Crystal Structures and Human Leukemia Cell Apoptosis Inducible

Jan 19, 2018 - At a concentration lower than 2.0 μM, both 1 and 2 induced approximately 50% of the cells to become apoptotic at a late stage of the c...
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Crystal Structures and Human Leukemia Cell Apoptosis Inducible Activities of Parthenolide Analogues Isolated from Piptocoma rufescens Yulin Ren,†,# Judith C. Gallucci,‡,# Xinxin Li,§,# Lichao Chen,§ Jianhua Yu,§,⊥ and A. Douglas Kinghorn*,†,⊥ †

Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, ‡Department of Chemistry and Biochemistry, §Division of Hematology, Department of Internal Medicine, College of Medicine, and ⊥Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, United States S Supporting Information *

ABSTRACT: The molecular structures of three parthenolide analogues, (−)-goyazensolide (1), (−)-15-deoxygoyazensolide (2), and (−)-ereglomerulide (3), isolated from the leaves of Piptocoma rufescens in a previous study were determined by X-ray analysis, and the absolute configuration of (−)-goyazensolide (1) was confirmed crystallographically using Cu Kα radiation at low temperature. Compounds 1−3, (+)-rufesolide A (4), and commercial parthenolide were found to be growth inhibitory toward MOLM-13 and EOL-1 human acute myeloid leukemia cells using PKC412 (midostaurin) as the positive control, with 1−3 being more active than parthenolide. Also, compounds 1−4 exhibited synergistic effects when tested with PKC412, but parthenolide did not show this type of activity. At a concentration lower than 2.0 μM, both 1 and 2 induced approximately 50% of the cells to become apoptotic at a late stage of the cell cycle, but no similar apoptotic effects were observed for 3, 4, or parthenolide. Leukemia cell apoptosis was induced by these compounds through the activation of caspase-3 and the inhibition of NF-κB, as indicated by immunoblotting analysis, and compounds 1 and 2 seem to be promising leads for development as potential antileukemic agents.

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cytotoxicity toward HT-29 cells by down-regulating IKKα and IKKβ and up-regulating ROS levels and caspase-3 expression.8 As a continuation of these previous studies, the single-crystal X-ray structures of compounds 1−3 are reported herein. Also, the leukemia cell growth inhibition of compounds 1−4 and parthenolide and their synergistic effects with PKC412 (midostaurin) toward MOLM-13 and EOL-1 cells, as well as their leukemia cell apoptosis inducible activities, caspase 3 activation, and NF-κB inhibition, have been investigated in the present study.

esquiterpenoid lactones occur widely in the plant family Asteraceae and exhibit multiple potent bioactivities.1 For example, artemisinin derivatives have been used for the treatment of malaria and found more recently to show anticancer efficacy.1 Parthenolide, an epoxylated germacranolide constituent of Tanacetum parthenium Sch. Bip. (synonym, Chrysanthemum parthenium Bernh.),2 has been shown to exhibit promising antitumor efficacy. One of its derivatives, dimethylaminoparthenolide, with improved activity and enhanced water solubility, has been evaluated in a phase I clinical trial for the treatment of human leukemia.1,3 In our continuing search for anticancer agents from higher plants,4 four sesquiterpenoid lactones, (−)-goyazensolide (1), (−)-15-deoxygoyazensolide (2), (−)-ereglomerulide (3), and (+)-rufesolide A (4), and several analogues were isolated as the cytotoxic principles toward the HT-29 human colon cancer cell line from the leaves of Piptocoma rufescens Cass. (Asteraceae), collected in the Dominican Republic.5,6 Among these, (−)-goyazensolide (1) was found to inhibit human NF2deficient schwannoma and meningioma cell growth by reducing the levels of cyclins A and E, phospho-AKT, and NF-κB.7 It also showed antitumor efficacy, when tested in an in vivo hollow fiber assay in an immunodeficient NCr nu/nu mouse model implanted with HT-29 cells, at a dose of 12.5 mg/kg (i.p.). Mechanistically, (−)-goyazensolide (1) mediated its © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Crystal Structures of 1−3. The isolation and identification of (−)-goyazensolide (1), (−)-15-deoxygoyazensolide (2), (−)-ereglomerulide (3), and (+)-rufesolide A (4) have been reported previously,5,9−11 with the 1H and 13C NMR data of 1− 4 assigned completely by analysis of their 1H, 13C, and 2D NMR spectra (Tables S1 and S2 and Figures S1−S3, Supporting Information).5,12,13 The molecular structure of 1 was established by X-ray crystallographic analysis in 1982, but H-8 was omitted from this previous report.14 The crystal Special Issue: Special Issue in Honor of Susan Horwitz Received: December 28, 2017

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lactone ring at C-6 and C-7, and an ester group located at C-8, of which the C-1′ carbonyl group is oriented toward the lactone ring, with the C-1′−C-2′ bond having an s-cis (single-bond) configuration. Also, an intermolecular hydrogen bond exists between the C-15 hydroxy and C-1 carbonyl groups. The planar furan and lactone rings of 1 have been proposed previously by analysis of the proton spin−spin coupling constants, NOE correlations, and molecular mechanics calculations using the MM3* force field, with the chair−boat central macro-ether ring adopted as the most stable conformation.15 As a confirmation of this previous work, the crystal structure of 1 shows that this molecule is composed of three ring systems, a planar five-membered furanone ring formed by C-1, C-2, C-3, C-10, and O-2, a planar trans-fused αmethylene-γ-lactone ring at C-6 and C-7, and a nine-membered ether ring in a boat−chair conformation, with C-3, C-4, C-5, C6, C-7, and O-2 constituting a boat conformation and with atoms C-6, C-7, C-8, C-9, C-10, and O-2 forming a chair conformation (Figure 2). Following a previous assignment, a (7R) absolute configuration of 1 could be defined by its negative electronic circular dichroism (ECD) Cotton effects around 230 and 270 nm, with the (6R,7R,8S,10R) absolute configuration assigned from its βoriented H-2, H-6, H-8, H-9a, and H-15 and the α-oriented H7, H-9b, and H-14, as shown by the NOESY 2D NMR spectrum.5 Such a determination has also been verified by the configuration of (−)-centratherin determined recently by analysis of its experimental ECD, electronic dissymmetry factor, optical rotatory dispersion, vibrational CD, and vibrational dissymmetry factor spectra.16 To confirm these

structure, conformation of the central ring system, and absolute configuration of (+)-rufesolide A (4) have been reported in our previous study,5 but the crystal structures and crystallographic data of 2 and 3 have not been published. In the present study, the molecular structure of 1 was determined by analysis of its single-crystal X-ray diffraction data collected at 180 K, using Mo Kα radiation, and this was confirmed by a different measurement using Cu Kα radiation at 90 K, with the absolute configuration established by analysis of the Flack and Hooft y parameters. As shown in Figure 1 and Tables S3−S8 and Figure S4 (Supporting Information), the crystal structure of 1 contains a C-1 carbonyl group, an oxygen atom between C-3 and C-10, a trans-fused α-methylene-γ-

Figure 1. ORTEP plots of 1−3 showing the atom-numbering schemes (oxygen atoms are red and carbon atoms are blue) and displacement ellipsoids at the 50% probability level. The two independent molecules, labeled as 2a and 2b, in the asymmetric unit of 2 are shown. The structures of 2 and 3 were determined using Mo Kα radiation, with that of 1 from Cu Kα radiation. B

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Δ2′(3′) bond, of which two methyl groups (Me-4′ and Me-5′) are trans with respect to this double bond. Comparison of the ECD spectrum of 3 with those of 1 and 2 shows that both of the positive Cotton effects around 210 and 320 nm disappeared in the spectrum of 3, due to loss of the α,β-unsaturated carbonyl chromophore of the cyclopentenone moiety, as indicated by the absence of an absorption maximum around 260 nm in its UV spectrum. A negative Cotton effect around 220 nm implied its (7S) configuration, as supported by the absolute configuration determinations of its close analogues.5,19 Thus, a (4R,6R,7S,8S,10R) absolute configuration could be assigned for 3, from its β-oriented H-2, H-3, H-6, H-8, H-14, and H-15 and α-oriented H-4 and H-7 evident in its NOESY 2D NMR spectrum and crystal structure (Figure 1). As discussed above, the absolute configuration of 1 established by analysis of its X-ray data is the same as that proposed previously from its ECD and NOESY 2D NMR spectra. This confirmed that the negative Cotton effects around 220 and 260 nm resulting from the trans-fused α-methylene-γlactone ring could indicate the absolute configuration of C-7. The positive Cotton effects at 210 and 310 nm, which were absent in the ECD spectra of 3 and 4, occurring from the exciton coupling between the α,β-unsaturated carbonyl chromophore of the cyclopentenone moiety conjugated with the Δ4(5) bond and the γ-lactone, could be useful in defining the C-10 absolute configuration of goyazensolide-like germacranolides. Apoptosis-Mediated Cytotoxicity toward Human Leukemia Cells of 1−4. Compounds 1−4, along with a commercially available parthenolide, were evaluated for their cell growth inhibitory activity against MOLM-13 and EOL-1 acute myeloid leukemia cells with a 72 h treatment,20 using PKC412 (midostaurin, Rydapt) as the positive control. PKC412 is a semisynthetic derivative of the microbial indolocarbazole alkaloid staurosporine, used as a multitargeted protein kinase inhibitor and approved by the U.S. FDA in 2017 for the treatment of acute myeloid leukemia.21 All these compounds showed activity toward these human cancer cell lines, with compounds 1−4 all showing micromolar IC50 values, in contrast to the nanomolar IC50 values of PKC412 and paclitaxel (Table 1). In addition, both 1 and 2 were more active

Figure 2. View of the central portion of compounds 1−3 showing the conformations of the nine- or 10-membered rings and the atomnumbering schemes.

previous assignments, the data set for (−)-goyazensolide (1) was collected with Cu Kα radiation at 90 K, and both the Flack parameter, which refined to a value of −0.04(13),17 and the Hooft y parameter determined as −0.03(4) after refinement by analysis of the Bijvoet pairs18 indicated that this model contains the correct (6R,7R,8S,10R) absolute configuration. The crystal structure of (−)-15-deoxygoyazensolide (2) contains two independent molecules (2a and 2b) in the asymmetric unit (Figure 1), of which the conformations are very similar, as indicated by an overlay of all non-hydrogen atoms that resulted in a root-mean-square derivation of 0.097 Å. This structure is essentially the same as that of 1, except for the absence of the C-15 hydroxy group, indicating that the conformations of 2 are not changed with the substitution of a hydroxy group at C-15. The crystal structure of ereglomerulide (3) shows that this molecule contains a C-1 carbonyl group, a cis double bond between C-2 and C-3, and a trans-fused α-methylene-γ-lactone ring at C-6 and C-7 (Figure 1). It comprises two rings, a 10membered ring adopting a boat−boat conformation, with C-1, C-2, C-3, C-4, C-5, and C-10 forming a boat conformation and with C-5, C-6, C-7, C-8, C-9, and C-10 comprising another boat conformation, and a trans-fused α-methylene-γ-lactone ring in a twist conformation. This twist occurs along a line from C-12 to the midpoint of the C-6−C-7 bond, which is an approximate local 2-fold rotation axis (Figure 2). This conformation has been indicated by its torsion angles for C7−C-11−C-12−O-3 (11.7°), C-7−C-6−O-3−C-12 (−25.2°), and O-3−C-6−C-7−C-11 (30.1°), which are larger than those of 1 and 2 (Table S6, Supporting Information). The carbonyl group in the angelate moiety is oriented toward the lactone ring and in an s-cis configurational relationship with the conjugated

Table 1. Cell Growth Inhibition of 1−4 and Parthenolide against the Human Leukemia and Colon Cancer Cell Linesa compound

MOLM-13b

EOL-1c

HT-29d

1 2 3 4 parthenolidee PKC412g paclitaxelg

0.86 1.1 4.3 10.0 10.0 0.0047

1.1 1.0 4.3 8.0 7.5 0.0038

0.56 0.26 1.2 3.0 NTf 0.0008

a

IC50 values are the concentration (μM) required for 50% inhibition of cell viability for a given test compound and were calculated using nonlinear regression analysis with measurements performed in triplicate and representative of three independent experiments, wherein the values generally agreed within 10%. bIC50 value toward the human MOLM-13 leukemia cell line with a 72 h treatment. cIC50 value toward the human EOL-1 leukemia cell line with a 72 h treatment. dIC50 value reported previously toward the HT-29 human colon cancer cell line with a 72 h treatment.5 eCommercially available. f Not tested. gPositive control. C

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Figure 3. Synergistic effects with the control compound PKC412 observed for 1−4 and parthenolide toward MOLM-13 and EOL-1 cells. Cells were treated with either a test sample, PKC412, or the test sample plus PKC412 at a concentration that resulted in around 75% cell survival. The viability of cells was tested by a commercial absorbance assay, CellTiter 96 AQueous One solution cell proliferation assay (Promega Corp.), with the IC50 values calculated from the vehicle control. The data shown are representative of three independent experiments. **p ≤ 0.05 (for significant differences).

of MOLM-13 cells with 1 (1.1 μM) or 2 (1.8 μM) for 24 h resulted in 94.6% or 87.1% cells undergoing late-stage apoptosis, while the analogous values for 3 (2.5 μM), 4 (6.0 μM), or parthenolide (4.0 μM) were 2.77%, 1.19%, and 1.02%, respectively (Figure 4). Closely comparable to these results, treatment of EOL-1 cells with 1 (1.1 μM), 2 (1.8 μM), 3 (2.5 μM), 4 (6.0 μM), or parthenolide (4.0 μM) resulted in 93.3%, 69.1%, 32.1%, 0.545%, or 0.697% cell apoptosis at the late stage (Figure 5). These results showed that compounds 1 and 2 exhibited a higher potency than 3, 4, or parthenolide in inducing MOLM-13 and EOL-1 cell apoptosis, which is consistent with their cell growth inhibitory activity, indicating that 1−4 mediated their activity toward these human leukemia cells probably through cell apoptosis induction. To test this hypothesis, immunoblotting was obtained for 1− 4, parthenolide, and midostaurin toward MOLM-13 and EOL-1 cells. Consistent with the previous observation for midostaurin,23 caspase-3 activity was found to be activated by all these compounds (Figure 6). Activation of nuclear factor kappa B (NF-κB) is associated with induction of antiapoptotic proteins required by cancer cells to maintain viability; thus inhibition of this protein has been regarded as a promising target for anticancer drug discovery.24 Also, it has been reported that inhibition of NF-κB can prohibit tumor cell resistance to the chemotherapeutic agents to sensitize cancer treatment.25 Compounds 1−4, parthenolide, and PKC412 were further tested for their NFκB inhibitory activity toward MOLM-13 and EOL-1 cells, using an immunoblotting method,26 and they all were found to be active. The NF-κB inhibitory activity of compounds 1 and 2 toward MOLM-13 cells was more potent than that toward EOL-1 cells, and both compounds were more active than 3 and 4 toward MOLM-13 cells (Figure 6). These results indicate that compounds 1−4 mediated their leukemia cell apoptotic

than 3, which, in turn, showed more potent activity than 4 and parthenolide, implying that either a 3,10-furan-3(2H)-one unit or a C-1−C-3 enone group plays an important role and is more influential than a C-4−C-5 epoxy group, which is similar to the C-8 and C-10 ester groups, in mediating the human leukemia cell growth inhibition of these germacranolides. Midostaurin (PKC 412) is an effective drug to treat acute myeloid leukemia. However, a dose reduction is required in some patients, owing to its serious adverse effects, including febrile neutropenia, nausea, mucositis, vomiting, headache, petechiae, and musculoskeletal pain.21 As an approach to overcome this problem, we here investigated the potential of the in vitro synergistic antileukemic effects of compounds 1−4 and parthenolide with midostaurin (2.3 nM), at concentrations of 0.42, 0.44, 1.9, 4.9, and 3.2 μM, respectively, toward MOLM13 cells, and those of 0.56, 0.59, 2.5, 2.5, and 3.2 μM, respectively, against EOL-1 cells, which resulted in up to 75% cell survival in the MTS assay used (Figure 3). These results showed that each of compounds 1−4 exhibited a synergistic effect against midostaurin in inhibiting MOLM-13 and EOL-1 cell growth, but parthenolide did not show this type of activity. This indicates that 1−4 possibly could be used to improve the therapeutic efficacy of midostaurin. In our previous study, (−)-goyazensolide (1) was found to induce HT-29 human colon cancer cell apoptosis in vitro and in vivo.8 Following this observation and using a reported procedure,22 compounds 1−4 and parthenolide were tested for their MOLM-13 and EOL-1 cell apoptosis inducible effects, at a concentration range covering their IC50 values. Approximately 50% of late-stage apoptotic cells were induced, when MOLM13 and EOL-1 cells, respectively, were treated with 1 or 2 for 24 h at a concentration lower than 2.0 μM, respectively, but no similar cell apoptotic effects were observed for 3, 4, or parthenolide at this concentration (Figures 4 and 5). Treatment D

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Figure 4. Apoptosis induced in MOLM-13 human leukemia cells by compounds 1−4 and parthenolide. Cells were treated for 24 h with different concentrations of compounds 1−4, parthenolide, or the vehicle control, followed by an annexin V staining procedure. Lower left quadrant: percentage of viable cells; lower right quadrant: percentage of apoptotic cells; upper left quadrant: percentage of necrotic cells; upper right quadrant: percentage of late-stage apoptotic cells or dead cells. **p ≤ 0.05 (for significant differences).

activity and synergistic antileukemic effect of PKC412 partially through inhibition of NF-κB. Investigation of the dose−response curves of the cell growth inhibition of 1−4, parthenolide, and PKC412 against MOLM13 and EOL-1 cells (Figures S5 and S6, Supporting Information) showed that the curves of each of these compounds toward MOLM-13 and EOL-1 cells were closely comparable, implying that each compound mediated its cytotoxicity toward these two leukemia cell lines in a similar manner. However, compounds 1−4 and parthenolide all exhibited different curves toward both cell lines, suggesting that these compounds mediated their cytotoxicity through a mechanism distinct from each other. Thus, the C-15 hydroxy group in 1, the 3,10-furan-3(2H)-one moiety in 1 and 2, the C1−C-3 enone group in 3, the C-8 and C-10 ester groups in 4, and the C-4−C-5 epoxy group in parthenolide, along with the diverse conformations of these molecules, all may play a role in mediating their resultant activity. Interestingly, the dose−

response curves of (−)-15-deoxygoyazensolide (2) were similar to those observed for PKC412, indicating that these two compounds might share the same mechanism of action, i.e., that of targeting caspase-3 and NF-κB, as supported by their activation of caspase-3 and inhibition of NF-κB (Figure 6). Parthenolide has been investigated thoroughly for its potent and promising anticancer activity.3 It induces human leukemia cell apoptosis specifically through the inhibition of NF-κB, the proapoptotic activation of p53, and an increased reactive oxygen species production, without affecting normal progenitor and stem cell activities. It targets leukemia stem cells (LSC), the parent cells for the initiation, growth, and potential relapse of acute or chronic myelogenous leukemia, and is regarded as a representative of a new class of LSC-targeted anticancer drugs.27 In addition, parthenolide was found to show a synergistic effect with 5-fluorouracil in a murine xenograft model implanted with SW620 human colorectal tumor cells.28 The present investigation has shown that both 1 and 2 possess E

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Figure 5. Apoptosis induced in EOL-1 human leukemia cells by compounds 1−4 and parthenolide. Cells were treated for 24 h with different concentrations of compounds 1−4, parthenolide, or the vehicle control, followed by an annexin V staining procedure. Lower left quadrant: percentage of viable cells; lower right quadrant: percentage of apoptotic cells; upper left quadrant: percentage of necrotic cells; upper right quadrant: percentage of late-stage apoptotic cells or dead cells. **p ≤ 0.05 (for significant differences).

Figure 6. Caspase-3 activation and NF-κB inhibition by 1−4 (10 μM for each), parthenolide (10 μM), and PKC412 (10 nM) in MOLM-13 and EOL-1 cells. Cells were incubated with each test compound for 72 h, and the activity of caspase-3 and NF-κB was determined by immunoblotting using rabbit monoclonal cleaved caspase-3 (Asp175) or NF-κB-p65 and goat polyclonal β-actin antibodies. The data shown are representative blots from three independent experiments with similar results.

potential antileukemic activity, in a more potent manner than parthenolide. Also, these agents showed a synergistic effect to midostaurin against both MOLM-13 and EOL-1 human

leukemia cells. Thus, both goyazensolide (1) and 15deoxygoyazensolide (2) could be promising leads for development as either antileukemic agents or combination therapies F

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MTS assay) for 72 h. The viability of cells was evaluated using a commercial absorbance assay (CellTiter 96). Apoptosis Assay. In a manner described previously,22 MOLM-13 or EOL-1 cells were treated with either the vehicle, compounds 1−4, or parthenolide at the appropriate concentration for 24 h. The cells were washed with annexin V binding buffer, centrifuged at 300g for 10 min, and suspended (1 × 106) in 100 μL of 1× annexin V binding buffer. Then, 10 μL of annexin V-FITC and 10 μL of 7aminoactinomycin (BD Biosciences, San Jose, CA, USA) were added. After each suspension was mixed and incubated in a dark room at room temperature for 15 min, the cells were centrifuged, and the cell pellet was resuspended (1 × 106) in 400 μL of 1× annexin V binding buffer. The percentage of apoptotic cells was calculated by flow cytometric analysis (BD Biosciences). Immunoblotting. Immunoblotting was conducted as described previously.22,26 Briefly, after a 72 h treatment, MOLM-13 or EOL-1 cells were harvested, washed once with ice-cold phosphate-buffered saline, and lysed (108 cells/mL lysis buffer) in hypertonic buffer {1% NP-40, 10 mM HEPES (pH 7.5), 0.5 M NaCl, 10% glycerol supplemented with protease and phosphatase inhibitors [(1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 50 mM NaF, 10 mM βglycerol phosphate, 1 mM EDTA], and protease inhibitor cocktail tablet (Roche Applied Science, Indianapolis, IN, USA]}. Cell lysates, after the addition of 2× laemmli buffer (Bio-Rad Laboratories, Hercules, CA, USA) by supplementing with 2.5% β-mercaptoethanol to give 1× SDS sample buffer, were boiled for 5 min and subjected to immunoblotting. To determine protein concentrations, samples were dissolved in NP-40 lysis buffer and protein levels were assessed using a BCA protein assay kit (Bio-Rad Laboratories), standardized with bovine serum albumin. Then, proteins were separated with SDSpolyacrylamide gel electrophoresis and were transferred onto a polyvinylidene difluoride membrane (Thermo Fisher Scientific, Waltham, MA, USA). The membrane was incubated with a primary antibody for 12 h, followed by incubation with a secondary antibody for 1 h. Visualization was performed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). The antibodies used were rabbit monoclonal cleaved caspase-3 (Asp175) or NF-κB-p65 (Cell Signaling Technology, Beverly, MA, USA) and goat polyclonal β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Statistical Analysis. The measurements were performed in triplicate and are representative of three independent experiments, where the values generally agreed within 10%. Dose−response curves were calculated for IC50 determinations using nonlinear regression analysis (Table Curve2DV4; AISN Software Inc., Mapleton, OR, USA). Differences among samples were assessed by one-way ANOVA followed by Tukey−Kramer’s test, and the significance level was set at p < 0.05.

with midostaurin to overcome the side effects observed for this recently approved anticancer agent.

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EXPERIMENTAL SECTION

Chemicals. (−)-Goyazensolide (1), (−)-15-deoxygoyazensolide (2), (−)-ereglomerulide (3), and (+)-rufesolide A (4) were isolated from the leaves of Piptocoma rufescens in a previous study,5 with their single crystals grown in a mixture of n-hexane and acetone by slow crystallization in each case. Parthenolide was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Paclitaxel, PKC412, and other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). All procedures were carried out using solvents obtained from commercial sources and employed without further purification. X-ray Crystallography of 1−3. Colorless crystals of 1−3 suitable for crystal structure analysis were grown from a mixture of n-hexane and acetone. The crystallographic details are shown in Table S3 (Supporting Information). Data sets for 1, 2, and 3 were measured on a Nonius Kappa CCD diffractometer with Mo Kα radiation, and that for 1 was also collected on a Bruker Kappa Apex-II Duo diffractometer using Cu Kα radiation. Reflections were collected at low temperature for 1 (180 or 90 K) and 3 (220 K), using an Oxford Cryosystems Cryostream Cooler, and at room temperature for 2. Data integration was performed with Denzo, and scaling and merging of the data were done with Scalepack for 1 (from Mo Kα radiation), 2, and 3.29 For the structure of 1 determined from Cu Kα radiation, data integration was done with SAINT30 using a wide-frame algorithm, with data scaled and corrected for absorption using the multiscan method of SADABS.31 The crystal structures of 1−3 were solved by direct methods in SHELXS97, with full-matrix least-squares refinements based on F2 performed in SHELXL9732 as incorporated in the WinGX package.33 For each methyl group, the hydrogen atoms were added at the calculated positions using a riding model with U(H) = 1.5Ueq (bonded carbon atom). The torsion angle that defines the orientation of the methyl group about the C−C bond was refined. The remaining hydrogen atoms were included in the model at the calculated positions using a riding model with U(H) = 1.2Ueq (bonded atom). For compound 1, the hydrogen atom bonded to O-5 is involved in an intermolecular hydrogen bond with atom O-1, and it was located on a difference electron density map and refined isotropically. For compound 2, the asymmetric unit contains two molecules labeled as 2a and 2b. The CIF files for structures 1−3 have been deposited in the Cambridge Crystallographic Data Centre [deposition numbers: CCDC 1581580 (1 from Mo Kα radiation), CCDC 1582580 (1 from Cu Kα radiation), CCDC 1581834 (2a and 2b), and CCDC 1581839 (3)]. Cell Lines. The MOLM-13 and EOL-1 human acute myeloid leukemia cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and they were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA), supplemented with fetal bovine serum (10%). Cells were incubated at 37 °C in 5% CO2 and maintained with penicillin (100 U/mL) and streptomycin (100 μg/mL). MTS Assay. Cell growth inhibition/cytotoxicity was evaluated after 72 h incubation using an MTS assay (CellTiter 96) (Promega Corp., Madison, WI, USA),20 with PKC412 (midostaurin) used as the positive control. Briefly, MOLM-13 or EOL-1 cells were seeded in 96well plates and incubated with the vehicle (DMSO), the test samples, or PKC (all dissolved in DMSO and diluted to the concentrations required) for 72 h. Then, 20 μL of the CellTiter 96 reagent was added to each well. After an additional 24 h incubation, the optical density at 490 nm was measured, with IC50 values calculated with respect to the control samples. Synergistic Effect Evaluation. Following the cytotoxicity assay (MTS assay) described above, MOLM-13 or EOL-1 cells in log phase growth were seeded in 96-well clear flat-bottomed plates. Cells were incubated at 37 °C in 5% CO2 overnight and then treated with either the vehicle (DMSO), a test sample, PKC412 (the positive control), or the test sample plus PKC412 (all dissolved in DMSO and diluted to the concentrations required for approximately 75% cell survival in the

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b01079. Crystal data of 1−3; copies of MS, 1H and 13C NMR spectra of compounds 1−3; crystal structures of 1 determined with different radiations; assignments of 1H and 13C NMR spectroscopic data of 1−3; and dose− response curves of compounds 1−4, parthenolide, and PKC412 for their in vitro cytotoxicity against MOLM-13 and EOL-1 cells (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +1 614-247-8094. Fax: +1 614-247-8642. E-mail: [email protected]. G

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ORCID

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A. Douglas Kinghorn: 0000-0002-6647-8707 Author Contributions #

Y. Ren, J. C. Gallucci, and X. Li are joint first-named coauthors. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants P01 CA125066, CA185301, and AI129582, funded by NIH, Bethesda, MD, USA. We are very thankful to Dr. F. R. Fronczek, X-ray Facility, Department of Chemistry, Louisiana State University, Baton Rouge, USA, for collecting the crystallographic data for compound 1 using copper radiation. We thank Ms. J. Zhang, Center for Biostatistics, The Ohio State University, for the supportive statistical analysis. We are very thankful to Dr. D. D. Soejarto (University of Illinois at Chicago, Chicago, IL), for the plant taxonomic identification. We thank Dr. J. C. Yalowich, Division of Pharmacology, College of Pharmacy, The Ohio State University, for providing the reference compound, parthenolide. Drs. A. Somogy and N. M. Kleinholz of the Mass Spectrometry and Proteomics of the Campus Chemical Instrument Center, The Ohio State University, are thanked for acquisition of the MS data. Drs. C. A. McElroy (College of Pharmacy) and C. Yuan (Nuclear Magnetic Resonance Laboratory, Campus Chemical Instrument Center), The Ohio State University, are acknowledged for access to key NMR instrumentation used in this investigation and for the 700 or 800 MHz NMR measurements, respectively.

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DEDICATION Dedicated to Dr. Susan Band Horwitz, of Albert Einstein College of Medicine, Bronx, NY, for her pioneering work on bioactive natural products.

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DOI: 10.1021/acs.jnatprod.7b01079 J. Nat. Prod. XXXX, XXX, XXX−XXX