(E)-1,3-diphenyl-1H-pyrazole derivatives containing O-benzyl oxime moiety as potential immunosuppressive agents: Design, synthesis, molecular docking and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2015.12.020

A series of novel (E)-1,3-diphenyl-1H-pyrazole derivatives containing O-benzyl oxime moiety were firstly synthesized and their immunosuppressive activities were evaluated. Among all the compounds, 4n exhibited the most potent inhibitory activity (IC₅₀ Combining double low line 1.18 μM for lymph node cells and IC₅₀ Combining double low line 0.28 μM for PI3Kγ3), which was comparable to that of positive control. Moreover, selected compounds were tested for their inhibitory activities against IL-6 released in ConA-simulated mouse lymph node cells, 4n exhibited the most potent inhibitory ability. Furthermore, in order to study the preliminary mechanism of the compounds with potent inhibitory activity, the RT-PCR experiment was performed to assay the effect of selected compounds on mRNA expression of IL-6. Among them, compound 4n strongly inhibited the expression of IL-6 mRNA.

Full text of DOI:10.1016/j.ejmech.2015.12.020

Accepted Manuscript
(E)-1,3-diphenyl-1H-pyrazole derivatives containing O-benzyl oxime moiety as
potential immunosuppressive agents: Design, synthesis, molecular docking and
biological evaluation
Xian-Hai Lv, Qing-Shan Li, Zi-Li Ren, Ming-Jie Chu, Jian Sun, Xin Zhang, Man Xing,
Hai-Liang Zhu, Hai-Qun Cao
PII:
S0223-5234(15)30406-2
10.1016/j.ejmech.2015.12.020
EJMECH 8260
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 June 2015
Revised Date: 1 December 2015
Accepted Date: 10 December 2015
Please cite this article as: X.-H. Lv, Q.-S. Li, Z.-L. Ren, M.-J. Chu, J. Sun, X. Zhang, M. Xing, H.-L. Zhu,
H.-Q. Cao, (E)-1,3-diphenyl-1H-pyrazole derivatives containing O-benzyl oxime moiety as potential
immunosuppressive agents: Design, synthesis, molecular docking and biological evaluation, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2015.12.020.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
(
E)-1,3-diphenyl-1H-pyrazole derivatives containing O-benzyl oxime
moiety as potential immunosuppressive agents: Design, synthesis,
molecular docking and biological evaluation
a
b
a
a
c
c
Xian-Hai Lv , Qing-Shan Li , Zi-Li Ren , Ming-Jie Chu , Jian Sun , Xin Zhang ,
c
c
a
Man Xing , Hai-Liang Zhu* , Hai-Qun Cao*
A series of novel pyrazole derivatives containing O-benzyl oxime moiety were
reported in this study for their significantly immunosuppressive activities. Compound
4
n exhibited the most potent inhibitory activity (IC =1.18 µM for lymph node cells
50
and IC = 0.28 µM for PI3Kγ).
5
0

ACCEPTED MANUSCRIPT
(
E)-1,3-diphenyl-1H-pyrazole derivatives containing O-benzyl
oxime moiety as potential immunosuppressive agents: Design,
synthesis, molecular docking and biological evaluation
a
b
a
a
c
c
Xian-Hai Lv , Qing-Shan Li , Zi-Li Ren , Ming-Jie Chu , Jian Sun , Xin Zhang ,
c
c
a
Man Xing , Hai-Liang Zhu* , Hai-Qun Cao*
a
b
College of Plant Protection, Anhui Agricultural University, Hefei 230036, P.R. China
School of Medical Engineering, Hefei University of Technology, Hefei 230009, P.R.
China
c
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
2
10093, P.R. China
Abstract: A series of novel (E)-1,3-diphenyl-1H-pyrazole derivatives containing
O-benzyl oxime moiety were firstly synthesized and their immunosuppressive
activities were evaluated. Among all the compounds, 4n exhibited the most potent
inhibitory activity (IC =1.18
µM for lymph node cells and IC50 = 0.28 µM for
5
0
PI3Kγ), which was comparable to that of positive control. Moreover, selected
compounds were tested for their inhibitory activities against IL-6 released in
ConA-simulated mouse lymph node cells, 4n exhibited the most potent inhibitory
ability. Furthermore, in order to study the preliminary mechanism of the compounds
with potent inhibitory activity, the RT-PCR experiment was performed to assay the
effect of selected compounds on mRNA expression of IL-6. Among them, compound
4
n strongly inhibited the expression of IL-6 mRNA.
Keywords: Immunosuppressant; Molecular docking; Oxime ethers; PI3Kγ; Pyrazole.
*
Corresponding author. Tel. & fax: +86 551 65786906, E-mail: haiquncao@163.com
Abbreviations: PI3K, Phosphoinositide 3-kinase; IL-6, interleukin-6; AKT, protein
kinase B; NaOAc, CH COONa; CsA, cyclosporin A; ConA, Concanavalin A; SAR,
3
1

ACCEPTED MANUSCRIPT
structure–activity relationships; CADD, computer assistant drug design; SI, selective
index, CC /IC ; mp, melting point; DMF, N,N-Dimethylformamide.
50
50
2

ACCEPTED MANUSCRIPT
1
. Introduction
Immunosuppressant has been one of the most prescribed drugs in the world,
widely used in the treatment of autoimmune diseases, including rheumatoid arthritis,
systemic lupus erythematosus, multiple sclerosis, type I diabetes mellitus, psoriasis,
and inflammatory bowel disease[1]. Although immunosuppressive drugs have been
used for the clinical treatment of autoimmune diseases, their side effects including
liver toxicity, nephro toxicity, infection, cardiovascular toxicity and others which limit
their clinical applications[2-4]. Thus, there is an urgent need for the design and
development of novel and less toxic anti-inflammatory agents.
Phosphoinositide 3-kinases (PI3Ks) phosphorylate the 3-hydroxyl of the head
group of phosphatidylinositol (PtdIns), and of phosphorylated derivatives of PtdIns
termed phosphoinositides[5]. PI3Ks are key components of the PI3K/AKT pathway
which plays essential roles in various cellular activities including cell proliferation,
cell survival, membrane trafficking, glucose transport, neurite outgrowth, membrane
ruffling, superoxide production[6, 7]. The dysregulation of the PI3K pathway is
associated with numerous cancers as well as inflammatory and autoimmune
diseases[8, 9]. PI3Ks are grouped into three classes (I, II, and III) according to their
structure preference and substrate specificity, class I PI3Ks are separated into two
subfamilies (IA and IB) depending on the receptors to which they couple[9-11].
Among these isoforms, PI3Kγ (the only isoform of class IB) plays a pivotal role in
inflammation, and it is involved in allergy, development of chronic inflammation,
autoimmune diseases[12, 13]. Therefore, there is important significance to discover
new agents that can influence PI3Kγ.
Figure 1.
The oxime ether moiety have received significant attention for this structural
scaffold makes up the core structure of numerous biologically active compounds in
chemical, food and pharmaceutical research. Some oxime-ether derivatives of
hydroxylated benzaldehydes and acetophenones were found to possess pronounced
nonsteroidal anti-inflammatory activities and favorable toxicities.[14-18]. On the
3

ACCEPTED MANUSCRIPT
other hand, it was found that a number of other 1,3-substituted 1H-pyrazole
derivatives (Figure 1. A, B and C) showed potent anti-inflammatory activity[19-21].
In continuation of our research work on the development of new anti-inflammatory
agents[22-24], we report the design and SAR of a series of novel
(E)-1,3-diphenyl-1H-pyrazole oxime ether derivatives (Figure 2) in this study.
Introducing the oxime ether moiety into the 1,3-substituted 1H-pyrazole skeleton
leads to a positive result in preliminary in silico screening. The binding model
generated by molecular modeling process revealed that the designed
(E)-1,3-diphenyl-1H-pyrazole oxime ether derivative was tightly embedded in the
binding sites of PI3Kγ via hydrogen bonds and π–cation interactions (Figure 3).
Figure 2.
Figure 3.
2
2
. Results and discussion
.1 Chemistry
Twenty
(E)-1,3-diphenyl-1H-pyrazole-4-carbaldehyde
O-benzyl
oxime
derivatives (4a–4t) were firstly synthesized and the general pathway outlined in
Scheme 1. The requisite intermediates 3 and 4 were prepared by the reaction of the
Vilsmeier–Haack reagent (DMF/POCl ) and phenyl hydrazones 1 and 2,
3
respectively[25]. Then target compounds 4a–4t were synthesized by direct
addition-elimination reactions of 1.0 equivalent of corresponding intermediates 3/4
and 1.2 equivalents of O-benzylhydroxylamine hydrochloride in the presence of
NaOAc as catalytic agent. Then compounds 4a–4t were obtained by subsequent
purification with recrystallisation in methanol. All of the synthetic compounds gave
satisfactory analytical and spectroscopic data, which were in full accordance with
their depicted structures. Compound 4m was successfully crystallized and its structure
was determined by single-crystal X-ray diffraction analysis. The structure was solved
2
by direct methods and refined on F by full-matrix least-squares methods using
[
26]
SHELXL-97 . The crystal data, data collection, and refinement parameter for the
4

ACCEPTED MANUSCRIPT
compound 4m are listed in Table 1, the crystal structure is shown in Figure 4.
Scheme 1
Table 1
Figure 4
2
.2 Cytotoxicity and inhibitory activity
The inhibitory activity of tested compound is sometimes a result of their toxic
effect and consequently might cause an erroneous conclusion, so we performed
cytotoxicity test before inhibitory activity assay of target compounds.
(E)-1,3-diphenyl-1H-pyrazole oxime ether derivatives 4a–4t were screened for their
cyotoxicities on lymph node cells with cyclosporin A (CsA) as the positive control,
the cytotoxicity of each compound was expressed as the CC values which were
5
0
summarized in Table 2. The results depicted that most of the compounds were low
toxic.
Table 2
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. Next, all the synthetic (E)-1,3-diphenyl-1H-pyrazole oxime ether derivatives
4
a–4t were tested for evaluated for their in vitro inhibitory activity on murine
lymphocyte proliferation induced by Concanavalin (ConA). The
A
immunosuppressive activity of each compound was expressed as the concentration of
the compound that inhibited ConA stimulated T cells proliferation to 50 % (IC ) of
5
0
the control value, and the results were summarized in Table 2. In general, it was
observed that a number of synthesized pyrazole oxime ether analogues displayed
potent immunosuppressive activities in the low micromolar range. Inspection of the
chemical structure of the compounds 4a–4t suggested that it could be divided into two
subunits: A and B rings. A comparison of the para substituents on the A-ring
demonstrated that an electron-withdrawing group have improved immunosuppressive
activity and the potency order is F > Cl > OMe >H > Me. SAR also suggested that
compounds bearing the same substituents on phenyl ring A exhibited distinct
5

ACCEPTED MANUSCRIPT
inhibitory activity due to the difference of the substituents on the ring B. Among these
compounds, compounds with ortho electron-withdrawing substitution (4m-4t)
showed stronger anti-inflammatory activities in the following order: F > Cl > H.
Furthermore, introduction of substituent to para-position of ring B results in less
active analog (4d, 4h, 4p and 4t). Among all the compounds, 4n and 4o exhibited the
most potent inhibitory activities (2.16 and 1.74
µM, respectively), which owing
para-F group in the A ring and para-F/2,4-Cl in the B ring. Furthermore, the
cytotoxicity and SI of the compound 4n (SI=219.59) and 4o (SI=108.35) have
absolute advantage compared to others, which demonstrated that these compounds
could selectively inhibited the proliferation of ConA stimulated T cells. Considering
the high inhibitory potency and low toxicity, it can be concluded that compound 4n
and 4o could be potential anti-inflammatory agents.
2
.3 PI3Kγ enzymatic activity
Based on the data of cellular immunosuppressive activities obtained before, the
top 7 compounds (4n, 4p, 4o, 4r, 4s, 4b and 4t) and the bottom 3 compounds (4g, 4h
and 4e) were selected for evaluate their PI3Kγ inhibitory activities compared with
LY294002[27]. The results were summarized in Table 3. Among the tested
compounds, 4n showed the most potent inhibitory activity with IC50 of 0.28
µM,
which was comparable to the positive control LY294002 with IC of 7.2 M. As
µ
5
0
shown in Table 3, for tested pyrazole oxime ether derivatives, the SAR of PI3Kγ
inhibitory activities were consistent with their inhibitory activities of ConA stimulated
T cells proliferation, all the bottom compounds showed poor PI3Kγ inhibitory
activities while most of the top compounds displayed potent inhibitory activities. The
results indicated that the potent anti-inflammatory activities of the synthetic
compounds were probably correlated to their PI3Kγ inhibitory activities.
2
.4 IL-6 inhibitory activity
IL-6 (interleukin-6), is an important multifunctional pro-inflammatory cytokine,
contributes to the initiation and extension of the inflammatory process and considered
6

ACCEPTED MANUSCRIPT
as a central mediator in a range of inflammatory diseases[28-30]. It has been found
that the activation of PI3K signaling pathway leading to up-regulation of IL-6
expression[31].
To determine whether target compounds suppress activation of AKT (a key
downstream effector of PI3K and important node in the PI3K/AKT signaling
pathway), selected compounds 4n, 4o, 4p and 4r were tested for their inhibitory
activities against IL-6 released in ConA-simulated mouse lymph node cells. As shown
in Figure 5.A, among these seven compounds, 4n exhibited the highest inhibitory
effect against ConA-induced IL-6 expression. Furthermore, in order to study the
preliminary mechanism of the compounds with potent inhibitory activity, the RT-PCR
experiment was performed to assay the effect of four selected compounds on mRNA
expression of IL-6. The RT-PCR results were summarized in Figure 5.B. Among them,
compound 4n strongly inhibited the expression of IL-6 mRNA. Combining with the
results of PI3Kγ inhibitory activity assay, it can be inferred that compound 4n might
inhibit IL-6 production through blocking activation of PI3K/AKT signaling pathway.
3
. Conclusions
In conclusion, a series of pyrazole derivatives containing O-benzyl oxime moiety
were firstly synthesized and evaluated for their immunosuppressive activity. It is
worth noticing that compound 4n demonstrated the most outstanding
immunosuppressive effect in vitro with IC of 1.18
µM which was comparable to
5
0
that of cyclosporin A (IC = 0.57
µM). Meanwhile, compounds 4n, 4p, 4o and 4r that
5
0
exhibited significant immunosuppressive activities were evaluated for their PI3Kγ
inhibitory activities. The results showed that compound 4n displayed the most potent
inhibitory activity (IC = 0.28 µM), which was more potent than that of LY294002.
5
0
Moreover, compounds 4n, 4p, 4o and 4r were assayed for PI3K/AKT signaling
pathway inhibition using the ELISA assay. We examined the compounds with potent
inhibitory activities against IL-6 released in ConA-simulated mouse lymph node cells.
4
n exhibited the highest inhibitory effect against ConA-induced IL-6 expression.
7

ACCEPTED MANUSCRIPT
Furthermore, in order to study the preliminary mechanism of the compounds with
potent inhibitory activity, the RT-PCR experiment was performed to assay the effect
of 4n, 4p, 4o and 4r on mRNA expression of IL-6.The results showed that the
apoptosis induced by 4p might be mediated by the inhibition of PI3K/AKT signaling
pathway. Molecular docking study indicated that compound 4n was nicely bound to
the PI3Kγ with two hydrogen bonds. All the results showed the great potential of
compound 4n as an immunosuppressant targeting PI3Kγ.
Acknowledgement
This work was supported by National Natural Science Foundation of China
(No.21302002) and Anhui Provincial Natural Science Foundation (1408085QB33,
1
508085MB33).
4
4
. Experimental section
.1. General
All of the synthesized compounds were chemically characterized by thin layer
1
chromatography (TLC), proton nuclear magnetic resonance ( H NMR) and elemental
1
microanalyses (CHN). H NMR spectra were measured on a Bruker AV-300 or a
o
Agilent DD2 600Hz spectrometer at 25 C. Chemical shifts were reported in ppm (δ)
using the residual solvent line as internal standard and referenced to Me Si. Splitting
4
patterns were designed as s, singlet; d, doublet; t, triplet; m, multiplet. ESI-MS spectra
were recorded on a Mariner System 5304 Mass spectrometer. Elemental analyses
were performed on a CHN-O-Rapid instrument and were within ±0.4% of the
theoretical values. Melting points were determined on a XT4 MP apparatus (Taike
Corp., Beijing, China) and were as read. Analytic thin-layer chromatography was
performed on the glass backed silica gel sheets (silica gel 60 A GF254). All
compounds were detected using UV light (254 nm or 365 nm).
4
.2. General method for the preparation of target compounds 3a-3e
The starting material 1,3-diphenyl-1H-pyrazole-4-carbaldehyde (3a-3e) were
8

ACCEPTED MANUSCRIPT
synthesized as following: para-substituted acetophenone (1a-1e) (20 mmol) interact
with phenylhydrazine hydrochloride (25 mmol) couple with sodium acetate (40 mmol)
in anhydrous ethanol to form 1-phenyl-2-(1- phenylethylidene) hydrazine (2a-2e),
which was then dissolved in a cold mixed solution of DMF (20 mL) and POCl (16
3
o
mL), stirred at 50-60 C for 5 h. The resulting mixture was poured into ice-cold water,
a saturated solution of sodium hydroxide was added to neutralize the mixture, and the
solid precipitate was filtered, washed with water, dried and recrystallized from
ethanol to give the compounds 3a-3e.
4
.3. General procedure for (E)-1,3-diphenyl-1H-pyrazole-4-carbaldehyde
O-benzyl oxime derivatives 4a-4t
To a solution of 3a-3e (1 mmol) in anhydrous ethanol (10 mL), corresponding
O-benzylhydroxylamine hydrochloride (1 mmol) and NaOAc (1.2 mmol) were added
and the resulting solution was stirred at the room temperate for 3 h. The mixture was
concentrated and taken in ethyl acetate (20 mL), washed with water (20 mL),
saturated sodium chloride solution (20 mL) and dried over sodium sulfate. The
resulting solution was concentrated and the purification of the residue by
recrystallization from ethanol yielded the desired compounds 4a–4t.
4
.3.1 (E)-1,3-diphenyl-1H-pyrazole-4-carbaldehyde O-benzyl oxime (4a)
o
1
White solid, yield: 80%. Mp: 73-75 C. H NMR (300 MHz, CDCl ): 5.34 (s, 2H);
3
7
8
7
.30-7.53 (m, 11H); 7.66 (d, J=6.39Hz, 2H); 7.78 (d, J=7.83Hz, 2H); 8.36 (s, 1H);
+
.84 (s, 1H). MS (ESI): 354.5 (C H N O, [M+H] ). Anal. Calcd for C H N O: C,
2
3
19
3
23 19
3
8.16; H, 5.42; N, 11.89; Found: C, 78.18; H, 5.46; N, 11.92%.
4
.3.2 (E)-1,3-diphenyl-1H-pyrazole-4-carbaldehyde O-(2-fluorobenzyl) oxime (4b)
o
1
White solid, yield: 73%. Mp: 55-57 C. H NMR (300 MHz, CDCl ): 5.40 (s, 2H);
3
7
8
.15 (m, 1H);7.31-7.52 (m, 9H); 7.65 (d, J=7.68Hz, 2H); 7.76 (d, J=7.68Hz, 2H);
+
.35 (s, 1H); 8.86 (s, 1H). MS (ESI): 372.8 (C H FN O, [M+H] ). Anal. Calcd for
2
3
18
3
C H FN O: C, 74.42; H, 4.83; N, 11.40; Found: C, 74.38; H, 4.88; N, 11.31%.
23
18
3
9

ACCEPTED MANUSCRIPT
4
.3.3 (E)-1,3-diphenyl-1H-pyrazole-4-carbaldehyde O-(2-chlorobenzyl) oxime (4c)
o
1
White solid, yield: 80%. Mp: 74-76 C. H NMR (300 MHz, CDCl ): 5.45 (s, 2H);
3
7
8
.27 (m, 1H);7.33-7.53 (m, 9H); 7.66 (d, J=6.39Hz, 2H); 7.78 (d, J=7.50Hz, 2H);
+
.36 (s, 1H); 8.93 (s, 1H). MS (ESI): 388.9 (C H ClN O, [M+H] ). Anal. Calcd for
2
3
18
3
C H ClN O: C, 71.22; H, 4.68; N, 10.83; Found: C, 72.35; H, 4.72; N, 10.96%.
23
18
3
4
.3.4 (E)-1,3-diphenyl-1H-pyrazole-4-carbaldehyde O-(2,4-dichlorobenzyl) oxime (4d)
o
1
White solid. Yield: 87%. Mp: 103-104 C. H NMR (300 MHz, CDCl ): 5.40 (s, 2H);
3
7
.24 (d, J=2.01Hz, 1H);7.28-7.52 (m, 8H); 7.65 (d, J=6.42Hz, 2H); 7.77 (d, J=7.68Hz,
+
2
H); 8.34 (s, 1H); 8.91 (s, 1H). MS (ESI): 422.7 (C H Cl N O, [M+H] ). Anal.
2
3
17
2
3
Calcd for (C H Cl N O: C, 65.41; H, 4.06; N, 9.95; Found: C, 65.57; H, 4.12; N,
2
3
17
2
3
1
0.03%.
4
.3.5 (E)-1-phenyl-3-(p-tolyl)-1H-pyrazole-4-carbaldehyde O-benzyl oxime (4e)
o
1
White solid. Yield: 79%. Mp: 70-72 C. H NMR (300 MHz, CDCl ): 2.42 (s, 3H);
3
5
.34 (s, 2H); 7.27-7.57 (m, 10H); 7.73 (d, J=8.25Hz, 2H); 7.78 (d, J=8.04Hz, 2H);
+
8
.35 (s, 1H); 8.83 (s, 1H). MS (ESI): 368.3 (C H N O, [M+H] ). Anal. Calcd for
24
21
3
C H N O: C, 78.45; H, 5.76; N, 11.44; Found: C, 78.55; H, 5.77; N, 11.56%.
24
21
3
4
.3.6 (E)-1-phenyl-3-(p-tolyl)-1H-pyrazole-4-carbaldehyde O-(2-fluorobenzyl) oxime
4f)
White solid. Yield: 69%. Mp: 40-42 C. H NMR (300 MHz, CDCl ): 2.47 (s, 3H);
(
o
1
3
5
.19 (s, 2H); 7.23 (m, 1H);7.30-7.60 (m, 8H); 7.88 (d, J=8.40Hz, 2H); 7.94 (d,
+
J=8.40Hz, 2H); 8.26 (s, 1H); 8.90 (s, 1H). MS (ESI): 386.9 (C H FN O, [M+H] ).
2
4
20
3
Anal. Calcd for C H FN O: C, 74.79; H, 5.23; N, 10.90; Found: C, 74.84; H, 5.00;
2
4
20
3
N, 11.15 %.
4
.3.7 (E)-1-phenyl-3-(p-tolyl)-1H-pyrazole-4-carbaldehyde O-(2-chlorobenzyl) oxime
(4g)
1
0

ACCEPTED MANUSCRIPT
o
1
White solid. Yield: 79%. Mp: 56-58 C. H NMR (300 MHz, DMSO-d ): 2.48 (s,
6
3
H); 5.24 (s, 2H); 7.28 (m, 1H); 7.30-7.61 (m, 8H); 7.89 (d, J=8.22Hz, 2H); 7.95 (d,
+
J=8.22Hz, 2H); 8.32 (s, 1H); 8.87 (s, 1H). MS (ESI): 403.0 (C H ClN O, [M+H] ).
2
4
20
3
Anal. Calcd for C H ClN O: C, 71.73; H, 5.02; N, 10.46; Found: C, 71.82; H, 4.98;
2
4
20
3
N, 10.62%.
4
.3.8 (E)-1-phenyl-3-(p-tolyl)-1H-pyrazole-4-carbaldehyde O-(2,4-dichlorobenzyl)
oxime (4h)
White solid. Yield: 85%. Mp: 103-104 C. H NMR (300 MHz, CDCl ): 2.42 (s, 3H);
o
1
3
5
.39 (s, 2H); 7.25 (d, J=2.02Hz, 1H);7.27-7.52 (m, 7H); 7.55 (d, J=7.68Hz, 2H); 7.77
(
d, J=7.86Hz, 2H); 8.32 (s, 1H); 8.87 (s, 1H). MS (ESI): 437.2 (C H Cl N O,
2
4
19
2
3
+
[
M+H] ). Anal. Calcd for C H Cl N O: C, 66.06; H, 4.39; N, 9.63; Found: C, 66.28;
24 19 2 3
H, 4.19; N, 9.75%.
4
.3.9 (E)-3-(4-methoxyphenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde O-benzyl oxime
(4i)
o
1
White solid. Yield: 82%. Mp: 78-80 C. H NMR (300 MHz, CDCl ): 3.86 (s, 3H);
3
5
.34 (s, 2H); 6.99 (t, J=8.79Hz, 2H); 7.30-7.61 (m, 8H); 7.73 (d, J=7.68Hz, 2H); 7.77
+
(
d, J=7.89Hz, 2H); 8.35 (s, 1H); 8.82 (s, 1H). MS (ESI): 384.3 (C H N O , [M+H] ).
2
4
21
3
2
Anal. Calcd for C H N O : C, 75.18; H, 5.52; N, 10.96; Found: C, 75.33; H, 5.65; N,
2
4
21
3
2
11.06%.
4
.3.10
O-(2-fluorobenzyl) oxime (4j)
White solid. Yield: 70%. Mp: 88-89 C. H NMR (300 MHz, CDCl ): 3.86 (s, 3H);
(E)-3-(4-methoxyphenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde
o
1
3
5
.40 (s, 2H); 6.99 (t, J=8.40Hz, 2H); 7.15 (m, 1H);7.32-7.50 (m, 6H); 7.59 (d,
J=8.61Hz, 2H); 7.75 (d, J=7.68Hz, 2H); 8.28 (s, 1H); 8.84 (s, 1H). MS (ESI): 402.7
+
(
C H FN O , [M+H] ). Anal. Calcd for C H FN O : C, 71.81; H, 5.02; N, 10.47;
2
4
20
3
2
24 20
3
2
Found: C, 71.92; H, 5.00; N, 10.63%.
1
1

ACCEPTED MANUSCRIPT
4
.3.11
(E)-3-(4-methoxyphenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde
O-(2-chlorobenzyl) oxime (4k)
White solid. Yield: 78%. Mp: 79-81 C. H NMR (300 MHz, CDCl ): 3.87 (s, 3H);
o
1
3
5
.44 (s, 2H); 7.02 (t, J=8.58Hz, 2H); 7.28 (m, 1H); 7.32-7.47 (m, 6H); 7.67 (d,
J=8.68Hz, 2H); 7.80 (d, J=8.58Hz, 2H); 8.42 (s, 1H); 8.91 (s, 1H). MS (ESI): 418.7
+
(
C H ClN O , [M+H] ). Anal. Calcd for C H ClN O : C, 68.98; H, 4.82; N, 10.06;
2
4
20
3
2
24 20
3
2
Found: C, 69.03; H, 4.78; N, 10.16%.
4
.3.12
O-(2,4-dichlorobenzyl) oxime (4l)
White solid. Yield: 87%. Mp: 108-110 C. H NMR (300 MHz, CDCl ): 3.86 (s, 3H);
(E)-3-(4-methoxyphenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde
o
1
3
5
.39 (s, 2H); 7.00 (t, J=8.76Hz, 2H); 7.27 (m, 1H);7.31-7.50 (m, 5H); 7.59 (d,
J=7.38Hz, 2H); 7.77 (d, J=7.50Hz, 2H); 8.30 (s, 1H); 8.86 (s, 1H). MS (ESI): 452.7
+
(
C H Cl N O , [M+H] ). Anal. Calcd for C H Cl N O : C, 63.73; H, 4.23; N, 9.29;
2
4
19
2
3
2
24 19
2
3
2
Found: C, 63.87; H, 4.09; N, 9.36%.
4
.3.13 (E)-3-(4-fluorophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde O-benzyl oxime
4m)
Colorless crystal. Yield: 81%. Mp: 73-75 C. H NMR (300 MHz, DMSO-d ): 5.13 (s,
(
o
1
6
2
H); 7.28 (t, J=8.97Hz, 2H); 7.34-7.55 (m, 8H); 7.75 (q, J=5.67Hz, 2H); 7.94 (t,
1
3
J=8.04Hz, 2H); 8.28 (s, 1H); 8.88 (s, 1H). C NMR (600 MHz, DMSO-d ):163.51;
6
1
9
61.88;150.47;142.41;139.33;130.88;130.83;130.05;128.77;128.66;128.24;127.45;11
.64;119.15;115.99;113.82;75.65. The spectra were presented in supplementary
+
material. MS (ESI): 372.8 (C H FN O, [M+H] ). Anal. Calcd for C H FN O : C,
2
3
18
3
23 18
3
2
7
4.38; H, 4.88; N, 11.31; Found: C, 74.42; H, 4.65; N, 11.46%.
4
.3.14
(E)-3-(4-fluorophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde
O-(2-fluorobenzyl) oxime (4n)
White solid. Yield: 65%. Mp: 89-90 C. H NMR (300 MHz, DMSO-d ): 5.18 (s, 2H);
o
1
6
7
.20 (m, 1H); 7.25 (t, J=8.79Hz, 2H); 7.32-7.59(m, 6H); 7.72 (d, J=8.68Hz, 2H); 7.94
1
2

ACCEPTED MANUSCRIPT
1
3
(d, J=7.68Hz, 2H); 8.28 (s, 1H); 8.87 (s, 1H).
C NMR (600 MHz,
DMSO-d ):163.52;161.89;160.07;150.48;142.75;139.31;131.66;131.63;130.89;130.8
6
4
;130.69;130.05;128.95;127.47;124.85;119.16;115.97;113.68;69.46. The spectra were
+
presented in supplementary material. MS (ESI): 390.7 (C H F N O, [M+H] ).
23
17
2
3
Anal. Calcd for C H F N O: C, 70.94; H, 4.40; N, 10.79; Found: C, 71.02; H, 4.23;
2
3
17
2
3
N, 10.83%.
4
.3.15
O-(2-chlorobenzyl) oxime (4o)
White solid. Yield: 78%. Mp: 75-77 C. H NMR (300 MHz, DMSO-d ): 5.22 (s, 2H);
(E)-3-(4-fluorophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde
o
1
6
7
7
1
9
.29 (t, J=8.58Hz, 2H); 7.37 (m, 3H); 7.47-7.55 (m, 4H); 7.77 (d, J=6.75Hz, 2H);
1
3
.94 (d, J=7.47Hz, 2H); 8.33 (s, 1H); 8.88 (s, 1H). C NMR (600 MHz, DMSO-d ):
6
63.52;161.90;150.54;142.95;139.31;132.88;130.90;130.88;130.85;130.10;130.05;12
.71;128.86;127.68;127.47;119.16;116.01;113.63;72.84. The spectra were presented
+
in supplementary material. MS (ESI): 406.2 (C H ClFN O, [M+H] ). Anal. Calcd
2
3
17
3
for C H ClFN O: C, 68.07; H, 4.22; N, 10.35; Found: C, 68.14; H, 4.37; N, 10.46%.
2
3
17
3
4
.3.16
O-(2,4-dichlorobenzyl) oxime (4p)
White solid. Yield: 81%. Mp: 135-136 C. H NMR (300 MHz, CDCl ): 5.39 (s, 2H);
(E)-3-(4-fluorophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde
o
1
3
7
.17 (t, J=8.58Hz, 2H); 7.27 (m, 1H); 7.35-7.51 (m, 5H); 7.63 (d, J=8.61Hz, 2H);
.76 (d, J=7.50Hz, 2H); 8.31 (s, 1H); 8.87 (s, 1H). MS (ESI): 441.9 (C H Cl FN O,
7
2
3
16
2
3
+
[
M+H] ). Anal. Calcd for (C H Cl FN O: C, 62.74; H, 3.66; N, 9.54; Found: C,
23 16 2 3
6
2.82; H, 3.50; N, 9.61%.
4
.3.17 (E)-3-(4-chlorophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde O-benzyl oxime
(4q)
o
1
White solid. Yield: 83%. Mp: 82-84 C. H NMR (300 MHz, DMSO-d ): 5.13 (s, 2H);
6
7
.32 (t, J=7.50Hz, 2H); 7.34-7.58 (m, 8H); 7.75 (d, J=8.40Hz, 2H); 7.94 (d, J=8.04Hz,
+
2
H); 8.30 (s, 1H); 8.88 (s, 1H). MS (ESI): 388.4 (C H ClN O, [M+H] ). Anal. Calcd
23
18
3
1
3

ACCEPTED MANUSCRIPT
for C H ClN O : C, 71.22; H, 4.68; N, 10.83; Found: C, 71.29; H, 4.75; N, 11.02%.
2
3
18
3
2
4
.3.18
O-(2-fluorobenzyl) oxime (4r)
White solid. Yield: 65%. Mp: 75-77 C. H NMR(300 MHz, CDCl ): 5.24 (s, 2H);
(E)-3-(4-chlorophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde
o
1
3
7
.15 (m, 1H); 7.32 (t, J=7.50Hz, 2H); 7.40-7.51(m, 6H); 7.62 (d, J=7.11Hz, 2H); 7.75
(
d, J=7.50Hz, 2H); 8.32 (s, 1H); 8.85 (s, 1H). MS (ESI): 406.2 (C H ClFN O,
2
3
17
3
+
[
M+H] ). Anal. Calcd for C H F N O: C, 68.07; H, 4.22; N, 10.35; Found: C, 68.16;
23 17 2 3
H, 4.07; N, 10.51%.
4
.3.19
O-(2-chlorobenzyl) oxime (4s)
White solid. Yield: 80%. Mp: 82-84 C. H NMR (300 MHz, DMSO-d ): 5.22 (s, 2H);
(E)-3-(4-chlorophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde
o
1
6
7
.35 (t, J=6.45Hz, 2H); 7.43 (m, 3H); 7.47-7.59 (m, 4H); 7.74 (d, J=8.61Hz, 2H);
.94 (d, J=7.68Hz, 2H); 8.35 (s, 1H); 8.89 (s, 1H). MS (ESI): 423.6 (C H Cl N O,
7
2
3
17
2
3
+
[
M+H] ). Anal. Calcd for C H Cl N O: C, 65.41; H, 4.06; N, 9.95; Found: C, 65.51;
23 17 2 3
H, 4.23; N, 10.07%.
4
.3.20
O-(2,4-dichlorobenzyl) oxime (4t)
White solid. Yield: 85%. Mp: 147-149 C. H NMR (300 MHz, CDCl ): 5.39 (s, 2H);
(E)-3-(4-chlorophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde
o
1
3
7
.35-7.49 (m, 8H); 7.61 (d, J=8.61Hz, 2H); 7.74 (d, J=7.50Hz, 2H); 8.31 (s, 1H); 8.88
+
(
s, 1H). MS (ESI): 457.4 (C H Cl N O, [M+H] ). Anal. Calcd for C H Cl N O: C,
2
3
16
3
3
23 16
3
3
6
0.48; H, 3.53; N, 9.20; Found: C, 60.56; H, 3.61; N, 9.33%.
4
.4 Crystal structure determination
Crystal structure determination of compound 4m was carried out on a Bruker D8
VENTURE PHOTON equipped with graphite-monochromated MoKα (λ = 0.71073
2
Ǻ) radiation. The structure was solved by direct methods and refined on F by
[
26]
full-matrix least-squares methods using SHELX-97 . All the non-hydrogen atoms
1
4

ACCEPTED MANUSCRIPT
were refined anisotropically. All the hydrogen atoms were placed in calculated
positions and were assigned fixed isotropic thermal parameters at 1.2 times the
equivalent isotropic U of the atoms to which they are attached and allowed to ride on
their respective parent atoms.
4
.5 Molecular docking
The PI3Kγ-LXX protein–ligand complex crystal structure (PDB code: 3L54)
was used as the target structure in this approach. The molecular docking procedure
was performed by using CDOCKER protocol for receptor-ligand interactions section
of DS 3.1 (Discovery Studio 3.1, Accelrys, Inc., San Diego, CA)[32, 33].
4
.6 Cellular activity
Cytotoxicities and inhibitory activities of compounds 4a-4t on lymph node cells
were evaluated, as described in previously study[22].
4
.7 PI3Kγ inhibitory assay
Kinase assays of selected compounds (4n, 4p, 4o, 4r, 4s, 4b, 4t, 4g, 4h and 4e)
for IC value determinations with PI3Kγ (Minneapolis, MN) were carried out, as
5
0
previously described[22].
4
.8 IL-6 inhibitory assay
Mouse lymph node cells were incubated in DMEM media (Gibco) supplemented
o
with 10% FBS, 100 U/mL penicillin, and 100 ꢀg/mL streptomycin at 37 C with 5%
CO . Cells were pretreated with 5 ꢀM of selected compounds 4n, 4o, 4p, 4r or vehicle
2
control for 2 h, then treated with ConA (5 ꢀg/mL) for 22 h. The culture media
collected were centrifuged at 1000 rpm for 10 min. The levels of IL-6 in the media
were determined by ELISA using mouse IL-6 ELISA Kits (BOSTER, USA) and
strictly according to the manufacturer’s instructions. Next, selected four compounds
were tested with potent inhibitory activities against mRNA expression of IL-6 in
ConA-simulated mouse lymph node cells using the RT-PCR assay
.
1
5

ACCEPTED MANUSCRIPT
Reference
[
1] J.H. Cho, P.K. Gregersen, Genomics and the Multifactorial Nature of Human Autoimmune Disease,
New Engl. J. Med., 365 (2011) 1612-1623.
2] S.J. Chen, Y.L. Wang, H.C. Fan, W.T. Lo, C.C. Wang, H.K. Sytwu, Current Status of the
[
Immunomodulation and Immunomediated Therapeutic Strategies for Multiple Sclerosis, Clin. Dev.
Immunol., (2012).
[3] C.A. Dinarello, Anti-inflammatory Agents: Present and Future, Cell, 140 (2010) 935-950.
[4] P.Q. Eichacker, C. Parent, A. Kalil, C. Esposito, X. Cui, S.M. Banks, E.P. Gerstenberger, Y. Fitz, R.L.
Danner, C. Natanson, Risk and the efficacy of antiinflammatory agents: retrospective and confirmatory
studies of sepsis, Am. J. Respir. Crit. Care. Med., 166 (2002) 1197-1205.
[5] B. Vanhaesebroeck, S.J. Leevers, K. Ahmadi, J. Timms, R. Katso, P.C. Driscoll, R. Woscholski, P.J.
Parker, M.D. Waterfield, Synthesis and function of 3-phosphorylated inositol lipids, Annu. Rev.
Biochem., 70 (2001) 535-602.
[6] L.C. Cantley, The phosphoinositide 3-kinase pathway, Science, 296 (2002) 1655-1657.
[7] S. Ward, Y. Sotsios, J. Dowden, I. Bruce, P. Finan, Therapeutic potential of review phosphoinositide
3
-kinase inhibitors, Chem. Biol., 10 (2003) 207-213.
[
(
[
8] T.L. Yuan, L.C. Cantley, PI3K pathway alterations in cancer: variations on a theme, Oncogene, 27
2008) 5497-5510.
9] J.G. Foster, M.D. Blunt, E. Carter, S.G. Ward, Inhibition of PI3K Signaling Spurs New Therapeutic
Opportunities in Inflammatory/Autoimmune Diseases and Hematological Malignancies, Pharmacol.
Rev., 64 (2012) 1027-1054.
[
10] M.P. Wymann, L. Pirola, Structure and function of phosphoinositide 3-kinases, Biochim. Biophys.
Acta., 1436 (1998) 127-150.
11] B. Vanhaesebroeck, J. Guillermet-Guibert, M. Graupera, B. Bilanges, The emerging mechanisms of
isoform-specific PI3K signalling, Nat. Rev. Mol. Cell Biol., 11 (2010) 329-341.
12] A.M. Condliffe, K. Davidson, K.E. Anderson, C.D. Ellson, T. Crabbe, K. Okkenhaug, B.
[
[
Vanhaesebroeck, M. Turner, L. Webb, M.P. Wymann, E. Hirsch, T. Ruckle, M. Camps, C. Rommel, S.P.
Jackson, E.R. Chilvers, L.R. Stephens, P.T. Hawkins, Sequential activation of class IB and class IA PI3K is
important for the primed respiratory burst of human but not murine neutrophils, Blood, 106 (2005)
1
432-1440.
[13] M.M. Al-Alwan, K. Okkenhaug, B. Vanhaesebroeck, J.S. Hayflick, A.J. Marshall, Requirement for
phosphoinositide 3-kinase p110 delta signaling in B cell antigen receptor-mediated antigen
presentation, J. Immunol., 178 (2007) 2328-2335.
[14] S.M. Johnson, H.M. Petrassi, S.K. Palaninathan, N.N. Mohamedmohaideen, H.E. Purkey, C. Nichols,
K.P. Chiang, T. Walkup, J.C. Sacchettini, K.B. Sharpless, J.W. Kelly, Bisaryloxime ethers as potent
inhibitors of transthyretin amyloid fibril formation, J. Med. Chem., 48 (2005) 1576-1587.
[
15] D.P. Jindal, R. Chattopadhaya, S. Guleria, R. Gupta, Synthesis and antineoplastic activity of
-alkylaminoethyl derivatives of various steroidal oximes, Eur. J. Med. Chem., 38 (2003) 1025-1034.
16] A. Balsamo, M.C. Breschi, M. Chini, P. Domiano, G. Giannaccini, A. Lucacchini, B. Macchia, M.
2
[
Macchia, C. Manera, A. Martinelli, C. Martini, E. Martinotti, P. Nieri, A. Rossello, CONFORMATIONALLY
RESTRAINED BETA-BLOCKING OXIME ETHERS - SYNTHESIS AND BETA-ADRENERGIC PROPERTIES OF
DIASTEREOISOMERIC ANTI AND SYN 2-(5'-ISOXAZOLIDINYL)-ETHANOLAMINES, Eur. J. Med. Chem., 27
1
6

ACCEPTED MANUSCRIPT
(
1992) 751-764.
17] R.A. Chapman, C.R. Harris, Determination of residues of methomyl and oxamyl and their oximes
[
in crops by gas-liquid chromatography of oxime trimethylsilyl ethers, J. Chrom., 171 (1979) 249-262.
18] C. Ramalingan, Y.T. Park, S. Kabilan, Synthesis, stereochemistry, and antimicrobial evaluation of
substituted piperidin-4-one oxime ethers, Eur. J. Med. Chem., 41 (2006) 683-696.
19] A.P. Keche, G.D. Hatnapure, R.H. Tale, A.H. Rodge, V.M. Kamble, Synthesis, anti-inflammatory and
[
[
antimicrobial evaluation of novel 1-acetyl-3,5-diaryl-4,5-dihydro (1H) pyrazole derivatives bearing urea,
thiourea and sulfonamide moieties, Bioorg. Med. Chem. Lett., 22 (2012) 6611-6615.
[
20] K.S. Girisha, B. Kalluraya, V. Narayana, Padmashree, Synthesis and pharmacological study of
-acetyl/propyl-3-aryl-5-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)-2- pyrazoline, Eur. J. Med.
Chem., 45 (2010) 4640-4644.
21] E. Bansal, V.K. Srivastava, A. Kumar, Synthesis and anti-inflammatory activity of
-acetyl-5-substitute daryl-3-(beta-aminonapthyl)-2-pyrazolines and beta-(substituted aminoethyl)
amidonaphthalenes, Eur. J. Med. Chem., 36 (2001) 81-92.
22] J.-F. Tang, X.-H. Lv, X.-L. Wang, J. Sun, Y.-B. Zhang, Y.-S. Yang, H.-B. Gong, H.-L. Zhu, Design,
1
[
1
[
synthesis, biological evaluation and molecular modeling of novel 1,3,4-oxadiazole derivatives based on
Vanillic acid as potential immunosuppressive agents, Bioorg. Med. Chem., 20 (2012) 4226-4236.
[23] R. Yan, Z.-M. Zhang, X.-Y. Fang, Y. Hu, H.-L. Zhu, Synthesis, molecular docking and biological
evaluation of 1,3,4-oxadiazole derivatives as potential immunosuppressive agents, Bioorg. Med.
Chem., 20 (2012) 1373-1379.
[24] Z.-M. Zhang, X.-W. Zhang, Z.-Z. Zhao, R. Yan, R. Xu, H.-B. Gong, H.-L. Zhu, Synthesis, biological
evaluation and molecular docking studies of 1,3,4-oxadiazole derivatives as potential
immunosuppressive agents, Bioorg. Med. Chem., 20 (2012) 3359-3367.
[25] T.D. Penning, J.J. Talley, S.R. Bertenshaw, J.S. Carter, P.W. Collins, S. Docter, M.J. Graneto, L.F. Lee,
J.W. Malecha, J.M. Miyashiro, R.S. Rogers, D.J. Rogier, S.S. Yu, G.D. Anderson, E.G. Burton, J.N. Cogburn,
S.A. Gregory, C.M. Koboldt, W.E. Perkins, K. Seibert, A.W. Veenhuizen, Y.Y. Zhang, P.C. Isakson, Synthesis
and biological evaluation of the 1,5-diarylpyrazole class of cyclooxygenase-2 inhibitors: Identification
of 4- 5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl benzenesulfona mide (SC-58635,
Celecoxib), J. Med. Chem., 40 (1997) 1347-1365.
[
26] G.M. Sheldrick, SHELXTL-97, Program for Crystal Structure Solution and Refinement, University of
Göttingen, Göttingen, Germany, 1997.
27] C.J. Vlahos, W.F. Matter, K.Y. Hui, R.F. Brown, A specific inhibitor of phosphatidylinositol 3-kinase,
-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), J. Biol. Chem., 269 (1994) 5241-5248.
28] H. Tilg, C.A. Dinarello, J.W. Mier, IL-6 and APPs: anti-inflammatory and immunosuppressive
mediators, Immunol. Today, 18 (1997) 428-432.
[
2
[
[29] S.M. Opal, V.A. DePalo, Anti-inflammatory cytokines, Chest, 117 (2000) 1162-1172.
[30] K. Ishihara, T. Hirano, IL-6 in autoimmune disease and chronic inflammatory proliferative disease,
Cytokine Growth Factor Rev., 13 (2002) 357-368.
31] C.H. Tang, D.Y. Lu, R.S. Yang, H.Y. Tsai, M.C. Kao, W.M. Fu, Y.F. Chen, Leptin-induced IL-6 production
[
is mediated by leptin receptor, insulin receptor substrate-1, phosphatidylinositol 3-kinase, Akt,
NF-kappa B, and p300 pathway in microglia, J. Immunol., 179 (2007) 1292-1302.
[32] Discovery Studio 3.1, Accelrys Software Inc., San Diego, 2011.
[33] G. Wu, D.H. Robertson, C.L. Brooks, M. Vieth, Detailed analysis of grid-based molecular docking: A
case study of CDOCKER—A CHARMm-based MD docking algorithm, J. Comput. Chem., 24 (2003)
1
7

ACCEPTED MANUSCRIPT
1
549-1562.
1
8

ACCEPTED MANUSCRIPT
Table 1. Crystallographic and Experimental Data for compound 4m.
Compound
Formula
4m
C H FN O
23
18
3
Crystal size(mm)
0.31×0.23×0.17
-1
MW(g mol )
Crystal system
α (°)
371.40
Triclinic
96.327(9)
β(°)
96.542(10)
γ(°)
108.859(7)
a(Å)
8.272(6)
b(Å)
10.477(8)
c(Å)
23.85(2)
3
V(Å )
1919(3)
Z
4
θ limits(°)
hkl limits
F(000)
2.13≤θ≤24.74
-16 ≤h≤16, -13≤k≤14, -13 ≤l≤12
776
6442/1/505
0.087
Data/restraints/parameters
-1
Absorption coefficient(mm )
Reflections collected
12975
Independent reflections
R / wR [I>2&Gs(I)]
6442 [R = 0.0392]
int
0.0596/ 0.1489
0.1408/ 0.1966
1.005
1
2
R / wR (all data)
1
2
2
Goodness-of-fit on F
1
9

ACCEPTED MANUSCRIPT
Table 2. Cytotoxicities and inhibitory activities of compounds 4a–4t on lymph node
cells.
Compound
R
1
R
2
CC ±SDꢀ
µ
Mꢁ IC ±SDꢀ
µMꢁ
SI
5
0
50
4
a
H
H
H
376.14±23.15
434.52±26.75
325.64±23.71
276.73±36.34
328.59±19.27
382.56±27.65
275.19±27.35
228.68±19.36
348.58±21.63
374.87±16.39
289.51±25.64
212.08±18.35
319.43±36.74
259.12±22.72
296.89±27.45
375.47±27.62
333.36±28.34
362.28±25.68
323.71±23.67
265.78±38.32
178.26±27.87
18.33±5.64
20.52
41.94
17.05
16.52
8.10
4
b
2-F
10.36±0.39
19.10±0.75
16.75±0.68
40.57±2.45
28.00±3.72
34.58±5.43
38.30±7.56
26.29±0.78
20.22±1.87
20.16±1.76
27.23±3.64
17.57±1.67
1.18±0.26
2.74±0.24
7.22±1.18
18.45±0.76
8.03±0.25
9.08±0.06
10.76±0.17
0.57±0.01
4
c
H
2-Cl
2,4-Cl
H
4
d
H
4
e
CH
CH
CH
CH
OCH
OCH
OCH
OCH
F
3
3
3
3
4
f
2-F
13.66
7.96
4
g
2-Cl
2,4-Cl
H
4
h
5.97
4
i
3
3
3
3
13.26
18.54
14.36
7.79
4
j
2-F
4
k
2-Cl
2,4-Cl
H
4
l
4
m
18.18
219.59
108.35
52.00
18.07
45.12
35.65
24.70
312.73
4
n
F
2-F
4
o
p
q
F
2-Cl
2,4-Cl
H
4
F
4
Cl
4
r
Cl
2-F
4
s
Cl
2-Cl
2,4-Cl
4
t
Cl
CsA
2
0

ACCEPTED MANUSCRIPT
Table 3. PI3Kγ inhibitory activity of the selected compounds
compound IC PI3Kγ (
µM)
5
0
4
4
n
p
0.28
1.26
3.12
12.36
13.78
6.57
9.36
21.45
>25
4
o
4
r
4
s
4
b
4
t
4
g
4
h
4
e
>25
LY294002
7.2
2
1

ACCEPTED MANUSCRIPT
Figure caption:
Figure 1. The structure of compounds A–C.
Figure 2. Title compounds.
Figure 3. Docking model of compound 4p with PI3Kγ. It is nicely bound to the
o
o
PI3Kγ via three H-bonds (Ser806 - F: 2.10 Å, 139.0 , Lys890 - O: 2.27 Å, 108.5 ,
o
Lys890 - N19: 2.33 Å, 126.8 ) and a Pi-cation interaction (3.2 Å).
Figure 4. The molecular structure of 4m showing 30% probability displacement
ellipsoids.
Figure 5. (a) Selected compounds inhibited ConA-induced IL-6 secretion in Mouse
lymph node cells. (b) The RT-PCR results of selected compounds (5 M) inhibit
mRNA expression of IL-6.
Scheme 1. General synthesis of compounds 4a–4t. R =H, CH , OCH , F, Cl; R =H,
1
3
3
2
o
o
2
-F, 2-Cl, 2, 4-Cl. iꢂethanol, 50-60 C, 3hꢃiiꢂDMF, POCl , 50-60 C, 5h; iiiꢂ
3
o
sub-benzylhydroxyamine hydrochloride, ethanol, NaOAc, 30 C, 3h
2
2

ACCEPTED MANUSCRIPT
Figure 1.
2
3

ACCEPTED MANUSCRIPT
R1
R2
N
O
N
N
4a-4t
Figure 2.
2
4

ACCEPTED MANUSCRIPT
Figure 3.
2
5

ACCEPTED MANUSCRIPT
Figure 4.
2
6

ACCEPTED MANUSCRIPT
Figure 5.
2
7

ACCEPTED MANUSCRIPT
Scheme 1.
2
8