Isolation, structure elucidation, and antitumor activity of spirohexenolides A and B

Article abstract of DOI:10.1021/jo901826d

(Chemical Equation Presented) In this report, we describe the discovery of a pair of bioactive spirotetronates, spirohexenolides A (1) and B (2), that arose from the application of mutagenesis, clonal selection techniques, and media optimization to strain

Full text of DOI:10.1021/jo901826d

pubs.acs.org/joc
Isolation, Structure Elucidation, and Antitumor Activity
of Spirohexenolides A and B
MinJin Kang, Brian D. Jones, Alexander L. Mandel, Justin C. Hammons,
Antonio G. DiPasquale, Arnold L. Rheingold, James J. La Clair, and Michael D. Burkart*
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive,
La Jolla, California 92093
mburkart@ucsd.edu
Received August 25, 2009
In this report, we describe the discovery of a pair of bioactive spirotetronates, spirohexenolides A (1)
and B (2), that arose from the application of mutagenesis, clonal selection techniques, and media
optimization to strains of Streptomyces platensis. The structures of spirohexenolides A (1) and B (2)
were elucidated through X-ray crystallography and confirmed by 1D and 2D NMR studies. Under all
examined culture conditions, spirohexenolide A (1) was the major metabolite with traces of spirohex-
enolide B (2) arising in cultures containing increased loads of adsorbent resins. Spirohexenolide A (1)
inhibited tumor cell growth with GI50 values spanning from 0.1 to 17 μM across the NCI 60 cell line
panel. An increased activity was observed in leukemia (GI50 value of 254 nM in RPMI-8226 cells), lung
cancer (GI50 value of 191 nMin HOP-92cells), and colon cancer (GI50 value of 565 nM inSW-620 cells)
tumor cells. Metabolite 1 was fluorescent and could be examined on a confocal fluorescent microscope
with conventional laser excitation and filter sets. Time lapse imaging studies indicated that spirohex-
enolide A (1) was readily taken up by tumor cells, appearing through the cell immediately after dosing
and subcellularly localizing in the lysosomes. This activity, combined with a unique selectivity in NCI
60 cancer cell line screening, indicates that 1 warrants further chemotherapeutic evaluation.
Introduction
including synthetic analogues,10 have demonstrated potent
activity against tumor progression, and an analogue of
Since its classification in 1956,1 Streptomyces platensis has
demonstrated a remarkable ability to produce biologically
active polyketides including the dorrigocins,2 the migrasta-
tins,3 the pladienolides,4 leustroducin B,5 TPU-0037,6 pla-
tensimideA,7 and platensimycin.8 Many of these compounds,9
(4) (a) Asai, N.; Kotake, Y.; Niijima, J.; Fukuda, Y.; Uehara, T.; Sakai, T.
J. Antibiot. 2007, 60, 364–369. (b) Mandel, A. L.; Jones, B. D.; La Clair, J. J.;
Burkart, M. D. Bioorg. Med. Chem. Lett. 2007, 17, 5159–5164. (c) Kanada,
R. M.; Itoh, D.; Nagai, M.; Niijima, J.; Asai, N.; Mizui, Y.; Abe, S.; Kotake,
Y. Angew. Chem., Int. Ed. 2007, 46, 4350–4355.
(5) Kohama, T.; Maeda, H.; Sakai, J. I.; Shiraishi, A.; Yamashita, K. J.
Antibiot. 1996, 49, 91–94.
(1) Tresner, H. D.; Backus, E. J. Appl. Microbiol. 1956, 4, 243–250.
(2) (a) Hochlowski, J. E.; Whittern, D. N.; Hill, P.; McAlpine, J. B. J.
Antibiot. 1994, 47, 870–874. (b) Karwowski, J. P.; Jackson, M.; Sunga, G.;
Sheldon, P.; Poddig, J. B.; Kohl, W. L.; Kadam, S. J. Antibiot. 1994, 47, 862–
869.
(3) (a) Woo, E. J.; Starks, C. M.; Carney, J. R.; Arslanian, R.; Cadapan,
L.; Zavala, S.; Licari, P. J. Antibiot. 2002, 55, 141–146. (b) Ju, J. H.; Lim, S.
K.; Jiang, H.; Shen, B. J. Am. Chem. Soc. 2005, 127, 1622–1623. (c) Gaul, C.;
Njardarson, J. T.; Shan, D.; Dorn, D. C.; Wu, K. D.; Tong, W. P.; Huang, X.
T.; Moore, M. A.; Danishefsky, S. J. J. Am. Chem. Soc. 2004, 126, 11326–
11337.
(6) Furumai, T.; Eto, K.; Sasaki, T.; Higuchi, H.; Onaka, H.; Saito, N.;
Fujita, T.; Naoki, H.; Igarashi, Y. J. Antibiot. 2002, 55, 873–880.
(7) Herath, K. B.; Zhang, C.; Jayasuriya, H.; Ondeyka, J. G.; Zink, D. L.;
Burgess, B.; Wang, J.; Singh, S. B. Org. Lett. 2008, 10, 1699–1702.
(8) Wang, J.; Soisson, S. M.; Young, K.; Shoop, W.; Kodali, S.; Galgoci,
A.; Painter, R.; Parthasarathy, G.; Tang, Y. S.; Cummings, R.; Ha, S.;
Dorso, K.; Motyl, M.; Hayasuriya, H.; Ondeyka, J.; Herath, K.; Zhang, C.;
Hernandez, L.; Allocco, J.; Basilio, A.; Tormo, J. R.; Genilloud, O.; Vicente,
F.; Pelaez, F.; Colwell, L.; Lee, S. H.; Michael, B.; Felcetto, T.; Gill, C.;
Silver, L. L.; Hermes, J. D.; Bartizal, K.; Barrett, J.; Schmatz, D.; Becker, J.
W.; Cully, D.; Singh, S. B. Nature 2006, 441, 358–361.
9054 J. Org. Chem. 2009, 74, 9054–9061
Published on Web 11/02/2009
DOI: 10.1021/jo901826d
r
2009 American Chemical Society

Kang et al.
JOCArticle
pladienolide D, E7107, has recently entered clinical trials.11
Given this track record, we were interested in evaluating
associated strains of S. platensis for the production of yet
undiscovered polyketides.
than 1 mg/L), we applied both UV irradiation and NTG
(N-methyl-N0-nitrosoguanidine) chemical mutagenesis for
strain improvement. From UV irradiation analysis, we
identified three mutant strains, MJ1A (1A, Figure 1a),
MJ2B (2B, Figure 1a), and MJ6 (6, Figure 1a), that displayed
an increased zone of inhibition over their parent S. platensis
strain MJ (wt, Figure 1a). Subsequent efforts led to the
production of two stable morphologies of MJ1A noted as
strains MJ1A1 and MJ1A2.18 16S rRNA gene sequence data
indicated that MJ1A strain showed high sequence identity to
S. platensis NBRC12901 (99%),19a S. hygroscopicus subsp.
glebosus LMG 19950 (99%),19b S. libani subsp. rufus NBRC
15424 (99%),19c and S. caniferus NBRC 15389 (99%).19d
Recently, genome sequencing studies suggest that
the bacterial secondary metabolomes are far more compli-
cated than previously recognized by evaluation of their
natural product content.12 This, combined with further
genetic screening programs, suggests that only a fraction of
the potential natural products produced in bacteria have
been identified.13 The cause for this lack in production is
complex. First, media and environmental stimuli can con-
tribute to bacterial secondary metabolism either up- or
down-regulating the production of specific metabolites
based on external cues or morphological reponses.14 Second,
evolutionary pressures are often key in regulating a mi-
crobe0s ability to access secondary metabolism.15 Mutagen-
esis offers a strong potential to circumvent the lack in
production,16 as mutant strains can be directed, through
associated screening efforts, to enhance production. In this
study, we demonstrate how applications of such strain
improvement techniques can be used to access the produc-
tion of new metabolites.
Results
Our studies began by evaluating a panel of S. platensis
strains from available culture collections. An antibiotic assay
using the inhibition of Bacillus subtilis growth was eventually
chosen (comparable methods have been used in the discovery
of spirotetronate natural products17). Due to the presence of
only traces of compound 1 from the parent strain (often less
(9) Ju, J. H.; Rajski, S. R.; Lim, S. K.; Seo, J. W.; Peters, N. R.;
Hoffmann, F. M.; Shen, B. Bioorg. Med. Chem. Lett. 2008, 18, 5951–5954.
(10) Metaferia, B. B.; Chen, L.; Baker, H. L.; Huang, X. Y.; Bewley, C. A.
J. Am. Chem. Soc. 2007, 129, 2434–2435.
(11) Kotake, Y.; Sagane, K.; Owa, T.; Mimori-Kiyosue, Y.; Shimizu, H.;
Uesugi, M.; Ishihama, Y.; Iwata, M.; Mizui, Y. Nat. Chem. Biol. 2007, 3,
570–575.
(12) (a) Li, M. H.; Ung, P. M.; Zajkowski, J.; Garneau-Tsodikova, S.;
Sherman, D. H. BMC Bioinf. 2009, 10, 185. (b) Gulder, T. A.; Moore, B. S.
Curr. Opin. Microbiol. 2009, 12, 252–260. (c) Jones, A. C.; Gu, L.; Sorrels, C.
M.; Sherman, D. H.; Gerwick, W. H. Curr. Opin. Chem. Biol. 2009, 13, 216–
223. (d) Udwary, D. W.; Zeigler, L.; Asolkar, R. N.; Singan, V.; Lapidus, A.;
Fenical, W.; Jensen, P. R.; Moore, B. S. Proc. Natl. Acad. Sci. U.S.A. 2007,
104, 10376–10381.
(13) (a) Gust, B. Methods Enzymol. 2009, 458, 159–180. (b) Wilkinson, B.;
Micklefield, J. Nat. Chem. Biol. 2007, 3, 379–386. (c) Baltz, R. H. Antonie Van
Leeuwenhoek. 2001, 79, 251–259.
(14) van Wezel, G. P.; Krabben, P.; Traag, B. A.; Keijser, B. J.; Kerste, R.;
Vijgenboom, E.; Heijnen, J. J.; Kraal, B. Appl. Environ. Microbiol. 2006, 72,
5283–5288.
(15) (a) te Poele, E. M.; Bolhuis, H.; Dijkhuizen, L. Antonie Van Leeu-
wenhoek 2008, 94, 127–143. (b) Demain, A. L. Int Microbiol. 1998, 1, 259–
264. (c) Hopwood, D. A. Philos. Trans. R. Soc. London, Ser. B 1989, 324,
549–562.
(16) (a) Wang, L. Y.; Yun, B. S.; George, N. P.; Wendt-Pienkowski, E.;
Galm, U.; Oh, T. J.; Coughlin, J. M.; Zhang, G.; Tao, M.; Shen, B. J. Nat.
Prod. 2007, 70, 402–406. (b) Malina, H.; Tempete, C.; Robert-Gero, M. J.
Antibiot. 1985, 38, 1204–1210. (c) Lee, S. D.; Park, S. W.; Oh, K. K.; Hong, S.
I.; Kim, S. W. Lett. Appl. Microbiol. 2002, 34, 370–375. (d) Lyutskanova, D.
G.; Stoilova-Disheva, M. A.; Peltekova, V. T. Appl. Biochem. Microbiol.
2005, 41, 165–168. (e) Kieser, T.; Bibb, M. J.; Buttner, M. J.; Chater, K. F.;
Hopwood, D. A. Practical Streptomyces Genetics; The John Innes Foundation:
Norwich, England, 2000. (f) Lalm, R.; Khanna, R.; Kaur, H.; Khanna, M.;
Dhingra, N.; Lal, S.; Gartemann, K. H.; Eichenlaub, R.; Ghosh, P. K. Crit. Rev
Microbiol. 1996, 22, 201–255. (g) Hopwood, D. A.; Chater, K. F. Philos. Trans.
R. Soc. London, Ser. B 1980, 290, 313–328.
FIGURE 1. Production of metabolite 1 from Streptomyces platen-
sis strains MJ1A1 and MJ1A2. (a) Ultraviolet light mutagenesis
provided mutants with an increased ability to inhibit the growth of
Bacllius subtilis 6633. An enhanced zone of growth inhibition was
observed from mutant strains MJ1A, MJ2B, and MJ6 as compared
to their parent strain (wt). (b) TLC analysis of extracts from
S. platensis strains cultures. A direct comparison of crude extracts
from these cultures indicates that metabolite 1 (lane 1) was enhanced
in S. platensis strain MJ1A1 (lane 2) and strain MJ1A2 (lane 3), as
compared to their parental strain (lane 4) or two morphologically
different colonies of S. platensis FERM BP-8442 (lanes 5 and 6). An
arrow denotes the position of metabolite 1 and stars denote the
position of lipids and acylglycerides. The TLC observations were
confirmed by preparative isolation, which after multiple repeats
failed to return traces of 1 from cultures of the strains in lanes 5 and 6.
(c) HPLC traces collected with UV detection at 254 nm confirmed
the presence of 1 in both parent S. platensis MJ and mutant
S. platensis MJ1A2 strains while not in S. platensis FERM BP-
8442. The MIC of pure 1 against Bacillus subtilis was determined to
be 12.25 μM (see the Experimental Section), therein supporting the
viability of the screening procedure.
(17) (a) Schindler, P. W.; Zahner, H. Eur. J. Biochem. 1973, 39, 591–600.
(b) Gary, N. D.; Bard, R. C. J. Bacteriol. 1952, 64, 501–512. (c) Sundaram,
T. K.; Cazzulo, J. J.; Kornberg, H. L. Biochim. Biophys. Acta 1969, 192,
355–357.
(18) Pang, X. H.; Aigle, B.; Girardet, J. M.; Mangenot, S.; Pernodet, J. L.;
Decaris, B.; Leblond, P. Antimicrob. Agents Chemother. 2004, 48, 575–588.
J. Org. Chem. Vol. 74, No. 23, 2009 9055

Kang et al.
JOCArticle
1H NMR-guided fractionation was applied to extracts
from cultures of the S. platensis MJ1A1 and S. platensis
MJ1A2. Metabolite 1, with a unique signature of olefinic
protons in the NMR spectrum, was identified in the ethyl
acetate (EtOAc) extract from both cultures. TLC analysis
indicated that metabolite 1 (lane 1, Figure 1b) was more
abundant in extracts from strains MJ1A1 (lane 2, Figure 1b)
and MJ1A2 (lane 3, Figure 1b) than their parent strain
S. platensis MJ (lane 4, Figure 1b). Control experimentation
indicated that 1 also did not appear in related strains such as
FERM BP-8442 (lanes 5 and 6, Figure 1b), indicating that
the production of 1 was restricted to strains MJ1A1 and
MJ1A2. Similarly, HPLC analyses using UV detection at
254 nm confirmed the presence of 1 in both parent
(S. platensis strain MJ) and mutant strains but not in other
strains of S. platensis (Figure 1c). While 1 was observed
in parent (traces) and mutant extracts, TLC evidence
(Figure 1b) indicates that the mutants offered a significant
increase in production of 1 relative to their lipid content
(lipids could not be detected under our HPLC methods).
After identification by 1H NMR and MS analyses, small
yellow needles of compound 1 were obtained by perfusion of
a chloroform solution of 1 with benzene. Yellow plates were
more effectively obtained by recrystallization from ethanol,
mp 280-285 °C dec. Samples of these crystals were then
evaluated by X-ray crystallography. The structure of 1 was
refined to a final R1 of 4.6%. With use of anomalous copper
dispersion effects,20 absolute stereochemical information
was obtained as depicted in Figure 2.
Spectroscopic methods confirmed the crystal structure
as follows. A molecular formula for 1 of C25H28O5 was
determined from high-resolution EI-MS analysis (m/z
408.1947, Mþ, Δ 3.7 ppm). Strong absorption bands at
1754 and 1702 cm-1 in the FT-IR spectrum confirmed the
presence of both ester and ketone groups, respectively.
1
An NMR data set including H, 13C, gCOSY, TOCSY,
NOESY, ROESY, HMQC, HSQC, DEPT, and HMBC
spectra was collected for spirohexenolide A (1) in CDCl3
(Table 1). Twenty-five resonances were observed in the 13
C
spectrum as expected from the HRMS data. The DEPT
spectrum indicated sixteen protonated carbons including
four methyl carbons, an oxymethylene, two aliphatic methy-
lene carbons, an aliphatic methine, an oxymethine, and seven
olefin methine carbons. Three of the nine quaternary car-
bons were observed in the olefin region for a total of ten
olefinic carbon resonances, indicating five double bonds.
Three of the six remaining quaternary carbons appeared in
the carbonyl region of the spectrum, one of which was the
conjugated ketone at δC 196.0 and two of which appeared in
the ester/lactone region at δC 169.3 and δC 165.7; this was
supported by the carbonyl peaks in the FT-IR spectrum. The
fourth was thought to be a quaternary center due to its
upfield shift at δC 44.3. The two quaternary carbons at δC
100.8 and δC 89.2 remained ambiguous.
1
Analysis of the H and gCOSY spectra of 1 (Figure 3a)
revealed four spin systems. The first system began with the two
downfield olefin methine protons H-4 (δH 7.44, d, 10.0 Hz) and
H-5 (δH 7.02, d, 10.0 Hz). H-5 showed allylic coupling to
oxymethylene proton H-21a (δH 4.73, d, 12.5 Hz), implicating a
four-carbon subunit for this spin system with a junction at
quaternary olefinic C-6. The J = 10.0 Hz coupling constant
between H-4 and H-5 was consistent with a cis-olefin.
The second spin system comprised a linear subunit including
olefinic methine H-7 (δH 5.72, d, 8.4 Hz), oxymethine H-8 (δH
4.60, m), aliphatic methylene pair H2-9 (δH-9a 2.60, m, δH-9b
2.17, dt, 12.3, 10.6 Hz), olefinic methine H-10 (δH 5.55, ddd, 5.4,
10.6, 15.5 Hz), and olefinic methine H-11 (δH 5.69, d, 15.5 Hz).
The J = 15.5 Hz coupling constant between H-10 and H-11
established the E configuration for the Δ10,11 olefin. The third
spin system was an isolated two-resonance spin system includ-
ing olefinic methine H-13 (δH 5.07, s) and vinyl methyl H3-22
(δH 1.76, s), presumably connected via quaternary olefinic C-12.
The fourth spin system was a branched subunit beginning
with olefinic methine H-15 (δH 5.29, s), which displayed
allylic coupling to vinyl methyl H3-24 (δH 1.76, s) and to
aliphatic methine H-17 (δH 2.39, m). H-17 also coupled to
methyl H3-25 (δH 1.34, d, 7.2 Hz) and methylene pair H2-18
(δH-18a 2.35, m, δH-18b 1.71, d, 13.6 Hz). The C-23 methyl
group was not in any of the spin systems, indicating that it
was attached to a quaternary center.
Figure 3a depicts several of the key HMBC correlations that
validated the structure. The HMBC data confirmed the assign-
ments of the C-3 and C-6 13C signals at δC 165.7 and δC 126.7,
respectively, based on the correlations from H-4, H-5, and H-
21a, all in the first spin system. Tethering of the first and second
spin systems hinged on the HMBC correlation from H-5 and
H-21b to olefinic methine C-7, suggesting quaternary C-6 as the
junction. This C-3 to C-11 segment could be extended to
include the CH-13/CH3-22 system based on reciprocal HMBC
correlations between olefinic H-11 and H-13 and their respec-
tive carbons. Quaternary olefinic C-12 (δC 136.2) was assigned
FIGURE 2. Structures of spirohexenolides A (1) and B (2), and
corresponding ORTEP drawings of their X-ray crystal structures with
ellipsoids drawn at the 50% probability level. The drawings represent
absolute configuration as the Flack x parameter was 0.0(3).20b
(19) (a) Shirling, E. B.; Gottlieb, D. Int. J. Syst. Bacteriol. 1968, 18, 279–
392. (b) Kumar, Y.; Goodfellw, M. Int. J. Syst. Evol. Microbiol. 2008, 58,
1369–1378. (c) Skerman, V. B. D.; Mcgowan, V.; Sneath, P. H. A. Int. J. Syst.
Bacteriol. 1980, 30, 225–420. (d) Preobrazhenskaya, T. P. Int. J. Syst.
Bacteriol. 1986, 36, 573–576.
€
(20) (a) For an example, see: Niu, X. M.; Li, S. H.; Gorls, H.; Scholl-
meyer, D.; Hilliger, M.; Grabley, S.; Sattler, I. Org. Lett. 2007, 9, 2437–2440.
(b) For a definition, see: Flack, H. D. Acta Crystallogr., Sect. A 1983, 39,
876–881.
9056 J. Org. Chem. Vol. 74, No. 23, 2009

Kang et al.
JOCArticle
TABLE 1. 1H, 13C, gCOSY, NOESY, and HMBC NMR Datag for Spirohexenolide A (1) in CDCl3
C/H no.
δ
H mult (J Hz)a
δ
C (mult)b
COSYc
NOESYc
HMBCc,f
1
2
3
4
5
6
7
8
169.3 (C)
100.8 (C)
165.7 (C)
120.3 (CH)
142.1 (CH)
126.7 (C)
139.3 (CH)
69.3 (CH)
7.44 d (10.0)
7.02 d (10.0, <1)
5
4,21a
5
4,7
3,6
3,6,7,21
5.72 d (8.4)
4.60 m
2.60 m
8
5,21b
9a,9b,10
5,21
9
8,10,11
8,10,11
9,11,12
9,10,13,22
7,9a,9b
8,9b,10
8,9a,10
9a,9b,11
9a,10
9a
9b
10
11
12
13
14
15
16
17
18a
18b
19
20
21a
21b
22
23
24
25
8,9b,10,11e
8,9a,10,11
2.17 dt (10.6, 12.3)
5.55 ddd (5.4, 10.6, 15.5)
5.69 d (15.5)
42.6 (CH2)
120.8 (CH)
140.9 (CH)
136.2 (C)
134.8 (CH)
44.3 (C)
128.0 (CH)
133.4 (C)
33.5 (CH)
33.3 (CH2)
8d,9a,9b,13e,21a,21bd,22d
9b,13
5.07 s
5.29 s
22
10,11,15e,23e
13,23,24
11,14,15,22,23
13,14,17,19,24
17,24
2.39 m
2.35 m
1.71 d (13.6)
15,18a,18b,25
17,18b
17,18a
18a,25
17,18b,23
18a,25
15,16,18,19,25
14,17,19,20,25
14,16,17,19,20,25
89.2 (C)
196.0 (C)
64.8 (CH2)
4.73 d (12.5)
4.57 d (12.5)
1.76 s
1.19 s
1.76 s
1.34 d (7.2)
5,21b
21a
13
10,21bd
7,10,21a
10
3,5,6,7
5,6,7
11,12
13,14,15,19
15,16,17,18
16,17,18
14.1 (CH3)
27.2 (CH3)
22.0 (CH3)
19.6 (CH3)
13,15,18a
15,25
15
17
17,18b,24d
a1H NMR data were collected at 500 MHz. b13C NMR data were collected at 100 MHz. 13C NMR multiplicities were determined by the DEPT
spectrum. cgCOSY, HMBC, and NOESY spectra were collected at 800 MHz. dOverlapping signals detected eA weak crosspeak was detected. fHMBC
2,3
data were collected with an evolution delay optimized for
J
= 8 Hz. gSpectra were collected at 296 K in CDCl3.
CH
FIGURE 3. Select NMR data. (a) Key gCOSY and HMBC correlations for spirohexenolide A (1) and (b) Nuclear Overhauser effects
identified through analysis of a NOESY spectrum as mapped on the X-ray crystal structure of 1. Both proximal (green) and transannluar (blue)
NOEs are shown. (c) ΔδS-R values for the Mosher esters 3a and 3b.
as the link due to correlations from H-10 and H3-22. Mutual
HMBC correlations between olefinic H-13 and H-15 and their
respective carbons combined with their additional correlation
to the upfield quaternary center C-14 (δC 44.3) indicated that
C-14 was the link to the fourth spin system. Correlation from
the isolated CH3-23 methyl group to C-14 established its
position. Quaternary C-16 (δC 133.4) was assigned due to
HMBC correlations from H-17, H-18b, H3-24, and H3-25.
Fourth spin system protons H-15, H-17, H2-18 and the isolated
methyl H3-23 correlated to the downfield quaternary carbon C-
19 (δC 89.2), placing it adjacent to CH2-18, indicating a bond to
quaternary C-14 and thus a cyclohexene ring. The ketone
carbonyl at C-20 (δC 196.0) was assigned adjacent to C-19
due to correlations from CH2-18. The chemical shift of C-19
suggested oxidation, which implicated it as the quaternary
center of a spirotetronate system due to its inclusion in the
cyclohexene ring. C-1 (δC 169.3) and C-2 (δC 100.8) were
assigned based on their chemical shifts since no protons were
within HMBC correlation distance to them.
The NOESY spectrum (Table 1) revealed an NOE correla-
tion between methylene proton H-18a and the H3-23 isolated
methyl group, providing additional support for the presence
of the cyclohexene ring. The transannular NOE correlation
between olefinic methine H-10 and oxymethylene H2-21 was
indicative of the macrocycle in 1. Key NOESY interactions
are shown in Figure 3b. Taken together, the NMR data were
consistent with the X-ray crystal structure. The absolute
configuration of 1 was confirmed by preparing (S)-MTPA
(3a) and (R)-MTPA (3b) esters (Figure 3c).21
(21) (a) Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95, 512–519.
(b) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc.
1991, 113, 4092–4096.
J. Org. Chem. Vol. 74, No. 23, 2009 9057

Kang et al.
JOCArticle
TABLE 2. 1H, 13C, gCOSY, and HMBC Datae for Spirohexenolide B (2) in C6D6
C/H no.
δ
H mult (J, Hz)a
δ
C (mult)b
COSYc
HMBCc,d
1
2
3
168.9 (C)
101.1 (C)
165.5 (C)
4
5
6
7.59 d (10.0)
6.20 d (10.0)
119.3 (CH)
142.2 (CH)
128.8 (C)
5
4,21b
3,6
3,6,7,21
7
8
4.97 t (8.5)
1.53 m
1.92 m
1.40 m
5.16 ddd (4.9, 10.9, 15.4)
5.46 d (15.4)
135.4 (CH)
27.8 (CH2)
32.3 (CH2)
8,21a
7,9a,9b
8,9b,10
8,9a,10
9a,9b,11
10
9a
9b
10
11
12
13
14
15
16
17
18a
18b
19
20
21a
21b
22
23
24
25
125.3 (CH)
139.7 (CH)
135.5 (C)
134.8 (CH)
44.5 (C)
129.0 (CH)
133.3 (C)
33.9 (CH)
33.7 (CH2)
12
13,22
5.13 s
5.33 s
22
11,14,15,22,23
17,19,24
17,24
2.10 m
2.30 dd (8.6, 14.6)
1.62 d (14.6)
15,18a,18b,25
17,18b
18a
16,25
14,17,19,20,25
14,16,17,19,20,25
88.4 (C)
195.3 (C)
63.4 (CH2)
4.13 d (12.6)
3.72 d (12.6)
1.96 s
1.30 s
1.65 s
1.43 d (6.9)
5,21b
7,21a
13
3,5,6,7
6,7
11,12
13,14,15,19
15,16,17
16,17
14.6 (CH3)
27.4 (CH3)
22.0 (CH3)
19.9 (CH3)
15
17
a1H NMR data were collected at 500 MHz. b13C NMR data were collected at 125 MHz. cgCOSY and HMBC spectra were collected at 800 MHz. dThe
2,3
HMBC spectrum was collected with an evolution delay of
J
= 6 Hz. eSpectra were collected at 296 K in C6D6. Due to a slow decomposition of 2 in
CH
CDCl3, C6D6 was required for extended times required to collect 13C NMR, HMBC, and HSQC data.
With structure elucidation studies complete, we returned
to culturing to produce additional quantities of compound 1
for biological studies. Using our optimized strains, we
screened for media that provided an optimal yield of 1. After
evaluating over 50 different liquid cultures, we found that
culturing S. platensis MJ1A in a fermentation media (6% w/v
soluble starch, 1% w/v dry yeast, 1% w/v β-cyclodextrin, 1%
w/v CaCO3) containing 2% of Amberlite XAD-16 resin
provided 1 at up to 325 mg/L (see the Experimental Section
for further details). By increasing the resin content to 10%,
we were able to obtain 15-20 mg/L of a second metabolite
spirohexenolide B (2) from these cultures. The structure of 2
was characterized by X-ray crystallography (Figure 2) and
subsequent NMR analyses (Table 2) indicating that 2 failed
to undergo oxidation at C-8, suggesting that 2 is a biosyn-
thetic precursor to 1.
With access to the natural product, we were able to characterize
its biological activity. While we identified 1 using an antibiotic
screen, the activity of 1 was more significant in tumor cell lines.
Initial activity studies used the human colon tumor HCT-116 cell
line, and 1 displayed cytotoxicity activity with a GI50 value of
36.0 (5.1 μM, using the MTT assay. Submission of 1to the single
and multiple dose screens NCI-60 human tumor cell line screen22
identified the enhanced activity as given by lower GI50 values in
leukemia (CCRF-CEM, MOLT-4, and RPMI-8226), lung cancer
(HOP-92), and colon cancer (SW-629) cell lines (complete data
provided in the Supporting Information). Subsequent COM-
PARE analysis failed to provide a match to a known compound
and any associated mechanism of action, suggesting a novel
anticancer action for 1. In vivo studies in athymic nude mice
produced toxicity after a single dose of 1(6-10 mg/kg), indicating
the threshold for further studies.
We then turned to evaluate the cellular uptake and localization
of 1 in HCT-116 tumor cells using fluorescence microscopy.
Fortunately, spirohexenolide A (1) was natively fluorescent, with
an excitation maximum λmax = 435 nm and emission maximum
at λmax = 466 nm. HCT-116 cells were treated with 10 μM 1
in DMEM containing 10% FCS, 100 U/mL penicillin-G, and
100 μg/mL streptomycin and analyzed by fluorescence micro-
scopy. Spirohexenolide A (1) was readily uptaken and appeared
within minutes throughout the cell (Figure 4a). Within 6-12 h,
fluorescence from 1 concentrated within vesicles surrounding the
nucleus and remained in these structures (Figure 4b). This staining
could not be washed from the cells by repetitive incubation with
media and remained consistent thereafter (Figure 4c). Co-staining
experiments with a panel of organelle probes provided a direct
correlation with LysoTracker Red DND-9923 (Figures 4d-4e)
indicating that the localization occurred in the lysosomes.
Discussion
Spirohexenolide A (1) belongs to a large class of spirote-
tronate natural products that includes A88696F,24 abysso-
micins,25 chlorothricin,26 decatromicins,27 pyrrolosporin A,28
(24) Bonjouklian, R. Tetrahedron Lett. 1993, 34, 7861–7864.
€
(25) Bister, B.; Bischoff, D.; Strobele, M.; Riedlinger, J.; Reicke, A.;
€
€
Wolter, F.; Bull, A. T.; Zahner, H.; Fiedler, H. P.; Sussmuth, R. D. Angew.
Chem., Int. Ed. 2004, 43, 2574–2576.
(26) Brufani, M.; Cerrini, S.; Muntwyle, R.; Fedeli, W.; Mazza, F. Helv.
Chim. Acta 1972, 55, 2094–2102.
(27) Momose, I.; Hirosawa, S.; Nakamura, H.; Naganawa, H.; Iinuma,
H.; Ikeda, D.; Takeuchi, T. J. Antibiot. 1999, 52, 787–796.
(28) Schroeder, D. R.; Colson, K. L.; Klohr, S. E.; Lee, M. S.; Matson,
J. A.; Brinen, L. S.; Clardy, J. J. Antibiot. 1996, 49, 865–872.
(22) Shoemaker, R. H. Nat. Rev. Cancer 2006, 6, 813–823.
(23) Lemieux, B.; Percival, M. D.; Falgueyret, J. P. Anal. Biochem. 2004,
327, 247–251.
9058 J. Org. Chem. Vol. 74, No. 23, 2009

Kang et al.
JOCArticle
FIGURE 4. Uptake and subcellular localization of spirohexenolide A (1) in HCT-116 cells. Confocal fluorescent images from HCT-116 cells
treated with 10 μM 1 for (a) 1 h, (b) 6 h, and (c) 12 h. Cells were washed twice with media prior to imaging. Live cell images were collected with
excitation from a laser at 488 nm (emission filtered at 524 ( 40 nm). Co-staining with LysoTracker Red DND-99 indicates that compound 1
localizes within the lysosomes. HCT-116 cells were treated with 10 μM 1 for 6 h and washed before staining with 10 μM LysoTracker Red DND-
9923 for 20 min. (d) Fluorescence from 1 collected with excitation from a laser at 488 nm (emission filtered at 524 ( 40 nm). (e) Fluorescence from
LysoTracker Red DND-99 collected with excitation from a laser at 568 nm (emission filtered at 624 ( 40 nm). (f) Two-color overlap depicting the
fluorescence from 1 (red) and LysoTracker Red DND-99 (green). Yellow denotes overlap of both probes. Bars denote 10 μm.
PA-46101-A,29 tetronomycin,30 and versipelostatin.31 While
structural similarities exist, spirohexenolide A (1) contains a
unique and functionally compact carbon framework and
offers a new carbon skeleton. Its salient features include a
unique pyran, a high degree of unsaturation, and a tetra-
substituted olefin juncture between its tetronic acid and
the adjacent pyran. This juncture may be the result of an
intramolecular dehydration reaction of an appropriately
spaced distal alcohol onto the 3-keto portion of the spirote-
tronate, such as in carolic acid.32
The biosynthesis of 1 may be derived through a late-stage
intramolecular Diels-Alder (IMDA) cycloaddition. Appli-
cation of IMDA reactions to the syntheses of spirotetronate
natural products is well established, such as in the total
synthesis of abyssomicin C by Sorensen33 and an approach
to chlorothricolide by Yoshii.34 To date, the biosynthetic
gene clusters of four metabolites of this family
(chlorothricin,35 kijanimicin,36 tetronomycin,37 and tetro-
carcin A38) have been elucidated and several of these path-
ways include a putative IMDA biogenesis. The isolation of
spirohexenolide B (2) suggests that oxidation at C-8 arose at
a late stage by oxidation via a cytochrome P450 or related
enzyme.39
In conclusion, we have discovered two new spirotetronate
polyketides, spirohexenolide A (1) and B (2), from S. pla-
tensis. We have elucidated their structures through spectro-
scopic and X-ray crystallographic analyses. We have shown
that mutagenesis can be used in conjunction with culture
optimization to provide viable quantities of trace metabo-
lites.40 Activity analyses indicated that 1 displayed signifi-
cant activity against tumor cell growth with a unique
specificity to select tumor cell lines (cf. NCI-60 cell line
screening data in the Supporting Information). The fact that
2 (GI50 value of 61.2 ( 7.8 μM in HCT116 cells) also
displayed comparable activity to 1 (GI50 value of 36.0 (
5.1 μM in HCT116 cells) when screened in house using the
MTT assay indicates that the C-8 hydroxyl group may serve
as a site for reporter attachment for identifying its cellular
targets.41 The combination of the unique structure and
activity of these spirohexenolides serve as the starting point
for the development of both chemical synthesis and mechan-
ism of action studies.
(29) Matsumoto, M.; Kawamura, Y.; Yosimura, Y.; Terui, Y.; Nakai,
H.; Yoshida, T.; Shoji, J. J. Antibiot. 1990, 43, 739–747.
Experimental section
ꢀ
(30) Keller-Juslen, C.; King, H. D.; Kuhn, M.; Loosli, H. R.; Pache, W.;
Mutagenesis of S. platensis. Spore suspensions were prepared
from glycerol stocks of S. platensis MJ. While a series of strains
were examined, we have only obtained compounds 1 and 2 from
this parent strain and its mutants. A 1 μL aliquot of these
suspensions was added to 1 mL of sterilized water and further
diluted by addition of 10 μL of this solution into 10 mL of water
to yield a solution containing approximately 6 ꢀ 105 spores/mL.
This solution was then poured onto a sterile 9 cm glass Petri dish
Petcher, T. J.; Weber, H. P.; Von Wartburg, A. J. Antibiot. 1982, 35, 142–150.
(31) Park, H. R.; Chijiwa, S.; Furihata, K.; Hayakawa, Y.; Shin-Ya, K.
Org. Lett. 2007, 9, 1457–1460.
(32) Clutterbuck, P. W.; Raistrick, H.; Reuter, F. Biochem. J. 1935, 29,
300–321.
(33) Zapf, C. W.; Harrison, B. A.; Drahl, C.; Sorensen, E. J. Angew.
Chem., Int. Ed. 2005, 44, 6533–6537.
(34) Takeda, K.; Igarashi, Y.; Okazaki, K.; Yoshii, E.; Yamaguchi, K. J.
Org. Chem. 1990, 55, 3431–3434.
(35) Jia, X. Y.; Tian, Z. H.; Shao, L.; Qu, X. D.; Zhao, Q. F.; Tang, J.;
Tang, G. L.; Liu, W. Chem. Biol. 2006, 13, 575–585.
(36) Zhang, H.; White-Phillip, J. A.; Melanc-on, C. E. 3rd; Kwon, H. J.;
Yu, W. L.; Liu, H. W. J. Am. Chem. Soc. 2007, 129, 14670–14683.
(37) Demydchuk, Y.; Sun, Y.; Hong, H.; Staunton, J.; Spencer, J. B.;
Leadlay, P. F. ChemBioChem 2008, 9, 1136–1145.
(38) Fang, J.; Zhang, Y.; Huang, L.; Jia, X.; Zhang, Q.; Zhang, X.; Tang,
Z. J. Bacteriol. 2008, 190, 6014–6025.
(40) Bode, H. B.; Bethe, B.; Hofs, R.; Zeeck, A. ChemBioChem 2002, 3,
619–627.
^
(41) (a) Hughes, C. C.; MacMillan, J. B.; Gaudencio, S. P.; Fenical, W.;
La Clair, J. J. Angew. Chem., Int. Ed. 2009, 48, 728–732. (b) Leslie, B. J.;
Hergenrother, P. J. Chem. Soc. Rev. 2008, 37, 1347–1360. (c) Alexander, M.
D.; Burkart, M. D.; Leonard, M. S.; Portonovo, P.; Liang, B.; Ding, X.;
ꢀ
(39) Related P450 oxidations: (a) Machida, K.; Aritoku, Y.; Tsuchida, T.
J. Biosci. Bioeng. 2009, 107, 596–598. (b) Lin, X.; Cane, D. E. J. Am. Chem.
Soc. 2009, 131, 6332–6333. (c) Henry, K. M.; Townsend, C. A. J. Am. Chem.
Soc. 2005, 127, 3724–3733.
Joullie, M. M.; Gulledge, B. M.; Aggen, J. B.; Chamberlin, A. R.; Sandler, J.;
Fenical, W.; Cui, J.; Gharpure, S. J.; Polosukhin, A.; Zhang, H. R.; Evans, P.
A.; Richardson, A. D.; Harper, M. K.; Ireland, C. M.; Vong, B. G.; Brady, T.
P.; Theodorakis, E. A.; La Clair, J. J. ChemBioChem 2006, 7, 409–416.
J. Org. Chem. Vol. 74, No. 23, 2009 9059

Kang et al.
JOCArticle
and UV irradiated (Stratalinker 1800) at 8000 μJ at a distance of
12 cm while being stirred. Samples were taken every 6 s over a
3 min period. After serial dilution of UV-irradiated spore
suspension in deionized H2O, the sample was spread onto
Bennett0s agar (1.0% w/v glucose, 0.2% w/v pancreatic digest
of casein, 0.1 w/v of yeast extract, 0.1% w/v beef extract, 1.5%
w/v of agar in deionized H2O at pH 7.0), YEMED agar (0.4% w/v
yeast extract, 1.0% w/v malt extract, 0.4% w/v glucose, 1.5%
w/v agar in deionized H2O at pH 7.2), and ISP4 agar (1.0% w/v
soluble starch, 0.2% w/v CaCO3, 0.1% w/v K2HPO4, 0.1% w/v
to sterilization. The fermentation media was shaken for 72 h at
220 rpm at 28 °C. The cultures were filtered through cheesecloth
to collect the resin. The resin was then returned to the baffled
flask and acetone (250 mL) and EtOAc (250 mL) were added.
The flask was shaken for 2 h at 220 rpm. The resin was filtered
again through cheesecloth, and the filtrate was concentrated on
a rotary evaporator until only insoluble solids and water
remained. EtOAc was added until most of the solids were
dissolved, and the mixture was poured into a separatory funnel.
The aqueous layer was extracted with additional EtOAc (2 ꢀ
100 mL), and the combined organic layers were concentrated to
provide a crude extract. Crude extract was dissolved in a
minimum amount of 1:1 hexanes:EtOAc (sonication was used
to facilitate dissolution). A 2 in. ID column containing silica gel
(EM Sciences) was packed with 1:1 hexanes:EtOAc, and the
solution of the crude extract was loaded. The column was run
with 1:1 hexanes:EtOAc for at least two column volumes before
EtOAc was used to elute 1 with Rf 0.29 (EtOAc). Compounds 1
and 2 could be visualized by ceric ammonium molybdate, 2,4-
dinitrophenylhydrazine, iodine, and potassium permanganate
stains, and short-wave UV (excitation at 254 nm). Pure spir-
ohexenolide A (1) was obtained after a second flash column,
using a gradient from hexanes to EtOAc or trituration with
small amounts of absolute ethanol.
Isolation of Spirohexenolide B (2) from Cultures of S. platensis
Strain MJ1A1. S. platensis strain MJ1A1 was cultured in the
same manner on the same scale used to produce spirohexenolide
A (1), (above), but the fermentation media was supplemented
with 10% w/v of Amberlite XAD-16 resin (Alfa Aesar) that was
washed repetitively with deionized water prior to sterilization.
The fermentation media was shaken for 72 h at 220 rpm at 28 °C.
The crude extract of the resin was processed in the same manner
as used for the isolation of spirohexenolide A (1), as described in
the preceding paragraph. A 2 in. i.d. column containing silica gel
(EM Sciences) was packed with 1:1 hexanes:EtOAc, and the
solution of the crude extract was loaded. The column was run
with 1:1 hexanes:EtOAc for two column volumes, and spiro-
hexenolide B (2) was obtained from the eluted and concentrated
material by subjecting it to a second Flash purification on a 2 in.
i.d. column with a gradient from hexanes to 1:1 hexanes:EtOAc
with elution of 2 in 1:1 hexanes:EtOAc with Rf 0.68 (EtOAc),
followed by crystallization from either EtOH or a mixture of
CH2Cl2 and hexanes to obtain yellow crystals.
Synthesis of Mosher Esters 3a and 3b. The (S)- and (R)-MTPA
derivatives 3a and 3b were prepared with use of a slight modifica-
tion of the standard procedure.21 (S)-MTPA ester 3a: To a
sample of compound 1 (30.3 mg, 0.0743 mmol) in a dry 25 mL
round-bottomed flask with a Teflon-coated magnetic stirbar
were added a few crystals of 4-dimethylaminopyridine and the
flask was sealed with a rubber septum and flushed with argon.
CH2Cl2 (3 mL) and pyridine (0.120 mL, 1.5 mmol) were added at
rt, and the mixture was stirred until a yellow solution was
achieved. Stirring was then continued as 70 μL of (R)-MTPA-
Cl (0.374 mmol) was added via syringe at rt. After 30 min the
solution turned dark green. After 50 min, TLC indicated a new
compound had formed with Rf 0.76 (EtOAc), and that com-
pound 1 had been consumed. The reaction mixture was then
poured into a separatory funnel containing half-saturated NaH-
CO3 (30 mL) and CH2Cl2 (20 mL), and the organic layer became
yellow again upon shaking. The aqueous layer was extracted with
another 20 mL of CH2Cl2 and the combined organic layers were
driedoverNa2SO4 andconcentratedunderreducedpressure. The
residue was dissolved in 3:1 hexanes:EtOAc (5 mL), and standard
flash chromatography with 3:1 hexanes:EtOAc provided pure
3a (16.3 mg, 35%). The same procedure was used on 1 and
(S)-MTPA-Cl to make the (R)-MTPA ester 3b (13.5 mg, 40%).
Spirohexenolide A (1):. yellow needles, mp 280-285 °C dec;
[R]25D þ551.3 (c 0.4, CHCl3); UV λmax (MeOH) 339 (ε 8650), 236
MgSO4 7H2O, 0.1% w/v NaCl, 0.2% w/v (NH4)2SO4, 0.001%
w/v FeSO4 7H2O, 0.001% w/v MnCl2 4H2O and 0.001% w/v
of ZnSO4 7H2O in deionized H2O at pH 7.2) for examining the
3
3
3
3
morphologically differentiating colonies. To prevent photoreac-
tivation, the plates were wrapped with foil for 24 h and then
incubated at 30 °C for 15 days.
Mutant Screening Identifies Producer Strains S. platensis
MJ1A1 and MJ1A2. After 15 days of incubation, survival
colonies were transferred onto R2YE media (10.3% w/v su-
crose, 0.5% w/v yeast extract (Difco), 0.01% w/v casaminoacids
(Difco), 0.025% w/v K2SO4, 1.01% w/v MgCl2 6H2O, 1% w/v
3
glucose, 0.025% w/v KH2PO4, 0.29% w/v CaCl2 2H2O,
0.0008% w/v ZnCl2, 0.004% w/v FeCl3 6H2O, 0.0004% w/v
3
3
CuCl2 2H2O, 0.0004% w/v MnCl2 4H2O, 0.0004% w/v Na2-
3
3
B4O 10H2O, 0.0004% w/v (NH4)5Mo7O24 4H2O 0.3% w/v
3
3
L-proline, 0.573% w/v N-tris(hydroxymethyl)methyl-2-ami-
noethane-sulfonic acid (TES), 0.005% v/v 1 N NaOH to provide
a pH 7.2). Once the mutants had sporulated, agar cones (3 mm
OD ꢀ 5 mm height) were excised containing a single colony and
stamped on top of a glucose basal salt (1% g of glucose, 0.01%
yeast extract, 1.5% agar, 0.02% MgSO4 7H2O, 0.001% NaCl,
0.001% FeSO4 7H2O, 0.001% MnSO 4H2O 0.2% NH4Cl,
3
3
3
0.465% K2HPO4, 0.09% of KH2PO4 at pH 7.0) agar seeded
with ∼8 ꢀ 107 of Bacillus subtilis 6633 per cm2. After incubation
at 37 °C for 24 h, colonies showing a zone of inhibition were
compared against their parent strain. Strains MJ1A, MJ2B and
MJ6 (Figure 1a) were obtained with this method.
Minimum Inhibitory Concentration (MIC) Assay of Spirohex-
enolide A (1), using Bacillus subtilis 6633. Spirohexenolide A (1)
in dimethyl sulfoxide (DMSO) was diluted to 10, 15, 30, 60, 120,
250, 500, and 1000 μg/mL stocks in tryptic soy broth with a final
concentration of 1% DMSO. B. subtilis, cultured for 18 h at
37 °C in tryptic soy broth, was inoculated at 1/10 000 to a final
volume of 200 μL per well on a 96 well plate and then treated
with 2 μL of a stock solution of 1 (10, 15, 30, 60, 120, 250, 500,
and 1000 μg/mL in tryptic soy broth containing 1% DMSO).
The plate was incubated in 37 °C for 18 h, indicating that pure 1
had an MIC value of 12.25 μM (no visible bacterial growth). The
compound was tested in duplicates. Negative control comprised
of DMSO solvent did not show any effect on the bacterial
growth.
Culturing of Spirohexenolide A (1) from S. platensis Strain
MJ1A1. A single colony of S. platensis MJ1A1 grown on yeast
extract-malt extract-dextrose (YEMED) agar was resuspended
in 50 μL of sterilized water, using a sterilized pellet pestle, and
inoculated into 3 mL of tryptic soy broth (BD Biosciences) and
shaken at 220 rpm at 28 °C for 40 h. An aliquot (2 mL) of this
starter culture was transferred into a 250 mL baffled Erlenmeyer
flask containing 100 mL of seed medium containing 1% w/v
glucose, 2.4% w/v soluble starch, 0.3% w/v beef extract, 0.5%
w/v tryptone, 0.5% w/v yeast extract, and 2.0% w/v CaCO3
adjusted to pH 7.2. After shaking the seed medium for 48 h at
220 rpm and 28 °C, a 50 mL aliquot was transferred to a 2.8 L
baffled Erlenmeyer flask containing 500 mL of fermentation
media (6% w/v soluble starch, 1% w/v dry yeast, 1% w/v
β-cyclodextrin, 0.2% w/v CaCO3 adjusted to pH 6.8 prior to
sterilization) and 2% w/v of Amberlite XAD-16 resin (Alfa
Aesar) that was washed repetitively with deionized water prior
9060 J. Org. Chem. Vol. 74, No. 23, 2009

Kang et al.
JOCArticle
(ε 25583) nm; IR (film) νmax 3469, 1754, 1702, 1582, 1550, 1059,
1043, 988, and 968 cm-1; ESIMS m/z 409.03 [M þ H]þ, 431.03,
[M þ Na]þ; HR-EI-MS m/z 408.1947, [M]þ (calcd for C25H28O5
[M]þ, 408.1931); 1H and 13C NMR (Table 1).
reflections were found to be symmetry independent, with an Rint
of 0.0366. Indexing and unit cell refinement indicated a primi-
tive, monoclinic lattice. The space group was found to be P2(1)
(No. 4). The data were integrated with the Bruker SAINT
software program and scaled with the SADABS software
program. Solution by direct methods (SIR-2004) produced a
complete heavy-atom phasing model consistent with the pro-
posed structure. All non-hydrogen atoms were refined aniso-
tropically by full-matrix least-squares (SHELXL-97). All
hydrogen atoms were placed with use of a riding model. Their
positions were constrained relative to their parent atom by using
the appropriate HFIX command in SHELXL-97.
Spirohexenolide B (2):. yellow rhomboid crystals recrystal-
lized from CH2Cl2 and hexanes, mp 219-221 °C dec; IR (film)
νmax 2922, 2852, 1735, 1707, 1587, 1551, 1466, 1410 cm-1
;
ESIMS m/z 392.91 [M þ H]þ; HR-ESI-MS m/z 415.1888,
[M þ Na]þ (calcd for C25H28O4Na [M þ Na]þ, 415.1885);
1H and 13C NMR (Table 2).
Spirohexenolide A (S)-MTPA derivative (3a):. yellow solid,
mp 208-211 °C dec; [R]23D þ159.3 (c 1, CH2Cl2); IR (film) νmax
2936, 1750, 1709, 1594, 1554, 1252, 1168, 1056, 1014, and
722 cm-1; ESI-MS m/z: 624.92 [M þ H]þ, 647.04 [M þ Na]þ;
HR-ESI-FT-MS (Orbit-trap-MS) m/z calcd for C35H35F3O7Na
[M þ Na]þ 647.2227, found 647.2218; see the Supporting
Information for 1H and 13C NMR spectroscopic data.
Spirohexenolide A (R)-MTPA derivative (3b):. yellow solid,
mp 246-250 °C dec; [R]23D þ223.5 (c 1, CH2Cl2); IR (film) νmax
A colorless plate of compound 2 0.33 ꢀ 0.28 ꢀ 0.08 mm3 in
size was mounted on a Cryoloop with Paratone oil. Data were
collected in a nitrogen gas stream at 100(2) K, using ψ and ω
scans. Crystal-to-detector distance was 50 mm and exposure
time was 10 s per frame with a scan width of 0.5°. Data collection
was 99.9% complete to 25.00° in θ. A total of 24117 reflections
were collected covering the indices, -8e h e 8, -15e k e 15,
-27e l e 27. A total of 7549 reflections were found to be
symmetry independent, with an Rint of 0.0363. Indexing and unit
cell refinement indicated a primitive, monoclinic lattice. The
space group was found to be P2(1). The data were integrated
with the Bruker SAINT software program and scaled with the
SADABS software program. Solution by direct methods (SIR-
2004) produced a complete heavy-atom phasing model consis-
tent with the proposed structure. All non-hydrogen atoms were
refined anisotropically by full-matrix least-squares (SHELXL-97).
All hydrogen atoms were placed with use of a riding model. Their
positions were constrained relative to their parent atom by using
the appropriate HFIX command in SHELXL-97.
2936, 1750, 1709, 1594, 1554, 1252, 1169, 1056, and 1014 cm-1
;
ESI-MS m/z: 625.18 [M þ H]þ, 647.21 [M þ Na]þ; HR-ESI-FT-
MS (Orbit-trap-MS) m/z calcd for C35H36F3O7 [M þ H]þ
625.2408, found 625.2419; see the Supporting Information for
1H and 13C NMR spectroscopic data.
Uptake and Localization in HeLa Cells. HCT-116 cells
(ATCC CCL-247) were cultured in Dulbecco0s modification of
Eagle0s medium (DMEM) with 4.5 g L-1 glucose, 4.5 g L-1
L-glutamine, and 5% heat inactivated fetal calf serum (FCS) in
glass-bottom dishes. Fluorescent images were collected on a
Leica (Wetzlar, Germany) DMI6000 inverted confocal micro-
scope with a Yokogawa (Tokyo, Japan) spinning disk confocal
head, Orca ER High Resolution B&W Cooled CCD camera
(6.45 μm/pixel at 1X) (Hamamatsu, Sewickley, PA), Plan Apoc-
hromat 40ꢀ/1.25 na and 63ꢀ/1.4 na objective, and a Melles
Griot (Carlsbad, CA) Argon/Krypton 100 mW air-cooled laser
for 488, 568, and 647 nm excitations. Confocal z-stacks were
acquired in all experiments. Co-staining was conducted by
treating cells exposed to 1 to either Syto 60 (nucleus), Lyso-
Tracker Red DND-99 (lysosomes), BODIPY TR glibenclamide
(endoplasmic reticulum), or MitoTracker Red 580 (mitocho-
ndria) for 20 min and washing the cells three times with media
and collecting images in two colors.
Acknowledgment. Financial support from the American
Cancer Society (RSG-06-011-01-CDD) is gratefully ack-
nowledged. We thank Dr. Yongxuan Su (UC San Diego)
for mass spectrometric analyses, and Drs. Anthony Mrse
and Xuemei Huang (UC San Diego) for assistance with
acquiring the NMR data.
1
Supporting Information Available: H, 13C, DEPT, gCOSY,
X-ray Crystallography. A yellow needle of compound 1
0.25 ꢀ 0.10 ꢀ 0.10 mm3 in size was mounted on a Cryoloop
with Paratone oil. Data were collected in a nitrogen gas stream
at 100(2) K using ψ and ω scans. Crystal-to-detector distance
was 50 mm and exposure time was 10 s per frame with a scan
width of 0.5°. Data collection was 99.3% complete to 67.00° in θ.
A total of 7195 reflections were collected covering the indices,
-8e h e 8, -15e k e 14, -13e l e 13. A total of 3065
TOCSY, NOESY, ROESY, HSQC, and HMBC spectra on
spirohexenolide A (1), 1H, 13C, DEPT, gCOSY, TOCSY,
NOESY, ROESY, HSQC, and HMBC spectra on spirohexe-
1
nolide B (2), H, 13C spectra of 3a and 3b, copies of crystal-
lographic information files (CIF) for compounds 1 and 2, as well
as copies of the NCI data on spirohexenolide A (1). This
material is available free of charge via the Internet at http://
pubs.acs.org.
J. Org. Chem. Vol. 74, No. 23, 2009 9061