Flavonoid analogues as urease inhibitors: Synthesis, biological evaluation, molecular docking studies and in-silico ADME evaluation

Article abstract of DOI:10.1016/j.bioorg.2020.104370

A series of novel flavonoid analogues were designed and synthesized. The aimed compounds for urease inhibitory activities were clearly superior to the control drug thiourea (more than 10 times). Among these compounds, L2 (IC₅₀ = 1.343 μM) and L12 (IC₅₀ = 1.207 μM) exhibited the most excellent urease inhibitory activity in vitro. The molecular dockings of L2, L12 and L22 into urease were performed to explore the binding modes and their structure-activity relationship. Furthermore, these aimed compounds showed good druggable properties.

Full text of DOI:10.1016/j.bioorg.2020.104370

Journal Pre-proofs
Flavonoid analogues as urease inhibitors: Synthesis, biological evaluation,
molecular docking studies and in-silico ADME evaluation
Honghui Liu, Yan Wang, Mingxia Lv, Yi Luo, Bu-Ming Liu, Yan Huang,
Mian Wang, Jianyi Wang
PII:
S0045-2068(20)31668-0
DOI:
https://doi.org/10.1016/j.bioorg.2020.104370
YBIOO 104370
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
4 August 2020
22 September 2020
8 October 2020
Please cite this article as: H. Liu, Y. Wang, M. Lv, Y. Luo, B-M. Liu, Y. Huang, M. Wang, J. Wang, Flavonoid
analogues as urease inhibitors: Synthesis, biological evaluation, molecular docking studies and in-silico ADME
evaluation, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104370
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Flavonoid analogues as urease inhibitors: Synthesis, biological
evaluation, molecular docking studies and in-silico ADME evaluation
Honghui Liu1,3, Yan Wang2, Mingxia Lv1, Yi Luo4, Bu-Ming Liu5,·Yan Huang5, Mian Wang2*,
Jianyi Wang1,3*
1. Medical College, Guangxi University, Nanning 530004, China.
*E-mail: jianyiwang@gxu.edu.cn
2. College of Life Science and Technology, Guangxi University, Nanning, 530004, China,
*E-mail: mianwang@gxu.edu.cn
3. School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, China
4. Guangxi Institute for Food and Drug Control, Nanning 530021, China
5. Guangxi Key Laboratory of Traditional Chinese Medicine, Quality Standards, Nanning
530022, China
Abstract
A series of novel flavonoid analogues were designed and synthesized. The aimed
compounds for urease inhibitory activities were clearly superior to the control drug
thiourea (more than 10 times). Among these compounds, L2 (IC50 = 1.343 µM) and
L12 (IC50 = 1.207 µM) exhibited the most excellent urease inhibitory activity in vitro.
The molecular dockings of L2, L12 and L22 into urease were performed to explore the
binding modes and their structure-activity relationship. Furthermore, these aimed
compounds showed good druggable properties.
Keywords
Urease inhibitor; Flavonoid analogues; Synthesis; Docking; Druggability
1. Introduction
Urease (urea amidohydrolase EC3.5.1.5) is nickel-dependent enzyme that can
specifically catalyze the hydrolysis of urea to release ammonia(NH3) and carbon
dioxide (CO2)[1]. The produced ammonia catalyzed by urease helps maintain the pH
balance of human intestinal microflora, providing Helicobacter pylori a suitable
environment for their growth and reproduction in the harsh pH conditions of the
stomach[2]. Clearly, Urease is closely related to colonization and survival of
Helicobacter pylori in the stomach[3], and considered as a therapeutic target of gastric
lymphoma, gastric carcinoma and peptic ulcer disease caused by Helicobacter pylori.
Some existing urease inhibitors were summarized in Fig. 1. The acetohydroxamic
1

acid (Compound 1) is commonly recognized as urease inhibitor via competitively
binding the metal center of the active site of the urease [4], and forming strong complex
with the metal center [5], [6].To date acetohydroxamic acid was the only urease
inhibitor that is approved by the US Food and Drug Administration for marketing.
However, acetohydroxamic acid only shows moderate activity, resulting in a relative
large dose [7]. Phosphoramidates (Compounds 2,3) exhibit high urease inhibitory
activities because phosphoramidates can tightly bind to the active metallocenter, which
is closely similar to the transition state of the enzymatic reaction[8]. But they were not
approved for marketing as drugs probably because of their rapid hydrolysis in low pH
of gastric juice[9]. The barbiturates or thiobarbiturates (Compounds 4,5,6) were
analogous compounds to urea or thiourea fragment, which can competitively bind to
the nickel (II) center of urease[10][11][12][13], showing some urease activities.
However, they appeared to be weak to moderate uncompetitive or mixed inhibitors, and
low practical value. The aromatic heterocyclic compounds (Compounds 7,8,9) bound
the active site of the urease were very significant urease inhibitors. Their inhibition
activity for urease results from interaction of side chain of cysteine or methionine with
π electrons of aromatic fragment of the compound [14][15][16][17]. Some metal
complexes can also produce good inhibition of urease[18]. However, the serious side
effects and toxicity limit further research of the aromatic heterocyclic compounds and
metal complexes. Therefore, it is an urgent need to develop novel urease inhibitors with
low toxicity and high efficiency.
O
O
NH NH
O
C
S
O
O
H2N
HO
P
NH
N
O
P
H2N
NH
NH
NO2
NH2
NH2
F
IC50=23.81.5M
IC50=nM
IC50=nM
IC50=13.01.2M
1
3
2
4
S
O
HN
NH
OH
N
N
O
O
O
O
N
Br
O
O
Br
N
NH
NH
OH
OH
O
OH
OH
IC50=10.57±0.M
IC50=12.6±0.M
IC50=1.61±0.M
IC50=91.83±0.M
7
8
6
5
OH
N
NH
OH
OH
O
S
HO
O
N
N
N
OH
O2N
H
OH
H3CO
O
OH
OCH3
IC50=11.2±0.9M
IC50=8.4±0.05M
IC50=45.6±0.M
10
11
9
Fig. 1. Some existing urease inhibitors
2

The natural product flavonoid had been demonstrated significant inhibitory activity
on urease[19], beside antioxidant [20], anti-inflammatory[21], antidiabetic [22], anti-
bacterial [23], antiviral[24], anti-protozoals [25] and anticancer[26]. A structural
resemblance of hydrazide moiety along with thiourea(semicarbazone [27],
benzoylhydrazide[28] and isoniazid derivatives[29] make them become good
pharmacophore for urease inhibitor.
However, regardless of the flavonoid or urea structure, any single group of urease
inhibitory activity is difficult to satisfy. To do so, in this work, we design and synthesize
a series of novel urease inhibitor of flavonoid analogues, in which flavonoids could
integrate the top of the active chamber, and the hydrazide moiety (semicarbazide,
benzoylhydrazide, isoniazid) could bind the active chamber(Fig. 2). On this basis, we
performed biological evaluation, molecular docking studies and in-silico ADME
evaluation to the aimed compounds. It is hopeful to further improve the inhibitory
activity of urease and obtain novel candidate compound for urease inhibitors.
OH
O
HO
O
OH
R1
OH
HN
N
O
OH
HO
O
O
R
O
R
N
The aimed compound
N
NH2
H
Fig. 2. The designed urease inhibitors
2. Results and discussion
2.1. The synthesis of the aimed compounds
HO
O
C,15h
O
HO
C,5h
O
R
K2CO3,
Cl
TBAB,60°
R
+
,130°
CH3COONa
R
R
Dry acetone
acid
acetic
Glacial
O
O
OH
O
OH
O
O
2(a-j)
A
4(a-j)
3(a-j)
O
R1
O
O
HN
HO
N
NH2
N
R1
HO
O
O
R
6(a-c)
H
O
O
R
Glacial acetic acid
C,8h
100°
HMTA
Absolute ethanol
C,8h
83°
5(a-j)
(L1-L10,L11-L20,L21-L30)
Scheme 1. Synthetic routes of the aimed compounds
3

As show in Scheme 1, Compound A firstly reacted with compound 2(a-j) to
afford an intermediate 3(a-j). Subsequently, the intermediate 3(a-j) was treated with
glacial acetic acid and sodium acetate to yield the cyclic intermediate 4(a-j). In the
presence of glacial acetic acid, 4(a-j) and hexamethylenetetramine (HMTA) underwent
a 8h Duff formylation reaction to provide 5(a-j). In the ethanol, Compound 5 (a-j) and
6 (a-c) further reacted to give the aimed product semicarbazide(L1-L10), benzoyl
hydrazide(L11-L20) and isoniazid(L21-L30). The synthesized compounds were
characterized by HR-EI-MS, 1H NMR, and 13C NMR spectroscopic techniques.
2.2 The inhibitory activity assays for Urease
Table 1. The inhibitory activities of the aimed compounds for urease.
O
R1
NH
N
HO
O
O
R
R
R1
Species
L1
R
R1
IC50(µM)a
IC50(µM)a
O
NH2-
1.565±0.102
L16
L17
L18
L19
L20
L21
L22
L23
L24
L25
1.663±0.429
NO2
F
L2
L3
L4
L5
L6
L7
L8
L9
L10
NH2-
NH2-
NH2-
NH2-
NH2-
NH2-
NH2-
NH2-
NH2-
1.343±0.288
1.356±0.381
1.907±0.065
1.965±0.635
1.833±0.232
1.936±0.576
ND
2.002±0.069
ND
O
Cl
S
Br
2.441±0.355
2.771±0.338
1.546±0.503
1.470±0.502
2.101±0.842
1.523±0.922
1.658±0.621
N
N
O
NO2
F
Cl
Br
O
N
S
N
N
1.621±0.351
2.205±1.199
4

O
N
L11
L12
L13
L14
1.378±0.616
1.207±0.186
1.475±0.434
1.643±0.415
L26
L27
L28
L29
L30
1.821±0.846
1.449±0.247
ND
NO2
N
F
Cl
Br
O
S
N
N
1.645±0.469
1.566±0.364
N
L15
1.869±0.734
21.902±0.696
thiourea
a
Values are the mean ± SEM. All experiments were performed at least three times, ND = Not determined.
Next, the synthesized compounds L1–L10, L11–L20 and L21-L30 were
performed inhibitory activity assays for urease. As shown in Table 1, thiourea served
as a positive control drug (IC50 values of 21.902 ± 0.696μM), the inhibitory activities
of semicarbazide, benzohydrazide and isoniazid compounds for urease were 10 times
more than the thiourea’s one.
A comparison of Compounds L2-L4 and L12-L14 show that the H atom in the B
ring replaced by an electron-withdrawing halogen atom (-F, -Cl, -Br) with similar size,
the inhibitory activities for urease gradually increased with the -Br, -Cl, -F trend. On
the other hand, the H atom in the B ring replaced by the electron-withdrawing group -
NO2 (L7, L17) or the electron-donated -CH3 or –OCH3 group (L6, L16) with larger
size, decrease the inhibitory activity against urease. These show that the polarity
(hydrophobicity) of compound and the pocket size of the active site of urease may
determine the inhibitory activities of compounds for urease. The H atom in the B ring
replaced by electron-withdrawing -F, -Cl, or –Br group was favorable for the formation
of the halogen-bond interaction between the B ring and the urease, and the active pocket
of urease is not large enough to accommodate the large bulk B ring.
When the B ring of the flavonoid became thiophene, furan, and naphthalene, the
urease activity of compounds (L8-L10, L18-L20 and L28-L30) are accordingly
decreased compared with compounds L1-L7，L11-L17 and L21- L27, showing that
the benzene is the optimal group in B ring. No clear activity difference between L1,
L11, and L21, imply that in these compounds the -NH2 group is not a key group, and
can be modified by benzene or pyridine ring.
5

All aimed compounds (semicarbazide, benzohydrazide and isoniazid) exhibited
better inhibitory activities for urease, showing that flavonoid analogues is significant
novel lead compounds for urease inhibitors or anti-Helicobacter pylori agents. It is
worth further structure modification of flavonoid analogues.
2.3. Docking studies
To demonstrate the binding mode and the structure-activity relationship of
flavonoid analogues L1-L10，L11-L20 and L21-L30 at the active site of urease, we
performed molecular docking studies on representative compounds L2, L12, L22 acting
on urease. It should be noticed that, L2, L12 and L22 were surrounded by similar
residues CYS322, ALA170, HIS323, Ni798, ASP224 and HIS222 (Fig. 3-5), and
presented similar interaction with the active site of urease, which may be the reason
why L2, L12 and L22 show inhibitory activity of the same order of magnitude toward
urease. In the other hand, the binding sites of urease was slightly changed by L2, L12
and L22, which may be the reason why L2, L12 and L22 show different inhibitory
activity toward urease.
Seen from Fig.3, the B ring in L2 surrounded by residues LYS169, GLU166 and
ALA170, could form pi-Alkly interaction with ALA170 and Vander waals interaction
with LYS169 and GLU166. Clearly, the benzene as B ring is more favorable for these
pi-Alkly and Vander waals interaction than the thiophene and the furan as B ring,
supporting that in the inhibitory activity for urease, L1, L11 and L21 were superiors to
L8-L9, L18-L19 and L28-L29. The benzene H in ring B replaced by a small bulk
bioisosteres F group (electron-withdrawing) can increase the halogen bond interaction
between B ring and the residues in the active site, and the interaction between B ring
and Ni metal center, supporting the inhibitory activities order for urease that the
benzene H in ring B replaced by small bulk and electron-withdrawing group are
favorable for the inhibitory activity for urease.
The semicarbazone moiety (NNH) in L2 can form hydrogen-bonded interaction
with residue ASP363. Beside one conventional hydrogen bonds between the NH group
and ASP224, the benzoyl hydrazide moiety in L12 (Fig.4) was observed to form π-π
interactions with residue TRP225. This may be the main reason that L12 is superiors to
L2 in the inhibitory activities for urease. When the benzene moiety of benzoyl
hydrazide in L12 was replaced by the pyridine to give L22(Fig.5), the π-π interactions
disappeared between the active site residues and the pyridine moiety of isoniazid,
6

correspondingly decreasing the inhibitory activity for urease.
HIS
C:139
NI
KCX
C:220
GLY
C:280
HIS
C:275
C:798
HIS
NI
C:249
ALA
C:366
C:799
HIS
C:222
ASP
C:363
O
O
CYS322
LYS
H
ASP
C:224
N
N
N
C:169
MET367
H
F
HIS323
GLU
C:166
ARG
C:339
O
ASP363
HIS
C:323
ALA170
ALA
C:170
O
MET
C:367
ASP224
CYS
C:322
HIS222
Pi-Pi T-shaped
Pi-Sulfur
van der Waals
Conventional Hydrogen Bond
Metal-Acceptor
Carbon Hydrogen Bond
Pi-Alkyl
Fig. 3. The interaction map of compound L2 docked into urease.
GLY
C:167
NI
C:799
HIS
C:249
TRP
C:225
HIS
C:275
NI
C:798
ASP
C:224
O
ARG
H
HIS323
C:339
N
KCX
HIS
CYS322
C:220
C:222
GLY
C:280
HN
H
F
ASP
O
O
O
C:363
ARG339
MET
C:367
ALA366
ALA
C:366
ASP224
ALA170
ALA
C:170
LYS
C:169
HIS
C:324
GLY280
TRP225
HIS
C:323
CYS
C:322
HIS222
Pi-Pi T-shaped
Pi-Pi Stacked
Metal-Acceptor
Pi-Cation
Carbon Hydrogen Bond
Pi-Alkyl
Halogen (Fluorine)
van der Waals
Conventional Hydrogen Bond
Fig.4. The interaction map of compound L12 docked into urease.
ARG339
HIS
C:249
HIS
C:222
GLY
O
O
HIS
C:275
ASP
C:224
C:280
GLY280
N
N
NI
C:799
HIS
C:324
CYS322
N
H
F
HIS323
NI
C:798
O
KCX
C:220
MET ALA
C:367 C:366
HIS
C:323
ALA366
ARG
C:339
ASP
O
CYS
ALA
C:170
C:363
LYS
C:169
C:322
ALA170
ASP224
Metal-Acceptor
Carbon Hydrogen Bond
Pi-Catio
Pi-Alkyl
Pi-Pi Stacked
Halogen (Fluorine)
van der Waals
n
Fig.5. The interaction map of compound L22 docked into urease.
2.4 In-silico ADME evaluation
To further evaluate druggability of these compounds, we performed in-silico
7

adsorption, distribution, metabolism, excretion and toxicity (ADME) evaluation using
Molinspiration and Molsoft online （ http://www.molinspiration.com/ and
http://www.molsoft.com/) toolkit[30]. Seen from Table 2, all compounds had 2–4
hydrogen bond donors (HBD) and 5–8 hydrogen bond acceptors (HBA), obeying the
Lipinski’s rule of 5[31]: The hydrogen bond donors are no more than 5, and the
hydrogen bond acceptors are no more than 10. The molecular mass is less than 500
Daltons, and the octanol-water partition coefficient log P is smaller than 5[32]. All
compounds had 3-5 rotatable bonds (nROTB) determining the molecular flexibility
(should be <10), and 90.90–131.06ꢀÅ2 (<140ꢀÅ2)topological polar surface area (TPSA)
values, showing good permeability of the aimed compounds in the cellular plasma
membrane and blood-brain barrier (BBB) [33]. Clearly, most of the aimed compounds
possess agreeable ADME properties for oral bio-availability, and good druggability.
Table 2. The druggability of the aimed compounds.
Compound
H-bond
H-bond
nROTB Lipinski, TPSA(Å2)
violation
Mol.wt
logP
donor
acceptor
L1
L2
4
4
4
4
4
4
4
4
4
4
2
2
2
2
2
2
2
2
2
5
5
5
5
5
6
7
6
6
5
5
5
5
5
6
6
7
6
6
3
3
3
3
3
4
3
3
3
3
4
4
4
4
4
5
4
4
4
Yes, 0
Yes, 0
Yes, 0
Yes, 0
Yes, 0
Yes, 0
Yes, 0
Yes, 0
Yes, 0
Yes, 0
Yes, 0
Yes, 0
Yes, 1
Yes, 1
Yes, 1
Yes, 0
Yes, 0
Yes, 0
Yes, 0
117.92
117.92
117.92
117.92
117.92
127.16
117.92
131.06
117.92
117.92
91.90
323.09
341.08
357.05
401.00
337.11
353.10
368.08
313.07
329.05
373.11
384.11
402.10
418.07
462.02
399.12
414.12
429.10
374.09
390.07
3.03
3.20
3.71
3.84
3.48
3.09
2.16
1.98
2.62
4.22
4.79
4.95
5.47
5.60
5.24
4.85
3.92
3.74
4.38
L3
L4
L5
L6
L7
L8
L9
L10
L11
L12
L13
L14
L15
L16
L17
L18
L19
91.90
91.90
91.90
91.90
101.14
91.90
105.04
91.90
8

L20
L21
L22
L23
L24
L25
L26
L27
L28
L29
L30
2
2
2
2
2
2
2
2
2
2
2
5
6
6
6
6
6
7
8
7
7
6
4
4
4
4
4
4
5
4
4
4
4
Yes, 1
Yes, 0
Yes, 0
Yes, 0
Yes, 0
Yes, 0
Yes, 0
Yes, 0
Yes, 0
Yes, 0
Yes, 0
91.90
104.79
104.79
104.79
104.79
104.79
114.03
104.79
117.93
104.79
104.79
434.13
385.11
403.10
419.07
463.02
399.12
415.12
430.09
375.09
391.06
435.12
5.97
3.67
3.73
4.18
4.31
3.95
3.56
2.63
2.45
3.09
4.68
MW: Molecular Weight, HBA: a number of H-bond acceptors, HBD: a number of H-bond donors, log P: the octanol-
water partition coefficient. nROTB: a number of rotatable bonds. TPSA: the polar surface area
3. Conclusion
In this work, we design and synthesize a series of novel urease inhibitor flavonoid
analogues(L1-L10,L11-L20,L21-L30) based on the combination principles. On this
basis, we performed inhibitory activity evaluation against urease, molecular docking
studies and in-silico ADME evaluation for the aimed compounds. These aimed
compounds for urease inhibitory activities were clearly superior to the control drug
thiourea (more than 10 times), and L2 and L12 exhibited optimal urease inhibitory
activity. The molecular docking studies demonstrated the binding modes of the aimed
compounds and their structure-activity relationship. The aimed compounds possess
good druggability.
4. Experimental part
4.1. Methods and materials
The chemicals and solvents were commercially available and used without
1
further purification. H NMR and 13C NMR spectra were obtained on a Bruker
500ꢀMHzꢀat 25ꢀ°C, with TMS as the internal standard in deuterium reagent. MS analysis
was performed with a Thermo Fisher Scientific LTQ ORBITRAP ELITE (USA).
4.2. General procedure for the preparation of compounds L1-L10,L11-L20 and L21-
L30, exemplified with L1
2,4-dihydroxyacetophenone (1.5g, 10mmol) and anhydrous potassium
9

carbonate(10g, 72mmol),TBAB (1.5g,10mmol ) was dissolved in 100 mL Dry acetone
at Room temperature. Then benzoyl chloride (2.3ml, 20mmol) , was added dropwise
with constant stirring, The resulting solution was stirred for 15ꢀh under reflux
condition.and completion of reaction is checked by TLC analysis. Later the solution
was cooled to RT and filtered. The filtrate was transferred into 200ꢀmL of 10% glacial
acetic acid (GAA) under vigorous stirring and obtained yellow precipitate was filtered
and dried. The product was purified by recrystallisation from acetone to give of 3a as a
yellow solid.Yellow solid 3a and 3.5g sodium acetate was dissolved in glacial acetic
acid(17ꢀmL) and refluxed at 130ꢀ°C for 5ꢀh give of 4a. then hexamethylenetetramine
(HMTA) (5.6ꢀg, 40mmol) was added and refluxed at 100ꢀ°C for 8ꢀh give of 5a. finally,
5a and semicarbazide were dissolved in anhydrous ethanol and refluxed at 83ꢀ°C
for 8ꢀh.The product was purified by recrystallisation from anhydrous ethanol to give of
L1.
4.2. Spectral data of the aimed compounds
(Z)-2-((7-hydroxy-4-oxo-2-phenyl-4H-chromen-8-yl)methylene)hydrazine-1-
carboxamide L1
Yield 63.74%; yellow solid; 1H NMR (500 MHz, DMSO-d6) δ 11.67 (s, 1H, NH), 10.70
(s, 1H, OH), 8.74 (s, 1H, CH=N), 8.18 – 8.12 (m, 2H, Ar), 7.91 (d, J = 8.8 Hz, 1H, Ar),
7.65 – 7.55 (m, 3H, Ar), 7.05 (d, J = 8.8 Hz, 1H, CH), 7.01 (s, 1H, Ar ), 6.53 (s, 2H,
NH2).13C NMR (151 MHz, DMSO-d6)δ176.68, 162.32, 161.80, 155.99, 154.91, 136.71,
132.24, 131.56, 129.58, 127.03, 126.87, 116.70, 115.74, 107.76, 107.22. HR-EI-MS:
m/z calcd for C17H13N3O4, [M]+324.09998; Found 324.09788;
7(Z)-2-((2-(4-fluorophenyl)-7-hydroxy-4-oxo-4H-chromen-8-
yl)methylene)hydrazine-1-carboxamide L2
Yield 64.96%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 10.51 (s, 1H, OH), 8.65
(s, 1H, CH=N), 8.29 – 8.21 (m, 2H, Ar), 7.82 (d, J = 8.9 Hz, 1H, Ar), 7.39 (t, J = 8.8
Hz, 2H, Ar), 6.93 (s, 1H, CH), 6.88 (d, J = 8.9 Hz, 1H, Ar), 6.42 (s, 2H, NH2).13C NMR
(151 MHz, DMSO-d6) δ 176.47, 165.25, 163.59, 160.97, 156.41, 155.51, 137.57,
129.48, 129.42, 128.43, 126.72, 116.62, 116.48, 107.79, 106.95. HR-EI-MS: m/z calcd
for C17H12FN3O4, [M]+Na364.07230; Found 364.07041;
(Z)-2-((2-(4-chlorophenyl)-7-hydroxy-4-oxo-4H-chromen-8-yl)methylene)hydrazine-
1-carboxamide L3
Yield 72.28%; yellow solid; 1H NMR (500 MHz, DMSO-d6) δ 10.53 (s, 1H, OH), 8.64
(s, 1H, CH=N), 8.21 (s, 2H, Ar), 7.83 (d, J = 8.6 Hz, 1H, Ar), 7.61 (d, J = 8.1 Hz, 2H,
Ar), 6.95 (d, J = 27.9 Hz, 2H, CH+Ar), 6.45 (s, 2H, NH2).13C NMR (151 MHz, DMSO-
d6) δ 176.62, 161.82, 161.21, 156.07, 154.83, 136.97, 136.57, 130.49, 129.57, 128.74,
127.02, 116.68, 115.76, 107.86, 107.54.HR-EI-MS: m/z calcd for C17HL26lN3O4,
[M]+Na380.04285; Found 380.04085;
(Z)-2-((2-(4-bromophenyl)-7-hydroxy-4-oxo-4H-chromen-8-yl)methylene)hydrazine-
10

1-carboxamide L4
Yield 63.50%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 10.70 – 10.51 (m, 1H,
OH), 8.65 (s, 1H, CH=N), 8.12 (d, J = 8.2 Hz, 2H, Ar), 7.85 (d, J = 8.8 Hz, 1H, Ar),
7.75 (d, J = 8.2 Hz, 2H, Ar), 7.03 – 6.93 (m, 2H, CH+Ar), 6.48 (s, 2H, NH2).13C NMR
(151 MHz, DMSO-d6) δ 176.54, 161.12, 155.12, 136.99, 132.48, 131.01, 128.88,
126.89, 125.76, 116.47, 107.90, 107.47. HR-EI-MS: m/z calcd for C17HL16rN3O4, [M]+
402.01083; Found 402.00839;
(Z)-2-((7-hydroxy-4-oxo-2-(p-tolyl)-4H-chromen-8-yl)methylene)hydrazine-1-
carboxamide L5
Yield 72.29%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 10.64 (s, 1H, OH), 8.71
(s, 1H, CH=N), 8.05 (d, J = 8.0 Hz, 2H, Ar), 7.88 (d, J = 8.8 Hz, 1H, Ar), 7.39 (d, J =
7.9 Hz, 2H, Ar), 6.99 (d, J = 8.8 Hz, 1H, CH, Ar), 6.93 (s, 1H, Ar), 6.49 (s, 2H, NH2),
2.42 (s, 3H, CH3).13C NMR (151 MHz, DMSO-d6) δ 176.55, 142.28, 130.16, 128.95,
126.88, 126.78, 107.75, 106.50, 21.55.HR-EI-MS: m/z calcd for C18H15N3O4, [M]+Na
360.09714; Found 360. 09548;
(Z)-2-((7-hydroxy-2-(4-methoxyphenyl)-4-oxo-4H-chromen-8-
yl)methylene)hydrazine-1-carboxamide L6
Yield 58.70%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 10.64 (s, 1H, OH), 8.71
(s, 1H, CH=N), 8.12 (d, J = 8.5 Hz, 2H, Ar), 7.88 (d, J = 8.8 Hz, 1H, Ar), 7.11 (d, J =
8.5 Hz, 2H, Ar), 7.00 (d, J = 8.9 Hz, 1H, CH), 6.90 (s, 1H, Ar), 6.51 (s, 2H, NH2), 3.88
(s, 3H, CH3).13C NMR (151 MHz, DMSO-d6) δ 176.53, 162.49, 136.95, 128.74, 126.96,
123.82, 114.96, 107.74, 105.68, 55.98. HR-EI-MS: m/z calcd for C18H15N3O5, [M]+
354.11069; Found 354.10845;
(Z)-2-((7-hydroxy-2-(4-nitrophenyl)-4-oxo-4H-chromen-8-yl)methylene)hydrazine-1-
carboxamide
L7
Yield 65.58%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 10.52 (s, 1H, OH), 8.62
(s, 1H, CH=N), 8.47 (d, J = 8.4 Hz, 2H, Ar), 8.33 (d, J = 8.5 Hz, 2H, Ar), 7.86 (d, J =
8.9 Hz, 1H, Ar), 7.16 (s, 1H, CH), 6.96 (d, J = 8.9 Hz, 1H, Ar), 6.46 (s, 2H, NH2).13C
NMR (151 MHz, DMSO-d6) δ 176.44, 159.71, 149.33, 137.83, 136.95, 128.33, 126.88,
124.36, 109.43, 108.07. HR-EI-MS: m/z calcd for C17H12N4O6, [M]+Na 391.06677;
Found 391.06491;
(Z)-2-((2-(furan-2-yl)-7-hydroxy-4-oxo-4H-chromen-8-yl)methylene)hydrazine-1-
carboxamide
L8
Yield 65.43%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 11.42 (s, 1H, NH), 10.55
(s, 1H, OH), 8.66 (s, 1H, CH=N), 8.08 – 8.02 (m, 1H, CH), 7.88 (d, J = 8.8 Hz, 1H,
Ar), 7.52 (d, J = 3.5 Hz, 1H, CH), 7.03 (d, J = 8.8 Hz, 1H, CH), 6.87 (dd, J = 3.6, 1.7
Hz, 1H, CH), 6.59 (d, J = 13.5 Hz, 3H, NH2+Ar).13C NMR (151 MHz, DMSO-d6) δ
175.87, 161.60, 154.46, 154.39, 147.61, 145.82, 136.41, 127.04, 116.86, 115.61,
114.43, 113.45, 107.60, 104.67. HR-EI-MS: m/z calcd for C15H11N3O5,
[M]+314.07941;Found 314.07715;
(Z)-2-((7-hydroxy-4-oxo-2-(thiophen-2-yl)-4H-chromen-8-yl)methylene)hydrazine-1-
carboxamide L9
Yield 62.34%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 11.75 (s, 1H, NH), 10.74
(s, 1H, OH), 8.64 (s, 1H, CH=N), 8.06 (dd, J = 3.7, 1.2 Hz, 1H, CH), 8.00 (dd, J = 4.9,
11

1.2 Hz, 1H, CH), 7.88 (d, J = 8.8 Hz, 1H, Ar), 7.32 (dd, J = 5.0, 3.8 Hz, 1H, CH), 7.03
(d, J = 8.8 Hz, 1H, CH), 6.79 (s, 1H, Ar), 6.51 (s, 2H, NH2). 13C NMR (151 MHz,
DMSO-d6) δ 176.09, 161.79, 158.27, 154.55, 136.41, 134.61, 132.04, 129.97, 129.44,
126.99, 116.59, 115.69, 107.50, 105.66. HR-EI-MS: m/z calcd for C15H11N3O4S,
[M]+330.05646; Found 330.05430;
(Z)-2-((7-hydroxy-2-(naphthalen-2-yl)-4-oxo-4H-chromen-8-yl)methylene)hydrazine-
1-carboxamide L10
Yield 56.59%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 11.71 (s, 1H, NH), 10.73
(s, 1H, OH), 8.84 – 8.72 (m, 2H, CH=N+Ar), 8.23 – 8.16 (m, 2H, Ar), 8.10 (d, J = 8.7
Hz, 1H, Ar), 8.06 – 8.02 (m, 1H, Ar), 7.95 (d, J = 8.8 Hz, 1H, Ar), 7.70 – 7.63 (m, 2H,
Ar), 7.17 (s, 1H, CH), 7.08 (d, J = 8.8 Hz, 1H, Ar), 6.58 (s, 2H, NH2).13C NMR (151
MHz, DMSO-d6) δ 176.70, 162.30, 161.86, 155.01, 134.67, 133.03, 129.61, 129.26,
128.90, 128.62, 128.22, 127.53, 127.24, 127.07, 123.30, 116.81, 115.73, 107.96,
107.63. HR-EI-MS: m/z calcd for C15H11N3O4S, [M]+374.11581;Found 374.11353;
(Z)-N'-((7-hydroxy-4-oxo-2-phenyl-4H-chromen-8-yl)methylene)benzohydrazide
L11
Yield 66.50%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 13.10 (s, 1H, NH), 12.51
(s, 1H, OH), 9.27 (s, 1H, CH=N), 8.19 (dd, J = 6.7, 2.9 Hz, 2H, Ar), 8.03 – 7.97 (m,
3H, Ar), 7.71 – 7.58 (m, 6H, Ar), 7.11 (d, J = 8.8 Hz, 1H, CH), 7.04 (s, 1H, Ar). 13C
NMR (151 MHz, DMSO-d6) δ 176.56, 163.46, 163.33, 162.40, 155.37, 143.70, 132.87,
132.83, 132.29, 131.59, 129.69, 129.19, 128.24, 126.89, 116.58, 116.10, 107.51,
107.04.HR-EI-MS: m/z calcd for C23H16N2O4, [M]+385.12039; Found 385.11828; (Z)-
N'-((2-(4-fluorophenyl)-7-hydroxy-4-oxo-4H-chromen-8-
yl)methylene)benzohydrazide L12
Yield 58.77%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 9.02 (s, 1H, OH), 8.27
(s, 2H, CH=N+Ar), 8.01 (d, J = 7.5 Hz, 2H, Ar), 7.70 – 7.50 (m, 3H, Ar), 7.41 (t, J =
8.6 Hz, 2H, Ar), 6.75 (s, 1H, CH), 6.46 (s, 1H, Ar).13C NMR (151 MHz, DMSO-d6) δ
176.42, 165.39, 163.73, 163.46, 161.39, 155.25, 143.57, 132.96, 132.75, 129.62,
129.57, 129.14, 128.23, 128.12, 116.75, 116.61, 116.35, 116.09, 107.31, 107.06.HR-
EI-MS: m/z calcd for C23H15FN2O4, [M]+403.11133; Found 403.10886;
(Z)-N'-((2-(4-chlorophenyl)-7-hydroxy-4-oxo-4H-chromen-8-
yl)methylene)benzohydrazide L13
Yield 57.43%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 12.93 (s, 1H, NH), 12.42
(s, 1H, OH), 9.22 (s, 1H, CH=N), 8.28 (d, J = 8.5 Hz, 2H, Ar), 7.99 (t, J = 8.2 Hz, 3H,
Ar), 7.67 (dd, J = 8.2, 3.1 Hz, 3H, Ar), 7.61 (t, J = 7.5 Hz, 2H, Ar), 7.11 (d, J = 8.9 Hz,
1H, CH), 7.08 (s, 1H, Ar) 13C NMR (151 MHz, DMSO-d6) δ 176.44, 163.43, 163.29,
161.22, 155.17, 143.48, 137.00, 132.95, 132.76, 130.45, 129.63, 129.14, 128.77,
128.23, 128.14, 116.56, 115.99, 107.71, 107.16.HR-EI-MS: m/z calcd for C23HL29lN2O4,
[M]+419.08194; Found 419.07931;
(Z)-N'-((2-(4-bromophenyl)-7-hydroxy-4-oxo-4H-chromen-8-
yl)methylene)benzohydrazide L14
Yield 62.58%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 12.65 (d, J = 219.8 Hz,
2H, NH+OH), 9.20 (s, 1H, CH=N), 8.19 (d, J = 8.2 Hz, 2H, Ar), 7.98 (dd, J = 17.6, 8.2
Hz, 3H, Ar), 7.79 (d, J = 8.3 Hz, 2H, Ar), 7.67 (t, J = 7.4 Hz, 1H, Ar), 7.60 (t, J = 7.5
12

Hz, 2H, Ar), 7.11 – 7.05 (m, 2H, CH+Ar).13C NMR (151 MHz, DMSO-d6) δ 176.47,
163.47, 163.35, 161.42, 155.24, 143.58, 132.99, 132.76, 132.59, 130.88, 129.16,
129.01, 128.24, 128.19, 125.95, 116.60, 116.03, 107.77, 107.23.HR-EI-MS: m/z calcd
for C23HL19rN2O4, [M]+463.03174; Found 463.02880;
(Z)-N'-((7-hydroxy-4-oxo-2-(p-tolyl)-4H-chromen-8-yl)methylene)benzohydrazide
L15
Yield 66.13%; yellow solid;1HNMR (500 MHz, DMSO-d6) δ 13.06 (s, 1H, NH), 12.48
(s, 1H, OH), 9.23 (s, 1H, CH=N), 8.06 (d, J = 8.0 Hz, 2H, Ar), 8.01 – 7.95 (m, 3H, Ar),
7.70 – 7.66 (m, 1H, Ar), 7.61 (dd, J = 8.3, 6.7 Hz, 2H, Ar), 7.41 (d, J = 8.2 Hz, 2H, Ar),
7.08 (d, J = 8.9 Hz, 1H, CH), 6.97 (s, 1H, Ar), 2.42 (s, 3H, CH3).13C NMR (151 MHz,
DMSO-d6) δ 176.50, 163.45, 163.25, 143.73, 142.53, 132.82, 130.27, 129.20, 128.24,
126.85, 115.99, 106.86, 21.57.HR-EI-MS: m/z calcd for C24H18N2O4, [M]+399.13467;
Found 399.13393;
(Z)-N'-((7-hydroxy-2-(4-methoxyphenyl)-4-oxo-4H-chromen-8-
yl)methylene)benzohydrazide L16
Yield 51.47%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 12.73 (d, J = 264.6 Hz,
2H, NH+OH), 9.22 (s, 1H, CH=N), 8.12 (d, J = 8.4 Hz, 2H, Ar), 8.00 (d, J = 7.6 Hz,
2H, Ar), 7.94 (d, J = 8.9 Hz, 1H, Ar), 7.67 (t, J = 7.4 Hz, 1H, Ar), 7.61 (t, J = 7.5 Hz,
2H, Ar), 7.12 (d, J = 8.4 Hz, 2H, Ar), 7.06 (d, J = 8.8 Hz, 1H, CH), 6.90 (s, 1H, Ar),
3.87 (s, 3H, CH3).13C NMR (151 MHz, DMSO-d6) δ 176.35, 163.39, 163.15, 162.52,
162.36, 155.19, 143.66, 132.91, 132.78, 129.16, 128.67, 128.24, 128.14, 123.71,
116.48, 115.82, 115.02, 106.94, 105.92, 55.97.HR-EI-MS: m/z calcd for C24H18N2O5,
[M]+415.13144; Found 415.12885;
(Z)-N'-((7-hydroxy-2-(4-nitrophenyl)-4-oxo-4H-chromen-8-
yl)methylene)benzohydrazide L17
Yield 62.77%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 12.74 (s, 1H, NH), 12.34
(s, 1H, OH), 9.20 (s, 1H, CH=N), 8.65 – 8.60 (m, 2H, Ar), 8.40 – 8.38 (m, 2H, Ar),
8.03 – 7.99 (m, 3H, Ar), 7.69 – 7.66 (m, 1H, Ar), 7.61 (dd, J = 8.2, 6.7 Hz, 2H, Ar),
7.26 (s, 1H, CH), 7.13 (d, J = 8.8 Hz, 1H, Ar).13C NMR (151 MHz, DMSO-d6) δ 176.46,
163.53, 163.43, 160.12, 155.22, 149.42, 143.38, 137.57, 133.09, 132.70, 129.13,
128.59, 128.24, 124.43, 116.76, 116.09, 109.63, 107.55.HR-EI-MS: m/z calcd for
C23H15N3O6, [M]+430.10583; Found 430.10336;
(Z)-N'-((2-(furan-2-yl)-7-hydroxy-4-oxo-4H-chromen-8-
yl)methylene)benzohydrazide L18
Yield 67.07%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 12.89 (s, 1H, NH), 12.37
(s, 1H, OH), 9.13 (s, 1H, CH=N), 8.06 (d, J = 1.7 Hz, 1H, CH), 8.00 – 7.96 (m, 2H,
Ar), 7.91 (d, J = 8.8 Hz, 1H, Ar), 7.67 (t, J = 7.2 Hz, 1H, Ar), 7.60 (dd, J = 8.3, 6.7 Hz,
2H, Ar), 7.49 (d, J = 3.5 Hz, 1H, CH), 7.04 (d, J = 8.8 Hz, 1H, CH), 6.87 (dd, J = 3.5,
1.7 Hz, 1H, CH), 6.56 (s, 1H, Ar).13C NMR (151 MHz, DMSO-d6) δ 175.66, 163.36,
163.14, 154.76, 154.22, 147.72, 145.80, 143.45, 132.89, 132.79, 129.18, 128.18,
128.17, 116.61, 115.87 , 114.40, 113.39, 106.70, 104.74.HR-EI-MS: m/z calcd for
C21H14N2O5, [M]+Na397.08139; Found 397.07949;
(Z)-N'-((7-hydroxy-4-oxo-2-(thiophen-2-yl)-4H-chromen-8-
yl)methylene)benzohydrazide L19
13

Yield 62.07%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 13.16 (s, 1H, NH), 12.57
(s, 1H, OH), 9.16 (s, 1H, CH=N), 8.07 (dd, J = 3.7, 1.2 Hz, 1H, Ar), 8.02 (dd, J = 4.9,
1.2 Hz, 1H, CH), 8.00 – 7.98 (m, 2H, Ar), 7.94 (d, J = 8.8 Hz, 1H, CH), 7.70 – 7.66 (m,
1H, CH), 7.61 (dd, J = 8.2, 6.7 Hz, 2H, Ar), 7.34 (dd, J = 5.0, 3.7 Hz, 1H, Ar), 7.07 (d,
J = 8.9 Hz, 1H, CH), 6.82 (s, 1H, Ar).13C NMR (151 MHz, DMSO-d6) δ 175.91, 163.53,
163.28, 158.19, 154.95, 143.32, 134.56, 132.85, 132.82, 132.04, 129.96, 129.51,
129.17, 128.26, 128.17, 116.43, 116.03, 106.66, 105.84. HR-EI-MS: m/z calcd for
C21H14N2O4S, [M]+391.07663; Found 391.107470;
(Z)-N'-((7-hydroxy-2-(naphthalen-2-yl)-4-oxo-4H-chromen-8-
yl)methylene)benzohydrazide L20
Yield 56.79%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 13.08 (s, 1H, NH), 12.54
(s, 1H, OH), 9.37 (s, 1H, CH=N), 8.82 (s, 1H, Ar), 8.32 – 7.96 (m, 7H, Ar), 7.67 (dt, J
= 21.9, 7.3 Hz, 5H, Ar), 7.25 – 7.10 (m, 2H, CH+Ar).13C NMR (151 MHz, DMSO-d6)
δ 176.47, 163.40, 163.32, 162.30, 155.38, 143.75, 134.68, 133.01, 132.90, 132.81,
129.61, 129.31, 129.20, 128.89, 128.60, 128.26, 128.22, 128.20, 127.53, 127.29,
123.23, 116.65, 116.02, 107.87, 107.12.HR-EI-MS: m/z calcd for C27H18N2O4,
[M]+435.13483; Found 435.13393;
(Z)-N'-((7-hydroxy-4-oxo-2-phenyl-4H-chromen-8-
yl)methylene)isonicotinohydrazide L21
Yield 59.42%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 13.03 – 12.56 (m, 2H,
NH+OH), 9.24 (s, 1H, CH=N), 8.86 (s, 2H, pyridine), 8.26 – 8.18 (m, 2H, pyridine),
8.00 (d, J = 8.7 Hz, 1H, Ar), 7.90 (d, J = 4.9 Hz, 2H, Ar), 7.63 (d, J = 5.4 Hz, 3H, Ar),
7.10 (d, J = 8.7 Hz, 1H, CH), 7.04 (s, 1H, Ar).13C NMR (151 MHz, DMSO-d6) δ 176.50,
163.37, 162.44, 162.01, 155.41, 151.00, 144.79, 140.07, 132.27, 131.55, 129.67,
128.56, 126.96, 122.06, 116.66, 116.01, 107.49, 106.99.HR-EI-MS: m/z calcd for
C22H15N3O4, [M]+386.11597; Found 386.11353;
(Z)-N'-((2-(4-fluorophenyl)-7-hydroxy-4-oxo-4H-chromen-8-
yl)methylene)isonicotinohydrazide L22
Yield 52.15%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 9.06 (s, 1H, CH=N),
8.77 (s, 2H, pyridine), 8.22 (s, 2H, pyridine), 7.93 (d, J = 5.0 Hz, 2H, Ar), 7.73 (d, J =
9.2 Hz, 1H, Ar), 7.43 (t, J = 8.7 Hz, 2H, Ar), 6.81 (s, 1H, CH), 6.57 (d, J = 9.1 Hz, 1H,
Ar).13C NMR (151 MHz, DMSO-d6) δ 176.40, 163.33, 161.97, 161.32, 155.21, 150.94,
144.55, 140.16, 136.98, 130.40, 129.60, 128.93, 128.48, 122.06, 116.69, 115.85,
107.65, 107.23. HR-EI-MS: m/z calcd for C22H14FN3O4, [M]+404.10638; Found
404.10411;
(Z)-N'-((2-(4-chlorophenyl)-7-hydroxy-4-oxo-4H-chromen-8-
yl)methylene)isonicotinohydrazide L23
Yield 56.73%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 12.59 (d, J = 52.2 Hz,
2H, NH+OH), 9.14 (s, 1H, CH=N), 8.85 (d, J = 5.0 Hz, 2H, pyridine), 8.29 (d, J = 8.3
Hz, 2H, pyridine), 7.96 (d, J = 8.8 Hz, 1H, Ar), 7.92 – 7.88 (m, 2H, Ar), 7.63 (d, J =
8.3 Hz, 2H, Ar), 7.08 – 7.03 (m, 2H, CH+Ar).13C NMR (151 MHz, DMSO-d6) δ 176.33,
165.37, 163.71, 150.93, 129.69, 128.39, 128.17, 122.06, 116.72, 116.57, 107.29. HR-
EI-MS: m/z calcd for C22HL28lN3O4, [M]+420.07709; Found 420.07456;
(Z)-N'-((2-(4-bromophenyl)-7-hydroxy-4-oxo-4H-chromen-8-
14

yl)methylene)isonicotinohydrazide L24
Yield 47.58%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 12.66 – 12.49 (m, 2H,
NH+OH), 9.13 (d, J = 2.2 Hz, 1H, CH=N), 8.86 (s, 2H, pyridine), 8.27 – 8.18 (m, 2H,
pyridine), 7.96 (dd, J = 8.8, 2.1 Hz, 1H, Ar), 7.91 (d, J = 4.3 Hz, 2H, Ar), 7.77 (dd, J =
8.5, 2.1 Hz,2H, , Ar), 7.09 – 7.02 (m, 2H, CH+Ar).13C NMR (126 MHz, DMSO-d6) δ
176.42, 163.33, 162.00, 161.46, 155.22, 150.95, 144.57, 140.19, 132.55, 130.79,
129.12, 128.50, 125.94, 116.73, 115.85, 107.66, 107.27.HR-EI-MS: m/z calcd for
C22HL18rN3O4, [M]+464.02502; Found 464.02405;
(Z)-N'-((7-hydroxy-4-oxo-2-(p-tolyl)-4H-chromen-8-
yl)methylene)isonicotinohydrazide L25
Yield 63.86%; yellow solid; 1H NMR (500 MHz, DMSO-d6) δ 12.73 (d, J = 76.9 Hz,
1H, NH+OH), 9.20 (s, 1H, CH=N), 8.92 – 8.82 (m, 2H, pyridine), 8.08 (d, J = 8.1 Hz,
2H, pyridine), 7.97 (d, J = 8.8 Hz,2H, Ar), 7.91 – 7.89 (m, 1H, Ar), 7.40 (d, J = 8.0 Hz,
1H, Ar), 7.08 (d, J = 8.8 Hz, 1H, CH), 6.96 (s, 1H, Ar), 2.41 (s, 3H, CH3). 13C NMR
(151 MHz, DMSO-d6) δ 176.45, 163.29, 162.59, 161.99, 155.33, 150.99, 144.78,
142.48, 140.09, 130.23, 130.17, 128.72, 128.52, 126.87, 122.07, 116.65, 115.89,
106.96, 106.78, 21.56. HR-EI-MS: m/z calcd for C23H17N3O4, [M]+400.13181; Found
400.12918;
(Z)-N'-((7-hydroxy-2-(4-methoxyphenyl)-4-oxo-4H-chromen-8-
yl)methylene)isonicotinohydrazide L26
Yield 55.63%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 12.70 (d, J = 73.5 Hz,
2H, NH+OH), 9.19 (s, 1H, CH=N), 8.91 – 8.82 (m, 2H, pyridine), 8.14 (d, J = 8.4 Hz,
2H, pyridine), 7.95 (d, J = 8.8 Hz, 1H, Ar), 7.89 (d, J = 5.0 Hz, 2H, Ar), 7.11 (d, J =
8.4 Hz, 2H, Ar), 7.06 (d, J = 8.8 Hz, 1H, CH), 6.91 (s, 1H, Ar), 3.86 (s, 3H, CH3).13C
NMR (151 MHz, DMSO-d6) δ 176.33, 163.20, 162.53, 162.46, 161.96, 155.26, 150.98,
144.80, 140.08, 128.78, 128.50, 123.67, 122.04, 116.58, 115.75, 115.01, 106.91,
105.90, 55.97. HR-EI-MS: m/z calcd for C23H17N3O5, [M]+416.12659; Found
416.12410;
(Z)-N'-((7-hydroxy-2-(4-nitrophenyl)-4-oxo-4H-chromen-8-
yl)methylene)isonicotinohydrazide L27
Yield 57.13%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 12.49 (s, 2H, NH+OH),
9.14 (s, 1H, CH=N), 8.85 (s, 2H, pyridine), 8.68 (d, J = 8.3 Hz, 2H, pyridine), 8.37 (d,
J = 8.4 Hz, 2H, Ar), 8.00 (d, J = 8.8 Hz, 1H, Ar), 7.92 (d, J = 4.9 Hz, 2H, Ar), 7.25 (s,
1H, CH), 7.11 (d, J = 8.8 Hz, 1H, Ar).13C NMR (151 MHz, DMSO-d6) δ 149.14, 144.18,
136.13, 129.67, 128.97, 124.28, 107.48 (d, J = 73.0 Hz).HR-EI-MS: m/z calcd for
C22H14N4O6, [M]+431.10129; Found 431.09861;
(Z)-N'-((2-(furan-2-yl)-7-hydroxy-4-oxo-4H-chromen-8-
yl)methylene)isonicotinohydrazide L28
Yield 66.62%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 12.72 (s, 1H, NH), 12.61
(s, 1H, OH), 9.21 (s, 1H, CH=N), 8.90 – 8.82 (m, 2H, pyridine), 8.09 (d, J = 1.7 Hz,
1H, pyridine), 7.99 (d, J = 8.8 Hz, 1H, pyridine), 7.93 – 7.89 (m, 2H, CH), 7.63 (d, J =
3.5 Hz, 1H, Ar), 7.11 (d, J = 8.9 Hz, 1H, CH), 6.90 (dd, J = 3.5, 1.8 Hz, 1H, CH), 6.63
(s, 1H, Ar).13C NMR (151 MHz, DMSO-d6) δ 175.71, 163.22, 162.03, 154.94, 154.41,
151.02, 147.78, 145.82, 144.76, 140.10, 128.62, 116.79, 115.88, 114.67, 113.48,
15

106.77, 104.83. HR-EI-MS: m/z calcd for C20H13N3O5, [M]+376.09503; Found
376.09280;
(Z)-N'-((7-hydroxy-4-oxo-2-(thiophen-2-yl)-4H-chromen-8-
yl)methylene)isonicotinohydrazide L29
Yield 60.24%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 13.06 – 12.68 (m, 2H,
NH+OH), 9.19 – 9.12 (m, 1H, CH=N), 8.85 (d, J = 4.9 Hz, 2H, pyridine), 8.10 – 7.87
(m, 5H, pyridine+CH), 7.34 (d, J = 4.4 Hz, 1H, Ar), 7.07 (d, J = 11.0 Hz, 1H, CH), 6.82
(t, J = 4.4 Hz, 1H, Ar) 13C NMR (151 MHz, DMSO-d6) δ 175.89, 163.33, 162.13,
158.25, 155.07, 151.00, 144.54, 140.03, 134.52, 132.08, 130.11, 129.54, 128.55,
122.08, 116.53, 116.02, 106.58, 105.88.HR-EI-MS: m/z calcd for C20H13N3O4S,
[M]+392.07230; Found 392.06995;
(Z)-N'-((7-hydroxy-2-(naphthalen-2-yl)-4-oxo-4H-chromen-8-
yl)methylene)isonicotinohydrazide L30
Yield 56.37%; yellow solid;1H NMR (500 MHz, DMSO-d6) δ 12.77 (d, J = 43.9 Hz,
2H, NH+OH), 9.32 (s, 1H, CH=N), 8.85 (d, J = 32.8 Hz, 3H, Ar), 8.28 – 8.19 (m, 2H,
Ar), 8.12 (d, J = 8.7 Hz, 1H, Ar), 8.02 (t, J = 7.7 Hz, 2H, Ar), 7.94 (d, J = 4.8 Hz, 2H,
Ar), 7.70 – 7.63 (m, 2H, Ar), 7.19 (s, 1H, CH), 7.12 (d, J = 8.8 Hz, 1H, Ar). 13C NMR
(151 MHz, DMSO-d6) δ 176.51, 163.38, 162.50, 162.02, 155.50, 151.04, 145.01,
134.71, 133.04, 129.64, 129.35, 128.88, 128.64, 128.24, 127.56, 127.38, 123.36,
122.06, 116.78, 115.99, 107.90, 107.15.HR-EI-MS: m/z calcd for C26H17N3O4,
[M]+436.13184; Found 436.12918;
4.3.Urease inhibitory activity
The reaction mixtures, comprising 25ꢀμL of enzyme (jack bean urease, 5ꢀU/mL)
solution and 55ꢀμL of buffers containing 100ꢀmM urea, were incubated with 5ꢀμL of the
test compounds (0.5ꢀmM concentration) at 30ꢀ°C for 15ꢀmin in 96-well
plates[34].Urease activity was determined by measuring ammonia production using the
indophenol method as described by Weatherburn[35]. Briefly, 45ꢀμL of phenol reagent
(1% w/v phenol and 0.005% w/v sodium nitroprussside) and, 70ꢀμL of alkali reagent
(0.5% w/v NaOH and 0.1% active chloride NaOCl) were added to each well. The
increasing absorbance at 630ꢀnm was measured after 50ꢀmin, using a microplate reader
(M200 PQO NanoQuant). All reactions were performed in triplicate in a final volume
of 200ꢀμL. The entire assays were performed at pH 6.8.Thiourea was used as the
standard inhibitor for urease.
4.4. Molecular docking study
Docking study was performed based on the crystal structure of urease in order to
reveal the binding modes of synthesized Flavonoid analogues. For the purpose of
docking studies, the crystal structure of the urease was optimized using protein
16

preparation module in Discovery Studio 2018 (Dassault Systemes BIOVIA, USA)[36].
The three-dimensional (3D) protein structure of Bacillus Pasteurii Urease (PDB ID:
4UBP, resolution 1.55 Å), determined by X-ray crystallography, was retrieved from the
RCSB Protein Databank (http://www.rcsb.org/pdb/home/home.do.). All the water
molecules, hetero atoms, and co-factors were removed from the protein structure. Then
hydrogen bonds, missing atoms, and charges were computed, and the energy
optimization was carried out using default force field. The synthesized flavonoid
analogues used in these docking studies was prepared and optimized using built and
Ligand Preparation module implemented in Discovery Studio 2018 (Dassault Systemes
BIOVIA, USA). The active site was defined with a radius of 26 Å around nickel atoms
in the urease enzyme. Lastly, the conformations with the lowest predicted binding free
energy were selected for the each test compound. Graphic manipulations were
visualized using Accelrys discovery studio visualizer software.
Notes. The authors declare no competing financial interest.
Acknowledgement: This work was supported by the National Natural Science
Foundation of China (No. 21967003), the National Natural Science Foundation of
China (No. 81960649), Guangxi Natural Science Foundation, China
(2018GXNSFAA281030).
Appendix A. Supplementary data.
Supplementary data to this article can be found online at.
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Graphical abstract
OH
OH
HO
O
O
HIS323
CYS322
O
OH
OH
HN N
HO
F
Docking
ALA366
IC50=11.2±0.9M
O
O
ARG339
GLY280
OH
ASP224
ALA170
O
TRP225
N
N
OH
IC50=1.207±0.186M
H
H3CO
HIS222
OCH3
IC50=8.4±0.05M
22

Highlights
1．A series of flavonoid analogues were designed and synthesized.
2．The aimed compounds for urease inhibitory activities were superior to thiourea.
3．Compound L2 and L12 show the most potent inhibitory activities
4．The action mode and druggability were explored.
23