Identification of new multi-substituted 1H-pyrazolo[3,4-c]pyridin-7(6H)-ones as soluble guanylyl cyclase (sGC) stimulators with vasoprotective and anti-inflammatory activities.

Herein, we describe the rational design, synthesis and in vitro functional characterization of new heme-dependent, direct soluble guanylyl cyclase (sGC) agonists. These new compounds bear a 1H-pyrazolo[3,4-c]pyridin-7(6H)-one skeleton, modified to enable efficient sGC binding and stimulation. To gain insights into structure-activity relationships, the N6-alkylation of the skeleton was explored, while a pyrimidine ring, substituted with various C5'-polar groups, was installed at position C3. Among the newly synthesized 1H-pyrazolo[3,4-c]pyridin-7(6H)-ones, derivatives 14b, 15b and 16a display characteristic features of sGC "stimulators" in A7r5 vascular smooth muscle cells in vitro. They strongly synergize with the NO donor, sodium nitroprusside (SNP) in inducing cGMP generation in a manner that requires the presence of a reduced heme moiety associated with sGC, and elevate the cGMP-responsive phosphorylation of the protein VASP at Ser239. In line with their sGC stimulating capacity, docking calculations of derivatives 16a, 15(a-c) on a cryo-EM structure of human sGC (hsGC) in an ΝΟ-activated state indicated the implication of 1H-pyrazolo[3,4-c]pyridin-7(6H)-one skeleton in efficient bonding interactions with the recently identified region that binds known sGC stimulators, while the presence of either a N6-H or N6-methyl group pointed to enhanced binding affinity. Moreover, the in vitro functional effects of our newly identified sGC stimulators were compatible with a beneficial role in vascular homeostasis. Specifically, derivative 14b reduced A7r5 cell proliferation, while 16a dampened the expression of adhesion molecules ICAM-1 and P/E-Selectin in Human Umbilical Vein Endothelial Cells (HUVECs), as well as the subsequent adhesion of U937 leukocytes to the HUVECs, triggered by tumor necrosis factor alpha (TNF-α) or interleukin-1 beta (IL-1ß). The fact that these compounds elevate cGMP only in the presence of NO may indicate a novel way of interaction with the enzyme and may make them less prone than other direct sGC agonists to induce characteristic hypotension in vivo.

Targeting androgen receptor for prostate cancer therapy: From small molecules to PROTACs.

Prostate cancer (PCa) remains a serious type of cancer for men worldwide. The majority of new PCa cases are associated with androgen receptor (AR) hyperactivity. Various AR-targeting molecules that suppress its activity have been discovered. In this review, we present the already marketed antiandrogens and a selection of structurally and chemically interesting AR-targeting compounds, from a pharmacochemical perspective. Focus has been placed on the applied design approaches, structural evolution and structure-activity relationships of the most prominent compound classes. Passing from the traditional steroidal AR antagonists to the modern AR-targeting proteolysis targeting chimeras (PROTACs), we intend to provide a comprehensive overview on AR-targeting molecules for PCa treatment.

Synthesis and cytotoxic activity of new artemisinin hybrid molecules against human leukemia cells.

A series of new artemisinin-derived hybrids which incorporate cholic acid moieties have been synthesized and evaluated for their antileukemic activity against sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 cells. The new hybrids 20-28 showed IC50 values in the range of 0.019µM-0.192µM against CCRF-CEM cells and between 0.345µM and 7.159µM against CEM/ADR5000 cells. Amide hybrid 25 proved the most active compound against both CCRF-CEM and CEM/ADR5000 cells with IC50 value of 0.019±0.001µM and 0.345±0.031µM, respectively. A relatively low cross resistance to hybrids 20-28 in the range of 5.7-fold to 46.1-fold was measured. CEM/ADR5000 cells showed higher resistance than CCRF-CEM to all the tested compounds. Interestingly, the lowest cross resistance to 23 was observed (5.7-fold), whereas hybrid 25 showed 18.2-fold cross-resistant to CEM/ADR5000 cells. Hybrid 25 which proved even more potent than clinically used doxorubicin against CEM/ADR5000 cells may serve as a promising antileukemic agent against both sensitive and multidrug-resistant cells.

Steroidal esters of the aromatic nitrogen mustard 2-[4-N,N-bis(2-chloroethyl)amino-phenyl]butanoic acid (2-PHE-BU): synthesis and in-vivo biological evaluation.

On the basis of the results of in-silico predictions and in an effort to extend our structure-activity relationship studies, the aromatic nitrogen mustard 2-[4-N,N-bis(2-chloroethyl) amino-phenyl]butanoic acid (2-PHE-BU) was synthesized and conjugated with various steroidal alcohols. The resulting steroidal esters were evaluated for their in-vivo toxicity and antileukemic activity in P388-leukemia-bearing mice. The new derivatives showed significantly reduced toxicity and marginally improved antileukemic activity compared with free 2-PHE-BU. Nevertheless, they did not prove to be superior either to the template steroidal ester used for in-silico predictions or to previously synthesized steroidal esters of aromatic nitrogen mustards. The results obtained indicate that in-silico design predictions may guide the design and synthesis of new bioactive steroidal esters, but further parameters should be considered aiming at the discovery of compounds with optimum activity.

Aneugenic potential of the anticancer drugs melphalan and chlorambucil. The involvement of apoptosis and chromosome segregation regulating proteins.

Previous findings showed that the anticancer drugs p-N,N-bis(2-chloroethyl) amino-l-phenylalanine (melphalan, MEL) and p-N,N-bis(2-chloroethyl)aminophenylbutyric acid (chlorambucil, CAB) belonging to the nitrogen mustard group, in addition to their clastogenic activity, also exert aneugenic potential, nondisjunction and chromosome delay. Their aneugenic potential is mainly mediated through centrosome defects. To further investigate their aneugenicity we (a) studied whether apoptosis is a mechanism responsible for the elimination of damaged cells generated by MEL and CAB and (b) investigated if proteins that regulate chromosome segregation are involved in the modulation of their aneugenic potential. Apoptosis was studied by Annexin-V/Propidium Iodide staining and fluorescence microscopy. The involvement of apoptosis on the exclusion of cells with genetic damage and centrosome disturbances was analyzed by DAPI staining and immunofluorescence of ß- and γ-tubulin in the presence of pan-caspase inhibitor. The expressions of Aurora-A, Aurora-B, survivin and γ-tubulin were studied by western blot. We found that (a) apoptosis is not the mechanism of choice for selectively eliminating cells with supernumerary centrosomes, and (b) the proteins Aurora-A, Aurora-B and survivin are involved in the modulation of MEL and CAB aneugenicity. These findings are important for the understanding of the mechanism responsible for the aneugenic activity of the anticancer drugs melphalan and chlorambucil.

Design, synthesis and biological evaluation of new 3,4-dihydroquinoxalin-2(1H)-one derivatives as soluble guanylyl cyclase (sGC) activators.

Herein, we present the structure-based design, synthesis and biological evaluation of novel mono- and di-carboxylic 3,4-dihydroquinoxalin-2(1H)-one derivatives as potential heme-independent activators of soluble guanylate cyclase (sGC). Docking calculations of several known sGC agonists by utilizing both a homology model of human sGC ß1 Η-ΝΟΧ domain and a recent cryo-EM structure of the same domain guided the structural optimization of various designed compounds. Among these, mono- and di-carboxylic 3,4-dihydroquinoxalin-2(1H)-one derivatives arose as promising candidate sGC activators. A series of such compounds was synthesized and assessed for their effect on sGC activity. None of them was able to trigger any detectable activation of native sGC in prostate cancer (LnCaP) or rat aortic smooth muscle (A7r5) cells, even after loss of heme by treatment with the heme oxidant ODQ. Furthermore, selected derivatives did not exhibit any antagonistic effect against the known heme-independent sGC activator BAY 60-2770 nor any additive or synergistic effect with the heme-dependent NO donor sodium nitroprusside (SNP) on heme-associated sGC in A7r5 cells. However, when tested in vitro using purified recombinant sGC enzyme, the dicarboxylic 3,4-dihydroquinoxalin-2(1H)-one derivative 30d was able to increase the enzymatic activity of both the wild-type α1/ß1 sGC dimer (by 4.4-fold, EC50 = 0.77 µΜ) as well as the heme-free α1/ß1 His105Ala mutant sGC (by 4.8-fold, EC50 = 1.8 µΜ). Notably, the activity of compound 30d towards the mutant α1/ß1 Η105A enzyme was comparable with that previously reported by us for the bona fide activator BAY 60-2770, using the functionally equivalent wild-type sGC preparation treated with ODQ. These results indicate that compound 30d can indeed act as a promising sGC activator and may serve as a basic structure in the design of novel, optimized analogues with enhanced sGC agonistic activity and improved efficiency in cell-based and in vivo systems.

20-Aminosteroids as a novel class of selective and complete androgen receptor antagonists and inhibitors of prostate cancer cell growth.

Here, the synthesis and the evaluation of novel 20-aminosteroids on androgen receptor (AR) activity is reported. Compounds 11 and 18 of the series inhibit both the wild type and the T877A mutant AR-mediated transactivation indicating AR antagonistic function. Interestingly, minor structural changes such as stereoisomers of the amino lactame moiety exhibit preferences for antagonism among wild type and mutant AR. Other tested nuclear receptors are only weakly or not affected. In line with this, the prostate cancer cell growth of androgen-dependent but not of cancer cells lacking expression of the AR is inhibited. Further, the expression of the prostate specific antigen used as a diagnostic marker is also repressed. Finally steroid 18 enhances cellular senescence that might explain in part the growth inhibition mediated by this derivative. Steroids 11 and 18 are the first steroids that act as complete AR antagonists and exhibit AR specificity.

Tricyclic indole and dihydroindole derivatives as new inhibitors of soluble guanylate cyclase.

The synthesis of new tricyclic fused indole and dihydroindole derivatives and preliminary results from their in vitro inhibitory activity against soluble guanylate cyclase (sGC) are presented.

Pyrrolo[2,3-a]carbazoles as potential cyclin dependent kinase 1 (CDK1) Inhibitors. Synthesis, biological evaluation, and binding mode through docking simulations.

Pyrrolo[2,3- a]carbazole derivatives were synthesized, and their effects on CDK1/cyclinB activity were evaluated. The most potent and efficacious inhibitor was found to be ethyl 9-chloro-1H-pyrrolo[2,3-alpha]carbazole-2-carboxylate (1e), exhibiting an IC50 in the low micromolar range and leading to 90% at higher concentrations. Using a computational model for CDK1-1e, binding we have observed that 1e exhibited two likely binding modes in the ATP-binding cleft that involve interactions with Lys130, Thr14, and Asp146 of the enzyme.

Rational design, synthesis, and in vivo evaluation of the antileukemic activity of six new alkylating steroidal esters.

The synthesis and the in vivo evaluation against leukemias P388 and L1210 of six new alkylating steroidal esters are described. The esteric derivatives incorporating the 17beta-acetamido-B-lactamic steroidal skeleton exhibited increased antileukemic activity and lower toxicity, compared to the 17beta-acetamido-7-keto analogs. Among the 17beta-acetamido-B-lactamic steroidal esters, the most potent compound afforded four out of six cures in leukemia P388 and was measured to be almost non-toxic, producing significant low levels of toxicity.

Synthesis, in vivo antileukemic evaluation and comparative study of novel 5alpha-7-keto steroidal esters of chlorambucil and its active metabolite.

Recent structure-antileukemic activity studies showed that the steroidal part of complex molecules containing DNA alkylators does not play only the role of the "biological carrier". New such compounds designed to possess an allylic 7-ketone showed enhanced antileukemic potency compared with derivatives with a simple steroidal skeleton. In order to investigate whether the enhancement of the antileukemic potency is attributed to the introduction of the 7-ketone or to the Delta5-7-keto conjugated steroidal system we decided to reduce the Delta5 double bond. The 5alpha-7-keto-steroidal skeletons synthesized were tethered to chlorambucil and phenyl acetic acid's nitrogen mustard and studied against leukemia P338 in vivo. The reduction of the double bond had a negative impact on the antileukemic potency since the comparative study of the novel derivatives showed that a series of very potent Delta 5-7-keto-steroidal esters were converted by this modification to compounds with marginally accepted activity.

2D NMR and conformational analysis of a prototype anti-tumour steroidal ester.

The synthetic 3beta-hydroxy-17alpha-aza-d-homo-5-androsten-7,17-dione-p-N-N-bis(2-chloroethyl)aminophenylacetate (SOT-19, I) was found to be a very potent anti-leukaemic agent candidate. Its high biological activity and low toxicity rationalize the study of its conformational properties. It can also serve as a prototype and therefore as a template for a series of congener compounds possessing a variety of toxicity and anti-leukaemic activity in subsequent 3D-QSAR studies. Its low energy conformers were identified through a combination of conformational search methods and 2D NOESY NMR spectroscopy. The low energy conformers were mainly compact, with the alkylating aromatic group orienting either to the alpha- or beta-surface of the steroidal plane. The preference in the orientation of the alkyl chain may be steroid dependent and related to the mechanism by which they produce their anti-leukaemic action. This hypothesis is supported by the fact that small chemical modifications of the conformation on the steroidal skeleton produce significant alterations on the anti-leukemic activity.

Optimization of the allylic oxidation in the synthesis of 7-keto-delta5-steroidal substrates.

A variety of delta5-steroids were converted into alpha, beta-unsaturated 7-ketones using a modification of the already known method of t-butyl hydroperoxide in the presence of copper iodide in acetonitrile. The same alteration was applied to another oxidative procedure, which had never been used before on steroidal substrates. The same oxidative agent was used in the presence of copper iodide, and tetra-n-butylammonium bromide was used as a phase-transfer catalyst in a two-phase system of water/methylene chloride. It was found that the allylic oxidation proceeded more efficiently when t-butyl hydroperoxide was added to the reaction mixture in portions. The initial addition of the total amount of oxidant or its dropwise addition afforded low yields. This observation contributes to the investigation of the reaction mechanism, and high-yield conversions of steroidal 5,6-enes into the corresponding conjugated 7-ones in short reaction times are reported.

Synthetic approaches for the synthesis of a cytostatic steroidal B-D bilactam.

A new synthetic procedure and a modification of the original method described in the literature for the synthesis of the steroidal B-D bilactam, 3 beta-hydroxy-7 alpha,17 alpha-diaza-B,D-dihomo-5-androsten-7,17-dione are reported. The key step in the modified method involved protection of the D-lactamic nitrogen atom of 3 beta-acetoxy-17 alpha-aza-D-homo-5-androsten-17-one using a reagent of specific electrophilicity (due to the stereoelectronic properties of the cyclic amide), as Beckmann rearrangement of the B-steroidal ring was hindered, possibly via long range effects, by the presence of the unprotected D-lactamic moiety. Using the 3 beta-acetoxy-5-androsten-17-one as starting material, a new synthetic procedure was developed through ketalization of the 17-ketone and allylic oxidation to the 7-ketone, which was subsequently followed by Beckmann rearrangement of the B- and D-steroid rings. Both approaches resulted in 45 and 67% yields of the desired B,D-bilactam, respectively, in contrast to the 15% yield, which has been reported in the literature.

Antileukemic and cytogenetic effects of modified and non-modified esteric steroidal derivatives of 4-methyl-3-bis(2-chloroethyl)amino benzoic acid (4-Me-CABA).

The increase of the damaging effects on specific DNA sequences and the reduction of the subsequent toxicity of nitrogen mustards has been achieved by their chemical conjugation with modified steroids through an esteric bond. In an attempt to study the structure-activity relationships of these compounds, we synthesized eight steroidal esters of 4-methyl-3-bis(2-chloroethyl)aminobenzoic acid (4-Me-CABA). The anti-leukemic and cytogenetic effects of the parent alkylating agent were compared with those produced by the steroidal compounds, in vivo against leukemias P388 and L1210 and in vitro for induction of Sister Chromatid Exchanges (SCE) and on proliferation rate indices (PRI). The results demonstrate that the existence of the NH-CO group, either as an endocyclic lactamic or as an out of the ring amidic one but at axial conformation, at the steroid-carrier moiety is necessary for the expression of the antileukemic activity. The synthetic route for the preparation of the steroidal esters and their physicochemical data are also reported.

A new approach for evaluating in vivo anti-leukemic activity using the SCE assay. An application on three newly synthesised anti-tumour steroidal esters.

Three newly synthesised steroidal esteric derivatives of nitrogen mustard (compounds 1-3) were comparatively studied on a molar basis regarding their ability to induce sister chromatid exchanges (SCEs) in normal human lymphocytes in vitro and therapeutic effects on leukemia P388 bearing mice. Compounds 1 and 3 are modified steroidal esters of p-methyl-m-N,N-bis(2-chloroethyl)amino benzoic acid, and compound 2 is a modified steroidal ester of chlorambucil. All compounds induced statistically significant increases in SCEs and decreases in proliferation rate indices (PRIs) of cultured human lymphocytes and significantly increased the life span of P388 bearing mice. In this study, the doses applied for therapeutic purposes upon leukemia P388 bearing mice in vivo were derived from cytogenetic observations in normal human lymphocytes in vitro. A substantially better therapeutic effect was obtained compared to the effect achieved after the use of quite higher doses related with LD(10) values. We have demonstrated that the order of anti-tumour effectiveness of the treatment schedules of the three newly synthesised compounds tested (at doses derived from cytogenetic observations) coincides with the order of the cytogenetic effects they induce. The SCE assay appears to have an application in the clinical prediction of tumour sensitivity to potential chemotherapeutics.

Synergistic cytogenetic and antineoplastic effects by the combined action of esteric steroidal derivatives of nitrogen mustards.

We studied the effect of five newly synthesized steroidal derivatives of nitrogen mustards. These derivatives have as alkylators either P-N, N-bis(2-chloroethyl)aminophenyl-butyrate (CHL) or P-N, N-bis(2-chloroethyl)aminophenyl-acetate (PHE) groups esterified with different modified steroidal nuclei. We examined them alone or in combination, on sister chromatid exchange rates and on human lymphocyte proliferation kinetics. The antitumor activity of these compounds, alone or in combination, was also tested on Leukemia P388-bearing mice. A pronounced cytogenetic and antineoplastic action was demonstrated by the compounds that contain either PHE or CHL as alkylators and are esterified with a steroidal nucleus having added a cholestene group in the 17 position of the D-ring. The exocyclical insertion of an -NHCO- group in the D-ring of the steroidal nucleus esterified with PHE (amide ester of PHE) yielded a compound demonstrating a distinct cytogenetic and antineoplastic effect. In contrast, the ketone group in the D-ring being inserted endocyclically in the steroidal nucleus (androstene) esterified with either CHL or with PHE gave negative cytogenetic and antineoplastic effects. However, the combined action of cholestene esterified with either CHL or with PHE in combination with either the androstene ester of PHE or with the androstene ester of CHL, respectively, gave synergistic cytogenetic and antineoplastic effects. Also the amide ester of PHE in combination with the androstene ester of CHL gave distinct cytogenetic and antineoplastic effects in a synergistic manner.

Genotoxicity of all-trans retinoic acid (ATRA) and its steroidal analogue EA-4 in human lymphocytes and mouse cells in vitro.

The aim of our study is to: (a) investigate whether ATRA and its steroidal analogue EA-4 enhance micronucleation in human lymphocytes and mouse cells in vitro and clarify the micronucleation mechanism by FISH and CREST analysis respectively, and (b) analyze their effect on spindle organization by immunofluorescence of ß- and γ-tubulin in mouse cells. We found that they: (a) induce micronucleation mainly via chromosome breakage and chromosome delay in a lesser extent, (b) disturb microtubule network, chromosome orientation and centrosome duplication/separation, (c) accumulate cell cycle at ana-telophases, which exert micronucleation, multiple γ-tubulin signals, nucleoplasmic bridges and multinucleation, and (d) generate multinucleated and multimicronucleated interphase cells.

Cytogenetic and antineoplastic effects of modified steroidal alkylators.

INTRODUCTION: The aim of this study was to design new potentially antineoplastic agents by combining nitrogen mustard with steroidal skeleton, in an effort to improve specificity and simultaneously to reduce systemic toxicity. The steroidal part is aimed to act as a biological platform enabling the alkylating moiety to approach its site of action by altering its physicochemical properties. MATERIALS AND METHODS: The compounds tested have, as alkylating agents, either p-N,N-bis(2-chloroethyl)aminophenyl-butyrate or p-N,N-bis(2-chloroethyl)aminophenyl-acetate esterified with a modified steroidal nucleus. The four newly synthesized compounds were compared on a molar basis, regarding their ability to induce sister chromatid exchanges and modify proliferation rate indices in cultured human lymphocytes. Life span of BDF1 mice inoculated with L1210 leukemia was also estimated (antileukemic activity). RESULTS: A compound having p-N,N-bis(2-chloroethyl)aminophenyl-acetate as the alkylator and two ketone groups in the steroidal part demonstrated the highest statistically significant enhancement of sister chromatid exchanges and suppression of proliferation rate indices, and also caused significant antineoplastic activity. The other compounds proved less active. CONCLUSION: These results suggest that cytogenetic and antileukemic activity of alkylating steroidal esters depends on the configuration of the whole molecule and the appropriate combination of the alkylator with the steroidal molecule.

3D QSAR/CoMFA and CoMSIA studies on antileukemic steroidal esters coupled with conformationally flexible nitrogen mustards.

Thirty-eight antileukemic steroidal esters possessing conformationally flexible nitrogen mustards were studied, and the 3D QSAR/CoMFA and CoMSIA methodologies were applied in order to derive the correlation between their structure and the in vivo antileukemic activity. These compounds show significantly reduced toxicity and possibly increased bioavailability compared to free nitrogen mustards and therefore constitute potent antileukemic drugs. Both the CoMFA and CoMSIA studies gave similar results indicating that the steric effect and the hydrophobic/hydrophilic balance especially in the steroidal part of the molecules probably determined their bioactivity. Of paramount interest is the observation that the orientation of the alkylating part of the SMEs toward the surface of ring B of the steroidal skeleton was related with increased activity. Concerning the steroidal part, the presence of hydrophobic groups in rings B and D was found to be important for enhanced activity. Enhancement of antileukemic potency is further observed if hydrophilic/H-bond acceptor groups are present at the positions 7 and 17 of the steroidal skeleton. Leapfrog simulations provided novel compounds which lead our future synthetic endeavor for obtaining SMEs with optimum bioactivity.