Orally bioavailable Syk inhibitors with activity in a rat PK/PD model

Article abstract of DOI:10.1016/j.bmcl.2015.08.037

Design and optimization of benzo- and pyrido-thiazoles/isothiazoles are reported leading to the discovery of the potent, orally bioavailable Syk inhibitor 5, which was found to be active in a rat PK/PD model. Compound 5 showed acceptable overall kinase se

Full text of DOI:10.1016/j.bmcl.2015.08.037

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Orally bioavailable Syk inhibitors with activity in a rat PK/PD model
a,
⇑
a
a
b
a
Gebhard Thoma c , Siem Veenstra , Ross Strang , Joachim Blanz , Eric Vangrevelinghe ,
e
d
Jörg Berghausen , Christian C. Lee , Hans-Günter Zerwes
a
Global Discovery Chemistry, Novartis Institutes for Biomedical Research, 4056 Basel, Switzerland
b
c
Analytical Sciences & Imaging, Novartis Institutes for Biomedical Research, 4056 Basel, Switzerland
Metabolism & Pharmacokinetics, Novartis Institutes for Biomedical Research, 4056 Basel, Switzerland
Autoimmunity, Transplantation and Inflammation Research, Novartis Institutes for Biomedical Research, 4056 Basel, Switzerland
Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA
d
e
a r t i c l e i n f o
a b s t r a c t
Article history:
Design and optimization of benzo- and pyrido-thiazoles/isothiazoles are reported leading to the discov-
ery of the potent, orally bioavailable Syk inhibitor 5, which was found to be active in a rat PK/PD model.
Compound 5 showed acceptable overall kinase selectivity. However, in addition to Syk it also inhibited
Aurora kinase in enzymatic and cellular settings leading to findings in the micronucleus assay. As a con-
sequence, compound 5 was not further pursued.
Received 5 August 2015
Revised 12 August 2015
Accepted 13 August 2015
Available online xxxx
Ó 2015 Published by Elsevier Ltd.
Keywords:
Spleen Tyrosine Kinase
Fostamatinib
BIIB-057
GS-9973
Kinase inhibitors
Spleen Tyrosine Kinase (Syk) is a non-receptor kinase discov-
ered in 1991. Syk is critical for the transduction of intracellular
both the high tissue exposure (volume of distribution was
Vss = 16.4 L/kg) and the safety issues observed in the toxicity stud-
1
signal cascades through various immune recognition receptors,
such as B cell receptor, Fc receptors, adhesion receptors, or C-type
lectin receptors. Following activation Syk phosphorylates a number
of substrates and participates in a multi-protein signaling complex,
ies. Thus, we set out to identify structurally unrelated back up
compounds devoid of an amino group.
Here we disclose the discovery of the potent, orally bioavailable
Syk inhibitor 5, which is uncharged at physiological pH and struc-
turally unrelated to 4. Compound 5 did not inhibit the hERG chan-
the ‘signalosome’, which leads to the activation of downstream
2
effector pathways such as PKC, MAPK and NF
j
B. Syk is an estab-
nel (IC50 >30 lM) and showed substantially reduced tissue
lished drug target for autoimmune diseases because aberrant acti-
exposure compared to 4. Due to good absorption and low in vivo
clearance, compound 5 exhibited favorable PK properties in rat
and was active in a rat PK/PD model. Unfortunately, compound 5
could not be further pursued because in addition to Syk it also
inhibited Aurora kinase in enzymatic and cellular settings leading
to findings in the micronucleus assay.
vation of immunoreceptor signaling can contribute to the initiation
3
and progression of chronic inflammation and autoimmunity.
4
Despite considerable efforts, only few Syk inhibitors have been
evaluated in clinical trials (for structures and data on clinical can-
5
6
7
didates such as compounds 1, 2 and 3 see Fig. 1 and Table 1). We
stopped early work on Syk inhibitors sharing the binding mode of 2
because of insurmountable PK issues.8 Furthermore, we had to
abandon the promising Syk inhibitor 4 (see Fig. 1 and Table 1)
due to liver findings and a hERG flag leading to an insufficient ther-
apeutic index for autoimmune indications.9 In rat compound 4
exhibited >100 fold higher exposure in liver and kidney compared
to blood. We hypothesized that the basic, primary amino group
required for potency and acceptable PK properties contributed to
A structure of the early, moderately active Boehringer Ingel-
11
heim Syk inhibitor 6 bound to Syk kinase domain solved at
Novartis revealed that the nitrogen in the 1-position of the
2
1,6-naphthyridine core and the adjacent sp -carbon form H-bonds
12
with the hinge sequence of Syk (Fig. 2A, Table 2). Recently, the
Boehringer group has shown that the basic amine of 6 can be
replaced with neutral groups, such as lactams and aromatic
1
3
amides, leading to more potent analogs. To mimic the binding
mode of 6, we decided to explore benzo- and pyrido-thiazole cores
as novel hinge binding motifs and prepared a set of neutral pilot
⇑
Corresponding author. Tel.: +41 61 3243342; fax: +41 61 3246735.
E-mail address: gebhard.thoma@novartis.com (G. Thoma).
http://dx.doi.org/10.1016/j.bmcl.2015.08.037
960-894X/Ó 2015 Published by Elsevier Ltd.
0
Please cite this article in press as: Thoma, G.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.08.037

2
G. Thoma et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
H
N
nature of the bicyclic core similar metabolites were observed
across species mainly affecting the pyrazole (hydroxylation,
N-demethylation) and the lactam substituent (hydroxylation).
However, in contrast to the thiazoles, the isothiazoles formed trace
quantities of glutathione conjugates indicating the formation of
reactive intermediates. Their molecular masses pointed to an
unexpected metabolic pathway involving addition of both GSH
N
OMe
OMe
N
N
N
N
N
F
HN
OMe
O
O
HN
N
H N
N
2
HN
Me
NH
Me
2
and H O. The trace quantities prevented us from isolation and
O
1
NH
2
structure elucidation. As a consequence, we decided not to con-
sider the isothiazoles for the selection of potential development
compounds.
2
active drug
of Fostamatinib
Rigel)
BIIB-057
(Biogen Idec)
(
However, we used the benzisothiazole core for the rapid opti-
mization of the PK properties. Replacement of the pyrazole by aryl
and 5 or 6-ring heteroaryl groups did not lead to derivatives with
reduced in vivo clearance. In addition, the favorable physicochem-
ical properties were compromised. Thus, we explored differently
substituted pyrazoles and found the difluoromethyl derivative 13
to show improved PK properties (Table 2; for the synthesis see
Scheme 3). However, the potency was limited. In efforts to reduce
the hydroxylation of the lactam residue we introduced an isosteric
cyclic carbamate (for the synthesis of the building block see
N
NH
HN
O
HN
N
N
H N
2
N
N
N
NH
N
NH
NH2
N
O
3
4
GS-9973
Gilead)
(
Figure 1. Structures of clinical Syk inhibitors 1–3 and reference compound 4.
compounds 7–10 lacking the basic amine of 6 (Table 2; for the syn-
theses of 7–10 see Schemes 1–4).14
We were pleased to learn that these compounds are equipotent
to our development candidate 4 in enzymatic, cellular and blood
1
0
assays (Table 2). Notably, thiazoles (7, 8) and isothiazoles (9,
0) were equally active. We obtained crystal structures of repre-
1
sentative compounds 11 (thiazole; Table 2; for the synthesis see
Scheme 2) and 12 (isothiazole; Table 2; for the synthesis see
Scheme 3) bound to Syk kinase domain, which confirmed our bind-
ing hypothesis indicating that the novel sulfur containing thiazole
and isothiazole scaffolds are bioisosteres of 1,6-naphthyridine
(Fig. 2B and C). Similarly to 6, the thiazole 11 forms two H-bonds
with the hinge sequence of Syk. Isothiazole 12 cannot form the sec-
ond H-bond; however, this is compensated for by a favorable inter-
action between sulfur and the backbone carbonyl oxygen of
1
5
E449.
Compounds 7–10 exhibited good solubility (>1 mM at pH 6.8),
À6
medium permeability (log PAMPA from À4.9 to À5.2Á10 cm/s),
medium plasma protein binding (human: 80–90%) and acceptable
stability against rat liver microsomes (half-life from 34–82 min). In
vivo rat PK studies revealed medium to high clearance
(
CL = 45–86 mL/min kg), low volumes of distribution (Vss ꢀ2 L/kg)
and short mean residence times (MRT <1 h) for all three series
tested (Table 2). The medium to high clearance and the short
MRT were confirmed in dog PK studies.
To identify metabolic weak spots we investigated the metabo-
lism of representative compounds from all four sulfur containing
scaffolds in human and rat liver microsomes. Independent of the
Table 1
Key in vitro data on reference compounds1
0
Compd
Syka (enzyme)
Kinaseb selectivity
Syka (cell)
Syka (blood)
1
2
3
4
68 ± 29
13 ± 4
377 ± 90
35 ± 4
18 (79)
2 (77)
0 (58)
0 (69)
457 ± 8
178 ± 8
878 ± 88
99 ± 7
3354 ± 475
952 ± 70
19,416 ± 1474
367 ± 27
a
IC50 in nM; n P 3 for Syk enzyme, cell and blood assays (SEM shown).
Number of kinases with IC50 <100 nM in addition to Syk (number of kinases
b
Figure 2. Crystal structures of 6, 11 and 12 bound to Syk kinase domain (H-bonds
with hinge sequence E449-M450-A451 shown).12
tested).
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G. Thoma et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Table 2
Key in vitro data1 and rat PK parameters
0
O
Thiazoles
NH
X
(R)
O
5: A=N
X=O
R=
7: A=CH
8: A=N
11: A=N
15: A=CH
X=O
R=
X=CH
2
X=CH
X=CH
2
S
N
2
Naphthyridine
A
F
R=
R=
R=
O
O
F
N
N
N
N
R
N
F
N
N
N
F
HN
NH
2
N
O
N
Isothiazoles
NH
X
6
N
(R)
(
R)
O
9: A=CH
10: A=N
12: A=CH
X=O
R=
13: A=CH
14: A=CH
X=O
R=
X=CH
R=
2
X=CH
R=
2
X=CH
2
A
F
F
R=
S
N
N
N
N
N
N
N
N
N
N
F
N
F
R
Compd Syka (enzyme) Kinaseb selectivity Syka (cell)
Syka (blood) CLc (mL/min kg)
V
c
ss (L/kg) MRTc (h) AUCc,d iv (nM * h) BAVc (%)
C
max (nM)
c,d
4
5
6
7
8
9
1
1
1
1
1
1
35 ± 4
38 ± 15
552 ± 150
33 ± 24
26 ± 5
46 ± 8
40 (n = 1)
5 ± 2
16 ± 3
57 ± 9
16 ± 2
9 ± 9
0 (69)
1 (60)
0 (86)
4 (40)
0 (61)
2 (63)
0 (20)
4 (66)
7 (60)
0 (54)
5 (60)
11 (60)
99 ± 7
367 ± 27
522 ± 37
n.t.
186 ± 31
538 ± 157
298 ± 43
31
4
16.4
1.3
n.t.
n.t.
2.3
2.4
2.4
1.2
1.5
2.8
1.4
1.3
8.7
6.0
n.t.
n.t.
0.7
0.9
0.5
0.4
2.0
2.6
2.8
4.7
1449
14,791
n.t.
n.t.
951
1056
577
858
3887
2341
5440
10,142
60
88
n.t.
n.t.
46
30
27
35
100
100
100
91
80
284 ± 20
951 ± 234
89 ± 34
284 ± 20
161 ± 52
917
n.t.
n.t.
103
94
109
178
477
263
614
803
n.t.
n.t.
54
45
86
53
12
18
7
0
1
2
3
4
5
247 (n = 1) 290 ± 49
132 ± 19
166 ± 69
340 ± 31
328 ± 87
107 ± 8
517 ± 95
737 ± 116
1018 ± 407
197 ± 38
230 ± 51
5
a
b
c
IC50 in nM; n P 3 for Syk enzyme, cell and blood assays (SEM shown).
Number of kinases with IC50 <100 nM in addition to Syk (number of kinases tested).
Cassette dosing in Sprague Dawley rats; iv 1 mg/kg, NMP/PEG200 (3:7); po 3 mg/kg, CMC/water/Tween (0.5:99:0.5).
Dose normalized.
d
Scheme 5). Compound 12 and its lactam analog 9 showed similar
potencies as well as similar key interactions of lactam and cyclic
carbamate with the enzyme (Fig. 3). The carbonyl oxygen and
the ring nitrogen form H-bonds with K402 and S511, whereas ben-
eficial van der Waals contacts were observed between the methyl
group and G378 and V385. These lipophilic interactions along with
an improved pre-organization of the bioactive conformation might
O
O
O
a, b
TsO
c
HS
HO
40%
Br (2 steps) O N
Br
O N
Br
2
2
O
O
N
O
O
NH
Ref. 13
e, f
N
OH
S
O
S
N
NH2
N
OH
d
a, b
c
S
N
O
O
4
4%
59%
N
7%
82%
Br
S
N
Br
(
2 steps)
(
2 steps)
N
O
N
Br
O
NH
O
N
O
B
O
NH
O
N
N
O
Ref. 13
OH
d,e
g
O
S
N
NH
O
6
0%
S
N
2
3%
S
N
N
N
(
2 steps)
N
N
7
N
N
N
8
Scheme 1. Synthesis of 7 (15 prepared similarly). Reagents and conditions: (a)
HNO , CH CO
H, À10 °C ? 25 °C, 1 h; (b) TsCl (1.2 equiv), pyridine (2.0 equiv),
CH Cl , 0 ? 25 °C, 16 h; (c) Na S Â 9H O (2.0 equiv), DMF, 0 °C, 1 h; (d) Zn powder
5.0 equiv), HCOOH, reflux, 16 h; (e) alcohol (1.1 equiv), (t-BuO
PPh (2.0 equiv), CH Cl , 25 °C, 1 h; (f) TFA, microwave, 100 °C, 20 min; (g) boronate
ester (3.0 equiv), Pd(PPh Cl (0.1 equiv), Na CO (3.0 equiv), dioxane, 100 °C, 16 h.
3
3
2
Scheme 2. Synthesis of 8 (5, 11 prepared similarly). Reagents and conditions: (a)
RC„CH (1.5 equiv), CuI (0.05 equiv), Pd(PPh (0.07 equiv), NEt (3.0 equiv), DMF,
70 °C, 0.5 h; (b) NH (12 equiv), MeOH, 70 °C, 16 h; (c) NaOtBu (1.4 equiv), THF,
2
2
2
2
)
3 4
3
1
3
(
2
C)
N
2 2
(2.0 equiv),
3
3
2
2
70 °C, 1 h; (d) alcohol (1.8 equiv), (t-BuO
25 °C, 2 h; (d) TFA, 100 °C, 15 min.
2 2 2 3 2 2
C) N (2.2 equiv), PPh (2.2 equiv), CH Cl ,
)
3 2
2
2
3
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4
G. Thoma et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
O
S
O
O
O
N
S
O
O
O
a
b
Ref. 19
O
O
H
2
N
Br
75%
38%
H
2
N
a
O
b
O
S
N
Br
HN
58%
steps
OH
Ref. 20
2
HO
O
O
N
OH
Ref. 13
O
OH
c
OH d, e
O
S
S
O
91%
68%
(2 steps)
N
N
Br
Br
c
O
O
O
O
N
O
N
91%
O
HO
O
O
O
B
NH
OH
NH
OH
O
N
f
N
Scheme 5. Synthesis of the protected (hydroxyethyl)oxazolidinone. Reagents and
conditions. (a) Epoxide2 (1.0 equiv), Amine (1.3 equiv), EtOH, 60 h, 25 °C;
CO (2.5 equiv), ClCO Me 1.2 equiv), CH Cl /H O (2.5:3.0), 5 ? 25 °C, 3 h;
2 3 2 2 2 2
c) NaOMe (2.5 equiv), MeOH, reflux, 40 min.
O
0
O
45%
(
(
b) Na
S
S
N
N
N
Br
9
N
promising PK properties, low clearance, good potency but limited
kinase selectivity. Next we transferred the learnings to the thiazole
series and prepared 5 and 15 (Table 2; for the syntheses see
Schemes 2 and 1, respectively). Both compounds exhibited low
clearance, good MRT and promising AUC. Because of its superior
kinase selectivity we selected 5 for further profiling against the
abandoned development candidate 4.
Compound 5 showed IC50 values above 30 lM in both hERG
binding and automated patch clamp assays. Compound 4 did not
exhibit substantial hERG channel inhibition in a manual patch
Scheme 3. Synthesis of 9 (12, 13, 14 prepared similarly). Reagents and conditions:
(
(
(
1
(
a) SOCl
1.6 equiv), pyridine (1.1 equiv), benzene, reflux, 24 h; (c) 48% aq HBr, 140 °C, 2.5 h;
d) alcohol (1.0 equiv), (t-BuO C) (1.5 equiv), PPh (1.5 equiv), THF, 0 ? 25 °C,
6 h; (e) TFA, 80 °C, 3.5 h; (f) boronate (1.5 equiv), Pd(DTBPF)Cl (0.06 equiv), K PO
2.0 equiv), Tol/EtOH/H O (7:2:1), 100 °C, 1 h.
(2.0 equiv), benzene, reflux, 18 h; (b) N-sulfinyl-methanesulfonamide19
2
2
2
N
2
3
2
3
4
2
explain that related compounds lacking the methyl group were
substantially less active.
Carbamate 12 showed improved PK properties compared to its
lactam analog 9 (Table 2; for the synthesis see Scheme 3).
However, the MRT of 2 h and the kinase selectivity were subopti-
mal. Combination of difluoromethylpyrazole and carbamate led
to compound 14 (Table 2; for the synthesis see Scheme 3) with
clamp assay at 30
lM and showed an IC50 value of 28 lM in an
automated patch clamp assay; however, under GLP conditions,
9
an IC50 value of 5 lM was established. Thus, the predictability of
the in vitro assays can be challenged, but based on the available
data the neutral compound 5 appeared promising.
The PK parameters of 4 and 5 differed substantially probably
due to the absence of a basic amine in 5 (Table 2). Compound 4
exhibited medium clearance and a very high Vss leading to a long
MRT of 8.7 h while compound 5 showed a low Vss and a low clear-
O
O
Br
S
Br
S
CN
Br
a
b
NH2
NH2
S
N
39%
N
53%
N
Br
Br
ance also resulting in a long MRT of 6 h. In line with the distinct Vss
,
the compounds showed very different distribution profiles in rat.
Comparable concentrations in blood, liver and kidney were
observed for 5, whereas compound 4 showed 100–200 fold
increased concentrations in tissue (Table 3). In light of the liver
findings observed with 4, we considered the PK profile of 5 to be
O
O
NH2
N
S
N
d
N
NH
c
S
N
2
2%
N
more desirable.
N
N
Compound 5 was tested in our rat PK/PD model (Fig. 4).9
N
Following oral administration, blood samples were taken at differ-
ent times for PK determination as well as assessment of inhibition
of Syk-dependent signaling events. For this, the extent of SLP76
phosphorylation in monocytes in response to stimulation by anti
CD32 was quantified. A dose of 3 mg/kg of 5 led to 78%, 79% and
O
NH
O
O
O
N
NH
e
6
0% inhibition after 2, 4 and 8 h post administration, respectively.
Ref. 13
O
43%
OH
OMs
No inhibition was observed after 24 h. Blood concentrations were
1930, 1341, 827 and 40 nM, respectively. The low exposure after
24 h was unexpected based on the PK profile (Table 2). A potential
explanation is the usage of different rat strains for PK experiment
f
N
S
N
45%
N
2
steps
1
0
N
(
Sprague Dawley) and PK/PD model (Lewis). Due to the limited
Scheme 4. Synthesis of 10. Reagents and conditions: (a) H
b) Zn powder (5.0 equiv), MeOH/HCOOH (1.3:1), 10 °C, 2 h; (c) RC„CH (1.4 equiv),
CuI (0.07 equiv), Pd(PPh (0.1 equiv), NEt (3.0 equiv), DMF, 70 °C, 0.5 h; (d)
NaOtBu (2.1 equiv), DMF/THF, 70 °C, 0.5 h; (e) (1) TFA, microwave, 100 °C, 14 min;
2
SO
4
(concd), 25 °C, 72 h;
AUC of compound 5 (10-fold lower), higher doses were required
(
to achieve substantial inhibition in this model. However, the
3
)
4
3
9
effects seen with 5 were more sustained.
Compound 5 showed acceptable overall kinase selectivity
(
3 2 2
2) MsCl (0.9 equiv), NEt (2.7 equiv), CH Cl , 0 ? 25 °C, 1 h; (f) mesylate
(Table 2). However, in addition to Syk it also inhibited Aurora A
(
2.0 equiv), K CO (3.0 equiv), DMF, microwave, 140 °C, 0.5 h.
2
3
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G. Thoma et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
Figure 3. Crystal structures of 11 and 12 bound to Syk kinase domain (key interactions of lactam and cyclic carbamate shown; H-bonds with K402 and S511as well as van der
1
2
Waals contacts with G378 and V385).
Table 3
Acknowledgments
Distribution of compounds 4 and 5 in different matrices following 1 mg/kg iv
administration in rat
We gratefully acknowledge the expert assistance of Valerie
Caballero, Thierry Délémonté, Mu-Yun Gao, Joanna Grant, Alice
Hauchard, Anne-Marie Jutzi, Alexandre Luneau, Patric Neubert,
Kenneth Ng, Jürg Peter, Marc Schäfer, Jennifer Shaffer and Phuoc
Thanh Thai. We thank Reiner Aichholz and Hong Liu for helpful dis-
cussions. The structural work in this Letter is based on experiments
conducted at beamline 5.0.3 of the Advanced Light Source (ALS).
The ALS is supported by the Director, Office of Science, Office of
Basic Energy Sciences, Material Sciences Division of the US
Department of Energy under contract DE-AC03-76SF00098 at
Lawrence Berkeley National Laboratory.
Matrix
4
5
Blood (ng/g)
Liver (ng/g)
Kidney (ng/g)
41
9175
3866
1014
2134
1433
References and notes
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c
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in our enzymatic assay (IC50 = 57 nM).16 Unfortunately, this was
confirmed in a cellular Aurora B assay (IC50 = 110 nM).17 The com-
pound was negative in the Ames test but positive in the micronu-
cleus assay. This was not unexpected as Aurora A inhibition causes
micronuclei formation and aneuploidy, Aurora B inhibition results
in a cytokinesis defect leading to binucleate cells, and inactivation
of both Aurora A and B causes an abrogation in chromosome seg-
2
784; (f) Lucas, M. C.; Goldstein, D. M.; Hermann, J. C.; Kuglstatter, A.; Liu, W.;
Luk, K. C.; Padilla, F.; Slade, M.; Villasenor, A. G.; Wanner, J.; Xie, W.; Zhang, X.;
Liao, C. J. Med. Chem. 2012, 55, 10414; (g) Padilla, F.; Bhagirath, N.; Chen, S.;
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Kuglstatter, A.; Liao, C.; Liu, W.; Lowrie, L. E., Jr.; Luk, K. C.; Lynch, S. M.; Menke,
J.; Niu, L.; Owens, T. D.; Yang, C.; Railkar, A.; Schoenfeld, R. C.; Slade, M.; Steiner,
S.; Tan, Y.-C.; Villasenor, A. G.; Wang, C.; Wanner, J.; Xie, W.; Xu, D.; Zhang, X.;
Zhou, M.; Lucas, M. C. J. Med. Chem. 2013, 56, 1677; (h) Lucas, M. C.; Bhagirath,
N.; Chiao, E.; Goldstein, D. M.; Hermann, J.; Hsu, P.-Y.; Kirchner, S.; Kennedy-
Smith, J.; Kuglstatter, A.; Lukacs, C.; Menke, J.; Niu, L.; Padilla, F.; Peng, Y.;
Polonchuk, L.; Railkar, A.; Slade, M.; Soth, M.; Xu, D.; Yadava, P.; Yee, C.; Zhou,
M.; Liao, C. J. Med. Chem. 2014, 57, 2683; (i) Liddle, J.; Atkinson, F. L.; Barker, M.
D.; Carter, P. S.; Curtis, N. R.; Davis, R. P.; Douault, C.; Dickson, M. C.; Elwes, D.;
1
8
regation and mitotic exit with a single quadroploid nucleus.
We have described the optimization of novel benzo- and pyr-
ido-thiazoles/isothiazoles leading to the discovery of the potent,
orally bioavailable Syk inhibitor 5, which was found to be active
in a rat PK/PD model. However, as a consequence of findings in
the micronucleus assay, compound 5 was not further pursued.
Please cite this article in press as: Thoma, G.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.08.037

6
G. Thoma et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
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Littlewood-Evans, A.; Lee, C. C.; Liu, H.; Zerwes, H.-G. J. Med. Chem. 2015, 58, 1950.
10. Potency and kinase selectivity of Syk inhibitors were assessed in enzymatic
assays based on the Caliper microfluidic mobility shift technology. Cellular
activity was measured in Ramos B-cells upon BCR stimulation with anti-IgM
which leads to phosphorylation of the adaptor protein BLNK (B cell linker
protein), a direct Syk substrate. Inhibition of Syk in presence of 90% human
K.; Lewis, H. D.; McCleary, S.; Neu, M.; Patel, V. K.; Preston, A. G. S.; Ramirez-
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H.; Vincent, S.; Chapman, R. W.; Kelly, N.; Hickey, E.; Abraham, W. M.;
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blood was monitored in monocytes following FccR stimulation with an anti
CD32 antibody. This leads to the phosphorylation of the adaptor protein SLP-76
which is also a direct substrate of Syk. For details on the assays see Ref. 9.
11. Cywin, C. L.; Zhao, B.-P.; McNeil, D. W.; Hrapchak, M.; Prokopowicz, A. S.;
Goldberg, D. R.; Morwick, T. M.; Gao, A.; Jakes, S.; Kashem, M.; Magolda, R. L.;
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2
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V.; Grossbard, E. Arthritis Rheum. 2008, 58, 2209; For a more comprehensive
analysis of the kinase selectivity of 1 see Davis, M. I.; Hunt, J. P.; Herrgard, S.;
Ciceri, P.; Wodicka, L. M.; Pallares, G.; Hocker, M.; Treiber, D. K.; Zarrinkar, P. P.
Nat. Biotechnol. 2011, 29, 1046. For the discontinuation of 1 in rheumatoid
arthritis see http://clinicaltrials.gov/show/NCT01264770.
12. Structures deposited in the RCSB Protein Data Bank under PDB ID: 5CXZ (6),
PDB ID: 5CXH (11) and PDB ID: 5CY3 (12).
13. (a) Fiegen, D.; Handschuh, S.; Hobbie, S.; Hoffmann, M.; Kono, T.; Sato, Y.;
Schnapp, A.; Schuler-Metz, A. PCT Int. Appl. WO 2,010,015,518.; (b) Bouyssou,
T.; Dahmann, G.; Engelhardt, H.; Fiegen, D.; Handschuh, S.; Hobbie, S.;
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Handschuh, S.; Klicic, J.; Linz, G.; Schaenzle, G.; Schnapp, A.; East, S. P.;
Mazanetz, M.P.; Scott, J.; Walker, Edward PCT Int. Appl. WO 2,011,092,128.; (d)
Hoffmann, M.; Bischoff, D.; Dahmann, G.; Klicic, J.; Schaenzle, G.; Wollin, S. L.
M.; Convers-Reignier, S. G.; East, S. P.; Marlin, F. J.; McCarthy, C.; Scott, J. PCT
Int. Appl. WO 2,013,014,060.
14. We also explored benzo[c][1,2,5]thiadiazoles and [1,2,5]thiadiazolo[3,4-c]
pyridines but the corresponding analogs were found to be less potent. Only
the R,R-configuration of the chiral lactam residue led to highly active
compounds.
15. Beno, B. R.; Yeung, K.-S.; Bartberger, M. D.; Pennington, L. D.; Meanwell, N. A. J.
Med. Chem. 2015, 58, 4383.
6
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Coffey, G.; DeGuzman, F.; Inagaki, M.; Pak, Y.; Delaney, S. M.; Ives, D.; Betz, A.;
Jia, Z. J.; Pandey, A.; Baker, D.; Hollenbach, S. J.; Phillips, D. R.; Uma Sinha, U. J.
Pharm. Exp. Ther. 2012, 340, 350. For the withdrawal of a planned phase II trial
in
rheumatoid
arthritis
see
http://www.clinicaltrials.gov/ct2/show/
NCT01652937?term=biib-057&rank=1.
16. Compounds 7, 11, 12, 14 and 15 also inhibited Aurora A with IC50 values of
6100 nM. Compounds 9 and 10 were less potrnt showing IC50 values of 576
and 800 nM, respectively.
7
.
.
Currie, K. S.; Kropf, J. E.; Lee, T.; Blomgren, P.; Xu, J.; Zhao, Z.; Gallion, S.;
Whitney, J. A.; Maclin, D.; Lansdon, E. B.; Maciejewski, P.; Rossi, A. M.; Rong, H.;
Macaluso, J.; Barbosa, J.; Di Paolo, J. A.; Mitchell, S. A. J. Med. Chem. 2014, 57,
17. The Aurora
B cell assay was performed by ProQinase GmbH, Freiburg,
3
856.
Germany; http://proqinase.com/.
8
Thoma, G.; Blanz, J.; Buhlmayer, P.; Druckes, P.; Kittelmann, M.; Smith, A. B.;
van Eis, M.; Vangrevelinghe, E.; Zerwes, H.-G.; Che, J.; He, X.; Jin, Y.; Lee, C. C.;
Michellys, P.-Y.; Uno, T.; Liu, H. Bioorg. Med. Chem. Lett. 2014, 24, 2278.
18. Hochegger, H.; Hegarat, N.; Pereira-Leal, J. B. Open Biol. 2013, 3. 120185.
19. Singerman, G. M. J. Heterocycl. Chem. 1975, 12, 877.
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Please cite this article in press as: Thoma, G.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.08.037