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The incorporation of Arg residues into position 6 of gonadotropin releasing hormone antagonists had resulted in compounds with increased in vivo potency but also made these analogues potent mast cell degranulators. We have focused on the substitution of position 8 by hArg(R)2 (NG,NG'-dialkylhomoarginine) substitutions, based on the hypotheses that the Arg-Pro sequence is of major importance for this side effect and that shielding of the charge may be an effective way to block degranulation. Analogues in four series were evaluated: (A) [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Pal-(3)3,6,Arg5,hArg(R)2(8),D-+ ++Ala10]GnRH, (B) [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Pal(3)3,6,hArg(R)2(5,8),D-Ala10 ]-GnRH, (C) [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Pal(3)3,6,hArg(R)2(8),D-Ala10]G nRH, (D) [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Pal(3)3,D-hArg(R)2(6),hArg(R)2( 8),D-Ala10]GnRH. Although substitution by hArg(Et)2, hArg(Bu), hArg(CH2)3, and hArg(CH2CF3)2 was tested, in each series the hArg(Et)2 residue was superior. Two compounds were considered for clinical evaluation: [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Pal(3)3,6,hArg(Et)2(8),D-Ala10] GnRH and [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Pal(3)3,D-hArg(Et)2(6),hArg(Et) 2(8),D- Ala10]GnRH (ganirelix acetate). These compounds had high potency for ovulation suppression and low histamine-releasing potency in vitro (ED50 = 0.6, 0.29 microgram/rat and EC50 = 196, 13 micrograms/mL, respectively). Ganirelix is currently in Phase II clinical trials and appears to be the most potent GnRH antagonist tested in humans (based upon ED50 for 24-h suppression of testosterone levels).

In order to improve the biological potency of cyclic gonadotropin releasing hormone (GnRH) antagonists, we have synthesized analogues, the conformations of which were restrained through internal side chain/side chain amide bridges linking aspartic acid or glutamic acid and L-2,3-diaminopropionic acid or L-ornithine. A disulfide bridge linking L-cysteine residues was also introduced. Residues belonging to the bridge spanned from position 4 to positions 9 or 10. Two series of analogues were synthesized and are characterized by residues at positions 1 [Ac-D-3-(2'-naphthyl)alanine], 2 [D-(4-chlorophenyl)alanine or D-(4-fluorophenyl)alanine], 3 [D-3-(3'-pyridyl)alanine or D-tryptophan], 5 (arginine or tyrosine), and 6 [D-3-(3'-pyridyl)alanine or D-arginine], respectively. These substitutions were selected in an effort to optimize high biopotency for inhibition of luteinizing hormone secretion, minimization of histamine release activity, and high (relative) hydrophilicity. The most potent analogues in the antiovulatory assay were cyclo(4-10) [Ac-DNal1,DCpa2,DPal3,(Asp4 or Glu4),Arg5,DPal]6,Dpr10]GnRH (compounds 5 and 7), which were fully active at ca. 12.5 micrograms/rat in the first series, and cyclo(4-10)[Ac-DNal1,DFpa2,DTrp3,Asp4,DArg6++ +,Dpr10]GnRH (compound 12), which was fully active at 2.5 micrograms/rat in the second.

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.

Conformational energy calculations have been carried out on analogues of luteinizing hormone-releasing hormone which have been shown to be potent inhibitors of the release of luteinizing hormone and follicle-stimulating hormone. The analogues included in this study have D-amino acid substitutions in the 2 and/or 3 positions, such as [D-X2]-LH-RH, [D-X2,D-Y3]-LH-RH, [D-X2,Pro3]-lh-rh, and [D-X2,Leu3]-LH-RH. A configurational property which was common to the low-energy conformers of all the analogues is the directional change of the cis-peptide bond of the pyroglutamate ring. Further, there was no overall structural change in the analogues relative to the conformation of native LH-RH, and the orientation of the aromatic side chains relative to one another remained the same throughout this series of analogues.

In the isolated rat pituitary assay, [Thr2,Leu3]-LH-RH, [Leu2,Ala3,D-Ala6]-LH-RH, and des-Gly10-[Abu2,Ala3,D-Ala6]-LH-RH ethylamide inhibited the LH release due to 0.3 ng/mL of added LH-RH at a 10 microgram/mL dosage. Under these same assay conditions, des-Gly10-[Ile2,Ala3,D-Ala6]-LH-RH ethylamide was about one-tenth as active, and no inhibition was observed by [Leu2,Ser3]-LH-RH or [Leu2,Asn3]-LH-RH at a 100 microgram/mL dosage. The corresponding results from FSH inhibition assays, in vitro, are also reported.

A series of reduced-size hexapeptide analogues of LH-RH were synthesized that contain the residues corresponding to amino acid positions 4-9 and are linked to various carboxylic acids in place of residue 3. These compounds were tested in vitro in the rat pituitary receptor binding and LH release assays. A wide range of binding affinities was obtained up to and exceeding that of LH-RH. Both agonists and antagonists were obtained. From the SAR studies, it appears that a very precise size, length, and shape of the substituent at position 3 is required to achieve agonist activity, whereas the structural requirements for antagonist activity appear to be much less stringent. Depending on the nature of the substituent at positions 6 and 4, the biological response switches from antagonist to agonist or vice versa. The results suggest that conformational changes at position 6 or 4 feed back to the substituent at position 3, which induces the change from agonist to antagonist. The most potent compounds in the series were tested in vivo and found to be active.

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.

A series of des-His2 octa- and nonapeptide analogues of luliberin (luteinizing hormone-releasing hormone) with modifications in the 1 and 6 positions, and in some instances the 10 position, has been prepared. Some of these analogues are potent inhibitors of luliberin in vitro and in vivo. The use of ultraviolet absorption measurements for evaluating peptides containing tyrosine and tryptophan is described. An efficient synthesis of O-methyl-d-tyrosine is reported.

Twenty-four thyrotropin-releasing hormone (TRH) analogues containing mainly aliphatic amino acids in position 2 were synthesized and tested for central nervous system (CNS) and hormonal (TSH) activity. Application of the pentafluorophenyl ester method in the syntheses resulted in optimal yields and high purity of the products. The neutral tripeptides pGlu- Nva -Pro-NH2 (9), pGlu-Nle-Pro-NH2 (10), and pGlu-Leu-Pro-NH2 (3) with a three- or four-membered straight or branched alkyl side chain in the position of the central amino acid had 2.5 to 10 times stronger anticataleptic effect than TRH, demonstrating that the presence of histidine is not essential for the CNS activity. Analogue 9 exhibited tenfold anticataleptic activity as compared to TRH, and it was found to be fully inactive in the release of TSH.

There is evidence that, in its receptor-binding conformation, the N and C terminus of LH-RH may be in close proximity and two cyclic analogues of the hormone were synthesized to test the hypothesis. Cyclic [beta-Ala1,D-Ala6,Gly10]- and [6-aminohexanoic acid1,D-Ala6,Gly10]-LH-RH were prepared by treatment of their linear precursor peptides with dicyclohexylcarbodiimide in the presence of 1-hydroxybenzotriazole in dilute dimethylformamide solution. Although the linear peptides possessed no detectable LH-releasing activity in ovariectomized rats, the cyclic beta-Ala analogue had 1.2% the activity of LH-RH, whereas the longer chain cyclic 6-aminohexanoic acid analogue had 0.65% activity. These results support the concept of an important interaction between the ends of the LH-RH molecule possibly involving hydrogen-bond formation between the pyrrolidone carbonyl group of pyroglutamic acid and the glycinamide group.

The incorporation of simple d-amino acids in place of glycine in position 6 of the LH-RH decapeptide produces analogs which have far greater gonadotropin-releasing activities in vivo and in vitro than the natural hormone. An investigation of the structural features of the d-amino acids responsible for this phenomenon suggests that an increase in the lipophilic character and perhaps the size and aromaticity of the side chain coincides with an increase in biological activity. This is demonstrated by the LH-releasing activities of the following series of peptides which were assayed over a period of 6 h in immature male rats: [d-Glu(6)]-,1.8;[d-Ala(6)]-,7.0;[d-Leu(6)]-,9,0;[d-Phe(6)]-,10;[d-Trp(6)]-LH-RH, 13 times more active than LH-RH itself. In contrast to previous results with [d-Ala(6)]-and [d-Leu(6)]-LH-RH, where the substitution of an ethylamide group for the glycine amide at the C-terminus produces large increases in LH/FSH releasing activity, the ethylamide derivatives of [d-Phe(6)]-and [d-Trp(6)]-LH-RH were actually less potent than their parent peptides. [(N-Me-d-Ala)(6)]-LH-RH was found to be approximately 70 times less active than [d-Ala(6)]-LH-RH which indicates that disruption of a preferred receptor-site binding conformation might be brought about by methylation of the amide linkage in this position.

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.