Pui-Fun Louisa Tang

Design, synthesis and bioassays of antagonists of LHRH which have high antiovulatory activity and release negligible histamine

Potent antagonists of the luteinizing hormone releasing hormone have been achieved which release negligible histamine. [N-Ac-D-2-Nal1, D-pClPhe2, D-3-Pal3, NicLys5, D-NicLys6, ILys8, D-Ala10]-LHRH showed 100%AOA/1 microgram and 36%/0. 5 micrograms; ED50 greater than 300. [N-Ac-D-2-Nal1,D-pClPhe2,D-3-Pal3, PicLys5, D-PicLys6, ILys8, D-Ala10]-LHRH showed 100% AOA/0.5 micrograms and 40%/0.25 micrograms; ED50, 93 +/- 11, and is the most potent of 52 new peptides. These antagonists feature designs with weakly basic acylated D-Lys6, notably D-NicLys6 and D-PicLys6 and alkylated Lys8 or Orn8, e.g., ILys8 and IOrn8, and NicLys5 and PicLys5. Concepts included balanced overall basicity, superiority of ILys8 and IOrn8 which are sequence dependent and sensitivity of positions 5 and 6 for potency.

Increased potency of antagonists of the luteinizing hormone releasing hormone which have D-3-Pal in position 6.

Ever increasing potency is an international goal for antagonists of the luteinizing hormone releasing hormone (LHRH). [N-Ac-D-2-Nal1,D-pClPhe2, D-3-Pal3,Ser4,Arg5,D-3-Pal6,Leu7,Arg8,Pro9,D- Ala10]-NH2 caused 60%/125 ng inhibition of ovulation in the rat, and appears to be the most potent antagonist yet described. Strategy of design was the replacement of D-Arg6 with D-3-Pal6 and of Tyr5 with Arg5. Replacing Arg5 with His5 reduced activity by 50% at 250 ng. Both the Arg5 and His5 analogs showed 100% inhibition of ovulation at 0.5 microgram. Of ten pairs of analogs with D-3-Pal6 and D-Arg6, 3/10 with D-3-Pal6 were more potent than those with D-Arg6. Histamine release was less for the D-3-Pal6 peptides of three pairs.

Increased potency of antagonists of substance P having asparagine in position 6.

The general structure of antagonists of substance P (SP) which was found with the development of Spantide and analogs based on Spantide served for further refinement. The antagonistic potency was tested in vitro on guinea pig ileum and taenia coli. It was unexpectedly found that introduction of Asn6 gave rise to a considerable increase in potency. The exchange of Gln6 for Asn6 entails the shortening of the side chain by one CH2 unit and seems slight for steric advantages and potency increase. The analog [D-Arg1,D-Cl2Phe5,Asn6,D-Trp7,9,Nle11]SP had pA2 values of 7.4 (ileum) and 8.0 (taenia coli). We then used this sequence as a new lead to introduce new changes, which were made in positions 1, 3, 5, 7 and 9. It was found that Arg1 is important, but Lys3 can be exchanged. The Pal3 derivative had pA2 values of 8.1 and 8.0 and the Nle3 counterpart had 7.7 and 7.4 D-Cl2Phe is an effective substituent in position 5. D-Trp in positions 7 and 9 were superior to other alternatives.

Specificity of Design to Achieve Antagonists of LHRH of Increasing Effectiveness in Therapeutic Activity

The pioneering work on TRH [1–4] inspired the staff of the Institute for Biomedical Research of the University of Texas at Austin to isolate concentrates of LHRH from tissue and conduct chemical and enzymic inactivation experiments toward determining certain amino acids in LHRH. These studies [5] resulted in the crucial conclusion that LHRH is a decapeptide rather than a nonapeptide [6]. These inactivation experiments not only provided accurate structural information, but were the basis for the synthesis of <Glu-Tyr-Arg-Trp-NH2 [7]. the first reported synthetic LHRH agonist. These early successes on TRH and LliRH were the basis for K. Folkers and C. Bowers to join in the transition to antagonists of LHRH.

Activities of Antagonists of the Luteinizing Hormone Releasing Hormone with Emphasis on Positions 1, 5 and 6 and on Positions 1, 2 and 3

Abstract Analogs of the luteinizing hormone releasing hormone (LHRH) which are antagonists for controlling ovulation require potency and negligible release of histamine as a side effect. Forty analogs were designed, synthesized and bioassayed in two groups with emphasis upon positions 1, 5 and 6 and upon positions 1, 2 and 3. N-Ac-D-2-Nal1, D-pClPhe2, D-3-Pal3, Ser4, Tyr5, D-Lys6, Leu7, Arg8, Pro9, D-Ala10-NH2 and N-Ac-D-CL2Phe1, D-α-Me-pCIPhe2, D-3-Pal3, Ser4, Tyr5 D-Arg6, Leu7, Arg8, Pro9, D-Ala10-NH2 caused 100% inhibition of ovulation at 0.5 μg in rats. The former analog showed 12.5% anh-ovulatory activity (AOA) and the latter analog showed 40% AOA at 0.25 (μg. The neutral Cit6 moiety is unique since it and the basic D-NicLys6 moiety provided peptides comparable in activity. Frequently, D-2-Nal, D-pClPhe and D-CL2Phe are comparable in position 1. Histamine release was substantially low for a D-NicLys6 analog.

Relative Potencies of Antagonists of the Luteinizing Hormone Releasing Hormone with Lys8 and Arg8 and Substitutions in Positions 3 ,5 ,6 ,7 and 8

Abstract Antagonists of the luteinizing hormone releasing hormone (LHRH) of increased potency is a goal for control of ovulation. In the design and synthesis of 26 decapeptides, emphasis was given to analogs with Lys8 and Arg8 and with various substitutions in positions 3, 5, 6, 7 and 8. Two antagonists, [N-Ac-ᴅ-2-Nal1,ᴅ-pClPhe2,ᴅ-3-Pal3,Ser4,Tyr5,ᴅ-Arg6,Leu7,Lys8,Pro9,ᴅ-Ala10] - NH2 and [N-Ac-ᴅ-2-Nal1.ᴅ-pClPhe2,ᴅ-3-Pal3,Ser4,Arg5,ᴅ-3-Pal6,Leu7,Arg8,Pro9,ᴅ-Ala10]-NH2 showed 80-85% antiovulatory activity (AOA) at 0.25 μg in the rat. The latter antagonist showed 60% AOA at 0.125 μg. Of four pairs of analogs with Arg8 and Lys8, respectively, two pairs favored Lys8 over Arg8 for potency. One pair showed negligible difference and another pair favored Arg8 over Lys8. There is specificity of substitution for potency. In other antagonists, ᴅ-3- Pal3, Tyr5 or Phe5, ᴅ-Arg6 and Leu7 or Nle7 or Val7 and Arg8 were variously effective substitutions for increase of potency and reduction of histamine release.