Design, synthesis and biological study of pinacolyl boronate-substituted stilbenes as novel lipogenic inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.05.124

A pilot library of novel 4,4,5,5-tetramethyl-2-(4-substitutedstyrylphenyl)- 1,3,2 dioxaborolane derivatives has been synthesized. 4-(4,4,5,5-Tetramethyl-1, 3,2-dioxaboratophenyl)-methyl triphenylphosphonium bromide 3 was treated with various aldehydes in the presence of 3 equiv of ^tBuONa in DMF, and stirred at room temperature for 4-6 h to yield the corresponding boron-containing stilbene derivatives in 71-94% yields. Several of them, including BF102 and BF175, have the lipogenesis inhibitory effect by suppressing lipogenic gene expression in mammalian hepatocytes. Further, BF102 also inhibits cholesterol biosynthesis by suppressing HMG-CoA reductase gene expression in hepatocytes. Interestingly, our preliminary in vivo data suggests that BF102 has no significant toxicity in mice at the highest possible dose we can administered. Thus, BF102 is a potential lead for the next generation of lipid-lowering drugs.

Full text of DOI:10.1016/j.bmcl.2011.05.124

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5638–5641
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological study of pinacolyl boronate-substituted
stilbenes as novel lipogenic inhibitors
Bhaskar C. Das a,b, , Xiaoping Zhao a,c, Xiang-Ying Tang a,b, Fajun Yang a,c,
⇑
⇑
a Department of Developmental & Molecular Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
b Department of Nuclear Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA
c Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A pilot library of novel 4,4,5,5-tetramethyl-2-(4-substitutedstyrylphenyl)-1,3,2 dioxaborolane derivatives
has been synthesized. 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaboratophenyl)-methyl triphenylphosphonium
bromide 3 was treated with various aldehydes in the presence of 3 equiv of BuONa in DMF, and stirred
at room temperature for 4–6 h to yield the corresponding boron-containing stilbene derivatives in 71–
94% yields. Several of them, including BF102 and BF175, have the lipogenesis inhibitory effect by sup-
pressing lipogenic gene expression in mammalian hepatocytes. Further, BF102 also inhibits cholesterol
biosynthesis by suppressing HMG-CoA reductase gene expression in hepatocytes. Interestingly, our pre-
liminary in vivo data suggests that BF102 has no significant toxicity in mice at the highest possible dose
we can administered. Thus, BF102 is a potential lead for the next generation of lipid-lowering drugs.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 29 April 2011
Revised 29 May 2011
Accepted 31 May 2011
Available online 28 June 2011
t
Keywords:
Lipogenic inhibitors
Boron-containing stilbene derivatives
Sterol regulatory element binding proteins
(SREBPs)
Wittig reaction
Lipid-lowering drugs
Heart disease is the number one cause of death in the United
States. A high level of lipids in blood (hyperlipidemia) greatly in-
creases the risk of heart disease. Thus, understanding the regula-
tion of lipid metabolism is important to public health. Recent
studies have emphasized that transcriptional control of lipogenic
and cholesterogenic enzymes plays a pivotal role in regulating lipid
metabolism.1 Among the transcription factors that are known to
stimulate lipid biosynthesis, the sterol regulatory element binding
proteins (SREBPs) transcription factors are considered as the ‘mas-
ter’ regulators, as they can critically activate the transcription of
several rate-limiting enzymes, such as fatty acid synthase (FAS)
and HMG-CoA reductase (HMGCR), in both fatty acid/triglyceride
and cholesterol biosynthesis pathways.2,3 In addition to genetic de-
fects, environmental factors, such as diets, can modulate the func-
tions of these regulators, causing changes in lipid metabolism
through altering gene expression.4 Moreover, gene transcription
requires not only transcription factors that bind to the specific
DNA sequences, but also transcriptional cofactors that can criti-
cally enhance or suppress the activities of transcription factors.5
The effectiveness of statins, which inhibit HMGCR activity, in pa-
tients with hyperlipidemia suggests that blocking cholesterol bio-
synthesis is an effective approach to treat hyperlipidemia.6
The current limitations for treating hyperlipidemia are that: (1) The
molecular mechanisms of lipid metabolism regulation are still poorly
understood; and (2) Some patients with hyperlipidemia are not
responsive to statins and the adverse effects of statins have been re-
ported.7 Therefore, it is necessary to develop better approaches for
treating hyperlipidemia. So we undertook this project to develop novel
boron-containing stilbene-based compounds and test their potential
as lipogenic inhibitors. Our hypothesis to develop boron-containing
stilbene derivatives is based on two expectations as follows.
First, it is expected that the boron atoms introduced into biologi-
cally active molecular frameworks may interact with a target protein
not only through hydrogen bonds but also through covalent bonds,
and this interaction would produce potent biological activity.8,9 The
use of boron atoms in pharmaceutical drug design possesses a high po-
tential for discovery of new biological activity.10 Among various boron
compounds synthesized, much attention has been paid to boronic acid
containing peptides such as Velcade and DPP-IV inhibitors.11,12 In these
boropeptides, a carboxylic acid has been replaced by a boronic acid
group. These boron-containing compounds could become the basis
for novel drugs for the treatment of heart disease and other health
problems caused by dysregulation of lipid homeostasis.
Second, identification of interacting proteins could lead to dis-
covery of novel regulators in the pathways of lipid biosynthesis
and thereby improve our basic knowledge on regulation of lipid
homeostasis.
In this study, we synthesized a pilot library of pinacolyl boro-
nate-substituted stilbene derivatives. It is noteworthy to mention
⇑
Corresponding authors.
E-mail addresses: bdas@aecom.yu.edu (B.C. Das), Fajun.Yang@Einstein.yu.edu
(F. Yang).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.124

B. C. Das et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5638–5641
5639
that, unlike the commonly employed approach of high throughput
screening of large libraries to identify lead compounds that elicit a
desired phenotypic effect, we are utilizing a Limited Rational
Design approach (LRD). In this approach Instead of screening
10,000–100,000 arrayed compounds, we will generate a small
library of only about 50 or less compounds to identify lead com-
pounds. We have perfected this strategy, and used our ongoing
chemical biology projects for biomarker identification, and for
developing new therapeutic and diagnostic agents.13–21
In the process of synthesizing boron-containing lipogenic
inhibitors, we developed a synthetic methodology to synthesize
boron-containing stilbene derivatives (Fig. 1).17 To synthesize pin-
acolylboronate-substituted stilbene derivatives, we first synthe-
sized compound 3.
this salt 3 using benzaldehyde 4 in presence of base to give com-
pound 5 in 86% yield (Fig. 1). It is noteworthy that the three equiv-
alents of sodium tert-butoxide in DMF at room temperature led to
the highest yield of 5 (Fig. 1).
With this optimized condition in hand, we examined the scope
of the Wittig reaction for the synthesis of pinacolyl boronate-
substituted stilbenes using various aryl aldehydes. Good E:Z stereo
selectivity was attained when lithium hydroxide used as a base to
synthesize our lead molecule BF102 (E:Z >99:1). Detail experimen-
tal procedures and analytical data’s are presented in reference.22
After generalizing the reaction conditions, we synthesized a
small library of pinacolatoester substituted stilbene derivatives
(Fig. 2).
We then tested this small library of boron-containing stilbene
derivatives for their biological activity as lipogenic inhibitors using
the mRNA level of FAS as the readout.23 Treatment of HepG2, a line
The salt 3 was synthesized from the corresponding 2-[40-
(bromomethyl) phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
2 in the presence of 1.01 equiv of triphenylphosphine in acetoni-
trile under refluxing conditions (Fig. 1). Compound 2 was prepared
by refluxing the 4,4,5,5-tetramethyl -2-p-tolyl-1,3,2-dioxaborolane
1, NBS and AIBN in carbon tetrachloride for 12 h. In our initial
attempt 4-(4,4,5,5-tetramethyl-1,3,2-dioxaboratophenyl)-methyl-
triphenylphosphonium bromide 3 was isolated as a white solid
in 92% yield. The minor excess of PPh3 was removed from the prod-
uct by trituration with ether (2 ꢀ 10 mL) and the product was
found to be stable under normal atmospheric conditions. Subse-
quently, we optimized the Wittig reaction of the ylide derived from
of human hepatoma cells, with some of the compounds at 30 lM
resulted in a significant decrease of FAS gene expression as mea-
sured by quantitative RT-PCR (qRT-PCR) (Fig. 3A), although some
compounds, such as BF62, were inactive in this assay (Fig. 3A).
We chose two compounds, BF102 and BF175, for further analysis,
as they appeared to be less toxic to HepG2 cells at the concentra-
tions tested (data not shown).
Since FAS is a rate-limiting enzyme in de novo lipogenesis, we
decided to determine whether inhibition of FAS gene expression
by BF102 could result in a decrease of insulin-induced fatty acid
O
B
O
O
B
tBuONa, DMF
NBS, AIBN,
B
O
O
CCl4
PPh3.Br
CH3CN,
O
reflux, 12 h
82%
3
CHO
2
90 oC, 6 h
92%
4
PPh3.Br
Br
1
r.t., 2 h, 86%
O
B
O
5, E:Z = 74: 26
Figure 1. Synthesis of 4-(4,4,5,5-tetramethyl-1,3,2-di oxaboratophenyl)-methyl triphenylphosphonium bromide 3.
O
B
O
B
NBS, AIBN,
CCl4
reflux, 12 h
O
B
tBuONa, DMF
R1
PPh3.Br
O
O
O
CH3CN,
Br
R1
90 oC, 6 h
2; 82%
1
R2
CHO
R3
r.t., 2 h, 72-85%
PPh3.Br
3; 92%
O
B
O
R2
R3
O
B
O
HO
BF-102
O
O
O
B
H3CO
B
O
H3C
Cl
O
H3CO
H3CO
BF-62
BF-145
O
O
Cl
B
O
B
O
Cl
OH
BF-117
BF-175
Cl
Figure 2. A small library of boron-containing compounds.

5640
B. C. Das et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5638–5641
BF102 to have potent effects at sub-lethal doses. Treatment with
BF102 in hepatocytes resulted in a significant decrease of rate-
limiting enzymes in the synthesis pathways for both fatty acids
and cholesterol at the mRNA levels. Consistent with gene expres-
sion data, BF102 has displayed an inhibitory effect on biosynthesis
of both palmitate and cholesterol. Since SREBP transcription factors
are the key activators of these genes, it is likely that BF102 func-
tions through interfering SREBP functions. However, the precise
mechanism(s) of BF102 actions are currently unclear. Experiments
are ongoing in our laboratory to identify the molecular target(s) of
BF102. According to our data, the IC50 of BF102 is about 30 lM in
cultured cells. Currently, we are designing and synthesizing
BF102 analogs in order to improve the inhibitory effects on lipo-
genic gene expression. Interestingly, our preliminary data in vivo
suggests that BF102 has no significant toxicity in mice at the high-
est possible dose administered. Thus, our novel boron-containing
compounds may become the next generation of lipid-lowering
drugs in treating cardiovascular diseases in humans.
Figure 3. Effects of boron-containing compounds on lipogenic gene expression and
de novo lipogenesis. A. Relative mRNA levels of fatty acid synthase (FAS) (detected
by quantitative RT-PCR) in HepG2 cells treated with 30 lM of the indicated
compounds for 6 h. Cyclophilin B was the invariant control. B. Relative synthesis
rate of palmitate (detected by the deuterium enrichment method) in FAO cells after
12 h of treatment with BF102 (20 lM). Data are the averages of three independent
samples. #p <0.001 versus DMSO (n = 3).
Acknowledgments
This work was supported by AECOM start-up funds (to B.F. & F.Y.)
and a DRTC pilot & feasibility grant (to B.F. & F.Y.) under P60
DK020541 (to Einstein DRTC). The authors thank Drs. Bhavapriya
Vaitheesvaran and Irwin Kurland for measuring biosynthesis of
palmitate and cholesterol, and Dr. Jeffrey E. Pessin for his support
and advice.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.05.124.
References and notes
Figure 4. BF102 inhibits cholesterol biosynthesis. A. Relative mRNA levels of HMG-
CoA reductase (HMGCR) (detected by quantitative RT-PCR) in HepG2 cells treated
with the indicated concentrations of BF102 for 6 h. Cyclophilin B was the invariant
control. B. The synthesis rate of cholesterol (detected by the deuterium enrichment
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synthesis. Consistent with our gene expression results, using the
deuterium enrichment method followed by gas chromatography
and by mass spectrometry (GC/MS), we found that 20 lM of
BF102 can significantly decrease the synthesis rate of palmitate,
the product of FAS, in rat hepatocytes, FAO cells in the presence
of 100 nM insulin (Fig. 3B). Thus, BF102 can inhibit de novo
lipogenesis.
Next, we wish to test whether BF102 also affect the cholesterol
biosynthesis pathway. Using qRT-PCR, we found that BF102 treat-
ment can dose-dependently decrease the mRNA levels of HMGCR, a
rate-limiting enzyme for cholesterol biosynthesis and the target of
well-known cholesterol lowering drug statins, in HepG2 cells,
while BF62 had no effect at all (Fig. 4A and data not shown). Using
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that 20 lM of BF102 can also significantly decrease the synthesis
rate of cholesterol in FAO cells induced by 100 nM insulin
(Fig. 4B). Thus, BF102 can inhibit de novo cholesterol synthesis.
Together, our datas strongly suggest that BF102 is an inhibitor for
both de novo lipogenesis and cholesterogenesis by down-regulating
SREBP-target genes.
In this study, we have designed and synthesized a series of
novel boron-containing stilbene derivatives and tested their bio-
logical activity as lipogenic inhibitors in mammalian hepatocytes.
From a screen of this small library, we identified one compound
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Tissue culture: HepG2 and FAO cells were cultured at 37 °C and 5% CO2 in
Dulbecco’s modified Eagle’s medium (DMEM; Sigma), supplemented with
100 mg/ml of penicillin–streptomycin (P/S, GIBCO-BRL), 10% fetal bovine
serum (FBS, Hyclone) and 20 mM glutamine.
22.
O
B
Quantitative RT-PCR assay: Total RNA was extracted from cells with Trizol
(Invitrogen) and quantified with an Agilent 2100 Bioanalyzer. For mRNA
quantitation, 2 lg of RNA was converted to cDNA with a High Capacity cDNA
O
B
O
O
LiOH.H2O
iPrOH
O
H
+
Reverse Transcription kit (Applied Biosystems, Foster City, CA), and transcript
levels of relevant genes were determined by real-time, quantitative PCR with
an ABI 7300 Real Time PCR machine according to the manufacturer’s
instructions. (PCR primer information is available upon request)
Quantitative measurement of fatty acids and cholesterol synthesis: Fatty acids and
cholesterol synthesis rate was determined using the deuterium enrichment
method followed by GC/MS analysis. Briefly, cells were cultured in regular
DMEM medium containing 10% 2H2O for 18 h. Fatty acids and cholesterol were
purified by extraction with organic solvents chloroform/methanol and
quantitatively measured by GC/MS at The Stable Isotope & Metabolomics
Core of the Einstein DRTC. The synthesis rates were presented as the
percentage of 2H-containing palmitate or cholesterol in total amount of
palmitate or cholesterol.
BrPh3
P
HO
HO
General procedure for the synthesis of compound BF102: LiOHꢁH2O (0.8 mmol, 34 mg)
was added to the solution of phosphonium salt (0.6 mmol, 335 mg) in iPrOH (3 mL).
The reaction mixture was stirred at room temperature for 15 min. Aldehyde
(0.55 mmol, 67 mg) was added and the reaction was kept stirring at reflux over
night. After the aldehyde was consumed completely, the reaction was quenched
by water, extracted with ethyl acetate. The combined organic layers were dried over
Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel
chromatography to give a white solid product BF102 (52 mg, 29%), E:Z >99:1. Mp
146–148 °C. 1H NMR (CDCl3, 300 MHz) d 1.40 (s, 12H, 4CH3), 5.84 (s, 1H, Ar–OH),
6.85 (d, J = 8.4 Hz, 2H, Ar), 7.00 (d, J = 16.2 Hz, 1H, C@CH), 7.15 (d, J = 16.2 Hz, 1H,
C@CH), 7.43 (d, J = 8.4 Hz, 2H, Ar), 7.50 (d, J = 8.4 Hz, 2H, Ar), 7.80 (d, J = 8.4 Hz,
2H, Ar); 13C NMR (CDCl3, 75 MHz) d 24.8, 83.9, 115.7, 125.6, 126.4, 128.0, 129.2,
130.0, 135.1, 140.4, 155.6.
Statistical analyzes: Data are presented as the means S.D. or averages. The
significance of differences between two groups was evaluated using Student’s t
test. The difference is considered as significant when the p value is less than
0.05.