Synthesis of novel coumarin derivatives and in vitro biological evaluation as potential ptp 1b inhibitors

Article abstract of DOI:10.3987/COM-13-12730

The aim of protein tyrosine phosphatase 1B (PTP 1B) inhibitors is to develop effective drug for diabetes and obesity. Coumarin becomes as a good skeleton, and is often applied in drug design and synthesis. In this paper, we have synthesized a series of novel coumarin derivatives to be as potential PTP 1B inhibitors. The inhibition rate of compound 9 was more than 80%, and the IC50 value was 49.2 μM, which would be considered for further study.

Full text of DOI:10.3987/COM-13-12730

HETEROCYCLES, Vol. 87, No. 8, 2013
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HETEROCYCLES, Vol. 87, No. 8, 2013, pp. 1711 - 1726. © 2013 The Japan Institute of Heterocyclic Chemistry
Received, 15th April, 2013, Accepted, 4th June, 2013, Published online, 10th June, 2013
DOI: 10.3987/COM-13-12730
SYNTHESIS OF NOVEL COUMARIN DERIVATIVES AND IN VITRO
BIOLOGICAL EVALUATION AS POTENTIAL PTP 1B INHIBITORS
Cheng Sun,2 Chune Peng,2 Jian Wang,2 Quan Wang,2 Wei Liu,3 Honggang
Zhou,1* and Cheng Yang1
1College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research,
NanKai University, Tianjin 300071, China
2College of Life Sciences, NanKai University, Tianjin 300071, China
3College of Sciences, Tianjin University of Science and Technology, Tianjin
300457, China
Corresponding author. Tel.: +86 22 65378878
E-mail address: honggang.zhou@nankai.edu.cn
Abstract – The aim of protein tyrosine phosphatase 1B (PTP 1B) inhibitors is to
develop effective drug for diabetes and obesity. Coumarin becomes as a good
skeleton, and is often applied in drug design and synthesis. In this paper, we have
synthesized a series of novel coumarin derivatives to be as potential PTP 1B
inhibitors. The inhibition rate of compound 9 was more than 80%, and the IC50
value was 49.2 μM, which would be considered for further study.
Insulin and leptin resistance is a major pathophysiological factor in the development of type 2 diabetes
and obesity.1,2 Currently diabetic patients are treated with various oral antihyperglycemic agents, however,
over a period of time, type 2 diabetes mellitus subjects lose their response to these agents and thereby
require insulin therapy.3,4 Except incretin therapies, most of the available antihyperglycemic agents,
including insulin promote weight gain, which further aggravates obesity associated cardiovascular risk
and insulin resistance.5,6 Thus, there is an urgent need to develop novel agents for glycemic control.7,8

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PTP 1B is an active player in insulin and leptin resistance. This enzyme acts as a negative regulator of
insulin and leptin signaling.9-11 The role of PTP 1B in diabetes and obesity is well documented and has
been reviewed extensively.12,13 Recently inhibitors for PTP 1B mainly includes antisense nucleotides,
thiophene-based inhibitors, heterocyclic carboxylic acid mimetics, oxidoreduction-type inhibitors,
monocarboxylic acid inhibitors and allosteric PTP-1B inhibitors.14
Coumarins are a general structural class of aromatic heterocycle defined by the naturally occurring parent
compound, which was first isolated in 1822 from Tonka beans (Dipteryx odorata).15 They possess a wide
range of biological activities and have long been considered attractive drug scaffolds being used in the
treatment of viral infections, neurodegenerative diseases, oedema, inflammation, cancer, and as
hepatoprotective agents and antioxidants.16 Barrios has described an elegant coumarin-based amino acid
that can be incorporated into peptides for substrate screening purposes.17 In this paper, our substrate
design uses the commercially available phenols to synthesize some coumarin-based derivatives referred
to reported coumarin compound 1 (Km= 224 μM) (Figure 1), which is a potential non-peptide PTP 1B
inhibitor.18
Figure 1. Coumarin compound 1
We applied simple synthetic methods to get twenty-six coumarin derivatives 5-24 and 26-31, which were
purified by column chromatography or recrystallization. Their structures have been demonstrated by
1
HRMS, IR and HNMR analyses. Title Compounds were synthesized following the synthetic pathway
described in Scheme 1.

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1713
Scheme 1. Reagents and conditions: (a) acetonitrile, MgCl2, (CH2O)n, triethylamine, reflux; (b)
triethylamine, reflux; (c) DCC, DMAP, CH2Cl2, rt; (d) CDI, CH2Cl2, rt; (e) amine, CH2Cl2, rt; (f) H2O,
75 °C; (g) CDI, CH2Cl2, amines, rt; (h) ethyl acetoacetate, piperidine, EtOH, reflux, 4 h.
Compounds 3 were synthesized by a published method.19 In brief, phenols 2 as starting materials reacted
with paraformaldehyde to yield salicylaldehydes 3. Compounds 3 reacted with succinic anhydride to yield
2-(2-oxo-2H-chromen-3-yl)acetic acids 4 referring to literature.20 By the method of acid-amine
condensation, reacting acids 4 by CDI activated with aromatic amines yielded compounds 5-22. Similarly,
acids 4 reacted with alcohols yielded compounds 23,24 in the presence of both DMAP and DCC. The
compound 25 was obtained using the literature procedures21 with minor modifications. Briefly,
salicylaldehyde 3a reacted with meldrum's acid to yield 2-oxo-2H-chromene-3-carboxylic acid 25.
Reacting compound 25 by CDI activated with aromatic amines yielded compounds 26-28. Compounds
29-31 were synthesized following the synthetic pathway described in Scheme 1.22
We postulated that coumarin could be used as a core structure allowing for the introduction of different
substituents to obtain potential non-peptide PTP 1B inhibitors. Although the coumarin core is a part of
many natural and synthetic compounds with wide pharmacological activity, there is only one paper

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reporting about coumarin structures that inhibit PTP 1B.18 All of these coumarin derivatives were
evaluated for inhibition of recombinant human PTP 1B in vitro. The biological activity was detected by
absorption spectrophotometry, and the substrate model was nitrobenzene phosphate (pNPP). Under the
action of PTP 1B, the phosphate group of pNPP was removed, and generated p-nitrophenol. With alkaline
condition, p-nitrophenol deprotonated to get yellow water soluble p-nitrophenolate, which had a strong
absorption peak under 405 nm (Figure 2). If drug has inhibition to the PTP 1B, the dephosphorylation
would be weaken or even disappeared. According to the changes of absorbance value, we could get the
inhibition data of coumarin derivatives. The positive control was Na3VO4 (IC50=0.7 μM),23 results were
shown in Tables 1-2.
Figure 2. The screening model of PTP 1B in vitro
Table 1. Structure and inhibitory activity of derivatives 5-24 against PTP 1B
inhibition
ratio (%) in
0.5 mM
inhibition
ratio (%) in
0.5 mM
Compd. R1 (R2)
Compd
.
R3
R1 (R2)
R3
H
5
6
H
H
41.7%
36.7%
15
16
66.1%
ND
H
H
H
7
8
H
H
39.3%
56.0%
17
18
74.6%
2.6%

HETEROCYCLES, Vol. 87, No. 8, 2013
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93.7%
9
H
H
(IC50=49.2
19
20
H
0.2%
μM)a
10
24.1%
MeO
78.1%
11
12
13
H
H
H
ND
21
22
23
MeO
MeO
H
ND
28.7%
ND
34.6%
76.2%
14
H
37.1%
24
H
75.6%
aThe IC50 value of compounds was tested when its inhibition ratio exceeded 80%.
ND =Not done.
Table 2. Structure and inhibitory activity of derivatives 26-31 against PTP 1B
inhibition
ratio (%) in
0.5 mM
inhibition
ratio (%) in
0.5 mM
Compd.
26
R4
Compd.
29
Structure
73.8%
ND
31.9%
29.7%
27
30
28
67.1%
31
70.9%

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The inhibitory activity of coumarin derivatives was tested toward recombinant human PTP-1B by
absorption spectrophotometry. As show in Table 1 to 2, firstly, we decided to modify the 3-position of the
coumarin ring, and got two types of coumarin derivatives, 5-24 and 26-28. There has no significant
improvement for PTP1B in vitro, the inhibition rate of most of compounds were less than 80%. Whereas,
compound 9 was the only one which had a modest inhibitory activity, which likely caused by the
introduction of large hydrophobic substituent containing benzene ring. In contrast, introduction of
hydrophilic group to 3-position, such as aldehyde, the inhibition rate for PTP1B dropped down to 0.19%.
Additionally, substitution at 6-position of coumarin derivatives 20-22 and 7–position of coumarin
derivatives 29-31 did not exhibit enhanced potency.
To get some insight to binding mode of coumarin derivatives, compound 9 (Figure 3) was docked into the
active site of PTP 1B. The CDOCKER program was used to predict the binding modes of 9 to PTP 1B.
As shown in Figure 4, the active site consists of S1 and S2 parts (left). Modeling of 9 into the active site
of PTP 1B showed that the molecular mainly occupied S2 part. As shown in Figure 4 (right), coumarin
ring could be interacting with Arg24, Arg254 amino acid residues in the S2 pocket and formed four
hydrogen bonds. The amide group also could be interacting with Gln262 in the S2 pocket and developed
one hydrogen bond. The benzene ring P2 developed an σ-π ligand with Arg24, however, other
compounds bearing hydrophobic N-substituents (such as 8 and 11) have not σ-π interaction with active
pocket, which.led to very weak potency. Overall, compound 9 fitted well within S2 part, and formed five
hydrogen bonds. Judging from what has been discussed above, we have come to recognize that the
activity of coumarin derivatives might be improved to modify the other benzene ring P1 in order to
extend it to the S1 part of PTP 1B activity pocket.
Figure 3. Structure of compound 9 for the docking study (left) and its detection curve of IC50 (right)

HETEROCYCLES, Vol. 87, No. 8, 2013
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Figure 4. PTP 1B-binding electrostatic potential groove (left) and interactive map (right). Hydrogen
bonds are shown as green dashed.
In conclusion, we have completed the synthesis and in vitro evaluation of a series of coumarin derivatives
having a modest potency for inhibiting the recombinant human PTP 1B in vitro. The biological activity
results told us that introduction of hydrophobic groups to 3-position of coumarin might improve the
inhibition activity for PTP 1B. We have found that IC50 value of compound 9 is 49.2 μM. According to
the docking results of computer simulation, modifying the benzene ring P1 of coumarin derivative 9 to
extend it to the S1 part of PTP 1B activity pocket might improve the inhibitory activity of coumarin
derivatives against PTP 1B in vitro. Further work is in progress to optimize the structure of compound 9
with the aim of increasing its potency as a potential PTP 1B inhibitor.
EXPERIMENTAL
Chemistry. General Procedures. All experiments involving water-sensitive compounds were conducted
under scrupulously dry conditions. Melting points were determined using an SGW-X4B digital melting
1
point apparatus and was uncorrected. H NMR spectra was recorded on 400 MHz Bruker Avance DPX
spectrometers. All NMR spectra were recorded in CDCl3 or DMSO-d6 at room temperature. Chemical
1
shifts for H spectra are quoted in ppm downfield from TMS. Coupling constants are referred to as J
values. ESI mass spectra were acquired using a Bruker ESQUIRELCTM ESI ion trap spectrometer.
HR-ESI mass spectra were obtained with an Agilent Technologies ESI-TOF spectrometer. FT-IR spectra
were recorded in the region of 4000–400 cm-1 with a PerkinElmer spectrum 65 FT-IR using KBr pellets.
Preparation of Compound 3. The dry paraformaldehyde (116.7 mmol) was added in portions to a

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HETEROCYCLES, Vol. 87, No. 8, 2013
mixture of phenol (33.3 mmol), Et3N (93.3 mmol) and anhydrous MgCl2 (103 mmol) in MeCN (300 mL).
The mixture was refluxed for 8 h, cooled to room temperature, acidified with aqueous 3 N HCl solution,
and extracted with Et2O. The ether layer was washed with water, and brine, and dried (MgSO4). Removal
of solvent yielded a crude material which was purified by column chromatography to yield the products 3.
Preparation of 2-(2-oxo-2H-chromen-3-yl)acetic acids 4.20 A mixture of 0.3 mol succinic anhydride,
0.1 mol of salicylaldehyde 3, and 0.13 mol of Et3N was heated under stirring to the boiling point. After
1-1.5 h, abundant solid separated. The mixture was cooled and treated with concentrated hydrochloric
acid, and the precipitate was filtered off and dried. The product was dissolved in a warm saturated
aqueous solution of NaHCO3, the solution was filtered, finely powdered activated charcoal was added to
the filtrate, the mixture was stirred for 15 min and filtered, and the filtrate was acidified with concentrated
hydrochloric acid. The precipitate 4 was filtered off, washed with water, and dried in air until constant
weight.
Standard procedure A for Compound 5-22: refer to the method of acid amine condensation：0.162 g
(10 mmol) CDI and 10 mmol acid 4 in 10 mL CH2Cl2 stirred 30 min at room temperature, then the
reaction mixture was dropwise added to amines (10 mmol) dissolved in 5 mL CH2Cl2 , stirred 2-3 h at
room temperature. The product was separated by column chromatography over silica gel (eluted with
MeOH/CH2Cl2= 1/20) to afford the product.
N-(3-Bromophenyl)-2-(2-oxo-2H-chromen-3-yl)acetamide (5). Compound 5 was prepared according to
standard procedure A by using compound 4 and 3-bromoaniline, obtained as a white solid in 67% yield.
1
mp116.7-118.1 °C; IR (KBr, cm-1): 3435, 1747, 1699, 1616, 1565, 1248, 998, 797, 758; HNMR (400
MHz, CDCl3): 8.52 (s, 1H), 7.86 (s, 1H), 7.80 (s, 1H), 7.58-7.51 (m, 2H), 7.42-7.31 (m, 3H), 7.21 (d, J =
8.0 Hz, 1H), 7.15 (t, J = 8.0 Hz, 1H), 3.64 (s, 2H); HRMS-ESI (m/z) calcd for C17H12BrNO3 [M+H]+
358.0079, found 358.0081.
N-(4-Bromophenyl)-2-(2-oxo-2H-chromen-3-yl)acetamide (6). Compound 6 was prepared according to
standard procedure A by using compound 4 and 4-bromoaniline, obtained as a white solid in 64% yield.
mp 246.5-248.2 °C; IR (KBr, cm-1): 3334, 1714, 1698, 1607, 1544, 1489, 1395, 1248, 1085, 819, 749;
1HNMR (400 MHz, DMSO-d6): 10.31 (s, 1H), 8.01 (s, 1H), 7.72 (d, J = 7.6 Hz, 1H), 7.63-7.56 (m, 3H),
7.49 (d, J = 8.8 Hz, 2H), 7.43 (d, J = 8.0 Hz, 1H), 7.37 (t, J = 7.6 Hz, 1H), 3.63 (s, 2H); HRMS-ESI (m/z)
calcd for C17H12BrNO3 [M+H]+ 358.0079, found 358.0083.
N-(4-Iodophenyl)-2-(2-oxo-2H-chromen-3-yl)acetamide (7). Compound 7 was prepared according to
standard procedure A by using compound 4 and 4-iodoaniline, obtained as a white solid in 68% yield. mp
1
240.2-241.8 °C; IR (KBr, cm-1): 3334, 1714, 1698, 1605, 1538, 1246, 820, 749; HNMR (400 MHz,
DMSO-d6): 10.28 (s, 1H), 8.01 (s, 1H), 7.73 (d, J = 7.6 Hz, 1H), 7.65-7.58 (m, 3H), 7.45-7.39 (m, 3H),
7.37 (t, J = 7.6 Hz, 1H), 3.63 (s, 2H); HRMS-ESI (m/z) calcd for C17H12INO3 [M+H]+ 405.9940, found

HETEROCYCLES, Vol. 87, No. 8, 2013
1719
405.9949.
N-(Naphthalen-2-yl)-2-(2-oxo-2H-chromen-3-yl)acetamide (8). Compound 8 was prepared according
to standard procedure A by using compound 4 and naphthalen-2-amine, obtained as a white solid in 64%
1
yield. mp 238.2-240.1 °C; IR (KBr, cm-1): 3336, 1719, 1697, 1507, 1432, 1280, 859, 740; HNMR (400
MHz, DMSO-d6): 10.38 (s, 1H), 8.29 (s, 1H), 8.05 (s, 1H), 7.88-7.74 (m, 4H), 7.61 (d, J = 7.6 Hz, 2H),
7.48-7.36 (m, 4H), 3.70 (s, 2H); HRMS-ESI (m/z) calcd for C21H15NO3 [M+H]+ 330.1130, found
330.1137.
N-Benzhydryl-2-(2-oxo-2H-chromen-3-yl)acetamide (9). Compound 9 was prepared according to
standard procedure A by using compound 4 and diphenylmethanamine, obtained as a white solid in 58%
1
yield. mp 240.8-242.6 °C; IR (KBr, cm-1): 3292, 1728, 1644, 1242, 1074, 752, 697; HNMR (400 MHz,
DMSO-d6): 9.01 (d, J = 8.4 Hz, 1H), 7.91 (s, 1H), 7.67 (dd, J = 7.6, 1.6 Hz, 1H), 7.58 (td, J = 7.6, 1.6 Hz,
1H), 7.31 (m, 12H), 6.13 (d, J = 8.4 Hz, 1H), 3.53 (s, 2H); HRMS-ESI (m/z) calcd for C24H19NO3
[M+H]+ 370.1443, found 370.1436.
N-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-(2-oxo-2H-chromen-3-yl)acetamide (10). Compound 10
was prepared according to standard procedure
A
by using compound
4
and
2,3-dihydrobenzo[b][1,4]dioxin-6-amine, obtained as a white solid in 61% yield. mp 200.5-202.6 °C; IR
1
(KBr, cm-1): 3348, 1709, 1671, 1611, 1512, 1221, 1068, 755; HNMR (400 MHz, CDCl3): 8.21 (s, 1H),
7.85 (s, 1H), 7.53 (m, 2H), 7.37-7.28 (m, 2H), 7.17 (d, J = 2.4 Hz, 1H), 6.89 (dd, J = 8.8, 2.4 Hz, 1H),
6.77 (d, J = 8.4 Hz, 1H), 4.24 (s, 4H), 3.61 (s, 2H); HRMS-ESI (m/z) calcd for C19H15NO5 [M+H]+
338.1028, found 338.1033.
N-Benzyl-2-(2-oxo-2H-chromen-3-yl)acetamide (11). Compound 11 was prepared according to
standard procedure A by using compound 4 and phenylmethanamine, obtained as a white solid in 71%
1
yield. mp 184.6-186.4 °C; IR (KBr, cm-1): 3283,1731, 1643, 1550, 1455, 1239, 1057, 765, 698; HNMR
(400 MHz, CDCl3): 7.81 (s, 1H), 7.54-7.48 (m, 2H), 7.35-7.24 (m, 7H), 4.43 (d, J = 5.6 Hz, 2H), 3.51 (s,
2H); HRMS-ESI (m/z) calcd for C18H15NO3 [M+H]+ 294.1130, found 294.1137.
N-(3-Methoxyphenyl)-2-(2-oxo-2H-chromen-3-yl)acetamide (12). Compound 12 was prepared
according to standard procedure A by using compound 4 and 3-methoxyaniline, obtained as a white solid
in 69% yield. mp 109.5-111.2 °C; IR (KBr, cm-1): 3449, 3030, 2930, 1728, 1679, 1559, 1453, 1366, 1203,
1
972, 778; HNMR (400 MHz, CDCl3): 8.39 (s, 1H), 7.86 (s, 1H), 7.56-7.50 (m, 2H), 7.38-7.26 (m, 3H),
7.19 (t, J = 8.0 Hz, 1H), 7.00 (d, J = 7.6 Hz, 1H), 6.64 (dd, J = 8.0, 1.6 Hz, 1H), 3.79 (s, 3H), 3.64 (s, 2H);
HRMS-ESI (m/z) calcd for C18H15NO4 [M+H]+ 310.1079, found 310.1073.
N-(4-Methoxybenzyl)-2-(2-oxo-2H-chromen-3-yl)acetamide (13). Compound 13 was prepared
according to standard procedure A by using compound 4 and (4-methoxyphenyl)methanamine, obtained
as a white solid in 72% yield. mp 182.2-184.1 °C; IR (KBr, cm-1): 3275, 1729, 1637, 1515, 1254, 1055,

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HETEROCYCLES, Vol. 87, No. 8, 2013
750; 1HNMR (400 MHz, CDCl3): 7.81 (s, 1H), 7.50 (s, 2H), 7.35-7.21 (m, 4H), 6.86 (s, 2H), 6.56 (s, 1H),
4.37 (s, 2H), 3.79 (s, 3H), 3.49 (s, 2H); HRMS-ESI (m/z) calcd for C19H17NO4 [M+H]+ 324.1236, found
324.1240.
N-(2,4-Dimethoxybenzyl)-2-(2-oxo-2H-chromen-3-yl)acetamide (14). Compound 14 was prepared
according to standard procedure A by using compound 4 and (2,4-dimethoxyphenyl)methanamine,
obtained as a yellow solid in 54% yield. mp 159.2-160.8 °C; IR (KBr, cm-1): 3290, 1714, 1604, 1567,
1
1242, 1057, 762; HNMR (400 MHz, DMSO-d6): 8.27 (t, J = 5.2 Hz, 1H), 7.93 (s, 1H), 7.69 (d, J = 8.0
Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.41 (d, J = 7.6 Hz, 2H), 7.36 (d, J = 8.4 Hz, 1H), 7.12 (d, J = 8.0 Hz,
1H), 6.53 (s, 1H), 6.48 (d, J = 8.0 Hz, 1H), 4.17 (d, J = 5.6 Hz, 2H), 3.78 (s, 3H), 3.74 (s, 3H), 3.43 (s,
2H); HRMS-ESI (m/z) calcd for C20H19NO5 [M+H]+ 354.1341, found 354.1348.
N-(Furan-2-ylmethyl)-2-(2-oxo-2H-chromen-3-yl)acetamide (15). Compound 15 was prepared
according to standard procedure A by using compound 4 and furan-2-ylmethanamine, obtained as a white
solid in 62% yield. mp 150.2-152.0 °C; IR (KBr, cm-1): 3290, 1729, 1713, 1646, 1614, 1458, 1242, 1060,
743; 1HNMR (400 MHz, DMSO-d6): 8.18 (t, J = 5.2 Hz, 1H), 7.91 (s, 1H), 7.69 (d, J = 7.6 Hz, 1H), 7.59
(t, J = 7.6 Hz, 1H), 7.42-7.32 (m, 3H), 6.95 (t, J = 3.6 Hz, 1H), 6.90 (s, 1H), 3.37 (s, 2H), 2.94 (t, J = 7.2
Hz, 2H); HRMS-ESI (m/z) calcd for C16H13NO4 [M+H]+ 284.0923, found 284.0921.
2-(2-Oxo-2H-chromen-3-yl)-N-(2-(thiophen-2-yl)ethyl)acetamide (16). Compound 16 was prepared
according to standard procedure A by using compound 4 and 2-(thiophen-2-yl)ethanamine, obtained as a
white solid in 56% yield. mp 149.2-151.1 °C, IR (KBr, cm-1): 3290, 1730, 1716, 1647, 1555, 1244, 1187,
1060, 760, 700; 1HNMR (400 MHz, DMSO-d6): 8.18 (t, J = 5.2 Hz, 1H), 7.91 (s, 1H), 7.70 (d, J = 8.0 Hz,
1H), 7.59 (t, J = 7.6 Hz, 1H), 7.42-7.32 (m, 3H), 6.95 (t, J = 3.6 Hz, 1H), 6.90 (s, 1H), 3.37 (s, 2H), 3.31
(t, J = 7.2 Hz, 2H), 2.94 (t, J = 7.2 Hz, 2H); HRMS-ESI (m/z) calcd for C17H15NO3S [M+H]+ 314.0851,
found 314.0855.
Methyl 3-(4-hydroxyphenyl)-2-(2-(2-oxo-2H-chromen-3-yl)acetamido)propanoate (17). Compound
17 was prepared according to standard procedure A by using compound 4 and methyl
2-amino-3-(4-hydroxyphenyl)propanoate, obtained as a white solid in 46% yield. mp 162.4-164.3 °C, IR
(KBr, cm-1): 3317, 3242, 1737, 1722, 1716, 1610, 1243; 1HNMR (400 MHz, CDCl3): 7.68 (s, 1H), 7.53 (t,
J = 7.6 Hz, 1H), 7.45 (d, J = 7.6 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.33 (d, J = 7.2 Hz, 1H), 6.94 (d, J =
8.4 Hz, 2H), 6.73 (d, J = 8.0 Hz, 1H), 6.59 (d, J = 8.0 Hz, 2H), 4.82 (dd, J = 13.2, 7.6 Hz, 1H), 3.89 (s,
1H), 3.74 (s, 1H), 3.73 (s, 3H), 3.52 (d, J = 14.0 Hz, 1H), 2.99 (d, J = 14.0 Hz, 1H); HRMS-ESI (m/z)
calcd for C21H19NO6 [M+H]+ 382.1291, found 382.1296.
3-(2-(4-Methylpiperazin-1-yl)-2-oxoethyl)-2H-chromen-2-one (18). Compound 18 was prepared
according to standard procedure A by using compound 4 and 1-methylpiperazine, obtained as a white
solid in 60% yield. mp 123.3-125.2 °C; IR (KBr, cm-1): 3009, 2935, 2858, 2800, 1726, 1659, 1226, 1063,

HETEROCYCLES, Vol. 87, No. 8, 2013
1721
1
758; HNMR (400 MHz, CDCl3): 7.75 (s, 1H), 7.52-7.46 (m, 2H), 7.33 (d, J = 8.0 Hz, 2H), 7.28 (d, J =
8.0 Hz, 2H), 3.72-3.63 (m, 6H), 2.45 (d, J = 16.4 Hz, 4H), 2.33 (s, 3H); HRMS-ESI (m/z) calcd for
C16H18N2O3 [M+H]+ 287.1396, found 287.1341.
3-(2-(4-(Furan-2-carbonyl)piperazin-1-yl)-2-oxoethyl)-2H-chromen-2-one. (19). Compound 19 was
prepared
according
to
standard
procedure
A
by
using
compound
4
and
4-(piperazine-1-carbonyl)furan-2-carbaldehyde, obtained as a white solid in 62% yield; mp
188.5-190.4 °C; IR (KBr, cm-1): 3107, 2918, 1732, 1630, 1483, 1434, 1232, 1011, 757 cm-1; 1HNMR
(400 MHz, DMSO-d6): 7.88 (d, J = 8.0 Hz, 2H), 7.71 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 7.2 Hz, 1H), 7.42
(d, J = 8.4 Hz, 1H), 7.36 (t, J = 7.6 Hz, 1H), 7.05 (d, J = 3.2 Hz, 1H), 6.65 (q, J = 1.6 Hz, 1H), 3.75-3.65
(m, 8H), 3.57 (m, 2H); HRMS-ESI (m/z) calcd for C20H18N2O5 [M+H]+ 367.1294, found 367.1299.
2-(7-Methoxy-2-oxo-2H-chromen-3-yl)-N-(4-methoxybenzyl)acetamide (20). Compound 20 was
prepared according to standard procedure A by using compound 4 and (4-methoxyphenyl)methanamine,
obtained as a yellow solid in 58% yield. mp 189.6-191.4 °C; IR (KBr, cm-1): 3282, 1725, 1640, 1611,
1
1513, 1240, 820; HNMR (400 MHz, DMSO-d6): 8.43 (t, J = 5.6 Hz, 1H), 7.86 (s, 1H), 7.60 (d, J = 8.4
Hz, 1H), 7.19 (d, J = 8.4 Hz, 2H), 7.00 (s, 1H), 6.96 (d, J = 8.8 Hz, 1H), 6.88 (d, J = 8.4 Hz, 2H), 4.22 (d,
J = 5.6 Hz, 2H), 3.85 (s, 3H), 3.73 (s, 3H), 3.37 (s, 2H); HRMS-ESI (m/z) calcd for C20H19NO5 [M+H]+
354.1341, found 354.1344.
N-(4-Hydroxyphenethyl)-2-(7-methoxy-2-oxo-2H-chromen-3-yl)acetamide (21). Compound 21 was
prepared according to standard procedure A by using compound 4 and 4-(2-aminoethyl)phenol, obtained
as a white solid in 48% yield. mp 180.1-182.0 °C; IR (KBr, cm-1): 3336, 1705, 1613, 1444, 1249, 1158,
1068, 829; 1HNMR (400 MHz, DMSO-d6): 9.16 (s, 1H), 8.03 (t, J = 5.6 Hz, 1H), 7.80 (s, 1H), 7.60 (d, J
= 8.4 Hz, 1H), 7.00-6.94 (m, 4H), 6.66 (d, J = 8.4 Hz, 2H), 3.86 (s, 3H), 3.29 (s, 2H), 3.21 (dd, J = 6.4,
13.6 Hz, 2H), 2.59 (t, J = 7.2 Hz, 2H); HRMS-ESI (m/z) calcd for C20H19NO5 [M+H]+ 354.1341, found
354.1347.
N-(4-Fluorobenzyl)-2-(7-methoxy-2-oxo-2H-chromen-3-yl)acetamide (22). Compound 22 was
prepared according to standard procedure A by using compound 4 and (4-fluorophenyl)methanamine,
obtained as a yellow solid in 48% yield. mp 236.4-238.3 °C; IR (KBr, cm-1): 3280, 1723, 1643, 1612,
1509, 1211, 836; 1HNMR (400 MHz, DMSO-d6): 8.51 (s, 1H), 7.87 (s, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.31
(t, J = 8.0 Hz, 2H), 7.14 (t, J = 8.0 Hz, 2H), 7.01 (s, 1H), 6.96 (d, J = 8.0 Hz, 1H), 4.27 (s, 2H), 3.85 (s,
3H), 3.39 (s, 2H); HRMS-ESI (m/z) calcd for C19H16FNO4 [M+H]+ 342.1142, found 342.1139.
Standard procedure B for Compound 23, 24: To a stirred solution of the appropriate acid 4 (5 mmol)
and alcohol (5.5 mmol) in CH2Cl2 (25 mL), DCC (5.5 mmol) and DMAP (0.5 mmol) were added. After
24 h stirring at room temperature, the reaction mixture was filtered and the solution washed with 10%
citric acid (2×20 mL), water (15 mL), and brine (30 mL), dried over anhydrous Na2SO4, filtered and

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HETEROCYCLES, Vol. 87, No. 8, 2013
concentrated under reduced pressure. The crude product was purified by column chromatography over
silica gel (eluted with MeOH/CH2Cl2= 1/20) to afford the product.
Benzyl 2-(2-oxo-2H-chromen-3-yl)acetate (23). Compound 23 was prepared according to standard
procedure B by using compound 4 and phenylmethanol, obtained as a white solid in 71% yield. mp
1
114.2-115.5 °C; IR (KBr, cm-1): 3087, 2931, 1727, 1639, 1608, 1452, 1338, 1194, 756; HNMR (400
MHz, CDCl3): 7.65 (s, 1H), 7.51 (t, J = 8.0 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 7.37-7.32 (s, 6H), 7.27 (t, J
= 7.2 Hz, 1H), 5.10 (s, 2H), 3.65 (s, 2H); HRMS-ESI (m/z) calcd for C18H14O4 [M+H]+ 295.0970, found
295.0974.
(5-Formylfuran-2-yl)methyl 2-(2-oxo-2H-chromen-3-yl)acetate (24). Compound 24 was prepared
according to standard procedure B by using compound 4 and 5-(hydroxymethyl)furan-2-carbaldehyde,
obtained as a white solid in 73% yield. mp 141.8-143.3 °C; IR (KBr, cm-1): 3095, 2963, 1740, 1712, 1677,
1610, 1343, 1268, 1190, 759; 1HNMR (400 MHz, DMSO-d6): 9.60 (s, 1H), 8.04 (s, 1H), 7.71 (d, J = 7.6
Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.53 (d, J = 3.2 Hz, 1H), 7.43 (d, J = 8.4 Hz, 2H), 7.38 (t, J = 7.6 Hz,
1H), 6.82 (d, J = 3.2 Hz, 1H), 5.23 (s, 2H), 3.69 (s, 2H); HRMS-ESI (m/z) calcd for C17H12O6 [M+H]+
313.0712, found 313.0714.
Preparation of 2-oxo-2H-chromene-3-carboxylic acid 25. Salicylaldehyde 3a with Meldrum’s acid in
water at 75 °C provided 3-carboxycoumarin derivatives. The reaction proceeds to completion in 2 h and
the product precipitates during the course of the reaction and was separated by filtration. Yield 80%.
1HNMR (400 MHz, CDCl3): 12.32 (s, 1H), 9.00 (s, 1H), 7.82-7.76 (m, 2H), 7.51-7.47 (m, 2H). ESI-MS
(m/z) [M+H]+ 191.2.
N-(4-Hydroxyphenyl)-7-methoxy-2-oxo-2H-chromene-3-carboxamide (26). Compound 26 was
prepared according to standard procedure A by using compound 25 and 4-aminophenol, obtained as a
white solid in 52% yield. 1HNMR (400 MHz, DMSO-d6): 9.83 (s, 1H), 8.52 (s, 1H), 7.63 (d, J = 8.0 Hz,
1H), 7.31 (d, J = 7.6 Hz, 1H), 7.22 (m, 4H), 6.73 (d, J = 8.0 Hz, 2H), 5.72 (s, 1H); HRMS-ESI (m/z) calcd
for C16H11NO4 [M+H]+ 282.0766, found 282.0770.
N-(3,5-Dimethoxyphenyl)-7-methoxy-2-oxo-2H-chromene-3-carboxamide (27). Compound 27 was
prepared according to standard procedure A by using compound 25 and 3,5-dimethoxyaniline, obtained as
1
a yellow solid in 54% yield. HNMR (400 MHz, CDCl3): 10.82 (s, 1H), 9.02 (s, 1H), 7.72 (dd, J = 7.2,
13.6 Hz, 2H), 7.52-7.40 (m, 2H), 7.00 (d, J = 2.0 Hz, 2H), 6.31 (s, 1H), 3.83 (m, 6H); HRMS-ESI (m/z)
calcd for C18H15NO5 [M+H]+ 326.1028, found 326.1033.
Methyl 3-(4-hydroxyphenyl)-2-(2-oxo-2H-chromene-3-carboxamido)propanoate (28). Compound 28
was prepared according to standard procedure A by using compound 25 and methyl
1
2-amino-3-(4-hydroxyphenyl)propanoate, obtained as a white solid in 45% yield. HNMR (400 MHz,
CDCl3): 9.22 (d, J = 7.2 Hz, 1H), 8.85 (s, 1H), 7.68-7.65 (m, 2H), 7.38 (dd, J = 8.4, 14.8 Hz, 2H), 7.09

HETEROCYCLES, Vol. 87, No. 8, 2013
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(d, J = 8.4 Hz, 2H), 6.76 (d, J = 8.4 Hz, 2H), 5.04 (s, 1H), 4.95 (dd, J = 7.2, 12.8 Hz, 1H), 3.77 (s, 3H),
3.20 (dd, J = 5.2, 14.0 Hz, 1H), 3.09 (dd, J = 7.2, 14.0 Hz, 1H); HRMS-ESI (m/z) calcd for C20H17NO6
[M+H]+ 368.1134, found 368.1139.
Standard procedure C for Compound 29-31. The corresponding salicylaldehyde (10 mmol) and ethyl
acetoacetate (10 mmol) were dissolved in EtOH. Piperidine (0.02 mmol) was added, and the mixture was
refluxed for 4 h. Filtration of the cooled mixture and the subsequent washing of product with cold ethanol
provided analytically pure crystals, or separated by column chromatography over silica gel (eluted with
MeOH/CH2Cl2= 1/20) to afford the product.
3-Acetyl-6,7,8,9-tetrahydro-2H-benzo[g]chromen-2-one (29). Compound 29 was prepared according to
standard procedure C by using ethyl acetoacetate and 3-hydroxy-5,6,7,8-tetrahydronaphthalene-
2-carbaldehyde, obtained as a yellow solid in 56% yield. mp 94.8-95.6 °C; IR (KBr, cm-1): 2939, 1729,
1683, 1622, 1551, 1365, 1219, 769; 1HNMR (400 MHz, CDCl3): 8.45 (s, 1H), 7.31 (s, 1H), 7.06 (s, 1H),
2.89-2.83 (m, 4H), 2.72 (s, 3H), 1.83 (m, 4H); HRMS-ESI (m/z) calcd for C15H14O3 [M+H]+ 243.1021,
found 243.1022.
3-Acetyl-7,8-dihydrocyclopenta[g]chromen-2(6H)-one (30). Compound 30 was prepared according to
standard procedure C by using ethyl acetoacetate and 6-hydroxy-2,3-dihydro-1H-indene-5-carbaldehyde,
obtained as a yellow solid in 60% yield. mp 162.3-164.2 °C; IR (KBr, cm-1): 2970, 1736, 1718, 1681,
1
1560, 1361, 1212, 954, 769; HNMR (400 MHz, CDCl3): 11.10 (s, 1H), 9.80 (s, 1H), 7.34 (s, 1H), 2.92
(m, 6H), 2.12 (s, 3H); HRMS-ESI (m/z) calcd for C14H12O3 [M+H]+ 229.0865, found 229.0869.
3-Acetyl-6-(phenylamino)-2H-chromen-2-one (31). Compound 31 was prepared according to standard
procedure C by using ethyl acetoacetate and 2-hydroxy-5-(phenylamino) benzaldehyde, obtained as a red
1
solid in 42% yield. mp 192.3-194.2 °C; IR (KBr, cm-1): 3328, 1700, 1686, 1562, 1232, 1001; HNMR
(400 MHz, DMSO-d6): 8.62 (s, 1H), 8.40 (s, 1H), 7.60 (d, J = 2.4 Hz, 1H), 7.42-7.36 (m, 2H), 7.26 (t, J =
7.6 Hz, 2H), 7.11 (d, J = 7.6 Hz, 2H), 6.87 (t, J = 7.2 Hz, 1H), 2.58 (s, 3H); HRMS-ESI (m/z) calcd for
C17H13NO3 [M+H]+ 280.0974, found 280.0979.
Preparation of active protein tyrosine Phosphatase 1B. The catalytic domain of PTP1B, consisting of
amino acids 1–321, was inserted into the EcoR I and Not I site of the pGEX-6P-1 vector R. Recombinant
human PTP 1B was produced according to a procedure published previously with minor modification.
Briefly, PTP 1B was expressed and processed in Transetta (DE3) cells, Bacterial cultures were grown in
800 mL LB medium at 37 °C for 4-5 h, which containing 100 µg/mL ampicillin until the OD600 reached
0.6-0.8. Then, the culture was transferred to 16 °C, and protein expression was induced for 12-16 h with
0.25 mol/L isopropyl-β-D-1-thiogalactopyranoside (IPTG). Harvested cells were resuspended in lysis
buffer containing 50 mM Hepes-NaOH (pH=7.5), 10 mM NaCl, 10% glycerol, 1% β-Me with a JN-3000
PLUS low temperature ultra-high pressure cell disrupter (JNBIO, Guangzhou). The lysate was

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centrifuged at 16000 rpm for 30 min at 4 °C to remove cell debris. The supernatant was then loaded twice
onto a GST agarose column pre-equilibrated with lysis buffer. The FLAG peptide of PTP 1B was cut off
by a PPase in the above buffer. Fractions containing PTP 1B were pooled and concentrated with a
Millipore Ultrafree filtration device. The concentrated PTP 1B was loaded onto a Hi-Trap heparin (GE
Healthcare) equilibrated with above buffer. Fractions containing PTP 1B were pooled and concentrated.
The PTP 1B obtained with this method shows 38 kDa on 10% SDS-polyacrylamide gel electrophoresis,
approximately 6-10 mg of PTP 1B were recovered from a 1000 mL culture and aliquots stored at -80 °C.
Biological activtity test23
Preliminary screening. PTP 1B purified are dissolved in enzyme activity buffer, made into 200 nM PTP
1B protein solution. And the substrate pNPP (bought from BIO BASIC INC) soluble are dissolved in
enzyme activity buffer, made into 4 mM substrate reserve liquid. All synthesized compounds are
dissolved in 95% DMSO respectively, made up to 50 mM for the test solution.
First of all, 50 μL PTP 1B protein solution is added to NUNC 96 orifice (Nunclon TM Surface) in turn,
and 1 μL compound solution to be tested, shakes 1 min, incubates 30 min in 30 °C, then add the 50 μL
pNPP, shakes 10 s. At the same time set positive and negative control, and join 50 μL PTP 1B protein
solution, 1 μL 95% DMSO to the negative control, concussion 1 min, incubate 30 min in 30 °C, then add
50 μL pNPP, shake 10 s. Join 50 μL PTP 1B protein solution, 1 μL (5 mM) Na3VO4 to the positive control,
shake 1 min, incubate 30 min in 30 °C, then add the 50 μL pNPP, shake 10 s. Use the VARIOSKAN
FLASH (Thermo SCITIFIC) to measure the changes of absorbance with reaction time under 405 nm
wavelength, make a measurement each 6 s, 60 times in all. Each compound takes parallel test for three
times, and draw the response curve to compute the inhibitory activity for PTP 1B. Make a further IC50
value measurement of compound with the residual activity less than 20%.
Compound IC50 value measurement. First of all, add 50 μL PTP 1B protein, 2 μL different concentrations
compound into the 96 orifice plate in turn, shake 1 min, incubate 30 min in 30 °C, then add the 50 μL
pNPP, shake 10 s. The changes of absorbance with reaction time under 405 nm wavelength, make a
measurement each 6 s, 60 points in all. Test it three times in parallel, and draw the response curve to
calculate the enzyme inhibitory activity of different concentrations compound. Take use of software
GraphPad Prism 5 to do the nonlinear fitting analysis, with the residual active value for ordinate,
compound concentration on the numerical for x axis, then draw the curve plotting to calculate the
compound's IC50 value at last.
Computational studies
In order to predict the interactions and binding modes of 9 synthesized inhibitors in the PTP 1B active site,
the Discovery Studio 3.0 was used to perform the molecular docking studies. The crystal structure of PTP
1B with small molecule was acquired from the Protein Data Bank (PDB code 2NT7). As for the

HETEROCYCLES, Vol. 87, No. 8, 2013
1725
preparation of the protein, the small molecule included in 2NT7 protein was extracted, hydrogen atoms
were added and the deletion of crystal waters using Prepare Protein Structure module.
ACKNOWLEDGEMENTS
This work was supported by the National Natural Science Foundation of China (Grant No. 81102374) and
National Biomedical Special Project of International Innovation Park (10ZCKFSY08600).
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