Design, Synthesis, and in Vitro Evaluation of Novel 3, 7-Disubstituted Coumarin Derivatives as Potent Anticancer Agents

Article abstract of DOI:10.1111/cbdd.12531

Twenty-seven 3, 7-disubstituted coumarin derivatives were designed, synthesized, and evaluated in vitro as anticancer agents. Most of the compounds showed moderate-to-potent antiproliferative activity against K562 cells. Compounds 7b and 7d were chosen to evaluate the concentration of 50% growth inhibition (GI₅₀) against SN12C, OVCAR, BxPC-3, KATO-III, T24, SNU-1, WiDr, HeLa, K562, and AGS cell lines. The most potent compound 7d was selected for further cell cycle arrest assay in the AGS cell line. The in vitro data indicated that methylation of benzimidazole moiety at the 3-position of coumarin exhibited significant enhancement of anticancer activity. This study should provide important information for further modification and optimization of coumarin derivatives as anticancer agents.

Full text of DOI:10.1111/cbdd.12531

Received Date : 09-Sep-2014
Revised Date : 12-Dec-2014
Accepted Date : 08-Jan-2015
Article type
: Research Article
Design, synthesis and in vitro evaluation of novel 3, 7-disubstituted coumarin derivatives
as potent anticancer agents
Yubin Wanga, Haitao Liub, Peng Lua, Rui Maob, Xiaojian Xuea, Chen Fana, Jinxiong Shea,b*
aSino-American Institute of Translational Medicine, School of Pharmaceutical Sciences, Nanjing Tech University, No 5.
Xinmofan Road, Nanjing 210009, PR China
b Center for Biotechnology and Genomic Medicine, Medicinal College of Georgia, Georgia Regents University, 1120 15th
ST ,Augusta, GA, 30912, USA
Twenty-seven 3, 7-disubstituted coumarin derivatives were designed, synthesized and evaluated in
vitro as anticancer agents. Most of the compounds showed moderate to potent anti-proliferative
activity against K562 cells. Compounds 7b and 7d were chosen to evaluate the concentration of 50%
growth inhibition (GI50) against SN12C, OVCAR, BxPC-3, KATO- III, T24, SNU-1, WiDr, HeLa,
K562, and AGS cell lines. The most potent compound 7d was selected for further cell cycle arrest
assay in the AGS cell line. The in vitro data indicated that methylation of benzimidazole moiety at
the 3-position of coumarin exhibited significant enhancement of anticancer activity. This study
should provide important information for further modification and optimization of coumarin
derivatives as anticancer agents.
Keywords: Coumarin; 3, 7-disubstituted; synthesis; anticancer activity
Natural products have historically been an extremely productive source for new medicines in all cultures
and continue to deliver a great variety of structural templates for drug discovery and development.
Although products derived from natural sources may not necessarily represent active ingredients in their
final form, the majority of all drugs in the market have their origin in nature. A primary aim in medicinal
chemistry is the design of structural analogues of bioactive natural products with an improved
pharmacological profile. Such examples have been provided with anticancer agents derived from natural
products, such as microtubule targeting drugs.
Coumarins (Fig. 1), which usually bear a 2H-1-benzopyran-2-one as part of their structure, are a very large
and important family of compounds which are mainly derived from a wide range of plants, fungi, and bacteria
(1). Furthermore, coumarin derivatives also can be synthesized in laboratory. As substitutions can occur at any
of the six available sites of their basic molecular moiety (1, 2-benzopyrone), these compounds are
extremely variable in structure. Structural diversity of coumarin derivatives leads to compounds displaying
*Corresponding author. Sino-American Institute of Translational Medicine, School of Pharmaceutical Sciences, Nanjing
Tech University, Nanjing 210009, PR China.Tel: +86 25 8317 2108; Fax: +86 25 8317 2105; E-mail: jshe@gru.edu.
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
an 'Accepted Article', doi: 10.1111/cbdd.12531
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multiple biological properties that promote human health and help reducing the risk of diseases. Coumarins
are of great interest because of their widespread application as anticoagulant (2), spasmolytic,
bacteriostatic, anti-mutagenic (3), antiviral (4), and antitumor agents (5, 6), and they have the ability to
inhibit human immuno-deficiency, virus-integrase and prevent free radical injury (7, 8). Among these
properties, the anticancer activities of coumarins have been the most extensively studied and as a result
coumarin compounds have served as valuable leads for further design and synthesis of more active
anticancer analogues (9-13). It has been widely reported that anticancer activity of coumarin derivatives
may be due to the inhibition of aromatase (14), carbonic anhydrase (15), steroid sulfatase (16), MEK1
kinase (17), Human telomerase reverse transcriptase (hTERT) (18), G-quadruplexes and so on (19).
Even though the application of coumarin derivatives in anticancer therapy constitutes an exploitable source
of new anticancer agents, the details of the relationship between the structure and biological activity of
these coumarins remain to be fully understood. Therefore the coumarin scaffold remains of great interest
for researchers. Recently, some 3-substituted coumarins, such as compounds Іa and Іb (Fig. 1), which can
inhibit telomerase activity and bind to G-quadruplexes respectively, exhibit significant potency in cancer
concerning targets (18, 19). Actually, warfarin and tecarfarin (Fig. 1) which have been successfully used in
the clinic to prevent thromboembolic disease are also 3-substituted coumarins (20). Furthermore,
7-position is also an attractive site in the modification of coumarins. For example, 7-hydroxycoumarin
derivatives have been investigated as potential lead structures for cancer drug development and the related
compounds scopoletin and esculetin (Fig. 1) show anti-proliferative effects in several tumor cell lines and
they have been proposed as potential anticancer agents (21, 22). However, rare examples are known for
structure activity relationship (SAR) of 3, 7-disubstituted coumarins as antitumor agents. In addition, the
benzimidazole moiety exists in many biologically active natural products and synthetic compounds. Some
of them show clinical value toward breast cancer, leukemia and other tumor cells (23-25). For these
reasons, we introduced benzimidazole moiety to the 3-position of coumarin scaffold which was substituted
at 7-position simultaneously to form compounds 7a-q. We also note that novobiocin derivative Ic (Fig. 1),
which has been identified as Hsp 90 inhibitor with anticancer activity, has an amide group at 3-position of
coumarin core through 3-amino group. Implied by these structure information and the idea of “overturn of
functional group”, we introduced an amide group to 3-position via 3-carboxyl group to design compounds
15a-j. Here we reported the design, synthesis, evaluation of 3, 7-disubstituted coumarines 7a-q and 15a-j
(Fig. 2) and SAR analysis found that methylation of benzimidazole moiety at 3-position of coumarin
exhibited significant enhance of anticancer activity.
Methods and Materials
Chemistry
General
All reagents were purchased from Shanghai Chemical Reagent Company. Column chromatography (CC):
silica gel 60 (200–300mesh). Thin-layer chromatography (TLC): silica gel 60 F254 plates (254 nm;
Qingdao Ocean Chemical Company, China). M.p.: capillarytube; uncorrected. IR spectra: Shimadzu
FTIR-8400Sspectrophotometer; in cm-1. 1H NMR spectra: Bruker ACF-500Q apparatus at 500 MHz or
Bruker AV-300 apparatus at 300MHz, in CDCl3 unless otherwise indicated; d in ppm rel. to Me4Si, J in
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Hz. Mass spectrometry (MS): Hewlett–Packard 1100 LC/MSD spectrometer; in m/z. Elemental analyses:
CHN-O-Rapid instrument and were within 0.4% of the theoretical values.
General procedure for the preparation of 7a-q
A mixture of 4-substituted salicylaldehyde 1 (41.4 mmol) and diethyl malonate (2) (48.1 mmol) in ethanol
(50 mL) was added 1mL piperidine and 50μL acetic acid and then heated to reflux overnight, cooled to
room temperature. Reaction mixture was poured into 200 mL water. The resulting precipitate was
collected via vacuum filtration, washed with cold water and dried under vacuum to give 7-substituted ethyl
2-oxo-2H-chromene-3-carboxylate 3, followed by hydrolysis with 25 mL aqueous NaOH (30%, w/w) in 50
mL ethanol at room temperature to give 26.8 mmol key intermediate 4 with 65% yield in two steps. A
mixture of EDCHCl (3.8 mmol), triethylamine (7.6 mmol) and HOBTH2O (5.7 mmol) in 15mL DMF
was added 4 (1.9 mmol) and after stirring at room temperature for 0.5 h, substituted o-phenylenediamine 5
(1.9 mmol) was added. Continued stirring for 2 h at 80 °C. Reaction mixture was poured into 50 mL water.
The resulting precipitate was collected via vacuum filtration, washed with cold water and dried under
vacuum to give crude product 6 without further purification. Refluxing intermediate 6 in 10 mL acetic acid
for 3 h afforded desired product 7a-q. Concentrated to dryness and the residue was purified by
chromatography column (1% methanol in dichloromethane) to give target compounds 7a-q with 80% yield
in two steps.
General procedure for the preparation of 15a-f
Substituted benzaldehyde 8 (100 mmol), hydroxylamine hydrochloride (200 mmol) and NaOH (200 mmol)
in 20 mL ethanol was refluxed for 4 h. Cooled down and precipitate was filtered off. Filtrate was
concentrated and residue was dissolved in 200 mL ethyl acetate, washed by water (50 mL×3), dried by
Na2SO4, and concentrated to give product 9 in 97% yield. Stirring intermediate 9 (50 mmol) and NCS (50
mmol) in 70mL DMF under room temperature for 4 h. Reaction mixture was poured into 250 mL water
and extracted by ethyl acetate (100 mL×2), washed by water (75 mL×3), dried by Na2SO4, and
concentrated to give 10 without further purification. Methyl acetoacetate (20 mmol) was stirred in 5.4 g
MeONa/MeOH (20%, w/w) under room temperature. After 30 min, the mixture was added dropwise to the
solution of 10 (20 mmol) in 20 mL THF under ice-water bath. After addition, the reaction was warmed up
to room temperature and stirred overnight, and concentrated to dryness, diluted in 100mL ethyl acetate and
200 mL water, extracted by ethyl acetate (50 mL×2). Combined organic layer was dried by Na2SO4,
purified by chromatography column (ethyl acetate/hexane=1/12) to give ester 11, followed by hydrolysis
in 30mL THF and 13mL aqueous NaOH (10%, w/w) to obtained acid 12 with 40% yield in two steps. A
mixture of EDCHCl (7.0 mmol), triethylamine (14.0 mmol) and HOBTH2O (10.5 mmol) in 10 mL DMF
was added 12 (3.5 mmol) and after stirring at room temperature for 0.5 h, N-Boc-piperazine (3.5 mmol)
was added. Continued stirring at room temperature overnight. Reaction mixture was poured into 70 mL
water. The resulting precipitate was collected via vacuum filtration, washed with cold water and dried
under vacuum to give crude product 13 without further purification (57% yield). Treatment 13 (2.0 mmol)
with 2 mL trifluoroacetic acid in 35 mL dichloromethane at room temperature for 2 h to give compound 14
in 81.0% yield. In the last step, target compounds 15a-f were obtained by coupling key intermediate 4 with
compound 14 under the same condition as preparation of 13 in 75% yield. Compounds 15g-h and 15i-j
were prepared in the similar way, but starting materials were 4-methyl-1, 2, 3-thiadiazole-5-carboxylic
acid and 4-oxo-4H-chromene-2-carboxylic acid, which were commercially available.
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7-(diethylamino)-3-(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)-2H-chromen-2-one (7a)
Yield: 80.0%, mp 283-285 °C.IR (KBr, cm-1): 3136, 1704, 1615, 1591, 1528. 1H NMR (500 MHz, CDCl3)
δ (ppm): 1.26 (t, 6H, J = 7.0 Hz, -CH3), 2.39 (s, 6H, -CH3), 3.47 (q, 4H, J = 7.0Hz, -CH2-), 6.56 (d, 1H, J =
2.5 Hz, Ar-H), 6.67 (dd, 1H, J1 = 2.5 Hz, J2 = 9.0 Hz, Ar-H), 7.26 (s, 1H, Ar-H, overlapped by solvent
peak), 7.45 (d, 1H, J = 9.0 Hz, Ar-H), 7.52 (s, 1H, Ar-H), 8.89 (s, 1H, CH), 11.06 (s, 1H, -NH). MS (ESI,
m/z): 362.9 ([M+H]+). Anal.calcd for C22H23N3O2: C 73.11; H 6.41; N 11.63. Found: C73.23; H 6.13; N
11.56.
7-(diethylamino)-3-(1, 5, 6-trimethyl-1H-benzo[d]imidazol-2-yl)-2H-chromen-2-one (7b)
1
Yield: 68.1%, mp 189-191 °C. IR (KBr, cm-1): 3136, 1707, 1611, 1527. H NMR (300 MHz, CDCl3) δ
(ppm): 1.26 (t, 6H, J = 7.2 Hz, -CH3), 2.40 (s, 3H, -CH3), 2.43 (s, 3H, -CH3), 3.47 (q, 4H, J = 7.2 Hz,
-CH2-), 3.83 (s, 1H, -NCH3), 6.53 (d, 1H, J = 2.4Hz, Ar-H), 6.65 (dd, 1H, Ar-H, J1 = 2.4 Hz, J2 = 9.0 Hz),
7.21 (s, 1H, Ar-H); 7.47 (d, 1H, J = 9.0 Hz, Ar-H), 7.66 (s, 1H, Ar-H), 8.46 (s, 1H, CH). MS (ESI, m/z):
376.6 ([M+H]+). Anal.calcd for C23H25N3O2: C 73.57; H 6.71; N 11.19. Found: C 73.72; H 6.43; N 11.33.
7-(diethylamino)-3-(5-methyl-1H-benzo[d]imidazol-2-yl)-2H-chromen-2-one (7c)
Yield: 82.0%, mp 212-214 °C. IR (KBr, cm-1): 3134, 1687, 1618, 1595, 1531. 1H NMR (500 MHz, CDCl3)
δ (ppm): 1.26 (t, 6H, J = 7.0 Hz, -CH3), 2.50 (s, 6H, -CH3), 3.51 (q, 4H, J = 7.0 Hz, -CH2-), 6.55 (d, 1H, J
= 2.0 Hz, Ar-H), 6.67 (dd, 1H, J1 = 2.5Hz, J2 = 9.0Hz, Ar-H), 7.10 (d, 1H, J = 7.5Hz, Ar-H), 7.26 (s, 1H,
Ar-H, overlapped by solvent peak), 7.46 (m, 1H, Ar-H), 7.50 (d, 1H,J = 9.0 Hz, Ar-H), 9.03 (s, 1H, CH),
11.61 (s, 1H, -NH). MS (ESI, m/z): 348.2 ([M+H]+). Anal.calcd for C21H21N3O2: C 72.60; H 6.09; N 12.10.
Found: C 72.87; H 5.83; N 12.24.
7-(diethylamino)-3-(1, 5-dimethyl-1H-benzo[d]imidazol-2-yl)-2H-chromen-2-one (7d)
Yield: 78.3%, mp 194-196 °C. IR (KBr, cm-1): 3135, 1697, 1609, 1590, 1535. 1HNMR (500 MHz, CDCl3)
δ (ppm):1.26 (t, 6H, J = 7.0 Hz, -CH3), 2.50 (s, 3H, -CH3), 3.47 (q, 4H, J = 7.0 Hz, -CH2-), 3.79 (s, 1H,
-NCH3), 6.54 (d, 1H, J = 2.5 Hz, Ar-H), 6.64 (dt, 1H, J1 = 2.5 Hz, J2 = 9.0 Hz, Ar-H), 7.12-7.41 (m, 3H,
Ar-H), 7.68 (d, 1H, Ar-H, J = 9.0 Hz), 8.24 (s, 1H, CH). 13CNMR (300MHz, CDCl3) δ (ppm): 157.5, 147.1,
147.0, 132.9, 131.9, 130.1, 130.0, 124.4, 123.8, 119.2, 119.0, 109.5, 109.4, 109.1, 108.4, 97.0, 45.0, 31.8,
31.6, 21.9, 21.5, 12.4. MS (ESI, m/z): 362.3 ([M+H]+). Anal.calcd for C22H23N3O2: C 73.11; H 6.41; N
11.63. Found: C 73.02; H6.69; N 11.74. HR-TOF-MS m/z 384.1681 ([M+ Na]+, C22H23N3O2Na+; Calcd
384.1688). Purity: 99.4% (determined by HPLC, area %).
7-(diethylamino)-3-(5-methoxy-1H-benzo[d]imidazol-2-yl)-2H-chromen-2-one (7e)
Yield: 82.9%, mp 220-223 °C. IR (KBr, cm-1): 3135, 1695, 1596, 1530, 1508. 1HNMR (300 MHz, CDCl3)
δ (ppm):1.19 (t, 6H, J = 7.2 Hz, -CH3), 3.40 (q, 4H, J = 7.2 Hz, -CH2-), 3.81 (s, 3H, -OCH3), 6.49 (d, 1H, J
= 2.4 Hz, Ar-H), 6.63 (dd, 1H, J1 = 9 Hz,J2=2.4 Hz,Ar-H), 6.87 (dd, 1H, J1 = 2.4 Hz, J2 = 9.0 Hz,Ar-H),
7.26 (s, 1H, Ar-H, overlapped by solvent peak), 7.42 (d, 1H, Ar-H, J =9.0 Hz), 8.92 (s, 1H, CH), 11.16 (s,
1H, -NH). MS (ESI, m/z): 364.3 ([M+H]+). Anal.calcd for C21H21N3O3: C 69.41; H 5.82; N 11.56. Found:
C 69.71; H 5.59; N 11.47.
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7-(diethylamino)-3-(5-hydroxy-1H-benzo[d]imidazol-2-yl)-2H-chromen-2-one (7f)
Yield: 94.0%, mp 232-235 °C. IR (KBr, cm-1): 3135, 1695, 1619, 1591, 1525. 1HNMR (300 MHz, CDCl3)
δ (ppm): 1.26 (t, 6H, J = 7.2 Hz, -CH3), 3.48 (q, 4H, -CH2, J = 7.2 Hz, -CH2-), 6.56 (d, 1H, J = 2.1 Hz,
Ar-H), 6.67 (dd, 1H, J1 = 9.0 Hz, J2 = 2.1 Hz, Ar-H), 6.83 (dd, 1H, J1 = 2.1 Hz, J2 = 8.4 Hz, Ar-H), 7.26 (s,
1H, ArH, overlapped by solvent peak), 7.45 (d, 1H, Ar-H, J = 9.0 Hz), 7.60 (s, 1H, Ar-H), 8.87 (s, 1H, CH),
11.16 (s, 1H, -NH). MS (ESI, m/z): 350.2 ([M+H]+). Anal.calcd for C20H19N3O3: C 68.75; H 5.48; N 12.03.
Found: C 69.04; H 5.37; N 11.88.
3-(6-benzoyl-1H-benzo[d]imidazol-2-yl)-7-(diethylamino)-2H-chromen-2-one (7g)
Yield: 62.0%, mp 263-267 °C. IR (KBr, cm-1): 3135, 1713, 1618, 1594, 1526. 1HNMR (300 MHz, CDCl3)
δ (ppm): 1.26 (t, 6H, J = 7.2 Hz, -CH3), 3.51 (q, 4H, J = 7.2 Hz, -CH2-), 6.56 (d, 1H, J = 2.4 Hz, Ar-H),
6.73 (dd, 1H, J1 = 2.4 Hz, J2 = 9.0 Hz, Ar-H), 7.47-7.63 (m, 5H, Ar-H), 7.82-7.91 (m, 4H, Ar-H), 8.89 (s,
1H, CH). MS (ESI, m/z): 438.2 ([M+H]+). Anal.calcd for C27H23N3O3: C 74.12; H 5.30; N 9.60. Found: C
74.35; H 5.21; N 9.48.
3-(6-bromo-1H-benzo[d]imidazol-2-yl)-7-(diethylamino)-2H-chromen-2-one (7h)
Yield: 83.0%, mp 285-289 °C. IR (KBr, cm-1): 3135, 1695, 1624, 1592, 1526. 1HNMR (300 MHz, CDCl3)
δ (ppm): 1.26(t, 6H, J = 7.2 Hz, -CH3), 3.47 (q, 4H, J = 7.2 Hz, -CH2-), 6.57 (d, 1H, Ar-H, J = 2.4 Hz),
6.68 (dd, 1H, J1 = 9.0 Hz, J2 = 2.4 Hz, Ar-H), 7.36-7.50 (m, 4H, Ar-H), 8.97 (s, 1H, CH), 11.34 (s, 1H,
-NH). MS (ESI, m/z): 412.0/414.1 ([M+H]+). Anal.calcd for C20H18BrN3O2: C 58.26; H 4.40; N 10.19.
Found: C 58.58; H 4.29; N 10.03.
3-(5,6-dichloro-1H-benzo[d]imidazol-2-yl)-7-(diethylamino)-2H-chromen-2-one (7i)
Yield: 77.6%, mp 267-270 °C. IR (KBr, cm-1): 3137, 1694, 1619, 1592, 1522. 1HNMR (300 MHz, CDCl3)
δ (ppm): 1.28 (t, 6H, J = 7.2 Hz, -CH3), 3.50 (q, 4H, J = 7.2 Hz, -CH2-), 6.56 (d, 1H, J = 2.1 Hz, Ar-H),
6.72 (dd, 1H, J1 = 2.1 Hz, J2 = 9.0 Hz, Ar-H), 7.60-7.85 (m, 3H , Ar-H), 9.46 (s, 1H, CH). MS (ESI, m/z):
404.5 ([M+H]+). Anal.calcd for C20H17Cl2N3O2: C 59.71; H 4.26; N 10.45. Found: C 59.39; H 4.53; N
10.57.
7-(diethylamino)-3-(6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)-2H-chromen-2-one (7j)
Yield: 47.4%, mp 305-307°C. IR (KBr, cm-1): 3132, 1708, 1620, 1596, 1532. 1HNMR (300 MHz, CDCl3)
δ (ppm): 1.27 (t, 6H, J = 7.2 Hz, -CH3), 3.50 (q, 4H, J = 7.2 Hz, -CH2-), 6.57 (d, 1H, J = 2.4 Hz, Ar-H),
6.70 (dd, 1H, J1 = 2.4 Hz, J2 = 9.0 Hz, Ar-H), 7.51-7.55 (m, 2H , Ar-H), 7.88-8.03 (m, 2H , Ar-H), 9.10 (s,
1H, CH), 11.58 (s, 1H, -NH). MS (ESI, m/z): 402.2 ([M+H]+). Anal.calcd for C21H18F3N3O2: C 62.84; H
4.52; N 10.47. Found: C 63.05; H 4.39; N 10.61.
7-(diethylamino)-3-(1-phenyl-1H-benzo[d]imidazol-2-yl)-2H-chromen-2-one (7k)
Yield: 43.4%, mp 198-200 °C. IR (KBr, cm-1): 3137, 1715, 1618, 1601, 1500. 1HNMR (300 MHz, CDCl3)
δ (ppm): 1.22 (t, 6H, J = 7.2 Hz, -CH3), 3.42 (q, 4H, J = 7.2 Hz, -CH2-), 6.42 (d, 1H, J = 2.4 Hz, Ar-H),
6.60 (dd, 1H, J1 = 2.4 Hz, J2 = 9.0 Hz, Ar-H), 6.87 (dd, 1H, J1 = 2.4 Hz, J2 = 9 Hz, Ar-H), 7.29-7.50 (m, 9H,
Ar-H), 7.89 (d, 1H, J = 7.5 Hz, Ar-H), 8.23 (s, 1H, CH). MS (ESI, m/z): 410.1 ([M+H]+). Anal.calcd for
C26H23F3N3O2: C 76.26; H 5.66; N 10.26. Found: C 76.59; H 5.43; N 10.08.
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3-(1-benzyl-1H-benzo[d]imidazol-2-yl)-7-(diethylamino)-2H-chromen-2-one (7l)
Yield: 37.6%, mp 201-203 °C. IR (KBr, cm-1): 3131, 1728, 1609, 1601, 1505. 1HNMR (300 MHz, CDCl3)
δ (ppm): 1.24 (t, 6H, J = 7.2 Hz, -CH3), 3.44 (q, 4H, J = 7.2 Hz, -CH2-), 5.52 (s, 2H, -CH2Ar), 6.50 (d, 1H,
J = 2.1 Hz, Ar-H), 6.61 (dd, 1H, J1 = 2.1 Hz, J2=9.0 Hz, Ar-H), 7.19-7.31 (m, 9H, Ar-H), 7.80 (d, 1H, J =
7.5 Hz, Ar-H), 8.06 (s, 1H, CH). MS (ESI, m/z): 424.4 ([M+H]+). Anal.calcd for C27H25N3O2: C 76.57; H
5.95; N 9.92. Found: C 76.73; H 5.64; N 9.98.
3-(1H-benzo[d]imidazol-2-yl)-7-(4-benzylpiperazin-1-yl)-2H-chromen-2-one (7m)
Yield: 63.0%, mp 230-232 °C. IR (KBr, cm-1): 3135, 1686, 1618, 1596, 1522. 1HNMR (300 MHz, CDCl3)
δ (ppm): 2.64-2.68 (m, 4H, -CH2-), 3.45-3.46 (m, 4H, -CH2-), 3.61 (s, 2H, -CH2Ar), 6.75-7.73 (m, 12H,
Ar-H), 8.07 (s, 1H, CH), 11.27 (s, 1H, NH). MS (ESI, m/z): 437.2 ([M+H]+). Anal.calcd for C27H24N4O2: C
74.29; H 5.54; N 12.84. Found: C 74.61; H 5.33; N 12.72.
7-(4-benzylpiperazin-1-yl)-3-(1-methyl-1H-benzo[d]imidazol-2-yl)-2H-chromen-2-one (7n)
Yield: 50.2%, mp 187-190 °C. IR (KBr, cm-1): 3128, 1677, 1630, 1588, 1519. 1HNMR (300 MHz, CDCl3)
δ (ppm): 2.65-2.69 (m, 2H, -CH2-), 3.43-3.348 (m, 4H, -CH2-), 3.61 (s, 2H, -CH2Bn), 3.82 (s, 3H, -NCH3),
7.64- 6.80 (m, 12H, Ar-H), 8.22 (s, 1H, CH). MS (ESI, m/z): 451.2 ([M+H]+). Anal.calcd for C28H26N4O2:
C 74.65; H 5.82; N 12.44. Found: C 74.60; H 5.53; N 12.76.
3-(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)-7-methoxy-2H-chromen-2-one (7o)
Yield: 67.6%, mp 178-180 °C. IR (KBr, cm-1): 3134, 1669, 1621, 1578, 1526. 1HNMR (300 MHz, CDCl3)
δ (ppm): 2.42(d, 6H, J = 9.0 Hz, Ar-CH3), 3.91(s, 3H, -OCH3), 6.90 (d, 1H, J = 2.4Hz, Ar-H), 6.98 (dd, 1H,
J1 = 2.4 Hz, J2 = 9.0 Hz, Ar-H), 7.51 (s, 2H, Ar-H), 7.75 (d, 1H, J = 9.0Hz, Ar-H), 9.66 (s, 1H, CH). MS
(ESI, m/z): 321.3 ([M+H]+). Anal.calcd for C19H16N2O3: C 71.24; H 5.03; N 8.74. Found: C 71.55; H 4.89;
N 8.59.
7-methoxy-3-(1,5,6-trimethyl-1H-benzo[d]imidazol-2-yl)-2H-chromen-2-one (7p)
Yield: 79.2%, mp 189-191 °C. IR (KBr, cm-1): 3139, 1657, 1633, 1587, 1530. 1HNMR (300 MHz, CDCl3)
δ (ppm): 2.41(d, 6H, J = 9.0 Hz, Ar-CH3), 3.77 (s, 3H, -NCH3), 3.91 (s, 3H, -OCH3), 6.90-6.93 (m, 2H,
Ar-H), 7.17 (s, 1H, Ar-H), 7.49-7.53 (m, 2H, Ar-H), 8.27 (s, 1H, CH). MS (ESI, m/z): 335.1 ([M+H]+).
Anal.calcd for C20H18N2O3: C 71.84; H 5.43; N 8.38. Found: C 71.65; H5.32; N 8.63.
3-(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)-2H-chromen-2-one(7q)
Yield: 73.8%, mp 171-173 °C. IR (KBr, cm-1): 3131, 1649, 1641, 1576, 1535. 1HNMR (300 MHz, CDCl3)
δ (ppm): 2.41 (d, 6H, J=9.0 Hz, Ar-CH3), 7.34-7.75 (m, 6H, Ar-H), 9.18 (s, 1H, CH), 11.2 (s, 1H, NH). MS
(ESI, m/z): 290.1 ([M+H]+). Anal.calcd for C18H14N2O2: C 74.47; H 4.86; N 9.65. Found: C 74.22; H 4.98;
N 9.53.
3-(4-(3-(2,6-dichlorophenyl)-5-methylisoxazole-4-carbonyl)piperazine-1-carbonyl)-7-(di-ethylamino)-
2H-chromen-2-one (15a)
Yield: 75.0%, mp 325-327 °C. IR (KBr, cm-1): 3135, 1701, 1618, 1596, 1522. 1HNMR (300 MHz, CDCl3)
δ (ppm): 1.22 (t, 6H, J = 7.2 Hz, -CH3), 2.60 (s, 3H, -CH3), 3.17-3.40 (m, 4H, -CH2-), 3.40-3.53 (m, 4H,
-CH2-), 3.44 (q, 4H, J = 7.2 Hz, -CH2-), 6.53 (d, 1H, J = 2.4 Hz, Ar-H), 6.67 (dd, 1H, J1 = 2.4 Hz, J2 = 8.7
Hz, Ar-H), 7.29-7.46 (m, 4H, Ar-H), 7.86 (s, 1H, CH). MS (ESI, m/z): 584.3 ([M+H]+). Anal.calcd for
C29H28Cl2N4O5: C 59.70; H 4.84; N 9.60. Found: C 59.42; H 4.95; N 9.51.
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3-(4-(3-(2,6-dichlorophenyl)-5-methylisoxazole-4-carbonyl)piperazine-1-carbonyl)-2H-c
-hromen-2-one (15b)
Yield: 62.3%, mp 289-391 °C. IR (KBr, cm-1): 3133, 1719, 1608, 1581, 1517. 1HNMR (300 MHz, CDCl3)
δ (ppm): 2.60 (s, 3H, -CH3), 3.17-3.20 (m, 2H, -CH2-), 3.40-3.55 (m, 6H, -CH2-), 7.32-7.40 (m, 3H, Ar-H),
7.42-7.46 (m, 2H, Ar-H), 7.53-7.55 (m, 1H, Ar-H), 7.60-7.64 (m, 1H, Ar-H), 7.94 (s, 1H, CH). MS (ESI,
m/z): 513.4 ([M+H]+). Anal.calcd for C25H19Cl2N3O5: C 58.61; H3.74; N8.20. Found: C 58.93; H3.49;
N8.11.
7-(diethylamino)-3-(4-(5-methyl-3-(2-(trifluoromethyl)phenyl)isoxazole-4-carbonyl)
piper-azine-1-carbonyl)-2H-chromen-2-one (15c)
1
Yield: 65.0%, mp 235-237 °C. IR (KBr, cm-1): 3135, 1705, 1643, 1620, 1600, 1518. HNMR (300 MHz,
CDCl3) δ (ppm): 1.23 (t, 6H, J = 6.9 Hz, -CH3), 2.53 (s, 3H, -CH3), 2.96-3.26 (m, 3H, -CH2-), 3.45 (q, 4H,
J = 6.9 Hz, -CH2-), 3.50-3.87 (m, 5H, -CH2-), 6.51-6.67 (m, 2H, Ar-H), 7.29-7.32 (m, 1H, Ar-H), 7.71-7.83
(m, 4H, Ar-H), 7.86 (s, 1H, CH). MS (ESI, m/z): 583.3 ([M+H]+). Anal.calcd for C30H29F3N4O5: C 61.85;
H 5.02; N 9.62. Found: C 61.66; H 5.17; N 9.86.
3-(4-(5-methyl-3-(2-(trifluoromethyl)phenyl)isoxazole-4-carbonyl)piperazine-1-carbonyl)-2H-chrom
en-2-one (15d)
1
Yield: 73.8%, mp 278-280 °C. IR (KBr, cm-1): 3139, 1715, 1633, 1629, 1615, 1530. HNMR (300 MHz,
CDCl3) δ (ppm): 2.57 (s, 3H, -CH3), 3.14-3.54 (m, 8H, -CH2-), 7.33-7.40 (m, 2H, Ar-H), 7.54-7.56 (m, 2H,
Ar-H), 7.61-7.68 (m, 3H, Ar-H), 7.82-7.85 (m, 1H, Ar-H),7.95 (s, 1H, CH). MS (ESI, m/z): 512.4
([M+H]+). Anal.calcd for C26H20F3N3O5: C 61.06; H 3.94; N 8.22. Found: C61.34; H 3.89; N 8.07.
7-(diethylamino)-3-(4-(5-methyl-3-(4-(trifluoromethyl) phenyl) isoxazole-4-carbonyl)-piper
-azine-1-carbonyl)-2H-chromen-2-one (15e)
1
Yield: 67.0%, mp 220-223 °C. IR (KBr, cm-1): 3135, 1705, 1643, 1620, 1600, 1518. HNMR (300 MHz,
CDCl3) δ (ppm): 1.25 (t, 6H, -CH3, J = 7.2 Hz), 2.55 (s, 3H, -CH3), 3.44 (q, 4h, J = 7.2Hz, -CH2-),
3.41-3.48 (m, 8H, -CH2-), 6.59 (d, 1H, Ar-H, J = 2.1 Hz), 6.74 (dd, 1H, Ar-H, J1 = 2.1 Hz, J2 = 9.0 Hz),
7.33 (d, 1H, J = 9.0 Hz, Ar-H), 7.52-7.55 (m, 1H, Ar-H), 7.62-7.66 (m, 2H, Ar-H), 7.80-7.83 (m, 1H, Ar-H),
7.86 (s, 1H, CH). MS (ESI, m/z): 583.3 ([M+H]+). Anal.calcd for C30H29F3N4O5: C 61.85; H 5.02; N 9.62.
Found: C 61.57; H5.21; N9.85.
3-(4-(5-methyl-3-(4-(trifluoromethyl)phenyl)isoxazole-4-carbonyl)piperazine-1-carbonyl)-2H-chrom
en-2-one (15f)
1
Yield: 59.1%, mp 251-253°C. IR (KBr, cm-1): 3135, 1710, 1644, 1625, 1600, 1515. HNMR (300 MHz,
CDCl3) δ (ppm):2.54 (s, 3H, -CH3), 3.26-3.38 (m, 4H, -CH2-), 3.80-3.86 (m, 4H, -CH2-), 7.31-7.36 (m, 2H,
Ar-H), 7.53-7.64 (m, 2H, Ar-H), 7.77 (dd, 4H, J = 8.1Hz, Ar-H), 7.94 (s, 1H, CH). MS (ESI, m/z): 512.1
([M+H]+). Anal.calcd for C26H20F3N3O5: C 61.06; H 3.94; N 8.22. Found: C 61.21; H 3.66; N 8.37.
7-(diethylamino)-3-(4-(4-methyl-1, 2, 3-thiadiazole-5-carbonyl) piperazine-1-carbonyl)-2H
-chromen-2-one (15g)
1
Yield: 76.0%, mp 195-197 °C. IR (KBr, cm-1): 3133, 1704, 1621, 1522. HNMR (300 MHz, CDCl3) δ
(ppm): 1.25 (t, 6H, J = 7.2Hz, -CH3), 2.76 (s, 3H, -CH3), 3.45 (q, 4H, J = 7.2Hz, -CH2-), 3.48-3.77 (m, 8H,
-CH2-), 6.52 (d, 1H, J = 1.8 Hz,Ar-H), 6.67 (dd, 1H, Ar-H, J1 = 2.4 Hz, J2 = 8.7 Hz), 7.33 (d, 1H, J = 8.7
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Hz, Ar-H), 7.93 (s, 1H, CH). MS (ESI, m/z): 456.2 ([M+H]+). Anal.calcd for C22H25N5O4S: C 58.01; H
5.53; N 15.37. Found: C 58.34; H 5.38; N 15.48.
3-(4-(4-methyl-1, 2, 3-thiadiazole-5-carbonyl) piperazine-1-carbonyl)-2H-chromen-2-one (15h)
1
Yield: 73.8%, mp204-206 °C. IR (KBr, cm-1): 3135, 1713, 1614, 1521. HNMR (300 MHz, CDCl3) δ
(ppm):2.76 (s, 3H, -CH3), 3.44-3.82 (m, 8H, -CH2-), 7.33-7.40 (m, 2H, Ar-H), 7.55-7.66 (m, 2H, Ar-H),
8.01 (s, 1H, CH). MS (ESI, m/z): 385.3 ([M+H]+). Anal.calcd for C18H16N4O4S: C 56.24; H 4.20; N 14.57.
Found: C 56.52; H 4.03; N 14.76.
7-(diethylamino)-3-(4-(4-oxo-4H-chromene-2-carbonyl) piperazine-1-carbonyl)-2H
-chro-men-2-one (15i)
1
Yield: 78.0%, mp 147-150 °C. IR (KBr, cm-1): 3135, 1712, 1649, 1619, 1590, 1518. HNMR (300 MHz,
CDCl3) δ (ppm): 1.25 (t, 6H, J = 7.5Hz, -CH3), 3.45 (q, 4H, J = 7.5Hz, -CH2-), 3.52-3.88 (m, 8H, -CH2-),
6.67-6.52 (m, 3H, Ar-H), 7.33 (d, 1H, Ar-H, J = 8.7 Hz), 7.43-7.50 (m, 2H, Ar-H), 7.71-7.75 (m, 1H, Ar-H),
7.94 (s, 1H, CH), 8.22 (d, 1H, Ar-H, J = 8.4 Hz). MS (ESI, m/z): 502.3 ([M+H]+). Anal.calcd for
C28H27N3O6: C 67.05; H 5.43; N 8.38. Found: C 67.38; H 5.22; N 8.19.
3-(4-(4-oxo-4H-chromene-2-carbonyl)piperazine-1-carbonyl)-2H-chromen-2-one (15j)
1
Yield: 68.5%, mp164-166 °C. IR (KBr, cm-1): 3135, 1713, 1645, 1585. HNMR (300 MHz, CDCl3) δ
(ppm): 3.50-3.91 (m, 8H, -CH2-), 6.60-6.61 (m, 1H, Ar-H), 7.34-7.75 (m, 7H, Ar-H), 8.04 (s, 1H, CH),
8.26 (d, 1H, Ar-H, J = 7.8 Hz). MS (ESI, m/z): 431.3 ([M+H]+). Anal.calcd for C24H18N2O6: C 66.97; H
4.22; N 6.51. Found: C 67.22; H 4.13; N 6.39.
In vitro anticancer activity
Evaluation of Growth inhibition rate against K562 cells
K562 cells were added into the wells at a density of 3000 cells/well. Drug (1.0 μL) was transferred into
100 μL of culture medium in 96-well plates and the final concentration was approximately 1.0 μM, 0.5 μM
and 0.25 μM for each drug. Plates were incubated for three days at 37 ˚C and 5% CO2. Viable cell number
was assessed using a Dojindo CCK-8 kit (Dojindo Molecular Technologies, Inc.) according to
manufacturer’s instructions. Plates were read using a Synergy HT Microplate Reader (BioTek Instruments,
Inc.), and optical density (OD) values were recorded.
OD values were converted to cell numbers by comparing the experimental OD values to OD values for
known cell numbers. For this purpose, a standard curve was constructed for each cell type. Briefly, cells
were counted and diluted to 5 x105 cells / mL. A total of 100 µL (5 x104 cells) were seeded into the first
well of a 96-well plate. Each successive well was seeded with half as many cells as the previous well and
supplemented with RPMI medium to a total volume of 100 µL. Plates were read using the same procedure
as the experimental assays. A standard curve was constructed using an excel spreadsheet. OD value for
each experimental well was converted to cell number based on the standard curve. Growth inhibition was
calculated by first subtracting the original number of cells seeded (3000), and then using the following
formula: GI = (control cell number – experimental cell number)/control cell number.
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Evaluation of GI50 value against various cancer cell lines
Cancer cells (SN12C, OVCAR, BxPC-3, KATO-III, T24, SNU-1, WiDr, HeLa, K562, and AGS) were
prepared and diluted to a concentration of 3.5 x 104 cells / mL. The center 6x10 wells of the 96-well plates
were seeded with 100 µL of the cell suspension, totaling 3.5 x 103 cells / well. The border wells of the
96-well plates were seeded with 150 µL of autoclaved water to minimize evaporation of the center wells.
The plates were incubated in 5% CO2 at 37˚C overnight, giving ample time for adherent cell lines to
adhere to the plates. The following day, 1.0 µL of the prepared compounds (7b and 7d) dilutions (2-fold
dilutions) was added to each well, so that the highest final concentration was 10 µM with 5 successive 2x
dilutions. Plates were then incubated for another three days. A Dojindo CCK-8 kit was used to estimate the
number of viable cells in each well according to the manufacturer’s instructions. The data was then
analyzed to construct a GI50 curve and calculate the GI50 value using Sigmaplot 10.0 software.
Cell cycle analysis
AGS cells were prepared and seeded in 6-well multidishes (Thermo Scientific) at a concentration of 5x105
cells/well in 3 mL RPMI medium. Compound (7d) was prepared and added to the wells at a concentration
of 5 µM. Cells were incubated in 5% CO2 at 37˚C for 24, 48, 72, 96 hours. Then, cells were digested with
0.25% Trypsin and pelleted then washed twice with 1.0 mL PBS. After removing the supernatant, cells
were fixed with 70% ethanol and stored in 4˚C until all samples were taken. A 10x Permeabilization Buffer
(eBioscience) was diluted to 1x with deionized water and 500 µL was added to each sample. Samples were
gently vortexed before centrifugation. The supernatant was removed and cells were treated with a PI /
RNase Staining Buffer (BD Pharmingen) and incubated for 15 minutes at room temperature in darkness.
Cells were analyzed using flow cytometry and data was analyzed using FACSDiva Version 6.1 software
(BD Biosciences).
Results and discussion
Chemistry
The synthetic strategy to obtain the target compounds 7a-q is depicted in Scheme 1 (26-28). In order to
prepare benzimidazole substituted coumarins 7a-q, we began with the commercially available starting
material 4-substituted salicylaldehyde 1 and diethylmalonate (2). In a typical protocol, 4-substituted
salicylaldehyde 1 was refluxed with diethylmalonate in the presence of piperidine, acetic acid and ethanol
for 5 h to give 7-substituted ethyl 2-oxo-2H-chromene-3-carboxylate 3 followed by hydrolysis with NaOH
at room temperature to obtain 7-substituted 2-oxo-2H-chromene-3-carboxylic acid 4 with 65% yield in two
steps. Intermediate 4 reacted with substituted o-phenylenediamine 5 under N-(3-dimethylaminopropyl)-
N’-ethyl carbodiimide hydrochloride (EDCHCl), 1-hydroxybenzotriazole hydrate (HOBTH2O) and
triethylamine in N, N-dimethylformamide (DMF) as solvent at 80 °C for 2 h to give intermediate 6.
Compounds 7a-q was obtained by reflux 6 in acetic acid for 3 h with 80% yield in two steps.
Compounds 15a-j were synthesized by coupling key intermediate 4 with various heteroaromatic
carboxylic acid using piperazine as link, as described in Scheme 2 (29). Treatment substituted
benzaldehyde with hydroxylamine hydrochloride in ethanol using sodium hydroxide as base at refluxing
condition for 4 h gave compound 9 (97% yield). Chlorination of 9 was accomplished by
N-chlorosuccinimide (NCS) in DMF under room temperature for 4 h with 91% yield. Reaction
intermediate 10 with methyl acetoacetate in methanol / tetrahydrofuran (THF) using sodium methoxide
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(20%, w/w in methanol) at room temperature gave substituted methyl oxazole-4-carboxylate 11 followed
by hydrolysis with NaOH to obtain acid 12 (40% yield in two steps). Compound 12 was coupled with
N-tert-butoxycarbonyl (Boc) protected piperazine under EDCHCl, HOBTH2O and triethylamine at room
temperature overnight to give amide 13 (57% yield). Protecting group Boc was removed easily by
trifluoroacetic acid. Target compounds 15a-f were obtained by coupling key intermediate 4 with the
product of deprotection 14 with 75% yield. Compounds 15g-h and 15i-j could be prepared in the similar
way, as depicted in Scheme 2, but starting materials (heteroaromatic carboxylic acid in the left hand) were
4-methyl-1,2,3-thiadiazole-5-carboxylic acid and 4-oxo-4H-chromene-2-carboxylic acid, which were
commercially available. The structures of synthesized compounds were described in Table 1 and 2. All
compounds were characterized by 1H-NMR as well as mass spectroscopy.
In vitro anticancer activity
In the screening assay studies, all title compounds were evaluated for their anti-proliferative activity
against K562 cell lines. The cells were allowed to proliferate inpresence of testedmaterial at three
concentrations (1.0 μM, 0.50 μM, 0.25 μM) for 72 h, and the results were reported interms of inhibition
rate (Fig. 3 and Fig. 4). It was obvious that most of compounds 7a-q, which were substituted with
benzimidazole at 3-position of coumarin, had higher activity against K562, compared with 15a-j.
Compounds 7b and 7d were the most potent compounds against K562 at concentration of 1.0 μM with
95% and 94% growth inhibition rates respectively. In addition, even though concentration decreased to
0.25 μM, compound 7d still showed strong growth inhibition (88%). From the data in Fig. 4, it was found
that most of compounds 15a-j, which had amide group in 3-position of coumarin scaffold, showed weak
inhibition activity and only 15f, 15g, 15h and 15i had moderate activity with growth inhibition values
ranging from 18%-24% at 1.0 μM. The in vitro data indicated that benzimidazole substitution in
3-positon was more effective than amide substitution in 3-position. Comparing 7a with 7b, 7c with 7d and
7m with 7n, we can conclude that methylation of the N atom of benzimidazole can obviously enhance
anticancer activity. In an attempt to measure the effect of a bulky group at N atom of benzimidazole,
phenyl and benzyl was introduced to form compound 7k, 7l, but sharply decrease of activity was observed.
The activity data of 7a was higher than 7m, 7o, 7p and 7q, although they only had weak anticancer activity
against K562. Therefore, diethylamino at 7-position of coumarin scaffold may be more suitable than
4-benzylpiperazin and methoxyl group.
Furthermore, the most potent compound 7b and 7d were chose to evaluate anti-proliferative activity
against SN12C, OVCAR, BxPC-3, KATO-III, T24, SNU-1, WiDr, HeLa, K562, and AGS cell lines. The
cells were allowed to proliferate in the presence of tested compounds for 72 h, and the results were
reported in terms of 50% growth inhibition (GI50) (Table. 3). Overall, 7d exhibited high anti-proliferative
activity against SN12C, OVCAR, BxPC-3, KATO-III, T24, SNU-1, WiDr, HeLa, K562, and AGS cell
lines with GI50 values of 2.18, 2.33, 1.3, 0.59, 0.99, 0.67, 1.31, 3.96, 0.12, 0.78 μM, surpassing that of the
compound 7b. From these in vitro data, we conclude that compound 7d should be further explored.
Cell cycle analysis
Proliferation of AGS cells was evaluated against selected compound 7d (with high activity against AGS
cells, GI50 = 0.78 μM). The results of in vitro assay showed that compound 7d could inhibit proliferation of
AGS cells. In order to understand whether the reduced cell proliferation was due to cell cycle arrest, we
used flow cytometric analyses to measure the effect of compound 7d on induction of cell cycle arrest. As
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shown in Fig. 5, the results indicated that compound 7d could inhibit AGS cell proliferation. Furthermore
the fraction of AGS cells staying in the sub G0 phase was distinctly increased, whereas cells in the S and
G0 / G1 phase decreased after treatment with compound7d. These results suggest that compound 7d
inhibits proliferation, at least partially, via inducing cell cycle arrest.
Conclusions
In summary, we designed and synthesized series novel 3, 7-disubstituted coumarins as anticancer agent,
and evaluated their anti-proliferative activity against K562 cells. The most potent compounds, 7b and 7d
were chosen to evaluate their GI50 against SN12C, OVCAR, BxPC-3, KATO-III, T24, SNU-1, WiDr,
HeLa, K562, and AGS cell lines. Compound 7d was selected to for further pharmacological screening
including cell proliferation and cell cycle assay in the AGS cell line. In brief, based on reasonable drug
design and the result of this study, it can be concluded that 3, 7-disubstituted coumarinis a promising
scaffold in anticancer drug design. Furthermore, methylation of benzimidazole at 3-position of coumarin
exhibits significant enhancement of anticancer activity and diethylamino group in 7-position is also helpful
to its anti-proliferative activity. The most potent compound 7d inhibits proliferation of AGS cells via
inducing cell cycle arrest. Based on these preliminary results, further modification and optimization of the
most potent compounds, as well as detailed study of the pharmacological target, are warranted based on
reasonable molecular design.
Acknowledgments
The authors wish to thank Damin Zhao for his discussion in chemical synthesis and Dongjun Chen from
China Pharmaceutical University for his help in 1H-NMR testing.
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Figure captions
1) Figure 1. Structures of natural and synthetic coumarin derivatives.
2) Figure 2. Proposed 3, 7-disubstituted coumarin derivatives.
3) Figure 3. Growth inhibition rates of compounds 7a-q against K562 cells.
4) Figure 4. Growth inhibition rates of compounds 15a-j against K562 cells.
5) Figure5. Cell cycle analysis by flow cytometry in AGS cells after incubation with 5 µM of compound
7d for 0 h, 24 h, 48 h, 72 h, 96 h.
Table 1. Structures of compounds 7a-q.
Compd
R1
R2
R3
-H
7a
5, 6-dimethyl
7b
7c
7d
7e
7f
5, 6-dimethyl
5-CH3
-CH3
-H
5- CH3
-CH3
-H
5-OCH3
5-OH
-H
7g
7h
6-benzoyl
6-Br
-H
-H
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7i
7j
7k
7l
5, 6-dichloro
-H
-H
6-CF3
-H
-Ph
-Bn
-H
7m
7n
-H
-H
-H
-CH3
7o
7p
7q
-OCH3
-OCH3
-H
5, 6-dimethyl
5, 6-dimethyl
5, 6-dimethyl
-H
-CH3
-H
Table 2. Structures of compounds 15a-j.
Compd
15a
15b
15c
15d
15e
15f
R1
-H
-H
-H
-H
-H
R2
2, 6-dichloro
2, 6-dichloro
2-CF3
2-CF3
4-CF3
4-CF3
15g
15h
15i
-
-
-
-
15j
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Table 3. GI50 valuesof compounds 7b and 7d against various cancer cell lines
GI50 (µM)
Cell ID
7b
7d
SN12C
OVCAR
BXPC-3
KATO-III
T24
2.85±0.13
3.46±0.11
2.88±0.09
1.50±0.06
2.60±0.15
2.83±0.12
2.72±0.10
7.20±0.28
0.40±0.01
3.50±0.12
2.18±0.10
2.33±0.12
1.30±0.08
0.59±0.02
0.99±0.03
0.67±0.03
1.31±0.08
3.96±0.20
0.12±0.01
0.78±0.05
SNU-1
WIDR
HELA
K562
AGS
Figure 1. Structures of bioactive natural and synthetic coumarin derivatives.
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Figure 2.Proposed 3, 7-disubstituted coumarin derivatives.
1μM
1.0
0.5
0.0
0.5μM
0.25μM
7i
7j
7l
7f
7a
7c
7e
7k
7b
7d
7g 7h
7n 7o 7p 7q
CN
7m
Compoun ID
Figure3. Growth inhibition rates of compounds 7a-q against K562 cells.
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1μM
0.3
0.2
0.5μM
0.25μM
0.1
0.0
15i
15j
15f
CN
15a
15c
15e
15b
15d
15g
15h
-0.1
Compoun ID
Figure 4. Growth inhibition rates of compounds 15a-j against K562 cells.
M1M2
M1M2
M1M2
M3
M4
M3
M3
M4
M4
0 h
48 h
24 h
M1M2
M1M2
M3
M4
M3
M4
96 h
72h
Figure 5. Cell cycle analysis by flow cytometry in AGS cells after incubation with 5 µM of compound 7d
for 0 h, 24 h, 48 h, 72 h, 96 h.
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Scheme 1. The synthesis of 3, 7-disubstituted coumarin derivatives 7a-q. Reaction conditions: (i ) acetic
acid, piperidine, EtOH, reflux; ( ii ) NaOH, r t; ( iii ) EDCHCl, Et3N, HOBT, DMF, 80°C; ( iv ) acetic acid,
reflux.
Scheme 2. The synthesis of 3, 7-disubstituted coumarin derivatives 7a-q. Reaction conditions: ( i )
hydroxylamine hydrochloride, NaOH, EtOH, reflux; ( ii ) NCS, DMF, r t; ( iii ) methyl acetate, MeONa,
MeOH/THF, r t; ( iv ) NaOH, THF / H2O, r t; ( v ) EDCHCl, Et3N, HOBT, DMF, r t; ( vi ) trifluoroacetic;
( vii ) EDCHCl, Et3N, HOBT, DMF, r t.
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