Furan- and thiophene-2-carbonyl amino acid derivatives activate hypoxia-inducible factor via inhibition of factor inhibiting hypoxia-inducible factor-1

Article abstract of DOI:10.3390/molecules23040885

Induction of a series of anti-hypoxic proteins protects cells during exposure to hypoxic conditions. Hypoxia-inducible factor-α (HIF-α) is a major transcription factor that orchestrates this protective effect. To activate HIF exogenously, without exposing

Full text of DOI:10.3390/molecules23040885

molecules
Article
Furan- and Thiophene-2-Carbonyl Amino Acid
Derivatives Activate Hypoxia-Inducible Factor via
Inhibition of Factor Inhibiting
Hypoxia-Inducible Factor-1
Shin-ichi Kawaguchi 1,2,3 ID , Yuhei Gonda 2, Takuya Yamamoto 2, Yuki Sato 2,
Hiroyuki Shinohara 2, Yohsuke Kobiki 2, Atsuhiko Ichimura 3,4, Takashi Dan 3,
Motohiro Sonoda 2,5, Toshio Miyata 3, Akiya Ogawa 2 and Tadayuki Tsujita 3,6,
*
ID
1
Center for Education and Research in Agricultural Innovation, Faculty of Agriculture, Saga University,
152-1 Shonan-cho, Karatsu, Saga 847-0021, Japan; skawa@cc.saga-u.ac.jp
2
3
Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University,
1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan; y.gonda.0402@gmail.com (Y.G.);
0tg38t18086577t@ezweb.ne.jp (T.Y.); su108027@edu.osakafu-u.ac.jp (Y.S.);
h.shinohara@katayamakagaku.co.jp (H.S.); yohsuke.kobiki@gmail.com (Y.K.);
sonoda@bioinfo.osakafu-u.ac.jp (M.S.); ogawa@chem.osakafu-u.ac.jp (A.O.)
Department of Molecular Medicine and Therapy, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan; ichimura.atsuhiko.2r@kyoto-u.ac.jp (A.I.);
dantks@med.tohoku.ac.jp (T.D.); miyata@med.tohoku.ac.jp (T.M.)
4
5
6
Keihanshin Consortium for Fostering the Next Generation of Global Leaders in Research (K-CONNEX),
Kyoto University, Kyoto 606-8501, Japan
Department of Applied Biosciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture
University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
Department of Applied Biochemistry and Food Science, Faculty of Agriculture, Saga University,
1 Honjyo-machi, Saga, 840-8502, Japan
*
Correspondence: tada@cc.saga-u.ac.jp; Tel.: +81-952-28-8771
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 9 March 2018; Accepted: 10 April 2018; Published: 11 April 2018
Abstract: Induction of a series of anti-hypoxic proteins protects cells during exposure to hypoxic
conditions. Hypoxia-inducible factor-α (HIF-α) is a major transcription factor that orchestrates
this protective effect. To activate HIF exogenously, without exposing cells to hypoxic conditions,
many small-molecule inhibitors targeting prolyl hydroxylase domain-containing protein have been
developed. In addition, suppression of factor inhibiting HIF-1 (FIH-1) has also been shown to
have the potential to activate HIF-α. However, few small-molecule inhibitors of FIH-1 have been
developed. In this study, we synthesized a series of furan- and thiophene-2-carbonyl amino acid
derivatives having the potential to inhibit FIH-1. The inhibitory activities of these compounds were
evaluated in SK-N-BE(2)c cells by measuring HIF response element (HRE) promoter activity. Several
furan- and thiophene-2-carbonyl amino acid derivatives inhibited FIH-1 based on correlations among
the docking score of the FIH-1 active site, the chemical structure of the compounds, and biological
HIF-α/HRE transcriptional activity.
Keywords: hypoxia inducible factor; factor inhibiting hypoxia inducible factor; furan; thiophene
1. Introduction
Hypoxia-inducible factors (HIFs) are pivotal transcription factors that contribute to the hypoxic
stress response [1–8]. HIFs are classified into HIF-α and HIF-β subfamilies. HIF-α is hydroxylated by
Molecules 2018, 23, 885; doi:10.3390/molecules23040885
www.mdpi.com/journal/molecules

Molecules 2018, 23, 885
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prolyl hydroxylase domain-containing proteins (PHD1, PHD2, and PHD3), and factor inhibiting
HIF (FIH-1) under normoxic conditions (21% O2) [ 12] (Figure 1). Hydroxylated HIF- is
polyubiquitinated by von Hippel–Lindau E3 ubiquitin ligase (VHL) and rapidly degraded by
the proteasome system. Enzymes that contribute to HIF- quenching require molecular oxygen,
2-oxoglutarate (2OG), and ferric (Fe) ions. When cells encounter severe hypoxic conditions, HIF-
hydroxylation is inhibited due to low oxygen levels. Thus, the PHD/HIF- molecular axis acts as an
oxygen sensor system [ ]. Under hypoxic conditions, HIF- is stabilized and translocated to the
nucleus, where it forms a heterodimeric complex with HIF- . The HIF complex binds to HIF response
9
–
α
α
α
α
1
–
5
α
β
element (HRE) ([A/G]CGTC) and transactivates genes that facilitate adaptation to hypoxic and/or
ischemic stress, such as vascular endothelial growth factor (VEGF), erythropoietin (EPO), and glucose
transporter-1 (GLUT-1).
Figure 1. Regulatory mechanisms mediating the HIF-related pathway.
To date, small molecules that can stabilize HIF-
to treat ischemic heart failure, chronic kidney disease, and stroke [13
normoxic conditions, Asn803 on HIF-1 is hydroxylated and inhibited by FIH-1 [12]. This Asn803
α
via PHD inhibition have been widely investigated
–28]. On the other hand, under
α
hydroxylation blocks the interaction with the CREB-binding protein/p300 (CBP/p300) transcriptional
coactivator. When cells are exposed to severe hypoxic conditions, FIH-1 fails to hydroxylate
Asn803. Consequently, dehydroxylated HIF-1
transactivation of anti-hypoxic proteins [ 10] (Figure 1). This mechanism has also been observed in
FIH-1-knockout mice [29] and cells [30], however, selective small-molecule FIH-1 inhibitors [31 34] are
still rare compared with PHD inhibitors [13 28]. Because HIF activation induced by PHD inhibition is
different from that induced by FIH inhibition [35 36], the development of FIH-1 inhibitors is necessary.
α can interact with CBP/p300 and contribute to the
9,
–
–
,
Accordingly, in this study, we aimed to develop novel small-molecule inhibitors of FIH-1.
2. Results and Discussion
2.1. Design and Synthesis of New FIH-1 Inhibitors
FIH-1 has an Fe atom in its active center, and this Fe is held in place by two histidine and aspartic
acid residues. 2OG is a required cofactor that mediates FIH-1 activity. Based on X-ray crystallography
data (PBD ID: 1MZF), 2OG binds the Fe atom bidentately, and forms a hydrogen bond with Lys214
and Tyr145 in human HIF-1 (Figure 2a) [37]. In this study, furan- and thiophene-2-carbonyl amino
acid derivatives were designed to antagonize 2OG binding [38] based on the X-ray crystallography
structure. The designed structure had a bidentate site for Fe atom binding and a carboxylic acid that
could form a hydrogen bond with Lys214 and/or Tyr145 residues (Figure 2b). The R1 moiety contained
0
0
an H, Me, phenyl, 4 -phenoxyphenyl, or 4 -biphenyl. The R2 moiety was prepared with either an H or

Molecules 2018, 23, 885
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phenyl group. The R3 moiety corresponded to an amino acid residue (glycine, alanine, phenylalanine,
tryptophan, or tyrosine). A Me or Et group was introduced as the R4 moiety to increase the lipophilicity
and cell membrane permeability. The R4 moiety was thought to be converted to H by hydrolysis in the
cell. Many inhibitors have been reported to act as antagonists of 2OG [15
–17,39]; however, the use of
furan- and thiophene-2-carbonyl amino acid derivatives as antagonists of 2OG has not been evaluated.
Figure 2.
(a) The coordination of 2OG in the active center of FIH-1 (PBD ID: 1MZF); (b) The structure
of the newly designed FIH-1 inhibitor structure based on the 2OG scaffold.
The synthetic strategy to design the compounds is summarized in Scheme 1. Furan- and
thiophene-2-carbonyl amino acid derivatives were synthesized using condensation reactions of
thiophene-2-carboxylic acids with amino acid salts. Nonsubstituted furan- or thiophene-2-carboxylic
acids and benzofuran- or benzothiophene-2-carboxylic acids were obtained from commercial supplies.
The other furan- or thiophene-2-carboxylic acids were synthesized as follows.
Scheme 1. Synthetic routes of 2-carbonylfuran and 2-carbonylthiophene derivatives.
0
2-([1,1 -Biphenyl]-4-yl)thiophene and 2-(4-phenoxyphenyl)thiophene were prepared in moderate
yields via a Negishi coupling reaction using the zinc salt of thiophene and aryl bromide (Scheme 2).
Next, 5-substituted thiophene-2-carboxylic acids and 5-methylfuran-2-carboxylic acid were synthesized
by carboxylating the corresponding furan or thiophene (Scheme 3).
Scheme 2. Preparation of 2-arylthiophene.

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Scheme 3.
Preparation of 5-substituted thiophene-2-carboxylic acid and 5-methylfuran-
2-carboxylic acid.
Preparation of 5-phenylfuran-2-carboxylic acid is shown in Scheme 4. Suzuki-Miyaura
cross-coupling of phenyl boronic acid and methyl 5-bromofuran-2-carboxylate gave the coupling
product, and subsequent hydrolysis produced the corresponding carboxylic acid in good yield.
Scheme 4. Preparation of 5-phenylfuran-2-carboxylic acid.
Since the starting material was commercially available, the method for the synthesis of
4-phenylthiophene-2-carboxylic acid differed from that of 5-phenylfuran-2-carboxylic acid (Scheme 5).
Suzuki-Miyaura cross-coupling of 4-bromo-2-thiophenecarbaldehyde and phenylboronic acid was
used to obtain the corresponding coupling product, which yielded the corresponding carboxylic acid
upon subsequent oxidation.
Scheme 5. Preparation of 4-phenylthiophene-2-carboxylic acid.
For the condensation reaction, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC) was used as a condensation agent in the presence of 1-hydroxybenzotriazole (HOBt) (Scheme 6).
The use of EDC/HOBt produced the condensation products in good yields and suppressed
racemization of amide units.
Scheme 6. Condensation reactions with EDC.

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2.2. Evaluation of HIF Activation by FIH-1 Inhibition
To evaluate HIF transactivation following FIH-1 inhibition in vivo, we employed a luciferase
reporter assay system in the human neuroblastoma cell line SK-N-BE(2)c. The cells were stably
transfected with a reporter vector possessing secretion-type luciferase (Metridia longa luciferase, MLuc)
under control of the 7-time-repeat human VEGFA regulatory sequence (40 bp length containing the
HRE) and mini TATA promoter. Hereafter these HRE reporter cells were designated SKN:HRE-MLuc
cells [40]. To evaluate the inhibitory activity of FIH-1, SKN:HRE-MLuc cells were cultured under mild
hypoxic conditions (3% O2) based on previous studies showing that under normoxic conditions (21%
O2), FIH-1 inhibition does not significantly affect for HIF activation, whereas under 3% O2 conditions,
FIH-1 inhibition elevates HIF transcriptional activity [1,30,41].
To confirm that our proposed system could be utilized to evaluate FIH-1 inhibitory activity, FIH-1
was transiently silenced by transfecting SKN:HRE-MLuc cells for 72 h with siRNA. After 24 h culture
with fresh medium under normoxic conditions, FIH-1 protein was analyzed by immunoblotting.
FIH-1 protein levels in FIH-1 siRNA transfectants were significantly reduced compared with that in
untreated or scrambled siRNA-transfected control cells (Figure 3a). Using this FIH-1 siRNA system,
we compared the efficacy of dimethyloxalyl glycine (DMOG) or FibroGen compound (FG4592) under
normoxic or hypoxic conditions (3% O2) for 24 h (Figure 3b). Under hypoxic conditions, PHD proteins
were mostly inactivated; therefore, the HIF-HRE top-up transcriptional activity under these conditions
can be measured as FIH-1 inhibitory activity [30].
According to the experimental design indicated in Figure 3b, SKN:HRE-MLuc cells were
transfected with the indicated siRNAs, after 72 h, the transfectants were treated with normoxic
or hypoxic conditions (3% O2) for 24 h, and the luciferase activities were determined (Figure 3c). HIF
transcriptional activity on SKN:HRE-MLuc cells was significantly elevated during hypoxia treatment
(compare A with B). Moreover, HIF was not stabilized in FIH-1 knockdown cells under normoxic
conditions (compare A with C). In contrast, under hypoxic conditions, HIF transcriptional activity
was enhanced in FIH-1 knockdown cells, supporting the inhibitory activity of FIH-1 (compare B with
D). Treatment with DMOG, which inhibits both PHDs and FIH-1, resulted in higher HIF stabilization
activity (compare A with E, F, G, or H). On the other hand, treatment with FG4592, which is a selective
inhibitor of PHD [36,42], stabilized HIF compared with vehicle-treated cells (compare A with I). The
difference between E and I was supported by changes in FIH-1 activity. Therefore, FG4592 treatment
under hypoxic conditions only slightly stabilized HIF (compare I with J). Importantly, FG4592 treatment
did not affect FIH-1 inhibitory activity. Additionally, SKN:HRE-MLuc cells were significantly activated
following FIH-1 knockdown treatment under mild hypoxic conditions, even in the presence of FG4592
(compare J with L). Taken together, these results suggested that measuring HIF-HRE transcriptional
activity with continuous mild hypoxia may reflect FIH-1 activity.
Next, to confirm that the proposed system could be used to evaluate FIH-1 inhibition, we
treated the cells with DMOG, which can inhibit both PHD and FIH as a positive control, or dimethyl
N-oxalyl-D-phenylalanine (DM-NOFD), which is a prodrug of N-oxalyl-D-phenylalanine, a reported
FIH-1-selective inhibitor (Figure 4b) [31,34]. Treatment with DM-NOFD (100 µM) significantly
enhanced HIF activity (Figure 4a). This result indicated that our FIH-1 evaluation system could
be utilized for quantification of further optimized small compounds derived from DM-NOFD.

Molecules 2018, 23, 885
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Figure 3. Establishment of the evaluation system for FIH-1 inhibitor activity. (a) Confirmation of
siRNA knockdown efficiency for FIH-1. Scrambled siRNA and siRNA against FIH-1 were introduced
into SK-N-BE(2)c cells. Transfected cells were cultured for 72 h with siRNA. After an additional 24 h,
total cell lysates were analyzed by immunoblotting to detect FIH-1 or
an internal control; ( ) Experimental design of the evaluation system for FIH-1 inhibitor activity;
) HIF transcriptional activities were measured with secretion-type luciferase (Metridia longa luciferase,
β-actin, which was used as
b
(c
MLuc) based HRE transcriptional reporter analysis in SKN:HRE-MLuc reporter cells. To confirm the
FIH-1 inhibitory activity, random target scrambled siRNA or siRNA against FIH-1 was transfected
into SKN:HRE-MLuc cells, as indicated. The transfected cells were also treated with normoxic or
mild hypoxic conditions (3% O2), DMOG (100 µM), or FG4592 (100 µM), as indicated. The degree
of induction is presented as relative luciferase units, with the value from control siRNA, normoxia
and DMSO treatment (column A) cells set as 1 for each treatment. All experiments were performed in
triplicate. Data are means
±
SEMs (n = 3). The statistical significance of results compared with data from
the control group was calculated using one-way analysis of variance (ANOVA) with Newman-Keuls
multiple-comparison test. ns, p > 0.05; * p = 0.05–0.01; ** p = 0.01–0.001; *** p < 0.001.
Figure 4.
(a) Evaluation of HIF activation using an FIH-1 inhibitor (DM-NOFD). SKN:HRE-MLuc
cells were treated with DMSO (1%), DMOG (100
µ
M) or DM-NOFD (100 M) for 24 h under hypoxic
µ
conditions (3% O2). The degree of induction is presented as relative luciferase units, with the value
from DMSO-treated cells set as 1 for each treatment. Experiments were performed in triplicate. Data are
means
±
SEMs (n = 3). The statistical significance of results compared with data from the control group
was calculated using one-way ANOVA with Newman-Keuls multiple-comparison tests. * p = 0.05 to
0.01; *** p < 0.001; (b) Chemical structure of DM-NOFD.
2.3. Evaluation of HIF Activation by Furan- and Thiophene-2-Carbonyl Amino Acid Derivatives under
Hypoxic Conditions
Next, we evaluated the activation of HIF by furan- and thiophene-2-carbonyl amino acid
derivatives using the evaluation methods described above. Simultaneously, we investigated
cellular toxicity for those derivatives with a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. To indicate cellular membrane permeability, the
partition coefficients, which are frequently represented by calculated logP (clogP) are listed in
Tables 1 and 2.

Molecules 2018, 23, 885
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Table 1. Evaluation of HIF activity by several furan- and thiophene-2-carbonyl amino acid derivatives, their toxicity, and their partition coefficients.
Entry
Y
R1
R2
R3
R4
R/S
Compound No.
25% Activity a
Toxicity IC50
clogP
c
100 µM Activity b
1
2
3
4
5
6
7
8
O
S
O
O
S
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Ph
Ph
Ph
Ph
Ph
Ph
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Ph
Ph
Ph
Ph
Ph
H
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
Et
Me
Et
H
-
-
1
2
3
4
5
6
7
8
25 µM
ND
ND
ND
ND
ND
25 µM
ND
25 µM
6.3 µM
25 µM
ND
20%
0%
0%
0%
0%
0%
22%
2%
22%
0%
23%
18%
0%
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
75 µM
0.04
0.70
1.03
1.03
2.39
2.39
1.82
1.82
1.37
1.37
0.49
1.25
1.85
3.19
3.53
2.47
3.07
3.07
2.96
3.22
2.65
3.25
4.32
4.58
4.13
2.36
4.87
4.07
4.07
4.14
4.09
6.60
3.96
4.45
6.12
5.66
-CH2-3-indolyl
-CH2-3-indolyl
-CH2-3-indolyl
-CH2-3-indolyl
-CH2-Ph
-CH2-Ph
-CH2-3-indolyl
-CH2-3-indolyl
H
S
R
S
R
S
R
S
R
-
S
S
R
S
R
S
R
S
S
S
S
S
S
S
-
S
S
R
S
-
S
-
S
S
S
S
O
O
O
O
S
S
S
S
S
S
S
S
O
O
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
9
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Me
Me
ND
ND
-CH2-Ph
-CH2-Ph
-CH2-3-indolyl
-CH2-3-indolyl
-CH2-3-indolyl
-CH2-Ph
-CH2-Ph
Me
0%
25 µM
100 µM
100 µM
100 µM
ND
ND
ND
ND
ND
ND
ND
ND
ND
25 µM
ND
6.3 µM
ND
ND
ND
12%
30%
49%
52%
0%
10%
0%
16%
0%
7%
0%
0%
2%
0%
0%
0%
0%
0%
0%
0%
0%
Et
Et
H
Me
H
Me
Et
H
-CH2-Ph
-CH2-Ph
-CH2-3-indolyl
H
-CH2-Ph
-CH2-3-indolyl
-CH2-3-indolyl
-CH2-C6H4-4-OH
H
Me
Me
Me
Et
Me
Me
Me
Me
Et
4-Ph-C6H4
4-Ph-C6H4
4-PhO-C6H4
4-PhO-C6H4
4-PhO-C6H4
4-PhO-C6H4
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
-CH2-Ph
H
Me
Me
Me
Et
ND
ND
ND
-CH2-Ph
-CH2-3-indolyl
S
Me
0%
a
25% activity represents the lower dose limit showing 25% of the RLU intensity of the positive control DMOG; b 100
µM activity represents the RLU intensity at 100 µM stimulation
compared with the positive control DMOG; c The values were calculated using the MTS assay results. ND = not determined.

Molecules 2018, 23, 885
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Table 2. Evaluation of HIF activity by benzofuran- and benzothiophene-2-carbonyl amino acid derivatives, their toxicity, and their partition coefficients.
Entry
Y
R3
R4
R/S
Compound No.
25% Activity a
Toxicity IC50
clogP
c
100 µM Activity b
37
38
39
40
41
42
43
44
O
O
S
S
S
S
S
S
-CH2-Ph
-CH2-3-indolyl
H
Et
Me
Me
Et
S
S
-
S
S
R
S
S
37
38
39
40
41
42
43
44
ND
25 µM
ND
ND
ND
ND
ND
ND
0%
25%
13%
0%
2%
0%
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
>100 µM
2.88
2.08
1.74
2.57
4.24
3.91
3.45
3.52
Me
-CH2-Ph
-CH2-Ph
Et
Me
Me
Me
-CH2-3-indolyl
-CH2-C6H4-4-OH
0%
0%
a
25% activity represents the lower dose limit showing 25% of the RLU intensity of the positive control DMOG; b 100
µM activity represents the RLU intensity at 100 µM stimulation
compared with the positive control DMOG; c The values were calculated using the MTS assay results. ND = not determined.

Molecules 2018, 23, 885
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Compounds
1,
7
,
9
–
11,
15
–
18,
28
,
30, and 38 stabilized HIF and transactivated the HRE-driven
MLuc reporter in SKN:HRE-MLuc cells. Some trends were observed among the active compounds.
For example, compounds with bulky R1 groups, such as Ph, 4-Ph-C6H4-, and 4-PhO-C6H4-, did not
enhance HIF activity (Entry 19–25, 31–36). Some active compounds were obtained when the R3 group
was an aromatic group, such as Ph-CH2- or 3-indolyl-CH2 (Compounds
7, 9–11, 15–18, 28, 30, 38).
In some cases, both the S and R isomer had activity (10 11 and 17 18, respectively). Both furan
,
,
and thiophene derivatives stabilized HIF activity. Only compound 30 showed cellular toxicity in the
MTS assays.
2.4. Docking Simulations Using Furan- and Thiophene-2-Carbonyl Amino Acid Derivatives with FIH-1
To understand the inhibitory tendencies observed in HRE-MLuc reporter assays on
SKN:HRE-MLuc cells, docking simulations with FIH-1 were performed with four compounds (7, 9
,
10, and 16); among the compounds that stabilized HIF activity (i.e., compounds 11 15 18 28,
7,
9–
,
–
,
30, and 38), these four compounds had characteristic scaffolds. Docking simulations were run on
Molegro Virtual Docker 6.0.0 [43]. The corresponding hydrolyzed compounds were used in the
docking simulations (compounds
Table 3. For compound
, a furan derivative with a bulky group (such as Ph-CH2-) in the R3 position,
and compound
, a furan derivative with a 3-indolyl-CH2 group in the R3 position, both dockings had
good scores (entries 1 and 2). The predicted binding models of compounds and within the active
center of FIH-1 are shown in Figure 5a,b. When compound , which had an L-tryptophan scaffold,
was compared with compound 10, which had a D-tryptophan scaffold, good docking scores were
7, 9, and 10). The results of the FIH-1 docking studies are shown in
7
9
7
9
9
obtained (Table 3, entries 2 and 3). The predicted binding models of compounds
9
and 10 showed that
and 10 were
both 3-indolyl-CH2 groups in the R3 position fit within the space, although compounds
enantiomers to each other (Figure 5b,c).
9
Table 3. The docking scores of FIH-1 with compounds
7,
9,
10, and 16 run on Molegro Virtual
Docker 6.0.0.
Entry
Compound
Score (Plants)
1 a
2 a
3 a
4
7 (hydrolysate)
9 (hydrolysate)
10 (hydrolysate)
16
−63.8
−70.8
−57.7
−29.9
a
Docking simulations of FIH-1 were performed with their carboxylic acid.
The thiophene compound 16, which was related to compound 9, also docked within FIH-1;
however, it had a lower score (Table 3, entry 4). The predicted binding model of compound 16 showed
a similar conformation (Figure 5d). These results supported the notion that compounds with bulky
groups in the R1 position did not show the activity because there was not sufficient space ahead of the
R1 position. In contrast, compound
that used 2OG as a cofactor because of its scaffold.
1 did not selectively inhibit FIH-1 and could inhibit other enzymes

Molecules 2018, 23, 885
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Figure 5. The predicted binding model of compound
center of FIH-1. ( ) Compound (hydrolysate); ( ) Compound
(hydrolysate); and (d) Compound 16.
7
,
9
,
10 (hydrolysate)
,
and 16 within the active
) Compound 10
a
7
b
9
(hydrolysate); (c
2.5. Inactibation of HIF by FIH-1 Inhibitors under Normoxic Conditions
We next measured HIF transactivity of FIH-1 inhibitors under normoxic conditions to confirm
whether these inhibitors stabilized HIF via inhibiting PHD. Under normoxic conditions, inhibition
of PHD dramatically activated HRE reporter. In contrast, when PHD was inhibited under hypoxic
conditions, the rate of activation of the HRE reporter was low because hypoxia already partially
activated the HRE reporter. The compounds showing HIF activation under hypoxic conditions
(compounds
cells under normoxic conditions (Figure 6). Treatment with 100
selective inhibitor of PHD, was used as a positive control and showed significant HRE reporter activity.
In contrast, compounds 11 15 18 28 30, and 38 did not elicit any HIF transactivation. This
7,
9
–11
,
15–
18
,
28,
30, and 38) were evaluated by the luciferase assays in SKN:HRE-MLuc
µM FG4592, a commonly used
7,
9–
,
–
,
,
result indicated that these compounds selectively inhibited FIH-1. At the same time, these compounds
(7, 9–11, 15–18, 28, 30, and 38) did not fit into the catalytic domain of the PHD2 (data not shown).
Figure 6. HIF activation using the identified compounds (7, 9–11, 15–18, 28, 30, and 38) under normoxic
conditions. The degree of induction is presented as relative luciferase units, with the value from DMSO
treatment of cells set as 1 for each treatment. Experiments were performed in triplicate. Data are means
±
SEMs (n = 3). The statistical significance of the results compared with data from the control group
was calculated using one-way ANOVA with Newman-Keuls multiple-comparison tests. ns, p > 0.05;
*** p < 0.001.

Molecules 2018, 23, 885
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2.6. mRNA Expression in SK-N-BE(2) Cells
To confirm transactivation of HIF by our optimized compounds, the expression of HIF target genes
was analyzed with quantitative polymerase chain reaction (PCR) in SK-N-BE(2)c cells. We attempted
to analyze several HIF target genes including CA9, VEGF-
Among these genes, as described previously [30], CA9 was significantly elevated when treated with
compound (Figure 7a). Notably, this induction of CA9 did not occur via HIF-1 mRNA transactivation
mRNA was slightly suppressed by DMOG
α, EPO, and PHD3, under 3% O2 conditions.
9
α
(Figure 7b). In addition, de novo translation of HIF-1
treatment but not by treatment with compound 9.
α
Figure 7. Expression levels of CA9 (
DMOG (100 M) and compound
presented as relative mRNA levels, with the qRT-PCR values from DMSO-treated cells set as 1 for each
treatment. Experiments were performed in triplicate. Data are means SEMs (n = 3). The statistical
a) and HIF-1
α mRNA. (b) SK-N-BE(2)c cells were stimulated with
µ
9
(100 M) under hypoxic conditions (3% O2). Gene induction is
µ
±
significance of the results compared with data from the control group was calculated using one-way
ANOVA with Newman-Keuls multiple-comparison tests. ns, p > 0.05; * p < 0.05–0.01; *** p < 0.001.
3. Conclusions
In this study, we used SKN:HRE-MLuc cells cultured under mild hypoxic conditions to reflect
FIH-1 activity even in the presence of PHD proteins. To evaluate FIH-1 function more specifically,
N to C transactivation domain of the HIF-1α (amino acids 531–826) and GAL4 fusion protein with
a GAL4-DNA binding domain-driven reporter system would be a powerful alternative evaluation
method and high-throughput screening system [44]. Additionally, we designed and synthesized
novel FIH-1 inhibitors having furan- or thiophene-2-carbonyl amino acids as the main scaffold.
Several compounds showed HIF activation potential using SKN:HRE-MLuc cells under mild hypoxic
conditions. These compounds tended to have bulky lipophilic moieties in the R3 position. The results
of docking simulations and luciferase assays under normoxic conditions supported the idea that the
identified compounds activated HIF transcription by FIH-1 inhibition. Selective FIH-1 inhibitors are
still rare; therefore, the identified compounds may provide alternative HIF activation tools.
4. Materials and Methods
4.1. General Information
DMSO, DMOG, and FG4592 were obtained from Merck (Darmstadt, Germany), and other general
reagents were purchased from Nacalai Tesque (Kyoto, Japan). DM-NOFD was synthesized according
to a literature method [45].
4.2. Preparation of 2-([1,10-biphenyl]-4-yl)thiophene and 2-(4-phenoxyphenyl)thiophene [46]
Under an N2 atmosphere, thiophene (1.5 g, 18 mmol) was placed in a three-necked flask with
distilled tetrahydrofuran (THF, 30 mL). The flask was cooled at 0 ◦C, and n-butyllithium (ca. 1.6 M
in hexane, 10 mL, 16 mmol) was added dropwise. After addition, the mixture was stirred at room

Molecules 2018, 23, 885
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temperature for 30 min. The flask was cooled at 0 ◦C again, and dried zinc chloride (2.0 g, 15 mmol)
suspended in THF (10 mL) was added dropwise. After addition, the mixture was stirred at room
temperature for 30 min., 4-bromobiphenyl or 4-bromodiphenyl ether (15 mmol) and Pd(PPh3)4 (0.69 g,
◦
0.6 mmol) in THF (10 mL) were added, and the mixture was stirred at 80 C. After stirring for
16 h, the mixture was cooled to room temperature and quenched with water. The resulting mixture
was extracted with diethyl ether and dried with MgSO4. After filtration, the solvent was removed
under reduced pressure. The desired pure thiophene was obtained after purification by column
chromatography on silica gel.
4.3. Preparation of 2-((1,10-biphenyl)-4-yl)thiophene-2-carboxylic acid, 2-(4-phenoxyphenyl)thiophene-
2-carboxylic acid, 5-phenylthiophene-2-carboxylic acid, and 5-methylfuran-2-carboxylic acid [47]
2-Methylfuran was obtained from commercial supplies. 2-Phenylthiophene was prepared
according to a previously described method [48]. Under an N2 atmosphere, 2-([1,10-biphenyl]-4-yl)
thiophene, 2-(4-phenoxyphenyl)thiophene, or 2-methylfuran (1.0 equiv.) was placed in a three-necked
flask with distilled THF or Et2O. The flask was cooled at 0 ◦C, and n-butyllithium (1.1 equiv.) was
added dropwise over a period of 15 min. The mixture was then stirred for 30 min at room temperature.
After the mixture was cooled at
vigorous stirring and cooling. After stirring for 2 h, the mixture was warmed to room temperature and
quenched with water. The ether layer was separated and washed with water. The combined solution
−
78 ◦C with a dry ice/acetone bath, CO2 gas was introduced under
◦
was acidified with a 3 N HCl solution at 0 C, and then extracted with EtOAc. The organic layer
was washed with brine, dried with MgSO4, and concentrated under reduced pressure. The desired
products were obtained without further purification.
4.4. Preparation of 5-phenylfuran-2-carboxylic acid
Methyl 5-bromofuran-2-carboxylate and phenylboronic acid were obtained from commercial
supplies. Under an N2 atmosphere, methyl 5-bromofuran-2-carboxylate (26.8 g, 131 mmol),
phenylboronic acid (20.7 g, 133 mmol), toluene (30 mL), Na2CO3 (2 M, 70 mL), and Pd(PPh3)4 (7.7 g,
6.5 mmol) were added to a three-necked flask. The mixture was refluxed for 18 h. The reaction mixture
was extracted with EtOAc, dried over MgSO4, filtered, and concentrated under reduced pressure. The
crude product was purified by column chromatography on silica gel (eluent: hexane/Et2O = 9/1) to
obtain the product. In a three-necked flask, methyl 5-phenylfuran-2-carboxylate (14.9 g, 63 mmol),
◦
toluene (40 mL), and KOH (2 M, 400 mL) were added. The mixture was heated at 70 C for 15 h.
EtOAcwas added to the reaction solution, and the water layer was extracted. HCl aq. (3 N) was
added to the water layer at 0 ◦C, and the solution was extracted with EtOAcand washed with brine.
The combined organic phases were dried over Na2SO4, filtered, and concentrated under reduced
pressure. 5-Phenylfuran-2-carboxylic acid was obtained as a white solid and its structure confirmed by
1H nuclear magnetic resonance (NMR) [49].
4.5. Preparation of 4-phenylthiophene-2-carboxylic acid
4-Bromo-2-thiophenecarbaldehyde and phenylboronic acid were obtained from commercial
suppliers. 4-Bromo-2-thiophenecarbaldehyde (1.9 g, 10 mmol) was added to a solution of
phenylboronic acid (1.8 g, 15 mmol), Na2CO3 (3.2 g, 30 mmol), Pd(PPh3)4 (0.35 g, 0.4 mmol) in
toluene (15 mL)/EtOH (15 mL), and the mixture was refluxed for 12 h. The reaction was filtered
with silica gel and concentrated. The resulting residue was purified by column chromatography
on silica gel to give 4-phenyl-2-thiophenecarbaldehyde (96%). The product was confirmed by
1H-NMR.[50] Then KMnO4 (1.7 g, 11 mmol) in water (15 mL) was added dropwise to a solution
of 4-phenyl-2-thiophenecarbaldehyde (1.8 g, 9.5 mmol) in acetone (10 mL), and the mixture was
refluxed for 12 h. After cooling, the reaction mixture was filtered with silica gel. The mixture was
extracted with Et2O and saturated NaHCO3 aq. (3
×
20 mL). The aqueous layer was neutralized with
3 N HCl aq. and extracted with Et2O (3 20 mL). The resulting organic layer was washed with brine,
×

Molecules 2018, 23, 885
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dried over Na2SO4, and concentrated. The crude product was purified by column chromatography
on silica gel to obtain 4-phenylthiophene-2-carboxylic acid (53%). The product was confirmed by
1H-NMR [51].
4.6. General Procedure for Furan- and Thiophene-2-Carbonyl Amino Acid Derivatives: Condensation Reaction
of Furan- and Thiophene-2-Carboxylic Acids with Amino Acid Ester Hydrochlorides
Furan-2-carboxylic acid, thiophene-2-carboxylic acid, 5-methylthiophene-2-carboxylic acid,
benzofuran-2-carboxylic acid, benzothiophene-2-carboxylic acid, and amino acid ester hydrochlorides
were obtained from commercial supplies and were used without further purification. To a two–necked
flask, an amino acid ester hydrochloride (1.1 mmol), 1–ethyl–3–dimethylaminopropylcarbodiimide
hydrochloride (EDC, 0.29 g, 1.5 mmol), HOBt (0.20 g, 1.5 mmol), Et3N (0.42 mL, 3.0 mmol), and CHCl3
◦
or DMF (2.2 mL) were added, and the mixture was stirred at 0 C for 10 min. Furan-2-carboxylic
acid or thiophene-2-carboxylic acid dissolved in DMF (2.2 mL) was added, and then stirred for
17 h. The reaction mixture was diluted with water and extracted with EtOAc (3
×
20 mL).
The organic phase was then washed with 3 N HCl aq.
(
3 × 20 mL), saturated NaHCO3 aq.
(
3 × 20 mL), and brine (20 mL). The combined organic phases were dried over Na2SO4, filtered,
and concentrated under reduced pressure. The residue was purified by column chromatography
on silica gel (eluent: hexane/EtOAc = 1/1) to obtain the product. The purity of the product was
1
confirmed by H-NMR. Stereochemistry of the final product was measured by high-performance
liquid chromatography (HPLC) with a chiral column (Compound 16). The chart is shown in the
supporting information.
Compounds 39
[52], 41 [53] were previously characterized. The analytical data of the final
products are described below.
Methyl (furan-2-carbonyl)glycinate (
ester hydrochloride; yield 76%; colorless oil; H-NMR (400 MHz, CDCl3)
1
) was synthesized from furan-2-carboxylic acid and glycine methyl
1
δ 3.80 (s, 3H), 4.23 (d,
J = 5.2 Hz, 2H), 6.51 (dd, J = 3.2 Hz, 1.6 Hz, 1H), 7.15 (d, J = 3.2 Hz, 1H), 7.47 (d, J = 1.6 Hz, 1H);
13C-NMR (100 MHz, CDCl3)
40.9, 52.6, 112.3, 114.9, 144.3, 147.4, 158.4, 170.3; HRMS (FAB) Calcd for
C8H10NO4 [M + H]+: 184.0610, Found: 184.0639.
δ
Methyl (thiophene-2-carbonyl)glycinate ( ) was synthesized from thiophen-2-carboxylic acid and glycine
methyl ester hydrochloride; yield 81%; white solid; 1H-NMR (400 MHz, CDCl3)
3.79 (s, 3H), 4.22 (d,
J = 5.2 Hz, 2H), 6.72 (br, 1H), 7.07 (dd, J = 4.0 Hz, 4.8 Hz, 1H), 7.49 (d, J = 5.2 Hz, 1H), 7.57 (d, J = 4.4 Hz
1H); 13C-NMR (100 MHz, CDCl3)
41.6, 52.6, 127.8, 128.7, 130.6, 138.1, 162.1, 170.5; HRMS (FAB) Calcd
for C8H10NO3S [M + H]+: 200.0381, Found: 200.0406.
(S)-Methyl (furan-2-carbonyl)tryptophanate ( ) was synthesized from furan-2-carboxylic acid and
L-tryptophan methyl ester hydrochloride; yield 88%; white solid; H-NMR (400 MHz, CDCl3)
2
δ
,
δ
3
1
δ
3.41 (dd, J = 5.6 Hz, 2.0 Hz 2H), 5.11 (dt, J = 8.4 Hz, 5.6 Hz, 1H), 6.46 (dd, J = 1.6 Hz, 3.2 Hz, 1H), 6.46
(dd, J = 1.6 Hz, 3.2 Hz, 1H), 6.89 (d, J = 7.6 Hz, 1H), 7.01 (d, J = 2.4 Hz, 1H), 7.07 (t, J = 7.6 Hz, 1H), 7.10
(d, J = 3.2 Hz, 1H), 7.18 (t, J = 7.6 Hz, 1H), 7.33–7.38 (m, 2H), 7.54 (d, J = 7.6 Hz, 1H), 8.24 (brs, 1H);
13C-NMR (100 MHz, CDCl3)
δ 28.1, 52.6, 52.7, 109.9, 111.4, 112.2, 114.8, 118.7, 119.7, 122.3, 123.0, 127.6,
136.3, 144.3, 147.5, 158.0, 172.2; HRMS (EI) HRMS (FAB) Calcd for C17H17N2O4 [M + H]+: 313.1188,
Found: 313.1163.
(R)-Methyl (furan-2-carbonyl)tryptophanate (
D-tryptophan methyl ester hydrochloride; yield 69%; white solid; H-NMR (400 MHz, CDCl3)
4
) was synthesized from furan-2-carboxylic acid and
1
δ
3.410 (d, J = 5.6 Hz, 1H), 3.414 (d, J = 5.2 Hz, 1H), 3.69 (s, 3H), 5.11 (dt, J = 8.0 Hz, 5.6 Hz, 1H), 6.46
(dd, J = 1.6 Hz, 3.2 Hz, 1H), 6.47 (dd, J = 1.2 Hz, 3.6 Hz, 1H), 6.89 (d, J = 7.6 Hz, 1H), 7.02 (d, J = 2.4 Hz,
1H), 7.08 (t, J = 7.6 Hz, 1H), 7.10 (d, J = 4.0 Hz, 1H), 7.17 (t, J = 7.6 Hz, 1H), 7.32–7.37 (m, 2H), 7.54 (d,
J = 7.6 Hz, 1H), 8.29 (s, 1H); 13C-NMR (100 MHz, CDCl3)
δ 27.9, 52.4, 52.5, 109.9, 111.2, 112.1, 114.7,

Molecules 2018, 23, 885
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118.6, 119.6, 122.2, 122.8, 127.5, 136.1, 144.2, 147.4, 157.9, 172.1; HRMS (FAB) Calcd for C17H17N2O4
[M + H]+: 313.1188, Found: 313.1189.
(S)-Methyl (thiophene-2-carbonyl)tryptophanate (
5
) was synthesized from furan-2-carboxylic acid and
3.42
L-tryptophan methyl ester hydrochloride; yield 68%; white solid; 1H-NMR (300 MHz, CDCl3)
δ
(d, J = 5.2 Hz, 2H), 3.70 (s, 3H), 5.11 (dt, J = 7.2 Hz, 5.2 Hz, 1H), 6.55 (d, 8.0 Hz, 1H), 6.98–7.01 (m,
2H), 7.07–7.11 (m, 1H), 7.16–7.20 (m, 1H), 7.33–7.36 (m, 2H), 7.45 (dd, J = 5.2 Hz, 1.2 Hz, 1H), 7.55
(d, J = 8.4 Hz, 1H), 8.31 (br, 1H); 13C-NMR (100 MHz, CDCl3)
δ 27.7, 52.4, 53.4, 109.8, 111.3, 118.6,
119.7, 122.3, 122.9, 127.6, 128.4, 130.4, 136.1, 138.3, 161.4, 172.2; HRMS (FAB) Calcd for C17H17N2O3S
[M + H]+: 329.0960, Found: 329.0944.
(R)-Methyl (thiophene-2-carbonyl)tryptophanate (
6) was synthesized from thiophene-2-carboxylic acid
and D-tryptophan methyl ester hydrochloride; yield 43%; white solid; 1H-NMR (400 MHz, CDCl3)
δ
3.42 (d, J = 5.2 Hz, 2H), 3.70 (s, 3H), 5.12 (dt, J = 8.4 Hz, 5.2 Hz, 1H), 6.54 (d, J = 8.0 Hz, 1H), 6.98–7.02
(m, 2H), 7.09 (t, J = 7.2 Hz, 1H), 7.19 (t, J = 7.2 Hz, 1H), 7.32–7.37 (m, 2H), 7.45 (d, J = 5.2 Hz, 1H), 7.55
(d, J = 7.6 Hz, 1H), 8.30 (br, 1H); 13C-NMR (100 MHz, CDCl3)
δ 27.8, 52.6, 53.5, 109.9, 111.5, 118.7, 119.9,
122.4, 123.0, 127.70, 127.73, 128.6, 130.5, 136.2, 138.4, 161.6, 172.3; HRMS (FAB) Calcd for C17H17N2O4S
[M + H]+: 329.0960, Found: 329.0973.
(S)-Methyl (5-methylfuran-2-carbonyl)phenylalaninate (
2-carboxylic acid and L-phenylalanine methyl ester hydrochloride; yield 84%; pale yellow oil; 1H NMR
(400 MHz, CDCl3) 2.33 (s, 3H), 3.18–3.28 (m, 2H), 3.74 (s, 3H), 5.05 (dt, J = 8.4 Hz, 5.2 Hz, 1H), 6.09
(br, 1H), 6.70 (d, J = 8.0 Hz, 1H), 7.01 (d, J = 3.2 Hz, 1H), 7.13–7.18 (m, 2H), 7.23–7.33 (m, 3H); 13C-NMR
(100 MHz, CDCl) 14.0, 38.3, 52.5, 52.9, 108.7, 116.1, 127.2, 128.7, 129.4, 135.9, 145.9, 155.0, 158.0, 172.0;
HRMS (FAB) Calcd for C16H18NO4 [M + H]+: 288.1236, Found: 288.1246.
7) was synthesized from 5-methylthiophene-
δ
δ
(R)-Methyl (5-methylfuran-2-carbonyl)phenylalaninate ( ) was synthesized from 5-methylfuran-
2-carboxylic acid and D-phenylalanine methyl ester hydrochloride; yield 81%; pale yellow oil; 1H-NMR
(400 MHz, CDCl3) 2.33 (s, 3H), 3.18–3.28 (m, 2H), 3.74 (s, 3H), 5.05 (dt, J = 8.4 Hz, 5.2 Hz, 1H), 6.09
(br, 1H), 6.70 (d, J = 8.0 Hz, 1H), 7.01 (d, J = 3.2 Hz, 1H), 7.13–7.18 (m, 2H), 7.23–7.33 (m, 3H); 13C-NMR
(100 MHz, CDCl3) 14.0, 38.3, 52.5, 52.9, 108.7, 116.1, 127.2, 128.7, 129.4, 135.9, 145.9, 155.0, 158.0, 172.0;
HRMS (FAB) Calcd for C16H18NO4 [M + H]+: 288.1236, Found: 288.1258.
(S)-Methyl (5-methylfuran-2-carbonyl)tryptophanate ( ) was synthesized from 5-methylfuran- 2-carboxylic
acid and L-tryptophan methyl ester hydrochloride; yield 78%; white solid; 1H-NMR (400 MHz, CDCl3)
2.26 (s, 3H), 3.35–3.45 (m, 2H), 3.69 (s, 3H), 5.11 (dt, J = 8.0 Hz, 5.2 Hz, 1H), 6.06 (br, 1H), 6.78 (d,
J = 8.4 Hz, 1H), 6.97–7.04 (m, 2H), 7.09 (t, J = 8.0 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 7.35 (d, J = 8.4 Hz,
1H), 7.56 (d, J = 8.0 Hz, 1H), 8.21 (br, 1H); 13C-NMR (100 MHz, CDCl3)
13.9, 28.1, 52.5, 52.6, 108.6,
8
δ
δ
9
δ
δ
110.2, 111.3, 116.0, 118.8, 119.7, 122.3, 122.9, 127.7, 136.2, 145.9, 155.0, 158.1, 172.4; HRMS (FAB) Calcd
for C18H19N2O4 [M + H]+: 327.1345, Found: 327.1331.
(R)-Methyl (5-methylfuran-2-carbonyl)tryptophanate (10) was synthesized from 5-methylfuran-
1
2-carboxylic acid and D-tryptophan methyl ester hydrochloride; yield 81%; white solid; H-NMR
(400 MHz, CDCl3)
(br, 1H), 6.78 (d, J = 8.4 Hz, 1H), 6.97–7.04 (m, 2H), 7.09 (t, J = 8.0 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 7.35
(d, J = 8.4 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 8.14 (br, 1H); 13C-NMR (100 MHz, CDCl3)
13.9, 28.1, 52.5,
δ 2.26 (s, 3H), 3.35–3.45 (m, 2H), 3.69 (s, 3H), 5.11 (dt, J = 8.0 Hz, 5.2 Hz, 1H), 6.06
δ
52.6, 108.6, 110.2, 111.3, 116.0, 118.8, 119.7, 122.3, 122.9, 127.7, 136.2, 145.9, 155.0, 158.1, 172.4; HRMS
(FAB) Calcd for C18H19N2O4 [M + H]+: 327.1345, Found: 327.1362.
(5-Methylthiophene-2-carbonyl)glycine (11) was synthesized by hydrolysis of methyl (5-methylthiophene-
1
2-carbonyl)glycinate; yield 36%; white solid; H-NMR (400 MHz, DMSO)
δ 2.49 (s, 3H), 3.94 (d,
J = 6.0 Hz, 2H), 6.87 (dd, J = 3.6 Hz, 1.2 Hz, 1H), 7.64 (d, J = 3.6 Hz, 1H), 8.75 (t, J = 6.0 Hz, 1H);
13C-NMR (100 MHz, DMSO)
15.4, 41.2, 126.6, 128.9, 136.9, 145.2, 161.8, 171.8; HRMS (FAB) Calcd for
C8H10NO3S [M + H]+: 200.0381, Found: 200.0379.
δ

Molecules 2018, 23, 885
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(S)-(5-Methylthiophene-2-carbonyl)alanine (12) was synthesized by hydrolysis of 13; yield 74%, white
1
solid; H-NMR (400 MHz, DMSO)
δ 1.41 (d, J = 7.2 Hz, 1H), 2.55 (t, J = 2.0 Hz, 3H), 4.32 (quint,
J = 7.2 Hz, 1H), 6.89 (dd, J = 3.6 Hz, 0.8 Hz, 1H), 7.70 (d, J = 3.6 Hz, 1H), 8.59 (d, J = 7.6 Hz, 1H);
13C-NMR (100 MHz, DMSO)
m/z = 214 ([M + H]+).
δ 15.8, 17.5, 48.5, 127.0, 129.4, 137.5, 145.5, 161.6, 174.8; LRMS (FAB)
(S)-Ethyl (5-methylthiophene-2-carbonyl)alaninate (13) was synthesized from 5-methylthiophene-
2-carboxylic acid and L-alanine ethyl ester hydrochloride; yield 65%; white solid; 1H-NMR (400 MHz,
CDCl3)
δ 1.30 (t, J = 4.8 Hz, 3H), 1.49 (d, J = 6.8 Hz, 3H), 2.50 (d, J = 0.8 Hz, 3H), 4.23 (q, J = 7.2 Hz, 2H),
4.73 (quint, J = 7.2 Hz, 1H), 6.56 (d, J = 6.4 Hz, 1H), 6.73 (dd, J = 3.6 Hz, 1.2 Hz, 1H), 7.36 (d, J = 4.0 Hz,
1H); 13C-NMR (100 MHz, CDCl3)
δ 14.0, 15.6, 18.6, 48.3, 61.5, 126.0, 128.7, 135.6, 145.5; HRMS (FAB)
Calcd for C11H16NO3S [M + H]+: 242.0851, Found: 242.0850.
(R)-Methyl (5-methylthiophene-2-carbonyl)phenylalaninate (14) was synthesized from 5-methylthiophene-
2-carboxylic acid and D-phenylalanine methyl ester hydrochloride; yield 83%; pale yellow solid;
1H-NMR (400 MHz, CDCl3)
δ
2.50 (s, 3H), 3.18–3.28 (m, 2H), 3.75 (s, 3H), 5.05 (dt, J = 7.6 Hz, 5.6 Hz,
1H), 6.32 (br, 1H), 6.72 (d, J = 3.6 Hz, 1H), 7.10–7.15 (m, 2H), 7.23–7.32 (m, 4H); 13C-NMR (100 MHz,
CDCl3) δ 15.8, 38.1, 52.5, 53.4,126.2, 127.3, 128.7, 128.9, 129.4, 135.5, 135.9, 145.9, 161.4, 172.0; HRMS
(FAB) Calcd for C16H18NO3S [M + H]+: 304.1007, Found: 304.1006.
(S)-Ethyl (5-methylthiophene-2-carbonyl)phenylalaninate (15) was synthesized from 5-methylthiophene-
1
2-carboxylic acid and L-phenylalanine ethyl ester hydrochloride; yield 37%; white solid; H-NMR
(400 MHz, CDCl3)
δ 1.26 (t, J = 7.6 Hz, 3H), 2.50 (s, 3H), 3.21 (d, J = 1.6 Hz, 2H), 4.19 (q, J = 7.6 Hz, 2H),
5.00 (dt, J = 7.6 Hz, 5.6 Hz, 1H), 6.35 (d, J = 7.6 Hz, 1H), 6.71 (d, J = 3.6 Hz, 1H), 7.14 (dd, J = 8.0 Hz
,
1.6 Hz, 2H), 7.24–7.30 (m, 4H); 13C-NMR (100 MHz, CDCl3)
δ 14.1, 15.6, 38.0, 53.3, 61.6, 126.1, 127.1,
128.5, 128.7, 129.4, 135.5, 135.8, 145.7, 161.2, 171.5; HRMS (FAB) Calcd for C17H20NO3S [M + H]+:
318.1164, Found: 318.1152.
(R)-(5-methylthiophene-2-carbonyl)tryptophan (16) was synthesized by hydrolysis of 18; yield 99%; pale
yellow solid; 1H-NMR (300 MHz, CDCl3)
δ
2.48 (s, 3H), 3.47 (t, J = 4.8 Hz, 2H), 3.49 (s, 1H), 5.02–5.04
(m, 1H), 6.36 (d, J = 6.8 Hz, 1H), 6.67 (d, J = 2.9 Hz, 1H), 7.07–7.29 (m, 4H), 7.39 (d, J = 8.0 Hz, 1H),
7.63 (d, J = 8.3 Hz, 1H), 8.18 (br, 1H); 13C-NMR (75 MHz, DMSO–d6)
15.5, 26.9, 53.7, 110.5, 111.6,
δ
118.3, 118.6, 121.2, 123.7, 126.6, 127.3, 129.0, 136.3, 137.0, 145.1, 161.3, 173.7; HRMS (FAB) Calcd for
C8H10NO3S [M + H]+: 200.0381, Found: 200.0379.
(S)-Ethyl (5-methylthiophene-2-carbonyl)tryptophanate (17) was synthesized from 5-methylthiophene-
1
2-carboxylic acid and L-phenylalanine ethyl ester hydrochloride; yield 86%; white solid; H-NMR
(300 MHz, CDCl3)
1H), 6.42 (d, J = 7.8 Hz, 1H), 6.68 (d, J = 2.9 Hz, 1H), 7.03–7.27 (m, 4H), 7.36 (d, J = 8.1 Hz, 1H), 7.55
(d, J = 8.0 Hz, 1H), 8.08 (br, 1H); 13C-NMR (100 MHz, CDCl3)
14.1, 15.6, 27.8, 53.3, 61.6, 110.0, 111.2,
δ 1.23 (t, J = 6.8 Hz, 3H), 2.50 (s, 3H), 3.42 (d, J = 5.1 Hz, 2H), 4.15 (m, 2H), 5.08 (m,
δ
118.7, 119.6, 122.2, 122.9, 126.0, 127.7, 128.8, 135.6, 136.1, 145.7, 161.5, 171.9; HRMS (FAB) Calcd for
C19H21N2O3S [M + H]+: 357.1273, Found: 357.1279.
(R)-Ethyl (5-methylthiophene-2-carbonyl)tryptophanate (18) was synthesized from 5-methylthiophene-
2-carboxylic acid and D-phenylalanine ethyl ester hydrochloride; yield 88%; white solid; 1H-NMR
(CDCl3, 400 MHz)
J = 8.2 Hz, 5.4 Hz, 1H), 6.48 (d, J = 7.8 Hz, 1H), 6.65 (d, J = 2.9 Hz, 1H), 6.98 (d, J = 2.2 Hz, 1H), 7.05–7.26
(m, 3H), 7.33 (d, J = 8.1 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 8.41 (br, 1H); 13C-NMR (75 MHz, CDCl3)
δ 1.21 (t, J = 7.3 Hz, 3H), 2.46 (s, 3H), 3.40 (d, J = 5.4 Hz, 2H), 4.13 (m, 2H), 5.07 (dt,
δ
14.4, 16.0, 28.2, 53.7, 62.0, 110.3, 111.7, 119.1, 120.0, 122.5, 123.4, 126.5, 128.1, 129.2, 136.0, 136.5, 146.1,
161.9, 172.3; HRMS (FAB) Calcd for C19H21N2O3S [M + H]+: 357.1273, Found: 357.1272.
(S)-(5-Phenylfuran-2-carbonyl)phenylalanine (19) was synthesized by hydrolysis of 20; yield 16%; yellow
solid; 1H-NMR (400 MHz, DMSO)
δ
3.11 (dd, J = 14.0 Hz, 10.4 Hz, 1H), 3.22 (dd, J = 14.0 Hz, 4.4 Hz,
1H), 4.60–4.66 (m, 1H), 7.08 (d, J = 3.6 Hz, 1H), 7.17 (d, J = 3.6 Hz, 1H), 7.18 (t, J = 6.8 Hz, 1H), 7.26 (t,

Molecules 2018, 23, 885
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J = 4.0 Hz, 2H), 7.31 (d, J = 6.8 Hz, 2H), 7.38 (t, J = 7.2 Hz, 1H), 7.48 (t, J = 4.0 Hz, 2H), 7.90 (d, J = 7.2 Hz
,
2H), 8.67 (d, J = 8.4 Hz, 1H); 13C-NMR (100 MHz, CDCl3)
δ 36.2, 53.3, 107.6, 116.0, 124.3, 126.4, 128.2,
128.6, 128.9, 129.0, 129.3, 138.0, 146.5, 154.6, 157.5, 172.9; HRMS (FAB) Calcd for C8H10NO3S [M + H]+:
200.0381, Found: 200.0379.
(S)-Methyl (5-phenylfuran-2-carbonyl)phenylalaninate (20) was synthesized from 5-phenylfuran-
2-carboxylic acid and L-phenylalanine methyl ester hydrochloride; yield 11%; colorless oil; 1H-NMR
(400 MHz, CDCl3)
δ 3.25 (d, J = 7.8 Hz, 2H), 3.76 (s, 3H), 5.04–5.10 (m, 1H), 6.72 (d, J = 4.9 Hz, 1H), 6.81
(d, J = 10.2 Hz, 1H), 7.15–7.46 (m, 9H), 7.66 (d, J = 9.8 Hz, 2H).
(S)-(5-Phenylthiophene-2-carbonyl)alanine (21) was synthesized by hydrolysis of 22; yield 72%; pale
yellow solid; 1H-NMR (400 MHz, DMSO)
δ
1.40 (d, J = 3.6 Hz, 3H), 4.40 (quint, J = 7.2 Hz, 1H), 7.37
(t, J = 7.2 Hz, 1H), 7.45 (t, J = 7.6 Hz, 2H), 7.56 (d, J = 3.6 Hz, 1H), 7.71 (d, J = 7.2 Hz, 2H), 7.87 (d,
J = 3.6 Hz, 1H); 13C-NMR (100 MHz, DMSO)
δ 16.9, 48.1, 124.3, 125.7, 128.5, 129.2, 129.7, 133.1, 138.3,
147.6, 160.8, 174.1; HRMS (FAB) Calcd for C14H14NO3S [M + H]+: 276.0694, Found: 276.0701.
(S)-Ethyl (5-phenylthiophene-2-carbonyl)alaninate (22) was synthesized from 5-phenylthiophene-
2-carboxylic acid and L-alanine ethyl ester hydrochloride; yield 53%; white solid; 1H-NMR (400 MHz,
CDCl3)
δ 1.32 (t, J = 7.1 Hz, 3H), 1.55 (m, 3H), 4.26 (q, J = 7.0 Hz, 2H), 4.76 (m, 1H), 6.56 (br, 1H),
7.19–7.43 (m, 4H), 7.51 (d, J = 7.4 Hz, 1H), 7.63 (m, 2H).
(S)-(5-Phenylthiophene-2-carbonyl)phenylalanine (23) was synthesized by hydrolysis of 24; yield 70%; pale
yellow solid; 1H-NMR (400 MHz, DMSO)
4.4 Hz, 1H), 4.59 (ddd, J = 11.2 Hz, 8.8 Hz, 4.4 Hz, 1H), 7.18 (dt, J = 7.2 Hz, 1.6 Hz, 1H), 7.27 (t, J = 7.6 Hz
δ
3.05 (dd, J = 14.0 Hz, 10.8 Hz, 1H), 3.19 (dd, J = 14.0 Hz
,
,
2H), 7.31 (d, J = 6.8 Hz, 2H), 7.44 (t, J = 7.6 Hz, 2H), 7.53 (d, J = 3.6 Hz, 1H), 7.69 (dt, J = 6.8 Hz, 1.6 Hz,
2H), 7.82 (d, J = 4.0 Hz, 1H); 13C-NMR (100 MHz, DMSO)
δ 36.3, 54.1, 124.3, 125.7, 126.4, 128.2, 128.5,
129.0, 129.2, 129.6, 133.0, 138.2, 147.7, 161.0, 173.0; HRMS (FAB) Calcd for C20H18NO3S [M + H+]:
352.1007, Found: 352.1001.
(S)-Methyl (5-phenylthiophene-2-carbonyl)phenylalaninate (24) was synthesized from 5-phenylthiophene-
2-carboxylic acid and L-phenylalanine methyl ester hydrochloride; yield 61%; white solid; 1H-NMR
(400 MHz, CDCl3)
δ 3.25 (t, J = 6.8 Hz, 2H), 3.77 (s, 3H), 5.07 (dt, J = 10.2 Hz, 5.3 Hz, 1H), 6.39(d,
J = 10.2 Hz, 1H), 7.14 (m, 2H), 7.24–7.44 (m, 8H), 7.62 (m, 2H).
(S)-Methyl (5-phenylthiophene-2-carbonyl)tryptophanate (25) was synthesized from 5-phenylthiophene-
2-carboxylic acid and L-tryptophan methyl ester hydrochloride; yield 41%; pale yellow solid; 1H-NMR
(400 MHz, CDCl3)
J = 8.0 Hz, 1H), 7.03 (d, J = 2.4 Hz, 1H), 7.11 (t, J = 7.6 Hz, 1H), 7.16–7.22 (m, 2H), 7.30–7.42 (m, 5H),
7.55–7.61 (m, 3H), 8.14 (br, 1H); 13C-NMR (100 MHz, CDCl3)
27.9, 52.6, 53.5, 110.1, 111.4, 118.8, 119.9,
δ 3.44 (d, J = 5.2 Hz, 2H), 3.72 (s, 3H), 5.13 (dt, J = 8.0 Hz, 4.8 Hz, 1H), 6.50 (d,
δ
122.5, 123.0, 123.5, 126.2, 127.7, 128.7, 129.1, 129.5, 133.5, 136.2, 136.4, 149.4, 161.4, 172.3; HRMS (FAB)
Calcd for C23H21N2O3S [M + H]+: 405.1273, Found: 405.1288.
Methyl (4-phenylthiophene-2-carbonyl)glycinate (26) was synthesized from 4-phenylthiophene-
2-carboxylic acid and glycine methyl ester hydrochloride; yield 75%; white powder; mp. 93–96 ◦C
;
1H-NMR (400 MHz, CDCl3)
1H), 7.40 (t, J = 7.8 Hz, 2H), 7.56 (d, J = 7.7 Hz, 2H), 7.58 (d, J = 1.4 Hz, 1H), 7.84 (d, J = 1.4 Hz, 1H);
δ 3.81 (s, 3H), 4.25 (d, J = 5.5 Hz, 2H), 6.65 (br, 1H), 7.31 (t, J = 7.8 Hz,
13C-NMR (100 MHz, CDCl3)
δ 41.7, 52.7, 125.1, 126.4, 127.7, 127.8, 128.7, 129.1, 135.0, 138.5, 143.1, 161.9,
170.5; HRMS (FAB) Calcd for C14H14NO3S [M + H]+: 276.0694, Found: 276.0701.
(S)-Ethyl (4-phenylthiophene-2-carbonyl)phenylalaninate (27) was synthesized from 4-phenylthiophene-
2-carboxylic acid and L-phenylalanine ethyl ester hydrochloride; yield 70%; white powder; mp.
94–96 ◦C; 1H-NMR (400 MHz, CDCl3)
δ 1.20 (t, J = 7.0 Hz, 3H), 3.16–3.27 (m, 2H), 4.15 (q, J = 7.0 Hz,
2H), 5.01–5.06 (m, 1H), 6.99 (d, J = 7.8 Hz, 1H), 7.18 (t, J = 7.3 Hz, 2H), 7.19–7.22 (m, 1H), 7.21–7.25 (m,
2H), 7.23–7.27 (m, 1H), 7.33 (t, J = 7.3 Hz, 2H), 7.59 (d, J = 7.3 Hz, 2H), 7.59 (d, J = 1.4 Hz, 1H), 7.95
(d, J = 1.4 Hz, 1H); 13C-NMR (100 MHz, CDCl3) δ 14.2, 38.1, 53.6, 61.8, 125.0, 126.4, 127.3, 127.5, 127.8,

Molecules 2018, 23, 885
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128.7, 129.1, 129.5, 135.0, 135.8, 138.9, 143.1; HRMS (FAB) Calcd for C22H22NO3S [M + H]+: 380.1320,
Found: 380.1333.
(S)-Methyl (4-phenylthiophene-2-carbonyl)tryptophanate (28) was synthesized from 4-phenylthiophene-
2-carboxylic acid and L-tryptophan methyl ester hydrochloride; yield 69%; flesh color powder; mp.
150–152 ◦C; 1H-NMR (400 MHz, CDCl3)
δ 3.43 (dd, J = 5.0 Hz, 2H), 3.70 (s, 3H), 5.12 (dt, J = 8.2 Hz,
5.0 Hz, 1H), 6.62 (d, J = 8.2 Hz, 1H), 6.99 (d, J = 2.3 Hz, 1H), 7.10 (dt, J = 6.8 Hz, 0.9 Hz, 1H), 7.19 (dt,
J = 6.8 Hz, 1.4 Hz, 1H), 7.30 (dt, J = 6.8 Hz, 0.9 Hz, 1H), 7.35–7.39 (m, 3H), 7.46 (dd, J = 6.8 Hz, 1.4 Hz,
1H), 7.46 (d, J = 8.2 Hz, 1H), 7.53 (dd, J = 9.5 Hz, 1.4 Hz, 2H), 7.58 (d, J = 8.2 Hz, 1H), 8.43 (s, 1H);
13C-NMR (100 MHz, CDCl3)
δ 27.7, 52.6, 53.6, 110.0, 111.5, 118.7, 120.0, 122.5, 123.0, 125.0, 126.4, 127.5,
127.8, 129.0, 134.9, 136.2, 139.0, 142.9, 161.4, 172.2; HRMS (FAB) Calcd for C23H21N2O3S [M + H]+:
405.1273, Found: 405.1272.
(R)-Methyl (4-phenylthiophene-2-carbonyl)tryptophanate (29) was synthesized from 4-phenylthiophene-
2-carboxylic acid and D-tryptophan methyl ester hydrochloride; yield 80%; pale yellow solid; mp.
◦
145–149 C; 1H-NMR (400 MHz, DMSO-d6)
δ3.18–3.30 (m, 2H), 3.60 (s, 3H), 4.63–4.69 (m, 1H), 6.95 (dd,
J = 8.2 Hz, 7.3 Hz, 1H), 7.03 (dd, J = 8.2 Hz, 7.7 Hz, 1H), 7.18 (d, J = 2.3 Hz, 1H), 7.30 (t, J = 7.3 Hz, 1H),
7.30 (d, J = 7.7 Hz, 1H), 7.42 (t, J = 7.7 Hz, 2H), 7.54 (d, J = 7.7 Hz, 1H), 7.66 (d, J = 7.7 Hz, 1H), 8.06
(d, J = 1.4 Hz, 1H), 8.30 (d, J = 1.4 Hz, 1H), 8.86 (d, J = 7.7 Hz, 1H), 10.8 (s, 1H); 13C-NMR (100 MHz,
DMSO-d6)
δ 26.9, 52.0, 53.7, 109.7, 111.5, 118.1, 118.4, 121.0, 123.7, 125.8, 125.9, 127.0, 127.4, 127.6, 129.0,
134.5, 136.1, 139.6, 141.7, 161.1, 172.3; HRMS (FAB) Calcd for C23H21N2O3S [M + H]+: 405.1273, Found:
405.1279.
(S)-Methyl (4-phenylthiophene-2-carbonyl)tyrosinate (30) was synthesized from 4-phenylthiophene-
2-carboxylic acid and l-tyrosine methyl ester hydrochloride; yield 70%; white solid; mp. 176–178 ◦C;
1H-NMR (400 MHz, DMSO-d6)
δ
2.90–3.07 (m, 2H), 3.63 (s, 3H), 4.50–4.60 (m, 1H), 6.65 (d, J = 8.2 Hz
2H), 7.08 (d, J = 8.2 Hz, 2H), 7.34 (t, J = 7.7 Hz, 1H), 7.47 (t, J = 7.7 Hz, 2H), 7.70 (d, J = 7.7 Hz, 2H), 8.10
(s, 1H), 8.33 (s, 1H), 8.87 (d, J = 8.2 Hz, 1H), 9.23 (br, 1H); 13C-NMR (100 MHz, DMSO-d6)
35.7, 52.0,
,
δ
54.5, 115.1, 125.8, 126.0, 127.3, 127.6, 127.9, 129.1, 130.0, 134.5, 139.6, 141.7, 156.0, 161.0, 172.1; HRMS
(FAB) Calcd for C21H20NO4S [M + H]+: 382.1113, Found: 382.1110.
Methyl (5-((1,10-biphenyl)-4-yl)thiophene-2-carbonyl)glycinate
5-((1,10-biphenyl)-4-yl)thiophene-2-carboxylic acid and glycine methyl ester hydrochloride;
yield 2%; yellow solid; mp. 183–185 ◦C; 1H-NMR (400 MHz, CDCl3)
3.82 (s, 3H), 4.26 (d, J = 5.2 Hz,
(
31
)
was synthesized from
δ
2H), 6.48 (t, J = 4.8 Hz, 1H), 7.32 (d, J = 4.0 Hz, 1H), 7.37 (t, J = 7.6 Hz, 1H), 7.46 (t, J = 7.6 Hz, 2H), 7.55
(d, J = 4.0 Hz, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 7.6 Hz, 2H), 7.71 (d, J = 8.0 Hz, 2H); 13C-NMR
(100 MHz, CDCl3) δ 41.7, 52.6, 123.6, 126.6, 127.0, 127.8, 127.8, 129.0, 129.7, 132.4, 136.5, 140.3, 141.5,
149.2, 161.9, 170.5; HRMS (FAB) Calcd for C20H18NO3S [M + H]+: 352.1007, Found: 352.1006.
(S)-Ethyl (5-((1,10-biphenyl)-4-yl)thiophene-2-carbonyl)phenylalaninate (32) was synthesized from
5-((1,10-biphenyl)-4-yl)thiophene-2-carboxylic acid and L-phenylalanine ethyl ester hydrochloride;
yield 15%; yellow solid; 1H-NMR (400 MHz, CDCl3)
δ 1.28 (t, J = 7.2 Hz, 3H), 3.24 (dd, J = 12.0 Hz,
5.2 Hz, 1H), 3.28 (dd, J = 12.0 Hz, 6.0 Hz, 1H), 5.05 (dt, J = 7.6 Hz, 5.2 Hz, 1H), 6.45 (d, J = 7.2 Hz,
1H), 7.16 (d, J = 6.4 Hz, 1H), 7.27–7.32 (m, 3H), 7.37 (t, J = 7.6 Hz, 1H), 7.45 (d, J = 4.8 Hz, 1H), 7.47
(d, J = 7.6 Hz, 2H), 7.62 (d, J = 7.6 Hz, 2H), 7.64 (d, J = 7.6 Hz, 2H), 7.69 (d, J = 7.6 Hz, 2H); 13C-NMR
(100 MHz, CDCl3)
δ 14.1, 38.1, 53.5, 61.7, 123.5, 126.5, 126.9, 127.2, 127.6, 127.7, 128.6, 128.9, 129.3,
129.4, 132.4, 135.8, 136.8, 140.2, 141.4, 149.0, 161.1, 171.5; HRMS (FAB) Calcd for C28H26NO3S [M + H]+:
456.1633, Found: 456.1637.
Methyl
5-(4-phenoxyphenyl)thiophene-2-carboxylic acid and glycine methyl ester hydrochloride; yield 22%;
yellow solid; 1H-NMR (400 MHz, CDCl3)
3.81 (s, 3H), 4.24 (d, J = 5.2 Hz, 2H), 6.47 (t, J = 4.8 Hz, 1H),
(5-(4-phenoxyphenyl)thiophene-2-carbonyl)glycinate
(
33
)
synthesized
from
δ
7.02 (dt, J = 8.8 Hz, 1.6 Hz, 2H), 7.05 (d, J = 7.6 Hz, 2H), 7.15 (t, J = 7.2 Hz, 1H), 7.20 (d, J = 3.6 Hz, 1H),
7.37 (t, J = 8.0 Hz, 2H), 7.51 (d, J = 4.0 Hz, 1H), 7.58 (dt, J = 8.8 Hz, J = 1.6 Hz, 2H); 13C-NMR (100 MHz,

Molecules 2018, 23, 885
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CDCl3)
δ 41.6, 52.5, 118.9, 119.3, 123.0, 123.8, 127.6, 128.4, 129.6, 129.9, 136.0, 149.0, 156.5, 158.0, 161.8,
170.4; HRMS (FAB) Calcd for C20H18NO4S [M + H]+: 368.0957, Found: 368.0962.
(S)-Methyl (5-(4-phenoxyphenyl)thiophene-2-carbonyl)alaninate 34 was synthesized from
5-(4-phenoxyphenyl)thiophene-2-carboxylic acid and L-alanine methyl ester hydrochloride;
yield 18%; pale yellow solid; 1H-NMR (400 MHz, CDCl3)
1.32 (t, J = 7.2 Hz, 3H), 1.52 (d, J = 6.8 Hz,
( )
δ
3H), 4.25 (q, J = 7.2 Hz, 2H), 4.76 (quint, J = 7.2 Hz, 1H), 6.61 (d, J = 7.2 Hz, 1H), 7.02 (dt, J = 8.8 Hz,
J = 2.4 Hz, 2H), 7.05 (d, J = 8.4 Hz, 2H), 7.19 (d, J = 4.0 Hz, 1H), 7.37 (tt, J = 8.4 Hz, 2.4 Hz, 2H), 7.50 (d,
J = 3.6 Hz, 1H), 7.57 (dt, J = 9.2 Hz, 2.4 Hz, 2H); 13C-NMR (100 MHz, CDCl3)
δ 14.1, 18.7, 48.5, 61.7,
118.9, 119.3, 123.0, 123.8, 127.6, 128.5, 129.3, 129.9, 136.5, 148.8, 156.6, 157.9, 161.2, 173.2; HRMS (FAB)
Calcd for C22H22NO4S [M + H]+: 396.1270, Found: 396.1245.
(S)-Ethyl (5-(4-phenoxyphenyl)thiophene-2-carbonyl)phenylalaninate
5-(4-phenoxyphenyl)thiophene-2-carboxylic acid and L-phenylalanine ethyl ester hydrochloride; yield
8%; yellow oil; 1H-NMR (400 MHz, CDCl3)
1.28 (t, J = 7.2 Hz, 2H), 3.25 (q, J = 7.2 Hz, 2H), 4.21 (q,
(35) was synthesized from
δ
J = 7.2 Hz, 2H), 5.04 (dt, J = 7.2 Hz, J = 6.0 Hz, 1H), 6.44 (d, J = 7.6 Hz, 1H), 7.02 (dt, J = 8.8 Hz, 2.4 Hz,
2H), 7.05 (d, J = 8.0 Hz, 2H), 7.25–7.31 (m, 3H), 7.37 (t, J = 8.4 Hz, 2H), 7.41 (d, J = 4.0 Hz, 1H), 7.57 (dt,
J = 8.4 Hz, 1.6 Hz, 2H); 13C-NMR (100 MHz, CDCl3)
δ 14.1, 38.1, 53.5, 61.7, 119.0, 119.3, 123.0, 123.8,
127.2, 127.6, 128.4, 128.6, 129.4, 129.9, 135.8, 136.3, 148.9, 156.5, 158.0, 161.2, 171.5; HRMS (FAB) Calcd
for C28H26NO4S [M + H]+: 472.1583, Found: 472.1587.
(S)-Methyl (5-(4-phenoxyphenyl)thiophene-2-carbonyl)tryptophanate
5-(4-phenoxyphenyl)thiophene-2-carboxylic acid and L-tryptophan methyl ester hydrochloride; yield
79%; white solid; 1H-NMR (400 MHz, CDCl3)
3.43 (d, J = 4.8 Hz, 2H), 3.71 (s, 3H), 5.12 (dt, J = 8.0 Hz
(36) was synthesized from
δ
,
J = 4.8 Hz, 1H), 6.51 (d, J = 7.6 Hz, 1H), 6.99 (d, J = 8.8 Hz, 2H), 7.00 (s, 1H), 7.04 (dd, J = 8.8 Hz,
1.2 Hz, 2H), 7.10 (td, J = 7.6 Hz, 1.2 Hz, 1H), 7.10 (d, J = 4.0 Hz, 1H), 7.14 (t, J = 8.8 Hz, 1H), 7.19
(t, J = 7.6 Hz, 1H), 7.27 (d, J = 4.4 Hz, 1H), 7.34 (d, J = 7.2 Hz, 1H), 7.36 (t, J = 7.2 Hz, 2H), 7.53 (dt,
J = 1.6 Hz, 2H), 7.57 (d, J = 7.6 Hz, 1H), 8.26 (s, 1H); 13C-NMR (100 MHz, CDCl3)
J = 8.4 Hz, δ 27.7,
52.4, 53.4, 109.9, 111.3, 118.6, 118.9, 119.3, 119.8, 122.3, 122.92, 122.94, 123.8, 127.5, 127.6, 128.4, 129.4,
129.9, 136.1, 136.4, 148.9, 156.5, 157.9, 161.3, 172.2; HRMS (FAB) Calcd for C29H25N2O4S [M + H]+:
497.1535, Found: 497.1538.
(S)-Ethyl (benzofuran-2-carbonyl)phenylalaninate (37) was synthesized from benzofuran-2-carboxylic acid
and L-phenylalanine ethyl ester hydrochloride; yield 8%; colorless oil; 1H-NMR (400 MHz, CDCl3)
δ
1.27 (t, J = 7.2 Hz, 3H), 3.25–3.28 (m, 2H), 4.21 (q, J = 7.2 Hz, 2H), 5.08 (dd, J = 6.0 Hz, 2.0 Hz, 1H), 7.12
(d, J = 8.0 Hz, 1H), 7.18 (d, J = 7.6 Hz, 2H), 7.23–7.32 (m, 4H), 7.41 (td, J = 8.0 Hz, 1.2 Hz, 1H), 7.47 (d,
J = 0.8 Hz, 1H), 7.50 (dd, J = 8.4 Hz, J = 0.8 Hz,), 7.66 (d, J = 8.0 Hz, 1H); 13C-NMR (100 MHz, CDCl3)
δ
14.1, 38.2, 53.1, 61.7, 110.8, 111.9, 122.7, 123.7, 127.0, 127.1, 127.5, 128.6, 129.3, 135.7, 148.1, 154.8, 158.2,
171.2; HRMS (FAB) Calcd for C20H20NO4 [M + H]+: 338.1392, Found: 338.1393.
(S)-Methyl (benzofuran-2-carbonyl)tryptophanate (38) was synthesized from benzofuran-2-carboxylic acid
and L-tryptophan methyl ester hydrochloride; yield 31%; white solid; 1H-NMR (400 MHz, CDCl3)
δ
3.45 (d, J = 5.2 Hz, 2H), 3.71 (s, 3H), 5.16 (dt, J = 8.0 Hz, 5.6 Hz, 1H), 7.04 (d, J = 2.4 Hz, 1H), 7.08
(t, J = 7.6 Hz, 1H), 7.18 (t, J = 7.2 Hz, 2H), 7.28 (t, J = 7.6 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.39 (dd,
J = 6.8 Hz, 1.2 Hz, 1H), 7.41 (d, J = 6.8 Hz, 1H), 7.46 (s, 1H); 13C-NMR (100 MHz, CDCl3)
27.9, 52.5,
δ
52.8, 109.8, 110.8, 111.3, 111.9, 119.7, 122.2, 122.7, 123.6, 127.0, 127.4, 127.5, 136.2, 148.2, 154.8, 158.4,
171.9; HRMS (FAB) Calcd for C21H19N2O4 [M + H]+: 363.1345, Found: 363.1341.
Methyl (benzothiophene-2-carbonyl)glycinate (39) was synthesized from benzothiophene-2-carboxylic
1
acid and glycine methyl ester hydrochloride; yield 45%; white solid; H-NMR (400 MHz, DMSO)
δ
3.67 (s, 3H), 4.05 (d, J = 6.0 Hz, 2H), 7.44–7.48 (m, 2H), 7.96 (dd, J = 6.4 Hz, 1.6 Hz, 1H), 8.03 (dd,
J = 6.8 Hz, 2.0 Hz, 1H), 8.13 (s, 1H), 9.25 (t, J = 2.0 Hz, 1H); 13C-NMR (100 MHz, DMSO)
δ
41.1, 51.8,
122.8, 125.0, 125.3, 125.4, 126.4, 139.0, 139.1, 140.3, 162.0, 170.2; HRMS (FAB) Calcd for C12H12NO3S
[M + H]+: 250.0538, Found: 250.0538.

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(S)-Ethyl (benzothiophene-2-carbonyl)alaninate (40) was synthesized from benzothiophene- 2-carboxylic
acid and L-alanine ethyl ester hydrochloride; yield 50%; white solid; 1H-NMR (400 MHz, DMSO)
δ
1.20 (t, J = 7.6 Hz, 3H), 1.43 (d, J = 7.6 Hz, 2H), 4.12 (qd, J = 7.6 Hz, 1.2 Hz, 2H), 4.46 (quint, J = 7.6 Hz,
1H), 7.42–7.49 (m, 2H), 7.97 (dd, J = 6.4 Hz, 2.4 Hz, 1H), 8.02 (dd, J = 6.8 Hz, 1.6 Hz, 1H), 8.21 (s,
1H), 9.08 (d, J = 6.8 Hz, 1H); 13C-NMR (100 MHz, DMSO)
δ 14.1, 16.7, 48.4, 60.6, 122.8, 124.9, 125.3,
125.5, 126.3, 139.1, 140.3, 161.5, 172.4; HRMS (FAB) Calcd for C14H16NO3S [M + H]+: 278.0851, Found:
278.0871.
(S)-Ethyl (benzothiophene-2-carbonyl)phenylalaninate (41) was synthesized from benzothiophene-
1
2-carboxylic acid and L-phenylalanine ethyl ester hydrochloride; yield 35%; white solid; H-NMR
(400 MHz, CDCl3)
2H), 5.07–5.12 (q, J = 7.3 Hz, 1H), 6.62 (d, J = 7.3 Hz, 1H), 7.18 (d, J = 7.8 Hz, 2H), 7.29–7.31 (m, 3H),
7.40–7.44 (m, 2H), 7.75 (s, 1H), 7.84 (t, J = 9.0 Hz, 2H); 13C-NMR (100 MHz, CDCl3)
14.2, 38.0, 53.6,
δ 1.31 (t, J = 7.1 Hz, 3H), 3.25–3.35 (dd, J = 8.6 Hz, 5.9 Hz, 2H), 4.23–4.28 (q, J = 7.2 Hz,
δ
61.8, 122.7, 125.0, 125.1, 125.5, 126.5, 127.2, 128.6, 129.5, 135.7, 137.9, 139.0, 141.0, 161.6, 171.3.
(R)-Ethyl (benzothiophene-2-carbonyl)phenylalaninate (42) was synthesized from benzothiophene-
2-carboxylic acid and D-phenylalanine ethyl ester hydrochloride; yield 23%; white solid; 1H-NMR
(400 MHz, CDCl3)
δ
3.21–3.33 (m, 2H), 3.77 (s, 3H), 5.09 (dt, J = 7.6 Hz, 5.6 Hz, 1H), 6.60 (d, J = 7.2 Hz
,
1H), 7.15 (dd, J = 7.6 Hz, 1.2 Hz, 2H), 7.24–7.32 (m, 3H), 7.37–7.44 (m, 2H), 7.72 (s, 1H), 7.83 (t, J = 8.0 Hz
,
2H); 13C-NMR (100 MHz, CDCl3)
δ
37.9, 52.5, 53.6, 122.7, 124.9, 125.1, 125.6, 126.5, 127.3, 128.7, 129.3,
135.6, 137.7, 140.0, 141.0, 161.7, 171.7; HRMS (FAB) Calcd for C19H18NO3S [M + H]+: 340.1007, Found:
340.1008.
(S)-Methyl (benzothiophene-2-carbonyl)tryptophanate (43) was synthesized from benzothiophene-
1
2-carboxylic acid and L-tryptophan methyl ester hydrochloride; yield 36%; white solid; H-NMR
(400 MHz, CDCl3)
δ 3.47 (d, J = 4.8 Hz, 2H), 3.73 (s, 3H), 5.15 (dt, J = 8.0 Hz, 4.8 Hz, 1H), 6.66 (d,
J = 8.0 Hz, 1H), 7.03 (d, J = 2.4 Hz, 1H), 7.10 (t, J = 8.4 Hz, 1H), 7.20 (t, J = 6.8 Hz, 1H), 7.36–7.43 (m,
3H), 7.58 (d, J = 8.0 Hz, 1H), 7.60 (s, 1H), 7.77 (d, J = 6.8 Hz, 1H), 7.84 (d, J = 7.6 Hz, 1H), 8.13 (s,
1H); 13C-NMR (100 MHz, CDCl3)
δ 27.7, 52.5, 53.5, 109.9, 111.3, 118.7, 119.9, 122.4, 122.7, 122.9, 124.9,
125.1, 125.5, 126.4, 127.7, 136.1, 137.9, 139.0, 141.0, 161.8, 172.0; HRMS (FAB) Calcd for C21H19N2O3S
[M + H]+: 379.1116, Found: 379.1110.
(S)-Methyl (benzothiophene-2-carbonyl)tyrosinate (44) was synthesized from benzothiophene- 2-carboxylic
acid and l-tyrosine methyl ester hydrochloride; yield 19%; white solid; 1H-NMR (400 MHz, DMSO)
δ
2.98–3.09 (m, 2H), 3.64 (s, 3H), 4.55–4.61 (m, 1H), 6.65 (d, J = 8.3 Hz, 2H), 7.08 (d, J = 8.7 Hz, 2H),
7.44–7.49 (m, 2H), 7.96–8.02 (m, 2H), 9.09 (d, J = 7.5 Hz, 1H), 9.20 (s, 1H); 13C-NMR (100 MHz, DMSO)
δ
35.5, 52.0, 54.7, 115.1, 122.8, 124.9, 125.3, 125.4, 126.3, 127.4, 130.0, 138.9, 139.0, 140.2, 155.9, 161.6,
172.0; HRMS (FAB) Calcd for C19H18NO4S [M + H]+: 356.0957, Found: 356.0956.
4.7. Docking Simulation
To calculate the binding affinity between FIH-1 and the identified compounds (compounds 7,
9–11, 15–18, 28, 30, 38), we used Molegro Virtual Docker 6.0.0 [44]. We obtained the crystal structure
data for FIH-1 (PDB ID: 1YCI [31]) from the RCSB Protein Data Bank. The binding motifs were also
predicted and drawn with Molegro Virtual Docker 6.0.0. To determine the binding affinity, we used
the MolDock score from MolDock Optimizer using default settings. The number of trial runs for
calculations was 20. The chemical structures of the ligands were drawn using ChemBioDraw Ultra
13.0, and optimized by MM2 calculations in Chem3D Pro 13.0.
4.8. Calculation of Solubility
The clogP values of each compounds were calculated by ChemBioDraw Ultra 13.0.

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4.9. Cell Culture
The human neuroblastoma cell line, SK-N-BE(2)c was purchased from American Type Culture
Collection (ATCC, Manassas, VA, USA). SKN:HRE-MLuc were stable transformants of SK-N-BE(2)c by
a reporter vector possessing secretion-type luciferase (Metridia luciferase, MLuc) under controlling 7
times repeated VEGFA regulatory sequence (40 bp length containing hypoxia response element (HRE,
0
0
indicated with underline, 5 -CCA CAG TGC ATA CGT GGG CTC CAA CAG GTC CTC TGG ATC T-3 )
and mini TATA promoter (50-CGA GCT CTA GAG GGT ATA TAA TGG AAG CTC GAA TTC CAG
0
CTT GGC ATT CCG GTA CTG TTG GTA AA-3 ) [41]. SK-N-BE(2)c and SKN:HRE-MLuc were cultured
in RPMI1640 medium (Nacalai, Kyoto, Japan) containing 10% charcoal/resin absorbed heat-inactivated
fetal bovine serum (FBS, MP Biomedicals, Santa Ana, CA, USA) and antibiotics (100 U/mL penicillin
and 100 µg/mL streptomycin) under a humidified atmosphere with 5% CO2 at 37 ◦C.
4.10. Evaluation of HIF Activity under Hypoxia Using a Luciferase Assay
SKN:HRE-MLuc (4
replaced with complete media and the test compounds (100, 25, 6.3 and 1.6
used as a vehicle control, DMOG (100 M) and FG4592 (100 M) were used as a PHDs or FIH-1
×
104) were incubated for 24 h on a 96 well culture plate. The medium was
M). DMSO (0.1%) was
µ
µ
µ
inhibitor. The cells were incubated for 24 h under a humidified atmosphere of 5% CO2 and 3% O2 at
37 ◦C. After incubation, the MLuc activity in culture media was measured with luciferase substrate
(Takara, Shiga, Japan) by LB960 luminometer (Berthold, Wildbad, Germany). The HIF activity was
calculated as the MLuc intensity, based on the vehicle and positive controls. The lower limit dose out
of 100 µM, 25 µM, 6.3 µM, 1.6 µM was listed in Tables 1 and 2, where 25% RLU intensity was measured
using DMOG as a positive control.
4.11. MTS Assay
Cellular toxicity was measured using the CellTiter 96® Aqueous One Solution Cell Proliferation
Assay kit (Promega, Madison, WI, USA). The cells were stimulated with each compound and treated
with the CellTiter solution. After incubation for 3 h, the absorbance of the solutions at 492 nm was
measured by Multiskan FC (Thermo Fisher Scientific, Waltham, MS, USA). As a negative control,
wells with culture media were used. Wells treated with DMSO (0.1%) were used as a positive control.
The IC50 values were determined using these controls.
4.12. Evaluation of HIF Activity under Normoxia Using a Luciferase Assay
SKN:HRE-MLuc (4
replaced with complete media and the tested compounds (100
×
104) were incubated for 24 h on a 96 well culture plate. The medium was
M). DMSO (0.1%) was used as a
µ
negative control, and DMOG (100
24 h under a humidified atmosphere of 5% CO2 at 37 C. After incubation, the MLuc activity in culture
media was measured with the method descried above.
µ
M) was used as◦a positive control. The cells were incubated for
4.13. Silence of FIH-1 by Small Interfering RNA
The siRNA reagents, including the transfection reagents, were obtained from Thermo Fisher
Scientific. Sequence: forward 50-GAA ACA UUG AGA AGA UGC UUG GAG A-30 and reverse
50-UCU CCA AGC AUC UUC UCA AUG UUU C-30. SKN: HRE-MLuc were transfected with siRNA
transiently, according to the protocol for Lipofectamine 2000 (Thermo Fisher Scientific) under normoxic
conditions. The siRNA-transfectants were incubated for 72 h. To confirm the FIH-1 knockdown effect
in cellular lysate was evaluated by immunoblotting after an additional 24 h culture under normoxic
condition. Briefly, cell lysate from siRNA transfectants were separated by SDS polyacrylamide gels,
transferred to polyvinylidene difluoride membranes. The blots were blocked with 5% skim milk
(Nacalai Tesque) in TBS-Tween, and treated with anti-human FIH-1 polyclonal goat antibody (sc-26219,
Santa Cruz Biotechnology, Dallas, TX, USA) or anti β-actin mouse monoclonal antibody (Merck)

Molecules 2018, 23, 885
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antibody, subsequently the blots were treated with horseradish peroxidase-conjugated anti- goat
or -mouse secondary antibody. Immunoreactivity was visualized with LAS400 image analyzer (GE
healthcare, Little Chalfont, UK) using the Western Lightning Chemiluminescence Reagent Plus kit
(PerkinElmer, Waltham, MS, USA).
4.14. Gene Expression Analysis
Total RNA was extracted from cells using ISOGEN (Nippon Gene, Tokyo, Japan). cDNA
syntheses were performed using random hexamer primers and SuperScript II Reverse Transcriptase
(Thermo Fisher Scientific) following the manufacturer’s instructions. Quantitative reverse transcription
polymerase-chain reaction (RT-PCR) analysis was performed as follows: amplification was done in an
◦
◦
◦
initial denaturing step at 96 C for 2 min, followed by 35 cycles of PCR (96 C for 1 min, 57 C for 30 s,
72 ◦C for 30 s) using recombinant Taq polymerase (Takara, Otsu, Japan) with the respective primer
pairs. For human carbonic anhydrase IX (CA9), 50-CCT GGC TGC TGG TGA CAT CC-30 and 50-AAG
GAA GTG GCA TAA TGA GC-30. Real time RT-PCR was performed on a LightCycler480 thermal
cycler system using a LightCycler480 SYBR Green I Master (Roche Applied Science, Indianapolis, IN)
according to the manufacturer’s instructions. Data were analyzed using the comparative Ct method
as means of relative quantification, and were normalized to an endogenous reference (
relative to a calibrator (normalized Ct value obtained from cells treated with vehicle only). Data were
expressed as 2−∆∆Ct
β-actin) and
.
4.15. Statistical Analyses
All data were analyzed using one-way ANOVA followed by Newman-Keuls Multiple Comparison
Test using GraphPad Prism 6 software (La Jolla, CA, USA).
Acknowledgments: We acknowledge Drs. Nobuo Watanabe and Norio Suzuki for technical suggestions and
valuable discussions. We also thank Ms. Sayaka Ito and Satomi Goto for technical assistance and the Biomedical
Research Core of Tohoku University Graduate School of Medicine for technical support. This work was supported
in part by Platforms for Drug Discovery, Informatics, and Structural Life Science (PDIS) from the MEXT, and
Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life
Science Research (BINDS)) from AMED under Grant Number JP17am0101095 (S.K., T.T., T.D., T.M., and A.O.),
Grant-in-Aid for JSPS Fellows 24-5784 (S.K.), JSPS KAKENHI grant 26860168 (S.K.), 16H05922 (T.T), 17K19916
(T.T.) and the Adaptable and Seamless Technology transfer Program through target driven R&D (A-STEP), Japan
Science and Technology Agency grant J120001909 (T.D. and A.O.).
Author Contributions: S.K., T.M., and T.T. conceived and designed the study. S.K. and T.T. wrote the manuscript.
M.S and A.O. contributed to the compound design. S.K., Y.G., T.Y., Y.S., H.S., and Y.K. synthesized the compounds.
S.K. A.I. T.D. and T.T. performed cellular analysis. S.K. performed the docking simulation.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1-44 are available from the authors.
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