Synthesis, receptor binding, and CNS pharmacological studies of new thyrotropin-releasing hormone (TRH) analogues

Abstract

As part of our search for selective and CNS-active thyrotropin-releasing hormone (TRH) analogues, we synthesized a set of 44 new analogues in which His and pGlu residues were modified or replaced. The analogues were evaluated as agonists at TRH-R1 and TRH-R2 in cells in vitro, and in vivo in mice for analeptic and anticonvulsant activities. Several analogues bound to TRH-R1 and TRH-R2 with good to moderate affinities, and are full agonists at both receptor subtypes. Specifically, analogue 21 a (R=CH3) exhibited binding affinities (Ki values) of 0.17 μM for TRH-R1 and 0.016 μM for TRH-R2; it is 10-fold less potent than TRH in binding to TRH-R1 and equipotent with TRH in binding to TRH-R2. Compound 21 a, the most selective agonist, activated TRH-R2 with a potency (EC50 value) of 0.0021 μM, but activated TRH-R1 at EC50=0.05 μM, and exhibited 24-fold selectivity for TRH-R2 over TRH-R1. The newly synthesized TRH analogues were also evaluated in vivo to assess their potencies in antagonism of barbiturate-induced sleeping time, and several analogues displayed potent analeptic activity. Specifically, analogues 21 a,b and 22 a,b decreased sleeping time by nearly 50% more than TRH. These analogues also displayed potent anticonvulsant activity and provided significant protection against PTZ-induced seizures, but failed to provide any protection in MES-induced seizures at 10 μmol kg(-1). The results of this study provide evidence that TRH analogues that show selectivity for TRH-R2 over TRH-R1 possess potent CNS activity.

Figure 1

General structures of the four series of synthesized TRH analogues.

Scheme 1

Reagents and conditions: a) 10 % DIPEA, CH₂Cl₂, RT, 5 min; b) Boc-L-Pro-OH (3), TBTU, DMF, 10 % DIPEA in CH₂Cl₂, RT, 2 h; c) 20 % TFA in CH₂Cl₂, RT, 20 min; d) Boc-L-His(1-methyl-2-alkyl)-OH (6 a–g), TBTU, DMF, 10 % DIPEA in CH₂Cl₂, RT, 2 h; e) Z-L-pGlu-OH (8), TBTU, 10 % DIPEA in CH₂Cl₂, RT, 2 h; f) TFMSA, EDT, thioanisole, TFA, 4 °C–RT, 2 h.

Scheme 2

Reagents and conditions: a) DIC, HONB, DMF, 4 °C, 36 h; b) 40 % TFA, CH₂Cl₂, 0 °C, 30 min; c) 7 N NH₃ in CH₃OH, 0 °C, 10 min; d) L-pGlu-OTcp (14) or L-pAad-OPfp (15), DMF, 4 °C, 36 h.

Scheme 3

Reagents and conditions: a) DIC, HONB, DMF, 4 °C, 36 h; b) 40 % TFA, CH₂Cl₂, 0 °C, 30 min; c) 7 N NH₃ in CH₃OH, 0 °C, 10 min; d) (1R)-3-Ocp-OH (20), DIC, HOBt, DMF, 4 °C, 36 h.

Scheme 4

Reagents and conditions: a) 40 % TFA, CH₂Cl₂, 0 °C, 30 min; b) 7 N NH₃ in CH₃OH, 0°C, 10 min; c) (1R)-3-Ocp-OH (20), DIC, HOBt, DMF, 4 °C, 36 h.