Synthesis, biological evaluation and molecular modeling studies of Schiff bases derived from 4-methylsalicylic acid as potential immunosuppressive agents

Article abstract of DOI:10.1007/s00044-013-0561-0

A series of Schiff bases derived from 4-methylsalicylic acid (4a-4s) were synthesized, 14 of which (4a-4h, 4j-4l, 4n, 4q, and 4s) were reported for the first time. All the synthesized compounds were evaluated for their immunosuppressive activities for the

Full text of DOI:10.1007/s00044-013-0561-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0561-0
ORIGINAL RESEARCH
Synthesis, biological evaluation and molecular modeling studies
of Schiff bases derived from 4-methylsalicylic acid as potential
immunosuppressive agents
•
Ru Yan Peng-Gang Liu Zhi-Ming Zhang Xian-Ying Fang
•
•
•
•
Xue-Wei Zhang Jia-Li Deng Hai-Liang Zhu
•
Received: 6 December 2012 / Accepted: 21 February 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of Schiff bases derived from 4-meth-
ylsalicylic acid (4a–4s) were synthesized, 14 of which (4a–
4h, 4j–4l, 4n, 4q, and 4s) were reported for the first time.
All the synthesized compounds were evaluated for their
immunosuppressive activities for the first time. Among
them, compound 4o displayed the most potent biological
activity against lymph node cells (IC50 = 1.02 lM for
lymph node cells and IC50 = 2.17 lM for PI3Kc). The
results of apoptosis and western-blot assays demonstrated
that the immunosuppressive activity of compound 4o might
be mediated by the inhibition of PI3K/AKT signaling
pathway. Docking simulation was performed to position
compound 4o into the PI3Kc structure active site to
determine the probable binding model.
glycocorticoids (e.g., cortisol), cyclosporin A (CsA),
tacrolimus (FK506), and sirolimus (rapamycin), etc. have
been clinically used for their undeniable clinical advanta-
ges, they were also proved to possess a number of unde-
sired side effects including liver toxicity, malignancy,
cosmetic effects, renal toxicity, infection, and other bad
responses (Smith et al., 2003). Therefore, there is a clinical
need for new and less toxic therapeutic agents with new
mechanisms of action.
The phosphatidylinositol 3-kinases (PI3Ks) belong to a
family of enzymes that phosphorylate the inositol ring at the
30-hydroxyl position of phosphatidylinositol and phosphoin-
ositides. They are evolutionarily conserved from yeast to high
mammals and are divided into class I, II, and III PI3Ks,
according to their molecular structure, cellular regulation, and
in vivo substrate specificities (Cantley, 2002; Rommel et al.,
2007; Foster et al., 2003). The best known PI3Ks are class I
PI3Ks, which include subclass IA (consisting of PI3Ka,
PI3Kb, and PI3Kd isoforms) and subclass IB (consisting of
PI3Kc only). Both subclasses phosphorylate phosphatidylin-
ositol-4,5-bisphosphate [PtdIns(4,5)P2] to produce phospha-
tidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3], a critical
second messenger (Deane and Fruman, 2004). PtdIns(3,4,5)P3
acts as a docking site at the plasma membrane, where it
recruits and activates proteins that contain phospholipid-
binding domains such as serine/threonine kinase AKT (also
known as PKB). At the plasma membrane, AKT is phos-
phorylated and activated by phosphoinositide-dependent
kinase 1 (PDK-1) and mammalian target of rapamycin com-
plex 2. Once activated, AKT mediates several functions such
as cell proliferation, survival, motility, and metabolism
(Marone et al., 2008; Roy et al., 2009). Class IA, which
comprises a p85 regulatory subunit and a p110 catalytic
subunit, is localized primarily in the cytoplasm and is acti-
vated by the growth factor receptor tyrosine kinases at the
Keywords Schiff base ꢀ Immunosuppressive activities ꢀ
PI3Kc ꢀ Molecular modeling ꢀ 4-Methylsalicylic acid
Introduction
Immunosuppressant agents are an important class of clin-
ical drugs for an array of medical processes, including
transplant rejection and treatment of autoimmune diseases
such as systemic lupus erythematosus, rheumatoid arthritis,
and psoriasis (Hackstein and Thomson, 2004; Kahan,
2003). Although immunosuppressive drugs such as
Ru Yan and Peng-Gang Liu equally contributed to this paper.
R. Yan ꢀ P.-G. Liu ꢀ Z.-M. Zhang ꢀ X.-Y. Fang ꢀ X.-W. Zhang ꢀ
J.-L. Deng ꢀ H.-L. Zhu (&)
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing
University, Nanjing 210093, People’s Republic of China
e-mail: zhuhl@nju.edu.cn
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Med Chem Res
plasma membrane; differently, class IB comprising a p101
regulatory subunit and a p110 catalytic subunit is activated by
Gbc subunits downstream of G protein-coupled receptors
(GPCRs) (Fougerat et al., 2008; Suire et al., 2005). Because of
the isoforms’ differential tissue distribution, they show dis-
tinct biological functions. PI3Ka and PI3Kb are ubiquitously
expressed and regulate functions such as cell proliferation and
survival, whereas PI3Kd and PI3Kc are mainly expressed in
the hematopoietic system and mediate immune responses.
PI3Kc can also be found in endothelium, heart, and brain
(Ferrandi et al., 2007; Camps et al., 2005). It has been found
that the PI3Kc isoform plays a pivotal role in inflammation,
and it is involved in allergy, cardiovascular disorders, devel-
opment of chronic inflammation, and autoimmune diseases.
Therefore, the inhibition of PI3Kc might represent a promis-
ing therapy for autoimmune and inflammatory diseases.
A large number of Schiff bases have been synthesized
and extensively studied because of their characteristic
properties like delivery of novel structures, thermal sta-
bility, relevant biological properties, high synthesis flexi-
bility, and medicinal utility (Siddiqui et al., 2006). Schiff
bases have been used in the preparation of many potential
drugs and are known to possess a broad spectrum of bio-
logical activities such as antiviral (Chavan and Sivappa,
2004), antiparasitic (Rathelot et al., 2002), antibacterial
(Lee et al., 2012), anti-inflammatory (Bekhit et al., 2003),
antiHIV (Pandeya et al., 1999), and anticancer (Demirbas
et al., 2004) activities.
active ingredient of agents used for the long term mainte-
nance therapy to prevent relapses of inflammatory bowel
diseases (Giannini et al., 2005). Introducing the structure of
the 4-methylsalicylic acid to the Schiff leads to a positive
result as initially designed by computer-aided molecular
design (CAMD). In order to study the structure–activity
relationships in anti-inflammatory activity, we introduced
different substituents on the other benzene ring which
connected with the azomethine moiety (–C=N–) with the
4-methylsalicylic acid moiety fixed. We can see that both
Schiff and salicylic acid have anti-inflammatory activities.
It is supposed to be worthful to synthesize a series of Schiff
bases derived from 4-methylsalicylic acid considering that
they might exhibit synergistic effect in anti-inflammatory
activities. PI3Kc inhibitory activity as the important target
was studied. Docking simulations were performed using
the X-ray crystallographic structure of the PI3Kc in com-
plex with the most potent inhibitor to explore the binding
model of the compound at the active site.
Results and discussion
Chemistry
In the present study, 19 Schiff base derivatives derived from
4-methylsalicylic acid (4a–s) were synthesized to screen for
the immunosuppressive activity. Among these compounds,
compounds 4a–4h, 4j–4l, 4n, 4q, and 4s were reported for the
first time. They were prepared in three steps (Scheme 1).
Firstly, esterification of 4-methylsalicylic acid 1 with ethanol
and concentrated sulfuric acid afforded the corresponding
ester 2. Secondly, the 2-hydroxy-4-methylbenzohydrazide 3
As we know, salicylic acid was identified in willow bark
extracts as an active anti-inflammatory compound over a
century ago. Some salicylic acid derivatives were reported
to show potent anti-inflammatory activity (Marra et al.,
2000). For example, 5-aminosalicylic acid (5-ASA) was an
O
O
COOH
OH
NH2
O
NH
i
ii
OH
OH
3
1
2
O
R4
N
N
H
iii
R1
R3
OH
R2
4a-4s
Scheme 1 General synthesis of Schiff bases derived from 4-methylsalicylic acid (4a–4s). Reagents and conditions: (i) ethanol, concentrated
sulfuric acid, reflux, 8–12 h; (ii) NH2NH2ꢀH2O (85 %), ethanol, reflux, 8–12 h; (iii) substituted benzaldehyde, ethanol, reflux, 2.5 h
123

Med Chem Res
Table 1 Structure of Schiff bases derived from 4-methylsalicylic
acid (4a–4s)
Table 2 Inhibitory effects of the synthetic compounds 4a–4s and
cyclosporin A on lymph node cells stimulated by ConA in vitro
Compounda
CCb50 (lM)
ICb50 (lM)
SIc
O
12
10
R4
11
16
6
8
N
4a
4b
4c
4d
4e
4f
578.34
362.12
124.65
643.66
166.21
636.48
113.44
672.42
732.47
567.57
116.23
733.89
722.54
291.34
760.56
559.65
1495.43
707.77
978.95
14.96
53.85
42.06
19.16
[100
31.24
24.35
28.26
20.60
[100
26.70
12.30
24.28
[100
9.78
10.74
13
14
7
5
N
9
8.61
6.51
1
H
R1
R3
15
2
\6.44
3.72
OH
4
3
R2
4a-4s
30.89
4.01
4g
4h
4i
32.64
\7.32
21.26
9.45
Compound
R1
R2
R3
R4
4a
4b
4c
4d
4e
4f
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Br
Cl
Br
4j
F
H
4k
4l
Cl
NO2
H
H
30.23
\7.23
29.79
745.65
433.84
1371.95
468.72
484.63
249.33
H
4m
4n
4o
4p
4q
4r
4s
F
H
Cl
Br
OCH3
H
1.02
4g
4h
4i
H
1.29
H
1.09
H
N(CH3)2
1.51
4j
H
H
F
2.02
4k
4l
H
H
Cl
NO2
OCH3
Cl
H
CsA
0.06
H
H
a
The compounds tested for immunosuppressive activity were con-
sistent with the description in the experimental section
4m
4n
4o
4p
4q
4r
4s
H
H
b
Cl
OH
OH
OH
OH
OH
H
Values are means of three experiments
c
H
Selectivity index (SI) is determined as the ratio of the concentration
H
H
of the compound that reduced cell viability to 50 % (CC50) to the
concentration of the compound needed to inhibit the proliferation to
50 % (IC50) of the control value
H
H
Cl
Br
H
H
as the concentration of compound that reduced cell via-
bility to 50 % (CC50). The immunosuppressive activity of
each compound was expressed as the concentration of the
compound that inhibited ConA stimulated T cells prolif-
eration to 50 % (IC50) of the control value. The selectivity
index (SI) value was used to evaluate the anti-inflammatory
activity of the compounds.
was obtained by reaction of the ester 2 with 85 % hydrazine
monohydrate in ethanol. Thirdly, the synthesis of compounds
4a–4s (Table 1) was accomplished by refluxing 3 with dif-
ferent substituted benzaldehydes in ethanol. All target com-
pounds were purified by recrystallization. All of the synthetic
compounds gave satisfactory analytic and spectroscopic data,
which were in full accordance with their depicted structures.
Studies were performed by modification of the phenyl
ring of the benzaldehyde derivatives to determine how the
substituents of the subunits affected the immunosuppres-
sive activity. As exhibited in Table 2, in general, most of
the synthesized Schiff base derivatives showed remarkable
effects on immunosuppressive activity. Among them,
compounds 4o–4s exhibited significant immunosuppres-
sive activities with IC50 ranging from 1.02 to 2.02 lM.
Meanwhile, the data of the cytotoxicity test showed that all
synthetic compounds had a rather low cytotoxicity.
The structure–activity relationships in these Schiff base
derivatives demonstrated that compounds with different
benzaldehyde substituents displayed different immunosup-
pressive activities. For the ortho, meta, and para position
Biological activity
T cells play a pivotal role in immune response. Excessive T
cell proliferation and activation has been implicated in the
pathogenesis of a variety of inflammatory diseases.
All the synthetic Schiff base derivatives were tested
in vitro for their cytotoxicity on lymph node cells and
inhibitory activity on concanavalin A (ConA) stimulated
lymph node cells with CsA as the control. The pharma-
cological results of these compounds were summarized in
Table 2. The cytotoxicity of each compound was expressed
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Med Chem Res
substituted compounds, replacement of H atom at ortho,
meta, or para position by one halogen atom (4b, 4c, 4e, 4f,
4g, 4j, and 4k) resulted in the improvement of the immu-
nosuppressive activity compared to 4a (IC50 = 53.85 lM),
with IC50 ranging from 12.30 to 42.06 lM. Meanwhile, a
comparison of the one-halogen-substituted derivatives
demonstrated that compounds with substituent Cl on the
phenyl ring (4c, 4f, and 4k) showed a little stronger immu-
nosuppressive activity than those with substituent F (4b, 4e,
and 4j). And the potent inhibitory order of compounds 4b, 4e,
and 4j was para [ meta [ ortho, while the potent inhibitory
order of compounds 4c, 4f, and 4k was para [ ortho [
meta. For compound 4n, in which two halogens were intro-
duced in the ortho and para position, the immunosuppressive
activity was remarkably increasing (4n IC50 = 9.78 lM)
compared to compounds 4c, 4f, and 4k. Besides, the SI
values of the compounds differed greatly, ranging from 4.01
to 1371.95. Some of the synthesized compounds (4k and 4n),
especially 4n (SI = 29.79) showed good immunosuppres-
sive activity but they are much less potent than CsA
(SI = 249.33).
inhibitory activity (SI = 745.65) than CsA (SI = 249.33).
Therefore, the IC50 and SI values suggested that compound
4o may be a promising lead for the further development of
novel immunosuppressive agents with low cytotoxicity.
PI3Kc inhibitory assay
Selected compounds 4o–4s that exhibited significant
immunosuppressive activities were evaluated for their PI3Kc
inhibitory activity with LY294002, a well-known class I
PI3K inhibitor, as the positive control (Pomel et al., 2006).
The results were presented in Table 3. All the selected
compounds displayed better PI3Kc inhibitory activity than
LY294002. Among them, compound 4o showed the most
potent inhibitory activity with IC50 of 2.17 lM. The results
of PI3Kc inhibitory activity of the tested compounds agree
with the structure relationships (SAR) of their immunosup-
pressive activities. This demonstrated that the potent
immunosuppressive activities of the synthetic compounds
were probably correlated to their PI3Kc inhibitory activities.
It was clear that the synthesized compounds with a
hydroxyl group in ortho position on the phenyl ring of the
benzaldehyde derivatives (4o, 4p, 4q, 4r, and 4s) showed
higher activity than others, with IC50 ranging from 1.02 to
2.02 lM. Among them, compounds 4o displayed the most
potent inhibitory activity with IC50 of 1.02 lM, which was
a little lower than that of cyclosporin A (IC50 = 0.06 lM).
However, compounds 4o exhibited reduced cytotoxicity
by over 50-fold compared with CsA and more potent
Apoptosis assay
Apoptosis is an essential mechanism used to eliminate
activated T cells during the shutdown process of excess
immune responses and maintains proper immune homeo-
stasis, while deficient apoptosis of activated T cells is
associated with a wide variety of immune disorders. As
representatives of these Schiff base derivatives, compounds
4o and 4r have been investigated in an in vitro experiment.
We detected the mechanism of compounds 4o and 4r
inhibition effects by flow cytometry (FCM) (Fig. 1), and
found that the compounds could induce the apoptosis of
activated lymph node cells in a dose-dependent manner. As
shown in Fig. 1, lymph node cells stimulated with ConA
were treated with 0, 3, 10, and 30 lM of compounds 4o
and 4r, respectively, for 24 h. The compounds increased
the percentage of apoptosis by Annexin V-FITC staining in
a dose-dependent manner. The result indicated that com-
pounds 4o and 4r could induce apoptosis of ConA stimu-
lated lymph node cells, especially the compound 4o.
Table 3 PI3Kc inhibitory activity of selected compounds 4o–4s
Compound
PI3Kc (ICa50, lM)
4o
2.17
3.69
2.60
4.81
7.12
7.26
4p
4q
4r
4s
LY294002
a
Values are means of three experiments
Fig. 1 Lymph node cells
isolated from naive mice were
cultured with ConA and various
concentrations of 4o and 4r,
respectively, for 24 h. Cells
were stained by Annexin
V-FITC and apoptosis was
analyzed by flow cytometry
123

Med Chem Res
benzoyl ring formed a hydrogen bond with the oxygen of
˚
ASP 964 (distance: 1.15 A), and the oxygen of hydrazide
group also formed an interaction with the amino group of
˚
LYS833 (distance: 2.47 A). This molecular docking result,
along with the biological assay data, suggested that com-
pound 4o was a potential inhibitor of PI3Kc.
Fig. 2 Compound 4o was examined by western blotting. Data are
representative of three independent experiments
Experimental section
Western-blot assay
Cytotoxicity assay
In an effort to study the preliminary mechanism of the
compound with potent inhibitory activity, the western-blot
experiment was performed to explore the effect of compound
4o. The western-blot results were summarized in Fig. 2,
confirming compound 4o’s inhibitory activity. As shown in
Fig. 2, p-AKT (pS473) was dose-dependently down-regu-
lated as a result of a 24 h treatment with 4o. The activity of
cleaved caspase 3 was dose-dependently up-regulated after
the treatment with 4o. It could be confirmed from the results
that the apoptosis induced by 4o might be mediated by the
inhibition of PI3K/AKT signaling pathway.
Fresh lymph node cells were obtained from BALB/C mice.
Cells were cultured in a 96-well plate at a density of
5 9 105 cells per well, and different concentrations of
compounds were added to each well, respectively. The
incubation was performed in a humidified, 37°, 5 % CO2-
containing incubator for 24 h. Twenty microliters of MTT
(Sigma, MO; 4 mg/mL in PBS) was added to each well 4 h
before the end of the incubation. MTT formazan produc-
tion was dissolved in 200 lL DMSO replacing the medium
at 37° for 30 min. The absorbance was measured at a
wavelength of 570 nm (OD570 nm) on an ELISA micro-
plate reader. Three replicate wells were used for each
concentration, and each assay was measured three times,
after which the average of CC50 and SD were calculated.
Molecular modeling
In an effort to elucidate the mechanism by which the title
compounds could induce immunosuppressive activity
against T cells and guide further SAR studies, molecular
modeling of the potent inhibitor 4o into ATP binding site of
PI3Kc was performed to simulate a binding model based on
PI3Kc structure (3L54.pdb). The docking result indicated
two hydrogen bond interactions between compound 4o and
PI3Kc (Figs. 3, 4): the hydrogen atom of 2-hydroxyl on
Cell proliferation assay
Fresh lymph node cells were obtained from BALB/C mice.
5 9 105 cells per well were cultured in a 96-well plate at
the same conditions as mentioned above. The cultures were
stimulated with 5 lg/mL of ConA to induce T cell prolif-
erative responses for 24 h. After that, various
Fig. 3 Molecular modeling of
compound 4o with PI3Kc:
compound 4o was nicely bound
to PI3Kc with its hydrogen
atom of 2-hydroxyl on benzoyl
ring forming a hydrogen bond
(shown as dotted green lines)
with the oxygen of ASP 964
˚
(distance: 1.15 A). The oxygen
of hydrazide group also formed
a hydrogen bond with the amino
group of LYS833 (distance:
˚
2.47 A) (Color figure online)
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Med Chem Res
Fig. 4 2D ligand interaction
diagram of compound 4o with
PI3Kc with the essential amino
acid residues at the binding site
tagged in circles. The purple
circles showed the amino acids
which participated in hydrogen
bonding, electrostatic or polar
interactions, and the green
circles showed the amino acids
which participated in the Van
der Waals interactions. Two
hydrogen bond interactions
between compound 4o and
PI3Kc were indicated (Color
figure online)
concentrations of compounds were added to each well and
the cultures were incubated for 72 h. Twenty microliters of
MTT (Sigma, MO; 4 mg/mL in PBS) was added to each
well 4 h before the end of the incubation. After removing
the supernatant, 200 lL DMSO was added to dissolve the
formazan crystals. The absorbance at 570 nm (OD570 nm)
was read on an ELISA reader (Tecan, Austria).
Nonidet P-40, 10 % glycerol, and phosphatase and protease
inhibitors). After centrifugation at 12,0009g for 5 min, the
protein content of the supernatant was determined by a
BCATM protein assay kit (Pierce, Rockford, IL, USA).
The protein samples were separated by 10 % SDS–PAGE
and subsequently electrotransferred onto a polyvinylidene
difluoride membrane (Millipore, Bedford, MA, USA). The
membrane was blocked with 5 % non-fat milk for 2 h at
room temperature. The blocked membrane was probed
with the indicated primary antibodies overnight at 4°, and
then incubated with a horse radish peroxidase (HRP)-
coupled secondary antibody. Detection was performed
using a LumiGLO chemiluminescent substrate system
(KPL,Guildford, UK).
PI3Kc inhibitory assay
A PI3Kc lipid kinase assay for compounds 4o–4s, based on
the neomycin-coated scintillation proximity assay (SPA)
bead technology (Amersham), was performed in a 96-well
plate using ATP/[c33P]ATP and phosphatidylinositol
(Sigma) as substrates. The PI3Kc lipid kinase inhibitory
assay method used was the same as previously described.
Experimental protocol of modeling study
Apoptosis assay
The PI3Kc-LXX protein–ligand complex crystal structure
(3L54.pdb) was chosen as the template for the modeling
study of compound 4o bound to PI3Kc (Knight et al., 2010).
The crystal structure was obtained from the RCSB Protein
Data Bank (http://www.rcsb.org/pdb/home/home.do).
The molecular docking procedure was performed by
using CDocker protocol within discovery studio 3.1. For
ligand preparation, the 3D structures of 4o were generated
and minimized using discovery studio 3.1. For enzyme
preparation, the hydrogen atoms were added, and the water
and impurities were removed. The whole PI3Kc enzyme was
defined as a receptor and the site sphere was selected based
on the ligand binding location of LXX; then, the LXX
molecule was removed and 4o was placed during the
molecular docking procedure. Types of interactions of the
Lymph node cells were incubated in a 24-well plate at a
density of 3 9 106 cells per well and stimulated with 5 lg/
mL of ConA for 24 h. Then different concentrations of
compounds were added to each well. Twenty four hours
later, the cells were harvested and stained with Annexin
V-FITC (fluorescein isothiocyanate). And then the samples
were analyzed by FACSCalibur flow cytometer (Becton–
Dickinson, San Jose, CA).
Western-blot analysis
After incubation, cells were washed with PBS and lysed in
lysis buffer (30 mm Tris, pH 7.5, 150 mm NaCl, 1 mm
phenylmethylsulfonyl fluoride, 1 mm Na3VO4, 1 %
123

Med Chem Res
docked enzyme with ligand were analyzed at the end of
molecular docking.
NMR (300 MHz, DMSO-d6) d: 2.31 (s, 3H, Me), 6.78–
6.80 (m, 2H, 3-H; 5-H), 7.46–7.48 (m, 3H, 13-H; 14-H;
15-H), 7.75 (d, J = 4.2 Hz, 2H, 12-H; 16-H), 7.82 (d,
J = 8.4 Hz, 1H, 6-H), 8.47 (s, 1H, 10-H), 11.80 (s, 1H,
NH), 11.99 (s, 1H, OH). MS (ESI): 255.11 (C15H15N2O2,
[M ? H]?). Anal. Calcd for C15H14N2O2: C, 70.85; H,
5.55; N, 11.02. Found: C, 70.89; H, 5.50; N, 11.07.
Chemistry
All chemicals and reagents used in the current study were of
analytic grade purchased from Aldrich (USA). The reactions
were monitored by thin layer chromatography (TLC) on
Merck pre-coated silica GF254 plates. Melting points
(uncorrected) were determined on an XT4MP apparatus (Tai
ke Corp., Beijing, China). ESI mass spectra were obtained on
(E)-N0-(2-fluorobenzylidene)-2-hydroxy-4-methylbenzo-
hydrazide (4b) Yellow crystal, yield: 83 %, mp: 203–
1
204°C. H NMR (300 MHz, DMSO-d6) d: 2.31 (s, 3H,
1
a Mariner System 5304 mass spectrometer, and H NMR
Me), 6.78–6.80 (m, 2H, 3-H; 5-H), 7.31 (t, J = 9.9 Hz, 2H,
13-H; 15-H), 7.48–7.55 (m, 1H, 14-H), 7.81 (d,
J = 8.6 Hz, 1H, 6-H), 7.95 (t, J = 7.5 Hz, 1H, 16-H), 8.71
(s, 1H, 10-H), 11.94 (s, 2H, NH; OH). MS (ESI): 273.10
(C15H14FN2O2, [M ? H]?). Anal. Calcd for C15H13FN2O2:
C, 66.17; H, 4.81; N, 10.29. Found: C, 66.13; H, 4.84; N,
10.21.
spectra were recorded on a DPX500 spectrometer at 25° with
TMS and solvent signals allotted as internal standards.
Chemical shifts are reported in ppm (d). Elemental analyses
were performed on a CHN-O-Rapid instrument and were
within 0.4 % of the theoretical values.
General procedure for synthesis of ethyl 2-hydroxy-4-
methylbenzoate (2)
(E)-N0-(2-chlorobenzylidene)-2-hydroxy-4-methylbenzohy-
drazide (4c) White crystal, yield: 79 %, mp: 209–210°C.
1H NMR (300 MHz, DMSO-d6) d: 2.31 (s, 3H, Me),
6.78–6.80 (m, 2H, 3-H; 5-H), 7.45–7.56 (m, 3H, 13-H;
14-H; 15-H), 7.82 (d, J = 8.4 Hz, 1H, 6-H), 8.03 (d,
J = 6.1 Hz, 1H, 16-H), 8.87 (s, 1H, 10-H), 11.96 (s, 1H,
NH), 12.04 (s, 1H, OH). MS (ESI): 289.07 (C15H14ClN2O2,
[M ? H]?). Anal. Calcd for C15H13ClN2O2: C, 62.40; H,
4.54; N, 9.70. Found: C, 62.48; H, 4.52; N, 9.75.
4-Methylsalicylic acid (7.6 g, 50 mmol) in ethanol
(50 mL) was treated with concentrated sulfuric acid
(2.5 mL) under reflux overnight. The solvent was removed
in vacuo leaving an oil. Water (50 mL) was added and the
organic phase was extracted with ethyl acetate (30 mL).
After washing the organic phase with saturated NaCl
(40 mL), drying it with anhydrous Na2SO4 and evaporating
the solvent under reduced pressure, product 2 appeared.
(E)-2-hydroxy-4-methyl-N0-(2-nitrobenzylidene)benzohyd-
razide (4d) Yellow crystal, yield: 69 %, mp: 234–235°C.
1H NMR (300 MHz, DMSO-d6) d: 2.31 (s, 3H, Me),
6.78–6.81 (m, 2H, 3-H; 5-H), 7.70 (t, J = 7.5 Hz, 1H, 14-H),
7.84 (t, J = 7.0 Hz, 2H, 6-H; 15-H), 8.11 (t, J = 9.2 Hz, 2H,
13-H; 16-H), 8.87 (s, 1H, 10-H), 11.90 (s, 1H, NH), 12.13 (s,
1H, OH). MS (ESI): 300.09 (C15H14N3O4, [M ? H]?).
Anal. Calcd for C15H13N3O4: C, 60.20; H, 4.38; N, 14.04.
Found: C, 60.28; H, 4.40; N, 14.00.
General procedure for synthesis of 2-hydroxy-4-
methylbenzohydrazide (3)
A stirred solution of compound 2 (10 mmol) in ethanol
(50 mL) was treated with 85 % hydrazine hydrate (2 mL)
under reflux for 8–10 h. After cooling, removal of the
solvent under reduced pressure gave a crude product which
was filtered off and purified with water and a small quantity
of ethanol to give compound 3.
(E)-N0-(3-fluorobenzylidene)-2-hydroxy-4-methylbenzohy-
drazide (4e) Light yellow crystal, yield: 84 %, mp:
General procedure for synthesis of Schiff bases derived
from 4-methylsalicylic acid (4a–4s)
1
183–184°C. H NMR (300 MHz, DMSO-d6) d: 2.30 (s,
3H, Me), 6.78–6.80 (m, 2H, 3-H; 5-H), 7.29 (t, J = 8.6 Hz,
1H, 14-H), 7.48–7.60 (m, 3H, 12-H; 15-H; 16-H), 7.82 (d,
J = 8.4 Hz, 1H, 6-H), 8.47 (s, 1H, 10-H), 11.88–11.90
(m, 2H, NH; OH). MS (ESI): 273.10 (C15H14FN2O2,
[M ? H]?). Anal. Calcd for C15H13FN2O2: C, 66.17; H,
4.81; N, 10.29. Found: C, 66.24; H, 4.78; N, 10.36.
The mixture of compound 3 (1 mmol) and different
substituted benzaldehydes (1.2 mmol) was refluxed in
ethanol (10 mL) for 2.5 h. After cooling to room temper-
ature, target compounds were filtered off, washed carefully
with ice water and cool ethanol, and then dried. All target
compounds were purified by recrystallization from ethanol
in refrigerator.
(E)-N0-(3-chlorobenzylidene)-2-hydroxy-4-methylbenzohy-
drazide (4f) Light yellow crystal, yield: 76 %, mp:
(E)-N0-benzylidene-2-hydroxy-4-methylbenzohydrazide
(4a) White crystal, yield: 81 %, mp: 232–233°C. 1H
1
183–184°C. H NMR (300 MHz, DMSO-d6) d: 2.31 (s,
123

Med Chem Res
3H, Me), 6.78–6.80 (m, 2H, 3-H; 5-H), 7.30 (t, J = 8.8 Hz,
1H, 15-H), 7.48–7.60 (m, 3H, 12-H; 14-H; 16-H), 7.82 (d,
J = 8.4 Hz, 1H, 6-H), 8.47 (s, 1H, 10-H), 11.89 (s, 1H,
NH), 11.91 (s, 1H, OH). MS (ESI): 289.07 (C15H14ClN2O2,
[M ? H]?). Anal. Calcd for C15H13ClN2O2: C, 62.40; H,
4.54; N, 9.70. Found: C, 62.34; H, 4.51; N, 9.76.
J = 8.6 Hz, 2H, 13-H; 15-H), 7.75–7.83 (m, 3H, 6-H;
12-H; 16-H), 8.46 (s, 1H, 10-H), 11.84 (s, 1H, NH), 11.93
(s, 1H, OH). MS (ESI): 289.07 (C15H14ClN2O2, [M ? H]?).
Anal. Calcd for C15H13ClN2O2: C, 62.40; H, 4.54; N, 9.70.
Found: C, 62.44; H, 4.51; N, 9.76.
(E)-2-hydroxy-4-methyl-N0-(4-nitrobenzylidene)benzohyd-
razide (4l) Yellow crystal, yield: 70 %, mp: 317–318°C.
1H NMR (300 MHz, DMSO-d6) d: 2.31 (s, 3H, Me),
6.79–6.82 (m, 2H, 3-H; 5-H), 7.82 (d, J = 8.4 Hz, 1H, 6-H),
8.01 (d, J = 8.6 Hz, 2H, 12-H; 16-H), 8.32 (d, J = 8.6 Hz,
2H, 13-H; 15-H), 8.57 (s, 1H, 10-H), 11.79 (s, 1H, NH),
12.03 (s, 1H, OH). MS (ESI): 300.09 (C15H14N3O4,
[M ? H]?). Anal. Calcd for C15H13N3O4: C, 60.20; H, 4.38;
N, 14.04. Found: C, 60.26; H, 4.39; N, 14.00.
(E)-N0-(3-bromobenzylidene)-2-hydroxy-4-methylbenzohy-
drazide (4g) Light brown crystal, yield: 79 %, mp: 198–
1
199°C. H NMR (300 MHz, DMSO-d6) d: 2.30 (s, 3H,
Me), 6.78–6.80 (m, 2H, 3-H; 5-H), 7.43 (t, J = 7.9 Hz, 1H,
15-H), 7.64 (d, J = 8.0 Hz, 1H, 14-H), 7.74 (d, J =
7.5 Hz, 1H, 16-H), 7.82 (d, J = 8.6 Hz, 1H, 6-H), 7.94 (s,
1H, 12-H), 8.43 (s, 1H, 10-H), 11.90 (s, 2H, NH; OH). MS
(ESI): 333.02 (C15H14BrN2O2, [M ? H]?). Anal. Calcd for
C15H13ClN2O2: C, 54.07; H, 3.93; N, 8.41. Found: C,
54.13; H, 3.90; N, 8.44.
(E)-2-hydroxy-N0-(4-methoxybenzylidene)-4-methylbenzohyd-
razide (4m) Light yellow crystal, yield: 73 %, mp: 223–
(E)-2-hydroxy-N0-(3-methoxybenzylidene)-4-methylbenzohyd-
razide (4h) Yellow crystal, yield: 74 %, mp: 180–181°C.
1H NMR (300 MHz, DMSO-d6) d: 2.30 (s, 3H, Me), 3.81
(s, 3H, OMe), 6.78–6.80 (m, 2H, 3-H; 5-H), 7.03 (d,
J = 7.0 Hz, 1H, 14-H), 7.30 (d, J = 7.0 Hz, 2H, 15-H;
16-H), 7.39 (t, J = 7.9 Hz, 1H, 12-H), 7.82 (d, J = 8.6 Hz,
1H, 6-H), 8.44 (s, 1H, 10-H), 11.81 (s, 1H, NH), 11.96 (s,
1H, OH). MS (ESI): 285.12 (C16H17N2O3, [M ? H]?).
Anal. Calcd for C16H16N2O3: C, 67.59; H, 5.67; N, 9.85.
Found: C, 67.63; H, 5.70; N, 9.80.
1
224°C. H NMR (300 MHz, DMSO-d6) d: 2.30 (s, 3H,
Me), 3.82 (s, 3H, OMe), 6.76–6.79 (m, 2H, 3-H; 5-H), 7.03
(d, J = 8.8 Hz, 2H, 13-H; 15-H), 7.69 (d, J = 8.6 Hz, 2H,
12-H; 16-H), 7.82 (d, J = 8.6 Hz, 1H, 6-H), 8.41 (s, 1H,
10-H), 11.70 (s, 1H, NH), 12.09 (s, 1H, OH). MS (ESI):
285.12 (C16H17N2O3, [M ? H]?). Anal. Calcd for C16H16
N2O3: C, 67.59; H, 5.67; N, 9.85. Found: C, 67.63; H, 5.69;
N, 9.79.
(E)-N0-(2,4-dichlorobenzylidene)-2-hydroxy-4-methylben-
zohydrazide (4n) White crystal, yield: 77 %, mp: 213–
(E)-N0-(4-(dimethylamino)benzylidene)-2-hydroxy-4-
methylbenzohydrazide (4i) Yellow crystal, yield: 70 %,
mp: 235–236°C. 1H NMR (300 MHz, DMSO-d6) d: 2.30 (s,
3H, Me), 2.98 (s, 6H, N(Me)2), 6.75–6.77 (m, 4H, 3-H; 5-H;
13-H; 15-H), 7.56 (d, J = 8.8 Hz, 2H, 12-H; 16-H), 7.81 (d,
J = 8.4 Hz, 1H, 6-H), 8.32 (s, 1H, 10-H), 11.56 (s, 1H, NH),
12.22 (s, 1H, OH). MS (ESI): 298.15 (C17H20N3O2,
[M ? H]?). Anal. Calcd for C17H19N3O2: C, 68.67; H, 6.44;
N, 14.13. Found: C, 66.74; H, 6.47; N, 14.03.
1
214°C. H NMR (300 MHz, DMSO-d6) d: 2.31 (s, 3H,
Me), 6.80 (s, 2H, 3-H; 5-H), 7.54 (d, J = 8.4 Hz, 1H,
15-H), 7.74 (d, J = 2.0 Hz, 1H, 13-H), 7.81 (d, J = 8.4 Hz,
1H, 6-H), 8.03 (d, J = 8.6 Hz, 1H, 16-H), 8.82 (s, 1H,
10-H), 11.90 (s, 1H, NH), 12.07 (s, 1H, OH). MS (ESI):
320.03 (C15H13Cl2N2O2, [M ? H]?). Anal. Calcd for
C15H12Cl2N2O2: C, 55.75; H, 3.74; N, 8.67. Found: C,
55.79; H, 3.71; N, 8.61.
(E)-2-hydroxy-N0-(2-hydroxybenzylidene)-4-methylbenzohyd-
razide (4o) Light yellow crystal, yield: 76 %, mp:
(E)-N0-(4-fluorobenzylidene)-2-hydroxy-4-methylbenzo-
hydrazide (4j) White crystal, yield: 84 %, mp: 217–
1
291–292°C. H NMR (300 MHz, DMSO-d6) d: 2.31 (s,
1
218°C. H NMR (300 MHz, DMSO-d6) d: 2.30 (s, 3H,
3H, Me), 6.79–6.81 (m, 2H, 3-H; 5-H), 6.91–6.96 (m, 2H,
13-H; 15-H), 7.32 (t, J = 8.1 Hz, 1H, 14-H), 7.56 (d,
J = 7.9 Hz, 1H, 16-H), 7.82 (d, J = 8.6 Hz, 1H, 6-H),
8.68 (s, 1H, 10-H), 11.19 (s, 1H, NH), 11.90 (s, 1H, 2-OH),
12.00 (s, 1H, 12-OH). MS (ESI): 271.103 (C15H15N2O3,
[M ? H]?). Anal. Calcd for C15H14N2O3: C, 66.66; H,
5.22; N, 10.36. Found: C, 66.69; H, 5.20; N, 10.31.
Me), 6.77–6.79 (m, 2H, 3-H; 5-H), 7.31 (t, J = 8.6 Hz, 2H,
13-H; 15-H), 7.80–7.83 (m, 3H, 6-H; 12-H; 16-H), 8.46 (s,
1H, 10-H), 11.81 (s, 1H, NH), 11.97 (s, 1H, OH). MS
(ESI): 273.10 (C15H14FN2O2, [M ? H]?). Anal. Calcd for
C15H13FN2O2: C, 66.17; H, 4.81; N, 10.29. Found: C,
66.23; H, 4.83; N, 10.24.
(E)-N0-(4-chlorobenzylidene)-2-hydroxy-4-methylbenzohy-
drazide (4k) Light yellow crystal, yield: 80 %, mp:
222–223°C. 1H NMR (300 MHz, DMSO-d6) d: 2.30
(s, 3H, Me), 6.78–6.80 (m, 2H, 3-H; 5-H), 7.54 (d,
(E)-N0-(5-chloro-2-hydroxybenzylidene)-2-hydroxy-4-
methylbenzohydr- azide (4p) Light yellow crystal, yield:
72 %, mp: 314–315°C. 1H NMR (300 MHz, DMSO-d6) d:
2.31 (s, 3H, Me), 6.78–6.81 (m, 2H, 3-H; 5-H), 6.96 (d,
123

Med Chem Res
J = 8.8 Hz, 1H, 13-H), 7.31–7.35 (m, 1H, 14-H), 7.66 (d,
J = 2.6 Hz, 1H, 16-H), 7.81 (d, J = 8.6 Hz, 1H, 6-H),
8.65 (s, 1H, 10-H), 11.21 (s, 1H, NH), 11.87 (s, 1H, 2-OH),
12.06 (s, 1H, 12-OH). MS (ESI): 305.06 (C15H14ClN2O3,
[M ? H]?). Anal. Calcd for C15H13ClN2O3: C, 59.12; H,
4.30; N, 9.19. Found: C, 59.02; H, 4.34; N, 9.22.
PI3Kc inhibitory activities. The results showed that com-
pound 4o displayed the most potent inhibitory activity
(IC50 = 2.17 lM), which was over threefold better than
that of LY294002 (IC50 = 7.26 lM). Moreover, the pre-
liminary mechanism of compounds 4o and 4r inhibition
effects was detected by FCM, and the compounds exerted
immunosuppressive activity via inducing the apoptosis of
activated lymph node cells in a dose-dependent manner.
The results of western-blot experiment also showed that the
apoptosis induced by 4o might be mediated by the inhibi-
tion of PI3K/AKT signaling pathway. Molecular docking
study indicated that compound 4o was nicely bound to the
PI3Kc with two hydrogen bonds. All the results showed the
great potential of compound 4o as an immunosuppressant
targeting PI3Kc.
(E)-N0-(5-bromo-2-hydroxybenzylidene)-2-hydroxy-4-
methylbenzohydr- azide (4q) Light yellow crystal, yield:
76 %, mp: 296–297°C. 1H NMR (300 MHz, DMSO-d6) d:
2.31 (s, 3H, Me), 6.78–6.81 (m, 2H, 3-H; 5-H), 6.91 (d,
J = 8.8 Hz, 1H, 13-H), 7.42–7.46 (m, 1H, 14-H), 7.79–7.83
(m, 2H, 6-H; 16-H), 8.64 (s, 1H, 10-H), 11.22 (s, 1H, NH),
11.87 (s, 1H, 2-OH), 12.06 (s, 1H, 12-OH). MS (ESI): 349.01
(C15H14BrN2O3, [M ? H]?). Anal. Calcd for C15H13BrN2
O3: C, 51.60; H, 3.75; N, 8.02. Found: C, 51.66; H, 3.72; N,
8.08.
Acknowledgments The study was financed by National Natural
Science Foundation of China (No. J1103512).
(E)-N0-(3,5-dichloro-2-hydroxybenzylidene)-2-hydroxy-4-
methylbenzo-hydrazide (4r) Light yellow crystal, yield:
1
69 %, mp: 260–261°C. H NMR (300 MHz, DMSO-d6) d:
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