Synthesis and cytotoxic activity of coumarin derivatives containing benzotriazole moieties

Article abstract of DOI:10.1080/10426500802561153

Three coumarin derivatives containing benzotriazole moieties, 3-(1-benzotriazolyl)-4-methyl-coumarin (BMC), 3-(1-benzotriazolyl)-4-methyl- benzo[7,8]coumarin (BMBC), and 3-(4-methyl-1-benzotriazolyl)-4-methyl-benzo[7,8] coumarin (MBMBC), were synthesized. Among them, 3-(4-methyl-1-benzotriazolyl)-4- methyl-benzo[7,8]coumarin has been structurally characterized by X-ray crystallography. The cytotoxic effect by Sulforhodamine B (SRB) assay with these compounds against the human hepatocellular carcinoma cell lines (HepG-2) was carried out in vitro. The preliminary results showed that all compounds inhibit HepG-2 growth effectively. Taylor & Francis Group, LLC.

Full text of DOI:10.1080/10426500802561153

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Synthesis and Cytotoxic
Activity of Coumarin
Derivatives Containing
Benzotriazole Moieties
Tianzhi Yu a , Peng Zhang a b , Yuling Zhao b , Hui
Zhang a , Duowang Fan a , Wenkui Dong b & Lan Ding
c
a Key Laboratory of Opto-Electronic Technology and
Intelligent Control (Lanzhou Jiaotong University),
Ministry of Education , Lanzhou, China
b School of Chemical and Biological Engineering ,
Lanzhou Jiaotong University , Lanzhou, China
c College of Life Science , Northwest Normal
University , Lanzhou, China
Published online: 21 Sep 2009.
To cite this article: Tianzhi Yu , Peng Zhang , Yuling Zhao , Hui Zhang , Duowang
Fan , Wenkui Dong & Lan Ding (2009) Synthesis and Cytotoxic Activity of Coumarin
Derivatives Containing Benzotriazole Moieties, Phosphorus, Sulfur, and Silicon and the
Related Elements, 184:10, 2655-2663, DOI: 10.1080/10426500802561153
To link to this article: http://dx.doi.org/10.1080/10426500802561153
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Phosphorus, Sulfur, and Silicon, 184:2655–2663, 2009
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500802561153
Synthesis and Cytotoxic Activity of Coumarin Derivatives
Containing Benzotriazole Moieties
Tianzhi Yu,1 Peng Zhang,1,2 Yuling Zhao,2 Hui Zhang,1
Duowang Fan,1 Wenkui Dong,2 and Lan Ding3
1Key Laboratory of Opto-Electronic Technology and Intelligent Control
(Lanzhou Jiaotong University), Ministry of Education, Lanzhou, China
2School of Chemical and Biological Engineering, Lanzhou Jiaotong
University, Lanzhou, China
3College of Life Science, Northwest Normal University, Lanzhou, China
Three coumarin derivatives containing benzotriazole moieties, 3-(1-benzotriazolyl)-
4-methyl-coumarin (BMC), 3-(1-benzotriazolyl)-4-methyl-benzo[7,8]coumarin
(BMBC),
and
3-(4-methyl-1-benzotriazolyl)-4-methyl-benzo[7,8]coumarin
(MBMBC), were synthesized. Among them, 3-(4-methyl-1-benzotriazolyl)-
4-methyl-benzo[7,8]coumarin has been structurally characterized by X-ray
crystallography. The cytotoxic effect by Sulforhodamine B (SRB) assay with these
compounds against the human hepatocellular carcinoma cell lines (HepG-2) was
carried out in vitro. The preliminary results showed that all compounds inhibit
HepG-2 growth effectively.
Keywords Benzotriazole; coumarin derivatives; cytotoxic activity; synthesis
INTRODUCTION
Coumarin and its derivatives are an important class of naturally occur-
ring benzopyrone derivatives with useful pharmacological activity,1–3
which are widely distributed in natural plants. Some of the coumarin
derivatives are also reported as antibacterial agents,4 and possess anti-
HIV5 and anticancer activities.1,5,6 The pharmacological and biochemi-
cal properties, and therapeutic applications of coumarins depend upon
the types of substituents in their basic structure. It was suggested
Received 25 September 2008; accepted 16 October 2008.
This work was supported by Program for Changjiang Scholars and Innovative Re-
search Team in University (IRT0629) and also supported by ‘Qing Lan’ talent engineer-
ing funds (QL-05–23A) by Lanzhou Jiaotong University. We also acknowledge financial
support from the NSFC (Grant 60776006).
Address correspondence to Tianzhi Yu, Key Laboratory of Opto-Electronic Technology
and Intelligent Control (Lanzhou Jiaotong University), Ministry of Education, Lanzhou
730070, China. E-mail: ytz823@hotmail.com
2655

2656
T. Yu et al.
that alterations in the chemical structure of coumarins could influ-
ence their biological properties. In addition, benzotriazole derivatives
are of biological, chemical, and industrial importance. The 1H-1,2,3-
Benzotriazole moiety has a documented prominence in a number of an-
titumor aromatase inhibitors.7 Benzotriazole derivatives exhibit good
pharmacological activities such as analgesic, anti-inflammatory, anti-
fungal, antineoplastic, antiviral, and antihypertensive activities.8
In view of the biological importance of the coumarin and 1H-1,2,3-
benzotriazole moieties, it prompted us to synthesize new heteroaro-
matic derivatives containing both these systems. Recently, we synthe-
sized three coumarin derivatives containing benzotriazole moieties (see
Figure 1) and tested them in vitro against the human hepatocellular
carcinoma cell lines (HepG-2).
RESULTS AND DISCUSSION
Synthesis
The coumarin derivatives 3-(4-methyl-1-benzotriazolyl)-4-methyl-
benzo[7,8]coumarin (MBMBC), 3-(1-benzotriazolyl)-4-methyl-benzo
[7,8]coumarin (BMBC), and 3-(1-benzotriazolyl)-4-methyl-coumarin
(BMC) were obtained from the starting substances 4-methyl-1H-
benzotriazole, 1H-benzotriazole, 2-acetyl-1-naphthol, and 2-acetyl-1-
phenol. The compounds were synthesized in three steps. The first
step was alkylation of benzotriazole derivatives with chloroacetic acid
in dry toluene. The second step was esterification of (4-methyl-1-
benzotriazolyl-) acetic acid or (1-benzotriazolyl-) acetic acid with 2-
acetyl-1-naphthol and 2-acetyl-1-phenol, respectively, in the presence
of phosphorus oxychloride in dry pyridine. The final step was cyclodehy-
dration of the intermediates using potassium hydroxide as catalyst in
dry pyridine. Noteworthy, in the same experimental conditions, Liu et
al. claimed that the esters took place in a rearrangement in third reac-
tion step and yielded 1, 3-diketones.11 However, due to the acidic char-
acter of the methylene group of the intermediate activated by strong
electron-withdrawing benzotriazolyl moiety, we suggest that the inter-
mediates were cyclodehydrated to form coumarin derivatives. When
treated with a base, the methylene group close to the benzotriazole is
easier to deprotonate, therefore, a stable carbanion forms. The carban-
ion connects to the carbonyl group, and the oxy anion attacks the hy-
drogen intramolecularly. An H-O− is then eliminated, and a new C C
bond is formed. The crystal structures of the compounds corroborate
our deductions.

Coumarin Derivatives Containing Benzotriazole Moieties
2657
FIGURE 1 Structures and the synthetic route of the coumarin derivatives.
X-Ray Crystallographic Analysis
The crystal structure and packing diagram of MBMBC are illustrated
in Figures 2 and 3, respectively. An acceptable crystal of MBMBC suit-
able for X-ray analysis was obtained by slow evaporation of an ethanol
solution, and the structure was determined by X-ray crystallography.
Crystal data: C21H15N3O2, M = 341.36, monoclinic, crystal dimensions
0.21 × 0.20 × 0.12 mm, space group P21/n, a = 7.3632(9) Å, b =

2658
T. Yu et al.
FIGURE 2 Crystal structure of MBMBC.
◦
14.0875(18) Å, c = 16.004(2) Å, α = 90◦, β = 93.699(2) , γ = 90◦, λ
= 0.71073 Å, T = 187(2) K, U = 1656.6(4) Å3, Z = 4, Dc = 1.369 g
cm−3, µ = 0.090 mm−1, F(000) = 712; 8679 reflections measured, 3032
unique (Rint = 0.0272), 237 refined parameters, transmission factors
0.9890 and 0.9815. The final discrepancy factors were R1 = 0.0523,
wR2 = 0.1329, goodness-of-fit S = 1.024 on F2, largest difference peak
and hole 0.510 and –0.237 eÅ−3. CCDC reference number 673252.
Because of the steric interaction between 4-positioned methyl group
and 3-positioned carbonyl oxygen in the coumarin ring and benzotria-
zole group, the benzotriazole skeleton is not coplanar with the benzo-
coumarin ring and the dihedral angle is 80.54◦ (Figure 2). The dihedral
angle of MBMBC is larger than that of BMBC (60.4◦)10 and BMC (72◦).9
It is shown that the mutual repulsion of the two electron-rich atoms,
the carbonyl oxygen of coumarin and the 2-positioned benzotriazolyl
nitrogen, is larger than that of other two compounds owing to the 4-
positioned electron-releasing CH3 of benzotriazole ring in MBMBC.
FIGURE 3 Packing diagram along a-axis. H atoms are omitted for clarity.

Coumarin Derivatives Containing Benzotriazole Moieties
2659
These results show that the benzotriazole and coumarin systems are
not coplanar, and not conjugated.
From Figure 3, the interplanar distance of coumarin rings between
two adjacent molecules along a-axis is approximately 6.911 Å, which
is far from the value of the intermolecular π-π stacking interaction.
It is means that there are not intermolecular π-π stacking interactions
between the MBMBC molecules in the crystal lattice. Weak intermolec-
ular π-π stacking interactions are noted in the BMBC molecules and in
the BMC molecules; the interplanar distance of the coumarin rings is
3.59 Å for BMBC and 3.93 Å for BMC.
Cytotoxic Activity
In the present study, we performed a comparative evaluation of the
cytotoxic effects of the three compounds against the human hepatocel-
lular carcinoma cell lines (HepG-2) using the standard Sulforhodamine
B (SRB) assay. The in vitro cytotoxicity assay was carried out accord-
ing to procedures described by Rubinstein et al.12 The values of IC50
(the concentration of test compounds required to reduce the cell sur-
vival fraction to 50% of the control) were calculated from dose response
curves and used as a measure of cellular sensitivity to a given treat-
ment. HepG-2 was incubated with RPMI-1640 medium containing 5%
fetal bovine serum and grown at 37◦C in a humidified atmosphere with
5% carbon dioxide. In order to maintain the cells in log-phase cellular
suspension, aliquots were re-fed with fresh RPMI-1640 medium two
or three times per week. The stock solutions of the tested compounds
were freshly prepared in dimethyl sulfoxide (DMSO) and consequently
diluted in RPMI-1640, and the final concentration never exceeded
0.5%.
The 48-h exposure of HepG-2 cells with the tested compounds re-
sulted in a concentration-dependent reduction of cell viability. Figure
4 shows the concentration-response curves of incubation of HepG-2
cells in the presence of various concentrations of the compounds after
48 h. The corresponding IC50 values are listed in Table I, which can
allow a quantitative merit for assessment of the relative potencies of
the compounds under investigation. The results indicated that these
compounds possess certain cytotoxicity at µg/mL scale.
From the results above, the in vitro studies indicated that all the
tested compounds exhibit higher cytotoxic activity against HepG-2
cells. In terms of the relative potency of the compounds, compound
BMC (IC50 value of 26.41 µg/mL) proved to be superior to the other
two compounds yielding the cytotoxic sequence BMC > MBMBC >
BMBC. The results clearly show that the conjugation of the coumarin

2660
T. Yu et al.
FIGURE 4 Cyctotoxic effects of MBMBC, BMBC, and BMC against HepG-2
cells after 48 h exposure as assessed by the standard Sulforhodamine B assay.
Each data point represents the arithmetic mean of at least six independent
experiments.
ring and the substitution on the benzotriazole ring substantially affect
the cell-killing ability of the molecules. The cell-killing ability of the
molecules decreased with increasing conjugation of the coumarin ring
of the compounds. Compared with BMBC, MBMBC has a 4-positioned
electron-releasing CH3 in the benzotriazole ring; the electron density
of triazole ring is higher, so the cell-killing ability of MBMBC is higher
than that of BMBC.
CONCLUSIONS
We have synthesized three coumarin derivatives containing ben-
zotriazole moieties, and their cytotoxic effect against the human
TABLE I Cytotoxic Activity of the Tested Compounds
Compounds
IC50 (µg/mL)
MBMBC
BMBC
BMC
27.78 2.37
36.84 3.55
26.41 2.28

Coumarin Derivatives Containing Benzotriazole Moieties
2661
hepatocellular carcinoma cell lines (HepG-2) was carried out in vitro.
It is clear from the results that these compounds are likely to have
much higher HepG-2 inhibitory activity. The relationship between the
structure and the cytotoxic property of the compounds was examined.
The results show that the conjugate degree of the coumarin ring and
the substitution on the benzotriazole ring can drastically alter the cyto-
toxic property of the compounds. Such results would lead us to modify
our new target molecules by introducing more potential groups on the
coumarin ring and the benzotriazole ring.
EXPERIMENTAL
Reagents and Chemicals
2-Acetyl-1-naphthol and 2-acetyl-1-phenol from Acros Organics and 4-
methyl-1H-benzotriazole and 1H-benzotriazole from Aldrich were used
without further purification. Chloroacetic acid and phosphorus oxy-
chloride were analytical grade reagents from Tanjin Fuchen Chemical
Reagent Factory. Phosphorus oxychloride was dried and redistilled be-
fore using. MBMBC, BMBC, and BMC were obtained by similar pro-
cedures. The synthesis of BMBC and BMC were described in earlier
articles.9,10
Apparatus
IR spectra (400–4000 cm−1) were measured on a Shimadzu IR Prestige-
1
21 FT-IR spectrophotometer. H-NMR spectra were obtained on Unity
Varian-500 MHz. Melting points were measured by using an X-4 mi-
croscopic melting point apparatus made by Beijing Taike Instrument
Limited Company, and the thermometer was uncorrected.
Procedure
Synthesis of (4-Methyl-1-benzotriazolyl-) Acetic Acid
50.00 g (0.376 mol) of 4-methyl-1H-benzotriazole and 32.12 g (0.342
mol) of chloroacetic acid were dissolved in dry toluene (300 mL), and
then the solution was heated to 120◦C and refluxed for 18 h with mag-
netic stirring. After cooling down to room temperature, it was poured
into a concentrated aqueous solution of NaHCO3. The water phase
was separated and extracted with dichloromethane three times. The
aqueous solution was adjusted to a pH of 4 by adding 2N HCl and al-
lowed to sit overnight. The precipitate was filtered, then washed succes-
sively with distilled water and methanol. The product was dried under

2662
T. Yu et al.
reduced pressure and purified with recrystallization from 90% ethanol
to yield 29.15 g (44.6%) of white crystalline solid; mp 201–203◦C. IR
(KBr pellet, cm−1): 3431 (νH-O-C O), 2862, 2918 (νCH2, νCH3), 1730 (νC O).
1H NMR (500 MHz, CDCl3, δ, ppm): 2.50 (s, 3H, CH3), 5.45 (s, 2H, CH2),
7.37–7.94 (m, 3H, Ar-H).
Synthesis of (2-Acetyl) Naphthol (4-Methyl-1-benzotriazolyl-)
Acetate
2.00 g (0.0107 mol) of 2-acetyl-1-naphthol and 2.21 g (0.0115 mol) of
(4-methyl-1-benzotriazolyl-) acetic acid were dissolved in dry pyridine
(20 mL). Then phosphorus oxychloride (1 mL) was added dropwise with
magnetic stirring at 5–10◦C, and the mixture was stirred for 10 h at
room temperature. After the reaction was complete, the mixture was
poured into an aqueous solution of HCl containing broken ice with vig-
orous stirring, and a white precipitate was produced. The precipitate
was filtered, and washed successively with a diluted aqueous solution
of NaHCO3 (10%) and distilled water, respectively. After ethanol re-
crystallization, filtration, and drying in vacuo, 1.82 g (47.1%) of white
flocculent crystals was obtained. mp 168–170◦C. IR (KBr pellet, cm−1):
1768 (ester C O), 1681 (ketone C O), 3057, 2985, 2953, 1465, 1357,
1
1180. H NMR (500 MHz, CDCl3, δ, ppm): 2.52 (s, 3H, CH3), 2.71 (s,
3H, CH3), 5.92 (s, 2H, CH2), 7.38–7.97 (m, 9H, Ar-H).
Synthesis of 3-(4-Methyl-1-benzotriazolyl)-4-methyl-benzo
[7,8]coumarin (MBMBC)
Into a one-neck, 100 mL round-bottomed flask, (2-acetyl) naphthol
(4-methyl-1-benzotriazolyl-) acetate (1.30 g (0.0036 mol)) and dry pyri-
dine (11 mL) were placed, then potassium hydroxide (0.30 g (0.0053
mol)) was added stepwise, and the reaction mixture was stirred vig-
orously for 5 h at room temperature. The mixture was poured into an
aqueous solution of HCl containing broken ice with vigorous stirring,
and a primrose yellow precipitate was formed. Then the crude prod-
uct was filtered and washed with distilled water, and purified with
recrystallization from chloroform:ethanol mixed solvent (5:1) to yield
0.81 g (65.9%) of a primrose yellow crystalline solid. mp > 250◦C. IR
(KBr pellet, cm−1): 1728 (νC O), 3056, 2924, 1637, 1395, 1284, 1116. 1H
NMR (500 MHz, CDCl3, δ, ppm): 2.42 (s, 3H, CH3), 2.57 (s, 3H, CH3),
7.33–8.66 (m, 9H, Ar-H).
Crystallography
A suitable single crystal of MBMBC was obtained by evaporation of
an ethanol solution. The diffraction data were collected with a Bruker

Coumarin Derivatives Containing Benzotriazole Moieties
2663
Smart Apex CCD area detector using a graphite monochromated MoKα
radiation (λ = 0.71073 Å) at 187(2) K.13 The structure was solved by
using the program SHELXL and Fourier difference techniques, and
refined by the full-matrix least-squares method on F2. All hydrogen
atoms were added theoretically.14
SUPPORTING INFORMATION
The crystallographic data (excluding structure factors) of MBMBC has
been deposited with the Cambridge Crystallographic Center as supple-
mentary publication no. CCDC-673252.
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