Total syntheses of neuroprotective mastigophorenes A and B

Article abstract of DOI:10.1016/S0040-4020(01)00631-7

(-)-Herbertenediol (3) which is regarded as a biosynthetic precursor of mastigophorenes A and B has been effectively synthesized from (R)-1,2-dimethyl-2-cyclopentene carboxylic acid by applying an intramolecular Heck reaction to the construction of the quaternary carbon center, and then horseradish peroxidase-catalyzed oxidative coupling of 3 has given rise to (-)-mastigophorenes A and B. Mastigophorenes A and B have been found to exhibit significant neuroprotective activity in primary cultures of fetal rat cortical neurons.

Full text of DOI:10.1016/S0040-4020(01)00631-7

TETRAHEDRON
Pergamon
Tetrahedron 57 %2001) 7127±7135
Total syntheses of neuroprotective mastigophorenes A and B
Yoshiyasu Fukuyama,a,p Keiji Matsumoto,a Yasutoshi Tonoi,a Ritsuko Yokoyama,a
Hironobu Takahashi,a Hiroyuki Minami,a Hiroshi Okazakib and Yasuhide Mitsumotob
aFaculty of Pharmaceutical Sciences, Institute of Pharmacognosy, Tokushima Bunri University, Yamashiro-cho,
Tokushima 770-8514, Japan
bTokushima New Drug Research Institute, Otsuka Pharmaceutical Co. Ltd., Tokushima 771-0192, Japan
Received 16 May 2001; accepted 11 June 2001
AbstractÐ%2)-Herbertenediol %3) which is regarded as a biosynthetic precursor of mastigophorenes A and B has been effectively syn-
thesized from %R)-1,2-dimethyl-2-cyclopentene carboxylic acid by applying an intramolecular Heck reaction to the construction of the
quaternary carbon center, and then horseradish peroxidase-catalyzed oxidative coupling of 3 has given rise to %2)-mastigophorenes A and B.
Mastigophorenes A and B have been found to exhibit signi®cant neuroprotective activity in primary cultures of fetal rat cortical neurons.
q 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
which features oxidative dimerization of %2)-3 and mono-
MOM derivative 18 by using HRP %horseradish peroxidase)
and %tert-BuO)2, respectively.
Mastigophorenes A %1) and B %2),1,2 isolated from the
Boruneo liverwort Mastigophora diclados, are unique
herbertane-type sesquiterpene dimers which exhibit
intriguing neurotrophic properties, i.e. promote neuronal
outgrowth and enhance choline acethyltransferase activity
in the primary cultures of fetal rat cerebral hemisphere.2
These sesquiterpene dimers 1 and 2 %Fig. 1) are presumably
formed by one-electron oxidative phenolic coupling
of %2)-herbertenediol %3)3 co-occurring in the above
liverwort.4 The ®rst synthesis of 1 and 2 which involved
the oxidative phenolic coupling of the mono-o-methyl
derivative 19 was recently communicated by Bringmann
et al.5,6 and the two unique syntheses were followed by
the atropo-diastereoselective constaction of the biaryl axis
of 1 and 2.7,8 These prompted us to account in detail for our
independent synthesis and biological evaluation of mastigo-
phorenes A %1) and B %2).9
2. Results and discussion
2.1. Synthesis of !2)-herbertenediol !3)
In the preceding paper,10 we reported the ef®cient synthesis
of the racemic herbertenediol %3) by employing the intra-
molecular Heck reaction for the crucial contraction of the
quaternary carbon directly bonded to the benzene ring. Thus
the synthesis of optical active %2)-3 started from the
preparation of %R)-carboxylic acid 7, to which the same
procedure used for the synthesis of the racemic 3 was
applied.
Koga et al.11 reported a convenient procedure for the syn-
thesis of both enantiomers of a,a-dialkyl-b-keto esters with
a predictable chirality depending on the solvent system.
Herein, we report the synthesis of the optical active 1 and 2,
Figure 1. Structures of mastigophorenes A %1) and B %2), and %2)-herbertenediol %3).
Keywords: mastigophorene; herbertane-type sesquiterpene; Heck reaction; horseradish peroxidase; neuroprotective activity.
Corresponding author. Tel.: 181-88-622-9611; fax: 181-88-655-3051; e-mail: fukuyama@ph.bunri-u.ac.jp
p
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020%01)00 631-7

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Y. Fukuyama et al. / Tetrahedron 57 32001) 7127±7135
Scheme 1. Reagents, conditions and yields: %a) %S)-valine tert-butyl ester, Et2O´BF3, benzene, 99%. %b) LDA, HMPA, MeI, 2788C and then 2258C, toluene,
40%. %c) MeMgI, Et2O. %d) P2O10, benzene. %e) KOH, MeOH/H2O, 33% over three steps.
Scheme 2. Reagents, conditions and yields: %a) 2,4,6-tri-ClBzCl, Et3N, THF. %b) 2-iodo-p-cresol, DMAP, benzene, 65%. %c) 10mol% Pd%OAc) 2, 20mol%
%o-tol)3P, n-Bu3N, DMF, 1208C, 94%. %d) 10% Pd/C, H2, EtOH, 95%. %e) LiAlH4, THF. %f) MeI, K2CO3, acetone, 98% over two steps. %g) 4-bromophenyl
isocyanate, DABCO, toluene.
According to the Koga protocol, lithiated chiral enamine 5
prepared from methyl 2-oxo-cyclopentanecarboxylate and
%S)-valine tert-butyl ester can be alkylated with methyl
iodide in a toluene solvent containing HMPA, after
hydrolysis, to give %R)-6, whereas %S)-6 can be prepared
from the same enamine 5 if THF is added to a toluene
solvent instead of HMPA %Scheme 1).
racemic one.10 Yamaguchi's coupling between 7 and
2-iodo-p-cresol gave an ester 8 in 65% yield. Intramolecular
Heck reaction of 8 was employed under the established
conditions such as 10mol% Pd%OAc) 2, 20mol% % o-tol)3P
and n-Bu3N in DMF giving rise to a d-lactone 9 in 94% yield
as a mixture of double bond isomers. Hydrogenation of 9,
followed by LiAlH4 reduction, yielded the diol 11, the
phenolic hydroxyl group of which was selectively methyl-
ated under the basic conditions giving rise to 12 in 93%
yield over three steps %Scheme 2).
Thus, the chiral enamine 5 was lithiated with LDA in the
presence of HMPA and then alkylated with methyl iodide at
2258C, after hydrolysis, to give 6 in 40% yield %98% ee).
The absolute con®guration of the newly formed quaternary
carbon was determined as R based on the optical rotation
which was identical with that of 6 prepared in the other way
by Fujiwara.12 On the other hand, the %S)-6 was obtained in
67% ee when the reaction was run in the presence of THF
instead of HMPA. This chirality was consistent with Koga's
prediction. Next, the %R)-6 was converted to an optical
active carboxylic acid 7 by the same procedures as the
To make sure of the con®guration of the quaternary carbon
newly formed by the intramolecular Heck reaction, 12 was
converted to a p-bromophenyl carbamate 13, which
provided single crystals suitable for X-ray crystallographic
analysis. The ORTEP drawing of 13 with the correct
absolute con®guration,13 as shown in Fig. 2, indicates the
favorable S con®guration on the C-7 carbon.
Swern oxidation of the primary alcohol in 12 afforded the
aldehyde 14 in 96% yield. Subsequent Hunag±Minlon
reduction of 14 yielded %2)-a-herbertenol %16)3 after
deprotection of the o-methyl group with BBr3. Matsuo et
al.3 already converted 16 to a mixture of %2)-herbertenediol
%3) and its positional isomer of the hydroxyl group by the
direct oxidation of 16 with benzoyl peroxide in poor yield.
Thus we exploited more ef®cient method of hydroxylation
on the benzene ring. The hydroxyl group of 16 was ®rst
converted to MOM-ether 17, which was lithiated with sec-
BuLi regioselectively at the ortho-position to the MOM
group and then treated with Davis reagent14 giving rise to
a hydoxylated product 18 in 51% yield. Treatment of 18
with 48% HBr gave %2)-herbertenediol %3), which was
identical in all respects with natural one %Scheme 3).
Figure 2. X-Ray ORTEP drawing of 13.

Y. Fukuyama et al. / Tetrahedron 57 32001) 7127±7135
7129
Scheme 3. Reagents, conditions and yields: %a) %COCl)2, DMSO, Et3N, CH2Cl2, 96%. %b) N2H4´H2O, NaOH, DEGL, 1808C, 83%. %c) BBr3, CH2Cl2, 2788C
and then rt, 66%. %d) MOMCl, i-Pr2NEt, CH2Cl2, 71%. %e)sec-BuLi, TMEDA, Davis reagent, THF, 51%. %f) 48% HBr, MeOH, 98%.
2.2. Syntheses of mastigophorenes A !1) and B !2)
oxidative dimerization of the monomethyl ether 19 to give
21 in 56% yield, much better than the reported yield %28%).6
Chemical oxidative couplings of 3 involving %tert-BuO)2,
however, failed to directly yield dimers 1 and 2. We were
previously succeeded in the formation of the benzodioxane-
type dimers from caffeic acid by using horseradish peroxi-
dase %HRP) catalyzed oxidative phenolic coupling.15 Thus
we applied this enzyme method to the oxidative coupling of
3 itself. A solution of 3 in acetonitrile was incubated with
HRP %200 units, type II) in phosphate buffer %pH 6.0) in the
presence of hydrogen peroxide for 6 days,16 resulting in the
Mastigophorenes A and B are biogenetically derived from
%2)-herbertenediol %3) by oxidative phenol coupling.2
Bringmann et al.6 already reported the oxidative dimeri-
zation of the mono-methly ether 19 using %tert-BuO)2. We
applied ®rst this method to oxidative coupling of the mono-
MOM ether 18, thereby giving rise to dimers 20 as an
inseparable diastereomeric mixture in 13.5% yield, which
led, after deprotection, to 1 and 2 in 34 and 29% yield,
respectively. On the other hand, we attempted the same
Scheme 4. Oxidative coupling of herbertenediol %3) and its derivatives; reagents and conditions: %a) 48% HBr, MeOH for 20. %b) BBr3, CH2Cl2 for 21.

7130
Y. Fukuyama et al. / Tetrahedron 57 32001) 7127±7135
formation of 1 %10%) and 2 %18%) with the recovery 3
%72%).
It should be noted that HRP-catalyzed oxidative coupling of
3 is superior to chemical oxidative ones in terms of simple
operation as well as of recycling 3 %Scheme 4).
3. Neuroprotective activity of 1 and 2
We reported that mastigophorenes A %1) and B %2) showed
an interesting neurotrophic activity in the primary neuronal
cultures in the serum-containing medium.1,2 With synthetic
1 and 2 in hand, we are now in position to be able to evaluate
more neurotrophic property of them. The present primary
cultures were performed using 18-day fetal rat cortical
neurons in the serum-free DMEM medium supplemented
with N2 as described by Abe et al.17 Morphorogical evalua-
tions of neurons, as shown in Fig. 3, indicated that
mastigophorenes A and B not only promoted signi®cantly
neurite outgrowth but also maintained the neuronal survival
at concentrations of 0.1 and 1 mM. However, they were
found to be toxic against neurons at a concentration of
10 mM. Neuronal survival was measured by the WST-8
reduction assay.18 As shown in Fig. 4, 1 and 2 exhibited
potent effects on the neuronal survival in primary cultures.
Particularly, they enhanced signi®cantly the survival of
neurons at a concentration of 1 mM in comparison of the
control cultures, but they lost their survival effect at 10 mM.
These results suggest that mastigophorenes A and B can
protect the neurons from being damaged by toxic substances
such as oxygen free radicals.
In conclusion, we have synthesized %2)-herbertenediol %3)
by following our previously developed procedure based on
the intramolecular Heck reaction, and then have found that
HRP catalyzed oxidative coupling serves as an effective
method for 3 being dimerized into mastigophorenes A %1)
and B %2). Moreover, our biological assay results indicate
that mastigophorenes A and B have potent neuroprotective
effect on the neuronal survival in the primary cultures in
addition to their outgrowth promoting activity. Further
Figure 3. Enhancement of neurite outgrowth by mastigophorenes A and B
in primary cultures of E18 SD rat cortical neurons. After the neuronal cells
%5£105 cells cm22) cultured for 5 days in the presence of 0.5% EtOH and
mastigophorenes A and B were ®xed by 4% paraformaldehyde-PBS, the
immunohistochemical staining for the microtuble associated protein-2 were
performed. Pictures taken with 150magni®cations. %a) .05% EtOH,
%b) 1 mM mastigophorene A, and %c) 1 mM mastigophorene B.
Figure 4. Enhancement of survival of rat cortical neurons by mastigophorenes A and B in primary cultures. Effects of survival were assessed by the WST-8
reduction assay. The data are expressed as means SE %nˆ4); pP,0.05, ppP,0.015, pppP,0.005 versus control.

Y. Fukuyama et al. / Tetrahedron 57 32001) 7127±7135
7131
biological studies on signi®cant neuroprotective effects are
under way.
dried over MgSO4, concentrated in vacuo to leave the
residue, which was puri®ed by column chromatography
%hexane/etherˆ10:1) to give 6 [%297 mg, 40% based on
54% recovery of 5 %1.19 g)] as an oil. The ee for 6 was
determined to be 98% by GC analysis with chiral column
%column: CYCLODEX-B; ®lm thickness: 0.25 mm; B
0.254 mm£30m; 50mL He/min; temperature: 70 8C;
4. Experimental
4.1. General
retention time R: 60.03 min, S: 63.17 min); [a]19
ˆ
D
1
Melting points were determined on a Yanagimoto micro
melting point apparatus without correction. IR, UV and
CD spectra were recorded on JASCO 5300 FT-IR, Shimazu
210.88 %c 1.28, CHCl3); H NMR %200 MHz, CDCl3) d
1.31 %3H, s), 1.83±2.06 %3H, m), 2.31±2.55 %3H, m), 3.70
%3H, s).
1
UV-300 and JASCO J-500 spectrometers, respectively. H-
and 13C-NMR spectra were taken on a Varian unity-200 or a
JEOL GX-400 spectrometer. Chemical shifts are expressed
in d units %part per million down®eld from Me4Si). Mass
spectra %MS) were recorded on a JEOL AX-500. Air- and
moisture-sensitive reagents were transferred via syringe or
cannula, and reactions involving these materials were
carried out in oven-dried ¯asks under a positive pressure
of argon. Silica gel %Wako, C-300) was used for column
chromatography. Analytical thin-layer chromatographies
%TLC) were performed with Merck precoated TLC plates
%Kiselgel 60F 254, 0.25 mm), and spots were visualized with
ultraviolet light, iodide, and 40% CeSO4/H2SO4.
4.2.3. !R)-1,2-Dimethyl-2-cyclopentenecarboxylate !7). A
solution of MeMgI was prepared from Mg %110mg,
4.54 mmol) and MeI %0.29 mL, 4.54 mmol) in ether
%5 mL). To the stirred Grignard solution, 6 %590mg,
3.78 mmol) in ether %5 mL) was added at room temperature
for 30min. The mixture quenched by the addition of
saturated NH4Cl solution, extracted with ether, washed
with saturated NaHCO3 solution, water and saturated
NaCl solution, dried over MgSO4. Evaporation of ether
afforded a residue. To a solution of the residue in benzene
%5 mL) was added phosphorus pentaoxide %4 g). The
mixture was stirred at room temperature for 2 h. Evapo-
ration of solvent gave the residue, which was dissolved in
a mixture of methanol/water %1:1, 10mL), added KOH
%636 mg, 11.34 mmol), and the mixture was stirred for
3 days. This solution was acidi®ed with 2N HCl, extracted
with ether. The organic layer was extracted with sat.
NaHCO3 solution. The resulting aqueous layer was acidi®ed
with 2N HCl, and then extracted with ether. The extract was
washed with water and saturated NaCl solution, dried over
MgSO4, concentrated, and puri®ed by column chroma-
tography %hexane/EtOAcˆ4:1) to give 7 %175 mg, 33%) as
4.2. Synthesis of !2)-herbertenediol
4.2.1. N-!2-Carbomethoxy-1-cyclopenten-1-yl)-!S)-valine
tert-butyl ester !5). To a solution of methyl 2-cyclopenta-
nonecarboxylate %1.56 g, 10.9 mmol) and %S)-valine tert-
butyl ester %3 g, 14.31 mmol) in benzene %60mL) was
added boron tri¯uroride diethyl etherate %0.14 mL,
0.55 mmol), and resulting solution was heated to re¯ux
using Dean±Stark apparatus for 12 h. This reaction mixture
was extracted with ether, washed with aqueous NaHCO3,
water and saturated NaCl solution and dried over MgSO4.
Evaporation of the solvent gave a crude mixture, which was
chromatographed on silica gel %hexane/EtOAcˆ10:1) to
an oil: [a]21 ˆ1128.88 %c 0.86, CHCl3); IR %®lm) 3400,
D
1698, 1448, 1408, 1282, and 1184 cm21
;
1H NMR
%200 MHz, CDCl3) d 1.30%3H, s), 1.72 %1H, t, Jˆ1.5 Hz),
1.80%1H, m), 2.44 %3H, m), and 5.51 %1H, m); EIMS m/z
%rel. int.) 140[M 1] %14), 95 %100), and 79 %10); HREIMS
m/z 140.0833 [M1] %Calcd 140.0837 for C8H12O2).
give 5 %3.24 g, 99%) as an oil: [a]22 ˆ196.88 %c 1.0,
D
CHCl3); IR %®lm) 3315, 1736, 1666, 1606, 1462, 1269,
1
and 1134 cm21; H NMR %200 MHz, CDCl3) d 0.97 %3H,
4.2.4. 2-Iodo-4-methylphenyl 1,2-dimethyl-2-cyclopente-
necarboxylate !8). 2,4,6-Trichlorobenzoyl chloride %0.28
mL, 1.8 mmol) was added to a mixture of 7 %250mg,
1.79 mmol) and triethylamine %0.25 mL, 1.8 mmol) in tetra-
hydrofuran %5 mL). The mixture was stirred for 40min at
room temperature. After the removal of triethylamine
hydrochloride by ®ltration, the ®ltrate was evaporated and
the residue was dissolved in benzene %10mL). To this solu-
tion was added a mixture of 2-iodo-p-cresol %632 mg,
2.7 mmol) and 4-dimethylaminopyridine %440mg, 3.6
mmol) and the resulting mixture was stirred at room
temperature for 41 h. The reaction mixture was extracted
with ether and the ether extract was washed with water,
Cu%NO3)2 solution, NaHCO3 solution and saturated NaCl
solution and dried over MgSO4. Evaporation of the solvent
gave a crude product, which was chromatographed on silica
gel %hexane/CH2Cl2ˆ9:1) to afford 8 %416 mg, 65%) as an
d, Jˆ2.3 Hz), 1.00 %3H, d, Jˆ2.3 Hz), 1.46 %9H, s), 1.82
%2H, q, Jˆ7.4 Hz), 2.10%1H, m), 2.47 %4H, m), 3.64 %1H,
dd, Jˆ10.9, 5.9 Hz), 3.70 %3H, s), and 7.52 %1H, m); EIMS
m/z %rel. int.) 297 [M1] %24), 266 %3), 241 %16), 210%6), 196
%100), and 164 %92); HREIMS m/z 297.1958 [M1] %Calcd
297.1940for C 16H27O4N).
4.2.2. Methyl !R)-1-methyl-2-cyclopentanonecarboxylate
!6). A n-BuLi %1.6 M solution in hexane, 6.57 mL,
10.5 mmol) was added to a solution of diisopropylamine
%1.47 mL, 10.5 mmol) in toluene %14 mL) at 2788C and
the mixture was stirred for 30min at 0 8C. To this in situ
prepared LDA solution was added a solution of 5 %2.60g,
8.75 mmol) in toluene %5 mL) at 2788C and the resulting
solution was stirred for 30min. HMPA %1.83 mL,
10.51 mmol) was added and the reaction mixture was stirred
for 30 min. Methyl iodide %0.67 mL, 10.5 mmol) was added
and the reaction mixture was stirred at 2258C for 5 h. The
reaction was quenched with 4% HCl %20mL) and the whole
mixture was stirred at 08C for 30min and was then extracted
with ether. The organic layer was washed with aqueous
NaHCO3, 5% Na2SO3, water and saturated NaCl solution,
oil: [a]23 ˆ146.38 %c 16.0, CHCl3); IR %®lm) 1751, 1599,
D
1481, 1217, 1130, and 1074 cm21; H NMR %200 MHz,
1
CDCl3) d 1.46 %3H, s, 1.82 %3H, d, Jˆ1.9 Hz), 1.91 %1H,
m), 2.30%3H, s), 2.49 %2H, m), 2.77 %1H, m), 5.57 %1H, m),
6.89 %1H, d, Jˆ8.1 Hz), 7.13 %1H, dd, Jˆ8.1, 1.8 Hz), and

7132
Y. Fukuyama et al. / Tetrahedron 57 32001) 7127±7135
7.63 %1H, d, Jˆ1.8 Hz); EIMS m/z %rel. int.) 356 [M1] %3),
328 %2), 234 %7), and 95 %100); HREIMS m/z 356.0258 [M1]
%Calcd for 356.0273 for C15H17O2I).
%92 mg, 0.39 mmol) and K2CO3 %85 mg, 0.61 mmol) in
acetone %10mL) were added iodomethane %0.20mL,
3.22 mmol) and reaction mixture was re¯uxed for 11 h.
The reaction mixture was diluted with water and extracted
with ether. The resulting organic layer was washed with
saturated NaCl, dried over MgSO4. Evaporation of the
organic solvent gave the residue, which was chromato-
graphed on silica gel %hexane/EtOAcˆ6:1) to afford 12
4.2.5. Intramolecular Heck reaction of 8. A mixture of 8
%110 mg, 0.31 mmol), palladium%II) acetate %7 mg, 0.03
mmol), tri%o-tolyl)phosphine %19 mg, 0.06 mmol) and tri-
buthyl amine %0.15 mL, 0.62 mmol) in DMF %31 mL) was
heated at 1208C for 20h. The solvent was removed in vacuo
to leave the residue, which was dissolved in ether. The
resulting organic solution was washed with 2N HCl, water
and saturated NaCl, dried over MgSO4. The solvent was
removed and the residue was chromatographed on silica
gel %hexane/EtOAcˆ20:1) to afford 9 %66 mg, 94%) as an
%95 mg, 98%) as an oil: [a]22 ˆ219.48 %c 0.68, CHCl3);
D
IR %®lm) 3435, 1604, 1496, 1242, and 1028 cm21
;
1H
NMR %200 MHz, CDCl3) d 1.20%3H, s), 1.42 %3H, s),
2.28 %3H, s), 2.44 %1H, m), 3.10%1H, bs), 3.13 %1H, bs),
3.80%3H, s), 6.81 %1H, d, Jˆ8.1 Hz), 7.01 %1H, dd, Jˆ8.1,
2.0Hz), and 7.09 %1H, d, Jˆ2.0Hz); EIMS m/z %rel. int.)
248 [M1] %45), 215 %9), 175 %29), 162 %26), and 149 %100);
HREIMS m/z 248.1774 [M1] %Calcd for 248.1776 for
C16H24O2).
oil: IR %®lm) 1751, 1614, 1496, 1248, 1221, and 1084 cm21
;
1H NMR %200 MHz, CDCl3) d 1.34 and 1.35 %total 3H, s),
1.37 and 1.38 %total 3H, s), 2.33 %3H, s), 2.57 %1H, dt,
Jˆ16.2, 1.8 Hz), 2.83 %1H, dt, Jˆ16.2, 1.8 Hz), 5.74±5.86
%2H, m), 6.88 %1H, d, Jˆ8.2 Hz), 7.02 %1H, dd, Jˆ8.2,
1.8 Hz), and 7.14 %1H, d, Jˆ1.8 Hz); EIMS m/z %rel. int.)
228 [M1] %100), 213 %27), 200 %85), 185 %76), and 159 %26);
HREIMS m/z 228.1145 [M1] %Calcd for 228.1150for
C15H16O2).
4.2.9. p-Bromophenylcarbamate of 12. To a mixture of 12
%12 mg, 0.05 mmol) and DABCO %8 mg, 0.07 mmol) in
toluene %1 mL) were added a solution of 4-bromophenyl
isocyanate %14 mg, 0.07 mmol) in toluene %0.5 mL) and
reaction mixture was stirred for 1 h at room temperature.
The reaction mixture was diluted with water and extracted
with ether. The organic layer was washed with water and
saturated NaCl solution, dried over MgSO4 and concen-
trated. Puri®cation of the residue by column chroma-
tography on silica gel %hexane/EtOAcˆ1:1) gave 13
%10mg): mp 143±145 8C %colorless plate from CH2Cl2 and
4.2.6. Hydorgenation of 9. A solution of 9 %60mg,
0.26 mmol) in ethanol %5 mL) containing palladium carbon
%Pd/C, Pd: 10%, 5 mg) was stirred under an atmospheric
pressure of hydrogen for 18 h. The reaction mixture was
®ltered, concentrated, and the residue was puri®ed by
column chromatography %hexane/EtOAcˆ4:1) to afford 10
hexane); [a]21 ˆ229.58 %c 0.38, CHCl3); IR %®lm) 3310,
D
%58 mg, 95%) as an oil: [a]21 ˆ132.78 %c 1.38, CHCl3); IR
1701, 1595, 1535, 1496, 1400, 1308, 1238, 1074 cm21; H
1
D
1
%®lm) 1755, 1614, 1495, 1238, and 1118 cm21; H NMR
NMR %200 MHz, CDCl3) d 1.23 %3H, s), 1.34 %3H, s), 2.22
%3H, s), 2.50%1H, m), 3.76 %1H, d, Jˆ8.6 Hz), 3.77 %3H, s),
3.79 %1H, d, Jˆ8.6 Hz), 6.24 %1H, m), 6.74 %1H, d,
Jˆ8.1 Hz), 6.96 %1H, dd, Jˆ8.1, 1.6 Hz), 7.08 %1H, d, Jˆ
1.6 Hz), 7.18 %2H, d, Jˆ8.8 Hz), and 7.37 %2H, d, Jˆ
8.8 Hz); EIMS m/z%rel. int.) 445[M1] %30), 447[M12]
%29), 248 %13), 230%22), 215 %17), 188 %26), 175 %52), and
149 %100); HREIMS found 445.1255[M1] %Calcd 445.1253
for C23H28O3NBr).
%200 MHz, CDCl3) d 1.24 %3H, s), 1.27 %3H, s), 1.38 %1H,
m), 2.34 %3H, s), 6.90%1H, d, Jˆ8.2 Hz), 7.03 %1H, dd,
Jˆ8.2, 2.0Hz), and 7.12 %1H, d, Jˆ2.0Hz); EIMS m/z
%rel. int.) 230[M 1] %100), 215 %15), 202 %30), 187 %65),
173 %13), and 159 %41); HREIMS m/z 230.1312 [M1]
%Calcd for 230.1307 for C15H18O2).
4.2.7. 1,2-Dimethyl-1-hydroxylmethyl-2-!2-hydroxy-5-
methylphenyl)cyclopentane !11). To a solution of 10
%55 mg, 0.24 mmol) in tetrahydrofuran %3 mL) was added
LiAlH4 %100 mg, 2.63 mmol) at 08C and the mixture was
stirred for 3 h at room temperature. The reaction mixture
was treated with water, and then was extracted with ether.
The ether layer was washed with saturated NaCl, dried over
MgSO4. Removal of the solvent gave 11 %56 mg) as a color-
less needle: mp 119.5±120.58C %colorless plate from
Crystal data: Triclinic, aˆ8.943 %2), bˆ9.5118 %9), cˆ
Ê
13.241 %3) A, aˆ105.53 %2), bˆ 90.3 %2), gˆ92.39 %1)8,
Ê
space group P1, Dcalˆ1.10, CuKa radiation, lˆ1.54178 A.
Diffraction measurement was made on a Mac Science
MXC18. The structure was solved by direct methods and
re®ned out by full matrix least-squares. Final
R
factorˆ0.0546. Crystallographic data have been deposited
at the CCDC %165776), 12 Union Road, Cambridge CB2
1EZ, UK.
CH2Cl2 and hexane); [a]27 ˆ115.58 %c 1.14, CHCl3); IR
D
%®lm) 3163, 1608, 1464, 1230, and 1024 cm21; EIMS m/z
%rel. int.) 234 [M1] %38), 216 %11), 201 %25), 173 %12), 161
%35), 148 %35), 135 %100); HREIMS m/z 234.1622 [M1]
4.2.10. 1,2-Dimethyl-1-formyl-2-!2-methoxy-5-methyl-
phenyl)cyclopentane !14). To a solution of dimethyl sulf-
oxide %0.02 mL, 0.28 mmol) in CH2Cl2 %0.5 mL) was added
oxalyl chloride %0.02 mL, 0.19 mmol) at 2788C, and
stirring was continued for 10min. A solution of 12
%23 mg, 0.1 mmol) in CH2Cl2 %1 mL) was added to this
solution, and the reaction mixture was stirred for 1 h at
the same temperature and then treated with Et3N
%0.09 mL, 0.65 mmol). After further stirring for 30 min at
08C, the reaction mixture was treated with saturated NH4Cl
solution and extracted with ether. The organic layer was
washed with water and saturated NaCl solution, dried over
1
%Calcd for 234.1620for C 15H22O2); H NMR %200 MHz,
CDCl3) d 1.23 %3H, s), 1.56 %3H, s), 1.93 %1H, m), 2.27
%3H, s), 2.45 %1H, m), 3.31 %1H, d, Jˆ11.1 Hz), 3.36 %1H,
d, Jˆ11.1 Hz), 6.74 %1H, d, Jˆ7.8 Hz), 6.92 %1H, dd, Jˆ7.8,
1.4 Hz), and 6.95 %1H, d, Jˆ1.4 Hz); 13C NMR %50MHz,
CDCl3) d 20.6, 21.0, 21.2, 24.1, 36.0, 42.4, 49.0, 51.0, 70.5,
117.2, 127.9, 129.1, 129.9, 132.9, and 152.9; Anal. Calcd
for C15H22O2: C, 76.88; H, 9.46. Found C, 76.91, H 9.66.
4.2.8. 1,2-Dimethyl-1-hydroxylmethyl-2-!2-methoxy-5-
methylphenyl)cyclopentane !12). To a mixture of 11

Y. Fukuyama et al. / Tetrahedron 57 32001) 7127±7135
7133
MgSO4. The solvent was removed in vacuo and the residue
was chromatographed on silica gel %hexane/EtOAcˆ15:1)
CH2Cl2ˆ9:1) to afford 17 %16 mg, 71%) as an oil and 16
%recovery, 14 mg): [a]21 ˆ250.38 %c 1.57, CHCl3); IR
D
to afford 14 %22 mg, 96%) as an oil: [a]22 ˆ249.28 %c 0.75,
%®lm) 1497, 1371, 1229, 1146, 1078,1 015, 922, and
D
CHCl3); IR %®lm) 1718, 1606, 1585, 1499, 1464, 1244, and
814 cm21; EIMS m/z %rel. int.) 262 [M1] %44), 217 %62),
173 %23), 161 %47), 145 %32), 135 %100), and 121 %31); H
1
1
1032 cm21; H NMR %200 MHz, CDCl3) d 1.30%3H, s),
1.33 %3H, s), 2.28 %3H, s), 2.38 %1H, m), 3.70%3H, s), 6.71
%1H, d, Jˆ8.1 Hz), 7.00 %1H, dd, Jˆ8.1, 1.6 Hz), 7.06 %1H,
d, Jˆ1.6 Hz), and 9.05 %1H, s); EIMS m/z%rel. int.) 246[M1]
%23), 229 %4), 175 %100), 162 %16), 149 %47), and 135 %17);
HREIMS m/z 246.1604 [M1] %Calcd 246.1620for
C16H22O2).
NMR %200 MHz, CDCl3) d 0.72 %3H, s), 1.16 %3H, s), 1.37
%3H, s), 2.28 %3H, s), 2.53 %1H, m), 3.48 %3H, s), 5.09 %1H, d,
Jˆ7.0Hz), 5.16 %1H, d, Jˆ7.0Hz), 6.93 %1H, dd, Jˆ1.8,
8.1 Hz), 7.03 %1H, d, Jˆ8.1 Hz), and 7.13 %1H, d, Jˆ
1.8 Hz); 13C NMR %50MHz, CDCl 3) d 20.5, 20.9, 23.2,
25.8, 27.4, 39.8, 41.8, 44.3, 51.3, 55.9, 94.6, 114.5, 127.3,
129.7, 130.0, 136.0, and 154.7; HREIMS m/z 262.1938
[M1] %Calcd 262.1933 for C17H26O2).
4.2.11. 1,1,2-Trimethyl-2-!2-methoxy-5-methylphenyl)-
cyclopentane !15). To a solution of 14 %96 mg, 0.39
mmol) in diethylene glycol %3 mL) was added NaOH
%290 mg, 6.55 mmol) and hydrazine monohydrate %0.02
mL, 0.43 mmol). The reaction mixture was stirred at
1508C for 4 h and at 1808C for additional 3 h. The reaction
mixture was extracted with ether, washed with water,
saturated NaCl solution, dried over MgSO4 and concen-
trated in vacuo. The residue was chromatographed on silica
gel %hexane/EtOAcˆ19:1) to afford 15 %78 mg, 83%) as an
4.2.14. 1,1,2-Trimethyl-2-!3-hydroxy-2-methoxymethyl-
oxy-5-methylphenyl)cyclopentane !18). To a solution of
17 %31 mg) and TMEDA %0.11 mL, 0.71 mmol) in THF
%1 mL) was added dropwise sec-BuLi %1.08 M in cyclo-
hexane, 0.66 mL, 0.71 mmol) at 2788C under argon atmos-
phere. After being stirred for 1 h at 2788C, %1R)-%2)-%10-
camphorsulfonyl)oxaziridine %136 mg, 0.60 mmol) was
added at 2158C and the reaction mixture was stirred for
50min and then at room temperature. The reaction mixture
was extracted with ether, washed with water and saturated
NaCl solution, dried over MgSO4, and concentrated in
vacuo. The residue was chromatographed on silica gel
%hexane/EtOAcˆ19:1) to afford 18 %12 mg, 51%) as an oil
1
oil: IR %®lm) 1606 1498, 1240, and 1035 cm21; H NMR
%200 MHz, CDCl3) d 0.68 %3H, s), 1.15 %3H, s), 1.35 %3H, s),
2.28 %3H, s), 2.53 %1H, m), 3.74 %3H, s), 6.75 %1H, d,
Jˆ8.2 Hz), 6.96 %1H, dd, Jˆ8.2, 2.2 Hz), and 7.11 %1H, d,
Jˆ2.2 Hz); EIMS m/z%rel. int.) 232 [M1] %81), 217 %12), 201
%5), 189 %1), 175 %43), 162 %73), 149 %100), 135 %40), and 119
%41); HREIMS m/z 232.1842 [M1] %Calcd 232.1827 for
C16H24O).
and 17 %recover, 8 mg): [a]22 ˆ293.18 %c 2.27, CHCl3); IR
D
%®lm) 3328, 1584, 1462, 1310, 1182, 1152, 1057, and
1
993 cm21; H NMR %200 MHz, CDCl3) d 0.72 %3H, s)
1.14 %3H, s), 1.29 %3H, s), 2.25 %3H, s), 3.65 %3H, s), 4.86
%1H, d, Jˆ6.3 Hz), 5.01 %1H, d, Jˆ6.3 Hz), 6.67 %1H, d, Jˆ
2.6 Hz), 6.68 %1H, d, Jˆ2.6 Hz), and 7.80%1H, s); 13C NMR
%50MHz, CDCl 3) d 20.3, 21.3, 24.4, 25.3, 26.8, 39.3, 40.9,
45.0, 51.3, 57.0, 100.3, 115.8, 120.8, 133.5, 140.1, 143.9,
and 149.1; EIMS m/z %rel. int.) 278 [M1] %84), 246 %35), 233
%22), 217 %7), 203 %12), 189 %35), 175 %48), 164 %31), 151
%100); HREIMS m/z 278.1886 [M1] %Calcd 278.1882 for
C17H26O3).
4.2.12. !2)-a-Herbertenol !16). To a solution of 14
%14 mg, 0.06 mmol) in CH2Cl2 %2 mL) was slowly added a
solution of BBr3 %1 M in CH2Cl2, 0.08 mL, 0.08 mmol) at
2788C for 40min, and the mixture was stirred at 0 8C for
40min and at room temperature for 3 h. The reaction
mixture was extracted with ether, washed with water and
saturated NaCl solution, and dried over MgSO4. After
evaporation of the solvent, the residue was chromato-
graphed on silica gel %hexane/EtOAcˆ19:1) to afford
%2)-a-herbertenol %16) %9 mg, 66%) as an oil: [a]24
ˆ
4.2.15. !2)-Herbertenediol !3). To a solution of 18 %20mg,
0.07 mmol) in MeOH %2 mL) was added one drop of 48%
hydrobromic acid and the mixture was stirred at 608 C for
15 min. After removal of solvent, the crude mixture was
extracted with ethyl acetate, washed with water and
saturated NaCl, dried over MgSO4, and concentrated in
vacuo. The residue was chromatographed on silica gel
%hexane/EtOAcˆ4:1) to afford 3 %16 mg, 98%): mp 88±
D
241.48 %c 0.43, CHCl3); IR %®lm) 3531, 2959, 2874,
1606, 1506, 1462, 1406, 1373, 1253, 1168, 1143, and
1
810cm 21; H NMR %200 MHz, CDCl3) d 0.76 %3H, s),
1.18 %3H, s), 1.41 %3H, s), 2.26 %3H, s), 2.59 %1H, m), 4.61
%1H, s), 6.57 %1H, d, Jˆ8.0Hz), 6.86 %1H, dd, Jˆ8.0,
2.0Hz), and 7.09 %1H, d, Jˆ2.0Hz); 13C NMR %50MHz,
CDCl3) d 20.4, 20.9, 23.0, 25.6, 27.0, 39.5, 41.3, 44.7, 51.0,
116.7, 127.3, 129.0, 130.1, 133.1, and 152.3; EIMS m/z%rel.
int.) 218 [M1] %70), 203 %8), 175 %10), 161 %34), 148 %100),
135 %85), 121 %29), 105 %13), 91 %11), and 77 %8); HREIMS
m/z 218.1664 [M1] %Calcd 218.1671 for C15H22O).
89.58C %colorless plate from CH2Cl2 and hexane); [a]22
ˆ
;
D
247.18 %c 1.0, CHCl3); IR %®lm) 3512 and 1597 cm21
EIMS m/z%rel. int.) 234 [M1] %78), 191 %7), 178 %11), 164
%52), 151 %100), and 137 %27); 1H NMR %400 MHz, CDCl3)
d 0.77 %3H, s), 1.19 %3H, s), 1.42 %3H, s), 2.23 %3H, s), 2.60
%1H, m), 6.56 %1H, s), and 6.69 %1H, s); 13C NMR
%100 MHz) d 20.3, 21.2, 22.9, 25.4, 26.9, 39.0, 41.0, 44.9,
51.5, 113.5, 121.9, 128.3, 133.5, 141.0, and 143.4; HREIMS
found 234.1618 [M1] %Calcd 234.1620for C 15H22O2).
4.2.13. 1,1,2-Trimethyl-2-!2-methoxymethyloxy-5-methyl-
phenyl)cyclopentane !17). To a solution of %2)-herbertenol
%16) %30mg, 0.14 mmol) in CH Cl2 %1.5 mL) was added
2
diisopropylethylamine %0.09 mL, 0.50 mmol) and chloro-
methyl methyl ether %0.04 mL, 0.50 mmol). The mixture
was stirred at room temperature for 23 h. The reaction
mixture was extracted with ether, washed with water and
saturated NaCl solution, dried over MgSO4, and concen-
trated in vacuo. Evaporation of organic solvent gave the
residue, which was chromatographed on silica gel %hexane/
4.3. Synthesis of mastigophrenes A and B
4.3.1. Oxidative dimerization of 18 with !tert-BuO)2. To a
solution of 18 %90mg, 0.32 mmol) in chlorobenzene %3 mL)
was added di-tert-butylperoxide %0.12 mL, 0.65 mmol)

7134
Y. Fukuyama et al. / Tetrahedron 57 32001) 7127±7135
under an argon atmosphere and the mixture was re¯uxed for
4 h. The solvent was removed and the residue was puri®ed
on silica gel %hexane/CH2Cl2ˆ2:1) to afford 20 %24 mg,
13.5%) as an oil and 18 %recovery, 70mg, 77.8%): EIMS
m/z %rel. int.) 554 [M1] %21), 522 %21), 490 %10), 478 %100),
408 %12), 396 %26), 261 %8), 111 %9); HREIMS m/z 554.3610
4.4.2. Mastigophorene B !2). Needles, mp 167±1708C;
[a]20 ˆ247.08 %c 1.85, CHCl3); IR %®lm) 3535, 2959,
D
1433, 1408, 1365, 1277, and 1226 cm21; UV lmax %EtOH)
284 %e 4300) and 238 %e 7500) nm; CD %EtOH) De
%217 nm) 213.4, De %203 nm) 112.3; EIMS m/z %rel. int.)
466 [M1] %100), 423 %14), 396 %9), 384 %59), 299 %7), 234
%3); HREIMS 466.3084 %Calcd 466.3083 for C30H42O4); 1H
NMR %200 MHz, CDCl3) d 0.78 %6H, s), 1.21 %6H, s), 1.47
%6H, s), 1.92 %6H, s), 4.71%2H, s), 5.57 %2H, s), and 6.85 %2H,
s); Anal. Calcd for C30H42O4 2H2O %502.68264): C, 71.31;
H, 8.92. Found C, 71.68, H 9.22.
1
[M1] %Calcd 554.3608 for C34H50O6); H NMR %200 MHz,
CDCl3) d 0.74 %3H, s), 0.75 %3H, s), 1.15 %3H, s), 1.16 %3H,
s), 1.33 %3H, s), 1.35 %3H, s), 1.93 %3H, s), 1.95 %3H, s), 3.59
%6H, s), 4.90%1H, d, Jˆ6.2 Hz), 4.93 %1H, d, Jˆ6.2 Hz),
5.02 %1H, d, Jˆ6.2 Hz), 5.20%1H, d, Jˆ6.2 Hz), 6.78 %1H,
s), 6.80%1H, s), 7.31 %1H, s), and 7.51 %1H, s). To a solution
of 20 %20 mg, 0.036 mmol) in MeOH %3.5 mL) was added
two drop of 48% hydrobromic acid and the mixture was
stirred at 608C for 15 min. After removal of solvent, the
crude mixture was extracted with ethyl acetate, washed
with water and saturated NaCl, dried over MgSO4, and
concentrated in vacuo. The residue was chromatographed
on silica gel %hexane/CH2Cl2ˆ1:1) to afford 1 %6 mg, 34%)
and 2 %5 mg, 29%) as needles.
4.5. Biological activity
4.5.1. Cell culture. Primary cell cultures were prepared as
described.17 All operations were carried out under sterile
conditions. The neuronal cells were separated from the
cerebral hemispheres of fetal 18-day SD rat %Japan SLC,
Inc.) and suspended in 10% FAB/MEM, then seeded at
5£105 cells cm22 into poly-l-lysine-coated 24 well-culture
plates. After 48 h, the medium was changed into the serum-
free medium, Dulbeco's modi®ed Eagle's medium
%DMEM) supplemented with N2, in the presence or absence
of the compounds at the concentrations of 0.01, 0.1, 1 and
10 mM. After being incubated for 5 days, the cells are ®xed
with 4% paraformaldehyde/PBS for anti-MAP-2 immuno-
histochemical stain. The neurite outgrowths affected by
samples were analyzed under microscope and photographs
taken with 150magni®cations.
4.3.2. Oxidative dimerization of 19 with !tert-BuO)2. To
a solution of 19 %47 mg, 0.19 mmol) in chlorobenzene
%1.5 mL) was added di-tert-butylperoxide %0.06 mL, 0.34
mmol) under an argon atmosphere and the mixture was
re¯uxed for 4 h. The solvent was removed and the residue
was puri®ed on silica gel %hexane/CH2Cl2ˆ2:1) to afford 21
%25 mg, 56%) as a mixture, which was separated by
chromatography on silica gel %hexane/CH2Cl2ˆ3:2) to
afford 21a %14 mg) and 21b %10mg), each of which was
treated with BBr3 %1 M in CH2Cl2,) at 2788C for 30min
and then at 08C for 1 h giving rise to 1 %9.4 mg, 70%) and 2
%4 mg, 41%), respectively.
4.5.2. Assay of neuronal survival. Neuronal survival was
determined by the WST-8 reduction assay, which was
performed using the cell counting kit-8 %Dojindo Co. Ltd.)
according to the manifucture's procedure.
4.4. Oxidative dimerization of !2)-herbertenediol !3)
with HRP
Acknowledgements
To a solution of 3 %23 mg, 0.1 mmol) in acetonitrile
%0.3 mL) and phosphate buffer %0.1 M, pH 6.0, 0.5 mL)
was added 0.45% hydrogen peroxide %0.55 mL, 0.75 eq.)
and a solution of HRP %1.0mg, 200 units, type II from
Sigma, USA) in phosphate buffer %0.1 M, pH 6.0, 0.5 mL).
The reaction mixture was stirred at 358C for 6 days. The
reaction was quenched by adding 1 M NaHSO3 and then
adjusted to pH 7.0with 1 M NaOH, ®nally to pH 3.0with
1 M NaHSO3. The reaction mixture was extracted with ethyl
acetate, washed with water and saturated NaCl, dried over
MgSO4, and concentrated in vacuo. The residue %23 mg)
was chromatographed on silica gel %hexane/CH2Cl2ˆ1:1)
to afford 1 %2.2 mg, 10%) and 2 %4.2 mg, 18%) and recovery
3 %16.5 mg, 72%).
We thank Dr Masami Tanaka, Miss Yasuko Okamoto and
Mr Shigeru Takaoka for measuring NMR, MS spectra and
X-ray. This work is supported by a Grant-in Aid for
Scienti®c Research %No. 12480175) from the Ministry of
Education, Culture, Sports, Science, and Technology,
Japan, and the High Technology Research Center Fund
from the Promotion and Mutual Aid Corporation for Private
School, Japan.
References
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4.4.1. Mastigophorene A !1). Needles, mp 217±2208C;
[a]20 ˆ269.08 %c 0.38, CHCl3); IR %®lm) 3530, 2955,
2. Fukuyama, Y.; Asakawa, Y. J. Chem. Soc., Perkin Trans.
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D
1431, 1404, 1365, 1282, and 1221 cm21; UV lmax %EtOH)
285 %e 8400) and 230 %e 20500) nm; CD %EtOH) De
%220nm) 17.1, De %206 nm) 212.1; EIMS m/z %rel. int.)
466 [M1] %100), 423 %13), 396 %10), 384 %56), 356, %4), 300
%6), and 273 %5); HREIMS 466.3076 %Calcd 466.3083 for
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%6H, s), 1.46 %6H, s), 1.94 %6H, s), 4.69 %2H, s), 5.57 %2H, s),
and 6.86 %2H, s); Anal. Calcd for C30H42O4 H2O
%484.66736): C, 74.70; H, 8.71. Found C, 74.34, H 9.15.
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