Illudalic acid as a potential LAR inhibitor: Synthesis, SAR, and preliminary studies on the mechanism of action

Article abstract of DOI:10.1016/j.bmc.2008.06.014

A novel synthesis of the human leukocyte common antigen-related (LAR) phosphatase inhibitor, illudalic acid, has been achieved by a route more amenable to structure modifications. A series of simpler analogues of illudalic acid was synthesized and evaluat

Full text of DOI:10.1016/j.bmc.2008.06.014

Bioorganic & Medicinal Chemistry 16 (2008) 7399–7409
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Illudalic acid as a potential LAR inhibitor: Synthesis, SAR, and preliminary
studies on the mechanism of action
Qing Ling a, , Yue Huang a, , Yueyang Zhou b, Zhengliang Cai a, Bing Xiong a, Yahui Zhang b, Lanping Ma a,
a,
Xin Wang a, Xin Li a, Jia Li b, , Jingkang Shen
*
*
a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, PR China
b National Center for Drug Screening, 189 Guoshoujing Road, Shanghai 201203, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel synthesis of the human leukocyte common antigen-related (LAR) phosphatase inhibitor, illudalic
acid, has been achieved by a route more amenable to structure modifications. A series of simpler ana-
logues of illudalic acid was synthesized and evaluated for potency in inhibiting LAR. The structure–activ-
ity relationship (SAR) study has shown that the 5-formyl group and the hemi-acetal lactone are crucial for
effective inhibition of LAR activity, and are the key pharmacophores of illudalic acid. The fused dimeth-
ylcyclopentene ring moiety evidently helps to enhance the potency of illudalic acid against LAR. A preli-
minary study of the mechanism of action of illudalic acid against LAR was conducted using electrospray
ionization mass spectrometry (ESI-MS) and molecular docking techniques. The results are in full agree-
ment with the described mechanism.
Received 24 April 2008
Revised 8 June 2008
Accepted 10 June 2008
Available online 13 June 2008
Keywords:
Human leukocyte common antigen-related
phosphatase (LAR)
Illudalic acid
Ó 2008 Elsevier Ltd. All rights reserved.
Inhibitor
Mechanism
Structure–activity relationship (SAR)
Synthesis
1. Introduction
compared with PTK inhibitors, which have been developed as
therapeutic agents, the task of identifying substrates for PTPs still
Protein tyrosine phosphatases (PTPs) remove phosphate from
tyrosine-phosphorylated proteins and protein tyrosine kinases
(PTKs) phosphorylate cellular substrates at tyrosine residues.1–5
PTPs, PTKs, and their corresponding substrates are integrated with-
in elaborate signal transducing networks that play significant roles
in regulating cellular growth, differentiation, metabolism, cell cy-
cles, cell–cell communication, cell migration, gene transcription,
ion-channel activity, the immune response, and survival.6–11 PTPs,
which contain a highly conserved active site (i.e., the pTyr-binding
site) with the signature motif (H/V)C(X)5R(S/T),12,13 are considered
to be a superfamily that consists of specific phosphatases, dual
specificity phosphatases, and the low-molecular-weight phospha-
tases. Tyrosine-specific phosphatases can be further divided into
two groups: receptor-like and intracellar PTPs. Following the pro-
gress in defining biological function, many PTPs have been pro-
posed as targets for therapeutic intervention, such as anticancer,
regulation of regenerative neurite outgrowth, immune response,
and treatment of type II diabetes, obesity, etc.14–24 However,
presents a challenge.25 Therefore, potent and selective PTP inhibi-
tors would be valuable as a means of enhancing our understanding
of the interaction between PTPs and substrates.
Among the known PTPs, human leukocyte common antigen-re-
lated phosphatase (LAR)26–30 is a receptor-like transmembrane
phosphatase whose extracellular structure includes three immu-
noglobulin-like domains and eight fibronectin type III-like do-
mains. The intracellular structure consists of two tandem
phosphatase catalytic domains, a membrane proximal domain
(D1), and a membrane distal domain (D2). Recently, LAR raised a
lot of interest as it was shown to regulate neurite growth and nerve
regeneration in a transgenic animal model.31–34 In addition, LAR is
potentially involved in the development of diabetes and cancer,
and may be an attractive target in the treatment of those dis-
eases.35–38 Although LAR is likely to become significant as a drug
target in various human pathologies, very little is known about
the mechanisms of action of LAR at present.29 Hence, the develop-
ment of potent enzyme-specific inhibitors is particularly important
because they may serve both as tools to study the role of LAR and
as therapeutic agents. However, no novel compounds that selec-
tively inhibit LAR have been reported39–44 despite the identifica-
tion of myriad inhibitors of other PTPs such as PTP1B44–57
(Fig. 1). By high throughput screening of a library of 44,145 syn-
thetic compounds and 19,960 natural product extracts, we have
* Corresponding authors. Tel.: +86 21 50801313; fax: +86 21 50801522 (J.L.); tel.:
+86 21 50806600; fax: +86 21 50807088 (J.S.).
E-mail addresses: jli@mail.shcnc.ac.cn (J. Li), jkshen@mail.shcnc.ac.cn, kxiao
simm123@163.com (J. Shen).
These two authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.06.014

7400
Q. Ling et al. / Bioorg. Med. Chem. 16 (2008) 7399–7409
O
HO
OH
O
S
CH3
O
O
HO
O
O
S
N
O
F3C
CF3
H3C
O
N
HO
S
O
N
CH3
N
H
Br
N
CF3
H
43
39
41
IC50
PTP1B 2.0
LAR 116
μ
M
IC50
PTP1B 55.4
LAR 18.2
μ
M
IC50
PTP1B 0.82
LAR 320
μ
μ
M
μ
M
μ
M
M
CD45
0.28
μ
M
TCPTP 0.68
μ
M
O
O
HO
O
O
OH
O
OH
O
OH
O
N
N
O
O
O
S
HO
O
H
O
O
HN
O
O
O
H3C
NH
O
HO
OH
O
O
O
42
40
Ki 44 PTP1B 0.022
LAR 1.3
TCPTP 0.049
μ
M
IC50
PTP1B 1.6
μ
M
IC50
PTP1B 13
LAR 41
μ
μ
M
M
μ
M
LAR 27 μ
M
μ
M
Yersinia PTPase 1.7 μM
Figure 1. Structures of some reported none-selective LAR inhibitors.
previously identified illudalic acid 1 isolated from the Basidiomy-
cete, Clitocybe illudens, as the first potent selective small molecular
The alkylation reaction of 2 with excess methyl iodide in the pres-
ence of NaH produced its dimethyl derivative 3, which was trans-
formed into bromo-dihydroindene 4 via Clemmensen reduction by
refluxing in an ethanolic solution of concentrated hydrochloric acid
in the presence of excess amalgamated zinc. Friedel–Crafts acyla-
tion of compound 4 with 3-chloropropionyl chloride gave the
chloro-containing ketone 5. Cyclization of 5 in concentrated sulfu-
ric acid at 90 °C for 8 h led to the key carbon skeleton 6.
The conversion of hydrindacen-1-one 6 to the target product 1
is shown in Scheme 2. The cyclic ketone 6 was reduced with so-
dium borohydride in methanol at 0 °C to give the acid-labile alco-
hol 7, the hydroxy group of which was protected by treatment with
tert-butyldimethylsilyl chloride (TBSCl), 4-dimethylaminopyridine
(DMAP), and imidazole in dry CH2Cl2 to give TBS ether 8. The hal-
ogen–metal exchange in 8 was accomplished using metalation by
treatment with n-BuLi at À78 °C, followed by addition of an excess
of methyl chloroformate to produce 9 in excellent yield.62,63 The
TBS protecting group was removed by treatment with HIO4 solu-
tion in THF–H2O giving 10.
inhibitor of LAR (IC50 = 1.1 l
M).58 However, the quantity of illuda-
lic acid isolated from natural extracts was very limited and resup-
plies of the compound for further biological evaluations had to be
accomplished via a total synthesis. Total synthesis of 1 was
achieved by Woodward starting from commercially available in-
dan,59 but we decided to take advantage of readily available start-
ing material 260 for SAR study. Herein, we give a full account of our
study of the design, synthesis, and structure–activity relationship
(SAR), as well as preliminary studies on the mechanism of action
of illudalic acid against LAR.
2. Results and discussion
2.1. Chemistry
In our synthetic approach, we took good advantage of the regio-
selective Friedel–Crafts acylation reaction of suitably substituted
bromo-dihydroindene with 3-chloropropanoyl chloride, followed
by Friedel–Crafts alkylation for the construction of the key carbon
skeleton 6 (Scheme 1).
Dihydroindan-1-one 2 equipped with methoxyl and bromo sub-
stituents was obtained according to the method of Michael et
al.,60,61 starting from readily available 3-methoxycinnamic acid.
The monohydroxyl product 10 dehydrated to afford olefin 11 as
white crystal in the presence of p-toluenesulfonic acid (p-TsOH).
Dihydroxylation of the olefin occurred efficiently by the treatment
of 11 with AD-mix-a in the presence of methanesulfonamide in t-
BuOH–H2O,64 followed by saponification with 2 N KOH in ethanol
to generate the requisite acid 13. Subjecting the above acid 13 to
O
O
O
O
O
O
O
O
O
c
b
a
d
Cl
Br
Br
Br
Br
Br
5
4
6
2
3
Scheme 1. Reagents and conditions: (a) CH3I, NaH, THF, 0 °C to rt; (b) Zn/Hg, concd HCl, C2H5OH, reflux; (c) SnCl4, ClCH2CH2COCl, CH3NO2, 0 °C to rt; (d) concd H2SO4, 90 °C.

Q. Ling et al. / Bioorg. Med. Chem. 16 (2008) 7399–7409
7401
O
O
O
O
O
O
TBSO
HO
O
HO
TBSO
e
d
a
b
c
COOCH3
COOCH3
Br
COOCH3
Br
Br
7
8
6
9
10
11
OH
O
O
O
HO
HO
O
O
h
i
g
HO
HO
f
COOH
HO
O
O
HO
O
O
COOCH3
1
13
12
14
Scheme 2. Reagents and conditions: (a) NaBH4, CH3OH, 0 °C; (b) TBSCl, imidazole, DMAP, CH2Cl2, rt; (c) n-BuLi, ClCO2CH3, THF, À78 °C to rt; (d) HIO4, THF–H2O, rt; (e) p-
TsOH, toluene, 80 °C; (f) AD-mix- , CH3SO2NH2, t-BuOH–H2O, 0 °C to rt; (g) 2 N KOH, C2H5OH, reflux; (h) NaIO4, 1,4-dioxane–H2O, rt; (i) BBr3, CH2Cl2, À78 °C to rt.
a
O
O
O
O
O
O
OH
OH
O
OTBS
OTBS
a
b
e
c
d
Br
COOCH3
Br
COOCH3
COOCH3
Br
18
19
2
15
16
17
O
O
O
OH
OH
O
f
g
h
OH
OH
COOH
HO
O
O
COOCH3
21
20
22
Scheme 3. Reagents and conditions: (a) NaBH4, CH3OH, 0 °C; (b) TBSCl, imidazole, DMAP, CH2Cl2, rt; (c) n-BuLi, ClCO2CH3, THF, À78 °C to rt; (d) HIO4, THF–H2O, rt; (e) p-
TsOH, toluene, 80 °C; (f) AD-mix-a, CH3SO2NH2, t-BuOH–H2O, 0 °C to rt; (g) 2 N KOH, C2H5OH, 80 °C; (h) NaIO4, 1,4-dioxane–H2O, rt.
O
O
O
O
O
b
c
a
d
HO
TBSO
TBSO
HO
HO
Br
Br
COOCH3
COOCH3
24
23
25
26
27
O
O
e
f
HO
COOH
HO
O
O
28
29
Scheme 4. Reagents and conditions: (a) Br2, CHCl3, 0 °C; (b) TBSCl, imidazole, DMAP, CH2Cl2, rt; (c) n-BuLi, ClCO2CH3, THF, À78 °C to rt; (d) HIO4, THF–H2O, rt; (e) 2 N KOH,
C2H5OH, 80 °C; (f) PCC, CH2Cl2, rt.
sodium metaperiodate furnished the dialdehyde, which existed as
the cyclized lactol 14 both in the solid state and in solution. Treat-
ment of illudalic acid methyl ether 14 with boron tribromide in
methylene chloride gave the major product, illudalic acid 1. The
route lends itself to the preparation of various analogues for a pos-
sible biological evaluation.
The simpler analogue 22 was prepared directly from 2 through
sequential manipulations similar to those performed in converting
6 to 14 (Scheme 3). The synthetic pathway affording the final com-
pound 29 is depicted in Scheme 4. Treatment of the starting mate-
rial 23 with bromine gave the product 24, which was then
protected as 25 following the procedure for 1. Likewise, the con-
version of 25 to 26 was performed by bromo-metal exchange reac-
tion. After deprotection and saponification, the corresponding
product 28 was subjected to oxidation over pyridinium
chlorochromate (PCC) to furnish 29. Scheme 5 illustrates the syn-
thesis of analogues 30–34 and 36 from their precursor 20. Firstly,
20 was converted to alcohol 35 by reduction with lithium alumin-
ium hydride (LAH), followed by oxidation cleavage to form the tar-
get compound 36. The same oxidation cleavage conditions applied
to 20 gave the desired 30, which was reduced by NaBH4 to gener-
ate the diol 31. The saponification of 31 converted 31 to 32, which,
in turn, was transformed into the lactone 33 in high yield by treat-
ment with p-TsOH. The aldehyde product 34 was derived from 33.
2.2. Biological results
All analogues of illudalic acid were measured for their potency
in inhibiting LAR with p-nitrophenyl phosphate (pNPP) as a
substrate at room temperature and pH 6.0 according to procedures

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Q. Ling et al. / Bioorg. Med. Chem. 16 (2008) 7399–7409
Table 1
CH3
O
CH3
HO
LAR-inhibiting activity of inhibitor illudalic acid analogues 14, 22, 29–32, 34, and 36
O
HO
f
b
HO
Compound
Structure
MW
IC50 (lM)
HO
OH
CH3
O
O
CH2OH
O
20
a
35
1
276
1.30 0.06
1.53 0.29
21.85 1.02
g
HO
O
O
O
O
O
O
CH3
CH3
CH3
O
O
OH
O
OCH3
O
O
b
HO
O
14
22
29
30
31
32
290
222
194
236
240
226
O
OCH3
HO
O
O
OCH3
HO
O
O
30
31
36
OCH3
c
CH3
e
CH3
CH3
O
O
OH
O
HO
O
d
HO
O
HO
OCH3
O
O
O
O
O
OH
32
33
34
<5% at 20
<5% at 20
<5% at 20
<5% at 20
l
l
l
l
g/mLa
g/mLa
g/mLa
g/mLa
Scheme 5. Reagents and conditions: (a) NaIO4, 1,4-dioxane–H2O, rt; (b) NaBH4,
CH3OH, 0 °C; (c) 2 N KOH, C2H5OH, 80 °C; (d) p-TsOH, CH2Cl2, rt; (e) PCC, CH2Cl2, rt;
(f) LAH, Et2O, reflux; (g) NaIO4, 1,4-dioxane–H2O, rt.
HO
O
O
O
described previously.58 The synthetic illudalic acid (IC50 = 1.3
lM)
displayed identical activity and selectivity as the natural product.
O
COOCH3
O
The results are shown in Table 1.
The methyl ether derivative 14 with an IC50 of 1.5 lM exhibited
HO
HO
almost the same potency against LAR as its parent 1. To understand
the SAR of illudalic acid, we initially prepared its simpler analogue
22, in which the fused dimethylcyclopentene ring of 14 was re-
moved, resulting in a 14-fold decrease in potency against LAR
COOCH3
O
(IC50 = 21.85 lM).
On the basis of the structure of compound 22, the 5-formyl and
3-hydroxyl groups were removed to afford compounds 29 and 34,
respectively, which led to a significant loss of activity. On the other
hand, compound 36 in which the 1-carbonyl group was removed
showed poor activity. To further understand the key structure scaf-
fold, compounds 30–32 were synthesized. In fact, structure 37 is
the open style of hemi-acetal lactone structure moiety found in
compound 22 (Scheme 6). With respect to the structure of 37,
we prepared compounds 30–32, in which two aldehyde groups
were reduced to two hydroxymethyl groups, the carboxylic acid
group was converted to a methyl ester or two aldehyde groups,
and the carboxylic acid groups were converted to two hydroxy-
methyl and methyl ester groups simultaneously. To our surprise,
none of these compounds showed any activities against LAR up
HO
HO
COOH
O
O
34
206
208
<5% at 20
l
g/ma
O
O
O
O
36
20% at 20
l
g/mLa
HO
O
to 100 lM. The above results strongly implied that the 5-formyl
a
% Inhibition at the given concentration.
IC50 values were determined by regression analyses and expressed as
means SD of three replicates.
group and the hemi-acetal lactone are crucial for effective inhibi-
tion of LAR activity, and are an absolute requirement for the activ-
ity of illudalic acid. The fused dimethylcyclopentene ring moiety
evidently helps to enhance the potency of illudalic acid against
LAR. The SAR of illudalic acid provides quite useful information
for designing analogues with more potent activity against LAR.
b
OCH3
OCH3
O
O
O
COOH
HO
O
O
2.3. Mechanism of action
37
22
In testing bioactivity, we found an interesting phenomenon:
compound 1 and its analogues showed inhibitory activity against
LAR only after these compounds were pre-incubated with LAR en-
zyme at pH 8 and the enzyme activity was then analyzed at pH 6.
Otherwise, none of these compounds show any LAR inhibitory
activity if they are incubated and tested at pH 6.
Scheme 6.
We performed two experiments to prove 1 as an irreversible
inhibitor of LAR. First, we plotted enzyme activity versus reaction
time. As shown in Figure 2A, without the inhibitor, the plot is a lin-

Q. Ling et al. / Bioorg. Med. Chem. 16 (2008) 7399–7409
7403
H
O
20μM
10μM
5μM
OCH3
R'
OCH3
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
O
OCH3
R'
A
R'
O
O
PH=6
O
PH=8
R
R
O
S
R
2.5μM
1.25μM
0.62μM
control
-O
O
HO
O
O
OH
I
II
III
Scheme 7.
nucleophilic attack by the thiol group of the Cys residue65,66 at
the base of the active site leading to the formation of a hemi-acetal.
The hydroxyl group of the hemi-acetal further attacks the 5-formyl
group to give a new hemi-acetal and results in a fused six-mem-
bered ring structure. The H-bond binding between the hydroxyl
group of the new hemi-acetal and the adjacent oxygen of the
methoxyl group on the aryl moiety produces a new fused six-
membered ring, thereby stabilizing the whole complex structure
III. The interaction between the Cys residue and the inhibitor plays
a critical role in irreversibly forming a covalent complex.
0
5
10
15
20
Time (min)
The employment of mass spectrometric techniques to illumi-
nate the mechanistic aspects of biological events is attracting
widespread attention.67–69 These techniques are useful since they
can simultaneously measure the masses of numerous products
accurately, rapidly, and with great sensitivity, thus enabling the
possibility of understanding the nature of intermediates and trans-
formations in biological events.70
To shed light on the above action mechanism, we investigated
the process by ESI-MS/MS studies of enzymatically and syntheti-
cally produced covalent complexes. The inhibitor 22 was incubated
45
40
35
30
25
20
15
10
5
B
E+DMSO
E+illudalic acid
with LAR and excess LAR simulacrum N-acetyl-L-cysteine at pH 8
for 10 min (Scheme 8), and the pH was then adjusted to 6 (the
same condition as that of the bioactivity assay). Then the product
22-1 of inhibitor 22 and simulacrum N-acetyl-L-cysteine were ana-
lyzed by ESI-MS/MS under low-resolution conditions (positive ion
mode) after the denatured LAR enzyme was filtered away.
The resulting spectra correlate extremely well, indicating that
the generated component is identical to the product 22-1. The pro-
posed fragmentation of the covalent complex 22-1 is illustrated in
Scheme 9. The prominent features are observed in the fragmenta-
tion of the molecule 22-1. Likewise, a synthetic version of the cova-
lent complex 30-1 was prepared by the treatment of compound 30
0
0
2
4
8
24
Dialysis time (hour)
Figure 2. Characterization of illudalic acid. (A) Time-dependent inhibition of LAR
by illudalic acid. Progress curves for LAR reaction with pNPP in the presence of
increasing concentrations of illudalic acid. The assay system containing 2% DMSO
was defined as control. (B) Irreversible inhibition of LAR by illudalic acid. The
activities of PTP-LAR pre-incubation with illudalic acid or DMSO were detected after
dialysis for the indicated time.
with N-acetyl-L-cysteine in CH2Cl2 (Scheme 10) and analyzed by
ear line. However, in the presence of various concentrations of 1,
the plot shows a curve for a while and a straight line later, and
the slope of the straight line is smaller than the plot without inhib-
itor and will decrease dependent on the increased concentrations.
This result suggested 1 as an irreversible or a slow tight-binding
inhibitor. To exclude the possibility that this compound is a slow
tight-binding inhibitor, we dialyzed the enzyme–inhibitor complex
and examined the recovery of the enzyme activity. As shown in
Figure 2B, the activity of LAR could not recover after 24 h of dialy-
sis. With these results, we can suggest that the inhibitor inhibits
LAR irreversibly.
ESI-MS/MS under low-resolution conditions. The proposed frag-
mentation processes illustrated in Scheme 11 are consistent with
the molecular structure of the covalent complex 30-1. ESI-MS/MS
OH OCH3
OCH3
OH
O
O
PH=6
PH=8
LAR
+
O
S
N-Acetyl-L-Cysteine
HN
CH3
O
OH
HO
O
O
O
LAR
22
22-1
This implies that the interaction of inhibitor and LAR involves
covalent binding. Enzymological and structural biological studies
have led to the explanation that phosphatases in the PTP super-
family share a common mechanism of catalysis that involves the
nucleophilic Cys residue in the PTP signature motif.65,66 At the
same time, the SAR of 1 revealed that the 5-formyl group and
the hemi-acetal lactone group are crucial for inhibitory activity
against LAR. On the basis of the above information we propose a
mechanism of action of illudalic acid and its analogues against
LAR (Scheme 7).
Scheme 8.
OH OCH3
OH OCH3
OH OCH3
OH
O
OH
O
H
O
O
S
O
+ S
+
HN
CH3
HN
CH3
O
OH
O
ONa
O
ONa
O
O
B
22-1
M = 385
A
The hemi-acetal lactone moiety of structure I firstly opens up
via hydrolysis when incubated with LAR enzyme at pH 8. The new-
ly produced free acetaldehyde group of structure II undergoes
m/z = 408
m/z = 245
Scheme 9.

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Q. Ling et al. / Bioorg. Med. Chem. 16 (2008) 7399–7409
OH OCH3
O
OCH3
OH
O
CH2Cl2
O
S
O
N-Acetyl-L-Cysteine
HN
CH3
O
OCH3
O
OCH3
O
30
30-1
Scheme 10.
OH OCH3
OH OCH3
OH OCH3
ONa
HN
O
OH
HN
O
O
+
H
S
O
O
S
+
CH3
CH3
O
OCH3
O
OCH3
O
OCH3
O
O
C
30-1
M = 399
B
m/z = 237
m/z = 422
H+
OH OCH3
OH
O
O
S
HN
CH3
Figure 3. Three-dimensional structural model of the inhibitor, illudalic acid 1,
O
OCH3
bound to LAR derived from the molecular docking simulation.
O
A
m/z = 400
Arg1528 and Glu1428, while the two oxygen atoms from the fused
hemi-acetal ring system are surrounded by the near amino acid
residues Ala1524, Gly1525, Val526, and Gly1527 and form several
H-bonding interactions. At the same time, the phenolic hydroxyl
group interacts with residue Gln1566 via a strong H-bond. A num-
ber of H-bonds efficiently enhance the enzyme–inhibitor interac-
tion and stabilize the LAR–illudalic acid complex within the
active site to a great extent. As expected, the molecular docking
analysis elucidates the inhibition mechanism of illudalic acid and
its analogues, and further validates our proposed mechanism of ac-
tion. Although the results of the above studies support our assump-
tion, the exact binding mode of the 1 with LAR should be obtained
from X-ray crystallography experiments.
Scheme 11.
studies of the enzymatically and synthetically produced covalent
complexes support this proposed mechanism of action of illudalic
acid and its analogues with LAR. Another important piece of evi-
dence also strongly sustained our proposal. The proton nuclear
magnetic resonance spectrum of compound 38 was measured in
CDCl3; it showed two aldehydic protons at d 10.48 ppm and d
9.77 ppm, respectively, which fully supported the dialdehyde struc-
ture 38. However, when it was measured in the aprotic polar sol-
vent, DMSO-d6, the chemical shift signals in the spectrum were
completely changed corresponding to a cyclic hemi-acetal struc-
ture 39 (Scheme 12), in which the two aldehydic protons disap-
peared and the two proton signals at d 5.92 ppm and d 5.40–
5.44 ppm were consistent with a hemi-acetal structure. It is sug-
gested that the formation of a cyclic thiohemiacetal intermediate
could be the driving force for the interaction between 1 and LAR.
To further reinforce our hypothesis and gain more insight into
the structural and mechanistic aspects of illudalic acid and its ana-
logues and their interaction with LAR, a complex model of the
inhibitor illudalic acid and LAR was constructed by molecular
docking based on the LAR crystal structure (Brookhaven Protein
Data Bank, http://www.rcsb.org./pdb).71 The active molecule
bound well into the LAR active site and formed favorable H-bond-
ing and steric interactions with various active site amino acid res-
idues inside the active site of LAR. The complex model and bound
conformation of 1 are shown in Figure 3.
3. Conclusions
In summary, we have successfully synthesized illudalic acid and
related simpler analogues to evaluate their SAR with respect to
LAR. It has been found that the 5-formyl group and the hemi-acetal
lactone play an extremely important role in effective inhibition of
LAR activity, and are the key pharmacophores of illudalic acid. The
phenolic hydroxy group seems to tolerate further modification,
while the fused dimethylcyclopentene ring moiety evidently helps
to enhance the potency of illudalic acid against LAR. The mecha-
nism of action of illudalic acid and its analogues against LAR was
elucidated by electrospray ionization mass spectrometry (ESI-MS)
studies and the molecular docking method. Based on SAR, further
efforts to modify illudalic acid with the aim of obtaining higher po-
tency as well as specificity are now in progress and will be reported
in due course.
According to the proposed mechanism of action, the structure of
the hemi-acetal lactone has changed to a new fused hemi-acetal
ring system upon binding to LAR. Therefore we used the active
structure version of 1 for docking purposes. The residue Cys1522
in the center of the LAR active site covalently binds with 3-C of
1. The carboxyl group interacts with the amino acid residues,
4. Experimental
4.1. Chemistry
OH OCH
3
OCH
3
The 1H NMR (300 MHz or 400 MHz) spectra were recorded on a
Varian Mercury-300 or -400 High Performance Digital FT-NMR
with TMS as internal standard. The chemical shifts were reported
in d (ppm) using the d 7.26 signal of CDCl3 (1H NMR) as internal
standard and spectra are given as shift (multiplicity, proton counts,
and coupling constants). The 13C NMR (100 MHz) spectra were
determined with a Varian Mercury-400 High Performance Digital
O
O
d6-DMSO
CDCl
HO
O
Br
3
Br
38
39
Scheme 12.

Q. Ling et al. / Bioorg. Med. Chem. 16 (2008) 7399–7409
7405
FT-NMR. The LC–MS was carried out on a Thermo Finnigan LCQDE-
CAXP and low-resolution EI-MS was measured on a MAT-95 spec-
trometer and HREI-MS on a MAT-77 spectrometer. TLC was carried
out with glass pre-coated silica gel GF254 plates. Spots were visu-
alized under UV light. All the solvents and reagents were used di-
rectly as obtained commercially unless otherwise noted.
The resulting mixture was stirred at 85 °C for 2.5 h. The mixture
was allowed to warm to room temperature and 10 mL of ice and
water were added, and the mixture was extracted with Et2O (3Â
10 mL). The combined organic layer was washed with saturated
sodium bicarbonate (10 mL) and brine (10 mL), dried over anhy-
drous Na2SO4, and concentrated in vacuo. The product was purified
by column chromatography over silica gel using petroleum ether/
EtOAc as eluent to afford 6 (18 mg, 25%) as an orange crystal.
Mp: 89–90 °C. 1H NMR (300 MHz, CDCl3): d 3.9 (s, 3H), 2.96 (m,
2H), 2.85 (s, 2H), 2.78 (s, 2H), 2.70 (m, 2H), 1.18 (s, 6H); 13C NMR
(400 MHz, CDCl3): d 203.3, 155.4, 153.8, 153.7, 135.4, 128.8,
112.3, 61.1, 49.1, 44.9, 39.7, 37.1, 28.7, 28.7, 26.5. EI-MS (m/z):
308 (M+).
4.1.1. 4-Bromo-7-methoxy-2,2-dimethyl-2,3-dihydroinden-1-
one (3)
To a suspension of NaH 60% (1000 mg, 25 mmol) in dry 1,2-
dimethoxyethane (5 mL) was added a solution of 4-bromo-7-
methoxy-2,3-dihydroinden-1-one 2 (2000 mg, 8.3 mmol) in dry
1,2-dimethoxyethane (5 mL). After stirring for 10 min, the reaction
mixture was cooled to 0 °C and CH3I (1.55 mL, 25 mmol) was
added dropwise. The mixture was allowed to warm to room tem-
perature and stirred overnight. The reaction solution was
quenched with 50 mL of water and extracted with Et2O (3Â 20
mL). The combined organic layer was washed with brine (20 mL),
dried over anhydrous Na2SO4, and evaporated to dryness in vac-
uum. The residue was purified by chromatography on silica gel
using petroleum ether/EtOAc to afford 3 (2000 mg, 90%) as needle
crystal. Mp: 172–175 °C. 1H NMR (300 MHz, CDCl3): d 7.67 (d, 1H,
J = 8.9 Hz), 6.75 (d, 1H, J = 8.9 Hz), 3.90 (s, 3H), 2.87 (s, 2H), 1.20 (s,
6H). EI-MS (m/z): 268 (M+).
4.1.5. 4-Bromo-8-methoxy-6,6-dimethyl-1,2,3,5,6,7-hexahydro-
s-indacen-1-ol (7)
A solution of 6 (250 mg, 0.81 mmol) in absolute MeOH (15 mL)
was cooled to 0 °C and NaBH4 (63 mg, 1.65 mmol) was added in
one portion. After stirring at 0 °C for 2 h, the reaction solution
was quenched with 20 mL of water and extracted with CH2Cl2
(3Â 15 mL). The combined organic layers were washed with brine
(10 mL), dried over anhydrous Na2SO4, and evaporated in vacuo to
give 7 (249 mg, 98%) as a colorless oil. 1H NMR (400 MHz, CDCl3): d
5.48 (m, 1H), 3.88 (s, 3H), 3.03 (m, 1H), 2.87 (s, 2H), 2.78 (m, 1H),
2.70 (s, 2H), 2.44 (m, 1H), 2.04 (m, 1H), 1.18 (s, 3H), 1.17 (s, 3H). EI-
MS (m/z): 310 (M+).
4.1.2. 4-Bromo-7-methoxy-2,2-dimethyl-2,3-dihydro-1H-
indene (4)
To newly prepared amalgamated zinc (40.4 g) were added dis-
tilled water (13.4 mL), EtOH (3.8 mL), and concd HCl (40.6 mL).
The mixture was stirred at a refluxing temperature. A solution of
3 (6200 mg, 23 mmol) in EtOH (48.7 mL) was added dropwise
slowly to the above refluxing mixture. The resulting reaction mix-
ture was refluxed overnight. The mixture was allowed to cool to
room temperature and filtered. The solid was washed with water
(3Â 20 mL) and Et2O (3Â 20 mL). The organic phase was separated
and the aqueous phase was extracted with Et2O (3Â 20 mL). The
combined organic layers were washed with water (2Â 20 mL), sat-
urated sodium bicarbonate (20 mL), and brine (20 mL), dried over
anhydrous Na2SO4, and evaporated to dryness under reduced pres-
sure. The product was purified by chromatography on silica gel
using petroleum ether/EtOAc to provide 4 (4900 mg, 84%) as a col-
orless oil. 1H NMR (300 MHz, CDCl3): d 7.20 (d, 1H, J = 8.5 Hz), 6.60
(d, 1H, J = 8.5 Hz), 3.80 (s, 3H), 2.76 (s, 2H), 2.75 (s, 2H), 1.18 (s, 6H).
EI-MS (m/z): 254 (M+).
4.1.6. (4-Bromo-8-methoxy-6,6-dimethyl-1,2,3,5,6,7-
hexahydro-s-indacen-1-yloxy)-tert-butyldimethylsilane (8)
A solution of 7 (100 mg, 0.32 mmol), TBSCl (97 mg, 0.64 mmol),
imidazole (88 mg, 1.28 mmol) and 1.7 mg of DMAP in 15 mL of dry
CH2Cl2 was stirred at room temperature for 36 h under argon
atmosphere. The reaction was quenched with 20 mL of water and
extracted with CH2Cl2 (3Â 15 mL). The combined organic phases
were washed with brine (10 mL), dried over anhydrous Na2SO4,
and concentrated to dryness. The residue was purified by chroma-
tography on silica gel using petroleum ether/EtOAc to obtain 8
(133 mg, 97%) as a colorless oil. 1H NMR (300 MHz, CDCl3): d
5.48 (m, 1H), 3.88 (s, 3H), 3.03 (m, 1H), 2.87 (s, 2H), 2.78 (m,
1H), 2.70 (s, 2H), 2.44 (m, 1H), 2.04 (m, 1H), 1.18 (s, 3H), 1.17 (s,
3H), 0.90 (s, 9H), 0.14 (s, 3H), 0.12 (s, 3H). EI-MS (m/z): 424 (M+).
4.1.7. Methyl 1-(tert-butyldimethylsilyloxy)-8-methoxy-6,6-
dimethyl-1,2,3,5,6,7-hexahydro-s-indacene-4-carboxylate (9)
Compound 8 (100 mg, 0.24 mmol) was dissolved in dry THF
(10 mL) and cooled to À78 °C. A solution of n-BuLi in hexane
(1.6 M, 0.222 mL, 0.36 mmol) was added dropwise by syringe un-
der argon atmosphere. Stirring was continued for an additional
20 min, and then the methyl chloroformate (0.055 mL, 0.72 mmol)
was added dropwise. After 30 min, the reaction mixture was al-
lowed to warm to room temperature, quenched with 10 mL of
H2O, and extracted with EtOAc (3Â 15 mL). The combined organic
layer was washed with saturated sodium bicarbonate (10 mL),
dried over anhydrous Na2SO4, and concentrated in vacuo. The
product was purified by column chromatography over silica gel
using petroleum ether/EtOAc as eluent to afford 9 (85 mg, 90%)
as a colorless oil. 1H NMR (300 MHz, CDCl3): d 5.37 (m, 1H), 3.88
(s, 3H), 3.83 (s, 3H), 3.29 (m, 1H), 3.10 (m, 1H), 3.00 (s, 2H), 2.77
(s, 2H), 2.21 (m, 1H), 2.04 (m, 1H), 1.16 (s, 3H), 1.11 (s, 3H), 0.90
(s, 9H), 0.14 (s, 3H), 0.12 (s, 3H). EI-MS (m/z): 404 (M+).
4.1.3. 1-(7-Bromo-4-methoxy-2,2-dimethyl-2,3-dihydro-1H-
inden-5-yl)-3-chloropropan-1-one (5)
Compound 4 (2000 mg, 7.8 mmol) was dissolved in the dry
CH3NO2 (20 mL) and cooled to 0 °C. A solution of 3-chloropropionyl
chloride (0.75 mL, 7.8 mmol) in dry CH3NO2 (5 mL) was added
dropwise under argon atmosphere. The reaction mixture was al-
lowed to warm to room temperature and stirring was continued
for an additional 8 h. The mixture was quenched with 2 mL of con-
cd HCl and 2 mL of ice and water and extracted with CH2Cl2 (3Â 15
mL). The combined organic layer was washed with 1 N HCl (15 mL)
and brine (10 mL), dried over anhydrous Na2SO4, and concentrated
in vacuo. The product was purified by column chromatography
over silica gel using petroleum ether/EtOAc as eluent to afford 5
(2050 mg, 76%) as a yellow oil. 1H NMR (300 MHz, CDCl3): d 7.64
(s, 1H), 3.85 (t, 2H, J = 6.7 Hz), 3.80 (s, 3H), 3.41 (t, 2H, J = 6.7 Hz),
2.90 (s, 2H), 2.70 (s, 2H), 1.20 (s, 6H). EI-MS (m/z): 344 (M+).
4.1.8. Methyl 1-hydroxy-8-methoxy-6,6-dimethyl-1,2,3,5,6,7-
hexahydro-s-indacene-4-carboxylate (10)
A solution of 9 (100 mg, 0.25 mmol) in 10 mL of THF was trea-
ted with 1 N HIO4 (1 mL) at room temperature. The reaction mix-
ture was diluted with 10 mL of H2O and extracted with EtOAc
4.1.4. 4-Bromo-8-methoxy-6,6-dimethyl-2,3,6,7-tetrahydro-s-
indacen-1(5H)-one (6)
A solution of 5 (100 mg, 0.29 mmol) in 2 mL of CH2Cl2 was
added dropwise to 0.237 mL of conc. H2SO4 at room temperature.

7406
Q. Ling et al. / Bioorg. Med. Chem. 16 (2008) 7399–7409
(3Â 15 mL). The combined organic extracts were washed with sat-
urated sodium bicarbonate (10 mL), dried over anhydrous Na2SO4,
and evaporated in vacuo. The crude product was purified by silica
chromatography using petroleum ether/EtOAc as eluent to give 10
(69 mg, 96%) as a colorless oil. 1H NMR (300 MHz, CDCl3): d 5.42
(m, 1H), 3.97 (s, 3H), 3.85 (s, 3H), 3.30 (m, 1H), 3.08 (m, 1H),
3.00 (s, 2H), 2.80 (s, 2H), 2.40 (m, 1H), 2.03 (m, 1H), 1.16 (s, 3H),
1.13 (s, 3H). EI-MS (m/z): 290 (M+).
164.2, 164.1, 157.7, 139.1, 134.2, 124.3, 117.4, 94.8, 61.1, 49.5,
44.9, 39.9, 31.6, 28.7. EI-MS (m/z): 290 (M+).
4.1.13. 3,6-Dihydroxy-8,8-dimethyl-1-oxo-1,3,4,7,8,9-
hexahydro-cyclopenta[h]isochromene-5-carbaldehyde (1)
Compound 14 (100 mg, 0.342 mmol) was dissolved in dry
CH2Cl2 (10 mL), cooled to À60 °C, and then BBr3 (100 mg,
0.342 mmol) was added under argon atmosphere. After stirring
at room temperature for 3 h, the reaction was quenched with
10 mL of ice and water and extracted with CH2Cl2 (3Â 15 mL).
The combined organic extracts were washed with brine (10 mL),
dried over anhydrous Na2SO4, and evaporated in vacuo. The crude
product was purified by silica chromatography using petroleum
ether/EtOAc as eluent to give 1 (41.8 mg, 44%) as a white solid.
Mp: 200–210 °C dec (lit. mp > 200 °C dec). 1H NMR (300 MHz,
CDCl3): d 12.41 (s, 1H), 10.25 (s, 1H), 5.95 (t, 1H, J = 3.9 Hz), 3.51
(m, 2H), 3.22 (s, 2H), 2.72 (s, 2H), 1.19 (s, 6H); 13C NMR
(400 MHz, DMSO-d6): d 196.2, 162.8.1, 161.3, 156.0, 142.3, 130.0,
116.6, 114.1, 94.4, 49.9, 42.5, 40.1, 30.4, 28.8. EI-MS (m/z): 276
(M+), 258, 243 (100%); HRMS-EI: m/z C15H16O5; Calcd: 276.0998,
found: 276.0999.
4.1.9. Methyl 8-methoxy-2,2-dimethyl-1,2,3,5-tetrahydro-s-
indacene-4-carboxylate (11)
p-Toluenesulfonic acid (0.50 mg) was added to a solution of 10
(100 mg, 0.34 mmol) in dry toluene (8 mL) under nitrogen and the
mixture was stirred at 80 °C for 10 min. After cooling to room tem-
perature, the reaction mixture was concentrated to a total volume
of 2 mL and applied to a short column of silica gel (petroleum
ether/EtOAc elution) to give 11 (84 mg, 95%) as a colorless crystal.
Mp: 78–79 °C. 1H NMR (300 MHz, CDCl3): d 7.03 (dt, 1H, J = 6 Hz
and 2 Hz), 6.52 (dt, 1H, J = 6 Hz and 2 Hz), 3.97 (s, 3H), 3.90 (s,
3H), 3.70 (s, 2H), 3.10 (s, 2H), 2.78 (s, 2H), 1.17 (s, 6H). EI-MS (m/
z): 272 (M+).
4.1.10. Methyl 1,2-dihydroxy-8-methoxy-6,6-dimethyl-
1,2,3,5,6,7-hexahydro-s-indacene-4-carboxylate (12)
4.1.14. 4-Bromo-7-methoxy-2,3-dihydro-1H-inden-1-ol (15)
Compound 15 was prepared according to the procedure as de-
scribed for 7 in 98% yield as a pale yellow solid. Mp: 79–81 °C.
1H NMR (300 MHz, CDCl3): d 7.34 (d, 1H, J = 8.7 Hz), 6.61 (d, 1H,
J = 8.7 Hz), 5.52 (m, 1H), 3.85 (s, 3H), 3.05 (m, 1H), 2.80 (m, 1H),
2.47 (m, 1H), 2.04 (m, 1H). EI-MS (m/z): 241 (M+).
To a solution of AD-mix-a (515 mg) in 2 mL of H2O and 2 mL of
t-BuOH was added methane sulfonamide (35 mg). The mixture
was cooled to 0 °C, 11 was added (100 mg, 0.36 mmol), and then
stirred overnight at room temperature. To the reaction mixture
was added solid sodium sulfite (551 mg) at 0 °C and the mixture
was allowed to warm to room temperature, stirred for another
30 min, and extracted with CH2Cl2 (3Â 15 mL). The combined or-
ganic layers were washed with 2 N KOH (2Â 10 mL) and brine
(10 mL), dried over Na2SO4, and evaporated to dryness under re-
duced pressure. The product was purified by chromatography on
silica gel using petroleum ether/EtOAc to obtain 12 (101 mg,
90%) as a white solid. Mp: 158–160 °C. 1H NMR (300 MHz, CDCl3):
4.1.15. (4-Bromo-7-methoxy-2,3-dihydro-1H-inden-1-
yloxy)(tert-butyl)dimethylsilane (16)
Compound 16 was prepared according to the procedure as de-
scribed for 8 in 98% yield as a colorless oil. 1H NMR (300 MHz,
CDCl3): d 7.32 (d, 1H, J = 8.7 Hz), 6.56 (d, 1H, J = 8.7 Hz), 5.43 (m,
1H), 3.78 (s, 3H), 3.07 (m, 1H), 2.78 (m, 1H), 2.30 (m, 1H), 2.00
(m, 1H), 0.90 (s, 9H), 0.14 (s, 3H), 0.08 (s, 3H). EI-MS (m/z): 356
(M+).
d
5.19 (m, 1H), 4.45 (m, 1H), 3.98 (s, 3H), 3.86 (s, 3H), 3.33 (m, 2H),
3.01 (s, 2H), 2.80 (s, 2H), 1.16 (s, 3H), 1.14 (s, 3H). EI-MS (m/z): 306
(M+).
4.1.16. Methyl 1-(tert-butyldimethylsilyloxy)-7-methoxy-2,3-
dihydro-1H-indene-4-carboxylate (17)
4.1.11. 1,2-Dihydroxy-8-methoxy-6,6-dimethyl-1,2,3,5,6,7-
hexahydro-s-indacene-4-carboxylic acid (13)
Compound 17 was prepared according to the procedure as de-
scribed for 9 in 88% yield as a colorless oil. 1H NMR (300 MHz,
CDCl3): d 7.95 (d, 1H, J = 8.7 Hz), 6.71 (d, 1H, J = 8.7 Hz), 5.37 (m,
1H), 3.85 (s, 3H), 3.84 (s, 3H), 3.40 (m, 1H), 3.16 (m, 1H), 2.24
(m, 1H), 1.98 (m, 1H), 0.90 (s, 9H), 0.14 (s, 3H), 0.08 (s, 3H). EI-
MS (m/z): 336 (M+).
Compound 12 (100 mg, 0.326 mmol) was dissolved in a metha-
nolic solution of 2 N KOH (5 mL). The solution was heated at 80 °C
with stirring for 2.5 h, most of the solvent was removed, and the
solution was diluted with saturated KH2PO4 solution (10 mL) and
extracted with EtOAc (5Â 10 mL). The combined organic extracts
were dried over anhydrous Na2SO4, concentrated to provide 13
(91 mg, 95%) as a pale yellow solid. Mp: 170–172 °C. 1H NMR
(300 MHz, CDCl3): d 5.19 (m, 1H), 4.45 (m, 1H), 4.02 (s, 3H), 3.38
(m, 2H), 3.07 (s, 2H), 2.80 (s, 2H), 1.16 (s, 3H), 1.14 (s, 3H). EI-MS
(m/z): 292 (M+).
4.1.17. Methyl 1-hydroxy-7-methoxy-2,3-dihydro-1H-indene-
4-carboxylate (18)
Compound 18 was prepared according to the procedure as de-
scribed for 10 in 96% yield as a colorless oil. 1H NMR (300 MHz,
CDCl3): d 7.98 (d, 1H, J = 8.7 Hz), 6.76 (d, 1H, J = 8.7 Hz), 5.45 (m,
1H), 3.95 (s, 3H), 3.91 (s, 3H), 3.43 (m, 1H), 3.16 (m, 1H), 2.24
(m, 1H), 2.02 (m, 1H). EI-MS (m/z): 222 (M+).
4.1.12. 3-Hydroxy-6-methoxy-8,8-dimethyl-1-oxo-1,3,4,7,8,9-
hexahydro-cyclopenta[h]isochromene-5-carbaldehyde (14)
Compound 13 (100 mg, 0.342 mmol) was dissolved in 30%
aqueous dioxane (6 mL) and treated with NaIO4 (88 mg,
0.411 mmol). This mixture was stirred for a few minutes at room
temperature and a white precipitate appeared. Stirring was contin-
ued for a total period of 2 h. The reaction mixture was diluted with
10 mL of H2O and extracted with CH2Cl2 (4Â 10 mL). The combined
organic phases were washed with brine, dried over anhydrous
Na2SO4, and evaporated to give 14 (99 mg, 98%) as a pale yellow
solid. Mp: 145–147 °C. 1H NMR (300 MHz, CDCl3): d 10.54 (s,1H),
5.84 (t, 1H, J = 3.9 Hz), 4.00 (s, 3H), 3.61 (m, 2H), 3.24 (q, 2H),
2.87 (s, 2H), 1.24 (s, 6H); 13C NMR (400 MHz, CDCl3): d 191.8,
4.1.18. Methyl 7-methoxy-3H-indene-4-carboxylate (19)
Compound 19 was prepared according to the procedure as de-
scribed for 11 in 95% yield as a colorless crystal. Mp: 49–51 °C.
1H NMR (300 MHz, CDCl3): d 7.91 (d, 1H, J = 8.8 Hz), 7.02 (m, 1H),
6.83 (d, 1H, J = 8.8 Hz), 6.56 (m, 1H), 3.94 (s, 3H), 3.91 (s, 3H),
3.76 (m, 2H). EI-MS (m/z): 204 (M+).
4.1.19. Methyl 1,2-dihydroxy-7-methoxy-2,3-dihydro-1H-
indene-4-carboxylate (20)
Compound 20 was prepared according to the procedure as de-
scribed for 12 in 90% yield as a white solid. Mp: 130–132 °C. 1H

Q. Ling et al. / Bioorg. Med. Chem. 16 (2008) 7399–7409
7407
NMR (300 MHz, CDCl3): d 8.01 (d, 1H, J = 8.8 Hz), 6.79 (d, 1H,
J = 8.8 Hz), 5.20 (m, 1H), 4.52 (m, 1H), 3.93 (s, 3H), 3.86 (s, 3H),
3.40 (m, 2H). EI-MS (m/z): 238 (M+).
silica gel using petroleum ether/EtOAc to provide 34 (3.6 mg,
30%). Mp: 80–81 °C. 1H NMR (400 MHz, CDCl3): d 8.07 (m, 1H),
6.90 (m, 1H), 6.76 (m, 1H), 5.90 (m, 1H), 3.87 (s, 3H), 3.16 (m,
2H). EI-MS (m/z): 194 (M+).
4.1.20. 1,2-Dihydroxy-7-methoxy-2,3-dihydro-1H-indene-4-
carboxylic acid (21)
4.1.28. Methyl 3-formyl-4-methoxy-2-(2-oxo-ethyl)benzoate
(30)
Compound 21 was prepared according to the procedure as de-
scribed for 13 in 95% yield as a pale yellow solid. Mp: 143–
145 °C. 1H NMR (300 MHz, DMSO):d 7.86 (d, 1H, J = 8.8 Hz), 6.91
(d, 1H, J = 8.8 Hz), 4.75 (m, 1H), 4.09 (m, 1H), 3.84 (s, 3H), 2.91
(m, 2H). EI-MS (m/z): 224 (M+).
Compound 30 was prepared according to the procedure as de-
scribed for 14 in 94% yield as a white solid. Mp: 163–164 °C.1H
NMR (300 MHz, CDCl3): d 10.56 (s, 1H), 9.80 (s, 1H), 8.20 (d, 1H,
J = 9 Hz), 7.00 (d, 1H, J = 9 Hz), 4.61 (s, 2H), 3.98 (s, 3H), 3.85 (s,
3H). EI-MS (m/z): 236 (M+).
4.1.21. 3-Hydroxy-6-methoxy-1-oxoisochroman-5-
carbaldehyde (22)
4.1.29. Methyl 2-(2-hydroxyethyl)-3-(hydroxymethyl)-4-
methoxybenzoate (31)
Compound 22 was prepared according to the procedure as de-
scribed for 14 in 99% yield as a pale yellow solid. Mp: 186–
188 °C. 1H NMR (300 MHz, CDCl3): d 10.60 (s, 1H), 8.35 (d, 1H,
J = 8.9 Hz), 7.05 (d, 1H, J = 8.9 Hz), 5.90 (t, 1H, J = 3.9 Hz), 4.00 (s,
3H), 3.62 (m, 2H). EI-MS (m/z): 222 (M+).
Compound 31 was prepared according to the procedure as de-
scribed for 7 in 60% yield as a white solid. Mp: 120–122 °C. 1H
NMR (300 MHz, CDCl3): d 8.12 (d, 1H, J = 8.8 Hz), 6.92 (d, 1H,
J = 8.8 Hz), 4.73 (s, 2H), 4.51 (t, 2H, J = 6 Hz), 3.94 (s, 3H), 3.82 (s,
3H), 3.15 (t, 2H, J = 6 Hz). EI-MS (m/z): 240 (M+).
4.1.22. 2-(2-Bromo-5-methoxyphenyl)ethanol (24)
2-(3-Methoxyphenyl)ethanol 23 (200 mg, 1.3 mmol) was dis-
solved in 4 mL of CHCl3 and cooled to 0 °C. To the solution was
added dropwise bromine (0.074 mL, 1.4 mmol). After stirring for
1 h at 0 °C, the reaction was quenched with 10% sodium metabisul-
fite, extracted with CHCl3 (3Â 5 mL). The combined organic ex-
tracts were washed with brine (10 mL) and dried over anhydrous
Na2SO4, and evaporated in vacuo to give 24 (279 mg, 92%). 1H
NMR (400 MHz, CDCl3): d 7.44 (m, 1H), 6.82 (m, 1H), 6.66 (m,
1H), 3.88 (t, 2H, J = 6.7 Hz), 3.78 (s, 3H), 2.98 (t, 2H, J = 6.7 Hz).
EI-MS (m/z): 229 (M+).
4.1.30. 2-(2-Hydroxyethyl)-3-(hydroxymethyl)-4-
methoxybenzoic acid (32)
Compound 32 was prepared according to the procedure as de-
scribed for 13 in 90% yield as a pale yellow solid. Mp: 150–
152 °C. 1H NMR (300 MHz, CDCl3): d 8.13 (d, 1H, J = 8.8 Hz), 6.93
(d, 1H, J = 8.8 Hz), 4.73 (s, 2H), 4.51 (t, 2H, J = 6 Hz), 3.94 (s, 3H),
3.14 (t, 2H, J = 6 Hz). EI-MS (m/z): 226 (M+).
4.1.31. 5-(Hydroxymethyl)-6-methoxy-1-oxoisochroman (33)
To a solution of 32 (18 mg, 0.079 mmol) in dry CH2Cl2 (4 mL)
was added p-toluenesulfonic acid (1 mg). After stirring at room
temperature for 1 h, the reaction was quenched with 10 mL of
water and extracted with CH2Cl2 (3Â 15 mL). The combined organ-
ic extracts were washed with brine (10 mL), dried over anhydrous
Na2SO4, and evaporated in vacuo. The crude product was purified
by silica chromatography using petroleum ether/EtOAc as eluent
to give 33 (16 mg, 96%) as a white solid. Mp: 96–98 °C. 1H NMR
4.1.23. (2-Bromo-5-methoxyphenethoxy)(tert-
butyl)dimethylsilane (25)
Compound 25 was prepared according to the procedure as de-
scribed for 8 in 96% yield as a colorless oil. 1H NMR (400 MHz,
CDCl3): d 7.41 (m, 1H), 6.81 (m, 1H), 6.63 (m, 1H), 3.84 (t, 2H,
J = 6.8 Hz), 3.77 (s, 3H), 2.92 (t, 2H, J = 6.8 Hz), 0.86 (s, 9H), 0.10
(s, 6H). EI-MS (m/z): 344 (M+).
(300 MHz, CDCl3):
d 8.12 (d, 1H, J = 8.8 Hz), 6.93 (d, 1H,
J = 8.8 Hz), 4.73 (s, 2H), 4.51 (t, 2H, J = 6.1 Hz), 3.95 (s, 3H), 3.14
4.1.24. Methyl 2-(2-(tert-butyldimethylsilyloxy)ethyl)-4-
methoxybenzoate (26)
(t, 2H, J = 6.1 Hz). EI-MS (m/z): 210 (M+).
Compound 26 was prepared according to the procedure as de-
scribed for 9 in 75% yield as a colorless oil. 1H NMR (400 MHz,
CDCl3): d 7.93 (m, 1H), 6.78 (m, 2H), 3.87–3.83 (m, 8H), 3.20 (t,
2H, J = 6.4 Hz), 0.85 (s, 9H), 0.10 (s, 6H). EI-MS (m/z): 324 (M+).
4.1.32. 6-Methoxy-1-oxoisochroman-5-carbaldehyde (34)
Compound 34 was prepared according to the procedure as de-
scribed for 29 in 78% yield. Mp: 137–138 °C. 1H NMR (300 MHz,
CDCl3): d 10.60 (s, 1H), 8.36 (d, 1H, J = 9 Hz), 7.05 (d, 1H,
J = 9 Hz), 4.46 (t, 2H, J = 6 Hz), 4.02 (s, 3H), 3.49 (t, 2H, J = 6 Hz).
EI-MS (m/z): 206 (M+).
4.1.25. Methyl 2-(2-hydroxyethyl)-4-methoxybenzoate (27)
Compound 27 was prepared according to the procedure as de-
scribed for 10 in 94% yield as a colorless oil. 1H NMR (400 MHz,
CDCl3): d 7.93 (m, 1H), 6.79 (m, 2H), 3.91 (t, 2H, J = 6.3 Hz), 3.86
(s, 3H), 3.84 (s, 3H), 3.22 (t, 2H, J = 6.3 Hz). EI-MS (m/z): 210 (M+).
4.1.33. 3-Hydroxy-6-methoxy-1H-isochroman-5-carbaldehyde
(36)
To the suspension of LAH (12.7 mg, 0.336 mmol) in dry THF
(5 mL) was added dropwise a solution of 20 (20 mg, 0.084 mmol)
in dry THF (3 mL). The mixture was refluxed for 2 h and then
was allowed to warm to room temperature. Two milliliters of
water, 2 mL of 1 N NaOH, and 3 mL of EtOAc were added dropwise
with ice bath cooling. The resulting precipitate was filtered and
washed with EtOAc (3Â 3 mL). The filtrate was extracted with
EtOAc (3Â 6 mL), washed with brine (10 mL), dried over Na2SO4,
and evaporated in vacuo. The residue was applied to silica gel chro-
matography using petroleum ether/EtOAc as eluent to provide 4-
(hydroxymethyl)-7-methoxy-2,3-dihydro-1H-indene-1,2-diol 35
(20 mg, 0.095 mmol). Compound 35 was dissolved in 30% aqueous
dioxane (5 mL) and treated with NaIO4 (25 mg, 0.116 mmol). This
mixture was stirred for a few minutes at room temperature and
a white precipitate appeared. Stirring was continued for a total
4.1.26. 2-(2-Hydroxyethyl)-4-methoxybenzoic acid (28)
Compound 28 was prepared according to the procedure as de-
scribed for 13 in 96% yield as a white solid. Mp: 131–132 °C. 1H
NMR (400 MHz, CDCl3): d 8.03 (m, 1H), 6.82 (m, 2H), 3.94 (t, 2H,
J = 6.3 Hz), 3.87 (s, 3H), 3.25 (t, 2H, J = 6.3 Hz). EI-MS (m/z): 196 (M+).
4.1.27. 3-Hydroxy-6-methoxyisochroman-1-one (29)
To a solution of PCC (27 mg, 0.124 mmol) in CH2Cl2 (5 mL) was
added dropwise a solution of 28 (12.1 mg, 0.062 mmol) in CH2Cl2
(3 mL). After stirring for 2 h, the reaction mixture was diluted with
anhydrous Et2O (10 mL) and filtered. The solid was washed with
5 mL of CH2Cl2 and the filtrate was evaporated to dryness under re-
duced pressure. The product was purified by chromatography on

7408
Q. Ling et al. / Bioorg. Med. Chem. 16 (2008) 7399–7409
period of 2 h. The reaction mixture was diluted with 10 mL of H2O
and extracted with CH2Cl2 (4Â 8 mL). The combined organic
phases were washed with brine, dried over anhydrous Na2SO4,
and evaporated to give 36 (19.4 mg) as a white solid. Mp: 180–
181 °C. 1H NMR (300 MHz, CDCl3): d 10.26 (s, 1H), 7.20 (d, 1H,
J = 8.6 Hz), 6.86 (d, 1H, J = 8.6 Hz), 5.37 (t, 1H, J = 4.2 Hz), 4.73 (m,
2H), 3.89 (s, 3H), 3.23 (m, 2H); 13C NMR (400 MHz, CDCl3): d
192.0, 162.3, 134.2, 130.9, 126.4, 122.4, 109.5, 91.7, 62.4, 55.8,
33.2. EI-MS (m/z): 210 (M+).
AutoDock472 was adopted to dock the ligand 1 into the binding site
of LAR which located around Cys1522. The Lamarckian genetic
algorithm (LGA) was applied to deal with the protein–ligand inter-
actions. A Solis and Wets local search was performed for the energy
minimization on a user-specified proportion of the population. Due
to the covalent interaction hypothesis we proposed, we first re-
moved the side chain of residue Cys1522 and performed the dock-
ing, then a list of conformations having 3-C of ligand 1 within 1.5 Å
of sulfur atom of Cys1522 were collected. Finally the conformation
with lowest predicted free energy was picked for later interaction
analysis. All the protein and ligand structures were prepared in
graphics software AutoDock tool according with default parame-
ters. To explore the conformational space of ligands, some param-
eters in AutoDock4 were set up as follows.
4.1.34. 3-Bromo-6-methoxy-2-(2-oxo-ethyl)-benzaldehyde (38)
Compound 38 was prepared from 2 according to the procedure
as described for 30. 1H NMR (300 MHz, CDCl3): d 10.48 (s, 1H), 9.77
(s, 1H), 7.75 (d, 1H, J = 9 Hz), 6.90 (d, 1H, J = 9 Hz), 4.37 (s, 2H), 3.93
(s, 3H). EI-MS (m/z): 255 (M+). 39 1H NMR (300 MHz, DMSO-d6): d
7.72 (d, 1H, J = 8.7 Hz), 6.84 (d, 1H, J = 8.7 Hz), 5.92 (s, 1H), 5.44–
5.40 (m, 1H), 3.76 (s, 3H), 2.95–2.72 (m, 2H).
The overall translation step was set to 0.2 Å, and the overall
rotation and torsion rotation step were set to 5° in the docking
study. The number of GA generations, the number of energy eval-
uations, and the number of docking runs were set to 370,000,
1,500,000, and 50, respectively.
4.2. Enzymatic assay
4.2.1. Inhibition assay
Acknowledgements
Recombinant LAR catalytic domain was expressed and purified
according to the previous report.58 The enzymatic activities of the
LAR catalytic domain were determined at 30 °C by monitoring the
hydrolysis of pNPP. Dephosphorylation of pNPP generates product
pNP, which was monitored at an absorbance of 405 nm by the
EnVision multilabel plate reader (PerkinElmer Life Sciences, Bos-
This work was supported by the Hi-tech Research and
Development Program of China through Grant Nos.
2006AA02Z315 (L.-P.M.) and 2007AA09Z402 (J.L.).
References and notes
ton, MA, USA). In a typical inhibition assay, 2
lL DMSO solution
with or without inhibitor pre-incubated with 20
tion containing 50 mM Tris, pH 8.0, 50 mM NaCl, 2 mM EDTA,
2 mM dithiothreitol, and 300 nM recombinant LAR for 10 min, then
the enzymatic reaction was initiated through adding 78 lL sub-
strate buffer containing 50 mM MES, pH 6.0, 2 mM EDTA, 2 mM
dithiothreitol, and 2 mM pNPP. Absorbance at 405 nm was contin-
uously monitored and the initial rate of the hydrolysis was deter-
mined using the early linear region of the enzymatic reaction
kinetic curve. The IC50 was calculated with Prism 4 software
(Graphpad, San Diego, CA, USA) from the non-linear curve fitting
of the percentage of inhibition (% inhibition) versus the inhibitor
concentration [I] by using the following equation: % Inhibi-
tion = 100/(1 + [IC50/[I]]k), where k is the Hill coefficient.
l
L enzyme solu-
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l
ken out, and their phosphatase activities were determined.
4.3. ESI-MS/MS studies
5
l
L inhibitor 22 (17 mM) was incubated with 90
M) and 5 L LAR simulacrum N-acetyl- -cysteine (30 mM) at
pH 8.0 for 10 min (Scheme 8), and the pH was then adjusted to 6
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(32
l
l
L
l
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