Discovery of novel simplified isoxazole derivatives of sampangine as potent anti-cryptococcal agents

Article abstract of DOI:10.1016/j.bmc.2019.01.029

Cryptococcus neoformans is the leading cause of cryptococcal meningitis, which is associated with high mortality due to lack of effective treatment. Herein a series of tricyclic isoxazole derivatives with excellent anti-cryptococcal activities were identi

Full text of DOI:10.1016/j.bmc.2019.01.029

Accepted Manuscript
Discovery of Novel Simplified Isoxazole Derivatives of Sampangine as potent
Anti-cryptococcal Agents
Zhuang Li, Na Liu, Jie Tu, Changjin Ji, Guiyan Han, Yan Wang, Chunquan
Sheng
PII:
S0968-0896(18)32120-5
https://doi.org/10.1016/j.bmc.2019.01.029
BMC 14721
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 December 2018
19 January 2019
23 January 2019
Please cite this article as: Li, Z., Liu, N., Tu, J., Ji, C., Han, G., Wang, Y., Sheng, C., Discovery of Novel Simplified
Isoxazole Derivatives of Sampangine as potent Anti-cryptococcal Agents, Bioorganic & Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.bmc.2019.01.029
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Discovery of Novel Simplified Isoxazole Derivatives of Sampangine as potent
Anti-cryptococcal Agents
Zhuang Li1, †, Na Liu2, †, Jie Tu2, Changjin Ji2, Guiyan Han2, Yan Wang2,*, Chunquan Sheng1,2,*
1 School of Pharmacy, Fujian University of Traditional Chinese Medicine, 1 Qiuyang Road, Fuzhou,
Fujian 350122, P. R. China
2
School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433,
People’s Republic of China
#These two authors contributed equally to this work.
* To whom correspondence should be addressed.
For C. Q. Sheng: Phone/Fax, 86-21-81871205, E-mail, shengcq@smmu.edu.cn;
For Y. Wang: Phone/Fax, 86-21-81871260, E-mail, wangyansmmu@126.com;
1

Abstract
Cryptococcus neoformans is the leading cause of cryptococcal meningitis, which is associated with
high mortality due to lack of effective treatment. Herein a series of tricyclic isoxazole derivatives
with excellent anti-cryptococcal activities were identified by structural simplification and scaffold
hopping of antifungal natural product sampangine. Particularly, compound 8a showed promising
features as an anti-cryptococcal lead compound. It was highly active against C. neoformans (MIC80 =
0.031 g/mL), which was more potent than fluconazole and voriconazole. Moreover, compound 8a
showed potent fungicidal activity and had potent inhibitory effects against important virulence
factors (i.e. biofilm, melanin and urease) of C. neoformans. Preliminary antifungal mechanism
investigation revealed that compound 8a induced apoptosis of C. neoformans cells and arrested the
cell cycle at the G1/S phase.
Keywords: structural simplification, anti-cryptococcal, fungicidal activity, virulence factors,
antifungal mechanism.
2

1. Introduction
In the past few decades, the incidence of life-threatening invasive fungal infections (IFIs) has
increased dramatically.1, 2 It was reported that serious fungal infections were mainly caused by
species of Candida, Cryptococcus, Aspergillus, and Pneumocystis.3 Cryptococcal meningitis (CM) is
an important mycosis worldwide caused by Cryptococcus neoformans (C. neoformans) and
Cryptococcus gattii (C. gattii) with over 1 million cases and 600,000 deaths per year.4-6 Moreover,
CM is a life-threatening disease in immunosuppressed patients and always associated with high
mortality due to the lack of effective treatment.7, 8 Currently, clinically available therapy and
maintenance regimens for CM mainly include amphotericin B (AMB), flucytosine and fluconazole
(FLC).9, 10 However, each of these antifungal agents has significant adverse effects. For example,
AMB is associated with infusional toxicity and renal impairment.11 Bone marrow suppression can
occur during 5-flucytosine therapy. FLC is fungistatic rather than fungicidal in the normal human
9
dose range, which may cause hepatotoxicity.4, The newly developed β-1,3-glucan synthase
inhibitors (i.e. echinocandins) have poor effect against Cryptococcus species.12, 13 Thus, there is an
urgent imperative to discover new molecules to treat cryptococcal infections.14, 15
Natural products are potential sources of new antifungal drugs.16 For example, polyenes and
echinocandins, two important types of antifungal agents, originated from natural products. However,
complex chemical structures of natural products often lead to difficulties in chemical synthesis and
unfavorable physicochemical properties.17 Thus, designing simplified derivatives of natural products
with similar or even better antifungal activity represents an effective but challenging strategy.
Previously, our group reported a successful example of structural simplification of natural product
sampangine.18-20 The simplified tricyclic pyridine derivatives (e.g. compound 3) showed excellent
3

antifungal activities against Candida albicans (C. albicans). However, structure-activity relationship
(SAR) for the anti-cryptococcal activity remains to be further investigated. Herein scaffold hopping
of tricyclic pyridines was performed by replacing pyridine with isoxazole. Interestingly, the novel
tricyclic isoxazole derivatives were highly potent and selective against C. neoformans. Particularly,
compound 8a (C. neoformans, MIC80 = 0.031 μg/mL, Fig. 1) showed potent anti-cryptococcal
activity, representing a promising lead compound for the development of novel CM therapeutic
agents. SAR and preliminary mechanism of action of compound 8a was also clarified.
O
O
O
N
N
N
Scaffold hopping
Structural simplification
S
S
O
N
N
2
Sampangine
1
C. neo. MIC80 = 8 g/mL
C. alb. MIC80 = 32 g/mL
O
N
O
Scaffold hopping
SAR
O
O2N
N
S
S
O
O
3
8a
C. neo. MIC80 = 0.031 g/mL
C. alb. MIC80 = 2 g/mL
C. neo. MIC80 = 1 g/mL
C. alb. MIC80 = 0.5 g/mL
Fig. 1 Structural simplification processes of sampangine.
2. Chemistry
The chemical synthesis of tricyclic isoxazole derivatives is depicted in Schemes 1. Intermediates (7)
were synthesized from substituted thienocyclohexanone (4) via three steps according to our reported
methods.20 Then, target compounds 8a-t were prepared by reacting intermediates 7 with substituted
aldoximes by a [1+2] cycloaddition reaction in the presence of NaClO (Scheme 1).
4

O
O
OH
O
O
Br
Br
Br
Br
a
b
c
S
S
S
S
4
5
1
6
7
O
7
O
8
4
d
2
6
N
S
5
3
OH
R1
N
R1
O
8a-t
R1
R1
R1
R1
8a
8b
8f
8k
8p
-C2H5
8g
8h
8i
8l
8m
8n
8q
8r
Br
-C3H7
Br
OCH3
8c
8d
-CH2OCH3
F
N
Br
Cl
8s
S
N
Cl
OCH3
8e
8j
8o
8t
N
S
OCH3
o
Scheme 1. Reagents and conditions: (a) CuBr2, EtOAc/CHCl3, 80 C, 12 h, 92.1%; (b) Li2CO3,
o
o
DMF, 100 C, 6 h, 90.3%; (c) PhI(OAc)2, HOAc/TFA, 0-25 C, 0.5 h, 69.7%; (d) NaClO, Et3N,
DCM, 0-25 oC, 12 h, 21.7% ~ 82.4%.
3. Results and discussion
3.1. Scaffold hopping, in vitro antifungal activities and SAR
In vitro antifungal activities of the isoxazole derivatives were assayed against two fungal strains C.
albicans SC5314 and C. neoformans H99 using triazole antifungal agents FLC and voriconazole
(VRC) as the positive controls (Table 1). The minimum inhibitory concentration that achieved 80%
inhibition (MIC80) is used to express the in vitro antifungal efficacy. Initially, compound 8a was
designed by scaffold hopping of compound 3, in which the pyridine group of the tricyclic scaffold
5

was replaced by isoxazole. Moreover, the aromatic nitro group was removed due to its potential risk
to cause toxicity. Interestingly, compound 8a was highly active against C. neoformans (MIC80 =
0.031 μg/mL), whereas its activity against C. albicans (MIC80 = 2 μg/mL) was slightly decreased as
compared with compound 3. The SAR was different from that observed in the tricyclic pyridine
derivatives, in which the nitro group was found to be essential for the antifungal activity.20 Inspired
by the results, a series of tricyclic isoxazole derivatives were synthesized and assayed. Most
compounds showed good to excellent antifungal activity against C. neoformans, whose activity was
significantly better than that against C. albicans (Table 1).
Various substitutions on the isoxazole ring (8a-t) were explored. Alkyl substitutions were more
favorable for the anti-cryptococcal activity than the heterocycle and substituted phenyl derivatives, in
which the ethyl substitution gave the best results. Among the substituted phenyl groups, ortho-
substitution was better than meta- and para-substitutions. Meta-substituted derivatives 8l, 8m and 8n
were totally inactive. Among the target compounds, compound 8a showed the best antifungal
activity against C. neoformans H99 (MIC80 = 0.031 μg/mL), which was 64-fold more potent than
FLC and 4-fold more potent than VRC (Table 1-2).
Table 1 In vitro antifungal activity of targets compounds 8a-t (MIC80, μg/mL) a
O
1
7
O
8
2
6
N
4
S
5
3
R1
O
C. neo.
(H99)
0.031
0.12
C. alb.
Compound
R1
(SC5314)
8a
8b
-C2H5
-C3H7
2
4
6

8c
-CH2OCH3
0.25
0.25
8
8
8d
N
N
8e
8f
0.25
0.25
0.25
0.25
0.25
0.5
1
8
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
Br
F
8g
8h
8i
Cl
OCH3
8j
8k
8l
>64
>64
>64
1
OCH3
8m
8n
8o
8p
8q
Br
Cl
OCH3
1
>64
Br
7

8r
8s
8t
4
2
1
16
N
>64
>64
S
S
FLC
_
2
0.25
0.12
VRC
_
0.12
a Abbreviations: C. neo., Cryptococcus neoformans; C. alb., Candida albicans.
3.2. Isoxazole derivatives showed excellent antifungal activities against C. neoformans var. grubii
and C. gattii
According to the results of MIC80 listed in Table 1, compounds 8a-d were chosen to further evaluate
the antifungal activity against C. neoformans var. grubii and C. gattii. These compounds had MIC80
values in the range of 0.12–0.5 μg/mL, which were superior to FLC and comparable to VRC (Table
2). On the basis of the anti-cryptococcal activity, compound 8a was chosen to further biological
evaluations.
Table 2 In vitro antifungal activity of part of the target compounds (MIC80, μg/mL)
C. neo. var. Grubii
C. gattii.
(ATCC 14116)
0.25
Compound
(ATCC 34877)
8a
8b
8c
0.12
0.12
0.25
0.25
0.5
8

8d
0.5
8
1
8
FLC
VRC
0.12
0.12
3.3. Compounds 8a and 8b showed effective inhibitory effect on the biofilm formation of C.
neoformans
Biofilm, an important virulence factor of fungi, is often related with drug resistance and repeated
fungal infections in clinic.21 Thus, effects of compounds 8a and 8b on the formation of cryptococcal
biofilms were evaluated. Considering that fluconazole had poor effects in inhibiting biofilm
formation assay, AmB, a potent biofilm inhibitor, was chosen as the positive control. The results
showed that they could significantly inhibit the biofilms formation, which was better than AMB. In
particular, compound 8a had an inhibition rate of more than 80% at the concentration of 0.062
μg/mL (Fig. 2).
Fig. 2. Effects of different concentrations of compounds 8a, 8b and AMB on biofilm formation. C.
neoformans H99 cells were treated with compound 8a, 8b and AMB (0.062-8 μg/mL). The results of
biofilms formation are represented the means ± standard deviations for three independent
experiments. Statistical differences among groups were determined by ANOVA. The comparison
9

between the two components was completed by t-test. * p < 0.05; ** p < 0.01; *** P < 0.001; **** P
< 0.0001 compared to the control group.
3.4. Compound 8a inhibited the production of melanin and urease of C. neoformans
Melanin and urease are important virulence factors of C. neoformans. Melanin plays an
indispensable role in protecting microorganisms from multiple toxic damages. It showed antioxidant
function to defense environmental impurities and oxidative stresses.22, 23 Urease can increase the
viability of C. neoformans in phagolysosomes.24 Different induction mediums were used to induce
melanin and urease production, respectively. As shown in Fig. 3A, if the cells produced melanin, the
colonies would appear black on the melanin induction medium. Conversely, the colonies appear
white. The results revealed that compound 8a inhibited the production of melanin at the
concentration of 16 μg/mL. As shown in Fig. 3B, if the cells produced urease, the medium would
appear red. The results also showed that compound 8a was able to inhibit the production of urease at
the concentration of 64 μg/mL. (Fig. 3B).
10

Fig. 3. Effects of compound 8a on melanin and urease production by C. neoformans H99 cells. (A)
C. neoformans H99 cells grown on L-DOPA medium were treated with different concentrations of
compound 8a to determine melanin production. (B) Strains grown on urea medium were used to
determine the production of urease.
3.5. Compound 8a exhibited fungicidal activity against C. neoformans
Time-growth curves were assayed to further evaluate the fungistatic activity of compound 8a. As
shown in Fig. 4A, different concentrations of compound 8a (0.062-1 μg/mL) were used in time-
growth curves assay. The results showed that the fungistatic activity of compound 8a at 0.25 μg/mL
was superior to FLC at 16 μg/mL. Compound 8a effectively inhibit C. neoformans H99 cells growth
in a concentration-dependent manner, and completely inhibit H99 cells growth at 1 μg/mL.
Meanwhile, time-kill curves assay was used to test whether compound 8a had fungicidal activity.
The FLC group (4 and 16 μg/mL) were served as the positive control. As shown in Figure 4B,
compound 8a exhibited fungicidal activity at 2 μg/mL. In contrast, FLC didn’t show fungicidal
activity at high concentration (16 μg/mL).
Fig. 4 (A) Time-growth curves of C. neoformans H99 obtained by using initial inoculums of 2 × 106
colony-forming units (CFU)/mL. (B) Time-kill curves of C. neoformans H99 obtained by using an
initial inoculum of 2 × 106 CFU/mL.
11

3.6. Compound 8a induced apoptosis of C. neoformans H99 cells and arrested cell cycle at the G1/S
phase
Apoptosis of C. neoformans H99 treated with compound 8a was investigated by flow cytometry. The
percentage of apoptotic cells in the control group was 1.20%. After treating with compound 8a at 0.5
μg/mL, 1 μg/mL and 2 μg/mL for 24 h, the percentage of apoptotic cells was 23.82%, 44.62%, and
57.48%, respectively (Fig. 5). The results suggested that compound 8a could significantly induce
apoptosis effect. Then, the effect of the compound 8a on the cell cycle was investigated using flow
cytometric analysis to further clarify the antifungal mechanism. As shown in Fig. 6, G1/S arrest was
occurred after a 24 h exposure to compound 8a. In contrast, control group showed 12.24% of cells in
G1 phase and 7.89% in S phase. After treated with compound 8a at 0.5, 1 and 2 μg/mL for 24 h, the
ratios in G1 phase were 50.21%, 49.14% and 48.61%, respectively (Fig. 6), while the S phase
decreased or disappeared. The results indicated that compound 8a could significantly increase the
subpopulation of cells in the G1 phase and reduce the number of cells in the G2 phase resulting in
cell cycle arrest at the G1/S phase.
12

Fig. 5 The effect of compound 8a on apoptosis in C. neoformans H99 cells. C. neoformans cells
were treated with different concentrations of compound 8a for 24 h. Flow cytometry was used to
examine apoptosis (n = 3). Representative photographs of three independent experiments were
shown.
13

Fig. 6 The effect of compound 8a on cell cycle in C. neoformans H99 cells. Cells treated with
different concentrations of compound 8a for 24 h were assayed by flow cytometry.
4. Conclusions
In summary, starting from antifungal natural product sampangine, a series of simplified tricyclic
isoxazole derivatives were designed and synthesized by scaffold hopping. As compared with the lead
compounds, the anti-cryptococcal activities of the target compounds were substantially improved.
Particularly, compound 8a showed excellent antifungal activity against C. neoformans (MIC80 =
0.031 g/mL). Moreover, it was fungicidal and had effective inhibitory effects against important
virulence factors (i.e. biofilm, melanin and urease) of C. neoformans. It was reported that o-vanillin
displayed fungicidal activity by triggering oxidative stress, which lead to the disruption of cellular
14

redox homeostasis and caused mitochondrial dysfunction.25 The melanin could act as a first line of
defense against ROS formation.26 It is suggested that inhibition of melanin biosynthesis by fungicide
compound 8a could be related to oxidative stress and mitochondrial dysfunction. In a preliminary
investigation of the antifungal mechanism, compound 8a induced apoptosis and arrest C. neoformans
cells at the G1/S phase. Similarly, triclosan was reported to induce apoptosis-like death in C.
neoformans cells.27 Taken together, compound 8a represents a novel anti-cryptococcal lead
compound with a new mode of action. Further lead optimization and target identification studies are
ongoing in our lab.
5. Experimental Section
5.1. Chemistry
Bruker AVANCE300 or AVANCE600 spectrometers (Bruker Company, Germany) is used to
1
13
recorded H NMR and C NMR spectra, using CDCl3 or DMSO-d6 as solvents and TMS as an
internal standard. The chemical shift are expressed as ppm (δ). Elemental analyses were performed
with a MOD-1106 instrument and were consistent with theoretical values within 0.4%. The mass
spectral data was recorded on an Esquire 3000 LC-MS mass spectrometer. However, the ion peaks
like [M+H]+ or [M-H]- cannot be determined for most target compounds. Only HRMS result of
compound 8d were reported herein. Silica gel thin-layer chromatography (TLC) was performed on
precoated plates GF-254 (Qingdao Haiyang Chemical, China). The target compound was isolated
and purified using a silica gel column (Silica gel 60G, Qingdao Haiyang Chemical, China). Purity of
the compounds was analyzed by HPLC (Agilent Technologies 1260 Infinity) using MeOH/H2O =
70/30 as the mobile phase with a flow rate of 0.5 mL/min. The purity of all compounds was greater
than 95%. All solvents and reagents were analytically pure, and no further purification was needed.
15

All starting materials were commercially available.
5.1.1. 5,5-Dibromo-6,7-dihydrobenzo[b]thiophen-4(5H)-one (5)
A solution of CuBr2 (11.3 g, 50.5 mmol) in EtOAc (30 mL) was stirred at 80 oC for 10 min. Then,
a solution of 6,7-dihydrobenzo[b]thiophen-4(5H)-one (1.918 g, 12.6 mmol) in CHCl3 (10 mL) was
added dropwise to the mixture solution (via addition funnel) and stirred for 12 h at 80 oC. Then, the
solvent was removed under reduced pressure. The mixture was filtered by celite pad after diluted
with EtOAc (40 mL). The filtrate was washed with sat. NaHCO3 (50 mL × 2), dried over Na2SO4,
filtrated and concentrated under reduced pressure. The crude product was purified by silica gel
chromatography eluted with Hexane/EtOAc = 50/1 to give target compound 6 (3.6 g, yield 92.1%) as
a white solid. 1H NMR (300 MHz, DMSO-d6) δ 7.55 (d, J = 5.3 Hz, 1H), 7.38 (d, J = 5.3 Hz, 1H),
3.15 (t, J = 3.0 Hz, 4H).
5.1.2. 5-Bromobenzo[b]thiophen-4-ol (6)
Li2CO3 (1.43 g, 19.32 mmol) was added into a solution of 5,5-dibromo-6,7-
dihydrobenzo[b]thiophen-4(5H)-one (1 g, 3.22 mmol) in DMF (30 mL). The mixture was heated at
o
100 C and stirred for 3 h under N2. Then, the reaction mixture was filtered after cooled to room
temperature. The filtrates were diluted with H2O (20 mL) and acidified with 1 N HCl to pH = 1. The
resulting solution was extracted with EtOAc (40 mL × 2). The separated organic layer was washed
with H2O (20 mL × 6), dried over Na2SO4, filtrated and concentrated under reduced pressure to
afford crude product. The residue was purified by column silica gel chromatography (Hexane/EtOAc
1
= 60/1) to give target compound 6 (0.66 g, yield 90.3%) as a white solid. H NMR (300 MHz,
DMSO-d6) δ 10.24 (s, 1H), 7.69-7.63 (m, 2H), 7.45 – 7.38 (m, 2H).
5.1.3. 5-Bromobenzo[b]thiophene-4,7-dione (7)
16

To a solution of 5-bromobenzo[b]thiophen-4-ol (0.5 g, 2.18 mmol) in AcOH (4 mL) and TFA (5
mL) and a few drops of water, PhI(OAc)2 (2.11 g, 6.55 mmol) was added in portions during a period
of 30 min at 0 oC. Then, the mixture was stirred at 0 oC for 10 min and then at room temperature for
20 min. Then, MeOH (12.5 mL) was added into the solution and stirred for 10 min. The mixture was
diluted with H2O (20 mL) and extracted by DCM (40 mL×2). The combined organic layer was
washed with sat. NaCl (20 mL × 2), dried over Na2SO4, filtrated and concentrated under reduced
pressure to afford crude product. The residue was purified by silica gel column chromatography
(Hexane/EtOAc = 60/1) to give target compound 7 (0.37 g, yield 69.7%) as a yellow solid. 1H NMR
(600 MHz, CDCl3) δ 7.70 (d, J = 5.1 Hz, 1H), 7.63 (d, J = 5.0 Hz, 1H), 7.39 (s, 1H).
5.1.4. 3-Methylthieno[3',2':4,5]benzo[1,2-d]isoxazole-4,8-dione (8a)
To a solution of 5-bromobenzo[b]thiophene-4,7-dione (0.125 g, 0.51 mmol) in DCM (10 mL) was
o
added dropwise Et3N (18 uL) and an aqueous solution of NaClO (15 mL, 7.5%) at 0 C. Then, a
solution of propionaldehyde oxime (53 mg, 0.72 mmol) in DCM (5 mL) was added dropwise into the
mixture during a period of 15 min. After the TLC showed the starting material was consumed
completely, the mixture was diluted with H2O (20 mL) and extracted by DCM (40 mL × 2). The
combined organic phase was washed with sat. NaCl (20 mL × 2). Then, the organic layer was
separated, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (Hexane/EtOAc = 60/1) to give target compound 8a
(70 mg, yield 61.6%) as a yellow solid. 1H NMR (300 MHz, CDCl3) δ 7.75 (d, J = 5.1 Hz, 1H), 7.67
(d, J = 5.1 Hz, 1H), 3.06 (q, J = 7.5 Hz, 2H), 1.41 (t, J = 7.5 Hz, 3H). 13C NMR (151 MHz, CDCl3) δ
174.17, 168.96, 166.12, 163.14, 145.89, 140.77, 134.35, 126.46, 120.38, 19.24, 11.84. HPLC purity:
99.3%.
17

The synthetic methods for compounds 8b-t were similar to the synthesis of compound 8a.
5.1.5. 3-Propylthieno[3',2':4,5]benzo[1,2-d]isoxazole-4,8-dione (8b)
Yellow solid (0.123 g, yield 60.9%). 1H NMR (300 MHz, CDCl3) δ 7.75 (d, J = 5.1 Hz, 1H), 7.67
13
(d, J = 5.1 Hz, 1H), 3.01 (t, J = 7.4 Hz, 2H), 1.92-1.78 (m, 2H), 1.04 (t, J = 7.4 Hz, 3H). C NMR
(151 MHz, CDCl3) δ 174.19, 168.95, 166.09, 161.95, 145.92, 140.75, 134.35, 126.46, 120.48, 27.20,
20.86, 13.72. HPLC purity: 95.8%.
5.1.6. 3-(Methoxymethyl)thieno[3',2':4,5]benzo[1,2-d]isoxazole-4,8-dione (8c)
Yellow solid (22 mg, yield 42.7%). 1H NMR (600 MHz, CDCl3) δ 7.77 (d, J = 5.1 Hz, 1H), 7.68
(d, J = 5.0 Hz, 1H), 4.86 (s, 2H), 3.54 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 173.43, 168.59, 166.22,
158.36, 145.61, 140.76, 134.65, 126.52, 120.34, 64.37, 59.33.
5.1.7. 3-(Pyridin-4-yl)thieno[3',2':4,5]benzo[1,2-d]isoxazole-4,8-dione (8d)
Yellow solid (84 mg, yield 36.6%). 1H NMR (300 MHz, CDCl3) δ 8.83 (d, J = 6.0 Hz, 2H), 8.12
(d, J = 6.1 Hz, 2H), 7.81 (d, J = 5.1 Hz, 1H), 7.71 (d, J = 5.1 Hz, 1H). 13C NMR (151 MHz, CDCl3)
δ 173.17, 168.39, 167.66, 158.80, 150.34, 146.54, 140.18, 135.18, 133.97, 126.41, 123.12, 119.66.
HRMS (ESI, negative) m/z: [M + H]+ calcd for C14H6N2O3S, 283.0172; found 283.0176, HPLC
purity: 99.4%.
5.1.8. 3-(Pyridin-2-yl)thieno[3',2':4,5]benzo[1,2-d]isoxazole-4,8-dione (8e)
Yellow solid (56 mg, yield 48.8%). 1H NMR (300 MHz, CDCl3) δ 8.87 (d, J = 4.8 Hz, 1H), 8.23
(d, J = 7.8 Hz, 1H), 7.96 (t, J = 7.7 Hz, 1H), 7.78 (d, J = 5.1 Hz, 1H), 7.70 (d, J = 5.1 Hz, 1H), 7.57 –
7.50 (m, 1H). 13C NMR (75 MHz, CDCl3) δ 172.95, 168.63, 167.24, 159.90, 150.10, 146.73, 145.77,
140.16, 137.25, 134.80, 126.32, 125.65, 125.30, 119.79.
5.1.9. 3-(o-Tolyl)thieno[3',2':4,5]benzo[1,2-d]isoxazole-4,8-dione (8f)
18

Yellow solid (0.168 g, yield 77.0%). 1H NMR (300 MHz, CDCl3) δ 7.76 (d, J = 5.1 Hz, 1H), 7.70
13
(d, J = 5.1 Hz, 1H), 7.48 – 7.43 (m, 2H), 7.38 – 7.30 (m, 2H), 2.35 (s, 3H). C NMR (151 MHz,
CDCl3) δ 173.23, 168.93, 166.37, 160.52, 146.35, 140.49, 137.66, 134.50, 130.68, 130.65, 130.32,
126.36, 125.77, 125.36, 120.58, 20.17.
5.1.10. 3-(2-Bromophenyl)thieno[3',2':4,5]benzo[1,2-d]isoxazole-4,8-dione (8g)
Yellow solid (0.217 g, yield 73.5%). 1H NMR (600 MHz, CDCl3) δ 7.77 -7.76 (m, 2H), 7.71 (d, J
= 5.1 Hz, 1H), 7.51 – 7.42 (m, 3H). 13C NMR (151 MHz, CDCl3) δ 172.69, 168.83, 166.10, 159.95,
146.05, 140.59, 134.60, 133.22, 132.06, 131.39, 127.64, 127.46, 126.44, 123.47, 120.92.
5.1.11. 3-(2-Fluorophenyl)thieno[3',2':4,5]benzo[1,2-d]isoxazole-4,8-dione (8h)
Yellow solid (64 mg, yield 52.1%). 1H NMR (600 MHz, CDCl3) δ 7.76 (d, J = 5.1 Hz, 1H), 7.70
13
(d, J = 5.1 Hz, 1H), 7.69 – 7.66 (m, 1H), 7.60 – 7.56 (m, 1H), 7.33 – 7.27 (m, 2H). C NMR (151
MHz, CDCl3) δ 172.70, 168.74, 166.45, 160.54 (d, J
= 253.3 Hz), 156.02, 146.34, 140.43,
C - F
134.53, 133.08 (d, J C - F = 8.4 Hz), 131.13, 126.37, 124.34 (d, J C - F = 3.3 Hz), 120.60, 116.24 (d, J C
- F = 20.9 Hz), 114.50 (d, J C - F = 14.2 Hz).
5.1.12. 3-(2-Chlorophenyl)thieno[3',2':4,5]benzo[1,2-d]isoxazole-4,8-dione (8i)
Yellow solid (0.1 g, yield 78.0%). 1H NMR (300 MHz, CDCl3) δ 7.77 (d, J = 5.1 Hz, 1H), 7.71 (d,
13
J = 5.1 Hz, 1H), 7.61 – 7.50 (m, 3H), 7.45 – 7.41 (m, 1H). C NMR (151 MHz, CDCl3) δ 172.70,
168.81, 166.16, 158.65, 146.11, 140.55, 134.56, 134.22, 132.01, 131.31, 130.05, 126.90, 126.42,
125.59, 121.02.
5.1.13. 3-(2-Methoxyphenyl)thieno[3',2':4,5]benzo[1,2-d]isoxazole-4,8-dione (8j)
19

Yellow solid (84 mg, yield 65.7%). 1H NMR (600 MHz, CDCl3) δ 7.71 (d, J = 5.0 Hz, 1H), 7.67
(d, J = 5.1 Hz, 1H), 7.55 – 7.51 (m, 1H), 7.49 (dd, J = 7.5, 1.6 Hz, 1H), 7.09 – 7.04 (m, 2H), 3.81 (s,
3H).
5.1.14. 3-(m-Tolyl)thieno[3',2':4,5]benzo[1,2-d]isoxazole-4,8-dione (8k)
Yellow solid (99 mg, yield 81.9%). 1H NMR (300 MHz, CDCl3) δ 7.97 – 7.91 (m, 2H), 7.76 (d, J
13
= 5.1 Hz, 1H), 7.69 (d, J = 5.1 Hz, 1H), 7.46 – 7.36 (m, 2H), 2.47 (s, 3H). C NMR (75 MHz,
CDCl3) δ 173.50, 168.85, 167.24, 160.84, 146.95, 140.07, 138.51, 134.56, 132.15, 129.76, 128.60,
126.53, 126.24, 125.88, 119.47, 21.44.
5.1.15. 3-(3-Methoxyphenyl)thieno[3',2':4,5]benzo[1,2-d]isoxazole-4,8-dione (8l)
Yellow solid (87 mg, yield 68.0%). 1H NMR (300 MHz, CDCl3) δ 7.79 – 7.75 (m, 3H), 7.70 (d, J
= 5.1 Hz, 1H), 7.45 (t, J = 8.2 Hz, 1H), 7.15 – 7.09 (m, 1H), 3.92 (s, 3H). 13C NMR (75 MHz, CDCl3)
δ 173.47, 168.81, 167.34, 160.62, 159.69, 147.00, 140.00, 134.63, 129.79, 127.16, 126.23, 121.63,
119.47, 117.84, 114.17, 55.51.
5.1.16. 3-(3-Bromophenyl)thieno[3',2':4,5]benzo[1,2-d]isoxazole-4,8-dione (8m)
Yellow solid (0.213 g, yield 72.3%). 1H NMR (300 MHz, CDCl3) δ 8.37 (s, 1H), 8.14 (d, J = 7.8
Hz, 1H), 7.80 (d, J = 5.1 Hz, 1H), 7.72 - 7.70 (m, 2H), 7.43 (t, J = 8.0 Hz, 1H). 13C NMR (75 MHz,
CDCl3) δ 173.37, 168.64, 167.41, 159.48, 146.73, 140.11, 134.92, 134.38, 132.14, 130.25, 127.95,
126.33, 122.74, 119.37.
5.1.17. 3-(3-Chlorophenyl)thieno[3',2':4,5]benzo[1,2-d]isoxazole-4,8-dione (8n)
Yellow solid (0.1 g, yield 78.2%). 1H NMR (300 MHz, CDCl3) δ 8.22 (s, 1H), 8.09 (d, J = 7.6 Hz,
13
1H), 7.80 (d, J = 5.1 Hz, 1H), 7.71 (d, J = 5.1 Hz, 1H), 7.58 – 7.45 (m, 2H). C NMR (75 MHz,
20

CDCl3) δ 173.37, 168.64, 167.42, 159.60, 146.74, 140.11, 134.90, 134.79, 131.47, 130.02, 129.32,
127.74, 127.49, 126.33, 119.38.
5.1.18. 3-(4-Methoxyphenyl)thieno[3',2':4,5]benzo[1,2-d]isoxazole-4,8-dione (8o)
Yellow solid (88 mg, yield 69.5%). 1H NMR (300 MHz, CDCl3) δ 8.15 (d, J = 8.9 Hz, 2H), 7.76
13
(d, J = 5.1 Hz, 1H), 7.69 (d, J = 5.0 Hz, 1H), 7.04 (d, J = 8.8 Hz, 2H), 3.90 (s, 3H). C NMR (75
MHz, CDCl3) δ 173.71, 168.89, 167.25, 162.07, 160.32, 147.02, 140.04, 134.49, 130.98, 126.22,
119.34, 118.37, 114.13, 55.43.
5.1.19. 3-(p-Tolyl)thieno[3',2':4,5]benzo[1,2-d]isoxazole-4,8-dione (8p)
Yellow solid (0.199 g, yield 82.4%). 1H NMR (300 MHz, CDCl3) δ 8.04 (d, J = 8.1 Hz, 2H), 7.76
13
(d, J = 5.1 Hz, 1H), 7.69 (d, J = 5.1 Hz, 1H), 7.34 (d, J = 8.0 Hz, 2H), 2.45 (s, 3H). C NMR (75
MHz, CDCl3) δ 173.58, 168.89, 167.23, 160.70, 146.97, 141.81, 140.06, 134.54, 129.43, 129.23,
126.24, 123.14, 119.45, 21.60.
5.1.20. 3-(4-Bromophenyl)thieno[3',2':4,5]benzo[1,2-d]isoxazole-4,8-dione (8q)
Yellow solid (0.206 g, yield 70.0%). 1H NMR (300 MHz, CDCl3) δ 8.08 (d, J = 8.5 Hz, 2H), 7.79
13
(d, J = 5.1 Hz, 1H), 7.72 - 7.67 (m, 3H). C NMR (75 MHz, CDCl3) δ 173.48, 168.66, 167.41,
159.85, 146.75, 140.11, 134.86, 132.04, 130.84, 126.33, 126.17, 124.99, 119.36.
5.1.21. 3-(1-Methyl-1H-pyrrol-2-yl)thieno[3',2':4,5]benzo[1,2-d]isoxazole-4,8-dione (8r)
Yellow solid (8 mg, yield 22.8%). 1H NMR (600 MHz, CDCl3) δ 7.75 (d, J = 5.1 Hz, 1H), 7.69 (d,
13
J = 5.1 Hz, 1H), 7.65 - 7.64 (m, 1H), 6.90 – 6.88 (m, 1H), 6.32 - 6.31 (m, 1H), 4.00 (s, 3H). C
NMR (151 MHz, CDCl3) δ 173.17, 168.91, 166.32, 153.47, 147.41, 139.76, 134.21, 128.93, 126.04,
119.14, 118.78, 118.55, 108.73, 37.88.
5.1.22. 3-(Thiophen-2-yl)thieno[3',2':4,5]benzo[1,2-d]isoxazole-4,8-dione (8s)
21

Yellow solid (56 mg, yield 47.8%). 1H NMR (300 MHz, CDCl3) δ 8.53 (d, J = 3.7 Hz, 1H), 7.78
(d, J = 5.1 Hz, 1H), 7.70 (d, J = 5.1 Hz, 1H), 7.57 (d, J = 5.1 Hz, 1H), 7.23 (dd, J = 5.0, 3.9 Hz 1H).
13C NMR (75 MHz, CDCl3) δ 173.36, 168.60, 167.14, 155.42, 146.71, 140.03, 134.70, 133.49,
130.21, 128.03, 127.03, 126.32, 118.82.
5.1.23. 3-(Thiophen-3-yl)thieno[3',2':4,5]benzo[1,2-d]isoxazole-4,8-dione (8t)
Yellow solid (23 mg, yield 39.1%). 1H NMR (300 MHz, CDCl3) δ 8.87 (d, J = 2.8 Hz, 1H), 7.87
(d, J = 5.0 Hz, 1H), 7.78 (d, J = 5.1 Hz, 1H), 7.70 (d, J = 5.1 Hz, 1H), 7.46 (dd, J = 5.1, 3.0 Hz, 1H).
13C NMR (151 MHz, DMSO-d6) δ 174.35, 169.29, 168.07, 155.65, 146.61, 140.73, 136.88, 131.17,
128.38, 127.16, 126.94, 126.35, 119.00.
5.2 Biology
5.2.1. In Vitro Antifungal Activity Assay
In vitro antifungal activity was measured by the serial dilution method in 96-well microtest plates.
C. albicans strains SC5314, C. neoformans strains H99, C. Gattii strains ATCC14116 and C. neo.
var. Grubii strains ATCC34877 was used in this study. MIC was measured according to the
requirements of National Committee for Clinical Laboratory Standards (NCCLS). RPMI 1640
(Gibco BRL) buffered with 0.165M MOPS (Sigma), NaHCO3 (0.2%) and NaOH (0.27%) was used
as the test medium. All strains were routinely grown in YPD with 1% yeast extract (BD), 2%
peptone (BD) and 2% D-Glucose (Sangon Biotech) liquid medium at 30 oC in a sharking incubator.
Briefly, the initial concentration of fungal suspension in RPMI 1640 medium was 103 CFU/mL, and
tested compounds were dissolved in DMSO. The yeast strains were incubated at 35 oC for 48 h.
5.2.2. In Vitro Biofilm Formation Assay
The experimental procedure was performed according to the reported method with a few
22

modification.28 The suspension of C. neoformans H99 cells (1.0 × 106 CFU/mL in RPMI 1640
medium) was added into a 96-well tissue culture plate (Corning, USA) and allowed to incubate at 37
°C for 90 minutes. After that, the upper culture medium and the non-adherent cells were removed.
Fresh RPMI 1640 medium with or without drugs was added into after the adherent cells had been
washed three times with PBS. A drug-free sample was served as a control. Concentrations for
compounds 8a and 8b and AMB were 0.062-8 μg/mL. Then the plates were incubated for a further
24 h at 37 oC. The biofilm formation rate was calculated by using an XTT reduction assay.
5.2.3. Melanin and Urease Production Assay
Exponentially growing C. neoformans H99 cells were harvested, washed three times with PBS,
and resuspended in RPMI 1640 medium at 1 × 106 cells/mL. Different concentrations of compound
8a and FLC were added into the suspension. Cell suspension containing different concentrations of
drugs was dropped onto the L-DOPA or urea medium and incubated at 37 oC to detect the production
of melanin and urease.
5.2.4. Time-growth Curve Assay
Time-growth curve assay was performed according to the reported method with a few
modifications.29 Briefly, exponentially growing C. neoformans H99 cells were harvested and
resuspended in fresh RPMI 1640 medium 5 mL to a concentration of 2 × 106 cells/mL. Various
concentrations of compound 8a were added. The sample were cultured at 30 °C under constant
shaking (200 rpm). A portion of the cell suspension was removed and counted at the indicated time
points (0h, 3 h, 6 h, 9 h, 12 h, 24 h, 48 h). DMSO was added into the control group. Three
independent experiments were performed.
5.2.5. Fungicidal Activity Assay
23

Time-kill curves were used to evaluate the fungicidal activity of compound 8a. The experimental
operation was carried out according to the reported method.28 Exponentially growing C. neoformans
H99 cells were washed with PBS (1 mL × 3), then resuspended with RPMI 1640 medium to 2 × 106
cells/mL in 5 mL. Different concentrations of the compound 8a and FLC were added and the sample
was cultured at 30 °C under constant shaking (200 rpm). A portion of the cell suspension was
removed at the indicated time points (0h, 6h, 12 h, 24 h, 48 h) and diluted to appropriate folds. The
diluent cell suspension was plated on SDA agar medium to determine the concentration after
incubation for 48 h at 30 °C.
5.2.6. Cell Cycle Analysis by Flow Cytometry
The experimental procedure was performed according to the reported method with a few
modification.30 Exponentially growing C. neoformans H99 cells were harvested, washed three times
with PBS, and resuspended in RPMI 1640 medium (20 mL) at 5 × 105 cells/mL. Different
concentrations of compound 8a were added into the suspension. After cultured at 30 °C under
constant shaking (200 rpm) for 24 h, each sample was centrifuged, washed with PBS three times.
Each 10 mg cells (wet weight) were resuspended with 100 mL PBS (containing 0.24% snailase) and
then incubated for 1 h at 30 °C to break the cell wall. The sample was washed with PBS twice and
fixed with 70% ethanol overnight. The cells were then stained with 50 μg/mL of propidium iodide
(PI) at 4 °C for 30 min. About 1 × 106 cells were measured using a FACSCalibur cytometer (Becton,
Dickinson, San Jose, CA).
5.2.7. Cell Apoptosis Detection by Flow Cytometry
The preparation procedure of the sample treated with different concentrations of compound 8a
was similar as the step of cell cycle assay. After the sample had been resuspended in binding buffer
24

400 μL, it was then added to 5 μL of annexin V-FITC and incubated at room temperature for 15 min.
Then, 10 μL of PI was added into the suspensions and incubated for another 15 min at room
temperature. The cells were analyzed by a flow cytometer (Becton, Dickinson, San Jose, CA).
Acknowledgments
This work was supported by the National Natural Science Foundation of China (Grants 81725020 to
C. S.), Science and Technology Commission of Shanghai Municipality (Grant 17XD1404700) and
National Natural Science Foundation of China (Grants 21502224 to N. L.).
A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at XXXX.
25

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Graphical Abstract
Discovery of Novel Simplified Isoxazole Derivatives of Sampangine as
potent Anti-cryptococcal Agents
Zhuang Li1,†, Na Liu2,†, Jie Tu2, Changjin Ji2, Guiyan Han2, Yan Wang2,*, Chunquan Sheng1,2,*
27

O
O
N
O
Structural
simplification
Scaffold
hopping
N
N
O
O2N
N
S
S
O
O
3
Sampangine
8a
C. neo. MIC80 = 1 g/mL
C. neo. MIC80 = 0.031 g/mL
Fungicidal activity
Inhibition of virulence factors
G1/S phase cell cycle arrest
28