylamino group significantly reduced the antagonistic activity. Thus, basic or bulky groups seem to be inappropriate recognition motifs for the antagonism. Compounds that were Boc-substituted at the R² position showed different antagonistic effects depending on phenyl sulfonyl ring para-substitution (i.e., R⁵-substituent) with the following order of potency: Br (30) > OCF₃ (33) > CN (32) >> CH₃ (35) > CH₂Br (34) > I (31) > F (29).

In the study of the effect of chirality of the tyrosine by comparing R- and S-isomers 35 and 36, the R-(d)-isomer was the more potent P2X₇ receptor antagonist. The substitution with a d-tyrosyl residue might add stability to the compound in biological systems. The dimeric compound 40 showed only marginal antagonism at a 10 µM concentration.

The functional antagonism of the tyrosine-based derivatives (9, 16–36, and 40) was evaluated with an ELISA for the detection of BzATP-activated IL-1β release in LPS/ IFNγ-differentiated human THP-1 cells (Table 2). Most of the compounds with low submicromolar IC₅₀ values in the ethidium accumulation assay (e.g., 9, 17, 18, 27, 28, etc.) except for 21, resulted in the parallel correlation with the inhibitory activity against the release of IL-1β by BzATP. However, compounds 24 and 31, which had very low antagonistic activity in the ethidium accumulation assay, showed a relatively high inhibitory effect on the release of IL-1β. The methyl group at the R⁴ position of 19 and the methyl group on the α-nitrogen of 20 seem to affect the inhibitory effect on the release of IL-1β as a comparison between them and compound 16 shows.

Table 2.

Antagonistic effects of tyrosyl derivatives against BzATP-stimulated IL-1β release in LPS/IFNγ-differentiated human THP-1 cells²⁹

Compound	% inhibitiona	Compound	% inhibition
KN-62	83 ± 9	26	65 ± 6
9	87 ± 8	27	68 ± 10
16	70 ± 14	28	78 ± 6
17	69 ± 12	29	56 ± 13
18	65 ± 3	30	68 ± 12
19	45 ± 4	31	72 ± 4
20	47 ± 12	32	60 ± 8
21	48 ± 7	33	46 ± 9
22	59 ± 11	34	52 ± 5
23	61 ± 6	35	72 ± 15
24	68 ± 7	36	56 ± 5
25	65 ± 12	40	37 ± 20

^aThe values represent the % inhibition at a 1 µM concentration expressed as means ± SD against 1 mM Bz-ATP (n = 3).

Compounds 9 and 28, which showed inhibitory effects similar to KN-62 against the release of IL-1β by BzATP, were further evaluated with full concentration–response curves to compare IC₅₀ values (Fig. 2). The IC₅₀ values of compounds 9 (481 nM) and 27 (881 nM) were 2- and 4-fold higher than that of KN-62 (188 nM).

Figure 2.

Concentration-dependent inhibition of BzATP-stimulated IL-1β release in LPS/IFNγ-differentiated human THP-1 cells by compounds 9, 28, and KN-62. Data points represent means ± SD of values obtained (n = 3).

In conclusion, a novel series of tyrosine-based P2X₇ receptor antagonists with modifications at the piperazine and arylsulfonyl groups were synthesized and evaluated in ethidium accumulation assays and ELISAs of the release of IL-1β. Substitution of the arylsulfonyl moiety with a nitro group, in comparison to a methyl group, increased antagonistic potency in compound 21, which was slightly more potent than KN-62. Several derivatives, including the 2,6-dimethylphenylsulfonate analog 9 and the methylpiperazine analogs 17 and 18, were nearly equipotent to the parent compound KN-62 and their functional inhibitory activity against the release of the inflammatory cytokine, IL-1β, was confirmed. Substitution with d-tyrosine in 36 and sterically bulky tyrosyl 2,6-dimethyl groups in 9 favored P2X₇ receptor antagonistic potency.

Scheme 2.

Synthesis of various P2X₇ receptor antagonists consisting of tyrosyl derivatives. Reagents and conditions: (a) substituted arylpiperazine or Boc-piperazine, HOBt, EDC·HCl, DMF, rt, 93– 98%; (b) substituted benzenesulfonyl chloride, DMAP, CH₂Cl₂, rt, 70– 90%.