Synthetic amatoxin analogue. A two-dimensional proton NMR study of S-deoxy-(Ile3)-(D-Ala7)-amaninamide

The effect of substitution of L and D amino acids in amatoxin analogues is discussed in this paper. The structure of the analog where D-alanine substitutes for glycine in position 7 has been worked out in solution by two-dimensional NMR methods using a 500 MHz instrument. The combined use of COSY and NOESY two-dimensional spectra allows a clear assignment of the resonances. The use of the coupling constants permits the calculation of the phi angles of the backbone. The NOE effects reveal the through-space contacts between protons of different peptide units, thus determining the rigidity of the amatoxin structure. On these grounds it has been possible to elucidate the conformation of the amatoxin analogue that resembles very closely that of beta-amanitin, thus explaining the high inhibitory activity toward RNA polymerase B.     

Ala analogues of the cyclolinopeptide A

Analogues of cyclolinopeptide A, due to the replacement of each amino acid in the Pro1-Pro2-Phe3-Phe4 sequences with an L-Ala residue, were synthesized by classical method in solution. Mixed anhydride and N,N'-dicyclohexylcarbodiimide coupling methods have been used for the synthesis of both linear and cyclic peptides. The products were characterized by Rf values and uv spectra, as well as by fast atom bombardment spectroscopy. 1H-nmr studies on [Ala2] analogues are also reported. Preliminary data in CDCl3 solution, at low temperature, seems more promising.     

Novel derivatives of 3-(dipropylamino)chroman. Interactions with 5-HT1A and D2A receptors.

Novel 8-aryl and 8-aroyl substituted derivatives of 3-(dipropylamino)chroman are described. The compounds have been prepared by a palladium catalyzed reaction of iodoarenes and a stannylated derivative of [eta6-3-(dipropylamino)chroman]Cr(CO)3. Several of the compounds have high affinity for 5-HT1A receptors whereas the affinity for D2A receptors is lower, the 8-arylated derivatives being slightly more potent than the 8-aroylated analogues.     

Synthesis of 2-(5-bromo-2,3-dimethoxyphenyl)-5-(aminomethyl)-1H-pyrrole analogues and their binding affinities for dopamine D2, D3, and D4 receptors

A series of 2-(5-bromo-2,3-dimethoxyphenyl)-5-(aminomethyl)-1H-pyrrole analogues was prepared and their affinity for dopamine D(2), D(3), and D(4) receptors was measured using in vitro binding assays. The results of receptor binding studies indicated that the incorporation of a pyrrole moiety between the phenyl ring and the basic nitrogen resulted in a significant increase in the selectivity for dopamine D(3) receptors. The most selective compound in this series is 2-(5-bromo-2,3-dimethoxyphenyl)-5-(2-(3-pyridal)piperidinyl)methyl-1H-pyrrole (6p), which has a D(3) receptor affinity of 4.3 nM, a 20-fold selectivity for D(3) versus D(2) receptors, and a 300-fold selectivity for D(3) versus D(4) receptors. This compound is predicted to be a useful ligand for studying the functional role of dopamine D(3) receptors in vivo.     

Chemoenzymatic synthesis and binding affinity of novel (R)- and (S)-3-aminomethyl-1-tetralones, potential atypical antipsychotics

A series of (R)- and (S)-3-aminomethyl-1-tetralones, conformationally constrained analogues of haloperidol, have been obtained by enzymatic resolution of the corresponding racemic 3-hydroxymethyl-1-tetralones using Pseudomonas fluorescens lipase. Their binding affinities at dopamine D(2) and serotonin 5-HT(2A) and 5-HT(2C) receptors were determined showing in some cases an atypical antipsychotic profile with Meltzer's ratio higher than 1.30.     

A comparative conformational study of thymidylyl(3'----5')-thymidine, thymidylyl(3'----5')-5'-thio-5'- deoxythymidine and thymidinylacetamido-[3'(O)----5'(C)]-5'-deoxythymidine

A comparative 270 MHz NMR spectroscopic study on the solution structure of the dimer d(TpT) 1, and its two analogues, namely, d(TpST) 2, and NH2d(TcmT) 4 has been reported. Analysis of chemical shifts and coupling constants indicate that: (i) The sugar moieties of the constituent nucleotides are not affected by modification of the internucleotide linkages and adopt preferentially an S-type conformation. (ii) The C4'-C5' bond in the pT part of the modified dimers 2 and 4 shows a large conformational freedom (gamma+ = 32% and 35%, respectively) compared to 1 (gamma+ = 75%). (iii) The population of the trans conformer about C5'-O5' is less important in d(TpST) 2 compared to d(TpT) 1. (iv) The C3'-O3' bond in 2 adopts a trans conformation as in 1. (v) The glycosidic bonds in the modified dimers 2 and 4 showed preferential syn conformation. UV and CD data show that the modified dimers 2 and 4 have poor tendency to stack intramolecularly, they also base pair less efficiently with d(ApA) as compared to d(TpT) 1.     

(R)-3-(N-methylpyrrolidin-2-ylmethyl)-5-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole derivatives as high affinity h5-HT1B/1D ligands

A series of (R)-3-(N-methylpyrrolidin-2-ylmethyl)-5-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole derivatives (2) have been prepared using parallel synthesis, and their structure-activity relationship studied. High affinity human 5-HT(1B/1D) (h5-HT(1B/1D)) ligands have been identified.     

Structure-activity studies on prolactin-releasing peptide (PrRP). Analogues of PrRP-(19-31)-peptide.

An investigation of a series of single replacement analogues of PrRP-(19-31)-peptide has shown that good functional activity was retained when Phe31 was replaced with His(Bzl), Phe(4Cl), Nle, Trp, Cys(Bzl) or Glu(OBzl); when Val28 or Ile25 was replaced with Phg; when Gly24 was replaced with D-Ala, L-Ala, Pro or Sar; when Ser22 was replaced with Gly and when Ala21 was replaced with Thr or MeAla. The results confirm that the functionally important residues are located within the carboxyl terminal segment, -Ile-Arg-Pro-Val-Gly-Arg-Phe-NH2.     

Escherichia coli tRNA (uracil-5-)-methyltransferase: Inhibition by analogues of adenosylhomocysteine.

Structural analogues of adenosylhomocysteine (AdoHcy) have been tested as inhibitors of a tRNA(uracil-5-)-methyltransferase preparation obtained from Escherichia coli. All analogues tested gave linear competitive inhibition kinetics with adenosylmethionine (AdoMet) as the variable substrate. Comparison of the Ki values obtained leads to the following conclusions concerning the specificity of the AdoMet-AdoHcy binding site on the enzyme: (i) the terminal amino group of the amino acid moiety is necessary for activity; (ii) both a chiral change of the asymmetric carbon atom of homocysteine and the presence of the terminal carboxyl group contribute little towards inhibitory activity; (iii) analogues in which the amino function of the adenyl moiety is modified or substituted are still potent inhibitors; (iv) inhibitor specificity is considerably reduced when adenine is replaced by a pyrimidine base.     

Spectroscopic studies of pyridoxamine (pyridoxine) 5'-phosphate oxidase. Equilibrium dissociation constants and spectra for riboflavin 5'-phosphate and analogues.

Pyridoxamine (pyridoxine) 5'-phosphate oxidase (EC 1.4.3.5) has been shown to bind 1 mol of riboflavin 5'-phosphate (FMN) per mol of apoenzyme and is active with or inhibited by numerous FMN analogues [Kazarinoff, M. N., & McCormick, D. B. (1975) J. Biol. Chem. 250, 3436--3442]. The KD values and spectra for selected apoenzyme--flavin complexes have been determined and used to elucidate some of the properties of the FMN-binding site of this flavoprotein. Alterations of the pyrimidinoid portion of the flavin ring decrease binding considerably. The absorption spectra for the protein complexes with 3-deaza-FMN and 8-hydroxy-FMN indicate the presence of a dipolar or positively charged protein group near N1 and O2. The substitution of methyl for hydrogen at N3 apparently causes distortion of the interaction between the flavin ring and an active-site aromatic amino acid residue. Although binding is also decreased somewhat by substitutions at postions 8 and 8 alpha, considerable bulk [e.g., 8-(diethylamino)-FMN and 8 alpha-S-(N-acetyl-cysteinyl)-FMN] is accommodated. Hence, this portion of the flavin ring is probably oriented toward, possibly in contact with, solvent, as has been found for the flavodoxins. The importance of optimum interactions between the flavin and the apoprotein is further emphasized by large differences in the activity of flavin analogues that have similar midpoint potentials in solution.     

N-Benzyl-1-heteroaryl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamides as inhibitors of co-activator associated arginine methyltransferase 1 (CARM1)

A series of N-benzyl-1-heteroaryl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamides targeting co-activator associated arginine methyltransferase 1 (CARM1) have been designed and synthesized. The potency of these inhibitors was influenced by the nature of the heteroaryl fragment with the thiophene analogues being superior to thiazole, pyridine, isoindoline and benzofuran based inhibitors.     

3-(2-pyrrolidin-1-ylethyl)-5-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole derivatives as high affinity human 5-HT(1B/1D) ligands

A series of 3-(2-pyrrolidin-1-ylethyl)-5-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole derivatives (2) has been prepared using parallel synthesis techniques, and their structure-activity relationships studied. High affinity human 5-HT(1B/1D) (h5-HT(1B/1D)) ligands have been identified.     

Synthesis and biological activity of (D)Ala2, Leu5-enkephalins containing hydrophilic or hydrophobic moieties

The synthesis in solution of some modified (D)Ala²,Leu⁵-enkephalins has been carried out. The lipophilic properties of the parent compound have been modified by amidation of the carboxyl function with alkylamines of increasing hydrophilicity to increase permeability of the blood-brain barrier. Attempts to reduce enzymatic degradation have been carried out either by reductive glucosamination or by amidation of the carboxyl function with 2-amino-2-deoxy-β-D-glucopyranose. Esterification of the carboxyl function of (D)Ala²,Leu⁵-enkephalin with polyethylenglycole 1000 has also been carried out. The effects induced by these modifications have been evaluated by in vitro and in vivo tests.     

A beta-turn is present in the 392-411 segment of the human fibrinogen gamma-chain. Effects of structural changes in this segment on affinity to antibody 4A5.

The interaction between fibrinogen gamma-peptide 392-411, LTIGEGQQHHLGGAKQAGDV, and monoclonal antibody 4A5, an antibody with a high affinity for both for the peptide and native fibrinogen, is being studied as a model for peptide-antibody interaction. Two-dimensional NMR studies of the free peptide at pH 5.2 indicated the presence of a significant population, about 60%, of type II beta-turn, spanning residues Gln407-Asp410. At pH 2.7, little, if any, turn structure is present. The D-Ala409 analog, which, for steric reasons, would be expected to preserve the beta-turn, and the L-Ala409 analog, which would not be expected to have this conformational feature, were synthesized, and NMR studies confirmed the respective structural predictions. The affinity of the D-Ala analog for antibody 4A5 is even greater than that displayed by native gamma 392-411, while the affinity of the L-Ala analog is less than one-tenth that of the native peptide. Both conformational and steric effects involving residues 407-410 may be important in recognition by antibody 4A5. Since gamma 392-411 includes a platelet receptor binding locus of fibrinogen, and this and related peptides are inhibitors of platelet aggregation, the D-Ala409 and L-Ala409 analogs were tested for platelet binding. Neither of the analogs displays any measurable platelet binding, indicating that the recognition requirements for the platelet receptor differ considerably from those for antibody 4A5.     

Resistance profiles of (+)2'-deoxy-3'-oxa-4'-thiocytidine and (-)2'-deoxy-3'-oxa-4'-thio-5-fluorocytidine.

Resistant variants were selected in vitro against two novel nucleoside analogues, (+) dOTC and (-) dOTFC using the HIV-1 molecular clone HXB2D. The variants obtained displayed 6.5-fold and 10-fold resistance to these compounds, respectively. Cloning and sequencing of the RT genes of the selected viruses identified two mutations, M184I for (+) dOTC and M184V for (-) dOTFC. Results with mutated recombinant clones of HXB2D confirmed the importance of these mutations in MT-4 cells. The resistance profiles of clinical samples with wild-type or 3TC-resistant phenotypes were also studied; low to moderate levels of cross-resistance were observed against the novel compounds.     

Synthesis of 2-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-5a-carba-alpha-D- mannopyranose, and 5a-carba-2-O- and 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoses.

Protected derivatives of the title carba-trisaccharide 1, and carba-disaccharides 4 and 6 have been synthesised by conventional glycosylation of the precursors 8, 9, or 22 of 5a-carba-alpha-D-mannopyranose with sugar halides. These are carba-sugar analogues of the oligosaccharide chains frequently occurring as structural units in biologically important glycoconjugates, and could be utilised as the building blocks for construction of higher carba-oligosaccharides of biological interest.     

Aminonucleosides and their derivatives. IV. Synthesis of the 3'-amino-3'-deoxynucleoside 5'-phosphates.

A new procedure has been developed for the synthesis of 3'-amino-3'-deoxyribonucleosides of adenine, cytosine and uracil by condensing the trimethylsilylated bases with peracylated 3-azido-3-deoxyribose derivative. The azido group could subsequently be reduced to amino. The 5'-phosphates of these nucleosides have been prepared and the analogues have been tested for their ability to stimulate the ribosome-catalyzed reaction of 3'(2')-O-(N-formylmethionyl) adenosine 5'-phosphate with phenylalanyl-tRNA.     

Synthesis and biofilm inhibition studies of 2-(2-amino-6-arylpyrimidin-4-yl)quinazolin-4(3H)-ones

Synthesis of novel 4(3H)-quinazolinonyl aminopyrimidine derivatives has been achieved via quinazolinonyl enones which in turn were obtained from 2-acyl-4(3H)-quinazolinone. They have been assayed for biofilm inhibition against Gram-positive (methicillin-resistant Staphylococcus aureus (MRSA)) and Gram-negative bacteria (Acinetobacter baumannii). The analogues with 2,4,6-trimethoxy phenyl, 4-methylthio phenyl, and 3-bromo phenyl substituents (5h, 5j & 5k) have been shown to inhibit biofilm formation efficiently in MRSA with IC₅₀ values of 20.7–22.4 μM). The analogues 5h and 5j have demonstrated low toxicity in human cells in vitro and can be investigated further as leads.     

Isomerization of (3S)-2,3-dihydro-5-fluoro-L-tryptophan and of 5-fluoro-L-tryptophan catalyzed by tryptophan synthase: studies using fluorine-19 nuclear magnetic resonance and difference spectroscopy

We are exploring the active site and the mechanism of the pyridoxal phosphate dependent reactions of the bacterial tryptophan synthase alpha 2 beta 2 complex by use of substrate analogues and of reaction intermediate analogues. Fluorine-19 nuclear magnetic resonance studies and absorption spectroscopy are used to study the binding and reactions of the D and L isomers of 5-fluorotryptophan, of tryptophan, and of (3S)- and (3R)-2,3-dihydro-5-fluorotryptophan. Tryptophan synthase specifically and tightly binds the 3S diastereoisomer of both 2,3-dihydro-5-fluoro-D-tryptophan and 2,3-dihydro-5-fluoro-L-tryptophan, whereas it binds 5-fluoro-D-tryptophan more tightly than 5-fluoro-L-tryptophan. Unexpectedly, we find that the D and L isomers of 5-fluorotryptophan, of tryptophan, and of (3S)-2,3-dihydro-5-fluorotryptophan are slowly interconverted by isomerization reactions. Since these isomerization reactions are 10(3)-10(5) times slower than the beta-replacement and beta-elimination reactions catalyzed by tryptophan synthase, they have no biochemical significance in vivo. However, the occurrence of these slow reactions does throw some light on the nature of the active site of tryptophan synthase and its requirements for substrate binding. Our results raise the interesting question of whether tryptophan synthase itself serves a catalytic role in these slow reactions or whether the enzyme simply binds the substrate and pyridoxal phosphate stereospecifically and thus promotes the intrinsic catalytic activity of pyridoxal phosphate.     

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.