Discovery of simplified sampangine derivatives as novel fungal biofilm inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.10.043

Lack of novel antifungal agents and severe drug resistance have led to high incidence and associated mortality of invasive fungal infections. To tackle the challenges, novel antifungal agents with new chemotype, fungicidal activity and anti-resistant potency are highly desirable. On the basis of our previously identified simplified analogue of antifungal natural product sampangine, systemic structure-activity relationships were clarified and two novel derivatives showed promising features as novel antifungal lead compounds. Compounds 22b and 22c showed good fungicidal activity against both fluconazole-sensitive and fluconazole-resistant Candida albicans strains. Moreover, they were proven to be potent inhibitors of Candida albicans biofilm formation and yeast-to-hypha morphological transition by down-regulating biofilm-associated genes. In a rat vaginal Candida albicans infection model, compounds 22b and 22c showed excellent therapeutic effects with low toxicity. The results highlighted the potential of sampangine derivatives to overcome fluconazole-related and biofilm-related drug resistance.

Full text of DOI:10.1016/j.ejmech.2017.10.043

Accepted Manuscript
Discovery of simplified sampangine derivatives as novel fungal biofilm inhibitors
Na Liu, Hua Zhong, Jie Tu, Zhigan Jiang, Yanjuan Jiang, Yan Jiang, Yuanyin Jiang,
Jian Li, Wannian Zhang, Yan Wang, Chunquan Sheng
PII:
S0223-5234(17)30843-7
10.1016/j.ejmech.2017.10.043
EJMECH 9835
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 July 2017
Revised Date: 28 September 2017
Accepted Date: 15 October 2017
Please cite this article as: N. Liu, H. Zhong, J. Tu, Z. Jiang, Y. Jiang, Y. Jiang, Y. Jiang, J. Li, W. Zhang,
Y. Wang, C. Sheng, Discovery of simplified sampangine derivatives as novel fungal biofilm inhibitors,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.10.043.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Discovery of Simplified Sampangine Derivatives as
Novel Fungal Biofilm Inhibitors
1
,#
1,#
1,#
1
1
1
Na Liu , Hua Zhong , Jie Tu , Zhigan Jiang , Yanjuan Jiang , Yan Jiang , Yuanyin
1
2
1,*
1,*
1,*
Jiang , Jian Li , Wannian Zhang , Yan Wang , Chunquan Sheng
Novel simplified analogues of natural product sampangine showed excellent in vitro
and in vivo antifungal activity with promising features to overcome
fluconazole-related and biofilm-related drug resistance.

ACCEPTED MANUSCRIPT
Discovery of Simplified Sampangine Derivatives as
Novel Fungal Biofilm Inhibitors
1
,#
1,#
1,#
1
1
1
Na Liu , Hua Zhong , Jie Tu , Zhigan Jiang , Yanjuan Jiang , Yan Jiang , Yuanyin
1
2
1,*
1,*
1,*
Jiang , Jian Li , Wannian Zhang , Yan Wang , Chunquan Sheng
1
School of Pharmacy, Second Military Medical University, 325 Guohe Road,
Shanghai 200433, People’s Republic of China
2
School of Pharmacy, East China University of Science & Technology, 130 Meilong
Road, Shanghai 200237, People’s Republic of China
#
These two authors contributed equally to this work.
*
8
8
8
To whom correspondence should be addressed. For C. Q. Sheng: Phone/Fax,
6-21-81871239, E-mail, shengcq@smmu.edu.cn; For Y. Wang: Phone/Fax,
6-21-81871260, E-mail, wangyansmmu@126.com
6-21-81871243, E-mail, zhangwnk@hotmail.com
; For W.N. Zhang: Phone/Fax,
.
Abstract：Lack of novel antifungal agents and severe drug resistance have led to high
incidence and associated mortality of invasive fungal infections. To tackle the
challenges, novel antifungal agents with new chemotype, fungicidal activity and
anti-resistant potency are highly desirable. On the basis of our previously identified
simplified analogue of antifungal natural product sampangine, systemic
structure-activity relationships were clarified and two novel derivatives showed
promising features as novel antifungal lead compounds. Compounds 22b and 22c
showed good fungicidal activity against both fluconazole-sensitive and
fluconazole-resistant Candida albicans strains. Moreover, they were proven to be
potent inhibitors of Candida albicans biofilm formation and yeast-to-hypha
morphological transition by down-regulating biofilm-associated genes. In a rat
vaginal Candida albicans infection model, compounds 22b and 22c showed excellent
therapeutic effects with low toxicity. The results highlighted the potential of
sampangine derivatives to overcome fluconazole-related and biofilm-related drug
resistance.
Keywords: Sampangine, antifungal activity, biofilm inhibition, anti-resistance
1

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1
. Introduction
Life-threatening invasive fungal infections (IFIs) are regarded as the “hidden
killers” of immunocompromised individuals such as patients with AIDS, cancer
and organ transplantation [1]. Recently, IFIs are associated with high incidence
and high mortality and become a serious health problem in clinic [2]. IFIs are
predominantly caused by Candidiasis (Candida albicans, mortality: 46% -
7
5%), cryptococcosis (Cryptococcus neoformans
,
mortality: 20% - 70%), and
aspergillosis (Aspergillus fumigates mortality: 30% - 95%) [1]. Despite the high
,
mortality of IFIs, effective and safe antifungal agents are rather limited [3].
Clinically, antifungal agents for the treatment of IFIs mainly include azoles (e.g.
fluconazole, voriconazole, itraconaole, posaconazole and isavuconazole),
polyenes (e.g. amphotericin B) and echinocandins (e.g. caspofungin and
micafungin) [4]. However, these antifungal drugs have only achieved modest
success in reducing the high mortality rates of IFIs [5]. Therapeutic limitations
of current antifungal agents mainly consist of low efficacy, narrow spectrum,
significant toxicity, drug-drug interactions and unfavorable pharmacodynamic
(
PD) and pharmacokinetic (PK) profiles [6]. Notably, severe resistance has
been observed in almost all of the clinically available antifungal agents [7, 8].
As a result, their therapeutic effects are significantly reduced, which is an
important contributing factor to the high mortality. Thus, discovery and
development of safer and more effective antifungal drugs is highly desirable
and urgently needed.
2

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Currently, only a few antifungal candidate drugs are currently in preclinical
or clinical development, most of which are structural analogues of azoles and
echinocandins [9]. However, these follow-on drugs with the same mode of
action can hardly solve the resistance and toxicity problems. Thus,
identification of novel antifungal molecular scaffold is of great importance for
discovery of new generation of antifungal agents [10]. Natural products provide
a rich and unique source of antifungal lead compounds [11]. For example,
polyenes and echinocandins are derived from natural products. However, it also
should be noted that these nature products-based antifungal agents are generally
limited by synthetic difficulty, undesired PK profiles and poor oral availability
due to their complex chemical structures [11]. To tackle the challenges,
clarifying structural determinants for antifungal activity and structural
simplification of antifungal natural products presents a promising strategy.
Nevertheless, it is difficult to identify a simplified scaffold with similar or even
better antifungal activity.
Previously, we reported a successful example of structural simplification of
antifungal natural product sampangine [12, 13]. As compared with sampangine,
simplified analogue 3 (ZG-20-7) showed better antifungal activity, improved
water solubility, low toxicity and promising anti-resistance profiles, which
represents a good starting point for the discovery of new generation of
antifungal agents [13]. However, detailed structure-activity relationship (SAR)
of compound
3
is still unknown. More importantly, its in vivo antifungal
3

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potency remains to be further improved. Herein, systemic SAR of lead
compound
3 was investigated. Two derivatives, 22b and 22c, showed excellent
in vitro and in vivo antifungal activity with promising features to overcome
fluconazole-related and biofilm-related drug resistance.
2
. Results and Discussion
2
.1 Chemistry
Chemical synthesis of the simplified sampangine derivatives is depicted in Schemes
−5. Compounds 3 and 9 were synthesized from 6,7-dihydrobenzo[b]
1
thiophen-4(5H)-one (4) via five steps [13]. Then, the nitro groups were reduced to
amine groups in the presence of NaBH , NiCl and MeOH/DMF to afford compounds
4
2
1
0a and 10b (Scheme 1).
Starting from compound 3, the carbonyl groups were reduced to the hydroxyl
groups (11a and 11b) in the presence of NaBH and DMF. Then, the two hydroxyl
4
groups were removed to form the aromatization product 12 under the acid condition.
Using hydrogen peroxide as the oxidant, the N-oxide derivative 13 was prepared.
o
Bromination of the methyl group of compound 3 by Br in HOAc at 110 C yielded
2
the di-bromo derivative 14. After further oxidation in the presence of DMSO, the
formyl derivative 15 was obtained, which was reacted with the Wittig reagent to form
the α, β-unsaturated esters 16a and 16b (Scheme 2).
Intermediate 20 was synthesized from 6,7-dihydrobenzo[b]thiophen-4(5H)-one (4)
via four steps [13]. Then, it was reacted with different hydrazones (21a-u) by the
Diels-Alder reaction to form various C7- and C8-substituted analogues (22a-u,
4

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Scheme 3).
.2 SAR of the Simplified Sampangine Derivatives
2
The SAR of lead compound 3 was focused on the nitro group, thiophene ring,
benzoquinone ring, pyridine ring and its substitutions. A number of derivatives were
synthesized and assayed, whose antifungal activity was expressed as the minimal
inhibitory concentration (MIC) that achieved 80% inhibition of the tested fungal
pathogens. First, the importance of the nitro group was investigated. The movement
of the nitro group from position 2 to 3 (compound 9) led to decrease of the antifungal
activity. When the nitro group was reduced to amine, compounds 10a and 10b were
almost inactive. Only compound 10a showed moderate activity against T. rubrum
(
MIC = 8 µg/mL). Second, the benzoquinone carbonyl groups were reduced to the
80
hydroxyl groups (11a and 11b). Interestingly, the antifungal activity of the cis isomer
1b was slightly decreased (MIC range: 2 to 16 µg/mL), whereas the trans isomer
1
8
0
11a was totally inactive. Further removal of the benzoquinone carbonyl groups also
resulted in general loss of the antifungal activity. Compound 12 were only moderately
active against C. glabrata and T. rubrum. Moreover, the benzoquinone containing
scaffolds did not possess antifungal activity. As described in Table 2, compounds
2
3-26 [12] were generally inactive against the tested fungal pathogens, indicating that
the benzoquinone substructure was not the determinant for the antifungal activity.
Intermediate 8 with the thiophene substructure showed moderate antifungal activity
and further incorporation of a nitro group led to significant improvement of the
antifungal activity (Table 1). Thus, the 2-nitrothirno[2,3-g]quinoline-4,9-dione played
5

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key roles for the antifungal activity.
Third, the effects of the pyridine ring and its substitutions were explored. Obvious
decrease of the antifungal activity was observed for N-oxide derivative 13.
Furthermore, a number of C7- and C8-substituted derivatives were synthesized and
assayed. Removal of the C8 methyl group had little effect on the antifungal activity
(compound 22f). In contrast, di-bromo derivative 14 and formyl derivative 15
generally showed decreased activity. Even though, compound 15 was highly active
against C. glabrata (MIC80 = 0.125 µg/mL), which was more potent than lead
compound 3, sampangine, fluconazole and itraconazole. However, further extension
of the formyl group by α, β-unsaturated esters (16a and 16b) were unfavorable for the
antifungal activity. When the C8-methyl group was extended to ethyl (22b), propyl
(
22c) or butyl (22d) group, good antifungal activity was retained. Similarly,
movement of the C8-methyl group to C7 position was also tolerable and compound
2e was comparable to lead compound 3. Replacement of the C7-methyl group by
2
ethyl (22g), propyl (22h), butyl (22i), isopropyl (22j) and tert-butyl (22k) led to slight
decrease of the antifungal activity. However, when more bulky groups, such as phenyl
groups and heterocycles, were attached on the C7 (22q-t) or C8 (22l-p) position,
antifungal activity was decreased obviously. In addition, compounds 26a-m with
larger C8-amide substitutions were totally inactive. Interestingly, compound 22a with
C7 and C8 dimethyl substitution showed excellent activity against C. albicans (MIC80
=
0.25 µg/mL), C.parapsilosis (MIC = 0.5 µg/mL) C. neoformans (MIC = 0.5
80 80
µg/mL), and A. fumigatus (MIC80 = 1 µg/mL). It’s activity was comparable or
6

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superior to that of flconazole and lead compound 3.
On the basis of the above analysis, primary SAR can be obtained (Fig. 2). For the
thiophene substitution, C2-nitro group was important for the antifungal activity. Small
alkyl substitution is favored at both C7 and C8 position on the pyridine and the
methyl group performed the best. Also, di-methyl substitution on C7 and C8 yielded a
highly active compound 22a. In contrast, bulky groups at C7 or C8 position led
significant decrease or loss of the antifungal activity. The hydrogen bond donor seems
to be important for the C4 and C9 position and carbonyl group was better than
hydroxyl group.
2
.3 Compounds 22b and 22c Shows Selective and Remarkable Fungicidal Activity
Including Fluconazole-resistant Clinical Isolates
Compounds 22a, 22b, 22c and 22e showed excellent antifungal activity with a
broad spectrum, which were subjected for further biological evaluations. According to
the results of MIC in 24 h listed in Table 1, we further evaluated the antifungal
8
0
activity of compounds 22a-c and 22e against C. albicans in 48 h. The four candidates
showed an MIC80 value of 4 µg/mL, 0.25 µg/mL, 0.5 µg/mL, and 0.5 µg/mL,
respectively, which was comparable or superior to fluconazole (MIC = 0.5 µg/mL).
8
0
Because 0.5 µg/mL of compounds 22a and 22e couldn’t completely inhibited C.
albicans hyphal growth in the following yeast-to-hypha morphological transition
assay (Fig. S1 in Supporting Information), only compounds 22b and 22c were further
evaluated.
Due to severe drug resistance of fluconazole, clinically resistant C. albicans
7

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strains, such as 032, 1010, J38, 103 and chang7, were used to evaluate the potency of
compounds 22b and 22c to treat resistant fungal infections (Table 3). To our delight,
both of them exhibited excellent inhibitory activity against those clinical
drug-resistant C. albicans with MIC80 values in the range of 0.25 to 1 µg/mL.
Moreover, time-kill curve assay was further carried out to evaluate their fungicidal
effects. In accordance with the MIC results, both of them exhibited dramatic
fungicidal activity. More specifically, 0.8 µg/mL of compounds 22b or 22c showed
remarkable fungicidal effect on C. albicans (Fig. 3A and B, P < 0.001).
Compounds 22b and 22c were assayed for antibacterial and antitumor activity.
Both compounds were inactive against two strains of Staphylococcus aureus (MIC >
1
28 µg/mL) and the Capan-1 cancer cell line (IC > 100 µM), indicating that they
50
had selective antifungal activity.
2
.4 Compounds 22b and 22b Effectively Inhibited C. albicans Biofilm Formation
C. albicans biofilms usually lead to drug resistance and repeated infection in clinic
14]. Fluconazole can inhibit biofilm formation for only 60% at the concentration as
[
high as 1024 µg/mL [15], and antifungal agents with anti-biofilm activity are urgently
needed [16]. Thus, we further evaluated the anti-biofilm activity of compounds 22b
and 22c. Interestingly, compounds 22b and 22c exhibited remarkable anti-biofilm
activity (Fig. 4A and B). Compounds 22b (6.4 µg/mL) or 22c (12.8 µg/mL) disrupted
the formation of C. albicans biofilms thoroughly.
Scanning electron microscopy (SEM) assay was conducted to confirm the
anti-biofilm activity of compounds 22b and 22c. In contrast to the proliferated C.
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albicans biofilm in the control group, the biofilm structure was apparently disrupted
by compounds 22b and 22c. 0.8 µg/mL of compound 22b or 22c disrupted the
formation of C. albicans biofilms thoroughly (Fig. 5).
2
.5 Compounds 22b and 22c Inhibited Yeast-to-hypha Morphological Transition of C.
albicans
Adhesion and hyphal formation are both essential for C. albicans biofilm formation
14]. Initially, we investigated the inhibition effect of compounds 22a-c and 22e on
[
the yeast-to-hypha morphological transition of C. albicans (Fig. S1 in Supporting
Information). Compounds 22b and 22c both exhibited inhibitory effect at 0.25 µg/mL,
and completely inhibited hyphal growth at 0.5 µg/mL, whereas compounds 22a and
2
2e couldn’t completely inhibited hyphal growth at the high concentration of 0.5
µg/mL. Since cellular surface hydrophobicity (CSH) can indicate the adhesion ability
of C. albicans [17-19], we examined the effect of compounds 22b and 22c on CSH of
C. albicans. Both compounds could decrease the CSH of C. albicans to some extent
(Fig. 6A and 6B). Moreover, we investigated the effect of compounds 22b and 22c on
hyphal formation of C. albicans in different inducing media. Five hypha-inducing
media were used, including YPD containing 10% fetal bovine serum (FBS), Lee,
Spider, SLAD and RPMI 1640 medium. Among these media, YPD containing FBS is
the most potent hypha inducer [20]. In our study, C. albicans could form long hyphae
as expected in all the media tested. Both compounds 22b and 22c inhibited hyphal
formation in a dose-dependent manner. 0.4 µg/mL of compound 22b or 22c could
inhibit hyphal formation obviously in Lee, Spider, SLAD and RPMI 1640 medium
9

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(Fig.S2A and S2B in Supporting Information). 1.6 µg/mL of compound 22b or 22c
disrupted hyphal formation thoroughly, even in the most potent hypha inducer YPD
containing FBS (Fig. 7).
2
.6 Anti-biofilm Mechanism of Compounds 22b and 22c by Real-time RT-PCR
To better understand the anti-biofilm mechanism of compounds 22b and 22c, we
conducted real-time RT-PCR to investigate the expression of some important
biofilm-associated genes (Fig. 8) [21-23]. Hypha-specific genes ECE1 [24], HGC1
[
25], HWP1 [26], UME6 [27] were down-regulated after treatment with 0.4 µg/mL of
compound 22b or 22c. Adhesion-specific genes, ALS3 [28] and EAP1 [29], were also
down-regulated. Interestingly, some important regulation genes in RAS/MAPK or
RAS/cAMP pathways, including RAS1 [21], CYR1 [21, 30], EFG1 [31], CEK1 [32]
and CPH1 [33] were down-regulated too. Collectively, both 22b and 22c could
down-regulate biofilm-associated genes.
2
.7 Compounds 22b and 22c Shows Potent in vivo Antifungal Activity with Low
Toxicity
The in vivo anti-fungal activities of compounds 22b and 22c were investigated
using a rat vaginal infection model. Vulvovaginal candidiasis (VVC) has high
incidence (about 75%) in women, which is mainly caused by C. albicans [34]. On the
basis of the antifungal mechanism of compounds 22b and 22c, this model is ideal for
evaluating the in vivo antifungal potency because VVC infections rely on fungal
morphogenesis and biofilm formation [35]. Rats were intravaginally inoculated with
C. albicans to cause vaginal infection, and then treated with compound 22b, 22c or
1
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fluconazole as a control. Both compounds 22b and 22c effectively reduced fungal
burden after infection. More specifically, at 1 mg/kg, both compounds 22b and 22c
could significantly alleviate vaginal fungal burden (Fig. 9A, P < 0.0001), and the
effect of compound 22b was comparable to fluconazole. Moreover, we evaluated the
toxicity of compounds 22b and 22c using the model organism Caenorhabditis elegans
-1
(
C. elegans). Under different concentrations (6.25 to 100 µg mL ), no toxicity was
observed (Fig. 9B), indicating a promising prospect for clinical applications.
3
. Conclusions
The treatment of resistant IFIs remains a challenging task. On the basis of our
previously identified thieno[2,3-g]quinoline-4,9-dione antifungal scaffold, systemic
SARs were clarified and highly potent sampangine derivatives were identified.
Compounds 22b and 22c showed remarkable antifungal activity both in vitro and in
vivo. Especially, they were fungicidal against fluconazole-resistant C. albicans strains
and were proven to be potent biofilm inhibitors. The results highlighted the potential
of the sampangine derivatives to overcome fluconazole-related and biofilm-related
drug resistance. Further structural optimization, antifungal mechanism and therapeutic
potential of these simplified sampangine derivatives are in progress.
4
. Experimental section
4
.1. Chemistry
General Methods. Nuclear magnetic resonance (NMR) spectra were recorded on a
Bruker 400 spectrometer at room temperature, with tetramethylsilane (TMS) as an
internal standard. Chemical shifts ( ) are reported in parts per million (ppm), and
δ
1
1

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signals are reported as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or
br s (broad singlet). High-resolution mass spectrometry data were collected on a
Kratos Concept mass spectrometer. TLC analysis was carried out on silica gel plates
GF254 (Qingdao Haiyang Chemical, China). Silica gel column chromatography was
performed with Silica gel 60 G (Qingdao Haiyang Chemical, China). Unless
otherwise noted, all materials were obtained from commercial suppliers and used
without further purification. Chemical purities were analyzed by HPLC using
MeOH/H O as the mobile phase with a flow rate of 0.6 mL/min on a C18 column. All
2
final compounds exhibited the purity greater than 95%.
Starting from 6,7-dihydrobenzo[b]thiophen-4(5H)-one (4), intermediates 8 and 20
were prepared via four steps according to our previously reported methods [12, 13].
4
.1.1. 8-Methyl-3-nitrothieno[2,3-g]quinoline-4,9-dione (9)
Compound 8 (229 mg, 1 mmol) was solved in con. H SO (2 mL). After cooling to
2
4
0
ºC, KNO (150mg, 1.5 mol, 1.5 eq) was added in portion and the mixture was
3
stirred at 0 ºC for 1.5 h, and then was poured into water (20 mL) and neutralized with
K CO to pH 8–9. The formed precipitate was filtered off, dried, and purified by
2
3
prep-HPLC to give 3 (140 mg, yield 51%) and 9 (93 mg, yield 34%) as yellow
crystals. Preparative HPLC was carried out using a C18 reverse phase column (150 ×
3
0 mm, 5um; Boston Green ODS) with a linear gradient of 0–100% CH CN in 0.05%
3
aqueous HCl over 15 min at 40 °C at a flow rate of 10.0 mL/min. The eluents were
1
collected at RT = 9.5 min for 3 and at RT = 12.0 min for 9. H NMR (400 MHz,
DMSO-d6) δ: 8.99 (s, 1 H), 8.87 (d, J = 4.8 Hz, 1 H), 7.72 (d, J = 4.8 Hz, 1 H), 2.78 (s,
1
2

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1
3
3
1
H). MS (ESI) m/z: 275.2 (M+1). C NMR (100 MHz, DMSO-d ) δ: 177.90, 173.65,
6
52.45, 150.10, 147.10, 144.70, 134.25, 131.80, 131.00, 128.15. HRMS (ESI) m/z:
+
calcd for C H N O S [M+H] 275.0048, Found 275.0125. MS (ESI) m/z: 274.8
1
2
7
2
4
(M+H).
4
.1.2. 2-Amino-8-methylthieno[2,3-g]quinoline-4,9-dione (10a)
To a stirred solution of 3 (28 mg, 0.1 mmol) in MeOH and DMF (5 mL, v/v=3/1),
NiCl ⋅6H O (47 mg, 2 eq) and NaBH (8 mg, 2 eq) were added at 0ºC. The resulting
2
2
4
black suspension was stirred at 0 ºC for 5 min. The reaction was quenched with
saturated aqueous NH Cl (5 mL), diluted with H O (5 mL) and extracted with CH Cl
2
4
2
2
(
3 × 10 mL). The combined organic layers were washed with H O (5 mL), dried over
2
Na SO , filtered and concentrated under reduced pressure to afford the crude product,
2
4
which was purified by flash column chromatography eluting with CH Cl / MeOH
2
2
1
(
90:10) to give 10a as a brown powder (7.5 mg. yield 32.2%). H NMR (400 MHz,
DMSO-d6) : 8.70 (d, J = 4.80 Hz, 1H), 7.57 (d, J = 4.80 Hz, 1H), 7.55 (br s, 2H),
δ
1
3
6
1
.34 (s, 1H), 2.75 (s, 3H). C NMR (100 MHz, DMSO-d ) δ: 177.25, 176.57, 165.90,
6
51.14, 150.08, 148.83, 143.28, 131.75, 129.40, 127.50, 101.61, 22.36. HRMS (ESI)
+
m/z: calcd for C H N O S [M+H] 245.0306, Found 245.2208. MS (ESI) m/z: 245.2
1
2
9
2
4
(M+H). Chemical synthesis of compound 10b was similar to that of compound 10a.
4
.1.3. 3-Amino-8-methylthieno[2,3-g]quinoline-4,9-dione (10b)
1
Yellow powder (5.5 mg, yield 20.2%). H NMR (400 MHz, DMSO-d )
δ
: 8.84 (d, J
6
1
3
=
4.40 Hz, 1H), 7.72 (d, J = 4.40 Hz, 1H), 6.93 (s, 1H), 2.81 (s, 3H). C NMR (100
: 178.82, 177.76, 162.11, 151.85, 149.86, 148.40, 143.63, 131.34,
MHz, DMSO-d6)
δ
1
3

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+
1
2
4
29.06, 127.70, 109.91, 17.17. HRMS (ESI) m/z: calcd for C H N O S [M+H]
1
2
9
2
4
45.0306, Found 245.2208. MS (ESI) m/z: 245.2 (M+H).
.1.4 8-Ethyl-2-nitrothirno[2,3-g]quinoline-4,9-dione(22b)
A solution of 5-bromo-2-nitrobenzo[b]thiophene-4,7-dione (20, 0.418 g, 1.45 mmol)
in absolute ethanol (10 mL) was stirred at 0℃ . solution of
E)-1,1-dimethyl-2-((E)-pent-2-en-1-ylidene)hydrazine (21b, 0.366 g, 2.90 mmol) in
A
(
absolute ethanol (5 mL) was added dropwise. Then, sodium bicarbonate (425 mg, 5.0
mmol) was added in one portion. Then the resulting mixture was stirred at 45℃ for 4 h.
The cooled mixture was concentrated in vacuo. The crude product was purified by
silica gel chromatography (hexane: EtOAc = 8: 1) to yield the compound 22b as a
1
green solid (0.13 g, 31.2%). H NMR (400 MHz, CDCl )
δ
: 8.96 (d, J = 5.00 Hz, 1H),
3
8
.44 (s, 1H), 7.58 (d, J = 5.00 Hz, 1H), 3.34 (q, J = 7.36 Hz, 2H), 1.31-1.40 (m, 3H).
1
3
C NMR (100 MHz, CDCl3) δ: 179.2, 176.15, 158.20, 154.60, 151.85, 150.45,
1
48.90, 139.30, 129.45, 128.55, 126.15, 27.60, 13.85. HRMS (ESI) m/z: calcd for
+
C H N O S [M+H] 289.0205, Found 289.0282.
13
9
2
4
Chemical synthesis of compounds 22a-u was similar to that of compound 22b. For
the synthesis of compounds 22l-p, toluene was used as the solvent instead of absolute
ethanol, and the temperature and reaction time were increased to 110 ºC and 16-24 h,
respectively.
4
.1.5. 7,8-Dimethyl-2-nitrothieno[2,3-g]quinoline-4,9-dione(22a)
1
Yellow solid (15 mg, yield 28.1%). H NMR (400 MHz, CDCl )
δ
: 8.82 (s, 1H),
: 179.5, 176.2,
3
1
3
8
.43 (s, 1H), 2.83 (s, 3H), 2.50 (s, 3H). C NMR (100 MHz, CDCl )
δ
3
1
4

ACCEPTED MANUSCRIPT
1
57.0, 154.6, 149.5, 149.0, 148.2, 139.0, 138.8, 128.5, 125.5, 18.0, 17.5. HRMS (ESI)
+
m/z: calcd for C H N O S [M+H] 289.0205, Found 289.0282. MS (ESI) m/z: 288.99
1
3
9
2
4
(M+H).
4
.1.6. 2-Nitro-8–propylthieno[2,3-g]quinoline-4,9-dione (22c)
1
Greyish green solid (166 mg, yield 32.4%). H NMR (400 MHz, CDCl )
δ
: 8.94 (d,
3
J = 5.04 Hz, 1H), 8.41-8.46 (m, 1H), 7.53 (d, J = 4.76 Hz, 1H), 3.22-3.31 (m, 2H),
1
3
1
.68-1.75 (m, 2H), 1.09 (t, J = 7.40 Hz, 3H). C NMR (100 MHz, CDCl3) δ: 179.07,
1
76.08, 157.16, 156.43, 153.75, 150.37, 148.93, 139.40, 130.37, 128.42, 125.84,
+
3
6.58, 23.45, 14.21. HRMS (ESI) m/z: calcd for C H N O S [M+H] 303.0361,
1
4
11
2
4
Found 303.0438. MS (ESI) m/z: 303.00 (M+H).
4
.1.7. 8-Buthy-2-nitrothie[2,3-g]quinoline-4,9-dione(22d)
1
Gray solid (12 mg, yield 20.0%). H NMR (400 MHz, DMSO-d )
δ
: 8.91 (d, J =
6
4
.88 Hz, 1 H), 8.50 (s, 1 H), 7.74 (d, J = 4.88 Hz, 1 H), 3.19-3.24 (m, 2 H), 1.59 (q, J
1
3
=
7.56 Hz, 2 H), 1.43 (t, J = 7.34 Hz, 2 H), 0.95 (t, J = 7.28 Hz, 3 H). C NMR (100
: 178.99, 176.01, 157.04, 156.81, 153.67, 150.25, 148.92, 139.34,
30.38, 128.34, 125.81, 34.43, 32.27, 22.88, 13.83. HRMS (ESI) m/z: calcd for
MHz, CDCl3)
δ
1
+
C H N O S [M+H] 317.0518, Found 317.2461.
15
13
2
4
4
.1.8. 7-Methyl-2-nitrothieno[2,3-g]quinoline-4,9-dione(22e)
1
Yellow solid (40 mg, yield 32.4%). H NMR (400 MHz, CDCl )
δ
: 8.95 (s, 1H),
: 176.95,
3
1
3
8
1
1
.48 (s, 1H), 8.37 (s, 1H), 2.60 (s, 3H). C NMR (100 MHz, CDCl3)
δ
75.57, 155.52, 146.05, 145.98, 140.43, 138.80, 137.92, 134.49, 129.69, 125.74,
+
8.45. HRMS (ESI) m/z: calcd for C H N O S [M+H] 275.0048, Found 275.0125.
1
2
7
2
4
1
5

ACCEPTED MANUSCRIPT
MS (ESI) m/z: 274.96 (M+H).
4
.1.9. 7-Ethyl-2-nitrothieno[2,3-g]quinoline-4,9-dione (22g)
1
Brown solid (32 mg, yield 65.2%). H NMR (400 MHz, CDCl )
δ
: 8.96 (s, 1H),
3
1
3
8
.49 (s, 1H), 8.39 (s, 1H), 2.90 (q, J = 7.52 Hz, 2H), 1.39 (t, J = 7.64 Hz, 3H). C
: 177.47, 176.05, 157.38, 155.41, 146.63, 146.60, 145.13,
40.92, 133.77, 130.38, 126.21, 26.32, 14.59. HRMS (ESI) m/z: calcd for
NMR (100 MHz, CDCl3)
δ
1
+
C H N O S [M+H] 289.0205, Found 289.0282.
13
9
2
4
4
.1.10. 2-Nitro-7-propylthieno[2,3-g]quinoline-4,9-dione (22h)
1
Gray solid (23 mg, yield 50.5%). H NMR (400 MHz, CD OD)
δ
: 8.90 (s, 1H), 8.51
3
(s, 1 H), 8.48 (s, 1H), 2.89 (t, J = 7.60 Hz, 2H), 1.77-1.83 (m, 2H), 1.04 (t, J = 7.2 Hz,
1
3
3
H). C NMR (100 MHz, CD OD) δ: 171.28, 169.01, 154.40, 151.76, 147.98, 146.94,
3
1
46.54, 144.04, 134.23, 128.65, 125.62, 34.44, 23.56, 12.47. HRMS (ESI) m/z: calcd
+
for C H N O S [M+H] 303.0361, Found 303.0438.
1
4
11
2
4
4
.1.11. 7-Butyl-2-nitrothieno[2,3-g]quinoline-4,9-dione (22i)
1
Gray solid (30 mg, yield 75.1%). H NMR (400 MHz, CDCl )
δ
: 8.96 (s, 1 H), 8.51
3
(s, 1H), 8.39 (s, 1 H), 2.87 (t, J = 7.60 Hz, 2H), 1.74 (q, J = 7.60 Hz, 2H), 1.39-1.48
1
3
(
m, 2H), 1.00 (t, J = 7.40 Hz, 3 H). C NMR (100 MHz, CDCl )
δ
: 176.83, 175.59,
3
1
2
3
4
56.45, 153.85, 146.43, 145.98, 143.79, 140.31, 133.66, 130.08, 125.10, 32.06, 31.72,
+
1.38, 12.21. HRMS (ESI) m/z: calcd for C H N O S [M+H] 317.0518, Found
1
5
13
2
4
17.2461.
.1.12. 7-Isopropyl-2-nitrothieno[2,3-g]quinoline-4,9-dione (22j)
1
Deep yellow solid (20 mg, 38.1%). H NMR (400 M Hz , CDCl )
δ
: 9.00 (d, J =
3
1
6

ACCEPTED MANUSCRIPT
2
1
1
.00 Hz, 1H), 8.49 (s, 1H), 8.42 (d, J = 2.00 Hz, 1H), 3.21 (td, J = 6.80, 13.9 Hz, 1H),
1
3
.41 (d, J = 7.00 Hz, 6H). C NMR (100 MHz, CDCl ) δ: 177.53, 176.05, 157.41,
3
54.68, 149.61, 146.76, 146.62, 140.93, 132.34, 130.49, 126.23, 32.16, 23.22. HRMS
+
(
ESI) m/z: calcd for C H N O S [M+H] 303.0361, Found 303.0438. MS (ESI) m/z:
14
11
2
4
3
4
02.99 (M+H).
.1.13. 7-(Tert-Butyl)-2-nitrothieno[2,3-g]quinoline-4,9-dione (22k)
1
Gray solid (18 mg, yield 55.2%). H NMR (400 MHz, CDCl )
δ
: 9.15 (d, J = 2.00
3
1
3
Hz, 1H), 8.51 (d, J = 2.00 Hz, 1H), 8.47 (s, 1H), 1.47 (s, 9H). C NMR (100 M Hz,
CDCl3)
δ
: 177.61, 176.05, 157.40, 153.54, 151.93, 146.69, 146.29, 140.94, 131.58,
+
1
3
4
30.05, 126.21, 34.66, 30.65. HRMS (ESI) m/z: calcd for C H N O S [M+H]
15 13 2 4
17.0518, Found 317.2461.
.1.14. 2-Nitro-8-(2-nitrophenyl)thieno[2,3-g]quinoline-4,9-dione (22m)
1
Gray solid (25 mg, yield 25.0 %). H NMR (400 MHz, CDCl )
δ
: 9.17 (d, J = 4.76
3
Hz, 1H), 8.49 (s, 1H), 8.39 (d, J = 8.28 Hz, 1H), 7.70-7.85 (m, 2H), 7.51 (d, J = 4.76
1
3
Hz, 1H), 7.30 (d, J = 1.24 Hz, 1H). C NMR (100 MHz, CDCl )
δ
: 177.64, 175.64,
57.50, 154.60, 149.78, 149.35, 147.48, 146.66, 140.04, 134.19, 133.99, 129.87,
29.75, 128.48, 125.93, 125.02, 100.05. HRMS (ESI) m/z: calcd for C H N O S
3
1
1
17
8
3
6
+
[
M+H] 382.0056, Found 382.2004.
4
.1.15. 8-(2-Methoxyphenyl)-2-nitrothieno[2,3-g]quindine-4,9-dione (22p)
1
Yellow solid (32.7 mg, yield 41.0%). H NMR (400 MHz, DMSO-d )
δ
: 8.98 (d, J
6
=
4.76 Hz, 1H), 8.46 (s, 1H), 7.59 (d, J = 4.76 Hz, 1H), 7.39 (t, J = 7.16 Hz, 1H),
1
3
7
.17 (d, J = 6.28 Hz, 1H), 6.97-7.08 (m, 2H), 3.57 (s, 3H). C NMR (100 MHz,
1
7

ACCEPTED MANUSCRIPT
DMSO-d6)
δ
: 177.2, 176.8, 157.2, 156.3, 154.2, 148.8, 148.5, 139.8, 130.3, 130.2,
1
28.5, 128.3, 127.0, 126.0, 121.2, 110.4, 55.2. HRMS (ESI) m/z: calcd for
+
C H N O S [M+H] 367.0310, Found 367.2297.
18
11
2
5
4
.2. Pharmacology
4
.2.1. Strains, culture and agents
Strains were routinely incubated in YPD (1% yeast extract, 2% peptone and 2%
dextrose) at 30 ℃ in a shaking incubator. All clinical strains are provided by
Changhai Hospital of Shanghai, China. All compounds were dissolved in DMSO at
6
.4 mg/mL as stock solutions. YPD+10 % (vol/ vol) Fetal Bovine Serum (FBS) [36],
Spider medium [37], Lee's medium (pH = 6.8) [36], Synthetic Low-Ammonium
Dextrose (SLAD) medium [38] and RPMI 1640 medium [39] are included as well.
Fluconazole was purchased from Pfizer Inc.
4
.2.2. Antifungal susceptibility testing
The test was performed as described previously with a few modifications [39]. The
3
initial concentration of fungal suspension in RPMI 1640 was 5×10 CFU/mL.
Different concentrations of compounds were added into the fungal suspension in
9
6-well plates and incubated at 35 ℃ for 24 h. The optical density at 600 nm was
detected by the spectrophotometer to indicate the inhibitory of the growth of C.
albicans. The MIC80 value is the lowest concentration of the group which the
compound inhibited the growth by 80 %. Each compound was tested in triplicate.
4
.2.3. Time-kill curve assay
C. albicans grown overnight was washed with PBS and resuspended in RPMI 1640.
1
8

ACCEPTED MANUSCRIPT
6
Then the suspension was diluted to 1×10 cells/mL, and treated with different
concentrations of compounds. The cells grew in a shaking incubator (200 rpm) at
3
0 ℃. CFUs were calculated at predetermined time points by plating suspension
dilutions on YPD agar after 48 hours’ incubation at 30 ℃. The assay was performed
for three times independently.
4
.2.4. Scanning Electron Microscopy
C. albicans cells were inoculated on glass disks coated with poly-L-lysine
hydrobromide (Sigma) and incubated statically at 37°C for 90 min for adhesion. Then
removed non-adherent cells and the disks were incubated in fresh RPMI 1640
medium with compound treatment at 37°C for 24 h. Then the supernatant was
removed and the biofilms were immersed in a fixative consisting of 2% (v/v)
glutaraldehyde in 0.15 M sodium cacodylate buffer (pH 7.2) for 2 h. The samples
were dehydrated in 30%, 50%, 70%, 90%, and 100% ethanol solution in turn, and
dried for 1 h. Then they were coated with gold and observed through a SEM (ZEISS,
MA10).
4
.2.5. Cellular surface hydrophobicity assay
C. albicans CSH was measured as described previously with a few modification
[
23, 40, 41]. Briefly, the C. albicans biofilms treated with compounds for 24 h were
removed from the plate surface to obtain a cell suspension in PBS (OD600 = 1.0). Then
.2 ml suspension and 0.3 ml octane was mixed by vortexing for 3 min. OD600 of the
1
aqueous phase was determined after the mixture staying at room temperature
statically for another 3 min for phase separation. OD600 for the group without the
1
9

ACCEPTED MANUSCRIPT
octane overlay was used as the control. Three replicates were performed for each
group. Relative hydrophobicity was obtained as [(OD600 of control - OD600 after
octane overlay)/ OD600 of the control] × 100%
4
.2.6. In vivo antifungal activity evaluation using a rat-vaginal model.
A rat vaginal model was used to demonstrate the antifungal effect of compounds in
vivo.[42] Briefly, female Oophorectomized Sprague-Dawley rats (Shanghai SLAC
Laboratory Animal Center, China) were injected subcutaneously with oestradiol
benzoate 0.5 mg (Hangzhou animal medicine factory, China) on day 2, 4, 6 and 8
7
after ovarian surgery. The intravaginal inoculation with 0.1mL of 1×10 CFU/mL C.
albicans suspension was conducted on day 10, 11 and 12 after ovarian surgery. The
suspension was delivered into the vaginal cavity through an inoculator. Compound
2
2B, 22C (dissovled in saline with 1% DMSO at 2, 4, and 8 mg/mL) and fluconazole
(dissolved in saline at 2 mg/mL, served as positive control) was delivered
intravaginally on day 1, 2 and 3 after inoculation. 100 µL vaginal fluid was picked
and cultured on the SDA agar containing chloramphenicol on the fourth day after
inoculation. The CFU was determined then. The animal experiments were approved
by the Animal Ethics Committee of the Second Military Medical University
(Shanghai, China). During the study, rats were observed and recorded at least two
times a day, measures were taken to ensure that the sorrow of animals were
minimized.
4
.2.7. Toxicity evaluation using C. elegans worms
C. elegans glp-4; sek-1 adult nematodes were used to assess the toxicity of
2
0

ACCEPTED MANUSCRIPT
compounds [22]. Briefly, the nematodes were transferred to sterile liquid medium
containing 6.25, 12.5, 25, 50 and 100 µg/mL compounds as treatment groups or
DMSO as control group. The worms were incubated at 25 ℃ for 2 d and observed
daily.
4
.2.8. Real-time RT-PCR assay
C. albicans biofilm was formed in 150 mm×25 mm cell culture dishes treated with
.4 µg/mL compounds or DMSO as the control for 1h. C. albicans cells were then
0
collected and total RNA was isolated using Fungal RNA-out kit (TIANDS, China).
Real-time quantitive PCR was performed with SYBR Green I (RR420A, TaKaRa
Biotechnology, China) using ABI 7500 Real-Time PCR system (Applied Biosystems
Co, USA). Primers are shown in Table S1. 18s rRNA was used as endogenous
reference. The relative expression of each target gene was calibrated against the
corresponding expression by control group (expression = 1). Triplicate independent
experiments were conducted to generate a mean value [23].
Acknowledgements
This work was supported by the National Natural Science Foundation of China
(
Grant 21502224 to N. L., 81573283 to C. S.), Science and Technology
Commission of Shanghai Municipality (Grant 17XD1404700), and the
Shanghai “ShuGuang” Project (Grant 14SG33).
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Figure Captions
2
5

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Fig. 1. Scaffold hopping and structural simplification of antifungal natural product
sampangine.
Fig. 2. Summary of SAR of thieno[2,3-g]quinoline-4,9-dione antifungal scaffold.
Fig. 3. Compounds 22b and 22c exhibit fungicidal activity. (A and B) Time-kill curve
of different concentrations of compounds 22b and 22c on standard C. albicans strain
SC5314.
Fig. 4. The anti-biofilm activity of different concentrations of compounds 22b and
2
2c on C. albicans biofilms. The results were presented as a percentage compared to
the control group without drug treatment. Biofilm formation results represent the
mean ± standard deviation from five independent experiments. ** P < 0.01 compared
with the treatment-free control group, *** P < 0.001.
Fig. 5. Effects of compounds 22b and 22c on C. albicans biofilm formation. The
SEM image indicated the anti-biofilm activity of different concentrations of
compound 22b (A) and 22c (B). Images in the dashed boxes are zoomed and shown
on the right.
Fig. 6. Compounds 22b and 22c reduced the CSH of C. albicans. CSH results
represent the mean ± standard deviation from three independent experiments. * P <
0
.05 compared with the treatment-free control group, ** P < 0.01, *** P < 0.001.
Fig. 7. Effect of different concentrations of compound 22b and 22c on the yeast-to-hypha
morphological transition of C. albicans in YPD containing FBS medium. The cellular morphology
was photographed after incubation at 37°C for 3 h.
Fig. 8. Gene expression changes of some important biofilm formation related genes.
-1
The concentration of compounds 22b and 22c was 0.4 µg mL . Gene expression was
2
6

ACCEPTED MANUSCRIPT
indicated as a fold change relative to that of the control group (C. albicans SC5314
without drug treatment). 18S rRNA was used to normalize the expression data. The
data shown are mean ± standard deviation from three independent experiments.
Fig. 9. Compounds 22b and 22c exhibit in vivo anti-fungal activity and low toxicity.
(A) Compounds 22b and 22c are effective to treat vaginal infection of rats. (B)
Compounds 22b and 22c show no toxicity to C. elegans nematodes. C. elegans
nematodes were pipetted into medium containing different concentrations of
compounds and observed daily. On day 2, the worms were photographed.
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ACCEPTED MANUSCRIPT
Table 1 In vitro antifungal activity of the simplified sampangine derivatives
a
(
MIC , µg/mL, 24 h)
80
C. alb.
C. par. C. gla. C. neo. A. fum.
T. rub
M. gyp
Compounds
SC5314 22019
537
4
32609
4
07544
>64
Cmccftla Cmccfmza
4
>64
>64
4
8
8
O
N
H2N
S
>64
>64
>64
>64
2
>64
>64
>64
4
>64
>64
>64
16
>64
O
1
0a
3
2
>64
>64
8
>64
>64
2
>64
>64
8
OH
OH
N
O N
2
S
>64
1
1a (trans)
OH
N
O N
2
S
2
OH
11b (cis)
N
O N
2
S
S
>64
8
>64
32
16
64
>64
4
>64
>64
4
8
>64
4
1
2
O
O
N
O N
2
O
13
32
32
16
32
64
32
16
2
8