Using ovality to predict nonmutagenic, orally efficacious pyridazine amides as cell specific spleen tyrosine kinase inhibitors

Article abstract of DOI:10.1021/jm401982j

Inhibition of spleen tyrosine kinase has attracted much attention as a mechanism for the treatment of cancers and autoimmune diseases such as asthma, rheumatoid arthritis, and systemic lupus erythematous. We report the structure-guided optimization of pyridazine amide spleen tyrosine kinase inhibitors. Early representatives of this scaffold were highly potent and selective but mutagenic in an Ames assay. An approach that led to the successful identification of nonmutagenic examples, as well as further optimization to compounds with reduced cardiovascular liabilities is described. Select pharmacokinetic and in vivo efficacy data are presented.

Full text of DOI:10.1021/jm401982j

Article
pubs.acs.org/jmc
Using Ovality to Predict Nonmutagenic, Orally Efficacious Pyridazine
Amides as Cell Specific Spleen Tyrosine Kinase Inhibitors
Matthew C. Lucas,*,†,§ Niala Bhagirath,† Eric Chiao,† David M. Goldstein,† Johannes C. Hermann,†
Pei-Yuan Hsu,† Stephan Kirchner,‡ Joshua J. Kennedy-Smith,† Andreas Kuglstatter,† Christine Lukacs,†
John Menke,† Linghao Niu,† Fernando Padilla,† Ying Peng,† Liudmila Polonchuk,‡ Aruna Railkar,†
Michelle Slade,† Michael Soth,† Daigen Xu,† Preeti Yadava,† Calvin Yee,† Mingyan Zhou,†
and Cheng Liao†
†Hoffmann-La Roche Inc., pRED, Pharma Research & Early Development, Small Molecule Research, 340 Kingsland Street, Nutley,
New Jersey 07110, United States
‡Hoffmann-La Roche Inc., pRED, Pharma Research & Early Development, Small Molecule Research, Grenzacherstrasse 124,
4070 Basle, Switzerland
S
* Supporting Information
ABSTRACT: Inhibition of spleen tyrosine kinase has attracted much attention as a
mechanism for the treatment of cancers and autoimmune diseases such as asthma,
rheumatoid arthritis, and systemic lupus erythematous. We report the structure-
guided optimization of pyridazine amide spleen tyrosine kinase inhibitors. Early
representatives of this scaffold were highly potent and selective but mutagenic in an
Ames assay. An approach that led to the successful identification of nonmutagenic
examples, as well as further optimization to compounds with reduced cardiovascular
liabilities is described. Select pharmacokinetic and in vivo efficacy data are presented.
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 it is
involved in bone resorption by osteoclasts.2−4 The activation of
Syk leads to a variety of downstream events including cytokine
release, differentiation, proliferation, reactive oxygen species
(ROS) production, cytoskeletal rearrangement, and cell
survival. Fostamatinib (1), a poorly selective Syk inhibitor
(vide infra), recently completed phase II trials for the treatment
Figure 1. Structures of Fostamatinib (1), PRT-062607 (2), and GSK-
143 (3).
of rheumatoid arthritis but subsequently failed in phase III.5
PRT-062607 (2)6 a somewhat more selective diaminopyr-
imidine derivative, recently completed phase I clinical trials and
Malignancies listed as a potential indication. It is being developed
both as a single agent as well as in combination with Idelalisib,
a PI3K delta inhibitor.7 GSK-143 (3) is reported to be a highly
is currently in phase II for allergic asthma and other inflammatory
disorders. Trials in Lupus as well as various oncology indications
are also planned or likely (Figure 1).6 The entry of GS-9973 into
phase I for the treatment for rheumatoid arthritis was reported,
although this indication is no longer included on the company
pipeline slide. The drug is currently in phase II with Hematological
Received: December 25, 2013
Published: February 12, 2014
© 2014 American Chemical Society
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Figure 2. Structures of Roche selective Syk inhibitors 4−9.
selective and potent Syk inhibitor and belongs to the same
pyrimidine scaffold as 2, although this compound did not advance
to the clinic due to mutagenicity.8
such as 2 bind in the Syk pocket with the aniline moiety
positioned above Gly454/Pro455 and that the diamino groups
occupy the ribose pocket of the ATP binding site. It is
reasonable to expect that 2 and 3 form similar interactions with
the protein. High levels of selectivity have been claimed for
these compounds. Our experience building Syk selectivity into
other chemical scaffolds (Figure 2) led us to contemplate that,
if the interaction with Pro455 could be enhanced further,
selectivity might also be superior. We felt that selective Syk
inhibitors would provide critical differentiation from the
competition by reducing the risk of off-target toxicities that
have been observed, for example, with 1 and any that emerge
during the phase II clinical trial of 2. We designed a pyridazine
carboxamide scaffold (13, Scheme 1) in which intramolecular
hydrogen bonding between the pyridazine core and a 2-
aminopyridine would favor one conformation and facilitate
exploration and optimization of hydrophobic interactions with
Pro455. An added advantage, we hoped, was that that we would
have also replaced a potentially problematic aniline group in
pyrimidines 1−3 with a safer 2-aminopyridine fragment.17
No pyridazines of this type were known in the literature, but
fortunately, we were able to synthesize our compounds based
on a three to four step procedure starting from commercially
available pyridazine 10 (Scheme 1). Regioselective nucleophilic
displacement of chlorine with the appropriate 2-aminopyridine
under thermal conditions gave 11. Simply stirring the resulting
ester in methanolic ammonia provided primary amide 12. A
second nucleophilic displacement with an alkylamine followed by
a Boc deprotection then gives the final product 13 (in some cases
where a protecting group was not present, the final product was
obtained directly from the nucleophilic displacement).
Compound 1 is not selective for Syk versus other kinase
enzymes.9 It has been reported that the off-target inhibition of
VEGFR2 may have been responsible for the hypertension
observed in patients receiving the drug in clinical trials.10,11
Portola and GlaxoSmithKline report that 2 and 3 each show
improved selectivity and have presented some data that support
these statements.6,8 Previously, we described a hypothesis
describing how highly selective Syk inhibitors can be designed
by targeting specific residues in and around the Syk active site that
appear only rarely among other kinases (Pro455 and Asn457, or
Pro455 alone).12 We have since disclosed additional scaffolds that
lend support to this hypothesis (Figure 2, 4−8).13,14
One of the challenges encountered in our earlier efforts was
pairing exceptionally high selectivity with good pharmaco-
kinetic (PK) properties. Herein, we describe the structure−
activity relationship (SAR) of a series of diaminopyridazine
amides (e.g., 8−9, Figure 2) that combine a binding interaction
with Pro455 to deliver the requisite selectivity, with exceptional
biopharmaceutical properties to convey high in vivo efficacy in
a mouse model of rheumatoid arthritis. To the best of our
knowledge, these represent the most selective and potent Syk
inhibitors reported to date. As also reported by the GSK group
for 3, we observed mutagenicity in an expanded Salmonella
mutagenicity test panel that included the Ames TA1537
salmonella strain.15 Mutagenicity was unexpected based on in
silico predictions. We provide our hypothesis for the cause of
the observed mutagenicity and how this led us to identify
nonmutagenic molecules. The ensuing optimization process
and delivery of candidate molecules possessing high potency,
high selectivity, and acceptable safety windows will be illustrated.
Our prototype compound 9 (Figure 2, Table 1) was a potent
Syk inhibitor and selective in a small panel of 30 kinases that
sampled all of the kinase families.18 When tested at 1 μM, only
Syk was inhibited greater than 90% (i.e., 1/30 kinases greater
than 90% at 1 μM). We are satisfied that this small 30 kinase
panel is an accurate predictor of selectivity based on results of
DISCUSSION
■
We have previously reported the crystal structures of several
inhibitors bound to Syk.16 We determined that compounds
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a
data as this represented the smallest substituent expected to
interact with Pro455, and it compared very favorably to our
benchmark Syk inhibitors. We used Fostamatinib (1) and
PRT-062607 (2) as our benchmarks, since these were the most
advanced Syk inhibitors currently progressing in clinical trials.
Compound 1 inhibited 6/30 kinases greater than 90% at 1 μM
and had a cell-based selectivity ratio of 0.5. Compound 2 was
somewhat more selective and inhibited 4/30 kinases greater
than 90% at 1 μM and had a cell-based selectivity ratio of 0.8.
We anticipated some potential hERG binding due to the
basic amine, but we hoped that the low clogP and high PSA
would reduce the propensity to bind and provide sufficient
therapeutic windows. Our goal was to achieve a greater than
30-fold window between the human whole blood IC50 and the
hERG IC50 corrected for protein binding. Compound 9 easily
met this criterion, displaying a 126-fold window. Thus, while it
remained important that we monitored hERG closely, it did not
prohibit us from examining the series further.
Scheme 1. General Synthesis of Pyridazines 13
Further profiling of 9 showed promising biopharmaceutical
properties; it was soluble, and despite a low predicted
permeability and high polar surface area, it was highly permeable
when measured in a CACO2 permeability assay and potent in
our cell assay (Tables 1−2). We rationalized that the good
permeability was due to the effective masking of some of the
donor and acceptor atoms through intramolecular hydrogen-
bond formation.19 The in vitro profile translated into good rat in
vivo PK (unpublished results). This was of critical importance to
us, since achieving suitable PK profiles had been a challenge in
some of our earlier scaffolds.12−14
We quickly expanded our SAR exploration and soon
obtained an X-ray crystal structure of 14 bound to the Syk
protein that confirmed the expected binding mode. The 6-ethyl
group of the pyridine projects out over the Pro455, and the cis-
cyclohexyldiamino moiety forms a hydrogen bond via its
secondary amine and a salt bridge via its primary amine to the
carboxy side chain of Asn512 (Figure 3). For our initial SAR
a
Reagents and conditions. (a) 2-aminopyridine, acetonitrile, 80 °C;
(b) 7 N ammonia in methanol, rt; (c) R3-NH2, NMP, 140 °C.
compounds tested previously in a larger, traditional kinase
binding assay panel and used by us previously.12−14 For
example, 19 inhibited 2/30 kinases greater than 90% in our
small panel (Syk and PDGFRβ) and 6/392 kinases greater than
90% in a larger panel. As well as assessing the biochemical
selectivity of our Syk inhibitors in the 30 kinase panel, we
determined a cell-based selectivity ratio. This was based on the
on-target effect of inhibitors of α-IgM antibody-mediated
intracellular calcium increase in human Ramos B cells versus
the off-target effect of T cell receptor induced calcium influx in
human Jurkat T cells. We set ourselves a goal of achieving good
biochemical selectivity as well as at least a 10-fold selectivity
ratio in the cell-based assays. The cell-based selectivity ratio for
9 was 9-fold. We were very encouraged by this intitial selectivity
Table 1. Inhibition of Syk Activity, Selectivity, Human Whole Blood Potency, and hERG Channel Inhibition by Pyridazine
Amides 9 and 14−23
b
Syk
pIC50
selectivity (no. kinase inhibited
T cell/B cell selectivity
HWB
d
hERG
e
a
c
compd
R1
Me
R2
R3
>90% at 1 μM)
ratio
pIC50
pIC50
9
H
H
H
H
H
F
H
H
H
H
H
H
H
H
Me
H
H
8.0
8.5
8.3
8.6
8.4
7.6
8.3
7.8
8.3
8.0
8.4
1
9
7.1
7.1
6.4
6.7
6.5
6.4
7.5
6.8
7.2
6.6
7.5
5.0
5.2
5.2
5.4
5.1
4.8
4.7
5.4
−
14
15
16
17
18
19
20
21
22
23
Et
−
4
3
nPr
cPr
3
3
2
iPr
1
4
Me
1
15
9
Me
Me
2
Me
Cl
2
14
1
Me
H
−
1
−CH2−
OMe
−CH2−
OMe
10
14
5.1
7
5.3
a
b
See Experimental Section (Supporting Information) for details. Carna Bioscience Inc. screening platform measurements employing an enzyme
c
binding assay to quantitatively measure interactions between test compounds at 1 μM and a panel of 30 kinases. Ratio of Jurkat T cell IC50 divided
by Ramos B cell IC50. Human whole blood pIC50. hERG channel pIC50.
d
e
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a
b
c
d
efg
Table 2. Solubility , Permeability , Human Plasma Protein Binding , CYP450 Inhibition , and Liver Microsomal Stability
for Pyridazine Amides 9, 18−20, 22, and 23
a
b
c
d
e
f
g
LYSA
CACO2 AB
(ER)
PPB
CYP450 IC50
HLM
RLM
MLM
compd
(μg/mL)
(% free)
(μM)
(mL/(min·kg))
(mL/(min·kg))
(mL/(min·kg))
9
471
390
329
345
404
522
7.5
9.6
−
15.4
>50
2
3
1
7
2
2
27
18
23
20
27
−
18
19
20
22
23
8.9
>50
20
47
22
−
1.8 (7)
3.9 (4)
2.7 (6)
>50
−
−
16 (2D6)
>50
0.5 (23)
33 (2D6)
14
−
a
b
c
Lyophilized solubility assay. Apical to basolateral CACO2 permeability, followed by measured efflux ratio. Human plasma protein binding % free
fraction. CYP450 inhibition at isoforms 3A4, 2C9, and 2D6. Lowest inhibition value indicated. Stability in human liver microsomes. Stability in rat
liver microsomes. Stability in mouse liver microsomes.
d
e
f
g
that the potency is driven primarily through the Syk activity: In
our 30 kinase panel, we included several kinases involved in
the B-cell signaling pathway that were not strongly inhibited
(e.g., Akt, Lyn, Btk, Erk). The increase in clogP accompanied
by the increased size also led to an increase in hERG channel
inhibition, and a slight loss of kinome selectivity, which is in line
with general reports that lipophilicity is often a driver of off-target
activity.20 We next investigated maintaining the 6-methyl substituent
and the effect of additional substitution on the pyridine ring. At the
5-position, fluorine (18), methyl (19), and chlorine (20) all
maintained similar potency in the enzyme and HWB assays, with
18 and 20 also improving cell-based selectivity. Substitution at the
4-position was generally detrimental to selectivity (21). This is
noteworthy as it suggests an advantage of our series. We believe that
the pyridine nitrogen strongly favors the conformation that projects
the R1 substituent over the Pro455, improving selectivity over the
kinome. If other conformations were sampled, as is likely the case
for 2 and 3, then the potential to bind to additional kinases is
increased. The lower selectivity of pseudosymmetric 4,6-
dimethylpyridine compound 21 supports this.
Figure 3. Structure of 14 bound to Syk.
Comfortable that we were able to achieve sufficient hERG
safety windows, cell selectivity, and good human whole blood
exploration, we kept the cis-cyclohexyldiamino portion constant
so that we could focus on optimizing selectivity and potency by
modifying the 2-aminopyridine portion of the scaffold.
potency, we profiled several molecules from the series in
biopharmaceutical and early safety assays. No significant
CYP450 inhibition or time-dependent inhibition was detected.
No GSH adducts were detected, and the compounds generally
had high to moderate stability in human and rat liver micro-
somes. Microsomal clearance correlated well with hepatocyte
clearance (unpublished results). With no mutagenicity or
carcinogenicity in silico alerts, the prospects for the series
seemed excellent. We obtained mouse PK on several
compounds with a good balance of cell selectivity and safety
profiles (Table 3) in order to assess the suitability of the
Highlights of our initial SAR study of substitution on the
2-aminopyridine moiety are shown in Table 1. As we increased
the size of the 6-alkyl substituent, we observed an increase in
potency. However, this did not translate into improved human
whole blood (HWB) activity. Since the HWB assay is not a
target-based assay but a functional assay, we could not
completely rule out other off-target activities imparting a
component of the observed activities. However, we do believe
a
Table 3. Pharmacokinetic Parameters after a Single Dosing in Mouse for Compounds 18−20
1
compd
species
CLiv (mL/(min·kg))
Vdss (L/kg)
t / (h)
Cmax (ng/mL)
PO dose (mg/kg)
AUC/dose ((ng/(mL·h))/(mg/kg))
F (%)
2
18
19
20
mouse
mouse
mouse
5.57
NA
1.44
NA
3.26
NA
1560
−
386
30
5
568
25.9
970
753
20
ND
51
8.65
2.74
3.92
5
1440
30
40
a
n = 2−3 per time point. IV dose = 0.5 mg/kg; PO dose = 5 or 30 mg/kg. NA = not available. ND = not determined. Blood samples were collected
from each animal at selected times postdose for the determination of compound levels in plasma. The samples were collected into tubes containing
potassium EDTA, as an anticoagulant and stabilizer, and were processed to plasma and kept in a freezer set to maintain −80 °C. The
pharmacokinetic parameters were estimated using the individual plasma concentration data for rat or pooled mean plasma concentration data for
mouse. Nominal sampling times were reported and were used for kinetic calculations. Concentrations below the limit of quantitation (BLQ) were
1
not used. The extrapolated area under the plasma concentration−time curve from zero to infinity (AUC), apparent terminal half-life (t / ), the total
2
plasma clearance (CL), the volume of distribution at steady state (Vdss), F%, bioavailability after a single oral dosing relative to a single IV dosing
corrected by doses, and dose normalized AUC (AUC/dose).
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scaffold for assessment in the mouse collagen induced arthritis
efficacy model of rheumatoid arthritis and further assess the
potential of the scaffold. 5-F-6-Me pyridine (18) and 5-Cl-6-Me
pyridine (20) both show a good PK profile. 5,6-dimethyl (19)
showed more moderate PK but showed good enzyme and cell-
based selectivity, was exceptionally potent in human whole
blood, and also displayed the largest hERG window of the
compounds that we had synthesized at that point. Thus, 19 was
selected to investigate as a representative to probe the in vivo
mCIA efficacy study (See the Experimental Section, Supporting
Information, for details). When tested in a standard mouse
model of collagen-induced arthritis, 19 displayed excellent
levels of efficacy, inhibiting arthritis scores in a dose-dependent
manner at the dose range 5−45 mg/kg by as much as 64%
(45 mg/kg, once a day), an efficacy that is comparable to that of
R788 (58%, 30 mg/kg, twice a day). That the compound
displayed comparable efficacy to R788, a nonselective Syk
inhibitor that has shown to be efficacious in patients with
rheumatoid arthritis, suggested to us that a selective Syk
inhibitor such as 19 may bring the same efficacy benefit to
patients as R788, while reducing potential for off-target toxicity
(Figure 5).
did not have an alert either. We promptly tested representatives
of our pyridazine series (9 and 14) in the Ames assay, and while
no signal was observed in our standard panel of five salmonella
strains (unpublished results), when we tested the compounds
in TA1537, a clear signal was observed, both in the presence
and absence of liver S9 fraction.
Every compound that we subsequently tested showed
mutagenicity in the TA1537 strain, including those in Table
1. To account for this, we immediately began further syntheses
to develop SAR and find nonmutagenic examples (Table 4).
Replacing the cis-diaminocyclohexyl moiety with ethylene
diamine (24) retained potency but did not eliminate the
mutagenicity flag. In contrast, the 2-aminoethyl ether (25),
simple cyclohexylamine (28), and ether linked phenyl (27)
were all clearly negative in Ames. Replacement of the 2-amino-
4,6-dimethyl-pyridine with 3,5-dimethylaniline provided non-
mutagenic (26). Unfortunately, these nonmutagenic com-
pounds showed diminished binding to the Syk protein (Table 4).
We also found it hard to rationalize the significant impact that these
apparently subtle changes had on the mutagenicity result, making it
challenging to create a design strategy.
The Ames assay is essentially binary, and even “weak”
mutagens are prohibited from entering clinical trials. However,
close inspection of the data (unpublished results) showed that
the number of reverting bacterial colonies trended downward as
the steric size of the 6-substituents on the pyridine ring
increased. Since mutagenicity was observed both in the presence
and in the absence of metabolic activation, we hypothesized that
it was the shape of the molecule that was driving mutagenicity.
We had designed the pyridazine scaffold with internal hydrogen
bonds to control the conformation and allow us to optimize
hydrophobic interactions with Pro455. A consequence of this
was also to lock the majority of the molecules into flat, pseudo-
polyaromatic fused ring systems, somewhat resembling the
anthraquinones, which are known DNA intercalators and
mutagens (Figure 4).21
Figure 4. Flat molecules are known to be DNA intercalators.
We speculated that the flat topology might promote dsDNA
intercalation and that subsequent frame shift mutations were
the cause of the observed Ames mutagenicity. Therefore, we
sought to incrementally increase the ovality (a computed
measure of thickness) of the molecules until the fit into the
DNA major groove was poor enough to switch off the
mutagenicity. We hoped that we would be able to retain the
potency and other good biopharmaceutical properties that had
made the scaffold so desirable at the outset.22
Compounds that exhibited greater “ovality” and were
nonmutagenic in the Ames assay were quickly identified: tert-
butyl derivative 29 and 5-methyl-6-isopropyl compound 31.
These provided early support for the DNA intercalation
hypothesis and suggested that the scaffold still offered great
potential to deliver clinical candidates. Next, compounds were
designed to better define the appropriate target ovality.
Retrospectively, we found that the ovality of the largest fragment
(subtracting the diamine right-hand piece) best predicted the
Ames mutagenicity result. We were able to use this predictive
model to prioritize our synthetic targets (Figure 6). To trigger
synthesis of our designed targets, we required that the predicted
ovality score was greater than 1.44.
Figure 5. Clinical scores from the assessment of 19 in the mouse
collagen-induced arthritis model. Collagen-induced arthritis was
induced in young adult male DBA1/J mice (7−8 weeks of age) as
described previously by immunizing them twice with bovine type II
collagen.24 Test compound was administered orally once a day for 14
days starting the day after the second immunization. Arthritis was
monitored by observing redness and swelling of the paws or digits and
given a clinical score based on a scale of 0−4 per paw with 4 being the
most severe.
While this work was in progress, we encountered a report
from GlaxoSmithKline in which they presented data for 3, a
member of the diaminopyrimidine scaffold and closely related
to Portola clinical candidate 2. We were surprised to learn that
3 showed mutagenicity in salmonella test strain TA1537. While
our series had not shown any mutagenicity flag in silico, we
noted that the in silico prediction for the pyrimidine 3 scaffold
We became confident in our ability to design compounds
that were nonmutagenic by increasing the ovality of the
molecules, and while not perfect, we were able to identify
multiple structures that were no longer mutagenic (∼67%
success rate). However, the increased ovality of the first
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a
b
c
Table 4. Inhibition of Syk Activity , Human Whole Blood Potency , and Ames Mutagenicity Results for Pyridazine Amides
24−28
a
b
c
See the Experimental Section (Supporting Information) for details; data shown is the average of at least n = 2. Human whole blood pIC50. Ames
mutagenicity assay category, in the presence (+S9) and absence (−S9) of liver S9 fraction.
Figure 6. Average ovality and relationship to mutagenicity. Ovality was measured based on the fragment shown. 74% of compounds mutagenic if
ovality <1.44, while 67% of compounds nonmutagenic or inconclusive had ovality >1.44.
nonmutagenic compounds led to encroachment on suboptimal
chemical property space,23 where the risk of off-target effects and
attrition rates increase. Not only was the measured HWB
potency negatively impacted, but the increased lipophilicity also
reduced enzyme panel and cell selectivity and often reduced the
therapeutic window between hERG binding and HWB potency
(e.g., 29−33, Tables 5 and 6).
Mining of the hERG data for the series suggested that we
needed to target a clogP of less than three in order to improve
our chances of achieving a sufficient therapeutic window over
hERG. Thus, we defined a hypothetical “safe zone” and used
this as a gate when designing new compounds (Figure 7).
Compounds were prioritized if they were predicted to be
nonmutagenic based on their thickness (ovality) and expected
to be poor hERG channel inhibitors with improved human
whole blood potency based on their lipophilicity (clogP). Any
molecule that fell outside of this zone was deprioritized or
refined further to shift it closer to the desired parameters.
Among the modifications that were able to reduce the clogP,
the 1,2-diaminoethyl and the 3,4-diaminotetrahydropyran
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a
b
,c
d
e
Table 5. Inhibition of Syk Activity , Selectivity , Human Whole Blood Potency , and hERG Channel Inhibition by
Pyridazine Amides 29−39
a
b
See the Experimental Section (Supporting Information) for details; data shown is the average of at least n = 2. Carna Bioscience Inc. screening
platform measurements employing an enzyme binding assay to quantitatively measure interactions between test compounds at 1 μM and a panel of
c
d
e
30 kinases. Ratio of Jurkat T cell IC50 divided by Ramos B cell IC50. Human whole blood pIC50. hERG channel pIC50.
groups generally maintained good potency and imparted a beneficial
effect on hERG windows relative to the 1,2-diaminocyclohexyl
moiety. This is likely due to the reduction in clogP in both
cases as well as a reduction in the pKa in the case of the
3,4-diaminotetrahydropyran group. Earlier SAR based on
matched pairs had identified that substitution in the 5-position
of the 2-aminopyridyl group, particularly with a small
hydrophobe such as fluorine or an alkyl (e.g., 18 vs 9 and 19
vs 9), also tended to reduce hERG binding. SAR also showed
methoxy substituents on the 2-aminopyridine tended to reduce
the clogP as well as improve potency. Thus, we concentrated
on generating targets that married these components together
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a
b
c
d
e,f,g
Table 6. Solubility , Permeability , Human Plasma Protein Binding , CYP450 Inhibition , and Liver Microsomal Stability
for Pyridazine Amides
a
b
c
d
e
f
g
compd LYSA (μg/mL) CACO2 AB (ER) PPB (% free) CYP450 IC50 (μM) HLM (mL/(min·kg)) RLM (mL/(min·kg)) MLM (mL/(min·kg))
29
31
32
33
34
35
36
37
38
39
36
9.3 (2)
5.1 (0.4)
3.1 (2)
4.0 (3)
7.5 (2)
3.8 (3)
1.3 (12)
0.8 (9)
7.4 (2)
1.0 (9)
1.6
25(2D6)
39 (2D6)
18 (2D6)
8 (3A4)
>50
4
11
8
17
35
5
−
−
83
35
2.2
134
106
1284
5
5.7
4.1
9
−
−
11.1
14.4
18.8
16.1
−
6
23
23
16
13
23
19
69
40
31
26
27
−
>50
3
16
>50
1
168
159
5
45 (2D6)
38 (2D6)
34 (2D6)
5
7
10.5
6
a
b
c
Lyophilized solubility assay. Apical to basolateral CACO2 permeability, followed by measured efflux ratio. Human plasma protein binding % free
fraction. CYP450 inhibition at isoforms 3A4, 2C9, and 2D6. Lowest inhibition value indicated. Stability in human liver microsomes. Stability in rat
liver microsomes. Stability in mouse liver microsomes.
d
e
f
g
a
Table 7. Pharmacokinetic Parameters after a Single Dosing in Mouse for Compounds 34, 36, and 37
1
compd
species
CLiv (mL/(min·kg))
t / (h)
Vdss (L/kg)
AUC/dose ((ng/(mL·h))/(mg/kg))
F (%)
2
34
36
37
Mouse
Mouse
Mouse
42.2
16.7
8.52
1.96
1.74
1.28
3.67
1.40
0.63
365
609
92
61
75
1470
a
n = 2−3 per time point. IV dose = 1 mg/kg; PO dose = 10 or 20 mg/kg.
SUMMARY
■
We have identified a highly efficacious and Syk selective scaffold
to enable further investigation of the merits of Syk inhibition as
a target to treat various diseases. To the best of our knowledge,
these molecules are superior to all Syk inhibitors that have been
published to date. On the basis of hypothesis and careful
prioritization, we identified several compounds that are
nonmutagenic and maintain excellent druglike properties and
whole blood potencies. Compounds 34, 36, and 37 are
noteworthy for their combination of human whole blood
potency, kinome selectivity, good therapeutic window over
hERG, and low risk of mutagenicity or carcinogenicity. They
represent excellent pharmacological tools for the further
investigation of selective Syk inhibitors in disease mitigation.
The fate of these molecules will be the subject of future
communications.
Figure 7. Plot of ovality, clogP, and Ames mutagenicity result (No =
not mutagenic in any of the five strains tested; Yes = mutagenic in at
least one of the five strains tested; Inconclusive = number of reverting
colonies just exceeded threshold of 2-fold increase over control).
Properties describing a hypothetical “safe zone” is depicted as a green
oval.
ASSOCIATED CONTENT
* Supporting Information
in an effort to find nonmutagenic compounds with a good
balance of in vitro properties.
■
S
In this regard, we identified a number of compounds that
offered an excellent balance of in vitro criteria (Tables 5 and 6,
34−39). This selection of top compounds was refined further by
PK profiling, and from this refinement, 34, 36, and 37 emerged as
excellent candidates (Table 7). All three showed very promising
PK profiles in mice, with high oral bioavailability and AUC values.
All three met our strict in vitro and in vivo requirements, and as
such, the compounds were difficult to segregate further.
Compound 34 was less selective in our cell-based assay but had
the advantage that it showed the best solubility and permeability.
Compounds 36 and 37 were essentially indistinguishable. To
further derisk the series, 37 was subjected to further safety
screening. We were pleased to find that this compound (in line
with expectations based on results of other scaffold representa-
tives, data not shown) was not carcinogenic in the MNT assay
(unpublished results) and as such confirmed for us that all three
remained excellent candidates and deserved further investigation
in vivo for potential progression toward the clinic.
Experimental procedures for synthesis of final compounds and
intermediates, and descriptions of assays performed. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +1-408-585-8051; e-mail: matthew.lucas@cubist.com.
■
Present Address
§Matthew Lucas: Cubist Pharmaceuticals, 65 Hayden Avenue,
Lexington, Massachusetts 02421, United States.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Karen Lackey, Satwant Narula, and Julie DeMartino
for their leadership and critical strategic discussions.
■
2690
dx.doi.org/10.1021/jm401982j | J. Med. Chem. 2014, 57, 2683−2691

Journal of Medicinal Chemistry
Article
has significant clinical activity in non-Hodgkin lymphoma and chronic
lymphocytic leukemia. Blood 2010, 115, 2578−2585.
ABBREVIATIONS USED
■
Syk, spleen tyrosine kinase; SLE, systemic lupus erythematous;
ATP, adenosine triphosphate; PLCγ, phospholipase Cγ; PI3K,
phosphoinositide 3-kinase; SLP, SH2 domain containing leukocyte
protein; ROS, reactive oxygen species; siRNA, small inhibitory
ribonucleic acid; BCR, B-cell receptor; VEGFR2, vascular
endothelial growth factor receptor 2; RET, rearranged during
transfection tyrosine kinase; MLK1, mixed-lineage kinase 1; Met,
methionine; Leu, leucine; Btk, Bruton’s tyrosine kinase; Jak, Janus
kinase; DIPEA, diisopropylethylamine; DMSO, dimthylsulfoxide; rt,
room temperature; THF, tetrahydrofuran; DMAP, 4-dimethylami-
nopyridine; DMF, dimethylformamide; EDCI, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide; X-Phos, 2-dicyclohexylphos-
phino-2′,4′,6′-triisopropylbiphenyl; Pd2(dba)3, tris-
(dibenzylideneacetone)dipalladium(0); NaOH, sodium hydroxide;
MeOH, methanol; HATU, (2-(7-aza-1H-benzotriazole-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate; PTSA, p-toluene-
sulfonic acid; MW, microwave
(11) Weinblatt, M. E.; Kavanaugh, A.; Burgos-Vargas, R.; Dikranian,
A. H.; Medrano-Ramirez, G.; Morales-Torres, J. L.; Murphy, F. T.;
Musser, T. K.; Straniero, N.; Vicente-Gonzales, A. V.; Grossbard, E.
Treatment of rheumatoid arthritis with a Syk kinase inhibitor: a
twelve-week, randomized, placebo-controlled trial. Arthritis Rheum.
2008, 58, 3309−3318.
(12) Lucas, M. C.; Goldstein, D.; Hermann, J. C.; Kuglstatter, A.;
Liao, C.; Liu, W.; Luk, K.-C.; Padilla, F.; Wanner, J.; Xie, W.; Zhang, J.
A hypothesis for the rational design of highly selective spleen tyrosine
kinase inhibitors. J. Med. Chem. 2012, 55, 10414.
(13) Padilla, F.; Bhagirath, N.; Chen, S.; Chiao, E.; Goldstein, D. M.;
Hermann, J. C.; Hsu, J.; Kennedy-Smith, J. J.; Kuglstatter, A.; Liao, C.;
Liu, W.; Lowrie, L. E., Jr; Luk, K.-C.; Lynch, S. M.; Menke, J.; Niu, L.;
Owens, T.; O-Yang, C.; Railkar, A.; Schoenfeld, R. C.; Slade, M.;
Steiner, S.; Tan, Y.-C.; Villasenor, A. G.; Wang, Ce.; Wanner, J.; Xie,
̃
W.; Xu, D.; Zhang, X.; Zhou, M.; Lucas, M. C. Pyrrolopyrazines as
selective spleen tyrosine kinase inhibitors. J. Med. Chem. 2013, 56,
1677.
(14) Liao, C.; Hsu, J.; Kim, Y.; Hu, D.-Q.; Xu, D.; Zhang, J.; Pashine,
A.; Menke, J.; Whittard, T.; Romero, N.; Truitt, T.; Slade, M.; Lukacs,
C.; Hermann, J.; Zhou, M.; Lucas, M.; Narula, S.; DeMartino, J.; Tan,
S.-L. Arthritis Res. Ther. 2013, 15, R146.
REFERENCES
■
(1) Mocsai, A.; Ruland, J.; Tybulewicz, V. L. J. The SYK tyrosine
kinase: A crucial player in diverse biological functions. Nat. Rev.
Immunol. 2010, 10, 387−402.
(2) Bajpai, M.; Chopra, P.; Dastidar, S. G.; Ray, A. Spleen tyrosine
kinase: A novel target for therapeutic intervention of rheumatoid
arthritis. Expert Opin. Invest. Drugs 2008, 17, 641−659.
(15) Maron, D. M.; Ames, B. N. Revised methods for the Salmonella
mutagenicity test. Mutat. Res. 1983, 113, 173−215.
(16) Villasenor, A. G.; Kondru, R.; Ho, H.; Wang, S.; Papp, E.; Shaw,
̃
D.; Barnett, J. W.; Browner, M. F.; Kuglstatter, A. Structural insights
for design of potent spleen tyrosine kinase inhibitors from crystallo-
graphic analysis of three inhibitor complexes. Chem. Biol. Drug Des.
2009, 73, 466−470.
(3) Zou, W.; Kitaura, H.; Reeve, J.; Long, F.; Tybulewicz, V. L.;
Shattil, S. J.; Ginsberg, M. H.; Ross, F. P.; Teitelbaum, S. L. Syk, c-Src,
the αvβ3 integrin, and ITAM immunoreceptors, in concert, regulate
osteoclastic bone resorption. J. Cell Biol. 2007, 176, 877−888.
(4) Koga, T.; Inui, M.; Inoue, K.; Kim, S.; Suematsu, A.; Kobayashi,
E.; Iwata, T.; Ohnishi, H.; Matozaki, T.; Kodama, T.; Taniguchi, T.;
Takayanagi, H.; Takai, T. Costimulatory signals mediated by the
ITAM motif cooperate with RANKL for bone homeostasis. Nature
2004, 428, 758−763.
(17) Zimmer, D.; Mazurek, J.; Petzold, G.; Bhuyan, B. K. Bacterial
mutagenicity and mammalian cell DNA damage by several substituted
anilines. Mutat. Res. 1980, 77, 317−326.
(18) The 30 kinase panel included the following: tyrosine kinases:
ALK, BTK, EPHB1, ITK, JAK1, JAK3, KDR, LCK, LYNa, MET,
PDGFRβ, SYK, ZAP70; serine/threonine kinases: AKT3, AurC,
CaMK2a, CK1δ (CSNK1d), CLK1, CLK2, DYRK1B, Erk2
(MAPK1), GSK3β, NEK7, p38α, p70S6K (S6K1, RPS6KB1), PAK4,
PEK (EIF2AK2, PRKR), PIM2, ROCK1; and other kinases: PIK3CA.
All kinases were tested at a single inhibitor concentration, in the
presence of ATP, in duplicate, and percent inhibition was measured.
The assay was performed at Carna Biosciences, Inc.
(19) Alex, A.; Millan, D. S.; Perez, M.; Wakenhut, F.; Whitlock, G. A.
Intramolecular hydrogen bonding to improve membrane permeability
and absorption in beyond rule of five chemical space. Med. Chem.
Commun. 2011, 2, 669−674.
(20) Waring, M. J. Lipophilicity in drug discovery. Expert Opin. Drug
Discovery 2010, 5, 235−248.
(21) Beckford, S. J.; Dixon, D. W. Molecular dynamics of
anthraquinone DNA intercalators with polyethylene glycol side chains.
J. Biomol. Struct. Dyn. 2012, 29, 1065−1080.
(22) Ovality is defined as the ratio of the molecular surface area to
the minimum surface area. The minimum surface area is the surface
area of a sphere having a volume equal to the solvent-excluded volume
of the molecule (computed from the Connolly molecular surface area
and solvent-excluded volume properties).
(5) Tam, F. W. K.; McAdoo, S. P. Fostamatinib disodium. Drugs
Future 2011, 36, 273−280.
(6) (a) Pandey, A. Discovery of PRT062607: A potent and selective
Syk inhibitor for the treatment of inflammatory, B cell proliferative
diseases and thrombosis. CHI 6th Annual Drug Discovery Chemistry
Conference, San Diego, CA, April 12−13, 2011. (b) 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.; Sinha,
U. Specific inhibition of spleen tyrosine kinase suppresses leukocyte
immune function and inflammation in animal models of rheumatoid
arthritis. J. Pharmacol. Exp. Ther. 2012, 34, 350−359.
(7) Gilead. Pipeline. http://www.gilead.com/pipeline (accessed
February 2014).
(8) Liddle, J.; Atkinson, F. L.; Barker, M. D.; Carter, P. S.; Curtis, N.
R.; Davis, R. P.; Douault, C.; Dickson, M. C.; Elwes, D.; Garton, N. S.;
Gray, M.; Hayhow, T. G.; Hobbs, C. I.; Jones, E.; Leach, S.; Leavens,
K.; Lewis, H. D.; McCleary, S.; Neu, M.; Patel, V. K.; Preston, A. G. S.;
Ramirez-Molina, C.; Shipley, T. J.; Skone, P. A.; Smithers, N.; Somers,
D. O.; Walker, A. L.; Watson, R. J.; Weingarten, G. G. Discovery of
GSK143, a highly potent, selective and orally efficacious spleen
tyrosine kinase inhibitor. Bioorg. Med. Chem. Lett. 2011, 21, 6188−
6194.
(9) Clemens, G. R.; Schroeder, R. E.; Magness, S. H.; Weaver, E. V.;
Lech, J. W.; Taylor, V. C.; Masuda, E. S.; Baluom, M.; Grossbard, E. B.
Developmental toxicity associated with receptor tyrosine kinase Ret
inhibition in reproductive toxicity testing. Birth Defects Res., Part A
2009, 85, 130−136.
(23) Hann, M. M.; Keseru, G. M. Finding the sweet spot: The role of
̈
nature and nurture in medicinal chemistry. Nat. Rev. Drug Discovery
2012, 11, 355−364.
(24) Xu, D.; Kim, Y.; Postelnek, J.; Vu, M. D.; Hu, D. Q.; Liao, C.;
Bradshaw, M.; Hsu, J.; Zhang, J.; Pashine, A.; Srinivasan, D.; Woods, J.;
Levin, A.; O’Mahony, A.; Owens, T. D.; Lou, Y.; Hill, R. J.; Narula, S.;
DeMartino, J.; Fine, J. S. RN486, a selective Bruton’s tyrosine kinase
inhibitor, abrogates immune hypersensitivity responses and arthritis in
rodents. J. Pharmacol. Exp. Ther. 2012, 341, 90−103.
(10) Friedberg, J. W.; Sharman, J.; Sweetenham, J.; Johnston, P. B.;
Vose, J. M.; Lacasce, A.; Schaefer-Cutillo, J.; De Vos, S.; Sinha, R.;
Leonard, J. P.; Cripe, L. D.; Gregory, S. A.; Sterba, M. P.; Lowe, A. M.;
Levy, R.; Shipp, M. A. Inhibition of Syk with fostamatinib disodium
2691
dx.doi.org/10.1021/jm401982j | J. Med. Chem. 2014, 57, 2683−2691