Design and syntheses of novel N′-((4-oxo-4H-chromen-3-yl)methylene) benzohydrazide as inhibitors of cyanobacterial fructose-1,6-/sedoheptulose-1,7- bisphosphatase

Article abstract of DOI:10.1016/j.bmc.2013.04.003

Cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphoshatase (Cy-FBP/SBPase) is an important target enzyme for finding inhibitors to solve harmful algal bloom (HAB). In this study, as potential inhibitors of Cy-FBP/SBPase, a series of novel chromone-connecting benzohydrazone compounds (Novel N′-((4-oxo-4H-chromen-3-yl)methylene)benzohydrazide) were designed and synthesized. Their inhibitory activities against Cy-FBP/SBPase were further examined in vitro. Some of these compounds, such as f6-f8, f11, f12 and f16, exhibit higher inhibitory activities (IC₅₀ = 11.2-16.1 μM), especially, the compound f7 was identified as the most potent inhibitor with IC₅₀ value of 11.2 μM. The probable binding-mode of compound f7 was further analyzed carefully by molecular docking methods. These results indicate that compound f7 could be used as a lead compound for further optimization and might have potential to be developed as a new algicide.

Full text of DOI:10.1016/j.bmc.2013.04.003

Bioorganic & Medicinal Chemistry 21 (2013) 2826–2831
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and syntheses of novel N0-((4-oxo-4H-chromen-3-yl)
methylene)benzohydrazide as inhibitors of cyanobacterial
fructose-1,6-/sedoheptulose-1,7-bisphosphatase
Qi-Dong Tu a,b,c, , Ding Li a,b, , Yao Sun a,b, , Xin-Ya Han a,b, Fan Yi a,b, Yibamu Sha a,b, Yan-Liang Ren a,b
,
Ming-Wu Ding a,b, Ling-Ling Feng a,b, , Jian Wan a,b,
⇑
⇑
a Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education, Wuhan 430079, China
b Department of Chemistry, Central China Normal University, Wuhan 430079, China
c School of Pharmacy, Jiangxi Science and Technology Normal University, Nanchang, Jiangxi 330013, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphoshatase (Cy-FBP/SBPase) is an important target
enzyme for finding inhibitors to solve harmful algal bloom (HAB). In this study, as potential inhibitors
Received 1 March 2013
Revised 4 April 2013
Accepted 5 April 2013
Available online 15 April 2013
of Cy-FBP/SBPase,
a series of novel chromone-connecting benzohydrazone compounds (Novel
N0-((4-oxo-4H-chromen-3-yl)methylene)benzohydrazide) were designed and synthesized. Their inhibi-
tory activities against Cy-FBP/SBPase were further examined in vitro. Some of these compounds, such
as f6–f8, f11, f12 and f16, exhibit higher inhibitory activities (IC50 = 11.2–16.1
lM), especially, the
Keywords:
Cyanobacteria harmful algal blooms
Benzohydrazone
Chromone
Inhibitory activity
compound f7 was identified as the most potent inhibitor with IC50 value of 11.2 M. The probable bind-
l
ing-mode of compound f7 was further analyzed carefully by molecular docking methods. These results
indicate that compound f7 could be used as a lead compound for further optimization and might have
potential to be developed as a new algicide.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
strated that the disruption of FBP/SBPase gene is a lethal mutation
in cyanobacteria Synechocystis PCC 6803 and Synechococcus PCC
Cyanobacteria harmful algal blooms (HABs) pose an increas-
ingly serious water pollution problem all over the world.1–3 It is
of general interest to find an efficient way to reduce its impacts
on human health, living resources, and water economies.
Approaches to control these HABs fall into three categories:
mechanical, physical/chemical, and biological controls.1–3
Recently, chemical control was regarded as one of the most effec-
tive method. Chemical control involves the use of compounds, such
as copper compounds, chemical oxidants and herbicides.3
However, none of these compounds are designed or developed
for specific target of cyanobacteria, thus a lot of them apparently
hazardous to human and animals.3
7942. Overexpression of Cy-FBP/SBPase in the chloroplasts of
transgenic tobacco significantly leads to increase photosynthetic
capacity in source leaves, carbonhydrate accumulation and acceler-
ated growth rate.9 These facts indicate that FBP/SBPase is actually
an optimized candidate target searching for specific algicides.
Chromone derivatives are an important class of compounds.
Most of chromone compounds possess a broad range of biological
activities, such as antimycobacterial, antifungal, anticancer, anti-
microbial, human-immunodeficiency virus (HIV-1) and mushroom
tyrosinase inhibition.10–15 These derivatives are also important
intermediates in the manufacture of agrochemicals, pharmaceuti-
cal. On the other hand, benzohydrazone derivatives have also
received much attention especially for their structures and biolog-
ical aspects, such as antiinflammatory activity,10 antimicrobial
activity,16 antioxidant activity17 and antimutagenicity activity.18
However, to our knowledge, no chromone and benzohydrazone
derivatives were used to solve the problem of HABs so far. Our pre-
liminary study found that some chromone-connecting benzohyd-
razone derivatives exhibited somewhat inhibitory activities
against cyanobacteria FBP/SBPase (Cy-FBP/SBPase). These results
encourage us to further design and synthesize chromone–ben-
zohydrazone derivatives as potential inhibitors of FBP/SBPase.
In cyanobacteria, fructose-1,6-/sedoheptulose-1,7-bisphospha-
tase (FBP/SBPase), an unique and important enzyme, can hydrolyze
both fructose-1,6-bisphosphate (FBP) and hydrolyze sedoheptu-
lose-1,7-bisphosphate (SBP).4,5 Previous studies6–8 have demon-
⇑
Corresponding authors. Tel./fax: +86 27 67862022.
E-mail addresses: fll708@mail.ccnu.edu.cn (L.-L. Feng), Jianwan@mail.ccnu.
edu.cn (J. Wan).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.04.003

Q.-D. Tu et al. / Bioorg. Med. Chem. 21 (2013) 2826–2831
2827
Table 1
In this study, we, therefore, synthesized a series of chromone-
Cy-FBP/SBPase inhibitory activities of title compounds
connecting benzohydrazone derivatives, some of them were in-
deed exhibit higher inhibitory activities against cyanobacteria
FBP/SBPase (Cy-FBP/SBPase). Especially, compound f7 exhibits
the highest inhibitory activities against Cy-FBP/SBPase. The rela-
tionship between structure of title compounds and their inhibitory
activities were analyzed as well. The rational binding mode of com-
pound f7 to Cy-FBP/SBPase was further examined by using molec-
ular docking.
R1
R2
R3
O
O
R4
N
N
H
O
Compounds
R1
R2
R3
R4
IC50 (lM)
f1
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CF3
F
P400
>200
>200
f2
f3
f4
f5
f6
f7
f8
f9
f10
f11
f12
f13
f14
f15
f16
f17
f18
f19
f20
f21
f22
f23
f24
H
H
H
H
Cl
Br
F
Me
Et
i-Pr
t-Bu
NO2
OH
Br
Br
Br
Br
Br
NO2
OH
Me
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
H
H
H
H
H
H
H
H
H
2. Results and discussion
2.1. Chemistry
137.1 6.4
22.1 5.5
12.1 2.0
11.2 0.8
12.8 3.9
46.3 1.5
33.5 1.1
13.2 4.2
16.1 5.4
>100
28.2 5.6
31.3 5.2
15.6 3.3
22.7 2.4
27.3 2.3
33.8 4.7
38.4 0.7
26.2 3.1
105.6 7.9
>200
As shown in Scheme 1, the N0-((4-oxo-4H-chromen-3-yl)meth-
ylene)benzohydrazide compounds f1–f24 were synthesized by
using substituted phenols as starting materials. Substituted phe-
nols a were reacted with acetic anhydride in the presence of sev-
eral drops of concentrated H2SO4, respectively, to afford the
corresponding esters b. This method is more convenient than pre-
vious methods.19–23 Subsequently, o-hydroxyacetophenone c was
generated by Fries-rearrangement reaction24,25 in moderate yields
(about 35%) under the condition of dry grinded AlCl3. The Vilsme-
NO2
OH
Br
Me
H
ier–Haack cyclization26–29 of o-hydroxyacetophenones
c were
Br
Br
Me
Me
Me
reacted with dry DMF and POCl3 to form substituted 3-formylchr-
omones d in a high yield. In addition, the nitro-substituted com-
pound d10 was prepared by the nitration of d1. The compounds
e (benzohydrazides) were purchased from Alfa Aesar Company.
The target molecules f1–f24 were synthesized by the condensation
of compounds d and e in the presence of acetic acid at room tem-
perature, which was similar to previous methods.17,30,31
H
H
H
>200
Subsequently, we fixed R3 group to bromine atom and only
changed the R1 group. Then compounds f6–f14 were synthesized.
As listed in Table 1, when the R3 position were substituted by bro-
mine atom, the corresponding compounds always exhibit high
inhibitory activities, such as compounds f6–f12 with IC50 values
2.2. Cy-FBP/SBPase inhibitory activities of title compounds
The IC50 values against Cy-FBP/SBPase of the synthesized title
compounds f1–f24 were listed in Table 1. Firstly, we only changed
R3 group, such as compounds f1–f5. As shown in Table 1, the inhib-
itory potencies of compounds f1–f5 against Cy-FBP/SBPase were
obviously different. Especially, when the R3 group was substituted
by bromine atom, the compound (f5) exhibit higher inhibitory
of 11.2–46.3 lM, except for the compound f13 (IC50 >100 lM). In
comparison, the compounds with bulky hydrophobic group (f11,
f12) and halogen groups (f6–f8) at R1 position exhibit higher inhib-
itory activities than those compounds with hydrophilic groups (f13
and f14). Especially, when both the R1 and R3 group were bromine
atom, the compound (f7) exhibit the most potent inhibitory activ-
activities (IC50 = 22.1 lM) than others. This result demonstrates
that, bromine atom is likely the optimum R3 group for these title
ities (IC50 = 11.2
l
M) against Cy-FBP/SBPase.
compounds.
O
OH
OH
O
DMF/POCl3
rt
anhydrous AlCl3
(CH3CO)2O
R1
CHO
conc. H2SO4, rt
°
C
120
R1
R1
O
R1
O
O
a1-a9
b1-b9
c1-c9
d1-d9
O
O
O
fuming HNO3,conc. H2SO4
0 °C
CHO
O2N
CHO
O
d1
d10
R2
R1
R2
R3
O
O
R3
CONHNH2
O
EtOH, HOAc, rt
O
+
R1
CHO
R4
N
R4
N
H
O
d1-d10
e
f 1-f 24
R1,R2,R3,R4 = H, Cl, Br, F, Me, Et, i-Pr, t-Bu, OH, NO2......
Scheme 1. Synthetic route for the title compounds f1–f24.

2828
Q.-D. Tu et al. / Bioorg. Med. Chem. 21 (2013) 2826–2831
In order to understand the probably interactions of compound
3. Conclusion
f7 and the active site of Cy-FBP/SBPase, a rigorous surflex docking
study of Cy-FBP/SBPase active site has been performed by using
Sybyl-X1.3, as shown in Figure 1. The hydrogen-bonds were
formed between compound f7 (the nitrogen atom of hydrazone
group and ketonic oxygen of amide group) and the surrounding
residues (R131, R176 and R178) of Cy-FBP/SBPase. Also, the R1
group of chromone of compound f7 might form the hydrogen-bond
with the carboxyl group of residue D175. From these analysis, we
can conclude that the potential inhibitory activity of compound
f7 likely result from these remarkable hydrogen bonds. Unexpect-
edly, when R1 group was substituted by –NO2, the inhibitory activ-
In the present study,
a
series of novel N0-((4-oxo-4H-
chromen-3-yl)methylene)benzohydrazide (f1–f24) were designed
and synthesized. The inhibitory activities of these compounds
against Cy-FBP/SBPase were further examined. Our results showed
that some of these new synthesized compounds had better Cy-FBP/
SBPase inhibitory activities. The inhibitory potency against Cy-FBP/
SBPase could be improved obviously by optimizing substituent
group of title compounds. When R3 was substituted by bromine
atom and R1 was replaced by the bulky hydrophobic (i-Pr, t-Bu)
and halogen groups (F, Cl, Br), the higher inhibitory activities
against Cy-FBP/SBPase could be noticed, such as compounds f6–
f8, f11 and f12. Additionally, the –CF3 group was the optimum
group for R4 position and –H group was the optimum group for
R2 position. Most interestingly, the compound (f7) with bromine
atom at R1 group and R3 group exhibited the most potent inhibitory
activity. The probable binding mode of compound f7 was further
examined carefully by using molecular docking methods. The con-
sistence between docking results and experimental results suggest
that compound f7 might have the potential to be developed as a
promising lead compound structure for novel algicides.
ity of compound (f13) was decreased remarkably (IC50 >100 lM).
According to the interaction mode of compound f7, it should be no-
ticed that the remarkable electrostatic repulsion effect between R1
group and D175 might be occurred when the R1 group was nega-
tive charged (–NO2), since D175 was a negative charged residue.
This confirmed to some extent that the compound f13 exhibited
lower inhibitory activities likely resulted from the larger electro-
static repulsion effect between nitro group in R1 position and car-
boxyl group of residue D175.
Subsequently, we fixed R1 group to bromine atom and only
changed R4 group, compounds f15–f21 were synthesized. As listed
in Table 1, all of these compounds (f15–f21) had lower IC50 values
4. Materials and methods
than 40
unfavorable, which decreased the inhibitory activities of title com-
pounds, such as compound f20 (IC50 = 38.4 M), while the –CF3
group at R4 position is favorable, which looks increase the inhibi-
tory activities, such as compound f16 (IC50 = 15.6 M).
l
M. In comparison, the bromine atom at R4 position is
All chemical reagents were commercially available and treated
with standard methods before use. Solvents were dried in a routine
way and distilled before use. 1H NMR spectra were recorded on a
VARIAN NMR System 600 or Mercury-Plus 400 spectrometer in
CDCl3 or DMSO-d6 with TMS as the internal reference. Mass spec-
tral data were obtained on a Thermo Fisher Mass platform DSQII by
electroionization (EI-MS). All MALDI experiments were performed
on a Synapt G2 HDMS system made by Waters Corp. (Milford, USA)
in sensitivity mode. Elemental analyses were performed on a Vario
EL III elemental analysis instrument. Conventional heating was car-
ried out on Corning stirrer/hotplates in oil baths. Melting points
were taken on an X-4 melting point apparatus and are uncorrected.
l
l
By fixing the bromine atom (most favorable group) in R1 posi-
tion and changing R4 group, several potential compounds have
been obtained in the previous studies. However, when R1 group
was an unfavorable group (–CH3), whether or not we also can im-
prove the inhibitory activities of hit compounds by substituting R2
group? For this purpose, we synthesized three compounds (f22–
f24). The comparison of the inhibitory activities of these three
compounds indicated clearly that the optimum R2 group was –H.
When the R2 group was replaced by –OH and –CH3, the inhibitory
activities of corresponding compounds (f23, f24) are always lower
4.1. General procedure for the preparation of substituted 3-
formylchromones (d1–d9)
(IC50 >200
Unfortunately, the IC50 values of compounds f22–f24 were always
l lM).
M) than those with –H at R2 position (IC50 = 105.6
larger than 100
activities.
lM, these compounds exhibit lower inhibitory
As shown in Scheme 1, the substituted 3-formylchromones d1–
d9 were prepared according to a three-step synthetic route using
substituted phenol as starting materials, which is similar to syn-
thesis method of 3-formylchromone.26
Step 1: Synthesis of substituted acetic acid phenyl ester (b1–
b9). To a mixture of phenol a1 (94.1 g, 1 mol) and acetic anhydride
(102 g, 1 mol) was added three drops of concentrated H2SO4 at
room temperature with stirring for 10 min. TLC monitoring indi-
cated that reaction was finished and acetic acid phenyl ester b1
was generated. The mixture was poured into water (500 mL) with
stirring, extracted with EtOAc (10 ꢀ 50 mL) and the layers were
separated. The organic layer was dried over anhydrous Na2SO4
and the solvent was removed under reduced pressure. Acetic acid
phenyl ester b1 (95.3 g, 70%) was obtained. The similar procedure
was adopted in making other acetic acid phenyl ester (b1–b9). b1–
b9 were not necessary for being dealt with for the next step.
Step 2: Synthesis of substituted o-hydroxyacetophenone (c1–
c9). Powdered anhydrous AlCl3 (186 g, 1.4 mol) was added little
by little to acetic acid phenyl ester b1 (95.3 g, 0.7 mol) in a
round-bottomed flask in an ice-water bath. The resultant mixture
was heated to 120 °C for 6 h in an oil bath. Then the reaction mix-
ture was added a lot of crushed ice for hydrolysis. The new formed
organic layer in the reaction mixture was extracted by EtOAc, dried
over anhydrous Na2SO4, and filtered off by suction filtration. The
Figure 1. The binding model of f7 at the active site of Cy-FBP/SBPase.

Q.-D. Tu et al. / Bioorg. Med. Chem. 21 (2013) 2826–2831
2829
solvent was removed under reduced pressure to give the crude
product, which was then purified by chromatography on silica
using petroleum ether/ethyl acetate as the eluent. o-hydroxyaceto-
phenone c1 (20.1 g, 21%) was obtained. The similar procedure was
adopted in making other substituted o-hydroxyacetophenone (c1–
c9).
Step 3: Synthesis of 6-substituted 4-oxo-4H-chromene-
3-carbaldehydes (d1–d9): POCl3 (112.6 g, 0.72 mol) was added
slowly to a well stirred and cooled (0–5 °C) mixture of N,N-dimeth-
ylformamide (53.6 g, 0.72 mol), o-hydroxyacetophenone c1 (20.1 g,
0.14 mol) for 30 min. The ice bath was removed and the mixture
was further stirred for 8–12 h at room temperature. The orange-
yellow colored organic mass was poured into crushed ice (400 g)
with stirring, extracted with EtOAc (8 ꢀ 50 mL) and the layers
were separated. The organic layer was dried over anhydrous
Na2SO4 and the solvent was removed under reduced pressure. Thus
obtained residue was subjected to column chromatography purifi-
cation on silica gel to give 4-oxo-4H-chromene-3-carbaldehyde d1
(11.4 g, 47%). The similar procedure was adopted in making other
chromone carbaldehydes (d1–d9).
Data for d6: 6-Ethyl-4-oxo-4H-chromen-3-carbaldehyde. Yield:
20%; light yellow powder; mp 98–101 °C; 1H NMR (400 MHz,
CDCl3) d 10.40 (s, 1H, CHO), 8.54 (s, 1H, @CH–O), 8.16–7.40 (m,
3H, ArH), 2.79 (q, J = 7.6 Hz, 2H, CH2), 1.30 (t, J = 7.6 Hz, 3H, CH3).
EI MS: m/z (%) 202.01 (M)+.
Data for d7: 6-Isopropyl-4-oxo-4H-chromen-3-carbaldehyde.
Yield: 17%; light yellow powder; mp 97–100 °C; 1H NMR
(600 MHz, CDCl3) d 10.39 (s, 1H, CHO), 8.52 (s, 1H, @CH–O),
8.18–7.40 (m, 3H, ArH), 3.15–2.95 (m, 1H, CH), 1.30 (d, J = 6.9 Hz,
6H, CH3). EI MS: m/z (%) 216.18 (M)+.
Data for d8: 6-Tertbutyl-4-oxo-4H-chromen-3-carbaldehyde.
Yield: 18%; light yellow powder; mp 90–93 °C; 1H NMR
(600 MHz, CDCl3) d 10.40 (s, 1H, CHO), 8.53 (s, 1H, @CH–O),
8.36–7.40 (m, 3H, ArH), 1.39 (s, 9H, CH3). EI MS: m/z (%) 230.17
(M)+.
Data for d9: 6-Hydroxy-4-oxo-4H-chromen-3-carbaldehyde.
Yield: 61%; gray powder; mp 223–226 °C; 1H NMR (600 MHz,
DMSO-d6) d 10.26 (s, 1H, CHO), 10.09 (s, 1H, OH), 8.80 (s, 1H,
@CH–O), 7.71–6.98 (m, 3H, ArH). EI MS: m/z (%) 190.08 (M)+.
Data for d10: 6-Nitro-4-oxo-4H-chromen-3-carbaldehyde. Yield:
83%; light yellow powder; mp 155–158 °C; 1H NMR (600 MHz,
CDCl3) d 10.38 (s, 1H, CHO), 9.21–7.67 (m, 4H, @CH–O, ArH). EI
MS: m/z (%) 219.12 (M)+.
4.2. Procedure for the preparation of 6-nitro-3-
formylchromone d10
As shown in Scheme 1, compound d1 (8.71 g, 50.0 mmol) was
added to 20 mL concentrated H2SO4 and the resulting reddish-or-
ange solution was cooled with ice water. To this stirred solution
was added 20 mL fuming HNO3 dropwise over the course of about
10 min. The mixture was poured over ice water (1.0 L liter) and the
precipitate was collected by filtration and recrystallized from ace-
tone to afford 9.02 g of light yellow powder d10.
The prepared substituted 3-formylchromones d1–d10 were
listed in Table 2.
Data for d1: 4-Oxo-4H-chromen-3-carbaldehyde. Yield: 47%;
light yellow powder; mp 150–154 °C; 1H NMR (400 MHz, CDCl3)
d 10.40 (s, 1H, CHO), 8.57 (s, 1H, @CH–O), 8.36–7.50 (m, 4H,
ArH). EI MS: m/z (%) 174.01 (M)+.
Data for d2: 6-Chloro-4-oxo-4H-chromen-3-carbaldehyde. Yield:
18%; light yellow powder; mp 167–169 °C; 1H NMR (400 MHz,
CDCl3) d 10.41 (s, 1H, CHO), 8.58 (s, 1H, @CH–O), 8.45–7.50 (m,
3H, ArH). EI MS: m/z (%) 207.96 (M)+.
Data for d3: 6-Bromo-4-oxo-4H-chromen-3-carbaldehyde.
Yield: 24%; light yellow powder; mp 181–185 °C; 1H NMR
(600 MHz, CDCl3) d 10.36 (s, 1H, CHO), 8.54 (s, 1H, @CH–O),
8.47–7.38 (m, 3H, ArH). EI MS: m/z (%) 253.90 (M)+.
4.3. Procedure for the preparation of N0-((4-oxo-4H-chromen-3-
yl)methylene)benzohydrazide (f1–f24)
As shown in Scheme 1, to a mixture solution of 3-formylchro-
mone d1 (174 mg, 1 mmol) and benzohydrazide e1 (136 mg,
1 mmol, all compounds e were purchased from Alfa Aesar Com-
pany) in 25 mL ethanol was added three drops of acetic acid with
stirring for 6 h at room temperature. Insoluble solid was gradually
generated, then filtered and washed several times with ethanol.
After drying, pure target compound f1 was afforded (227 mg, yield
78%). The similar procedure was adopted in making other title
compounds (f1–f24).
The prepared N0-((4-oxo-4H-chromen-3-yl)methylene)ben-
zohydrazide (f1–f24) were listed in Table 1. The property data of
these compounds were as follows.
Data for f1: N0-((4-oxo-4H-chromen-3-yl)methylene)benzo
hydrazide. White powder. Yield 78%. Mp: 169–173 °C. 1H NMR
(600 MHz, DMSO-d6) d 11.95 (s, 1H, NH), 8.85–7.45 (m, 11H,
@CH–O, CH@N, ArH). HRMS (MALDI): m/z = 293.0961, calcd for
C
17H12N2O3 [M+H]+: 293.0926.
Data for f2: 3-Nitro-N0-((4-oxo-4H-chromen-3-yl)methylene)
Data for d4: 6-Floro-4-oxo-4H-chromen-3-carbaldehyde. Yield:
63%; light yellow powder; mp 136–140 °C; 1H NMR (400 MHz,
CDCl3) d 10.36 (s, 1H, CHO), 8.55 (s, 1H, @CH–O), 8.10–7.35 (m,
3H, ArH). EI MS: m/z (%) 193.07 (M)+.
benzohydrazide. Light yellow powder. Yield 82%. Mp: 151–
161 °C. 1H NMR (600 MHz, DMSO-d6) d 12.25 (s, 1H, NH), 8.92–
7.50 (m, 10H, @CH–O, CH@N, ArH). MS (EI) m/z: 337.01 (M)+. Anal.
calcd for C17H11N3O5: C, 60.54; H, 3.29; N, 12.46. Found: C, 60.38;
H, 3.47; N, 12.20.
Data for d5:
6-Methy-4-oxo-4H-chromen-3-carbaldehyde.
Yield: 20%; white powder; mp 173–175 °C; 1H NMR (400 MHz,
CDCl3) d 10.39 (s, 1H, CHO), 8.55 (s, 1H, @CH–O), 8.20–7.40 (m,
3H, ArH), 2.50 (s, 3H, CH3). EI MS: m/z (%) 188.02 (M)+.
Data for f3: 3-Hydroxy-N0-((4-oxo-4H-chromen-3-yl)methy-
lene)benzohydrazide. White powder. Yield 80%. Mp: 199–203 °C.
1H NMR (600 MHz, DMSO-d6) d 11.90 (s, 1H, NH), 9.79 (s, 1H,
OH), 8.88–6.87 (m, 10H, @CH–O, CH@N, ArH). HRMS (MALDI):
m/z = 331.0661, calcd for C17H12N2O4 [M+Na]+: 331.0695.
Data for f4: 3-Methyl-N0-((4-oxo-4H-chromen-3-yl)methy-
lene)benzohydrazide.White powder. Yield 52%. Mp: 162–165 °C.
1H NMR (600 MHz, DMSO-d6) d 11.91 (s, 1H, NH), 8.89–7.35 (m,
10H, @CH–O, CH@N, ArH), 2.40 (s, 3H, CH3). MS (EI) m/z: 306.10
(M)+. Anal. calcd for C18H14N2O3: C, 70.58; H, 4.61; N, 9.15. Found:
C, 70.33; H, 4.39; N, 9.32.
Table 2
Substituted 3-formylchromone d1–d10
O
R1
CHO
Data for f5: 3-Bromo-N0-((4-oxo-4H-chromen-3-yl)methy-
lene)benzohydrazide. White powder. Yield 90%. Mp: 166–170 °C.
1H NMR (600 MHz, DMSO-d6) d 12.03 (s, 1H, NH), 8.89–7.39 (m,
10H, @CH–O, CH@N, ArH). HRMS (MALDI): m/z = 371.0038, calcd
for C17H11Br N2O3 [M+H]+: 371.0031.
O
d1-d10
Compounds
d1
d2
d3
d4
d5
d6
d7
d8
d9
d10
R1
H
Cl
Br
F
Me
Et
i-Pr
t-Bu
OH
NO2

2830
Q.-D. Tu et al. / Bioorg. Med. Chem. 21 (2013) 2826–2831
Data for f6: 3-Bromo-N0-((6-chloro-4-oxo-4H-chromen-3-yl)
NH), 8.90–7.30 (m, 9H, @CH–O, CH@N, ArH). MS (EI) m/z: 390.15
(M)+. Anal. Calcd for C17H10BrFN2O3: C, 52.47; H, 2.59; N, 7.20.
Found: C, 52.28; H, 2.49; N, 7.43.
methylene)benzohydrazide. White powder. Yield 98%. Mp:
164–168 °C. 1H NMR (600 MHz, DMSO-d6) d 12.03 (s, 1H, NH),
8.89–7.36 (m, 9H, @CH–O, CH@N, ArH). HRMS(MALDI):
m/z = 404.9643, calcd for C17H10BrClN2O3 [M+H]+: 404.9642.
Data for f7: 3-Bromo-N0-((6-bromo-4-oxo-4H-chromen-3-yl)
methylene)benzohydrazide. Light yellow powder. Yield 92%. Mp:
156–158 °C. 1H NMR (600 MHz, DMSO-d6) d 12.03 (s, 1H, NH),
8.90–7.40 (m, 9H, @CH–O, CH@N, ArH). HRMS (MALDI):
m/z = 450.9138, calcd for C17H10Br2N2O3 [M+H]+: 450.9116.
Data for f8: 3-Bromo-N0-((6-fluoro-4-oxo-4H-chromen-3-yl)
methylene)benzohydrazide. White powder. Yield 72%. Mp:
165–168 °C. 1H NMR (600 MHz, DMSO-d6) d 12.04 (s, 1H, NH),
8.90–7.36 (m, 9H, @CH–O, CH@N, ArH). HRMS (MALDI):
m/z = 388.9935, calcd for C17H10BrFN2O3 [M+H]+: 388.9937.
Data for f9: 3-Bromo-N0-((6-methyl-4-oxo-4H-chromen-3-yl)
methylene)benzohydrazide. White powder. Yield 95%. Mp:
156–159 °C. 1H NMR (600 MHz, DMSO-d6) d 12.00 (s, 1H, NH),
8.85–7.35 (m, 9H, @CH–O, CH@N, ArH), 2.43 (s, 3H, CH3). HRMS
Data for f18: N0-((6-Bromo-4-oxo-4H-chromen-3-yl)methy-
lene)-4-nitrobenzohydrazide. Yellow powder. Yield 94%. Mp:
182–184 °C. 1H NMR (600 MHz, DMSO-d6) d 12.25 (s, 1H, NH),
8.95–7.70 (m, 9H, @CH–O, CH@N, ArH). MS (EI) m/z: 415.08 (M)+.
Anal. Calcd for C17H10BrN3O5: C, 49.06; H, 2.42; N, 10.10. Found:
C, 49.30; H, 2.49; N, 10.31.
Data for f19: N0-((6-Bromo-4-oxo-4H-chromen-3-yl)methy-
lene)-4-hydroxybenzohydrazide. White powder. Yield 79%. Mp:
200–204 °C. 1H NMR (600 MHz, DMSO-d6) d 11.78 (s, 1H, NH),
10.16 (s, 1H, OH), 8.89–6.76 (m, 9H, @CH–O, CH@N, ArH). MS
(EI) m/z: 388.02 (M)+. Anal. Calcd for C17H11BrN2O4: C, 52.74; H,
2.86; N, 7.24. Found: C, 52.49; H, 2.98; N, 7.45.
Data for f20: 4-Bromo-N0-((6-bromo-4-oxo-4H-chromen-3-
yl)methylene)benzohydrazide. White powder. Yield 84%. Mp:
193–196 °C. 1H NMR (600 MHz, DMSO-d6) d 12.04 (s, 1H, NH),
8.91–7.62 (m, 9H, @CH–O, CH@N, ArH). HRMS (MALDI):
m/z = 448.9130, calcd for C17H10Br2N2O3 [M+H]+: 448.9136.
Data for f21: N0-((6-Bromo-4-oxo-4H-chromen-3-yl)methy-
lene)-4-methylbenzohydrazide. White powder. Yield 91%. Mp:
179–182 °C. 1H NMR (600 MHz, DMSO-d6) d 11.89 (s, 1H, NH),
8.89–7.29 (m, 9H, @CH–O, CH@N, ArH), 2.38 (s, 3H, CH3). MS (EI)
m/z: 384.01 (M)+. Anal. Calcd for C18H13BrN2O3: C, 56.12; H, 3.40;
N, 7.27;. Found: C, 56.33; H, 3.53; N, 7.02.
(MALDI): m/z = 385.0194, calcd for
C
18H13BrN2O3 [M+H]+:
385.0188.
Data for f10: 3-Bromo-N0-((6-ethyl-4-oxo-4H-chromen-3-yl)
methylene)benzohydrazide. White powder. Yield 79%. Mp: 128–
132 °C. 1H NMR (600 MHz, DMSO-d6) d 12.01 (s, 1H, NH), 8.85–
7.41 (m, 9H, @CH–O, CH@N, ArH), 2.75 (q, J = 7.4 Hz, 2H, CH2),
1.22 (t, J = 7.4 Hz, 3H, CH3). HRMS (MALDI): m/z = 399.0340, calcd
for C19H15BrN2O3 [M+H]+: 399.0344.
Data for f22: N0-((6-Methyl-4-oxo-4H-chromen-3-yl)methy-
lene)benzohydrazide. White powder. Yield 82%. Mp: 170–173 °C.
1H NMR (600 MHz, DMSO-d6) d 11.95 (s, 1H, NH), 8.92–7.40 (m,
10H, @CH–O, CH@N, ArH), 2.44 (s, 3H, CH3). HRMS (MALDI): m/
z = 329.0834, calcd for C18H14N2O3 [M+Na]+: 329.0902.
Data for f11: 3-Bromo-N0-((6-isopropyl-4-oxo-4H-chromen-3-
yl)methylene)benzohydrazide. White powder, yield 79%, mp:
158–162 °C. 1H NMR (600 MHz, DMSO-d6) d 12.00 (s, 1H, NH),
8.80–7.40 (m, 9H, @CH–O, CH@N, ArH), 3.01 (m, 1H, CH), 1.21 (d,
J = 7.2 Hz, 6H, CH3). MS (EI) m/z: 413.76 (M)+. Anal. calcd for
Data for f23: 2-Hydroxy-N0-((6-methyl-4-oxo-4H-chromen-3-
yl)methylene)benzohydrazide. White powder. Yield 89%. Mp:
198–204 °C. 1H NMR (600 MHz, DMSO-d6) d 11.96 (s, 1H, NH),
11.87 (s, 1H, OH), 8.87–6.90 (m, 9H, @CH–O, CH@N, ArH), 2.45
(s, 3H, CH3). MS (EI) m/z: 322.13 (M)+. Anal. Calcd for
C
20H17BrN2O3: C, 58.13; H, 4.15; N, 6.78. Found: C, 58.01; H,
4.30; N, 6.91.
Data for f12: 3-Bromo-N0-((6-(tert-butyl)-4-oxo-4H-chromen-3-
yl)methylene)benzohydrazide. White powder, yield 19%, mp: 150–
154 °C. 1H NMR (600 MHz, DMSO-d6) d 12.04 (s, 1H, NH), 8.89–7.40
(m, 9H, @CH–O, CH@N, ArH), 1.36 (s, 9H, CH3). MS (EI) m/z: 427.82
(M)+. Anal. Calcd for C21H19BrN2O3: C, 59.03; H, 4.48; N, 6.56.
Found: C, 58.90; H, 4.30; N, 6.75.
C
18H14N2O4: C, 67.07; H, 4.38; N, 8.69. Found: C, 66.96; H,4.49;
N,8.86.
Data for f24: 2-Methyl-N0-((6-methyl-4-oxo-4H-chromen-3-
yl)methylene)benzohydrazide. Light yellow powder. Yield 63%.
Mp: 157–160 °C. 1H NMR (600 MHz, DMSO-d6) d 11.85 (s, 1H,
NH), 8.83–7.28 (m, 9H, @CH–O, CH@N, ArH), 2.45 (s, 3H, CH3),
2.38 (s, 3H, CH3). HRMS (MALDI): m/z = 321.1256, calcd for
Data for f13: 3-Bromo-N0-((6-nitro-4-oxo-4H-chromen-3-yl)
methylene)benzohydrazide. White powder, yield 96%. Mp:
186–190 °C. 1H NMR (600 MHz, DMSO-d6) d 12.06 (s, 1H, NH),
9.05–7.10 (m, 9H, @CH–O, CH@N, ArH). HRMS (MALDI):
m/z = 453.9442, calcd for C17H10BrN3O5 [M+K]+: 453.9441.
Data for f14: 3-Bromo-N0-((6-hydroxy-4-oxo-4H-chromen-3-
yl)methylene)benzohydrazide. White powder, yield 76%, mp:
181–185 °C. 1H NMR (600 MHz, DMSO-d6) d 12.01 (s, 1H, NH),
10.18 (s, 1H, OH), 8.82–7.17 (m, 9H, @CH–O, CH@N, ArH). HRMS
C
19H16N2O3 [M+H]+: 321.1239.
4.4. Enzyme expression, purification, activity assay
Cy-FBP/SBPase were expressed in Escherichia coli BL21 (DE3)
cells as described previously.32–34 Enzyme activity were measured
by phosphate release described previously.35–38 For the assay, the
reaction was started by adding 2.5 mM FBP to assay buffer
(MALDI): m/z = 386.9980, calcd for
C
17H11BrN2O4 [M+H]+:
386.9980.
Data for f15: N0-((6-bromo-4-oxo-4H-chromen-3-yl)methy-
lene)benzohydrazide. White powder. Yield 86%. Mp: 159–163 °C.
1H NMR (600 MHz, DMSO-d6) d 11.98 (s, 1H, NH), 9.04–7.30 (m,
10H, @CH–O, CH@N, ArH). MS (EI) m/z: 371.94 (M)+. Anal. Calcd
for C17H11BrN2O3: C, 55.01; H, 2.99; N, 7.55. Found: C, 55.21; H,
2.83; N, 7.72.
(50 mM Tris–HCl pH 8.0, 15 mM MgCl2, 10 mM DTT, 0.04
fied enzyme) and incubated at 28 °C for 5 min. The reaction was
stopped by adding 12.5 L of 1 M perchloric acid and centrifuged
for 5 min at 4 °C. Sample (10 L) and standards (10 L) were
incubated for 30 min at 25 °C with 100 L of malachite green solu-
tion (0.035% malachite green, 0.35% polyvinyl alcohol) and the
A620 was measured using a microplate reader (Bioteck Synergy2,
USA). One unit (1U) of FBP/SBPase activity was defined as the
lg puri-
l
l
l
l
Data for f16: N0-((6-Bromo-4-oxo-4H-chromen-3-yl)methy-
lene)-4-(trifluoromethyl)benzohydrazide. Yellow powder. Yield
81%. Mp: 168–174 °C. 1H NMR (600 MHz, DMSO-d6) d 12.14 (s,
1H, NH), 8.91–7.65 (m, 9H, @CH–O, CH@N, ArH). MS (EI) m/z:
439.77 (M)+. Anal. Calcd for C18H10BrF3N2O3: C, 49.23; H, 2.30; N,
6.38. Found: C, 49.00; H, 2.49; N, 6.54.
amount of enzyme that hydrolyzes the substrate to release 1
of inorganic phosphate.
lmol
For inhibition constant (IC50) determination, a standard reaction
mixture contained 50 mM Tris–HCl pH 8.0, 15 mM MgCl2, 10 mM
DTT, 0.04 lg purified enzyme. Increasing concentration of each
compound were incubated for 5 min with Cy-FBP/SBPase before
2.5 mM FBP was added. Kinetic and inhibition constants were
Data for f17: N0-((6-Bromo-4-oxo-4H-chromen-3-yl)methy-
lene)-4-fluorobenzohydrazide. Light yellow powder. Yield 87%.
Mp: 172–175 °C. 1H NMR (600 MHz, DMSO-d6) d 12.00 (s, 1H,

Q.-D. Tu et al. / Bioorg. Med. Chem. 21 (2013) 2826–2831
2831
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Acknowledgments
This work was supported by the National Basic Research Pro-
gram of China (973 Program, No. 2010CB126100), the Natural Sci-
ence Foundation of China (Nos. 21202056, 21272089, 21172089,
21072073, 20873049), PCSIRT (No. IRT0953), Natural Science
Foundation of Hubei Province (No. 2010CDB01202), the Program
for Academic leader in Wuhan Municipality (No. 201150530150),
and the self-determined research funds of CCNU from the colleges’
basic research and operation of MOE (Nos. CCNU10C01002 and
CCNU10A02006).
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