Synthesis and biological evaluation of B-ring analogues of (-)-rhazinilam

Three macrocyclic analogues of rhazinilam 1 having a 11- or 12-membered B-ring with an endocyclic carbamate group or an amino-acid residue were synthesized from the natural product. These analogues 3 and 4 displayed a very low activity on tubulin. Thirty N-1 and C-16 substituted analogues of rhazinilam were also synthesized regioselectively from rhazinilam. Stereochemical analyses showed that N-1 and C-16alpha analogues have the same conformation as rhazinilam, whereas C-16beta analogues adopt a different conformation for rings B and D. All N-1 and C-16 analogues were less active than rhazinilam on tubulin, though analogues 5a, 6aalpha, 6balpha, and 6f having the less bulky substituents retained close affinities. A few analogues either active (like 6f) or inactive (like 5o) on tubulin showed significant inhibition of the growth of KB cancer cells.     

Direct photoaffinity labeling by dolastatin 10 of the amino-terminal peptide of beta-tubulin containing cysteine 12

Tubulin with bound [5-3H]dolastatin 10 was exposed to ultraviolet light, and 8-10% of the bound drug cross-linked to the protein, most of it specifically. The primary cross-link was to the peptide spanning amino acid residues 2-31 of beta-tubulin, but the specific amino acid could not be identified. Indirect studies indicated that cross-link formation occurred between cysteine 12 and the thiazole moiety of dolastatin 10. An equipotent analog of dolastatin 10, lacking the thiazole ring, did not form an ultraviolet light-induced cross-link to beta-tubulin. Preillumination of tubulin with ultraviolet light, known to induce cross-link formation between cysteine 12 and exchangeable site nucleotide, inhibited the binding of [5-3H]dolastatin 10 and cross-link formation more potently than it inhibited the binding of colchicine or vinblastine to tubulin. Conversely, binding of dolastatin 10 to tubulin inhibited formation of the cross-link between cysteine 12 and the exchangeable site nucleotide. Dithiothreitol inhibited formation of the beta-tubulin/dolastatin 10 cross-link but not the beta-tubulin/exchangeable site nucleotide cross-link. Modeling studies revealed a highly favored binding site for dolastatin 10 at the + end of beta-tubulin in proximity to the exchangeable site GDP. Computational docking of an energy-minimized dolastatin 10 conformation at this site placed the thiazole ring of dolastatin 10 8-9 A from the sulfur atom of cysteine 12. Dolastatin 15 and cryptophycin 1 could also be docked into positions that overlapped more extensively with the docked dolastatin 10 than with each other. This result was consistent with the observed binding properties of these peptides.     

Peptide conformations--49(1): synthesis and structure-activity relationships of side chain modified peptides of cyclo(-D-Pro-Phe-Thr-Lys-Trp-Phe.)

Cyclic hexapeptide analogues representing the modified retro sequence of the amino acid residues 7-11 of natural somatostatin are known to protect liver cells from phalloidin poisoning. To determine the influence of steric, lipophilic, and charge effects on (a) the conformation of the backbone and the aromatic side chains and (b) the biological response, the side chains of Phe2, Lys4, and Phe6 of cyclo(-D-Pro1-Phe2-Thr3-Lys(Z)4-Trp5-Phe6-), 1a, one of the most active peptides found so far, were modified by various residues. The discussion of conformationally relevant parameters proves that neither backbone conformations nor populations of aromatic side chain rotamers were altered by these substitutions. The potency of these derivatives in a cytoprotection assay varies by at most one order of magnitude (more or less active than the parent peptide 1a). A qualitative evaluation of lipophilic, steric, and charge effects reveals the dominance of lipophilic effects of aromatic residues; the most potent compounds contain aromatic substructures in the side chain of Lys4.     

Probing peptide backbone function in bombesin. A reduced peptide bond analogue with potent and specific receptor antagonist activity

Each peptide bond CONH group in the most important COOH-terminal octapeptide region of [Leu14]bombesin was replaced by a CH2NH group using recently developed rapid solid-phase methods. The resulting analogues were then examined for amylase releasing activity in guinea pig pancreatic acini and for their ability to inhibit binding of [125I-Tyr4]bombesin to acinar cells. Replacement of the Trp8-Ala9, Gly11-His12, and His12-Leu13 peptide bonds resulted in about 1000-, 200-, and 300-fold losses in both amylase releasing activity and binding affinity. The Val10-Gly11 replacement, however, retained 30% potency relative to the parent peptide. Ala9-Val10 and Leu13-Leu14 bond replacement analogues exhibited no detectable amylase releasing activity but were still able to bind to acini with Kd values of 1060 and 60 nM, respectively (compared to 15 nM for [Leu14]bombesin itself). Subsequently, both analogues were demonstrated to be competitive inhibitors of bombesin-stimulated amylase release with IC50 values of 937 and 35 nM, respectively. [Leu14-psi-CH2NH-Leu13]Bombesin exhibits a 100-fold improvement in binding affinity compared to previously reported bombesin receptor antagonists and showed no affinity for substance P receptors. It was also a potent inhibitor of bombesin-stimulated growth of murine Swiss 3T3 cells with an IC50 of 18 nM. In terms of a bombesin receptor-binding conformation, these results may aid in the delineation of intramolecular hydrogen-bonding points and the eventual design of improved, conformationally restricted analogues.     

A comparison of the effects of substance P and shorter analogues on the synaptosomal ATPases activities in the rat brain

The influence in vitro of SP and C-terminal fragments of analogues SP(5-11) (pyroGlu5, Tyr8); SP(6-11) (pyroGlu6, Tyr8); SP(6-11) (pyroGlu6, D-Phe7); SP(6-11) (pyroGlu6, D-Phe8) on the (Ca, Mg) and (Na, K) ATPases activities from synaptosomal membranes of cerebral cortex and hippocampus of rat brain were compared. The data obtained in this study indicate the following: 1. Substance P stimulates the activities of (Na, K) and (Ca, Mg) ATPases more effectively in synaptosomal membranes from hippocampus than cerebral cortex. 2. Heptapeptide SP(5-11) (pyroGlu5, Tyr8) causes a more distinct increase of (Ca, Mg) ATPase activity in cortical synaptosomal membranes than SP does. 3. The change of L-Phe conformation to D in position 7 in hexapeptide induces reduction of enzymes activities in hippocampus. 4. Especially important for the maintenance of biological activity of drugs is the replacement of Gln5 with pyroGlu6 and conformation of Phe residues. 5. SP and shorter analogues of fragments SP C-terminal SP regulate the active cation transport in synaptosomal membranes of cerebral cortex and hippocampus.     

Structural motifs of pituitary adenylate cyclase-activating polypeptide (PACAP) defining PAC1-receptor selectivity

Pituitary adenylate cyclase-activating polypeptide (PACAP) interacts with three types of PACAP/VIP-receptors. The PAC1-receptor accepts PACAP as a high affinity ligand but not vasoactive intestinal peptide (VIP) similarly binding to VPAC1- and VPAC2-receptors. To identify those amino acids not present in VIP defining PAC1-receptor selectivity of PACAP, radio receptor binding assays on AR4-2J cells were performed. It could be shown that PACAP(1-27) exhibited a distinct and much higher susceptibility to VIP-amino acid substitutions, compared to PACAP(1-38). Positions 4 and 5 seem to be most important for receptor binding of PACAP(1-27), whereas position 13 was identified to be crucial for maximal affinity of PACAP(1-38). PACAP(29-38) extension analogues of VIP revealed a stabilizing effect of the C-terminus of PACAP(1-38) on the optimal peptide conformation. The substitution analogues were also checked for their capacity to stimulate IP3 and cAMP formation in AR4-2J cells. Compared to PACAP(1-27) and PACAP(1-38), most analogues revealed potencies reduced congruously to their lower binding affinities. However, one of the analogues, PACAP(1-27) substituted in position 5, may represent a weak antagonist since this peptide was less potent in inducing second messengers than in label displacement. Our findings indicate that PACAP(1-27) and PACAP(1-38) differ in terms of their requirement of the amino acids in positions 4, 5, 9, 11 and 13 for maximal interaction with the PAC1-receptor.     

The synthesis and tubulin binding activity of thiophene-based analogues of combretastatin A-4

A number of analogues of combretastatin A-4 (1), containing a thiophene ring interposed between the two phenyl groups, have been prepared. The synthesis of these compounds employed a combination of palladium-mediated coupling and iodocyclization techniques. The thiophene compounds 11, 14, 18, and 19 also represent non-benzofused analogues of some recently described tubulin binding benzo[b]thiophenes 3-5. The most active thiophene compounds identified in this study were 11, 14, and 18. Overall they are less active than 1 but exhibit comparable activity to the most active of the benzo[b]thiophenes 3-5. A structure-activity relationship of these compounds is considered.     

Rat liver iodothyronine monodeiodinase. Evaluation of the iodothyronine ligand-binding site

Ligand binding characteristics of rat liver microsomal type I iodothyronine deiodinase were evaluated by measuring dose-response inhibition and apparent Michaelis-Menten or inhibitor constants of iodothyronine analogues to compete as substrates or inhibitors for the natural substrate L-thyroxine. These data show strong correlations with the binding requirements of hormone analogues to serum thyroxine-binding prealbumin since iodothyronine analogues with a negatively charged side chain, a negative charge or hydrogen bonding function in the 4'-position, tetraiodo ring substitution, and a skewed hormone conformation are structural features shared in common which markedly affect enzyme activity and protein binding affinity. 3,3',5'-Triiodo-L-thyronine is the most potent natural substrate (IC50 = 0.3 microM) and tetraiodothyroacetic acid is the most potent inhibitor (IC50 = 0.2 microM). Both thyroxine (T4)-5'- and T4-5-deiodination pathways are inhibited by these potent analogues, providing further evidence for a single enzyme catalyzing the rat liver microsomal deiodination reactions. These data also show that L-hormone analogues are preferentially deiodinated via the T4-5'-deiodination pathway, whereas D-analogues produce products via the T4-5-deiodination pathway. The thyroxine-binding prealbumin complex was used to model the interaction of thyroid hormones with the deiodinase active site. Computer graphic modeling of the prealbumin complex showed that only those analogues which are potent deiodinase inhibitors or substrates can be accommodated in the hormone binding site. This model suggests the design of functionally specific ligands which can modulate peripheral thyroid hormone metabolism and act as antithyroidal drugs.     

Enantiomers of cis- and trans-3-(4-propyl-cyclopent-2-enyl) propyl acetate. A study on the bioactive conformation and chiral recognition of a moth sex pheromone component

The enantiomers of cis- and trans-3-(4-propyl-cyclopent-2-enyl) propyl acetate, which are conformationally constrained analogues of (Z)-5-decenyl acetate (1), a sex pheromone component of the turnip moth, Agrotis segetum, have been synthesized and tested using the electrophysiological single-sensillum technique. The analogues mimic a cisoid and transoid conformation of 1, respectively. In addition, the enantiomers of each of the cis- and trans-isomers are conformationally constrained analogues of enantiomeric cisoid and transoid conformations of 1. Thus, the compounds prepared and tested are well suited to investigate the nature of the bioactive conformation of the natural pheromone component 1 and the chiral sense of its interaction with the receptor. Electrophysiological single-sensillum recordings show that the activity of the most active cis-isomer, which has a (1S,4R)-configuration, is more than two orders of magnitude higher than that of the most active trans-isomer. Furthermore, the (1S,4R)-isomer is at least 100 times more active than its enantiomer. These results strongly support a previously proposed cisoid bioactive conformation of 1. Furthermore, the (1S,4R)-configuration of most active stereoisomer identifies the chiral sense of the interaction between the natural pheromone component 1 and its receptor.     

Conformation of the polar headgroup of sphingomyelin and its analogues

The conformation of the polar headgroup of synthetic D-erythro-stearoylsphingomyelin (1), its L-threo-isomer (2) and phosphorothioyl analogues of 1 (3 and 4) has been studied in detail by high-resolution NMR spectroscopy. In both monomeric and aggregated states the phosphocholine function of 1 adopts the synclinal conformation (alpha 5 torsional angle), in analogy with phosphatidylcholine (Hauser, H., Guyer, W., Pascher, I., Skrabal, P. and Sundell, S. (1980) Biochemistry 19, 366-373). The conformation about the C1-C2 bond (theta 1 angle) of the sphingosine backbone is predominantly -synclinal, analogously to the conformation of the crystalline galactosyl cerebroside (Pascher, I. and Sundell, S. (1977) Chem. Phys. Lipids 20, 175-191). In contrast, the L-threo-isomer displays unrestricted rotation about C1-C2 bond. The possibility of the existence of a hydrogen bond between the 3-hydroxyl function and the bridged oxygen atom of sphingosine responsible for the different conformation of 1 and 2 is discussed. The modification of the phosphate function in 1 with sulfur has no significant effect on the conformation of the resulting analogues. The conformation of all studied compounds about the C-O phosphoester bonds (alpha 1 and alpha 4 torsion angles) is mainly antiperiplanar. Similar to other double-chain phospholipids, sphingomyelin shows a preference towards the antiperiplanar conformation about the C2-C3 bond.     

Conformation-activity relations of dermorphin and its pentapeptide analogs

Low-energy peptide backbone structures of dermorphin (DM), amide of its N-terminal pentapeptide (DM 1-5) and DM 1-5 analogues with substitutions of Gly4 for Leu, D-Gln, Aal or Tal were determined by energy calculations. The above analogues were shown to possess different affinities toward opiate receptors of mu-type. The comparison of low-energy backbone structures of DM, DM 1-5 and its analogues resulted in development of the dermorphin "biologically active" conformation being characteristic of its binding with mu-type receptors. The specific binding of dermorphin to this receptor apparently depends on the conformation of the whole N-terminal pentapeptide.     

Two photoaffinity analogues of the tripeptide, hemiasterlin, exclusively label alpha-tubulin

A synthetic analogue of the tripeptide hemiasterlin, designated HTI-286, depolymerizes microtubules, is a poor substrate for P-glycoprotein, and inhibits the growth of paclitaxel-resistant tumors in xenograft models. Two radiolabeled photoaffinity analogues of HTI-286, designated 4-benzoyl-N,beta,beta-trimethyl-l-phenylalanyl-N(1)-[(1S,2E)-3-carboxy-1-isopropylbut-2-enyl]-N(1),3-dimethyl-l-valinamide (probe 1) and N,beta,beta-trimethyl-l-phenylalanyl-4-benzoyl-N-[(1S,2E)-3-carboxy-1-isopropyl-2-butenyl]-N,beta,beta-trimethyl-l-phenylalaninamide (probe 2), were made to help identify HTI-286 binding sites in tubulin. HTI-286, probe 1, and probe 2 had similar affinities for purified tubulin [apparent K(D(app)) = 0.2-1.1 microM], inhibited polymerization of purified tubulin approximately 80%, and were potent inhibitors of cell growth (IC(50) = 1.0-22 nM). Both radiolabeled probes labeled exclusively alpha-tubulin. Labeling by [(3)H]probe 1 was inhibited by probe 1, HTI-286, vinblastine, or dolastatin 10 (another peptide antimitotic agent that depolymerizes microtubules) but was either unaffected or enhanced (at certain temperatures) by colchicine or paclitaxel. [(3)H]Probe 1 also labeled exclusively tubulin in cytosolic extracts of whole cells. The major, if not exclusive, contact site for probe 1 was mapped to residues 314-339 of alpha-tubulin and corresponds to the sheet 8 and helix 10 region. This region is known to (1) have longitudinal interactions with beta-tubulin across the interdimer interface, (2) have lateral interactions with adjacent protofilaments, and (3) contact the N-terminal region of stathmin, a protein that induces depolymerization of tubulin. Binding of probe 1 to this region may alter the conformation of tubulin outside the labeling domain, since enzymatic removal of the C-terminus of only alpha-tubulin by subtilisin after, but not before, photolabeling is blocked by probe 1. These results suggest that hemiasterlin is in close contact with alpha-tubulin and may span the interdimer interface so that it contacts the vinblastine- and dolastatin 10-binding sites believed to be in beta-tubulin. In addition, we speculate that antimitotic peptides mimic the interaction of stathmin with tubulin.     

Structural requirements of sphingosine molecules for inhibition of DNA primase: biochemical and computational analyses

Using 28 chemically well-defined compounds containing D-erythro-sphingosine and its analogues, we analyzed structure-activity relationships for DNA primase inhibition. Biochemical studies demonstrated a positively charged amino group at C2 and a long aliphatic chain to be absolutely required for inhibition. Whereas C2-amino group is intact, sphingosine 1-phosphate was totally inactive. This result could be due to cancellation of positive charge of the amino group by the interaction with negatively charged C1-phosphate, since simulations with the software INSIGHT II showed these two groups to be close enough to interact. The hydroxyl group at C3 and trans-double bond at C4-C5 were also found to be important for the inhibition. Dehydroxylation of C3, as well as saturation or cis-conversion of the trans-double bond led to decrease of inhibitory activity. Despite saturation of the double bond, introduction of a hydroxyl group into C4 of dihydrosphingosine resulted in restoration of inhibition. Conversion of the double bond into a triple bond did not abolish but rather enhanced the inhibitory activity. Among sphingosine stereoisomers, the naturally occurring D-erythro-sphingosine proved to be the strongest inhibitor. To ascertain the contribution of the total conformation to the inhibition, especially of the long aliphatic chain, we constructed a 3D-quantitative structure-activity relationship model using the computer program Catalyst/HipHop on the basis of information described above. Analysis of the hypothesis model for active compounds revealed that the orientation of aliphatic chain, represented by the dihedral angle of C2-3-4-5, correlated well with the inhibition. Modifications such as deletion of the hydroxyl group at C3 or saturation of the C4-C5 double bond caused shifts in the dihedral angle of C2-3-4-5, with concomitant decrease in inhibitory activity.     

A study of the biologically active conformation of the cholecystokinin-4 dipeptide analogue GB-115

The biologically active conformation of N-(6-phenylhexanoyl)glycyl-tryptophan amide (GB-115), a highly active cholecystokinin-4 retro dipeptide analogue with the anxiolytic activity, has been studied using the conformational analysis by ¹H NMR spectroscopy in solution and the method of sterically restricted analogues. A study of the relationship between the preferable conformation in solution and the anxiolytic activity in the series of GB-115 derivatives showed that the biologically active conformation of this compound is the β-turn. Based on the data on the nuclear Overhauser effect ¹H NMR spectroscopy, this structure was identified as the β-turn of type II. Subsequent synthesis and study of the pharmacological activity of novel sterically restricted analogues of dipeptide GB-115: (2S)-2-{(3R)-3-[(6-phenylhexanoyl)amino]-2-oxopyrrolidine-1-yl}-3-(1H-indole-3-yl)propionic acid ethyl ester, N-(6-phenylhexanoyl)glycyl-N ^α-methyltryptophan ethyl ester, (2S)-2-[(10,11-dihydro-5H-dibenzo[b, f]azepin-5-ylcarbonyl)amino]-3-(1H-indole-3-yl)propionic acid methyl ester, and (2S)-2-[({3-[(ethoxycarbonyl)amino]-10,11-dihydro-5H-dibenzo[b, f]azepin-5-yl}carbonyl)amino]-3-(1H-indole-3-yl)propionic acid methyl ester confirmed that the β-turn of type II is the active conformation of GB-115.     

Bioactive N-terminal undecapeptides derived from parathyroid hormone: the role of alpha-helicity.

The N-terminal 1-34 segment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it can reproduce all biological responses in bone characteristic of the native intact PTH. Recent studies have demonstrated that N-terminal fragments presenting the principal activating domain such as PTH(1-11) and PTH(1-14) with helicity-enhancing substitutions yield potent analogues with PTH(1-34)-like activity. To further investigate the role of alpha-helicity on biological potency, we designed and synthesized by solid-phase methodology the following hPTH(1-11) analogues substituted at positions 1 and/or 3 by the sterically hindered and helix-promoting C(alpha)-tetrasubstituted alpha-amino acids alpha-amino isobutyric acid (Aib), 1-aminocyclopentane-1-carboxylic acid (Ac(5)c) and 1-aminocyclohexane-1-carboxylic acid (Ac(6)c): Ac(5)c-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (I); Aib-V-Ac(5)c-E-I-Q-L-M-H-Q-R-NH(2) (II); Ac(6)c-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (III); Aib-V-Ac(6)c-E-I-Q-L-M-H-Q-R-NH(2) (IV); Aib-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (V); S-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (VI), S-V-Ac(5)c-E-I-Q-L-M-H-Q-R-NH(2) (VII); Ac(5)c-V-S-E-I-Q-L-M-H-Q-R-NH(2) (VIII); Ac(6)c-V-S-E-I-Q-L-M-H-Q-R-NH(2) (IX); Ac(5)c-V-Ac(5)c-E-I-Q-L-M-H-Q-R-NH(2) (X); Ac(6)c-V-Ac(6)c-E-I-Q-L-M-H-Q-R-NH(2) (XI). All analogues were biologically evaluated and conformationally characterized in 2,2,2-trifluoroethanol (TFE) solution by circular dichroism (CD). Analogues I-V, which cover the full range of biological activity observed in the present study, were further conformationally characterized in detail by nuclear magnetic resonance (NMR) and computer simulations studies. The results of ligand-stimulated cAMP accumulation experiments indicated that analogues I and II are active, analogues III, VI and VII are very weakly active and analogues IV, V, VIII-XI are inactive. The most potent analogue, I exhibits biological activity 3500-fold higher than that of the native PTH(1-11) and only 15-fold weaker than that of the native sequence hPTH(1-34). Remarkably, the two most potent analogues, I and II, and the very weakly active analogues, VI and VII, exhibit similar helix contents. These results indicate that the presence of a stable N-terminal helical sequence is an important but not sufficient condition for biological activity.     

Crystal-state structural analysis of two gamma-lactam-restricted analogs of Pro-Leu-Gly-NH2

The crystal structures of two analogs of Pro-Leu-Gly-NH2 (1), containing a gamma-lactam conformational constraint in place of the -Leu-Gly- sequences, are described. The highly biologically active (S,R)-diastereomer 2a is semi-extended at the C-terminus, with the N-terminal Pro residue in the unusual "C5" conformation [psi 1 = -0.8(15) degrees] stabilized by a (peptide)N-H...N(amino) intramolecular H-bond [the N(3)...N(4) separation is 2.687(11)A]. Conversely, the N,N'-isopropylidene aminal trihydrate of the (S,S)-diastereomer 2b, compound 3, adopts a beta-bend conformation at the C-terminus, as already reported for 1. However, the backbone torsion angles [phi 2 = 57.4(4), psi 2 = -129.9(3) degrees; psi 3 = -92.3(4), phi 3 = 6.4(5) degrees] lie close to the values expected for the corner residues of an ideal type-II' beta-bend. A weak intramolecular 4----1 H-bond is seen between the Gly carboxyamide anti-NH and Pro C = O groups. In the newly formed 2,2,3,4-tetraalkyl-5-oxo-imidazolidin-1-yl moiety the psi 1 torsion angle is 12.9(4) degrees and the intramolecular N(3)...N(4) separation is 2.321(4)A.     

Prostaglandin receptors on human platelets. Structure-activity relationships of stimulatory prostaglandins.

1. Synthetic analogues of prostaglandins E2 or F2a (monocyclic bisenoic prostaglandins), like the endogenous prostaglandin endoperoxides (prostaglandins G2 and H2) from platelets, and like synthetic analogues of prostaglandin H2 (bicyclic bisenoic prostaglandins), can induce aggregation of human platelets, although prostaglandins E2 and F2a themselves are inactive. 2. All the prostanoid compounds that induce platelet aggregation release 5-hydroxytryptamine from platelet dense bodies, but do not release beta-N-acetylglucosaminidase from lysosomal granules. Arachidonic acid evokes a similar response. 3. All endoperoxide analogues tested (bicyclic compounds) were powerful platelet stimulants, and all active compounds (whether mono- or bi-cyclid) apparently acted via the same receptor as the endogenous prostaglandin endoperoxides. 4. The nature and stereospecificity of substituents at positions 11 and 15 (or 16) on prostaglandin E2 are critical determinants for platelet-stimulating activity: deoxy substitution at position 11 plus methylation at position 15 (or 16) produces a potent stimulant, particularly if the groups around C-15 are in the S configuration. 5. The effects of these structural modifications are apparently due to, at least in part, a change in side-chain conformation.     

Structure-function relationship studies of PTH(1-11) analogues containing sterically hindered dipeptide mimetics.

The N-terminal 1-34 fragment of parathyroid hormone (PTH) is fully active in vitro and in vivo and reproduces all biological responses characteristic of the native intact PTH. In order to develop safer and non-parenteral PTH-like bone anabolic agents, we have studied the effect of introducing conformationally constrained dipeptide mimetics into the N-terminal portion of PTH in an effort to generate miniaturized PTH-mimetics. To this end, we have synthesized and conformationally and biologically characterized PTH(1-11) analogues containing 3R-carboxy-6S-amino-7,5-bicyclic thiazolidinlactam (7,5-bTL), a rigidified dipeptide mimetic unit. The wild type sequence of PTH(1-11) is H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-NH(2). The following pseudo-undecapeptides were prepared: [Ala(1), 7,5-bTL(3, 4), Nle(8), Arg(11)]hPTH(1-11)NH(2) (I); [Ala(1), 7,5-bTL(6, 7), Nle(8), Arg(11)]hPTH(1-11)NH(2) (II); [Ala(1), Nle(8), 7,5-bTL(9, 10), Arg(11)]hPTH(1-11)NH(2) (III). In aqueous solution containing 20% TFE, only analogue I exhibited the typical CD pattern of the alpha-helical conformation. NMR experiments and molecular dynamics calculations located the alpha-helical stretch in the sequence Ile(5)-His(9). The dipeptide mimetic unit 7,5-bTL induces a type III beta-turn, occupying the positions i - 1 and i of the turn. Analogue II exhibited an equilibrium between a type I beta-turn and an alpha-helix, and analogue III did not show any ordered structure. Biological tests revealed poor activity for all analogues (EC(50) > 0.1 mM). Apparently, the relative side-chain orientation of Val(2), Ile(5) and Met(8) can be critical for effective analogue-receptor interaction. Considering helicity as an essential property to obtain active PTH agonists, one must decorate the correctly positioned dipeptide mimetic azabicycloalkane scaffold with substitutions corresponding to the displaced amino acids.     

Conformationally-restricted analogues and partition coefficients of the 5-HT3 serotonin receptor ligands meta-chlorophenylbiguanide (mCPBG) and meta-chlorophenylguanidine (mCPG)

The present investigation examined two features of arylbiguanide and arylguanidine 5-HT(3) ligands: conformation and partition coefficients. Several conformationally-constrained analogues of mCPBG (2) and mCPG (11; K(i)=32 nM) were prepared and of these only 2-amino-5-chloro-3,4-dihydroquinazoline (14; K(i)=34 nM) retained high affinity. The partition coefficient of compound 11 (LogP(app)=-0.64) was less than that of its corresponding arylbiguanide 2 (LogP(app)=-0.38). The quinazoline structure may represent a pharmacologically-active conformation of these agents, and the arylbiguanides were found more lipid soluble than their arylguanidine counterparts at physiological pH.     

Cyclic lactam analogues of Ac-[Nle4]alpha-MSH4-11-NH2

Two side-chain cyclic lactam analogues of the 4-11 fragment of alpha-melanocyte-stimulating hormone (alpha-MSH), Ac-[Nle4,D-Orn5,Glu8]alpha-MSH4-11-NH2 and Ac-[Nle4,D-Orn5,D-Phe7,Glu8]alpha-MSH4-11-NH2, were prepared on p-methylbenzhydrylamine resin by using a combination of N alpha-Boc and N alpha-Fmoc synthetic strategies with diphenyl phosphorazidate mediated cyclization. The melanotropin activities of these two analogues were examined and compared relative to those of alpha-MSH, Ac-[Nle4]alpha-MSH4-11-NH2, and Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. In the frog (Rana pipiens) skin bioassay, the L-Phe7 17-membered ring cyclic analogue was slightly more potent than the linear Ac-[Nle4]alpha-MSH4-11-NH2 and exhibited prolonged melanotropic bioactivity (greater than or equal to 4 h). In this same assay, the D-Phe7 cyclic analogue was more than 100-fold less potent than the L-Phe cyclic analogue and was 10,000 times less potent than linear Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. In the lizard skin (Anolis carolinensis) bioassay, the L-Phe7 cyclic analogue was 100-fold less potent than Ac-[Nle4]alpha-MSH4-11-NH2, while the D-Phe7 cyclic analogue was 10,000-fold less potent than both Ac-[Nle4]alpha-MSH4-11-NH2 and the D-Phe7 linear derivative Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. The solution conformation of these two cyclic analogues in dimethyl sulfoxide-d6 was examined by 1D and 2D 500-MHz 1H NMR spectroscopy. Our analysis suggests an H bond stabilized C10 (or C13) turn for the D-Phe7 cyclic structure while the L-Phe7 analogue is more conformationally flexible.(ABSTRACT TRUNCATED AT 250 WORDS)