Scaffold hopping of sampangine: Discovery of potent antifungal lead compound against Aspergillus fumigatus and Cryptococcus neoformans

Article abstract of DOI:10.1016/j.bmcl.2014.07.064

Discovery of novel antifungal agents against Aspergillus fumigatus and Cryptococcus neoformans remains a significant challenge in current antifungal therapy. Herein the antifungal natural product sampangine was used as the lead compound for novel antifungal drug discovery. A series of D-ring scaffold hopping derivatives were designed and synthesized to improve antifungal activity and water solubility. Among them, the thiophene derivative S2 showed broad-spectrum antifungal activity, particularly for Aspergillus fumigatus and Cryptococcus neoformans. Moreover, compound S2 also revealed better water solubility than sampangine, which represents a promising antifungal lead compound for further structural optimization.

Full text of DOI:10.1016/j.bmcl.2014.07.064

Bioorganic & Medicinal Chemistry Letters 24 (2014) 4090–4094
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Scaffold hopping of sampangine: Discovery of potent antifungal lead
compound against Aspergillus fumigatus and Cryptococcus
neoformans
Zhigan Jiang a,b, , Na Liu a, , Guoqiang Dong a, Yan Jiang a, Yang Liu a, Xiaomeng He a, Yahui Huang a,
Shipeng He a, Wei Chen a, Zhengang Li a, Jianzhong Yao a, Zhenyuan Miao a, Wannian Zhang a,
,
⇑
Chunquan Sheng a,
⇑
a School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China
b WuXi AppTec (Shanghai) Co., Ltd, 288 FuTe Zhong Road, Shanghai 200131, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Discovery of novel antifungal agents against Aspergillus fumigatus and Cryptococcus neoformans remains a
significant challenge in current antifungal therapy. Herein the antifungal natural product sampangine
was used as the lead compound for novel antifungal drug discovery. A series of D-ring scaffold hopping
derivatives were designed and synthesized to improve antifungal activity and water solubility. Among
them, the thiophene derivative S2 showed broad-spectrum antifungal activity, particularly for Aspergillus
fumigatus and Cryptococcus neoformans. Moreover, compound S2 also revealed better water solubility
than sampangine, which represents a promising antifungal lead compound for further structural
optimization.
Received 8 June 2014
Revised 20 July 2014
Accepted 23 July 2014
Available online 1 August 2014
Keywords:
Sampangine
Scaffold hopping
Antifungal activity
Ó 2014 Elsevier Ltd. All rights reserved.
Recently, the rapid increase in immunocompromised patients
has led to severe invasive fungal infections (IFIs).1 IFIs are often
life-threatening and the associated mortality is very high. Candida
albicans (mortality: 20–40%), Cryptococcus neoformans (mortality:
20–70%), and Aspergillus fumigatus (mortality: 50–90%) represent
three major human fungal pathogens.2,3 Despite the high mortality
of IFIs, effective and safe antifungal agents are very limited. Clini-
cally available antifungals (Fig. 1) for IFIs can be classified into:
polyenes (e.g., amphotericin B), fluorinated pyrimidines (e.g., 5-flu-
orocytosine), azoles (e.g., fluconazole and voriconazole), and echi-
nocandins (e.g., caspofungin, micafungin and anidulafungin).4,5
However, clinical efficacy of them is far from satisfactory. These
antifungal agents generally suffered from limited efficacy, narrow
spectrum, low bioavailability, high toxicity and particularly severe
drug resistance.6 Therefore, it is highly desirable to discover and
develop antifungal agents with novel chemotype and fungal-
specific mode of action.
O
7
6
N
8
7a
6a
5
4
9
11b
11a
10
11c
3a
3
11
N
1
2
Sampangine
Figure 1. Chemical structure of sampangine.
important classes of antifungal agents, are derived from natural
products.7 Discovery of effective and safe antifungal agents from
natural products is becoming an active area of research interests.6,8
Sampangine (Fig. 1) is an azaoxoaporphine alkaloid extracted from
the stem bark of Cananga odorata.9 Sampangine showed potent
antifungal strong inhibitory activity against a variety of human
fungal pathogens including Candida albicans, Cryptococcus neofor-
mans, and Aspergillus fumigates.10 Although the molecular target
of sampangine was still unknown, inhibition of heme biosynthesis
and production of reactive oxygen species (ROS) was reported to be
its primary mode of action.11,12 Due to the broad-spectrum
antifungal activity of sampangine, it is of particular interest as an
Natural products provide a rich source for the discovery of novel
antifungal agents.7,8 For example, polyenes and echinocandins, two
⇑
Corresponding authors. Fax: +86 21 81870243 (W.Z.); tel./fax: +86 21 81871239
(C.S.).
E-mail addresses: zhangwnk@hotmail.com (W. Zhang), shengcq@hotmail.com
(C. Sheng).
These two authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2014.07.064
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

Z. Jiang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4090–4094
4091
antifungal lead compound. Peterson et al. reported several A- and
B-ring-substituted sampangine derivatives and found that the
introduction of a 3-methoxy, 3-methyl, or 4,5-benzo group was
favorable for the antifungal activity.13 Mink and Bracher designed
and synthesized hetero analogues of sampangine.14 These deriva-
tives showed similar antifungal activity to sampangine when
replacing its C7-carbonyl group with hetero atoms (O, S, N, SO).
The results indicated that the scaffold of sampangine could be
modified without loss of the antifungal activity. More recently,
Kimpe and co-workers reported novel sampangine derivatives
with excellent anti-tuberculosis activities, which expanded the
therapeutic application of sampangine.15
Although sampangine has shown promising future as an anti-
fungal lead compound, information of its structure–activity
relationship (SAR) is still limited. Moreover, sampangine showed
poor water solubility because of its aromatic tetracyclic scaffold.
Continuing our efforts on novel antifungal drug discovery,6,16–18
herein scaffold hopping studies of sampangine were performed.
A potent derivative S2 with improved antifungal activity and
aqueous solubility was successfully identified.
As shown in Figure 2, scaffold hopping of sampangine was
focused on the D-ring. Various heterocycles were used to replace
the D-ring phenyl group of sampangine to improve the water
solubility and provide new SAR information. As a result, a series
O
O
N
O
N
N
N
H3C
H3C
N
H3C
S
O
O
N
N
N
B
Scaffold hopping
D
C
S1
S2
S3
A
N
O
O
O
N
N
N
O
Sampangine
N
O
H3C
N
N
N
S5
S6
S4
Figure 2. Scaffold hopping design of sampangine.
Me
H3C
N
OH
Br
O
O
N
OH
O
Br
Br
H3C
O
O
H3C
H3C
4
a
H3C
H3C
b
H3C
H3C
c
1
2
3
O
O
O
N
N
N
O
O
H3C
H3C
d
H3C
H3C
CH3
5
S1
Scheme 1. Reagents and conditions: (a) Br2/CHCl3, 25 °C, 2 h, 67%; (b) CrO3, HOAc/H2O, 25 °C, 1 h, 51%; (c) xylene, 140 °C, 6 h, 48%; (d) (1) DMFDMA, DMF, 120 °C, 2 h, (2)
NH4Cl, HOAc, 120 °C, 1 h, 68% (2 steps).
O
O
O
OH
Br
Br
Br
Br
a
b
c
S
S
S
S
O
9
6
7
8
Me
H3C
O
N
O
N
N
N
N
H3C
4
e
S
S
d
O
CH3
10
S2
Scheme 2. Reagnents and conditions: (a) CuBr2/EtOAc/CHCl3, 80 °C, 12 h, 97%; (b) Li2CO3, DMF, 100 °C, 6 h, 96%; (c) PhI(OAc)2, HOAc/TFA, 0–25 °C, 0.5 h, 81%; (d) NaHCO3,
EtOH, 4 h, 73%; (e) (1) DMFDMA, DMF, 120 °C, 2 h, (2) NH4Cl, HOAc, 120 °C, 1 h, 58% (2 steps).

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Z. Jiang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4090–4094
O
of furan (S1, S4, S5), thiophene (S2) and pyrrole (S3, S6) derivatives
were designed and synthesized. Sampangine was synthesized
according to the procedure of Peterson et al.13 The synthetic route
of sampangine derivatives were depicted in Schemes 1–5. Starting
from 2,2-dimethyl-2,3-dihydrobenzofuran-7-ol (1), it was brom-
ized by Br2 to give dibromo intermediate 2, which was subse-
quently oxidized to quinone intermediate 3. BCD ring
intermediate 5 was obtained through the hetero Diels–Alder reac-
tion of quinone 3 with hydrazone 4, followed by an in situ elimina-
tion of dimethylammonium bromide from the cycloadduct. The
H3C
N
H3C
N
H3C
N
H
N
b
c
a
OBn
OBn
21
OH
22
O
23
20
O
N
H3C
O
H3C
N
N
N
N
d
e
4
condensation of
5 with dimethylformamide dimethyl acetal
O
24
S6
provided furan derivative S1 in good yield. Other sampangine
derivatives were obtained by a similar synthetic strategy using
various starting materials (Schemes 2–5). Based on compound
S2, bromination and chlorination using N-bromosuccinimide
(NBS) and N-chlorosuccinimide (NCS) afforded target compounds
S2a–d (Scheme 6). Subsequently, treating compound S2b with
1-methylpiperazine or CH3ONa yielded compounds S2e and S2f,
respectively. Catalyzed by HOAc, 2-substituted derivatives S2g–j
were obtained by condensation of intermediate 10 with various
aromatic aldehydes and NH4Cl (Scheme 6).
Scheme 5. Reagents and conditions: (a) MeI, NaH, DMF, 25 °C, 5 h, 55%; (b) 5% Pd-C,
MeOH, 40 psi, 25 °C, 4 h, 91%; (c) Fremy’s salt, KH2PO4 aq, acetone/H2O, 3 h, 52%; (d)
xylene, 140 °C, 6 h, 45%; (e) (1) DMFDMA, DMF, 120 °C, 2 h, (2) NH4Cl, HOAc, 120 °C,
1 h, 42% (2 steps).
and sampangine. Compound S2 was more active than fluconazole
and sampangine for Trichophyton rubrum (MIC = 2
lg/mL) and
Microsporum gypseum (MIC = 2 g/mL). SAR analysis revealed that
l
the replacement of D-ring phenyl group of sampangine by 2,2-
dimethyl-2,3-dihydrofuran and thiophene was favorable for the
antifungal activity. Particularly, the thiophene derivative S2
showed potent fungistatic activity with a broad spectrum (MIC
In vitro antifungal assay18 revealed that most of the target com-
pounds, except compound S3, showed broad-spectrum inhibitory
activity against the tested human fungal pathogens (Table 1). Clin-
ically, the treatment of Aspergillus fumigatus infection still remains
a significant challenge in current antifungal therapy. For example,
the first-line antifungal agent fluconazole is inactive against Asper-
range: 0.25–8 lg/mL). In contrast, other furan and pyrrole ana-
logues generally showed decreased antifungal activity. Moreover,
water solubility was also determined for several scaffold hopping
derivatives (Table 1). Most of the compounds showed better solu-
bility than sampangine. Particularly, compound S2, the most active
gillus fumigatus (MIC >64
showed good inhibitory activity (MIC = 1
l
g/mL). In contrast, compound S1 and S2
g/mL), which was also
l
more potent than lead compound sampangine. Cryptococcus neo-
formans infections frequently involve the brain and are often life-
threatening. Compound S2 revealed excellent inhibitory activity
one, also exhibited the best solubility (48
almost four fold higher than that of sampangine (12.6
l
g/mL), which was
l
g/mLÀ1).
Due to potent and broad-spectrum antifungal activity of thio-
phene derivative S2, it was subjected to further SAR analysis
(Table 2). First, various groups were introduced at positions 4
and 9 (S2a–S2f). The results of antifungal assay revealed that the
attachment of bromine on position 4 (S2b) or 9 (S2a) resulted in
the decreased antifungal activity. Similarly, the presence of 4-
methylpiperazinyl (S2e) or 4-methyloxyl (S2f) groups at position
4 was also unfavorable for the antifungal activity. Interestingly,
against Cryptococcus neoformans with a MIC value of 0.25
lg/mL,
whose activity was comparable to fluconazole (MIC = 0.25
lg/mL)
and superior to sampangine (MIC = 2 lg/mL). On the strains of Can-
dida spp., the sampangine derivatives generally showed decreased
activity. Only compound S1 was highly active against Candida glab-
rata (MIC = 0.125 lg/mL). For the dermatophytes, most of the com-
pounds were moderately active, which was similar to fluconazole
O
O
O
O
N
N
c
a
b
H3C
N
H3C
N
H3C
CH3
N
H
N
4
HO2C
N
O
O
CH3
O
S3
11
13
12
Scheme 3. Reagents and conditions: (a) sarcosine, paraformaldehyde, toluene, reflux (Dean Stark), 4.5 h, 15%; (b) xylene, 140 °C, 6 h, 39%; (c) (1) DMFDMA, DMF, 120 °C, 2 h,
(2) NH4Cl, HOAc, 120 °C, 1 h, 62% (2 steps).
O
c
a
b
d
HO2C
O
O
O
4
O
OCH3
OH
N
OCH3
O
14
15
16
17
O
O
O
O
N
N
O
N
O
e
+
+
O
O
N
N
O
CH3
O
CH3
19
S4
S5
18
Scheme 4. Reagents and conditions: (a) Cu, quinoline, 240 °C, 2 h, 64%; (b) BBr3, DCM, À78 °C, 2 h, 50%; (c) Fremy’s salt, KH2PO4 aq, acetone/H2O, 1 h, 42%; (d) xylene, 140 °C,
6 h, 35%; (e) (1) DMFDMA, DMF, 120 °C, 2 h, (2) NH4Cl, HOAc, 120 °C, 1 h, 58% (2 steps).

Z. Jiang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4090–4094
4093
O
O
a: R1 = Br, R2 = H
b: R1 = H, R2 = Br
N
N
S2a
S2b
S2c
S2d
a, b, c, d
R1
9
R
1 = H, R2 = Cl
R2
4
S
S
c:
d: R1 = Cl, R2 = Cl
N
N
2
S2
O
N
O
N
O
N
N
N
e
f
S
S
OCH3
Br
S
N
N
N
CH3
S2b
S2f
S2e
O
O
g: R =
i: R=
j: R =
N
N
g,h, i, j
H3C
S2g
S2h
S
S
S2i
S2j
O
CH3
N
h: R =
NHAc
N
10
R
Scheme 6. Reagents and conditions: (a) NBS, DMF, 140 °C, 5 h, 25%; (b) NBS, HOAc, CHCl3, 80 °C, 2 h, 57%; (c/d) NCS, HOAc, CHCl3, 80 °C, 3 h, 49%; (e) 1-methylpiperazine,
K3PO4.H2O, CuI, Me2NCH2CH2OH, 25 °C, 12 h, 38%; (f) Cs2CO3, MeOH, 40 °C, 1 h, 25%. (g) benzaldehyde, NH4Cl, HOAc, DMF, 120 °C, 3 h, 45%; (h) nicotinaldehyde, NH4Cl, HOAc,
DMF, 120 °C, 3 h, 36%; (i) 2-methyl benzaldehyde, NH4Cl, HOAc, DMF, 120 °C, 3 h, 40%; (j) N-(4-formylphenyl)acetamide, NH4Cl, HOAc, DMF, 120 °C, 3 h, 42%.
Table 1
In vitro antifungal activity (MIC80
,
l
g/mL) and kinetic solubility (l
g/mL) of D-ring modified sampangine analoguesa
Compounds A. fum.
C. neo.
C. alb.
C. par.
C. gla.
T. rub
M. gyp
Solubilityb
S1
S2
S3
S4
S5
S6
1
1
2
>64
4
>64
32
32
64
>64
8
0.125
4
>64
32
64
32
8
2
>64
8
8
16
16
4
>64
2
>64
16
27
48
17
-
0.25
>64
16
8
8
2
>64
32
32
64
16
>64
>64
>64
>64
>64
4
16
-
>64
>64
16
22
12.6
-
Sampangine
Fluconazole
0.5
0.5
0.125
0.25
0.25
2
a
Abbreviations: A. fum., Aspergillus fumigatus; C. neo., Cryptococcus neoformans; C. alb., Candida albicans; C. par., Candida parapsilosis; C. gla., Candida glabrata; T. rub.,
Trichophyton rubrum; M. gyp., Microsporum gypseum.
b
Kinetic solubility (PBS buffer, pH = 7.4).
In summary, a series of D-ring modified sampangine derivatives
were designed and synthesized and new SAR results were
obtained. After two rounds of drug design, several compounds
(e.g., S1, S2 and S2c) showed potent antifungal activity with a
broad spectrum and improved water solubility. Particularly, com-
pound S2 was highly active against Aspergillus fumigatus and Cryp-
tococcus neoformans, which are difficult to treat in current
antifungal therapy. Compound S2 has a novel scaffold that differs
from that of all reported antifungal agents and represents a prom-
ising antifungal lead compound for further structural optimization.
Table 2
In vitro antifungal activity of S2 derivatives (MIC80, l )
g mLÀ1
Compounds C. neo. A. fum. C. alb. C. par. C. gla. T. rub M. gyp
S2a
S2b
S2c
S2d
S2e
S2f
S2g
S2h
4
4
8
64
4
2
2
2
1
4
8
4
16
4
0.5
0.125
8
>64
>64
>64
32
>64
>64
16
0.5
>64
64
>64
4
32
>64
>64
1
2
32
32
>64
>64
32
32
32
32
0.5
>64
>64
>64
>64
>64
>64
>64
>64
1
0.5
>64
64
>64
>64
>64
>64
>64
2
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
16
>64
32
>64
0.5
S2i
S2j
Fluconazole
Acknowledgments
This work was supported in part by National Natural Science
Foundation of China (Grant 81222044), Key Project of Science
and Technology of Shanghai (Grant 13431900301), and the 863
Hi-Tech Program of China (Grant 2014AA020525) for financial
support.
Abbreviations: C. neo., Cryptococcus neoformans; A. fum., Aspergillus fumigatus; C. alb.,
Candida albicans; C. par., Candida parapsilosis; C. Gla., Candida glabrata; T. rub.,
Trichophyton rubrum; M. gyp., Microsporum gypseum.
the 4-chloro derivative S2c showed excellent activity against Cryp-
tococcus
(MIC = 0.5
neoformans
(MIC = 0.5
l
g/mL),
Candida
glabrata
g/mL),
l
g/mL), Microsporum gypseum (MIC = 0.125 l
Supplementary data
which was comparable or superior to S2, sampangine and fluco-
nazole. In contrast, almost loss of antifungal activity was observed
when introducing aromatic groups (S2g–j) on the position 2 of S2.
Supplementary data associated (experimental protocols and
structural characterization of target compounds.) with this article

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Z. Jiang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4090–4094
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