Coumarins-lipophilic cations conjugates: Efficient mitocans targeting carbonic anhydrases.

Being aware of the need to develop more efficient therapies against cancer, herein we disclose an innovative approach for the design of selective antiproliferative agents. We have accomplished the conjugation of a coumarin fragment with lipophilic cations (triphenylphosphonium salts, guanidinium) for providing mitochondriotropic agents that simultaneously target also carbonic anhydrases IX and XII, involved in the development and progression of cancer. The new compounds prepared herein turned out to be strong inhibitors of carbonic anhydrases IX and XII of human origin (low-to-mid nM range), also endowed with high selectivity, exhibiting negligible activity towards cytosolic CA isoforms. Key interactions with the enzyme were analysed using docking and molecular dynamics simulations. Regarding their in vitro antiproliferative activities, an increase of the tether length connecting both pharmacophores led to a clear improvement in potency, reaching the submicromolar range for the lead compounds, and an outstanding selectivity towards tumour cell lines (S.I. up to >357). Cytotoxic effects were also analysed on MDR cell lines under hypoxic and normoxic conditions. Chemoresistance exhibited by phosphonium salts, and not by guanidines, against MDR cells was based on the fact that the former were found to be substrates of P-glycoprotein (P-gp), the pump responsible for extruding foreign chemicals; this situation was reversed by administrating tariquidar, a third generation P-gp inhibitor. Moreover, phosphonium salts provoked a profound depolarization of mitochondria membranes from tumour cells, thus probably compromising their oxidative metabolism. To gain insight into the mode of action of title compounds, continuous live cell microscopy was employed; interestingly, this technique revealed two different antiproliferative mechanisms for both families of mitocans. Whereas phosphonium salts had a cytostatic effect, blocking cell division, guanidines led to cell death via apoptosis.

Conformationally Restricted Glycoconjugates Derived from Arylsulfonamides and Coumarins: New Families of Tumour-Associated Carbonic Anhydrase Inhibitors.

The involvement of carbonic anhydrases (CAs) in a myriad of biological events makes the development of new inhibitors of these metalloenzymes a hot topic in current Medicinal Chemistry. In particular, CA IX and XII are membrane-bound enzymes, responsible for tumour survival and chemoresistance. Herein, a bicyclic carbohydrate-based hydrophilic tail (imidazolidine-2-thione) has been appended to a CA-targeting pharmacophore (arylsulfonamide, coumarin) with the aim of studying the influence of the conformational restriction of the tail on the CA inhibition. For this purpose, the coupling of sulfonamido- or coumarin-based isothiocyanates with reducing 2-aminosugars, followed by the sequential acid-promoted intramolecular cyclization of the corresponding thiourea and dehydration reactions, afforded the corresponding bicyclic imidazoline-2-thiones in good overall yield. The effects of the carbohydrate configuration, the position of the sulfonamido motif on the aryl fragment, and the tether length and substitution pattern on the coumarin were analysed in the in vitro inhibition of human CAs. Regarding sulfonamido-based inhibitors, the best template turned out to be a d-galacto-configured carbohydrate residue, meta-substitution on the aryl moiety (9b), with Ki against CA XII within the low nM range (5.1 nM), and remarkable selectivity indexes (1531 for CA I and 181.9 for CA II); this provided an enhanced profile in terms of potency and selectivity compared to more flexible linear thioureas 1-4 and the drug acetazolamide (AAZ), used herein as a reference compound. For coumarins, the strongest activities were found for substituents devoid of steric hindrance (Me, Cl), and short linkages; derivatives 24h and 24a were found to be the most potent inhibitors against CA IX and XII, respectively (Ki = 6.8, 10.1 nM), and also endowed with outstanding selectivity (Ki > 100 µM against CA I, II, as off-target enzymes). Docking simulations were conducted on 9b and 24h to gain more insight into the key inhibitor-enzyme interactions.

Synthesis, antiproliferative evaluation and in silico studies of a novel steroidal spiro morpholinone.

Estrogens play a pivotal role in the development of estrogen-dependent breast cancer and other hormone-dependent disorders. A common strategy to overcome the pathological effects of estrogens is the use of aromatase inhibitors (AIs), which bind to the enzyme and prevent the union with the natural substrate, decreasing the amount of estrogens produced. Several AIs have been developed, including inhibitors with a steroidal backbone and a nitrogen heterocycle in their structure. Encouraged by the notable results presented by current and clinical steroidal drugs, herein we present the synthesis of a steroidal spiro morpholinone derivative as a plausible aromatase inhibitor. The morpholinone derivative was synthesized over a six-step methodology starting from estrone. The title compound and its hydroxychloroacetamide derivative precursor were evaluated for their antiproliferative profile against estrogen-dependent and independent solid tumor cell lines: A549, HBL-100, HeLa, SW1573, T-47D and WiDr. Both compounds exhibited a potent antiproliferative activity in the micromolar range against the six cancer cell lines, with the hydroxychloroacetamide derivative precursor being a more potent inhibitor (GI50 = 0.25-2.4 µM) than the morpholinone derivative (GI50 = 2.0-11 µM). Furthermore, both compounds showed, in almost all cases, better GI50 values than the steroidal anticancer drugs abiraterone and galeterone. Docking simulations of the derivatives were performed in order to explain the experimental biological activity. The results showed interactions with the iron heme (derivative 3) and important residues of the steroidal binding-site (Met374) for the inhibition of human aromatase. A correlation was found between in vitro assays and the score obtained from the molecular docking study.

Carbohydrate-derived bicyclic selenazolines as new dual inhibitors (cholinesterases/OGA) against Alzheimer's disease.

Concerned by the urgent need to explore new approaches for the treatment of Alzheimer's disease, we herein describe the synthesis and evaluation of new multitarget molecules. In particular, we have focused our attention on modulating the activity of cholinesterases (AChE, BuChE) in order to restore the levels of the neurotransmitter acetylcholine, and of O-GlcNAcase (OGA), which is associated with hyperphosphorylation of tau protein, in turn related to the formation of neurofibrillary tangles in the brain. Specifically, we considered the possibility of using carbohydrate-fused 1,3-selenazolines, decorated with a 2-alkylamino or 2-alkoxy moieties. On the one hand, the presence of a selenium atom might be useful in modulating the intrinsic oxidative stress in AD. On the other hand, such bicyclic structure might behave as a transition state analogue of OGA hydrolysis. Moreover, upon protonation, it could mimic the ammonium cation of acetylcholine. The lead compound, bearing a propylamino moiety on C-2 position of the selenazoline motif, proved to be a good candidate against AD; it turned out to be a strong inhibitor of BuChE (IC50 = 0.46 µM), the most prevalent cholinesterase in advanced disease stages, with a roughly 4.8 selectivity index in connection to AChE (IC50 = 2.2 µM). This compound exhibited a roughly 12-fold increase in activity compared to galantamine, one of the currently marketed drugs against AD, and a selective AChE inhibitor, and virtually the same activity as rivastigmine, a selective BuChE inhibitor. Furthermore, it was also endowed with a strong inhibitory activity against human OGA, within the nanomolar range (IC50 = 0.053 µM for hOGA, >100 µM for hHexB), and, thus, with an outstanding selectivity (IC50(hHexB)/IC50(hOGA) > 1887). The title compounds also exhibited an excellent selectivity against a panel of glycosidases and a negligible cytotoxicity against tumor and non-tumor cell lines. Docking simulations performed on the three target enzymes (AChE, BuChE, and OGA) revealed the key interactions to rationalize the biological data.

Preparation and cytotoxic evaluation of new steroidal oximes and aza-homosteroids from diosgenin and cholesterol.

Using cholesterol and diosgenin as starting materials, we have designed a straightforward methodology to prepare in a reduced number of steps a novel series of steroidal oximes and their aza-homolactam analogs with four types of side chains: cholestane, spirostane, 22-oxocholestane and 22,26-epoxycholestene. The products were evaluated for their cytotoxic activity against the MCF-7 breast cancer cell line. Moreover, the selectivity of the most active compounds was determined against peripheral blood lymphocytes. Compounds 5, 8 and 13 were found to be the most active derivatives, exhibiting IC50 values in the low micromolar range (7.9-9.5 µM) and excellent selectivities (IC50 > 100 µM) against the non-tumor cell line.

Thio- and selenosemicarbazones as antiprotozoal agents against Trypanosoma cruzi and Trichomonas vaginalis.

Herein, we report the preparation of a panel of Schiff bases analogues as antiprotozoal agents by modification of the stereoelectronic effects of the substituents on N-1 and N-4 and the nature of the chalcogen atom (S, Se). These compounds were evaluated towards Trypanosoma cruzi and Trichomonas vaginalis. Thiosemicarbazide 31 showed the best trypanocidal profile (epimastigotes), similar to benznidazole (BZ): IC50 (31)=28.72 µM (CL-B5 strain) and 33.65 µM (Y strain), IC50 (BZ)=25.31 µM (CL-B5) and 22.73 µM (Y); it lacked toxicity over mammalian cells (CC50 > 256 µM). Thiosemicarbazones 49, 51 and 63 showed remarkable trichomonacidal effects (IC50 =16.39, 14.84 and 14.89 µM) and no unspecific cytotoxicity towards Vero cells (CC50 ≥ 275 µM). Selenoisosters 74 and 75 presented a slightly enhanced activity (IC50=11.10 and 11.02 µM, respectively). Hydrogenosome membrane potential and structural changes were analysed to get more insight into the trichomonacidal mechanism.

2-Aminobenzoxazole-appended coumarins as potent and selective inhibitors of tumour-associated carbonic anhydrases.

We have carried out the design, synthesis, and evaluation of a small library of 2-aminobenzoxazole-appended coumarins as novel inhibitors of tumour-related CAs IX and XII. Substituents on C-3 and/or C-4 positions of the coumarin scaffold, and on the benzoxazole moiety, together with the length of the linker connecting both units were modified to obtain useful structure-activity relationships. CA inhibition studies revealed a good selectivity towards tumour-associated CAs IX and XII (Ki within the mid-nanomolar range in most of the cases) in comparison with CAs I, II, IV, and VII (Ki > 10 µM); CA IX was found to be slightly more sensitive towards structural changes. Docking calculations suggested that the coumarin scaffold might act as a prodrug, binding to the CAs in its hydrolysed form, which is in turn obtained due to the esterase activity of CAs. An increase of the tether length and of the substituents steric hindrance was found to be detrimental to in vitro antiproliferative activities. Incorporation of a chlorine atom on C-3 of the coumarin moiety achieved the strongest antiproliferative agent, with activities within the low micromolar range for the panel of tumour cell lines tested.

Tuning the activity of iminosugars: novel N-alkylated deoxynojirimycin derivatives as strong BuChE inhibitors.

We have designed unprecedented cholinesterase inhibitors based on 1-deoxynojirimycin as potential anti-Alzheimer's agents. Compounds are comprised of three key structural motifs: the iminosugar, for interaction with cholinesterase catalytic anionic site (CAS); a hydrocarbon tether with variable lengths, and a fragment derived from 2-phenylethanol for promoting interactions with peripheral anionic site (PAS). Title compounds exhibited good selectivity towards BuChE, strongly depending on the substitution pattern and the length of the tether. The lead compounds were found to be strong mixed inhibitors of BuChE (IC50 = 1.8 and 1.9 µM). The presumptive binding mode of the lead compound was analysed using molecular docking simulations, revealing H-bond interactions with the catalytic subsite (His438) and CAS (Trp82 and Glu197) and van der Waals interactions with PAS (Thr284, Pro285, Asn289). They also lacked significant antiproliferative activity against tumour and non-tumour cells at 100 µM, making them promising new agents for tackling Alzheimer's disease through the cholinergic approach.

Synthesis and Evaluation of Pyrimidine Steroids as Antiproliferative Agents.

A small and focused library of steroidal non-fused and fused pyrimidines was prepared from pregnenolone acetate and diosgenin, respectively. The key step was the cycloaddition reaction of nitrogen-containing 1,3-binucleophiles with the steroidal α,ß-unsaturated ketone. Urea, thiourea and guanidine reacted in a similar manner and afforded the steroidal pyrimidines in good yields. The antiproliferative tests against human tumor cell lines gave GI50 values in the micromolar range and had no effect on healthy fibroblasts. Additional experiments indicated that the compounds did not act as P-glycoprotein substrates, thus avoiding the rise of drug resistance. The fused steroidal pyrimidinethione was selected as drug lead for further testing due to its strong antiproliferative activities within the low micromolar range.

Bioactivities of Flavonoids from Lopezia racemosa.

Lopezia racemosa Cav. (Onagraceae) has been used in Mexican traditional medicine to alleviate stomachache, biliary colic, urine retention, stomach cancer, and skin, dental, buccal, and urinary infections. The objective of this study was to determine the bioactivities of specific parts of the plant to scientifically confirm its traditional use. Aerial parts and flowers were macerated and subsequently extracted with hexane, chloroform, and methanol. This study was focused on the analysis of polar components, and thus the methanolic fractions were selected for further investigations. These fractions were evaluated for their antimicrobial activity using a panel of bacterial Gram-positive and -negative strains, as well as fungal strains, including filamentous fungi and yeasts. In addition, the cytotoxic activity of the extract was assessed by MTT using the human-derived monocytic THP-1 and the normal human fibroblast cell lines. Various fractions showed antimicrobial activity against various pathogens, although the most relevant were against Pseudomonas aeruginosa and Trichophyton mentagrophytes. No inhibition of yeasts was recorded. Only four fractions showed cytotoxic effects when the human-derived THP-1 and fibroblast cells were assessed. The four flavonoids isolated from the extract were luteolin, luteolin-6-C-hexoside, luteolin-8-C-hexoside, and hyperoside. The biological activities presented in this study validate some traditional uses of the plant.

Synthesis of unprecedented steroidal spiro heterocycles as potential antiproliferative drugs.

Herein we report the straightforward preparation of novel conformationally-restricted steroids from trans-androsterone and estrone, decorated with spiranic oxazolidin-2-one or 2-aminooxazoline motifs at C-17 as potential antiproliferative agents. Such unprecedented pharmacophores were accessed using an aminomethylalcohol derivative at C-17 as the key intermediate; reaction of such functionality with triphosgene, or conversion into N-substituted thioureas, followed by an intramolecular cyclodesulfurization reaction promoted by yellow HgO, furnished such spirocycles in excellent yields. Title compounds were tested in vitro against a panel of six human tumor cell lines, named A549 (non-small cell lung), HBL-100 (breast), HeLa (cervix), SW1573 (non-small cell lung), T-47D (breast) and WiDr (colon), and the results were compared with steroidal chemotherapeutic agents (abiraterone and galeterone); the A-ring of the steroidal backbone, the nature of the heterocycle and the N-substituents proved to be essential motifs for establishing structure-activity relationships concerning not only the potency but also the selectivity against tumor cell lines. Estrone derivatives, particularly those bearing a spiranic 2-aminooxazoline scaffold were found to be the most active compounds, with GI50 values ranging from the low micromolar to the submicromolar level (0.34-1.5 µM). Noteworthy, the lead compounds showed a remarkable increase in activity against the resistant cancer cell lines (T-47D and WiDr) compared to the anticancer reference drugs (up to 120-fold).

New selenosteroids as antiproliferative agents.

Starting from natural steroids (diosgenin, hecogenin, smilagenin, estrone), we have prepared a wide panel of selenoderivatives, including benzoselenazolones, selenosemicarbazones, isoselenocyanates, selenoureas, selenocyanates and diselenides, with the aim of developing new families of potential chemotherapeutic agents. The modification of the organoselenium moieties, and their position on the steroid provided valuable information concerning the antiproliferative activities. Among all the families accessed herein, the best profile was achieved for selenoureas on the A ring of estrone, which exhibited GI50 values in the range 2.0-4.1 µM for all the tested tumor cell lines, with increased potency compared with commonly used chemotherapeutic agents, like 5-fluorouracil and cisplatin. Cell cycle analysis revealed that selenoureas induced accumulation of cells in the G1 phase of the cell cycle in the breast cancer cell lines HBL-100 and T-47D; therefore, a different mechanism than cisplatin, that induces cell cycle accumulation in the S phase as a result of DNA damage, must be involved. In the rest of the tumor cells, a slight increase of the S compartment was observed. Moreover, selenosteoids turned out to be excellent glutathione peroxidase (GPx) mimics for the catalytic removal of deleterious H2O2 (t1/2 8.0-22.5 min) and alkyl peroxides (t1/2 23.0-38.9 min) when used in substoichiometric amounts (1% molar ratio), thus providing a valuable tool for reducing the intrinsic oxidative stress in tumor progression.

Novel synthesis of steroidal oximes and lactams and their biological evaluation as antiproliferative agents.

A novel three-step methodology to obtain 6a-aza-B-homo steroidal lactams has been developed starting from the easily available cholesterol and pregnenolone. In addition, a new procedure for the synthesis of a 6a-aza-B-homo steroidal lactam analog of vespertilin, starting from diosgenin has been established. In both synthetic pathways, the key intermediate is a hydroxyimino derivative obtained in a one- or two-step sequence from the starting materials. These methods avoid the use of hazardous oxidant agents in the process. The new steroidal oximes and lactams were examined for their antiproliferative activities against several tumor cell lines. The 6,23-dihydroxyimino derivative exhibited the highest activity with GI50 values of 11-22µM.

Synthesis of monomeric and dimeric steroids containing [1,2,4]triazolo[1,5-a]pyrimidines.

The synthesis of several monomeric and dimeric steroidal [1,2,4]triazolo[1,5-a]pyrimidines (TPs) derived from steroids are described. These derivatives were prepared from α,ß-unsaturated carbonyl compounds through a Claisen Schmidt condensation and rearrangement of the spiro moiety followed by a cycloaddition with 3-amino-1,2,4-triazole. The antiproliferative activity of compounds 7, 13-15 was tested against human cancer cells; several IG50 values were below 10µM.

Diosgenin-based thio(seleno)ureas and triazolyl glycoconjugates as hybrid drugs. Antioxidant and antiproliferative profile.

The stereoselective preparation of diosgenin-derived thio(seleno)ureas and glycomimetics bearing a 1,2,3-triazolyl tether on C-3 has been accomplished. The key steps in the synthetic pathway are the incorporation of an amino moiety and its further transformation into thio- and selenoureas, and also a click chemistry reaction involving a propargyl residue and an azido moiety to afford carbohydrate-derived 1,2,3-triazoles; subsequent BF3-promoted acetolysis of the spiranic moiety afforded the corresponding 22-oxocholestanic structure. The N-phenyl selenourea, an hitherto unknown steroidal derivative, turned out to be a potent ROS scavenger, in particular against free radicals (EC50 = 29.47 ± 2.33 µM, DPPH method), and as a glutathione peroxidase mimic in the elimination of H2O2 (t1/2 = 4.8 min, 1% molar ratio). 22-Oxocholestane structures bearing a C-3 azido, propargyl, thioureido, and particularly selenoureido moiety behaved as strong antiproliferative agents against HeLa cells (IC50 1.87-11.80 µM). N-phenyl selenourea also exhibited IC50 values lower than 6.50 µM for MDA-MB-231, MCF-7 and HepG2 cancer cells; apoptosis was found to be involved in its mode of action. Such compound was also capable of efficiently eliminating ROS endogenously produced by HeLa cells. Antiproliferative properties of thioxo and selenoxo derivatives were stronger than diosgenin.

Epimerization of C-22 in (25R)- and (25S)-sapogenins.

Most of the naturally occurring steroidal sapogenins (C-23 non-substituted frameworks), possess an R configuration at the spiro C-22 center. Their C-22 epimers have become important targets in biological research. This paper describes a procedure to obtain 22S-spirostans from 22R-sapogenins and pseudosapogenin skeletons, without affecting the chirality at either C-25 or C-20. An optimal way to synthesize the pair of C-22 stereoisomers of 23-acetyldiosgenin is also reported. The latter was obtained from a 22,26-epoxycholestane or from 23-acetylfurostene compounds.

Synthesis of steroidal derivatives containing substituted, fused and spiro pyrazolines.

An efficient and facile synthesis of fused, substituted and spiro pyrazoline steroid derivatives through a cycloaddition reaction of different α,ß-unsaturated ketones with hydrazine acetate in acetic acid is reported. Depending on the starting material, the ring closure reaction provided a mixture of two steroidal pyrazoline epimers that were separated and studied by NMR techniques. In one case it was possible to isolate and characterize the hydrazone derivative as the reaction intermediate, which confirms the mechanism proposed in the literature [11,25,26].

Synthetic pathway to 22,23-dioxocholestanic chain derivatives and their usefulness for obtaining brassinosteroid analogues.

Recognizing the functionality of the pentacyclic steroidal derivative 7a as important synthon to obtain new brassinosteroid analogs, we have accomplished the derivatization of hecogenin, a sapogenin from the 25R serie containing a carbonyl group at C-12, to a 22,23-dioxocholestanic chain derivative. Starting from hecogenin acetate (5a) or hecogenin tosylate (5b), we obtained two pentacyclic derivatives (7a and 7b) which were subjected to an oxidation reaction on the double bond at C-12(23) to obtain a 22,23-dioxocholestanic chain, with the regeneration of the carbonyl group at C-12. Reduction of the carbonyl groups lead to the 20-epi-12,23-dihydroxy-22-oxo system 11a-b. The absolute configuration of compound 11a was established by X-ray diffraction analysis.

Rapid conversion of spirostans into furostan skeletons at room temperature.

We report a facile protocol to obtain 22-substituted furostans and pseudosapogenins in high yields from (25R)- and (25S)-sapogenins. This method involves the treatment of the sapogenin with acetic-trifluoroacetic mixed anhydride and BF(3)·OEt(2) at room temperature, followed by the addition of a nucleophile (H(2)O, MeOH or KSeCN). In the case of 22-hydroxyfurostans, they can be transformed to pseudosapogenins by treatment with p-toluensulfonic acid.

3-tert-Butyl 5-methyl (2R,4S,5R)-2-(4-methoxyphenyl)-4-(3-nitrophenyl)-1,3-oxazolidine-3,5-dicarboxylate.

The title mol-ecule, C(23)H(26)N(2)O(8), was synthesized in three steps starting from m-nitro-cinnamic acid. The central oxazolidine ring adopts an almost perfect envelope conformation with the O atom as the flap [puckering parameter ϕ = 0.3 (6)°]. The dihedral angle formed by the benzene rings is 61.81 (9)°. In the crystal, mol-ecules are connected into double chains parallel to [010] by C-H⋯O hydrogen bonds. The absolute configuration was assigned from the synthetic procedure.