Active core structure of terfestatin A, a new specific inhibitor of auxin signaling

Article abstract of DOI:10.1016/j.bmc.2008.02.085

The auxins, plant hormones, regulate many aspects of the growth and development of plants. Terfestatin A (TrfA), a novel auxin signaling inhibitor, was identified in a screen of Streptomyces sp. F40 extracts for inhibition of the expression of an auxin-in

Full text of DOI:10.1016/j.bmc.2008.02.085

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5331–5344
Active core structure of terfestatin A, a new specific inhibitor
of auxin signaling
Ken-ichiro Hayashi,a,* Atsushi Yamazoe,a Yuki Ishibashi,a Naoyuki Kusaka,a
Yutaka Oonob and Hiroshi Nozakia
aDepartment of Biochemistry, Okayama University of Science, 1-1 Ridai-cho, Okayama City 700-0005, Japan
bRadiation-Applied Biology Division, Japan Atomic Energy Agency, Takasaki 370-1292, Japan
Received 17 January 2008; revised 26 February 2008; accepted 27 February 2008
Available online 4 March 2008
Abstract—The auxins, plant hormones, regulate many aspects of the growth and development of plants. Terfestatin A (TrfA), a
novel auxin signaling inhibitor, was identified in a screen of Streptomyces sp. F40 extracts for inhibition of the expression of an
auxin-inducible gene. However, the mode of action of TrfA has not been elucidated. To identify the active core structure, 25 deriv-
atives of TrfA were synthesized and their inhibitory activities against auxin-inducible gene expression were evaluated. The structure–
activity relationships revealed the essential active core structure of TrfA, 3-butoxy-4-methylbiphenyl-2,6-diol, which will lead to the
design of biotin-tagged active TrfA.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
scription regulators. This heterodimerization represses
the expression of primary auxin-responsive genes by
interrupting the interaction of ARF with auxin-respon-
sive promoters.3 It has been demonstrated that ubiqui-
tin–proteasome-dependent proteolysis of AUX/IAA
repressor is crucial for primary auxin-responsive gene
expression and various subsequent developmental pro-
cesses, including lateral root growth, hypocotyl elonga-
tion, gravitropism, and photomorphogenesis.1 TIR1
encodes an F-box protein that interacts with the Skp1
and Cdc53 (cullin) proteins to form ubiquitin ligase
complexes called SCFs. SCFTIR1 complex is involved
in the proteolytic pathway that leads to auxin-dependent
degradation of AUX/IAA repressors.4 Auxins promote
the interaction between AUX/IAAs and TIR1 protein
and enhance the ubiquitination of AUX/IAA proteins
and the subsequent degradation rate of repressor, ulti-
mately enhancing auxin-responsive gene expression.4–6
Recent reports have demonstrated that TIR1 is one of
the six auxin receptors (TIR1 and AFBs 1-5)7 and that
auxins enhance the interaction between TIR1 and
AUX/IAA repressor.8,9
The plant hormones regulate the growth and develop-
ment of plants. These hormones, such as the auxins, gib-
berellin, cytokinin, abscisic acid, and ethylene, are low
molecular weight organic compounds, as distinct from
the proteinaceous hormones found in mammals. In-
dole-3-acetic acid (IAA: 2), an auxin, plays a crucial role
in many aspects of plant development including cell divi-
sion, elongation, and differentiation. At the whole plant
level, auxins regulate tropisms, apical dominance and
root development, and ultimately control the architec-
ture of adult plants.1
Recent molecular biological and genetic studies of auxin
signaling have revealed that three major families of
genes (Aux/IAAs, GH3s and SUARs) are induced in re-
sponse to auxins.2 Of the three families, it has been dem-
onstrated that the AUX/IAA family plays a critical role
in auxin signaling. AUX/IAAs encode a nuclear localized
transcriptional repressor capable of heterodimerization
with auxin-responsive factors (ARFs) families of tran-
In the course of screening for specific inhibitors of auxin
signaling, we have found a novel and specific inhibitor
of auxin signaling, designated terfestatin A (TrfA (1),
Fig. 1A).10,11 TrfA was identified in a screen of Strepto-
myces sp. F40 extracts for inhibition of the expression of
Keywords: Auxin; Structure–activity relationships; Terfestatin A;
Terphenyl; TIR1.
*
Corresponding author. Tel.: +81 86 256 9661; fax: +81 86 256
9559; e-mail: hayashi@dbc.ous.ac.jp
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.085

5332
K. Hayashi et al. / Bioorg. Med. Chem. 16 (2008) 5331–5344
OH
O
A
A
R1
R2
R3
HO
HO
OH
-O-β-D-glucose
-OCH -OH
Ph
Ph
1a
1b
3
4
HO
O
HO
O
HO
O
R1
R2
R3
O
-O-β-D-glucose
-O-β-D-tetraacetyl
glucose
-OH
-OH
-OCH
3
C
1
2'
HO
1c
9
-OH
-H
3'
1'
HN
B
O5H'
-OH
-O-β-D-glucose
1"
A
4''
IAA (2)
NAA (3) PAA (4)
TrfA (1)
B
B
Figure 1. Structures of terfestatin A (1) and auxins (2–4) and the stable
confomations of 1 and NAA.
an auxin-responsive GUS reporter construct.10 TrfA
specifically blocks auxin-responsive gene expression
and physiological auxin responses in roots.11 Conforma-
tional analysis of the TrfA structure revealed structural
similarities between the auxins and TrfA (Fig. 1A and
B). The positions of the aromatic ring and the carboxyl
group in auxins such as indole-3-acetic acid (IAA: 2)
and 1-naphthaleneacetic acid (NAA: 3) closely match
the positions of the aromatic A ring and the phenolic hy-
droxyl group in TrfA (Fig. 1B). In addition, phenylace-
tic acid (4) also was known to display weak auxin
activity. Therefore, we expected that TrfA mimics auxin
and acts as an auxin antagonist. However, biological
investigation of TrfA demonstrated that TrfA does not
bind to TIR1, leaving two possible explanations of TrfA
action.11 One is that TrfA selectively interacts with other
auxin receptors such as AFBs 1–5. The second is that
TrfA does not act on AFB receptors, but acts on the sig-
naling components regulating auxin-dependent AUX/
IAA proteolysis. The identification of the target protein
of TrfA would provide direct evidence for the inhibitory
mechanism of action of TrfA. Biotin-tagged TrfA is the
critical probe that would allow us to identify the target
protein by affinity chromatography.
Figure 2. (A) Structures of the derivatives 1a–c and 9. (B) Effects of
derivatives on the auxin-induced gene expression. Arabidopsis
BA3::GUS transgenic reporter line was treated with 50 lM solutions
of the derivatives in the presence of 1 lM IAA for 5 h. IAA-induced
GUS expression was visualized by histochemical staining using X-Gluc
for 3–4 h. The photos of the root tips were taken on three
representatives.
2,5-Diphenylbenzoquinone (5) was reduced by sodium
borohydrate to yield phenol (6a). Selective alkaline
hydrolysis of the diacetate (6b) gave a monoacetate
(7). This monoacetate was glycosylated by treatment
with acetobromo-a-glucose and cesium bicarbonate, fol-
lowed by alkaline hydrolysis to yield the dehydroxylated
TrfA derivative (9). Monomethylated derivatives (1a
and 1b) were synthesized according to essentially the
same procedure described previously.10 The biological
activities of the derivatives were evaluated by their
inhibitory activities against auxin-induced reporter gene
expression using Arabidopsis BA3::GUS transgenic
plants.12 The BA3::GUS line carries the GUS reporter
gene under the control of an auxin-responsive promoter,
and it rapidly expresses GUS enzyme in response to aux-
in.12 The inhibition of auxin-induced GUS enzymatic
activity was visualized by histochemical staining using
X-Gluc as a chromogenic substrate or was fluorometri-
cally quantified using 4-methyl umbelliferyl-b-D-glucu-
ronide as a fluorogenic substrate.
To design biotin-tagged TrfA, we investigated the struc-
ture–activity relationships of TrfA by examining the
characteristics of its derivatives. Here, we propose an ac-
tive core structure of TrfA based on its structure–activ-
ity relationships and report the design of biologically
active biotin-tagged TrfA.
Loss of the hydroxyl group in the R2 position of TrfA
(derivative 9) completely abolished the inhibitory effect
on BA3::GUS expression (Fig. 2). Methylation of the
hydroxyl groups at R1 or R2 (1a: R1 = OCH3, 1b:
R2 = OCH3) also caused complete inactivation (Fig. 2).
These results suggest that both hydroxyl groups are
essential to retain the activity of TrfA, and contributes
to binding TrfA to the target protein.
2. Results and discussion
2.1. The structure–activity relationships of the phenolic
hydroxyl group and the glucoside moiety in TrfA
To assess the role of hydroxyl groups in the inhibitory
activity, TrfA derivatives (1a–c and 9) were synthesized
(Fig. 2). The synthesis of 9 is summarized in Scheme 1.
TrfA has a b-glucoside moiety, and aglycon itself did not
show inhibitory activity (data not shown). In addition,

K. Hayashi et al. / Bioorg. Med. Chem. 16 (2008) 5331–5344
5333
OAc
AcO
AcO
OAc
O-β-D-glucose
OH
OH
O
a
Ph
Ph
Ph
e
Ph
b, c
d
O
O
Ph
Ph
Ph
Ph
Ph
OH
OAc
O
OH
Ph
OAc
5
8
6a
7
9
Scheme 1. Reagents and conditions: (a) NaBH4, MeOH, rt, 1 h, 70%; (b) Ac2O, DMAP, CH2Cl2, 0 ꢁC, 1 h, 93%; (c) KOH, MeOH, ꢀ18 ꢁC, 1 h,
81%; (d) Cs2CO3, acetobromo-a-D-glucose, CH3CN, rt, 24 h, 79%; (e) KOH, MeOH, rt, 1 h, 90%.
TrfA derivatives having tetraacetylglucoside (1c) were
also found to be inactive (Fig. 2A). This suggests the gly-
coside residue is essential for inhibitory activity. To fur-
ther elucidate the role of the glucoside moiety of TrfA,
we substituted an alkyl ether chain in place of the gluco-
side moiety (Fig. 3A). The alkyl ether derivatives (12a–k)
were synthesized by reaction of the intermediate (10)
with the corresponding alkyl bromide, TBAI, and so-
dium hydride, followed by the deprotection of the benzyl
groups (Fig. 3A and B). The inhibitory activities of 12a–
k are shown in Figure 3B. The ethyl ether and propyl
ether derivatives (12a and 12b) displayed weak inhibitory
activities toward BA3::GUS expression. The butyl ether
derivative (12c) retained inhibitory activity. The intro-
duction of 4-chlorobutyl ether (12f), and methoxylmeth-
yl ether groups (12g) produced the same inhibitory
activity as the butyl ether derivative (12c). On the other
hand, the pentyl ether derivative (12d) was less active.
The introduction of isopropyl ether (12h), isobutyl ether
(12i), cyclopropylmethyl ether (12j), hexyl ether (12e)
and cyclohexylmethyl ether (12k) completely abolished
the activity. From these results, we conclude the gluco-
side moiety is not essential for the binding to the target
protein, but the size and length of the carbon chain are
crucial to the inhibitory activity. Therefore, the glucoside
moiety in TrfA acts as an anchor to hold TrfA at the
binding site of the target protein. As a result of investi-
gating the structure–activity relationships for the sugar
moiety, we found that the introduction of a biotin-tagged
linkage into the sugar moiety would abolish the activity.
2.2. The structure–activity relationships of the phenyl A
ring in TrfA
We next synthesized A ring-substituted TrfA derivatives
to investigate the role of the phenyl A ring in the activity
(Scheme 2). Suzuki coupling was used as the key step to
introduce the various substituents into phenyl A ring of
TrfA. 2,5-Dihydroxybenzaldehyde (13) was protected
with benzyl groups to afford dibenzylaldehyde (14).
Monobenzylether (15) was obtained by selective depro-
tection using magnesium bromide.13 Compound 15
A
Ph
Ph
Ph
alkyl halide
TBAI
Ph
10 % Pd/C
H2
HO
RO
OH
BnO
HO
OBn
BnO
RO
OBn
NaH
EtOAc
rt
24h
THF
rt
24h
Ph
10
Ph
11a-k
12a-k
−Ο−β-D-glucose
TrfA
12a
B
12b
12c
12d
12e
12f
Cl
12g
12h
O
12i
12j
12k
Figure 3. (A) Synthetic scheme for the 20-alkyl ether derivatives (12a–k). (B) Effects of 12a–k on the auxin-induced gene expression. Arabidopsis
BA3::GUS transgenic reporter line was treated with 20 or 50 lM solutions of the derivatives in the presence of 1 lM IAA for 5 h. The photos of the
root tips were taken on three representatives.

5334
K. Hayashi et al. / Bioorg. Med. Chem. 16 (2008) 5331–5344
MOMO
MOMO
OHC
OBn
OH
OH
OH
a
d
b
c
e
OHC
OHC
Br
OHC
OHC
OHC
Br
OBn
14
OBn
16
OBn
18a
OH
13
OBn
15
OBn
17
O-β-D-glucose
OMOM
Ph
OMOM
Ph
OMOM
Ph
l, m, n, o
h, i, j
BnO
R
OHC
Br
BnO
Br
HO
R
Ph
f, g
k
OBn
OBn
OBn
OH
21a-d
22a-d
19a
20
O-β-D-glucose
Ph
OMOM
Ph
OMOM
Ph
s, t, u, v
r
HO
BnO
p, q
HO
18a
OBn
OBn
OH
23
24a
25
Scheme 2. Reagents and conditions: (a) BnBr, K2CO3, CH3CN, reflux, 4 h; (b) MgBr, benzene/Et2O = 7:1, reflux, 8 h; (c) Br2, AcONa, AcOH, rt,
3 h; (d) MOMCl, i-Pr2EtN, CH2Cl2, 6 h; (e) PhB(OH)2, Pd(PPh3)4, 2 M Na2CO3, DME/EtOH = 5:1, reflux, 3 h; (f) Br2, AcONa, AcOH, 50 ꢁC, 4 h;
(g) MOMCl, i-Pr2EtN, CH2Cl2, 6 h; (h) m-CPBA, CH2Cl2, 0 ꢁC–rt, 24 h; (i) KOH, MeOH, rt, 1 h; (j) BnBr, K2CO3, reflux, 2 h; (k) R-B(OH)2,
Pd(PPh3)4, 2 M Na2CO3, DME/EtOH = 5:1, reflux, 3 h; (l) AcCl, MeOH, 0 ꢁC–rt, 2 h; (m) Cs2CO3, acetobromo-a-D-glucose, CH3CN, rt, 24 h; (n)
10% Pd/C, H2, EtOAc, rt, 12 h; (o) KOH, MeOH, rt, 1 h; (p) m-CPBA, CH2Cl2, 0 ꢁC–rt, 8 h; (q) same as I; (r) BnBr, K2CO3, reflux, 1 h; (s) AcCl,
MeOH, 0 ꢁC–rt, 2 h; (t) same as m, CH3CN, rt, 24 h; (u) same as n; (v) same as o.
was treated with bromine to yield 16.14 The hydroxyl
group in 16 was protected by the MOM group and then
A
C-ring substituted TrfA b)
R2
A-ring substituted TrfA a)
R1
coupled with various phenyl boronic acids by Suzuki
coupling to yield biphenyl (18a). In this coupling, the
reaction did not proceed unless the hydroxyl group of
16 was protected. Therefore, biphenyl 18a was bromi-
nated and then protected by an MOM group. The alde-
hyde group in 19a was converted to a hydroxyl group by
Baeyer–Villiger oxidation, followed by alkaline hydroly-
sis. The corresponding boronic acids (Fig. 4A) were cou-
pled with the key intermediate (20) by Suzuki coupling
after protection with benzyl groups. The MOM group
in 21a–d was deprotected, and then the glycoside was
introduced by treatment with acetobromoglucose and
cesium bicarbonate. The deprotection of the benzyl
and acetyl groups yielded the A-ring-substituted TrfA
derivatives (22a–d). The biphenyl derivative of TrfA
(25) was synthesized from the intermediate (18a) as
shown in Scheme 2. The synthetic schemes for the A-
ring-substituted TrfA derivatives (Fig. 4A) are summa-
rized in Scheme 2.
O
CH
22a
3
34a
O
O
37a
37b
22b
22c
OCH
3
OC
H
5
2
O
O
22d
25
34b
41
CH3
H
CH3
H
a: A-ring substituted TrfA: R2=phenyl.
b: C-ring substituted TrfA: R1=phenyl.
R1
R2
HO
OH
O-β-D-glucose
TrfA: R1, R2 =phenyl
B
Derivatives 22d and 25, which lack the aromatic A-ring,
were found to have lost the ability to inhibit BA3::GUS
expression (Fig. 4A and B). This suggests that the aro-
matic A-ring of TrfA is important for this activity.
The introduction of a methyl group at the p-position
of the A-ring (22a) did not affect the activity, whereas
the introduction of a butylether group (22b) caused con-
siderable reduction of the activity (Fig. 4A and B). Con-
formational analysis implied that TrfA mimics auxin
(Fig. 1B). NAA (3) is a synthetic auxin. Derivative
22c, which has a naphthalene ring as an A-ring, was de-
signed to mimic the NAA molecule (Figs. 1A and B).
However, the inhibitory activity of 22c was dramatically
decreased. This suggests that the large naphthalene
A-ring could not fit into the binding pocket of target
protein. Alternatively, 1H and 13C NMR spectra of
Figure 4. (A) Structures of the A-ring-substituted derivatives and the
B-ring-substituted derivatives. (B) Effects of the derivatives on the
auxin-induced gene expression. Arabidopsis BA3::GUS transgenic
reporter line was treated with 10 and 20 lM solutions of the derivatives
in the presence of 1 lM IAA for 5 h. IAA-induced GUS expression
was determined fluorometrically by the fluorogenic substrate, 4-methyl
umbelliferyl b-D-glucuronide. Values are means SD of three-inde-
pendent experiments.

K. Hayashi et al. / Bioorg. Med. Chem. 16 (2008) 5331–5344
5335
22c showed two sets of signals from two rotational iso-
mers due to restricted rotation of the naphthalene ring
in 22c, whereas there is free rotation between the phenyl
ring and the hydroxyl group in TrfA. Therefore, deriva-
tive 22c would not be able to adopt the conformation re-
quired between the A-ring and the hydroxyl group to
enable binding to the target protein. These results indi-
cate it would be impossible to introduce the biotin-
tagged linker at the phenyl A-ring without loss of the
original activity of TrfA.
biphenyl aldehyde (30). Aldehyde (30) was protected
by the MOM group, and then converted to the key inter-
mediate (32) by Baeyer–Villiger oxidation and alkaline
hydrolysis, followed by protection with a benzyl group.
Suzuki coupling of 32 with the corresponding boronic
acids (Fig. 4A) gave terphenyls (33a–b and 35a–b).
The deprotection and glycosylation of terphenyls (33a–
b and 36a–b) were performed via essentially the same
procedures described above to yield C-ring-substituted
TrfA derivatives (34a–b and 37a–b). Biphenyl TrfA
derivative (41) was synthesized from intermediate (29)
by the same procedures as used for 34a (Scheme 4).
2.3. The structure activity–relationships of the phenyl C-
ring in TrfA
Biphenyl derivative 41, which lacks a phenyl C-ring, was
completely inactive (Fig. 4A and B). On the other hand,
biphenyl 34b with a methyl group instead of a phenyl
C-ring exhibited inhibitory activity to the same extent
as TrfA. These results suggest that the aromatic C-ring
is not essential for activity but the C1 chain (methyl
group), at least, is required for activity. It is possible that
the methyl group in 34b might contribute to hold 34b in
the binding site of the target protein. Derivative 34a (4-
To address the role of the C-ring of TrfA in the inhibi-
tory activity, C-ring-substituted TrfA derivatives were
synthesized (Schemes 3 and 4). 2,5-Dihydroxybenzalde-
hyde (13) was brominated15 and then protected with
benzyl groups to afford aldehyde (27). This aldehyde
was coupled with phenyl boronic acid by Suzuki cou-
pling to give a biphenyl (28). The selective deprotection
of the benzyl group, followed by bromination, yielded
OBn
OH
OH
OH
OH
OBn
e
a
OHC
d
OHC
Ph
Br
OHC
Br
OHC
Ph
OHC
b
OHC
Br
c
OBn
28
OBn
30
OH
26
OBn
29
OH
13
OBn
27
OMOM
R
O-β-D-glucose
OMOM
Br
OMOM
Br
BnO
Ph
j
HO
R
k, l, m, n
g, h, i
BnO
Ph
OHC
Ph
f
Ph
OBn
33a-b
OH
34a-b
OBn
32
OBn
31a
Scheme 3. Reagents and conditions: (a) Br2, CHCl3, rt, 3 h; (b) BnBr, K2CO3, reflux, 4 h; (c) PhB(OH)2, Pd(PPh3)4, 2 M Na2CO3, DME/
EtOH = 5:1, reflux, 2 h; (d) MgBr, benzene/Et2O = 7:1, reflux, 4 h; (e) Br2, AcONa, AcOH, rt, 3 h; (f) MOMCl, i-Pr2EtN, CH2Cl2, 6 h; (g) m-CPBA,
CH2Cl2, 0 ꢁC–rt, 24 h; (h) KOH, MeOH, rt, 1 h; (i) BnBr, K2CO3, CH3CN, reflux, 1 h; (j) R-B(OH)2, Pd(PPh3)4, 2 M Na2CO3, DME/EtOH = 5:1,
reflux, 3 h; (k) AcCl, MeOH, 0 ꢁC–rt, 2 h; (l) Cs2CO3, acetobromo-a-D-glucose, CH3CN, rt, 24 h; (m) 10% Pd/C, H2, EtOAc, rt, 12 h; (n) KOH,
MeOH, rt, 1 h.
OBn
OMOM
OBn
OMOM
OH
Ph
Ph
Ph
O-β-D-glucose
c, d, e, f
i, j, k,l
a
b
32
BnO
BnO
HO
OH
OBn
O
O
35a
36a
37a
O
O
OH
OBn
OMOM
OBn
OMOM
Ph
HO
O-β-D-glucose
Ph
Ph
BnO
g
h
32
BnO
O
O
O
35b
36b
37b
OH
O
O
O
OMOM
OMOM
OMOM
O-β-D-glucose
HO
Ph
OHC
Ph
BnO
Ph
HO
Ph
n, o
p
q, r, s, t
m
29
OBn
OBn
OBn
OH
41
40a
38
39
Scheme 4. Reagents and conditions: (a) 4-carboxy-PhB(OH)2, Pd(PPh3)4 2 M NaOH aq, DME/EtOH = 5:1, reflux, 3 h; (b) BnBr, K2CO3, CH3CN,
reflux, 1 h; (c) TFA, CH2Cl2, rt, 2 h; (d) Cs2CO3, acetobromo-a-D-glucose, CH3CN, rt, 24 h; (e) 10% Pd/C, H2, EtOAc, rt, 9 h; (f) AcCl, MeOH,
0 ꢁC–rt, 2 h; (g) 4-hydroxy-PhB(OH)2, Pd(PPh3)4 2 M NaOH aq, DME/EtOH = 5:1, reflux, 2 h; (h) 5-bromo-valerate, K2CO3, CH3CN, reflux, 6 h;
(i) TFA, CH2Cl2, rt, 1 h; (j) same as d; (k) 10% Pd/C, H2, EtOAc, rt, 6 h; (l) KOH, MeOH, rt, 1 h; (m) MOMCl, i-Pr2EtN, CH2Cl2, 3 h; (n) m-CPBA,
CH2Cl2, 0 ꢁC–rt, 8 h; (o) same as l; (p) BnBr, K2CO3, reflux, 2 h; (q) AcCl, MeOH, 0 ꢁC–rt, 1 h; (r) same as d; (s) same as k; (t) same as l.

5336
K. Hayashi et al. / Bioorg. Med. Chem. 16 (2008) 5331–5344
the indicated hormone and/or chemicals and then incu-
bated for the indicated time to induce each responsive
gene.
4.1.2. Histochemical and quantitative measurements of
GUS reporter activity. For GUS histochemical analysis,
the BA3::GUS seedlings were washed with a GUS-stain-
ing buffer (100 mM sodium phosphate, pH 7.0, 10 mM
EDTA, 0.5 mM K4Fe(CN)6, 0.5 mM K3Fe(CN)6, and
0.1% Triton X-100) and transferred to GUS-staining
buffer containing 1 mM 5-bromo-4-chloro-3-indolyl b-
D-glucuronide (X-Gluc), the substrate for histochemical
staining. They were then incubated at 37 ꢁC until suffi-
cient staining developed (3–4 h). For quantitative mea-
surement, the excised roots after reporter gene
induction (n = 15–20) were homogenized in an extrac-
tion buffer as previously described.12 After centrifuga-
tion to remove cell debris, GUS activity was measured
using 1 mM 4-methyl umbelliferyl b-D-glucuronide as
a fluorogenic substrate at 37 ꢁC. The protein concentra-
tion was determined by Bradford protein assay (Bio-
Rad Japan, Japan). The experiments were repeated at
least three times with three replications.
Figure 5. (A) Active core structure of terfestatin A (1). (B) Proposed
binding model of active core structure of 1 to the target protein.
butoxyphenyl as a C-ring) had almost the same activity
as TrfA, in contrast to 22b (4-butoxyphenyl as an A-
ring). Consistent with the model, the C-ring would be lo-
cated outside of the binding site in the target protein
(Fig. 1B). To design an active biotin-tagged TrfA affinity
probe for the identification of target proteins, deriva-
tives 37a and 37b would be useful intermediates to link
to a biotin-tagged probe. As expected, derivatives 37a
and 37b showed potent activity similar to that of TrfA.
Therefore, the introduction of a biotin linker would be
possible at the o-position on the phenyl C-ring without
loss of activity.
4.2. General experimental conditions
All melting points were measured on a melting-point
apparatus (Gallenkamp, UK) and are uncorrected.
Optical rotations were measured with a SEPT-200
polarimeter (Horiba, Japan). IR spectra on a model
3. Conclusions
1
The binding model of TrfA was proposed based on the
structure–activity study of TrfA. To find active core
structure, 25 derivatives of TrfA were synthesized and
evaluated with regard to inhibitory activity against aux-
in-responsive gene expression. We identified the essential
active core structure of TrfA as 3-butoxy-4-methylbi-
phenyl-2,6-diol (Fig. 5A). The results of structure–activ-
ity relationships propose the binding model of essential
active core structure to the target protein as shown in
our model (Fig. 5B). In our proposed model, A- and
B-rings of TrfA are recognized in binding pocket of tar-
get protein. On the other hand, C-ring would not be rec-
ognized by target protein and the sugar moiety is not
essential for the binding of TrfA. In addition, we dem-
onstrated that 37a and 37b, key intermediates for bio-
tin-tagged TrfA, maintain the original activity of TrfA.
In a future study, these results will enable us to design
biotin-tagged TrfA or solid support-linked TrfA for
affinity chromatography of the target protein.
1720 spectrometer (Horiba). H and 13C NMR spectra
were recorded on a JEOL lambda 500 NMR spectrom-
eter (JEOL, Japan) or Bruker ARX 400 NMR spec-
trometer (Bruker, Germany). Chemical shifts are
shown as d values from TMS as the internal reference.
Peak multiplicities are quoted in Hz. 13C NMR data
of compounds are listed in Supplemental data. Mass
spectra were measured on a JMS-700 spectrometer
(JEOL, Japan). Column chromatography was carried
out on columns of silica gel 60 (230–400 mesh, Merck,
Japan) and ODS gel (ODS DM1020T, Fuji Silysia
Chemical, Japan).
4.3. Modification of phenolic hydroxyl in TrfA (1a, 1b,
and 9)
4.3.1. 20,30-Dihydroxy-50-methoxy-p-terphenyl 20-b-D-
glucoside (1a). The synthetic procedure of 1a was essen-
tially same as described in Yamazoe et al.10 The syn-
thetic procedures, yields, physico-chemical and
spectroscopic data of 1a were described in Supporting
information.
4. Experimental
4.1. Biological assay
4.3.2. 20,50-Dihydroxy-30-methoxy-p-terphenyl 20-b-D-
glucoside (1b). The synthetic procedure of 1b was essen-
tially same as described in Yamazoe et al.10 The syn-
thetic procedures, yields, physico-chemical and
spectroscopic data of 1b were described in Supporting
information.
4.1.1. Hormone induction. Arabidopsis transgenic BA3::
GUS line was used in this assay.12 The seedlings (n = 10–
15) were grown vertically for 5 days in continuous light.
They were transferred to a 12-well micro-titer plate con-
taining 1 mL of a germination medium (GM, 0.5·
Murashige and Skoog salts (Gibco BRL, Gaithersburg,
MD), 1% sucrose, 1· B5 vitamins, and 0.2 g/L 2-(4-mor-
pholino)-ethane sulfonic acid (MES), pH 5.8) containing
4.3.3. 20,50-Dihydroxy-p-terphenyl (6a). To a suspension
of 5 (200 mg, 0.77 mmol) in MeOH (5.0 mL) was added
sodium borohydride (116 mg, 3.1 mmol), the mixture

K. Hayashi et al. / Bioorg. Med. Chem. 16 (2008) 5331–5344
5337
was stirred for 1 h at room temperature. The resulting
solution was added into water (100 mL), and then ex-
tracted with EtOAc (3· 100 mL). The organic layer
was washed with saturated NH4Cl solution and brine.
After drying over Na2SO4, the layer was concentrated
in vacuo. The residue was purified by a silica gel column
chromatography with hexane/EtOAc (7:3) to give 6a
(141 mg, yield 70%) as a white powder; 1H NMR
(400 MHz, acetone-d6/CD3OD = 1:1): d 7.64–7.61 (m,
4H), 7.41–7.37 (m, 4H), 7.30–7.26 (m, 2H), 6.90 (s,
2H); HR-EIMS: m/z [M]+; Calcd for C18H14O2:
262.0994. Found: 262.0973.
tetraacetylglucoside 8 (82 mg, yield 79%) as a white
powder: H NMR (400 MHz, CDCl3): d 7.50 (d, 2H,
1
J = 7.5 Hz), 7.46–7.42 (m, 3H), 7.41–7.32 (m, 5H), 7.28
(s, 1H), 7.13, (s, 1H), 5.19–5.15 (m, 2H), 5.10–5.06 (m,
1H), 5.02–5.00 (m, 1H), 4.15 (d, 1H, J = 4.3 Hz), 3.85
(quint, 1H, J = 4.8 Hz), 2.11 (s, 3H), 2.02 (s, 3H), 1.96
(s, 3H), 1.74 (s, 3H), 1.53 (s, 3H); Anal. Calcd for
C34H34O12: C, 64.35; H, 5.40. Found: C, 64.18; H,
5.63; HR-FABMS: m/z [M+Na]+; Calcd for
C34H34O12Na: 657.1948. Found: 657.1929.
Compound 8 (50 mg, 79 lmol) was hydrolyzed by
MeOH solution of KOH (31 mg, 0.55 mmol, in 5 mL
MeOH) for 1 h at room temperature. The resulting solu-
tion was added to 1 N HCl, (25 mL) and extracted with
EtOAc (3· 25 mL). The organic layer was washed with
saturated NH4Cl solution and brine. The solvent was re-
moved in vacuo after drying over Na2SO4. The residue
was purified by a silica gel column chromatography with
4.3.4. 20-Acetoxy-50-hydroxy-p-terphenyl (7). To a solu-
tion of 6a (100 mg, 0.38 mmol) in CH2Cl2 (5.0 mL) were
added acetic anhydride (84 lL, 0.76 mmol) and 4-
dimethylaminopyridine (93 mg, 0.76 mmol). The mix-
ture was stirred for 1 h at room temperature. The result-
ing solution was added into water (50 mL) and extracted
with EtOAc (3· 50 mL). The organic layer was washed
successively with saturated Na2CO3 solution, saturated
NH4Cl solution, and brine. After drying over Na2SO4,
the solvent was removed in vacuo. The residue was puri-
fied by a silica gel column chromatography with hexane/
EtOAc (4:1) to give the intermediate, 20,50-diacetoxy-p-
terphenyl 6b (123 mg, yield 93%) as a yellow powder;
1H NMR (400 MHz, CDCl3): d 7.48–7.45 (m, 4H),
7.43–7.39 (m, 4H), 7.37–7.23 (m, 2H), 7.20 (s, 2H),
2.08 (s, 6H); HR-EIMS: m/z [M]+; Calcd for
C22H18O4: 346.1205. Found: 346.1211.
CHCl3/MeOH (4:1) to give 9 (30 mg, yield 90%) as a
25
D
pale yellow powder: mp 103–104 ꢁC; ½aꢁ +47.2ꢁ (c
0.1, MeOH); IR (KBr): mmax 3379, 2923, 2853, 1600,
1
1483, 1404, 1262, 1190, 1072, 828, 701 cmꢀ1; H NMR
(400 MHz, CD3OD): d 7.65–7.62 (m, 4H), 7.41–7.37
(m, 4H), 7.31–7.28 (m, 2H), 7.26 (s, 1H), 6.88 (s, 1H),
4.92 (d, 1H, J = 7.24 Hz), 3.84 (d, 1H, J = 11.6 Hz),
3.66 (dd, 1 H, J = 12.0, 5.0 Hz), 3.43–3.30 (m, 3H);
HR-FABMS: m/z [M+Na]+; Calcd for C24H24O7Na:
447.1420. Found: 447.1415.
4.4. General method for the synthesis of 20-alkyl ether
derivatives (12a–k)
To a suspension of 6b (100 mg, 0.29 mmol) in MeOH
(5.0 mL) was added potassium hydroxide (18 mg,
0.32 mmol) at ꢀ18 ꢁC and, the mixture was stirred for
30 min at ꢀ18 ꢁC. The resulting solution was added to
1 N HCl (50 mL), and extracted with EtOAc (3·
50 mL). The organic layer was washed with saturated
Na2CO3 solution and saturated NH4Cl solution. After
drying over Na2SO4, the solvent was removed in vacuo.
The residue was purified by a silica gel column chroma-
tography with hexane/EtOAc (3:1) to give 7 (72 mg,
yield 81%) as a white powder: 1H NMR (400 MHz,
CDCl3): d 7.52–7.50 (m, 4H), 7.47–7.33 (m, 6H), 6.91
(s, 2H), 5.26 (s, 1H, OH), 2.08 (s, 3H); Anal. Calcd for
C20H16O3: C, 78.93; H, 5.30. Found: C, 79.11; H, 5.41;
HR-EIMS: m/z [M]+; Calcd for C20H16O3: 304.1099.
Found: 304.1073.
30,50-Dibenzyloxy-20-hydroxy-p-terphenyl (10) was pre-
pared as previously described.13 To a solution of 10
(50 mg, 0.11 mmol) in THF (5.0 mL) were added alkyl
halide (0.22 mmol), tert-butyl ammonium iodide
(81 mg, 0.22 mmol) and sodium hydride (5 mg,
0.22 mmol), and then stirred for 24 h at room tempera-
ture. The resulting solution was added to water (50 mL)
and extracted with EtOAc (3· 50 mL). The organic layer
was washed with saturated NH4Cl solution and brine
and then dried over Na2SO4. After removal of solvent
in vacuo, the residue was purified by a silica gel column
chromatography to give 11a–k. Compounds 11a–k in
EtOAc (4.0 mL) were hydrogenolyzed by H2 (1 atm)
and catalytic amounts of 10% Pd/C at room temperature
for 24 h to yield compounds 12a–k, respectively. The
yields, physico-chemical and spectroscopic data of com-
pounds 12a–k were described in Supporting
information.
4.3.5. 20,50-Dihydroxy-p-terphenyl 20-b-D-glucoside (9).
The glycosylation of 7 was performed by essentially the
same procedure as previously described.10 Cesium car-
bonate (107 mg, 0.32 mmol: 2 equiv) and acetobromo-
a-D-glucose (200 mg, 0.48 mmol: 3 equiv) were added
to a solution of 7 (50 mg, 0.16 mmol) in CH3CN
(5.0 mL). The mixture was stirred for 24 h at room tem-
perature. The resulting solution was added into water
(100 mL) and extracted with EtOAc (3· 100 mL). The
organic layer was washed with saturated NH4Cl solu-
tion and brine. The solvent was removed in vacuo after
drying over Na2SO4. The residue was purified by a silica
gel column chromatography with hexane/EtOAc (3:2)
and ODS column chromatography with MeOH/H2O
(9:1) to give 50-acetoxy-20-hydroxy-p-terphenyl 20-b-D-
4.5. Synthesis of A-ring-substituted derivatives (22a–d and
25)
4.5.1. 2,5-Dibenzyloxybenzaldehyde (14). To a suspen-
sion of 13 (3.00 g, 21.7 mmol) in CH3CN (130 mL), ben-
zyl bromide (6.40 mL, 54.4 mmol) and potassium
carbonate (7.50 g, 54.4 mmol) were added. The mixture
was refluxed for 4 h. After cooling at room temperature,
the resulting solution was added to water (500 mL), and
extracted with EtOAc (3· 500 mL). The organic layer
was washed with saturated NH4Cl solution and brine,

5338
K. Hayashi et al. / Bioorg. Med. Chem. 16 (2008) 5331–5344
and then dried over Na2SO4. After concentration of the
EtOAc layer in vacuo, the residue was purified by a sil-
ica gel column chromatography with hexane/EtOAc
(9:1) to give 14 (6.01 g, yield 87%) as a pale yellow crys-
fied by a silica gel column chromatography with CHCl3
to give 17 (4.78 g, yield 97%) as a pale yellow powder:
mp 61–63 ꢁC; IR (KBr): m 3068, 2990, 2942, 1689,
1592, 1453, 1380, 1309, 1226, 1158, 1023 cmꢀ1 1H
;
1
tal: mp 88–89 ꢁC; H NMR (400 MHz, CDCl3): d 10.05
NMR (400 MHz, CDCl3): d 10.27 (s, 1H), 7.47 (d, 1H,
J = 3.1 Hz), 7.41–7.33 (m, 6H), 5.12 (s, 1H), 5.04 (s,
1H), 3.59 (s, 3H); HR-EIMS: m/z [M]+; Calcd for
C16H15O479Br and C16H15O481Br: 350.0154 and
352.0135. Found: 350.0163 and 352.0144.
(s, 1H), 7.44–7.16 (m, 10H), 7.17 (dd, 1H, J = 9.1,
3.3 Hz), 6.97 (d, 1H, J = 9.1 Hz), 5.12 (s, 2H), 5.02 (s,
2H); Anal. Calcd for C21H18O3: C, 79.22; H, 5.70.
Found: C, 79.12; H, 5.56; HR-EIMS: m/z [M]+; Calcd
for C21H18O3: 318.1252. Found: 318.1262.
4.5.5. 5-Benzyloxy-2-methoxymethoxy-biphenyl-3-carb-
aldehyde (18a). To a solution of 17 (4.70 g, 13.4 mmol)
in DME/EtOH (5:1, 75 mL) were added phenyl boronic
acid (3.28 g, 26.9 mmol), tetrakis (triphenylphosphine)-
palladium (0) (776 mg, 0.67 mmol) and 2 M Na2CO3
solution (30 mL), the mixture was refluxed for 3 h. After
cooling at room temperature, the reaction solution was
filtered on the ceilte, and added to 1N HCl (300 mL),
and then extracted with EtOAc (3· 300 mL). The organ-
ic layer was washed with saturated Na2CO3 solution and
brine. The organic layer was dried over Na2SO4and con-
centratedin vacuo. The residue was purified by a silica
gel column chromatography with CHCl3 to give 18a
(4.53 g, yield 97%) as a pale yellow powder: mp 56–
58 ꢁC; IR (KBr): m 2947, 2876, 1681, 1596, 1496, 1376,
4.5.2. 5-Benzyloxy-2-hydroxybenzaldehyde (15). Selective
removal of 2-benzyl group of 14 was performed as pre-
viously described.13 To a suspension of 14 (6.00 g,
18.9 mmol) in benzene/diethyl ether (7:1, 130 mL) was
added magnesium bromide (4.17 g, 22.64 mmol), the
mixture was refluxed for 8 h. After cooling at room tem-
perature, to the resulting solution was added 1 N HCl,
and extracted with EtOAc. The organic layer was
washed with saturated Na2CO3 solution and brine.
The solvent was removed in vacuo after drying over
Na2SO4. The residue was purified by a silica gel column
chromatography with hexane/EtOAc (9:1) to give 16
(3.62 g, yield 84%) as a yellow powder: mp 85–87 ꢁC;
IR (KBr): m 3105, 2930, 2852, 1664, 1618, 1589, 1485,
1
1387, 1271, 1158, 1019 cmꢀ1
;
1H NMR (400 MHz,
1326, 1221, 1023 cmꢀ1; H NMR (400 MHz, CDCl3):
CDCl3): d 10.67 (s, 1H), 9.80 (s, 1H), 7.43–7.37 (m,
4H), 7.35–7.32 (m, 1H), 7.21 (dd, 1H, J = 9.2, 3.1 Hz),
7.05 (d, 1H, J = 3.1 Hz), 6.92 (d, 1H, J = 9.1 Hz), 5.04
(s, 1H); HR-EIMS: m/z [M]+; Calcd for C14H12O3:
228.0786. Found: 228.0775.
d 10.41 (s, 1H), 7.49 (d, 2H, J = 7.0 Hz), 7.40-7.37 (m,
5H), 7.35–7.31 (m, 3H), 7.29–7.26 (m, 1H), 7.23 (d,
1H, J = 5.4 Hz); Anal. Calcd for C22H20O4: C, 75.84;
H, 5.79. Found: C, 75.86; H, 5.98; HR-EIMS: m/z
[M]+; Calcd for C22H20O4: 348.1362. Found: 348.1379.
4.5.3. 5-Benzyloxy-3-bromo-2-hydroxybenzaldehyde (16).
To a solution of 15 (3.50 g, 15.4 mmol) in acetic acid
(100 mL) were added sodium acetate (1.89 g, 23.0 mmol)
and bromine (0.60 mL, 23.0 mmol), the mixture was stir-
red for 3 h at room temperature. 5% Na2S2O3 solution
(300 mL) was added to the reaction solution and then
extracted with EtOAc (3· 300 mL). The organic layer
was washed with 5% Na2S2O3 solution, saturated
Na2CO3 solution, and brine. The organic layer was
dried over Na2SO4 and then concentrated in vacuo.
The residue was purified by a silica gel column chroma-
tography with hexane/EtOAc (4:1) to give 15 (4.35 g,
yield 93%) as a yellow powder: mp 92–93 ꢁC; IR
(KBr): m 3087, 3060, 2898, 2864, 1649, 1613, 1450,
4.5.6. 5-Benzyloxy-4-bromo-2-methoxymethoxy-biphe-
nyl-3-carbaldehyde (19a). To a solution of 18a (4.50 g,
12.9 mmol) in acetic acid (100 mL) were added sodium
acetate (1.59 g, 19.4 mmol) and bromine (0.50 mL,
19.4 mmol), the mixture was stirred for 6 h at 50 ꢁC.
The solution was added to 5% Na2S2O3 solution
(300 mL), and then extracted with EtOAc (3·
300 mL). The organic layer was washed with saturated
Na2CO3 solution and brine. The EtOAc layer was dried
over Na2SO4 and concentrated in vacuo. The residue
was purified by a silica gel column chromatography with
hexane/EtOAc (4:1) to give a 5-benzyloxy-4-bromo-2-
hydroxy-biphenyl-3-carbaldehyde (18b: 3.46 g, yield
70%) as a yellow powder: mp 99–100 ꢁC; IR (KBr): m
3437, 2920, 2854, 1630, 1586, 1496, 1403, 1341, 1287,
1
1269, 1132, 1028 cmꢀ1; H NMR (400 MHz, CDCl3):
d 11.15 (s, 1H), 9.77 (s, 1H), 7.49 (d, 1H, J = 2.7 Hz),
7.41–7.38 (m, 4H), 7.37–7.34 (m, 1H), 7.08 (d, 1H,
J = 3.1Hz), 5.05 (s, 1H); Anal. Calcd for C14H11O3Br:
C, 54.75; H, 3.61. Found: C, 54.88; H, 3.79; HR-EIMS:
m/z [M]+; Calcd for C14H11O379Br and C14H11O381Br:
305.9892 and 307.9873. Found: 305.9889 and 307.9842.
1123, 1029 cmꢀ1 1H NMR (400 MHz, CDCl3): d
;
12.20 (s, 1H), 10.44 (s, 1H), 7.50–7.30 (m, 10H), 7.24
(s, 1H), 5.11 (s, 2H); HR-EIMS: m/z [M]+; Calcd for
C20H15O379Br and C20H15O381Br: 382.0205 and
384.0187. Found: 382.0189 and 384.0196.
Thereafter, to a solution of 18b (3.40 g, 8.90 mmol) in
CH2Cl2 (100 mL), chloromethyl methyl ether (1.0 mL,
13.4 mmol) and N-ethyl diisopropylamine (3.1 mL,
17.8 mmol) were added. The mixture was stirred for
6 h at room temperature. The resulting solution was
added to saturated NH4Cl solution (250 mL), and ex-
tracted with EtOAc (300 mL). The organic layer was
washed with brine and concentrated in vacuo after dry-
ing over Na2SO4. The residue was purified by a silica gel
column chromatography with CHCl3 to give 19a (3.56 g,
4.5.4. 5-Benzyloxy-3-bromo-2-methoxymethoxybenzalde-
hyde (17). To a solution of 16 (4.30 g, 14.1 mmol) in
CH2Cl2 (100 mL) were added chloromethyl methyl ether
(1.58 mL, 21.2 mmol) and N-ethyl diisopropylamine
(4.85 mL, 28.2 mmol), the mixture was stirred for 6 h
at room temperature. The resulting solution was washed
with saturated NH4Cl solution and extracted with
CH2Cl2 (3· 300 mL). The organic layer was dried over
Na2SO4 and evaporated in vacuo. The residue was puri-

K. Hayashi et al. / Bioorg. Med. Chem. 16 (2008) 5331–5344
5339
yield 94%) as a yellow oil: IR (KBr): m 2935, 2890, 1701,
1576, 1496, 1375, 1239, 1157, 1123, 1028 cmꢀ1 1H
and 2 M Na2CO3 solution (1.5 mL), the mixture was re-
fluxed for 3 h. After cooling at room temperature, the
resulting solution was filtered on Celite and added to
1 N HCl (25 mL), and extracted with EtOAc (3·
25 mL). The organic layer was washed with saturated
Na2CO3 solution and brine. After drying over Na2SO4,
the solvent was removed in vacuo. The residue was puri-
fied by a silica gel column chromatography with hexane/
EtOAc (5:1) to give 21a (47 mg, yield 76%) as a pale yel-
;
NMR (400 MHz, CDCl3): d 10.42 (s, 1H), 7.49–7.46
(m, 4H), 7.44–7.35 (m, 6H), 7.13 (s, 1H), 5.18 (s, 2H),
4.65 (s, 2H), 3.05 (s, 3H); HR-EIMS: m/z [M]+; Calcd
for C22H19O479Br and C22H19O481Br: 426.0467 and
428.0449. Found: 426.0464 and 428.0462.
4.5.7.
3,5-Dibenzyloxy-4-bromo-2-methoxymethoxy-
1
biphenyl (20). 3-Chloroperoxybenzoic acid (4.25 g,
24.7 mmol) was added to a solution of 19a (3.50 g,
8.22 mmol) in CH2Cl2 (100 mL) under ice cooling. The
reaction mixture was kept in ice bath for 1 h and then
stirred for 24 h at room temperature. The resulting solu-
tion was added to 5% Na2S2O3 solution (300 mL) and
extracted with EtOAc (3· 300 mL). The organic layer
was successively washed with saturated Na2CO3 solu-
tion, saturated NH4Cl solution, and brine. After drying
over Na2SO4, solvent was removed in vacuo. The resi-
due was suspended in methanolic KOH solution
(920 mg, 16.4 mmol in 75 mL of MeOH). The suspen-
sion was stirred for 1 h at room temperature. The result-
ing solution was added to 1 N HCl (300 mL) and
extracted with EtOAc (3· 300 mL). The EtOAc layer
was washed with saturated Na2CO3 solution and brine.
The solvent was removed in vacuo and dried over
Na2SO4. The residue was purified by a silica gel column
chromatography with hexane/EtOAc (4:1) to give 5-ben-
zyloxy-3-hydroxy-4-bromo-2-methoxymethoxy-biphe-
nyl (19b: 2.05 g, two steps yield 64%) as a yellow
powder: mp 68–70 ꢁC; IR (KBr): m 3336, 2943, 1593,
low powder; H NMR (400 MHz, CDCl3): d 7.57 (d,
2H, J = 7.9 Hz), 7.44–7.20 (m, 14H), 7.02–7.00 (m,
2H), 6.80 (s, 1H), 4.99 (s, 2H), 4.73 (s, 2H), 4.73 (s,
2H), 2.92 (s, 3H), 2.42 (s, 3H); HR-EIMS: m/z [M]+;
Calcd for C35H32O4: 516.2301. Found: 516.2287.
4.5.9. 20,30,50-Trihydroxy-400-methyl-p-terphenyl 20-b-D-
glucoside (22a). Acetyl chloride (9.0 lL, 0.13 mmol)
was added dropwise to a suspension of 21a (44 mg,
85 lmol), in MeOH (3.0 mL) under ice cooling, and
the mixture was stirred for 2 h at room temperature.
The resulting solution was poured into water, and ex-
tracted with EtOAc (3· 25 mL). The organic layer was
washed with saturated Na2CO3 solution and brine,
and then dried over Na2SO4. After removal of solvent
in vacuo, the residue was purified by a silica gel column
chromatography with hexane/EtOAc (4:1) to give the
intermediate
30,50-dibenzyloxy-20-hydroxy-400-methyl-
p-terphenyl (21e: 40 mg, yield 99%) a pale yellow pow-
der; 1H NMR (400 MHz, CDCl3): d 7.60 (d, 2H,
J = 7.7 Hz), 7.52 (d, 2H, J = 8.0 Hz), 7.42 (t, 2H,
J = 7.8 Hz), 7.35–7.22 (m, 11H), 7.11–7.09 (m, 2H),
6.84 (s, 1H), 5.75 (s, 1H, OH), 4.94 (s, 2H), 4.43 (s,
2H), 2.44 (s, 3 H); HR-EIMS: m/z [M]+; Calcd for
C33H28O3: 472.2038. Found: 472.2044.
1572, 1500, 1452, 1398, 1275, 1145, 1066 cmꢀ1 1H
;
NMR (400 MHz, CDCl3): d 7.47 (d, 2H, J = 7.0 Hz),
7.44–7.29 (m, 8H), 6.50 (s, 1H), 5.11 (s, 2H), 4.72 (s,
2H), 3.34 (s, 3H); HR-EIMS: m/z [M]+; Calcd for
C21H19O479Br and C21H19O481Br: 414.0467 and
416.0449. Found: 414.0480 and 416.0450.
Thereafter, to a solution of 21e (38 mg, 81 lmol) in
CH3CN (4.0 mL) were added acetobromo-a-D-glucose
(99 mg, 0.24 mmol: 3 equiv) and cesium carbonate
(52 mg, 0.16 mmol: 2 equiv), and the mixture was stirred
for 24 h at room temperature. The resulting solution was
added to water (20 mL), and extracted with EtOAc (3·
20 mL). The organic layer was washed with saturated
NH4Cl solution and brine. The solvent was removed
in vacuo after drying over Na2SO4. The residue was
purified by a silica column chromatography with hex-
ane/EtOAc (3:2) and ODS column chromatography
with MeOH/H2O (9:1) to give 30,50-dibenzyloxy-2-hy-
droxy-400-methyl-p-terphenyl 20-b-D-tetraacetylglucoside
(21f: 46 mg, yield 70%) as a pale yellow powder; 1H
NMR (400 MHz, CDCl3): d 7.51 (d, 2H, J = 7.0 Hz),
7.41 (t, 2H, J = 7.6 Hz), 7.37–7.19 (m, 13H), 6.99–6.96
(m, 2H), 6.77 (s, 1H), 5.14 (d, 1H, J = 7.2 Hz), 5.05–
4.97 (m, 5H), 4.85 (d, 1H, J = 10.7 Hz), 4.58 (d, 1H,
J = 10.7 Hz), 3.96 (dd, 1H, J = 12.3, 3.5 Hz), 3.73 (dd,
1H, J = 12.3, 2.3 Hz), 3.40–3.36 (m, 1H), 2.42 (s, 3H),
1.95 (s, 6 H), 1.93 (s, 3H), 1.84 (s, 3H); HR-FABMS:
m/z [M+Na]+; Calcd for C47H46O12Na: 825.2887.
Found: 825.2883.
To a solution of 19b (2.00 g, 4.83 mmol) in CH3CN
(40 mL) were added benzyl bromide (0.69 mL,
5.80 mmol) and potassium carbonate (0.80 g,
5.80 mmol), the mixture was refluxed for 2 h. After cool-
ing at room temperature, the reaction mixture was
added to water (200 mL) and then extracted with EtOAc
(3· 300 mL). The organic layer was washed with satu-
rated NH4Cl solution and dried over Na2SO4. After
evaporation in vacuo, the residue was purified by a silica
gel column chromatography with hexane/EtOAc (9:1) to
give 20 (2.31 g, yield 95%) as a pale yellow crystal: mp
108–109 ꢁC; IR (KBr): m 2940, 1573, 1496, 1415, 1367,
1
1250, 1157, 1087 cmꢀ1; H NMR (400 MHz, CDCl3):
d 7.63–7.27 (m, 15H), 6.76 (s, 1H), 5.13 (s, 2H), 5.13
(s, 2H), 4.83 (s, 2H), 2.92 (s, 3H); Anal. Calcd for
C28H25O4Br: C, 66.54; H, 4.99. Found: C, 66.31; H,
4.68; HR-EIMS: m/z [M]+; Calcd for C28H25O479Br
and C28H25O481Br: 504.0926 and 506.0920. Found:
504.0938 and 506.0915.
4.5.8. 30,50-Dibenzyloxy-400-methyl-20-methoxymethoxy-
p-terphenyl (21a). To
a
solution of 20 (61 mg,
Intermediate 21f (40 mg, 50 lmol) was hydrogenolyzed
by H2 (1 atm) and catalytic amounts of 10% Pd/C
(2 mg) in EtOAc (3 mL) for 12 h at room temperature.
The reaction mixture was filtered on Celite, and the fil-
0.12 mmol) in DME/EtOH (5:1, 4.0 mL) were added p-
tolyl boronic acid (32 mg, 0.24 mmol), tetrakis (triphen-
ylphosphine)-palladium(0) (7 mg, 6 lmol: 0.05 equiv)

5340
K. Hayashi et al. / Bioorg. Med. Chem. 16 (2008) 5331–5344
trate was concentrated in vacuo. The residue without
further purification was hydrolyzed by methanolic
KOH solution (17 mg, 0.3 mmol: 6 equiv in 3 mL of
MeOH) at room temperature for 1 h to give 22a
(18 mg, two steps yield 80%) as a pale yellow crystal:
J = 2.7 Hz), 5.13–4.94 (m, 7H), 3.86 (dd, 1H, J = 12.2,
4.0 Hz), 3.70 (dd, 1H, J = 12.1, 2.1 Hz), 3.36–3.32 (m,
1H), 1.95 (s, 6H), 1.95 (s, 3H), 1.73 (s, 3H); Anal. Calcd
for C40H40O12: C, 67.41; H, 5.66. Found: C, 67.31; H,
5.52; HR-FABMS: m/z [M+Na]+; Calcd for
C40H40O12Na, 735.2417. Found, 735.2390.
1
mp 145–147 ꢁC; H NMR (400 MHz, CD3OD): d 7.53
(d, 2H, J = 7.3 Hz), 7.38 (t, 2H, J = 7.3 Hz), 7.32–7.29
(m, 3H), 7.20 (d, 2H, J = 8.0 Hz), 6.38 (s, 1H), 4.28 (d,
1H, J = 7.8 Hz), 3.48 (dd, 1H, J = 11.8, 2.7 Hz), 3.40
(dd, 1H, J = 11.5, 4.9 Hz), 3.36–3.33 (m, 2H), 3.25–
3.18 (m, 1H); HR-FABMS: m/z [M+Na]+; Calcd for
C25H26O8Na: 477.1525. Found: 477.1513.
Thereafter, the same procedure described at 22a (21f–
22a) was employed with 24c (80 mg, 112 lmol). With
the hydrogenolysis and alkaline hydrolysis of 24c, the ti-
tle compound 25 (32 mg, two steps yield 80%) was ob-
tained as a pale yellow powder: mp 70–71 ꢁC; 1H
NMR (400 MHz, CD3OD): d 7.48 (d, 2H, J = 7.6 Hz),
7.36 (t, 2H, J = 7.4 Hz), 7.29 (t, 1H, J = 7.5 Hz), 6.35
(d, 1H, J = 2.7 Hz), 6.24 (d, 1H, J = 2.7 Hz), 4.29 (d,
1H, J = 7.7 Hz), 3.50 (d, 1H, J = 14.6 Hz), 3.42 (dd,
1H, J = 11.8, 4.9 Hz), 3.34 (bs, 1H), 3.26–3.19 (m,
2H), 3.02–2.96 (m, 1H); HR-FABMS: m/z [M+Na]+;
Calcd for C18H20O8Na, 387.1056. Found, 387.1061.
4.5.10. A-ring TrfA derivatives (22b–d) and the corre-
sponding intermediates (21b–d). The synthetic procedure
of 21b–d and 22b–d were essentially same as 21a and
22d, respectively. The procedures, yields, physico-chem-
ical and spectroscopic data of c 21b–d and 22b–d were
described in Supporting information.
4.5.11.
5-Benzyloxy-3-hydroxy-2-methoxymethoxy-
4.6. SynthesisofC-ring-substituted derivatives(34aand34b)
biphenyl (23). The same procedure described at 20
(19a–b) was employed with 18a (350 mg, 1.00 mmol).
With Baeyer–Villiger oxidation and alkaline hydrolysis
of 18a, the title compound 23 (306 mg, two steps yield
90%) was obtained as a yellow crystal; 1H NMR
(400 MHz, CDCl3): d 7.47 (d, 2H, J = 7.0 Hz), 7.44–
7.38 (m, 5H), 7.36–7.34 (m, 1H), 7.33–7.29 (m, 2H),
7.07 (s, 1H), 6.64 (d, 1H, J = 3.0Hz), 6.51 (d, 1H,
J = 3.0 Hz), 5.02 (s, 2H), 4.68 (s, 2H), 3.42 (s, 3H); Anal.
Calcd for C21H20O4: C, 74.98; H, 5.99. Found: C, 75.13;
H, 6.12; HR-EIMS: m/z [M]+; Calcd for C21H20O4:
336.1362. Found: 336.1372.
4.6.1. 2-Bromo-3,6-dihydroxybenzaldehyde (26). To a
suspension of 2,5-dihydroxybenzaldehyde (13: 3.00 g,
21.7 mmol) in CHCl3 (150 mL) were added sodium ace-
tate (2.67 g, 32.6 mmol) and bromine (0.84 mL,
32.6 mmol), the mixture was stirred for 3 h at room tem-
perature. The resulting solution was added to 10%
Na2S2O3 solution (150 mL), and extracted with CHCl3
(2· 100 mL). The organic layer was washed with satu-
rated Na2CO3 solution and saturated NH4Cl solution.
The solvent was removed in vacuo after drying over
Na2SO4. The residue was purified by a silica gel column
chromatography with hexane/EtOAc (7:3) to give 26
(3.76 g, yield 80%) as a yellow needle crystal: mp 88–
89 ꢁC; IR (KBr): m 3266, 2907, 2829, 1632, 1609, 1573,
4.5.12.
3,5-Dibenzyloxy-2-methoxymethoxy-biphenyl
(24a). The same procedure described at 20 (19b–20)
was employed with 23 (250 mg, 0.74 mmol). With the
introduction of benzyl group into 23, the title compound
24a (312 mg, yield 99%) was obtained as a yellow oil; 1H
NMR (400 MHz, CDCl3): d 7.54 (d, 2H, J = 7.2 Hz),
7.44–7.35 (m, 10H), 7.33–7.30 (m, 3H), 6.65 (d, 1H,
J = 2.8 Hz), 6.57 (d, 1H, J = 2.8 Hz), 5.09 (s, 2H), 5.01
(s, 2H), 4.81 (s, 2H), 2.92 (s, 3H); HR-EIMS: m/z
[M]+; Calcd for C28H26O4: 426.1831. Found: 426.1831.
1459, 1288, 1240, 1171 cmꢀ1 1H NMR (400 MHz,
;
CDCl3): d 11.64 (s, 1H), 10.27 (s, 1H), 7.24 (d, 1H,
J = 9.1 Hz), 6.91 (d, 1H, J = 9.1 Hz), 5.46 (s, 1H); Anal.
Calcd for C7H5O3 Br: C, 38.74; H, 2.32. Found: C,
38.89; H, 2.31; HR-EIMS: m/z [M]+; Calcd for
C7H5O379Br and C7H5O381Br: 215.9422 and 217.9401.
Found: 215.9423 and 217.9422.
4.6.2. 3,6-Dibenzyloxy-2-bromobenzaldehyde (27). The
same procedure described at 15 was employed with 26
(3.70 g, 17.1 mmol). The title compound 27 (4.88 g, yield
72%:) was obtained as a yellow needle crystal: mp 79–
80 ꢁC; IR (KBr): m 3029, 2900, 2869, 1682, 1563, 1496,
4.5.13. 2,3,5-Trihydroxy-biphenyl 2-b-D-glucoside (25).
The same procedure described at 22a (21a–e) was em-
ployed with 24a (200 mg, 0.47 mmol). With removal of
methoxymethyl group in 24a, the intermediate 3,5-diben-
zyloxy-2-hydroxy-biphenyl (24b: 172 mg, yield 96%) was
1376, 1265, 1159, 1011 cmꢀ1 1H NMR (400 MHz,
;
1
obtained as a pale yellow powder; H NMR (400 MHz,
CDCl3): d 10.44 (s, 1H), 7.43–7.32 (m, 10H), 7.00 (d,
1H, J = 9.1 Hz), 6.88 (d, 1H, J = 9.1 Hz), 5.07 (s, 4H);
HR-EIMS: m/z [M]+; Calcd for C21H17O379Br and
C21H17O381Br: 396.0361 and 398.0341. Found:
396.0373 and 398.0339.
CDCl3): d 7.60 (d, 2H, J = 7.5 Hz), 7.42–7.29 (m, 13H),
6.63 (d, 1H, J = 2.8 Hz), 6.60 (d, 1H, J = 2.8 Hz), 5.56
(s, 1H), 5.06 (s, 2H), 4.98 (s, 2H); HR-EIMS: m/z [M]+;
Calcd for C26H22O3: 382.1569. Found: 382.1564.
Thereafter, the same procedure described at 22a (21e–f)
was employed with 24b (100 mg, 0.26 mmol). With the
glycosylation of 24b, the intermediate 3,5-dibenzyloxy-
2-hydroxy-biphenyl 2-b-D-tetracetylglucoside (24c:
114 mg, yield 61%) was obtained as a pale yellow powder;
1H NMR (400 MHz, CDCl3): d 7.46 (d, 4H, J = 7.4 Hz),
7.43–7.30 (m, 11H), 6.64 (d, 1H, J = 2.7 Hz), 6.55 (d, 1H,
4.6.3. 3,6-Dibenzyloxy-biphenyl-2-carbaldehyde (28). The
same procedure described at 18a was employed with 27
(4.80 g, 12.1 mmol). The title compound 28 (4.39 g, yield
92%:) was obtained as a yellow oil: IR (KBr): m 3030,
2933, 2866, 1695, 1584, 1497, 1378, 1260, 1186,
1024 cmꢀ1 1H NMR (400 MHz, CDCl3): d 10.05 (s,
;

K. Hayashi et al. / Bioorg. Med. Chem. 16 (2008) 5331–5344
5341
1H), 7.47–7.35 (m, 7H), 7.32–7.28 (m, 3H), 7.29–7.22
(m, 2H), 7.11–7.08 (m, 3H), 6.95 (d, 1H, J = 9.1 Hz),
5.14 (s, 2H), 4.89 (s, 2H); HR-EIMS: m/z [M]+; Calcd
for C27H22O3: 394.1569. Found: 394.1567.
tection by benzyl group of 31b, the title compound 32
(2.86 g, yield 84%) was obtained as a pale yellow oil:
IR (KBr): m 3031, 2935, 1578, 1497, 1365, 1310, 1251,
1234, 1159, 1124, 1028 cmꢀ1 1H NMR (400 MHz,
;
CDCl3): d 7.38–7.30 (m, 5H), 7.24–7.13 (m, 8H), 7.01
(s, 1H), 6.92–6.90 (m, 2H), 5.17 (s, 2H), 4.88 (s, 2H),
4.61 (s, 2H), 3.61 (s, 3H); Anal. Calcd for C28H25O4Br:
C, 66.54; H, 4.99. Found: C, 66.23; H, 4.63; HR-EIMS:
m/z [M]+; Calcd for C28H25O479Br and C28H25O481Br:
504.0936 and 506.0915. Found: 504.0946 and 506.0927.
4.6.4. 6-Benzyloxy-3-hydroxy-biphenyl-2-carbaldehyde
(29). The same procedure described at 15 was employed
with 28 (4.30 g, 10.9 mmol). The title compound 29
(2.91 g, yield 88%) was obtained as a yellow needle crys-
tal: mp 62–64 ꢁC; IR (KBr): m 3100, 2878, 1650, 1579,
1460, 1384, 1281, 1171, 1027 cmꢀ1
;
1H NMR
(400 MHz, CDCl3): d 11.51 (s, 1H), 9.63 (s, 1H), 7.44–
7.42 (m, 3H), 7.33–7.31 (m, 2H), 7.26–7.22 (m, 4H),
7.08–7.06 (m, 2H), 6.92 (d, 1H, J = 9.1 Hz), 4.85 (s,
2H); Anal. Calcd for C20H16O3: C, 78.93; H, 5.30.
Found: C, 78.88; H, 5.23; HR-EIMS: m/z [M]+; Calcd
for C20H16O3: 304.1099. Found: 304.1089.
4.6.8. 30,50-Dibenzyloxy-4-butoxy-20-methoxymethoxy-p-
terphenyl (33a). Compound 32 (78 mg, 0.16 mmol) was
coupled with 4-buthoxyphenyl boronic acid (60 mg,
0.31 mmol) by the same procedure described at 21a to
give 33a (69 mg, yield 78%) as a pale yellow powder;
1H NMR (400 MHz, CDCl3):
d
7.51 (d, 2H,
J = 8.7 Hz), 7.47 (d, 2H, J = 7.3 Hz), 7.41 (t, 2H,
J = 7.0 Hz), 7.37–7.34 (m, 1H), 7.29–7.18 (m, 8H),
6.99–6.96 (m, 8H), 6.80 (s, 1H), 4.99 (s, 2H), 4.91 (s,
2H), 4.74 (s, 2H), 4.01 (t, 2H, J = 6.5 Hz), 3.01 (s,
3H), 1.80 (quint, 2H, J = 6.5 Hz), 1.52 (sext, 2H,
J = 7.4 Hz), 1.00 (t, 3H, J = 7.3 Hz); HR-EIMS: m/z
[M]+; Calcd for C38H38O5, 574.2719. Found: 574.2733.
4.6.5. 6-Benzyloxy-4-bromo-3-hydroxy-biphenyl-2-carb-
aldehyde (30). The same procedure described at 16 was
employed with 29 (2.80 g, 9.21 mmol). The title com-
pound 30 (3.30 g, yield 94%:) was obtained as a yellow
needle crystal: mp 123–124 ꢁC; IR (KBr): m 3422, 3028,
2942, 2894, 1643, 1566, 1438, 1395, 1270, 1133,
1028 cmꢀ1 1H NMR (400 MHz, CDCl3): d 12.06 (s,
;
1H), 9.59 (s, 1H), 7.53 (s, 1H), 7.46–7.44 (m, 3H), 7.32–
7.30 (m, 2H), 7.26–7.24 (m, 3H), 7.09–7.07 (m, 2H), 4.88
(s, 2H); 13C NMR (100 MHz, CDCl3): d 196.7, 153.7,
148.4, 136.1, 135.8, 132.0, 130.9, 128.4, 128.4, 128.1,
127.9, 127.8, 127.0, 119.0, 109.8, 72.5; HR-EIMS: m/z
[M]+; Calcd for C20H15O379Br and C20H15O381Br:
382.0205 and 384.0184. Found: 382.0222 and 382.0209.
4.6.9. 20,30,50-Trihydroxy-4-butoxy-p-terphenyl 20-b-D-
glucoside (34a). The same procedure described at 22a
(21a–e) was employed with 33a (67 mg, 0.12 mmol).
With deprotection of methoxymethyl group of 33a, the
intermediate, 30,50-dibenzyloxy-4-butoxy-20-hydroxy-p-
terphenyl (33c: 61 mg, yield 98%) was obtained as a yellow
1
powder; H NMR (400 MHz, CDCl3): d 7.61 (d, 2H,
J = 7.2 Hz), 7.54 (d, 2H, J = 8.6 Hz), 7.45 (t, 2H,
J = 7.7 Hz), 7.28–7.24 (m, 6H), 7.20 (d, 2H, J = 7.3 Hz),
7.08–7.06 (m, 2H), 6.97 (d, 2H, J = 8.7 Hz), 6.84 (s, 1H),
5.74 (s, 1H, OH), 4.93 (s, 2H), 4.42 (s, 2H), 4.01 (t, 2H,
J = 6.5 Hz), 1.79 (quint, 2H, J = 6.6 Hz), 1.51 (sext, 2H,
J = 7.6 Hz), 0.99 (t, 2H, J = 7.4 Hz); HR-EIMS: m/z
[M]+; Calcd for C36H34O4, 530.2457. Found, 530.2460.
4.6.6. 6-Benzyloxy-4-bromo-3-methoxymethoxy-biphe-
nyl-2-carbaldehyde (31a). The same procedure described
at 17 was employed with 30 (3.20 g, 8.38 mmol). The ti-
tle compound (3.56 g, yield 94%) was obtained as a yel-
low oil: IR (KBr): m 3031, 2928, 2830, 1699, 1650, 1563,
1497, 1453, 1372, 1253, 1129, 1028 cmꢀ1 1H NMR
;
(400 MHz, CDCl3): d 9.76 (s, 1H), 7.43–7.40 (m, 4H),
7.29–7.25 (m, 5H), 7.14–7.12 (m, 2H), 5.14 (s, 2H),
4.98 (s, 2H), 3.62 (s, 3H); HR-EIMS: m/z [M]+; Calcd
for C22H19O479Br and C22H19O481Br: 426.0467 and
428.0446. Found: 426.0485 and 428.0439.
Thereafter, the same procedure described at 22a (21e–f)
was employed with 33c, (60 mg, 0.11 mmol). With glyco-
sylation of 33c, the intermediate, 30,50-dibenzyloxy-4-
butoxy-p-terphenyl-20-yl-b-D-glucose 2,3,4,6-tetraace-
tate (33d: 80 mg, yield 82%) as a pale yellow powder;
1H NMR (400 MHz, CDCl3): d 7.46–7.32 (m, 7H),
7.30–7.19 (m, 8H), 6.96–6.93 (m, 4H), 6.77 (s, 1H),
5.16 (d, 1H, J = 7.6 Hz), 5.08–5.02 (m, 3H), 4.99 (d,
2H, J = 5.1 Hz), 4.86 (d, 1H, J = 10.7 Hz), 4.57 (d, 1H,
J = 10.7 Hz), 4.04 (t, 2H, J = 7.4 Hz), 3.99 (dd, 1H,
J = 12.6, 3.9 Hz), 3.79 (dd, 1H, J = 12.6, 2.4 Hz), 3.44–
3.40 (m, 1H), 1.96 (s, 3H), 1.96 (s, 3H), 1.94 (s, 3H),
1.86 (s, 3H), 1.83 (quint, 2H, J = 6.3 Hz), 1.54 (sext,
2H, J = 7.6 Hz), 1.02 (t, 3H, J = 7.3 Hz); HR-FABMS:
m/z [M+Na]+; Calcd for C50H52O13Na, 883.3306.
Found, 883.3279. Thereafter, the tetraacetate 33d
(30 mg, 41 lmol) was hydrogenolyzed (H2 [1 atm] and
10% Pd/C) and hydrolyzed (KOH in MeOH) by a sim-
ilar procedure described at 22a (21f–22a) to yield the ti-
tle compound 34a (18.6 mg, two steps yield 62%) as a
pale yellow powder: mp 188–189 ꢁC; 1H NMR
(400 MHz, CD3OD): d 7.46 (d, 2H, J = 8.6 Hz), 7.43
(d, 2H, J = 8.3 Hz), 7.36 (t, 2H, J = 7.5 Hz), 7.21 (t,
4.6.7.
2,6-Dibenzyloxy-4-bromo-3-methoxymethoxy-
biphenyl (32). The same procedure described at 20
(19a–b) was employed with 31a (3.40 g, 7.98 mmol).
With Baeyer–Villiger oxidation and alkaline hydrolyses
of 31a, the intermediate 6-benzyloxy-4-bromo-2-hydro-
xy-3-methoxymethoxy-biphenyl (31b: 2.84 g, two steps
yield 86%) was obtained as a pale yellow powder: mp
168–169 ꢁC; IR (KBr): m 3298, 3032, 2940, 2830, 1605,
1573, 1476, 1395, 1277, 1155, 1027 cmꢀ1 1H NMR
;
(400 MHz, CDCl3): d 7.42 (d, 4H, J = 4.3 Hz), 7.37–
7.31 (m, 1H), 7.27–7.22 (m, 3H), 7.16 (d, 2H,
J = 7.3 Hz), 7.04 (s, 1H), 6.76 (s, 1H), 5.07 (s, 2H),
4.93 (s, 2H), 3.57 (s, 3H); HR-EIMS: m/z [M]+; Calcd
for C21H19O479Br and C21H19O481Br: 414.0467 and
416.0446. Found: 414.0438 and 416.0421.
Thereafter, the same procedure described at 20 (19b–20)
was employed with 31b (2.80 g, 6.76 mmol). With pro-

5342
K. Hayashi et al. / Bioorg. Med. Chem. 16 (2008) 5331–5344
1H, J = 7.1 Hz), 6.92 (d, 2H, J = 8.5 Hz), 6.39 (s, 1H),
4.29 (d, 1H, J = 7.8 Hz), 3.99 (t, 2H, J = 6.3 Hz), 3.53
(d, 1H, J = 11.8 Hz), 3.45 (dd, 1H, J = 11.5, 4.8 Hz),
3.39–3.34 (m, 1H), 3.27–3.20 (m, 2H), 3.01–2.99 (m,
1H), 1.76 (quint, 2H, J = 7.2 Hz), 1.51 (sext, 2H,
J = 7.5 Hz), 0.99 (t, 3H, J = 7.4 Hz); HR-FABMS: m/z
[M+Na]+; Calcd for C28H32O9Na, 535.1944. Found,
535.1920.
carbonyl-p-terphenyl 36c (81 mg, yield 90%) as a yellow
powder; H NMR (400 MHz, CDCl3): d 8.13 (d, 2H,
1
J = 8.4 Hz), 7.68 (d, 2H, J = 8.3 Hz), 7.61 (d, 2H,
J = 7.7 Hz), 7.45 (q, 2H, J = 6.8 Hz), 7.41–7.32 (m, 4H),
7.30–7.24 (m, 5H), 7.23–7.19 (m, 3H), 7.08–7.05 (m,
2H), 6.85 (s, 1H), 5.84 (s, 1H, OH), 5.38 (s, 2H), 4.94 (s,
2H), 4.41 (s, 2H); HR-FABMS: m/z [M+H]+; Calcd for
C40H33O5, 593.2328. Found, 593.2350.
4.6.10. 3,5-Dibenzyloxy-2-methoxymethoxy-4-methylbi-
phenyl (33b) and 2,3,5-trihydroxy-4-methylbiphenyl 2-b-
D-glucoside (34b). The synthetic procedure of 33b and
34b was essentially same as 33a and 34a, respectively.
The procedures, yields, physico-chemical and spectro-
scopic data of 33b and 34b were described in Supporting
information.
Thereafter, to a solution of phenol 36c (75 mg, 0.13 mmol)
was added acetobromo-a-D-glucose (162 mg, 0.395 mmol)
and cesium carbonate (86 mg, 0.26 mmol). The mixture
was stirred for 24 h at room temperature. The resulting
solution was added to water (50 mL) and extracted with
EtOAc (3· 50 mL). The organic layer was washed with
saturated NH4Cl solution and brine, dried over Na2SO4,
The solvent was removed in vacuo after drying over
Na2SO4. The residue was purified by a silica gel column
chromatography with hexane/EtOAc (3:2) to give 30,50-
dibenzyloxy-20-hydroxy-4-benzyloxycarbonyl-p-terphenyl
20-b-D-tetraacetylglucoside (36d: 102 mg, yield 84%) as a
pale yellow powder. This tetraacetate 36d was hydrogeno-
lyzed (H2 [1 atm] and 10% Pd/C) by a similar procedure
described at 22a (21f–22a) to yield 20,30,50-trihydroxy-p-
terphenyl 4-carboxylic acid 20-b-D-tetraacetylglucoside
(36e) as a yellow powder; 1H NMR (400 MHz,
CDCl3:CD3OD = 1:1): d 8.08 (d, 2H, J = 8.3 Hz), 7.57
(d, 2H, J = 8.3 Hz), 7.49–7.43 (m, 4H), 7.37–7.33 (m,
1H), 6.45 (s, 1H), 5.13 (dt, 1H, J = 7.2, 2.6 Hz), 5.04
(dd, 2H, J = 7.1, 2.6 Hz), 4.65 (d, 1H, J = 7.9 Hz), 4.13
(dd, 1H, J = 12.3, 5.2 Hz), 3.97 (dd, 1H, J = 12.3,
2.4 Hz), 3.65-3.61 (m, 1H), 1.99 (s, 3H), 1.97 (s, 3H),
1.93 (s, 3H), 1.83 (s, 3H); HR-FABMS: m/z [M+Na]+;
Calcd for C33H32O14Na, 675.1690. Found, 675.1675.
4.7. Synthesis of B-ring-substituted derivatives (37a, 37b
and 41)
4.7.1. 30,50-Dibenzyloxy-20-methoxymethoxy-p-terphenyl-
4-carboxylic acid (35a). Compound 32 (438 mg,
0.869 mmol) was coupled with 4-carboxyphenyl boronic
acid (286 mg, 1.74 mmol) by the same procedure de-
scribed at 21a to give 35a (409 mg, yield 86%) as a yellow
1
powder; H NMR (400 MHz, CDCl3): d 8.21 (d, 2H,
J = 8.3 Hz), 7.72 (d, 2H, J = 8.3 Hz), 7.48–7.40 (m, 5H),
7.29–7.19 (m, 8H), 7.00–6.98 (m, 2H), 6.83 (s, 1H), 5.01
(s, 2H), 4.95 (s, 2H), 4.74 (s, 2H), 2.96 (s, 3H); HR-FAB-
MS: m/z [M+Na]+; Calcd for C35H30O6Na, 569.1940.
Found, 569.1918.
4.7.2. 30,50-Dibenzyloxy-20-methoxymethoxy-p-terphenyl-
4-carboxylic acid benzyl ester (36a). To a solution of
35a (100 mg, 0.183 mmol) in CH3CN (6.0 mL) were
added benzyl bromide (33 lL, 0.27 mmol) and potas-
sium carbonate (38 mg, 0.275 mmol), and the mixture
was refluxed for 1 h. After cooling at room temperature,
the resulting solution was added to water (50 mL), and
extracted with EtOAc (3· 50 mL). The organic layer
was washed with saturated NH4Cl solution and brine.
The solvent was removed in vacuo and dried over
Na2SO4. The residue was purified by a silica gel column
chromatography with hexnane/EtOAc (7:1) to give 36a
(106 mg, yield 91%) as a yellow powder; 1H NMR
(400 MHz, CDCl3): d 8.15 (d, 2 H, J = 8.4Hz), 7.66 (d,
2H, J = 8.4 Hz), 7.47–7.43 (m, 4H), 7.42–7.31 (m, 6H),
7.29–7.18 (m, 8H), 6.99–6.96 (m, 2H), 6.79 (s, 1H),
5.39 (s, 2H), 4.99 (s, 2H), 4.92 (s, 2H), 4.73 (s, 2H),
2.93 (s, 3H); HR-FABMS: m/z [M+Na]+; Calcd for
C42H36O6Na, 659.2410. Found, 659.2430.
Tetraacetate 36e was transesterified in anhydrous meth-
anolic HCl solution (a drop of acetyl chloride into 2 mL
of anhydrous MeOH) at room temperature for 2 h. The
reaction mixture was added into saturated NH4Cl solu-
tion (25 mL) and extracted with EtOAc (3· 25 mL). The
solvent was removed in vacuo and then purified by a sil-
ica gel column chromatography with CHCl3/MeOH
(4:1) to give 37a as a pale yellow powder (20 mg, three
1
steps yield 65%): mp 177–178 ꢁC; H NMR (400 MHz,
CD3OD): d 8.04 (d, 2H, J = 8.3 Hz), 7.66 (d, 2H,
J = 8.3 Hz), 7.43 (d, 2H, J = 7.6 Hz), 7.38 (t, 2H,
J = 7.4 Hz), 7.30–7.25 (m, 1H), 6.42 (s, 1H), 4.29 (d,
1H, J = 7.9 Hz), 3.92 (s, 3H), 3.46 (dd, 1H, J = 11.7,
2.9 Hz), 3.41 (dd, 1H, J = 11.7, 4.6 Hz), 3.23 (t, 1H,
J = 8.5 Hz), 3.23 (m, 2H, J = 9.1 Hz), 3.02 (m, 1H);
HR-FABMS: m/z [M+Na]+; Calcd for C26H26O10Na,
521.1424. Found, 521.1450.
4.7.3. 30,50-Dibenzyloxy-20-hydroxy-4-methoxycarbonyl-
p-terphenyl 20-b-D-glucoside (37a). A solution of 36a
(100 mg, 0.157 mmol) in trifluoroacetic acid (3.0 mL)
was stirred for 1 h at room temperature. The resulting
solution was added to saturated Na2CO3 solution
(100 mL) and extracted with EtOAc (3· 100 mL). The or-
ganic layer was washed with saturated NH4Cl solution
and brine. The solvent was removed in vacuo after drying
over Na2SO4. The residue was purified by a silica gel col-
umn chromatography with hexane/EtOAc (3:2) to give
intermediate, 30,50-dibenzyloxy-20-hydroxy-4-benzyloxy-
4.7.4. 30,50-Dibenzyloxy-20-methoxymethoxy-p-terphenyl-
4-ol (35b). The same procedure described at 21a was em-
ployed with 32 (254 mg, 0.501 mmol), 4-hydroxyphenyl
boronic acid (139 mg, 1.01 mmol). The title compound
35b (230 mg, yield 88%) was obtained as a yellow
1
powder; H NMR (400 MHz, CDCl3): d 7.49–7.16 (m,
7H), 7.25–7.15 (m, 8H), 6.98–6.96 (m, 2H), 6.84 (d, 2H,
J = 8.5 Hz), 6.78 (s, 1H), 4.93 (s, 2H), 4.93 (s, 2H), 4.73
(s, 2H), 3.04 (s, 3H); HR-EIMS: m/z [M]+; Calcd for
C34H30O5: 518.2093. Found: 518.2090.

K. Hayashi et al. / Bioorg. Med. Chem. 16 (2008) 5331–5344
5343
4.7.5. 30,50-Dibenzyloxy-20-methoxymethoxy-p-terphenyl-
4-yloxy-pentanoic acid ethyl ester (36b). To a solution of
35b (150 mg, 0.19 mmol) in CH3CN (8.0 mL) was added
ethyl 5-bromo-valerate (60 lL, 0.38 mmol) and potas-
sium carbonate (52 mg, 0.38 mmol), and the mixture
was refluxed for 4 h. After cooling at room temperature,
the resulting solution was added to water, and extracted
with EtOAc. The organic layer was washed with saturated
NH4Cl solution and brine. After drying over Na2SO4, the
solvent was removed in vacuo. The residuewas purified by
a silica gel column chromatography with hexane/EtOAc
(7:3) to give 36b (178 mg, yield 95%) as a pale yellow pow-
der; 1H NMR (400 MHz, CDCl3): d 7.51 (d, 2H,
J = 8.6 Hz), 7.47 (d, 2H, J = 8.6 Hz), 7.41 (t, 2H,
J = 6.9 Hz), 7.38–7.34 (m, 1H), 7.30–7.18 (m, 8H), 6.99–
6.97 (m, 2H), 6.96 (d, 2H, J = 8.6 Hz), 6.80 (s, 1H), 4.99
(s, 2H), 4.91 (s, 2H), 4.74 (s, 2H), 4.15 (q, 2H,
J = 7.1 Hz), 4.03–4.01 (m, 2H), 3.02 (s, 3H), 2.42–2.39
(m, 2H), 1.87–1.85 (m, 4H), 1.27 (t, 3H, J = 7.1 Hz);
HR-FABMS: m/z [M+Na]+; Calcd for C41H42O7Na,
669.2828. Found, 669.2826.
(q, 2H, J = 7.1 Hz), 4.03 (br. s, 2H), 3.53 (dd, 1H,
J = 11.8, 2.6 Hz), 3.45 (dd, 1H, J = 11.8, 5.0 Hz), 3.37–
3.35 (m, 1H), 3.28–3.20 (m, 2H), 3.03–2.99 (m, 1H),
2.45–2.40 (m, 2H), 1.83–1.81 (m, 4H), 1.26 (t, 3H,
J = 7.1 Hz); HR-FABMS: m/z [M+Na]+; Calcd for
C31H36O11Na, 607.2155. Found, 607.2162.
4.7.7. 6-Benzyloxy-3-methoxymethoxy-biphenyl-2-carb-
aldehyde (38). Compound 29 (300 mg, 0.987 mmol)
was protected by methoxymethyl group as described at
17 to give the title compound 38 (316 mg, yield 92%)
as a yellow powder; 1H NMR (400 MHz, CDCl3): d
9.94 (s, 1H), 7.45–7.39 (m, 3H), 7.31–7.24 (m, 5H),
7.19–7.11 (m, 4H), 5.23 (s, 2H), 4.94 (s, 2H), 3.53 (s,
3H); HR-EIMS: m/z [M]+; Calcd for C22H20O4:
348.1362. Found: 348.1370.
4.7.8. 6-Benzyloxy-2-hydroxy-3-methoxymethoxy-biphe-
nyl (39). The same procedure was employed at 20 (19a–
b) with 38 (302 mg, 0.868 mmol). With Baeyer–Villiger
oxidation and alkaline hydrolyses of 38, the title com-
pound 39 (262 mg, two steps yield 90%) was obtained
as a yellow powder; 1H NMR (400 MHz, CDCl3): d
7.49–7.42 (m, 4H), 7.37–7.33 (m, 1H), 7.29–7.18 (m,
5H), 7.01 (d, 1H, J = 8.8 Hz), 6.49 (d, 1H, J = 8.8 Hz),
5.98 (s, 1H, OH), 5.16 (s, 2H), 4.94 (s, 2H), 3.53 (s,
3H); HR-EIMS: m/z [M]+; Calcd for C21H20O4:
336.1362. Found: 336.1366.
4.7.6. 20,30,50-Trihydroxy-p-terphenyl-4-yloxy-pentanoic
acid ethyl ester 20-b-D-glucoside (37b). The same proce-
dure described at 37a (36a–c) was employed with 36b
(151 mg, 234 lmol). The intermediate, 50-dibenzyloxy-
20hydroxy-p-terphenyl-4-yloxy-pentanoic acid ethyl es-
ter (36f: 113 mg, yield 80%) was obtained as a yellow
1
powder; H NMR (400 MHz, CDCl3): d 7.61 (d, 2H,
J = 7.2 Hz), 7.54 (d, 2H, J = 8.7 Hz), 7.47 (t, 2H,
J = 7.7 Hz), 7.40–7.36 (m, 1H), 7.28–7.23 (m, 6H), 7.20
(d, 2H, J = 7.9 Hz), 7.08–7.05 (m, 2H), 6.96 (d, 2H,
J = 8.8 Hz), 6.84 (s, 1H), 5.77 (s, 1H, OH), 4.93 (s,
2H), 4.42 (s, 2H), 4.14 (q, 2H, J = 7.1 Hz), 4.03–4.00
(m, 2H), 2.41–2.38 (m, 2H), 1.86–1.83 (m, 4H), 1.26 (t,
3H, J = 7.1 Hz); HR-FABMS: m/z [M+Na]+; Calcd
for C39H38O6Na, 625.2566. Found, 625.2557.
4.7.9.
2,6-Dibenzyloxy-3-methoxymethoxy-biphenyl
(40a). The same procedure was employed at 20 (19b–
20) with 39 (252 mg, 0.751 mmol). With protection by
benzyl group of 39, the title compound 40a (259 mg,
yield 80%), was obtained as a yellow powder; 1H
NMR (400 MHz, CDCl3): d 7.44–7.33 (m, 5H), 7.30–
7.17 (m, 8H), 7.09 (d, 1H, J = 9.1Hz), 7.00–6.97 (m,
2H), 6.73 (d, 1H, J = 9.1 Hz), 5.16 (s, 2H), 4.94 (s,
2H), 4.67 (s, 2H), 3.53 (s, 3H); HR-EIMS: m/z [M]+;
Calcd for C28H26O4: 426.1831. Found: 426.1832.
Thereafter, phenol 36f (111 mg, 184 lmol) was glycosyl-
ated by the same procedure at 37a (36c–d) to give 30,50-
dihydroxy-p-terphenyl-4-yloxy-pentanoic acid ethyl es-
ter-20-O-b-D-tetraacetyl glucoside (36g: 148 mg, yield
86%) as a pale yellow powder; 1H NMR (400 MHz,
CDCl3): d 7.46–7.34 (m, 7H), 7.30–7.18 (m, 8H), 6.96–
6.94 (m, 2H), 6.93 (d, 2H, J = 8.7 Hz), 5.17 (d, 1H,
J = 7.2 Hz), 5.08–5.05 (m, 3H), 5.00 (d, 2H,
J = 5.1 Hz), 4.85 (d, 1H, J = 10.6 Hz), 4.57 (d, 1H,
J = 10.6 Hz), 4.16 (q, 2H, J = 7.1 Hz), 4.05 (br s, 2H),
3.98 (dd, 1H, J = 12.3, 3.8 Hz), 3.78 (dd, 1H, J = 12.3,
2.3 Hz), 3.44–3.40 (m, 1H), 2.44–2.41 (m, 2H), 1.96 (s,
6H), 1.93 (s, 3H), 1.89–1.88 (m, 4H), 1.87 (s, 3H), 1.28
(t, 3H, J = 7.3 Hz); HR-FABMS: m/z [M+Na]+; Calcd
for C53H56O15Na, 955.3517. Found, 955.3516.
4.7.10. 2,6-Dibenzyloxy-biphenyl-3-ol 3-b-D-glucoside
(41). The same procedure was employed at 22a (21a–e)
with 40a (200 mg, 0.469 mmol). With the removal of
methoxymethyl group of 40a, the intermediate, 2,6-
dibenzyloxy-3-hydroxy-biphenyl 40b (158 mg, yield
1
88%) was obtained as a pale yellow powder: H NMR
(400 MHz, CDCl3): d 7.56 (d, 2H, J = 7.6 Hz), 7.46 (t,
2H, J = 7.3 Hz), 7.39 (t, 1H, J = 7.2 Hz), 7.30–7.22 (m,
6H), 7.18 (br d, 2H, J = 6.8 Hz), 7.07–7.05 (m, 2H),
6.87 (d, 1H, J = 8.9 Hz), 6.74 (d, 1H, J = 8.9 Hz), 5.42
(s, 1H, OH), 4.90 (s, 2H), 4.37 (s, 2H); HR-EIMS: m/z
[M]+; Calcd for C26H22O3: 382.1569. Found: 382.1549.
Thereafter, same procedure described at 22a (21e–f) was
employed with 40b (100 mg, 0.261 mmol). With the gly-
cosylation of 21h, the intermediate 2,6-dibenzyloxy-
biphenyl-3-yl-b-D-glucose 2,3,4,6-tetraacetate (40c:
151 mg, yield 81%) was obtained as a white powder:
1H NMR (400 MHz, CDCl3): d 7.41–7.35 (m, 5H),
7.30–7.21 (m, 4H), 7.18–7.14 (m, 4H), 7.09 (d, 1H, J =
9.1 Hz), 6.90–6.87 (m, 2H), 6.67 (d, 1H, J = 9.1 Hz),
5.36–5.18 (m, 4H), 5.07 (d, 1H, J = 7.7 Hz), 4.99–4.92
(m, 2H), 4.75 (d, 1H, J = 10.5 Hz), 4.52 (d, 1H,
Compound 36 g (148 mg, 155 lmol) was hydrogeno-
lyzed (H2 (1 atm) and 10% Pd/C) and hydrolyzed
(KOH in MeOH) at 0 ꢁC by the same procedure de-
scribed at 22a (21f–22a) to yield the title compound
37b (20 mg, two steps yield 22%) as a yellow powder:
mp 98.0 ꢁC; H NMR (400 MHz, CDCl3): d 7.47 (d,
2H, J = 8.7 Hz), 7.42 (d, 2H, J = 6.9 Hz), 7.37 (t, 2H,
J = 7.5 Hz), 7.27 (t, 1H, J = 7.2 Hz), 6.94 (d, 2H,
J = 8.6 Hz), 6.37 (s, 1H), 4.29 (d, 1H, J = 7.9 Hz), 4.13
1

5344
K. Hayashi et al. / Bioorg. Med. Chem. 16 (2008) 5331–5344
J = 10.5 Hz), 4.31 (dd, 1H, J = 12.3, 5.1 Hz), 4.16 (dd,
1H, J = 12.3, 2.3 Hz), 3.85–3.80 (m, 1H), 2.06 (s, 3H),
2.04 (s, 6H), 2.01 (s, 3H), 1.80 (s, 3H); HR-FABMS:
m/z [M+Na]+; Calcd for C40H40O12Na, 735.2417.
Found, 735.2426.
References and notes
1. Woodward, A. W.; Bartel, B. Ann. Bot. (Lond.) 2005, 95,
707–735.
2. Hagen, G.; Guilfoyle, T. Plant Mol. Biol. 2002, 49, 373–
385.
3. Liscum, E.; Reed, J. W. Plant Mol. Biol. 2002, 49, 387–
400.
4. Gray, W. M.; Kepinski, S.; Rouse, D.; Leyser, O.; Estelle,
M. Nature 2001, 414, 271–276.
5. Dharmasiri, N.; Dharmasiri, S.; Jones, A. M.; Estelle, M.
Curr. Biol. 2003, 13, 1418–1422.
6. Kepinski, S.; Leyser, O. Proc. Natl. Acad. Sci. U.S.A.
2004, 101, 12381–12386.
7. Dharmasiri, N.; Dharmasiri, S.; Weijers, D.; Lechner, E.;
Yamada, M.; Hobbie, L.; Ehrismann, J. S.; Jurgens, G.;
Estelle, M. Dev. Cell 2005, 9, 109–119.
8. Dharmasiri, N.; Dharmasiri, S.; Estelle, M. Nature 2005,
435, 441–445.
Thereafter, the same procedure described at 22a (21f–
22a) was employed with 40c (100 mg, 140 lmol). With
the hydrogenolysis and alkaline hydrolysis of 40c, the ti-
tle compound 41 (33 mg, two steps yield 60%) was ob-
tained as a pale yellow powder: mp 118–119 ꢁC; 1H
NMR (400 MHz, CD3OD): d 7.36–7.13 (m, 4H), 7.29–
7.23 (m, 1H), 7.06 (d, 1H, J = 8.8 Hz), 6.34 (d, 1H,
J = 8.8 Hz), 4.60 (d, 1H, J = 7.3 Hz), 3.89 (d, 1H,
J = 12.2 Hz), 3.72 (dd, 1H, J = 12.2, 4.9 Hz), 3.47–3.34
(m, 4H); HR-FABMS: m/z [M+Na]+; Calcd for
C18H20O8Na, 387.1056. Found, 387.1037.
9. Kepinski, S.; Leyser, O. Nature 2005, 435, 446–451.
10. Yamazoe, A.; Hayashi, K.; Kuboki, A.; Ohira, S.; Nozaki,
H. Tetrahedron Lett. 2004, 45, 8359–8362.
11. Yamazoe, A.; Hayashi, K.; Kepinski, S.; Leyser, O.;
Nozaki, H. Plant Physiol. 2005, 139, 779–789.
12. Oono, Y.; Chen, Q. G.; Overvoorde, P. J.; Kohler, C.;
Theologis, A. Plant Cell 1998, 10, 1649–1662.
13. Baldwin, J. E.; Haraldsson, G. G. Acta Chem. Scand. B
1986, 40, 400–401.
14. Ji, B.-M.; Ding, K.-L.; Meng, J.-B. Chin. J. Chem. 2003,
21, 727–730.
15. Li, C.; Johnson, R. P.; Porco, J. A. J. Am. Chem. Soc.
2003, 125, 5095–5106.
Acknowledgments
This work was supported by JSPS Grant-in Aid for sci-
entific research to KH (18510197).
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2008.02.085.