Functionalized Congeners of Tyrosine-Based P2X7 Receptor Antagonists: Probing Multiple Sites for Linking and Dimerization

Wangzhong Chen; R Gnana Ravi; Sylvia B Kertesy; George R Dubyak; Kenneth A Jacobson

doi:10.1021/bc020025i

. Author manuscript; available in PMC: 2017 Aug 31.

Published in final edited form as: Bioconjug Chem. 2002 Sep-Oct;13(5):1100–1111. doi: 10.1021/bc020025i

Functionalized Congeners of Tyrosine-Based P2X₇ Receptor Antagonists: Probing Multiple Sites for Linking and Dimerization

Wangzhong Chen ^†, R Gnana Ravi ^†, Sylvia B Kertesy ^‡, George R Dubyak ^‡, Kenneth A Jacobson ^†,^*

PMCID: PMC5577561 NIHMSID: NIHMS818595 PMID: 12236792

Abstract

Chemically funtionalized analogues of antagonists of the P2X₇ receptor, an ATP-gated cation channel, were synthesized as tools for biophysical studies of the receptor. These functionalized congeners were intended for use in chemical conjugation with retention of biological potency. The antagonists were L-tyrosine derivatives, related to [N-benzyloxycarbonyl-O-(4-arylsulfonyl)-L-tyrosyl]benzoylpiperazine (such as MRS2409, 2). The analogues were demonstrated to be antagonists in an assay of human P2X₇ receptor function, consisting of inhibition of ATP-induced K⁺ efflux in HEK293 cells expressing the recombinant receptor. The analogues were of the general structure R₁-Tyr(OR₂)-piperazinyl-R₃, in which three positions (R₁–R₃) were systematically varied in structure through introduction of chemically reactive groups. Each of the three positions was designed to incorporate a 3- or 4-nitrophenyl group. The nitro groups were reduced using NaBH₄–copper(II) acetylacetonate to amines, which were either converted to the isothiocyanate groups, as potential affinity labels for the receptor, or acylated, as models for conjugation. An alternate route to N^α-3-aminobenzyloxycarbonyl functionalization was devised. The various positions of functionalization were compared for effects on biological potency, and the R₂ and R₃ positions were found to be most amenable to derivatization with retention of high potency. Four dimeric permutations of the antagonists were synthesized by coupling each of the isothiocyanate derivatives to either the precursor amine or to other amine congeners. Only dimers linked at the R₂-position were potent antagonists. In concentration–response studies, two derivatives, a 3-nitrobenzyloxycarbonyl derivative 18 and a 4-nitrotoluenesulfonate 26b, displayed IC₅₀ values of roughly 100 nM as antagonists of P2X₇ receptor-mediated K⁺ flux.

INTRODUCTION

P2X receptors are ligand-gated cation channels activated by ATP (adenosine 5′-triphosphate) and other purine nucleotides. Seven mammalian subtypes of P2X receptors (termed P2X₁ through P2X₇) have been cloned (1, 2). Each functional ion channel consists of an oligomeric assembly, probably a trimer. Both homo- and heterooligomerization seem to occur commonly; however the P2X₇ subtype exists exclusively as a homomer, and the P2X₆ subtype always occurs as a homomer. The cationic selectivity of the P2X receptors is Na⁺, K⁺ > Ca²⁺ (3).

The P2X₇ receptor (formerly known as the P_2Z receptor) is expressed primarily in the immune/inflammatory system (4–9), i.e., in blood cells (monocytes, macrophages, and lymphocytes) and in the brain on microglial cells, and in the salivary gland (10). Its activation is associated with apoptosis in the immune system and release of inflammatory cytokines, such as IL-1β (8, 9, 11). The P2X₇ receptor is distinct from the other P2X subunits in that at high μM concentrations of agonists, it forms or activates a large pore in addition to a cation channel (12, 13). This pore increases permeability indiscriminately to molecules having MW ≤ 900, such as ethidium bromide, which is used as a marker for pore activity. The P2X₇ receptor also has a long C-terminal segment which contributes to pore formation (13). 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 (14). Affinity labeling of the P2X₇ receptor in mast cells has been carried out using [³H]-BzATP (15).

An objective of the present study was to design antagonist ligand probes based on tyrosine derivatives, to be used for biophysical characterization of the P2X₇ receptor. The isoquinoline derivative of tyrosine 1-(N,O-bis[5-isoquinolinesulfonyl]-N-methyl-L-tyrosyl)-4-phenylpiperazine (1, KN-62), is a potent noncompetitive antagonist at P2X₇ receptors in the submicromolar range (6,7,12). It is also an antagonist of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) in the micromolar range (16). We recently reported a series of potent P2X₇ receptor antagonists derived from KN-62, of the general structure R₁-Tyr(OR₂)-piperazinyl-R₃, in which three positions (R₁, R₂, and R₃) were systematically varied (17). The derivative MRS 2409 ([N-benzyloxycarbonyl-O-[4-(isoquinolinylsulfonyl)]-L-tyrosyl]benzoylpiperazine), 2, in which R₁ and R₃ differed from the structure of KN-62, was among the most potent analogues prepared (IC₅₀ of 200 nM, for release of K⁺ stimulated by 3 mM ATP in HEK293 cells expressing the human P2X₇ receptor). We now extend the SAR (structure activity relationship) analysis in this series to include more varied functionalization of congeners (18–20) of MRS 2409, directed toward probing multiple sites on the antagonist molecules for sites of chain attachment that preserve the biological properties. This functionalization is intended for the purpose of cross-linking, either with the receptor and/or with reporter moieties such as fluorescent dyes or radioisotopes. Dimerization of the antagonists through various linkage points has also been carried out in order to explore the spatial relationship between possible multiple binding sites on the large extracellular loop (13, 21) of P2X₇ receptor homomers.

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MATERIALS AND METHODS

Chemical Synthesis

Protected tyrosine compounds are from Novabiochem (San Diego, CA). Other chemicals are from Aldrich (Milwaukee, WI). ¹H NMR spectra were recorded using a Varian Gemini-300 spectrometer. High-resolution FAB (fast atom bombardment) mass spectra were taken with a JEOL SX102 spectrometer using nitrobenzoic acid as matrix. Elemental analysis was performed by Atlantic Microlab Inc. (Norcross, GA).

General Procedure for the Synthesis of 46, 51, 62

A mixture of Fmoc-Tyr-OH or Cbz-Tyr-OH (3.0 mmol), Boc-piperazine, or benzoyl-piperazine (3.0 mmol), Bis(2-oxo-3-oxoazolidinyl)phosphinic chloride (Bop-Cl) (0.77 g, 3.0 mmol), and CH₂Cl₂ (15 mL) was treated with Et₃N (0.96 mL, 6.8 mmol) and stirred at room temperature for 1 h. The solvent was removed in vacuo, and the crude product was purified with silica gel chromatography eluting with methanol-chloroform (5:95) to furnish a white solid foam (yield 75–85%).

[N-Fmoc-L-tyrosyl]benzoylpiperazine (46)

¹H NMR (CDCl₃): δ 7.77 (d, J = 7.4 Hz, 2H), 7.59 (d, J = 7.4 Hz, 2H), 7.50–7.28 (m, 5H), 7.05 (d, J = 8.0 Hz, 2H), 6.75 (d, J = 8.0 Hz, 2H), 5.70 (d, J = 8.5 Hz, 1H), 4.84(b, 1H), 4.50–4.25 (m, 2H), 4.25–4.10 (t, J = 7.0 Hz, 1H), 3.80–2.80 (m, 10H).

[N-Fmoc-L-tyrosyl]-Boc-piperazine (51)

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 = 8.4 Hz, 1H), 4.90–4.78 (m, 1H), 4.44–4.28 (m, 2H), 4.24–4.16 (t, J = 7.0 Hz, 1H), 3.60–2.80 (m, 10 H), 1.45 (s, 9H).

[N-Benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl]pip-erazine (62)

¹H NMR (CDCl₃): δ 7.30–7.40 (m, 5H), 7.02 (d, J = 8.2 Hz, 2H), 6.72 (d, J = 8.2 Hz, 2H), 5.65 (d, J = 8.5 Hz, 1H), 5.45 (br s, 1H), 5.14–5.00 (ABq, J = 12.4 Hz, 2H), 4.90–4.75 (m, 1H), 3.60–2.80 (m, 10H), 1.45 (s, 9H).

General Procedure for the Synthesis of 23a, 23b, 47, 52a, 52b, 55

N-Substituted tyrosine (46, 51, or 62; 2.0 mmol) was dissolved in 10 mL of anhydrous CH₂Cl₂ and cooled to 0 °C. A solution of benzenesulfonyl chloride or related sulfonyl chloride (4.0 mmol), Et₃N (0.3 mL, 2.2 mmol), DMAP (214 mg, 1.7 mmol), and CH₂Cl₂ (5.0 mL) was added dropwise and stirring continued for 30 min at 0 °C. TLC showed the completion of the reaction. The solvent was removed under reduced pressure. Column chromatography (silica gel, CH₃OH:CHCl₃ = 5:95) yielded a white solid foam (70–75% yield).

[N-Benzyloxycarbonyl-O-(3-nitrobenzenesulfonyl)-L-tyrosyl]-Boc-piperazine (23a)

¹H NMR (CDCl₃): δ 8.71 (t, J = 1.9 Hz, 1H), 8.52 (ddd, J = 1.1, 2.2, 8.2 Hz, 1H), 8.12 (m, 1H), 7.78 (t, J = 8.0 Hz, 1H), 7.40–7.30 (m, 5H), 7.14 (d, J = 8.5 Hz, 1H), 6.93 (m. 1H), 5.58 (d, J = 8.8 Hz, 1H), 5.14–5.00(ABq, J = 12.4 Hz, 2H), 4.88–4.77 (m, 1H), 3.60–2.84 (m, 10H), 1.44 (s, 9H). FAB–HRMS for C₃₂H₃₇N₄O₁₀S (MH⁺): calcd 669.2230, found 669.2231. Anal. (C₃₂H₃₆N₄O₁₀S·0.1H₂O).

[N-Benzyloxycarbonyl-O-(4-nitrobenzenesulfonyl)-L-tyrosyl]-Boc-piperazine (23b)

¹H NMR (CDCl₃): δ 8.39 (d, J = 9.0 Hz, 2H), 8.02 (d, J = 9.1 Hz, 2H), 7.42–7.28 (m, 5H), 7.13 (d, J = 8.5 Hz, 2H), 6.90 (d, J = 8.5 Hz, 2H), 5.60 (d, J = 8.5 Hz, 1H), 5.14–5.00 (ABq, J = 13.7 Hz, 2H), 4.90–4.77 (m, 1H), 3.60–2.78 (m, 10H), 1.45 (s, 9H). FAB-MS: m/z (relative intensity) 669.1 (M + 1, 6), 613.1 (16), 91 (100). Anal. (C₃₂H₃₆N₄O₁₀S·0.1H₂O).

[N-Fmoc-O-toluenesulfonyl-L-tyrosyl]benzoylpiperazine (47)

¹H NMR (CDCl₃): δ 7.86–7.64 (m, 4H), 7.60 (d, J = 7.7 Hz, 2H), 7.50–7.28 (m, 11H), 7.13 (d, J = 8.2 Hz, 2H), 6.84–6.88 (d, J = 8.2 Hz, 2H), 5.66 (d, J = 8.2 Hz, 1H), 4.82 (b, 1H), 4.50–4.25 (m, 2H), 4.25–4.10 (m, 1H), 3.8–2.7 (m, 10H), 2.45 (s, 3H).

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

¹H NMR (CDCl₃): δ 7.88–7.72 (m, 4H), 7.70–7.60 (m, 2H), 7.46–7.27 (m, 7H), 7.10 (d, J = 8.5 Hz, 2H), 6.90 (d, J = 8.5 Hz, 2H), 5.66 (d, J = 8.2 Hz, 1H), 4.90–4.70 (m, 1H), 4.50–4.30 (m, 2H), 4.25–4.10 (m, 1H), 3.8–2.7 (m, 10H), 1.45 (s, 9H).

[N-Fmoc-O-(4-nitrobenzenesulfonyl)-L-tyrosyl]-Boc-piperazine (52b)

¹H NMR (CDCl₃): δ 8.38 (d, J = 8.8 Hz, 2H), 8.02 (d, J = 8.8 Hz, 2H), 7.77 (d, J = 7.7 Hz, 2H), 7.56 (d, J = 7.4 Hz, 2H), 7.47–7.27 (m, 4H), 7.14 (d, J = 7.4 Hz, 2H), 6.91 (d, J = 8.5 Hz, 2H), 5.61 (d, J = 8.5 Hz, 1H), 4.90–4.70 (m, 1H), 4.50–4.28 (m, 2H), 4.26–4.10 (m, 1H), 3.58–2.70 (m. 10H), 1.45 (s, 9H).

[N-Fmoc-O-toluenesulfonyl-L-tyrosyl]-Boc-piperazine (55)

¹H NMR (CDCl₃): δ 7.76 (d, J = 7.1 Hz, 2H), 7.70 (d, J = 8.0 Hz, 2H), 7.57 (d, J = 6.9 Hz, 2H), 7.46–7.27 (m, 6H), 7.10 (d, J = 7.9 Hz, 2H), 6.90 (d, J = 7.8 Hz, 2H), 5.65 (d, J = 8.2 Hz, 1H), 4.85–4.70 (m, 1H), 4.50–4.25 (m, 2H), 4.24–4.15 (m, 1H), 3.80–2.70 (m, 10H), 2.43 (s, 3H), 1.45 (s, 9H).

General Procedure for the Synthesis of 17, 19, 20b, 25, 26a, 26b, 28, 31a, 31b, 33, 53a, 53b, 53c, 53d, 57a, 57b

A tyrosine derivative containing a free amine (20a, 24, 27, 32, 52a, 52b, 56, 58a, 58b, or 63; 3.0 mmol) was dissolved in CH₂Cl₂ (8.0 mL). DMAP (300 mg) was added, and the solution was cooled to 0 °C. A solution of a benzoyl chloride (4.2 mmol) (alternatively a benzyl chloroformate or acetyl chloride), Et₃N (0.45 mL), and CH₂Cl₂ (5.0 mL) was added dropwise and stirring continued at 0 °C for 40 min. The solvent was removed, and the residue was washed with petroleum ether three times. The resulting crude product was purified by column chromatography (silica gel, CH₃OH:CHCl₃ = 5:95) to yield a white solid foam (80–90% yield).

[N-(4-Nitrobenzyloxycarbonyl)-O-toluenesulfonyl-L-tyrosyl]benzoylpiperazine (17)

¹H NMR (CDCl₃): δ 8.22 (d, J = 8.8 Hz, 2H), 7.73 (d, J = 8.2 Hz, 2H), 7.60–7.30 (m, 9H), 7.13 (d, J = 8.2 Hz, 2H), 6.93 (d, J = 8.5 Hz, 2H), 5.70 (d, J = 8.5 Hz, 1H), 5.26–5.10 (b, 2H), 4.92–4.70 (b, 1H), 3.90–2.70 (m, 10H), 2.46 (s, 3H). FAB-MS: m/z (relative intensity) 687.2 (M + 1, 35), 391.3 (70), 149.1 (100). Anal. (C₃₅H₃₄N₄O₉S·0.4EtOAc).

[N-(3-Nitrobenzyloxycarbonyl)-O-toluenesulfonyl-L-tyrosyl]benzoylpiperazine (19)

¹H NMR (CDCl₃): δ 8.22–8.16 (m, 2H), 7.72 (d, J = 8.5 Hz, 2H), 7.68–7.62 (m, 1H), 7.54 (t, J = 8.0 Hz, 1H), 7.46–7.30 (m, 7H), 7.13 (d, J = 8.5 Hz, 2H), 6.92 (d, J = 8.5 Hz, 2H), 5.70 (d, J = 8.5 Hz, 1H), 5.22–5.12 (m, 2H), 4.92–4.70 (b, 1H), 4.00–2.70 (m, 10H), 2.43 (s, 3H). FAB–HRMS for C₃₅H₃₅N₄O₉S (MH⁺): calcd 687.2125, found 687.2136.

[N-(3-Acetylaminobenzyloxycarbonyl)-O-toluenesulfonyl-L-tyrosyl]benzoylpiperazine (20b)

¹H NMR (CDCl₃): δ 7.71 (d, J = 8.5 Hz, 2H), 7.52–7.28 (m, 10H), 7.16–6.98 (m, 3H), 6.91 (d, J = 8.5 Hz, 2H), 5.88–5.52 (b, 1H), 5.05 (br s, 2H), 4.88–4.68 (b, 1H), 3.80–2.70 (m, 10H), 2.46 (s, 3H), 2.18 (s, 3H). FAB–HRMS for C₃₇H₃₉N₄O₈S (MH⁺): calcd 699.2489, found 699.2507.

[N-Benzyloxycarbonyl-O-(4-acetylamino)benzenesulfonyl-L-tyrosyl]-Boc-piperazine (25)

¹H NMR (CDCl₃): δ 7.72 (d, J = 8.8 Hz, 2H), 7.63 (d, J = 8.6 Hz, 2H), 7.40–7.28 (m, 5H), 7.10 (d, J = 8.2 Hz, 2H), 6.94 (d, J = 8.6 Hz, 2H), 5.64 (d, J = 8.5 Hz, 1H), 5.20–5.00 (ABq, J = 12.3 Hz, 2H), 4.90–4.70 (m, 1H), 4.00–2.70 (m, 10H), 2.23 (s, 3H), 1.47 (s, 9H). FAB–HRMS for C₃₄H₄₁N₄O₉S (MH⁺): calcd 681.2594, found 681.2610. Anal. (C₃₄H₄₀N₄O₉S).

[N-Benzyloxycarbonyl-O-(3-nitrobenzenesulfonyl)-L-tyrosyl]benzoylpiperazine (26a)

¹H NMR (CDCl₃): δ 8.76–8.64 (b, 1H), 8.58–8.30 (dq, J = 8.3, 1.1 Hz, 1H), 8.20–8.14 (m, 1H), 7.80 (t, J = 8.0 Hz, 1H), 7.48–7.28 (m, 10H), 7.16 (d, J = 8.5 Hz, 2H), 7.00–6.92 (m, 2H), 5.57 (d, J = 8.5 Hz, 1H), 5.14–5.02 (m, 2H), 4.92–4.70 (b, 1H), 3.80–2.70 (m, 10H). FAB–HRMS for C₃₄H₃₃N₄O₉S (MH⁺): calcd 673.1968, found 673.1981. Anal. (C₃₄H₃₂N₄O₉S).

[N-Benzyloxycarbonyl-O-(4-nitrobenzenesulfonyl)-L-tyrosyl]benzoylpiperazine (26b)

¹H NMR (CDCl₃): δ 8.40 (d, J = 8.8 Hz, 2H), 8.05 (d, J = 8.5 Hz, 2H), 7.48–7.28 (m, 10H), 7.16 (d, J = 8.2 Hz, 2H), 6.92 (d, J = 8.5 Hz, 2H), 5.76 (d, J = 8.5 Hz, 1H), 5.18–5.00 (m, 2H), 4.94–4.72 (b, 1H), 3.70–2.80 (m, 10H). Compound 26b (R_f = 0.85, CH₃OH/CHCl₃ = 1: 9) was shown to be pure by TLC.

[N-Benzyloxycarbonyl-O-(4-acetylamino)benzenesulfonyl-L-tyrosyl]benzoylpiperazine (28)

¹H NMR (CDCl₃): δ 7.72 (q, J = 9.1, 14.8 Hz, 4H), 7.50–7.30 (m, 10H), 7.10 (d, J = 7.7 Hz, 2H), 7.04–6.90 (b, 2H), 5.68–5.52 (b, 1H), 5.16–5.00 (ABq, J = 12.1, 16.2 Hz, 2H), 4.88–4.68 (b, 1H), 3.90–2.70 (m, 10H), 2.17 (s, 3H). FAB-MS: m/z (relative intensity) 685.4 (M + 1, 100), 641.4 (5), 91.1 (70).

[N-Benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl]-(3-nitrobenzoyl) piperazine (31a)

¹H NMR (CDCl₃): δ 8.34–8.22 (m, 2H), 7.80–7.70 (m, 3H), 7.68–7.36 (m, 1H), 7.42–7.30 (m, 7H), 7.16 (d, J = 8.5 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 5.58 (d, J = 8.8 Hz, 1H), 5.26–5.00 (m, 2H), 4.90–4.70 (b, 1H), 4.00–2.70 (m, 10H), 2.45 (s, 3H). FAB-MS: m/z (relative intensity) 687.2 (M + 1, 40), 643.3 (10), 91.0 (90). FAB–HRMS (NOBA/CsI) for C₃₅H₃₄N₄O₉SCs [M + Cs]⁺: calcd 819.1101, found 819.1096.

[N-Benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl]-(4-nitrobenzoyl)piperazine (31b)

¹H NMR (CDCl₃): δ 8.36–8.18 (m, 3H), 7.76 (d, J = 8.2 Hz, 2H), 7.56 (d, J = 8.5 Hz, 2H), 7.42–7.28 (m, 6H), 7.16 (d, J = 8.2 Hz, 2H), 6.96 (d, J = 8.0 Hz, 2H), 5.70–5.56 (m, 1H), 5.18–5.00 (m, 2H), 4.92–4.70 (b, 1H), 4.00–2.70 (m, 10H), 2.48 (s, 3H). FAB-MS: m/z (relative intensity) 687.2 (M + 1, 20), 643.3 (2), 91.0 (50). FAB–HRMS for C₃₅H₃₅N₄O₉S [MH]⁺: calcd 687.2125, found 687.2139. Anal. (C₃₅H₃₄N₄O₉S·0.2EtOAc).

[N-Benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl]-(4-acetylaminobenzoyl) piperazine (33)

¹H NMR (CDCl₃): δ 7.72 (d, J = 8.2 Hz, 2H), 7.53 (d, J = 8.5 Hz, 2H), 7.45 (s, 1H), 7.40–7.28 (m, 9H), 7.13 (d, J = 8.2 Hz, 2H), 6.91 (d, J = 7.7 Hz, 2H), 5.80–5.56 (b, 1H), 5.20–5.00 (m, 2H), 4.90–4.72 (b, 1H), 3.90–2.70 (m, 10H), 2.45 (s, 3H), 2.19 (s, 3H). FAB-HRMS for C₃₇H₃₉N₄O₈S [MH]⁺: calcd 699.2489, found 699.2505. Compound 33 (R_f = 0.35, CH₃OH/CHCl₃ = 5: 95) was shown to be pure by TLC. Anal. (C₃₇H₃₈N₄O₈S·0.5EtOAc).

[N-Fmoc-O-benzenesulfonyl)-L-tyrosyl]benzoylpiperazine (53a)

¹H NMR (CDCl₃). δ 7.80–7.28 (m, 18H), 7.16 (d, J = 8.2 Hz, 2H), 6.93 (d, J = 8.2 Hz, 2H), 5.64 (d, J = 8.5 Hz, 1H), 4.83 (br s, 1H), 4.50–4.28 (m, 2H), 4.20 (m, 1H), 3.85–2.70 (m, 10H).

[N-Fmoc-O-(4-nitrobenzene)sulfonyl-L-tyrosyl]-(4-nitrobenzoyl)piperazine (53b)

¹H NMR (CDCl₃): δ 8.42 (d, J = 8.3 Hz, 2H), 8.27 (d, J = 7.7 Hz, 2H), 8.10 (d, J = 8.8 Hz, 2H), 7.78 (d, J = 7.7 Hz, 2H), 7.56 (m, 4H), 7.41 (t, J = 7.4 Hz, 2H), 7.36–7.27(m, 2H), 7.19 (d, J = 8.2 Hz, 2H), 6.98 (d, J = 7.4 Hz, 2H), 5.58 (d, J = 8.8 Hz, 1H), 4.83 (b, 1H), 4.50–4.26 (m, 2H), 4.19(m, 1H), 3.80–2.55 (m, 10H).

[N-Fmoc-O-benzenesulfonyl-L-tyrosyl]-(4-nitrobenzoyl)-piperazine (53c)

¹H NMR (CDCl₃): δ 8.40–8.16 (b, 2H), 7.90 (d, J = 7.7 Hz, 2H), 7.78 (d, J = 7.4 Hz, 2H), 7.70 (d, J = 7.1 Hz, 1H), 7.64–7.48 (m, 6H), 7.46–7.28 (m, 4H), 7.17 (d, J = 8.2 Hz, 2H), 7.06–6.90 (m, 2H), 5.75–5.50 (b, 1H), 4.90–4.70 (b, 1H), 4.50–4.30 (m, 2H), 4.25–4.10 (m, 1H), 4.00–2.70 (m, 10H).

[N-Fmoc-O-(4-nitrobenzene)sulfonyl-L-tyrosyl]benzoylpiperazine (53d)

¹H NMR (CDCl₃): δ 8.38 (d, J = 8.5 Hz, 2H), 8.05 (d, J = 8.2 Hz, 2H), 7.77 (d, J = 7.4 Hz, 2H), 7.56 (d, J = 7.4 Hz, 2H), 7.50–7.28 (m, 9H), 7.16 (d, J = 8.2 Hz, 2H), 6.94 (d, J = 8.2 Hz, 2H), 5.63 (d, J = 8.5 Hz, 1H), 4.90–4.47 (b, 1H), 4.50–4.25 (m, 2H), 4.25–4.10 (m, 1H), 3.80–2.70 (m, 10H).

[N-(4-Nitrobenzyloxycarbonyl)-O-toluenesulfonyl-L-tyrosyl]Boc-piperazine (57a)

¹H NMR (CDCl₃): δ 8.21 (d, J = 8.5 Hz, 2H), 7.76 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 8.5 Hz, 2H), 7.36 (d, J = 8.2 Hz, 2H), 7.18 (d, J = 8.4 Hz, 2H), 7.02 (d, J = 8.5 Hz, 2H), 5.62(d, J = 8.5 Hz, 1H), 5.20 (ABq, J = 12.3, 15.6 Hz, 2H), 4.60–4.40 (m, 1H), 4.00–2.80 (m, 10H), 2.46 (s, 3H), 1.45 (s, 9H).

[N-Benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl]Bocpiperazine (57b)

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, 15.4 Hz, 2H), 4.90–4.76 (m, 1H), 3.60–2.70 (m, 10H), 2.45 (s, 3H), 1.44 (s, 9H).

General Procedure for the Synthesis of 3–16

The appropriate tyrosine derivative (48, 54a, 54b, 54c, or 54d; 1.0 mmol), Bop-Cl (1.0 mmol) and appropriate cinnamic acid derivative (1.0 mmol) were dissolved in CH₂Cl₂ (5.0 mL). The solution was treated with Et₃N (2.0 mmol) and stirred at room temperature for 5 h. The solvent was removed, and the residue was purified using silica gel chromatography eluting with methanolchloroform (5:95) to get a solid foam (40–60% yield).

[N-(trans-Cinnamoyl)-O-benzenesulfonyl-L-tyrosyl]benzoylpiperazine (3)

¹H NMR (CDCl₃): δ 7.85 (d, J = 7.7 Hz, 2H), 7.74–7.60 (m, 2H), 7.60–7.30 (m, 11H), 7.17 (d, J = 8.5 Hz, 2H), 6.94 (d, J = 8.5 Hz, 2H), 6.58–6.46 (m, 1H), 6.40 (d, J = 15.7 Hz, 1H), 5.40–5.10 (b, 1H), 3.90–2.90 (m, 10H). FAB-MS: m/z (relative intensity) 624.3 (M + 1, 40), 434.2 (20), 191.2 (100), 131.1 (100), 105.0 (100). Compound 3 (R_f = 0.60, CH₃OH/CHCl₃ = 5: 95) was shown to be pure by TLC.

[N-(trans-2-Methoxycinnamoyl)-O-toluenesulfonyl-L-tyrosyl]benzoylpiperazine (4)

¹H NMR (CDCl₃): δ 7.72 (d, J = 8.1 Hz, 2H), 7.52–7.32 (m, 10H), 7.16–6.80 (m, 5H), 6.34 (d, J = 8.4 Hz, 1H), 6.00 (d, J = 15.7 Hz, 1H), 5.20–5.00 (m, 1H), 4.00–2.70 (m, 13H), 2.45 (s, 3H).

[N-(cis-2-Methoxycinnamoyl)-O-toluenesulfonyl-L-tyrosyl]-benzoylpiperazine (5)

¹H NMR (CDCl₃): δ 7.70 (d, J = 7.7 Hz, 2H), 7.50–7.30 (m, 10H), 7.14–6.82 (m, 5H), 6.34 (d, J = 8.5 Hz, 1H), 5.96 (d, J = 12.4 Hz, 1H), 5.20–5.00 (m, 1H), 4.00–2.60 (m, 13H), 2.46 (s, 3H).

[N-(trans-3-Chlorocinnamoyl)-O-toluenesulfonyl-L-tyrosyl]benzoylpiperazine (6)

¹H NMR (CDCl₃): δ 7.72 (d, J = 8.0 Hz, 2H), 7.61–7.29 (m, 11H), 7.16 (d, J = 8.5 Hz, 2H), 6.94 (d, J = 8.2 Hz, 2H), 6.57 (d, J = 8.3 Hz, 1H), 6.45–6.37 (d, J = 15.7 Hz, 1H), 5.37–5.07 (b, 1H), 4.00–2.70 (m, 10H), 2.46 (s, 3H).

[N-(trans-2,4,5-Trimethoxycinnamoyl)-O-toluenesulfonyl-L-tyrosyl]benzoylpiperazine (7)

¹H NMR (CDCl₃): δ 7.88 (s, 1H), 7.82 (s, 1H), 7.71 (d, J = 7.7 Hz, 2H), 7.56–7.30 (m, 5H), 7.17 (d, J = 8.2 Hz, 2H), 7.02–6.86 (m, 4H), 6.56 (d, J = 15.9 Hz, 1H), 6.43 (d, J = 8.3 Hz, 1H), 5.40–5.05 (b, 1H), 4.00–2.70 (m, 19H), 2.45 (s, 3H).

[N-(trans-4-Nitrocinnamoyl)-O-benzenesulfonyl-L-tyrosyl]-benzoylpiperazine (8)

¹H NMR (CDCl₃): δ 8.24 (d, J = 8.5 Hz, 2H), 7.86 (d, J = 8.0 Hz, 2H), 7.76–7.50 (m, 6H), 7.48–7.30 (m, 4H), 7.17 (d, J = 8.2 Hz, 2H), 6.96 (d, J = 8.5 Hz, 2H), 6.64 (d, J = 8.2 Hz, 1H), 6.52 (d, J = 15.9 Hz, 1H), 5.40–5.32 (b, 1H), 3.90–2.60 (m, 10H). FAB-MS: m/z (relative intensity) 669.2 (M + 1, 25), 251.1 (40), 119.0 (100). Compound 8 (R_f = 0.47, CH₃OH/CHCl₃ = 5: 95) was shown to be pure by TLC.

[N-(trans-Cinnamoyl)-O-(4-nitrobenzenesulfonyl)-L-tyrosyl]-benzoylpiperazine (9)

¹H NMR (CDCl₃): δ 8.40 (d, J = 8.5 Hz, 2H), 8.06 (d, J = 8.3 Hz, 2H), 7.70–7.30 (m, 10H), 7.20 (d, J = 8.2 Hz, 2H), 6.95 (d, J = 8.5 Hz, 2H), 6.52 (d, J = 8.2 Hz, 1H), 6.40 (d, J = 15.7 Hz, 1H), 5.40–5.34 (b, 1H), 3.90–2.80 (m, 10H). FAB-MS: m/z (relative intensity) 669.2 (M + 1, 20), 191.2 (85), 131.1 (100), 119.0 (75). Compound 9 (R_f = 0.59, CH₃OH/CHCl₃ = 5: 95) was shown to be pure by TLC.

[N-(trans-Cinnamoyl)-O-benzenesulfonyl-L-tyrosyl]-(4-nitrobenzoyl)piperazine (10)

¹H NMR (CDCl₃): δ 8.26 (d, J = 7.7 Hz, 2H), 7.89 (d, J = 7.7 Hz, 2H), 7.78–7.30 (m, 10H), 7.21 (d, J = 8.5 Hz, 2H), 7.00 (d, J = 7.4 Hz, 2H), 6.60–6.46 (m, 1H), 6.44–6.34 d, J = 15.7 Hz, 1H), 5.40–5.06 (b, 1H), 4.00–2.80 (m, 10H). FAB-MS: m/z (relative intensity) 669.2 (M + 1, 20), 434.2 (35), 131.1 (100). Compound 10 (R_f = 0.56, CH₃OH/CHCl₃ = 5: 95) was shown to be pure by TLC.

[N-(trans-4-Nitrocinnamoyl)-O-(4-nitrobenzenesulfonyl)-L-tyrosyl]-benzoylpiperazine (11)

¹H NMR (CDCl₃): δ 8.41 (d, J = 8.5 Hz, 2H), 8.24 (d, J = 8.5 Hz, 2H), 8.07 (d, J = 8.5 Hz, 2H), 7.74–7.58 (m, 3H), 7.50–7.34 (m, 4H), 7.20 (d, J = 8.5 Hz, 2H), 6.97 (d, J = 8.5 Hz, 2H), 6.64 (d, J = 8.0 Hz, 1H), 6.52 (d, J = 15.7 Hz, 1H), 5.40–5.16 (b, 1H), 3.84–2.80 (m, 10H). FAB-MS: m/z (relative intensity) 714.2 (M + 1, 2), 119.0 (100), 91.0 (50). Compound 11 (R_f = 0.50, CH₃OH/CHCl₃ = 5: 95) was shown to be pure by TLC.

[N-(trans-4-Nitrocinnamoyl)-O-benzenesulfonyl-L-tyrosyl]-(4-nitrobenzoyl)piperazine (12)

¹H NMR (CDCl₃): δ 8.36–8.14 (m, 4H), 7.90 (d, J = 7.4 Hz, 2H), 7.80–7.50 (m, 7H), 7.22 (d, J = 8.5 Hz, 2H), 7.10–6.92 (m, 2H), 6.72–6.40 (m, 2H), 5.40–5.08 (b, 1H), 4.00–2.80 (m, 10H). FAB-MS: m/z (relative intensity) 714.2 (M + 1, 10), 119.0 (100). Compound 12 (R_f = 0.47, CH₃OH/CHCl₃ = 5: 95) was shown to be pure by TLC.

[N-(trans-Cinnamoyl)-O-(4-nitrobenzenesulfonyl)-L-tyrosyl]-(4-nitrobenzoyl)piperazine (13)

¹H NMR (CDCl₃): δ 8.43 (d, J = 8.8 Hz, 2H), 8.28 (d, J = 8.2 Hz, 2H), 8.10 (d, J = 8.8 Hz, 2H), 7.70–7.46 (m, 4H), 7.44–7.34 (m, 3H), 7.23 (d, J = 8.5 Hz, 2H), 7.00 (d, J = 8.0 Hz, 2H), 6.49 (d, J = 8.2 Hz, 1H), 6.40 (d, J = 15.7 Hz, 1H), 5.40–5.16 (b, 1H), 3.90–2.80 (m, 10H). FAB-MS: m/z (relative intensity) 714.2 (M + 1, 5.5), 119.0 (100). Compound 13 (R_f = 0.56, CH₃OH/CHCl₃ = 5: 95) was shown to be pure by TLC.

[N-(trans-4-Nitrocinnamoyl)-O-(4-nitrobenzenesulfonyl)-L-tyrosyl]-(4-nitrobenzoyl)piperazine (14)

¹H NMR (CDCl₃): δ 8.43 (d, J = 8.8 Hz, 2H), 8.34–8.20(m, 4H), 8.10 (d, J = 8.8 Hz, 2H), 7.78–7.50 (m, 4H), 7.24 (d, J = 8.5 Hz, 2H), 7.01 (d, J = 7.7 Hz, 2H), 6.64 (d, J = 8.2 Hz, 1H), 6.54 (d, J = 15.6 Hz, 1H), 5.40–5.10 (b, 1H), 3.90–2.90 (m, 10H). FAB-MS: m/z (relative intensity) 759.2 (M + 1, 1.5), 119.0 (100). Compound 14 (R_f = 0.45, CH₃OH/CHCl₃ = 5: 95) was shown to be pure by TLC.

[N-Benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl]-benzoylpiperazine (15)

¹H NMR (CDCl₃): δ 7.72 (d, J = 8.2 Hz, 2H), 7.48–7.28 (m, 12H), 7.12 (d, J = 8.2 Hz, 2H), 6.90 (d, J = 8.2 Hz, 2H), 5.61 (d, J = 8.5 Hz, 1H), 5.16–4.96 (m, 2H), 4.92–4.74 (m, 1H), 3.80–2.60 (m, 10H), 2.45 (s, 3H). Anal. (C₃₅H₃₅N₃O₇S·0.1 H₂O).

[N-(2-Chlorobenzyloxycarbonyl)-O-toluenesulfonyl-L-tyrosyl]-benzoylpiperazine (16)

¹H NMR (CDCl₃): δ 7.72 (d, J = 7.7 Hz, 2H), 7.48–7.30 (m, 11H), 7.13 (d, J = 8.2 Hz, 2H), 6.92 (d, J = 8.2 Hz, 2H), 5.56 (d, J = 8.2 Hz, 1H), 5.28–5.10 (ABq, J = 13.1 Hz, 2H), 4.94–4.74 (m, 1H), 3.90–2.70 (m, 10H), 2.46 (s, 3H). Compound 16 (R_f = 0.64, CH₃OH/CHCl₃ = 5: 95) was shown to be pure by TLC.

General Procedure for the Synthesis of 20a, 24, 27, 32

To a solution of an aromatic nitro compound (19, 23b, 26b, or 31b; 2.0 mmol) in methanol (10 mL), sodium borohydride (10.0 mmol) was added and the solution stirred at room temperature for 5 min. Then copper(II) acetylacetonate was added and stirring continued for 1 h. TLC showed the completion of the reaction. The solvent was removed, and the residue was washed with petroleum ether two times. Then, the crude product was purified using silica gel chromatography eluting with methanol–chloroform (5:95) to provide a white solid foam (80–85% yield).

[N-(3-Aminobenzyloxycarbonyl)-O-toluenesulfonyl-L-tyrosyl]benzoylpiperazine (20a)

¹H NMR (CDCl₃): δ 7.72 (d, J = 8.5 Hz, 2H), 7.46–7.28 (m, 7H), 7.18–7.06 (m, 3H), 6.92 (d, J = 8.8 Hz, 2H), 6.74–6.58 (m, 3H), 5.57 (d, J = 9.3 Hz, 1H), 5.04–4.92 (m, 2H), 4.88–4.74 (b, 1H), 3.95–2.70 (m, 10H), 2.45 (s, 3H). FAB–MS: m/z (relative intensity) 657.3 (M + 1, 45), 658.3 (M + 2, 15), 85 (100). FAB–HRMS for C₃₅H₃₇N₄O₇S [M + H]⁺: calcd 657.2383, found 657.2385.

[N-Benzyloxycarbonyl-O-(4-aminobenzenesulfonyl)-L-tyrosyl]Boc-piperazine (24)

¹H NMR (CDCl₃): δ 7.53 (d, J = 8.5 Hz, 2H), 7.40–7.28 (m, 5H), 7.08 (d, J = 8.5 Hz, 2H), 6.92 (d, J = 8.4 Hz, 2H), 6.61 (d, J = 8.8 Hz, 2H), 5.61 (d, J = 8.5 Hz, 1H), 5.02–4.96 (ABq, J = 12.3, 16.0 Hz, 2H), 4.81 (b, 1H), 4.38 (br s, 2H), 3.80–2.60 (m, 10H), 1.45 (s, 9H). FAB-MS (NOBA): m/z (relative intensity) 639.3 (M + 1, 1), 661.3 (M + Na, 7), 583.2 (8), 539.3 (10). 91.0 (100). FAB-MS (NOBA/LiCl): m/z (relative intensity) 645.3 (M + Li, 15), 687.3 (M + Li⁺₂Cl, 5).

[N-Benzyloxycarbonyl-O-(4-aminobenzenesulfonyl)-L-tyrosyl]benzoylpiperazine (27)

¹H NMR (CDCl₃): δ 7.55 (d, J = 8.5 Hz, 2H), 7.48–7.28 (m, 10H), 7.10 (d, J = 8.0 Hz, 2H), 6.94 (d, J = 8.0 Hz, 2H), 6.64 (d, J = 8.5 Hz, 2H), 5.63 (d, J = 8.8 Hz, 1H), 5.16–5.00 (ABq, J = 12.4, 15.9 Hz, 2H), 4.92–4.70 (b, 1H), 4.50–4.20 (b, 2H), 4.00–2.80 (m, 10H). FAB-MS: m/z (relative intensity) 643.4 (M + 1, 24), 391.4 (20), 251.2 (60), 91.1 (100). FAB–HRMS for C₃₄H₃₅N₄O₇S [M + H]⁺: calcd 643.2226, found 643.2244.

[N-Benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl]-(4-aminobenzoyl)piperazine (32)

¹H NMR (CDCl₃): δ 7.70 (d, J = 8.2 Hz, 2H), 7.40–7.30 (m, 7H), 7.23 (d, J = 8.2 Hz, 2H), 7.12 (d, J = 8.5 Hz, 2H), 6.90 (d, J = 8.5 Hz, 2H), 6.70 (d, J = 8.4 Hz, 2H), 5.61 (d, J = 8.5 Hz, 1H), 5.12–5.02 (ABq, J = 12.3, 15.1 Hz, 2H), 4.88–4.76 (m, 1H), 3.89 (s, 2H), 3.90–2.80 (m, 10H), 2.45 (s, 3H). FAB-MS: m/z (relative intensity) 657.2 (M + 1, 95), 679.2 (M+ Na, 25), 120.1 (100), 91.0 (85). FAB–HRMS for C₃₅H₃₇N₄O₇S [M + H]⁺: calcd 657.2383, found 657.2396. Anal. (C₃₅H₃₆N₄O₉S·0.1H₂O).

General Procedure for the Synthesis of 21, 29, 34

An amine compound (20a, 27, or 32; 0.08 mmol) was mixed with chloroform (2.0 mL), water (1.0 mL), and sodium bicarbonate (20 mg. 0.24 mmol) and stirred vigorously for 10 min. Thiophosgene (2.0 mmol) was added and stirring continued for 1 h. The solvent was removed, and the residue was washed two times with petroleum ether. The crude product was purified with a preparative TLC plate to provide a white solid foam (70–85% yield).

[N-(3-Isothiocyanato)benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl]benzoylpiperazine (21)

¹H NMR (CDCl₃): δ 7.72 (d, J = 8.8 Hz, 2H), 7.46–7.30 (m, 8H), 7.24–7.08 (m, 5H), 6.92 (d, J = 8.8 Hz, 2H), 5.64 (d, J = 8.5 Hz, 1H), 5.05 (s, 2H), 4.90–4.72 (b, 1H), 4.00–2.70 (m, 10H), 2.45 (s, 3H). FAB–HRMS for C₃₆H₃₅N₄O₇S₂ [M + H]⁺: calcd 699.1947, found 699.1948. Anal. (C₃₆H₃₄N₄O₇S₂).

[N-Benzyloxycarbonyl-O-(4-isothiocyanato)benzenesulfonyl-L-tyrosyl]benzoylpiperazine (29)

¹H NMR (CDCl₃): δ 7.82 (d, J = 8.5 Hz, 2H), 7.48–7.28 (m, 12H), 7.14 (d, J = 8.5 Hz, 2H), 6.92 (d, J = 8.5 Hz, 2H), 5.59 (d, J = 8.5 Hz, 1H), 5.16–5.00 (ABq, J = 12.1, 14.8 Hz, 2H), 4.92–4.72 (b, 1H), 3.80–2.70 (m, 10H). FAB-HRMS for C₃₅H₃₃N₄O₇S₂ [M + H]⁺: calcd 685.1791, found 685.1810.

[N-Benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl]-(4-isothiocyanatobenzoyl)piperazine (34)

¹H NMR (CDCl₃): δ 7.42 (d, J = 8.5 Hz, 2H), 7.40–7.30 (m, 9H), 7.24 (d, J = 8.2 Hz, 2H), 7.14 (d, J = 8.5 Hz, 2H), 6.93 (d, J = 8.5 Hz, 2H), 5.60 (d, J = 8.5 Hz, 1H), 5.16–5.00 (ABq, J = 12.4, 15.4 Hz, 2H), 4.90–4.72 (b, 1H), 3.80–2.60 (m, 10H), 2.47 (s, 3H). FAB-MS (NOBA): m/z (relative intensity) 699.2 (M + 1, 22), 721.2 (M+ Na, 14), 91.1 (100). FAB–HRMS (CsI) for C₃₆H₃₄N₄O₇S₂Cs [M + Cs]⁺: calcd 831.0923, found 831.0895. Anal. (C₃₆H₃₄N₄O₇S₂·0.4EtOAc).

General Procedure for the Synthesis of 22, 30, 35a, 35b

1,4-Diaminobutane (100 mg, 1.1 mmol) or 1,2-diaminoethane (100 mg, 1.7 mmol) was added in the flask. Then, a solution of isothiocyanate compound (21 or 29 or 34) (0.01 mmol) and CH₂Cl₂ (0.1 mL) was added dropwise and stirring continued at room temperature for 1 h. The solvent was removed and the residue was washed two times with petroleum ether. The crude product was purified on a preparative TLC plate using methanol–chloroform (1:9) as mobile phase to afford a white solid foam (40–45% yield).

[N-3-{3-(4-Aminobutyl)thioureido}benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl]benzoylpiperazine (22a)

¹H NMR (CDCl₃): δ 7.71 (d, J = 8.2 Hz, 2H), 7.48–7.30 (m, 10H), 7.18–7.10 (m, 2H), 7.08–7.00 (m, 1H), 6.90 (d, J = 8.2 Hz, 2H), 5.40–4.60 (m, 4H), 3.80–2.80 (m, 14H), 2.45 (s, 3H), 1.30–1.20 (m,4H). FAB-HRMS for C₄₀H₄₇N₆O₇S₂ [M + H]⁺: calcd 787.2948, found 787.2957.

[N-Benzyloxycarbonyl-O-4-{3-(4-aminobutyl)thioureido} benzenesulfonyl-L-tyrosyl]benzoylpiperazine (30)

¹H NMR (CDCl₃): δ 7.80–7.56 (m, 4H), 7.54–7.30 (m, 10H), 7.20–6.90 (m, 4H), 5.64 (d, J = 8.5 Hz, 1H), 5.18–5.00 (ABq, J = 12.4, 15.0 Hz, 2H), 4.88–4.72 (b, 1H), 3.80–2.25 (m, 14H), 1.30–1.20 (m, 4H). FAB-HRMS (CsI) for C₃₉H₄₄N₆O₇S₂Cs [M + Cs]⁺: calcd 905.1767, found 905.1771.

[N-Benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl](4-{3-[(2-aminoethyl)thioureido})benzoylpiperazine (35a)

¹H NMR (CDCl₃): δ 7.71 (d, J = 8.0 Hz, 2H), 7.52–7.28 (m, 11H), 7.13 (d, J = 8.0 Hz, 2H), 6.91 (d, J = 8.0 Hz, 2H), 5.70 (d, J = 7.7 Hz, 1H), 5.16–4.86 (ABq, J = 12.4, 17.0 Hz, 2H), 4.90–4.72 (b, 1H), 3.84–2.56 (m, 14H), 2.45 (s, 3H). FAB-HRMS (CsI) for C₃₈H₄₂N₆O₇S₂Cs [M + Cs]⁺: calcd 891.1611, found 891.1641.

[N-Benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl](4-{3-[(4-aminobutyl)thioureido})benzoylpiperazine (35b)

¹H NMR (CDCl₃): δ 7.73 (d, J = 8.5 Hz, 2H), 7.48–7.28 (m, 11H), 7.15 (d, J = 8.2 Hz, 2H), 6.93 (d, J = 8.5 Hz, 2H), 5.61 (d, J = 9.1 Hz, 1H), 5.14–5.02 (ABq, J = 12.4, 15.9 Hz, 2H), 4.92–4.74 (b, 1H), 3.80–2.60 (m, 14H), 2.43 (s, 3H), 1.40–1.20 (m, 4H). FAB–HRMS (CsI) for C₄₀H₄₇N₆O₇S₂ [M + H]⁺: calcd 787.2948, found 787.2919.

General Procedure for the Synthesis of 64

A solution of isothiocyanate compound 21 (20 mg, 0.029 mmol) in dry DMF (0.5 mL) was stirred under nitrogen for 4 days at 80 °C. The solvent was removed with nitrogen stream, and the residue was purified with preparative TLC (methanol: chloroform = 3:97) to afford a white solid (4.5 mg, 10% yield).

1,3-Bis{[3-N-benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl]benzoylpiperazinethiourea (64)

¹H NMR (CDCl₃): δ 8.02 (b, 2H), 7.70 (d, J = 8.0 Hz, 4H), 7.50–7.30 (m, 20H), 7.22–7.02 (m, 6H), 6.90 (d, J = 8.2 Hz, 4H), 5.88 (b, 2H), 5.18–4.94 (m, 4H), 4.81 (b, 2H), 3.80–2.80 (m, 20H), 2.45 (s, 6H).). FAB-MS: m/z (relative intensity) 1377.3 (M + Na, 15), 1355.3 (M + H, 5), 261.2 (100). FAB-HRMS (CsI) for C₇₁H₇₀N₈O₁₄S₃Cs [M + Cs]⁺: calcd 1487.3228, found 1487.3258. FAB-HRMS (CsI) for C₇₁H₆₉N₈O₁₄S₃Cs₂ {[(M − H)Cs]Cs}⁺: calcd 1619.2204, found 1619.2222.

General Procedure for the Synthesis of 65, 66, 67

Mixing equimolar amounts of isothiocyanate and amine component (Scheme 7) in dry DMF, and following the same synthetic procedure as for compound 64, the correspondence dimer was obtained in 5–10% yield.

1,3-Bis{[N-benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl]-(benzo-4-yl)piperazine}thiourea (65)

¹H NMR (CDCl₃): δ 8.38 (b, 2H), 7.72 (d, J = 8.0 Hz, 4H), 7.50–7.28 (m, 20H), 7.24–7.06 (m, 6H), 6.94 (d, J = 8.2 Hz, 4H), 5.68–5.58 (m, 2H), 5.14–5.00 (m, 4H), 4.90–4.74 (b, 2H), 3.80–2.80 (m, 20H), 2.45 (s, 6H). FAB-MS: m/z (relative intensity) 1355.41 (M + H, 7), 1356.41 (M + 2H, 5.5), 1377.38 (M + Na, 1), 1393.35 (M + K, 2.5), 119.0 (100). FAB–HRMS (CsI) for C₇₁H₇₀O₁₄N₈S₃Cs [M + Cs]⁺, calcd; 1487.3228; found, 1487.3210.

1-[(N-Benzyloxycarbonyl-O-(benzenesulfon-4-yl)-L-tyrosyl)benzoylpiperazine]-3-[(N-benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl)(benzo-4-yl)piperazine]thiourea (66)

¹H NMR (CDCl₃): δ 7.80 (b, 2H), 7.72 (d, J = 8.2 Hz, 4H), 7.50–7.28 (m, 10H), 7.22–7.28 (m, 6H), 6.93 (d, J = 8.0 Hz, 4H), 5.60 (b, 2H), 5.16–4.98 (m, 4H), 4.80 (b, 2H), 3.80–2.80 (m, 20H), 2.45 (s, 3H). FAB-MS: m/z (relative intensity) 1341.4 (M + H, 5), 119.0 (100). FAB–HRMS (CsI) for C₇₀H₆₈O₁₄N₈S₃Cs [M + Cs]⁺, calcd; 1473.3071; found, 1473.3105.

1,3-Bis{[N-benzyloxycarbonyl-O-(benzenesulfon-4-yl)-L-tyrosyl]benzoylpiperazine}thiourea (67)

¹H NMR (CDCl₃): δ 9.30 (b, 2H), 7.90–7.55 (m, 8H), 7.50–7.30 (m, 20H), 7.20–6.80 (m, 8H), 5.65 (d, J = 8.5 Hz, 2H), 5.25–4.90 (m, 4H), 4.80 (b, 2H), 3.80–2.50 (m, 20H). FAB–HRMS (CsI) for C₆₉H₆₆N₈O₁₄S₃Cs [M + Cs]⁺: calcd 1459.2915, found 1459.2919.

Pharmacological Analysis. P2X₇ Receptor 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 of 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 1000× 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, 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 wells in the absence of antagonist.

RESULTS

Chemical Synthesis

The analogues consisted of L-tyrosine derivatives, of the general structure R₁-Tyr-(OR₂)-piperazinyl-R₃ (Table 1), in which three variable regions were systematically varied in structure (compounds 3–35) through facile acylation reactions and other procedures. The lead compound MRS 2409 (17), 2, contained an isoquinolinyl sulfonate at the tyrosyl hydroxyl group. For the present study, this group was first substituted with a phenyl sulfonate ester, a structural simplification known to preserve affinity for the receptor (17). Each of the three regions (R₁–R₃) was probed systematically for possbile attachment sites, through synthesis alternating with biological evaluation, which consisted of screening at a single concentration (initially 3 μM). This process led to the identification and partial optimization of potent, functionalized P2X₇ antagonists.

Table 1.

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


compound	R₁	R₂	R₃	% inhibition	n
Monomeric
3	Ph-CH=CH (trans)	H	Ph	62 ± 4	2
4	2-OCH₃-Ph-CH=CH (trans)	4-CH₃	Ph	27 ± 2	2
5	2-OCH₃-Ph-CH=CH (cis)	4-CH₃	Ph	17 ± 1	2
6	3-Cl-Ph-CH=CH (trans)	4-CH₃	Ph	57 ± 5	2
7	2,4,5-(OCH₃)₃-Ph-CH=CH (trans)	4-CH₃	Ph	−2 ± 6	2
8	4-NO₂-Ph-CH=CH (trans)	H	Ph	43 ± 20	2
9	Ph-CHdCH (trans)	4-NO₂	Ph	72 ± 4	3
10	Ph-CHdCH (trans)	H	Ph-4-NO₂	41 ± 1	2
11	4-NO₂-Ph-CH=CH (trans)	4-NO₂	Ph	58 ± 1	2
12	4-NO₂-Ph-CH=CH (trans)	H	Ph-4-NO₂	30 ± 2	2
13	Ph-CH=CH (trans)	4-NO₂	Ph-4-NO₂	43 ± 14	3
14	4-NO₂-Ph-CH=CH (trans)	4-NO₂	Ph-4-NO₂	4 ± 9	2
15	Ph-CH₂O	4-CH₃	Ph	71 ± 21	2
16	2-Cl-Ph-CH₂O	4-CH₃	Ph	43 ± 17	3
17	4-NO₂-Ph-CH₂O	4-CH₃	Ph	69 ± 25	3
18	3-NO₂-Ph-CH₂O	4-CH₃	O-C(CH₃)₃	94 ± 28	3
19	3-NO₂-Ph-CH₂O	4-CH₃	Ph	72 ± 6	2
20a	3-NH₂-Ph-CH₂O	4-CH₃	Ph	50 ± 6	2
20b	3-CH₃CONH–Ph-CH₂O	4-CH₃	Ph	9 ± 13	2
21	3-SCN-Ph-CH₂O	4-CH₃	Ph	−4 ± 5	2
22	3-[H₂N(CH₂)₄NH-CSNH]-Ph-CH₂O	4-CH₃	Ph	17 ± 4	2
23a	Ph-CH₂O	3-NO₂	O-C(CH₃)₃	74 ± 2	2
23b	Ph-CH₂O	4-NO₂	O-C(CH₃)₃	30 ± 28	2
24	Ph-CH₂O	4-NH₂	O-C(CH₃)₃	64 ± 17	2
25	Ph-CH₂O	4-NHCOCH₃	O-C(CH₃)₃	64 ± 27	2
26a	Ph-CH₂O	3-NO₂	Ph	75 ± 10	2
26b	Ph-CH₂O	4-NO₂	Ph	78 ± 18	3
27	Ph-CH₂O	4-NH₂	Ph	56 ± 9	2
28	Ph-CH₂O	4-NHCOCH₃	Ph	31 ± 41	2
29	Ph-CH₂O	4-NCS	Ph	72 ± 1	2
30	Ph-CH₂O	4-[H₂N(CH₂)₄NH-CSNH]-	Ph	80 ± 11	3
31a	Ph-CH₂O	4-CH₃	Ph-3-NO₂	60 ± 16	2
31b	Ph-CH₂O	4-CH₃	Ph-4-NO₂	61 ± 6	2
32	Ph-CH₂O	4-CH₃	Ph-4-NH₂	93 ± 3	2
33	Ph-CH₂O	4-CH₃	Ph-4-NHCOCH₃	45 ± 21	2
34	Ph-CH₂O	4-CH₃	Ph-4-NCS	30 ± 16	2
35a	Ph-CH₂O	4-CH₃	Ph-4-NHCS-NH(CH₂)₂NH₂	14 + 19	2
35b	Ph-CH₂O	4-CH₃	Ph-4-NHCS-NH(CH₂)₄NH₂	63 ± 16	2
Dimeric
	linkage
64	R₁–R₁			−5 ± 4	2
65	R₃–R₃			26	1
66	R₂–R₃			61 ± 29	2
67	R₂–R₂			72 ± 41	2

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All experiments were performed using adherent HEK293 cells stably transfected with cDNA encoding the human P2X₇ receptor. Cells were preincubated with antagonists (3 μM final concentration) for 15 min prior to stimulation for 10 min with 3 mM ATP (final concentration). Antagonist function was measured by the percent inhibition of the K⁺ release triggered by 3 mM ATP in paired wells in the absence of antagonist. Data points represent the mean ± SD of values obtained. 15, MRS 2427; 18, MRS 2464; 26b, MRS 2447; 30, MRS 2483; 32, MRS 2452; 35b, MRS 2484; 66, MRS 2454; 67, MRS 2455.

The synthetic routes for systematic substitution at three positions on the molecule are shown in Schemes 1–5. Subsequent functional group replacement beginning with nitro groups is shown in Scheme 6. Synthetic yields are listed in Materials and Methods. General probing of the SAR at each position was carried out. Scheme 1 indicates substitutions made at the R₁ position, including both N-cinnamoyl (4–7) and N-benzyloxycarbonyl (15 and 16) derivatives. Each of the three ring-substitution positions shown in Table 1 (R₁–R₃) was designed for the initial incorporation of a nitro group (at the meta-position for R₁, meta- or para-positions for R₂ and R₃). For a series of N-cinnamoyl derivatives (Scheme 2), multiple nitro groups (11–14) were also included. At R₂ and R₃ the nitro groups were introduced as shown in Schemes 3 and 4.

Scheme 1 — Synthesis of P2X₇ Receptor Antagonists Consisting of L-Tyrosine Derivatives Differing in the Cinnamoyl or Urethane Substituent Present at the Nα Position

Scheme 5 — Synthesis of P2X₇ Receptor Antagonists Consisting of L-Tyrosine Derivatives Containing 3-Nitro Substitution at the Nα-Benzyloxycarbonyl Substituent

Scheme 6 — Functional Group Replacement in P2X₇ Receptor Antagonists

Scheme 2 — Synthesis of P2X₇ Receptor Antagonists Consisting of L-Tyrosine Derivatives Containing a Nitro Substitution at Either the Nα-Cinnamoyl Substituent, the Aryl Sulfonate Group Present on the Tyrosyl Side Chain, or the Cα-Benzoylpiperazine Substituent

Scheme 3 — Synthesis of P2X₇ Receptor Antagonists Consisting of L-Tyrosine Derivatives Containing a Nitro Substitution at the Nα-Benzyloxycarbonyl Substituent or of the Cα-Benzoylpiperazine Substituent

Scheme 4 — Synthesis of P2X₇ Receptor Antagonists Consisting of L-Tyrosine Derivatives Containing a Nitro Substitution at the Aryl Sulfonate Group Present on the Tyrosyl Side Chain

The synthesis of N-(nitrobenzyloxycarbonyl)-L-tyrosine derivatives (Scheme 5) required a modification of the original route based on simple reduction of 4-nitro groups. Since the N-(4-aminobenzyloxycarbonyl)tyrosine derivative of 17 proved to be unstable, we sought to develop an alternate synthetic method, aimed instead at the 3-nitro derivative, 18. Usually, benzyl chloroformates are synthesized through the reaction of benzyl alcohol with phosgene (22). Here, an interesting alternative for the incorporation of N-(3-nitrobenzyloxycarbonyl) into tyrosine was the transformation of the α-amine of the tyrosyl moiety into an isocyanate group first with the aid of 1,1′-carbonyldiimidazole (23) to yield 49, and subsequent addition of the alcohol to yield 18.

The nitro groups at R₁, R₂, and R₃ were directly reduced to amines (Scheme 6), which were either converted to the isothiocyanate groups, in 21, 29, and 34, as potential affinity labels for the receptor, or acylated, as models for conjugation. The isothiocyanate groups also reacted with alkyl diamines to produce the primary amine congeners 22, 30, and 35. Several methods were compared for reduction of the aromatic nitro compounds. Many reducing reagents have been used to reduce aromatic nitro compounds (24) with the most classic being zinc, tin, or iron in the presence of an acid. Other reagents include hydrazine, TiCl₄–dialkyl telluride and sulfide (25). However, most of them lack the desired chemoselectivity over the functional groups that are present in our compounds such as amide, tert-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), and benzyloxycarbonyl (Cbz). Though aromatic nitro compounds are seldom reduced by sodium borohydride in alcoholic solution, we found NaBH₄–copper(II) acetylacetonate (26) to be the most effective and a mild catalyst for the reduction of nitro groups on these tyrosine derivatives. This reaction occurred nearly quantitatively as judged using TLC.

Four dimer permutations (64–67) were created by coupling each of the isothiocyanate derivatives to either the precursor amine or to other amine congeners (Scheme 7). These coupling reactions required elevated temperature and were carried out in moderate yield.

Biological Activity

The effects of substitution at R₁, R₂, and R₃ on inhibition of P2X₇ receptor-mediated ion flux (Table 1) were compared. Experiments were performed using adherent HEK293 cells stably transfected with cDNA encoding the human P2X₇ receptor. Cells were preincubated with each antagonist at a fixed concentration (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.

The three structural regions for conjugation were compared in their effects on biological potency. Among N-cinnamoyl derivatives, more favorable potency was associated with the unsubstituted derivative 9 than with methoxy substitution. Multiple methoxy substitution, e.g., 7, eliminated the antagonistic effect at the P2X₇ receptor. The 4-nitro substitution of the cinnamoyl group also reduced potency (cf. 14 vs 13). The Cbz derivative 15 was a potent antagonist of the human P2X₇ receptor, and 4- or 3-nitro substitution preserved that property, as in 18 and 19. However, reduction and acetylation of the 3-nitro group, 20a and 20b, reduced potency. The corresponding isothiocyanate 21 and amine congener 22 were inactive or weakly potent, respectively.

Nitro subsitution of the tyrosyl side chain (R₂ substituent), at the 3- or 4-position, as in benzoylpiperazine (at R₃) derivatives 26a and 26b, respectively, maintained potency at the human P2X₇ receptor. There was no difference in biological potency between 3- and 4-nitro derivatives. The corresponding t-Boc derivatives (at the R₃ position, e.g., 23a and 23b) similarly antagonized the human P2X₇ receptor. This antagonism was preserved when the 4-nitro group was reduced and acetylated (e.g. 24 and 25; 27 and 28). A primary amine congener linked at the R₂ 4-position, 30, was a potent antagonist of the human P2X₇ receptor. The corresponding isothiocyanate 29 displayed considerable potency as a P2X₇ receptor antagonist and warranted examination as an irreversible receptor inhibitor.

For the R₃ substituent, 3- or 4-nitrobenzoyl subsitution, 31a and 31b, respectively, preserved antagonistic properties at the human P2X₇ receptor. As for the R₂ substituent, there was no difference in biological potency between 3- and 4-nitro derivatives. The P2X₇ receptor antagonism was increased when the 4-nitro group was reduced but diminished upon acetylation (e.g., 32 and 33). An isothiocyanate 34 only weakly antagonized the effects of ATP at the P2X₇ receptor. A primary amine congener containing four methylenes linked at the R₂ 4-position, 35b, was a potent antagonist of the human P2X₇ receptor, while a shorter homologue 35a (two methylenes) antagonized weakly.

Full concentration–response curves provided a more precise means of comparison among some selected, potent compounds, including the 3-nitro-Cbz derivative 18, a 4-nitrotoluenesulfonate 26b, a primary amine congener 30, and 4-aminobenzoyl derivative 32 (Figure 1). Compounds 18 and 26b, were roughly as potent as 1 as P2X₇ receptor antagonists, and appeared to have IC₅₀ values in the 0.1 μM range. The other compounds appeared to have IC₅₀ values in the 0.3 μM range (30) or > 0.3 μM (32).

Effects of tyrosyl derivatives on P2X₇ receptor-activation in hP2X₇–HEK cells. Cells were preincubated with or without the indicated concentrations of selected antagonists (**26b** MRS2447; 32 MRS2452; 18 MRS2464; and 30 MRS2483) prior to stimulation with 3 mM ATP. Data points represent the mean (± SD) K⁺ contents from triplicate wells in a single experiment. IC₅₀ values mentioned in the text 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) represses P2X₇ receptor function via complex mechanisms that are not readily amenable to standard ligand-binding analyses.

Among antagonist dimers, only the R₂–R₃- and R₂–R₂-linked dimers, 66 and 67, respectively, displayed significant antagonistic properties at the P2X₇ receptor.

DISCUSSION

The binding site for KN-62, 1, resides within the first 335 residues of the human P2X₇ receptor, likely within the large extracellular loop (13, 21). While it is not feasible to model this binding interaction, due to lack of a high-resolution template for this ion channel and uncertainty about the oligomeric nature of the channel (27), chemical probes of the receptor may be very useful in studying the structure. The stoichiometry of association of subunits in P2X receptors has been studied suggesting a trimeric organization (2, 27, 28), but little is known about the correspondence of antagonist binding sites to the overall oligomeric ion channel structure. By functionalizing derivatives (17) of the widely used P2X₇ antagonist KN-62 it may be possible to locate the binding site (in conjunction with mutagenesis studies). Mutagenesis studies of the P2X₇ receptor aimed at structural and functional elucidation have already been carried out (29, 32). Peptide mapping of receptor digests (30) using electrophoretic or mass spectroscopic methods following affinity labeling of the receptor would also be useful in this regard.

Since there may be multiple binding sites on the P2X₇ receptor homomer, it may be possible to detect the preferred orientation of covalently linked antagonist dimers that may occupy two distinct binding sites, based on divergent biological potencies for a variety of linkage combinations. Since the dimerization of the antagonists in the present study through the tyrosyl side chain, alone, provided retention of antagonist properties, it is likely that this region of the antagonist occurs in a portion of the binding site that is somewhat relaxed in its steric requirements. Whether the dimers 66 and/or 67 are able to bridge two putative binding sites on the P2X₇ homomer will be the object of further studies. The species selectivity of the new functionalized congeners also should be investigated, since differences have been noted for KN-62 (33).

Receptor ligands that bridge two subunits of a multimeric receptor may also potentially be of use for tissue selectivity, if there are differences between tissues in the organization of subunits. Such differences have been demonstrated for native P2X₇ receptors (34), which in peritoneal macrophage and bone marrow cells exist as a strongly bound multimeric complex, and in brain glia and/or astrocytes appear to form only as monomeric subunits.

In conclusion, we have systematically varied substituent groups in a series of tyrosyl analogues, resulting in analogues, among which are antagonists that are equipotent to the lead structure 1 and also contain chemical functional groups for linking purposes. Such functionalized congeners have been used effectively for the structural investigations of other purine receptors (18, 19, 31), including adenosine A₁, A_2A, and A₃ receptors. Suitable sites and chemical approaches to cross-linking these antagonists have been found on the tyrosyl side chain (R₂) and on the CR-substituent (R₃). The more versatile region for functionalization appears to be the R₂ substituent, since even dimerization through this substituent preserves the receptor binding property. We have prepared several amine functionalized congeners, that may potentially be conjugated to fluorescent probes for receptor characterization, or immobilized on a solid support for isolation of the receptor by affinity chromatography. Among P2X receptors, only the P2X₁ subtype has so far been isolated in this manner (35).

Acknowledgments

We thank Gilead Sciences (Foster City, CA) for financial support to W. Z. Chen. This work was supported by NIH grant GM36387 to G.D. We thank Mr. Noel Whittaker and Mr. Victor Livengood for determination of mass spectra.

Abbreviations

Bop-Cl: bis(2-oxo-3-oxazolidinyl)phosphinic chloride
Boc: tert-butyloxycarbonyl
BSA: bovine serum albumin
Cbz: benzyloxycarbonyl
DMAP: 4-(dimethylamino)pyridine
DMF: dimethylformamide
DMSO: dimethyl sulfoxide
EtOAc: ethyl acetate
FAB: fast atom bombardment
Fmoc: 9-fluorenylmethyloxycarbonyl
HEK: human embryonic kidney
HEPES: N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)
HRMS: high-resolution mass spectrometry
KN-62: 1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine
MRS 2409: [N-benzyloxycarbonyl-O-[4-(isoquinolinylsulfonyl)]-L-tyrosyl]benzoylpiperazine
TFA: trifluoroacetic acid
THF: tetrahydrofuran
TLC: thin-layer chromatography

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PERMALINK

Functionalized Congeners of Tyrosine-Based P2X7 Receptor Antagonists: Probing Multiple Sites for Linking and Dimerization

Wangzhong Chen

R Gnana Ravi

Sylvia B Kertesy

George R Dubyak

Kenneth A Jacobson

Abstract

INTRODUCTION

MATERIALS AND METHODS

Chemical Synthesis

General Procedure for the Synthesis of 46, 51, 62

[N-Fmoc-L-tyrosyl]benzoylpiperazine (46)

[N-Fmoc-L-tyrosyl]-Boc-piperazine (51)

[N-Benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl]pip-erazine (62)

General Procedure for the Synthesis of 23a, 23b, 47, 52a, 52b, 55

[N-Benzyloxycarbonyl-O-(3-nitrobenzenesulfonyl)-L-tyrosyl]-Boc-piperazine (23a)

[N-Benzyloxycarbonyl-O-(4-nitrobenzenesulfonyl)-L-tyrosyl]-Boc-piperazine (23b)

[N-Fmoc-O-toluenesulfonyl-L-tyrosyl]benzoylpiperazine (47)

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

[N-Fmoc-O-(4-nitrobenzenesulfonyl)-L-tyrosyl]-Boc-piperazine (52b)

[N-Fmoc-O-toluenesulfonyl-L-tyrosyl]-Boc-piperazine (55)

General Procedure for the Synthesis of 17, 19, 20b, 25, 26a, 26b, 28, 31a, 31b, 33, 53a, 53b, 53c, 53d, 57a, 57b

[N-(4-Nitrobenzyloxycarbonyl)-O-toluenesulfonyl-L-tyrosyl]benzoylpiperazine (17)

[N-(3-Nitrobenzyloxycarbonyl)-O-toluenesulfonyl-L-tyrosyl]benzoylpiperazine (19)

[N-(3-Acetylaminobenzyloxycarbonyl)-O-toluenesulfonyl-L-tyrosyl]benzoylpiperazine (20b)

[N-Benzyloxycarbonyl-O-(4-acetylamino)benzenesulfonyl-L-tyrosyl]-Boc-piperazine (25)

[N-Benzyloxycarbonyl-O-(3-nitrobenzenesulfonyl)-L-tyrosyl]benzoylpiperazine (26a)

[N-Benzyloxycarbonyl-O-(4-nitrobenzenesulfonyl)-L-tyrosyl]benzoylpiperazine (26b)

[N-Benzyloxycarbonyl-O-(4-acetylamino)benzenesulfonyl-L-tyrosyl]benzoylpiperazine (28)

[N-Benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl]-(3-nitrobenzoyl) piperazine (31a)

[N-Benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl]-(4-nitrobenzoyl)piperazine (31b)

[N-Benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl]-(4-acetylaminobenzoyl) piperazine (33)

[N-Fmoc-O-benzenesulfonyl)-L-tyrosyl]benzoylpiperazine (53a)

[N-Fmoc-O-(4-nitrobenzene)sulfonyl-L-tyrosyl]-(4-nitrobenzoyl)piperazine (53b)

[N-Fmoc-O-benzenesulfonyl-L-tyrosyl]-(4-nitrobenzoyl)-piperazine (53c)

[N-Fmoc-O-(4-nitrobenzene)sulfonyl-L-tyrosyl]benzoylpiperazine (53d)

[N-(4-Nitrobenzyloxycarbonyl)-O-toluenesulfonyl-L-tyrosyl]Boc-piperazine (57a)

[N-Benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl]Bocpiperazine (57b)

General Procedure for the Synthesis of 3–16

[N-(trans-Cinnamoyl)-O-benzenesulfonyl-L-tyrosyl]benzoylpiperazine (3)

[N-(trans-2-Methoxycinnamoyl)-O-toluenesulfonyl-L-tyrosyl]benzoylpiperazine (4)

[N-(cis-2-Methoxycinnamoyl)-O-toluenesulfonyl-L-tyrosyl]-benzoylpiperazine (5)

[N-(trans-3-Chlorocinnamoyl)-O-toluenesulfonyl-L-tyrosyl]benzoylpiperazine (6)

[N-(trans-2,4,5-Trimethoxycinnamoyl)-O-toluenesulfonyl-L-tyrosyl]benzoylpiperazine (7)

[N-(trans-4-Nitrocinnamoyl)-O-benzenesulfonyl-L-tyrosyl]-benzoylpiperazine (8)

[N-(trans-Cinnamoyl)-O-(4-nitrobenzenesulfonyl)-L-tyrosyl]-benzoylpiperazine (9)

[N-(trans-Cinnamoyl)-O-benzenesulfonyl-L-tyrosyl]-(4-nitrobenzoyl)piperazine (10)

[N-(trans-4-Nitrocinnamoyl)-O-(4-nitrobenzenesulfonyl)-L-tyrosyl]-benzoylpiperazine (11)

[N-(trans-4-Nitrocinnamoyl)-O-benzenesulfonyl-L-tyrosyl]-(4-nitrobenzoyl)piperazine (12)

[N-(trans-Cinnamoyl)-O-(4-nitrobenzenesulfonyl)-L-tyrosyl]-(4-nitrobenzoyl)piperazine (13)

[N-(trans-4-Nitrocinnamoyl)-O-(4-nitrobenzenesulfonyl)-L-tyrosyl]-(4-nitrobenzoyl)piperazine (14)

[N-Benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl]-benzoylpiperazine (15)

[N-(2-Chlorobenzyloxycarbonyl)-O-toluenesulfonyl-L-tyrosyl]-benzoylpiperazine (16)

General Procedure for the Synthesis of 20a, 24, 27, 32

[N-(3-Aminobenzyloxycarbonyl)-O-toluenesulfonyl-L-tyrosyl]benzoylpiperazine (20a)

[N-Benzyloxycarbonyl-O-(4-aminobenzenesulfonyl)-L-tyrosyl]Boc-piperazine (24)

[N-Benzyloxycarbonyl-O-(4-aminobenzenesulfonyl)-L-tyrosyl]benzoylpiperazine (27)

[N-Benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl]-(4-aminobenzoyl)piperazine (32)

General Procedure for the Synthesis of 21, 29, 34

[N-(3-Isothiocyanato)benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl]benzoylpiperazine (21)

[N-Benzyloxycarbonyl-O-(4-isothiocyanato)benzenesulfonyl-L-tyrosyl]benzoylpiperazine (29)

[N-Benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl]-(4-isothiocyanatobenzoyl)piperazine (34)

General Procedure for the Synthesis of 22, 30, 35a, 35b

[N-3-{3-(4-Aminobutyl)thioureido}benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl]benzoylpiperazine (22a)

[N-Benzyloxycarbonyl-O-4-{3-(4-aminobutyl)thioureido} benzenesulfonyl-L-tyrosyl]benzoylpiperazine (30)

[N-Benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl](4-{3-[(2-aminoethyl)thioureido})benzoylpiperazine (35a)

[N-Benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl](4-{3-[(4-aminobutyl)thioureido})benzoylpiperazine (35b)

General Procedure for the Synthesis of 64

1,3-Bis{[3-N-benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl]benzoylpiperazinethiourea (64)

General Procedure for the Synthesis of 65, 66, 67

Scheme 7.

1,3-Bis{[N-benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl]-(benzo-4-yl)piperazine}thiourea (65)

1-[(N-Benzyloxycarbonyl-O-(benzenesulfon-4-yl)-L-tyrosyl)benzoylpiperazine]-3-[(N-benzyloxycarbonyl-O-toluenesulfonyl-L-tyrosyl)(benzo-4-yl)piperazine]thiourea (66)

1,3-Bis{[N-benzyloxycarbonyl-O-(benzenesulfon-4-yl)-L-tyrosyl]benzoylpiperazine}thiourea (67)

Pharmacological Analysis. P2X7 Receptor Channel Activation

RESULTS

Chemical Synthesis

Table 1.

Scheme 1.

Functionalized Congeners of Tyrosine-Based P2X₇ Receptor Antagonists: Probing Multiple Sites for Linking and Dimerization

Pharmacological Analysis. P2X₇ Receptor Channel Activation