Identification of N,N-arylalkyl-picolinamide derivatives targeting the RNA-binding protein HuR, by combining biophysical fragment-screening and molecular hybridization.

Hu proteins are members of the RNA-binding protein (RBP) family and play a pivotal role in the regulation of post-transcriptional processes. Through interaction with selected mRNAs, RBPs regulate their function and stability; as a consequence, RBP dysregulation can cause abnormal translation of key proteins involved in several pathologies. In the past few years, this observation has sparked interest to develop new treatments against these pathologies by using small molecules able to modulate RBP activity. Among the four Hu proteins, we have directed our efforts towards the isoform HuR, which is mainly involved in cancer, inflammation and retinopathy. Aimed at developing compounds able to modulate the stability of HuR-mRNA complexes, in the present work, we applied a biophysical fragment screening by assessing a library of halogen-enriched heterocyclic fragments (HEFLibs) via Surface Plasmon Resonance (SPR) and Saturation Transfer Difference (STD) NMR to select promising fragments able to interact with HuR. One selected fragment and a few commercially available congeners were exploited to design and synthesize focused analogues of compound N-(3-chlorobenzyl)-N-(3,5-dihydroxyphenethyl)-4-hydroxybenzamide (1), our previously reported hit. STDNMR spectroscopy, molecular modeling, and SPR offered further insight into the HuR-small molecule interaction and showed that fragment-based approaches represent a promising and yet underexplored strategy to tackle such unusual targets. Lastly, fluorescence polarization (FP) studies revealed the capability of the new compounds to interfere with the formation of the HuR-mRNA complex. This is, to our knowledge, the first fragment-based campaign performed on the Hu protein class, and one of the few examples in the larger RBP field and constitutes an important step in the quest for the rational modulation of RBPs and related RNA functions by small molecules.

Lipid metabolites with free-radical scavenging activity from Euphorbia helioscopia L.

The methanolic extract of the plant Euphorbia helioscopia L. exhibited an interesting free-radical scavenging activity. From the aerial parts of Euphorbia helioscopia L. (Euphorbiaceae), a complex mixture of seven cerebrosides together with glucoclionasterol, a digalactosyldiacylglycerol and a diacylmonogalactosylglycerol were identified. The structures of the cerebrosides were characterized as 1-O-ß-D-glucosides of phytosphingosines, which comprised (2S, 3S, 4E, 8E)-2-amino-4(E),8(E)-octadecadiene-1,3-diol, (2S, 3S, 4E, 8Z)-2-amino-4(E),8(Z)-octadecadiene-1,3-diol, (2S, 3S, 4R, 8Z)-2-amino-8(Z)-octadecene-1,3,4-triol as long chain bases with seven 2-hydroxy fatty acids of varying chain lengths (C16, C24:1, C26:1, C24, C26, C28:1) linked to the amino group. The glycosylglycerides were characterized as (2S)-2,3-O-di-(9,12,15-octadecatrienoyl)-glyceryl-6-O-(α-D-galactopyranosyl)-ß-D-galactopyranoside and (2S)-2,3-O-di-(9,12,15-octadecatrienoyl)-glyceryl-1-O-ß-D-galactopyranoside. The structures were established on the basis of spectroscopic data and chemical reactions.

Syllabic effects in Italian lexical access.

Two cross-modal priming experiments tested whether lexical access is constrained by syllabic structure in Italian. Results extend the available Italian data on the processing of stressed syllables showing that syllabic information restricts the set of candidates to those structurally consistent with the intended word (Experiment 1). Lexical access, however, takes place as soon as possible and it is not delayed till the incoming input corresponds to the first syllable of the word. And, the initial activated set includes candidates whose syllabic structure does not match the intended word (Experiment 2). The present data challenge the early hypothesis that in Romance languages syllables are the units for lexical access during spoken word recognition. The implications of the results for our understanding of the role of syllabic information in language processing are discussed.

The retrieval of syntactic and semantic information in the production of verbs.

In Italian, there are two aspectual auxiliaries: essere (to be) and avere (to have). According to the syntactic hypothesis, aspectual auxiliary assignment in Italian is a syntactic phenomenon. Using the picture-word interference paradigm, Experiment 1 tested the predictions of this view. Results failed to support the syntactic hypothesis but could be explained within a semantic framework: A verb's thematic grid is involved in aspectual auxiliary assignment. Experiment 2 tested this hypothesis. The findings corroborated the predictions. The evidence is interpreted as suggesting the involvement of thematic information in the assignment of the aspectual auxiliary during the production of Italian verbs.

Enantiomers of 2-[(Acylamino)ethyl]-1,4-benzodiazepines, potent ligands of kappa-opioid receptor: chiral chromatographic resolution, configurational assignment and biological activity.

Compounds 2a and 3a-e are racemic 2-[(acylamino)ethyl]-1,4-benzodiazepines, tifluadom analogs, with high affinity and selectivity towards the kappa-opioid receptor. We describe the enantiomeric separation of all compounds through liquid chromatography with chiral stationary phases, as well as the resolution of the enantiomers of the most interesting compounds, 2a and 3a, by the semipreparative column Chiralpak AD. The configuration of the resolved enantiomers was investigated: the comparative study of CD and (1)H NMR spectra shows that compounds (-)-2a and (-)-3a have the same absolute configuration of (+)-(S)-tifluadom. A study on the stereoselective interaction with opiate receptors is reported.

The role of argument structure in the production of nouns and verbs.

The study reports an investigation on the role of argument structure complexity in the production of nouns and verbs in three Italian agrammatic patients. The patients, all showing a clear noun/verb dissociation in the assessment tests, were requested to produce either nouns or verbs in a picture naming task. Four sets of words were selected to be produced in the naming task: non-argumental nouns (e.g., medaglia-medal), argumental nouns (e.g., pianto-crying), one-place argument verbs (e.g., dormire-to sleep), and two-place argument verbs (e.g., sparare-to shoot). The performance of the three patients was almost identical. All made reliably fewer errors in the production of non-argumental nouns than in the production of verbs, thus replicating with the new materials the initially observed dissociation. Moreover, they made fewer errors with one-place than with two-place argument verbs, and with non-argumental than with argumental nouns. Finally, in contrast with the previously observed noun/verb dissociation, when their ability to produce argumental nouns and verbs was compared, no reliable difference was found. The results indicate that argument complexity may affect the ability of agrammatic patients to produce words. It is argued that since argument complexity is necessarily associated with verbs and only rarely with nouns, unless special attention is paid, argument complexity effects are easily confounded with grammatical class effects, possibly leading to erroneous interpretations of patients' performance.

Synthesis and antinociceptive activity of pyrrolidinylnaphthalenes.

In this paper the synthesis of the racemates (2R,3S/2S,3R)-1,2-dimethyl-3-[2-(6-substituted naphthyl)]-3-hydroxypyrrolidine 1b-d [(2R,3S/2S,3R)-1b-d] are reported. Compounds 1b-d were prepared by reaction of the racemic 1,2-dimethyl-3-pyrrolidone 2 with the lithiation product obtained from 2-bromo-6-substituted naphthalene 3b-d. Pharmacological properties of (2R,3S/2S,3R)-1a-d are also described. Analgesic activity was investigated by the hot plate test and binding affinities towards mu, delta and kappa opioid receptors were evaluated. A preliminary evaluation of the in vivo side-effects was also accomplished using the rota-rod test. Interesting antinociceptive activity was shown by all compounds and in particular by 1d, which is the most active compound, since it is six-fold more potent than morphine and has lower side effects on the locomotory activity.

Dialkylaminoalkylnaphthalenes as novel opioid-like analgesics.

The study of dialkylaminoalkylnaphthalenes as novel opioid-like analgesics is reported. In particular, the synthesis of (1R,2R/1S,2S)-1-ethyl-1-[2-(6-hydroxynaphthyl)]-1-hydrox-2-m ethyl-2-dimethylaminoethane and its structural analogue (1R,2R/1S,2S)-1-ethyl-1-[2-(6-fluoronaphthyl)]-1-hydroxy-2-methyl- 2-dimethylaminoethane and the configurational analysis by X-ray and 1H NMR spectroscopy are described. Pharmacological profiles are discussed on the basis of the experimental results of analgesia tests (hot plate and writhing test) and rota-rod test, which was performed to distinguish analgesia from drug-induced motor changes. The compounds showed dose-dependent antinociception, with less potency than morphine. Motor coordination appeared to be less involved.

Syllables in the processing of spoken Italian.

Five experiments explored the role of the syllable in the processing of spoken Italian. According to the syllabic hypothesis, the sublexical unit used by speakers of Romance languages to segment speech and access the lexicon is the syllable. However, languages with different degrees of acoustic-phonetic transparency give rise to syllabic effects that vary in robustness. It follows from this account that speakers of phonologically similar languages should behave in a similar way. By exploiting the similarities between Spanish and Italian, the authors tested this prediction in Experiments 1-4. Indeed, Italian listeners were found to produce syllabic effects similar to those observed in Spanish listeners. In Experiment 5, the predictions of the syllabic hypothesis with respect to lexical access were tested. The results corroborated these predictions. The findings are discussed in relation to current models of speech processing.

Synthesis of pyrrolinylnaphthalenes and evaluation of their antinociceptive activity.

In this paper the regioselective preparation of (R/S)-1,2-dimethyl-3-[2-(6-substituted naphthyl)]-2H,5H-pyrrolines 2a-d is reported. These compounds were prepared by thermal dehydration of the corresponding alcohols (2R,3S/2S,3R)-1,2-dimethyl-3-[2-(6-substituted naphthyl)]-3-hydroxy-pyrrolidines (2R,3S/2S,3R)-1a-d with anhydrous FeCl3-SiO2, under vacuum. Pharmacological properties of (R/S)-2a-d are also described. Analgesic activity was investigated by the hot plate test, also in the presence of selective antagonists of mu, delta and kappa opioid receptors. Preliminary analysis of the side-effects was also accomplished using the rota-rod test. Interesting antinociceptive activity was shown by all compounds and in particular by (R/S)-2a (AD50 = 0.31 mg/kg); delta opioid receptors were found to be mainly involved in the pharmacological process and, in general, it was found that the compounds influenced locomotory activity to a much lesser extent than did morphine.

The neural correlates of verb and noun processing. A PET study.

The hypothesis that categorical information, distinguishing among word classes, such as nouns, verbs, etc., is an organizational principle of lexical knowledge in the brain, is supported by the observation of aphasic subjects who are selectively impaired in the processing of nouns and verbs. The study of lesion location in these patients has suggested that the left temporal lobe plays a crucial role in processing nouns, while the left frontal lobe is necessary for verbs. To delineate the brain areas involved in the processing of different word classes, we used PET to measure regional cerebral activity during tasks requiring reading of concrete and abstract nouns and verbs for lexical decision. These tasks activated an extensive network of brain areas, mostly in the left frontal and temporal cortex, which represents the neural correlate of single word processing. Some left hemispheric areas, including the dorsolateral frontal and lateral temporal cortex, were activated only by verbs, while there were no brain areas more active in response to nouns. Furthermore, the comparison of abstract and concrete words indicated that abstract word processing was associated with selective activations (right temporal pole and amygdala, bilateral inferior frontal cortex), while no brain areas were more active in response to concrete words. There were no significant interaction effects between word class and concreteness. Taken together, these findings are compatible with the view that lexical-semantic processing of words is mediated by an extensive, predominantly left hemispheric network of brain structures. Additional brain activations appear to be related to specific semantic content, or, in the case of verbs, may be associated with the automatic access of syntactic information.

Preparation and configuration of racemic and optically active analgesic cycloaminoalkylnaphthalenes.

Cycloaminoalkylnaphthalene 3 shows interesting opioid-like analgesic properties. It possesses two chiral centers and can exist as two racemic pairs and four diastereomers. Since the binding of opioids with receptors is stereoselective, it was important to have the two racemic pairs as well as the four diastereomers. In this paper the synthesis of the (2R,3S/2S,3R) racemate and the (2R,3S) and (2S,3R) enantiomers of the 1,2-dimethyl-3-[2-(6-hydroxynaphthyl)]-3-hydroxypyrrolidine 3 is considered and the determination of absolute configuration is described. The (2R,3S/2S,3R)-3 racemate and the (2R,3S)-3 and (2S,3R)-3 enantiomers were prepared by reaction of the racemic and optically active 1,2-dimethyl-3-pyrrolidone 2, respectively, with the lithiation product obtained from 2-bromo-6-tetrahydropyranyloxy-naphthalene 1 and acidic hydrolysis. The above-mentioned enantiomers of 3 were also obtained by optical resolution via fractional crystallization of the salts with D- and L-tartaric acids. The configuration of the optically active compounds was determined by X-ray analysis of a crystal of (-)-(2S,3R)-3.HCl.H2O. The pharmacological test HPT showed that (-)-(2S,3R)-3.HCl.H2O enantiomer is able to induce opioid-like analgesia with a relative potency 1.5 times that of (2R,3S/2S,3R)-3 and approximately 1.5 times that of morphine.

Chiral resolution, configurational study and pharmacological profile of 2-phenoxypropionic acids.

The racemates and several enantiomers of 2-phenoxypropionic acids, bearing alkyl, acetyl, benzyl, benzoyl, phenyl, difluorophenyl, Cl, NO2 groups on the aromatic moiety, were investigated as potential analgesic-antiinflammatory drugs. The enantiomers, whose absolute configuration has been previously determined by us, were prepared by chiral resolution of the diastereoisomeric salts of the racemates with cynchonidine. The enantiomeric excess was determined by chiral chromatography. The chiroptical properties of the dextroisomers were investigated by CD. The pharmacological properties of the racemates and the enantiomers were monitored by analgesic-antiinflammatory activity tests as well as by gastrotolerability and acute toxicity tests. Some compounds were shown to be superior to ASA and ketoprofen because they have higher or similar analgesic properties, with less gastroulcerogenetic activity. Furthermore low acute toxicity was found for the compounds with high values of ED50. Correlations between the configuration of the enantiomers and their activity are not evident. For the most active compounds, the activity of one of the enantiomers is superior to that of the racemates. This is particularly true for (S)-3, (R)-15 and (S)-18.

Chiral resolution and absolute configuration of the enantiomers of 5-acetyl-2-methyl-4-methylsulfinyl-6-phenyl-3(2H)-pyridazinone and evaluation of their platelet aggregation inhibitory activity.

In a series of 5-acyl-6-phenyl-2,4-substituted-3(2H)-pyridaziones the derivative 1a, with a sulfur stereogenic center, had the most potent activity as human platelet aggregation inhibitor. The resolution of rac-1a was successfully performed by chiral chromatography on Chiralcel OD-R, OD-H, and Chiralpak AD columns and scaled up to a preparative level. The absolute configuration of (-)-(S)-1a was determined by X-ray crystallographic analysis. In vitro human platelet aggregation inhibitory activity was evaluated. Both the enantiomers showed IC50 values in the same micromolar range, but the (-)-(S) isomer was slightly more potent [(S)/(R) potency ratio was 4/1].

Preparation and configuration of racemic and optically active analgesic dialkylaminoalkylnaphthalenes.

The alkylaminoalkylnaphthalene 3 shows interesting opioid-like analgesic properties, mu-selective ligand competition, and enkephalin hydrolyzing enzyme inhibition. 3 possesses two chiral centers and can exist as two racemic pairs and four diastereomers. Since the binding of opioids with the receptor is stereoselective, it was important to have the two racemic pairs as well as the four diastereomers. In this paper the synthesis of the (1R,2R/1S,2S)- and (1R,2S/1S,2R)-racemates and the (1R,2R)- and (1S,2S)-enantiomers of the 1-ethyl-1-hydroxy-1-[2-(6-hydroxynaphthyl)]-2-methyl-3- dimethylaminopropane 3 is considered and the determination of absolute configuration is described. The (1R,2R/1S,2S)-3 and (1R,2S/1S,2R)-3 racemates and the (1R,2R)-3 and (1S,2S)-3 enantiomers were prepared by reaction of the racemic and optically active 1-dimethylamino-2-methylpentan-3-one 2, respectively, with the lithiation product obtained from 2-bromo-6-tetrahydropyranyloxynaphthalene and acidic hydrolysis. The optical resolution of aminoketone 2 was carried out via fractional crystallization of salts (+)- and (-)-dibenzoyltartrates. The configuration of the optically active compounds was determined by X-ray analysis of a crystal of (+)-(1R,2R)-3.HCl.H2O. Preliminary pharmacological tests showed that (+)-(1R,2R)-3 enantiomer is able to induce opioid-like analgesia with a relative potency 2.5 times that of (1R,2R/1S,2S)-3 and about 4 times that of morphine.