Synthesis of novel chemical probes for the study of tanshinone binding proteins

Article abstract of DOI:10.1016/j.bmcl.2006.07.019

Novel diazirine or biotin-labeled tanshinone probes were synthesized and evaluated for TRAP inhibitory activity against RANKL-induced osteoclastogenesis in RAW264.7 cells. We found that diazirine-labeled derivatives (18 and 20) are potent inhibitors of RA

Full text of DOI:10.1016/j.bmcl.2006.07.019

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4733–4737
Synthesis of novel chemical probes for the study of
tanshinone binding proteins
Jin-Soo Lee,a,b Sun-Young Han,a Myong Sang Kim,a Chan-Mo Yu,b
Myung Hee Kim,a Seong Hwan Kim,a Yong Ki Mina,* and Bum Tae Kima
aLaboratory of Chemical Genomics, Bio-Organic Science Division, Korea Research Institute of Chemical Technology,
Deajeon 305-600, Republic of Korea
bDepartment of Chemistry, Sungkyunkwan University, 300 Cheoncheon-dong, Jangan-gu, Suwon,
Gyeonggido 440-746, Republic of Korea
Received 10 April 2006; revised 19 June 2006; accepted 5 July 2006
Available online 25 July 2006
Abstract—Novel diazirine or biotin-labeled tanshinone probes were synthesized and evaluated for TRAP inhibitory activity against
RANKL-induced osteoclastogenesis in RAW264.7 cells. We found that diazirine-labeled derivatives (18 and 20) are potent inhib-
itors of RANKL-induced osteoclastogenesis. IC50 values were 18.02 and 15.00 lM, respectively. These probes will be useful reagents
for investigating tanshinone–proteins interactions.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Tanshinones are natural products isolated from Salvia
miltiorrhiza Bunge, a traditional Chinese medicinal herb,
by Nakao and Fukushima in 1934.1 The major
components of tanshinones are tanshinone IIA (1),
3-hydroxytanshinone (2), tanshinone IIB (3), and cryp-
totanshinone (4) (Fig. 1). These compounds have been
observed to possess various pharmacological activities
including antibacterial, antidermatophytic, antioxidant,
anti-inflammatory, antineoplastic, and antiplatelet
aggregation activities.2–9 Recently, tanshinones have
been reported to inhibit the osteoclast differentiation
and bone resorption by blocking RANKL/RANK sig-
naling pathway, which indicates that tanshinones can
be used for the treatment and prevention of osteoporosis
and other bone-resorptive diseases.10,11 Several inhibi-
tors that include estrogens, selective estrogen-receptor
modulators (SERMs), and bisphosphonates have been
used for the regulation of osteoclast generation and
function. However, the effects of these compounds in
bone biology are still under investigation.12 Despite
various biological activities, the mechanism of action
of tanshinones remains unknown. This led to the design
of chemical probes for the study of tanshinone
O
C
O
O
O
D
O
O
A
B
HO
1
2
O
O
O
O
O
O
HO
3
4
Figure 1. Chemical structures of tanshinone IIA (1), 3-hydroxytan-
shinone (2), tanshinone IIB (3), and cryptotanshinone (4).
binding proteins, which can provide the useful direct
probing method for defining target proteins. Here, we
investigated the synthesis of tanshinone derivatives with
a photoactive moiety or a biotin group at the A-ring of
tanshinone.
Keywords: Tanshinone derivatives; Osteoclastogenesis; Tanshinone–
protein(s) interactions; Probes.
Trifluoromethylaryldiazirine was selected as a photo-
phore due to its many useful features; (a) chemical sta-
bility in various reaction conditions that may facilitate
*
Corresponding author. Tel.: +82 42 860 7029; fax: +82 42 861
0307; e-mail: ykmin@krict.re.kr
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.019

4734
J.-S. Lee et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4733–4737
the use of a diazirine group as a carbene precursor; (b)
photoactivatability under long-wavelength UV light
(wavelength 350–360 nm) that minimize damage to pro-
teins; (c) generation of highly reactive carbene that react
with virtually proteins with no intramolecular rear-
rangements.13 Biotin labeling is a powerful technique
for nonradioactive detection of proteins based on the
avidin affinity chromatography because the biotin–avi-
din interaction is the strongest noncovalent, biological
interaction (Kd = 10À15 M) between protein and
ligand.14 These methods are especially useful for the
identification of ligand-binding sites of target proteins
and the investigation of ligand–receptor interactions.15
biotin or a photoactive moiety at the hydroxyl group
of the A-ring of tanshinone. The side chain at 3-hydroxyl
group of tanshinone (2) was introduced by the reaction
of (S)-5-hydroxy-6,6-dimethyl-1-vinylcyclohexene (5)
with TBDMSOCH2CH2Br under microwave irradiation
at 120 ꢁC for 10 min (Scheme 1). The diene 5 was syn-
thesized from 2,2-dimethyl-1,3-dicyclohexanone, as de-
scribed in the literature.16a The tert-butyldimethylsilyl
(TBDMS) ether 9 was obtained by the ultrasound-pro-
moted Diels–Alder reaction of diene 6 with o-quinone
8, which was synthesized by the oxidation of 7 using
Fremy’s salt, followed by the aromatization with
DDQ. Finally, the key intermediate 10 was prepared
by the deprotection of TBDMS ether 9 using 48%
hydrogen fluoride in THF in moderate yield. As illus-
trated in Scheme 2, we introduced a side chain at
hydroxyl group of tanshinone IIB (3). Ethyl ester 12
was obtained by the reaction of TBDMS-protected eth-
ylene glycol 11 with ethyl bromoacetate under micro-
wave irradiation. Compound 13 was prepared by the
saponification of compound 12 under 1 N NaOH/
MeOH condition. The key intermediate 15 was synthe-
sized by the condensation of tanshinone IIB (3) and acid
The natural products tanshinones 2 and 3 were prepared
as described previously.16 3-Hydroxytanshinone (2) was
synthesized from (S)-3-hydroxy-2,2-dimethylcyclohexa-
none by enantioselective reduction of 2,2-dimethylcyc-
lohexan-1,3-dione with Baker’s yeast. Tanshinone IIB
(3) was prepared from ethyl 2-methyl-1-oxocyclohex-
ane-2-carboxylate as a racemate. Based on the results
from previous structure–activity relationships (SAR)
study,17 we synthesized tanshinone derivatives with a
O
i)
iii)
O
+
TBDMSO
HO
O
O
5
6
8
ii)
HO
7
O
O
O
O
O
iv)
O
O
TBDMSO
O
HO
O
9
10
Scheme 1. Reagents and conditions: (i) NaH, TBDMSOCH2CH2Br, THF, microwave, 120 ꢁC, 10 min, 50%; (ii) Fremy’s salt, CH3OH, 0.07 M
KH2PO4 (pH 7.0); (iii) a—ultrasound, CH3OH, 35 ꢁC, 1 h, b—DDQ, benzene, reflux, overnight, 29% in three steps; (iv) 48% HF/THF (1:1), rt, 1 h,
68%.
O
O
i)
ii)
OH
TBDMSO
11
O
O
TBDMSO
O
TBDMSO
OH
12
13
O
O
O
O
iii)
iv)
O
O
TBDMSO
O
HO
O
O
O
O
O
15
14
Scheme 2. Reagents and conditions: (i) NaH, ethyl bromoacetate, THF, microwave, 120 ꢁC, 10 min, 40%; (ii) 1 N NaOH, CH3OH, rt, 30 min, 67%;
(iii) 3, DCC, DMAP, CH2Cl2, rt, overnight, 94%; (iv) 48% HF/THF (1:1), rt, 10 min, 98%.

J.-S. Lee et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4733–4737
4735
Table 1. The effect of tanshinone derivatives on the cell proliferation
and TRAP activity in RANKL-induced osteoclastogenesis in
RAW264.7 cells
13 in the presence of N,N-dicyclohexycarbodiimide
(DCC) in CH2Cl2/DMF (4:1) followed by the deprotec-
tion of silyl ether 14 in a solution of 48% HF/THF (1:1,
v/v) in good yield. Finally, 3-hydroxytanshinone deriva-
tives (16 and 18) were prepared by the condensation of
tanshinone derivative 10 with (+)-biotin or 3-(trifluoro-
methyl)-3H-diazirine derivative (17)18 in the presence of
DCC in good yields (Scheme 3).19 Tanshinone IIB deriv-
atives (19 and 20) were also obtained from intermediate
15 by the coupling reaction using 1-ethyl-3-(3-dimeth-
ylaminopropyl)carbodiimide hydrochloride (EDCI) as
shown in Scheme 4.19
Compound
Cell proliferation
(IC50, lM)a
TRAP activity
(IC50, lM)a
2
>20
12.85
9.56
16
18
3
18.72
>20
>20
18.02
15.55
12.37
15.00
19
20
18.34
>20
a IC50 values were calculated by using mean values presented as % of
control.
To estimate whether biotin- or diazirine-tagged tanshi-
none derivatives possess potent biological activity simi-
lar to the natural products (2 and 3), we determined
their inhibitory effects on TRAP (tartrate resistant acid
phosphatase; a biomarker of osteoclastogenesis) activity
in RANKL-induced osteoclastogenesis in RAW264.7
cells.20 All compounds (16 and 18–20) were found to
inhibit the TRAP activity as shown in Table 1 and
Figure 2. 3-Hydroxytanshinone (2) and tanshinone IIB
(3) inhibited the TRAP activity in RANKL-induced
osteoclastogenesis with IC50 values of 12.85 and
15.55 lM, respectively. Biotin-tagged derivatives (16
and 19) inhibited the TRAP activity, whereas they atten-
uated the proliferation of RAW264.7 cells with IC50 val-
ues of 18.72 and 18.34 lM, respectively.21 It did not
mean the cytotoxicity of 16 or 19, since the number of
cells cultured with each compound for 4 day was abso-
lutely higher than that (4000 cells/well) in cell seeding
step. On the other hand, diazirine-tagged derivatives
(18 and 20) were found to be approximately equipotent
O
O
O
O
NH
O
O
HN
HN
NH
i)
+
O
O
OH
O
S
S
HO
O
O
O
O
10
10
16
18
O
O
O
O
O
N
N
ii)
O
+
O
HO
CF3
O
F3C
O
HO
O
O
O
N
N
O
17
Scheme 3. Reagents and conditions: (i) DCC, DMAP, CH2Cl2/DMF (1:1), ultrasound, 40 ꢁC, 2 h, 84%; (ii) DCC, DMAP, DMF, rt, overnight, 73%.
O
O
O
O
O
O
HN
NH
HN
S
i)
NH
O
O
+
OH
S
HO
O
O
O
O
O
O
O
O
O
19
15
O
O
O
O
O
N
N
CF3
ii)
O
O
O
+
H O
HO
O
F3C
O
O
O
O
O
N
N
O
O
O
15
17
20
Scheme 4. Reagents and conditions: (i) EDCI, DMAP, DMF, rt, overnight, 40%; (ii) EDCI, DMAP, DMF, rt, 3 h, 28%.

4736
J.-S. Lee et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4733–4737
Compound (20μM) treatment
-
-
-
+3
+19
+20
+
+
+
+
RANKL treatment
Figure 2. The effect of tanshinone derivatives on the osteoclast formation.
7. Lee, A. R.; Wu, W. L.; Chang, W. L.; Lin, H. C.; King, M.
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inhibitors of TRAP activity compared to mother com-
pounds (2 and 3), with IC50 values of 18.02 and
15.00 lM, respectively. We determined that the applied
doses of inhibitors (18 and 20) did not have any effect
on cell proliferation. The results show that diazirine-
tagged probes functionalized at the A-ring of tanshinone
can maintain the biological activities of the natural
products (2 and 3) and can be powerful photoaffinity re-
agents to fish out the target protein(s), which is involved
in the cellular process of osteoclastogenesis.
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In summary, we synthesized novel tanshinone probes
(16 and 18–20) and investigated inhibitory effects on
TRAP activity in RANKL-induced osteoclastogenesis
in RAW264.7 cells. Diazirine-labeled derivatives (18
and 20) are potent inhibitors of RANKL-induced osteo-
clastogenesis. Despite the inhibitory effect of biotinyla-
ted tanshinones (16 and 19) on the rate of cell
proliferation, it can still be valuable bioprobes for inves-
tigating ligand–protein interactions. Further studies for
the identification of target proteins using these probes
are now in progress and results will be published in
due course.
Acknowledgments
This study was supported by ‘Chemical Genomics
Research Project’, Korea Research Institute of Chemical
Technology and ‘Chemical Genomics R&D Project’,
Ministry of Science and Technology (MOST), Republic
of Korea.
References and notes
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Masuda, K.; Tomohiro, T.; Nakano, T.; Hatanaka, Y.
ChemBioChem 2005, 6, 814.
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1988, 36, 408.
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(m, 2H), 4.50 (m, 1H), 5.30 (br s, 1H), 5.75 (br s, 1H),
7.25 (s, 1H), 7.59 (d, J = 8.2 Hz, 1H), 7.62 (d, J = 8.2 Hz,
1H). HRMS-FAB (m/z) [M+H]+ calcd for C33H39N2O9S,
639.2376; found, 639.2379.
17. Tanshinone IIA (1) analogues were synthesized and
evaluated for the TRAP activity and cell proliferation of
RAW264.7 cells in RANKL-induced osteoclastogenesis.
The applied doses of inhibitors (1, 2, and 2a–c) did not
have any effect on cell proliferation.
O
Compound 20: 1H NMR (500 MHz, CDCl3) d 1.33 (s,
3H), 1.60–1.64 (m, 1H), 1.78–1.91 (m, 3H), 2.26 (s, 3H),
3.18–3.22 (m, 2H), 3.73 (t, J = 4.6 Hz, 2H), 4.07 (m, 2H),
4.17 (d, J = 11.1 Hz, 1H), 4.33 (d, J = 11.1 Hz, 1H), 4.36
(t, J = 4.6 Hz, 2H), 4.66 (s, 2H), 6.72 (s, 1H), 6.81 (d,
J = 7.8 Hz, 1H), 6.93 (dd, J = 8.1, 2.0 Hz, 1H), 7.24 (s,
1H), 7.31 (t, J = 8.1 Hz, 1H), 7.57 (d, J = 8.2 Hz, 1H),
7.61 (d, J = 8.2 Hz, 1H). HRMS-FAB (m/z) [M+H]+
calcd for C33H30F3N2O9, 655.1903; found, 655.1906.
20. TRAP staining and activity assay—All materials used for
cell culture were purchased from HyClone (UT).
RAW264.7 cells were maintained in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% fetal
bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml
streptomycin with a change of medium every 3 days in
humidified atmosphere of 5% CO2 at 37 ꢁC. To induce the
osteoclast formation, cells were plated in a 96-well plate at
the density of 1 · 103 cells/well and cultured in a-minimal
essential medium (MEM) supplemented with 10% FBS in
the presence of 100 ng/ml RANKL (R&D Systems Inc.,
MN). Next day, chemicals were treated and at day 4,
multinucleated osteoclasts were visualized by TRAP
staining using a leukocyte acid phosphatase kit 387-A
(Sigma, MO) and observed under a microscope. To
measure the TRAP activity, the multinucleated cells were
fixed with 10% formalin for 10 min and 95% ethanol for
1 min, and then dried. One hundred microliters of citrate
buffer (50 mM, pH 4.6) containing 10 mM sodium tartrate
and 5 mM p-nitrophenylphosphate (Sigma) was added to
the dried cells. After incubation for 30 min, the enzyme
reaction mixtures were transferred into the well of fresh
plates containing 100 ll of 0.1 N NaOH. Absorption was
measured at 410 nm with Wallac EnVision HTS micro-
plate reader (Perkin-Elmer, Finland). All experiments
were performed in triplicate.
O
1
2
R=H
R=OH
O
2a R=CH3CH2CO2
2b R=PhCO2
2c R=HOCH2CH2O
R
TRAP activity (IC50, lM)a
Compound
1
18.17
12.85
16.68
19.13
10.24
2
2a
2b
2c
aIC50 values were calculated by using mean values presented as %
of control.
18. Hatanaka, Y.; Hashimoto, M.; Kurihara, H.; Nakayama,
H.; Kanoka, Y. J. Org. Chem. 1994, 59, 383.
19. Compound 16: 1H NMR (500 MHz, CDCl3) d 1.32 (s,
3H), 1.34 (s, 3H), 1.42–1.47 (m, 2H), 1.63–1.77 (m, 4H),
1.97–2.00 (m, 2H), 2.25 (s, 3H), 2.34 (t, J = 7.6 Hz, 2H),
2.74 (d, J = 12.7 Hz, 1H), 2.99 (dd, J = 12.7, 5.0 Hz, 1H),
3.13–3.24 (m, 2H), 3.28–3.35 (m, 2H), 3.61–3.65 (m, 1H),
3.83–3.87 (m, 1H), 4.21 (m, 2H), 4.34 (m, 1H), 4.50 (m,
1H), 5.41 (br s, 1H), 5.85 (br s, 1H), 7.22 (s, 1H), 7.55 (d,
J = 8.2 Hz, 1H), 7.61 (d, J = 8.2 Hz, 1H). HRMS-FAB
(m/z) [M+H]+ calcd for C31H37N2O7S, 581.2321; found,
581.2325.
Compound 18: 1H NMR (500 MHz, CDCl3) d 1.31 (s,
6H), 1.92–2.02 (m, 2H), 3.25 (s, 3H), 3.12–3.21 (m, 1H),
3.30–3.35 (m, 2H), 3.61–3.65 (m, 1H), 3.88–3.92 (m, 1H),
4.33–4.42 (m, 2H), 4.62 (m, 2H), 6.71 (s, 1H), 6.80 (d,
J = 7.8 Hz, 1H), 6.91 (dd, J = 8.3, 2.5 Hz, 1H), 7.22 (s,
1H), 7.30 (t, J = 8.3 Hz, 1H), 7.56 (d, J = 8.2 Hz, 1H),
7.61 (d, J = 8.2 Hz, 1H). HRMS-FAB (m/z) [M+H]+
calcd for C31H28F3N2O7, 597.1849; found, 597.1849.
Compound 19: 1H NMR (500 MHz, CDCl3) d 1.33 (s,
3H), 1.45 (m, 2H), 1.62–1.90 (m, 8H), 2.27 (s, 3H), 2.36
(t, J = 7.1 Hz, 2H), 2.74 (d, J = 12.7 Hz, 1H), 2.90 (dd,
J = 12.7, 5.0 Hz, 1H), 3.14–3.22 (m, 3H), 3.70 (t,
J = 4.7 Hz, 2H), 4.10 (m, 2H), 4.16–4.23 (m, 3H), 4.32
21. Cell proliferation assay—RAW264.7 cells were plated at
96-well plates at the density of 4 · 103 cells/well in a-MEM
containing 10% FBS in the presence of 100 ng/ml RANKL.
Next day, cells were treated with serially diluted chemicals
and incubated for 3 days. Cell proliferation was then
measured with a Cell Counting Kit-8 (Dojindo Molecular
Technologies, ML) according to the manufacturer’s pro-
tocol. All experiments were performed in triplicate.