Modified tetra-oxygenated xanthones analogues as anti-MRSA and P. aeruginosa agent and their synergism with vancomycin

Article abstract of DOI:10.1016/j.bmcl.2020.127494

Five isolated xanthones from the C. cochinchinense and G. mangostana were evaluated and tested for antibacterial activities. Isolated 4 and 5 exhibited potent anti-MRSA and P. aeruginosa activity, but showed poor pharmacokinetic properties via ADMET prediction. It led us to improve pharmacokinetic properties of 4 and 5 by partially modifying them in acidic condition yielding fourteen analogues. It was found that analogues 4b, 4d and 5b possessed proper pharmacokinetic properties, while only 4b exhibited the best anti-MRSA and P. aeruginosa activity. The SEM results indicated that 4b may interact with or damage the cell wall of MRSA and P. aeruginosa. Moreover, a combination of 4b and vancomycin exhibits synergistic effect against both MRSA and P. aeruginosa at MIC value of 4.98 (MIC = 18.75 μg/mL for 4b) and 9.52 μg/mL (MIC = 75 μg/mL for 4b), respectively.

Full text of DOI:10.1016/j.bmcl.2020.127494

Journal Pre-proofs
Modified tetra-oxygenated xanthones analogues as anti-MRSA and P. aerugi‐
nosa agent and their synergism with vancomycin
Nawong Boonnak, Suchada Chantrapromma, Korbtham Sathirakul, Chutima
Kaewpiboon
PII:
S0960-894X(20)30605-3
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127494
BMCL 127494
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Bioorganic & Medicinal Chemistry Letters
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29 June 2020
10 August 2020
Please cite this article as: Boonnak, N., Chantrapromma, S., Sathirakul, K., Kaewpiboon, C., Modified tetra-
oxygenated xanthones analogues as anti-MRSA and P. aeruginosa agent and their synergism with vancomycin,
Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127494
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1
Modified tetra-oxygenated xanthones analogues as anti-MRSA
and P. aeruginosa agent and their synergism with vancomycin
Nawong Boonnak,a,*Suchada Chantrapromma,b Korbtham Sathirakul,c
and Chutima Kaewpiboond
aDepartment of Basic Sciences and Mathematics, Faculty of Science, Thaksin University,
Songkhla, 90000, Thailand
bDepartment of Chemistry, Faculty of Science, Prince of Songkla University, Hat-Yai,
Songkhla 90112, Thailand.
cDepartment of Pharmacy, Faculty of Pharmacy, Mahidol University, Bangkok, 10400,
Thailand
dDepartment of Biology, Faculty of Science, Thaksin University, Phatthalung, 93210,
Thailand
*Corresponding Author: nawong.b@psu.ac.th
Abstract
Five isolated xanthones from the C. cochinchinense and G. mangostana were
evaluated and tested for antibacterial activities. Isolated 4 and 5 exhibited potent anti-MRSA
and P. aeruginosa activity, but showed poor pharmacokinetic properties via ADMET
prediction. It led us to improve pharmacokinetic properties of 4 and 5 by partially modifying
them in acidic condition yielding fourteen analogues. It was found that analogues 4b, 4d and
5b possessed proper pharmacokinetic properties, while only 4b exhibited the best anti-MRSA
and P. aeruginosa activity. The SEM results indicated that 4b may interact with or damage
the cell wall of MRSA and P. aeruginosa. Moreover, a combination of 4b and vancomycin
exhibits synergistic effect against both MRSA and P. aeruginosa at MIC value of 4.98 (MIC
= 18.75 g/mL for 4b) and 9.52 g/mL (MIC = 75 g/mL for 4b), respectively.

2
GRAPHICAL ABSTRACT
KEYWORDS: Xanthone, MRSA, P. aeruginosa, vancomycin, synergism and ADMET
Bacterial infections are common causes of various diseases such as sepsis, pneumonia,
bloodstream infection, gastrointestinal infection, urinary tract infection; however they are
successfully cured by treatment with antibiotics. Nowadays, many reports1 reveal that many
microorganisms develop themselves to be the drug-resistance microbes such as Staphylococcus
aureus shows resistance to the methicillin (MRSA),1A Neisseria gonorrhoeae1B resists to the
quinolone, while Enterobacteriaceae1C resists to the carbapenam. These become a serious
public health problem. Moreover, there have been several reports of colistin-resistance which
is a last resort treatment for infection caused by drug-resistance bacteria.2,3 Many antibiotic
resistances from various reports around the world showed increment, hence we need a new
promising candidate of antibiotics, which should be highly effective but possess low toxicity.
Nature has been the important source to provide several medicines for treatment of various
types of diseases in humans and animals for many years. Therefore, the natural source should
be considered as a good candidate for a new frame of antibiotics. From the previous works on
the Cratoxylum genus,4 it revealed that oxygenated xanthones exhibited potent antibacterial
activity against both Gram-positive and Gram-negative bacteria. This result led us to
investigate tetraoxygenated xanthone, a major bioactive component from C. cochinchinense
and G. mangostana.

3
The crude CH2Cl2 extracts of the C. cochichinense resin and G. mangostana hulls were
subjected to chemical investigation leading to the isolation of five known 1,3,6,7-tetra-
oxygenated xanthones identified as norathyriol (1),5A -mangostin (2),5B dulxisxanthone F
(3),5C -mangostin (4)5B and -mangostin (5)5B as shown in Scheme 1. Their structures were
elucidated by NMR analysis and comparison of their spectroscopic data with those reported
in the literatures. Compounds 15 have been evaluated for their antibacterial activity against
both Gram-positive and Gram-negative bacteria.
O
OH
O
OH
O
O
OH
8
1
HO
HO
HO
HO
R2O
R3O
O
OH
6
3
OH
O
OR1
1
2
3
1
: norathyriol
2
2a
2b
: mangostin
: R = R = H, R = CH3
1
2
3
: R = H, R = R = CH3
1
2
3
2c
: R = R = R = CH3
O
O
OH
O
O
OH
O
O
OH
H3CO
HO
HO
HO
H3CO
HO
OCH3
OCH3
OH
4
: -mangostin
3
: dulxisxanthone
5
: -mangostin
Scheme 1 The structures of compounds 15 and their derivatives 2a2c
From the anti-bacterial activity result as shown in Table 1, it showed that compound
(1) was inactive against both Gram-positive and Gram-negative bacteria except moderately
active against MRSA (MIC = 18.75 µg/mL), whereas 2, an isoprenyl substitute of 1,
exhibited greater anti-bacterial activity against MRSA than 1 (MIC = 4.68 µg/mL).
Compound (4), a methylated substitute of 2, showed more potent activity against MRSA, B.
subtilis and P. aeruginosa with the same MIC value of 2.34 g/mL, as compare to 2 (MIC =
4.68, 18.75, 18.75 g/mL, respectively), whereas 5, a dimethylated substitute of 2, showed
moderate activity against MRSA and P. aeruginosa with MIC values of 4.68 and 9.37

4
g/mL, respectively. We further attempted to partially modify the structure of compounds 2,
4 and 5 by treatment with MeI/K2CO3 in methanol to yield 2a6A (monomethylated of 2), 2b6B
(dimethylated of 2) and 2c6A (trimethylated of 2), respectively. Compound 2a showed potent
activity against P. aeruginosa (MIC = 4.67 µg/mL) and moderate activity against MRSA
(MIC = 9.37 µg/mL). Compound 2b showed moderate activity against MRSA and P.
aeruginosa with the same MIC values of 18.75 µg/mL while compound 2c was inactive. It
could be suggested that a methoxyl group at C-7 and on tetra-oxygenated xanthone skeleton
should be responsible for the effectiveness as anti-bacterial agent as seen in compound 4.
Table 1 Antibacterial activity (MIC, g/mL) of 15, 2a2c, 4a4f and 5a5e
Gram-positive bacteriaa
Gram-negative bacteriab
Compound
s
B.
MRSA
E. faecalis
VRE
S. typhi
S. sonei P. aeruginosa
subtilis
37.5
18.75
18.75
2.34
2.34
75
1
2
3
4
5
2a
2b
2c
4a
4b
4c
4d
4e
4f
5a
5b
18.75
4.68
9.37
2.34
4.68
9.37
18.75
>300
2.34
18.75
9.37
37.5
>300
>300
37.5
18.75
>300
150
300
9.37
300
150
300
75
75
150
>300
>300
>300
>300
300
>300
75
150
>300
NT
300
150
300
NT
NT
300
>300
>300
>300
>300
NT
>300
18.75
150
18.75
>300
37.5
150
300
300
150
>300
18.75
150
150
>300
75
150
>300
300
37.5
18.75
18.75
2.34
9.37
4.67
18.75
>300
9.37
75
75
75
2.34
18.75
9.37
37.5
>300
>300
150
9.37
75
150
300
300
300
75
300
300
300
>300
>300
150
>300
>300
150
>300
>300
150
NT
NT
>300
150
>300
>300
2.34
300
300
300
300
18.75
300
5c
5d
5e
75
>300
>300
2.34
300
300
300
>300
2.34
300
2.34
300
2.34
300
2.34
>300
2.34
vancomycin
aMethicillin-Resistant Staphylococcus aureus ATCC 43300 (MRSA); Bacillus subtilis;
Enterococcus faecalis TISTR 459; Vancomycin-Resistant Enterococcus faecalis ATCC
51299 (VRE); bSalmonella typhi; Shigella sonei and Pseudomonas aeruginosa
From the in vitro result (Table 1), it revealed that among the isolated xanthones (15)
and partially modified 2a2c, compound (4) exhibited the most potent activity against

5
MRSA, B. subtilis and P. aeruginosa with the same MIC value of 2.34 µg/mL, therefore
suitable for further study on the pharmacokinetic properties. In this study, the
pharmacokinetic properties were conducted via ADMET PredictorTM software.7 For
instance, a compound with low ADMET problem should be considered to further undergo the
process of a new drug development.
From the ADMET result in (Tables 2 and S5), it revealed that 1 has a low
permeability (Pf = 0.0839) in the gut model, whereas compounds 25 and 2a2c containing
isoprenyl side chains on a xanthone nucleus, showing no serious problem about permeability,
suggesting that the isoprenyl side chain should be responsible for its proper permeability in
the gut tissue and also its anti-bacterial activity. According to the ADMET result, it revealed
that compound 4 and the rest of the compounds failed to be used in the in vivo model because
of the hepatotoxicity (Hp), low fraction unbound of these compounds in the plasma (Fu) and
mutagenicity (Mu) problems. Since in Table 1 compounds 4 and 5 exhibited potent
antibacterial activity, thus, further study should be focussed on the structural modification of
compounds 4 and 5 in order to improve their pharmacokinetic and pharmacodynamic
properties.
To increase the pharmacodynamic properties of 4, the isoprenyl side chain of 4 was
partially modified to 3-hydroxyl-3-methyl butyl group and dimethylchromane ring in the
acidic medium to yield compounds 4a4d (Scheme 2). The antibacterial activity of 4a4d
were shown in Table 1. Only 4a showed strong activity against MRSA and B. subtilis with
MIC value of 2.34 g/mL, the same as those of 4, while compound 4b showed moderate
activity against MRSA and B. subtilis, whreas inactive against P. aeruginosa with MIC
values of 18.75, 18.75 and 75 g/mL, respectively.

6
Table 2 Prediction of pharmacokinetic properties and toxicity risks of 15 and 2a2c by ADMET prediction TM software
Absorption
Distribution
Metabolism and Excretion
Toxicity risks
ADMET
riski
Vde
Rah
No.
CYPf
ADMET code
Pfa
Swb
Owc
Fud
Hpg
(L/kg)
0.53
1.11
2.26
1.08
2.88
1.05
3.39
1.20
(mg/kg)
MET_3A4
0.182
MET_2C9
1.492
3.415
2.487
4.36
1
2
3
4
0.0839
0.8353
1.0458
0.9307
1.1383
0.9872
1.2881
1.0863
0.1390
0.0353
0.0160
0.0177
0.0051
0.0190
0.0031
0.0155
1.79
4.28
4.24
4.58
4.58
4.51
4.97
4.92
3.48
1.42
3.36
1.21
3.17
1.34
3.62
1.22
2
2
2
2.5
2
2.5
3
1205.53
497.26
583.39
336.11
477.29
396.64
452.71
319.06
3
2
3
2
3
2
3
2
Pf, Fu, Hp
Fu, Hp
2.031
2.732
3.433
Fu, Ow, Hp
Fu, Muj
5
4.103
3.006
5.171
4.404
3.156
2.439
1.964
2.672
Fu, Ow, Hp
Fu, Mu
Sw, Ow, Mu
Fu, Ow
2a
2b
2c
2
aPf = low permeability if < 0.1; bSw = low solubility if <0.005; cOw = excessive lipophobicity if <-0.891; dFu = low fraction unbound in plasma if <3.5%; eVd = high steady-
state volume of distribution if > 5.5; fCYP = metabolic risk if and MET_3A4 > 30 and MET_2C9 > 30; gHp = human liver hepatotoxicity if TOX_AlkPhos = Toxic or
TOX_GGT = Toxic or TOX_LDH = Toxic and TOX_SGOT = Toxic or TOX_SGPT = Toxic; hRa = acute toxicity in rats if <300; ieach score related with potential ADMET
problem; jMu = mutagenicity if TOX_MUT_risk > 2
OR
OH
O
O
OH
O
O
O
OH
H3CO
HO
H3CO
HO
H3CO
HO
O
O
OH
O
O
4a
4d
4b
4c
: R = H
: R = CH3
O
OH
O
OH
H3CO
H3CO
O
O
O
O
O
S
S
O
O
O
O
O
Br
Br
4e
4f
Scheme 2 The structures of compounds 4a4f

7
In addition, the pharmacokinetic properties of 4a4d were conducted via ADMET
Predictor (Tables 3 and S5), it revealed that potent compound 4a possessed low solubility
(Sw = 0.0020), excessive lipophobicity (Ow = 4.75) and showed toxicity to the rat (Ra =
299.60 mg/kg). Surprisingly, compounds 4b and 4d were moderately active for antibacterial
activity against MRSA and B. subtilis but they have no ADMET risk (Table 3). To increase a
permeability of 4 (Pf = 0.9307), a hydroxyl group of 4 was replaced with the isoprenyl and p-
bromobenzenesulfonyl side chains to yield 4e and 4f, respectively. The ADMET result
revealed that they have better permeability than 4, but along with high risk of the other
ADMET parameters. From the structure relationship between compounds 4 and 4a4d, it
could be suggested that the replacement of an isoprenyl side chain with a 3-hydroxy-3-
methylbutyl group and a chromane ring can obviously improve the pharmacokinetic
properties of 4 to be more drug-likeness. It can be summarized that 4b and 4d with no
ADMET risk have the proper pharmacokinetic properties. However, compound 4b showed
better antibacterial activity than 4d, see Table 1.
We further studied the possible mode of action of 4, 4a, and 4b against MRSA and P.
aeruginosa by observing the bacteria cell morphology through scanning electron microscope
(SEM) for 24 h after treatment with 4, 4a, and 4b.
From the SEM results (see Figs. 1 and 2), it was clearly indicated that the cell
morphology of MRSA and P. aeruginosa after treatment with 4, 4a, and 4b, most cells were
completely deformed whose results were correlated to their good antibacterial activity (Table
1). Therefore, it can be suggested that 4, 4a, and 4b may interact with or damage the cell wall
of MRSA and P. aeruginosa as seen by the formation of pores on the cell wall of MRSA and
P. aeruginosa (Figs. 1 and 2).

8
Table 3 Prediction of pharmacokinetic properties and toxicity risks 4 and 4a4f by ADMET prediction TM software
Absorption
Sw
Distribution
Metabolism and Excretion
CYP
Toxicity risks
ADMET
risk
No.
ADMET code
Vd
(L/kg)
Ra
(mg/kg)
Pf
Ow
Fu
Hp
MET_3A4
3.433
MET_2C9
4.360
4
0.9307 0.0177
0.5622 0.0020
0.0069
0.4029 0.0032
0.0178
4.58
4.75
3.64
4.45
3.25
7.07
1.21
4.64
7.98
6.68
6.56
2.02
1.08
3.24
3.89
3.97
3.34
5.55
2.5
2
2
2
2
336.11
299.60
383.85
307.02
368.89
2
3
0
2
0
Fu, Mu
Sw, Ow, Ra
4a
4b
4c
4d
4e
2.823
3.509
3.289
1.833
0.1949

4.167
2.044
Sw, Ow
3.332
1.921
0.1965

2.1528 0.0012
60.88
3.903
1.5
394.62 10
Sw, Fu, Ow, Vd, 3A,a
Hp, ti,c Sz,d Rb,e Chf
4f
2.0880 0.0030
6.72
0.32
0.77
74.348
46.604
0
208.07 11
Sw, Fu, Ow, 3A,a C9,b ti,c
Ra, Sz,d Rb,e Ch,f HAg
a3A = CYP_3A4_Substr =Yes and MET_3A4_CLint > 30; bC9 = CYP_2C9_Substr = Yes and MET_2C9_CLint >30; cti = specific inhibition
constant for the CYP 3A4-mediated metabolism of testosterone; dSz = large size; eRb = too flexible; fCh = excessive partial atomic charge; gHA
= too many H-bond acceptors
a) control
b) 4
c) 4a
d) 4b
e) vancomycin
Figure 1 SEM images of MRSA after treatment with 4, 4a and 4b

9
a) control
b) 4
c) 4a
d) 4b
e) vancomycin
Figure 2 SEM images of P. aeruginosa after treatment with 4, 4a and 4b
control
4b+VCM
vancomycin
Figure 3 SEM images of MRSA after co-treatment with 4b and vancomycin

10
Further, we decided to extend the study on synergism of potent antibacterial
compounds 4 and 4a with vancomycin (commercially available antibiotic), and also a
combination between a moderate antibacterial compound 4b (no ADMET risk) and
vancomycin against MRSA and P. aeruginosa strains with the hope of enhancing their
activity. FIC index results were shown in Tables 4 and 5. From this result in Table 4,
synergism between 4 and vancomycin (entry 1) against MRSA was observed at MIC value of
0.5850 g/mL, lower than MIC value of 4 (MIC value of 2.34 µg/mL, Table 1). While, a
combination of 4b and vancomycin (entry 3) exhibited synergistic effect against MRSA at
MIC value of 4.6875 g/mL (MIC value of 18.75 g/mL for 4b).
Table 4 MIC (g/mL) and fractional inhibitory concentration (FIC) index of combination of
4, 4a, 4b and vancomycin against MRSA
Total MIC
(g/mL) of
combination
MIC (g/mL)
Entry
FIC index
4
4a
4b
VCMc
1
2
3
0.5850
0.2925
0.5850
0.2925
0.8775
1.7550
4.9800
0.375a
0.750b
0.375a

1.1700





4.6875
asynergism if FIC = 0.300.70,8A bmoderate synergism if FIC = 0.700.858A, cthe
concentration of VCM start at 1.17 g/mL.
We further studied the possible mode of action of a combination of 4b and
vancomycin at MIC value of 4.6875 g/mL against MRSA by observing the cell
morphology. From the SEM results (see Fig. 3), it was clearly indicated that most cells of
MRSA were completely deformed
From the result in Table 5, all combinations of 4, 4a, 4b with vancomycin exhibit
synergistic effect against P. aeruginosa, especially for 4 and 4b exhibit significant synergistic
effect against P. aeruginosa with FIC index at 0.187. Moreover, a combination of 4b and
vancomycin exhibits strong antibacterial activity against P. aeruginosa with MIC value of
9.375 g/mL compare to the pure form of 4b (MIC value = 75 g/mL, Table 1).

11
Table 5 MIC (g/mL) and FIC index of combination of 4, 4a, 4b and vancomycin against
P. aeruginosa
Total MIC
(g/mL) of
combination
MIC (g/mL)
Entry
FIC index
4
4a
4b
VCMc
1
2
3
0.2925
0.1462
0.2925
0.1462
0.4387
2.6350
9.5210
0.187a
0.375b
0.187b

2.3425





9.3750
astrong synergism if FIC = 0.100.30,8A bsynergism if FIC = 0.300.708A, cthe concentration
of VCM start at 1.17 g/mL.
The possible mode of action against P. aeruginosa of 4b and vancomycin was further
investigated by observing the cell morphology from SEM (see Fig. 4), which was clearly
indicated that most cells of MRSA were completely deformed.
a) control
b) 4b+ vancomycin
c) vancomycin
Figure 4 SEM images of P. aeruginosa after treatment with 4b and vancomycin
From the result of antibacterial activity and ADMET, it revealed that 4b has the
proper pharmacokinetic and pharmacodynamics properties that meet criteria for further study
on in vivo model. For in vivo study, a 4b would be used in gram scale to complete the project.
We also report a partial modifying of 4b from 4 in two-steps with 49% overall yield (Scheme
S1), of which compound 4 was mainly found as a major constituent in the hulls of
G.mangostana a bio-waste after the agricultural product processing in Thailand.

12
The partial modification of 5 was shown in Scheme 3. From the antibacterial activity
in Table 1, it was shown that only 5b showed moderate activity against MRSA and P.
aeruginosa with the same MIC value of 18.75 g/mL as compare to those of 5 (MIC value of
4.68 and 9.37 g/mL, respectively), and no ADMET risk (Tables 6 and S5), whereas
compounds 5a, 5c-5f were inactive for antibacterial activity. In this work, we have no further
study on compound 5b because of its lower polarity than 4b and 4d.
OH
O
OH
O
OH
H3CO
HO
H3CO
HO
O
OCH3
O
OCH3
5b
5a
OR
O
O
O
O
H3CO
HO
H3CO
HO
O
OCH3
O
OCH3
5c
5d
5e
: R = H
: R = Ac
Scheme 3 The structures of compounds 5a5e
In conclusion, we have isolated xanthones from the C. cochinchinense and G.
mangostana were tested for antibacterial activities. Isolated 4 and 5 exhibited potent anti-
MRSA and P. aeruginosa activity, but showed poor pharmacokinetic properties. It led us to
improve pharmacokinetic properties of 4 and 5 by partially modifying them in acidic
condition yielding fourteen analogues. It was found that analogues 4b, 4d and 5b possessed
proper pharmacokinetic properties, while only 4b exhibited the best anti-MRSA and P.
aeruginosa activity. Moreover, a combination of 4b and vancomycin exhibits synergistic
effect against both MRSA and P. aeruginosa.

13
Table 6 Prediction of pharmacokinetic properties and toxicity risks 5 and 5a5e by ADMET prediction TM software
Absorption
Sw
Distribution
Metabolism and Excretion
CYP
Toxicity risks
ADMET
risk
No.
ADMET code
Vd
(L/kg)
Ra
(mg/kg)
Pf
Ow
Fu
Hp
MET_3A4
4.103
MET_2C9
3.156
5
1.1383
1.1299
0.6370
0.7958
0.3250
0.3052
0.0051
0.0023
0.0150
0.0012
0.0045
0.0017
4.58
5.36
3.5
4.72
3.54
3.94
3.17
5.67
5.34
5.25
8.51
7.84
2.88
4.23
3.33
4.29
4.87
4.56
2
2
2
2
2
2
477.29
517.04
581.27
275.20
334.10
273.52
3
2
0
3
1
2
Ow, Fu, Hp
Sw, Ow

Sw, Ow, Ra
Sw
5a
5b
5c
5d
5e
6.478
1.398
2.954
1.760
3.327
2.508
3.810
1.346
3.799
1.649
Sw, Ra

14
Acknowledgements
We are greatly indebted to IPST for research funds for DPST graduate with first replacement
(No.03/2557). The authors thank Assoc. Prof. Chanita Ponglimanont for useful suggestions.
N.B. gratefully acknowledges Dr.Theerasak Anantapong for antibacterial testing.
Supplementary data
Experimental section, Synthesis of xanthone analogues, Antibacterial assay, Synergistic study
and 1H and 13C NMR spectra (Figures S1S19) of selected compounds. All compounds were
predicted under 24 criteria (Tables S1S4) in pH 7.4 condition described in Table S5.
References and notes
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