Note pubs.acs.org/jnp
The Cephalostatins. 24. Isolation, Structure, and Cancer Cell Growth Inhibition of Cephalostatin 20 George R. Pettit,* Jun-Ping Xu, Jean-Charles Chapuis, and Noeleen Melody Cancer Research Institute and Department of Chemistry and Biochemistry, Arizona State University, P.O. Box 871604, Tempe, Arizona 85287-1604, United States S Supporting Information *
ABSTRACT: For the purpose of advancing knowledge of the structural variations available in the natural cephalostatins contained in the marine worm Cephalodiscus gilchristi, the isolation and structure of the 20th member (1) has been accomplished (10−7 % yield). In turn cephalostatin 20 (1) proved to be enough for an initial SAR study comprising six important human cancer cell lines. A parallel objective was aimed at the possible discovery of a natural cephalostatin with a more accessible structure for total synthesis and/or synthetic modifications, but with powerful cancer cell growth inhibition.
A
Cephalostatin 20 (1) was isolated as an amorphous powder employing a series of solvent partitioning, gel-permeation chromatographic separations on Sephadex LH-20, followed by successive partition chromatographies using the LH-20 gel and final purification by reversed-phase HPLC.3,9a The new member 20 (1) exhibited UV maximum absorption in MeOH appearing at λmax 289 (log ε 4.04) and 307 (shoulder) nm, which was a characteristic UV spectroscopic pattern corresponding to other cephalostatins. HR-FABMS analysis of the disteroidal alkaloid afforded a protonated molecule with an m/z of 945.5446 ([M + H]+) corresponding to C54H76N2O12. In comparison with other cephalostatins, it was apparent that cephalostatin 20 (1) contained one more H 2 O than cephalostatin 2 (2, C54H74N2O11) and one more oxygen than cephalostatin 9 (3, C54H76N2O11). The structure of cephalostatin 20 (1) was further determined by using 1D- and 2DNMR spectroscopic interpretations. On the basis of the chemical shift values and coupling patterns of the observed signals in the 1H and 13C NMR spectra, the right-side moiety structure was determined to be the same as the corresponding unit in cephalostatin 2 (2) and cephalostatin 9 (3). The rightside structure was also confirmed by the C−H correlations of 2D-HMBC (Table 1). Hence, cephalostatin 20 (1) differed from both 2 and 3 in the left-side steroid unit. That new structural unit of cephalostatin 20 (1) was primarily elucidated by analysis of the signal information obtained through the 2D-NMR spectra. The assignment of 2DHMQC and -HMBC correlations established the left-side structure corresponding to the same steroidal A′/B′/C′/D′ rings of cephalostatin 2 (2), with a hydroxy group at C-8′, a ketone at C-12′, and a double bond between C-14′ and C-15′.
fter 16 years of challenges we succeeded in the discovery of the powerful cancer cell growth inhibitor cephalostatin 1.1a,b,2 Subsequently, we extended this series of marine worm anticancer constituents by 18 new and structurally related steroidal alkaloids.3 Overall this is a promising field of marine organism constituents, providing important leads to new therapeutic drugs.4 Meanwhile, interest in developing such encouraging leads to useful antitumor drugs offered by the cephalostatins continues to expand.3,5 The remarkable levels (ED50 < nm) of cancer cell growth inhibition and the discovery by Vollmar6 of their unique ability to cause cancer cell apoptosis are quite important. The mechanism proceeds by promoting the release of Smac/ DiABLo (a mitochondrial sequencing transporter) and not by cytochrome c or AIF from mitochondria causing morphological collapse instead of swelling.6 In addition the cephalostatins provide a unique molecule for investigating apoptotic signaling.6c In order to increase supply of the most promising anticancer cephalostatins, considerable effort has been devoted to total synthesis7,3 as well as synthesis of more accessible major modifications.3,7b,8 To possibly assist with both approaches, we undertook research aimed at expanding the 19 cephalostatins already in hand that we isolated from the South African marine tube worm Cephalodiscus gilchristi.3 For those purposes we further explored bioassay (P388 murine lymphocyte leukemia)-active, albeit very small, fractions from 1981 (166 kg) and 1990 (450 kg) collections (by scuba) of C. gilchristi. The majority of these fractions were obtained from the isolation of cephalostatins 10−19,9 and the combined total was 286.6 mg. From this starting point, we isolated 6.6 mg (1 × 10−7 %) and elucidated the structure of a new cephalostatin designated the 20th (1) as follows. © XXXX American Chemical Society and American Society of Pharmacognosy
Received: February 6, 2015
A
DOI: 10.1021/acs.jnatprod.5b00129 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
Chart 1
The E′-pyran ring was found between C-18′ and C-22′ as with cephalostatin 2 (2), but the 1H and 13C chemical shifts around the E′ ring differed. Furthermore, a linear chain corresponding to −CH(OH)−CH2−C(OH)−(CH3)2 was bonded to ring E′ or confirmed from a proton−proton relay system of 2D-COSY and -TOCSY couplings. The chemical shift of C-22′ was 100.5
ppm and was moved upfield by 10.4 ppm; the data for C23′and C-25′ (a quaternary carbon in the linear chain) were also shifted upfield by 9.9 and 11.0 ppm, respectively, in comparison with the corresponding data for C-22′ (81.6 ppm) and C-23′ (81.1 ppm) in cephalostatin 2 (2). This evidence clearly pointed to the furan type F′ ring of cephalostatin 2 (2) B
DOI: 10.1021/acs.jnatprod.5b00129 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
Table 1. 1H (400 MHz) and 13C NMR (100 MHz) Spectroscopic Data of Cephalostatin 20 (1) and Their Assignments (in C5D5N) right side δC, type 1
46.0, CH2
2 3 4
148.6, C 148.5, C 35.7, CH2
5 6
41.7, CH 28.1, CH2
7
28.6, CH2
8 9 10 11
33.7, 53.1, 36.3, 28.9,
CH CH C CH2
12 13 14 15
75.6, 55.3, 152.7, 122.2,
CH C C CH
16
93.1, CH
17 18
91.6, C 12.5, CH3
19 20 21 22 23 24
11.6, 44.4, 9.0, 117.1, 71.5, 39.5,
CH3 CH CH3 C CH CH2
25 26
82.8, C 69.4, CH2
27 12-OH 17-OH
29.4, CH3
δH (J in Hz) 2.60, m 3.90, m
left side
2.78, m 1.54, m 1.41, m 1.66 m 1.30, m 1.60, m 2.03, m 0.82, m 1.75, m 2.03, m 4.04, dd (15.0, 4.2)
5.62, brs 5.23, brs
9
18
8, 13, 14, 16, 17 8′, 13′, 16′, 17′ 14, 15, 17, 20
1.32, s
12, 13, 14, 17
0.74, s 2.75, m 1.34, d (7.0)
1, 5, 9, 10 13, 17, 21, 22, 23 17, 20, 22
4.80, m 2.68, m 2.35, m
23, 27 23, 25, 26
3.70, 3.82, 1.64, 4.70, 6.23,
m m s s s
δC, type
δH (J in Hz)
HMBC (H to C)
1′
39.5, CH2
2.94, m 3.71, m
5′, 10′, 19′
2′ 3′ 4′
148.2, C 148.2, C 36.1, CH2
5′ 4′
5′ 6′
34.1, CH 28.1, CH2
7′
24.5, CH2
8′ 9′ 10′ 11′
39.0, 78.6, 41.1, 45.5,
CH C C CH2
2.70, 3.04, 2.56, 1.41, 1.66, 1.85, 2.02, 2.73,
12′ 13′ 14′ 15′
211.4, 61.4, 148.8, 124.5,
C C C CH
HMBC (H to C) 9 9
25 23, 24, 25, 26 11, 12 13, 17
16′
32.2, CH2
17′ 18′
43.8, CH 63.4, CH2
19′ 20′ 21′ 22′ 23′ 24′
15.0, 31.7, 14.7, 100.5, 71.7, 44.3,
CH3 CH CH3 C CH CH2
25′ 26′
70.2, C 29.8, CH3
27′ 9′-OH
31.2, CH3
m m m m m m m m
2.89, m 3.32, d (14.0)
8′, 9′, 10′, 13′ 12′
5.59, brs 2.27, 3.16, 2.78, 4.34, 4.48, 0.90, 2.95, 1.17,
m m m d (15.0) d (15.0) s m d (6.9)
12′, 13′, 15′, 17′ 12′, 13′, 16′ 12′, 14′, 17′, 22′ 12′, 14′, 17′, 22′ 1′, 5′, 10′ 22′ 20′
4.51, d (9.5) 2.38, m
24′, 25′ 25′
1.48, s
24′, 27′
1.50, s 5.98, s
24′, 27′ 8′, 9′, 11′
As the first phase of this SAR analysis, comparisons of cephalostatins 1 (4), 2 (2), 9 (3), and 20 (1) were made against a panel of six human solid cancer cell lines (Table 2), originating from patients. In the comparative cancer bioassay with the same panel of human carcinoma cell lines, cephalostatins 1 (4) and 2 (2), as expected, displayed the most powerful cancer cell growth inhibition in vitro. Except for the equal result (GI50 0.11 nM) found using the DU-145 prostate cancer cells, cephalostatin 2 (2) exerted more potent growth-inhibitory effects against the other cell lines compared to cephalostatin 1 (4). This was especially true for the NCI-H460 non-small-cell lung cancer, KM20L2 colon carcinoma, and SF-268 glioblastoma cells, where cephalostatin 1 (4) was 10 times less active than cephalostatin 2 (2). The NCI-H460 lung cancer cells and KM20L2 colon cancer cells were the most sensitive to cephalostatin 2 (2), whereas the SF-268 cells showed more than 10 times less sensitivity to cephalostatin 1 (4). Given the
being opened in 1 to form a linear chain connected at C-22′, similar to that in cephalostatin 9 (3).10a An NOE correlation between H-20′ (2.95 ppm) and H-23′ (4.51 ppm) was detected to support the linkage from C-22′ to C-23′, although the 2DHMBC weakly provided the related cross-peaks between the two positions. The summarized assignments of 1H and 13C NMR data for cephalostatin 20 (1) appear in Table 1. The spectroscopic results and other evidence noted above established the structure of cephalostatin 20 as 1. While the cephalostatin 20 structure did not materially change the cephalostatin total synthesis challenges, it did add to the SAR relationship foundation. With respect to SAR we next undertook a detailed cancer cell growth evaluation of all 20 cephalostatins to obtain a uniform comparison beyond our early research using primarily the murine P388 lymphocyte leukemia cell line, where especially cephalostatins 1-4 proved to be superb cancer cell growth inhibitors. C
DOI: 10.1021/acs.jnatprod.5b00129 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
Table 2. Cancer Growth Inhibitory Activity of the 20 Cephalostatins against Six Human Tumor Cell Lines Expressed as GI50 μg/mL and Nanomolar (nM)a cell lineb cephalostatin 1 (4) 2 (2) 3 4 5 6 7 8 9 (3) 10 11 12 13 14 15 16 17 18 19 20 (1)
BXPC-3
MCF-7
SF-268
NCI-H460
KM20L2
DU-145
0.000040 (0.044) 0.000020 (0.022) 0.000040 (0.043) 0.034 (36) 0.30 (330) 0.34 (380) 0.0046 (5.0) 0.024 (26) 0.013 (14) 0.0012 (1.30) 0.0047 (4.9) 0.029 (31) 0.30 (310) 0.033 (35) 0.048 (50) 0.00040 (0.44) 0.00033 (0.36) 0.0031 (3.30) 0.0037 (3.90) 0.015 (16)
0.000090 (0.099) 0.000020 (0.022) 0.000030 (0.032) 0.041 (43) 0.31 (340) 0.49 (550) 0.046 (50) 0.024 (26) 0.10 (110) 0.0014 (1.40) 0.0039 (4.10) 0.031 (33) 0.28 (290) 0.026 (28) 0.043 (45) 0.0010 (1.10) 0.00039 (0.43) 0.0031 (3.30) 0.0070 (7.40) 0.021 (22)
0.0015 (1.60) 0.00011 (0.12) 0.00058 (0.58) 0.020 (21) 0.40 (440) 0.55 (610) 0.10 (110) 0.14 (150) 0.14 (150) 0.0035 (3.70) 0.026 (27) 0.14 (150) 3.30 (340) 0.075 (80) 0.060 (63) 0.0047 (5.20) 0.0025 (2.70) 0.0073 (7.80) 0.073 (78) 0.034 (36)
0.000040 (0.044) 0.0000052 (0.0056) 0.00040 (0.043) 0.042 (44) 0.14 (150) 0.29 (320) 0.0076 (8.20) 0.014 (15) 0.036 (39) 0.00057 (0.60) 0.0052 (5.40) 0.035 (37) 0.36 (370) 0.030 (32) 0.065 (68) 0.00058 (0.64) 0.00042 (0.46) 0.0026 (2.80) 0.0050 (5.30) 0.0057 (6.00)
0.000060 (0.066) 0.0000056 (0.0060) 0.000090 (0.096) 0.039 (39) 0.091 (100) 0.35 (390) 0.045 (48) 0.047 (47) 0.054 (58) 0.00067 (0.70) 0.0060 (6.30) 0.036 (0.038) 0.45 (470) 0.033 (35) 0.060 (63) 0.00055 (0.60) 0.00053 (0.58) 0.0029 (3.10) 0.0057 (6.10) 0.0068 (7.20)
0.00010 (0.11) 0.00010 (0.11) 0.00015 (0.16) 0.29 (31) 0.12 (120) >1 (>1100) 0.44 (470) >1 (>1100) >1 >1100 0.0081 (8.50) 0.022 (23) 0.42 (440) 1.10 (1100) 0.12 (130) 0.13 (140) 0.011 (12) 0.0030 (3.30) 0.019 (20) 0.028 (30) 0.20 (210)
a
Cytotoxicity concentrations as nanomolar values are given in parentheses. bCancer cell lines in order: pancreas (BXPC-3); breast (MCF-7); CNS (SF-268); lung (NCI-H460); colon (KM20L2); prostate (DU-145). size 25−100 pm) used in gel permeation and partition column chromatographic separations was obtained from Pharmacia Fine Chemicals. Fractionations and separations were monitored by thinlayer chromatography (TLC) using Analtech silica gel GHLF Uniplates under shortwave UV light. For HPLC separations, a Phenomenex Prepex (particle size 5−20 μm, 10.0 mm × 250 mm) C-8 column was used in reversed-phase mode with Altex (model 110A) solvent metering pumps and Gilson Holochrome HM UV detection at 288 nm. All organic solvents were freshly distilled. Isolation and Purification. A murine P388 lymphocytic leukemia cell line active fraction (286.6 mg) was obtained (P388 bioassay) from the fractionations of the extracts (166 kg from the 1981 collection and 450 kg from the 1990 collection) derived from the marine worm Cephalodiscus gilchristi.2,9 The initial further separations of this large bioassay-active fraction afforded cephalostatins 2 and 12. Extended separations of the remaining active fractions by Sephadex LH-20 column chromatography was achieved using n-hexane−CH2Cl2− MeOH (5:1:1) and n-hexane−toluene−MeOH (3:1:1) as the mobile phase. Finally repeat bioassay separation/purification employing reversed-phase HPLC with CH3CN−MeOH−H2O (18:10:14) as an eluent led to a pure amorphous solid specimen of cephalostatin 20 (1); 6.6 mg. Cephalostatin 20 (1): colorless, amorphous powder; mp >320 °C; UV (MeOH) λmax 289 (log ε 4.04) and 307 (shoulder) nm; the 1H NMR, 13C NMR, and 1H−13C long-range correlations from HMBC assignments have been recorded in Table 1; HRFABMS m/z of 945.5446 [M + H]+ (calcd for C54H76N2O12, 945.5477; −3.2 ppm). Its ED50 value in P388 cells was 0.0082 μg/mL. Cancer Cell Line Procedures. Inhibition of human cancer cell growth was assessed using the National Cancer Institute’s standard sulforhodamine B assay as previously described.12 Briefly, cells in a 5% fetal bovine serum/RPMI1640 medium were inoculated in 96-well plates and incubated for 24 h. Serial dilutions of the compounds were then added. After 48 h, the plates were fixed with trichloroacetic acid, stained with sulforhodamine B, and read with an automated microplate reader. A growth inhibition of 50% (GI50, or the drug concentration causing a 50% reduction in the net protein increase) was calculated from optical density data with Immunosoft software.
very minor difference in the structures of the two cephalostatins, the hydroxy substitution at C-8′ in 2 appears to be an obvious source of enhancement of the antineoplastic potency. Similarly, the presence of a hydroxy group at C-8′ potentiated the 10 times greater cancer cell growth inhibition by cephalostatin 20 (1) compared to cephalostatin 9 (3) except for the BXPC-3 pancreas adenocarcinoma cells, where both cephalostatins 2 (2) and 9 (3) showed essentially the same level of inhibition (GI50 0.013 and 0.015 μg/mL). Importantly, the inhibition by cephalostatins 20 (1) and 9 (3) was remarkably diminished by 100−1000 times compared to those of the corresponding cephalostations 2 (2) and 1 (4). From this evidence, it is clear that the spirostanol structure must be intact and is a critical center for the antineoplastic activities. The opening of the left-side spiro-ring dramatically attenuated the suppressive power on these carcinoma cells. In addition it is clear that cephalostatins 10, 11, 16, and 17, and to a lesser extent 18 and 19, provide important templates for maintaining potent inhibition of cancer cell growth. These overall SAR results should be very helpful for future anticancer drug design and mechanism investigations. Increasing interest continues to grow in diverse scientific areas in further development of the cephalostatins, ritterazines, and the partially related, but also exceptional cancer cell inhibitors schweinfurthin A and OSW-1.3,6,7,8,11c Evidence has been found that these all target oxysterol bending protein (OSBP) involved in signal transduction.11d
■
EXPERIMENTAL SECTION
General Experimental Procedures. The UV spectra were obtained using a Hewlett-Packard 8450 UV−vis spectrometer. The 1 H NMR and 13C NMR spectra were recorded employing Bruker 400 instruments using C5D5N (TMS internal reference) as solvent unless otherwise noted; high-resolution FABMS spectra were obtained employing a Kratos MS-50 spectrometer. Sephadex LH-20 (particle D
DOI: 10.1021/acs.jnatprod.5b00129 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
■
Note
(9) (a) Pettit, G. R.; Xu, J.-P.; Williams, M. D.; Christie, N. D.; Doubek, D. L.; Schmidt, J. M.; Boyd, M. R. J. Nat. Prod. 1994, 57, 52− 63. (b) Pettit, G. R.; Ichihara, Y.; Xu, J.-P.; Boyd, M. R.; Williams, M. D. Bioorg. Med. Chem. Lett. 1994, 4, 1507−1512. (c) Pettit, G. R.; Xu, J.-P.; Ichihara, Y.; Williams, M. D.; Boyd, M. R. Can. J. Chem. 1994, 72, 2260−2267. (d) Pettit, G. R.; Xu, J.-P.; Schmidt, J. M.; Boyd, M. R. Bioorg. Med. Chem. Lett. 1995, 5, 2027−2032. (10) (a) Pettit, G. R.; Kamano, Y.; Inoue, M.; Dufresne, C.; Boyd, M. R.; Herald, C. L.; Schmidt, J. M.; Doubek, D. L.; Christie, N. D. J. Org. Chem. 1992, 57, 429−31. (b) Nawasreh, M. Bioorg. Med. Chem. 2008, 16, 255−265. (11) (a) Covell, D. G. PLoS One 2012, 7, e44631. (b) Rodriguez, E. M.; Rudy, A.; del Rosario, R. C. H.; Vollmar, A. M.; Mendoza, E. R. Nat. Comput. 2011, 10, 993−1015. (c) Burgett, A. W. G.; Poulsen, T. B.; Wangkanont, K.; Anderson, D. R.; Kikuchi, C.; Shimada, K.; Okubo, S.; Fortner, K. C.; Mimaki, Y.; Kuroda, M.; Murphy, J. P.; Schwalb, D. J.; Petrella, E. C.; Cornella-Taracido, I.; Schirle, M.; Tallarico, J. A.; Shair, M. D. Nat. Chem. Biol. 2011, 7, 639−647. (12) Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.; Vistica, D.; Hose, C.; Langley, J.; Cronise, P.; Viagro-Wolff, A.; GrayGoodrich, M.; Campbell, H.; Mayo, J.; Boyd, M. J. Natl. Cancer Inst. 1991, 83, 757−766.
ASSOCIATED CONTENT
S Supporting Information *
The authors declare no competing financial interest. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00129.
■
AUTHOR INFORMATION
Corresponding Author
*Tel: (480) 965-3351. Fax: (480) 965-2747. E-mail: bpettit@ asu.edu. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We are pleased to thank for financial assistance grants RO1CA90441-02-05 and 5RO1CA90441-07 from the Division of Cancer Treatment Diagnosis and Centers, National Cancer Institute, DHHS; the Arizona Biomedical Research Commission; Dr. Alec D. Keith; the J. W. Kieckhefer Foundation; the Margaret T. Morris Foundation; the Robert B. Dalton Endowment Fund; and Dr. W. Crisp and Mrs. A. Crisp.
■
REFERENCES
(1) (a) Pettit, G. R.; Moser, B. R.; Herald, D. L.; Knight, J. C.; Chapuis, J.-C.; Zheng, X. J. Nat. Prod. 2015, 78, 1067−1072. (b) Pettit, G. R.; Moser, B. R.; Mendonca, R. F.; Knight, J. C.; Hogan, F. J. Nat. Prod. 2012, 75, 1063−1069. (2) Pettit, G. R.; Inoue, M.; Kamano, Y.; Herald, D. L.; Arm, C.; Dufresne, C.; Christie, N. D.; Schmidt, J. M.; Doubek, D. L.; Krupa, T. S. J. Am. Chem. Soc. 1988, 110, 2006−2007. (3) (a) Pettit, G. R.; Mendonca, R. F.; Knight, J. C.; Pettit, R. K. J. Nat. Prod. 2011, 74, 1922−1930. (b) Kumar, K. A.; La Clair, J. J.; Fuchs, P. L. Org. Lett. 2011, 13, 5334−5337. (c) Shawakeh, K. Q.; AlSaid, N. H. Steroids 2011, 76, 232−237. (d) Pettit, G. R.; Tan, R.; Xu, J.-P.; Ichihara, Y.; Williams, M. D.; Boyd, M. R. J. Nat. Prod. 2008, 71, 487−491. (4) Nadkarni, D. H.; Murugesan, S.; Velu, S. E. Tetrahedron 2013, 69, 4105−4113. (5) von Schwarzenberg, K.; Vollmar, A. M. Cancer Lett. 2013, 332, 295−303. (6) (a) Dirsch, V. M.; Muller, I. M.; Eichhorst, S. T.; Pettit, G. R.; Kamano, Y.; Inoue, M.; Xu, J.-P.; Ichihara, Y.; Wanner, G.; Vollmar, A. M. Cancer Res. 2003, 63, 8869−8876. (b) Rudy, A.; Lopez-Anton, N.; Dirsch, V. M.; Vollmar, A. M. J. Nat. Prod. 2008, 71, 482−486. (c) Rudy, A.; Lopez-Anton, N.; Barth, N.; Pettit, G. R.; Dirsch, V. M.; Schulze-Osthoff, K.; Rehm, M.; Prehn, J. H. M.; Vogler, M.; Fulda, S.; Vollma, A. M. Cell Death Differ. 2008, 15, 1930−1940. (7) (a) Shi, Y.; Jia, L.; Xiao, Q.; Lan, Q.; Tang, X.; Wang, D.; Li, M.; Ji, Y.; Zhou, T.; Tian, W. Chem.−Asian J. 2011, 6, 786−790. (b) Fortner, K. C.; Kato, D.; Tanaka, Y.; Shair, M. D. J. Am. Chem. Soc. 2010, 132, 275−280. (c) Taber, D. F.; Joerger, J.-M. J. Org. Chem. 2008, 73, 4155−4159. (d) Lee, S.; Fuchs, P. L. Org. Lett. 2002, 4, 317−318. (e) LaCour, T. G.; Guo, C.; Bhandaru, S.; Fuchs, P. L.; Boyd, M. R. J. Am. Chem. Soc. 1998, 12, 692−707. (8) (a) Poza, J. J.; Rodriguez, J.; Jimenez, C. Bioorg. Med. Chem. 2010, 18, 58−63. (b) Kanduluru, A. K.; Banerjee, P.; Beutler, J. A.; Fuchs, P. L. J. Org. Chem. 2013, 78, 9085−9092. (c) Iglesias-Arteaga, M. A.; Morzycki, J. W. Alkaloids (San Diego, CA, U.S.) 2013, 72, 153−279. (d) Cheun, Y.; Koag, M. C.; Kou, Y.; Warnken, Z.; Lee, S. Steroids 2012, 77, 276−281. (e) Kumar, K. A.; La Clair, J. J.; Fuchs, P. L. Org. Lett. 2011, 13, 5334−5337. (f) Koag, M.; Lee, S. Org. Lett. 2011, 13, 4766−4769. (g) Yunus, U.; Iqbal, R.; Winterfeldt, E. J. Heterocycl. Chem. 2005, 42, 1079−1084. (h) Gryszkiewicz-Wojtkielewicz, A.; Jastrzebska, I.; Morzycki, J. W.; Romanowska, D. B. Curr. Org. Chem. 2003, 7, 1257−1277. E
DOI: 10.1021/acs.jnatprod.5b00129 J. Nat. Prod. XXXX, XXX, XXX−XXX
