Synthesis of 8-hydroxy-2-iminochromene derivatives as selective and potent inhibitors of human carbonyl reductase 1

Article abstract of DOI:10.1039/c5ob00847f

Human carbonyl reductase 1 (CBR1), a member of the short-chain dehydrogenase/reductase superfamily, reduces anthracycline anticancer drugs to their less potent anticancer C-13 hydroxy metabolites, which are linked with pathogenesis of cardiotoxicity, a side effect of the drugs. CBR1 inhibitors are thought to be promising agents for adjuvant therapy with a twofold beneficial effect in prolonging the anticancer efficacy of the anthracyclines while decreasing cardiotoxicity. In order to search for new potential inhibitors of CBR1, we synthesized a series of des-methoxyphenyl derivatives of (Z)-2-(4-methoxyphenylimino)-7-hydroxy-N-(pyridin-2-yl)-2H-chromene-3-carboxamide (1) that was developed previously as a potent inhibitor of aldo-keto reductase (AKR) 1B10 and AKR1B1. Among the newly synthesized inhibitors, 8-hydroxy-2-imino-2H-chromene-3-carboxylic acid (2-chlorophenyl)amide (13h) was the most potent competitive inhibitor of CBR1, showing a K_i value of 15 nM. 13h also showed high selectivity to CBR1 over its isozyme CBR3 and other enzymes with CBR activity (AKR1B1, AKR1B10, AKR1C1, AKR1C2, AKR1C4, DXCR and DHRS4). Furthermore, 13h inhibited the cellular metabolism by CBR1 at its concentration of 4 μM. The structure-activity relationship of the derivatives, site-directed mutagenesis of putative binding residues (Met141 and Trp229) and molecular docking of 13h in CBR1 revealed that the interactions of 13h with the substrate-binding residues (Ser139, Met141, Tyr193 and Trp229) are important for the tight binding.

Full text of DOI:10.1039/c5ob00847f

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Synthesis of 8-hydroxy-2-iminochromene
derivatives as selective and potent inhibitors of
Cite this: DOI: 10.1039/c5ob00847f
human carbonyl reductase 1†
Dawei Hu,a Namiki Miyagi,b Yuki Arai,b Hiroaki Oguri,b Takeshi Miura,‡c
Toru Nishinaka,c Tomoyuki Terada,c Hiroaki Gouda,d Ossama El-Kabbani,e
Shuang Xia,f Naoki Toyooka,a,f Akira Hara,g Toshiyuki Matsunaga,b Akira Ikarib and
Satoshi Endo*b
Human carbonyl reductase 1 (CBR1), a member of the short-chain dehydrogenase/reductase superfamily,
reduces anthracycline anticancer drugs to their less potent anticancer C-13 hydroxy metabolites, which
are linked with pathogenesis of cardiotoxicity, a side effect of the drugs. CBR1 inhibitors are thought to be
promising agents for adjuvant therapy with a twofold beneficial effect in prolonging the anticancer
efficacy of the anthracyclines while decreasing cardiotoxicity. In order to search for new potential inhibi-
tors of CBR1, we synthesized a series of des-methoxyphenyl derivatives of (Z)-2-(4-methoxyphenyl-
imino)-7-hydroxy-N-(pyridin-2-yl)-2H-chromene-3-carboxamide (1) that was developed previously as a
potent inhibitor of aldo-keto reductase (AKR) 1B10 and AKR1B1. Among the newly synthesized inhibitors,
8-hydroxy-2-imino-2H-chromene-3-carboxylic acid (2-chlorophenyl)amide (13h) was the most potent
competitive inhibitor of CBR1, showing a Ki value of 15 nM. 13h also showed high selectivity to CBR1 over
its isozyme CBR3 and other enzymes with CBR activity (AKR1B1, AKR1B10, AKR1C1, AKR1C2, AKR1C4, DXCR
and DHRS4). Furthermore, 13h inhibited the cellular metabolism by CBR1 at its concentration of 4 μM. The
structure–activity relationship of the derivatives, site-directed mutagenesis of putative binding residues
(Met141 and Trp229) and molecular docking of 13h in CBR1 revealed that the interactions of 13h with the
substrate-binding residues (Ser139, Met141, Tyr193 and Trp229) are important for the tight binding.
Received 27th April 2015,
Accepted 20th May 2015
DOI: 10.1039/c5ob00847f
www.rsc.org/obc
belong to the short-chain dehydrogenase/reductase (SDR)
family. Among them, the gene for CBR2 is not present in the
1. Introduction
Carbonyl reductase (CBR, EC 1.1.1.184) catalyzes the NADPH- human genome and human CBR4 is reported to act as a
linked reduction of various aldehydes, ketones and quinones reductase for quinones and 3-ketoacyl-acyl carrier protein.4,5
to their corresponding alcohols.1–3 The enzyme exists in two Human CBR1 and CBR3 are monomeric proteins sharing 72%
forms in both the cytosol (CBR1 and CBR3) and the mitochon- amino acid sequence identity, but are clearly different in their
dria (CBR2 and CBR4) of mammalian cells, and the four CBRs enzymatic properties.3,6,7 CBR1 reduces a wide range of bio-
logically active carbonyl compounds, while CBR3 exhibits
limited carbonyl reductase activity. Since homozygous null
aGraduate School of Innovative Life Science, University of Toyama,
(Cbr1 −/−) mice are fetal lethal,8 CBR1 has been suggested to
Toyama 930-8555, Japan
bLaboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196, Japan.
E-mail: sendo@gifu-pu.ac.jp; Fax: +81 58 230 8105; Tel: +81 58 230 8100
cLaboratory of Biochemistry, Faculty of Pharmacy, Osaka Ohtani University,
Osaka 584-8540, Japan
dSchool of Pharmacy, Showa University, Tokyo 142-8555, Japan
eNagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
fGraduate School of Science and Technology, University of Toyama, 3190 Gofuku,
play pivotal roles in metabolism of endogenous carbonyl com-
pounds. Such endogenous substrates of the enzyme have been
reported to be isatin,3,9 prostaglandins,10 6-pyruvoyltetra-
hydropterin,11 S-nitrosoglutathione12 and lipid peroxidation-
derived aldehydes.2,13,14 However, these substrates are also
reduced or metabolized by several aldo-keto reductases
(AKRs)15–17 and other enzymes,18 and the physiological roles
of CBR1 have not yet been fully elucidated.
Toyama 930-8555, Japan
gFaculty of Engineering, Gifu University, Gifu 501-1193, Japan
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
CBR1 also reduces a wide variety of xenobiotic carbonyl
compounds to their corresponding alcohols, which are easier
to be conjugated and eliminated.1–3 Thus, the enzyme plays a
c5ob00847f
‡Present address: School of Pharmacy and Pharmaceutical Sciences, Mukogawa
Women’s University, Hyogo 663-8179, Japan.
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major role in the phase I metabolism of xenobiotic com- and flavonoids23,25–27 of which a flavonoid luteolin most
pounds including drugs, but its carbonyl reduction of anthra- potently inhibits CBR1 (IC50 0.095 μM).27 However, the inhibi-
cycline drugs, doxorubicin and daunorubicin, into their tory selectivity of the above synthetic CBR1 inhibitors has not
13-alcohol metabolites leads to unfavorable bioactivation. The been studied, and curcumin and flavonoids are reported to
anthracyclines are highly effective anticancer drugs for the inhibit other enzymes such as human aldose reductase
treatment of many cancers such as leukemia, soft tissue sarco- (AKR1B1), aldose reductase-like protein (AKR1B10)28,29 and/or
mas and breast cancer, but their dosages are strictly limited dehydrogenase/reductase SDR family member 4 (DHRS4).30
due to increased risk for congestive heart failure depending on
The aim of this study is to find novel and potent inhibitors
the cumulative dose.19 The pathogenesis of anthracycline- that are selective to human CBR1. We previously discovered
related cardiotoxicity has been linked to the synthesis of (Z)-2-(4-methoxyphenylimino)-7-hydroxy-N-(pyridin-2-yl)-2H-
anthracycline C-13 alcohol metabolites by CBR1.19,20 The chromene-3-carboxamide (1) as a potent competitive inhibitor
involvement of this enzyme in the development of chemoresis- of AKR1B10 and AKR1B1,31 and subsequently elucidated the
tance and cardiotoxicity during therapy with doxorubicin is structure–activity relationship by synthesis of 1-derivatives.32
also supported by experiments in heterozygous null (Cbr1 +/−) As a preliminary study to search for structurally new CBR1
mice8 and transgenic mice overexpressing CBR1.21 Therefore, inhibitors, we examined the inhibitory activities of these chro-
inhibitors for human CBR1 are thought to be promising mene derivatives, and found that a des-methoxyphenyl deriva-
agents for adjuvant therapy with a twofold beneficial effect in tive of 1, 2-(imino)-8-hydroxy-N-(pyridin-2-yl)-2H-chromene-3-
improving the therapeutic response to the anthracyclines carboxamide (9), inhibits CBR1, but not AKR1B10 and
while reducing the cardiotoxic side effects in patients under- AKR1B1. To obtain more potent and selective CBR1 inhibitors,
going chemotherapy. In addition, selective inhibitors of derivatives of 9 were synthesized, and evaluated for their
CBR1 may be useful in elucidating the physiological roles of inhibitory activity and selectivity towards CBR1 by comparing
the enzyme and its contribution to metabolism of newly deve- their effects on human CBR3, DHRS4 and dicarbonyl/L-xylu-
loped carbonyl-containing drugs.
lose reductase (DCXR)33 that are members of the SDR super-
Human CBR1 is reported to be inhibited by several family with CBR activity. Since compound 13h with the
synthetic and natural compounds (Fig. 1). Representative 2-chlorophenyl moiety instead of the 2-pyridyl moiety of 9 was
synthetic inhibitors are ethacrynic acid, indomethacin,10 found to be the most potent competitive inhibitor of CBR1
3-(1-tert-butyl-4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenol (IC50 34 nM), docking simulation and site-directed muta-
(hydroxy-PP),22 triclosan and zearalenone analogues,23 of genesis studies were also carried out to investigate the binding
which a zearalenone analogue shows the most potent inhi- mode of the most potent inhibitor 13h in the active site
bition (IC50 0.21 μM). The natural inhibitors are curcumin24 of CBR1.
2. Results and discussion
2.1. Inhibition of human CBR1 by chromene-2-carboxamide
derivatives
CBR1 is potently inhibited by flavonoids with 7-hydroxylated
chromene rings.27 Since compound 1, an inhibitor of
AKR1B10 and AKR1B1,31 has a 7-hydroxylated chromene ring,
we first examined the inhibition of the isatin reductase activity
of human CBR1 by 1 and its derivatives hydroxylated at
different positions on the chromene ring (2–4), in our search
for new inhibitors of this enzyme (Table 1). Compounds 1 and
2 inhibited CBR1, and their IC50 values are comparable to
those of zealarenone analogues23 and flavonoids other than
luteolin.27 Next, we tested the inhibitory activities of com-
pounds 6–9, which are synthetic intermediates of 1–4 and lack
the 4-methoxyphenyl moiety. While 6 and 7 showed less
inhibitory potency than the corresponding compounds with
the 4-methoxyphenyl moiety (1 and 2, respectively), 9 with the
8-hydroxychromene ring inhibited CBR1 with an IC50 value of
0.32 μM, and did not show significant inhibition for AKR1B10
and AKR1B1 at its high concentration of 10 μM. The results
suggest that 9 binds to CBR1 differently from 1 and 2 due to
Fig. 1 Structures and inhibitory potency of known inhibitors of CBR1.
the lack of the 4-methoxyphenyl moiety and its 8-hydroxyl
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Table 1 Inhibitory effects of the chromene derivatives with or without the 4-methoxyphenyl moiety on CBR1, AKR1B10 and AKR1B1
IC50 (μM)
Entry
R1
R2
CBR1
AKR1B10
AKR1B1
1
2
3
4
5
6
7
8
9
7-OH
5-OH
6-OH
8-OH
H
7-OH
5-OH
6-OH
8-OH
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
0.25 0.021
0.53 0.062
>10b
0.006a
0.29a
0.011a
>10b
>10b
>10b
>10b
>10b
>10b
4-Methoxyphenyl
>10b
3.0 0.27
5.3 1.1
>10b
>10b
>10b
H
H
H
H
0.099 0.010
1.5 0.045
>10b
0.52 0.078
>10b
>10b
0.32 0.0070
>10b
>10b
a The values are taken from ref. 31 and 32. b Less than 33% inhibition at 10 μM.
group is a structural requisite for the selective inhibition of 3-chlorobenzyl rings was the most potent inhibitor, although
CBR1.
its IC50 value (0.090 μM) was higher than that of 13h. For the
8-hydroxycoumarin derivatives (14a–14q), their IC50 values
were higher than those of the 8-hydroxy-2-iminochromene
derivatives having the corresponding substituents on the car-
boxamide moiety. For example, the most potent coumarin
derivative was 14h, but its IC50 value was 7-fold higher than
that of the corresponding chromene derivative 13h. Since the
8-hydroxy-2-iminochromene and 8-hydroxycoumarin deriva-
tives differ only in the substituent at 2-position on the benzo-
furan ring (i.e., imino or carbonyl group), the above results
suggest that the 2-imino group is a structural requisite for the
tight binding of 13h and 13p. It should be noted that an
8-methoxylated derivative of 13h (15) did not inhibit CBR1 at
its high concentration of 10 μM, indicative of the most critical
prerequisite of the 8-hydroxyl group for the CBR1 inhibition by
these inhibitors.
Among the newly synthesized inhibitors, 13h was the most
potent, followed by 13o and 13p. The IC50 value of 13h is
3-fold lower than that of luteolin that was previously reported
as the most potent CBR1 inhibitor,27 although those of 13o
and 13p are comparable to those of luteolin. We examined the
inhibitory selectivity of 13h, 13o and 13p by comparing their
effects on the activities of CBR3, DCXR, DHRS4 and AKRs
(1B1, 1B10, 1C1, 1C2 and 1C4) that metabolize endogenous
and xenobiotic carbonyl compounds1,2,15 (Table 3). These
inhibitors did not significantly inhibit the eight enzymes at a
high concentration of 10 μM, with the exception of moderate
inhibition of CBR3 (by 13h and 13o) and AKR1B10 (by 13o and
13p). With respect to the selectivity ratio (IC50 for the eight
enzymes/that for CBR1), 13h is the most selective inhibitor
among the three synthesized compounds. To compare the
inhibitory selectivity of 13h with that of luteolin, we examined
its inhibitory effects on the activities of the five enzymes.
Luteolin inhibited all the eight enzymes, and its inhibitory
2.2. Synthesis of 8-hydroxychromene and 8-hydroxycoumarin
derivatives
Using the selective CBR1 inhibitor 9 as the lead compound, we
synthesized 8-hydroxy-2-iminochromene (13a–13q) and
8-hydroxycoumarin (14a–14q) derivatives by replacing the pyri-
dine moiety bound to the carboxamide of the chromene ring
with substituted phenyl or benzyl rings according to Scheme 1.
The cyanoacetoamides (12l) and (12m) were prepared by the
deprotection of the corresponding MOM-protected cyano-
acetoamides, which were prepared by the condensation of
benzylamines (10l) and (10m) with cyanoacetic acid by using
EDC. The Knoevenagel condensation of cyanoacetoamides
(12a–12q) with 2,3-dihydroxybenzaldehyde (11) gave rise to the
corresponding chromenes (13a–13q),34 which were converted
to the coumarins (14a–14q) by acid hydrolysis. To confirm the
importance of the 8-hydroxy group on the chromene ring, com-
pound 15 (i.e., 8-methoxy analog of 13h) was also synthesized
using 2-hydroxy-3-methoxybenzaldehyde instead of 11.
2.3. Inhibitory potency and selectivity of the synthesized
compounds
The structures of the synthesized compounds and their inhibi-
tory activities for CBR1 are summarized in Table 2. In the
8-hydroxy-2-iminochromene derivatives, 13a with a phenyl ring
on the carboxamide showed a slightly lower IC50 value than
the lead 9, but other derivatives having hydroxyphenyl and
fluorophenyl rings (13b, 13d, 13e–13g), except for 13c, did not
improve the inhibitory potency. In contrast, the derivatives
having chlorophenyl rings (13h–13j) potently inhibited CBR1,
and particularly the IC50 value (0.034 μM) of 13h having
2-chlorophenyl rings was the lowest. Among the derivatives
having benzyl and substituted benzyl rings, 13p having
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Scheme 1 Synthesis of chromene derivatives (13a–13q and 15) and coumarin derivatives (14a–14q).
selectivity was much lower than that of 13h. Thus, 13h is not suggest that 13h and 13p bind to the substrate-binding site of
only the most potent inhibitor among the known CBR1 inhibi- CBR1.
tors, but also highly selective.
The crystallographic and site-directed mutagenesis studies
of human CBR1 and CBR3 have shown that despite their high
sequence identity, the active sites of the two enzymes differ in
shape and surface properties.7 The residue difference in the
substrate binding sites of the two enzymes is suggested to be
related to their differences in substrate specificity. Such resi-
dues of CBR1 are Met141, Trp229 and Ala235 (Fig. 2), which
correspond to Gln, Pro and Asp, respectively, in CBR3. The
crystal structure of the CBR1–NADP+–hydroxy-PP complex has
also shown that the pyrazolopyrimidine of the inhibitor is sur-
rounded by hydrophobic residues (Ile140, Met141 and Trp229)
and its phenolic hydroxyl group interacts with Ser139 and
Tyr193 of the catalytic triad (ESI Fig. S1†).22 To ensure the
binding of 13h to the substrate-binding site of CBR1, Met141
and Trp229 were chosen among the residues in the site and
were subjected to site-directed mutagenesis (Table 4). Met141
2.4. Structural insight into the high inhibitory potency and
selectivity of 13h
The inhibition patterns of 13h were examined in both NADPH-
linked isatin reduction and NADP+-linked (S)-(+)-1,2,3,4-tetra-
hydro-1-naphthol (S-tetralol) oxidation by CBR1. As reported
for the inhibition patterns of luteolin,27 the inhibition by
13h was mixed-type with respect to isatin in the reduction
reaction (Ki 64 7 nM and K′ 18 2 nM), and was competitive
i
with respect to S-tetralol in the oxidation reaction. The inhi-
bition by 13p was also competitive with respect to the sub-
strate in the S-tetralol oxidation. The Ki values for 13h and 13p
estimated in the S-tetralol oxidation were 15 and 47 nM,
respectively (Table 4), of which the value for 13h is 4-fold lower
than that for luteolin (59 nM).27 The inhibition patterns
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Table 2 Inhibition of CBR1 by 2-carboxamide derivatives of 8-hydroxy-
Table 4 Effects of mutations of CBR1 on the Ki values of 12h and 12p
2-iminochromene and 8-hydroxycoumarin
13h
13p
Enzyme
Kia (nM)
Mu/Wtb
Kia (nM)
Mu/Wtb
Wild type
15 1.9
180 17
47 2.1
69 8.5
2400 330
—
12
3
5
160
47 2.9
530 21
97 8.1
110 5.3
1600 160
—
11
2
2
34
Met141Gln
Met141Val
Trp229Phe
Trp229Leu
R3
Entry
IC50 (μM)
Entry
IC50 (μM)
a The inhibition patterns in the NADP+-linked S-tetralol dehydrogenase
activity were all competitive with respect to the substrate. b Mu/Wt
represents the ratio of the Ki value for the mutant enzyme to that for
the wild type enzyme.
Phenyl
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
13m
13n
13o
13p
13q
0.21 0.012
0.33 0.025
0.15 0.011
0.88 0.045
0.31 0.037
0.37 0.045
0.44 0.086
0.034 0.0035
0.12 0.015
0.22 0.015
0.33 0.03
0.35 0.026
0.11 0.0011
0.17 0.022
0.10 0.013
0.090 0.00064
0.26 0.0072
14a
14b
14c
14d
14e
14f
14g
14h
14i
14j
14k
14l
14m
14n
14o
14p
14q
1.9 0.16
0.47 0.028
0.37 0.030
1.3 0.066
1.8 0.42
2.5 0.15
2.5 0.11
0.26 0.037
1.5 0.082
0.45 0.0082
0.92 0.0091
1.3 0.18
1.1 0.051
0.82 0.013
0.41 0.019
1.1 0.072
1.0 0.0011
2-Hydroxyphenyl
3-Hydroxyphenyl
4-Hydroxyphenyl
2-Fluorophenyl
3-Fluorophenyl
4-Fluorophenyl
2-Chlorophenyl
3-Chlorophenyl
4-Chlorophenyl
Benzyl
2-Hydroxybenzyl
3-Hydroxybenzyl
4-Hydroxybenzyl
2-Chlorobenzyl
3-Chlorobenzyl
4-Chlorobenzyl
was mutated to Gln (the corresponding residue of CBR3) and
Val (a smaller hydrophobic residue), and Trp229 was mutated
to Phe (a smaller aromatic residue) and Leu (a non-aromatic
and hydrophobic residue). The Met141Gln mutation decreased
the Ki (i.e., affinity) for 13h by 12-fold compared to wild-type
CBR1, although the Met141Val mutation gave a smaller effect.
The affinity for 13h was impaired greatly by the Trp229Leu
mutation, but the Trp229Phe mutation resulted in only a
5-fold decrease. The results demonstrated that 13h indeed
binds to the substrate-binding site of CBR1, and suggest that
the residue differences at positions 141 and 229 are related to
the high inhibitory selectivity of 13h to CBR1 over CBR3. The
affinity for 13p was similarly decreased by these mutations,
but the effects of the mutagenesis of Trp229 were lower than
those on the affinity for 13h.
Fig. 2 13h-docked CBR1 model. NADP+ (yellow) and residues (grey)
within 4.0 Å from 13h (pink) are depicted with possible H-bonds (dotted
line) and their corresponding distances are shown in Å. Only side-chains
are shown for Ser139, Met141, Cys226, Tyr193, and Trp229.
Table 3 Inhibitory effects of 13h, 13p and 13o on human enzymes in the SDR and AKR superfamilies
13h
13p
13o
Luteolin
SRb
IC50
SRb
IC50
SRb
IC50
SRb
a
a
a
a
Enzyme
IC50
CBR1
CBR3
DCXR
DHRS4
AKR1B10
AKR1B1
AKR1C1
AKR1C2
AKR1C4
0.034
1.5 0.16
>10
>10
>10
>10
>10
>10
>10
—
0.090
>10
>10
>10
3.7 0.010
>10
>10
>10
>10
—
0.10
2.5 0.026
>10
>10
4.8 0.31
>10
>10
>10
>10
—
0.095c
—
6
69
56
8
44
>110
>110
>110
34
>110
>110
>110
>110
25
0.53 0.052
6.6 0.39
5.3 0.38
0.73 0.058
0.18 0.010
1.9 0.11
6.7 0.31
1.7 0.10
>290
>290
>290
>290
>290
>290
>290
>100
>100
48
>100
>100
>100
>100
2
20
71
18
a IC50 (μM) was determined as described in section 3.2.2. >10: less than 36% inhibition at 10 µM. b SR: selectivity ratio of the IC50 value for other
enzymes to that for CBR1. c The value is taken from ref. 27.
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The underlying structural reasons for the high inhibitory exposed to the solvent. Such interactions of this moiety might
potency and selectivity of 13h were also examined by construct- not be properly formed in the case of other less potent deriva-
ing models of docked 13h in the CBR1–NADP+ complex. In the tives with 3- and 4-chlorophenyl rings, and thereby affects the
model with the lowest IFDScore (Fig. 2), 13h was held in the interactions of the other parts with the residues described
substrate-binding site through a number of contacts including above.
the catalytic residues, Tyr193 and Ser139, of which the follow-
ing interactions may be involved in the inhibitory potency and
selectivity. (1) The 2-imino group on the chromene ring of 13h
2.5. Inhibitory effects of 13h and 13p on cellular metabolism
by CBR1
formed an H-bond with Tyr193, which is probably related to
the higher inhibitory potency of the 2-iminochromene deriva-
tives than the coumarin derivatives. (2) An H-bond network
was observed between 13h (its 8-hydroxy group and 1-ether
oxygen of the chromene ring) and CBR1 residues (the side
chain of Ser139 and the main chain nitrogen of Ile140). In par-
ticular, the H-bond interaction of the 8-hydroxy group with the
residues is probably important for the high inhibitory potency
of 13h, because its 8-methoxylated derivative (15) did not
inhibit CBR1. (3) The chromene ring of 13h was surrounded
by hydrophobic side chains of Trp229, Met141 and Leu140, of
which the side chain of Trp229 formed hydrophobic and
π-stacking interactions. The indole nitrogen of Trp229 was in
close proximity to the carbonyl group in the carboxamide of
13h (2.9 Å), and can form an H-bond interaction. The impor-
tance of the interactions of Trp229 and Met141 with the chro-
mene ring for the inhibitory potency of 13h is supported by
larger impairment of its affinity by the Trp229Leu and
Met141Gln mutations. (4) The 2-chlorophenyl moiety of 13h
was surrounded by the side and/or main chains of Ala93,
Phe94, Lys95, Val96, Met141, Met234 and Ala235, and its
chlorine atom was accommodated in a space composed of the
side-chains Met234 and Ala235, although it was partially
The efficacies of 13h and 13p as CBR1 inhibitors were investi-
gated by analyzing their effects on cellular metabolism of 9,10-
phenanthrenequinone (9,10-PQ) by human CBR1 over-
expressed in bovine aorta endothelial cells (BAEC). 9,10-PQ is
an efficient substrate of human CBR1,9 and reduction of 9,10-
PQ generates its semiquinone, superoxide anion and hydrogen
peroxide through the reaction of the reduced hydroquinone
with molecular oxygen followed by redox cycling.9,35,36 CBR4
and DCXR reduce 9,10-PQ and their overexpression in BAEC
accelerates the generation of reactive oxygen species and cyto-
toxicity by 9,10-PQ.4,37 However, there has been no report on
the involvement of human CBR1 in the toxic effect of 9,10-PQ
at cellular levels. Therefore, we first examined the effect of
overexpression of CBR1 on the cytotoxicity of 9,10-PQ using
BAEC transfected with the cDNA. Compared to the control
cells, the cytotoxicity of 9,10-PQ was clearly increased in the
CBR1-transfected cells (Fig. 3A), indicating that the reduction
of 9,10-PQ by the enzyme is indeed responsible for the cyto-
toxicity. Next, the inhibitory effects of 13h and 13p on the
cellular metabolism of 9,10-PQ were assessed by determining
the viability of the cells treated with 5 μM 9,10-PQ (Fig. 3B).
While the two compounds had no effect on the viability of the
control cells at 10 μM, they decreased the 9,10-PQ-induced
Fig. 3 Inhibitory effects of 13h and 13p on 9,10-PQ cytotoxicity exacerbated by CBR1 overexpression in BAEC. (A) Dose-dependent effect of 9,10-
●
○
PQ on viability of the cells transfected with CBR1 cDNA ( , cDNA) and control cells transfected with the vector alone ( , vector). The overexpression
of CBR1 in the transfected cells was analyzed by Western blotting (inset). The cells were treated for 24 h with various concentrations of 9,10-PQ,
and the cell viability was expressed as % S.D. (n = 3) of control culture conditions. *Significant difference from the control cells, p < 0.05. (B)
Effects of 13h and 13p on cell viability. The control and CBR1-overexpressing cells were pretreated for 2 h with the vehicle alone (0), 13h and 13p (4
and 10 μM), and then treated for 24 h with 5 μM 9,10-PQ. The viability of the control cells pretreated with the vehicle is taken as 100%. *Significant
difference from the CBR1-overexpressing cells pretreated with vehicle alone (0) inhibitors, p < 0.05.
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cytotoxicity of the CBR1-overexpressing cells at their lower 2.5 Hz), 7.28 (1H, t, J = 6.5 Hz); 13C-NMR (125 MHz, DMSO-d6)
concentrations of 4 μM. Thus, the results indicate that, δ 25.5, 43.7, 55.8, 94.1, 114.8, 115.1, 115.4, 120.8, 129.6, 138.6,
although 13h and 13p inhibited the cellular metabolism of 157.2, 161.9; IR (KBr): 3288, 1646, 1555, 1155, 1030 cm−1; MS
9,10-PQ by CBR1, 13p is more effective than 13h in the cells in (EI): m/z 234 (M+); HRMS: calcd for C12H14N2O3 234.2512,
contrast to their in vitro inhibitory potencies. This inconsis- found: 234.0999.
tency between the results of in vitro and ex vivo experiments Deprotection of the MOM group
might have resulted from the differences in lipophilicities of
To a stirred solution of the acetoamides obtained above
13h and 13p, although further studies on cell permeability are (1 mmol) in THF (5 mL) was added 10% HCl (5 drops), and
needed.
then the resulting mixture was heated to 40 °C for 24 h. After
cooling, the reaction was quenched by H2O (5 mL) and the
aqueous mixture was extracted with EtOAc (5 mL × 3). The
organic extracts were combined, dried over Na2SO4, and evapo-
rated under reduced pressure. The residue was chromato-
graphed on silica gel (15 g, hexane : acetone = 2 : 1) to give the
corresponding amide.
3. Experimental section
3.1. Chemistry
The chromenes (13a–13q), 15, and the coumarins (14a–14q)
were synthesized as shown in Scheme 1.
3.1.2.3. 2-Cyano-N-(2-hydroxybenzyl)acetamide (12l). Yield:
91%; mp: 108–110 °C; 1H-NMR (500 MHz, CDCl3) δ 3.43 (2H, s),
4.43 (2H, d, J = 6.5 Hz), 6.88 (1H, td, J = 6.5, 1.0 Hz), 6.93 (1H,
dd, J = 6.5, 1.0 Hz), 6.98 (1H, br), 7.14 (1H, dd, J = 6.5, 1.0 Hz),
7.23 (1H, td, J = 6.5, 1.0 Hz), 7.88 (1H, br); 13C-NMR (125 MHz,
CDCl3) δ 25.6, 40.8, 114.3, 117.3, 120.4, 123.0, 130.2, 130.8,
155.1, 163.2; IR (KBr): 3291, 1674, 1458 cm−1; MS (EI): m/z 190
(M+); HRMS: calcd for C10H10N2O3 190.0742, found: 190.0741.
3.1.2.4. 2-Cyano-N-(3-hydroxybenzyl)acetamide (12m). Yield:
3.1.1. General. Melting points were determined with a
Yanaco micro melting apparatus and were uncorrected. Flash
chromatography was performed on Kanto Kagaku silica gel
60N. NMR spectra were recorded on JEOL GX 400 and JEOL
JNX-ECX500 spectrometers. Chemical shifts (δ) are given in
ppm downfield from TMS and referenced to CHCl3 (7.26 ppm)
or DMSO (2.50 ppm) for 1H-NMR spectra and the centre line of
CDCl3 (77.0 ppm) for 13C-NMR spectra as an internal standard.
Peak multiplicities are designated by the following abbrevi-
ations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
br, broad and coupling constants in (J) Hz. High-resolution
mass spectral data were obtained using a JEOL MStation
JMS-700. All commercial reagents were used as received unless
otherwise noted. The amides 12c, 12i, 12j, 12k, 12n, 12o, 12p,
and 12q are commercially available from UORSY Building
Blocks, Ukraine.
3.1.2. General procedure for the preparation of 12. To a
stirred solution of cyanoacetic acid (1 mmol) in CH2Cl2 (5 mL)
were added EDC (2 mmol), DMAP (0.2 mmol), and the corres-
ponding amine (1 mmol), and the resulting mixture was
stirred for 24 h at room temperature. The volatiles were
removed under reduced pressure, and the residue was
chromatographed on silica gel (15 g, hexane : acetone = 5 : 1) to
give the corresponding amide. The amides 12a, 12d, 12e, 12f,
12g, and 12h are known compounds,38 and the 1H-NMR
spectra of our synthetic materials were identical with those for
the reported values.
1
74%; mp: 85–87 °C; H-NMR (500 MHz, CDCl3) δ 3.41 (2H, s),
4.43 (2H, d, J = 6.0 Hz), 6.40 (1H, br), 6.77 (1H, s), 6.78 (1H,
dd, J = 7.5, 1.0 Hz), 6.83 (1H, d, J = 7.5 Hz), 7.22 (1H, t, J = 7.5
Hz); 13C-NMR (125 MHz, CDCl3) δ 25.8, 43.2, 114.5, 114.8,
116.8, 118.5, 129.9, 140.4, 158.0, 162.6; IR (KBr): 3209, 1653,
1558 cm−1; MS (EI): m/z 190 (M+); HRMS: calcd for C10H10N2O2
190.0742, found 190.0741.
3.1.3. General procedure for the preparation of 13. To a
stirred solution of cyanoamide 12 (1 mmol) in ethanol (5 mL)
were added 11 (1 mmol) and piperidine (3 drops), and the
resulting mixture was stirred for 24 h at room temperature.
The solid product was filtered, washed with ethanol, and dried
to give the corresponding chromene 13.
3.1.3.1. 8-Hydroxy-2-imino-2H-chromene-3-carboxylic
acid
phenylamide (13a). Yield: 82%; mp: 235–237 °C; 1H-NMR
(500 MHz, DMSO-d6) δ 7.07 (1H, d, J = 3.0 Hz), 7.08 (1H, d, J =
4.0 Hz), 7.11 (1H, t, J = 7.5 Hz), 7.23 (1H, dd, J = 4.0, 3.0 Hz),
7.37 (2H, t, J = 7.5 Hz), 7.67 (2H, dd, J = 7.5, 1.0 Hz), 8.50 (1H,
d, J = 1.5 Hz), 9.09 (1H, s), 10.23 (1H, br); 13C-NMR (125 MHz,
DMSO-d6) δ 119.9, 120.1, 120.2, 120.4, 120.5, 124.5, 124.6,
129.6, 138.8, 142.5, 142.6, 144.5, 156.5, 160.3; IR (KBr): 3298,
1674, 1598, 1473 cm−1; MS (EI): m/z 280 (M+); HRMS: calcd for
C16H12N2O3 280.0848, found 280.0847.
3.1.2.1. 2-Cyano-N-(2-methoxymethoxybenzyl)acetamide. Yield:
45%; mp: 86–88 °C; H-NMR (500 MHz, CDCl3) δ 3.33 (2H, s),
3.48 (3H, s), 4.47 (2H, d, J = 6.5 Hz), 5.24 (2H, s), 6.82 (1H, br),
6.97 (1H, td, J = 6.5, 1.0 Hz), 7.13 (1H, dd, J = 6.5, 1.0 Hz), 7.25
(1H, d, J = 6.5 Hz), 7.26 (1H, td, J = 6.5, 1.0 Hz); 13C-NMR
1
3.1.3.2. 8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid (2-
(125 MHz, CDCl3) δ 25.9, 40.6, 56.5, 94.8, 114.3, 122.1, 125.7, hydroxyphenyl)amide (13b). Yield: 48%; mp: 253–255 °C;
126.5, 129.7, 130.2, 155.6, 160.5; IR (KBr): 3291, 1653, 1559, 1H-NMR (500 MHz, DMSO-d6) δ 6.80 (1H, td, J = 6.0, 2.0 Hz),
1155 cm−1; MS (EI): m/z 234 (M+); HRMS: calcd for C12H14N2O3 6.88–6.92 (1H, 1H, and 1H, each d), 7.06 (1H, d, J = 2.5 Hz),
234.1004, found: 234.1003.
7.07 (1H, d, J = 4.5 Hz), 7.21 (1H, dd, J = 4.5, 2.5 Hz), 8.37 (1H,
d, J = 8.0 Hz), 8.48 (1H, s), 8.98 (1H, s), 9.96 (1H, br), 10.20
(1H, br); 13C-NMR (125 MHz, DMSO-d6) δ 115.23, 119.5,
119.97, 120.05, 120.5, 121.0, 124.5, 124.6, 127.5, 142.0, 142.4,
142.5, 144.4, 147.7, 155.8, 160.1; IR (KBr): 3313, 1668, 1558,
3.1.2.2. 2-Cyano-N-(3-methoxymethoxybenzyl)acetamide. Yield:
1
51%; mp: 83–85 °C; H-NMR (500 MHz, CDCl3) δ 3.41 (2H, s),
3.48 (3H, s), 4.45 (2H, d, J = 5.5 Hz), 5.17 (2H, s), 6.35 (1H, br),
6.93 (1H, d, J = 6.5 Hz), 6.96 (1H, s-like), 7.00 (1H, dd, J = 6.5,
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1471 cm−1; MS (EI): m/z 296 (M+); HRMS: calcd for C16H12N2O4 1218 cm−1
;
MS (EI): m/z 298 (M+); HRMS: calcd for
296.0797, found: 296.0798.
C16H11FN2O3 298.0754, found 298.0754.
3.1.3.3. 8-Hydroxy-2-imino-2H-chromene-3-carboxylic
acid
3.1.3.8. 8-Hydroxy-2-imino-2H-chromene-3-carboxylic
acid
(3-hydroxyphenyl)amide (13c). Yield: 40%; mp: 202–204 °C; (2-chlorophenyl)amide (13h). Yield: 67%; mp: 282–283 °C;
1H-NMR (500 MHz, DMSO-d6) δ 6.52 (1H, dd, J = 7.0, 2.0 Hz), 1H-NMR (500 MHz, DMSO-d6) δ 7.08–7.13 (1H, 1H, and 1H,
6.97 (1H, dd, J = 7.0, 1.0 Hz), 7.08 (1H, d, J = 2.5 Hz), 7.09 (1H, each d), 7.16 (1H, td, J = 6.5, 1.5 Hz), 7.24 (1H, dd, J = 5.0, 2.0
d, J = 4.5 Hz), 7.14 (1H, t, J = 7.0 Hz), 7.22 (1H, dd, J = 4.5, 2.5 Hz), 7.39 (1H, td, J = 7.0, 1.0 Hz), 7.54 (1H, dd, J = 6.5, 1.5 Hz),
Hz), 7.28 (1H, t, J = 2.0 Hz), 8.48 (1H, s), 9.08 (1H, s), 9.50 (1H, 8.52 (1H, dd, J = 7.0, 1.0 Hz), 8.55 (1H, s), 9.13 (1H, s);
br), 10.20 (1H, br); 13C-NMR (125 MHz, DMSO-d6) δ 107.1, 13C-NMR (125 MHz, DMSO-d6) δ 119.8, 120.2, 120.4, 120.7,
110.8, 111.7, 119.9, 120.2, 120.5, 120.6, 124.6, 130.3, 139.8, 122.6, 123.6, 124.6, 125.5, 128.2, 129.9, 136.0, 142.5, 143.3,
142.5, 144.5, 156.5, 158.4, 160.1; IR (KBr): 3303, 1676, 1577, 144.5, 156.0, 160.8; IR (KBr): 3305, 1684, 1471, 1224 cm−1; MS
1473 cm−1; MS (EI): m/z 296 (M+); HRMS: calcd for C16H12N2O3 (EI): m/z 314 (M+); HRMS: calcd for C16H11ClN2O3 314.0458,
296.0797, found: 296.0799.
found: 314.0459.
3.1.3.4. 8-Hydroxy-2-imino-2H-chromene-3-carboxylic
acid
3.1.3.9. 8-Hydroxy-2-imino-2H-chromene-3-carboxylic
acid
(4-hydroxyphenyl)amide (13d). Yield: 46%; mp: 231–233 °C; (3-chlorophenyl)amide (13i). Yield: 65%; mp: 252–254 °C;
1H-NMR (500 MHz, DMSO-d6) δ 6.76 (2H, d, J = 7.8 Hz), 7.07 1H-NMR (500 MHz, DMSO-d6) δ 7.07–7.13 (1H and 1H, each
(1H, d, J = 3.0 Hz), 7.08 (1H, d, J = 3.5 Hz), 7.21 (1H, dd, J = 3.5, d), 7.18 (1H, d, J = 8.0 Hz), 7.23 (1H, d, J = 8.0 Hz), 7.39 (1H, t,
3.0 Hz), 7.47 (2H, d, J = 7.8 Hz), 8.46 (1H, s), 9.03 (1H, s), 9.34 J = 8.0 Hz), 7.44 (1H, d, J = 8.0 Hz), 7.95 (1H, s), 8.50 (1H, s),
(1H, br), 12.58 (1H, br); 13C-NMR (125 MHz, DMSO-d6) δ 116.0, 9.12 (1H, s); 13C-NMR (125 MHz, DMSO-d6) δ 118.6, 119.6,
119.96, 120.03, 120.5, 120.7, 121.7, 124.6, 130.5, 142.1, 142.4, 119.8, 120.1, 120.3, 120.6, 124.2, 124.6, 131.2, 133.9, 140.1,
144.5, 154.5, 156.5, 159.6; IR (KBr): 3287, 1669, 1513, 142.5, 142.9, 144.5, 156.4, 160.7; IR (KBr): 3301, 1681, 1595,
1473 cm−1; MS (EI): m/z Calcd for C16H12N2O4 296.0797, 1482 cm−1
;
MS (EI): m/z 314 (M+); HRMS: calcd for
found: 296.0796.
C16H11ClN2O3 314.0458, found: 314.0458.
3.1.3.5. 8-Hydroxy-2-imino-2H-chromene-3-carboxylic
acid
3.1.3.10. 8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid
(2-fluorophenyl)amide (13e). Yield: 82%; mp: 247–249 °C; (4-chlorophenyl)amide (13j). Yield: 91%; mp: 272–273 °C;
1H-NMR (500 MHz, DMSO-d6) δ 7.10 (1H, d, J = 8.0 Hz), 7.11 1H-NMR (500 MHz, DMSO-d6) δ 7.07–7.11 (1H and 1H, each
(1H, d, J = 10.0 Hz), 7.13–7.18 (1H, m), 7.23 (1H, d, J = 8.0 Hz), d), 7.23 (1H, dd, J = 5.0, 2.5 Hz), 7.43 (2H, dd, J = 5.0, 2.0 Hz),
7.26 (1H, dd, J = 8.0, 1.5 Hz), 7.33 (1H, dd, J = 10. 0, 8.0 Hz), 7.70 (2H, dd, J = 5.0, 2.0 Hz), 8.50 (1H, d, J = 1.5 Hz), 9.11 (1H,
8.48 (1H, td, J = 8.0, 1.5 Hz), 8.55 (1H, d, J = 1.5 Hz), 9.14 (1H, s); 13C-NMR (125 MHz, DMSO-d6) δ 119.8, 120.2, 120.3, 120.6,
br); 13C-NMR (125 MHz, DMSO-d6) δ 115.6 (d, J = 23.1 Hz), 121.7, 124.6, 128.1, 129.5, 137.7, 142.5, 142.7, 144.5, 156.4,
119.8, 120.2 (d, J = 23.1 Hz), 120.7, 121.9, 124.6, 125.0 (d, J = 160.5; IR (KBr): 3302, 1675, 1473, 1230 cm−1; MS (EI): m/z 314
8.5 Hz), 125.2 (d, J = 3.6 Hz), 127.0 (d, J = 8.5 Hz), 142.5, 143.1, (M+); HRMS: calcd for C16H11ClN2O3 314.0458, found:
144.5, 153.0 (d, J = 241.8 Hz), 156.3, 160.6; IR (KBr): 3310, 314.0459.
1607, 1569, 1473 cm−1; MS (EI): m/z 298 (M+); HRMS: calcd for
C16H11FN2O3 298.0754, found 298.0754.
3.1.3.11. 8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid
benzylamide (13k). Yield: 55%; mp: 206–208 °C; 1H-NMR
(500 MHz, DMSO-d6) δ 4.53 (2H, d, J = 6.0 Hz), 7.04–7.08 (1H
3.1.3.6. 8-Hydroxy-2-imino-2H-chromene-3-carboxylic
acid
(3-fluorophenyl)amide (13f). Yield: 51%; mp: 225–227 °C; and 1H, each d), 7.18 (1H, dd, J = 8.0, 6.0 Hz), 7.25–7.28 (1H,
1H-NMR (500 MHz, DMSO-d6) δ 6.96 (1H, td, J = 7.0, 1.5 Hz), m), 7.32–7.36 (4H, m), 8.39 (1H, s), 8.82 (1H, s), 10.74 (1H, t,
7.06 (1H, d, J = 2.5 Hz), 7.08 (1H, d, J = 4.5 Hz), 7.22 (1H, dd, J = 6.0 Hz); 13C-NMR (125 MHz, DMSO-d6) δ 43.3, 119.9, 120.4,
J = 4.5, 2.5 Hz), 7.28 (1H, d, J = 9.0 Hz), 7.39 (1H, q, J = 7.0 Hz), 120.5, 124.4, 127.5, 127.9, 128.8, 129.0, 139.3, 141.9, 142.4,
7.74 (1H, d, J = 9.0 Hz), 8.50 (1H, s), 9.10 (1H, br), 10.22 (1H, 144.4, 156.1, 162.2; IR (KBr): 3320, 1696, 1558, 1469 cm−1; MS
br); 13C-NMR (125 MHz, DMSO-d6) δ 107.0 (d, J = 23.1 Hz), (EI): m/z 294 (M+); HRMS: calcd for C17H14N2O3 294.1004,
110.0 (d, J = 23.1 Hz), 116.0, 119.8, 120.1, 120.3, 120.6, found: 294.1000.
3.1.3.12. 8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid
2-hydroxybenzylamide (13l). Yield: 62%; mp: 140–141 °C;
1H-NMR (500 MHz, DMSO-d6) δ 4.30 (2H, d, J = 6.0 Hz), 6.76
(1H, t, J = 7.0 Hz), 6.82 (1H, d, J = 7.0 Hz), 7.03–7.06 (1H and
1H, each d), 7.09 (1H, dd, J = 5.0, 2.5 Hz), 7.17 (2H, dd, J = 7.0,
4.0 Hz), 8.38 (1H, s), 8.79 (1H, s), 10.66 (1H, t, J = 6.0 Hz);
124.6 (d, J = 1.3 Hz), 131.1 (d, J = 10.3 Hz), 140.4 (d, J = 10.3
Hz), 142.5, 142.9, 144.5, 156.4, 160.7, 162.8 (d, J = 239.4
Hz); IR (KBr): 3305, 1609, 1579, 1473 cm−1; MS (EI): m/z
298 (M+); HRMS: calcd for C16H11FN2O3 298.0754, found
298.0754.
3.1.3.7. 8-Hydroxy-2-imino-2H-chromene-3-carboxylic
acid
(4-fluorophenyl)amide (13g). Yield: 65%; mp: 240–242 °C; 13C-NMR (125 MHz, DMSO-d6) δ 39.4, 115.4, 115.6, 119.4,
1H-NMR (500 MHz, DMSO-d6) δ 7.09–7.10 (1H and 1H, each 124.4, 125.7, 128.89, 128.94, 129.6, 141.9, 144.4, 145.0, 148.7,
d), 7.22–7.24 (2H and 1H, each dd), 7.71 (1H, dd, J = 5.0, 155.8, 161.1, 161.7, 162.1; IR (KBr): 3309, 1669, 1457,
4.5 Hz), 8.50 (1H, s), 9.09 (1H, br); 13C-NMR (125 MHz, DMSO- 1240 cm−1; MS (EI): m/z 310 (M+); HRMS: calcd for C17H14N2O4
d6) δ 116.1 (d, J = 23.1 Hz), 119.8, 120.2, 120.3, 120.5, 121.9 (d, 310.0954, found: 310.0951.
3.1.3.13. 8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid
3-hydroxybenzylamide (13m). Yield: 46%; mp: 205–207 °C;
J = 7.4 Hz), 124.6 (d, J = 3.6 Hz), 135.2, 142.4, 142.6, 144.5,
156.4, 158.9 (d, J = 239.0 Hz), 160.3; IR (KBr): 3298, 1674, 1511,
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1H-NMR (500 MHz, DMSO-d6) δ 4.44 (2H, d, J = 6.0 Hz), 6.64
Yield: 98%; mp: 237–238 °C; H-NMR (500 MHz, DMSO-d6)
(1H, dd, J = 6.0, 2.0 Hz), 6.69–6.74 (1H and 1H, d and s), δ 3.89 (3H, s), 7.16 (1H, t, J = 7.0 Hz), 7.22 (1H, t, J = 7.0 Hz),
7.04–7.08 (1H and 1H, each d), 7.12 (1H, t, J = 8.0 Hz), 7.19 7.31 (1H, d, J = 7.0 Hz), 7.36–7.40 (3H, m), 7.53 (1H, d, J = 7.3
(1H, dd, J = 7.0, 4.0 Hz), 8.39 (1H, s), 8.82 (1H, br), 10.70 (1H, Hz), 8.49 (1H, d, J = 7.3 Hz), 8.56 (1H, s), 9.36 (1H, br); 13C-NMR
t, J = 5.5 Hz); 13C-NMR (125 MHz, DMSO-d6) δ 43.2, 114.5, (125 MHz, DMSO-d6) δ 56.3, 115.0, 119.0, 120.9, 121.2, 122.6,
114.7, 118.4, 119.8, 119.9, 120.4, 124.4, 130.0, 140.7, 141.9, 124.0, 124.4, 124.8, 127.3, 129.3, 135.8, 142.6, 143.3, 146.3,
142.4, 144.4, 156.1, 158.0, 162.1; IR (KBr): 3291, 1669, 1596, 156.8, 160.5; IR (KBr): cm−1 MS (EI): m/z 328 (M+); HRMS: calcd
1473 cm−1; MS (EI): m/z 310 (M+); HRMS: calcd for C17H14N2O4 for C17H13ClN2O3 328.0615, Found: 328.0618.
310.0954, found: 310.0954.
3.1.4. General procedure for the preparation of 14. To a
stirred solution of chromene 13 (1 mmol) in THF (5 mL) was
added 10% HCl (5 drops), and the resulting mixture was
heated at 40 °C for 24 h. The reaction was quenched by H2O
(5 mL), and the aqueous mixture was extracted with EtOAc
(5 mL × 3), the organic extracts were combined, and dried over
Na2SO4, and the solvent was removed under reduced pressure.
The residue was chromatographed on silica gel (15 g, CH2Cl2)
to give the corresponding 14.
3.1.3.14. 8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid
4-hydroxybenzylamide (13n). Yield: 24%; mp: 187–188 °C;
1H-NMR (500 MHz, DMSO-d6) δ 4.38 (2H, d, J = 6.0 Hz), 6.72
(2H, d, J = 8.0 Hz), 7.05–7.07 (1H and 1H, each d), 7.12 (2H, d,
J = 8.0 Hz), 7.18 (1H, dd, J = 3.5, 3.0 Hz), 8.39 (1H, s), 8.78 (1H,
br), 10.61 (1H, t, J = 6.0 Hz); 13C-NMR (125 MHz, DMSO-d6)
δ 42.9, 115.5, 115.8, 119.9, 120.4, 120.6, 124.4, 129.4, 141.9,
142.4, 144.4, 156.1, 157.0, 160.6, 161.9; IR (KBr): 3293, 1675,
1473, 1240 cm−1; MS (EI): m/z 310 (M+); HRMS: calcd for
C17H14N2O3 310.0954, found: 310.0951.
3.1.4.1. 8-Hydroxy-2-oxo-2H-chromene-3-carboxylic acid phenyl-
1
amide (14a). Yield: 81%; mp: 251–253 °C; H-NMR (500 MHz,
3.1.3.15. 8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid
2-chlorobenzylamide (13o). Yield: 85%; mp: 258–259 °C;
1H-NMR (500 MHz, DMSO-d6) δ 4.59 (2H, d, J = 6.0 Hz),
7.04–7.08 (1H and 1H, each d), 7.18 (1H, dd, J = 4.0, 3.0 Hz),
7.32–7.35 (2H, m), 7.41–7.43 (1H, m), 7.47–7.49 (1H, m), 8.38
(1H, s), 8.84 (1H, s), 10.81 (1H, t, J = 6.0 Hz); 13C-NMR
(125 MHz, DMSO-d6) δ 41.4, 119.8, 120.0, 120.37, 120.43,
124.5, 127.9, 129.5, 129.8, 129.9, 132.9, 136.4, 142.1, 142.5,
DMSO-d6) δ 7.15 (1H, td, J = 7.5, 1.5 Hz), 7.24–7.28 (1H and
1H, each d), 7.37–7.42 (3H, m), 7.72 (2H, dd, J = 7.5, 1.5 Hz),
8.86 (1H, s), 10.50 (1H, br), 10.69 (1H, s); 13C-NMR (125 MHz,
DMSO-d6) δ 120.0, 120.1, 120.4, 120.6, 120.8, 124.8, 125.8,
129.5, 138.5, 143.1, 145.0, 148.4, 160.4, 161.0; IR (KBr): 3424,
1707, 1598, 1472 cm−1; MS (EI): m/z 281 (M+); HRMS: calcd for
C16H11NO4 281.0688, found 281.0689.
3.1.4.2. 8-Hydroxy-2-oxo-2H-chromene-3-carboxylic
acid
144.4, 156.1, 162.3; IR (KBr): 3301, 1674, 1472, 1225 cm−1; MS (2-hydroxyphenyl)amide (14b). Yield: 81%; mp: 224–226 °C;
(EI): m/z 328 (M+); HRMS: calcd for C17H13ClN2O3 328.0615, 1H-NMR (500 MHz, DMSO-d6) δ 6.83 (1H, td, J = 7.0, 1.5 Hz),
found: 328.0618.
6.92 (1H, dd, J = 7.0, 1.5 Hz), 6.97 (1H, td, J = 7.0, 1.5 Hz),
7.25–7.28 (1H and 1H, each d), 7.45 (1H, dd, J = 4.0, 3.0 Hz),
8.39 (1H, dd, J = 7.0, 1.5 Hz), 8.99 (1H, s), 10.02 (1H, br), 10.51
(1H, br), 11.14 (1H, br); 13C-NMR (125 MHz, DMSO-d6) δ 115.1,
119.0, 119.7, 120.1, 120.4, 120.7, 120.9, 124.8, 125.7, 127.1,
143.2, 145.1, 147.2, 149.3, 159.4, 161.4; IR (KBr): 3177, 1718,
1558, 1457 cm−1; MS (EI): m/z 297 (M+); HRMS: calcd for
C16H11NO5 297.0637, found 297.0634.
3.1.3.16. 8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid
3-chlorobenzylamide (13p). Yield: 90%; mp: 229–230 °C;
1H-NMR (500 MHz, DMSO-d6) δ 4.53 (2H, d, J = 5.5 Hz),
7.04–7.08 (1H and 1H, each d), 7.18 (1H, dd, J = 4.0, 3.0 Hz),
7.30 (1H, d-like, J = 7.0 Hz), 7.33 (1H, d-like, J = 7.0 Hz),
7.36–7.40 (2H, m), 8.39 (1H, s), 8.85 (1H, br), 10.77 (1H, t, J =
6.0 Hz); 13C-NMR (125 MHz, DMSO-d6) δ 42.7, 119.8, 120.0,
120.4, 124.4, 126.6, 127.4, 127.5, 127.7, 130.8, 133.6, 142.1,
3.1.4.3. 8-Hydroxy-2-oxo-2H-chromene-3-carboxylic
acid
142.2, 142.5, 144.5, 156.1, 162.4; IR (KBr): 3299, 1674, 1472, (3-hydroxyphenyl)amide (14c). Yield: 83%; mp: 248–250 °C;
1225 cm−1 MS (EI): m/z 328 (M+); HRMS: calcd for 1H-NMR (500 MHz, DMSO-d6) δ 6.55 (1H, dd, J = 6.5, 1.5 Hz),
;
C17H13ClN2O3 328.0615, found: 328.0612.
7.02 (1H, dd, J = 6.5, 1.5 Hz), 7.16 (1H, t, J = 6.5 Hz), 7.24–7.28
(1H and 1H, each d), 7.32 (1H, t, J = 3.0 Hz), 7.41 (1H, dd, J =
4.5, 2.5 Hz), 8.84 (1H, s), 9.54 (1H, s), 10.49 (1H, br), 10.61 (1H,
s); 13C-NMR (125 MHz, DMSO-d6) δ 107.3, 111.0, 111.9, 120.0,
120.2, 120.6, 120.8, 125.8, 130.2, 139.5, 143.0, 145.0, 148.3,
158.3, 160.2, 161.0; IR (KBr): 3288, 1714, 1609, 1472 cm−1; MS
(EI): m/z 297 (M+); HRMS: calcd for C16H11NO5 297.0637,
found 297.0639.
3.1.3.17. 8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid
4-chlorobenzylamide (13q). Yield: 93%; mp: 241–242 °C;
1H-NMR (500 MHz, DMSO-d6) δ 4.53 (2H, d, J = 6.0 Hz),
7.03–7.08 (1H and 1H, each d), 7.18 (1H, dd, J = 4.0, 2.5 Hz),
7.35 (2H, d, J = 7.0 Hz), 7.40 (2H, d, J = 7.0 Hz), 8.39 (1H, s),
8.94 (1H, s), 10.75 (1H, t, J = 6.0 Hz); 13C-NMR (125 MHz,
DMSO-d6)
δ
42.6, 119.8, 120.0, 120.4, 120.5, 124.4,
128.9, 129.8, 132.1, 138.6, 142.0, 142.5, 144.5, 156.2, 162.3;
3.1.4.4. 8-Hydroxy-2-oxo-2H-chromene-3-carboxylic
acid
IR (KBr): 3299, 1675, 1473, 1229 cm−1; MS (EI): m/z 328 (4-hydroxyphenyl)amide (14d). Yield: 52%; mp: 254–256 °C;
(M+); HRMS: calcd for C17H13ClN2O3 328.0615, found: 1H-NMR (500 MHz, DMSO-d6) δ 6.75 (2H, d, J = 6.5 Hz),
328.0612.
7.23–7.25 (1H and 1H, each d, m), 7.39 (1H, dd, J = 5.0, 2.5
Hz), 7.50 (2H, d, J = 6.5 Hz), 8.83 (1H, s), 9.36 (1H, s), 10.47
(1H, s), 10.48 (1H, br); 13C-NMR (125 MHz, DMSO-d6) δ 115.9,
120.00, 120.03, 120.6, 120.7, 122.1, 125.7, 130.1, 143.0, 145.0,
148.2, 154.7, 159.7, 161.1; IR (KBr): 3113, 1705, 1513,
3.1.3.18. 8-Methoxy-2-imino-2H-chromene-3-carboxylic acid
(2-chlorophenyl)amide (15). This compound was prepared by
the same procedure as that for 13 but using 2-hydroxy-3-methoxy-
benzaldehyde instead of 11.
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1473 cm−1; MS (EI): m/z 297 (M+); HRMS: calcd for C16H11NO5
3.1.4.10. 8-Hydroxy-2-oxo-2H-chromene-3-carboxylic
acid
(4-chlorophenyl)amide (14j). Yield: 54%; mp: 299–300 °C;
297.0637, found 297.0638.
3.1.4.5. 8-Hydroxy-2-oxo-2H-chromene-3-carboxylic
acid 1H-NMR (500 MHz, DMSO-d6) δ 7.24–7.28 (1H and 1H, each
(2-fluorophenyl)amide (14e). Yield: 98%; mp: 248–250 °C;
1H-NMR (500 MHz, DMSO-d6) δ 7.20 (1H, m), 7.25–7.29 (3H
m), 7.36 (1H, dd, J = 5.0, 2.5 Hz), 7.48 (1H, d, J = 6.5 Hz), 8.40
(1H, t, J = 6.5 Hz), 9.00 (1H, s), 10.53 (1H, br), 11.08 (1H, br);
13C-NMR (125 MHz, DMSO-d6) δ 115.7 (d, J = 18.3 Hz), 118.7,
120.0, 120.9, 121.2, 122.1, 125.4 (d, J = 2.4 Hz), 125.5 (d, J = 8.4
Hz), 125.9, 126.5 (d, J = 8.6 Hz), 143.1, 145.1, 149.6, 152.9 (d,
J = 241.9 Hz), 160.2, 161.7; IR (KBr): 3386, 1704, 1554,
d), 7.41 (1H, dd, J = 5.0, 2.5 Hz), 7.44 (2H, d, J = 7.0 Hz), 7.77
(2H, d, J = 7.0 Hz), 8.84 (1H, s), 10.76 (1H, br); 13C-NMR
(125 MHz, DMSO-d6) δ 119.9, 120.0, 120.5, 120.9, 122.0, 125.8,
128.4, 129.4, 137.4, 143.1, 145.2, 148.5, 160.7, 160.8; IR (KBr):
3233, 1733, 1472, 1207 cm−1; MS (EI): m/z 315 (M+); HRMS:
calcd for C16H10ClNO4 315.0298, found: 315.0301.
3.1.4.11. 8-Hydroxy-2-oxo-2H-chromene-3-carboxylic
acid
benzylamide (14k). Yield: 99%; mp: 262–264 °C; 1H-NMR
(500 MHz, DMSO-d6) δ 4.54 (2H, d, J = 6.0 Hz), 7.22–7.40 (8H,
m), 8.82 (1H, s), 9.14 (1H, t, J = 6.0 Hz), 10.45 (1H, br);
1471 cm−1
;
MS (EI): m/z 299 (M+); HRMS: calcd for
C16H10FNO4 299.0594, found 299.0591.
3.1.4.6. 8-Hydroxy-2-oxo-2H-chromene-3-carboxylic
acid 13C-NMR (125 MHz, DMSO-d6)
δ 119.3, 119.9, 120.56,
(3-fluorophenyl)-amide (14f). Yield: 85%; mp: 247–249 °C;
1H-NMR (500 MHz, DMSO-d6) δ 6.99 (1H, tt, J = 6.5, 1.5 Hz),
7.26–7.29 (1H and 1H, each d), 7.42–7.46 (3H, m), 7.78
(1H, dt, J = 6.5, 1.5 Hz), 8.86 (1H, s), 10.52 (1H, br), 10.83
(1H, br); 13C-NMR (125 MHz, DMSO-d6) δ 107.3 (d, J =
120.62, 125.6, 127.4, 127.9, 128.9, 139.4, 143.1, 144.9, 148.5,
160.8, 161.8; IR (KBr): 3320, 1672, 1583, 1473 cm−1; MS (EI):
m/z 295 (M+); HRMS: calcd for C17H13NO4 295.0845, found:
295.0845.
3.1.4.12. 8-Hydroxy-2-oxo-2H-chromene-3-carboxylic
acid
2-hydroxybenzylamide (14l). Yield: 50%; mp: 256–257 °C;
1H-NMR (500 MHz, DMSO-d6) δ 4.47 (2H, d, J = 6.0 Hz), 6.76
(1H, td, J = 7.0, 0.5 Hz), 6.83 (1H, dd, J = 7.0, 0.5 Hz), 7.10 (1H,
td, J = 7.0, 1.5 Hz), 7.19 (1H, dd, J = 7.0, 1.5 Hz), 7.22–7.24 (1H
and 1H, each d), 7.39 (1H, dd, J = 4.0, 2.5 Hz), 8.84 (1H, s),
9.16 (1H, t, J = 6.0 Hz), 9.70 (1H, br), 10.45 (1H, br); 13C-NMR
23.8 Hz), 111.3 (d,
J = 23.8 Hz), 116.3, 119.9, 120.0,
120.6, 120.9, 125.8 (d, J = 1.25 Hz), 131.1 (d, J = 10.3 Hz), 140.1
(d, J = 10.3 Hz), 143.1, 145.0, 148.6, 160.7, 160.8, 162.7 (d, J =
239.3 Hz); IR (KBr): 3436, 1701, 1555, 1471 cm−1; MS (EI): m/z
299 (M+); HRMS: calcd for C16H10FNO4 299.0594, found
299.0591.
3.1.4.7. 8-Hydroxy-2-oxo-2H-chromene-3-carboxylic
acid (125 MHz, DMSO-d6) δ 39.4, 115.6, 119.0, 119.4, 120.0, 120.6,
(4-fluorophenyl)amide (14g). Yield: 89%; mp: 241–243 °C;
1H-NMR (500 MHz, DMSO-d6) δ 7.21–7.27 (4H, m), 7.41 (1H,
dd, J = 4.5, 2.5 Hz), 7.56 (2H, dd, J = 6.5, 4.5 Hz), 8.85 (1H, s),
10.70 (1H, br); 13C-NMR (125 MHz, DMSO-d6) δ 116.1 (d, J =
23.1 Hz), 119.9, 120.1, 120.6, 120.9, 122.3 (d, J = 8.5 Hz), 125.8
(d, J = 3.6 Hz), 134.9, 143.1, 145.1, 148.4, 159.1 (d, J = 239.4
Hz), 160.5, 160.8; IR (KBr): 3372, 1611, 1508, 1472 cm−1; MS
(EI): m/z 299 (M+); HRMS: calcd for C16H10FNO4 299.0594,
found 299.0591.
120.7, 124.9, 125.6, 128.9, 129.6, 143.1, 145.0, 148.7, 155.8,
161.0, 161.6; IR (KBr): 3315, 1685, 1555, 1293 cm−1; MS (EI):
m/z 311 (M+); HRMS: calcd for C17H13NO5 311.0794, found:
311.0793.
3.1.4.13. 8-Hydroxy-2-oxo-2H-chromene-3-carboxylic
acid
3-hydroxybenzylamide (14m). Yield: 81%; mp: 208–210 °C;
1H-NMR (500 MHz, DMSO-d6) δ 4.45 (2H, d, J = 6.0 Hz), 6.64
(1H, dd, J = 6.5, 2.5 Hz), 6.75 (1H, s), 6.77 (1H, d, J = 6.5 Hz),
7.12 (1H, t, J = 6.5 Hz), 7.21–7.26 (1H and 1H, each d), 7.39
3.1.4.8. 8-Hydroxy-2-oxo-2H-chromene-3-carboxylic
acid (1H, dd, J = 5.0, 2.5 Hz), 8.82 (1H, s), 9.08 (1H, t, J = 6.0 Hz),
9.39 (1H, s), 10.47 (1H, br); 13C-NMR (125 MHz, DMSO-d6)
δ 114.4, 114.7, 118.5, 119.3, 119.9, 120.6, 120.7, 125.6, 129.9,
140.7, 143.1, 145.0, 148.6, 157.9, 160.8, 161.7; IR (KBr): 3320,
1700, 1539, 1473 cm−1; MS (EI): m/z 311 (M+); HRMS: calcd for
C17H13NO5 311.0794, found: 311.0793.
(2-chlorophenyl)amide (14h). Yield: 95%; mp: 286–287 °C;
1H-NMR (500 MHz, DMSO-d6) δ 7.20 (1H, td, J = 7.0, 1.5 Hz),
7.28–7.29 (1H and 1H, each d), 7.42 (1H, td, J = 7.0, 1.5 Hz),
7.48 (1H, t, J = 5.5 Hz), 7.59 (1H, dd, J = 7.0, 1.5 Hz), 8.53 (1H,
dd, J = 7.0, 1.0 Hz), 9.03 (1H, s), 10.56 (1H, br), 11.31 (1H, br);
13C-NMR (125 MHz, DMSO-d6) δ 118.5, 120.0, 121.0, 121.3,
122.3, 123.3, 125.9, 128.5, 130.0, 135.3, 137.7, 143.2, 145.1,
150.0, 160.3, 161.7; IR (KBr): 3200, 1733, 1473, 1202 cm−1; MS
(EI): m/z 315 (M+); HRMS: calcd for C16H10ClNO4 315.0298,
found: 315.0301.
3.1.4.14. 8-Hydroxy-2-oxo-2H-chromene-3-carboxylic
acid
4-hydroxybenzylamide (14n). Yield: 99%; mp: 234–235 °C;
1H-NMR (500 MHz, DMSO-d6) δ 4.41 (2H, d, J = 6.0 Hz),
6.72 (2H, d, J = 8.0 Hz), 7.16 (2H, d, J = 8.0 Hz), 7.20–7.25
(1H and 1H, each d), 7.38 (1H, dd, J = 5.0, 2.0 Hz), 8.82
(1H, s), 8.99 (1H, t, J = 6.0 Hz), 9.33 (1H, br), 10.44 (1H, br);
13C-NMR (125 MHz, DMSO-d6) δ 31.2, 42.9, 115.7, 119.3,
120.0, 120.60, 120.65, 125.65, 129.5, 143.1, 145.0, 148.5, 157.0,
160.9, 161.6; IR (KBr): 3321, 1699, 1472, 1214 cm−1; MS (EI):
m/z 311 (M+); HRMS: calcd for C17H13NO5 311.0794, found:
311.0795.
3.1.4.9. 8-Hydroxy-2-oxo-2H-chromene-3-carboxylic
acid
(3-chlorophenyl)amide (14i). Yield: 47%; mp: 266–268 °C;
1H-NMR (500 MHz, DMSO-d6) δ 7.21 (1H, ddd, J = 7.0, 2.5, 1.0
Hz), 7.24–7.28 (1H and 1H, each d), 7.38–7.42 (2H, m), 7.57
(1H, ddd, J = 7.0, 2.5, 1.0 Hz), 7.90 (1H, d, J = 2.0 Hz), 8.83 (1H,
s), 10.78 (1H, s); 13C-NMR (125 MHz, DMSO-d6) δ 119.0, 119.8,
119.9, 120.0, 120.6, 120.9, 124.5, 125.8, 131.2, 133.8, 139.9,
143.1, 145.0, 148.6, 160.6, 160.9; IR (KBr): 3238, 1733, 1604,
3.1.4.15. 8-Hydroxy-2-oxo-2H-chromene-3-carboxylic
acid
2-chlorobenzylamide (14o). Yield: 71%; mp: 248–249 °C;
1H-NMR (500 MHz, DMSO-d6) δ 4.61 (2H, d, J = 6.0 Hz)
7.22–7.26 (1H and 1H, each d), 7.31–7.34 (2H, m), 7.39 (1H,
1473 cm−1
;
MS (EI): m/z 315 (M+); HRMS: calcd for
C16H10ClNO4 315.0298, found: 315.0301.
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dd, J = 4.0, 2.5 Hz), 7.42 (1H, dd, J = 7.0, 2.5 Hz), 7.47 (1H, d- 0.1 M potassium phosphate buffer, pH 7.0, 0.1 mM NADPH,
like, J = 7.0 Hz), 8.82 (1H, s), 9.21 (1H, t, J = 6.0 Hz), 10.45 (1H, substrate and enzyme, in a total volume of 2.0 mL. The sub-
br); 13C-NMR (125 MHz, DMSO-d6) δ 119.1, 119.9, 120.6, 120.7, strates were 50 μM isatin (for CBR1), 0.1 mM menadione (for
125.7, 127.8, 129.3, 129.5, 129.7, 132.6, 136.4, 143.1, 145.0, CBR3), 0.4 mM diacetyl (for DCXR), 25 μM 1-phenylisatin (for
148.7, 160.9, 162.1; IR (KBr): 3329, 1701, 1470, 1248 cm−1; MS DHRS4) and 0.2 mM pyridine-3-aldehyde (for AKR1B10 and
(EI): m/z 329 (M+); HRMS: calcd for C17H12ClNO4 329.0455, AKR1B1). The dehydrogenase activity of CBR1 was assayed
found: 329.0457.
3.1.4.16. 8-Hydroxy-2-oxo-2H-chromene-3-carboxylic
using 0.25 mM NADP+ and an appropriate amount of S-tetralol
as the coenzyme and the substrate, respectively, in the above
reaction mixture. The kinetic studies in the presence of inhibi-
tors were carried out in both isatin reduction and S-tetralol oxi-
dation over a range of five or six substrate concentrations at
the above saturating concentration of NADPH or NADP+.27 The
inhibition patterns were judged from the Lineweaver–Burk
acid
3-chlorobenzylamide (14p). Yield: 90%; mp: 207–208 °C;
1H-NMR (500 MHz, DMSO-d6) δ 4.54 (2H, d, J = 6.0 Hz),
7.21–7.25 (1H and 1H, each d), 7.31–7.34 (2H, m),
7.37–7.39 (2H, m), 7.42 (1H, t-like, J = 2.0 Hz), 8.81 (1H, s), 9.21
(1H, t, J = 6.0 Hz), 10.44 (1H, br); 13C-NMR (125 MHz, DMSO-d6)
δ 42.7, 119.3, 119.9, 120.6, 120.7, 125.6, 126.6, 127.4, 127.8,
130.7, 133.5, 142.3, 143.1, 145.0, 148.6, 160.7, 162.1; IR (KBr):
3334, 1696, 1472, 1211 cm−1; MS (EI): m/z 329 (M+); HRMS:
calcd for C17H12ClNO4 329.0455, found: 329.0454.
plots, and inhibition constants, Ki (slope effect) and K′ (inter-
i
cept effect), were determined from replots of the slopes and
intercepts, respectively, versus inhibitor concentration.42 The
IC50 and Ki values are expressed as the means SD for at least
three determinations.
3.1.4.17. 8-Hydroxy-2-oxo-2H-chromene-3-carboxylic
acid
4-chlorobenzylamide (14q). Yield: 73%; mp: 269–270 °C;
1H-NMR (500 MHz, DMSO-d6) δ 4.52 (2H, d, J = 6.0 Hz),
7.20–7.25 (1H and 1H, each d), 7.36–7.41 (5H, m, including at
7.39 (4H, d, J = 7.0 Hz)), 8.81 (1H, s), 9.18 (1H, t, J = 6.0 Hz),
10.45 (1H, br); 13C-NMR (125 MHz, DMSO-d6) δ 42.6, 119.3,
119.9, 120.6, 120.7, 125.6, 128.8, 129.8, 132.0, 138.6, 143.1,
145.0, 148.6, 160.7, 162.0; IR (KBr): 3320, 1694, 1468,
3.2.3. Molecular modeling. All calculations were per-
formed using the Schrödinger suite 2013-2 (Schrödinger, LLC,
New York, NY). The 2D structure of 13h was first converted
into the 3D structure using the LigPrep2.7 program. The proto-
nation states of the compounds were predicted using the
Epik2.5 program.43 Then, the conformational search of the
compound was carried out using the ConfGen2.5 program,44
and the resulting conformers were used in the following
docking calculations.
1204 cm−1
;
MS (EI): m/z 329 (M+); HRMS: calcd for
C17H12ClNO4 329.0455, found: 329.0457.
In the docking calculations, a total of five X-ray structures
of CBR1 with the following PDB codes were used in order to
3.2. Biological assays
3.2.1. Preparation of recombinant enzymes. The recombi- consider protein flexibility as much as possible: 1WMA,7
nant CBR1,6 CBR3,6 DHRS4,30 DCXR,33 AKR1B1,16 AKR1B10,39 2PFG,45 3BHI, 3BHJ, and 3BHM.12 The structures were pre-
AKR1C1,40 AKR1C4,40 and AKR1C2,41 were prepared and pared using the Protein Preparation Wizard in Maestro9.5. The
purified to homogeneity, as described previously. Site- missing atoms for side chains were compensated for using the
directed mutagenesis was performed using a QuickChange Prime3.3 program.46 Finally, the structures were minimized
site-directed mutagenesis kit (Stratagene, La Jolla, CA) and using force-field OPLS 2005. We removed all HETATM mole-
the pET28a expression plasmid harboring the cDNA for CBR16 cules except NADP+ (i.e. ligands, ions, and water molecules)
as the template, according to the protocol described by the before docking calculations. The docking calculation of 13h
manufacturer. The primer pair used for the mutagenesis against five X-ray structures of CBR1 was performed using the
was composed of sense and antisense oligonucleotides to IFD 2006 protocol as implemented in Schrödinger suite
alter one codon of the cDNA. The 26- to 30-mer primers 2013-2.47 The box center for the “Receptor Grid Generation”
were synthesized to give the Met141Gln, Met141Val, protocol was set to a centroid of Ser139, Met141, Trp229, and
Trp229Phe and Trp229Leu mutant enzymes (ESI Table S1†). Ala235 of CBR1, which were residues forming the active site.
The coding regions of the cDNAs in the expression plasmids Initial Glide docking was performed using reduced van der
were sequenced using a Beckman CEQ8000XL DNA sequencer Waals radii for proteins and ligands to allow minor readjust-
in order to confirm the presence of the desired mutation ments. The van der Waals radii of both molecules were scaled
and ensure that no other mutation had occurred. The expression by 80%. The maximum number of poses per conformer was
constructs were transformed into Escherichia coli BL21 (DE3) set to 2. Re-docking was performed in Glide using standard
pLysS cells (Life Technologies, Carlsbad, CA). The mutant settings. The generated poses were ranked according to IFD-
enzymes were expressed in Escherichia coli cells, and purified to Score, and we finally selected a pose with the lowest IFDScore
homogeneity as described above for the wild-type CBR1.6
as the interaction model. In order to validate our procedure,
3.2.2. Assay of enzyme activity. The reductase and de- we applied it for the hydroxy-PP–CBR1 complex. The result
hydrogenase activities of CBR1 were assayed at 25 °C by is shown in ESI Fig. S1.† The resulting model, i.e., the
measuring the rate of change in NADPH absorbance (at top-ranked pose, reproduced all interactions between hydroxy-
340 nm) and fluorescence (at 455 nm with an excitation wave- PP and CBR1 observed in the crystal structure. The positional
length of 340 nm), respectively.27 The IC50 values for inhibitors and conformational RMSDs were 0.48 and 0.13 Å, respectively.
were determined in the reaction mixture that consisted of These results suggested that our procedure is appropriate for
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producing reliable interaction models of 13h as well as hydroxy- strate-binding residues, Ser139, Met141, Tyr193 and Trp229,
PP. The docked model shown in Fig. 2 was generated using of which the interactions with Met141 and Trp229 contribute
PyMOL (DeLano Scientific, San Carlos, CA, USA).
to the high selectivity to CBR1 over CBR3. We also provided a
3.2.4. Cell culture experiments. BAEC were a generous gift method that is useful to evaluate the efficacy of the
from Dr Junichi Nakagawa (Tokyo University of Agriculture, inhibitor at the cellular level. These findings may contribute
Abashiri, Japan). The cells were grown in Dulbecco’s modified to future development of potent and selective CBR1 inhibitors.
Eagle’s medium (DMEM) supplemented with 10% fetal bovine
serum, 100 U mL−1 penicillin and 100 µg mL−1 streptomycin
at 37 °C in a humidified incubator containing 5% CO2. In
order to overexpress CBR1 in the cells, the cDNA for the
Author contributions
enzyme was amplified by PCR from the bacterial expression Participated in research design: Hu, Toyooka, and Endo.
vector6 using primers (ESI Table S1†). The amplified cDNA was
Conducted experiments: Hu, Miyagi, Arai, Oguri, Xia, Hara,
subcloned at the EcoRI and SalI sites into the mammalian Matsunaga, and Endo.
pGW1 expression vector, and the sequence of the insert was
Contributed new reagents or analytic tools: Miura, Nishi-
verified by DNA sequencing. The pGW1 expression vector har- naka, Terada, and Gouda.
boring the cDNA for CBR1 was transfected into BAEC using
Performed data analysis: Hu, Miyagi, El-Kabbani, Matsu-
Lipofectamine 2000 (Invitrogen, Carlsbad, CA) when the cells naga, Ikari and Endo.
were grown to 80% confluence in microplates. The empty
Wrote or contributed to the writing of the manuscript: Hu,
vector was similarly transfected into the cells, which were used El-Kabbani, Toyooka, Hara, Matsunaga, Ikari, and Endo.
as control cells. The transfection efficacy was evaluated by
Western blotting using the antibodies against CBR148 and
human β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), of
which β-actin was used as a loading control. After 48 h culture,
Abbreviations
the cells were washed twice with DMEM containing 2% fetal AKR
Aldo-keto reductase
bovine serum and the antibiotics, and then treated for 24 h AKR1B1
Human aldose reductase
with 9,10-PQ. In the experiments with 13h and 13p, the cells AKR1B10 Human aldose reductase-like protein
were pretreated with or without the inhibitors 2 h before the BAEC
treatment of 5 μM 9,10-PQ. Dimethylsulfoxide was the vehicle CBR
of the agents. The cell viability was evaluated by the tetra- DCXR
zolium dye-based cytotoxicity assay using 2-(4-iodophenyl)-3- DHRS4
Bovine aorta endothelial cells
Carbonyl reductase
Dicarbonyl/L-xylulose reductase
Dehydrogenase/reductase SDR family member 4
(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium.49
Data Hydroxy- 3-(1-tert-Butyl-4-amino-1H-pyrazolo[3,4-d]pyrimi-
are expressed as means SD for at least three independent PP
experiments. Statistical evaluation of the data was performed 9,10-PQ
using the unpaired Student’s t-test and ANOVA followed by SDR
din-3-yl)phenol
9,10-Phenanthrenequinone
Short-chain dehydrogenase/reductase
Fisher’s test. A p value <0.05 was considered statistically S-Tetralol (S)-(+)-1,2,3,4-Tetrahydro-1-naphthol
significant.
Acknowledgements
This work was supported in part by a JSPS Grant-in-Aid for
Scientific Research (C) from the Japan Society for the Pro-
4. Conclusion
In this study,
a series of 3-carboxamide derivatives of motion of Science grant number 26460149.
8-hydroxy-2-iminochromene (13a–13q) and 8-hydroxycoumarin
(14a–14q) were synthesized, and their inhibitory activities for
CBR1 were evaluated. The structure–activity relationship of the
synthesized compounds revealed that the 8-hydroxyl and
2-imino groups on the chromene ring were essential to main-
tain the potent inhibitory effect. In addition, substituents in
the 3-carboxamide chain of the 8-hydroxy-2-iminochromenes
affected the inhibitory potency, and 13h with the 2-chloro-
phenyl ring as the substituent showed the lowest IC50 value,
which is superior to those for the previously reported inhibi-
tors of CBR1. 13h also showed high selectivity to CBR1 over
other SDRs (CBR3, DCXR and DHRS4) and AKRs (1B1, 1B10,
1C1, 1C2 and 1C4). Site-directed mutagenesis and molecular
docking of 13h in CBR1 demonstrated that the high inhibitory
potency is attributed to the interactions of 13h with the sub-
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