Exploration and biological evaluation of 20-vinyl pregnenes: A step forward toward selective modulators of the estrogen receptor α signaling for breast cancer treatment.

A series of D-ring modified steroids bearing a vinyl ketone pendant were synthesized and evaluated for antiproliferative activity against breast cancer cell line and cytochromes P450. The lead compound, 21-vinyl 20-keto-pregnene (2f) (IC50 = 2.4 µM), was shown to be a promising candidate for future anticancer drug design, particularly against estrogen receptor α (ERα)-positive breast cancer. The lead compound was found to have a significant effect on the signaling pathways in parental and 4-hydroxytamoxifen-resistant cells. Compound 2f modulated the ERK, cyclin D1, and CDK4 pathways and blocked the expression of ERα, the main driver of breast cancer growth. Compound 2f significantly reduced 17ß-estradiol-induced progesterone receptor expression. Accumulation of cleaved poly(ADP-ribose) polymerase in cells treated with compound 2f indicated induction of apoptosis. The selectivity analysis showed that lead compound 2f produces no significant effects on cytochromes P450, CYP19A1, CYP21A2, and CYP7B1.

Novel pentacyclic derivatives and benzylidenes of the progesterone series cause anti-estrogenic and antiproliferative effects and induce apoptosis in breast cancer cells.

The promising antitumor effects of progesterone derivatives have been identified in many studies. However, the specific mechanism of action of this class of compounds has not been fully described. Therefore, in this study, we investigated the antiproliferative and (anti)estrogenic activities of novel pentacyclic derivatives and benzylidenes of the progesterone series. The antiproliferative effects of the compounds were evaluated on hormone-dependent MCF7 breast cancer cells using the MTT test. Estrogen receptor α (ERα) activity was assessed by a luciferase-based reporter assay. Immunoblotting was used to evaluate the expression of signaling proteins. All benzylidenes demonstrated inhibitory effects with IC50 values below 10 µM, whereas pentacyclic derivatives were less active. These patterns may be associated with the lability of the geometry of benzylidene molecules, which contributes to an increase in the affinity of interaction with the receptor. The selected compounds showed significant anti-estrogenic potency. Benzylidene 1d ((8 S,9 S,10R,13 S,14 S,17 S)-17-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-10,13-dimethyl-1,2,6,7,8,9,11,12,14,15-decahydrocyclopenta[a]phenanthren-3-one) was the most active in antiproliferative and anti-estrogenic assays. Apoptosis induced by compound 1d was accompanied by decreases in CDK4, ERα, and Cyclin D1 expression. Compounds 1d and 3d were characterized by high inhibitory potency against resistant breast cancer cells. Apoptosis induced by the leader compounds was confirmed by PARP cleavage and flow cytometry analysis. Compound 3d caused cell arrest in the G2/M phase. Further analysis of novel derivatives of the progesterone series is of great importance for medicinal chemistry, drug design, and oncology.

Effects of Progesterone and Selective Ligands of Membrane Progesterone Receptors in HepG2 Cells of Human Hepatocellular Carcinoma.

Progesterone exerts multiple effects in different tissues through nuclear receptors (nPRs) and through membrane receptors (mPRs) of adiponectin and progestin receptor families. The effect of progesterone on the cells through different types of receptors can vary significantly. At the same time, it affects the processes of proliferation and apoptosis in normal and tumor tissues in a dual way, stimulating proliferation and carcinogenesis in some tissues, suppressing them and stimulating cell death in others. In this study, we have shown the presence of high level of mPRß mRNA and protein in the HepG2 cells of human hepatocellular carcinoma. Expression of other membrane and classical nuclear receptors was not detected. It could imply that mPRß has an important function in the HepG2 cells. The main goal of the work was to study functions of this protein and mechanisms of its action in human hepatocellular carcinoma cells. Previously, we have identified selective mPRs ligands, compounds LS-01 and LS-02, which do not interact with nuclear receptors. Their employment allows differentiating the effects of progestins mediated by different types of receptors. Effects of progesterone, LS-01, and LS-02 on proliferation and death of HepG2 cells were studied in this work, as well as activating phosphorylation of two kinases, p38 MAPK and JNK, under the action of three steroids. It was shown that all three progestins after 72 h of incubation with the cells suppressed their viability and stimulated appearance of phosphatidylserine on the outer surface of the membranes, which was detected by binding of annexin V, but they did not affect DNA fragmentation of the cell nuclei. Progesterone significantly reduced expression of the proliferation marker genes and stimulated expression of the p21 protein gene, but had a suppressive effect on the expression of some proapoptotic factor genes. All three steroids activated JNK in these cells, but had no effect on the p38 MAPK activity. The effects of progesterone and selective mPRs ligands in HepG2 cells were the same in terms of suppression of proliferation and stimulation of apoptotic changes in outer membranes, therefore, they were mediated through interaction with mPRß. JNK is a member of the signaling cascade activated in these cells by the studied steroids.

Synthesis of Phosphorus(V)-Substituted Six-Membered N-Heterocycles: Recent Progress and Challenges.

Heterocycles functionalized with pentavalent phosphorus are of great importance since they include a great variety of biologically active compounds and pharmaceuticals, advanced materials, and valuable reactive intermediates for organic synthesis. Significant progress in synthesis of P(O)R2-substituted six-membered heterocycles has been made in the past decade. This review covers the synthetic strategies towards aromatic monocyclic six-membered N-heterocycles, such as pyridines, pyridazines, pyrimidines, and pyrazines bearing phosphonates and phosphine oxides, which were reported from 2012 to 2022.

Synthesis and evaluation of avermectin-imidazo[1,2-a]pyridine hybrids as potent GABAA receptor modulators.

The γ-aminobutyric acid type A (GABAA) receptors are pentameric transmembrane protein complexes. They have attracted extensive attention from the scientific community due to their significant pharmacological potential. Here we report the first synthesis of avermectin-imidazo[1,2-a]pyridine hybrids promising as GABAA receptor positive allosteric modulators (PAMs). An efficient multi-step protocol was elaborated for the installation of the 6-methyl-2-(p-tolyl)imidazo[1,2-a]pyridine pendant to the Avermectin B1a and Ivermectin skeletons through a linker. A variety of linkers were used in order to study the effect of disturbances in the hybrid structure on the GABAA receptor affinity. In vitro experiments showed that the lead compounds exhibited high potency (IC50 = 207 and 359 nM) for binding at the benzodiazepine site of GABAA receptors. In silico studies suggest that the hybrids are able to bind at the Ivermectin binding site of the GABAA receptor. The functional properties of the highest-affinity hybrid (compound 15e) as GABAAR PAM were evaluated by patch-clamp electrophysiological recordings of GABA-mediated currents in rat cerebellar Purkinje neurons. The results obtained suggest that the potentiating effect of hybrid compound 15e is due to its interaction both with benzodiazepine- and Ivermectin-binding sites of GABAARs. Drug-induced behavioral responses in adult zebrafish for hybrids correlate with an alternative mode of action of avermectin and imidazo[1,2-a]pyridine pharmacophores. The investigation of avermectin-imidazo[1,2-a]pyridine hybrid molecules with activity as GABAA receptor modulators is important for the discovery of safe and effective drugs for the treatment of neurological disorders and pest control agents.

Cytotoxic Effects of the Selective Ligands of Membrane Progesterone Receptors in Human Pancreatic Adenocarcinoma Cells BxPC3.

Progesterone and its synthetic analogues act on cells through different types of receptors, affecting proliferation and apoptosis. These compounds exert their effect through the nuclear receptors and the insufficiently studied membrane progesterone receptors (mPRs) belonging to the progestin and adiponectin Q receptor (PAQR) family. We have identified two selective ligands of mPRs that activate only this type of progesterone receptors - 19-hydroxypregn-4-en-20-one (LS-01) and 19-hydroxy-5ß-pregn-3-en-20-one (LS-02). The goal of this work is to study the effect of these compounds on proliferation and death of human pancreatic adenocarcinoma cells BxPC3 and involvement of the two kinases (p38 MAPK and JNK) in signaling pathways activated by progestins through mPRs. It was shown that progesterone and the compound LS-01 significantly (p < 0.05) inhibited the BxPC3 cell viability, with JNK serving as a mediator. The identified targets of these two steroids are the genes of the proteins Ki67, cyclin D1, PCNA, and p21. Progesterone and the compound LS-01 significantly (p < 0.05) stimulate DNA fragmentation, enhancing the cell death. The p38 mitogen-activated protein kinase (MAPK) is a key mediator of this process. The BCL2A1 protein gene was identified as a target of both steroids. The compound LS-02 significantly (p < 0.05) alters membrane permeability and changes the exposure of phosphatidylserine on the outer membrane leaflet, also enhancing the cell death. This compound acts on these processes by activating both kinases, JNK and p38 MAPK. The compound LS-02 targets the genes encoding the proteins HRK, caspase 9, and DAPK.

Osmolytes and membrane lipids in adaptive response of thermophilic fungus Rhizomucor miehei to cold, osmotic and oxidative shocks.

In contrast to mesophiles, in which levels of trehalose and phosphatidic acids (PA) increased only under heat shock (HS), in thermophiles trehalose and PA were predominant under optimal growth conditions. To study the role of trehalose protection in the adaptation of thermophiles to various stressors, the composition of osmolytes and membrane lipids in the thermophilic fungus Rhizomucor miehei was studied under cold (CS), osmotic (OS) and oxidative (OxS) shocks. CS resulted in no accumulation of glycerol in the mycelium, while the amount of trehalose decreased. The main lipid changes were the increase in the PA proportion with simultaneous decrease of sterols (St), the increase of the unsaturation degree of polar lipids and the decrease of the ergosterol proportion in total St. OS did not cause changes in the lipid composition, but led to the decrease of ergosterol proportion too. Despite the low ability of Mucorales to produce polyols, increase in the level of arabitol and glycerol was observed under OS. OxS led to the decrease of trehalose level and had no effect on the lipid composition. Thus, our results show the similarity (OS) and the difference (CS and OxS) between adaptation mechanisms of thermophiles and mesophiles.

Sulfur-mediated synthesis of unsymmetrically substituted N-aryl oxalamides by the cascade thioamidation/cyclocondensation and hydrolysis reaction.

A facile and straightforward synthesis of unsymmetrically substituted N-aryl oxalamides from 2,2'-biphenyldiamines, 2-chloroacetic acid derivatives, elemental sulfur, and water has been developed. This protocol is distinguished by efficiency in water under metal-free conditions for N-aryl oxalamides bearing a side-chain NH2-group; it can be adapted for scale-up synthesis. The scope and limitations of this transformation have been investigated.

Fe(iii)-Catalyzed synthesis of steroidal imidazoheterocycles as potent antiproliferative agents.

An efficient and practical method has been developed for the synthesis of steroidal imidazoheterocycles via cost-effective and environmentally benign FeCl3-catalyzed oxidative amination. A library of steroidal imidazo[1,2-a]pyridines was directly synthesized from readily available 2-aminopyridines and steroidal ketones in aerobic conditions. The synthesized compounds were screened for activity on human microsomal cytochrome P450s CYP7, CYP17 and CYP21. Antiproliferative activity of two lead compounds 3ia and 3la was additionally evaluated against the human MCF-7 (breast cancer), SKOV3 (ovarian cancer), and 22Rv1 (prostate cancer) cell lines. Steroidal imidazo[1,2-a]pyridine 3la which is a substrate molecule for CYP17A1 with IC50 = 1.7 µM (MCF-7), 3.0 (SKOV3), and 6.0 µM (22Rv1) has proved to be more active than reference drug cisplatin.

Development of 1,3-thiazole analogues of imidazopyridines as potent positive allosteric modulators of GABAA receptors.

Structure-activity relationship studies were conducted in the search for 1,3-thiazole isosteric analogs of imidazopyridine drugs (Zolpidem, Alpidem). Three series of novel γ-aminobutyric acid receptor (GABAAR) ligands belonging to imidazo[2,1-b]thiazoles, imidazo[2,1-b][1,3,4]thiadiazoles, and benzo[d]imidazo[2,1-b]thiazoles were synthesized and characterized as active agents against GABAAR benzodiazepine-binding site. In each of these series, potent compounds were discovered using a radioligand competition binding assay. The functional properties of highest-affinity compounds 28 and 37 as GABAAR positive allosteric modulators (PAMs) were determined by electrophysiological measurements. In vivo studies on zebrafish demonstrated their potential for the further development of anxiolytics. Using the OECD "Fish, Acute Toxicity Test" active compounds were found safe and non-toxic. Structural bases for activity of benzo[d]imidazo[2,1-b]thiazoles were proposed using molecular docking studies. The isosteric replacement of the pyridine nuclei by 1,3-thiazole, 1,3,4-thiadiazole, or 1,3-benzothiazole in the ring-fused imidazole class of GABAAR PAMs was shown to be promising for the development of novel hypnotics, anxiolytics, anticonvulsants, and sedatives drug-candidates.

Novel d-Annulated Pentacyclic Steroids: Regioselective Synthesis and Biological Evaluation in Breast Cancer Cells.

The acid-catalyzed cyclization of benzylidenes based on 16-dehydropregnenolone acetate (16-DPA) was studied. It was found that these compounds readily undergo regioselective interrupted Nazarov cyclization with trapping chloride ion and an efficient method of the synthesis of d-annulated pentacyclic steroids based on this reaction was proposed. The structures of the synthesized pentacyclic steroids were determined by NMR and X-ray diffraction. It was found that the reaction affords a single diastereomer, but the latter can crystallize as two conformers depending on the structure. Antiproliferative activity of synthesized compounds was evaluated against two breast cancer cell lines: MCF-7 and MDA-MB-231. All tested compounds showed relatively high antiproliferative activity. The synthetic potential of the protocol developed was illustrated by the gram-scale experiment.

Synthesis of Dibenzo[d,f][1,3]Diazepines via Elemental Sulfur-Mediated Cyclocondensation of 2,2'-Biphenyldiamines with 2-Chloroacetic Acid Derivatives.

The three-component reaction of 2,2'-biphenyldiamines with 2-chloroacetic acid derivatives and elemental sulfur was developed for the practical synthesis of unknown 2-carboxamide-substituted dibenzo[d,f][1,3]diazepines. This protocol is distinguished by efficiency in water and good tolerance to functional groups and can be adapted to a large-scale synthesis. The chemoselective preparation of a variety of 2-S,N,O-substituted dibenzo[d,f][1,3]diazepines was accomplished using the developed method.

Flexible Synthesis of Phosphoryl-Substituted Imidazolines, Tetrahydropyrimidines, and Thioamides by Sulfur-Mediated Processes.

The solvent-free sulfur-mediated reactions of phosphinic chlorides with alkyl diamines were developed for the practical synthesis of unknown phosphoryl-substituted 4,5-dihydro-1H-imidazoles, 1,4,5,6-tetrahydropyrimidines, and thioamides. Their good tolerance to functional groups, broad substrate scope, and easy scalability were shown. The chemoselective preparation of a variety of phosphoryl-substituted bis(thioamides) was accomplished via the adjustment of a solvent.

Novel steroidal 1,3,4-thiadiazines: Synthesis and biological evaluation in androgen receptor-positive prostate cancer 22Rv1 cells.

A flexible approach to previously unknown spirofused and linked 1,3,4-thiadiazine derivatives of steroids with selective control of heterocyclization patterns is disclosed. (N-Arylcarbamoyl)spiroandrostene-17,6' [1,3,4]thiadiazines and (N-arylcarbamoyl)17-[1',3',4']thiadiazine-substituted androstenes, novel types of heterosteroids, were prepared from 16ß,17ß-epoxypregnenolone and 21-bromopregna-5,16-dien-20-one in good to high yields by the treatment with oxamic acid thiohydrazides. The synthesized compounds were screened for antiproliferative activity against the human androgen receptor-positive prostate cancer cell line 22Rv1. Most of (N-arylcarbamoyl)17-[1',3',4']thiadiazine-substituted androstenes exhibit better antiproliferative potency (IC50 = 2.1-6.6 µM) than the antiandrogen bicalutamide. Compounds 7d with IC50 = 3.0 µM and 7j with IC50 = 2.1 µM proved to be the most active in the series under study. Lead synthesized compound 7j downregulates AR expression and activity in 22Rv1 cells. NF-κB activity is also blocked in 7j-treated 22Rv1 cells. Apoptosis is considered as a possible mechanism of 7j-induced cell death.

Mesoporous particle-based microcontainers for intranasal delivery of imidazopyridine drugs.

The aim of this study was to develop mesoporous containers for entrapment of imidazopyridines, such as sedative-hypnotic medicine zolpidem, anxiolytic agent alpidem and their derivatives. For this purpose, calcium carbonate (size 1.2 µm (PDI: 0.6), zeta potential: -10 mV), manganese carbonate (2.5 µm (PDI: 0.5), zeta potential: -12 mV) and titanium dioxide particles (3.7 µm (PDI: 0.4), zeta potential: -15 mV) were used. The compounds were encapsulated applying two techniques: adsorption on the preformed particles and co-precipitation during the synthesis of the particles. The polymer shell of the containers was formed by electrostatic adsorption of polyelectrolytes on the surface of the particles. The best encapsulation efficacy was shown for zolpidem incorporated into calcium carbonate (5.4%) and manganese carbonate (4.6%) by adsorption. Release of the compounds from the containers based on the proposed particles were characterised by the short time burst effect (<10 min) followed by desorption prolongation by formation of polymer shell. X-ray microtomography results demonstrate the prolonged retention of the containers with the mucoadhesive shell in the nasal cavity.

Synthesis of Imidazo[2,1-b]thiazoles via Copper-Catalyzed A3-Coupling in Batch and Continuous Flow.

A straightforward method for the synthesis of functionalized imidazo[2,1-b]thiazoles starting from benzaldehydes, 2-aminothiazoles, and alkynes under copper(I,II) catalysis was developed. The protocol allows the construction of a variety of aryl-substituted imidazo[2,1-b]benzothiazoles, -[2,1-b]thiazoles, and -[2,1-b][1,3,4]thiadiazoles. The reactions were easy to perform affording most of the desired products in 33-93% yields. The intensification of the process in a continuous-flow reactor increases the products' yields up to quantitative.

Copper(II)-Mediated Aerobic Synthesis of Imidazo[1,2-a]pyridines via Cascade Aminomethylation/Cycloisomerization of Alkynes.

A single copper(II)-catalyzed three-component cascade aminomethylation/cycloisomerization of propiolates to form imidazo[1,2-a]pyridines was explored. A straightforward method was developed for the practical synthesis of functionalized imidazo[1,2-a]pyridines from benzaldehydes, 2-aminopyridines, and propiolate derivatives catalyzed by Cu(OAc)2 hydrate in the presence of air. The protocol is marked by excellent yields, functional group tolerance, and, above all, adaptability to synthesize imidazo[1,2-a]pyridine-based drug molecules such as Alpidem.

General Photoinduced Sequential Electrocyclization/[1,9]-Sigmatropic Rearrangement/Ring-Opening Reaction of Diarylethenes.

A novel and efficient photochemical transformation of diarylethenes comprising a five-membered heterocyclic ring and phenyl moiety is described. This reaction provides a simple method for the preparation of functionalized naphthalene derivatives via photorearrangement reaction of diarylethenes, and the process is characterized by high efficiency that was determined by NMR monitoring. Some mechanistic aspects of this process have been also explored. It was found that the reaction includes tandem transformation of three basic processes: the photocyclization of the hexatriene system, [1,9]-sigmatropic rearrangement, and heterocyclic ring opening. Diarylethenes with different heterocycle moieties (thiophene, benzo[b]thiophene, furan, indole, imidazole, thiazole, oxazole, pyrazole) have been involved into this process, and the target naphthalenes with good yields have been obtained. The opportunity for use in the transformation of diarylethenes with different heterocyclic residues permits synthesis of naphthalenes with desired functional groups. The general character and high efficiency of the reaction promise that the transformation can be an effective synthetic route for the annulation of benzene rings to various aromatic systems, including heterocycles.

Difluoroboron complexes as versatile precursors for steroid modification. Lumiestrone-based steroid derivatives: synthesis and structural studies.

A preparation method for steroid-based difluoroboron complexes has been developed using lumiestrone as a steroid example. Previously inaccessible lumiestrone-based difluoroboron complexes annulated at positions 16 and 17 of the D ring have been prepared. Such difluoroboron complexes may have large synthetic potential for heterofunctionalization of steroids at the D ring. An application of a borylation mixture Ac2O-BF3•OEt2 significantly simplify the preparation of steroid "dimers" bearing two estrone moieties connected at positions 2 and 2' via a linker. Crystal structures of key representatives have been determined by X­ray diffraction.

Design and synthesis of phosphoryl-substituted steroidal pyridazines (Pho-STPYRs) as potent estrogen receptor alpha inhibitors: targeted treatment of hormone-dependent breast cancer cells.

Estrogen receptor alpha (ERα) is an important target for the discovery of new therapeutic drugs against hormone-dependent breast cancer. A series of phosphoryl-substituted steroidal pyridazines (Pho-STPYRs) were synthesized and biologically evaluated as potent ERα inhibitors. Pho-STPYRs showed cytotoxicity against breast cancer cells with IC50 values of 5.9 µM and higher. Pho-STPYRs 33 and 34 [IC50 (MCF7) = 6.5 and 5.9 µM, respectively] were found to block the expression of ERα, the main driver of breast cancer growth, and modulate the ERK, cyclin D1, and CDK4 pathways. Compound 34 showed selectivity, anti-estrogenic potency and high antiproliferative efficacy in combination with the AKT inhibitor. Molecular docking was used to more accurately define the binding mode of lead compounds 33 and 34 to ERα. The selectivity analysis showed that lead compounds 33 and 34 produce no effects on cytochromes P450, including CYP7A1, CYP7B1, CYP17A1, CYP19A1, and CYP21A2. In a word, Pho-STPYRs 33 and 34 are promising ERα inhibitors for the treatment of hormone-dependent breast cancer.