Synthesis and biological evaluation of 1,2-dithiol-3-thiones and pyrrolo[1,2-a]pyrazines as novel hypoxia inducible factor-1 (HIF-1) inhibitor

Article abstract of DOI:10.1016/j.bmc.2016.04.054

Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor which is strongly associated with tumor survival, progression, and therapeutic resistance. Accordingly, it has been suggested that the inhibition of the HIF-1 pathway can suppress tumor, and it has become an important therapeutic target. In present study, oltipraz, its metabolite M2, and their derivatives were synthesized and evaluated as HIF-1α inhibitors. Among the synthesized, benzyl-substituted pyrrolo[1,2-a]pyrazine 2g most potently inhibited HIF-1α protein accumulation (81% at 10 μM) and VEGF, GLUT-1 transcription (77% and 92% at 10 μM, respectively).

Full text of DOI:10.1016/j.bmc.2016.04.054

Accepted Manuscript
Synthesis and biological evaluation of 1,2-dithiol-3-thiones and pyrrolo[1,2-
a]pyrazines as novel hypoxia inducible factor-1 (HIF-1) inhibitor
Young Hun Lee, Jung Min Lee, Sang Geon Kim, Yong Sup Lee
PII:
S0968-0896(16)30297-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.04.054
BMC 12969
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
12 March 2016
24 April 2016
25 April 2016
Please cite this article as: Lee, Y.H., Lee, J.M., Kim, S.G., Lee, Y.S., Synthesis and biological evaluation of 1,2-
dithiol-3-thiones and pyrrolo[1,2-a]pyrazines as novel hypoxia inducible factor-1 (HIF-1) inhibitor, Bioorganic &
Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.04.054
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3
a
b
b
a,c
Young Hun Lee , Jung Min Lee , Sang Geon Kim , Yong Sup Lee *
a
Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 02447, Republic of Korea
b
Department of Pharmacy, College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea
c
Medicinal Chemistry Laboratory, Department of Pharmacy, College of Pharmacy, Kyung Hee University, Seoul 02447, Republic of Korea

1
Bioorganic & Medicinal Chemistry
Synthesis and biological evaluation of 1,2-dithiol-3-thiones and pyrrolo[1,2-
a]pyrazines as novel hypoxia inducible factor-1 (HIF-1) inhibitor
a
b
b
a,c
Young Hun Lee , Jung Min Lee
,
Sang Geon Kim and Yong Sup Lee 
a
Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 02447, Republic of Korea
Department of Pharmacy, College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea
b
c
Medicinal Chemistry Laboratory, Department of Pharmacy, College of Pharmacy, Kyung Hee University, Seoul 02447, Republic of Korea
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor which is strongly associated
with tumor survival, progression, and therapeutic resistance. Accordingly, it has been suggested
that the inhibition of the HIF-1 pathway can suppress tumor, and it has become an important
therapeutic target. In present study, oltipraz, its metabolite M2, and their derivatives were
synthesized and evaluated as HIF-1α inhibitors. Among the synthesized, benzyl-substituted
pyrrolo[1,2-a]pyrazine 2g most potently inhibited HIF-1α protein accumulation (81% at 10 μM)
and VEGF, GLUT-1 transcription (77% and 92% at 10 μM, respectively).
Available online
Keywords:
Hypoxia inducible factor-1
VEGF
2
009 Elsevier Ltd. All rights reserved.
GLUT-1
oltipraz
pyrrolopyrazine
1
. Introduction
protein using a wide range of growth-promoting stimuli and
oncogenic signals for their survival and proliferation.
7
Inadequate vascularization associated with fast proliferating
solid tumors limits efficient oxygen supply and renders the tumor
hypoxic, which eventually leads to tumor necrosis. In hypoxic
condition, however, many of cancer cells promote appropriate
responses such as anaerobic metabolism and increased oxygen
delivery so that they can survive and even proliferate in a
hypoxic environment. These processes hinder the effectiveness of
chemotherapy and radiotherapy, and they also contribute to
HIF-1 have been evaluated as a cancer therapeutic target via a
variety of approaches. Clinically, overexpression of HIF-1α has
8
–10
been found in many cancer cells,
treatment failure and increased patient mortality.
inhibition of the HIF-1 pathway significantly inhibits tumor
and this is associated with
11–14
The
15
growth in animal models. Accordingly, HIF-1 represents an
attractive molecular target for the development of novel
1
,2
16
aggressive tumor behavior.
anticancer agents.
Hypoxia-inducible factor-1 (HIF-1) is a major modulator
expressed in many tumor cells and responsible for orchestrating
diverse cellular responses such as angiogenesis and glycolysis
3
that help cancer cells adapt to hypoxic conditions. At normal
oxygen levels, HIF-1α is continually degraded by the pVHL (von
Hippel-Lindau protein)-mediated ubiquitination and proteasomal
degradation and the degradation pathway requires O . Therefore,
2
under hypoxic conditions, HIF-1α rapidly accumulates in the cell
and dimerizes with HIF-1β to form the active transcription factor
4,5
HIF-1. HIF-1 binds to hypoxia-responsive elements (HRE)
with co-activator p300 and CBP. The binding activates the
transcription of variety of genes involved in angiogenesis,
glycolysis, growth factor signaling, tumor invasion, and
Figure 1. Chemical structures of oltipraz metabolites M1–4
6
metastasis. Moreover, many cancer cells induce HIF-1 pathway
2
O -independently via enhancing de novo synthesis of HIF-1α
—
——

Corresponding author. Tel.: +82-2-961-0370; e-mail: kyslee@khu.ac.kr

2
A growing number of studies have reported that a series of
compounds, such as YC-1, rapamycin, PX-478, and 17-AAG,
from other reported HIF-1 inhibitors and may lead to significant
insight into novel approaches to tumor suppression.
17–20
inhibit HIF-1α pathway in cellular and animal models.
In the present study, we synthesized new 4-substituted 1,2-
dithiol-3-thiones 1a–k (oltipraz derivatives) and 7-substituted
pyrrolo[1,2-a]pyrazines 2a–l (M2 derivatives), in order to study
the structure-activity relationships, and thereby, to provide new
leads possessing enhanced HIF-1 inhibitory activity in
comparison to oltipraz and M2. We introduced various alkyl
chain to determine the effects of the length of alkyl group, and
various phenyl, benzyl, phenethyl group to determine the effects
of the additional aromatic ring linked to the parent compound.
All the pyrrolopyrazine compounds 2a–l were synthesized as
HCl salt to increase stability and water solubility. (Figure 2)
However, because of their toxicity and other adverse effects,
2
2
. Results and discussion
.1. Chemistry
The syntheses of 1,2-dithiol-3-thione derivatives 1a–k were
Figure 2. Structures of oltipraz, M2, and their derivatives 1a–k, 2a–l.
accomplished using the procedures shown in Scheme 1. Mixed
Claisen condensation of methyl pyrazine-2-carboxylate 3 with
various ester compounds 4a–k yielded the 2-substituted 3-
oxoester intermediates 5a–k. Since the yields of thionation
reactions are largely affected by the purity of 3-oxoester
current pharmacologic interventions in HIF-1α activity are
limited and expected to be advanced by tailor-made drugs.
Oltipraz is a synthetic dithiolethione (5-(2-pyrazinyl)-4-
methyl-1,2-dithiol-3-thione) that is structurally similar to the
dithiolethiones found in cruciferous vegetables. It originally was
marketed by Rhone-Poulenc (Vitry-sur-Seine, France) for the
treatment of schistosomiasis, and extensively evaluated as a
intermediates,
compounds
5a–k
were
purified
by
recrystallization in EtOAc/n-Hexane. The 1,2-dithiol-3-thione
compounds are usually synthesized from 3-oxoesters using
2
5
thionation reaction, which suffers from low yield. There are
many efforts to improve the thionation reaction, but the
thionation reaction of 3-oxoesters which have pyrazinyl moiety at
C-3 still shows low yield. Among the reported procedure, we
chose the recent and improved procedure reported by Curphey et
6,21
chemopreventive agent in the 1990s.
Recent studies revealed that oltipraz inhibits HIF-1 pathway
by affecting both synthesis and degradation of HIF-1α. Oltipraz
was found to inhibit HIF-1α synthesis by suppressing
mammalian target of rapamycin (mTOR) and p70 ribosomal S6
kinase-1 (S6K1) pathway and promote degradation by facilitating
26
al. and the modification of the procedure was used to synthesize
,2-dithiol-3-thione compounds 1a–k. Compounds 5a–k were
reacted with P 10 in refluxing toluene and recrystallized in
1
4
S
2
2
ubiquitylation. Separate pharmacokinetic studies revealed that
oltipraz is metabolized by the two major pathways: first,
oxidative desulfuration of the thione to yield M1, which does not
seem to be metabolized further; and second, desulfuration,
methylation, and molecular rearrangement to yield M2 (7-
methyl-6,8-bis(methylthio)pyrrolo[1,2-a]pyrazine), which can be
acetonitrile to give 1a–k in acceptable yields.
The pyrrolo[1,2-a]pyrazine derivatives 2a–l were synthesized
by using the modification of Largeron’s method. (Scheme 2)
Lageron et al. reported that M2 could be synthesized from
oltipraz by rearrangement using sodium thiomethoxide
2
7
23
(NaSMe).
The key features of the rearrangement are
metabolized to other oxidized forms, M3 and M4 (Figure 1).
-
nucleophilic attack of MeS at S-2 position and intramolecular
ring closure with elimination of molecular sulfur. Compounds
M2 was also reported to suppress HIF-1 pathway by inhibiting de
novo synthesis of HIF-1α via induction of microRNAs 199a-5p
and 20a indicating that M2 has distinct mechanism of HIF-1α
1b–k were reacted with NaSMe to undergo rearrangement that
24
formulates pyrrolopyrazine structure, and then reacted with CH
3
I
inhibition in comparison to oltipraz. Such a mechanism differs
Scheme 1. Synthesis of compounds 1a–k

3
Scheme 2. Synthesis of compounds 2a–l
to yield compounds 2b–k as free base form. The resulting
Insulin also promotes HIF1A mRNA translation and thus
compounds were dissolved in 1.25 M HCl methanol solution,
increases de novo synthesis of HIF-1α through the mTOR/ S6K1
3
0
stirred for 1 h and then precipitated in MeOH/Et
yield compounds 2b–k as HCl salt. However, reaction of 1a with
NaSMe followed by methylation using CH I yielded 7-
2
O solution to
pathway.
Accordingly, the HIF-1α protein levels were
determined using Western blot assay to evaluate the effects of the
synthesized compounds 1a–k and 2a–l on insulin induced HIF-
1α accumulation. The effects on HIF-1α expression were
monitored at proper concentration (1a–k: 30 μM, 2a–l: 10 μM) in
which most of the compounds showed effective HIF-1α
inhibition and did not show significant cytotoxicities. Few
compounds (1a, 2h–j) exhibited severe cytotoxicity at the assay
condition and their effects on HIF-1α accumulation were not
determined. Topotecan and YC-1 were used as positive control
and their effects were determined at 3 μM in which the
compounds showed effective inhibitory activities without
significant cytotoxicities. The results are listed in Figure 3.
3
methylthio substituted compound 2l instead of 2a. It was
supposed that the thiomethoxide attacked C-4 instead of S-2 and
the following ring closure resulted the compound 2l. Finally,
compound 2a could be synthesized using t-BuOK as nucleophile
in t-BuOH.
2.2. Inhibitory effects of 1a–k and 2a–l on insulin-induced
HIF-1α accumulation
It has been known that insulin decreases both synthesis and
stability of HIF-1α. Insulin affects the stability of the HIF-1α
2
8,29
protein by generating ROS, in particular H
2
O
2
.
2 2
H O
As shown in Figure 3, insulin induced HIF-1α accumulations
were potently inhibited by several dithiolethiones (1d: 76%; 1e:
inactivates proline hydroxylase-domain enzymes, which initiate
29
ubiquitylation and degradation, thereby stabilizing HIF-1α.
80%) and pyrrolopyrazines (2c: 86%; 2d: 90%; 2g: 81%) at 30
Figure 3. Inhibitory effects of 1a–k (Figure 3a) and 2a–l (Figure 3b) on insulin-induced HIF-1α accumulation assessed
by protein immunoblot (Western blot) analysis. The values above the figures represent relative density of the bands
normalized to HIF-1β. The values are mean ± S.E.M. from three independent experiments. **significantly different from
#
##
vehicle-treated control (P < 0.01); significantly different from insulin-treated control (P < 0.05); significantly different
†
from insulin-treated control (P < 0.01); not determined due to cytotoxicity; To, topotecan (positive control); YC, YC-1
(positive control)

4
μM, 10 μM respectively. The inhibitory activities of these
compounds were higher than those of their parent compounds
1b: 48%; 2b: 37%). Generally, pyrrolopyrazine derivatives
analysis results are well correlated with the Western blot data
(Figure 4). Compounds with long alkyl substituents (1d, 2d)
potently inhibited target gene expression and phenyl substituted
compounds (1f, 2f) showed weak inhibition. The propyl-
substituted 1d exhibited the most potent inhibition (VEGF: 70%,
GLUT-1: 86%, at 30 μM) among the dithiolethione derivatives,
and benzyl-substituted 2g showed the most potent inhibition
(VEGF: 77%, GLUT-1: 92%, at 10 μM) among the
pyrrolopyrazine derivatives. The inhibitory activities of these two
compounds were markedly increased in comparison to their
parent compound 1b (VEGF: 36%, GLUT-1: 59%, at 30 μM)
and 2b (VEGF: no inhibition, GLUT-1: 14%, at 10 μM). These
results indicate that the synthesized compounds can inhibit target
gene expression as well as HIF-1α accumulation showing the
promising anticancer activities of the compounds.
(
showed similar or higher inhibitory activities at lower
concentration in comparison to dithiolethione compounds. It is
noteworthy that the analogues with the longer alkyl moiety
showed the more potent inhibitory activities. The inhibitory
activities of phenyl substituted analogues 1f (8%) and 2f (3%)
dramatically diminished. This is likely due to an inappropriate
positioning of phenyl ring which induces undesired interaction
with target protein. A comparison of the phenyl moiety of 1f
(8%), the benzyl moiety of 1g (41%), and the phenethyl moiety
of 1k (65%) showed that additional aromatic substituents with
appropriate alkyl linker could enhance HIF-1 inhibitory
activities. The similar trend can be seen with the pyrrolopyrazine
derivatives (2f: 3%; 2g: 81%; 2k: 87%). The 7-methylthio
substitution (2l: 6%) resulted in a significant decrease in HIF-1α
inhibition.
3. Conclusion
In summary, we have synthesized 11 dithiolethione
compounds and 12 pyrrolopyrazine compounds based on oltipraz
and its metabolite M2, respectively. These compounds were
evaluated for their inhibitory activities on insulin induced HIF-1α
accumulation and HIF-1 target gene VEGF and GLUT-1
expressions. The introduction of long alkyl moiety enhanced
HIF-1α inhibition in both dithiolethione and pyrrolopyrazine
derivatives. Benzyl and phenethyl substitutions also increased
inhibitory activities indicating that additional aromatic
substituents with proper alkyl linker could improve HIF-1α
2
.3. Inhibitory effects of 1a–k and 2a–l on insulin-induced
HIF-1 target gene transcription
A subset of these compounds was analyzed by RT-PCR to
confirm that inhibition of HIF-1α results in decreased expression
of its target genes. The effects of selected compounds on the
mRNA expression of VEGF and GLUT-1 were determined, both
of which are well-known HIF-1 target genes and associated with
3
the aggressive tumor phenotype. Generally, the RT-PCR
Figure 4. Inhibitory effects of the selected subset of compounds on insulin-induced HIF-1 target gene expression. The values are mean ± S.E.M. from three
#
independent experiments. **significantly different from vehicle-treated control (P < 0.01); significantly different from insulin-treated control (P < 0.05);
#
#
significantly different from insulin-treated control (P < 0.01); To, topotecan (positive control); YC, YC-1 (positive control)

5
1
inhibition. Among the synthesized compounds, compound 2g
containing benzyl moiety at 7-position most potently inhibited
HIF-1α accumulation (81%) and their target gene expressions
3
for 5a. Yield 79%. H NMR (CDCl , 400 MHz) δ 9.28 (d, 1H, J
= 1.4 Hz), 8.78 (d, 1H, J = 2.4 Hz), 8.64 (dd, 1H, J = 2.4, 1.4
Hz), 4.68 (q, 1H, J = 7.1 Hz), 3.68 (s, 3H), 1.52 (d, 3H, J = 7.1
Hz).
(VEGF: 77%, GLUT-1: 92%) at 10 μM without severe
cytotoxicity showing marked enhancement of inhibitory activity
in comparison to those of M2 (HIF-1α: 37%, VEGF: no
inhibition, GLUT-1: 14%). These results provided an insight into
structure-activity relationship of 4-substituted dithiolethiones and
4
.2.4. Methyl 2-(pyrazine-2-carbonyl)butanoate (5c)
The compound 5c was prepared from methyl pyrazine-2-
carboxylate and methyl butyrate using the procedure described
for 5a. Yield 64%. H NMR (CDCl , 400 MHz) δ 9.28 (d, 1H, J
=
Hz), 4.57 (t, 1H, J = 7.3 Hz), 3.67 (s, 3H), 2.06 (m, 2H), 1.02 (t,
3
1
7
4
4
-substituted pyrrolopyrazines.
3
1.4 Hz), 8.78 (d, 1H, J = 2.4 Hz), 8.65 (dd, 1H, J = 2.4, 1.4
. Experimental.
H, J = 7.3 Hz).
.1. Instrumentation and reagents
4.2.5. Methyl 2-(pyrazine-2-carbonyl)pentanoate (5d)
Nuclear magnetic resonance (NMR) spectral analyses were
performed using a Brucker Avance 400 spectrometer operating at
The compound 5d was prepared from methyl pyrazine-2-
1
13
400 MHz for H NMR and 100 MHz for C NMR spectra.
carboxylate and methyl pentanoate using the procedure described
1
Chemical shifts (δ) are reported in ppm, downfield from internal
tetramethylsilane (TMS) standard. High resolution mass spectra
for 5a. Yield 75%. H NMR (CDCl , 400 MHz) δ 9.27 (d, 1H, J
3
= 1.5 Hz), 8.78 (d, 1H, J = 2.4 Hz), 8.65 (dd, 1H, J = 2.4, 1.5
Hz), 4.66 (t, 1H, J = 7.1 Hz), 3.67 (s, 3H), 2.00 (m, 2H), 1.41 (m,
2H), 0.95 (t, 3H, J = 7.3 Hz).
(HRMS) were recorded on a Jeol accuTOF (JMS-T100TD)
equipped with a DART (direct analysis in real time) ion source
from Ionsense (Tokyo, Japan) in the positive modes. Analytical
thin layer chromatography (TLC) was carried out using precoated
silica gel (E. Merck Kiesegel 60F254, layer thickness 0.25 mm)
and flash column chromatography was performed with using
Merck Kiesegel 60 Art 9385 (230–400 mesh). All solvents,
chemicals and reagents were purchased from Sigma-Aldrich or
Tokyo chemical industry (TCI), and used without further
purification. Antibodies specifically directed against HIF-1α and
HIF-1β were purchased from Becton-Dickinson Biosciences,
Anti-ubiquitin antibody was obtained from Sigma-Aldrich
chemical. Antibodies recognizing S6K1, p-S6K1, lamin A/C and
HSP70 were purchased from Cell Signaling Technology.
4
.2.6. Methyl 2-(pyrazine-2-carbonyl)hexanoate (5e)
The compound 5e was prepared from methyl pyrazine-2-
carboxylate and methyl pentanoate using the procedure described
1
for 5a. Yield 49%. H NMR (CDCl
1.5 Hz), 8.78 (d, 1H, J = 2.4 Hz), 8.65 (dd, 1H, J = 2.4, 1.5
Hz), 4.64 (t, 1H, J = 7.1 Hz), 3.67 (s, 3H), 2.06–1.97 (m, 2H),
3
, 400 MHz) δ 9.27 (d, 1H, J
=
1
.41–1.29 (m, 4H), 0.93–0.86 (m, 3H).
4
.2.7. Methyl 3-oxo-2-phenyl-3-(pyrazin-2-yl)propanoate (5f)
The compound 5f was prepared from methyl pyrazine-2-
carboxylate and methyl 2-phenylacetate using the procedure
1
described for 5a. Yield 41%. H NMR (CDCl
3
, 400 MHz) δ 9.28
4
4
.2. Synthesis
(
2
d, 1H, J = 1.4 Hz), 8.76 (d, 1H, J = 2.4 Hz), 8.65 (dd, 1H, J =
.4, 1.4 Hz), 7.45–7.30 (m, 5H), 6.11 (s, 1H), 3.74 (s, 3H).
.2.8. Methyl 2-benzyl-3-oxo-3-(pyrazin-2-yl)propanoate (5g)
The compound 5g was prepared from methyl pyrazine-2-
.2.1 Methyl pyrazine-2-carboxylate
4
Pyrazine-2-carboxylic acid (5 g, 40.3 mmol) was dissolved in
MeOH (150 ml), and a few drops of H SO were added. The
2 4
resulting reaction mixture was refluxed for 2 h. Methanol was
evaporated and the resulting reaction mixture was extracted with
carboxylate and methyl 3-phenylpropanoate using the procedure
1
described for 5a. Yield 60%. H NMR (CDCl
3
, 400 MHz) δ 9.22
EtOAc, washed with saturated NaHCO
and dried over anhydrous Na SO . The solvent was removed in
vacuo to give the methyl pyrazine-2-carboxylate (5.5 g, yield
3
solution, then with brine
(
d, 1H, J = 1.5 Hz), 8.75 (d, 1H, J = 2.4 Hz), 8.62 (dd, 1H, J =
2
4
2
3
1
.4, 1.5 Hz), 7.26–7.14 (m, 5H), 5.10 (dd, 1H, J = 7.8, 6.8 Hz),
.64 (s, 3H), 3.37 (dd, 1H, J = 13.7, 7.8 Hz), 3.32 (dd, 1H, J =
3.7, 6.8 Hz).
1
9
8
3
3
8%). H NMR (CDCl , 400 MHz) δ 9.32 (d, 1H, J = 1.5 Hz),
.78 (d, 1H, J = 2.4 Hz), 8.73 (dd, 1H, J = 2.4, 1.5 Hz), 4.04 (s,
H).
4.2.9.
Methyl
2-(4-methylbenzyl)-3-oxo-3-(pyrazin-2-
yl)propanoate (5h)
4.2.2 Methyl 3-oxo-3-(pyrazin-2-yl)propanoate (5a)
The compound 5h was prepared from methyl pyrazine-2-
To a mixture of NaH (60% in mineral oil, 0.68 g, 28.2 mmol),
carboxylate and methyl 3-p-tolylpropanoate using the procedure
methyl pyrazine-2-carboxylate (3 g, 21.7 mmol) and dry DMF
30 mL), methyl acetate (2.09 g, 28.2 mmol) was added dropwise
under N atmosphere. After being stirred at rt for 5 h, the reaction
mixture was concentrated in vacuo, and the residue treated with
saturated aqueous NaHCO solution followed by extraction with
EtOAc. The combined organic phase were dried with MgSO
1
described for 5a. Yield 51%. H NMR (CDCl
3
, 400 MHz) δ 9.23
(
(
d, 1H, J = 1.5 Hz), 8.76 (d, 1H, J = 2.4 Hz), 8.63 (dd, 1H, J =
2
2
1
3
.4, 1.5 Hz), 7.17–7.12 (m, 2H), 7.09–7.04 (m, 2H), 5.08 (dd,
H, J = 7.8, 6.8 Hz), 3.66 (s, 3H), 3.35 (dd, 1H, J = 14.2, 7.8 Hz),
.30 (dd, 1H, J = 14.2, 6.8 Hz), 2.29 (s, 3H).
3
4
,
filtered, and concentrated in vacuo. Column chromatographic
4.2.10.
Methyl
2-(4-methoxybenzyl)-3-oxo-3-(pyrazin-2-
purification (n-hexane / EtOAc = 8 : 1) yielded 5a as white solid
yl)propanoate (5i)
1
(1.95 g, 50%). H NMR (CDCl
3
, 400 MHz) δ keto form: 9.28 (d,
The compound 5i was prepared from methyl pyrazine-2-
carboxylate and methyl 3-(4-methoxyphenyl)propanoate using
the procedure described for 5a. Yield 44%. H NMR (CDCl
1
4
=
H, J = 1.5 Hz), 8.80 (d, 1H, J = 2.4 Hz), 8.66 (overlapped, 1H),
.18 (s, 2H), 3.74 (s, 3H); enol form: 12.30 (s, 1H), 9.15 (d, 1H, J
1.5 Hz), 8.66 (overlapped, 1H), 8.60 (dd, 1H, J = 2.3, 1.5 Hz),
1
3
, 400
MHz) δ 9.21 (d, 1H, J = 1.5 Hz), 8.74 (d, 1H, J = 2.4 Hz), 8.61
dd, 1H, J = 2.4, 1.5 Hz), 7.17–7.13 (m, 2H), 6.79–6.75 (m, 2H),
5.05 (dd, 1H, J = 7.8, 6.8 Hz), 3.74 (s, 3H), 3.63 (s, 3H), 3.31
dd, 1H, J = 14.2, 7.8 Hz), 3.26 (dd, 1H, J = 14.2, 6.8 Hz).
6.34 (s, 1H), 3.85 (s, 3H).
(
4.2.3. Methyl 2-methyl-3-oxo-3-(pyrazin-2-yl)propanoate (5b)
(
The compound 5b was prepared from methyl pyrazine-2-
carboxylate and methyl propionate using the procedure described

6
4.2.11.
Methyl
2-(4-chlorobenzyl)-3-oxo-3-(pyrazin-2-
4.2.17. 4-Butyl-5-(pyrazin-2-yl)-3H-1,2-dithiole-3-thione (1e)
yl)propanoate (5j)
The compound 1e was prepared from compound 5e using the
1
The compound 5j was prepared from methyl pyrazine-2-
carboxylate and methyl 3-(4-chlorophenyl)propanoate using the
procedure described for 5a. Yield 36%. H NMR (CDCl
procedure described for 1a. Yield 7%, red solid. mp 67 °C;
NMR (CDCl
(m, 2H), 2.97–2.91 (m, 2H), 1.64–1.54 (m, 2H), 1.48–1.36 (m,
H
3
, 400 MHz) δ 8.97 (d, 1H, J = 1.0 Hz), 8.76–8.73
1
3
, 400
13
MHz) δ 9.23 (d, 1H, J = 1.5 Hz), 8.76 (d, 1H, J = 2.4 Hz), 8.62
dd, 1H, J = 2.4, 1.5 Hz), 7.23–7.16 (m, 4H), 5.06 (dd, 1H, J =
2H), 0.93 (t, 3H, J = 7.3 Hz); C NMR (CDCl
3
, 100 MHz) δ
(
217.1, 163.4, 148.1, 147.5, 146.1, 144.9, 143.4, 30.3, 30.2, 23.0,
+
7
7
.8, 6.8 Hz), 3.74 (s, 3H), 3.63 (s, 3H), 3.33 (dd, 1H, J = 14.2,
.8 Hz), 3.29 (dd, 1H, J = 14.2, 6.8 Hz).
12 2 3
14.0; HRMS-DART (m/z): [M+H] calcd for C11H N S ,
269.0235, found, 269.0169.
4.2.12. Methyl 4-phenyl-2-(pyrazine-2-carbonyl)butanoate
4.2.18. 4-Phenyl-5-(pyrazin-2-yl)-3H-1,2-dithiole-3-thione (1f)
(
5k)
The compound 1f was prepared from compound 5f using the
procedure described for 1a. Yield 14%, red solid. mp 213 °C; H
1
The compound 5k was prepared from methyl pyrazine-2-
carboxylate and methyl 4-phenylbutanoate using the procedure
NMR (CDCl
3
, 400 MHz) δ 8.62 (dd, 1H, J = 2.4, 1.5 Hz), 8.56
1
described for 5a. Yield 40%. H NMR (CDCl
s, 1H), 8.76 (s, 1H), 8.62 (s, 1H), 7.29–7.15 (m, 5H), 4.66 (t, 1H,
J = 6.9 Hz), 3.67 (s, 3H), 2.83–2.62 (m, 2H), 2.43–2.28 (m, 2H).
3
, 400 MHz) δ 9.24
(d, 1H, J = 2.4 Hz), 7.88 (d, 1H, J = 1.5 Hz), 7.55–7.49 (m, 3H),
7.28–7.23 (m, 2H); C NMR (CDCl
147.4, 146.3, 145.7, 144.0, 143.4, 134.1, 130.1, 129.8 (2C), 129.7
13
(
3
, 100 MHz) δ 216.3, 165.7,
+
8 2 3
(2C); HRMS-DART (m/z): [M+H] calcd for C13H N S ,
4.2.13. 5-(Pyrazin-2-yl)-3H-1,2-dithiole-3-thione (1a)
288.9922, found, 288.9873.
4
P S
10 (12 g, 27.0 mmol), toluene (90 ml) and xylene (90 ml)
4
.2.19. 4-Benzyl-5-(pyrazin-2-yl)-3H-1,2-dithiole-3-thione (1g)
were added to a reactor and heated to a temperature of 120 °C.
Compound 5a (8.1 g, 45.1 mmol) was added to the reactor. The
reaction mixture was allowed to proceed under reflux at 135 °C
for 4 h and then cooled to 20 °C and filtered through Celite. The
solvent was then removed under reduced pressure and the
The compound 1g was prepared from compound 5g using the
1
procedure described for 1a. Yield 16%, red solid. mp 167 °C; H
NMR (CDCl
(m, 2H), 7.27–7.06 (m, 5H), 4.41 (s, 2H); C NMR (CDCl
3
, 400 MHz) δ 8.77 (d, 1H, J = 1.0 Hz), 8.69–8.66
13
3
, 100
resultant red solid was extracted with CH
2
Cl
2
. The organic layer
SO
MHz) δ 216.9, 165.8, 147.6, 146.3, 144.9, 144.8, 143.6, 137.3,
was washed with aqueous K CO solution, dried over Na
2
3
2
4
,
129.1 (2C), 128.1 (2C), 126.9, 35.8; HRMS-DART (m/z):
+
and filtered. The solvent was removed under reduced pressure
and the crude solid was dried in vacuo. The dried solid was
washed with cold EtOAc, and then recrystallized in acetonitrile
[M+H] calcd for C14
10
H N
2 3
S , 303.0079, found, 303.0016.
4.2.20. 4-(Methylbenzyl)-5-(pyrazin-2-yl)-3H-1,2-dithiole-3-
1
thione (1h)
to give 1a (0.9 g, 10%) as a red solid. mp 202 °C; H NMR
(
2
(
1
2
CDCl
.4 Hz), 8.66 (dd, 1H, J = 2.4, 1.4 Hz), 7.76 (s, 1H); C NMR
CDCl , 100 MHz) δ 216.7, 168.3, 146.9, 146.1, 144.7, 141.5,
3
, 400 MHz) δ 9.05 (d, 1H, J = 1.4 Hz), 8.74 (d, 1H, J =
The compound 1h was prepared from compound 5h using the
procedure described for 1a. Yield 16%, red solid. mp 158 °C; H
13
1
3
NMR (CDCl , 400 MHz) δ 8.78 (d, 1H, J = 1.0 Hz), 8.69–8.66
3
+
36.0; HRMS-DART (m/z): [M+H] calcd for C
7 4
H N
2
S
3
,
(m, 2H), 7.08–7.02 (m, 2H), 7.00–6.95 (m, 2H), 4.36 (s, 2H),
13
12.9609, found, 212.9575.
3
2.28 (s, 3H); C NMR (CDCl , 100 MHz) δ 216.8, 165.5, 147.4,
1
3
3
46.1, 144.8, 144.5, 143.4, 136.3, 133.9, 129.5 (2C), 127.7 (2C),
5.2, 21.0; HRMS-DART (m/z): [M+H] calcd for C15H N S ,
12 2 3
17.0235, found, 317.0206.
4
.2.14.
4-Methyl-5-(pyrazin-2-yl)-3H-1,2-dithiole-3-thione
+
(1b)
The compound 1b was prepared from compound 5b using the
1
4.2.21. 4-(Methoxybenzyl)-5-(pyrazin-2-yl)-3H-1,2-dithiole-3-
thione (1i)
procedure described for 1a. Yield 14%, red solid. mp 165 °C; H
NMR (CDCl , 400 MHz) δ 9.01 (d, 1H, J = 1.2 Hz), 8.76–8.72
3
13
(
1
(
m, 2H), 2.51 (s, 3H); C NMR (CDCl
3
, 100 MHz) δ 216.8,
The compound 1i was prepared from compound 5i using the
1
63.0, 148.0, 145.9, 144.7, 143.6, 142.5, 17.5; HRMS-DART
procedure described for 1a. Yield 13%, red solid. mp 122 °C; H
+
m/z): [M+H] calcd for C
8
6
H N
2
S
3
, 226.9766, found, 226.9714.
NMR (CDCl , 400 MHz) δ 8.79 (d, 1H, J = 1.0 Hz), 8.69–8.66
3
(
3
m, 2H), 7.06–6.96 (m, 2H), 6.84–6.72 (m, 2H), 4.33 (s, 2H),
4.2.15. 4-Ethyl-5-(pyrazin-2-yl)-3H-1,2-dithiole-3-thione (1c)
13
.75 (s, 3H); C NMR (CDCl
3
, 100 MHz) δ 216.8, 165.4, 158.3,
The compound 1c was prepared from compound 5c using the
procedure described for 1a. Yield 5%, red solid. mp 102 °C; H
147.4, 146.1, 145.0, 144.5, 143.5, 129.0 (2C), 128.9 (2C), 114.3,
55.2, 34.8; HRMS-DART (m/z): [M+H] calcd for C15H N OS ,
12 2 3
1
+
NMR (DMSO-d
6
, 400 MHz) δ 9.10 (d, 1H, J = 1.5 Hz), 8.90 (d,
333.0185, found, 333.0095.
1
7
2
H, J = 2.4 Hz), 8.89 (dd, 1H, J = 2.4, 1.5 Hz), 2.87 (q, 2H, J =
13
4.2.22. 4-(Chlorobenzyl)-5-(pyrazin-2-yl)-3H-1,2-dithiole-3-
thione (1j)
.3 Hz), 1.11 (t, 3H, J = 7.3 Hz); C NMR (CDCl
3
, 100 MHz) δ
16.9, 163.6, 148.4, 148.0, 146.2, 145.0, 143.4, 24.1, 12.8;
+
HRMS-DART (m/z): [M+H] calcd for C
found, 240.9879.
9
8
H N
2
S
3
, 240.9922,
The compound 1j was prepared from compound 5j using the
1
procedure described for 1a. Yield 15%, red solid. mp 152 °C; H
NMR (CDCl
3
, 400 MHz) δ 8.78 (s, 1H), 8.72 (s, 2H), 7.25–7.21
4.2.16. 4-Propyl-5-(pyrazin-2-yl)-3H-1,2-dithiole-3-thione (1d)
13
(m, 2H), 7.08–7.04 (m, 2H), 4.38 (s, 2H); C NMR (CDCl , 100
3
The compound 1d was prepared from compound 5d using the
MHz) δ 216.6, 165.8, 147.5, 146.5, 144.9, 144.5, 143.5, 135.8,
1
procedure described for 1a. Yield 7%, red solid. mp 111 °C; H
NMR (CDCl , 400 MHz) δ 8.96 (d, 1H, J = 1.0 Hz), 8.76–8.73
m, 2H), 2.91 (m, 2H), 1.65 (m, 2H), 1.00 (t, 3H, J = 7.3 Hz);
NMR (CDCl , 100 MHz) δ 217.1, 163.6, 148.1, 147.3, 146.1,
132.8, 129.5 (2C), 129.1 (2C), 35.2; HRMS-DART (m/z):
+
3
[M+H] calcd for C14
9
H ClN
2 3
S , 336.9689, found, 336.9731.
1
3
(
C
4.2.23.
4-(Phenethyl)-5-(pyrazin-2-yl)-3H-1,2-dithiole-3-
3
+
thione (1k)
1
44.9, 143.4, 32.5, 21.7, 14.4; HRMS-DART (m/z): [M+H]
calcd for C10
H
10
N
2
S
3
, 255.0079, found, 255.0022.
The compound 1k was prepared from compound 5k using the
1
procedure described for 1a. Yield 11%, red solid. mp 68 °C;
H

7
NMR (CDCl
3
, 400 MHz) δ 8.70–8.65 (m, 3H), 7.24–7.13 (m,
8.99 (d, 1H, J = 1.5 Hz), 8.21 (dd, 1H, J = 4.9, 1.5 Hz), 7.72 (d,
3
7
1
3
3
H), 7.11–7.07 (m, 2H), 3.25 (t, 2H, J = 7.7 Hz), 2.93 (t, 2H, J =
1H, J = 4.9 Hz), 2.90 (t, 2H, J = 7.8 Hz), 2.30 (s, 3H), 2.22 (s,
3H), 1.75–1.61 (m, 2H), 1.02 (t, 3H, J = 7.3 Hz); C NMR
13
13
.7 Hz); C NMR (CDCl
3
, 100 MHz) δ 216.5, 163.9, 148.0,
46.2, 145.9, 144.8, 143.6, 140.7, 128.8 (2C), 128.7 (2C), 126.6,
3
(CDCl , 100 MHz, determined as free base) δ 143.4, 140.7,
+
12 2 3
3.8, 32.3; HRMS-DART (m/z): [M+H] calcd for C15H N S ,
131.7, 128.8, 116.5, 115.8, 108.1, 27.9, 25.0, 21.6, 18.3, 14.5;
+
17.0235, found, 317.0294.
2 2
HRMS-DART (m/z): [M-Cl] calcd for C12H17ClN S , 253.0828,
found, 253.0792.
4.2.24.
6,8-Bis(methylthio)pyrrolo[1,2-a]pyrazine
hydrochloride (2a)
4.2.28.
7-Butyl-6,8-bis(methylthio)pyrrolo[1,2-a]pyrazine
hydrochloride (2e)
To a solution of 1a (106 mg, 0.5 mmol) in t-BuOH (500 ml)
was added t-BuOK (0.7 g, 10 mmol) under N atmosphere at 45
C. After 90 min, the solution was methylated with an excess
CH I (7.1 g, 50 mmol). The solution was neutralised with dry-ice
2
The compound 2e was prepared from compound 1e using the
°
procedure described for 2b. Yield 85%, yellow solid. mp 153 °C
1
3
(dec.); H NMR (CDCl
3
, 400 MHz, determined as free base) δ
and evaporated to dryness in vacuo at 60 °C. The residue was
poured into water (50 ml) and extracted with EtOAc (50 ml). The
organic phase was dried over anhydrous Na SO and evaporated
2 4
to dryness giving a brown oil. Column chromatographic
purification (n-hexane / EtOAc = 15 : 1) yielded 2a as free base
from. The purified compound was added to ~1.25 M HCl
methanol solution and stirred at rt for 1 h. The solvent was
8.99 (d, 1H, J = 1.5 Hz), 8.20 (dd, 1H, J = 4.9, 1.5 Hz), 7.71 (d,
1H, J = 4.9 Hz), 2.92 (t, 2H, J = 7.8 Hz), 2.30 (s, 3H), 2.22 (s,
3H), 1.69–1.60 (m, 2H), 1.44–1.37 (m, 2H), 0.98 (t, 3H, J = 7.3
13
3
Hz); C NMR (CDCl , 100 MHz, determined as free base) δ
143.4, 141.0, 131.8, 128.8, 116.5, 115.7, 108.0, 34.0, 25.6, 23.1,
+
21.6, 18.3, 14.2; HRMS-DART (m/z): [M-Cl] calcd for
13 2 2
C H19ClN S , 267.0984, found, 267.0974.
removed and the residue was precipitated in Et
mg, 10%) as a yellow HCl salt. mp 165 °C (dec.); H NMR
2
O to afford 2a (12
1
4.2.29. 6,8-Bis(methylthio)-7-phenyl-pyrrolo[1,2-a]pyrazine
hydrochloride (2f)
3
(CDCl , 400 MHz, determined as free base) δ 8.97 (s, 1H), 8.14
(
2
C
dd, 1H, J = 4.9, 1.5 Hz), 7.71 (d, 1H, J = 4.9 Hz), 7.09 (s, 1H),
.42 (s, 3H), 2.32 (s, 3H); HRMS-DART (m/z): [M-Cl] calcd for
The compound 2f was prepared from compound 1f using the
+
procedure described for 2b. Yield 59%, yellow solid. mp 188 °C
1
9
H11ClN
2 2
S , 211.0358, found, 211.0369.
(dec.); H NMR (CDCl , 400 MHz, determined as free base) δ
3
9
7
1
.09 (s, 1H), 8.28 (d, 1H, J = 4.9 Hz), 7.77 (d, 1H, J = 4.9 Hz),
4
.2.25.
7-Methyl-6,8-bis(methylthio)pyrrolo[1,2-a]pyrazine
13
.61–7.40 (m, 5H), 2.13 (s, 3H), 2.11 (s, 3H); C NMR (CDCl
3
,
hydrochloride (2b)
00 MHz, determined as free base) δ 144.5, 139.1, 133.3, 131.5,
A mixture of 1b (100 mg, 0.4 mmol) and NaSMe (309 mg, 4.4
mmol) was dissolved in MeOH (10 ml) and 0.01 M aqueous
130.7 (2C), 129.3, 128.3 (2C), 128.0, 116.7, 116.3, 108.4, 20.9,
18.4; HRMS-DART (m/z): [M-Cl] calcd for C15H15ClN S ,
2 2
+
solution of NH
4
OAc (10 ml). The mixture was stirred at rt for 3
287.0671, found, 287.0657.
h. CH I (627 mg, 4.4 mmol) was added to the reaction mixture
3
4
.2.30.
7-Benzyl-6,8-bis(methylthio)pyrrolo[1,2-a]pyrazine
and stirred at rt for 3 h. After completing the reaction (TLC
monitoring), MeOH was removed in vacuo. The mixture was
extracted with EtOAc twice, and the extract was washed with
hydrochloride (2g)
The compound 2g was prepared from compound 1g using the
brine, dried over anhydrous Na
2
SO
4
and then filtered. The filtrate
procedure described for 2b. Yield 27%, yellow solid. mp 171 °C
(dec.); H NMR (CDCl , 400 MHz, determined as free base) δ
9.02 (d, 1H, J = 1.5 Hz), 8.22 (dd, 1H, J = 4.9, 1.5 Hz), 7.73 (d,
1H, J = 4.9 Hz), 7.29–7.12 (m, 5H), 4.33 (s, 2H), 2.12 (s, 3H),
2.01 (s, 3H); C NMR (CDCl , 100 MHz, determined as free
base) δ 143.5, 140.8, 139.2, 131.7, 128.9, 128.7 (2C), 128.3 (2C),
126.0, 116.4, 116.1, 108.3, 31.3, 21.0, 17.7; HRMS-DART (m/z):
[M-Cl] calcd for C H ClN S , 301.0828, found, 301.0857.
1
was concentrated and purified by chromatography on silica gel to
yield 2b as free base form. The purified compound was added
3
~
1.25 M HCl methanol solution and stirred at rt for 1 h. The
solvent was removed and the residue was precipitated in Et O to
afford 2b (57 mg, 53%) as a yellow HCl salt. mp 162 °C (dec.);
13
2
3
1
H NMR (CDCl
3
, 400 MHz, determined as free base) δ 8.99 (s,
+
1
H), 8.21 (dd, 1H, J = 4.9, 1.5 Hz), 7.70 (d, 1H, J = 4.9 Hz), 2.50
16
17
2 2
13
(s, 3H), 2.30 (s, 3H), 2.22 (s, 3H); C NMR (CDCl
3
, 100 MHz,
4.2.31.
7-(4-Methylbenzyl)-6,8-bis(methylthio)pyrrolo[1,2-
determined as free base) δ 143.2, 136.0, 131.3, 128.5, 116.3,
+
a]pyrazine hydrochloride (2h)
115.9, 108.1, 20.5, 17.8, 10.6; HRMS-DART (m/z): [M-Cl]
calcd for C10 , 225.0515, found, 225.0492.
H
2 2
13ClN S
The compound 2h was prepared from compound 1h using the
procedure described for 2b. Yield 92%, yellow solid. mp 190 °C
4
.2.26. 7-Ethyl-6,8-bis(methylthio)pyrrolo[1,2-a]pyrazine
1
(dec.); H NMR (CDCl
3
, 400 MHz, determined as free base) δ
hydrochloride (2c)
9.01 (d, 1H, J = 1.5 Hz), 8.21 (dd, 1H, J = 4.9, 1.5 Hz), 7.73 (d,
The compound 2c was prepared from compound 1c using the
1H, J = 4.9 Hz), 7.15 (m, 2H), 7.05 (m, 2H), 4.29 (s, 2H), 2.28 (s,
13
procedure described for 2b. Yield 77%, yellow solid. mp 157 °C
3H), 2.13 (s, 3H), 2.03 (s, 3H); C NMR (CDCl , 100 MHz,
3
1
(dec.); H NMR (CDCl
3
, 400 MHz, determined as free base) δ
determined as free base) δ 143.7, 139.6, 137.9, 135.6, 131.9,
8
1
3
.99 (d, 1H, J = 1.5 Hz), 8.21 (dd, 1H, J = 4.9, 1.5 Hz), 7.71 (d,
H, J = 4.9 Hz), 2.96 (q, 2H, J = 7.5 Hz), 2.31 (s, 3H), 2.23 (s,
129.3 (2C), 129.1, 128.8 (2C), 116.6, 116.3, 108.5, 31.1, 21.4,
+
21.3, 18.0; HRMS-DART (m/z): [M-Cl] calcd for C H ClN S ,
1
7
19
2 2
13
H), 1.28 (t, 3H, J = 7.5 Hz); C NMR (CDCl
3
, 100 MHz,
315.0984, found, 315.0967.
determined as free base) δ 143.5, 142.4, 131.8, 128.9, 116.5,
1
Cl] calcd for C11
4.2.32. 7-(4-Methoxybenzyl)-6,8-bis(methylthio)pyrrolo[1,2-
15.5, 107.7, 21.7, 19.2, 18.5, 16.5; HRMS-DART (m/z): [M-
+
a]pyrazine hydrochloride (2i)
2 2
H15ClN S , 239.0662, found, 239.0638.
The compound 2i was prepared from compound 1i using the
4
.2.27. 6,8-Bis(methylthio)-7-propyl-pyrrolo[1,2-a]pyrazine
procedure described for 2b. Yield 77%, yellow solid. mp 181 °C
hydrochloride (2d)
1
(
dec.); H NMR (CDCl
3
, 400 MHz, determined as free base) δ
The compound 2d was prepared from compound 1d using the
9.02 (s, 1H), 8.22 (d, 1H, J = 4.4 Hz), 7.73 (d, 1H, J = 4.4 Hz),
7.21–7.16 (m, 2H), 6.82–6.76 (m, 2H), 4.27 (s, 2H), 3.75 (s, 3H),
procedure described for 2b. Yield 65%, yellow solid. mp 155 °C
1
(dec.); H NMR (CDCl
3
, 400 MHz, determined as free base) δ

8
1
3
2
.13 (s, 3H), 2.03 (s, 3H); C NMR (CDCl
3
, 100 MHz,
detection kit (Amersham Biosciences). β-Actin served as an
internal control to verify equal loading of proteins. Band intensity
of the immunoblots was scanned with the Image Scan and
Analysis System (Alpha Innotech Co.). The total area of each
lane was integrated, and band intensity was determined using
Alpha Ease version 5.5 software, followed by background
subtraction.
determined as free base) δ 157.8, 143.3, 139.6, 132.8, 131.6,
1
2
C
29.6 (2C), 128.6, 116.4, 116.1, 113.7 (2C), 108.3, 55.2, 30.4,
+
1.1, 17.7; HRMS-DART (m/z): [M-Cl]
OS , 331.0933, found, 331.0949.
calcd for
17
H19ClN
2
2
4.2.33.
7-(4-Chlorobenzyl)-6,8-bis(methylthio)pyrrolo[1,2-
a]pyrazine hydrochloride (2j)
4.3.3. Real-time RT-PCR assay
The compound 2j was prepared from compound 1j using the
procedure described for 2b. Yield 35%, yellow solid. mp 182 °C
Total RNA was isolated from cells using Trizol (Invitrogen),
1
(dec.); H NMR (CDCl
3
, 400 MHz, determined as free base) δ
and cDNA was synthesized by reverse transcription using an
oligo (dT) primer. Then, RT-PCR was conducted with a Light
Cycler 1.5 (Roche) using a Light Cycler DNA master SYBR
green-I kit, following the manufacturer's instructions. PCR was
conducted using primers specifically directed against the genes
9
1
2
.02 (d, 1H, J = 1.5 Hz), 8.22 (dd, 1H, J = 4.9, 1.5 Hz), 7.75 (d,
H, J = 4.9 Hz), 7.24–7.17 (m, 4H), 4.29 (s, 2H), 2.14 (s, 3H),
13
3
.03 (s, 3H); C NMR (CDCl , 100 MHz, determined as free
base) δ 143.5, 139.3, 138.5, 131.8, 130.0 (2C), 129.0, 128.8,
28.4 (2C), 116.4, 116.0, 108.3, 30.7, 21.1, 17.7; HRMS-DART
1
encoding VEGF (sense, 5′-CCAAGAGTTTGTCTTTCAAC-3′;
+
33
(m/z): [M-Cl] calcd for C16
H
2
16Cl N
2
S
2
, 335.0438, found,
antisense, 5′-AGAGGCGTTGACATAGGCTT-3′),
GLUT-1
3
35.0430.
(sense, 5′-CGGGCCAAGAGTGTGCTAAA-3′; antisense, 5′-
34
TGACGATACCGGAGCCAATG-3′),
HIF-1α (sense, 5′-
4.2.34.
6,8-Bis(methylthio)-7-phenethyl-pyrrolo[1,2-
GTCGGACAGCCTCACCAAACAGAGC-3′; antisense, 5′-
a]pyrazine hydrochloride (2k)
35
GTTAACTTGATCCAAAGCTCTGAG-3′), and β-actin (sense,
The compound 2k was prepared from compound 1k using the
5′-CTCTTCCAGCCTTCCTTCCTG-3′;
CAGCACTGTGTTGGCGTACAG-3′).
antisense,
5′-
procedure described for 2b. Yield 55%, yellow solid. mp 173 °C
1
(
9
1
dec.); H NMR (CDCl
3
, 400 MHz, determined as free base) δ
4.3.4. Immunoprecipitation assay
.01 (d, 1H, J = 1.5 Hz), 8.19 (dd, 1H, J = 4.9, 1.5 Hz), 7.73 (d,
H, J = 4.9 Hz), 7.32–7.17 (m, 5H), 3.24 (t, 2H, J = 7.6 Hz), 2.99
To determine HIF-1α ubiquitylation, cells were transfected
13
(
t, 2H, J = 7.6 Hz), 2.28 (s, 3H), 2.11 (s, 3H); C NMR (CDCl
3
,
with the plasmid of His-Ubi. The lysates were incubated with
anti-His antibody overnight at 4 C. After immunoprecipitation,
the samples were subjected to SDS-PAGE and were
immunoblotted with anti-ubiquitin antibody. The antigen-
antibody complex was precipitated following incubation for 2 h
at 4 C with protein G-agarose. The immune complex was
solubilized in Laemmli 2 x concentrate buffer and heated for 5
min. The samples were immunoblotted with anti-ubiquitin
antibody.
1
1
2
00 MHz, determined as free base) δ 143.5, 142.0, 139.7, 131.8,
28.9, 128.8 (2C), 128.6 (2C), 126.3, 116.6, 116.0, 108.2, 37.8,
+
8.1, 21.5, 18.1; HRMS-DART (m/z): [M-Cl] calcd for
C
17
H19ClN
2 2
S , 315.0984, found, 315.0987.
4
.2.35.
6,7,8-Tris(methylthio)pyrrolo[1,2-a]pyrazine
hydrochloride (2l)
The compound 2l was prepared from compound 1a using the
procedure described for 2b. Yield 51%, yellow solid. mp 161 °C
1
4.3.5. Purification and quantification of miRNAs
(dec.); H NMR (CD
3
OD, 400 MHz, determined as free base) δ
9
.22 (s, 1H), 8.76 (dd, 1H, J = 5.4, 1.0 Hz), 7.75 (d, 1H, J = 5.4
Total RNA was prepared as described above. The cDNA was
synthesized by using the miScript Reverse Transcription kit
(QIAGEN) following the manufacturer’s instructions. Real-time
RT-PCR assay was conducted using the primers selective for
13
3
Hz), 2.70 (s, 3H), 2.57 (s, 3H), 2.54 (s, 3H); C NMR (CDCl ,
100 MHz, determined as free base) δ 143.3, 134.4, 128.5, 124.8,
121.3, 116.9, 114.0, 20.8, 19.6, 18.6; HRMS-DART (m/z): [M-
+
Cl] calcd for C10
H13ClN
2 3
S , 257.0235, found, 257.0229.
miRNAs
of
interest:
hsa-miRNA-199a-5p,
5′-
CCCAGTGTTCAGACTACCTGTTC-3′; hsa-miRNA-20a, 5′-
TAAAGTGCTTATAGTGCAGGTAG-3′; hsa-miRNA-20b, 5′-
CAAAGTGCTCATAGTGCAGGTAG-3′; hsa-miRNA-17, 5′-
CAAAGTGCTTACAGTGCAGGTAG-3′; hsa-miRNA-18a, 5′-
TAAGGTGCATCTAGTGCAGATAG-3′; hsa-miRNA-19a, 5′-
TGTGCAAATCTATGCAAAACTGA-3′; hsa-miRNA-19b-1, 5′-
TGTGCAAATCCATGCAAAACTGA-3′; hsa-miRNA-92a-1, 5′-
TATTGCACTTGTCCCGGCCTGT-3′; hsa-miRNA-210, 5′-
CTGTGCGTGTGACAGCGGCTGA-3′ and hsa-miRNA-519c-
3p, 5′-AAAGTGCATCTTTTTAGAGGAT-3′.
4
4
.3. Biological activity screening
.3.1. Cells and cell culture conditions
Human colon cancer cell line HCT116, were supplied from
American Type Culture Collection. The cells were maintained in
growth medium containing 10% fetal bovine serum, Dulbecco’s
modified Eagle’s medium and 5% penicillin-streptomycin at 37

2
C in a humidified atmosphere containing 5% CO . For all
experiments, HCT116 cells were grown to 80%–90% confluency
and were deprived of serum for 16 h before treatment.
4.3.6. Transfection of miRNAs
4.3.2. Immunoblot analysis
36
miRNA duplexes were prepared as described previously.
Cell lysates were prepared according to previously published
The following sequences were used to synthesize mimics:
miRNA-199a-5p, 5′-CCCAGUGUUCAGACUACCUGUUC-3′
31
methods. Briefly, cells were lysed in buffer containing 10 mM
tris(hydroxymethyl)aminomethane hydrochloride (pH 7.1), 0.1 M
NaCl, 1 mM ethylenediaminetetraacetic acid, 0.5% Nonidet P-
(guide)
(passenger)
and
5′-ACAGGUAGUCUGAACACUGGGUA-3′
and miRNA-20a, 5′-
4
0, 0.5% Triton X-100, 10% glycerol, 1 mM compound 1a–k, or
UAAAGUGCUUAUAGUGCAGGUAG-3′ (guide) and 5′-
ACCUGCACUAUAAGCACUUUAAG-3′ (passenger) (Bioneer
Co.). Cells were transiently transfected with 100 nM of control
mimics (Dharmacon CO.), miRNA-199a-5p mimics or miRNA-
20a mimics or a mixture of miRNA 199a-5p and 20a mimics (50
nM each) using FuGENE_HD Reagent (Roche) according to the
manufacturer’s instruction.
2a–l, and 0.5 mM phenylmethylsulfonyl fluoride, which was
supplemented with
Millipore). Sodium dodecyl sulfate - polyacrylamide gel
electrophoresis (SDS-PAGE) and immunoblot analyses were
performed as described previously. Immunoreactive protein
was visualized using the enhanced chemiluminescence ECL
a protease inhibitor cocktail (Merck
32

9
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