Total synthesis and dual PPARα/γ agonist effects of Amorphastilbol and its synthetic derivatives

Article abstract of DOI:10.1016/j.bmcl.2012.04.062

Amorphastilbol (APH-1), isolated from a Robinia pseudoacacia var. umbraculifer seed extract, is a biologically interesting natural trans-stilbene compound with dual peroxisome proliferator-activated receptor (PPAR) α/γ agonist activity. After total synthesis of APH-1 and its derivatives by Pd-catalyzed Suzuki-Miyaura cross-coupling of a common (E)-styryl bromide intermediate and various aromatic trifluoroborate compounds, we biologically evaluated APH-2-APH-12 for PPAR agonist activity. APH-4 and APH-11 were effective PPARα/γ transcriptional activators, compared with APH-1. Therefore, we suggest that APH-4 and APH-11 are novel dual PPARα/γ agonists and are potentially useful for treating type 2 diabetes by enhancing glucose and lipid metabolism.

Full text of DOI:10.1016/j.bmcl.2012.04.062

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4122–4126
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Total synthesis and dual PPAR
and its synthetic derivatives
a/c agonist effects of Amorphastilbol
Taejung Kim a, , Woojung Lee a, , Kyu Hyuk Jeong a, Jung Ho Song a, Soon-Hye Park a, Pilju Choi a,
Su-Nam Kim a, Seokjoon Lee b, Jungyeob Ham a,
⇑
a Natural Medicine Center, Korea Institute of Science and Technology, Gangneung 210-340, Republic of Korea
b Department of Basic Science, Kwandong University College of Medicine, Gangneung 210-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Amorphastilbol (APH-1), isolated from a Robinia pseudoacacia var. umbraculifer seed extract, is a biolog-
ically interesting natural trans-stilbene compound with dual peroxisome proliferator-activated receptor
Received 30 December 2011
Revised 10 April 2012
Accepted 11 April 2012
Available online 19 April 2012
(PPAR)
a/c agonist activity. After total synthesis of APH-1 and its derivatives by Pd-catalyzed Suzuki–
Miyaura cross-coupling of a common (E)-styryl bromide intermediate and various aromatic trifluorobo-
rate compounds, we biologically evaluated APH-2–APH-12 for PPAR agonist activity. APH-4 and APH-11
were effective PPAR
APH-4 and APH-11 are novel dual PPAR
by enhancing glucose and lipid metabolism.
a/c transcriptional activators, compared with APH-1. Therefore, we suggest that
Keywords:
a/c
agonists and are potentially useful for treating type 2 diabetes
Natural product
Amorphastilbol
Type 2 diabetes
Ó 2012 Elsevier Ltd. All rights reserved.
Dual PPARa/c agonists
Suzuki–Miyaura cross-coupling
Type 2 diabetes (T2DM) is characterized by hyperglycemia,
insulin resistance, and relative insulin deficiency and is usually
associated with dyslipidemia, hypertension, atherosclerosis, and
obesity.1 Although its pathophysiology remains incompletely
understood, metabolic defects in the liver, pancreatic b-cells, adi-
pose tissue, and skeletal muscle contribute to the development of
T2DM. Recently, the members of the peroxisome proliferator-acti-
vated receptor (PPAR) subfamily of nuclear receptors have emerged
as key pharmacological targets in the management of T2DM, be-
cause their activation can normalize metabolic dysfunctions and re-
duce some cardiovascular risk factors associated with this disease.
PPAR agonists are effective drugs for improving the metabolic
abnormalities linking hyperlipidemia to diabetes, hyperglycemia,
insulin resistance, and atherosclerosis. Especially, dual PPARa/c
agonists, such as muraglitazar4 and tesaglitazar,5 combine the
plasma triglyceride- and very low-density lipoprotein-lowering
and high-density lipoprotein cholesterol-raising effects of a PPAR
activator with the insulin resistance- and blood glucose level-
reducing and insulin-sensitizing properties of a PPAR activator.
a
c
Therefore, dual PPARa/c agonists are expected to be effective
hypoglycemic and hypolipidemic agents.6
There is growing interest in the therapeutic use of natural prod-
ucts for metabolic syndrome, because such products are consid-
ered to be less toxic and cause fewer side effects. In the field of
drug discovery, natural products and their analogs can be devel-
oped into useful lead compounds by highly efficient bioactivity-
guided fractionation, followed by synthesis of natural product-
PPARs occur as 3 isoforms: PPAR
expressed predominantly in the liver and regulates genes involved
in fatty acid uptake and oxidation. PPAR agonists such as fibrates
have been used for treating hyperlipidemia and ameliorating car-
diovascular disease. PPAR is expressed mainly in adipose tissue
and regulates genes involved in fatty acid uptake and storage
and in glucose homeostasis. PPAR agonists such as thiazolidined-
a, PPARc a is
, and PPARd.2 PPAR
a
c
based libraries.7 Among the dual PPAR
a/c activators, however, nat-
c
ural ligands are generally less potent than biochemically optimized
iones or glitazones have been used for treating insulin resistance
and hyperglycemia. PPARd is expressed in most tissues and regu-
lates genes involved in fatty acid catabolism and macrophage lipid
homeostasis. PPARd agonists such as GW501516 may have a ben-
eficial effect on lipid and lipoprotein metabolism.3
synthetic ligands.8
We previously discovered amorphastilbol (APH-1), having a
C-geranylated 40-dehydroxyresveratrol structure (Fig. 1), as the
most active compound of the Robinia pseudoacacia var. umbraculif-
er (RPU) extract, and explored its effects on the transcriptional
activities of PPARa and PPARc, adipogenesis in 3T3-L1 cells, insulin
resistance-associated disorders in db/db mice (mouse model of
⇑
Corresponding author. Tel.: +82 33 650 3502; fax: +82 33 650 3629.
E-mail address: ham0606@kist.re.kr (J. Ham).
These authors are equally contributed on this work.
T2DM), and hypolipidemia in a high-fat diet model.9 Especially,
APH-1 stimulates PPARa/c transcriptional activity at the lower
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.062

T. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4122–4126
4123
The synthesis of APH-1 and its derivatives (APH-2–APH-12) is
described in Scheme 1. Initially, conversion to the known benzyl
alcohol (6) was readily accomplished starting from methyl 3,
5-dihydroxybenzoate (1) using the literature methods.10a,b After
protection of phenolic alcohols by using MOMCl, general reduction
of the MOM-protected ester (2) produced benzyl alcohol (3). TBS-
mediated protection of benzyl alcohol completed the silyl ether
synthesis. Geranylation of the silyl ether (4) was investigated
under several well-known conditions in the presence of copper
catalysts such as CuI, CuCN, and CuBr-DMS. However, the best re-
sult was obtained only by using geranyl bromide and n-BuLi, with-
out a copper catalyst.10 TBAF-mediated deprotection of the TBS
ether yielded the corresponding alcohol (6) in 2 steps (83% yield).
To construct the trans-stilbene skeleton, (E)-styryl bromide (9), a
common precursor of APH derivatives, was also obtained from
OH
OH
HO
HO
OH
Amorphastilbol
Resveratrol
Figure 1. Structure of amorphastilbol and resveratrol
concentration than previously reported natural PPARs ligands.8
Therefore, we focused on the total synthesis of APH-1 from RPU
seed extract (0.04% yield). Here, we describe the total synthesis
of APH-1 and its derivatives and their biological evaluation as PPAR
transcriptional activators.
OMOM
OMOM
OMOM
OH
b
a
c
OH
OTBS
OH
MOMO
CO2Me
MOMO
MOMO
MOMO
HO
CO2Me
1
2
3
4
OMOM
OMOM
d
e
OTBS
MOMO
5
6
OMOM
OMOM
f
g
Br
Br
MOMO
MOMO
CHO
MOMO
MOMO
7
8
Suzuki−Miyaura
OMOM
cross-coupling
OMOM
i
h
Br
Ar
10
9
j
OH
HO
Ar
CF3
OMe
CF3
CN
F
APH-1
APH-2
APH-8
APH-3
APH-4
APH-5
APH-6
F
O
S
Cl
CHO
OH
APH-10
F
APH-7
APH-9
APH-11
APH-12
Amorphastilbol and its derivatives
Scheme 1. Reagents and conditions: (a) MOMCl, NaH, DMF, rt, 12 h, 97%; (b) LAH, THF, rt, 2 h, 94%; (c) TBSCl, imidazole, DMAP, rt, 5 h, 99%; (d) n-BuLi, THF, 0 °C, 0.5 h, then
geranylbromide, rt, 6 h, 83%; (e) TBAF, THF, rt, 1 h, 99%; (f) PCC, MS 4 Å, CH2Cl2, rt, 3 h, 98%; (g) CBr4, PPh3, Zn dust, CH2Cl2, rt, 1.5 h, 78%; (h) diethyl phosphite, Et3N, DMF, rt,
2 h, 98%; (i) potassium aryltrifluoroborate, 5 mol % of Pd(PPh3)4, K2CO3, EtOH, 60 °C, 6 h; (j) CSA, MeOH, rt, 10 h, 45–75% (2 steps).

4124
T. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4122–4126
Table 1
In vitro PPAR binding and transactivation activity of APH derivatives
a
b
Entry
Compound
TA EC50
(
l
M)
Binding IC50
h-PPAR
(lM)
h-PPARac (%max)
h-PPARcc (%max)
h-PPARc (%max)
NA
a
h-PPARc
1
2
3
4
5
6
7
8
9
10
11
12
Amorphastilbol (APH 1)
APH 2
APH 3
APH 4
APH 5
APH 6
APH 7
APH 8
APH 9
12
19
(66)
(63)
5
14
(83)
(66)
(21)
(20)
(ND)
(ND)
(15)
(17)
(ND)
(ND)
(7)
1.52
2.50
3.33
1.21
3.47
2.48
2.08
7.47
2.82
1.21
2.03
5.41
10.11
ND
0.85
0.63
8.10
0.53
1.20
2.32
0.90
0.54
0.89
0.11
0.65
1.01
ND
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
ND
4
20
6
(ND)
(ND)
(54)
ND
5
ND
11
7
ND
ND
8
(ND)
(ND)
(41)
(68)
(ND)
(ND)
(27)
(ND)
(98)
(68)
(88)
ND
ND
ND
8
(ND)
(ND)
(35)
(ND)
(87)
APH 10
APH 11
APH 12
(ND)
(34)
(24)
6
5
6
14
0.13
(56)
(100)
Wy14643
Troglitazone
GW501516
0.40
(100)
0.30
ND
0.0012
(100)
ND
a
TA (PPARa/c/d transactivation assay) measuring the ligand-mediated luminescence resulting from PPARa/c/d-induced transcription of a luciferase reporter. PPARa/c/d
and the luciferase reporter genes are transfected into CV-1 cells, then treated with test compound. The EC50 values are the mean of two independent experiments in triplicate.
NA denotes inactive where compounds did not shows any fold induction above the basal level shown by vehicle and ND denotes not determined.
b
Binding affinity for PPAR
a
and PPAR
The maximum percent efficacy (%max) of all compound 30
PPARd) normalized to 100%.
c
using TR-FRET (time-resolved fluorescence resonance energy transfer).
c
lM compared to reference compounds (Wy14643 for PPARa, Troglitazone for PPARc and GW501516 for
the alcohol (6). After oxidation with a PCC suspension and by using
molecular sieves (4 Å), geranylated benzaldehyde (7) was con-
verted to dibromoolefine (78% yield) by the Ramirez olefination
reaction. Subsequent treatment with diethyl phosphite followed
by quenching with water produced (E)-styryl bromide (9) with
excellent yield (98%).
Suzuki–Miyaura cross-coupling reaction with various aromatic
trifluoroborate compounds for fixation of the trans-stilbene moiety
and removal of the MOM-protected groups.
The PPAR agonist activity of the APH derivatives was evaluated by
both cell-based and cell-free assays. Wy14643 (a potent PPAR
a li-
gand), troglitazone (a PPAR ligand), and GW501516 (a PPARd
c
Finally, (E)-styryl bromide (9) was cross-coupled with potas-
sium phenyltrifluoroborate under the standard Suzuki–Miyaura
reaction conditions to produce a trans-stilbene. Deprotection of
the MOM ether by treatment with 10-camphorsulfonic acid (CSA)
yielded APH-1. The derivatives, i.e., APH-2 to APH-12, were
similarly synthesized. Their synthesis required only 2 steps from
the common (E)-styryl bromide (9) intermediate: Pd-catalyzed
ligand) were used as positive controls, strongly and dose-depen-
dently activated reporter gene. However, these synthetic ligands
are generally more potent than natural product-derived ligands.8
We therefore attempted the PPAR-agonist activity test of APH
derivatives compared with APH-1 which has reported natural
product-derived dual PPAR
rized in Table 1. All the compounds seemed to have little effect on
a/c
agonists.9 The results are summa-
Figure 2. The effects of APH derivatives on adipocyte differentiation. 3T3-L1 cells were grown in the IDX condition and treated with Wy14643 (10
lM), troglitazone (10 lM)
and APH derivatives (10 M). (A) Morphological changes associated with adipogenesis were assessed by staining the cellular triglyceride deposition with Oil Red O. (B) The
l
stained oil droplets were dissolved in isopropanol and were quantified by measuring the absorbance 520 nm. Relative Oil red O levels were expressed as the fold increase
relative to the control. Data were expressed as mean SD.

T. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4122–4126
4125
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PPARd. However, APH-4 (PPAR
and APH-11 (PPAR , EC50 = 6
tive transcriptional activators of both PPAR
ited similar PPAR agonist activity to but better PPAR
activity than APH-1 (PPAR , EC50 = 4 M; PPAR , EC50 = 5
30 M, APH-11 had higher agonist efficacy (PPAR , 87%; PPAR
98%) than APH-1 (PPAR , 66%; PPAR , 83%), but APH-4 induced cyto-
toxicity. We then examined whether the APH derivatives could di-
rectly bind to PPAR or PPAR by using LanthaScreen™ technology
(Invitrogen) in a TR-FRET PPAR competitive binding assay (Invit-
rogen).11 APH-4 exhibited increased binding affinity (IC50 = 1.21
and 0.53 M, respectively) for PPAR and PPAR compared with
APH-1 (IC50 = 1.52 M and 0.85 M, respectively); APH-11 exhibited
increased binding affinity only for PPAR (IC50 = 0.65 M).
To explore the potential effects of the APH derivatives on adipo-
genesis, 3T3-L1 preadipocytes were differentiated with the deriva-
tives for 8 days in combination with the adipogenic IDX cocktail
(insulin, dexamethasone, and IBMX).12 When preadipocytes differ-
entiated into adipocytes, morphological changes were observed
because of the cytoplasmic accumulation of lipid droplets. Adipo-
cyte differentiation was confirmed by Oil Red O staining to observe
lipid droplet accumulation. Lipid droplet accumulation in adipo-
cytes was significantly greater with the APH-1 treatment than
without the APH-1 treatment (Fig. 2). However, the APH-4 and
APH-11 treatments decreased lipid droplet accumulation to a
greater extent than the APH-1 treatment. APH-4 and APH-11 had
a
l
, EC50 = 4
M; PPAR , EC50 = 5
and PPAR
l
M; PPAR
c
, EC50 = 5
M) were effec-
; they exhib-
agonist
M). At
lM)
a
c
l
a
c
c
a
a
l
c
l
l
a
c,
a
c
a
c
a/c
lM
l
a
c
l
l
c
l
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better PPARa agonist activity but not PPARc agonist activity than
APH-1, leading to the relative reduction in lipid droplet accumula-
tion in 3T3-L1 cells.13
In conclusion, we synthesized APH-1 and its derivatives by Pd-
catalyzed Suzuki–Miyaura cross-coupling of a common (E)-styryl
bromide intermediate and various aromatic trifluoroborate com-
pounds. Synthetic APH-1 stimulated PPAR
ity at a lower concentration than natural APH-1. Further, the APH-1
derivatives APH-4 and APH-11 were effective PPAR transcrip-
tional activators.14,15 Therefore, we suggest that APH-4 and APH-
11 are novel dual PPAR agonists and are potentially useful for
a/c transcriptional activ-
9. Kim, S. N.; Lee, W. J.; Kwon, H. C.; Ham, J.; Ahn, H. R.; Kim, M. S.; Nam, C.-W.;
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metabolism.
Acknowledgment
This research was supported by the KIST Institutional Program
(2Z03550), Republic of Korea.
13. PPARa target genes have been mainly concentrated on hepatocytes and PPARa
activation upregulates CPT1 (carnitine palmitoyltransferase 1) and ACS (acyl-
CoA synthetase) gene expression involved in mitochondrial fatty acid uptake
and oxidation. APH-4 treatment strongly increased CPT1 mRNA expression and
APH-11 treatment strongly increased ACS mRNA expression involved in fatty
acid b-oxidation in HepG2 cells than APH-1. Therefore we suggested that APH-
References and notes
1. (a) Ohtsubo, K.; Chen, M. Z.; Olefsky, J. M.; Marth, J. D. Nat. Med. 2011, 17, 1067;
(b) Donath, M. Y.; Shoelson, S. E. Nat. Rev. Immunol. 2011, 11, 98; (c) Reaven, G.
4 and APH-11 had better PPARa agonist activity than APH-1.

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T. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4122–4126
137.6, 135.9, 133.8, 130.7, 129.0, 128.6, 127.3, 126.3, 124.3, 123.2, 105.2, 105.1,
39.7, 26.6, 24.9, 22.2, 16.8, 15.4. FT-IR (ATR): 3376, 2907, 2850, 1612, 1577,
1448, 1425, 1261, 1156, 1039, 960, 827, 748, 688 cmꢀ1. ESI-MS: m/z calcd for
14. Luciferase reporter gene assay description: CV-1 cells were seeded into 24-well
plates and cultured for twenty four hour before transfection. CV-1 cells were
grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10%
charcoal dextran treated fetal bovine serum (CDT-FBS) and 1% antibiotics.
C
24H28O2 [M+H]+ 348.21, found 348.00. APH 4: 1H NMR (400 MHz, CDCl3) d
7.70 (s, 1H), 7.62 (d, 1H, J = 7.5 Hz), 7.47 (m, 2H), 7.01 (d, 1H, J = 16.0 Hz), 6.98
(d, 1H, J = 16.0 Hz), 6.59 (s, 2H), 5.27 (m, 3H), 5.05 (t, 1H, J = 7.0 Hz), 3.44 (d, 2H,
J = 7.0 Hz), 2.09 (m, 4H), 1.82 (s, 3H), 1.68 (s, 3H), 1.59 (s, 3H). 13C NMR
(100 MHz, CDCl3) d 155.3, 139.6, 138.0, 136.2, 132.2, 129.9, 129.5, 129.1, 127.1,
124.1, 124.0, 123.7, 123.1, 123.0, 121.1, 113.9, 106.7, 39.7, 26.4, 25.7, 22.6,
17.7, 16.2. FT-IR (ATR): 3338, 2917, 1850, 1622, 1574, 1441, 1324, 1163, 1115,
1068, 1039, 1011, 944, 817, 691, 653 cmꢀ1. ESI-MS: m/z calcd for C25H27F3O2
[M+H]+ 416.20, found 416.00. APH 11: 1H NMR (400 MHz, CDCl3) d 7.50 (s, 1H),
7.39 (s, 1H), 6.97 (d, 1H, J = 20.0 Hz), 6.85 (d, 2H, J = 20.0 Hz), 6.62 (s, 1H), 6.51
(s, 2H), 5.27 (t, 1H, J = 5.5 Hz), 5.20 (s, 2H), 5.05 (m, 1H), 3.42 (d, 2H, J = 8.5 Hz),
2.08 (m, 4H), 1.81 (s, 3H), 1.68 (s, 3H), 1.59 (s, 3H). 13C NMR (100 MHz, CDCl3) d
155.2, 143.7, 141.0, 139.4, 136.9, 132.1, 127.8, 124.4, 123.7, 121.3, 118.4, 113.0,
107.4, 106.2, 39.7, 26.4, 25.7, 22.5, 17.7, 16.2. FT-IR (ATR): 3392, 2913, 1584,
1422, 1267, 1153, 1017, 960, 868, 824, 779, 650, 586 cmꢀ1. ESI-MS: m/z calcd
for C22H26O3 [M+H]+ 338.19, found 337.75.
24 h later, a DNA mixture containing PPRE-luciferase reporter plasmid (0.3
lg),
pcDNA3-hPPAR- /- /-d (30 ng) and internal control plasmid pRL-SV-40 (5 ng)
c
a
was transfected using TransFast™ transfection reagent (Promega, Madison,
WI). After 24 h of transfection, the cells were incubated for an additional 24 h
following treatment with positive control or the indicated concentrations of
APHs. The luciferase activity of the cell lysates was measured using Dual-
LuciferaseÒ Reporter Assay System (Promega, Madison, WI), according to the
manufacturer’s instruction. Relative luciferase activity was normalized for
transfection efficiency using the corresponding Renilla luciferase activity.
15. Spectroscopic data of representative compounds. Amorphastilbol (APH 1): 1H NMR
(400 MHz, CDCl3) d 7.48 (d, 2H, J = 7.1 Hz), 7.34 (t, 2H, J = 7.4 Hz), 7.25 (m, 1H),
7.01 (d, 1H, J = 16.5 Hz), 6.94 (d, 1H, J = 16.5 Hz), 6.59 (s, 2H), 5.28 (td, 1H,
J = 7.5, 1.5 Hz), 5.08 (s, 2H), 5.05 (m, 1H), 3.44 (d, 2H, J = 7.1 Hz), 2.10 (m, 4H),
1.82 (s, 3H), 1.68 (s, 3H), 1.59 (s, 3H). 13C NMR (100 MHz, CDCl3) d 155.2, 139.4,
137.2, 136.9, 132.1, 128.7, 128.1, 127.6, 126.5, 123.7, 121.3, 113.3, 106.6, 39.7,
26.4, 25.7, 22.5, 17.7, 16.3. 13C NMR (100 MHz, Acetone-d6) d 156.2, 156.1,