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1 alpha-beta-carboxypropionyl-cyclo(9----1 epsilon)-[Lys1, Gly6]bradykinin (Suc-c[Lys1, Gly6]B), 1 alpha-beta-carboxypropionyl-cyclo(10----1 epsilon)kallidin (Suc-cK), cyclo(10 gamma----1 epsilon)-[Glu10]kallidin (c[Glu10]K) and cyclo(11 gamma----1 epsilon)kallidylglutamic acid (cKG) were synthesized. Suc-c[Lys1, Gly6]B and Suc-cK were prepared by acylating the appropriate cyclopeptides with succinic anhydride. c[Glu10]K and cKG were obtained by the classic peptide synthesis, the cyclization being carried out with 61 and 42% yields, respectively. The protecting groups were then eliminated by catalytic hydrogenation. c[Glu10]K and cKG exerted myotropic action on isolated rat uterus (alpha 0.73 and 0.89, pD2 6.61 and 8.61, respectively). cKG displayed direct myotropic activity with respect to electrically stimulated rat vas deferens and guinea-pig ileum, potentiating the contractions (by 100%) in response to electric stimuli. c[Glu10]K and cKG elicit histamine release in isolated rat mast cells (EC30 4.91.10(-5) and 1.47.10(-6) M, respectively). Both cyclopeptides alter arterial pressure following intravenous administration to anaesthetized rats, cats and dogs and affect heart rate. In all assays cKG is more active than c[Glu10]K. Suc-c[Lys1, Gly6]B and Suc-cK do not possess myotropic, histamine-releasing or hypotensive activity, though they were found to elicit a transient increase of bloodflow in cats and dogs.

The histamine-releasing activity of some linear and cyclic analogues of bradykinin (BK) and kallidin (K) was studied on rat peritoneal mast cells and compared with that of angiotensin (AT) cycloanalogues assayed earlier. Peptide cyclization, irrespective of the main pharmacological effect of the linear precursors (hypotensive for BK and K, hypertensive for AT), considerably enhanced their histamine-releasing activity. The activity of the tested compounds was found to depend on their amphiphilicity and cycle size. Linear AT and BK and their cycloanalogues bind to different receptor structures on rat mast cells. These findings suggest that BK, K and AT cycloanalogues belong to the same group of nonimmunological mast cell activators whose specific mechanism of action is based on the common structural features resulting from cyclization.

Linear and cyclic analogues of the specific active center of the ACTH molecule have been synthesized, viz. [Lis5]ACTH-(5-10)-, [Lys5, cyclo (Gly10----epsilon Lys5)]-ACTH-(5-10)-hexapeptides, [Lys5 (Gly)]ACTH-(5-10)- and [Lys5, Gly11, cyclo (Gly11----epsilon Lys5)]-ACTH-(5-11)-heptapeptides. The cyclic structures are fixed by covalent bond between the COOH-group of the C-terminal glycine and epsilon-amino group of a lysine residue. Azide method, DCC/HOBT or pentafluorophenyl esters are used for fragment coupling, while cyclization is achieved by means of diphenylphosphoryl azide or pentafluorophenyl esters. Cyclic compounds are 2-3 orders of magnitude more active than their linear counterparts as revealed by assaying their melanocyte-stimulating activity in vitro on frog skin. Only the title heptapeptide possesses a steroidogenic activity similar to that of ACTH-(5-10)-hexapeptide. The results obtained are in accord with the idea implying the formation in the hormone-receptor complexes of quasi-cyclic structures in the region of the specific active center of ACTH molecule.

The synthesis and biological properties of 10 angiotensin cyclic analogues are described. These compounds exhibit prolong depressor effects and act as potent histamine-releasing agents.

Possible hormonal activity of ACTH4-7, a long-acting ACTH4-10 analog (ProGly Pro) was studied. Unlike ACTH5-10 the peptide did not reveal steroidogenic and melanocyte-stimulating activity.

A cyclic analogue and the corresponding linear segment of the corticotropin molecule, namely ACTH-(5-14)- and [cyclo (Glu gamma-epsilon Lys)]ACTH-(5-14)-decapeptide, both including the specific and unspecific active centers of the ACTH molecule, have been synthesized and studied. The cyclic structure is fixed by amide bond between the glutamic acid and lysine side chains. Condensation of fragments has been realized by azide or DCC/HOBT methods. Cyclization has been achieved using diphenylphosphorylazide. The cyclic analogue has full steroidogenic activity, while its melanotropic activity is 3 orders of magnitude higher than that of the linear decapeptide.

A comparative study of structural and functional organization of the polypeptides -- ACTH and wasp kinin was made. The effects of fragments Lys 17, 18-ACTH11(-18)-NH2--(I) and WK4(-12)--(II), possessing "common" fragments and a cluster of basic amino-acids, on the lipolytic and steroidogenic effects of ACTH and myotropic effects of bradykinin were studied. Both fragments I and II potentiate ACTH-induced lipolysis and steroidogenesis in isolated rat fat and adrenal cells but suppress the myotropic effect of bradykinin on guinea pig ileum. The similarity of biological effects of ACTH and WK fragments support our supposition on the similarity in structurally functional organization of these peptides.

The steroidogenic and lipolytic activities of ACTH fragments (ACTH11-24--I, ACTH11-19--II, ACTH11-16--III and ACTH 17-24--IV) were studied. Fragments I--IV exert a steroidogenic effect in isolated fasciculata rat adrenal cells at concentrations of 1--500 micrograms/ml. The inner activity (alpha) and concentration at which a half-maximum effect is achieved (EC50) for fragments I and IV are 0.64+/-0.09 and 0.5--2.0 micrograms/ml, for fragment III--0.49+/-0.07 and 0.7 microgram/ml, respectively. Fragments I--IV have no effect on the lipolysis in isolated rat fat cells. The results obtained are indicative of the functional importance of fragment ACTH11-24 in manifestation of steroidogenic action of ACTH and suggest that the second active site of ACTH is enclosed within this amino acid sequence.

The binding of some modified angiotensin (AT) analogs and fragments to isolated rat adrenal glomerular cells was studied by radioreceptor analysis with a view of clarifying the role of C- and N-terminal amino acids in the binding of AT molecules to cell receptors. It was demonstrated that Arg2 and Val3 residues are of great importance for effective binding of the AT molecule to cell receptors. The presence of a free C-terminal carboxylic group in position 8 in the vicinity of the bulky lipophilic residue is a necessary condition for this process. The Asp and Asn residues located in position 1 of the AT molecule are not essential for the binding of the hormone molecule to adrenal cell receptors.

The influence of ACTH fragments, possessing structural elements, common for certain groups of peptide hormones and kinins--"common" fragments and cluster of basic amino-acids--(Lys 17,18-ACTH 11-18-NH2--I; ACTH 11-13-NH2--II; NH2CO-ACTH18-20-NH2--III) on lipolytic effect of ACTH in rat isolated fat cells and on the steroidogenic effect of ACTH in isolated rat adrenal cells was studied. Fragment I exerts a steroidogenic effect (alpha=0,84) at concentrations of 1--100 microng/ml. At low concentrations (10(-8)--10(-3) microng/ml) fragment I potentiates ACTH-induced steroidogenesis. Fragment I has no effect on the lipolysis;however, it potentiates ACTH-induced lipolysis at concentrations of 10--100 microng/ml. The results obtained support our previous supposition that "common" fragments are essential secondary non-specific active sites of hormones.

Modified corticotropin fragment - [Lys11 (Gly)]ACTH-(5-14)- and its cyclic analogue - [cyclo (Glu gamma----epsilon Lys (Gly)] ACTH-(5-14)-undecapeptides have been synthesized by classical approach. The cyclic structure has been fixed by amide bond between gamma-COOH group of glutamic acid and alpha-NH2 group of glycine coupled to the epsilon-NH2 group of lysine. Fragment condensation has been achieved by azide or dicyclohexylcarbodiimide methods. Cyclization has been performed using diphenylphosphorylazide. The melanotropic activity of the cyclicanalogue on isolated frog skin exceeds by two orders of magnitude that of the linear undecapeptide, however the steroidogenic activity in isolated cells of rat adrenal cortex is diminished by an order of magnitude as compared with that of the linear precursor. A similarity of the CD spectra for the cyclic ACTH peptides and their linear counterparts in water and trifluoroethanol points to the similarity and relative rigidity of their structures.

Linear and cyclic analogues of angiotensin were studied to clarify the structural properties of peptides possessing a histamine-releasing action. It was shown that an increase in the angiotensin basicity or its cyclization leads to the appearance of the histamine-releasing activity which is not characteristic of the natural hormone. This increase in the basicity of the angiotensin cyclic analogs results in highly active compounds with the EC50 exceeding by 2 to 3 orders of magnitude that of polymyxin B or substance 48/80. The data obtained confirm the hypothesis postulating a high degree of amphiphilicity for histamine-releasing peptides. As a result of cyclization of angiotensin analogues, a block of positively charged amino acids with an oppositely located hydrophobic region is formed. This finding can be of importance for the effective interaction of peptides with cellular structures as well as for the stimulation of secretory processes.

The influence of fragment ACTH 11-14 analogues with amino acid sequences H-Lys-Pro-Val-Gly-OH (fragment I) and H-Lys-Pro-Val-Gly-NH2 (fragment II), possessing structural elements, similar for certain groups of peptide hormones and kinins, on lipolytic effect of ACTH in adipose tissue and isolated epydidymal fat cells of rat, was studied. Both fragments have no effect on the lipolysis; they potentiate the ACTH-induced lipolysis 1,5--2,0 fold, but do not alter the maximal effect at concentrations 0,1--1,0 mkg/ml in tissue and fragment I--at concentrations from 0,01 to 0,1 mkg/ml in isolated fat cell system. The role of "common" fragments in hormone-receptor interactions as well as mechanism of their potentiating effect is discussed. It is assumed that the "common" fragment of ACTH--ACTH11-14--is a second, non-specific active site of hormone directly involved in secondary signal formation.

Biological properties of five novel angiotensin analogues synthesized, using the conventional methods of peptide chemistry, have been studied. Cyclization was attained by means of amide linkage with the aid of diphenylphosphorylazide or pentafluorophenyl esters. Unlike the natural hormone, the cyclic analogues of angiotensin show no pressor activity, but elicit a depressor effect untypical of angiotensin. A slight pressor activity was exhibited by the compound containing aspartic acid in position 1. The cyclic analogues in question release histamine from peritoneal mast cells in rats.

Four cyclic derivatives of des-Arg9[Leu8]bradykinin have been obtained by classical methods of peptide chemistry. They are cyclo-(-X-Arg-Pro-Pro-Gly-Phe-Gly-Pro-Leu-), where X=Lys or none, and cyclo-(Y-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu-), where Y= Lys or Orn. Peptide bonds have been formed by the pentafluorophenylester method, and cyclization has been carried out in a diluted dioxane solution with 40% yield. Subsequent cleavage of protecting groups was made by treatment with hydrogen fluoride. The products obtained were purified by droplet counter-current chromatography. These substances liberate histamine from the rat mast cells comparably to bradykinin and fail to produce myotripic and vascular effects.