Discovery and SAR of novel 4-thiazolyl-2-phenylaminopyrimidines as potent inhibitors of spleen tyrosine kinase (SYK)

Article abstract of DOI:10.1016/j.bmcl.2008.09.106

A series of SYK inhibitors based on the phenylamino pyrimidine thiazole lead 4 were prepared and evaluated for biological activity. Lead optimization provided compounds with nanomolar K_i's against SYK and potent inhibition in mast cell degranulation assays.

Full text of DOI:10.1016/j.bmcl.2008.09.106

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6231–6235
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and SAR of novel 4-thiazolyl-2-phenylaminopyrimidines
as potent inhibitors of spleen tyrosine kinase (SYK)
*
Luc J. Farmer , Guy Bemis, Shawn D. Britt , John Cochran, Martin Connors, Edmund M. Harrington ,
Thomas Hoock, William Markland, Suganthini Nanthakumar, Paul Taslimi , Ernst Ter Haar, Jian Wang,
Darshana Zhaveri, Francesco G. Salituro à
Department of Medicinal Chemistry, Vertex Pharmaceuticals, Inc., 130 Waverly Street, Cambridge, MA 02139, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2008
Revised 25 September 2008
Accepted 29 September 2008
Available online 7 October 2008
A series of SYK inhibitors based on the phenylamino pyrimidine thiazole lead 4 were prepared and eval-
uated for biological activity. Lead optimization provided compounds with nanomolar Ki’s against SYK and
potent inhibition in mast cell degranulation assays.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
SYK
Spleen tyrosine kinase
Asthma
MeO
Asthma and allergic disorders remain an area of high interest
for drug discovery. Drugs, such as bronchodilators or anti-inflam-
matory agents, are currently available to treat either acute or per-
sistent symptoms of asthma. While these therapies provide
effective and safe treatments for quick relief of asthma, many of
these treatments for persistent symptoms have either side effects,
low efficacy or show little efficacy in refractory patients.1 Activa-
tion of Spleen tyrosine kinase (SYK) was shown to be the earliest
detectable signaling response to cross-linking of the high affinity
CF3
HN
NH2
N
N
NH
N
N
CONH2
H2NO2S
N
O
H2N
N
H
N
H
O
2 IC50 = 8 nM
3 IC50 = 41 nM
1 IC50 = 5 nM
In our effort to discover potent and selective SYK inhibitors, we
identified a small molecule lead from a HTS campaign, N-(3-phen-
oxyphenyl)-4-(thiazol-2-yl)pyrimidin-2-amine (4), shown below
that demonstrated good affinity for SYK (Ki = 630 nM). The novel
phenyl amino pyrimidine pharmacophore-based analog served as
the starting point for further optimization.
IgE receptor (FceRI) cross-linking on mast cells, an event which
subsequently leads to mast cell degranulation.2–4 Such a critical
role for SYK makes it a good target for therapeutic intervention
in allergic diseases such as asthma.5a,b Interestingly, other roles
have recently been reported for SYK as a tumor suppressor in
breast cancer,6 and a regulator of metastatic behavior in human
melanoma cells7a and as an attractive target for B-cell malignan-
cies.7b Over the last few years, SYK inhibition4 has been an area
of extensive research in asthma resulting in the disclosure of sev-
eral potent SYK inhibitors of varied chemotypes. Lai et al.8 and Cy-
win et al.9 have reported on a novel series of oxindoles 1 and
[1,6]naphthyridines 2 which were very potent SYK inhibitors,
and Hisamichi et al.10 published on a potent series with the disclo-
sure of pyrimidine-5-carboxamides 3 based inhibitors.
O
HN
N
N
N
S
4
Based on this lead, a novel series of SYK inhibitors was prepared
and evaluated (Tables 1–3). The syntheses of thiazole precursors
7a–c and 9a,c are described in Scheme 1. Lithiation of thiazoles11
5a–d with n-butylithium in ether at À78 °C, followed by the
addition of methoxymethy acetamide, afforded good yields of
* Corresponding author. Tel.: +1 617 444 6657.
E-mail address: luc_farmer@vrtx.com (L.J. Farmer).
Present address: Novartis, Cambridge, MA, USA.
à
Present address: Wyeth Reserch, Collegeville, PA, USA.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.106

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L. J. Farmer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6231–6235
Table 1
Binding affinities and cellular activity from aniline variation
R3
HN
R4
R5
N
N
N
S
Compound R3
R4
R5 SYK Ki (
l
M) CDK-2 Ki (
l
M) ROCK Ki (
l
M) SRC Ki (lM) ZAP-70 Ki (lM) Mast Cell Degran IC50
(lM) Rat Microsome Stability.
(% unchanged after 30 min.)
17
18
34
19
35
20
21
22
Me
OMe
OH
Me
H
H
H
H
H
H
H
0.008
0.028
0.044
0.11
0.026
0.014
0.082
>5
>5
1.652
>5
>5
>5
2.452
>5
>2.5
>2.5
>2.5
>2.5
>2.5
>2.5
>.5
1.062
1.833
0.991
1.357
0.422
0.862
2.984
0.153
0.4191
2.145
0.754
0.891
0.677
0.318
1.551
NA
0.55
0.49
0.41
0.12
0.09
0.07
0.14
NA
24
54
24
N.A.
68
12
N.A.
N.A.
CF3
CF3
CF3
Me
O(CH2)3OH CF3
OMe
Me
OMe
H
H
Me
Me 1.817
NA
Table 2
Substituted thiazoles: aniline mix/match
R3
HN
R4
N
N
S
R1
N
R2
VRT
number
R1
R2
R3
R4
SYK Ki
CDK-2 Ki
ROCK Ki
M)
SRC Ki
M)
ZAP70 Ki
M)
Mast cell degran. IC50
(lM)
Rat microsome stability
(% unchanged after
30 min)
(l
M)
(
lM)
(
l
(
l
(
l
23
24
25
26
27
28
29
30
31
32
Me
H
H
Me
H
Me
Me
Me
Me 0.023
Me 0.056
Me 0.004
Me 0.01
Me 0.032
CF3 0.022
1.1
0.1
>2.5
2.4
>2.5
>2.5
>2.5
>2.5
>2.5
>2.5
>2.5
>2.5
>2.5
>2.5
0.348
2.03
2
2.23
3.5
3.5
3.5
4
0.41
1.03
0.27
0.45
0.56
3.5
0.2
15
5
1.55
0.04
0.05
0.07
0.18
0.18
0.09
0.22
0.07
CH2OH
H
(CH2)2OH Me
(CH2)2OH Me
H
H
37
35
65
80
N.A.
50
N.A.
64
CH2OH Me
Me
Me
2.27
>2.5
>2.5
1.5
>2.5
1.5
CH2OH OMe CF3 0.033
CH2OH OH CF3 0.05
OMe CF3 0.023
CH2OH Me CF3 0.01
1
0.68
1.14
0.75
Me
H
H
3.7
1.8
the desired 2-acetyl thiazoles 6a–c. Bromination of 4- and 5-
methyl-2-acetyl thiazoles 6b,c with NBS and a catalytic amount
of AIBN provided, after treatment with potassium acetate in ace-
tic acid, the desired 4- and 5-hydroxymethyl thiazoles intermedi-
ates 8a,b in good overall yield. 2-Acethyl thiazoles 6a–d and 8a,b
were treated with neat DMF–DMA at 80 °C to afford the key
intermediate enaminones 7a–d and the corresponding alcohols
9a,b in moderate to high yield.
5-Carboethoxy-4-methyl-thiazole enaminone intermediate 12
was prepared according to Scheme 2. Diazotization of 2-amino thi-
azole 10 followed by treatment with hypophosphorous acid12 affor-
ded intermediate 11 in good yield. Deprotonation with lithium
hexamethyldisilazide in the presence of methoxymethyl acetamide
gave, after treatment with Bredereck’s reagent,13 enaminone 12.
The phenylamino pyrimidines 17–33 were prepared according
to Scheme 3. The enaminones 7, 9 and 12 were cyclized with guan-
idines 13–16 to provide pyrimidine analogs 17–33 in moderate to
good yields.
Pyrimidines 34–37 were prepared as depicted in Scheme 4. De-
methylation of compound 18 with TMSI in quinoline at 180 °C pro-
ceeded smoothly to give intermediate phenol 34 in high yield.
Alkylation with bromopropanol in the presence of potassium car-
bonate in DMF gave hydroxyalkyl ether 35. Saponification of ester
33 with potassium hydroxide in methanol provided the corre-
sponding intermediate acid, which was coupled with both enanti-
omers of alanol to give, after deprotection with TFA, the desired
amides 36 and 37 in good yields.
Compounds 17–32 and 34–37 were tested for SYK inhibition
and selectivity against a small panel of other key kinases. Further-
more, their ability to inhibit signaling activity through the high
affinity IgE receptor (FceRI) was evaluated in the mast cell degran-
ulation assay, a more physiologically relevant endpoint for asthma.
Screening numerous aniline aryl substitutions led us to the 3,5-
disubstituted patterns as being optimal for SYK inhibition. As an
example, 3,5-dimethyl aniline based compound 17 was a good
starting point with a potent SYK Ki of 8 nM and an IC50 of

L. J. Farmer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6231–6235
6233
Table 3
Addition of chiral motifs
HN
N
N
O
S
N
X
Compound
X
SYK Ki (
l
M)
CDK-2 Ki (
lM)
ROCK Ki (
lM)
SRC Ki (
l
M)
ZAP70 Ki (
l
M)
Mast cell degran. IC50
0.07
(lM)
0.009
>3.33
2.3
>3.33
0.24
36
OH
OH
HN
0.21
>3.33
>2.5
>3.33
1.5
4.2
37
HN
R1
R1
a, b
N
R2
N
R2
O
S
R1
S
N
5a R1 = R2 = H
R2
6a-d
c
O
N
5b R1 = Me; R2 = H
5c R1 = H; R2 = Me
S
5d R1 = Me; R2 = (CH2)2OH
R1
R1
7a R1 = R2 = H
d, e
N
7b R1 = Me; R2 = H
7c R1 = H; R2 = Me
N
R2
R2
O
S
O
S
7d R1 = Me; R2 = (CH2)2OH
9a R1 = H; R2 = CH2OH
9b R1 = CH2OH; R2 = H
8a R1 = H; R2 = CH2OAc
8b R1 = CH2OAc; R2 = H
6b, c
Scheme 1. Reagents and conditions: (a) n-BuLi, À78 °C, ether, 15 min; (b) AcN(OMe)Me, THF, À78 °C to rt (70–85%); (c) DMF–DMA, 85 °C, 16 h (60–90%); (d) NBS, CCl4 70 °C,
16 h (73%); (e) KOAc, HOAc, rt, 16 h (67%).
N
a
b, c
CO2Et
N
N
O
S
CO2Et
CO2Et
S
S
H2N
11
12
N
10
Scheme 2. Reagents and conditions: (a) NaNO2, H2O, À5 °C then H3PO2, À5 °C then NaOH, rt (70%); (b) LiHMDS, THF, À78 °C, 30 min then AcN(OMe)Me, THF, À78 °C to rt
(61%); (c) Bredereck’s reagent (74%).
550 nM. Changing the position of the two methyl groups on the
aniline moiety from 3,5- to 2,5-, as in compound 22, resulted in
lower activity against SYK activity with a Ki of 1820 nM. A first
round of optimization explored variations of aniline 3,5-substitu-
tions (Table 1). Removal of one of the methyl groups in compound
17, as in compound 21, led to a 10-fold decrease in SYK affinity. The
replacement of a methyl by a trifluoromethyl group such as com-
pound 19 also resulted in a 12-fold decrease in activity against
SYK. 3-Methoxy-5-trifluoromethyl aniline based analogs 18 and
the de-methylated methyl ether counterpart 34 also retained
activity in enzyme assay for SYK with respect to lead compound
17, the latter being more selective against ZAP70. The most notable
improvement in cellular activity was observed for the 3,5-dime-
thoxy aniline and 3-hydroxy propyloxy-5-trifluoromethyl analogs
20 and 35 with IC50’s of 70 and 90 nM, respectively. Unfortunately,
a reduced selectivity against Zap70 was observed for both com-
pounds. All of the other 3,5-analogs were very selective against
CDK-2 and ROCK, and moderately selective against SRC and ZAP70.
The poor correlation between IC50 and Ki for compounds 17 and
its des-methyl analog 21 was attributed to the very poor solubility

6234
L. J. Farmer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6231–6235
R3
29, 31 and 32 led to an improved selectivity against Zap70 while
maintaining moderate to good cellular potency.
R1
R3
R4
R5
Based on the high homology observed among kinases near the
hinge-binding region, it was also hypothesized that the addition of
chiral motifs may interact with amino acid residues distal to that re-
gion and may result in an improvement in kinase selectivity. Results
are shown in Table 3. (S)-Alanol based compound 36 exhibited good
SYK activity with a Ki of 9 nM and an IC50 of 70 nM. However, com-
pound 36 showed no improvement over 25 in selectivity against
Zap70. The (R)-enantiomer 37 was 20-fold less active than 36 for
SYK and again with no improvement observed for Zap70 selectivity.
Compounds 17, 23 and 24 only exhibited poor rat liver microsome
stability after 30 min incubation with only 24%, 15% and 5% remain-
ing, respectively. Variation of substitutions on either the phenyl or
thiazole ring (Tables 1 and 2) led to some improvements in meta-
bolic stability. The replacement of the benzylic alcohol of 25 by an
alkanol side chain, shown in compound 27, led to an improvement
of metabolic stability (from 37% to 65%). Furthermore, the substitu-
tion of a methyl group by a trifluoromethyl group on the phenyl ring,
as for compound 28 improved stability to 80%.
N
R2
R4
a
HN
+
O
S
R5
N
N
N
H2N
NH
NH
S
R2
N
R1
7a-d
9a-b
12
13a R3 = R4 = Me; R5 = H
= R5 = Me; R4 = H
13b R3
17-33
14 R3 = R4 = OMe; R5 = H
15 R3 = CF3; R4 = OMe; R5 = H
16 R3 = CF3; R4 = Me; R5 = H
Scheme 3. Reagents and conditions: (a) K2CO3, DMF, 85 °C, 16 h (40–80%).
CF3
CF3
R
HN
O
OH
HN
O
b
In order to better understand and improve selectivity for our
inhibitor series, the X-ray crystal structure14 for compound 36
bound to the kinase domain of SYK was solved. As Fig. 1 shows, res-
idue A451, in the hinge region of SYK, acts as an H-bond donor for
the pyrimidine ring nitrogen and as an H-bond acceptor for the –
NH– group of compound 36. Based on this (Fig. 1), it was suggested
that the planarity of the aniline ring with respect to the pyrimidine
core might be important for its activity. This may explain the dra-
matic decrease in activity for compound 22 which bears an ortho-
methyl thereby forcing the aniline out of the plane.
The environment of the 3,5-dimethyl phenyl moiety is primarily
hydrophobic, being approximately bounded by residues L377, V385,
K386, and K387 above and residues G454, P455, and K458 below. As
a consequence, removal of one of the meta-methyl groups (com-
pound 17–21) from this series results in an approximately 10-fold
loss in activity. The orientation of the 3,5-dimethylphenyl pocket
relative to the hinge-binding elements, forces the pyrimidine-NH-
phenyl unit to remain almost completely planar. Moving a methyl
group from the 3- to the 2-position (compound 17–22) stabilizes a
non-planar arrangement of this unit resulting in SYK inhibition that
is approximately 200-fold reduced. Interestingly, the same change
results in SRC inhibition that is about 10-fold better. This is likely
resulting from the smaller serine residue (S345) for SRC compared
to (Pro 455 in SYK; shown in red) providing additional space for
the phenyl ring to twist out of the plane of the pyrimidine.
N
N
N
N
S
S
N
N
35
18 R = Me
34 R = H
CH3
a
CH3
HN
CH3
HN
CH3
c, d
N
N
N
N
O
S
N
S
CH3
CO2Et
CH3
HN
CH3
N
OH
36 (S)-methyl
37 (R)-methyl
33
Scheme 4. Reagents and conditions: (a) TMSI, quinoline, 180 °C (80%); (b)
Br(CH2)3OH, K2CO3, DMF, 68 °C (75%); (c) KOH, MeOH, H2O, 60 °C, 5 h; (d) EDC,
HOBT, DIEA, DCM, H2NCH(Me)CH2OH (70–73%).
of the latter. This hypothesis was further supported by the correla-
tion from methyl-thiazole regioisomers 23 and 24. Both com-
pounds had high affinity for SYK while only compound 23
elicited moderate cellular activity which was explained by the
The X-ray crystal structure for the complex of (S)-alanol 36 in the
vicinity of salt-bridge residue K402 is shown in Fig. 2. Here we ob-
better solubility of 23 (100 lM) compared to 24 (<2.5 lM). The
most important increase in cellular activity was obtained via the
functionalization of the 2-thiazole moiety, which resulted in
compounds with cellular potency below 100 nM (Table 2). The
5-hydroxymethyl thiazolyl phenylaminopyrimidine 25 showed a
2-fold increase in SYK potency, and more importantly, a 14-fold in-
crease in cellular activity over unsubstituted thiazole 17. The
4-regioisomer 26 showed comparable activity, and both com-
pounds exhibited weak residual activity against ZAP70. The
4-hydroxyethyl analog 27 was also active in cells, albeit with
weaker inhibition of SYK (Ki of 32 nM). Selective SYK inhibitors
for the potential treatment of asthma should be devoid of activity
against kinases involved in cellular proliferation, such as CDK-2, or
the immune response, such as SRC and ZAP70. Since the most po-
tent cell active compounds were also associated with sub-micro
molar activity for ZAP70, further selectivity optimization was
needed. Replacement of the 3,5-dimethyl substituents for a 3-tri-
fluoro methyl-5-methyl or methoxy groups as for compounds 28,
Figure 1.

L. J. Farmer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6231–6235
6235
Further optimization with branched amide capped aminoacids
analogs to achieve both an improvement in SYK biological activity
and greater selectivity will be subsequently reported.
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Figure 2.
served the amide carbonyloxygen accepting an H-bond from theside
chain of K402. Interestingly, alanol based enantiomer 37 was 20-fold
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the terminal OH group within H-bonding distance of the catalytic
aspartic acid residue (D512). Our crystallographic analysis suggests
that the CH2OH terminal group is conformationally mobile.
Selectivity against Zap70 in our most potent cell active com-
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also currently underway to improve the solubility of this scaffold.
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