Chemical study and absolute configuration of a new marine secospatane from the brown alga Dilophus okamurae

Article abstract of DOI:10.1021/jo9902190

A new diterpene, dilkamural (1), of the secospatane class has been isolated from the brown alga Dilophus okamurae Dawson, and its structure, including absolute stereochemistry, has been determined by NMR spectroscopic and chemical means.

Full text of DOI:10.1021/jo9902190

5436
J . Org. Chem. 1999, 64, 5436-5440
Ch em ica l Stu d y a n d Absolu te Con figu r a tion of a New Ma r in e
Secosp a ta n e fr om th e Br ow n Alga Diloph u s oka m u r a e
Masayori Ninomiya,*,†,‡ Hideo Hirohara,†,§ J un-ichi Onishi,†,| and Takenori Kusumi*,
Marine Greens Laboratory Company, Ltd., Mori, Iyo, Ehime 799-3192, J apan, and Faculty of
Pharmaceutical Sciences, Tokushima University, Tokushima 770-8505, J apan
Received February 5, 1999
A new diterpene, dilkamural (1), of the secospatane class has been isolated from the brown alga
Dilophus okamurae Dawson, and its structure, including absolute stereochemistry, has been
determined by NMR spectroscopic and chemical means.
Marine organisms produce unique secondary metabo-
lites, some of which have important biological and phar-
maceutical activities.1 In the course of our screening for
bioactive components of the marine algae growing around
Shikoku Island of J apan, we isolated from the brown alga
Dilophus okamurae Dawson (Dictyotaceae), a secospa-
tane-type diterpenoid 1, designated dilkamural, having
moderate antimicrobial activity against a Gram-positive
microorganism. We herein describe the structure and
absolute configuration of the new compound, elucidated
by the chemical and spectroscopic analyses of 1 and its
derivatives.
D. okamurae Dawson2 was collected in 1994 at Ehime,
J apan, and immediately freeze-dried. The dried algal
mass yielded a new secospatane diterpenoid 1 as a color-
less oil in 1.9% yield. The molecular formula of C24H34O6
was established by high-resolution mass spectrometry
and analysis of the 13C NMR data. The presence of two
acetoxyl groups attached to secondary carbons was indi-
cated by the 1H NMR (C6D6) signals at δ 1.60 (s, 3H),
1.61 (s, 3H), 4.80 (dd, 1H), and 5.59 (dd, 1H) and the 13C
NMR signals at δ 169.3 (s), 169.8 (s), 75.3 (d), 76.2 (d),
20.6 (q), and 20.7 (q). The NMR showed the signals due
to a secondary methyl group (δ 0.69, d, 3H), an aldehyde
group [δ 9.86 (1H, d); δ 197.8 (d)], three olefinic methyl
groups [δ 1.41, 1.53, 1.66 (each bs, 3H)], and two olefinic
protons [δ 5.09, 5.16 (each bt, 1H)]. The 13C NMR showed
a ketone carbonyl signal at δ 214.9 (s). The existence of
a cyclopentanone and acetoxy groups was supported by
the IR bands at 1739 and 1240 cm-1. Extensive NMR
work (H-H COSY, C-H COSY, NOESY, HMBC) on this
compound led to the structure (1) with the secospatane
framework.
rapidly around the single bond connecting them on the
NMR time scale.
When 1 was allowed to stand with silica gel and
CH2Cl2,3 (5R,13Z)-5-acetoxy-10-oxo-4,10-secospata-2,13-
(15),17-trien-12-al (2) was formed. This compound has
been isolated from Dilophus marginata Okamura by an
Australian group,4 and its NMR data and stereochemical
features were identical with those of the present de-
acetoxyl compound. However, neither the relative ster-
eochemistry of the A versus B rings nor its absolute
configuration had been established for 2. Therefore, we
attempted to elucidate the absolute configuration of the
respective rings.5
Treatment of 2 with carboxylesterase in 100 mM
phosphate buffer (pH 7.0) yielded deacetylated product
(3). To establish the absolute configuration of the 5-OH
group, the (S)- and (R)-MTPA esters (4) were prepared.6
The relative stereochemistry of the substituents on
each of the A and B rings was determined by NOEs and
coupling constants of the ring protons. The stereochem-
istry of the A ring relative to the B ring, however, could
not be established, because the rings apparently rotate
* To whom correspondence should be addressed. FAX: +81-886-
33-7288. E-mail: tkusumi@ph2.tokushima-u.ac.jp.
† Marine Greens Laboratory.
The ∆δ () δS - δR; ppm) values were calculated for the
respective protons, and they are summarized in the
formula 4′. No systematic arrangement of the ∆δ values
was observed in 4′; the modified Mosher’s method failed
‡ Present address: Marutomo Co., Ltd., Kominato, Iyo, Ehime 799-
31, J apan.
§ Present address: Department of Material Science, The University
of Shiga Prefecture, Hassaka, Hikone 522, J apan.
| Present address: Sumitomo Chemical Co., Ltd., Takatsukasa,
Takarazuka, Hyogo 655, J apan.
(3) Ishitsuka, O. M.; Kusumi, T.; Ichikawa, A.; Kakisawa, H.
Phytochemistry 1990, 29, 2605.
Tokushima University.
(1) Faulkner, D. J . Nat. Prod. Rep. 1998, 15, 259 and references
therein.
(2) The voucher specimen is preserved in this laboratory.
(4) Ravi, B. N.; Wells, R. J . Aust. J . Chem. 1982, 35, 129.
(5) Kusumi, T.; Hamada, T.; Ishitsuka, M. O.; Ohtani, I.; Kakisawa,
H. J . Org. Chem. 1992, 57, 1033.
10.1021/jo9902190 CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/09/1999

Study of a New Marine Secospatane from Dilophus
J . Org. Chem., Vol. 64, No. 15, 1999 5437
in this case. This anomaly seemed to be caused by the
flexibility of the molecule due to the single-bond linkage
(C8-C9) between the two rings, which might result in
the different conformations of the (R)- and (S)-MTPA
diastereomers (4).
To resolve this difficulty, we fixed the conformation by
building a new linkage utilizing the aldehyde at C-12 and
the carbonyl oxygen at C-10. Reduction of 2 with NaBH4
in methanol gave diol 5 (the stereochemistry of 5 is
commented on at the end of the text), in which the
conjugated double bond was concomitantly reduced as a
single isomer. The intramolecular cyclization of 5 was
carried out with TsCl in pyridine to give 6, which was
saponified (NaOMe/MeOH) into 7.
The modified Mosher’s method was applied to this
compound (see 8′). Although the R configuration of 5-OH
was easily deduced by these results, another problem
arose: The 1H NMR spectra (600 MHz, CDCl3) of the
MTPA esters (8) exhibited heavily overlapped signals in
the aliphatic region 2.2-1.2 ppm, which made the as-
signment of the proton signals and, therefore, the ster-
eochemistry of the substituents extremely difficult.
esters (13). The ∆δ values of the protons summarized in
the formula (10′′) are systematically arranged on the
right- and left-hand sides of the MTPA plane, confirming
the R configuration of the hydroxyl group at C-5. This
finding automatically established the absolute stereo-
chemistry of dilkamural (1) as shown.
The reaction course of the Mitsunobu reaction of 9
should be considered here. The reagent (DEAD-PPh3)
would activate the primary alcohol (12-OH) preferentially
rather than the secondary alcohol (10-OH). In this case,
since the free secondary alcohol would attack at C-12
(10S -9-prim: route A), the secondary hydroxyl group (10-
OH) should have the S configuration considering the
stereochemistry of 10. It turned out, however, that the
benzoate (15) of 9 showed a negative Cotton effect at 230
nm (EtOH), indicating the R configuration of 10-OH. The
modified Mosher’s method carried out on 9 (see 9′) also
supported the R configuration of the 10-OH. These
results imply that in the Mitsunobu reaction the acti-
vated secondary alcohol (C-10) of 10R-9-sec was attacked
by the primary alcohol (12-OH), which resulted in inver-
sion of the configuration at C-10 (route B).
On the basis of these facts, we propose the following
reaction course: The phosphonium activator will attach
to either the secondary (10R-9-sec) or primary (10R-9-
prim) alcohols, the latter being sterically preferable.
These two species would be in equilibrium.9 In 10R-9-
prim, attack of the secondary alcohol to the primary
carbon (route C) would give sterically strained trans-anti-
trans product 17, while the cyclized trans-anti-cis product
10 from 10R-9-sec is sterically less compressed (route
A).10 Here, one should recall that the tosylation of 5
To overcome this problem, we turned to cyclization of
allylic alcohol 9 (the stereochemistry of 10-OH was not
determined at this stage), which was successfully ob-
tained by reduction with NaBH4 in the presence of cerium
chloride7 in MeOH. The attempted intramolecular cy-
clization of diol 9 with TsCl, however, was unsuccessful
and only gave C-12 tosylate (11). Apparently, the C-10
hydroxyl group was not positioned for a backside attack
at the C-12 tosylate carbon (vide infra). We were de-
lighted to find that the Mitsunobu reaction8 carried out
on 9 resulted in the formation of cyclization product (10)
in a good yield.
1
The H NMR spectrum of the cyclized product exhib-
ited well-separated signals of the protons, and the
relative stereochemistry of this compound was firmly
established by the NOEs as shown in 10′. The cyclized
compound 10 was then saponified, giving 12, and the
alcohol was further converted to the (S)- and (R)-MTPA
(6) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J . Am. Chem.
Soc. 1991, 113, 4092.
(7) Luche, J .-L. J . Am. Chem. Soc. 1978, 29, 2226.
(8) Mitsunobu, O.; Eguchi, M. Bull. Chem. Soc. J pn. 1971, 44, 3472.
(9) Robinson, P. L.; Barry, C. N.; Bass, S. W.; J arvis, S. E.; Evans,
S. A. J . Org. Chem. 1983, 48, 5396.

5438 J . Org. Chem., Vol. 64, No. 15, 1999
Ninomiya et al.
ported as feeding-deterrent substances against the young
abalone.11 Diterpenoid 1, present in a large amount (1.9%
in freeze-dried alga), might therefore serve as a chemical
defense substance against mollusks and fish.
Exp er im en ta l Section
1H NMR (13C NMR) spectra were recorded at 270 (67.5) and
600 (150) MHz. Chemical shifts are reported relative to TMS
(δ 0), and coupling constants are given in hertz.
Alga l Collect ion a n d Isola t ion of Dilk a m u r a l 1. D.
okamurae Dawson was collected in May 1994 at the intertidal
zones around the Honai Beach, Ehime Prefecture, J apan. The
collection was immediately washed with seawater and freeze-
dried. Dried alga (100 g) was powdered and soaked in 1 L of
EtOAc for 1 day at room temperature with shaking. The EtOAc
was evaporated in vacuo to give a dark-green residue (3.0 g)
that was applied to a column (2.5 × 20 cm) of COSMOSIL
PREP C18 (Nakarai Tesque), and the column was eluted with
500 mL of acetonitrile to remove the pigments. The eluate was
concentrated under reduced pressure and applied to a column
(4 × 45 cm) of Kieselgel 60 (Merck), and the column was eluted
with a stepwise gradient solvent system of EtOAc-hexane.
The eluate from 20% EtOAc/hexane was charged on a normal-
phase HPLC column (2 × 25 cm) of SIL-06 (YMC), and it was
eluted with 5% EtOH/hexane to yield pure dilkamural (1) (1.9
g): [R]27 +32° (c ) 0.30, CHCl3); IR (film) 1738, 1374, 1238,
D
1174, 1024 cm-1; HRFABMS m/z 359.2222 (MH+ - AcOH, 0
mamu deviation, C22H31O4); low-resolution FABMS obsd m/z
359 (MH+ - AcOH, 20), 299 (MH+ - 2AcOH, 100), 281 (21);
1H NMR (270 MHz, C6D6) δ 9.86 (1H, d, J ) 1.7 Hz, H-12),
5.59 (1H, ddd, J ) 7.6, 6.2, 4.3 Hz, H-5), 5.16 (1H, bt, J ) 6.2
Hz, H-15), 5.09 (1H, bt, J ) 7.0 Hz, H-17), 4.80 (1H, dd, J )
5.4, 1.4 Hz, H-2), 3.48 (1H, ddd, J ) 7.6, 6.5, 1.7 Hz, H-4),
3.38 (1H, dt, J ) 8.4, 8.1, 5.9 Hz, H-7), 3.06 (1H, m, H-8), 2.67
(2H, m, H-16), 2.48 (1H, m, H-1), 2.31 (1H, dd, J ) 9.7, 7.3
Hz, H-9), 2.11 (1H, dd, J ) 5.4, 19.4 Hz, H-3R), 1.99 (1H, dd,
J ) 19.4, 1.4 Hz, H-3â), 1.90 (1H, ddd, J ) 14.6, 6.2, 5.9, H-6R),
1.77 (1H, ddd, J ) 14.6, 8.1, 4.3 Hz, H-6â), 1.66 (3H, s, H-19),
1.61 (3H, s, H-22), 1.60 (3H, s, H-24), 1.53 (3H, s, H-20), 1.41
(3H, s, H-14), 0.69 (3H, d, J ) 7.3 Hz, H-11); 13C NMR (67.5
MHz, C6D6) δ 214.9 (s, C-10), 197.8 (d, C-12), 169.8 (s, C-23),
169.3 (s, C-21), 134.5 (s, C-13), 132.0 (s, C-18), 129.0 (d, C-15),
123.0 (d, C-17), 76.2 (d, C-5), 75.3 (d, C-2), 57.9 (d, C-4), 50.6
(d, C-9), 41.6 (t, C-3), 40.5 (d, C-7), 40.0 (d, C-1), 37.5 (d, C-8),
37.1 (t, C-6), 27.5 (t, C-16), 25.8 (q, C-14), 22.2 (q, C-19), 20.7
(q, C-24), 20.6 (q, C-22), 17.7 (q, C-20), 13.6 (q, C-11).
afforded the cyclized product (6: trans-anti-cis), whereas
the reaction of 9 with TsCl afforded only the tosylate 11
instead of the expected cyclized trans-anti-trans product
18. This fact is also compatible with the retarded cycliza-
tion of 10S-9-prim and the instability of the trans-anti-
trans system.
Dea cetoxyla tion of 1. A solution of dilkamural (1; 0.5 g,
1.20 mmol) in CH2Cl2 (10 mL) was allowed to stand with 2 g
of Kieselgel 60 (Merck) overnight. The silica gel was washed
with CH2Cl2, and the washing was concentrated to give 2
quantitatively. Compound 2: [R]27 +33° (c ) 0.33, CHCl3);
D
IR (film) 1738, 1703, 1460, 1374, 1342, 1237, 1067, 1024 cm-1
;
HRFABMS m/z 359.2223 (MH+, 0.1 mamu deviation, C22H30O4);
low-resolution FABMS obsd m/z 359 (MH+, 8), 299 (MH+
-
Hydrogenation of the benzoate 15 by using a homoge-
1
1
neous catalyst afforded 19, the H NMR data of which
AcOH, 13); H NMR (270 MHz, C6D6) δ 9.82 (1H, d, J ) 1.5
Hz, H-12), 6.77 (1H, dd, J ) 5.9, 3.0 Hz, H-2), 5.77 (1H, dd, J
) 5.9, 1.9 Hz, H-3), 5.64 (1H, ddd, J ) 7.3, 6.8, 3.5, H-5), 5.18
(1H, bt, J ) 7.3 Hz, H-15), 5.08 (1H, bt, J ) 6.3 Hz, H-17),
3.73 (1H, ddd, J ) 10.0, 7.3, 1.5 Hz, H-4), 3.46 (1H, m, H-7),
3.17 (1H, m, H-8), 2.63 (3H, m, H-1, 16), 2.14 (2H, m, H-6R,
9), 1.82 (1H, m, H-6â), 1.65 (3H, s, H-14), 1.59 (3H, s, H-24),
1.54 (3H, s, H-20), 1.45 (3H, s, H-19), 0.88 (3H, d, J ) 7.3 Hz,
H-11).
were different from those of 20, which was obtained by
monoacetylation of 5 followed by benzoylation. This
finding unambiguously establishes the stereochemistry
of 5.
Diterpenoids 1 and 2 exhibited an antimicrobial activ-
ity against Bacillus subtilis, a Gram-positive microorgan-
ism: 1 and 2 showed 12 and 9 mm of clear zone diameter
at 10 µg/ disk, respectively, in the agar-disk diffusion
method. Compound 1 also showed weak inhibitory activ-
ity against a species of plant pathogenic mold (Colletot-
richum lagenarium). Some secospatanes have been re-
Hyd r olysis of 2. A solution of 2 (0.107 g, 0.30 mmol) in
MeOH (1 mL) was added into 200 mM phosphate buffer (pH
7.0; 50 mL) containing 12 mg of carboxyl esterase (EC 3.1.1.1,
crude product from Streptomyces rochei) (Wako Pure Chemi-
cal), and the mixture was incubated at 30 °C for 24 h. The
mixture was extracted with EtOAc (3 × 50 mL), and the
combined EtOAc extract was washed with water (3 × 100 mL)
(10) Substituted cis-hydroindans are known to be more thermody-
namically stable than trans-hydroindans. The presence of two trans-
fused cyclopentane units in 17 would make the tricyclic system less
favorable than 10 (or 6). Validation of this assumption by MM
calculation is in progress.
(11) Taniguchi, K.; Kurata, K.; Suzuki, M. Kagaku to Seibutsu 1994,
32, 434.

Study of a New Marine Secospatane from Dilophus
J . Org. Chem., Vol. 64, No. 15, 1999 5439
and dried over anhydrous MgSO4. Evaporation of the solvent
afforded an oil, which was purified by HPLC [YMC-pack SIL-
06 (2 × 25 cm) (YMC)]. Elution with 20% EtOH/hexane yielded
the hydrolyzed product 3 (0.061 g, 0.19 mmol, 63%). Compound
3: IR (film) 3423, 1704, 1381, 1344, 1077 cm-1; HRFABMS
m/z 317.2113 (MH+, 0.3 mamu deviation, C20H29O3); 1H NMR
(270 MHz, CDCl3) δ 9.88 (1H, d, J ) 1.9 Hz, H-12), 7.59 (1H,
dd, J ) 5.7, 3.0 Hz, H-2), 6.02 (1H, dd, J ) 5.7, 1.6 Hz, H-3),
5.26 (1H, bt, J ) 7.4 Hz, H-15), 5.06 (1H, bt, J ) 6.2 Hz, H-17),
4.80 (1H, dt, J ) 5.9, 3.0 Hz, H-5), 3.67 (1H, ddd, J ) 8.6, 8.4,
6.2 Hz, H-7), 3.55 (1H, ddd, J ) 11.1, 5.9, 1.9 Hz, H-4), 3.00
(2H, m, H-1,8), 2.76 (2H, m, H-16), 2.33 (1H, dd, J ) 9.2, 5.9
Hz, H-9), 2.13 (1H, m, H-6R), 1.88 (1H, ddd, J ) 14.2, 8.4, 3.0
Hz, H-6â), 1.71 (3H, s, H-14), 1.66 (3H, s, H-20), 1.65 (3H, s,
H-19), 1.17 (3H, d, J ) 7.3 Hz, H-11).
MTP A Ester s of 3. The hydrolyzed compound 3 (30 mg,
0.09 mmol) was dissolved in 1 mL of CH2Cl2, into the solution
were added DMAP (0.1 mmol), DCC (0.1 mmol) and (S)- or
(R)-R-methoxy-R-(trifluoromethyl)phenylacetic acid (MTPA)
(0.2 mmol), and the reaction mixture was allowed to stand at
room temperature for 18 h. The solvent was evaporated in
vacuo, and the crude product was purified by HPLC (see
above). Elution with 5% EtOH/hexane yielded the MTPA
esters 4 [(S)-MTPA ester, 12 mg, 0.023 mmol, 25%; (R)-MTPA
ester, 9 mg, 0.017 mmol, 19%). 4 [(R)-MTPA ester]: IR (film)
1749, 1700, 1452, 1273, 1171, 1123, 1021 cm-1; HRFABMS
m/z 532.2441 (M+, 0.5 mamu deviation, C30H35F3O5); 1H NMR
(270 MHz, CDCl3) δ 9.58 (1H, d, J ) 2.2 Hz, H-12), 7.58 (1H,
dd, J ) 5.9, 3.0 Hz, H-2), 7.42 (5H, m, MTPA), 6.01 (1H, dd, J
) 5.9, 1.9 Hz, H-3), 5.86 (1H, dt, J ) 6.5, 2.7 Hz, H-5), 5.29
(1H, bt, J ) 6.6 Hz, H-15), 5.02 (1H, bt, J ) 7.3 Hz, H-17),
3.71 (1H, ddd, J ) 10.7, 6.5, 2.2 Hz, H-4), 3.49 (3H, s, MTPA),
3.44 (1H, m, H-7), 2.96 (2H, m, H-1, 8), 2.68 (2H, m, H-16),
2.30 (2H, m, H-6R, 9), 1.92 (1H, ddd, J ) 14.8, 8.4, 2.7 Hz,
H-6â), 1.71 (3H, s, H-14), 1.65 (3H, s, H-20), 1.63 (3H, s, H-19),
1.11 (3H, d, J ) 7.3 Hz, H-11). 4 [(S)-MTPA ester]: δ 9.50
(1H, d, J ) 1.6 Hz, H-12), 7.57 (1H, dd, J ) 5.9, 2.0 Hz, H-2),
7.44 (5H, m, MTPA), 6.01 (1H, dd, J ) 5.9, 1.6 Hz, H-3), 5.92
(1H, dt, J ) 6.3, 3.5 Hz, H-5), 5.31 (1H, bt, J ) 7.6 Hz, H-15),
5.03 (1H, bt, J ) 7.3 Hz, H-17), 3.77 (1H, m, H-4), 3.54 (1H,
m, H-7), 3.48 (3H, s, MTPA), 3.01 (2H, m, H-1, 8), 2.71 (2H,
m, H-16), 2.36 (2H, m, H-9, 6R), 2.00 (1H, ddd, J ) 14.3, 8.1,
3.5 Hz, H-6â), 1.70 (3H, s, H-14), 1.67 (3H, s, H-20), 1.64 (3H,
s, H-19), 1.14 (3H, d, J ) 7.6 Hz, H-11).
6 (26 mg, 0.075 mmol, 29%). 6: IR (film) 1738, 1375, 1358,
1238, 1100, 1078 cm-1; HREIMS m/z 346.2509 (M+, 0.1 mamu
deviation, C22H34O3); H NMR (270 MHz, CDCl3) δ 5.27 (1H,
1
dt, J ) 5.7, 1.9 Hz, H-5), 5.15 (1H, bt, J ) 7.6 Hz, H-15), 5.06
(1H, bt, J ) 7.3 Hz, H-17), 3.92 (1H, m, H-10), 3.80 (1H, dd, J
) 10.5, 4.6 Hz, H-12â), 3.60 (1H, dd, J ) 10.5, 9.2 Hz, H-12R),
3.49 (1H, dt, J ) 8.6, 5.1 Hz, H-7), 2.73 (2H, m, H-16), 2.07
(4H, m, H-1, 6, 8), 2.10 (3H, s, H-24), 1.89 (2H, m, H-3R, 9),
1.69 (3H, s, H-14), 1.66 (3H, s, H-20), 1.63 (2H, m, 2R, 3â),
1.62 (3H, s, H-19), 1.48 (1H, m, H-2â), 0.99 (3H, d, J ) 7.0
Hz, H-11).
Hyd r olysis of 6. The cyclized product 6 (30 mg, 0.087
mmol) was dissolved in MeOH (150 µL), and the solution was
treated with a 1 M solution of sodium methoxide in methanol
(15 µL). The mixture was allowed to stand for 4 h at room
temperature and poured onto a 200 mM phosphate buffer
solution (pH 7.5; 5 mL). The product was extracted with EtOAc
(3 × 50 mL). The organic extract was washed with water and
dried over anhydrous MgSO4, and the solution was concen-
trated in vacuo. Purification by HPLC (see above) eluted with
20% EtOH/hexane afforded the deacetylated product 7 (17 mg,
0.056 mmol, 64%). This product was directly used for the next
reaction.
MTP A Ester s of 7. The (S)- and (R)-MTPA esters of 7 (each
8 mg, 0.026 mmol) were prepared according to the aforemen-
tioned method, yielding the MTPA esters 8 [(S)-MTPA ester,
12 mg, 0.023 mmol, 89%; (R)-MTPA ester, 11 mg, 0.021 mmol,
81%]. 8 [(S)-MTPA ester]: IR (film) 1746, 1275, 1240, 1170,
1123, 1022 cm-1; HRFABMS m/z 520.2805 (M+, 0.5 mamu
deviation, C30H39F3O4); 1H NMR (600 MHz, CDCl3; several
signals are overlapping) δ 7.55 (2H, m, MTPA), 7.42 (3H, m,
MTPA), 5.16 (1H, m, H-5), 5.46 (1H, bt, J ) 7.5 Hz, H-15),
5.05 (1H, bt, J ) 7.2 Hz, H-17), 3.97 (1H, m, H-10), 3.76 (1H,
dd, J ) 10.5, 4.9 Hz, H-12â), 3.56 (3H, s, MTPA), 3.45 (2H, m,
H-7, 12R), 2.77 (1H, m, H-16a), 2.63 (1H, m, H-16b), 2.06 (3H,
m, H-4, 6, 8), 1.99 (1H, m, H-1), 1.90 (1H, m, H-9), 1.82 (1H,
m, H-3R), 1.69 (3H, s, H-14), 1.67 (3H, s, H-20), 1.62 (3H, s,
H-19), 1.58 (2H, m, H-2R, 3â), 1.36 (1H, m, H-2â), 0.93 (3H, d,
J ) 7.2 Hz, H-11). 8 [(R)-MTPA ester]: 1H NMR (600 MHz,
CDCl3; several signals are overlapping) δ 7.52 (2H, m, MTPA),
7.42 (3H, m, MTPA), 5.41 (1H, dt, J ) 4.2, 1.7 Hz, H-5), 5.15
(1H, bt, J ) 7.2 Hz, H-15), 5.03 (1H, bt, J ) 6.3 Hz, H-17),
3.98 (1H, m, H-10), 3.80 (1H, dd, J ) 10.8, 7.2 Hz, H-12R),
3.55 (1H, dd, J ) 10.8, 3.0 Hz, H-12â), 3.54 (3H, s, MTPA),
3.35 (1H, dt, J ) 9.0, 6.0 Hz, H-7), 2.74 (1H, m, H-16a), 2.59
(1H, m, H-16b), 2.09 (1H, m, H-4), 1.97 (1H, m, H-1), 1.88 (1H,
m, H-9), 1.82 (1H, m, H-3R), 1.68 (3H, s, H-14), 1.66 (3H, s,
H-20), 1.61 (3H, s, H-19), 1.58 (2H, m, H-3â, 2R), 1.36 (1H, m,
H-2â), 0.89 (3H, d, J ) 7.6 Hz, H-11).
Sod iu m Bor oh yd r id e Red u ction of 2 in th e P r esen ce
of Cer iu m Ch lor id e. To a methanolic solution (15 mL) of 2
(0.462 g, 1.29 mmol) and CeCl3‚7H2O (0.961 g, 2.58 mmol) was
added a methanol solution (2 mL) of NaBH4 (0.097 g, 2.58
mmol) with stirring at 0 °C. The reaction mixture was allowed
to stand for 10 min, and 100 mL of water was added. The
mixture was extracted with EtOAc (3 × 100 mL), and the
extract was washed with water, dried over anhydrous MgSO4,
and concentrated in vacuo. After purification by HPLC (see
above) eluted with 20% EtOH/hexane, diol 9 (0.145 g, 0.40
mmol, 31%) was obtained. 9: IR (film) 3370, 1738, 1440, 1376,
1245, 1107, 1042 cm-1; HRFABMS m/z 345.2423 (MH+ - H2O,
0.6 mamu deviation, C22H33O3); 1H NMR (270 MHz, CDCl3) δ
5.90 (1H, m, H-2), 5.64 (1H, bd, J ) 5.7 Hz, H-3), 5.52 (1H, dt,
J ) 6.2, 4.1 Hz, H-5), 5.15 (1H, bt, J ) 7.3 Hz, H-15), 5.06
(1H, bt, J ) 7.0 Hz, H-17), 4.67 (1H, bd, J ) 7.8 Hz, H-10),
3.87 (1H, dd, J ) 11.9, 4.1 Hz, H-12a), 3.70 (1H, dd, J ) 11.9,
6.5 Hz, H-12b), 3.50 (1H, ddd, J ) 11.3, 9.7, 6.5 Hz, H-7), 2.71
(5H, m, H-1, 4, 9, 16), 2.34 (1H, m, H-8), 2.08 (3H, s, H-24),
2.04 (1H, m, H-6R), 1.67 (1H, m, H-6â), 1.71 (3H, s, H-14), 1.70
(3H, s, H-20), 1.62 (3H, s, H-19), 0.91 (3H, d, J ) 7.3 Hz, H-11).
Cycliza tion of 9 by th e Mitsu n obu Rea ction . A solution
of 9 (0.110 g, 0.30 mmol), diethyl azodicarbonate (DEAD as a
40% toluene solution, 150 µL, 0.34 mmol), and triphenylphos-
phine (90 mg, 0.34 mmol) in THF (2 mL) was allowed to stand
at room temperature overnight. The reaction mixture was
Sod iu m Bor oh yd r id e Red u ction of 2. A solution of 2
(0.212 g, 0.59 mmol) in MeOH (5 mL) was treated with a
MeOH solution (1 mL) of NaBH4 (0.045 g, 1.18 mmol) with
stirring at 0 °C. The mixture was stirred for 10 min, and water
(50 mL) was added. The product was extracted with EtOAc (3
× 50 mL). The EtOAc extract was washed with water, dried
over anhydrous MgSO4, and concentrated in vacuo. Purifica-
tion was performed by HPLC (see above) eluted with 20%
EtOH/hexane, yielding the 1,4-reduced diol 5 (95 mg, 0.26
mmol, 44%). 5: IR (film) 3370, 1738, 1450, 1376, 1244, 1025
cm-1; HREIMS m/z 364.2599 (M+, 1.4 mamu deviation,
C
22H36O4); 1H NMR (270 MHz, CDCl3) δ 5.45 (1H, dt, J ) 6.8,
3.5 Hz, H-5), 5.12 (1H, bt, J ) 7.8 Hz, H-15), 5.07 (1H, bt, J )
7.3 Hz, H-17), 4.34 (1H, t, J ) 4.3 Hz, H-10), 3.92 (1H, dd, J
) 12.2, 4.1 Hz, H-12), 3.88 (1H, dd, J ) 12.2, 5.1 Hz), 3.53
(1H, dt, J ) 8.1, 7.8, 4.9 Hz, H-7), 2.74 (3H, m, H-8, 16), 2.36
(2H, m, H-4, 9), 2.23 (1H, m, H-1), 2.09 (3H, s, H-24), 2.04
(1H, m, H-6R), 1.83 (3H, m, H-2R, 3R, 6â), 1.69 (3H, s, H-14),
1.66 (3H, s, H-20), 1.63 (3H, s, H-19), 1.52 (2H, m, H-2â, 3â),
1.11 (3H, d, J ) 7.3 Hz, H-11).
Cycliza tion of 5 w ith Tosyl Ch lor id e. The diol 5 (0.095
g, 0.26 mmol) was dissolved in pyridine (6 mL), and the
solution was cooled in an ice bath. Tosyl chloride (0.05 g, 0.27
mmol) was added to this solution in two portions at 1 h
intervals with stirring. The reaction mixture was allowed to
warm to room temperature, and after 24 h, an aqueous
NaHCO3 solution (100 mL) was added. The mixture was
extracted with EtOAc (3 × 50 mL). The EtOAc extract was
washed with water, dried over anhydrous MgSO4, and con-
centrated in vacuo. Purification was performed by HPLC (see
above), eluted with 5% EtOH/hexane to yield the cyclic ether

5440 J . Org. Chem., Vol. 64, No. 15, 1999
Ninomiya et al.
concentrated, and the product was purified by HPLC eluted
IR (film) 1741, 1717, 1366, 1272, 1248, 1112, 1070, 1027 cm-1
;
with 5% EtOH/hexane to yield the cyclic ether 10 (56 mg, 0.163
HRFABMS m/z 386.2455 (M+ - C7H6O2, 0.2 mamu deviation,
mmol, 54%). 10: IR (film) 1738, 1372, 1237, 1073, 932 cm-1
;
C24H34O4); H NMR (270 MHz, CDCl3) δ 8.01 (2H, d, J ) 8.1
1
HREIMS m/z 344.2342 (M+, 0.9 mamu deviation, C22H32O3);
1H NMR (270 MHz, CDCl3) δ 5.95 (1H, m, H-2), 5.73 (1H, m,
H-3), 5.26 (1H, bt, J ) 4.3 Hz, H-5), 5.19 (1H, bt, J ) 7.0 Hz,
H-15), 5.06 (1H, bt, J ) 7.0 Hz, H-17), 4.33 (1H, bd, J ) 3.5
Hz, H-10), 3.88 (1H, dd, J ) 10.3, 6.8 Hz, H-12R), 3.54 (2H,
m, H-7, 12â), 2.76 (3H, m, H-1, 16), 2.11 (5H, m, H-4, 6, 8, 9),
2.05 (3H, s, H-24), 1.69 (3H, s, H-14), 1.67 (3H, s, H-20), 1.62
(3H, s, H-19), 1.07 (3H, d, J ) 7.3 Hz, H-11).
Hz, 10-OBz), 7.52 (3H, m, 10-OBz), 6.04 (1H, m, H-2), 5.85
(1H, bd, J ) 5.9 Hz, H-3), 5.74 (1H, bd, J ) 7.8 Hz, H-10),
5.50 (1H, m, H-5), 5.24 (1H, bt, J ) 7.6 Hz, H-15), 5.08 (1H,
bt, J ) 7.0 Hz, H-17), 4.11 (1H, dd, J ) 11.1, 8.6 Hz, H-12a),
3.97 (1H, dd, J ) 11.1, 4.6 Hz, H-12b), 3.51 (1H, m, H-7), 2.76
(4H, m, H-1, 4, 16), 2.39 (2H, m, H-8, 9), 2.09 (1H, m, H-6R),
2.03 (3H, s, H-24), 1.87 (1H, m, H-6â), 1.80 (3H, s, H-14), 1.70
(3H, s, H-20), 1.69 (3H, s, 12-OAc), 1.64 (3H, s, H-19), 0.98
(3H, d, J ) 7.0 Hz, H-11).
MTP A Ester s of 12. The cyclized compound 10 (14 mg,
0.041 mmol) was saponified (NaOH/MeOH) to give the deacet-
ylated product 12 (12 mg, 0.039 mmol, 95%). Halves of 12 (6
mg, 0.017 mmol) were converted to (S)- and (R)-MTPA esters
by the aforementioned method to yield the diastereomeric
MTPA esters 13 [(S)-MTPA ester, 8 mg, 0.015 mmol, 88%; (R)-
MTPA ester, 7 mg, 0.013 mmol, 76%]. 13 [(S)-MTPA ester]:
IR (film) 1748, 1452, 1272, 1180, 1125, 1019 cm-1; HRFABMS
m/z 518.2650 (M+, 0.6 mamu deviation, C30H37F3O4); 1H NMR
(270 MHz, CDCl3) δ 7.51 (2H, m, MTPA), 7.40 (3H, m, MTPA),
5.90 (1H, m, H-2), 5.68 (1H, m, H-3), 5.44 (1H, bs, H-5), 5.21
(1H, bt, J ) 7.3 Hz, H-15), 5.05 (1H, bt, J ) 7.3 Hz, H-17),
4.36 (1H, bd, J ) 3.8 Hz, H-10), 3.84 (1H, m, H-12R), 3.49 (1H,
m, H-12â), 3.54 (3H, s, MTPA), 3.44 (2H, m, H-7), 2.80 (1H,
m, H-16R), 2.65 (2H, m, H-1, 16â), 2.13-2.17 (5H, m, H-4, 6,
8, 9), 1.70 (3H, s, H-14), 1.69 (3H, s, H-20), 1.65 (3H, s, H-19),
1.00 (3H, d, J ) 7.3 Hz, H-11). 13 [(R)-MTPA ester]: 1H NMR
(270 MHz, CDCl3) δ 7.49 (2H, m, MTPA), 7.40 (3H, m, MTPA),
5.96 (1H, m, H-2), 5.69 (1H, m, H-3), 5.40 (1H, dt, J ) 3.5 Hz,
H-5), 5.19 (1H, bt, J ) 7.3 Hz, H-15), 5.04 (1H, bt, J ) 7.3 Hz,
H-17), 4.36 (1H, bd, J ) 5.7 Hz, H-10), 3.89 (1H, m, H-12R),
3.60 (1H, m, H-12â), 3.53 (3H, s, MTPA), 3.34 (1H, m, H-7),
2.76 (1H, m, H-16R), 2.61 (2H, m, H-1, 16â), 2.12-2.18 (5H,
m, H-4, 6, 8, 9), 1.69 (3H, s, H-14), 1.67 (3H, s, H-20), 1.61
(3H, s, H-19), 0.97 (3H, d, J ) 7.3 Hz, H-11).
Attem p ted Cycliza tion of 9. To a solution of 9 (25 mg,
0.07 mmol) in pyridine (1 mL), cooled in an ice bath, was added
TsCl (26 mg, 0.14 mmol) in two portions at 1 h intervals with
stirring. The reaction mixture was allowed to warm to room
temperature, and after 24 h, 100 mL of aqueous NaHCO3 was
added. The mixture was extracted with EtOAc (3 × 50 mL),
and the EtOAc extract was washed with water, dried over
anhydrous MgSO4, and concentrated. HPLC purification by
elution with 20% EtOH/hexane gave C12-tosylate 11 (15 mg,
0.029 mmol, 41%). 11: IR (film) 3517, 1738, 1452, 1362, 1242,
1177, 1108, 948 cm-1; HRFABMS m/z 439.2312 (MH+ - H2O
- CH3COOH, 0.6 mamu deviation, C27H35O3S); 1H NMR (270
MHz, CDCl3) δ 7.80 (2H, d, J ) 8.6 Hz, 12-OTs), 7.34 (2H, d,
J ) 7.8 Hz, 12-OTs), 5.88 (1H, m, H-2), 5.58 (1H, bd, J ) 5.7
Hz, H-3), 5.40 (1H, dt, J ) 6.2, 4.1 Hz, H-5), 5.13 (1H, bt, J )
7.0 Hz, H-15), 5.03 (1H, bt, J ) 7.0 Hz, H-17), 4.47 (1H, dd, J
) 9.2, 4.3 Hz, H-12a), 4.40 (1H, bt, J ) 7.3 Hz, H-10), 4.23
(1H, t, J ) 9.2 Hz, H-12b), 3.44 (1H, m, H-7), 2.70 (5H, m,
H-1, 4, 9, 16), 2.44 (3H, s, ArMe), 2.11 (1H, m, H-8), 1.97 (1H,
m, H-6R), 1.97 (3H, s, H-24), 1.80 (1H, m, H-6â), 1.69 (3H, s,
H-14), 1.64 (3H, s, H-20), 1.61 (3H, s, H-19), 1.43 (1H, bd, J )
7.3 Hz, 10-OH), 0.86 (3H, d, J ) 7.0 Hz, H-11).
P r ep a r a tion of MTP A Ester s of 14. The (S)- and (R)-
MTPA esters of 14 (each 15 mg, 0.038 mmol) were prepared
according to the aforementioned method. MTPA 16 [(S)-MTPA
ester, 14 mg, 0.023 mmol, 61%; (R)-MTPA ester, 14 mg, 0.023
mmol, 61%]. 16 [(S)-MTPA ester]: IR (film) 1743, 1367, 1246,
1170, 1025 cm-1; HRFABMS m/z 386.2451 (M+ - C7H6O2, 0.6
mamu deviation, C24H34O4); 1H NMR (270 MHz, CDCl3) δ 7.55
(2H, m, MTPA), 7.40 (3H, m, MTPA), 6.08 (1H, m, H-2), 5.81
(1H, m, H-3), 5.74 (1H, bd, J ) 5.4 Hz, H-10), 5.18 (2H, m,
H-5, 15), 5.05 (1H, bt, J ) 7.0 Hz, H-17), 4.06 (1H, dd, J )
11.3, 7.6 Hz, H-12a), 3.94 (1H, dd, J ) 11.3, 5.4 Hz, H-12b),
3.58 (3H, s, MTPA), 3.38 (1H, m, H-7), 2.77 (1H, m, H-1), 2.71
(2H, m, H-16), 2.28 (1H, m, H-9), 2.27 (1H, m, H-8), 2.03 (3H,
s, H-24), 2.01 (3H, s, 12-OAc), 1.84 (1H, m, H-6R), 1.71 (1H,
m, H-6â), 1.69 (3H, s, H-14), 1.62 (3H, s, H-20), 1.59 (3H, s,
H-19), 0.92 (3H, d, J ) 6.8 Hz, H-11). 16 [(R)-MTPA ester]:
1H NMR (270 MHz, CDCl3) δ 7.52 (2H, m, MTPA), 7.42 (3H,
m, MTPA), 6.03 (1H, m, H-2), 5.76 (1H, m, H-3), 5.73 (1H, bd,
J ) 5.4 Hz, H-10), 5.31 (1H, m, H-5), 5.19 (1H, bt, J ) 6.8 Hz,
H-15), 5.05 (1H, m, H-17), 4.10 (1H, dd, J ) 11.3, 8.6 Hz,
H-12a), 3.97 (1H, dd, J ) 11.3, 5.4 Hz, H-12b), 3.46 (3H, s,
MTPA), 3.41 (1H, m, H-7), 2.79 (1H, m, H-1), 2.72 (2H, m,
H-16), 2.40 (1H, m, H-4), 2.28 (1H, m, H-9), 2.28 (1H, m, H-8),
2.02 (3H, s, H-24), 2.01 (3H, s, 12-OAc), 1.92 (1H, m, H-6R),
1.77 (1H, ddd, J ) 13.8, 7.6, 4.3 Hz, H-6â), 1.69 (3H, s, H-14),
1.62 (6H, s, H-19, 20), 0.92 (3H, d, J ) 7.0 Hz, H-11).
P r ep a r a tion of 19 w ith Wilk in son ’s Ca ta lyst. A solution
of 15 (8 mg, 0.015 mmol) in MeOH (5 mL) was stirred with
Wilkinson’s catalyst [chlorotris(triphenylphosphine)rhodium-
(I)] (0.05 g) under hydrogen at room temperature for 1 h. The
mixture was applied onto a silica gel column (0.5 × 25 cm)
[Kieselgel 60 (Merck)], and the column was eluted with EtOAc/
hexane (1:3). Purification was performed by HPLC (see above)
eluted with 2% EtOAc/hexane, yielding 19 (4 mg, 0.008 mmol,
53%): 1H NMR (270 MHz, CDCl3) δ 8.00 (2H, m), 7.56 (1H,
m), 7.45 (2H, m), 5.51 (1H, m), 5.22 (1H, bt, J ) 7.3 Hz), 5.08
(2H, m), 4.07 (1H, dd, 11.0, 8.9 Hz), 3.89 (1H, dd, J ) 11.0,
4.3 Hz), 3.43 (1H, m), 2.02 (3H, s), 1.80 (3H, s), 1.70 (3H, s),
1.64 (6H, s), 0.88 (3H, d, J ) 7.0 Hz).
P r ep a r a tion of 20. A solution of 5 (20 mg, 0.07 mmol) and
Ac2O (300 µL) in pyridine (1 mL) was stirred at 0 °C for 15
min. Workup and HPLC separation gave C12-acetate of 5. This
acetate (13 mg, 0.03 mmol) was treated with benzoyl chloride
to yield 20 (3 mg, 0.006 mmol, 20%); 1H NMR (270 MHz,
CDCl3) δ 7.99 (2H, m), 7.56 (1H, m), 7.44 (2H, m), 5.85 (1H,
m), 5.25 (1H, bt, J ) 6.8 Hz), 5.13 (2H, m), 4.81 (1H, bd, J )
6.2 Hz), 4.49 (1H, bs), 3.43 (1H, m), 2.01 (3H, s), 1.90 (3H, s),
1.83 (3H, s), 1.70 (3H, s), 1.63 (3H, s), 0.92 (3H, d, J ) 7.6
Hz).
Ben zoyla tion of 14. A solution of 9 (32 mg, 0.09 mmol)
and Ac2O (300 µL) in pyridine (1 mL) was stirred in an ice
bath for 15 min. The usual workup and purification by HPLC
yielded the C12-acetate 14 (24 mg, 0.047 mmol, 52%), which
was dissolved in 1 mL of pyridine. The solution was cooled in
an ice bath and treated with 100 µL of benzoyl chloride, and
the mixture was stirred for 30 min. An aqueous NaHCO3
solution (10 mL) was added, and the mixture was extracted
with EtOAc (3 × 10 mL). The EtOAc extract was washed with
water, dried over anhydrous MgSO4, and concentrated in
vacuo. Purification by HPLC eluted with 5% EtOH/hexane
yielded the C10-benzoate 15 (24 mg, 0.047 mmol, 52%). 15:
Ack n ow led gm en t. We are grateful to Dr. K. Wa-
tanabe, Sumitomo Chemical Co., Ltd., for his helpful
discussion on the structure of dilkamural (1) and to Mr.
T. Suzuki, Industrial Research Center of Ehime Pre-
fecture, for his help of obtaining the low- and high-
resolution mass spectra and optical rotation data.
J O9902190