Synthesis and antitumor activity of novel phenylhydrazonopyrazolone derivatives and molecular dynamics simulations

Article abstract of DOI:10.1007/s11164-018-3412-2

SHP2 is a new promising target for anti-cancer drug discovery. A series of novel phenylhydrazonopyrazolone derivatives was synthesized by a more convenient method, and their chemical structures were characterized by various spectroscopic methods. The inhibitory effects of these compounds on SHP2 enzyme and SHP2-dependent cancer cell H1975 were evaluated. The compound 11f with IC₅₀ value of 3.38?μmol/L exhibited more potent antitumor activity against H1975 cell than GS-493 (IC₅₀ = 20.92?μmol/L). Molecular dynamics simulation of compound 11f displayed a possible mode of interaction between this compound and SHP2 enzyme.

Full text of DOI:10.1007/s11164-018-3412-2

Res Chem Intermed
https://doi.org/10.1007/s11164-018-3412-2
Synthesis and antitumor activity of novel
phenylhydrazonopyrazolone derivatives and molecular
dynamics simulations
1
,2
2
2
2
•
Xia-min Hu
Cheng-zhi Gao
•
Zhi-wen Cui
•
•
Wei Dong
•
•
Yue Zhu
2,3,4
2
2
2
•
Shi-qiang Xu
Qiong Yuan
•
Zhi-jun Yu
2
,3,4
Zhen-li Min
Received: 31 October 2017 / Accepted: 26 March 2018
Ó Springer Science+Business Media B.V., part of Springer Nature 2018
Abstract SHP2 is a new promising target for anti-cancer drug discovery. A series
of novel phenylhydrazonopyrazolone derivatives was synthesized by a more con-
venient method, and their chemical structures were characterized by various spec-
troscopic methods. The inhibitory effects of these compounds on SHP2 enzyme and
SHP2-dependent cancer cell H1975 were evaluated. The compound 11f with IC50
value of 3.38 lmol/L exhibited more potent antitumor activity against H1975 cell
than GS-493 (IC50 = 20.92 lmol/L). Molecular dynamics simulation of compound
11f displayed a possible mode of interaction between this compound and SHP2
enzyme.
Keywords Phenylhydrazonopyrazolone Á Anticancer activity Á SHP2
inhibitor Á GS-493 Á Molecular dynamics simulation
Xia-min Hu and Zhi-wen Cui have contributed equally to this work.
&
Zhen-li Min
mchust@126.com
1
2
3
4
College of Pharmacy, Shanghai University of Medicine and Health Sciences, Shanghai 201318,
China
Department of Pharmacy, College of Medicine, Wuhan University of Science and Technology,
Wuhan 430065, Hubei Province, China
Hubei Province Key Laboratory of Occupational Hazard Identification and Control, Wuhan
University of Science and Technology, Wuhan 430065, Hubei Province, China
New Drugs Innovation and Development Institute, Department of Pharmacy, College of
Medicine, Wuhan University of Science and Technology, Wuhan 430065, Hubei Province,
China
123

X. Hu et al.
Introduction
Phenylhydrazonopyrazolone is an important class of heterocyclic compounds with a
broad spectrum of biological activities such as antibacterial (1, Fig. 1) [1], apoptosis
inducer (2, Fig. 1), cholesterol-lowering [2], and some can be used as disperse dyes
[
3]. Recently, two phenylhydrazonopyrazolones containing a sulfonic acid group (3,
, Fig. 1) were reported to possess inhibition activity toward SHP2 [4, 5]. SHP2 is a
4
non-receptor enzyme that contains two tandem Src homology-2 (SH2) domains and
one protein tyrosine phosphatase (PTP) catalytic domain. It is ubiquitously
expressed in cell and plays crucial roles in signal transduction to regulate cell
survival and proliferation [6–8]. SHP2 is the first reported oncogenic tyrosine
phosphatase and increasing findings indicate that reduction of SHP2 activity
suppresses tumor cell growth, which makes SHP2 a new promising target for anti-
cancer drug discovery [9–12].
Several synthetic protocols have been reported for the synthesis of phenylhy-
drazonopyrazolone [1–3, 13–16]. The most frequently used method for synthesis of
the molecule mainly comprises of two steps: (1) preparation of the intermediate,
ethyl 2-phenylhydrazono-3-substituted-3-oxo-propionate; (2) condensation reaction
between the intermediate and corresponding hydrazine. This method was applied to
synthesize compounds 3 and 4, but some expensive and not easily available reagents
were used to prepare the intermediate including dimethyldioxirane (DMDO) and
1
-(2-mesitylenesulfonyl)-3-nitro-1H-1, 2, 4-triazol (MSNT) [4, 5], which led to
inconvenience. In view of the significant bioactivities of SHP2 inhibitor, this
method should be improved.
On the other hand, although 3 and 4 had exhibited good potency as a SHP2
inhibitor, there was still a problem of crossing membrane barriers due to the high
polar sulfonic acid group. In this series of phenylhydrazonopyrazolone sulfonic
acids, the preliminary structure–activity relationships (SARs) research indicated that
the sulfonic acid group bound to the phosphotyrosine pocket, the catalytic domain
of SHP2, and the N–H and C=O groups of hydrazonopyrazolone motif could form
EtO
R1
S
OH
N
R2
N
N
N
N
N
N
O
O
O
N
NH
N
NH
O
N
NH
HO S
3
3
4
PHPS1 (R =H, R =NO )
1 2 2
1
2
GS493 (R =NO , R =NO )
1
2
2
2
Fig. 1 Structures of some phenylhydrazonopyrazolone derivatives
1
23

Synthesis and antitumor activity of novel…
hydrogen bonds with some amino acid residues of SHP2, which rendered these
compounds primary inhibitory activity on SHP2. Meanwhile, the modifications on
the R and R positions improved inhibitory activity on SHP2, which left room for
1
2
further structural optimization [4, 5].
In an effort to discover chemical targeted entities active against tumors, we
decided to optimize these leads further by exploring additional modifications on the
R and R positions and keeping unchanged the scaffold of phenylhydrazonopy-
1
2
razolone sulfonic acid responsible for the main SHP2 inhibition profile. Considering
hydrogen bonds can be formed between the R substitutions of the compounds and
2
SHP2, several amide and lactone groups were introduced to the R2 position.
Meanwhile, these substitutions with lipophilic groups such as dimethyl and tert-
butyl groups were expected to be helpful for improving the ability to cross
membrane barriers. As for R substitutions, different electrical groups were used.
1
Here we described a convenient method to synthesize 12 new phenylhydrazonopy-
razolone sulfonic acid derivatives and their preliminary evaluations of inhibition
activities against SHP2 enzyme and non-small cell lung cancer (NSCLC) cell line
H1975. Molecular dynamics (MD) simulation were performed for further inves-
tigating interaction of the most potent compound with SHP2.
Experimental
Chemistry
All the reagents and solvents were obtained from commercially available sources
and were used without further purification. Melting points were determined on a
WRS-2B digital melting point apparatus and uncorrected. IR spectra were recorded
using a Nicolet 380 Fourier-transform infrared (FTIR) spectrophotometer (Thermo
1
13
Scientific, USA) from KBr pellets. H NMR and C NMR spectra were recorded on
a Bruker AVANCE III-600 NMR spectrometer (Bruker Biospin Co., Switzerland)
with tetramethylsilane (TMS) as the internal standard and CDCl or DMSO-d as
3
6
the solvent and known chemical shifts of residual proton signals of deuterated
1
solvents ( H NMR: d 2.50 for DMSO-d and d 3.33 for H O) or carbon signals of
6
2
1
3
deuterated solvents ( C NMR: d 39.52 for DMSO-d ) as internal standard. MS
6
(
ESI) measurement was conducted on an Agilent 1100 LC–MS spectrometer
Agilent, Palo Alto, CA, USA).
(
Synthesis of diazonium salt 8
To the mixture of conc. hydrochloric acid (3.0 mL) and water (3.0 mL) was added
4
0
-aminobenzene sulfonic acid (1.73 g, 10 mmol), and the solution was cooled to
°C. Sodium nitrite solution (0.73 g, 10.10 mmol) dissolved in water (3.0 mL) was
added dropwise to the mixture under stirring, and then starch-iodide paper was used
to check the presence of excess nitrous acid. After the reaction mixture stirring at
0
–5 °C for 0.5 h, the desired diazonium salt 8 was obtained.
123

X. Hu et al.
Synthesis of intermediates 10a–10d
The corresponding 4-substituted ethyl benzoylacetate 9a–9d (10.0 mmol) and
sodium acetate (4.60 g, 50 mmol) were added into ethanol (50 mL) and stirred. The
temperature was cooled to 0–5 °C. Subsequently, the prepared diazonium salt 8
(
10 mmol) solution was slowly added to the mixture. The reaction mixture was
stirred at 0–5 °C for 5 h then allowed to reach room temperature. The yellow
precipitate was collected by suction filtration and washed with water and
crystallized in ethanol to yield the desired compound 10a–10d.
Ethyl 3-phenyl-3-oxo-2-[(4-sulfophenyl)hydrazono]-propionate (10a) Yield 66%.
1
H-NMR (DMSO-d , 600 MHz), d: 8.32 (s, 1H, NH), 7.93 (d, J = 8.40 Hz, 2H,
6
ArH), 7.47 (d, J = 7.92 Hz, 2H, ArH), 7.10–7.03 (m, 5H, ArH), 4.21(q,
J = 7.08 Hz, 2H, OCH ), 1.21 (t, J = 7.02 Hz, 3H, CH ).
2
3
Ethyl 3-(4-fluorophenyl)-3-oxo-2-[(4-sulfophenyl)hydrazono]-propionate (10b)
1
Yield: 65%. H-NMR (DMSO-d , 600 MHz), d: 8.29 (s, 1H, NH), 7.98 (d,
6
J = 8.34 Hz, 2H, ArH), 7.44 (d, J = 7.92 Hz, 2H, ArH), 7.09–7.01 (m, 5H, ArH),
4
.19(q, J = 7.14 Hz, 2H, OCH ), 1.22 (t, J = 7.08 Hz, 3H, CH ).
2 3
Ethyl 3-(4-nitrophenyl) -3-oxo-2-[(4-sulfophenyl)hydrazono]-propionate (10c)
1
Yield: 62%. H-NMR (DMSO-d , 600 MHz), d: 8.45 (s, 1H, NH), 8.22 (d,
6
J = 8.34 Hz, 2H, ArH), 7.95 (d, J = 8.64 Hz, 2H, ArH), 7.41 (d, J = 7.86 Hz, 2H,
ArH), 7.05 (d, J = 8.04 Hz, 2H, ArH), 4.16(q, J = 6.96 Hz, 2H, OCH ), 1.22 (t,
J = 7.02 Hz, 3H, CH3).
2
Ethyl 3-(4-methoxylphenyl) -3-oxo-2-[(4-sulfophenyl)hydrazono]-propionate (10d)
1
Yield: 75%. H-NMR (DMSO-d , 600 MHz), d: 8.26 (s, 1H, NH), 7.88 (d,
6
J = 8.04 Hz, 2H, ArH), 7.50 (d, J = 8.46 Hz, 2H, ArH), 7.17(d, J = 7.86 Hz, 2H,
ArH), 7.04 (d, J = 8.28 Hz, 2H, ArH), 4.25(q, J = 7.02 Hz, 2H, OCH ), 3.83(s, 3H,
2
OCH ), 1.20 (t, J = 7.14 Hz, 3H, CH ).
3
3
Synthesis of intermediates 13
A mixture of compounds 12 (10 mmol) and 10% (mass fraction) Pd/C (0.20 g) in
MeOH (20 mL) was subjected to standard hydrogenolysis condition at atmospheric
pressure (balloon) and room temperature. The progress of the reaction was
monitored by TLC. After completion of the reactions, the reaction mixture was
filtered off and the filtrate was evaporated on a rotary evaporator to afford the crude
products 13, which were used directly in the next step without further purification.
1
6
-Aminoisobenzofuran-1(3H)-one (13) Yield: 87%, Mp: 186–187 °C. H-NMR
(
J = 8.1 Hz, 2.1 Hz, 1H, ArH), 5.25(s, 2H), 3.95(s, 2H, NH2).
CDCl , 600 MHz), d: 7.28(s, 1H, ArH), 7.11–7.07(m, 1H, ArH), 7.05(dd,
3
1
23

Synthesis and antitumor activity of novel…
Synthesis of intermediates 14
Compounds 13 (10 mmol) was added to a mixed solution of conc. hydrochloric acid
(
3.0 mL) and water (3.0 mL) under stirring, and the solution was cooled to 0 °C.
Sodium nitrite (0.73 g, 10.10 mmol) dissolved in water (3.0 mL) was added
dropwise to the mixture while the internal temperature was maintained at 0–5 °C.
After a check by using starch-iodide paper indicated the presence of excess nitrous
acid, the reaction mixture was stirred at 0–5 °C for 0.5 h. A solution of stannous
chloride dihydrate (6.76 g, 30 mmol) in 1:1 conc. hydrochloric acid/water (10 mL)
was added dropwise to the diazonium salt solution over 0.5 h maintaining an
internal reaction temperature at 0–5 °C. The heterogeneous orange mixture was
stirred at 0 °C for 1 h before being allowed to warm to ambient temperature and
vigorous stirring was maintained for 6 h. The orange precipitate was collected by
suction filtration and dissolved in water (50 mL) under stirring at 0 °C, and then
ether (50 mL) was added. The biphasic mixture was basified with 10 M sodium
hydroxide to pH 13 before it was transferred to a separatory funnel. The organic
layer was separated and dried over magnesium sulfate and filtered. Then it was
evaporated on a rotary evaporator to afford the crude products 14, which were used
directly in the next step without further purification.
General procedure for the synthesis of target compounds 11a–11l
A mixture of compounds 7a–7b (10.0 mmol) or compound 14 (10.0 mmol) and
4
-substituted ethyl benzoylacetate 10a–10d (10.0 mmol) in acetic acid (40 mL) was
heated to 120 °C and stirred for 3–4 h. Subsequently, the mixture was cooled to
room temperature. The yellow precipitate was collected by suction filtration and
washed with water and crystallized in acetone to yield the target compound 11a–
1
1l. The Spectral data of all target compounds were summarized as followed and the
melting points of all target compounds are more than 300 °C.
4-{(2Z)-2-[1-((4- dimethylcarbamoyl) phenyl)-3-phenyl-5-oxo-1, 5-dihydro-4H-
pyrazol-4-ylidene]hydrazinyl}benzenesulfonic acid (11a) An orange-red solid,
-
yield 34%.IR (KBr) cm :3415, 2967, 1650, 1545, 1505, 1119,1033; HNMR
1
1
(
DMSO-d , 600 MHz), d: 13.70 (s, 1H, NH), 8.19 (dt, J = 7.02, 1.44 Hz, 2H, ArH),
6
8
7
1
1
.08 (dt, J = 8.76, 2.34 Hz, 2H, ArH), 7.68 (dt, J = 8.76, 2.22 Hz, 2H, ArH),
1
3
.50–7.60 (m, 5H, ArH), 2.96 (s, 6H, 2CH3); CNMR (DMSO-d , 150 MHz), d:
6
70.00, 157.58, 146.89, 146.70, 141.43, 138.90, 133.35, 130.50, 130.30, 129.32,
?
28.66, 127.69, 127.56, 127.12, 118.00, 116.30, 35.60; ESI–MS:490.13 [M–H] .
4-{(2Z)-2-[1-((4- dimethylcarbamoyl) phenyl)-3-(4-nitrophenyl)-5-oxo-1, 5-dihy-
dro-4H-pyrazol-4-ylidene]hydrazinyl}benzenesulfonic acid (11b) An orange-red
-
1
solid, yield 28%.IR (KBr) cm : 3418, 2968, 1657, 1540, 1502, 1115,1031;
1
HNMR (DMSO-d , 600 MHz), d: 13.70 (s, 1H, NH), 8.41–8.47 (m, 4H, ArH), 8.08
6
(
d, J = 8.58 Hz, 2H, ArH), 7.65–7.70 (m, 4H, ArH), 7.56 (d, J = 8.58 Hz, 2H,
1
3
ArH), 2.96 (s, 6H, 2CH3); CNMR (DMSO-d , 150 MHz), d: 169.92, 157.37,
6
123

X. Hu et al.
1
1
48.20, 146.88, 144.57, 141.32, 138.70, 136.31, 133.68, 128.66, 128.53, 127.55,
?
26.64, 124.52, 118.16, 116.68, 35.23; ESI–MS:535.11 [M–H] .
4-{(2Z)-2-[1-((4- dimethylcarbamoyl) phenyl)-3-(4-methoxyphenyl)-5-oxo-1, 5-
dihydro-4H-pyrazol-4-ylidene] hydrazinyl}benzenesulfonic acid (11c) An
-
orange-red solid, yield 39%. IR (KBr) cm :3419, 2969, 1653, 1543, 1502,
1
1
1
116,1037; HNMR (DMSO-d , 600 MHz), d: 13.67 (s, 1H, NH), 8.15 (d,
6
J = 8.88 Hz, 2H, ArH), 8.07 (d, J = 8.40 Hz, 2H, ArH), 7.67 (d, J = 8.46 Hz, 2H,
ArH), 7.57 (d, J = 8.58 Hz, 2H, ArH), 7.53 (d, J = 8.52 Hz, 2H, ArH), 7.12 (d,
1
J = 8.76 Hz, 2H, ArH), 3.83(s, 3H, OCH ), 2.96 (s, 6H, 2CH ); CNMR (DMSO-
3
3
3
d6, 150 MHz), d: 170.02, 161.18, 157.52, 146.61, 146.30, 141.55, 138.92, 133.71,
29.19, 128.63, 127.56, 127.34, 122.68, 117.85, 116.17, 114.79, 55.78, 35.68; ESI–
1
MS:520.14 [M–H] .
?
4-{(2Z)-2-[1-((4- dimethylcarbamoyl) phenyl)-3-(4-fluorophenyl)-5-oxo-1, 5-dihy-
dro-4H-pyrazol-4-ylidene] hydrazinyl}benzenesulfonic acid (11d) An orange-red
-
solid, yield 35%.IR (KBr) cm : 3422, 2969, 1654, 1541, 1502, 1116,1032;
1
1
HNMR (DMSO-d , 600 MHz), d: 13.68 (s, 1H, NH), 8.24 (td, J = 5.64, 2.04 Hz,
6
2H, ArH), 8.07 (d, J = 8.64 Hz, 2H, ArH), 7.67 (d, J = 8.58 Hz, 2H, ArH), 7.59 (d,
J = 8.64 Hz, 2H, ArH), 7.54 (d, J = 8.64 Hz, 2H, ArH), 7.39–7.42 (m, 2H, ArH),
1
3
2
1
1
.96 (s, 6H, 2CH3); CNMR (DMSO-d , 150 MHz), d: 169.99, 164.38, 162.74,
6
57.46, 146.68, 145.97, 141.41, 138.86, 133.36, 130.01, 128.65, 127.53,
?
26.99,126.85, 117.97, 116.48, 35.23; ESI–MS:508.12 [M–H] .
4-{(2Z)-2-[1-((4- tert-butylcarbamoyl) phenyl)-3-phenyl-5-oxo-1, 5-dihydro-4H-
pyrazol-4-ylidene] hydrazinyl}benzenesulfonic acid (11e) An orange-red solid,
-
1
1
yield 43%. IR (KBr) cm :3423, 2954, 1653, 1547, 1503, 1120,1033; H NMR
(
DMSO-d , 600 MHz), d: 13.70 (s, 1H, NH), 8.18 (d, J = 7.14 Hz, 2H, ArH), 8.08
6
(
d, J = 8.16 Hz, 2H, ArH), 7.93 (d, J = 8.76 Hz, 2H, ArH), 7.77(s, 1H, NHCO),
1
3
7
.68(d, J = 8.70 Hz, 2H, ArH), 7.50–7.60 (m, 5H, ArH), 1.38 (s, 9H, 3CH3);
C
NMR (DMSO-d , 150 MHz), d: 165.96, 157.60, 146.98, 146.57, 141.47, 140.05,
6
1
32.60, 130.50, 129.29, 128.87, 127.72, 127.55, 127.08, 117.47, 116.31, 51.26,
?
9.07; ESI–MS:518.16 [M–H] .
2
4-{(2Z)-2-[1-((4- tert-butylcarbamoyl) phenyl)-3-(4-nitrophenyl)-5-oxo-1, 5- dihy-
dro-4H-pyrazol-4-ylidene]hydrazinyl}benzenesulfonic acid (11f) An orange-red
-
1
1
solid, yield 36%. IR (KBr) cm :3427, 2957, 1655, 1545 1504, 1121,1036; H
NMR (DMSO-d , 600 MHz), d: 13.71 (s, 1H, NH), 8.41–8.47 (m, 4H, ArH), 8.07
6
(
d, J = 8.76 Hz, 2H, ArH), 7.94 (d, J = 8.76 Hz, 2H, ArH), 7.79 (s, 1H, NHCO),
1
3
7
.65–7.69 (m, 4H, ArH), 1.38 (s, 9H, 3CH3); CNMR (DMSO-d , 150 MHz),
6
d:165.91, 157.45, 148.25, 146.93, 144.76, 141.30, 139.84, 136.31, 132.94, 128.90,
28.62, 127.53, 126.65, 124.54, 117.66, 116.68, 51.28, 29.06; ESI–MS:563.14 [M–
1
H] .
?
4-{(2Z)-2-[1-((4- tert-butylcarbamoyl) phenyl)-3-(4-methoxyphenyl)-5-oxo-1, 5-
dihydro-4H-pyrazol-4-ylidene]hydrazinyl}benzenesulfonic acid (11g) An orange-
-
red solid, yield 47%. IR (KBr) cm :3420, 2956, 1656, 1542, 1117; HNMR
1
1
1
23

Synthesis and antitumor activity of novel…
(
DMSO-d , 600 MHz), d: 13.66 (s, 1H, NH), 8.13 (d, J = 8.58 Hz, 2H, ArH), 8.05
6
(
d, J = 8.52 Hz, 2H, ArH), 7.91 (d, J = 8.52 Hz, 2H, ArH), 7.75(s, 1H, NHCO),
7
J = 8.58 Hz, 2H, ArH), 3.82(s, 3H, OCH ), 1.38 (s, 9H, 3CH ); CNMR (DMSO-
.68 (d, J = 8.28 Hz, 2H, ArH), 7.56 (d, J = 8.34 Hz, 2H, ArH), 7.10 (d,
1
3
3
3
d6, 150 MHz), d: 165.98, 161.19, 157.59, 146.69, 146.43, 141.50, 140.08, 132.44,
29.21, 128.83, 124.54, 127.27, 122.65, 117.34, 116.16, 114.7, 55.75, 51.25, 29.08;
1
ESI–MS:548.17 [M–H] .
?
4-{(2Z)-2-[1-((4- tert-butylcarbamoyl) phenyl)-3-(4-fluorophenyl)-5-oxo-1, 5- dihy-
dro-4H-pyrazol-4-ylidene]hydrazinyl}benzenesulfonic acid (11h) An orange-red
-
solid, yield 41%. IR (KBr) cm :3426, 2959, 1658, 1543, 1119; HNMR (DMSO-
1
1
d6, 600 MHz), d: 13.69 (s, 1H, NH), 8.23–8.26 (m, 2H, ArH), 8.13 (d, J = 8.76 Hz,
2H, ArH), 7.93 (d, J = 8.58 Hz, 2H, ArH), 7.76 (s, 1H, NHCO), 7.67 (d,
J = 8.58 Hz, 2H, ArH), 7.60 (d, J = 8.58 Hz, 2H, ArH), 7.39–7.42 (m, 2H, ArH),
1
3
1
1
1
.37 (s, 9H, 3CH3); CNMR (DMSO-d , 150 MHz), d:165.94, 162.75, 157.54,
6
46.74, 146.10, 141.40, 140.02, 132.63, 130.06, 130.00, 128.88, 127.52, 126.96,
?
26.81, 117.47, 116.48, 51.25, 29.07; ESI–MS:536.15 [M–H] .
4
4
-{(2Z)-2-[1-(1,3-dihydro-1-oxoisobenzofuran-6-yl)-3-phenyl-5-oxo-1,5-dihydro-
H-pyrazol-4-ylidene] hydrazinyl}-benzenesulfonic acid (11i) An orange-red
-
1
1
solid, yield 53%. IR (KBr) cm :3436, 2924, 1751, 1655, 1558, 1121; HNMR
(
J = 7.68 Hz, 2H, ArH), 7.79 (d, J = 8.16 Hz, 2H, ArH), 7.68 (d, J = 8.52 Hz, 2H,
DMSO-d , 600 MHz), d: 13.68 (s, 1H, NH), 8.40–8.43(m, 2H, ArH), 8.19 (d,
6
1
3
ArH), 7.05–7.59(m, 5H, ArH),5.43(s, 2H, OCH2); CNMR (DMSO-d , 150 MHz),
6
d:170.74, 157.52, 146.98, 146.72, 143.90, 138.85, 130.52, 130.18, 129.27, 127.71,
1
27.54, 126.95, 126.24, 124.24, 124.23, 116.35, 113.64, 70.39; ESI–MS:475.08
?
M–H] .
[
4-{(2Z)-2-[1-(1,3-dihydro-1-oxoisobenzofuran-6-yl)-3-(4-nitrophenyl)-5-oxo-1,5-di-
hydro-4H-pyrazol-4-ylidene] hydrazinyl}benzenesulfonic acid (11j) An orange-
-
red solid, yield 45%. IR (KBr) cm :3432, 2928, 1753, 1654, 1553, 1123; HNMR
1
1
(
J = 8.28 Hz, 2H, ArH), 7.67 (d, J = 8.58 Hz, 2H, ArH), 7.61 (d, J = 8.58 Hz, 2H,
DMSO-d , 600 MHz), d: 13.62 (s, 1H, NH), 8.35–8.42 (m, 5H, ArH), 7.74 (d,
6
1
3
ArH), 5.40 (s, 2H, OCH2); CNMR (DMSO-d , 150 MHz), d: 170.54, 157.10,
6
1
47.94, 146.70, 144.07, 143.97, 141.04, 138.36, 135.85, 128.22, 127.41, 126.18,
?
24.28, 123.72, 122.00, 116.57, 113.29, 108.42, 70.28; ESI–MS:520.06 [M–H] .
1
4
1
-{(2Z)-2-[1-(1,3-dihydro-1-oxoisobenzofuran-6-yl)-3-(4-methoxyphenyl)-5-oxo-
,5-dihydro-4H-pyrazol-4-ylidene] hydrazinyl}benzenesulfonic acid (11k) An
-
1
orange-red solid, yield 63%.IR (KBr) cm :3442, 2928, 1753, 1652, 1557, 1121;
1
HNMR (DMSO-d , 600 MHz), d: 13.59 (s, 1H, NH), 8.33–8.36 (m, 2H, ArH), 8.09
6
(
ArH), 7.06 (d, J = 8.52 Hz, 2H, ArH), 5.38 (s, 2H, OCH ), 3.80 (s, 3H, OCH );
d, J = 8.52 Hz, 2H, ArH), 7.66–7.14 (m, 3H, ArH), 7.52 (d, J = 8.28 Hz, 2H,
2
3
1
3
CNMR (DMSO-d , 150 MHz), d: 170.76, 161.22, 157.54, 146.73, 146.64, 143.76,
6
1
1
41.41, 138.89, 129.24, 127.53, 127.16, 126.22, 124.31, 124.14, 122.58, 116.20,
?
14.77, 113.53, 70.39, 55.75; ESI–MS:505.09 [M–H] .
123

X. Hu et al.
4-{(2Z)-2-[1-(1,3-dihydro-1-oxoisobenzofuran-6-yl)-3-(4-fluorophenyl)-5-oxo-1,5-
dihydro-4H-pyrazol-4-ylidene] hydrazinyl}benzenesulfonic acid (11l) An orange-
-
red solid, yield 59%. IR (KBr) cm :3439, 2927, 1753, 1653, 1554, 1123; HNMR
1
1
(
J = 5.58, 1.08 Hz, 2H, ArH), 7.81 (d, J = 8.22 Hz, 1H, ArH), 7.68 (d, J = 8.58 Hz,
DMSO-d , 600 MHz), d: 13.68 (s, 1H, NH), 8.41–8.43 (m, 2H, ArH), 8.26 (td,
6
2
2
1
1
H, ArH), 7.61 (d, J = 8.58 Hz, 2H, ArH), 7.41 (t, J = 8.82 Hz, 2H, ArH), 5.44 (s,
1
3
H, OCH2); CNMR (DMSO-d , 150 MHz), d: 170.63, 164.33, 162.68, 157.29,
6
46.44, 145.65, 143.69, 141.27, 138.63, 129.82, 127.47, 126.68, 126.52, 126.10,
?
24.14, 123.78, 116.26, 113.27, 70.29; ESI–MS:493.07 [M–H] .
Biological evaluation
Material and instrument
DMEM high-sugar medium, bovine serum, penicillin and streptomycin were
obtained from Hangzhou GNM Company. FBS was obtained from Sigma-Aldrich.
Recombinant human SHP2 PTP domain protein was purchased from Abcam. H1975
Ò
(
NCI-H1975, ATCC ) was purchased from BeiNa Culture Collection, Beijing,
China. Cells were treated with 0.1% DMSO as the negative control group. The
positive control drugs PHPS1 and GS-493 were synthesized by us.
SHP2 activity assay
SHP2 inhibitory activity was measured according to reported method [17] by
incubating recombinant SHP2 PTP domain proteins with reaction buffer (50 mmol/
L Bis–Tris, 50 mmol/L NaCl, 2 mmol/L EDTA, 2 mmol/L DTT, pH 6.0) and 1%
volume of dimethyl sulfoxide containing synthesized compounds in a 96-well plate
with a final volume of 100 lL at 30 °C. The mixture was incubated for 30 min, and
then p-nitrophenyl phosphate (pNPP) was added. The amount of the p-nitrophenol
product was determined from the absorbance at 405 nm detected using a Spectra
Max i3x microplate spectrophotometer. The nonenzymatic hydrolysis of pNPP was
corrected by measuring the control without addition of the SHP2 proteins.
MTT-assay
The anti-proliferative activities of compounds 11a–11l were assessed against non-
small cell lung cancer (NSCLC) cell line H1975 by MTT assay. The H1975 was
cultured at 37 °C and 5% CO in DMEM supplemented with 10% fetal bovine
2
3
serum and 1% penicillin and streptomycin for 10 h. 5 9 10 cells were seeded in
each well of 96-well plates, and then treated with the synthesized compounds
dissolved in DMSO at concentrations 40 lmol/L using PHPS1 and GS-493 as
positive drugs. After treating with compounds for 96 h, cells were incubated with
20 lL of 5 mg/mL MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide) for 3–4 h. Then the culture medium was removed, DMSO was added to
dissolve the formazan crystals. Wells containing only media were used for
1
23

Synthesis and antitumor activity of novel…
background correction. The optical density was measured spectrophotometrically at
490 nm. The percentage of inhibition was calculated according to formula:
percentage of inhibition (%) = (Asample - Anegative)/(Ablank - Anegative) 9 100%.
Molecular docking and molecular dynamics simulation
To study the structural basis of the inhibitive effects on SHP2, 11f and the SHP2
PTP domain (PDB ID: 3O5X, residue 262–527) were introduced to the molecular
model. In addition, GS-493 was selected as the positive control in the molecular
docking and molecular dynamics (MD) simulations. Molecular docking was
1
performed by AutoDock Vina software. The PDB file of SHP2 PTP domain was
2
obtained from RCSB PROTEIN DATA BANK. To determine the binding pocket
3
and the grid coordinate, AutoGridFR software was applied, which generated the
grid configuration with center x = 31.100, y = 3.510 and z = 6.290, size
˚
0 9 20 9 26 A. The critical search algorithm with exhaustiveness parameter
2
was set to 8.
4
The MD simulations were performed using GROMACS software in 30 ns scale.
The initial structures of GS-493 and 11f were selected with the best shapes based on
the least binding scores among the docking results. Each SHP2-ligand complex was
soaked in a cubic box of simple point charge (SPC) water molecules with a margin
˚
of at least 10 A from the complex surface. Sodium and chloride counter ions were
added to preserve electro-neutrality. Energy minimization was done with a 50,000
steps of steepest descent minimization approach. NVT and NPT equilibration
phases were applied using a 2.0 fs integration time step for 500 ps, respectively.
The MD production phase was conducted using NPT ensemble at 300 K with the
pressure of 1 atm. The trajectories and the binding energy were analyzed.
Results and discussion
Chemistry
A novel series of 12 phenylhydrazonopyrazolone derivatives were synthesized as
shown in Scheme 1 and Scheme 2. Initially, various phenylhydrazines 7a–7b and
1
4 were prepared through reported procedure. 4-Nitrobenzoic acid was reacted with
thionyl chloride followed by acetylations with dimethylamine or tert-butylamine to
afford amides 5a–5b. Compound 5a–5b and 12 were reduced to yield amine 6a–6b
and 13 which were oxidized by nitrous acid and then reduced by stannous chloride
dihydrate to generate phenylhydrazines 7a–7b and 14. Secondly, 4-aminobenzene
sulfonic acid was oxidized to diazonium salt intermediate 8, which was then
1
http://vina.scripps.edu/, version 1.1.2, platform independent, Apache license.
2
http://www.rcsb.org/pdb/home/home.do.
3
http://adfr.scripps.edu/AutoDockFR/agfr.html, version 1.0, platform independent, LGPL OpenSource
software license.
4
http://www.gromacs.org/, version 5.1.2, platform independent, GNU LGPL 2.1.
123

X. Hu et al.
O
O
a
b
c
H N
2
O N
COOH
O N
2
2
R3
R3
5
a-5b
6a-6b
O
d
H NHN
2
H N
SO H
ClN2
SO H
2
3
3
R3
8
7
a-7b
O
O
O
O
e
OC H
2
5
OC H
2
5
N
R4
NH
R4
9
a-9d
1
0a-10d
4
a-4c, 5a-5c, 6a-6c R =(CH ) N, (CH ) CNH,
3
3 2
3 3
SO H
9
a-9d, 10a-10d
R =H, F, NO , OCH
4
2
3
3
O
R3
N
R4
N
O
11a R3=(CH3)2N,
1b R =(CH ) N,
R4=H
4
N
1
R =NO2
3
3 2
f
NH
11c R =(CH ) N,
R =OCH3
3
3 2
4
1
1
1
1
1
1d R =(CH ) N,
R =F
3
3 2
4
1e R =(CH ) CNH, R =H
3
3 3
4
1f R =(CH ) CNH, R =NO2
3
3 3
4
1g R =(CH ) CNH, R =OCH3
3
3 3
4
4
HO S
3
1h R =(CH ) CNH, R =F
3
3 3
1
1a-11h
Scheme 1 Synthetic routes of target compounds 11a–11h. Reagents and conditions: a. (1) SOCl
2
, 75 °C,
CNH , HCl, 0 °C,
2 5
, HCl, 0 °C, 0.5 h; e. 8, NaAc, C H OH. 0 °C; f. 6a–6c, HAc, 120 °C,
2
0
3
h; (2) (CH
.5 h; (2) SnCl
–4 h
3
)
2
NH.HCl, (CH
Á2H O; d. NaNO
3
)
3
2 2 5 3 2 3 2
, N(C H ) ; b. H , 10%Pd/C, CH OH; c. (1) NaNO
2
2
2
coupled with the substituted ethyl benzoylacetate 9a–9d to yield the intermediates
0a–10d. Finally, the phenylhydrazine derivatives 7a–7b and 14 were condensed
1
with 10a–10d to afford the corresponding target compounds 11a–11l upon refluxing
in glacial acetic acid. In this method, reagents were easily available and reaction
conditions were mild, which provided a convenient way for the preparation of
phenylhydrazonopyrazolone-based SHP2 inhibitors.
The structure of newly synthesized target compounds 11a–11l was confirmed by
various spectral techniques such as NMR, mass, and IR. The IR spectra of the
-
1
compounds revealed a band at around 3420–3440 cm , corresponding to sulfonic
acid group. The two carbonyl groups for benzamide and pyrazolone moieties in
1
23

Synthesis and antitumor activity of novel…
O
O
O
H NHN
O N
2
a
H2N
2
b
O
O
O
12
13
14
O
O
O
N
N
R4
OC H
O
2
5
N
O
R4
NH
N
c
14
NH 11i R4=H
1
1
1j R =NO2
4
1k R =OCH3
4
1
1l R =F
4
SO H
3
HO S
3
1
0a-10d
1
1i-11l
Scheme 2 Synthetic routes of target compounds 11i–11l. Reagents and conditions: a. H
CH OH; b. (1) NaNO Á2H O; c. HAc, 120 °C, 3–4 h
, HCl, 0 °C, 0.5 h; (2) SnCl
2
, 10%Pd/C,
3
2
2
2
-
1
compounds 11a–11h showed similar absorption at around 1650 cm , while in
compounds 11i–11l the distinction was obvious, showing absorption at 1750 and
-
1
650 cm corresponding to the C=O functionality for lactone and pyrazolone
1
1
3
moieties, respectively. The C NMR spectra of target compounds showed the two
resonating signals between 166–170 and 157–159 ppm corresponding to the C=O
functionality for benzamide or lactone and pyrazolone moieties, respectively. The
hydrogen-bonded NH group at the b-nitrogen appeared at about 13.70 ppm in the
1
H NMR spectra of the target compounds, which also confirmed that the compounds
prepared existed in the hydrazone form C=N–NH.
Biological evaluation
The inhibitory activities of synthesized compounds and PHPS1 against SHP2
catalytic domain were assessed at 400 nM final concentrations while GS-493 at
2
00 nM final concentrations. The results obtained were reported in Table 1, which
showed that six compounds exhibited similar or more potent activities than PHPS1.
Than the IC50 value of these compounds, PHPS1 and GS-493 against SHP2 were
determined and calculated by GraphPad Prism 6.0 software. Compounds 11b, 11e,
1
2
1f, 11g and 11j inhibited SHP2 with IC50 values of 2.14, 1.86, 0.23, 2.25 and
.51 lmol/L, respectively. Compound 11f exhibited excellent inhibitory activity
against SHP2 with IC50 value of 0.23 lmol/L but less potent than GS-493.
SHP2 is reported to be required for the full activation of the Ras-ERK1/2
pathway that can lead to the proliferation of non-small cell lung cancer (NSCLC)
123

X. Hu et al.
Table 1 Inhibition of SHP2 by synthesized compounds in vitro enzyme assay
a
Inhibition (%)
b
a
Inhibition (%)
b
Compd
IC50 (lmol/L)
Compd
IC50(lmol/L)
1
1
1
1
1
1
1
1a
1b
1c
1d
1e
1f
4.32 ± 0.46
42.67 ± 2.13
13.59 ± 0.46
30.73 ± 1.89
35.36 ± 2.64
67.12 ± 3.76
39.24 ± 2.38
ND
11h
16.49 ± 1.62
7.69 ± 1.46
37.82 ± 3.37
20.37 ± 2.16
19.85 ± 1.25
34.25 ± 1.56
65.45 ± 3.34
ND
ND
2.51
ND
ND
2.73
0.18
2.14
ND
11i
11j
2.65
1.86
0.23
2.25
11k
11l
PHPS1
GS-493
1g
ND not determined
a
Inhibition rate was calculated as the mean of 3 assays ± standard deviations
b
Each experiment was carried out in triplicate
cell line H1975, so the tumor cell line H1975 has been used as cellular model in
some bioactivity tests of inhibiting SHP2 [18, 19]. Therefore, we further evaluated
the ability of the synthesized compounds to inhibit proliferation of the SHP2-
dependent tumor cell line H1975. The preliminary bioactivity evaluations of all
target compounds at 40 lmol/L in DMSO were conducted by MTT assay while
PHPS1 at 40 lmol/L in DMSO was used as positive control drug and another
positive control drug GS-493 was at 20 lmol/L in DMSO due to its poor solubility.
As shown in Fig. 2, most of the tested compounds were able to reduce H1975 cell
Fig. 2 Anti-proliferation assays against H1975 cell for target compounds 11a–11l (40 lmol/L), PHPS1
(40 lmol./L) and GS-493 (20 lmol/L) as positive drugs. After 96 h of administration, the MTT assay
was preformed to detect the cell viability of H1975 cell. Results are presented as mean ± SD (n = 3) and
analyzed by GraphPad Prism 6.0 followed by the Student t test. **p \ 0.01 or ***p \ 0.001 versus GS-
493
1
23

Synthesis and antitumor activity of novel…
proliferation. Compounds 11b, 11e, 11f, 11j were found to be more potent than
PHPS1 (40 lmol/L) and compounds 11b, 11e, 11f were potent than GS-493
(
inhibited 50% of the H1975 cells proliferation (IC ) were tested by MTT assay.
20 lmol/L). Furthermore, the concentrations of the compounds 11b, 11e, 11f that
5
0
The results were summarized in Table 2 and it indicated that 11f with IC50 value of
.38 lmol/L was more potent than GS-493 with IC50 value of 20.92 lmol/L.
3
Subsequently, the SARs could be inferred. The 4-nitro group at R position of
4
target compounds seemed to be definitely playing a significant role in determining
activity, which was consistent with that reported [5]. Comparison of substitutions at
R position of target compounds indicated the importance of the tert-butylamine
3
group and replacement of it with the other amine groups and lactone group rendered
the compound reduction in activity. Being a bulky substituent, the tert-butylamine
group might increase interaction with SHP2, on the other hand, it was a lipophilic
moieties which could enhance partly cell permeability and resulted in improved
cellular efficacy.
Molecular docking and molecular dynamics simulation
The structure-based docking of SHP2 PTP domain with GS-493 and 11f gave the
best poses with the binding energy values of - 7.5 and - 8.3 kacl/mol,
respectively. A further study on the SHP2-ligand interaction was elucidated by
molecular dynamics (MD) simulations.
Root mean square deviations (RMSD) is a measure of the stability of the
structures. The RMSD of backbone atoms were plotted in Fig. 3. Binding with 11f
or GS-493, the backbone atoms of SHP2 PTP domain showed the dynamics
stabilities after 17 ns of the simulations phrases, with nearly the same RMSD values
of 0.21 ± 0.02 nm. The protein without ligand showed to be less stable compared to
the one with ligand. The variations of the hydrogen bonds between the ligands and
SHP2 PTP domain during the last 2 ns of the simulations were record and plotted in
Fig. 4. According to the plots, the number of hydrogen bonds between 11f and
SHP2 PTP domain was stable after 22 ns, ranging from 2 to 8.
The energy of SHP2 PTP domain binding to GS-493 and 11f during the MD
simulation was calculated by g_mmpbsa program [20], with the values of
- 88.628 ± 8.446 and - 167.832 ± 1.311 kacl/mol, respectively. 11f displayed
potent affinity to SHP2 PTP domain. Per residue contribution of the binding energy
Table 2 Anti-proliferative
activities of selected compounds
against H1975 cell line
a
Compd
IC50 (lmol/L)
PHPS1
GS-493
[ 100
20.92
16.63
8.46
1
1
1
1e
1b
1f
a
Each experiment was carried
out in triplicate
3.38
123

X. Hu et al.
Fig. 3 RMSD between SHP2
with ligand and without ligand
Fig. 4 Number of hydrogen
bonds between SHP2 and 11f in
time scale
Fig. 5 Per residue binding
energy contribution
was plotted in Fig. 5. As plotting, the similar residues impacted or contributed in the
protein–ligand interactions. Residue Lys274, Arg278, Lys280, Lys358, Arg362,
Lys366, Arg465, Arg498, Arg501 and Arg512 contributed the most both in the two
protein–ligand systems. Slight differences were observed among the residue
Ser460-Gly464, which were important residue group showing strong interactions
with 11f. Thr507 and Gln510 showed the strong interaction with GS-493; however,
their energy contribution was not significant in interaction with 11f.
1
23

Synthesis and antitumor activity of novel…
Fig. 6 The interactions between SHP2 PTP domain and 11f after 30 ns molecular dynamic simulation.
Hydrogen bonds are marked in green dashed lines, hydrophobic interactions are marked in red
semicircles. Carbon, nitrogen and oxygen atoms have been shown in black, blue and red, respectively.
The violet lines represent the ligand bond. (Color figure online)
The protein–ligand interactions are plotted in 2D diagrams after 30 ns simulation
by LigPlot [21] in Fig. 6. The sulfonic acid and nitro groups of 11f were observed to
format 8 hydrogen bonds with residue Tyr279, Ser460, Ala461, Gly462, Ile463,
Gly464, Arg465 and Glu508, and His 426, Gln506 and Thr507 showed hydrophobic
interactions with 11f.
123

X. Hu et al.
Conclusions
A series of novel phenylhydrazonopyrazolone derivatives was synthesized by a
more convenient method, and their preliminary evaluations of inhibitions on the
SHP2 enzyme and SHP2-mutated cancer cell H1975 were conducted. The
compound 11f was found to be more potent than PHPS1 and GS-493 in anti-
proliferation activity against H1975 cell. Molecular dynamics simulation showed
interaction mode of compound 11f with SHP2 including strong hydrophobic
interactions and formation of hydrogen bond.
Acknowledgements This work was supported by the National Youth Innovative Training Fund for
College Students of Wuhan University of Science and Technology [Grant No. 201510488010].
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