The design, synthesis, and biological evaluation of novel YC-1 derivatives as potent anti-hepatic fibrosis agents

Article abstract of DOI:10.1039/c5ob00710k

1-Benzyl-3-(substituted aryl)-5-methylfuro[3,2-c]pyrazole (YC-1) is a well-known synthetic compound with various satisfactory pharmacological activities, such as the activation of soluble guanylate cyclase (sGC) and the inhibition of hypoxia-induced facto

Full text of DOI:10.1039/c5ob00710k

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The design, synthesis, and biological evaluation of novel YC-1
derivatives as potent anti-Hepatic fibrosis agents #
Juan Xiao,†a Chunmei Jin,†b Zhixue Liu,a Shujing Guo,a Xiaochuan Zhang,b Xin Zhou*a, c and Xue Wu*a
Received 00th January 20xx,
Accepted 00th January 20xx
1-benzyl-3-(substituted aryl)-5-methylfuro[3, 2-c]pyrazoles (YC-1) is a well-known synthetic compound with various
satisfactory pharmacological activities, such as the activation of soluble gualylate cyclase (sGC) and the inhibition of
hypoxia-induced factor-1α (HIF-1α). Recently, YC-1 has been demonstrated to have a potent activity on anti-fibrotic.
However, the mechanism underlying its anti-fibrotic activity is still largely unknown. To this end, we presented here the
design and synthesis of YC-1 and its novel derivatives, as well as the evaluation of their anti-fibrotic effects on activated
human hepatic stellate cells (HSCs) LX-2. Moreover, the possible underlying mechanism of anti-fibrotic was also
investigated for the first time by means of CCK-8 assay, cell apoptosis analysis, and western blot. Our study revealed that
YC-1 and its derivatives suppressed activated LX-2 cell viability and induced cell apoptosis in a time and dose-dependent
DOI: 10.1039/x0xx00000x
www.rsc.org/
manner. Western blot data demonstrated that these derivatives not only decreased the expression of α-smooth muscle
actin (α-SMA), but also increased the expression of caspase-3, resulting in cell apoptosis. These findings strongly indicated
that YC-1 and its derivatives, especially AC could significantly inhibit LX-2 cell activation and induce LX-2 cell apoptosis by
inhibiting
α-SMA protein expression and promoting caspase-3 expression, respectively. In summary, our findings
suggested that YC-1 derivatives might be potential agents for hepatic fibrosis therapy.
expression. It means that YC-1 may play an important role in
Introduction
controlling liver fibrosis by regulating the apoptosis of HSC.
Nevertheless, the mechanism underlying its anti-fibrotic
activity is still largely unknown.
1-benzyl-3-(5'-hydroxymethyl-2'-furyl) indazole
(YC-1) is a
synthetic compound that has been demonstrated with various
potent biological and pathological activities. For example, YC-1
was first developed as an activator of soluble gualylate cyclase
(sCG), and played an important role in inhibiting platelet
aggregation, vascular contraction, and ATP release.1, 2 After
then, various biological functions and pharmacological actions
of YC-1 have been achieved to date. These discrete actions
include antiplatelet activity,3 sGC activity,4 suppression of
Based on the previous studies, it is known that liver fibrosis
is a dynamic process that results from an imbalance between
the production and dissolution of the extracellular matrix. It is
caused by
a variety of chronic stimuli, including viral,
autoimmune, drug induced, cholestatic and metabolic
diseases.15-17 Generally speaking, activation of hepatic stellate
cells (HSCs) is recognized as the primary cellular source of
matrix components in chronic liver disease as it plays a critical
role in the development and maintenance of liver fibrosis.18
Upon activation, HSCs change their phenotype into
myofibroblast-like cells along with increasing production of
extracellular matrix (ECM) components (such as α-SMA), and
up-regulating expression of several plasma membrane
receptors (such as PDGF-receptors).19 Therefore, apoptosis of
activated HSC could effectively control fibrosis in the liver. In
such an apoptosis process, the mitochondrial-induced
apoptosis members of the bcl-2 and bax families, and the
casepase families (such as caspase-3), are always involved.20-23
Taking these factors into account, we hypothesized that
YC-1 and its analogues may also provide an effective effect for
antifibrosis of HSCs through the similar pathways (inhibiting
the activation or inducing activated apoptosis of HSCs). To
verify our above mentioned hypothesis, we presented herein
the design and synthesis of YC-1 and its three novel derivatives
(Fig. 1). For the first time, we explored their capability of anti-
hypoxia-induced factor-1
α
(HIF-1α
),5-7 and anticancer
activity.8-11 In addition, YC-1 also exhibited an anti-proliferative
effect through arresting the cell cycle in the G0-G1 phase in
hepatocellular carcinoma cells.12 Many evidences suggested
that YC-1 attenuated hypoxia-induced pulmonary arterial
hypertension in mice13 and induced lipolysis through a PKA
pathway.14 Beside above mentioned features of YC-1, latest
research from our collaborated team indicated that YC-1 could
decrease pro-casepase-3 and nuclear factor kB (NF-kB)
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hepatic fibrosis and the corresponding underlying mechanism. designed and synthesized. In addition, the introduction of
Our findings indicated that all derivatives significantly inhibited proper alkyl chain can not only add drug bulk, but also increase
LX-2 cell activation and induced LX-2 cell apoptosis by drug’s lipid solubility and membrane permeability, thereby
inhibiting α-SMA protein expression and promoting pro- improving drug absorption and bioavailability. To this end, 4-
apoptotic protein caspase-3 expression, respectively. pentylbicycle[2.2.2]-octane containing derivative 1-(4'-
Moreover, we also demonstrated the structure-activity pentylbicyclo[2.2.2]octan-1'-formoxyl)-3-(5'-hydroxymethyl-2'-
relationship of YC-1 analogues. It is found that bulky furyl) indazole
(POC) was synthesized. Moreover, the
substituted benzyl group is of significant benefit for anti- molecular shape and electrostatic distribution of YC-1 play a
hepatic fibrosis. Compared with YC-1 and other derivatives, AC crucial role in enzyme recognition and contribute extensively
has the strongest capability of preventing the development of to binding affinity.31 To further investigate the biological
liver fibrosis, and thus could be treated as a potent anti- activity of hydroxymethyl moiety of YC-1 and the electrostatic
hepatic fibrosis agent.
distribution of 3-furylindazole skeleton, we induced an
acylamino group to substitute hydroxymethyl moiety. We
expected that such a modification could not only increase
interactions and hydrogen bondings, but also improve their
water solubility. Therefore, acylamino group containing
Results and discussion
derivative
5-(1-benzyl-1H-indazol-3-yl)-N-(prop-2-yn-1-yl)
furan-2-carboxamide (MPa) was synthesized. Moreover, MPa
was designed with a premeditated terminal alkynyl group that
can be used for tracking through cell imaging by click reaction.
Synthesis
The synthetic routes of YC-1 and its derivatives are
summarized as follows (Scheme 1-3). The commercially
available material 1H-indazole reacted with iodine and
potassium hydride in DMF to give the key intermediate
compound 3-iodoindazole 1. Then 1 was treated with benzyl
bromide or appropriate acyl chloride in the presence of
potassium tert-butoxide in absolute THF to give the
Figure 1. The structure of YC-1 and its analogues.
corresponding 3-iodo-1-substituted-1H-indazoles
yields. And then Suzuki–Miyaura coupling of
2
in high
2
with (5-
Design concepts
formylfuran-2-yl)boronic acid proceeded in the presence of
palladium catalyst (Pd(PPh3)4, 5 mM%) and Na2CO3 in dimethyl
formamide (DMF) at 80 °C to give the corresponding 5-
YC-1 has been well demonstrated with various biological
activities.24 To further investigate the structure-activity
relationship of YC-1, its synthetic analogues through chemical
modification are urgently needed. Generally, the structural
modifications of YC-1 are mainly focused on the benzyl or
hydroxymethyl moiety.25 Previous studies shown that furan
moiety was considered as an optimal heterocyclic
pharmacophore for its capability of potent HIF-1 inhibition.7
Meanwhile, when the benzyl or hydroxymethyl group was
altered appropriately, their activity toward sGC, antiplatelet
activities and HIF-1 inhibitory activity were maintained or
improved.26-28 These YC-1 analogues have guided us in
developing activity-based skeleton for the investigation of the
anti-fibrotic.
formylfuranyl-indazoles
3. The formyl group of
3
was reduced
by sodium borohydride in methanol to give YC-1
.
AC and POC
were synthesized in the same methods. The formyl group of
was oxidized by potassium permanganate in acetone and
water (v/v, 1:1) at 0°C to give oxidation product which
reacted with propynlamine could give MPa in 45% yield.
3
4
Considering these factors, we set to introduce bulky
substituents containing carbonyl group in the benzyl position
of YC-1. Their corresponding structures are documented in
Fig.1. It is well known that adamantine is often used in
modification of many bioactive compounds in which it is
viewed as providing just the critical lipophilicity for known
pharmacophores. The steric bulk of adamantane modifications
Scheme 1. The synthetic routes of YC-1 and MPa. Reagents and conditions: (a)
KOH, I2, DMF, rt.; (b) benzyl bromide, t-BuOK, THF, rt.; (c) (5-formylfuran-2-yl)
were chosen to not only enhance lipophilicity and stability of boronic acid, 5 mM% Pd(PPh3)4, Na2CO3, DMF, 80-90°C; (d) NaBH4, MeOH, rt.; (e)
KMnO4, H2O, Me2CO, 0°C; (f) Propynylamine, EDC·HCl, HOBt, DIPEA, DMF, rt..
the drugs; but also restrict or modulate intramolecular
reactivity; and impede the access of hydrolytic enzymes,
thereby improving their pharmacokinetics and plasma half-
life.29,
30
Hence, adamantane containing derivative AC was
2 | J. Name., 2012, 00, 1-3
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perhaps also induce cell apoptosis. To this end, we next
examined the effects of YC-1 analogues on LX-2 apoptosis by
using PI and FITC conjugated annexin-V staining. PI-positive
cells and FITC-positive/Pi-negative cells were measured by
flow cytometry. First, LX-2 exhibited significant morphology
alteration following treatment of 25 uM YC-1 and its analogues
at 18h. As shown in Fig. 3A inset, cell shrinkage and plasma
membrane blebbing were distinctly observed. The cell death
Scheme 2. The synthetic routes of AC. Reagents and conditions: (g) t-BuOK, THF, rt.; (c)
(5-formylfuran-2-yl) boronic acid, 5 mM % Pd(PPh3)4, Na2CO3, DMF, 80-90 °C; (d)
NaBH4, MeOH, rt..
rate after treatment with 25 uM of YC-1, AC, POC or MPa was
determined as 14.7%, 22.9%, 20.1% and 29.4%, respectively
(Fig. 3B light gray bar). While lengthening the exposure time
from 24 h to 48 h, the cell death rate was increased (32.7%,
53.9%, 36.0%, 32.2%, respectively, Fig. 3C dark gray bar). After
FITC conjugated annexin-V/PI double staining, the average
data of early apoptosis rate was measured as 29.5%, 32.1%,
Scheme 3. The synthetic routes of POC. Reagents and conditions: (h) 1. SOCl2, 28.9%, 28.4% and later apoptosis rate was detected as 5.5%,
DCM, 0 °C, 100%; 2. t-BuOK, THF, rt.; (c) (5-formylfuran-2-yl)boronic acid, 5 mM
11.8%, 7.6%, 7.4%, respectively (Fig.3C, the raw flow
% Pd(PPh3)4, Na2CO3, DMF, 80-90 °C; (d) NaBH4, MeOH, rt..
cytometry data shown in figure S1). These results were in
keeping with the PI simple staining. Therefore, these data
Biological evaluation
indicated that YC-1, AC, POC, and MPa could induced LX-2
apoptosis in a time-dependent manner.
Inhibitory effect on cellular viability of LX-2
To evaluate the anti-fibrotic effects of the YC-1 analogues.
LX-2 cells (a stable and unlimited source of human HSC) were
employed as they have been extensively characterized as a
valuable cell-based model for studies of human hepatic
fibrosis.32 Firstly, the viability of LX-2 cells following respective
treatment with YC-1, AC, POC, and MPa, were investigated by
CCK-8 assay. After direct pretreatment with different
concentrations of YC-1 and its analogues (0, 5, 25, 50, and 100
μM) for 24h, respectively, living LX-2 cells were found to be
decreased in a dose-dependent manner (Fig. 2a). The viability
rate at the concentration of 100 uM was measured as 42.2%,
32.0%, 33.5%, and 60.0%, respectively. This result suggested
that YC-1, AC, POC, and MPa exhibited cytotoxicity to LX-2
cells and could induce LX-2 cells apoptosis. Moreover, in the
case of AC and POC, The concentration at 25 μM exhibited a
significant cytotoxicity towards LX-2 cells compared with YC-1
.
Next, in order to investigate whether the doses showing
toxicity in HSC LX-2 cells are also toxic in non-liver cell types,
we checked CCK-8 assay with Hela cells under the same
conditions. As shown in Fig.2b, both YC-1 and its analogues
showed a certain but much weaker cytotoxicity towards Hela
cells compared with LX-2 cells. The cell viability after
treatment with YC-1 and its analogs were 72.7%, 68.7%, 91.1%,
69.8%, respectively.
Figure 2. Inhibitory effect of YC-1 and its analogues on LX-2 (a) or Hela (b) cell
The effect on LX-2 apoptosis
viability measured by the CCK-8 assays. Cells were incubated with YC-1, AC, POC,
HSC apoptosis is thought to be essential for the resolution
phase of fibrosis. Thus, induction of HSC apoptosis would be
expected to be with anti-fibrogenic effect by inhibiting the
accumulation of the activated HSCs within the liver.33 From the
and MPa at different concentrations (0, 5, 25, 50, and 100 μM) in DMEM for 24
h, respectively. Data were presented as mean ±S.D. of three independent
experiments with n = 3.
cell viability assay data, we observed that YC-1, AC, POC and
MPa at a concentration level of 100 uM significantly induced
cell death. Thus, we hypothesized that YC-1 and its derivatives
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Figure 3. The effects of YC-1, AC, POC and MPa on LX-2 apoptosis. Activated LX-2 (2 ×105 cells/well in 6-well plate) were incubated with 25 uM YC-1, AC and MPa for 24 h or 48 h,
respectively. Cellular morphology were observed by confocal microscope at 18 h (A inset). Cells were then harvested and incubated with propidium iodide (PI) (A) or FITC
conjugated annexin-V and PI. PI-positive cells (B) and FITC-positive/PI-negative cell (C) were measured by flow cytometry. Data are presented as mean ± standard deviation (SD) (n
= 3/group).*P < 0.05, **P < 0.01, ***P< 0.001, compared with the control group, ###P < 0.001, significantly different compared with YC-1.
The effect on the levels of α-SMA expression
its analogues may inhibit the activation of HSCs, then prevent
the development of liver fibrosis.
The effect on the levels of caspase-3 expression
The activation of HSC cells is considered as one of the
central pathophysiological mechanisms of liver fibrosis, and
HSC activation is characterized by the overexpression of a
During the cell apoptosis assay, we found that YC-1 and its
smooth muscle actin (α-SMA) and collagen I. Herein, we analogues could induce LX-2 cell apoptosis. Such an apoptosis
measured α-SMA expression by western blot assay. We found is believed to be caused by the pro-apoptosis protein-caspase-
that the level of α-SMA protein underwent a significantly 3.34 Herein, we detected the expression of pro-apoptosis
down-regulation after treatment with 25 uM YC-1, AC, POC, protein caspase-3 by western blot assay. Afther pretreatment
and MPa respectively for 3 h (Fig. 4). The results suggested of LX-2 cells with 25 uM YC-1 and its analogues for 12 h, the
that YC-1 and its derivatives could inhibit α-SMA protein cleaved substrate of caspase-3 (it was not observed when
expression in a time-dependent manner. In summary, YC-1 and treated for 3 h or 6 h) was obviously detected (Fig. 5).
Compared with the control group, the protein level of cleaved
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caspase-3 were obviously increased, especially for the inducing activated HSCs apoptosis. In addition, from the cell
compound AC, which indicated that AC was more potency apoptosis assay data we found that AC was more potent
than YC-1. These results indicated that YC-1 and its analogues efficient than YC-1 on inducing activate LX-2 cell apoptosis.
AC
, POC, MPa could promote caspase-3 expression. And the This may attributed to the introduction of bulky substituents
effects of YC-1 and its analogues on LX-2 cell apoptosis maybe adamantane that has been found to increase drug-like
depended on caspase-3 expression and cleavage.
qualities of a lead compound. From what has been discussed
above, we can concluded that the introduction of bulky
substituents to the benzyl group was benefit for inhibiting LX-2
cell activity which is closely associated with anti-liver fibrosis.
Besides, hydroxymethyl moiety of YC-1 was efficient but not
prerequisite for anti-liver fibrosis. Moreover, the introduction
of conjugated carbonyl may be useful for improving the
analogues biological activity.
Conclusions
We designed and synthesized YC-1 and its three derivatives
AC, POC, and MPa. Their anti-fibrosis activity and the
corresponding underlying mechanism were investigated in
detail for the first time. Our findings indicated that all of them
showed significant inhibition of LX-2 cell activation through
inhibiting α-SMA protein, and induction of LX-2 cell apoptosis
through promoting caspase-3 expression. More important, we
also demonstrated that bulky substituted benzyl group is of
significant benefit for anti-hepatic fibrosis. All YC-1 derivatives
Figure 4. Effects of YC-1
,
AC
,
POC, and MPa on α-SMA expression. LX-2 cells
AC POC, and MPa for 1 h or 3 h,
were pretreated with or without 25 uM YC-1
,
,
respectively. α-SMA proteins were detected via western blotting with specific especially AC has the capability of preventing the development
antibodies. β-Actin was used as a loading control. Data are presented as mean ±
of liver fibrosis, and thus could be treated as a potent anti-
standard deviation (SD) (n = 3/group). ∗∗ p < 0.01, ∗∗∗p < 0.001.
hepatic fibrosis agent. Further studies on the effects of YC-1
analogues associated proteins is still undergoing in our lab.
Such a research can supply further understanding about
molecular and cellular mechanisms responsible for their
inhibitory effects on liver fibrosis, which can provide
importance basis for the development of new efficient anti-
Hepatic Fibrosis agent.
Experimental section
Materials and general methods
All chemical reagents were purchased from commercial
suppliers (Aladdin Industrial Corporation or J&K Scientific,
Beijing, China); Human HSC LX-2 cell line was graciously
provided by Professor Nan from college of pharmacy, Yanbian
University (Jilin province, China). The biological reagents
Figure 5. Effects of YC-1
were pretreated with or without 25 uM YC-1
respectively. Caspase-3 proteins were detected via western blotting with specific
antibodies. -Actin was used as a loading control. Data are presented as mean ±
standard deviation (SD) (n = 3/group). ∗∗∗p < 0.001, compared with control
group, ###p < 0.001, compared with YC-1
,
AC
,
POC, and MPa on caspase-3 expression. LX-2 cells
Dulbecco's modified Eagle's medium (DMEM), fetal bovine
serum (FBS), 0.25% Trypsin and penicillin streptomycin were
purchased from Gibco (USA). Cell counting kit-8 (CCK-8),
dimethyl sulfoxide (DMSO), Annexin V-FITC apoptosis
detection kit, Prodium Iodide (PI), α-smooth muscle actin (α–
SMA) antibody, caspase-3 antibody, anti-β-actin antibodies,
anti-rabbit IgG antibody conjugated with horseradish
peroxidase were obtained from Cell signaling technology Inc.
(USA). Western blot detection reagents enhanced
chemiluminescence (ECL) and nitrocellulose (NC) membrane
were purchased from Millipore (USA). Proton nuclear magnetic
resonances (1H NMR) and 13C NMR spectra were recorded in
,
AC, POC and MPa for 12 h,
β
.
The relationship between chemical structures and bioactivity
With these bioactivity data in our hand, we further
summarized and discussed here the structure-bioactivity
relationship between YC-1 and its derivatives. According to the
results of CCK-8 assay and the cell apoptosis assay, it is clear
that YC-1, AC, POC, and MPa could be useful for anti-hepatic
fibrosis by inhibiting the activation and proliferation of HSCs or
deuterated solvents on
a
Brucker (300 MHz) NMR
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spectrometer. Chemical shifts are reported in parts per million General Procedure for Synthesis of 3, 6, 8: To a mixture of 1-
1
(ppm, δ). H NMR splitting patterns are designated as singlet benzyl-3-iodoindazole
2 or 5 or 7 (0.334 g, 1.0 mM) and
(s), doublet (d), triplet (t), and quartet (q). Splitting patterns Pd(PPh3)4 (0.08 g, 0.07 mM) in DMF (5 ml), then (5-
that could not be interpreted or easily visualized were formylfuran-2-yl) -boronic acid (0.168 g, 1.2 mmol) was added
recorded as multiplet (m) or broad (br). MALDI-TOF mass followed by the addition of sodium carbonate (0.318 g, 3.0
spectra were recorded by using a Shimadzu MALDI AXIMA- mM). The reaction mixture was refluxed with vigorous stirring
CFR+ spectrometer. Analytical thin layer chromatography (TLC) under nitrogen atmosphere and the rate of the reaction was
was performed on a glass plates of silica gel 60 GF254 followed by TLC. After the starting materials were consumed,
(Qingdao Haiyang chemical Co., Ltd, China) with visualization the reaction mixture was filtered, the filtrate was poured into
accomplished with phosphomolybdic acid, iodine, or with a water and extracted with DCM (3×50 ml). The combined DCM
UV-visible lamp. Column chromatography was conducted on layers were washed with water and brine, dried (anhydrous
silica gel (Qingdao Haiyang chemical Co., Ltd, 100-200 mesh). Na2SO4), filtration and the solvent was removed under reduced
Most reagents were purchased from commercial suppliers and pressure. The crude product was purified by column
used without further purification. Some solvents were purified chromatography (silica gel, ethyl acetate/Petroleum ether 1 : 4)
and dried by standard methods prior to use: tetrahydrofuran to give 3 as an orange solid (0.182g, yield 60 %). 5-(1′-benzyl-
1
): H NMR (300 MHz,
(THF) was distilled from sodium/benzophenoneketyl. All the 1H-indazol-3′-yl) furan-2-carbaldehyde (
3
end-products finally were further purified by reversed-phase CDCl3) δ 9.77 (s, 1H), 8.30 (d, J = 8.1Hz, 1H), 7.43 - 7.28 (m, 9H),
HPLC.
7.14 (d, J = 3.6Hz, 1H), 5.69 (s, 2H); Calc. for C19H14N2O2: 302.33,
Tof-MS found: 302.3.
Synthesis
5-(1′-adamantaneformoxyl-1H-indazol-3′-yl)furan-2-
1
carbaldehyde (6): Yield 49%; a light yellow solid; H NMR (300
3-iodoindazole (1): Iodine (0.86 g, 3.4 mM) and potassium
hydroxide pellets (0.2 g, 3.4 mM) were successively added into
a DMF solution (5 ml) of indazole (0.2 g, 1.7 mM) at room
temperature under stirring. After 2 h, the reaction mixture was
poured into 10% aqueous NaHSO3 (100 ml) and extracted with
Et2O (3 x 150 ml). The combined organic layers were washed
with water and brine, dried (anhydrous Na2SO4), filtration and
MHz, CDCl3) δ 9.82 (s, 1H), 8.54 (dd, J = 8.4, 0.9 Hz, 1H), 8.35
(dd, J = 8.4, 0.9 HZ, 1H), 7.63 (td, J = 7.2, 0.9 Hz, 1H), 7.51(dd, J
= 7.2, 0.9 Hz, 1H),7.46(d, J = 3.9 Hz, 1H), 7.29 (d, J = 3.9 Hz, 1H),
2.44 (s, 6H), 2.17 (s, 3H), 1.83 (s, 6H). Calc. for C23H22N2O3:
474.16, Tof-MS found: 374.2.
5-(4'-pentylbicyclo[2.2.2]octan-1'-formoxyl-1H-indazol-3′-
yl)furan-2-carbaldehyde (8): Yield 54%; a light yellow solid; 1H
NMR (300 MHz, CDCl3) δ 9.82 (s, 1H), 8.52 (dd, J = 8.7, 0.9 Hz,
1H), 8.34 (dd, J = 8.1, 0.9 Hz, 1H), 7.63 (td, J = 8.7, 0.8 Hz, 1H),
7.50 (dd, J = 8.1, 0.9 Hz,1H), 7.29 (d, J = 4.5 Hz, 1H), 2.40-2.19
(m ,6H), 1.60-1.46(m, 6H), 1.37-1.14 (m 8H), 0.91 (t, J = 6.9 Hz,
3H); Calc. for C26H30N2O3: 418.23, Tof-MS found: 418.2.
the solvent evaporated under reduced pressure to give
1 as
1
white solid (0.410 g, yield 99.2%). H NMR (300 MHz, CDCl3) δ
9.12 (s, 1H), 8.25 (dd, J = 7.6, 1.8 Hz, 1H), 7.46 (dt, J = 7.8, 2.1
Hz, 1H), 7.41 (dd, J = 7.8, 2.1 Hz, 1H), 7.36 (dd, J = 7.0, 1.8 Hz,
1H); Calc. for C7H5IN2: 244.03, Tof-MS found: 245.2.
General Procedure for Synthesis of 2, 5, 7: To a solution of 3-
iodoindazole
1 (0.201 g, 0.82 mM) in anhydrous THF (6 ml)
General Procedure for Synthesis of YC-1 and AC, POC
：To a
cooled at 0 °C was added potassium tert-butoxide (0.141 g,
1.30 mM). After 1 h at 0°C, benzyl bromide or appropriate acyl
chloride (approximately 0.1 ml, 0.82 mM) was added drop
solution of 3 or 6 or 8 (0.12 g, 0.4 mM) dissolved in MeOH (8
ml) was added NaBH4 (0.02 g, 0.5 mM). After stirred 1 h at
room temperature, the resulting mixture was evaporated. The
residue was dissolved with EA (20 ml), washed with water and
saturated sodium bicarbonate, dried (anhydrous Na2SO4),
filtration and the solvent evaporated under reduced pressure.
Chromatography (silica gel, ethyl acetate/Petroleum ether 1:1)
followed by recrystallization from hexane gave YC-1 as a white
solid (0.11 g, yield 92%). 1-benzyl-3-(5'-hydroxymethyl-2'-
wise. The resulting mixture was stirred
4 h at room
temperature then evaporated. The residue was dissolved with
EA (50 ml), washed with water and brine, dried (MgSO4) ,
filtration and the solvent evaporated under reduced pressure
to give
2 as a light yellow oil (0.260g, yield 95%). 1-Benzyl-3-
1
): H NMR (300 MHz, CDCl3) δ7.51(dd, J = 8.1,
iodoindazole (
2
1.2Hz, 1H), 7.40 (td, J = 8.1, 1.2Hz, 1H), 7.36-7.29(m, 4H), 7.26 -
7.19 (m, 3H), 5.63 (s, 2H); Calc. for C14H11IN2 :334.16, Tof-MS
found: 334.6.
1
furyl)indazole (YC-1): H NMR (300 MHz, CDCl3) δ 8.09 (d, J =
8.4 Hz, 1H), 7.38-7.22 (m, 8H), 6.90 (d, J = 3.3 Hz, 1H), 6.51 (d, J
= 3.3 Hz, 1H), 5.68 (brs, 2H), 4.77 (s, 2H), 1.92 (brs, 1H); Calc.
for C19H16N2O2: 304.12, Tof-MS found: 304.2.
1-Adamantaneformoxyl-3-iodoindazole (5): Yield 98%; a white
1
solid; H NMR (300 MHz, CDCl3) δ 8.43 (dd, J = 8.4, 1.2Hz, 1H),
1-adamantaneformoxyl-3-(5'-hydroxymethyl-2'-furyl)indazole
(AC): Yield 90%; a white solid; 1H NMR (300 MHz, CDCl3) δ 8.53
(dd, J = 8.4, 0.9 Hz, 1H), 8.20 (dd, J = 7.2, 0.9 Hz, 1H), 7.58 (td, J
= 8.4, 0.9 Hz, 1H), 7.43 (td, J = 7.2, 0.9 Hz, 1H), 7.08 (d, J = 3.4
Hz, 1H), 6.55 (d, J = 3.4 Hz, 1H), 4.81 (s, 2H), 2.44 (d, J = 2.6 Hz,
6H), 2.16 (s, 3H), 1.94-1.72 (m, 7H); 13C NMR (75 MHz, CDCl3) δ
177.64 (s), 155.26 (s), 148.11 (s), 141.39 (s), 140.07 (s), 129.48
(s), 124.69 (s), 122.57 (s), 121.51(s), 116.36(s), 110.17(s),
109.86(s), 57.76 (s), 44.56 (s), 38.79 (s, 3C), 36.79 (s, 3C), 28.41
7.61 (td, J = 8.4, 1.2Hz, 1H), 7.51 (dd, J = 7.5, 0.6Hz, 1H), 7.42
(td, J = 7.5, 0.6Hz, 1H), 2.38 (d, J = 2.4Hz, 6H), 2.15(s, 3H),
1.85(s, 6H); Calc. for C18H19IN2O: 406.26, Tof-MS found: 406.8.
1-(4'-pentylbicyclo[2.2.2]octan-1'-formoxyl)-3-iodoindazole
1
(7): Yield 95%; a white solid; H NMR (300 MHz, CDCl3) δ 8.43
(dd, J = 8.4, 1.3 Hz, 1H), 7.61 (td, J = 8.4, 1.3 Hz, 1H), 7.50 (dd, J
= 8.1, 0.9 Hz, 1H), 7.41 (td, J = 8.4, 0.9 Hz,1H), 2.29-2.16 (m,
6H), 1.60-1.46 (m, 6H), 1.33-1.15 (m, 8H), 0.91 (t, J = 6.9 Hz,
3H); Calc. for C21H27IN2O : 450.36, Tof-MS found: 450.9.
6 | J. Name., 2012, 00, 1-3
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(s, 3C); Calc. for C23H24N2O3: 376.18, HRMS found: [M+Na]+ Cell Culture Conditions: LX-2 cells were cultured in DMEM
399.1677.
supplemented with 10% FBS, 100 IU/ml penicillin and
streptomycin. Cultures were incubated at 37 °C in a humidified
1-(4'-pentylbicyclo[2.2.2]octan-1'-formoxyl)-3-(5'-
hydroxymethyl-2'-furyl)indazole (POC): Yield 67%; a white atmosphere of 5% CO2 and the medium was changed every
solid; 1H NMR (300 MHz, CDCl3) δ 8.51 (dd, J = 8.4, 1.2 Hz, 1H), other day.
8.18 (dd, J = 7.8, 0.9 Hz, 1H), 7.58 (td, J = 8.4, 1.2 Hz,1H), 7.42 Cellular viability of LX-2: Cell viability was determined by CCK-
(td, J = 7.8, 0.9 Hz,1H), 7.07 (d, J = 3.3 Hz, 1H), 6.55 (d, J = 3.3 8 assay. A total of 1
×
104 cells were seeded in 96 well plates
Hz, 1H), 4.81 (s, 2H), 2.36-2.22 (m, 6H), 1.88 (brs, 1H), 1.61- for 24h. YC-1, AC, POC or MPa was dissolved in DMSO and
1.49 (m, 6H), 1.38-1.15 (m, 8H), 0.92 (t, J = 6.9 Hz, 3H); 13C reached the final culture concentration of 0.1% in 200 ul
NMR (75 MHz, CDCl3) δ 178.31 (s), 155.26 (s), 148.03 (s), DMEM respectively. After treating with different
141.30 (s), 139.99 (s), 129.48 (s), 124.70 (s), 122.65 (s), 121.52 concentrations (0, 5, 25, 50, and 100 μM) of YC-1 ( AC, POC or
(s), 116.35 (s), 110.29 (s), 109.89 (s), 57.74 (s), 42.89 (s), 41.57 MPa) for 24h, 20 ul CCK-8 was added to the wells and
(s), 32.87 (s), 30.81 (s), 30.61(s, 3C), 28.64(s, 3C), 23.44 (s), incubated for additional 4h at 37 °C. The optical density of the
22.75 (s), 14.14 (s); Calc. for C26H32N2O3: 420.24, HRMS found: dissolved material was measured at 450 nm. The assays were
[M+Na]+ 443.2299.
performed in three independent experiments with n = 3.
5-(1′-benzyl-1H-indazol-3′-yl)furan-2-carboxylic acid (4): To a LX-2 apoptosis analysis: The apoptosis of cells were analyzed
solution of 3 (0.1 g, 0.33 mmol) in acetone (3 ml) was added a with Annexin V-FITC apoptosis detection kit and PI simple
solution of KMnO4 (0.06 g, 0.38 mM) in water (2 ml) at 0°C. staining. Activated HSCs LX-2 cells were plate in 6-well plates 2
The reaction mixture was stirred about 1 h and then filtered.
The residue was washed and the filtrate was acidified with 1 M YC-1
HCl, then extracted with EA (3 20 mL), the combined DCM observed by confocal microscope at 18 h. Cells were collected
×
105 cells/well and cultured 24 h. Then treated with 25 uM
AC, or MPa respectively. Cellular morphology were
,
×
layers were washed with water and brine, dried (anhydrous by trypsinization after 24 and 48 h, followed by repeated
Na2SO4), filtration and the solvent was removed under reduced washes with PBS. Cells were then incubated with propidium
pressure. The crude product was purified by recrystallization iodide (PI) or FITC conjugated annexin-V and propidium iodide
from acetonitrile to give 4 as a light yellow solid (0.034 g, yield (PI) away from light for 20 min at room temperature. PI-
32%). 1H NMR (300 MHz, DMSO) δ 13.19 (s, 1H), 8.16 (d, J = 8.1 positive cells and FITC-positive / Pinegative cells were analyzed
Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.50 (td, J = 8.4, 0.9 Hz, 1H), using a Becton-Dickinson FAC Scan flow cytometer respectively.
7.39 (d, J = 3.6 Hz, 1H), 7.36-7.25 (m, 6H), 7.19 (d, J = 3.6 Hz, Western blot analysis: Whole cell proteins were lysed on ice
1H), 5.77 (s, 2H). 13C NMR (75 MHz, DMSO) δ 159.76 (s), 151.82 by RIPA lysis buffer with 1% phenylmethylsulfonyl fluoride
(s), 144.50 (s), 140.84 (s), 137.52 (s), 134.78 (s), 129.12 (s, 2C)), (PMSF), centrifuged at 15,000
g for 15 min and the
128.14 (s), 127.81 (s, 2C), 127.63 (s), 122.71 (s), 121.31 (s), supernatant was collected. Protein concentration was
120.99 (s), 119.98 (s), 111.05 (s), 109.22 (s), 52.64 (s); Calc. for measured using enhanced BCA protein assay kit. Equal
C19H14N2O3: 318.33, Tof-MS found: 318.8.
amounts of the protein (60 μg/lane) were separated by SDS-
PAGE and transferred to NC membrane. The membranes were
5-(1-benzyl-1H-indazol-3-yl)-N-(prop-2-yn-1-yl)furan-2-
carboxamide (MPa): To a solution of 4 (0.030 g, 0.094 mM) in blocked with 5% milk containing 0.1% Tween 20 for 1 h at
anhydrous THF (5 ml) was added EDC·HCl (0.022 g, 0.115 mM) room temperature. Blots were probed overnight at 4 °C with
under an argon atmosphere, followed by the addition of HOBT the following primary antibodies: anti-α–SMA and anti-β-actin
(0.013 g, 0.096 mM) and DIEA (10 ul). The reaction mixture antibodies at a dilution of 1:1000. This was followed by
was stirred for 30 min followed by addition of propynylamine incubation with secondary antibody: anti-rabbit IgG antibody
(0.006 g, 0.1 mM). The resulting mixture was stirred 24 h at conjugated with horseradish peroxidase 1:5000 for 1 h.
room temperature then saturated aqueous NaHCO3 (20 ml) Immuno-detection was visualized using the ECL detection
was added, and the mixture was extracted with EA (3
×20 ml). system according to the manufacturer's instructions.
The combined organic layers washed with water and brine,
dried (Na2SO4), filtration and the solvent was removed under
reduced pressure. The crude product was purified by column
chromatography (silica gel, ethyl acetate/ Petroleum ether 1 :
2) to give MPa as a white solid (0.047 g, yield 45%). 1H NMR
(300 MHz, CDCl3) δ 8.02 (d, J = 8.1 Hz, 1H), 7.49-7.39 (m, 2H),
7.38-7.23 (m, 6H), 7.02 (d, J = 3.6 Hz, 1H), 6.87 (d, J = 3.6 Hz,
1H), 5.69 (s, 2H), 4.31 (d, J = 2.5 Hz, 2H), 2.31 (t, J = 2.5 Hz, 1H),
1.98 (brs, 1H). 13C NMR (75 MHz, CDCl3) δ 157.93 (s), 150.22 (s),
146.59 (s), 140.62 (s), 136.32 (s), 135.15 (s), 128.85 (s, 2C),
128.00 (s), 127.18 (s), 127.12 (s, 2C), 122.10 (s), 121.48 (s),
120.98 (s), 116.77 (s), 109.97 (s), 109.40 (s), 79.40 (s), 71.77 (s),
53.40 (s), 28.88 (s); Calc. for C22H17N2O2: 356.39, HRMS found:
356.1382.
Acknowledgements
We thank the Natural Science Foundation of China (NSFC)
(grant nos. 21165019) for support of this work.
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