Anand S. Dutta

Polypeptides. Part 15. Synthesis and biological activity of α-aza-analogues of luliberin modified in positions 6 and 10

Analogues of luliberin (luteinising hormone-releasing hormone) and 2-dehistidyl-luliberin containing α-azaglycine (–NHNHCO–) or α-aza-alanine (–NHNMeCO–) residues in either position 6 or 10 have been synthesised by classical procedures of peptide synthesis. The agonist and antagonist activities of these compounds were evaluated in androgen-sterilised constant-oestrus rats; [10-Azgly]-, [6-Azgly]-, and [6-Azala]-luliberin were marginally less active than the parent peptide. When the aza-change in position 6 was combined with an ethylamide substitution in position 10 the resulting compounds, [6-Azgly-10-de-Gly-NH2-9-Pro-ethylamide]- and [6-Azala-10-de-Gly-NH2-9-Pro-ethylamide]-luliberin, were twice as active as luliberin. [6-D-Ala-10-Azgly]luliberin was also twice as active as luliberin. [2-De-His-10-Azgly]-luliberin inhibited completely ovulation induced by luliberin (0.5 µg/rat) at a dose of 250 µg/rat, but 2-de-His-analogues of all the other compounds were devoid of antagonist activity in this test system.

Enkephalin analogues eliciting analgesia after intravenous injection

Abstract Extensive study of structure-activity relations in enkephalin-like peptides has led to analogues which are up to 70 times more potent than Leu-enkephalin in vitro in the electrically-stimulated guinea pig ileum preparation, and which are analgesic in the mouse hot-plate test at doses as low as 5mg/kg following intravenous administration.

Potent agonist and antagonist analogues of luliberin containing an azaglycine residue in position 10.

Potent agonist and antagonist analogues of luliberin containing an azaglycine residue in position 10 were synthesised and tested in androgen-sterilised constant-oestrus rats. The agonist, [D-Ser(But)6, Azgly10]-luliberin, induced ovulation at a dose of 6ng/rat i.v., 10μg/rat p.o. and was at least five times as potent as [D-Ser(But)6, des-Gly-NH210, Pro-ethylamide9]-luliberin. [D-Ser(But)6, Azgly10]-luliberin (1μg/rat) also prevented HCG-induced increases in ovarian and uterine weight in immature rats and was a highly potent antitumour agent when given to rats bearing DMBA-induced mammary tumours. The antagonist, [D-Phe2, D-Phe6, Azgly10]-luliberin at a dose of 15μg/rat completely inhibited ovulation induced by luliberin (0.5μg/rat), whereas [D-Phe2, D-Phe6]-luliberin lost activity below 125μg/rat.

Polypeptides. Part XIII. Preparation of α-aza-amino-acid (carbazic acid) derivatives and intermediates for the preparation of α-aza-peptides

Esters and amides of several α-aza-amino-acids (carbazic acids), and intermediates of use in the introduction into peptides of α-aza-glycyl, -alanyl, -valyl, -leucyl, -isoleucyl, -phenylalanyl, -tyrosyl, -tryptophyl, -prolyl, -aspartyl, -asparaginyl, -glutamyl, -glutaminyl, and -pyroglutaminyl residues are described. t-Butyl 3-alkyl- or -aralkyl carbazates, obtained by catalytic hydrogenation of the corresponding hydrazones, were the most versatile intermediates; they were converted in high yield into α-aza-amino-acid esters and amides, gave α-aza-dipeptide esters when treated with α-isocyanato-esters, and afforded ‘active esters’ of N-t-butoxycarbonyl-α-aza-amino-acids when treated with 2,4,5-trichlorophenyl chloroformate.

N-isobutyryl-His-Trp-Ala-Val-D-Ala-His-Leu-NHMe (ICI 216140) a potent in vivo antaconist analogue of bombesin/gastrin releasing peptide (BN/GRP) derived from the C-terminal sequence lacking the final methionine residue.

The GRP receptor mediated growth response in Swiss 3T3 cells has been used to identify BN/GRP antagonists. Analysis of bombesin antagonism by substance P analogues and by truncated GRP analogues revealed that deletion of the C-terminal methionine residue was important for antagonism. Des-Met analogues showing potent antagonist activity in the in vitro 3T3 system (IC50 approximately 2nM) were synthesized. Further structural modification of these peptides led to the identification of (CH3)2CHCO-His-Trp-Ala-Val-D-Ala-His-Leu-NHCH3 (ICI 216140) which reduced bombesin-stimulated rat pancreatic amylase secretion to basal levels when administered subcutaneously at 2.0 mg per kg.

Anti-inflammatory activity of c(ILDV-NH(CH2)5CO), a novel, selective, cyclic peptide inhibitor of VLA-4-mediated cell adhesion.

Small, N‐ to C‐terminal cyclized peptides containing the leucyl‐aspartyl‐valine (LDV) motif from fibronectin connecting segment‐1 (CS‐1) have been investigated for their effects on the adhesion of human T‐lymphoblastic leukaemia cells (MOLT‐4) to human plasma fibronectin in vitro mediated by the integrin Very Late Antigen (VLA)‐4 (α4β1, CD49d/CD29). Cyclo(‐isoleucyl‐leucyl‐aspartyl‐valyl‐aminohexanoyl‐) (c(ILDV‐NH(CH2)5CO)) was approximately 5 fold more potent (IC50 3.6±0.44 μM) than the 25‐amino acid linear CS‐1 peptide. Cyclic peptides containing two more or one less methylene groups had similar potency to c(ILDV‐NH(CH2)5CO) while a compound containing three less methylene groups, c(ILDV‐NH(CH2)2CO), was inactive at 100 μM. c(ILDV‐NH(CH2)5CO) had little effect on cell adhesion mediated by two other integrins, VLA‐5 (α5,β1, CD49e/CD29) (K562 cell adhesion to fibronectin) or Leukocyte Function Associated molecule‐1 (LFA‐1, αLβ2, CD11a/CD18) (U937 cell adhesion to Chinese hamster ovary cells transfected with intercellular adhesion molecule‐1) at concentrations up to 300 μM. c(ILDV‐NH(CH2)5CO) inhibited ovalbumin delayed‐type hypersensitivity or oxazolone contact hypersensitivity in Balb/c mice when dosed continuously from subcutaneous osmotic mini‐pumps (0.1–10 mg kg−1 day−1). Maximum inhibition (approximately 40%) was similar to that caused by the monoclonal antibody PS/2 (7.5 mg kg−1 i.v.) directed against the α4 integrin subunit. c(ILDV‐NH(CH2)5CO) also inhibited oxazolone contact hypersensitivity when dosed intravenously 20 h after oxazolone challenge (1–10 mg kg−1). Ear swelling was reduced at 3 h and 4 h but not at 1 h and 2 h post‐dose (10 mg kg−1). Small molecule VLA‐4 inhibitors derived from c(ILDV‐NH(CH2)5CO) may be useful as anti‐inflammatory agents.

Chapter 21. Luteinizing Hormone Releasing Hormone (LHRH) Analogues

Publisher Summary This chapter discusses the luteinizing hormone releasing hormone (LHRH) agonists and antagonists and their pharmacological activities and clinical applications. A number of LHRH agonists have entered clinical trial. Potent antagonists of LHRH show good in vivo activity. A few LHRH antagonists have recently been reported to be active by the oral route. Because LHRH analogues are poorly absorbed by the oral route, considerable effort has been expended to devise suitable formulations for clinical trials. LHRH agonists can stimulate gonadal function if given at low dose as a series of pulses. The combination of LHRH agonists with pure anti-androgens has also been studied in prostate cancer and preliminary results suggest that survival may be improved. The clinical utility of LHRH analogues in other forms of cancer remains to be determined. After treatment with LHRH analogues, responses have also occurred in post-menopausal women with breast cancer. The gonadal suppressing action of LHRH agonists as also been used to treat successfully precocious puberty in both boys and girls; preliminary results suggest that it has advantages over other available therapies. There has been widespread interest in the use of LHRH analogues as contraceptive agents and numerous studies have been conducted in primates and man. As many highly potent and safe LHRH agonists are available, there is room for improvement in the potency of LHRH antagonists and ideally in the identification of the orally active agents.

Conformationally restrained cyclic peptides as antaconists of luteinizing hormone-releasing hormone

Using minimum energy calculations and molecular dynamics techniques the preferred conformational states of LHRH and its analogues have been reported to involve a modified beta-bend between residues 5 to 8. Based on some of these models cyclic peptide analogues of LHRH antagonists were synthesised using solid phase peptide synthesis methodology. The analogues were tested for their ability to inhibit ovulation in normal cycling rats. Some analogues were also tested in receptor binding and in vitro LH release assays. The most potent cyclic peptide analogue, Ac-D-Phe(p-C1)-D-Phe(p-C1)-D-Trp-Ser-Glu-D-Arg-Leu-Lys-Pro-D-Ala-NH2 (V), had an ED50 value of 91.9 micrograms/kg in the inhibition of ovulation test. The corresponding linear peptide (IV) was about three times less potent. Analogues with smaller or larger ring sizes or with modifications within the ring were also prepared but these were either less potent or inactive, up to a dose of 1000 micrograms/kg, in inhibiting ovulation in normal cycling rats.

Polypeptides. Part 16. Synthesis and biological activity of α-aza-analogues of luliberin with high antagonist activity

Analogues of luliberin (luteinising hormone-releasing hormone) were synthesised where the glycine residue in position 10 was replaced by either azaglycine (–NH–NH–CO–) or aza-alanine (–NH–NMe–CO–), the histidine residue in position 2 was either omitted, or replaced by D-phenylalanine or D-tryptophan, and the glycine residue in position 6 was substituted either by D-phenylalanine or D-tryptophan. These compounds were evaluated for their ability to prevent ovulation induced by luliberin in androgen-sterilised constant-oestrus rats. Compounds with the azaglycine residue in position 10 and other modifications in positions 2 and 6 showed good antagonist activity, whereas aza-alanine replacement in position 10 together with modifications in position 2 resulted in inactive compounds. The most potent analogue, [2-D-Phe-6-D-Phe-10-Azgly]-luliberin, completely inhibited ovulation induced by luliberin (0.5 µg/rat) at a dose of 15 µg/rat.

Polypeptides. Part XIV. A comparative study of the stability towards enzymes of model tripeptides containing α-aza-amino-acids, L-amino-acids, and D-amino-acids

The stability of three model tripeptides, L-lysyl-α-aza-phenylalanyl-L-leucine, L-lysyl-L-phenylalanyl-L-leucine, and L-lysyl-D-phenylalanyl-L-leucine, towards aminopeptidase, carboxypeptidase, thermolysin, and trypsin has been investigated. These tripeptides were synthesised by the classical method of peptide synthesis. The α-aza-phenylalanine-containing tripeptide was stable to the first three of these enzymes, but the L-lysyl–α-aza-phenylalanine bond was readily cleaved by trypsin. The tripeptide containing D-phenylalanine was stable to all these enzymes, whereas the tripeptide with L-phenylalanine was completely cleaved in 72 h.