Napabucasin and Related Heterocycle-Fused Naphthoquinones as STAT3 Inhibitors with Antiproliferative Activity against Cancer Cells

Napabucasin (<b>6</b>) and its angularly anellated isomer (<b>7</b>), for which the synthesis is described, together with related plant-derived naphthoquinones, were evaluated in vitro against human breast cancer (MDA-MB-231) and chronic myelogenous leukemia (K562) cells. As observed for β-lapachone (<b>3</b>), the active naphthoquinones all induced apoptosis in a cell-cycle-independent fashion. In contrast to the pyran-fused β-lapachone (<b>3</b>), however, the most potent furan-fused naphthoquinones were able to redox cycle and generate superoxide in cell-based assays, which was independent of NAD­(P)­H:quinone oxido-reductase 1. In a homogeneous time-resolved fluorescence (HTRF) assays with MDA-MB-231 cells, both napabucasin (<b>6</b>) and isonapabucasin (<b>7</b>) were identified as targeting STAT3 phosphorylation. In addition, drug affinity responsive target stability assays were performed to validate a direct interaction of the naphthoquinones with STAT3. Isonapabucasin (<b>7</b>) turned out to be twice as potent against STAT3 as napabucasin (<b>6</b>) in the HTRF assay, with an EC₅₀ in the submicromolar range, which was in excellent agreement with the potency of both agents to inhibit the growth of MDA-MB-231 cells. Moreover, molecular docking experiments predicted different binding modes to the STAT3 SH2 domain for the linearly anellated napabucasin (<b>6</b>) and its angularly anellated isomer (<b>7</b>)

Synthesis and Structure–Activity Relationships of Lapacho Analogues. 1. Suppression of Human Keratinocyte Hyperproliferation by 2‑Substituted Naphtho[2,3‑<i>b</i>]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-<i>b</i>]­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 IC₅₀ 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 <b>18</b>, 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

<i>N</i>‑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)-10<i>H</i>-phenoxazine-3-carbonitrile (<b>16o</b>), showed excellent antiproliferative properties, with low nanomolar GI₅₀ values (<b>16o</b>, 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 β-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

Synthesis and Structure–Activity Relationships of Lapacho Analogues. 2. Modification of the Basic Naphtho[2,3‑<i>b</i>]furan-4,9-dione, Redox Activation, and Suppression of Human Keratinocyte Hyperproliferation by 8‑Hydroxynaphtho[2,3‑<i>b</i>]thiophene-4,9-diones

The basic structure of linearly anellated lapacho quinones, naphtho­[2,3-<i>b</i>]­furan-4,9-dione (<b>7</b>), 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-<i>b</i>]­thiophene-4,9-dione (<b>8a</b>), are described. Out of a total of 71 analogues, particularly 2-thenoyl-substituted <b>26l</b>, 2-nicotinoyl-substituted <b>26m</b>, and 2-oxadiazole-substituted <b>35a</b> 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

Fluorination Patterning: A Study of Structural Motifs That Impact Physicochemical Properties of Relevance to Drug Discovery

The synthesis of a collection of 3-substituted indole derivatives incorporating partially fluorinated <i>n</i>-propyl and <i>n</i>-butyl groups is described along with an in-depth study of the effects of various fluorination patterns on their properties, such as lipophilicity, aqueous solubility, and metabolic stability. The experimental observations confirm predictions of a marked lipophilicity decrease imparted by a <i>vic</i>-difluoro unit when compared to the <i>gem</i>-difluoro counterparts. The data involving the comparison of the two substitution patterns is expected to benefit molecular design in medicinal chemistry and, more broadly, in life as well as materials sciences