Synthesis and evaluation of a series of homologues of lobelane at the vesicular monoamine transporter-2

A series of lobelane homologues has been synthesized and evaluated for their [(3)H]DTBZ binding affinity at the vesicular monoamine transporter-2 (VMAT2). The structure-activity relationships (SAR) indicate that for retention of binding affinity at VMAT2, the lengths of the methylene linkers should be no shorter than one methylene unit at C-6 of the piperidine ring, and no shorter than two methylene units at C-2 of the piperidine ring. These results indicate that the intramolecular distances between the piperidine ring and two phenyl rings in lobelane analogues are an important criterion for retention of high affinity at VMAT2.     

Synthesis and evaluation of a series of tropane analogues as novel vesicular monoamine transporter-2 ligands

A series of tropane derivatives has been synthesized as lobelane analogues and evaluated for their binding affinity at the vesicular monoamine transporter-2 (VMAT2), and at alpha4beta2* and alpha7* nicotinic acetylcholine receptors. The trop-2-ene analogues 4a and 4b exhibited good affinity and high selectivity for VMAT2.     

Lobelane analogues as novel ligands for the vesicular monoamine transporter-2

A series of lobelane analogues has been synthesized and their structure-activity relationships at the vesicular monoamine transporter-2 (VMAT2) have been evaluated. The most potent analogues in this series were the cis-2,6-piperidino analogues, 25b, 27b, 28b, and 30b, with K(i) values ranging from 430 to 580 nM.     

Synthesis of CMP-9'-modified-sialic acids as donor substrate analogues for mammalian and bacterial sialyltransferases

Cytidine-5'-monophospho-sialic acid (CMP-Neu5Ac) derivatives bearing a phenyl group in which the tether length between the phenyl group and the 9-position of Neu5Ac varied were synthesized and evaluated as substrates for sialyltransferases. In the synthesis of the compounds, a coupling reaction between methyl 5-acetamido-4,7,8-tri-O-acetyl-9-azido-3,5,9-trideoxy-beta-D-glycero-D-galacto-2-nonulopyranosonate and 2-cyanoethyl 2',3'-O,N4, triacetylcytidine-5'-yl N,N-diisopropylphosphoramidite was carried out and the phosphite derivative thus obtained was oxidized and then deprotected to yield CMP-9'-azido-Neu5Ac. Modification of the 9-amino group prepared by reduction of the azido groups was performed by the use of several phenyl-substituted alkylcarboxylic acid derivatives. Using these CMP-9'-modified-Neu5Ac analogues bearing the phenyl-substituted alkyl-amide group, sialyltransferase assays were performed with both rat liver alpha-(2-->6)-sialyltransferase and Photobacterium alpha-(2-->6)-sialyltransferase. These 9-modified analogues could be transferred to disaccharide acceptors, and a practical enzymatic synthesis using CMP-9'-modified-Neu5Ac yielded sialoside analogues and sialylglycoproteins in good yield. These experiments demonstrate that the Photobacterium sialyltransferase can be used in the synthesis of sialoside analogues having a large substituent at the 9-position of Neu5Ac.     

Structure-activity relationship on human serum paraoxonase (PON1) using substrate analogues and inhibitors

Substrate analogues based on the parent compounds paraoxon and phenyl acetate were tested on human serum paraoxonase (PON1) to explore the active site of the enzyme. Replacement of the nitro group of paraoxon with an amine or hydrogen, as well as electronic changes to the parent compound, converted these analogues into inhibitors. Introduction of either electron-withdrawing or donating groups onto phenyl acetate resulted in reduction in their rate of hydrolysis by PON1.     

Influence of the separation of the charged groups and aromatic ring on interaction of tyrosine and phenylalanine analogues and derivatives with beta-cyclodextrin

Interactions of tyrosine and phenylalanine analogues with beta-cyclodextrin have been examined in terms of structural features of the ligand such as the separation of the charged amino group and aromatic ring, the presence of additional functional group attached to the amino or phenyl ring, and the presence of a charge on amino or carboxyl group, and steric effects using steady-state and time-resolved fluorescence spectroscopy and microcalorimetry. The studied aromatic amino acids possess low binding constant to beta-cyclodextrin, diversified with respect to the presence or absence of a substituent in para position of the phenyl ring. However, calculated, based on the global analysis of the fluorescence intensity decays, binding constants do not allow to estimate unequivocally the influence of the distance between the charged groups and phenol/phenyl ring on the inclusion complex stability because of their low diversification.     

Synthesis and anticancer activity of novel quinoline–docetaxel analogues

A series of novel quinoline–docetaxel analogues (6a–6g, 13a–13g) were designed and synthesized by introducing bioactive quinoline scaffold to C2′-OH of docetaxel. The anticancer activities of these novel analogues were investigated against different human cancer cell lines including Hela, A549, A2780, MCF-7 and two resistant strains A2780-MDR and MCF-7-MDR. The data showed these analogues possessed similar to better cytotoxicity than docetaxel. Compound 6c was found to be the most potent one, and its IC₅₀ value against MCF-7-MDR was 8.8 nM (IC₅₀ of docetaxel was 180 nM). The work indicated that the introduction of quinolyl group in docetaxel could enhance cytotoxicity and reduce drug-resistance.     

Removal of the nitro and phenyl groups from NPPB decreases its inhibitory effect on cytoplasmic streaming in the alga Nitella hookeri.

Structural analogues of the arylaminobenzoate 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), prepared using a simple reductive amination sequence, were tested for their effects on cytoplasmic streaming rates in the alga Nitella hookeri. Cytoplasmic streaming was sensitive to NPPB, with an IC50 value of 24 micromol/L. Removal of the nitro group from the benzoate ring decreased the IC50 to 455 micromol/L. The introduction of an extra carbon or double bond into the aliphatic chain had no effect on activity. Loss of the phenyl group decreased potency, with an IC50 of 6.4 mmol/L. These data are the first documenting the relative inhibitory effects of structural changes to arylaminobenzoates in algae. Patch-clamp data and the effects of tetrapentyl ammonium chloride on streaming suggest that the nitro and phenyl groups may act by inhibiting both K+ and Cl- channels. This is likely, through changes in the membrane potential, to affect Ca2+ fluxes and action potentials, thereby slowing cytoplasmic streaming.     

N-Alkoxypyrazoles as biomimetics for the alkoxyphenyl group in tamoxifen

The preparation of a series of novel analogues of the selective antiestrogen tamoxifen is reported. 1Z-alkoxyphenyl group in tamoxifen has been replaced by a N-alkoxypyrazole, while functionalised phenyl groups or heteroaromatics were introduced at the 2Z-position using sequential Suzuki cross coupling of 1,2-(bis)borylpinacol 1-phenylbutene with 4- or 5-iodo-1-N,N-dimethylaminoethyl or propyl-pyrazoles. Approximately 50 tamoxifen analogues were obtained and tested in an estrogen receptor (ERalpha) affinity assay. Several compounds exhibited binding affinities 2-5-fold lower than tamoxifen. Dose-response experiments with six selected compounds were carried out using two different human breast cancer cell lines, MCF-7 and the tamoxifen resistant cell line MCF-/TAM(R)-1. Both cell lines exhibited growth inhibition upon treatment with the tamoxifen analogues. Co-treatment of the cells, with estradiol and the individual compounds, were also performed. The results indicated that the observed growth inhibitory effect was mediated by the ERalpha. Analogues of the potent antiestrogen 4-hydroxytamoxifen (4-OHT) were synthesised where the 1E-4-hydroxyphenyl was replaced by a 1-hydroxypyrazol-4-yl group. However, modest growth inhibition of MCF-7 cells was observed upon treatment with these analogues. In contrast, 1Z-, 2Z-ringclosed tamoxifen analogue (59) was shown to possess antiproliferative effects on MCF-7 and MCF-/TAM(R)-1 cells in lower doses than tamoxifen.     

Trifunctional agents as a design strategy for tailoring ligand properties: irreversible inhibitors of A1 adenosine receptors

The 1,3-phenylene diisothiocyanate conjugate of XAC (8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]- oxy]phenyl]-1,3-dipropylxanthine, a potent A1 selective adenosine antagonist) has been characterized as an irreversible inhibitor of A1 adenosine receptors. To further extend this work, a series of analogues were prepared containing a third substituent in the phenyl isothiocyanate ring, incorporated to modify the physiochemical or spectroscopic properties of the conjugate. Symmetrical trifunctional cross-linking reagents bearing two isothiocyanate groups were prepared as general intermediates for cross-linking functionalized congeners and receptors. Xanthine isothiocyanate derivatives containing hydrophilic, fluorescent, or reactive substituents, linked via an amide, thiourea, or methylene group in the 5-position, were synthesized and found to be irreversible inhibitors of A1 adenosine receptors. The effects of the 5-substituent on water solubility and on the A1/A2 selectivity ratio derived from binding assays in rat brain membranes were examined. Inhibition of binding of [3H]-N6-(2-phenylisopropyl)-adenosine and [3H] CGS21680 (2-[2-[4-carboxyethyl)phenyl]ethyl]amino] adenosine-5'-N-ethylcarboxamide) at central A1 and A2 adenosine receptors, respectively, was measured. A conjugate of XAC and 1,3,5-triisothiocyanatobenzene was 894-fold selective for A1 receptors. Reporter groups, such as fluorescent dyes and a spin-label, were included as chain substituents in the irreversible binding analogues, which were designed for spectroscopic assays, histochemical characterization, and biochemical characterization of the receptor protein.     

Perturbing the hydrophobic pocket of mandelate racemase to probe phenyl motion during catalysis

Mandelate racemase (MR, EC 5.1.2.2) from Pseudomonas putida catalyzes the Mg(2+)-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. Crystal structures of MR reveal that the phenyl group of all ground-state ligands is located within a hydrophobic cavity, remote from the site of proton abstraction. MR forms numerous electrostatic and H-bonding interactions with the alpha-OH and carboxyl groups of the substrate, suggesting that these polar groups may remain relatively fixed in position during catalysis while the phenyl group is free to move between two binding sites [i.e., the R-pocket and the S-pocket for binding the phenyl group of (R)-mandelate and (S)-mandelate, respectively]. We show that MR binds benzilate (K(i) = 0.67 +/- 0.12 mM) and (S)-cyclohexylphenylglycolate (K(i) = 0.50 +/- 0.03 mM) as competitive inhibitors with affinities similar to that which the enzyme exhibits for the substrate. Therefore, the active site can simultaneously accommodate two phenyl groups, consistent with the existence of an R-pocket and an S-pocket. Wild-type MR exhibits a slightly higher affinity for (S)-mandelate [i.e., K(m)(S)(-)(man) < K(m)(R)(-)(man)] but catalyzes the turnover of (R)-mandelate slightly more rapidly (i.e., k(cat)(R)(-->)(S) > k(cat)(S)(-->)(R)). Upon introduction of steric bulk into the S-pocket using site-directed mutagenesis (i.e., the F52W, Y54W, and F52W/Y54W mutants), this catalytic preference is reversed. Although the catalytic efficiency (k(cat)/K(m)) of all the mutants was reduced (11-280-fold), all mutants exhibited a higher affinity for (R)-mandelate than for (S)-mandelate, and higher turnover numbers with (S)-mandelate as the substrate, relative to those with (R)-mandelate. (R)- and (S)-2-hydroxybutyrate are expected to be less sensitive to the additional steric bulk in the S-pocket. Unlike those for mandelate, the relative binding affinities for these substrate analogues are not reversed. These results are consistent with steric obstruction in the S-pocket and support the hypothesis that the phenyl group of the substrate may move between an R-pocket and an S-pocket during racemization. These conclusions were also supported by modeling of the binary complexes of the wild-type and F52W/Y54W enzymes with the substrate analogues (R)- and (S)-atrolactate, and of wild-type MR with bound benzilate using molecular dynamics simulations.     

Synthesis and testing of novel phenyl substituted side-chain analogues of classical cannabinoids

A series of novel phenyl substituted side-chain analogues of classical cannabinoids were synthesized and their CB1 and CB2 binding affinities were evaluated relative to Delta(8)-THC and compound 2. CB1 and CB2 binding assays indicate that the dimethyl and ketone analogues (3) and (6) display selectivity for the CB2 receptor in comparison to delta(8)-THC and compound 2. This study provides newer insights into the geometrical and functional group requirements of the ligand binding pockets of the CB1 and the CB2 receptors.     

Synthesis and biological activity of analogues of the C-terminal hexapeptide of substance P with modifications at glutaminyl and methioninyl residues. Structure-activity studies.

Analogues of [Orn6]-SP6-11 have been synthesized in which the methionyl residue is replaced by glutamine gamma-carboxamide substituted derivatives. These analogues where tested in three in vitro preparations representative of NK-1, NK-2 and NK-3 receptor types. Substitution of the SCH3 group of the Met11 side chain by CONHCH3, CON(CH3)2, CONHPh and CONCH3Ph groups results in analogues which are full agonists in NK-1 and NK-2 preparations with the exception of the Glu[N(CH3)2]11 and the Glu(NHCH3)11 analogues, which are partial agonists at NK-1 and NK-2 receptors respectively. The Glu(NHCH3)11 analogue shows selectivity for the NK-1 receptor type and is equipotent to the Glu(NCH3Ph)11 analogue in the same receptor type. The latter analogue is 2.84 times more potent than the parent hexapeptide in the NK-2 preparation. The Glu(NHPh)11 analogue is a full agonist in the NK-3 preparation and equipotent to the parent hexapeptide, in contrast to the other analogues in which Met has been replaced by glutamine gamma-carboxamide substituted residues. It is concluded that for NK-1 receptor type the lipophilic character of Met11 side chain is not a determining factor for activity but it is an important factor for activity in the NK-2 receptor type and has a stronger effect when a phenyl group is present, thus leading to analogues which are full agonists and more potent than the parent hexapeptide.     

New synthetic analogues of N-acyl homoserine lactones as agonists or antagonists of transcriptional regulators involved in bacterial quorum sensing

A series of 22 novel synthetic N-acyl-homoserine lactone analogues has been evaluated for both their inducing activity and their ability to competitively inhibit the action of 3-oxo-hexanoyl-L-homoserine lactone, the natural inducer of bioluminescence in the bacterium Vibrio fischeri. In the newly synthesized analogues, the extremity of the acyl chain was modified by introducing ramified alkyl, cycloalkyl or aryl substituents at the C-4 position. Most of the analogues bearing either acyclic or cyclic alkyl substituents showed inducing activity. In contrast, the phenyl substituted analogues displayed significant antagonist activity. We hypothesized that the antagonist activity of the phenyl compounds may result from the interaction between the aryl group and aromatic amino acids of the LuxR receptor, preventing it from adopting the active dimeric form.     

Structural features of 5,10-dideaza-5,6,7,8-tetrahydrofolate that determine inhibition of mammalian glycinamide ribonucleotide formyltransferase

We have investigated the structural features of 5,10-dideaza-5,6,7,8-tetrahydrofolate (DDATHF) that determine the activity of this compound as an inhibitor of glycinamide ribonucleotide formyltransferase (GARFT) purified from mouse L1210 cells. 5-Deazatetrahydrofolate was as good an inhibitor of GARFT as DDATHF, indicating that isosteric replacement of nitrogen by carbon at the 5-position of tetrahydrofolate is sufficient for inhibition of GARFT. 5,10-Dideazafolic acid, 5,8,10-trideazatetrahydrofolate, and 2-desamino-5,10-dideazatetrahydrofolate were poor inhibitors of GARFT, indicating that a reduced pyridopyrimidine ring, N-8, and the 2-amino group of DDATHF, respectively, play an important role in the binding of tetrahydrofolate analogues to this enzyme. DDATHF analogues in which the phenyl ring was replaced either by a cyclohexyl ring or by methylene groups retained activity as inhibitors. 5,10-Dideazatetrahydrohomofolate was about 6 times more potent as an inhibitor of GARFT than DDATHF, but 5,10-dideazatetrahydronorfolate had about one-fifth of the activity of DDATHF. An analogue of DDATHF in which the glutamic acid side chain was replaced by aspartic acid (which was not a substrate for polyglutamation and was only weakly cytotoxic) was equiactive with DDATHF as an inhibitor of purified GARFT. Surprisingly, 5,10-dideazatetrahydropteroic acid was about as active as DDATHF as an inhibitor of GARFT, an indication that the glutamic acid in the side chain of DDATHF does not play a role in this ligand-enzyme interaction. The polyglutamate derivatives of DDATHF bound up to 100 times tighter to GARFT than DDATHF itself; longer chain polyglutamates conformed to Goldstein's zone B behavior under experimental conditions and were projected to be in zone C, i.e., stoichiometric inhibition, in vivo. We conclude that the presence of carbon at the 5-position of tetrahydrofolate analogues is sufficient for inhibition of GARFT, that N-8 and the 2-amino group are involved in binding of DDATHF to GARFT, probably through hydrogen bonds, and that the structures of the phenyl ring and amino acid side chain of DDATHF analogues are not primary determinants of GARFT inhibition by monoglutamate forms of these compounds. We also conclude that polyglutamation plays a major role in the potent cytotoxicity of DDATHF.     

Design and synthesis of 2-oxo-imidazolidine-4-carboxylic acid hydroxyamides as potent matrix metalloproteinase-13 inhibitors

A novel series of imidazolidinone-based matrix metalloproteinase (MMP) inhibitors was discovered by structural modification of pyrrolidinone la. Potent inhibition of MMP-13 was exhibited by the analogues having 4-(4-fluorophenoxy)phenyl (4a, IC50 = 3 nM) and 4-(naphth-2-yloxy)phenyl (4h, IC50 = 4 nM) as P1' groups.     

Novel C-terminal gastrin antagonists. Synthesis and biological activity

The C-terminal tetrapeptide, Trp-Met-Asp-Phe-NH2, is a full agonist of gastrin, but des-Phe analogues, including Boc-Trp-Met-Asp-NH2, are antagonists. To ascertain the minimum structural requirement for an antagonist, we used conventional solution phase methodology to synthesize analogues with further modifications including removal of the alpha-amino group of Trp, conversion of the indole to a phenyl ring, and methylation of amide bonds. These analogues were tested for their effect on pentagastrin-stimulated acid release in dogs surgically prepared with a gastric fistula. When infused intravenously at a dose of 20 pmol kg-1 h-1, the peptides significantly inhibited acid secretion. The extent of inhibition ranged from 12% to 60%. Thus, tripeptide analogues based on the C-terminal sequence of gastrin act as potent and specific antagonists of gastrin-stimulated acid secretion.     

Expression of the two isoforms of the vesicular monoamine transporter (VMAT1 and VMAT2) in the endocrine pancreas and pancreatic endocrine tumors.

The uptake of monoamines into the secretory granules of monoamine-storing neuroendocrine cells is mediated by vesicular monoamine transporter protein 1 or 2 (VMAT1 or VMAT2). This study analyzed the expression of VMAT1 and VMAT2 in endocrine cells of normal human and monkey pancreas. The expression of VMAT1 and VMAT2 was also examined in infants with hyperinsulinemic hypoglycemia and in adults with pancreatic endocrine tumors (PETs). Using immunohistochemistry (IHC) and in situ hybridization (ISH), we demonstrated the mutually exclusive expression of VMAT1 in endocrine cells of the duct system and of VMAT2 in many cells of the islets of Langerhans. By confocal laser scanning microscopy, VMAT1-positive cells were identified as enterochromaffin (EC) cells and VMAT2-positive cells as beta-cells. In PETs, VMAT1 was found exclusively in all serotonin-containing tumors. In contrast, VMAT2 expression was lost in many insulinomas, independent of their biological behavior. VMAT2 was expressed by some non-insulin-producing tumors. The mutually exclusive expression of VMAT1 in EC cells and of VMAT2 in beta-cells suggests that both cell types store monoamines. Monoamine storage mediated by VMAT1 in EC cells is apparently maintained in EC cell tumors. In contrast, many insulinomas appear to lose their ability to accumulate monoamines via VMAT2.     

Structure of mandelate racemase with bound intermediate analogues benzohydroxamate and cupferron

Mandelate racemase (MR, EC 5.1.2.2) from Pseudomonas putida catalyzes the Mg(2+)-dependent interconversion of the enantiomers of mandelate, stabilizing the altered substrate in the transition state by 26 kcal/mol relative to the substrate in the ground state. To understand the origins of this binding discrimination, we determined the X-ray crystal structures of wild-type MR complexed with two analogues of the putative aci-carboxylate intermediate, benzohydroxamate and Cupferron, to 2.2-? resolution. Benzohydroxamate is shown to be a reasonable mimic of the transition state and/or intermediate because its binding affinity for 21 MR variants correlates well with changes in the free energy of transition state stabilization afforded by these variants. Both benzohydroxamate and Cupferron chelate the active site divalent metal ion and are bound in a conformation with the phenyl ring coplanar with the hydroxamate and diazeniumdiolate moieties, respectively. Structural overlays of MR complexed with benzohydroxamate, Cupferron, and the ground state analogue (S)-atrolactate reveal that the para carbon of the substrate phenyl ring moves by 0.8-1.2 ? between the ground state and intermediate state, consistent with the proposal that the phenyl ring moves during MR catalysis while the polar groups remain relatively fixed. Although the overall protein structure of MR with bound intermediate analogues is very similar to that of MR with bound (S)-atrolactate, the intermediate-Mg(2+) distance becomes shorter, suggesting a tighter complex with the catalytic Mg(2+). In addition, Tyr 54 moves closer to the phenyl ring of the bound intermediate analogues, contributing to an overall constriction of the active site cavity. However, site-directed mutagenesis experiments revealed that the role of Tyr 54 in MR catalysis is relatively minor, suggesting that alterations in enzyme structure that contribute to discrimination between the altered substrate in the transition state and the ground state by this proficient enzyme are extremely subtle.     

Stimulation by thyroid hormone analogues of red blood cell Ca2+-ATPase activity in vitro. Correlations between hormone structure and biological activity in a human cell system

Human red blood cell membrane Ca2+-ATPase activity is stimulated in vitro by physiological concentrations (10(-10) M) of L-thyroxine (L-T4) and 3,5,3'-triiodo-L-thyronine (L-T3). This human cell system has been utilized to examine a series of iodothyronine and iodotyrosine analogues for structure-activity relationships. Analogue purity was verified by high pressure liquid chromatography. Analogues were studied at a concentration of 10(-10) M and the stimulatory effect of each analogue was compared with that of L-T4 in this system. Essential to Ca2+-ATPase stimulation were occupation of the 3 and 5 phenyl positions by iodide, bromide, or methyl groups, the L-configuration of the alanine side chain, side chain length equal to that of alanine, and a perpendicular (skewed) conformation of the two rings. The 4'-hydroxyl group is not essential to Ca2+-ATPase stimulation in this model system. T3 was 76% as active as T4 in stimulating Ca2+-ATPase activity. The stimulatory effect of 3,5-dimethyl-3'-isopropyl-L-thyronine and 3,5,3',5'-tetrabromo-L-thyronine approximated that of L-T4. Selected tyrosine analogues also stimulated the enzyme. The bioactivities of hormone analogues in this human model of extra-nuclear thyroid hormone action differ in several ways from results obtained previously in other animal model systems in vitro and in vivo.