Synthesis, biological evaluation and computational studies of acrylohydrazide derivatives as potential Staphylococcus aureus NorA efflux pump inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.104225

The NorA efflux pump decreases the intracellular concentration of fluoroquinolones (ciprofloxacin, norfloxacin) by effluxing them from Staphylococcus aureus cells. The synthesis of novel acrylohydrazide derivatives was achieved using well-known reactions and were characterized by various spectroscopy techniques. The synthesized 50 compounds were evaluated for the NorA efflux pump inhibition activity against S. aureus SA-1199B (norA++) and K1758 (norA-) strains. The study provided two most active compounds viz. 19 and 52. Compound 19 was found to be most active in potentiating effect of norfloxacin and also it showed enhanced uptake, efflux inhibition in ethidium bromide assay. Further compound 19 also enhanced post antibiotic effect and reduced mutation prevention concentration of norfloxacin. The homology modeling study was performed to elucidate three-dimensional structure of NorA. Docking studies of potent molecules were done to find the binding affinity and interaction with active site residues. Further, all the tested compounds exhibited good ADME and drug-likeness properties in- silico. Based on the in-silico studies and detailed in vitro studies, acrylohydrazides derivatives may be considered as potential NorA EPI candidates.

Full text of DOI:10.1016/j.bioorg.2020.104225

Journal Pre-proofs
Synthesis, biological evaluation and computational studies of acrylohydrazide
derivatives as potential Staphylococcus aureus NorA efflux pump inhibitors
Gautam Kumar, Ambati Goutami Godavari, Rushikesh Tambat, Siva Kumar,
Hemraj Nandanwar, M. Elizabeth Sobhia, Sanjay M. Jachak
PII:
S0045-2068(20)31522-4
DOI:
https://doi.org/10.1016/j.bioorg.2020.104225
YBIOO 104225
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Bioorganic Chemistry
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16 July 2020
30 July 2020
Please cite this article as: G. Kumar, A. Goutami Godavari, R. Tambat, S. Kumar, H. Nandanwar, M. Elizabeth
Sobhia, S.M. Jachak, Synthesis, biological evaluation and computational studies of acrylohydrazide derivatives
as potential Staphylococcus aureus NorA efflux pump inhibitors, Bioorganic Chemistry (2020), doi: https://
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Synthesis, biological evaluation and computational studies of
acrylohydrazide derivatives as potential Staphylococcus aureus NorA
efflux pump inhibitors
Gautam Kumara, Ambati Goutami Godavaria, Rushikesh Tambatb, Siva Kumarc, Hemraj
Nandanwarb, M. Elizabeth Sobhiac, and Sanjay M. Jachaka
aDepartment of Natural Products, National Institute of Pharmaceutical Education and Research,
Sector-67, S.A.S Nagar, Punjab 160062, India.
bCSIR-Institute of Microbial Technology, Sector-39A, Chandigarh-160036, India.
cDepartment of Pharmacoinformatics, National Institute of Pharmaceutical Education and
Research, Sector-67, S.A.S Nagar, Punjab 160062, India.
Abstract
The NorA efflux pump decreases the intracellular concentration of fluoroquinolones
(ciprofloxacin, norfloxacin) by effluxing them from Staphylococcus aureus cells. The
synthesis of novel acrylohydrazide derivatives was achieved using well-known reactions
and were characterized by various spectroscopy techniques. The synthesized 50
compounds were evaluated for the NorA efflux pump inhibition activity against S. aureus
SA-1199B (norA++) and K1758 (norA-) strains. The study provided two most active
compounds viz. 19 and 52. Compound 19 was found to be most active in potentiating
effect of norfloxacin and also it showed enhanced uptake, efflux inhibition in ethidium
bromide assay. Further compound 19 also enhanced post antibiotic effect and reduced
mutation prevention concentration of norfloxacin. The homology modeling study was
performed to elucidate three-dimensional structure of NorA. Docking studies of potent
molecules were done to find the binding affinity and interaction with active site residues.
Further, all the tested compounds exhibited good ADME and drug-likeness properties in-
silico. Based on the in-silico studies and detailed in vitro studies, acrylohydrazides
derivatives may be considered as potential NorA EPI candidates.
Keywords: Acrylohydrazides; Efflux pump inhibitors; NorA efflux pump;
fluoroquinolones;MRSA
1. Introduction
The emergence of the antibiotic resistance in the pathogenic microorganisms leads to
ineffectiveness of several antibiotics and infections associated with multi-drug resistance
pathogens leads to high mortality and morbidity [1,2]. The extensive and un-restricted use
of antibiotics not only imposed evolutionary pressure upon bacteria, but also augmented
virulence in bacteria [3]. Among the antibiotic-resistant bacteria, methicillin-resistant
Staphylococcus aureus (MRSA) is responsible for various community and hospital
acquired infections [4,5]. MRSA cause invasive infections at the multiple body sites, it
mainly affects skin and soft tissues, also affects lung, bones, joints and blood stream and
urinary tract [6]. Infections associated with MRSA strains are bacteremia, endocarditis,
meningitis, arthritis, liver abscess and spinal epidural abscess. Vancomycin and
1

teicoplanin are used clinically for the treatments of the MRSA infection. It is reported that
MRSA strains have acquired resistance to the vancomycin [7,8]. The reasons for acquired
antibiotic resistance in bacteria are (a) modification or alteration of the antibiotics targets
(b) chemical modification or enzymatic degradation of the antibiotics (c) reduction in the
concentration of antibiotics inside the bacterial cell via membrane permeability or
activation or over expression of the efflux pumps [9-11]. The factors responsible for the
resistance of antibiotics by over expression of the efflux pump are most studied. Multidrug
efflux pumps are the membrane-based proteins present in bacteria which utilizes cellular
energy to extrude antibiotics and thus results in sub-lethal concentrations of antibiotics at
the active site and further it helps the bacteria in acquiring resistance.
NorA gene encode NorA efflux pump of S.aureus belongs to major facilitator superfamily
(MFS), and this pump is over expressed in MRSA strains [12]. NorA pump can efflux out
fluoroquinolones (ciprofloxacin, norfloxacin), [13,14] biocides (acriflavine, cetrimide),
dyes (ethidium bromide, rhodamines and acridines) [15] by using the energy generated
from the proton motive force and thus it lowers the required effective concentrations at
the active sites.The use of Efflux pump inhibitors (EPIs) in combination with antibiotics is
a promising strategy and will have beneficial effects viz.(i) it will block drug extrusion from
the bacterial cell, and so it will reduce the rate of antibiotic resistance (ii) it will revitalize
the use of antibiotics and (iii) the dose of the antibiotics will decrease [16].The well-known
non-antibiotic inhibitors of NorA efflux pump are reserpine [17,18], verapamil, omeprazole
[19], paroxetine [20] selective serotonin inhibitors such as chlorpromazine, elacridar and
tariquidar [21]. Few natural product inhibitors of NorA efflux pump are piperine [22],
tetramethylscutellarein, sarothrin [23], citral [24], capsaicin [25] etc. (Figure 1). Among
these NorA EPIs reserpine is used as a reference standard but it is not used clinically
because required concentration is too high for EPI activity, and at high concentration it
shows neurotoxicity [26].
Due to non-availability of crystal structure of NorA efflux pump; the major challenge for
medicinal chemist is to design specific and efficient inhibitors for NorA pump. Based on
the homology modeling study 3D structure of NorA was studied. Natural and semi-
synthetic acrylic acid derivatives possessing medicinally importance scaffold are well
reported in the literature [27,28]. Herein we report the convenient synthesis of
acrylohydrazides with in-silico studies and NorA efflux pump inhibition activity in vitro.
2

Figure 1 Non-antibiotic compounds asNorA EPIs.
2. Results and discussion
2.1 Chemistry
Total 50 acrylohydrazides were synthesized as depicted in scheme-1.The synthesized
acrylohydrazides were evaluated for NorA efflux pump inhibition activity. The synthesis
of aromatic acrylic acids (1a-1d) was achieved by knoevenagel-Doebner condensation
reaction. The aromatic aldehyde was refluxed with malonic acid in pyridine and piperidne
at 110 °C to give substituted aromatic acrylic acids [29]. Aromatic acrylic hydrazides were
synthesized by reacting aromatic acrylic acid with ethanol and sulphuric acid. Further, the
aromatic acrylic ester was treated with hydrazine hydrate in ethanol, but desired product
acrylic hydrazides were not obtained. By using carbodiimide, the aromatic acrylic acids
were successfully converted into acrylic hydrazides. The aromatic acrylic acids (2a-2d)
were coupled with hydrazine hydrate using EDC.HCl and HOBt to give acrylic hydrazides
[30]. Further aromatic acrylic hydrazides were reacted with substituted aromatic
aldehydes in presence of catalytic amount of HCl in ethanol to give acrylic hydrazide
imines in yields ranging from 57-92%.
The aromatic acrylic hydrazide imines were characterized using 1H NMR, 13C NMR and
HRMS techniques. Purity of the synthesized hydrazides were determined using ultra-
performance liquid chromatography (UPLC). All the synthesized hydrazides showed
purity in the range 95-99%.
Due to solubility problems of synthesized hydrazides different deuterated solvents such
as chloroform (CDCl3) or dimethyl sulfoxide (DMSO-d6), Pyridine-d5, CF3COOD were
used for recording NMR spectra. In DMSO-d6 and Pyridine-d5 the compounds tautomerize
into amide and iminol form and showed signals of tautomers in 1H and 13C-NMR spectra.
Whereas in solvents like CDCl3 and CF3COOD, the signals obtained in 1H and 13CNMR
were of combined form of tautomers. The compound 43 was dissolved separately in
1
CDCl3, DMSO-d6, Pyridine-d5, and CF3COOD and H NMR spectra were recorded
(supporting information). qNMR study of Compound 14 was performed to determine the
percentage of amide and iminol tautomer. Compound 14 was dissolved in DMSO-d6 with
internal standard,1,3,5-trimethoxybenzene and the NMR spectrum was analyzed. It was
observed that compound 14 exist in the proportion of 54.89% and 45.11% for amide and
iminol tautomers respectively. The formula used for qNMR analysis is depicted below [31].
WAnalyteA; weight of the analyte A, IAnalyte A; Integral of the analyte A signal, IStandard; Integral of the standard
signal, NStandard; Number of standard protons, NAnalyte A; Number of analyte protons, MAnalyte A Molecular mass
of the analyte A, MStandard: Molecular mass of standard, WStandard; Weight of standard sample.
3

Figure 2. Solution structure of acrylic hydrazide imine
The structure of compound 14 was used for the theoretical calculations of energy of
amide-iminol tautomers. The relative energy difference of both tautomers were computed
in the presence of gas phase, solvent phases (CHCl3, DMSO). From the theoretical in-
silico study, it was observed that amide form is more stable than the iminol form. The
relative free energy in DMSO for amide ⇌ iminol is 10.46 kcal/mol and in chloroform found
to be 8.4 kcal/mol. The energy difference between amide and iminol is higher in DMSO
solvent as compared to chloroform, therefore in DMSO both the tautomer signals were
observed in 1H and 13C NMR. Whereas in chloroform the rate of inter-conversion between
1
13
tautomer’s is fast due to less energy barrier, and thus signals observed in H and C
NMR are due to combined form of both tautomers.
4

O
O
O
O
i
Ar1
+
OH
Ar1
H
OH
O
HO
81-84 %
1a-1d
ii
NH2NH2 .2H2O
63-79 %
Ar1 = 1a = 2a =
O
O
Ar1 = 1b = 2b =
Ar1 = 1c = 2c =
NO2
O
NH2
Ar1
N
H
2a-2d
iii
O
O
O
H
47-85 %
Ar2
O
Ar1 = 1d = 2d =
O
N
Ar2
Ar1
N
H
3-52
Reagents and conditions (i) Pyridine, Piperidine, 110 °C, 16-24 h
(ii) HOBT, EDC.HCl, ACN, rt to 0-5 °C to rt (iii) HCl, EtOH, 80 °C, 2-5 h
Scheme 1. Synthesis of acrylic hydrazide imines
O
N
Ar2
Ar1
N
H
3-53
Table 1. The compounds (3-53) synthesized from scheme1.
S.No. Ar1
Ar2
3.
4.
5.
6.
7.
8.
5

9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
6

27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
7

40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
8

52.
2.2 In vitro modulation studies
The MIC of ciprofloxacin, norfloxacin and all the synthesized molecules were determined
so as to use these molecules at concentration devoid of antibacterial activity, a
prerequisite of any compound to be used as EPI [32]. Of the 50 molecules tested, most
active molecules were used in combination with ciprofloxacin (Table 2) and norfloxacin
(Table 3) in dose-dependent manner and evaluated against S. aureus SA-1199B
(norA++) and K1758 (norA-). The modulation factor (MF) that is fold reduction in MIC of
ciprofloxacin was observed to be ranging from 4-16-fold when tested in combination with
9 most active compounds (10, 15, 19, 26, 27, 29, 49, 50, and 52) at 1/4th of their MIC and
all these represented synergy (FICI≤0.5). The MF varies with concentration of EPIs,
suggested dose-dependent NorA efflux pump inhibition. The additive effect (4>FICI>0.5)
was observed with compound 19 and compound 52 at 1/16th of their MIC as well as with
compound 49 and compound 50 at 1/8th of their MIC (Table 2). Similarly, the MF that is
fold reduction in MIC of norfloxacin was observed to be ranging from 8-32-fold when
tested in combination with 9 most active EPIs enlisted above at 1/4thof their MIC. All the
above concentrations mentioned represented synergy (FICI≤0.5). The additive effect
(4>FICI>0.5) was observed with compound 50 and compound 52 at 1/16th and 1/4th of
their MIC, respectively (Table 3).Compound19 and compound52were shown to be more
prominent in ciprofloxacin as well as norfloxacin potentiation. On the other hand S. aureus
SA-K1758 (norA-) did not show any reduction in the MIC of ciprofloxacin as well as
norfloxacin that validated the role of the tested compounds in NorA efflux pump inhibition.
Table 2In vitro ciprofloxacin/synthetic EPI’s combination studiesa (n)-Fold reduction of Ciprofloxacin MIC;
*- FICI≤0.5 indicates synergy
Compounds
Intrinsic activity
against modified
S. aureus (MIC in
µg/ml)
Concentration Ciprofloxacin MICs
Fractional
Inhibitory
Concentration
index (FICI)
of Inhibitor
(µg/ml)
in µg/ml
SA-
1199B
(norA+
+)
K1758
(norA-)
SA-
1199B
(norA++)
K1758
(norA-)
SA-
1199B
(norA++)
K1758
(norA-)
10
32
32
8
4
32
16
8
1 (8)
2 (4)
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.38*
0.38*
0.38*
0.31*
0.38*
0.56
-
-
-
-
-
15
19
128
64
128
64
1 (8)
0.5 (16)a
2 (4)
4 (2)
2 (4)
4
8
26
32
32
0.50*
-
27
29
64
64
64
64
16
8
16
1 (8)
2 (4)
1 (8)
0.125
0.125
0.125
0.38*
0.38*
0.38*
-
-
9

49
50
128
64
128
64
32
16
16
1 (8)
4 (2)
2 (4)
0.125
0.125
0.125
0.38*
0.75
0.50*
-
-
8
4 (2)
0.125
0.63
52
64
64
16
8
4
0.5 (16)
1 (8)
2 (4)
0.125
0.125
0.125
0.125
0.31*
0.25*
0.56
-
Reserpine
128
-
128
-
32
2 (4)
0.50*
-
-
Ciprofloxacin
-
8
0.125
-
Table 3In vitro Norfloxacin/synthetic EPI’s combination studiesa(n)-Fold reduction of Norfloxacin MIC; *-
FICI≤0.5 indicates synergy.
Compounds Intrinsic activity Concentrat
Norfloxacin MICS in
µg/ml
Fractional
Inhibitory
Concentration
index (FICI)
against modified
S. aureus (MIC
in µg/ml)
ion of
Inhibitor
(µg/ml)
SA-
K1758
SA-
K1758
SA-
K1758
1199B (norA-)
1199B
(norA-)
1199B
(norA-)
(norA+
(norA++)
(norA++)
+)
10
32
32
8
4
32
16
8
2 (16)
4 (8)
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.31*
0.25*
0.38*
0.28*
0.18*
0.31*
0.38*
0.31*
0.38*
0.38*
0.31*
0.25*
0.31*
-
-
-
-
-
-
-
-
15
19
128
64
128
64
4 (8)
1 (32)a
2 (16)
8 (4)
4
8
26
27
32
64
32
64
4 (8)
2 (16)
8 (4)
4 (8)
2 (16)
4 (8)
16
8
16
32
16
16
29
49
64
128
64
128
-
-
2 (16)
50
52
64
64
64
64
-
8
16 (2)
0.25
0.63
16
8
4
32
-
1 (32)
4 (8)
16 (2)
4 (8)
32
0.25
0.25
0.25
0.25
0.25
0.28*
0.25*
0.56
0.38*
-
-
-
-
-
-
Reserpine
Norfloxacin
128
-
128
-
2.3 Cell cytotoxicity studies
The most active 9 compounds (10, 15, 19, 26, 27, 29, 49, 50, and 52) observed in
modulation study mentioned above were tested for their cytotoxicity before evaluation of
their EPI activity. The cytotoxicity was evaluated using MTT (3-(4, 5- dimethylthiazol-2-
yl)-2, 5- diphenyltetrazolium bromide) assay in HEK 293 cells (Human Embryonic Kidney)
with different concentrations. The tested 9 compounds showed high IC50 values and
selectivity index (Supporting information: Table 1) revealing that they do not possess
cytotoxicity at the tested concentrations.
2.4Ethidium Bromide (EtBr) uptake assay
10

The most active 9 compounds (10, 15, 19, 26, 27, 29, 49, 50, and 52) observed in
modulation study mentioned above were tested for their cytotoxicity before evaluation of
their EPI activity. The cytotoxicity was evaluated using MTT assay in HEK 293 cells (with
different concentrations. The tested 9 compounds showed high IC50 values and selectivity
index (Supporting information) revealing that they do not possess cytotoxicity at the tested
concentrations.
The ability of compounds to facilitate the uptake of EtBr in S. aureus SA-1199B and SA-
K1758 was evaluated by using a fluorescence assay. The cells were loaded with EtBr
with and without tested EPIs and placed in fluorimeter containing a fresh medium. EtBr
fluoresces only when it is bound to nucleic acid inside bacterial cells. There was negligible
amount of accumulation observed due to NorA mediated EtBr efflux (Figure3), illustrating
that there is hardly any accumulation in control samples which are devoid of any EPI. The
accumulation potential of reserpine (positive control) [17, 32–35] was evaluated at sub-
inhibitory concentration of 20 µg/ml. The accumulation activity of compounds10, 19, 27,
49, 50 and 52 were evaluated at sub-inhibitory concentrations ranging from 4-16 µg/ml.
The compounds 10, 49 and 52 showed significant effect, which was comparable to
reserpine. Compound 19 showed most potent effect on EtBr accumulation whereas
compounds 27 and 50 showed moderate potential on accumulation of EtBr against S.
aureus SA-1199B strain. On the contrary, in the NorA deficient strain, there was almost
no effect on EtBr accumulation was observed in the presence or absence of any of the
above-mentioned compounds and reserpine (Figure 4). These results revealed that
reserpine and other tested compounds have specific ability for the inhibition of NorA efflux
pump. (Cautionary Note: EtBr can bind with DNA; it is highly toxic as a mutagen. It is
suspected to cause carcinogenic or teratogenic effects. Exposure routes of EtBr are
inhalation, ingestion, and skin absorption.)
Figure 3 Real-time fluorescence-based determination of ethidium bromide uptake by S. aureus SA-1199B
in the presence and absence of synthetic EPI’s tested at concentration of 1/8th MIC. Reserpine at 20 µg/ml
was used as a positive control.
2.5 Ethidium bromide efflux inhibition assay
11

The ability of reserpine (positive control) and compounds (10, 15, 19, 26, 27, 29, 49, 50
and 52) to inhibit S. aureus efflux was confirmed by real time fluorometry using EtBr, a
broad efflux pump substrate. We evaluated the efflux pump inhibitory potential of
compounds at sub-inhibitory concentrations (1/4th, 1/8th and1/16th MIC) to promote the
intracellular accumulation of EtBr and calculated the RFFs (Table 4). The relative final
fluorescence (RFF) value is a measure of how effective the compound is on the inhibition
of the EtBr efflux (at a given concentration) by comparison of the final fluorescence at the
last time point (45 min) of the treated cells with the cells treated only with EtBr. An index
of activity above zero indicated that the cells accumulate more EtBr under the condition
used than those of the control (non-treated cells taken as 0). In case of negative RFF
values, these indicated that the treated cells accumulated less EtBr than those of the
control condition. Values above 1 in the presence of the EPIs were considered as
enhanced accumulation of EtBr inside the cells [36,37]. At 1/4th MIC compound 19 and
52 showed RFF of 7.63 and 7.64, respectively indicating a strong ability to interfere with
EtBr efflux compared to reserpine that showed RFF value of 4.49. The result showed that
phenomenon of observed efflux inhibition is dose-dependent. RFF value started declining
as sub-inhibitory concentration decreases. Compounds 15, 26, 29, 49, 50 and 52 at
1/16thof their MIC displayed significantly lower RFF values of 1.51, 1.16, 1.46, 1.74, 1.23
and 1.55 respectively, being less effective in the inhibition of efflux activity (Table 4).
Figure 4 Real-time fluorescence-based determination of ethidium bromide uptake by S. aureus SA-K1758
in the presence and absence of synthetic EPI’s tested at concentration of 1/8th MIC. Reserpine at 20 µg/ml
was used as a known EPI control.
Table 4. RFF based on the EtBr efflux inhibition for S. aureus SA-1199B in the presence of synthetic library
of efflux inhibitors at varied concentrations. Reserpine at 20 µg/ml was used as a known EPI control. Results
were represented as mean ±SD of three biological replicates and considered significant when *P< 0.05 and
highly significant when **P< 0.01 and ***P<0.001.
Compounds Concentration Relative
Final
of inhibitor Fluorescence
(µg/ml)
(RFF) ± SD
10
8
4
6.26±0.09***
4.7±0.07***
12

2
3.06±0.12***
4.74±0.15***
2.39±0.12**
1.51±0.09**
7.63±0.16***
6.37±0.11***
3.93±0.12***
4.75±0.1***
2.3±0.14***
1.16±0.09*
15
32
16
8
16
8
4
8
4
2
19
26
27
16
8
4
16
8
6.22±0.07***
3.93±0.05***
2.49±0.14**
4.71±0.08***
2.2±0.11**
29
4
1.46±0.08**
6.11±0.07***
4.8±0.09***
1.74±0.2*
49
32
16
8
50
16
8
4
16
8
4
6.07±0.13***
1.61±0.14**
1.23±0.08**
7.64±0.06***
4.34±0.15***
1.55±0.15**
4.49±0.09***
52
Reserpine
20
2.6Time-kill kinetics
The time-kill kinetics study of S. aureus SA-1199B was performed to examine the
bactericidal effect of the combination of norfloxacin with compound 19. Norfloxacin was
used at its sub-inhibitory concentration of 1 µg/ml (1/64th MIC) alone as well as in
combination with compound 19. As expected, norfloxacin at 1 µg/ml did not show any
inhibitory activity over the period of 24 h. The sub-inhibitory concentration of norfloxacin
(1 µg/ml) resulted in bactericidal activity in presence of compound 19 (16 µg/ml). Re-
growth of bacteria was not observed after 24 h in the combination tested. However, even
after 24 h, the combination of norfloxacin with compound 19 retained bactericidal activity
and maintained the 3 log10cfu difference between the initial log cfu at 0 h (Figure 5).
Further, the surviving cells from each experiment recovered after 24 h exhibited the same
norfloxacin MIC (64 µg/ml) and similarly exhibited a reduced norfloxacin MIC in the
presence of compound 19 (data not shown).
13

12
10
8
Control
Norfloxacin (1 µg/mL) (Synergistic MIC)
19 (16 µg/mL)
6
Norfloxacin (1 µg/mL) + 19 (16 µg/mL)
4
2
0
0
4
8
12
16
20
24
Time (Hours)
Figure 5. Time-kill curves of S. aureus SA-1199B showing the bactericidal effect of norfloxacin (1 µg/ml) in
combination with compound19 (16 µg/ml). Each time point represents the mean of log10 ±SD of three
biological replicates.
2.7 Post Antibiotic Effect (PAE)
The PAE is the phenomenon of continued suppression of bacterial growth after a short
exposure of bacteria to antimicrobial agents [33,38]. The PAE of norfloxacin alone and in
combination with the most active compound 19 was determined against S. aureus SA-
1199B. Norfloxacin alone exhibited a PAE of 0.3, 0.97 and 1.43 h at 0.25 MIC (8 µg/ml),
0.5 MIC (16 µg/ml) and MIC (32 µg/ml), respectively. The same concentrations of
norfloxacin in combination with compound 19 (16 µg/ml) resulted in significantly
prolonged PAEs of 1.6, 1.97 and 3.15 h, respectively (Table 5).
Table 5. PAE of norfloxacin alone and in combination with 19 against S. aureus SA-1199B after exposure
of 2h. Results were represented as mean ±SD of three biological replicates.
Regimen
Mean PAE (h) ± SD
0.5 MIC (16 µg/ml)
0.97 ± 0.16
0.25 MIC (8 µg/ml)
0.3 ± 0.08
1 MIC (32 µg/ml)
1.43 ± 0.08
Norfloxacin
Norfloxacin +
Compound19(16
µg/ml)
1.6 ± 0.12
1.97 ± 0.12
3.15 ± 0.19
2.8Frequency of appearance of norfloxacin resistance in the presence of compound 19
The minimum concentration of a drug at which no mutant is selected is defined as its
mutation prevention concentration (MPC) [33,39]. A mutant selection study was
performed on S. aureus ATCC 25923, which is wild type strain with no reported mutation
in the regulatory domain of NorA as well as drug target domain (DNA gyrase and
topoisomerase IV). Norfloxacin at 8 µg/ml (16×MIC), at which no mutant was selected,
has been defined as the MPC. When tested in combination with compound 19 at 4 and 8
µg/ml the MPC of norfloxacin was reduced to 4 and 2 µg/ml, respectively (Table 6). The
14

MPC of the combination was found to be lower than the Cmaxof norfloxacin, indicating the
clinical significance of these combinations for the selection of resistant mutants.
Table 6. Mutation frequency of S. aureus ATCC 25923.
Compound-19
Mutation frequency with norfloxacin
(µg/ml)
2×MIC
4×MIC
8×MIC
16×MIC (8
µg/ml)
<10-8
(1 µg/ml) (2 µg/ml) (4 µg/ml)
0
4
8
1.5×10-8
8.1×10-8
2.8×10-8
6.9×10-8
3.5×10-8
<10-8
4.1×10-8
<10-8
<10-8
<10-8
<10-8
2.9 NorA model construction
The protein sequence of NorA was collected from UniProt (ID: P0A0J7) database to build
the 3D-structure of NorA protein [40]. Lactose permease transporter (PDBID: 1PV6)
belonging to E. coli organism was searched with the help of PSI-BLAST from structural
database (PDB). The PSI-BLAST returned with a set of 8 template structures with an ‘e
value’ above the threshold. The final template structure was made on the basis of score
values, e value, query cover, similarities and identities. The structure of Lactose
permease from E. coli was selected as the template (PDB ID- 1PV6) with 43% query
cover in which 27% of the residues were identical and 48% were positive substitutions.
In the context of alignments displayed in the PSI-BLAST output, positives are those non-
identical substitutions that receive a positive score in the underlying scoring matrix,
BLOSUM62 by default. Most often positives indicate a conservative substitution or
substitutions that are often observed in related proteins. NorA structure was then
subjected to prime module to refine the missing side chains, loops of protein structure
(Supporting information: Section 6). On analyzing the constructed structure, it was
observed that NorA efflux pump is a single polypeptide chain, which exhibits 12 TM α-
helices topology with N- and C-terminal domains, arranged as pseudo-two-fold symmetry.
Both N- and C-terminal domains are connected by a long cytoplasmic loop between helix-
VI and helix-VII. N-terminal domain consists of six TM α-helices: TM helix-I (3-29), TM
helix-II (38-65), TM helix-III (72-85), TM helix-IV (112-122), TM helix-V (143-153), TM
helix-VI (164-173) and C-terminal domain consist of TM α-helices: TM helix-VII (198-235),
TM helix-VIII (242-274), TM helix-IX (276-295), TM helix-X (300-328), TM helix-XI (334-
364), TM helix-XII (366-387). Finally, the constructed structure was subjected to protein
preparation wizard of maestro module to get the lower energy state of NorA structure.
The refined model was processed for quality check using PROCHECK and ERRAT2 plot.
2.10 Identification of novel Binding site
Modelled NorA protein was prepared and subjected to SiteMap for identifying and
analyzing binding sites and for predicting target druggability [41]. SiteMap predicted five
binding sites and the parameters of these sites are shown in Table 7. From theSiteMap
analysis, site2 (N-terminal domain) was chosen for molecular docking studies based on
site score and drug score. Both scores are found to be above one which indicates that
15

site2 has components that are reasonable in size, enclosure, hydrophobic and hydrophilic
parameters to confirm it as a ligand binding and druggable target site. The Figure 6 shows
the top ranked binding site components (Yellow colour maps-hydrophobic, Red maps-
HBA, Blue maps-HBD) within the NorA protein for site 2. On close examination, it is clear
that SiteMap finds essential information about the druggability and ligand binding pocket
as the N-terminal domain of NorA has the essential complementary elements to
accommodate ligands or therapeutic agents binding to block it’s mechanism of action.
The main purpose of SiteMap is to identify the probable ligand binding pockets in a
macromolecular structure and in the present study, the identified site2 was used for the
receptor grid generation for molecular docking studies.
Figure 6. Novel binding pocket of NorA from N-terminal domain.
Table 7. SiteMap results of NorA efflux transporter protein.
Site
Site score Drug score
Volume
Å3
Size
Site2
Site3
Site1
Site4
Site5
1.209
1.095
1.070
0.842
0.722
1.267
1.127
1.117
0.885
0.739
488.08
253.82
1775.71
85.06
219
92
703
42
52.47
25
2.11 Molecular docking studies of NorA
Molecular docking studies were carried out to investigate the binding affinity of NorA
inhibitors using the identified site2. Compound 19, One of the most potent NorA EPI
molecules, showed binding affinity of -6.084 kcal/mol and one hydrogen bond interaction
with Gly106, one pi-pi stacking interaction with Phe47 residue (Figure 7).The binding
pose of molecule19 with NorA complex revealed that activity of the molecule 19 is mainly
due to hydrophobic interactions as aryl group of molecule 19 make interactions with
hydrophobic residues (Figure 7). Another potent NorA EPI molecule 52 showed binding
16

affinity of -4.646 kcal/mol. Though the molecule 52 did not show good binding score, it
displayed important interactions that are maintained within the active site. The molecule
52 exhibited one hydrogen bond interaction with Val108, one aromatic H-bond interaction
with Ala105. It has three methoxy groups attached to aromatic ring which formed
hydrophobic interaction with Leu26, Ile23 in the active site of NorA (Figure 8). The
conformation and binding mode of molecule 52 makes it possible to form an hydrogen
bond and an aromatic hydrogen bond interaction with Val108 and Ala 105, respectively
that are missing in the molecule 19.This could be the main reason why molecule 52
showed promising activity in the experimental study as compared to 19.
From the binding affinity studies, it is evident that hydrophobic interactions play a vital
role in both the molecules and the residues involved in these hydrophobic interactions
are listed in Table 8.
Figure 7. The molecule 19 showing intermolecular interaction with active site residues of NorA protein.
17

Figure 8. The molecule 52 showing intermolecular interaction with active site residues of NorA protein.
Table 8. NorA inhibitors and their hydrogen bonding and pi-pi interactions and hydrophobic interactions
with the active site residues of NorA protein.
Molecule ID
H-bond
interactions
Gly106
π-π interactions
Hydrophobic
interactions
Molecule 19
Phe47
Ile15, Val22, Ile23,
Leu26, Ile43, Pro27,
Met103, Ala105,
Met107, Val108,
Met109.
Molecule 52
Val108
-
Leu14, Phe16, Ile19,
Ile23, Leu26, Val112,
Ile116.
2.12 Prediction of ADME properties of acrylohydrazide derivatives
For about 95% of FDA approved drugs, physicochemical properties are observed as
molecular weight (M.W) 130-725, hydrogen bond donor (donors HB) 0-6, hydrogen bond
acceptor (acceptors HB) 2-20 and predicted QPlogPo/w -2-6.5 [42]. The predicted
properties of all the studied molecules are found to be within the above-mentioned values.
According to Lipinski’s rule, physicochemical parameters should be within the range like
M.W ≤500, lop P ≤5, H-bond donor ≤5 and H-bond acceptor ≤10. These physiochemical
parameters are responsible for aqueous solubility and intestinal permeability [43]. A
compound is more likely to exhibit poor absorption or poor permeability, when two or more
parameters are not within the range [44,45]. All the 50 molecules were studied for Lipinski
rule of five. It was observed that 39 molecules did not violate any parameters, whereas
11 molecules violated 1 parameter, viz., QPPCaco predict Caco-2 cell permeability (<25
is poor absorption, >500 is great absorption). Caco-2 cell permeability study provides
information about the human absorption process by the non-active transport mechanism
through gut-blood barrier [46,47]. When all the 50 molecules were studied for Caco-2 cell
permeability, it is observed that 40 molecules possess better gut-blood permeability,
whereas 10 molecules showed intermediate property of absorption. The molecules
having log BB >0.3 cross BBB, while those with logBB<-1.0 are poorly distributed to the
brain. For the molecules to be CNS active its value must be ≥2 [46,48]. The predicted
logBB value suggested that, all the molecules will not able to cross the brain and hence
molecules will not show any central nervous system (CNS) activity. In addition, the
molecules exhibited good predicted pharmacological properties like HERG K+ (HERG K+
channel), QPlogKhsa (human serum albumin), QPPMDCK (MDCK cell permeability), and
skin permeability (Supporting information:Table 2).
3. Conclusion
In the present study we described synthesis of 50 acrylohydrazides as NorA EPIs in
S.aureus1199B strain. Of the 50 compounds nine have been identified as potent EPIs
which are capable of potentiating the activity of norfloxacin and ciprofloxacin by 4-32 fold
against NorA over-expressing strain. These compounds were also tested against NorA
18

deletion strain (S. aureus SA-K1758). The MICs of norfloxacin and ciprofloxacin in
combination with these compounds remain unchanged, due to loss of functional NorA
protein. This confirms the role of newly synthesized acrylohydrazides as NorA EPIs. The
involvement of efflux pump in the resistance mechanism was further confirmed by EtBr
accumulation and efflux inhibition studies. The fluorescence-based assay results showed
reduced efflux in the presence of EPIs. The Compound 19 displayed most prominent EtBr
efflux inhibition. In time-kill studies, the combination of norfloxacin (1 μg/mL) and
compound 19 (16 μg/mL) produced a bactericidal effect. The reduction in CFU at 24 h
was >3 Log10. This combination reduced the MPC of norfloxacin as well as emergence of
norfloxacin-resistant mutants. Furthermore, concentration-dependent enhancement in
the PAE of norfloxacin was observed. The most significant extension of PAE was
observed at 32 μg/mL (1 × MIC) of norfloxacin in combination with compound 19.
The NorA structure was modeled as there was no crystal structure of efflux protein
subjected to accuracy testing followed by optimization and energy minimization to get low
energy state of NorA protein. The binding affinity studies of the 50 molecules showed that
biologically potent molecules 52 and 19 were recognized as top rank molecules from the
molecular docking studies. These two molecules were identified as potent inhibitors of
NorA efflux pump as they showed strong hydrogen bonds and hydrophobic intermolecular
interactions with NorA protein, which makes them ideal therapeutic candidate to block
NorA function. Further the active compounds were (10, 15, 19, 26, 27, 29, 49, 50, and
52) found to be non-toxic in MTT assay (Supporting information: Table 1). The ADME
study of 50 molecules revealed that 40 molecules showed better gut-blood permeability,
whereas these molecules were not able to penetrate the brain and central nervous system
as they are having log BB >0.3 and ≤2 respectively.
In conclusion for the first time, we report acrylohydrazides as a novel class of NorA EPIs.
By combining the most potent compounds 19 and 52 with clinically used antibiotics will
re-allow the use of the antibiotics by reducing their antibacterial resistance.
4. Experimental
4.1 General experimental: All the chemicals and reagents required for the synthesis and
biological activity were procured from Sigma Aldrich (St. Louis, MO, USA) and Alfa Aesar
(Johnson Matthey Company, Ward Hill, MA, USA).The LR grade solvents were used for
reactions and column chromatography. The progress of reactions was monitored using
pre-coated with silica gel 60 F254TLC plates (E-Merck, Germany). Column
chromatography was performed on silica gel 60-120 and 100-200 mesh (E-Merck,
Germany). Melting points were determined in open capillaries using Veego LX-300
melting point apparatus and are uncorrected. 1H and 13C NMR were recorded on Avance
DPX 400 spectrometer (Brukers, Germany) at 400 MHz and 100 MHz, respectively. NMR
spectra were recorded in CDCl3 and/or DMSO-d6 at room temperature. The mass spectra
were recorded on Bruker Maxis High resolution LCMS/MS, and Thermo LTQ-XL
LCMS/MS. S. aureus ATCC 25923 was obtained from the HiMedia®, India. NorA-
19

overproducing S. aureus strain SA-1999B, S. aureus SA-1199 (wild type) and norA
knockout S. aureus SA-K1758 were obtained as a kind gift by Dr. Inshad A. Khan (CSIR-
IIIM, Jammu, India with kind permission of Dr. G.W. Kaatz (Wayne state university, USA).
BBLTM Cation Adjusted Mueller Hinton II Medium was used for combination studies,
mutation studies and kill kinetic experiments. Mueller-Hinton Agar (HiMedia®, India) was
used for culturing the bacteria and colony counts.
4.2 Synthesis of substituted acrylic acids (1) Aromatic aldehyde (1 mole), malonic acid
(1.2 mole) and piperidine (0.1 mole) were dissolved in pyridine and refluxed at 110 °C for
16- 24 h in oil bath. After the completion of reaction, the reaction mixtures were quenched
by pouring it into cold dilute HCl solution. The crude product was adsorbed and loaded
on the silica gel (100-200 mesh) column, and the compound was eluted with (20 %) ethyl
acetate in hexane to give pure compounds with 81-84 % yield.
4.2.1.(E)-3-(furan-2-yl)acrylic acid (1a). Yield 84 %; mp: 147-149 °C;1H NMR (400 MHZ,
CDCl3) δ ppm: 11.43 (s, 1H), 7.56-7.53 (m, 2H), 6.69 (d, J = 3.40 HZ, 1H), 6.51 (dd, JL =
3.40, JS = 1.80 HZ, 1H), 6.34 (d, J = 15.68 HZ, 1H); 13C NMR (100 MHz, CDCl3) δ ppm:
172.73, 150.65, 145.33, 133.08, 115.85, 114.90, 112.48; HRMS (ESI) m/z; calcd for
+
C7H6NaO3 ([M + Na]+) 161.0209, found 161.0214.
4.2.2.(E)-3-(4-(benzyloxy)phenyl)acrylic acid (1c).Yield 81 %;mp: 197-200 °C;1H NMR
(400 MHZ, DMSO-d6) δ ppm: 7.63 (d, J = 8.64 HZ, 2H), 7.53 (d, J = 15.96 HZ, 1H), 7.45
(d, J = 7.32 HZ, 2H), 7.39 (t, J = 7.12 HZ, 2H), 7.33 (t, J = 7.08 HZ, 1H), 7.04 (d, J = 8.64
HZ, 2H), 6.38 (d, J = 15.96 HZ, 1H), 5.15 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ ppm:
173.06, 165.21, 148.92, 141.94, 135.17, 133.69, 133.15, 132.98, 132.23, 121.83, 120.38,
+
74.52; ESI-MS m/z; calcd for C16H15O3 ([M + H]+) 255.10, found 255.03.
4.3Synthesis of substituted aromatic acrylohydrazides (2a-2d) The acid (1 equiv.) was
dissolved in CH3CN (15 ml) and to this solution, HOBt (1.2 equiv.) was added in one
portion followed by addition of EDC. HCl(1.2 equiv.). The resultant mixture was stirred at
room temperature and TLC monitoring was done to check the progress of the reaction,
until the acid completely consumed in the reaction mixture. The resulting mixture was
then slowly added to a solution of hydrazine (2 equiv.) and cyclohexene (1 ml) in CH CN
3
(20 ml) while the temperature was maintained at 0-5 °C. The resultant mixture was stirred
for 15 min. After completion of the reaction, acetonitrile was removed under vacuum.
Then reaction mixture was extracted with EtOAc and passed over anhydrous sodium
sulfate. The crude compound was adsorbed and loaded on to the silica gel (100-200
mesh) column, and compound was eluted with (25-45%) ethyl acetate in hexane to give
pure compounds with 63-79% yield.
4.3.1.(E)-3-(furan-2-yl)acrylohydrazide (2a) Yield 71 %; mp: 102-105 °C;1H NMR (400
MHZ, DMSO-d6) δ ppm: 9.37 (s, 1H), 7.76 (s, 1H), 7.25 (d, J = 15.60 HZ, 1H), 6.75 (d, J =
20

3.16 HZ, 1H), 6.57 (s, 1H), 6.34 (d, J = 15.60 HZ, 1H), 4.45 (s, 2H); 13C NMR (100 MHz,
DMSO-d6) δ ppm: 164.74, 151.4, 145.0, 126.0, 118.00, 114.0; HRMS (ESI) m/z; calcd for
+
C7H8N2NaO2 ([M + Na]+) 175.0478, found 175.0478.
4.3.2.(E)-3-(5-nitrofuran-2-yl)acrylohydrazide (2b)Yield 63 %; mp: 233-236°C;1H NMR
(400 MHZ, DMSO-d6) δ ppm: 9.64 (s, 1H), 7.74 (d, J = 3.84 HZ, 1H), 7.35 (d, J = 15.72
HZ, 1H), 7.12 (d, J = 3.80 HZ, 1H), 6.68 (d, J = 15.72 HZ, 1H), 4.61 (s, 2H); 13C NMR (100
MHz, DMSO-d6) δ ppm: 163.13, 153.8, 151.9, 124.4, 124.3, 116.4, 115.2; ESI-MS m/z;
+
calcd for C7H7N3NaO4 ([M + H]+) 220.03, found 220.97.
4.3.3.(E)-3-(3,4,5-trimethoxyphenyl)acrylohydrazide (2d)Yield 79 %; mp: 156-159 °C; 1H
NMR (400 MHZ, DMSO-d6) δ ppm: 9.25 (s, 1H), 7.39 (d, J = 15.76 HZ, 1H), 6.89 (s, 2H),
6.50 (d, J = 15.76 HZ, 1H), 4.44 (s, 2H), 3.81 (s, 6H), 3.68 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ ppm: 165.0, 153.5, 139.0, 138.8, 131.0, 120.0, 105.3, 60.5, 56.3; ESI-MS
+
m/z; calcd for C12H17N2O4 ([M + H]+) 253.12, found 253.03.
4.4.Synthesis of substituted (2E,N'E)-N'-benzylidene-3-(furan-2-yl)acrylohydrazide (3-52)
Equimolar hydrazide and aldehyde were taken in ethanol and to this catalytic amount of
HCl was added and the resultant reaction mixture was refluxed at 80°C for 2-5 h. The
crude compound was adsorbed and loaded onto the silica gel (100-200 mesh) column,
and compound was eluted with (30-60 %) ethyl acetate in hexane to give pure compounds
with 47-85 % yield.
4.4.1.(2E,N'E)-N'-(4-chlorobenzylidene)-3-(furan-2-yl)acrylohydrazide(3)Yield
85 %;mp:173-175 °C;1H NMR (400 MHZ, DMSO-d6) δ ppm: 11.73 (s, 1H), 11.57 (s, 1H),
8.22 (s, 1H), 8.05 (s, 1H), 7.85 (d, J = 18.60 HZ, 2H), 7.73 (t, J = 8.68 HZ, 4H), 7.52 (d, J
= 7.48 HZ, 4H), 7.47-7.42 (m, 2H), 7.31 (d, J = 15.76 HZ, 1H), 6.96 (d, J = 3.00 HZ, 1H),
6.87 (d, J = 3.08 HZ,1H), 6.64 (d, J = 7.44 HZ, 2H), 6.50 (d, J = 15.52 HZ, 1H); 13C NMR
(100 MHz, DMSO-d6) δ ppm: 166.4,161.9, 151.6, 151.3, 146.0, 145.8, 145.7, 142.4, 134.9,
134.7, 133.7, 133.6, 129.6, 129.5, 129.4, 129.2, 128.8, 128.3, 117.6, 115.9, 115.4, 114.4,
+
113.1; HRMS (ESI) m/z; calcd for C14H11ClN2NaO2 ([M + Na]+) 297.0401, found
297.0402.
4.4.2.(2E,N'E)-N'-(4-fluorobenzylidene)-3-(furan-2-yl)acrylohydrazide (4)Yield 74 %;
mp: 175-177 °C;1H NMR (400 MHZ, DMSO-d6) δ ppm: 11.68 (s, 1H), 11.52 (s, 1H), 8.23
(s, 1H), 8.06 (s, 1H), 7.85 (d, J = 17.68 HZ, 2H), 7.80-7.74 (m, 4H), 7.50-7.42 (m, 2H),
7.34-7.27 (m, 5H), 6.95 (d, J = 3.20 HZ, 1H), 6.87 (d, J = 3.24 HZ, 1H), 6.65-6.62 (m, 2H),
6.50 (d, J = 15.52 HZ, 1H); 13C NMR (100 MHz, DMSO-d6) δ ppm: 166.3, 164.8, 162.3,
161.8, 151.6, 151.4, 145.9, 145.7, 142.6, 131.4, 131.2, 129.8, 129.7, 129.4, 129.4, 129.3,
128.2, 117.7, 116.5, 116.3, 116.2, 115.8, 115.3, 114.5, 113.1; HRMS (ESI) m/z; calcd for
+
C14H11FN2NaO2 ([M + Na]+) 281.0697, found 281.0697.
21

4.4.3.(2E,N'E)-3-(furan-2-yl)-N'-(4-(trifluoromethyl)benzylidene)acrylohydrazide (5) Yield
80 %;mp: 206-208 °C;1H NMR (400 MHZ, CDCl3) δ ppm: 10.18 (s, 1H), 7.93 (s, 1H), 7.85
(d, J = 8.12 HZ, 2H), 7.69-7.62 (m, 3H), 7.56 (s, 1H), 7.43 (d, J = 15.68 HZ, 1H), 6.69 (d,
J = 3.04 HZ, 1H), 6.51 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ ppm: 166.5, 162.1, 151.5,
151.3, 146.1, 145.9, 145.3, 142.1, 138.8, 138.6, 129.8, 128.6, 128.1, 127.8, 126.2, 125.9,
123.2, 117.5, 116.0, 115.6, 115.6, 114.2, 113.2, 113.1; HRMS (ESI) m/z; calcd for
+
C15H11F3N2NaO2
([M
+
Na]+)
331.0065,
found
331.0658.
4.4.4.
(2E,N'E)-N'-(3-bromobenzylidene)-3-(furan-2-yl)acrylohydrazide
(6)
Yield
79 %;mp: 192-194 °C;1H NMR (400 MHZ, DMSO-d6) δ ppm: 11.80 (s, 1H), 11.62 (s, 1H),
8.19 (s, 1H), 8.03 (s, 1H), 7.92-7.83 (m, 4H), 7.72 (t, J = 7.48 HZ, 2H), 7.61 (d, J = 8.16
HZ, 2H), 7.51-7.39 (m, 4H), 7.30 (d, J = 15,76 HZ, 1H), 6.97 (d, J = 3.12 HZ, 1H), 6.88 (d,
13
J = 3.24 HZ, 1H), 6.65-6.53 (m, 2H), 6.50 (d, J = 15.56 HZ, 1H); C NMR (100 MHz,
DMSO-d6) δ ppm: 166.4, 161.9, 151.5, 151.3, 146.1, 145.8, 145.3, 142.1, 137.3, 137.1,
132.9, 132.8, 131.5, 131.4, 129.7, 129.5, 128.5, 126.2, 122.7, 122.6, 117.5, 115.9, 115.5,
+
114.3, 113.1; HRMS (ESI) m/z; calcd for C14H11BrN2NaO2 ([M + Na]+) 340.9896, found
340.9890.
4.4.5. (2E,N'E)-3-(furan-2-yl)-N'-(naphthalen-2-ylmethylene)acrylohydrazide (7) Yield
67 %;mp: 192-194 °C;1H NMR (400 MHZ, DMSO-d6) δ ppm: 11.78 (s, 1H), 11.61 (s, 1H),
8.39 (s, 1H), 8.24 (s, 1H), 8.13 (d, J = 28.08 HZ, 2H), 8.01-7.93 (m, 8H), 7.87 (d, J = 23.72
HZ, 2H), 7.58-7.54 (m, 4H), 7.50-7.38 (m, 3H), 6.98 (d, J = 3.24 HZ, 1H), 6.88 (d, J = 3.28
HZ, 1H), 6.67-6.63 (m, 2H), 6.54 (d, J = 15.56 HZ, 1H);13C NMR (100 MHz, DMSO-d6) δ
ppm:166.3, 161.9, 151.6, 151.4, 147.0, 146.0, 145.8, 143.9, 134.2, 134.1, 133.4, 133.3,
132.5, 132.3, 129.5, 129.2, 129.1, 128.9, 128.8, 128.3, 127.6, 127.5, 127.2, 123.2, 122.6,
+
117.8, 115.8, 115.3, 114.6, 113.2, 113.1; HRMS (ESI) m/z; calcd for C18H14N2NaO2 ([M
+ Na]+) 313.0947, found 313.0943.
4.4.6.(2E,N'E)-3-(furan-2-yl)-N'-(3-nitrobenzylidene)acrylohydrazide (8)Yield 59 %;mp:
194-196 °C;1H NMR (400 MHZ, DMSO-d6) δ ppm: 11.92 (s, 1H), 11.74 (s, 1H), 8.55 (s,
1H), 8.48 (s, 1H), 8.35 (s, 1H), 8.25 (d, J = 8.12 HZ, 2H), 8.18-8.15 (m, 3H), 7.89 (s, 1H),
7.84 (s, 1H), 7.77-7.73 (m, 2H), 7.53-7.45 (m, 2H), 7.32 (d, J = 15,76 HZ, 1H), 6.97-6.89
(m, 2H), 6.66-6.64 (m, 2H), 6.53 (d, J = 15.52 HZ, 1H); 13C NMR (100 MHz, DMSO-d6) δ
ppm: 166.7, 162.2, 151.4, 151.2, 148.2, 148.1, 146.1, 145.9, 144.7, 141.5, 141.1, 140.9,
130.0, 128.9, 128.5, 128.1, 124.6, 124.5, 117.2, 116.2, 115.8, 113.9, 113.3, 113.2; HRMS
+
(ESI) m/z; calcd for C14H11N3NaO4 ([M + Na]+) 308.0642, found 308.0641.
4.4.7.(2E,N'E)-N'-(3,5-di-tert-butyl-4-hydroxybenzylidene)-3-(furan-2-
yl)acrylohydrazide(9)Yield 59 %;mp: 226-228 °C;1H NMR (400 MHZ, CDCl3) δ ppm: 9.60
(s, 1H), 7.79 (s, 1H), 7.61 (d, J = 15.68 HZ, 1H), 7.55 (s, 2H), 7.48 (d, J = 7.24 HZ, 2H),
6.64 (d, J = 3.12 HZ, 2H), 6.49 (s, 1H), 5.49 (s, 1H), 1.48 (s, 18H); 13C NMR (100 MHz,
CDCl3) δ ppm: 167.8, 155.9, 151.9, 144.9, 144.5, 136.3, 129.6, 125.3, 124.5, 114.9, 114.3,
22

+
112.3, 34.4, 30.2; HRMS (ESI) m/z; calcd for C22H29N2O3 ([M + H]+) 369.2173, found
369.2166.
4.4.8.(2E,N'E)-3-(furan-2-yl)-N'-(3,4,5-trimethoxybenzylidene)acrylohydrazide (10) Yield
62 %;mp: 160-162 °C;1H NMR (400 MHZ, CDCl3) δ ppm: 10.10 (s, 1H), 7.85 (s, 1H), 7.66
(d, J = 15.68 HZ, 1H), 7.54 (s, 1H), 7.46 (d, J = 15.72 HZ, 1H), 6.99 (s, 2H), 6.68 (d, J =
13
2.96 HZ, 1H), 6.52 (s, 1H), 3.96 (s, 6H), 3.92 (s, 3H); C NMR (100 MHz, DMSO-d6) δ
ppm:166.3, 161.7, 153.7, 153.6, 151.6, 151.4, 147.1, 145.9, 145.7, 143.6, 139.6, 139.5,
130.3, 130.2, 129.4, 128.1, 117.8, 115.5, 115.3, 114.7, 113.1, 104.8, 104.5, 60.6, 56.4;
+
HRMS (ESI) m/z; calcd for C17H19N2O5 ([M + H]+) 331.1288, found 331.1286.
4.4.9.(2E,N'E)-N'-(4-(dimethylamino)benzylidene)-3-(furan-2-yl)acrylohydrazide
(11)
Yield 48 %; mp: 188-190 °C;1H NMR (400 MHZ, DMSO-d6) δ ppm: 11.38 (s, 1H), 11.23
(s, 1H), 8.07 (s, 1H), 7.92 (s, 1H), 7.82 (d, J = 18.04 HZ, 1H), 7.54-7.46 (m, 4H), 7.42-
7.31 (m, 3H), 6.92 (d, J = 3.28 HZ, 1H), 6.92 (d, J = 3.28 HZ, 1H), 6.83 (d, J = 3.32 HZ,
1H), 6.77-6.74 (m, 4H), 6.65-6.61 (m, 2H), 6.48 (d, J = 15.52 HZ, 1H), 2.97 (s, 12H);13C
NMR (100 MHz, DMSO-d6) δ ppm:165.8, 161.3, 151.9, 151.8, 151.7, 151.5, 147.9, 145.8,
145.5, 144.5, 128.9, 128.8, 128.4, 127.5, 122.0, 118.2, 115.4, 115.1, 114.8, 113.1, 113.0,
+
112.4, 112.2, 40.2; HRMS (ESI) m/z; calcd for C16H17N3NaO2 ([M + Na]+) 306.1213,
found 306.1214.
4.4.10.(2E,N'E)-3-(furan-2-yl)-N'-(4-nitrobenzylidene)acrylohydrazide (12) Yield 47 %;
mp: 229-232 °C;1H NMR (400 MHZ, DMSO-d6) δ ppm: 12.07 (s, 1H), 11.87 (s, 1H), 8.36
(d, J = 8.88 HZ, 5H), 8.21 (s, 1H), 8.03 (t, J = 8.84 HZ, 4H), 7.92 (d, J = 17.08 HZ, 2H),
7.60-7.52 (m, 2H), 7.37 (d, J = 15.76 HZ, 1H), 7.05 (d, J = 2.40 HZ, 1H), 6.96 (d, J = 2.72
HZ, 1H), 6.72 (d, J = 8.92 HZ, 2H), 6.57 (d, J = 15.48 HZ, 1H); 13C NMR (100 MHz, DMSO-
d6) δ ppm: 166.7, 162.3, 151.4, 151.2, 148.2, 148.1, 146.1, 145.9, 144.7, 141.5, 141.1,
140.9, 130.0, 128.9, 128.5, 128.1, 124.6, 124.5, 117.2, 116.2, 115.8, 113.9, 113.3, 113.2;
+
HRMS (ESI) m/z; calcd for C14H11N3NaO4 ([M + Na]+) 308.0642, found 308.0637.
4.4.11.(2E,N'E)-3-(furan-2-yl)-N'-(4-(methylthio)benzylidene)acrylohydrazide (13)Yield
68 %;mp: 184-186 °C;1H NMR (400 MHZ, DMSO-d6) δ ppm: 11.68 (s, 1H), 11.47 (s, 1H),
8.15 (s, 1H), 8.01 (s, 1H), 7.83 (d, J = 17.96 HZ, 2H), 7.66-7.41 (m, 4H), 7.49-7.41 (m,
2H), 7.33-7.29 (m, 5H), 6.94 (d, J = 3.32 HZ, 1H), 6.86 (d, J = 3.30 HZ, 1H), 6.64-6.62 (m,
13
2H), 6.48 (d, J = 15.52 HZ, 1H), 2.50 (s, 6H); C NMR (100 MHz, , DMSO-d6) δ ppm:
166.3, 161.8, 151.5, 151.3, 146.8, 145.9, 145.7, 143.4, 141.5, 141.1, 131.1, 131.0, 129.4,
128.2, 128.0, 127.6, 126.2, 126.0, 117.7, 115.8, 115.3, 114.5, 113.2, 113.1, 14.7; HRMS
(ESI) m/z; calcd for C15H14N2NaO2S+ ([M + Na]+) 309.0668, found 309.0665.
4.4.12.(2E,N'E)-N'-(4-bromobenzylidene)-3-(furan-2-yl)acrylohydrazide
(14)Yield
66 %;mp: 187-190 °C;1H NMR (400 MHZ, CDCl3) δ ppm: 10.74 (s, 1H), 7.93 (s, 1H), 7.67-
7.62 (m, 3H), 7.58-7.56 (m, 3H), 7.45 (d, J = 15.72 HZ, 1H), 6.70 (d, J = 3.16 HZ, 1H),
23

6.53 (s, 1H); 13C NMR (100 MHz, CDCl3) δ ppm: 168.2, 151.7, 144.7, 142.9, 132.9, 131.9,
+
130.2, 128.7, 124.3, 115.0, 114.0, 112.4; HRMS (ESI) m/z; calcd for C14H11BrN2NaO2
([M + Na]+) 340.9896, found 340.9898.
4.4.13.(2E,N'E)-3-(furan-2-yl)-N'-(4-methoxybenzylidene)acrylohydrazide
(15)Yield
63 %;mp: 189-192 °C;1H NMR (400 MHZ, CDCl3) δ ppm: 9.72 (s, 1H), 7.84 (s, 1H), 7.70
(d, J = 8.64 HZ, 2H), 7.63 (d, J = 15.27 HZ, 1H), 7.55 (s, 1H), 7.46 (d, J = 15.72 HZ, 1H),
6.97 (d, J = 8.68 HZ, 2H), 6.68 (d, J = 3.20 HZ, 1H), 6.52-6.51(m, 1H), 3.88 (s, 3H); 13C
NMR (100 MHz, CDCl3) δ ppm: 167.7, 161.2, 151.9, 144.5, 143.7, 129.8, 128.8, 126.7,
+
114.7, 114.5, 114.2, 112.3, 55.4; HRMS (ESI) m/z; calcd for C15H14N2NaO3 ([M + Na]+)
293.0897, found 293.0892.
4.4.14.(2E,N'E)-3-(furan-2-yl)-N'-(4-hydroxybenzylidene)acrylohydrazide
(16)
Yield
60 %;mp: 261-263 °C;1H NMR (400 MHZ, DMSO-d6) δ ppm: 11.65 (s, 1H), 11.36 (s, 1H),
10.18 (s, 2H), 8.15 (s, 1H), 8.02 (s, 1H), 7.87 (d, J = 16.84 HZ, 2H), 7.64-7.58 (m, 4H),
7.53-7.45 (m, 2H), 7.36 (d, J = 15.80 HZ, 1H), 6.98 (d, J = 3.24 HZ, 1H), 6.91-6.88 (m,
5H), 6.70-6.66 (m, 2H), 6.53 (d, J = 15.48 HZ, 1H); 13C NMR (100 MHz, DMSO-d6) δ ppm:
166.2, 161.8, 159.9, 159.6, 151.5, 151.3, 147.7, 145.8, 145.6, 144.3, 129.5, 129.2, 128.9,
127.9, 125.6, 125.6, 117.7, 116.2, 115.7, 115.2, 114.7, 113.2, 113.1; HRMS (ESI) m/z;
+
calcd for C14H12N2NaO3 ([M + Na]+) 279.0740, found 279.0738.
4.4.15.(2E,N'E)-3-(furan-2-yl)-N'-(pyridin-4-ylmethylene)acrylohydrazide(17)Yield
64 %;mp:172-174 °C;1H NMR (400 MHZ, DMSO-d6) δ ppm: 11.98 (s, 1H), 11.78 (s, 1H),
8.64 (s, 4H), 8.20 (s, 1H), 8.03 (s, 1H), 7.85 (d, J = 16.68 HZ, 2H), 7.65 (t, J = 6.04 HZ,
4H), 7.53-7.45 (m, 2H), 7.31 (d, J = 15.76 HZ, 1H), 6.98 (d, J = 3.12 HZ, 1H), 6.89 (d, J =
3.20 HZ, 1H), 6.65-6.63 (m, 2H), 6.50 (d, J = 15.52 HZ, 1H); 13C NMR (100 MHz, DMSO-
d6) δ ppm: 166.7, 162.2, 151.4, 151.2, 150.7, 146.1, 145.9, 144.7, 141.9, 141.8, 141.3,
130.0, 121.2, 117.2, 116.2, 115.8, 113.9, 113.2; HRMS (ESI) m/z; calcd for
+
C13H11N3NaO2 ([M + Na]+) 264.0743, found 264.0740.
4.4.16.(2E,N'E)-N'-benzylidene-3-(furan-2-yl)acrylohydrazide (18) Yield 86 %;mp: 225-
227 °C;1H NMR (400 MHZ, DMSO-d6) δ ppm: 11.72 (s, 1H), 11.51 (s, 1H), 8.21 (s, 1H),
8.06 (s, 1H), 7.83 (d, J = 17.16 HZ, 2H), 7.73-7.68 (m, 4H), 7.50-7.40 (m, 8H), 7.33(d, J =
15.76 HZ, 1H), 6.95 (d, J = 3.24 HZ, 1H), 6.87 (d, J = 3.24 HZ, 1H), 6.65-6.61 (m, 2H), 6.49
(d, J = 15.52 HZ, 1H); 13C NMR (100 MHz, DMSO-d6) δ ppm: 166.4, 161.9, 151.5, 151.3,
147.2, 145.9, 145.7, 143.8, 134.7, 134.6, 130.6, 130.3, 129.5, 129.4, 129.3, 127.6, 127.2,
+
117.6, 115.9, 114.5, 113.2, 113.1; HRMS (ESI) m/z; calcd for C14H13N2O2 ([M + H]+)
241.0972, found 241.0876.
4.4.17.(2E,N'E)-N'-((5-bromofuran-2-yl)methylene)-3-(furan-2-yl)acrylohydrazide
(19)
Yield 52 %; mp: 162-164 °C;1H NMR (400 MHZ, CDCl3) δ ppm: 10.13 (s, 1H), 7.72-7.52
(m, 3H), 7.39 (d, J = 15.68 HZ, 1H), 6.75-6.67 (m, 2H), 6.51-6.45 (m, 2H); 13C NMR (100
24

MHz, DMSO-d6) δ ppm: 166.3, 161.9, 151.8, 151.6, 151.4, 151.3, 146.1, 145.8, 135.7,
132.7, 129.6, 128.4, 125.1, 124.8, 117.3, 116.8, 116.5, 116.2, 115.5, 114.7, 114.1, 113.1;
+
HRMS (ESI) m/z; calcd for C12H9BrN2NaO3 ([M + Na]+) 330.9689, found 330.9685.
4.4.18.(2E,N'E)-3-(furan-2-yl)-N'-(2-(trifluoromethyl)benzylidene)acrylohydrazide
(20)
Yield 79 %; mp: 182-184 °C;1H NMR (400 MHZ, CDCl3) δ ppm: 10.03 (s, 1H), 8.29 (d, J
= 7.80 HZ, 1H), 8.25 (s, 1H), 7.72 (d, J = 7.76 HZ, 1H), 7.68-7.64 (m, 2H), 7.55-7.50 (m,
13
2H), 7.43 (d, J = 15.96 HZ, 1H), 6.70 (d, J = 2.92 HZ, 1H), 6.52 (s, 1H); C NMR (100
MHz, CDCl3) δ ppm: 167.7, 151.7, 144.7, 139.4, 132.0, 130.6, 129.5, 128.5, 128.2, 127.3,
+
125.9, 125.5, 122.7, 115.3, 113.7, 112.4; HRMS (ESI) m/z; calcd for C15H11F3N2NaO2
([M + Na]+) 331.0665, found 331.0664.
4.4.19.(2E,N'E)-3-(furan-2-yl)-N'-(pyridin-3-ylmethylene)acrylohydrazide
(21)
Yield
61 %;mp: 215-217°C;1H NMR (400 MHZ, DMSO-d6) δ ppm: 11.88 (s, 1H), 11.67 (s, 1H),
8.84 (s, 2H), 8.60-8.58 (m, 2H), 8.26 (s, 1H), 8.14-8.09 (m, 3H), 7.84 (d, J = 13.20 HZ,
2H), 7.51-7.43 (m, 4H), 7.33 (d, J = 15.80 HZ, 1H), 6.97 (d, J = 3.24 HZ, 1H), 6.88 (d, J =
3.28 HZ, 1H), 6.64-6.62 (m, 2H), 6.50 (d, J = 15.52 HZ, 1H); 13C NMR (100 MHz, DMSO-
d6) δ ppm: 166.5, 162.1, 151.5, 151.2, 151.1, 150.9, 149.2, 148.8, 146.0, 145.8, 144.4,
140.9, 133.9, 133.7, 130.7, 130.6, 129.7, 128.6, 124.5, 117.4, 115.9, 115.6, 114.3, 113.2;
+
HRMS (ESI) m/z; calcd for C13H11N3NaO2 ([M + Na]+) 264.0743, found 264.0741.
4.4.20.(2E,N'E)-N'-(3,4-dihydroxybenzylidene)-3-(furan-2-yl)acrylohydrazide (22)Yield
59 %;mp: 228-230°C;1H NMR (400 MHZ, DMSO-d6) δ ppm: 11.43 (s, 1H), 11.26 (s, 1H),
9.33 (s, 4H), 8.02 (s, 1H), 7.87-7.81 (m, 3H), 7.46 (d, J = 15.80 HZ, 1H), 7.40 (d, J = 15.48
HZ, 1H), 7.29 (d, J = 15.80 HZ, 1H), 7.22-7.18 (m, 2H), 6.94-6.88 (m, 3H), 6.84 (d, J =
3.28 HZ, 1H), 6.79 (d, J = 3.48 HZ, 1H), 6.77 (d, J = 3.56 HZ, 1H), 6.65-6.64 (m, 1H), 6.62-
6.61 (m, 1H), 6.47 (d, J = 15.52 HZ, 1H); 13C NMR (100 MHz, DMSO-d6) δ ppm: 165.9,
161.4, 151.6, 151.4, 148.5, 148.2, 147.6, 146.2, 145.8, 145.6, 144.3, 129.0, 127.7, 126.2,
126.1, 121.1, 120.6, 118.1, 116.1, 115.9, 115.7, 114.9, 114.8, 113.1, 113.0, 112.8;
+
HRMS (ESI) m/z; calcd for C14H12N2NaO4 ([M + Na]+) 295.0695, found 295.0688.
4.4.21.(2E,N'E)-3-(furan-2-yl)-N'-(3-hydroxy-4-methoxybenzylidene)acrylohydrazide
(23)Yield 61 %;mp: 201-203 °C;1H NMR (400 MHZ, DMSO-d6) δ ppm: 11.50 (s, 1H), 11.34
(s, 1H), 9.31 (s, 1H), 9.30 (s, 1H), 8.06 (s, 1H), 7.91 (s, 1H), 7.84 (d, J = 19.64 HZ, 2H),
7.49-7.39 (m, 2H), 7.32-7.23 (m, 3H), 7.07-7.04 (m, 1H), 7.02-6.96 (m, 3H), 6.92 (d, J =
3.32 HZ, 1H), 6.85 (d, J = 3.32 HZ, 1H), 6.65-6.64 (m, 1H), 6.63-6.61 (m, 1H), 6.48 (d, J =
15.52 HZ, 1H); 13C NMR (100 MHz, DMSO-d6) δ ppm: 166.0, 161.5, 151.6, 151.4, 150.3,
150.1, 147.3, 147.2, 145.9, 145.6, 143.9, 129.1, 127.8, 127.6, 127.5, 120.8, 120.5, 117.9,
115.7, 115.1, 114.7, 113.1, 113.0, 112.8, 112.4, 112.3, 112.2, 56.0; HRMS (ESI) m/z;
+
calcd for C15H14N2NaO4 ([M + Na]+) 309.0846, found 309.0843.
25

4.4.22.(2E,N'E)-N'-((5-bromothiophen-2-yl)methylene)-3-(furan-2-yl)acrylohydrazide
(24)Yield 72 %;mp: 208-210 °C;1H NMR (400 MHZ, DMSO-d6) δ ppm: 11.71 (s, 1H), 11.59
(s, 1H), 8.39 (s, 1H), 8.14 (s, 1H), 7.85 (d, J = 21.44 HZ, 2H), 7.48-7.40 (m, 2H), 7.32 (d,
J = 3.80 HZ, 1H), 7.27 (d, J = 4.12 HZ, 3H), 7.17 (d, J = 15.76 HZ, 1H), 6.93 (d, J = 3.16
HZ, 1H), 6.88 (d, J = 3.20 HZ, 1H), 6.65-6.63 (m, 2H), 6.45 (d, J = 15.52 HZ, 1H); 13C NMR
(100 MHz, DMSO-d6) δ ppm: 166.0, 161.8, 151.5, 151.3, 146.1, 145.8, 141.5, 141.3,
137.8, 131.9, 131.8, 131.2, 129.6, 128.3, 117.5, 116.1, 115.5, 115.2, 114.5, 113.9, 113.1;
HRMS (ESI) m/z; calcd for C12H9BrN2NaO2S+ ([M + Na]+) 346.9460, found 346.9456.
4.4.23.(2E,N'E)-N'-(4-(benzyloxy)benzylidene)-3-(furan-2-yl)acrylohydrazide (25) Yield
87 %; mp: 226-228°C;1H NMR (400 MHZ, DMSO-d6) δ ppm: 11.57 (s, 1H), 11.41 (s, 1H),
8.16 (s, 4H), 8.00 (s, 1H), 7.84 (d, J = 18.52 HZ, 2H), 7.68-7.63 (m, 4H), 7.48-7.44 (m,
5H), 7.40 (t, J = 7.40 HZ, 5H), 7.36-7.30 (m, 3H), 7.10 (d, J = 7.36 HZ, 4H), 6.94 (d, J =
3.16 HZ, 1H), 6.86 (d, J = 3.16 HZ, 1H),6.64-6.62 (m, 2H), 6.49 (d, J = 15.52 HZ, 1H), 5.16
(s, 4H); 13C NMR (100 MHz, DMSO-d6) δ ppm: 166.1, 161.6, 160.4, 160.2, 151.6, 151.4,
146.9, 145.9, 145.6, 143.5, 137.2, 129.2, 128.9, 128.7, 128.4, 128.3, 127.9, 127.5, 127.4,
+
117.9, 115.7, 115.6, 115.2, 114.7, 113.1, 69.8; HRMS (ESI) m/z; calcd for C21H18N2NaO3
([M + Na]+) 369.1210, found 369.1209.
4.4.24.(2E,N'E)-N'-(4-(difluoromethoxy)benzylidene)-3-(furan-2-yl)acrylohydrazide (26)
Yield 72 %;mp: 164-166 °C;1H NMR (400 MHZ, CDCl3) δ ppm: 10.40 (s, 1H), 7.93 (s, 1H),
7.77 (d, J = 8.20 HZ, 2H), 7.67-7.57 (m, 2H), 7.46 (d, J = 15.72 HZ, 1H), 7.19 (d, J = 8.16
HZ, 2H), 6.77-6.40 (m, 3H); 13C NMR (100 MHz, CDCl3) δ ppm: 168.2, 152.4, 151.7, 144.7,
142.8, 131.2, 130.2, 128.8, 119.5, 118.2, 115.7, 114.9, 114.1, 113.1, 112.4; HRMS (ESI)
+
m/z; calcd for C15H12F2N2NaO3 ([M + Na]+) 329.0708, found 329.0709.
4.4.25.(2E,N'E)-3-(5-nitrofuran-2-yl)-N'-(3,4,5-trimethoxybenzylidene)acrylohydrazide
(27)Yield 79 %;mp: 251-253 °C;1H NMR (400 MHZ, DMSO-d6) δ ppm: 11.91 (s, 1H), 11.85
(s, 1H), 8.14 (s, 1H), 7.99 (s, 1H), 7.78-7.74 (m, 3H), 7.75-7.51 (m, 2H), 7.29 (d, J= 3.72
HZ, 1H), 7.20 (d, J = 3.76 HZ, 1H), 7.06 (s, 4H), 6.87 (d, J = 15.68 HZ, 1H), 3.85 (d, J =
12.04 HZ, 12H), 3.70 (s, 6H); 13C NMR (100 MHz, DMSO-d6) δ ppm: 165.2, 160.6, 153.5,
148.1, 143.8, 139.8, 130.0, 126.4, 124.3, 117.3, 115.2, 104.9, 104.3, 60.6, 60.3, 56.4;
+
HRMS (ESI) m/z; calcd for C17H17N3NaO7 ([M + Na]+) 398.0959, found 398.0952.
4.4.26.(2E,N'E)-N'-(2-hydroxy-5-methoxybenzylidene)-3-(5-nitrofuran-2-
yl)acrylohydrazide (28) Yield 63 %; mp: 252-254 °C;1H NMR (400 MHZ, DMSO-d6) δ ppm:
12.08 (s, 2H), 10.47 (s, 2H), 8.39 (d, J = 22.6 HZ, 2H), 7.77-7.66 (m, 2H), 7.55-7.50 (m,
2H), 7.29-7.16 (m, 4H), 6.92-6.81 (m, 6H), 3.77 (s, 3H), 3.72 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ ppm: 164.9, 160.4, 153.7, 153.4, 152.7, 152.6, 152.2, 151.9, 151.1, 147.4,
141.1, 127.4, 126.6, 123.9, 121.6, 120.9, 119.4, 119.1, 118.7, 117.8, 117.4, 117.2, 115.2,
+
112.2, 108.9, 59.9, 55.6; HRMS (ESI) m/z; calcd for C15H13N3NaO6 ([M + Na]+) 354.0697,
found 354.0694.
26

4.4.27.(2E,N'E)-N'-(4-fluorobenzylidene)-3-(5-nitrofuran-2-yl)acrylohydrazide (29) Yield
66 %; mp: 240-242 °C;1H NMR (400 MHZ, DMSO-d6) δ ppm: 11.94 (s, 1H), 11.79 (s, 1H),
8.22 (s, 1H), 8.09 (s, 1H), 7.83-7.77 (m, 6H), 7.63-7.52 (m, 3H), 7.36-7.28 (m, 5H), 7.20
(d, J = 3.84 HZ, 1H), 6.85 (d, J = 15.68 HZ, 1H); 13C NMR (100 MHz, DMSO-d6) δ ppm:
164.6, 164.4, 161.9, 160.1, 153.2, 152.9, 151.8, 151.6, 146.4, 143.0, 130.6, 130.4, 129.4,
129.3, 129.0, 128.9, 127.3, 125.9, 123.7, 120.7, 116.8, 115.9, 115.7, 114.7; HRMS (ESI)
+
m/z; calcd for C14H10FN3NaO4 ([M + Na]+) 326.0548, found 326.0545.
4.4.28.(2E,N'E)-N'-(2-hydroxy-4-methoxybenzylidene)-3-(5-nitrofuran-2-
yl)acrylohydrazide (30)Yield 47 %;mp: 262-264 °C;1H NMR (400 MHZ, DMSO-d6) δ ppm:
12.00 (s, 2H), 11.35 (s, 2H), 8.33 (d, J = 13.04 HZ, 2H), 7.79-7.76 (m, 2H), 7.65-7.46 (m,
4H), 7.31-7.20 (m, 2H), 6.82 (d, J = 15.68 HZ, 2H), 6.54-6.46 (m, 4H); 13C NMR (100 MHz,
DMSO-d6) δ ppm: 162.8, 160.1, 159.8, 153.8, 153.5, 152.2, 148.7, 142.3, 131.5, 127.4,
126.4, 123.9, 121.5, 117.3, 115.2, 112.2, 107.1, 101.6, 101.4, 55.8, 55.6; HRMS (ESI)
+
m/z; calcd for C15H13N3NaO6 ([M + Na]+) 354.0697, found 354.0701.
4.4.29.(2E,N'E)-N'-(4-fluoro-2-hydroxybenzylidene)-3-(5-nitrofuran-2-yl)acrylohydrazide
(31) Yield 62 %;mp: 288-290 °C;1H NMR (400 MHZ, DMSO-d6) δ ppm: 12.08 (s, 1H),
11.51 (s, 1H), 8.40 (s, 2H), 7.77-7.74 (m, 2H), 7.64 (t, J = 7.12 HZ, 2H), 7.52 (d, J = 15.60
13
HZ, 2H), 7.32-7.19 (m, 2H), 6.83-6.68 (m, 6H); C NMR (100 MHz, DMSO-d6) δ ppm:
165.6, 164.9, 163.1, 160.4, 159.7, 159.5, 153.8, 153.4, 152.3, 152.2, 147.2, 140.9, 131.5,
131.4, 127.6, 126.6, 123.8, 121.4, 117.3, 117.2, 116.3, 115.1, 107.4, 107.2, 103.9, 103.7;
+
HRMS (ESI) m/z; calcd for C14H10FN3NaO5 ([M + Na]+) 342.0497, found 342.0492.
4.4.30.(2E,N'E)-N'-(4-(benzyloxy)benzylidene)-3-(5-nitrofuran-2-yl)acrylohydrazide (32)
Yield 64 %;mp: 224-226°C;1H NMR (400 MHZ, DMSO-d6) δ ppm: 11.79 (s, 1H), 11.64 (s,
1H), 8.16 (s, 1H), 8.04 (s, 1H), 7.80-7.77 (m, 2H), 7.70-7.60 (m, 5H), 7.54 (d, J = 5.08 HZ,
1H), 7.51-7.46 (m, 5H), 7.40 (t, J = 7.12 HZ, 4H), 7.35 (t, J = 6.84 HZ, 3H), 7.19 (d, J =
3.80 HZ, 1H), 7.11 (t, J = 5.60 HZ, 4H), 6.85 (d, J = 15.64 HZ, 1H), 5.17 (s, 4H); 13C NMR
(100 MHz, DMSO-d6) δ ppm: 160.6, 160.4, 153.6, 152.1, 147.9, 144.51, 137.2, 129.4,
128.9, 128.4, 128.3, 127.3, 126.2, 124.6, 117.2, 115.7, 115.6, 115.2, 69.8; HRMS (ESI)
+
m/z; calcd for C21H17N3NaO5 ([M + Na]+) 414.1060, found 414.1054.
4.4.31.(2E,N'E)-3-(5-nitrofuran-2-yl)-N'-(4-
(trifluoromethoxy)benzylidene)acrylohydrazide (33)Yield 67 %;mp: 180-182°C;1H NMR
(400 MHZ, DMSO-d6) δ ppm: 12.01 (s, 1H), 11.86 (s, 1H), 8.25 (s, 1H), 8.12 (s, 1H), 7.87
(t, J = 8.00 HZ, 4H), 7.81-7.77 (m, 2H), 7.63-7.53 (m, 3H), 7.47 (t, J = 8.04 HZ, 4H), 7.36
(d, J = 3.72 HZ, 1H), 7.21 (d, J = 3.76 HZ, 1H), 6.86 (d, J = 15.68 HZ, 1H); 13C NMR (100
MHz, DMSO-d6) δ ppm: 165.3, 160.7, 153.7, 153.4, 152.2, 149.8, 146.5, 143.2, 133.8,
133.7, 129.6, 129.2, 128.0, 126.6, 124.2, 121.9, 121.8, 121.1, 119.2, 117.4, 115.2; HRMS
+
(ESI+) m/z; calcd for C15H10F3N3NaO5 ([M + Na]+) 392.0465, found 392.0461.
27

4.4.32.(2E,N'E)-3-(4-(benzyloxy)phenyl)-N'-(3,4,5-
trimethoxybenzylidene)acrylohydrazide (34) Yield 74 %;mp: 196-198 °C;1H NMR (400
MHZ, CDCl3) δ ppm: 10.01 (s, 1H), 7.86 (t, J = 10.24 HZ, 2H), 7.61 (t, J = 8.48 HZ, 2H),
7.48-7.36 (m, 7H), 7.04-6.96 (m, 3H), 5.14 (s, 2H), 3.96 (s, 6H), 3.92 (s, 3H);13C NMR
(100 MHz, CDCl3) δ ppm: 167.9, 160.5, 153.5, 143.7, 143.5, 139.9, 136.5, 129.9, 129.3,
128.7, 128.2, 128.1, 127.5, 115.2, 114.1, 104.4, 70.1, 61.0, 56.3; HRMS (ESI) m/z; calcd
+
for C26H26N2NaO5 ([M + Na]+) 469.1734, found 469.1719.
4.4.33.(2E,N'E)-3-(4-(benzyloxy)phenyl)-N'-(4-
(trifluoromethoxy)benzylidene)acrylohydrazide (35) Yield 69 %;mp: 227-229 °C;1H NMR
(400 MHZ, DMSO-d6) δ ppm: 11.72 (s, 1H), 11.45 (s, 1H), 8.21 (s, 1H), 8.06 (s, 1H), 7.78-
7.70 (m, 6H), 7.65-7.56 (m, 4H), 7.45 (d, J = 7.92 HZ, 9H), 7.40 (t, J = 7.28 HZ, 4H), 7.33
(t, J = 7.12 HZ, 2H), 7.07 (d, J = 8.40 HZ, 4H), 6.55 (d, J = 15.68 HZ, 1H), 5.16 (s, 4H); 13C
NMR (100 MHz, DMSO-d6) δ ppm: 166.9, 162.3, 160.4, 145.2, 142.5, 141.7, 141.0, 137.2,
134.1, 130.5, 129.9, 129.4, 129.2, 128.9, 128.4, 128.2, 121.8, 119.2, 118.0, 115.8, 115.7,
+
69.8; HRMS (ESI) m/z; calcd for C24H19F3N2NaO3 ([M + Na]+) 463.1240, found 463.1235.
4.4.34.(2E,N'E)-N'-(4-(benzyloxy)benzylidene)-3-(4-(benzyloxy)phenyl)acrylohydrazide
(36) Yield 75 %;mp: 221-223 °C;1H NMR (400 MHZ, DMSO-d6) δ ppm: 11.48 (s, 1H),
11.32 (s, 1H), 8.18 (s, 1H), 7.99 (s, 1H), 7.72-7.64 (m, 6H), 7.60-7.54 (m, 4H), 7.47 (d, J
= 7.04 HZ, 8H), 7.41 (t, J = 7.12 HZ,9H), 7.36-7.32 (m, 4H), 7.09 (t, J = 6.60 HZ, 8H), 6.55
(d, J = 15.72 HZ, 1H), 5.16 (s, 8H); 13C NMR (100 MHz, DMSO-d6) δ ppm: 166.6, 162.0,
160.3, 160.2, 160.1, 146.6, 143.1, 142.1, 140.5, 137.2, 130.4, 129.8, 129.1, 128.9, 128.4,
128.2, 127.9, 127.6, 127.5, 118.4, 115.8, 115.7, 115.2, 69.8; HRMS (ESI) m/z; calcd for
+
C30H26N2NaO3 ([M + Na]+) 485.1836, found 485.1823.
4.4.35.(2E,N'E)-3-(4-(benzyloxy)phenyl)-N'-(4-
(difluoromethoxy)benzylidene)acrylohydrazide (37) Yield 57 %;mp: 206-208 °C;1H NMR
(400 MHZ, DMSO-d6) δ ppm: 11.63 (s, 1H), 11.45 (s, 1H), 8.24 (s, 1H), 8.05 (s, 1H), 7.85-
7.78 (m, 4H), 7.72 (d, J = 8.44 HZ, 2H), 7.66-7.57 (m, 4H), 7.51-7.46 (m, 5H), 7.40 (t, J
= 7.24 HZ,4H), 7.34 (t, J = 7.12 HZ, 3H), 7.26-7.24 (m, 4H), 7.14-7.07 (m, 5H), 6.57 (d, J
= 15.76 HZ, 1H), 5.16 (s, 4H); 13C NMR (100 MHz, DMSO-d6) δ ppm: 166.8, 162.3, 160.4,
160.2, 152.5, 145.7, 142.4, 142.2, 140.8, 137.2, 131.7, 130.4, 129.9, 129.3, 129.1, 128.9,
128.4, 128.2, 128.1, 127.9, 119.2, 118.1, 116.6, 115.8, 115.7, 115.0, 114.1, 69.8; HRMS
+
(ESI) m/z; calcd for C24H20F2N2NaO3 ([M + Na]+) 445.1334, found 445.1336.
4.4.36.(2E,N'E)-3-(4-(benzyloxy)phenyl)-N'-(2,3,4-
trimethoxybenzylidene)acrylohydrazide (38) Yield 70 %; mp: 164-166 °C;1H NMR (400
MHZ, CDCl3) δ ppm: 9.25 (s, 1H), 8.10 (s, 1H), 7.83 (d, J = 15.96 HZ, 1H), 7.71 (d, J =
8.80 HZ, 1H), 7.61 (d, J = 8.60 HZ, 2H), 7.48-7.34 (m, 6H), 7.02 (d, J = 8.60 HZ, 2H), 6.79
(d, J = 8.84 HZ, 1H), 5.13 (s, 2H), 3.96 (s, 3H), 3.94 (s, 3H), 3.91 (s, 3H); 13C NMR (100
28

MHz, CDCl3) δ ppm: 167.4, 160.4, 155.5, 153.1, 143.2, 141.9, 139.3, 136.5, 129.9, 128.7,
128.2, 127.5, 121.2, 120.4, 115.1, 114.3, 107.9, 70.1, 61.9, 60.9, 56.1; HRMS (ESI) m/z;
+
calcd for C26H26N2NaO5 ([M + Na]+) 469.1734, found 469.1722.
4.4.37.(2E,N'E)-3-(4-(benzyloxy)phenyl)-N'-((5-bromothiophen-2-
yl)methylene)acrylohydrazide (39)Yield 77 %;mp: 211-213°C;1H NMR (400 MHZ, DMSO-
d6) δ ppm: 11.63 (s, 1H), 11.52 (s, 1H), 8.40 (s, 1H), 8.13 (s, 1H), 7.65 (d, J = 7.88 HZ,
2H), 7.60-7.54 (m, 4H), 7.46 (d, J = 6.60 HZ, 4H), 7.40 (t, J = 7.24 HZ, 4H), 7.35 (d, J =
7.08 HZ, 2H), 7.31 (d, J = 4.00 HZ, 1H), 7.29-7.26 (m, 4H), 7.09 (t, J = 8.92 HZ, 4H), 6.52
(d, J = 15.72 HZ, 1H), 5.16 (s, 4H); 13C NMR (100 MHz, DMSO-d6) δ ppm: 166.4, 162.2,
160.4, 160.3, 142.5, 141.6, 141.5, 141.2, 140.9, 137.5, 137.2, 131.8, 131.0, 130.3, 129.9,
128.9, 128.4, 128.2, 127.9, 127.8, 117.9, 115.8, 115.1, 114.5, 69.8; HRMS (ESI) m/z;
calcd for C21H17BrN2NaO2S+ ([M + Na]+) 463.0086, found 463.0079.
4.4.38.(2E,N'E)-N'-((5-bromofuran-2-yl)methylene)-3-(3,4,5-
trimethoxyphenyl)acrylohydrazide (40)Yield 72 %;mp: 213-216°C;1H NMR (400 MHZ,
DMSO-d6) δ ppm: 11.68 (s, 1H), 11.50 (s, 1H), 8.04 (s, 1H), 7.88 (s, 1H), 7.58 (d, J =
15.52 HZ, 2H), 7.38 (d, J = 15.88 HZ, 1H), 7.02-6.96 (m, 6H), 6.76 (d, J = 3.44 HZ, 2H),
6.61 (d, J = 15.68 HZ, 1H), 3.85 (s, 6H), 3.83 (s, 6H), 3.70 (s, 6H); 13C NMR (100 MHz,
DMSO-d6) δ ppm: 162.0, 153.6, 151.9, 141.5, 139.5, 135.5, 130.7, 125.0, 119.7, 116.6,
+
114.7, 106.1, 105.7, 60.6, 56.4; HRMS (ESI) m/z; calcd for C17H17BrN2NaO5 ([M + Na]+)
431.0203, found 431.0261.
4.4.39.(2E,N'E)-N'-((5-bromothiophen-2-yl)methylene)-3-(3,4,5-
trimethoxyphenyl)acrylohydrazide(41) Yield 78 %;mp: 221-223 °C;1H NMR (400 MHZ,
CDCl3) δ ppm: 10.21 (s, 1H), 8.01 (s, 1H), 7.79 (d, J = 15.84 HZ, 1H), 7.39 (d, J = 15.80
HZ, 1H), 7.04 (d, J = 2.76 HZ, 2H), 6.89 (s, 2H), 3.96 (s, 6H),3.93 (s, 3H); 13C NMR (100
MHz, DMSO-d6) δ ppm: 161.9, 153.6, 142.9, 141.5, 141.4, 141.3, 139.5, 137.7, 131.9,
131.8, 131.2, 130.9, 130.6, 119.7, 116.6, 115.1, 106.0, 105.7, 60.6, 56.4; HRMS (ESI)
m/z; calcd for C17H17BrN2NaO4S+ ([M + Na]+) 446.9985, found 446.9979.
4.4.40.(2E,N'E)-N'-(thiophen-2-ylmethylene)-3-(3,4,5-trimethoxyphenyl)acrylohydrazide
(42)Yield 56 %;mp: 205-207°C;1H NMR (400 MHZ, CDCl3) δ ppm: 10.29 (s, 1H), 8.15 (s,
1H), 7.79 (d, J = 15.88 HZ, 1H), 7.48 (d, J = 15.88 HZ, 1H), 7.41 (d, J = 4.92 HZ, 1H), 7.32
(d, J = 3.24 HZ, 1H), 7.10 (t, J = 3.92 HZ, 1H), 6.90 (s, 2H), 3.96 (s, 6H), 3.92 (s, 3H); 13C
NMR (100 MHz, CDCl3) δ ppm: 167.8, 153.4, 143.7, 139.9, 138.9, 138.8, 130.7, 129.9,
128.0, 127.6, 115.8, 105.5, 61.0, 56.2; HRMS (ESI) m/z; calcd for C17H18N2NaO4S+ ([M
+ Na]+) 369.0879, found 369.0877.
4.4.41.(2E,N'E)-N'-([1,1'-biphenyl]-4-ylmethylene)-3-(3,4,5-
trimethoxyphenyl)acrylohydrazide (43)Yield 82 %;mp: 194-196°C;1H NMR (400 MHZ,
CDCl3) δ ppm: 9.96 (s, 1H), 7.99 (s, 1H), 7.86-7.81 (m, 3H), 7.71-7.65 (m, 4H), 7.53-7.47
29