Design, synthesis and biological evaluation of novel 3,4-dihydro-2(1H)-quinolinone derivatives as potential chitin synthase inhibitors and antifungal agents

Article abstract of DOI:10.1016/j.ejmech.2020.112278

A series of 3,4-dihydro-2(1H)-quinolinone derivatives contained butenediamide fragment were designed and synthesized. Their inhibition potency against chitin synthase and antimicrobial activities were screened in vitro. The enzymatic assays showed that all the synthesized compounds had inhibition potency against chitin synthase at concentration of 300 μg/mL. Compound 2d displayed excellent potency with inhibition percentage (IP) value of 82.3%, while IP value of the control polyoxin B was 87.5%. Compounds 2b, 2e and 2s whose IP values were above 70% showed good inhibition potency against chitin synthase. Moreover, the IC₅₀ value of 2b was comparable with that of polyoxin B (0.09 mM). The K_i of compound 2b was 0.12 mM and the result from Lineweaver-Burk plot showed that 2b was non-competitive inhibitor to bind chitin synthase. The antifungal experiment showed that these compounds had excellent antifungal activity against fungal strains, especially for candida albicans. The antifungal activities against C .albicans of compounds 2b, 2d, 2e and 2l were comparable with that of fluconazole and were superior to that of polyoxin B. Meanwhile, the other compounds against C. albicans showed better antifungal activity (MIC 2 μg/mL) than polyoxin B except for compound 2n (MIC 4 μg/mL). The trial of drug combination use showed that these synthesized compounds had synergistic effects with fluconazole and polyoxin B. It indicated that these compounds were not competing with polyoxin B to bind with chitin synthase, which was also consistence with the result of enzymatic assays. The antibacterial experiment showed that these compounds had no activity against selected strains including three Gram-positive and three Gram-negative bacteria. These results showed that the designed compounds were chitin synthase inhibitors and had selective antifungal activity.

Full text of DOI:10.1016/j.ejmech.2020.112278

European Journal of Medicinal Chemistry 195 (2020) 112278
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and biological evaluation of novel 3,4-dihydro-
2(1H)-quinolinone derivatives as potential chitin synthase inhibitors
and antifungal agents
Baihui Li, Yangli Shen, Hu Wu, Xiaobo Wu, Lvjiang Yuan, Qinggang Ji*
School of Chemistry and Chemical Engineering, Southwest University, Chongqing, 400715, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3,4-dihydro-2(1H)-quinolinone derivatives contained butenediamide fragment were designed
and synthesized. Their inhibition potency against chitin synthase and antimicrobial activities were
screened in vitro. The enzymatic assays showed that all the synthesized compounds had inhibition po-
Received 10 January 2020
Received in revised form
25 March 2020
Accepted 25 March 2020
Available online 4 April 2020
tency against chitin synthase at concentration of 300 mg/mL. Compound 2d displayed excellent potency
with inhibition percentage (IP) value of 82.3%, while IP value of the control polyoxin B was 87.5%.
Compounds 2b, 2e and 2s whose IP values were above 70% showed good inhibition potency against
chitin synthase. Moreover, the IC50 value of 2b was comparable with that of polyoxin B (0.09 mM). The Ki
of compound 2b was 0.12 mM and the result from Lineweaver-Burk plot showed that 2b was non-
competitive inhibitor to bind chitin synthase. The antifungal experiment showed that these com-
pounds had excellent antifungal activity against fungal strains, especially for candida albicans. The
antifungal activities against C .albicans of compounds 2b, 2d, 2e and 2l were comparable with that of
fluconazole and were superior to that of polyoxin B. Meanwhile, the other compounds against C. albicans
Keywords:
Quinolinone
3,4-Dihydro-2(1H)-Quinolinone
Chitin synthase inhibitor
Antifungal activity
showed better antifungal activity (MIC 2 mg/mL) than polyoxin B except for compound 2n (MIC 4 mg/mL).
The trial of drug combination use showed that these synthesized compounds had synergistic effects with
fluconazole and polyoxin B. It indicated that these compounds were not competing with polyoxin B to
bind with chitin synthase, which was also consistence with the result of enzymatic assays. The anti-
bacterial experiment showed that these compounds had no activity against selected strains including
three Gram-positive and three Gram-negative bacteria. These results showed that the designed com-
pounds were chitin synthase inhibitors and had selective antifungal activity.
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
urgent need to explore novel antifungal agents to overcome these
disadvantages.
Fungal infection which range from non-fatal skin and mucous
membrane disease to invasive infection involving many organs [1]
is a significant worldwide health problem [2]. More than a billion
people are affected by fungal infections [3]. In addition, the
morbidity and mortality are very high due to invasive fungal
infection for immunosuppressive patients [4,5]. The main anti-
fungal drugs used in clinic include echinocandins, polyenes, azoles,
pyrimidines and acrylamides [6]. However, these drugs are
hampered notoriously with potential toxicity, adverse effects and
the increasing drug resistance of the fungi [7]. Therefore, it is an
Chitin synthase (CHS) is a fundamental enzyme in the biosyn-
thesis of chitin which is a polymer of N-acetylglucosamine [8]
connected by
b-(1, 4) glycosidic bonds. Chitin is the scaffold
structure of fungal cell walls and absent in mammalian cells [9].
Chitin synthase inhibitors can cause abnormality of cell wall
structure, resulting in cell rupture and death [10]. So, chitin syn-
thase is a safe and highly selective target to design new antifungal
drugs [11]. Nikkomycins and polyoxins which are two natural-
occupying substances possess inhibition potency against chitin
synthase owning to their structures similar to that of UDP-GlcNAc
which is the substrate of chitin synthase (Fig. 1) [12].
However, despite excellent in vitro results, the weak efficacy and
high inhibitory concentrations resulting from poor cellular uptake
and metabolic instability compromise these natural compounds in
* Corresponding author.
E-mail address: jiqing@swu.edu.cn (Q. Ji).
https://doi.org/10.1016/j.ejmech.2020.112278
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
B. Li et al. / European Journal of Medicinal Chemistry 195 (2020) 112278
Fig. 1. Structural comparision of Polyoxins, Nikkomycins and UDP-GlcNAc.
clinical utility [13]. Nikkomycin Z is currently the only chitin syn-
thase inhibitor that has undergone clinical trials for curing Valley
Fever [14].Therefore, many efforts have been devoted to the syn-
thesis of analogs to improve the pharmacokinetic properties of
these compounds since nikkomycins and polyoxins were discov-
ered [15,16]. These efforts have no progress in finding analogs
befitting to develop as drug candidates, however, these in-
vestigations extended our knowledge of chitin synthase and its
binding mode. It also provides information that it is necessary to
consider the mechanism of action of chitin synthase to develop
new chitin synthase inhibitors.
The quinolinone scaffold is seen as a privileged heterocycle-
fused structure [17] and its derivatives exhibited numerous amus-
ingly biological activities such as antipsychotic, antiallergic, anti-
histaminic or anti-inflammatory [18e20]. These broad biological
activities have been a great concern in the field of pharmaceutical
chemistry, thus numerous synthetic quinolinone antibacterial
agents have been developed and various diseases have been treated
with these drugs in clinical trials [21], for example, fluo-
roquinolones are widely used as antibacterial drugs (Fig. 2). 3-
Amino-3,4-dihydro-quinolin-2(1H)-one is an interesting core of
quinolinones. The amino substituent makes it coordination better
and selective with metal ions, and it also be used as an inlay of
biological macromolecules and fluorescent probes [22,23]. It is an
essential scaffold of many natural products and synthetic products
which have a wide range of biological activities. As shown in Fig. 2,
compounds I and IV are potent glycogen phosphorylase inhibitors,
and compounds II are inhibitors of angiotensin converting enzyme,
and compound III can exert a radioprotective effect [24e27].
a,b-Unsaturated carbonyl fragment which can bind with the
DNA, RNA, or enzymes through Michael addition reaction is func-
tional group of many natural products [28]. Precisely because it
contains such a special structure, chalcone has excellent biological
activity and clinical potential. For example, licochalcone possessed
the activity of anticancer and anti-inflammation, while iso-
bavachalcone exerted antifungal activity (Fig. 3) [29e33].
Aromatic ring can generate rigid scaffold and could bind with
sulfur, hydrogen or oxygen atoms of DNA or enzyme through
p
interactions, so it is the most frequently encountered ring system in
small molecule drugs. Moreover, various substituents on the aro-
matic rings can adjust the hydrophilic lipophilic coefficient of
molecules, which is very important for the absorption and
Fig. 2. Representative quinolinones.

B. Li et al. / European Journal of Medicinal Chemistry 195 (2020) 112278
3
Fig. 3. Representative chalcones and the designed compounds.
transportation of drugs [34]. Consideration of the mechanism of
action of chitin synthase, the -unsaturated carbonyl was hy-
The results showed that all compounds exhibited moderate to
excellent inhibitory to CHS. Compound 2d exhibited the highest
inhibitory potency among all compounds with the inhibition per-
centage value of 82.3% which was approximately equal to that of
polyoxin B (P.B whose value is 87.5%). Moreover, compounds 2b, 2e
and 2s showed significant inhibition against CHS with IP over 70%.
The compounds 2a, 2h and 2p exhibited moderated potency whose
IP ranged from 60% to 70% (Inhibition percentage ¼ 67.3%, 65.2%
and 66.5%, respectively).
In this series of compounds, the structural difference is the type
and the position of the substituents on the aromatic benzene ring.
The compounds possessing methyl or the methoxyl substituents had
excellent IP values, such as 2b, 2d, 2e and 2s. The nitro-substituted
compounds 2f, and 2g showed moderate potency, while the para-
substitution compound 2h showed good potency with IP value of
65.2%. The most of the halo-substituted compounds revealed poor to
moderate potency except for the difluoro-substituted compound 2p
which exerted good potency with IP value of 66.5%.
a,b
bridized with 3-amino-3,4-dihydro-quinolin-2(1H)-one and
substituted anilines to form a novel series of quinlinone derivatives
(Fig. 3). Two amide fragments were introduced into this series
compounds. It is well known that amide is a hydrophilic pharma-
cophore and it is employed in clinical drug extensively to improve
bioavailability [35]. We expected that this series of compounds
should have good CHS inhibitory activity and excellent antifungal
activity. Herein, we reported the synthesis, biological evaluation of
this series of novel 3-amino-3,4-dihydroquinoline-2(1H)-one
derivatives.
2. Result and discussion
2.1. Chemistry
The 3-amino-3,4-dihydroquinolin-2(1H)-one is rarely commer-
cially available in market, but it can be produced from an easily
obtained compound o-nitrotoluene. o-Nitrotoluene reacted with
NBS in carbon tetrachloride to produce 1-(bromomethyl)-2-
nitrobenzene, and the product combined with diethyl acetylamino-
malonate under strong alkaline condition to afford diethyl 2-(2-
nitrobenzyl)-2-acetamidomalonate which then was decarboxyl-
ation in acidic solution at 100 ꢀC to produce compound 2-amino-3-
(2-nitrophenyl) propanoic acid hydrochloride which was esterified
with methanol at presence of sulfoxide chloride. The obtained
methyl 2-amino-3-(2-nitrophenyl) propanoate hydrochloride was
reduced by iron powder to offer the desired compound 3-amino-3,4-
dihydroquinolin-2 (1H)-one with good yield (see supplementary
material).
The compounds 3-(arylcarbamoyl) acrylic acid 1a-s were pre-
pared by dissolving maleic anhydride and various substituted ani-
line in dichloromethane and stirring at room temperature. The
compounds 1a-s combined with 3-amino-3,4-dihydroquinolin-2
(1H)-one by catalyzation of triethyl phosphite and iodine to pro-
duce the target compounds (see Scheme 1).
To further evaluate the inhibitory effects of these compounds,
the IC50 (semi-inhibitory concentration) values of compounds
whose inhibition percentage (IP) were above 50% were measured.
(Fig. 4). The results showed that the IC50 values of these test com-
pounds were distributed between 0.09 and 0.40 mM. Among them,
compound 2b had an IC50 value of 0.09 mM, which had similar
potency to polyoxin B (IC50 ¼ 0.09 mM). The IC50 values of the
compounds 2d, 2s were 0.16, 0.19 mM respectively, exhibiting
moderate potency against CHS.
2.2.2. Kinetic parameters of chitin synthase
The Michaelis-Menten equation v ¼ Vmax½Sꢁ can be expressed the
relationship between the enzymatic reKamctþio½Snꢁ speed and the sub-
strate concentration. Km can be used to reflect the affinity between
the enzyme and various substrates. The smaller the Km value is, the
stronger the affinity become between the enzyme and the
substrate.
2.2.2.1. Determination of Michaelis-Menten constant (Km). The
Michaelis constant Km is equal to half of the substrate concentra-
tion when the reaction rate reaches its maximum. In this experi-
2.2. Biological analysis
ment, the values of Km and Vmax were 4.25 mM, 0.76D ,
OD450Chꢂ1
2.2.1. CHS inhibitory activity
The inhibition percentages (IP) of compounds 2a-s against chitin
synthase were screened at concentration of 300 mg/mL (Table 1).
respectively. As shown in Fig. 5, the effect of substrate concentra-
tion on the enzymatic activities indicated that it was well suited to
the Michaelis-Menten model.

4
B. Li et al. / European Journal of Medicinal Chemistry 195 (2020) 112278
Scheme 1. Synthetic route of 3-amino-3,4-dihydroquinoline-2(1H)-one derivatives.
Table 1
The chitin synthase inhibition percent (IP) of designed compounds at 300
m
g/mL.
IP(%)
Compound
IP(%)
Compound
IP(%)
Compound
2a
2b
2c
2d
2e
2f
67.3
78.3
58.7
82.3
74.6
53.3
49.3
2h
2i
2j
2k
2l
2m
2n
65.2
46.7
50.5
39.9
45.2
40.3
48.9
2o
2p
2q
2r
51.2
66.5
35.7
27.8
71.5
87.5
2s
Polyoxin B
2g
Fig. 5. The Lineweaver-Burk plot of UDP-GlcNAc.
2.2.3. Antifungal activity
Attempts had been made to evaluate the antifungal properties
of all synthesized compounds using two-fold broth dilution method
in 96-well microplate. Five pathogenic fungal strains which were
the common pathogen fungi in clinic were selected as the test fungi
and fluconazole and polyoxin B were used as positive controls. Each
of groups was tested triply. The minimum inhibition concentration
(MIC) value of each compound was measured to evaluate the
antifungal efficacy. As shown in Table 2, these compounds showed
moderate to excellent antifungal activity to the five pathogenic
fungi strains. To C. albicans (ATCC 90023), compounds 2b, 2d, 2e
Fig. 4. The IC50 values of some compounds.
and 2l displayed the most excellent potency with MIC values (1 mg/
mL) similar to that of fluconazole. All compounds showed superior
activity compared with polyoxin B. To C. albicans (ATCC 76615),
2.2.2.2. Determination of Ki. The Ki value was determined in the
presence of different concentrations of compound 2b and the Ki
value was 0.12 mM, which was the intersection of the straight lines
(Fig. 6b). The results proved that compound 2b was non-
competitive inhibitors (Fig. 6a) and had stronger affinity than
UDP-GlcNAc.
compounds 2a, 2d with MIC values of 1
mg/mL exhibited most
potency, whose MIC values were equal to that of fluconazole and
superior to that of polyoxin B. Compounds 2b, 2e, 2l, 2m, 2o and 2r
with MIC values of 2
mg/mL respectively exhibited similar potency
comparable with fluconazole. To A. fumigatus (GIMCC 3.19), the

B. Li et al. / European Journal of Medicinal Chemistry 195 (2020) 112278
5
Fig. 6. Inhibition constant Ki of compound 2b were determined by Lineweaver-Burk plot.
Table 2
The MIC value (mg/mL) of compounds 2a-s against fungi in vitro.
Compounds
Fungi
C. albicans (ATCC 76615)
A. fumigatus (GIMCC 3.19)
C. albicans (ATCC 90023)
C. neoformans (ATCC 32719)
A. flavus (ATCC 16870)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
1
2
4
1
2
4
4
8
4
4
4
2
2
8
2
4
4
2
4
1
2
4
4
4
4
4
8
8
4
8
8
8
4
4
1
4
8
4
4
2
2
2
2
1
2
1
1
2
2
2
2
2
2
1
2
4
2
2
2
2
2
1
4
16
64
16
8
16
4
4
8
16
4
32
32
16
16
8
16
4
4
4
2
4
2
2
4
4
2
8
4
2
4
4
8
8
8
4
8
1
2
4
2q
2r
2s
16
2
4
Fluconazole
Polyoxin B
compounds 2n with MIC value of 1
m
g/mL showed more excellent
These compounds of this work were designed to target the chitin
synthase and destroy the cell wall of fungi, which are not same as
fluconazole, so they can enhance the efficacy of antifungal activity
by different modes of action [36] when combination use. Further-
more, when they were used with the polyoxin B, the result can
provide the information that whether these compounds were
competitive or non-competitive agents. The representative com-
pounds 2b, 2d were chosen to combination use with fluconazole
and polyoxin B respectively, using the checkerboard method. The
results were depicted in Table 4 and Table 5.
The combination 2b-fluconazole showed strongest synergism
effects against C.albicans (ATCC 90023) (FIC ¼ 0.5) and this com-
bination displayed synergistic or additive effects against other
strains. The combination 2d-fluconazole displayed moderate to
excellent synergistic effect for selected strains whose FIC values
ranged from 0.375 to 1. The combinations 2b-polyoxin B, 2d-pol-
yoxin B exhibited strongest synergistic effects against C.albicans
(ATCC 90023) with FIC values of 0.375, 0.375 respectively. The 2d-
polyoxin B showed excellent synergistic effects against A. fumigatus
(GIMCC 3.19) with FIC value 0.375. The 2d-polyoxin B displayed
good inhibition on other selected strains. The results suggested that
these compounds were not competitive with polyoxin B for they
can enhance the efficacy of polyoxin B.
potency when compared with fluconazole. To C. neoformans (ATCC
32719), compounds 2f, 2g, 2j, 2q and 2r with MIC values of 4 g/mL
showed the equal potency of polyoxin B. To A. flavus (ATCC 16870),
compound 2s with MIC of 1 g/mL showed good potency and
m
m
others exhibited moderate activity.
2.2.4. Antibacterial activity
All target molecules were evaluated the antibacterial activity
using three Gram-positive organisms and three Gram-negative
organisms. Streptomycin and norfloxacin were selected as control
drugs. Each of groups was tested triply. The minimum inhibition
concentration (MIC) value of each compound was measured to
evaluate the antimicrobial efficacy. As shown in Table 3, the MIC
value of the strongest compound was several-fold compared with
referenced drugs. It indicated that they were several-fold weaker
than Streptomycin and Norfloxacin. Therefore, these compounds
had no antibacterial activity against the selected strains.
2.3. Drug combination study
Studies have shown drug combination use had synergistic roles
in many aspects, including expanding antibacterial spectrum,
sterilizing, reducing toxicity, reducing and delaying drug resistance.

6
B. Li et al. / European Journal of Medicinal Chemistry 195 (2020) 112278
Table 3
The MIC value (
m
g/mL) of compounds 2a-s against bacterial in vitro.
Gram-positive bacteria
Compounds
Gram-negative bacteria
MRSA (N. 315)
S. aureus (ATCC 25923)
B. subtilis (ATCC 6633)
C. coli (JM 109)
P. aeruginosa (ATCC 9027)
B. proteus (ATCC 8427)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
256
512
256
256
256
256
256
256
512
512
256
512
256
512
512
512
256
256
512
16
128
128
128
128
128
128
128
128
128
128
128
128
128
256
128
256
256
256
128
32
512
256
512
256
256
256
256
256
512
256
512
256
512
256
512
512
256
256
256
8
256
512
256
256
512
256
128
256
256
256
512
256
512
512
256
512
512
512
256
32
512
512
512
512
512
256
512
512
256
512
512
256
512
512
256
512
256
256
256
8
256
256
256
256
256
256
256
256
512
512
512
256
512
512
256
512
256
256
512
16
2q
2r
2s
Streptomycin
Norfloxacin
4
2
4
8
8
2
Table 4
Combination effects of 2b, 2d with fluconazole.
Fungi
MIC (
m
g/mL)a
FIC
FICb
Index(2b)
Index(2d)
Alone
2b
In combination
2d
FCAc
2b
2d
FCA (with 2b)
FCA (with 2d)
ATCC 76615
GIMCC 3.19
ATCC 90023
ATCC 32719
ATCC 16870
2
4
1
64
2
1
4
1
8
2
1
2
1
2
2
2
2
0.25
32
2
0.5
1
0.25
2
0.5
0.5
0.25
2
0.25
0.5
0.25
0.5
1
1.5
0.75
0.5
1.5
1.5
0.75
0.375
0.5
0.5
1
1
1
a
Each of groups was tested independently triply.
FIC index ¼ (MIC of compound A combined/MIC of compound A alone) þ (MIC of compound B combined/MIC of compound B alone). FIC index >2 represents antagonistic
b
effect, 1 < FIC index ꢃ2 represents additive effect, FIC index & 1 represents synergistic effect.
c
C. albicans (ATCC 76615), A. fumigatus (GIMCC 3.19), C. albicans (ATCC 90023), C. neoformans (ATCC 32719), A. flavus (ATCC 16870). FCA (fluconazole), P.B (Polyoxin B).
Table 5
Combination effects of 2b, 2d with polyoxin B.
Fungi
MIC (
mg/mL)
FIC
FIC
Index(2b)
Index(2d)
Alone
2b
In combination
2d
P.Ba
2b
2d
P. B (with 2b)
P. B (with 2d)
ATCC 76615
GIMCC 3.19
ATCC 90023
ATCC 32719
ATCC 16870
2
4
1
64
2
1
4
1
8
2
2
2
4
4
4
1
2
0.25
64
1
0.25
0.5
0.125
4
1
0.5
0.5
1
2
2
1
0.5
0.5
0.5
4
0.75
0.375
2
0.375
0.375
1
0.5
2
1
0.75
a
C. albicans (ATCC 76615), A. fumigatus (GIMCC 3.19), C. albicans (ATCC 90023), C. neoformans (ATCC 32719), A. flavus (ATCC 16870). FCA (fluconazole), P.B (Polyoxin B).
3. Conclusion
2b had stronger affinity binding with chitin synthase than UDP-
GlcNAc and was non-competitive inhibitor. The results of anti-
fungal evaluation revealed that these compounds had good po-
tency against the selected fungi (except C. neoformans) and their
A series of N1-(2-oxo-1,2,3,4-tetrahydroquinolin-3-yl)-N4-phe-
nylfumaramide derivatives were designed and synthesized. Their
inhibitory potency of chitin synthase and antimicrobial activity
were evaluated in vitro as well. The results showed that all com-
pounds exhibited moderate to excellent inhibitory potency against
CHS. Compounds 2b, 2d, 2e and 2s displayed excellent potency
with IP over 70% and their IC50 values are 0.09, 0.16, 0.21 and
0.19 mM respectively. 2b was the strongest among them whose IC50
value was 0.09 mM which was equal to that of polyoxin B whose
IC50 value was 0.09 mM. Kinetic parameters indicated compound
MIC values were between 1 and 4 mg/mL. Compounds 2a, 2b, 2d, 2e,
2l, 2m, 2o, 2r showed excellent potency against C. albicans (ATCC
76615), and their MIC values were equal to these of polyoxin B and
fluconazole. Compound 2n was strongest agent against A. fumigatus
(GIMCC 3.19) with MIC value 1
excellent antifungal activities against C. albicans (ATCC 90023), and
the MIC values of them exceeded polyoxin B (MIC ¼ 4 g/mL).
Compounds 2b, 2d, 2e, 2h, and 2k with MIC values of 2 g/mL
mg/mL. All compounds exhibited
m
m

B. Li et al. / European Journal of Medicinal Chemistry 195 (2020) 112278
7
respectively, had similar potency to fluconazole against A. flavus
C
20H19N3NaO3 [MþNa]þ, 372.1319, found, 372.1317.
(ATCC 16870). In addition, drug combination provided that FIC
values were ranging from 0.375 to 1, showing that these com-
pounds had synergistic or additive effects with fluconazole or
polyoxin B against these selected strains. The result indicated that
these compounds had different antifungal action mode from
reference drugs. Antibacterial experiments confirmed that these
compounds had little bactericidal potency against the selected
bacteria. These results suggested that these designed compounds
were chitin synthase inhibitors and had selective antifungal
activities.
4.1.2.2. N1-(2-oxo-1,2,3,4-tetrahydroquinolin-3-yl)-N4-(m-tolyl)
fumaramide (2b). White powder, yield 82%, m.p. 188e190 ꢀC. 1H
NMR (400 MHz, DMSO‑d6)
d 11.01 (s, 1H), 10.41 (s, 1H), 9.12 (s, 1H),
7.47e7.38 (m, 2H), 7.25e7.14 (m, 3H), 6.95 (t, J ¼ 7.4 Hz, 1H), 6.89 (d,
J ¼ 7.7 Hz, 2H), 6.37 (s, 2H), 4.62e4.50 (m, 1H), 3.18e3.08 (m, 1H),
2.97e2.86 (m, 1H), 2.28 (s, 3H).13C NMR (101 MHz, DMSO‑d6)
d
168.64, 165.06, 163.79, 139.16, 138.42, 137.90, 134.30, 130.42,
129.07, 128.63, 128.07, 124.76, 122.79, 122.70, 120.34, 117.09, 115.63,
48.63, 31.66, 21.64. HRMS (ESI): calcd for C20H19N3NaO3 [MþNa]þ,
372.1319, found, 372.1318.
4. Experimental protocols
4.1.2.3. N1-(2-oxo-1,2,3,4-tetrahydroquinolin-3-yl)-N4-(p-tolyl)
4.1. Chemistry
fumaramide (2c). White powder, yield 80%, m.p. 188e190 ꢀC. 1H
NMR (400 MHz, DMSO‑d6) d 10.99 (s, 1H), 10.40 (s, 1H), 9.16 (s, 1H),
All chemicals were commercial grade and no further purifica-
tion. 1H NMR and 13C NMR spectra were recorded on a Bruker AV
400 or 600 spectrometer (Bruker Company, Germany) using TMS as
an internal standard and DMSO‑d6, CDCl3 or D2O as solvent.
7.51 (d, J ¼ 8.1 Hz, 2H), 7.24e7.17 (m, 2H), 7.12 (d, J ¼ 8.1 Hz, 2H),
6.95 (t, J ¼ 7.4 Hz,1H), 6.89 (d, J ¼ 7.8 Hz,1H), 6.37 (s, 2H), 4.59e4.50
(m, 1H), 3.17e3.08 (m, 1H), 2.97e2.87 (m, 1H), 2.26 (s, 3H). 13C NMR
(101 MHz, DMSO‑d6) d 168.65, 165.08, 163.59, 137.90, 136.70, 134.11,
Chemical shift values were given in ppm (
d
values), coupling con-
133.04, 130.62, 129.61, 128.62, 128.06, 122.78, 122.71, 119.88, 115.63,
48.66, 31.65, 20.95. HRMS (ESI): calcd for C20H19N3NaO3 [MþNa]þ,
372.1319, found, 372.1317.
stants were given in Hz (J values). The following abbreviations were
used to set multiplicities: s ¼ singlet, d ¼ doublet, t ¼ triplet,
q ¼ quartet, m ¼ multiplet, br ¼ broad. The mass spectra were
acquired in positive mode, scanning over the mass range, it were
recorded on Shimadzu LC-MS-2010A/EV and the ESI-HRMS spectra
were recorded on Bruker impact II. Melting point (m.p.) was ob-
tained on amicroscopic melting point apparatus(x-4 type). Reaction
process was monitored by TLC and UV detection was monitored at
254 nm.
4 .1. 2 . 4 . N 1 - ( 2 - m e t h o x y p h e n y l ) - N 4 - ( 2 - o x o - 1, 2 , 3 , 4 -
tetrahydroquinolin-3-yl) fumaramide (2d). White powder, yield
81%, m.p. 189e191 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d 10.99 (s, 1H),
10.42 (s, 1H), 9.21 (d, J ¼ 6.8 Hz, 1H), 8.10 (d, J ¼ 7.5 Hz, 1H),
7.24e7.16 (m, 2H), 7.13e7.06 (m, 1H), 7.04 (d, J ¼ 7.7 Hz, 1H),
6.99e6.86 (m, 3H), 6.40 (s, 2H), 4.63e4.52 (m, 1H), 3.81 (s, 3H),
3.17e3.09 (m, 1H), 2.99e2.88 (m, 1H). 13C NMR(101 MHz, DMSO‑d6)
4.1.1. General method for preparation 1a-s. [37]
d 168.59, 165.20, 163.30, 150.11, 137.92, 134.59, 130.93, 128.60,
Substituted aniline (10 mmol) was added to a solution of maleic
anhydride (0.98 g, 10 mmol) in methane dichloride. The reaction
was stirred at room temperature for approximately 2 h. The process
of the reaction was monitored by TLC until the reaction was
completed. The resulting suspension was filtered and then the
corresponding amide was dried at 50 ꢀC, yielding 96e99% of the
desired products 1a-s.
128.08, 127.63, 125.13, 122.79, 122.69, 122.08, 120.73, 115.64, 111.72,
56.24, 48.62, 31.59. HRMS (ESI): calcd for C20H20N3O4 [MþH]þ,
366.1448, found, 366.1449. C20H19N3NaO4 [MþNa]þ, 388.1268,
found, 388.1267.
4 .1. 2 . 5 . N 1 - ( 4 - m e t h o x y p h e n y l ) - N 4 - ( 2 - o x o - 1, 2 , 3 , 4 -
tetrahydroquinolin-3-yl) fumaramide (2e). White powder, yield
81%, m.p. 189e191 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d 10.92 (s, 1H),
4.1.2. General method for preparation 2a-s. [38]
10.40 (s, 1H), 9.21 (d, J ¼ 7.3 Hz, 1H), 7.54 (d, J ¼ 8.5 Hz, 2H),
7.25e7.15 (m, 2H), 6.99e6.85 (m, 4H), 6.42e6.31 (m, 2H), 4.59e4.50
(m, 1H), 3.72 (s, 3H), 3.16e3.09 (m, 1H), 2.97e2.86 (m, 1H). 13C NMR
Iodines (2.54 g, 10 mmol) were added to a solution of triethyl
phosphite (1.66 g, 10 mmol) in methane dichloride (50 mL) cooled
with ice bath. One compound of 1a-s (10 mmol) was added to the
solution after the iodine dissolved completely. The triethylamine
(6.96 mL, 50 mmol) was added to the mixture and stirred for
15 min at a cooling bath. 3-Amino-3,4-dihydroquinolin-2(1H)-one
(1.95 g, 12 mmol) was added and the mixture was stirred at room
temperature to complete the reaction (reaction monitored by TLC).
The solution was washed with saturated aqueous sodium bicar-
bonate (3 ꢄ 15 mL),1 M HCl solution (3 ꢄ 15 mL), water (3 ꢄ 15 mL),
brine (3 ꢄ 15 mL). The combined organic layer was dried with
Na2SO4, filtered and concentrated. The crude product was purified
by silica column chromatography (hexanes/EtOAc).
(101 MHz, DMSO‑d6) d 168.66, 165.08, 163.33, 155.98, 137.91, 133.98,
132.32, 128.62, 128.07, 122.77, 121.43, 115.63, 114.39, 55.65, 48.66,
31.65. HRMS (ESI): calcd for C20H19N3NaO4 [MþNa]þ, 388.1268,
found, 388.1269.
4.1.2.6. N1-(2-nitrophenyl)-N4-(2-oxo-1,2,3,4-tetrahydroquinolin-3-
yl) fumaramide (2f). White powder, yield 84%, m.p. 190e193 ꢀC. 1H
NMR (400 MHz, DMSO‑d6) 1H NMR (400 MHz, DMSO)
d 11.20 (s,
1H), 10.42 (s, 1H), 8.98 (d, J ¼ 7.7 Hz, 1H), 7.98 (dd, J ¼ 6.6, 5.6 Hz,
1H), 7.78 (d, J ¼ 8.0 Hz, 1H), 7.75e7.70 (m, 1H), 7.42e7.36 (m, 1H),
7.25e7.15 (m, 2H), 6.95 (t, J ¼ 7.4 Hz, 1H), 6.89 (d, J ¼ 7.8 Hz, 1H),
6.50e6.41 (m, 2H), 4.59e4.48 (m,1H), 3.15e3.05 (m,1H), 2.97e2.86
4.1.2.1. N1-(2-oxo-1,2,3,4-tetrahydroquinolin-3-yl)-N4-(o-tolyl)
(m, 1H). 13C NMR(101 MHz,DMSO‑d6)
d168.59, 164.74,164.52,
fumaramide (2a). White powder, yield 80%, m.p. 188e190 ꢀC. 1H
142.50, 137.88, 134.56, 133.39, 131.50, 130.92, 128.59, 128.07, 125.85,
125.80, 125.40, 122.80, 122.67, 115.64, 48.62, 31.60. HRMS (ESI):
calcd for C20H19N3NaO3 [MþNa]þ, 403.1013, found, 403.1014.
NMR (400 MHz, DMSO‑d6)
d 10.78 (s, 1H), 10.41 (s, 1H), 9.23 (d,
J ¼ 7.7 Hz, 1H), 7.62 (d, J ¼ 7.8 Hz, 1H), 7.24e7.14 (m, 4H), 7.08 (t,
J ¼ 7.2 Hz, 1H), 6.95 (t, J ¼ 7.4 Hz, 1H), 6.89 (d, J ¼ 7.8 Hz, 1H), 6.41 (s,
2H), 4.62e4.51 (m, 1H), 3.16e3.07 (m, 1H), 2.99e2.87 (m, 1H), 2.25
4.1.2.7. N1-(3-nitrophenyl)-N4-(2-oxo-1,2,3,4-tetrahydroquinolin-3-
(s, 3H).13C NMR (101 MHz, DMSO‑d6)
d
168.58, 165.13, 163.72,
yl) fumaramide (2g). White powder, yield 83%, m.p. 190e193 ꢀC. 1H
137.90, 136.49, 134.80, 131.39, 130.79, 130.40, 128.62, 128.09, 126.41,
125.51, 124.62, 122.79, 122.67, 115.64, 48.66, 31.61, 18.40. HRMS
(ESI): calcd for C20H20N3O3 [MþH]þ, 350.1499, found, 350.1499.
NMR (400 MHz, DMSO‑d6) d 11.19 (s, 1H), 10.42 (s, 1H), 8.98 (d,
J ¼ 7.6 Hz, 1H), 7.98 (d, J ¼ 8.0 Hz, 1H), 7.80e7.69 (m, 2H), 7.39 (t,
J ¼ 7.5 Hz, 1H), 7.24e7.13 (m, 2H), 6.95 (t, J ¼ 7.4 Hz, 1H), 6.88 (d,

8
B. Li et al. / European Journal of Medicinal Chemistry 195 (2020) 112278
J ¼ 7.8 Hz, 1H), 6.51e6.41 (m, 2H), 4.58e4.48 (m, 1H), 3.15e3.07 (m,
1H), 2.96e2.85 (m, 1H). 13C NMR(101 MHz, DMSO‑d6)
168.59,
164.73, 164.52, 142.51, 137.87, 134.56, 133.39, 131.50, 130.91, 128.59,
128.08, 125.86, 125.81, 125.40, 122.80, 122.67, 115.64, 48.62, 31.60.
7.25e7.13 (m, 2H), 6.95 (t, J ¼ 7.4 Hz, 1H), 6.89 (d, J ¼ 7.8 Hz, 1H),
6.46e6.35 (m, 2H), 4.59e4.48(m, 1H), 3.16e3.06 (m, 1H), 2.96e2.85
d
(m, 1H). 13C NMR (151 MHz, DMSO‑d6)
d 168.60, 164.95, 164.19,
138.65, 137.91, 133.89, 132.03, 130.51, 128.61, 128.05, 122.78, 122.69,
121.83, 115.65, 115.59, 48.68, 31.69. HRMS (ESI): calcd for
4.1.2.8. N1-(4-nitrophenyl)-N4-(2-oxo-1,2,3,4-tetrahydroquinolin-3-
C
19H17BrN3O3 [MþH]þ,414.0448, found, 414.0443. C19H16BrN3NaO3
yl) fumaramide (2h). White powder, yield 83%, m.p. 190e193 ꢀC. 1H
[MþNa]þ, 436.0267, found, 436.0265.
NMR (400 MHz, DMSO‑d6)
d 11.38 (s, 1H), 10.41 (s, 1H), 8.93 (d,
J ¼ 4.9 Hz, 1H), 8.23 (d, J ¼ 8.7 Hz, 2H), 7.87 (d, J ¼ 8.8 Hz, 2H),
7.24e7.14 (m, 2H), 6.95 (t, J ¼ 7.4 Hz, 1H), 6.89 (d, J ¼ 7.7 Hz, 1H),
6.52e6.41 (m, 2H), 4.58e4.49 (m,1H), 3.15e3.07 (m,1H), 2.97e2.87
4.1.2.14. N1-(2-fluorophenyl)-N4-(2-oxo-1,2,3,4-tetrahydroquinolin-
3-yl) fumaramide (2n). White powder, yield 81%, m.p.187e190 ꢀC.
1H NMR (400 MHz, DMSO‑d6)
d 11.04 (s, 1H), 10.41 (s, 1H), 9.02 (d,
(m, 1H).13C NMR (101 MHz, DMSO‑d6)
d
168.65, 167.02, 165.43,
J ¼ 7.6 Hz, 1H), 7.65 (d, J ¼ 8.8 Hz, 2H), 7.37 (d, J ¼ 8.8 Hz, 2H),
7.24e7.15 (m, 2H), 6.95 (t, J ¼ 7.4 Hz, 1H), 6.89 (d, J ¼ 7.8 Hz, 1H),
6.45e6.34 (m, 2H), 4.59e4.49 (m, 1H), 3.16e3.07 (m, 1H),
164.66, 145.61, 142.72, 137.87, 133.83, 132.73, 130.13, 128.62, 128.07,
125.49, 125.46, 122.79, 122.67, 119.47, 119.42, 48.58, 31.68.
2.96e2.87 (m, 1H). 13C NMR (101 MHz, DMSO‑d6)
d 168.49, 165.28,
4.1.2.9. N1-(2-chlorophenyl)-N4-(2-oxo-1,2,3,4-tetrahydroquinolin-
163.67, 137.88, 134.53, 130.94, 128.63, 128.09, 124.86, 124.83, 123.87,
122.80, 122.62, 115.94, 115.75, 115.64, 48.67, 31.54. HRMS (ESI):
calcd for C19H16FN3NaO3 [MþNa]þ, 376.1068, found, 376.1067.
3-yl) fumaramide (2i). White powder, yield 81%, m.p. 189e191 ꢀC.
1H NMR (400 MHz, DMSO‑d6)
d 11.27 (s, 1H), 10.44 (s, 1H), 9.24 (d,
J ¼ 7.1 Hz, 1H), 7.92 (d, J ¼ 7.7 Hz, 1H), 7.50 (d, J ¼ 7.8 Hz, 1H),
7.38e7.32 (m, 1H), 7.23e7.14 (m, 3H), 6.98e6.87 (m, 2H), 6.54e6.38
(m, 2H), 4.63e4.49 (m, 1H), 3.15e3.07 (m, 1H), 3.00e2.89 (m, 1H).
4.1.2.15. N1-(4-fluorophenyl)-N4-(2-oxo-1,2,3,4-tetrahydroquinolin-
3-yl) fumaramide (2o). White powder, yield 80%, m.p. 187e190 ꢀC.
13C NMR (101 MHz, DMSO‑d6)
d
168.49, 165.13, 163.86, 137.90,
1H NMR (400 MHz, DMSO‑d6)
d 10.98 (s, 1H), 10.41 (s, 1H), 9.06 (d,
135.22, 134.26, 131.13, 129.95, 128.60, 128.07, 127.85, 126.65, 126.19,
125.89, 122.78, 122.63, 115.66, 48.64, 31.54. HRMS (ESI): calcd for
C
J ¼ 7.5 Hz, 1H), 7.64 (dd, J ¼ 8.9, 5.0 Hz, 2H), 7.24e7.13 (m, 4H), 6.95
(t, J ¼ 7.4 Hz, 1H), 6.89 (d, J ¼ 7.8 Hz, 1H), 6.43e6.34 (m, 2H),
4.59e4.48 (m, 1H), 3.16e3.07 (m, 1H), 2.97e2.85 (m, 1H). 13C NMR
19H16ClN3NaO3 [MþNa]þ, 392.0772, found, 392.0772.
(101 MHz, DMSO‑d6)
d 168.66,164.94,163.99,137.90,135.69,133.90,
4.1.2.10. N1-(4-chlorophenyl)-N4-(2-oxo-1,2,3,4-tetrahydroquinolin-
130.42, 128.62, 128.06, 122.78, 122.71, 121.68, 121.61, 115.91, 115.69,
3-yl) fumaramide (2j). White powder, yield 80%, m.p. 189e191 ꢀC.
115.63, 48.62, 31.67. HRMS (ESI): calcd for C19H16FN3NaO3
1H NMR (400 MHz, DMSO‑d6)
d
11.57 (s, 1H), 10.43 (s, 1H), 9.21 (s,
[MþNa]þ, 376.1068, found, 376.1069.
1H), 8.15e8.05 (m, 1H), 7.27e7.13 (m, 5H), 6.96 (t, J ¼ 7.4 Hz, 1H),
6.89 (d, J ¼ 7.8 Hz, 1H), 6.50e6.36 (m, 2H), 4.62e4.53 (m, 1H),
3.18e3.08 (m, 1H), 3.00e2.88 (m, 1H). 13C NMR (101 MHz,
4 .1. 2 .16 . N 1 - ( 3 , 4 - d i flu o ro p he nyl ) - N 4 - ( 2 - o x o - 1, 2 , 3 , 4 -
tetrahydroquinolin-3-yl) fumaramide (2p). White powder, yield
DMSO‑d6)
d
168.66, 164.90, 164.27, 138.26, 137.89, 135.23, 133.94,
79%, m.p.186e189 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d 11.01 (s, 1H),
130.31, 129.38, 129.13, 128.87, 128.62, 128.07, 127.51, 122.79, 122.69,
121.39, 115.63, 48.62, 31.67. HRMS (ESI): calcd for C19H17ClN3O3
[MþH]þ, 370.0953, found, 370.0950. C19H16ClN3NaO3 [MþNa]þ,
392.0772, found, 392.0770.
10.40 (s, 1H), 9.03 (s, 1H), 7.85e7.77 (m, 1H), 7.44e7.35 (m, 1H), 7.30
(d, J ¼ 8.9 Hz, 1H), 7.23e7.15 (m, 2H), 6.98e6.92 (m, 1H), 6.89 (d,
J ¼ 7.7 Hz, 1H), 6.44e6.36 (m, 2H), 4.57e4.45 (m, 1H), 3.15e3.05 (m,
1H), 2.97e2.84 (m, 1H). 13C NMR (101 MHz, DMSO‑d6)
d 168.68,
164.76, 164.61, 137.89, 136.52, 133.88, 130.19, 128.62, 128.06, 122.78,
122.70, 118.00, 117.83, 116.18, 115.62, 108.91, 108.70, 48.58, 31.69.
HRMS (ESI): calcd for C19H15F2N3NaO3 [MþNa]þ, 394.0974, found,
394.0972.
4.1.2.11. N1-(2-bromophenyl)-N4-(2-oxo-1,2,3,4-tetrahydroquinolin-
3-yl) fumaramide (2k). White powder, yield 82%, m.p. 188e190 ꢀC.
1H NMR (400 MHz, DMSO‑d6)
d 11.00 (s, 1H), 10.42 (s, 1H), 9.19 (d,
J ¼ 6.0 Hz, 1H), 7.79 (d, J ¼ 7.7 Hz, 1H), 7.66 (d, J ¼ 7.9 Hz, 1H), 7.39 (t,
J ¼ 7.6 Hz, 1H), 7.26e7.10 (m, 3H), 7.00e6.84 (m, 2H), 6.53e6.38 (m,
2H), 4.62e4.51 (m, 1H), 3.16e3.06 (m, 1H), 3.00e2.85(m, 1H). 13C
4.1.2.17. N1-(2-oxo-1,2,3,4-tetrahydroquinolin-3-yl)-N4-phenyl-
fumaramide (2q). White powder, yield 81%, m.p. 188e190 ꢀC. 1H
NMR (101 MHz, DMSO‑d6)
d
168.54, 165.05, 163.90, 137.89, 136.46,
NMR (400 MHz, DMSO‑d6) d 11.12 (s, 1H), 10.44 (s, 1H), 9.19 (d,
134.06, 133.15, 131.20, 128.61, 128.42, 128.08, 127.40, 126.93, 122.79,
122.65, 115.64, 48.65, 31.58. HRMS (ESI): calcd for C19H16BrN3NaO3
[MþNa]þ, 436.0267, found, 436.0263.
J ¼ 6.1 Hz, 1H), 7.63 (d, J ¼ 7.9 Hz, 2H), 7.31 (t, J ¼ 7.7 Hz, 2H),
7.24e7.15 (m, 2H), 7.07 (t, J ¼ 7.3 Hz, 1H), 6.99e6.86 (m, 2H),
6.45e6.34 (q, J ¼ 12.6 Hz, 2H), 4.60e4.49 (m, 1H), 3.16e3.07 (m,
1H), 2.98e2.87 (m, 1H). 13C NMR (101 MHz, DMSO‑d6)
d 168.65,
4.1.2.12. N1-(3-bromophenyl)-N4-(2-oxo-1,2,3,4-tetrahydroquinolin-
165.07, 163.94, 139.28, 137.92, 133.92, 130.71, 129.20, 128.61, 128.05,
124.02, 122.76, 122.72, 119.90, 115.65, 48.64, 31.64. HRMS (ESI):
calcd for C19H17N3NaO3 [MþNa]þ, 358.1162, found, 358.1163.
3-yl) fumaramide (2l). White powder, yield 83%, m.p. 188e190 ꢀC.
1H NMR (400 MHz, DMSO‑d6)
d 10.99 (s, 1H), 10.41 (s, 1H), 8.96 (d,
J ¼ 7.6 Hz,1H), 7.98 (s,1H), 7.50 (d, J ¼ 7.4 Hz,1H), 7.34e7.15 (m, 4H),
6.95 (t, J ¼ 7.4 Hz, 1H), 6.89 (d, J ¼ 7.8 Hz, 1H), 6.47e6.36 (m, 2H),
4.62e4.48 (m, 1H), 3.16e3.07 (m, 1H), 2.96e2.86 (m, 1H). 13C NMR
4 .1. 2 .18 . N 1 - ( n a p h t h a l e n - 1 - y l ) - N 4 - ( 2 - o x o - 1, 2 , 3 , 4 -
tetrahydroquinolin-3-yl) fumaramide (2r). White powder, yield
(151 MHz, DMSO‑d6)
d
168.62, 164.85, 164.52, 140.89, 137.91, 133.89,
84%, m.p. 188e190 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d 11.38 (s, 1H),
131.20, 130.34, 128.61, 128.06, 126.56, 122.79, 122.69, 122.21, 122.01,
118.68, 115.65, 48.66, 31.72. HRMS (ESI): calcd for C19H17BrN3O3
[MþH]þ,414.0448, found, 414.0451. C19H16BrN3NaO3 [MþNa]þ,
436.0267, found, 436.0265.
10.43 (s, 1H), 9.23 (d, J ¼ 7.5 Hz, 1H), 8.21 (d, J ¼ 5.8 Hz, 1H),
7.98e7.91 (m, 1H), 7.87 (d, J ¼ 7.3 Hz, 1H), 7.77 (d, J ¼ 8.1 Hz, 1H),
7.59e7.46 (m, 3H), 7.26e7.13 (m, 2H), 7.00e6.83 (m, 2H), 6.60e6.43
(m, 2H), 4.68e4.56 (m, 1H), 3.19e3.09 (m, 1H), 3.01e2.88 (m, 1H).
13C NMR (101 MHz, DMSO‑d6)
d 168.60, 165.25, 164.46, 137.91,
4.1.2.13. N1-(4-bromophenyl)-N4-(2-oxo-1,2,3,4-tetrahydroquinolin-
134.83, 134.16, 133.67, 130.36, 128.61, 128.09, 127.90, 126.49, 126.46,
126.05, 125.75, 123.13, 122.80, 122.67, 121.47, 115.64, 48.67, 31.63.
HRMS (ESI): calcd for C23H19FN3NaO3 [MþNa]þ, 408.1319, found,
408.1313.
3-yl) fumaramide (2m). White powder, yield 84%, m.p. 188e190 ꢀC.
1H NMR (400 MHz, DMSO‑d6)
d
11.04 (s, 1H), 10.41 (s, 1H), 9.01 (d,
J ¼ 7.5 Hz, 1H), 7.60 (d, J ¼ 8.6 Hz, 2H), 7.50 (d, J ¼ 8.6 Hz, 2H),

B. Li et al. / European Journal of Medicinal Chemistry 195 (2020) 112278
9
4.1. 2.19. N1 -(3, 5-dimethylphenyl)-N4 -(2-oxo-1, 2, 3, 4-
with modified Sabouraud chloramphenicol agar containing 1%
peptone, 2% glucose and solid medium and 15% agar. Incubating for
48 h at 37 ꢀC, inhibition was tested and the minimum inhibitory
tetrahydroquinolin-3-yl) fumaramide (2s). White powder, yield
78%, m.p. 189e192 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d 11.02 (s, 1H),
10.41 (s, 1H), 9.14 (d, J ¼ 7.7 Hz, 1H), 7.27e7.16 (m, 4H), 6.95 (t,
J ¼ 7.4 Hz, 1H), 6.89 (d, J ¼ 7.8 Hz, 1H), 6.72 (s, 1H), 6.36 (s, 2H),
4.61e4.52 (m, 1H), 3.16e3.08 (m, 1H), 2.98e2.88 (m, 1H), 2.23 (s,
concentration (MIC, mg/mL) was recorded. Polyoxin B and flucon-
azole were used as positive controls.
6H). 13C NMR (151 MHz, DMSO‑d6)
d 168.60, 165.16, 163.58, 139.02,
4.2.3.2. Antibacterial assay. In this experiment, six strains were
tested, three Gram-positive bacteria and three Gram-negative
bacteria, such as Methicillin-resistant staphylococcus aureus N 3.15,
Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633,
Escherichia coli JM 109, Pseudomonas aeruginosa ATCC 9027, Bacillus
proteus ATCC 8427. The test compounds were prepared at different
concentrations. The medium used for this assay was beef extract
peptone medium containing 1% peptone, 0.3% beef extract, 0.5%
sodium chloride, solid medium and 15% agar in distilled water.
138.22, 137.90, 134.38, 130.58, 128.62, 128.07, 125.67, 122.80, 122.69,
117.69, 115.66, 48.69, 31.67, 21.52. HRMS (ESI): calcd for
C
21H21N3NaO3 [MþNa]þ, 386.1475, found, 386.1475.
4.2. Biological activity assay
4.2.1. Inhibition against yeast CHS
The procedure of evaluating the inhibition potency against
chitin synthase which was described by Lucero [39] was adopted
with little modifications. In this experiment, the chitin synthase
was extracted from the tropical yeast C. tropicalis ATCC 750 by low-
speed and high-speed centrifugation. All tested compounds were
prepared as stock solutions by dissolving in DMSO at a concen-
tration of 10 mg/mL and then it were diluted to 1200, 600, 300, 150,
4.2.4. Drug combination assay
The checkerboard method and the two-fold broth dilution method
were used to do drug combination studies that combined 2b, 2d
with fluconazole and polyoxin B, respectively. Each set of experi-
ments was repeated three times independently. The effects of drug
combination were evaluated by the fractional inhibitory concen-
tration (FIC), which were calculated by formula as following: FIC
index ¼ (MIC of compound A combined/MIC of compound A
alone) þ (MIC of compound B combined/MIC of compound B
alone). FIC index >2 represents antagonistic effect, 1 < FIC index ꢃ2
represents additive effect, FIC index & 1 represents synergistic
effect.
75
centration of the enzyme solution were diluted to 300, 150, 75.0,
37.5, 18.75, 9.375 g/mL.
mg/mL with 50 mM of pH 7.5 Tris-HCl buffer, and the test con-
m
There were several simple steps in the experiment. Firstly, the
biosynthesis of chitin was catalyzed by chitin synthase in 96 well
plates coated with WGA. Secondly, the WGA-HRP which was con-
jugated WGA with HRP was added to bind to the immobilized
chitin. Lastly, the 96 well plates were washed by distilled water to
eliminate unbound components. TMB was oxidized by hydrogen
peroxide and it was monitored the HRP activity. The reaction was
Funding
stopped by adding 50 mL 2 M H2SO4 to each well. Polyoxin B was
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
selected as a positive control. The OD values were measured by
Biotek ELx808 microplate reader. Each group was tested in parallel
for two groups. The calculation formula of Inhibition percentage:
Inhibition percentage ¼ (B0 - Bn)/(B0 - OD0).
Declaration of competing interest
I would like to declare on behalf of my co-authors that the work
described was original research that has not been published pre-
viously, and not under consideration for publication elsewhere, in
whole or in part. I declare that we have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
4.2.2. Kinetic parameters of chitin synthase
In this experiment, UDP-GlcNAc was used as the substrate
which ranged from 0.8 to 4.0 mM to measure the Km and the Vmax
.
The values of 1/Km and Vmax of UDP-GlcNAc were the intersection of
the cross axle and vertical axis, respectively. The Ki value was
determined by intersection of the straight lines whose abscissa and
ordinate were fixed by concentrations of substrate UDP-GlcNAc and
1/v, respectively. The concentrations of UDP-GlcNAc were 0.5, 1.0,
1.5 and 2.0 mM, respectively and the concentrations of 2b were 0,
0.032, 0.126 and 0.25 mM, respectively. Graphs of different in-
hibitor’s concentrations were drawn by the Lineweaver-Burk plot.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112278.
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