Synthesis of Rhodamine-Conjugated Lupane Type Triterpenes of Enhanced Cytotoxicity.

Various conjugates with rhodamines were prepared by starting with betulinic acid (BA) and platanic acid (PA). The molecules homopiperazine and piperazine, which were identified in earlier research, served as linkers between the rhodamine and the triterpene. The pentacyclic triterpene's ring A was modified with two acetyloxy groups in order to possibly boost its cytotoxic activity. The SRB assays' cytotoxicity data showed that conjugates 13-22, derived from betulinic acid, had a significantly higher cytotoxicity. Of these hybrids, derivatives 19 (containing rhodamine B) and 22 (containing rhodamine 101) showed the best values with EC50 = 0.016 and 0.019 µM for A2780 ovarian carcinoma cells. Additionally, based on the ratio of EC50 values, these two compounds demonstrated the strongest selectivity between malignant A2780 cells and non-malignant NIH 3T3 fibroblasts. A375 melanoma cells were used in cell cycle investigations, which showed that the cells were halted in the G1/G0 phase. Annexin V/FITC/PI staining demonstrated that the tumor cells were affected by both necrosis and apoptosis.

Synthesis and evaluation of pentacyclic triterpenoids conjugates as novel HBV entry inhibitors targeting NTCP receptor.

Chronic liver diseases caused by hepatitis B virus (HBV) are the accepted main cause leading to liver cirrhosis, hepatic fibrosis, and hepatic carcinoma. Sodium taurocholate cotransporting polypeptide (NTCP), a specific membrane receptor of hepatocytes for triggering HBV infection, is a promising target against HBV entry. In this study, pentacyclic triterpenoids (PTs) including glycyrrhetinic acid (GA), oleanolic acid (OA), ursolic acid (UA) and betulinic acid (BA) were modified via molecular hybridization with podophyllotoxin respectively, and resulted in thirty-two novel conjugates. The anti-HBV activities of conjugates were evaluated in HepG2.2.15 cells. The results showed that 66% of the conjugates exhibited lower toxicity to the host cells and had significant inhibitory effects on the two HBV antigens, especially HBsAg. Notably, the compounds BA-PPT1, BA-PPT3, BA-PPT4, and UA-PPT3 not only inhibited the secretion of HBsAg but also suppressed HBV DNA replication. A significant difference in the binding of active conjugates to NTCP compared to the HBV PreS1 antigen was observed by SPR assays. The mechanism of action was found to be the competitive binding of these compounds to the NTCP 157-165 epitopes, blocking HBV entry into host cells. Molecular docking results indicated that BA-PPT3 interacted with the amino acid residues of the target protein mainly through π-cation, hydrogen bond and hydrophobic interaction, suggesting its potential as a promising HBV entry inhibitor targeting the NTCP receptor.

Synthesis and biological evaluation of celastrol derivatives as potential anti-glioma agents by activating RIP1/RIP3/MLKL pathway to induce necroptosis.

Celastrol, a quinone methide triterpenoid, possesses potential anti-glioma activity. However, its relatively low activity limit its application as an effective agent for glioma treatment. In search for effective anti-glioma agents, this work designed and synthesized two series of celastrol C-3 OH and C-20 COOH derivatives 4a-4o and 6a-6o containing 1, 2, 3-triazole moiety. Their anti-glioma activities against four human glioma cell lines (A172, LN229, U87, and U251) were then evaluated using MTT assay in vitro. Results showed that compound 6i (IC50 = 0.94 µM) exhibited substantial antiproliferative activity against U251 cell line, that was 4.7-fold more potent than that of celastrol (IC50 = 4.43 µM). In addition, compound 6i remarkably inhibited the colony formation and migration of U251 cells. Further transmission electron microscopy and mitochondrial depolarization assays in U251 cells indicated that the potent anti-glioma activity of 6i was attributed to necroptosis. Mechanism investigation revealed that compound 6i induced necroptosis mainly by activating the RIP1/RIP3/MLKL pathway. Additionally, compound 6i exerted acceptable BBB permeability in mice and inhibited U251 cell proliferation in an in vivo zebrafish xenograft model, obviously. In summary, compound 6i might be a promising lead compound for potent celastrol derivatives as anti-glioma agents.

Synthesis and biological evaluation of celastrol derivatives as potent antitumor agents with STAT3 inhibition.

Using STAT3 inhibitors as a potential strategy in cancer therapy have attracted much attention. Recently, celastrol has been reported that it could directly bind to and suppress the activity of STAT3 in the cardiac dysfunction model. To explore more effective STAT3 inhibiting anti-tumour drug candidates, we synthesised a series of celastrol derivatives and biologically evaluated them with several human cancer cell lines. The western blotting analysis showed that compound 4 m, the most active derivative, could suppress the STAT3's phosphorylation as well as its downstream genes. SPR analysis, molecular docking and dynamics simulations' results indicated that the 4m could bind with STAT3 protein more tightly than celastrol. Then we found that the 4m could block cell-cycle and induce apoptosis on HCT-116 cells. Furthermore, the anti-tumour effect of 4m was verified on colorectal cancer organoid. This is the first research that discovered effective STAT3 inhibitors as potent anti-tumour agents from celastrol derivatives.

Synthesis and Biological Evaluation of C-17-Amino-Substituted Pyrazole-Fused Betulinic Acid Derivatives as Novel Agents for Osteoarthritis Treatment.

A series of pyrazole-fused betulinic acid (BA) derivatives were designed and synthesized by replacing the carboxyl group at C-17 with aliphatic amine, amide, and urea groups. The suppressive effects of the compounds on osteoclast (OC) formation and inflammatory cytokine production were evaluated on murine macrophages, RAW264.7 cells, conditioned with receptor activator for nuclear factor-κB ligand (RANKL)/macrophage colony stimulating factor (M-CSF) or lipopolysaccharide (LPS), respectively. Results showed that, compared with betulinic acid, most of these compounds exhibited significant improvements in inhibitory potency. Compound 25 exhibited distinguished activities on inhibiting OC differentiation with an IC50 value of 1.86 µM. Meanwhile, compound 25, displaying the most promising suppression on IL-1ß secretion from RAW264.7 cells, was further found to possess therapeutic effects in the sodium monoiodoacetate (MIA)-induced osteoarthritis rat model. Dose-dependent benefits were observed in MIA-elicited rats with ameliorated joint pain as well as decreased cartilage damage and bone changes after compound 25 treatment.

The Effect of the New Lupeol Derivatives on Human Skin Cells as Potential Agents in the Treatment of Wound Healing.

Skin barrier damage can be the result of various external factors including heat, radiation, chemicals and many others. Any interruption of the skin barrier integrity causes the exposure of the organism to harmful environmental factors. Therefore, there is an urgent need to develop novel therapeutics characterized by high bioavailability and effectiveness in skin damage recovery. Birch bark is known as a clinically proven, traditional medicinal remedy to accelerate wound healing. Lupeol, one of the main birch bark ingredients, shows a wide range of biological activity beneficial to the skin. The purpose of the research was to determine the influence of new lupeol derivatives on keratinocyte and fibroblast migration and proliferation, as well as to investigate various mechanisms of their antioxidant activity. The chemical modification of lupeol structure was intended to obtain more effective therapeutics characterized by higher bioavailability, permeability and safety of use. The novel triterpenes presented in this study were evaluated as the potential active ingredients preventing skin tissue degradation. Lupeol esters influence skin cells' motility and proliferation. Importantly, they are able to reduce reactive oxygen species and act indirectly by protecting the skin protein structure from being oxidized by free radicals.

Design, synthesis of novel celastrol derivatives and study on their antitumor growth through HIF-1α pathway.

Four series of hypoxia-inducible factor-1 alpha (HIF-1α) functioning derivatives stemming from modifications to the C-29 carboxyl group of celastrol were designed and synthesized, and their anticancer activities were evaluated. To address the structure and activity relationship of each derivative, extensive structural changes were made. HRE luciferase reporter assay demonstrated that 12 modified compounds showed superior HIF-1α inhibitory activity. Among them, compound C6 exhibited the best features: firstly, the strongest HIF-1α inhibitory activity (IC50 = 0.05 µM, 5-fold higher than that of celastrol); secondly, lower cytotoxicity (22-fold lower, C6-16.85 µM vs celastrol-0.76 µM). Thus, the safety factor of C6 was about 112 times higher than that of celastrol. Western blot assay indicated that C6 may inhibit the expression of HIF-1α protein in cells. Additionally, C6 hindered tumor cell cloning, migration and induced cell apoptosis. It is worth mentioning that in the mouse tumor xenograft model, C6 (10 mg/kg) displayed good antitumor activity in vivo, showing a better inhibition rate (74.03%) than the reference compound 5-fluorouracil (inhibition rate, 59.58%). However, the celastrol treatment group experienced collective death after four doses of the drug. Moreover, C6 minimally affected the mouse weight, indicating that its application in vivo has little toxic effect. H&E staining experiments show that it could also exacerbate the degree of tumor cell damage. The results of water solubility experiment show that the solubility of C6 is increased by 1.36 times than that of celastrol. In conclusion, C6 is a promising antitumor agent through HIF-1α pathway.

Natural and Semisynthetic Pentacyclic Triterpenes for Chronic Myeloid Leukemia Therapy: Reality, Challenges and Perspectives.

Chronic myeloid leukemia (CML) is a neoplasm characterized by BCR-ABL1, an oncoprotein with vital role in leukemogenesis. Its inhibition by tyrosine kinase inhibitors represents the main choice of treatment. However, therapeutic failure is worrying given the lack of pharmacological options. Pentacyclic triterpenes are phytochemicals with outstanding antitumoral properties and have also been explored as a basis for the design of potential leads. In this review, we have gathered and discuss data regarding both natural and semisynthetic pentacyclic triterpenes applied to CML cell treatment. We found consistent evidence that the class of pentacyclic triterpenes in general exerts promising pro-apoptotic and antiproliferative activities in sensitive and resistant CML cells, and thus represents a rich source for drug development. We also analyze the predicted drug-like properties of the molecules, discuss the structural changes with biological implications and show the great opportunities this class represents, as well as the perspectives they provide on drug discovery for CML treatment.

Design, Synthesis and In Vitro Investigation of Novel Basic Celastrol Carboxamides as Bio-Inspired Leishmanicidal Agents Endowed with Inhibitory Activity against Leishmania Hsp90.

The natural triterpene celastrol (CE) is here used as lead compound for the design and synthesis of a panel of eleven CE carboxamides that were tested in vitro for their growth inhibitory activity against Leishmania infantum and L.tropica parasites. Among them, in vitro screening identified four basic CE carboxamides endowed with nanomolar leishmanicidal activity, against both the promastigotes and the intramacrophage Leishmania amastigotes forms. These compounds also showed low toxicity toward two human (HMEC-1 and THP-1) and one murine (BMDM) cell lines. Interestingly, the most selective CE analogue (compound 3) was also endowed with the ability to inhibit the ATPase activity of the Leishmania protein chaperone Hsp90 as demonstrated by the in vitro assay conducted on a purified, full-length recombinant protein. Preliminary investigations by comparing it with the naturally occurring Hsp90 active site inhibitor Geldanamycin (GA) in two different in vitro experiments were performed. These promising results set the basis for a future biochemical investigation of the mode of interaction of celastrol and CE-inspired compounds with Leishmania Hsp90.

Synthesis and Biological Evaluation of Celastrol Derivatives as Potential Immunosuppressive Agents.

Celastrol, a friedelane-type triterpenoid isolated from the genus Triperygium, possesses antitumor, anti-inflammatory, and immunosuppressive activities. A total of 42 celastrol derivatives (1a-1t, 2a-2l, and 3a-3j) were synthesized and evaluated for their immunosuppressive activities. Compounds 2a-2e showed immunosuppressive effects, with IC50 values ranging from 25 to 83 nM, and weak cytotoxicity (CC50 > 1 µM). Compound 2a, with a selectivity index value 31 times higher than that of celastrol, was selected as a lead compound. Further research showed that 2a exerted its immunosuppressive effects by inducing apoptosis and inhibiting cytokine secretion via Lck- and ZAP-70-mediated signaling pathways.

New 30-norlupane derivatives through chemical-microbial semi-synthesis of betulinic acid and their neuroprotective effect.

In this study, seven 30-norlupane derivatives (2-8) wasobtained from the chemical oxidation ofbetulinic acidfollowed bybiotransformationviaBacillus megateriumCGMCC 1.1741. And metabolites 2-4 and 6-8 were newly identified products. In the first step, betulinic acid was chemically oxidizedto platanic acid (1). Following the chemical oxidation, B. megaterium catalyzed the hydroxylation at C-7, C-11, C-15 and C-23 of platanic acid (1) as well as the oxidation of C-3 hydroxyl group. Compared to the labor-intensive isolation from natural plants, this chemical-microbial semi-synthesis is more capable to provide increased structural diversity of oxygenated 30-norlupane. Finally, the potential neuroprotective effect of the derivatives was assessed on neuron-like PC12 cells induced by cobalt chloride (CoCl2). Metabolite 6 showed a potent neuroprotective activity.

Enhancing Betulinic Acid Dissolution Rate and Improving Antitumor Activity via Nanosuspension Constructed by Anti-Solvent Technique.

PURPOSE: The aim of this study was to prepare and evaluate betulinic acid nanosuspension (BA-NS) for new drug delivery to enhance its solubility and in vitro anti-tumor activity. METHODS: BA-NS was formulated by an anti-solvent precipitation method using the Box-Behnken design (BBD). Particle size (PS) and Zeta potential were measured by laser particle size analysis. The drug solid state after freeze drying was characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD) and Fourier transform infrared spectroscopy (FTIR) after freeze drying. The saturation solubility and dissolution rate were determined by solubility assay and in vitro dissolution studies, respectively. The in vitro cytotoxicity assay was performed using 3-(4,5-dimethylthiazole)-2,5-diphenltetraazolium bromide (MTT) method. RESULTS: The PS was 129.7±12.2 nm having a Zeta potential of -28.1±4.5 mV and the polydispersity index (PDI) was 0.231±0.013, which confirmed that the nanosuspension was in the stable amorphous state. A series of characterization experiments demonstrated that nanoparticles retained original effective structure and existed as spherical or near-spherical nanoparticles in the nanosuspension, but the drug transferred from the crystal state to the amorphous state. The form of lyophilized BA-NS was very successful in enhancing the dissolution rate in PH-dependent way. The cytotoxicity assay revealed that BA-NS could significantly enhance the in vitro anti-proliferation against tumor cells compared to the BA suspension (BA-S). CONCLUSION: The BA-NS can remarkably improve solubility and in vitro antitumor activity, which seems very promising for the treatment of cancers in practical application.

Betulinic Acid and Brosimine B Hybrid Derivatives as Potential Agents against Female Cancers.

BACKGROUND: Cancer is a multifactorial disease, representing one of the leading causes of death worldwide. On a global estimate, breast cancer is the most frequently occurring cancer in women and cervical cancer, the fourth most common. Both types of cancer remain the major cause of cancer-related mortality in developing countries. A strategy for rational drug design is hybridization, which aims to bring together in one molecule, two or more pharmacophores in order to reach several biological targets. OBJECTIVE: The objective of this work was to develop new hybrids based on natural pharmacophores: Betulinic acid (1) and brosimine b (2), active in female cancer cell lines. METHODS: The coupling reactions were carried out by Steglich esterification. Different compounds were designed for the complete and simplified structural hybridization of molecules. The anticancer activities of the compounds were evaluated in human cervical adenocarcinoma (HeLa), human cervical metastatic epidermoid carcinoma (ME-180), and human breast adenocarcinoma (MCF-7) cell lines. RESULTS: Hybrid 3 presented higher potency (IC50 = 9.2 ± 0.5µM) and SI (43.5) selectively in MCF-7 cells (in relation to Vero cells) with its cytotoxic effect occurring via apoptosis. In addition, compound 6 showed activity in MCF-7 and HeLa cells with intermediate potency, but with high efficacy, acting via apoptosis as well. CONCLUSION: In this context, we showed that the combination of two complex structures generated the development of hybrids with differing inhibitory profiles and apoptotic modes of action, thus representing potential alternatives in female cancer treatment.

Design, Synthesis and Biological Evaluation of Pentacyclic Triterpene Derivatives: Optimization of Anti-ABL Kinase Activity.

Imatinib, an Abelson (ABL) tyrosine kinase inhibitor, is a lead molecular-targeted drug against chronic myelogenous leukemia (CML). To overcome its resistance and adverse effects, new inhibitors of ABL kinase are needed. Our previous study showed that the benzyl ester of gypsogenin (1c), a pentacyclic triterpene, has anti-ABL kinase and a subsequent anti-CML activity. To optimize its activities, benzyl esters of carefully selected triterpenes (PT1-PT6), from different classes comprising oleanane, ursane and lupane, and new substituted benzyl esters of gypsogenin (GP1-GP5) were synthesized. All of the synthesized compounds were purified and charachterized by different spectroscopic methods. Cytotoxicity of the parent triterpenes and the synthesized compounds against CML cell line K562 was examined; revealing three promising compounds PT5, GP2 and GP5 (IC50 5.46, 4.78 and 3.19 µM, respectively). These compounds were shown to inhibit extracellular signal-regulated kinase (ERK) downstream signaling, and induce apoptosis in K562 cells. Among them, PT5 was identified to have in vitro activity (IC50 = 1.44 µM) against ABL1 kinase, about sixfold of 1c, which was justified by molecular docking. The in vitro activities of GP2 and GP5 are less than PT5, hence they were supposed to possess other more mechanisms of cytotoxicity. In general, our design and derivatizations resulted in enhancing the activity against ABL1 kinase and CML cells.

Treatment of obesity-related inflammation with a novel synthetic pentacyclic oleanane triterpenoids via modulation of macrophage polarization.

BACKGROUND: Obesity leads to the chronic inflammation in the whole body and triggers the macrophage polarization to the pro-inflammatory phenotype. Targeting macrophage polarization provides a promising therapeutic strategy for obesity-related metabolic disorders and inflammation. Here, we show that SO1989, a derivative of natural occurring compound oleanolic acid, restores the balance between M1-polarized and M2-polarized macrophages in high fat diets (HFD)-induced obese mice resulting in the improvement of adipose inflammation and the metabolic dysfunctions. METHODS: Histological analysis, magnetic cell sorting and FACS, in vitro cell model of adipose inflammation, Western blotting, HFD mice model. FINDINGS: SO1989 exhibits similar or even stronger activity in inhibiting inflammation and M1 polarization of macrophages both in vitro and in vivo compared to its analogue CDDO-Me, previously known as a powerful anti-inflammation chemical small molecule. In addition, SO1989 can significantly increase the level of fatty acid oxidation in macrophages which can efficiently facilitate M2 polarization of macrophages. Unlike CDDO-Me, SO1989 shows less adverse effects on obese mice. INTERPRETATION: Taken all together, our findings identify SO1989 as a modulator in macrophage polarization and a safer potential leading compound for pro-resolution of inflammation treatment in metabolic disorders. FUND: Supported by grants from the National Key Research and Development Plan (2017YFA0506000, 2017YFA0205400) and National Natural Science Foundation of China (81673439) and Natural Science Fund project in Jiangsu Province (BK20161408).

Synthesis, structure activity relationship and in vitro anti-influenza virus activity of novel polyphenol-pentacyclic triterpene conjugates.

It is urgently necessary to develop more effective anti-influenza agents due to the continuous emergence of drug-resistant strains of influenza virus. Our earlier studies have identified that certain pentacyclic triterpene derivatives are effective inhibitors of influenza virus infection. In the present study, a series of C-28 modified pentacyclic triterpene derivatives via conjugation with a series of polyphenols were synthesized, and their antiviral activities against influenza A/WSN/33 (H1N1) virus in MDCK (Madin-Darby canine kidney) cells were evaluated. Four compounds 23m, 23o, 23q and 23s displayed robust anti-influenza potency with averaged IC50 values at the low-micromole level, surpassing the potency of oseltamivir. In addition, the in vitro cytotoxic activity of the four conjugates against MDCK cells showed no toxicity at 100 µM. Further mechanism studies of compound 23s, one of the best representative conjugates with IC50 value of 5.80 µM and a selective index (SI) value of over 17.2, by hemagglutination inhibition (HI), surface plasmon resonance and molecular modeling indicated that this conjugate bound tightly to the viral envelope hemagglutinin (KD = 15.6 µM), thus blocking the invasion of influenza viruses into host cells.

Transformation of asiatic acid into a mitocanic, bimodal-acting rhodamine B conjugate of nanomolar cytotoxicity.

Based on their biological activity natural products continue to represent optimal lead structures for the development of novel drug candidates. We focused on the syntheses of several derivatives of the triterpene asiatic acid and on the evaluation of their cytotoxic activity in a photometric sulforhodamin B assay. Especially, benzamide 2 and rhodamine B conjugate 11 show a distinct cytotoxicity for several human tumor cell lines, e.g. EC50 (A2780) = 110 ±â€¯1 nM and EC50 (A2780) = 8 ±â€¯2 nM, respectively. Interestingly, compound 11 showed for two human tumor cell lines (HT29 and 518A2) non-linear, bimodal dose-response relationships.

Assessment of Toxicity and Absorption of the Novel AA Derivative AA-Pme in SGC7901 Cancer Cells In Vitro and in Zebrafish In Vivo.

BACKGROUND Asiatic acid (AA; 2α,3ß,23-trihydroxyurs-12-ene-28-oic acid) is an active compound derived from Centella asiatica, a traditional medicinal plant used widely in many Asian countries, particularly for the treatment of cancer. However, the modified AA derivative N-(2α,3ß,23-acetoxyurs-12-en-28-oyl)-l-proline methyl ester (AA-PMe) has shown markedly better anti-tumor activity than AA. MATERIAL AND METHODS We evaluated the toxicity of AA and AA-PMe on zebrafish morphology, mortality, and hatching rate and determined the effect on SGC7901 cancer cells by acute toxicity assay. AA-PMe absorption in vitro in SGC7901 cells and in vivo in zebrafish was determined by establishing a highly accurate and reproducible HPLC protocol. RESULTS In zebrafish, the toxicity of AA-PMe was lower than AA, with an acute toxic dose of AA-PMe above 25 µM, compared to acute toxicity at doses above 10 µM for AA. However, chronic toxicity of AA-PMe began occurring at doses below 25 µM but became apparent for AA at doses below 10 µM. Although low doses of AA-PMe were tolerated acutely, it became chronically toxic during zebrafish development, resulting in morphological abnormalities, including peripheral and abdominal edema, hemorrhage, abnormal body shape, enlarged yolk sac, and reduced motility. At low concentrations, absorption of AA-PMe by cells and zebrafish embryos occurred in a dose-dependent manner, but this stabilized as the concentration increased. CONCLUSIONS This pharmacokinetic study outlines the cellular and organismal effects of AA-PMe and suggests a theoretical basis that may underlie its mechanism of action.

Regulation of Signaling Pathways Involved in the Anti-proliferative and Apoptosis-inducing Effects of M22 against Non-small Cell Lung Adenocarcinoma A549 Cells.

The compound 29-(4-methylpiperazine)-luepol (M22), a novel derivative of lupeol has shown anti-proliferative effects against the human non-small cell lung cancer A549 cell line. M22 showed significant anti-proliferative activity at 6.80 µM and increased accumulation of G1 cells and effectively suppressed expression of the G1 arrest-related genes cyclins D1 and E1, CDK2 and CDC25A. This was further confirmed by Western blotting demonstrating decreased cyclin D1 and CDC25A protein levels. Furthermore, M22 caused induction of apoptosis that downregulated the anti-apoptotic BCL-2 gene and increased expression of BAX, CASP3 and CASP9 as well as the APAF1 gene. The effect of caspase-induced apoptosis was confirmed by an increase in reactive oxygen species (ROS), loss of mitochondrial membrane potential (MMP). Taken together, our findings indicated that M22 possessed potent anti-proliferative and apoptotic activities.

Synthesis and anticancer activity of novel fluorinated asiatic acid derivatives.

A series of novel fluorinated Asiatic Acid (AA) derivatives were successfully synthesized, tested for their antiproliferative activity against HeLa and HT-29 cell lines, and their structure activity relationships were evaluated. The great majority of fluorinated derivatives showed stronger antiproliferative activity than AA in a concentration dependent manner. The most active compounds have a pentameric A-ring containing an α,ß-unsaturated carbonyl group. The compounds with better cytotoxic activity were then evaluated against MCF-7, Jurkat, PC-3, A375, MIA PaCa-2 and BJ cell lines. Derivative 14 proved to be the most active compound among all tested derivatives and its mechanism of action was further investigated in HeLa cell line. The results showed that compound 14 induced cell cycle arrest in G0/G1 stage as a consequence of up-regulation of p21(cip1/waf1) and p27(kip1) and down-regulation of cyclin D3 and Cyclin E. Furthermore, compound 14 was found to induce caspase driven-apoptosis with activation of caspases-8 and caspase-3 and the cleavage of PARP. The cleavage of Bid into t-Bid, the up-regulation of Bax and the down-regulation of Bcl-2 were also observed after treatment of HeLa cells with compound 14. Taken together, these mechanistic studies revealed the involvement of extrinsic and intrinsic pathways in the apoptotic process induced by compound 14. Importantly, the antiproliferative activity of this compound on the non-tumor BJ human fibroblast cell line is weaker than in the tested cancer cell lines. The enhanced potency (between 45 and 90-fold more active than AA in a panel of cancer cell lines) and selectivity of this new AA derivative warrant further preclinical evaluation.