Bridged Piperidine Analogues of a High Affinity Naphthalene-Based P2Y14R Antagonist

Abstract

High affinity phenyl-piperidine P2Y₁₄R antagonist 1 (PPTN) was modified with piperidine bridging moieties to probe receptor affinity and hydrophobicity. Various 2-azanorbornane, nortropane, isonortropane, isoquinuclidine, and ring-opened cyclopentylamino derivatives preserved human P2Y₁₄R affinity (fluorescence binding assay), and their pharmacophoric overlay was compared. Enantiomeric 2-azabicyclo[2.2.1]hept-5-en-3-one precursors assured stereochemically unambiguous, diverse products. Pure (S,S,S) 2-azanorbornane enantiomer 15 (MRS4738) displayed higher affinity than 1 (3-fold higher affinity than enantiomer 16) and in vivo antihyperallodynic and antiasthmatic activity. Its double prodrug 143 (MRS4815) dramatically reduced lung inflammation in a mouse asthma model. Related lactams 21-24 and dicarboxylate 42 displayed intermediate affinity and enhanced aqueous solubility. Isoquinuclidine 34 (IC₅₀ 15.6 nM) and isonortropanol 30 (IC₅₀ 21.3 nM) had lower lipophilicity than 1. In general, rigidified piperidine derivatives did not lower lipophilicity dramatically, except those rings with multiple polar groups. P2Y₁₄R molecular modeling based on a P2Y₁₂R structure showed stable and persistent key interactions for compound 15.

Figure 1.

(A) Alignment of piperidine 1 (purple) and quinuclidine 5 (gray) reference compounds. (B) Pharmacophore hypothesis built on compound 5, with the blue, red and green spheres representing, respectively, positive ionic, negative ionic and hydrophobic features. The orange toroids represent two aromatic ring features defining the naphthalene scaffold. (C−H) Alignment to the pharamcophore hypothesis and docking poses at an hP2Y₁₄R model of compounds 15 (lime) and 16 (pink) (C, D), 17 (cyan) and 18 (orange) (E, F), and 32 (yellow) (G, H).

Figure 2.

Representative fluorescence binding inhibition curves (mean ± SEM, n = 3−5). (A) CHO-hP2Y₁₄R cells. (B) HEK293-mP2Y₁₄R cells.

Figure 3.

Compound 15 (10 μmol/kg, i.p.) reverses established mechano-allodynia in the CCI mouse model at the time of peak pain, i.e., 7 days post-injury. Mean ± SD is shown, n = 3, ANOVA, *P < 0.05 vs day 0 and †P < 0.05. The vehicle consisted of 5% aqueous DMSO (0.2 mL injected).

Figure 4.

Activity of P2Y₁₄R antagonists 15 and its ester prodrug 141, carbamate prodrug 142, and double prodrug 143 in a mouse model of asthma (dose: 20 μmol/kg (9.8−12.2 mg/kg); concentration: 2 μmol/mL (2 mM) in 10% DMSO, 30% PEG-400, 60% H₂O; administration: 10 μL/g of body weight). 7-Day sensitization with ovalbumin/aspergillus was followed by a single challenge (ovalbumin, aerosol) at day 14. Antagonists were given 30 min prior to challenge. Harvesting of cells and cell counting were on day 16.

Figure 5.

(A) Docking pose of compound 15 at the predicted P2Y₁₄R structure obtained though homology modeling. (B) RMSD of compound 15 heavy atoms during three replicates of 30 ns MD simulations. (C) Percentage of H-bonds between compound 15 and selected P2Y₁₄R residues during three replicates of 30 ns MD simulations. Only residues with an average number of H-bonds >5% are reported.

Chart 1.

Key Reported P2Y₁₄R Antagonists Related to Prototypical Antagonist 1^{a a}IC₅₀ values were determined using a whole-cell fluorescence binding assay with 4 as a tracer.^, Compound 3 was reported by Zhang et al.

Scheme 1.

Preparation of 4-Bromophenyl Intermediates Containing Modified Piperidine Rings for Suzuki Coupling^{a a}Reagents and conditions: (a) Br₂, AcOH, rt., 10 min, 30−62%; (b) Et₂Zn, CH₂I₂, DCM, Ar, rt., overnight, 60−62%; (c) TMSCF₃, NaI, THF, 65 °C, overnight, 26%; (d) TFA, DCM, rt., 1 h, 75%; (e) (+) Pt−Pt (−) (4.5 V, 2 F/mol), MeOH, Et₄NOTs, 95%; (f) NH₄Cl, 100 °C, 2 h, 73%; (g) 4-bromophenylmagnesium bromide (in-situ generated: dibromobenzene, magnesium, THF, sonication, rt., 2 h), THF, 0 °C, 4 h, 42−86%; (h) NaBH₄, H₂SO₄, rt., 30 min, 54%.

Scheme 2.

General Scheme for Preparation of Derivatives of the Lead P2Y₁₄R Antagonist, Piperidine 1, via Suzuki Coupling Followed by Ester Hydrolysis^{a a}Reagents and conditions: (a) Pd(PPh₃)₄, K₂CO₃, DMF, MW, 150 °C, 30 min, 37−76%; (b) for substrate 60 and 61: Pd(PPh₃)₄, aq. Na₂CO₃, DME-EtOH, 80 °C, 30 min, 25−40%; (c) MeOH, aq. KOH, overnight, 25−83%; (d) H₂, DMF, Pd/C, 150 psi, 7 h, 58−85%.

Scheme 3.

Preparation of Bridged Piperidine Analogues 15 and 17 Containing a 2-Azanorbornane Moiety, 21 and 23 Containing a Lactam, Ring-opened 38 and 40 Containing 1-Amino-3-hydroxymethylcyclopentane, and 42 and 44 Containing 1-Amino-3-carboxylcyclopentane^{a a}Reagents and conditions: (a) 1-bromo-4-iodobenzene, (PPh₃)₂PdCl₂, formic acid, TEA, THF, 65 °C overnight, quantitative; (b) TEA, DMAP, (Boc)₂O, DCM, rt., overnight. 46% 91 and 36% 92; (c) TFA, DCM, rt., 1 h, 88−95%; (d) H₃N-BH₃, B(C₆F₅)₃, BF₃Et₂O, DCE, 75 °C, 24 h, 25−52% 95/100 and 12−48% 96/101; (e) boronic acid pinacol ester 68, Na₂CO₃, Pd(PPh₃)₄, DME-H₂O (4:1), 85 °C, overnight, 56−100%; (f) LiOH, THF-MeOH-H₂O (3:1:1), rt., 3 h, 30−66%; (g) LiOH, THF-MeOH-H₂O (3:1:1), rt., 1.5 h, 40−62%; (h) LiOH, THF-MeOH-H₂O (3:1:1), rt., overnight, 73−77%. The synthesis maintained absolute stereochemistry originating from chiral bicyclic precursor 89.

Scheme 4.

Preparation of Nortropane Derivatives from Nortropanol^{a a}Reagents and conditions: (a) TFA, 90 °C, 2 h, 27%; (b) H₂, Pd/C, MeOH, 100 psi, 3 h, 92%.

Scheme 5.

Preparation of Isoquinuclidine Derivatives^{a a}Reagents and conditions: (a) K₂CO₃, PhCH₂Br, DMF, 70 °C, 4 h, 62%; (b) 4-bromophenylmagnesium bromide (in-situ generated: dibromobenzene, magnesium, THF, sonication, rt., 2 h), THF, 0 °C, 3 h, yield 46% 131 and 27% 132; (c) boronic acid pinacol ester 68, Na₂CO₃, Pd(PPh₃)₄, DME-H₂O (4:1), 85 °C, overnight, 65−67%; (d) LiOH, THF-MeOH-H₂O (3:1:1), rt., 3 h; (e) H₂, Pd/C, DMF, 5 h, 11−56% overall yield from 133/135; (f) TFA, 90 °C, 2 h, 51% overall yield from 133; (g) H₂, Pd/C, DMF, 3 h, 5.4% overall yield from 133.

Scheme 6.

Preparation of Amidomethyl Esters 141, 146, 144, and 145, of 15, 20, 22, and 23, and Reported Amidomethyl Ester of 1, i.e., 147, and the Carbamate (142) and Double Prodrug (143) Derivatives of Potent 2-Azanorbornane Analogue 15^{a a}Reagents and conditions: (a) 2-chloro-N,N-dimethylacetamide, Cs₂CO₃, DMF, 45 °C, 3 h (except 12 h at rt for compound 140), 76−98%; (b) TFA, DCM, rt., 1 h, 99%; (c) ethyl chloroformate, N,N-diisopropylethylamine, DMF, rt., overnight, 71%; (d) ethyl chloroformate, NaOH, DMF, rt., overnight, 71%.