10-(4-Phenylpiperazine-1-carbonyl)acridin-9(10H)-ones and related compounds: Synthesis, antiproliferative activity and inhibition of tubulin polymerization.

As part of our continuing search for potent inhibitors of tubulin polymerization, two novel series of 42 10-(4-phenylpiperazine-1-carbonyl)acridin-9(10H)-ones and N-benzoylated acridones were synthesized on the basis of a retrosynthetic approach. All newly synthesized compounds were tested for antiproliferative activity and interaction with tubulin. Several analogs potently inhibited tumor cell growth. Among the compounds tested, 10-(4-(3-methoxyphenyl)piperazine-1-carbonyl)acridin-9(10H)-one (17c) exhibited excellent growth inhibitory effects on 93 tumor cell lines, with an average GI50 value of 5.4 nM. We were able to show that the strong cytotoxic effects are caused by disruption of tubulin polymerization, as supported by the EBI (N,N'-Ethylenebis(iodoacetamide)) assay and the fact that the most potent inhibitors of cancer cell growth turned out to be the most efficacious tubulin polymerization inhibitors. Potencies were nearly comparable or superior to those of the antimitotic reference compounds. Closely related to this, the most active analogs inhibited cell cycling at the G2/M phase at concentrations down to 30 nM and induced apoptosis in K562 leukemia cells. We believe that our work not only proves the excellent suitability of the acridone scaffold for the design of potent tubulin polymerization inhibitors but also enables synthetic access to further potentially interesting N-acylated acridones.

Small molecule-induced degradation of the full length and V7 truncated variant forms of human androgen receptor.

The androgen receptor (AR) is a hormone-activated transcription factor that regulates the development and progression of prostate cancer (PCa) and represents one of the most well-established drug targets. Currently clinically approved small molecule inhibitors of AR, such as enzalutamide, are built upon a common chemical scaffold that interacts with the AR by the same mechanism of action. These inhibitors eventually fail due to the emergence of drug-resistance in the form of AR mutations and expression of truncated AR splice variants (e.g. AR-V7) that are constitutively active, signalling the progression of the castration-resistant state of the disease. The urgent need therefore continues for novel classes of AR inhibitors that can overcome drug resistance, especially since AR signalling remains important even in late-stage advanced PCa. Previously, we identified a collection of 10-benzylidene-10H-anthracen-9-ones that effectively inhibit AR transcriptional activity, induce AR degradation and display some ability to block recruitment of hormones to the receptor. In the current work, we extended the analysis of the lead compounds, and used methods of both ligand- and structure-based drug design to develop a panel of novel 10-benzylidene-10H-anthracen-9-one derivatives capable of suppressing transcriptional activity and protein expression levels of both full length- and AR-V7 truncated forms of human androgen receptor. Importantly, the developed compounds efficiently inhibited the growth of AR-V7 dependent prostate cancer cell-lines which are completely resistant to all current anti-androgens.

Oxadiazole-substituted naphtho[2,3-b]thiophene-4,9-diones as potent inhibitors of keratinocyte hyperproliferation. Structure-activity relationships of the tricyclic quinone skeleton and the oxadiazole substituent.

Novel analogues of oxadiazole-substituted naphtho[2,3-b]thiophene-4,9-diones were synthesized in which the tricyclic quinone skeleton was systematically replaced with simpler moieties, such as structures with fewer rings and open-chain forms, while the oxadiazole ring was maintained. In addition, variants of the original 1,2,4-oxadiazole ring were explored. Overall, the complete three-ring quinone was essential for potent suppression of human keratinocyte hyperproliferation, whereas analogous anthraquinones were inactive. Also, the oxadiazole ring per se was not sufficient to elicit activity. However, rearrangement of the heteroatom positions in the oxadiazole ring resulted in highly potent inhibitors with compound 24b being the most potent analogue of this series showing an IC50 in the nanomolar range. Furthermore, experiments in isolated enzymatic assays as well as in the keratinocyte-based hyperproliferation assay did not support a major role of redox cycling in the mode of action of the compounds.

N-Heterocyclic (4-Phenylpiperazin-1-yl)methanones Derived from Phenoxazine and Phenothiazine as Highly Potent Inhibitors of Tubulin Polymerization.

We report here a series of 27 10-(4-phenylpiperazin-1-yl)methanones derived from tricyclic heterocycles which were screened for effects on tumor cell growth, inhibition of tubulin polymerization, and induction of cell cycle arrest. Several analogues, among them the 10-(4-(3-methoxyphenyl)piperazine-1-carbonyl)-10H-phenoxazine-3-carbonitrile (16o), showed excellent antiproliferative properties, with low nanomolar GI50 values (16o, mean GI50 of 3.3 nM) against a large number (93) of cancer cell lines. Fifteen compounds potently inhibited tubulin polymerization. Analysis of cell cycle by flow cytometry revealed that inhibition of tumor cell growth was related to an induction of G2/M phase cell cycle blockade. Western blotting and molecular docking studies suggested that these compounds bind efficiently to ß-tubulin at the colchicine binding site. Our studies demonstrate the suitability of the phenoxazine and phenothiazine core and also of the phenylpiperazine moiety for the development of novel and potent tubulin polymerization inhibitors.

8-Halo-substituted naphtho[2,3-b]thiophene-4,9-diones as redox-active inhibitors of keratinocyte hyperproliferation with reduced membrane-damaging properties.

A series of 8-chloronaphtho[2,3-b]thiophene-4,9-diones and also some 8-bromo analogues were prepared. The compounds were evaluated for their potencies to suppress keratinocyte hyperproliferation using the human keratinocyte line HaCaT as the primary test system. Structure-activity relationship studies revealed that replacement of the phenolic group of an earlier series with an 8-halogen atom retained the inhibitory potency against keratinocyte hyperproliferation with IC50 values in the submicromolar range. 8-Chloro-substitution led to inhibitors that were more potent than their bromo analogues. Concomitantly, halo-substitution eliminated membrane-damaging properties as assessed by LDH release from the cytoplasm of the keratinocytes which, in contrast, were induced by the corresponding phenolic analogues. Finally, selective compounds were characterized for their ability to participate in redox cycling to generate superoxide in enzymatic and keratinocyte-based studies.

Inhibitory effect of phenothiazine- and phenoxazine-derived chloroacetamides on Leishmania major growth and Trypanosoma brucei trypanothione reductase.

A number of phenothiazine-, phenoxazine- and related tricyclics-derived chloroacetamides were synthesized and evaluated in vitro for antiprotozoal activities against Leishmania major (L. major) promastigotes. Several analogs were remarkably potent inhibitors, with antileishmanial activities being comparable or superior to those of the reference antiprotozoal drugs. Furthermore, we explored the structure-activity relationships of N-10 haloacetamides that influence the potency of such analogs toward inhibition of L. major promastigote growth in vitro. With respect to the mechanism of action, selected compounds were evaluated for time-dependent inactivation of Trypanosoma brucei trypanothione reductase. Our results are indicative of a covalent interaction which could account for potent antiprotozoal activities.

Synthesis and structure-activity relationships of lapacho analogues. 2. Modification of the basic naphtho[2,3-b]furan-4,9-dione, redox activation, and suppression of human keratinocyte hyperproliferation by 8-hydroxynaphtho[2,3-b]thiophene-4,9-diones.

The basic structure of linearly anellated lapacho quinones, naphtho[2,3-b]furan-4,9-dione (7), was modified in the search for novel agents against keratinocyte hyperproliferation. The synthesis and structure-activity relationships of several heterocycle-fused naphthoquinones as well as a full range of 2- and 7-substituted derivatives of one of these, 8-hydroxynaphtho[2,3-b]thiophene-4,9-dione (8a), are described. Out of a total of 71 analogues, particularly 2-thenoyl-substituted 26l, 2-nicotinoyl-substituted 26m, and 2-oxadiazole-substituted 35a compared favorably with the antipsoriatic agent anthralin. Their potency for suppression of keratinocyte hyperproliferation, which was evaluated using HaCaT cells as a model, was combined with comparably low membrane-damaging effects toward keratinocytes, as established by the release of lactate dehydrogenase activity from the cytoplasm of the cells. With respect to the mechanism of action, redox activation of lapacho quinones by one- and two-electron reduction in isolated enzymatic assays was studied, and their potential to generate superoxide was confirmed in the keratinocyte-based hyperproliferation assay.

Characterization of a new class of androgen receptor antagonists with potential therapeutic application in advanced prostate cancer.

The human androgen receptor plays a major role in the development and progression of prostate cancer and represents a well-established drug target. All clinically approved androgen receptor antagonists possess similar chemical structures and exhibit the same mode of action on the androgen receptor. Although initially effective, resistance to these androgen receptor antagonists usually develops and the cancer quickly progresses to castration-resistant and metastatic states. Yet even in these late-stage patients, the androgen receptor is critical for the progression of the disease. Thus, there is a continuing need for novel chemical classes of androgen receptor antagonists that could help overcome the problem of resistance. In this study, we implemented and used the synergetic combination of virtual and experimental screening to discover a number of new 10-benzylidene-10H-anthracen-9-ones that not only effectively inhibit androgen receptor transcriptional activity, but also induce almost complete degradation of the androgen receptor. Of these 10-benzylidene-10H-anthracen-9-one analogues, a lead compound (VPC-3033) was identified that showed strong androgen displacement potency, effectively inhibited androgen receptor transcriptional activity, and possesses a profound ability to cause degradation of androgen receptor. Notably, VPC-3033 exhibited significant activity against prostate cancer cells that have already developed resistance to the second-generation antiandrogen enzalutamide (formerly known as MDV3100). VPC-3033 also showed strong antiandrogen receptor activity in the LNCaP in vivo xenograft model. These results provide a foundation for the development of a new class of androgen receptor antagonists that can help address the problem of antiandrogen resistance in prostate cancer.

Natural product derived antiprotozoal agents: synthesis, biological evaluation, and structure-activity relationships of novel chromene and chromane derivatives.

Various natural products with the chromane and chromene scaffold exhibit high antiprotozoal activity. The natural product encecalin (7) served as key intermediate for the synthesis of different ethers 9, amides 11, and amines 12. The chromane analogues 14 and the phenols 15 were obtained by reductive amination of ketones 13 and 6, respectively. Angelate 3, ethers 9, and amides 11 did not show considerable antiprotozoal activity. However, the chromene and chromane derived amines 12, 14, and 15 revealed promising antiprotozoal activity and represent novel lead compounds. Whereas benzylamine 12a and α-methylbenzylamine 12g were active against P. falciparum with IC50 values in the range of chloroquine, the analogous phenols 15a and 15b were unexpectedly 10- to 25-fold more potent than chloroquine with selectivity indexes of 6760 and 1818, respectively. The phenylbutylamine 14d based on the chromane scaffold has promising activity against T. brucei rhodesiense and L. donovani.

Synthesis and structure-activity relationships of lapacho analogues. 1. Suppression of human keratinocyte hyperproliferation by 2-substituted naphtho[2,3-b]furan-4,9-diones, activation by enzymatic one- and two-electron reduction, and intracellular generation of superoxide.

A series of linearly anellated lapacho quinone analogues substituted at the 2-position of the tricyclic naphtho[2,3-b]furan-4,9-dione system were synthesized and evaluated for their ability to suppress keratinocyte hyperproliferation using HaCaT cells as the primary test system. While very good in vitro potency with IC(50) values in the submicromolar range was attained with electron-withdrawing substituents, some compounds were found to induce plasma membrane damage, as evidenced by the release of LDH activity from cytoplasm of the keratinocytes. The most potent analogue against keratinocyte hyperproliferation was the 1,2,4-oxadiazole 18, the potency of which was combined with comparably low cytotoxic membrane damaging effects. Structure-activity relationship studies with either metabolically stable or labile analogues revealed that the quinone moiety was required for activity. Selected compounds were studied in detail for their capability to generate superoxide radicals both in isolated enzymatic one- and two-electron reduction assays as well as in a HaCaT cell-based assay.

Phenylimino-10H-anthracen-9-ones as novel antimicrotubule agents-synthesis, antiproliferative activity and inhibition of tubulin polymerization.

A novel series of phenylimino-10H-anthracen-9-ones and 9-(phenylhydrazone)-9,10-anthracenediones were synthesized and evaluated for interaction with tubulin and for cytotoxicity against a panel of human tumor cell lines. The 10-(3-hydroxy-4-methoxy-phenylimino)-10H-anthracen-9-one 15h and its dichloro analog 16b were identified as potent inhibitors of tumor cell growth (16b, IC(50) K562 0.11 µM), including multidrug resistant phenotypes. Compound 15h had excellent activity as an inhibitor of tubulin polymerization. Concentration-dependent cell cycle analyzes by flow cytometry confirmed that KB/HeLa cells treated by 15h and 16b were arrested in the G2/M phases of the cell cycle. In competition experiments, 15h strongly displaced radiolabeled colchicine from its binding site on tubulin, showing IC(50) values similar to that of colchicine. The results obtained demonstrate that the antiproliferative activity is related to the inhibition of tubulin polymerization.

N-benzoylated phenoxazines and phenothiazines: synthesis, antiproliferative activity, and inhibition of tubulin polymerization.

A total of 53 N-benzoylated phenoxazines and phenothiazines, including their S-oxidized analogues, were synthesized and evaluated for antiproliferative activity, interaction with tubulin, and cell cycle effects. Potent inhibitors of multiple cancer cell lines emerged with the 10-(4-methoxybenzoyl)-10H-phenoxazine-3-carbonitrile (33b, IC(50) values in the range of 2-15 nM) and the isovanillic analogue 33c. Seventeen compounds strongly inhibited tubulin polymerization with activities higher than or comparable to those of the reference compounds such as colchicine. Concentration-dependent flow cytometric studies revealed that inhibition of K562 cell growth was associated with an arrest in the G2/M phases of the cell cycle, indicative of mitotic blockade. Structure-activity relationship studies showed that best potencies were obtained with agents bearing a methoxy group placed para at the terminal phenyl ring and a 3-cyano group in the phenoxazine. A series of analogues highlight not only the phenoxazine but also the phenothiazine structural scaffold as valuable pharmacophores for potent tubulin polymerization inhibitors, worthy of further investigation.

Synthesis and biological evaluation of novel 10-benzyl-substituted 4,5-dichloro-10H-anthracen-9-ones as inhibitors of keratinocyte hyperproliferation.

A series of 10-substituted 4,5-dichloro-10H-anthracen-9-ones were synthesized in the search for novel agents against keratinocyte hyperproliferation. The antiproliferative activity of these novel anthrones was evaluated using the human keratinocyte line HaCaT as the primary test system. Structure-activity relationships with respect to the nature and position of the substituents at the benzyl moiety were studied, with a 3-hydroxy-4-methoxy-substitution pattern being the most potent (IC(50) = 0.7 µM) and comparable to the potency of the antipsoriatic anthralin. In contrast to anthralin, inhibition of keratinocyte hyperproliferation was not mediated by damage to the keratinocyte membrane, as the activity of lactate dehydrogenase released from the cytoplasm was in the control range. These findings may be rationalized as a benefit of the ineffectiveness of the novel anthrones to interact with the free radical 2,2-diphenyl-1-picrylhydrazyl.

Structure-activity relationship studies of acridones as potential antipsoriatic agents. 2. Synthesis and antiproliferative activity of 10-substituted hydroxy-10H-acridin-9-ones against human keratinocyte growth.

A series of 10-substituted hydroxy-10H-acridin-9-ones were synthesized and studied as potential antipsoriatic agents. The antiproliferative activity of the novel derivatives, which can be considered as aza-analogues of the antipsoriatic drug anthralin, was determined using the human keratinocyte cell line HaCaT. Structure-activity relationships with respect to the nature of the N-substituent at the acridone scaffold were delineated. Release of lactate dehydrogenase (LDH) was used to exclude non-specific cytotoxic effects. As compared to anthralin, N-substitution of the acridone scaffold in the target compounds provided agents devoid of radical producing properties, which was documented by their ineffectiveness to interact with the free radical 2,2-diphenyl-1-picrylhydrazyl. This was in excellent agreement with the data obtained from the LDH assay in which the novel compounds did not induce membrane damage. Benzyl substitution at the 10-position yielded keratinocyte growth inhibitory activity in the low micromolar range. The most potent inhibitor of keratinocyte hyperproliferation was compound 8a having an N-methyl group and a 1,3-dihydroxy arrangement at the acridone scaffold, with an IC(50) value comparable to that of anthralin.

Synthesis, antiproliferative activity and inhibition of tubulin polymerization by 1,5- and 1,8-disubstituted 10H-anthracen-9-ones bearing a 10-benzylidene or 10-(2-oxo-2-phenylethylidene) moiety.

A novel series of 1,5- and 1,8-disubstituted 10-benzylidene-10H-anthracen-9-ones and 10-(2-oxo-2-phenylethylidene)-10H-anthracen-9-ones was synthesized to assess the substituent effects on biological activity. The 3-hydroxy-2,4-dimethoxy-benzylidene analogue 16 h displayed strong antiproliferative activity against several tumor cell lines, including multi-drug resistant phenotypes. Flow cytometric studies showed that KB/HeLa cells treated by elected compounds were arrested in the G2/M phases of the cell cycle. Among the compounds tested for inhibition of tubulin polymerization, 14 compounds proved to be exceptionally active with IC(50) values < 1 microM. In the 1,5-dichloro-derived series of benzylideneanthracenones, E/Z isomers were separated and biological effects were monitored. We found that the olefinic geometry had no significant effect on biological activity. Furthermore, the E isomeric 1,5-dichloro-substituted phenacylidenes entirely proved to be more potent inhibitors of tubulin polymerization than the recently described 10-(2-oxo-2-phenylethylidene)-10H-anthracen-9-ones. In conclusion, the present study improves understanding of the action of anthracenone-based tubulin polymerization inhibitors and contributes to the design of further potent anti-tubulin drugs.

Synthesis, antiproliferative activity and inhibition of tubulin polymerization by anthracenone-based oxime derivatives.

A series of anthracenone-based oxime ethers and -esters were synthesized in order to evaluate their antiproliferative activity. Several investigated compounds displayed strong antiproliferative activity against K562 leukemia cells and proved to be strong inhibitors of tubulin polymerization. In this context, anthracenone-based oxime ethers and -esters are considered to contribute to the development of novel antiproliferative drugs, based on tubulin interaction.

Synthesis of 2,6-aceanthrylenedione, a cyclic vinylog of anthraquinone.

The first synthesis of 2,6-aceanthrylenedione (6), a cyclic vinylog of anthraquinone and a useful starting material for the synthesis of 1-phenylaceanthrylene-2,6-diones such as 7, 8, and 9, is described. (10-Oxo-10H-anthracen-9-ylidene) acetyl chloride (5) cyclizes intramolecularly at room temperature in the presence of AlCl(3) to give 6. We found that 6 is a cytotoxic compound that inhibits tubulin polymerization.

Structure-activity relationship studies of acridones as potential antipsoriatic agents. 1. Synthesis and antiproliferative activity of simple N-unsubstituted 10H-acridin-9-ones against human keratinocyte growth.

A series of N-unsubstituted hydroxy-10H-acridin-9-ones were synthesized and evaluated for inhibitory action against HaCaT keratinocyte growth, in order to explore their potential as antipsoriatic agents. For structure-activity relationship studies, the number and position of the hydroxyl groups were modified, the oxygen functions substituted or replaced, or additional functional groups were introduced into the acridone scaffold. 1,8-Dihydroxy-10H-acridin-9-one (4), which is an aza-analogue of the antipsoriatic anthralin, was only marginally active. However, 1,3-dihydroxy-substituted 5ee was the most potent acridone within this series and inhibited keratinocyte growth with an IC(50) value comparable to that of anthralin. In contrast to anthralin, nearly all members of the acridone series were devoid of radical generating properties, which were determined by their capability to interact with the free radical 2,2-diphenyl-1-picrylhydrazyl. Structures with a phenolic hydroxyl or an aromatic amine arranged ortho or para to the acridone NH group were exceptions. Also in contrast to anthralin, membrane-damaging effects as documented by the release of lactate dehydrogenase into the culture medium were not observed for acridones.

10-(2-oxo-2-phenylethylidene)-10H-anthracen-9-ones as highly active antimicrotubule agents: synthesis, antiproliferative activity, and inhibition of tubulin polymerization.

A series of 10-(2-oxo-2-phenylethylidene)-10H-anthracen-9-ones were synthesized and evaluated for interactions with tubulin and for antiproliferative activity against a panel of human and rodent tumor cell lines. The 4-methoxy analogue 17b was most potent, displaying IC(50) values ranging from 40 to 80 nM, including multidrug resistant phenotypes, and had excellent activity as an inhibitor of tubulin polymerization (IC(50) = 0.52 microM). Concentration-dependent flow cytometric studies showed that KB/HeLa cells treated with 17b were arrested in the G2/M phases of the cell cycle (EC(50) = 90 nM). In competition experiments, 17b strongly displaced [(3)H]-colchicine from its binding site in the tubulin. The results obtained demonstrate that the antiproliferative activity is related to the inhibition of tubulin polymerization.

Sulfonate derivatives of naphtho[2,3-b]thiophen-4(9H)-one and 9(10H)-anthracenone as highly active antimicrotubule agents. Synthesis, antiproliferative activity, and inhibition of tubulin polymerization.

Benzenesulfonate derivatives of naphtho[2,3-b]thiophen-4(9H)-one and 9(10H)-anthracenone were prepared and found to inhibit microtubule formation by an in vitro tubulin polymerization assay. Several analogues showed potent cytotoxic activity in an assay based on K562 leukemia cells with IC50 values of <100 nM. The methylamino analogue 14i was the most active compound in this assay (14i, IC50 K562: 0.05 muM). Antiproliferative activities of selected compounds were additionally evaluated against a panel of 12 tumor cell lines, including multi-drug-resistant phenotypes. All resistant cell lines were sensitive to these compounds. Concentration-dependent flow cytometric studies showed that KB/HeLa cells treated with selected compounds were arrested in the G2/M phases of the cell cycle. In competition experiments, these compounds strongly displaced radiolabeled colchicine from its binding site in the tubulin, showing IC50 values lower than that of colchicine. The results demonstrate that the antiproliferative activity is related to the inhibition of tubulin polymerization.