Abietane-Type Diterpenoid Amides with Highly Potent and Selective Activity against <i>Leishmania donovani</i> and <i>Trypanosoma cruzi</i>

Dehydroabietylamine (<b>1</b>) was used as a starting material to synthesize a small library of dehydroabietyl amides by simple and facile methods, and their activities against two disease-causing trypanosomatids, namely, <i>Leishmania donovani</i> and <i>Trypanosoma cruzi,</i> were assayed. The most potent compound, <b>10</b>, an amide of dehydroabietylamine and acrylic acid, was found to be highly potent against these parasites, displaying an IC₅₀ value of 0.37 μM against <i>L. donovani</i> axenic amastigotes and an outstanding selectivity index of 63. Moreover, compound <b>10</b> fully inhibited the growth of intracellular amastigotes in <i>Leishmania donovani</i>-infected human macrophages with a low IC₅₀ value of 0.06 μM. This compound was also highly effective against <i>T. cruzi</i> amastigotes residing in L6 cells with an IC₅₀ value of 0.6 μM and high selectivity index of 58, being 3.5 times more potent than the reference compound benznidazole. The potent activity of this compound and its relatively low cytotoxicity make it attractive for further development in pursuit of better drugs for patients suffering from leishmaniasis and Chagas disease

Primary 3D screen.

<p>PC-3 cells were cultured in 3D Matrigel ECM for 4 days and treated for 6 days with 25 betulin derivatives (from 2D high throughput screens), DMSO/vehicle control, and three reference compounds. A) Representative maximum intensity projections of confocal microscope stack images for selected compound treatments at 300 nM concentration (5× objective, scale 100 μm). B) Three graphs showing the relative impact on three morphometric parameters for 7 betulin derivatives, DMSO control, and one control compound (paclitaxel). Data scaling: displays the relative difference between median of Area/Complexity/Area Ratio, to DMSO control. Paclitaxel treatments and DMSO controls have been assigned values of -100 and 0, respectively. C) Hierarchical clustering was done using three morphological parameters derived from PC3 organoids: spheroid size (area), complexity and the number of dead cells. D) Wound healing curves of the two betulin derivatives <b>4</b> and <b>20</b>, highlighting the 50% cut-off level (orange dashed line).</p

Chemical structures of the fused 5-membered ring derivatives of betulinic acid.

<p>Chemical structures of the fused 5-membered ring derivatives of betulinic acid.</p

Estimated EC₅₀ values for cell proliferation.

<p>Values have been calculated using Harmony High-Content Imaging and Analysis Software.</p><p>Estimated EC₅₀ values for cell proliferation.</p

Chemical structures of the parental compound betulinic acid and its derivatives.

<p>Chemical structures of the parental compound betulinic acid and its derivatives.</p

Secondary 3D screens.

<p>Experimental betulin derivatives and control compounds were tested across a panel of prostate cancer lines (LNCaP, LAPC-4) and non-transformed, prostate epithelial cells (EP156T) in 3D culture. <b>A</b>) The dendrogram is based on three main morphological parameters and combines all data from primary and secondary 3D screens. Effective compound treatments are indicated in red and yellow, yellow being the most invasion-specific. <b>B</b>) Boxplots showing the impact of selected compounds on spheroid size (Area). Data scaling: as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126111#pone.0126111.g004" target="_blank">Fig 4</a><b>C</b>) Effects of all betulin derivatives and control compounds on cell death (number of dead cells).</p

Mechanisms of action.

<p>PC-3 spheroids were exposed to two representative betulin derivatives, <b>5</b> and <b>20</b>, for 4 h (at 1 μM) and 6 days (at 0.3 μM). A) Computational quantification of signal intensities from the kinase arrays, aligned by fold changes observed (left to right). B) Western blot of total and phospho-Akt (S473) shown for both short and long-term exposure to <b>20</b> and <b>5</b>.</p

Cytotoxicity tests performed in 2D monolayer culture.

<p><b>A</b>) Cell proliferation/cell number, <b>B</b>) programmed cell death (apoptosis), and <b>C</b>) number of dead cells were assessed by conventional assays, combined with high-content microscopy (Operetta). Cells were treated with the five most effective betulin derivatives, including paclitaxel control for 72h. Proliferation was measured as the total number of nuclei (= cells), apoptosis as ratio of caspase-3 positive versus all cells, and cell death as ethidium homodimer-2 positive nuclei versus all cells.</p

Chemical structures of the fused 6-membered ring derivatives of betulinic acid.

<p>Chemical structures of the fused 6-membered ring derivatives of betulinic acid.</p