Ana S. Leal

Ursane-type pentacyclic triterpenoids as useful platforms in anticancer drug discovery

This review highlights the potential of natural and semisynthetic ursane-type triterpenoids as candidates for the design of multi-target bioactive compounds, with focus on their anticancer effects. A brief illustration of the biosynthesis, sources, and general biological effects of the main classes of naturally occurring pentacyclic triterpenoids (PTs) are provided.

Synthesis of novel ursolic acid heterocyclic derivatives with improved abilities of antiproliferation and induction of p53, p21waf1 and NOXA in pancreatic cancer cells

A series of new heterocyclic derivatives of ursolic acid 1 were synthesized and evaluated for their antiproliferative activity against AsPC-1 pancreatic cancer cells. Compounds 24-32, with an α,β unsaturated ketone in conjugation with an heterocyclic ring in ring A have improved antiproliferative activities. Compound 32 is the most active compound with an IC(50) of 1.9 μM which is sevenfold more active than ursolic acid 1. Compound 32 arrests cell cycle in G1 phase and induces apoptosis in AsPC-1 cells with upregulation of p53, p21(waf1) and NOXA protein levels.

Semisynthetic Ursolic Acid Fluorolactone Derivatives Inhibit Growth with Induction of p21waf1 and Induce Apoptosis with Upregulation of NOXA and Downregulation of c‐FLIP in Cancer Cells

A series of ursolic acid ((1S,2R,4aS,6aR,6aS,6bR,8aR,10S,12aR,14bS)‐10‐hydroxy‐1,2,6a,6b,9,9,12a‐heptamethyl‐2,3,4,5,6,6a,7,8,8a,10,11,12,13,14b‐tetradecahydro‐1H‐picene‐4a‐carboxylic acid) derivatives with a 12‐fluoro‐13,28β‐lactone moiety were synthesized using the electrophilic fluorination reagent Selectfluor. The antiproliferative effects of these novel compounds were evaluated in AsPC‐1 pancreatic cancer cells, and the structure–activity relationships (SARs) were evaluated. Of the compounds synthesized, ursolic acid derivatives carrying a heterocyclic ring, such as imidazole or methylimidazole, and cyanoenones were among the more potent inhibitors of AsPC‐1 pancreatic cancer cell growth. 2‐Cyano‐3‐oxo‐12α‐fluoro‐urs‐1‐en‐13,28β‐olide, compound 20, was the most effective inhibitor with IC50 values of 0.7, 0.9 and 1.8 μM in pancreatic cancer cell lines AsPC‐1, MIA PaCa‐2 and PANC‐1, respectively. This compound also exhibited better antiproliferative activities against breast (MCF7), prostate (PC‐3), hepatocellular (Hep G2) and lung (A549) cancer cell lines, with IC50 values lower than 1 μM. The mechanism of action by which these compounds exert their biological effect was evaluated in AsPC‐1 cells using the most potent inhibitor synthesized, compound 20. At 1 μM, the cell cycle arrested at the G1 phase with upregulation of p21waf1. Apoptosis was induced at an inhibitor concentration of 8 μM with upregulation of NOXA and downregulation of c‐FLIP. These data indicate that fluorolactone derivatives of ursolic acid have improved antiproliferative activity, acting through arrest of the cell cycle and induction of apoptosis.

Efficient oxidation of oleanolic acid derivatives using magnesium bis(monoperoxyphthalate) hexahydrate (MMPP): A convenient 2-step procedure towards 12-oxo-28-carboxylic acid derivatives

Summary A new, straightforward and high yielding procedure to convert oleanolic acid derivatives into the corresponding δ-hydroxy-γ-lactones, by using the convenient oxidizing agent magnesium bis(monoperoxyphthalate) hexahydrate (MMPP) in refluxing acetonitrile, is reported. In addition, a two-step procedure for the preparation of oleanolic 12-oxo-28-carboxylic acid derivatives directly from Δ12-oleananes, without the need for an intermediary work-up, and keeping the same reaction solvent in both steps, is described as applied to the synthesis of 3,12-dioxoolean-28-oic acid.

Abstract A23: Novel ursolic acid derivatives with potent antitumor activity

Cancer is the fourth leading cause of death worldwide, within pancreatic cancer is the fourth leading cause of cancer death and without effective medical therapy. 1 Natural products provide a promising source for developing effective anticancer agents. 2 Ursolic acid is a pentacyclic triterpenoid present in plants, vegetables, and fruits that has been found to have several biological activities, including antitumor activity. 3 In order to improve the preexisting antitumor activity of ursolic acid, a panel of semisynthetic derivatives were prepared, characterized, and evaluated for the antiproliferative activities in several cancer cell lines. Ursolic acid was chemically modified by the introduction of heterocyclic rings in several points of the backbone structure and was found that modifications at C 2 had improved activities against proliferation of pancreatic cancer cell lines, inducing apoptosis with upregulation of p53 and NOXA levels, and downregulation of XIAP levels. Fluor is a highly desirable atom, in key positions of active molecules can improve metabolic and chemical stability, membrane permeability and binding affinity. 4 We have introduced fluor into the ring C of ursolic acid and a series of ursane-type fluor-derivatives were synthesized. The most potent derivative was 19-fold more active than ursolic acid to inhibit Aspc-1 cell growth and to arrest cell cycle at G1 phase with upregulation of p21 waf1 , and apoptosis at higher concentrations with upregulation of NOXA and downregulation of c-FLIP. These new semisynthetic derivatives of ursolic acid may have potential for drug discovery to treat cancer. Citation Format: Ana S. Leal, Jorge A.R. Salvador, Jing Yongkui. Novel ursolic acid derivatives with potent antitumor activity [abstract]. In: Proceedings of the AACR Special Conference on Chemical Systems Biology: Assembling and Interrogating Computational Models of the Cancer Cell by Chemical Perturbations; 2012 Jun 27-30; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2012;72(13 Suppl):Abstract nr A23.

Dehydroabietic oximes halt pancreatic cancer cell growth in the G1 phase through induction of p27 and downregulation of cyclin D1

Low 5-year survival rates, increasing incidence, as well as the specific challenges of targeting pancreatic cancer, clearly support an urgent need for new multifunctional drugs for the prevention and treatment of this fatal disease. Natural products, such as abietane-type diterpenoids, are widely studied as promiscuous anticancer agents. In this study, dehydroabietic oximes were identified as potential compounds to target pancreatic cancer and cancer-related inflammation. The compounds inhibited the growth of human pancreatic cancer Aspc-1 cells with IC50 values in the low micromolar range and showed anti-inflammatory activity, measured as the inhibition of nitric oxide production, an important inflammatory mediator in the tumour microenvironment. Further studies revealed that the compounds were able to induce cancer cell differentiation and concomitantly downregulate cyclin D1 expression with upregulation of p27 levels, consistent with cell cycle arrest at the G1 phase. Moreover, a kinase profiling study showed that one of the compounds has isoform-selective, however modest, inhibitory activity on RSK2, an AGC kinase that has been implicated in cellular invasion and metastasis.

Abstract 5082: The selective bromodomain inhibitor, INCB054329 targets both cancer cells and the tumor microenvironment in the KC inflammatory preclinical model of ductal pancreatic cancer

Pancreatic cancer is expected to become the second most deadly cancer by 2030, with few effective therapeutic options available to improve patient survival. The Kras gene is mutated in over 90% of pancreatic cancers, but the Kras protein is considered to be an undruggable target. p-Erk is a downstream effector of Kras and has been shown to be essential for the progression and maintenance of pancreatic cancer. The LSL-KrasG12D/+; Pdx-1-Cre (KC) mouse model is used to study pancreatic cancer, especially cancer progression, after stimulation with an inflammatory agent, such as caerulein or LPS. Bromodomain inhibitors are a new generation of drugs that target BET (bromodomain and extra-terminal) proteins, impairing their ability to bind to acetylated lysines and therefore interfering with transcriptional initiation and elongation. BET proteins regulate several genes responsible for cell growth, apoptosis and inflammation. INCB54329 is a novel, orally bioavailable BET inhibitor that is currently being investigated in Phase 1 clinical trials.INCB054329 inhibits cell growth in murine pancreatic cancer cells harboring Kras mutations, derived from a KrasG12D/+; Trp53R172H/+; Pdx-1-Cre (KPC) mouse with an IC50 values less than 100 nM. Moreover, INCB054329 reduces the levels of p-ERK 1/2 in these cells lines. When KC mice are injected with LPS to induce inflammation and pancreatitis, p-Stat3 protein levels are significantly increased, but this pro-survival protein is reduced by more than 50%, in mice that were pre-treated with INCB054329. Tumor cells can use epigenetic modulation to evade immune recognition and to shape the TME (tumor microenvironment) toward an immunosuppressive phenotype. Since epigenetic modulation is a mechanism used by tumors to regulate the TME, using small molecules as epigenetic modulators to activate immune recognition is a therapeutically desirable approach. Within the TME, several cytokines and chemokines play crucial roles, recruiting and regulating inflammatory cells [macrophages, T cells and Myeloid derived suppressor cells]. KC mice stimulated with LPS have higher levels of CCL2 (1947 ± 591 ng/mL) and IL-6 (2459 ± 577 pg/mL) than unstimulated mice; these increased levels of inflammatory cytokines and the subsequent immune cell infiltration can be modulated by treatment with INCB054329, with INCB054329 demonstrating >50% inhibition of both cytokines (IL-6: 1063 ± 502 pg/mL; CCL2: 713 ± 134 ng/mL). These data suggest that INCB054329 has a dual activity, targeting cancer cells and modulating the TME; both activities may prove beneficial for the treatment of pancreatic cancer. Moreover, this work suggests that additional mechanisms may underlie the beneficial effects of bromodomain inhibitors. Citation Format: Ana Sofia Leal, Karen T. Liby, Phillip Liu, Bruce Ruggeri. The selective bromodomain inhibitor, INCB054329 targets both cancer cells and the tumor microenvironment in the KC inflammatory preclinical model of ductal pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5082. doi:10.1158/1538-7445.AM2017-5082

Abstract 4234: Ursolic acid induces apoptosis in pancreatic cancer cells associated with inhibition of STAT3 and down-regulation of Bcl-XL

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Pancreatic cancer is the fourth leading cause of cancer death and without effective medical therapy. New agents acting on novel targets need to be developed. Natural products provide a promising source for developing effective anticancer agents. Oleanolic acid (OA) and ursolic acid (UA) are two natural triterpenoids with reported antitumor activities in several types of cancer. The antitumor effects of OA, oleanolic acid methyl ester (OAME) and UA were determined in pancreatic cancer Aspc-1 cells. Cell growth inhibition of the three compounds was determined using the MTT assay. The IG50s of OA, OAME and UA after treatment with UA for 4 days are >30, 8.9, 12.5 μM, respectively. OA is a weak cell growth inhibitor and methyl esterification improves its antitumor activity. UA is more effective than OA to inhibit cell growth in Aspc-1 cells and was used to study the mechanism of action. Aspc-1 cells treated with UA at a concentration higher than 25 uM induced apoptosis as determined by PARP cleavage detection. Western blot analysis revealed that the levels of procaspase-3, -9 and Bcl-XL were markedly decreased while the levels of Mcl-1, XIAP and survivin were weakly or not decreased. Correlated with the decrease in the level of Bcl-XL, the level of phosphorylated STAT3 was significantly inhibited. STAT3 is activated in pancreatic cancer cells and contributes to pancreatic tumorigenesis. Activated STAT3 increases the levels of antiapoptotic protein such as Bcl-XL, Mcl-1 and survivin, are being considered as a target for developing agents for pancreatic cancer treatment. Our data suggest that UA is a lead candidate for targeting STAT3 and structural modification of UA may result in a new group of compounds with improved antitumor activity in pancreatic cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4234. doi:10.1158/1538-7445.AM2011-4234