Synthesis and Biology of New Thyrotropin-Releasing Hormone (TRH) Analogues y Rahul Jain, a, * Jatinder Singh, a Jeffery H. Perlman b and Marvin C. Gershengorn b a Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S. Nagar, Punjab 160 062, India b Division of Molecular Medicine, Department of Medicine, Weill Medical College of Cornell University, New York, NY 10021, USA Received 7 May 2001; accepted 18 July 2001 Abstract—We report synthesis and biological activities of several thyrotropin-releasing hormone (TRH) analogues in which the N- terminal pyroglutamic acid residue has been replaced with various carboxylic acids and the central histidine is modified with sub- stituted-imidazole derivatives. # 2001 Elsevier Science Ltd. All rights reserved. Introduction Thyrotropin-releasing hormone (TRH, l-pGlu-l-His-l- Pro-NH 2 ), a tripeptide synthesized in the hypothala- mus, 1 operates in the anterior pituitary to control levels of TSH (thyroid-stimulating hormone) and prolactin. 2 This peptide was the first hypothalamic releasing factor characterized, establishing the fundamental proof for the existence of a neuroendocrine regulation of pituitary function by hypothalamic neuronal structures. 1,3 The same peptide is found in many other tissues and appears to be involved in a wide variety of physiological activ- ities. 4 Elucidation of its mechanism of action, identifi- cation of critical features of the molecule, separation of its multiple activities through design of selective analo- gues and affinity labels, and stabilization to enzymatic degradation have been elusive goals for 30 years. Results and Discussion Isolation and cloning of the pituitary receptor, as well as replacement by point mutation of predicted key amino acids, have permitted Perlman et al. 5 to for- mulate the first model of the TRH peptide–receptor complex. Since the first residue of TRH, pyroglutamic acid, is known to be responsible for at least half of the peptide’s binding energy, its role was first investigated by Perlman et al. 5 Removal of the carbonyl group of the five-membered ring (replacement with proline) results in loss of 100,000-fold in binding affinity (based on loss of activity). An equivalent loss in affinity is observed on replacement of Tyr-106 by Phe in helix 3 of the receptor. It is possible, therefore, that a strong H-bond exists between the hydroxyl of Tyr-106 and the ring carbonyl group. Parallel manipulations show that the ring nitro- gen of pyroglutamic acid forms a critical H-bond with Asparagine-110 of helix 3. 6 However, Tashjian et al. 7 have concluded by single residue mutations at the other amino acids that the role of Tyr106 in TRH binding is indirect, and proposed a hydrogen bonding interaction of Asn-289 in the third extracellular loop (EL3) with lactam moiety of the pyroglutamyl ring of TRH. In a paper published recently, 8 we find that retention of the carbonyl, but replacement of the ring N–H group of pGlu by a methylene group, provides an analogue (RJ- 601) whose binding affinity and signal-transducing potency are approximately 100-fold less than those of TRH itself. Hence, we concluded that the H-bond interaction to Tyr-106 is considerably more important than that to Asn-110, and the loss of NH binding is partially compensated by the strong H-bonding ability of the ketonic carbonyl group. To further validate this hypothesis, and in continuation of our earlier work, this paper describes synthesis and biological activities of several TRH analogues, where pGlu has been replaced with carboxylic acids (2–5) of varying hydrogen-bond donating abilities (Fig. 1). 0968-0896/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0968-0896(01)00265-6 Bioorganic & Medicinal Chemistry 10 (2002) 189–194 *Corresponding author. Tel.: +91-172-214682; fax: +91-172-214692; e-mail: rahuljain@mailmetoday.com y NIPER communication no. 106