Adenine derivatives invert high glucose-induced thioredoxin-interacting protein overexpression

Article abstract of DOI:10.1111/cbdd.13371

Overexpression of thioredoxin-interacting protein (TXNIP) is associated with reduced insulin sensitivity and β-cell apoptosis. We have previously shown that W2476 inhibited high glucose-induced TXNIP expression at both mRNA and protein levels in INS-1E ce

Full text of DOI:10.1111/cbdd.13371

DR QING LIU (Orcid ID : 0000-0003-1825-6760)
Article type
: Research Article
Adenine derivatives invert high glucose-induced thioredoxin-interacting
protein (TXNIP) overexpression
Li Zhong1,2,3,§, Qing Liu1,2, §, Yan Sie Ting 4, Vun Yien Thien 4, Nurwafa Syafiqah Binti
Kalong4, Dehua Yang1,2 and Ming-Wei Wang1,2,3,5*
1The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai 201203, China; 2The National Center for Drug
Screening, Shanghai 201203, China; 3University of Chinese Academy of Sciences, Beijing
100049, China; 4Faculty of Science and Natural Resources, Universiti Malaysia Sabah,
88400, Kota Kinabalu, Sabah, Malaysia; 5School of Pharmacy, Fudan University, Shanghai
201203, China
§These authors contributed equally to this work.
*To whom correspondence should be addressed: mwwang@simm.ac.cn
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13371
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ABSTRACT
Overexpression of thioredoxin-interacting protein (TXNIP) is associated with reduced insulin
sensitivity and β-cell apoptosis. We have previously shown that W2476 inhibited high
glucose-induced TXNIP expression at both mRNA and protein levels in INS-1E cells. In this
paper, we describe structural modification and optimization of W2476 leading to three more
active derivatives, 8d, 8g and 9h, capable of suppressing TXNIP expression in BG73 and
INS-1E cells, increasing insulin production and reducing high glucose-induced apoptosis in
INS-1E cells.
Keywords: Thioredoxin-interacting protein (TXNIP); adenine derivative; structural
optimization; thioredoxin binding protein-2 (TBP-2); vitamin D3-upregulated protein-1
(VDUP1).
1. Introduction
Thioredoxin-interacting protein (TXNIP), also known as thioredoxin binding protein-2
(TBP-2) or vitamin D3-upregulated protein-1 (VDUP1), is expressed ubiquitously.[1-3]
TXNIP down-regulates the activity of thioredoxin (TRX) redox-dependently and plays vital
roles in various cancers and metabolic disorders.[4-7] Several studies[8-10] showed that mRNA
and protein levels of TXNIP were substantially up-regulated at high concentrations of
glucose in multiple β-cell lines,[11] cultured islets of Langerhans,[11] retina,[12] sensorial
neurons,[13] mesangial cells[14] and podocytes. [15]
The promoter region on TXNIP contains a unique nonpalindromic 17-bp E-box repeat.[9]
The two E-boxes are highly conserved between different species and act as cis-acting
carbohydrate response element (ChoRE) to recruit carbohydrate response element-binding
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protein (ChREBP) and Max-like protein X (Mlx) thereby forming a protein complex
(ChREBP/Mlx), which activates glucose-induced txnip transcription. Another
glucose-responsive transcription factor, forkhead box O1 (FOXO1), competes with
ChREBP/Mlx and down-regulates TXNIP expression in pancreatic cells.[16]
TXNIP has been implicated in glucose homeostasis. The expression of TXNIP is
modulated by extracellular glucose levels, and this glucose-induced TXNIP expression
negatively regulates glucose uptake.[17] Overexpression of TXNIP was shown to be
associated with reduced insulin sensitivity[18] and β-cell apoptosis, involving apoptosis signal
regulating kinase 1 (ASK1) and the apoptotic signaling cascade.[18,19] This is coupled with an
increased expression of pro-apoptotic miR-200 and down-regulation of zinc finger
E-box-binding homeobox1 (Zeb1).[20] TXNIP is capable of up-regulating islet amyloid
polypeptide (IAPP), which aggregates into insoluble amyloid fibrils and promotes
inflammation and β-cell toxicity.[21-25] In addition, TXNIP inhibits glucose uptake by
regulating the expression and localization of glucose transporter (Glut1).[26] It thus suppresses
glycolysis through down-regulation of hypoxia-induced transcription factor HIF1α
expression,[27] activation of phosphatase and tensin homolog (PTEN) lipid phosphatase,[28]
and elevation of oxidative metabolism of pyruvate.[29] Islets from diabetic BTBR/ob mice
incubated at high glucose demonstrated increased TXNIP and apoptosis while the islets of
TXNIP-deficient HcB-19 mice were protected against glucose-induced apoptosis.[18, 30]
We have previously described a small molecule compound (W2476) that was able to
inhibit high glucose-induced TXNIP expression at mRNA and protein levels in INS-1E cells
and rat pancreatic islets.[31] Here we report our follow-up studies focusing on structural
modification and optimization of this compound and bioactivity evaluation of its analogues.
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2. Results and Discussion
2.1 Chemistry
Preliminary structure modification around W2476 was performed from three angles: ethoxy
linker, adenine and 4-acetylphenyl part (Figure 1). We first extended the length of ethoxy
linker to propyloxy and butyloxy. 4-Acetylphenol 1 was alkylated with 1,2-dibromoethane
2a, or 1,3-dibromopropane 2b, or 1,4-dibromobutane 2c to obtain 3a-c, respectively, which
further condensed with adenine in DMF to obtain W2476, 4a and 4b, respectively.
Compounds 4c-d were also prepared according to the same procedures of 4a-b with
2-chloroadenine instead. Next, modification around adenine covering three positions (2-, 8-
and N6-adenine) was completed with 2-halogen (5a-b), 2-methyl (5e), N6-furan-2-ylmethyl
(5d) substituted adenine, 6-dimethylaminopurine (5c), or 8-azaadenine (5f) reacting with
4-(2-bromoethoxy)acetophenone 3a to obtain compound 5. Modification around
4-acetylphenyl part was started from 2-bromoethoxybenzene derivatives 7a-h, which further
condensed with adenine, 2-fluoro/chloro-adenine, or 6-dimethylaminopurine to obtain 8-11.
2.2 Repressive effects on TXNIP in BG73 cells
Initial screening of the above compounds was conducted (5 μmol/L) in DMSO (1% final
concentration) with BG73 cells, which were stably transfected with a human TXNIP
proximal glucose response element connected to a luciferase reporter plasmid in rat
insulinoma (INS-1) cells. Expression of the luciferase reporter gene was fully stimulated by
glucose at 19.4 mmol/L. Dose-response characteristics study was performed at concentrations
of 200, 66.67, 22.22, 7.41, 2.47, 0.82, 0.27 and 0.09 μmol/L. The results (Table 1 and Figure
2) showed that the length of ethoxy linker in W2476 is necessary for its bioactivity with the
optimal length of two carbons (W2476 vs. 4a and 4b, 5b vs. 4c and 4d). Modification around
the adenine part covered three positions: 2-, 8- and N6-adenine. Introduction of
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electron-withdrawing group such as halogen (2-F or 2-Cl) to 2-adenine retained the inhibitory
activity against TXNIP expression, with 2-chlorine derivatives showing a better potency
(5a-b vs. W2476). Inversely, introduction of electron-donating group such as methyl
decreased its inhibition on TXNIP by 15% (5e vs. W2476). Substitution of nitrogen instead
of carbon at the 8-adenine position led to ~40% loss of inhibition (5f vs. W2476).
Monosubstitution on N6-NH2 of adenine did not affect the inhibition greatly (5d vs. W2476).
Conversely, disubstitution derivative exemplified by compound 5c exhibited about 95%
inhibition on TXNIP expression, which may be caused by its high toxicity in BG73 cells
(Figure S3). Subsequent modification on the 4-acetylphenyl part showed a great fluctuation
on TXNIP expression. The position of acetyl group was important with 2- or 4-acetyl
substituted derivatives exhibiting the greatest efficacy (W2476 and 8b vs. 8a). Also, with the
substitution of acetyl group at the 2-phenyl, additional substitution of electron-withdrawing
group, such as chlorine or bromine, on the 4-phenyl enhanced its inhibitory activity in
comparison to that of electron-donating group such as methyl (8b and 8d vs. 8e). Other
electron-withdrawing groups such as trifluoromethyl or 2,2,2-trifluoroacetyl substituted at the
4-phenyl instead of acetyl decreased (by ~60%) or kept the bioactivity (8f and 8h vs.
W2476). Similar phenomenon was observed for 9h in comparison with 8h that introduction
of chlorine to 2-adenine exhibited a better activity than adenine derivatives. Among all the
compounds modified on the 4-acetylphenyl part, 2-phenyl displayed more tolerance with
2-trifluoromethoxy and 2-acetyl derivatives showing equal effects on TXNIP expression (8g
vs. 8b). Finally, compounds 9-11b and 9-11d were prepared with an acetyl group at the
2-phenyl position and Cl/Br at the 4-phenyl position. Similar inhibition was observed that
Cl/F at the 2-adenine position (9-10b, 9-10d) could maintain or increase the activity slightly
except 6-dimethylaminopurine derivatives (11b and 11d).
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2.3 Inhibition of TXNIP expression and toxicity assessment in INS-1E cells
Compounds 8-10b, 8-10d, 8g and 9h were further investigated in INS-1E cells. INS-1E cells
were treated with 5 mmol/L or 25 mmol/L glucose for 24 h in the presence or absence of
W2476 or its analogues (20 μmol/L). Western blot data demonstrate that 25 mmol/L glucose
significantly increased TXNIP expression which was suppressed by W2476 and its analogues
(Figures 3, S1 and S2). Compared with W2476 (35.2% inhibition, P<0.01), compounds 8d,
8g and 9h displayed better inhibitory effects, i.e., 80.4% (P<0.0001), 64.9% (P<0.001) and
73.2% (P<0.0001), respectively after 24 h exposure at the concentration of 20 μmol/L.
Compounds 8d, 8g, 9h also suppressed high-glucose induced TXNIP overexpression at 6 h,
12 h and 48 h (Figure S4). No toxicity was noted for concentrations below 30 μmol/L, an
improvement from the parent compound W2476. [27] Compounds 8b, 9-10b and 9-10d
showed equal or less inhibition of TXNIP expression in INS-1E cells with higher toxicity
blow the concentration of 30 μmol/L compared with W2476.
2.4 Effects on insulin production and INS-1E cells apoptosis
We have previously found that W2476 could reverse the impact of high glucose on insulin
sensitivity via reduction of TXNIP expression. Here, we continued to investigate whether
compounds had better effects on insulin expression than W2476. Compounds 8d, 8g and 9h
with high inhibition of TXNIP expression and low toxicity in INS-1E cells were thus
selected. INS-1E cells were incubated with 5 mmol/L or 25 mmol/L glucose in the presence
or absence of W2476 and its derivatives for 24 h to measure insulin expression. The results
showed that high glucose significantly attenuated insulin protein levels accompanied by
up-regulated TXNIP (Figures 4A and 3A). In the high glucose groups, production of insulin
was increased by 1.6, 3.7 (P<0.01), 3.4 (P<0.001) and 3.5 times (P<0.001) for compounds
W2476, 8d, 8g and 9h, respectively (Figures 4A and 4B).
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TXNIP is critical for high glucose-induced β-cell apoptosis. Our previous work [31]
indicates that W2476 is able to inhibit TXNIP overexpression and reverse high
glucose-induced INS-1E cell apoptosis. Western blotting thus was performed by incubating
INS-1E cells at low (5 mmol/L) or high (33.3 mmol/L) glucose in the presence of W2476 or
its derivatives (20 μmol/L) for 48 h. All four compounds were capable of suppressing the
level of cleaved caspase-9 at high glucose (Figure 4C) with the effects of 8d and 8g being
more pronounced.
3. Materials and methods
3.1 Chemistry
General Statement. Reagents were commercial grades and used as received unless otherwise
noted. The structures of all new compounds were consistent with their 1H, 13C NMR and
mass spectra, and were judged to be ≥95% pure by HPLC. NMR spectra were recorded on
Varian Mercury 300, AVANCE III 500 spectrometers. Chemical shifts were reported in parts
per million (ppm), with the solvent resonance as the internal standard (CD3OD 3.31 ppm for
1H NMR, 49.15 ppm for 13C NMR; CDCl37.27 ppm for 1H NMR, 77.23 ppm for 13C NMR;
DMSO-d6 2.50 ppm for 1H NMR, 39.51 ppm for 13C NMR). Low resolution mass spectral
data (electrospray ionization) were acquired on a Finnigan LCQ-DECA mass spectrometer.
High resolution mass spectral data (TOF) were acquired on an Agilent 6224 mass
spectrometer. Samples were analyzed for purity on a HP1100 series equipped with a Zorbax
SB-C18 column (5 μm, 4.6 mm × 250 mm). Purities of final compounds were determined
using a 5 μL injection with quantitation by AUC at 210 and 254 nm.
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3.1.1 Procedure for compounds 3 and 7
The solution of 4'-hydroxyacetophenone (1, 0.037 mol, 1 eq) or phenol derivatives (6, 0.037
mol, 1 eq) in ethyl acetate (50 mL) was slowly added into the mixture of 1,2-, 1,3-, or
1,4-dibromoalkane (0.278 mol, 7.5 eq) and potassium carbonate (17.87 g, 0.129 mol, 3.5 eq)
and triethylbenzylammonium chloride (TEBA, catalytic amount) in ethyl acetate (50 mL).
The reaction was refluxed overnight. After cooling, water was added and the mixture was
extracted with ethyl acetate. The organic phase was combined, washed subsequently with 1 N
NaOH solution, water, and dried over anhydrous sodium sulphate. After filtration, the solvent
was removed in vacuo and the residue was separated on the Biotage SNAP cartridge KP-Sil
340 g column eluting with 0-30% ethyl acetate/petroleum ether. The product was obtained as
yellowish oil which solidified after cooling.
3a
Yield: 89.6%. The purity was 95% by HPLC analysis. LR-ESI: 243.0 (M+1), 265.0 (M+Na).
HRMS (TOF) m/z calcd for C10H12BrO2 [M+H]+ 243.0021, found 243.0028. 1H NMR
(CDCl3, 300 MHz) 2.57 (s, 3H), 3.67 (t, J = 6.3 Hz, 2H), 4.36 (t, J = 6.3 Hz, 2H), 6.95 (d, J =
8.4 Hz, 2H), 7.95 (d, J = 8.7 Hz, 2H). 13C NMR (CDCl3, 75 MHz) 26.5, 29.8, 68.0, 114.4
(3C), 130.8 (2C), 162.0, 196.8.
3b
Yield: 87.6%. The purity was 95% by HPLC analysis. LR-ESI: 257.1 (M+1), 279.1 (M+Na).
HRMS (TOF) m/z calcd for C11H14BrO2 [M+H]+ 257.0177, found 257.0179. 1H NMR
(CDCl3, 300 MHz) 2.34 (m, 2H), 2.58 (s, 3H), 3.60 (t, J = 6.3 Hz, 2H), 4.18 (t, J = 6.0 Hz,
2H), 6.94 (d, J = 9.0 Hz, 2H), 7.93 (d, J = 9.0 Hz, 2H). 13C NMR (CDCl3, 75 MHz) 26.6,
29.9, 32.3, 65.7, 114.4 (3C), 130.9 (2C), 162.8, 197.9.
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3c
Yield: 93.2%. The purity was 95% by HPLC analysis. LR-ESI: 271.1 (M+1), 293.1 (M+Na).
HRMS (TOF) m/z calcd for C12H16BrO2 [M+H]+ 271.0334, found 271.0336. 1H NMR
(CDCl3, 300 MHz) 2.059 (m, 4H), 2.56 (s, 3H), 3.49 (t, J = 6.3 Hz, 2H), 4.07 (t, J = 5.7 Hz,
2H), 6.92 (d, J = 9.0 Hz, 2H), 7.93 (d, J = 9.0 Hz, 2H). 13C NMR (CDCl3, 75 MHz) 26.6,
28.0, 29.6, 33.5, 67.4, 114.4 (3C), 130.8 (2C), 144.9, 195.8.
7a
Yield: 73.7%. The purity was 95% by HPLC analysis. LR-ESI: 243.0 (M+1), 265.0 (M+Na).
HRMS (TOF) m/z calcd for C10H12BrO2 [M+H]+ 243.0021, found 243.0019. 1H NMR
(CDCl3, 300 MHz) 2.56 (s, 3H), 3.64 (t, J = 6.0 Hz, 2H), 4.32 (t, J = 6.0 Hz, 2H), 7.11 (dd, J
= 2.1 Hz, J = 8.4 Hz , 1H), 7.36 (t, J = 8.1 Hz, 1H), 7.47 (d, J = 2.4 Hz, 1H), 7.55 (d, J = 7.8
Hz, 1H). 13C NMR (CDCl3, 75 MHz) 26.9, 29.2, 68.1, 113.4, 120.3, 121.9, 129.9, 138.7,
158.5, 197.9.
7b
Yield: 62.1%. The purity was 95% by HPLC analysis. LR-ESI: 277.0 (M+1), 299.0 (M+Na).
HRMS (TOF) m/z calcd for C10H11BrClO2 [M+H]+ 276.9631, found 276.9630. 1H NMR
(CDCl3, 300 MHz) 2.66 (s, 3H), 3.69 (t, J = 5.4 Hz, 2H), 4.37 (t, J = 6.0 Hz, 2H), 6.83 (d, J =
9.0 Hz, 1H), 7.37 (dd, J = 2.7 Hz, J = 8.7 Hz, 1H), 7.69 (d, J = 2.7 Hz, 1H). 13C NMR
(CDCl3, 125 MHz) 28.9, 32.4, 68.8, 113.9, 126.8, 129.6, 130.4, 133.3, 156.0, 198.2.
7c
Yield: 35.0%. The purity was 95% by HPLC analysis. LR-ESI: 286.0 (M+1), 308.0 (M+Na).
HRMS (TOF) m/z calcd for C11H13BrNO3 [M+H]+ 286.0079, found 286.0077. 1H NMR
(CDCl3, 300 MHz) 2.63 (s, 3H), 3.79 (t, J = 5.4 Hz, 2H), 4.54 (t, J = 5.4 Hz, 2H), 7.01 (d, J =
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8.7 Hz, 1H), 8.14 (d, J = 2.1 Hz, J = 8.7 Hz, 1H), 8.80 (d, J = 2.7 Hz, 1H). 13C NMR (CDCl3,
125 MHz) 26.8, 29.2, 68.8, 112.4, 121.2, 131.4, 133.4, 134.3, 159.7, 166.0, 196.8.
7d
Yield: 61.9%. The purity was 95% by HPLC analysis. LR-ESI: 320.9 (M+1), 342.9 (M+Na).
HRMS (TOF) m/z calcd for C10H11Br2O2 [M+H]+ 320.9126, found 320.9123. 1H NMR
(CDCl3, 300 MHz) 2.67 (s, 3H), 3.70 (t, J = 5.4 Hz, 2H), 4.39 (t, J = 5.7 Hz, 2H), 6.80 (d, J =
8.7 Hz, 1H), 7.53 (dd, J = 2.7 Hz, J = 9.0 Hz, 1H), 7.86 (d, J = 2.4 Hz, 1H). 13C NMR
(CDCl3, 125 MHz) 28.8, 32.4, 68.8, 114.1, 114.3, 130.1, 133.5, 136.3, 156.5, 198.2.
7e
Yield: 34.5%. The purity was 95% by HPLC analysis. LR-ESI: 257.0 (M+1), 279.0 (M+Na).
HRMS (TOF) m/z calcd for C11H14BrO2 [M+H]+ 257.0177, found 257.0179. 1H NMR
(CDCl3, 300 MHz) 2.30 (s, 3H), 2.67 (s, 3H), 3.69 (t, J = 5.7 Hz, 2H), 4.37 (t, J = 6.0 Hz,
2H), 6.80 (d, J = 8.4 Hz, 1H), 7.25 (d, J = 8.1 Hz, 1H), 7.55 (s, 1H). 13C NMR (CDCl3, 125
MHz) 14.3, 20.5, 29.2, 68.6, 112.5, 128.5, 131.0, 131.1, 134.3, 155.5, 200.1.
7f
Yield: 61.1%. The purity was 95% by HPLC analysis. LR-ESI: 269.0 (M+1), 291.0 (M+Na).
HRMS (TOF) m/z calcd for C9H9BrF3O [M+H]+ 268.9789, found 268.9791. 1H NMR
(CDCl3, 300 MHz) 3.64 (t, J = 7.2 Hz, 2H), 4.32 (t, J = 6.3 Hz, 2H), 6.98 (d, J = 8.7 Hz, 2H),
7.56 (d, J = 8.1 Hz, 2H). 13C NMR (CDCl3, 75 MHz) 28.8, 68.2, 114.9 (2C), 127.3 (4C),
160.8 (C-1).
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7g
Yield: 90.8%. The purity was 95% by HPLC analysis. LR-ESI: 285.0 (M+1), 307.0 (M+Na).
HRMS (TOF) m/z calcd for C9H9BrF3O2 [M+H]+ 284.9738, found 284.9739. 1H NMR
(CDCl3, 300 MHz) 3.65 (t, J = 6.3 Hz, 2H), 4.34 (t, J = 6.6 Hz, 2H), 7.02 (d, J = 7.8 Hz, 2H),
7.26 (t, J = 7.5 Hz, 2H). 13C NMR (CDCl3, 125 MHz) 28.5, 69.2, 115.1, 122.0, 123.6, 128.2,
138.8, 150.8 (2C).
7h
Yield: 76.8%. The purity was 95% by HPLC analysis. LR-ESI: 297.0 (M+1), 319.0 (M+Na).
HRMS (TOF) m/z calcd for C10H9BrF3O2 [M+H]+ 296.9738, found 296.9737. 1H NMR
(CDCl3, 300 MHz) 3.68 (t, J = 6.0 Hz, 2H), 4.40 (t, J = 6.3 Hz, 2H), 7.02 (d, J = 9.0 Hz, 2H),
8.07 (d, J = 7.2 Hz, 2H). 13C NMR (CDCl3, 75 MHz) 28.5, 68.3, 115.2 (3C), 117.3, 133.1
(2C), 164.0, 177.7.
3.1.2 Procedure for compounds W2476, 4-5 and 8-11
Potassium carbonate (0.27 g, 1.98 mmol, 2 eq) was added into the mixture of adenine
derivative (0.99 mmol, 1 eq) and 3 or 7, (1.23 mmol, 1.25 eq) in anhydrous DMF (30 mL).
Then the reaction was stirred at 80oC overnight. After cooling, the mixture was filtered and
the solution was evaporated in vacuo. The residue was separated on the Biotage SNAP
cartridge KP-Sil 100 g column eluting successively with 0-50% ethyl acetate/petroleum ether
and ethyl acetate to obtain the product as yellowish powder.
W2476
Yield: 79.3%. The purity was 95% by HPLC analysis. LR-ESI: 298.1 (M+1), 320.1 (M+Na).
HRMS (TOF) m/z calcd for C15H16N5O2 [M+H]+ 298.1304, found 298.1305. 1H NMR
(DMSO-d6) 2.50 (s, 3H), 4.47 (t, J = 5.1 Hz, 2H), 4.57 (t, J = 4.8 Hz, 2H), 7.03 (d, J = 8.4
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Hz, 2H), 7.20 (brs, 2H, NH2), 7.90 (d, J = 8.4 Hz, 2H), 8.16 (s, 1H), 8.19 (s, 1H). 13C NMR
(DMSO-d6), 26.4, 42.4, 66.0, 114.4 (2C), 118.6, 130.3, 130.5 (2C), 141.2, 149.6, 152.5,
156.0, 161.8, 196.3.
4a
Yield: 68.3%. The purity was 95% by HPLC analysis. LR-ESI: 312.1 (M+1), 334.1 (M+Na).
HRMS (TOF) m/z calcd for C16H18N5O2 [M+H]+ 312.1460, found 312.1463. 1H NMR
(DMSO-d6) 2.32 (m, 2H), 2.50 (s, 3H), 4.07 (t, J = 6.0 Hz, 2H), 4.33 (t, J = 6.6 Hz, 2H), 6.96
(d, J = 8.7 Hz, 2H), 7.17 (brs, NH2), 7.89 (d, J = 8.4 Hz, 2H), 8.11 (s, 1H), 8.12 (s, 1H). 13C
NMR (DMSO-d6) 26.5, 28.8, 40.4, 65.4, 114.3 (2C), 118.8, 130.0, 130.5 (2C), 141.0, 149.6,
152.4, 156.0, 162.3, 196.5.
4b
Yield: 81.2 %. The purity was 95% by HPLC analysis. LR-ESI: 326.2 (M+1), 348.2 (M+Na).
HRMS (TOF) m/z calcd for C17H20N5O2 [M+H]+ 326.1617, found 326.1615. 1H NMR
(DMSO-d6) 1.70 (m, 2H), 1.97 (m, 2H), 2.50 (s, 3H), 4.08 (t, J = 6.6 Hz, 2H), 4.21 (t, J = 6.9
Hz, 2H), 7.00 (d, J = 9.0 Hz, 2H), 7.16 (brs, NH2), 7.90 (d, J = 9.0 Hz, 2H), 8.13 (s, 1H), 8.16
(s, 1H). 13C NMR (DMSO-d6), 25.7, 26.1, 26.4, 42.6, 67.3, 114.3 (2C), 118.7, 129.8, 130.5
(2C), 140.8, 149.6, 152.4, 156.0, 162.4, 196.3.
4c
Yield: 88.9%. The purity was 95% by HPLC analysis. LR-ESI: 346.1 (M+1), 368.1 (M+Na).
HRMS (TOF) m/z calcd for C16H17ClN5O2 [M+H]+ 346.1071, found 346.1073. 1H NMR
(DMSO-d6, 300 MHz) 2.28 (t, J = 6.3 Hz, 2H), 2.50 (s, 3H), 4.08 (t, J = 5.7 Hz, 2H), 4.29 (t,
J = 6.9 Hz, 2H), 6.95 (d, J = 8.7 Hz, 2H), 7.72 (brs, NH2), 7.89 (d, J = 8.4 Hz, 2H), 8.14 (s,
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1H). 13C NMR (DMSO-d6, 125 MHz) 26.4, 28.6, 40.6, 65.3, 114.2 (2C), 117.9, 130.0, 130.4
(2C), 141.6, 150.7, 152.9, 156.7, 162.2, 196.3.
4d
Yield: 96.2%. The purity was 95% by HPLC analysis. LR-ESI: 360.1 (M+1), 382.1 (M+Na).
HRMS (TOF) m/z calcd for C17H19ClN5O2 [M+H]+ 360.1227, found 360.1225. 1H NMR
(DMSO-d6, 300 MHz) 1.70 (t, J = 6.6 Hz, 2H), 1.95 (t, J = 7.2 Hz, 2H), 2.50 (s, 3H), 4.08 (t,
J = 6.3 Hz, 2H), 4.17 (t, J = 6.9 Hz, 2H), 7.00 (d, J = 9.0 Hz, 2H), 7.73 (brs, NH2), 7.91 (d, J
= 8.4 Hz, 2H), 8.18 (s, 1H). 13C NMR (DMSO-d6, 125 MHz) 25.7, 26.0, 26.4, 42.9, 67.3,
114.3 (2C), 117.8, 129.8, 130.5 (2C), 141.5, 150.6, 153.0, 156.8, 162.4, 196.3.
5a
Yield: 95.0%. The purity was 95% by HPLC analysis. LR-ESI: 316.1 (M+1), 338.1 (M+Na).
HRMS (TOF) m/z calcd for C15H15FN5O2 [M+H]+ 316.1210, found 316.1208. 1H NMR
(DMSO-d6, 300 MHz) 2.50 (s, 3H), 4.45 (t, J = 3.6 Hz, 2H), 4.49 (t, J = 4.5 Hz, 2H), 7.02 (d,
J = 8.7 Hz, 2H), 7.76 (brs, NH2), 7.90 (d, J = 8.7 Hz, 2H), 8.16 (s, 1H). 13C NMR (DMSO-d6,
75 MHz) 26.4, 42.5, 65.9, 114.4 (2C), 117.0, 130.3, 130.5 (2C), 141.6, 150.8, 157.5, 157.7,
161.7, 196.3.
5b
Yield: 90.4%. The purity was 95% by HPLC analysis. LR-ESI: 332.1 (M+1), 354.1 (M+Na).
HRMS (TOF) m/z calcd for C15H15ClN5O2 [M+H]+ 332.0914, found 332.0913. 1H NMR
(DMSO-d6, 300 MHz) 2.50 (s, 3H), 4.44 (t, J = 4.5 Hz, 2H), 4.53 (t, J = 5.1 Hz, 2H), 7.03 (d,
J = 8.1 Hz, 2H), 7.76 (brs, NH2), 7.90 (d, J = 7.8 Hz, 2H), 8.20 (s, 1H). 13C NMR (DMSO-d6,
125 MHz) 26.4, 42.6, 66.0, 114.4 (2C), 117.7, 130.3, 130.5 (2C), 141.8, 150.7, 153.0, 156.8,
161.7, 196.3.
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5c
Yield: 71.4%. The purity was 95% by HPLC analysis. LR-ESI: 326.2 (M+1), 348.2 (M+Na).
HRMS (TOF) m/z calcd for C17H20N5O2 [M+H]+ 326.1617, found 326.1619. 1H NMR
(DMSO-d6, 300 MHz) 2.50 (s, 3H), 3.38 (s, 6H), 4.46 (t, J = 4.5 Hz, 2H), 4.59 (t, J = 4.5 Hz,
2H), 7.02 (d, J = 8.7 Hz, 2H), 7.89 (d, J = 8.7 Hz, 2H), 8.21 (s, 1H), 8.23 (s, 1H). 13C NMR
(DMSO-d6, 125 MHz) 26.4, 39.8 (2C), 42.4, 66.0, 114.4 (2C), 119.1, 130.3, 130.5 (2C),
140.1, 143.1, 151.9, 154.3, 161.8, 196.4.
5d
Yield: 88.0%. The purity was 95% by HPLC analysis. LR-ESI: 378.2 (M+1), 400.2 (M+Na).
HRMS (TOF) m/z calcd for C20H20N5O3 [M+H]+ 378.1566, found 378.1565. 1H NMR
(DMSO-d6, 300 MHz) 2.50 (s, 3H), 4.47 (t, J = 5.1 Hz, 2H), 4.59 (t, J = 4.8 Hz, 2H), 6.23 (d,
J = 2.7 Hz, 1H), 6.35 (t, J = 3.0 Hz, 1H), 7.03 (d, J = 8.4 Hz, 2H), 7.53 (m, 1H), 7.89 (d, J =
8.7 Hz, 2H), 8.23 (s, 1H), 8.25 (s, 1H). 13C NMR (DMSO-d6, 125 MHz) 26.4, 42.4 (2C),
66.0, 106.6, 110.4, 114.4 (2C), 119.1, 130.3, 130.5 (2C), 141.3 (2C), 141.8, 143.1, 152.4,
153.0, 161.8, 196.4.
5e
Yield: 80.0%. The purity was 97% by HPLC analysis. LR-ESI: 312.1 (M+1). HRMS (TOF)
m/z calcd for C16H18N5O2 [M+H]+ 312.1460, found 312.1459. 1H NMR (DMSO-d6, 300
MHz) 2.52 (s, 6H), 4.44 (t, J = 4.8 Hz, 2H), 4.64 (t, J = 5.1 Hz, 2H), 6.98 (d, J = 8.7 Hz, 2H),
7.92 (d, J = 8.7 Hz, 2H), 8.12 (s, 1H).
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5f
Yield: 78.0%. The purity was 95% by HPLC analysis. LR-ESI: 299.1 (M+1). HRMS (TOF)
m/z calcd for C14H15N6O2 [M+H]+ 299.1256, found 299.1255. 1H NMR (DMSO-d6, 300
MHz) 2.51 (s, 3H), 4.66 (t, J = 5.1 Hz, 2H), 4.95 (t, J = 4.8 Hz, 2H), 6.98 (d, J = 8.1 Hz, 2H),
7.88 (d, J = 8.4 Hz, 2H), 8.31 (s, 1H).
8a
Yield: 84.2%. The purity was 95% by HPLC analysis. LR-ESI: 298.1 (M+1), 320.1 (M+Na).
HRMS (TOF) m/z calcd for C15H16N5O2 [M+H]+ 298.1304, found 298.1305. 1H NMR
(DMSO-d6, 300 MHz) 2.50 (s, 3H), 4.40 (t, J = 4.8 Hz, 2H), 4.52 (t, J = 5.1 Hz, 2H), 7.15
(brs, 2H, NH2), 7.37 (m, 3H), 7.49 (d, J = 8.1 Hz, 1H), 8.11 (s, 1H), 8.15 (s, 1H). 13C NMR
(DMSO-d6, 75 MHz), 26.9, 42.5, 66.0, 113.5, 118.6, 119.6, 121.1, 130.0, 138.2, 141.2, 149.6,
152.5, 156.0, 158.1, 197.7.
8b
Yield: 94.4%. The purity was 95% by HPLC analysis. LR-ESI: 332.1 (M+1), 354.1 (M+Na).
HRMS (TOF) m/z calcd for C15H15ClN5O2 [M+H]+ 332.0914, found 332.0916. 1H NMR
(DMSO-d6, 300 MHz) 2.25 (s, 3H), 4.57 (t, J = 4.2 Hz, 2H), 4.60 (t, J = 4.2 Hz, 2H), 7.22
(brs, 3H), 7.46 (d, J = 1.5 Hz, 1H), 7.53 (dd, J = 1.5 Hz, J = 8.7 Hz, 1H), 8.13 (s, 1H), 8.20
(s, 1H). 13C NMR (DMSO-d6, 125 MHz) 31.1, 42.4, 66.8, 115.3, 118.7, 124.8, 128.7, 129.3,
133.0, 141.0, 149.6, 152.5, 155.6, 156.0, 197.6.
8c
Yield: 94.4%. The purity was 95% by HPLC analysis. LR-ESI: 341.1 (M+1), 363.1 (M+Na).
HRMS (TOF) m/z calcd for C16H17N6O3 [M+H]+ 341.1362, found 341.1359. 1H NMR
(DMSO-d6, 300 MHz) 2.51 (s, 3H), 4.54 (t, J = 4.8 Hz, 2H), 4.65 (t, J = 4.5 Hz, 2H), 7.20
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(brs, NH2), 7.72 (d, J = 8.4 Hz, 1H), 7.99 (dd, J = 2.1 Hz, J = 8.4 Hz, 1H), 8.15 (s, 1H), 8.16
(s, 1H), 8.31 (d, J = 2.4 Hz, 1H). 13C NMR (DMSO-d6, 125 MHz) 26.4, 42.2, 67.9, 112.7,
118.5, 123.1, 129.8, 131.3, 132.6, 141.0, 149.6, 152.7, 156.0, 159.3, 165.4, 196.1.
8d
Yield: 94.1%. The purity was 95% by HPLC analysis. LR-ESI: 376.0 (M+1), 398.0 (M+Na).
HRMS (TOF) m/z calcd for C15H15BrN5O2 [M+H]+ 376.0409, found 376.0410. 1H NMR
(DMSO-d6, 300 MHz) 2.24 (s, 3H), 4.57 (d, J = 4.2 Hz, 2H), 4.61 (d, J = 4.2 Hz, 2H), 7.17
(d, J = 9.3 Hz, 1H), 7.21 (brs, 2H, NH2), 7.58 (d, J = 3.0 Hz, 1H), 7.66 (dd, J = 2.4 Hz, J =
8.7 Hz, 1H), 8.13 (s, 1H), 8.20 (s, 1H). 13C NMR (DMSO-d6, 125 MHz) 31.2, 42.4, 66.8,
112.4, 115.8, 118.7, 129.8, 131.6, 135.9, 141.0, 149.6, 152.5, 156.0, 156.1, 197.6.
8e
Yield: 37.8%. The purity was 95% by HPLC analysis. LR-ESI: 312.1 (M+1), 334.1 (M+Na).
HRMS (TOF) m/z calcd for C16H18N5O2 [M+H]+ 312.1460, found 312.1457. 1H NMR
(DMSO-d6, 300 MHz) 2.23 (s, 6H), 4.51 (t, J = 5.1 Hz, 2H), 4.60 (t, J = 5.1 Hz, 2H), 7.05 (d,
J = 9.0 Hz, 1H), 7.20 (brs, NH2), 7.31 (d, J = 7.2 Hz, 2H), 8.14 (s, 1H), 8.21 (s, 1H). 13C
NMR (CDCl3, 125 MHz) 19.8, 31.3, 42.6, 66.3, 113.2, 118.7, 127.7, 129.7, 134.1, 141.1,
149.6, 152.5, 155.0, 156.0, 198.9.
8f
Yield: 44.4%. The purity was 95% by HPLC analysis. LR-ESI: 324.1 (M+1), 346.1 (M+Na).
HRMS (TOF) m/z calcd for C14H13F3N5O [M+H]+ 324.1072, found 324.1070. 1H NMR
(DMSO-d6, 300 MHz) 4.46 (t, J = 4.8 Hz, 2H), 4.57 (t, J = 5.1 Hz, 2H), 7.10 (d, J = 8.4 Hz,
2H), 7.21(brs, NH2), 7.61 (d, J = 8.7 Hz, 2H), 8.16 (s, 1H), 8.19 (s, 1H). 13C NMR
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(DMSO-d6, 75 MHz) 42.3, 66.1, 115.0 (2C), 118.6, 121.7, 123.4, 127.0 (2C), 141.2, 149.6,
152.5, 156.0, 160.8.
8g
Yield: 95.8%. The purity was 95% by HPLC analysis. LR-ESI: 340.1 (M+1), 362.1 (M+Na).
HRMS (TOF) m/z calcd for C14H13F3N5O2 [M+H]+ 340.1021, found 340.1022. 1H NMR
(DMSO-d6, 300 MHz) 4.44 (t, J = 5.4 Hz, 2H), 4.57 (t, J = 5.4 Hz, 2H), 7.00 (t, J = 7.5 Hz,
1H), 7.18 (brs, NH2), 7.23 (d, J = 8.4 Hz, 1H), 7.31 (m, 2H), 8.07 (s, 1H), 8.13 (s, 1H). 13C
NMR (DMSO-d6, 125 MHz) 42.4, 66.6, 114.6, 118.6, 121.5, 123.0, 128.8, 136.9, 141.0,
149.5, 150.2, 150.3, 152.4, 155.9.
8h
Yield: 77.8%. The purity was 95% by HPLC analysis. LR-ESI: 352.1 (M+1), 374.1 (M+Na).
HRMS (TOF) m/z calcd for C15H13F3N5O2 [M+H]+ 352.1021, found 352.1022. 1H NMR
(DMSO-d6, 300 MHz) 4.56 (m, 4H), 6.93 (d, J = 8.7 Hz, 2H), 7.20 (brs, NH2), 7.47 (d, J =
8.7 Hz, 2H), 7.99 (d, J = 8.1 Hz, 2H), 8.15 (s, 1H), 8.18 (s, 1H). 13C NMR (DMSO-d6, 75
MHz) 42.8, 65.8, 92.5, 113.6 (2C), 118.6, 128.8 (3C), 141.2, 149.6, 152.5, 156.0, 158.5,
164.1.
9b
Yield: 80.7%. The purity was 95% by HPLC analysis. LR-ESI: 366.0 (M+1), 388.0 (M+Na).
HRMS (TOF) m/z calcd for C15H14Cl2N5O2 [M+H]+ 366.0525, found 366.0524. 1H NMR
(DMSO-d6, 500 MHz) 2.29 (s, 3H), 4.52 (t, J = 4.5 Hz, 2H), 4.58 (t, J = 4.5 Hz, 2H), 7.22 (d,
J = 9.0 Hz, 1H), 7.47 (d, J = 3.0 Hz, 1H), 7.54 (dd, J = 9.0 Hz, J = 3.0 Hz, 1H), 7.74 (brs,
NH2), 8.22 (s, 1H). 13C NMR (DMSO-d6, 125 MHz) 31.1, 42.6, 66.9, 115.4, 117.7, 124.9,
128.7, 129.4, 133.0, 141.6, 150.7, 155.6, 156.8, 197.7.
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9d
Yield: 88.9%. The purity was 95% by HPLC analysis. LR-ESI: 410.0 (M+1), 432.0 (M+Na).
HRMS (TOF) m/z calcd for C15H14BrClN5O2 [M+H]+ 410.0019, found 410.0021. 1H NMR
(DMSO-d6, 500 MHz) 2.28 (s, 3H), 4.52 (t, J = 5.0 Hz, 2H), 4.58 (t, J = 4.5 Hz, 2H), 7.17 (d,
J = 9.0 Hz, 1H), 7.59 (d, J = 2.5 Hz, 1H), 7.66 (dd, J = 2.5 Hz, J = 9.0 Hz, 1H), 7.74 (brs, 2H,
NH2), 8.22 (s, 1H). 13C NMR (DMSO-d6, 125 MHz) 31.1, 42.5, 66.8, 112.4, 115.8, 117.7,
129.8, 131.6, 135.8, 141.6, 150.7, 156.0, 156.7, 197.6.
9f
Yield: 72.6%. The purity was 95% by HPLC analysis. LR-ESI: 358.1 (M+1), 380.1 (M+Na).
HRMS (TOF) m/z calcd for C14H12ClF3N5O [M+H]+ 358.0682, found 358.0680. 1H NMR
(DMSO-d6, 300 MHz) 4.43 (t, J = 4.8 Hz, 2H), 4.53 (t, J = 4.5 Hz, 2H), 7.11 (d, J = 8.1 Hz,
2H), 7.62 (d, J = 8.7 Hz, 2H), 7.75 (brs, NH2), 8.20 (s, 1H). 13C NMR (DMSO-d6, 125 MHz)
42.6, 66.1, 115.2 (2C), 117.7, 123.5, 125.6, 127.1 (2C), 141.9, 150.7, 153.1, 156.8, 160.8.
9h
Yield: 82.5%. The purity was 95% by HPLC analysis. LR-ESI: 386.1 (M+1), 408.1 (M+Na).
HRMS (TOF) m/z calcd for C15H12ClF3N5O2 [M+H]+ 386.0632, found 386.0633. 1H NMR
(DMSO-d6, 300 MHz) 4.53 (m, 4H), 7.18 (d, J = 8.7 Hz, 2H), 7.75 (brs, NH2), 7.99 (d, J =
8.7 Hz, 2H), 8.21 (s, 1H). 13C NMR (DMSO-d6, 125 MHz) 42.7, 65.7, 113.7 (2C), 117.7,
122.2, 124.7, 128.8 (2C), 141.8, 150.7 153.0, 156.8, 158.4, 164.1.
10b
Yield: 60.2%. The purity was 95% by HPLC analysis. LR-ESI: 350.1 (M+1), 372.1 (M+Na).
HRMS (TOF) m/z calcd for C15H14ClFN5O2 [M+H]+ 350.0820, found 350.0822. 1H NMR
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(DMSO-d6, 300 MHz) 2.28 (s, 3H), 4.54 (brs, 4H), 7.22 (d, J = 8.7 Hz, 1H), 7.48 (s, 1H),
7.55 (d, J = 9.0 Hz, 1H), 7.78 (brs, NH2), 8.18 (s, 1H).
10d
Yield: 45.8%. The purity was 95% by HPLC analysis. LR-ESI: 394.0 (M+1), 416.0 (M+Na).
HRMS (TOF) m/z calcd for C15H14BrFN5O2 [M+H]+ 394.0315, found 394.0313. 1H NMR
(DMSO-d6, 300 MHz) 2.27 (s, 3H), 4.54 (brs, 4H), 7.17 (d, J = 9.0 Hz, 1H), 7.59 (s, 1H),
7.66 (d, J = 8.7 Hz, 1H), 7.77 (brs, NH2), 8.18 (s, 1H).
10h
Yield: 90.0%. The purity was 95% by HPLC analysis. LR-ESI: 370.1 (M+1), 392.1 (M+Na).
HRMS (TOF) m/z calcd for C15H12F4N5O2 [M+H]+ 370.0927, found 370.0926. 1H NMR
(DMSO-d6, 300 MHz) 4.52 (brs, 4H), 7.20 (d, J = 8.1 Hz, 2H), 7.75 (brs, 2H, NH2), 7.99 (d,
J = 8.1 Hz, 2H), 8.17 (s, 1H). 13C NMR (DMSO-d6, 125 MHz) 42.7, 65.7, 113.7 (2C), 117.0,
122.2, 124.7, 128.8 (2C), 141.6, 150.9, 157.5, 158.5, 164.1, 178.0.
11b
Yield: 98.0%. The purity was 95% by HPLC analysis. LR-ESI: 360.1 (M+1), 382.1 (M+Na).
HRMS (TOF) m/z calcd for C17H19ClN5O2 [M+H]+ 360.1227, found 360.1228. 1H NMR
(DMSO-d6, 500 MHz) 2.28 (s, 3H), 3.32 (s, 6H), 4.56 (t, J = 5.0 Hz, 2H), 4.63 (t, J = 5.0 Hz,
2H), 7.22 (d, J = 9.0 Hz, 1H), 7.47 (d, J = 2.5 Hz, 1H), 7.54 (dd, J = 2.5 Hz, J = 8.5 Hz, 1H),
8.21 (s, 1H), 8.22 (s, 1H). 13C NMR (DMSO-d6, 125 MHz) 31.2, 39.8 (2C), 42.4, 66.8, 115.3,
119.2, 124.8, 128.7, 129.3, 132.9, 139.8, 150.4, 151.8, 154.2, 155.6, 197.6.
11d
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Yield: 98.0%. The purity was 95% by HPLC analysis. LR-ESI: 404.1 (M+1), 426.1 (M+Na).
HRMS (TOF) m/z calcd for C17H19BrN5O2 [M+H]+ 404.0722, found 404.0721. 1H NMR
(DMSO-d6, 500 MHz) 2.28 (s, 3H), 3.32 (s, 6H), 4.55 (t, J = 5.0 Hz, 2H), 4.63 (t, J = 5.0 Hz,
2H), 7.17 (d, J = 9.0 Hz, 1H), 7.58 (d, J = 3.0 Hz, 1H), 7.65 (dd, J = 2.5 Hz, J = 9.0 Hz, 1H),
8.21 (s, 1H), 8.22 (s, 1H). 13C NMR (DMSO-d6, 125 MHz) 31.2, 39.5 (2C), 42.4, 66.7, 112.4,
115.8, 119.2, 129.7, 131.6, 135.8, 139.8, 150.4, 151.8, 154.2, 156.1, 197.6.
3.2 Repressive effects on TXNIP expression and toxicity assessment in BG73 cells
The screening assay was constructed according to the previous paper. [31] Expression of the
luciferase reporter gene was fully stimulated by the glucose at the concentration of 19.4
mmol/L in BG73 cells. This assay system showed coefficient of variation (CV) values of 5.94
% for high glucose (HG, 19.4 mmol/L) and 7.12 % for basal glucose (BG, 3.3 mmol/L), a
signal-to-background ratio of 6.91 and a Z’ factor of 0.72. BG73 cells were treated with HG
in the presence or absence of compounds for 24 h. Dose-response characteristics study was
performed at concentrations of 200, 66.67, 22.22, 7.41, 2.47, 0.82, 0.27, 0.09 μmol/L for
W2476 and its analogues (1% DMSO final concentration).BG73 cells were treated with BG
without compounds for 24 h. The inhibition ratio was calculated by A/B which was defined
as: A = average value of HG without compound - average value of HG with compound, B =
average value of HG without compound - average value of BG without compound. The
concentration for inhibition screening was 5 μmol/L in DMSO (1% DMSO final
concentration). We also investigated cytotoxicity in BG73 cells following 24 h treatment with
W2476 and its analogues at concentrations of 8.78, 13.17, 19.75, 29.63, 44.44, 66.67 and
100.00 μmol/L and the absorbance at 450 nm was recorded.
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3.3 Inhibition of TXNIP expression and toxicity assessment in INS-1E cells
INS-1E cells were treated with 5 mmol/L or 25 mmol/L glucose in the presence or absence of
W2476 and its analogues at the concentration of 20 μmol/L for 24 h (0.5% DMSO final
concentration). Western blot was preformed to analyze TXNIP expression. In addition,
cytotoxicity of the compounds in INS-1E cells was evaluated at concentrations of 1, 5, 10,
20, 25, 50, 60 and 70 μmol/L for 24 h (0.5% DMSO final concentration) and the absorbance
at 450 nm was recorded.
3.4 Effects on insulin production and INS-1E cells apoptosis
INS-1E cells were incubated with 5 or 25 mmol/L glucose in the presence or absence of
W2476 and its derivatives for 24 h (0.5% DMSO final concentration) followed by Western
blotting to analyze insulin expression. The compound effect on high glucose-induced
apoptosis was also investigated by incubating INS-1E cells at LG (5 mmol/L) or HG (33.3
mmol/L) for 48 h, respectively and Western blot was applied to measure cleaved caspase-9
expression.
3.5 Statistical analysis
Data presented are means ± SEM of three independent experiments performed in triplicate.
Statistical analysis was conducted using Graphpad Prism® software version 6.0 (San Diego,
CA).
4. Conclusion
Derivatives of W2476 (8d, 8g and 9h) showed a better bioactivity spectrum in terms of
regulation of TXNIP and insulin expression, and reverse of high glucose-induced apoptosis in
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INS-1E cells. These results suggest that compounds 8d, 8g and 9h may have the potential for
pharmaceutical development against metabolic disorders.
ACKNOWLEDGEMENTS
We gratefully acknowledge the financial support from the “Personalized Medicines ‒
Molecular Signature-based Drug Discovery and Development Strategic Priority Research
Program” of Chinese Academy of Sciences grants (XDA12020347 to M.-W.W.;
XDA12040308 to D.Y.) and the Thousand Talents Program in China.
CONFLICT OF INTEREST
The authors have declared no conflict of interest.
Legends
Scheme 1. Synthesis of compounds 3-11.
(i) K2CO3/TEBA in ethyl acetate, reflux; (ii) K2CO3/adenine or 2-chloroadenine in DMF,
reflux; (iii) K2CO3/2-fluoro or 2-chloroadenine or 6-(dimethylamino)purine or
N-(furan-2-ylmethyl)-9H-purin-6-amine or 2-methyladenine or 8-azaadenine in DMF, reflux;
(iv) K2CO3/adenine in DMF, reflux; (v) K2CO3/2-chloroadenine in DMF, reflux; (vi)
K2CO3/2-fluoroadenine in DMF, reflux; and (vii) K2CO3/6-(dimethylamino)purine in DMF,
reflux.
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Figure 1. Structure-activity relationship studies around W2476.
Figure 2. Repressive effects of compounds 8d, 8g, 9h and W2476 on TXNIP in BG73
cells.
Dose-response characteristics were studied at concentrations of 200, 66.67, 22.22, 7.41, 2.47,
0.82, 0.27 and 0.09 μmol/L. Each value represents mean ± SEM of three independent
experiments performed in triplicate.
Figure 3. Inhibition of TXNIP expression by W2476 and its analogues in INS-1E cells.
(A) Western blot analysis of TXNIP expression and (B) TXNIP/GAPDH ratio after treatment
with W2476 or its derivatives at the concentration of 20 μmol/L. Each bar represents mean ±
SEM of three independent experiments performed in triplicate. (C) Cytotoxicity of W2476
and its analogues at concentrations of 1, 5, 10, 20, 25, 50, 60 and 70 μmol/L.
Figure 4. Effects of W2476 and its analogues on insulin production and INS-1E cells
apoptosis.
Western blot was performed by incubating INS-1E cells in 25 or 5 mmol/L glucose in the
presence of W2476 or its derivatives at the concentration of 20 μmol/L for 24 h or 48 h to
analyze insulin (A and B) and cleaved caspase-9 (C; high glucose at 33.3 mmol/L)
expression. Each bar represents mean ± SEM of three independent experiments performed in
triplicate.
Figure S1. Inhibition of TXNIP expression by W2476 and its analogues in INS-1E cells.
(A)/(C) Western blot analysis of TXNIP expression and (B)/(D) TXNIP/GAPDH ratio after
treatment with W2476 and its derivatives at the concentration of 20 μmol/L. Each bar
represents mean ± SEM of three independent experiments performed in triplicate. (E)
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Cytotoxicity of W2476 and its analogues at concentrations of 5, 10, 20, 25, 50, 60 and 70
μmol/L in INS-1E cells.
Figure S2. Cytotoxicity of W2476 and its analogues in INS-1E cells at concentrations of
5, 10, 20, 25, 50, 60 and 70 μmol/L.
Figure S3. Cytotoxicity of W2476 and its analogues in BG73 cells at concentrations of
8.78, 13.17, 19.75, 29.63, 44.44, 66.67 and 100.00 μmol/L.
Figure S4. Western blot analysis of the effects of 8d, 8g and 9h on TXNIP expression in
INS-1E cells.
INS-1E cells were treated with 5 mmol/L or 25 mmol/L glucose in the presence or absence of
8d, 8g and 9h at the concentration of 20 μmol/L for 6 h, 12 h, 24 h and 48 h.
Table 1. Repressive effects of W2476 and its analogues on TXNIP in BG73 cells.
aInhibition of TXNIP expression by W2467 and its analogues was assessed at the concentration of 5
μmol/L. Each value represents mean ± SEM of three independent experiments performed in triplicate.
bIC50 value represents mean ± SEM of three independent experiments performed in triplicate.
cN.D., not determined. IC50 values of compounds with <55% inhibition at the concentration of 5 μmol/L
could not be established.
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Scheme 1.
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Figure 1.
Figure 2.
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Figure 3.
Figure 4.
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