Effect of new carbonyl cyanide aromatic hydrazones on biofilm inhibition against methicillin resistant: Staphylococcus aureus

Article abstract of DOI:10.1039/d0ra03124k

Carbonyl cyanide m-chlorophenylhydrazone (CCCP), as a protonophore, in combination with antibiotics exhibited potentiating antibacterial activity. To improve CCCP's potency and toxicity, a series of aromatic hydrazones were synthesized and their antimicrobial activity was evaluated; amongst them, compounds 2e and 2j with a strong para-electron-withdrawing substituent (-NO₂ and -CF₃) at the phenyl ring had the lowest MICs against both S. aureus and methicillin resistant Staphylococcus aureus (1.56 and 1.56 μM, respectively). Some compounds in combination with antibiotics exhibited potentiate Gram-positive antibacterial activity; compound 2e was found to display unaided or synergistic efficacy against MRSA. In particular, when compound 2e is combined with ofloxacin, it has a good synergistic effect against MRSA. Moreover, electron microscopy revealed that compound 2e inhibits biofilm formation and effectively eradicates preformed biofilm. MTT assay showed that compound 2e displays as low toxicity as CCCP. Overall, our data showed that the aromatic hydrazone is a promising scaffold for anti-staphylococcal drug development.

Full text of DOI:10.1039/d0ra03124k

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PAPER
Effect of new carbonyl cyanide aromatic
hydrazones on biofilm inhibition against methicillin
resistant Staphylococcus aureus†
Cite this: RSC Adv., 2020, 10, 17854
c
Xueer Lu,ab Ziwen Zhang,c Yingying Xu,c Jun Lu,a Wenjian Tang
ab
*
and Jing Zhang
Carbonyl cyanide m-chlorophenylhydrazone (CCCP), as a protonophore, in combination with antibiotics
exhibited potentiating antibacterial activity. To improve CCCP's potency and toxicity, a series of aromatic
hydrazones were synthesized and their antimicrobial activity was evaluated; amongst them, compounds
2e and 2j with a strong para-electron-withdrawing substituent (–NO2 and –CF3) at the phenyl ring had
the lowest MICs against both S. aureus and methicillin resistant Staphylococcus aureus (1.56 and 1.56
mM, respectively). Some compounds in combination with antibiotics exhibited potentiate Gram-positive
antibacterial activity; compound 2e was found to display unaided or synergistic efficacy against MRSA. In
particular, when compound 2e is combined with ofloxacin, it has a good synergistic effect against MRSA.
Moreover, electron microscopy revealed that compound 2e inhibits biofilm formation and effectively
eradicates preformed biofilm. MTT assay showed that compound 2e displays as low toxicity as CCCP.
Overall, our data showed that the aromatic hydrazone is a promising scaffold for anti-staphylococcal
drug development.
Received 7th April 2020
Accepted 29th April 2020
DOI: 10.1039/d0ra03124k
rsc.li/rsc-advances
uncoupling of the proton gradient, acts essentially as an iono-
phore and reduces the ability of ATP synthase to function
1. Introduction
optimally. CCCP causes the gradual destruction of living cells
and death of the organism by affecting the respiration and
respiration-dependent phosphorylation.8,9 Antibiotic accumu-
lation in Gram-negative bacteria is one of the major causes of
AMR. CCCP was widely used to study cellular accumulation in
Gram-negative bacteria for many small molecules10–12 due to its
ability to collapse the proton motive force.13 As AMR spreads,
a promising approach is to restore the effectiveness of existing
drugs via co-administration with adjuvants that inhibit the
growth of drug-sensitive pathogens.4,14,15 CCCP in combination
with small molecules showed synergistic effect against most of
the MDR pathogenic bacterial strains.16–20 CCCP in combination
with antibiotics could potentiate antibacterial activity.
Since CCCP acts as a protonophore which disperses the
membrane proton motive force by modifying the transmembrane
electrochemical potential, it simultaneously causes toxicity to the
cell of the host.21,22 Moreover, the concentration of synergistic
antibacterial effect of CCCP is so high (at least 50 mM) that the
effective dose may disrupt mitochondrial function to lead to
toxicity.23–25 Therefore, in this work, a series of aromatic hydra-
zones were synthesized and evaluated for their antibacterial
activity to try and improve antibacterial potency and reduce
toxicity. The preliminary screening showed that aromatic hydra-
zones exhibited potential Gram-positive antibacterial activities.
New compounds alone or in combination with antibiotics
exhibited potentiate Gram-positive antibacterial activities.
Antimicrobial resistance (AMR) is an increasingly serious threat
to global public health that requires a collaborative global
approach across sectors. AMR largely reduces the antibiotic
efficacies and increases health care costs, and the situation is
getting worse due to the emergence of multidrug-resistant
(MDR) bacterial pathogens, such as extended spectrum beta-
lactamase Enterobacteriaceae, methicillin resistant Staphylo-
coccus aureus (MRSA) and vancomycin-resistant enterococci
(VRE).1,2 Therefore, there is now an urgent need to develop new
antibacterial agents with novel targets and new approaches,
which could be addressed by developing new antibacterial
agents with unique chemical scaffolds.3–5
Mitochondria are well-known for their role as biosynthetic
and bioenergetic organelles, which play a critical role in the
innate immune response against viral and bacterial infec-
tions.6,7 As a chemical inhibitor of oxidative phosphorylation in
the mitochondria, carbonyl cyanide m-chlorophenylhydrazone
(CCCP) affects mitochondrial protein synthesis, causes an
aSchool of Medicine, Anhui University of Science and Technology, Huainan 232001,
China
bAnhui Prevention and Treatment Center for Occupational Disease, Anhui No. 2
Provincial People's Hospital, Hefei 230041, China. E-mail: hfzj2552@163.com
cSchool of Pharmacy, Anhui Medical University, Hefei 230032, China
† Electronic supplementary information (ESI) available: The copies of
representative 1H and 13C NMR spectra. See DOI: 10.1039/d0ra03124k
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Fig. 1 The general strategy in this work.
Therefore, the aromatic residue is a promising scaffold for further hydrochloric acid) led to aromatic diazonium salts, which can
antibacterial modications. Further, a plausible antibacterial be used to next reaction without purication. The diazonium
mechanism was proposed and investigated via scanning electron salt as the key intermediate underwent a condensation reaction
microscopy (SEM) and transmission electron microscopy (TEM) with methylene of malononitrile to yield the title compound 2.
(Fig. 1).
2.2. Antibacterial activity of compounds 2a–2q
In order to determine the antimicrobial potential of aromatic
hydrazones, they were evaluated in either Mueller–Hinton (MH)
broth or Sabouraud Dextrose Agar (SDA) using a micro-broth
2. Results and discussion
2.1. Chemistry
The synthetic route to compounds 2a–2q is illustrated in dilution method against a panel of bacteria and fungi,
Scheme 1. The nitrosation of the aromatic amines (1) with including two Gram-positive bacteria: Staphylococcus aureus
nitrous acid (in situ from sodium nitrite and concentrated ATCC 25923 (SA) and Methicillin Resistant Staphylococcus
Scheme 1 Synthesis of aromatic hydrazones 2a–2q. Reaction conditions and reagents: (i) HCl, NaNO2, 0 ꢀC, 1 h; (ii) CH2(CN)2, CH3COONa, 0 ꢀC,
2 h.
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Table 1 MIC (mM)a of aromatic hydrazones against Gram-positive
bacteria
compounds exhibited the moderate to high level of antibacte-
rial activity against Gram-positive bacteria (Table 1). Amongst
them, compounds 2e and 2j showed better activity than CCCP
(2a) against both S. aureus and MRSA (MICs ¼ 1.56 mM), which
are even better than cefoxitin and linezolid, and similar with the
MICs of ooxacin. The growth inhibition effects of compounds
2a, 2e and 2j were further investigated against both S. aureus
and MRSA. The results conrmed that both compounds 2e and
2j were able to inhibit the growth of S. aureus and MRSA
effectively at the MIC or higher concentrations. Once the
concentration drops down to half or less of the MICs, they could
only slow down the growing rate of S. aureus and MRSA during
logarithmic period, while the growth could be recovered aer
being incubated for longer time (Fig. 2).
Compoundsb
SA
MRSA
2a
2b
2c
2d
2e
2f
3.12
>200
50
100
1.56
25
6.25
>200
100
100
1.56
50
2g
2h
2i
100
6.25
50
100
12.50
100
2j
2k
2l
2m
2n
2o
2p
2q
A
1.56
>200
200
200
25
100
50
100
25
1.56
>200
>200
>200
100
100
100
50
100
2.3. SAR analysis
The structure–activity relationship (SAR) analysis are as fol-
lowed: (i) when the aromatic ring of carbonyl cyanide m-chlor-
ophenylhydrazone (CCCP) was substituted by heterocycle, the
antibacterial activity against Gram-positive bacteria was signif-
icantly decreased (such as 2b, 2c and 2k); (ii) the phenyl ring
with strong electron-withdrawing substituent (–NO2 and –CF3)
showed the moderate antibacterial activity (such as 2d–2h, 2j
and 2p); (iii) further, para-substituted group (–NO2 and –CF3)
exhibited better activity than meta-substituted group, e.g. for S.
aureus and MRSA (MIC values), 2e (1.56, 1.56 mM), 2h (6.25, 12.5
mM) > 2f (25, 100 mM), 2g (100, 100 mM); 2j (1.56, 1.56 mM) > 2d
(100, 100 mM), 2p (50, 100 mM).
B
C
0.63
7.50
1.25
7.50
a
b
MICs representing mean values of at least three replicates. A:
cefoxitin, B: ooxacin, C: linezolid.
aureus (MRSA); two Gram-negative bacteria: Pseudomonas aeru-
ginosa ATCC 9027 (PA) and Escherichia coli ATCC 8739 (EC); and
Candida albicans ATCC 10231 (CA), respectively. The results
showed that compounds 2a–2q showed no anti-microbial
activity against Gram-negative bacteria and fungi (MICs > 200
mM, except for 2a, MIC ¼ 50 mM for fungi), while some of
2.4. Cytotoxicity assays
The human hepatic L02 cells were treated with different
concentrations of tested compound (3.125, 6.25, 12.5, 25, 50
Fig. 2 S. aureus and MRSA growth inhibition curves. Titration curves showing the effect of different concentrations of compounds 2e and 2j on
the growth of S. aureus (A and B) and MRSA (C and D). Each OD point presented is the average values of three tests and all experiments are
internally controlled. Data are presented as the mean ꢁ standard deviation (n ¼ 3).
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and 100 mM), and cell viability was measured aer 24 h using
MTT method. As shown in Fig. 3, compounds 2a, 2e and 2j at
the test concentrations (3.125–50 mM) had no obvious cytotox-
icity against L02 cells, and the relative cell viabilities of treated
cells were all more than 70%.
2.5. Checkerboard assay
To develop a feasible medical application, active compounds 2a,
2e and 2j were tested in combination with clinical antibiotics on
SA and MRSA by checkerboard assay in order to evaluate their
ability to improve the anti-bacterial activity.20 Each checker-
board test generates many different combinations and, by
convention, the FIC value of the most effective combination was
used in calculating the fractional inhibitory concentration
index (FICI). FICI was calculated by adding both FICs:
Fig. 3 Cell viability assay of tested compounds to L02 cells. Data are
presented as the mean ꢁ standard error (n ¼ 3), one-way ANOVA (vs.
control), *P < 0.05, **P < 0.01.
Table 2 Synergistic activity assays on MRSA
Compounds
MIC alone (mM)
MIC combination (mM)
A
B
A
B
A
B
FICI
Mode of action
2a + ooxacin
2e + ooxacin
2j + ooxacin
2a + cefoxitin
2e + cefoxitin
2j + cefoxitin
2a + linezolid
2e + linezolid
2j + linezolid
6.25
1.56
1.56
6.25
1.56
1.56
6.25
1.56
1.56
1.25
1.25
1.25
50.00
50.00
50.00
7.50
3.12
0.39
0.78
3.12
0.78
0.78
3.12
0.78
0.78
0.04
0.04
0.04
1.56
1.56
1.56
0.47
0.47
1.90
0.53
0.28
0.53
0.53
0.53
0.53
0.56
0.56
0.75
Additive
Synergy
Additive
Additive
Additive
Additive
Additive
Additive
Additive
7.50
7.50
Fig. 4 Electronic microscopies of MRSA (A–C). Scanning electronic microscopic images of MRSA and (D–F) Transmission electronic micro-
scopic images of MRSA. (A and D) represent untreated bacteria. (B and E) represent bacteria treated with compound 2e at 1/2 MIC. (C and F)
represent bacteria treated with compound 2e at 1/2 MIC and ofloxacin of 1/8 MIC.
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ꢀ
ꢀ
FICI ¼ FICA þ FICB ¼ Ccomb MICaAlone þ CBcomb MICaBlone
exhibited better Gram-positive antibacterial activities, espe-
cially when compound 2e was used in combination with oox-
acin, in which the synergistic effect was observed. MTT assay
showed that the toxicity of compound 2e was low as that of
CCCP. Further, electron microscopy showed that compound 2e
possesses the capability to inhibit the formation of biolm and
eradicate already existing biolm. In sum, compound 2e dis-
played antibacterial activity against MRSA through inhibiting
biolm, especially improved the bactericidal effects of clinical
antibiotics by synergistic effect. Therefore, the aromatic residue
is a promising scaffold for further antibacterial modications.
A
where MICAalone and MICaBlone are the MICs of compound A and B
when acting alone and CcAomb and CcBomb are concentrations of
compounds A and B at the isoeffective combinations. The FICI
was interpreted as synergistic when it was #0.5, additional
effects when 0.5 < FICI # 1.0, indifferent when 1.0 < FICI # 2.0,
and antagonistic when FICI > 2.0, and any value between was
interpreted as indifferent.
As shown in Table 2, when compound 2a was used in
combination with antibiotics, MIC was reduced 2-fold, but for
compound 2e, MIC was reduced 4-fold. Moreover, compounds
2a, 2e and 2j could signicantly improve the performance of
clinical antibiotics, for example, ooxacin, cefoxitin and line-
zolid lowered their MIC values from 1.25, 50.0 and 7.5 mM to
0.04, 1.56 and 0.47 mM, respectively. Calculations of FIC and
FICI (always less than 1.0) obtained by checkerboard assays on
SA and MRSA showed at least additive effects of active
compounds (2a, 2e and 2j) with clinical antibiotics. When
compound 2e was combined with ooxacin, FICI value of 0.28
suggested a synergistic effect. Therefore, worthy of note is the
prophylactic purpose that low doses of clinical antibiotics plus
a protonophore may be developed as an anti-MRSA therapy by
inhibiting biolm.
4. Materials and methods
4.1. Chemistry
All reagents were purchased from commercial sources and were
used without further purication. Melting points (uncorrected)
were determined on a XT4MP apparatus (Taike Corp., Beijing,
China). 1H NMR and 13C NMR spectra were recorded on Bruker
AV-600 or AV-400 MHz instruments in CDCl3. Chemical shis
are reported in parts per million (d) downeld from the signal of
tetramethylsilane (TMS) as internal standards. Coupling
constants are reported in Hz. The multiplicity is dened by s
(singlet), d (doublet), t (triplet), or m (multiplet). High resolu-
tion mass spectra (HRMS) were obtained on an Agilent 1260-
6221 TOF mass spectrometry. Column and thin-layer chroma-
tography (CC and TLC, resp.) were performed on silica gel (200–
300 mesh) and silica gel GF254 (Qingdao Marine Chemical
Factory) respectively.
2.6. Electron microscope
To elucidate the effects of compound 2e on MRSA, both the
Scanning Electron Microscopy (SEM) and Transmission Elec-
tron Microscopy (TEM) were used to observe the bacteria aer
being treated with either compound 2e alone or in combination
with ooxacin. As shown in Fig. 4, SEM results revealed that
untreated MRSA form biolms in normal growth condition,
while the biolms were eradicated when treated with
compound 2e at 1/2 MIC concentration or in its combination
with 1/8 MIC of ooxacin. Furthermore, TEM results revealed
that the regular cell conformation was destructed and the
leakage of cellular substances under the treatment of
compound 2e in combination with ooxacin.
4.2. General procedures for synthesis of 2-(2-arylhydrazono)
malononitriles 2a–2q
To a solution of the aromatic amine (15 mmol) and concen-
trated HCl (37%, 13.8 mL) in H2O (75 mL) was dropwise added
NaNO2 (15 mmol 1.04 g) in H2O (50 mL) for 1 h in an ice bath,
and the mixture was stirred for 30 min. Then, the reaction
solution was added to a solution of CH2(CN)2 (20 mmol, 1.26
mL) and NaOAc (31 mmol, 38.1 g) in H2O (130 mL) under
ꢀ
continuous stirring at 0 C. Aer 2 hours, the reaction mixture
was ltrated, washed twice with water, and the residue was
recrystallized from ethanol to give the title compounds 2a–2q.
N-(3-Chlorophenyl)carbonohydrazonoyl dicyanide (2a).
3. Conclusion
In summary, a series of aromatic hydrazones were synthesized
and evaluated for their antibacterial activities. Some Yellow-green powder, yield, 80%; mp 131–133 ꢀC; 1H NMR (400
compounds showed potential antimicrobial activity against MHz, DMSO-d6) d 7.49–7.40 (m, 3H), 7.26 (d, J ¼ 6.9 Hz, 1H). 13
C
Gram-positive bacteria, amongst them, compounds 2e and 2j NMR (151 MHz, CDCl3) d 142.71, 135.01, 130.30, 125.54, 116.69,
had the lowest MICs against both S. aureus and MRSA (1.56 mM), 114.73, 113.44, 109.53, 85.54.
and the growth inhibition assay conrmed the inhibition
N-(2-Chloropyridin-4-yl)carbonohydrazonoyl dicyanide (2b).
1
ꢀ
effects. SAR showed that (i) for aromatic hydrazones containing Yellow powder, yield, 83%; mp 92–94 C; H NMR (400 MHz,
heterocycles, the antibacterial activity against Gram-positive DMSO-d6) d 8.25 (d, J ¼ 5.3 Hz, 1H), 7.29 (d, J ¼ 5.1 Hz, 2H). 13
C
bacteria was signicantly decreased (2a > 2b, 2c, 2k); (ii) the NMR (151 MHz, CDCl3) d 153.60, 151.86, 149.81, 114.60, 111.33,
phenyl ring with strong electron-withdrawing substituent (–NO2 110.86, 110.60, 86.25. TOF-HRMS: m/z [M + H]+ calcd for
and –CF3) showed the moderate antibacterial activity (2d–2h, 2j C8H4ClN5: 206.0155; found: 206.0156.
and 2p); (iii) further, para-substituted group (–NO2 and –CF3)
N-(2-Methoxypyridin-4-yl)carbonohydrazonoyl
dicyanide
exhibited better activity than meta-substituted group, e.g. for S. (2c). Orange powder, yield, 78%; mp 42–43 ꢀC; 1H NMR (400
aureus and MRSA (MIC values), 2e, 2h > 2f, 2g; 2j > 2d, 2p. MHz, CDCl3) d 8.17 (d, J ¼ 5.8 Hz, 1H), 6.90 (dd, J ¼ 5.7, 1.7 Hz,
Aromatic hydrazones in combination with clinical antibiotics 1H), 6.67 (s, 1H), 3.97 (s, 3H). 13C NMR (151 MHz, DMSO-d6)
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d 163.06, 115.16, 110.59, 106.22 (2C), 95.33 (2C), 87.73, 54.98. 137.82, 137.56, 120.18 (2C), 117.56 (2C), 115.42, 110.94, 83.40,
TOF-HRMS: m/z [M + H]+ calcd for C9H7N5O: 202.0651; found: 24.43.
202.0649.
N-(4-Fluoro-3-(triuoromethyl)phenyl)carbonohydrazonoyl
N-(4-Carbamoylphenyl)carbonohydrazonoyl dicyanide (2m).
Dark yellow green powder, yield, 82%; mp 155–157 ꢀC; 1H NMR
dicyanide (2d). Earthy red powder, yield, 78%; mp 30 ꢀC; 1H (400 MHz, DMSO-d6) d 7.96 (s, 1H), 7.90 (d, J ¼ 8.8 Hz, 2H), 7.46
NMR (400 MHz, CDCl3) d 10.06 (s, 1H), 7.82–7.42 (m, 2H), 7.31 (d, J ¼ 8.8 Hz, 2H), 7.33 (s, 1H). 13C NMR (101 MHz, DMSO-d6)
(t, J ¼ 9.1 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) d 157.75, d 167.69, 146.72, 131.09 (2C), 129.38 (2C), 116.90, 116.29,
138.80, 122.89, 122.78, 119.27, 117.72, 115.36, 114.46, 110.15, 111.71, 84.09.
86.63.
N-(4-Acetylphenyl)carbonohydrazonoyl
dicyanide
(2n).
N-(4-Nitrophenyl)carbonohydrazonoyl dicyanide (2e). Bright Bright yellow powder, yield, 91%; mp 254–255 ꢀC; 1H NMR (400
yellow powder, yield, 81%; mp 40–41 ꢀC; 1H NMR (400 MHz, MHz, DMSO-d6) d 7.93 (d, J ¼ 8.6 Hz, 1H), 7.46 (d, J ¼ 8.6 Hz,
DMSO-d6) d 8.19 (d, J ¼ 9.1 Hz, 2H), 7.49 (d, J ¼ 9.1 Hz, 2H). 13
C
1H), 2.51 (s, 1H). 13C NMR (101 MHz, DMSO-d6) d 197.03,
NMR (101 MHz, DMSO-d6) d 142.71, 125.19 (2C), 118.14 (2C), 150.90, 133.24, 130.23 (2C), 117.73 (2C), 113.00, 99.98, 82.71,
117.98, 113.20, 94.76, 82.41. TOF-HRMS: m/z [M + H]+ calcd for 26.99.
C9H6N5O2: 230.0547; found: 230.0550.
N-(4-Cyanophenyl)carbonohydrazonoyl
dicyanide
(2o).
N-(3-Nitrophenyl)carbonohydrazonoyl dicyanide (2f). Yellow Yellow-green powder, yield, 88%; mp 116–117 ꢀC; 1H NMR (400
powder, yield, 82%; mp 144–145 ꢀC; 1H NMR (400 MHz, DMSO- MHz, DMSO-d6) d 7.78 (d, J ¼ 8.4 Hz, 2H), 7.51 (d, J ¼ 8.4 Hz,
d6) d 8.21 (t, J ¼ 2.1 Hz, 1H), 8.08–7.96 (m, 1H), 7.91–7.77 (m, 2H). 13C NMR (101 MHz, DMSO-d6) d 149.82, 134.03 (2C),
1H), 7.68 (t, J ¼ 8.2 Hz, 1H). 13C NMR (151 MHz, DMSO-d6) 119.57, 118.29 (2C), 116.88, 112.26, 106.62, 84.34.
d 148.91, 144.78, 131.33, 123.26, 119.80, 115.30, 111.59, 110.85,
N-(3,5-Bis(triuoromethyl)phenyl)carbonohydrazonoyl
85.83.
dicyanide (2p). Light yellow powder, yield, 70%; mp 113–115 ꢀC;
N-(4-Chloro-3-nitrophenyl)carbonohydrazonoyl
(2g). Yellow powder, yield, 80%; mp 40–41 ꢀC; 1H NMR (400
dicyanide 1H NMR (400 MHz, CDCl3) d 7.86 (s, 2H), 7.73 (s, 1H).
N-(5-Acetyl-4-hydroxy-2-methoxyphenyl)carbonohydrazonoyl
MHz, DMSO-d6) d 7.73 (s, 1H), 7.56 (d, J ¼ 8.7 Hz, 1H), 7.48 (d, J dicyanide (2q). Dark green powder, yield, 70%; mp 132–134 ꢀC;
¼ 8.7 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) d 158.90, 153.42, 1H NMR (400 MHz, DMSO-d6) d 12.58 (s, 1H), 7.79 (s, 1H), 6.69
136.88, 129.41, 126.08, 122.79, 121.01, 119.43, 81.46.
(s, 1H), 3.95 (s, 3H), 2.59 (s, 3H). 13C NMR (101 MHz, DMSO-d6)
N-(2-Methyl-4-nitrophenyl)carbonohydrazonoyl
dicyanide d 203.23, 162.86, 156.87, 123.39, 122.67, 114.81, 113.71, 110.20,
1
ꢀ
(2h). Dark green powder, yield, 85%; mp 41 C; H NMR (400 100.94, 85.12, 57.21, 27.67.
MHz, CDCl3) d 9.57 (s, 1H), 8.18 (dt, J ¼ 27.6, 12.5 Hz, 2H), 7.70
(d, J ¼ 8.9 Hz, 1H), 2.51 (s, 3H). 13C NMR (101 MHz, CDCl3) 4.3. Culture conditions and treatments
d 146.56, 144.00, 129.46, 126.58, 122.61, 118.70, 114.81, 110.23,
L02 (normal human liver) cell lines were purchased from the
87.24, 17.47.
Russian Cell Culture Collection (Institute of Cytology Russian
Academy of Science, Saint Petersburg, Russia). L02 cells were
maintained in Dulbecco's modied Eagle's medium (DMEM)
(Invitrogen, USA) supplemented with 2 mM L-glutamine (Sigma-
Aldrich, UK), 10% fetal bovine serum (Invitrogen, USA), 50 mg
mLꢂ1 gentamicin sulfate (Invitrogen, USA) at 37 ꢀC and 5% CO2.
All compounds were dissolved in 100% DMSO (Sigma-Aldrich,
UK) to 100 mM stock solutions and diluted in completed DMEM
immediately before addition to the assay plates. DMSO was
maintained at a nal concentration of 0.1%.
N-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)carbonohydrazonoyl
1
ꢀ
dicyanide (2i). Earth orange powder, yield, 84%; mp 90 C; H
NMR (400 MHz, DMSO-d6) d 6.99–6.94 (m, 1H), 6.92–6.87 (m,
1H), 4.25 (s, 4H). 13C NMR (101 MHz, DMSO-d6) d 144.21,
142.25, 136.12, 118.20, 115.24, 110.81, 110.17, 105.94, 83.35,
64.68, 64.49.
N-(2-Fluoro-5-(triuoromethyl)phenyl)carbonohydrazonoyl
dicyanide (2j). Orange powder, yield, 90%; mp 33–35 ꢀC; 1H
NMR (400 MHz, DMSO-d6) d 7.80 (d, J ¼ 6.9 Hz, 1H), 7.68–7.51
(m, 2H). 13C NMR (101 MHz, DMSO-d6) d 155.03, 132.74, 126.42,
124.09, 124.06, 118.49, 117.50, 115.37, 110.80, 87.05. TOF-
HRMS: m/z [M + Na]+ calcd for C10H4F4N4Na: 279.0264; found:
279.0261.
4.4. Minimum inhibitory concentrations (MICs)
The MICs of tested compounds were determined using Muel-
ler–Hinton (MH) broth micro-broth dilution assay established
by the Clinical Laboratory Standards Institute (CLSI) in 96-well
micro-test plates. The nal test concentration ranged from 0.39
to 200 mM and the bacterial inocula was 108 CFU mLꢂ1. Aer 18–
20 hours of incubation at 37 ꢀC, the MICs were determined to be
the lowest concentration of tested compound that inhibited the
apparent increase in microorganisms. Each experiment was
repeated at least 3 times to report the MIC value.26
N-(5-Phenyl-1H-pyrazol-3-yl)carbonohydrazonoyl dicyanide
1
ꢀ
(2k). Yellow powder, yield, 89%; mp 138–139 C; H NMR (400
MHz, DMSO-d6) d 9.33 (s, 2H), 8.17 (d, J ¼ 7.5 Hz, 2H), 7.66 (s,
1H), 7.53 (dt, J ¼ 13.8, 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO-
d6) d 156.98, 150.06, 143.24, 131.62, 130.41, 129.41 (2C), 127.14
(2C), 116.37, 105.62, 96.13. TOF-HRMS: m/z [M + H]+ calcd for
C
12H8N6: 237.0801; found: 237.0805.
N-(4-Acetamidophenyl)carbonohydrazonoyl dicyanide (2l).
1
ꢀ
Yellow powder, yield, 70%; mp 45–46 C; H NMR (400 MHz,
DMSO-d6) d 10.07 (s, 1H), 7.63 (d, J ¼ 8.8 Hz, 2H), 7.40 (d, J ¼ 8.8
4.5. Inhibition of bacterial growth
Hz, 2H), 2.05 (s, 3H). 13C NMR (101 MHz, DMSO-d6) d 168.77, The effect of concentrations ranging from 0.5 to 4 times MIC of
the active compounds on the growth of S. aureus or MRSA was
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quantied aer incubation at 35 ꢀC for 0, 4, 8, 10, 18, 22 and 26 with uranyl acetate and lead citrate before examine with Hitachi
hours. At each time point, an aliquot (100 mL) was pipetted and TEM system at an accelerating voltage of 80 kV. The SEM model
measured for the A450 nm. The experiment was performed in used is the Hitachi su8100 at 3.0 kV voltage.27
three biologically independent assays, each tested in triplicate.
4.9. Statistical analysis
4.6. Checkerboard assays
All results were expressed as mean values ꢁ standard deviation.
The synergistic effect of the combination of clinical antibacte-
rials with the tested compounds was determined by checker-
board microdilution assays. In brief, checkerboards were set up
One-way analysis of variance followed by Dunnett's post hoc test
was used for all comparisons.
with double dilutions of compounds 2a (0–12.5 mM) or 2e (0–
3.12 mM) or 2j (0–3.12 mM) in the horizontal wells and ooxacin
Conflicts of interest
(0–2.5 mM) or cefoxitin (0–100 mM) or linezolid (0–14 mM) in the
vertical wells. Then 50 mL each was arranged on the rows and
columns of the plate, and 100 mL of MRSA was added to the
There are no conicts of interest to declare.
wells and bacteria inocula of 5 ꢃ 108 CFU mLꢂ1. Aer incuba-
tion at 35 ꢀC for 20 hours in 96-well micro-test plates. Aromatic
hydrazones were further tested to determine their nature of
interaction (synergy, antagonism, additive or no interaction)
with ooxacin, cefoxitin and linezolid and expressed as the
fractional inhibitory concentration index (FICI) for each agent.
Acknowledgements
Financial support was provided by the Natural Science Foun-
dation of Anhui provincial Department of Education
(KJ2019ZD21, KJ2018B12), and Provincial Natural Science
Foundation of Anhui (2008085MH261).
4.7. Cell viability
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