Isobutylhydroxyamides from the pericarp of Nepalese Zanthoxylum armatum inhibit NF1 -defective tumor cell line growth

Article abstract of DOI:10.1021/np300696g

A neurofibromatosis type 1 (NF1)-based bioassay-guided phytochemical investigation on Zanthoxylum armatum collected in Nepal led to the isolation of new timuramides A-D (1-4) and six known sanshools (5-10). The structures of all compounds were established by using modern spectroscopic techniques, including 1D and 2D NMR analysis and comparison with previously reported data. Most of the compounds inhibited growth of an Nf1- and p53-deficient mouse glioma cell line at noncytotoxic concentrations.

Full text of DOI:10.1021/np300696g

Article
pubs.acs.org/jnp
Isobutylhydroxyamides from the Pericarp of Nepalese Zanthoxylum
armatum Inhibit NF1-Defective Tumor Cell Line Growth
Krishna P. Devkota,† Jennifer Wilson,† Curtis J. Henrich,†,‡ James B. McMahon,† Karlyne M. Reilly,§
and John A. Beutler*,†
†Molecular Targets Laboratory, Center for Cancer Research, Frederick National Laboratory for Cancer Research, Frederick, Maryland
21702, United States
‡SAIC-Frederick, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States
§Mouse Cancer Genetics Program, Center for Cancer Research, Frederick National Laboratory for Cancer Research, Frederick,
Maryland 21702, United States
S
* Supporting Information
ABSTRACT: A neurofibromatosis type 1 (NF1)-based bioassay-guided
phytochemical investigation on Zanthoxylum armatum collected in Nepal
led to the isolation of new timuramides A−D (1−4) and six known
sanshools (5−10). The structures of all compounds were established by
using modern spectroscopic techniques, including 1D and 2D NMR
analysis and comparison with previously reported data. Most of the
compounds inhibited growth of an Nf1- and p53-deficient mouse glioma
cell line at noncytotoxic concentrations.
efects in the tumor suppressor gene NF1 have been
carminative, stomachic, and anthelmintic.10 In Nepal, pericarps
Drecognized as important factors in the development of of Z. armatum are used for abdominal pain, cold and cough,
tonsillitis, fever, altitude sickness, diarrhea, and dysentery.11,12
Bioassay-guided fractionation of the pericarp extract of Z.
armatum led to the isolation of four new (1−4) and six known
(5−10) isobutylhydroxyamides classified as sanshools. More
than 50 sanshool structures have been reported from
Zanthoxylum species, some of which possess antioxidant and
anthelmintic properties.13,14 Compounds 1 and 2 contain a
novel endoperoxide ring. This is the first report of NF1-related
and glioma-related activity from this class of compounds.
both sporadic and familial cancers.1 Both direct genetic
inactivation of NF1 and excessive proteasomal degradation of
its gene product, neurofibromin, can lead to the development
of glioblastomas,2,3 while inherited defects in NF1 lead to
higher rates of astrocytoma, malignant peripheral nerve sheath
tumors (MPNST), and neuroblastoma, as well as gastro-
intestinal stromal tumors, breast cancer, pheochromocytoma,
and other tumors.1
Two recent studies4,5 have found preparations made from
the spice of Asian Zanthoxylum spp. to be active against
xenografted MDA-MB-231 breast cancer cells, which are
known to be deficient in NF1,6 as well as against cultured
MPNST cells from an NF1 patient. This stimulated us to
isolate, characterize, and evaluate NF1-related activity of the
compounds obtained from Zanthoxylum spp. using a recently
published bioassay system in which the mouse NF1 homologue,
Nf1, and the gene encoding the tumor suppressor p53 (Trp53)
are deleted.7 For this study, Zanthoxylum armatum DC
(Rutaceae) of Nepalese origin, locally known as Timur, was
selected. Z. armatum, an important medicinal and spice plant, is
a small tree distributed throughout the Himalayas, predom-
inantly in Nepal, India, and Bhutan up to 2500 m elevation.8 In
traditional folk medicine, Zanthoxylum plants are referred to as
“toothache trees” because their anesthetic or counterirritant
properties render them useful in the treatment of pain.9 The
characteristic biting or numbing taste of the pericarp of the dry
seeds makes it an indispensable spice among the people of
South and Southeast Asia. The stem bark and pericarps are
extensively used in indigenous systems of medicine as a
RESULTS AND DISCUSSION
■
Dried pericarps of Z. armatum were extracted in MeOH to
yield four new (1−4) and six known (5−10) isobutylhydrox-
yamides using diol batch elution, LH-20 Sephadex chromatog-
raphy, and C18-HPLC. All compounds possessed maximal UV
absorption near 250 nm, consistent with the presence of a
conjugated amide.15 The IR spectra also showed characteristic
bands for the amidocarbonyl group near 3300 cm−1.13
Compound 1 was isolated as a colorless oil. The molecular
formula, deduced to be C16H25NO4 by HREIMS, was further
supported by 13C NMR data showing five double-bond
1
equivalents. The H and 13C NMR spectra (Tables 1 and 2,
respectively) showed the polyunsaturated fatty acid amide
nature of compound 1 bearing an N-isobutylhydroxyl moiety.
1
In the H NMR spectrum, a 6H singlet resonating at δH 1.22
was assigned to H-3′ and H-4′. A broad 2H doublet at δH 3.31
Received: October 5, 2012
Published: December 26, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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Table 1. 1H NMR Data (600 MHz) for Compounds 1 and 2
(CDCl3) and 3 and 4 (CD3OD)
position
1
2
3
4
1
2
5.85, d (15.4) 5.80, brs
6.04, d (15.4) 6.00, d
(15.4)
3
6.75, dt
(15.4, 6.5)
6.78, dt (15.4, 6.5) 6.79, dt
(15.1, 6.7)
6.81, dt
(15.1, 6.6)
4
5
6
2.25, m
2.33, m
2.26, m
2.20, m
2.31, m
2.46, q (6.9)
1.30, t (7.2)
2.46, m
5.62, dt
(10.7, 6.9)
5.71, ddd (15.4,
8.9, 6.3)
5.71, dd
(10.3, 7.7)
7
5.47, dd
(11.1, 10.5)
5.56, dd (15.5,
7.0)
6.14, t (11.1)
8
5.19, brd
(7.7)
4.79, brd (5.2)
7.41, dd
(15.0, 11.6)
9
5.88, dt
5.87, dt (8.4, 1.8) 5.90, d (15.1)
5.83, dt (7.8, 2.3)
(10.1, 2.1)
10
11
5.78, dt
(10.2, 2.5)
4.62, m
4.64, dddd (8.5,
6.6, 4.3, 1.8)
Figure 1. Δδ values (δS − δR) in ppm × 1000 (1d) for the two MTPA
esters 1b and 1c.
12
1.28, d (6.6) 1.23, d (6.6)
1′
3.31, brd
(6.1)
3.29, brd (6.2)
3.25, brs
1.17, s
3.24, brs
3′ and 4′ 1.22, s
NH 6.02, brs
1.21, s
1.17, s
ND
a
5.96, brs
ND
a
ND: Not detected.
Table 2. 13C NMR Data (δ, 150 MHz) for Compounds 1 and
2 (CDCl3) and 3 and 4 (CD3OD)
Figure 2. Important HMBC and COSY correlations in compound 1.
position
1
2
3
4
1. The cis-olefinic protons of the ring resonated at δH 5.88 (dt, J
= 10.1, 2.1 Hz, H-9) and δH 5.78 (dt, J = 10.2, 2.5 Hz, H-10).
Similarly, two methine protons of the ring geminal to the
peroxide resonated at δH 5.19 (brd, J = 7.7 Hz, H-8) and δH
4.62 (m, H-11). The 11.1 and 10.5 Hz couplings of H-7
indicated cis-vicinal protons at C-6 and C-8. In the HMBC
spectrum (Figure 2), the correlation of H-8 with C-7 (δC
127.0) and C-10 (δC 126.6), H-9 with C-8 (δC 74.3), C-10, and
C-11 (δC 74.6), H-10 with C-8 and C-11, H-11 with C-10 and
C-12 (δC 18.6), and H-12 (δH 1.28) with C-9 (δC 129.3), C-10,
and C-11 were observed. In the ROESY spectrum, H-8 showed
a correlation with H-11, indicating that these protons were on
the same face of the ring.
We attempted to assign the absolute configuration of
compound 1 by converting it into a secondary diol (1a) by
hydrogenolysis of the peroxide with Lindlar’s catalyst.17 The
reaction of 1a with (R)- and (S)-methoxytrifluoromethylphe-
nylacetyl chloride (MTPA-Cl) produced the corresponding
(S)- and (R)-MTPA diesters 1b and 1c (Figure 1); however,
we were unable to assign the configurations due to lack of
consistent observed shift changes, presumably due to the close
proximity of the two MTPA moieties to each other. While the
absolute configurations of 1,2-, 1,3-, and 1,4-saturated diols
have been assigned using a sign distribution model,18,19 such
assignments for unsaturated diols have not yet been explored.
Thus the lack of supporting literature data and limited
experimental evidence does not allow us to assign the absolute
configuration.
1
167.2
125.4
143.4
31.6
167.1
124.2
144.0
31.4
169.1
125.4
144.8
33.0
169.3
124.7
145.5
30.1
2
3
4
5
26.7
31.1
27.8
37.4
6
134.2
127.0
74.3
135.2
128.0
78.6
137.6
129.3
136.9
128.2
174.7
180.7
7
8
9
129.3
126.6
74.6
129.5
126.2
74.5
10
11
12
18.6
18.3
1′
2′
50.7
50.6
51.2
71.8
27.3
51.2
71.8
27.3
71.3
71.2
3′ and 4′
27.5
27.5
(J = 6.6 Hz, H-1′) showed a 1H−1H COSY correlation with δH
6.02 (NH) and HMBC correlations with δC 167.2 (C-1), 71.3
(C-2′), and 27.5 (C-3′ and C-4′) consistent with the presence
of an N-isobutylhydroxyl moiety attached to the amide
1
carbonyl. The H NMR spectrum also showed signals for one
pair of protons on trans-olefinic carbons at δH 5.85 (d, J = 15.4,
H-2) and δH 6.75 (dt, J = 15.4, 6.5 Hz, H-3) and another pair of
protons on cis-olefinic carbons at δH 5.62 as overlapped doublet
and triplet (d and t, J = 10.7, 6.9 Hz, H-6) and 5.47 (dd, J =
11.1, 10.5 Hz, H-7) separated by two methylenes at C-4 (δC
31.6) and C-5 (δC 26.7).16 The H−1H COSY correlations for
1
all consecutive protons from H-2 to H-12 (Figure 2)
demonstrated an extended spin system in the molecule. The
combined analysis of the NMR spectra, molecular formula, and
degree of unsaturation suggested the presence of an
endoperoxide ring containing one olefinic bond in compound
Very few endoperoxide compounds are reported from
terrestrial plants, mainly from Artemisia spp.;20 however no
compounds of the sanshool series have been previously
reported to have an endoperoxide ring, making compound 1
the most novel structure of this series. According to the above
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Compound 3 was obtained as a colorless oil. The molecular
formula C14H21NO4 for this compound was deduced from
1
HREIMS. The H and 13C NMR spectra of compound 3
(Tables 1 and 2) showed many similarities with compounds 1
and 2 except that it lacked an endoperoxide moiety and a
terminal carboxylic acid functionality at C-10 appeared in 3.
The carbonyl group of the carboxylic acid was supported by IR
1
(1730 cm−1) and 13C NMR (δC 174.7). In the H NMR
spectrum, a pair of trans-olefinic protons resonated at δH 7.41
(dd, J = 15.0, 11.6 Hz) and δH 5.90 (d, J = 15.1 Hz) and were
assigned to H-8 and H-9, respectively. In the HMBC spectrum,
correlations of H-8 with C-7 (δC 129.3), C-9 (δC 128.2), and C-
10 and of H-9 with C-8 (δC 136.9) and C-10 were observed.
1
Like compounds 1 and 2, compound 3 also exhibited H−1H
COSY correlations, supporting an extended spin system from
H-2 to H-9. The above data show the structure of 3 to be
(2E,6Z,8E)-N-(2-hydroxy-2-methylpropyl)-2,6,8-decatriena-
mid-10-oic acid, trivially named timuramide C.
Compound 4 was obtained as a colorless oil. The molecular
formula C10H17NO4 for this compound was deduced from
1
HREIMS. The H and 13C NMR spectra of compound 4
(Tables 1 and 2) showed that this compound was a shorter
chain sanshool distinctly similar to compound 3 except that it
lacked both the C-6/C-7 and C-8/C-9 pairs of olefinic carbons.
The terminal carboxylic acid functionality of compound 4 was
supported by IR (1738 cm−1) and 13C NMR (δC 180.7). In the
1H NMR spectrum, a pair of trans-olefinic protons resonated at
δH 6.00 (d, J = 15.4 Hz) and δH 6.81 (dt, J = 15.1, 6.6 Hz) and
were assigned to H-2 and H-3, respectively. The methylene
protons appeared at δH 2.46 (q, J = 6.9 Hz, H-4) and δH 2.30 (t,
J = 7.2 Hz, H-5), showing HMBC correlations with C-6 and C-
3 (δC 145.5). Compound 4 possessed an extended spin system
between H-2 and H-5. According to the above data, the
structure of 4 is proposed as (2E)-N-(2-hydroxy-2-methyl-
propyl)-2-hexenamid-6-oic acid, trivially named timuramide D.
Together with four new timuramides, A−D, six known
compounds, namely, ZP-amide C (5),22 ZP-amide D (6),22
hydroxy-α-sanshool (7),16 hydroxy-β-sanshool (8),21 ZP-amide
A (9),22 and ZP-amide E (10),22 were also isolated and
characterized by comparing their spectroscopic data with
literature values.
All of the purified compounds were tested in an Nf1- and
p53-defective mouse malignant glioma tumor cell line
engineered to express a dual reporter, in which cell viability
and growth inhibition are reported separately in the same cells
using filtered red and green luminescence, respectively (Table
3). Loss of cell viability is measured using a red luminescent
reporter of all living cells comparing compound-treated cells in
growth media relative to untreated controls in growth-retarding
media. Growth inhibition is measured using a green
luminescent reporter only expressed in actively dividing cells,
comparing compound-treated cells in growth media to vehicle-
treated cells in growth media. High growth inhibition scores
indicate that cells stop dividing in the presence of the
compound.
data, the structure of 1 was assigned as (2E,6Z,9Z)-8,11-
endoperoxy-N-(2-hydroxy-2-methylpropyl)-2,6,9-dodecatriena-
mide, trivially named timuramide A.
Compound 2 was obtained as a colorless oil. The molecular
formula, deduced to be C16H25NO4 by HREIMS, was further
1
supported by 13C NMR data and was isomeric with 1. The H
and 13C NMR spectra of compound 2 (Tables 1 and 2,
respectively) showed distinct similarities with compound 1
except that it contained a C-6/C-7 trans-olefinic bond in
contrast to the cis-configured bond in 1. The C-6/C-7 trans-
olefinic bond in 2 was confirmed by the 13C NMR values of the
adjacent carbons C-5 and C-8, which resonated at δC 31.1 and
78.6, significantly higher21 than in compound 1. Similarly, in
1
the H NMR spectrum, the H-5 methylene protons and H-8
methine proton resonated at δH 2.20 (m) and 4.79 (brd, J = 5.2
Hz), noticeably lower than in compound 1. Like that of 1, the
1H−1H COSY correlations between H-2 through H-12 in
compound 2 indicated an extended spin system in the
molecule. In the ROESY spectrum, H-8 showed a correlation
with H-11, indicating that they were on the same face of the
ring. According to the above data, the structure of 2 was
assigned as (2E,6E,9Z)-8,11-endoperoxy-N-(2-hydroxy-2-meth-
ylpropyl)-2,6,9-dodecatrienamide, trivially named timuramide
B.
All compounds exhibited moderate cytotoxicity; however
inhibition of cell growth varied at identical concentrations.
Compound 7 showed the best activity among the series of
compounds, with a cell growth inhibition percentage of 53%, at
2 μg/mL, without observed toxicity. The novel endoperoxide 1,
which possesses the cis-configuration at C-6, showed superior
activity to compound 2, the corresponding trans-geometric
isomer at C-6. Similarly, cis-isomer 7 was superior to its trans-
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eluted with hexane/CH2Cl2/MeOH (2:5:1, v/v), with 250 drop
fractions collected in each tube. Upon the basis of the UV absorption
(254 nm), 24 fractions (A2−X2) were collected. Among those,
fractions G2 and H2 showed activity and were further purified by
HPLC. HPLC separation was performed on 18.0 mg of fraction G2
dissolved in 2.4 mL of MeOH and injected in 200 μL aliquots
(approximately 1.5 mg/injection) onto a 250 × 10 mm Varian
Dynamax Microsorb 60-8 C18 HPLC column. The detector wave-
length was 225 nm, and solvent flow rate was 4.0 mL/min. Elution
began with 70% MeOH/H2O, isocratic for 3 min, then a linear
gradient of 70% to 100% MeOH at 25 min. The column was flushed
with 100% MeOH for 5 min. Three major UV-absorbing peaks were
collected and evaporated in vacuo to yield compound 1 (4.9 mg),
compound 2 (2.2 mg), and compound 8 (1.4 mg) eluting at
approximately 6.4, 6.1, and 12.1 min, respectively. Similar HPLC was
employed for fraction H2 (24.0 mg) to yield compounds 7 (2.8 mg)
and 8 (4.7 mg), eluting at approximately 11.2 and 12.0 min,
respectively.
Table 3. Cell Viability and Growth Inhibition (%) in Nf1-
and p53-Defective Mouse CNS Tumor Cell Line by
Compounds 1−10 at 2.0 μg/mL
compound cell viability (untreated controls = 100) % growth inhibition
1
2
3
4
5
6
7
8
9
10
97
65
66
70
79
66
106
56
75
67
38
13
47
42
31
56
53
65
52
41
isomer 8. This is similar to the pattern observed with the
pungency of the compounds, as the trans-amides are tasteless,
while the cis-amides are strongly pungent.23 Testing of
compounds 1, 3, and 4 in the NCI 60 cell screening assay,
however, found negligible potency and selectivity when tested
at a 10 μM single concentration.
Our results support the original observation that compounds
found in Z. armatum can inhibit tumor cells mutant for NF1,
but do not preclude the possibility that these compounds may
also have activity against tumors cells lacking the p53 signaling
pathway. Future studies will examine the specificity of these
compounds for different tumor suppressor mutations important
in human cancer.
Fraction C (75.0 mg) was chromatographed on a 2.5 × 40 cm
Sephadex LH-20 column and eluted with CH2Cl2/MeOH (1:1), with
200 drop fractions collected in each tube to give eight fractions, A3−
H3. Among the eight fractions, fractions B3 and C3 were found to
have activity. HPLC purification was performed on 21.0 mg of fraction
B3 dissolved in 2.8 mL of MeOH and injected in 200 μL aliquots
(approximately 1.5 mg/injection) onto a 250 × 10 mm Varian
Dynamax Microsorb 60-8 C18 HPLC column. The detector wave-
length was 225 nm, and solvent flow rate was 4.0 mL/min. Elution
began with 40% MeOH/H2O, isocratic for 3 min, then a linear
gradient of 40% to 100% MeOH at 25 min. The column was flushed
with 100% MeOH for 5 min. Four major UV-absorbing peaks were
collected and evaporated in vacuo, yielding compounds 9 (1.1 mg), 10
(0.9 mg), 5 (0.9 mg), and 6 (1.2 mg), eluting at approximately 7.8, 8.1,
9.7, and 11.3 min, respectively. Similar HPLC of fraction C3 (8.0 mg)
yielded compounds 4 (1.2 mg) and 3 (1.8 mg), eluting at
approximately 5.5 and 10.8 min, respectively.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations ([α]D)
were measured on a Perkin-Elmer 241 polarimeter in a 100 × 2 mm
cell (units 10−1 deg cm2g−1). UV absorption spectra were obtained
using a Varian Cary 50 Bio UV−visible spectrophotometer. IR spectra
were measured using a Perkin-Elmer FT-IR spectrometer, Spectrum
2000. LCMS were obtained using a Hewlett-Packard Series 1100
MSD, whereas HRMS were acquired on an Agilent 6520 Accurate
Mass Q-TOF instrument with internal reference masses at 121.05087
and 922.00979, both within 5 ppm. The NMR experiments were
Timuramide A (1), NSC#764085: colorless oil; [α]20 −20.6 (c
D
0.2, MeOH); UV (MeOH) λmax (log ε) 215 (3.2), 230 (4.1), and 250
1
(4.5) nm; IR (CHCl3) νmax 3308, 1670, and 1632 cm−1; H (600
MHz, CDCl3) and 13C (150 MHz, CDCl3) NMR data, see Tables 1
and 2; HREIMS [M + H]+ at m/z 296.1859 (calcd for C16H26NO4
296.1856).
Hydrogenolysis of 1 with Lindlar’s Catalyst. Compound 1 was
reduced by hydrogenolysis using Lindlar’s catalyst (5% Pd/CaCO3,
lead poisoned). A 20 mL RB flask containing a magnetic stir bar was
charged with Lindlar’s catalyst (2.0 mg). Then 2 mL of THF was
added to compound 1 (3.5 mg, 0.0118 mmol). The solution was then
transferred to a RB flask in a dropwise manner with constant stirring.
The flask was flushed with argon and fitted with a balloon of hydrogen
gas. It was then stirred vigerously with a constant supply of hydrogen
gas (1 atm.) at room temperature. After 12 h, the mixture was filtered
through a plug of silica gel and flushed with 50 mL of CH2Cl2. The
residue was loaded in a 250 × 10 mm Varian Dynamax Microsorb 60-8
C18 HPLC column and eluted with a gradient of 70−100% MeOH/
H2O. Eluting at approximately 7.15 min was a diol product (1a, 2.1
performed on a Bruker 600 MHz NMR spectrometer. H and 13C
1
spectra were referenced to deuterated solvent peaks. The diol DIO
Spe-ed SPE cartridge and Sephadex LH-20 columns attached to a
model UA-6 UV detector and Foxy 200 fraction collector (Teledyne
Isco) were used for fractionation of the extract, whereas purification of
the compounds was performed using a Varian ProStar 210/215 HPLC
equipped with a Varian ProStar 325 UV−vis detector, operating under
Star 6.41 chromatography workstation software. All solvents and
chemicals were of analytical grade.
Plant Material. The pericarp of Z. armatum (Rutaceae) was
collected from Khara of Kaski District, Nepal, in November 2010 at an
altitude of 1600 m. A voucher specimen (No. KD 109/2010) was
deposited in the Central Department of Botany, Tribhuvan University,
Kathmandu, Nepal, and identified by taxonomist Prof. Dr. Krishna K.
Shrestha of the same department.
Extraction and Isolation. Dried pericarp of Z. armatum, 25.0 g,
was ground and soaked in 2 L of MeOH overnight. It was then heated
to 70 °C for 5 min and filtered. The filtrate was concentrated in a
vacuum and yielded 3.2 g of crude extract N192131. The extract, 2.0 g,
was loaded on 10 diol DIO Spe-ed SPE cartridges (200.0 mg in each
cartridge) and eluted with 6.0 mL each of hexane/CH2Cl2 (9:1),
CH2Cl2/EtOAc (20:1), EtOAc, EtOAc/MeOH (5:1), and MeOH to
give five fractions (A−E) of 815.8, 181.6, 75.9, 224.8, and 253.8 mg,
respectively. The activity against NF1-deficient cells of fractions B and
C was higher than other fractions; thus these were selected for further
fractionation and isolation of pure compounds. Fraction B (180.0 mg)
was chromatographed on a 2.5 × 40 cm Sephadex LH-20 column and
1
mg, 69%): H NMR (CDCl3, 600 MHz) δ 6.73 (1H, dt, J = 15.3, 6.5
Hz, H-3), 5.84 (1H, dt, J = 9.8, 2.2 Hz, H-9), 5.82 (1H, d, J = 15.3 Hz,
H-2), 5.79 (1H, dt, J = 9.9, 2.4 Hz, H-10), 5.59 (1H, dd, J = 10.7, 6.9
Hz, H-6), 5.44 (1H, dd, J = 11.0, 10.5 Hz, H-7), 5.15 (1H, t, J = 7.4
Hz, H-8), 4.85 (1H, ddd, J = 6.5, 4.5, 2.1 Hz, H-11), 3.32 (2H, brd, J =
6.1 Hz, H-1′), 2.19 (2H, m, H-4), 2.28 (2H, m, H-5), 1.25 (3H, d, J =
6.6 Hz, H-12), 1.22 (6H, s, H-3′); HREIMS [M + Na]+ at m/z
320.1828 (calcd for C16H27NNaO4 320.1832).
Preparation of (S)- and (R)-MTPA Esters (1b and 1c) of 1a. To
a solution of compound 1a (1.0 mg, 3.3 μmol) in pyridine (0.1 mL)
was added (R)-MTPA-Cl (3 μL, 13.2 μmol) and a small crystal of
DMAP. The resultant mixture was stirred for 12 h at room
temperature. The reaction mixture was worked up by passing through
a small silica column and flushing with EtOAc. The crude product was
then injected on a 250 × 10 mm Varian Dynamax Microsorb 60-8 C18
HPLC column and eluted with a gradient of 70−100% MeOH/H2O.
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The elution at approximately 17.08 min yielded the corresponding
(S)-MTPA ester 1b (0.4 mg): partial H NMR data of H-6 to H-12
imply endorsement by the U.S. Government. We gratefully
acknowledge K. Woerpel and C. Rieder (NYU) for helpful
suggestions on endoperoxide cleavage, and H. Bokesch (MTL),
S. Tarasov, and M. Dyba (Biophysics Resource Core, Structural
Biophysics Laboratory, CCR) for acquiring high-resolution
mass spectra.
1
(CDCl3, 600 MHz), δ 6.5399 (1H, dd, J = 11.2, 10.4 Hz, H-7), 5.8574
(1H, dt, J = 10.0, 2.3 Hz, H-9), 5.4952 (1H, dd, J = 10.4, 6.6 Hz, H-6),
5.3740 (1H, dt, J = 9.3, 2.5 Hz, H-10), 5.2312 (1H, ddd, J = 6.4, 4.0,
2.1 Hz, H-11), 1.2342 (3H, d, J = 6.6 Hz, H-12); HREIMS [M + H]+
at m/z 730.2813 (calcd for C36H42F6NO8 730.2809). Treatment of 1a
(1.0 mg) in a similar way with (S)-MTPA chloride in pyridine and
DMAP gave the corresponding (R)-MTPA ester 1c (0.3 mg): partial
1H NMR data of H-6 to H-12 (CDCl3, 600 MHz), δ 6.5569 (1H, dd, J
= 11.2, 10.3 Hz, H-7), 5.8391 (1H, dt, J = 9.9, 2.2 Hz, H-9), 5.5026
(1H, dd, J = 10.4, 6.6 Hz, H-6), 5.3686 (1H, dt, J = 9.2, 2.5 Hz, H-10),
5.2181 (1H, ddd, J = 6.3, 4.1, 2.0 Hz, H-11), 1.2515 (3H, d, J = 6.6 Hz,
H-12); HREIMS [M + H]+ at m/z 730.2813 (calcd for C36H42F6NO8
730.2809).
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ASSOCIATED CONTENT
* Supporting Information
NMR spectra for compounds 1−4 are available free of charge
via the Internet at http://pubs.acs.org.
■
S
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AUTHOR INFORMATION
Corresponding Author
*Tel: +1 301-846-1942. Fax: +1 301-846-6177. E-mail:
beutlerj@mail.nih.gov.
1295−1298.
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Notes
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The authors declare no competing financial interest.
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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, as well as
by funds from the NIH Office of Dietary Supplements, Grant
OD-Y2-OD-1557-01. This research was supported, in part, by
the Intramural Research Program of the NIH, National Cancer
Institute, Center for Cancer Research. 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
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