Discovery of novel Syk/PDGFR-α/c-Kit inhibitors as multi-targeting drugs to treat rheumatoid arthritis

Article abstract of DOI:10.1016/j.bmc.2018.06.029

Due to the complex biological pathways involved in rheumatoid arthritis, discovery of multi-targeting small molecules provides an effective strategy to achieve better efficacy and lower toxicity. Herein the first Syk/PDGFR-α/c-Kit inhibitors were designed and evaluated. Dihydrofuropyrimidine derivative 13 showed potent inhibitory activity against the three targets. Importantly, compound 13 exhibited good cellular efficacy against fibroblast-like synoviocytes (IC₅₀ = 3.21 μM) and mouse bone marrow-derived mast cells (IC₅₀ = 2.03 μM) and significantly decreased the secretion of inflammatory cytokines. Thus, Syk/PDGFR-α/c-Kit triple inhibitor 13 represented a promising lead compound for the treatment of RA.

Full text of DOI:10.1016/j.bmc.2018.06.029

Accepted Manuscript
Discovery of Novel Syk/PDGFR-α/c-Kit Inhibitors as Multi-targeting Drugs to
Treat Rheumatoid Arthritis
Xiaokang Li, Yahui Huang, Junfei Chen, Lingling Zhang, Fei Mao, Jin Zhu,
Chunquan Sheng, Jian Li
PII:
S0968-0896(18)30668-0
https://doi.org/10.1016/j.bmc.2018.06.029
BMC 14421
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
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Revised Date:
Accepted Date:
3 April 2018
14 June 2018
21 June 2018
Please cite this article as: Li, X., Huang, Y., Chen, J., Zhang, L., Mao, F., Zhu, J., Sheng, C., Li, J., Discovery of
Novel Syk/PDGFR-α/c-Kit Inhibitors as Multi-targeting Drugs to Treat Rheumatoid Arthritis, Bioorganic &
Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.06.029
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Discovery of Novel Syk/PDGFR-α/c-Kit Inhibitors as Multi-targeting Drugs to
Treat Rheumatoid Arthritis
Xiaokang Li,1, † Yahui Huang,2, † Junfei Chen, 2, † Lingling Zhang,1 Fei Mao,1 Jin Zhu,1 Chunquan
Sheng,2,* Jian Li1,*
1 Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of
Science & Technology, 130 Meilong Road, Shanghai 200237, People’s Republic of China
2 Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical University, 325
Guohe Road, Shanghai 200433, People’s Republic of China
#These three authors contributed equally to this work.
* To whom correspondence should be addressed.
For C. Sheng: Phone/Fax, 86-21-81871205, E-mail, shengcq@smmu.edu.cn;
For J. Li: Phone/Fax 86-21-64252584, E-mail, jianli@ecust.edu.cn
1

Abstract
Due to the complex biological pathways involved in rheumatoid arthritis, discovery of
multi-targeting small molecules provides an effective strategy to achieve better efficacy and lower
toxicity. Herein the first Syk/PDGFR-α/c-Kit inhibitors were designed and evaluated.
Dihydrofuropyrimidine derivative 13 showed potent inhibitory activity against the three targets.
Importantly, compound 13 exhibited good cellular efficacy against fibroblast-like synoviocytes (IC50
= 3.21 μM) and mouse bone marrow-derived mast cells (IC50 = 2.03 μM) and significantly decreased
the secretion of inflammatory cytokines. Thus, Syk/PDGFR-α/c-Kit triple inhibitor 13 represented a
promising lead compound for the treatment of RA.
Keywords: Rheumatoid arthritis; Syk; PDGFR-α; c-Kit; multi-targeting inhibitors;
dihydrofuropyrimidines
2

1. Introduction
Rheumatoid arthritis (RA) is a chronic and multi-factorial autoimmune disorder, affecting
approximately 0.5-1% of the adult population.1 RA is characterized by the accumulation and
proliferation of inflammatory cells in the synovial lining, resulting in the formation of pannus tissue
and the erosion of cartilage and bone. In RA patients, mast cells and fibroblast-like synoviocytes
(FLSs) are activated and contribute to synovial inflammation and joint destruction. The relationship
of FLS and mast cells with joint damage and the propagation of inflammation has been well
established.2 Fibroblasts express platelet-derived growth factor receptor (PDGFR) and proliferate in
response to a variety of PDGF ligands. Both PDGFR and its ligands are overexpressed in RA
synovial tissue, and PDGF is a potent stimulant of synovial hyperplasia in RA.3 In addition,
increased number of mast cells were found in the synovium of RA patients.2 The activation of mast
cell resulted in the release of mediators, such as tumor necrosis factor-α (TNF-α), contributing to the
inflammatory and destructive processes of RA. Stem cell factor (SCF) is essential for the growth and
survival of mast cells, which also functions as the ligand of c-Kit receptor.4 Thus, the inhibition of
c-Kit signaling might prevent mast cell activity in the synovium of RA patients. Imatinib5 and
masitinib (1)6 are multi-targeted kinase inhibitors (Fig. 1) with potent inhibitory activities against
c-Kit and PDGFR, which also showed considerable antirheumatic efficacy. Masitinib is currently
evaluated as a RA therapeutic drug in phase II/III clinical trials.7
Fig. 1. Chemical structures of the masitinib (1), R406 (2) and fostamatinib disodium (3).
3

Spleen tyrosine kinase (Syk) represents a promising target for the treatment of RA.8 As an
intracellular cytoplasmic non-receptor tyrosine kinase, Syk plays an important role in the production
of cytokine and metalloproteinase in RA FLSs. A number of Syk inhibitors were developed and
several of them, such as R406 (2)9 and fostamatinib disodium (3)10, have been evaluated in clinical
trials (Fig. 1).11 However, clinical development of Syk inhibitors was mainly hampered by adverse
effects including diarrhea, upper respiratory tract infection, neutropenia, hypertension and so on.12 To
overcome the drawbacks of the unsuccessful clinical candidates, discovery of new Syk inhibitors
with improved efficiency and selectivity is still an active area.13 Alternatively, Syk-based
multi-targeting inhibitors offer a new opportunity for the treatment of RA.
Autoimmune disorders are regulated by complex biological pathways. Rather than inhibiting a
single target, blocking multi-targets would be advantageous to achieve better efficacy and lower
toxicity. Herein, the first Syk/PDGFR-α/c-Kit inhibitors were discovered, which showed potent
anti-inflammatory activity. The proof-of-concept study provided a new strategy for the treatment of
RA.
2. Chemistry
The general procedure for the synthesis of thiazolopyrimidine diamine derivatives was outlined in
Scheme 1. Starting from commercially available 4, intermediate 5 was prepared through a
nucleophilic aromatic substitution reaction with various primary amines (R-NH2). In the presence of
DIPEA and NMP, compound 5 reacted with tert-butyl pyrrolidin-3-ylcarbamate under the microwave
condition to afford intermediate 6. Then, it was transformed into intermedate 9 by the condensation
reaction with mono-benzylbenzoate 8, which was synthesized by the subsituion reaction between
acid 7 and benzyl bromide. Finally, target compounds 10-23 were prepared by deprotection of the
4

benzyl group in the presence of Pd/C and methanol.
Scheme 1. Synthesis of dihydrofuropyrimidine diamine derivatives 10-23. Reagents and conditions:
o
(a) DIPEA, DMSO, 60 C, overnight, 65%; (b) tert-butyl pyrrolidin-3-ylcarbamate, DIPEA, NMP,
o
M.W., 80 W, 140 C, 1 h, 49%; (c) (i) Triethylamine, methanol, overnight, r.t., (ii) Benzyl bromide,
o
DMF, 100 C, 2 h, 17% over two steps; (d) HATU, DIPEA, DMAP, EDCI, DMF, 24 h, 63%; (e)
5

Pd/C, methanol, overnight, 34%
3. Results and discussion
3.1 Design of Syk/PDGFR-α/c-Kit Inhibitors
Thiazolopyrimidine diamines were selective Syk inhibitors developed by Roche.13 However, poor
physicochemical properties hampered them from further development. Herein the thiazolopyrimidine
diamine scaffold was used as the starting point for drug design (Fig. 2). The thiazolopyrimidine core
was replaced by dihydrofuropyrimidine to form favorable interactions with Syk/PDGFR-α/c-Kit (for
detail see Fig. 5). Also, the dihydrofuropyrimidine scaffold and the benzene/pyridine replacement
were favorable for improving the water solubility (Table S2 in supporting information). As a result,
dihydrofuropyrimiddine derivatives 10-23 were designed and synthesized. Besides, two
cyclopentapyrimidine diamine derivatives (29 and 31) were designed and synthesized to investigate
the importance of the scaffold oxygen atom and the terminal COOH (Scheme S1 in supporting
information).
Fig. 2. The design rationale of dihydrofuropyrimidine diamine Syk/PDGFR-α/c-Kit inhibitors.
3.2 Syk/PDGFR-α/c-Kit Inhibitory Activities and Structure-Activity Relationships
Syk, PDGFR-α and c-Kit inhibitory activities of the target compounds were assayed (Table 1).
Compound 10 showed moderate inhibitory activity against Syk (IC50 = 23.7 M) and PDGFR-α
(IC50 = 28.1 M). However, it was inactive against c-Kit. Interestingly, after the introduction of an
6

additional methoxyl group at position 5, trimethoxyl derivative 11 was proven to be a Syk (IC50 =
14.2 M), PDGFR-α (IC50 = 2.2 M) and c-Kit (IC50 = 31.3 M) triple inhibitor. Then, a series of
derivatives (12-23) were designed and synthesized by varying the amine substitutions.
Syk/PDGFR-α/c-Kit inhibitory activity was retained for 3,5-dimethoxyl derivative 13. When the
3,4-methoxyl group was fused into 1,4-dioxane, compound 12 was a Syk/c-Kit dual inhibitor,
whose PDGFR-α inhibitory activity was lost. Removal of 3-methoxyl group of compound 10 led to
the loss of PDGFR-α and c-Kit inhibitory activity (compound 14). Similarly, 4-ethyl (16) and
3,4-dimethyl (17) derivatives were only effective against Syk. When the phenyl group of compound
10 was replaced by various alkyl groups, compounds 18-23 were inactive against PDGFR-α and
c-Kit. Among them, compounds 21-23 lost the activity against all the three targets. In addition,
removing the oxygen atom of the scaffold (compound 31) resulted in less potent Syk inhibitory
activity (IC50 = 25.3 μM) than compound 10 (IC50 = 23.7 μM). Moreover, after the esterification of
carboxyl group, compound 29 was totally inactive against Syk (IC50 > 50 μM), indicating that the
importance of the carboxyl group for the Syk inhibitory activity (Table S1 in supporting
information).
Table 1 Chemical structures and anti-Syk assay results of compounds 10-23.
Syk
IC50 (μM)
23.7
PDGFR-α
IC50 (μM)
28.1
c-Kit
IC50 (μM)
> 50
Compounds
10
R
3,4-dimethoxyphenyl
7

3,4,5-trimethoxyphenyl
2,3-dihydrobenzo[b][1,4]dioxin-6-yl
3,5-dimethoxyphenyl
4-methoxyphenyl
4-methylphenyl
4-ethylphenyl
14.2
20.3
20.9
32.1
20.9
19.2
15.6
13.5
9.7
2.2
31.3
49.2
18.6
> 50
> 50
> 50
> 50
> 50
> 50
25.8
> 50
> 50
> 50
11
12
13
14
15
16
17
18
19
20
21
22
23
> 50
7.5
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
3,4-dimethylphenyl
cyclohexyl
hexyl
heptyl
11.4
> 50
> 50
> 50
pentyl
3-methoxypropyl
3-ethoxypropyl
3.3 Cellular Potency of the Selected Compounds
According to the Syk, PDGFR-α and c-kit inhibitory activity, seven compounds (11, 13, 16-20)
were selected to evaluate their potency on FLSs-RA proliferation, using compound 2 as the
reference drug. As shown in Table 2, most compounds were capable of inhibiting FLSs
proliferation and five of them (13, 16, 18-20) showed potent cellular inhibitory activity with IC50
value lower than 10 M. On the basis of the cellular potency, two compounds (13 and 19) were
selected to evaluate whether the addition of inhibitors could block the production of inflammatory
cytokines, such as interleukin-6 (IL-6) and metalloproteinase-3 (MMP-3). The results indicated that
both compounds decreased the production of IL-6 and MMP-3 in a concentration-dependent
manner (Fig. 3). In particular, compound 13 exhibited comparable potency to positive control 2 at
8

the concentration of 15 μM.
Table 2 Effects of preferred compounds on FLSs-RA proliferation.
Compounds
2
11
13 16
17
18
19
20
14.6 31.0 3.2 9.4 11.2 5.4 4.0 8.1
IC50 (μM)
(B)100
**
(A)12
**
2 (14.62 μM)
15 μM
5 μM
0.5 μM
0
***
***
**
80
60
40
20
0
*
***
***
9
***
***
**
*
**
***
***
6
3
0
**
***
**
ns
**
**
*
13
19
13
19
Fig. 3. Effects of compounds 13 and 19 on inflammatory cytokine production of IL-6 (A) and
MMP-3 (B) from FLSs.
Three compounds (11, 13 and 20) were subjected to the mast cell assay using masitinib as the
positive control. Masitinib exhibited strong potency to inhibit the proliferation of mouse bone
marrow-derived mast cells (mBMMCs). However, all of our three compounds were less effective
(Table 3), possibly due to moderate inhibitory activity against the three kinases. The most
promising compound 13 exhibited the inhibitory activity with an IC50 value of 2 μM. Compounds
13 and 20 were further evaluated the ability to decrease the release of TNF-α. Two compounds both
concentration-dependently decreased the release of TNF-α, and compound 13 showed better
potency (Fig. 4).
Table 3 Effects of compounds 11, 13 and 20 on mBMMCs proliferation.
Compounds
1
11
13
20
0.1
21.1
2.0
13.0
IC50 (μM)
9

***
350
280
210
140
70
ns
****
****
1 (0.1 μM)
1 μM
0.1 μM
0
**
****
*
*
ns
ns
0
13
20
Fig. 4. Effects of compounds 13 and 20 on TNF-α production from mBMMCs.
3.4 Binding Mode of Compound 13 with Syk/PDGFR-α/c-Kit
In order to investigate the binding modes of compound 13 with Syk, PDGFR-α and c-Kit,
molecular docking studies were performed. As shown in Fig. 5A, the dihydrofuropyrimidine diamine
scaffold of compound 13 formed a hydrogen bond to the backbone of Ala451 in the hinge region of
Syk (PDB ID: 1XBC14). Moreover, its carboxyl group formed additional hydrogen bonding
interaction with Lys533. For c-Kit (PDB ID: 1T4615), the carbamoylnicotinic acid group formed two
hydrogen bonds with Glu640 and Thr670 in the hinge region of c-Kit (Fig. 5B). Furthermore, an
additional hydrogen bond was observed between the 5-methoxyl group and Ile571, which might be
the reason why 13 was more potent than 3,4-dimethyoxyl derivative 10 against c-Kit. For the binding
mode of 13 with PDGFR-α (PDB ID: 5GRN16), Asp836 formed two hydrogen bonds with the
dihydrofuropyrimidine diamine scaffold and the terminal carboxyl group (Fig. 5C). The pyrimidine
moiety formed π-π interactions with Lys627.
10

Fig. 5. Proposed binding mode of compound 13 in the active site of Syk (PDB ID: 1XBC, A), c-Kit
(PDB ID: 1T46, B) and PDGFR-α (PDB ID: 5GRN, C). Hydrogen bonds are indicated with dashed
lines. The figure was generated using PyMol (http://www.pymol.org/).
11

4. Conclusions
In summary, novel dihydrofuropyrimidine Syk/PDGFR-α/c-Kit triple inhibitors were designed and
synthesized. Compound 13 showed relatively balanced inhibitory activity against the three targets.
Molecular docking studies revealed that it bound with the three kinases mainly through hydrogen
bonding and hydrophobic interactions. Importantly, compound 13 exhibited potent cellular efficacy.
It possessed good anti-proliferative activities against FLSs and mBMMCs with IC50 values of 3.21
μM and 2.03 μM, respectively, and significantly decreased the secretion of inflammatory cytokine.
Thus, compound 13 represented a promising lead compound for further investigation. The discovery
of Syk/PDGFR-α/c-Kit inhibitors could serve as a new strategy for the treatment of RA. Further
structural optimization will focus on improving the inhibitory activity against Syk/PDGFR-α/c-Kit
and adjusting the physicochemical properties.
5. Experimental Section
5.1 Chemistry
Synthetic starting materials, reagents and solvents were purchased from Alfa Aesar, Acros,
Adamas-beta, Energy Chemical, J&K, Shanghai Chemical Reagent Co. and TCI at the highest
commercial quality and used without further purification. Analytical thin-layer chromatography
(TLC) was performed on HSGF 254 (150-200 μm thickness; Yantai Huiyou Co., China), and the
components were visualized by observation under UV light (254 nm and 365 nm). Melting points
were determined on a SGW X-4 melting point apparatus without correction. The products were
purified by recrystallization or column chromatography on silica gel (200-300 mesh). Reaction
yields were not optimized. Nuclear magnetic resonance (NMR) spectroscopy was performed on a
Bruker AMX-400 NMR spectrometer using deuterated chloroform (CDCl3), deuterated methanol
12

(CD3OD), or deuterated dimethyl sulfoxide (DMSO-d6) as the solvent. Chemical shifts were reported
in parts per million (ppm, δ) downfield from tetramethylsilane. Proton coupling patterns were
described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br). Low- and
high-resolution mass spectra (LRMS and HRMS) were given with electron spray ionization (ESI)
produced by a Finnigan MAT-95 and a LCQ-DECA spectrometer. HPLC data analysis of compounds
2-31 were performed on an Agilent 1100 with a quaternary pump and diode-array detector (DAD).
The peak purity was verified with UV spectra. All analogs were confirmed to be > 95% pure.
5.1.1 2-Chloro-N-(3,4-dimethoxyphenyl)-5,7-dihydrofuro[3,4-d]pyrimidin-4-amine (5).
A solution of 2,4-dichloro-5,7-dihydrofuro[3,4-d]pyrimidine (4, 1.89 g, 10 mmol) in DMSO (10
mL) was added commercially available 3,4-dimethoxyaniline (1.53 g, 10 mmol) and DIPEA (2.58 g,
20 mmol). The solution was heated to 60 ºC and stirred overnight. Then the solution was poured into
water and extracted with EtOAc, the organic layer was washed by brine, dried with anhydrous
Na2SO4, and filtered and concentrated under reduced pressure. The crude product was further
purified by flash chromatography on silica gel (EtOAc/hexane = 1:1) to afford compound 5 (2.00 g,
yield 65%) as an off-white solid. 1H NMR (400 MHz, CDCl3) δ: 6.90-6.81 (m, 3H), 4.88 (s, 2H),
4.42 (s, 2H), 3.92 (s, 3H), 3.89 (s, 3H).
5.1.2
2-(3-Aminopyrrolidin-1-yl)-N-(3,4-dimethoxyphenyl)-5,7-dihydrofuro[3,4-d]pyrimidin-4-
amine (6).
A solution of compound 5 (1.85 g, 6.02 mmol) in 1-methyl-2-pyrrolidinone (2 mL) was added
tert-butyl pyrrolidin-3-ylcarbamate (1.68 g, 0.90 mmol) and DIPEA (1.55 g, 12.04 mmol). The
reaction was conducted under the microwave condition. The solution was heated to 140 ºC under 80
W for 1 h, and then the mixture was concentrated under reduced pressure. The crude product was
13

purified by flash chromatography on silica gel (MeOH/CH2Cl2 = 1: 8) to afford compound 6 (1.05 g,
yield 49%) as a brown solid. 1H NMR (400 MHz, DMSO-d6) δ: 8.53 (s, 1H), 7.41-7.34 (m, 1H), 7.30
(dd, J = 8.7, 2.4 Hz, 1H), 6.91 (t, J = 7.6 Hz, 1H), 4.24 (q, J = 7.0 Hz, 2H), 3.72 (t, J = 11.1 Hz, 6H),
2.71 (dd, J = 14.2, 7.1 Hz, 4H), 2.08-1.96 (m, 2H), 1.27 (dd, J = 9.0, 5.2 Hz, 3H).
5.1.3 5-((Benzyloxy)carbonyl)picolinic acid (8).
A solution of pyridine-2,5-dicarboxylic acid (7, 1.67 g, 10 mmol) in MeOH (50 mL) was added
triethylamine (1.52 mL, 11 mmol) dropwisely, and the solution was stirred overnight. The solvent
was evaporated under the reduced pressure. The residue was resolved in DMF (10 mL) and added
benzyl bromide (1.2 mL) dropwisely. The solution was heated to 100 ºC for 2 h. Then the mixture
was poured into water, acidified with diluted HCl and extracted by EtOAc. The organic layer was
washed by brine, dried with anhydrous Na2SO4, filtered and concentrated under the reduced pressure.
The crude product was further purified by flash chromatography on silica gel (EtOAc/hexane = 1: 1)
to afford compound 8 (0.44 g, yield 17%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ: 9.39 (s,
1H), 8.43 (d, J = 7.5 Hz, 1H), 8.12 (d, J = 7.4 Hz, 1H), 7.53 (d, J = 7.2 Hz, 2H), 7.41 (dt, J = 23.2,
7.2 Hz, 3H), 5.41 (s, 2H).
5.1.4
6-((1-(4-((3,4-Dimethoxyphenyl)amino)-5,7-dihydrofuro[3,4-d]pyrimidin-2-yl)pyrrolidin
-3-yl)carbamoyl)nicotinic acid (10).
A solution of compound 6 (0.21 g, 0.58 mmol) in DMF (5 mL) was added intermediate 8 (0.15 g,
0.58 mmol), DIPEA (0.20 mL, 1.16 mmol), HATU (0.21 g, 1.16 mmol). After stirring for 2 h, EDCI
(0.22 g, 1.16 mmol) and DMAP (0.14 g, 1.16 mmol) were added into the solution and the resulting
mixture was stirred overnight. The solution was poured into water and extracted by EtOAc and the
organic layer was washed by brine, dried with anhydrous Na2SO4, filtered and concentrated under
14

reduced pressure to obtain the crude product 9 (0.22 g, yield 63%) without further purification. Then
compound 9 was dissolved in MeOH (20 mL) followed by the addition of 10% Pd/C (0.001 mmol).
The mixture was stirred under the H2 atmosphere overnight. The catalyst was filtered off and the
combined organic solution was concentrated under reduced pressure to afford target compound 10
(0.046 g, yield 44%) as an off-white solid. 1H NMR (400 MHz, DMSO) δ: 9.15 (s, 1H), 8.94 (s, 1H),
8.59 (s, 1H), 8.42 (s, 1H), 8.09 (s, 1H), 7.62 (s, 1H), 7.21 (s, 1H), 6.89 (s, 1H), 4.89 (s, 2H), 4.68 (s,
2H), 4.51 (s, 1H), 4.37 (s, 1H), 3.71 (s, 6H), 3.41 (d, J = 20.6 Hz, 2H), 2.87 (d, J = 64.1 Hz, 1H),
2.20 (s, 1H), 1.99 (s, 1H); HRMS (ESI) m/z calcd C25H27N6O6 [M+H]+ 507.1992, found 507.1992.
5.1.5
6-((1-(4-((3,4,5-Trimethoxyphenyl)amino)-5,7-dihydrofuro[3,4-d]pyrimidin-2-yl)
pyrrolidin-3-yl)carbamoyl)nicotinic acid (11).
1
Yield 22%, off-white solid. H NMR (400 MHz, DMSO) δ: 8.95 (s, 1H), 8.59 (d, J = 14.8 Hz, 2H),
8.43 (s, 1H), 8.10 (s, 1H), 7.28 (s, 2H), 4.92 (s, 2H), 4.70 (s, 2H), 4.50 (s, 1H), 3.75 (d, J = 7.8 Hz,
7H), 3.60 (s, 5H), 3.48-3.40 (m, 1H), 2.20 (s, 1H), 2.02 (s, 1H); HRMS (ESI) m/z calcd C26H29N6O7
[M+H]+ 537.2098, found 537.2101.
5.1.6
6-((1-(4-((2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)amino)-5,7-dihydrofuro[3,4-d]pyrimidin
-2-yl)pyrrolidin-3-yl)carbamoyl)nicotinic acid (12).
Yield 30%, off-white solid. 1H NMR (400 MHz, DMSO) δ: 8.94 (s, 1H), 8.88 (s, 1H), 8.59 (s, 1H),
8.23 (d, J = 7.3 Hz, 1H), 7.97 (d, J = 8.1 Hz, 1H), 7.61-7.47 (m, 1H), 7.15 (d, J = 8.8 Hz, 1H), 6.77
(d, J = 8.6 Hz, 1H), 4.87 (s, 2H), 4.68 (s, 2H), 4.58 (s, 1H), 4.20 (d, J = 6.5 Hz, 4H), 3.73 (d, J = 32.8
Hz, 2H), 3.54 (s, 2H), 2.25 (s, 1H), 2.04 (s, 1H); HRMS (ESI) m/z calcd C25H25N6O4 [M+H]+
505.1836, found 505.1839.
5.1.7
6-((1-(4-((3,5-Dimethoxyphenyl)amino)-5,7-dihydrofuro[3,4-d]pyrimidin-2-yl)pyrrolidin
15

-3-yl)carbamoyl)nicotinic acid (13).
1
Yield 21%, off-white solid. H NMR (400 MHz, DMSO) δ: 8.96 (s, 1H), 8.89 (s, 1H), 8.68 (s, 1H),
8.24 (d, J = 7.5 Hz, 1H), 7.98 (s, 1H), 7.19 (s, 2H), 6.12 (s, 1H), 4.90 (d, J = 25.4 Hz, 2H), 4.71 (s,
13
2H), 4.56 (s, 1H), 3.74 (d, J = 18.9 Hz, 8H), 3.57 (s, 2H), 2.24 (s, 1H), 2.07 (s, 1H); C NMR (150
MHz, DMSO) δ 168.95, 166.17, 163.92, 161.08, 160.72, 154.97, 153.25, 149.55, 142.54, 139.17,
129.27, 122.48, 102.12, 98.31, 72.57, 70.96, 55.51, 49.53, 30.67; HRMS (ESI) m/z calcd
C25H27N6O4 [M+H]+ 507.1992, found 507.1997. HPLC purity: 97.8%.
5.1.8
6-((1-(4-((4-Methoxyphenyl)amino)-5,7-dihydrofuro[3,4-d]pyrimidin-2-yl)pyrrolidin
-3-yl)carbamoyl)nicotinic acid (14).
1
Yield 28%, off-white solid. H NMR (400 MHz, DMSO) δ: 9.00 (s, 1H), 8.62 (d, J = 32.9 Hz, 2H),
8.40 (d, J = 32.9 Hz, 1H), 8.10 (s, 1H), 7.69 (s, 2H), 6.88 (d, J = 7.7 Hz, 2H), 4.86 (s, 2H), 4.67 (s,
2H), 4.53 (s, 1H), 3.72 (s, 4H), 3.58 (s, 1H), 3.55 (s, 2H), 2.18 (s, 1H), 1.99 (s, 1H); HRMS (ESI)
m/z calcd C24H25N6O5 [M+H]+ 477.1886, found 477.1887.
5.1.9
6-((1-(4-(p-Tolylamino)-5,7-dihydrofuro[3,4-d]pyrimidin-2-yl)pyrrolidin-3-yl)carbamoyl)
nicotinic acid (15).
1
Yield 28%, off-white solid. H NMR (400 MHz, MeOD) δ: 8.97 (s, 1H), 8.43 (s, 1H), 8.21 (s, 1H),
7.62 (d, J = 8.0 Hz, 2H), 7.11 (d, J = 8.0 Hz, 2H), 4.91 (s, 2H), 4.76 (s, 2H), 4.67 (s, 1H), 3.90 (s,
1H), 3.74 (s, 1H), 3.63 (s, 2H), 2.34 (s, 1H), 2.29 (s, 3H), 2.15 (s, 1H); HRMS (ESI) m/z calcd
C24H25N6O4 [M+H]+ 461.1937, found 461.1940.
5.1.10
6-((1-(4-((4-Ethylphenyl)amino)-5,7-dihydrofuro[3,4-d]pyrimidin-2-yl)pyrrolidin
-3-yl)carbamoyl)nicotinic acid (16). Yield 30%, off-white solid. 1H NMR (400 MHz, DMSO) δ: 9.26
(s, 1H), 8.94 (s, 1H), 8.68 (s, 1H), 8.50-8.29 (m, 1H), 8.16-7.97 (m, 1H), 7.71 (s, 2H), 7.13 (d, J =
16

7.8 Hz, 2H), 4.89 (s, 2H), 4.68 (s, 2H), 4.59 (s, 1H), 3.79 (s, 1H), 3.57 (s, 3H), 2.55 (d, J = 6.1 Hz,
2H), 2.22 (s, 1H), 2.05 (s, 1H), 1.16 (dd, J = 8.6, 5.8 Hz, 3H); HRMS (ESI) m/z calcd C25H27N6O4
[M+H]+ 475.2094, found 475.2094.
5.1.11
6-((1-(4-((3,4-Dimethylphenyl)amino)-5,7-dihydrofuro[3,4-d]pyrimidin-2-yl)pyrrolidin-3
-yl)carbamoyl)nicotinic acid (17).
1
Yield 25%, off-white solid. H NMR (400 MHz, DMSO) δ: 8.96 (s, 1H), 8.60 (d, J = 18.9 Hz, 2H),
8.39 (d, J = 24.9 Hz, 1H), 8.09 (d, J = 8.2 Hz, 1H), 7.66 (s, 1H), 7.50 (s, 1H), 7.04 (s, 1H), 4.88 (s,
2H), 4.68 (s, 2H), 4.52 (s, 1H), 3.77 (s, 1H), 3.57 (s, 3H), 2.28-2.11 (m, 7H), 1.99 (s, 1H); HRMS
(ESI) m/z calcd C25H27N6O4 [M+H]+ 475.2094, found 475.2094.
5.1.12 6-((1-(4-(Cyclohexylamino)-5,7-dihydrofuro[3,4-d]pyrimidin-2-yl)pyrrolidin-3-yl) carbamoyl)
nicotinic acid (18).
1
Yield 29%, off-white solid. H NMR (400 MHz, DMSO) δ: 9.24 (s, 1H), 8.93 (s, 1H), 8.65-8.36 (m,
1H), 8.08 (d, J = 10.4 Hz, 1H), 6.58 (t, J = 11.2 Hz, 1H), 4.76 (s, 2H), 4.59 (s, 2H), 4.56-4.44 (m,
1H), 3.86 (s, 1H), 3.74 (s, 1H), 3.55 (d, J = 20.0 Hz, 2H), 3.47 (d, J = 13.6 Hz, 1H), 2.19 (s, 1H),
1.96 (s, 1H), 1.90 (s, 3H), 1.70 (s, 3H), 1.59 (d, J = 11.7 Hz, 2H), 1.18-1.05 (m, 2H); HRMS (ESI)
m/z calcd C23H29N6O4 [M+H]+ 453.2250, found 453.2252.
5.1.13
6-((1-(4-(Hexylamino)-5,7-dihydrofuro[3,4-d]pyrimidin-2-yl)pyrrolidin-3-yl)carbamoyl)
nicotinic acid (19).
1
Yield 25%, off-white solid. H NMR (400 MHz, DMSO) δ: 8.91 (s, 1H), 8.86 (d, J = 6.4 Hz, 1H),
8.23 (d, J = 7.7 Hz, 1H), 7.97 (d, J = 8.1 Hz, 1H), 6.86 (s, 1H), 4.76 (s, 2H), 4.61 (s, 2H), 4.53 (d, J =
6.3 Hz, 1H), 3.76 (s, 1H), 3.62 (s, 1H), 3.50 (s, 1H), 3.47-3.42 (m, 1H), 3.32-3.25 (m, 2H), 2.18 (dd,
J = 11.3, 7.1 Hz, 1H), 2.01 (dd, J = 11.2, 5.6 Hz, 1H), 1.53 (s, 2H), 1.30 (d, J = 26.1 Hz, 6H), 0.84 (d,
17

J = 6.6 Hz, 3H); HRMS (ESI) m/z calcd C23H31N6O4 [M+H]+ 455.2407, found 455.2406.
5.1.14 6-((1-(4-(Heptylamino)-5,7-dihydrofuro[3,4-d]pyrimidin-2-yl)pyrrolidin-3-yl)carbamoyl)
nicotinic acid (20).
1
Yield 29%, off-white solid. H NMR (400 MHz, DMSO) δ: 9.05 (s, 1H), 8.93 (d, J = 6.6 Hz, 1H),
8.33 (d, J = 7.5 Hz, 1H), 8.06 (d, J = 8.1 Hz, 1H), 6.84 (s, 1H), 4.76 (s, 2H), 4.61 (s, 2H), 4.54 (d, J =
6.5 Hz, 1H), 3.87 (d, J = 23.0 Hz, 1H), 3.78 (s, 1H), 3.62 (s, 1H), 3.53 (s, 1H), 3.46 (dd, J = 11.5, 4.5
Hz, 2H), 2.21 (dd, J = 12.4, 6.5 Hz, 1H), 2.04-1.97 (m, 1H), 1.53 (s, 2H), 1.26 (d, J = 18.4 Hz, 8H),
0.82 (s, 3H); HRMS (ESI) m/z calcd C24H33N6O4 [M+H]+ 469.2563, found 469.2563.
5.1.15
6-((1-(4-(Pentylamino)-5,7-dihydrofuro[3,4-d]pyrimidin-2-yl)pyrrolidin-3-yl)carbamoyl)
nicotinic acid (21).
1
Yield 31%, off-white solid. H NMR (400 MHz, DMSO) δ: 9.04 (s, 1H), 8.92 (d, J = 6.2 Hz, 1H),
8.31 (d, J = 8.0 Hz, 1H), 8.05 (d, J = 8.1 Hz, 1H), 6.83 (d, J = 5.2 Hz, 1H), 4.76 (s, 2H), 4.61 (s, 2H),
4.54 (d, J = 5.2 Hz, 1H), 3.77 (s, 1H), 3.62 (s, 1H), 3.46 (dd, J = 11.4, 4.5 Hz, 2H), 3.33 (s, 2H), 2.21
(dd, J = 13.0, 7.0 Hz, 1H), 2.05-1.99 (m, 1H), 1.54 (d, J = 6.5 Hz, 2H), 1.28 (d, J = 13.2 Hz, 4H),
0.86 (s, 3H); HRMS (ESI) m/z calcd C22H29N6O4 [M+H]+ 441.2250, found 441.2248.
5.1.16
6-((1-(4-((3-Methoxypropyl)amino)-5,7-dihydrofuro[3,4-d]pyrimidin-2-yl)pyrrolidin
-3-yl)carbamoyl)nicotinic acid (22).
1
Yield 20%, off-white solid. H NMR (400 MHz, DMSO) δ: 9.06 (s, 1H), 8.95 (d, J = 6.4 Hz, 1H),
8.34 (d, J = 8.0 Hz, 1H), 8.08 (d, J = 8.1 Hz, 1H), 6.86 (s, 1H), 4.77 (s, 2H), 4.61 (s, 2H), 4.54 (d, J =
5.8 Hz, 1H), 3.79 (s, 1H), 3.62 (s, 1H), 3.53 (s, 1H), 3.49-3.45 (m, 1H), 3.36 (t, J = 5.9 Hz, 4H), 3.22
(s, 3H), 2.21 (dd, J = 12.7, 6.4 Hz, 1H), 2.05-2.00 (m, 1H), 1.79 (dd, J = 13.1, 6.5 Hz, 2H); HRMS
(ESI) m/z calcd C21H27N6O5 [M+H]+ 443.2043, found 443.2040.
18

5.1.17
6-((1-(4-((3-Ethoxypropyl)amino)-5,7-dihydrofuro[3,4-d]pyrimidin-2-yl)pyrrolidin-3-yl)
1
carbamoyl)nicotinic acid (23). Yield 30%, off-white solid. H NMR (400 MHz, DMSO) δ: 9.08 (s,
1H), 8.96 (d, J = 6.5 Hz, 1H), 8.36 (d, J = 8.1 Hz, 1H), 8.10 (d, J = 8.1 Hz, 1H), 6.85 (t, J = 5.3 Hz,
1H), 4.77 (s, 2H), 4.61 (s, 2H), 4.55 (d, J = 6.1 Hz, 1H), 3.80 (d, J = 16.7 Hz, 1H), 3.63 (s, 1H), 3.52
(s, 1H), 3.47 (dd, J = 11.4, 4.8 Hz, 1H), 3.39 (dd, J = 13.1, 6.5 Hz, 6H), 2.21 (dd, J = 12.7, 6.5 Hz,
1H), 2.02 (dd, J = 13.4, 7.4 Hz, 1H), 1.84-1.70 (m, 2H), 1.08 (t, J = 6.9 Hz, 3H); HRMS (ESI) m/z
calcd C22H29N6O5 [M+H]+ 457.2199, found 457.2199.
5.2 Biology
5.2.1 Kinase inhibition assays
The kinase assays were performed in the kinases buffer. Syk kinase: 50 mM HEPES pH 7.5, 0.015%
Brij-35, 10 mM MgCl2, 10 mM MnCl2, 2 mM DTT; PDGFR-α/c-Kit kinase buffer: 50 mM HEPES
pH 7.5, 0.01% Brij-35, 10 mM MgCl2, 10 mM MnCl2, 2 mM DTT. A 5 μL volume of tested
compounds were pre-diluted for dose response in 384-well plates. A 10 μL volume of diluted enzyme
solution was sequentially added and the assay plates were incubated at room temperature for 10 min,
and then. And then a 10 μL volume of a mixture of peptide solution containing FAM-labeled peptide
(Cat. No.112396, Lot. No. P100804-XZ112396; GL Biochem, China) and ATP (Cat. No. A7699-1G,
CAS No. 987-65-5; Sigma, America) was incubated at 28 °C for 25 min. Reaction was stopped with
the addition of 50 mM EDTA containing 25 μL of 100 mM HEPES, pH 7.5, 0.015% Brij-35 and 0.2%
Coating Reagent #3, and the data were collected on a caliper. Half-maximal inhibition (IC50) values
were calculated using a non-linear curve fit with XLfit software.
5.2.2 FLSs assay
Fibroblast-liked synoviocytes (FLS; MEXN, Shanghai, China) were separated from synovial
19

tissues of RA patients. FLS-RA cells were maintained in high-glucose Dulbecco’s Modified Eagle’s
Medium (DMEM; Corning, America) containing 10% fetal bovine serum (FBS; Gibco, America) at
37 °C in humidified environment containing 5% CO2. FLS-RA cells from a homogeneous population
were cultured for 3 generations and were used for subsequent experiments.
Antiproliferation assay was evaluated using the MTT assay performed according to the
manufacturer’s protocol. FLS-RA cells were seeded at a density of 2 × 104 cells/well into 96-well
plates. A 1 μL volume of tested compounds of different concentrations were added into a 96-well
plate (the final volume was 100 μL). Meanwhile, FLS-RA cells without inhibitors were evaluated as
a control. FLS-RA cells were incubated in the medium under 5% CO2 in an incubator maintained at
37 °C for 72 h, respectively. Then, a 10 μL volume of the MTT was added to each well of a 96-well
incubated for additional 4 h. The absorbance was measured at 450 nm by spectrophotometer readings.
All wells were incubated for 72 h at 37 °C. Half-maximal inhibition (IC50) values were calculated
from 6-point dose-response curves using non-linear regression analysis.
IL-6 and MMP-3 examined by ELISA. FLS-RA cells were seeded at a density of 2 × 104
cells/well into 6-well plate, and then incubated with compounds 2, 13, 19 or vehicle for 30 min. After
a 72 h incubation time, supernatants were collected for the determination of the IL-6 and MMP-3
levels using enzyme-linked immunoassay (ELISA) kits (Dakewe Biotech Co. Ltd., China) according
to kit manufacturer’s protocol.
5.2.3 mBMMCs assay
mBMMCs from the tibia and the femur of BALB/c mouse were purchased from Shanghai Slack
laboratory animal co., LTD (Shanghai, China). mBMMCs were maintained at 37 °C in Roswell Park
Memorial Institute medium (RPMI 1640 with 1% non-essential amino acid; Corning, America)
20

containing 10% FBS in humidified environment containing 5% CO2. Antiproliferation assay was
evaluated using MTT performed according to the manufacturer’s protocol. mBMMCs were seeded at
a density of 2 × 104 cells/well into 96-well plates. A 1 μL volume of tested compounds of different
concentrations were added into a 96-well plate (the final volume was 100 μL). Meanwhile,
mBMMCs without inhibitors were evaluated as a control. mBMMCs were incubated in the medium
under 5% CO2 in an incubator maintained at 37 °C for 48 h, respectively. Then, a 10 μL volume of
the MTT was added to each well of a 96-well incubated for additional 4 h. The absorbance was
measured at 450 nm by spectrophotometer readings. All wells were incubated for 48 h at 37 °C. IC50
values were calculated from 9-point dose-response curves using non-linear regression analysis.
TNF-α was examined by ELISA kit according to the manufacturer’s instructions. mBMMCs were
seeded at a density of 2 × 104 cells/well into 6-well plate, and then incubated with compounds 1, 13,
20 or vehicle for 30 min. After a 48 h incubation time, supernatants were collected for the
determination of the TNF-α levels using ELISA kits (Dakewe Biotech Co. Ltd., China) according to
kit manufacturer’s protocol.
5.3 Molecular docking
The crystal structure of Syk in complex with staurosporin (a Syk inhibitor) was obtained from
protein database bank (PDB ID: 1XBC14), which was prepared for docking using the protein
preparation tool in Discovery Studio 3.0.17 During this process, the ligands and waters were removed
and hydrogens were added to the structure. Staged minimization was performed with default setting.
The docking studies were carried out using GOLD 5.0.18 Binding site was defined as whole residues
within a 10 Å radius subset encompassing the staurosporin. Conformations were generated by
genetic algorithm and scored using GoldScore as fitness function. The best conformation was chosen
21

to analyse the ligand-protein interaction. The image representing the best pose was prepared using
PyMOL. Docking analysis was performed on PDGFR-α and c-Kit (PDB ID: 5GRN, 1T46,
respectively)15, 16 using a similar procedure as described above.
5.4 Statistical Analysis
Data was represented as the mean ± SD with at least three independent experiments. Comparisons
between two groups were analyzed for statistical significance using Student’s t test. The levels of
significance were set at P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***).
Acknowledgments
This work was supported by the National Key R&D Program of China (2017YFA0506000 to C.
S.), National Natural Science Foundation of China (81725020 to C. S.), and the National Basic
Research Program of China (2014CB541800 to C. S.).
A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at XXXX.
22

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Graphical Abstract
Discovery of Novel Syk/PDGFR-α/c-Kit Inhibitors as Multi-targeting Drugs to
Treat Rheumatoid Arthritis
Xiaokang Li,1, † Yahui Huang,2, † Junfei Chen, 2, † Lingling Zhang,1 Fei Mao,1 Jin zhu,1 Chunquan
Sheng,2,* Jian Li1,*
25