Derek E. Brundish

CGS 8216: Receptor binding characteristics of a potent benzodiazepine antagonist

CGS 8216 is a novel nonbenzodiazepine that inhibited 3H-flunitrazepam (3H-FLU) binding to rat synaptosomal membranes in vitro at subnanomolar concentrations. It prevented the in vivo labeling of brain benzodiazepine receptors by 3H-FLU with the same potency as diazepam when given orally to mice. Pharmacologic tests showed that it was devoid of benzodiazepine-like activity but it antagonized the actions of diazepam in these tests. It did not interact with alpha- or beta- adrenergic, H1-histaminergic or GABA receptors but it inhibited adenosine-activation of cyclic AMP formation. Studies with 3H-CGS 8216 demonstrated that it bound specifically and with high affinity to rat forebrain membranes and was displaced by drugs with an order of potencies similar to that observed when 3H-diazepam and 3H-FLU were used as radioligands. The regional distribution of 3H-CGS 8216 binding sites in the brain was also similar to that of 3H-FLU. Dissociation of 3H-CGS 8216 binding was slow at 0 degrees C but increased with temperature and was almost complete within 1 min at 37 degrees C. Scatchard analyses were linear, yielding KD values of 0.044, 0.11 and 0.18 nM at 0, 25 and 37 degrees C, respectively; the Bmax value did not change appreciably with temperature and was approximately 1000 fmoles/mg protein. Using 3H-FLU, the value for Bmax as well as for the KD increased with temperature. The total number of binding sites determined for 3H-FLU was greater than that for 3H-CGS 8216 at each temperature. CGS 8216 exhibited mixed-type inhibition of 3H-FLU binding. GABA did not stimulate 3H-CGS 8216 binding whereas it enhanced 3H-FLU binding. CGS 8216 may be a useful ligand for probing the antagonist properties of the benzodiazepine receptor and is likely to exhibit interesting therapeutic effects.

Synthesis and metabolic stability of a tritium-labelled substance P analogue.

We have described the synthesis of a substance P analogue, <Glu-Gln-Phe-MePhe-MeGly-LeuMet-NH2 [ 11. The peptide, referred to herein as DiMeC7, was designed to be resistant to a substance P degrading enzyme purified from brain [2]. Evidence for the increased stability of DiMeC7 in rat brain preparations has been presented [l]. Moreover, DiMeC7 exhibited a high potency in competing for tritiated substance P binding to rat brain membranes as well as retaining biological activities in peripheral substance P bioassays [ 11. This report describes the synthesis of the precursor, production of the radiolabelled peptide by catalytic dehalogenation and further evidence for enhanced resistance of DiMeC7 to in vitro and in vivo digestion by rat brain compared with substance P.

Rapid conversion of somatostatin to active metabolite in human plasma

Immunoreactive somatostatin occurs naturally in many parts of the body and is known to inhibit the secretion of a large number of peptide hormones [ 11. Whether or not its physiological actions are entirely local is an open question [2] but it has been demonstrated by radioimmunoassay that biologically active concentrations [3 ] are present in normal human plasma after a meal [4]. Somatostatin is known to be converted rapidly to a closely related product, [desAla’]-somatostatin, in rat plasma in vitro and in vivo [5]. We can now report that this conversion also occurs in human plasma and that the product is equipotent with somatostatin in several biological assay systems. trifluoroacetic acid (99: 1, by vol.) to acetonitrile/ water/trifluoroacetic acid (80: 19: 1, by vol.). The eluate was monitored for absorbance at 280 nm and peaks were film dried and subjected to amino acid analysis after acid hydrolysis.

Synthesis of [3,5-3H2-Tyr23]-β-corticotrophin-(1–24)-tetracosapeptide

The synthesis is described of β-corticotrophin-(1–24)-tetracosapeptide labelled with tritium in the tyrosine residue at position 23 to a specific radioactivity of 46 Ci mmol–1 by reductive deiodination of a protected precursor. Evidence for the integrity of the final product is provided by amino-acid analysis, column and thin-layer chromatography, and bioassay, supported by chemical and enzymic analytical data on the protected precursor and the derived tetracosapeptide containing di-iodotyrosine.

Synthesis of 5- and 7-bromotryptophan and of [5-bromotryptophan9]-β-corticotrophin-(1–24)-tetracosapeptide, a highly potent corticotrophin analogue

The synthesis of 5- and 7-bromo-L-tryptophans via Fischer cyclisation of the appropriate bromophenylhydrazone of 4-acetamido-4,4-bis(ethoxycarbonyl)butanal is described. The title tetracosapeptide was synthesised using standard methods of stepwise and fragment condensation. The product was pure as judged by amino-acid analysis after acidic or enzymic hydrolysis and by high pressure liquid chromatography. In an isolated adrenal cell bioassay, the peptide had a steroidogenic potency 2.4 times that of Synacthen.

The design and synthesis of thrombin inhibitors: analogues of MD805 containing non-polar surrogates for arginine at the P1 position

A series of monocyclic and bicyclic amino acids have been synthesised and incorporated into thrombin inhibitors based on CGH728, an analogue of the Mitsubishi compound MD805. Benzthiazolylalanine (Bta) was found to be a good non-polar substitute for arginine at the P1 position, yielding compounds with low nanomolar potency and good selectivity for thrombin.

Tritiated peptides. Part 3. Synthesis of [4-3H-Phe7]-β-corticotrophin-(1–24)-tetracosapeptide

The synthesis is described of β-corticotrophin-(1–24)-tetracosapeptide labelled with tritium in the phenylalanine residue at position 7 to a specific radioactivity of 27 Ci mmol–1 by reductive deiodination of a protected precursor. Evidence for the integrity of the final product is provided by amino-acid analysis, column chromatography, and bioassay, supported by chemical and enzymic analytical data on the protected precursor and the derived free tetracosapeptide containing iodophenylalanine.

Tritiated peptides. Part 7. Synthesis of [2,5-3H2-His6]-β-corticotrophin-(1–24)-tetracosapeptide

The synthesis is described of β-corticotrophin-(1–24)-tetracosapeptide ‡ labelled with tritium in the histidine residue at position 6 at a specific radioactivity of 30 Ci mmol–1 by reductive dehalogenation of a protected precursor. Evidence for the integrity of the final product is provided by amino-acid analysis, column chromatography, and bioassay, supported by chemical and enzymic analytical data on the protected precursor and the derived free peptide containing di-iodohistidine.

Tritiated peptides. Part 15. Synthesis of tritium labelled biologically active analogues of somatostatin

The syntheses are described of cyclo-{[D-Trp8,Gaba12]somatostatin-(5–12)-peptide}(77), and (79)–(82) labelled singly at positions 6, 7, 8, and 11 and doubly at residues 6 and 11 to specific radioactivities of between 4.8 and 22.4 Ci mmol–1. The linear sequence (5–12) and the related cyclo-{[D-Trp8,Nag12]somatostatin-(5–12)-peptide}(78) and (83) were also prepared to specific radioactivities of 19.2 and 19.6 Ci mmol–1 respectively. The syntheses of the labelled hexapeptide cyclo-{[4-3H-Phe7,D-Trp8,Pro12] somatostatin-(7–12)-peptide}(84) and the full sequences [4-3H-Phe6,D- Trp8,D-Cys14]somatostatin (94) and [D-Trp8,4-3H-Phe11,D-Cys14]somatostatin (95) labelled at ca. 13.0 Ci mmol–1 are described. Labelling was effected by reductive dehalogenation in the presence of tritium of the fully protected precursors and the purity of the final products was assessed by amino acid analysis after acidic hydrolysis following purification by ion-exchange and h.p.l.c. as appropriate.

Synthesis of analogues of bradykinin with replacement of the arginine residues by 4-guanidinophenyl-L-alanine

The synthesis is described of three analogues of bradykmin in which the terminal arginine residues were separately or together replaced by 4-guanidinophenyl-L-alanine. The analogues were considerably less potent than the parent peptide in their action on smooth muscle in vitro