6,8-Dibromo- and 6,8-Diiodo-5,7-dihydroxyflavones as New Potent Antibacterial Agents

Article abstract of DOI:10.1246/cl.171089

Thirteen flavones including chrysin, three natural and nine synthesized compounds, were examined for antibacterial activity. 6,8-Dibromo- (11) and 6,8-diiodo-5,7-dihydroxyflavone (12) exhibited the highest activity against all bacteria with MIC 31.2562.5

Full text of DOI:10.1246/cl.171089

CL-171089
Received: December 5, 2017 | Accepted: December 18, 2017 | Web Released: February 23, 2018
6,8-Dibromo- and 6,8-Diiodo-5,7-dihydroxyflavones as New Potent Antibacterial Agents
Krongkan Kingkaew,1 Ritbey Ruga,1,2 and Warinthorn Chavasiri*1
1Center of Excellence in Natural Products Chemistry, Department of Chemistry, Faculty of Science,
Chulalongkorn University, Bangkok 10330, Thailand
2Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
E-mail: warinthorn.c@chula.ac.th
Thirteen flavones including chrysin, three natural and
nine synthesized compounds, were examined for antibacterial
activity. 6,8-Dibromo- (11) and 6,8-diiodo-5,7-dihydroxyflavone
(12) exhibited the highest activity against all bacteria with MIC
31.25-62.5 ¯M. For Propionibacterium acnes and Staphylo-
coccus aureus, these compounds were bacteriostatic agents,
while for Streptococcus sobrinus, S. mutans, and Salmonella
typhi, they were bactericidal agents. The combination of 11 and
12 with known antibiotics displayed synergistic effect. The
combination of 11 with streptomycin (16) revealed the most
synergistic effect with rate in increasing antibacterial activity of
streptomycin in combination was sixteen folds against P. acnes,
S. sobrinus, and S. mutans.
investigated in order to enhance their biological activities. The
ether and halogenated derivatives of chrysin have been evaluated
for their biological activities as hypoglycemic,14 anticancer,15
and anti-inflammatory agents,16 respectively. Moreover, chrysin
derivatives were synthesized by alkylation and acetylation to
evaluate their cytotoxicity,17 antitumor activity,10 and inhibitory
activity of prostaglandin production.18
Thirteen flavones were collected including 5-hydroxy-7-
methoxyflavone (1), 5-hydroxy-3,7-dimethoxyflavone (2), and
5-hydroxy-3,7,4¤-trimethoxyflavone (3) from the rhizomes of
Kaempferia parviflora Wall. Ex Baker (Thai name: Kra-chai-
dum, Zingiberaceae). The others with different substituents
were synthesized by functionalization of chrysin (13) (Figure 1).
5,7-Dimethoxyflavone (4) was prepared by refluxing 13 with
(CH3)2SO4 in the presence of K2CO3 in acetone.19 Six ether
analogues of flavones (5-10) were synthesized by reacting 13
with selected bromoalkane in the presence of K2CO3.20 6,8-
Dibromo- (11) and 6,8-diiodo-5,7-dihydroxychrysin (12) were
manipulated from the reaction of 13 in acetone with NaBr/
oxone,16 and that in acetic acid with I2/CH2Cl2.19 All com-
pounds were purified by silica gel column and characterized by
1H NMR.
Keywords: Flavone
| Antibacterial agent | Synergistic effect
Increasing resistance of pathogenic bacteria against avail-
able antibacterial agents is a major concern among scientists and
clinicians worldwide. Nowadays, pathogenic microorganisms
are more difficult to treat with existing drugs.1 Numerous efforts
have been made to find new and potent antibacterial compounds
from various sources including nature and synthesis with
expectation to decrease the resistance of pathogenic bacteria.2
Several studies demonstrated that certain compounds revealed
promising antibacterial activity such as carnosic acid,3 α-
mangostin,4 epigallocatechin gallate,5 and erybraedin A.6 In
addition, synergistic effect of natural and synthesized com-
pounds with commercial antibiotics in combination could
contribute to increase the antibacterial activity of either anti-
biotics or compounds. The potential chalcone analogues (2¤-
bromo-2-hydroxychalcone and 4-hydroxychalcone) in combina-
tion with non-β-lactam antibiotic (ciprofloxacin) were previ-
ously reported to possess synergistic effects against methicillin-
resistant Staphylococcus aureus (MRSA) at very low MICs for
ciproflaxocin in both combination with eight-fold increased
susceptibility of MRSA.7 Another report demonstrated the
synergistic effect of the combination between tetrandrine
(bisbenzylisoquinoline alkaloid) and cefazolin reduced 75-
94%/75-88% compared with the agents alone against 90% of
the tested pathogenic strains (SCCmec III type MRSA isolates).8
Flavones belong to a class of flavonoid widely distributed in
leaves, flowers, and fruits as aglycones or their glycosides.
Celery, parsley, and ginkgo biloba are among the major sources
of flavones. There have been a lot of studies concerning the
inhibitory activities of flavones. Natural flavones such as chrysin
(5,7-dihydroxyflavone), apigenin (4¤,5,7-trihydroxyflavone),
luteolin (3¤,4¤,5,7-tetrahydroxyflavone) have been reported to
possess various biological activities as anti-inflammatory,9
antitumor,10 anticancer,11 antibacteria,12 and antipruritic.13 Fur-
thermore, the structural modification of flavones has been
The preliminary screening of antibacterial activity of
thirteen flavones against 5 pathogenic bacteria including
P. acnes (KCCM41747), S. aureus (ATCC25923), S. sobrinus
(KCCM11898), S. mutans (ATCC25175), and S. typhi (ATCC
A)
B)
Figure 1. A) Structures of natural flavones isolated from the
rhizomes of K. parviflora. B) Synthesis of flavone analogues: a)
dimethyl sulfate, K2CO3, acetone, reflux, 24 h; b) RBr, K2CO3,
acetone, reflux, overnight; c) NaBr, oxone, acetone or I2, acetic
acid.
358 | Chem. Lett. 2018, 47, 358–361 | doi:10.1246/cl.171089
© 2018 The Chemical Society of Japan

Table 1. Inhibition zone (mm) of thirteen flavone analogues (1-13)
Inhibition zone average (mm) « SD
Compounda
P. acnes
S. aureus
S. sobrinus
S. mutans
S. typhi
KCCM41747
ATCC25923
KCCM11898
ATCC25175
ATCC442
1
2
3
4
5
6
7
8
9
10
11
12
13
8.67 « 0.47
8.00 « 0.00
9.00 « 0.00
9.33 « 0.47
7.00 « 0.00
7.00 « 0.00
8.67 « 0.47
9.00 « 0.00
9.00 « 0.00
9.67 « 0.47
21.00 « 0.82
20.33 « 0.94
10.33 « 0.47
10.67 « 0.47
9.00 « 0.00
9.00 « 0.00
9.33 « 0.94
¯6.00
9.67 « 0.47
11.00 « 0.82
11.00 « 0.00
12.33 « 0.47
8.67 « 0.47
9.00 « 0.82
10.67 « 0.47
11.67 « 0.47
13.33 « 0.47
8.00 « 0.00
19.67 « 0.47
18.67 « 0.47
13.67 « 0.94
10.67 « 0.94
11.67 « 0.47
9.33 « 0.47
11.33 « 0.47
7.00 « 0.00
7.00 « 0.00
9.67 « 0.47
13.00 « 0.00
11.33 « 0.47
10.33 « 0.47
19.67 « 0.47
18.67 « 1.25
15.33 « 0.47
8.00 « 0.00
11.00 « 0.82
9.67 « 0.47
8.00 « 0.00
¯6.00
7.00 « 0.00
10.67 « 0.47
9.00 « 0.00
8.67 « 0.94
8.67 « 0.47
19.67 « 0.47
18.33 « 1.25
11.67 « 0.47
¯6.00
9.67 « 0.47
9.33 « 0.94
10.33 « 0.47
9.00 « 0.00
20.00 « 0.82
20.67 « 0.47
11.00 « 0.82
a1 mM; criteria of inhibition zone activity (mm): inhibition zone >15.0: excellent, 13.1-15.0: very good, 10.1-13.0: good, 8.1-10.0:
moderate, 6.1-8.0: weak, ¯6.0: no activity.
Table 2. MICs and MBCs of 11, 12 and known antibiotics (14-17)a
P. acnes
S. aureus
S. sobrinus
S. mutans
S. typhi
KCCM 41747
ATCC 25923
KCCM 11898
ATCC 25175
ATCC 477
Compound
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
11
12
14
15
16
17
31.25
31.25
15.62
7.81
15.62
62.50
250
250
250
125
125
31.25
31.25
62.50
3.91
15.62
125
250
250
500
250
125
62.50
62.50
15.62
1.95
3.91
125
250
250
250
250
125
62.5
62.5
15.62
0.98
1.95
125
250
250
250
62.5
62.5
15.62
0.49
3.91
125
250
250
250
125
250
250
15.625
>1000
1000
>1000
>1000
>1000
aSerial concentration of compounds (1000-0.488 ¯M) by two-fold serial dilution. MIC and MBC (¯M).
442) was conducted by diffusion method (Table 1).21 Among
the tested compounds, halogen-substituted flavones (11 and 12)
displayed the strongest activity against all bacteria with an
excellent inhibition zone followed by 1, 2, 4, 8, 9, and 10 with
good activity against S. mutans. Between bromo- and iodo-
substituents of 5,7-dihydroxyflavone, 11 showed slightly higher
antibacterial activity than 12 except for S. aureus. Comparing
between 13 with 5,7-dihydroxy group and 5,7-dimethoxyflavone
(4), 13 exhibited higher activity than 4. The former exhibited
good to excellent activity against five bacteria, especially
S. mutans, with inhibition zone about 15 mm while 4 showed
weak activity against S. typhi. For these flavones, the hydroxy
groups at C-5 and C-7 played an important role for this activity.
In addition, 9 revealed a very good activity while 2, 3, 4, 7,
and 8 showed good inhibitory activity against S. sobrinus. Good
activity was also observed by 2 and 7 against S. typhi, whereas 1
and 9 exhibited good activity against S. aureus. 11 and 12 were
selected to further examine their potent growth inhibition by
determination of the minimum inhibitory concentration (MIC)
and minimum bactericidal concentration (MBC).
Determination of antibacterial susceptibility with two-fold serial
dilution (250-7.81 ¯M) disclosed the MIC of 11 and 12 at 31.25
and 31.25 ¯M against P. acne and S. aureus, respectively, while
their MBCs were 250 ¯M.
Both compounds showed the same activity against S.
sobrinus, S. mutans, and S. typhi with MIC 62.5 ¯M and MBC
250 ¯M. MIC and MBC of four antibiotics were found to be
in the range of 125-0.488 ¯M. The MIC index was calculated
by MBC/MIC to determine if the compounds possessed
bactericidal or bacteriostatic properties.24 When MIC index is
¯4, the compound is bactericidal while when the MIC index
>4, the compound is bacteriostatic. For P. acnes and S. aureus,
the MIC index of 11 and 12 were 8. Thus, for these bacteria,
both compounds were bacteriostatic agents. Whereas their MIC
index on S. sobrinus, S. mutans, and S. typhi were 4 revealing
that these compounds were bactericidal agent. The results of
these compounds are presented in Table 2.
In certain cases, the use of some antibiotics alone is not
effective to inhibit pathogenic bacteria even some bacteria
become resistance. In addition, the use of antibiotics at high
doses may affect normal cells in humans. Therefore, there is a
need to search for new antibacterial agents and test their activity
alone and in combination. Generally, the antibacterial activity of
Antibacterial tests of 11 and 12 with four antibiotics (14-17)
were conducted by determining MIC and MBC using colori-
metric assay according to Sarker et al.22 and Koochak et al.23
Chem. Lett. 2018, 47, 358–361 | doi:10.1246/cl.171089
© 2018 The Chemical Society of Japan | 359

Table 3. MICs of 11 and 12 in combination with known antibiotics (14-17)a
P. acnes
S. aureus
S. sobrinus
S. mutans
S. typhi
KCCM 41747
ATCC 25923
KCCM 11898
ATCC 25175
ATCC 477
Antibiotics
11
12
11
12
11
12
11
12
11
12
MICb Actc MICb Actc MICb Actc MICb Actc MICb Actc MICb Actc MICb Actc MICb Actc MICb Actc MICb Actc
14
15
16
17
1.953
0.977
0.977
7.813
S
S
S
S
1.953
0.977
0.977
7.813
S
S
S
S
7.8125
0.488
1.953
S
S
S
S
3.906
0.488
1.953
S
S
S
S
1.953
0.244
0.244
S
S
S
S
1.953
0.244
0.488
S
S
S
S
1.953
0.122
0.122
S
S
S
S
1.953
0.122
0.244
S
S
S
S
1.953
0.122
0.488
S
S
S
S
1.953
0.122
0.488
S
S
S
S
15.625
15.625
15.625
15.625
15.625
15.625
15.625
15.625
b
c
aSerial concentration of compounds (125-0.488 ¯M) by two-fold serial dilution. Combination. Activity. S (Synergis); The FICI was
interpreted as follows: synergy, FICI ¯ 0.5; additive, 0.5 < FICI 1; indifference, 1 < FICI ¯ 2; antagonism, FICI > 2. Each test was run
in duplicate.
flavonoid derivatives against Methicillin-resistant Staphylococ-
cus aureus (MRSA) is weak. Nonetheless, they could contribute
to enhance antibacterial activity of antibiotics when they are in
combination.7 In this research, halogenated flavones (11 and 12)
were selected for testing their antibacterial activity in combina-
tion with known antibiotics namely chloramphenicol (14),
tetracycline (15), streptomycin (16), and ampicillin (17) using
broth checkerboard method.25 The range of tested compounds
and antibiotics in combination was started from a half of MIC
of each compound in two serial dilutions. Synergistic was
determined by measuring the fractional inhibitory concentration
index (FICI).7
supported by East Kalimantan Government under Kaltim
Cemerlang Scholarship. The Thailand Research Fund via
Directed Basic Research Grant (Grant No. DBG 5980001) for
financial support is also acknowledged.
Supporting Information is available on http://dx.doi.org/
10.1246/cl.171089.
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© 2018 The Chemical Society of Japan | 361