Potent small molecule inhibitors of spleen tyrosine kinase (Syk)

Article abstract of DOI:10.1016/S0960-894X(03)00658-9

A series of oxindoles demonstrating inhibition of the phosphorylation of biotinylated substrates of Syk and IgE/FcεRI triggered basophil cell degranulation has been identified. A study of the SAR around sulfonamide 31 (IC₅₀=5 nM, EC₅₀=1400 nM) is discussed. The modest cellular activity representative of the sulfonamide series was overcome when the Polar Surface Area was lowered to <110 A², leading to the identification of amide 32 (IC₅₀=145 nM, EC₅₀=100 nM).

Full text of DOI:10.1016/S0960-894X(03)00658-9

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3111–3114
Potent Small Molecule Inhibitors of Spleen Tyrosine
Kinase (Syk)
Justine Y. Q. Lai,y Paul J. Cox,* Rajesh Patel, Shazia Sadiq, David J. Aldous,
Sukanthini Thurairatnam, Keith Smith, Darren Wheeler, Savita Jagpal, Sofia Parveen,
Gary Fenton, Trevor K. P. Harrison, Clive McCarthy and Paul Bamborough
Aventis Pharmaceuticals, Route 202/206, Brigdewater, NJ 08807, USA
Received 24 April 2003; accepted 20 May 2003
Abstract—A series of oxindoles demonstrating inhibition of the phosphorylation of biotinylated substrates of Syk and IgE/FceRI
triggered basophil cell degranulation has been identified. A study of the SAR around sulfonamide 31 (IC50=5 nM, EC50=1400
nM) is discussed. The modest cellular activity representative of the sulfonamide series was overcome when the Polar Surface Area
was lowered to <110 A2, leading to the identification of amide 32 (IC50=145 nM, EC50=100 nM).
# 2003 Published by ElsevierLtd.
Introduction
of therapeutic use for the treatment of asthma and other
inflammatory diseases. High-throughput screening led
to the identification of oxindoles 1–3 (Table 1). Further
optimisation led to the identification of 31 with IC50=5
nM.
Syk (Spleen Tyrosine Kinase), a double SH2 domain
containing cytoplasmic protein, plays an important role
in mediating inflammatory cell signalling. In mast cells
and basophils, Syk plays a key role in IgE/FceRI trig-
gered cell degranulation,1 resulting in the release of a
variety of inflammatory mediators that orchestrate the
allergic response. In macrophages, Fcg-receptor medi-
ated phagocytosis and mediatorerlease is also depen-
dent on the activation and phosphorylation of Syk.2
Furthermore, Syk is involved in B-cell receptor signal-
ling and activation,3 as well as IL5 and GM-CSF medi-
ated survival of eosinophils.4 More recently, a critical
role for Syk has been identified in Fc receptor mediated
antigen presentation and induction of dendritic cell
maturation.5 Collectively, these data suggest that inhi-
bitors of Syk kinase may be effective in inhibiting the
function of a variety of cell populations involved in the
inflammatory response.
Results andDiscussion
A study of the importance of the double bond revealed
that saturation to give 4 led to loss in potency whilst the
Z-analogue 5 was also inactive.
As such, the aim of ourstudy was to identify oarlly
available inhibitors of Syk in the belief that they may be
*Corresponding author. E-mail: paul.cox@aventis.com
yPresent address: Millennium Pharmaceuticals Research and Devel-
opment Limited, Oncology Discovery Chemistry, Granta Park, Great
Abington, Cambridge CB1 6ET, UK.
0960-894X/$ - see front matter # 2003 Published by ElsevierLtd.
doi:10.1016/S0960-894X(03)00658-9

3112
J. Y. Q. Lai et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3111–3114
Compounds 6 and 7 (Table 1) indicate that one sulfo-
namide NH is essential forpotency. Increasing the bulk
of the substituent at the sufonamide (7–10) led to a drop
in potency. Flexible chains with acidic groups are less
tolerated than carboxamide groups (11—13). Substitu-
tion of the indole nitrogen showed that, given the cor-
rect chain length, acids and polar groups such as
alcohols and carboxamide groups are tolerated (14–19).
However, introduction of the ester group led to a loss in
potency (20–23).
Although potent inhibitors of Syk were identified in the
primary screen, the compounds were all weakly active
orinactive in the cellularassays, with the exception of 2
and 7. It had been observed on a number of occasions
that the compounds were very insoluble, and would
precipitate out at low concentrations. Hence, it was
speculated that the poorcellularactivity was related to
the solubility of the compounds. Attempts to improve
the solubility were made by adding hydrophilic groups
to the molecule. Since substitution off the indole NH
tolerated amides (see 19), hydrophilic amides were syn-
thesised (24–25). They demonstrated a 10–50-fold loss
in potency, but suggest that the solubility could be
improved.
Figure 1. Correlation between EC50 and PSA. The labels show IC50
(nM) values.
Table 1. Comparison of the physicochemical properties and inhibitory activities of oxindoles in kinase and whole cell based assays
Compd
R1
R2
R3
R4
IC50
(nM)
EC50
(nM)
Solubility
(mg/L)
PSA
1
2
3
31
6
7
8
9
10
11
12
13
14
15
16
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
Et
iBu
H
Me
H
Me
Me
Me
Me
Me
H
H
128
14
28
5
121
108
162
117
94
105
104
101
116
148
151
152
163
163
163
313
>10,000
1400
>10,000
110
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
<0.1
<0.1
<0.1
Inactive @ 80 mM
20
100
937
20% @ 4 mM
658
p-MeOPhCH2
CH2COOH
Me
Me
Me
1940
>10,000
>10,000
30
8
1
70
CH2CH2C(¼O)NH2
60
98
850
14
CH2C(¼O)NH2
Me
H
H
H
CH2COOH piperidine salt
(CH2)3COOH
(CH2)4COOH
11
20,700
<0.1
17
H
H
OMe
27
149
18
19
20
21
22
23
24
25
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
(CH2)3OH
(CH2)3CONH2
CH2COOtBu
(CH2)3COOEt
(CH2)4COOEt
OMe
OMe
OMe
OMe
OMe
OMe
H
32
12
>10,000
>10,000
<0.1
5
142
164
140
147
147
147
153
156
1000
204
678
465
133
616
6960
6300
5600
>30,000
<0.1
<0.1
<0.1
10
(CH2)5COOEt
(CH2)3CO-morpholino
(CH2)3CO-piperidine
H

J. Y. Q. Lai et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3111–3114
3113
Scheme 1. (a) HSO3Cl, 60 ꢀC, 2 h; (b) concd NH3, rt, 20 h; (c) NaH, MeI, DMF, rt, 20 h; (d) piperidine, EtOH, 70 ꢀC, 30 min.
A calculation method fordetemr ining the physico-
chemical properties6 demonstrated that the compounds
obeyed Lipinski’s rules.7 However, in the list of com-
pounds tested in the cellular assay, apart from 31, 2 and
7, the polar surface area (PSA)6 of the oxindoles were in
excess of the recommended maximum value of 140 A2.
Hence compounds with lowerPSA were sought. As the
sulfonamide group contributed to ca. 40 A2 to the PSA
value, other functional groups were examined. It was
observed that although replacement with an acid
group8 26 led to a loss in potency, the compound
demonstrated moderate cellular activity. The methyl-
amide group 32 led to a 2-fold increase in potency in
the kinase assay. Despite a reduced solubility compared
to 26, good cellularactivity (EC 50=100 nM) was
observed.
piperidine,10 where the desired product precipitated
out of solution.
Biological Data
Binding assay
Syk kinase activity was measured using a homogeneous
time-resolved fluorescence assay, essentially as described
by Park et al.11 N-Flag tagged catalytic domain of
human SYK (Ala340-Asn635) was expressed in the
yeast Klyveromyces lactis and purified using standard
procedures. Kinase assays were performed at 2ÂKm for
ATP using the biotinylated substrate, Biot-(BA)3-
DEEEYEIPP-NH2 (capped). Phosphorylated products
were detected, upon termination of reactions with EDTA
and the addition of streptavidin-labelled allophycocyanin
(streptavidin-XL665; Cis bio) and europium cryptate
labelled anti-phosphotyrosine antibody (PY20; Cis bio) as
fluorescence acceptor and donor respectively (above ref).
Upon excitation at 337 nm, a long-lived, 665 nm energy
transfer signal, measured on the Packard Discovery, corre-
lates with the amount of phosphorylated peptide formed.
Functional assay
Syk kinase inhibitors were evaluated in an in vitro
model of IgE/FceRI triggered basophil cell degranula-
tion, modified from that previously described by Beaven
et al.12 Briefly, rat-basophilic cells (RBL-2H3) were
incubated overnight with IgE anti-DNP (1 mg/mL) and
50-hydroxytryptamine binoxylate (5-HT; 0.5 mCi/mL).
Cells were then washed and incubated for 30 min with
various concentrations of compound, prior to the addi-
tion of antigen (DNP; 100 ng/mL) fora further30 min.
Degranulation was assessed by measuring the amount
of 5-HT release in the supernatant, using a liquid scin-
tillation counter.
These preliminary results suggested that when the PSA
is <110 A2, good cellularpenetartion is achieved.
However, poor cellular activity would be predicted if
the PSA is >110 A2 (Fig. 1).
Chemistry
The synthesis of the oxindoles, as exemplified by 31, is
outlined in Scheme 1. Commercially available oxindole
27 was treated portion-wise with an excess of
chlorosulfonic acid at 0 ꢀC over 1 h and then stirred
at 60 ꢀC for 2 h. The precipitate obtained from
quenching in ice was subjected to overnight treat-
ment of concd NH3 at rt to furnish sulfonamide
28. Alkylation of the indole 29 was carried out using
NaH in DMF and MeI. The condensation9 of sulfon-
amide 28 with aldehyde 30 was carried out under
refluxing conditions in the presence of catalytic
Conclusion
This study demonstrated that oxindoles are potent
inhibitors of Syk. Sulfonamide 31 inhibited Syk kinase
activity with IC50=5 nM, EC50=1400 nM. Its modest
cellular activity is representative of the sulfonamide ser-
ies where the PSA is >140 A2. When the PSA was
lowered to <110 A2, as exemplified with carboxamide
32, good cellularactivity (EC 50=100 nM) was achieved.

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J. Y. Q. Lai et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3111–3114
7. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney,
P. J. Adv. Drug Deliv. Rev. 1997, 23, 3.
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