Chlorinated englerins with selective inhibition of renal cancer cell growth

Article abstract of DOI:10.1021/np200905u

The chlorinated englerins (3-9) were isolated from Phyllanthus engleri and shown to selectively inhibit the growth of renal cancer cells. The compounds were shown to be extraction artifacts produced by exposure to chloroform decomposition products during

Full text of DOI:10.1021/np200905u

Article
pubs.acs.org/jnp
Chlorinated Englerins with Selective Inhibition of Renal Cancer Cell
Growth
Rhone K. Akee,† Tanya Ransom,‡ Ranjala Ratnayake,‡,§ James B. McMahon,‡ and John A. Beutler*,‡
†Natural Products Support Group, SAIC-Frederick National Cancer Institute, Frederick, Maryland 21702, United States
‡Molecular Targets Laboratory, Molecular Discovery Program, Center for Cancer Research, National Cancer Institute, Frederick,
Maryland 21702, United States
S
* Supporting Information
ABSTRACT: The chlorinated englerins (3−9) were isolated from Phyllanthus engleri and shown to selectively inhibit the
growth of renal cancer cells. The compounds were shown to be extraction artifacts produced by exposure to chloroform
decomposition products during their isolation. The most active compound, 3, was synthesized from englerin A (1).
RESULTS AND DISCUSSION
ur group recently reported the isolation of two new
■
Osesquiterpenes, englerins A (1) and B (2), from the root
An organic extract of stem bark was separated using batch
elution from a diol-bonded phase medium to give five fractions
of increasing polarity. The methylene chloride-soluble fraction
was separated via flash chromatography over silica gel, using
mixtures of chloroform and methanol, to yield three fractions,
which potently inhibited the growth of UO-31 renal cancer
cells, but were inactive against SF-295 CNS cancer cells. It was
determined that it was at this stage when the natural
compounds were modified by chloroform that had apparently
spontaneously generated Cl2 on standing, since later attempts
to purify the same fractions with newly purchased chloroform
yielded only 1 and 2.
The modified fractions still possessed the selectivity of the
parent extract toward renal cancer cells and were therefore
subjected to HPLC to yield a total of seven compounds, 3−9.
The structures were elucidated by spectroscopic techniques in
comparison to data obtained for 1.
bark and stem bark of Phyllanthus engleri Pax (Euphorbiaceae).1
Englerin A displayed remarkable potency and selectivity in its
inhibition of renal cancer cell line growth. Consequently,
englerin A (1) has been under intensive preclinical inves-
tigation at the National Cancer Institute, and several groups
have published the synthesis of englerin A2−7 and analogues.8,9
At an early stage of this project, seven related bioactive
compounds (3−9) were isolated, with these considered to be
artifacts produced during the isolation procedure. Reported
here are their structures and biological activity and the synthesis
from 1 of the most active compound.
HRMS established the molecular formula of 3 as
1
C26H33O6Cl. The H and 13C NMR data (Tables 1 and 2)
differed only slightly from 1, with the only differences being
replacement of the C-2′, C-3′ vinyl doublet with a 1H singlet at
7.94 ppm and alterations of the corresponding shifts for the
three carbons of the cinnamate. This argued for substitution of
chlorine at the C-2′ position, which was confirmed by HMBC
correlation of H-3′ with C-4′, C-5′, and C-6′. The Z
configuration of the double bond was established by analogy
with the chemical shift of H-3′ being δ 7.91 for Z-ethyl α-
chlorocinnamate,10 while for 3 it was δ 7.94. In addition, ACD
Special Issue: Special Issue in Honor of Gordon M. Cragg
Received: November 14, 2011
Published: January 26, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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Journal of Natural Products
Article
Table 1. 13C NMR Data for 3−9 (125 MHz, d4-methanol)
carbon no.
3
4
5
6
7
8
9
1
48.9
25.5
48.9
25.3
48.9
25.2
48. 6
25.5
49.0
25.4
48.8
25.2
48.8
25.3
2
3
32.0
31.7
31.9
31.9
32.0
31.7
31.9
4
32.4
31.8
31.9
32.3
32.4
31.8
31.3
5
47.9
47.4
47.5
47.8
47.9
47.5
47.6
6
74.8
74.3
74.3
73.9
73.9
73.9
73.8
7
86.5
86.2
86.0
86. 5
40.3
86.5
86.3
86.2
8
40.8
40.1
39.9
40.2
40.2
39.9
9
76.6
76.4
76.4
76.5
76.6
76. 5
86.0
76.5
10
11
12
13
14
15
1′
2′
3′
4′
5′
6′
7′
8′
9′
1″
2″
86.2
85.9
85.1
86.1
86.0
85.9
17.2
17.0
16.8
17.2
17.2
17.1
18.2
34.1
31.9
31.2
32.7
32.2
32.4
32.2
17.7
17.5
17.3
17.5
17.5
17.5
17.0
18.6
18.2
18.1
18.4
18.3
18.3
17.3
19.2
19.1
18.6
19.2
19.2
19.1
19.1
163.5
122.9
138.9
134.2
131.8
129.7
131.6
129.7
131.8
174.0
61.1
167.2
62.9
167.1
62.8
169.1
61.4
169.1
60.9
168.3
63.5
168.2
63.5
64.7
64.6
76.5
76.3
76.1
76.0
138.7
129.3
130.0
130.6
130.0
129.3
173.9
61.0
138.6
129.3
130.0
130.7
130.0
129.3
174.0
61.0
141.7
128.4
129.3
129.4
129.3
128.4
174.0
61.0
141.6
128.3
128.5
129.4
128.5
128.3
174.0
61.0
141.1
128.7
129.6
129.7
129.6
128.7
174.0
61.0
141.0
128.7
129.6
129.8
129.6
128.7
174.0
61.0
1
Table 2. H NMR Data for 3−9 (500 MHz, d4-methanol, δH (m, J in Hz))
position
3
4
5
6
7
8
9
1
1.76 (m)
1.75 (m)
1.34 (m)
2.02 (m)
1.28 (m)
2.14 (m)
1.75 (m)
1.58 (m)
1.63 (m)
1.20 (m)
1.77 (m)
1.09 (m)
1.34 (m)
1.34 (m)
1.61 (m)
1.66 (m)
1.25 (m)
1.85 (m)
1.16 (m)
1.63 (m)
1.39 (m)
1.72 (m)
1.72 (m)
1.28 (m)
1.98 (m)
1.26 (m)
2.19 (m)
1.55 (m)
1.74 (m)
1.74 (m)
1.33 (m)
2.00 (m)
1.29 (m)
2.23 (m)
1.64 (m)
1.61 (m)
1.18 (m)
1.60 (m)
1.76 (m)
1.05 (m)
1.32 (m)
1.25 (m)
1.62 (m)
2a
2b
3a
3b
4
1.67 (m)
1.21 (m)
1.86 (m)
1.18 (m)
1.27 (m)
1.47 (m)
4.81 (d, 9.5)
5
6
5.14 (d, 9.5)
4.80 (d, 9.5)
4.79 (d, 10.0)
5.02 (d, 10.0)
5.07 (d, 10.0)
4.81 (d, 7.0)
8a
8b
9
2.73 (dd, 14.5, 8.0) 2.44 (dd, 14.5, 8.0) 2.43 (dd, 14.5, 8.0) 2.45 (dd, 15.0, 8.0) 2.57 (dd, 15.0, 8.0) 2.42 (dd, 15.0, 8.0) 2.40 (dd, 14.5, 8.0)
1.89 (dd, 14.5, 3.0) 1.80 (dd, 14.5, 3.0) 1.82 (dd, 14.5, 3.0) 1.81 (dd, 15.0, 3.0) 1.90 (dd, 15.0, 3.0) 1.81 (dd, 15.0, 3.0) 1.75 (dd, 14.5, 3.0)
5.27 (dd, 8.0, 3.0) 5.12 (dd, 8.0, 3.0)
5.15 (dd, 8.0, 3.0)
0.72 (d, 7.0)
1.27 (m)
5.18 (dd, 8.0, 3.0) 5.23 (dd, 8.0, 3.0) 5.11 (dd, 8.0, 3.0) 5.15 (dd, 8.0, 3.0)
11
12
13
14
15
2′
0.93 (d, 7.0)
1.87 (m)
0.56 (d, 6.5)
1.46 (m)
0.89 (d, 7.0)
1.87 (m)
0.93 (d, 7.0)
1.88 (m)
0.62 (d, 7.0)
1.66 (m)
0.64 (d, 7.0)
1.82 (m)
0.97 (d, 7.0)
1.02 (d, 6.5)
1.19 9 (s)
0.84 (d, 7.0)
0.89 (d, 7.0)
1.10 (s)
0.64 (d, 7.0)
0.80 (d, 6.5)
1.11 (s)
0.95 (d, 7.5)
0.98 (d, 7.0)
1.16 (s)
0.99 (d, 6.5)
1.01 (d, 6.5)
1.19 (s)
0.89 (d, 7.0)
0.92 (d, 6.5)
1.11 (s)
0.77 (d, 6.5)
0.78 (d, 7.0)
1.11 (s)
5.02 (d, 9.5)
5.31 (d, 9.5)
4.98 (d, 10.0)
5.27 (d, 10.0)
4.40 (d, 9.0)
4.88 (d, 9.0)
4.36 (d, 9.0)
4.90 (d, 9.0)
4.58 (d, 8.5)
4.93 (d, 8.5)
4.54 (d, 9.0)
4.92 (d, 9.0)
3′
7.94 (s)
5′
7.87 (brd, 7.5)
7.45 (m)
7.47 (brdd, 8.0, 3.0) 7.48 (brdd, 7.5, 2.0) 7.43 (d, 7.0)
7.44 (dd, 8.0, 2.0) 7.39 (dd, 8.0, 2.0) 7.40 (dd, 8.0, 1.5)
7.36 (dd, 8.0, 7.0) 7.36 (dd, 8.0, 7.0) 7.36 (dd, 8.0, 7.0) 7.35 (m)
7.31 (brd, 8.0) 7.33 (d, 7.0) 7.31 (d, 7.0) 7.35 (m)
7.36 (dd, 8.0, 7.0) 7.36 (dd, 8.0, 7.0) 7.36 (dd, 8.0, 7.0) 7.35 (m)
7.44 (dd, 8.0, 2.0) 7.39 (dd, 8.0, 2.0) 7.40 (dd, 8.0, 1.5)
4.13 (s) 4.12 (s) 4.11 (s)
6′
7.37 (m)
7.37 (m)
7.37 (m)
7.37
7.37
7.37
7′
7.45 (m)
8′
7.45 (m)
9′
7.87 (brd, 7.5)
4.15 (s)
7.47 (brdd, 8.0, 3.0) 7.48 (brdd, 7.5, 2.0) 7.43 (d, 7.0)
4.12 (s) 4.12 (s) 4.13 (s)
2″
predictions of shifts for this proton were δ 7.13 (E) and 7.85
(Z), respectively. The UV maximum at 284 nm was consistent
with an intact cinnamate moiety, showing only a small shift
from the 279 nm maximum of 1. Thus, 3 was assigned as [Z]-
2′-chloroenglerin A.
Compounds 4 and 5 gave identical molecular formulas of
C26H34O6Cl2, as determined by HRMS. The isotope
abundances for the M + H + 1, M + H + 2, and M + H + 3
ions were indicative of a formula with two chlorine atoms. As
1
with 3, the core sesquiterpene signals in the H and 13C NMR
spectra were very similar to those of 1, with the only
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substantive differences at C-1′, C-2′, and C-3′, where the 13C
NMR signals for C-2′ and C-3′ were changed to δ 62.9 and 64.7
for 4 and δ 62.8 and 64.6 for 5, respectively, with a pair of
Following dichlorination, dehydrohalogenation with Et3N
yielded 3 in modest yield.10 While it is not possible to specify
the conditions that inadvertently led to dehydrohalogenation, it
is likely that molecular chlorine was generated in stored CHCl3
and reacted with the biosynthetic product englerin A (1) to
form the dichloro products 4 and 5, which then yielded 3
(Scheme 1). The generation of molecular chlorine by photo-
oxidation has long been recognized due to chloroform’s
importance in 19th century anesthesia, with phosgene and
hydrochloric acid among the other major products gener-
ated.11−13
1
corresponding 1H H NMR doublets at δ 5.02 and 5.31 for 4
and δ 4.98 and 5.27 for 5 also observed, each with ca. 10 Hz
couplings. This suggested compounds 4 and 5 to be a pair of
epimeric dichloro derivatives, namely, 2′,3′-dichlorodihydroen-
glerin A. The absolute configuration of the C-2′ and C-3′
centers was not determined for either of these compounds.
Compounds 6−9 were found to all share the same molecular
formula, C26H35O7Cl, based on HRMS measurements. The
chemical shifts of the carbon signals for C-3′ were observed
from ca. δ 64 to ca. δ 76 for these four compounds. This
information, coupled with the addition of water compared to 3,
indicated that these compounds are epimeric chlorohydrins. As
with the previous compound 3, HMBC correlations of H-3′
with carbons C-4′, C-5′, and C-6′ established that the hydroxy
group was at C-3′, thereby locating the chlorine at C-2′. The
absolute configurations of these epimers were not determined.
All of the compounds were tested in the NCI 60-cell assay,
and most showed some selectivity for renal cancer cell lines,
with 3 having the best potency and renal cancer selectivity,
being approximately 2.5-fold less active than 1. The mean GI50
values and individual GI50 values for eight renal cancer cell lines
are tabulated in Table 3. It is notable that the pairs of epimers 4
1
In support of this mechanism, new H NMR peaks were
observed in samples of 4 and 5 stored dry at −20 °C for
extended periods, which precisely matched those of 3,
indicating that spontaneous conversion of both 4 and 5 to 3
can occur. These new peaks were ca. 30% of the area of the
parent. A UV absorbance maximum at 284 nm was also
observed in the stored samples of 4 and 5, which may be
accounted for by the formation of 3. It is also possible that the
bioactivity observed for 4 and 5 may be due to in situ
production of 3 in cell culture. In contrast, the chlorohydrins
6−9, as well as 3, were stable under the storage conditions
used.
Our group is attempting to produce other 2′-halogenated
products via this route, since direct iodination or fluorination
could be used to synthesize isotopically labeled tracer
compounds that may be useful in understanding the
distribution and metabolism of englerin A (1) in animals and
humans.
Table 3. Selected NCI 60-Cell-Line Data for Compounds 3−
7
1,
n = 3
3,
n = 1
4,
5,
6,
7,
n = 1 n = 1 n = 1 n = 1
EXPERIMENTAL SECTION
mean GI50 (μM)
3.7
5.6
8.3
12
6.9
12
■
range at GI50 (log
units)
3.30
3.48
2.65
3.42
3.42
2.43
General Experimental Procedures. Solvents were of HPLC
grade, with chloroform stabilized with a hydrocarbon (amylene)
stabilizer. Optical rotations ([α]D) were obtained on a Perkin-Elmer
241 polarimeter in a 100 by 2 mm cell (units 10−1 deg cm2 g−1), while
ultraviolet (UV) absorption spectra were obtained using a Varian Cary
50 Bio UV−visible spectrophotometer. NMR experiments were
Individual Renal Cancer Cell Line GI50 Values (μM)
786−0
A498
1.1
10
12
12
1.7
11
0.011
0.017
16
0.028
0.041
0.39
0.059
0.56
21
0.14
0.14
0.42
0.067
0.049
0.16
0.18
0.32
0.46
0.51
0.63
17
0.66
1.4
5.0
0.39
4.7
27
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
1
performed on a Varian INOVA 500 MHz NMR spectrometer. H
and 13C NMR spectra were referenced to the deuterated solvent peaks.
High-resolution mass spectrometric measurements were acquired on
an Agilent 6520 Accurate Mass Q-TOF instrument with internal
reference masses at 121.05087 and 922.00979, both to within 5 ppm.
High-performance liquid chromatography (HPLC) was performed
using a Varian ProStar 210/215 solvent delivery module equipped
with a Varian ProStar 325 UV−vis detector, operating under Star 6.41
chromatography workstation software. HPLC-DAD-MS( ) analysis
was carried out on an Agilent 1100 series separation module binary
pump system with a vacuum degasser, thermostat control (40 °C)
column compartment, well-plate autosampler, diode array detector,
and quadrupole mass detector. Equipment was under the control of
Agilent ChemStation (Revision A.10.02) software. Standard HPLC-
DAD-MS gradient conditions were 0.5 mL/min gradient elution from
90% H2O (5% CH3CO2H)−MeCN to 100% MeCN over 20 min
a
a
0.011
1.1
n.t.
n.t.
0.93
18
1.0
25
1.9
0.015
0.035
0.39
0.16
0.25
1.9
a
Assay failure; no data for this cell line.
and 5, as well as 6 and 7, showed significant differences in
potency for many of the cell lines. Compounds 8 and 9 were
less potent and did not meet criteria in the one-dose NCI 60-
cell test for further testing in the full screen.
The identity and likely origin of 3 was confirmed by synthesis
from englerin A (1), using a known method, wherein molecular
chlorine is generated from HCl by treatment with oxone.
Scheme 1
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Journal of Natural Products
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followed by a 5 min flush with 100% MeCN, using a Phenomenex
Jupiter C18 150 × 2 mm, 5 μm column.
CH3OH step in the gradient to give 4.3 mg of 4. The middle peak was
rechromatographed on a 10 × 250 mm C8 Dynamax column, isocratic
at 80% aqueous MeCN for 25 min, to 100% MeCN at 30 min to give
4.8 mg of 5, eluting at 16 min. The earliest cluster of peaks in the
primary HPLC separation yielded 6 (3.6 mg, 10 min), 7 (3.5 mg, 12
min), 8 (3.8 mg, 8 min), and 9 (4.0 mg, 9 min).
Plant Material. Specimens of Phyllanthus engleri were collected on
February 6, 1989, by Roy Gereau and James Lovett of the Missouri
Botanical Garden and C. O. Kyalawa and Z. H. Mbwambo near
Ilembula, Njombe District, Iringa Region, Tanzania (longitude 8°50′ S,
latitude 34°31′ E), at an elevation of 1380 m. The plant (voucher
specimen Q66T-3055, http://www.tropicos.org/Specimen/149907)
was identified definitively by A. Radcliffe-Smith of the Royal Botanical
Garden, Kew, Richmond, UK. The specimen collected was a 3 m tall
tree of 15 cm dbh in a Brachystegia-Combretum woodland. Leaf (377 g
dry wt.), stem bark (387 g), stem wood (255 g), root bark (201 g),
and root wood (312 g) samples were collected separately and dried in
the field.
Extraction and Isolation. Dried plant material was ground and
extracted using the standard NCI extraction proptocol.14 The root
bark yielded 14.85 g of an organic solvent extract (N042029). A 2.61 g
portion of this material was dissolved in CH2Cl2−CH3OH (1:1) and
coated on 27 g of diol-bonded phase media using a rotary evaporator.
The medium was resuspended in hexane and evaporated to remove all
of the initial solvent to produce a dry flowable powder, which was
packed in a Buchner funnel over an equal amount of uncoated diol
medium, then eluted batchwise with 150 mL of hexane, CH2Cl2,
EtOAc, acetone, and CH3OH in succession. Evaporation gave 612 mg
of a CH2Cl2 fraction.
A subsample of 515 mg of the CH2Cl2 fraction was chromato-
graphed on a 5 × 14 cm flash column of silica gel. The sample was
dissolved in CHCl3 and eluted with increasing amounts of CH3OH in
CHCl3 [CHCl3, CHCl3−CH3OH (4:1, v/v), CHCl3−CH3OH (1:1),
CH3OH]. Fractions were combined on the basis of TLC, and fraction
E (194 mg) was found to have excellent selective activity in the two-
cell assay (IC50 0.18 μg/mL in UO-31, 93 μg/mL in SF-295). Adjacent
fractions had lesser activity (D: 187 mg, IC50 0.91 μg/mL in UO-31,
>100 μg/mL in SF-295; F: 57 mg, IC50 14 μg/mL in UO-31, >100 μg/
mL in SF-295). Recovery of biological activity was judged acceptable
within the limits of assay precision. To guard against loss of activity in
the next step, C18 HPLC, an experiment was conducted wherein 10
mg of the active fraction from a parallel separation fraction from the
stem bark was applied to a prewashed Bondesil C18 SPE cartridge and
eluted with CH3OH, 50% CH3OH−THF, and then THF. The
fractions were tested in the tracking assay, and the methanol eluate was
found active against UO-31 cells (IC50 0.03 μg/mL), but not against
SF-295 cells (IC50 >100 μg/mL), while the THF eluates were entirely
inactive against either cell line. The parent fraction, tested in tandem,
was also extremely active against UO-31 but not SF-295 (IC50's 0.04
μg/mL, >100 μg/mL, respectively). This established that the bioactive
compounds could be eluted from a C18 column and that the conditions
of separation were unlikely to alter the active compounds in such a
way that activity would be destroyed.
[Z]-2′-Chloroenglerin A (3). NSC#746567: white solid; [α]D −60
1
(c 0.1, EtOH); UV (EtOH) λmax (log ε) 284 (4.14) nm; H and 13C
NMR data in d4-MeOH, see Tables 1 and 2; in CDCl3, see Supporting
Information; ESIMS m/z 477 [M + H]+ 477 (100%), 478 (27%), 479
(35%), 480 (9%); HRESIMS m/z 477.2054 (calcd for C26H34ClO6,
477.2038).
2′,3′-Dichlorodihydroenglerin A (epimer 1) (4). NSC#746565:
white solid; 1H and 13C NMR data in d4-MeOH, see Tables 1 and 2; in
d6-DMSO, see Supporting Information; ESIMS m/z 513 [M + H]+
513 (100%), 514 (29%), 515 (65%), 516 (19%); HRESIMS m/z
513.1771 (calcd for C26H35Cl2O6, 513.1805).
2′,3′-Dichlorodihydroenglerin A (epimer 2) (5). NSC#746566:
white solid; 1H and 13C NMR data in d4-MeOH, see Tables 1 and 2; in
d6-DMSO, see Supporting Information; ESIMS m/z 513 [M + H]+
513 (100%), 514 (33%), 515 (80%), 516 (15%); HRESIMS m/z
513.1814 (calcd for C26H35Cl2O6, 513.1805).
2′-Chloro-3′-hydroxydihydroenglerin A (epimer 1) (6).
NSC#746563: white solid; [α]D −47 (c 0.3, EtOH); 1H and 13C
NMR data in d4-MeOH, see Tables 1 and 2; in d6-DMSO, see
Supporting Information; ESIMS m/z 495 [M + H]+ 495 (100%), 496
(31%), 497 (35%), 498 (11%); HRESIMS m/z 495.2138 (calcd for
C26H35ClO7, 495.2144).
2′-Chloro-3′-hydroxydihydroenglerin A (epimer 2) (7).
NSC#746564: white solid; [α]D −10 (c 0.3, EtOH); 1H and 13C
NMR data in d4-MeOH, see Tables 1 and 2; in d6-DMSO, see
Supporting Information; ESIMS m/z 495 [M + H]+ 495 (100%), 496
(30%), 497 (35%), 498 (10%); HRESIMS m/z 495.2134 (calcd for
C26H35ClO7 495.2144).
2′-Chloro-3′-hydroxydihydroenglerin A (epimer 3) (8).
NSC#746858: white solid; [α]D −37 (c 0.3, EtOH); 1H and 13C
NMR data in d4-MeOH, see Tables 1 and 2; ESIMS m/z 495 [M +
H]+ 495 (100%), 496 (26%), 497 (36%), 498 (10%); HRESIMS m/z
495.2149 (calcd for C26H35ClO7, 495.2144).
2′-Chloro-3′-hydroxydihydroenglerin A (epimer 4) (9).
NSC#746859: white solid; [α]D −28 (c 0.4, EtOH); 1H and 13C
NMR data in d4-MeOH, see Tables 1 and 2; ESIMS m/z 495 [M +
H]+ 495 (100%), 496 (25%), 497 (34%), 498 (8%); HRESIMS m/z
495.2112 (calcd for C26H35ClO7, 495.2144).
Synthesis of (Z)-2′-Chloroenglerin A (3). To a mixture of
englerin A (1) (26 mg, 58 μmol) and oxone (178 mg, 292 μmol) in
CH2Cl2 (0.5 mL) was added 2 N aqueous HCl (64 μL, 128 μmol of
HCl(aq)), in a single portion at room temperature. After stirring for 2
h at rt, Et3N (49 μL, 350 μmol) was added, and the solution was left
on the magnetic stir pad overnight, ∼12 h. The reaction mixture was
partitioned against 10 mL of CH2Cl2 and 10 mL of water, and the
aqueous layer was washed two more times with 10 mL of CH2Cl2. The
CH2Cl2 layers were combined and partitioned against a concentrated
brine solution. The organic layer was dried over anhydrous Na2SO4,
and the supernatant was dried under vacuum. The crude product was
passed through a silica gel column to give a fraction containing a
mixture of Z and E isomers of 2′-chloroenglerin A as a clear oil (0.14 g,
40% yield, Z:E ratio of 9:1 by HPLC/ELSD). Purification of (Z)-2′-
chloroenglerin A by reversed-phase C18 preparative HPLC, using
methanol−water, gave 2 mg of the Z isomer 3 as well as several
products with molecular mass corresponding to the chlorohydrins 6−
9.
The active fraction E was then chromatographed by HPLC using a
Varian Microsorb 60−8 C18 column (250 × 21.4 mm) using a
CH3OH−H2O gradient starting at 75% CH3OH for 5 min, linearly to
85% at 32 min, to 100% CH3OH at 36 min, held, and returned to
original conditions at 40 min, with a flow rate of 32 mL/min.
Injections were made with 40 mg of sample in 400 μL of DMSO per
injection. Detection was at 225 nm. Consistent separations were
obtained, with three groups of peaks separated (see Supporting
Information). The first, earliest eluting peaks appeared at 7−12 min,
while a larger series of more abundant peaks eluted from 20 to 30 min,
and the third group eluted after 35 min. This late group of peaks
1
showed no cytotoxic activity and by H NMR spectroscopy appeared
to be composed of phytosterols, consistent with the report of
phyllanthol from the plant many years ago.15 The peaks in the two
(Z)-2′-Chloroenglerin A (3): 1H NMR (d4-MeOH) δ 7.96 (1H, s,
H-3′), 7.89 (2H, m, H-5′, H-9′), 7.46 (3H, m, H-6′, H-7′, H-8′), 5.29
(1H, dd, J = 3.0, 8.0 Hz, H-9), 5.15 (1H, d, J = 9.3 Hz, H-6), 4.16 (2H,
s, H-2″), 2.75 (1H, dd, J = 8.0, 14.6 Hz, H-8a), 2.15 (1H, m, H-4), 2.03
(1H, m, H-3a), 1.90 (1H, m, H-8b), 1.88 (1H, m, H-12), 1.76 (3H, m,
H-1, H-2a, H-5), 1.32 (2H, m, H-2b, H-3b), 1.20 (3H, s, H-15), 1.03
(3H, d, J = 6.8 Hz, H-14), 0.98 (3H, d, J = 7.1 Hz, H-13), 0.94 (3H, d,
J = 7.1 Hz, H-11); 13C NMR (d4-MeOH) δ 174.0 (C-1″), 163.5 (C-
1
earlier eluting clusters were subjected to HPLC-MS and H NMR
spectroscopic analysis and appeared to be a related series of C26
compounds.
Eluting at 27 min was compound 3 (8.9 mg), well separated from
other peaks (chromatogram in Supporting Information). At 25 min,
the first of a poorly resolved set of three compounds was further
purified by rechromatography in the same solvent without the 100%
462
dx.doi.org/10.1021/np200905u | J. Nat. Prod. 2012, 75, 459−463

Journal of Natural Products
Article
1′), 138.9 (C-3′), 134.2 (C-4′), 131.8 (C-9′), 131.8 (C-5′), 131.6 (C-
7′), 129.7 (C-8′), 129.7 (C-6′), 122.8 (C-2′), 86.5 (C-7), 86.2 (C-10),
76.6 (C-9), 74.7 (C-6), 61.0 (C-2″), 49.0 (C-1), 47.8 (C-5) 40.8 (C-
8), 34.1 (C-12), 32.4 (C-4), 32.0 (C-3), 25.5 (C-2), 19.2 (C-15), 18.6
(C-13), 17.7 (C-14), 17.2 (C-11).
Biological Assays. Fractionation was guided by a 48 h cell growth
assay using either XTT or SRB end points with the UO-31 or A498
renal cancer cell lines. NCI 60-cell tests were conducted as described
previously.16
(7) Zhou, Q.; Chen, X.; Ma, D. Angew. Chem., Int. Ed. 2010, 49,
3513−3515.
(8) Chan, K. P.; Chen, D. Y. ChemMedChem. 2011, 6, 420−423.
(9) Radtke, L.; Willot, M.; Sun, H.; Ziegler, S.; Sauerland, S.;
Strohmann, C.; Frohlich, R.; Habenberger, P.; Waldmann, H.;
Christmann, M. Angew. Chem., Int. Ed. 2011, 50, 1−6.
(10) Kim, K.-M.; Park, I.-H. Synthesis 2004, 2641−2644.
(11) Morson, T. N. R. Pharm. J. 1848, 8, 69.
(12) Baskerville, C.; Hamor, W. A. J. Ind. Eng. Chem. 1912, 4, 278−
288.
(13) Chapman, A. T. J. Am. Chem. Soc. 1935, 57, 419−422.
(14) McCloud, T. G. Molecules 2010, 15, 4526−4563.
(15) Barton, D. H. R.; Page, J. E.; Warnhoff, E. W. J. Chem. Soc. 1954,
2715−2719.
(16) Shoemaker, R. H. Nat. Rev. Cancer 2006, 6, 813-823. See also
http://dtp.nci.nih.gov/branches/btb/ivclsp.html.
ASSOCIATED CONTENT
■
S
* Supporting Information
Full NMR data for 3−9, NCI 60-cell testing data for 3−9, and
an HPLC chromatogram. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: beutlerj@mail.nih.gov.
Present Address
§Department of Medicinal Chemistry, College of Pharmacy,
University of Florida, Gainesville, Florida 32610, United States.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This project has been funded in whole or in part with federal
funds from the National Cancer Institute, National Institutes of
Health, under Contract No. HHSN261200800001E. This
research was supported, in part, both by the Intramural
Research Program of the NIH, National Cancer Institute,
Center for Cancer Research, and, in part, by the Developmental
Therapeutics Program in the Division of Cancer Treatment and
Diagnosis of the National Cancer Institute. The content of this
publication does not necessarily reflect the views or policies of
the Department of Health and Human Services, nor does
mention of trade names, commercial products, or organizations
imply endorsement by the U.S. Government. We gratefully
acknowledge J. Schneider and P. Grothaus for helpful
comments, and H. Bokesch (MTL), S. Tarasov, and M. Dyba
(Biophysics Resource Core, Structural Biophysics Laboratory,
CCR) for acquiring high-resolution mass spectra.
DEDICATION
■
Dedicated to Dr. Gordon M. Cragg, formerly Chief, Natural
Products Branch, National Cancer Institute, Frederick, Mary-
land, for his pioneering work on the development of natural
product anticancer agents.
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