Synthesis, biological evaluation, and molecular modeling of glycyrrhizin derivatives as potent high-mobility group box-1 inhibitors with anti-heart-failure activity in vivo

Article abstract of DOI:10.1021/jm301248y

Novel glycyrrhizin (GL) derivatives were designed and synthesized by introducing various amine or amino acid residues into the carbohydrate chain and at C-30. Their inhibitory effects on high-mobility group box 1 (HMGB1) were evaluated using a cell-based lipopolysaccharide (LPS) induced tumor necrosis factor α (TNF-α) release study. Compounds 10, 12, 18-20, 23, and 24, which had substituents introduced at C-30, demonstrated moderate HMGB1 inhibition with ED₅₀ values ranging from 337 to 141 μM, which are values comparable to that of the leading GL compound (1) (ED₅₀ = 70 μM). Compounds 23 and 24 emerged as novel and interesting HMGB1 inhibitors. These compounds were able to extend the survival of mice with chronic heart failure (CHF) and acute heart failure (AHF), respectively. In addition, molecular modeling studies were performed to support the biological data.

Full text of DOI:10.1021/jm301248y

Article
pubs.acs.org/jmc
Synthesis, Biological Evaluation, and Molecular Modeling of
Glycyrrhizin Derivatives as Potent High-Mobility Group Box‑1
Inhibitors with Anti-Heart-Failure Activity in Vivo
Dan Du,† Jun Yan,† Jinhong Ren,† Haining Lv, Yong Li, Song Xu, Yadan Wang, Shuanggang Ma, Jing Qu,
Weibin Tang, Zhuowei Hu,* and Shishan Yu*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing 100050, China
S
* Supporting Information
ABSTRACT: Novel glycyrrhizin (GL) derivatives were designed
and synthesized by introducing various amine or amino acid
residues into the carbohydrate chain and at C-30. Their inhibitory
effects on high-mobility group box 1 (HMGB1) were evaluated
using a cell-based lipopolysaccharide (LPS) induced tumor
necrosis factor α (TNF-α) release study. Compounds 10, 12,
18−20, 23, and 24, which had substituents introduced at C-30,
demonstrated moderate HMGB1 inhibition with ED50 values
ranging from 337 to 141 μM, which are values comparable to that of the leading GL compound (1) (ED50 = 70 μM).
Compounds 23 and 24 emerged as novel and interesting HMGB1 inhibitors. These compounds were able to extend the survival
of mice with chronic heart failure (CHF) and acute heart failure (AHF), respectively. In addition, molecular modeling studies
were performed to support the biological data.
High-mobility group box 1 protein (HMGB1) is localized in
the nucleus and can be either passively released from necrotic
cells or actively secreted by activated macrophages/monocytes
to serve as an extracellular signaling molecule involved in acute
and chronic inflammation.6 The investigation of HMGB1 as a
regenerative and proliferative agent in the myocardium was first
suggested by Roberta et al.7,8 HMGB1 has been reported to
stimulate angiogenesis and promote cardiac stem cell growth
and differentiation.9−11 Notably, several HMGB1-inhibiting
agents (e.g., ethyl pyruvate, green tea, and adrenomedullin) can
preserve cardiac function after myocardial ischemia, which is
consistent with the pathogenic role of HMGB1 in cardiovas-
cular diseases.12 HMGB1 has three major structural domains:
two DNA binding motifs, called the A box and B box, and an
acidic carboxyl terminus. It has been revealed that the
proinflammatory activity of HMGB1 localizes to the B box
with the most significant cytokine functionality mapping to the
first 20 amino acid residues of this domain (HMGB1 amino
acids 89−108).13 The A box exerts an antagonistic, anti-
inflammatory effect, perhaps by binding unproductively to
glycation end products (RAGE) and competing with functional
HMGB1.10−12 Hence, the appropriate inhibition of HMGB1 by
targeted binding to the B box has arisen as an efficient
therapeutic strategy for cardiovascular diseases.13,16,17
INTRODUCTION
■
Heart failure (HF) is a serious public health problem that is
characterized by the inability of the heart to sufficiently
distribute blood to meet the needs of the body.1 Common
causes of HF include myocardial infarction and other forms of
ischemic heart disease, hypertension, valvular heart disease, and
cardiomyopathy.2 Echocardiography is commonly used to
support the clinical diagnosis of HF. This modality uses
ultrasound to determine the stroke volume, end diastolic
volume, and ejection fraction. Chronic heart failure (CHF)
represents a long-term condition that usually shows stabilized
symptomatology. Acute heart failure (AHF) refers to episodes
in which a patient displays a change in heart failure signs and
symptoms, resulting in a need for urgent therapy or
hospitalization.3 Although various agents, devices, and surgical
treatments are currently used for HF, few are ideal for the
treatment of its associated pathologies. HF is a common, costly,
disabling, and potentially deadly condition.2 Postmyocardial
infarction remodeling, morbidity, and mortality are highly
dependent on the number of cardiomyocytes lost during
ischemic injury. However, emerging data now provide
convincing evidence that the heart possesses a regenerative
capacity from both endogenous and exogenous sources.4,5
Inflammation, which has emerged as a critical biological process
that contributes to nearly all aspects of cardiovascular diseases
including HF, plays a paradoxical role in cardiac repair and
regeneration. An array of cytokines has been implicated in the
activation of the inflammatory response as well as in the healing
and repair process.4
Natural products play a major role in drug discovery, and
nearly half of the new drugs introduced into the market over
Received: August 29, 2012
© XXXX American Chemical Society
A
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a
Scheme 1. Synthesis of Glycyrrhizin Intermediates 2−4
a
(a) 2% methanolic HCl, rt, 2h, Et3N, 90%; (b) Ac2O−C5H5N, rt, 4h, 72%; (c) (i) ClCOOC2H5, Et3N, THF, −5 °C, 30 min; (ii) NaBH4, H2O, rt,
4h, two steps 58%.
a
Scheme 2. Synthesis of Glycyrrhizin Derivatives 5−8
a
(a) Propylamine, sealed tube, 80 °C, 4 h, 47%; (b) benzylamine, sealed tube, 80 °C, 4 h, 65%; (c) ethanolamine, sealed tube, 80 °C, 4 h, 49%; (d)
butylamine, sealed tube, 80 °C, 4 h, 47%.
the past 2 decades are natural products or their derivatives.18
GL (1) is the major bioactive triterpene glycoside of licorice
root extract and possesses a wide range of pharmacological
properties, especially anti-inflammatory properties. In a recent
study by Mollica et al., GL was found to inhibit HMGB1 by
directly binding (KD ≈ 150 μM) to each of the two HMG
boxes and thus prohibiting its chemoattractant and mitogenic
activities. Furthermore, the moiety of GL that is responsible for
this binding is its triterpene ring, and a molecular model of the
HMGB1−GL interaction has been created.19 In Japan, Sitia et
al. reported that treatment with GL as a HMGB1 inhibitor
diminishes cytotoxic T lymphocyte induced liver disease in
hepatitis B virus (HBV) transgenic mice.20 Recently, we also
found that GL can be used at low concentrations to treat heart
failure by inhibiting HMGB1 (Supporting Information).
Although excessive HMGB1 may play a pathogenic role in
cardiovascular diseases, low levels of HMGB1 might be
beneficial. To the best of our knowledge, the anti-HF activity
of GL in vivo has not been investigated to date. It is thus
important to pharmacologically attenuate (but not completely
abrogate) HMGB1 release to interfere with the progression of
various cardiovascular diseases and to facilitate the recovery of
cardiovascular functions. Therefore, this profile prompted us to
synthesize and evaluate derivatives related to GL to explore the
structural requirements for the appropriate inhibition and
selective binding of the B box.
To identify more potent HMGB1 inhibitors with increased
water solubility, we designed and synthesized a series of GL
derivatives by introducing amino acids into the carbohydrate
chain and at C-30, and their biological activities are described in
B
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a
Scheme 3. Synthesis of Glycyrrhizin Derivatives 9−24
a
(a) PCC, CH2Cl2, 0 °C, rt, 2 h, 73%; (b) (i) NaBH3CN, propylamine, MeOH, rt, 3 h, 53%; (ii) 5% KOH, EtOH/H2O = 1:1, Amberlite 120 H+
resin, 91%; (c) (i) NaBH3CN, isopropylamine, rt, 3 h, 35%; (ii) 5% KOH, EtOH/H2O = 1:1, Amberlite 120 H+ resin, 89%; (d) (i) NaBH3CN,
cyclohexylamine, rt, 3 h, 48%; (ii) 5% KOH, EtOH/H2O = 1:1, Amberlite 120 H+ resin, 94%; (e) (i) NaBH3CN, benzylamine, rt, 3 h, 61%; (ii) 5%
KOH, EtOH/H2O = 1:1, Amberlite 120 H+ resin, 87%; (f) (i) NaBH3CN, cyclopentylamine, rt, 3 h, 38%; (ii) 5% KOH, EtOH/H2O = 1:1,
Amberlite 120 H+ resin, 98%; (g) (i) NaBH3CN, 4-(2-aminoethyl) morpholine, rt, 3 h, 47.3%; (ii) 5% KOH, EtOH/H2O = 1:1, Amberlite 120 H+
resin, 91%; (h) (i) NaBH3CN, 2-pyridylethylamine, rt, 3 h, 38%; (ii) 5% KOH, EtOH/H2O = 1:1, Amberlite 120 H+ resin, 83%; (i) (i) NaBH3CN,
L-Gly-OEt-HCl, MeOH, rt, 3 h, 62%; (ii) 5% KOH, EtOH/H2O = 1:1, Amberlite 120 H+ resin, 96%; (j) (i) NaBH3CN, L-Ala-OMe-HCl, MeOH, rt,
3 h, 45%; (ii) 5% KOH, EtOH/H2O = 1:1, Amberlite 120 H+ resin, 94%; (k) (i) NaBH3CN, L-Ser-OMe-HCl, MeOH, rt, 3 h, 49%; (ii) 5% KOH,
EtOH/H2O = 1:1, Amberlite 120 H+ resin, 86%; (l) (i) NaBH3CN, L-Try-OMe-HCl, MeOH, rt, 3 h, 59%; (ii) 5% KOH, EtOH/H2O =1:1,
Amberlite 120 H+ resin, 94%; (m) (i) NaBH3CN, L-Phe-OMe-HCl, MeOH, rt, 3 h, 66%; (ii) 5% KOH, EtOH/H2O = 1:1, Amberlite 120 H+ resin,
96%; (n) (i)NaBH3CN, L-Tyr-OMe-HCl, MeOH, rt, 3 h, 37%; (ii) 5% KOH, EtOH/H2O = 1:1, Amberlite 120 H+ resin, 89%; (o) (i) (i)
NaBH3CN, L-Glu-(OMe)2-HCl, MeOH, rt, 3 h, 48%; (ii) 5% KOH, EtOH/H2O = 1:1, Amberlite 120 H+ resin, 97%; (p) (i) NaBH3CN, L-Asp-
(OMe)2-HCl, MeOH, rt, 3 h, 44%; (ii) 5% KOH, EtOH/H2O = 1:1, Amberlite 120 H+ resin, 96%.
this report. The inhibition of LPS-induced TNF-α secretion in
RAW264.7 cells by these GL derivatives was tested in vitro.
The results obtained demonstrated that compounds 23 and 24
were potent inhibitors of HMGB1. The anti-HF effects of the
C
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promising GL derivatives were also evaluated based on their
protective effects on cardiac dysfunctions and survival in
doxorubicin (DOX) induced acute and chronic Doll Capital
Management (DCM) mice in vivo.
Table 1. Inhibitory Activities of Selective HMGB1
Antagonists against LPS-Induced TNF-α Release in RAW
264.7 Cells
compound
glycyrrhizin
ED50 (μM)
95% confidence interval (μM)
70−90
70
RESULTS AND DISCUSSION
■
a
a
HMGB1 antibody
2.29
1.7−3.4
Chemistry. The series was designed based on the improved
binding properties between GL and HMGB1 reported by
Mollica et al.19 as well as the chemical modifications of GL
reviewed by Baltina.21 In Mollica’s study, the triterpene
establishes favorable van der Waals interactions with the
HMG boxes; the C-11 carbonyl group and C-30 carboxyl group
of GL create a hydrogen bond or an electrostatic interaction
with the amino acid group of the HMG boxes, and the sugar
moieties point away from the protein. According to Baltina’s
review, elongation of the GL carbohydrate chains or
introduction of amino acids or heterocyclic fragments has
previously been shown to significantly affect the anti-
inflammatory activities of glycosides. Because the carbonyl at
C-11 is an inert site that is sterically hindered, it is not suitable
for derivatization. In this regard, we chose to investigate
whether chemical modifications of the carbohydrate chain and
the C-30 carboxyl group affect the HMGB1 inhibitory activity.
Consequently, amides 5−8 were synthesized and compounds
10−25 contain an amine or amino acid residue with a C−N
bond through the amino group and C-30.
10
12
18
19
20
23
24
226
193
337
161
144
141
144
110−370
150−220
200−660
120−210
80−330
120−180
100−200
a
μg/mL.
GL derivatives 10, 12, 18−20, 23, and 24, ranging from 337 to
141 μM, as compared to the leading compound 1, which has
ED50 = 70 μM. However, GL amides 5−8 showed either low
solubility levels in PBS or high cell cytotoxicity. Briefly, the
introduction of amine residues in the carbohydrate region of
the GL molecule resulted in a loss of activity, indicating that the
free carboxyl at the carbohydrate is necessary. In contrast,
significantly different inhibitory activities were found for
compounds 10−24. Interestingly, the activity of the targeted
compounds 10−16, which contain the alkylamino substituents,
differed and only amines 10 and 12 showed reduced inhibition.
The incorporation of amino acid residues at C-30 resulted in
compounds with reduced anti-HMGB1 properties (18−20, 23,
and 24). No inhibition against HMGB1 was found for
compounds 17, 21, and 22. Of the seven bioactive GL
derivatives, 23 exhibited the highest level of inhibition and 18
showed the lowest level. We speculated that substituents at C-
30 with a certain length and electrical property might be
necessary for bioactivity. It was suggested that a small change at
the C-30 side chain highly impacts the HMGB1 inhibitory
activity.
Pharmacological Activities in Mice. The in vivo profiles
of selected compounds 17−20, 23, and 24 were evaluated using
DOX-induced acute heart failure DCM mice after the ligands
had been administered intraperitoneally (ip) (Figure 1).
Therapeutic blockade of HMGB1 induced protective effects
on cardiac dysfunctions, and the animal survival rate by the GL
derivatives was measured. GL derivative 17 did not possess
anti-HF activity, which was supported by its lack of efficacy in
the HMGB1 release assay, as shown previously (Table 1).
Compound 18 (ED50 = 161 μM) displayed an ip potency
similar to that of the parent compound 1. It did not increase the
survival rate or ejection fraction significantly, but it elicited an
improvement in the maximum ascending and descending rate
of the left ventricle (LV) pressure. As observed in Figure 1A
and Figure 1B, the protective effects on cardiac dysfunctions
and the animal survival rate of GL derivatives 19, 20, and 23
were not significantly higher than that of 1. Treatment of DCM
mice with 24 significantly increased the animal survival rate
(Figure 1A) compared with the DOX plus phosphate-buffered
saline (PBS) treated group and improved cardiac dysfunction as
measured by different parameters (Figure 1B).
Compound 4, the key intermediate for the synthesis of the
target compounds (Scheme 1), was produced by treating the
pentaacetate of the GL dimethyl ester at the diglucuronide
moiety (3) with ethyl chloroformate and subsequently reducing
it with NaBH4 as previously described.22
The synthesis of the amide derivatives of GL (5−8), which
contain two amine residues in the carbohydrate region of the
GL molecule, was carried out by transesterification of the GL
dimethyl ester (4) with the corresponding aqueous amine at 80
°C in a sealed tube as outlined in Scheme 2.
To synthesize amines 10−25, compound 4 was treated with
pyridinium chlorochromate (PCC) in CH2Cl2 as shown in
Scheme 3 to yield the key aldehyde 9. Compounds 10−16 were
obtained from 9 by addition of the corresponding amine and
treatment with NaBH3CN in anhydrous MeOH with the pH
adjusted to ∼6. Furthermore, deprotection of the sugar moiety
provided the desired derivatives. Following the same procedure,
compounds 17−24 were prepared from 9 using the
corresponding alkyl (methyl, ethyl) ester of the amino acid
hydrochloride salt.
In Vitro LPS-Induced HMGB1 Release Studies. The GL
derivatives were tested for the inhibition of HMGB1 stimulated
by LPS. Several studies have demonstrated independently that
HMGB1 can be actively released from a variety of cells,
including macrophage-like RAW 264.7 cells.13 Once released,
HMGB1 can bind to cell-surface receptors [e.g., the receptor
for advanced RAGE, Toll-like receptor (TLR) 2, and TLR4]
and mediates various cellular responses, including chemotactic
cell movement and the release of proinflammatory cytokines
(e.g., TNF and IL-1).6 In other words, HMGB1 can induce
TNF-α release from RAW 264.7 cell cultures activated by LPS.
All of the GL derivatives and antibodies were screened and
assayed for the inhibition of TNF-α production by ELISA
according to the manufacturer’s instructions. All compounds
were tested at least twice in triplicate.
GL derivatives 23 and 24 were evaluated using the DOX-
induced chronic heart failure DCM mouse model. Treatment
with 23 increased the animal survival rate and markedly
ameliorated DOX-induced LV dysfunction, including affecting
the diastolic interventricular septum, systolic interventricular
From a preliminary analysis of the results, which are
summarized in Table 1, general inhibition was observed for
D
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Figure 1. Therapeutic blockade of HMGB1 by GL or its derivatives induces protective effects on cardiac dysfunctions and animal survival in acute
DCM mice. Ten days after DCM (injection of one high dose of DOX) was induced, the animals were treated with the indicated agent once per day.
Fifty days later, the mice were studied and sacrificed. (A) Survival rate curves were generated using the Kaplan−Meier method and compared using a
log rank test (n = 15/group). (B) Effects of GL derivatives on DOX-induced cardiac dysfunctions. EF = ejection fraction; FS = fractional shortening;
+dp/dtmax = maximum ascending rate of LV pressure; −dp/dtmax = maximum descending rate of LV pressure. Data are the mean SEM: (##) P <
0.01 versus PBS-treated group; (∗) P < 0.05, (∗∗) P < 0.01 versus DOX plus PBS-treated group.
septum, ejection fraction, fractional shortening, and LV
diastolic anterior wall (Figure 2). Conversely, 24 did not
show any cardioprotective effect.
consistent with that from the study of Mollica and co-workers,
which found higher chemical-shift differences for these residues
in an NMR assay.19 Derivatives 23 and 24 displayed binding
patterns similar to that of GL because the triterpene scaffold of
23 made hydrophobic interactions with Met12, Tyr15, Ala16,
Val19, Gln20, Arg23, Lys27, and Phe37, and a specific
hydrogen bond was formed between the C-30 carboxyl group
of 23 and the hydroxyl group of Ser41. However, the
electrostatic interaction and hydrogen bonding contact between
the carbonyl group at C-11 and the guanidinium group of
Arg23 were not observed, which may be one of the reasons for
the weaker bonding force of 23 with the A box compared with
GL (Figure 3B). For 24, hydrogen bonding with the hydroxyl
group of Ser41 and hydrophobic interactions with Gly10,
Met12, Ser13, Tyr15, Ala16, Gln20, Arg23, Phe37, and Ser38
were detected. In addition, no electrostatic interactions were
predicted in the model (Figure 3C), which was similar to 23.
Therefore, GL performed best in the interaction with the A
box.
Molecular Docking with the B Box. Phe101, Phe102,
Lys113, Ile121, Ala125, and Lys126 in the active site of the B
box formed hydrophobic interactions with the triterpene
scaffold of GL. Moreover, the carbonyl group at C-11 of GL
formed a hydrogen bond with the guanidinium group of
Arg109 (Figure 4A). Compounds 23 and 24 displayed similar
bonding modes, including hydrogen bonds with Arg109 and
hydrophobic contacts with Phe101, Phe102, Ile121, Val 124,
Ala125, and Lys126, except for additional hydrogen bonds
between their sugar moieties and the amino group of Lys113
(Figure 4B and Figure 4C), which further stabilized these
complexes. Therefore, 23 and 24 exhibited much stronger
bonding forces with the B box than GL, which is the domain of
HMGB1 that is important in cardiovascular disease treatment.
From the molecular modeling study, we can speculate that
GL possesses a stronger bonding force to the A box and a
Compounds 23 and 24 were found to be potent antagonists
of CHF and AHF, respectively, as indicated by the increased
survival rate and amelioration of the cardioprotective effect.
However, both of these compounds showed comparable
inhibition of HMGB1 in vitro. Indeed, the observation of
neither high nor low HMGB1 inhibitory activity in vitro with
cell-based results was associated with anti-HF activity. Thus,
molecular modeling studies were performed to explain the
subtle difference in the interactions between the GL derivatives
and HMG boxes.
Modeling Studies. To get deep insight into the mechanism
for the HMGB1 inhibitory activities of GL, 23, and 24 and the
anti-HF effects of 23 and 24, molecular modeling studies were
performed to explore the detailed interactions of these
compounds with the A box and B box, respectively. The
crystal structures of the A box [Protein Data Bank (PDB) entry
1aab]23 and B box (PDB entry 1hmf)24 were downloaded from
the Protein Data Bank (http://www.rcsb.org/pdb/). Molecular
dockings were performed with Gold 5.1.25 Molecular dynamics
(MD) simulations were then performed on the docking
complexes using the GROMACS package26 to refine the
models.
Molecular Docking with the A Box. The molecular
modeling results revealed that in addition to hydrophobic
interactions between the cycloalkane skeleton of GL with
Met12, Tyr15, Ala16, Val19, Gln20, Lys27, and Phe37, the
carbonyl group at C-11 and the carboxyl group at C-30 of GL
formed two hydrogen bonds with the guanidinium group of
Arg23 and the hydroxyl group of Ser41, respectively. Moreover,
the complex was further stabilized by an electrostatic
interaction between the C-30 carboxyl group of GL and the
hydroxyl group of Tyr15 (Figure 3A). This result is generally
E
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Figure 2. Inhibition of HMGB1 improves cardiac function and animal survival in DOX-induced chronic DCM. (A) Therapeutic protocol for animals
with chronic DCM. Mice were treated with low doses of DOX (3.5 mg/kg) weekly for 8 weeks. Two weeks after the last DOX treatment, the
animals were treated with the indicated agent once per day. (B) Survival rate curves were generated using the Kaplan−Meier method and compared
using a log rank test (n = 20/group). (C) Representative images of LV M-mode echocardiograms. (D) Inhibition of HMGB1 markedly ameliorated
DOX-induced LV dysfunction. IVS-d = diastolic interventricular septum; IVS-s = systolic interventricular septum; EF = ejection fraction; FS =
fractional shortening; LVAW-d = left ventricular diastolic anterior wall. Data are the mean SEM (n = 8/group): (##) P < 0.01, (###) P < 0.001
versus PBS-treated group; (∗) P < 0.05 versus DOX plus PBS-treated group.
Figure 3. Predicted binding modes of GL, 23, and 24 in the active site of the A box. (A) GL (CPK) establishes favorable van der Waals interactions
with the hydrophobic side chains of Met12, Tyr15, Ala16, Val19, Gln20, Lys27, and Phe37 (green). The carbonyl group and carboxyl group of GL
form two stable hydrogen bonds with the guanidinium group of Arg23 and the hydroxyl group of Ser41, respectively (magenta). The complex is
further stabilized by an electrostatic interaction between the carboxyl group of GL and the hydroxyl group of Tyr15. The secondary structure of the
A box is represented in green. (B) The model predicts hydrophobic interactions of the triterpene scaffold of 23 (CPK) with the side chains of
Met12, Tyr15, Ala16, Val19, Gln20, Arg23, Lys27, and Phe37 (green). The carboxyl group of 23 forms a hydrogen bond with the hydroxyl group of
Ser41 (magenta). (C) Similar to 23, hydrogen bonding contacts with the hydroxyl group of Ser41 and hydrophobic interactions with Gly10, Met12,
Ser13, Tyr15, Ala16, Gln20, Arg23, Phe37, and Ser38 were detected for 24 (CPK).
F
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Figure 4. Predicted binding modes of GL, 23, and 24 in the active site of the B box. (A) GL (CPK) establishes favorable van der Waals interactions
with the hydrophobic side chains of Phe101, Phe102, Lys113, Ile121, Ala125, and Lys126 (green). The carbonyl group of GL at C-11 forms
hydrogen bonds with the guanidinium group of Arg109 (magenta). The secondary structure of the B box is represented in green. (B, C) The two
models predict the hydrophobic interactions of the cycloalkane skeletons of 23 and 24 (CPK) with the side chains of Phe101, Phe102, Ile121,
Val124, Ala125, and Lys126 (green). In addition to the hydrogen bond formed between the carbonyl group of GL and the guanidinium group of
Arg109 in the receptor, the sugar moieties of 23 and 24 are predicted to create stable hydrogen bonds with the amino group of Lys113 (magenta).
used to monitor the progression of the reactions. Flash column
chromatography was performed using silica gel (200−400 mesh size,
Qing Dao Marine Chemical Factory, Qingdao, China). The purity of
the test compounds was determined by HPLC analysis using an
Agilent 1100 series instrument equipped with a DAD detector and a
YMC-Pack ODS column (100 mm × 4.6 mm, 5 μm). The intensity of
the major peak in the analytical HPLC trace of each target compound
was ≥95% that of the combined intensities of all of the peaks detected
at 254 nm. Preparative HPLC was performed on a Shimadzu
instrument equipped with a LC-6AD pump and a SPD-10A detector
using a YMC-PACK ODS-A column (250 mm × 20 mm, 5 μm).
Compound 3. The pentaacetate of the GL dimethyl ester at its
diglucuronide moiety was prepared according to the method of Sasaki
relatively weaker bonding force to the B box compared with
compounds 23 and 24. It has been shown that the appropriate
inhibition of HMGB1 by targeted binding to the B box is
efficient for cardiovascular diseases treatment.13,16,17 Thus, 23
and 24 should show a stronger cardioprotective effect than GL,
which is in accordance with the in vivo activity results. Because
the A box exerts an antagonistic effect on the B box, excess
interaction with the A box will weaken the inhibition of the B
box.13−15 GL, which possesses a stronger bonding force to the
A box than 23 and 24, exhibits a higher inhibitory activity on
HMGB1 in vitro and a relatively weaker in vivo effect. In
conclusion, a moderate bonding force with the HMG box A
and relatively higher binding to the B box should result in the
moderate inhibition of HMGB1 and thus a higher animal
survival rate and stronger cardioprotective effect, reflecting the
dual role of HMGB1 in promoting cardiac stem cell growth and
differentiation and inducing myocardial cell apoptosis.
et al.27 White powder (72% yield); H NMR (500 MHz, pyridine) δ
1
5.91(1H, s, H-12), 5.34 (1H, d, J = 8 Hz, H-1″), 4.94 (1H, d, J = 7.5
Hz, H-1′), 3.79 (3H, s, OCH3), 3.67 (3H, s, OCH3), 3.23 (1H, br d, J
= 9 Hz, H-3), 3.03 (1H, br d, J = 12.5 Hz, H-1a), 2.46 (1H, br d, J =
12 Hz, H-18), 2.15, 2.08, 2.01, 1.99, 1.98 (each 3H, s, Ac) 1.36, 1.30,
1.21, 1.18, 1.05, 1.05, 0.75 (each 3H, s, CH3).
Compound 4. This compound was prepared as previously
reported:22 A solution of ethyl chloroformate (1 mL) in THF (5
mL) was added to a solution of the dimethyl ester pentaacetate of GL
(5.4 g) and Et3N (1.4 mL) in THF (25 mL) over the course of 30 min
at −5 °C, and the mixture was stirred for 30 min at the same
temperature. The precipitate was filtered and washed with THF, and
the combined filtrates were added over 30 min to a solution of NaBH4
(0.47 g) in H2O (5 mL) at 10−15 °C. Then the reaction mixture was
stirred at room temperature for 4 h and acidified with diluted HCl; the
two layers of the reaction mixture were subsequently separated. The
organic layer was washed with 10% aqueous NaOH and then dried
over anhydrous Na2SO4. The solvent was evaporated to yield a residue,
which was chromatographed on silica gel (solvent, CHCl3/MeOH =
CONCLUSIONS
■
We have synthesized a series of novel GL derivatives with a C−
N bond through the amino group of amines or amino acids and
C-30. Compounds 23 and 24 emerged as promising HMGB1
inhibitors, facilitating the recovery of the cardiovascular
functions, supported by their strong bonding forces with the
B box. The finding that a small change in the GL structure
produces a profound change in effectiveness of the HF
treatment exemplifies the complexity of the structure−activity
relationships in this series. In light of the potential applications
of the effective inhibitors, it appears worthwhile to further
investigate this new class of compounds with anti-HF activities.
1
100/1) to obtain the product. White powder (58% yield); H NMR
(500 MHz, pyridine) δ 5.78 (1H, s, H-12), 5.38 (1H, d, J = 8 Hz, H-
1″), 4.99 (1H, d, J = 7.5 Hz, H-1′), 3.82 (3H, s, OCH3), 3.78 (1H, dd,
J = 11, 4.5 Hz, H-30a), 3.71 (1H, dd, J = 10.5, 5.5 Hz, H-30b), 3.69
(3H, s, OCH3), 3.26 (1H, dd, J = 11, 4.5 Hz, H-3), 3.11 (1H, br d, J =
13.5 Hz, H-1a), 2.42 (1H, s, H-9), 2.18, 2.06, 2.03, 2.01, 2.00 (each
3H, s, Ac), 1.38, 1.26, 1.23, 1.15, 1.12, 1.09, 0.82 (each 3H, s, CH3);
13C NMR (125 MHz, pyridine) δ 199.5, 170.3, 170.3, 170.1, 169.9,
169.9, 169.7, 168.3, 167.6, 128.5, 103.4, 101.2, 90.2, 77.9, 74.5, 73.1,
73.0, 72.6, 72.0, 70.6, 70.3, 65.4 (C-30), 62.0, 55.4, 52.7, 52.6, 47.3,
45.5, 43.6, 40.8, 39.8, 39.4, 37.2, 36.4, 36.0, 32.9, 32.5, 30.0, 28.7, 28.2,
27.8, 26.9, 26.7, 26.4, 23.5, 20.7, 20.5, 20.5, 20.4, 20.4, 18.8, 17.6, 16.7,
16.5. ESI-MS: m/z = 1067 [M + Na]+.
EXPERIMENTAL SECTION
■
Chemistry. All the starting materials were of reagent grade. The
solvents used for the isolation/purification of the compounds were
obtained from Fisher Scientific (Pittsburgh, PA, U.S.). All reactions
were carried out in oven-dried glassware under an argon atmosphere
unless otherwise noted. All yields reported refer to the yields of the
1
isolated compounds. H NMR and 13C NMR spectra were recorded
using a Varian Inova-500 spectrometer (500 MHz). High-resolution
mass spectra were obtained with an AccuTOF CS (JMS-T100CS,
JEOL) mass spectrometer. ESI-MS was performed with an Agilent
1100 series LC-MSD-Trap-SL mass spectrometer. Silica gel TLC
plates (Qing Dao Marine Chemical Factory, Qingdao, China) were
General Procedures for Converting the Ester to an Amide
for Compounds 5−8. The GL ester (0.1 mmol) was dissolved in
G
dx.doi.org/10.1021/jm301248y | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
amine (2 mL), and the solution was allowed to stand at 80 °C for 4 h
in a sealed tube. The solvent was then evaporated, and the resulting
product was purified by preparative HPLC (CH3CN/H2O = 60:40,
flow rate = 5 mL/min).
The reaction mixture was filtered over a celite pad and evaporated
under reduced pressure to dryness. The residue was purified by flash
column chromatography (petroleum/EtOAc = 1/1, v/v), which
afforded 9 (1.1 g, 73% yield) as a white powder. 1H NMR (500
MHz, pyridine) δ 9.50 (1H, s, CHO), 5.82 (1H, s, H-12), 5.38 (1H, d,
J = 8 Hz, H-1″), 4.99 (1H, d, J = 8 Hz, H-1′), 3.82 (3H, s, OCH3),
3.69 (3H, s, OCH3), 3.26 (1H, dd, J = 11.5, 4.5 Hz, H-3), 3.10 (1H, br
d, J = 13 Hz, H-1a), 2.42 (1H, s, H-9), 2.18, 2.07, 2.03, 2.01, 2.00
(each 3H, s, Ac), 1.41, 1.26, 1.23, 1.12, 1.06, 0.88, 0.72 (each 3H, s,
CH3); 13C NMR (125 MHz, pyridine) δ 206.0 (CHO), 199.4, 170.3,
170.3, 169.9, 169.7, 168.7, 168.3, 167.6, 128.8, 103.4, 101.2, 90.3, 77.9,
74.6, 73.1, 73.0, 72.6, 72.0, 70.6, 70.3, 62.1, 55.4, 52.7, 52.6, 48.0, 47.0,
45.6, 43.4, 39.8, 39.4, 38.5, 37.4, 37.2, 32.8, 32.1, 28.6, 28.4, 27.8, 26.7,
26.4, 26.3, 23.9, 23.6, 20.7, 20.5, 20.5, 20.4, 20.4, 18.8, 17.7, 16.7, 16.5.
General Procedures for Reductive Amination Using Sodium
Cyanoborohydride for Compounds 10−16. The aldehyde
precursor (9, 250 mg) was dissolved in methanol (10 mL), and an
amount of 2 equiv of amine was added. The pH of the solution was
adjusted to ∼6 by adding glacial acetic acid. Then an amount of 1.2
equiv of NaBH3CN in methanol was added. The mixture was stirred at
room temperature for 3 h and quenched with H2O (10 mL). The
solution was extracted with EtOAc, and the combined organic layers
were washed with water, dried over anhydrous Na2SO4, and
concentrated. Purification by preparative HPLC (MeCN/H2O =
9:1) gave the corresponding intermediate. A solution of the
intermediate in 5% potassium hydroxide in EtOH/H2O (1:1, 10
mL) was stirred at room temperature for 1 h. The reaction mixture
was then neutralized with ion-exchange resin (Amberlite IR-120 H+),
and the filtrate was concentrated in vacuo to yield a residue that was
subjected to Sephadex LH-20 (MeOH/H2O = 1:1) to afford the
product.
1
Compound 5. White powder (47% yield); H NMR (500 MHz,
pyridine) δ 8.25 (1H, t, J = 6 Hz, H−N), 8.12 (1H, t, J = 6 Hz, H−N),
5.77 (1H, s, H-12), 5.43 (1H, d, J = 8 Hz, H-1″), 4.84 (1H, d, J = 7.5
Hz, H-1′), 3.78 (1H, d, J = 10.5 Hz, H-30a), 3.72 (1H, d, J = 10.5 Hz,
H-30b), 3.49−3.29 (4H, m, N−CH2 × 2), 3.23 (1H, dd, J = 12, 4.5
Hz, H-3), 3.12 (1H, br d, J = 13.5 Hz, H-1a) 2.45 (1H, s, H-9), 2.26
(1H, dd, J = 13.5, 3.5 Hz), 1.42, 1.29, 1.23, 1.16, 1.10, 1.10, 0.82 (each
3H, s, CH3); 13C NMR (125 MHz, pyridine) δ 199.5, 170.7, 170.2,
170.0, 128.5, 105.6, 104.7, 88.7, 82.2, 77.6, 77.4, 76.5, 76.3, 76.2, 73.8,
73.4, 65.4, 62.1, 55.3, 47.3, 45.6, 43.7, 41.2 (CH2−N), 41.0 (CH2−N),
40.8, 39.9, 39.4, 37.3, 36.4, 36.0, 32.9, 32.5, 30.0, 28.7, 28.3, 28.0, 26.9,
26.8, 26.7, 23.5, 23.4 (CH2−CH3), 23.2 (CH2−CH3), 18.8, 17.6, 16.7,
16.7, 11.7 (CH3), 11.4 (CH3). ESI-MS: m/z = 913 [M + Na]+, 925.5
[M + Cl]−. HRESIMS: m/z = 889.5428 [M − H]− (calcd for
C48H78N2O13, 889.5431).
1
Compound 6. White powder (65% yield); H NMR (500 MHz,
pyridine) δ 9.08 (1H, t, J = 6 Hz, H−N), 8.83 (1H, t, J = 6 Hz, H−N),
7.53 (2H, d, J = 6 Hz, Ar−H), 7.46 (2H, d, J = 6 Hz, Ar−H), 7.34−
7.24 (4H, m, Ar−H), 7.17 (2H, m, Ar−H), 5.78 (1H, s, H-12), 5.41
(1H, d, J = 7.5 Hz, H-1″), 4.85 (1H, d, J = 7.5 Hz, H-1′), 3.78 (1H, d, J
= 10.5 Hz, H-30a), 3.71 (1H, d, J = 10.5 Hz, H-30b), 3.14 (1H, dd, J =
12, 4.5 Hz, H-3), 3.09 (1H, br d, J = 13.5 Hz, H-1a), 2.43 (1H, s, H-9),
2.26 (1H, dd, J = 13.5, 3.5 Hz), 1.42, 1.22, 1.17, 1.15, 1.10, 1.00, 0.83
(each 3H, s, CH3); 13C NMR (125 MHz, pyridine) δ 199.5, 170.7,
170.1, 170.0, 139.8 (Ar−C), 139.6 (Ar−C), 128.9 (Ar−C), 128.9
(Ar−C), 128.9 (Ar−C), 128.9 (Ar−C), 128.5, 128.1 (Ar−C), 128.1
(Ar−C), 127.6 (Ar−C), 127.6(Ar−C), 127.4 (Ar−C), 127.3 (Ar−C),
105.9, 104.8, 88.7, 82.7, 77.7, 77.4, 77.1, 76.9, 76.3, 73.7, 73.2, 65.4,
62.0, 55.3, 47.3, 45.5, 43.6, 43.2 (−CH2−Ar), 42.9 (−CH2−Ar), 40.8,
39.8, 39.5, 37.2, 36.4, 36.0, 32.9, 32.5, 30.0, 28.7, 28.2, 27.9, 26.9, 26.8,
26.8, 23.6, 18.9, 17.6, 16.8, 16.7. ESI-MS: m/z = 1009 [M + Na]+, 985
[M − H]−. HRESIMS: m/z = 985.5417 [M − H]− (calcd for
C56H77N2O13, 985.5431).
1
Compound 10. White powder (48% yield); H NMR (500 MHz,
DMSO) δ 5.57 (1H, s, H-12), 4.39 (1H, d, J = 7.5 Hz, H-1″), 4.28
(1H, d, J = 7.5 Hz, H-1′), 2.27 (1H, s, H-9), 2.10−2.05 (2H, m), 1.30,
1.02, 1.02, 0.94, 0.94 (each 3H, s, CH3), 0.87 (3H, t, J = 7 Hz), 0.81,
0.73 (each 3H, s, CH3); 13C NMR (125 MHz, DMSO) δ 199.0, 172.4,
171.6, 169.3, 127.7, 103.9, 103.4, 87.9, 81.7, 76.3, 75.9, 75.3, 73.6, 72.0,
72.0, 61.2, 54.5, 51.8 (N−CH2), 50.6 (C-30), 45.7, 44.9, 43.0, 41.2,
40.8, 38.9, 36.4, 35.3, 33.5, 32.2, 31.9, 28.1, 27.7, 27.4, 26.1, 26.1, 26.0,
25.4, 23.2, 23.2 (CH2−CH3), 18.4, 17.0, 16.2, 16.2, 11.4 (CH3).
HRESI-MS: m/z = 850.4967 (calcd for C45H72NO14, 850.4947). ESI-
MS: m/z = 850 [M + H]+, 848 [M − H]−.
1
Compound 7. White powder (49% yield); H NMR (500 MHz,
pyridine) δ 8.41 (1H, t, J = 5.5 Hz, H−N), 8.37 (1H, t, J = 5.5 Hz, H−
N), 5.76 (1H, s, H-12), 5.40 (1H, d, J = 8 Hz, H-1″), 4.85 (1H, d, J =
7.5 Hz, H-1′), 3.95−3.69 (10H, m), 3.23 (1H, dd, J = 11.5, 4.5 Hz, H-
3), 3.08 (1H, br d, J = 13.5 Hz), 2.40 (1H, s, H-9), 2.26 (1H, dd, J =
13.5, 4 Hz), 1.39, 1.27, 1.24, 1.16, 1.13, 1.07, 0.82 (each 3H, s, CH3);
13C NMR (125 MHz, pyridine) δ 199.42, 171.0, 170.7, 169.9, 128.5,
105.8, 104.6, 88.8, 82.6, 77.5, 77.3, 76.4, 76.2, 75.7, 73.8, 73.4, 65.4,
62.0, 61.1 (O−CH2), 60.8 (O−CH2), 55.3, 47.3, 45.5, 43.6, 42.7 (N−
CH2), 42.3 (N−CH2), 40.8, 39.9, 39.5, 37.2, 36.4, 36.0, 32.9, 32.5,
30.0, 28.7, 28.2, 27.9, 26.9, 26.8, 26.7, 23.5, 18.8, 17.6, 16.7, 16.6. ESI-
MS: m/z = 893.5 [M − H]−. HRESIMS: m/z = 893.5004 [M − H]−
(calcd for C46H73N2O15, 893.5016).
1
Compound 11. White powder (31% yield); H NMR (500 MHz,
DMSO) δ 5.50 (1H, s, H-12), 4.43 (1H, d, J = 7.5 Hz, H-1″), 4.28
(1H, d, J = 7.5 Hz, H-1′), 2.29 (1H, s, H-9), 1.32, 1.05 (each 3H, s,
CH3), 1.03 (6H, overlap, CH3 × 2), 1.02, 0.96, 0.86, 0.80, 0.72 (each
3H, s, CH3); 13C NMR (125 MHz, DMSO) δ 199.1, 172.8, 171.6,
169.9, 127.4, 103.5, 103.2, 88.0, 80.4, 76.4, 76.1, 75.1, 73.7, 72.2, 72.2,
61.1, 54.4, 49.7 (N−CH), 49.4 (C-30), 46.2, 44.9, 43.0, 41.50, 40.8,
38.7, 36.4, 35.6, 33.8, 32.2, 31.9, 30.7, 28.3, 28.1, 27.5, 26.1, 26.0, 25.4,
23.14, 21.7 (CH3), 21.7 (CH3), 18.4, 17.0, 16.20, 16.2. HRESI-MS: m/
z = 850.4960 (calcd for C45H72NO14, 850.4947). ESI-MS: m/z = 850
[M + H]+, 848 [M − H]−.
1
Compound 8. White powder (47% yield); H NMR (500 MHz,
pyridine) δ 8.22 (1H, t, J = 6 Hz, H−N), 8.09 (1H, t, J = 6 Hz, H−N),
5.77 (1H, s, H-12), 5.43 (1H, d, J = 7.5 Hz, H-1″), 4.85 (1H, d, J = 8
Hz, H-1′), 3.77 (1H, d, J = 10.5 Hz, H-30a), 3.71 (1H, d, J = 10.5 Hz,
H-30b), 3.51−3.34 (4H, m, N−CH2 × 2), 3.23 (1H, dd, J = 12, 4.5
Hz, H-3), 3.13 (1H, br d, J = 13.5 Hz, H-1a), 2.46 (1H, s, H-9), 2.25
(1H, dd, J = 13, 3.5 Hz), 1.42, 1.30, 1.24, 1.15, 1.11, 1.10 (each 3H, s,
CH3), 0.85 (3H, t, J = 7.5 Hz, CH3), 0.82 (3H, s, CH3) 0.78 (3H, t, J =
7.5 Hz, CH3); 13C NMR (125 MHz, pyridine) δ 199.4, 170.6, 170.1,
170.0, 128.5, 105.7, 104.7, 88.7, 82.2, 77.6, 77.4, 76.5, 76.3, 76.2, 73.8,
73.4, 65.4 (30), 62.1, 55.3, 47.3, 45.6, 43.6, 40.8, 39.9, 39.5, 39.2 (N−
CH2), 39.0 (N−CH2), 37.3 (22), 36.4 (20), 36.0, 32.9, 32.5, 32.3 (N−
CH2−CH2−), 31.9 (N−CH2−CH2−), 30.0 (21), 28.7, 28.2, 28.0,
26.9, 26.9, 26.7, 23.6, 20.5 (CH3−CH2−), 20.2 (CH3−CH2−), 18.8,
17.6, 16.8, 16.8, 14.0 (CH3), 13.9 (CH3). ESI-MS: m/z = 917.6 [M −
H]−. HRESIMS: m/z = 917.5740 [M − H]− (calcd for C50H81N2O13,
917.5744).
1
Compound 12. White powder (45% yield); H NMR (500 MHz,
DMSO) δ 5.58 (1H, s, H-12), 4.41 (1H, d, J = 7.5 Hz, H-1″), 4.30
(1H, d, J = 7.5 Hz, H-1′), 2.27 (1H, s, H-9), 1.31, 1.02, 1.02, 0.95,
0.93, 0.82, 0.70 (each 3H, s, CH3); 13C NMR (125 MHz, DMSO) δ
199.0, 171.8, 170.7, 169.3, 127.7, 104.6, 103.5, 88.0, 82.5, 76.2, 75.9,
75.7, 75.2, 74.1, 71.8, 71.8, 61.2, 57.6 (N−CH), 54.5, 48.4, 45.6, 43.0,
40.9, 40.8, 38.8, 36.3, 35.2, 33.2, 32.1, 31.9, 30.1, 28.6, 28.0, 27.3
(CH2), 27.3 (CH2), 26.0, 25.9, 25.5, 25.0 (CH2), 24.5 (CH2), 24.5
(CH2), 23.2, 18.4, 17.0, 16.3, 16.1. HRESI-MS: m/z = 890.5275 (calcd
for C48H76NO14, 890.5260). ESI-MS: m/z = 890 [M + H]+, 888 [M −
H]−.
1
Compound 13. White powder (53% yield); H NMR (500 MHz,
pyridine) δ 7.63 (2H, m, Ar−H), 7.39 (2H, m, Ar−H), 7.33 (1H, m,
Ar−H), 5.66 (1H, s, H-12), 5.43 (1H, d, J = 7.5 Hz, H-1″), 5.04 (1H,
d, J = 7.5 Hz, H-1′), 4.63−4.26 (10H, m), 3.31 (1H, br d, J = 11.5 Hz,
H-3), 3.06 (1H, br d, H-30a), 3.01 (1H, br d, J = 13 Hz, H-1a), 2.87
(1H, br d, J = 11.5 Hz, H-30), 2.35 (1H, s, H-9), 2.28 (1H, m), 1.39,
Compound 9. To a solution of 4 (1.5 g) in CH2Cl2 (8 mL) was
added PCC (659 mg) at 0 °C. After being stirred for 1 h at 0 °C, the
reaction mixture was warmed to room temperature and stirred for 1 h.
H
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Journal of Medicinal Chemistry
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1.30, 1.23, 1.18, 1.13, 0.97, 0.69 (each 3H, s, CH3); 13C NMR (125
MHz, pyridine) δ 199.4, 172.5, 172.2, 168.6, 134.8 (Ar−C), 130.1
(Ar−C), 130.1 (Ar−C), 129.2 (Ar−C), 129.2 (Ar−C), 128.8 (Ar−C),
128.8, 106.9, 105.1, 89.1, 84.5, 78.5, 77.7, 77.6, 77.5, 76.9, 73.3, 73.1,
62.0, 55.4, 52.8 (Ar−CH2−N), 51.7 (C-30), 46.8, 45.4, 43.5, 42.1,
39.9, 39.4, 37.2, 35.9, 34.1, 32.8, 32.2, 30.6, 28.4, 28.1, 27.8, 26.7, 26.6,
26.5, 23.4, 18.7, 17.6, 16.8, 16.7. HRESI-MS: m/z = 898.4924 (calcd
for C49H72NO14, 898.4947). ESI-MS: m/z = 898 [M + H]+, 896 [M −
H]−.
(each 3H, s, CH3); 13C NMR (125 MHz, DMSO) δ 199.2, 170.5,
170.1, 169.2, 167.5 (CO), 127.9, 104.8, 103.6, 88.2, 82.7, 76.3, 76.0,
75.7, 75.5, 75.0, 71.6, 71.4, 61.1, 54.4, 52.4 (N−CH2−CO), 50.1 (C-
30), 45.3, 44.9, 42.9, 39.9, 39.0, 38.6, 36.3, 35.2, 33.4, 32.2, 31.8, 31.0,
28.1, 27.3, 27.2, 26.0, 26.0, 25.7, 23.2, 18.4, 17.0, 16.2, 16.0. HRESI-
MS: m/z = 866.4545 (calcd for C44H68NO16, 866.4533). ESI-MS: m/z
= 866 [M + H]+, 864 [M − H]−.
1
Compound 18. White powder (42% yield); H NMR (500 MHz,
DMSO) δ 5.82 (1H, s, H-12), 4.45 (1H, d, J = 7.5 Hz, H-1″), 4.33
(1H, d, J = 7.5 Hz, H-1′), 2.84 (1H, br d, J = 12.5 Hz), 2.58 (1H, m),
2.28 (1H, s, H-9), 1.32 (3H, s, CH3), 1.31 (3H, d, J = 8 Hz, CH-CH3),
1.02, 1.02, 0.95, 0.93, 0.79, 0.70 (each 3H, s, CH3); 13C NMR (125
MHz, DMSO) δ 199.2, 170.3, 170.1, 169.2, 167.9 (CO), 127.9, 104.7,
103.6, 88.2, 82.6, 76.1, 75.7, 75.5, 75.0, 72.7, 71.5, 61.2, 58.3 (N−CH−
CO), 54.4, 51.2 (C-30), 45.2, 44.9, 42.9, 39.9, 39.0, 38.6, 36.3, 35.4,
33.4, 32.2, 31.8, 31.6, 28.2, 27.4, 27.2, 26.0, 26.0, 25.6, 23.2, 18.4, 17.0,
16.4 (CH3), 16.2, 16.0. HRESI-MS: m/z = 880.4687 (calcd for
C45H70NO16, 880.4689). ESI-MS: m/z = 880 [M + H]+, 878 [M −
H]−.
1
Compound 14. White powder (37% yield); H NMR (500 MHz,
pyridine) δ 5.77 (1H, s, H-12), 5.42 (1H, d, J = 7.5 Hz, H-1″), 5.03
(1H, d, J = 7.5 Hz, H-1′), 4.62−4.26 (8H, m), 3.35 (1H, br d, J = 8
Hz, H-3), 3.28 (1H, m, H-30a), 3.04 (1H, br d, J = 12.5 Hz, H-1a),
2.77 (1H, m), 2.72 (1H, m), 2.40 (1H, s, H-9), 2.20−2.00 (5H, m),
1.38, 1.35, 1.24, 1.20, 1.07, 1.04, 0.85 (each 3H, s, CH3); 13C NMR
(125 MHz, pyridine) δ 199.5, 173.4, 172.2, 167.3, 128.7, 107.0, 105.1,
88.9, 84.7, 78.4, 77.8, 77.8, 77.6, 76.9, 73.4, 73.3, 62.1, 60.8 (N−CH),
55.4, 52.4 (C-30), 47.1, 45.5, 43.6, 42.1, 39.9, 39.6, 37.2, 36.3, 34.4,
32.9, 32.4, 32.4 (CH2), 32.2 (CH2), 31.1, 28.7, 28.5, 28.1, 26.8, 26.7,
26.6, 24.3 (CH2), 24.3 (CH2), 23.5, 18.7, 17.6, 16.9, 16.8. HRESI-MS:
m/z = 876.5100 (calcd for C47H74NO14, 876.5104). ESI-MS: m/z =
876 [M + H]+, 874 [M − H]−.
1
Compound 19. White powder (42% yield); H NMR (500 MHz,
DMSO) δ 5.72 (1H, s, H-12), 4.46 (1H, d, J = 7.5 Hz, H-1″), 4.37
(1H, d, J = 7.5 Hz, H-1′), 2.90 (1H, br d, J = 12 Hz), 2.64 (1H, br d, J
= 12.5 Hz), 2.54 (1H, br d, J = 13 Hz), 2.29 (1H, s, H-9), 2.21 (1H,
m), 2.07 (1H, m), 1.32, 1.02, 1.02, 0.95, 0.93, 0.80, 0.70 (each 3H, s,
CH3); 13C NMR (125 MHz, DMSO) δ 199.2, 170.6, 170.1, 169.6,
169.3 (CO), 127.7, 104.7, 103.5, 88.2, 82.7, 76.2, 76.0, 75.7, 75.2, 75.0,
71.6, 71.4, 64.6 (CH2OH), 61.1, 60.6 (N−C−CO), 54.3, 51.7 (C-30),
45.4, 44.9, 42.9, 39.9, 39.0, 38.6, 36.3, 35.4, 33.6, 32.2, 31.8, 31.3, 28.2,
27.5, 27.2, 26.0, 26.0, 25.7, 23.2, 18.4, 17.0, 16.3, 16.0. HRESI-MS: m/
z = 896.4653 (calcd for C45H70NO16, 896.4638). ESI-MS: m/z = 896
[M + H]+, 894 [M − H]−.
1
Compound 15. White powder (42% yield); H NMR (500 MHz,
pyridine) δ 5.77 (1H, s, H-12), 5.44 (1H, d, J = 7.5 Hz, H-1″), 5.05
(1H, d, J = 7.5 Hz, H-1′), 4.67−4.25 (8H, m), 3.64 (2H, br s), 3.46
(2H, m), 3.32 (1H, br d, J = 11.5), 3.16 (1H, br s), 3.01 (1H, br d, J =
13.5 Hz), 2.79 (2H, br s), 2.43 (2H, m), 2.37 (1H, s, H-9), 2.30−2.21
(2H, m), 2.09−1.97 (2H, m), 1.84−1.03 (4H, m), 1.40, 1.32, 1.24,
1.18, 1.00, 1.00, 0.83 (each 3H, s, CH3); 13C NMR (125 MHz,
pyridine) δ 199.3, 172.4, 172.1, 168.3, 128.9, 107.0, 105.1, 89.1, 84.6,
78.5, 77.7, 77.6, 77.5, 76.9, 73.3, 73.1, 66.8 (CH2−O−CH2), 66.8
(CH2−O−CH2), 62.0, 55.3, 54.0 (HN−CH2−CH2−N), 53.7 (CH2−
N−CH2), 53.7 (CH2−N−CH2), 52.5 (HN−CH2−CH2−N), 46.7,
45.5, 45.2 (C-30), 43.5, 42.4, 39.9, 39.4, 37.1, 35.9, 34.1, 32.8, 32.3,
30.1, 28.3, 28.0, 27.7, 26.7, 26.5, 26.4, 23.4, 18.7, 17.5, 16.8, 16.7.
HRESIMS: m/z = 921.5227 (calcd for C55H73N2O10, 921.5260). ESI-
MS: m/z = 921 [M + H]+, 919 [M − H]−.
1
Compound 20. White powder (55% yield); H NMR (500 MHz,
DMSO) δ 10.8 (1H, s, N−H), 7.55 (1H, d, J = 8 Hz, Ar−H), 7.31(1H,
d, J = 8 Hz, Ar−H), 7.15 (1H, br s, Ar−H), 7.04 (1H, t, J = 7, 8 Hz,
Ar−H), 6.96 (1H, t, J = 7, 8 Hz, Ar−H), 5.55 (1H, s, H-12), 4.46 (1H,
d, J = 7.5 Hz, H-1″), 4.36 (1H, d, J = 7.5 Hz, H-1′), 2.64 (1H, br d, J =
11.5 Hz), 2.54 (1H, br d, J = 13 Hz), 2.27 (1H, s, H-9), 2.02 (1H, m),
1.94 (1H, m), 1.29, 1.01, 1.00, 0.93, 0.81, 0.70, 0.70 (each 3H, s, CH3);
13C NMR (125 MHz, DMSO) δ 199.1, 173.0 (CO), 170.6, 170.1,
169.5, 136.2 (Ar−C), 127.5, 127.3 (Ar−C), 123.8 (Ar−C), 121.0
(Ar−C), 118.4 (Ar−C), 118.4 (Ar−C), 111.5 (Ar−C), 110.1 (Ar−C),
104.2, 103.5, 88.2, 82.6, 76.2, 76.0, 75.7, 75.2, 75.0, 71.6, 71.4, 63.1
(N−CH−CO), 61.1, 54.4, 51.7 (C-30), 45.7, 44.9, 42.9, 39.9, 39.0,
38.5, 36.3, 35.3, 33.8, 32.2, 31.7, 30.9 (CH−CH2), 28.1, 27.6, 27.5,
27.2, 26.0, 26.0, 25.7, 23.1, 18.4, 17.0, 16.3, 16.0. HRESI-MS: m/z =
995.5143 (calcd for C53H75N2O16, 995.5151). ESI-MS: m/z = 995 [M
+ H]+, 993 [M − H]−.
1
Compound 16. White powder (31% yield); H NMR (500 MHz,
pyridine) δ 8.64 (1H, m, Ar−H), 7.12 (1H, m, Ar−H), 5.76 (1H, s, H-
12), 5.42 (1H, d, J = 7.5 Hz, H-1″), 5.04 (1H, d, J = 7.5 Hz, H-1′),
4.62−4.28 (8H, m), 3.33 (3H, m), 3.17 (3H, m), 3.03 (1H, br d, J =
11.5 Hz), 2.82 (1H, m), 2.39 (1H, s, H-9), 1.37, 1.35, 1.24, 1.20, 1.08,
1.03, 0.81 (each 3H, s, CH3); 13C NMR (125 MHz, pyridine) δ 199.4,
172.3, 172.2, 169.3, 161.9 (Ar−C), 149.4 (Ar−C), 136.9 (Ar−C),
128.6, 123.9 (Ar−C), 121.9 (Ar−C), 107.0, 105.1, 89.0, 84.7, 78.4,
77.7, 77.6, 77.5, 77.0, 73.4, 73.1, 62.1, 55.4, 53.4 (N−CH2), 47.0, 46.2
(C-30), 45.5, 43.5, 41.9, 40.0, 39.6, 37.2 (CH2−Ar), 37.2, 36.2, 34.6,
32.9, 32.4, 32.0, 28.6, 28.6, 28.1, 26.8, 26.8, 26.6, 23.6, 18.8, 17.6, 16.9,
16.8. HRESIMS: m/z = 913.5055 (calcd for C49H73N2O14, 913.5056).
ESI-MS: m/z = 913 [M + H]+, 911 [M − H]−.
1
Compound 21. White powder (63% yield); H NMR (500 MHz,
pyridine) δ 7.51 (2H, t, J = 6 Hz, Ar−H), 7.29 (2H, m, Ar−H), 7.23
(1H, overlap, Ar−H), 5.91 (1H, s, H-12), 5.44 (1H, d, J = 7.5 Hz, H-
1″), 5.06 (1H, d, J = 7.5 Hz, H-1′), 4.67−4.56 (4H, m), 4.42 (1H, t),
4.35−4.25 (3H, m), 3.33 (3H, m), 3.03 (2H, m), 2.38 (1H, s, H-9),
2.29 (1H, dd, J = 10, 4 Hz), 2.23 (1H, br d, J = 14 Hz), 1.40, 1.32,
1.24, 1.18, 1.02, 0.98, 0.72 (each 3H, s, CH3); 13C NMR (125 MHz,
pyridine) δ 199.5, 176.7 (CO), 172.4, 172.1, 169.5, 135.8 (Ar−C),
130.0 (Ar−C), 130.0 (Ar−C), 128.6, 128.6 (Ar−C), 128.6 (Ar−C),
126.8 (Ar−C), 107.0, 105.1, 89.2, 84.5, 78.5, 77.7, 77.6, 77.5, 76.9,
73.3, 73.1, 64.7 (N−CH−CO), 62.0, 55.4, 52.9 (C-30), 46.6, 45.5,
43.4, 41.1, 40.0 (CH−CH2), 39.9, 39.5, 37.2, 36.2, 34.6, 32.9, 32.3,
31.9, 28.6, 28.6, 28.1, 26.8, 26.8, 26.7, 23.5, 18.7, 17.6, 16.8, 16.7.
HRESI-MS: m/z = 956.4999 (calcd for C51H74NO16, 956.5002). ESI-
MS: m/z = 954 [M − H]−.
General Procedures for Reductive Amination Using Sodium
Cyanoborohydride for Compounds 17−24. The aldehyde
precursor (9, 250 mg) was dissolved in methanol (10 mL), and an
amount of 2 equival of L-amino acid methyl (ethyl) ester hydro-
chloride was added, followed by the addition of 1.2 equiv of
NaBH3CN in methanol. The mixture was stirred at room temperature
for 3 h and quenched with water (10 mL). The solution was extracted
with CH2Cl2, and the combined organic layers were washed with
water, dried over anhydrous Na2SO4, and concentrated. Purification by
preparative HPLC (MeCN/H2O = 9:1) gave the corresponding
intermediate. A solution of the intermediate in 5% potassium
hydroxide in EtOH/H2O (1:1, 10 mL) was stirred at room
temperature for 1 h. The reaction mixture was then neutralized with
ion-exchange resin (Amberlite IR-120 H+), and the filtrate was
concentrated in vacuo to yield a residue that was subjected to
Sephadex LH-20 (MeOH/H2O = 1:1) to afford the product.
1
Compound 22. White powder (33% yield); H NMR (500 MHz,
pyridine) δ 7.45 (2H, d, J = 8 Hz, Ar−H), 7.12 (2H, d, J = 8 Hz, Ar−
H), 5.97 (1H, s, H-12), 5.44 (1H, d, J = 7.5 Hz, H-1″), 5.05 (1H, d, J =
7.5 Hz, H-1′), 4.67−4.56 (4H, m), 4.42 (1H, t), 4.35−4.25 (3H, m),
3.40−3.30 (3H, m), 3.04 (2H, m), 2.79 (1H, m), 2.37 (1H, s, H-9),
2.27 (2H, m), 2.09−1.95 (2H, m), 1.39, 1.31, 1.23, 1.18, 1.10, 0.97,
0.72 (each 3H, s, CH3); 13C NMR (125 MHz, pyridine) δ 199.6, 176.3
(CO), 172.4, 172.1, 169.6, 157.7 (Ar−C), 135.1 (Ar−C), 131.2 (Ar−
1
Compound 17. White powder (59% yield); H NMR (500 MHz,
DMSO) δ 5.73 (1H, s, H-12), 4.45 (1H, d, J = 7.5 Hz, H-1″), 4.37
(1H, d, J = 7.5 Hz, H-1′), 2.74(1H, br d, J = 11 Hz), 2.54 (1H, br d, J
= 12 Hz), 2.28 (1H, s, H-9), 1.31, 1.02, 1.02, 0.96, 0.93, 0.80, 0.70
I
dx.doi.org/10.1021/jm301248y | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
C), 131.2 (Ar−C), 128.7, 116.2 (Ar−C), 116.2 (Ar−C), 107.0, 105.1,
89.1, 84.6, 78.5, 77.7, 77.6, 77.5, 76.9, 73.3, 73.0, 65.0 (N−C−CO),
62.0, 55.4, 52.9 (C-30), 46.6, 45.5, 43.4, 41.3, 39.9, 39.4, 38.7 (CH−
CH2), 37.2, 36.2, 34.7, 32.8, 32.3, 31.9, 28.5, 28.4, 28.1, 26.8, 26.7,
26.6, 23.5, 18.7, 17.6, 16.9, 16.8. HRESI-MS: m/z = 972.4949 (calcd
for C51H74NO17, 972.4951). ESI-MS: m/z = 972 [M + H]+, 970 [M −
H]−.
Echocardiographic and Hemodynamic Measurements. At
the end of experiment, the mice were anesthetized and transthoracic
echocardiography was performed using a Vevo 770 high resolution
imaging system (VisualSonics) with a 30 MHz image transducer.
Measurements were taken as described previously.30 After the cardiac
echocardiography, a PE-10 catheter (Becton Dickinson, U.S.) was
inserted into the aorta and LV through the right common carotid
artery for continuous registration of arterial and LV pressure using a
MPA2000 physiological signal acquiring system (Alcolt Biotech). The
maximum ascending and descending rate of the LV pressure ( dp/
dtmax) were measured as the representative index of the LV systolic and
diastolic function.31
Modeling Studies. The target proteins were prepared by
removing water molecules and adding all hydrogen atoms and
Amber7 charges. Then energy minimizations were performed in
SYBYL (Tripos Inc., St. Louis, MO), using steepest descent (SD)
calculations with an embedded Amber7 force field for 5000
iterations.32 In this procedure, heavy atoms were tethered to relieve
possible steric clashes and overlaps of the hydrogen atoms. Conjugated
gradient (CG) minimizations were then carried out until the systems
converged to less than 0.05 kcal mol−1 Å−1 for the gradient. Other
miscellaneous parameters were assigned to their default values in
SYBYL. In the CG minimization procedure, heavy atoms and the
backbone were tethered successively, and then the whole structure was
set to free.
1
Compound 23. White powder (47% yield); H NMR (500 MHz,
pyridine) δ 5.93 (1H, s, H-12), 5.44 (1H, d, J = 7.5 Hz, H-1″), 5.06
(1H, d, J = 7.5 Hz, H-1′), 4.64−4.56 (4H, m), 4.42 (1H, t), 4.35−4.25
(3H, m), 3.73 (1H, m), 3.33 (1H, br d, J = 11.5 Hz, H-3), 3.07−2.96
(5H, m), 2.57 (1H, m), 2.40 (1H, s, H-9), 2.29 (2H, m), 2.08−1.99
(2H, m), 1.40, 1.34, 1.24, 1.19, 1.10, 0.99, 0.75 (each 3H, s, CH3); 13
C
NMR (125 MHz, pyridine) δ 199.6, 177.6 (CH2−CO), 175.9 (CH−
CO), 172.4, 172.1, 169.7, 128.6, 107.0, 105.1, 89.2, 84.6, 78.5, 77.8,
77.6, 77.5, 76.9, 73.3, 73.1, 62.7 (N−CH−CO), 62.0, 55.4, 53.0 (C-
30), 46.7, 45.5, 43.5, 41.5, 40.0, 39.5, 37.2, 36.3, 34.8, 32.9, 32.3, 32.1
(CH2−CO), 31.9, 28.6, 28.6 28.1 (CH−CH2), 28.1, 26.8, 26.8, 26.7,
23.5, 18.7, 17.6, 16.9, 16.8. HRESI-MS: m/z = 938.4769 (calcd for
C47H72NO18, 938.4744). ESI-MS: m/z = 938 [M + H]+, 936 [M −
H]−.
1
Compound 24. White powder (42% yield); H NMR (500 MHz,
pyridine) δ 5.99 (1H, s, H-12), 5.44 (1H, d, J = 7.5 Hz, H-1″), 5.05
(1H, d, J = 7.5 Hz, H-1′), 4.67−4.56 (4H, m), 4.42 (1H, t), 4.35−4.24
(4H, m), 3.40 (1H, dd, J = 11, 4.5 Hz), 3.32 (2H, m), 3.20 (1H, br d, J
= 11 Hz), 3.03 (1H, br d, J = 13.5 Hz), 2.85 (1H, d, J = 10.5 Hz), 2.38
(1H, s, H-9), 2.35 (1H, m), 2.30 (1H, br d, J = 10 Hz), 2.03 (2H, m)
1.39, 1.32, 1.23, 1.18, 1.12, 0.98, 0.76 (each 3H, s, CH3); 13C NMR
(125 MHz, pyridine) δ 199.6, 175.6 (CH2−CO), 174.0 (CH−CO),
172.4, 172.1, 169.5, 128.7, 106.9, 105.1, 89.1, 84.5, 78.5, 77.7, 77.6,
77.5, 76.9, 73.3, 73.0, 62.0, 59.6 (N−C−CO), 55.4, 52.8 (C-30), 46.6,
45.5, 43.4, 41.3, 39.9, 39.4, 38.1 (CH−CH2), 37.2, 36.2, 34.6, 32.8,
32.3, 31.9, 28.5, 28.4, 28.1, 26.7, 26.7, 23.4, 18.7, 17.6, 16.9, 16.8.
HRESI-MS: m/z = 924.4581 (calcd for C46H70NO18, 924.4587). ESI-
MS: m/z = 924 [M + H]+, 922 [M − H]−.
The 3D structures of GL, 23, and 24 were built with the SYBYL-
Sketch module. Hydrogen atoms and Gasteiger−Hukel atomic charges
̈
were added. Then energy minimizations were performed with the
SYBYL compute/minimize module using POWELL calculations with a
Tripos force field for rms less than 0.01 kcal mol−1.33 The final
coordinates were stored in mol2 files.
GOLD utilizes a genetic algorithm (GA) to create putative poses for
a single ligand. The program considers side chain flexibility and local
backbone movement during docking. In this procedure, the molecular
dockings were centered on (−0.85, −6.44, −0.87) for the A box and
(−3.37, 7.47, 4.85) for the B box. The maximum number of energy
evaluations was set to 25 000 000, and 50 conformational binding
modes for each ligand were generated at the active site. The top-10
ranked poses identified by the native scoring functions of the tested
programs were chosen for further analysis. Other parameters were set
as defaults. The default radius defined the grid box. To further
accelerate the calculation, GA docking was stopped when the top three
solutions were within 1.5 Å rmsd of each other.
In the MD procedure, the simulations were carried out using
periodic boundary conditions and the models were immersed in a
cubic box (0.80 × 0.80 × 0.80 nm3) of extended SPC216 water
molecules. The solvated system was neutralized by adding chloride
ions whose positions were randomly chosen in the simulation.
Standard GROMACS amino acid residue topology and parameters
based on the Gromacs force field were used. The topology and
parameter files for GL, 23, and 24 were obtained using the Dundee
ProDrg Server (http://davapc1.bioch.dundee.ac.uk/prodrg/index.
html). After energy minimization with the steepest decent method,
the system was subjected to equilibration at 300 K and normal
pressure for 100 ps under the conditions of position restraints for
heavy atoms. Next, 100 ps MD simulations with restrained protein
main chains and Cα atoms were performed step by step. Finally, the
systems were subjected to free simulations under the same conditions
for 1000 ps. Then the average structures were further refined by
steepest decent and conjugate gradient energy minimizations
sequentially.
Biological Assays/Pharmacology. Reference compounds, me-
dium, and other cell culture reagents were purchased from Sigma-
Aldrich (Milan, Italy).
In Vitro HMGB1 Inhibitory Activities. Cell Culture and
Stimulation. Murine macrophage-like RAW 264.7 cells (American
Type Culture Collection, U.S.) were cultured in RPMI 1640 medium
supplemented with 10% heat inactivated fetal bovine serum (Gemini,
Calabasas, U.S.) and 2 mM glutamine in a humidified incubator with
5% CO2 and 95% room air. To evaluate the effect of HMGB1, the
RAW 264.7 cells were stimulated with LPS (0.01 ng/mL) for 16 h.28,29
The TNF-α released into the cell culture supernatants was quantitated
using a commercially available ELISA kit (catalog no. MTA00, R&D
Systems) according to the manufacturer’s instructions. A HMGB1
neutralization antibody and the compounds were added to the
medium 2 h before LPS stimulation at the indicated concentrations.
Anti-Heart-Failure Activities. Animal Model and Treatments.
Male C57BL/6J mice were purchased from Vital River Lab Animal
Technology, Co. Ltd. (Beijing, China) and maintained under standard
conditions in an animal facility at the Institute of Materia Medica.
Animal care and experimentation were conducted in accordance with
guidelines of the Institutional Committee for the Ethics of Animal
Care and Treatment in Biomedical Research of the Chinese Academy
of Medical Sciences.
Acute heart failure was induced with a single ip injection of DOX at
a dose of 10 mg/kg. The mice were treated with GL, its derivatives,
captopril, or vehicle once per day from day 10 after the injection of
DOX. Sixty days after the DOX injection, the mice were anesthetized,
and the LV structural and functional changes were observed by
echocardiography and hemodynamic measurements. Chronic heart
failure was induced by ip injection of DOX at a dose of 3.5 mg/kg for
8 weeks. The mice were treated with GL, its derivatives, captopril, or
vehicle once per day from day 70. Thirty-five days later, the mice were
anesthetized, and the LV structural and functional changes were
observed by echocardiography.
ASSOCIATED CONTENT
■
S
* Supporting Information
NMR (1H and 13C) spectra of compounds, spectroscopic data
for intermediates, inhibitory data, HPLC results, and
therapeutic blockade information. This material is available
free of charge via the Internet at http://pubs.acs.org.
J
dx.doi.org/10.1021/jm301248y | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(9) Mitola, S.; Belleri, M.; Urbinati., C.; Coltrini, D.; Sparatore, B.;
Pedrazzi, M.; Melloni, E.; Presta, M. Cutting edge: extracellular high
mobility group box-1 protein is a proangiogenic cytokine. J. Immunol.
2006, 176, 12−15.
AUTHOR INFORMATION
Corresponding Author
*For S.Y.: phone, +86-10-63165326; fax, 86-10-63017757; e-
mail, yushishan@imm.ac.cn. For Z.H.: phone, +86-10-
63165207; fax, 86-10-63017757; e-mail, huzhuowei@imm.ac.
cn.
■
(10) Limana, F.; Gemani, A.; Zacheo, A.; Kajstura, J.; Di Carlo, A.;
Borsellino, G.; Leoni, O.; Palumbo, R.; Battistini, L.; Rastaldo, R.;
Muller, S.; Pompilio, G.; Anversa, P.; Bianchi, M. E.; Capogrossi, M. C.
̈
Exogenous high-mobility group box 1 protein induces myocardial
regeneration after infraction via enhanced cardiac C-kit+ cell
proliferation and differentiation. Circ. Res. 2005, 97, e73−e83.
(11) Germani, A.; Limana, F.; Capogrossi, M. C. Pivotal advances:
high-mobility group box 1 proteina cytokine with a role in cardiac
repair. J. Leukocyte Biol. 2007, 81, 41−45.
(12) Li, W.; Sama, A. E.; Wang, H. C. Role of HMGB1 in
cardiovascular diseases. Curr. Opin. Pharmacol. 2006, 6, 130−135.
(13) Li, J.; Kokkola, R.; Tabibzadeh, S.; Yang, R.; Ochani, M.; Qiang,
X.; Harris, H. H.; Czura, C. J.; Wang, H. C.; Ulloa, L.; Wang, H.;
Warren, H. S.; Moldawer, L. L.; Fink, M. P.; Andersson, U.; Tracey, K.
J.; Yang, H. Structure basis for the proinflammatory cytokine activity of
high mobility group box 1. Mol. Med. 2003, 9, 37−45.
(14) Yang, H.; Ochani, M.; Li, J.; Qiang, X.; Tanovic, M.; Harris, H.
E.; Susarla, S. M.; Ulloa, L.; Wang, H.; DiRaimo, R.; Czura, C. J.;
Wang, H.; Roth, J.; Warren, H. S.; Fink, M. P.; Fenton, M. J.;
Andersson, U.; Tracey, K. J. Reversing established sepsis with
antagonists of endogenous high-mobility group box1. Proc. Natl.
Acad. Sci. U.S.A. 2004, 101, 296−301.
Author Contributions
†These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was financially supported by National Natural
Science Foundation of China (Grant No. 201072234, Grant
No. 21132009) and the National Science and Technology
Project of China (Grant No. 2012ZX09301002-002).
ABBREVIATIONS USED
■
GL, glycyrrhizin; HMGB1, high-mobility group box 1; LPS,
lipopolysaccharide; HF, heart failure; AHF, acute heart failure;
CHF, chronic heart failure; RAGE, glycation end products;
HBV, hepatitis B virus; DOX, doxorubicin; DCM, Doll Capital
Management; PCC, pyridinium chlorochromate; TLR, Toll-like
receptor; ip, intraperitoneal; LV, left ventricle; PBS, phosphate
buffered saline; PDB, Protein Data Bank; MD, molecular
dynamics; THF, tetrahydrofuran; SD, steepest descent; CG,
conjugated gradient; GA, genetic algorithm
(15) Dumitriu, I. E.; Baruah, P.; Manfredi, A. A.; Bianchi, M. E.;
Rovere-Querini, P. HMGB1: guiding immunity from within. Trends.
Immunol. 2005, 26, 381−387.
(16) Andrassy, M.; Volz, H. C.; Igwe, J. C.; Funke, B.; Eichberger, S.
N.; Kaya, Z.; Buss, S.; Autschbach, F.; Pleger, S. T.; Lukic, I. K.; Bea,
F.; Hardt, S. E.; Humpert, P. M.; Bianchi, M. E.; Mairbaurl, H.;
̈
REFERENCES
■
Nawroth, P. P.; Remppis, A.; Katus, H. A.; Bierhaus, A. High-mobility
group box-1 in ischemia−reperfusion injury of the heart. Circulation
2008, 117, 3216−3226.
(1) Lindenfield, J. Excutive summary: 2010 HFSA comprehensive
practice guideline. J. Card. Failure 2010, 16, 475−539.
(2) Jessup, M.; Abraham, W. T.; Casey, D. E.; Feldman, A. M.;
Francis, G. S.; Ganiats, T. G.; Konstam, M. A.; Mancini, D. M.; Rahko,
P. S.; Silver, M. A.; Stevenson, L. W.; Yancy, C. W. 2009 focused
update: ACCF/AHA guidelines for the diagnosis and management of
heart failure in adults: a report of the American College of Cardiology
Foundation/American Heart Association Task Force on Practice
Guidelines: developed in collaboration with the International Society
for Heart and Lung Transplantation. Circulation 2009, 119, 1977−
2016.
(17) Takahashi, K.; Fukushima, S.; Yamahara, K.; Yashiro, K.;
Shintani, Y.; Coppen, S. R.; Salem, H. K.; Brouilette, S. W.; Yacoub, M.
H.; Suzuki, Ken. Modulated inflammation by injection of high-mobility
group box 1 recovers post-infarction chronically failing heart.
Circulation 2008, 118, S106−S114.
(18) (a) Newman, D. J.; Cragg, G. M.; Snader, K. M. The influence
of natural products upon drug discovery. Nat. Prod. Rep. 2000, 17,
215−234. (b) Newman, D. J.; Cragg, G. M.; Snader, K. M. Natural
products as sources of new drugs over the period 1981−2002. J. Nat.
Prod. 2003, 66, 1022−1037. (c) Newman, D. J.; Cragg, G. M. Natural
products as sources of new drugs over the last 25 years. J. Nat. Prod.
2007, 70, 461−477. (d) Newman, D. J.; Cragg, G. M. Natural
products as sources of new drugs over the 30 years from 1981−2010. J.
Nat. Prod. 2012, 75, 311−335.
(3) McMurray, J. J.; Pfeffer, M. A. Heart failure. Lancet 2005, 365,
1877−1889.
(4) Lloyd-Jones, D.; Adams, R.; Carnethon, M.; De Simone, G.;
Ferguson, T. B.; Flegal, K.; Ford, E.; Furie, K.; Go, A.; Greenlund, K.;
Haase, N.; Hailpern, S.; Ho, M.; Howard, V.; Kissela, B.; Kittner, S.;
Lackland, D.; Lisabeth, L.; Marelli, A.; McDermott, M.; Meigs, J.;
Mozaffarian, D.; Nichol, G.; O’Donnell, C.; Roger, V.; Rosamond, W.;
Sacco, R.; Sorlie, P.; Stafford, R.; Steinberger, J.; Thom, T.;
Wasserthiel-Smoller, S.; Wong, N.; Wylie-Rosett, J.; Hong, Y. Heart
disease and stroke statistics2009 update: a report from the American
Heart Association Statistics Committee and Stroke Statistics
Subcommittee. Circulation 2009, 119, e21−e181.
(19) Mollica, L.; Marchis, F. D.; Spitaleri, A.; Dallacosta, C.;
Pennacchini, D.; Zamai, M.; Agresti, A.; Trisciuoglio, L.; Musco, G.;
Bianchi, M. E. Glycyrrhizin binds to high-mobility group box 1 protein
and inhibits its cytokine activities. Chem. Biol. 2007, 14, 431−441.
(20) Sitia, G.; Lannacone, M.; Muller, S.; Bianchi, M. E.; Guidotti, L.
̈
G. Treatment with HMGB1 inhibitors diminishes CTL-induced liver
disease in HBV transgenic mice. J. Leukocyte Biol. 2007, 81, 100−107.
(21) Baltina, L. A. Chemical modification of glycyrrhizic acid as a
route to new bioactive compounds for medicine. Curr. Med. Chem.
2003, 10, 155−171.
(5) Jiang, B. B.; Liao, R. The paradoxical role of inflammation in
cardiac repair and regeneration. J. Cardiovasc. Transl. Res. 2010, 3,
410−416.
(6) Yang, H.; Wang, H. C.; Czura, C. J.; Tracey, K. J. The cytokine
activity of HMGB1. J. Leukocyte Biol. 2005, 78, 1−8.
(7) Rosenberg, H. F.; Oppenheim, J. J. Interview with Dr. Maurizio
C. Capogrossi regarding pivotal advance: high-mobility group box 1
proteina cytokine with a role in cardiac repair. J. Leukocyte Biol.
2007, 81, 38−40.
(8) Palumbo, R.; Sampaolesi, M.; De Marchis, F.; Tonlorenzi, R.;
Colombetti, S.; Mondino, A.; Cossu, G.; Bianchi, M. E. Extracellular
HMGB1, a signal of tissue damage, induces mesoangioblast migration
and proliferation. J. Cell Biol. 2004, 164, 441−449.
(22) Hirabayashi, K.; Iwata, S.; Matsumoto, H.; Mori, T.; Shibata, S.;
Bara, M.; Ito, M.; Shigeta, S.; Nakashima, H.; Yamamoto, N. Antiviral
activities of glycyrrhizin and its modified compounds against human
immunodeficiency virus type 1 (HIV-1) and herpes simplex virus type
1 (HSV-1) in vitro. Chem. Pharm. Bull. 1991, 39, 112−115.
(23) Hardman, C. H.; Broadhurst, R. W.; Raine, A. R.; Grasser, K. D.;
Thomas, J. O.; Laue, E. D. Structure of the A-domain of HMG1 and its
interaction with DNA as studied by heteronuclear three- and four-
dimensional NMR spectroscopy. Biochemistry 1995, 34, 16596−16607.
K
dx.doi.org/10.1021/jm301248y | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(24) Weir, H. M.; Kraulis, P. J.; Hill, C. S.; Raine, A. R.; Laue, E. D.;
Thomas, J. O. Structure of the HMG box motif in the B-domain of
HMG1. EMBO J. 1993, 12, 1311−1319.
(25) Jones, G. Development and validation of a genetic algorithm for
flexible docking. J. Mol. Biol. 1997, 267, 727−748.
(26) Berendsen, H. J. C.; van der Spoel, D.; van Drunen, R.
GROMACS: a message-passing parallel molecular dynamics imple-
mentation. Comput. Phys. Commun. 1995, 91, 43−56.
(27) Sasaki, Y.; Mizutani, K.; Kasai, R.; Tanaka, O. Solubilizing
properties of glycyrrhizin and its derivatives: solubilization of
saikosaponin-a, the saponin of Bupleuri Radix. Chem. Pharm. Bull.
1988, 36, 3491−3495.
̈
(28) Ostberg, T.; Wahamaa, H.; Palmblad, K.; Ito, N.; Stridh, P.;
̈
̈
Shoshan, M.; Lotze, M. T.; Harris, H. E.; Andersson, U. Oxaliplatin
retains HMGB1 intranuclearly and ameliorates collagen type II-
induced arthritis. Arthritis Res. Ther. 2008, 10, R1−R9.
(29) Yang, H.; Hreggvidsdottir, H. S.; Palmblad, K.; Wang, H. C.;
Ochani, M.; Li, J. H.; Lu, B.; Chavan, S.; Rosas-Ballina, M.; Al-Abed,
Y.; Akira, S.; Bierhaus, A.; Erlandsson-Harris, H.; Andersson, U.;
Tracey, K. J. A critical cysteine is required for HMGB1 binding to
Toll-like receptor 4 and activation of macrophage cytokine release.
Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 11942−11947.
(30) Liu, Y. Y.; Cai, W. F.; Yang, H. Z.; Cui, B.; Chen, Z. H; Liu, H.
Z.; Yan, J.; Jin, W.; Yan, H. M.; Xin, B. M.; Yuan, B.; Hua, F.; Hu, Z.
́
W. Bacillus calmette-guerin and TLR4 agonist prevent cardiovascular
hypertrophy and fibrosis by regulating immune microenviroment. J.
Immunol. 2008, 180, 7349−7357.
(31) Cai, W. F.; Zhang, X. W.; Yan, H. M.; Ma, Y. G.; Wang, X. X.;
Yan, J.; Xin, B. M.; Lv, X. X.; Wang, Q. Q.; Wang, Z. Y.; Yang, H. Z.;
Hu, Z. W. Intracellular or extracellular heat shock protein 70
differentially regulates cardiac remodeling in pressure overload mice.
Cardiovasc. Res. 2010, 88, 140−149.
(32) Weiner, S. J; Kollman, P. A.; Case, D. A.; Singh, U. C.; Ghio, C.;
Alagona, G.; Profeta, S.; Weiner, P. K. A new force field for molecular
mechanical simulation of nucleic acids and proteins. J. Am. Chem. Soc.
1984, 106, 765−784.
(33) Clark, M.; Cramer, R. D.; Van, O. N. Validation of the general
purpose Tripos 5.2 force field. J. Comput. Chem. 1989, 10, 982−1012.
L
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