Anders Ljungqvist

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.

Antagonists of LHRH superior to antide ; effective sequence/activity relationships

Abstract Twenty new analogs of LHRH featured acylated aminocyclohexylalanines and acylated lysines in positions 5,6. (N-Ac-D-2-Nal1,DpC1Phe2,D-3-Pal3,PicLys5, pzACAla6,Val7,Ilys8,D-Ala1O)-LHRH (l00% AOA/0.5ug) and (N-Ac-D-2-Nal1, DpClPhe2 D-3-Pal,PicLys5,D-Piclys6Abu7,D-Ala10-LHRH (50% AOA/0.25ug) were most potent.

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.

Blockade of sensory nerve mediated contraction of the rabbit iris sphincter by a series of novel tachykinin antagonists

Electrical stimulation of the isolated rabbit iris sphincter muscle in the presence of atropine gives rise to a contraction that can be blocked by tachykinin antagonists. The ability of a series of novel tachykinin antagonists to inhibit the contractile effect of SP on the guinea-pig taenia coli and to suppress the electrically evoked contraction of the atropinized rabbit iris sphincter was tested. Several of the novel antagonists were found to be more potent in terms of pA2 and pIC50 values than the two previously described analogs, [D-Pro2, D-Trp7,9]SP9(1-11) and [D-Arg1, D-Trp7,9, Leu11]SP-(1-11) (Spantide). Apart from D-Trp in positions 7 and 9 the characteristic features of the potent novel antagonists were D-Cl2Phe (or D-Cys(Bzl] in position 5, Asn in position 6 and Nle in position 11. In addition Pal in position 3 seemed to offer an enhanced potency.

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.

Metabolic stability of the LHRH antagonist antide to cell-surface peptidases

The susceptibility to hydrolysis of LHRH and the decapeptide analogue Antide has been compared. The hydrolysis of LHRH by pig kidney brush border membranes is inhibited by phosphoramidon (I50 = 5.6 nM) implicating endopeptidase-24.11 in the initiation of hydrolysis. Under conditions in which LHRH is fully degraded by brush border membranes, Antide was completely resistant to hydrolysis. Similar results were obtained with purified preparations of both endopeptidase-24.11 and angiotensin converting enzyme. These data confirm that the remarkable duration of action of Antide is due principally to its stability to hydrolysis by cell-surface peptidases.

Design, Synthesis and Biological Evaluation of Antagonists of LHRH by Criteria of Potency, Safety and Solubility

Some analogs of Antide and congeners with higher water solubility have been synthesized by substitutions in positions 1, 5 or 6 with hydrophilic residues. In position 1, D-3-Qal has been incorporated in four peptides and D-3-Pal in one peptide. In positions 5 and 6, D and L-3-Pal, PzAla and (DSer)Lys have been tried. In one peptide, D—(AcDSer)Lys was substituted in position 6. Most of the new analogs had lower AOA (antiovulatory activity) than the parent compounds but three potent analogs were identified. The first one, [N — Ac— D — 3-Qal1,DpClPhe2,D— 3-Pal3,c-PzACAla5,D — PicLys6,ILys8,D — Ala10]— LHRH, had 55% AOA at 0.25 μg and 100% at 0.5 μg. Its ED50 for in vitro histamine release was 171 ± 17 μg/ml which is an increase from 49±4.8 μg/ml for the parent compound with N—Ac-D-2-Nal [1]. The second analog, [N — Ac— D — 2-Nal1,DpClPhe2,D — 3-Pal3,PicLys5,D — (DSer)Lys6,ILys8,D—Ala10] — LHRH, had 69% AOA at 0.25 μg and 95% at 0.5 μg. This analog released somewhat more histamine than the parent analog featuring D-PicLys6, the ED50 being 18 μg/ml compared to 93 ± 11 for the parent analog. The third analog is: [N — Ac— D— 2-Nal1,DpClPhe2,D— 3-Pal3,c-PzACAla5,D—PzAla6,ILys8,DAla1]— LHRH. The AOA for this analog was 63% at 0.25 μg and the ED50 for histamine release 88±6.4 μg/ml.

Design, synthesis and biological evaluation of antagonists of LHRH including substitutions with L-azetidine 2-2-carboxylic acid

A new series of luteinizing hormone-releasing hormone (LH-RH) antagonists containing L-azetidine-22-carboxylic acid has been synthesized and tested for safety and initial trails in laboratory animals. L-azetidine-2-carboxylic acid is the lower homolog of proline and derivatives of LH-RH containing it have not previously been reported. These new drugs have been designed to improve potency solubility in aqueous media and safety in terms of local histamine release. Antide has the substitutions D-2-Nal(1) NicLys(5) D-NicLys(6) Leu(7). Antide has N-nicotinoyl-D-lysine at position 6 and N-isopropyl-lysine at position 8 instead of the basic arginines which in combination with a cluster of hydrophobic aromatic amino acids at the N-terminus are thought to elicit histamine release. Antide had 36% antiovulatory activity at 0.5 mcg an ED50 for histamine release of >300 mcg/ml and a wheal area in square mm/10 mcg of 132.7. 3 of 21 other analogs of Antide also showed significant antiovulatory activity at 0.125 mcg and 13 others had some antiovulatory activity at 0.250 mcg.

POSITION 10 IS CRITICAL FOR ANTAGONISTS OF THE LUTEINIZING HORMONE-RELEASING HORMONE AND FOR INHIBITION OF OVULATION IN RATS

Eleven analogs of the luteinizing hormone-releasing hormone (LHRH) have been designed, synthesized, bioassayed and compared for antiovulatory activity (AOA) in rats. The emphasis of design was on analogs with D–Ala10, Sar10, D–Ser10, (desGly10, NHEt), D–Abu10, Gly10, and with substitutions in position 5, 6, and 8. High antiovulatory activity was obtained with analogs having D– Ala in position 10. Four earlier analogs, 1, 4, 11 and 13, with D– Ala in position 10, showed 67-100% AOA at 0.25 μg in rats. Analog 1 showed 60% AOA at 0.125 μg. The replacement of D–Ala10 in analog 11 by Sar10 in analog 12, retained activity, 62% vs. 67% AOA at 0.25μg, 100% vs. 90% AOA at 0.5 μg. This analog with Sar10 was the only one that did not show substantial loss of activity upon replacement of D–Ala10.

Study of hydrogenolytic cleavage of peptide-resin benzyl ester bonds for synthesis of protected fragments of the human leukocyte interferon

Three Roe-protected fragments of the human leukocyte interferon, LeIFA, have been synthesized: I, BOC LeIFA (99-105) (BOC-Val-Ile-Gln-Gly-Val-Gly-Val); II, BOC Lei FA (116-119) (BOC-Ile-Leu-Ala-Val); III, BOC LeIFA (123-126) (BOC-Phe-Gln-Arg(Tos)-Ile). The presence of the Boc-group was desired so that these peptides could serve as intermediates in fragment condensation toward larger segments of interferon. To achieve these intermediates, a study was desirable on three methods of cleavage of the benzyl ester-polymer bonds. It was found that hydrogenolysis with hydrogen and palladium generated in situ was distinctly superior to transfer hydrogenation with ammonium formate or cyclohexene. An effective swelling of the peptide-resin to allow penetration of palladium acetate into the matrix and mobility of the peptide chains on the resin to expose the bonzyl ester bond to hydrogenolysis appear to be essential conditions for the best cleavage.