Synthesis and evaluation of antibacterial activities of 5,7-dihydroxycoumarin derivatives

Article abstract of DOI:10.1002/ardp.201000233

This study examines the synthesis and antibacterial activities of 5,7-dihydroxycoumarin derivatives, whose structures were confirmed using analytical and spectral data. Twenty compounds were tested for their antibacterial activities against five microbial species such as E. coli, S. aureus, K. pneumonia, P. aeruginosa, and S. typhimurium were studied. Compounds 5 and 12 exhibited the most potent activity against Staphylococcus aureus with a MIC value of 2.5 μg/mL for each of the compounds. Copyright

Full text of DOI:10.1002/ardp.201000233

386
Arch. Pharm. Chem. Life Sci. 2011, 11, 386–393
Full Paper
Synthesis and Evaluation of Antibacterial Activities of
5,7-Dihydroxycoumarin Derivatives
Yi-Ping Chin1, Wei-Jan Huang2, Feng-Lin Hsu2, Yuh-Ling Lin3, and Mei-Hsiang Lin4
1 Department of Microbiology and Immunology, Taipei Medical University, Taipei, Taiwan, ROC
2 Graduate Institute of Pharmacognosy Science, College of Pharmacy, Taipei Medical University, Taipei,
Taiwan, ROC
3 School of Medicine, Fu Jen Catholic University, Taiwan, ROC
4 School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan, ROC
This study examines the synthesis and antibacterial activities of 5,7-dihydroxycoumarin derivatives,
whose structures were confirmed using analytical and spectral data. Twenty compounds were tested
for their antibacterial activities against five microbial species such as E. coli, S. aureus, K. pneumonia,
P. aeruginosa, and S. typhimurium were studied. Compounds 5 and 12 exhibited the most potent activity
against Staphylococcus aureus with a MIC value of 2.5 mg/mL for each of the compounds.
Keywords: Antibacterial activity / 5,7-Dihydroxycoumarin / Methicillin-resistance / Serratin
Received: August 13, 2010; Revised: November 15, 2010; Accepted: November 26, 2010
DOI 10.1002/ardp.201000233
Introduction
perform various pharmacological functions involving a num-
ber of antibacterial [2, 3], antiviral [4], antitumor [5–9], anti-
inflammatory [10–14], and DNA-repair properties [15, 16].
Previous studies have focused on the inhibition of bacterial
growth and antifungal activity using naturally occurring
coumarins (e.g., umbelliferone, xanthoxin, herniarin, and
scopoletin) [17–21] and synthetic coumarins and angular
furanocoumarins derived from natural coumarins [22]. A
number of coumarin derivatives (e.g., novobiocin and
analogues) have also been shown to be highly effective anti-
biotics [23, 24]. Synthetic derivatives of coumarins possessing
antibacterial properties include 3-acyl [2, 25–28], 7-acetohy-
drazide [29], 3-carbamoyl-4-hydroxycoumarins [28, 30], 6,7-di-
substituted, and 6,7,8-tri-substituted coumarin derivatives
[22]. A [7-hydroxy-4-methyl-8-coumarinyl] glycine complex
with metals (Cu(II), Co(II), Ni(II), Zn(II), and Cd(II)) has been
synthesized, demonstrating greater antimicrobial effective-
ness than the free ligand [31].
Coumarins belong to a class of phenolic substances, charac-
terized by a benzene moiety fused with a-pyrone rings.
Coumarin derivatives are widely distributed throughout
the plant kingdom; they are structurally diverse and can
be roughly classified into five major groups. Simple cou-
marins are those with substituents in the benzene ring.
The second group is furocoumarins, containing a five-mem-
bered furan ring attached to the coumarin structure with the
furan oxygen in the 7-position. Furocoumarins can be either
linear or angular, based on the position of the attached furan
ring [1]. The third group is pyranocoumarins. They have a six-
membered oxygen heterocyclic ring, and can be found in
either linear or angular forms. The fourth group includes
coumarins substituted in the pyrone ring in either position 3
or 4. The last group is the coumarin dimers, which are usually
made up of two coumarin units linked together. Coumarins
Simple coumarins with two hydroxyls in the benzene are
known for their antifungal properties, and the free 6-OH has
been identified as an important element in antifungal
activity [22]. However, the free hydroxyl group at position
7 of the coumarin nucleus is also important in the antibac-
terial activity [32]. Coumarins substituted with dihydroxyl
in the benzene ring demonstrate antibacterial activity
Correspondence: Mei-Hsiang Lin, School of Pharmacy, College of
Pharmacy, Taipei Medical University, 250 Wu-Xing Street, Taipei 110,
Taiwan, ROC.
E-mail: mh100001@tmu.edu.tw
Fax: þ886-2-27361661-3164.
Additional correspondence: Professor Yuh-Ling Lin.
E-mail: yllin054230@mail.fju.edu.tw
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Arch. Pharm. Chem. Life Sci. 2011, 11, 386–393
Antibacterial Activities of 5,7-Dihydroxycoumarins
387
enhanced through the substitution of I and 4-methyl in the
benzene ring [21]. For example, 8-iodo-5,7-dihydroxycou-
marin shows strong to very strong antibacterial character-
istics with an MIC value in the range of 1.56 mg/mL to
50 mg/mL, whereas 5,7-dihydroxycoumarin displays antibac-
terial characteristics with an MIC value in the range of
1000 mg/mL to >1000 mg/mL. 7,8-Dihydroxy-4-methylcou-
marin exhibits good antibacterial activity with an MIC value
in the range of 100 mg/mL to 500 mg/mL.
Four substituted coumarins have been tested for antibac-
terial activity limited to methyl and hydroxyl groups. A few
coumarins were discussed with regard to other substituent
alkyl and aryl simple groups (e.g., allyl, alkyl, methoxy, hal-
ogens) substituted in the benzene ring. In this study, we were
interested in the antibacterial activity of 4-alkyl/aryl-6- or 8-
acyl-5,7-dihydroxycoumarins. In a previous study, 5,7-dihy-
droxy-4-phenylcoumarin [2, serratin, (Fig. 1)] was isolated
from the aerial parts of Passiflora serratodigitata (Passi-
floraceae) [33]. However, no antibacterial activity was
observed. Considering the biological aspect of coumarin,
we prepared compound 2 as mentioned in [34] and sub-
sequently tested the antibacterial activities against five
microbial species including E. coli, S. aureus, K. pneumonia,
P. aeruginosa, and S. typhimurium. The results showed moderate
antibacterial activity against Staphylococcus aureus (MIC ¼
160 mg/mL). We synthesized a series of 4-alkyl/aryl-6-acyl-
5,7-dihydroxycoumarin derivatives (3–21, Figs. 1 and 2)
from phloroglucinol for their antibacterial characteristics.
Out of these, compounds 5 and 12 showed the best
antibacterial activity against Staphylococcus aureus with a
MIC value of 2.5 mg/mL for each of the compounds. In
this study, we described our effort regarding the synthesis
Figure 2. Structure of coumarin derivatives 14–21.
of 5,7-dihydroxycoumarin derivatives and investigated their
structure–activity relationship (SAR).
Results and discussion
Chemistry
Compounds 3–13 were obtained as per previous protocols
reported in [35]. It has been documented that the Pechmann
reaction is able to provide an efficient synthesis of benzopyr-
anone moiety from the condensation of phenols with b-keto
esters in the presence of acid [36–38]. We incorporated the
Pechmann reaction in our protocol to prepare coumarin
derivatives 2 and 14–21. The synthesis of the 6-benzoylcou-
marins (14–17) was achieved as described in Scheme 1. Mono-
acylation of phloroglucinol (22) with the appropriate benzoyl
chlorides gave benzoylphloroglucinols (23–26) in 15–37%
Figure 1. Structure of coumarin (1), serratin (2), and derivatives 3–13.
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Arch. Pharm. Chem. Life Sci. 2011, 11, 386–393
Scheme 1. Synthesis of coumarin derivatives 14–17. Reagents and conditions: a) Substituted benzoyl chloride, AlCl3, nitrobenzene,
608C, 15–37%; b) ethyl benzoylacetate, rt, 22–41%.
yields. Compounds 14–17 were obtained by benzoylphloro-
glucinols (23–26) reaction with either ethyl benzoylacetate or
ethyl propionylacetate in 22–41% yields.
the range of 160–639 mg/mL. Three coumarins (9, 10, and 14)
displayed good antibacterial activity, with MIC values in the
range of 80–159 mg/mL. Two coumarins (15 and 17) showed
strong antibacterial activity, with MIC values in the range of
10–79 mg/mL. Two coumarins (5 and 12) exhibited very strong
antibacterial activity with MIC values <10 mg/mL.
Using the same approach as above, attempts to synthesize
the 6-benzoylcoumarins (29–30) from 40-CN or 40-NO2-benzoyl-
phloroglucinols (27–28) failed (Scheme 2). The phloroglucinol
22 reaction with ethyl benzoylacetate gave 4-phenylcou-
marin (2) in a 28% yield. Compounds 23–26 were treated
under similar condition as 6-benzoyl-4-phenylcoumarin 18.
This resulted in a 16% yield. In addition, diacetylation of 4-
phenylcoumarin (2) using acetic anhydride produced acetyl-
ated 4-phenylcoumarin 21. Furthermore, the reaction of 21
with appropriate benzoyl chlorides produced selective 5-O-
benzoyl-4-phenylcoumarin (19–20).
Coumarin 6 exhibited mild activity only against PS, with a
MIC value of 640 mg/mL. Coumarin 7 showed moderate
activity only against SA, with a MIC value of 160 mg/mL.
Eight coumarins were active against all five microorganisms;
four of these (18, 19, 20, and 21) had MIC values ranging
between 160 mg/mL and 640 mg/mL; three of these (14, 15,
and 17) had MIC values ranging between 20 mg/mL and
640 mg/mL; and one coumarin 5 had a MIC value of 2.5–
320 mg/mL. Coumarin 5 exhibited very strong antibacterial
activity against SA (MIC ¼ 2.5 mg/mL) and moderate activity
against EC, KP, and PA (MIC ¼ 160 mg/mL). Coumarin 12
displayed very strong bioactivity against the entire range
of bacteria tested, demonstrating MIC values against the
Gram-positive bacterium SA with a MIC value of 2.5 mg/mL.
Against the Gram-negative bacterium EC, the value was
640 mg/mL. MIC values for the other two Gram-negative bac-
teria were KP 320 mg/mL and PA 80 mg/mL.
Biological activity
The antibacterial activity of 5,7-dihydroxycoumarins (2–21) is
shown in Table 1. The MIC values of 2–21 were screened
against five strains of bacteria including, Escherichia coli (EC)
ATCC 25922, Staphylococcus aureus (SA) ATCC 25923, Klebsiella
pneumoniae (KP) ATCC 13883, Pseudomonas aeruginosa (PA) ATCC
27853, and Salmonella typhimurium (ST) ATCC 14028. The
potency was defined as following:
From these results, it was also possible to make a number of
correlations regarding the relationship between the struc-
ture of the coumarins and their antimicrobial activities.
ꢀ No bioactivity with MIC > 640 mg/mL
ꢀ Mild bioactivity with MIC in the range of 320–640 mg/mL
ꢀ Moderate bioactivity with MIC in the range of 160–
319 mg/mL
ꢀ 4-Phenyl substitution appears to enhance bioactivity
with 5,7-dihydroxycoumarin and coumarin 2.
Protection of 5-hydroxyl and 5,7-dihydroxyl reduces the
antibacterial activity as with coumarin 19, 20, and 21. It
appears that, as far as the antibacterial activity is con-
cerned, 5- and 7-OH are essential, while protected 5-OH
and 7-OH have less bioactivity.
ꢀ 6-(3-Methyl-butyryl)-4-phenyl-5,7-dihydroxycoumarin (3)
and 8-(3-methyl-butyryl)-4-phenyl-5,7-dihydroxycoumarin
(4) both demonstrate mild bioactivity and 6-(3-methyl-
butyryl)-4-(p-methylphenyl)-5,7-dihydroxycoumarin (13)
enhances bioactivity, showing moderate antibacterial
activity. 6-Benzoyl-4-phenyl-5,7-dihydroxycoumarin (5)
ꢀ Good bioactivity with MIC in the range of 80–159 mg/mL
ꢀ Strong bioactivity with MIC in the range of 10–79 mg/mL
ꢀ Very strong bioactivity with MIC < 10 mg/mL.
These compounds showed a broad range of antimicrobial
activity. A number of the coumarins exhibited activity
against all five microorganisms, and a few of them were
selectively active against one or two strains. Eight coumarins
(3, 4, 6, 11, 16, 19, 20, and 21) showed mild antibacterial
characteristics in general, with MIC values in the range of
320 mg/mL to >640 mg/mL. Five coumarins (2, 7, 8, 13, and 18)
exhibited moderate antibacterial activity, with MIC values in
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Arch. Pharm. Chem. Life Sci. 2011, 11, 386–393
Antibacterial Activities of 5,7-Dihydroxycoumarins
389
Scheme 2. Synthesis of coumarin derivatives 18–21. Reagents and conditions: a) 4-Cyano or 4-nitrobenzoyl chloride, AlCl3, nitroben-
zene, 608C, 23–29%; b) ethyl benzoylacetate, rt, 28%; c) p-anisoyl chloride, AlCl3, nitrobenzene, 608C, 3 h, 16%; d) acetic anhydride,
sodium acetate, 1608C, 1 h, 86%; e) p-cyanobenzoyl chloride or p-nitrobenzoyl chloride, AlCl3, nitrobenzene, 608C, 3 h, 16%.
displays strong antibacterial activity. This suggests that
introducing a 6-benzoyl group to 4-phenylcoumarin has
a positive influence on antibacterial activity.
ꢀ It has been found that the antibacterial activity of 6-acyl-
4-alkyl-5,7-dihydroxycoumarins decreases in the follow-
decreases in the following sequence: 15 > 17 > 18 > 16
(i.e., o-Cl > p-CH3 > p-OH > p-Br). In this study, substi-
tutions with electron-attracting, electron-donating,
hydrophilic and lipophilic substituents on the benzene
of the 6-benzoyl group showed a decrease in bioactivity.
ing sequence: 12 > 14 > 9 ꢁ 10 > 7 ꢁ 8 > 11 ꢁ 6 (i.e., ꢀ Comparing the bioactivities of compound 5 with com-
cyclohexylacyl-4-ethyl > 6-benzoyl-4-ethyl > 6-propionyl-
4-ethyl ꢁ acetyl-4-ethyl > 3-methyl-butyryl-4-ethyl ꢁ
phenylacetoyl-4-ethyl > 3,3-dimethyl-butyryl-4-ethyl ꢁ 3-
methyl-butyryl-4-methyl).
ꢀ The antibacterial activity of 4-phenyl-5,7-dihydroxycou-
marins substituted in the benzene of the 6-benzoyl
group is not enhanced through the substitution of o-
Cl, p-Br, p-CH3 or p-OH, and the antibacterial activity
pound 14 revealed that compounds with a 4-phenyl
group have a higher level of activity, suggesting that
the p-donor of the benzene makes a greater contribution
to the antibacterial activity than inductive electron-
donating ethyl substituent on position 4 of the coumarin.
To conclude, serratin, a naturally occurring coumarin
showing antibacterial activity, is considered an essential
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Arch. Pharm. Chem. Life Sci. 2011, 11, 386–393
Table 1. Antibacterial activity of coumarin derivatives 2–21 obtained from the standard dilution techniques of various compounds tested
against numerous bacteria.
Compound
E. coli
(ATCC 25922)
S. aureus
(ATCC 25923)
K. pneumonia
(ATCC 13883)
P. aeruginosa
(ATCC 27853)
S. typhimurium
(ATCC 14028)
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
640
>640
320
160
>640
>640
2.5
>640
>640
160
>640
>640
640
>640
640
>640
160
>640
>640
640
>640
>640
>640
320
>640
640
640
640
640
640
640
640
640
>640
>640
>640
160
640
>640
640
128
128
640
80
160
640
640
640
640
640
640
640
640
>640
640
640
160
320
>640
>640
>640
>640
640
>640
640
>640
640
640
640
640
640
640
640
640
>640
160
160
320
>640
>640
>640
640
640
640
640
640
640
640
2.5
>640
80
20
>640
40
160
640
320
640
640
640
Minimum inhibitory concentrations (MICs) are reported in mg/mL.
structure in synthetic coumarins. Twenty coumarins were
synthesized and evaluated for antibacterial activity against
EC, SA, KP, PA, and ST, to investigate their structure–activity
Further experiments regarding antibacterial mechanisms
of these 5,7-dihydroxycoumarins are under way.
relationship (SAR). Of these twenty coumarins, compound 5 Experimental chemistry
showed good selectivity against SA with very strong activity
(MIC ¼ 2.5 mg/mL). Compound 12 also showed very strong
activity (MIC ¼ 2.5 mg/mL) against SA as well as good activity
(MIC ¼ 80 mg/mL) against PA. This suggests that the cyclo-
hexylacyl group contributed to action against SA and PA,
while the benzoyl group contributed to selectivity for SA.
Furthermore, compounds 15 and 17 designed from com-
pound 5 displayed a lower level of bioactivity than compound
5, suggesting o-chloro- and p-methyl-substituents to the 6-
benzoyl group had a negative contribution on antibacterial
activity. Compounds 15 and 17 were tested against methicil-
lin-resistant Staphyloccocus aureus (MRSA) (Table 2). Compound
15 also showed good activity against MRSA. In many of the
synthesized coumarins, the free 7-OH was found to be an
important contributor to the antibacterial activity [32].
General
We measured the melting points in a capillary tube using a
MEL-TEMP II melting point apparatus produced by Laboratory
Devices. We also recorded the nuclear magnetic resonance
(NMR) spectra on Bruker DMX-500 FT-NMR spectrometers.
Similarly we recorded chemical shifts in parts per million
downfield from Me4Si. We determined the IR spectra with a
Perkin-Elmer 1760-X FT-IR spectrometer. We recorded mass
spectra on Jeol JMS-D300 and FINNIGAN TSQ-46C mass spec-
trometers and obtained high-resolution mass spectrometry
(HRMS) data with a Jeol JMS-HX110 spectrometer. We per-
formed thin layer chromatography (TLC) on Merck (Art. 5715)
silica gel plates and visualized it under UV light (254 nm).
This was done upon treatment with iodine vapor or upon
heating after treatment with 5% phosphomolybdic acid in
ethanol. We also performed Flash column chromatography
with Merck (Art. 9385) 40–63 mm silica gel 60. We prepared
compound 2 abiding by a method reported in the literature
earlier [34].
Table 2. Minimum inhibitory concentrations (mg/mL) of coumarin
derivatives 15 and 17 were tested against meticillin-resistant
Staphylococcus aureus (MRSA).
Compound
S. aureus methicillin resistance
(ATCC 33591)
General procedure for preparing acylphloroglucinols 23–28
Aluminum trichloride (48 mmol) was added to a suspension
of anhydrous phloroglucinol (22, 12 mmol) in nitrobenzene
15
17
80
160
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Antibacterial Activities of 5,7-Dihydroxycoumarins
391
(30 mL) and was stirred for 30 min at room temperature.
Then, acyl chloride (12 mmol) was added and the mixture
was heated to 608C for 2 h. We then allowed the reaction
mixture to cool to room temperature. Thereafter, the mix-
ture was poured into ice water and extracted with ethyl
acetate. We could then extract the organic layer with 10%
NaOH(aq) and neutralized the alkaline layer with concen-
trated HCl followed by extraction with ethyl acetate. We then
washed the ethyl acetate extract with water and brine. We
dried (MgSO4), filtered, and evaporated the resulting mixture.
We then chromatographed (silica gel, EtOAc/n-hexane 1:5)
the residue to produce solid acylphloroglucinol.
(4-Nitrophenyl)(2,4,6-trihydroxyphenyl)methanone 28
p-Nitrobenzoyl chloride (0.54 mL, 4.0 mmol) was the starting
material from which 28 (0.37 g, 36%) was obtained as a yellow
solid. 1H-NMR (500 MHz, DMSO-d6) d 5.84 (s, 2H), 7.77 (d,
J ¼ 8.5 Hz, 2H), 8.25 (d, J ¼ 8.5 Hz, 2H), 10.24 (s, 1H), 10.66
(s, 2H); 13C-NMR (125 MHz, DMSO-d6) d 94.65, 104.56, 123.27,
128.97, 146.29, 148.59, 161.22, 164.05, 195.37.
General procedure for preparing 5,7-dihydroxycoumarins
2, 14–20
Concentrated H2SO4 (0.2 mL) was added to a mixture of
acylphloroglucinol (1 mmol) and ethyl benzoylacetate or
ethyl propionylacetate (1.1 mmol) in glacial HOAc (3 mL)
and stirred for 2 h at room temperature. The obtained pre-
cipitate was filtered, washed with water, and crystallized
with MeOH to yield the final product.
Phenyl(2,4,6-trihydroxyphenyl)methanone 23
Benzoyl chloride (0.46 mL, 4.0 mmol) was the starting
material from which 23 (0.14 g, 15%) was obtained as a yellow
solid. 1H-NMR (500 MHz, DMSO-d6) d 5.83 (s, 2H), 7.42
(t, J ¼ 7.5 Hz, 2H), 7.52 (t, J ¼ 7.4 Hz, 1H), 7.60–7.61
(m, 2H), 9.83 (s, 1H), 10.08 (s, 2H).
5,7-Dihydroxy-4-phenyl-2H-chromen-2-one 2
22 (1.0 g, 7.9 mmol) and ethyl benzoylacetate (1.53 mL,
7.9 mmol) yielded in 2 (0.56 g, 28%), yellow solid. 1H-NMR
(500 MHz, DMSO-d6) d 5.73 (s, 1H), 6.15 (d, J ¼ 2.0 Hz, 2H), 6.25
(d, J ¼ 2.0 Hz, 2H), 7.30–7.36 (m, 5H), 10.10 (s, 1H), 10.38
(s, 2H); 13C-NMR (125 MHz, DMSO-d6) d 94.64, 99.11, 100.56,
110.15, 127.24, 127.38, 127.76, 139.56, 156.00, 156.75, 157.09,
159.89, 161.70; MS (EI, 70 eV) m/z 254 (Mþ), 226 (base
peak); HRMS (EI) calcd. for C15H10O4þ: 254.0574, found
254.0580.
(2-Chlorophenyl)(2,4,6-trihydroxyphenyl)methanone 24
o-Chlorobenzoyl chloride (0.50 mL, 4.0 mmol) was the start-
ing material from which 24 (0.39 g, 37%) was obtained as a
yellow solid. 1H-NMR (500 MHz, DMSO-d6) d 5.76 (s, 2H), 7.27–
7.43 (m, 4H), 10.63 (s, 1H), 11.69 (s, 2H); 13C-NMR (125 MHz,
DMSO-d6) d 94.58, 104.04, 126.84, 127.01, 128.46, 128.70,
129.72, 142.39, 164.39, 166.55, 196.07.
(4-Bromophenyl)(2,4,6-trihydroxyphenyl)methanone 25
p-Bromobenzoyl chloride (0.90g, 4.0 mmol) was the starting
material from which 25 (0.45 g, 37%) was obtained as a yellow
solid. 1H-NMR (500 MHz, DMSO-d6) d 5.83 (s, 2H), 7.52
(d, J ¼ 8.5 Hz, 2H), 7.62 (d, J ¼ 8.5 Hz, 2H), 9.93 (s, 1H),
10.19 (s, 2H); 13C-NMR (125 MHz, DMSO-d6) d 94.61, 105.41,
125.64, 130.50, 131.22, 139.14, 159.74, 162.40, 195.58.
6-Benzoyl-4-ethyl-5,7-dihydroxy-2H-chromen-2-one 14
23 (0.14g, 0.6 mmol) and ethyl propionylacetate (8.3 mL,
0.6 mmol) gave compound 14 (0.05 g, 27%), yellow solid. IR
(neat) 3000–3500, 1667, 1608 cmꢂ1
;
1H-NMR (500 MHz,
DMSO-d6) d 1.18 (t, 3H), 2.95 (q, 2H), 5.87 (s, 1H), 6.46
(s, 1H), 7.51 (m, 2H), 7.63 (m, 1H), 7.74 (m, 2H), 10.66
(s, 1H), 10.99 (s, 1H); 13C-NMR (125 MHz, DMSO-d6) d 13.88,
28.59, 98.95, 101.24, 107.39, 107.64, 128.87, 129.02, 133.65,
137.27, 153.64, 158.00, 159.48, 160.43, 192.70; MS (EI, 70 eV)
p-Tolyl(2,4,6-trihydroxyphenyl)methanone 26
þ
m/z 310 (Mþ), 309 (base peak); HRMS (EI) calcd. for C18H14O5
:
p-Toluoyl chloride (0.54 mL, 4.0 mmol) was the starting
material from which 26 (0.37 g, 36%) was obtained as a yellow
solid. 1H-NMR (500 MHz, DMSO-d6) d 2.34 (s, 3H), 5.84 (s, 2H),
7.23 (d, J ¼ 7.9 Hz, 2H), 7.53 (d, J ¼ 7.9 Hz, 2H), 9.75 (s, 1H),
9.93 (s, 2H); 13C-NMR (125 MHz, DMSO-d6) d 21.09, 94.43,
106.12, 128.65, 128.83, 137.12, 142.21, 158.94, 161.38, 195.01.
310.0841, found 310.0850.
6-(2-Chlorobenzoyl)-5,7-dihydroxy-4-phenyl-2H-chromen-
2-one 15
24 (0.39g, 1.5 mmol) was the starting material from which 15
(0.24 g, 41%) was obtained as a yellow solid. Mp: 243–2448C;
4-(2,4,6-Trihydroxybenzoyl)benzonitrile 27
p-Cyanobenzoyl chloride (0.54 mL, 4.0 mmol) was the start-
ing material from which 27 (0.37 g, 36%) was obtained as a
IR (neat) 3000–3500, 1687, 1593 cmꢂ1 1H-NMR (500 MHz,
;
DMSO-d6) d 5.80 (s, 1H), 6.24 (s, 1H), 7.32–7.45 (m, 6H),
7.52–7.57 (m, 3H), 10.97 (s, 1H), 11.47 (s, 1H); 13C-NMR
(125 MHz, DMSO-d6) d 98.78, 100.90, 106.89, 110.90, 127.31,
127.90, 130.03, 130.26, 130.43, 132.24, 139.05, 139.28, 155.38,
155.73, 158.36, 160.06, 161.91, 192.19; MS (EI, 70 eV) m/z 392
1
yellow solid. H-NMR (500 MHz, DMSO-d6) d 5.83 (s, 2H), 7.68
(d, J ¼ 8.2 Hz, 2H), 7.89 (d, J ¼ 8.2 Hz, 2H), 10.19 (s, 1H), 10.59
(s, 2H); 13C-NMR (125 MHz, DMSO-d6) d 95.75, 105.83, 114.35,
119.53, 129.47, 133.17, 145.13, 161.71, 164.37, 197.09.
(Mþ), 357(base peak); HRMS (EI) calcd. for C22H13ClO5
:
þ
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Arch. Pharm. Chem. Life Sci. 2011, 11, 386–393
þ
392.0446, found 392.0450; calcd. for C22H1337ClO5
394.0417, found 394.0427.
:
131.32, 132.02, 137.50, 148.16, 153.36, 155.94, 159.05,
161.18, 162.66; MS (EI, 70 eV) m/z 383 (Mþ), 130 (base peak);
HRMS (EI) calcd. for C23H13NO5þ: 383.0788, found 383.0793.
6-(4-Bromobenzoyl)-5,7-dihydroxy-4-phenyl-2H-
chromen-2-one 16
7-Hydroxy-2-oxo-4-phenyl-2H-chromen-5-yl 4-
nitrobenzoate 20
2 (0.92g, 3.6 mmol) and p-nitrobenzoyl chloride (0.68 mL,
25 (0.45g, 1.5 mmol) was the starting material from which 16
(0.14 g, 22%) was obtained as a yellow solid. Mp: 273–2748C;
IR (neat) 3000–3500, 1703, 1604 cmꢂ1 1H-NMR (500 MHz,
;
3.6 mmol) gave compound 20 (0.24 g, 16%), yellow solid.
Mp: 260–2618C; IR (neat) 3000–3500, 1736, 1702 cmꢂ1 1H-
;
DMSO-d6) d 5.78 (s, 1H), 6.33 (s, 1H), 7.37 (m, 5H), 7.74 (m,
4H), 10.57 (s, 1H), 10.83 (s, 1H); 13C-NMR (125 MHz, DMSO-d6) d
98.82, 100.57, 106.76, 110.61, 127.36, 127.41, 127.86, 127.96,
131.03, 132.05, 136.42, 139.39, 153.77, 156.00, 157.94, 159.01,
159.07, 191.67; MS (EI, 70 eV) m/z 435 (Mþ, base peak); HRMS
(EI) calcd. for C22H13BrO5þ: 435.9931, found 435.9941.
NMR (500 MHz, DMSO-d6) d 5.96 (s, 1H), 6.70 (d, J ¼ 2.4 Hz,
1H), 6.80 (t, J ¼ 7.7 Hz, 1H), 6.83 (d, J ¼ 2.4 Hz, 1H), 7.06
(t, J ¼ 7.7 Hz, 2H), 7.22 (d, J ¼ 7.7 Hz, 2H), 7.71 (d,
J ¼ 8.2 Hz, 2H), 8.17 (d, J ¼ 8.2 Hz, 1H), 11.07 (s, 1H);
13C-NMR (125 MHz, DMSO-d6)
d 101.56, 104.98, 108.52,
113.46, 123.06, 126.90, 127.90, 126.85, 127.81, 128.07, 130.14,
131.32, 132.02, 137.50, 148.16, 153.36, 155.94, 159.05, 161.18,
162.66; MS (EI, 70 eV) m/z 403 (Mþ), 150 (base peak); HRMS (EI)
calcd. for C22H13NO7þ: 403.0687, found 403.0697.
5,7-Dihydroxy-6-(4-methylbenzoyl)-4-phenyl-2H-
chromen-2-one 17
26 (0.36g, 1.5 mmol) was the starting material from which 17
(0.11 g, 29%) was obtained as a yellow solid. Mp: 265–2668C;
IR (neat) 3000–3500, 1706 cmꢂ1; 1H-NMR (500 MHz, DMSO-d6)
d 2.34 (s, 3H), 5.76 (s, 1H), 6.33 (s, 1H), 7.33–7.38 (m, 7H), 7.70
(d, J ¼ 8.1 Hz, 2H), 10.47 (s, 1H), 10.71 (s, 1H); 13C-NMR
(125 MHz, DMSO-d6) d 21.26, 98.79, 100.44, 107.55, 110.47,
127.33, 127.41, 127.91, 129.26, 129.44, 134.95, 139.45, 144.22,
153.55, 156.02, 157.50, 158.88, 159.12, 192.05; MS (EI, 70 eV)
2-Oxo-4-phenyl-2H-chromene-5,7-diyl diacetate 21
Initially, a mixture of 2 (0.2 g, 0.80 mmol) and sodium
acetate (19 mg, 0.24 mmol) in acetic anhydride (1.0 mL)
refluxed for 1 h, and then the reaction mixture cooled to
room temperature. The mixture was poured into water and
extracted with ethyl acetate. The ethyl acetate extract was
thereafter washed with water and brine, dried (MgSO4), fil-
tered, and evaporated to give 21 (0.23 g, 86%) as white solid.
þ
m/z 372 (Mþ), 371 (base peak); HRMS (EI) calcd. for C23H16O5
:
372.0992, found 372.0985.
Mp: 184–1858C; IR (neat) 1772, 1734 cmꢂ1
;
1H-NMR
5,7-Dihydroxy-6-(4-hydroxybenzoyl)-4-phenyl-2H-
chromen-2-one 18
(500 MHz, CDCl3) d 1.33 (s, 3H), 2.33 (s, 3H), 6.20 (s, 1H),
6.76 (d, J ¼ 2.4 Hz, 1H), 7.15 (d, J ¼ 2.4 Hz, 1H), 7.31–7.33
(m, 2H), 7.45–7.48 (m, 3H); 13C-NMR (125 MHz, CDCl3) d 19.44,
21.10, 108.72, 110.60, 113.62, 117.48, 127.66, 128.36, 128.73,
137.65, 147.85, 152.79, 153.00, 155.34, 159.29, 168.17, 168.61;
MS (EI, 70 eV) m/z 338 (Mþ), 226 (base peak); HRMS (EI) calcd.
for C19H14O6þ: 338.0790, found 338.0785.
2 (0.56g, 2.2 mmol) was the starting material from which 18
(0.13 g, 16%) was obtained as a yellow solid. Mp: 266–2678C;
IR (neat) 3000–3500, 1683 cmꢂ1; 1H-NMR (500 MHz, DMSO-d6)
d 5.72 (s, 1H), 6.31 (s, 1H), 6.85 (d, J ¼ 8.1 Hz, 2H), 7.34–7.37
(m, 5H), 7.66 (d, J ¼ 8.1 Hz, 2H), 10.43 (s, 1H), 10.48 (s, 1H),
10.67 (s, 1H); 13C-NMR (125 MHz, DMSO-d6) d 98.86, 100.45,
107.98, 110.46, 127.36, 127.41, 127.86, 127.96, 131.03, 132.05, Biological activity evaluation
136.42, 139.39, 153.77, 156.00, 157.94, 159.01, 159.07, 191.67;
MS (EI, 70 eV) m/z 374 (Mþ), 373 (base peak); HRMS (EI) calcd.
for C22H14O6þ: 374.0785, found 374.0776.
General procedure for minimum inhibitory
concentration (MICs)
We used microtiter plates to determine the MICs for bacteria
of 2–21. A 2-fold serial dilution of the tested compound in
Mueller-Hinton broth or yeast nitrogen base with glucose
(YNBG) broth was added to the test organism. The microtiter
plates were then incubated for 16–20 h [39] and the test tubes
were capped at 378C for 48 h [40]. The tested bacteria grew in
Muller-Hinton broth for 4 h and were diluted to OD600 ¼ 0.1.
Microtiter plates thereafter were covered with a sterile lid
and incubated at 378C for 18–20 h. The MICs were deter-
mined by examining the wells for bacterial growth at
OD600. We found that growth was present in the medium
control and absent in the inoculum control [39]. The MIC
7-Hydroxy-2-oxo-4-phenyl-2H-chromen-5-yl 4-
cyanobenzoate 19
2 (0.50 g, 2.0 mmol) and p-cyanobenzoyl chloride (0.34 g,
2.0 mmol) reacted to form compound 19 (0.12 g, 15%), yellow
solid. Mp: 241–2428C; IR (neat) 3000–3500, 2228, 1738,
1
1703 cmꢂ1; H-NMR (500 MHz, DMSO-d6) d 5.95 (s, 1H), 6.67
(d, J ¼ 2.4 Hz, 1H), 6.80 (t, J ¼ 7.7 Hz, 1H), 6.82 (d, J ¼ 2.4 Hz,
1H), 7.06 (t, J ¼ 7.7 Hz, 2H), 7.21 (d, J ¼ 7.7 Hz, 2H), 7.59
(d, J ¼ 8.2 Hz, 2H), 7.83 (d, J ¼ 8.2 Hz, 1H), 11.06 (s, 1H);
13C-NMR (125 MHz, DMSO-d6) d 101.48, 104.96, 108.49,
113.42, 115.70, 117.98, 126.85, 127.81, 128.07, 130.14,
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2011, 11, 386–393
Antibacterial Activities of 5,7-Dihydroxycoumarins
393
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This study was supported by the National Science Council, Taiwan (96–
2113-M-038–002). We are thankful to Sun, S.-Y., Instrumentation Center,
National Taiwan University, for assistance in MS Experiments.
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ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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