Synthesis and Evaluation of in Vivo Anti-hypothermic Effect of the N- and C-Terminus Modified Thyrotropin-Releasing Hormone Mimetic: [(4S,5S)-(5-Methyl-2-oxooxazolidine-4-yl)carbonyl]-[3-(thiazol-4-yl)-L-alanyl]-L-prolinamide.

We explored orally effective thyrotropin-releasing hormone (TRH) mimetics, which show high central nervous system effects in structure-activity relationship studies based on in vivo antagonistic activity on reserpine-induced hypothermia (anti-hypothermic effect) in mice starting from TRH. This led us to the TRH mimetic: [(4S,5S)-(5-methyl-2-oxooxazolidine-4-yl)carbonyl]-[3-(thiazol-4-yl)-L-alanyl]-L-prolinamide 1, which shows a higher anti-hypothermic effect compared with that of TRH after oral administration. We next attempted further chemical modification of the N- and C-terminus of 1 to find more orally effective TRH mimetics. As a result, we obtained several N- and C-terminus modified TRH mimetics which showed high anti-hypothermic effects.

Synthesis of CNS active thyrotropin-releasing hormone (TRH)-like peptides: Biological evaluation and effect on cognitive impairment induced by cerebral ischemia in mice.

Thyrotropin-releasing hormone (TRH)-like peptides were synthesized by replacing critical histidine and pGlu residues in the native peptide. The peptides were evaluated in vitro for receptor binding activity assay and in the cell functional assay; the peptides exhibit selective basal signaling agonist behavior toward TRH-R2. For example, peptides 8a, 8b, 8c, 8 f, 8 h, 8 l and 12 d activated TRH-R2 with potency (EC50) of 0.53 µM, 0.048 µM, 0.05 µM, 0.006 µM, 0.31 µM, 0.034 µM and 0.004 µM, respectively. In contrast for signaling activation of TRH-R1, the same peptide required higher concentration of 19.35 µM, 3.98 µM, 2.54 µM, 0.287 µM, 11.28 µM, 0.986 µM and 0.944 µM, respectively. The results showed that peptides were 36.5, 82.9, 50.8, 47.8, 36.3, 32.6 and 235-fold selective to TRH-R2 receptor subtype. The peptides were investigated for CNS activity at 10 µmol/kg in pentobarbital-induced sleep assay study. Peptides 8c (16.5 ± 1.4 min) and 8l (16.5 ± 2.1 min) displayed excellent CNS activity. In an in vivo study, peptide 8c did not cause significant change in the rat plasma TSH levels. The peptide 8c was further investigated for neuroprotective potential, and significantly reduced infracts volume and neurological score in the focal cerebral ischemia model in mice. Peptide 8c also significantly lowered MDA levels, indicating reduction of oxidative and enhanced percentage cell survival in CA1 region, when compared to ischemic brain.

Quantification of thyrotropin-releasing hormone by liquid chromatography-electrospray mass spectrometry.

Thyrotropin-releasing hormone (TRH) is involved in a wide range of biological responses. It has a central role in the endocrine system and regulates several neurobiological activities. In the present study, a rapid, sensitive and selective liquid chromatography-mass spectrometry method for the identification and quantification of TRH has been developed. The methodology takes advantage of the specificity of the selected-ion monitoring acquisition mode with a limit of detection of 1 fmol. Furthermore, the MS/MS fragmentation pattern of TRH has been investigated to develop a selected reaction monitoring (SRM) method that allows the detection of a specific b2 product ion at m/z 249.1, corresponding to the N-terminus dipeptide pyroglutamic acid-histidine. The method has been tested on rat hypothalami to evaluate its suitability for the detection within very complex biological samples.

Thyrotropin secretion in rats after hypothalamic electrical stimulation or injection of synthetic TSH-releasing factor.

Plasma thyroid-stimulating hormone (TSH) levels, as measured by radioimmunoassay, begin to rise within 5 minutes after initiation of electrical stimulation of the medial-basal hypothalamus, become significantly elevated at 10 minutes, and reach a peak at 10 to 25 minutes. Intravenous administration of synthetic thyrotropin-releasing factor induces a marked rise in plasma thyroidstimulating hormone which is maximal within 5 minutes after administration. These data are interpreted to indicate that there are neuronal structures within the medial-basal hypothalamus which release preformed thyrotropin-releasing factor. The claim (based on bioassay data) that pyroglutamyl-histidyl-proline amide is a potent thyrotropin-releasing factor has also been confirmed by using a highly specific immunoassay system.

A novel TRH analog, Glp-Asn-Pro-D-Tyr-D-TrpNH2, binds to [3H][3-Me-His2]TRH-labelled sites in rat hippocampus and cortex but not pituitary or heterologous cells expressing TRHR1 or TRHR2.

Glp-Asn-Pro-D-Tyr-D-TrpNH(2) is a novel synthetic peptide that mimics and amplifies central actions of thyrotropin-releasing hormone (TRH) in rat without releasing TSH. The aim of this study was to compare the binding properties of this pentapeptide and its all-L counterpart (Glp-Asn-Pro-Tyr-TrpNH(2)) to TRH receptors in native rat brain tissue and cells expressing the two TRH receptor subtypes identified in rat to date, namely TRHR1 and TRHR2. Radioligand binding studies were carried out using [(3)H][3-Me-His(2)]TRH to label receptors in hippocampal, cortical and pituitary tissue, GH4 pituitary cells, as well as CHO cells expressing TRHR1 and/or TRHR2. In situ hybridization studies suggest that cortex expresses primarily TRHR2 mRNA, hippocampus primarily TRHR1 mRNA and pituitary exclusively TRHR1 mRNA. Competition experiments showed [3-Me-His(2)]TRH potently displaced [(3)H][3-Me-His(2)]TRH binding from all tissues/cells investigated. Glp-Asn-Pro-D-Tyr-D-TrpNH(2) in concentrations up to 10(-5)M did not displace [(3)H][3-Me-His(2)]TRH binding to membranes derived from GH4 cells or CHO-TRHR1 cells, consistent with its lack of binding to pituitary membranes and TSH-releasing activity. Similar results were obtained for the corresponding all-L peptide. In contrast, both pentapeptides displaced binding from rat hippocampal membranes (pIC(50) Glp-Asn-Pro-D-Tyr-D-TrpNH(2): 7.7+/-0.2; pIC(50) Glp-Asn-Pro-Tyr-TrpNH(2): 6.6+/-0.2), analogous to cortical membranes (pIC(50) Glp-Asn-Pro-D-Tyr-D-TrpNH(2): 7.8+/-0.2; pIC(50) Glp-Asn-Pro-Tyr-TrpNH(2): 6.6+/-0.2). Neither peptide, however, displaced [(3)H][3-Me-His(2)]TRH binding to CHO-TRHR2. Thus, this study reveals for the first time significant differences in the binding properties of native and heterologously expressed TRH receptors. Also, the results raise the possibility that Glp-Asn-Pro-D-Tyr-D-TrpNH(2) is not displacing [(3)H][3-Me-His(2)]TRH from a known TRH receptor in rat cortex, but rather a hitherto unidentified TRH receptor.

Synthesis of the phosphinic analogue of thyrotropin releasing hormone.

The synthesis of the phosphinic analogue of thyrotropin releasing hormone (TRH) GlpPsi[P(O)(OH)]HisProNH2, where the scissile peptide bond of TRH has been replaced by the hydrolytically stable phosphinic bond, has been achieved by a multistep synthetic strategy, providing thus one of the most potent synthetic inhibitors of pyroglutamyl peptidase II (PPII) reported to date (170 nM). The key synthetic step, an Ugi-type condensation reaction, produced directly the suitably protected for solid-phase peptide synthesis pseudodipeptidic block FmocGlu(OMe)Psi[P(O)(OH)]His(Tr)OH. Formation of the pyroglutamic ring was performed on solid phase, providing thus a general method for synthesizing pyroglutamyl phosphinic peptides on solid phase. Using this strategy, the phosphinic analogue of TRH has been synthesized for the first time.

Thyrotropin-releasing hormone (TRH) analogues that exhibit selectivity to TRH receptor subtype 2.

Thyrotropin-releasing hormone (TRH) analogues in which the C-2 position of the imidazole ring of the centrally placed histidine residue is substituted with various alkyl groups were synthesized and studied as agonists for TRH receptor subtype 1 (TRH-R1) and subtype 2 (TRH-R2). Several analogues were found to be selective agonists for TRH-R2 exhibiting no activation of TRH-R1. For example, analogue 4 (R= c-C3H5) was found to activate TRH-R2 with a potency (EC50) of 0.41 microM but did not activate TRH-R1 (potency > 100 microM). This study describes the first discovery of TRH-R2-specific agonists and provides impetus to design predominately CNS-effective TRH peptides.

Synthesis of thyrotropin-releasing hormone analogues with selective central nervous system effects.

Thyrotropin-releasing hormone (TRH) analogues which show relative selectivity for action in the central nervous system have been recognized. Practical syntheses for three of these TRH analogues which show the greatest selectivity, less than Aad-His-Tzl-NH2 (5), less than Glu-His-Pip-OMe (2), and less than Aad-His-Pro-NH2 (6), are described. The first two were prepared by solution methods of peptide synthesis. Compound 6 was prepared by the solid-phase method. Problems of histidine racemization, facile diketopiperazine formation, and instability of acylated thiazolidine carboxylic acid derivatives under acidic conditions have been minimized in order to attain optimal yields. Physical properties such as pK, NMR shifts, and circular dichroism have been examined as they might relate to biological activity and peptide conformation.

Synthesis of thyrotropin-releasing hormone analogues. 2. Tripeptides structurally greatly differing from TRH with high central nervous system activity.

A new series of thyrotropin-releasing hormone (TRH) analogues, obtained by further modifications of our most potent central nervous system (CNS) stimulating neutral tripeptides at both termini, were synthesized by the pentafluorophenyl ester method and tested for CNS and thyrotropin (TSH) releasing activity. Replacement of pyroglutamic acid by pyro-2-aminoadipic acid, 2-oxoimidazolidine-4-carboxylic acid or gamma-butyrolactone-gamma-carboxylic acid and that of proline by pipecolic acid, thiazolidine-4-carboxylic acid, or homoproline in [Leu2]- and [Nva2]TRH led to tripeptides structurally widely different from TRH. In spite of this fact, 7 of the 17 analogues (1, 2, 8-10, 16, and 17) have stronger anticataleptic effect than TRH, with negligible or no hormonal potency. The highest CNS activity was achieved when pyroglutamic acid was replaced by pyro-2-aminoadipic acid at the N-terminus [pAad-Leu-Pro-NH2, 1 (RGH 2202), and pAad-Nva-Pro-NH2,2]. A novel synthesis of L-2-aminoadipic acid suitable for large-scale preparation is also described.

Synthesis and biological properties of the 2-L-beta-(pyrazolyl-1)alanine analogs of luteinizing hormone-releasing hormone and thyrotropin-releasing hormone.

The luteinizing hormone-releasing hormone (LH-RH) analog, less thanGlu-Pyr(1)Ala-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, and the thyrotropin-releasing hormone (TRH) analog, less thanGlu-Pyr(1)Ala-Pro-NH2, were synthesized by azide couplings of the dipeptide hydrazide, less thanGlu-Pyr(1)Ala-NHNH2, to the C-terminal octapeptide of LH-RH and to proline amide, respectively. In an ovariectomized, steroid-blocked rat assay, the LH-RH analog was found to have only 1% of the LH-releasing activity of the natural hormone. The TRH analog was 1.5 times more effective than TRH itself in releasing TSH in vivo from the anterior pituitary of mice. This peptide is one of two synthetic peptides so far discovered which are more potent than TRH.

Synthesis and central nervous system actions of thyrotropin-releasing hormone analogues containing a dihydroorotic acid moiety.

A series of thyrotropin-releasing hormone (TRH) analogues in which the pyroglutamic acid residue was replaced by (S)-4,5-dihydroorotic acid (Dio-OH) and the related derivatives were prepared. Their central nervous system actions based on spontaneous locomotor activity, antagonistic effect on reserpine-induced hypothermia, and antagonistic effect on pentobarbital anesthesia were evaluated and the structure-activity relationships are discussed. Of these, (1-methyl-(S)-4,5-dihydroorotyl)-L-histidyl-L-prolinamide (14b) showed the most potent activities, which were 30-90 times greater than those of TRH. Moreover, the thyrotropin-releasing activity of 14b was about 50 times weaker than that of TRH, and compound 14b (TA-0910) was selected as a potent candidate.

Centrally acting and metabolically stable thyrotropin-releasing hormone analogues by replacement of histidine with substituted pyridinium.

Metabolically stable and centrally acting thyrotropin-releasing hormone (TRH) analogues were designed by replacing the central histidine with substituted pyridinium moieties. Their analeptic and acetylcholine-releasing actions were evaluated to assess their potency as central nervous system (CNS) agents. A strong experimental connection between these two CNS-mediated actions of the TRH analogues was obtained in subject animals. The analogue 3-(aminocarbonyl)-1-(3-[2-(aminocarbonyl)pyrrolidin-1-yl]-3-oxo-2-[[(5-oxopyrrolidin-2-yl)carbonyl]amino]propyl)pyridinium (1a) showed the highest (TRH-equivalent) potency and longest, dose-dependent duration of action from a series of homologous compounds in antagonizing pentobarbital-induced narcosis when administered intravenously in its CNS-permeable prodrug form (2a) obtained via reduction of the pyridinium moiety to the nonionic dihydropyridine. The maximum change in hippocampal acetylcholine concentration upon perfusion of the pyridinium-containing tripeptides into the hippocampus of rats was also achieved with 1a. No binding to the endocrine TRH receptor was measured for the TRH analogues reported here; therefore, our design afforded a novel lead for centrally acting TRH analogues. We have also demonstrated the benefits of the prodrug approach on the pharmacokinetics and brain uptake/retention of pyridinium-containing TRH analogues (measured by in vivo microdialysis sampling) upon systemic administration.

Metabolism-based brain-targeting system for a thyrotropin-releasing hormone analogue.

Gln-Leu-Pro-Gly, a progenitor sequence for the thyrotropin-releasing hormone (TRH) analogue [Leu(2)]TRH (pGlu-Leu-Pro-NH(2)), was covalently and bioreversibly modified on its N- and C-termini (by a 1,4-dihydrotrigonellyl and a cholesteryl group, respectively) to create lipoidal brain-targeting systems for the TRH analogue. The mechanism of targeting and the recovery of the parent peptide at the target site involve several enzymatic steps, including the oxidation of the 1,4-dihydropyridine moiety. Due to the lipid insolublity of the peptide pyridinium conjugate obtained after this reaction, one of the rudimentary steps of brain targeting (i.e., trapping in the central nervous system) can be accomplished. Our design also included spacer amino acid(s) inserted between the N-terminal residue of the progenitor sequence and the dihydrotrigonellyl group to facilitate the posttargeting removal of the attached modification. The release of the TRH analogue in the brain is orchestrated by a sequential metabolism utilizing esterase/lipase, peptidyl glycine alpha-amidating monooxygenase (PAM), peptidase cleavage, and glutaminyl cyclase. In addition to in vitro experiments to prove the designed mechanism of action, the efficacy of brain targeting for [Leu(2)]TRH administered in the form of chemical-targeting systems containing the embedded progenitor sequence was monitored by the antagonistic effect of the peptide on the barbiturate-induced anesthesia (measure of the activational effect on cholinergic neurons) in mice, and considerable improvement was achieved over the efficacy of the parent peptide upon using this paradigm.

Analogs of thyrotropin-releasing hormone using an aminoglycine-based template.

Novel thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2) analogs, made by solid phase, were derived from the general scaffold pGlu-(D/L)Agl(X)-Pro-NH2 where Agl = aminoglycine. Analogs ranged from X being a proton to an acylating agent derived from substituted (aromatic heterocyclic rings) formic or acetic acids or an aminotriazolyl moiety (3'-amino-1H-1',2',4'-triazolyl) built on N(alpha) of aminoglycine or Nbeta of alpha,beta-diaminoproprionic acid (Dpr). X was expected to mimic the electronic and structural characteristics of the imidazole ring of histidine. Analogs were purified by HPLC, characterized by mass spectrometry and isolated as either diastereoisomeric mixtures or pure isomers. Analogs, tested for their binding affinity to mouse pituitary TRH receptors, have apparent equilibrium inhibitory constants > 1 microM.

A novel peptide synthesis using fluorous chemistry.

Three new fluorous supports for peptide synthesis, i.e., the trialkoxybenzhydryl-type (6), the Wang-type (7) and the tert-butyl-type support (8), were prepared. A bioactive peptide TRH was easily synthesized by an Fmoc strategy using the benzhydryl-type fluorous support with fluorous chemistry.

Potential thyroliberin affinity labels II: Chloroacetyl substituted phenylalanyl prolineamides.

Three analogs of thyroliberin (I) were prepared. These compounds, N-m-chloroacetylbenzoyl-phenylalanyl-prolineamide (VIa), N-p-chloroacetylbenzoyl-phenylalanyl-prolineamide (VIb) and N-chloroacetyl-alanyl-phenylalanyl-prolineamide (IX), were designed as potential I antagonist affinity labels. However, no significant antagonist activity was observed. Compounds VIa and IX were found to have weak agonist activity. Cyclo (Phe-Pro) an analog of the I metabolite, cyclo (His-Pro), was found, however, to have significant I antagonist activity, but no agonist activity.

Synthesis and biological evaluation of a novel pyroglutamyl-modified TRH analogue.

The TRH analogue 3, incorporating the (S)-isothiazolidine-1,1-dioxide-3-carboxylic acid (1) moiety in place of the native L-pyroglutamic acid (pGlu) residue, has been synthesized and fully characterized by 1H and 13C NMR. The effects of replacing pGlu with its sulphonamido counterpart on biological activity have been investigated. This peptide, which is significantly stabilized towards hydrolysis by pyroglutamyl peptidase type I (PP I, EC 3.4.19.3), has shown to maintain in vitro prolactin-releasing activity.

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

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.