Design and synthesis of novel desfluoroquinolone-aminopyrimidine hybrids as potent anti-MRSA agents with low hERG activity

Article abstract of DOI:10.1016/j.bioorg.2020.104176

Despite the fact that the introduction of a fluorine atom at the C-6 position has resulted in the evolution of fluoroquinolones, fluoroquinolone-induced cardiac toxicity has drawn considerable attention. In this context, desfluoroquinolone-based hybrids w

Full text of DOI:10.1016/j.bioorg.2020.104176

BioorganicChemistry103(2020)104176
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Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design and synthesis of novel desfluoroquinolone-aminopyrimidine hybrids
as potent anti-MRSA agents with low hERG activity
Runzhe Songa, Yue Wanga, Minghui Wangb, Ruixuan Gaob, Teng Yangc,d, Song Yangc,
⁎
⁎
⁎
Cai-Guang Yangd, Yongsheng Jine, Siyuan Zoua, Jianfeng Caib, , Renhua Fana, , Qiuqin Hea,
a Department of Chemistry, Fudan University, 2005 Songhu Road, Yangpu District, Shanghai 200438, China
b Department of Chemistry, University of South Florida, Tampa, FL 33620, United States
c State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of
Education, Center for R&D of Fine Chemicals, Guizhou University, Guiyang 550025, China
d State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
e School of Pharmacy, The Second Military Medical University, Shanghai 200433, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Despite the fact that the introduction of a fluorine atom at the C-6 position has resulted in the evolution of
fluoroquinolones, fluoroquinolone-induced cardiac toxicity has drawn considerable attention. In this context,
desfluoroquinolone-based hybrids with involvement of C-7 aminopyrimidine functional group were designed
and synthesized. The biological results showed majority of these hybrids still demonstrated potent anti-MRSA
activity with MIC values between 0.38 and 1.5 μg/mL, despite the lack of the typical C-6 fluorine atom.
Particularly, the most active B14 exhibited activities at submicromolar concentrations against a panel of MRSA
strains including vancomycin-intermediate strains, levofloxacin-resistant isolates, and linezolid-resistant iso-
lates, etc. As expected, it also displayed highly selective toxicity toward bacterial cells and low hERG inhibition.
Further resistance development study indicated MRSA is unlikely to acquire resistance against B14. The docking
study revealed that two hydrogen bonds were formed between the C-7 substituent and the surrounding DNA
bases, which might contribute to overcome resistance by reducing the dependence on the magnesium-water
bridge interactions with topoisomerase IV. These results indicate a promising strategy for developing new an-
tibiotic quinolones to combat multidrug resistance and cardiotoxicity.
Desfluoroquinolone
Aminopyrimidine
Hybrids
Anti-MRSA
hERG activity
1. Introduction
resort for the treatment of MRSA infections. Unfortunately, since the
first strain of MRSA with reduced susceptibility to vancomycin was
The ever-increasing bacterial resistance to the existing antibiotics
poses a global threat to public health. According to newly updated es-
timates reported by the Centers for Disease Control and Prevention
(CDC), more than 2.8 million infections are caused by antibiotic-re-
sistant pathogens annually in the United Sates, leading to at least
35,000 deaths [1]. Among them, the fatalities attributed to methicillin-
resistant Staphylococcus aureus (MRSA) are particularly high as com-
pared with other antibiotic-resistant pathogens. MRSA infections are
difficult to treat because they have acquired resistance to a variety of
classes of antibacterial drugs, including β-lactam antibiotics [2], mac-
rolides [3], fluoroquinolones [4], glycopeptides [5], and ox-
azolidinones. [6] Vancomycin is currently used as an antibiotic of last
reported in 1997 [7], there has been an increase in the number of cases
with both vancomycin-intermediate MRSA (VISA) and vancomycin-re-
sistant MRSA (VRSA) [8]. Therefore, the search for new anti-MRSA
agents with improved resistance profiles continues to be a major chal-
lenge in antibacterial chemotherapy [9].
Quinolones are one of the most widely prescribed antibiotics used to
treat various bacterial infections. They can be divided into four gen-
erations. The first-generation agents without a substituent at the C-6
position of the quinolone nucleus only exhibit weak to moderate ac-
tivity against Gram-negative bacteria. A remarkable breakthrough in
the development of quinolone antibiotics came with the introduction of
a fluorine atom at the C-6 position, resulting in the evolution of
Abbreviations: CDC, Centers for Disease Control and Prevention; E. Coli., Escherichia coli; MRSA, Methicillin-resistant Staphylococcus aureus; VISA, vancomycin-
intermediate MRSA; VRSA, vancomycin-resistant MRSA; hEGR, human ether-a-go-go related gene; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-
bromide; DA5, deoxyadenosine monophosphate 5; DG1, deoxyguanosine monophosphate 1
⁎ Corresponding authors.
E-mail addresses: jianfengcai@usf.edu (J. Cai), rhfan@fudan.edu.cn (R. Fan), qqhe@fudan.edu.cn (Q. He).
https://doi.org/10.1016/j.bioorg.2020.104176
Received 27 May 2020; Received in revised form 4 August 2020; Accepted 11 August 2020
Availableonline26August2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

R. Song, et al.
BioorganicChemistry103(2020)104176
Fig. 3. Design of the desfluoroquinolone-aminopyrimidine hybrids.
surrounding DNA bases in the binding sites to produce strong hydrogen
and/or π-π interactions. The additional drug-enzyme contacts mediated
by this C-7 substituent would offer the possibility to overcome quino-
lone resistance by reducing the dependence on the magnesium-water
bridge interactions with topoisomerase IV. The phenyl moiety with li-
pophilic property directly linked on the aminopyrimidine ring would
enhance the cell penetration and thus compensate the loss caused by
the absence of the C-6 fluorine atom. Structural optimization resulted
into hybrid compounds B (Fig. 3) in order to further enhance their
potency.
Fig. 1. Representative anti-MRSA fluoroquinolone antibiotics 1–3 and anti-
biotics 4–6 withdrawn from the market.
fluoroquinolones with expanding antibacterial spectrum from Gram-
negative to -positive bacteria [10]. Consequently, the 6-fluoro sub-
stituent has been a fundamental structural feature of all the latter
generations. In addition, some of the fourth-generation drugs such as
moxifloxacin [11] (1, Fig. 1), delafloxacin [12] (2, Fig. 1), and sita-
floxacin [13] (3, Fig. 1) can also demonstrate strong potency against
MRSA. Despite their wide usage in clinical practice, fluoroquinolone-
induced cardiac toxicity due to the blockage of the human Ether-a-go-
go Related Gene (hEGR) potassium channel has drawn considerable
attention. [14] This issue had been further stressed by the withdrawal
of temafloxacin (4, Fig. 1), sparfloxacin (5, Fig. 1) and grepafloxacin (6,
Fig. 1) from the market because of pro-arrhythmic side-effects. [15]
Thus the development of this class of new antibiotics to overcome
multidrug resistance and cardiotoxicity has become increasingly ur-
gent.
The synthesis of the desfluoroquinolone-aminopyrimidine hybrids A
and B is depicted in Scheme 1. The benzoyl chlorides 10 was coupled
with ethyl 3-(dimethylamino)acrylate to give the acrylates 11. Sub-
stitution with appropriate aliphatic amines followed by cyclization with
potassium carbonate afforded quinolones 13. Hydrolysis of 13 and then
treatment with 2, 4-dimethoxybenzylamine furnished 15. Removal of 2,
4-dimethoxybenzyl group and subsequent esterification with
1.1. Design and chemistry
Recently, ozenoxacin [16] (7, Fig. 2), garenoxacin [17] (8, Fig. 2)
and nemonoxacin [18] (9, Fig. 2), the antibiotics lacking the typical 6-
fluoro substituent on quinolone nucleus, have been approved in many
regions for the treatment of bacterial infections including those caused
by MRSA. Despite the particular interest generated by these antibiotics,
few examples on desfluoroquinolones as potent antibacterial agents are
described in the literature. Based on our understanding of quinolone-
enzyme interactions, in this context, we exploited “merging” approach
[19] by combining the structural features of antibiotic quinolones and
another antibacterial aminopyrimidines [20] to generate a novel series
of desfluoroquinolone-aminopyrimidine hybrids A (Fig. 3), with the
aim to reduce their cardiotoxic potential while maintaining anti-MRSA
properties.
Genetic evidence strongly suggests that the primary target of qui-
nolones in Staphylococcus aureus is topoisomerase IV [21]. Quinolones
act by stabilizing the topoisomerase IV-cleaved DNA complexes via a
critical water-metal ion interaction, thus inhibiting DNA synthesis [22].
Target-mediated quinolone resistance is caused by specific mutations in
topoisomerase IV that disrupt this water-metal ion bridge interaction
[23]. Our newly designed hybrids A are characterized by the in-
corporation of an aminopyrimidine ring at the C-7 position of quino-
lones, which contains both hydrogen-accepting and -donating func-
tionalities and is expected to be sufficiently close in space to the
Scheme 1. Synthesis of desfluoroquinolone-aminopyrimidine hybrids A and B.a
aReagents and conditions: (a) ethyl 3-(dimethylamino)acrylate, Et3N, toluene,
90 °C, 4 h; (b) aliphatic amines, THF, 50 °C, 3 h; (c) K2CO3, DMF, 60–90 °C,
1–6 h; (d) 5 M NaOH, THF, 50 °C, 3.5 h (e) 2,4-dimethoxybenzylamine; DMSO,
85 °C, 6 h; (f) CF3COOH, DCM, rt, 5 h; (g) TMSCH2N2, THF, MeOH, rt, 24 h; (h)
Pd(OAc)2, K2CO3, BINAP, DMF, 90 °C, 12–18 h; (i) 5 M NaOH, THF, 50 °C,
3.5 h.
Fig. 2. Desfluoroquinolone antibiotics.
2

R. Song, et al.
BioorganicChemistry103(2020)104176
Table 1
Table 2
Antibacterial activities of desfluoroquinolone-aminopyrimidine hybrids
A
Antibacterial activities of desfluoroquinolone-aminopyrimidine hybrids B
against MRSA and E. Coli.a
against MRSA and E. Coli.a
Compd R1
R2
MICa (μg/mL)
Compd R1
R2
MICb (μg/mL)
MRSA
E. coli
MRSA
E. coli
(ATCC33591)
(ATCC25922)
(ATCC33591)
(ATCC25922)
B1
Cyclopropyl
H
12.5
1.5
1.5
0.38
1.5
3
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
0.5
A1
Cyclopropyl
H
> 50
> 50
6
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
0.5
B2
Cyclopropyl 2-Methyl
Cyclopropyl 3-Methyl
Cyclopropyl 4-Methyl
Cyclopropyl 4-Ethyl
Cyclopropyl 4-n-Butyl
Cyclopropyl 4-i-propyl
Cyclopropyl 4-t-Butyl
A2
Cyclopropyl 2-Methyl
Cyclopropyl 3-Methyl
Cyclopropyl 4-Methyl
B3
A3
B4
A4
0.75
B5
A5
Cyclopropyl 2,4-diMethyl 1.5
Cyclopropyl 3,4-diMethyl 0.75
B6
A6
B7
6
A7
Cyclopropyl 4-n-Propyl
12.5
25
B8
6
A8
B9
Cyclopropyl 4-Cyclohexyl 12.5
16a
Cip.
Van.
> 50
0.5
B10
B11
B12
B13
B14
B15
B16
B17
B18
B19
Cip.
Van.
Cyclopropyl 2-Methoxyl
Cyclopropyl 3-Methoxyl
Cyclopropyl 4-Methoxyl
6
3
3
1.5
> 50
a
Cyclopropyl 2,4-diMethyl 1.5
Cyclopropyl 3,4-diMethyl 0.38
Data provided by Prof. Jianfeng Cai. All experiments were performed in at
least triplicates.
n-Propyl
i-Propyl
i-Butyl
4-Methyl
4-Methyl
4-Methyl
4-Methyl
4-Methyl
1.5
1.5
1.5
1.5
1.5
0.5
1.5
b
MIC: minimum inhibitory concentration. Cip.: Ciprofloxacin. Van.:
Vancomycin.
t-Butyl
Cyclohexyl
trimethylsilyldiazomethane yielded 17. Buchwald-Hartwig amination
with 2-chloro-4-arylpyrimidines and subsequent hydrolysis provided
the target hybrids A1-7. For comparison, A8 was also prepared. Buch-
wald-Hartwig amination with 2-chloro-4-aryloxypyrimidines and sub-
sequent hydrolysis afforded the target hybrids B1-22.
> 50
a
Data provided by Prof. Jianfeng Cai. All experiments were performed in at
least triplicates.
and the pyrimidine moiety with the aim to increase the structural
flexibility, thus enhancing the interactions with DNA topoisomerase IV.
A series of new hybrids B were prepared and assessed in parallel with
ciprofloxacin and vancomycin against MRSA and E. coli.
2. Results and discussion
2.1. Inhibitory activities against MRSA ATCC33591
As presented in Table 2, all the hybrids with an oxygen-linker ex-
hibited moderate to excellent anti-MRSA potency with MIC values
ranging from 0.38 to 12.5 μg/mL. Interestingly, a loss of activity in
inhibiting E. coli with these new hybrids B was observed as well,
showing Gram-positive specific inhibitory activity. The introduction of
electron-donating groups into the benzene ring (B2-15) dramatically
increased the anti-MRSA activity regardless of the features of the sub-
stituted groups, except for B9. The positive effect of introducing a
substituent at the para position was greater than that at the ortho or
meta position (B4 vs B2-3, B12 vs B10). Additionally, the introduction
of a linear alkyl group at the para position of the benzene ring showed a
decrease in activity with the length of the alkyl group (B4-6). The 4-
dimethyl (B4) and 3,4-dimethyl (B14) compounds appeared to be the
most active ones against MRSA with a MIC value of 0.38 μg/mL, being
approximately 4-fold more potent than vancomycin and comparable to
ciprofloxacin. N-substituent variation was extended to other more
substituents (B15-19), and the N-cyclopropyl was confirmed to be op-
timal for anti-MRSA activity.
At the outset of our study, desfluoroquinolone-aminopyrimidine
hybrids A1-7 as well as parent compounds A8 and 16a were synthe-
sized and evaluated for antibacterial activities against two bacterial
cells (MRSA ATCC33591 and E. coli ATCC25922). FDA-approved drugs
including commercial ciprofloxacin and vancomycin were used as re-
ference according to the same procedure.
The summarized results are shown in Table 1. Hybrid A1, char-
acterized by a cyclopropyl moiety at N-1 and a phenyl group directly
linked to the pyrimidine ring, was devoid of anti-MRSA activity even at
a concentration of 50 μg/mL. Although the introduction of a methyl
group at the ortho position (A2) of the benzene ring still remained no
inhibitory activity, the introduction of a methyl group at the meta or
para position of the benzene ring resulted in A3 and A4, exhibiting MIC
values of 6 and 0.75 μg/mL, respectively. Particularly, A4 was proved
to be slightly less active than ciprofloxacin. We next introduced di-
methyl groups into the benzene ring and found that both 2,4-dimethyl
compound (A5) and 3,4-dimethyl compound (A6) showed good in-
hibitory activities, whereas the parent compound 16a were completely
inactive. Removal of the substituted benzene ring afforded A8, resulting
in dramatically decreased activity. These observations highlight the
importance of the hybrid scaffold for the anti-MRSA activity. All the
synthesized compounds proved to be inactive in inhibiting Gram-ne-
gative E. coli at a concentration of 50 μg/mL, suggesting that these
hybrids may exhibit Gram-positive specific inhibitory activity.
2.2. Inhibitory activities against clinical isolates of MRSA
The antibacterial activities of B4 and B14 were further evaluated
against S. aureus Newman strain and a panel of clinical isolates of MRSA
(NRS-1, NRS-70, NRS-100, NRS-108, NRS-271). As shown in Table 3,
both B4 and B14 were more potent against S. aureus Newman strain
than vancomycin. In particular, B14 strongly exhibited antibacterial
activity against all five clinical isolates of MRSA, with MIC values be-
tween < 0.17 and 0.69 μg/mL, whereas B4 had comparable activity as
vancomycin with a MIC value of 1.38 μg/mL.
With this first set of compounds in our hands, we decided to make
further modification in an attempt to obtain more potent anti-MRSA
agents. Considering the rigidity of the structure of hybrids A, an
oxygen-linker was introduced to connect the substituted benzene ring
3

R. Song, et al.
BioorganicChemistry103(2020)104176
Table 3
Antibacterial activity of B4 and B14 against S. aureus Newman strain and a
panel of clinical isolates of MRSA.a
Compd
MICa (μg/mL)
Newman
NRS-1
NRS-70
NRS-100
NRS-108
NRS-271
B4
0.8
1.38
0.35
3.13
1.38
< 0.17
0.78
1.38
0.35
1.56
1.38
0.69
0.78
1.38
0.35
0.78
B14
Van.
0.2
1.56
a
Data provided by Prof. Caiguang Yang. All experiments were performed in
at least triplicates. MRSA strains: NRS-1 (resistant to aminoglycosides and tet-
racycline), NRS-70 (resistant to erythromycin), NRS-100 (resistant to oxacillin
and tetracycline), NRS-108 (resistant to gentamicin), NRS-271 (resistant to
linezolid).
Fig. 4. Bacterial resistance study of B14 against MRSA ATCC43300. Data
provided by Sichuan Primed Shines Bio-tech Co., Ltd.
2.3. Inhibitory activities against fluoroquinolone-resistant MRSA and VISA
control. Starting MIC values for B14 and vancomycin were found to be
0.5 μg/mL. ATCC43300 was exposed to B14 and vancomycin from MIC
for sustained passages and the new MIC values were determined every
passage after propagation of MRSA cultures with fresh media, respec-
tively. As shown in Fig. 4, the MIC value of B14 toward MRSA did not
change even after 15 passages, whereas MIC of vancomycin increased
by 4-fold. It indicates that MRSA is less likely to acquire resistance
against B14 compared with vancomycin.
In addition to the five MRSA above, three clinical isolates of fluor-
oquinolone-resistant MRSA (19–25, 19–26, 19–27) and two VISA
standard strains (ATCC700788, ATCC700699) were also selected to
explore the antibacterial activities of B4 and B14, as presented in
Table 4. To our pleasure, both quinolone-based hybrids B4 and B14
were able to inhibit all three clinical isolates of fluoroquinolone-re-
sistant MRSA. Notably, B4 displayed comparable activity as vanco-
mycin, whereas B14 had approximately 16-fold more potent than this
reference drug. Moreover, B14 also exhibited good potency against
VISA, while vancomycin showed reduced susceptibility.
2.6. Docking study
In an attempt to investigate the potential binding mode of the newly
synthesized hybrids, molecular simulation was conducted by using
Sybyl-X 2.0. The model of S. aureus topoisomerase IV was homology
constructed based on the original X-ray crystal structures of Topo IV
from S. pneumoniae (pdb: 4KPF) [24].
2.4. Initial safety evaluation
As an initial safety evaluation, we assessed hERG inhibition and
assayed for potential mammalian cytotoxicity with lung carcinoma
A549, breast adenocarcinoma MDA-MB-231, prostate adenocarcinoma
PC-3, as well as bonvine aorta endothelial ABAE. The results of the
cytotoxicity assay showed that both B4 and B14 were unable to inhibit
the growth of the three tested human cancer cell lines or ABAE cells at
the highest tested concentration (100 μM), indicating its highly selec-
tive toxicity toward bacterial cells. Inhibition of cardiac potassium
channels encoded by hERG is frequently associated with QT interval
prolongation and life-threatening arrhythmia [14]. Hence, B4 and B14
were tested for their abilities to inhibit hERG potassium channel and
cisapride was used as a positive control. The results showed that B4 and
B14 demonstrated marginal inhibitory activities with an IC50 exceeding
40 μM, while the reference cisapride exhibited strong inhibitory activity
with an IC50 value of 0.035 μM.
Hybrids A6 and B14 were chosen as representatives to be docked
into the homology constructed topoisomerase IV/DNA complex. As
shown in Fig. 5a and b, A6 and B14 established similar ligand-receptor
2+
interactions: (i) A Mg
chelation formed with the quinolone C3/C4
keto acid, (ii) Three hydrogen bonds between the nitrogen atom of the
NH linker and DA5, the nitrogen atom of the pyrimidine ring and DG1,
the quinolone 3-carboxylic acid and Arg118, (iii) Favorable π-π inter-
action of the quinolone ring with DG1 and DA5. The difference in the
ligand-enzyme interactions is that 3,4-dimethylphenyl group of B14
(Score 13.2917) is positioned deeply in a pocket, as defined by the side
chains of LYS455, VAL456, LIE457, to engage hydrophobic interac-
tions. Conversely, the 3,4-dimethylphenyl moiety of A6 (Score
11.1128) is positioned toward the solvent accessible area (Fig. 5c).
2.5. Propensity to induce bacterial resistance
3. Conclusions
A common concern for newly obtained antibiotics is the emergence
of rapid resistance. Hence, B14 was further subjected to the bacterial
resistance study using MRSA ATCC43300 as the test pathogen due to its
superior antibacterial profile. Vancomycin was used as a positive
Based on the understanding of quinolone-enzyme interactions, a
series of desfluoroquinolone-aminopyrimidine hybrids was designed
and synthesized with the aim to combat multidrug resistance and car-
diotoxicity. Despite the lack of the typical C-6 fluorine atom on the
Table 4
Antibacterial activity of B4 and B14 against fluoroquinolone-resistant MRSA and VISA.a
Compd
MICa (μg/mL)
Standard strain
ATCC29213
Levofloxacin-resistant strain
Vancomycin-intermediate strain
ATCC700788
19–25
19–26
19–27
ATCC700699
B4
1
2
2
2
2
2
B14
Van.
Lev.
0.125
1
0.125
2
0.125
2
0.125
2
0.25
4
1
8
0.125
> 64
> 64
> 64
64
16
a
Data provided by Sichuan Primed Shines Bio-tech Co., Ltd. All experiments were performed in at least triplicates. Lev. = Levofloxacin.
4

R. Song, et al.
BioorganicChemistry103(2020)104176
reducing the dependence on the magnesium-water bridge interactions
with topoisomerase IV. These results suggested a promising strategy via
enhancing functionality of C-7 substituent for developing new anti-
MRSA quinolones with improved efficacy, resistance profile, and low
cardiotoxicity.
4. Experimental section
4.1. Chemistry
1H NMR and 13C NMR spectra were recorded on a Bruker Avance
Neo 400 MHz spectrometer. Chemical shifts are reported in δ (ppm)
units relative to the internal standard tetramethylsilane (TMS). HRMS
were obtained on a Bruker micrOTOF II or Bruker Compact instrument
using electrospray ionization (ESI) techniques. Melting points were
measured with a SGW X-1 microscopic melting-point apparatus and are
uncorrected. All chemicals and solvents used were of reagent grade and
were purified and dried by standard methods before use. All the reac-
tions were monitored by thin layer chromatography (TLC) on pre-
coated silica gel G plates at 254 nm under a UV lamp using di-
chloromethane/methanol or ethyl acetate/hexane as eluent. Column
chromatography separations were obtained on silica gel (300–400
mesh) using dichloromethane and methanol as eluents. Analysis of
sample purity was performed on a Shimadzu LC-20AD series HPLC
system with C18 Inertsil ODS-3 (4.6 mm × 250 mm × 5 μm). HPLC
conditions were the following: solvent A = water (0.03% TFA), solvent
B = MeCN; Gradient: 5% B increase to 95% B within 10.5 min, 95% B
decrease to 5% B within 5 min, 5% B for 5 min; flow rate = 1 mL/min.
Purity was determined by the absorbance at 254 nm. All tested com-
pounds have a purity of > 95%.
General procedure for the preparation of methyl 7-amino-1-alkyl-4-
oxo-1,4-dihydroquinoline-3-carboxylate (17). To a mixture of 16
(2.0 mmol), which was prepared according to our previously reported
protocol [25], in a mixture solution of dichloromethane (30 mL) and
methanol (10 mL) was added TMSCHN2 (3.0 mmol, 2 M in cyclo-
hexane) at 0 °C. The resulting mixture was allowed to warm to room
temperature. After stirring at room temperature for 24 h, 1% aqueous
acetic acid (0.5 mL) was added slowly to the reaction solution. The
solvents were removed under reduced pressure. The resulting pre-
cipitate was filtered, washed by water, and dried. The crude was pur-
ified by column chromatography on silica gel (dichloromethane/ me-
thanol 25:1, v/v) to afford 17 as a solid.
Methyl 7-amino-1-cyclopropyl-4-oxo-1,4-dihydro-quinolone-3-
carboxylate (17a). Yield 72% (from 16); white solid; m.p.
274.7–275.9 °C; 1H NMR (400 MHz, DMSO‑d6) δ 8.34 (s, 1H), 7.88 (d,
J = 8.7 Hz, 1H), 7.03 (s, 1H), 6.72 (d, J = 8.7 Hz, 1H), 6.22 (s, 2H),
4.16–4.10 (m, 1H), 3.18 (s, 3H), 1.22–1.05 (m, 4H).
Fig. 5. (a) Predicted binding mode of hybrid A6, (b) predicted binding mode of
hybrid B14, c) predicted binding mode of A6 (cyan) and B14 (orange) with
topoisomerase IV/DNA complex (derived from PDB code 4KPF) for comparison.
Hydrogen bonds are indicated with dashed lines in yellow. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
Methyl
7-amino-1-propyl-4-oxo-1,4-dihydroquinoline-3-car-
boxylate (17b). Yield 72% (from 16); white solid; m.p.
288.2–289.0 °C; 1H NMR (400 MHz, DMSO‑d6) δ 8.48 (s, 1H), 7.89 (d,
J = 8.8 Hz, 1H), 6.67 (d, J = 9.2 Hz, 1H), 6.63 (s, 1H), 6.08 (s, 2H),
4.11 (t, J = 7.2 Hz, 2H), 3.76 (s, 3H), 1.81–1.72 (m, 2H), 0.91 (t,
J = 7.2 Hz, 3H).
quinolone nucleus, majority of these hybrids still demonstrated potent
anti-MRSA activity with MIC values between 0.38 and 1.5 μg/mL.
Among them, B14 exhibited powerful inhibitory activity against a
panel of MRSA strains including vancomycin-intermediate strains,
aminoglycosides and tetracycline-resistant isolates, linezolid-resistant
isolates, etc. Notably, this quinolone-based agent even can inhibit le-
vofloxacin-resistant isolates. As expected, B14 also showed highly se-
lective toxicity toward bacterial cells and no significant hERG toxicity.
Further bacterial resistance study suggested that MRSA is unlikely to
acquire resistance against B14. The docking study revealed that the
introduced C-7 aminopyrimidine functional group established two hy-
drogen bonds with the surrounding DNA bases. The additional drug-
enzyme contacts might contribute to overcome quinolone resistance by
Methyl
7-amino-1-isopropyl-4-oxo-1,4-dihydro-quinoline-3-
carboxylate (17c). Yield 73% (from 16); white solid; m.p.
308.3–309.1 °C; 1H NMR (400 MHz, DMSO‑d6) δ 8.44 (s, 1H), 7.95 (d,
J = 8.7 Hz, 1H), 6.82 (s, 1H), 6.72 (d, J = 8.7 Hz, 1H), 6.10 (s, 2H),
4.78–4.72 (m, 1H), 3.73 (s, 3H), 1.49 (d, J = 6.5 Hz, 6H).
Methyl 7-amino-1-isobutyl-4-oxo-1,4-dihydroquino-line-3-car-
boxylate (17d). Yield 73% (from 16); white solid; m.p.
243.8–244.6 °C; 1H NMR (400 MHz, DMSO‑d6) δ 8.42 (s, 1H), 7.89 (d,
J = 8.8 Hz, 1H), 6.67 (d, J = 8.7 Hz, 1H), 6.62 (s, 1H), 6.08 (s, 2H),
3.97 (d, J = 7.4 Hz, 2H), 3.70 (s, 3H), 2.19–2.13 (m, 1H), 0.89 (d,
J = 6.4 Hz, 6H).
Methyl 7-amino-1-(tert-butyl)-4-oxo-1,4-dihydro-quinoline-3-
carboxylate (17e). Yield 74% (from 16); white solid; m.p.
5

R. Song, et al.
BioorganicChemistry103(2020)104176
221.8–222.3 °C; 1H NMR (400 MHz, CDCl3) δ 8.99 (s, 1H), 8.37 (d,
J = 8.7 Hz, 1H), 7.00 (d, J = 1.5 Hz, 1H), 6.75 (dd, J = 8.7 Hz, 1.5 Hz,
1H), 3.89 (s, 3H), 1.85 (s, 9H).
2H), 1.31–1.25 (m, 5H), 1.15–1.10 (m, 2H).
General procedure for the preparation of 1-alkyl-4-oxo-7-
quinoline-3-car-
((phenylpyrimidin-2-yl)amino)-1,4-dihydro
Methyl 7-amino-1-cyclohexyl-4-oxo-1,4-dihydro-quinoline-3-
carboxylate (17f). Yield 64% (from 16); white solid; m.p.
226.0–226.9 °C; 1H NMR (400 MHz, DMSO‑d6) δ 8.63 (s, 1H), 8.04 (d,
J = 8.8 Hz, 1H), 6.94(s, 1H), 6.88 (d, J = 8.8 Hz, 1H), 6.48 (s, 2H),
5.76–5.72 (m, 1H), 4.45–4.40 (m, 1H), 3.88 (s, 3H), 2.05–1.26 (m,
10H).
boxylic acid (A1-7). To a solution of 18 (1.0 mmol) in THF (8 mL) was
added satd. aq. NaOH (5 mL, 5 M). After being stirred at 50 °C for 3 h,
the mixture was cooled, poured into ice-water and acidified with 4 M
HCl to pH ~ 2. The precipitate was filtered off, washed by water, and
dried. The crude was purified by column chromatography on silica gel
(dichloromethane/methanol 40:1 to 25:1, v/v) to afford the desired
product as a solid.
General procedure for the preparation of methyl 1-alkyl-4-oxo-
7-((4-phenylpyrimidin-2-yl)amino)-1,4-dihydroquinoline-3-car-
boxylate (18). A Schlenk vessel was charged with 17 (2 mmol), 2-
chloro-4-phenylpyrimidine (3 mmol), Pd(OAc)2 (0.2 mmol), K2CO3
(3 mmol), BINAP (0.3 mmol) in DMF (20 mL). The reaction vessel was
sealed and the contents were stirred and heated at 90 °C for 12–18 h
under N2. After filtration, ethyl acetate (30 mL) was added and the
organic layer was washed with brine (15 mL × 3) and water (15 mL),
dried over MgSO4, and then filtered. The filtrate was evaporated and
the residue was purified by column chromatography on silica gel (di-
chloromethane/methanol 30:1, v/v) to give 18 as a solid.
1-Cyclopropyl-4-oxo-7-((4-phenylpyrimidin-2-yl) amino)-1,4-
dihydroquinoline-3-carboxylic acid (A1). Yield 83%; white solid;
m.p. 207.8–208.9 °C; 1H NMR (400 MHz, DMSO‑d6) δ 15.23 (s, 1H),
8.82 (d, J = 5.3 Hz, 1H), 8.70 (s, 1H), 8.34 (d, J = 8.8 Hz, 1H),
8.07–8.05 (m, 4H), 7.95 (d, J = 5.3 Hz, 1H), 7.56–7.47 (m, 4H),
3.71–3.66 (m, 1H), 1.00–0.94 (m, 4H); 13C NMR (100 MHz, DMSO‑d6) δ
166.3, 164.8, 162.4, 160.1, 149.2, 148.4, 142.2, 136.0, 131.7, 129.4,
129.3, 127.6, 127.3, 126.7, 124.2, 117.2, 113.6, 113.4, 35.9, 7.8;
HRMS m/z calcd for C23H18N4O3 ([M + H]+): 399.1452, found
399.1455.
Methyl 1-cyclopropyl-4-oxo-7-((4-phenylpyrimidin-2-yl)amino)-
1,4-dihydroquinoline-3-carboxylate (18a). Yield 38%; 1H NMR
(400 MHz, CDCl3) δ 8.84 (s, 1H), 8.71 (d, J = 5.3 Hz, 1H), 8.52 (d,
J = 8.8 Hz, 1H), 8.06 (s, 1H), 7.95–7.98 (m, 3H), 7.49–7.41 (m, 5H), 3.90
(s, 3H), 3.47–3.40 (m, 1H), 1.11–1.07 (m, 4H).
1-Cyclopropyl-4-oxo-7-((4-(o-tolyl)pyrimidin-2-yl) amino)-1,4-
dihydroquinoline-3-carboxylic acid (A2). Yield 85%; yellow solid;
m.p. > 320 °C; 1H NMR (400 MHz, DMSO- d6) δ 15.50 (s, 1H), 10.65
(s, 1H), 9.25 (s, 1H), 8.73 (d, J = 5.0 Hz, 1H), 8.66 (s, 1H), 8.24 (d,
J = 9.0 Hz, 1H), 7.86 (d, J = 8.6 Hz, 1H), 7.55 (d, J = 7.3 Hz, 1H),
7.42–7.33 (m, 3H), 7.21 (d, J = 5.0 Hz, 1H), 3.69–3.63 (m, 1H), 2.42
(s, 3H), 1.13–1.09 (m, 2H), 1.00–0.99 (m, 2H); 13C NMR (100 MHz,
DMSO‑d6) δ 177.4, 167.7, 166.7, 159.8, 159.0, 148.8, 146.4, 143.1,
138.4, 136.0, 131.3, 129.9, 129.7, 126.8, 126.4, 119.0, 118.5, 114.3,
107.3, 104.9, 36.2, 20.6, 7.8; HRMS m/z calcd for C24H20N4O3
([M + H]+): 413.1608, found 413.1604.
Methyl
1-cyclopropyl-4-oxo-7-((4-(o-tolyl)pyrimidin-2-yl)
amino)-1,4-dihydroquinoline-3-carboxylate (18b). Yield 42%; 1H
NMR (400 MHz, CDCl3) δ 9.06 (s, 1H), 8.57 (d, J = 5.0 Hz, 1H), 8.54 (s,
1H), 8.38 (d, J = 8.7 Hz, 1H), 8.22 (s, 1H), 7.48 (d, J = 7.5 Hz, 1H),
7.36 (d, J = 6.8 Hz, 1H), 7.31–7.25 (m, 3H), 6.99 (d, J = 4.9 Hz, 1H),
3.90 (s, 3H), 3.37–3.31 (m, 1H), 2.44 (s, 3H), 1.02–0.97 (m, 4H).
Methyl
1-cyclopropyl-4-oxo-7-((4-(m-tolyl)pyrimidin-2-yl)
1-Cyclopropyl-4-oxo-7-((4-(m-tolyl)pyrimidin-2-yl) amino)-1,4-
dihydroquinoline-3-carboxylic acid (A3). Yield 82%; white solid;
m.p. 302.6–304.0 °C; 1H NMR (400 MHz, DMSO- d6) δ 15.49 (s, 1H),
10.58 (s, 1H), 9.17 (d, J = 1.9 Hz, 1H), 8.72 (d, J = 5.2 Hz, 1H), 8.69
(s, 1H), 8.27 (d, J = 8.9 Hz, 1H), 8.05 (s, 1H), 8.00 (d, J = 7.7 Hz, 1H),
7.95 (dd, J = 9.0, 1.9 Hz, 1H), 7.58 (d, J = 5.2 Hz, 1H), 7.45 (t,
J = 7.5 Hz, 1H), 7.40 (d, J = 7.6 Hz, 1H), 3.86–3.82 (m, 1H), 2.41 (s,
3H), 1.23–1.19 (m, 4H); 13C NMR (100 MHz, DMSO‑d6) δ 177.3, 166.6,
164.6, 160.0, 159.4, 148.7, 146.4, 143.0, 138.6, 136.7, 132.2, 129.1,
127.8, 126.7, 124.7, 118.9, 118.4, 110.3, 107.2, 104.6, 36.0, 21.3, 8.0;
HRMS m/z calcd for C24H20N4O3 ([M + H]+): 413.1608, found
413.1598.
amino)-1,4-dihydroquinoline-3-carboxylate (18c). Yield 48%; 1H
NMR (400 MHz, DMSO‑d6) δ 10.34 (s, 1H), 8.96 (d, J = 2.0 Hz, 1H),
8.68 (d, J = 5.2 Hz, 1H), 8.47 (s, 1H), 8.13 (d, J = 8.8 Hz, 1H), 8.04 (s,
1H), 8.00 (d, J = 7.7 Hz, 1H), 7.80 (dd, J = 8.9, 2.0 Hz, 1H), 7.54 (d,
J = 5.2 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.39 (d, J = 7.5 Hz, 1H),
3.74 (s, 3H), 3.69–3.65 (m, 1H), 2.41 (s, 3H), 1.17–1.14 (m, 2H),
1.12–1.10 (m, 2H).
Methyl
1-cyclopropyl-4-oxo-7-((4-(p-tolyl)pyrimidin-2-yl)
amino)-1,4-dihydroquinoline-3-carboxylate (18d). Yield 57%; 1H
NMR (400 MHz, CDCl3) δ 8.88 (s, 1H), 8.61 (s, 1H), 8.54 (d, J = 5.3 Hz,
1H), 8.44 (d, J = 8.8 Hz, 1H), 8.00 (d, J = 7.8 Hz, 2H), 7.42 (d,
J = 8.9 Hz, 1H), 7.31 (d, J = 8.1 Hz, 2H), 7.26 (d, J = 5.2 Hz, 1H),
3.94 (s, 3H), 3.54–3.49 (m, 1H), 2.46 (s, 3H), 1.30–1.26 (m, 2H),
1.15–1.13 (m, 2H).
1-Cyclopropyl-4-oxo-7-((4-(p-tolyl)pyrimidin-2-yl) amino)-1,4-
dihydroquinoline-3-carboxylic acid (A4). Yield 91%; white solid;
m.p. 314.6–315.6 °C; 1H NMR (400 MHz, DMSO‑d6) δ 15.50 (s, 1H),
10.57 (s, 1H), 9.15 (s, 1H), 8.71–8.70 (m, 2H), 8.28 (d, J = 8.9 Hz, 1H),
8.14 (d, J = 7.9 Hz, 2H), 7.98 (d, J = 9.2 Hz, 1H), 7.59 (d, J = 5.2 Hz,
1H), 7.38 (d, J = 7.9 Hz, 2H), 3.88–3.82 (m, 1H), 2.41 (s, 3H),
1.26–1.21 (m, 4H); 13C NMR (100 MHz, DMSO‑d6) δ 177.3, 166.6,
164.3, 159.4, 148.7, 146.4, 143.0, 141.6, 130.0, 129.8, 127.4, 126.8,
126.7, 118.9, 118.4, 109.9, 107.2, 104.7, 36.1, 21.4, 8.0; HRMS m/z
calcd for C24H20N4O3 ([M + H]+): 413.1608, found 413.1617.
Methyl 1-cyclopropyl-4-oxo-7-((4-(2, 4-dimethylphenyl) pyr-
imidin-2-yl)amino)-1,4-dihydroquinoline-3-carboxylate
(18e).
1
Yield 55%; H NMR (400 MHz, CDCl3) δ 9.02 (s, 1H), 8.55–8.53 (m,
2H), 8.37 (d, J = 8.8 Hz, 1H), 8.11 (s, 1H), 7.40 (d, J = 7.7 Hz, 1H),
7.25 (dd, J = 8.8, 2.0 Hz, 1H), 7.11–7.08 (m, 2H), 6.97 (d, J = 5.1 Hz,
1H), 3.90 (s, 3H), 3.38–3.33 (m, 1H), 2.42 (s, 3H), 2.37 (s, 3H),
1.03–1.02 (m, 4H).
Methyl 1-cyclopropyl-4-oxo-7-((4-(3,4-dimethylphenyl) pyr-
1-Cyclopropyl-7-((4-(2,
4-dimethylphenyl)pyrimidin-2-yl)
imidin-2-yl)amino)-1,4-dihydroquinoline-3-carboxylate
(18f).
amino)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (A5). Yield
79%; yellow solid; m.p. 288.6–289.8 °C; 1H NMR (400 MHz, DMSO‑d6)
δ 10.60 (s, 1H), 9.23 (d, J = 1.9 Hz, 1H), 8.71 (d, J = 5.1 Hz, 1H), 8.67
(s, 1H), 8.25 (d, J = 8.9 Hz, 1H), 7.89 (dd, J = 8.9, 2.0 Hz, 1H), 7.47
(d, J = 7.7 Hz, 1H), 7.18 (d, J = 5.0 Hz, 2H), 7.16 (d, J = 7.9 Hz, 1H),
3.70–3.66 (m, 1H), 2.41 (s, 3H), 2.36 (s, 3H), 1.13–1.11 (m, 2H),
1.06–1.04 (m, 2H); 13C NMR (100 MHz, DMSO‑d6) δ 177.3, 167.5,
166.6, 159.6, 158.7, 148.6, 146.3, 142.9, 139.3, 138.1, 135.8, 135.4,
131.8, 129.7, 126.9, 126.6, 118.3, 114.2, 107.1, 104.7, 36.1, 21.1,
20.5, 7.7; HRMS m/z calcd for C25H22N4O3 ([M + H]+): 427.1765,
found 427.1762.
Yield 46%; 1H NMR (400 MHz, CDCl3) δ 8.92 (s, 1H), 8.60 (s, 1H), 8.52
(d, J = 5.2 Hz, 1H), 8.43 (d, J = 8.8 Hz, 1H), 7.86 (s, 1H), 7.82–7.80
(m, 2H), 7.38 (dd, J = 8.8, 1.9 Hz, 1H), 7.26 (s, 1H), 7.24 (d,
J = 2.6 Hz, 1H), 3.93 (s, 3H), 3.78–3.74 (m, 1H), 2.35 (s, 6H),
1.28–1.23 (m, 2H), 1.16–1.10 (m, 2H).
Methyl 1-cyclopropyl-4-oxo-7-((4-(4-propylphenyl) pyrimidin-
2-yl)amino)-1,4-dihydroquinoline-3-carboxylate (18 g). Yield 40%;
1H NMR (400 MHz, CDCl3) δ 8.89 (s, 1H), 8.61 (s, 1H), 8.53 (d,
J = 5.2 Hz, 1H), 8.44 (d, J = 8.7 Hz, 1H), 8.01 (d, J = 8.1 Hz, 2H),
7.68 (s, 1H), 7.41 (d, J = 8.2 Hz, 1H), 7.35 (d, J = 8.1 Hz, 2H), 7.25 (s,
1H), 3.93 (s, 3H), 3.76–3.73 (m, 2H), 3.54–3.48 (m, 1H), 1.87–1.84 (m,
1-Cyclopropyl-7-((4-(3,4-dimethylphenyl)pyrimidin-2-yl)
6

R. Song, et al.
BioorganicChemistry103(2020)104176
amino)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (A6). Yield
85%; yellow solid; m.p. 316.9–318.8 °C; 1H NMR (400 MHz, DMSO‑d6)
δ 10.55 (s, 1H), 9.18 (d, J = 1.9 Hz, 1H), 8.70–8.68 (m, 2H), 8.27 (d,
J = 9.0 Hz, 1H), 8.02 (s, 1H), 7.97–7.94 (m, 2H), 7.57 (d, J = 5.3 Hz,
1H), 7.32 (d, J = 7.9 Hz, 1H), 3.88–3.83 (m, 1H), 2.32 (s, 3H), 2.31 (s,
3H), 1.23–1.20 (m, 4H); 13C NMR (100 MHz, DMSO‑d6) δ 177.3, 166.6,
164.5, 160.0, 159.3, 148.6, 146.4, 143.0, 140.4, 137.3, 134.2, 130.3,
128.2, 126.7, 125.0, 118.8, 118.4, 109.9, 107.2, 104.6, 36.1, 29.3,
19.8, 8.0; HRMS m/z calcd for C25H22N4O3 ([M + H]+): 427.1765,
found 427.1766.
126.8, 126.6, 122.5, 119.2, 119.1, 118.3, 107.3, 105.4, 100.8, 36.0,
21.3, 8.1; HRMS m/z calcd for C24H20N4O4 ([M + H]+): 429.1557,
found 429.1540.
1-Cyclopropyl-4-oxo-7-((4-(p-tolyloxy)pyrimidin-2-yl) amino)-
1,4-dihydroquinoline-3-carboxylic acid (B4). Yield 82%; white
solid; m.p. 301.8–302.4 °C; 1H NMR (400 MHz, CDCl3) δ 15.19 (s, 1H),
8.85 (s, 1H), 8.80 (s, 1H), 8.37 (d, J = 5.2 Hz, 1H), 8.33 (d, J = 9.0 Hz,
1H), 7.53 (s, 1H), 7.33 (d, J = 9.4 Hz, 1H), 7.23 (d, J = 7.9 Hz, 2H),
7.07 (d, J = 7.9 Hz, 2H), 6.46 (d, J = 5.3 Hz, 1H), 3.32–3.20 (m, 1H),
2.42 (s, 3H), 1.30–1.24 (m, 2H), 1.18–1.07 (m, 2H); 13C NMR
(100 MHz, CDCl3) δ 177.8, 170.1, 167.3, 159.4, 159.0, 150.2, 147.8,
144.4, 142.7, 135.6, 130.2, 127.7, 121.2, 120.2, 117.5, 108.4, 104.2,
100.9, 35.1, 20.9, 8.1; HRMS m/z calcd for C24H20N4O4 ([M + H]+):
429.1557, found 429.1565.
1-Cyclopropyl-4-oxo-7-((4-(4-propylphenyl)
pyrimidin-2-yl)
amino)-1,4-dihydro quinoline-3-carboxylic acid (A7). Yield 72%;
yellow solid; m.p. 299.6–300.2 °C; 1H NMR (400 MHz, DMSO‑d6) δ
15.50 (s, 1H), 10.57 (s, 1H), 9.15 (d, J = 2.0 Hz, 1H), 8.71 (s, 1H), 8.70
(s, 1H), 8.29 (d, J = 8.9 Hz, 1H), 8.16 (d, J = 8.3 Hz, 2H), 8.00 (dd,
J = 9.0, 1.9 Hz, 1H), 7.58 (d, J = 5.3 Hz, 1H), 7.44 (d, J = 8.3 Hz, 2H),
3.86–3.82 (m, 1H), 3.03–2.96 (m, 2H), 2.03–1.97 (m, 2H), 1.27–1.20
(m, 7H); 13C NMR (100 MHz, DMSO‑d6) δ 177.3, 166.6, 164.4, 160.0,
159.4, 152.3, 148.6, 146.4, 142.9, 134.4, 127.6, 127.2, 126.7, 118.9,
118.4, 110.0, 107.2, 104.7, 36.1, 33.7, 26.9, 24.0, 8.0; HRMS m/z calcd
for C26H24N4O3 ([M + H]+): 441.1921, found 441.1926.
1-Cyclopropyl-7-((4-(4-ethylphenoxy)pyrimidin-2-yl) amino)-
4-oxo-1,4-dihydroquinoline-3-carboxylic acid (B5). Yield 71%;
white solid; m.p. 289.0–290.7 °C; 1H NMR (400 MHz, DMSO‑d6) δ
10.42 (s, 1H), 8.75 (s, 1H), 8.63 (s, 1H), 8.50 (d, J = 5.6 Hz, 1H), 8.08
(d, J = 9.0 Hz, 1H), 7.83 (dd, J = 9.0, 1.7 Hz, 1H), 7.32 (d, J = 8.5 Hz,
2H), 7.17 (d, J = 8.5 Hz, 2H), 6.58 (d, J = 5.6 Hz, 1H), 3.46–3.41 (m,
1H), 2.66 (q, J = 7.6 Hz, 2H), 1.25–1.21 (m, 5H), 1.15–1.11 (m, 2H);
13C NMR (100 MHz, DMSO‑d6) δ 177.4, 169.9, 166.6, 160.5, 159.6,
150.7, 148.7, 146.1, 142.7, 141.6, 129.6, 126.5, 121.9, 119.1, 118.3,
107.2, 105.3, 100.8, 35.9, 28.0, 16.1, 8.0; HRMS m/z calcd for
1-Cyclopropyl-4-oxo-7-(pyrimidin-2-ylamino)-1,4-dihy-
droquinoline-3-carboxylic acid (A8)
The preparation for A8 was similar to A1-7. Yield 44%; white solid;
m.p. 325.7–327.2 °C; 1H NMR (400 MHz, DMSO‑d6) δ 10.56 (s, 1H),
9.14 (s, 1H), 8.68–8.66 (m, 3H), 8.24 (d, J = 9.5 Hz, 1H), 7.90 (dd,
J = 8.9, 2.0 Hz, 1H), 7.05 (td, J = 4.7, 1.6 Hz, 1H), 3.75–3.73 (m, 1H),
1.36–1.35 (m, 2H), 1.29–1.22 (m, 2H); 13C NMR (100 MHz, DMSO‑d6)
δ 177.3, 166.6, 159.8, 158.6, 148.7, 146.2, 142.9, 130.0, 126.6, 118.2,
114.5, 107.2, 104.8, 36.0, 7.9; HRMS m/z calcd for C17H13N4O3
([M−H]−): 321.0993, found 321.0981.
C
25H22N4O4 ([M + H]+): 443.1714, found 443.1702.
7-((4-(4-Butylphenoxy)pyrimidin-2-yl)amino)-1-cyclopropyl-4-
oxo-1,4-dihydroquinoline-3-carboxylic acid (B6). Yield 72%; white
1
solid; m.p. 237.6–238.4 °C; H NMR (400 MHz, DMSO‑d6) δ 10.40 (s,
1H), 8.71 (s, 1H), 8.62 (s, 1H), 8.49 (d, J = 5.6 Hz, 1H), 8.07 (d,
J = 9.2 Hz, 1H), 7.82 (d, J = 9.1 Hz, 1H), 7.29 (d, J = 7.5 Hz, 2H),
7.15 (d, J = 7.6 Hz, 2H), 6.57 (d, J = 5.2 Hz, 1H), 3.44–3.39 (m, 1H),
2.62 (t, J = 7.5 Hz, 2H), 1.63–1.55 (m, 2H), 1.38–1.29 (m, 2H),
1.24–1.20 (m, 2H), 1.15–1.10 (m, 2H), 0.91 (t, J = 7.3 Hz, 3H); 13C
NMR (100 MHz, DMSO‑d6) δ 177.5, 170.0, 166.6, 160.6, 159.7, 150.8,
148.8, 146.2, 142.7, 140.3, 130.1, 126.6, 121.8, 119.1, 118.3, 107.3,
105.4, 100.8, 36.0, 34.7, 33.7, 22.3, 14.2, 8.0; HRMS m/z calcd for
General procedure for the preparation of 1-alkyl-4-oxo-7-((4-
phenylpyrimidin-2-yl)amino)-1,4-dihydro-quinoline-3-carboxylic
acid (B1-24). The preparation for B1-22 was similar to A1-7.
1-Cyclopropyl-4-oxo-7-((4-phenoxypyrimidin-2-yl) amino)-1,4-
dihydroquinoline-3-carboxylic acid (B1). Yield 70%; yellow solid;
m.p. 295.5–296.8 °C; 1H NMR (400 MHz, DMSO‑d6) δ 10.43 (s, 1H),
8.75 (s, 1H), 8.65 (s, 1H), 8.54 (d, J = 5.4 Hz, 1H), 8.08 (d, J = 9.0 Hz,
1H), 7.84 (d, J = 9.0 Hz, 1H), 7.51 (t, J = 7.2 Hz, 2H), 7.35 (t,
J = 7.3 Hz, 1H), 7.30 (d, J = 7.8 Hz, 2H), 6.61 (d, J = 5.4 Hz, 1H),
3.50–3.45 (m, 1H), 1.25–1.21 (m, 2H), 1.17–1.13 (m, 2H); 13C NMR
(100 MHz, DMSO‑d6) δ 177.5, 169.9, 166.6, 160.7, 159.7, 152.9, 148.8,
146.2, 142.8, 130.5, 126.6, 126.1, 122.1, 119.2, 118.3, 107.3, 105.4,
100.9, 36.0, 8.0; HRMS m/z calcd for C23H18N4O4 ([M + H]+):
415.1401, found 415.1408.
C
27H26N4O4 ([M + H]+): 471.2027, found 471.2036.
1-Cyclopropyl-7-((4-(4-isopropylphenoxy)pyrimidin-2-yl)
amino)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (B7). Yield
72%; white solid; m.p. 286.9–288.8 °C; 1H NMR (400 MHz, DMSO‑d6) δ
10.41 (s, 1H), 8.72 (s, 1H), 8.62 (s, 1H), 8.50 (d, J = 5.3 Hz, 1H), 8.07
(d, J = 8.9 Hz, 1H), 7.83 (d, J = 9.0 Hz, 1H), 7.35 (d, J = 7.8 Hz, 2H),
7.17 (d, J = 7.8 Hz, 2H), 6.58 (d, J = 5.7 Hz, 1H), 3.46–3.39 (m, 1H),
2.99–2.92 (m, 1H), 1.24 (d, J = 6.9 Hz, 6H), 1.22–1.20 (m, 3H),
1.15–1.10 (m, 2H); 13C NMR (100 MHz, DMSO‑d6) δ 177.5, 170.0,
166.7, 160.6, 159.7, 150.8, 148.7, 146.2, 146.2, 142.7, 128.1, 126.6,
121.8, 119.2, 118.3, 107.3, 105.4, 100.8, 36.0, 33.4, 24.5, 8.1; HRMS
m/z calcd for C26H24N4O4 ([M + H]+): 457.1870, found 457.1851.
7-((4-(4-(tert-Butyl)phenoxy)pyrimidin-2-yl)amino)-1-cyclo-
propyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (B8). Yield
69%; white solid; m.p. 302.1–303.2 °C; 1H NMR (400 MHz, DMSO‑d6) δ
10.42 (s, 1H), 8.71 (s, 1H), 8.62 (s, 1H), 8.51 (d, J = 5.5 Hz, 1H), 8.06
(d, J = 8.9 Hz, 1H), 7.83 (d, J = 9.0 Hz, 1H), 7.49 (d, J = 8.3 Hz, 2H),
7.18 (d, J = 8.1 Hz, 2H), 6.59 (d, J = 5.5 Hz, 1H), 3.46–3.39 (m, 1H),
1.33 (s, 9H), 1.23–1.22 (m, 2H), 1.16–1.11 (m, 2H); 13C NMR
(100 MHz, DMSO‑d6) δ 177.4, 169.9, 166.6, 160.6, 159.6, 150.5, 148.7,
148.4, 146.1, 142.6, 127.0, 126.5, 121.4, 119.1, 118.2, 107.2, 105.4,
100.8, 36.9, 34.7, 31.7, 8.0; HRMS m/z calcd for C27H26N4O4
([M + H]+): 471.2027, found 471.2016.
1-Cyclopropyl-4-oxo-7-((4-(o-tolyloxy)pyrimidin-2-yl) amino)-
1,4-dihydroquinoline-3-carboxylic acid (B2). Yield 83%; white
1
solid; m.p. 266.7–268.6 °C; H NMR (400 MHz, DMSO‑d6) δ 10.41 (s,
1H), 8.72 (s, 1H), 8.64 (s, 1H), 8.51 (d, J = 5.5 Hz, 1H), 8.03 (d,
J = 9.0 Hz, 1H), 7.80 (d, J = 8.9 Hz, 1H), 7.39 (d, J = 7.3 Hz, 1H),
7.32 (t, J = 6.9 Hz, 1H), 7.26 (t, J = 6.8 Hz, 1H), 7.20 (d, J = 7.7 Hz,
1H), 6.57 (d, J = 5.6 Hz, 1H), 3.57–3.50 (m, 1H), 2.12 (s, 3H),
1.26–1.22 (m, 2H), 1.17–1.12 (m, 2H); 13C NMR (100 MHz, DMSO‑d6)
δ 177.4, 169.6, 166.6, 160.6, 159.7, 151.2, 148.8, 146.1, 142.7, 131.9,
130.6, 127.9, 126.5, 126.4, 122.5, 119.1, 118.1, 107.2, 105.4, 100.2,
36.0, 16.3, 8.0; HRMS m/z calcd for C24H20N4O4 ([M + H]+):
429.1557, found 429.1541.
1-Cyclopropyl-4-oxo-7-((4-(m-tolyloxy)pyrimidin-2-yl) amino)-
1,4-dihydroquinoline-3-carboxylic acid (B3). white solid; m.p.
1
285.1–286.7 °C; H NMR (400 MHz, DMSO‑d6) δ 15.41 (s, 1H), 10.43
7-((4-(4-Cyclohexylphenoxy)pyrimidin-2-yl)amino)-1-cyclo-
propyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (B9). Yield
74%; white solid; m.p. 297.5–299.0 °C; 1H NMR (400 MHz, DMSO‑d6) δ
10.42 (s, 1H), 8.72 (s, 1H), 8.62 (s, 1H), 8.50 (d, J = 5.6 Hz, 1H), 8.07
(d, J = 9.0 Hz, 1H), 7.82 (d, J = 8.9 Hz, 1H), 7.32 (d, J = 7.9 Hz, 2H),
7.16 (d, J = 7.7 Hz, 2H), 6.58 (d, J = 5.7 Hz, 1H), 3.46–3.41 (m, 1H),
2.59–2.53 (m, 1H), 1.82–1.70 (m, 5H), 1.48–1.29 (m, 5H), 1.24–1.20
(s, 1H), 8.79 (s, 1H), 8.66 (s, 1H), 8.53 (d, J = 5.6 Hz, 1H), 8.10 (d,
J = 9.0 Hz, 1H), 7.88 (dd, J = 9.0, 1.9 Hz, 1H), 7.39 (t, J = 7.8 Hz,
1H), 7.17 (d, J = 7.5 Hz, 1H), 7.12 (s, 1H), 7.09 (d, J = 8.0 Hz, 1H),
6.59 (d, J = 5.6 Hz, 1H), 3.56–3.51 (m, 1H), 2.36 (s, 3H), 1.26–1.24
(m, 2H), 1.18–1.16 (m, 2H); 13C NMR (100 MHz, DMSO‑d6) δ 177.5,
169.9, 166.6, 160.6, 159.7, 152.8, 148.8, 146.2, 142.8, 140.3, 130.2,
7

R. Song, et al.
BioorganicChemistry103(2020)104176
(m, 2H), 1.17–1.10 (m, 2H); 13C NMR (100 MHz, DMSO- d6) δ 177.4,
169.9, 166.7, 160.6, 159.6, 150.8, 148.7, 146.1, 145.4, 142.6, 128.4,
126.6, 121.8, 119.1, 118.3, 107.2, 105.4, 100.8, 43.6, 36.0, 34.5, 26.8,
26.0, 8.0; HRMS m/z calcd for C29H28N4O4 ([M + H]+): 497.2183,
found 497.2186.
1H), 8.94 (s, 1H), 8.47 (d, J = 5.5 Hz, 1H), 8.32 (s, 1H), 8.10 (d,
J = 8.9 Hz, 1H), 7.85 (d, J = 8.9 Hz, 1H), 7.29 (d, J = 7.9 Hz, 2H),
7.14 (d, J = 7.9 Hz, 2H), 6.55 (d, J = 5.5 Hz, 1H), 4.24 (t, J = 6.9 Hz,
2H), 2.36 (s, 3H), 1.84–1.79 (m, 2H), 0.90 (t, J = 7.2 Hz, 3H); 13C NMR
(100 MHz, DMSO‑d6) δ 177.2, 170.0, 166.8, 160.5, 159.6, 150.5, 149.6,
146.2, 140.7, 135.4, 130.8, 126.9, 121.9, 119.6, 118.2, 107.3, 104.8,
100.7, 55.4, 21.9, 20.9, 11.1; HRMS m/z calcd for C24H22N4O4
([M + H]+): 431.1714, found 431.1710.
1-Cyclopropyl-7-((4-(2-methoxyphenoxy)pyrimidin-2-yl)
amino)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (B10). Yield
75%; white solid; m.p. 265.5–267.2 °C; 1H NMR (400 MHz, DMSO‑d6) δ
15.40 (s, 1H), 10.38 (s, 1H), 8.69 (s, 1H), 8.66 (s, 1H), 8.50 (d,
J = 5.6 Hz, 1H), 8.06 (d, J = 9.0 Hz, 1H), 7.85 (dd, J = 9.0, 1.8 Hz,
1H), 7.36 (td, J = 7.7, 1.3 Hz, 1H), 7.29–7.25 (m, 2H), 7.07 (td,
J = 7.7, 1.3 Hz, 1H), 6.58 (d, J = 5.6 Hz, 1H), 3.74 (s, 3H), 3.60–3.54
(m, 1H), 1.30–1.25 (m, 2H), 1.19–1.15 (m, 2H); 13C NMR (100 MHz,
DMSO‑d6) δ 177.4, 169.7, 166.6, 160.3, 159.6, 151.7, 148.8, 146.2,
142.7, 141.2, 127.4, 126.5, 123.4, 121.5, 119.0, 118.0, 113.8, 107.2,
105.4, 100.2, 56.2, 36.0, 8.0; HRMS m/z calcd for C24H20N4O5
([M + H]+): 445.1506, found 445.1502.
1-Isopropyl-4-oxo-7-((4-(p-tolyloxy)pyrimidin-2-yl) amino)-1,4-
dihydroquinoline-3-carboxylic acid (B16). Yield 76%; white solid;
m.p. 279.4–280.6 °C; 1H NMR (400 MHz, DMSO‑d6) δ 15.54 (s, 1H),
10.27 (s, 1H), 8.78 (s, 1H), 8.50 (d, J = 5.6 Hz, 1H), 8.46 (s, 1H), 8.14
(d, J = 9.0 Hz, 1H), 7.92 (d, J = 8.2 Hz, 1H), 7.31 (d, J = 8.3 Hz, 2H),
7.16 (d, J = 8.4 Hz, 2H), 6.59 (d, J = 5.6 Hz, 1H), 4.83–4.79 (m, 1H),
2.37 (s, 3H), 1.58 (d, J = 6.5 Hz, 6H); 13C NMR (100 MHz, DMSO‑d6) δ
176.9, 170.0, 166.8, 160.4, 159.6, 150.6, 146.3, 141.0, 135.3, 130.8,
130.1, 126.9, 121.8, 119.7, 118.2, 107.5, 104.5, 100.8, 29.4, 21.6,
20.9; HRMS m/z calcd for C24H22N4O4 ([M + H]+): 431.1714, found
431.1701.
1-Cyclopropyl-7-((4-(3-methoxyphenoxy)pyrimidin-2-yl)
amino)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (B11). Yield
76%; white solid; m.p. 270.8–272.4 °C; 1H NMR (400 MHz, DMSO‑d6) δ
10.43 (s, 1H), 8.78 (s, 1H), 8.65 (s, 1H), 8.52 (d, J = 5.6 Hz, 1H), 8.09
(d, J = 9.0 Hz, 1H), 7.87 (dd, J = 9.0, 1.5 Hz, 1H), 7.39 (t, J = 8.1 Hz,
1H), 6.92–6.89 (m, 2H), 6.84 (d, J = 8.3 Hz, 1H), 6.59 (d, J = 5.6 Hz,
1H), 3.77 (s, 3H), 3.54–3.48 (m, 1H), 1.24–1.22 (m, 2H), 1.16–1.12 (m,
2H); 13C NMR (100 MHz, DMSO‑d6) δ 177.4, 169.8,166.6, 161.0, 160.6,
159.7, 153.8, 148.8, 146.1, 142.7, 130.9, 126.5, 119.1, 118.3, 114.1,
111.9, 108.0, 107.2, 105.3, 100.8, 55.9, 36.0, 8.0; HRMS m/z calcd for
1-Isobutyl-4-oxo-7-((4-(p-tolyloxy)pyrimidin-2-yl)amino)-1,4-
dihydroquinoline-3-carboxylic acid (B17). Yield 78%; white solid;
m.p. 279.4–279.9 °C; 1H NMR (400 MHz, CDCl3) δ 15.26 (s, 1H), 8.73
(s, 1H), 8.64 (s, 1H), 8.39–8.34 (m, 3H), 7.57–7.51 (m, 1H), 7.35–7.29
(m, 2H), 7.06 (d, J = 8.4 Hz, 2H), 6.41 (d, J = 5.6 Hz, 1H), 4.01 (d,
J = 8.0 Hz, 2H), 2.49–2.44 (m, 2H), 2.42 (s, 3H), 1.34–1.23 (m, 3H),
1.03 (d, J = 6.6 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 177.6, 170.2,
167.4, 159.2, 159.0, 150.0, 148.5, 144.5, 141.0, 135.8, 130.4, 127.8,
121.2, 120.8, 117.4, 108.1, 104.0, 100.7, 62.3, 27.5, 20.9, 19.9; HRMS
m/z calcd for C25H24N4O4 ([M + H]+): 445.1870, found 445.1860.
1-(tert-Butyl)-4-oxo-7-((4-(p-tolyloxy)pyrimidin-2-yl)amino)-
1,4-dihydroquinoline-3-carboxylic acid (B18). Yield 90%; white
solid; m.p. 188.9–189.1 °C; 1H NMR (400 MHz, CDCl3) δ 15.38 (s, 1H),
9.46 (s, 1H), 9.09 (s, 1H), 8.43–8.36 (m, 2H), 7.21–7.18 (m, 2H), 7.27
(s, 1H), 7.05–7.06 (m, 3H), 6.38 (d, J = 4.4 Hz, 1H), 2.41 (s, 3H), 1.98
(s, 9H); 13C NMR (100 MHz, CDCl3) δ 177.1, 170.3, 167.8, 159.1,
150.0, 145.4, 143.1, 140.8, 135.8, 130.4, 127.9, 122.0, 121.2, 116.9,
108.1, 107.4, 100.5, 64.6, 30.5, 20.9; HRMS m/z calcd for C25H24N4O4
([M + H]+): 445.1870, found 445.1860.
C
24H20N4O5 ([M + H]+): 445.1506, found 445.1502.
1-Cyclopropyl-7-((4-(4-methoxyphenoxy)pyrimidin-2-yl)
amino)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (B12). Yield
73%; white solid; m.p. 282.8–284.5 °C; 1H NMR (400 MHz, DMSO‑d6) δ
10.40 (s, 1H), 8.79 (s, 1H), 8.66 (s, 1H), 8.51 (d, J = 5.6 Hz, 1H), 8.11
(d, J = 9.0 Hz, 1H), 7.88 (dd, J = 9.0, 1.9 Hz, 1H), 7.22 (d, J = 9.1 Hz,
2H), 7.05 (d, J = 9.1 Hz, 2H), 6.56 (d, J = 5.6 Hz, 1H), 3.82 (s, 3H),
3.57–3.52 (m, 1H), 1.27–1.24 (m, 2H), 1.18–1.14 (m, 2H); 13C NMR
(100 MHz, DMSO‑d6) δ 177.3, 170.1, 166.5, 160.3, 159.6, 157.2, 148.7,
146.0, 142.6, 130.0, 126.5, 123.0, 119.0, 118.2, 115.2, 107.1, 105.2,
100.5, 55.9, 35.9, 7.9; HRMS m/z calcd for C24H20N4O5 ([M + H]+):
445.1506, found 445.1498.
1-Cyclohexyl-4-oxo-7-((4-(p-tolyloxy)pyrimidin-2-yl) amino)-
1,4-dihydroquinoline-3-carboxylic acid (B19). Yield 81%; white
solid; m.p. 157.8–158.3 °C; 1H NMR (400 MHz, CDCl3) δ 15.42 (s, 1H),
8.87 (s, 2H), 8.41–8.37 (m, 2H), 7.32–7.29 (m, 4H), 7.08 (d,
J = 7.8 Hz, 2H), 6.42 (d, J = 5.1 Hz, 1H), 4.69–4.38 (m, 1H), 2.44 (s,
3H), 2.31–2.28 (m, 2H), 2.10–2.07 (m, 2H), 1.86–1.80 (m, 4H),
1.37–1.28 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 177.2, 170.2, 167.8,
159.2, 150.6, 150.0, 144.6, 143.9, 141.2, 135.8, 130.4, 127.9, 121.2,
120.9, 117.3, 108.4, 103.3, 100.6, 77.2, 32.7, 25.9, 25.3, 20.9; HRMS
m/z calcd for C27H26N4O4 ([M + H]+): 471.2027, found 471.2029.
1-Cyclopropyl-7-((4-(2,
4-dimethylphenoxy)pyrimidin-2-yl)
amino)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (B13). Yield
86%; white solid; m.p. 290.4–291.9 °C; 1H NMR (400 MHz, DMSO‑d6) δ
10.38 (s, 1H), 8.76 (s, 1H), 8.64 (s, 1H), 8.49 (d, J = 5.6 Hz, 1H), 8.06
(d, J = 9.0 Hz, 1H), 7.81 (dd, J = 9.0, 1.6 Hz, 1H), 7.18 (s, 1H), 7.11
(d, J = 8.3 Hz, 1H), 7.06 (d, J = 8.1 Hz, 1H), 6.54 (d, J = 5.6 Hz, 1H),
3.54–3.50 (m, 1H), 2.33 (s, 3H), 2.07 (s, 3H), 1.26–1.22 (m, 2H),
1.16–1.14 (m, 2H); 13C NMR (100 MHz, DMSO‑d6) δ 177.4, 169.7,
166.6, 160.5, 159.7, 148.9, 148.8, 146.1, 142.7, 135.5, 132.3, 130.1,
128.3, 126.5, 122.2, 119.1, 118.2, 107.2, 105.4, 100.2, 36.0. 20.8,
16.3, 8.0; HRMS m/z calcd for C25H22N4O4 ([M + H]+): 443.1714,
found 443.1708.
4.2. In vitro antibacterial assay [26]
1-Cyclopropyl-7-((4-(3,4-dimethylphenoxy)pyrimidin-2-yl)
amino)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (B14). Yield
82%; white solid; m.p. 287.7–289.7 °C; 1H NMR (400 MHz, DMSO‑d6) δ
10.40 (s, 1H), 8.83 (s, 1H), 8.66 (s, 1H), 8.51 (d, J = 5.6 Hz, 1H), 8.11
(d, J = 9.0 Hz, 1H), 7.87 (dd, J = 9.0, 1.7 Hz, 1H), 7.25 (d, J = 8.2 Hz,
1H), 7.08 (d, J = 2.3 Hz, 1H), 6.99 (dd, J = 8.1, 2.4 Hz, 1H), 6.56 (d,
J = 5.6 Hz, 1H), 3.52–3.47 (m, 1H), 2.28 (s, 3H), 2.25 (s, 3H),
1.26–1.23 (m, 2H), 1.17–1.14 (m, 2H); 13C NMR (100 MHz, DMSO‑d6) δ
177.3, 170.0, 166.5, 160.3, 159.6, 150.6, 148.7, 146.1, 142.6, 138.5,
134.0, 130.9, 126.4, 122.7, 119.0, 118.2, 107.1, 105.2, 100.6, 35.9,
19.8, 19.1, 7.9; HRMS m/z calcd for C25H22N4O4 ([M + H]+):
443.1714, found 443.1704.
Prior to the experiment, a single colony was picked from TSA plate.
All strains were grown at 37 °C overnight in TSB without the antibiotic.
1000-fold dilution of overnight cultures were grown at 37 °C for 2–3 h
until A600 = 0.6. Then bacteria were diluted 1:400 into fresh TSB
medium, compounds were dissolved in DMSO to a concentration of to a
concentration of 10 mg/mL, and the solutions were diluted with
medium before antibacterial activity test. Then, the stock solution was
stocked at −20 °C. The commercial ciprofloxacin and vancomycin were
used as the reference drugs. Equal volume of bacteria and compounds
were added to 96 well plates and mixed well by shaking. Plates were
incubated at 37 °C for 16–18 h followed by observations of MIC values
by the absence or presence of visible growth. All experiments were done
in duplicates and repeated for at least three times. MIC was determined
as the lowest concentration needed to inhibit bacterial growth.
4-Oxo-1-propyl-7-((4-(p-tolyloxy)pyrimidin-2-yl)amino)-1,4-di-
hydroquinoline-3-carboxylic acid (B15). Yield 84%; light yellow
1
solid; m.p. 274.0–274.4 °C; H NMR (400 MHz, DMSO‑d6) δ 10.28 (s,
8

R. Song, et al.
BioorganicChemistry103(2020)104176
4.3. Cytotoxicity assay [27]
bacterial dilution was prepared by using the bacterial suspension from
sub-MIC concentration of the compound (at MIC/2) and assayed for the
other MIC experiment. The process was repeated for 15 passages, and
the fold increase in MIC was determined. The results indicate the fold of
increase in MIC every day.
The cell lines were maintained at 37 °C in 5% CO2 in culture flasks
in RPMI-1640 medium, supplemented with 10% fetal calf serum, 100
U/mL penicillin, and 0.1 mg/mL streptomycin. Upon reaching con-
fluence, the cells were trypsinized with 0.25% trypsin containing 0.01%
EDTA for 5 min at 37 °C and then stopped by the addition of complete
medium. About 5 × 105 of the viable cells were then re-suspended in
complete medium.
4.6. Homology model of topoisomerase IV/DNA complex
The protein sequence of S. aureus topoisomerase IV was obtained
from UniProt Knowledgebase. The protein consists of four subunits in
the form of AABB. The entry of the subunit A and subunit B in UniProt
Knowledgebase were Q6GH50 and Q6GH51 [29]. Only the following
portions of subunit A and B which contained both the DNA- and ligand-
binding sites were modelled, respectively: 1–497 in subunit A and
412–637 in subunit B. Sequence identities between S. aureus topoi-
somerase IV and the template in subunit A and B were 64.1% and
77.9%, respectively. Sequence alignment was performed in ORCHES-
TRAR panel. Structurally conserved regions (SCRs) were built by using
one chain of 4KPF as model.
The cell lines were seeded in 96-well plates and incubated for 24 h
to allow the cells to attach. A stock solution of 400 mM xanthone de-
rivatives was prepared in DMSO and stored at −20 °C. The stock so-
lution was diluted to the appropriate concentrations with culture
medium. The final concentration of DMSO was less than 0.1% (v/v).
The same amount of DMSO was used as the vehicle control throughout
this study. Desfluoroquinolone-aminopyrimidine hybrids were serial
diluted (final concentration 0–400 μM) and added to the microtiter
plate. Commercial 5-FU was added as positive control. The cells were
incubated with compounds for 48 h. MTT assay was conducted fol-
lowing the protocol described previously. The cell viability was de-
termined at 560 nm absorbance using a Spectra Max M2 plate reader.
Experiment was performed in triplicates.
The loop regions comprised between residues 1–4, 483–497 in
subunit A and 540–553, 564–574, 636–637 in subunit B were con-
structed by using default settings. Then the sidechains were added by
using Borrow Chin1 + 2 + 3/Restrict Chin1 + 2 as borrow option. The
model was energy minimized using MMFF94 as force filed. The other
three chains were modeled by the same method and energy optimized
by means of staged minimization to homology construct the overall
model of S. aureus topoisomerase IV.
4.4. hERG assay
hERG-CHO cells (constructed in-house) were cultured in T75 flasks
to maximum 70–80% confluence at 37 °C in 5% CO2 incubator. The
culture media ((F-12 medium (Invitrogen 11765062, ThermoFisher,
USA) supplemented with 10% fetal bovine serum (Invitrogen
10099141, ThermoFisher, USA), 100 g/mL G418 (Invitrogen
The co-crystallized ligand was extracted and the protein was pre-
pared by adding hydrogens. Gasteiger-Hückel charges were assigned to
the ligand atoms. The structures of A6 and B14 were individually en-
ergy minimized using the conjugate gradient method.
11811023, ThermoFisher, USA) and 100 g/mL Hygromycin
B
(Invitrogen 10687010, ThermoFisher, USA) was removed and the
hERG-CHO cells were washed with 7 mL PBS. Then the cells were
dissociated with 3 mL Detachine reagent in 37 °C incubator for 3 min,
and 7 mL medium was added to gently suspend cells by pipetting up
and down several times. Finally, the cells were harvested by 800 rpm/
min centrifuge and adjusted to 2–5 million cells/mL for automated
Qpatch 16× (Qpatch 16x, Sophion, Denmark) experiments. Before
assay, all the tested compounds stock solutions (20 mM in DMSO) were
diluted with external solution to the desired concentrations and final
concentration of DMSO is 0.2%, which has no effects on hERG currents.
External solution was prepared as follows (pH 7.4): 140 mM NaCl,
5 mM KCl, 1 mM CaCl2, 1.25 mM MgCl2, 10 mM Glucose, 10 mM
HEPES.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgment
This work was supported by the National Natural Science
Foundation of China (21971043, 81861138046) and the Science and
Technology Commission of Shanghai Municipality (17XD1404400,
18XD1400800, 19ZR1403400). The technical assistance of Dr. Chaomei
Liu and Mrs. Mei Zhang from University of Florida for the cytotocixity
assay, Dr. Zhaobing Gao from Shanghai Institute of Materia Medica for
hERG assay is gratefully acknowledged.
The hERG current was recorded in the whole-cell patch clamp
configuration on QPatch 16× using single-hole QPlate. The cells were
voltage-clamped at a holding potential of −80 mV. Then the hERG
current was activated by depolarizing at +20 mV for 5 s, after which
the current was taken back to −50 mV for 5 s to remove the in-
activation. Finally, the deactivating tail current was observed. The peak
size of tail current was used to quantify hERG current amplitude. Raw
data included membrane resistance Rm > 100 MΩ and tail current
amplitude > 300 pA. Internal solution was prepared as follows (pH
7.2): 140 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM
EGTA.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104176.
References
[1] Centers for Disease Control and Prevention. Antibiotic resistance threats in the
United States; 2019. https://www.cdc.gov/drugresistance/pdf/threats-report/
2019-ar-threats-report-508.pdf.
4.5. Propensity to induce bacterial resistance [28]
[2] H.F. Chambers, Community-associated MRSA-resistance and virulence converge, N.
Engl. J. Med. 352 (2005) 1485–1487.
At beginning, the MIC value of compound B14 and vancomycin was
determined against MRSA ATCC43300 in a 96-well plate. Here, the
visual end point where there is no bacterial growth was considered to
be the MIC of the tested compound. For the next-day MIC experiment,
the bacterial dilution was made by using the bacteria from sub-MIC
concentration of the compounds (at MIC/2). Then, the concentration of
this bacterium was adjusted to ~105 CFU/mL on the basis of OD600 and
subjected to the next MIC assay. After a 24 h incubation period, again
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