Identification of pyrazolopyridine derivatives as novel spleen tyrosine kinase inhibitors

Article abstract of DOI:10.1002/ardp.201800083

Inhibition of spleen tyrosine kinase (Syk) is a promising strategy for the treatment of various allergic and autoimmune disorders such as asthma, rheumatoid arthritis, and allergic rhinitis. Previously, a Syk inhibitor with novel indazole scaffold was discovered by structure-based virtual screening. Herein, the structure–activity relationship of the indazole Syk inhibitors was investigated. Several new inhibitors demonstrated potent activity against Syk. In particular, compound 18c showed good Syk inhibitory activity (IC₅₀ = 1.2 μM), representing a good lead compound for further optimization.

Full text of DOI:10.1002/ardp.201800083

Received: 19 March 2018
Revised: 3 June 2018
Accepted: 8 June 2018
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DOI: 10.1002/ardp.201800083
FULL PAPER
Identification of pyrazolopyridine derivatives as novel spleen
tyrosine kinase inhibitors
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Yahui Huang
Yu Li
Guoqiang Dong
Wannian Zhang
Na Liu
Chunquan Sheng
School of Pharmacy, Second Military Medical
University, Shanghai, People's Republic of
China
Abstract
Inhibition of spleen tyrosine kinase (Syk) is a promising strategy for the treatment of
various allergic and autoimmune disorders such as asthma, rheumatoid arthritis, and
allergic rhinitis. Previously, a Syk inhibitor with novel indazole scaffold was discovered
by structure-based virtual screening. Herein, the structure–activity relationship of the
indazole Syk inhibitors was investigated. Several new inhibitors demonstrated potent
activity against Syk. In particular, compound 18c showed good Syk inhibitory activity
(IC50 = 1.2 µM), representing a good lead compound for further optimization.
Correspondence
Prof. Chunquan Sheng and Na Liu, School of
Pharmacy, Second Military Medical University,
325 Guohe Road, Shanghai 200433, People's
Republic of China.
Email: shengcq@smmu.edu.cn (C.S.),
liuna66@aliyun.com (N.L.)
Funding information
The National Key R&D Program of China,
Grant number: 2017YFA0506000; National
Basic Research Program of China,
Grant number: 2014CB541800
K E Y W O R D S
pyrazolopyridines inhibitors, spleen tyrosine kinase, structure–activity relationship,
structure-based drug design
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1
INTRODUCTION
activated immunoreceptor to downstream events which orchestrates
various cellular responses (e.g., proliferation, differentiation, cell
Inflammation is generally the body's first immune response to irritation
and infection, which can be classified into acute inflammation and
chronic inflammation. The latter is the result of many autoimmune and
inflammatory diseases (e.g., rheumatoid arthritis, RA), which is the
consequence of imbalance between the innate and the adaptive
immune system. Although non-steroidal anti-inflammatory drugs
(NSAIDs) and disease-modifying antirheumatic drugs (DMARDs) are
widely used in the treatment of pain and inflammation, long-time use
usually results in adverse side effects.[1,2] Therefore there is an urgent
need for novel therapeutics with better pharmacological features.
Recently, progress in the signal path of autoimmune and allergic
diseases has identified several new targets,[3] and spleen tyrosine
kinase (Syk) is one of them with promising therapeutic value.
adhesion, cytokine production, mast cell degranulation). Thus, target-
ing Syk is a promising strategy for the treatment of inflammatory and
autoimmune disorders.[5] To date, a number of Syk inhibitors were
developed for treating various diseases ranging from arthritis to
leukemia. Fostamatinib (2, Figure 1) is a prodrug of R406 (1) and has
demonstrated efficacy in several animal models including RA,[6]
nephrotoxic nephritis,[7] asthma,[8] systemic lupus erythramatosis
(SLE),[9] autoimmune diabetes,[10] and autoimmune hemolytic anemia
(AHA).[11] However, compound 2 failed in phase 3 clinical trials
because of severe side effects,[12,13] which putatively attributed to the
poor kinase selectivity of its parent compound 1.[14] A more selective
inhibitor 3 (Figure 1) is currently in phase 2 clinical trials, but safety data
have not yet been reported. Previously, we identified compound 4 as a
novel Syk inhibitor through structure-based virtual screening.[15]
However, its Syk inhibitory activity remains to be further improved and
its structure–activity relationship (SAR) is still unknown. Therefore, a
series of novel indazole Syk inhibitors were rationally designed,
synthesized, and assayed.
Syk, a member of the non-receptor protein tyrosine kinase family,
is mainly expressed in hematopoietic cells, including B cells, mast cells,
macrophages, and neutrophils.[4] Syk plays a key role in coupling
Yahui Huang and Yu Li contributed equally to this work.
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Arch Pharm Chem Life Sci. 2018;1–8.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
1

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HUANG ET AL.
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FIGURE 1 Chemical structures of the Syk inhibitors
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2
RESULTS AND DISCUSSION
2.3 Syk inhibitory activity
The Syk inhibitory activities of the target compounds were evaluated
using the assay as previously described.[15] As shown in Table 1,
various substitutions were introduced on the benzene group including
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2.1 Design rationale
As shown in Figure 2, the superposition of binding conformation
of compounds 1 and 4 was performed to guide structural modification
of compound 4. On the basis of the binding mode, a series of
pyrazolopyridine inhibitors were rationally designed (Figure 3). First,
aromatic groups were introduced on the indazole scaffold to form
additional hydrophobic interactions with Syk. Second, the replacement
of benzene of the indazole by pyridine/pyrazine formed additional
hydrogen bond with the Syk hinge region. Third, various amide side
chains were designed to adjust the hydrophobic and hydrogen bonding
interactions with Syk.
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2.2 Chemical synthesis
The synthetic routes of the target compounds are shown in Schemes 1
and 2. Commercially available compound 5 reacted with hydrazine
hydrate under the microwave conditions, affording compound 6
(Scheme 1). Then Suzuki coupling reaction was applied for the
synthesis of the key intermediate 8, which was treated with the
substituted carboxylic acids 9 or 10 in the presence of HBTU and
Et3N to give compounds 11a–c and 12. Finally, the removal of the Boc
protecting group and/or methyl group in the presence of BBr3 gave
target compounds 13a,b and 14a,b. Compounds 18a–e were
synthesized by a similar procedure using compound 15[16] as the
starting material (Scheme 2).
FIGURE 2 Superimposition of the binding conformation of
compounds 1 and 4 in the active site of Syk (PDB ID: 3FQS[17]).
Compound carbon atoms colored in purple for compound 1 and in
cyan for compound 4. The figure was generated using PyMol
(http://www.pymol.org/)

HUANG ET AL.
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FIGURE 3 Design rationale of the indazole Syk inhibitors
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4-fluoro (11a), 3-ethyl ester (11b, 11c), 3-amino (13a, 13b) and
3-hydroxyl (14a, 14b) groups with various substituted amides on the
indazole scaffold. However, only a few compounds (11b, 14a, and 14b)
showed moderate inhibitory Syk activity at 5 µM.
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CONCLUSIONS
In summary,
a series of novel indazole, pyrazolopyridine, and
pyrazolopyrazine Syk inhibitors were designed and synthesized.
Several derivatives exhibited good Syk inhibitory potency. Especially,
compound 18c showed improved inhibitory activity against Syk
(IC50 = 1.2 µM) compared to the hit compound 4 (IC50 = 3.2 µM).
Taken together, the present study provides a good starting point for
the development of novel Syk inhibitors. Further structural optimiza-
tion is in process.
Owing to the poor activity of the indazole derivatives, the
pyrazolopyridine and pyrazolopyrazine derivatives (18a–e) were
further synthesized and evaluated (Table 2). Interestingly, the
pyrazolopyridine analogues (18a and 18b) showed improved activity
as compared to the corresponding indazole compounds. Moreover, the
introduction of meta-benzoic acid (18c) led to significant increase of
the Syk inhibitory activity (IC50 = 1.2 µM), which was more potent
compound 4 (IC50 = 3.2 µM). Unfortunately, the replacement of
pyridine by pyrazine group on the indazole skeleton (18d and 18e)
resulted in decreased activity.
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4
EXPERIMENTAL
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4.1 Chemistry
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2.4 Molecular docking
4.1.1 General procedures
Molecular docking studies were performed using GOLD 5.0[18] to
investigate the binding mode of compound 18c with Syk. The crystal
structure of Syk in complex with compound 1 was obtained from
protein database bank (PDB ID: 3FQS[17]) and prepared for molecular
docking analysis in Discovery Studio 3.0. As depicted in Figure 4, the
pyrazolopyridine scaffold formed two hydrogen bonds with Ala451
and Glu449 in the Syk hinge region. In contrast, compound 1 only
formed a hydrogen bond with Ala451. The 4-fluorophenyl formed
hydrophobic and van der Waals interaction with Pro455, which was
identified as a reliable residue to achieve high levels of Syk specificity
and potency.[19] Moreover, the carboxyl interacted with Asp512
through a hydrogen bond. On the basis of the binding mode and SAR,
the three hydrogen bonds were essential for the Syk inhibitory activity.
Reagents were purchased from commercial sources and were used
without further purification. Oxygen or water sensitive reactions were
performed under the nitrogen atmosphere. 13C NMR and 1H NMR
spectra were recorded on Bruker AVANCE600, or AVANCE300
spectrometer (Bruker Company, Germany), operating at the indicated
frequencies and CDCl3 or DMSO-d6 as solvents. Chemical shift was
expressed in ppm (δ). The mass spectra were recorded on an Esquire
3000 LC-MS mass spectrometer. Flash chromatography was per-
formed on 200–300 mesh silica gel with the indicated solvent systems
(Qingdao Haiyang Chemical, China).
The InChI codes of the investigated compounds together
with some biological activity data are provided as Supporting
Information.

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HUANG ET AL.
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SCHEME 1 Chemical synthesis of compounds 11a–c, 13a, 13b, 14a, and 14b. Reagents and conditions: (a) hydrazine hydrate, CH3CH2OH,
microwave, 150°C, 20 min, 95%; (b) Pd(PPh3)4, Na2CO3, 1,4-dioxane, H2O, reflux, 12 h, 35%; (c) HBTU, Et3N, DMF, rt, 2 h, 46–85%; (d)
CF3COOH, CH2Cl2, rt, 1 h, 67%; (e) BBr3, CH2Cl2, −78°C, 4 h, 54%
SCHEME 2 Synthesis route for compounds 18a–e. Reagents and conditions: (a) hydrazine hydrate, CH3CH2OH, microwave, 150°C, 20 min,
95%; (b) Pd(PPh3)4, Na2CO3, 1,4-dioxane, H2O, reflux, 12 h, 35%; (c) HBTU, Et3N, DMF, rt, 2 h, 46–85%

HUANG ET AL.
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TABLE 1 Syk inhibition activity of target compounds 11a–c, 13a, 13b, 14a, and 14b
Compound
11a
R1
R2
Inhibition rate at 5 µM (%)
4-F
−8.1
11b
11c
3-COOCH2CH3
3-COOCH2CH3
28
0.2
13a
13b
3-NH2
3-NH2
−3.8
−2.4
14a
14b
3-OH
3-OH
30.0
33.7
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4.1.2 Synthetic procedures
column chromatography (PE/EA = 1:1) to afford 8 (0.51 g, yield 57%)
as an off-white solid. To a stirred solution of compound 8 (0.2 g,
0.88 mmol) and 3-aminobenzoic acid (9, 0.18 g, 1.32 mmol) in DMF
(5 mL) was added HBTU (0.5 g, 1.32 mmol) and DIPEA (0.23 mL,
1.32 mmol). Then the reaction was stirred at room temperature for
4 h, diluted with water (100 mL), and extracted with EtOAc
(3 × 30 mL). The organic layer was dried over Na2SO4, filtrated and
the solvent was evaporated under the reduced pressure. The residue
was purified by column chromatography (CH2Cl2/CH3OH = 100:8)
to give target compound 11a as a yellow solid (0.11 g, yield 35%).
1H NMR (DMSO-d6, 600 MHz) δ: 8.34 (d, J = 8.83 Hz, 1H), 8.22 (d,
J = 1.02 Hz, 1H), 7.87 (dd, J1 = 8.64 Hz, J2 = 1.80 Hz, 1H), 7.72–7.75
(m, 2H), 7.33 (t, J = 9.36 Hz, 2H), 7.10 (t, J = 7.20 Hz, 1H), 7.03–7.05
(m, 2H), 6.71–6.73 (m, 1H), 6.48 (s, 2H). 13C NMR (150 MHz, DMSO-
d6, TMS) δ: 167.54, 166.10, 161.54, 153.78, 146.32, 140.60, 140.47,
140.22, 135.32, 133.22, 131.88, 131.43, 130.21, 128.54, 128.23,
127.63, 125.34, 119.33, 117.78, 116.78. MS (ESI positive): m/z
[M+H]+: 346.10.
4-Amino-N-(5-(4-fluorophenyl)-1H-indazol-3-yl)benzamide
(11a)
A solution of 2-fluoro-5-iodobenzonitrile (5, 1.0 g, 4 mmol) and
hydrazine hydrate (1 mL) in ethanol (5 mL) was reacted under
microwave condition at 150°C for 20 min. The solvent was
concentrated in vacuo, and the mixture was purified by column
chromatography (CH2Cl2/CH3OH = 100:2) to give compound
6
(1.0 g, yield 97%) as off-white solid. A mixture of compound 6
(1.0 g, 3.86 mmol) and 7 (1.1 g, 7.7 mmol) in 1 M aqueous sodium
carbonate (8 mL) and 1,4-dioxane (24 mL) was stirred under nitrogen
atmosphere for 5 min. The resulting mixture was treated with
tetrakis(triphenylphosphine)palladium(0) (0.2 g, 0.18 mmol) and
stirred at reflux for 8 h. Then, the reaction was cooled and diluted
with EtOAc (100 mL), and washed with brine (100 mL). The aqueous
layers were extracted with EtOAc (100 mL), the combined layer was
dried over Na2SO4 and filtered, and the filtrate was concentrated
under the reduced pressure. The residue obtained was purified by
The synthetic method for compounds 11b, 11c, 13a, 13b, and
18a–e was similar to that of compound 11a.

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HUANG ET AL.
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TABLE 2 In vitro Syk inhibition activity of the target compounds 18a–e
Compound
18a
X
R1
R2
Inhibition rate at 5 µM (%)
IC50 (µM)
C
3-NH2
14.7
NT
18b
C
C
3-NH2
4-F
2.9
77
NT
18c
1.2 0.04
18d
18e
N
4-F
20
6 0.54
N
4-F
23
5.5 0.36
3.2 0.11
4
–
–
–
50.3
NT, not tested.
Ethyl 3-(3-(3-aminobenzamido)-1H-indazol-5-yl)benzoate (11b)
Yellow solid (0.07 g, yield 60%). 1H NMR (DMSO-d6, 600 MHz) δ: 8.39
(d, J = 8.59 Hz, 1H), 8.33 (s, 1H), 8.28 (s, 1H), 8.05 (d, J = 8.13 Hz, 1H),
7.98 (d, J = 8.54 Hz, 1H), 7.95–7.99 (m, 2H), 7.67 (t, J = 7.7 Hz, 1H),
7.12 (t, J = 8.18 Hz, 1H), 6.74 (d, J = 7.78 Hz, 1H), 6.55 (s, 2H), 5.26 (s,
2H), 4.37 (d, J = 7.0 Hz, 2H), 1.34–1.37 (m, 3H). 13C NMR (150 MHz,
DMSO-d6, TMS) δ: 167.67, 166.13, 153.58, 148.61, 140.71, 140.26,
135.41, 132.01, 131.20, 130.09, 129.99, 128.62, 127.57, 121.39,
119.35, 117.91, 116.91, 116.56, 115.44, 61.44, 14.67. MS (ESI
positive): m/z [M+H]+: 401.34.
Ethyl 3-(3-(3-(methoxycarbonyl)benzamido)-1H-indazol-5-yl)-
benzoate (11c)
Yellow solid (0.07 g, yield 43%). 1H NMR (DMSO-d6, 600 MHz) δ:
8.43–8.48 (m, 2H), 8.16 (d, J = 7.75 Hz, 1H), 8.13 (d, J = 7.75 Hz, 1H),
7.98 (s, 1H), 7.93 (s, 1H), 7.67 (dd, J1 = 5.88 Hz, J2 = 7.84 Hz, 1H),
7.62 (t, J = 7.84 Hz, 1H), 6.66 (s, 1H), 4.36 (dd, J1 = 7.03 Hz,
J2 = 7.03 Hz, 1H), 3.88 (s, 2H), 3.86 (s, 3H), 1.20 (s, 3H). 13C NMR
(150 MHz, DMSO-d6, TMS) δ: 167.14, 166.14, 162.84, 154.11,
140.58, 140.13, 135.91, 135.33, 135.17, 134.23, 133.31, 132.07,
131.21, 131.21, 130.21, 131.00, 129.70, 129.32, 128.97, 128.62,
119.48, 116.67, 61.47, 52.85, 36.26. MS (ESI positive): m/z [M+H]+:
444.16.
FIGURE 4 Superimposition of the binding conformation of
compound 18c and 1 in the active site of Syk-kinase. Compound
carbon atoms colored in yellow for compound 18c and in purple for
compound 1. The figure was generated using PyMol (http://www.
pymol.org/)

HUANG ET AL.
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3-Amino-N-(5-(3-aminophenyl)-1H-indazol-3-yl)propanamide
(13a)
118.05, 117.14, 116.36, 114.93, 114.12. MS (ESI positive): m/z
[M+H]+: 346.35.
Off-white solid (0.10 g, yield 85%). 1H NMR (DMSO-d6, 600 MHz) δ:
8.25 (d, J = 8.75 Hz, 1H), 8.13 (s, 1H), 7.74 (d, J = 8.52 Hz, 1H), 7.13 (t,
J = 7.83 Hz, 1H), 6.87 (s, 1H), 6.81 (d, J = 7.79 Hz, 1H), 6.53–6.59 (m,
3H), 5.20 (s, 2H), 2.94–3.12 (m, 4H). 13C NMR (150 MHz, DMSO-d6,
TMS) δ: 170.74, 153.37, 150.02, 149.44, 142.35, 141.29, 131.34,
129.70, 125.96, 118.32, 114.81, 112.65, 110.01, 40.56, 35.21, 28.33.
MS (ESI positive): m/z [M+H]+: 296.27.
3-Amino-N-(5-(3-aminophenyl)-1H-pyrazolo[3,4-b]pyridin-3-
yl)propanamide (18a)
Yellow solid (0.06 g, yield 68%). 1H NMR (DMSO-d6, 600 MHz) δ: 8.32
(d, J = 9.39, 1H), 8.12 (s, 1H), 7.73 (d, J = 9.39 Hz, 1H), 7.13, (t,
J = 7.84 Hz, 1H), 6.87 (s, 1H), 6.81 (d, J = 8.39, 1H), 6.57 (d, J = 7.95 Hz,
1H), 6.48 (s, 2H), 5.20 (s, 2H), 2.83 (s, 2H), 2.71 (d, J = 10.6 Hz, 2H), 1.63
(t, J = 8.45 Hz, 3H), 1.52 (s, 3H), 1.23 (s, 2H). 13C NMR (150 MHz,
DMSO-d6, TMS) δ: 152.33, 149.67, 148.93, 147.97, 139.49, 130.02,
127.88, 127.55, 114.77, 113.22, 112.49, 108.69, 38.56, 36.25. MS
(ESI positive): m/z [M+H]+: 297.23.
N-(5-(3-Aminophenyl)-1H-indazol-3-yl)-4-methylpiperidine-4-
carboxamide (13b)
Off-white solid (0.11 g, yield 88%). 1H NMR (DMSO-d6, 600 MHz) δ:
8.32 (d, J = 8.36 Hz, 1H), 8.16 (s, 1H), 7.76 (d, J = 8.86 Hz, 1H), 7.13 (t,
J = 7.75 Hz, 1H), 6.87 (s, 1H), 6.81 (6, J = 8.49 Hz, 1H), 6.61 (s, 1H), 5.77
(s, 1H), 5.24 (s, 2H), 3.21 (s, 2H), 2.72 (d, J = 13.6 Hz, 2H), 1.89 (t,
J = 12.2 Hz, 2H), 1.56 (s, 2H). 13C NMR (150 MHz, DMSO-d6, TMS) δ:
173.20, 153.04, 149.26, 140.87, 140.16, 137.67, 129.93, 129.04,
120.41, 118.85, 116.55, 115.23, 113.84, 112.91, 45.71, 42.48, 31.88,
24.36, 8.81. MS (ESI positive): m/z [M+H]+: 350.27.
N-(5-(3-Aminophenyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)-4-
methylpiperidine-4-carboxamide (18b)
Yellow solid (0.1 g, yield 52%). 1H NMR (DMSO-d6, 600 MHz) δ: 11.95
(s, 1H), 8.52 (d, J = 1.68 Hz, 1H), 8.27 (d, J = 1.68 Hz, 1H), 7.10 (t,
J = 10.1 Hz, 1H), 6.81 (s, 1H), 6.75 (d, J = 10.1 Hz, 1H), 6.53 (d,
J = 8.39 Hz, 1H), 5.61 (s, 2H), 5.17 (s, 2H), 2.57–2.71 (m, 2H), 1.45 (d,
J = 4.25), 1.21 (s, 7H), 0.83 (t, J = 6.33 Hz, 1H). 13C NMR (150 MHz,
DMSO-d6, TMS) δ: 152.36, 149.67, 148.92, 147.96, 139.40, 130.01,
127.92, 127.53, 114.75, 113.17, 112.48, 106.72, 29.46. MS (ESI
positive): m/z [M+H]+: 351.18.
3-Amino-N-(5-(3-hydroxyphenyl)-1H-indazol-3-yl)benzamide
(14a)
To a stirred solution of N-(5-(3-methoxyphenyl)-1H-indazol-3-yl)-
pivalamide (12a, 0.15 g, 0.42 mmol) in CH2Cl2, BBr3 (0.2 mL) was
added in one portion and stirred for 6 h at −78°C. After the addition of
methanol (0.4 mL) and CH2Cl2 (50 mL), the solution was washed with
saturated NaHCO3 aqueous solution (60 mL) and brine (40 mL) and
dried over Na2SO4, and the solvent was removed under reduced
pressure. The residue was purified by silica gel column chromatogra-
phy (CH2Cl2/CH3OH = 100:5) to afford 14a (0.08 g, yield 55%) as
yellow solid. 1H NMR (DMSO-d6, 600 MHz) δ: 9.58 (s, 1H), 8.33 (d,
J = 8.47 Hz, 1H), 8.20 (d, J = 1.58 Hz, 1H), 7.82 (dd, J1 = 8.57 Hz,
J2 = 1.80 Hz, 1H), 7.28 (t, J = 7.95 Hz, 1H), 7.08–7.13 (m, 3H), 7.03 (d,
J = 7.53 Hz, 2H), 6.78 (dd, J1 = 7.95 Hz, J2 = 2.09 Hz, 1H), 6.71 (d,
J = 7.53 Hz, 1H), 6.52 (s, 2H), 5.27 (s, 2H). 13C NMR (150 MHz, DMSO-
d6, TMS) δ: 158.37, 153.61, 148.58, 141.64, 139.98, 136.78, 135.57,
130.49, 128.85, 128.60, 121.26, 118.96, 118.02, 117.91, 116.86,
116.32, 115.43, 114.89, 114.12. MS (ESI positive): m/z [M+H]+:
345.36.
3-((5-(4-Fluorophenyl)-1H-pyrazolo[3,4-b]pyridin-3-
yl)carbamoyl)benzoic acid (18c)
Yellow solid (0.08 g, yield 52%). 1H NMR (DMSO-d6, 600 MHz) δ: 8.62
(d, J = 2.30 Hz, 1H), 8.12 (t, J = 6.41 Hz, 2H), 8.06 (d, J = 7.21 Hz, 1H),
7.78 (t, J = 6.61 Hz, 2H), 7.68 (t, J = 7.51 Hz, 1H), 7.62 (t, J = 7.51 Hz,
1H), 7.37 (t, J = 8.73 Hz, 2H), 6.75 (d, J = 2.90 Hz, 1H), 6.65 (s, 2H).
13C NMR (150 MHz, DMSO-d6, TMS) δ: 167.47, 152.29, 151.64,
149.38, 134.26, 133.94, 133.55, 132.41, 131.25, 130.95, 129.58,
129.47, 128.60, 127.93, 126.01, 116.75, 116.47, 116.20, 113.21.
HRMS (ESI, positive) m/z calcd. for C20H14FN4O3 (M+H): 377.1044;
found 377.1058.
3-((5-(4-Fluorophenyl)-1H-pyrazolo[3,4-b]pyrazin-3-
yl)carbamoyl)benzoic acid (18d)
Yellow solid (0.05 g, yield 38%). 1H NMR (DMSO-d6, 600 MHz) δ:
12.47 (s, 1H), 9.05 (s, 1H), 8.22 (dd, J1 = 3.55 Hz, J2 = 5.75 Hz, 2H), 7.39
(t, J = 9.12 Hz, 2H), 5.81 (s, 2H). 13C NMR (150 MHz, DMSO-d6, TMS)
δ: 167.19, 166.12, 152.19, 150.83, 144.54, 134.24, 133.44, 130.41,
130.21, 129.77, 128.52, 127.63, 126.10, 116.45, 116.23, 116.10,
113.43, 91.35. MS (ESI positive): m/z [M+H]+: 378.36.
The synthetic method for compounds 14b was similar to the
synthesis of compound 14a.
3-Hydroxy-N-(5-(3-hydroxyphenyl)-1H-indazol-3-yl)benzamide
(14b)
Yellow solid (0.07 g, yield 48%). 1H NMR (DMSO-d6, 600 MHz) δ:
9.71 (s, 1H), 9.59 (s, 1H), 8.35 (d, J = 8.58 Hz, 1H), 8.21 (s, 1H),
7.84 (dd, J1 = 8.58 Hz, J2 = 1.07 Hz, 1H), 7.35 (d, J = 7.51 Hz, 1H),
7.28 (t, J = 8.04 Hz, 2H), 7.12 (d, J = 7.51 Hz, 1H), 7.09 (s,1H), 6.94
(dd, J1 = 8.08 Hz, J2 = 2.33 Hz, 1H), 6.78 (dd, J1 = 8.12 Hz,
J2 = 2.08 Hz, 1H), 6.56 (s, 2H). 13C NMR (150 MHz, DMSO-d6,
TMS) δ: 166.73, 158.34, 157.06, 153.78, 141.58, 139.95, 136.96,
136.02, 130.52, 129.31, 128.98, 121.13, 121.18, 118.99, 118.59,
3-Amino-N-(5-(4-fluorophenyl)-1H-pyrazolo[3,4-b]pyrazin-3-
yl)benzamide (18e)
Yellow solid (0.06 g, yield 65%). 1H NMR (DMSO-d6, 600 MHz) δ:
13.85 (s, 1H), 10.51 (s, 1H), 9.19 (s, 1H), 8.16–8.19 (m, 2H), 7.33 (t,
J = 9.40 Hz, 2H), 7.20–7.23 (m, 2H), 7.15 (t, J = 8.05 Hz, 1H), 6.77 (d,
J = 7.55 Hz, 1H), 5.30 (s, 2H). 13C NMR (150 MHz, DMSO-d6, TMS) δ:
167.74, 158.70, 149.32, 144.08, 142.15, 134.60, 131.01, 133.60,

8
HUANG ET AL.
|
130.94, 129.77, 129.59, 129.41, 117.96, 116.53, 116.25, 115.57,
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ACKNOWLEDGMENTS
SUPPORTING INFORMATION
This work was supported by the National Key R&D Program of China
(2017YFA0506000) and the National Basic Research Program of
China (2014CB541800).
Additional supporting information may be found online in the
Supporting Information section at the end of the article.
CONFLICT OF INTEREST
The authors have declared no conflict of interest.
How to cite this article: Huang Y, Li Y, Dong G, Zhang W,
Liu N, Sheng C. Identification of pyrazolopyridine derivatives
as novel spleen tyrosine kinase inhibitors. Arch Pharm Chem
Life Sci. 2018;1–8. https://doi.org/10.1002/ardp.201800083
ORCID
Chunquan Sheng
http://orcid.org/0000-0001-9489-804X