Synthetic approaches towards the marine alkyl purines.

Agelasines, asmarines and related compounds are natural products with a hybrid terpene-purine structure isolated from numerous genera of sponges (Agela sp, Raspailia sp). Nuttingins and malonganenones are tetraprenylated purine alkaloids from gordonian (Eplexura sp, Leptogorgia sp). Some of these alkaloids displayed broad spectrum activity including cytotoxic activity against several cancer cells. The review summarizes the synthesis of mono- or bi-cyclic diterpenoids usually having a 9-methyladenine moiety.

First analytical evidences of precious colourants on Mediterranean illuminated manuscripts.

Two Byzantine VI century manuscripts known as Vienna Dioskurides and Vienna Genesis, held in the Austrian National Library at Vienna, were analysed with in situ non-invasive techniques. Raman spectroscopy, UV-Vis diffuse reflectance spectrophotometry with optic fibres and X-ray fluorescence spectrometry were used to characterise the palette of these early Middle Ages manuscripts. The analytical study was performed to have a better knowledge on the colourants used by ancient miniature painters, a subject known more on the basis of traditional sources (i.e. medieval treatises) than of analytical evidences. Indeed these illuminated manuscripts are, to the authors' knowledge, among the oldest ever being analysed, so that the colourants found in them can be considered among the oldest evidences of their use. The main feature of Vienna Dioskurides and Vienna Genesis palettes is their richness, exemplified by the simultaneous presence of gold and ultramarine blue; in Vienna Dioskurides cinnabar is also present. Information regarding ultramarine blue is surprising, being the analytical evidence of the use of this precious pigment at least three centuries before its use in Western manuscripts, a feature justified by the fact that the Byzantine Empire was the dominant culture in early Middle Ages in the Mediterranean World. Other colourants include azurite and indigo, red lead, orpiment, red and yellow ochres, while a mixture of blue and yellow colourants, known as vergaut, was used to render green hues. Organic colourants were also used, such as madder and Tyrian purple, the latter employed to dye the parchment of Vienna Genesis.

Quantification of carbamazepine and its active metabolite by direct injection of human milk serum using liquid chromatography tandem ion trap mass spectrometry.

This work reports the use of a liquid chromatography ion trap tandem mass spectrometry (LC-IT-MS/MS) system for quantification in human milk samples of both carbamazepine (CBZ) and its active metabolite, carbamazepine 10,11-epoxide (CBZE). An octadecyl restricted-access media bovine serum albumin column (RAM-BSA C(18)) was used in single-column mode. Selectivity, extraction efficiency, accuracy and precision were achieved employing 100 µL of the sample, without preparation, with detection limits of 20.0 ng/mL for CBZ and 40.0 ng/mL for CBZE. The matrix effect was investigated for the compounds by post-column infusion (qualitative) and by on-line extraction (quantitative). It was observed suppression effect for CBZ and CBZE by post-column infusion, ion suppression of 0.80 for CBZ, and enhancement of 1.28 for CBZE by on-line extraction. The developed method was validated and applied to analyze breast milk samples from one nursing mother. CBZ and CBZE were quantified in the concentrations of 2.26 µg/mL and 1.54 µg/mL, respectively. To our knowledge, this is the first report on the simultaneous determination of CBZ and its active metabolite by direct injection of human milk serum.

Spectral analysis of pharmaceutical formulations prepared according to ancient recipes in comparison with old museum remains.

A study of the composition of the remains of ancient ointments from museums was undertaken to enable understanding of the preparation techniques. Comparison of ancient recipes from different historical periods and spectroscopic characteristics of inorganic and/or organic remains recovered in museum vessels enabled preparation of ancient pharmaceutical-cosmetic formulations. Farmacopea Augustana by Occo was one the most important books studied for the 14 formulations prepared in the laboratory. Three formulations are discussed in detail and raw materials and new preparations were proposed for ozone ageing. The most important micro Raman results are discussed. The spectra of the raw materials lipids, beeswax, and resins are discussed; beeswax and pig suet (axungia) Raman spectra were found to be similar, but different from those of the aged oils. SERS was applied to ancient ointments and galbanum and the Raman spectra are reported and discussed for the first time.

Allosteric enhancers of A1 adenosine receptors: state of the art and new horizons for drug development.

Adenosine is an important autocoid, exerting its physiological effects on the human body by activation of four different G-protein-coupled-receptors (GPCRs) classified as A(1), A(2A), A(2B), and A(3). These receptors are coupled to secondary messenger systems including adenylate cyclase, inositol phosphate metabolism, and K(+), K(ATP) and Ca(2+) channels. Pharmacological agents that increase the activation of A(1) adenosine receptors in response to adenosine would be useful for treatment of cardiovascular, central nervous system, and inflammatory pathologies. Compounds that are able to enhance the activity of the A(1)-adenosine receptors by the endogenous ligand within specific tissues may have potential therapeutic advantages over non-endogenous agonists. Such an opportunity for intervention is provided by the concept of allosteric modulation of GPCRs. Therefore the use of allosteric enhancers to increase the responsiveness of the A(1) receptors to endogenous adenosine at sites of its production is an appealing alternative to activation by exogenous agonists. This approach minimizes side effects because allosteric enhancers amplify the action of the agonist by stabilizing the agonist-A(1)-receptor-G protein ternary complex. The allosteric enhancement of the GABA(A) receptor by benzodiazepines is the most famous and successful example of this strategy. The aim of this article is to give an overview of the results obtained in this field and discuss the opportunities and challenges that this class of ligands might offer for medicinal chemistry and pharmacology.

Transient receptor potential ankyrin receptor 1 is a novel target for pro-tussive agents.

BACKGROUND AND PURPOSE: The transient receptor potential ankyrin receptor 1 (TRPA1) is a cation channel, co-expressed with the pro-tussive transient receptor potential vanilloid type 1 (TRPV1) channel in primary sensory neurons. TRPA1 is activated by a series of irritant exogenous and endogenous alpha,beta-unsaturated aldehydes which seem to play a role in airway diseases. We investigated whether TRPA1 agonists provoke cough in guinea pigs and whether TRPA1 antagonists inhibit this response. EXPERIMENTAL APPROACH: Animals were placed in a Perspex box, and cough sounds were recorded and counted by observers unaware of the treatment used. KEY RESULTS: Inhalation of two selective TRPA1 agonists, allyl isothiocyanate and cinnamaldehyde, dose-dependently caused cough in control guinea pigs, but not in those with airway sensory nerves desensitized by capsaicin. Coughs elicited by TRPA1 agonists were reduced by non-selective (camphor and gentamicin) and selective (HC-030031) TRPA1 antagonists, whereas they were unaffected by the TRPV1 antagonist, capsazepine. Acrolein and crotonaldehyde, two alpha,beta-unsaturated aldehydes recently identified as TRPA1 stimulants and contained in cigarette smoke, air pollution or produced endogenously by oxidative stress, caused a remarkable tussive effect, a response that was selectively inhibited by HC-030031. Part of the cough response induced by cigarette smoke inhalation was inhibited by HC-030031, suggesting the involvement of TRPA1. CONCLUSIONS AND IMPLICATIONS: A novel pro-tussive pathway involves the TRPA1 channel, expressed by capsaicin-sensitive airway sensory nerves and is activated by a series of exogenous (cigarette smoke) and endogenous irritants. These results suggest TRPA1 may be a novel target for anti-tussive medicines.

Isotopic analysis of lead present in the cinnabar of Roman wall paintings from the Xth Regio "(Venetia et Histria)" by ICP-MS.

The analysis of Roman wall paintings coming from Verona, Vicenza, Padova, Pordenone and Trieste evidentiated the presence of cinnabar. In this work, the isotopic composition (206)Pb, (207)Pb and (208)Pb of trace of lead present in cinnabar is reported, via ICP-MS measurements. The isotopic ratio values are compared with the values obtained from samples coming from Roman wall paintings of Pompeii and from the mines of Almaden (Spain) Monte Amiata (Grosseto Italy) and Idria (Slovenia). All the isotopic data can be represented by a "field" ranging from Huelva, to Almeria Spanish provinces. This trend could be due to the mixing of cinnabar products treated in Rome.

Touch or pain? Spatio-temporal patterns of cortical fMRI activity following brief mechanical stimuli.

Most imaging studies on the human pain system have concentrated so far on the spatial distribution of pain-related activity. In the present study, we investigated similarities and differences between the spatial and temporal patterns of brain activity related to touch vs. pain perception. To this end, we adopted an event-related functional magnetic resonance imaging (fMRI) paradigm allowing us to separately assess the activity related to stimulus anticipation, perception, and coding. The fMRI signal increases following brief mechanical noxious or non-noxious stimulation of the hand dorsum were largely overlapping in the contralateral and ipsilateral hemispheres, including portions of the parietal, insular, frontal and cingulate cortices. Higher activity following noxious stimulation was found in the contralateral mid-anterior insular cortex, in the anterior mid-cingulate cortex (aMCC) and in the adjacent dorso-medial frontal cortex. Significant decreases in fMRI signals following both tactile and painful stimuli were found in perigenual cingulate (pACC)/medial prefrontal cortex (MPF) and in the posterior cingulate/precuneus/paracentral lobule; more intense decreases were found in the pACC/MPF following painful stimuli. fMRI signal increases in the contralateral insula and in aMCC, but not in the parietal cortex, were more prolonged following painful than tactile stimuli. Moreover, a second peak of signal increases (albeit of lower intensity) was found in anterior insula and aMCC during pain intensity rating. These results show specific spatio-temporal patterns of cortical activity related to processing noxious vs. non-noxious mechanical stimuli.

Effects of ketamine anesthesia on central nociceptive processing in the rat: a 2-deoxyglucose study.

Ketamine is a dissociative anesthetic with complex actions on the CNS. We investigated here the effects of ketamine anesthesia on somatosensory processing in the rat spinal cord, thalamus, and cerebral cortex, using the quantitative 2-deoxyglucose mapping technique. Unanesthetized or ketamine-anesthetized male Sprague-Dawley rats received a s.c. injection of a dilute formaldehyde solution (5%, 0.08 ml) into a forepaw, inducing prolonged noxious afferent input, or an equal volume of isotonic saline as a control stimulus. The 2-deoxyglucose experiments started 30 min after the injection. In the cervical enlargement of the spinal cord, ketamine had no significant effect on glucose metabolic rates in saline-injected animals, whereas it prevented the metabolic increases elicited by prolonged noxious stimulation in unanesthetized animals. At the thalamic level, ketamine increased glucose uptake in both saline- and formalin-injected rats in the lateral posterior, lateral dorsal, medial dorsal, gelatinosus, antero-ventral and antero-medial thalamic nuclei, whereas it decreased metabolic activity in the ventro-basal complex. At the cortical level, the drug increased metabolic activity in both control and formalin groups in the lacunosus-molecularis layer of the dorsal hippocampus, posterior parietal, retrosplenial, cingulate and frontal cortex; significant metabolic decreases were found in the CA1 region of the dorsal hippocampus and in the parietal 1 and 2 cortical areas. In the investigated brain regions, ketamine did not abolish noxious-evoked increases in glucose uptake, which were in fact enhanced in the forelimb cortex and in the lacunosus-molecularis layer of the hippocampus. The dissociation between the spinal and supraspinal effects of ketamine suggests a specific antinociceptive action on spinal circuits, in parallel with complex changes of the activity of brain circuits involved in somatosensory processing. More generally, this study shows that functional imaging techniques are able to quantitatively assess the effects of anesthetic drugs on nociceptive processing at different levels of the neuraxis.

Fluorosulfonyl- and bis-(beta-chloroethyl)amino-phenylamino functionalized pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives: irreversible antagonists at the human A3 adenosine receptor and molecular modeling studies.

A series of pyrazolotriazolopyrimidines was previously reported to be highly potent and selective human A(3) adenosine receptor antagonists (Baraldi et al. J. Med. Chem. 2000, 43, 4768-4780). A derivative having a methyl group at the N(8) pyrazole combined with a 4-methoxyphenylcarbamoyl moiety at N(5) position, displayed a K(i) value at the hA(3) receptor of 0.2 nM. We now describe chemically reactive derivatives which act as irreversible inhibitors of this receptor. Electrophilic groups, specifically sulfonyl fluoride and nitrogen mustard (bis-(beta-chloroethyl)amino) moieties, have been incorporated at the 4-position of the aryl urea group. Membranes containing the recombinant hA(3) receptor were preincubated with the compounds and washed exhaustively. The loss of ability to bind radioligand following this treatment indicated irreversible binding. The most potent compound in irreversibly binding to the receptor was 14, which contained a sulfonyl fluoride moiety and a propyl group at the N(8) pyrazole nitrogen. The bis-(beta-chloroethyl)amino derivatives displayed a much smaller degree of irreversible binding than the sulfonyl fluoride derivatives. A computer-generated model of the human A(3) receptor was built and analyzed to help interpret these results. The model of the A(3) transmembrane region was derived using primary sequence comparison, secondary structure predictions, and three-dimensional homology building, using the recently published crystal structure of rhodopsin as a template. According to our model, sulfonyl fluoride derivatives could dock within the hypothetical TM binding domain, adopting two different energetically favorable conformations. We have identified two amino acids, Ser247 and Cys251, both in TM6, as potential nucleophilic partners of the irreversible binding to the receptor.

Pharmacological and biochemical characterization of A3 adenosine receptors in Jurkat T cells.

1. The present work was devoted to the study of A3 adenosine receptors in Jurkat cells, a human leukemia line. 2. The A3 subtype was found by means of RT-PCR experiments and characterized by using the new A3 adenosine receptor antagonist [3H]-MRE 3008F20, the only A3 selective radioligand currently available. Saturation experiments revealed a single high affinity binding site with K(D) of 1.9+/-0.2 nM and B(max) of 1.3+/-0.1 pmol mg(-1) of protein. 3. The pharmacological profile of [3H]-MRE 3008F20 binding on Jurkat cells was established using typical adenosine ligands which displayed a rank order of potency typical of the A3 subtype. 4. Thermodynamic data indicated that [3H]-MRE 3008F20 binding to A3 subtype in Jurkat cells was entropy- and enthalpy-driven, according with that found in cells expressing the recombinant human A3 subtype. 5. In functional assays the high affinity A3 agonists Cl-IB-MECA and IB-MECA were able to inhibit cyclic AMP accumulation and stimulate Ca(2+) release from intracellular Ca(2+) pools followed by Ca(2+) influx. 6. The presence of the other adenosine subtypes was investigated in Jurkat cells. A1 receptors were characterized using [3H]-DPCPX binding with a K(D) of 0.9+/-0.1 nM and B(max) of 42+/-3 fmol mg(-1) of protein. A2A receptors were studied with [3H]-SCH 58261 binding and revealed a K(D) of 2.5+/-0.3 nM and a B(max) of 1.4+/-0.2 pmol mg(-1) of protein. 7. In conclusion, by means of the first antagonist radioligand [3H]-MRE 3008F20 we could demonstrate the existence of functional A3 receptors on Jurkat cells.

Explicit and incidental facial expression processing: an fMRI study.

Considerable evidence indicates that processing facial expression involves both subcortical (amygdala and basal ganglia) and cortical (occipito-temporal, orbitofrontal, and prefrontal cortex) structures. However, the specificity of these regions for single types of emotions and for the cognitive demands of expression processing, is still unclear. This functional magnetic resonance imaging (fMRI) study investigated the neural correlates of incidental and explicit processing of the emotional content of faces expressing either disgust or happiness. Subjects were examined while they were viewing neutral, disgusted, or happy faces. The incidental task required subjects to decide about face gender, the explicit task to decide about face expression. In the control task subjects were requested to detect a white square in a greyscale mosaic stimulus. Results showed that the left inferior frontal cortex and the bilateral occipito-temporal junction responded equally to all face conditions. Several cortical and subcortical regions were modulated by task type, and by facial expression. Right neostriatum and left amygdala were activated when subjects made explicit judgements of disgust, bilateral orbitofrontal cortex when they made judgement of happiness, and right frontal and insular cortex when they made judgements about any emotion.

Design, synthesis, DNA binding, and biological evaluation of water-soluble hybrid molecules containing two pyrazole analogues of the alkylating cyclopropylpyrroloindole (CPI) subunit of the antitumor agent CC-1065 and polypyrrole minor groove binders.

We have synthesized and evaluated a series of hybrids, denoted 22--27, for in vitro cytotoxic activity against a variety of cancer cell lines. These hybrids represent a molecular combination of polypyrrole minor groove binders structurally related to the natural antitumor agent distamycin A and two pyrazole analogues of the left-hand segment called cyclopropylpyrroloindole (CPI) of the potent antitumor antibiotic (+)-CC-1065. These novel water-soluble hybrids have been designed to enhance the minor groove binding ability of alkylating units 20 and 21, which should increase their clinical appeal by overcoming the administration problems of (+)-CC-1065 derivatives. The DNA alkylating and cytotoxic activities against several tumor cell lines are reported and discussed in terms of their structural differences in relation to both the number of N-methyl pyrrole rings and the type of the alkylating unit tethered to the oligopeptidic frame. It may be noted that, in general, and especially for 22--24, the cytotoxicity of the hybrids was much greater than that of the alkylating units alone. In only one case, compound 27, did the hybrid have cytotoxic activity comparable to that of the alkylating unit alone against FM3A/0 cells. The broadest spectrum of activity and greatest potency was shown by the hybrid 24, in which the alkylating unit 20 and the deformyl distamycin A are tethered by 1-methyl 2,5-dicarbonyl pyrazole, with IC(50) values for the different tumor cell lines ranging from 7 to 71 nM. For compounds 22--24, the increase of the length of the pseudopeptidic moiety from one to three N-methylpyrrole residues led to an increased cytotoxicity. Among the hybrids tested for their inhibitory effects on the proliferation of murine L1210 leukemia cell line, compound 24 proved to be the most active (IC(50) = 7.4 nM), and in the sequencing gel experiments, it showed the strongest and most highly sequence-specific DNA alkylation activity. For compounds 22-24, the sequence specificity of DNA alkylation appears to be affected by the modification of the number of pyrrole rings, and the correlation between cytotoxicity and alkylation pattern suggests that 24 exerts its cytotoxicity through DNA sequence-specific alkylation of the third adenine located in the sequence 5'-ACAAAAATCG-3'. The two other hybrids 22 and 23 were slightly less active for tumor cell proliferation, with IC(50) values of 58 and 19 nM, respectively. With only one exception, none of the compounds was endowed with antiviral activity at subtoxic concentrations. Compound 24 inhibited the effect of vaccinia virus at a concentration that was significantly lower than its minimum cytotoxic concentration for the E(6)SM host cells. These compounds gave distinct patterns of alkylation in AT-rich sequences, indicating that minor structural changes produced marked alterations in sequence selectivity.

Design, synthesis and enzymatic activity of highly selective human mitochondrial thymidine kinase inhibitors.

Highly selective arabinofuranosyl nucleosides, which inhibit the mitochondrial thymidine kinase (TK-2) without affecting the closely related herpes simplex virus type 1 thymidine kinase (HSV-1 TK), varicella-zoster virus thymidine kinase (VZV-TK), cytosolic thymidine kinase (TK-1) or the multifunctional Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK), have been obtained. SAR studies indicate a close relation between the length of the substituent at the 2' position of the arabinofuranosyl moiety and the inhibitory activity.

Selective adenosine A2A receptor antagonists.

In the early 1990s it became clear that the A2A adenosine receptor had characteristics that made it distinct from the other A1, A2B and A3 adenosine receptors. Great progress has been made with the discovery of selective A2A receptor antagonists. A variety of synthetic substitutions on the xanthine moiety led the chemists of Kyowa-Hakko to discover that introduction of the styryl group in the 8 position of xanthines was critical in achieving compounds endowed with selective A2A receptor antagonistic properties. One compound, KW 6002, (E)1,3-diethyl-8-(3,4-dimethoxystyryl)-7-methylxanthine, is currently being developed for treatment of Parkinson's disease. A number of non-xanthine heterocycles have also been synthesized starting from the non-selective adenosine antagonist CGS 15943, a triazoloquinazoline. Thus, replacement of the phenyl ring of CGS 15943 with a heterocyclic ring such as pyrazole or imidazole, led to a series of interesting compounds whose prototype, SCH 58261, 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine, has become a reference A2A receptor antagonist. Modification of N7 substituents has progressed to optimize A2A receptor selectivity and pharmacokinetic characteristics. A related class of compounds having a bicyclic instead of the tricyclic ring structure is also of interest. The prototype of these triazintriazolo derivatives, ZM 241385, is a potent A2A receptor antagonist; however, it also shows interactions with A2B receptors. The relevance of the A2A receptors in specific disease states, especially in the central nervous system, makes this class of adenosine receptor blockers of interest for treatment of neurodegenerative disorders such as Parkinson's disease.

Study of the vibrational spectrum of cuprorivaite.

In this research, after tracing a history of the pigment, the preparation and analysis of samples of Egyptian blue have been reported. The main reactions taking place during its preparation are indicated. X-ray diffractions show that, starting from mixtures of precursors with stoichiometric composition, the product is practically only cuprorivaite. The kind of flux and cristallinity of silica does not seem determining factors for the pigment formation. Infrared and Raman spectra and an assignment of the bands are reported. Impurities ascertained are discussed on the basis on the data available.

Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives as highly potent and selective human A(3) adenosine receptor antagonists: influence of the chain at the N(8) pyrazole nitrogen.

An enlarged series of pyrazolotriazolopyrimidines previously reported, in preliminary form (Baraldi et al. J. Med. Chem. 1999, 42, 4473-4478), as highly potent and selective human A(3) adenosine receptor antagonists is described. The synthesized compounds showed A(3) adenosine receptor affinity in the sub-nanomolar range and high levels of selectivity evaluated in radioligand binding assays at human A(1), A(2A), A(2B), and A(3) adenosine receptors. In particular, the effect of the chain at the N(8) pyrazole nitrogen was analyzed. This study allowed us to identify the derivative with the methyl group at the N(8) pyrazole combined with the 4-methoxyphenylcarbamoyl moiety at the N(5) position as the compound with the best binding profile in terms of both affinity and selectivity (hA(3) = 0.2 nM, hA(1)/hA(3) = 5485, hA(2A)/hA(3) = 6950, hA(2B)/hA(3) = 1305). All the compounds proved to be full antagonists in a specific functional model where the inhibition of cAMP generation by IB-MECA was measured in membranes of CHO cells stably transfected with the human A(3) receptor. The new compounds are among the most potent and selective A(3) antagonists so far described. The derivatives with higher affinity at human A(3) adenosine receptors proved to be antagonists, in the cAMP assay, capable of inhibiting the effect of IB-MECA with IC(50) values in the nanomolar range, with a trend strictly similar to that observed in the binding assay. Also a molecular modeling study was carried out, with the aim to identify possible pharmacophore maps. In fact, a sterically controlled structure-activity relationship was found for the N(8) pyrazole substituted derivatives, showing a correlation between the calculated molecular volume of pyrazolo[4,3-e]1,2, 4-triazolo[1,5-c]pyrimidine derivatives and their experimental K(i) values.

[2,1-c][1,4]benzodiazepine (PBD)-distamycin hybrid inhibits DNA binding to transcription factor Sp1.

We designed and synthesized the hybrid 6, prepared combining the minor groove binders distamycin A and pyrrolo [2,1-c][1,4] benzodiazepine (PBD) 4, related to the natural occurring anthramycin (2) and DC-81 (3). In this paper, the effects of the compound 6 on molecular interactions between DNA and transcription factor Sp1 were studied. The results obtained demonstrate that PBD-distamycin hybrid is a powerful inhibitor of Sp1/DNA interactions.