Synthesis of riccardin D derivatives as potent antimicrobial agents

Article abstract of DOI:10.1016/j.bmcl.2016.06.006

We describe the synthesis and biological evaluation of riccardin D derivatives, a novel class of antimicrobial molecules. Structural diversification of these derivatives was achieved by introducing hydroxy, methoxy, and bromine into the aromatic rings of riccardin D. The antimicrobial evaluation of these compounds was performed as in vitro assays against clinically isolated bacteria and fungi. The introduction of bromine atom into the arene B of riccardin D led to several strongly active antibacterial compounds with a MIC value ranging from 0.5 to 4 μg/mL for Staphylococcus aureus, both methicillin-sensitive and -resistant strains. Antifungal tests found compound 34 was the most potent molecule with a MIC value of 2 μg/mL against Candida albicans. This initial biological evaluation suggests that these novel molecules merit further investigation as potential antimicrobial agents.

Full text of DOI:10.1016/j.bmcl.2016.06.006

Bioorganic & Medicinal Chemistry Letters 26 (2016) 3617–3620
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of riccardin D derivatives as potent antimicrobial agents
Bin Sun a,b, Ming Zhang b, Ying Li b, Qing-wen Hu b, Hong-bo Zheng b, Wen-qiang Chang b,
Hong-xiang Lou a,b,
⇑
a National Glycoengineering Research Center, Shandong University, Jinan 250012, PR China
b School of Pharmaceutical Sciences, Shandong University, Jinan 250012, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe the synthesis and biological evaluation of riccardin D derivatives, a novel class of antimicro-
bial molecules. Structural diversification of these derivatives was achieved by introducing hydroxy,
methoxy, and bromine into the aromatic rings of riccardin D. The antimicrobial evaluation of these com-
pounds was performed as in vitro assays against clinically isolated bacteria and fungi. The introduction of
bromine atom into the arene B of riccardin D led to several strongly active antibacterial compounds with
Received 3 February 2016
Revised 23 May 2016
Accepted 3 June 2016
Available online 4 June 2016
a MIC value ranging from 0.5 to 4
tant strains. Antifungal tests found compound 34 was the most potent molecule with a MIC value of 2
mL against Candida albicans. This initial biological evaluation suggests that these novel molecules merit
further investigation as potential antimicrobial agents.
l
g/mL for Staphylococcus aureus, both methicillin-sensitive and -resis-
Keywords:
l
g/
Bisbibenzyl
Derivatives
Antimicrobial
Anti-MRSA
Anti-fungal
Ó 2016 Elsevier Ltd. All rights reserved.
Macrocyclic bisbibenzyls are a series of phenolic natural prod-
ucts that are almost exclusively found in liverworts.1–4 These nat-
ural products exhibit versatile and often potent biological
activities, including cyclooxygenase, calmodulin, and 5-lipoxyge-
nase inhibitory effects and antifungal, antibacterial, antioxidative,
antivirus, anti-mitotic, cytotoxic, muscle-relaxing, NOS-inhibiting,
and LXR-modulating activities.1,3,5–13 Bisbibenzyls, therefore, are
of great interest to natural product researchers for their potential
applications as pharmacological agents.5,14–18
on arene A and arene C to determine the influence of these groups
on the biological properties of riccardin D. We also methylated the
hydroxyl group on the arene B and arene D to evaluate the effect of
methoxyl group on the antimicrobial activity. In addition, Huigens
et al. reported brominated phenazines exhibited potent anti-MRSA
activity by eradicating biofilms and killing MRSA persister cells.29
In consideration of these results, the bromination of riccardin D
and the evaluation of these derivatives as antimicrobial agents
were also studied.
Riccardin D, a macrocyclic bisbibenzyl isolated from Marchantia
polymorpha L., has been the focus of considerable biological inves-
tigation and pharmacological testing in our group19–25 and has
been shown to possess potent antifungal activity, with a MIC value
The synthesis of 13-hydroxyl riccardin D (25) was achieved in
10 steps, as shown in Scheme 1. The synthetic route began with
the Ullmann coupling of the protected 3-hydroxy-4,5-
dimethoxyl-benzaldehyde (2) with methyl 4-bromobenzoate (3),
resulting in the formation of the diphenyl ether 5. Compound 5
was then reduced with lithium aluminum hydride, followed by
treatment with triphenylphosphonium bromide to afford AC frag-
ment 8 in three steps. The BD fragment 15 was prepared from com-
pounds 13 and 14 by a standard Suzuki reaction, following the
approach reported previously.11 The building blocks 8 and 15 were
coupled by a Wittig reaction in the presence of potassium carbon-
ate and 18-crown-6. The resulting stilbene, a mixture of geometri-
cal isomers, was then hydrogenated over Pd/C, followed by acidic
hydrolysis to afford compound 16. The distal hydroxyl group was
directly treated with triphenylphosphine HBr to afford compound
19 for cyclization. The intramolecular cyclization was then
achieved via Wittig reaction in the presence of sodium methoxide,
followed by hydrogenation over Pd/C to yield compound 22.
Derivative 25 was obtained after the methyl ether cleavage of com-
of 16 l
g/mL against Candida albicans.26–28 Moreover, it was
recently reported that riccardin D also exhibits in vitro antibacte-
rial activity against methicillin-resistant Staphylococcus aureus
(MRSA), with MIC values in the 4–8 l
g/mL range.7–9
These encouraging antifungal and antibacterial properties of
riccardin D motivated us to synthesize additional derivatives to
discover more potent molecules as potential new antimicrobial
agents. Recently, Miyachi et al. reported the number and position
of hydroxyl groups on the benzene ring could impact the anti-
MRSA activity of riccardin C.7,8 These results inspired our group
to prepare riccardin D derivatives bearing more hydroxyl groups
⇑
Corresponding author. Tel.: +86 531 88382501; fax: +86 531 88382019.
E-mail address: louhongxiang@sdu.edu.cn (H.-x. Lou).
http://dx.doi.org/10.1016/j.bmcl.2016.06.006
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

3618
B. Sun et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3617–3620
Scheme 1. Synthesis of riccardin D derivatives. Reagents and conditions: (a) CuO, K2CO3, Py, reflux, 71–83%; (b) (i) LiAlH4, THF, ꢀ40 °C to rt; (ii) PPh3HBr, MeCN, reflux, 98%;
(c) (i) LiAlH4, THF, ꢀ40 °C to rt; (ii) 2,3-dihydropyran, p-toluenesulfonic acid, DCM, 77%; (d) n-BuLi, B(OMe)3, KH2PO4, THF, ꢀ30 °C to rt, 78%; (e) Pd(PPh3)4, toluene, EtOH,
Na2CO3, reflux, 81%; (f) K2CO3, 18-crown-6, DCM, reflux, 91%; (g) (i) Pd/C (10%), H2, Et3N, EtOAc, rt; (ii) HCl/THF (1:1), rt, 93%; (h) PPh3HBr, MeCN, reflux, 98%; (i) (i) NaOMe,
DCM, rt; (ii) Pd/C (10%), H2, EtOAc, rt, 81–89%; (j) BBr3, DCM, ꢀ40 °C to rt, 80–86%.
pound 22 by boron tribromide. The derivatives 26 and 27 were
synthesized by the method similar to that described for compound
25 (Scheme 1).
isolates from The Second Hospital of Shandong University China.
The minimum inhibitory concentration (MIC, g/mL) was deter-
l
mined by the liquid microdilution method. Cefotaxime, proc-
taphlin, ceftazidime and cloxacillin were used as positive
antimicrobial agents. The antifungal activity of the synthesized
compounds was evaluated against Candida albicans SC5314, and
fluconazole served as the positive antifungal agent. The suscepti-
bility test was performed according to the guidelines of CLSI.
Antibacterial activities of riccardin D and its derivatives are
summarized in Table 1. The lead riccardin D exhibited moderate
antibacterial activity and inhibited the growth of S. aureus
The preparation method for the methylated derivatives is
shown in Scheme 2. Treatment of riccardin D and iodomethane
in the presence of potassium carbonate in acetone afforded com-
pounds 28 and 29. The methylated position of riccardin D was
determined by NOE spectrum as shown in Figure 1. Correlations
of H3-15 with H-40 and H-2 were determined in compound 28
and compound 29, respectively (Figs. S5–S6 and S9–S10).
We have previously synthesized two brominated derivatives of
riccardin D with N-bromosuccinimide (NBS) and evaluated their
anticancer activity.11 In this Letter, a more convenient and efficient
method was applied for the bromination. Riccardin D was treated
with HBr in the presence of DMSO in ethyl acetate (Scheme 2),30
and derivatives 30–38 were provided by HPLC separation.
The antibacterial activity of the riccardin D derivatives were
assessed in vitro against Gram-positive Staphylococcus aureus
strains SDS15016 and MRSA SDSM1503, both strains were clinical
SDS15016 and MRSA SDSM1503 at 4 lg/mL and 8 lg/mL, respec-
tively. Most of the derivatives demonstrated a two- to eight-fold
improvement in MIC values. Compound 32, with a bromine atom
at C-40 of arene B, displayed the most potent antibacterial activities
against S. aureus and MRSA with MIC values of 0.5 lg/mL and 1 lg/
mL, respectively, and was eight-fold better than the parent ric-
cardin D. Its activity against S. aureus was comparable to that of
cloxacillin, and the anti-MRSA activity of compound 32 was much

B. Sun et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3617–3620
3619
Scheme 2. Synthesis of riccardin D derivatives. Reagents and conditions: (a) MeI, K2CO3, acetone; (b) HBr, DMSO, EtOAc.
Figure 1. Key NOESY correlations (dashed blue arrows) for compounds 28 and 29.
Table 1
In vitro antimicrobial activity of riccardin D derivativesa
Structures
S. aureus
MASA
C. albicans
Structures
S. aureus
MASA
C. albicans
25
26
27
28
29
30
31
32
2
2
8
2
2
2
4
0.5
4
8
4
16
4
8
8
16
1
>128
>128
>64
>64
>64
>64
>64
>64
>64
>64
N/Tb
N/Tb
33
34
35
36
37
38
2
1
2
2
2
8
4
4
4
2
4
4
4
16
8
>128
>128
N/Tb
8
2
>64
>64
8
>64
16
Riccardin D
Ceftazidime
Cloxacillin
Fluconazole
N/Tb
N/Tb
2
Cefotaxime
Proctaphlin
0.5
8
N/Tb
a
Mean values based on three independent experiments; all data are minimum inhibitory concentrations (MIC), given as
N/T means not tested.
lg/mL.
b
better than the reference drugs cefotaxime, proctaphlin, cef-
tazidime and cloxacillin. In addition, introduction of bromine at
C-60 of arene B led to another excellent antibacterial compound
34, which showed four-fold enhancement of the MIC. Compounds
30 and 35, both with a bromine atom on arene D, also exhibited
slightly improved antibacterial activity compared with riccardin
D. Unexpectedly, di-bromination of riccardin D did not signifi-
cantly enhance the antibacterial activity and the MIC values of all
di-brominated derivatives 31, 33, 36, 37, and 38 were higher than
the values of mono-brominated derivatives 32 and 34. This finding
indicates that bromination improves antibacterial activity, and the
mono-brominated derivatives, especially with the bromine atom
on arene B, exhibit optimum potency against both S. aureus and
MRSA. Furthermore, the MIC for 25, 26 (containing four hydroxyl

3620
B. Sun et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3617–3620
groups) and 28, 29 (containing one methoxyl group and two
hydroxyl groups) matched or exceeded the MIC attained for ric-
cardin D. The presence of five hydroxyl groups reduced the
antibacterial activity of riccardin derivative 27, which is most
likely due to poor lipophilicity.
Riccardin D and its derivatives were demonstrated to inhibit the
growth of Candida albicans, and their MICs are shown in Table 1.
Compound 34 exhibited excellent antifungal activity with a MIC
References and notes
1. Keseru, G. M.; Nogradi, M. Bioorg. Med. Chem. 1995, 3, 1511.
2. Asakawa, Y. Phytochemistry 2001, 56, 297.
3. Harrowven, D. C.; Kostiuk, S. L. Nat. Prod. Rep. 2012, 29, 223.
4. Asakawa, Y.; Ludwiczuk, A.; Nagashima, F. Phytochemistry 2013, 91, 52.
5. Speicher, A.; Holz, J.; Hoffmann, A. Nat. Prod. Commun. 2011, 6, 393.
6. Huang, W. J.; Wu, C. L.; Lin, C. W.; Chi, L. L.; Chen, P. Y.; Chiu, C. J.; Huang, C. Y.;
Chen, C. N. Cancer Lett. 2010, 291, 108.
7. Sawada, H.; Onoda, K.; Morita, D.; Ishitsubo, E.; Matsuno, K.; Tokiwa, H.;
Kuroda, T.; Miyachi, H. Bioorg. Med. Chem. 2013, 23, 6563.
8. Sawada, H.; Okazaki, M.; Morita, D.; Kuroda, T.; Matsuno, K.; Hashimoto, Y.;
Miyachi, H. Bioorg. Med. Chem. Lett. 2012, 22, 7444.
9. Onoda, K.; Sawada, H.; Morita, D.; Fujii, K.; Tokiwa, H.; Kuroda, T.; Miyachi, H.
Bioorg. Med. Chem. 2015, 23, 3309.
10. Sun, L. M.; Lv, B. B.; Cheng, A. X.; Wu, X. Z.; Lou, H. X. Biol. Pharm. Bull. 2009, 32,
36.
11. Jiang, J.; Sun, B.; Wang, Y. Y.; Cui, M.; Zhang, L.; Cui, C. Z.; Wang, Y. F.; Liu, X. G.;
Lou, H. X. Bioorg. Med. Chem. 2012, 20, 2382.
12. Noguchi-Yachide, T.; Miyachi, H.; Aoyama, H.; Aoyama, A.; Makishima, M.;
Hashimoto, Y. Chem. Pharm. Bull. 2007, 55, 1750.
13. Noguchi-Yachide, T.; Aoyama, A.; Makishima, M.; Miyachi, H.; Hashimoto, Y.
Bioorg. Med. Chem. Lett. 2007, 17, 3957.
14. Aoyama, A.; Endo-Umeda, K.; Kishida, K.; Ohgane, K.; Noguchi-Yachide, T.;
Aoyama, H.; Ishikawa, M.; Miyachi, H.; Makishima, M.; Hashimoto, Y. J. Med.
Chem. 2012, 55, 7360.
value of 2 lg/mL, eight-fold better than riccardin D. Its activity
against C. albicans is comparable to that of the positive control drug
fluconazole. Compounds 33 and 37 both display moderate antifun-
gal activity with MIC values of 8
lg/mL, two-fold better than ric-
cardin D. The MIC values of other derivatives exceeded the MIC
for riccardin D and were more than 64 lg/mL. This suggests the
addition of bromine at the 60- position of arene B is critical for
increased the antifungal activity.
In conclusion, we have prepared a series of riccardin D deriva-
tives and evaluated their in vitro antimicrobial activity against S.
aureus SDS15016, MRSA SDSM1503, and C. albicans SC5314. Com-
pound 32 exhibited excellent antibacterial activity against both
methicillin-sensitive and -resistant S. aureus, with MIC values of
15. Speicher, A.; Backes, T.; Hesidens, K.; Kolz, J. Beilstein J. Org. Chem. 2009, 5, 71.
16. Hioki, H.; Shima, N.; Kawaguchi, K.; Harada, K.; Kubo, M.; Esumi, T.; Nishimaki-
Mogami, T.; Sawada, J.; Hashimoto, T.; Asakawa, Y.; Fukuyama, Y. Bioorg. Med.
Chem. Lett. 2009, 19, 738.
0.5
l
g/mL and 1
l
g/mL, respectively. Compound 34 also displayed
g/mL. Further
potent antifungal activity with a MIC value of 2
l
explanation of the molecular target of these novel riccardin D
derivatives will be presented in a future publication. The character-
ization of these novel compounds presents an intriguing step in the
development of a novel class of therapeutic agents effective for
treating bacterial and fungal infections.
17. Makino, K.; Harada, K.; Kubo, M.; Hioki, H.; Fukuyama, Y. Nat. Prod. Commun.
2013, 8, 915.
18. Morita, D.; Sawada, H.; Ogawa, W.; Miyachi, H.; Kuroda, T. Biochim. Biophys.
Acta 2015, 1848, 2057.
19. Yue, B.; Zhang, Y. S.; Xu, H. M.; Zhao, C. R.; Li, Y. Y.; Qin, Y. Z.; Wang, R. Q.; Sun,
D.; Yuan, Y.; Lou, H. X.; Qu, X. J. Cancer Lett. 2013, 328, 104.
20. Niu, C.; Qu, J. B.; Lou, H. X. Chem. Biodivers. 2006, 3, 34.
21. Sun, C. C.; Xu, H. M.; Yuan, Y.; Gao, Z. H.; Lou, H. X.; Qu, X. J. Basic Clin.
Pharmacol. Toxicol. 2014, 115, 488.
Acknowledgments
22. Sun, C. C.; Zhang, Y. S.; Xue, X.; Cheng, Y. N.; Liu, H. P.; Zhao, C. R.; Lou, H. X.; Qu,
X. J. Eur. J. Pharmacol. 2011, 667, 136.
23. Wang, Y.; Ji, Y.; Hu, Z.; Jiang, H.; Zhu, F.; Yuan, H.; Lou, H. Toxical. In vitro 2013,
27, 1928.
24. Xue, X.; Qu, X. J.; Gao, Z. H.; Sun, C. C.; Liu, H. P.; Zhao, C. R.; Cheng, Y. N.; Lou, H.
X. Invest. New Drug 2012, 30, 212.
25. Qu, J.; Lou, H. J. Pharm. Res. 2014, 8, 435.
This work was supported by grant from the National Natural
Science Foundation (NNSF) of China (No. 81273383 and No.
81102319) and Shandong Provincial Natural Science Foundation,
China (No. ZR2011HQ024).
26. Wu, X. Z.; Cheng, A. X.; Sun, L. M.; Sun, S. J.; Lou, H. X. Biochim. Biophys. Acta
2009, 1790, 770.
Supplementary data
27. Wu, X. Z.; Cheng, A. X.; Sun, L. M.; Lou, H. X. Acta Pharmacol. Sin. 2008, 29, 1478.
28. Cheng, A.; Sun, L.; Wu, X.; Lou, H. Biol. Pharm. Bull. 2009, 32, 1417.
29. Garrison, A. T.; Abouelhassan, Y.; Kallifidas, D.; Bai, F.; Ukhanova, M.; Mai, V.;
Jin, S.; Luesch, H.; Huigens, R. W. Angew. Chem., Int. Ed. 2015, 54, 14819.
30. Song, S.; Sun, X.; Li, X.; Yuan, Y.; Jiao, N. Org. Lett. 2015, 17, 2886.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.06.
006.