Synthesis and biological evaluation of gamma-aminophosphonates as potent, subtype-selective sphingosine 1-phosphate receptor agonists and antagonists

Abstract

The synthesis of N-arylamide phosphonates and related arylether and arylamine analogues provided potent, subtype-selective agonists and antagonists of the five known sphingosine 1-phosphate (S1P) receptors (S1P(1-5)). To this end, the syntheses of phosphoserine mimetics-selectively protected and optically active phosphonoserines-are described. In vitro binding assays showed that the implementation of phosphonates as phosphate mimetics provided compounds with similar receptor binding affinities as compared to their phosphate precursors. meta-substituted arylamide phosphonates were discovered to be antagonists of the S1P(1) and S1P(3) receptors. When administered to mice, an antagonist blocked the lymphopenia evoked by a S1P receptor agonist and caused capillary leakage in both lung and kidney.

Figure 1

Structures of endogenous sphingosine 1-phosphate (S1P) and the S1P receptor ligands, FTY720, VPC22173 and VPC23019.

Figure 2

The lymphopenia evoked by the S1P agonist, 12b (VPC44152) is blocked by co-administration of the S1P receptor antagonist 12d (VPC44116). Groups of 3 C57BL/6 x sv129/J mice injected with vehicle (2% hydroxypropyl β-cyclodextrin), VPC44116 (22 mg/kg) and/or VPC44152 (18 mg/kg). After 16 hours, blood was drawn from the orbital sinuses and lymphocytes were measure with a Hemavet blood analyzer. Data are presented as mean ± S.E.

Figure 3

Effect of VPC44116 on vascular permeability. C57BL/6 mice were treated with vehicle (2% hydroxypropyl β-cyclodextrin) or VPC44116 (25 mg/kg) two hours prior to injection 2% Evans Blue dye (EBD) (20 mg/kg) into the jugular vein 30 min. before harvesting tissues. EBD was extracted into formamide, measured in a spectrometer and the amount of extravasated EBD in tissues was calculated from a standard curve. Values are means ± SE; n = 4 for each group. **P < 0.05, **P < 0.01 compared with vehicle treatment.

Scheme 1

Synthesis of (3R)-4a,b – Method A: a.) i. SOCl₂, MeOH, rt, 16h ii. Boc₂O, Et₃N, CH₂Cl₂, rt, 12h iii. 2,2-dimethoxypropane, p-TsOH, CH₂Cl₂, 0 °C to rt, 2h 62% (3 steps) iv. NaBH₄, LiCl, 3:2 EtOH/THF, 0 °C to rt, 4h 89% v. DMSO, (COCl)₂, CH₂Cl₂, −78 °C, then Et₃N −78 °C to rt, 2–4 h, 97% b.) tetraethyl methylenebisphosphonate, n-BuLi, THF, −78 °C, rt, overnight, 75% (2a) or tetraethyl 2-fluoromethynebisphosphonate, n-BuLi, THF, -78 °C, rt, overnight, 31% (2b) c.) H₂, Pd/C, rt., 12h, EtOH, 99% (3a) and 88% (3b) d.) Jones reagent, acetone, 0 °C to rt., 12h then, isopropyl alcohol, celite, rt, 15min., 59% (4a) and 48% (4b).

Scheme 2

Synthesis of (3R)-[or (3S)-]4 – Method B: a.) BnBr, DMF, 60% NaH, 0 °C to rt, 3h, 78% b.) CH₃PO₃Et₂, n-BuLi, BF₃·OEt₂, THF, −78 °C to rt, 3h then, NH₄Cl, 1h, 96% c.) DPPA, DIAD, 3%-polymer-bound PhPPh₂, CH₂Cl₂, 0 °C to rt, 20h, 96%, d.) Boc₂O, H₂ (balloon), 20%^w/w Lindlar’s catalyst, MeOH, rt, 24h 77% e.) H₂ (balloon), Pd/C, EtOH, rt, 24h, 94% f.) TEMPO, bis(acetoxy)iodosobenzene, NaHCO₃, 1:1 CH₃CN/H₂O, rt, 3h., 38% or RuCl₃·hydrate, NaIO₄, 3:2:2 H₂O/CH₃CN/CCl₄, rt, 3h, 76%.

Scheme 3

Synthesis of arylamide phosphonates 12 a–f & 13: a) PyBOP, 10 a–d, di-iso- propyl ethylamine (DIEA), CH₂Cl₂, 24–70% b.) TMSBr, CH₂Cl₂, rt, 4-6h then, 95:5 MeOH/H₂O, rt, 1–4h, 45-100%.

Scheme 4

Efficient synthesis of 4a from 2a: a.) H₂, 10% Pd/C (20 ^w/_w% followed by 10 ^w/_w%), EtOH, rt, 3d, >95% b.) RuCl₃·H₂O_x, NaIO₄, 2:2:3 CCl₄/CH₃CN/H₂O, rt, 1–3h, 79%.

Scheme 5

Synthesis of arylether phosphonate analogues 18 a–b and 19: a.) DIAD, PPh₃, THF, 0 °C to rt, overnight, 80–84% b.) CH₃PO₃Et₂, n-BuLi, BF₃·OEt₂, THF, −78 °C to rt, 3h then, NH₄Cl, 1h, 72–87% c.) DPPA, DIAD, PPh₃, CH₂Cl₂, 0 °C to rt, 20h, 67–98% d.) H₂ (balloon), Pd/C, EtOH, formic acid cat., rt, 3.5h, quantitative e.) TMSBr, CH₂Cl₂, rt, 4–6h then, 95:5 MeOH/H₂O, rt, 2–4h, 79–100%.

Scheme 6

Synthesis of arylamine phosphonate analogue 26: a.) DIAD, PPh₃, THF, 0 °C to rt, overnight, 69% b.) CH₃PO₃Et₂, n-BuLi, BF₃·OEt₂, THF, –78 °C to rt, 2h then, NH₄Cl, 2h, 97% c.) DPPA, DIAD, PPh₃, CH₂Cl₂, 0 °C to rt, 20h, 85% (containing 5% OPPh₃) d.) H₂ (balloon), 20%^w/^w Pd(OH)₂, 20:1 MeOH/conc. HCl, 1h, 100% e.)Na_(s), NH_3(l), –78 °C, 5 min.then EtOH, 25% (recovered 28% starting material) f.) TMSBr, CH₂Cl₂, rt, 4–6h then, 95:5 MeOH/H₂O, 4h, rt, 95%.