Synthesis of somatostatin mimetics based on the 1-deoxymannojirimycin scaffold

A novel synthesis of somatostatin mimetics based on the 1-deoxymannojirimycin (DMJ) scaffold has been developed. This involved development of a route suitable for the strategic grafting of pharmacophoric tryptophan and lysine side chains to the nitrogen atom of the piperidine ring and to the primary hydroxyl group of DMJ, respectively. The novel peptidomimetics were found to bind with higher affinity to sst4 receptors than to sst5 receptors.     

Synthesis and binding affinities of novel SRIF-mimicking beta-D-glucosides satisfying the requirement for a pi-cloud at C1

[reaction: see text] The synthesis of four bioactive analogues of the somatostatin (SRIF-14) mimetic, beta-d-glucoside (+)-2, in which the C1 indole side chain is replaced with indole surrogates, has been achieved. These congeners, possessing the naphthyl, benzothiophene, benzyl, and benzofuran substituents, were predicted to satisfy the electrostatic requirements of the tryptophan binding pocket of SRIF. Unlike the previously described C4 picolyl and pyrazinyl congeners, these ligands bind the hSST4 receptor.     

Synthesis of macrolide-saccharide hybrids by ring-closing metathesis of precursors derived from glycitols and benzoic acids

The benzomacrolactone structural motif is a privileged or evolutionarily selected scaffold that codes properties required for binding to proteins and novel analogues thereof may provide a source of new bioactive compounds. Saccharides are also privileged structures, with (amino)sugars, iminosugars, and sugar amino acids being applied as scaffolds for the development of nonpeptidal peptidomimetics. The syntheses of novel polyhydroxylated oxamacrolides, structural analogues of natural polyketide derived macrolides, are described herein, providing a basis for their development as scaffolds. The syntheses were carried out from benzoic acids and appropriately protected D-mannitol or D-sorbitol (D-glucitol). Ring-closing metathesis was applied in the macrocyclization step with high E-alkene selectivities being observed. X-ray crystal structures, for two polyhydroxylated derivatives, show that the macrocyclic rings display similar conformations. In addition, intermolecular hydrogen-bonding networks are observed in the lattices.     

Carbohydrate-Based Peptido Mimetics. Synthesis of Two New Scaffolds for Combinatorial Libraries.

The recent utilisation of the glucopyranose ring as scaffold for the synthesis of a potent somatostatin agonist demonstrated the use of monosaccharides as viable templates in drug design.^2,3 Monosaccharide-based mimics provide enantiomerically pure, rigid moieties (able to give precise orientation of functional groups), with a high degree of oxygenation to assure water solubility.⁴ Moreover, carbohydrates exhibit a high combinatorial density. These advantages prompted us to synthesise new monosaccharide derivatives as carbohydrate scaffolds for potential drug design.     

Catechol: A minimal scaffold for non-peptide peptidomimetics of the i + 1 and i + 2 positions of the beta-turn of somatostatin

The design, synthesis, and evaluation of a series of catechol-based non-peptide peptidomimetics of the peptide hormone somatostatin have been achieved. These ligands comprise the simplest known non-peptide mimetics of the i + 1 and i + 2 positions of the somatostatin beta-turn. Incorporation of an additional side chain to include the i position of the beta-turn induces a selective 9-fold affinity enhancement at the sst2 receptor.     

Synthesis of a Type-VIβ-Turn Peptide Mimetic and Its Incorporation into a High-Affinity Somatostatin Receptor Ligand

The synthesis of a cis-Phe-Pro dipeptide mimetic is described, which adopts a type-VIβ-turn conformation. In this mimetic, the α-positions of Phe and Pro are joined by a CH₂CH₂ bridge, thereby forming a fused bicyclic system, and fixing a geometry similar to that seen in cis-Phe-Pro units in protein crystal structures. The dipeptide mimetic 20 was synthesized in optically pure form starting from (R)-α-allylproline ( 6 ; Schemes 1, 3, and 4), with a free carboxylic acid and an Fmoc-protected N-terminus, thereby allowing its incorporation into linear and cyclic peptides using standard solid-phase methods. The mimetic 20 was incorporated into the cyclic somatostatin analogue cyclo(-Phe = Pro-Phe-D -Trp-Lys-Thr-), where Phe = Pro represents the mimetic. This analogue shows a high affinity (pIC₅₀ 8.6) for somatostatin receptors on rat-brain cortex membranes. Based on NMR studies in aqueous solution, likely low-energy conformations for this analogue were deduced using restrained dynamic simulated annealing. The conformations found, which include a distorted type-II′ turn at D -Trp-Lys, are similar to low-energy conformations deduced elsewhere for cyclo(-Phe-Pro-Phe-D -Trp-Lys-Thr-), as well as to those seen in crystal structures of the somatostatin analogue octreotide.     

Synthesis of trifunctional somatostatin based derivatives for improved cellular and subcellular uptake

It is now well established that the biological effects of Auger-emitting radionuclides are critically dependent on their subcellular location. Therefore, for their use in molecular imaging and targeted radionuclide therapy, attempts should be made to increase the nuclear specificity of the carriers. In the present paper the synthesis of novel trifunctional somatostatin derivatives containing a nuclear localization motif is described. These derivatives of [DOTA⁰, Tyr³]-octreotide (DOTATOC, DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) were obtained in high yields using Fmoc peptide synthesis in solid and in solution phase.     

A structure-based approach to understanding somatostatin receptor-4 agonism (sst4)

It has been reported that somatostatin receptor subtypes 4 and 5 would be high-impact templates for homology modeling if their 3D structures became available. We have generated a homology model of the somatostatin receptor subtype 4 (sst4), using the newest active state β(2) adrenoreceptor crystal structure, and subsequently docked a variety of agonists into the model-built receptor to elucidate the binding modes of reported agonists. Using experimental restraints, we were able to explain observed activity profiles. We propose two binding modes that can consistently explain findings for high-affinity agonists and reason why certain structures display low affinities for the receptor.     

Hypothalamus as a possible modulator of the rates of development and aging of mammals

The review analyzes the information concerning the possibility to delay aging by slowing down development. It is supposed that the rates of development and aging of mammals are modulated by the circadian rhythm generated by the suprachiasmatic nuclei of the hypothalamus. Reversible arrest of growth in certain mammal species can be triggered by enhanced somatostatin synthesis with an inhibition of growth hormone, thyroid-stimulating hormone, and melatonin and with a loss of circadian function in the suprachiasmatic nucleus of the hypothalamus.     

Decarboxylative Peptide Macrocyclization through Photoredox Catalysis

A method for the decarboxylative macrocyclization of peptides bearing N‐terminal Michael acceptors has been developed. This synthetic method enables the efficient synthesis of cyclic peptides containing γ‐amino acids and is tolerant of functionalities present in both natural and non‐proteinogenic amino acids. Linear precursors ranging from 3 to 15 amino acids cyclize effectively under this photoredox method. To demonstrate the preparative utility of this method in the context of bioactive molecules, we synthesized COR‐005, a somatostatin analogue that is currently in clinical trials.     

Design, synthesis, and binding affinities of pyrrolinone-based somatostatin mimetics

[structure: see text] Tetrapyrrolinone somatostatin (SRIF) mimetics (cf. 1), based on a heterochiral (D,L-mixed) pyrrolinone scaffold, were designed, synthesized, and evaluated for biological activity. The iterative synthetic sequence, incorporating the requisite functionalized coded and noncoded amino acid side chains, comprised a longest linear synthetic sequence of 23 steps. Binding affinities at two somatostatin receptor subtypes (hsst 4 and 5) reveal micromolar activity, demonstrating that the d,l-mixed pyrrolinone scaffold can be employed to generate functional mimetics of peptide beta-turns.     

Kinetics and Equilibria of the Thiol/Disulfide Exchange Reactions of Somatostatin with Glutathione

Rate and equilibrium constants are reported for the thiol/disulfide exchange reactions of the peptide hormone somatostatin with glutathione (GSH). GSH reacts with the disulfide bond of somatostatin to form somatostatin-glutathione mixed disulfides (Cys(3)-SH, Cys(14)-SSG and Cys(3)-SSG, Cys(14)-SH), each of which can react with another molecule of GSH to give the reduced dithiol form of somatostatin and GSSG. The mixed disulfides also can undergo intramolecular thiol/disulfide exchange reactions to re-form the disulfide bond of somatostatin or to interconvert to the other mixed disulfide. Analysis of the forward and reverse rate constants indicates that, at physiological concentrations of GSH, the intramolecular thiol/disulfide exchange reactions that re-form the disulfide bond of somatostatin are much faster than reaction of the mixed disulfides with another molecule of GSH, even though the intramolecular reaction involves closure of a 38-membered ring. Thus, even though the disulfide bond of somatostatin is readily cleaved by thiol/disulfide exchange, it is rapidly reformed by intramolecular thiol/disulfide exchange reactions of the somatostatin-glutathione mixed disulfides. By comparison with rate constants reported for analogous reactions of model peptides measured under random coil conditions, it is concluded that disulfide bond formation by intramolecular thiol/disulfide exchange in the somatostatin-glutathione mixed disulfides is not completely random, but rather it is directed to some extent by conformational properties of the mixed disulfides that place the thiol and mixed disulfide groups in close proximity. A reduction potential of -0.221 V was calculated for the disulfide bond of somatostatin from the thiol/disulfide exchange equilibrium constant.     

A combinatorial approach toward the discovery of non-peptide, subtype-selective somatostatin receptor ligands

The tetradecapeptide somatostatin is widely distributed throughout the body and is thought to be involved with a variety of regulatory functions. Recently, five human somatostatin receptors (hSSTR1-5) have been cloned and characterized. Several selective peptidal agonists of the hSSTR receptors are known, and we sought to apply this information to the design of novel non-peptide small molecule ligands for each receptor. Initial computational methods identified a 200 nM murine SSTR2 active compound via a database search of our sample collection. A combinatorial library was designed around the structural class of the compound with the goal of rapidly developing this initial lead into the desired subtype-selective small molecules in order to characterize the pharmacology of each of the receptor subtypes. The library was synthesized using the resin-archive, iterative deconvolution format. The total number of unique compounds in the library was expected to be 131,670, present in 79 mixtures of 1330 or 2660 compounds per mixture. Through sequences of screening and mixture deconvolution, the components of selective and highly active (Ki = 50 pM to 200 nM) non-peptide small molecule ligands for somatostatin subtypes 1, 2, 4, and 5 were identified. In addition to discovering compounds with the desired activity and selectivity, useful structure/activity information was generated which can be used in the design of new compounds and second-generation combinatorial libraries.     

Direct inhibition of pepsinogen secretion from rat gastric chief cells by somatostatin

Effects of somatostatin on pepsinogen secretion was investigated in the rat in vivo and in vitro. In the perfused rat stomach, somatostatin inhibited secretagogue-induced acid secretion in dose-dependent manner. However, effects of somatostatin on secretagogue-induced pepsinogen secretion were obscure. To clarify the effects of somatostatin on the chief cells, gastric mucosal cells were isolated by a proteolytic enzyme. Somatostatin inhibited carbachol- and cholecystokinin octapeptide-induced pepsinogen secretion from dispersed gastric mucosal cells in a dose-dependent manner. Histamine-induced pepsinogen secretion, which was recovered by culturing, was also inhibited by somatostatin. These results suggest that somatostatin inhibits secretagogue-induced pepsinogen secretion directly.     

Synthesis of Peptide Hormones using Recombinant DNA Techniques

Recombination of genetic material enables the creation of new bacterial strains which can synthesize specific proteins in large amounts. Such bacteria permit the production of previously inaccessible proteins. They can therefore be used as starting materials for the production of drugs which will open up new paths for therapy. Several proteins produced by bacteria after DNA recombination are presently undergoing clinical trials while others are already being produced on a large scale. Thus, in the area of recombinant DNA techniques the transition from the research laboratory to industrial exploitation has occurred much faster than was anticipated several years ago. The methods, possibilities and problems encountered in the synthesis of peptide hormones by bacteria after DNA recombination are outlined, using insulin, somatostatin, and growth hormone as examples. Great emphasis is placed on the molecular biological aspects of this approach.     

Bioactive unnatural somatostatin analogues through bioorthogonal iodo- and ethynyl-disulfide intercalators

Iodo- and ethynyl-containing bisalkylating bioconjugation agents 5 and 8 were achieved and allow the introduction of reactive unnatural substituents into proteins and peptides whilst the bioactive 3D structure is retained. Derivatives of the peptide hormone somatostatin bearing a single iodo or ethynyl group were prepared through intercalation into the disulfide bridge. For the first time, the exact reaction mechanism of the intercalation was elucidated by applying 2D NMR experiments and it was shown that, during the reaction, somatostatin diastereomers were formed. Site-directed modification of the ethynyl-modified peptide with a coumarin chromophore was achieved through a [1,3] dipolar Huisgen cycloaddition reaction; this suggests that such a derivative could serve as an attractive platform to prepare artificial somatostatin compound libraries. The biological activity and specificity of a representative modified somatostatin derivative was demonstrated and efficient receptor-mediated cell uptake occurred in a dose-dependent manner into receptor positive cells only. The iodo and ethynyl bioconjugation reagents presented herein could be applied for introducing such substituents into alternative peptides and proteins and, in principle, could facilitate the efficient design of a broad variety of artificial protein and peptide analogues with previously unknown bioactivities.     

侧脑室注射氯化镧对大鼠血清生长素和甲状腺素的影响

西方研究了侧脑室注入ＬａＣｌ３对大鼠血清中生长系,甲状腺素,促甲状腺素和下丘脑中生长抑素的影响。侧脑室注射０．００１和０．０１ｍｏｌ．ｌ＾－１ＬａＣｌ３,血清中Ｔ４和ＧＨ含量明显高于对照组,０．１和０．５ｍｏｌ．ｌ＾－１ＬａＣｌ３组血清Ｔ４和ＧＨ未见明显变化。     

Formulation and radiochemical evaluation of a freeze-dried mixed peptide kit for the preparation of 68Ga-labeled peptides for PET imaging of somatostatin receptor positive neuroendocrine cancers

⁶⁸Ga-labeled DOTA-coupled somatostatin analogue peptides are regularly being used for the PET imaging of patients suspected to be suffering from various types of neuroendocrine cancers over-expressing somatostatin receptors. The article describes the formulation of a freeze-dried mixed peptide kit containing equal amounts of DOTA-TATE and DOTA-NOC and the radiochemical evaluation of the kit for the easy and convenient preparation of ⁶⁸Ga-labeled mixed radiopeptides. The simultaneous use of two different peptides in the kit is expected to perceive more cancerous lesions and may have a wider applicability in the diagnosis of neuroendocrine cancers.     

Synthesis of novel 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA) derivatives for chemoselective attachment to unprotected polyfunctionalized compounds

A convenient synthesis of novel bifunctional poly(amino carboxylate) chelating agents allowing chemoselective attachment to highly functionalized biomolecules is described. Based on the well known chelator 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA), we synthesized novel bifunctional chelating agents bearing additional functional groups by alkylating 1,4,7,10-tetraazacyclododecane (cyclen) with one equivalent of para-functionalized alkyl 2-bromophenyl-acetate and three equivalents of tert-butyl 2-bromoacetate. The resulting compounds, which contain an additional carbonyl or alkyne functionality, allow site specific labeling of appropriately functionalized unprotected biomolecules in a rapid manner via click reactions. This was demonstrated by the attachment of our new DOTA derivatives to the somatostatin analogue Tyr3-octreotate by chemoselective oxime ligation and CuI-catalyzed azide-alkyne cycloaddition. Initial biodistribution studies in mice with the radiometalated compound demonstrated the applicability of the described DOTA conjugation.     

High-Performance Liquid Chromatography of the Tetradecapeptide Somatostatin

Abstract

Optimum reversed-phase systems for qualitative and quantitative determinations of somatostatin are evaluated. Temperature, pH, buffer concentration, type and concentration of organic modifier and the presence of ion-pairing agents more or less influence the retention and the separating efficiency. The retention behaviour of some analogues is described to illustrate the selectivity of the system. Down to 10-20 ng of somatostatin can be determined, even in the presence of a large excess of albumin, by using 210 nm as the detection wavelength.