Anacolosins A-F and Corymbulosins X and Y, Clerodane Diterpenes from Anacolosa clarkii Exhibiting Cytotoxicity toward Pediatric Cancer Cell Lines

Article abstract of DOI:10.1021/acs.jnatprod.8b01015

An extract of the plant Anacolosa clarkii was obtained from the NCI Natural Products Repository, and it showed cytotoxic activity toward several types of pediatric solid tumor cell lines. Bioassay-guided fractionation led to the purification of eight new clerodane diterpenes [anacolosins A-F (1-6) and corymbulosins X and Y (7 and 8)] and two known compounds (9 and 10) that contained an isozuelanin skeleton. The structures of the new natural products were determined using 1D and 2D NMR and HRESIMS data, while the relative and absolute configurations of the compounds were assessed using a combination of ¹H NMR coupling constant data, ROESY experiments, ECD (electronic circular dichroism) and VCD (vibrational circular dichroism) spectroscopy, chemical methods (including Mosher and 2-naphthacyl esterification), and chiral HPLC analyses. The purified natural products exhibited a range of cytotoxic activities against cell lines representing four pediatric cancer types (i.e., rhabdomyosarcoma, Ewing sarcoma, medulloblastoma, and hepatoblastoma) with total growth inhibitory (TGI) values in the range 0.2-4.1 μM. The rhabdomyosarcoma and medulloblastoma cell lines showed higher sensitivity to compounds 1-4, which are the first compounds reported to contain an isozuelanin skeleton and feature keto carbonyl groups at the C-6 positions. In contrast, the hepatoblastoma cell line was modestly more sensitive to 7-10, which contained a C-6 hydroxy group moiety.

Full text of DOI:10.1021/acs.jnatprod.8b01015

Article
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Anacolosins A−F and Corymbulosins X and Y, Clerodane Diterpenes
from Anacolosa clarkii Exhibiting Cytotoxicity toward Pediatric
Cancer Cell Lines
Shengxin Cai,†,‡ April L. Risinger,§,⊥ Cora L. Petersen,§ Tanja Grkovic,∥ Barry R. O’Keefe,#,¶
Susan L. Mooberry,*,§,⊥ and Robert H. Cichewicz*,†,‡
‡
†Department of Chemistry and Biochemistry, Stephenson Life Sciences Research Center, and Natural Products Discovery Group
and Institute for Natural Products Applications and Research Technologies, University of Oklahoma, Norman, Oklahoma 73019,
United States
⊥
§Department of Pharmacology and Mays Cancer Center, University of Texas Health Science Center, San Antonio, Texas 78229,
United States
∥Natural Products Support Group, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick,
Maryland 21702, United States
#Natural Products Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer
Institute, Frederick, Maryland 21702, United States
¶Molecular Targets Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, United States
S
* Supporting Information
ABSTRACT: An extract of the plant Anacolosa clarkii was obtained from the NCI Natural Products Repository, and it showed
cytotoxic activity toward several types of pediatric solid tumor cell lines. Bioassay-guided fractionation led to the purification of
eight new clerodane diterpenes [anacolosins A−F (1−6) and corymbulosins X and Y (7 and 8)] and two known compounds (9
and 10) that contained an isozuelanin skeleton. The structures of the new natural products were determined using 1D and 2D
NMR and HRESIMS data, while the relative and absolute configurations of the compounds were assessed using a combination
1
of H NMR coupling constant data, ROESY experiments, ECD (electronic circular dichroism) and VCD (vibrational circular
dichroism) spectroscopy, chemical methods (including Mosher and 2-naphthacyl esterification), and chiral HPLC analyses. The
purified natural products exhibited a range of cytotoxic activities against cell lines representing four pediatric cancer types (i.e.,
rhabdomyosarcoma, Ewing sarcoma, medulloblastoma, and hepatoblastoma) with total growth inhibitory (TGI) values in the
range 0.2−4.1 μM. The rhabdomyosarcoma and medulloblastoma cell lines showed higher sensitivity to compounds 1−4,
which are the first compounds reported to contain an isozuelanin skeleton and feature keto carbonyl groups at the C-6
positions. In contrast, the hepatoblastoma cell line was modestly more sensitive to 7−10, which contained a C-6 hydroxy group
moiety.
that enable the separation of childhood cancers into distinct
groups and subtypes. These results also demonstrate that many
of the molecular drivers found in childhood cancers are
espite numerous clinical advances, cancer remains a
Dsignificant cause of death among children and
adolescents across the world.1 In the United States alone,
>10,000 cases of cancer are reported in children each year.2
While childhood solid cancers are often grouped based solely
on patient age, studies reveal a growing list of genomic features
Received: December 1, 2018
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.8b01015
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different than those observed in adult cancers. While high
mutational burdens define adult solid cancers, few mutations
are found in childhood cancers, highlighting the significant
differences between pediatric and adult cancers.2,3 The
implication of these findings is that childhood cancers can be
targeted pharmacologically based on their unique genomic and
molecular features. A key consequence of the improved
molecular characterization of pediatric cancers is the
identification of molecular targets and cognate inhibitors that
can be used to selectively affect the often unique defects that
drive childhood solid cancers. Therefore, efforts invested in
identifying compounds that selectively target specific child-
hood cancer types offer new opportunities to develop drugs
that are potentially more effective at treating younger cancer
patients.
Chart 1
Acting on this premise, our consortium of investigators from
the University of Oklahoma, the University of Texas Health
Science Center at San Antonio, and the Natural Products
Branch of NCI has investigated plant and fungal natural
products as a resource for compounds that exhibit selective
activity profiles against pediatric cancer cell lines. As part of
this drug discovery process, we have identified 10 compounds
from the plant Anacolosa clarkii Pierre (Olacaceae) that display
cytotoxic activities against pediatric solid tumor cell lines. An
investigation of the literature showed that the genus Anacolosa
is a relatively unexplored group of plants, with only two
published accounts appearing in SciFinder that reported
secondary metabolites from this species.4,5 This report
provides details of the metabolites obtained from A. clarkii
and describes their differential activities against pediatric
cancer cell lines representing four distinct pediatric solid
tumor types.
unsaturation that were confirmed in 1 (two acetoxy groups,
a propionate group, and three double bonds), as well as 13C
NMR-derived evidence for the presence of a keto carbonyl (δC
207.9, C-6), it was concluded that the remaining three units of
unsaturation could be accounted for as three rings. Bearing this
in mind, an analysis of the 1H−1H COSY and HMBC
correlation data (Figure 1) led to the proposal that 1 featured a
tricyclic clerodane diterpene core.6−12 More specifically,
compound 1 belongs to a subgroup of clerodane diterpenes
that possess an isozuelanin skeleton.10 However, in contrast to
the reported isozuelanin-containing natural products that bear
a methylene, a hydroxy, or an alkoxy group at C-6, metabolite
1 is the first member of this class to feature a C-6 keto carbonyl
group.10
RESULTS AND DISCUSSION
■
Examination of the LC-PDA-MS data collected for the A.
clarkii extract revealed that the sample contained an abundance
of metabolites that exhibited λmax values occurring near 220 nm
and molecular weights averaging ∼500 Da. Bioassay-guided
fractionation using a combination of vacuum-liquid-chroma-
tography (HP20ss), preparative HPLC (C18), and semi-
preparative HPLC (C18 and pentafluorophenyl) led to the
purification of metabolites 1−10 (Chart 1).
The relative configuration of 1 was determined based on a
1
combination of H−1H ROESY correlations (Figure 1) and
coupling constants between key protons (Table 1). We
prioritized establishing the relative configuration of ring B
(Figure 1) because ascertaining the relative configuration of
this portion of the compound was crucial to mapping the
spatial relationships of all the protons throughout the
molecule. The two large J values measured for H-7ax (14.7
and 15.1 Hz) were interpreted as having arisen from a geminal
coupling with H-7eq, as well as a vicinal (trans-diaxial)
coupling with H-8. Additionally, a ROESY correlation from H-
7ax across ring B to H-11 indicated that both groups were on
the same face of the cyclohexanone system. Expanding on
these observations, a series of ROESY correlations was
detected that extended from H-8 to the H-20 methyl protons
(attached to ring B) and H-1ax (part of ring A). Proton H-1ax
was determined to be at a critical junction point in the
molecule since it presented diagnostic couplings with one
Anacolosin A (1) was purified as a colorless oil. Its
molecular formula was determined to be C27H36O8 based on
HRESIMS data (m/z 511.2304 [M + Na]+, calcd for
511.2302), which indicated 10 degrees of unsaturation.
Based on the HMBC correlations between the singlets at δH
1.84 and 2.05 (Table 1) and the carbonyl carbons at δC 168.6
and 170.0 (Table 2), the presence of two acetoxy groups was
proposed. The occurrence of a propionate group was
1
confirmed by the H−1H COSY correlations between H-2′
(δH 2.36, m) and H-3′ (δH 1.06, t, J = 7.5 Hz) and the HMBC
correlations from H-2′/3′ and H-2 (δH 5.31, m) to C-1′ (δC
173.0) (Figure 1). Beyond the aforementioned resonances, an
additional 20 carbon spins remained (Table 2), which led to an
initial conjecture that this metabolite might be a diterpenoid.
The carbon signals at δC 122.4 (C-3), 141.7 (C-4), 144.8 (C-
13), 140.1 (C-14), 112.8 (C-15), and 115.9 (C-16), as well as
the proton resonances at δH 6.05 (brd, J = 3.8 Hz, H-3), 6.45
(dd, J = 10.8, 17.6 Hz, H-14), 5.23 (d, J = 17.5 Hz, H-15a),
5.06 (brd, J = 10.7 Hz, H-15b), 5.11 (s, H-16a), and 5.05 (s,
H-16b), supported the presence of three double bonds in the
structure. Taking into consideration the six units of
2
3
geminal (H-1eq, J = 14.8 Hz) and two vicinal (H-10, Jtrans
=
3
15.1 and H-2, Jcis = 3.9 Hz) protons that were key to
establishing the relative configuration of ring A. Returning to
H-7ax, an additional ROESY correlation was identified with H-
19, and this in turn displayed a ROESY correlation with H-18,
which helped finalize the relative configuration of the ring C
system. The geometry of the conjugated diene was determined
B
DOI: 10.1021/acs.jnatprod.8b01015
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1
1
Figure 1. Selected H−1H COSY, HMBC, and H−1H ROESY correlations used to determine the structure of compound 1.
1
Table 1. H NMR Data for Compounds 1−8 (δ in ppm, J in Hz)
a
b
c
d
d
d
d
d
no.
1ax
1
2
3
4
5
6
7
8
1.42 ddd (3.9,
14.8, 15.1)
1.53 ddd (4.0,
14.8, 14.9)
1.54 ddd (4.0,
14.2, 14.3)
1.53 dd (3.9,
13.7, 14.5)
1.54 ddd (4.0,
14.7, 14.8)
1.51 ddd (3.9,
14.9, 14.9)
1.94 m
1.94 m
1eq
2
1.86 d (14.8)
5.31 m
2.04 brd (14.0) 2.05 brd (11.7) 2.06 brd (14.9) 2.06 brd (14.9) 2.04 m
2.03 m
5.42 m
2.03 m
5.46 m
5.37 m
5.36 brs
5.36 m
5.42 brs
5.35 m
3
6.05 brd (3.8)
6.12 brd (4.6)
6.14 brd (4.6)
6.13 brd (4.8) 6.15 brd (4.7)
6.11 brd (4.8)
6.02 brd (4.5)
6.02 brd (4.3)
6
3.82 dd (4.3,
12.1)
3.84 dd (4.3,
12.1)
7ax
7eq
3.18 dd (14.7,
15.1)
2.04 m
2.95 dd (14.0,
14.0)
2.19 m
2.96 dd (14.0,
13.7)
2.19 m
2.96 dd (14.1,
13.7)
2.20 m
2.97 dd (13.6,
14.1)
2.20 m
2.95 dd (13.7,
14.0)
2.19 m
1.72 ddd (12.9,
12.6, 12.6)
1.63 ddd (3.7,
4.0, 13.4)
1.73 ddd (12.8,
12.6, 12.6)
1.64 ddd (3.7,
3.9, 13.3)
8
10
2.06 m
2.40 m
2.15 m
2.59 dd (2.5,
13.7)
2.15 m
2.62 dd (2.3,
13.6)
2.17 m
2.63 dd (2.4,
13.6)
2.16 m
2.68 dd (2.4,
13.6)
2.15 m
2.62 dd (2.5,
13.7)
1.87 m
2.36 dd (3.6,
13.7)
1.86 m
2.41 dd (3.5,
13.6)
11a
11b
12
1.62 m
1.62 m
2.10 m
1.78 m
1.70 m
2.25 m
1.78 m
1.71 m
2.24 m
1.79 m
1.71 m
2.25 m
1.79 m
1.71 m
2.26 m
1.78 m
1.70 m
2.24 m
1.53 m
1.28 m
2.12 m
1.54 m
1.30 m
2.13 m
14
6.45 dd (10.8,
17.6)
6.50 dd (11.0,
17.6)
6.51 dd (11.0,
17.6)
6.51 dd (11.1,
17.8)
6.52 dd (11.1,
17.7)
6.50 dd (11.1,
17.7)
6.48 dd (10.9,
17.6)
6.48 dd (10.9,
17.6)
15a
5.23 d (17.5)
5.27 brd (17.4) 5.26 d (17.6)
5.24 dd (0.9,
17.5)
5.25 dd (1.0,
17.7)
5.24 dd (1.0,
17.6)
5.23 dd (1.1,
17.6)
5.22 dd (1.1,
17.6)
15b
16a
16b
17
5.06 d (10.7)
5.11 s
5.05 s
0.90 d (6.4)
6.45 brs
5.08 d (10.0)
5.07 brs
5.07 brs
1.02 d (6.2)
6.59 t (1.6)
5.08 d (10.2)
5.07 brs
5.07 brs
1.03 d (6.7)
6.59 brs
5.08 d (10.3)
5.08 brs
5.08 brs
1.03 d (6.7)
6.61 t (1.6)
5.08 brd (11.5) 5.06 brd (10.1) 5.05 brd (11.0)
5.05 brd (10.9)
5.03 brs
4.95 brs
0.95 d (6.8)
6.68 dd (1.7,
1.7)
5.08 brs
5.07 brs
5.02 brs
5.08 brs
5.07 brs
4.94 brs
1.04 d (6.7)
6.61 t (1.6)
1.03 d (6.7)
6.60 t (1.6)
0.95 d (6.8)
6.66 dd (1.6,
1.7)
18
19
20
OAc-18
(Me)
6.58 s
0.93 s
2.05 s
6.54 s
1.06 s
2.05 s
6.54 s
1.06 s
2.04 s
6.55 s
1.05 s
2.06 s
6.55 s
1.06 s
2.07 s
6.54 s
1.05 s
2.06 s
6.44 s
0.96 s
2.04 s
6.44 s
0.95 s
2.06 s
OAc-19
(Me)
1.84 s
1.90 s
1.90 s
1.90 s
1.91 s
1.90 s
1.84 s
1.85 s
2′
3′
2.36 m
1.06 t (7.5)
2.37 t (7.2)
1.70 m
2.26 m
2.11 m
2.46 m
2.62 m
1.22 d (7.0)
2.26 m
2.12 m
1.69 m; 1.58 m 6.91 qq (7.0,
1.5)
6.90 qq (1.5, 7.2)
4′
1.01 t (7.4)
1.03 d (6.7)
0.99 t (7.5)
1.85 dq (7.1,
1.2)
1.88 m
1.19 d (6.7)
1.03 d (6.7)
1.02 d (6.7)
1.84 dq (7.1,
1.2)
1.88 m
5′
1.02 d (6.7)
1.18 d (6.9)
a
b
Obtained on a 400 MHz Varian instrument in a 5 mm NMR tube; solvent was DMSO-d6. Obtained on a 600 MHz Varian instrument in a 5 mm
c
d
NMR tube; solvent was MeOH-d4. Obtained on a 600 MHz Varian instrument in a 3 mm NMR tube; solvent was MeOH-d4. Obtained on a 500
MHz Varian instrument in a 3 mm NMR tube; solvent was MeOH-d4.
based on the ROESY correlations between H-14 and H-16b, as
well as between H-12 and H-15a.
generated at the CAM-B3LYP/SVP level, and the results
compared to an experimentally derived spectrum, revealing a
strong match between both data sets. Similarly, a theoretical
VCD spectrum was prepared using optimized geometries of
the molecule obtained from a conformational search employ-
ing ComputeVOA (BioTool, Inc.) with MMFF and further
optimized at the density functional theory (DFT) level
With the relative configuration of 1 established, we
proceeded to evaluate the absolute configuration of the
molecule, which included comparing the experimental and
calculated (theoretical) ECD and VCD spectra for 1 (Figures 2
and 3, respectively). A theoretical ECD spectrum for 1 was
C
DOI: 10.1021/acs.jnatprod.8b01015
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Journal of Natural Products
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Table 2. 13C NMR Data for Compounds 1−8 (δ in ppm)
a
b
c
d
d
e
d
e
no.
1
2
3
4
5
6
7
8
1
2
3
4
26.7 CH2
65.2 CH
122.4 CH
141.7 C
28.1 CH2
67.3 CH
123.8 CH
143.4 C
28.0 CH2
67.4 CH
123.8 CH
143.4 C
28.0 CH2
67.3 CH
123.8 CH
143.5 C
28.2 CH2
67.3 CH
123.9 CH
143.4 C
27.6 CH2
67.0 CH
123.7 CH
143.1 C
27.7 CH2
68.2 CH
122.5 CH
147.6 C
27.7 CH2
67.9 CH
122.2 CH
147.3 C
5
65.1 C
67.0 C
66.9 C
67.0 C
67.1 C
66.7 C
55.1 C
55.4 C
6
7
8
9
207.9 C
209.0 C
209.0 C
209.0 C
209.0 C
208.9 C
73.4 CH
37.8 CH2
38.4 CH
38.6 C
73.0 CH
37.5 CH2
38.2 CH
38.4 C
43.2 CH2
38.8 CH
37.0 C
44.4 CH2
40.9 CH
38.8 C
44.4 CH2
40.9 CH
38.8 C
44.4 CH2
41.0 CH
38.8 C
44.4 CH2
41.0 CH
38.8 C
44.0 CH2
40.4 CH
37.9 C
10
11
12
13
14
15
16
17
18
19
20
38.4 CH
27.9 CH2
23.3 CH2
144.8 C
140.1 CH
112.8 CH2
115.9 CH2
15.7 CH3
94.0 CH
96.9 CH
24.3 CH3
170.0 C
21.0 CH3
168.6 C
21.0 CH3
173.0 C
27.3 CH2
9.2 CH3
40.4 CH
29.6 CH2
24.8 CH2
146.6 C
141.6 CH
112.9 CH2
116.1 CH2
16.3 CH3
96.0 CH
98.8 CH
25.2 CH3
171.7 C
20.9 CH3
170.8 C
21.5 CH3
174.5 C
37.2 CH2
19.8 CH2
14.0 CH3
40.4 CH
29.6 CH2
24.8 CH2
146.6 C
141.6 CH
112.9 CH2
116.1 CH2
16.2 CH3
96.0 CH
98.8 CH
25.1 CH3
171.6 C
20.9 CH3
170.7 C
21.5 CH3
173.9 C
44.5 CH2
27.4 CH
22.8 CH3
22.8 CH3
40.3 CH
29.5 CH2
24.8 CH2
146.5 C
141.6 CH
112.8 CH2
116.1 CH2
16.2 CH3
96.0 CH
98.8 CH
25.2 CH3
171.7 C
20.9 CH3
170.7 C
21.4 CH3
177.3 C
42.5 CH
28.2 CH2
12.2 CH3
17.1 CH3
40.5 CH
29.5 CH2
24.8 CH2
146.5 C
141.7 CH
112.8 CH2
116.1 CH2
16.2 CH3
95.9 CH
98.7 CH
25.3 CH3
171.7 C
20.9 CH3
170.7 C
21.4 CH3
168.5 C
129.9 C
138.9 CH
14.5 CH3
40.0 CH
29.3 CH2
24.4 CH2
146.5 C
141.4 CH
112.7 CH2
116.0 CH2
15.9 CH3
95.7 CH
98.7 CH
24.9 CH3
171.3 C
20.5 CH3
170.3 C
21.1 CH3
177.3 C
34.9 CH
19.3 CH3
18.6 CH3
37.9 CH
29.2 CH2
25.1 CH2
147.0 C
141.7 CH
112.8 CH2
115.7 CH2
16.1 CH3
97.1 CH
99.8 CH
25.8 CH3
172.0 C
21.1 CH3
171.5 C
21.9 CH3
174.1 C
44.6 CH2
27.4 CH
22.8 CH3
22.8 CH3
37.8 CH
29.0 CH2
24.8 CH2
146.3 C
141.4 CH
112.3 CH2
115.5 CH2
15.8 CH3
96.9 CH
99.3 CH
25.7 CH3
171.7 C
OAc-18 (CO)
OAc-18 (Me)
20.8 CH3
171.2 C
21.7 CH3
167.9 C
OAc-19 (CO)
OAc-19 (Me)
1′
2′
3′
4′
5′
129.5 C
138.5 CH
14.2 CH3
11.9 CH3
12.1 CH3
a
b
Obtained on a 100 MHz Varian instrument in 5 mm NMR tube; solvent was DMSO-d6. Obtained on a 100 MHz Varian instrument in a 5 mm
NMR tube; solvent was MeOH-d4. Obtained on a 150 MHz Varian instrument in a 3 mm NMR tube; solvent was MeOH-d4. Obtained on a 100
MHz Varian instrument in a 3 mm NMR tube; solvent was MeOH-d4 Extracted from HSQC and HMBC spectra which were obtained on a 500
c
d
e
MHz Varian instrument in a 3 mm NMR tube; solvent was MeOH-d4.
Figure 2. Calculated and experimental ECD curves for compounds 1 and 4.
(B3LYP/6-31+G(d,p)) with Gaussian software (a computa-
tional package for electronic structure modeling).13 The
calculated and experimentally derived VCD data revealed a
strong match between the two data sets obtained. Therefore,
the absolute configuration of the metabolite was established as
shown for compound 1.
however, some modest differences were observed correspond-
ing to the chemical shifts attributable to the propionate group.
Specifically, both an additional methylene carbon signal and
proton resonance were found that were determined to be part
of a butyrate group [δC 174.5 (C-1′, CO), 37.2 (C-2′, CH2),
19.8 (C-3′, CH2), and 14.0 (C-4′, CH3); δH 2.37 (2H, t, J =
7.2, H-2′), 1.70 (2H, m, H-3′), and 1.01 (3H, t, J = 7.4 Hz, H-
4′)]. The placement and identity of the butyrate were
Anacolosin B (2) was purified as a colorless oil. The
1
HRESIMS (m/z 525.2464), together with the H and 13C
1
NMR data (Tables 1 and 2), led to its proposed molecular
formula, C28H38O8. This corresponded to one additional
methylene unit in 2 compared to 1. The 1H and 13C NMR data
for 2 (Tables 1 and 2) were similar to those obtained for 1;
established using H−1H COSY and HMBC correlation data
(Figure S1, Supporting Information). The relative and absolute
1
configurations of 2 were also confirmed by H−1H ROESY
data (Figure S2, Supporting Information), coupling constants,
D
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Figure 3. Calculated and experimental VCD/IR spectra for compound 1.
Scheme 1. Synthesis of 2-Naphthacyl Esters (S- and R/S-13) of S- and R/S-2-Methylbutyric Acid (S- and R/S-12)
and ECD and VCD analyses (Figures S3 and S4, Supporting
Information). Based on these experiments, compound 2 was
confirmed to have the same absolute configuration as 1.
Anacolosin C (3) was obtained as a colorless oil, and its
molecular formula was established to be C29H40O8 based on
the HRESIMS data (m/z 539.2613 [M + Na]+, calcd for
were appropriate for determining the absolute configuration of
the molecule’s isozuelanin core, the configuration of the C-2′
stereocenter remained undefined. To probe this last stereo-
center, compound 4 was subjected to hydrolysis to liberate 2-
methylbutyric acid, which was converted to its corresponding
naphthacyl ester with α-bromo-2-acetonaphthone (11)
(Scheme 1).14,15 Similarly, the naphthacyl esters made from
authentic samples of S- and R/S-2-methylbutyric acid (12)
were also prepared, and the products were analyzed by chiral
HPLC. The retention times for S- and R-13 were determined
to be 12.8 and 13.6 min, respectively, on a Phenomenex Lux 5
μm Cellulose-3 chiral column. With these data in hand, the
product generated from the hydrolysis of compound 4 was
analyzed and found to match the S isomer. Thus, the structure
of the new metabolite was secured as shown for 4.
1
539.2615). Focusing on the small changes in the H and 13C
NMR resonances for 3 versus 2 (Tables 1 and 2), our attention
was guided to the ester group moiety with which a new methyl
signal was associated. The 1H−1H COSY and HMBC
correlation data (Figure S1, Supporting Information) con-
firmed the position of the methyl group belonging to a
methylbutanoate moiety in 3. The relative configuration of 3
1
was confirmed by H−1H ROESY and coupling constants
analyses, whereas its absolute configuration was established
based on the significant similarity between the ECD spectra of
1 and 2 to data obtained for 3.
Anacolosin E (5) was obtained as a colorless oil. The
sodiated molecular ion observed by HRESIMS at m/z
537.2455 (calcd 537.2459) suggested the metabolite’s
Anacolosin D (4) was determined to have the same
molecular formula as metabolite 3, which suggested the new
compound was isomeric. Analysis of the 1H and 13C NMR data
(Tables 1 and 2) led to the conclusion that the structural
changes in metabolite 4 were once again confined to the ester
moiety. In this case, a 2-methylbutanoate group was proposed,
which was supported by 1H−1H COSY and HMBC correlation
data (Figure S1, Supporting Information). Whereas ECD data
1
molecular formula was C29H38O8. Examination of the H and
13C NMR data (Tables 1 and 2) focused our attention on the
ester moiety as the site where this compound varied from other
metabolites in the series. In this case, the ester group exhibited
rather substantial changes including an upfield carbonyl group
(δC 168.5, C-1′), two olefinic carbons (δC 129.9, C-2′; δC
138.9, CH-3′), and two methyl carbons (δC 14.5, CH3-4′; δC
E
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a
Table 3. TGI Values of Compounds 1−10 in Pediatric Cancer Cell Lines
cell line
compound
A-673
SJCRH30
D283
Hep293TT
1
2
3
4
5
6
7
8
1.1 0.1
1.0 0.1
1.1 0.1
1.2 0.1
3.1 0.1
4.1 0.5
0.7 0.1
1.0 0.2
1.7 0.2
0.9 0.1
0.006 0.002
0.52 0.03
0.50 0.02
0.67 0.02
0.73 0.02
1.9 0.1
0.7 0.1
0.6 0.1
0.66 0.04
0.66 0.04
2.0 0.2
2.3 0.1
0.36 0.02
0.7 0.1
1.1 0.2
0.5 0.1
∼0.08
1.0 0.1
0.9 0.2
1.0 0.1
0.8 0.1
1.8 0.2
3.2 0.6
0.22 0.02
0.28 0.02
0.6 0.1
0.3 0.1
∼0.08
2.3 0.2
0.34 0.01
0.44 0.04
0.8 0.1
0.36 0.03
0.0060 0.0004
9
10
vinorelbine
a
Concentration (μM) that caused total growth inhibition (TGI) for each pediatric cancer cell line as determined by the SRB assay. n = 3; mean
SEM.
Figure 4. Concentration−response data from the SRB assay for compounds 1−10 and vinorelbine against A-673 (blue), SJCRH30 (red), D283
(green), and Hep293TT (orange) cell lines. n = 3; mean SEM.
F
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12.1, CH3-5′). Analysis of the chemical shift data and HMBC
correlations confirmed the identity of the new ester group as a
(SJCRH30), medulloblastoma (D283), and hepatoblastoma
(Hep293TT). The microtubule-destabilizing chemotherapeu-
tic vinorelbine, which has demonstrated particular efficacy in
sarcomas from among a panel of patients with pediatric solid
tumors,16 was used as a positive control. As expected,
vinorelbine had potent and selective activity against the
Ewing and rhabdomyosarcoma cells, with only cytostatic
effects observed in the hepatoblastoma and medulloblastoma
cells (Table 3, Figure 4). Compounds 1−10 demonstrated a
distinct profile of efficacy with cytotoxicity observed in each of
the 4 cell lines with TGI values ranging from 0.2 to 4.1 μM
(Table 3, Figure 4); however, certain cell lines exhibited
greater sensitivities to one or more of the natural products.
The A-673 cells were the least sensitive to each of the 10
compounds, whereas SJCRH30 and D283 cells were more
sensitive to each compound throughout the entire concen-
tration response curve (Figure 4). This shift was associated
with a modest, but consistent, 1.4−2.5-fold difference in TGI
values between these cell lines (Table 3). Among the C-6 keto-
carbonyl-group-containing compounds (i.e., compounds 1−6),
compounds 5 and 6 were the least potent, with TGI values 2−
5-fold higher than 1−4.
The Hep293TT hepatoblastoma cells were some of the most
resistant to compounds 1−6. However, the cell line selectivity
profile was distinct for the C-6 hydroxy-group-containing
compounds 7−10, with the Hep293TT cells being the most
sensitive (TGI values of 0.2−0.6 μM) followed by the
SJCRH30 (TGI values 0.3−0.8 μM), D283 (TGI values
0.4−1.1 μM), and A-673 (TGI values 0.7−1.7 μM) cells lines.
The finding that Hep293TT cells were selectively sensitive to
each of the C-6 hydroxy compounds (i.e., 7, 8, 9, and 10), but
not the analogous C-6 keto carbonyl compounds (i.e., 3, 5, 4,
and 1), demonstrated the importance of this moiety in
conferring sensitivity for hepatoblastoma cells as compared to
cells representing other pediatric cancer types. Importantly,
although the windows of selectivity for these compounds is
narrow, each of these compounds was highly effective, with
maximal efficacy values of essentially 100% in each of the
pediatric cancer cell lines. The results were reproducible and
significant, which suggests that medicinal chemistry efforts
would have the potential to identify analogues with even
greater selectivity based on this scaffold.
1
2-methylbut-2-enoate. H−1H ROESY correlation data (δH
6.91, H-3′ and δH 1.88, H-5′) supported a Z configuration for
the alkene. A combination of 1H−1H ROESY and ECD helped
verify that compound 5 shared the same absolute configuration
with other compounds in this series.
Anacolosin F (6) was determined to have the molecular
formula C28H38O8 based on HRESIMS, which yielded a [M +
Na]+ ion at m/z 525.2456 (calcd 525.2459). An examination
1
of the H and 13C NMR data (Tables 1 and 2) for the ester
moiety led to the conclusion that 6 was a constitutional isomer
of compound 2, with the former compound bearing an
isobutyrate group. The limited quantity of 6 obtained from the
extract precluded the collection of a ROESY data set; however,
we propose that this molecule shared the same absolute
configuration as metabolites 1−5 based on (i) its co-
occurrence with these metabolites, (ii) an ECD spectrum
that was similar to the previously described compounds, and
1
(iii) H NMR coupling constants that were a good match to
the isozuelanin portions of the other natural products.
Purified corymbulosin X (7) appeared as a colorless oil and
was found to possess the molecular formula C29H42O8 as
determined by HRESIMS, which indicated that the metabolite
contained nine degrees of unsaturation. An examination of the
1H and 13C NMR data for 7 and 3 (Tables 1 and 2,
respectively) showed the two compounds to be quite similar,
with one major exception: the C-6 keto carbonyl signal in 3
was missing and it was replaced by a new oxygenated methine
(δC 73.4). The relative configuration of ring B was established
1
based on an analysis of J-coupling (Table 1) and H−1H
ROESY correlation data (Figure S2, Supporting Information).
The experimental ECD spectrum (Figure S3, Supporting
Information) was consistent with data calculated for 7, as well
as published data for similar metabolites,8 leading to the
proposed absolute configuration as depicted. Mosher ester
analysis was carried out on 7 (Figure S7, Supporting
Information) confirming its 6S configuration.
Corymbulosin Y (8) appeared as a colorless oil, and its
HRESIMS data (m/z 539.2638 [M + Na]+, calcd 539.2615)
supported a molecular formula of C29H40O8, which meant the
1
compound contained 10 degrees of unsaturation. The H and
13C NMR data indicated that 7 and 8 shared the same
clerodane diterpene skeleton, but the two molecules differed in
EXPERIMENTAL SECTION
■
1
their respective ester groups. The H−1H COSY and HMBC
General Experimental Procedures. Optical rotation data were
obtained on a Rudolph Research AUTOPOL III automatic
polarimeter. ECD data were obtained on a JASCO J-715 CD
instrument. VCD spectra were recorded on a BioTools, Inc. ChiralIR-
2X VCD spectrometer, equipped with a dual PEM accessory. NMR
data were collected on Varian 400, 500, and 600 MHz NMR
spectrometers. LC-MS data were obtained on a Shimadzu LC-MS
2020 system (ESI quadrupole) coupled to a photodiode array
detector, with a Phenomenex Kintex 2.6 μm C18 column (100 Å, 75 ×
3.0 mm, 0.4 mL/min). The preparative HPLC system utilized SCL-
10A VP pumps and a system controller with a Phenomenex Gemini 5
μm C18 column (110 Å, 250 × 21.2 mm, 10 mL/min), and the
analytical and semipreparative HPLC systems utilized Waters 1525
binary pumps with Waters 2998 photodiode array detectors and
Phenomenex Gemini 5 μm Gemini C18, Phenomenex Kinetex 5 μm
biphenyl, and Phenomenex Kinetex 5 μm pentafluorophenyl (250 ×
4.6 mm, 1 mL/min and 250 × 10 mm, 4 mL/min) columns. Accurate
mass data were collected on an Agilent 6538 HRESI QTOF MS
coupled to an Agilent 1290 HPLC. Chiral HPLC analyses were
performed on a system composed of Waters 1525 binary pumps, a
Waters 2998 photodiode array detector, and a Phenomenex Lux 5 μm
data confirmed that 8 and 5 shared the same pendant (Z)-2-
methylbut-2-enoate ester moiety. Based on its ECD data
(Figure S3, Supporting Information), compound 8 was
proposed to have the same absolute configuration as the
other metabolites purified from this plant.
Two previously described metabolites, 9 and 10,8 were also
purified from the bioactive extract. The identities of both
compounds were confirmed based on comparisons of the
experimental and reported NMR chemical shift data, as well as
analyses of their ECD spectroscopic data. The configuration of
the C-2′ stereocenter in compound 9 was determined to be S
as previously reported in the literature.8 To the best of our
knowledge, a trivial name had not been previously assigned to
compound 9, whereas compound 10 had been assigned the
name caseamembrin S.8
The antiproliferative and cytotoxic effects of metabolites 1−
10 were evaluated in cell lines representing four pediatric
cancer types: Ewing sarcoma (A-673), rhabdomyosarcoma
G
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Cellulose-3 column (250 × 4.6 mm, 1 mL/min). The S- and R/S-2-
methylbutyric acid (12) and α-bromo-2-acetonaphthone (11) were
purchased from Sigma-Aldrich. All solvents were of ACS grade or
better.
Plant Material. Fruit, leaf, and twigs of the plant Anacolosa clarkii
were collected in the Bana Forest Preserve in Danang, Vietnam, by
Dr. Djaja D. Soejarto (University of Illinois at Chicago) under
contract to the Natural Products Branch for the National Cancer
Institute, on December 3, 2003. Voucher specimens for this collection
[Voucher #0GHA5499] are housed at the Smithsonian Institution,
Washington, DC.
Extraction and Isolation. An organic extract of the plant A.
clarkii (N135363) was produced as reported previously17 and
provided by NCI Natural Products Branch (Frederick, MD, USA).
The crude extract (1.0 g) was fractionated by vacuum-liquid
chromatography (HP20ss) eluted with a MeOH−H2O step gradient
(30:70, 50:50, 70:30, 90:10, and 100:0) and washed with CH2Cl2−
MeOH (50:50). The fourth fraction (MeOH−H2O, 90:10) was
found to be bioactive, and it was further fractionated by C18
preparative HPLC (gradient elution using MeOH−H2O, 30:70 to
100% MeOH in 30 min) followed by semipreparative HPLC using a
pentafluorophenyl column (isocratic CH3CN−H2O, 55:45 or 60:40),
to obtain 1 (20.0 mg), 2 (8.6 mg), 3 (3.7 mg), 4 (1.0 mg), 5 (0.6
mg), 6 (0.3 mg), 7 (0.6 mg), 8 (0.4 mg), 9 (0.7 mg), and 10 (0.5
product (13) was purified from the reaction residue by semi-
preparative HPLC.
The esters from the hydrolysate of 4, S-13, and R/S-13 were
analyzed by chiral HPLC (Phenomenex Lux 5 μm Cellulose-3
column, 1 mL/min, 60:40 acetonitrile−water), and the retention
times of the compounds were recorded as 12.8 min (ester from
compound 4), 12.8 min (S-13), and 12.8 and 13.6 min (R/S-13),
which revealed a match between the natural-product-derived material
and the S-form of the authentic standard. This conclusion was further
confirmed by co-injection of the material prepared from metabolite 4
and S-13, which showed both compounds eluted at the same time and
had superimposable PDA chromatograms.
VCD Calculations for Compounds 1 and 2. Anacolosins A and
B (1 and 2) were dissolved separately in CDCl3 (50 mg/mL for 1 and
43 mg/mL for 2) and placed in a 100 μm path-length cell with BaF2
windows. IR and VCD spectra were recorded on a ChiralIR-2X VCD
spectrometer (BioTools, Inc.) coupled with a single PEM accessory,
with 4 cm−1 resolution, and instrument optimization at 1400 cm−1 (5
h data collection for each sample, as well as a CDCl3 blank). A
preliminary conformational search was executed with ComputeVOA
(BioTools, Inc.) at the molecular mechanics level (MMFF). The
geometry, frequency, IR, and VCD intensity calculations of the low-
energy conformers were carried out at the B3LYP/6-31+G(d,p) level
with Gaussian 09 (Gaussian Inc., Wallingford, CT, USA).13 The
calculated IR and VCD spectra of the stable conformers with
populations greater than 1% were added together after Boltzmann
statistical weighting using SpecDis 1.71 (scaled by 0.97 and a half-
bandwidth of 6 cm−1). The resulting calculated IR and VCD spectra
were compared against the experimental VCD curves obtained for 1
and 2 and are shown in Figures 3 and S4 (Supporting Information),
respectively.
ECD Calculations for Compounds 1−5, 7, and 8. A
preliminary conformational search was executed with ComputeVOA
(BioTools, Inc.) at the molecular mechanics level (MMFF). The
conformers obtained were further optimized at the B3LYP/6-
31+G(d,p) level with Gaussian 09 (Gaussian Inc.).13 Stable
conformers with populations greater than 1% were submitted to
ECD calculation by time-dependent DFT at the CAM-B3LYP/SVP
level. The ECD spectra were added together after a Boltzmann
statistical weighting using SpecDis 1.71 (sigma value of 0.3 eV). After
applying a UV-shift correction, the computed ECD spectra were
compared with the experimentally derived ECD curves (Figures 2 and
S3, Supporting Information).
Pediatric Cancer Cell Lines. The rhabdomyosarcoma
(SJCRH30), Ewing sarcoma (A-673), and medulloblastoma (D283)
cell lines were purchased from the ATCC (Manassas, VA, USA). The
hepatoblastoma cell line (Hep293TT) was obtained directly from Dr.
Gail Tomlinson.18 The SJCRH30, A-673, and Hep293TT cells were
grown in RPMI-1640 medium (Corning) with 5% fetal bovine serum
(Corning) and 0.5% gentamycin (Gibco), while the D283 cells were
grown in IMEM (Gibco) with 5% fetal bovine serum and 0.3%
gentamycin. Cells were grown at 37 °C in a humidified incubator with
5% CO2. Cells were used within four months of retrieval from liquid
nitrogen. The SJCRH30, D283, and A-673 cells lines were validated
by STR profiling (Genetica).
mg).
1
Anacolosin A (1): colorless oil; [α]20 +2.6 (c 1.02, MeOH); H
D
and 13C NMR, see Tables 1 and 2; HRESIMS m/z 511.2304 [M +
Na]+ (calcd for C27H36O8Na, 511.2302).
Anacolosin B (2): colorless oil; [α]20 +7.4 (c 0.43, MeOH); H
1
D
and 13C NMR, see Tables 1 and 2; HRESIMS m/z 525.2464 [M +
Na]+ (calcd for C28H38O8Na, 525.2459).
Anacolosin C (3): colorless oil; [α]20 +9.7 (c 0.12, MeOH); H
1
D
and 13C NMR, see Tables 1 and 2; HRESIMS m/z 539.2613 [M +
Na]+ (calcd for C29H40O8Na, 539.2615).
Anacolosin D (4): colorless oil; 1H and 13C NMR, see Tables 1 and
2; HRESIMS m/z 539.2610 [M + Na]+ (calcd for C29H40O8Na,
539.2615).
Anacolosin E (5): colorless oil; 1H and 13C NMR, see Tables 1 and
2; HRESIMS m/z 537.2455 [M + Na]+ (calcd for C29H38O8Na,
537.2459).
Anacolosin F (6): colorless oil; 1H and 13C NMR, see Tables 1 and
2; HRESIMS m/z 525.2456 [M + Na]+ (calcd for C28H38O8Na,
525.2459).
Corymbulosin X (7): colorless oil; [α]20D +16 (c 0.05, MeOH); 1H
and 13C NMR, see Tables 1 and 2; HRESIMS m/z 541.2798 [M +
Na]+ (calcd for C29H42O8Na, 541.2772).
Corymbulosin Y (8): colorless oil; H and 13C NMR, see Tables 1
1
and 2; HRESIMS m/z 539.2638 [M + Na]+ (calcd for C29H40O8Na,
539.2615).
Analysis of the Ester Group Chirality for Compound 4. α-
Bromo-2-acetonaphthone (11, 45 mg, 0.18 mmol) was added to an
acetonitrile solution of S-2-methylbutyric acid (S-12) [20 μL (18.8
mg, 0.18 mmol)] in 0.8 mL of acetonitrile, followed by the addition of
0.2 mL of an aqueous solution of LiOH (1.0 mg/mL). The solution
was stirred at room temperature for 3 h before the solvent was
removed. The residue was analyzed by LC-MS, and analytical chiral
HPLC was used to confirm the identity of the product [S-2-
methylbutyric acid 2-naphthacyl ester (S-13)]. S-13 (27 mg) was
purified via semipreparative HPLC, and its structure was confirmed by
1H and 13C NMR (Figures S67 and S68, Supporting Information) and
ESIMS m/z 271 [M + H]+. Similarly, the R/S-2-methylbutyric acid
(R/S-12) was used as described above to prepare the R/S-2-
methylbutyric acid 2-naphthacyl esters (R/S-13) (NMR: Figures S65
and S66, Supporting Information, and ESIMS m/z 271 [M + H]+).
Compound 4 (0.2 mg) was added to 0.2 mL of an aqueous LiOH
solution (1 mg/mL) and stirred for 3 h followed by the addition of α-
bromo-2-acetonaphthone (5 mg dissolved in 0.8 mL of acetonitrile).
The mixture was stirred overnight at room temperature, at which
point the presence of 13 was confirmed by analysis of the extracted
ion chromatogram (Figure S5, Supporting Information). The target
Determination of TGI Values against Pediatric Cancer Cell
Lines Using a Sulforhodamine B Cytotoxicity Assay. Com-
pounds were evaluated for antiproliferative and cytotoxic activities
using a sulforhodamine B (SRB) assay as previously described.19,20
Cells were plated at a predetermined density of 4000−6000 cells per
well in 96-well tissue culture plates and allowed to adhere overnight.
Compounds were dissolved in DMSO and added to triplicate wells
with final DMSO volumes of 0.5% v/v with vehicle controls included
on each plate. Cells were treated for 48 h, after which the medium was
removed, and the adherent cells were fixed with 10% TCA. Due to the
low adherence of the D283 cells, 180 μL of 20% TCA was added to
the tissue culture medium of each well for these cells. At the time of
compound addition, a separate “time 0” plate was fixed to allow
calculations of TGI and cytotoxicity. The absorbance values were
measured and compared to vehicle and time 0 controls to determine
H
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concentrations that caused total growth inhibition for each experi-
ment using the nonlinear regression function in Prism 6 (GraphPad).
Kuhlen, M.; Eggert, A.; Fulda, S.; Gessler, M.; Wegert, J.; Kappler, R.;
Baumhoer, D.; Burdach, S.; Kirschner-Schwabe, R.; Kontny, U.;
Kulozik, A. E.; Lohmann, D.; Hettmer, S.; Eckert, C.; Bielack, S.;
Nathrath, M.; Niemeyer, C.; Richter, G. H.; Schulte, J.; Siebert, R.;
Westermann, F.; Molenaar, J. J.; Vassal, G.; Witt, H.; Burkhardt, B.;
Kratz, C. P.; Witt, O.; van Tilburg, C. M.; Kramm, C. M.; Fleischhack,
G.; Dirksen, U.; Rutkowski, S.; Fruhwald, M.; von Hoff, K.; Wolf, S.;
Klingebiel, T.; Koscielniak, E.; Landgraf, P.; Koster, J.; Resnick, A. C.;
Zhang, J.; Liu, Y.; Zhou, X.; Waanders, A. J.; Zwijnenburg, D. A.;
Raman, P.; Brors, B.; Weber, U. D.; Northcott, P. A.; Pajtler, K. W.;
Kool, M.; Piro, R. M.; Korbel, J. O.; Schlesner, M.; Eils, R.; Jones, D.
T. W.; Lichter, P.; Chavez, L.; Zapatka, M.; Pfister, S. M. Nature
2018, 555, 321−327.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.8b01015.
Summaries of 2D NMR correlation data, experimental
and calculated ECD curve comparisons, comparison of
experimental and calculated VCD curves for compound
2, Mosher’s reaction results for compound 7, LC-MS
analysis of the reaction-product residue for compound 4,
C18 and chiral HPLC analysis of synthetic product for
compound 4 and the synthetic standards, 1D and 2D
NMR spectra for compounds 1−8, 1D NMR spectra for
synthetic standards, and the HRESIMS data (PDF)
(4) Bourjot, M.; Leyssen, P.; Eydoux, C.; Guillemot, J. C.; Canard,
́
B.; Rasoanaivo, P.; Gueritte, F.; Litaudon, M. Fitoterapia 2012, 83,
1076−1080.
(5) Alimboyoguen, A. B.; Castro-Cruz, K. A. D.; Shen, C.-C.;
Ragasa, C. Y. Res. J. Pharm. Biol. Chem. Sci. 2014, 5, 1189−1191.
(6) Kanokmedhakul, S.; Kanokmedhakul, K.; Kanarsa, T.;
Buayairaksa, M. J. Nat. Prod. 2005, 68, 183−188.
(7) Kanokmedhakul, S.; Kanokmedhakul, K.; Buayairaksa, M. J. Nat.
Prod. 2007, 70, 1122−1126.
AUTHOR INFORMATION
Corresponding Authors
*Phone: 210-567-4788. Fax: 210-567-4300. E-mail:
Mooberry@uthscsa.edu.
■
(8) Aimaiti, S.; Suzuki, A.; Saito, Y.; Fukuyoshi, S.; Goto, M.;
Miyake, K.; Newman, D. J.; O’Keefe, B. R.; Lee, K.-H.; Nakagawa-
Goto, K. J. Org. Chem. 2018, 83, 951−963.
(9) Suzuki, A.; Saito, Y.; Fukuyoshi, S.; Goto, M.; Miyake, K.;
Newman, D. J.; O’Keefe, B. R.; Lee, K.-H.; Nakagawa-Goto, K. J. Nat.
Prod. 2017, 80, 1065−1072.
*Phone: 405-325-6969. Fax: 405-325-6111. E-mail:
rhcichewicz@ou.edu.
ORCID
April L. Risinger: 0000-0002-4363-3268
Tanja Grkovic: 0000-0002-6537-3997
Barry R. O’Keefe: 0000-0003-0772-4856
Robert H. Cichewicz: 0000-0003-0744-4117
(10) Li, R.; Morris-Natschke, S. L.; Lee, K.-H. Nat. Prod. Rep. 2016,
33, 1166−1226.
(11) Xu, J.; Zhang, Q.; Wang, M.; Ren, Q.; Sun, Y.; Jin, D.-Q.; Xie,
C.; Chen, H.; Ohizumi, Y.; Guo, Y. J. Nat. Prod. 2014, 77, 2182−
2189.
́
(12) Calderon, C.; Ford, C. D.; Castro, V.; Merfort, I.; Murillo, R. J.
Notes
Nat. Prod. 2014, 77, 455−463.
The authors declare no competing financial interest.
(13) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci,
B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H.
P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;
Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.;
Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.;
Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.;
Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi,
R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar,
S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox,
J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.;
Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.;
Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.;
Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A.
ACKNOWLEDGMENTS
■
Support for this project was provided by NIH grant
R01GM107490 to R.H.C. and S.L.M. and the Greehey
Endowment (S.L.M.). The LC-MS instrument used for this
project was provided in part by a Challenge Grant from the
Office of the Vice President for Research, University of
Oklahoma, Norman Campus, and an award through the
Shimadzu Equipment Grant Program (R.H.C.). Computa-
tional data for ECD and VCD experiments were obtained
through the University of Oklahoma Supercomputing Center
for Education & Research (OSCER). This project has been
funded in whole or in part with federal funds from the National
Cancer Institute, National Institutes of Health, under contract
HHSN261200800001E. 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 endorse-
ment by the U.S. Government.
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