Discovery of Benzylidene Derivatives as Potent Syk Inhibitors: Synthesis, SAR Analysis, and Biological Evaluation

Article abstract of DOI:10.1002/ardp.201500096

Four scaffolds of varied benzylidene derivatives were synthesized and evaluated as Syk inhibitors for the treatment of rheumatoid arthritis (RA). Among these 31 compounds, 3-benzylidene pyrrolidine-2,5-dione derivatives (including 12k) universally showed good Syk inhibitory activities in the low micromolar to submicromolar range. In the cellular profiling, compound 12k, the most efficient compound, showed excellent antiproliferative activity against fibroblast-like synoviocytes (FLS)-RA, and demonstrated potencies for suppression of IL-6 and MMP-3 secretion almost equal to R406 (positive control). The oral efficacy of 12k in the murine collagen-induced arthritis model was significant, despite being weaker than R406. Taken together, all preliminary pharmacological results supported 12k as a potential small-molecule inhibitor targeting Syk for the treatment of RA.

Full text of DOI:10.1002/ardp.201500096

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Full Paper
Discovery of Benzylidene Derivatives as Potent Syk Inhibitors:
Synthesis, SAR Analysis, and Biological Evaluation
Lingling Zhang1, Wei Liu2, Fei Mao1, Jin Zhu1, Guoqiang Dong3, Hualiang Jiang1, Chunquan Sheng3,
Liyan Miao4, Lixin Huang2, and Jian Li1
1
Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and
Technology, Shanghai, China
Department of Orthopaedic, The First Affiliated Hospital of Soochow University, Suzhou, China
School of Pharmacy, Second Military Medical University, Shanghai, China
Department of Clinical Pharmacology Research Laboratory, The First Affiliated Hospital of Soochow
2
3
4
University, Suzhou, China
Four scaffolds of varied benzylidene derivatives were synthesized and evaluated as Syk inhibitors for the
treatment of rheumatoid arthritis (RA). Among these 31 compounds, 3-benzylidene pyrrolidine-2,5-dione
derivatives (including 12k) universally showed good Syk inhibitory activities in the low micromolar to
submicromolar range. In the cellular profiling, compound 12k, the most efficient compound, showed
excellent antiproliferative activity against fibroblast-like synoviocytes (FLS)-RA, and demonstrated
potencies for suppression of IL-6 and MMP-3 secretion almost equal to R406 (positive control). The oral
efficacy of 12k in the murine collagen-induced arthritis model was significant, despite being weaker than
R406. Taken together, all preliminary pharmacological results supported 12k as a potential small-
molecule inhibitor targeting Syk for the treatment of RA.
Keywords: Antiproliferation / Benzylidene derivatives / Biological evaluation / Rheumatoid arthritis / Syk
inhibitors
Received: March 16, 2015; Revised: April 17, 2015; Accepted: April 17, 2015
DOI 10.1002/ardp.201500096
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
mediated signaling in inflammatory cells [1], such as B-cells,
T-cells, monocytes, macrophages, mast cells, neutrophils,
Introduction
Spleen tyrosine kinase (Syk), a 72-kDa multiple-domain
intracellular cytosolic non-receptor protein tyrosine kinase,
serves as a key mediator of Fc receptor- and B-cell receptor-
basophils, dendritic cells (DC), as well as noninflammatory
cells [2], such as osteoclasts and synoviocytes. Syk is located
upstream in the cell signaling pathway, and possesses
Correspondence: Prof. Jian Li, Shanghai Key Laboratory of New
Drug Design, School of Pharmacy, East China University of Science
and Technology, 130 Mei Long Road, Shanghai 200237, China
E-mail: jianli@ecust.edu.cn
piperazineethanesulfonic acid; HRMS, high resolution mass
spectrometer; IC50, half maximal inhibitory concentration; IgA,
immunoglobulin A; IL-6, interleukin-6; MMP-3, matrix metal-
loproteinase-3; MTT, 3-4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-H-
tetrazolium bromide; PAINS, pan-assay interference compounds;
RA, rheumatoid arthritis; SAR, structure–activity relationship; Syk,
spleen tyrosine kinase; TNFa, tumor necrosis factor a; TZD,
thiazolidinedione; UV, ultraviolet; YLD, years lived with disability.
Fax: þ86-21-64252584
Abbreviations: ATP, adenosine triphosphate; Brij-35, polyoxye-
thyleneglycol dodecyl ether; CCK-8, cell counting kit-8; CFA,
complete Freund’s adjuvant; CIA, collagen-induced arthritis; DTT,
dithiothreitol; EDTA, ethylene diamine tetraacetic acid; EI, electric
ionization; ELISA, enzyme-linked immunoassay; ESI, electrospray
ionization; FAM, fast auxiliary memory; FBS, fetal bovine serum;
FLS, fibroblast-like synoviocytes; H-DMEM, high-glucose Dulbec-
co’s modified Eagle’s medium; HEPES, 4-2-hydroxyethyl-1-
*Additional correspondence: Prof. Lixin Huang,
E-mail: szhuanglx@yeah.net;
Prof. Liyan Miao, E-mail: miaolysuzhou@163.com
Lingling Zhang and Wei Liu contributed equally to this work.
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multiple phosphorylation sites, several of which serve as
docking sites for downstream signaling proteins that orches-
trate overall cellular process [3]. Therefore, therapies target-
ing Syk might be more advantageous than that acting on a
single downstream event. Despite the compelling result,
blocking the activation of inflammatory cells (e.g., macro-
phages and B cells) may raise the possibility of side effects
owing to the shutting down of the whole inflammatory
process.
Rheumatoid arthritis (RA) is a chronic and multifactorial
autoimmune disorder that is associated with the release of
autoantibodies, pannus formation, the erosion of cartilage
and bone, synovial hyperplasia, and various extra-articular
manifestations. A report from the World Health Organization
releases that RA is the 31st leading reason of years lived with
disability (YLD) accounting for 0.8% of total global YLD [4].
Several biologic agents targeting at proinflammatory cyto-
kines have had a high effect on the treatment of RA [5].
However, these therapies are expensive and will produce
significant side effects. The majority of patients relapse when
the treatment is withdrawn [6]. In addition, approximately
one-third of the patients fail to respond adequately to these
agents [7]. Consequently, the use of small molecule inhibitors
(e.g., Syk-targeting inhibitors) against the signaling pathways
has been a recent focus for development of drugs for RA.
Nowadays, numerous Syk chemtypes (Fig. 1) including
1 (R406) [8], 2 (fostamatinib disodium) [9], 3 (R112) [10], 4
(GS-9973) [11], 5 (BAY 61-3606) [12], 6 (P505-15) [13], and 7
(GSK-143) [14] have been reported by famous pharmaceutical
companies such as Rigel, Gilead Sciences, Bayer, Portola, and
GlaxoSmithKline. Fostamatinib disodium (2) is the disodium
phosphate prodrug form of R406 (1) and is the best-
characterized Syk inhibitor in patient studies. With good
solubility but poor selectivity, 2 has completed phase II trials in
human patients for RA, but failed in phase III trials [15]. The
drug is currently in phase I for IgA nephropathy and phase II
for idiopathic thrombocytopenic purpura (ITP) [16]. R112 (3) is
an intranasal inhibitor tested for the alleviation of seasonal
allergies and now under phase II for allergic rhinitis. GS-9973
(4) failed in phase I for RA and now is in phase II as a potential
indication for hematological malignancies. BAY 61-3606 (5) is
an imidazopyrimdine analog that selectively inhibits Syk
activity to treat allergic diseases in vitro. Diaminopyrimidine
carboxamides P505-15 (6) and GSK-143 (7) are both highly
selective Syk inhibitors. Compound 6 is currently in phase II for
allergic asthma and other inflammatory disorders, while 7 is
Figure 1. Examples of Syk inhibitors. R406 (1), fostamatinib disodium (2), R112 (3), GS-9973 (4), BAY 61-3606 (5), P505-15 (6), and
GSK-143 (7).
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in Schemes 2–5. The exocyclic double bond was Z-configura-
tion in series of 2,4-thiazolidinedione (TZD, 8a–g, 9a–f), 6,7-
dihydro-2H-thiazolo[3,2-a]pyrimidin-3(5H)-one (10a–d) and
2H-benzo[b][1,4]oxazin-3(4H)-one (11a,b) or E-configuration
in the system of pyrrolidine-2,5-dione (12a–l) which may
be ascribed to the thermodynamic stability of these config-
urations. The key intermediates 16 were synthesized in
one or two steps (Scheme 1). 3-Bromo-4-ethoxy-5-hydroxy-
benzaldehyde (14a) was obtained by halogenation following
O-alkylation of commercially available 3,4-dihydroxybenzal-
dehyde (13a). In parallel, 3-bromo-5-ethoxy-4-hydroxy-
benzaldehyde (14b) was acquired by halogenation of the
commercial 3-ethoxy-4-hydroxybenzaldehyde (13b). Subse-
quent O-alkylation coupling reaction of 14 with varied benzyl
halide 15 afforded 16. All of the heterocycle parts (17, 19, 20,
22) of our structural classes were either purchased from
reagent companies or prepared according to the literature
procedures.
Scheme 1. Reagents and conditions: a) (i) Br2, acetic acid, rt, 2 h;
(ii) CH3CH2I, Cs2CO3, DMF, 40°C, overnight; b) K2CO3, DMF, 40°C,
4 h; c) Br2, acetic acid, rt, 2 h.
under further modification due to a mutagenicity risk [17].
However, none of them is put into practical use. Thus,
research on Syk inhibitors for the treatment of autoimmune
(RA) is necessary and has a great significance.
SAR analysis
Herein, we carried out a random screening of our internal
chemical library and identified compound 9a which had a
promising inhibitory activity in Syk kinase inhibition assay.
Further design and synthesis led to four structural classes
of benzylidene compounds, and the biological activities
of them were evaluated in vitro and in vivo. A number of
3-benzylidene pyrrolidine-2,5-dione analogs were found
to be potent Syk inhibitors with single-digit micro-molar
inhibitory activity except 12b. A promising compound 12k
demonstrated almost equal potencies for the suppression of
IL-6 and MMP-3 to R406 in the cellular assay. Oral efficacy in
the murine collagen-induced arthritis (CIA) model supported
12k as a good lead compound for potential treatment for RA.
The 5-benzylidene thiazolidine-2,4-dione derivative 9a, which
was discovered by a random screening of our internal
chemical library, exhibited promising Syk inhibitory activity,
with an IC50 value of 3.6 mM. Maintaining the structure of
5-benzylidene thiazolidine-2,4-dione (8, 9) or replacing the
thiazolidine-2,4-dione with other heterocycle moieties (10–
12), four structural classes of small molecules were designed
and synthesized.
For the first series of compounds, we focused on the
structure of 5-benzylidene thiazolidine-2,4-dione relating to
the parent compound 9a and synthesized five derivatives (9b–
f). Initially we sought to vary the R2 substituent while fixing
the R3 group of the TZD part and the R1 group on oxygen
atom. Compound 9b without the carboxyl substituent at the
R2 group did not show any Syk inhibitory activity. Similarly,
esterification of carboxyl group with methanol or benzyl
alcohol (9e,f) resulted in the loss of potency. Replacement of
the carboxylbenzyl group with a methyl (9c) produced a slight
decrease (IC50 ¼ 7.8 mM, Table 1). In addition, removal of the
whole 4-carboxylbenzyl group (9d) was about threefold less
potent compared with the hit. Compound 8a, which was
Results and discussion
Chemistry
Four structural classes of compounds (8–9, 10–12) were
prepared by Knoevenagel reaction or Wittig reaction starting
from the intermediates 16 (see Scheme 1), as shown
Scheme 2. Reagents and conditions: a)
piperdine, EtOH, reflux, 36 h; b) 2-bromo-1-
phenylethanone, K2CO3, acetone, reflux, 8 h.
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Scheme 3. Reagents and conditions: a) (i)
carbon disulphide, EtOH, 40°C, overnight; (ii)
H2O, 40°C, 10 h; (iii) BrCH2COOH, absolute
ethanol, reflux, 6 h; b) piperdine, EtOH,
reflux, 48 h.
obtained by removing the 2-oxo-2-phenylethyl part of 9a,
exerted a comparative activity with an IC50 value of 2.9 mM.
Compounds 8b–f, the truncated TZDs bearing a pyridin-2-yl,
2-chlorobenzyl, 3,5-dimethylbenzyl, 3,4,5-trimethoxybenzyl,
or 2,4-dichlorobenzyl groups at the R2 position, exhibited
inhibitory activity in a range of 0.5–50.0 mM. Notably, analog
8f, which possessed a 2,4-dichlorobenzyl group at the R2
position, displayed an excellent activity by near sevenfold
more potent compared with the hit. Interestingly, compound
8g, in which the position of the ethyl and 2,4-dichlorobenzyl
groups of 8f was transposed, was tolerated with a moderate
increase (IC50 ¼ 0.4 mM, Table 1). Considering “pan-assay
interference compounds (PAINS)” feature of TZD moiety [18],
we hoped to replace the TZD with other heterocycle moieties.
With the optimized R1 and R2 substituents, subsequently,
we worked on the optimization of the heterocycle moieties
selected from 6,7-dihydro-2H-thiazolo[3,2-a]pyrimidin-3(5H)-
one, 2H-benzo[b][1,4]oxazin-3(4H)-one, or pyrrolidine-2,5-
dione. Finally, pyrrolidine-2,5-dione (12a,b, Table 1) as
preferred heterocycle moiety was selected to do further
structural modification (IC50 up to 1.3 mM, Table 1), whereas
6,7-dihydro-2H-thiazolo[3,2-a]pyrimidin-3(5H)-ones (10a–d,
Table 1) showed no better beneficial effect on Syk inhibition
(IC50 up to 3.7 mM, Table 1), and 2H-benzo[b][1,4]oxazin-
3(4H)-ones (11a,b, Table 1) abolished activities (IC50 up to
50 mM, Table 1). Furthermore, we varied the positions of the
chlorine atoms or increased the number of chlorine atoms,
and gained compounds 12c–I (Table 1). All of them showed
significant inhibition against Syk. Especially, 12d, 12f, and 12g
exerted IC50 values of 0.8, 1.0, and 0.7 mM, respectively
(Table 1). Based on the good results, the positions of ethyl and
substituted benzyl in compounds 12d, 12f, and 12g were
transposed to form analogs 12j–l (Table 1), which also showed
comparable activities (12j, IC50 ¼ 2.9 mM; 12f, IC50 ¼ 1.3 mM;
12g, IC50 ¼ 4.6 mM, Table 1).
Biology
Effects of compounds on FLS cells
Compounds including 9a (the hit), 8f, 8g, 12a, 12d, 12f, 12g,
and 12k, which showed high Syk inhibitory activities
(expressed as IC50 under 1.5 mM), were selected for
a
preliminary test in cellular profiling. Firstly, the antiprolifer-
ative activity against fibroblast-like synoviocytes (FLS) was
evaluated with CCK-8 assay (Fig. 2a), and then 9a, 12a, 12g,
and 12k emerged as excellent compounds showing promising
cell inhibition efficiencies. These four compounds were
further evaluated with MTT-assay. The results are shown in
Fig. 2b. R406 was included as a comparator in MTT-assay with
an IC50 value of 18.1 mM. Compounds 12a and 12g resulted in
less-potent activities with IC50 values of 21.3 and 32.2 mM,
respectively (Table 1). While compounds 9a and 12k were able
to inhibit FLS-RA proliferation with more superior activities
(9a, IC50 ¼ 14.8 mM, ns; 12k, IC50 ¼ 11.8 mM, p < 0.01, Fig. 2b)
compared with R406. As a member of PAINS [18], compound
9a was no longer an alternative despite its excellent
Scheme 4. Reagents and conditions: a) Et3N, acetic anhydride,
reflux, 9 h.
Scheme 5. Reagents and conditions: a) acetone, reflux, 4 h; b)
MeOH, reflux, 6 h.
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Figure 2. Effect of compounds on FLS-RA proliferation (n ¼ 3). a) Preliminary test of eight compounds was determined by the
difference of the absorbance with CCK-8. FLS-RA cells were cultured in 96-well plates and treated with Syk inhibitors for 24, 48, and
72 h at a concentration of 10 mM. b) The IC50 values were determined by MTT assay and fitted by nonlinear regression analysis to
calculate. Statistical comparisons with control were performed by t-tests using GraphPad Prism 5.0 software. Data are the
mean ꢀ SEM, **p < 0.01 versus R406, ns, not significant.
antiproliferative activity. From this refinement, compound
12k which showed the highest antiproliferative activity was
chosen to be a lead candidate. The levels of IL-6 and MMP-3
production suppressed by compound 12k then were tested. IL-
6 and MMP-3 play key roles in the pathogenesis of RA, and are
the representative RA-related proinflammatory cytokines. In
vitro, compound 12k promoted the reduction of IL-6 and
MMP-3 secretion from FLS-RA stimulated from by TNFa
(Fig. 3). The IL-6 levels in FLS-RA cells treated with different
concentrations of compound 12k (15 mM or 50 mM) were
decreased by 80.9 and 85.7%, respectively (Fig. 3a), compared
with the vehicle group. Similarly, the MMP-3 levels in FLS-RA
cells treated with compound 12k (15 or 50 mM) were
decreased by 42.0 and 46.6%, respectively (Fig. 3b), compared
with those treated with vehicle alone. Compound 12k
demonstrated almost equal potencies for suppression of IL-
6 and MMP-3 production to R406 dose dependently.
Once-daily dosing was initiated on day 14 when chronic
inflammation was well established, and 12k was highly
effective at reversing established ankle swelling with a dose
of 10 mg/kg (Fig. 4). From macroscopic evidences, administra-
tion of 12k ameliorated the typical signals of acute
inflammation such as swelling and edema compared with
vehicle group (Fig. 4a). Ankle thickness of mice was markedly
reduced from day 25 through day 43 (Fig. 4b) after
administration of 12k. However, the efficacy of R406 was
more potent than that of 12k, and paws from mice treated
with R406 were similar to those of disease-free mice.
Experimental
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 purifi-
cation. Melting points were determined on a SGW X-4 melting
point apparatus without correction. Analytical thin-layer
chromatography (TLC) was performed on HSGF 254 (150–
Effects of 12k on CIA model
The efficient inhibitory activity of 12k on the cell proliferation
assay as well as inflammatory mediators prompted us to
examine the compound for the effect on murine CIA model,
which recapitulated the clinical development of human RA.
Figure 3. Effects of compound 12k on inflammatory cytokine production (n ¼ 3). a) Effects on IL-6. b) Effects on MMP-3.
Inflammatory cytokines in the cultured supernatants were measured with ELISA. Data are the mean ꢀ SEM, **p < 0.01, ***p < 0.001
versus vehicle control.
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Figure 4. Effects of compound 12k and R406
in murine collagen-induced arthritis model
(n ¼ 5). Collagen-induced arthritis was in-
duced in young male DBA/1 mice as
described in the Experimental section. a)
Macroscopic evidences of arthritis such as
edema and swelling were markedly ob-
served in model mice (day 43), while dose of
10 mg/kg/day compound 12k or positive
control (R406) significantly attenuated ar-
thritis severity. b) Once-daily oral dosing
with vehicle, R406, or compound 12k was
commenced from days 14 to 43 after the
onset of the disease. Calipers were used to
measure ankle thickness every three days.
Data are the mean ꢀ SEM, **p < 0.01, ***p
< 0.001 versus vehicle control.
200 mm thickness; Yantai Huiyou Co., China), and components
were visualized by observation under UV light (254 and
365 nm). The products were purified by column chromatog-
raphy on silica gel (200–300 mesh). Reaction yields were not
optimized.
was stirred overnight. After cooling to room temperature, the
mixture was extracted with ethyl acetate (3 ꢁ 50 mL). The
combined organic layers were washed with brine (20 mL),
dried over Na2SO4, filtered, and concentrated. The crude
material was then purified via flash chromatography (silica
gel, ethyl acetate/petroleum ether ¼ 1:2 to 1:1) to afford 14a
as a white solid (0.20 g, 35%). 1H NMR (400 MHz, CDCl3): d 9.83
(s, 1H), 7.62 (d, J ¼ 2 Hz, 1H), 7.42 (d, J ¼ 2 Hz, 1H), 5.98 (s, 1H),
4.24 (q, J ¼ 7.2 Hz, 2H), 1.48 (t, J ¼ 7.2 Hz, 3H).
Low- and high-resolution mass spectra (LRMS and HRMS)
were given with electric and electrospray ionization (EI and
ESI) produced by a Finnigan MAT-95 and a LCQ-DECA
spectrometer. 1H NMR spectra were performed on a Bruker
AMX-400 spectrometer in CDCl3 or DMSO-d6 solutions, and
chemical shifts were reported in parts per million (ppm)
downfield from tetramethylsilane (TMS). Proton coupling
patterns were described as singlet (s), doublet (d), triplet (t),
quartet (q), doublet of doublets (dd), triplet of doublets (td),
and multiplet (m). The purity of compounds used for
biological evaluation was determined to be higher than
95% using Agilent-1100 with an Agilent Eclipse plus-C18
column (4.6 mm ꢁ 250 mm, 5 mM particle size, column tem-
perature: 20°C), quaternary pump, and diode-array detector
(DAD). The compounds were eluted with CH3OH/H2O in ratios
of 80:20 or 90:10 v/v at a flow rate of 0.5 mL/min (Supporting
Information Table S1).
3-Bromo-5-ethoxy-4-hydroxybenzaldehyde (14b)
Bromine (2.32 mL, 45.2 mmol) was added dropwise to a
solution of commercial 3-ethoxy-4-hydroxybenzaldehyde 13b
(10.00 g, 60.2 mmol) in acetic acid (30 mL) at room tempera-
ture. After completion of addition, the solution was stirred for
2 h. Then, the yellow solids that had formed were removed by
filtration and washed with acetic acid (30 mL). After dried in
vacuum, crude 14b (7.23 g, 49%) was acquired. 1H NMR
(400 MHz, CDCl3): d 9.78 (s, 1H), 7.63 (s, 1H), 7.35 (s, 1H), 6.56
(s, 1H), 4.22 (q, J ¼ 7.0 Hz, 2H), 1.50 (t, J ¼ 7.0 Hz, 3H).
General procedure for the synthesis of 16
K2CO3 (1.5 equiv) was added to a solution of 14 (1 equiv) in dry
DMF (10 mL). The mixture was stirred for 30 min, and then the
commercial substituted benzyl chloride 15 (1 equiv) was
added and stirred for another 4 h. Finally, the reaction
mixture was poured into water (30 mL) and the precipitate
was isolated by filtration, washed with water (20 mL), and
dried in vacuum resulting in 16.
3-Bromo-4-ethoxy-5-hydroxybenzaldehyde (14a)
(i) Bromine (2.78 mL, 54.3 mmol) was added dropwise to a
solution of commercial 3,4-dihydroxybenzaldehyde 13a
(10.00 g, 72.4 mmol) in acetic acid (30 mL) at room tempera-
ture. After completion of addition, the solution was stirred for
2 h. Then, the yellow solids (7.25 g, 46%) that had formed
were removed by filtration and washed using acetic acid
(30 mL), which were used for the next step without further
purification. (ii) Cs2CO3 (1.13 g, 3.4 mmol) was added to a
solution of 3-bromo-4,5-dihydroxybenzaldehyde (0.50 g,
2.3 mmol) in dry DMF (10 mL), and then the mixture was
heated to 40°C. After stirring over 30 min, iodoethane
(0.18 mL, 2.3 mmol) was added portionwise and the mixture
General procedure for the synthesis of 8b–g
Thiazolidine-2,4-dione 17 (1 equiv) and piperidine (1.5 equiv)
were added to a solution of 16 (1 equiv) in ethanol (15 mL),
and then the solution was refluxed for 36 h. When the mixture
was cooled to room temperature, the products formed were
removed by filtration to give homogeneous compounds 8b–g.
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(Z)-4-((2-Bromo-4-((2,4-dioxothiazolidin-5-ylidene)-
methyl)-6-ethoxyphenoxy)methyl)benzoic acid (8a)
J ¼ 6.8 Hz, 3H); HRMS (EI): m/z [Mþ] calcd. for C19H14BrCl2NO4S:
502.9184, found 502.9200.
(Z)-Methyl-4-((2-bromo-4-((2,4-dioxothiazolidin-5-ylidene)-
methyl)-6-ethoxyphenoxy)methyl)benzoate was obtained in
the manner described above. Li2CO3 (0.03 g, 0.4 mmol) was
added to a stirred solution of methyl 8a (0.13 g, 0.3 mmol)
in the solution (10 mL) of tetrahydrofuran/methol/water
(3:3:1, v/v/v). The mixture was stirred at 40°C for 4 h, and then
the mixture was concentrated in vacuum and the residue
resuspended in tetrahydrofuran (5 mL). The mixture was
acidified to pH 3–4. The resulting brown precipitate 8a
(0.11 g, 87%) was collected by filtration and washed with
water (10 mL) and air dried; mp: 275–277°C; 1H NMR (400 MHz,
DMSO-d6): d 12.98 (s, 1H), 12.67 (s, 1H), 7.97 (d, J ¼ 8.2 Hz, 2H),
7.75 (s, 1H), 7.62 (d, J ¼ 8.2 Hz, 2H), 7.41 (s, 1H), 7.28 (s, 1H), 5.16
(s, 2H), 4.16 (q, J ¼ 6.9 Hz, 2H), 1.40 (t, J ¼ 6.9 Hz, 3H); HRMS (EI):
m/z [Mþ] calcd. for C20H16BrNO6S: 476.9882, found 476.9883.
(Z)-5-(3-Bromo-5-((2,4-dichlorobenzyl)oxy)-4-
ethoxybenzylidene)thiazolidine-2,4-dione (8g)
Yellow solid (61%); mp: 170–172°C; 1H NMR (400 MHz, DMSO-
d6): d 7.75 (s, 1H), 7.70–7.66 (m, 2H), 7.50 (dd, J1 ¼ 7.6Hz,
J2 ¼ 1.6 Hz, 1H), 7.41 (s, 1H), 7.29 (s, 1H), 5.18 (s, 2H), 4.14 (q,
J ¼ 7.2 Hz, 2H), 1.37 (t, J ¼ 6.8 Hz, 3H); HRMS (EI): m/z [Mþ]
calcd. for C19H14BrCl2NO4S: 502.9184, found 502.9167.
General procedure for the synthesis of 9b–f
K2CO3 (1.2 equiv) was added to a solution of 8 (1 equiv) in
acetone (10 mL). The mixture was stirred for 15 min, and
then the commercial 2-bromo-1-phenylethanone (1.5 equiv)
was added and refluxed for another 8 h. The mixture was
concentrated in vacuum and the residue was purified by flash
column chromatography (silica gel, tetrahydrofuran/petro-
leum ether ¼ 1:4) to afford 9b–f.
(Z)-5-(3-Bromo-5-ethoxy-4-(pyridin-2-ylmethoxy)-
benzylidene)thiazolidine-2,4-dione (8b)
White solid (43%); mp: 225–227°C; 1H NMR (400 MHz, DMSO-
d6): d 12.70 (s, 1H), 8.54 (d, J ¼ 4.8 Hz, 1H), 7.88 (td, J1 ¼ 7.6 Hz,
J2 ¼ 1.6 Hz, 1H), 7.74 (d, J ¼ 4.8 Hz, 1H), 7.72 (s, 1H), 7.42 (d,
J ¼ 1.6 Hz, 1H), 7.39–7.35 (m, 1H), 7.30 (d, J ¼ 2.0 Hz, 1H), 5.16 (s,
2H),4.15(q,J ¼ 6.8 Hz, 2H),1.35(t, J ¼ 6.8 Hz,3H);HRMS(EI):m/z
[Mþ] calcd. for C18H15BrN2O4S: 435.9915, found 435.9938.
(Z)-4-((2-Bromo-4-((2,4-dioxo-3-(2-oxo-2-phenylethyl)-
thiazolidin-5-ylidene)methyl)-6-ethoxyphenoxy)methyl)-
benzoic acid (9a)
(Z)-Methyl-4-((2-bromo-4-((2,4-dioxo-3-(2-oxo-2-phenylethyl)-
thiazolidin-5-ylidene)methyl)-6-ethoxyphenoxy)methyl)benzo-
ate was obtained in the manner described above. A stirred
solution of methyl 9a (0.15 g, 0.2 mmol) in hydrobromic acid
(5mL) was refluxed overnight. The mixture was concentrated in
vacuum to afford 9a (0.07 g, 52%); mp: 247–249°C; 1H NMR
(400MHz, DMSO-d6): d 8.09 (d, 4H), 8.00–8.01 (m, 2H), 7.46–7.55
(m, 6H), 5.17 (s, 2H), 4.52 (d, 2H), 3.74 (q, 2H), 1.24 (t, 3H); HRMS
(ESI): m/z [MꢂH]ꢂ calcd. for C28H21BrNO7S: 594.0222, found
594.0208.
(Z)-5-(3-Bromo-4-((2-chlorobenzyl)oxy)-5-ethoxybenzylid-
ene)thiazolidine-2,4-dione (8c)
Beige solid (60%); mp: 213°C; 1H NMR (400 MHz, DMSO-d6):
d 7.68–7.71 (m, 1H), 7.48–7.51 (m, 1H), 7.44 (s, 1H), 7.39–7.41
(m, 2H), 7.36 (d, 1H), 7.27 (d, 1H), 5.17 (s, 2H), 4.14 (q, 2H), 1.37
(t, 3H); HRMS (ESI): m/z [MꢂH]ꢂ calcd. for C19H14BrClNO4S:
465.9515, found 465.9520.
(Z)-5-(4-(Benzyloxy)-3-bromo-5-ethoxybenzylidene)-3-(2-
oxo-2-phenylethyl)thiazolidine-2,4-dione (9b)
(Z)-5-(3-Bromo-4-((3,5-dimethylbenzyl)oxy)-5-
ethoxybenzylidene)thiazolidine-2,4-dione (8d)
Yellow solid (71%); mp: 173–174°C; 1H NMR (400 MHz, DMSO-
d6):d 8.12–8.08(m,1H),7.97(s,1H),7.75(m,1H),7.61(t,J¼ 8.0 Hz,
2H), 7.53–7.49 (m, 3H), 7.44–7.34 (m, 5H), 5.34 (s, 2H), 5.12 (s, 2H),
4.20 (q, J ¼ 6.9 Hz, 2H), 1.44 (t, J ¼ 6.9 Hz, 3H); HRMS (EI): m/z [Mþ]
calcd. for C27H24BrNO5S: 553.0559, found 553.0392.
Yellow solid (46%); mp: 224°C; 1H NMR (400 MHz, DMSO-d6): d
12.67 (s, 1H), 7.74 (s, 1H), 7.41 (d, J ¼ 1.6 Hz, 1H), 7.27 (d,
J ¼ 1.7 Hz, 1H), 7.11 (s, 2H), 6.98 (s, 1H), 4.97 (s, 2H), 4.16 (q,
J ¼ 6.8 Hz, 2H), 2.28 (s, 6H), 1.42 (t, J ¼ 6.8 Hz, 3H); HRMS (EI):
m/z [Mþ] calcd. for C21H20BrNO4S: 463.0254, found 463.0295.
(Z)-5-(3-Bromo-5-ethoxy-4-methoxybenzylidene)-3-(2-
oxo-2-phenylethyl)thiazolidine-2,4-dione (9c)
(Z)-5-(3-Bromo-5-ethoxy-4-((3,4,5-trimethoxybenzyl)oxy)-
benzylidene)thiazolidine-2,4-dione (8e)
Yellow solid (52%); mp: 156–158°C; 1H NMR (400 MHz, DMSO-
d6): d 8.00 (d, J ¼ 7.2 Hz, 2H), 7.79 (s, 1H), 7.65 (t, J ¼ 7.2 Hz, 1H),
7.53 (t, J ¼ 7.2 Hz, 2H), 7.33 (d, J ¼ 2.0 Hz, 1H), 7.00 (d,
J ¼ 2.0 Hz, 1H), 5.18 (s, 2H), 4.14 (q, J ¼ 7.0 Hz, 2H), 3.94 (s,
3H), 1.51 (t, J ¼ 7.0 Hz, 3H); HRMS (EI): m/z [Mþ] calcd. for
Yellow solid (50%); mp: 165–167°C; 1H NMR (400 MHz, DMSO-
d6): d 12.67 (s, 1H), 7.74 (s, 1H), 7.41 (s, 1H), 7.28 (s, 1H), 6.80
(s, 2H), 5.04 (s, 2H), 4.17 (q, J ¼ 6.8 Hz, 2H), 3.78 (s, 6H), 3.66
(s, 3H), 1.44 (t, J ¼ 6.8 Hz, 3H); HRMS (EI): m/z [Mþ] calcd. for
C
22H22BrNO7S: 525.0280, found 525.0270.
C21H20BrNO5S: 477.0246, found 477.0068.
(Z)-5-(3-Bromo-4-((2,4-dichlorobenzyl)oxy)-5-
(Z)-5-(3-Bromo-5-ethoxy-4-hydroxybenzylidene)-3-(2-oxo-
2-phenylethyl)thiazolidine-2,4-dione (9d)
ethoxybenzylidene)thiazolidine-2,4-dione (8f)
Yellow solid (43%); mp: 245–247°C; 1H NMR (400 MHz, DMSO-
d6): d 7.75 (s, 1H), 7.69 (s, 2H), 7.51 (d, J ¼ 7.9 Hz, 1H), 7.42 (s,
1H), 7.29 (s, 1H), 5.19 (s, 2H), 4.15 (d, J ¼ 6.8 Hz, 2H), 1.38 (t,
Yellowsolid(79%);mp:200–201°C;1HNMR(400 MHz,DMSO-d6):
d
10.32 (s, 1H), 8.09 (d, J¼ 7.7 Hz, 2H), 7.91 (s, 1H), 7.75 (t,
J¼ 7.3 Hz, 1H), 7.61 (t, J ¼ 7.5 Hz, 2H), 7.45 (s, 1H), 7.24 (s, 1H), 5.33
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(s, 2H), 4.17 (q, J¼ 6.8 Hz, 2H), 1.40 (t, J¼ 6.8 Hz, 3H); HRMS (EI):
m/z [Mþ] calcd. for C20H18BrNO5S: 463.0089, found 462.9919.
(Z)-2-(3-Bromo-4-((2,4-dichlorobenzyl)oxy)-5-
ethoxybenzylidene)-6,6-dimethyl-6,7-dihydro-2H-
thiazolo[3,2-a]pyrimidin-3(5H)-one (10b)
(Z)-Methyl-4-((2-bromo-4-((2,4-dioxo-3-(2-oxo-2-
phenylethyl)thiazolidin-5-ylidene)methyl)-6-ethoxy-
phenoxy)methyl)benzoate (9e)
Yellow solid (68%); mp: 145–157°C; 1H NMR (400 MHz, DMSO-
d6): d 7.73 (d, J ¼ 7.9 Hz, 2H), 7.43 (s, 1H), 7.33 (d, J ¼ 5.7 Hz,
2H), 7.02 (s, 1H), 5.23 (s, 2H), 4.15 (dd, J1 ¼ 13.8 Hz, J2 ¼ 6.7 Hz,
2H), 3.58 (s, 2H), 3.48 (s, 2H), 1.47 (t, J ¼ 6.9 Hz, 3H), 1.14 (s, 6H);
HRMS (EI): m/z [Mþ] calcd. for C24H25BrCl2N2O3S: 570.0146,
found 569.9935.
Brown oil (40%); 1H NMR (400 MHz, DMSO-d6): d 8.09 (d, 2H),
8.02 (d, 2H), 7.83 (s, 1H), 7.63–7.70 (m, 3H), 7.56 (t, 2H), 7.36 (m,
1H), 7.04 (m, 1H), 5.21 (d, 5H), 4.16 (q, 2H), 3.96 (s, 2H), 1.52 (t,
3H); HRMS (EI): m/z [Mþ] calcd. for C29H24BrNO7S: 611.0436,
found 611.0452.
(Z)-2-(3-Bromo-5-((2,4-dichlorobenzyl)oxy)-4-
ethoxybenzylidene)-6,7-dihydro-2H-thiazolo[3,2-a]-
pyrimidin-3(5H)-one (10c)
(Z)-Benzyl-4-((2-bromo-4-((2,4-dioxo-3-(2-oxo-2-
phenylethyl)thiazolidin-5-ylidene)methyl)-6-ethoxy-
phenoxy)methyl)benzoate (9f)
White solid (62%); mp: 146°C; 1H NMR (400 MHz, DMSO-d6): d
7.76–7.71 (m, 2H), 7.43 (s, 1H), 7.33 (t, J ¼ 7.2 Hz, 2H), 7.02
(s,1H), 5.23 (s, 2H), 4.15 (dd, J1 ¼ 14.1 Hz, J2 ¼ 6.9 Hz, 2H), 3.97–
3.90 (m, 2H), 3.81 (dd, J1 ¼ 7.8 Hz, J2 ¼ 3.1 Hz, 2H), 2.18–2.09
(m, 2H), 1.47 (t, J ¼ 6.9 Hz, 3H); HRMS (EI): m/z [Mþ] calcd. for
Brown oil (37%); 1H NMR (400 MHz, DMSO-d6): d 8.12 (d, 2H),
8.02 (d, 2H), 7.82 (s, 1H), 7.66–7.70 (m, 3H), 7.56–7.64 (m, 2H),
7.39–7.50 (m, 2H), 7.36–7.38 (m, 4H), 7.03 (s, 1H), 5.40 (s, 2H),
5.12 (d, 4H), 4.16 (q, 2H), 1.51(t, 3H); HRMS (EI): m/z [Mþ] calcd.
for C35H30BrNO7S: 687.0926, found 687.0748.
C
22H19BrCl2N2O3S: 541.9656, found 541.9642.
(Z)-2-(3-Bromo-5-((2,4-dichlorobenzyl)oxy)-4-
ethoxybenzylidene)-6,6-dimethyl-6,7-dihydro-2H-
thiazolo[3,2-a]pyrimidin-3(5H)-one (10d)
General procedure for the synthesis of 19
(i) Carbon disulfide (4 equiv) was added dropwise to a solution
of propane-1,3-diamine or 2,2-dimethylpropane-1,3-diamine
18 (1 equiv) in ethanol (4 mL) below 40°C. The reaction was
stopped when the solid was no longer produced and filtrated.
(ii) The cake was dissolved in water (4 mL) and reacted for
another 10 h under 40°C. The precipitate was filtered and
used for the next step without further purification. (iii)
Bromoacetic acid (2 equiv) was added to a solution of
appropriate tetrahydropyrimidine-2(1H)-thione (1 equiv) in
ethanol (5 mL). The reaction mixture was refluxed for 6 h. The
precipitate was filtered and the cake was washed with
ethanol (5 mL). The crude was extracted with dichloro-
methane (3 ꢁ 15 mL), washed with brine (10 mL), dried over
Na2SO4, and concentrated to give 19.
White solid (66%); mp: 179–180°C; 1H NMR (400 MHz, DMSO-
d6): d 7.82–7.75 (m, 1H), 7.73 (d, J ¼ 8.0 Hz, 1H), 7.43 (s, 1H),
7.34–7.31(m, 2H), 7.02 (s, 1H), 5.24 (s, 2H), 4.15 (q, J ¼ 6.8 Hz,
2H), 3.61 (s, 2H), 3.50 (s, 2H), 1.47 (t, J ¼ 6.8 Hz, 3H), 1.16 (s, 6H);
HRMS (EI): m/z [Mþ] calcd. for C24H25BrCl2N2O3S: 570.0146,
found 569.9971.
General procedure for the synthesis of 11a,b
2,4-Dichlorobenzyl substituted 16 (1 equiv) and triethylamine
(1 equiv) were added to a solution of commercial 2H-benzo[b]-
[1,4]oxazin-3(4H)-one 20 (1 equiv) in acetic anhydride (3 mL).
Thereaction mixture was refluxed for9 h. Afterstirring atroom
temperature overnight, the mixture was poured into crushed
ice. The crude product was collected by filtration and extracted
with dichloromethane (3 ꢁ 15mL), washed with brine (10 mL),
dried over Na2SO4, and concentrated to give 11a,b.
General procedure for the synthesis of 10a–d
2,4-Dichlorobenzyl substituted 16 (1 equiv) and piperidine (1
equiv) were added to a solution of 19 (1 equiv) in ethanol
(10 mL), and then the solution was refluxed for 48 h. When the
mixture was cooled to room temperature, the resulting
precipitate was collected by filtration and washed with
ethanol (10 mL) and air dried. The residue was purified by
flash chromatography (silica gel, tetrahydrofuran/petroleum
ether ¼ 1:3) to afford 10a–d.
(Z)-2-(3-Bromo-4-((2,4-dichlorobenzyl)oxy)-5-
ethoxybenzylidene)-2H-benzo[b][1,4]oxazin-3(4H)-one
(11a)
Beigesolid(4%);mp:225–227°C;1HNMR(400MHz,DMSO-d6):d
11.19 (s, 1H), 7.72–7.67 (m, 3H), 7.64 (s, 1H), 7.51 (dd, J1 ¼ 8.0
Hz, J2 ¼ 2.0 Hz, 1H), 7.21–7.13 (m, 1H), 7.07–7.04 (m, 2H), 6.99
(dd, J1 ¼ 6.8 Hz, J2¼ 2.4 Hz, 1H), 6.75 (s, 1H), 5.15 (s, 2H), 4.18 (q,
J ¼ 6.8 Hz, 2H), 1.39 (t, J ¼ 6.8 Hz, 3H); HRMS (ESI): m/z [MþNa]þ
calcd. for C24H18BrCl2NNaO4: 555.9694, found 555.9713.
(Z)-2-(3-Bromo-4-((2,4-dichlorobenzyl)oxy)-5-
ethoxybenzylidene)-6,7-dihydro-2H-thiazolo[3,2-a]-
pyrimidin-3(5H)-one (10a)
Yellow solid (70%); mp: 144–145°C; 1H NMR (400 MHz, DMSO-
d6): d 7.73 (d, J ¼ 8.0 Hz, 2H), 7.43 (s, 1H), 7.34–7.32 (m, 2H),
7.02 (s, 1H), 5.24 (s, 2H), 4.15 (q, J ¼ 7.2 Hz, 2H), 3.94 (t,
J ¼ 4.4 Hz, 2H), 3.81 (t, J ¼ 4.4 Hz, 2H), 2.22–2.10 (m, 2H), 1.47
(t, J ¼ 6.8 Hz, 3H); HRMS (EI): m/z [Mþ] calcd. for
(Z)-2-(3-Bromo-5-((2,4-dichlorobenzyl)oxy)-4-
ethoxybenzylidene)-2H-benzo[b][1,4]oxazin-3(4H)-one
(11b)
White solid (3%); mp: 213–215°C; 1H NMR (400 MHz, DMSO-
d6): d 11.20 (s, 1H), 7.79–7.74 (m, 2H), 7.72 (s, 1H), 7.67 (d,
J ¼ 8.4 Hz, 1H), 7.55 (dd, J1 ¼ 8.3 Hz, J2 ¼ 2.0 Hz, 1H), 7.09–6.96
C
22H19BrCl2N2O3S: 541.9656, found 541.9656.
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Archiv der Pharmazie
(m, 4H), 6.75 (s, 1H), 5.30 (s, 2H), 4.07 (q, J ¼ 7.2 Hz, 2H), 1.30 (t,
J ¼ 7.2 Hz, 3H); HRMS (ESI): m/z [MþNa]þ calcd. for
(E)-3-(3-Bromo-4-((3,5-dichlorobenzyl)oxy)-5-
ethoxybenzylidene)pyrrolidine-2,5-dione (12e)
C
24H18BrCl2NNaO4: 555.9694, found 555.9670.
White solid (88%); mp: 270–271°C; 1H NMR (400 MHz, DMSO-
d6): d 11.48 (s, 1H), 7.77 (d, J ¼ 2.3 Hz, 1H), 7.55 (d, J ¼ 8.6 Hz,
1H), 7.51–7.44 (m, 2H), 7.34 (s, 1H), 7.30 (s, 1H), 5.16 (s, 2H),
4.17 (q, J ¼ 6.9 Hz, 2H), 3.72 (d, J ¼ 1.7 Hz, 2H), 1.36 (t,
J ¼ 6.9 Hz, 3H). HRMS (EI): m/z [Mþ] calcd. for C20H16BrCl2NO4:
484.9619, found 484.9615.
3-(Triphenylphosphoranylidene)pyrrolidine-2,5-dione (22)
Triphenylphosphine (2.70 g, 10.3 mmol) was added to a
solution of maleimide (1.00 g, 10.3 mmol) in anhydrous
acetone (10 mL) and refluxed for 4 h. After cooling, the
precipitates formed were filtered and the filter cake was
washed with cold acetone (10 mL). Drying under reduced
vacuum afforded 22 (3.40 g, 92%) as a white solid. The
solid 22 was used for the next step without further
purification.
(E)-3-(3-Bromo-5-ethoxy-4-((2,3,5-trichlorobenzyl)oxy)-
benzylidene)pyrrolidine-2,5-dione (12f)
White solid (85%); mp: 279°C; 1H NMR (400 MHz, DMSO-d6): d
11.48 (s, 1H), 7.89 (d, J ¼ 2.4 Hz, 1H), 7.76 (d, J ¼ 2.4 Hz, 1H), 7.46
(s, 1H), 7.34 (s, 1H), 7.30 (s, 1H), 5.20 (s, 2H), 4.16 (q, J ¼ 6.8 Hz,
2H), 3.72 (s, 2H), 1.33 (t, J ¼ 6.8 Hz, 3H); HRMS (ESI): m/z [MꢂH]–
calcd. for C20H14BrCl3NO4: 515.9172, found 515.9157.
General procedure for the synthesis of 12a–l
Varied 16 (1 equiv) was added to a solution of 22 (1 equiv) in
methanol (10 mL), and the solution was refluxed for 6 h. When
the mixture was cooled to room temperature, the product
precipitated out of the solution to afford 12a–l.
(E)-3-(3-Bromo-5-ethoxy-4-((2,3,6-trichlorobenzyl)oxy)-
benzylidene)pyrrolidine-2,5-dione (12g)
White solid (79%); mp: 279–280°C; 1H NMR (400 MHz, DMSO-d6):
d 11.48 (s, 1H), 7.89 (d, J ¼ 2.4 Hz, 1H), 7.76 (d, J ¼ 2.4 Hz, 1H), 7.46
(s, 1H), 7.34 (s, 1H), 7.30 (s, 1H), 5.20 (s, 2H), 4.16 (q, J¼ 6.8 Hz, 2H),
3.72 (s, 2H), 1.33 (t, J ¼ 6.8 Hz, 3H); HRMS (ESI): m/z [MꢂH]ꢂ calcd.
for C20H14BrCl3NO4: 515.9172, found 515.9158.
(E)-3-(3-Bromo-4-((2,4-dichlorobenzyl)oxy)-5-
ethoxybenzylidene)pyrrolidine-2,5-dione (12a)
White solid (90%); mp: 273–274°C; 1H NMR (400 MHz,
DMSO-d6): d 11.48 (s, 1H), 7.71–7.66 (m, 2H), 7.50 (dd,
J1 ¼ 8.0 Hz, J2 ¼ 2.0 Hz, 1H), 7.44 (d, J ¼ 1.6 Hz, 1H), 7.33 (t,
J ¼ 2.0 Hz, 1H), 7.29 (d, J ¼ 1.6 Hz, 1H), 5.15 (s, 2H), 4.17 (q,
J ¼ 6.8 Hz, 2H), 3.71 (d, J ¼ 2.4 Hz, 2H), 1.36 (t, J ¼ 6.8 Hz, 3H);
HRMS (EI): m/z [Mþ] calcd. for C20H16BrCl2NO4: 484.9619,
found 484.9613.
(E)-3-(3-Bromo-4-((3,4-dichlorobenzyl)oxy)-5-
ethoxybenzylidene)pyrrolidine-2,5-dione (12h)
White solid (77%); mp: 258–260°C; 1H NMR (400 MHz, DMSO-
d6): d 11.47 (s, 1H), 7.79 (d, J ¼ 1.2 Hz, 1H), 7.67 (d, J ¼ 8.4 Hz,
1H), 7.49 (dd, J1 ¼ 8.4 Hz, J2 ¼ 1.6 Hz, 1H), 7.44 (s, 1H), 7.33 (s,
1H), 7.29 (s, 1H), 5.07 (s, 2H), 4.18 (q, J ¼ 6.8 Hz, 2H), 3.72 (d,
J ¼ 2.0 Hz, 2H), 1.39 (t, J ¼ 6.8 Hz, 3H); HRMS (ESI): m/z [MꢂH]–
calcd. for C20H15BrCl2NO4: 481.9562, found 481.9546.
(E)-3-(3-Bromo-5-((2,4-dichlorobenzyl)oxy)-4-
ethoxybenzylidene)pyrrolidine-2,5-dione (12b)
White solid (88%); mp: 233–234°C; 1H NMR (400 MHz, DMSO-
d6): d 11.48 (s, 1H), 7.74 (d, J ¼ 2.0 Hz, 1H), 7.66 (d, J ¼ 8.4 Hz,
1H), 7.56–7.51 (m, 1H), 7.49 (s, 1H), 7.42 (s, 1H), 7.34 (s, 1H),
5.28 (s, 2H), 4.05 (q, J ¼ 7.0 Hz, 2H), 3.70 (d, J ¼ 2.0 Hz, 2H), 1.26
(t, J ¼ 7.0 Hz, 3H); HRMS (EI): m/z [Mþ] calcd. for
(E)-3-(3-Bromo-4-((2,6-dichlorobenzyl)oxy)-5-
ethoxybenzylidene)pyrrolidine-2,5-dione (12i)
Beige solid (85%); mp: 271–273°C; 1H NMR (400 MHz, DMSO-
d6): d 11.47 (s, 1H), 7.51 (d, J ¼ 1.6 Hz, 1H), 7.49 (s, 1H), 7.45–
7.41 (m, 1H), 7.38 (d, J ¼ 2.0 Hz, 1H), 7.32 (t, J ¼ 2.4 Hz, 1H), 7.25
(d, J ¼ 1.6 Hz, 1H), 5.40 (s, 2H), 4.13 (q, J ¼ 6.8 Hz, 2H), 3.70 (d,
J ¼ 2.4 Hz, 2H), 1.37 (t, J ¼ 6.8 Hz, 3H); HRMS (ESI): m/z [MꢂH]ꢂ
calcd. for C20H15BrCl2NO4: 481.9562, found 481.9544.
C
20H16BrCl2NO4: 482.9640, found 482.9646.
(E)-3-(3-Bromo-4-((2,3-dichlorobenzyl)oxy)-5-
ethoxybenzylidene)pyrrolidine-2,5-dione (12c)
White solid (91%); mp: 257–259°C; 1H NMR (400 MHz,
DMSO-d6): d 11.48 (s, 1H), 7.74 (d, J ¼ 2.0 Hz, 1H), 7.66 (d,
J ¼ 8.4 Hz, 1H), 7.53 (dd, J1 ¼ 8.4 Hz, J2 ¼ 2.0 Hz, 1H), 7.49 (s,
1H), 7.42 (s, 1H), 7.34 (s, 1H), 5.28 (s, 2H), 4.05 (q, J ¼ 6.8 Hz,
2H), 3.70 (d, J ¼ 2.0 Hz, 2H), 1.26 (t, J ¼ 7.2 Hz, 3H); HRMS (EI):
m/z [Mþ] calcd. for C20H16BrCl2NO4: 484.9619, found
484.9621.
(E)-3-(3-Bromo-5-((2,5-dichlorobenzyl)oxy)-4-
ethoxybenzylidene)pyrrolidine-2,5-dione (12j)
White solid (78%); mp: 189–191°C; 1H NMR (400 MHz, DMSO-
d6): d 11.48 (s, 1H), 7.74 (d, J ¼ 2.4 Hz, 1H), 7.60 (d, J ¼ 8.4 Hz,
1H), 7.54–7.48 (m, 2H), 7.45 (s, 1H), 7.35 (s, 1H), 5.27 (s, 2H),
4.06 (q, J ¼ 6.8 Hz, 2H), 3.71 (d, J ¼ 2.0 Hz, 2H), 1.29 (t,
J ¼ 6.8 Hz, 3H); HRMS (EI): m/z [Mþ] calcd. for C20H16BrCl2NO4:
482.9640, found 482.9644.
(E)-3-(3-Bromo-4-((2,5-dichlorobenzyl)oxy)-5-
ethoxybenzylidene)pyrrolidine-2,5-dione (12d)
White solid (92%); mp: 270–272°C; 1H NMR (400 MHz, DMSO-
d6): d 11.48 (s, 1H), 7.60 (m, 3H), 7.46 (s, 1H), 7.34 (s, 1H), 7.30 (s,
1H), 5.08 (s, 2H), 4.18 (q, J ¼ 6.8 Hz, 2H), 3.72 (d, J ¼ 2.0 Hz, 2H),
1.38 (t, J ¼ 6.8 Hz, 3H); HRMS (EI): m/z [Mþ] calcd. for
(E)-3-(3-Bromo-4-ethoxy-5-((2,3,5-trichlorobenzyl)oxy)-
benzylidene)pyrrolidine-2,5-dione (12k)
White solid (82%); mp: 230–231°C; 1H NMR (400 MHz, DMSO-
d6): d 11.49 (s, 1H), 7.93 (d, J ¼ 2.4 Hz, 1H), 7.73 (d, J ¼ 2.4 Hz,
C
20H16BrCl2NO4: 484.9619, found 484.9622.
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2015, 348, 463–474
Benzylidene Derivatives as Potent Syk Inhibitors
Archiv der Pharmazie
1H), 7.52 (s, 1H), 7.46 (s, 1H), 7.36 (s, 1H), 5.32 (s, 2H), 4.06 (q,
J ¼ 7.2 Hz, 2H), 3.72 (d, J ¼ 2.0 Hz, 2H), 1.31 (t, J ¼ 7.2 Hz, 3H);
HRMS (EI): m/z [Mþ] calcd. for C20H15BrCl3NO4: 516.9250,
found 516.9248.
Antiproliferation examination by MTT
Based on our previously established CCK-8 assays, an MTT
assay was established in a similar format. All wells were
incubated for 72 h at 37°C. IC50 values were calculated from
10-point dose–response curves using nonlinear regression
analysis.
(E)-3-(3-Bromo-4-ethoxy-5-((2,3,6-trichlorobenzyl)oxy)-
benzylidene)pyrrolidine-2,5-dione (12l)
White solid (87%); mp: 226–227°C; 1H NMR (400 MHz, DMSO-
d6): d 11.48 (s, 1H), 7.81 (d, J ¼ 8.8 Hz, 1H), 7.68–7.50 (m, 5H),
7.36 (s, 1H), 5.41 (s, 2H), 3.99 (q, J ¼ 7.2 Hz, 2H), 3.77 (s, 2H),
1.18 (t, J ¼ 7.2 Hz, 3H); HRMS (EI): m/z [Mþ] calcd. for
IL-6 and MMP-3 examination by ELISA
FLS-RA cells were seeded at a density of 2 ꢁ 105 cells/well into
6-well plate for 48 h, and then incubated with R406,
compound 12k or vehicle for 30 min, and following by
stimulation by TNFa. After an 18 h incubation time, super-
natants were collected for the determination of the
Interleukin-6 (IL-6) and metalloproteinase-3 (MMP-3) levels
using enzyme-linked immunoassay (ELISA) kits (Dakewe
Biotech Co. Ltd., China) according to kit manufacturer’s
protocol.
C
20H15BrCl3NO4: 516.9250, found 516.9247.
Biology
Syk kinase inhibition assay
The kinase assay was performed in the buffer (50 mM
HEPES pH 7.5, 0.015% Brij-35, 10 mM MgCl2, 10 mM MnCl2,
2 mM DTT). A 5 mL volume of tested compounds were
prediluted for dose response in 384-well plates. A 10 mL
volume of diluted enzyme solution was sequentially added
and the assay plates were incubated at room temperature for
10 min. And then a 10 mL 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 mL 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 nonlinear
curve fit with XLfit software.
Collagen-induced arthritis
CIA was elicited in Male DBA/1 mice (mean mass 20 g; Beijing
HFK Bioscience Co. Ltd., China) by immunization with
subcutaneous injection of 100 mL bovine type II collagen
(Beijing Biolead Biology SCI
& TECH CO. Ltd., China)
emulsified with an equal volume complete Freund’s adjuvant
(CFA) at the base of the tail on day 1 and again on day 22.
After the onset of arthritis (on day 15), the mice were
randomly divided into three groups (n ¼ 5): compound 12k
group (10 mg/kg), R406 group (10 mg/kg), and CIA control
group (vehicle) [19]. This dose for R406 as positive control
was set according to Ref. [19], in order to facilitate
comparison, the dose for 12k was also set in the same dose.
Another normal 5 mice were selected as blank group. Each
group was given an oral administration intragastrically using
syringe. The treatment was performed once a day from
days 15 to 43 after the first immunization. The progression of
CIA was evaluated by ankle thickness, which was measured
by a caliper.
Primary culture of FLS
FLS from synovial tissues of RA patients were purchased from
Shanghai Zhonghua Biological Co. (Shanghai, China). FLS-RA
cells were maintained in high-glucose Dulbecco’s Modified
Eagle’s Medium (H-DMEM; Thermo, America) containing 10%
fetal bovine serum (FBS; Gibco, America) at 37°C in humidified
environment containing 5% CO2. FLS-RA cells from a homoge-
neous population (phenotypes <1.5% CD55, and >94% CD68,
as determined by flow cytometry) were cultured for three
generations and were used for subsequent experiments.
Financial support of this research provided by the National
Natural Science Foundation of China (grant nos. 21222211,
21372001, 91313303), the Program for New Century Excellent
Talents in University (grant no. NCET-12-0853), the National
Basic Research Program of China (grant no. 2014CB541800),
and the Fundamental Research Funds for the Central
Universities is gratefully acknowledged.
Antiproliferation examination by CCK-8
Antiproliferation assay was evaluated using the Cell Counting
Kit-8 (CCK-8). CCK-8 assays were performed according to the
manufactural instructions. FLS-RA cells were seeded at a
density of 2 ꢁ 103 cells/well into 96-well plates. Tested
compounds were added into a 96-well plate (the final
concentration was 10 mM). 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 24, 48, and 72 h, respectively. Then,
10 mL of the CCK-8 was added to each well of a 96-well
incubated for additional 4 h. The absorbance was measured at
450 nm by spectrophotometer readings.
The authors have declared no conflict of interest.
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