Small molecular inhibitors of miR-1 identified from photocycloadducts of acetylenes with 2-methoxy-1,4-naphthalenequinone

Article abstract of DOI:10.1016/j.bmc.2013.04.058

Small molecules which can modulate endogenous microRNAs are important chemical tools to study microRNA regulational network. In this Letter we screened the [2+2] photocycloadducts of 2-methoxy-1,4-naphthalenequinone with a series of aryl acetylenes on the

Full text of DOI:10.1016/j.bmc.2013.04.058

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Small molecular inhibitors of miR-1 identified from photocycload-
ducts of acetylenes with 2-methoxy-1,4-naphthalenequinone
b,
Su-Bee Tan a, Chengmei Huang b, Xuejiao Chen b, Yihan Wu b, Mi Zhou b, Chenyu Zhang a, , Yan Zhang
⇑
⇑
a School of Life Science, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
b School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online xxxx
Small molecules which can modulate endogenous microRNAs are important chemical tools to study
microRNA regulational network. In this Letter we screened the [2+2] photocycloadducts of 2-methoxy-
1,4-naphthalenequinone with a series of aryl acetylenes on their activity to modulate endogenous
microRNAs. A potent inhibitor of the muscle-specific miR-1 which is closely related with cardiac devel-
opment and disease was identified. The small molecular inhibitor was the cyclobutene type product
derived from the photocycloaddition of 2-methoxy-1,4-naphthalenequinone with tert-butyl (5-(phenyl-
ethynyl)quinolin-8-yl) carbonate. Analogues of the small molecular inhibitor were then prepared using
similar photocycloaddition reactions for evaluation on inhibition activity on miR-1 to provide struc-
ture–activity relationship of the miR-1 inhibitor.
Keywords:
Inhibitor of miR-1
Photocycloaddition
2-Methoxy-1,4-naphthalenequinone
Acetylene
Cyclobutene
Ó 2013 Published by Elsevier Ltd.
1. Introduction
Small molecules which can regulate the expression or function
of miRNAs are important tools for elucidating the regulatory mech-
MicroRNAs (miRNAs) are a class of endogeneous noncoding
small RNA which have emerged recently as a new class of modula-
tors of gene expression at the post-transcriptional level.1,2 The bio-
genesis pathway of mature miRNA from primary miRNAs has been
elucidated.2 Mature miRNAs can silence their target mRNA through
binding to its 30 untranslated region (30-UTR) upon loading into the
RNA-induced silencing complex (RISC) to repress mRNA transcrip-
tion or to trigger mRNA degradation.3 Over the past several years,
more and more evidences have indicated that dysregulation in the
expression of miRNAs contributes to the pathogenesis of many hu-
man diseases.4,5 For example, miRNAs such as miR-21,6–8 miR-25,
miR-214, miR-221,9,10 miR-17-92 cluster,11,12 were found to over-
express in various tumor tissue, while miRNAs such as miR-34,13
miR-15, miR-16,14,15 are down-regulated in tumorgenesis.
The regulation of gene expression by miRNAs which are abun-
dant in specific tissues also plays fundamental roles in diverse bio-
logical processes including cell proliferation and differentiation,
apoptosis, immune response, etc.2 For example, miR-1 is enriched
in skeletal muscle and is believed to mediate myogenesis, muscle
development and heart development.16–19 Due to the wide regula-
tory functions of miRNAs on gene expression, they have become
attractive biomarkers and therapeutic targets for various dis-
eases.20–22
anism of miRNAs and hold the promise to develop novel therapeu-
tic agents. By far only a few type of small molecules of miRNAs
have been identified.23 Dieters et al. identified small molecules that
can specifically regulate the expression of miR-21 or miR-122 from
established chemical libraries.24,25 Maiti and co-workers, discov-
ered a tuberculosis drug, streptomycin from 15 aminoglycosides
that can down-regulates miR-21 by binding to pre-miRNA (precur-
sor miRNA) and blocking the miRNA maturation process.26 Other
molecules such as enoxacin27 and our previously established pho-
toreaction product28 have been proved to activate the function of
miRNAs by enhancing TRBP activity or upregulating TRBP (TAR
RNA binding protein) expression.
Photocycloaddition reactions of carbonyl compounds with al-
kenes or acetylenes have been used by us as efficient method to
generate structurally diversified compounds.29–35 In the photore-
actions of naphthalene-1,4-dione and acetylenes, both the car-
bonyl group and the C@C bond between the two carbonyl groups
could react with the acetylene. Therefore both the benzoanthrace-
none product and the cyclobutene type product were generated.28
Successful identification of a universal activator of miRNAs from
the photoreaction products of naphthalene-1,4-dione and acety-
lenes encouraged us to evaluate systematically on the modulation
activity of different photocycloadducts on various endogeneous
miRNAs. In this Letter, we synthesized a series of substituted 2a-
methoxy-cyclobuta[b]naphthalene-3,8(2aH,8aH)-diones via che-
mo- and regio-selectively [2+2] photocycloadditions of 2-meth-
oxy-1,4-naphthalenequinone with various aryl acetyelens and
⇑
Corresponding authors. Tel.: +86 25 83686234; fax: +86 25 83686234 (C.Z); tel.:
+86 25 83593072; fax: +86 25 83685976 (Y.Z.).
E-mail addresses: cyzhang@nju.edu.cn (C. Zhang), njuzy@nju.edu.cn (Y. Zhang).
0968-0896/$ - see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2013.04.058
Please cite this article in press as: Tan, S.-B.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.058

2
S.-B. Tan et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
evaluated the activity of these compounds to modulate endoge-
nous miRNAs.
OBoc
N
O
O
O
O
OMe
OMe
hv
2. Results and discussion
+
2.1. Photocycloaddition of 2-methoxy-1,4-naphthalenequinone
with aryl acetylenes
N
OBoc
1
14
4a
With the methoxy group substituted on the 2-position of 1,4-
naphthalenequinone, photoreactions of 2-methoxy-1,4-naphtha-
lenequinone 1 with biphenylacetylene 2a happened exclusively
on the C@C bond to give cyclobutene type product. We therefore
synthesized a series of acetylenes including 2-phenyl or 2-azaaryl
phenylacetylenes 2a–2c, aza-aryl cyclopropyl acetylenes 3a–3f
and 5-ethynylquinolin-8-ol derivatives 4a–4c (Chart 1) to react
with 2-methoxy-1,4-naphthalenequinone to get compounds with
the same fused cyclobutene framework but different substitution
groups on the cyclobutene ring.
Irradiation of 1 with 2-phenyl or 2-azaaryl phenylacetylenes
2a–2c in benzene gave cyclobutenes 5–7 with yields ranging from
60% to 80% (Scheme 1). The photocycloaddition proceeded with
high chemo- and regio-selectivity. The photoreaction proceeded
via the pp⁄ excited state and the C@C bond conjugated with two
carbonyl groups reacted with the CBC bond in the acetylenes to
form the cyclobutene ring. For the acetylenes substituted with
different aryl groups, only one regio-isomer was obtained.
We used NOESY spectra to determine the relative position of
the aryl rings to the methoxy group (Supplementary data). In the
Scheme 3. Photoreaction of 1 with 4a.
NOESY spectrum of 6, the relevance between the methoxy proton
and the proton on the phenyl group was observed. In the NOESY of
compound 7, the proton on the pyrazine ring showed relevance
with the proton on the cyclobutene ring. The regio-selectivity of
the photocycloaddition can be rationalized by the fact that phenyl
ring is more electron-sufficient than the aza-aryl ring.
The photoreaction of 2-methoxy-1,4-naphthalenequinone 1
with cyclopropylethynyl aza-aryls 3a–3f also proceeded with high
regio-selectivity to give one regio-isomer predominantly
(Scheme 2). We also used NOESY spectra to determine the relative
position of the cyclopropyl ring to the methoxy group (Supplemen-
tary data). In the NOESY spectra of the products, relevance be-
tween the cyclopropyl proton and the proton on the cyclobutene
ring was also detected. The regio-selectivity in the photocycloaddi-
tion may be rationalized by the ability of the aryl ring to stabilize
the conjugated radical center in the diradical intermediates.
To get the acetylene substituted with 8-hydroxyquinoneline
ring, we used 5-bromoquinolin-8-yl tert-butyl carbonate to couple
with phenylacetylene catalyzed by Pd(PPh3)4. Then we tested the
photoreaction of 4a with 2-methoxy-1,4-naphthalenequinone 1.
Irradiation of 1 and 4a in benzene gave 14 as the only product with
a yield of 80% (Scheme 3). According to the NOESY spectrum of 14,
the quinoline ring resides on the carbon adjacent to the methoxy
substituted carbon. The regio-selectivity can also be rationalized
by the most stable diradical intermediates.
O
R1
2a
2b
2c
R1
=
O
N
N
O
1
N
2
N
N
N
N
R3
R2
3a
R2
=
3d
3e
4a
2.2. Preliminary assay of compounds 5–14 on different
endogenous miRNAs
R3
=
N
N
3b
3c
4b
4c
N
N
With compounds 5–14 obtained from the photocycloaddition of
2-methoxy-1,4-naphthalenequinone with different aryl substi-
tuted acetylenes, we were able to perform preliminary evaluation
on their effects on endogenous miRNAs. The cell-based evaluation
system was similar to that reported previously by us and oth-
N
SiMe3
3f
3
OBoc
4
Chart 1.
R1
O
O
5
6
7
R1
=
O
OMe
OMe
N
N
hv
+
R1
O
N
1
2a-2c
Scheme 1. Photoreactions of 2-methoxynaphthalene-1,4-dione 1 with 1-phenyl-2-arylethynes 2a–2c.
N
N
N
O
O
11
12
O
O
8
R2
=
R2
OMe
OMe
R2
hv
N
+
9
N
N
N
10
N
13
1
3a-3f
Scheme 2. Photoreactions of 1 with cyclopropyl aza-arylethynes 3a–3f.
Please cite this article in press as: Tan, S.-B.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.058

S.-B. Tan et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
ers.24,28 In general, luciferase reporter gene with the complemen-
tary sequence of the miRNAs of interest was tranfected into the
cells. b-Gal was used as internal reference for transfection efficacy
and cell viability. The disturbed expression or function of the end-
ogeneous miRNAs upon incubation with the compounds could
therein be read out from the increased or decreased luciferase sig-
nal from the cells relative to cells treated by DMSO as control.
We selected miR-1, miR-214, miR-25 and miR-150 as miRNAs
of our interest and constructed the corresponding cellular reporter
systems. MiR-1 is expressed specifically in muscle tissue and its
misregulation has been reported in coronary artery diseases and
heart failing.36,37 MiR-25 is highly conserved and is one of the
key modulator of TGFB signaling in many tumors, interfering with
cell cycle arrest and apoptosis.38 Expression of miR-214 was signif-
icantly deregulated in various types of cancer and miR-214 has
been reported to play distinct roles in different tumors.39,40 MiR-
150 is stably expressed in most immune cells, including macro-
phage, monocyte, mature T and B cell.41–43 It has been reported
closely related to inflammation, immune-response and autoim-
mune diseases.44,45
activation activity on miR-1 since the cellular luciferase signal
was only 45% to DMSO control.
Then we measured the activity of compounds 5–14 in modulat-
ing miR-150 in mouse leukaemic mococyte macrophage
RAW267.4 cells. The cells transfected with miR-150 reporter
showed no distinct change on luciferase expression upon treat-
ment with all these compounds compared with DMSO control (col-
umn 3 in Table 1). We further measured the activity of compounds
5–14 in the modulation of the oncogenic miR-25 and miR-214 in
A549 cells. No significant change was observed on the luciferase
signal of the reporter cells upon treatment of compounds 6–14
(column 4 and 5 in Table 1). The reporter cells for miR-25 and
miR-214 upon treatment of compound 5 showed significantly de-
creased luciferase signal, which indicated that this compound
could also activate miR-25 and miR-214.
Based on the preliminary screening on the 10 photocloadducts
with respect to their modulation activity on the four types of miR-
NAs, we were able to identify compound 14 as a potent inhibitor of
miR-1. This small molecule selectively inhibits the endogenous
miR-1 in muscle cells but shows no influence on other miRNAs
we tested. Due to the importance of miR-1 in coronary artery dis-
eases, we explored further on this types of small molecular inhib-
itors of miR-1.
According to the abundance of the four miRNAs of our interests,
we selected three different types of cell-lines to construct the cel-
lular assay systems. Mouse myoblast cell-line C2C12 was transfec-
ted with luciferase reporter for miR-1. Mouse leukaemic mococyte
macrophage cell-line RAW267.4 was transfected with luciferase
reporter for miR-150. Human lung adenocarcinoma cell-line
A549 was transfected with luciferase reporter for miR-25 and
miR-214 respectively. According to reports concerning the identifi-
2.3. Inhibition of compound 14 on miR-1
Upon identification of compound 14 as selective inhibitor of
miR-1, we investigated further the selectivity, dose-dependence
and mechanism of action on its inhibition of miR-1. C2C12 cells
transfected with control vector containing luciferase gene without
the complimentary sequence of miRNA were constructed as nega-
tive control. Reporter systems for several other miRNAs were also
constructed, including miR-21 in HeLa cells (cervical cancer cell-
line) and miR-9 in MCF-7 cells (breast cancer cell-line). Figure 1
showed the relative luciferase signal from various reporter cells
cation of small molecules for miRNA regulator, 10 lM of com-
pounds were normally used for screening. Therefore, activity
assay of our photocycloadducts were carried out under this con-
centration as well.
Modulation of compounds 5–14 on the endogenous miR-1,
miR-150, miR-25 and miR-214 was then evaluated. The relative
luciferase signal from the reporter cells upon treatment of com-
pounds 5–14 at 10
lM relative to that from the same cells treated
upon treatment with compound 14 at 10 lM for 48 h. In contrast
with DMSO control were summarized in Table 1. Firstly, we inves-
tigated the regulatory effect of the compounds on miR-1 which is
abundant in differentiated C2C12 cells. Since miR-1 is expressed
mainly in myotube, we incubated our reporter C2C12 cell-lines
in culture medium containing 2% horse serum after transfection
of reporter plasmid to induce differentiation. In the meanwhile,
to the potent inhibition on miR-1, the compound showed no signif-
icant alteration on the relative luciferase signal from the cells with
control vector and reporters for miR-21, miR-9, miR-25, miR-150
and miR-214.
10 lM of compounds were added and the cells were further incu-
bated for 48 h. Luciferase assay results indicated that compound 14
had potent inhibition on miR-1 since an increase of luciferase sig-
nal in C2C12 cells by ꢀ7 fold was observed in reporter cell treated
by 14 compared with that of DMSO control. Compared with com-
pound 14, the effect of other photocycloadducts 6–13 showed
much lower modulation activity on miR-1. Compound 5 showed
Table 1
Relative luciferase signals from reporter cells upon treatment with compounds 5–14a
Compound
miR-1
miR-150
miR-25
miR-214
5
6
7
8
0.45 0.01
0.90 0.04
1.12 0.09
1.55 0.17
0.93 0.16
0.84 0.25
2.06 0.61
1.36 0.02
0.87 0.23
7.11 1.03
0.91 0.27
1.16 0.40
1.32 0.44
1.20 0.39
1.04 0.40
1.63 0.47
1.02 0.27
0.83 0.15
1.75 0.60
1.35 0.34
0.63 0.01
0.95 0.05
1.01 0.05
0.92 0.05
0.97 0.09
0.83 0.08
1.16 0.11
1.00 0.04
1.16 0.25
0.95 0.01
0.58 0.07
0.95 0.01
0.93 0.08
0.82 0.10
0.91 0.11
0.74 0.08
1.28 0.06
0.93 0.04
1.10 0.03
1.01 0.11
9
10
11
12
13
14
Figure 1. Selective inhibition of compound 14 on miR-1. The luciferase signal from
various reporter cells was detected after 48 h incubation with compound 14. All
experiments were conducted in triplicates and normalized to DMSO control and the
data are presented as mean SE (n = 3).
a
All experiments were conducted in triplicates and normalized to DMSO control.
The relative luciferase signals are presented as mean SE (n = 3).
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4
S.-B. Tan et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
The dose-dependence of the compound on inhibition activity of
2.4. Inhibition activity of compound 14 analogues
miR-1 was then studied according to the relative luciferase signal
from reporter cells treated by compound 14 at different concentra-
tions. As shown in Figure 2, at concentrations between 0 and 5 lM,
the inhibition activity of the compound on miR-1 increased rapidly
with the increase of compound concentration in the cell culture
The distinct activity of compound 14 with other photocycload-
ducts 5–13 suggested that the 8-hydroxylquinoline ring substi-
tuted on the cyclobutene ring was crucial for its inhibition
activity on miR-1. To get more information on the structure–activ-
ity relationship, we synthesized a series of 14 analogues with the
same 8-hydroxylquinoline ring substituted cyclobutene framework
(Chart 2). Change of the substitution group on the 8-hydroxyquin-
oline ring was straight-forward as described in the experimental
part. The preparation of the cyclopropyl ring or trimethylsilyl group
substituted cyclobutenes 18 and 22 was based on similar photocy-
cloaddition reactions of 2-methoxy-1,4-naphthalenequinone with
the 5-cyclopropylethynyl or 5-(trimethylsilyl)ethynyl 8-tert-but-
oxyquinolines, respectively. Removal of the trimethylsilyl group
in 22–25 with TBAF gave 26–29, respectively.
media. While increase of concentration from 5 to 10 lM showed
no further increase on the inhibition activity. The dose-dependent
curve suggests that the inhibition of 14 on miR-1 was based on
specific interaction between the compound and its target.
The selective inhibition of miR-1 by compound 14 is most likely
due to the down-regulation of miR-1 expression in C2C12 cells. We
then checked the expression level of mature miR-1 in differentiated
C2C12 cells treated by the compound for 48 h using quantitative
RT-PCR. As shown in Figure 3, the expression level of miR-1 in com-
pound 14 treated cells decrease by ꢀ4 fold compared with that in
DMSO treated cells. As contrast, compound 14 did not significantly
change the expression level of other miRNAs we tested. The results
on the regulation of compound 14 on miRNA were consistent with
the luciferase assay results. Therefore, we conclude that compound
14 decreases the cytoplasmic levels of mature miR-1.
According to the dose-dependence curve of the inhibition activ-
ity of compound 14 on miR-1 in differentiated C2C12 cells, com-
pound 14 reached its maxima effect at concentration of 5
Therefore we evaluated the inhibition activity of compounds 15–
29 on miR-1 in C2C12 cells at the concentration of 5 M. Prior to
lM.
l
activity evaluations, cytotoxicity of compounds were examined
by MTT assay, and results showed that C2C12 cells were compati-
ble with most of these compounds at the concentration of 5 lM.
(Supplementary data) The relative luciferase signal from the repor-
ter cells for miR-1 treated by these compounds was shown in Fig-
ure 4. It seemed that the majority of the analogues had comparable
inhibition activity as compound 14 on miR-1.
OR4
N
OR5
N
O
O
O
O
OMe
18
19
Boc
H
OMe
R5
=
=
H
15
16
17
R4
=
Me
Ac
Me 20
Ac
21
OR7
OR6
N
Boc
H
26
R7
O
O
Boc
H
22
R6
=
O
N
27
28
29
23
Me
Ac
Me
24
Figure 2. Dose-dependence curve of the inhibition activity of compound 14 on
miR-1 in differentiated C2C12 cells. The values are presented as means SE (n = 3).
OMe
SiMe3
OMe
Ac 25
O
Chart 2.
Figure 3. Influence of compound 14 on the expression level of different endoge-
nous miRNAs. The expressions of mature miR-1 in differentiated C2C12 cells, miR-
21 in HeLa cells, miR-150 in RAW267.4 cells, miR-9 in MCF-7 cells, and miR-25/
miR-214 in A549 cells were determined by quantitative RT-PCR after treatment
with compound 14 for 48 h. All experiments were conducted in triplicates and
normalized to DMSO control and the data are presented as mean SE (n = 3).
Figure 4. Inhibition activity of compounds 15–29 on miR-1. The luciferase signals
from C2C12 transfected with Luc-miR-1 reporter were detected after 48 h incuba-
tion with compounds 15–29. All experiments were conducted in triplicates and
normalized to DMSO control. The relative luciferase signals are presented as
mean SE (n = 3).
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S.-B. Tan et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
The structural features of the compounds which lost their activ-
ity provided us information on the structure–activity relationship
on this type of miR-1 inhibitors. For the substitution groups on
the hydroxyl group, it seemed that Ac or Boc substitution did not
have significant influence on the inhibition activity. However,
methylation of the hydroxyl group easily diminished the activity.
Substitution group on the C@C of the cyclobutene seemed to be
necessary since compounds 26–29 with H group at this position
showed much lower activity compared with their counterparts
with phenyl, cyclopropyl or trimethylsilyl substitution groups.
7.37–7.30 (m, 6H), 4.49 (s, 1H), 3.50 (s, 3H); 13C NMR (75 MHz,
CDCl3): d 196.0, 195.1, 143.9, 141.2, 134.7, 134.4, 133.7, 133.6,
132.2, 131.6, 129.7, 129.2, 128.7 (4C), 128.2, 127.4 (2C), 127.0
(3C), 84.1, 56.9, 53.4; HRMS (ESI): m/z calcd for C25H18NaO3
[M+Na]+ 389.1148, found 389.1150.
Photocycloaddition of 1 with 2b gave 6 (58%), pale yellow crys-
tal; MP: 144–145 °C; 1H NMR (300 MHz, CDCl3): d 8.7–8.68 (m,
1H), 8.39 (d, 1H, J = 1.8 Hz), 8.37 (d, 1H, J = 1.2 Hz), 8.06–8.03 (m,
1H), 7.86–7.83 (m, 1H), 7.77 (td, 1H, J = 7.8, 1.8 Hz), 7.70–7.63
(m, 3H), 7.43–7.34 (m, 3H), 7.26–7.22 (m, 1H), 4.56 (s, 1H), 3.53
(s, 3H); 13C NMR (75 MHz, CDCl3): d 196.1, 194.5, 151.1, 149.7,
145.2, 141.4, 136.7, 134.6, 134.4, 133.7, 133.6, 131.3, 130.0, 129.1
(2C), 128.4 (2C), 128.1, 126.9, 123.6, 123.5, 84.2, 55.9, 53.3; HRMS
(ESI): m/z calcd for C24H18NO3 [M+H]+ 368.1281, found 368.1286.
Photocycloaddition of 1 with 2c gave 7 (74%), white crystal; MP:
142–143 °C; 1H NMR (300 MHz, CDCl3): d 8.93 (s, 1H), 8.62 (s, 1H),
8.48 (d, 1H, J = 2.1 Hz), 8.31–8.28 (m, 2H), 8.04–7.99 (m, 1H), 7.88–
7.85 (m, 1H), 7.67–7.64 (m, 2H), 7.42–7.37 (m, 3H), 4.56 (s, 1H),
3.52 (s, 3H); 13C NMR (75 MHz, CDCl3): d 195.4, 194.0, 148.2,
146.9, 144.5, 144.2, 143.9, 138.3, 134.9, 134.6, 133.6, 133.5,
130.9, 130.6, 129.2 (2C), 128.5 (2C), 128.2, 127.1, 84.6, 55.6, 53.5;
HRMS (ESI): m/z calcd for C23H16N2NaO3 [M+Na]+ 391.1053, found
391.1062.
3. Conclusions
In summary, we have identified one type of miR-1 inhibitors
from the cyclobutenes derived from the [2+2] photocycloaddition
of aryl acetylenes with 2-methoxy-1,4-naphthalenequinone. This
type of miR-1 inhibitors have the common framework as the
photocycloadduct of 8-(tert-butoxy)-5-(phenylethynyl)quinoline
with 2-methoxy-1,4-naphthalenequinone. The inhibitors were
able to decrease the cytoplasmic levels of mature miR-1 inside
C2C12 cells. Preparation of structural analogues and derivatives
of the active compounds allowed us to analyze some structure–
activity relationship. Due to the importance of miR-1 in the regu-
lation of signal transduction pathway in muscle cells and its wide
relevance with coronary artery diseases and heart failing, the small
molecular inhibitor of miR-1 identified in this paper should pro-
vide useful information for the construction of miR-1 related small
molecular probes. Exploration on the detailed mechanism for this
type of compounds to inhibit miR-1 is underway in our group.
Photocycloaddition of 1 with 3a gave 8 (46%), white crystal;
MP: 167–168 °C; 1H NMR(300 MHz, CDCl3):
d 8.51 (d, 1H,
J = 4.2 Hz), 8.04 (dd, 1H, J = 7.5, 1.2 Hz), 7.92 (d, 1H, J = 7.2 Hz),
7.76–7.60 (m, 4H), 7.09 (t, 1H, J = 5.7 Hz), 3.94 (s, 1H), 3.42 (s,
3H), 2.78–2.73 (m, 1H), 1.13–1.02 (m, 2H), 0.87–0.73 (m, 2H);
13C NMR (75 MHz, CDCl3): d 197.0, 194.5, 155.5, 151.2, 149.6,
139.7, 136.6, 134.8, 134.4, 133.9, 133.8, 128.2, 126.8, 122.4,
122.2, 82.9, 56.1, 53.4, 13.4, 7.4, 6.8; HRMS (ESI): m/z calcd for
C
21H18NO3 [M+H]+ 332.1281, found 332.1282.
4. Experimental
4.1. Synthesis
Photocycloaddition of 1 with 3b gave 9 (86%), yellow crystal;
MP: 173–174 °C; 1H NMR (300 MHz, CDCl3): d 8.88 (s, 1H), 8.48
(s, 1H), 8.08 (dd, 1H J = 7.5, 1.5 Hz), 8.00–7.96 (m, 2H), 7.82–7.71
(m, 2H), 7.32–7.28 (m, 1H), 3.95 (s, 1H), 3.42 (s, 3H), 1.96–1.89
(m, 1H), 1.08–1.00 (m, 2H), 0.86–0.71 (m, 2H); 13C NMR
(75 MHz, CDCl3): d 196.6, 194.5, 151.5, 148.7, 147.8, 138.4, 134.9,
134.8, 134.5, 133.9, 133.7, 133.6, 128.2, 126.9, 123.7, 83.2, 56.5,
53.5, 12.8, 6.9, 6.3; HRMS (ESI): m/z calcd for C21H18NO3 [M+H]+
332.1281, found 332.1283.
4.1.1. General
Commercial reagents were used as supplied or purified by stan-
dard techniques where necessary. Benzene was refluxed with Na
overnight before use. Dichloromethane (DCM) and triethylamine
was freshly distillated over calcium hydride prior use. NMR spectra
were recorded on Bruker 300 MHz spectrometers or Bruker
400 MHz spectrometers. Chemical shifts for 1H NMR spectra are re-
corded in parts per million from tetramethylsilane with the solvent
resonance as the internal standard (chloroform, 7.262 ppm). Data
are reported as follows: chemical shift, multiplicity (s = singlet,
d = doublet, t = triplet, q = quartet, m = multiplet and br = broad),
integration and coupling constant in Hz. All the NOESY spectrum
were measured on Bruker 400 MHz spectrometers. High resolution
mass spectra (HRMS) were recorded on a Agilent 6540Q-TOF high
resolution LC-MS spectrometer (ESI). Reagents:
Photocycloaddition of 1 with 3c gave 10 (70%), white crystal;
MP: 156–157 °C; 1H NMR (300 MHz, CDCl3): d 8.89 (s, 1H), 8.49–
8.47 (m, 1H), 8.40 (d, 1H, J = 2.1 Hz), 8.11–8.08 (m, 1H), 7.79–
7.96(m, 1H), 7.81–7.71 (m, 2H), 3.98 (s, 1H), 3.44 (s, 3H), 2.67–
2.61 (m, 1H), 1.26–1.04 (m, 2H), 0.87–0.78 (m, 2H); 13C NMR
(75 MHz, CDCl3): d 196.3, 193.9, 158.4, 146.9, 144.2, 143.5, 143.3,
142.9, 137.3, 134.9, 134.6, 133.7, 128.3, 126.9, 82.5, 56.6, 53.6,
13.6, 7.9, 7.1; HRMS (ESI): m/z calcd for
C20H16N2NaO3
[M+Na]+355.1053, found 355.1051.
Photocycloaddition of 1 with 3d gave 11 (68%), white crystal;
MP: 158–159 °C; 1H NMR (300 MHz, CDCl3): d 9.08 (s, 1H), 9.01
(s, 2H), 8.12–7.98 (m, 1H), 8.01–7.98 (m, 1H), 7.84–7.73 (m, 2H),
3.96 (s, 1H), 3.41 (s, 3H), 1.93–1.85 (m, 1H), 1.13–1.05 (m, 2H),
0.91–0.74 (m, 2H); 13C NMR (75 MHz CDCl3): d 195.9, 194.0,
157.3, 154.4 (3C), 153.9, 135.1, 134.7, 133.5, 133.4, 128.3, 127.1,
126.2, 82.7, 56.8, 53.8, 13.1, 7.3, 6.7; HRMS (ESI): m/z calcd for
4.1.2. Photocycloaddition reactions
General: the light source was a high-pressure mercury lamp
(500 W) in a cooling water jacket which was further surrounded
by a layer of filter solution (1 cm thick, 15% aqueous NaNO2) to
cut off light of wavelength shorter than 400 nm. The solution of
2-methoxynaphthalene-1,4-dione 1 (0.04 M) and corresponding
alkynes (0.08 M) in anhydrous benzene was irradiated under con-
tinuous nitrogen purging. The reaction course was monitored by
TLC. At the end of the reaction, the solvent was removed under re-
duced pressure and the crude products were further purified by
flash column chromatography with petroleum ether/ethyl acetate
to give the final products.
C
20H17N2O3 [M+H]+ 333.1234, found 333.1240.
Photocycloaddition of 1 with 3e gave 12 (85%), pale yellow crys-
tal; MP: 172–173 °C; 1H NMR (300 MHz, CDCl3): d 9.19 (d, 1H,
J = 2.1 Hz), 8.50 (d, 1H, J = 1.5 Hz), 8.10 (d, 1H, J = 8.4 Hz), 8.07
(dd, 1H, J = J = 7.8, 1.2 Hz), 8.00 (dd, 1H, J = 7.8, 1.2 Hz), 7.94 (d,
1H, J = 8.1 Hz), 7.80 (td, 1H, J = 7.2, 1.5 Hz), 7.76–7.70 (m, 2H),
7.62–7.56 (m, 1H), 4.02 (s, 1H), 3.47 (s, 3H), 2.06–1.99 (m, 1H),
1.13–1.06 (m, 2H), 0.93–0.76 (m, 2H); 13C NMR (75 MHz, CDCl3):
d 196.6, 194.4, 151.3, 148.6, 147.1, 138.6, 134.9, 134.5, 133.6,
Photocycloaddition of 1 with 2a gave 5 (81%), pale yellow crys-
tal; MP: 124–125 °C; 1H NMR (300 MHz, CDCl3): d 8.15–8.12 (m,
1H), 7.87–7.84 (m, 1H), 7.75–7.65 (m, 4H), 7.57–7.52 (m, 2H),
Please cite this article in press as: Tan, S.-B.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.058

6
S.-B. Tan et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
133.3, 129.9, 129.2, 128.5, 128.2, 127.7, 127.0, 126.9, 125.3, 83.3,
Compound 17, pale yellow powder; 181–183 °C; 1H NMR
(400 MHz, CDCl3) d 7.86 (dd, 1H, J = 4.0, 1.2 Hz), 8.11–8.09 (m,
1H), 8.01–7.98 (m, 1H), 7.93 (d, 1H, J = 8.4 Hz), 7.79–7.74 (m,
2H), 7.53 ((d, 1H, J = 7.6 Hz), 7.49 (d, 1H, J = 8.0 Hz), 7.25–7.15
(m, 6H), 4.59 (s, 1H), 3.50 (s, 3H), 2.52 (s, 3H); 13C NMR(75 MHz,
CDCl3) d 196.2, 194.1, 169.4, 150.5, 148.2, 148.1, 141.4, 138.6,
134.9, 134.6, 134.1, 133.8, 133.6, 131.7, 130.1, 128.7 (2C), 127.8,
127.5 (3C), 126.7, 126.6, 121.9, 121.4, 85.6, 56.7, 54.3, 21.0; HRMS
(ESI): m/z calcd for C30H22NO5 [M+H]+ 476.1492, found 476.1495.
Photocycloaddition of 1 with 4b gave 18 (90%), white pow-
56.5, 53.5, 12.9, 7.0, 6.5; HRMS (ESI): m/z calcd for
C
25H20NO3[M+H]+ 382.1438, found 382.1440.
Photocycloaddition of 1 with 3f gave 13 (84%), pale yellow crys-
tal; MP: 125–126 °C; 1H NMR (300 MHz, CDCl3): d 9.19 (s, 1H), 8.45
(s, 1H), 8.08–8.04 (m, 2H), 8.00–7.96 (m, 2H), 7.84 (td, 1H, J = 7.5,
1.5 Hz), 7.77(td, 1H, J = 7.5, 1.5 Hz), 7.68–7.57 (m, 2H), 4.06 (s,
1H), 3.47 (s, 3H), 1.56–1.54 (m, 1H), 1.0–0.97 (m, 2H), 0.91–0.85
(m, 2H); 13C NMR (75 MHz, CDCl3): d 196.8, 194.4, 155.6, 152.9,
142.7, 138.5,134.8, 134.6, 133.9, 133.6, 130.8, 128.3, 128.0 (3C),
127.4, 127.1, 124.8, 123.1, 84.9, 56.8, 54.3, 12.7, 6.6, 5.9; HRMS
(ESI): m/z calcd for C25H20NO3 [M+H]+ 382.1438, found 382.1437.
Photocycloaddition of 1 with 4a gave 14 (80%), white powder
from acetone/petroleum ether; 175–177 °C; 1H NMR (300 MHz,
CDCl3) d 8.88 (dd, 1H, J = 4.0, 1.4 Hz), 8.11–8.08 (m, 1H), 8.01–
7.98 (m, 1H), 7.90 (dd, 1H, J = 8.6, 1.4 Hz), 7.78–7.75 (m, 2H),
7.57 (d, 1H, J = 7.9 Hz), 7.51 (d, 1H, J = 7.9 Hz), 7.25–7.14 (m, 6H),
4.59 (s, 1H), 3.50 (s, 3H), 1.61 (s, 9H); 13C NMR(75 MHz, CDCl3) d
196.4, 194.1, 151.8, 150.5, 148.2, 148.1, 141.5, 138.6, 134.9,
134.6, 133.9, 133.8, 133.6, 131.7, 130.1, 128.7 (2C), 127.8, 127.7,
127.5(3C), 126.7, 126.6, 121.9, 120.8, 85.6, 83.9, 56.7, 54.3, 27.7
(3C); HRMS (ESI): m/z calcd for C33H28NO6 [M+H]+ 534.1911, found
534.1919.
der;161–163 °C; 1H NMR (300 MHz, CDCl3)
d 8.87 (d, 1H,
J = 3.0 Hz), 8.27 (d, 1H, J = 8.4 Hz), 8.03 (d, 2H, J = 7.5 Hz), 7.84–
7.72 (m, 2H), 7.56 (d, 1H, J = 7.8 Hz), 7.49 (d, 1H, J = 7.8 Hz), 7.33
(dd, 1H, J = 8.4, 3.9 Hz), 4.01 (s, 1H), 3.45 (s, 3H), 1.58 (s, 9H),
1.52–1.44 (m, 1H), 1.01–0.94 (m, 1H), 0.90–0.82 (m, 1H), 0.75–
0.61 (m, 2H);13C NMR(75 MHz, CDCl3) d 196.8, 194.4, 154.1,
151.8, 150.2, 147.7, 141.3, 139.5, 134.9, 134.6, 134.0, 133.8,
133.6, 128.0, 127.4, 127.3, 127.0, 126.7, 121.6, 120.7, 84.9, 83.8,
56.5, 54.2, 27.6 (3C), 12.8, 6.5, 5.8; HRMS (ESI): m/z calcd for
C
30H28NO6 [M+H]+ 498.1911, found 498.1912.
Treatment of 18 according to procedure A gave 19 (86%), pale
yellow powder; 158–160 °C; 1H NMR (400 MHz, CDCl3) d 8.73
(dd, 1H, J = 4.2, 1.4 Hz), 8.33 (dd, 1H, J = 8.4, 1.2 Hz), 8.04 (s, 1H),
8.02 (s,1H), 7.83–7.79 (m, 1H), 7.76–7.72 (m, 1H), 7.51 (d, 1H,
J = 8.0 Hz), 7.37 (dd, 1H, J = 8.6, 4.2 Hz), 7.14 (d, 1H, J = 8.0 Hz),
4.02 (s, 1H), 3.46 (s, 3H); 1.56–1.49 (m, 1H), 1.00–0.93 (m, 1H),
0.89–0.82 (m, 1H), 0.75–0.67 (m, 1H), 0.66–0.59 (m, 1H); 13C
NMR(75 MHz, CDCl3) d 197.1, 194.6, 152.8, 152.3, 147.7, 140.2,
138.2, 134.7, 134.5(2C), 133.9, 133.6, 128.5, 127.9, 127.0, 126.4,
121.8, 119.8, 109.7, 85.0, 56.4, 54.1, 12.7, 6.3, 5.6; HRMS (ESI):
m/z calcd for C25H20NO4 [M+H]+ 398.1387, found 398.1392.
Treatment of 19 according to procedure B gave 20 (78%), yellow
powder;164–166 °C; 1H NMR (400 MHz, CDCl3) d 8.88 (d, 1H,
J = 2.8 Hz), 8.24 (d, 1H, J = 8.8 Hz), 8.04 (s, 1H), 8.02 (s,1H), 7.82
(t, 1H, J = 7.6 Hz), 7.74 (t, 1H, J = 7.6 Hz), 7.54 (d, 1H, J = 8.0 Hz),
7.33 (dd, 1H, J = 8.4, 4.0 Hz), 7.04 (d, 1H, J = 8.0 Hz), 4.08 (s, 3H),
4.02 (s, 1H), 3.46 (s, 3H), 1.53–1.45 (m, 1H), 0.99–0.92 (m, 1H),
0.87–0.82 (m, 1H), 0.74–0.69 (m, 1H), 0.65–0.60 (m, 1H); 13C
NMR(75 MHz, CDCl3) d 197.0, 194.6, 155.8, 152.7, 149.0, 140.1,
139.9, 134.8, 134.4, 133.9, 133.6, 127.9, 127.4, 127.1, 127.0,
121.6, 121.1, 107.3, 85.0, 56.4, 56.0, 54.1, 12.7, 6.3, 5.6; HRMS
(ESI): m/z calcd for C26H22NO4 [M+H]+ 412.1543, found 412.1546.
Treatment of 19 according to procedure C gave 21 (93%), white
powder; 163–165 °C; 1H NMR (400 MHz, CDCl3) d 8.77 (dd, 1H,
J = 4.0, 1.6 Hz), 8.28(dd, 1H, J = 8.6, 1.4 Hz), 8.06–8.02 (m, 2H),
7.82 (td, 1H, J = 7.6, 1.2 Hz), 7.76 (td, 1H, J = 7.6, 1.2 Hz), 7.59 (d,
1H, J = 7.6 Hz), 7.42 (d, 1H, J = 7.6 Hz), 7.33 (dd, 1H, J = 8.6,
4.2 Hz), 4.02 (s, 1H), 3.46 (s, 3H), 2.49 (s, 3H). 1.54–1.47 (m, 1H),
1.04–0.98 (m, 1H), 0.92–0.85 (m, 1H), 0.76–0.70 (m, 1H), 0.69–
0.62 (m, 1H); 13C NMR (75 MHz, CDCl3) d 196.8, 194.4, 169.6,
154.1, 150.2, 147.7, 141.2, 139.4, 134.9, 134.6, 134.1, 133.8,
133.6, 128.0, 127.6, 127.3, 127.0, 126.7, 121.6, 121.2, 84.9, 56.5,
54.2, 21.0, 12.8, 6.5, 5.8; HRMS (ESI): m/z calcd for C27H22NO5
[M+H]+ 440.1492, found 440.1490.
4.1.3. Preparation of analogues of compound 14
Procedure A: to a solution of compound 14 in DCM (0.1 M) was
added 20 equiv of TFA. The mixture was stirred at room tempera-
ture and monitored by TLC. Upon complete conversion, the reac-
tion mixture was poured into saturated NaHCO3 solution. The
mixture was extracted with DCM. The organic layer was dried over
Na2SO4. The solvent was removed under vacum, and the crude
products were separated by flash column chromatography with
petroleum ether/ethyl acetate to give 15 (91%).
Compound 15, yellow powder;176–178 °C; 1H NMR (400 MHz,
CDCl3) d 8.72 (dd, 1H, J = 4.0, 1.6 Hz), 8.11–8.07 (m, 1H), 8.00–
7.96 (m, 1H), 7.89 (dd, 1H, J = 8.6, 1.4 Hz), 7.77–7.72 (m, 2H),
7.48 (d, 1H, J = 8.0 Hz), 7.24–7.14 (m, 7H), 4.57 (s, 1H), 3.50 (s,
3H); 13C NMR(75 MHz, CDCl3) d 196.6, 194.3, 153.3, 147.9, 146.9,
139.5, 138.3, 134.8, 134.5 (2C), 133.8, 133.7, 132.1, 129.7, 128.5
(3C), 127.8, 127.4 (3C), 125.6, 122.1, 120.0, 110.1, 85.7, 56.5,
54.2; HRMS (ESI): m/z calcd for C28H20NO4 [M+H]+ 434.1387, found
434.1388.
Procedure B: to the solution of 15 in acetone(0.1 M) was added
10 equiv of CH3I and 3 eq. of K2CO3. The mixture was stirred at re-
flux temperature and monitored by TLC until complete conversion.
After cooling to the room temperature, the mixture was poured
into water and extracted with DCM. The organic layer was dried
over Na2SO4. Then, the solvent was removed and the residue was
separated by flash column chromatography with petroleum
ether/ethyl acetate to give the final products 16 (84%).
Compound 16, pale yellow powder; 179–181 °C; 1H
NMR(400 MHz, CDCl3) d 8.87 (dd, 1H, J = 4.0, 1.6 Hz), 8.08–8.06
(m, 1H), 8.00–7.98 (m, 1H), 7.82 (dd, 1H, J = 8.4, 1.6 Hz), 7.78–
7.71 (m, 2H), 7.51 (d, 1H, J = 8.0 Hz), 7.24–7.12 (m, 6H), 7.09 (d,
1H, J =8.0 Hz), 4.57 (s, 1H), 4.12 (s, 3H), 3.50 (s, 3H); 13C
NMR(75 MHz, CDCl3) d 196.5, 194.2, 156.2, 149.2, 147.1, 140.2,
139.3, 134.8, 134.5, 133.9, 133.8, 133.6, 132.0, 129.8, 128.5 (2C),
127.7, 127.5, 127.4 (3C), 126.3, 121.8, 121.3, 107.5, 85.7, 56.6,
Photocycloaddition of 1 with 4c gave 22 (75%), pale yellow oil;
1H NMR (400 MHz, CDCl3) d 8.90 (dd, 1H, J = 4.4, 1.6 Hz), 8.20 (dd,
1H, J = 8.8, 1.6 Hz), 8.08–8.04 (m, 2H), 7.85 (dt, 1H, J = 7.6, 1.6 Hz),
7.79 (dt, 1H, J = 7.6, 1.2 Hz), 7.45 (d, 1H, J = 8.0 Hz), 7.33 (dd, 1H,
J = 8.6, 4.2 Hz), 7.23 (d, 1H, J = 8.0 Hz), 4.17 (s, 1H), 3.44 (s, 3H),
1.57 (s, 9H), –0.13 (s, 9H); 13C NMR(75 MHz, CDCl3) d 196.4,
194.5, 160.8, 159.1, 151.5, 150.3, 147.9, 141.0, 135.0, 134.5,
134.1, 133.7, 133.3, 129.3, 127.7, 127.1, 126.4, 121.7, 120.1, 87.8,
83.6, 58.0, 54.1, 27.6 (3C), –2.2 (3C); HRMS (ESI): m/z calcd for
56.1, 54.2; HRMS (ESI): m/z calcd for
C
29H22NO4 [M+H]+
448.1543, found 448.1550.
Procedure C: to a solution of 15 in anhydrous DCM (0.1 M) was
added 1.2 equiv of anhydrous TEA and 1.1 eq. of acetyl chloride
drop wise at 0 °C. The mixture solution was left to be stirrers at
rt for 5 min. The solvent was removed and the crude product
was separated by flash column chromatography with petroleum
ether/ethyl acetate to give the final products to give 17 (96%).
C
30H22NO6Si [M+H]+ 530.1993, found 530.1995.
Treatment of 22 according to procedure A gave 23 (88%), pale
yellow powder; 162–164 °C; 1H NMR (400 MHz, CDCl3) d 8.73
Please cite this article in press as: Tan, S.-B.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.058

S.-B. Tan et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
7
(dd, 1H, J = 4.0, 1.6 Hz), 8.27 (dd, 1H, J = 8.6, 1.4 Hz), 8.05 (dd, 1H,
J = 7.6, 1.2 Hz), 8.01 (dd, 1H, J = 7.6, 0.8 Hz), 7.81 (td, 1H, J = 7.2,
1.2 Hz), 7.74 (td, 1H, J = 7.6, 1.6 Hz), 7.34 (dd, 1H, J = 8.4, 4.4 Hz),
7.18 (d, 1H, J = 8.0 Hz), 7.08 (d, 1H, J = 8.0 Hz), 4.16 (s, 1H), 3.44
(s, 3H), –0.11 (s, 9H); 13C NMR(100 MHz, CDCl3) d 198.9, 197.0,
162.0, 161.5, 155.1, 150.0, 140.0, 137.0, 136.9, 136.5, 135.9,
135.4, 130.5, 129.8, 129.2, 129.0, 124.1, 124.0, 111.6, 59.9, 56.2, –
7.73 (t, 1H, J = 7.6 Hz), 7.49 (d, 1H, J = 8.0 Hz), 7.43 (dd, 1H, J =8.4,
4.0 Hz), 7.00 (s, 1H), 4.29 (s, 1H), 3.53 (s, 3H), 2.51 (s, 3H); 13C
NMR(100 MHz, CDCl3) d 195.0, 194.3, 169.5, 149.9, 148.2, 135.7,
134.9, 134.7, 133.3, 133.2, 128.5, 128.4, 127.4, 127.1, 125.7,
122.2, 121.5, 87.0, 56.4, 53.4, 21.0; HRMS (ESI): m/z calcd for
C
24H18NO5 [M+H]+ 400.1179, found 400.1176.
0.01 (3C); HRMS (ESI): m/z calcd for
C
25H24NO4Si [M+H]+
4.2. Biological assay
430.1469, found 430.1469.
Treatment of 23 according to procedure B gave 24 (80%), yellow
oil; 1H NMR (400 MHz, CDCl3) d 8.90 (dd, 1H, J = 4.2, 1.4 Hz), 8.16
(d, 1H, J = 8.4 Hz), 8.07 (dd, 1H, J = 7.8, 1.0 Hz), 8.03 (dd, 1H,
J = 7.8, 1.0 Hz), 7.84 (td, 1H, J = 7.6, 1.6 Hz), 7.77 (td, 1H, J = 7.6,
1.2 Hz), 7.32 (dd, 1H, J = 8.6, 4.2 Hz), 7.25 (d, 1H, J = 8.0 Hz), 6.99
(d, 1H, J = 8.0 Hz), 4.17 (s, 1H), 4.08 (s, 3H), 3.44 (s, 3H), –0.12 (s,
9H); 13C NMR(75 MHz, CDCl3) d 196.7, 194.8, 159.7, 159.5, 155.8,
149.1, 139.8, 134.9, 134.4, 133.7, 133.3, 127.7, 127.6, 127.2,
127.1, 123.1, 121.7, 106.9, 87.9, 57.9, 56.0, 54.1, –2.26 (3C); HRMS
(ESI): m/z calcd for C26H26NO4Si [M+H]+ 444.1626, found 444.1630.
Treatment of 23 according to procedure C gave 25 (94%), color-
less foam; 1H NMR (400 MHz, CDCl3) d 8.88 (dd, 1H, J = 4.0,
1.6 Hz), 8.21 (dd, 1H, J = 8.6, 1.4 Hz), 8.08 – 8.05 (m, 2H), 7.85 (td,
1H, J = 7.6, 1.2 Hz), 7.79 (td, 1H, J = 7.6, 1.2 Hz), 7.38 (d, 1H,
J = 8.0 Hz), 7.33 (dd, 1H, J = 8.6, 4.2 Hz), 7.25 (d, 1H, J = 8.0 Hz),
4.18 (s, 1H), 3.44 (s, 3H), 2.48 (s, 3H), –0.13 (s, 9H); 13C
NMR(75 MHz, CDCl3) d 196.4, 194.6, 169.2, 160.8, 159.0, 150.3,
147.9, 141.0, 135.0, 134.5, 134.2, 133.7, 133.3, 129.4, 127.7, 127.1,
126.4, 121.7, 120.8, 87.8, 58.0, 54.1, 20.9, –2.2 (3C); HRMS (ESI):
m/z calcd for C25H20NO4 [M+H]+ 398.1387, found 398.1392. HRMS
(ESI): m/z calcd for C27H26NO5Si [M+H]+ 472.1575, found 472.1570.
Procedure D: To a solution of 22–25 in DCM (0.1 M) was added
10 eq. of TBAF. The solution was stirred at rt for 3 h. When 22–25
was completely conversed, the solvent was removed and the resi-
due was septerated by flash column chromatography with petro-
leum ether/ethyl acetate to give 26–29, respectively.
4.2.1. Cell culture and reagents
HeLa, C2C12, MCF-7, RAW267.4 and A549 cells were cultured in
high glucose DMEM (Dulbecco’s Modified Eagle’s Medium, Invitro-
gen) containing 10% fetal bovine serum (FBS) and 1% penincilin/
streptomycin at 37 °C and 5% CO2. C2C12 cells were induced to dif-
ferentiate in DMEM containing 2% horse serum (Invitrogen). Com-
pounds were pre-dissolved in DMSO; the final concentration of
DMSO in culture medium was 1:1000.
4.2.2. Toxicity test of compounds
The cultured cells were seeded on 96 wells plates with around
10,000 cells/well. The next day, they were incubated with 10uM
compounds in high glucose DMEM containing 2% FBS. MTT assay
was performed after 48 h of incubation. Briefly, cells were incu-
bated with 0.5 mg/mL of MTT (Sigma–Aldrich) for 4 h in a CO2
incubator at 37 °C. After that, the medium was removed com-
pletely and the purple precipitates were dissolved in DMSO for
10 min at room temperature. The absorbance of solution was mea-
sured at 490 nm. Each experiment was performed in triplicates.
4.2.3. Reporter plasmids construction and isolation
The luciferase reporter plasmid was sequentially digested with
HindIII and SpeI restriction endonuclease (Takara) and was gel
purified. The complementary DNA sequences of mature miR-1,
miR-150, miR-25 and miR-214 containing HindIII and SpeI ‘sticky’
end were individually cloned into the purified luciferase reporter
by using T4 ligase (Takara) at 16 °C overnight. The plasmids were
transformed into chemically competent Escherichia coli by incubat-
ing the plasmid and competent E. coli on ice for 30 min, followed
by a pulse of heat shock (42 °C, 90 s). The E. coli was concentrated
and plate on agar plate containing 100 ug/ml Ampicillin. The agar
plate was incubated at 37 °C for 16 h. Positive colonies were se-
lected by PCR colony screens, and the construction of Luciferase
miRNA reporters were confirmed by sequencing (Invitrogen). All
mature miRNAs complementary sequences were purchased from
Invitrogen Company and were 30 phosphorylated. The followings
were their corresponding sequences:
Compound 26 (95%), pale yellow powder; 73–75 °C; 1H
NMR(400 MHz, CDCl3) d 8.87 (d, 1H, J = 4.0 Hz), 8.46 (d, 1H,
J = 8.8 Hz), 8.09 (d, 1H, J = 8.0 Hz), 8.03–8.00 (m, 2H), 7.75 (t, 1H,
J = 7.6 Hz), 7.69 (t, 1H, J = 7.6 Hz), 7.53 (d, 1H, J = 8.0 Hz), 7.39
(dd, 1H, J = 8.0, 4.0 Hz), 6.98 (s, 1H), 4.26 (s, 1H), 3.50 (s, 3H),
1.56 (s, 9H); 13C NMR(100 MHz, CDCl3) d 195.0, 194.3, 151.6,
150.0, 148.4, 148.2, 141.3, 135.6, 134.9, 134.6, 133.3, 133.2,
132.4, 128.4, 128.3, 127.3, 127.1, 125.5, 122.2, 120.7, 87.0, 83.9,
56.3, 53.3, 27.6 (3C); HRMS (ESI): m/z calcd for C27H23NNaO6
[M+Na]+ 480.1418, found 480.1417.
Compound 27 (87%), pale yellow powder; 178–180 °C; 1H
NMR(400 MHz, CDCl3) d 8.76 (d, 1H, J = 3.2 Hz), 8.49 (d, 1H,
J = 8.4 Hz), 8.07–8.02 (m, 3H), 7.77 (td, 1H, J = 7.6, 1.2 Hz), 7.70
(td, 1H, J = 7.6, 1.2 Hz), 7.45 (dd, 1H, J = 8.4, 4.0 Hz), 7.19 (d, 1H,
J = 8.0 Hz), 6.84 (d, 1H, J = 1.6 Hz), 4.26 (d, 1H, J = 1.2 Hz), 3.52 (s,
3H); 13C NMR(100 MHz, CDCl3) d 195.5, 194.7, 153.5, 148.7,
147.6, 138.2, 134.8, 134.5, 133.3, 133.2, 133.0, 131.9, 130.5,
128.2, 127.1, 126.5, 122.5, 118.4, 109.7, 87.0, 56.2, 53.2; HRMS
(ESI): m/z calcd for C22H16NO4 [M+H]+ 358.1074, found 358.1073.
Compound 28 (83%) yellow oil; 1H NMR (400 MHz, CDCl3) d
8.94 (s, 1H), 8.48 (d, 1H, J = 8.4 Hz), 8.12(d, 1H, J = 8.0 Hz),8.05 (s,
1H), 8.03(s, 1H), 7.77 (t, 1H, J = 7.6 Hz), 7.71(t, 1H, J = 7.6 Hz),
7.45 (dd, 1H, J = 8.0, 3.2 Hz), 7.10 (d, 1H, J = 8.0 Hz), 6.88 (s, 1H),
4.28 (d, 1H, J = 1.2 Hz), 4.12 (s, 3H), 3.53 (s, 3H);13C NMR(100 MHz,
CDCl3) d 195.5, 194.7, 156.3, 148.8, 148.4, 134.8, 134.5, 133.3,
133.2, 132.7,129.7, 128.2, 127.3, 127.1, 122.2, 119.6, 107.4, 87.0,
56.3, 56.2, 53.2; HRMS (ESI): m/z calcd for C23H18NO4 [M+H]+
372.1230, found 372.1226.
miR-1: 50-ATACATACTTCTTTACATACCA-30
miR-25: 50-TCAGACCGAGACAAGTGCAATG-30
miR-214: 50-ACTGCCTGTCTGTGCCTGCTGT-30
miR-150: 50-CACTGGTACAAGGGTTGGGAGA-30
Positive plasmids were isolated from E. coli by using plasmid
isolation kit (Invitrogen).
4.2.4. Screening of photocycloadducts for miRNAs function
modifier
Luciferase assays were performed to identify effect of com-
pounds on function of endogeneous miRNAs: For miR- miR-1,
C2C12 cells at 70% confluence in 48-well plates were co-transfec-
ted with miR-1 luciferase reporter (0.25
ase reporters and b-galactosidase expressing plasmid (0.25
by using lipo-fectamine 2000 (Invitrogen) according to manufac-
turer’s protocol. The b-galactosidase was used as transfection con-
trol. After 6 h of transfection, the medium was changed to high
glucose DMEM containing 2% horse serum, 1% penincilin/strepto-
l
g) or respective lucifer-
l
g)
Compound 29 (96%), white powder; 88–90 °C; 1H NMR
(400 MHz, CDCl3) d 8.91 (d, 1H, J = 2.8 Hz), 8.52 (d, 1H, J = 8.8 Hz),
8.12 (d, 1H, J = 8.0 Hz), 8.06–8.03(m, 2H), 7.78 (t, 1H, J = 7.6 Hz),
mycin and 10
lM of compounds. The cells were further incubated
for 48 h at 37 °C, 5% CO2, and then assayed using luciferase assay
Please cite this article in press as: Tan, S.-B.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.058

8
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Acknowledgments
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Financial support the National Basic Research Program of China
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Supplementary data
Supplementary data (Viability tests on C2C12 cells. Copies of
NMR spectra of all the photocycloadducts and their derivatives.)
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Please cite this article in press as: Tan, S.-B.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.058