Synthesis and biological evaluation of novel N-aryl-ω-(benzoazol-2-yl)-sulfanylalkanamides as dual inhibitors of α-glucosidase and protein tyrosine phosphatase 1B

Article abstract of DOI:10.1111/cbdd.13331

α-Glucosidase is known to catalyze the digestion of carbohydrates and release free glucose into the digestive tract. Protein tyrosine phosphatase 1B (PTP1B) is engaged in the dephosphorylation of the insulin receptor and regulation of insulin sensitivity.

Full text of DOI:10.1111/cbdd.13331

DR XIAN-CHAO CHENG (Orcid ID : 0000-0001-6420-1045)
DR RUN-LING WANG (Orcid ID : 0000-0001-7914-3060)
Article type
: Research Article
Synthesis and Biological Evaluation of Novel
N-Aryl--(Benzoazol-2-yl)-Sulfanylalkanamides as Dual Inhibitors of
α-Glucosidase and Protein Tyrosine Phosphatase 1B
Mei-Yan Wanga, §, Xian-Chao Chengb, §, Xiu-Bo Chenb, c, Yu Lib, Lan-Lan Zangb, Yu-Qing Duanb, Ming-Zhu
Chend, Peng Yud, Hua Sund, * and Run-Ling Wangb, *
aTianjin Key Laboratory of Food and Biotechnology, School of Biotechnology and Food Science, Tianjin
University of Commerce, Tianjin 300134, China
bTianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics
(Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin 300070, China
cTianjin Medical University Eye Hospital, Tianjin 300384, China
dCollege of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China
Running Title: dual α-glucosidase/PTP1B inhibitory activities of
N-aryl--(benzoazol-2-yl)-sulfanylalkanamides.
Keywords: (azole-2-yl)-sulfanylalkanamides; α-glucosidase; PTP1B; dual inhibitor.
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lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13331
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*Correspondence should be addressed to:
Run-Ling Wang, Ph.D.
Professor of Pharmacology
School of Pharmacy, Tianjin Medical University
No. 22 Qixiangtai Rd., Heping Dist.
Tianjin, 300070, China
Tel: +86-22-8333-6690
Fax: +86-22-8333-6690
Email: wangrunling@tjmu.edu.cn
Hua Sun, Ph.D.
Professor of Biotechnology
College of Biotechnology, Tianjin University of Science and Technology
No. 29 13th. Aveue, Tianjin Economic and Technological Development Area (TEDA)
Tianjin, 300457, China
E-mail: sunhua@tust.edu.cn
§ These authors contributed equally to this work.
Abstract
α-Glucosidase is known to catalyze the digestion of carbohydrates and release free glucose
into the digestive tract. Protein tyrosine phosphatase 1B (PTP1B) is engaged in the
dephosphorylation of the insulin receptor and regulation of insulin sensitivity. Therefore, dual
antagonists by targeting both α-glucosidase and PTP1B may be potential candidates for type
2 diabetes therapy. In this work, three series of novel
N-aryl--(benzoazol-2-yl)-sulfanylalkanamides were synthesized and assayed for their
α-glucosidase and PTP1B inhibitory activities, respectively. Compound 3l, exhibiting the
most effective α-glucosidase inhibitory activity (IC50 = 10.96 μM (3l), IC50 = 51.32 μM
(Acarbose), IC50 = 18.22 μM (Ursolic acid)) and potent PTP1B inhibitory activity (IC50 =
13.46 μM (3l), IC50 = 14.50 μM (Ursolic acid)), was identified as a novel dual inhibitor of
α-glucosidase and PTP1B. Furthermore, 3l is a highly selective PTP1B inhibitor since no
inhibition was showed by 3l at 100 μM against PTP-MEG2, TCPTP, SHP2, or SHP1.
Subsequent kinetic analysis revealed 3l inhibited α-glucosidase in a reversible and mixed
manner. Molecular docking study indicated that hydrogen bonds, van der Waals, charge
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interactions and Pi-cation interactions all contributed to interactions between 3l and
α-glucosidase/PTP1B.
1. Introduction
Type 2 Diabetes Mellitus (T2DM) is closely associated with a diverse range of vascular
complications as a result of insulin resistance of the human organism.1 As of today, over 300
million people are suffering from T2DM and its incidence rate is still on the rise globally.2,3
α-Glucosidase (EC 3.2.1.20), a carbohydrate-hydrolase commonly found in small
intestine, can specifically hydrolyze 1, 4-α-glycosidic bond and release α-D-glucose into the
blood stream during food digestion.4 The proper reduction of digestion and absorption of
glucose by inhibiting α-glucosidase may be an effective strategy to treat T2DM,
hyperlipidemia and obesity.5,6 Therefore, numerous α-glucosidase inhibitors were explored in
hope of finding more excellent anti-diabetic drugs,7-9 in which acarbose, voglibose and
miglitol have been used as clinical drugs for the therapy of T2DM.10,11
PTP1B plays as a primary negative regulator in the insulin signaling pathway by
dephosphorylating insulin receptor as well as its substrate, and thus is closely related to the
onset and development of T2DM.12-14 Related studies disclosed PTP1B knockout mice
displayed a significant improvement in insulin sensitivity and resistance to obesity,15 which
indicated that PTP1B might be a promising target for T2DM treatment.
For some diseases with complicated nosogenesis, like psychiatric disorders, cancer and
T2DM, the modulation of a single drug target might be therapeutically insufficient.16,17
Herein, Besnard et al.18 proposed the concept of “multi-target therapeutics”, which means a
drug can be simultaneously engaged in two or more targets for better therapeutic efficacy.17,19
Given the key roles that α-glucosidase and PTP1B play in blood glucose control, we hope to
design an inhibitor that target both α-glucosidase and PTP1B to achieve better glycemic
control to T2DM patients. The dual α-glucosidase/PTP1B inhibitor can suppress postprandial
hyperglycemia on one hand by inhibiting α-glucosidase, and on the other hand, it can
increase insulin sensitivity and decrease insulin resistance by inhibiting PTP1B. Thus, the
dual α-glucosidase/PTP1B inhibitor could be a valuable therapeutic strategy for not only
T2DM patients, but also individuals with impaired glucose tolerance. Despite some plant
extracts were found to show α-glucosidase and PTP1B inhibitory activities,20-22 synthesizing
small molecules as dual α-glucosidase/PTP1B inhibitors to treat T2DM has been few
explored23,24. Moreover, there is no report about
N-aryl--(benzoazol-2-yl)-sulfanylalkanamides as dual inhibitors of α-glucosidase and
PTP1B.
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Benzoxazole/benzothiazole scaffold is a privileged structure which possess
anti-tumor,25 anti-HIV,26 anti-inflammatory,27 antimicrobial,26 antitubercular,28 and
anti-diabetic29 activities. As the representative of benzoxazole derivatives, calcimycin and
prinaberel30 have entered preclinical or clinical trials. Interestingly,
benzoxazole/benzothiazole scaffold was disclosed in 2013 as potential PTP1B inhibitors by
two studies.31 Inspired by these researches, a series of (azole-2-yl)-sulfanylalkanamides were
designed and synthesized as potential dual anti-diabetic candidates by targeting both
α-glucosidase and PTP1B. Considering structural diversity, benzothiazole and thiazoline
were chosen to replace the benzoxazol nucleus with the anticipation to further improve the
bioactivities of these α-glucosidase/PTP1B antagonists. In this paper, we wish to report the
synthesis and dual α-glucosidase/PTP1B inhibitory activities of three series of novel
compounds including 2-mercaptobenzoxazole, 2-mercaptobenzothiazole and
2-mercaptothiazoline derivatives. The docking studies on α-glucosidase- and
PTP1B-inhibitor complexes and kinetic analysis of α-glucosidase-3l were further employed
to investigate protein-drug interaction details.
2. Methods and materials
2.1 Chemistry
All the reagents and solvents were purchased from commercial suppliers and used
without further purification unless otherwise indicated. The processes of reactions were
monitored by thin layer chromatography (TLC) on silica gel pre-coated F254 Merck plates
with observation under UV. The column chromatography was performed on 200-300 mesh
silica gel. 1H NMR spectra and 13C NMR spectra were taken on Bruker Avance NMR
Spectrometer with CDCl3 or DMSO-d6 as the solvent. All chemical shifts (δ H and δ C) were
reported in parts per million (ppm) and the coupling constants were measured in hertz (Hz).
The mass spectrometry analyses were performed with an Agilent 6310 Ion Trap mass
spectrometer (Agilent, Santa Clara, CA, USA) for the ESI-MS spectra. High-resolution mass
spectra were recorded on a Bruker MicrOTOF-Q II ESI-Qq-TOF spectrometer (Bruker
Daltonics, Karlsruhe, Germany).
2.1.1 General procedure for the synthesis of compounds 2a-2g
The saturated potassium carbonate aqueous solution (55 mmol) was added to a well
stirred solution of 1a-1d (50 mmol) in acetone (or DMF). The solution was cooled to 0 °C,
and then chloroacetyl chloride (or 4-chlorobutyryl chloride) (55 mmol) was added dropwise.
The mixture was slowly warmed to rt and stirred until the TLC analysis showed reaction
endpoint. The mixture was poured into ice-water and extracted by ethyl acetate. The organic
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layer was combined, washed by saturated brine, dried over anhydrous Na2SO4, filtered, and
concentrated to give compound 2a (white solid, 9.0 g, 82%), 2b (white solid, 3.8 g, 40%), 2c
(pale yellow solid, 7.9 g, 94%), 2d (pale grey solid, 7.9 g, 64%), 2e (white solid, 3.6 g, 34%),
and 2f (pale yellow solid, 6.6 g, 67%). The product 2g was included in the tan filtrate and was
used directly for the next reaction.
2.1.2 General procedure for the synthesis of compounds 3a-3l and 4a-4c
A mixture of benzo[d]oxazole-2-thiol (or benzo[d]thiazole-2-thiol, or
4,5-dihydrothiazole-2-thiol) (5 mmol), 2a-2g (5 mmol; The yield of 2g was calculated by
100%), K2CO3 (5.5 mmol), and KI (0.5 mmol) in 10 mL DMF was stirred at rt for 3-12 h.
After the reaction was complete detected by TLC analysis, the mixture was extracted by ethyl
acetate. The combined organic layers were washed with saturated brine, dried over anhydrous
Na2SO4, filtered, and concentrated under vacuum. The residue was purified by silica gel
column chromatography (petroleum ether: ethyl acetate = 7:1 - 4:1) to give the target
compounds 3a-3l and 4a-4c.
2-(benzo[d]oxazol-2-ylthio)-N-(naphthalen-1-yl)acetamide (3a)32: pale yellow solid, 55%
yield; 1H NMR (400 MHz, CDCl3) δ 10.12 (br s, 1H), 8.21 (d, J = 7.2 Hz, 1H), 8.09 (d, J =
8.4 Hz, 1H), 7.86 (d, J = 7.6 Hz, 1H), 7.75 (d, J = 7.6 Hz, 1H), 7.68 (d, J = 8.4 Hz, 1H),
7.57-7.31 (m, 6H), 4.20 (s, 2H) (Fig.S1); 13C NMR (101 MHz, CDCl3) δ 152.4, 140.9, 134.0,
132.8, 128.8, 126.3, 126.1, 125.9, 125.3, 124.9, 124.8, 120.8, 119.2, 118.2, 110.5, 36.3
(Fig.S2); ESI-MS (m/z): 357.7 [M+Na]+.
2-(benzo[d]oxazol-2-ylthio)-N-(4-fluorophenyl)acetamide (3b): pale grey solid, 31% yield;
1H NMR (400 MHz, CDCl3) δ 9.88 (br s, 1H), 7.70 (d, J = 7.6 Hz, 1H), 7.58-7.45 (m, 3H),
7.42-7.33 (m, 2H), 7.01 (t, J = 8.4 Hz, 2H), 4.02 (s, 2H) (Fig.S3); 13C NMR (101 MHz,
CDCl3) δ 166.0, 165.7, 159.4 152.3, 140.8, 134.0, 125.0, 124.8, 121.3, 118.1, 115.7, 110.5,
36.0; ESI-MS (m/z): 325.1 [M+Na]+ (Fig.S4); HRMS-ESI (m/z): 325.0409 [M+Na]+
[calculated for C15H11FN2O2S : 325.0417].
2-(benzo[d]thiazol-2-ylthio)-N-(naphthalen-1-yl)acetamide (3c)32: white solid, 72% yield; 1H
NMR (400 MHz, CDCl3) δ 10.05 (br s, 1H), 8.14 (d, J = 8.0 Hz, 1H), 8.00 (d, J = 8.0 Hz,
1H), 7.95 (d, J = 8.0 Hz, 1H), 7.87-7.80 (m, 2H), 7.65 (d, J = 8.0 Hz, 1H), 7.52-7.30 (m, 5H),
4.26 (s, 2H) (Fig.S5); 13C NMR (101 MHz, CDCl3) δ 166.2, 166.1, 152.6, 134.9, 133.7
133.2, 128.1, 127.8, 126.4, 126.1, 125.9, 125.6, 125.6, 124.6, 122.7, 121.0, 37.3 (Fig.S6);
ESI-MS (m/z): 373.3 [M+Na]+.
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2-(benzo[d]thiazol-2-ylthio)-N-(4-fluorophenyl)acetamide (3d)33: pale yellow solid, 47%
yield; 1H NMR (400 MHz, CDCl3) δ 10.08 (br s, 1H, NH), 7.91 (d, J = 8.0 Hz, 1H), 7.78 (dd,
J = 8.0, 0.4 Hz, 1H), 7.52-7.40 (m, 3H), 7.39-7.32 (m, 1H), 6.99-6.91 (m, 2H), 4.02 (s, 2H)
(Fig.S7); 13C NMR (101 MHz, CDCl3) δ 167.6, 166.5, 159.3, 152.0, 135.5, 134.2, 126.8,
125.2, 121.5, 121.1, 121.0, 115.7, 37.2 (Fig.S8); ESI-MS (m/z): 341.7 [M+Na]+.
2-(benzo[d]thiazol-2-ylthio)-N-phenylacetamide (3e)32: yellow solid, 25% yield; 1H NMR
(400 MHz, CDCl3) δ 10.07 (br s, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H),
7.54-7.50 (m, 3H), 7.39 (t, J = 7.7 Hz, 1H), 7.31-7.27 (m, 2H), 7.08 (t, J = 7.4 Hz, 1H), 4.07
(s, 2H) (Fig.S9); 13C NMR (101 MHz, CDCl3) δ 167.6, 166.6, 152.0, 138.2, 135.5, 129.0,
126.7, 125.1, 124.3, 121.5, 121.1, 119.5, 37.3 (Fig.S10); ESI-MS (m/z): 323.1 [M+Na]+.
4-(benzo[d]oxazol-2-ylthio)-N-(naphthalen-1-yl)butanamide (3f): white solid, 40% yield; 1H
NMR (400 MHz, CDCl3) δ 8.38 (br s, 1H), 7.96-7.84 (m, 3H), 7.74 (d, J = 8.0 Hz, 1H),
7.51-7.48 (m, 3H), 7.42 (d, J = 7.2 Hz,1H), 7.29-7.17 (m, 3H), 3.52 (t, J = 6.8 Hz, 2H), 2.77
(t, J = 6.8 Hz, 2H), 2.53-2.34 (m, 2H) (Fig.S11); 13C NMR (101 MHz, CDCl3) δ 170.9,
164.3, 151.2, 141.3, 133.7, 133.6, 128.1, 127.8, 125.9, 125.7, 125.5, 125.1, 124.5, 124.2,
122.7, 121.7, 118.2, 110.1, 34.4, 31.5, 25.1 (Fig.S12); ESI-MS (m/z): 385.5 [M+Na]+;
HRMS-ESI (m/z): 385.0962 [M+Na]+ [calculated for C21H18N2O2S : 385.0981].
4-(benzo[d]oxazol-2-ylthio)-N-(4-fluorophenyl)butanamide (3g): grey solid, 24% yield; 1H
NMR (400 MHz, CDCl3) δ 8.21 (br s, 1H), 7.55-7.48 (m, 3H), 7.46-7.43 (m, 1H), 7.32-7.21
(m, 2H), 7.07-6.98 (m, 2H), 3.43 (t, J = 6.8 Hz, 2H), 2.59 (t, J = 6.8 Hz, 2H), 2.35-2.80 (m,
2H) (Fig.S13); 13C NMR (101 MHz, CDCl3) δ 170.2, 165.4, 159.5, 151.87, 141.5, 134.0,
124.5, 124.2, 122.1, 118.2, 115.6, 110.1, 35.4, 31.3, 25.6 (Fig.S14); ESI-MS (m/z): 353.3
[M+Na]+; HRMS-ESI (m/z): 353.0726 [M+Na]+ [calculated for C17H15FN2O2S : 353.0730].
4-(benzo[d]oxazol-2-ylthio)-N-phenylbutanamide (3h): grey solid, 24% yield; 1H NMR (400
MHz, CDCl3) δ 8.10 (br s, 1H), 7.55-7.42 (m, 3H), 7.40-7.35 (m, 1H), 7.29-7.24 (m, 1H),
7.24-7.15 (m, 3H), 7.06 (t, J = 7.2 Hz, 1H), 3.37 (t, J = 6.8 Hz, 2H), 2.53 (t, J = 6.8 Hz, 2H),
2.29-2.21 (m, 2H) (Fig.S15); 13C NMR (101 MHz, CDCl3) δ 170.2, 165.3, 151.9, 141.5,
138.0, 129.0, 124.5, 124.4, 124.1, 120.2, 118.3, 110.0, 35.6, 31.3, 25.6 (Fig.S16); ESI-MS
(m/z): 335.3 [M+Na]+; HRMS-ESI (m/z): 335.0833 [M+Na]+ [calculated for C17H16N2O2S :
335.0824].
4-(benzo[d]thiazol-2-ylthio)-N-(naphthalen-1-yl)butanamide (3i)34: white solid, 26% yield;
1H NMR (400 MHz, CDCl3) δ 8.21 (br s, 1H), 7.88-7.86 (m, 3H), 7.74-7.72 (m, 2H), 7.59 (d,
J = 8.0 Hz, 1H), 7.54-7.38 (m, 3H), 7.36-7.21 (m, 2H), 3.58 (t, J = 6.8 Hz, 2H), 2.75 (t, J =
6.8 Hz, 2H), 2.36 (m, 2H) (Fig.S17); 13C NMR (101 MHz, CDCl3) δ 171.1, 167.1, 153.0,
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135.2, 134.2, 132.4, 128.6, 127.8, 126.3, 126.2, 126.1, 126.0 125.6, 124.4, 121.9, 121.4,
121.3, 121.0, 35.4, 32.8, 25.6 (Fig.S18); ESI-MS (m/z): 401.3 [M+Na]+.
4-(benzo[d]thiazol-2-ylthio)-N-(4-fluorophenyl)butanamide (3j): white solid, 53% yield; 1H
NMR (400 MHz, CDCl3) δ 7.94 (br s, 1H), 7.76-7.73 (m, 2H), 7.49-7.46 (m, 2H), 7.40-7.36
(m, 1H), 7.32-7.27 (m, 1H), 7.03-6.98 (m, 2H), 3.49 (t, J = 6.8 Hz, 2H), 2.58 (t, J = 6.8 Hz,
2H), 2.32-2.25 (m, 2H) (Fig.S19); 13C NMR (101 MHz, CDCl3) δ 170.2, 167.3 159.4, 152.7,
135.1, 133.8, 126.2, 124.5, 122.1, 121.3, 121.1, 115.6, 35.5, 32.6, 25.6 (Fig.S20); ESI-MS
(m/z): 369.4 [M+Na]+; HRMS-ESI (m/z): 369.0467 [M+Na]+ [calculated for C17H15FN2OS2 :
369.0502].
4-(benzo[d]thiazol-2-ylthio)-N-phenylbutanamide (3k): white solid, 25% yield; 1H NMR
(400 MHz, CDCl3) δ 7.92-7.63 (m, 3H), 7.52 (d, 2H), 7.40-7.27 (m, 3H), 7.12 (t, J =7.6 Hz,
1H), 3.50 (t, J = 6.8 Hz, 2H), 2.59 (t, J = 6.8 Hz, 2H), 2.33-2.27 (m, 2H) (Fig.S21); 13C NMR
(101 MHz, CDCl3) δ 170.3, 167.2, 152.9, 137.8, 135.2, 129.0, 126.2, 124.4, 124.4, 121.4,
121.0, 120.2, 35.7, 32.7, 30.9, 25.6 (Fig.S22); ESI-MS (m/z): 351.3 [M+Na]+; HRMS-ESI
(m/z): 351.0606 [M+Na]+ [calculated for C17H16N2OS2 : 351.0596].
4-(4-(benzo[d]thiazol-2-ylthio)butanamido)benzenesulfonic acid (3l): grey solid, 35% yield;
1H NMR (400 MHz, CDCl3) δ 10.04 (br s, 1H), 8.01 (d, J = 8.0 Hz, 1H),7.83 (d, J = 8.0 Hz,
1H), 7.59-7.49 (m, 4H), 7.46 (t, J = 8.0 Hz, 1H), 7.36 (t, J = 7.6 Hz, 1H), 3.43 (t, J = 7.2 Hz,
2H), 2.58-2.42 (m, 2H), 2.14-2.06 (m, 2H) (Fig.S23); 13C NMR (101 MHz, CDCl3) δ 170.4,
166.6, 152.7, 134.5, 126.3, 126.1, 124.4, 121.7, 121.1, 118.1, 34.9, 32.5, 24.7 (Fig.S24);
ESI-MS (m/z): 407.2 [M-H]-; HRMS-ESI (m/z): 407.0182 [M-H]- [calculated for
C17H16N2O4S3 : 407.0188].
2-((4,5-dihydrothiazol-2-yl)thio)-N-(naphthalen-1-yl)acetamide (4a)35: white solid, 54%
yield; 1H NMR (400 MHz, CDCl3) δ 10.10 (br s, 1H), 8.13 (d, J = 7.6 Hz, 1H),7.95 (d, J =
7.6 Hz, 1H), 7.90-7.84 (m, 1H), 7.67 (d, J = 8.4 Hz, 1H),7.57-7.45 (m, 3H), 4.33 (t, J = 8.0
Hz, 2H), 3.96 (s, 2H), 3.53 (t, J = 8.0 Hz, 2H) (Fig.S25); 13C NMR (101 MHz, CDCl3) δ
169.3 167.6, 134.1, 133.0, 128.8, 126.6, 126.0, 125.9, 125.1, 120.9, 119.4, 63.3, 37.0, 36.6
(Fig.S26); ESI-MS (m/z): 325.2 [M+Na]+.
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4-((4,5-dihydrothiazol-2-yl)thio)-N-(naphthalen-1-yl)butanamide (4b): white solid, 28%
yield; 1H NMR (400 MHz, CDCl3) δ 8.38 (br s, 1H), 7.96-7.84 (m, 3H), 7.74 (d, J = 8.0 Hz,
1H), 7.51-7.48 (m, 3H), 7.42 (d, J = 7.2 Hz, 1H), 7.29-7.17 (m, 3H), 3.52 (t, J = 6.8 Hz, 2H),
2.77 (t, J = 6.8 Hz, 2H), 2.53-2.34 (m, 2H) (Fig.S27); 13C NMR (101 MHz, CDCl3) δ 171.3,
167.1, 134.2, 132.7, 128.6, 127.9, 126.1, 126.0, 126.0, 125.7, 121.9, 121.5 63.9, 35.5, 35.4,
32.0, 26.0 (Fig.S28); ESI-MS (m/z): 331.3 [M+H]+; HRMS-ESI (m/z): 353.0727 [M+Na]+
[calculated for C17H18N2OS2 : 353.0753].
4-((4,5-dihydrothiazol-2-yl)thio)-N-(4-fluorophenyl)butanamide (4c): white solid, 33% yield;
1H NMR (400 MHz, CDCl3) δ 8.59 (br s, 1H), 7.50 (dd, J = 8.8, 4.8 Hz, 2H), 7.02 (t, J = 8.4
Hz, 2H), 4.23 (t, J = 8.0 Hz, 2H), 3.43 (t, J = 8.0 Hz, 2H), 3.22 (t, J = 6.8 Hz, 2H), 2.50 (t, J
= 8.0 Hz, 2H), 2.22-2.10 (m, 2H) (Fig.S29); 13C NMR (101 MHz, CDCl3) δ 170.6, 159.3,
134.2, 122.0, 115.5, 63.9, 35.48, 35.44, 31.8, 26.0 (Fig.S30); ESI-MS (m/z): 321.9
[M+Na]+; HRMS-ESI (m/z): 321.0478 [M+Na]+ [calculated for C13H15FN2OS2 : 321.0502].
2.2 Biological evaluation
2.2.1 α-Glucosidase inhibition assay
The α-glucosidase inhibition assay was conducted according to the literature protocol.36
The α-glucosidase (EC 3.2.1.20) from baker’s yeast (Sigma, G5003) and pNPG (Sigma,
N1377) were dissolved in 0.05 M potassium phosphate buffer with pH 6.8. The enzymatic
reaction mixture composed of α-glucosidase buffer (20 μL, 0.04 U/mL), substrate (30 μL, 0.5
mM), test compound (10 μL) and potassium phosphate buffer (0.1 M, 140 μL) was incubated
at 37 °C for 30 min. The enzymatic activity of α-glucosidase was detected by
spectrophotometer at the wavelength of 405 nm.
2.2.2 PTP1B, PTP-MEG2, TCPTP, SHP2, and SHP1 inhibition assays
The enzymatic assay of PTP1B, PTP-MEG2, TCPTP, SHP2, and SHP1 was performed
based on the literature procedure.37-39 The regents used in this step are as follows: buffer,
consisting of 50 mM citrate (pH 6.0), 0.1 M NaCl, 1 mM EDTA and 1 mM dithiothreitol
(DTT); substrate, 2 mM pNPP in buffer; stopping solution, 0.2 M sodium hydroxide. The
human recombinant GST-PTP1B40 (or PTP-MEG234, TCPTP39, SHP241, or SHP141) and
tested compounds with multifarious concentrations mixed in 50 μL buffer were added to each
well of a 96-well plate. PTP1B or other PTPs was replaced by an equivalent volume of buffer
in blank. After pre-incubation for 15 min at rt, 50 μL of substrate was added and incubated at
37 °C for 30 min, and then 10 μL of stopping buffer was poured into each well on ice. The
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enzymatic activity of PTPs was detected by spectrophotometer at the wavelength of 405 nm
against blank.
2.2.3 Kinetics of α-glucosidase inhibition
Compound 3l (0, 5, 10 and 20 μM) was added to α-glucosidase buffer (20 μL, 0.04
U/mL) at 37 °C and incubated for 30 min. The substrate pNGP at different concentrations
(0.15, 0.3, 0.6, and 1.2 mM) was then added to initiate the enzyme reaction. The inhibitory
kinetics of compound 3l on α-glucosidase was analyzed using the Lineweaver-Burk plot of
the substrate concentration and velocity.
2.2.4 Dialysis experiment of α-glucosidase
The dialysis experiment was conducted as previously literature with small
modifications.42 Briefly, using α-glucosidase alone (400 μL, 0.01 U/mL) or α-glucosidase
(400 μL, 0.01 U/mL) treated with 3l (20 μL, 20 μM) were dialyzed in phosphate buffer (0.05
M, pH 6.8) with dialysis bags (MD 34-3.5-5, Solarbio, Beijing, Solarbio Science &
Technology Co., Ltd., China) at 4 °C for 24 h. After dialysis, one of the α-glucosidase alone
group was treated with 3l for 30 min. The contents in the dialysis bags were determined for
residual enzyme activity as described above.
2.3 Molecular docking
Molecular docking analysis was performed via the module of “flexible docking”
embedded in Accelrys Discovery Studio package 3.5 (http://accelrys.com/). Isomaltase (EC
3.2.1.10, oligo-1,6-glucosidase) from baker’s yeast was found to be the most suitable
template since it shares 71% identity and 84% similarity with the target α-glucosidase.43 The
crystal structures of isomaltase from baker’s yeast (PDB code: 3A4A) and PTP1B (PDB
code: 2QBQ) were downloaded from the Protein Data Bank (PDB).44 The docking results had
been visualized using Discovery Studio visualizer 3.5.
3. Results and discussion
3.1 Chemistry
Synthesis of the 15 designed compounds was accomplished using known synthetic
approaches.23,31 Synthetic routes of all the 15 target compounds were shown in Scheme 1.
Using aromatic primary amines and acyl chlorides as the starting materials, the nucleophilic
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substitution reaction was employed to afford intermediates 2a, 2b and 2d-2f in 34-82% yield.
Subsequent nucleophilic substitution with benzo[d]oxazole-2-thiol using K2CO3/KI as base in
DMF resulted in the target compounds 3a, 3b and 3f-3h. Benzothiazole- and
thiazolidine-2-thiol derivatives were synthesized to diversify the molecular nucleus.
Corresponding target compounds 4a-4c, 3c-3e and 3i-3l were obtained in a similar manner to
compounds 3a, 3b and 3f-3h.
3.2 Biological evaluation
3.2.1 Enzymatic assays
The dual α-glucosidase/PTP1B in vitro inhibitory activities and PTPs selectivity of all
the 15 newly synthesized compounds were summarized in Table 1 and Table 2. Acarbose
was selected as the positive control for α-glucosidase inhibition. Considering ursolic acid was
reported not only as PTP1B inhibitor45, but also as α-glucosidase inhibitor46, we used ursolic
acid as the positive control of dual inhibition against α-glucosidase/PTP1B. Apparently,
compounds 3c, 3f, 3i, 3j and 3l exhibited more effective α-glucosidase inhibition than
acarbose, among of which 3l (IC50 = 10.96 μM) was 5-fold stronger than that of the positive
control acarbose. To PTP1B inhibitory activity, compound 3c (IC50 = 5.28 μM) was the most
potent molecule while 3l (IC50 = 13.46 μM) was ranked third, which were both stronger than
the positive control ursolic acid (IC50 = 14.50 μM). According to the results of selectivity
experiments of PTPs, we found that compound 3l showed no inhibition at 100 μM against
mammalian PTP-MEG2, TCPTP, SHP2, or SHP1, which indicated that 3l achieved more
than 7.7-fold selectivity over above four PTPs. The IC50 of 3e and 3f for PTP1B were 18.31
μM and 15.17 μM, respectively, which were almost 6-fold stronger than that of other four
PTPs. Compound 3c, the most potent PTP1B inhibitor in this work, exhibited more than
18-fold selectivity over TCPTP, SHP2 and SHP1. In addition, 3c displayed an IC50 of 12.87
μM for PTP-MEG2, 2.4-fold lower than PTP1B in potency.
As an obvious result of Table 1 and Table 2, the inhibitory activities of
2-mercaptothiazoline derivatives (4a-4c) for both targets α-glucosidase and PTP1B were
significantly weaker than the other two series derivatives (2-mercaptobenzoxazole and
2-mercaptobenzothiazole derivatives), which indicated that the benzoazole scaffold was more
beneficial to α-glucosidase/PTP1B dual inhibition than thiazolines. Moreover, benzothiazole
derivatives (3c, 3d, 3i and 3j) generally achieved stronger α-glucosidase inhibition than
corresponding benzoxazole derivatives (3a, 3b, 3f and 3g).
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In the synthesis process of 15 target molecules, acyl chlorides provided the spacer
connecting aromatic primary amines and azole-2-thiols, and the resulting aliphatic chain
formed between amides nitrogen and sulfur atom was regarded as a linker in this study. As
displayed in Table 1, compounds 3f, 3g, 3i and 3j with a 4-carbon linker were all more active
to α-glucosidase than their 2-carbon linker counterparts 3a, 3b, 3c and 3d, which suggested
that appropriately extending the linker was favorable to α-glucosidase inhibition.
It is interesting to point out that the introduction of electron-withdrawing substituents
such as naphth-1-yl (3c, 3f and 3i) and p-fluorophenyl (3d, 3g and 3j) improved the
inhibitory activity towards α-glucosidase compared to phenyl (3e, 3h and 3k). The influence
order of different aromatic substituents on α-glucosidase inhibition was naphth-1-yl >
p-fluorophenyl > Ph, and the bioactivities rank of corresponding compounds were 3c > 3d >
3e, 3f > 3g > 3h, and 3i > 3j > 3k. It is noteworthy that compound 3l, bearing strong
electron-withdrawing substituent 4-SO3H in aniline ring, showed the most potent
α-glucosidase inhibitory activity. The results above demonstrated the pattern substitution in
the aniline ring was closely related to the bioactivity of this kind of α-glucosidase inhibitors.
3.2.2 Kinetics of α-glucosidase inhibition
In order to obtain enzyme inhibition properties of the representative compound 3l, a
kinetic analysis on the α-glucosidase inhibition was performed through Lineweaver-Burk plot
analysis, using 4 concentrations of 3l (0, 5, 10 and 20 M), as illustrated in Figure 1.
Analysis on these data shown that Vmax (the y-intercept) decreased with changing 1/Km (the
x-intercept) in the presence of increasing concentrations of inhibitor 3l, which revealed that 3l
inhibited α-glucosidase in a mixed type.
3.2.3 Dialysis experiment
We further investigated α-glucosidase inhibition properties of 3l using the dialysis
experiment. The results were presented in Figure 2. After dialysis, the activity of
α-glucosidase preincubated with 3l reverted to 98%, similar to that of the untreated
α-glucosidase, which indicated that the enzymatic activity was recovered after dialysis. In
contrast, α-glucosidase incubated with 3l but without further dialysis showed relatively low
activity (17%). These results demonstrated that the inhibition of α-glucosidase by 3l was
reversible.
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3.3 Docking study
Flexible Docking (Discovery Studio 2016) was employed to investigate the
pharmacophore mapping between inhibitors and α-glucosidase/PTP1B, and results are
depicted in Figure 3 and Figure 4, respectively. Figures 3A, 4A and 4B respectively
represent the surfaces of complex α-glucosidase-acarbose/3l, complex PTP1B-4B3/3l and
complex PTP1B-3l/3d/4c. The drug-protein interactions of α-glucosidase-acarbose,
α-glucosidase-3l, PTP1B-3l, PTP1B-3d, and PTP1B-4c are illustrated in Figures 3B, 3C, 4C,
4D and 4E, respectively. H-bond interactions between the inhibitor and protein backbone or
amino acid side chains are respectively represented by green and blue dashed arrow to the
electron donor. A pink dashed arrow with heads on both sides marks the charge interactions,
while an orange line represents Pi interactions. Pink plates describe or show the residues
involved in H-bonding, charge or polar interactions, while green plates mark residues that are
involved in van der Waals interactions.
As depicted in Figure 3A, the 4-sulfophenyl group of 3l entered the deep and narrow
active pocket, whereas benzothiazole ring of 3l was positioned at the entrance of the pocket,
which made 3l spirally matching well with the steric conformation of acarbose. However, the
thiazoline ring of compounds 4a-4c was too small to occupy the entire space of the long and
narrow active pocket, which might be an explanation why 2-mercaptothiazoline derivatives
exhibited weak α-glucosidase inhibitory potency. Detailed analysis in Figure 3C showed that
3l formed six H-bonds with key residues His112, Gln182, Glu277 and Arg442, respectively.
In addition, charge and polar interactions were observed between 3l and the binding site of
α-glucosidase. Importantly, 3l drew intense van der Waals interactions with key residues of
binding site. All above interactions helped 3l achieve high α-glucosidase inhibitory potency.
However, despite the existence of strong H-bonding interactions, there were significantly less
van der Waals interactions observed between acarbose and α-glucosidase (Figure 3B), which
should be responsible for its lower α-glucosidase inhibition than 3l. Furthermore, the
calculated docking score of 3l (39.47 kcal/mol) was significantly higher than score of
acarbose (31.84 kcal/mol), which was consistent with the experimental result (Table 1).
As illustrated in Figure 4A, compound 3l was found to anchor in the binding pocket of
PTP1B in a similar conformation with its co-crystallized ligand 4B3, which made our
docking study in this paper more conceivable. Based on the former crystal structure,
compounds 3l, 3d and 4c were docked into the binding pocket to study the binding modes
between inhibitors with different bioactivities and their receptor PTP1B. As previously
reported, the binding pocket of PTP1B contains the catalytic site and an allosteric site, both
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of which are two important regions involved in PTP1B inhibition47. The catalytic site,
designated site A as well, is constructed by key residues Lys120, Asp181, Phe182, Cys215,
Ser216, Gly220, and Arg221. On the other hand, an allosteric region known as site B is
composed by residues Arg24, Tyr46, Asp48, Val49, Ile219, Arg254, Met258, and Gln262. In
Figure 4B, the 4-sulfophenyl group of 3l was observed to occupy the core of the catalytic site
and the benzothiazole ring of 3l was located at the allosteric site, which allowed 3l to match
perfectly with the spatial structure of PTP1B. Conversely, the p-fluorophenyl groups of both
3d and 4c were positioned at the entry of site A while the benzothiazole ring and thiazoline
ring of both compounds were oriented towards the deep catalytic site of PTP1B. Detailed
interactions in Figure 4C illustrated that the hydrophilic sulfonic acid group of 3l established
four H-bonds with residues Phe182, Cys215, Ser216 and Arg221, which contributed the most
interactions with the catalytic site of PTP1B. The aliphatic linker provided torsional degree of
freedom that substantially lowered the entropic penalty for the formation of receptor-inhibitor
complex and improve the inhibition activity. On the other hand, the benzothiazole ring of 3l
drew Pi-cation interaction with the residue Arg254 in site B, which further enhanced the
bioactivity of 3l. In addition, polar interaction with residues Met258 and Gln262, and van der
Waals interaction with residues Arg24, Tyr46, Asp48, Val49, and Ile219, were found to
contribute to the close affinity between 3l and the allosteric site of PTP1B. In Figure 4D, the
most significant interaction between 3d and the catalytic site of PTP1B was the Pi-Pi
stacking, established between benzothiazole ring of 3d and residue Phe182. The benzene ring
of 3d was implicated in additional Pi-cation interaction with Arg254. However, only one
H-bond formed by 3d and Gln262 was observed, which might be a reason for the lower
bioactivity of 3d than 3l. Besides some van der Waals, charge and polar interactions, the
H-bond with Gln262 was the only interaction to combine 4c to PTP1B, which might explain
the poor activity of 4c (Figure 4E). Furthermore, the calculated docking scores were 3l
(43.77 kcal/mol) > 3d (39.52 kcal/mol) > 4c (29.68 kcal/mol), which was in line with their
experimental biactivities (Table 1 and Table 2).
4. Conclusions
In conclusion, three series of novel N-aryl--(benzoazol-2-yl)-sulfanylalkanamides
were synthesized and evaluated for their α-glucosidase and PTP1B inhibitory activities,
respectively. The compound 3l was identified as a potent dual α-glucosidase/PTP1B inhibitor
with an IC50 value of 10.96 μM/13.46 μM, and was found to show excellent PTP1B
selectivity. Molecular docking analysis indicated that 3l matched perfectly with the binding
pockets of both receptors α-glucosidase and PTP1B via intense interactions including
hydrogen bonds, van der Waals, charge and Pi-cation interactions. In addition, kinetic
analysis demonstrated that the α-glucosidase inhibition of 3l operates by a mixed reversible
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mechanism. The above results suggested that 3l is a dual α-glucosidase/PTP1B inhibitor and
deserves further research.
Acknowledgments
This work was supported by the Natural Science Foundations of China (Grant No.
81273361 and 21502138), the Natural Science Foundation of Tianjin (Grant No.
16JCZDJC32500) and the International (Regional) Cooperation and Exchange Project of the
Natural Science Foundation of China (Grant No. 81611130090).
Conflict of interest
All authors declare no conflict of interests.
Figure legends
Scheme 1. Synthesis of 15 target compounds. Reagents and conditions: (a) K2CO3, acetone, 0-5 °C,
34-94%; (b) K2CO3/KI, DMF, rt, 3-12 h, 24-72%.
Figure 1. Lineweaver-Burk plots of compound 3l on α-glucosidase. The concentrates of 3l were 0, 5,
10, and 20 μM.
Figure 2. α-Glucosidase activity after dialysis. α-Glucosidase (400 μL, 0.01 U/mL) was dialyzed or
non-dialyzed in absence of DMSO (20 μL) or compound 3l (20 μL, 20 μM) at 4 °C for 24 h, and the
residual enzyme activities were measured.
Figure 3. Binding modes of 3l and acarbose with α-glucosidase. (A) Surface representation of
α-glucosidase in complex with 3l (green) and acarbose (orange). (B) Docking interactions of acarbose
with α-glucosidase active site. (C) Docking interactions of compound 3l with α-glucosidase active site.
Figure 4. Binding modes of 3l, 3d, 4c, and co-crystallized ligand 4B3 with PTP1B. (A) Surface
representation of PTP1B in complex with 3l (green) and co-crystallized ligand 4B3 (magenta). (B)
Surface representation of PTP1B in complex with 3l (green), 3d (cyan), and 4c (yellow). (C) Docking
interactions of 3l with PTP1B binding site. (D) Docking interactions of 3d with PTP1B binding site. (E)
Docking interactions of 4c with PTP1B binding site.
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Supporting Information
Supplementary data including 1H NMR and 13C NMR spectra for the synthesized
compounds can be found on the online version.
Table 1. PTP1B and α-glucosidase inhibitory activities of benzoxazole-2-thiol and
benzothiazoles-2-thiol derivatives.
IC50 (μM)
No.
R
X
n
α-Glucosidase
PTP1B
PTP-MEG2 TCPTP SHP2 SHP1
3a
Naphth-1-yl
p-F-C6H4-
Naphth-1-yl
p-F-C6H4-
Ph
O
O
S
1
1
1
1
1
3
3
3
3
3
3
3
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
—
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
3b
3c
28.11  0.50
5.28  0.68 12.87  0.84
3d
S
77.69  5.03 30.78  1.92
>100
18.31  1.36
>100
>100
>100
>100
>100
3e
S
3f
Naphth-1-yl
p-F-C6H4-
Ph
O
O
O
S
18.67  0.65 15.17  1.24
68.22  3.07 30.27  3.18
3g
3h
>100
53.46  3.68
3i
Naphth-1-yl
p-F-C6H4-
Ph
11.00  1.59 10.04  0.79 8.64  1.18
3j
3k
S
38.53  3.05
>100
>100
>100
>100
>100
—
S
>100
3l
p-SO3H-C6
S
10.96  1.46 13.46  1.20
Acarbosea
Ursolic acidb
—
—
—
—
—
51.32  2.75
18.22 1.38
—
—
14.50  0.90
a Reference compound of α-glucosidase inhibition.
b Reference compound of dual α-glucosidase/PTP1B inhibition.
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Table 2. PTP1B and α-glucosidase inhibitory activities of thiazolidine-2-thiol derivatives.
IC50 (μM)
PTP-MEG2 TCPTP SHP2 SHP1
No.
R
n
α-Glucosidase
PTP1B
4a
4b
Naphth-1-yl
Naphth-1-yl
p-F-C6H4-
1
3
3
>100
>100
>100
>100
>100
>100
—
>100
>100
>100
—
>100
>100
>100
—
>100 >100
>100 >100
>100 >100
4c
Acarbosea
—
—
—
—
51.32  2.75
18.22 1.38
Ursolic acidb
—
—
14.50  0.90
a Reference compound of α-glucosidase inhibition.
b Reference compound of dual α-glucosidase/PTP1B inhibition.
Scheme 1
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Figure 1
3000
2000
1000
0
0 μM
5 μM
10 μM
20 μM
-20
-10
0
10
1/(S, mM)
Figure 2
α-Glucosidase/3l
α-Glucosidase
Dialyzed
Dialyzed
α-Glucosidase/3l
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Figure 3
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Figure 4
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