Synthesis and antimicrobial activities of 4-aryl-3,4-dihydrocoumarins and 4-arylcoumarins

Article abstract of DOI:10.1007/s10600-012-0149-9

A new series of 4-aryl-3,4-dihydrocoumarins and 4-arylcoumarins were synthesized by the reaction of substituted cinnamic acids and 3-arylpropiolic acid with the corresponding phenols. These compounds were evaluated for antibacterial activity in vitro. The

Full text of DOI:10.1007/s10600-012-0149-9

Chemistry of Natural Compounds, Vol. 48, No. 1, March, 2012 [Russian original No. 1, January–February, 2012]
SYNTHESIS AND ANTIMICROBIAL ACTIVITIES
OF 4-ARYL-3,4-DIHYDROCOUMARINS AND 4-ARYLCOUMARINS
Jie Sun,1,3 Wei-Xian Ding,2 Xiao-Ping Hong,2
Ke-Yun Zhang,2* and Yong Zou1*
UDC 547.587.51
A new series of 4-aryl-3,4-dihydrocoumarins and 4-arylcoumarins were synthesized by the reaction of
substituted cinnamic acids and 3-arylpropiolic acid with the corresponding phenols. These compounds were
evaluated for antibacterial activity in vitro. The synthesized compounds displayed different degrees of
antimicrobial activity against Staphylococcus aureus, Escherichia coli, and Bacillus dysenteriae, and Candida
albicans (a fungus). Compounds with catechol moieties and 7,8-substituted dihydroxyls in the A ring were
the most active antimicrobial agents.
Keywords: 4-aryl-3,4-dihydrocoumarin, 4-arylcoumarin, antimicrobial activity.
4-Arylcoumarin and 4-aryl-3,4-dihydrocoumarin constitute a specific branch of coumarin derivatives and are also
regarded as a rare subgroup of flavonoids. In recent years, several 4-arylcoumarins and 4-aryl-3,4-dihydrocoumarins have
been shown to possess antidiabetic [1], anti-inflammatory [2, 3], cytotoxic [4, 5], andantifungal [6] effects. Notably, antibacterial
activity has also been reported for 4-arylcoumarins isolated from Mesua ferrea blossoms [7]. As part of our ongoing research
into bioactive arylcoumarins, we report herein the synthesis and antimicrobial activity of five 4-arylcoumarins and twelve
4-aryl-3,4-dihydrocoumarins, some of which displayed a broad spectrum of activities.
The synthetic methods for 4-arylcoumarins have mainly involved Pechmann reaction, Perkin reaction, Suzuki or
Stille reaction, Houben-Hoesch reaction, and Wittig reaction. Most of the reported methods suffered from one or more drawbacks,
such as harsh reaction conditions, long reaction times, low yields, and inconvenient workup procedures [8–11]. We herein
report a phenylpropiolic acid-based Ponndorf reaction with phloroglucinol to afford 4-arylcoumarins in mild conditions with
satisfactory yields.
COOCH
Br
3
Br
COOH
COOCH
3
COOH
ii
R = OCH
3
OCH
3
OCH
3
v
i
COOCH
COOCH
3
3
R
R
Br
R
Br
OCH
OCH
3
3
OCH
3
iii
R = OH
iv
21, 22
21:R = OCH
22:R = OH
OCOCH
OCOCH
3
3
3
OCH
OCH
3
3
i. MeOH, SOCl , reflux, 4 h; ii. Br , CH Cl , 0ꢀC, 20 min; iii. CH COOH, SOCl , reflux, 2 h; iv. Br , CH Cl , 0ꢀC, 20 min;
2
2
2
2
3
2
2
2
2
v. KOH, EtOH, reflux, 6 h.
Scheme 1
1) Guangzhou Institute of Chemistry, Chinese Academy of Sciences, Guangzhou, 510650, P. R. China, e-mail:
zouyong@gic.ac.cn; 2) Department of Zoology, College of Life Sciences, Nanjing Agricultural University, Nanjing, 210095,
P. R. China, e-mail: keyunzhang@njau.edu.cn; 3) Graduate School of the Chinese Academy of Science, Beijing 100039,
P. R. China. Published in Khimiya Prirodnykh Soedinenii, No. 1, pp. 20–25, January–February, 2012. Original article submitted
October 18, 2010.
16
0009-3130/12/4801-0016 02012 Springer Science+Business Media, Inc.

OCH
OCH
3
3
COOH
COOH
R
1
R
2
R
1
OH
OH
R
R
1
R
R
1
i
ii
R
R
R
2
3
+
+
2
R
1
R
2
R
2
1
O
9 - 20
O
R
3
O
O
4
R
3
OCH
R
3
OCH
3
3
R
5
R
4
21, 22
23 - 26
1 - 4
5 - 8
5, 6, 8
1: R = R = H; 2: R = H, R = OH; 3: R = H, R = OCH ; 4: R = R = OCH ; 5: R = R = H, R = OH
1
2
1
2
1
2
3
1
2
3
1
3
2
6: R = R = OH, R = H; 7: R = R = H, R = OH; 8: R = R = H, R = OCH ; 9: R = R = R = H, R = R = OH
1
2
3
1
2
3
1
3
2
3
1
3
5
2
4
10: R = R = R = H, R = OCH , R = OH; 11: R = R = H, R = R = R = OH; 12: R = R = H, R = OCH , R = R = OH
1
3
5
2
3
4
1
3
2
4
5
1
3
2
3
4
5
13: R = R = OCH , R = R = H, R = OH; 14: R = R = OCH , R = H, R = R = OH; 15: R = R = R = H, R = R = OH
1
2
3
3
5
4
1
2
3
3
4
5
1
2
3
4
5
16: R = R = R = H, R = OCH , R = OH; 17: R = R = OCH , R = OH, R = R = H; 18: R = R = R = R = H, R = OH
1
4
5
2
3
3
1
2
3
3
4
5
1
2
4
5
3
19: R = R = R = R = H, R = OCH ; 20: R = R = R = OCH , R = R = H; 21: R = OCH ; 22: R = OH
1
2
3
5
4
3
1
2
4
3
3
5
1
3
1
23: R = OCH , R = R = H, R = OH; 24: R = OCH , R = H, R = R = OH; 25: R = OCH , R = R = H, R = OCH
1
3
2
4
3
1
3
2
3
4
1
3
2
4
3
3
26: R = R = R = OH, R = H
1
3
4
2
i. POCl and BF –Et O, 0ꢀꢂroom temperature, 4–18 h; ii. CF COOH, room temperature, 4–8 h.
3
3
2
3
Scheme 2
OCH
OCH
3
3
i
H CO
3
O
O
H CO
3
O
O
19
27
i. DDQ in dioxane, room temperature.
Scheme 3
The 3-arylpropiolic acid derivatives 21 and 22 were synthezised as previously described [12] in three or four steps
with 37–44% yields (Scheme 1). Twelve 4-aryl-3,4-dihydrocoumarins 9–20 were synthesized by the condensation of substituted
cinnamic acids 1–4 with the corresponding phenols 5–8 in the presence of BF3–Et2O and POCl3, resulting in 52–73% yields
(Scheme 2). Four 4-arylcoumarins 23–26 were synthesized by the condensation of substituted propargyl acids 21 and 22 with
the corresponding phenols 5, 6, and 8 in the presence of CF3COOH with 53–58% yields (Scheme 2). Compound 19 was
dehydrogenated with 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) in dioxane at room temperature to yield compound 27
with 69% yield (Scheme 3). To date, we have attempted to transform the dihydrocoumarin by several methods; compound 19
was treated with SeO2, Pd-C/PhOPh, MnO2, or I2/pyridine to introduce unsaturation into the lactone ring. However, compound
27 was not formed under these conditions. Dehydrogenation with DDQ finally yielded 4-arylcoumarin, and this proved to be
a mild method for the synthesis of 4-arylcoumarins [5].
The structures of all the synthesized compounds were supported by FT-IR, MS, 1H NMR, and 13C NMR spectral data.
In the IR spectrum of 4-aryl-3,4-dihydrocoumarins, a prominent absorption band at 1714–1761cm–1 indicated the presence of
a carbonyl group in the compound. The other absorption band at 3320–3513 cm–1 in the IR spectrum corresponded to a
hydroxy group. The 1H NMR spectrum of 4-aryl-3,4-dihydrocoumarins showed a multiplet at ꢁ 2.96–3.03 due to the C-3
hydrogen and a quartet at 4.16–4.24 due to the C-4 hydrogen, and the aromatic hydrogens of rings A and B appeared as
multiplets at 6.33–7.11. The 13C NMR spectrum of 4-aryl-3,4-dihydrocoumarins showed the C-3 carbon at ꢁ 36.8–37.9, the C-4
carbon at 39.2–41.0, the aromatic carbons at 104.0–160.0, and the carbonyl carbon at 166.9–168.5. In addition, the mass
spectra of 4-aryl-3,4-dihydrocoumarins showed molecular ion peaks corresponding to their molecular formulas. The infrared
spectra of 4-arylcoumarins showed an absorption band at 1695–1738 cm–1, corresponding to a carbonyl group. The 1H NMR
spectra of 4-arylcoumarins showed a singlet at ꢁ 6.03–6.20 due to H-3, and the aromatic hydrogens of rings A and B appeared
as multiplets at 6.74–7.55. In the 13C NMR spectrum, the C-3 carbon was detected at ꢁ 110.6–111.8, the C-4 carbon around
112.7, and the carbonyl carbon at 161.2–163.8. The mass spectra of these compounds showed corresponding molecular ion
peaks.
17

TABLE 1. In vitro Minimum Inhibitory Concentrations (MIC in ꢃg/mL), Minimum Bactericidal Concentrations (MBC in ꢃg/mL),
and Minimum Fungicidal Concentrations (MFC in ꢃg/mL) for the Synthesized Compounds
MIC, g/mL
MBC (or MFC), g/mL
ꢃ
ꢃ
Compound
S. aureus
E. coli
B. dysenteriae
C. albicans
S. aureus
E. coli
B. dysenteriae
C. albicans
625
937.5
234
469
–
937.5
312.5
156
469
117
937.5
–
117
156
<1.95
<1.95
469
–
58.5
39
–
58.5
78
117
–
39
–
234
937.5
156
469
937.5
117
117
117
469
14.5
29
234
29.3
4.9
29
<1.95
469
1250
117
469
1250
234
312.5
469
312.5
117
–
117
1250
–
–
1250
–
–
625
625
–
312.5
–
–
–
–
–
625
–
–
–
625
1250
–
–
–
–
1250
–
–
–
–
–
312.5
–
–
–
–
3.9
125
9
10
11
12
–
13
312.5
625
1250
–
–
–
–
–
–
–
14
625
625
–
156
–
–
–
78
125
31
15
16
17
18
19
20
24
26
–
–
78
156
23
<1.95
58.5
<1.95
2.9
–
Ampicilin
__G_e_n_ta_micin
<1.95
7.8
125
<1.95
The negative control, DMSO, showed no activity.
–: totally inactive (MIC > 1250 ꢃg/mL); compounds 23, 25, and 27 were totally inactive.
All the synthesized compounds were evaluated for their antimicrobial activities against four microorganisms:
Staphylococcus aureus (ATCC2592) (Gram-positive), Escherichia coli (ATCC25922) (Gram-negative), Bacillus dysenteriae
(Bacillaceae), and Candida albicans (ATCC2002) (fungus). The antimicrobial activities of the seventeen 4-arylcoumarins
derivatives, as indicated by the minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC)
values, are listed in Table 1. These compounds exhibited a broad range of antimicrobial activities. Some of the 4-arylcoumarin
derivatives exhibited activity against all four microorganisms tested, and a few of them were selectively active against one or
two strains, while others displayed little or no activity against any of the microorganisms, even at the highest test concentration
of 1250 ꢃg/mL. In general, the activity against Bacillus dysenteriae was the highest of the four microorganisms tested.
Compound 26 showed the highest activity against Bacillus dysenteriae, with an MIC value of 4.9 ꢃg/mL (Table 1) and an
MBC value of 78 ꢃg/mL (Table 1). Compound 18 (MIC = 14.5 ꢃg/mL, MBC = 156 ꢃg/mL) and compound 24 (MIC = 29.3 ꢃg/mL,
MBC > 1250 ꢃg/mL) (Table 1) also displayed strong activity against Bacillus dysenteriae. For most compounds, the MBC
value was fourfold greater than the MIC value.
From these results, it is also possible to establish tentative relations between the structures of 4-arylcoumarin derivatives
and their antimicrobial activities. The presence of a catechol moiety in the A ring increased the antimicrobial activity of the
tested compounds. As a result, compounds 11, 12, 14, 15, 24, and 26 displayed moderate to good activity. Derivatives containing
6-substituted hydroxy moieties showed higher activity than their corresponding 7-substituted hydroxy analogues. For example,
compounds 16 and 17 with a 6-substituted hydroxyl group were more active than compounds 10 and 13 with a 7-substituted
hydroxyl group. Simultaneously, 4ꢄ-methoxy substitution with a 3ꢄ-hydroxy group gave good antimicrobial activity; consequently,
compounds 9, 11, and 26 all display antimicrobial activity. Finally, it is noteworthy that double bonds in the C ring were not a
definite factor determining antimicrobial activity. While 4-arylcoumarins 24 and 26 were more active than 4-aryl-3,4-
dihydrocoumarins with the same substituted group (11 and 12), 4-aryl-3,4-dihydrocoumarins 10 and 20 were more active than
4-arylcoumarins 23 and 27.
In this study, the antimicrobial potential of a number of 4-aryl-3,4-dihydrocoumarin and 4-arylcoumarin derivatives
has been demonstrated. The presence of 7,8-substituted dihydroxyls in the A ring increased the antimicrobial activity of the
tested compounds. Derivatives containing a 6-substituted hydroxy moiety demonstrated higher activity than the corresponding
7-substituted hydroxy analogues. In general, it has been demonstrated that the nature and position of substituent groups can
change the antimicrobial activity of these compounds. Therefore, 4-arylcoumarins could be considered as candidates for
further research to prove that they are potentially novel antimicrobial agents.
18

EXPERIMENTAL
Melting points were determined using a Thiele tube and were uncorrected. The FT-IR spectra were recorded using an
Analect RFX-65A spectrometer with KBr pellets. The 1H NMR and 13C NMR spectra were recorded with a Bruker AM-400
spectrometer with TMS as the internal standard. Chemical shifts were reported on a ꢁ scale (ppm) with CDCl3 or CD3COCD3
as the solvents. Spin multiplets are given as s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). Mass spectra were
obtained with a Shimadzu QP-5050A spectrometer.
Synthesis of 3-Arylpropargyl Acids 21 and 22. A mixture of cinnamic acid (10 mM) and SOCl2 (1 mL) in MeOH
(20 mL) was refluxed for 4 h, followed by removal of the solvent in vacuo. A mixture of the ester (10 mmol) and acetic acid
(20 mL) in the presence of SOCl2 (2 mL) was refluxed for 2 h, poured into ice water, and filtered to obtain the crude product.
To a solution of the ester (10 mmol) in CH2Cl2 (20 mL), Br2 (0.5 mL) was added dropwise at 0ꢀC for 20 min. The solvent was
removed under reduced pressure and the residue washed with water to obtain the crude product. A mixture of dibromide (10 mmol)
and KOH (3 N) in EtOH (50 mL) was heated for 6 h, followed by removal of the solvent in vacuo. The residue was treated with
2 N HCl, washed with water, and then purified by chromatography (Scheme 1) [12].
General Procedure for the Synthesis of 4-Aryl-3,4-dihydrocoumarins 9–20 [13]. To a mixture of POCl3 (10 mmol)
and BF3–Et2O (20 mmol) at 0ꢀC, a substituted cinnamic acid (5 mmol) was added and the reaction mixture was stirred for
15 min at 0ꢀC. A substituted phenol (5 mmol) was added and the mixture stirred at room temperature for 4–18 h. The reaction
mixture was poured onto ice water, extracted with ethyl acetate (2 ꢅ 150 mL), washed with water (150 mL), and dried. The
solvent was evaporated under reduced pressure to obtain the crude product, which was purified by chromatography on silica
gel or by crystallization from acetone and water. The physical properties of the synthesized compounds are listed below.
(ꢆ)-7-Hydroxy-4-(3-hydroxy-4-methoxyphenyl)-3,4-dihydrocoumarin (9). White solid, mp 165–168ꢀC, yield 55.2%.
IR (KBr, ꢇ, cm–1): 3513 (OH), 3334, 1733 (CO), 1627, 1597, 1513, 1450, 1300, 1273, 1223, 1151 and 815. 1H NMR (400 MHz,
CDCl3, ꢁ, ppm, J/Hz): 2.98 (2H, m, H-3), 3.86 (3H, s, OCH3), 4.16 (1H, t, J = 6.4, H-4), 6.54 and 6.59 (each H, dd, J = 2.4 and
8.4, H-6ꢄ and H-6), 6.61 and 6.69 (each H, d, J = 2.4, H-2ꢄ and H-8), 6.77, 6.83 (each H, d, J = 8.4, H-5ꢄ and H-5). 13C NMR
(100 MHz, CDCl3, ꢁ): 37.9, 39.9, 56.2, 104.2, 112.3, 112.7, 114.9, 117.9, 119.1, 129.9, 135.6, 147.4, 147.6, 153.4, 158.5,
168.2. MS m/z (%): 286 [M]+, 268 [M – H2O]+, 253 [M – CH3 – H2O]+, 243, 227, 213, 176, 115.
(ꢆ)-7-Hydroxy-4-(3,4-dimethoxyphenyl)-3,4-dihydrocoumarin (10). Yellow solid, mp 144–146ꢀC, yield 60.7%.
IR (KBr, ꢇ, cm–1): 3429 (OH), 1761 (CO), 1626, 1597, 1516, 1462, 1419, 1335, 1271, 1244, 1159, 1103, 1024, 991, 847, and
812. 1H NMR (400 MHz, CDCl3, ꢁ, ppm, J/Hz): 2.98 (2H, m, H-3), 3.79 and 3.84 (each 3H, s, OCH3), 4.20 (1H, t, J = 6.0, H-4),
6.56 (1H, dd, J = 2.4 and 8.4, H-6), 6.63–6.67 (3H, m, H-6ꢄ, H-5ꢄ, H-5), 6.80 and 6.82 (each H, d, J = 2.4, H-8 and H-2ꢄ). 13C NMR
(100 MHz, CDCl3, ꢁ): 37.5, 39.6, 55.9, 104.2, 110.5, 111.6, 112.0, 117.7, 119.7, 129.1, 133.1, 148.3, 149.3, 152.2, 156.3,
168.5. MS m/z (%): 300 [M]+, 282 [M – H2O]+, 267 [M – CH3 – H2O]+, 257, 243, 227, 190 ,163, 129.
(ꢆ)-7,8-Dihydroxy-4-(3-hydroxy-4-methoxyphenyl)-3,4-dihydrocoumarin (11). White solid, mp 188–190ꢀC,
yield 69.5%. IR (KBr, ꢇ, cm–1): 3498 (OH), 3417, 1753 (CO), 1516, 1468, 1271, 1198, 1174 and 1126. 1H NMR (400 MHz,
CDCl3, ꢁ, ppm, J/Hz): 2.99 (2H, m, H-3), 3.86 (3H, s, OCH3), 4.19 (1H, t, J = 6.4, H-4), 6.43 and 6.65 (each H, d, J = 8.4, H-6ꢄ and
H-5ꢄ), 6.59 and 6.78 (each H, d, J = 8, H-6 and H-5), 6.69 (1H, s, H-2). 13C NMR (100 MHz, CD3COCD3, ꢁ): 37.9, 40.4, 56.2,
111.7, 112.7, 115.0, 118.6, 119.1, 119.1, 133.8, 135.5, 141.3, 146.3, 147.5, 147.6, 168.0. MS m/z (%): 302 [M]+, 284 [M – H2O]+,
269 [M – CH3 – H2O]+, 259, 243, 229, 194, 179.
(ꢆ)-4-(3,4-Dimethoxyphenyl)-7,8-dihydroxy-3,4-dihydrocoumarin (12). White solid, mp 174–176ꢀC, yield 72.8%.
1
IR (KBr, ꢇ, cm–1): 3423 (OH), 1755 (CO), 1516, 1468, 1275, 1238, 1192, 1169, and 1020. H NMR (400 MHz, CDCl3, ꢁ,
ppm, J/Hz): 3.03 (2H, m, H-3), 3.81 and 3.85 (each 3H, s, OCH3), 4.24 (1H, dd, J = 7.6 and 6.4, H-4), 6.41, 6.66, and 6.80 (4H,
4 ꢅ d, J = 8.4, H-6ꢄ, H-5ꢄ, H-6, and H-5), 6.64 (1H, s, H-2). 13C NMR (100 MHz, CD3COCD3, ꢁ): 37.4, 40.0, 55.9, 110.6,
111.3, 111.5, 118.1, 118.9, 119.7, 131.1, 132.7, 139.3, 144.2, 148.5, 149.4, 166.9. MS m/z (%): 316 [M]+, 298 [M – H2O]+, 283
[M – CH3 – H2O]+, 273, 259, 243, 190.
(ꢆ)-7-Hydroxy-4-(3,4,5-trimethoxyphenyl)-3,4-dihydrocoumarin (13). White solid, mp 176–178ꢀC, yield 70.0%.
1
IR (KBr, ꢇ, cm–1): 3381 (OH), 1747 (CO), 1626, 1597, 1510, 1456, 1358, 1230, 1271, 1240, 1122, 987, and 968. H NMR
(400 MHz, CDCl3, ꢁ, ppm, J/Hz): 3.01 (2H, m, H-3), 3.78 and 3.82 (9H, 2s, 3H and 6H each, 3 ꢅ OCH3), 4.18 (1H, t, J = 6.0,
H-4), 6.33 (2H, s, H-2ꢄ and H-6ꢄ), 6.56 (1H, dd, J = 2.4 and 8.4, H-6), 6.64 (1H, d, J = 2.4, H-8), 6.84 (1H, d, J = 8.4, H-5). 13C NMR
(100 MHz, CDCl3, ꢁ): 37.4, 40.4, 56.1, 60.9, 104.3, 104.5, 112.0, 117.4, 129.1, 136.4, 137.2, 152.3, 153.6, 156.3, 167.9.
MS m/z (%): 330 [M]+, 315 [M – CH3]+, 297 [M – CH3 – H2O]+, 287, 273, 257, 213, 128, 107.
19

(
ꢆ
)-7,8-Dihydroxy-4-(3,4,5-trimethoxyphenyl)-3,4-dihydrocoumarin (14). White solid, mp 221–223ꢀC, yield 52.6%.
IR (KBr, ꢇ, cm–1): 3471 (OH), 1751 (CO), 1597, 1512, 1466, 1311, 1246, 1122, and 1007. H NMR (400 MHz, CDCl3, ꢁ,
ppm, J/Hz): 3.03 (2H, m, H-3), 3.78 (6H, s, 2 ꢅ OCH3), 3.82 (3H, s, OCH3), 4.22 (2H, dd, J = 6 and 7.6), 6.33 (2H, s, H-2ꢄ and
H-6ꢄ), 6.45 and 6.67 (each H, d, J = 8.8, H-5 and H-6). 13C NMR (100 MHz, CD3COCD3, ꢁ): 37.3, 40.7, 56.2, 60.9, 104.5,
111.3, 117.6, 119.0, 131.1, 136.0, 139.3, 144.3, 153.6, 166.7. MS m/z (%): 346 [M]+, 328 [M – H2O]+, 313 [M – CH3 – H2O]+,
273, 257, 243, 229, 181, 115.
1
(ꢆ)-7,8-Dihydroxy-4-(4-methoxyphenyl)-3,4-dihydrocoumarin (15). White solid, mp 166–168ꢀC, yield 62.2%.
IR (KBr, ꢇ, cm–1): 3413 (OH), 1751 (CO), 1612, 1514, 1311, 1284, 1247, 1180, 1144, and 1007. 1H NMR (400 MHz, CDCl3, ꢁ):
3.01 (2H, m, H-3), 3.78 (3H, s, OCH3), 4.24 (1H, t, J = 6.8, H-4), 6.39 (2H, 2 ꢅ d, J = 8.4, H-5 and H-6), 6.85 and 7.04 (4H,
2 ꢅ d, J = 8.8, H-2ꢄ, H-6ꢄ, H-3ꢄ, and H-5ꢄ). 13C NMR (100 MHz, CD3COCD3, ꢁ): 37.9, 40.2, 55.4, 111.7, 114.9, 114.9, 118.5,
119.1, 129.3, 133.8, 134.5, 141.2, 146.3, 159.6, 167.9. MS m/z (%): 286 [M]+, 268 [M – H2O]+, 243, 229, 200, 115.
(ꢆ)-6-Hydroxy-4-(3,4-dimethoxyphenyl)-3,4-dihydrocoumarin (16). White solid, mp 168–170ꢀC, yield 62.3%.
IR (KBr, ꢇ, cm–1): 3273 (OH), 1714 (CO), 1521, 1487, 1247, 1244, and 1186. 1H NMR (400 MHz, CDCl3, ꢁ, ppm, J/Hz):
3.0 (2H, m, H-3), 3.79 and 3.82 (each 3H, s, OCH3), 4.17 (1H, dd, J = 6 and 8.4, H-4), 6.41 (1H, d, J = 2.8, H-2ꢄ), 6.63 (1H, d,
J = 2.4, H-5), 6.68 (1H, dd, J = 2 and 8, H-7), 6.72 (1H, dd, J = 2.8 and 8.8, H-6ꢄ), 6.79 (1H, d, J = 8, H-8), 6.96 (1H, d, J = 8.8,
H-5ꢄ). 13C NMR (100 MHz, CDCl3, ꢁ): 36.9, 40.4, 55.9, 110.6, 111.6, 114.5, 115.3, 118.0, 119.9, 127.4, 132.3, 145.4, 148.5,
149.4, 152.5, 168.2. MS m/z (%): 300 [M]+, 282 [M – H2O]+, 267 [M – CH3 – H2O]+, 257, 243, 227, 151, 115.
(ꢆ)-6-Hydroxy-4-(3,4,5-trimethoxyphenyl)-3,4-dihydrocoumarin (17). White solid, mp 174–176ꢀC, yield 71.4%.
IR (KBr, ꢇ, cm–1): 3421 (OH), 1757 (CO), 1504, 1454, 1232, 1195, and 1122. 1H NMR (400 MHz, CDCl3, ꢁ, ppm, J/Hz): 2.98
(2H, m, H-3), 3.77 and 3.80 (9H, 2s, 6H and 3H each, 3 ꢅ OCH3), 4.17 (1H, t, J = 6.4, H-4), 6.33 (2H, s, H-2ꢄ and H-6ꢄ), 6.55
(1H, dd, J = 2.4 and 8.6, H-6), 6.64 (1H, d, J = 2.4, H-8), 6.85 (1H, d, J = 8.6, H-5). 13C NMR (100 MHz, CDCl3, ꢁ): 36.8, 41.0,
56.1, 60.8, 104.6, 114.5, 115.5, 118.0, 126.9, 135.7, 137.2, 145.3, 152.6, 153.6, 168.1. MS m/z (%): 330 [M]+, 312 [M – H2O]+,
297 [M – H2O – CH3]+, 257, 213, 181, 128.
(ꢆ)-6-Hydroxy-4-(4-methoxyphenyl)-3,4-dihydrocoumarin (18). White solid, mp 170–172ꢀC, yield 52.2%.
IR (KBr, ꢇ, cm–1): 3320 (OH), 1732 (CO), 1510, 1456, 1309, 1252, 1194, 1155, and 831. 1H NMR (400 MHz, CDCl3, ꢁ, ppm,
J/Hz): 2.96 (2H, m, H-3), 3.75 (3H, s, OCH3), 4.19 (1H, dd, J = 6.0 and 8.4, H-4), 6.38 (1H, d, J = 2.4, H-5), 6.71 (1H, dd,
J = 3.2 and 8.8, H-7), 6.85 (2H, dd, J = 2 and 6.0, H-2ꢄ and H-6ꢄ), 6.96 (1H, dd, J = 8.8, H-5), 7.05 (2H, dd, J = 2 and 6.0, H-3ꢄ and
H-5ꢄ). 13C NMR (100 MHz, CDCl3, ꢁ): 37.0, 39.9, 55.3, 76.7, 77.0, 77.2, 77.3, 114.5, 114.6, 114.7, 115.3, 118.0, 127.5, 128.7,
131.9, 145.5, 152.3, 159.0, 168.2. MS m/z (%): 270 [M]+, 252 [M – H2O]+, 227 [M – H2O – CH3]+, 213, 197, 128.
(ꢆ)-7-Methoxy-4-(4-methoxyphenyl)-3,4-dihydrocoumarin (19). White solid, mp 136–137ꢀC, yield 52.1%.
IR (KBr, ꢇ, cm–1): 2937, 1761 (CO), 1510, 1252, 1124, 1030, and 831. 1H NMR (400 MHz, CD3COCD3, ꢁ, ppm, J/Hz): 3.03
(2H, m, H-3), 3.76 and 3.81 (6H, 2s, 3H and 3H each, 2 ꢅ OCH3), 4.37 (1H, t, J = 6.4, H-4), 6.66 (1H, d, J = 2.4, H-8), 6.69
(1H, dd, J = 2.4 and 8.4, H-6), 6.89 and 7.11 (each 2H, d, H-2ꢄ, H-3ꢄ, H-5ꢄ, H-6ꢄ), 6.95 (1H, d, J = 8.4, H-5). 13C NMR
(100 MHz, CDCl3, ꢁ): 37.4, 39.2, 55.2, 55.4, 102.4, 110.6, 114.3, 117.9, 128.4, 128.7, 132.6, 152.3, 158.8, 159.8, 167.7.
MS m/z (%): 284 [M]+, 256 [M – CO]+, 241 [M – CO – CH3]+, 227, 211, 128. The physical/spectral data of the compound
matched well with those reported earlier [5, 14].
(ꢆ)-7-Methoxy-4-(3,4,5-trimethoxyphenyl)-3,4-dihydrocoumarin (20). White solid, mp 131–133ꢀC, yield 57.2%.
IR (KBr, ꢇ, cm–1): 3456, 1761 (CO), 1593, 1506, 1460, 1130. 1H NMR (400 MHz, CDCl3, ꢁ, ppm, J/Hz): 3.00 (2H, m, H-3),
3.78, 3.79, and 3.81 (12H, 3s, 6H, 3H, and 3H each, 4 ꢅ OCH3), 4.19 (1H, t, J = 7.6, H-4), 6.33 (1H, s, H-2ꢄ and H-6ꢄ), 6.64
(1H, dd, J = 2.4 and 8.4, H-6), 6.66 (1H, d, H-8), 6.89 (1H, d, J = 8.4, H-5). 13C NMR (100 MHz, CDCl3, ꢁ): 37.4, 40.5, 55.5,
56.1, 60.8, 102.5, 104.4, 110.8, 117.4, 128.9, 136.4, 137.3, 152.4, 153.6, 160.0, 167.6. MS m/z (%): 344 [M]+, 329 [M –
CH3]+, 311 [M – CO – CH3] +, 271, 220, 177, 121, 77. The physical/spectral data of the compound matched well with those
reported earlier [5, 14].
Synthesis of 4-Arylcoumarins 23, 24, 25, 26. A solution of the appropriate phenol (11 mmol) and the appropriate
arylpropargyl acid (10 mmol) in 10 mLCF3COOH was stirred at room temperature for 4–8 h. The reaction mixture was poured
onto ice water, extracted with ethyl acetate (2 ꢅ 150 mL), washed with water (150 mL), and dried. The solvent was removed
under reduced pressure to obtain the crude product, which was then purified by chromatography on silica gel.
7-Hydroxy-4-(3,4-dimethoxyphenyl)coumarin (23). White solid, mp 233–234ꢀC, yield 56.4%. IR (KBr, ꢇ, cm–1):
3194 (OH), 1695 (CO), 1624, 1599, 1518, 1263, and 1140. 1H NMR (400 MHz, CD3COCD3, ꢁ, ppm, J/Hz): 3.90 and 3.94
(6H, s, 3H and 3H, 2 ꢅ OCH3), 6.20 (1H, s, H-3), 6.74 (1H, s, J = 2.4 and 8, H-6ꢄ), 6.91 (1H, s, J = 2.4, H-2ꢄ), 6.97 (1H, d, J = 8,
H-5ꢄ), 6.98 (1H, d, J = 2, H-8), 7.02 (1H, dd, J = 2 and 8.8, H-6ꢄ), 7.45 (1H, d, J = 8.8, H-5ꢄ). 13C NMR (100 MHz, CD3COCD3,
20

ꢁ): 56.1, 56.2, 103.5, 111.0, 112.3, 112.5, 112.9, 113.6, 122.0, 128.9, 129.3, 150.3, 151.4, 156.5, 157.0, 161.3, 162.2.
MS m/z (%): 298 [M]+, 283 [M – CH3]+, 270, 255, 227, 199, 184, 113.
7,8-Dihydroxy-4-(3,4-dimethoxyphenyl)coumarin (24). White solid, mp 271–272ꢀC, yield 58.0%. IR (KBr, ꢇ, cm–1):
2987, 1726 (CO), 1618, 1518, 1379, 1255, 1147, and 814. 1H NMR (400 MHz, CD3COCD3, ꢁ, ppm, J/Hz): 3.87 and 3.88
(each 3H, s, OCH3), 6.07 (1H, s, H-3), 6.83 and 7.00 (2H, 2 ꢅ d, J = 8.8, H-5 and H-6), 7.06 (1H, dd, J = 2 and 8.4, H-5ꢄ), 7.10
(1H, d, J = 2, H-2ꢄ), 7.11 (1H, d, J = 8.4, H-6ꢄ). 13C NMR (100 MHz, CD3COCD3, ꢁ): 56.1, 56.2, 110.8, 112.5, 112.7, 113.0,
118.7, 122.1, 129.1, 133.3, 144.7, 149.9, 150.2, 151.4, 157.2, 161.2. MS m/z (%): 314 [M]+, 286 [M – CO]+, 271 [M – CO – CH3]+,
161, 133, 115, 103.
7-Methoxy-4-(3,4-dimethoxyphenyl)coumarin (25). White solid, mp 148–149ꢀC, yield 53.8%. IR (KBr, ꢇ, cm–1):
3411 (CO), 1695 (CO), 1606, 1518, 1448, 1173, and 1140. 1H NMR (400 MHz, CD3COCD3, ꢁ, ppm, J/Hz): 3.88, 3.89, and
3.93 (9H, s, 3H, 3H, and 3H, 3 ꢅ OCH3), 6.14 (1H, s, H-3), 6.90 and 7.08 (each H, dd, J = 8.8, 2.4 and J = 8.4, 2, H-6 and H-6ꢄ),
6.94 and 7.12 (each H, d, J = 2.4, H-8 and H-2ꢄ), 7.12 and 7.55 (2H, 2 ꢅ d, J = 8.4 and 8.8, H-5 and H-5ꢄ). 13C NMR (100 MHz,
CDCl3, ꢁ): 56.1, 56.2, 56.3, 101.8, 111.8, 112.6, 112.9, 113.0, 113.2, 122.1, 128.7, 129.0, 150.4, 151.5, 156.3, 157.0, 161.1,
163.8. MS m/z (%): 312 [M]+, 297 [M – CH3]+, 284 [M – CO – CH3]+, 269, 213, 183, 139.
7,8-Dihydroxy-4-(3-hydroxy-4-methoxyphenyl)coumarin (26). Yellow solid, mp 176–177ꢀC, yield 53.7%. IR (KBr,
ꢇ, cm–1): 3371 (OH), 1695 (CO), 1653, 1599, 1448, 1240, 1178, and 1130. 1H NMR (400 MHz, CD3COCD3, ꢁ, ppm, J/Hz):
3.91 (3H, s, OCH3), 6.03 (1H, s, H-3), 6.83 (1H, d, J = 8.8, H-6), 6.95–6.99 (3H, m, H-5, H-2ꢄ, and H-6ꢄ), 7.10 (1H, d, J = 8, H-5ꢄ).
13C NMR (100 MHz, CD3COCD3, ꢁ): 56.2, 110.6, 112.4, 112.7, 113.0, 116.1, 118.7, 120.9, 129.3, 133.2, 144.6, 147.4, 149.6,
149.9, 157.2, 161.3. MS m/z (%): 300 [M]+, 272 [M – CO]+, 257 [M – CO – CH3]+, 229, 115.
Synthesis of 7-Methoxy-4-(4-methoxyphenyl)coumarin (27) [5]. To a solution of compound 19 (1 mmol) in 4.5 mL
of dioxane, DDQ (1.5 mmol) was added. The mixture was stirred at room temperature until the starting material completely
disappeared. After elimination of dioxane in vacuo, the crude product was purified by flash column chromatography on silica
gel to yield compound 27.
White solid, mp 155–157ꢀC, yield 69%. IR (KBr, ꢇ, cm–1): 3440, 3064, 2993, 1738 (CO), 1612, 1510, 1375, 1250,
825. 1H NMR (400 MHz, CDCl3, ꢁ, ppm, J/Hz): 3.86 (6H, s, 2 ꢅ OCH3), 6.18 (1H, s, H-3), 6.78 (1H, dd, J = 8.8 and 2.4, H-6),
6.87 (1H, d, J = 2.4, H-8), 7.01 and 7.37 (each 2H, d, J = 8.8, H-2ꢄ, H-3ꢄ, H-5ꢄ, H-6ꢄ), 7.43 (1H, d, J = 8.8, H-5).13C NMR (100 MHz,
CDCl3, ꢁ): 55.4, 55.8, 101.1, 111.4, 112.2, 112.7, 114.3, 127.8, 128.0, 129.9, 155.5, 156.0, 160.7, 161.4, 162.7. MS m/z (%):
282 [M]+, 254 [M – CO]+, 239 [M – CO – CH3]+, 211, 196, 168, 152, 140, 127.
Antibacterial Activity Assay. The synthesized compounds were tested for their antimicrobial activity against four
microorganisms. The minimal inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), and minimum
fungicidal concentrations (MFCs) of the tested compounds were determined by the dilution method. Each of the test compounds
and standards were dissolved in DMSO at concentrations of 10 mg/mL. Further dilutions of the compounds and standards
were made in test medium [15]. Standard bacterial and fungal strains were used in the antimicrobial assays. Staphylococcus
aureus (ATCC2592) (Gram-positive), Escherichia coli (ATCC25922) (Gram-negative), Bacillus dysenteriae (Bacillaceae),
and Candida albicans (ATCC2002) (fungus) were obtained from Kunming Medical College and were grown in Luria Bertani
(LB) broth for 24 h at 37ꢀC.
Microorganisms were first grown in LB broth to an OD600 of 0.8. Then, 10 ꢃL was inoculated into 8 mL of fresh LB
medium with 0.7% agar and poured over a 90 mm Petri dish containing 25 mL of 1.5% agar in LB medium. When the top agar
hardened, 5 ꢃL of filtered sample (50 mg/mL) was dropped onto the surface of the top agar and completely dried before
incubation overnight at 37ꢀC. If a clear zone formed on the surface of the top agar, growth of microorganism was
inhibited [16].
Minimum inhibitory concentrations (MICs) were determined by the LB broth dilution method according to Tamokou
et al. [17] with slight modifications. Samples in DMSO were diluted in LB to 0.049–25 mg/mL. Microorganisms were cultured
for 12 h and then adjusted with LB to 106 colony-forming units (CFU) per mL. The 96-well cell culture plates were prepared
by dispensing 180 ꢃL of inoculum (106 CFU/mL for bacteria and 5 ꢅ 105 spores/mL for yeasts) and 20 ꢃL test substances into
each well. The plates were covered with their sterile sealer and incubated at 37ꢀC for 24 h. The MIC was the lowest concentration
of the test sample that prevented visible growth of microorganisms [17].
The minimum bactericidal concentrations (MBCs) or minimum fungicidal concentrations (MFCs) were determined by
dropping 10 ꢃL from each negative well and from the positive growth control onto LB agar and incubating them at 37ꢀC for 24 h.
The MBCs or MFCs were defined as the lowest concentration yielding negative subcultures or only one colony. For every
experiment, sterility check (200 ꢃL medium in one well), negative controls (adding 20 ꢃL DMSO to medium), and positive
controls (20 ꢃL ampicilin or gentamicin at 400–0.78 ꢃg/mL) were included. All the experiments were performed in triplicate.
21

ACKNOWLEDGMENT
This work was financially supported by the Science and Technology Program of Guangdong Province, Strategic
Cooperation Program between Guangdong Province and Chinese Academy of Sciences and Core Technology Program for
Strategic Emerging Industries of Guangdong Province (2006B35604002, 2009B091300125, and 2011A081401002).
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