Targeting urokinase and the transferrin receptor with novel, anti-mitotic N-alkylisatin cytotoxin conjugates causes selective cancer cell death and reduces tumor growth.

Tumor-specific delivery of ligand-directed prodrugs can increase the therapeutic window of chemotherapeutics by maintaining efficacy whilst decreasing toxic side effects. We have previously described a series of synthetic N-alkylated isatin cytotoxins that destabilize microtubules and induce apoptosis with 10-fold greater potency than conventional anti-mitotics in vitro. Here, we report the characterization, in vitro cytotoxicity and in vivo efficacy of a lead compound, 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin (N-AI) conjugated via an esterase-labile linker (N-AIE) to two proven targeting ligands, transferrin (Tf) and plasminogen activator inhibitor type 2 (PAI-2/serpinB2). N-AI was released from N-AIE and the targeting ligands Tf/PAI-2 in an esterase-dependent manner at 37 C and both Tf- and PAI-2-N-AIE conjugates were stable at physiological pH. Human cancer cell lines which vary in their expression levels of Tf receptor (TfR/CD71) and PAI-2 target, receptor bound urokinase (uPA) selectively internalized the conjugates. Tf-N-AIE was up to 24 times more active than the free drug and showed clear selectivity patterns based on TfR levels. PAI-2-N-AIE showed equivalent activity compared to the parent drug and strong selectivity patterns for uPA levels. In preliminary in vivo experiments, the PAI-2- and Tf-N-AIE conjugates were efficacious at 1/20(th) and 1/10(th) of the dose of the free N-AI, respectively, in a metastatic, orthotopic human breast tumor xenograft mouse model. Thus, this strategy specifically delivers and concentrates a novel class of isatin-based, tubulin destabilizing agents to tumors in vivo and warrants further detailed preclinical investigation.

An adsorption study of Al(III) ions onto chitosan.

The adsorption of Al(III) from aqueous solutions onto chitosan was studied in a batch system. The isotherms and the kinetics of adsorption with respect to the initial Al(III) concentration and temperature were investigated. Langmuir and Freundlich adsorption models were applied to describe the experimental isotherms. Equilibrium data fitted very well to the Langmuir model in the entire concentration range (5-40 mg/L). The negative values of free energy (DeltaG degrees ) and enthalpy (DeltaH degrees ) for the adsorption of Al(III) onto chitosan indicated that the adsorption process is a spontaneous and exothermic one. Two simplified kinetic models, based on pseudo first-order and pseudo second-order equations, were tested to describe the adsorption mechanism. The pseudo second-order kinetic model resulted in an activation energy of 56.4 kJ/mol. It is suggested that the overall rate of Al(III) ion adsorption is likely to be controlled by the chemical process. The values of the enthalpy (DeltaH(#)) and entropy (DeltaS(#)) of activation were 53.7 kJ/mol and -164.4 J/molK, respectively. The free energy of activation (DeltaG(#)) at 30 degrees C was 103.5 kJ/mol.

A new flavonoid from Chromolaena odorata.

Fractionation of the aerial parts of Chromolaena odorata afforded a new flavonoid, 5,7-dihydroxy-6,4'-dimethoxyflavanone, in addition to 14 known flavonoid compounds, six of which had not been isolated previously from this plant. The structure of the new compound was determined by spectroscopic methods particularly 2D-NMR analysis.

Constituents of the essential oil from aerial parts of Chromolaena odorata from Thailand.

The chemical composition of the essential oil from the aerial parts of Chromolaena odorata, collected from Phitsanulok, Thailand was analyzed by means of GC-(FID) and GC-MS. Twenty-two constituents were identified. The major components were pregeijerene (17.6%), germacrene D (11.1%), alpha-pinene (8.4%), beta-caryophyllene (7.3%), vestitenone (6.5%), beta-pinene (5.6%), delta-cadinene (4.9%), geijerene (3.1%), bulnesol (2.9%), and trans-ocimene (2.2%).

Isolation and NMR spectroscopic clarification of the alkaloid 1,3,7-trimethylguanine from the ascidian Eudistoma maculosum.

The alkaloid 1,3,7-trimethylguanine was isolated from the ascidian Eudistoma maculosum, and differences in the NMR spectroscopic data for this compound compared with data in the literature are due to ammonium salt formation.

5-substituted 3,4-dihydro-3-amino-2H-1-benzopyran derivatives: synthesis and interaction with serotoninergic receptors.

A new series of 3,4-dihydro-3-amino-2H-1-benzopyran derivatives (1 and 2) bearing various substituents on the 5-position was successfully prepared via palladium-mediated cross-coupling reactions. Some of the new compounds showed high affinity for 5-HT1A and 5-HT7 receptors. The best affinity for the 5-HT1A and 5-HT7 receptors was obtained for 2b (Ki = 0.3 nM for 5-HT1A and 3.1 nM for 5-HT7). The anxiolytic activity of compound 2b was evaluated.

Ligand design for alpha(1) adrenoceptors.

An area of continuing interest in medicinal chemistry is the design, synthesis and pharmacological evaluation of ligands which bind at adrenoceptor subtypes, which include alpha(1A), alpha(1B), alpha(1D); alpha(2A), alpha(2B), alpha(2C); beta(1), beta(2), beta(3) and possibly beta(4) subtypes. The selective blockade or stimulation of these receptor subtypes is of on-going pharmacological and medicinal interest. However, the design principles for ligand differentiation at these subtypes still need further development. This review focuses on alpha(1) adrenoceptors with a concentration on literature over the past five years. Structural, physiological and therapeutic aspects of the alpha(1A), alpha(1B) and alpha(1D) subtypes are discussed together with ligands binding to these receptor subtypes. Approaches to alpha(1) adrenoceptor ligand design based on known ligands and on receptor docking are evaluated. A new combined approach using pharmacophores and receptor docking affords possibilities for deeper insights into achieving small molecule binding selectivity.

Chemical defense in the egg masses of benthic invertebrates: an assessment of antibacterial activity in 39 mollusks and 4 polychaetes.

Many marine invertebrates deposit benthic egg masses that are potentially vulnerable to microbial infection. To help counter this threat these species may have evolved some form of chemical protection for their encapsulated embryos. In this study the egg masses from 7 marine mollusks were tested for antibacterial activity against 4 marine pathogens: Enterococcus sericolicida, Vibrio anguillarum, Vibrio alginolyticus, and Vibrio harveyi. Extracts from all of these egg masses were found to inhibit the growth of at least 1 marine bacterium at concentrations that approximate the natural concentration of extract in the egg masses. The egg masses of 39 mollusks and 4 polychaetes were then tested for antibacterial activity against 3 human pathogenic bacteria; Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Activity was detected in the egg masses from 34 species, including 2 polychaetes and mollusks from two classes and 18 families. Antibacterial activity in molluskan egg masses was found to extend across the marine, estuarine, freshwater, and terrestrial environments. Both gelatinous egg masses and tough egg capsules were found to inhibit microbial growth, suggesting that physical protection alone may not be sufficient to protect the eggs. Antimicrobial activity was observed in the fresh egg masses but not in the well-developed egg masses of a subset of species. The results of this study indicate that a wide range of invertebrates use chemical defense to protect their early stage embryos against bacterial infection.

Ligand design for alpha1 adrenoceptor subtype selective antagonists.

Alpha1 adrenoceptors have three subtypes and drugs interacting selectively with these subtypes could be useful in the treatment of a variety of diseases. In order to gain an insight into the structural principles governing subtype selectivity, ligand based drug design (pharmacophore development) methods have been used to design a novel 1,2,3-thiadiazole ring D analogue of the aporphine system. Synthesis and testing of this compound as a ligand on cloned and expressed human alpha1 adrenoceptors is described. Low binding affinity was found, possibly due to an unfavourable electrostatic potential distribution. Pharmacophore models for antagonists at the three adrenoceptor sites (alpha1A, alpha1B, alpha1D) were generated from a number of different training sets and their value for the design of new selective antagonists discussed. The first preliminary antagonist pharmacophore model for the alpha1D adrenoceptor subtype is also reported.

Antibacterial compounds from Carissa lanceolata R.Br.

The dichloromethane extract of the wood of Carissa lanceolata R.Br. (Apocynaceae) afforded the eudesmanes carissone, dehydrocarissone and carindone. This is the first account of carissone being isolated from the wood of C. lanceolata, and of carindone being isolated from this Carissa species. Dehydrocarissone has not been isolated previously from any Carissa species. The antibacterial activity of these natural products were examined against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. All three compounds showed activity, with dehydrocarissone and carindone having a minimum inhibitory concentration less than 0.5 mg/ml against S. aureus and E. coli.

Modelling of adrenoceptor ligand targets based on novel medium- or macro-sized fused nitrogen heterocyclic systems.

Novel medium- and macro-sized heterocyclic compounds were assessed for their potential as subtype-selective adrenergic ligands. Their conformational flexibilities were investigated and their geometric shapes were compared to rigid lead compounds of known selectivity. In the case of alpha 1A selective antagonists, interesting potential targets for synthesis and evaluation were identified by 'opening up' various rings of the fused-ring lead compound 1 by shared-bond cleavage. For alpha 2 selective ligands, compound 6 was the lead compound and the possibility of mimicking the fused-ring system via intramolecular hydrogen bonding was investigated. None of the potential targets were closely enough related in this case to the lead compound to warrant synthesis.

A quantitative investigation of the transannular amine-ketone (N...C=O) interaction in medium-sized heterocycles.

The transannular N...C=O interaction in several medium-sized heterocycles has been investigated by force-field methods. Conformational searching has been performed at the molecular mechanics level using different methods and the conformers so generated have been reoptimized at RHF/6-31G(d). For selected conformers, N...C distances are reported, together with transannular bond orders and atomic charges. Good agreement with available X-ray crystallographic data is obtained for the transannular bond distance in cryptopine. Changes in the partial atomic charges derived from the electrostatic potential provide good support for the donor-acceptor model of transannular interactions. Partial charges derived with other methods do not give satisfactory results. Some force fields do not reproduce the transannular interaction very well. This is demonstrated and rationalized, and modifications are suggested and tested for these force fields with good results when comparing diagnostic geometric features with X-ray data.

Pharmacophore development for antagonists at alpha 1 adrenergic receptor subtypes.

Many receptors, including alpha 1 adrenergic receptors, have a range of subtypes. This offers possibilities for the development of highly selective antagonists with potentially fewer detrimental effects. Antagonists developed for alpha 1A receptors, for example, would have potential in the treatment of benign prostatic hyperplasia. As part of the molecular design process, structural features necessary for the selective affinity for alpha 1A and alpha 1B adrenergic receptors have been investigated. The molecular modelling software (particularly the Apex module) of Molecular Simulations, Inc. was used to develop pharmacophore models for these two subtypes. Low-energy conformations of a set of known antagonists were used as input, together with a classification of the receptor affinity data. The biophores proposed by the program were evaluated and pharmacophores were proposed. The pharmacophore models were validated by testing the fit of known antagonists, not included in the training set. The critical structural feature for selectivity between the alpha 1A and alpha 1B adrenergic receptor sites is the distance between the basic nitrogen atom and the centre of an aromatic ring system. This will be exploited in the design and synthesis of structurally new selective antagonists for these sites.

Antigenotoxic spinasterol from Cucurbita maxima flowers.

The antigenotoxic constituent of squash flowers was isolated by solvent partitioning and repeated vacuum liquid chromatography. The micronucleus test, an in vivo method, was used to monitor the antigenotoxicity of the various fractions during the isolation process. Isolate SQFwB2D from the chloroform extract of squash flowers is the most antigenotoxic isolate. It decreased the mutagenicity of tetracycline by 64.7% at a dosage of 100 mg/kg mouse. Statistical analysis using Kruskall Wallis one-way analysis of variance by Ranks showed that SQFw2D is different from the control group (tetracycline + corn oil) at alpha = 0.001. GC-MSD of isolate SQFwB2D shows 2 peaks at Rt = 19.860 (SQFwB2D-1) and 20.242 min (SQFwB2D-2) with relative peak heights of 16:1, respectively. Spectral analyses show that SQFwB2D-1 is 24 alpha-ethyl-5 alpha-cholesta-7,trans-22-dien-3 beta-ol or spinasterol.

The action of 1,2,3,5,6,11b-hexahydro-[1]benzothieno [3,2-g]indolizine hydrochloride (ADT 16) on peripheral and central responses mediated by 5-hydroxytryptaminergic and adrenergic systems of the rat.

Some acute pharmacological effects have been examined of racemic ADT 16 (1,2,3,5,6,11b-hexahydro[1]benzothieno[3,2-g]indolizine hydrochloride), on peripheral and central responses mediated by 5-HT and adrenergic systems in the rat. In-vitro, ADT 16 (10-1000 nM), similarly to mianserin, antagonized the inhibitory responses to B-HT 920 of the electrically-stimulated rat isolated prostatic vas deferens. High concentrations of ADT 16 (10 microM), also resembled those of mianserin by potentiating twitch responses to electrical stimulation of the tissue. Contractile responses to phenylephrine of rat isolated epididymal vas deferens were antagonized by ADT 16 (0.3-1 microM). In the rat stomach fundus strip, ADT 16 (1-3 microM) antagonized contractions due to 5-HT. ADT 16 (0.1-1 microM) had no effect on responses to acetylcholine of the guinea-pig isolated ileum. In-vivo, in spinalized, decerebrated rats, fenfluramine- or clonidine-induced facilitation of flexor reflex activity of the anterior tibialis muscle was attenuated by ADT 16 (3 and 10 mg kg-1, i.v., and 3 mg kg-1, i.v. respectively). In the anaesthetized rat, L-3,4-dihydroxyphenylalanine (L-dopa)- or L-5-hydroxytryptophan (L-5-HTP)-induced increases in the frequency of spontaneous twitches of the anterior digastricus muscle were attenuated by ADT 16 (1 and 3 mg kg-1, i.v.; n = 4). It is concluded that ADT 16, similarly to mianserin, is a novel peripherally and centrally active antagonist of 5-HT and adrenergic responses in the rat.

Epi-sarsasapogenin and epi-smilagenin: two sapogenins isolated from the rumen content of sheep intoxicated by Brachiaria decumbens.

Spectroscopic examination of purified extracts of the rumen content of sheep intoxicated by Brachiaria decumbens revealed the presence of two spirostanes, identified as epi-sarsasapogenin and epi-smilagenin. Sarsasapogenone was obtained by the oxidation of sarsasapogenin. The reduction of sarsasapogenone using lithium aluminum hydride yielded isomeric products, sarsasapogenin (20%) and epi-sarsasapogenin (80%).

Structure of an antimutagen from Carmona retusa leaves.

An antimutagenic compound was isolated from the leaves of Carmona retusa (Vahl) Masam. Its structure has been elucidated by spectral analysis to be 4-hydroxy-7,8,11,12,15,7',8',11',12',15'-decahydro-beta, psi-carotene. The results of the Micronucleus Test, an in vivo method, showed that the isolated antimutagenic compound reduced by approximately 68.4% the number of micronucleated polychromatic erythrocytes induced by tetracycline, a known mutagen.

Hepatotoxic constituents in the rumen of Brachiaria decumbens intoxicated sheep.

Spectroscopic examinations of purified extracts of the rumen content of sheep intoxicated by Brachiaria decumbens revealed the presence of a mixture of sapogenins, identified as 3-spirostanols. These isomeric steroid sapogenins (C27H44O3) are believed the toxic principles in causing toxicity in sheep after feeding on B. decumbens.

The effects of replacing ester by amide on the biological properties of compounds related to acetylcholine.

1 Replacement of ester by amine in series of derivatives of diphenylacetic acid reduces the affinity for muscarine-sensitive acetylcholine receptors of the guinea-pig ileum from 40- to 100-fold. With similar series of phenylacetic acid the reduction is only 2- to 4-fold. In both series changes in the composition of the onium group produce similar changes in the affinity of amides and esters and it appears that the stiffness of the amide bond reduces the binding of the phenyl groups at the far end of the molecule from the onium atom. 2 Replacement of ester by amide in similar series of acetyl compounds reduces activity on the guinea-pig ileum over 1000-fold and on the frog rectus over 50-fold. Compounds with larger onium groups are antagonists on both preparations with log affinity constant around 3. The amides have similar affinity for electric eel acetylcholinesterase. 3 The amides are slightly bigger than the esters in solution and slightly more hydrophilic. 4 Replacement of ester by amide in acetylcholine reduces the proportion of gauche conformer about the C--C--bond from 100% to 39%. 5 The ability of acetylcholine to activate receptors is thought to depend on some degree of flexibility in the --CO--0--bond, though the hydration of the bond may also be important.