N-Hydroxy-Phe-Phe, a new dipeptide, and cytotoxic macrocyclic trichothecenes from the lethal toxic mushroom Podostroma cornu-damae.

Podostroma cornu-damae, commonly referred to as the red deer's horn mushroom due to its distinct resemblance to the antlers of a deer, is a lethal toxic mushroom that causes vomiting, dehydration, diarrhea, disturbance of consciousness, and even death. In continuation of our research aiming to investigate the novel structural and/or biological principles present in Korean wild mushrooms, a new N-hydroxyphenylalanine-phenylalanine dipeptide, N-hydroxy-Phe-Phe (1), and three known macrocyclic trichothecenes, satratoxin H (2), 12'-episatratoxin H (3), and roridin F (4), were isolated from the MeOH extract of a plate culture of the poisonous mushroom P. cornu-damae. The chemical structure of the new dipeptide (1) was determined by analyzing 1D and 2D NMR spectra and high-resolution (HR)-electrospray ionization mass spectroscopy (ESIMS), along with a computational method combined with a statistical procedure (DP4+), and its absolute configuration was unambiguously assigned by quantum chemical ECD calculations. To the best of our knowledge, compound 1 is the first dipeptide found in P. cornu-damae. Upon evaluating the cytotoxicity of compounds 1-4 against four human-derived cancer cell lines namely SK-OV-3, SK-MEL-2, A549, and HCT15, 12'-episatratoxin H (3) displayed potent cytotoxic effects toward all four cell lines tested, with IC50 values ranging from 0.7 to 2.8 nM, which was found to be stronger than that of doxorubicin. Satratoxin H (2) also demonstrated moderate cytotoxic potency against all four cell lines, with IC50 values ranging from 1.93 to 4.22 µM. Our findings provide experimental data supporting the potential of the poisonous mushroom P. cornu-damae as a source of anticancer agents.

T Cells Expressing CAR Equipped With Extracellular Domain of BTLA Are Effective Against HVEM-over-expressing Melanoma Cell Lines.

BACKGROUND/AIM: Several chimeric antigen receptor (CAR) T cells have been used to treat melanoma but have not shown favorable results. This study investigated whether Herpes virus entry mediator (HVEM), which is overexpressed in melanoma, is a potential novel antigen for CAR T cell therapy. MATERIALS AND METHODS: A CAR construct, composed of the BTLA extracellular domain for HVEM recognition (BTLA-28z), was developed and tested. RESULTS: Jurkat cells transduced with BTLA-28z exhibited enhanced IL-2 secretion when incubated with HVEM-over-expressing melanoma cells. KHYG-1 cells transduced with BTLA-28z also lysed melanoma cell lines. Using primary T cells, we generated CAR T cells targeting HVEM. BTLA-28z CAR T cells exhibited excellent lytic activities against melanoma cell lines. CONCLUSION: HVEM-targeting CAR T cells may be useful for the treatment of melanoma.

Anti-Inflammatory, Neurotrophic, and Cytotoxic Oxylipins Isolated from Chaenomeles sinensis Twigs.

Oxylipins are important biological molecules with diverse roles in human and plants such as pro-/anti-inflammatory, antimicrobial, and regulatory activity. Although there is an increasing number of plant-derived oxylipins, most of their physiological roles in humans remain unclear. Here, we describe the isolation, identification, and biological activities of four new oxylipins, chaenomesters A-D (1-4), along with a known compound (5), obtained from Chaenomeles sinensis twigs. Their chemical structures were determined by spectroscopic (i.e., NMR) and spectrometric (i.e., HRMS) data analysis including 1H NMR-based empirical rules and homonuclear-decoupled 1H NMR experiments. Chaenomester D (4), an omega-3 oxylipin, showed a potent inhibitory effect on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated BV-2 cells (NO production, 8.46 ± 0.68 µM), neurotrophic activity in C6 cells through the induction of the secretion of nerve growth factor (NGF, 157.7 ± 2.4%), and cytotoxicity in A549 human cancer cell lines (IC50 = 27.4 µM).

Triterpenoids from the leaves of Abies koreana and their biological activities.

Seven undescribed triterpenoids, abikoranes A-G, along with three known triterpenoids were isolated from the leaves of Abies koreana E. H. Wilson. The structures of compounds were elucidated by 1D and 2D NMR, HRMS, ECD, specific rotation, and DP4+ analysis. Abikorane A represents the second example of nor-3,4-seco-17,14-friedo-lanostane triterpenoid. Among the isolates, some compounds showed strong cytotoxic activities against some of four tested cancer cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-116) with the values of IC50 0.89-9.62 µM, inhibited lipopolysaccharide-stimulated nitric oxide production with IC50 values of 11.57-15.16 µM, and exhibited significant nerve growth factor release effect (192.54 ± 12.33%) from C6 glioma cells.

Meyeroguilline E, a New Isoindolinone Alkaloid from the Poisonous Mushroom Chlorophyllum molybdites, and Identification of Compounds with Multidrug Resistance (MDR) Reversal Activities.

Three isoindolinone alkaloids (1-3), including one new isoindolinone-type alkaloid, meyeroguilline E (1), and six other known compounds (4-9) were isolated from the poisonous mushroom Chlorophyllum molybdites (Agaricaceae). The structure of the new compound was determined using extensive spectroscopic analyses via one-dimensional (1D) and two-dimensional (2D) NMR data interpretation and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). To the best of our knowledge, compound 1 is the first example of a natural isoindolinone with a butanoic acid moiety, and this study is the first to detect the other known compounds (2-9) in C. molybdites. The isolated compounds (1-9) were examined for their multidrug resistance (MDR) reversal activity against MES-SA, MES-SA/DX5, HCT15, and HCT15/CL02 human cancer cells. Based on the results, 20 µM of compounds 3 and 6 slightly potentiated paclitaxel (TAX)-induced cytotoxicity in MES-SA/DX5, HCT15, and HCT15/CL02 cells; however, the compounds had no effect on the cytotoxicity against MES-SA and nonMDR cells.

Terpenoids from Glechoma hederacea var. longituba and their biological activities.

Glechoma hederacea var. longituba (common name: ground ivy) has been used for the treatment of asthma, bronchitis, cholelithiasis, colds, and inflammation. In the present study, three new sesquiterpene glycosides (1-3), two new diterpene glycosides (4 and 5), and four known compounds (6-9) were isolated from its MeOH extract. A structure elucidation was performed for the five new compounds (1-5) using 1D and 2D NMR, HRESIMS, DP4+ and ECD calculations, and chemical methods. All the isolates (1-9) were assessed for their antineuroinflammatory activities on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated BV-2 cells, nerve growth factor (NGF) secretion stimulation activities in C6 glioma cells, and cytotoxic activities against four human cancer cell lines (A549, SK-OV-3, SK-MEL-2, and HCT15). Compounds 2 and 5-7 exhibited inhibitory effects on the NO production with IC50 values of 52.21, 47.90, 61.61, and 25.35 µM, respectively. Compound 5 also exhibited a significant stimulating effect on NGF secretion (122.77 ± 8.10%). Compound 9 showed potent cytotoxic activity against SK-OV-3 (IC50 = 3.76 µM) and SK-MEL-2 (IC50 = 1.48 µM) cell lines, while 7 displayed a strong cytotoxic activity against the SK-MEL-2 (IC50 = 9.81 µM) cell line.

Anticancer and Anti-Neuroinflammatory Constituents Isolated from the Roots of Wasabia japonica.

Wasabi (Wasabia japonica (Miq.) Matsum.) is a pungent spice commonly consumed with sushi and sashimi. From the roots of this plant, a new 2-butenolide derivative (1) and 17 previously reported compounds (2-18) were isolated and structurally characterized. Their chemical structures were characterized based on the conventional NMR (1H and 13C, COSY, HSQC, and HMBC) and HRESIMS data analysis. All of these phytochemicals (1-18) were evaluated for their antiproliferative effects on the four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and MKN-1), for their inhibitory activity on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated BV-2 microglia cells, and for their nerve growth factor (NGF)-releasing effect from C6 glioma cells. Among the isolated compounds, compound 15 showed powerful antiproliferative activities against A549 and SK-MEL-2 cell lines with IC50 values of 2.10 and 9.08 µM, respectively. Moreover, the new compound 1 exhibited moderate NO inhibition activity with IC50 value of 45.3 µM.

c-Met-Specific Chimeric Antigen Receptor T Cells Demonstrate Anti-Tumor Effect in c-Met Positive Gastric Cancer.

Chimeric antigen receptor (CAR) technology has been highlighted in recent years as a new therapeutic approach for cancer treatment. Although the impressive efficacy of CAR-based T cell adoptive immunotherapy has been observed in hematologic cancers, limited effect has been reported on solid tumors. Approximately 20% of gastric cancer (GC) patients exhibit a high expression of c-Met. We have generated an anti c-Met CAR construct that is composed of a single-chain variable fragment (scFv) of c-Met antibody and signaling domains consisting of CD28 and CD3ζ. To test the CAR construct, we used two cell lines: the Jurkat and KHYG-1 cell lines. These are convenient cell lines, compared to primary T cells, to culture and to test CAR constructs. We transduced CAR constructs into Jurkat cells by electroporation. c-Met CAR Jurkat cells secreted interleukin-2 (IL-2) only when incubated with c-Met positive GC cells. To confirm the lytic function of CAR, the CAR construct was transduced into KHYG-1, a NK/T cell line, using lentiviral particles. c-Met CAR KHYG-1 showed cytotoxic effect on c-Met positive GC cells, while c-Met negative GC cell lines were not eradicated by c-Met CAR KHYG-1. Based on these data, we created c-Met CAR T cells from primary T cells, which showed high IL-2 and IFN-γ secretion when incubated with the c-Met positive cancer cell line. In an in vivo xenograft assay with NSG bearing MKN-45, a c-Met positive GC cell line, c-Met CAR T cells effectively inhibited the tumor growth of MKN-45. Our results show that the c-Met CAR T cell therapy can be effective on GC.

Phenolic Constituents of Chinese Quince Twigs (Chaenomeles sinensis Koehne) and Their Anti-Neuroinflammatory, Neurotrophic, and Cytotoxic Activities.

Chaenomeles sinensis has been used as a food and traditional medicines. However, most of research on discovering bioactive constituents from this plant have been focused on its yellow fruit, Chinese quince, due to its wide usage. Here, we isolated and characterized three new phenolic compounds (1, 9, and 11) and 21 known compounds (2-8, 10, and 12-24) from the twigs of C. sinensis. Their chemical structures were established by spectroscopic and spectrometric data analysis including 1D and 2D NMR, high-resolution mass spectrometry (HRMS), electronic circular dichroism (ECD), and LC-MS analysis. Some of the isolated compounds (1-24) showed anti-neuroinflammatory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated BV-2 cells, neurotrophic activity in C6 cells through the secretion of nerve growth factor (NGF) and/or cytotoxicity against four human cancer cell lines (A549, SK-OV-3, SK-MEL-2, MKN-1).

Novel brd4 inhibitors with a unique scaffold exhibit antitumor effects.

Since bromodomain containing 4 (brd4) has been considered as a prominent cancer target, numerous attempts have been made to develop potent brd4 bromodomain inhibitors. The present study provided a novel chemical scaffold which inhibited brd4 activity. Mid-throughput screening against brd4 bromodomain was performed using alpha-screen and homogeneous time-resolved fluorescence assays. Furthermore, cell cytotoxicity and xenograft assays were performed to examine if the compound was effective both in vitro and in vivo. As a result, it was revealed that compounds having naphthalene-1,4-dione scaffold inhibited the binding of bromodomain to acetylated histone. The compounds with naphthalene-1,4-dione had cytotoxic effects against the Ty82 cell line, a NUT midline carcinoma cell line, whose proliferation is dependent on brd4 activity. A10, one of the compounds with naphthalene-1,4-dione scaffold, also exhibited tumor growth inhibition effects in the xenograft assay. In addition, the compounds exhibited cytotoxic effects against gastric cancer cell lines which were resistant to I-BET-762, a BET bromodomain inhibitor. In conclusion, the novel scaffold to suppress brd4 activity was effective against cancer cells both in vitro and in vivo.

Development of Antigen-specific Chimeric Antigen Receptor KHYG-1 Cells for Glioblastoma.

BACKGROUND/AIM: Glioblastoma is the most common cancer among primary brain tumors, however, its prognosis and treatment advances are very poor. Here, we investigated whether c-Met, FOLR1, and AXL proteins are promising targets for chimeric antigen receptor (CAR) T-cell therapy, for they are known to be over-expressed in a variety of solid tumors. MATERIALS AND METHODS: CAR constructs were prepared and CAR KHYG-1 cells targeting c-Met, FOLR1, or AXL were made by lentiviral transduction. The activity of CAR KHYG-1 cells against cancer cells was measured by cytokine secretion and cell lysis assays. RESULTS: c-Met and AXL were over-expressed in most glioblastoma cell lines (11/13), but not in neuroblastoma cell lines (0/8). FOLR1 was over-expressed only in one among 16 glioblastoma cell lines. Our antigen-specific CAR KHYG-1 cells eradicated target positive glioblastoma cells selectively. CONCLUSION: Anti-c-Met and anti-AXL CAR NK or T cells could be effective in glioblastoma cells.

Identification of Potent CD19 scFv for CAR T Cells through scFv Screening with NK/T-Cell Line.

CD19 is the most promising target for developing chimeric-antigen receptor (CAR) T cells against B-cell leukemic cancer. Currently, two CAR-T-cell products, Kymriah and Yescarta, are approved for leukemia patients, and various anti-CD19 CAR T cells are undergoing clinical trial. Most of these anti-CD19 CAR T cells use FMC63 single-chain variable fragments (scFvs) for binding CD19 expressed on the cancer cell surface. In this study, we screened several known CD19 scFvs for developing anti-CD19 CAR T cells. We used the KHYG-1 NK/T-cell line for screening of CD19 scFvs because it has advantages in terms of cell culture and gene transduction compared to primary T cells. Using our CAR construct backbone, we made anti-CD19 CAR constructs which each had CD19 scFvs including FMC63, B43, 25C1, BLY3, 4G7, HD37, HB12a, and HB12b, then made each anti-CD19 CAR KHYG-1 cells. Interestingly, only FMC63 CAR KHYG-1 and 4G7 CAR KHYG-1 efficiently lysed CD19-positive cell lines. In addition, in Jurkat cell line, only these two CAR Jurkat cell lines secreted IL-2 when co-cultured with CD19-positive cell line, NALM-6. Based on these results, we made FMC63 CAR T cells and 4G7 CAR T cells from PBMC. In in vitro lysis assay, 4G7 CAR T cells lysed CD19-positive cell line as well as FMC63 CAR T cells. In in vivo assay with NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, 4G7 CAR T cells eradicated NALM-6 as potently as FMC63 CAR T cells. Therefore, we anticipate that 4G7 CAR T cells will show as good a result as FMC63 CAR T cells for B-cell leukemia patients.

Discovery of a Novel Chemical Scaffold Against Mutant Isocitrate Dehydrogenase 1 (IDH1).

BACKGROUND: Mutations in the isocitrate dehydrogenase 1 (IDH1) gene are frequently found in various cancer types. IDH1 mutants produce 2-hydroxyglutarate (2-HG), an oncometabolite, from alpha-ketoglutarate (α-KG). This 2-HG plays a key role in tumorigenesis via inhibition of α-KG dependent enzymes. For this reason, IDH1 mutant could be an ideal target for the treatment of cancer. MATERIALS AND METHODS: To find a new IDH1 inhibitor, 8,364 compounds were obtained from Korea Chemical Bank. Using high-throughput screening (HTS) of a chemical library, we unveiled a compound that could inhibit the IDH1 mutant. RESULTS: According to the enzyme assay, our compound (KRC-09) effectively inhibited the activity of IDH1 R132H mutant. In addition, KRC-09 decreased the concentration of intracellular 2-HG in the U-87 MG cell line harboring IDH1 R132H. CONCLUSION: In this article, we present a novel chemical scaffold that suppresses the activity of an IDH1 mutant.

Three New Oleanane-Type Triterpenoidal Glycosides from Impatiens balsamina and Their Biological Activity.

Three new oleanane-type triterpenoidal glycosides, imbalosides A-C (1-3), were isolated from the white flowers of Impatiens balsamina. The structures of these phytochemical constituents (1-3) were elucidated through 1D and 2D Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS) data analyses followed by chemical methods. All the characterized compounds (1-3) were evaluated for their antiproliferative activity against four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and BT549) and their anti-neuroinflammatory activity on the basis of inhibition levels of nitric oxide (NO) in the lipopolysaccharide (LPS)-stimulated murine microglia BV-2 cell lines.

A Chemical Switch System to Modulate Chimeric Antigen Receptor T Cell Activity through Proteolysis-Targeting Chimaera Technology.

Despite the excellent efficacy of chimeric antigen receptor (CAR T) cell therapy, concerns about its safety have been constantly raised. The side effects of CAR T cells result from an aberrantly upregulation of CAR T cell activity. Therefore, it is crucial to control the CAR T cell activity whenever the patient is at risk. For this purpose, the iCas9 system, which induces apoptosis in CAR T cell through caspase-9 dimerization by compound, has been invented and is currently going under clinical trial. However, the iCas9 system is irreversible, as the entire CAR T cell population is removed from the patient. Thus, CAR T cells, which are very expensive, should be reinfused to the patients after they recovered from the side-effect. Here, we propose a new CAR T cell safety strategy, which targets CAR "protein", not CAR "T cell". In this system, the CAR construct is modified to bear a bromodomain (BD). The addition of a BD in the CAR protein did not interfere with the original CAR functions, such as cytokine secretion and target cell lysis. Our data showed that the use of a proteolysis-targeting chimaera (PROTAC) compound against BD successfully degraded the BD-containing CAR protein. Moreover, the CAR expression is recovered when the PROTAC compound is removed from the cell, demonstrating that our system is reversible. In a target cell lysis assay, the PROTAC compound successfully suppressed the lytic activity of CAR T cells by degrading the CAR protein. In conclusion, we developed a new safety system in which CAR T cells can be "reversibly" controlled by a compound.

Novel nickel(II), palladium(II), and platinum(II) complexes having a pyrrolyl-iminophosphine (PNN) pincer: Synthesis, crystal structures, and cytotoxic activity.

A pyrrolyl-iminophosphine (PNNH) which would act as a potential terdentate ligand has been prepared by Schiff base reaction. Complexes [M(PNN)X] (M = Ni; X = Cl (1), Pd; X = Cl (2), Br (3), I (4), M = Pt; X = Cl (5)) were prepared. The title complexes were characterized by various spectroscopic (IR, 1H, 13C, and 31P NMR) and elemental analyses. The molecular structures of 1, 2, and 5 have been established by single-crystal X-ray crystallography, demonstrating a distorted square planar geometry comprising two 5-membered metallacyclic rings. Complexes 1 and 2 were found to crystallize in the orthorhombic while complex 5 crystallizes in the monoclinic. Cytotoxicities of the complexes along with PNNH were evaluated against A549 (lung), SK-OV-3 (ovarian), SM-MEL-2 (skin), and HCT15 (colon) human cancer cell lines by sulforhodamine B assay. Notably, the palladium(II) complex (2) shows the highest activity. Apoptosis activity along with the caspase inhibitor Z-VAD (Z-Val-Ala-Asp-fluoromethyl ketone) assay of 2 and 5 against A549 and HCT15 cancer cell lines were investigated to learn a mechanistic pathway for the observed cytotoxicity, practically eliminating an apoptotic cell-death route. Complexes 2 and 5 were studied to DNA cleavage assay and molecular docking simulation. The DNA (pcDNA3.0) cleavage experiment evaluates complex 5 interacting with DNA, more effectively, in comparison to complex 2. Molecular docking simulation of 2 and 5 toward DNA and GRP78 (glucose-regulated protein 78) was performed to predict binding sites of ligand-receptors and a plausible mechanistic aspect of metallodrug-action.

Chemical constituents of Chaenomeles sinensis twigs and their biological activity.

A new megastigmane-type norsesquiterpenoid glycoside, chaemeloside (1), was isolated from the twigs of Chaenomeles sinensis together with 11 known phytochemicals through chromatographic methods. The chemical structure of the new isolate 1 was determined by conventional 1D and 2D NMR data analysis, ECD experiment, hydrolysis followed by a modified Mosher's method, and LC-MS analysis. The characterized compounds' biological effects including cytotoxicity against cancer cell lines, antineuroinflammatory activity, and potential neurotrophic effect were evaluated.

Radiosensitizing Effect of Novel Phenylpyrimidine Derivatives on Human Lung Cancer Cells via Cell Cycle Perturbation.

Radiotherapy is one of the most common treatments for cancer, but radioresistance and injury to normal tissue are considered major obstacles to successful radiotherapy. Thus, there is an urgent need to develop radiosensitizers to improve the therapeutic outcomes of radiotherapy in cancer patients. Our previous efforts to identify novel radiosensitizers, using high-throughput screening targeting p53 and Nrf2 revealed a promising N-phenylpyrimidin-2-amine (PPA) lead compound. In the present study, 17 derivatives of this lead compound were examined, and it was found that 4-(4-fluorophenyl)-N-(4-nitrophenyl)-6-phenylpyrimidin-2-amine (PPA5), 4-((4-(4-fluorophenyl)pyrimidin-2-yl)amino)-3-methoxy-N-methyl -benzamide (PPA13), 4-((4-(4-fluorophenyl)pyrimidin-2-yl)amino)benzenesulfonamide (PPA14), 4-((4-(2-chlorophenyl)pyrimidin-2-yl)amino)benzenesulfonamide (PPA15), and 4-((4-(2-chlorophenyl)pyrimidin-2-yl)amino)-N-methylbenzamide (PPA17) inhibited cell viability by more than 50%, with a marked increase in the proportion of cells arrested at the G2/M phase of cell cycle. Among these compounds, PPA15 markedly increased the sub-G1 cell population and increased the levels of cyclin B1 and the phosphorylation levels of cyclin-dependent kinase (CDK) 1. Combined treatment with radiation and PPA14 or PPA15 significantly decreased clonogenic survival. An in vitro kinase assay revealed that PPA15 inhibited multiple CDKs involved in cell cycle regulation. Compared with drug or radiation treatment alone, combined treatment with PPA15 and radiation resulted in the suppression of A549 tumor growth in mice by 59.5% and 52.7%, respectively. Treatment with PPA15 alone directly inhibited tumor growth by 25.7%. These findings suggest that the novel pan CDK inhibitor, PPA15, may be a promising treatment to improve the effectiveness of radiotherapy for the treatment of cancer. SIGNIFICANCE STATEMENT: Several inhibitors of CDK have been successfully evaluated in combination with other chemotherapeutics in clinical trials, but negative side effects have partially restricted their clinical use. In this study, we identified a novel pan-CDK inhibitor to increase radiosensitivity, and we hope this work will encourage the development of promising small-molecule radiosensitizers.

Securinega Alkaloids from the Twigs of Securinega suffruticosa and Their Biological Activities.

Seven new Securinega alkaloids, securingines A-G (1-7), together with seven known analogues (8-14), were isolated from the twigs of Securinega suffruticosa. Their chemical structures were elucidated by a combined approach of spectroscopic analysis, chemical methods, ECD calculations, and DP4+ probability analysis. The full NMR assignments and the absolute configuration of compound 8 are also reported. In addition, all the isolated phytochemicals (1-14) were assessed for their cytotoxic, anti-inflammatory, and potential neuroprotective activities. Compound 4 showed cytotoxic activity (IC50 values of 1.5-6.8 µM) against four human cell lines (A549, SK-OV-3, SK-MEL-2, and HCT15). Compounds 3, 10, 12, and 13 showed potent inhibitory effects on nitric oxide production (IC50 values of 12.6, 12.1, 1.1, and 7.7 µM, respectively) in lipopolysaccharide-stimulated murine microglia BV-2 cells. Compound 5 exhibited a nerve growth factor production effect (172.6 ± 1.2%) in C6 glioma cells at 20 µg/mL.

Cytotoxic Withanolides from the Roots of Indian Ginseng ( Withania somnifera).

Withania somnifera, commonly known as "Indian ginseng" or "ashwagandha", is popular as a functional food because of its diverse purported therapeutic efficacies including invigorating, improvement of cognitive ability, and stress release activities. Chemical investigation of the MeOH extract of W. somnifera roots combined with LC/MS-based analysis resulted in the identification of six new withanolides, withasilolides A-F (1-6), as well as seven known compounds (7-13). The structures of the new compounds were established by application of spectroscopic methods, including 1D and 2D NMR, HRMS, and ECD measurements. The cytotoxicity of the isolated compounds was evaluated against four human cancer cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15). Compounds 1, 2, 4, 6, and withanone (11) each showed cytotoxicity for one or more of the four cancer cell lines used.