Development of hydroxy-based sphingosine kinase inhibitors and anti-inflammation in dextran sodium sulfate induced colitis in mice

Article abstract of DOI:10.1016/j.bmc.2016.05.047

Sphingosine kinase (SphK)-catalyzed production of sphingosine-1-phosphate (S1P) regulates cell growth, survival and proliferation as well as inflammatory status in animals. In recent study we reported the N′-(3-(benzyloxy)benzylidene)-3,4,5-trihydroxybenzohydrazide scaffold as a potent SphK inhibitor. As a continuation of these efforts, 51 derivatives were synthesized and evaluated by SphK1/2 inhibitory activities for structure-activity relationship (SAR) study. Among them, 33 was identified as the most potent SphK inhibitor. Potency of 33 was also observed to efficiently decrease SphK1/2 expression in human colorectal cancer cells (HCT116) and significantly inhibit dextran sodium sulfate (DSS)-induced colitis as well as the decreased expression of interleukin (IL)-6 and cyclooxygenase-2 (COX-2) in mouse models. Collectively, 33 was validated as an effective SphK inhibitor, which can be served as anti-inflammatory agent to probably treat inflammatory bowel diseases in human.

Full text of DOI:10.1016/j.bmc.2016.05.047

Bioorganic & Medicinal Chemistry 24 (2016) 3218–3230
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of hydroxy-based sphingosine kinase inhibitors and
anti-inflammation in dextran sodium sulfate induced colitis in mice
a
b
b
a
Meiyang Xi a,y, Jun Ge b,y, Xiaojian Wang a,b, , Chenbin Sun , Tianqi Liu , Liang Fang , Qiong Xiao ,
⇑
Dali Yin a,b,
⇑
a State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of
Medical Sciences, 1st Xian Nong Tan Street, Beijing 100050, PR China
b Department of Medicinal Chemistry, Beijing Key Laboratory of Active Substances Discovery and Drugability Evaluation, Institute of Materia Medica, Peking Union Medical College and
Chinese Academy of Medical Sciences, 1st Xian Nong Tan Street, Beijing 100050, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Sphingosine kinase (SphK)-catalyzed production of sphingosine-1-phosphate (S1P) regulates cell growth,
survival and proliferation as well as inflammatory status in animals. In recent study we reported the N0-
(3-(benzyloxy)benzylidene)-3,4,5-trihydroxybenzohydrazide scaffold as a potent SphK inhibitor. As a
continuation of these efforts, 51 derivatives were synthesized and evaluated by SphK1/2 inhibitory activ-
ities for structure–activity relationship (SAR) study. Among them, 33 was identified as the most potent
SphK inhibitor. Potency of 33 was also observed to efficiently decrease SphK1/2 expression in human col-
orectal cancer cells (HCT116) and significantly inhibit dextran sodium sulfate (DSS)-induced colitis as
well as the decreased expression of interleukin (IL)-6 and cyclooxygenase-2 (COX-2) in mouse models.
Collectively, 33 was validated as an effective SphK inhibitor, which can be served as anti-inflammatory
agent to probably treat inflammatory bowel diseases in human.
Received 11 April 2016
Revised 20 May 2016
Accepted 21 May 2016
Available online 24 May 2016
Keywords:
SphK
DSS
Inflammation
Colitis
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
Studies in mice indicate a redundant role of SphK1 and SphK2
because the individual knockout remains viable, but double knock-
The conserved lipid kinase, sphingosine kinase (SphK), catalyzes
the transfer of phosphate from ATP to the 1-OH in sphingosine
(Sph) to form sphingosine 1-phosphate (S1P), which plays an
important role in regulating cell growth, survival, proliferation,
neovascularization, and migration.1–3 There are two known iso-
forms of SphK exist in mammals, SphK1 and SphK2. The amino acid
sequence of these two kinases is 80 percent similar and 45 percent
overall identical.4 SphK has been involved with a diverse range of
disease including sickle cell disease,5,6 cancer,7,8 atherosclerosis,9,10
asthma,11,12 and inflammation,13,14 among others.
SphK1’s role in colitis is widely studied, where correlation
between elevated expression and severity of disease has been
reported. Deletion of SphK1 reduces colitis severity.15 However,
SphK2 knockout induces increased severe colitis. It is thought that
the two enzymes have some compensatory mechanism. SphK2
knockout up-regulating SphK1 expression is one main reason.16
outs are embryonically lethal.4,17 In addition, there are also differ-
ences in their subcellular localization and structure elucidation:
SphK1 is mainly localized in cytosol while SphK2 is mainly in
nuclei;18 a high resolution X-ray crystal structure of human SphK1
was announced but the structure of SphK2 was not resolved.19,20
At present, there are several classes of SphK inhibitors reported:
(1) SphK-nonselective inhibitors, such as N,N-dimethylsphingosine
(DMS),21 SKI-II22 and ABC294735;21 (2) SphK1-selective inhibitors,
such as PF-543,19 Compound 56 (derived from VPC94075),18,23
RB00524,25 and SLP7111228;17 (3) SphK2-selective inhibitors, such
as ABC294640,26 SG-1227 and SLP12070117 (Fig. 1). Some inhibitors
are reported to exert anti-inflammatory activities in animal mod-
els.28–30 For example, ABC747080, a derivative of known SKI-II,
was reported to have influence on the expression of S1P levels
and on inflammation progression including colonic levels of tumor
necrosis factor (TNF) a, interleukin (IL)-1b, IL-6 in dextran sodium
sulfate (DSS)-induced colitis in mice.4,31 In this model, decreased
severity of DSS-induced colitis was observed when SphK was
inhibited. Actually, this model is often used to evaluate potency
against colitis and elucidate mechanisms involved in colitis, being
considered as one of the most widely used models for understand-
⇑
Corresponding authors. Tel.: +86 010 63165248 (X.W.), +86 010 63037952
(D.Y.).
E-mail addresses: wangxiaojian@imm.ac.cn (X. Wang), yindali@imm.ac.cn
(D. Yin).
y
These two authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2016.05.047
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.

M. Xi et al. / Bioorg. Med. Chem. 24 (2016) 3218–3230
3219
SphK-nonselective
OH
Cl
Cl
OH
N
O
N
HO
C13H27
NH
HN
ABC294735
N
S
SKI-II
DMS
Ki (μM), SphK1: 3.2
Ki (μM), SphK1: 17
Ki (μM), SphK1: 16
SphK2: 4.2
SphK2: 50
SphK2: 14
SphK1-selective
H2N
NH HCl
O
OH
O
O
N
N
S
O
N
N
C8H17
PF-543
O
RB005
Compound 56
Ki (μM), SphK1: 0.047
OH
IC50 (μM), SphK1: 3.6
Ki (μM), SphK1: 0.0036
SphK2: ---
SphK2: ---
SphK2: 4.2
SphK2-selective
O
N
NH2
OH
Cl
N
N
N
O
H2N
C8H17
NH HCl
OH
HN
ABC294640
SLP120701
SG-12
IC50 (μM), SphK1: >10
IC50 (μM), SphK1: ---
SphK2: 22
Ki (μM), SphK1: ---
SphK2: 1.2
SphK2: 9.8
Figure 1. Representative structures and potencies of SphK inhibitors.
ing inflammatory bowel disease in human.32,33 Herein, targeting
the SphK pathway can treat colitis progression with concomitant
decrease in inflammatory cytokines. Considering the importance
of SphK inhibition in the therapy of colitis, novel SphK inhibitors
are urgently needed to develop and attract the attention of medic-
inal chemists throughout the world.
2.2. Inhibitory activity on SphK1 and SphK2 and preliminary
SAR
In an effort to discover new SphK inhibitors, we performed an
enzyme-based screening for each compound to evaluate their abil-
ity to decrease SphK1 and SphK2 activity in 384-well plates.40–42
Compound 33 showed the best inhibitory effect on SphK1 and
SphK2 activity followed by 37 and 20. In comparison, the positive
control DMS displayed modest activity (Table 1).
In this study, we report a series of 51 compounds derived
from
N0-(3-(benzyloxy)benzylidene)-3,4,5-trihydroxybenzohy-
drazide scaffold. Among these compounds, 1 was first identified
via structure-based hierarchical screening in recent study.34 It
showed potent activity against SphK in vitro. Thus 1 was selected
as a hit for further optimization. Other 50 derivatives were
designed, synthesized, and evaluated their ability to decrease
SphK1 and SphK2 levels. Structure–activity relationship (SAR)
studies indicated a more promising compound 33 for further anal-
ysis. It showed potent activity against SphK in cellular level and
suppressed colitis in DSS mouse models. As far as we know, these
compounds are first confirmed as SphK inhibitors by us, exhibiting
favorable suppressive activity and serving as an ideal lead com-
pound for further development of anti-inflammatory agents.
To determine the effect of different alkyl or halogen substituent
on ring A toward SphK1 activity, a set of compounds (1–21) around
the phenyl ring were afforded to assess the extent of SphK1 inhibi-
tion. First, several examples incorporating either –F or –Cl atom
were prepared. The great lack of inhibitory activity displayed by
–F or –Cl substitution against SphK1 indicated that the halogen
group was not tolerated except 2-Cl substituent (11). We also
assessed the role of electron-drawing groups in the lipophilic tail
of compounds. Changing the –CF3 to –CN substitution at ortho-
or para-position, the inhibitory activity against SphK1 was gener-
ally eliminated. The result confirmed that –CF3 substituent played
a more important role in increasing activity. However, it is worth
mentioning that the –CF3 substituents (6) at meta-position was
inactive and –CN substituents (16) showed little inhibition. At this
position, the introduction of electron-drawing groups exerted a
negative influence on inhibition.
2. Results and discussion
2.1. Chemical synthesis
To further evaluate the effect of the electron-donating group on
the ring A against SphK1 activity, we designed and synthesized
some derivatives with –CH3, –C(CH3)3 or –OCH3 substitution. The
compound with 3-OCH3 substitution (20) increased SphK1 inhibi-
tion remarkably, suggesting that the 3-OCH3 substituent main-
tained an efficient binding with SphK1 and had a positive effect
on activity. When 4-OCH3 group (21) was substituted, the activity
was abolished completely, indicating that the substitution position
was responsible for mediating the SphK1 activity. At para-position,
when a –C(CH3)3 substituent (14) was further shortened to a –CH3
The synthetic route we employed to synthesize a class of aro-
matic compounds 1–51 was shown in Scheme 1. Accordingly, 3-
or 4-(substituted benzyloxy)benzaldehyde (III) was synthesized
via substitution reaction of 3- or 4-hydroxybenzaldehyde (II) with
substituted benzyl bromide (I).35,36 Treatment of substituted car-
boxylate (IV) with hydrazine monohydrate (V) in refluxing ethanol
afforded the common intermediate or substituted carbohydrazide
(VI).37 Subsequent condensation was accomplished with III, which
produced the corresponding desired target compounds 1–51 at
reflux in excellent yield.38,39

3220
M. Xi et al. / Bioorg. Med. Chem. 24 (2016) 3218–3230
Br
CHO
O
R
a
CHO
+
R
O
O
OH
II
O
R
N
I
III
N
H
OH
1-34, 36-51
c
OH
N
O
OH
N
O
NH
NH
N
b
H2N
VI
O
N
H
O
O
NH2NH2·2H2O
or
35
+
IV
N
V
or
HN
O
N
H
O
N
H
H2N
Scheme 1. Synthetic route of 1–51. (Reagents and conditions: (a) K2CO3, DMF, 80 °C, 3 h; (b) EtOH, reflux, 36 h; (c) EtOH, reflux, 6 h.)
one (4), a slightly potency loss was observed indicating that a lar-
ger bulk of a substituent was beneficial.
observed in the DSS group. Body weight in compound administra-
tion groups was increased by approximately 12% average com-
pared to the DSS group at the end of the study, showing
apparent preventive effect on DSS treated weight loss (Table 2).
In addition, DSS treatment induced a reduction in colon length in
4 groups on day 10. However, colon length in compound treated
groups was significantly longer than in the DSS group (Fig. 4B).
In a second step, we paid attention to the substitution position
of ring B in the linker. Substituted benzyloxy groups attached to
the para-position on ring B gave rise to representative analogues
(22–34). Their inhibitory activity toward SphK1 was evaluated as
to facilitate exploration of SAR. Among these monosubstituted
derivatives, the slightly changed activity between para- and
meta-position except 3-CF3 substituted benzyloxy group (6, 24)
implied that the substitution position may be not the key factor
in affecting the potency of SphK1 inhibition.
To exam the role of –OH groups on ring C, an imidazole group
(bioisosterism, 35), 3,5-OH, 3,4-OH, 3-OH or 4-OH (35–51) was
introduced to get access to a last set of compounds. As it turned
out, most of the compounds were inactive except 3,4-OH sub-
stituents (37, 41, 45 and 49). The result suggested that the polar
head of 3,4-OH was essential for mediating SphK1 inhibitory activ-
ity but the 5-OH group was dispensable. For SphK2 inhibition,
most of these compounds showed similar activity to SphK1. In a
word, these compounds can be considered as SphK inhibitors. A
brief description of SAR was presented in Figure 2. On the basis
of these data, the derivative 33 can serve as an ideal lead com-
pound for further optimization and evaluation.
2.5. Pathology findings of DSS mice treated with 33
To evaluate the colitis, we observed the cellular infiltration of
the intestinal wall, mucosal lesion and crypt damage on the day
of dissection.47–49 Histological signs of colitis indicated cellular
infiltrations of predominantly mononuclear macrophages and neu-
trophils, mucosal and submucosal lesion, degeneration and necro-
sis of crypt cells were severe in the DSS group when compared
with the DSS + 5-ASA or 33 treated mice. Consequently, mean his-
tologic score in mice treated with 5-ASA or 33 was significantly
lower than in the DSS group. In a word, the inflammation level of
33 at a dose of 10 mg/kg treated mice was the lowest among the
4 DSS treated groups, which revealed that 33 effectively attenuated
the colitis (Fig. 5A and HE).
2.6. Suppression of 33 on the overexpression of IL-6 and COX-2
in DSS induced colitis in mice
2.3. Potency of compound 33 to inhibit the expression of SphK1
and SphK2
Some of the inflammatory mediators such as interleukin-6 (IL-
6) and cyclooxygenase-2 (COX-2) were characterized recently.
The importance of pro-inflammatory cytokines IL-6 in colon tissue
has been demonstrated in animal models of colitis. It showed that
inhibiting IL-6 efficiently ameliorated the colitis.50,51 COX-2 was
also overexpressed in DSS induced colitis.52,53 Therefore, we evalu-
ated anti-inflammatory activity by IL-6 and COX-2 immunohisto-
chemistry54,55 in mice. Overexpression of IL-6 and COX-2 in the
DSS group was observed. After treated with 5-ASA or 33, the evi-
dently decreased expression was observed, indicating that 33
inhibiting this factor in the inflammatory environment was able
to suppress the colitis effectively (Fig. 5B, C, IL-6 and COX-2).
Through enzyme-based activity studies, 33 was identified as the
most potent SphK inhibitor. In order to exam the inhibitory effect
for this compound in cellular level, human colorectal cancer cells
(HCT116) were used to evaluate the expression of the target
enzymes. Treatment with 33 decreased the expression of SphK1
and SphK2 in a time dependent manner by measuring protein
levels in HCT116 (Fig. 3). At 24 h, SphK levels were significantly
lower compared to the Con panel (p < 0.01).43,44
2.4. Effect of administration with identified compound 33 on
development of DSS mouse models
As described above, it has been showed that pharmacological
SphK inhibitors can inhibit DSS-induced colitis. To confirm 33
was able to inhibit colon inflammation, mice were measured for
their body weight, colon length and histological score. 5-aminosal-
icylic acid (5-ASA) was used as the positive control.45,46 In Fig-
ure 4A, no obvious change of body weight was observed in the
DSS group and 3 other compound administration groups until
the 7th day. After day 8, a significant body weight decrease was
3. Conclusion
We have identified a series of SphK inhibitors derived from N0-
(3-(benzyloxy)benzylidene)-3,4,5-trihydroxybenzohydrazide scaf-
fold via structure-based hierarchical screening in recent study.
According to this finding, we made a structural optimization and
SAR Study. The result indicated that electrostatic state and substi-
tution position in lipophilic ring was responsible for mediating the

M. Xi et al. / Bioorg. Med. Chem. 24 (2016) 3218–3230
3221
Table 1
SphK1 and SphK2 inhibitory activity
O
4
O
B
2
N
R
3
A
N
H
1
3
C
OH
4
5
Tail
LInker
Head
IC50 (l
M)a
3
4
R
A
O
B
OH
C
Compd ID
SphK1
SphK2
4
1
3
2
5
1
2
3
4
5
6
7
8
H
3-O–
3-O–
3-O–
3-O–
3-O–
3-O–
3-O–
3-O–
3-O–
3-O–
3-O–
3-O–
3-O–
3-O–
3-O–
3-O–
3-O–
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
40.21 3.05
63.24 5.61
52.10 3.28
45.79 4.15
19.57 2.43
>100
16.26 1.25
>100
>100
26.33 2.55
81.17 4.29
78.69 6.04
65.38 3.72
16.32 2.31
>100
22.15 1.96
98.90 5.21
>100
2-CH3
3-CH3
4-CH3
2-CF3
3-CF3
4-CF3
2-F
3-F
4-F
2-Cl
3-Cl
9
10
11
12
13
14
15
16
17
>100
36.80 4.83
>100
>100
58.79 6.52
67.37 5.97
61.20 4.28
62.17 5.16
68.55 3.71
>100
4-Cl
>100
4-C(CH3)3
2-CN
3-CN
4-CN
35.22 3.24
45.38 2.31
85.26 4.64
69.44 3.85
35.11 4.58
18
3-O–
3,4,5-OH
31.30 2.74
40.58 5.16
19
20
21
22
23
24
25
26
27
28
29
30
2-OCH3
3-OCH3
4-OCH3
H
2-CF3
3-CF3
4-CF3
2-CN
3-CN
4-CN
4-CH3
4-C(CH3)3
3-O–
3-O–
3-O–
4-O–
4-O–
4-O–
4-O–
4-O–
4-O–
4-O–
4-O–
4-O–
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
20.52 2.61
8.57 2.50
>100
22.02 2.71
15.01 2.12
54.72 0.98
37.40 2.81
>100
45.76 5.82
27.74 1.09
32.73 1.85
40.55 7.06
19.53 1.74
8.70 0.14
>100
28.51 1.88
15.08 1.16
23.83 1.42
15.64 1.06
44.54 3.70
22.59 4.53
17.79 0.87
44.50 4.91
43.28 5.79
31
4-O–
3,4,5-OH
20.61 3.38
30.23 2.14
32
33
34
2-OCH3
3-OCH3
4-OCH3
4-O–
4-O–
4-O–
3,4,5-OH
3,4,5-OH
3,4,5-OH
18.44 4.25
4.36 1.08
>100
26.62 3.29
2.17 0.77
>100
H
N
35
H
3-O–
>100
>100
N
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
DMS
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
2-CF3
2-CF3
2-CF3
2-CF3
2-CF3
3-O–
3-O–
3-O–
3-O–
4-O–
4-O–
4-O–
4-O–
3-O–
3-O–
3-O–
3-O–
4-O–
4-O–
4-O–
4-O–
3,5-OH
3,4-OH
3-OH
>100
5.00 0.65
>100
>100
>100
25.32 3.11
>100
>100
>100
15.53 0.97
>100
>100
>100
16.48 2.08
>100
>100
>100
4.60 0.67
>100
>100
>100
10.15 1.07
>100
>100
>100
13.84 1.21
>100
>100
>100
11.40 0.70
>100
>100
4-OH
3,5-OH
3,4-OH
3-OH
4-OH
3,5-OH
3,4-OH
3-OH
4-OH
3,5-OH
3,4-OH
3-OH
2-CF3
2-CF3
2-CF3
4-OH
50.56 3.12
26.29 4.66
a
Values shown are mean SD (n = 3, 1 h of treatment in triplicate). Each compound was exposed to human SphK, Sph and ATP measured their inhibitory ability.
activity. It was worth noting that there was no long lipophilic tail
position was not the key factor for mediating the inhibitory activ-
ity. In an effort to discover a surrogate for polar head, 3,4,5-OH was
replaced with imidazole according to the biosostere theory as well
as 3,5-OH, 3,4-OH, 3-OH, 4-OH. The result suggested that the 3,4-
compared with
a large portion of previous reported com-
pounds.56–58 It broke the limit of long-chain aliphatic hydrocar-
bons and was a notable exception. In the linker, the substitution

3222
M. Xi et al. / Bioorg. Med. Chem. 24 (2016) 3218–3230
The substitution position may be
not the key factor in affecting the
potency of SphK1 inhibition except
3-CF3 substituted benzyloxy group.
The electrostatic state and
O
substitution position were
important for activity.
≅
3-OCH3 > 2-CF3, 4-CF3
O
> 2-CN, 4-CN > H > -CH3, -F, -Cl
4
O
3,4,5-OH, 3,4-OH
O
B
2
N
> 3-OH, 4-OH, 3,5-OH
R
3
A
N
H
1
3
C
OH
3,4-OH substitution
was essential for SphK1 inhibitory
activity but
4
5
Tail
Linker
Head
the 5-OH group was
dispensable.
1. For SphK2 inhibition, most of these compounds
showed similar activity to SphK1.
2. These compounds can be considered as
SphK inhibitors.
Figure 2. SAR of compounds 1–51.
OH group was essential for potent SphK inhibitory activity but the
5-OH was dispensable. In the future plan, amidine, guanidine,
animo alcohol or other groups will be introduced to measure the
inhibitory activity to probe the polar head.
According to SAR, a potent compound 33 was discovered as the
most active SphK inhibitor. The examination of protein expression
was used to prove that 33 was a potent SphK inhibitor in cellular
level. In DSS treated mouse models, 33 diminished loss in body
weight and reduction in colon length. Furthermore, 33 significantly
inhibited colitis as well as decreased expression of pro-inflamma-
tory mediators IL-6 and COX-2. It was believed that SphK inhibitors
with micromolar level were effective in vivo in suppressing inflam-
mation response.18 However, in further plan, there is still a need for
optimizing this structure that could be active at submicromolar or
nanomolar level. In summary, we demonstrated the efficacy of 33
notonlyin vitrobutalsoin vivo. TargetingSphK notonlygaveinsight
into the treatment of colitis in mice, but also a possibly novel thera-
peutic strategy to treat inflammatory bowel diseases in human.
4. Experimental procedures
4.1. Synthetic chemistry
Figure 3. 33 inhibited SphK1 and SphK2 activity in vitro. (A) HCT116 cells were
treated with vehicle or 33 (30 lM) for different time points and analyzed by
western blot for total SphK1, SphK2 and b-actin. (B) Quantification of expression of
SphK1 and SphK2. b-Actin was used for normalization. *p < 0.05, **p < 0.01,
statistically significant difference from the vehicle group.
All of reagents were purchased from commercial sources.
Organic solutions were concentrated in a rotary evaporator (Büchi)
Figure 4. Changes in (A) body weight and (B) colon length at the end of the study in DSS-induced colitis in C57BL/6 mice. The following groups of mice were used: (1) Con:
water + vehicle; (2) DSS: 2.5% DSS + vehicle; (3) DSS + 5-ASA (50 mg/kg): 2.5% DSS + 5-ASA (50 mg/kg, ig); (4) DSS + 33 (5 mg/kg): 2.5% DSS + 33 (5 mg/kg, ip); (5) DSS + 33
(10 mg/kg): 2.5% DSS + 33 (10 mg/kg, ip). Data are shown as mean SD (n = 10). *p < 0.05, **p < 0.01, statistically significant difference from the DSS group. #P < 0.05,
##P < 0.01, compared with the Con group.

M. Xi et al. / Bioorg. Med. Chem. 24 (2016) 3218–3230
3223
Table 2
(4.6 ꢀ 150 mm, 5.0
l
m) column using a mixture of solvent metha-
Overview of DSS modelsa
nol/water or acetonitrile/water with 0.5% TFA at the flow rate of
1 mL/min and peak detection at 210 nm under UV. 51 compounds
were synthesized as follows:
Group
Body weight (g) day 10
Colon length (mm)
Con
DSS
18.98 0.84
15.69 0.65
18.32 0.58
17.10 0.82
17.50 0.77
55.64 2.31
45.68 1.34##
53.40 2.53**
53.04 2.14**
51.41 2.67*
To a solution of II (8.2 mmol) in DMF (15 mL), K2CO3 (1.35 g,
9.8 mmol) and I (9.0 mmol) was added. The resulting mixture
was stirred at 80 °C for 3 h, then it was cooled to room temperature
and poured into EtOAc (40 mL). The organic layer was washed with
H2O (15 mL) and brine (15 mL), then dried over Na2SO4 and con-
centrated in vacuo to remove solvents. The crude was purified by
flash column chromatography (eluent: 10–15% EtOAc/petroleum
ether) to afford III as a white solid. Yield 75.5–86.8%.
DSS + 5-ASA (50 mg/kg)
DSS + 33 (5 mg/kg)
DSS + 33 (10 mg/kg)
a
Data are shown as mean SD (n = 10).
p <0.05,
*
**
p <0.01, statistically significant difference from the DSS group.
P <0.01, compared with the Con group.
##
To a solution of IV (15.12 mmol) in ethanol (35 mL), Hydrazine
monohydrate (8.6 ml, 120.9 mmol) was added. The resulting mix-
ture was heated under reflux for 36 h. After being cooled to room
temperature, the mixture was filtered. The filter cake was washed
with EtOAc (45 mL) to afford VI as a white solid. Yield 70.1–93.3%.
below 50 °C under reduced pressure. Reactions were monitored by
thin layer chromatography silica gel slabs on 0.2 0.03 mm and
visualized under UV light at 254 nm. Melting points were deter-
mined with a Yanaco MP-J3 microscope melting point apparatus.
The 1H NMR and 13C NMR spectra were measured on a Bruker
AV-400 instrument using deuterated solvents with tetramethylsi-
lane (TMS) as internal standard. High resolution mass spectra
(HRMS) and ESI-mass were recorded on a Thermo Exactive Orbi-
trap plus spectrometer. The purity (P95%) of the compounds is
verified by the HPLC study performed on YMC-Pack ODS-A
To
a solution of VI (4.89 mmol) in ethanol (60 mL), I
(4.08 mmol) was added. The resulting mixture was heated under
reflux for 6 h. After being cooled to room temperature, the mixture
was filtered. The filtrate was evaporated in vacuo and the crude
was purified by flash column chromatography (eluent: 5–10%
MeOH/DCM) to afford 1–51. Yield 56.7–92.0%.
33 (50 mg-scale) was synthesized for ig administration of DSS
treated mice.
Figure 5. Histological evaluation of the DSS-induced colitis and 33 inhibited the expression of IL-6 and COX-2. (A) Histological score on day 10. Values represent the
individual scores of each mouse (n = 10). (B and C) Quantification of IL-6 and COX-2 staining. Data are shown as mean SD value for each group. Asterisk represents
significant difference from the DSS group (*p < 0.05, **p < 0.01, ***p < 0.001). ###P < 0.001, compared with the Con group. p < 0.05, p < 0.01, significant difference between the
DSS + 33 (5 mg/kg) group and the DSS + 33 (10 mg/kg) one. (HE) Representative pictures of colon from groups in C57BL/6 mice. (IL-6 and COX-2) Representative pictures of IL-
6 and COX-2 immunohistochemistry staining in indicated treatment. Bars inserted indicate magnification (50 lm).

3224
M. Xi et al. / Bioorg. Med. Chem. 24 (2016) 3218–3230
4.1.1. N0-(3-(Benzyloxy)benzylidene)-3,4,5-trihydroxybenzohy-
drazide (1)
4.1.6. 3,4,5-Trihydroxy-N0-(3-((3-(trifluoromethyl)benzyl)oxy)
benzylidene)benzohydrazide (6)
Yield 35.0%; White solid; mp 213–215 °C; 1H NMR (400 MHz,
DMSO) d 11.54 (s, 1H), 9.04 (s, 3H), 8.36 (s, 1H), 7.46 (d,
J = 7.5 Hz, 2H), 7.38 (t, J = 7.3 Hz, 2H), 7.36–7.30 (m, 2H), 7.25 (d,
J = 7.7 Hz, 1H), 7.05 (d, J = 8.2 Hz, 1H), 6.92 (s, 2H), 5.14 (s, 2H);
13C NMR (100 MHz, DMSO) d 163.62, 159.10, 146.87, 146.00,
137.44, 137.39, 136.56, 130.40, 128.90, 128.32, 128.15, 123.75,
120.40, 117.06, 112.73, 107.63, 69.70; HRMS calcd. For
Yield 41.5%; White solid; mp 208–210 °C; 1H NMR (400 MHz,
DMSO) d 11.55 (s, 1H), 9.06 (s, 3H), 8.37 (s, 1H), 7.83 (s, 1H),
7.79 (d, J = 7.4 Hz, 1H), 7.70 (d, J = 7.6 Hz, 1H), 7.64 (m, 1H), 7.37
(m, 2H), 7.27 (d, J = 7.3 Hz, 1H), 7.08 (d, J = 8.0 Hz, 1H), 6.91 (s,
2H), 5.25 (s, 2H); 13C NMR (125 MHz, DMSO) d 163.60, 158.86,
146.75, 146.00, 138.93, 137.44, 136.62, 132.13, 130.46, 130.03,
129.61 (q, J = 31.5 Hz), 125.03 (q, J = 3.6 Hz), 124.65 (q,
J = 270.8 Hz), 124.49(q, J = 3.9 Hz), 123.71, 120.64, 117.02, 112.64,
107.59, 68.78; HRMS calcd. For C22H18F3N2O5 [M+H]+ 447.1162,
found 447.1159; HPLC (63% methanol and 0.5% TFA in water):
tR = 9.745 min, 99.72%.
C
21H19N2O5 [M+H]+ 379.1288, found 379.1282; HPLC (40% acetoni-
trile and 0.5% TFA in water): tR = 6.500 min, 98.66%.
4.1.2. 3,4,5-Trihydroxy-N0-(3-((2-methylbenzyl)oxy)benzylidene)
benzohydrazide (2)
Yield 37.8%; White solid; mp 206–208 °C; 1H NMR (400 MHz,
DMSO) d 11.53 (s, 1H), 9.13 (s, 2H), 8.82 (s, 1H), 8.37 (s, 1H),
7.42 (d, J = 7.2 Hz, 1H), 7.37 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 4.4 Hz,
1H), 7.23 (m, 4H), 7.07 (m, 1H), 6.91 (s, 2H), 5.12 (s, 2H), 2.33 (s,
4.1.7. 3,4,5-Trihydroxy-N0-(3-((4-(trifluoromethyl)benzyl)oxy)
benzylidene)benzohydrazide (7)
Yield 46.4%; White solid; mp 129–131 °C; 1HNMR (400 MHz,
DMSO) d 11.54 (s, 1H), 9.12 (s, 3H), 8.36 (s, 1H), 7.75 (d,
J = 8.0 Hz, 2H), 7.69 (d, J = 8.0 Hz, 2H), 7.36 (m, 2H), 7.26 (d,
J = 7.6 Hz, 1H), 7.07 (m, 1H), 6.91 (s, 2H), 5.27 (s, 2H); 13C NMR
3H); 13C NMR (100 MHz, DMSO)
d 163.23, 158.82, 146.49,
145.61, 137.08, 136.73, 136.15, 134.85, 130.19, 130.00, 128.68,
128.17, 125.83, 123.32, 120.02, 116.59, 112.22, 107.20, 68.05,
18.53; HRMS calcd. For C22H21N2O5 [M+H]+ 393.1445, found
393.1442; HPLC (63% methanol and 0.5% TFA in water): tR =
6.956 min, 99.25%.
(100 MHz, DMSO)
d 163.65, 158.84, 146.78, 146.02, 142.35,
137.46, 136.65, 130.49, 128.76 (q, J = 31.6 Hz), 128.46, 125.81 (q,
J = 3.7 Hz), 124.71 (q, J = 270.4 Hz), 123.75, 120.71, 117.03,
112.71, 107.64, 68.80; HRMS calcd. For C22H18F3N2O5 [M+H]+
447.1162, found 447.1159; HPLC (63% methanol and 0.5% TFA in
water): tR = 10.849 min, 99.34%.
4.1.3. 3,4,5-Trihydroxy-N0-(3-((3-methylbenzyl)oxy)benzylidene)
benzohydrazide (3)
Yield 36.0%; White solid; mp 156–158 °C; 1HNMR (400 MHz,
DMSO) d 11.53 (s, 1H), 9.03 (s, 3H), 8.36 (s, 1H), 7.34 (m, 2H),
7.26 (m, 4H), 7.13 (d, J = 6.7 Hz, 1H), 7.05 (d, J = 8.1 Hz, 1H), 6.91
(s, 2H), 5.09 (s, 2H), 2.31 (s, 3H); 13C NMR (100 MHz, DMSO) d
162.69, 159.15, 158.98, 146.86, 146.02, 139.05, 138.08, 137.50,
137.32, 136.57, 132.31, 130.41, 128.96, 128.81, 128.72, 128.06,
125.25, 123.73, 120.38, 117.03, 112.72, 110.34, 107.62, 70.53,
21.47; HRMS calcd. For C22H21N2O5 [M+H]+ 393.1445, found
393.1441; HPLC (63% methanol and 0.5% TFA in water): tR =
7.554 min, 99.67%.
4.1.8. N0-(3-((2-Fluorobenzyl)oxy)benzylidene)-3,4,5-trihydro-
xybenzohydrazide (8)
Yield 38.6%; White solid; mp 228–230 °C; 1HNMR (400 MHz,
DMSO) d 11.54 (s, 1H), 8.98 (s, 3H), 8.36 (s, 1H), 7.57 (t,
J = 7.4 Hz, 1H), 7.37 (m, 3H), 7.24 (m, 3H), 7.07 (d, J = 8.0 Hz, 1H),
6.91 (s, 2H), 5.18 (s, 2H); 13C NMR (100 MHz, DMSO) d 162.10,
159.66, 158.96, 146.78, 146.02, 137.47, 136.65, 131.18 (d,
J = 3.0 Hz), 130.90 (d, J = 8.0 Hz) 130.47, 125.03 (d, J = 3.0 Hz),
124.19 (d, J = 14.0 Hz), 123.75, 120.52, 116.94, 11.88 (d,
J = 21.0 Hz), 112.78, 107.63, 64.10 (d, J = 3.0 Hz); HRMS calcd. For
C
21H18FN2O5 [M+H]+ 397.1194, found 397.1194; HPLC (60% metha-
4.1.4. 3,4,5-Trihydroxy-N0-(3-((4-methylbenzyl)oxy)benzylidene)
benzohydrazide (4)
nol and 0.5% TFA in water): tR = 6.592 min, 96.11%.
Yield 39.6%; White solid; mp 213–215 °C; 1H NMR (400 MHz,
DMSO) d 11.52 (s, 1H), 9.13 (s, 2H), 8.81 (s, 1H), 8.35 (s, 1H),
7.35 (m, 3H), 7.29 (s, 1H), 7.24 (d, J = 7.4 Hz, 1H), 7.19 (d,
J = 7.7 Hz, 2H), 7.04 (d, J = 7.9 Hz, 1H), 6.90 (s, 2H), 5.09 (s, 2H),
4.1.9. N0-(3-((3-Fluorobenzyl)oxy)benzylidene)-3,4,5-trihydro-
xybenzohydrazide (9)
Yield 35.3%; White solid; mp 211–213 °C; 1H NMR (400 MHz,
DMSO) d 11.52 (s, 1H), 8.94 (s, 3H), 8.36 (s, 1H), 7.51 (m, 2H),
7.34 (m, 2H), 7.22 (m, 3H), 7.05 (d, J = 7.9 Hz, 1H), 6.91 (s, 2H),
2.29 (s, 3H); 13C NMR (100 MHz, DMSO)
d 163.16, 160.03,
158.68, 146.42, 145.57, 137.10, 137.04 136.09, 133.90, 129.93,
129.00, 127.79, 127.69, 123.27, 119.87, 116.62, 112.32, 107.16,
69.16, 20.80; HRMS calcd. For C22H21N2O5 [M+H]+ 393.1445, found
393.1441; HPLC (63% methanol and 0.5% TFA in water): tR =
7.598 min, 98.57%.
5.12 (s, 2H); 13C NMR (100 MHz, DMSO)
d 163.16, 162.99,
160.57, 158.56, 146.36, 145.57, 137.07, 136.14, 133.9 (d,
J = 3.0 Hz), 129.98 (d, J = 9.0 Hz),129.96, 123.21, 120.02, 116.61,
115.37, 115.16, 112.24, 107.15, 68.54; HRMS calcd. For C21H18FN2-
O5 ([M+H]+ 397.1194, found 397.1191; HPLC (60% methanol and
0.5% TFA in water): tR = 6.997 min, 97.74%.
4.1.5. 3,4,5-Trihydroxy-N0-(3-((2-(trifluoromethyl)benzyl)oxy)
benzylidene)benzohydrazide (5)
4.1.10. N0-(3-((4-Fluorobenzyl)oxy)benzylidene)-3,4,5-trihydro-
xybenzohydrazide (10)
Yield 43.3%; White solid; mp 206–207 °C; 1H NMR (400 MHz,
DMSO) d 11.54 (s, 1H), 9.11 (s, 3H), 8.37 (s, 1H), 7.79 (m, 2H),
7.72 (t, J = 7.6 Hz, 1H), 7.58 (t, J = 7.6 Hz, 1H), 7.37 (t, J = 7.8 Hz,
1H), 7.29 (m, 2H), 7.05 (d, J = 7.8 Hz, 1H), 6.90 (s, 2H), 5.27 (s,
Yield 36.8%; White solid; mp 202–204 °C; 1H NMR (400 MHz,
DMSO) d 11.54 (s, 1H), 9.03 (s, 3H), 8.36 (s, 1H), 7.57–7.47 (m,
2H), 7.35 (m, 2H), 7.23 (m, 3H), 7.05 (d, J = 8.1 Hz, 1H), 6.91 (s,
2H), 5.13 (s, 2H); 13C NMR (125 MHz, DMSO) d 163.21, 162.79,
160.85, 158.60, 146.42, 145.61, 137.06, 136.16, 133.21(d,
J = 3.0 Hz), 130.03 (d, J = 8.1 Hz), 123.30, 120.07, 116.66, 115.31
(d, J = 21.3 Hz), 112.25, 107.19, 68.56; HRMS calcd. For C21H18FN2-
O5 [M+H]+ 397.1194, found 397.1193; HPLC (60% methanol and
0.5% TFA in water): tR = 6.992 min, 97.39%.
2H); 13C NMR (100 MHz, DMSO)
d 163.20, 158.36, 146.30,
145.57, 137.05, 136.24, 134.80, 132.88, 130.57, 130.10, 128.83,
128.83 (q, J = 30.3 Hz), 126.20 (q, J = 5.4 Hz), 124.35 (q,
J = 272.3 Hz), 123.28, 120.32, 116.41, 112.20, 107.17, 66.28; HRMS
calcd. For C22H18F3N2O5 [M+H]+ 447.1162, found 447.1162; HPLC
(63% methanol and 0.5% TFA in water): tR = 8.722 min, 98.59%.

M. Xi et al. / Bioorg. Med. Chem. 24 (2016) 3218–3230
3225
4.1.11. N0-(3-((2-Chlorobenzyl)oxy)benzylidene)-3,4,5-
trihydroxybenzohydrazide (11)
(d, J = 7.5 Hz, 1H), 7.09 (m, 1H), 6.93 (s, 2H), 5.22 (s, 3H); 13C
NMR (100 MHz, DMSO) d163.27, 158.39, 145.60, 138.77, 137.03,
136.23, 132.48, 131.73, 131.08, 130.06, 129.80, 123.34, 120.29,
118.74, 116.66, 112.27, 111.49, 107.24, 68.12; HRMS calcd. For
Yield 35.2%; White solid; mp 208–210 °C; 1H NMR (400 MHz,
DMSO-d6) d 11.53 (s, 1H), 9.13 (s, 2H), 8.81 (s, 1H), 8.37 (s, 1H),
7.62 (t, J = 4.8 Hz, 1H), 7.51 (t, J = 5.2 Hz, 1H), 7.43–7.36 (m, 3H),
7.34 (d, J = 9.6 Hz, 1H), 7.27 (d, J = 7.7 Hz, 1H), 7.07 (d, J = 8.0 Hz,
1H), 6.90 (s, 2H), 5.19 (s, 2H); 13C NMR (100 MHz, DMSO) d
163.61, 158.94, 146.78, 146.01, 137.44, 136.65, 134.65, 133.10,
130.63, 130.50, 130.38, 129.87, 127.86, 123.75, 120.68, 116.98,
112.65, 107.64, 67.38; HRMS calcd. For C21H18N2 O5Cl [M+H]+
413.0899, found 413.0886; HPLC (63% methanol and 0.5% TFA in
water): tR = 7.883 min, 99.69%.
C
25H26N2O5 [M+H]+ 404.1236, found 404.1241; HPLC (55% metha-
nol and 0.5% TFA in water): tR = 11.906 min, 95.60%.
4.1.17. N0-(3-((4-Cyanobenzyl)oxy)benzylidene)-3,4,5-
trihydroxybenzohydrazide (17)
Yield 43.9%; White solid; mp 243–245 °C; 1H NMR (400 MHz,
DMSO) d 11.53 (s, 1H), 9.13 (s, 2H), 8.81 (s, 1H), 8.36 (s, 1H),
7.86 (d, J = 7.8 Hz, 2H), 7.66 (d, J = 7.9 Hz, 2H), 7.42–7.21 (m, 3H),
7.06 (d, J = 8.1 Hz, 1H), 6.90 (s, 2H), 5.27 (s, 2H); 13C NMR
(100 MHz, DMSO-d6) d 163.61, 158.75, 146.74, 146.00, 143.26,
137.44, 136.65, 132.89, 130.48, 128.53, 123.74, 120.76, 119.23,
117.04, 112.64, 110.95, 107.63, 68.75; HRMS calcd. For
4.1.12. N0-(3-((3-Chlorobenzyl)oxy)benzylidene)-3,4,5-
trihydroxybenzohydrazide (12)
Yield 36.4%; White solid; mp 140–142 °C; 1H NMR (400 MHz,
DMSO) d 11.54 (s, 1H), 9.02 (s, 3H), 8.36 (s, 1H), 7.53 (s, 1H),
7.42 (m, 2H), 7.40–7.29 (m, 3H), 7.26 (d, J = 7.3 Hz, 1H), 7.06 (m,
1H), 6.91 (s, 2H), 5.16 (s, 2H); 13C NMR (100 MHz, DMSO) d
163.17, 158.43, 146.34, 145.57, 139.57, 137.07, 136.18, 133.12,
130.40, 130.00, 127.79, 127.31, 126.19, 123.24, 120.13, 116.60,
112.26, 107.16, 68.32; HRMS calcd. For C21H18ClN2O5 [M+H]+
413.0899, found 413.0898; HPLC (63% methanol and 0.5% TFA in
water): tR = 8.717 min, 97.80%.
C
22H18N3O5 [M+H]+ 404.1241, found 404.1229; HPLC (55% metha-
nol and 0.5% TFA in water): tR = 6.476 min, 99.98%.
4.1.18. 3,4,5-Trihydroxy-N0-(3-(naphthalen-1-ylmethoxy)
benzylidene)benzohydrazide (18)
Yield 41.1%; White solid; mp 128–130 °C; 1H NMR (400 MHz,
DMSO) d 11.55 (s, 1H), 9.15 (s, 2H), 8.83 (s, 1H), 8.39 (s, 1H),
8.11 (d, J = 7.7 Hz, 1H), 7.96 (m, 2H), 7.70 (d, J = 6.7 Hz, 1H), 7.56
(m, 3H), 7.40 (m, 2H), 7.30 (s, 1H), 7.15 (d, J = 7.4 Hz, 1H), 6.92 (s,
2H), 5.60 (s, 2H); 13C NMR (100 MHz, DMSO) d 163.19, 158.76,
146.44, 145.57, 137.00, 136.18, 133.32, 132.40, 131.20, 130.00,
128.78, 128.51, 126.83, 126.52, 126.02, 125.41, 123.97, 123.33,
119.95, 116.59, 112.57, 107.20, 67.94; HRMS calcd. For
4.1.13. N0-(3-((4-Chlorobenzyl)oxy)benzylidene)-3,4,5-
trihydroxybenzohydrazide (13)
Yield 32.6%; White solid; mp 200–202 °C; 1H NMR (400 MHz,
DMSO) d 11.54 (s, 1H), 9.10 (s, 2H), 8.36 (s, 1H), 7.49 (d,
J = 8.5 Hz, 2H), 7.44 (d, J = 8.5 Hz, 2H), 7.34 (m, 2H), 7.25 (d,
J = 7.4 Hz, 1H), 7.05 (d, J = 8.0 Hz, 1H), 6.91 (s, 2H), 5.14 (s, 2H);
13C NMR (100 MHz, DMSO) d 163.26, 158.52, 146.41, 145.59,
137.02, 136.19, 136.07, 132.46, 130.02, 129.55, 128.49, 123.34,
120.13, 116.65, 112.30, 107.23, 68.44; HRMS calcd. For C21H18ClN2-
O5 [M+H]+ 413.0899, found 413.0898; HPLC (63% methanol and
0.5% TFA in water): tR = 8.912 min, 99.88%.
C
25H21N2O6 [M+H]+ 429.1445, found 429.1445; HPLC (63% metha-
nol and 0.5% TFA in water): tR = 10.578 min, 99.33%.
4.1.19. 3,4,5-Trihydroxy-N0-(3-((2-methoxybenzyl)oxy)
benzylidene)benzohydrazide (19)
Yield 39.8%; White solid; mp 163–165 °C; 1H NMR (400 MHz,
DMSO) d 11.52 (s, 1H), 9.13 (s, 2H), 8.81 (s, 1H), 8.36 (s, 1H),
7.41 (d, J = 7.5 Hz, 1H), 7.36 (d, J = 7.9 Hz, 1H), 7.32 (d, J = 7.1 Hz,
2H), 7.23 (d, J = 7.6 Hz, 1H), 7.09–7.00 (m, 1H), 6.96 (t, J = 7.4 Hz,
1H), 6.90 (s, 2H), 5.10 (s, 2H), 3.84 (s, 3H); 13C NMR (100 MHz,
4.1.14. N0-(3-((4-(tert-Butyl)benzyl)oxy)benzylidene)-3,4,5-
trihydroxybenzohydrazide (14)
Yield 35.6%; White solid; mp 126–128 °C; 1H NMR (400 MHz,
DMSO) d 11.53 (s, 1H), 8.98 (s, 3H), 8.36 (s, 1H), 7.44–7.28 (m,
6H), 7.25 (s, 1H), 7.04 (d, J = 7.1 Hz, 1H), 6.91 (s, 2H), 5.09 (s, 2H),
DMSO-d6) d 163.61, 159.22, 157.34, 146.91, 146.01, 137.44,
136.55, 130.41, 129.85, 129.46, 124.95, 123.76, 120.77, 120.49,
117.16, 112.18, 111.35, 107.63, 64.95, 55.95; HRMS calcd. For
1.26 (s, 9H); 13C NMR (100 MHz, DMSO)
d
163.27, 158.77,
C
22H21N2O6 [M+H]+ 409.1394, found 409.1383; HPLC (60% metha-
150.39, 146.47, 145.61, 137.06, 136.16, 133.97, 130.00, 127.66,
125.24, 123.34, 119.94, 116.64, 112.23, 107.21, 69.10, 34.35,
31.18; HRMS calcd. For C25H26N2O5 [M+H]+ 435.1915, found
435.1902; HPLC (70% methanol and 0.5% TFA in water):
tR = 10.454 min, 99.20%.
nol and 0.5% TFA in water): tR = 6.988 min, 97.30%.
4.1.20. 3,4,5-Trihydroxy-N0-(3-((3-methoxybenzyl)oxy)
benzylidene)benzohydrazide (20)
Yield 36.7%; White solid; mp 181–183 °C; 1H NMR (400 MHz,
DMSO) d 11.52 (s, 1H), 9.04 (s, 3H), 8.35 (s, 1H), 7.39–7.21 (m,
4H), 7.09–6.97 (m, 3H), 6.96–6.85 (m, 3H), 5.12 (s, 2H), 3.75 (s,
4.1.15. N0-(3-((2-Cyanobenzyl)oxy)benzylidene)-3,4,5-
trihydroxybenzohydrazide (15)
3H); 13C NMR (100 MHz, DMSO)
d 163.61, 159.79, 159.06,
Yield 37.7%; White solid; mp 239–240 °C; 1H NMR (400 MHz,
DMSO) d 11.54 (s, 1H), 9.07 (s, 3H), 8.37 (s, 1H), 7.90 (d,
J = 7.7 Hz, 1H), 7.75 (d, J = 4.3 Hz, 2H), 7.62–7.51 (m, 1H), 7.45–
7.24 (m, 3H), 7.09 (d, J = 8.1 Hz, 1H), 6.90 (s, 2H), 5.28 (s, 2H);
13C NMR (100 MHz, DMSO) d 163.62, 158.84, 146.72, 146.01,
140.36, 137.45, 136.68, 133.92, 133.79, 130.52, 130.19, 129.62,
123.75, 120.85, 117.70, 116.97, 112.78, 107.64, 68.12; HRMS calcd.
For C22H18N3O5 [M+H]+ 404.1241, found 404.1228; HPLC (55%
methanol and 0.5% TFA in water): tR = 5.328 min, 99.77%.
146.84, 146.01, 138.98, 137.48, 136.56, 130.40, 130.02, 123.73,
120.42, 120.15, 117.07, 113.77, 113.54, 112.70, 107.63, 69.55,
55.52; HRMS calcd. For C22H21N2O6 [M+H]+ 409.1394, found
409.1382; HPLC (60% methanol and 0.5% TFA in water): tR = 6.301
min, 98.67%.
4.1.21. 3,4,5-Trihydroxy-N0-(3-((4-methoxybenzyl)oxy)
benzylidene)benzohydrazide (21)
Yield 36.1%; White solid; mp 127–129 °C; 1H NMR (400 MHz,
DMSO) d 11.52 (s, 1H), 9.12 (s, 3H), 8.35 (s, 1H), 7.42–7.31 (m,
3H), 7.29 (s, 1H), 7.24 (d, J = 7.5 Hz, 1H), 7.03 (m, 1H), 6.96–6.89
(m, 4H), 5.05 (s, 2H), 3.74 (s, 3H); 13C NMR (100 MHz, DMSO) d
163.37, 159.02, 158.71, 146.42, 145.55, 136.97, 136.07, 129.91,
129.51, 128.79, 123.31, 119.80, 116.64, 113.83, 112.32, 107.17,
69.04, 55.10; HRMS calcd. For C22H21N2O6 [M+H]+ 409.1394, found
4.1.16. N0-(3-((3-Cyanobenzyl)oxy)benzylidene)-3,4,5-
trihydroxybenzohydrazide (16)
Yield 39.1%; White solid; mp 168–170 °C; 1H NMR (400 MHz,
DMSO) d 11.49 (s, 1H), 8.95 (s, 3H), 8.37 (s, 1H), 7.93 (s, 1H),
7.81 (t, J = 8.7 Hz, 2H), 7.62 (t, J = 7.8 Hz, 1H), 7.37 (m, 2H), 7.28

3226
M. Xi et al. / Bioorg. Med. Chem. 24 (2016) 3218–3230
409.1381; HPLC (60% methanol and 0.5% TFA in water): tR = 5.915
min, 96.92%.
4.1.27. N0-(4-((3-Cyanobenzyl)oxy)benzylidene)-3,4,5-
trihydroxybenzohydrazide (27)
Yield 38.1%; White solid; mp >250 °C; 1H NMR (400 MHz,
DMSO) d 11.40 (s, 1H), 9.11 (s, 2H), 8.78 (s, 1H), 8.33 (s, 1H),
7.93 (s, 1H), 7.80 (s, 2H), 7.62 (s, 3H), 7.09 (s, 2H), 6.89 (s, 2H),
4.1.22. N0-(4-(Benzyloxy)benzylidene)-3,4,5-trihydroxybenzo-
hydrazide (22)
Yield 34.2%; White solid; mp 214–215 °C; 1H NMR (400 MHz,
DMSO) d 11.38 (s, 1H), 8.94 (s, 3H), 8.33 (s, 1H), 7.61 (d,
J = 8.2 Hz, 2H), 7.45 (d, J = 7.4 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H),
7.36–7.28 (m, 1H), 7.07 (d, J = 8.1 Hz, 2H), 6.89 (s, 2H), 5.14 (s,
5.21 (s, 3H); 13C NMR (100 MHz, DMSO)
d 163.47, 159.78,
146.80, 145.99, 139.02, 137.29, 132.96, 132.19, 131.58, 130.24,
128.96, 128.15, 123.95, 119.13, 115.62, 111.92, 107.55, 68.60;
HRMS calcd. For C22H18O5N3 [M+H]+ 404.1241, found 404.1227;
HPLC (50% methanol and 0.5% TFA in water): tR = 9.628 min,
95.30%.
2H); 13C NMR (100 MHz, DMSO)
d
163.47, 160.15, 146.90,
145.99, 137.28, 137.25, 128.92, 128.38, 128.23, 127.85, 123.98,
115.59, 107.56, 69.80; HRMS calcd. For
21H19N2O5 [M+H]+
C
379.1288, found 379.1277; HPLC (55% methanol and 0.5% TFA in
water): tR = 8.813 min, 99.42%.
4.1.28. N0-(4-((4-Cyanobenzyl)oxy)benzylidene)-3,4,5-
trihydroxybenzohydrazide (28)
Yield 37.5%; White solid; mp >250 °C; 1H NMR (400 MHz,
DMSO) d 11.40 (s, 1H), 9.12 (s, 2H), 8.78 (s, 1H), 8.33 (s, 1H),
7.86 (s, 2H), 7.64 (s, 4H), 7.08 (s, 2H), 6.89 (s, 2H), 5.26 (s, 3H);
13C NMR (100 MHz, DMSO) d 163.48, 159.73, 146.79, 145.99,
143.10, 137.29, 132.91, 128.98, 128.59, 128.17, 123.94, 119.21,
115.62, 111.00, 107.55, 68.82; HRMS calcd. For C22H18O5N3 [M
+H]+ 404.1241, found 404.1226; HPLC (50% methanol and 0.5%
TFA in water): tR = 9.605 min, 95.65%.
4.1.23. 3,4,5-Trihydroxy-N0-(4-((2-(trifluoromethyl)benzyl)oxy)
benzylidene)benzohydrazide (23)
Yield 32.5%; White solid; mp 190–192 °C; 1H NMR (400 MHz,
DMSO) d 11.40 (s, 1H), 9.12 (s, 2H), 8.78 (s, 1H), 8.34 (s, 1H),
7.85–7.68 (m, 3H), 7.68–7.54 (m, 3H), 7.08 (d, J = 8.3 Hz, 2H),
6.90 (s, 2H), 5.27 (s, 2H); 13C NMR (100 MHz, DMSO) d 163.47,
159.79, 146.79, 145.99, 137.29, 135.09, 133.34, 131.07, 129.33,
129.01, 128.25, 127.40 (q, J = 30.7 Hz), 126.66 (q, J = 5.5 Hz),
124.77 (q, J = 275.0 Hz), 123.95, 115.50, 107.55, 66.80; HRMS calcd.
For C22H18O5N2F3 [M+H]+ 447.1162, found 447.1147; HPLC (60%
methanol and 0.5% TFA in water): tR = 11.426 min, 99.82%.
4.1.29. 3,4,5-Trihydroxy-N0-(4-((4-methylbenzyl)oxy)
benzylidene)benzohydrazide (29)
Yield 32.7%; White solid; mp 243–245 °C; 1H NMR (400 MHz,
DMSO) d 11.38 (s, 1H), 9.11 (s, 2H), 8.77 (s, 1H), 8.32 (s, 1H),
7.60 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 7.6 Hz, 2H), 7.19 (d, J = 7.6 Hz,
2H), 7.05 (d, J = 8.3 Hz, 2H), 6.89 (s, 2H), 5.09 (s, 2H), 2.29 (s,
4.1.24. 3,4,5-Trihydroxy-N0-(4-((3-(trifluoromethyl)benzyl)oxy)
benzylidene)benzohydrazide (24)
Yield 32.0%; White solid; mp 188–190 °C; 1H NMR (400 MHz,
DMSO) d 11.40 (s, 1H), 9.11 (s, 2H), 8.80 (s, 1H), 8.34 (s, 1H),
7.82 (s, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.63
(d, J = 7.8 Hz, 3H), 7.10 (d, J = 8.3 Hz, 2H), 6.89 (s, 2H), 5.26 (s,
3H); 13C NMR (100 MHz, DMSO)
d 163.44, 160.19, 146.91,
145.99, 137.63, 137.27, 134.20, 129.46, 128.91, 128.33, 127.78,
123.98, 115.60, 107.54, 69.71, 21.25; HRMS calcd. For
C
22H21O5N2 [M+H]+ 393.1445, found 393.1431; HPLC (60% metha-
2H); 13C NMR (100 MHz, DMSO)
d
163.46, 159.87, 146.79,
145.99, 138.82, 137.28, 132.20, 130.12, 129.65 (q, J = 31.8 Hz),
128.96, 128.11, 125.09 (q, J = 4.0 Hz), 124.64 (q,
J = 273.1 Hz),124.57 (q, J = 3.8 Hz), 123.96, 115.61, 107.55, 68.89;
HRMS calcd. For
22H18O5N2F3 [M+H]+ 447.1162, found
nol and 0.5% TFA in water): tR = 8.514 min, 98.09%.
4.1.30. N0-(4-((4-(tert-Butyl)benzyl)oxy)benzylidene)-3,4,5-
trihydroxybenzohydrazide (30)
C
Yield 38.4%; White solid; mp 232–234 °C; 1H NMR (400 MHz,
DMSO) d 11.39 (s, 1H), 9.11 (s, 2H), 8.78 (s, 1H), 8.33 (s, 1H),
7.61 (d, J = 8.2 Hz, 2H), 7.38 (d, J = 6.3 Hz, 4H), 7.06 (d, J = 8.2 Hz,
2H), 6.89 (s, 2H), 5.09 (s, 2H), 1.26 (s, 9H); 13C NMR (100 MHz,
DMSO) d 163.45, 160.22, 150.86, 146.91, 145.98, 137.27, 134.23,
128.93, 128.14, 127.77, 125.66, 123.98, 115.55, 107.54, 69.61,
34.77, 31.59; HRMS calcd. For C25H27O5N2 [M+H]+ 435.1914, found
435.1899; HPLC (70% methanol and 0.5% TFA in water): tR = 9.100
min, 98.31%.
447.1148; HPLC (45% acetonitrile and 0.5% TFA in water):
tR = 7.386 min, 99.86%.
4.1.25. 3,4,5-Trihydroxy-N0-(4-((4-(trifluoromethyl)benzyl)oxy)
benzylidene)benzohydrazide (25)
Yield 38.8%; White solid; mp >250 °C; 1H NMR (400 MHz,
DMSO) d 11.40 (s, 1H), 9.11 (s, 2H), 8.78 (s, 1H), 8.33 (s, 1H),
7.76 (d, J = 7.9 Hz, 2H), 7.67 (d, J = 8.2 Hz, 2H), 7.63 (d, J = 8.2 Hz,
2H), 7.09 (d, J = 8.3 Hz, 2H), 6.89 (s, 2H), 5.27 (s, 2H). 13C NMR
(100 MHz, DMSO)
d
163.47, 159.82, 146.79, 145.99, 142.19,
4.1.31. 3,4,5-Trihydroxy-N0-(4-(naphthalen-2-ylmethoxy)
benzylidene)benzohydrazide (31)
137.29, 128.97, 128.82 (q, J = 31.8 Hz), 128.52, 128.12, 125.83 (q,
J = 3.8 Hz), 124.64 (q, J = 273.1 Hz), 123.95, 115.62, 107.55, 68.87;
Yield 39.4%; White solid; mp 246–248 °C; 1H NMR (400 MHz,
DMSO) d 11.39 (s, 1H), 9.11 (s, 2H), 8.78 (s, 1H), 8.33 (s, 1H),
7.99 (s, 1H), 7.97–7.89 (m, 3H), 7.63 (d, J = 8.3 Hz, 2H), 7.58 (d,
J = 8.5 Hz, 1H), 7.56–7.46 (m, 2H), 7.13 (d, J = 8.3 Hz, 2H), 6.89 (s,
2H), 5.32 (s, 2H); 13C NMR (100 MHz, DMSO) d 163.46, 160.17,
146.88, 145.99, 137.28, 134.89, 133.23, 133.03, 132.29, 128.96,
128.57, 128.27, 128.08, 127.92, 126.82, 126.64, 126.18, 123.98,
HRMS calcd. For
C
22H18O5N2F3 [M+H]+ 447.1162, found
447.1148; HPLC (60% methanol and 0.5% TFA in water):
tR = 12.108 min, 99.97%.
4.1.26. N0-(4-((2-Cyanobenzyl)oxy)benzylidene)-3,4,5-
trihydroxybenzohydrazide (26)
Yield 33.3%; White solid; mp >250 °C; 1H NMR (400 MHz,
DMSO) d 11.41 (s, 1H), 9.12 (s, 2H), 8.78 (s, 1H), 8.35 (s, 1H),
7.91 (s, 1H), 7.74 (s, 2H), 7.65 (s, 2H), 7.58 (s, 1H), 7.12 (s, 2H),
6.90 (s, 2H), 5.29 (s, 2H); 13C NMR (100 MHz, DMSO) d 163.48,
159.80, 146.76, 145.99, 140.20, 137.29, 133.95, 133.81, 130.21,
129.67, 128.98, 128.36, 123.95, 117.66, 115.60, 111.83, 107.56,
68.13; HRMS calcd. For C22H18O5N3 [M+H]+ 404.1241, found
404.1227; HPLC (50% methanol and 0.5% TFA in water): tR = 8.778
min, 95.94%.
115.68, 107.56, 69.93; HRMS calcd. For C
25H21O5N2 [M+H]+
429.1445, found 429.1431; HPLC (60% methanol and 0.5% TFA in
water): tR = 13.448 min, 99.89%.
4.1.32. 3,4,5-Trihydroxy-N0-(4-((2-methoxybenzyl)oxy)
benzylidene)benzohydrazide (32)
Yield 35.4%; White solid; mp 238–240 °C; 1H NMR (400 MHz,
DMSO) d 11.38 (s, 1H), 9.11 (s, 2H), 8.78 (s, 1H), 8.33 (s, 1H),
7.61 (s, 2H), 7.36 (d, J = 24.5 Hz, 2H), 7.05 (s, 3H), 6.95 (s, 1H),
6.89 (s, 2H), 5.09 (s, 2H), 3.82 (s, 4H); 13C NMR (100 MHz, DMSO)

M. Xi et al. / Bioorg. Med. Chem. 24 (2016) 3218–3230
3227
d 163.45, 160.32, 157.43, 146.91, 145.99, 137.26, 129.99, 129.70,
4.1.38. 3-Hydroxy-N0-(3-((3-methoxybenzyl)oxy)benzylidene)
128.95, 127.75, 124.78, 123.99, 120.77, 115.43, 111.39, 107.54,
65.19, 55.92; HRMS calcd. For C22H21O6N2 [M+H]+ 409.1394, found
409.1374; HPLC (60% methanol and 0.5% TFA in water): tR = 6.318
min, 95.82%.
benzohydrazide (38)
Yield 66.1%; White solid; mp 183–185 °C; 1H NMR (400 MHz,
DMSO) d 11.76 (s, 1H), 9.74 (s, 1H), 8.40 (s, 1H), 7.43–7.23 (m,
7H), 7.08 (s, 1H), 7.03 (d, J = 4.8 Hz, 2H), 6.96 (d, J = 7.1 Hz, 1H),
6.88 (d, J = 8.3 Hz, 1H), 5.12 (s, 2H), 3.75 (s, 3H); 13C NMR
4.1.33. 3,4,5-Trihydroxy-N0-(4-((3-methoxybenzyl)oxy)
benzylidene)benzohydrazide (33)
(100 MHz, DMSO) d 163.87, 160.03, 159.31, 158.11, 148.18,
139.17, 136.53, 135.49, 130.66, 130.24, 130.20, 120.81, 120.38,
119.40, 118.80, 117.55, 115.23, 113.99, 113.78, 113.09, 69.81,
55.73; HRMS calcd. For C22H21N2O4 [M+H]+ 377.1423, found
377.1491; HPLC (65% methanol and 0.5% TFA in water): tR = 7.558
min, 99.86%.
Yield 33.6%; White solid; mp 202–204 °C; 1H NMR (400 MHz,
DMSO) d 11.39 (s, 1H), 9.11 (s, 2H), 8.78 (s, 1H), 8.33 (s, 1H),
7.61 (d, J = 8.3 Hz, 2H), 7.30 (t, J = 8.0 Hz, 1H), 7.07 (d, J = 8.4 Hz,
2H), 7.02 (s, 2H), 6.89 (s, 3H), 5.12 (s, 2H), 3.75 (s, 3H); 13C NMR
(100 MHz, DMSO)
d 163.44, 160.11, 159.80, 146.88, 145.98,
4.1.39. 4-Hydroxy-N0-(3-((3-methoxybenzyl)oxy)benzylidene)
benzohydrazide (39)
138.83, 137.27, 130.05, 128.92, 127.86, 123.97, 120.24, 115.60,
113.80, 113.66, 107.54, 69.65, 55.52; HRMS calcd. For
C
22H21O6N2 [M+H]+ 409.1394, found 409.1379; HPLC (58% metha-
Yield 45.7%; White solid; mp 174–176 °C; 1H NMR (400 MHz,
DMSO) d 11.62 (s, 1H), 10.09 (s, 1H), 8.38 (s, 1H), 7.79 (d,
J = 8.2 Hz, 2H), 7.40–7.20 (m, 4H), 7.06 (d, J = 8.2 Hz, 1H), 7.02 (d,
J = 4.9 Hz, 2H), 6.89 (s, 1H), 6.84 (d, J = 8.2 Hz, 2H), 5.12 (s, 2H),
nol and 0.5% TFA in water): tR = 6.446 min, 99.60%.
4.1.34. 3,4,5-Trihydroxy-N0-(4-((4-methoxybenzyl)oxy)
benzylidene)benzohydrazide (34)
3.75 (s, 3H); 13C NMR (100 MHz, DMSO)
d 163.23, 161.16,
Yield 38.7%; White solid; mp196–198 °C; 1H NMR (400 MHz,
DMSO) d 11.38 (s, 1H), 9.11 (s, 2H), 8.78 (s, 1H), 8.33 (s, 1H),
7.60 (d, J = 8.3 Hz, 2H), 7.38 (d, J = 8.2 Hz, 2H), 7.05 (d, J = 8.3 Hz,
2H), 6.94 (d, J = 8.3 Hz, 2H), 6.89 (s, 2H), 5.05 (s, 2H), 3.74 (s,
159.80, 159.07, 147.12, 138.96, 136.46, 130.42, 130.16, 130.02,
124.30, 120.45, 120.16, 117.12, 115.47, 113.76, 113.56, 112.78,
69.56, 55.51; HRMS calcd. For C22H21O4N2 [M+H]+ 377.1496, found
377.1481; HPLC (63% methanol and 0.5% TFA in water): tR = 8.086
min, 99.24%.
3H); 13C NMR (100 MHz, DMSO)
d 163.45, 160.23, 159.52,
146.94, 145.99, 137.27, 130.08, 129.08, 128.90, 127.72, 123.99,
115.59, 114.29, 107.55, 69.59, 55.56; HRMS calcd. For
4.1.40. 3,5-Dihydroxy-N0-(4-((3-methoxybenzyl)oxy)
benzylidene)benzohydrazide (40)
C
22H21O6N2 [M+H]+ 409.1394, found 409.1378; HPLC (55% metha-
nol and 0.5% TFA in water): tR = 7.797 min, 99.99%.
Yield 59.6%; White solid; mp 214–216 °C; 1H NMR (400 MHz,
DMSO) d 11.76 (s, 1H), 9.74 (s, 1H), 8.40 (s, 1H), 7.43–7.19 (m,
7H), 7.07 (d, J = 8.5 Hz, 1H), 7.03 (d, J = 4.6 Hz, 2H), 6.96 (d,
J = 7.1 Hz, 1H), 6.88 (d, J = 8.3 Hz, 1H), 5.12 (s, 2H), 3.75 (s, 3H);
13C NMR (100 MHz, DMSO) d 163.85, 160.49, 160.03, 159.06,
147.99, 139.03, 136.32, 130.26, 129.29, 127.90, 120.46, 115.84,
114.02, 113.88, 106.41, 106.20, 69.89, 55.73; HRMS calcd. For
4.1.35. N0-(3-(Benzyloxy)benzylidene)-1H-benzo[d]imidazole-5-
carbohydrazide (35)
Yield 31.6%; White solid; mp 222–224 °C; 1H NMR (400 MHz,
DMSO) d 12.74 (s, 1H), 11.86 (s, 1H), 8.44 (s, 1H), 8.36 (s, 1H),
8.23 (s, 1H), 7.80 (d, J = 7.9 Hz, 1H), 7.67 (d, J = 8.2 Hz, 1H), 7.47
(m, 2H), 7.36 (m, 5H), 7.08 (m, 1H), 5.15 (s, 2H); 13C NMR
C
22H21N2O5 [M+H]+ 393.1372, found 393.1459; HPLC (62% metha-
(100 MHz, DMSO)
136.00, 129.99, 128.45, 127.87, 127.71, 126.93, 121.84, 120.01,
116.72, 112.42, 69.26; HRMS calcd. For
22H19N4O2 [M+H]+
d 163.56, 158.68, 147.00, 144.18, 136.94,
nol and 0.5% TFA in water): tR = 9.417 min, 98.41%.
C
4.1.41. 3,4-Dihydroxy-N0-(4-((3-methoxybenzyl)oxy)
benzylidene)benzohydrazide (41)
371.1503, found 371.1496; HPLC (35% acetonitrile and 0.5% TFA
in water): tR = 7.777 min, 99.77%.
Yield 61.5%; White solid; mp 198–200 °C; 1H NMR (400 MHz,
DMSO) d 11.42 (s, 1H), 9.57 (s, 1H), 9.19 (s, 1H), 8.34 (s, 1H),
7.62 (d, J = 8.3 Hz, 2H), 7.33 (s, 1H), 7.29 (d, J = 8.1 Hz, 1H), 7.26
(d, J = 8.4 Hz, 1H), 7.07 (d, J = 8.4 Hz, 2H), 7.01 (d, J = 4.8 Hz, 2H),
6.89 (d, J = 8.4 Hz, 1H), 6.79 (d, J = 8.2 Hz, 1H), 5.12 (s, 2H), 3.75
(s, 3H); 13C NMR (100 MHz, DMSO) d 163.42, 160.36, 160.02,
149.57, 147.21, 145.70, 139.04, 130.26, 129.16, 128.04, 125.18,
120.45, 120.15, 116.04, 115.83, 115.63, 114.01, 113.87, 69.88,
55.73; HRMS calcd. For C22H21N2O5 [M+H]+ 393.1372, found
393.1445; HPLC (57% methanol and 0.5% TFA in water):
tR = 10.171 min, 99.17%.
4.1.36. 3,5-Dihydroxy-N0-(3-((3-methoxybenzyl)oxy)
benzylidene)benzohydrazide (36)
Yield 62.3%; White solid; mp 103–105 °C; 1H NMR (400 MHz,
DMSO) d 11.72 (s, 1H), 9.59 (s, 2H), 8.40 (s, 1H), 7.35 (s, 1H),
7.27 (d, J = 7.9 Hz, 2H), 7.04 (q, J = 6.9, 5.5 Hz, 3H), 6.87 (d,
J = 8.2 Hz, 1H), 6.77 (s, 2H), 6.45 (s, 1H), 5.10 (s, 2H), 3.74 (s, 3H);
13C NMR (100 MHz, DMSO) d 164.09, 160.02, 159.29, 159.11,
148.06, 139.17, 136.57, 136.12, 130.64, 130.24, 120.83, 120.37,
117.53, 113.98, 113.76, 113.01, 109.99, 106.50, 69.79, 55.70; HRMS
calcd. For C22H21N2O5 [M+H]+ 393.1372, found 393.1450; HPLC
(57% methanol and 0.5% TFA in water): tR = 11.483 min, 99.75%.
4.1.42. 3-Hydroxy-N0-(4-((3-methoxybenzyl)oxy)benzylidene)
benzohydrazide (42)
4.1.37. 3,4-Dihydroxy-N0-(3-((3-methoxybenzyl)oxy)
benzylidene)benzohydrazide (37)
Yield 67.2%; White solid; mp 174–176 °C; 1H NMR (400 MHz,
DMSO) d 11.61 (s, 1H), 9.71 (s, 1H), 8.36 (s, 1H), 7.64 (d,
J = 8.3 Hz, 2H), 7.31 (d, J = 7.8 Hz, 3H), 7.27 (d, J = 8.6 Hz, 3H),
7.08 (d, J = 8.4 Hz, 2H), 7.01 (d, J = 4.7 Hz, 2H), 6.95 (d, J = 6.6 Hz,
1H), 6.89 (d, J = 8.4 Hz, 1H), 5.12 (s, 2H), 3.75 (s, 3H); 13C NMR
Yield 62.2%; White solid; mp 170–172 °C; 1H NMR (400 MHz,
DMSO) d 11.56 (s, 1H), 9.60 (s, 1H), 9.21 (s, 1H), 8.37 (s, 1H),
7.40–7.33 (m, 2H), 7.33–7.20 (m, 4H), 7.09–6.99 (m, 3H), 6.88
(dd, J = 8.3, 2.4 Hz, 1H), 6.80 (d, J = 8.2 Hz, 1H), 5.12 (s, 2H), 3.75
(s, 3H); 13C NMR (100 MHz, DMSO) d 163.66, 160.03, 159.30,
149.76, 147.24, 145.75, 139.19, 136.74, 130.63, 130.25, 124.99,
120.70, 120.38, 117.34, 116.14, 115.68, 113.99, 113.78, 112.95,
69.79, 55.73; HRMS calcd. For C22H21N2O5 [M+H]+ 393.1372, found
393.1436; HPLC (57% methanol and 0.5% TFA in water):
tR = 12.434 min, 99.23%.
(100 MHz, DMSO)
d 163.69, 160.53, 160.03, 158.09, 148.18,
139.01, 135.67, 130.26, 130.16, 129.34, 127.85, 120.45, 119.26,
118.74, 115.85, 115.19, 114.01, 113.88, 69.89, 55.72; HRMS calcd.
For C22H21N2O4 [M+H]+ 377.1423, found 377.1497; HPLC (55%
methanol and 0.5% TFA in water): tR = 14.280 min, 98.29%.

3228
M. Xi et al. / Bioorg. Med. Chem. 24 (2016) 3218–3230
4.1.43. 4-Hydroxy-N0-(4-((3-methoxybenzyl)oxy)benzylidene)
benzohydrazide (43)
415.1263; HPLC (65% methanol and 0.5% TFA in water):
tR = 12.879 min, 97.38%.
Yield 50.2%; White solid; mp 188–190 °C; 1H NMR (400 MHz,
DMSO) d 11.49 (s, 1H), 10.07 (s, 1H), 8.35 (s, 1H), 7.78 (d,
J = 8.3 Hz, 2H), 7.63 (d, J = 8.2 Hz, 2H), 7.29 (t, J = 8.0 Hz, 1H), 7.07
(d, J = 8.4 Hz, 2H), 7.01 (d, J = 4.9 Hz, 2H), 6.89 (s, 1H), 6.84 (d,
J = 8.2 Hz, 2H), 5.12 (s, 2H), 3.74 (s, 3H); 13C NMR (100 MHz, DMSO)
d 163.04, 161.04, 160.17, 159.80, 147.13, 138.82, 130.05, 128.98,
127.77, 124.48, 120.24, 115.62, 115.44, 113.80, 113.66, 69.65,
55.52; HRMS calcd. For C22H21O4N2 [M+H]+ 377.1496, found
377.1478; HPLC (43% acetonitrile and 0.5% TFA in water):
tR = 8.276 min, 99.87%.
4.1.48. 3,5-Dihydroxy-N0-(4-((2-(trifluoromethyl)benzyl)oxy)
benzylidene)benzohydrazide (48)
Yield 64.0%; White solid; mp 218–220 °C; 1H NMR (400 MHz,
DMSO) d 11.54 (s, 1H), 9.53 (s, 2H), 8.35 (s, 1H), 7.83–7.75 (m,
2H), 7.72 (t, J = 7.6 Hz, 1H), 7.65 (d, J = 8.3 Hz, 2H), 7.59 (t,
J = 7.6 Hz, 1H), 7.08 (d, J = 8.4 Hz, 2H), 6.71 (s, 2H), 6.39 (s, 1H),
5.27 (s, 2H); 13C NMR (100 MHz, DMSO)
d 163.90, 160.17,
159.07, 147.91, 136.30, 135.28, 133.50, 131.24, 129.50, 129.38,
128.29, 127.63 (d, J = 30.7 Hz), 126.85 (q, J = 5.5 Hz), 124.97 (d,
J = 274.2 Hz), 115.73, 106.43, 106.24, 67.02; HRMS calcd. For
4.1.44. 3,5-Dihydroxy-N0-(3-((2-(trifluoromethyl)benzyl)oxy)
benzylidene)benzohydrazide (44)
C
22H18F3N2O4 [M+H]+ 431.1140, found 431.1203; HPLC (62%
methanol and 0.5% TFA in water): tR = 11.616 min, 99.98%.
Yield 61.5%; White solid; mp 239–241 °C; 1H NMR (400 MHz,
DMSO) d 11.68 (s, 1H), 9.55 (s, 2H), 8.39 (s, 1H), 7.83–7.76 (m,
2H), 7.72 (t, J = 7.6 Hz, 1H), 7.58 (t, J = 7.7 Hz, 1H), 7.38 (t,
J = 7.9 Hz, 1H), 7.35–7.25 (m, 2H), 7.07 (d, J = 8.2 Hz, 1H), 6.72 (s,
2H), 6.41 (s, 1H), 5.27 (s, 2H); 13C NMR (100 MHz, DMSO) d
164.08, 159.11, 159.04, 147.90, 136.71, 136.11, 135.44, 133.48,
131.19, 130.77, 129.44, 127.62 (d, J = 30.4 Hz), 126.83 (q,
J = 5.6 Hz), 125.00 (d, J = 274.1 Hz), 121.14, 117.30, 112.95,
106.49, 106.37, 66.96; HRMS calcd. For C22H18F3N2O4 [M+H]+
431.1140, found 431.1191; HPLC (62% methanol and 0.5% TFA in
water): tR = 12.727 min, 96.04%.
4.1.49. 3,4-Dihydroxy-N0-(4-((2-(trifluoromethyl)benzyl)oxy)
benzylidene)benzohydrazide (49)
Yield 63.4%; White solid; mp 223–225 °C; 1H NMR (400 MHz,
DMSO) d 11.45 (s, 1H), 9.58 (s, 1H), 9.20 (s, 1H), 8.35 (s, 1H),
7.83–7.75 (m, 2H), 7.72 (t, J = 7.6 Hz, 1H), 7.65 (d, J = 8.2 Hz, 2H),
7.58 (t, J = 7.7 Hz, 1H), 7.34 (s, 1H), 7.27 (d, J = 8.2 Hz, 1H), 7.08
(d, J = 8.3 Hz, 2H), 6.80 (d, J = 8.2 Hz, 1H), 5.27 (s, 2H); 13C NMR
(100 MHz, DMSO)
d 166.85, 163.48, 160.05, 149.61, 147.12,
145.71, 135.31, 133.52, 131.25, 129.52, 129.26, 128.44, 127.62 (d,
J = 30.3 Hz), 126.88, 125.17, 124.99 (d, J = 272.2 Hz), 120.17,
116.06, 115.72. 67.05; HRMS calcd. For C22H18F3N2O4 [M+H]+
431.1140, found 431.1214; HPLC (62% methanol and 0.5% TFA in
water): tR = 12.624 min, 97.35%.
4.1.45. 3,4-Dihydroxy-N0-(3-((2-(trifluoromethyl)benzyl)oxy)
benzylidene)benzohydrazide (45)
Yield 62.1%; White solid; mp 193–195 °C; 1H NMR (400 MHz,
DMSO) d 11.57 (s, 1H), 9.60 (s, 1H), 9.21 (s, 1H), 8.38 (s, 1H),
7.79 (t, J = 6.3 Hz, 2H), 7.72 (t, J = 7.6 Hz, 1H), 7.58 (t, J = 7.7 Hz,
1H), 7.43–7.21 (m, 5H), 7.06 (d, J = 8.5 Hz, 1H), 6.80 (d, J = 8.2 Hz,
1H), 5.27 (s, 2H); 13C NMR (100 MHz, DMSO) d 163.63, 159.03,
149.76, 147.09, 145.74, 136.86, 135.45, 133.49, 131.19, 130.76,
129.45, 127.60 (d, J = 30.3 Hz), 126.83 (q, J = 5.5 Hz), 125.00 (d,
J = 272.2 Hz), 124.97, 121.01, 120.30, 117.11, 116.12, 115.67,
112.89, 67.13; HRMS calcd. For C22H18F3N2O4 [M+H]+ 431.1140,
found 431.1218; HPLC (62% methanol and 0.5% TFA in water):
tR = 13.959 min, 96.97%.
4.1.50. 3-Hydroxy-N0-(4-((2-(trifluoromethyl)benzyl)oxy)
benzylidene)benzohydrazide (50)
Yield 65.6%; White solid; mp 200–202 °C; 1H NMR (400 MHz,
DMSO) d 11.63 (s, 1H), 9.71 (s, 1H), 8.37 (s, 1H), 7.84–7.76 (m,
2H), 7.75–7.69 (m, 1H), 7.67 (d, J = 8.2 Hz, 2H), 7.59 (t, J = 7.6 Hz,
1H), 7.30 (s, 1H), 7.28 (d, J = 11.0 Hz, 2H), 7.09 (d, J = 8.3 Hz, 2H),
6.95 (s, 1H), 5.28 (s, 2H); 13C NMR (100 MHz, DMSO) d 163.72,
160.21, 158.09, 148.09, 135.64, 135.27, 133.51, 131.24, 130.16,
129.51, 129.42, 128.23, 127.63 (q, J = 30.3 Hz), 126.85 (q,
J = 5.5 Hz), 124.98 (d, J = 273.8 Hz), 119.27, 118.73, 115.74,
115.19, 67.03; HRMS calcd. For C22H18F3N2O3 [M+H]+ 415.1191,
found 415.1245; HPLC (65% methanol and 0.5% TFA in water):
tR = 11.832 min, 99.86%.
4.1.46. 3-Hydroxy-N0-(3-((2-(trifluoromethyl)benzyl)oxy)
benzylidene)benzohydrazide (46)
Yield 64.6%; White solid; mp 175–177 °C; 1H NMR (400 MHz,
DMSO) d 11.76 (s, 1H), 9.74 (s, 1H), 8.41 (s, 1H), 7.79 (t,
J = 5.9 Hz, 2H), 7.72 (t, J = 7.6 Hz, 1H), 7.59 (t, J = 7.7 Hz, 1H), 7.39
(t, J = 7.8 Hz, 1H), 7.37–7.25 (m, 5H), 7.08 (d, J = 8.1 Hz, 1H), 6.96
(d, J = 6.9 Hz, 1H), 5.28 (s, 2H); 13C NMR (100 MHz, DMSO) d
163.90, 159.05, 158.12, 148.07, 136.67, 135.47, 133.50, 131.19,
130.79, 130.21, 129.46, 129.09, 127.61 (q, J = 30.3 Hz), 126.84 (d,
J = 6.0 Hz), 125.01 (d, J = 273.8 Hz), 121.16, 119.42, 118.80,
117.35, 115.23, 113.01, 66.96; HRMS calcd. For C22H18F3N2O3 [M
+H]+ 415.1191, found 415.1258; HPLC (65% methanol and 0.5%
TFA in water): tR = 13.108 min, 96.54%.
4.1.51. 4-Hydroxy-N0-(4-((2-(trifluoromethyl)benzyl)oxy)
benzylidene)benzohydrazide (51)
Yield 40.7%; White solid; mp 191–193 °C; 1H NMR (400 MHz,
DMSO) d 11.50 (s, 1H), 10.07 (s, 1H), 8.36 (s, 1H), 7.79 (t,
J = 7.9 Hz, 4H), 7.72 (t, J = 7.6 Hz, 1H), 7.66 (d, J = 8.1 Hz, 2H), 7.59
(t, J = 7.7 Hz, 1H), 7.08 (d, J = 8.3 Hz, 2H), 6.84 (d, J = 8.2 Hz, 2H),
5.27 (s, 2H); 13C NMR (100 MHz, DMSO)
d 163.05, 161.04,
159.85, 147.03, 135.08, 133.33, 131.06, 130.05, 129.32, 129.06,
128.15, 127.41 (q, J = 30.5 Hz), 126.65 (q, J = 5.6 Hz), 124.77 (q,
J = 274.7 Hz), 124.46, 115.51, 115.44, 66.80; HRMS calcd. For C22
-
H
18O3N2F3 [M+H]+ 415.1264, found 415.1249; HPLC (65% metha-
4.1.47. 4-Hydroxy-N0-(3-((2-(trifluoromethyl)benzyl)oxy)
benzylidene)benzohydrazide (47)
nol and 0.5% TFA in water): tR = 10.552 min, 99.26%.
1H and 13CNMR spectral data of representative compounds for
characterization were presented in the supporting information.
Yield 55.67%; White solid; mp 175–177 °C; 1H NMR (400 MHz,
DMSO) d 11.63 (s, 1H), 10.10 (s, 1H), 8.39 (s, 1H), 7.79 (d, J = 8.3 Hz,
4H), 7.72 (t, J = 7.6 Hz, 1H), 7.58 (t, J = 7.7 Hz, 1H), 7.38 (t, J = 7.9 Hz,
1H), 7.31 (d, J = 11.0 Hz, 2H), 7.06 (d, J = 8.1 Hz, 1H), 6.84 (d,
J = 8.2 Hz, 2H), 5.27 (s, 2H); 13C NMR (100 MHz, DMSO) d 163.50,
161.41, 159.04, 147.24, 136.81, 135.45, 133.49, 131.16, 130.76,
130.38, 129.44, 127.59 (q, J = 30.2 Hz), 126.82 (q, J = 5.5 Hz),
125.00 (q, J = 272.2 Hz), 124.51, 121.03, 117.13, 115.70, 112.91,
66.93; HRMS calcd. For C22H18F3N2O3 [M+H]+ 415.1191, found
4.2. Biology
4.2.1. Enzyme-based screening
Human recombinant SphK1 (PV5214, 0.37 mg/mL), SphK2
(PV5216, 0.39 mg/mL), Sph (PV5372, 400 nmol), ATP (10 mM)
were supplied from Life technologies (NY, USA). ADP QuestTM Assay
Kit were provided by DiscoveRx (Fremont, CA, USA). A typical reac-

M. Xi et al. / Bioorg. Med. Chem. 24 (2016) 3218–3230
3229
tion contained 5
l
L compound solution, 5
l
L sph, 5
l
L ATP and
inflammation was assigned a value of 0, sligt inflammation was
assigned a value of 1, moderate inflammation was assigned a value
of 2, and severe inflammation was assigned a value of 3; (2) for
extent of inflammation, normal colonic mucosa was scored as 0,
mucosal lesion was scored as 1; mucosal and submucosal lesion
was scored as 2; transmural lesion was scored as 3; (3) for crypt
damage, no crypt damage was assigned as 0, 1/3 of crypt damage
was assigned as 1, 2/3 of crypt damage was assigned as 2, complete
loss of crypts and normal surface epithelium were assigned as 3,
complete loss of crypt and surface epithelium were assigned as
4; (4) for percent involvement, the proportion of involvement
being 0% was scored as 0, 1–25% was scored as 1, 26–50% was
scored as 2, 51–75% was scored as 3, 76–100% was scored as 4.
The score of percent involvement (0–4) was multiplied by the
sum of former three parameters (0–10) to yield the final score.
5
l
L SphK. The mixture was incubated at 28 °C for 1 h with a
10 min preincubation of SphK and compounds. All reactions were
proceeded by the addition of ADP QuestTM reagent A and B, then
incubated at room temperature for 30 min. For fluorescence inten-
sity measurement, the PerkinElmer envision enspire multimode
reader (Waltham, USA) was used at 530 nm excitation and
590 nm emission.
4.2.2. Cell culture conditions
HCT116 (China Infrastructure of Cell Line Resources, Beijing,
China) were maintained in IMDM medium (GiBco, Invitrogen
Corp., USA) with 10% fetal bovine serum (FBS) (GiBco, Invitrogen
Corp., USA) in a humidified atmosphere of 5% CO2 and 95% air at
37 °C.
4.2.3. Western blot
4.2.6. Immunohistochemistry
Anti-SphK2 (sc-22704, goat, polyclonal, 1:500) were from Santa
Cruz Biotechnology (CA, USA). Anti-SphK1 (#3297, rabbit, poly-
clonal, 1:1000) and b-actin (#4970S, rabbit, monoclonal, 1:1000)
were purchased from cell signaling technology (Boston, USA).
HCT116 (5 ꢀ 106 cells/well) were washed once with cold PBS and
driven down with 1 mL of trypsin. Cells were centrifuged at
1000 rpm and resuspended in NP-40 lysis buffer (Beyotime, NT,
China) and 1 mM PMSF for 1 h on ice. Then cells were centrifuged
again at 12000 rpm for 15 min at 4 °C. The protein concentration
was determined by the BCA assay at 562 nm. Samples were stored
at ꢁ80 °C. The extracts were separated by SDS–PAGE (12% gel) and
transferred to a PVDF membrane (Perkin-Elmer, Northwalk, CT,
USA) followed by blocking with 1% BSA, detecting via a primary
antibody and an anti-rabbit HRP-labeled secondary antibody using
the UVP EC3 Imaging System (UVP, USA). The intensity data was
analyzed and quantified to the comparable value according to
the protocol of VisionWorks LS software (The imaging system itself
contains this software).
Tissues were prepared from formalin fixed, paraffin-embedded
colon tissue. Stains against IL-6 (ab6672, rabbit, polyclonal, 1:500)
and COX-2 (sc-1746, goat, polyclonal, 1:200) were performed
according to the kit protocol (KeyGEN, NanJing, China). Briefly,
the slides were deparaffinized. Antigen unmasking was carried
out by incubation in 100 °C water bath in 10 mM sodium citrate
buffer with 0.1% Tween 20 for 20 min. Slides were incubated with
primary antibodies in PBS containing 5% BSA and 10% goat serum.
HRP Polymer were added and incubated at room temperature for
30 min. And the sections were stained with DAB substrate and
counterstained with hematoxylin after 40 min. Image Pro Plus
6.0 software (Media Cybernetics, Silver Spring, USA) was used for
automatic counting of labeled inflammatory cytokines to quantify
the experimental data.
4.2.7. Statistical analysis
All results are shown as mean SD. Each study was of 3 sepa-
rate experiments and performed in parallel experiments for tripli-
cate unless otherwise indicated. P-Values less than 0.05 (p < 0.05)
were considered statistically significant. They were undertaken
using the GraphPad Prism software (GraphPad Software Inc., Ave-
nida, CA).
4.2.4. DSS mouse models
Animal studies were conducted according to protocols
approved by Institutional Animal Care and Use Committee of Pek-
ing Union Medical College and Chinese Academy of Medical
Sciences. All animals were appropriately used on a scientifically
valid and ethical manner. DSS (molecular weight at 36–50 kDa)
(MP Biomedicals, Morgan Irvine, CA) was dissolved at 2.5% with
distilled water. 5-ASA was purchased from Energy Chemical (SH,
China). C57BL/6 female mice (6–8 weeks old) were divided into
five groups in random: Con group, DSS group, DSS + 5-ASA group
(50 mg/kg, ig), DSS + 33 group (5 mg/kg, ip) and DSS + 33 group
(10 mg/kg, ip). Colitis was induced by 2.5% w/v DSS in drinking
water for 5 days followed by another 5 days with normal water.
Mice were weighted every day, and administration of 5-ASA or
33 lasted until the end of the experiment. After 10 days, mice were
sacrificed. The colon was fixed in 10% buffered formalin (pH 7.4)
for at least 24 h for further histopathological assessment and
immunohistochemical study.
Acknowledgments
This work was financially supported by National Natural
Science Foundation of China (NSFC No. 81473096), Peking Union
Medical College Youth Fund (No.2013CHX12, 2015CX10).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.05.047.
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