Caged xanthones: Potent inhibitors of global predominant MRSA USA300

Article abstract of DOI:10.1016/j.bmcl.2016.05.030

Total of 22 caged xanthones were subjected to susceptibility testing of global epidemic MRSA USA300. Natural morellic acid showed the strongest potency (MIC of 12.5 μM). However, its potent toxicity diminishes MRSA therapeutic potential. We synthetically modified natural morellic acid to yield 13 derivatives (3a-3m). Synthetically modified 3b retained strong potency in MRSA growth inhibition, yet the toxicity was 20-fold less than natural morellic acid, permitting the possibility of using caged xanthones for MRSA therapeutic.

Full text of DOI:10.1016/j.bmcl.2016.05.030

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Caged xanthones: Potent inhibitors of global predominant MRSA
USA300
a
b
a
a,
⇑
Pongkorn Chaiyakunvat , Natthinee Anantachoke , Vichai Reutrakul , Chutima Jiarpinitnun
a
Department of Chemistry and Center for Innovation in Chemistry (PERCH-CIC), Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand
Department of Pharmacognosy and Center for Innovation in Chemistry (PERCH-CIC), Faculty of Pharmacy, Mahidol University, Sri-Ayudhaya Road, Bangkok 10400, Thailand
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Total of 22 caged xanthones were subjected to susceptibility testing of global epidemic MRSA USA300.
Received 28 November 2015
Revised 21 April 2016
Accepted 11 May 2016
Available online xxxx
Natural morellic acid showed the strongest potency (MIC of 12.5
ishes MRSA therapeutic potential. We synthetically modified natural morellic acid to yield 13 derivatives
3a–3m). Synthetically modified 3b retained strong potency in MRSA growth inhibition, yet the toxicity
lM). However, its potent toxicity dimin-
(
was 20-fold less than natural morellic acid, permitting the possibility of using caged xanthones for MRSA
therapeutic.
Keywords:
Ó 2016 Elsevier Ltd. All rights reserved.
Caged xanthones
Methicillin resistant Staphylococcus aureus
USA300
Bacterial invasion
Garcinia hanburyi hook. f. belongs to Guttiferae family, com-
monly found in Southeast Asia and China. In traditional medicine,
that the potent cytotoxicity of these compounds hinders the poten-
tial of caged xanthones as anti-bacterial agent.
1
the resin is applied as a laxative as well as therapeutic of infected
wounds. The prenylated caged xanthones 1–9 were isolated from
We are interested in developing antibacterial agents for global
epidemic methicillin-resistant Staphylococcus aureus (MRSA), par-
ticularly MRSA PFGE strain type USA300 (SF8300). USA300 is
known as community-acquired MRSA; its rapid transmission and
resistance towards antibiotics lead to global healthcare prob-
2
resins and fruits of Garcinia hanburyi hook. f. (Fig. 1). The structures
were elucidated based on spectroscopic analyses.3 Besides their
unique chemical structure, caged xanthones have received a high
level of attention due to their potent cytotoxicity against various
cultured mammalian cancer cell lines as well as drug-resistant cell
lines at low micromolar concentration.2 The mechanisms under-
lying the anticancer and antitumor activities were extensively
2
2,23
lem.
Natural caged xanthones 1–9 isolated from G. hanburyi
hook. f. were first quantitatively screened for their ability to inhibit
growth of MRSA USA300 strain SF8300 via disk diffusion assay.
Methicillin susceptible S. aureus (MSSA) ATCC 25923 was used as
control strain. Out of 9 caged xanthones, 5 compounds (2–5, 8)
exhibited inhibition zones against both MSSA and MRSA strains
while beta-lactam antibiotics such as ampicillin and oxacillin, as
anticipated did not inhibit MRSA USA300. These 5 caged xanthones
(2–5, 8) were further subjected to microdilution assay to quantita-
tively access their minimum inhibitory concentration (MIC) and
minimum bactericidal concentration (MBC). Among the natural
caged xanthones assayed, morellic acid (3) was the most potent
–7
8
–12
studied.
Multiple targets and mechanisms were suggested to
be accounting for the potent cytotoxicity of caged xanthones.
These includes the induction of apoptosis via regulation of key pro-
teins such as caspase3, Bax, and Bcl-2, suppression of telomerase
activity, inhibition of cell metastasis by down-regulating the
expression of metalloproteinases and integrin, and affecting angio-
1
3–17
genesis.
In addition to potent cytotoxicity in cancer cells, nat-
urally isolated caged xanthones from G. hanburyi also showed
potent anti-HIV activities in reverse transcriptase assay with IC50
MSSA and MRSA growth inhibitors with MIC of 12.5 lM (Table 1).
3
less than 50
thones were previously reported.
l
g/mL. Antimicrobial activity of several caged xan-
Gambogic acid (4) inhibited growth with comparable extent.
The results indicated that prenyl moiety at C-2 position was not
significant for growth inhibition. The potency of isomorellinol
(2), 2-isoprenylforbesione (5) and forbesione (8) were dramatically
5
,10,18–21
Caged xanthones were
highlighted their potent growth inhibition in experimental staphy-
lococcal infection in mice.21 However, treatment of caged xan-
thones at high concentration resulted in side effects including
the decline in cell growth and reduced blood cell count, suggesting
decrease (MIC > 200 lM) while the other caged adducts (1, 6–7, 9)
tested could not inhibit growth of neither MRSA USA300 strain
SF8300 nor MSSA control strain. Deoxymorellin (1) and morellic
acid (3) share similarity in their structures with only minor
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2016.05.030
960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
0
Please cite this article in press as: Chaiyakunvat, P.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.030

2
P. Chaiyakunvat et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
R2
R
1
OHC
3
0
O
O
O
O
O
O
O
2
O
O
O
OH
R5
O
O
O
O
R3
R4
OH
OH
5
OH
1
2
2
2
R3 = -H
3
4
4
4
4
1
2
3
4
: R = -CH
1
3
,
R
R
= -CH
3
,
5: R = -H,
R
R
= Prenyl
= Geranyl
= -H
9
: R = -CH ,
3
= -CH OH,
R
= -H
6: R = -H,
5
2
1
3
3
5
5
: R = -COOH,
R
= -CH
= -CH
3
,
,
R
R
= -H
7: R = Prenyl,
R
1
2
: R = -COOH,
R
3
= Prenyl
8: R = -H,
R
= H
Figure 1. Natural isolated caged xanthones (1–9).
Table 1
Antibacterial activities of caged-xanthones and their synthetic derivatives determined by disk diffusiona and microdilution susceptibility testb
Compounds
Diameter of clear zones (mm)
MIC (
ATCC29213
lM)
MBC (
lM)
IC50 (lM)
ATCC25923
SF8300
SF8300
ATCC 29213
SF8300
Ampicillin (10
l
g)
16.97 ± 1.19c
28.86 ± 3.68c
6
7.75 ± 0.13
19.23 ± 0.35
16.59 ± 0.51
7.56 ± 0.12
6
6
6
6
1.56
0.62
—
>100
>19.9
—
1.56
1.25
—
>100
>19.9
—
—
—
Oxacillin (1
lg)
Deoxymorellin (1)
Isomorellinol (2)
Morellic acid (3)
Gambogic acid (4)
1.42
2.69
3.50
3.40
—
8.57 ± 0.35
19.52 ± 0.49
17.29 ± 0.77
7.20 ± 0.53
6
—
—
—
—
12.5
12.5
400
—
12.5
25
>400
—
12.5
25
>400
—
25
50
>400
—
2
-Isoprenylforbesione (5)
Deoxygamboginin (6)
Hanburin (7)
—
—
6
6
—
—
—
—
Forbesione (8)
Dihydroisomorellin (9)
Methyl morellate (3a)
7.86 ± 0.12
6
6
7.97 ± 0.11
6
6
200
—
—
>400
—
—
200
—
—
>400
—
—
—
—
—
3
3
3
3
3
3
3
3
3
3
3
b
c
d
e
f
g
h
i
19.99 ± 0.43
17.07 ± 2.03
6.53 ± 0.92
6
7.09 ± 1.13
16.52 ± 2.49
6.09 ± 0.18
15.91 ± 0.79
13.08 ± 0.37
6.89 ± 1.19
7.50 ± 0.45
22.24 ± 2.06
16.27 ± 2.33
6.27 ± 0.46
6
9.53 ± 1.13
18.34 ± 2.43
6.28 ± 0.56
19.35 ± 1.60
15.91 ± 1.60
8.21 ± 1.60
9.09 ± 0.45
12.5
12.5
—
—
—
25
—
25
50
—
25
25
—
—
—
25
—
25
100
—
12.5
12.5
—
—
—
25
—
25
100
—
25
25
—
—
—
50
—
50
>100
—
29.8
12.2
—
—
—
15.5
—
24.5
21.1
—
j
k
l
—
—
—
—
—
a
30 lg of test compounds in DMSO were used for disk diffusion testing. At least three independent experiments were performed and the average diameters of complete
inhibition zones were reported with standard errors. Whatman disk has a diameter of 6 mm, therefore, average inhibition zone of 6 mm indicates inability to inhibit growth.
b
Each compound was 2-fold diluted based on its stock concentration.
The number indicates the diameter of visually complete inhibition zone. However, the compound also exhibited incomplete zone of inhibition.
c
exception at C-30. Interestingly, the anti-bacterial activities of
these compounds were drastically different, highlighting the
importance of carboxyl functionality at C-30 position. To verify
the significance of carboxylic acid moiety, morellic acid 3 was
methylated to the methyl ester (3a). The modified ester 3a resulted
in no inhibition activity. Hence, the carboxylic functionality is cru-
cial for anti-bacterial activity. Even though the natural morellic
acid (3) and gambogic acid (4) possessed potent antibacterial activ-
ities, their high cytotoxicity hinder their potentials as bacterial
infection therapeutics. The reported IC50 of morellic acid (3) and
morellic acid 3 and gambogic acid 4 exhibited high cytotoxicity;
however, deoxymorellin 1, isomorellinol 2 were slightly more toxic
than morellic acid 3 and gambogic acid 4. These results along the
susceptibility experiments suggested that replacing polar COOH
substituent with less polar groups could drastically alter the
antibacterial activity and could perhaps increase the cytotoxicity.
Interestingly, N-(carboxymethyl) gambogiamide, GA3, was previ-
ously reported as potential candidate in cancer therapy.24 GA3 is
a modification of –COOH functional group with natural amino acid
glycine; this alteration in structure showed slightly reduce in cyto-
toxicity of gambogic acid yet enhanced solubility in aqueous solu-
tion. The declining trend in cytotoxicity with glycine conjugation at
C-30 led us to further explore other amino acids conjugate at this
particular position. We envisioned that amino acid conjugation
analogues 3b–m, which still possess the carboxylic functionality,
could perhaps retain potent antibacterial activity against MSSA
and MRSA strains with less toxicity (Fig. 2).
gambogic acid (4) are 3.5 lM and 3.4 lM, respectively. These IC50
values are above MIC and MBC values, suggesting that these com-
pounds potentially would be toxic to treated cells (see Supporting
information). We, therefore, modified morellic acid (3) in an effort
to acquire the derivative of caged xanthones that are potent
antibacterial activities with low toxicity. Based on our SAR analy-
sis, the key structure for retaining anti-bacterial activity is car-
boxylic functionality at C-30. To determine whether carboxylic
functionality at C-30 is required for toxicity of the caged xan-
thones, deoxymorellin 1 and isomorellinol 2 were evaluated in
comparison to morellic acid 3 and gambogic acid 4 as natural ana-
logues 1 and 2 lack of –COOH substituent while 3 and 4 do contain
The synthetically modified morellic (3b–3m) were achieved via
solid-phase synthesis and subjected to susceptibility testing with
MRSA: USA300 and MSSA strains. The results showed that
compound 3b, 3c, 3g, 3i, and 3j could retain growth inhibitory
activity in both strains (Table 1). Interestingly, all of the morellic
derivatives that inhibit bacterial growth were those that modified
–
COOH at C-30. All compounds—deoxymorellin 1, isomorellinol 2,
Please cite this article in press as: Chaiyakunvat, P.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.030

P. Chaiyakunvat et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Figure 2. Syntheses of morellic acid derivatives (3b–3m).
with amino acid that contain hydrophobic side chains. The inhibi-
tory activity of compound 3e, which contains additional carboxylic
acid functionality from glutamic acid side chain, was surprisingly
diminished. The MIC of 3b and 3c are equivalent to that of natural
morellic acid 3, indicating that these two derivatives could inhibit
growth of MSSA and MRSA strains in comparable extent. The cyto-
toxicity of derivatives that exhibited growth inhibitory activity (3b,
1
00
8
6
4
0
0
0
3
c, 3g, 3i, and 3j) was determined. When treated with human
epithelial A549 cell line, the toxicity of these compounds was 10-
fold lower than natural morellic acid. For instance, at MIC concen-
tration, the derivative 3b could inhibit growth of bacteria and did
not alter morphology of A549 cells. Meanwhile, the natural pro-
duct morellic acid 3 evidently damaged A549 cells at its MIC con-
centration (see Supporting information). We also performed the
cytotoxicity experiments with non-transformed cell line HEK293.
Compound 3b also exhibited approximately 10-fold decrease in
toxicity when compared with natural analogues, including morel-
lic acid 3 (see Supporting information). This raises the potential for
developing morellic acid derivatives as therapeutics for MRSA
infection.
20
0
Control
3 (1.5 µM) 0.5MIC of 3b MIC of 3b 2MIC of 3b
6.25 µM) (12.5 µM) (25 µM)
(
Figure 3. Ability of caged xanthones (3b) to inhibit bacterial invasion.
indicated that, when bacterial were exposed to caged xanthone
analogue 3b at MIC or even higher concentration for 2 h, com-
pound 3b did not perturb bacterial growth (see Supporting infor-
mation). The mechanism underlying the inhibitory activity of
caged xanthone 3b in the invasion pathway is under investigation
(Fig. 3).
In conclusion, in this study we determined the susceptibility of
global epidemic MRSA USA300 strain SF8300 to series of caged
xanthones isolated from resins and fruit of G. hanburyi hook. f.
Morellic acid (3) and gambogic acid (4) showed high potency in
The ability of bacterial cells to adhere and to invade into host
cells is important for infection. Even though S. aureus is classically
considered as extracellular bacteria, many evidence suggested that
they could also persist inside host cells.25 Interestingly, once they
are uptaken inside host cells, not many current antibiotics could
2
6
kill the bacteria. Therefore, inhibition at the adhesion and inva-
sion steps is essential. Several natural products, for instance Cur-
cuma longa L., could effectively prevent bacterial invasion to host
cells.2 Therefore, we further explored whether morellic derivative
growth inhibitory activity with MIC of 12.5 lM. However, due to
7
the high toxicity of these compounds, host cells could potentially
be damaged when treated at MIC concentration, reducing their
potentials as anti-bacterial agents for MRSA infection. We further
modified the caged xanthone synthetically. Morellic acid derivative
3b was discovered to retain high potency in global epidemic MRSA:
USA300 growth inhibitory activity. Synthetic modification in 3b
led to significant reduction in toxicity that host cells could no
longer be impaired when treated with 3b at its MIC. We discovered
that caged xanthone 3b could disrupt intracellular invasion of S.
aureus. How caged xanthone 3b could perturb intracellular inva-
sion of bacteria should be further studied.
3
b that exhibited potent anti-bacterial activity (MIC = 12.5 lM)
against MSSA and MRSA could affect the ability of bacterial to
invade host cells. S. aureus could invade into host cells within
2
8
1
5–90 min. Human epithelial A549 cells were infected with S.
aureus in the presence of testing compound 3b at various concen-
trations for 2 h. We also attempted to compare the intracellular
invasion inhibitory activity between the synthetic derivative 3b
with the natural caged xanthone morellic acid 3. However, due
to the high toxicity of morellic acid 3, the concentration tested
was necessitated to reduce to the concentration that does not dam-
age A549 cells within 2 h of incubation. At 1.5 lM, morellic 3 could
not inhibit invasion of S. aureus into host cells. Synthetic modified
morellic acid derivative 3b was found to inhibit invasion ability of
S. aureus into host cells in concentration-dependent manner with-
out perturbing morphology of host cells (see Supporting informa-
tion). To determine whether the invasion results were not due to
the effect of the compound on bacterial growth, the non-invading
bacteria as well as invading bacteria were cultured. The results
Acknowledgments
This work was supported in part by the Thailand Research Fund
and the Office of the Higher Education Commission (TRG5780211
to C.J.), Mahidol University under the National Research Universi-
ties Initiative, the Center of Excellence for Innovation in Chemistry
(PERCH-CIC).
Please cite this article in press as: Chaiyakunvat, P.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.030

4
P. Chaiyakunvat et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
0. Ren, Y.; Lantvit, D. D.; De Blanco, E. J. C.; Kardono, L. B. S.; Riswan, S.; Chai, H.;
1
Supplementary data
Cottrell, C. E.; Farnsworth, N. R.; Swanson, S. M.; Ding, Y.; Li, X. C.; Marais, J. P.
J.; Ferreira, D.; Kinghorn, A. D. Tetrahedron 2010, 66, 5311.
11. Wang, X.; Lu, N.; Yang, Q.; Dai, Q.; Tao, L.; Guo, X.; Guo, Q.; You, Q. Bioorg. Med.
Chem. Lett. 2010, 20, 2438.
Supplementary data (synthetic methods and experimental
details, including morphological changes of A549 and HEK293 in
the presence of caged xanthones) associated with this article can
be found, in the online version, at http://dx.doi.org/10.1016/j.
bmcl.2016.05.030.
1
1
1
1
1
1
1
1
2. Chantarasriwong, O.; Batova, A.; Chavasiri, W.; Theodorakis, E. A. Chem. Eur. J.
2010, 16, 9944.
3. Zhang, H.-Z.; Kasibhatla, S.; Wang, Y.; Herich, J.; Guastella, J.; Tseng, B.; Drewe,
J.; Cai, S. X. Bioorg. Med. Chem. 2004, 12, 309.
4. Qin, Y.; Meng, L.; Hu, C.; Duan, W.; Zuo, Z.; Lin, L.; Zhang, X.; Ding, J. Mol. Cancer
Ther. 2007, 6, 2429.
5. Qi, Q.; Gu, H.; Yang, Y.; Lu, N.; Zhao, J.; Liu, W.; Ling, H.; You, Q. D.; Wang, X.;
Guo, Q. J. Mol. Med. 2008, 86, 1367.
6. Zhao, J.; Qi, Q.; Yang, Y.; Gu, H.-Y.; Lu, N.; Liu, W.; Wang, W.; Qiang, L.; Zhang,
L.-B.; You, Q.-D.; Guo, Q.-L. Eur. J. Pharmacol. 2008, 589, 127.
7. Yi, T.; Yi, Z.; Cho, S. G.; Luo, J.; Pandey, M. K.; Aggarwal, B. B.; Liu, M. Cancer Res.
References and notes
1.
2.
3.
Anantachoke, N.; Tuchinda, P.; Kuhakarn, C.; Pohmakotr, M.; Reutrakul, V.
Pharm. Biol. 2012, 50, 78.
Panthong, A.; Norkaew, P.; Kanjanapothi, D.; Taesotikul, T.; Anantachoke, N.;
Reutrakul, V. J. Ethnopharmacol. 2007, 111, 335.
Reutrakul, V.; Anantachoke, N.; Pohmakotr, M.; Jaipetch, T.; Sophasan, S.;
Yoosook, C.; Kasisit, J.; Napaswat, C.; Santisuk, T.; Tuchinda, P. Planta Med.
2008, 68, 1843.
8. Rukachaisirikul, V.; Kaewnok, W.; Koysomboon, S.; Phongpaichit, S.; Taylor, W.
C. Tetrahedron 2000, 56, 8539.
9. Sukpondma, Y.; Rukachaisirikul, V.; Phongpaichit, S. Chem. Pharm. Bull. 2005,
2
007, 73, 33.
5
3, 850.
4
5
.
.
Asano, J.; Chiba, K.; Tada, M.; Yoshii, T. Phytochemistry 1996, 41, 815.
Rukachaisirikul, V.; Phainuphong, P.; Sukpondma, Y.; Phongpaichit, S.; Taylor,
W. C. Planta Med. 2005, 71, 165.
2
2
2
2
2
0. Nageswara Rao, K. V.; Narasimha Rao, P. L. Experientia 1961, 17, 213.
1. Rao, K. V.; Rao, P. L. Indian J. Exp. Biol. 1967, 5, 101.
2. Tenover, F. C.; Goering, R. V. J. Antimicrob. Chemother. 2009, 64, 441.
3. Otto, M. Cell. Microbiol. 2012, 14, 1513.
4. Xie, H.; Qin, Y. X.; Zhou, Y. L.; Tong, L. J.; Lin, L. P.; Geng, M. Y.; Duan, W. H.;
Ding, J. Acta Pharmacol. Sin. 2009, 30, 346.
5. Lowy, F. D. Trends Microbiol. 2000, 8, 341.
6. Krut, O.; Sommer, H.; Krönke, M. J. Antimicrob. Chemother. 2004, 53, 167.
7. Kim, K. J.; Yu, H. H.; Cha, J. D.; Seo, S. J.; Choi, N. Y.; You, Y. O. Phytother. Res.
6.
7.
8.
9.
Tao, S. J.; Guan, S. H.; Wang, W.; Lu, Z. Q.; Chen, G. T.; Sha, N.; Yue, Q. X.; Liu, X.;
Guo, D. A. J. Nat. Prod. 2009, 72, 117.
Han, Q.; Yang, L.; Liu, Y.; Wang, Y.; Qiao, C.; Song, J.; Xu, L.; Yang, D.; Chen, S.;
Xu, H. Planta Med. 2006, 72, 281.
Han, Q. B.; Wang, Y. L.; Yang, L.; Tso, T. F.; Qiao, C. F.; Song, J. Z.; Xu, L. J.; Chen, S.
L.; Yang, D. J.; Xu, H. X. Chem. Pharm. Bull. 2006, 54, 265.
Wang, J.; Zhao, L.; Hu, Y.; Guo, Q.; Zhang, L.; Wang, X.; Li, N.; You, Q. Eur. J. Med.
Chem. 2009, 44, 2611.
2
2
2
2005, 19, 599.
2
8. Edwards, A. M.; Massey, R. C. J. Vis. Exp. 2011, 49, 2693.
Please cite this article in press as: Chaiyakunvat, P.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.030