SYNTHESIS OF THIOPHENE-CONTAINING HETEROCYCLES AND THEIR APPLICATION AS ANTICANCER AGENTS

Five-membered heterocycles represent a privileged scaffold in drug discovery and are the focus of analog design. The first chapter presents the synthesis and biological evaluation of analogs of NSC 652287, which targets the renal carcinoma cell line A498. The Pd-catalyzed Suzuki-Miyaura cross-coupling with halogenated furan, thiophene, and selenophene led to generally high overall yields for the construction of heterocyclic triads. C-H bond activation provided an efficient strategy for the Pd-catalyzed cross-coupling at C(2) of oxazoles. Further analog variation was achieved with a copper-catalyzed azide-alkyne cycloaddition to form a 1,4-substituted 1,2,3-triazole. Potency and selectivity of the final hydroxymethyl products were determined in the NCI-60 cell assay. Two lead compounds were tested in vivo and compared to NSC 652287. The evaluation of these compounds continues with a thermal shift assay coupled with differential mass spectrometry for biological target identification. The chemical stability of select triads are also discussed. The second chapter details the synthesis of thienopyridone and thienopyrimidine dione analogs as inhibitors of protein tyrosine phosphatase 4A3 (PTP4A3), an attractive anticancer target. Derivatization by photooxygenation led to a compound with a unique structural motif, high potency, and excellent selectivity towards PTP4A3. An automated synthesis strategy using Lilly’s Automated Synthesis Lab was employed for analog synthesis

Targeting Ovarian Cancer and Endothelium with an Allosteric PTP4A3 Phosphatase Inhibitor

Overexpression of protein tyrosine phosphatase PTP4A oncoproteins is common in many human cancers and is associated with poor patient prognosis and survival. We observed elevated levels of PTP4A3 phosphatase in 79% of human ovarian tumor samples, with significant overexpression in tumor endothelium and pericytes. Furthermore, PTP4A phosphatases appear to regulate several key malignant processes, such as invasion, migration, and angiogenesis, suggesting a pivotal regulatory role in cancer and endothelial signaling pathways. While phosphatases are attractive therapeutic targets, they have been poorly investigated because of a lack of potent and selective chemical probes. In this study, we disclose that a potent, selective, reversible, and noncompetitive PTP4A inhibitor, JMS-053, markedly enhanced microvascular barrier function after exposure of endothelial cells to vascular endothelial growth factor or lipopolysaccharide. JMS-053 also blocked the concomitant increase in RhoA activation and loss of Rac1. In human ovarian cancer cells, JMS-053 impeded migration, disrupted spheroid growth, and decreased RhoA activity. Importantly, JMS-053 displayed anticancer activity in a murine xenograft model of drug resistant human ovarian cancer. These data demonstrate that PTP4A phosphatases can be targeted in both endothelial and ovarian cancer cells, and confirm that RhoA signaling cascades are regulated by the PTP4A family

Head-to-head comparison of BAM15, semaglutide, rosiglitazone, NEN, and calorie restriction on metabolic physiology in female <i>db/db</i> mice

Metabolic disorders such as type 2 diabetes, fatty liver disease, hyperlipidemia, and obesity commonly co-occur but clinical treatment options do not effectively target all disorders. Calorie restriction, semaglutide, rosiglitazone, and mitochondrial uncouplers have all demonstrated efficacy against one or more obesity-related metabolic disorders, but it currently remains unclear which therapeutic strategy best targets the combination of hyperglycaemia, liver fat, hypertriglyceridemia, and adiposity. Herein we performed a head-to-head comparison of 5 treatment interventions in the female db/db mouse model of severe metabolic disease. Treatments included ∼60 % calorie restriction (CR), semaglutide, rosiglitazone, BAM15, and niclosamide ethanolamine (NEN). Results showed that BAM15 and CR improved body weight and liver steatosis to levels superior to semaglutide, NEN, and rosiglitazone, while BAM15, semaglutide, and rosiglitazone improved glucose tolerance better than CR and NEN. BAM15, CR, semaglutide, and rosiglitazone all had efficacy against hypertriglyceridaemia. These data provide a comprehensive head-to-head comparison of several key treatment strategies for metabolic disease and highlight the efficacy of mitochondrial uncoupling to correct multiple facets of the metabolic disease milieu in female db/db mice.</p

Scope of first-generation SAR studies on SD-208.

<p>2-Zone model for SD-208 analog synthesis.</p

Overview of structures and yields of intermediates 3 and 4 (shown in “S1 Fig.”) and analogs 5 (as described in “<b><i>Supporting Information</i></b>”) with PKD1 inhibition data for analogs 5.

<p>^aPiperazine is directly connected to the pteridine core without a secondary amine.</p><p>^bVia the PyBOP route.</p><p>^cVia the chlorination/substitution route (2-step yield).</p><p>Overview of structures and yields of intermediates 3 and 4 (shown in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119346#pone.0119346.s001" target="_blank">S1 Fig.</a>”) and analogs 5 (as described in “<b><i>Supporting Information</i></b>”) with PKD1 inhibition data for analogs 5.</p

SD-208 inhibited prostate cancer cells proliferation, survival, and invasion and the anti-proliferative effect of SD-208 was mediated through the inhibition of PKD.

<p><b>A-B. SD-208 inhibited PC3 (A) and LNCaP (B) prostate cancer cell proliferation</b>. PC3 and LNCaP cells were plated in triplicates in 24-well plates. Cells were allowed to attach overnight. A cell count at day 1 was made, and then either a vehicle (DMSO) or SD-208 at 30 μM was added. Cells were counted daily for a total of 6 days. Data are the mean ± S.E. of two independent experiments with triplicate determinations at each data point in each experiment. <b>C</b>. <b>SD-208 inhibited PC3 prostate cancer cell survival</b>. PC3 cells were seeded into 96-well plates (3000 cells/well) and were then incubated in media containing 0.3–100 μM inhibitors for 72 h. MTT solution was added to each well and incubated for 4 h. Optical density was read at 570 nm to determine cell viability. The IC₅₀ was determined as the mean of three independent experiments for each compound. <b>D. SD-208 inhibited prostate cancer cell invasion</b>. DU145 cells were incubated with 30 μM SD 208 in Matrigel inserts. After 20 h, noninvasive cells were removed and invasive cells were fixed in 100% methanol, stained in 0.4% hematoxylin solution, and photographed. The number of cells that invaded the Matrigel matrix was determined by cell counts in 6 fields relative to the number of cells that migrated through the control insert. Percentage invasion was calculated as the percent of the cells invaded through Matrigel inserts vs. the total cells migrated through the control inserts. Data are the mean ± S.E. of three independent experiments with triplicate determinations at each data point in each experiment. Statistical significance was determined using the unpaired t-test. ***, p<0.001. <b>E-F. Overexpression of PKD1 and PKD3 in prostate cancer cells rescued the anti-proliferative effects of SD-208</b>. PC3 (0.5 million) cells were seeded in a 60 mm dish and infected the next day with 50 and 100 MOI of PKD1 and PKD3 adenoviruses (Adv-PKD1 and Adv-PKD3). Empty adenovirus (Adv-null) was used as control. After 24 h, 3000 cells/well were plated in 96-well plates and treated with and without 10 and 30 μM SD-208 for 72 h. MTT solution was added to each well and incubated for 4 h. Optical density was read at 570 nm to determine cell viability. The overexpression of PKD1 and PKD3 was confirmed by Western blotting analysis. This experiment was repeated three times and data are the mean ± S.E. of all three independent experiments. Statistical significance between DMSO and inhibitor treatment was determined using the unpaired t-test.*, p<0.05; **, p<0.01; ***, p<0.001. <b>G. PKD mediated Hsp27 activity in prostate cancer cells was inhibited by SD-208</b>. DU145 cells were pretreated with different doses of inhibitors for 45 min, followed by PMA stimulation at 10 nM for 20 min. Cell lysates were subjected to immunoblotting for p-S⁹¹⁰-PKD1 and p-S^738/742-PKD1. GAPDH was blotted as loading control. The experiment was repeated two times and the representative blots are shown.</p

SD-208 arrested cells in G2/M and regulated the levels and activities of cell cycle regulatory proteins at the G2/M phase of cell cycle in prostate cancer cells.

<p><b>A-B. SD-208 induced G2/M cell cycle arrest in prostate cancer cells</b>. DU145 cells <b>(A)</b> and PC3 cells <b>(B)</b> were treated with either vehicle (DMSO) or 30 μM SD 208 for 48 h. Cell cycle distribution was determined by flow cytometry after propidium iodide labeling of fixed cells. The cell cycle plots are representative of three independent experiments (<i>left</i>). Data in the bar graph are the mean ± SEM of three independent experiments (<i>right</i>). Statistical significance was determined using the unpaired t-test and is indicated. **, p<0.01; ***, p<0.001. <b>C-D. Effects of SD-208 on the expression and activities of G2/M cell cycle regulatory proteins</b>. DU145 (<b>C</b>) and PC3 (<b>D</b>) cells were treated with DMSO or 5–30 µM SD-208 for 24 and 48 h. At the end of each treatment, cells were harvested and subjected to immunoblotting for the cell cycle regulatory proteins as indicated. GAPDH was blotted as loading control. The densitometry data (mean ± SEM from four experiments) were plotted as ‘fold change’ over the control after normalization with GAPDH. The experiments were repeated four times and representative blots from one experiment are shown.</p

SD-208 was a cell-active, ATP-competitive PKD inhibitor and did not inhibit PKC and CAMK.

<p><b>A. Determination of PKD kinase activity <i>in vitro</i></b>. Inhibition of recombinant human PKD1, 2 and 3 was assayed in the presence of 10 different concentrations of SD-208 by an <i>in vitro</i> radiometric PKD kinase assay. The IC₅₀ values were calculated as the mean ± SEM of at least three independent experiments with triplicate determinations at each compound concentration in each experiment. The data were plotted as a function of inhibitor concentration and a representative graph is shown. <b>B</b>. <b>SD-208 inhibited PMA-induced PKD1 activation in prostate cancer cells</b>. LNCaP cells were pretreated with different doses of inhibitors for 45 min, followed by PMA stimulation at 10 nM for 20 min. Cell lysates were subjected to immunoblotting for p-S⁹¹⁰-PKD1 and p-S^738/742-PKD1. Tubulin was blotted as loading control. The experiment was repeated three times and the representative blots are shown. <b>C. Determination of cellular IC</b>_<b>50</b><b> </b>. Western blots from ‘B” were quantified using densitometry analysis. The data were plotted and IC₅₀ values were derived from the concentration-response curves using GraphPad. One of the three concentration-response curves is shown. <b>D. SD-208 is an ATP-competitive kinase inhibitor</b>. PKD1 kinase activity was measured as a function of increasing concentrations of ATP in the presence of varying concentrations of SD-208. Lineweaver-Burke plots of the data are shown. Data presented were the mean ± S.E. of three independent experiments with triplicate determinations at each data point in each experiment. <b>E-F. Selectivity of SD-208 against related kinases</b>. Inhibition of PKCα (<b>B</b>) or PKCδ (<b>C</b>) was determined at 10 nM, 100 nM, 1μM, and 10 μM. As controls, the PKC inhibitor GF109203X potently inhibited PKCα and PKCδ activity. Data are the mean ± SEM of two independent experiments. <b>G</b>. Inhibition of CAMKIIα was measured by the radiometric CAMK kinase assay. Data are the mean ± S.E. of two independent experiments with triplicate determinations at each data point in each experiment. Statistical significance was determined using the unpaired t-test. ns, not significantly significant; *, p<0.05; **, p<0.01; ***, p<0.001.</p