Potent P2X₇ Receptor Antagonists: Tyrosyl Derivatives Synthesized Using a Sequential Parallel Synthetic Approach

Abstract

Novel analogs of 1-(N,O-bis[5-isoquinolinesulfonyl]-N-methyl-L-tyrosyl)-4-phenylpiperazine (KN-62,1) were synthesized and found to be potent antagonists in a functional assay, inhibition of ATP-induced K⁺ efflux in HEK293 cells expressing recombinant human P2X₇ receptors. Antagonism of murine P2X₇ receptors was also observed. The analogs consisted of L-tyrosine derivatives, of the general structure R₁-Tyr(OR₂)-piperazinyl-R₃, in which three positions were systematically varied in structure through facile acylation reactions. Each of the three positions was optimized in sequence through parallel synthesis alternating with biological evaluation, leading to the identification and optimization of potent P2X₇ antagonists. The optimal groups at R₁ were found to be large hydrophobic groups, linked to the α-amino position through carbamate, amide, or sulfonamide groups. The benzyloxycarbonyl (Cbz) group was preferred over most sulfonamides and other acyl groups examined, except for quinoline sulfonyl. At R₂, an arylsulfonate ester was preferred, and the order of potency was p-tolyl, p-methoxyphenyl, phenyl > α-naphthyl, β-naphthyl. A benzoyl ester was of intermediate potency. Aliphatic esters and carbonate derivatives at the tyrosyl phenol were inactive, while a tyrosyl O-benzyl ether was relatively potent. The most potent P2X₇ receptor antagonists identified in this study contained Cbz at the R₁ position, an aryl sulfonate at the R₂ position, and various acyl groups at the R₃ position. At R₃, t-butyloxycarbonyl- and benzoyl groups were preferred. The opening of the piperazinyl ring to an ethylene diamine moiety abolished antagonism. In concentration-response studies, a di-isoquinolinyl, Boc derivative, 4 (MRS2306), displayed an IC₅₀ value of 40 nM as an antagonist of P2X₇ receptor-mediated ion flux and was more potent than the reference compound 1. N^α-Cbz, Boc-piperazinyl derivatives, 11 (MRS2317), 22 (MRS2326), and 41 (MRS2409) were less potent than 1, with IC₅₀ values of 200–300 nM.

INTRODUCTION

P2 receptors, which are activated by ATP (adenosine 5′-triphosphate) and other purine/pyrimidine nucleotides, consist of two families: G-protein-coupled receptors termed P2Y, of which seven mammalian subtypes have been cloned, and ligand-gated cation channels termed P2X, of which seven mammalian subtypes have been cloned [Fredholm et al., 1997; North and Barnard, 1997; Jacobson et al., in press]. The nomenclature of P2 receptors and their various ligand specificities have been reviewed previously [Jacobson and Knutsen, 2001; Jacobson et al., 1997; Bhagwat and Williams, 1997; Fischer, 1999].

The P2X₇ receptor (formerly P2Z receptor) is expressed primarily in blood cells (monocytes, macrophages, and lymphocytes), in the brain (on microglial cells) [Ferrari et al., 1999], and in the salivary gland. Characteristic of the P2X₇ receptor is that at high µM concentrations of agonists it forms or activates a large pore in addition to a cation channel. This pore increases permeability indiscriminately to molecules having MW = 900, such as ethidium bromide, which is used as a marker for pore activity. 2′- and 3′-O-(4-benzoylbenzoyl)-ATP (BzATP) is among the most potent agonists at P2X₇ receptors, but also has nanomolar potency at P2X₁ receptors [Bianchi et al., 1999]. Affinity labeling of the P2X₇ receptor in mast cells has been carried out using [³H]-BzATP [Erb et al., 1990].

In macrophages, activation of the P2X₇ receptor triggers the processing and release of IL-1β. In the immune system, activation of the P2X₇ receptor leads to apoptosis or programmed cell death [Ferrari et al., 1997; Coutinho-Silva et al., 1999; Humphreys et al., 2000]. BzATP (5 mM) caused apoptosis in dendritic cells, which play a significant role in T-cell activation [Coutinho-Silva et al., 1999]. BzATP (1 mM) was very effective in activating the transcription factor NFAT in N9 microglial cells, suggesting purinergic modulation of early inflammatory gene expression in the nervous and immune systems [Ferrari et al., 1999]. Activation of the P2X₇ receptor in rat microglia triggers the release of TNF-α [Hide et al., 2000].

Recent reports have emphasized the importance of P2X₇ receptors in the immune system and inflammatory processes. It would be very useful to design selective antagonists of high affinity for this receptor. A P2X₇ receptor antagonist may be useful in treating septic shock [Hu et. al., 1998] or neurodegenerative diseases, since the receptor activates astrocytes [Sun et al., 1999] and microglial cells [Ferrari et al., 1999; Visentin et al., 1999]. Modulation of the P2X₇ receptor may also be beneficial in ophthalmic diseases [Bringmann et al., 2001]. The P2X₇ receptor is expressed in high levels in dendritic cells and ATP acting at this site might serve as a signal to downmodulate the immune response [Coutinho-Silva et al., 1999; Ferrari et al., 2000]. A mouse line engineered to have null expression of the P2X₇ receptor has been reported [Solle et al., 2001] and may aid in the identification of therapeutic targets for P2X₇ receptor antagonists.

The isoquinoline derivatives (Fig. 1) 1-(N,O-bis[5-isoquinolinesulfonyl]-N-methyl-L-tyrosyl)-4-phenylpiperazine (1, KN-62), and 5-isoquinolinesulfonic acid, 4-[2-[(5-isoquinolinesulfonyl)amino]-3-(4-phenylpiperazine)propyl]phenyl ester (2, KN-04) are potent noncompetitive antagonists at P2X₇ receptors [Gargett and Wiley, 1997; Wiley et al., 1998; Humphreys et al., 1998]. Compound 1, but not 2, is also an antagonist of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) in the micromolar range [Tokumitsu et al., 1990]. Compound 1 displayed noncompetitive antagonism at P2X₇ receptors in HEK293 cells, with an IC₅₀ value of approximately 15 nM [Chessell et al., 1998]. In human leukemic B lymphocytes, compound 1 reduced the rate of permeability increase to larger permeant cations, like ethidium, induced by Bz-ATP with an IC₅₀ of 13.1 nM. Complete inhibition of the flux was observed at 500 nM [Wiley et al., 1998]. Compound 1 had no effect on responses mediated by the P2Y₂ receptor of human neutrophils [Gargett and Wiley, 1997] or on calcium mobilization induced by the P2Y₁ and P2Y₂ receptors naturally expressed in HEK293 human fibroblasts (G. Dubyak, unpubl. obs.). Thus, compound 1 has considerable selectivity for P2X₇ receptors within the P2 family.

Fig. 1.

Structures of isoquinoline derivatives reported to be P2X₇ receptor antagonists.

Species differences in P2X₇ receptor antagonism have been reported [Humphreys et al., 1998; Bianchi et al., 1999]. For example, Brilliant blue G was reported to be a selective antagonist of IC₅₀ values of 10 and 200 nM at the rat and human P2X₇ receptors, respectively [Jiang et al., 2000]. Compound 1 at a concentration of 3 µM had no effect on ATP-induced ethidium influx through the rat P2X₇ receptor, while the IC₅₀ at the human P2X₇ receptor was 0.1 µM [Humphreys et al., 1998].

The binding site for compound 1 resides within the first 335 residues of the human P2X₇ receptor [Humphreys et al., 1998]. The precise modeling of this binding site is not feasible at present, due to a lack of a high-resolution template for this ion channel and uncertainty about the oligomeric nature of the channel [Torres et al., 1999]. P2X₇ receptors do not form heteromeric receptors with any of the other six P2X subunits and it is not known what types of homomeric assemblies of P2X₇ subunits occur [Kim et al., 2001]. P2X₇ receptors in brain glia and/or astrocytes appear to be expressed only as monomeric subunits [Kim et al., 2001].

In a study of conformationally constrained analogs of 1 by Baraldi et al. [2000], only one compound showed appreciable activity as a P2X₇ antagonist (30-fold weaker than 1) in human macrophage cells. In the present study, we systematically varied all of the substituent groups in a series of tyrosyl analogs of 1, resulting in a qualitative elucidation of structure–activity relationships and an enhancement of potency.

MATERIALS AND METHODS

Chemical Synthesis

Synthetic reagents were purchased from Sigma Chemical Co. (St. Louis, MO) and Aldrich (Milwaukee, WI). ¹H-NMR spectra were obtained with a Varian Gemini-300 spectrometer using CD₃OD or CDCl₃ as a solvent. Low-resolution CI-NH₃ (chemical ionization) mass spectra were carried out with a Finnigan 4600 mass spectrometer and high-resolution EI (electron impact) mass spectrometry with a VG7070F mass spectrometry at 6 kV. High-resolution FAB (fast atom bombardment) mass spectrometry was performed with a JEOL SX102 spectrometer using 6-kV Xe atoms following desorption from a glycerol matrix. Compounds 51 and 55a–c were obtained from Calbiochem-Novabiochem (La Jolla, CA).

[N-Fmoc-L-tyrosyl]-Boc-piperazine (52)

A mixture of Fmoc-Tyr-OH (51) (0.4 g, 1 mmol) Bocpiperazine (0.186 g, 1 mmol) and BOP-Cl (0.255 g, 1 mmol) in CH₂Cl₂ (5 mL) was treated with Et₃N (0.28 mL, 2 mmol) and stirred at rt for 5 h. The solvent was removed and the residue obtained was purified using flash chromatography eluting with 10% MeOH in CHCl₃ to furnish 52 (0.38 g, 67%) as a solid foam.

¹H NMR (CDCl₃): δ 7.77 (d, J = 7.3 Hz, 2H), 7.59 (d, J = 7.3 Hz, 2H), 7.40 (t, J = 7.3 Hz, 2H), 7.31 (t, J = 7.3 Hz, 2H), 7.04 (d, J = 7.7 Hz, 2H), 6.74 (d, J = 7.7 Hz, 2H), 5.88 (s, 1H), 5.72 (d, J = 8.4Hz, 1H), 4.90-4.78 (m, 1H), 4.44-4.28 (m, 2H), 4.24-4.16 (m, 2H), 3.6-2.8 (m, 9H), 1.45 (s, 9H).

[L-Tyrosyl]-Boc-piperazine (53)

Compound 52 (0.37 g, 0.65 mmol) was treated with 20% piperidine in DMF (N,N-dimethylformamide) at rt for 10 min for complete reaction. DMF was removed under high vacuum and the residue obtained was purified using flash chromatography eluting with 15% MeOH in CHCl₃ to furnish 53 (0.19 g, 54%) as a gum.

¹H NMR (CDCl₃): δ 6.99 (d, J = 8.2 Hz, 2H), 6.72 (d, J = 8.2 Hz, 2H), 3.9 (t, J = 7.1 Hz, 1H), 3.62-3.20 (m, 6H), 3.10-2.74 (m, 4H), 1.45 (s, 9H).

[N,O-Bis-(quinolinesulfonyl)-L-tyrosyl]-Boc-piperazine and [N,O-Bis-(5-isoquinolinesulfonyl)-L-tyrosyl]-Boc-piperazine (4 and 6)

To a suspension of 8-quinoline sulfonyl chloride or 5-isoquinoline sulfonyl chloride (0.274 g, 1.2 mmol) in anhydrous CH₂Cl₂ (5 mL) was added a solution of 53 (0.175 g, 0.5 mmol) and Et₃N in CH₂Cl₂ (3 mL) at 0°C, and the mixture stirred at rt for 4 h. The solvent was removed from the reaction mixture under vacuum and the residue obtained was purified using flash chromatography using 5% MeOH in CHCl₃ to furnish 0.26 g of 4 and 0.25 g of 6 as a solid foam.

4: ¹H NMR (CDCl₃): δ 9.43 (s, 1H), 9.34 (s, 1H), 8.84 (d, J = 5.9 Hz, 1H), 8.69 (d, J = 5.4 Hz, 1H), 8.52 (d, J = 5.9 Hz, 1H), 8.54-8.10 (m, 5H), 7.70-7.52 (m, 2H), 6.85 (d, J = 8.2 Hz, 2H), 6.61 (d, J = 8.2 Hz, 2H), 6.00 (bs, 1H), 4.20-4.38 (m, 1H), 3.12-2.50 (m, 10H), 1.45 (s, 9H).

6: ¹H NMR (CDCl₃): δ 9.25 (d, J = 3.9 Hz, 1H), 9.08 (d, J = 3.9 Hz, 1H), 8.34-8.20 (m, 4H), 8.13 (d, J = 8.1 Hz, 1H), 8.02 (d, J = 8.1 Hz, 1H), 7.68-7.52 (m, 4H), 7.17 (d, J = 9.9 Hz, 1H), 6.90 (d, J = 8.2 Hz, 2H), 6.79 (d, J = 8.2 Hz, 2H), 4.42–4.58 (m, 1H), 3.02-2.26 (m, 10H), 1.44 (s, 9H).

[N,O-Bis-(quinolinesulfonyl)-L-tyrosyl]piperazine and [N,O-Bis-(5-isoquinolinesulfonyl)-L-tyrosyl]piperazine (3 and 5)

Compound 4 or 6 (0.17 g, 0.23 mmol) was treated with 10% TFA in CH₂Cl₂ at rt for 6 h for complete reaction. Solvent was removed under vacuum and the residue obtained was purified using flash chromatography using 10% MeOH in CHCl₃ to furnish 0.12 g of 3 and 5 as a solid foam.

3: ¹H NMR (CD₃OD): δ 9.41 (s, 1H), 9.30 (s, 1H), 8.73 (d, J = 5.9 Hz, 1H), 8.55-8.05 (m, 7H), 7.66 (ABq, J = 8.1 Hz, 2H), 6.74 (d, J = 8.4 Hz, 2H), 6.27 (d, J = 8.4 Hz, 2H), 4.38-4.22 (m, 1H), 3.80-3.38 (m, 5H), 3.18-2.58 (m, 6H).

5: ¹H NMR (CD₃OD): δ 9.14 (d, J = 4.3 Hz, 1H), 8.97 (d, J = 4.1 Hz, 1H), 8.52 (d, J = 8.4 Hz, 1H), 8.44-8.22 (m, 4H), 8.17 (d, J = 8.2 Hz, 1H), 7.82-7.58 (m, 4H), 6.94 (d, J = 8.4 Hz, 2H), 6.74 (d, J = 8.4 Hz, 2H), 3.64-2.45 (m, 12H).

[N-Fmoc-O-quinolinesulfonyl-L-tyrosyl]-Boc-piperazine (8)

A suspension of 8-quinoline sulfonyl chloride (0.16 g, 0.71 mmol) in CH₂Cl₂ was treated with a solution of 52 (0.34 g, 0.60 mmol) and Et₃N in CH₂Cl₂ at 0°C and the mixture was stirred at rt for 6 h. The solvent was removed under vacuum and the crude product was purified using flash chromatography using 5% MeOH in CHCl₃ to furnish 0.35 g of 8.

8: ¹H NMR (CDCl₃): δ 9.26 (s, 1H), 8.3 (t, J = 6.3 Hz, 2H), 8.12 (d, J = 7.9 Hz, 1H), 7.75 (d, J = 7.1 Hz, 2H), 7.64-7.48 (m, 4H), 7.44-7.20 (m, 4H), 7.03 (d, J = 8.3 Hz, 2H), 6.91 (d, J = 8.3 Hz, 2H), 5.61 (d, J = 8.2 Hz, 1H), 4.85-4.65 (m, 1H), 4.45-4.05 (m, 4), 3.55-3.12 (m, 9H), 1.47 (s, 9H).

[O-Quinolinesulfonyl-L-tyrosyl]-Boc-piperazine (9)

Compound 8 (0.3 g, 0.39 mmol) was treated with 20% piperidine in DMF (10 mL) at rt for 10 min. DMF was removed under vacuum and the residue obtained was purified using flash chromatography using 10% MeOH in CHCl₃ to furnish 0.19 g of 9 as a solid foam.

9: ¹H NMR (CDCl₃): δ 9.26 (d, J = 2.8 Hz, 1H), 8.42-8.25 (m, 2H), 8.15 (d, J = 7.9 Hz, 1H), 7.70-7.56 (m, 2H), 7.03 (d, J = 8.3 Hz, 2H), 6.92 (d, J = 8.3 Hz, 2H), 3.92-2.62 (m, 13H), 1.47 (s, 9H).

General Procedure for the Synthesis of 7, 10–18

To a solution of respective R₁Cl (0.11 mmol) in anhydrous CH₂Cl₂ (1 mL) at 0°C was added a solution of 9 (0.03 g, 0.055 mmol), Et₃N (0.015 mL, 0.11 mmol) and DMAP (0.007 g, 0.055 mmol) in CH₂Cl₂ (1 mL) and the mixture stirred for 30 min for complete reaction. The solvent was removed and the crude product was purified by preparative TLC using 5% MeOH in CHCl₃ to furnish 7, 10–18.

[N-(5-Isoquinolinesulfonyl)-O-quinolinesulfonyl-L-tyrosyl]-Boc-piperazine (7)

¹H NMR (CDCl₃): δ 9.45-9.20 (m, 2H), 8.40-8.18 (m, 6H), 7.70-7.52 (m, 4H), 6.83 (d, J = 8.5 Hz, 2H), 6.74 (d, J = 8.5 Hz, 2H), 5.99 (d, J = 9.3 Hz, 1H), 4.40-4.20 (m, 1H), 3.15-2.49 (m, 10H), 1.45 (s, 9H).

[N-Ethyloxycarbonyl-O-quinolinesulfonyl-L-tyrosyl]-Boc-piperazine (10)

¹H NMR (CDCl₃): δ 9.27 (d, J = 2.4 Hz, 1H), 8.32 (d, J = 7.4 Hz, 2H), 8.15 (d, J = 7.9 Hz, 1H), 7.72-7.52 (m, 2H), 7.04 (d, J = 8.2 Hz, 2H), 6.91 (d, J = 8.2 Hz, 2H), 5.47 (d, J = 7.9 Hz, 1H), 4.82-4.64 (m, 1H), 4.06 (q, J = 7.1 Hz, 2H), 3.52-2.64 (m, 10H), 1.47 (s, 9H), 1.21 9(t, J = 7.1 Hz, 3H).

[N-Cbz-O-quinolinesulfonyl-L-tyrosyl]-Boc-piperazine (11)

¹H NMR (CDCl₃): δ 9.27 (d, J = 3.9 Hz, 1H), 8.31 (d, J = 8.2 Hz, 2H), 8.14 (d, J = 7.9 Hz, 1H), 7.68-7.52 (m, 2H), 7.44-7.20 (m, 5H), 7.02 (d, J = 7.9 Hz, 2H), 6.90 (d, J = 7.9 Hz, 2H), 5.63 (d, J = 8.5 Hz, 1H), 5.05 (s, 2H), 4.82-4.68 (m, 1H), 3.71 (dd, J = 13.7, 6.87 Hz, 1H), 3.50-2.60 (m, 9H), 1.48 (s, 9H).

[N-Benzoyl-O-quinolinesulfonyl-Ltyrosyl]-Boc-piperazine (12)

¹H NMR (CDCl₃): δ 9.27 (d, J = 3.8 Hz, 1H), 8.31 (d, J = 7.9 Hz, 2H), 8.14 (d, J = 8.2 Hz, 1H), 7.74 (d, J = 7.6 Hz, 2H), 7.68-7.54 (m, 2H), 7.54-7.34 (m, 3H), 7.08 (d, J = 8.5 Hz, 2H), 6.92 (d, J = 8.2 Hz, 2H), 5.25 (m, 1H), 3.56-2.68 (m, 10H), 1.47 (s, 9H).

[N-Methanesulfonyl-O-quinolinesulfonyl-L-tyrosyl]-Boc-piperazine (13)

¹H NMR (CDCl₃): δ 9.27 (d, J = 3.9 Hz, 1H), 8.33 (d, J = 7.9 Hz, 2H), 8.15 (d, J = 9.0 Hz, 1H), 7.70-7.55 (m, 2H), 7.06 (d, J = 8.2 Hz, 2H), 6.97 (d, J = 8.2 Hz, 2H), 5.50 (bs, 1H), 4.47 (bs, 1H), 3.66-3.44 (m, 1H), 3.42-2.80 (m, 9H), 2.62 (s, 3H), 1.47 (s, 9H).

[N-Benzenesulfonyl-O-quinolinesulfonyl-L-tyrosyl]-Boc-piperazine (14)

¹H NMR (CDCl₃): δ 9.25 (s, 1H), 8.36-8.24 (m, 2H), 8.14 (d, J = 7.9 Hz, 1H), 7.74 (d, J = 7.4 Hz, 2H), 7.68-7.38 (m, 5H), 6.97 (d, J = 7.9 Hz, 2H), 6.87 (d, J = 7.9 Hz, 2H), 5.80 (d, J = 9.3 Hz, 1H), 4.34-4.18 (m, 1H), 3.10-2.48 (m, 10H), 1.45 (s, 9H).

[N-Toluenesulfonyl-O-quinolinesulfonyl-L-tyrosyl]-Boc-piperazine (15)

¹H NMR (CDCl₃): δ 9.26 (d, J = 2.5 Hz, 1H), 8.38-8.24 (m, 2H), 8.15 (d, J = 8.2 Hz, 1H), 7.70-7.54 (m, 4H), 7.32-7.18 (m, 2H), 6.96 (d, J = 8.2 Hz, 2H), 6.88 (d, J = 8.2 Hz, 2H), 5.72 (bs, 1H), 4.24 (s, 1H), 3.18-2.44 (m, 10H), 2.38 (s, 3H), 1.46 (s, 9H).

[N-(4-Methoxybenzenesulfonyl)-O-quinolinesulfonyl-L-tyrosyl]-Boc-piperazine (16)

¹H NMR (CDCl₃): δ 9.25 (d, J = 3.9 Hz, 1H), 8.31 (d, J = 8.2 Hz, 2H), 8.15 (d, J = 8.2 Hz, 1H), 7.72-7.56 (m, 4H), 7.06-6.80 (m, 6H), 5.70 (d, J = 12.6 Hz, 1H), 4.30-4.14 (m, 1H), 3.83 (s, 3H), 3.22-2.42 (m, 10H), 1.46 (s, 9H).

[N-(1-Naphthylsulfonyl)-O-quinolinesulfonyl-L-tyrosyl]-Boc-piperazine (17)

¹H NMR (CDCl₃): δ 9.25 (d, J = 2.5 Hz, 1H), 8.52 (d, J = 8.5 Hz, 1H), 8.38-8.22(m, 2H), 8.20-8.08 (m, 2H), 8.04 (d, J = 8.2 Hz, 1H), 7.90 (d, J = 7.7 Hz, 1H), 7.76-7.52 (m, 4H), 7.47 (t, J = 7.7 Hz, 1H), 6.83 (d, J = 8.5 Hz, 2H), 6.75 (d, J = 8.5 Hz, 2H), 5.94 (bs, 1H), 4.30-4.15 (m, 1H), 3.0-2.32 (m, 10H), 1.45 (s, 9H).

[N-(2-Naphthylsulfonyl)-O-quinolinesulfonyl-L-tyrosyl]-Boc-piperazine (18)

¹H NMR (CDCl₃): δ 9.25 (d, J = 2.2 Hz, 1H), 8.38-8.22 (m, 3H), 8.13 (d, J = 8.2 Hz, 1H), 7.96-7.80 (m, 3H), 7.76-7.52 (m, 5H), 6.95 (d, J = 8.2 Hz, 2H), 6.86 (d, J = 8.2 Hz, 2H), 5.83 (bs, 1H), 4.28 (m, 1H), 3.0-2.70 (m, 5H), 2.70-2.25 (m, 5H), 1.41 (s, 9H).

[N-Cbz-L-tyrosyl]-Boc-piperazine (19)

A mixture of Cbz-Tyr-OH (54) (0.4 g, 1.3 mmol, Aldrich), Boc-piperazine (0.24 g, 1.3 mmol) and BOP-Cl (0.33 g, 1.3 mmol) in anhydrous CH₂Cl₂ (5 mL) was treated with Et₃N (0.36 mL, 2.6 mmol) at rt and stirred for 5 h. The reaction mixture was concentrated under vacuum and the crude product obtained was purified using flash chromatography using 10% MeOH in CHCl₃ to furnish 0.4 g of 19.

19: ¹H NMR (CDCl₃): δ 7.34 (s, 5H), 7.01 (d, J = 8.2 Hz, 2H), 6.72 (d, J = 8.2 Hz, 2H), 6.16 (bs, 1H), 5.70 (d, J = 8.5 Hz, 1H), 5.09 (ABq, J = 12.4 Hz, 2H), 4.90–7.77 (m, 1H), 3.60-3.40 (m, 2H), 3.40-3.14 (m, 4H), 3.08-2.80 (m, 4H), 1.45 (s, 9H).

General Procedure for the Synthesis of 20–31

To a solution of respective R₁Cl (0.124 mmol) in anhydrous CH₂Cl₂ (1 mL) at 0°C was added a solution of 19 (0.03 g, 0.062 mmol), Et₃N (0.017 mL, 0.124 mmol) and DMAP (0.007 g, 0.055 mmol) in CH₂Cl₂ (1 mL) and the mixture stirred for 30 min for complete reaction. The solvent was removed and the crude product was purified by preparative TLC using 5% MeOH in CHCl₃ to provide compounds 20–31.

[N-Cbz-O-Methanesulfonyl-L-tyrosyl]-Boc-piperazine (20)

¹H NMR (CDCl₃): δ 7.4-7.3 (m, 5H), 7.23 (d, J = 8.8 Hz, 2H), 7.19 (d, J = 8.8 Hz, 2H), 5.70 (d, J = 8.2 Hz, 1H), 5.07 (ABq, J = 12.1 Hz, 2H), 4.92-4.80 (m, 1H), 3.60-3.12 (m, 8H), 3.11 (s, 3H), 3.06-2.72 (m, 2H), 1.45 (s, 9H).

[N-Cbz-O-Benzenesulfonyl-L-tyrosyl]-Boc-piperazine (21)

¹H NMR (CDCl₃): δ 7.82 (d, J = 7.7 Hz, 2H), 7.62- 7.48 (m, 3H), 7.34 (s, 5H), 7.10 (d, J = 7.9 Hz, 2H), 6.88 (d, J = 7.9 Hz, 2H), 5.65 (d, J = 8.5 Hz, 1H), 5.07 (ABq, J = 12.4 Hz, 2H), 4.88-4.76 (m, 1H), 3.60-2.65 (m, 10H), 1.44 (s, 9H).

[N-Cbz-O-Toluenesulfonyl-L-tyrosyl]-Boc-piperazine (22)

¹H NMR (CDCl₃): δ 7.69 (d, J = 7.9 Hz, 2H), 7.42-7.28 (m, 7H), 7.09 (d, J = 7.9 Hz, 2H), 6.88 (d, J = 7.9 Hz, 2H), 5.65 (d, J = 8.5 Hz, 1H), 5.07 (ABq, J = 12.6 Hz, 2H), 4.90-4.76 (m, 1H), 3.60–3.70 (m, 10H), 2.45 (s, 3H), 1.44 (s, 9H).

[N-Cbz-O-(4-Methoxylbenzenesulfonyl)-L-tyrosyl]-Boc-piperazine (23)

¹H NMR (CDCl₃): δ 7.83 (d, J = 8.8 Hz, 2H), 7.50-7.40 (m, 5H), 7.20 (d, J = 8.3 Hz, 2H), 7.09 (d, J = 8.8 Hz, 2H), 6.99 (d, J = 8.8 Hz, 2H), 5.75 (d, J = 8.51 Hz. 1H), 5.18 (ABq, J = 12.4 Hz, 2H), 5.0-4.84 (m, 1H), 3.99 (s, 3H), 3.66-2.80 (m, 10H), 1.55 (s,9H).

[N-Cbz-O-(1-Naphthylsulfonyl)-L-tyrosyl]-Boc-piperazine (24)

¹H NMR (CDCl₃): δ 8.81 (d, J = 8.5 Hz, 1H), 8.13 (d, J = 8.2 Hz, 1H), 8.11-7.94 (m, 2H), 7.92-7.64 (m, 2H), 7.49 (t, J = 7.7 Hz, 1H), 7.32 (s, 5H), 6.99 (d, J = 8.2 Hz, 2H), 6.74 (d, J = 8.2 Hz, 2H), 5.62 (d, J = 8.2 Hz, 1H), 5.05 (ABq, J = 12.1 Hz, 2H), 4.86-4.70 (m, 1H), 3.58-2.64 (m, 10H), 1.47 (s, 9H).

[N-Cbz-O-(2-Naphthylsulfonyl)-L-tyrosyl]-Boc-piperazine (25)

¹H NMR (CDCl₃): δ 8.38 (s, 1H), 8.05-7.86 (m, 3H), 7.86-7.76 (m, 1H), 7.75-7.45 (m, 2H), 7.32 (s, 5H), 5.61 (d, J = 8.5 Hz, 1H), 5.05 (ABq, J = 12.6 Hz, 2H), 4.88-4.68 (m, 1H), 3.54-2.68 (m, 10H), 1.45 (s, 9H).

[N-Cbz-O-(5-Isoquinolinesulfonyl)-L-tyrosyl]-Boc-piperazine (26)

¹H NMR (CDCl₃): δ 9.43 (s, 1H), 8.83 (d, J = 6.0 Hz, 1H), 8.55 (d, J = 6.0 Hz, 1H), 8.30-8.21 (m, 2H), 7.67 (t, J = 7.7 Hz, 1H), 7.37-7.30 (m, 5H), 7.02 (d, J = 8.2 Hz, 2H), 6.74 (d, J = 8.2 Hz, 2H), 5.58 (d, J = 8.8 Hz, 1H), 5.05 (ABq, J = 3.3 Hz, 2H), 4.81-4.73 (m, 1H), 3.76-2.75 (m, 10H), 1.46 (s, 9H).

[N,O-Bis-Cbz-L-tyrosyl]-Boc-piperazine (27)

¹H NMR (CDCl₃): δ 7.48-7.28 (m, 10 H), 7.19 (d, J = 8.2 Hz, 2H), 7.19 (d, J = 8.2 Hz, 2H), 5.68 (d, J = 8.5 Hz, 1H), 5.25 (s, 2H), 5.08 (ABq, J = 12.36 Hz, 2H), 4.94-3.78 (m, 1H), 3.60-2.68 (m, 10H), 1.44 (s, 9H).

[N-Cbz-O-Benzoyl-L-tyrosyl]-Boc-piperazine (28)

¹H NMR (CDCl₃): δ 8.18 (d, J = 7.1 Hz, 2H), 7.68-7.42 (m, 3H), 7.25 (d, J = 8.2 Hz, 2H), 7.13 (d, J = 8.2 Hz, 2H), 5.78 (d, J = 8.2 Hz, 1H), 5.10 (ABq, J = 12.4 Hz, 2H), 4.88-4.80 (m, 1H), 3.62-2.80 (m, 10H), 1.45 (s, 9H).

[N-Cbz-O-Ethoxycarbonyl-L-tyrosyl]-Boc-piperazine (29)

¹H NMR (CDCl₃): δ 7.34 (s, 5H), 7.19 (d, J = 8.5 Hz, 2H), 7.09 (d, J = 8.2 Hz, 2H), 5.79 (d, J = 8.5 Hz, 1H), 5.09 (ABq, J = 12.3 Hz, 2H), 4.94-4.74 (m, 1H), 4.29 (q, J = 7.1 Hz, 2H), 3.58-2.70 (m, 10H), 1.44 (s, 9H), 1.38 (t, J = 7.1 Hz, 3H).

[N-Cbz-O-Acetyl-L-tyrosyl]-Boc-piperazine (30)

¹H NMR (CDCl₃): δ 7.34 (s, 5H), 7.19 (d, J = 8.2 Hz, 2H), 6.99 (d, J = 8.2 Hz, 2H), 5.70 (d, J = 8.5 Hz, 1H), 5.09 (ABq, J = 12.3 Hz, 2H), 4.88-4.84 (m, 1H), 3.51-2.76 (m, 10H), 2.27 (s, 3H), 1.44 (s, 9H).

[N-Cbz-O-Propionyl-L-tyrosyl]-Boc-piperazine (31)

¹H NMR (CDCl₃): δ 7.34 (s, 5H), 7.18 (d, J = 8.2 Hz, 2H), 6.99 (d, J = 8.2 Hz, 2H), 5.71 (d, J = 8.2 Hz, 1H), 5.09 (ABq, J = 12.6 Hz, 2H), 4.88-4.84 (m, 1H), 3.48-2.78 (m, 10H), 2.57 (q, J = 7.4 Hz, 2H), 1.44 (s, 9H), 1.24 (t, J = 7.4 Hz, 3H).

General Procedure for the Synthesis of 32–40, 42, 43

A solution of 21 (0.03 g, 0.057 mmol), Et₃N (0.017 mL, 0.124 mmol) and DMAP (0.007 g, 0.055 mmol) in CH₂Cl₂ (1 mL) was added to a solution of the respective R₁Cl (0.124 mmol) in anhydrous CH₂Cl₂ (1 mL) at 0° C. The mixture was stirred for 30 min to achieve complete reaction. The solvent was removed and the crude product was purified by preparative TLC using 5% MeOH in CHCl₃ to provide compounds 32–40, 42, 43.

[N-Cbz-O-Benzenesulfonyl-L-tyrosyl]toluenesulfonylpiperazine (32)

¹H NMR (CDCl₃): δ 7.90-7.76 (m, 2H), 7.76-7.64 (m, 1H), 7.64-7.48 (m, 4H), 7.42-7.28 (m, 7H), 7.02 (d, J = 8.5 Hz, 2H), 6.81 (d, J = 8.5 Hz. 2H), 5.57 (d, J = 8.5 Hz, 1H), 5.03 (s, 2H), 4.80-4.64 (m, 1H), 3.90-3.68 (m, 1H), 3.50-2.52 (m, 9H), 2.45 (s, 3H).

[N-Cbz-O-Benzenesulfonyl-L-tyrosyl]methanesulfonylpiperazine (33)

¹H NMR (CDCl₃): δ 7.85 (d, J = 7.4 Hz, 2H), 7.69 (t, J = 7.4 Hz, 1H), 7.62-7.52 (m, 2H), 7.40-7.28 (m, 5H), 7.14 (d, J = 8.3 Hz, 2H), 6.95 (d, J = 8.5 Hz, 2H), 5.61 (d, J = 8.8 Hz, 1H), 5.08 (t, J = 12.9 Hz, 2H), 4.88-4.72 (m, 1H), 3.70-2.80 (m, 9H), 2.73 (s, 3H), 2.60-2.44 (m, 1H).

[N-Cbz-O-Benzenesulfonyl-L-tyrosyl]benzenesulfonylpiperazine (34)

¹H NMR (CDCl₃): δ 7.83 (d, J = 7.9 Hz, 2H), 7.76-7.50 (m, 8H), 7.40 (m, 5H), 7.01 (d, J = 8.2 Hz, 2H), 6.79 (d, J = 8.2 Hz, 2H), 5.03 (t, J = 12.6 Hz, 2H), 4.78-4.62 (m, 1H), 3.84-3.64 (m, 1H), 3.44-2.45 (m, 9H).

[N-Cbz-O-benzenesulfonyl-L-tyrosyl]-(4-methoxybenzenesulfonyl)piperazine (35)

¹H NMR (CDCl₃): δ 7.90-7.80 (m, 2H), 7.74-7.52 (m, 5H), 7.38-7.28 (m, 5H), 7.03 (d, J = 9.1 Hz, 4H), 6.82 (d, J =8.5 Hz, 2H), 5.58 (d, J = 8.5 Hz, 1H), 5.03 (t, J = 12.4 Hz, 2H), 4.78-4.64 (m, 1H), 3.89 (s, 3H), 3.84-3.70 (m, 1H), 3.50-2.52 (m, 9H).

[N-Cbz-O-Benzenesulfonyl-L-tyrosyl]-(1-naphthylsulfonyl)piperazine (36)

¹H NMR (CDCl₃): δ 8.78 (d, J = 8.24 Hz, 1H), 8.32-8.16 (m, 2H), 8.06 (d, J = 8.2 Hz, 1H), 7.92 (d, J = 8.2 Hz, 2H), 7.84-7.60 (m, 6H), 7.46-7.32 (m, 5H), 7.10 (d, J = 8.2 Hz, 2H), 6.90 (d, J = 8.2 Hz, 2H), 5.64 (d, J = 8.2 Hz, 1H), 5.11 (q, J = 12.4 Hz, 2H), 4.88-4.72 (m, 1H), 3.94-2.82 (m, 10H).

[N-Cbz-O-Benzenesulfonyl-L-tyrosyl]-(2-naphthylsulfonyl)piperazine (37)

¹H NMR (CDCl₃): δ 8.39 (s, 1H), 8.18-8.0 (m, 3H), 7.91 (d, J = 8.5 Hz, 2H), 7.84-7.70 (m, 4H), 7.70-7.62 (m, 2H), 7.46-7.30 (m, 5H), 7.11 (d, J = 8.5 Hz, 2H), 6.90 (d, J = 8.5 Hz, 2H), 5.63 (d, J = 8.5 Hz, 1H), 5.08 (ABq, J = 12.4 Hz, 2H), 4.86-4.74 (m, 1H), 4.02-3.86 (m, 1H), 3.60-3.15 (m, 4H), 3.10-2.78 (m, 5H).

[N-Cbz-O-Benzenesulfonyl-L-tyrosyl]-Cbz-piperazine (38)

¹H NMR (CDCl₃): δ 7.80 (d, J = 7.69 Hz, 2H), 7.69-7.58 (m, 1H), 7.58-7.44 (m, 2H), 7.44-7.26 (s, 10H), 7.09 (d, J = 8.2 Hz, 2H), 6.89 (d, J = 8.2 Hz, 2H), 5.62 (d, J = 8.2 Hz, 1H), 5.22-5.0 (m, 4H), 4.88-4.74 (m, 1H), 3.62-2.74 (m, 10H).

[N-Cbz-O-Benzenesulfonyl-L-tyrosyl]ethoxycarbonylpiperazine (39)

¹H NMR (CDCl₃): δ 7.83 (d, J = 7.4 Hz, 2H), 7.72-7.62 (m, 1H), 7.60-7.48 (m, 2H), 7.42-7.28 (m, 5H), 7.10 (d, J 8.5 Hz, 2H), 6.89 (d, J = 8.5 Hz, 2H), 5.63 (d, J = 8.5 Hz, 1H), 5.07 (ABq, J = 12.4 Hz, 2H), 4.88-4.76 (m, 1H), 4.13 (q, J = 7.1 Hz, 2H), 3.62-2.78 (m, 10H), 1.25 (t, J = 7.1 Hz, 3H).

[N-Cbz-O-Benzenesulfonyl-L-tyrosyl]benzoylpiperazine (40)

¹H NMR (CDCl₃): δ 7.85 (d, J = 7.7 Hz, 2H), 7.72-7.50 (m, 3H), 7.46-7.28 (m, 10H), 7.12 (d, J = 8.2 Hz, 2H), 6.91 (d, J = 8.2 Hz, 2H), 5.65 (d, J = 8.8 Hz, 1H), 5.07 (ABq, J = 12.4 Hz, 2H), 4.82 (bs, 1H), 3.65-2.85 (m, 10H).

[N-Cbz-O-Benzenesulfonyl-L-tyrosyl]acetylpiperazine (42)

¹H NMR (CDCl₃): δ 7.83 (d, J = 7.7 Hz, 2H), 7.72-7.64 (m, 1H), 7.55 (t, J = 7.7 Hz, 2H), 7.34 (s, 5H), 7.13 (t, J = 8.7 Hz, 2H), 6.91 (t, J = 8.7 Hz, 2H), 5.64 (bs, 1H), 5.08 (s, 2H), 4.86-4.80 (m, 1H), 3.53-2.94 (m, 10H), 2.08 (s, 3H).

[N-Cbz-O-Benzenesulfonyl-L-tyrosyl]propionylpiperazine (43)

¹H NMR (CDCl₃): δ 7.82 (d, J = 7.1 Hz, 2H), 7.66 (t, J = 7.1 Hz, 1H), 7.54 (t, J = 7.1 Hz, 2H), 7.33 (s, 5H), 7.12 (t, J = 8.8 Hz, 2H), 6.90 (t, J = 8.8 Hz, 2H), 5.62 (bs, 1H), 5.07 (s, 2H), 4.86-4.78 (m, 1H), 3.58-2.89 (m, 10H), 2.31 (q, J = 7.4 Hz, 2H), 1.12 (t, J = 7.4 Hz, 3H).

[N-Cbz-O-Benzyl-L-tyrosyl]Boc-piperazine (44)

To a mixture of Cbz-Tyr(Bzl)OH (55a) (0.5 g, 1.23 mmol), Boc-piperazine (0.23g, 1.23 mmol), Bop-Cl (0.31 g, 1.23 mmol) in CH₂Cl₂ (10 mL) was added Et₃N (0.343 mL, 2.46 mmol) and stirred at rt for 5 h. The solvent was removed under vacuum and the crude material obtained was purified by flash chromatography using 5% MeOH in CHCl₃ to furnish 0.5 g of 44 as a solid foam. ¹H NMR (CDCl₃): δ 7.46 (m, 5H), 7.08 (d, J = 8.3 Hz, 2H), 6.87 (d, J = 8.5 Hz, 2H), 5.66 (d, J = 8.5 Hz, 1H), 5.08 (ABq, J = 12.1 Hz, 2H), 5.04 (s, 2H), 4.90-4.74 (m, 1H), 3.62-2.70 (m, 10H), 1.44 (s, 9H).

[N-Boc-O-Benzyl-L-tyrosyl]-Boc-piperazine (45)

A mixture of Boc-Tyr(Bzl)OH (55b) (0.03 g, 0.08 mmol), Boc-piperazine (0.015 g, 0.08 mmol), BOP-Cl (0.02 g, 0.08 mmol) in CH₂Cl₂ (2 mL) was treated with Et₃N (0.022 mL, 0.16 mmol) and stirred at rt for 5 h. The solvent was removed under vacuum and the crude material obtained was purified using flash chromatography to furnish 0.30 g of 45 as a solid foam. ¹H NMR (CDCl₃): δ 7.46-7.28 (m, 5H), 7.09 (d, J = 8.5 Hz, 2H), 6.88 (d, J = 8.5 Hz, 2H), 5.37 (d, J = 8.5 Hz, 1H), 5.04 (s, 2H), 4.84-4.66 (m, 1H), 3.60-2.70 (m, 10H), 1.44 (s, 9H), 1.42 (s, 9H).

[N-Boc-O-Benzenesulfonyl-L-tyrosyl]-Boc-piperazine (46)

Compound 45 (0.02 g, 0.037 mmol) was hydrogenated using Pd/C (0.005 g), H₂ at 40 psi for 6 h. The reaction mixture was filtered and concentrated to dryness. A solution of this material (0.012 g, 0.026 mmol), Et₃N (0.008 mL, 0.54 mmol) and DMAP (0.003 g, 0.027 mmol) in CH₂Cl₂ was treated with benzenesulfonyl chloride (0.007 mL, 0.054 mmol) at 0° C and stirred at rt for 30 min. The reaction mixture was concentrated and purified by preparative TLC using 5% MeOH in CHCl₃ to furnish 0.01 g of 46 as a solid foam. ¹H NMR (CDCl₃): δ 7.93 (d, J = 8.2 Hz, 2H), 7.84-7.60 (m, 3H), 7.22 (d, J = 8.2 Hz, 2H), 6.99 (d, J = 8.2 Hz, 2H), 5.44 (s, 1H), 4.94-4.80 (m, 1H), 3.68-3.24 (m, 10H), 1.54 (s, 9H), 1.51 (s, 9H).

[N-Boc-O-Benzyl-N-methyl-L-tyrosyl]-Boc-piperazine (47)

A mixture of Boc-MeTyr(Bzl)OH (55c) (0.5 g, 1.29 mmol), Boc-piperazine (0.24 g, 1.29 mmol), BOP-Cl (0.33 g, 1.29 mmol) in CH₂Cl₂ (10 mL) was treated with Et₃N (0.36 mL, 2.58 mmol) and stirred at rt for 5 h. The solvent was removed under vacuum and the crude material obtained was purified by flash chromatography using 5% MeOH in CHCl₃ to furnish 0.5 g of 47 as a solid foam. ¹H NMR (CDCl₃): δ 7.48-7.22 (m, 5H), 7.14 (d, J = 8.5 Hz, 2H), 6.87 (d, J = 8.5 Hz, 2H), 5.26-5.14 (m, 1H), 5.02 (s, 2H), 3.88-2.84 (m, 10H), 2.79 (s, 3H), 1.45 (s, 9H), 1.35 (s, 9H).

[N-Cbz-O-Benzyl-N-methyl-L-tyrosyl]-Cbz-piperazine (48)

Compound 47 (0.05 g, 0.09 mmol) was treated with 10% TFA in CH₂Cl₂ (2 mL) at rt for 5 h. The solvent was removed under vacuum and the crude material was purified by preparative TLC to furnish 0.027 g compound. This product (0.027 g, 0.076 mmol) and Et₃N (0.042 ml, 0.18 mmol) and DMAP (0.009 g, 0.073 mmol)) in CH₂Cl₂ (2 mL) was cooled to 0°C and Cbz-Cl was added (0.026 mL, 0.18 mmol) and stirred at rt for 30 min. Solvent was removed under vacuum and the crude material was purified by preparative TLC using 5% MeOH in CHCl₃ to furnish 0.025 g of 48 as a solid foam. ¹H NMR (CDCl₃): δ 7.48-7.24 (m, 15H), 7.13 (d, J = 8.5 Hz, 2H), 6.86 (d, J = 8.5 Hz, 2H), 5.32-5.19 (m, 1H), 5.12 (s, 2H), 5.05 (s, 2H), 5.02 (s, 2H), 3.76-2.98 (m, 10H), 2.92 (s, 3H).

[N-Boc-O-Benzyl-D-tyrosyl]-Boc-piperazine (49)

This compound was prepared starting from Boc-D-Tyr(Bzl)OH (Bachem Bioscience, King of Prussia, PA), by the same method as for the corresponding L-enantiomer, 45.

[N-Cbz-O-Benzyl-L-tyrosyl]-Boc-ethylenediamine (50)

To a mixture of Cbz-Tyr(Bzl)OH (55a) (0.03 g, 0.074 mmol), Boc-ethylenediamine (0.012 mL, 0.074 mmol), Bop-Cl (0.019 g, 0.074 mmol) in CH₂Cl₂ (2 mL) was added Et₃N (0.01 mL, 0.072 mmol) and stirred at rt for 5 h. The solvent was removed under vacuum and the crude material obtained was purified by flash chromatography using 5% MeOH in CHCl₃ to furnish 50 as a solid foam. ¹H NMR (CDCl₃): δ 7.46-7.28 (m, 10H), 709 (d, J = 8.2 Hz, 2H), 6.90 (d, J = 8.2 Hz, 2H), 6.23 (bs, 1H), 5.40-5.22 (m, 1H), 5.18-4.98 (m, 4H), 4.82-4.64 (m, 1H), 4.40-4.22 (m, 1H), 3.38-2.85 (m, 6H), 1.41 (s, 9H).

Pharmacological Analyses

P2X₇Receptor Channel Activation

All experiments were performed using adherent HEK293 cells stably transfected with cDNA encoding the human P2X₇ receptor. Adherent cells on 12-well polylysine-coated plates were incubated at 37°C in 1 mL physiological salt solution (125 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1.5 mM CaCl₂, 25 mM NaHEPES (pH 7.5), 10 mM D-glucose, 1 mg/mL BSA). Antagonists were added from 1,000× stock solutions dissolved in DMSO. Cells were preincubated with antagonists for 15 min prior to stimulation for 10 min with 3 mM ATP (final concentration). Reactions were terminated by rapid aspiration of the extracellular medium in each well. The adherent cells in each well were then extracted overnight with 1 ml 10% HNO₃. K⁺ content in these nitric acid extracts was assayed by atomic absorbance spectrophotometry. Duplicate or triplicate wells were run for all test conditions in each separate experiment.

Several compounds were tested in a similar manner using adherent Bac1.2F5 mouse macrophages that natively express the murine P2X₇ receptor.

RESULTS AND DISCUSSIONS

Chemical Synthesis

The analogs consisted of L-tyrosine derivatives, of the general structure R₁-Tyr(OR₂)-piperazinyl-R₃ (Tables 1, 2), in which three positions were systematically varied in structure through facile acylation reactions. Each of the three positions was optimized in sequence through parallel synthesis alternating with biological evaluation, consisting of screening at a single concentration (initially 3 µM), leading to the identification and optimization of potent P2X₇ antagonists.

TABLE 1.

Synthetic Yields and Characterizations of Tyrosyl Derivatives

Compound	Formula	% Yield	Analysis*
3	C31H29N5O6S2	82	HRMS
4	c36H37N5O8S2.2H2O	72	CHN
5	C31H29N5O6S2	82	HRMS
6	C36H37N5O8S2.2H2O	69	CHN
7	C36H37N5O8S2	60	HRMS
8	C42H42N4O8S	71	HRMS
9	C27H32N4O6S	90	HRMS
10	C30H36N4O8S.1.5H2O	84	CHN
11	C35H38N4O8S.0.5H2O	73	CHN
12	C34H36N4O7S	71	CHN
13	C28H34N4O8S2.1.9H2O	60	CHN
14	C33H36N4O8S2.1.5H2O	73	CHN
15	C34H38N4O8S2.2.1H2O	65	CHN
16	C34H38N4O9S2.2.5H2O	69	CHN
17	C37H38N4O8S2.3.5H2O	75	CHN
18	C37H38N4O8S2.1.5H2O	85	CHN
19	C26H33N3O6	63	HRMS
20	C27H35N3O8S.0.5H2O	88	CHN
21	C32H37N3O8S.0.75H2O	68	CHN
22	C33H39N3O8S.0.5H2O	66	CHN
23	C33H39N3O9S	55	HRMS
24	C36H39N3O8S.0.5H2O	63	CHN
25	C36H39N3O8S	58	CHN
26	C35H38N4O8S	62	HRMS
27	C34H39N3O8	80	CHN
28	C33H37N3O7	80	CHN
29	C29H37N3O8	68	CHN
30	C28H35N3O7	66	HRMS
31	C29H37N3O7	66	CHN
32	C34H35N3O8S2.1H2O	73	CHN
33	C28H31N3O8S2	76	HRMS
34	C33H33N3O8S2.H2O	75	CHN
35	C34H35N3O9S2.H2O	67	CHN
36	C37H35N3O8S2.1.5H2O	70	CHN
37	C37H35N3O8S2.1.8H2O	70	CHN
38	C35H35N3O8S	70	HRMS
39	C30H33N3O8S.0.5H2O	72	CHN
40	C34H33N3O7S.H2O	77	CHN
41	C37H34N4O7S	60	HRMS
42	C29H31N3O7S.H2O	68	CHN
43	C30H33N3O7S	69	CHN
44	C33H39N3O6.0.2H2O	71	CHN
45	C30H41N3O6.0.2H2O	69	CHN
46	C29H39N3O8S.1H2O	63	CHN
47	C31H43N3O6	70	CHN
48	C37H39N3O6	53	HRMS
49	C30H41N3O6.0.2H2O	70	CHN
50	C31H37N3O6	60	CHN

^*Elemental analyses of ± 0.4% were considered acceptable. Acceptable tolerance of ± 50 ppm was applied to the high-resolution mass spectral determination.

TABLE 2.

Antagonistic Effects of Tyrosine Derivatives on Function of Human P2X₇ Receptors Expressed in HEK293 Cells^a

Compound	Structure R1R2R3	% Inhibition of ATP-induced K releasea
1 (KN-62)		85 ± 9	(13)
3	IqIqH	14 ± 12	(3)
4	IqIqB	77 ± 24	(3)
5	QuQuH	5± 9	(3)
6	QuQuB	61 ± 30	(3)
7	IqQuB	37 ± 21	(5)
8	FQuB	34 ± 28	(3)
9	HQuB	1± 1	(3)
10	EQuB	5± 7	(3)
11	ZQuB	53 ± 23	(4)
12	BzQuB	13 ± 8	(3)
13	MsQuB	4± 4	(3)
14	BsQuB	8± 3	(3)
15	TsQuB	32 ± 16	(3)
16	MoQuB	26 ± 20	(4)
17	NQuB	16 ± 2	(3)
18	NbQuB	0	(1)
19	ZHB	0	(1)
20	ZMsB	0	(1)
21	ZBsB	59 ± 14	(3)
22	ZTsB	71 ± 30	(3)
23	ZMoB	62 ± 15	(3)
24	ZNB	25 ± 15	(3)
25	ZNbB	43 ± 16	(4)
26	ZIqB	43	(1)
27	ZZB	1	(1)
28	ZBzB	47 ± 21	(3)
29	ZEB	0	(1)
30	ZAB	0	(1)
31	ZPB	4	(1)
32	ZBsTs	0	(1)
33	ZBsMs	0	(1)
34	ZBsBs	0	(1)
35	ZBsMo	0	(1)
36	ZBsN	0	(1)
37	ZBsNb	0	(1)
38	ZBsZ	48 ± 29	(3)
39	ZBsE	24 ± 11	(3)
40	ZBsBz	78 ± 22	(3)
41	ZIqBz	85 ± 10	(3)
42	ZBsA	0	(1)
43	ZBsP	0	(1)
44	ZBnB	45	(1)
45	BBnBc	14 ± 18	(3)
46	BBsB	6	(1)
47	BBnBbc	0± 0	(3)
48	ZBnZbc	8± 7	(3)
49	BBnBbc	4± 3	(3)
50	ZBnBb	0	(1)

^aAll experiments were performed using adherent HEK293 cells stably transfected with cDNA encoding the human P2X₇ receptor. Adherent cells on 12-well polylysine-coated plates were incubated at 37°C in 1 ml physiological salt solution (125 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1.5 mM CaCl₂, 25 mM NaHEPES (pH 7.5), 10 mM D-glucose, 1 mg/ml BSA). Antagonists (3 µM final concentration) were added from 1,000× stock solutions dissolved in DMSO. Cells were preincubated with antagonists for 15 min prior stimulation for 10 min with 3 mM ATP (final concentration). Reactions were terminated by rapid aspiration of the extracellular medium in each well. The adherent cells in each well were then extracted overnight with 1 ml 10% HNO₃ and the K⁺ content in the extracts was assayed by atomic absorbance spectrophotometry. Duplicate or triplicate wells were run for all test conditions in each separate experiment and the measured K⁺ contents were averaged. Antagonist function was measured by the percent inhibition of the K⁺ release triggered by 3 mM ATP in paired cells in the absence of antagonist. Data points represent the mean ± SD values obtained; the number of separate experiments is indicated in parentheses. 4, MRS 2306; 6, MRS 2300; 11, MRS 2317; 21, MRS 2328; 22, MRS 2326; 23, MRS 2329; 28, MRS 2333; 38, MRS 2359; 40, MRS 2361; 41, MRS 2409.

^b47 = N^α-methyl derivative of 45. 48 = N^α-methyl derivative. 49 = D-isomer of 45. 50 = Boc-ethylene diamine (instead of Boc-piperazine) derivative of 44.

^cNo inhibition detected at 30 µM.

The choice of target compounds and the versatile synthetic routes for systematic substitution around the molecule (Figs. 2–4) were made possible through the ability to replace groups at two positions on the structure of the lead compound, 1. These two positions were the tyrosyl N^α -methyl group, which could be omitted without loss of antagonism, and the N-phenyl-piperazinyl group, which could be replaced conveniently with an N-t-butyloxycarbonyl- (Boc-) -piperazinyl group (see below, potency of 4). Key intermediates that lacked the N^α-Me group, therefore, were the Boc-piperazinyl derivatives, bearing either a N-(9-fluorenylmethyloxycarbonyl-) (Fmoc-), 52 (Fig. 2), or a N-benzyloxycarbonyl (Cbz-), 19 (Fig. 3), protecting group at the N^α-Tyr position. Thus, it was possible to use the Boc both as a group favorable for biological screening, common to many analogs, and also as a protecting group for synthetic intermediates. Amides were formed readily using BOP-Cl (bis(2-oxo-3-oxazolidinyl)phosphinic chloride) as condensing agent. Other amides and sulfonamides were prepared from the corresponding acyl chlorides or sulfonyl chlorides in the presence of DMAP (4-dimethylaminopyridine). Synthetic yields are listed in Table 1.

Fig. 2.

Synthesis of tyrosyl derivatives: introduction of quinoline- and isoquinolinesulfonyl groups and variation of the R₁ group.

Fig. 4.

Synthesis of tyrosyl derivatives: miscellaneous variations including N-methyl, D-configuration, and opening of the piperazinyl ring.

Fig. 3.

Synthesis of tyrosyl derivatives: variation of R₂ and R₃ groups.

The analogs were synthesized in sets, in which one of the “R” positions was varied and the other two were kept constant. Since the reference compound 1 was an isoquinoline derivative, initially a set of quinoline- and isoquinolinesulfonyl derivatives (3–7) was prepared and tested (Fig. 2). An extension of this set (8–18), in which R₁ was further varied, contained quinolinesulfonyl at R₂, Boc at R₃, and various acyl groups at R₁. A second set (19–31), in which R₂ was varied, contained Cbz at R₁ and Boc at R₃ (Fig. 3). A third set (32–40, 42, 43), in which R₃ was varied, contained Cbz at R₁ and benzenesulfonyl at R₂. Finally, related derivatives containing O-benzene-sulfonyl tyrosine (44–50) were included (Fig. 4). These derivatives were designed to test the effects of D-Tyr, reintroduction of the N^α -methyl group, and opening the piperazinyl ring, seven analogs containing O-benzyl tyrosine and combinations of Boc and Cbz at R₁ and R₃. A D-tyrosyl derivative, 49, was prepared by the same method as for the corresponding L-enantiomer, 45. Chemical yields and elemental analyses of the analogs synthesized are shown in Table 1

Biological Activity

The effects of substitution at .R₁, R₂, and R₃ on inhibition of P2X₇ receptor-mediated ion flux (Table 2) were compared. Experiments were performed using adherent HEK293 cells stably transfected with cDNA encoding the human P2X₇ receptor. Cells were preincubated with antagonists (3 µM) prior to stimulation for 10 min with 3 mM ATP. The percent inhibition of the K⁺ release was the parameter used to indicate antagonist function.

At the R₁ position there was a preference for large hydrophobic groups, linked to the α -amino position through carbamate, amide, or sulfonamide groups. Within the group of 6–18, containing quinolinesulfonyl at R₂ and Boc at R₃, the derivatives containing quinolinesulfonyl, 6, and Cbz, 11, at the N^α -position were preferred over all other acyl groups and sulfonamides examined and inhibited by >50%. A free NH at the N^α-position, in 9, abolished activity. There appeared to be a sensitivity of the percent inhibition to the precise structure at the R₁ position. For example, a toluenesulfonamide, 15, produced greater percent inhibition than the corresponding benzensulfonamide.

At the R₂ position, both arylsulfonyl and benzoyl groups led to antagonism, while in the unsubstituted case a sulfonyl group was preferred over an acyl group (cf. 21 and 28). A benzyl ether, 44, having the same substituents at R₁ and R₃ inhibited to a comparable degree. A free hydroxyl, 19, and a methanesulfonate, 20, were inactive, while a variety of substitution of aryl sulfonates (21–26), including bicyclics, were generally tolerated for antagonism. The approximate rank order of percent inhibition for aryl sulfonates was p-tolyl, 22; p-methoxyphenyl, 23; phenyl, 21 > α -naphthyl, 24; β -naphthyl, 25. A benzoyl ester at the R₂ position, 28, inhibited to an intermediate degree. Acyl substitutions at the R₂ position that resulted in inactivity included a small carbonate, 29, and small alkyl ester groups, 30 and 31.

A free NH at the R₃ position, 3 and 5, resulted in inactivity, thus mainly acylated species were evaluated. At the R₃ position, a Boc group, present in many of the derivatives, including 6–31, was well tolerated at the receptor site. Subsequently, for the compounds in which the R₃ position was systematically varied, the order of potency was: benzoyl, 40 = Boc, 21, Cbz, 38 > ethyloxycarbonyl, 39. Other analogs in which R₁ was Cbz and R₂ was benzenesulfonyl were inactive, i.e., R₃ consisted of alkyl and aryl sulfonamides, 32–37, and small alkyl amides, 42 and 43. Thus, the structural requirements for the R₃ position in order to produce potent antagonists were relatively narrowly defined.

Compounds 45–50 were essentially inactive as human P2X₇ receptor antagonists. Thus, the opening of the piperazinyl group to an ethylene diamine moiety, 50, greatly reduced the percent inhibition in comparison to 44. However, it was not possible to adequately evaluate the effects of inversion of configuration of Tyr or N^α -methylation, since the reference compound, 45, was inactive.

Full concentration-response curves provided a more precise means of comparison among some selected, potent compounds, including the reference compound 1 (Fig. 5A). A di-isoquinolinyl, piperazinyl-Boc derivative, 4, displayed an IC₅₀ of ~40 nM as an antagonist of P2X₇ receptor-mediated ion flux (Fig. 5B), and appeared to be more potent than the reference compound, 1. N^α-Cbz-Boc derivatives, 11 and 22, were nearly as potent as 1 as a P2X₇ receptor antagonist, with IC₅₀ values of ~200 and ~300 nM, respectively. Compound 41 was also nearly as potent as 1. The IC₅₀ values of 1 and 41 were roughly 100 and 200 nM, respectively.

Fig. 5.

Effects of tyrosyl derivatives on P2X₇ receptor-activation in hP2X₇-HEK cells. The adherent cells were preincubated with antagonists for 15 min prior to stimulation for 10 min with 3 mM ATP (final concentration). K⁺ content in these nitric acid extracts was assayed by atomic absorbance spectrophotometry. Duplicate or triplicate wells were run for all test conditions in each separate experiment. A: hP2X₇-HEK cells were preincubated with or without 3 µM 1 prior to stimulation with 3 mM ATP. Data points represent the mean (± SD) K⁺ content from nine separate experiments. B: hP2X₇-HEK cells were preincubated with or without the indicated concentrations of selected antagonists prior to stimulation with 3 mM ATP. Data points represent the mean (± SD) K⁺ contents from triplicate wells in a single experiment. The dashed horizontal lines illustrate the mean K⁺ content in control cells incubated in the absence of antagonist or ATP. The dotted horizontal lines illustrate the mean K⁺ content in ATP-stimulated cells that were not preincubated with antagonist. IC₅₀ values are rough estimates from visual inspection of the concentration-response relationships. Hill coefficients were not determined, since previous studies have shown that the lead compound (KN-62,1) represses P2X₇ receptor function via complex mechanisms that are not readily amenable to standard ligand-binding analyses.

Several compounds were tested in preliminary experiments as antagonists at the murine P2X₇ receptor, expressed natively in Bac1.2F5 mouse macrophages. The degree of inhibition by the tyrosyl derivatives examined was generally greater at murine than at human P2X₇ receptors. Percent inhibition measured at 3 µM in the mouse macrophages was: 35 (32), 34 (33), 74 (38), 80 (39), 94 (40), 27 (42), and 28 (43). Thus, four compounds that were inactive at human P2X₇ receptors (32, 33, 42, and 43) displayed antagonism at murine P2X₇ receptors.

The compounds were not measured as antagonists at other subtypes of P2 receptors. The lead compound, 1, however, is inactive at various other P2 receptors, including P2X₄ [Jacobson et al., 2000], P2Y1, and P2Y2 (G. Dubyak, unpubl.). To assess P₂ vs. P₁ receptor selectivity, binding to adenosine receptors was measured in competitive binding assays [Kim et al., 1998]. Compounds 39 and 41 at 100 µM did not displace radioligand from rat brain adenosine A1 or A2A receptors.

The ability to use Cbz and Boc both as protecting groups and as substituents favoring biological activity suggested a convenient approach to optimizing permutations of acyl groups appended at the three “R” positions. This approach may be described as “sequential parallel synthesis,” of which we have carried out one complete cycle. In subsequent studies the optimization process may be continued in the order: R₁-, R₂-, R₃-, and then back to R₁-, and so forth.

Eight novel ligands displaying high percent inhibition (>40% at 3 µM) have been identified, in order of decreasing potency: 41, 40, 4, 22, 23, 6, 21, 11. Modifications at R₁ and R₂ may not be independent in their effect on potency, since the IqIqB and QuQuB analogs, 4 and 6, were equipotent and both were more potent than the mixed analog, 7.

Since the parent compound, 1, was also reported to be an inhibitor of CaMKII [Tokumitsu et al., 1990] and at other sites [Puhl et al., 1997], activity of selected analogs at these enzymes will have to be examined to establish selectivity for the P2X₇ receptor. It will also be interesting to prepare reduced peptide analogs, similar to 2, to examine the role of this carbonyl group in antagonism of both the P2X₇ receptor and CaMKII.

Several Tyr derivatives in this study inhibited ion flux to a greater degree at murine vs. human P2X₇ receptors. This is the opposite of results reported for the reference compound 1, which was more potent at human than at mouse (BAC1.2f5) P2X₇ receptors [Humphreys et al., 1998].

In summary, the present analysis of structure-activity relationships of tyrosyl derivatives as noncompetitive antagonists of the P2X₇ receptor has demonstrated that the Tyr N^α -methyl group is unnecessary for antagonism. The three positions selected for extensive modification, i.e., R₁ (at N^α-amine), R₂ (on Tyr side chain), and R₃ (an extension of the C^α -position), were amenable to substitution with various acyl-type groups. The most potent P2X₇ receptor antagonists identified in this study contained Cbz at the R₁ position, an aryl sulfonate at the R₂ position, and various acyl groups at the R₃ position. At R₁ and R₂ groups, aryl substituents were preferred over alkyl. At R₃ the structural requirements were the most restrictive of the three positions. Carbonyl, but not sulfonyl, attachment to the piperazinyl ring was allowed and t-butyloxycarbonyl- and benzoyl groups were preferred. Further structure-activity studies are now warranted using a wider range of chemical functionality at these three positions.