Chemical studies on the chiral indanone derivatives as the inhibitor of Renilla luciferase

Article abstract of DOI:10.1016/S0040-4020(01)00980-2

The bioluminescence reaction of coelenterazine involves an oxidative process. To investigate the reaction mechanism, we synthesized three mechanism-based inhibitors with an indanone core structure. The inhibitors exhibited the competitive inhibition of the Renilla luciferase reaction. The (-)-4-benzyl-2-(4-hydroxybenzyl)-2-hydroxymethyl-6-(4-hydroxyphenyl)-indan- 1-one showed the significant enantio-selectivity of the inhibition and its absolute configuration was assigned as the R-configuration. These inhibitors could be useful probes to study the catalytic environment in the coelenterazine-luciferase reaction.

Full text of DOI:10.1016/S0040-4020(01)00980-2

TETRAHEDRON
Pergamon
Tetrahedron 57 -2001) 9575±9583
Chemical studies on the chiral indanone derivatives as the
inhibitor of Renilla luciferase
Chun Wu,a,p Hideshi Nakamura,a,² Akio Muraib and Satoshi Inouyec
aDivision of Biomodeling, Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University,
Nagoya 464-8601, Japan
bDivision of Chemistry, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan
cYokohama Research Center, Chisso Co., 5-1 Okawa, Kanazawa-ku, Yokohama 236-8605, Japan
Received 22 August 2001; accepted 2 October 2001
AbstractÐThe bioluminescence reaction of coelenterazine involves an oxidative process. To investigate the reaction mechanism, we
synthesized three mechanism-based inhibitors with an indanone core structure. The inhibitors exhibited the competitive inhibition of the
Renilla luciferase reaction. The -2)-4-benzyl-2--4-hydroxybenzyl)-2-hydroxymethyl-6--4-hydroxyphenyl)-indan-1-one showed the signi®-
cant enantio-selectivity of the inhibition and its absolute con®guration was assigned as the R-con®guration. These inhibitors could be useful
probes to study the catalytic environment in the coelenterazine±luciferase reaction. q 2001Elsevier Science Ltd. All rights reserved.
1. Introduction1
catalyzed by luciferase is higher than that of chemilumi-
nescence in polar aprotic solvent. To investigate the inter-
mediates of chemiluminescence, the C-2 peroxide of
coelenterazine analog was synthesized by the sensitized
photooxidation at 2958C.5 The synthesis of a 13C-labelled
coelenterazine analog was also reported6 and its photo-
oxidation products have been analyzed by the 13C NMR
spectroscopy, which suggest that the C-2 peroxide and diox-
etanone are present at low temperature. The model studies
Coelenterazine -1a) is well known as a substrate -luciferin)
in various bioluminescence reactions of marine organisms,
such as jelly®shes,2 sea pansies3 and deep-sea shrimps.4 The
bio- and chemiluminescence reaction of coelenterazine -1a)
is an oxidation process that could involve several inter-
mediates -2a,b), and then results in light emission -Fig.
1). The ef®ciency of bioluminescence of coelenterazine
Figure 1. The luminescence mechanism of coelenterazine -1a) and possible intermediates -2a-b).
Keywords: bioluminescence; coelenterazine; peroxide; inhibitor; transition-state.
Corresponding author. Tel./fax: 181-52-789-4280; e-mail: i993001d@mbox.media.nagoya-u.ac.jp
p
²
Deceased November 9, 2000.
0040±4020/01/$ - see front matter q 2001Elsevier Science Ltd. All rights reserved.
PII: S0040-4020-01)00980-2

9576
C. Wu et al. / Tetrahedron 57 52001) 9575±9583
to emit blue light.3 The gene encoding Renilla luciferase has
been cloned and expressed in bacterial cells.9 A highly
puri®ed recombinant Renilla luciferase was obtained by
the Ni-chelate af®nity chromatography.9 The quantum
yield in the luminescence reaction of recombinant Renilla
luciferase with coelenterazine is 0.10±0.11.9 The substrate
speci®city and inhibitory studies on Renilla luciferase were
reported using several coelenterazine analogs.3,9
In this study we designed and synthesized three mechanism-
based inhibitors of coelenterazine -3a±c), having an inda-
none core structure to overcome the unstable properties of
intermediates -Fig. 2). Separation of the enantiomers by a
chiral column, determination of their con®guration and
examination of the inhibition of the recombinant Renilla
luciferase reaction are also described.
Figure 2. Structure of three mechanism-based inhibitors.
on the chemiluminescence of coelenterazine analogs have
suggested that the formation of the C-2 peroxide could be
the rate-determining step in the luminescence reaction.7 The
18O2 labeling studies of the intermediates in the biolumi-
nescence reaction with the shrimp luciferase has been
reported.4 Recently, the structure of the C-2 peroxide of
coelenterazine with S-con®guration in aequorin, which is
a stable complex in the absence of Ca21, has been
determined by the X-ray crystal structure analysis.8 This
result implied that the catalytic site of luciferase could be
chiral and one enantiomer of the peroxide intermediate can
®t it properly. However, no investigation of the chiral
environment of luciferase has been reported.
2. Results and discussions
2.1. Preparation of the chiral mechanism-based
inhibitors
The synthesis of the inhibitors -3a,b) was shown in Scheme
1. Under the Suzuki-coupling conditions, the starting
material 3-bromobenzamide -4) was treated with a boronic
reagent to give the biphenyl -5) in good yield.10 A benzyl
group was introduced to 5 by the ortho-metalation with
benzaldehyde and then the product was converted to the
lactone -6) in 80% overall yield.11 Reduction of 6 with
Renilla luciferase was isolated from sea pansy, Renilla
reniformis, and it catalyzes the oxidation of coelenterazine
Scheme 1. Synthesis of the inhibitors 3a and 3b.

C. Wu et al. / Tetrahedron 57 52001) 9575±9583
9577
Scheme 2. Synthesis of the inhibitor 3c.
DIBAL provided the hemiacetal -7). After the Wittig
reaction, catalytic hydrogenation and hydrogenolysis of
the product 8 were accomplished in one step to give the
acid -9) in 94% yield. Acid halide formation of 9 was
followed by the intramolecular Friedel±Crafts acylation to
give the indanone structure -10). A p-hydroxybenzyl moiety
was introduced by the aldol condensation with p-methoxy-
benzaldehyde to form the E-exo ole®n -11). Hydrogenation
of 11 was performed with Pt2O to give 12. Deprotection of
methyl ether with BBr3 afforded 3a, which has a chiral
center at C-2 position. The second Aldol condensation of
3a was performed with the formaldehyde solution to give
3b. The enantiomers of 3a and 3b were separated on a chiral
column.
with p-methoxybenzaldehyde in a basic condition provided
16. Hydrogenation using Pt2O followed by the Aldol
reaction gave 17. The Aldol condensation of 17 with the
formaldehyde solution gave 18. Unfortunately, the depro-
tection of the methyl ether of 18 using BBr3 led to the retro-
Aldol reaction. After the deprotection of 17, the Aldol
condensation afforded -^)-3c in 35% overall yield. Resolu-
tion of -^)-3c was carried out by the same chiral column to
give -1)- and -2)-3c.
2.2. Assignment of the absolute con®gurations of
inhibitors
The absolute con®guration of each enantiomers was deter-
mined by the circular dichroic -CD) methods. The conju-
gated ketone exhibits CD cotton effect in the range of 320±
370 nm, which is due to n!pp transition. Snatzke has
introduced the sector rule to indanone derivatives.12 In the
case of the chiral indanone derivatives with a substituent at
C-2 position, the substituent prefers the pseudoequatorial
orientation to the pseudoaxial orientation. The 2S-con®gu-
ration exhibits the negative cotton effect, which is explained
To study the effect of the substituent at the C-4 position of
inhibitor on the luminescence reaction, we synthesized the
debenzyl analog 3c. The synthesis of 3c was accomplished
in 7 steps from a commercially available indanone
compound -13). The synthetic route was shown in Scheme
2. The Suzuki-coupling reaction of the aryl tri¯ate -14) with
a boronic acid gave 15 in 86% yield. Condensation of 15
Figure 3. Separation and assignment of the absolute con®gurations of -1)- and -2)-3a.

9578
C. Wu et al. / Tetrahedron 57 52001) 9575±9583
Scheme 3. Preparation of -R)-18.
by the octant type projection -Fig. 3).12 The enantiomer of
3a showing a negative cotton effect at 350 nm in the CD
spectra was determined as the S-con®guration.
the other hand -2)-19 was oxidized by using MnO2 to give
-R)-18. The enantiomers -2)-3b and -2)-3c having a long
retention time on the chiral HPLC exhibited the CD spectra
matching -R)-18; therefore, these enantiomers could be
determined as the R-con®guration -Fig. 4).
To assign the absolute con®gurations of 3b and 3c, we
compared the CD spectra with -R)-18, which was prepared
from its racemic form -Scheme 3). Treatment of -^)-18
with NaBH4 gave the major diol 19 and the corresponding
diastereomer. The trans conformation of 19 was character-
ized by the nOe and HMBC experiments. The both enantio-
mers -1)- and -2)-19 were separated by HPLC on the chiral
column, and -1)-19 was treated with 4-dimethylamino-
benzoyl chloride to give the dibenzoate derivative 20. The
CD spectrum exhibited negative ®rst and positive second
cotton effects, implying that the chiral center at the C-2
position of 20 and -1)-19 could be determined as the
S-con®guration by the CD dibenzoate chirality rule.13 On
The cotton effect of the indanone derivatives with a hydroxy-
methyl substituent at the C-2 position affected by the solvent
polarity has been discussed.14 The cotton effect of these
indanone derivatives in ethanol gives opposite sign to that
of chloroform as a solvent. This observation suggests
ethanol interrupted the hydrogen bonding between ketone
and hydroxymethyl moiety. We thought that the pseudo-
equatorial conformation of the benzyl group of 3b and 3c
in ethanol like the conformation of 3a could be predominant
due to its bulkiness. Thus, these enantiomers of -2)-3b and
-2)-3c could be also determined as the R-con®guration
according to the sector rule of indanone derivatives.
Interestingly, all inhibitors having S-con®guration exhibited
a short retention time on the same chiral column.
2.3. Inhibition of the luminescence with Renilla
luciferase
Renilla luciferase catalyzes the luminescence reaction of
coelenterazine -1a) or 2-deoxy-coelenterazeine -1b)
ef®ciently.9 According to the previous report for the
inhibitory study on Renilla luciferase,3 we chose 2-deoxy-
coelenterazine -1b) as the substrate. Measurement of the
luminescence activity was started by adding a substrate to
a mixture of the inhibitor and luciferase. The Ki values were
determined from the Lineweaver±Burk plots. The inhibitors
3a and 3b showed very strong competitive inhibition.
Interestingly, -R)-3b -Kiˆ9.3£1029 M) showed much
Figure 4. CD spectra of the enantiomers of 3b and 3c in comparison with
-R)-18.

C. Wu et al. / Tetrahedron 57 52001) 9575±9583
Table 1. Inhibition of the recombinant Renilla luciferase
9579
a Brucker ARX-400 -100 MHz). Chemical shifts -d) were
given in parts per million relative to CDCl3 -d 77.0) or
CD3OD -d 49.0) as an internal standard. Low-resolution
mass spectra were recorded on a JEOL JMS-D 100 -EI), a
JEOL DX-705L -FAB) or a JEOL Mstation spectrometer.
High-resolution mass spectra -HRMS) were recorded on
JEOL DX-705L and JEOL Mstation spectrometers.
Elemental analyses were tested in the Analytical Laboratory
at the school of Bioagricultural Sciences, Nagoya Uni-
versity. Reactions were monitored by thin-layer chroma-
tography -TLC) on 0.25 mm silica gel coated glass plates
60F-254 -Merck, Art 5715) and visualized using UV light
and 7% ethanolic phosphomolybdic acid or 2.5% p-anisal-
dehyde solution as developing reagents by heating. Prepara-
tive thin-layer chromatography separations were carried out
on 0.5 mm silica gel plate 60F-254 -Merck, Art 5774).
Silica gel for chromatography was obtained from Cica±
Merck -Silica Gel 60, particle size 0.063±0.22 mm A
STM). Optical rotations were measured on a JASCO DIP-
370 digital polarimeter. Circular dichroism -CD) spectra
were measured on a JASCO J-720WN. Anhydrous solvent
of ethyl ether, tetrahydrofuran and dichloromethane were
purchased from Kanto Chemical. N,N,N,N-tetramethylethyl-
ene-diamine was purchased from Aldrich. All other
commercially available reagents were used without puri®-
cation. HPLC was carried out using a JASCO PU-880 pump
system equipped with a JASCO UV-875 UV/Vis detector.
The peak intensity was measured with a photomultiplier
tube -Hamamatsu photonics, R105UH) and ampli®ed output
of the tube was introduced to an integrator -Atto, Digimini
recorder AC 5150). The peak intensities of bioluminescence
Compound
KI/M
-S)-3a
-S)-3b
-S)-3c
4.0£1028
2.1£1027
1.2£1027
-R)-3a
-R)-3b
-R)-3c
5.0£1028
9.3£1029
0.9£1027
stronger inhibitory effect than its enantiomer -S)-3b
-Kiˆ2.1£1027 M). These results suggested that the recog-
nition of the peroxide intermediates could be enantio-
selective in the catalytic site of Renilla luciferase. On the
other hand, -R)-and -S)-3a showed almost same Ki values in
the range of 1028 M. This inhibitory result of 3a resembled
the ®nding in the acetylcholine esterase inhibition.15 The Ki
values for -S)- and -R)-3c were 1.2£1027 and 0.9£1027 M,
respectively, which is implying no enantioselectivity of
inhibition between the enantiomers of 3c. In our previous
work, the substituent at the C-8 position of coelenterazine
corresponding to the C-4 position of indanone could
in¯uence the reaction rate and total light with Renilla
luciferase.16 These results suggested that the benzyl group
at the C-4 of the inhibitor should be necessary to bind on the
chiral catalytic site. The moderate inhibitory effect of 3c
suggested that it could be used to develop an af®nity column
for luciferase. The Ki values of all chiral inhibitors were
shown in Table 1. The result of the inhibitor -R)-3b showed
the strongest inhibition, which was the opposite enantio-
selectivity to the observation from aequorin.9
were measured at ®nal substrate concentration of 1£1027
,
2£1028, 3.3£1028 M in presence of the recombinant Renilla
luciferase -500 ng ml21) in 25 mM Tris±HCl buffer pH 7.5
containing 0.1M NaCl at 23 8C with or without inhibitor
-1£1027 M). The inhibitory data of Lineweaver±Burk
plots was obtained from Eq. -1) and was summarized in
Table 1
3. Conclusion
In conclusion, we prepared three mechanism-based inhibi-
tors in enantiomerically pure form. The result of inhibition
of recombinant Renilla luciferase indicated that the stable
inhibitor -R)-3b gave the strongest inhibition with a high
enantioselectivity, suggesting that this inhibitor could
re¯ect the chiral environment in the catalytic site of
luciferase. It is remarkable that these compounds could be
usable as a probe to study on the bioluminescence mecha-
nism of coelenterazine including the structural analysis for
the inhibitor±luciferase complex.
Vmax‰SŠ
v ˆ
ꢀ1†
Kmꢀ1 1 ‰IŠ=Ki† 1 ‰SŠ
4.1.1. N,N-diethyl-40-Methoxybiphenyl-4-carboxamide /5).
4-Bromo-N,N-diethyl-benzamide -4)17 -4.0 g, 0.015 mol)
was treated with 4-methoxyphenyl-boronic acid -2.83 g,
0.018 mol), 2 M aqueous sodium carbonate solution
-9.0 ml, 0.018 mol), tetrakis-triphenylphosphine)palla-
dium-0) -880 mg, 0.75 mmol) in a mixture of benzene
-80 ml) and methanol -40 ml) at 808C for 17 h. The solution
was dried over Na2SO4 and evaporated. The residue was
puri®ed by silica gel column chromatography -CH2Cl2,
then 1:1 EtOAc±hexane) to give 4.07 g of yellow needles
4. Experimental
4.1. General
1
All melting points were recorded on a Yanaco MP-J3
melting point apparatus and were not corrected. UV spectra
were taken on a JASCO V-530 spectrometer. Infrared
spectra were determined with a JASCO FT/IR-8300 spec-
trophotometer and were reported in wave number -cm21).
Proton nuclear magnetic resonance -1H NMR) spectra were
recorded on a Varian Gemini-2000 -300 MHz) or a Brucker
ARX-400 -400 MHz) spectrometer. Chemical shifts -d)
were given in parts per million relative to tetramethylsilane
-d 0.00) or CD3OD -d 3.30) as an internal standard and
coupling constants -J) in Hz. 13C NMR were recorded on
-99%). 5: mp 96±988C; H NMR -300 MHz, CD3OD) d
1.15±1.26 -6H, br), 3.34±3.36 -2H, br), 3.55±3.57 -2H,
br), 3.83 -3H, s), 7.01-2H, d, Jˆ8.5 Hz), 7.42 -2H, d, Jˆ
8.5 Hz), 7.59 -2H, d, Jˆ8.5 Hz), and 7.67 -2H, d, Jˆ
8.5 Hz); HRMS -EI) Calcd for C15H21O2N m/z 283.1572,
found m/z 283.1546; IR -Nujol) nmax 2925, 2856, 1626,
1604, 1580, 1530, 1500, 1462, 1378, 1310, 1294, 1258,
1200, 1184, 1096, 1064, 1040, 1016, 856, 830, 770, 722
and 660 cm21
.
4.1.2. 5-/4-Methoxyphenyl)-3-phenyl-3H-isobenzofuran-

9580
C. Wu et al. / Tetrahedron 57 52001) 9575±9583
1-one /6)To a solution of TMEDA -1.32 ml, 8.4 mmol) in
dry THF -40 ml) was added dropwise sec-BuLi -8.1ml,
8.4 mmol) at 2788C. After several minutes, a solution of
5 -1.01 g, 3.5 mmol) in dry THF -40 ml) was added drop-
wise over 30 min period. After another 20 min, a solution of
benzaldehyde -877 ml, 8.4 mmol) in dry THF -2 ml) was
added and the mixture was stirred at 2788C for 20 min.
After saturated aqueous ammonium chloride -10 ml) was
added, the bath was removed and the solution was allowed
to warm up to ambient temperature. THF was removed in
vacuo, the aqueous residue was extracted with EtOAc, and
the extract was dried over MgSO4. The solvent was removed
in vacuo and the residual yellow liquid -1.2 g) was puri®ed
by silica gel column chromatography -CH2Cl2, then 1:1
hexane±EtOAc) to yield a yellow solid -1.0 g). The product
was heated in toluene at re¯ux for 12 h. Solvent removal in
vacuo left a slightly yellowish solid. Recrystallization from
Et2O±hexane gave 900 mg of 6 as a colorless solid -80%).
6: mp 131±1328C; 1H NMR -300 MHz, CDCl3) d 3.85 -3H,
4.1.5. 3-/3-Benzyl-40-methoxybiphenyl-4-yl)-propionic
acid /9). A solution of 8 -680 mg, 1.5 mmol) in 20 ml of
ethanol containing 10% palladium on activated carbon
-200 mg) was stirred under hydrogen using a balloon for
36 h at room temperature. The catalyst was removed by
®ltration. The ®ltrate was evaporated under reduced
pressure. The residue was puri®ed by silica gel column
chromatography using EtOAc±hexane -1:1) as a eluent to
give 490 mg of a colorless amorphous solid -94%). 9: mp
1
121±1238C; H NMR -300 MHz, CDCl3) d2.50 -2H, t,
Jˆ8.7 Hz), 2.96 -2H, t, Jˆ8.7 Hz), 3.83 -3H, s), 4.09 -2H,
s), 6.95 -2H, d, Jˆ8.9 Hz), 7.49 -2H, d, Jˆ8.9 Hz), 7.15±
7.27 -5H, m), 7.35 -1H, s), and 7.40 -1H, d, Jˆ8.9 Hz);
HRMS -EI) Calcd for C23H22O3 m/z 346.1569 Found m/z
346.1581; IR -KBr) nmax 3020, 2915, 2833, 1707, 1605,
1496, 1250 and 825 cm21
.
4.1.6. 4-Benzyl-6-/4-methoxyphenyl)-indan-1-one /10). A
stirred solution of 9 -200 mg, 0.58 mmol) and 0.4 ml of
SOCl2 in dry CH2Cl2 -1ml) was heated at 60 8C over 3 h.
The mixture was evaporated in vacuo and the residue was
redissolved in CH2Cl2 -2 ml). The crude acid chloride
solution was added to a cooled solution -08C) of TiCl4
-0.126 ml, 1.16 mmol) in dry CH2Cl2 over 30 min. The
mixture was stirred for 12 h below 58C and then added
into a rapidly stirred mixture of ice, 0.6 ml of conc. HCl
and 2 ml CH2Cl2. After 15 min the mixture was partitioned
and the aqueous layer was extracted with CH2Cl2. The
organic layers were combined, washed successively with
3 M HCl, aqueous sat. NaHCO3, and brine, and concen-
trated at reduced pressure. The residue was puri®ed by silica
gel column chromatography using CH2Cl2 as an eluent to
give 85 mg of a colorless amorphous solid -50%). 10: mp
79±818C; 1H NMR -400 MHz, CDCl3) d 2.69 -2H, t,
Jˆ6.8 Hz), 2.97 -2H, t, Jˆ6.8 Hz), 3.82 -3H, s), 4.10 -2H,
s), 6.95 -2H, d, Jˆ8.9 Hz), 7.50 -2H, d, Jˆ8.9 Hz), 7.16±
7.28 -5H, m), 7.61 -1H, s) and 7.82 -1H, s); IR -KBr) nmax
3040, 2980, 2960, 1709, 1609, 1519, 1474, 1250 and
827 cm21; HRMS -EI) Calcd for C23H20O2 m/z 328.1463,
found m/z 328.1463.
s), 6.41-H1 , s), 6.96 -2H, d,
Jˆ8.7 Hz), 7.50 -2H, d,
Jˆ8.7 Hz), 7.72 -1H, d, Jˆ8.7 Hz), 7.96 -1H, d, Jˆ
8.7 Hz), 7.43 -1H, s) and 7.31±7.39 -5H, m); HRMS -EI)
Calcd for C21H12O3 m/z 316.1100, found m/z 316.1096; IR
-Nujol) nmax 2942, 2852, 1750, 1600, 1522, 1464, 1378,
1312, 1182, 1068, 996, 822, 762 and 722 cm21
.
4.1.3. 5-/4-Methoxyphenyl)-3-phenyl-1,3-dihydroiso-
benzofuran-1-ol /7). Under nitrogen atmosphere, to a
solution of 6 -730 mg, 2.31mmol) in 10 ml of dry CH Cl2
2
at 2788C was added DIBAL -2.6 ml of 1M hexane solution
2.6 mmol) over 30 min. Methanol -10 ml) was added to the
reaction mixture, which was then allowed to warm up to
ambient temperature. The solvent was removed under
reduced pressure. The residue was puri®ed by silica gel
column chromatography using CH2Cl2 as an eluent to give
654 mg of a white amorphous solid -90%). 7: mp 83±858C;
1H NMR -300 MHz, CDCl3) d 3.02 -0.5H, d, Jˆ7.8 Hz),
3.18 -0.5H, d, Jˆ7.8 Hz), 3.82 -3H, s), 6.18 -0.5H, s), 6.39
-0.5H, s), 6.62 -0.5H, d, Jˆ9.8 Hz), 6.76 -0.5H, d, Jˆ
9.8 Hz), 6.95 -2H, d, Jˆ7.8 Hz) 7.40 -2H, d, Jˆ7.8 Hz),
7.28±7.40 -5H, m), and 7.42±7.48 -3H, m); HRMS -EI)
Calcd for C21H18O3 m/z 318.1256, found m/z 318.1230; IR
4.1.7. /E)-4-Benzyl-2-/4-methoxybenzylidene)-6-/4-meth-
oxyphenyl)-indan-1-one /11). A solution of 10 -200 mg,
0.12 mmol), 2.5 M aqueous sodium hydroxide solution
-5 ml, 12.5 mmol) and benzaldehyde -0.15 ml, 1.2 mmol)
in 120 ml of 99.5% ethanol was stirred at room temperature
for 24 h. The reaction mixture was cooled and ®ltered. The
cake was washed with water until the alkali was entirely
removed. The cake was dried to give 85 mg of a colorless
amorphous solid -74%). 11: mp 152±1548C; 1H NMR
-400 MHz, CDCl3) d 3.83 -2H, s), 3.84 -3H, s), 3.86 -3H,
s), 4.19 -2H, s), 6.97 -2H, d, Jˆ8.0 Hz), 6.98 -2H, d, Jˆ
8.0 Hz), 7.55 -2H, d, Jˆ8.0 Hz), 7.57 -2H, d, Jˆ8.0 Hz),
7.18±7.34 -5H, m), 7.61 -1H, s), 7.65 -1H, s) and 7.98 -1H,
s); HRMS -EI) Calcd for C31H26O3 m/z 446.1882, found m/z
446.1893; IR -KBr) nmax 3040, 2925, 1706, 1654, 1609,
-KBr) 3391, 2930, 1608, 1520, 1249, 1180 and 820 cm21
;
Anal. Calcd for C21H18O3; C, 79.20%, H, 5.60%. Found C,
79.18%; H, 5.41%.
4.1.4. Benzyl 3-[3-/hydroxyphenylmethyl)-40-methoxy-
biphenyl-4-yl]-acrylate /8). A mechanically stirred solu-
tion of 7 -610 mg, 1.9 mmol) and benzyl -triphenyl-
phosphoranylidene) acetate -1.02 g, 2.48 mmol) in 20 ml
of benzene was heated at 808C for 1h. The solvent was
removed in vacuo. The residue was puri®ed by a short silica
gel column chromatography using CH2Cl2 as an eluent to
give 815 mg of a colorless amorphous solid -95%). 8: mp
1
133±1358C; H NMR -400 MHz, CDCl3) d 2.30 -1H, s),
3.83 -3H, s), 5.21 -2H, s), 6.25 -1H, s), 6.33 -1H, d,
Jˆ15.6 Hz), 6.96 -2H, d, Jˆ8.4 Hz), 7.54 -2H, d, Jˆ
8.4 Hz), 7.79 -1H, s), 7.38±7.24 -10H, m), 7.59 -1H, d,
Jˆ8.0 Hz), 7.48 -1H, d, Jˆ8.0 Hz) and 8.11 -1H, d, Jˆ
15.6 Hz); HRMS -EI) Calcd for C30H26O4 m/z 450.1855,
found m/z 450.1830; IR -KBr) nmax 3466, 3052, 3030,
2966, 1686, 1626, 1597, 1491, 1311, 1173, 825 and
1584, and 1251 cm21
.
4.1.8. 4-Benzyl-2-/4-methoxybenzyl)-6-/4-methoxy-
phenyl)-indan-1-one /12). A solution of 11 -40 mg,
0.09 mmol) in a mixture of 99.5% ethanol -2 ml) and
EtOAc -2 ml) containing 2 mg of platinum oxide was stirred
in hydrogen atmosphere using a balloon. The mixture was
705 cm21
.

C. Wu et al. / Tetrahedron 57 52001) 9575±9583
9581
stirred at room temperature for 15 h. The catalyst was
removed by ®ltration and the solvent was removed under
reduced pressure. The residue was chromatographed on
TLC plates -1:3 EtOAc±hexane) to give 28 mg of 12 as
4.1.11. 6-Tri¯uoromethanesulfonyloxyindan-1-one /14).
To a solution of BBr3 -25 ml of 1M CH 2Cl2 solution,
0.025 mol) in 50 ml of dry CH2Cl2 at 2788C was added
dropwise 13 -2 g, 12 mmol, Aldrich) in 50 ml of dry
CH2Cl2. After 5 h, the bath was removed and the reaction
mixture was stirred at room temperature for 12 h. The reac-
tion was quenched with water and the aqueous layer was
extracted with CH2Cl2. The organic layers were combined,
dried over Na2SO4, and concentrated in vacuo. The product
was dissolved in 50 ml of dry CH2Cl2 at 2788C. Et3N -1.6 g,
6.2 mmol) and DMAP -15 mg, cat.) were added to the
solution. After complete dissolution had occurred, tri¯uoro-
methanesulfonic anhydride -1.6 g, 6.2 mmol) was added
dropwise over 5 min. The solution was stirred at 2788C
for 30 min then warmed to room temperature over 2 h.
The reaction mixture was poured into saturated NH4Cl
solution -20 ml), the layers were separated and the aqueous
layer was extracted with CH2Cl2. The combined organic
layers were washed with water, dried over MgSO4 and
®ltered. The solvent was removed in vacuo. The residue
was pass though a short column of silicagel with CH2Cl2
1
an oil -70%). H NMR -300 MHz, CDCl3) d 2.63±2.71
-2H, m), 2.95±3.05 -2H, m), 3.24±3.30 -1H, m), 3.79
-3H, s), 3.82 -3H, s), 4.03 -2H, s), 6.95 -2H, d, Jˆ8.8 Hz),
6.97 -2H, d, Jˆ8.8 Hz), 7.07±7.24 -5H, m), 7.24 -2H, d,
Jˆ8.8 Hz), 7.44 -2H, d, Jˆ8.8 Hz), 7.58 -1H, s) and 7.83
-1H, s); HRMS -EI) Calcd for C31H28O3 m/z 448.2039,
found m/z 448.2046; IR -KBr) nmax 3020, 2925, 1706,
1609, 1514, 1251, 1032 and 832 cm21
.
4.1.9. 4-Benzyl-2-/4-hydroxybenzylidene)-6-/4-hydroxy-
phenyl)-indan-1-one /3a). To a solution of BBr3 -0.12 ml
of 1M CH 2Cl2 solution, 0.12 mmol) in 0.5 ml of dry CH2Cl2
at 2788C was added dropwise 12 -14 mg, 0.05 mmol) in
0.5 ml of dry CH2Cl2. After 5 h, the bath was removed
and the reaction mixture was stirred at room temperature
for 48 h. The reaction was quenched with water and the
aqueous layer was separated and extracted with CH2Cl2.
The organic layers were combined, dried over Na2SO4,
and concentrated in vacuo. The residue was puri®ed by
1
to give 1.4 g of a red oil -87%). 14: H NMR -300 MHz,
CDCl3) d 2.78 -2H, t, Jˆ6.0 Hz), 3.20 -2H, t, Jˆ6.0 Hz),
7.64 -1H, d, Jˆ2.0 Hz) 7.49 -1H, dd, Jˆ8.4, 2.0 Hz) and
7.27 -1H, d, Jˆ8.4 Hz); IR -CHCl3) nmax 1720, 1686, 1610,
1498, 1296 and 1144 cm21; HRMS -EI) Calcd for
C10H7O4F3S m/z 280.0017, found m/z 280.0018.
silica gel column chromatography to give 6.6 mg of a
±
1
yellow oil -52%). 3a: H NMR -400 MHz, 9:1CDCl
3
CD3OD) d 2.59±2.72 -2H, m), 2.96±3.05 -2H, m), 3.19±
3.25 -1H, m), 4.03 -2H, s), 6.75 -2H, d, Jˆ8.8 Hz), 6.90
-2H, d, Jˆ8.8 Hz), 7.12±7.34 -5H, m), 7.00 -2H, d, Jˆ
8.8 Hz), 7.44 -2H, d, Jˆ8.8 Hz), 7.59 -1H, s) and 7.79
-1H, s); IR -KBr) nmax 3337, 2922, 1716, 1683, 1598,
1248 and 1172 cm21; HRMS -FAB) Calcd for -M1H)
C29H25O3 m/z 421.1820, found m/z 421.1804; Resolution
of 3a was performed on a HPLC DAICEL OC column
-mobile phase: 2:1hexane±EtOH, ¯ow rate: 0.5 ml
min21); -S)-3a: rtˆ20 min; -R)-3a: rtˆ24 min; -S)-3a: CD
-EtOH) 263 nm -17.4) 330 -0.0) 350 -20.6); -R)-3a: CD
-EtOH) 263 nm -7.4) 330 -0.0) 350 -11.1).
4.1.12. 6-/4-Methoxyphenyl)-indan-1-one /15). 14 -2.0 g,
5 mmol) was treated with 4-methoxyphenylboronic acid
-1g, 6 mmol), 2 M aqueous sodium carbonate solution
-3 ml, 6 mmol), tetrakis-triphenylphosphine)palladium-0)
-238 mg, 0.2 mmol) in a mixture of benzene -10 ml) and
methanol -5 ml) at 808C for 1h. The solution was dried
over Na2SO4 and evaporated. The residue was puri®ed by
silica gel column chromatography -CH2Cl2) to give 1g of a
white amorphous solid -86%). 15: mp 139±1418C. 1H NMR
-300 MHz, CDCl3) d 2.74 -2H, t, Jˆ6.0 Hz), 3.18 -2H, t,
Jˆ6.0 Hz), 3.87 -3H, s), 6.99 -2H, d, Jˆ8.4 Hz), 7.54 -2H,
d, Jˆ8.4 Hz) 7.80 -1H, dd, Jˆ8.4, 2.0 Hz) 7.93 -1H, d,
Jˆ2.0 Hz) and 7.52 -1H, d, Jˆ8.4 Hz); HRMS -EI) Calcd
for C16H14O2 m/z 238.0993, found m/z 238.0987; IR
4.1.10. 4-Benzyl-2-/4-hydroxybenzyl)-2-hydroxymethyl-
6-/4-hydroxyphenyl)-indan-1-one /3b). A solution of 3a
-4 mg, 0.009 mmol), 2.5 M aqueous sodium hydroxide
solution -0.01ml, 0.025 mmol) and 37% formaldehyde
solution -0.003 ml, 0.027 mmol) in 5 ml of 99.5% ethanol
was stirred at room temperature for 2 h. The ethanol was
removed in vacuo. The residue in CH2Cl2 was washed with
aqueous HCl and dried over Na2SO4. The solvent was
removed in vacuo. The residue was chromatographed on
-CHCl3) nmax 1702, 1608, 1482, 1269 and 828 cm21
.
4.1.13. 2-/4-Methoxybezylidene)-6-/4-methoxyphenyl)-
indan-1-one /16). A solution of 15 -560 mg, 2.3 mmol),
2.5 M aqueous sodium hydroxide solution -2.3 ml,
2.3 mmol) and benzaldehyde -0.15 ml, 1.2 mmol) in
150 ml of 99.5% ethanol was stirred at room temperature
for 22 h. The reaction mixture was ®ltered. The cake was
washed with water until the alkali was entirely removed.
The cake was dried to give 630 mg of a colorless amorphous
TLC plates -9:1CH Cl2±CH3OH) to give 2.5 mg of an oil
2
-60%).3b: 1H NMR -400 MHz, 9:1CDCl 3±CD3OD)d 2.80
-1H, d, Jˆ14.8 Hz), 2.81 -1H, d, Jˆ17.6 Hz), 2.94 -1H, d,
Jˆ17.6 Hz), 3.41 -1H, d, Jˆ14.8 Hz), 3.81 -1H, d,
Jˆ10.4 Hz), 3.58 -1H, d, Jˆ10.4 Hz), 4.20 -2H, s), 6.59
-2H, d, Jˆ8.4 Hz), 6.81-2H, d, Jˆ8.4 Hz), 6.88 -2H, d,
Jˆ8.4 Hz), 7.06±7.40 -5H, m), 7.41-2H, d, Jˆ8.4 Hz),
7.55 -1H, d, Jˆ1.2 Hz) and 7.72 -1H, d, Jˆ1.2 Hz),
HRMS -EI) Calcd for C30H27O4 m/z 451.1910 found m/z
451.1905 -M11); Resolution of 3b was performed on a
HPLC DAICEL OC column -mobile phase: 6:1hexane±
EtOH, ¯ow rate: 0.5 ml min21); -S)-3b: rtˆ33 min;
-R)-3b: rtˆ38 min; -S)-3b: CD -EtOH) 263 nm -110) 330
-0.0) 350 -20.2); -R)-3b CD -EtOH) 263 nm -210) 330
1
powder -75%). 16: mp 129±1318C; H NMR -300 MHz,
CDCl3) d 3.87 -3H, s), 3.88 -3H, s), 4.05 -2H, s), 6.99
-4H, d, Jˆ8.4 Hz), 7.58 -2H, d, Jˆ8.4 Hz), 7.80 -1H, dd,
Jˆ8.4, 2.0 Hz), 8.09 -1H, d, Jˆ2.0 Hz), 7.61-1H, d, Jˆ
8.4 Hz), and 7.68 -1H, s); IR -CHCl3) nmax 1700, 1635,
1601, 1484 and 1268 cm21
;
Anal. Calcd for
C24H20O3´0.5H2O; C, 78.88; H, 5.79%. Found C, 79.11%;
H, 5.43.
-0.0) 350 -10.5); -S)-3b: [a]25 ˆ1110, -c 0.02, EtOH);
4.1.14. 2-/4-Methoxybenzyl)-6-/4-methoxyphenyl)-indan-
1-one /17). A solution of 16 -200 mg, 0.56 mmol) in a
D
-R)-3b: [a]25 ˆ2107, -c 0.02, EtOH).
D

9582
C. Wu et al. / Tetrahedron 57 52001) 9575±9583
mixture of 99.5% ethanol -5 ml) and EtOAc -5 ml) contain-
ing 5 mg of platinum oxide was stirred in hydrogen
atmosphere using a balloon at room temperature for 15 h.
The catalyst was removed by ®ltration and the solvent was
removed under reduced pressure. The residue was puri®ed
by silica gel chromatography -CH2Cl2) to give 150 mg of a
¯ow rate: 1.0 ml min21); -S)-19: rtˆ9 min; -R)-19: rtˆ
7 min; -R)-19 [a]25 ˆ255, -c 0.02, EtOH); Anal. Calcd
D
for C25H26O4´0.8H2O; C,74.16%; H, 6.87%. Found C,
74.38%; H, 6.83%.
4.2. Compound /R)-18
1
white amorphous solid -73%). 17: mp 91 ±938C; H NMR
-300 MHz, CDCl3) d 2.67 -1H, dd, Jˆ7.8, 17.1 Hz), 3.17
-1H, dd, Jˆ7.8, 17.1 Hz), 2.84 -1H, dd, Jˆ4.2, 14.1 Hz),
3.34 -1H, dd, Jˆ4.2, 14.1 Hz), 3.00 -1H, m), 3.78 -3H, s),
3.85 -3H, s), 6.84 -2H, d, Jˆ8.4 Hz), 6.99 -2H, d, Jˆ
8.4 Hz), 7.79 -1H, dd, Jˆ8.4, 2.0 Hz), 7.94 -1H, d, Jˆ
2.0 Hz), 7.56 -2H, d, Jˆ8.4 Hz), 7.44-1H, d, Jˆ8.4 Hz)
and 7.18 -2H, d, Jˆ8.4 Hz); IR -CHCl3) nmax 1705, 1609,
1582, 1490, 1301 and 1135 cm21; Anal. Calcd for
C24H22O3´0.5 H2O; C, 78.45; H, 5.91. Found C, 78.17; H,
6.30.
A solution of -2)-19 -1mg, 0.003 mmol), MnO 2 -1mg,
excess) in 0.5 ml of THF solution was stirred at room
temperature for 1h. The solvent was removed in vacuo.
The residue was puri®ed by silica gel plate -CH2Cl2) to
give 0.76 mg -75%) of -R)-18. [a]25 ˆ291, -c 0.04,
D
1
EtOH); H NMR was same as -^)-18; CD -EtOH) 263 nm
-211) 330 -0.0) 350 -0.2).
4.2.1. Di-4-dimethylaminobenzoate 20. A mixture solu-
tion of -1)-19 -10 mg, 0.025 mmol), 4-dimethylamino-
benzoyl chloride in 0.5 ml of pyridine was stirred at
1108C for 24 h. The mixture was quenched with water and
aqueous layer was extracted with CH2Cl2. The organic layer
was combined and dried over Na2SO4. The solvent was
removed in vacuo. The residue was puri®ed on a silica gel
4.1.15. 2-Hydroxymethyl-2-/4-methoxybenzyl)-6-/4-meth-
oxyphenyl)-indan-1-one /18). A solution of 17 -100 mg,
0.27 mmol), 2.5 M aqueous sodium hydroxide solution
-0.11 ml, 0.27 mmol) and 37% formaldehyde solution
-0.025 ml, 0.27 mmol) in 5 ml of 99.5% ethanol was stirred
at room temperature for 0.5 h. The solvent was removed in
vacuo. The residue in CH2Cl2 was washed with aqueous
HCl and dried over Na2SO4. The solvent was removed in
vacuo. The residue was puri®ed by silica gel chroma-
tography -CH2Cl2) to give 80 mg of a white amorphous
solid -77%). 18: mp 91 ±983C; 1H NMR -300 MHz,
CDCl3) d 2.92 -1H, d, Jˆ17.8 Hz), 3.05 -2H, m), 3.16
-1H, d, Jˆ17.8 Hz), 3.62 -1H, d, Jˆ10.1 Hz), 3.85 -1H, d,
Jˆ10.1 Hz), 3.76 -3H, s), 3.84 -3H, s), 6.73 -2H, d,
Jˆ9.0 Hz), 6.98 -2H, d, Jˆ9.0 Hz), 7.07 -2H, d, Jˆ
9.0 Hz), 7.41-1H, d, Jˆ9.0 Hz), 7.52 -2H, d, Jˆ9.0 Hz),
7.75 -1H, dd, Jˆ9.0, 1.8 Hz) and 7.87 -1H, d, Jˆ1.8 Hz);
HRMS -FAB) Calcd for C25H25O4 -M1H) m/z 389.1753,
found m/z 389.1754; IR -CHCl3) nmax 2932, 1702, 1611,
1
plate to give 2.5 mg of 20 as a yellow oil -20%). H NMR
-400 MHz, CDCl3) d 2.64 -1H, d, Jˆ16 Hz), 2.98 -6H, s),
3.02 -6H, s), 3.23 -2H, d, Jˆ16 Hz), 3.75 -3H, s), 3.81 -3H,
s), 3.76±3.83 -1H, m), 4.04 -1H, d, Jˆ10 Hz), 4.16 -1H, d,
Jˆ10 Hz), 6.52 -2H, d, Jˆ8.8 Hz), 6.6 -1H, s), 6.64 -2H, d,
Jˆ8.8 Hz), 6.77 -2H, d, Jˆ8.8 Hz), 6.91-2H, d, Jˆ8.8 Hz),
7.08 -2H, d, Jˆ8.8 Hz), 7.25 -1H, d, Jˆ8.0 Hz), 7.44 -1H, d,
Jˆ8.0 Hz), 7.47 -2H, d, Jˆ8.8 Hz), 7.81-2H, d, Jˆ8.8 Hz),
7.98 -2H, d, Jˆ8.8 Hz), and 7.61-1H, s); HRMS -EI) Calcd
for C43H44O6N2 m/z 684.3200, found m/z 684.3215; IR
-CHCl3) nmax 1701, 1608, 1278 and 1183 cm21; [a]22
151- c 0.02, EtOH).
ˆ
D
4.2.2.
2-/4-Hydroxy-benzyl)-2-hydroxymethyl-6-/4-
hydroxyphenyl)-indan-1-one /3c). To a solution of BBr3
-0.15 ml, 0.15 mmol) in 5 ml of dry CH2Cl2 at 2788C was
added dropwise 17 -50 mg, 0.14 mmol) in 5 ml of dry
CH2Cl2. After 3 h, the bath was removed and the reaction
mixture was stirred at room temperature for 15 h. The
reaction was quenched with water and the aqueous layer
was extracted with CH2Cl2. The organic layers were
combined, dried over Na2SO4, and concentrated in vacuo.
A solution of the crude product -36 mg, 0.11 mmol), 2.5 M
aqueous sodium hydroxide solution -0.044 ml, 0.11 mmol)
and 37% formaldehyde solution -0.01ml, 0.11mmol) in
2 ml of 99.5% ethanol was stirred at room temperature for
0.5 h. The solvent was removed in vacuo. The residue in
CH2Cl2 was washed with aqueous HCl and dried over
Na2SO4. The solvent was removed in vacuo. The residue
was puri®ed by silica gel chromatography -CH2Cl2) to give
36 mg of a white amorphous solid -60% in two steps). 3c:
1513, 1250 and 1036 cm21
.
4.1.16. 2-Hydroxymethyl-2-/4-methoxybenzyl)-6-/4-
methoxyphenyl)indan-1-ol /19). To a solution of 18
-75 mg, 0.19 mmol) in 1 ml of 99.5% ethanol solution was
added dropwise NaBH4 -21mg, 0.57 mmol) suspend in 1ml
of THF at 08C. The reaction mixture was stirred at 48C for
12 h. The mixture was quenched with water and aqueous
layer and extracted with CH2Cl2. The organic layers were
combined and dried over Na2SO4. The solvent was removed
in vacuo. The residue was puri®ed by silica gel column
chromatography -CH2Cl2) to give diol 19 -40 mg) and its
diastereomers -28 mg). 19: mp 80±828C; 1H NMR
-400 MHz, 9:1CDCl 3±CD3OD) d 2.34 -1H, d, Jˆ
16.0 Hz), 2.49 -1H, d, Jˆ12.4 Hz), 2.81 -1H, d, Jˆ
16.0 Hz), 3.10 -1H, d, Jˆ12.4 Hz), 3.45 -1H, d, Jˆ
10.4 Hz), 3.62 -1H, d, Jˆ10.4 Hz), 3.75 -3H, s), 3.80 -3H,
s), 5.22 -1H, s) 6.84 -2H, d, Jˆ8.8 Hz), 6.98 -2H, d,
Jˆ8.8 Hz), 7.14 -1H, d, Jˆ8.8 Hz), 7.22 -1H, d, Jˆ
7.8 Hz), 7.48 -1H, d, Jˆ7.8 Hz), 7.54-2H, d, Jˆ8.8 Hz)
and 7.59 -1H, s); 13C NMR -75 MHz, CDCl3) d 35.8,
53.8, 55.2, 55.3, 66.1, 80.5, 114.1, 113.4, 122.5, 125.1,
126.6, 128.0, 130.6, 131.5, 133.9, 138.8, 139.6, 144.7,
157.9 and 158.9 ppm; IR -CHCl3) nmax 3422, 1610, 1511
and 1248 cm21; resolution of 19 was performed on a HPLC
DAICEL OC column -mobile phase: 1:1 hexane±EtOH,
1
mp 91±938C; H NMR -300 MHz, 9:1CDCl 3±CD3OD)
2.87 -1H, d, Jˆ13.5 Hz), 2.94 -1H, d, Jˆ13.5 Hz), 2.95
-1H, d, Jˆ16 Hz), 3.15 -1H, d, Jˆ16 Hz), 3.61 -1H, d, Jˆ
10.8 Hz), 3.84 -1H, d, Jˆ10.8 Hz), 6.66 -2H, d, Jˆ8.4 Hz),
7.00 -2H, d, Jˆ8.4 Hz), 6.88 -2H, d, Jˆ8.4 Hz), 7.42 -2H, d,
Jˆ8.4 Hz), 7.39 -1H, d, Jˆ8.1Hz), 7.71-1H, d, Jˆ8.1Hz)
and 7.82 -1H, s); HRMS -EI) Calcd for C23H20O4 m/z
360.1362, found m/z 360.1389; IR -CHCl3) nmax 3278,
2918, 1691, 1662, 1614, 1514, 1485, 1479, 1440, 1276
and 1223 cm21; Resolution of 3c was performed on a

C. Wu et al. / Tetrahedron 57 52001) 9575±9583
9583
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D
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D
330 -0.0) 350 -0.4).
Acknowledgements
8. Head, J. F.; Inouye, S.; Teranishi, K.; Shimomura, O. Nature
2000, 405, 372±376.
We thank ®nancial supports from Grants-in-Aid for
Scienti®c Research on Priority Areas -A) from the Ministry
of Education, Sciences, Sports and Culture of Japan, from
the Naito Foundation, and a JSPS fellowship for C. Wu. We
also thank Professor M. Isobe and Dr Y. Oba of Nagoya
University for his helpful discussions and suggestions.
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