Conformationally restricted macrocyclic analogues of combretastatins

New analogues of combretastatins have been evaluated as inhibitors of tubulin polymerization. These compounds present a macrocyclic structure, in which the para positions of the aromatic moieties have been linked by a 5- or 6-atoms chain, in order to produce a conformational restriction. This could contribute to determine the active conformation for these ligands. Such a conformational restriction and/or the steric hindrance makes them less potent inhibitors than the model compound CA-4.     

Conformationally restricted mimics of vitamin D rotamers

Drug developments in the vitamin D field have continued to focus on structure-function studies of analogs produced by chemically modifying the structure of 1alpha,25-dihydroxyvitamin D(3) (1,25-D3) and its metabolites. Direct structural information gleaned from X-ray crystallographic or NMR studies regarding the ligand-receptor complex and other guest-host systems, which are likely involved in initiating biologic responses, also offers potential insight into drug design. Evidence has accrued suggesting that topologically different conformers of 1,25-D3 may bind to proteins in different ways, including the induction of different conformations of protein. This paper concerns our progress on the chemical synthesis of analogs (e.g. ansa-steroids, suprasterols, vinylallenes and other analogs) conformationally locked or at least rotationally restricted to mimic higher energy conformers of 1,25-D3.     

Conformationally restricted TRH analogues: constraining the pyroglutamate region.

A modified synthetic route has been developed so that the steric size of constraints added to the pyroglutamate region of TRH (pGluHisProNH(2)) can be varied. Both an analogue with a smaller ethylene bridge and a larger, more flexible propane bridge in this region have been synthesized. These analogues were synthesized in order to probe why the initial incorporation of an ethane bridge into this region of the molecule had led to an analogue with a binding constant and potency three times lower than that of an directly analogous unconstrained analogue. The data for both analogues indicated that the fall off in activity caused by the ethane bridge in the initial analogue was not caused by the size of the bridge.     

Conformationally restricted analogs of oxytocin; stabilization of inhibitory conformation

Analogs of oxytocin containing tetrahydroisoquinoline carboxylic acid (Tic) of L or D configuration in position 2 were synthesized and their biological activities were tested. Both analogs showed negligible agonist activity in uterotonic, galactogogic, and pressor assays, but they are in vitro uterotonic inhibitors. In comparison with oxytocin analogs containing L- or D-phenylalanine in position 2, the analog with the D-configuration of the conformationally fixed aromatic residue has significantly increased inhibitory activity which suggests that the proper conformation for the interaction with the receptor, but not for its activation, was stabilized. 1H NMR and CD studies, supported by theoretical calculations, suggest that the conformational properties of the analog containing D-tetrahydroisoquinoline carboxylic acid are similar to those of [2-D-phenylalanine]oxytocin.     

Conformationally restricted cyclic analogues of substance P: insight into the receptor binding process

Three new cyclic substance P analogues were prepared to examine the possible role of a pseudocyclic turn structure for receptor recognition. In the guinea pig isolated ileum [Cys5, Cys11]-SP5-11-NH2 and [Cys6, Cys11]-SP5-11-NH2 were inactive at concentrations up to 100 microM, while [Cys5, Cys6, Nle11]-SP was a weak agonist. The order of relative affinities on the rat brain radioreceptor assay was as follows: [Cys5, Cys6, Nle11]-SP greater than [Cys5, Cys11]-SP5-11-NH2 greater than [Cys6, Cys11]-SP5-11-NH2. We interpret these results to indicate that a pseudocyclic structure of the 5-11 sequence may not be an important factor involved in the receptor recognition of substance P.     

Conformationally restricted analogs of deoxynegamycin

Deoxynegamycin (1b) is a protein synthesis inhibitor with activity against Gram-negative (GN) bacteria. A series of conformationally restricted analogs were synthesized to probe its bioactive conformation. Indeed, some of the constrained analogs were found to be equal or better than deoxynegamycin in protein synthesis assay (1b, IC(50)=8.2 microM; 44, IC(50)=6.6 microM; 35e(2), IC(50)=1 microM). However, deoxynegamycin had the best in vitro whole cell antibacterial activity (Escherichia coli, MIC=4-16 microg/mL; Klebsiella pneumoniae, MIC=8 microg/mL) suggesting that other factors such as permeation may also be contributing to the overall whole cell activity. A new finding is that deoxynegamycin is efficacious in an E. coli murine septicemia model (ED(50)=4.8 mg/kg), providing further evidence of the favorable in vivo properties of this class of molecules.     

Conformationally restricted analogues designed for selective inhibition of GAR Tfase versus thymidylate synthase or dihydrofolate reductase

The synthesis and evaluation of a series of conformationally restricted analogues of 10-formyl-tetrahydrofolate as potential inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) or aminoimidazole carboxamide transformylase (AICAR Tfase) are reported.     

Conformationally constrained analogues of 2-arachidonoylglycerol

Novel monocyclic analogues of 2-arachidonoylglycerol (2-AG) were designed in order to explore the pharmacophoric conformations of this endocannabinoid ligand at the key cannabinergic proteins. All 2-arachidonoyl esters of 1,2,3-cyclohexanetriol [meso-7 (AM5504), (+/-)-8 (AM5503), and meso-9 (AM5505)] were synthesized by regioselective acylation of 2,3-dihydroxycyclohexanone followed by selective reductions. The optically active isomers (+)-8 (AM4434) and (-)-8 (AM4435) were synthesized from (2S,3S)- and (2R,3R)-2,3-dihydroxycyclohexanone, respectively, via a chemoenzymatic route. These head group constrained and conformationally restricted analogues of 2-AG as well as the 1-keto precursors were evaluated as substrates for the endocannabinoid deactivating hydrolytic enzymes monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH), and also were tested for their affinities for CB1 and CB2 cannabinoid receptors. The observed biochemical differences between these ligands can help define the conformational requirements for 2-AG activity at each of the above endocannabinoid protein targets.     

Conformationally constrained tachykinins: N-methylated analogues of neurokinin A

Limited conformational constraints have been introduced in the sequence of the C-terminal fragment of the peptide neurotransmitter neurokinin A by N-methylation of individual peptide bonds, and the biological activities of the peptides thus obtained were evaluated in order to assess the effect of such conformational constraint on receptor selectivity. The analogue methylated in position 7 shows enhanced selectivity toward NK-1 receptor.     

Conformationally restricted dinucleotides: tandem ring-closing metathesis and hydrogenation approach

Cyclic dinucleotides with saturated connections between a nucleobase and the phosphate are synthesised using a tandem ring-closing metathesis (RCM) and hydrogenation protocol and found to be significantly stabilised towards ammonia.     

Conformationally restricted analogs of the direct thrombin inhibitor FM 19

The serine protease thrombin plays several key roles in the clotting cascade within the hemostatic system, such as in fibrin formation and platelet activation. Thus, development of an inhibitor that binds to the enzyme’s active site (a direct thrombin inhibitor) offers an approach for the treatment of thrombus-associated diseases. Previous structure–activity relationship studies originally based on the bradykinin breakdown product Arg-Pro-Pro-Gly-Phe (RPPGF) led to the development of lead compound FM 19 (d-Arg-Oic-Pro-d-Ala-Phe(p-Me)-NH₂). The recently determined X-ray structure of FM 19 in the active site of thrombin has revealed sites of modification to potentially improve inhibition. In this study, we report the synthesis and biological characterization of nine peptides that replace only the d-Arg residue of the FM 19 sequence, investigating ways to add conformational restriction, modification of the basic moiety at the end of the side chain, and removal of the charge from the N-terminus. Two of these peptides, 6 and 7 (IC₅₀ values of 0.51 and 0.45 μM, respectively), show similar potency to the best compounds in the FM 19 series reported thus far.     

Synthesis and properties of oligonucleotides containing carbocyclic L-nucleoside analogues with a restricted glycosyl conformation.

To construct nuclease-resistant oligonucleotides, we designed novel carbocyclic L-nucleoside analogues (1-4) whose glycosyl conformation is fixed at chi = 180 degrees by an oxygen-bridge between the base and the cyclopentane ring. We have already achieved the racemic synthesis of these analogues. In this study, we succeeded in synthesizing an optically active form of these analogues. The properties of oligonucleotides containing them will be shown.     

Syntheses and bioactivities of macrocyclic paclitaxel bis-lactones

Five macrocyclic paclitaxel bis-lactones and their corresponding open chain taxoids were synthesized as models of the tubulin-binding conformation of paclitaxel. Macrocyclic lactones with a 19-21-membered ring underwent isomerization to form smaller rings. The lactones were evaluated for cytotoxicity and tubulin-polymerization ability. All five macrocyclic paclitaxel lactones were active, but less so than paclitaxel, while the rearranged macrocyclic lactones and the corresponding open-chain taxoids were much less active or inactive.     

Conformationally constrained single-chain peptide mimics of relaxin B-chain secondary structure.

Relaxin is a member of the insulin superfamily and has many biological actions including angiogenesis and collagen degradation. It is a 6 kDa peptide hormone consisting of two peptide chains (A and B) tethered by two disulphide bonds. Past structure-function relationship studies have shown the key receptor binding site of relaxin to be principally situated within the B-chain alpha-helix. Molecular dynamic simulations were performed to aid the design of conformationally constrained relaxin B-chain analogues that possess alpha-helical structure and relaxin-like activity. Restraints included disulphide bonds, both single and double, and lactam bonds. Each peptide was prepared by solid phase synthesis and, following purification, subjected to detailed conformational analysis by circular dichroism spectroscopy. Of 15 prepared relaxin B-chain mimetics, one was able to mimic the secondary structure of the native ligand as indicated by biomolecular recognition/interaction analysis using surface enhanced laser desorption ionization mass spectroscopy together with a relaxin antibody. However, none of the mimetics possess characteristic relaxin-like biological activity which strongly indicates that the pharmacophore comprises additional structural elements other than the relaxin B-chain alpha-helix. These findings will assist in the design and preparation of novel relaxin agonists and antagonists.     

Conformationally restricted nucleotides as a probe of structure-function relationships in RNA

We introduce the use of commercially available locked nucleic acids (LNAs) as a functional probe in RNA. LNA nucleotides contain a covalent linkage that restricts the pseudorotation phase of the ribose to C3'-endo (A-form). Introduction of an LNA at a single site thus allows the role of ribose structure and dynamics in RNA function to be assessed. We apply LNA probing at multiple sites to analyze self-cleavage in the lead-dependent ribozyme (leadzyme), thermodynamic stability in the UUCG tetraloop, and the kinetics of recognition of U1A protein by U1 snRNA hairpin II. In the leadzyme, locking a single guanosine residue into the C3'-endo pucker increases the catalytic rate by a factor of 20, despite the fact that X-ray crystallographic and NMR structures of the leadzyme ground state reported a C2'-endo conformation at this site. These results strongly suggest that a conformational change at this position is critical for catalytic function. Functional insights obtained in all three systems demonstrate the highly general applicability of LNA probing in analysis of the role of ribose orientation in RNA structure, dynamics, and function.     

Synthesis of metabolically blocked paclitaxel analogues

The stereospecific syntheses of the metabolically blocked 6-alpha-F, Cl, Br paclitaxel, and 6-alpha-F-10-acetyldocetaxel are described and in vitro and in vivo activity is presented.     

Conformationally constrained diketopimelic acid analogues as inhibitors of dihydrodipicolinate synthase

Dihydrodipicolinate synthase (DHDPS) is a key enzyme in lysine biosynthesis and a potential antibiotic target. The enzyme catalyses the condensation of (S)-aspartate semi-aldehyde (ASA) and pyruvate to form dihydrodipicolinate. Constrained diketopimelic acid derivatives have been designed as mimics of the acyclic enzyme-bound condensation product of ASA and pyruvate. Several of the compounds are shown to be active, slow-binding inhibitors with improved inhibition of DHDPS.     

Conformationally restricted analogs of Combretastatin A-4 derived from SU5416

A series of compounds originally derived from the vascular endothelial growth factor receptor tyrosine kinase inhibitor, SU5416, was synthesized and evaluated. The most potent compound in this series, compound 7, structurally resembles the potent anti-microtubule agent Combretastatin A-4, inhibited tubulin polymerization, and showed potent growth inhibitory activities on both prostate and breast cancer lines with IC₅₀ values in low to subnanomolar range.     

Synthesis and biological activity of macrocyclic taxane analogues

A series of paclitaxel analogues possessing a macrocyclic structure between the 7 and 10 positions has been prepared. These compounds possess in vitro activity against a paclitaxel resistant cell line and have in vivo activity comparable to paclitaxel.