3-nitro-2H-chromenes as a new class of inhibitors against thioredoxin reductase and proliferation of cancer cells

Article abstract of DOI:10.1002/ardp.201200121

A series of 3-nitrochromenes were designed and synthesized. These compounds showed good inhibitory activity against thioredoxin reductase (TrxR) and the proliferation of A549 cancer cells. The structure-activity relationship analysis indicates that the 3-nitrochromene scaffold is the crucial pharmacophore for achieving good inhibitory activity. The bromo-substitutions at the 6- and 8-position of 3-nitrochromene significantly increase the inhibitory activity. A series of 3-nitrochromenes were designed and synthesized. They showed good inhibitory activity against thioredoxin reductase and the proliferation of A549 cancer cells. Structure-activity relationship analysis revealed that the 3-nitrochromene scaffold is the crucial pharmacophore for achieving good inhibitory activity. Bromo-substitutions at the 6- and 8-position of 3-nitrochromene significantly increase the inhibitory activity. Copyright

Full text of DOI:10.1002/ardp.201200121

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–4
1
Full Paper
3-Nitro-2H-chromenes as a New Class of Inhibitors against
Thioredoxin Reductase and Proliferation of Cancer Cells
Guo-Qiang Xiao, Bao-Xia Liang, Shu-Han Chen, Tian-Miao Ou, Xian-Zhang Bu, and Ming Yan
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China
A series of 3-nitrochromenes were designed and synthesized. These compounds showed good
inhibitory activity against thioredoxin reductase (TrxR) and the proliferation of A549 cancer cells.
The structure–activity relationship analysis indicates that the 3-nitrochromene scaffold is the crucial
pharmacophore for achieving good inhibitory activity. The bromo-substitutions at the 6- and
8-position of 3-nitrochromene significantly increase the inhibitory activity.
Keywords: 3-Nitro-2H-chromene / Cancer cell / Inhibitory activity / Synthesis / Thioredoxin reductase
Received: March 26, 2012; Revised: June 4, 2012; Accepted: June 29, 2012
DOI 10.1002/ardp.201200121
Introduction
Chromenes and chromans are widespread in plants
and are metabolites of microorganisms. They possess a
range of interesting biological activities [12]. The 3-nitro-
2H-choromenes can be readily prepared from salicylalde-
hydes and 2-nitroethylene [13]. They have been used as
Michael acceptors in a couple of reactions [14, 15]. In
addition, nitro compounds were reported to exert strong
interactions with TrxRs [16]. We speculate that 3-nitro-2H-
chromenes are efficient inhibitors against TrxRs. In this
paper, we report the synthesis of 3-nitro-2H-chromenes and
their biological evaluations as a new class of inhibitors
against TrxRs as well as the proliferation of A549 human
cancer cell lines.
Mammalian thioredoxin reductases (TrxRs) are a family of
NADPH-dependent oxidoreductases with a special seleno-
cysteine residue. They catalyze the reduction of the redox
protein thioredoxins, as well as other endogenous and
exogenous compounds [1]. Biochemical, virological, and
clinical evidences suggest that the overactivation/dysfunc-
tion of TrxRs play a pathophysiologic role in cancers and
chronic diseases such as rheumatoid arthritis, Sjo¨gren’s syn-
drome, and AIDS [2–3]. Inhibiting the activity of TrxR may
lead to new treatments for these diseases. Besides, TrxRs from
different organisms such as Escherichia coli, Mycobacterium lep-
rae, Plasmodium falciparum, Drosophila melanogaster, and human
show a surprising diversity in the reduction mechanism. The Results and discussion
significant species difference provides the possibility to
develop specific TrxR inhibitors for chemotherapeuty [4, 5].
In recent years, extensive efforts have been made to develop
TrxR inhibitors. Nitroureas, organochalcogenides (S, Se, and
Te), nitroaromatic compounds, polyphenols, and metal com-
plexes were identified as effective TrxR inhibitors. Due to the
existence of nucleophilic cysteine and selenocysteine resi-
dues in their structure, TrxRs can react with Michael accept-
ors to form covalent adducts. Several Michael acceptors such
as curcumins [6–8], cinnamaldehydes [9], enals [10], and pros-
taglandins [11] were found to be efficient inhibitors of TrxRs.
Synthesis
3-Nitro-2H-chromenes (1a–i) were prepared from substituted
salicylaldehydes and 2-nitroethanol according to the
reported procedure (Scheme 1) [13]. For a comparison, the
structurally related compounds 2–5 were also prepared
(Scheme 2). 6-Chloro-2H-chromene-3-carbonitrile (2a) was
obtained from 5-chlorosalicylaldehyde and acrylonitrile in
the presence of 1,4-diazabicyclo[2.2.2]octane (DABCO). The
basic hydrolysis of 2a provided 6-chloro-2H-chromene-3-car-
boxylic acid (2b). 3-Nitrochromans (3a–3b) were prepared by
the reduction of 1a–b with NaBH4. N-(6-Chlorochroman-3-
yl)acetamide (4) was obtained via the reduction of 3b with
zinc powder in the mixed solvent of acetic acid and acetic
anhydride. 3-Nitro-2H-chromen-2-one (5) was prepared from
salicylaldehyde and methyl nitroacetate.
Correspondence: Ming Yan, School of Pharmaceutical Sciences, Sun
Yat-sen University, Guangzhou 510006, China.
E-mail: yanming@mail.sysu.edu.cn
Fax: þ86 20 39943049; þ86 20 39943054
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2
G.-Q. Xiao et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–4
R1
NO2
Table 1. Inhibitive activities (IC50) of 3-nitro-2H-chromenes 1a–i
and their analogs 2–5 against TrxR
O
Entry
Compound
IC50 (mM)
R2
_
1a 1i
1
2
3
4
5
6
7
8
1a
1b
1c
1d
1e
1f
1g
1h
1i
2a
2b
3a
3b
4
4.01
2.18
1.68
1.17
1.79
1.04
2.41
2.82
4.52
>50
>50
2.58
3.88
>50
>50
1a: R1 = H, R2 = H; 1b: R1 = Cl, R2 = H;
1c: R1 = F, R2 = H; 1d: R1 = Br, R2 = H;
1e: R1 = Cl, R2 = Cl; 1f: R1 = Br, R2 = Br
1g: R1 = NO2, R2
1
2
;
1h: R = MeO, R = H
= H;
1i: R1 = H, R2 = MeO
Scheme 1. 3-Nitro-2H-chromenes 1a–i.
9
10
11
12
13
14
15
Biological activities
The inhibitive activities of compounds 1–5 against TrxR were
determined by incubation with DTNB, TrxR, NADPH, and the
test compound (25 mM). The initial increase of the absorb-
ance at 405 nm was recorded and IC50 was calculated by the
standard method. The results are listed in Table 1. The 3-nitro-
2H-chromenes 1a–i showed potent inhibitory activities
(Table 1, entries 1–9), however the corresponding 3-cyano-
and 3-carboxylic acid analogues 2a–b were inactive (Table 1,
entries 10–11). The 3-nitro-chromans 3a–b showed compara-
tive inhibitory activities with 1a and b (Table 1, entries 12–
13). The fact implicates that the 3-nitro group is the crucial
function group interacting with TrxR. 6-Bromo-3-nitro-2H-
chromene 1d showed better inhibitory activity than 6-
chloro-3-nitro-2H-chromene 1b, 6-fluoro-3-nitro-2H-chromene
1c and 3-nitro-2H-chromene 1a (Table 1, entry 4 vs. entries
5
1–3). Further increase of inhibitive activity was observed for
6,8-dibromo-3-nitro-2H-chromene 1f (Table 1, entry 6). These
results demonstrate that the bromo substitution at the
benzene ring is beneficial for the activity. On the other hand,
3-acetamido-chroman 4 and 3-nitrochromenone 5 were
found to be ineffective (Table 1, entries 14–15).
Furthermore, the inhibitive activities of compounds 1–5
against the A549 cancer cell line were studied. Chlormethine
was used as the positive control and the results are listed in
Table 2. Generally compounds 1a–i showed good inhibitory
activities (Table 2, entries 1–9). The 6-halogen substitutions
exerted a beneficial effect on the activity (Table 2, entries 2–
4), however 6,8-di-halogen substitutions could not further
improve the activity (Table 2, entries 5–6). 6-Methoxyl-3-
Cl
CHO
OH
Cl
Cl
CN
CN
DABCO
re ux
+
H
C
N
3C
,
fl
O
a
2
Table 2. Inhibition against the proliferation of A549 cancer cells by
3-nitro-2H-chromenes 1a–i and their analogs 2–5
COOH
a
N OH
H
2O
Entry
Compound
Inhibition of A549
O
2b
IC50 (mM)
R
C
N
R
N
O2
O2
1
2
3
4
5
6
7
8
1a
1b
1c
1d
1e
1f
1g
1h
1i
2a
2b
3a
3b
4
5.84
1.52
1.42
1.91
2.62
3.19
1.81
5.64
10.46
>100
>100
>100
>100
>100
19.27
3.80
a
N BH4
-
r
CH2Cl2/i P OH
O
H
O
H
l
C
a:
:
a:
:
=
=
=
=
1
1b
3
3b
R
R
R
R
l
C
l
N
O2
c
l
NHA
C
n
Z
ower
p
H A
/
c
c
2O
A
O
O
O
9
3b
4
10
11
12
13
14
15
16
CHO
e
NO2
M
OOC
er ne
idi
i
p p
C6H6 fl
+
re ux
,
H
O
O2
N
O
O
5
5
Chlormethine
Scheme 2. Synthesis of compounds 2–5.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–4
Inhibitors Against TrxR and Cancer Cells
3
DABCO (0.112 g, 1 mmol). The reaction mixture was refluxed for
20 h and cooled down to room temperature. After aqueous NaOH
(2 M, 10 mL) was added, the mixture was extracted with CH2Cl2
(25 mL ꢁ 3). The combined organic layer was dried over anhy-
drous Na2SO4 and concentrated under reduced pressure. The
crude product was purified by flash chromatography (petroleum
ether/CH2Cl2 ¼ 7:1) to give the product 2a as a white solid.
Yield 72%, mp 135.0–136.58C; 1H NMR (400 MHz, CDCl3):
d ¼ 7.22 (dd, J ¼ 8.7, 2.5 Hz, 1H), 7.12–7.08 (m, 2H), 6.82
(d, J ¼ 8.7 Hz, 1H), 4.83 (d, J ¼ 1.4 Hz, 2H); 13C NMR (100 MHz,
CDCl3): d ¼ 152.76, 137.56, 132.31, 127.73, 127.40, 121.11,
nitro-2H-chromene 1h and 8-methoxyl-3-nitro-2H-chromene
1i showed significantly lower inhibitory activities than 3,6-
dinitro-2H-chromene 1g and halogen substituted compounds
1b–f (Table 2, entries 8–9 vs. entries 2–7). The replacement the
3-nitro group with CN and COOH resulted in the complete
loss of the activity (Table 2, entries 10–11). In addition,
the reduction of the 3,4-double bond of 3-nitro-2H-chromenes
also led to inactive compounds 3a and b (Table 2, entries 12–
13). 3-Acetoamido-chroman 4 was also found to be ineffective
(Table 2, entry 14). On the other hand, 3-nitrochromenone 5
showed much lower inhibitory activity (Table 2, entry 15).
These results undoubtedly demonstrate the importance of
the 3-nitro-2H-chromene scaffold. Since 3-nitrochromenes
are efficient Michael acceptors in a couple of organic reactions
[14, 15], we reason that their inhibitory activity against the
A549 cell line originates from the electrophilic reactivity with
DNA. It should be noted that 3-nitro-2H-chromenes 1b–g
showed more potent inhibitory activities than chlormethine
(Table 2, entries 2–7 vs. entry 16). They have the potential as a
new class of alkylating reagents for cancer treatment.
118.01, 115.93, 104.89, 64.50; IR (KBr) n/cmꢀ1
: 3062 (m),
2925 (w), 2213 (s), 1840 (m), 1451 (s), 1178 (w), 815 (s), 628 (m);
HRMS (ESI) calcd for C10H5ClNOꢀ [MꢀH]ꢀ: 191.0138, found:
191.0133.
Synthesis of 6-chloro-2H-chromene-3-carboxylic acid (2b)
A mixture of 6-chloro-2H-chromene-3-carbonitrile 1j (0.575 g,
3 mmol) in aqueous NaOH (10%, 15 mL) was refluxed for 5 h.
The reaction mixture was placed in an ice bath and acidified with
concentrated HCl to pH 10. The precipitate was filtered and
washed with hexane. The crude product was purified by flash
chromatography (CH2Cl2/MeOH ¼ 9:1) to give 2b as a yellow
solid. Yield 95%, mp 240.1–241.28C; 1H NMR (400 MHz, DMSO-
d6): d ¼ 7.46–7.44 (m, 2H), 7.29 (dd, J ¼ 8.6, 2.6 Hz, 1H), 6.88
(d, J ¼ 8.6 Hz, 1H), 4.93 (d, J ¼ 1.5 Hz, 2H); 13C NMR (100 MHz,
DMSO-d6): d ¼ 165.19, 153.07, 130.97, 130.92, 128.13, 125.17,
124.73, 122.44, 117.34, 64.38; IR (KBr) n/cmꢀ1: 3287 (w), 1700 (s),
1478 (s), 1097 (w), 1018 (w), 820 (s), 758 (s); HRMS (ESI) calcd
Conclusion
In summary, a series of 3-nitro-2H-chromenes were found to
show good inhibitory activities against TrxR and the prolifer-
ation of the A549 cell line. The structure–activity relationship
analysis undoubtedly demonstrates that the 3-nitro-2H-chro-
mene scaffold is the crucial pharomacophore. Further studies
are currently under way to improve the inhibitory activity
and to understand the interaction mechanism.
for C10H6ClO3 [MꢀH]ꢀ: 209.0005, found: 209.0004.
ꢀ
Synthesis of 3-nitrochromans (3a)
To a solution of 3-nitro-2H-chromene 1a (0.531 g, 3 mmol) in
CH2Cl2 (40 mL) and i-PrOH (16 mL) was added silica gel (2.4 g) at
room temperature. Then NaBH4 (0.227 g, 6 mmol) was added
slowly over 15 min. The reaction mixture was stirred for 0.5 h
and quenched with acetic acid (6 mL). After stirring for another
0.5 h, the reaction mixture was filtered. The insoluble solid was
washed thoroughly with CH2Cl2 (3 mL ꢁ 2). The filtrate was
concentrated and the residue was treated with EtOAc (25 mL)
and H2O (25 mL). The organic layer was separated, washed
with brine, and dried over anhydrous Na2SO4. After the solvent
was evaporated under vacuum, 3-nitrochroman (3a) was
obtained as a white solid. Yield 98%, mp 86.2–87.58C; 1H NMR
(400 MHz, CDCl3): d ¼ 7.18–7.12 (m, 2H), 6.95 (td, J ¼ 7.5, 1.2 Hz,
1H), 6.88–6.86 (m, 1H), 4.95 (m, 1H), 4.62–4.43 (m, 2H),
3.45 (dd, J ¼ 16.9, 5.8 Hz, 2H); 13C NMR (100 MHz, CDCl3):
d ¼ 153.32, 129.53, 128.21, 121.90, 117.60, 117.02, 77.62, 65.83,
28.30; IR (KBr) n/cmꢀ1: 1583 (w), 1549 (s), 1489 (m), 1359 (w), 1259
(s), 1117 (wꢀ), 1047 (m), 883 (m), 767 (s); HRMS (ESI) calcd
for C9H8NO3 [MꢀH]ꢀ: 178.0504, found: 178.0506.
Experimental
General
All solvents were used as commercial anhydrous grade without
further purifications. The flash column chromatography was
performed over silica gel (230–400 mesh), purchased from
Qingdao Haiyang Chemical Co., Ltd. Melting points were
recorded on an OptiMelt melting point apparatus. 1H and
13C NMR spectra were recorded on a Bruker AVANCE 400 MHz
spectrometer. Chemical shifts in 1H NMR spectra are reported in
parts per million (ppm, d) downfield from the internal standard
Me4Si (TMS, d ¼ 0.00 ppm). Chemical shifts in 13C NMR spectra
are reported relative to the central line of the chloroform signal
(d ¼ 77.0 ppm). High-resolution mass spectra were obtained with
a Shimadzu LCMS-IT-TOF spectrometer. Infrared (IR) spectra were
recorded on a Bruker Tensor 37 spectrophotometer. Data are
represented as follows: frequency of absorption (cmꢀ1), intensity
of absorption (vs, very strong; s, strong; m, medium; w, weak).
3-Nitro-2H-chromenes (1a–i) were prepared according to the
reported procedure [13].
Synthesis of 6-chloro-3-nitrochroman (3b)
3b was prepared by a similar procedure starting from 6-chloro-
3-nitro-2H-chromene (1b). Yellow solid, yield 96%, mp 80.0–
81.28C; 1H NMR (400 MHz, CDCl3): d ¼ 7.13–7.11 (m, 2H), 6.81
(d, J ¼ 8.9 Hz, 1H), 4.95 (m, 1H), 4.71–4.39 (m, 2H), 3.43
(dd, J ¼ 17.2, 5.2 Hz, 2H); 13C NMR (100 MHz, CDCl3):
d ¼ 151.93, 129.05, 128.29, 126.74, 119.18, 118.42, 77.14,
65.96, 27.85; IR (KBr) n/cmꢀ1: 2997 (w), 2925 (m), 1547 (s),
1482 (s), 1240 (m), 1215 (s), 1021 (w), 925 (w), 818 (s), 638 (m);
Synthesis of 6-chloro-2H-chromene-3-carbonitrile (2a)
To a solution of 5-chloro-salicylaldehyde (0.783 g, 5 mmol) in
acetonitrile (5 mL) was added acrylonitrile (1.33 g, 25 mmol) and
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G.-Q. Xiao et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–4
HRMS (ESI) calcd for C9H7ClNO3 [MꢀH]ꢀ: 212.0114, found:
ꢀ
Growth inhibition assay of A549 cell line
212.0121.
A549 carcinoma human alveolar basal epithelial cell suspensions
were prepared and diluted to cell density about 10 000 cells/
well. Cells were added by pipette into 96-well microtiter plates.
The inoculates were allowed after a pre-incubation period of 24 h
at 378C with 5% CO2. All tested compounds were used as solution
in DMSO. The incubation of the cells with the test compounds
lasted for 72 h at 378C under 5% CO2 atmosphere and 100%
humidity. At the end of the incubation period, MTT stock
solution (20 mL, 2.5 mg/mL) was added to each well. The plate
was incubated for a further 4 h. The generated formazan was
dissolved by the addition of 100 mL/well of DMSO. The optical
density (OD) was measured at 490 nm with a BioTek PowerWave
XS2 microplate spectrophotometer. The IC50 values were calcu-
lated from the dose–response curves by Microsoft Excel.
Synthesis of N-(6-chlorochroman-3-yl)acetamide (4)
To a solution of 6-chloro-3-nitrochroman 3b (0.214 g, 1 mmol) in
acetic anhydride (5 mL) and acetic acid (5 mL) at 08C was added
activated zinc powder (0.523 g, 8 mmol). The reaction mixture
was stirred for 8 h at room temperature. The mixture was fil-
tered to remove excess zinc and the filtrate was concentrated
under vacuum. The residue was triturated with water to remove
zinc acetate. The resulted precipitate was purified by flash
chromatography (petroleum ether/EtOAc ¼ 3:1) to give 4 as a
white solid. Yield 62%, mp 117.0–118.58C; 1H NMR (400 MHz,
CDCl3): d ¼ 7.08 (dd, J ¼ 8.5, 2.5 Hz, 1H), 7.03 (s, 1H), 6.78
(d, J ¼ 8.7 Hz, 1H), 5.89 (s, 1H), 4.46 (m, 1H), 4.14 (dd, J ¼ 17.1,
6.6 Hz, 2H), 3.11–2.71 (m, 2H), 1.96 (s, 3H); 13C NMR (100 MHz,
CDCl3): d ¼ 169.95, 152.47, 129.96, 127.79, 125.96, 121.00,
118.20, 68.21, 41.98, 30.60, 23.28; IR (KBr) n/cmꢀ1: 3280 (m),
1480 (s), 1214 (m), 1021 (w), 914 (w), 815 (s), 628 (w); HRMS
(ESI) calcd for C11H11ClNO2ꢀ [MꢀH]ꢀ: 224.0478, found: 224.0476.
We thank the National Natural Science Foundation of China (Nos.
20972195, 21172270) and Guangdong Engineering Research Center of
Chiral Drugs for the financial support of this study.
The authors have declared no conflicts of interest.
Synthesis of 3-nitro-2H-chromen-2-one (5)
To a solution of salicylaldehyde (0.366 g, 3 mmol) in dry benzene
(30 mL) was added methyl nitroacetate (0.426 g, 3.6 mmol) and
piperidine (0.06 mL, 0.6 mmol). The reaction mixture was
refluxed overnight. The water generated in the reaction was
removed with a Dean-Stark apparatus. The mixture was then
cooled to 08C and the yellow precipitate was collected with a
sintered glass funnel. The precipitate was dissolved in DMF
(70 mL) and the solution was cooled to 08C. After ice-water
(60 mL) was added, the bright yellow precipitate was collected,
washed with cold water (20 mL ꢁ 2) and dried under vacuum.
Yield 83%, mp 142.5–144.08C; 1H NMR (400 MHz, CDCl3):
d ¼ 8.75 (s, 1H), 7.80 (ddd, J ¼ 8.6, 7.4, 1.6 Hz, 1H), 7.74 (dd,
J ¼ 8.1, 1.6 Hz, 1H), 7.48–7.45 (m, 2H); 13C NMR (100 MHz,
CDCl3): d ¼ 154.92, 151.97, 142.40, 136.24, 134.93, 130.74,
125.98, 117.18, 116.22; IR (KBr) n/cmꢀ1: 3057 (m), 2283 (w),
1756 (s), 1609 (ꢀm), 1521 (m), 1348 (s), 770 (s); HRMS (ESI) calcd
for C9H4NNaO4 [MþNa]ꢀ: 214.0116, found: 214.0112.
References
[1] D. Mustacich, G. Powis, Biochem. J. 2000, 346, 1–8.
[2] K. Becker, S. Gromer, R. H. Schirmer, S. Mu¨ller, Eur. J.
Biochem. 2000, 267, 6118–6125.
[3] S. Gromer, S. Urig, K. Becker, Med. Res. Rev. 2003, 24, 40–89.
[4] W. Cai, L. Zhang, Y. Song, B. Wang, B. Zhang, X. Cui, G. Hu,
Y. Liu, J. Wu, J. Fang, Free Radic. Bio. Med. 2012, 52, 257–265.
[5] H. H. Zeng, L. H. Wang, Med. Chem. 2010, 6, 286–297.
[6] J. G. Fang, J. Lu, A. Holmgren, J. Biol. Chem. 2005, 280, 25284–
25290.
[7] X. Qiu, Z. Liu, W. Y. Shao, X. Liu, D. P. Jing, Y. J. Yu, L. K. An,
S. L. Huang, X. Z. Bu, Z. S. Huang, L. Q. Gu, Bioorg. Med. Chem.
2008, 16, 8035–8041.
[8] Z. Liu, Z. Y. Du, Z. S. Huang, K. S. Lee, L. Q. Gu, Biosci.
Biotechnol. Biochem. 2008, 72, 2214–2218.
Biological evaluations
TrxR inhibition assay
[9] E. H. Chew, A. A. Nagle, Y. C. Zhang, S. Scarmagnani,
P. Palaniappan, T. D. Bradshaw, A. Holmgren, A. D. Westwell,
Free Radic. Biol. Med. 2010, 48, 98–111.
Commercially available rat liver TrxR (Sigma), DTNB (Sigma), and
NADPH (Roche) were used. Compound stocks (10 mM) were pre-
pared in dimethyl sulfoxide (DMSO). All assays were conducted in
a total volume of 40 mL in 384-well microplate. In each measure-
ment, the compound stock solution in the indicated volume was
added to potassium phosphate buffer (100 mM, pH ¼ 7.0) con-
taining recombinant rat TrxR (2 nM), NADPH (120 mM), and
EDTA (1.25 mM). The resulting mixture was incubated for
5 min at 378C. Then the enzyme reaction was initiated by the
addition of DTNB (3.2 mL, 63 mM in ethanol). The first 2 min
absorbance increase at 412 nm (De412 ¼ 13.6 mMꢀ1 cmꢀ1) was
recorded by a Tecan Infinite M200 PRO microplate reader. The
same volume of DMSO was used instead of the compound
solution as the blank control. The inhibitive ratio of TrxR
activity was obtained as a percentage of enzyme activity of the
control. The IC50 values were calculated from the concentration-
inhibitory ratio curves by Microsoft Excel.
[10] J. G. Fang, A. Holmgren, J. Am. Chem. Soc. 2006, 128, 1879–
1885.
[11] P. J. Moos, K. Edes, P. Cassidy, E. Massuda, F. A. Fitzpatrick,
J. Biol. Chem. 2003, 278, 745–750.
[12] G. X. Wang, N. X. Wang, T. Shi, J. L. Yu, X. L. Tang, Prog. Chem.
2008, 20, 518–524.
[13] M. Alneirabeyeh, R. Koussini, G. Guillaumet, Synth. Commun.
1990, 20, 783–788.
[14] H. G. C. Evin, L. A. Maldonado, J. Org. Chem. 2009, 74, 5097–
5099.
[15] V. Y. Korotaev, V. Y. Sosnovskikh, I. B. Kutyashev, A. Y.
Barkov, Y. V. Shklyaev, Tetrahedron Lett. 2008, 49, 5376–5379.
[16] N. Cenas, S. Prast, H. Nivinskas, J. Sarlauskas, E. S. J. Arner,
J. Biol. Chem. 2006, 281, 5593–5603.
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