Serotoninergic receptor ligands improve Tamoxifen effectiveness on breast cancer cells.

BACKGROUND: Serotonin (or 5-Hydroxytryptamine, 5-HT) signals in mammary gland becomes dysregulated in cancer, also contributing to proliferation, metastasis, and angiogenesis. Thus, the discovery of novel compounds targeting serotonin signaling may contribute to tailor new therapeutic strategies usable in combination with endocrine therapies. We have previously synthesized serotoninergic receptor ligands (SER) with high affinity and selectivity towards 5-HT2A and 5-HT2C receptors, the main mediators of mitogenic effect of serotonin in breast cancer (BC). Here, we investigated the effect of 10 SER on viability of MCF7, SKBR3 and MDA-MB231 BC cells and focused on their potential ability to affect Tamoxifen responsiveness in ER+ cells. METHODS: Cell viability has been assessed by sulforhodamine B assay. Cell cycle has been analyzed by flow cytometry. Gene expression of 5-HT receptors and Connective Tissue Growth Factor (CTGF) has been checked by RT-PCR; mRNA levels of CTGF and ABC transporters have been further measured by qPCR. Protein levels of 5-HT2C receptors have been analyzed by Western blot. All data were statistically analyzed using GraphPad Prism 7. RESULTS: We found that treatment with SER for 72 h reduced viability of BC cells. SER were more effective on MCF7 ER+ cells (IC50 range 10.2 µM - 99.2 µM) compared to SKBR3 (IC50 range 43.3 µM - 260 µM) and MDA-MB231 BC cells (IC50 range 91.3 µM - 306 µM). This was paralleled by accumulation of cells in G0/G1 phase of cell cycle. Next, we provided evidence that two ligands, SER79 and SER68, improved the effectiveness of Tamoxifen treatment in MCF7 cells and modulated the expression of CTGF, without affecting viability of MCF10A non-cancer breast epithelial cells. In a cell model of Tamoxifen resistance, SER68 also restored drug effect independently of CTGF. CONCLUSIONS: These results identified serotoninergic receptor ligands potentially usable in combination with Tamoxifen to improve its effectiveness on ER+ BC patients.

Synthesis, docking studies, and pharmacological evaluation of 5HT2C ligands containing the N'-cyanoisonicotinamidine or N'-cyanopicolinamidine nucleus.

N'-Cyanoisonicotinamidine and N'-cyanopicolinamidine derivatives, linked to an arylpiperazine moiety, were prepared and their affinities to the 5-HT1A , 5-HT2A , and 5-HT2C receptors were evaluated. Several of the newly synthesized compounds, tested by binding studies, showed nanomolar affinity at the 5-HT1A and 5-HT2C receptors and moderate or no affinity for other relevant receptors (D1 , D2 , α1 , and α2 ). Compound 8e (Ki = 21.4 nM) was the most affine for the 5-HT2C receptor, showing, at the same time, a high selectivity with respect to the other receptors analyzed. Compounds 4a and 4c, instead, showed an interesting mixed 5-HT1A /5-HT2C activity with Ki values of 21.3/11.5 and 23.2/6.48 nM, respectively. The compounds with better affinity and selectivity binding profiles toward 5-HT2C (4a, 4c, 8b, and 8e) were selected for further in vivo assays to determine their functional activity. Finally, to rationalize the obtained results, molecular docking studies were performed. The results of the pharmacological studies showed that compounds 4a, 8b, and 8e exerted antidepressant-like effects and 4a and 8e revealed also significant anxiolytic properties. Among the developed derivatives, the most promising compound seems to be 4a, which displayed antipsychotic-, antidepressant- and anxiolytic-like properties. No side effects, like catalepsy, motor-impairment or ethanol-potentiating effects, were observed after the injection of the tested compounds.

New 5-HT1A, 5HT2A and 5HT2C receptor ligands containing a picolinic nucleus: Synthesis, in vitro and in vivo pharmacological evaluation.

Picolinamide derivatives, linked to an arylpiperazine moiety, were prepared and their affinity to 5-HT1A, 5-HT2A and 5-HT2C receptors was evaluated. The combination of structural elements (heterocyclic nucleus, alkyl chain and 4-substituted piperazine), known to play critical roles in affinity for serotoninergic receptors, and the proper selection of substituents led to compounds with high specificity and affinity towards serotoninergic receptors. In binding studies, several molecules showed high affinity in nanomolar and subnanomolar range at 5-HT1A, 5-HT2A and 5-HT2C receptors and moderate or no affinity for other relevant receptors (D1, D2, α1 and α2). N-(2-(4-(pyrimidin-2-yl)piperazin-1-yl)ethyl)picolinamide (3o) with Ki=0.046nM, was the most affine and selective derivative for the 5-HT1A receptor compared to other serotoninergic dopaminergic and adrenergic receptors. N-(2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl)picolinamide (3b), instead, showed a subnanomolar affinity towards 5-HT2A with Ki=0.0224nM, whereas N-(2-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)ethyl)picolinamide (3s) presented an attractive 5-HT2C affinity with Ki=0.8nM. Moreover, the compounds having better affinity and selectivity binding profiles towards 5-HT2A were selected and tested on rat ileum, to determine their effect on 5HT induced contractions. Those more selective towards 5-HT1A receptors were studied in vivo on several behavioral tests.

5-HT2 receptor affinity, docking studies and pharmacological evaluation of a series of 1,3-disubstituted thiourea derivatives.

A series of 10 thiourea derivatives have been synthesized by the reaction of aromatic amine with a substituted aryl (compounds 1-3, 6-8) and alkylphenyl (4, 5, 9, 10) isothiocyanates. Their in vitro and in vivo pharmacological properties were studied. Among the evaluated compounds, two displayed very high affinity for the 5-HT2A receptor (1-0.043 nM and 5-0.6 nM), being selective over the 5-HT2C receptor. Derivatives 3, 5, 9, 10 by 70-89% diminished L-5-HTP-induced head twitch episodes. Compounds 1 and 5 as the 5-HT2A receptor antagonists produced a dose-dependent decrease in the number of DOI-elicited HTR. Compounds 1-5 strongly reduced amphetamine-evoked hyperactivity in rodents. In another test, 1 and 2 caused hyperthermia in mice, whereas 9 and 10 led to hypothermia. Antinociceptive and anticonvulsant properties of selected derivatives were demonstrated. Molecular docking studies using a homology model of 5-HT2A revealed a significant role of hydrogen bonds between both thiourea NH groups and Asp155/Tyr370 residues, as well as π-π interaction with Phe339.

Synthesis, in vitro and in vivo pharmacological evaluation of serotoninergic ligands containing an isonicotinic nucleus.

Isonicotinamide derivatives, linked to an arylpiperazine moiety, were prepared and their affinity to 5-HT1A, 5-HT2A and 5-HT2C receptors were evaluated. The combination of structural elements (heterocyclic nucleus, alkyl chain and 4-substituted piperazine) known to play critical roles in affinity for serotoninergic receptors and the proper selection of substituents led to compounds with high specificity and affinity towards serotoninergic receptors. In binding studies, several molecules showed high affinity in nanomolar and subnanomolar range at 5-HT1A, 5-HT2A and 5-HT2C receptors and moderate or no affinity for other relevant receptors (D1, D2, α1 and α2). N-(3-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)propyl)isonicotinamide (4s) with Ki = 0.130 nM, was the most active and selective derivative for the 5-HT1A receptor compared to other serotoninergic, dopaminergic and adrenergic receptors. Compound 4o, instead, showed 5-HT2A affinity values in subnamolar range. Moreover, the compounds having better affinity and selectivity binding profile towards 5-HT1A and 5-HT2A receptors were selected in order to be tested by in vitro and in vivo assays to determine their functional activity.

Predicting targets of compounds against neurological diseases using cheminformatic methodology.

Recently developed multi-targeted ligands are novel drug candidates able to interact with monoamine oxidase A and B; acetylcholinesterase and butyrylcholinesterase; or with histamine N-methyltransferase and histamine H3-receptor (H3R). These proteins are drug targets in the treatment of depression, Alzheimer's disease, obsessive disorders, and Parkinson's disease. A probabilistic method, the Parzen-Rosenblatt window approach, was used to build a "predictor" model using data collected from the ChEMBL database. The model can be used to predict both the primary pharmaceutical target and off-targets of a compound based on its structure. Molecular structures were represented based on the circular fingerprint methodology. The same approach was used to build a "predictor" model from the DrugBank dataset to determine the main pharmacological groups of the compound. The study of off-target interactions is now recognised as crucial to the understanding of both drug action and toxicology. Primary pharmaceutical targets and off-targets for the novel multi-target ligands were examined by use of the developed cheminformatic method. Several multi-target ligands were selected for further study, as compounds with possible additional beneficial pharmacological activities. The cheminformatic targets identifications were in agreement with four 3D-QSAR (H3R/D1R/D2R/5-HT2aR) models and by in vitro assays for serotonin 5-HT1a and 5-HT2a receptor binding of the most promising ligand (71/MBA-VEG8).

Synthesis and in vitro pharmacological evaluation of novel 2-hydroxypropyl-4-arylpiperazine derivatives as serotoninergic ligands.

This paper reports the synthesis of new norbornene and exo-N-hydroxy-7-oxabicyclo[2.2.1]hept-5-ene-2,3-dicarboximide derivatives and their binding to the 5-HT1A , 5-HT2A , and 5-HT2C receptors, in order to identify selective ligands for these 5-hydroxytryptamine (5-HT, serotonine) receptor subtypes. The combination of structural elements (heterocyclic nucleus, hydroxyalkyl chain, and 4-substituted piperazine) known to be critical for affinity to 5-HT1A receptors and the proper selection of substituents led to compounds with high specificity and affinity toward serotoninergic receptors. The most active compounds were selected and further evaluated for their binding affinities to D1 , D2 dopaminergic and α1 , α2 adrenergic receptors. 4-[3-[4-(2-Furoyl)piperazin-1-yl]propoxy-2-ol]-4-aza-tricyclo[5.2.1.02,6]dec-8-ene-3,5-dione 3e with Ki = 5.04 ± 0.227 nM was the most active and selective derivative for the 5-HT2C receptor with respect to other serotonin receptors, and the most selective derivative versus dopaminergic and adrenergic receptors.

Anti-tumor activity of the methanolic extracts of Salvia menthifolia

In the present research we investigated the anti-proliferative activity of Salvia menthifolia Ten. (formerly Salvia menthaefolia), Lamiaceae, on a glioblastoma cell line, since up to date poor therapeutic results have been reported for treatment of malignant glioblastoma. Methanol extracts from different anatomical parts of S. menthifolia were tested on DBTRG-05MG cell line by MTT assay. The most active primary stems extract was also evaluated for apoptosis induction. Results confirmed the anti-tumor property of all the organs and demonstrated that the primary stems extract induced apoptosis after 4 h with the highest values of DNA fragmentation after 6 to 24 h. Some extracts were also HPLC analyzed for polyphenols, althought activities could be due also to other constituents and to synergistic interactions. Rosmarinic acid, caffeic acid, luteolin-7-O-glucosyde and quercitrin were found in all the extracts. The good performance revealed for S. menthifolia towards this extremely aggressive human glioblastoma cell line confirms that the genus Salvia is a natural source of anti-tumor agents though there are great differences among the various species.

New potent 5-HT(2A) receptor ligands containing an N'-cyanopicolinamidine nucleus: Synthesis and in vitro pharmacological evaluation.

N'-cyanopicolinamidine derivatives, linked to an arylpiperazine moiety, were prepared and their affinity to serotonin 5-HT(1A), 5-HT(2A) and 5-HT(2C) receptors were evaluated. The combination of structural elements (heterocyclic nucleus, alkyl chain and 4-substituted piperazine) known to be critical for affinity to 5-HT(1A) receptors and the proper selection of substituents led to compounds with high specificity and affinity towards serotoninergic receptors. In binding studies, several molecules showed affinity in nanomolar and subnanomolar range at 5-HT(2A) and moderate to no affinity for other relevant receptors (5-HT(1A), 5-HT(2C), D(1), D(2), α(1) and α(2)). N'-cyano-N-(3-(4-(3-chlorophenyl)piperazin-1-yl)propyl)-picolinamidine (4l) with K(i)=0.000185nM, was the most active and selective derivative for the 5-HT(2A) receptor compared to other serotoninergic, dopaminergic and adrenergic receptors.

Synthesis of 1-naphtylpiperazine derivatives as serotoninergic ligands and their evaluation as antiproliferative agents.

Serotonin (5-hydroxytryptamine, 5-HT) is one of the most important neuromediator involved in numerous physiological and pathophysiological processes. In addition it is well established that 5-HT acts as a growth factor on several types of non-tumoral and tumoral cells, and recently it was also related to oncogenes. 5-HT1A receptor expression was identified in prostatic tumor cell lines (PC3 cells) and in human hormone refractory prostate cancer tissue. Based on these observations, development of 5-HT1A antagonists could be useful in inhibiting the growth of cancer cells. In order to investigate on potential use of 5-HT1A ligands as antiproliferative agents, we have analyzed a new set of 1-naphtylpiperazine derivatives. In binding studies, several molecules showed affinity in nanomolar and subnanomolar range at 5-HT1A and moderate to no affinity for other relevant receptors (5-HT2A, 5-HT2C, D1, D2, α1 and α2). All compounds were then evaluated in order to assess their antiproliferative activity using PC3 cells and the most active compounds (1 and 2) were fully characterized to define the mechanism responsible for the observed antiproliferative effect.

New 5-HT(1A) receptor ligands containing a N'-cyanoisonicotinamidine nucleus: synthesis and in vitro pharmacological evaluation.

N'-cyanoisonicotinamidine derivatives, linked to an arylpiperazine moiety, were prepared to identify highly selective and potent 5-HT(1A) ligands as potential pharmacological tools in studies of wide spread psychiatric disorders. The combination of structural elements (heterocyclic nucleus, alkyl chain and 4-substituted piperazine) known to be critical in order to have affinity on 5-HT(1A) receptor and the proper selection of substituents led to compounds with high specificity and affinity towards serotoninergic receptors. In binding studies, several molecules showed affinity in nanomolar and subnanomolar range at 5-HT(1A) and moderate to no affinity for other relevant receptors (5-HT(2A), 5-HT(2C), D(1), D(2), alpha(1) and alpha(2)). N'-cyano-N-(3-(4-(pyridin-2-yl)piperazin-1-yl)propyl)isonicotinamidine (4o) with K(i)=0.038 nM, was the most active and selective derivative for the 5-HT(1A) receptor with respect to other serotoninergic, dopaminergic and adrenergic receptors.

Efficient microwave combinatorial synthesis of novel indolic arylpiperazine derivatives as serotoninergic ligands.

An easy and convenient microwave-assisted synthesis of a small library of indolic arylpiperazine derivatives is described. Parallel and mixed pool combinatorial methods are reported and compared. The described reactions are nucleophilic substitutions of several aromatic piperazines in presence of K(2)CO(3). Good yields and short reaction times are the main aspect of these procedures. Binding assays shed additional light on the influence of the LCAPs on the 5-HT(1A), 5-HT(2A) and 5-HT(2C) receptors affinity and allowed to disclose three interesting compounds as 5-HT(2C), mixed 5-HT(2A)/5-HT(2C) and 5-HT(1A)/5-HT(2C) ligands (4i, 4l and 4d, respectively), with potential antiepileptic, anxiolytic or atypical antipsychotic agent therapeutical profiles.

Synthesis and in-vitro pharmacological evaluation of new 5-HT1A receptor ligands containing a benzotriazinone nucleus.

This paper reports the microwave-assisted synthesis and the binding assays on the 5-HT(1A), 5-HT(2A) and 5-HT(2C) receptors of new benzotriazinone derivatives, in order to identify selective ligands for the 5-HT(1A) subtype receptor. Conventional and microwave heating of the reactions were compared. Good yields and short reaction times are the main advantages of our synthetic route. More active compounds were selected and further evaluated for their binding affinities on D(1), D(2) dopaminergic and alpha(1), alpha(2) adrenergic receptors. The 3-(2-(4-(naphthalen-1-yl)piperazin-1-yl)ethyl)benzo[d][1,2,3]triazin-4(3H)-one 5 with K(i)= 0.000178 nM was the most active and selective derivative for the 5-HT(1A)receptor with respect to other serotonin receptors and the most selective derivative compared to dopaminergic and adrenergic receptors.

Anti-inflammatory and analgesic effects of Spartium junceum L. flower extracts: a preliminary study.

Our objective was to study the anti-inflammatory and analgesic activity of extract of Spartium junceum L. flowers. Samples of flowers were collected from wild plants, dried, powdered, and extracted with hexane and methanol. The extracts were evaporated to dryness and then suspended in suitable solvent. They were then tested for anti-inflammatory activity in the carrageenin rat paw edema test and for analgesic activity in the Randall and Selitto mechanical pressure test and in the tail-flick test. Twenty-four hours after treatment, the gastric mucosa of each rat was observed macroscopically. Based on these results the hexane extract was fractioned by column chromatography, and the fractions obtained were tested in the same way. The results showed good anti-inflammatory activity only for a single fraction of the hexane extract, while all the extracts and all the other hexane fractions showed both peripheral and central analgesic activity. In rats treated with the tested compounds hyperemia and ulcers were absent. The data from this preliminary study reveal interesting pharmacological properties of S. junceum L. flowers extract related to the marked analgesic activity and the absence of gastric ulcerogenic activity.

Preliminary evaluation on anti-inflammatory and analgesic effects of Sideritis syriaca L. herba extracts.

We studied the anti-inflammatory and analgesic activity of extracts of Sideritis syriaca L. herba. Samples were collected from the wild plant, dried, powdered, and extracted with hexane and methanol. The extracts were evaporated to dryness and then suspended in suitable solvent. They were then tested for anti-inflammatory activity in the carrageenin rat paw edema test and for analgesic activity in the Randall and Selitto test and in the tail-flick test. At 24 hours after the treatment, the gastric mucosa of each rat was observed macroscopically. Based on these results the hexane extract was fractionated by column chromatography, and the fractions were obtained tested in the same way. Results showed interesting anti-inflammatory activity only for the hexane extract and all the fractions obtained from it. All the extracts and all the other fractions showed both peripheral and central analgesic activity. In rats treated with the tested compounds hyperemia and ulcers were absent. The data from this preliminary study reveal interesting pharmacological properties of S. syriaca L. herba extracts related to the marked analgesic activity and the absence of gastric ulcerogenic activity. The same is for anti-inflammatory activity, but in this case it seems to be related only to the apolar fraction.

New 5-hydroxytryptamine(1A) receptor ligands containing a norbornene nucleus: synthesis and in vitro pharmacological evaluation.

New arylpiperazine derivatives were prepared to identify highly selective and potent ligands for the 5-hydroxytryptamine 1A (5-HT(1A)) receptor as potential pharmacological tools in studies of central nervous system (CNS) disorders. The combination of structural elements (heterocyclic nucleus, oxyalkyl chain, and arylpiperazine) known to introduce 5-HT(1A) receptor affinity and the proper selection of substituents led to compounds with higher receptor specificity and affinity. In binding studies, several molecules showed affinity in the nanomolar and subnanomolar ranges at 5-HT(1A) and moderate to no affinity for other relevant receptors (5-HT(2A), 5-HT(2C), D(1), D(2), alpha(1), and alpha(2)). The 4-[3-[4-(o-methoxyphenyl)piperazin-1-yl]propoxy]-4-aza-tricyclo[5.2.1.02,6]dec-8-ene-3,5-dione, with K(i) = 0.021 nM, was the most active and selective derivative for the 5-HT(1A) receptor with respect to other serotonin receptors, whereas the most selective derivative for dopaminergic and adrenergic receptors was a CF(3)-substituted arylpiperazine. As a general trend, compounds with a piperazinylpropoxy chain showed a preferential affinity for the 5-HT(1A) receptor, suggesting that the alkyl chain length represents a critical structural feature in determining 5-HT(1A) receptor affinity and selectivity, as confirmed by the molecular modeling invoked for explaining the differential binding affinities of the new arylpiperazines.

Synthesis of 4-(1-oxo-isoindoline) and 4-(5,6-dimethoxy-1-oxo-isoindoline)-substituted phenoxypropanolamines and their beta1-, beta2-adrenergic receptor binding studies.

Phenoxypropanolamines with 1-oxo-isoindoline and 5,6-dimethoxy-1-oxo-isoindoline groups at the para position were synthesized. beta1, beta2-Adrenergic receptor binding affinities for the synthesized compounds were tested and compared with propranolol and atenolol. It was found that the incorporation of para-amidic functionality within the 1-oxo-isoindoline ring and 5,6-dimethoxy-1-oxo-isoindoline ring system led to a high degree of cardioselectivity in the phenoxypropanolamines. Two of the compounds and possessed beta1-adrenergic receptor affinity comparable with that of atenolol and both showed a better cardioselectivity than atenolol. Both and are undergoing further pharmacological evaluation.

Synthesis, beta-adrenergic blocking activity and beta-receptor binding affinities of 1-substituted-3-(2-isopropyl-5-methyl-phenoxy)-propan-2-ol oxalates.

The compounds 1-isopropylamino-3-(2-isopropyl-5-methyl-phenoxy)-propan-2-ol oxalate (5) and 1-tert-butylamino-3-(2-isopropyl-5-methyl-phenoxy)-propan-2-ol oxalate (6) were synthesized from thymol (1), a naturally occurring agent in Thymus vulgaris L. Pharmacological evaluation of 5 and 6 were carried out using mouse ECG and isolated rat uterus models. Pretreatment of 5 (100 microg/kg, i.v.) and 6 (50 microg/kg, i.v.) antagonized isoprenaline (2 microg/kg, i.v.) induced tachycardia, similar to that of atenolol (CAS 29122-68-7, 20 microg/kg, i.v.) pretreatment in mouse ECG experiments as measured by R-R interval. Pretreatment of 5 and 6 blocked isoprenaline and adrenaline induced relaxation of isolated rat uterus (unprimed). Also the compounds 5 and 6 were subjected to in vitro beta1- and beta2-adrenergic receptor binding assay using turkey erythrocyte membrane (beta1) and lung homogenate of rats (beta2). Both 5 and 6 showed beta-adrenergic receptor affinity comparable with that of propranolol (propranolol hydrochloride, CAS 318-98-9) with out selectivity to any one beta-adrenergic receptor. These results suggest that both the compounds possess non-selective beta-adrenergic blocking activity, with the tert-butyl derivative 6 being more active than the isopropyl derivative 5.

Synthesis and beta 1-, beta 2-adrenergic receptor binding studies of 4-acylamino-substituted phenoxypropanolamine and 5-acylamino-substituted naphthyloxypropanolamine derivatives.

The object of this study was to investigate the beta-adrenergic receptor binding affinity of 4-acylaminophenoxypropanolamine (10-15) and 5-acylaminonaphthyloxypropanolamine (21-24) derivatives, which were prepared from 4-aminophenol (5) and 5-amino-1-naphthol (16), respectively. The in vitro beta 1- and beta 2-adrenergic receptor binding affinities of the newly synthesized compounds were assessed in turkey erythrocyte membrane (beta 1) and lung homogenates of rats (beta 2). The binding affinities were compared with that of propranolol (3) (propranolol hydrochloride, CAS 318-98-9). The compound N-[5-(3-tert-butylamino-2-hydroxy-propoxy)-naphthalen-1-yl]-acetamide (22) has beta-adrenergic receptor affinity comparable with that of propranolol and shows selectivity to beta 1-adrenergic receptors.