384 Bull. Korean Chem. Soc. 1998, Vol. 19, No. 3 Notes
Reinvestigation of the Structure of Galbanic Acid by 2D NMR
Techniques Including 2D INADEQUATE
Sueg-Geun Lee*, Shi Yong Ryu, and Jong Woong Ahn
Korea Research Institute of Chemical Technology, P.O. Box 107, Yusung, Taejon 305-606, Korea
Received November 18, 1997
The galbanic acid (1) is a sesquiterpene coumarin ether
occurred in the resin of Ferula assa-foetida L. (Umbelliferae)
and also found in various species of the Ferula genus. It
had been isolated for the first time from the gum resin of F.
microloba in 19721 and its structure (2) was postulated by
Borisov et al. in 1973.2 However, the proposed structure
was completely revised as 1 in 1980 by an analysis of 300
MHz 'H NMR spectrum? On the other hand, Kajimoto and
his co-worker had isolated a compound named asacoumarin
B (3) from F. assa-foetida in 1989, and suggested a structure
which had been quite different from that of I.4 They
had proposed the structure of asacoumarin B particularly by
the investigation of the ozonolysis product. However, the
asacoumarin B was revealed recently to be a galbanic acid
(1) by Appendino et al.5 They had investigated the structure
of 3 by the aid of 2-D NMR techniques, which was not depicted
in detail in literature, and confirmed the structure of
galbanic acid as 1 suggested by Bagirov et al. in 1980?
In the process of searching for new compounds from the
gum resin of F. assafoetida, we found the presumed same
compound as 1 at least on the basis of the patterns and
나lemical 아)ifts of and 13C NMR. Although there are
numerous indirect ways6 to elucidate the carbon skeleton of
organic molecules, indirect approach fails and some ambiguities
remain further in the assignment of carbon resonances
when the proton spectra do not exhibit well resolved signals.
All of these limitations could be solved if we could measure
the direct carbon-carbon correlations.
Because the importance of correct chemical shifts assignments
even can never be overstated to unlock all the information
present in the spectrum, it is worth reinvestigating
the controversial structure of galbanic acid (1).
Consequently, we reinvestigate the structure of 1 by 2D
NMR techniques including 2D INADEQUATE and herein
report the unambiguous results and correct stereochemistry
of 1.
Experimental
Isolation of galbanic acid. The gum resin of F. assafoetida
was obtained from Chien Yuen Herbal Medicinal
Co., Taipei, Taiwan and was identified by the Herbarium of
Natural Products Research Institute, Seoul National University,
Korea, where the voucher specimen is deposited.
The gum resin of F. assafoetida (1.2 kg) was refluxed
with MeOH for 4 hours. The extract was collected and concentrated
to dryness to give a crude extract (ca. 460 g), which
was suspended in water and subjected to partition with hexane
(56 g), CH2C12 (290 g) and n-butanol (50 g), successively.
Five grams of CH2C12 soluble part was purified by
silica gel column chromatography and eluted with hexane
and EtOAc mixture by gradient manner (R广0.3, hexane/
EtOAc=3 :1) to give ca. 150 mg of galbanic acid, which
was further purified by preparative TLC on silica gel.
NMR Measurements. All NMR spectra were recorded
on Bruker AMX-500 spectrometer. In all cases, a 5 mm
Willmad NMR tube was used and 80-90 mg of galbanic
acid (1) was dissolved in 0.5 mL of CDC13.
Proton and carbon chemical shifts are reported in ppm relative
to TMS, but were measured against the central solvent
peak at 7.24 and 77.00 ppm, respectively. The spectrum
was collected as 32 K data points over an 7500 Hz spectral
width using a 30 °C pulse (Normal ID) and data were processed
using exponential multiplication with a 1.5 Hz line
broadening.
The COSY spectrum was obtained using COSY-45
pulse sequence. The spectral widths were both set at 7500
Hz. An initial matrix of 1 K (256 data points was zero-filled
to give 1 Kx 1 K and was processed by sinusoidal multiplication
in each dimension followed by symmetrization of
the final data matrix. Eight transients with a 1 s delay were
accumulated for each of the 256 increments. The total acquisition
time was 40 min.
The long range COSY was obtained by the cosylr program
with the 0.12 s dalay for evolution of long range couplings.
For each increment, 32 scans were accumulated and
the total acquisition time was 3 h.
The two dimensional NOESY spectrum in phase-sensitive
mode was recorded using the noesytp program, where the
mixing time and the relaxation delay time used were 0.3 s
and 1 s, respectively. Scans were accumulated for each of
Notes Bull. Korean Chem. Soc. 1998, Vol. 19, No. 3 385
the 512 increments, and the total acquisition time was 6.5 h.
The collected 1 Kx 1 K was processed by squared sine-bell
window shifted by k/2 in both dimensions.
In the heteronuclear correlation (HETCOR) experiments,
the spectral widths were 35 KHz in F2 and 7.5 KHz in Fl.
The initial matrix of 1 Kx 128 was zero-filled to 2 Kx256
to give the final spectrum. For each increment, 16 transients
were accumulated; the relaxation delay was 1 s and the total
acquisition time was 40 min.
The long-range 0-*。HETCOR spectra were obtained
with the parameter optimized by JC-h=4, 6, and 9 Hz, respectively.
Other acquisition parameters were the same as in the
normal HETCOR experiment except that for each increment
256, 192, and 128 scans respectively; the total acquisition
time for each experiment was 13, 9, and 6 h, respectively.
In the 2D INADEQUATE experiment, the spectral width
of Fl dimension was the half of the F2=22730 Hz to increase
the digital resolution of Fl dimension (vide infra).
The initial matrix of 4 Kx 242 was zero-filled to 4 Kx 256
to give the final spectrum and was processed by exponential
multiplication (LB=2.5) in F2 dimension and n/2 shifted
sine-bell window in Fl dimension. For each increment, 512
transients were accumulated; the relaxation delay was 2 s
and the 13C-13C coupling constant was set to 45 Hz and the
total acquisition time was 74 h.
Results and Discussion
High Resolution EIMS revealed the formula C24H30O5 (m/
z 398.2130, 3.7 mmu error) for 1. A combination of DEPT7
and HETCOR experiments8 of 1 confirmed that there were
four CH3 groups, five CH2 groups, seven CH groups, and
eight quaternary carbons in the molecule. A large carbon
peak corresponding to two carbons at 8 20.06 was found to
be two methyl carbons linked to 8 1.56 and 8 1.39 methyl
protons in HETCOR spectrum, respectively. The remaining
one proton was concluded to be a carboxylic exchangeable
proton and observed as a broad signal at 8 9.8-8.8.
Without the aid of 13C-13C connectivity experiment, the
presence of a coumarin moiety was easily confirmed by the
proton COSY, long-range COSY, and HETCOR experiments.
The results of long-range HETCOR correlations of
nine 13C chemical signals were in good agreement with the
coumarin moiety. However, the remaining moiety of the
molecule could not be established by the usual and
connectivity because of the incomplete analysis of
proton spectrum in which the signals at 8 1.9-1.8 consist of
four protons; one methine proton, one of methylene protons,
and two of other methylene protons.
Therefore, we first determined direct 13C-13C connectivities
by using two-dimensional INADEQUATE experiment.9
The pulse sequence employed was the 90°-T-l800-t-90°-tr
90°-t2 with quadrature detection in Fl dimension.
The partial carbon-13 2D INADEQUATE spectrum of 1
obtained by the condition of F2=2F1 (folded twice) is shown
in the Figure 1. Beginning with the carbon resonating at
71.53 ppm (Figure 1), we can easily identify that this peak
is responsible for the carbon 11 referring to the multiplicities
of carbon spectrum determined by DEPT experiment8
and proton chemical shift determined by C-H HETCOR experiment.9
This methylene carbon has a correlation with the
quaternary carbon C-9 at 40.59 ppm. Then this quaternary
carbon C-9 has additional correlations peaks with two methine
carbons at C-8 (8, 34.68) and C-10 (6, 42.47), and
methyl carbon at C-15 (8, 22.35). The methine carbon C-8
has a correlation peak with the C-7 resonating at 31.79 ppm
which has an additional correlation peak with the C-6 at
24.32 ppm. Then both C-6 and C-10 have a correlation
peak with the C-5 at 129.36 ppm. By doing this way, all
the correlations are drawn parallel to F2 axis as shown in
the Figure 1 except those between the C-2 and C-3 and
between the C-4 and C-5.
The strongly coupled AB connection between the C-4
and C-5 was confirmed by the long range C-H correlation
peaks between the C-5 and methyl protons at C-14 and the
C-4 and the methine protons at C-10 and one of methylene
proton (6a) at C-6.
j ■ I ' 1
ppm 60 50 30
5
Figure 1. Partial 2D-INADEQUATE spectrum of 1.
PPM 3 2
Figure 2. 2D-NOESY spectrum of 1.
386 Bull. Korean Chem. Soc. 1998, Vol. 19, No. 3 Notes
Figure 3. Revised configuration of galbanic acid.
The carboxylic group was also confirmed by the long
range C-H correlation peaks between the carbonyl carbon 3
at 180.02 ppm and the methylene protons at C-2.
With this carbon fragment information and the C-H
HETCOR experiment, the phase sensitive NOESY experiment
was done for stereochemical analysis. The expanded
partial NOESY spectrum of 1 is shown in Figure 2.
Based on the fact that the carbons at C-4, C-5, C-6, C-10,
C-13, and C-14 are in the same plane, we may easily
predict the NOE cross peaks between protons at C-13 and
C-10 or C-l.
Figure 2 shows clearly the NOE cross peak between the
protons at C-10 and C-13 which says that the previous
chemical shift assignments of methyl protons at C-13 and C-
14 are reversed.5 On the other hand, the methyl protons at
C-14 show the NOE peak with the a-proton at C-6. Thus,
the NOE results of the methyl protons at C-13 and C-14
not only can correct the previous chemical shift assignments
of these protons, but can support the pseudo-equatorial position
of the protons at C-10 and H-6a. '
With these fixed two positions and the coupling constants
(J) 13.7, 13.0, 13.0 and 4.7 Hz in dddd splitting pattern of
H-7a, we can arrange the stereochemistry of H-6p and H-8
to have anti-parallel axial positions with respect to H-7a,
respectively. The further streochemical arrangements in C-8
and C-9 are supported by the NOE peaks between the H-
7a and one proton at C-ll, and between the protons at C-12
and the other proton at C-ll. Thus, the two methyl groups,
which were assigned as a and p previously, at C-8 and C-9
should have a and 0 positions in the twisted boat conformation,
respectively. The revised configuration of 1 is
shown in Figure 3 with partial conformations. The final assignments
of chemical shifts of the a and。protons at C-6
and C-7, which belong to equatorial or axial protons of cyclohexane
depending on position, are valid for relationship,
5 Ha<8 氏
In summary, we have shown that unambiguous structural
analysis of galbanic acid (1) was done by using modem 2D
NMR techniques including the 2D INADEQUATE experiment.
It is also evident that the stereochemistry at C-8 and
C-9 determined previously should be revised.
References
1. Kamilov, Kh. M.; Nikonov, G. K. Khim. Prirodn.
Soedin, 1972, 114.
2. Borisov, V. N.; Ban'kovski, A. L; Sheichenko, V. L; Pimenov,
M. G.; Zakharov, P. I. Khim, Prirodn. Soedin.
1973, 429.
3. Bagirov, V. Y.; Sheichenoco, V. I.; Veselovskaya, N. V.;
Skylar, Y. E.; Savina, A. A.; Kir'yanova, I. A. Khim.
Prirodn. Soedin. 1980, 620. Chem. Abstr. 1981, 95,
25279.
4. Kajimoto, T.; Yahiro, K.; Nohara, T. Phytochemistry
1989, 28, 1761.
5. Appendino, G.; Tagliapietra, S.; Nano, G. M.; Jakupovic,
J. Phytochemistry 1994, 35, 183.
6. Two-Dimensional NMR spectroscopy. Application for
Chemists and Biochemists; W. R. Croasman and R. M.
K. Carlson, Eds.; Methods in stereochemical Analysis 6;
VCH Publisher, 1987 and references cited therein.
7. Doddrell, D. M.; Pegg, D. T.; Bendall, M. R. J. Magn.
Res. 1982, 48, 323.
8. Bax, A.; Morris, G. A. J. Magn. Res. 1981, 42, 501.
9. Bax, A.; Freeman, R.; Frenkiel, T. A. J. Am. Chem. Soc.
1981, 103, 2102.
Enantioselective Synthesis of the C20-C25 Portion
of the Cytotoxic Natural Product, Amphidinolide Bl
Duck-Hyung Lee* and Man-Dong Rho
Department of Chemistry, Sogang University, Seoul 121-742, Korea
Received November 18, 1997
Amphidinolides A-Q have recently been isolated from dinoflagellates,
genus Amphidinium, symbiotic with the Okinawan
marine flatworms and generally exhibited potent toxicities
against cancer tumor cell lines.1,2 Although the chemical
structures of them were elucidated mai끼y on the basis
of extensive spectroscopic studies including 2D NMR experiments,
their configurations still remain unclear except
for amphidinolides B, J, and L. The relative stereochemical
relationship of amphidinolide B group (1), the most representative
and important group of amphidinolides family, was
unambiguously solved by single crystal X-ray diffraction
method* and the absolute configuration of amphidinolide Bl
(la) was established on the basis of enantiospecific synthesis
of a degradation product* The absolute configurations
of amphidinolide J and L were determined similarly.4a,4b
In our program toward the total synthesis of amphidin이ide
Bl (la), the target molecule was initially divided
into three components 2a, 2b, and 2c and enantioselective