Rational design of highly selective spleen tyrosine kinase inhibitors

Article abstract of DOI:10.1021/jm301367c

A novel approach to design selective spleen tyrosine kinase (Syk) inhibitors is described. Inhibition of spleen tyrosine kinase has attracted much attention as a mechanism for the treatment of autoimmune diseases such as asthma, rheumatoid arthritis, and SLE. Fostamatinib, a Syk inhibitor that successfully completed phase II clinical trials, also exhibits some undesirable side effects. More selective Syk inhibitors could offer safer, alternative treatments. Through a systematic evaluation of the kinome, we identified Pro455 and Asn457 in the Syk ATP binding site as a rare combination among sequence aligned kinases and hypothesized that optimizing the interaction between them and a Syk inhibitor molecule would impart high selectivity for Syk over other kinases. We report the structure-guided identification of three series of selective spleen tyrosine kinase inhibitors that support our hypothesis and offer useful guidance to other researchers in the field.

Full text of DOI:10.1021/jm301367c

Article
pubs.acs.org/jmc
Rational Design of Highly Selective Spleen Tyrosine Kinase Inhibitors
Matthew C. Lucas,*,†,§ David M. Goldstein,†,∥ Johannes C. Hermann,† Andreas Kuglstatter,†
Wenjian Liu,‡ Kin Chun Luk,† Fernando Padilla,† Michelle Slade,† Armando G. Villasenor,†
̃
Jutta Wanner,† Wenwei Xie,‡ Xiaohu Zhang,‡,⊥ and Cheng Liao†
†Small Molecule Research, Discovery Chemistry, pRED, Pharma Research and Early Development, Hoffmann-La Roche Inc., 340
Kingsland Street, Nutley, New Jersey 07110, United States
‡BioDuro(Beijing)Co., Ltd., Building E, No. 29, Life Science Park Road, Changping District, Beijing 102206, P. R. China
S
* Supporting Information
ABSTRACT: A novel approach to design selective spleen tyrosine kinase (Syk)
inhibitors is described. Inhibition of spleen tyrosine kinase has attracted much
attention as a mechanism for the treatment of autoimmune diseases such as
asthma, rheumatoid arthritis, and SLE. Fostamatinib, a Syk inhibitor that
successfully completed phase II clinical trials, also exhibits some undesirable side
effects. More selective Syk inhibitors could offer safer, alternative treatments.
Through a systematic evaluation of the kinome, we identified Pro455 and Asn457
in the Syk ATP binding site as a rare combination among sequence aligned
kinases and hypothesized that optimizing the interaction between them and a Syk
inhibitor molecule would impart high selectivity for Syk over other kinases. We
report the structure-guided identification of three series of selective spleen
tyrosine kinase inhibitors that support our hypothesis and offer useful guidance to
other researchers in the field.
II clinical trial in asthma in September 2009 (inhaled
administration).7 No adverse effects were noted in the phase
I trial. PRT062607, reported to be a Syk specific inhibitor,7 is
currently in phase II, but no data related to adverse events have
been described.
INTRODUCTION
■
Spleen tyrosine kinase (Syk) is an intracellular protein tyrosine
kinase whose signaling pathway is largely involved in the
adaptive immune response but also in some innate and
nonimmune functions.1 Syk is most widely expressed in
hematopoietic cells but is also expressed in other tissues. It
serves as a key mediator of B-cell receptor- and Fc receptor-
mediated signaling in inflammatory cells such as B cells, mast
cells, macrophages, dendritic cells, and neutrophils and is
involved in bone resorption by osteoclasts.2−4 Upon receptor
stimulation, Syk becomes activated primarily by SRC family
tyrosine kinase Lyn and subsequently transduces the signal
through VAV family members, phospholipase Cγ (PLCγ), the
regulatory subunits of phosphoinositide 3-kinases (PI3Ks) and
SH2 domain containing leukocyte proteins (SLP) SLP76 and
SLP65. The resulting activation of various downstream
components leads to cellular responses including cytokine
release, differentiation, proliferation, reactive oxygen species
(ROS) production, cytoskeletal rearrangement, and survival.
No human patients with a spontaneous mutation in Syk have
been described, although recently there was a description of a
family carrying a 5.3 Mb microdeletion. The deletion involves
30 genes, including the immunologically active SYK gene.5
Fostamatinib 1, a poorly selective Syk inhibitor, recently
completed phase II trials for the treatment of rheumatoid
arthritis (Figure 1).6 In these studies adverse effects included
hypertension, gastrointestinal effects (nausea and diarrhea), and
neutropenia. In addition, a Syk selective siRNA (Excellair)
successfully completed phase I trials and advanced into a phase
The SYK gene has also been disrupted in mice. The absence
of the viable kinase results in perinatal lethality (due to a defect
in lymphatic vascular development) and a lack of mature B
cells.8 Syk−/− reconstituted mice are unable to promote B-cell
maturation because of failure of the ProB to PreB transition.9
These mice are protected from autoantibody-induced arthritis
in a K/BxN model.10 Collectively these data support a role for
Syk in B-cell signaling and rheumatoid arthritis. Indeed, Syk
inhibitors are efficacious in rodent arthritis models and have
been shown to suppress arthritis scores, bone erosions, pannus
formation, and synovitis. Syk inhibitors have also shown
efficacy in in vivo animal models of asthma, idiopathic
thrombocytopenic purpura, systemic lupus erythematous, and
autoimmune diabetes. In human cell lines, Syk inhibitors are
able to inhibit BCR-mediated B-cell activation, FcεR-mediated
mast cell and basophil responses, and Fcγ-mediated mono-
cytes/macrophage response and block osteoclast differentia-
tion; these cell types are implicated in the onset and
propagation of autoimmune disease and the cartilage and
bone destruction associated with rheumatoid arthritis.11−20
Received: July 5, 2012
© XXXX American Chemical Society
A
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Figure 1. Structure of fostamatinib 1 and Syk high-throughput screening hit 2.
Collectively, the clinical trial data and the critical role of B
cells in human autoimmune and inflammatory diseases make
Syk a compelling drug target in the therapeutic areas of
rheumatoid arthritis, SLE, B-cell malignancies, asthma, and
allergic upper airway disorders.21−23
Table 1
Ramos
B cell
Syk
LYSA
selectivity (no. of
IC
IC
solubility
kinases inhibited
50a
b
50 c
d
e
compd
(nM)
LE
(μM)
(μg/mL)
>90% at 10 μM)
PRT062607
1
0.41
0.31
0.29
0.31
0.32
0.223
0.267
0.156
0.444
0.482
<1
<1
<1
<1
<1
116/385
145/290
14/386
34/386
While there has been significant effort to find Syk inhibitors
in recent years, few of these have advanced into clinical trials.
The limited or unknown selectivity over the human kinome of
those molecules that have made it as far as the clinic makes
interpretation of the clinical benefit of Syk inhibition
challenging and ambiguous. The most advanced of these, 1,
has a poor selectivity profile, and it is likely that some of the
reported safety concerns and even some of the efficacy
observed are a consequence of off-target kinase activity. For
example, 1 is a potent inhibitor of the kinase VEGFR2; it is
widely reported that such inhibitors cause elevated blood
pressure.24 It is also a strong inhibitor of the kinase c-RET. This
particular kinase is critical for renal and ureteric development,
and its inhibition may have resulted in the developmental
toxicity of 1 observed in rabbits.25
f
1
17
7
3
4
3
5
1
a
See Supporting Information for details. Data shown are the average of
b
at least n = 2. Ligand efficiency (in energy units/atom ≈ (kcal/mol)/
c
d
atom). See Supporting Information for details. Lyophilized solubility
e
assay data. DiscoveRx KINOMEscan screening platform measure-
ments employing an active site-directed competition binding assay to
quantitatively measure interactions between test compounds and 290−
386 kinase assays. Data shown are for the active entity R406,
f
(fostamatinib is a prodrug).
a
Scheme 1. Synthesis of Thienopyrimidine 3
As a result, we were compelled to develop rational
approaches to generate highly selective Syk inhibitors that
could help to improve and advance our understanding of the
relevance of Syk in disease and enable the determination of the
potential of a selective Syk inhibitor to replicate the clinical
benefits displayed by fostamatinib while minimizing the off-
target effects that may limit or prevent its use in a clinical
setting. Our ultimate vision was to create a safer, efficacious
drug candidate for the treatment of autoimmune diseases.
Recently, Portola Pharmaceuticals reported detailed results of
selective Syk inhibitors, and the presented biochemical, cellular,
and animal data support our aim to identify highly selective Syk
inhibitors to maximize clinical benefit.7 We have confirmed
some of the Portola data to allow us to benchmark our own
efforts in this regard (Table 1). Herein we present an approach
that enabled the rational design of selective Syk inhibitors and
disclose selective in vitro tools.
SYNTHESIS
■
The syntheses of the compounds described are shown
(Schemes 1−6; see Supporting Information). The thiazolopyr-
imidines were all synthesized from the commercially available
dichlorothiazolopyrimidine 15. This was converted to 16 via a
regioselective nucleophilic aromatic substitution of the C7-
chlorine. Suzuki coupling gave ester 17, which could be easily
converted to 3 in three steps via saponification, amide coupling,
and a final saponification (Scheme 1). Compounds 6−9 were
synthesized from 16 through a Buchwald−Hartwig amination
followed by deprotection, amide bond formation, and a final
deprotection to give the final compounds (Schemes 2 and 3).
The triazolopyridine 4 was synthesized via a Suzuki and
Buchwald−Hartwig coupling sequence (Scheme 4). In the case
a
Reagents and conditions: (a) 3,4-dimethoxyaniline, DIPEA, DMSO,
rt, 2 h, 64%; (b) 3-(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-yl)-
benzoic acid methyl ester, 1,4-dioxane, H2O, Na2CO3, Pd(PPh3)4,
100 °C, 16 h, 81%; (c) NaOH, THF, H2O, rt, 16 h, 98%; (d) tert-butyl
4-aminobenzoate, EDCI, DMAP, DMF, rt, 2 h, 70%; (e) CF3CO2H,
CH2Cl2, rt, 15 h, 66%.
of imidazopyridazines 5 and 10−14, the 2-aminopyridine was
installed first, via a regioselective nucleophilic aromatic
B
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a
a
Scheme 2. Synthesis of Thienopyrimidines 6 and 7
Scheme 3. Synthesis of Thienopyrimidines 8 and 9
a
Reagents and conditions: (a) n = 1, methylpiperidine 3-carboxylate,
Cs2CO3, X-Phos, Pd2(dba)3, 110 °C, 15 h, 56%; n = 0,
methylpyrrolidine 3-carboxylate, Cs2CO3, X-Phos, Pd2(dba)3, 95 °C,
24 h, 89%; (b) NaOH, THF, MeOH, rt, 24 h, 98% (n = 1); NaOH,
MeOH, rt, 24 h, 61% (n = 0); (c) HATU, DIPEA, DMF, rt, 72 h, 92%
(n = 1), 77% (n = 0); (d) CF3CO2H, CH2Cl2, rt, 24 h, 37% (n = 1),
59% (n = 0).
a
Reagents and conditions: (a) n = 1, tert-butyl piperidin-3-yl-
carbamate, X-Phos, Cs2CO3, dioxane, Pd2(dba)3, 95 °C, 24 h, 93%;
n = 0, tert-butyl pyrrolidin-3-yl carbamate, X-Phos, Cs2CO3, dioxane,
Pd2(dba)3, 95 °C, 24 h, 88%; (b) HCl, dioxane, rt, 24 h, 100%; (c) 4-
methoxybenzoic acid, HATU, DIPEA, DMF, rt, 2 h, then EDCI,
DMAP, 4 days, 96% (n = 1), 88% (n = 0); (d) LiOH, H2O, MeOH,
THF, 2 h, rt, 18% (n = 1), 47% (n = 0).
displacement of the bromine, and this was followed by the
aryl−aryl bond creation through a Suzuki coupling (Schemes 5
and 6).
favorable hydrophobic interactions with Pro455 or formed
attractive electrostatic interactions with Asn457 would result in
selective Syk inhibition. Furthermore, simultaneously achieving
favorable interactions with Pro455 and Asn457 (an incidence
that is present in only one other kinase, MLK1) seemed to have
the potential to afford exquisite selectivity over the kinome. We
were encouraged by the observation that the X-ray crystal
structure of the active component of nonselective Syk kinase
inhibitor 1 showed a suboptimal interaction with Pro455 and
no discernible interaction with Asn457 or Met450 (Figure
2a).26
Following a high throughput screening campaign, we had
identified the imidazopyrazine scaffold (e.g., 2, Figure 1) and
selected this as a starting point for our investigation.
Imidazopyrazines as kinase hinge binders were familiar to us
from our own, and others’, effort to develop kinase
inhibitors.27,28 While Btk inhibitors containing the imidazopyr-
azine hinge binding motif have been reported to bind to the
kinase active site in an extended “N”-conformation,28 this
conformation seemed awkward and unsatisfactory when
applied to the Syk ATP binding pocket. An X-ray crystal
structure obtained of 2 bound to the Syk kinase domain (Figure
3) suggested to us that vectors from the 6-position of the
imidazopyrazine might offer the ability to project toward the
Asn457 residue via a bent, “inverted U-conformation”. The
aniline projecting out of the front of the binding site already
appeared to make some hydrophobic interactions with the
RESULTS AND DISCUSSION
■
We began our endeavor by analyzing the Syk ATP binding site
structure and comparing it with other aligned kinase structures
to identify commonality with respect to amino acid residue
incidences (Figure 2). Four amino acid residues were identified
that rarely appeared in other kinase active sites: Met450,
Leu453, Pro455, and Asn457 (each having an incidence in less
than 5% of all kinases, Figure 2b). On the basis of our
examination of the binding pocket, we felt that Pro455 and
Asn457 were the most attractive as targets for optimization of
the interaction between them and putative ATP-competitive
spleen tyrosine kinase inhibitors. The spatial proximity of
Pro455 and Asn457 suggested we might be able to design
inhibitors that interacted with these two simultaneously. On the
other hand, Leu453, with its side chain projecting out and away
from the ATP-binding site did not offer as many opportunities
for targeting. Met450, while proximal, offered a smaller
accessible surface area with which to interact, so we chose
not to focus on this residue in our initial exploration.
Pro455 is present in only 9 aligned kinases (of a total of
433), while Asn457 is present in 21 aligned kinases. Pro455, a
hydrophobic residue, is most commonly replaced with
hydrophilic or charged residues, while Asn457, an acidic
residue, is most commonly replaced by hydrophobic residues.
Thus, we predicted that a small molecule that either had
C
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a
a
Scheme 4. Synthesis of Triazolopyridine 4
Scheme 6. Synthesis of Imidazopyridazines 10−14
a
Reagents and conditions: (a) tert-butyl 4-(3-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)benzamido)benzoate, Na2CO3, H2O, dioxane,
Pd(PPh3)4, 96 °C, 24 h, 44%; (b) 5,6-dimethoxypyridin-2-amine, X-
Phos, Cs2CO3, dioxane, Pd2(dba)3, 95 °C, 24 h, 44%; (c) CF3CO2H,
CH2Cl2, rt, 24 h, 59%.
a
Reagents and conditions: (a) n = 1, R1 = H, 6-(piperidin-1-yl)pyridin-
2-amine, NaH, DMF, rt, 16 h, 24%; (b) 6-fluoropyridin-2-amine, NaH,
DMF, rt, 16 h, 99%; (c) pyrrolidine, H2O, 205 °C, microwave, 30 min,
18% (n = 0, R1 = H); 2-methylpyrrolidine, H2O, 205 °C, microwave,
30 min, 64% (n = 0, R1 = CH3); (d) PhB(OH)2, Pd2(dba)3, X-Phos,
K2CO3, dioxane, water, 100 °C, 4 h, 58% (n = 1, R1 = H, R2 = H);
PhB(OH)2, Pd2(dba)3, X-Phos, K2CO3, dioxane, water, 100 °C, 4 h,
78% (n = 0, R1 = H, R2 = H); PhB(OH)2, Pd2(dba)3, X-Phos, K2CO3,
dioxane, water, 100 °C, 4 h, 40% (n = 0, R1 = CH3, R2 = H); R2-
PhB(OH)2, X-Phos, K2CO3, dioxane, H2O, Pd2(dba)3, 100 °C, 3 h,
81% (n = 0, R1 = H, R2 = CH2OH); R2-PhB(OH)2, X-Phos, Na2CO3,
dioxane, H2O, Pd2(dba)3, 95 °C, 15 h, 52% (n = 0, R1 = H, R2 = CN);
(e) LiAlH4, THF, 2 h, rt, 53%.
a
Scheme 5. Synthesis of Imidazopyridazine 5
Thus, we initiated an effort to support our selectivity
hypothesis and to deliver selective tool molecules to enable
internal biological investigation and validation of Syk as a
worthy target for pharmacological intervention.
The crowded chemical space surrounding the imidazopyr-
azine screening hit 2 led us to investigate alternative hinge
binding cores from which we could expand our hit to lead
activities. Multiple heterocyclic alternatives were explored based
on their ability to mimic the same hinge binding interactions.
Ultimately, we focused on the thiazolopyrimidines, triazolopyr-
idines, and imidazopyridazines described herein (Figure 4). The
thiazolopyrimidines had the advantage that chemical manipu-
lation was more facile and that when A is phenyl, very few
literature or patent examples were uncovered. The triazolopyr-
idine Markush structure was novel, and to the best of our
knowledge, this remains an under-represented kinase binding
motif in the literature. Finally, the imidazopyridazine Markush
structure also appeared only sparsely in the patent literature at
that time, and we had in-house experience with it. The majority
of our SAR work focused on the thiazolopyrimidines that could
be easily substituted in the 6-position, which in our view offered
the best vector to reach and interact with both the Pro455 and
the Asn457 residues (vide infra).
a
Reagents and conditions: (a) 2-chloro- 1,1-diethoxyethane, PTSA, i-
PrOH, 80 °C, 20 h, 98%; (b) 5,6-dimethoxypyridin-2-amine, DMF,
NaH, rt, 15 h, 91%; (c) ethyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxabor-
olan-2-yl)benzoate, K3PO4, X-Phos, dioxane, water, Pd2(dba)3, 125 °C,
MW, 60 min, 34%; (d) LiOH, dioxane, H2O, rt, 4 h, 88%; (e) tert-
butyl 4-aminobenzoate, 1-methyl-1H-imidazole, EDCI, DMF, 16 h, rt,
55%; (f) CF3CO2H, CH2Cl2, rt, 24 h, 39%.
Pro455 and was expected to provide a good basis for
optimization.
D
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Figure 3. X-ray crystal structure of high throughput screening hit,
imidazopyrazine 2, bound to Syk (2.3 Å resolution, PDB accession
number 4FYN). The scaffold provides the appropriate vectors from
the core heterocycle to project toward the Pro455 and Asn457
residues (highlighted in orange).
Figure 4. Markush structures of thiazolopyrimidines, triazolopyridines,
and imidazopyridazines, substituted in the 6-position with generic
cyclic group A.
ways of extending downward and adjacent to the Pro455, as
well as reaching the Asn457. Indeed, hit expansion around our
initial HTS hit had shown that several related compounds,
where A was phenyl, showed submicromolar Syk inhibition. We
chose to carry out our initial exploration using dimethox-
yphenylaniline or 5,6-dimethoxy-2-aminopyridine functionality
at the left-hand side of the molecule (i.e., Y = OMe, n = 2,
Figure 4). The reason for this was twofold: First, the methoxy
functional group is fairly innocuous and was unlikely to
interfere with late stage chemical transformations, and second,
our own past experience as well as extensive Syk literature
(including R406) had taught us that this group would likely
offer good intrinsic Syk potency. We expected that later, once
potent, selective Syk inhibitors had been identified, we would
be able to manipulate these solvent exposed groups to modify
the biopharmaceutical properties of the scaffold, if needed.
On the basis of our continuous monitoring and analysis of
emerging imidazopyrazine and Syk literature, we identified a
number of potential R1 groups that might be of the correct
topology to interact with the Pro455 and the Asn457 amino
acid side chains.29 Ultimately, benzoylaminobenzoic acid
containing groups were identified that provided highly potent
inhibitors of Syk (3−5, Figure 6). When tested in our Ramos B-
cell assay, we were encouraged that these compounds exhibited
potencies commensurate with that of 1. Yet when the kinome
selectivity of 3 and 4 against a panel of 386 kinases was assessed
Figure 2. (a) Schematic of Syk ATP-binding site illustrating incidence
of individual residues in a total of 433 aligned kinases. Incidence key is
as follows: red, 1−21; yellow, 22−46; green, 50−92; blue, 93−433.
The figure is based on the crystal structure of Syk in complex with 1
(PDB accession number 3FQS, 1 is colored orange and shown as
sticks).26 The residues with lowest incidence, Met450, Leu453,
Pro455, and Asn457 are labeled. (b) Incidence of Met450, Leu453,
Pro455, and Asn457 residues in a total of 433 aligned kinases and
comparison to other residues. Yellow bars indicate aromatic residues.
Gray bars indicate hydrophobic residues. Blue indicates basic residues.
Red indicates acidic residues, and green represents polar residues.
The evolution of our Hit to lead effort is summarized in
Figure 5. As mentioned above, we had extensive experience
working with imidazopyrazine containing Btk inhibitor
scaffolds, and we expected that similar scaffolds, where cycle
A was a phenyl group, would offer opportunities to explore
E
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Figure 5. Evolution of Hit to lead.
Figure 6. Representative, potent examples of new Syk inhibitors.
(DiscoveRx ScanMax), it was found to be far superior to 1,
suggesting that the desired interaction between Pro455 and
Asn457 could be giving rise to enhanced selectivity (Figure 7,
panels a, b, c, e). We obtained an X-ray crystal structure that
confirmed the interactions (unpublished results). Encouraged,
we began to focus on assessing the biopharmaceutical
properties of the compounds. In general, the compounds
were stable in liver microsomes; however, the permeability of
these carboxylic acids was poor (data not shown). The kinetic
solubility for 3−5 was also poor, all showing LYSA solubility of
<1 (Table 1).
Since the ratio of sp3 to sp2 centers in a molecule is predictive
of druglikeness and developability,30 we addressed this in our
new Syk inhibitor scaffolds by replacing some of the sp2 centers,
anticipating that this would also improve the solubility.
Modeling highlighted the potential of pyrrolidines and
piperidines to replace the central phenyl while retaining a
conformation that could maintain simultaneous interactions
with Pro455 and Asn457. The preferred replacement was not
obvious from modeling, as we were unsure of the requirement
for any hydrogen bonding interactions between the linker
amide and the binding pocket. The thiazolopyrimidine series
was selected for further exploration because of its synthetic
tractability. Deterioration of potency occurred with all four
modifications, and yet 6 and 9 kept respectable potency (Table
2). The kinase selectivity was measured and was excellent. An
X-ray crystal structure of 9 bound to Syk was obtained and
confirmed that hydrophobic interactions between the two
phenyl groups of 9 with Pro455 and a polar interaction
between the carboxylic acid and Asn457 were occurring (Figure
8). This provided excellent support for our selectivity
hypothesis. We were pleased to find that in most cases (e.g.,
7−9, Table 2) our measured solubility also significantly
improved. However, the improved solubility did not translate
into improved cellular potency, suggesting that permeability,
not solubility, was the prevalent driver of the cellular readout.
Thus, we considered ways of improving the intrinsic
permeability of the molecules and focused in particular on
replacing the carboxylic acid functional group.
Figure 7. DiscoveRx kinase screening dendrograms at 10 μM
compound. The KINOMEscan screening platform employs an active
site-directed competition binding assay to quantitatively measure
interactions between test compounds and 386 kinase assays. Images
were generated using TREEspot software tool and reprinted with
permission from KINOMEscan, a division of DiscoveRx Corporation,
Copyright DISCOVERX CORPORATION 2010: (a) fostamatinib
parent R406; (b) thiazolopyrimidine 3; (c) triazolopyridine 4; (d)
imidazopyridazine 13; (e) legend showing circle size and its
correlation to the percent inhibition described for inhibition of
kinases represented in dendrograms.
Unfortunately, our SAR studies demonstrated that deleting
the carboxylate moiety led to significant loss of binding
potency. Without the critical ionic interaction to act as an
“anchor” it is likely that other binding conformations occur and
that the close interaction with Pro455 and the distal phenyl is
also lost. Simple alternative functional groups to replace the
carboxylic acid (e.g., a methylamide), as well as more complex
F
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Table 2. Inhibition of Syk Activity by Thiazolopyrimidines and Kinase Selectivity
a
b
See Supporting Information for details. Data shown are the average of at least n = 2. Ligand efficiency (in energy units/atom ≈ (kcal/mol)/atom).
c
d
e
See Supporting Information for details. Lyophilized solubility assay data. DiscoveRx KINOMEscan screening platform measurements employing
an active site-directed competition binding assay to quantitatively measure interactions between test compounds and 386 kinase assays.
heterocyclic bioisosteres of carboxylic acids that covered a
calculated pKa range of 4−9 (e.g., 4-methyl-4H-[1,2,4]triazole-
3-thiol, [1,3,4]oxadiazole-2-thiol, 1,2,4-oxadiazol-5-one), were
investigated but generally led to some deterioration in potency
(data not shown). The doggedly low ligand efficiency, high
molecular weight, and restricted opportunities to replace the
carboxylic acid prompted us to investigate whether disregarding
the Asn457 and focusing instead on optimizing interactions
with only the Pro455 residue could still provide Syk selective
compounds. Reducing the molecular weight and addressing the
poor permeability that seemed to accompany the carboxylate
moiety were also expected to improve the biopharmaceutical
properties of the scaffolds. We also chose to return to structures
containing a phenyl linker, despite the high sp2 ratio, since
these had provided our most cell potent examples. Pro455 was
most readily accessed from vectors extending from the left-
hand side 2-aminopyridine or aniline moiety. We excluded the
thiazolopyrimidine core from this exercise, as it did not offer
the ability to lock the conformation of the aniline ring and may
have led to ambiguous data as we sought to project groups over
Figure 8. X-ray cocrystal structure of thiazolopyrimidine 9 (depicted
in green) bound to Syk (1.4 Å resolution, PDB accession number
4FYO). The Pro455 and Asn457 are highlighted in red.
the proline ring. In contrast, the triazolopyridine and
imidazopyridazine series, each possessing 2-aminopyridine
front groups, were conformationally rigid and thus more
suitable. We ultimately selected the imidazopyridazine core for
G
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Table 3. Inhibition of Syk Activity by Imidazopyridazine Analogues 10−14
a
b
See Supporting Information for details. Data shown are the average of at least n = 2. Ligand efficiency (in energy units/atom ≈ (kcal/mol)/atom).
c
d
e
See Supporting Information for details. Lyophilized solubility assay data. DiscoveRx KINOMEscan screening platform measurements employing
an active site-directed competition binding assay to quantitatively measure interactions between test compounds and 386 kinase assays.
this investigation, since it represented the most ligand efficient
starting point.
interactions and result in improved potency, thus enabling a
better comparison of kinome selectivity profiles. Among the
compounds investigated, substitution on the phenyl linker
provided some of our most promising compounds. For
example, benzyl alcohol 13 and benzylamine 14 retained
good ligand efficiencies (Table 3). Benzyl alcohol 13 was highly
selective (Figure 7d and Table 3). The X-ray structure was
solved for benzylamine 14 and confirmed that the proline
remained in van der Waals contact with the pyrrolidine ring
(not shown). This provided excellent evidence that optimiza-
tion of hydrophobic interactions with the Pro455 alone is
sufficient to achieve highly selective Syk inhibition. All crystal
structures solved with a methylpyrrolidine showed a single
enantiomer bound, with the chiral methyl group pushing up
and interacting with the glycine loop. This was somewhat
unexpected, and we cannot rule out that this could also be
contributing to the good selectivity profile. Indeed, inhibitors of
other kinases such as Pfizer’s Jak3 inhibitor tofacitinib gain
selectivity by making a Gly loop interaction, albeit in this case
through hydrophilic substructure interactions rather than the
hydrophobic interaction reported here.31 Thus, we were able to
We prioritized pyridine substituents that appeared to interact
well with Pro455 during computational modeling. Among
these, pyridines substituted in the 6-position with N-linked
pyrrolidine and piperidine emerged as particularly promising
groups to target the proline aliphatic ring (Table 3, 10−12).
Substituents on the pyrrolidine ring were chosen for their
potential to “surround” the proline residue and further optimize
van der Waals interactions. The crystal structure of 12 bound to
the Syk protein was solved and indicated a close contact
between the 2-methylpyrrolidine and the Pro455 side chain
(Figure 9). However, enzyme potency of 10−12 was
suboptimal, and despite the significant reduction in molecular
weight, measured solubility remained poor, likely because of the
high clogP for these compounds.
Follow-up compounds were prioritized that were expected to
reduce the clogP of the molecules and thus improve their
biopharmaceutical properties. We hoped that in addition to
improving physicochemical properties, the polar functionality
might introduce additional opportunities to form polar
H
dx.doi.org/10.1021/jm301367c | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
∥Principia BioPharma, Inc., 400 E. Jamie Court, No. 302, South
San Francisco, CA 94080, U.S.
⊥School of Pharmaceutical Sciences, Soochow University, 199
Ren Ai Road, Suzhou, Jiangsu, P. R. China.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Joshua Kennedy-Smith for careful proof-reading of
this manuscript.
■
ABBREVIATIONS USED
■
BCR, B-cell receptor; Btk, Bruton’s tyrosine kinase; DIPEA,
d i i s o p r o p y l e t h y l a m i n e ; E D C I , 1 - e t h y l - 3 - ( 3 -
dimethylaminopropyl)carbodiimide; HATU, (2-(7-aza-1H-ben-
zotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophos-
phate; Jak, Janus kinase; MeOH, methanol; MLK1, mixed-
lineage kinase 1; MW, microwave; Pd2(dba)3, tris-
(dibenzylideneacetone) dipalladium(0); PLCγ, phospholipase
Cγ; PTSA, p-toluenesulfonic acid; RET, rearranged during
transfection tyrosine kinase; siRNA, small inhibitory ribonucleic
acid; SLE, systemic lupus erythematous; SLP, SH2 domain
containing leukocyte protein; Syk, spleen tyrosine kinase;
VEGFR2, vascular endothelial growth factor receptor 2; X-
Phos, 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl
Figure 9. Crystal structure of 12 bound to Syk (1.85 Å resolution,
PDB accession number 4FZ6). The hydrophobic interaction between
the pyrrolidine ring and Pro455 (surface shaded orange) is apparent.
Note that only a single enantiomer was observed in the crystal
structure.
identify molecules that allowed us to drastically reduce the
molecular weight and obtain cell potent Syk selective inhibitors.
However, despite respectable cell potency, we were unable to
achieve suitable combinations of permeability and solubility,
and ultimately the pharmacokinetics of these compounds did
not allow full in vivo characterization.
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■
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ASSOCIATED CONTENT
* Supporting Information
■
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AUTHOR INFORMATION
Corresponding Author
*Phone: (408) 585-8051. E-mail: matthew.lucas@cubist.com.
■
́ ́
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