Discovery of GS-9973, a selective and orally efficacious inhibitor of spleen tyrosine kinase

Article abstract of DOI:10.1021/jm500228a

Spleen tyrosine kinase (Syk) is an attractive drug target in autoimmune, inflammatory, and oncology disease indications. The most advanced Syk inhibitor, R406, 1 (or its prodrug form fostamatinib, 2), has shown efficacy in multiple therapeutic indications, but its clinical progress has been hampered by dose-limiting adverse effects that have been attributed, at least in part, to the off-target activities of 1. It is expected that a more selective Syk inhibitor would provide a greater therapeutic window. Herein we report the discovery and optimization of a novel series of imidazo[1,2-a]pyrazine Syk inhibitors. This work culminated in the identification of GS-9973, 68, a highly selective and orally efficacious Syk inhibitor which is currently undergoing clinical evaluation for autoimmune and oncology indications.

Full text of DOI:10.1021/jm500228a

Article
pubs.acs.org/jmc
Discovery of GS-9973, a Selective and Orally Efficacious Inhibitor of
Spleen Tyrosine Kinase
Kevin S. Currie,† Jeffrey E. Kropf,† Tony Lee,† Peter Blomgren,† Jianjun Xu,† Zhongdong Zhao,†
Steve Gallion,⊥ J. Andrew Whitney,‡ Deborah Maclin,§ Eric B. Lansdon,∥ Patricia Maciejewski,‡
Ann Marie Rossi,‡ Hong Rong,‡ Jennifer Macaluso,‡ James Barbosa,‡ Julie A. Di Paolo,‡
and Scott A. Mitchell*,†
‡
§
†Department of Chemistry, Department of Biology, and Department of Drug Metabolism, Gilead Sciences, Inc., Branford,
Connecticut 06405, United States
∥Department of Structural Chemistry, Gilead Sciences, Inc., Foster City, California 94404, United States
⊥Department of Informatics & Modeling, Atheon Pharma Inc., Waltham, Massachusetts 02451, United States
S
* Supporting Information
ABSTRACT: Spleen tyrosine kinase (Syk) is an attractive drug target in autoimmune,
inflammatory, and oncology disease indications. The most advanced Syk inhibitor,
R406, 1 (or its prodrug form fostamatinib, 2), has shown efficacy in multiple
therapeutic indications, but its clinical progress has been hampered by dose-limiting
adverse effects that have been attributed, at least in part, to the off-target activities of 1.
It is expected that a more selective Syk inhibitor would provide a greater therapeutic
window. Herein we report the discovery and optimization of a novel series of
imidazo[1,2-a]pyrazine Syk inhibitors. This work culminated in the identification of
GS-9973, 68, a highly selective and orally efficacious Syk inhibitor which is currently
undergoing clinical evaluation for autoimmune and oncology indications.
and deformation of peripheral joints with a detrimental impact
on the quality of life. The underlying mechanisms leading to
the disease are not completely understood, but several biologic
therapeutics targeting proinflammatory cytokines such as tumor
necrosis factor α (TNFα), interleukin-1, and interleukin-6 have
had a tremendous impact on its treatment.10 Despite this
therapeutic advance, approximately one-third of all patients fail
to respond adequately to these agents or experience treatment
intolerance that limits their use.11
The therapeutic potential of Syk inhibition in autoimmune as
well as oncology indications has stimulated increased research
interest in recent years,2,12−14 and several Syk inhibitor
chemotypes have been reported (Figure 1), including
diaminopyrimidines15 1 and 2, heteroaryl carboxamides16−18
3−5, aminopyrimidine19 6, and naphthyridine20 7. Several of
these compounds have demonstrated efficacy in animal models
of inflammation. Compounds 2 and 3 dose-dependently
inhibited disease scores and suppressed joint inflammation
and bone erosion in rodent models of arthritis.15,16,21
Compound 4 is reported to be a highly selective Syk inhibitor
and was efficacious in the rat reversed passive arthus (RPA)
model of inflammation.17 Preclinical studies with 1, or its
prodrug 2 (R788, fostamatinib), have demonstrated efficacy in
INTRODUCTION
■
Spleen tyrosine kinase (Syk) is a 72 kDa multiple-domain
intracellular cytoplasmic tyrosine kinase that is expressed
primarily in hematopoietic cells (e.g., B-cells, monocytes,
macrophages, mast cells, and neutrophils), where it is
recognized as an important mediator of immunoreceptor
signaling and has been identified as a potential therapeutic
target in allergic, autoimmune, and oncology indications.1−3
Immunoreceptor engagement triggers phosphorylation of a pair
of tyrosine residues in the cytoplasmic immunoreceptor
tyrosine-based activation motifs (ITAMs) by Src family
members, and Syk is recruited to the phosphorylated ITAMs
via its SH2 domain.4−6 Syk critically regulates immune cell
function by propagating signaling cascades through phosphor-
ylation of direct targets (such as BLNK/SLP65), leading to
activation of downstream pathways, including PI3K, MAPK,
Btk, and PLCγ.5,7,8 The activation of these pathways in immune
cells leads to proliferation, differentiation, cytoskeletal remodel-
ing, and cytokine release. Importantly, since Syk is not
expressed in mature T-cells, the immunosuppression that is
associated with therapeutics that inhibit T-cell signaling should
be avoided.
Rheumatoid arthritis (RA) is a chronic, multifactorial disease
that is characterized by the production of autoantibodies,
synovial inflammation, pannus formation, and the erosion of
cartilage and bone9 that manifests itself by marked destruction
Received: February 18, 2014
Published: April 6, 2014
© 2014 American Chemical Society
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Figure 1. Syk inhibitor chemotypes.
a
Scheme 1
a
Reagents and conditions: (a) benzyl alcohol, Et3N, CH2Cl2, rt, 22 h; (b) Fe powder, EtOH, H2SO4, H2O, 60 °C, 90 min; (c) 3-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)benzoyl chloride, DIPEA, CH2Cl2, rt; (d) 3,4-dimethoxyaniline, DIEA, IPA, 100 °C, 22 h; (e) Pd(PPh3)4, Na2CO3, 1,4-
dioxane, H2O, reflux, 2.5 h; (f) Pd/C, H2, EtOH, EtOAc, 30 min.
several additional animal models of autoimmune/inflammatory
disease, including systemic lupus erythramatosis (SLE),22,23
nephrotoxic nephritis,24 autoimmune diabetes,25 idiopathic
thrombocytopenia purpura (ITP),26 autoimmune hemolytic
anemia (AHA),26 and asthma.27,28 Furthermore, 2 has shown
activity in various B-cell leukemia and lymphoma models.29−31
Fostamatinib has been evaluated in several phase II RA
studies,32−35 where it has demonstrated dose-dependent
efficacy as measured by reduction in ACR20 and ACR50
scores. However, gastrointestinal upset, neutropenia, and
hypertension were commonly occurring adverse effects. The
adverse events seen in the initial TASKI trial have prohibited
the use of the most efficacious dose (150 mg bid) in
subsequent RA studies, and fostamatinib failed to meet primary
efficacy end points in some of these trials.33,36−38 Additional
clinical trials in B-cell malignancies,39 ITP,26 and solid
tumors40,41 have been reported with promising efficacy results,
but with a similar adverse effect profile. It appears that the dose-
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limiting adverse effects of 2 have restricted the ability to achieve
maximum efficacy and may limit its clinical application in
chronic indications such as RA. The adverse event profile of 2
has been putatively attributed to the poor kinase selectivity of
its parent compound 1 (R406).42,43
Table 1. Optimization of the D-Ring Acid of 9
Together, these data validate the therapeutic potential of Syk
inhibition in autoimmune and oncology indications and
highlight the need for compounds with improved safety
profiles. A key advantage of such compounds would be the
ability to achieve levels of in vivo Syk inhibition that are not
currently achievable due to dose-limiting toxicities. Accordingly,
we sought to identify Syk inhibitors with significantly improved
selectivity profiles over 1. Herein we describe the discovery of
GS-9973, a selective and orally efficacious imidazopyrazine-
based Syk inhibitor that has the potential for an improved
efficacy and tolerability in patients.
SYNTHESIS
■
Our initial lead compound 9 was prepared as shown in Scheme
1. Commercially available 3,4-dimethoxyaniline regiospecifically
displaced the 6-bromide of 6,8-dibromoimidazo[1,2-a]pyrazine
(12), which was either purchased or synthesized according to
the literature procedures, to provide the bromide intermediate
13 in high yield.44 Reaction of 3-nitrobenzoyl chloride with
benzyl alcohol followed by the iron-mediated reduction of the
nitro group gave aniline 16, which was acylated to afford the
corresponding boronic ester 17. Subsequent Suzuki−Miyaura
coupling reaction of 13 with the boronic ester 17 afforded 18,
which was then debenzylated by hydrogenolysis to provide 9.
All of the analogues in Table 1 were prepared analogously from
variously substituted anilines.
The syntheses of disubstituted coupling partners 21e−l for
the analogues described in Table 2 are shown in Scheme 2.
Commercially available m- and p-benzoic acids were efficiently
converted to the corresponding amides 20e−k by treatment
with amines in the presence of pyBOP or EDCI. Then 20e−l
were transformed to boronic esters 21e−l using a catalytic
amount of PdCl2(dppf). It was important to apply an excess
amount of bis(pinacolato)diboron to ensure a complete
consumption of 20e−l to provide 21e−l in good yields.
The bicyclic heteroaromatic analogues described in Table 3
were prepared using the route exemplified by compound 24 in
Scheme 3. From the commercially available bromide 22, the
boronic ester 23 was synthesized in one step, and Suzuki−
Miyaura coupling reaction with the intermediate 13 afforded 24
in good yield. All boronic esters used to prepare the
compounds in Table 3 were either purchased or synthesized
using the procedures described above.
The synthesis of 30 (Scheme 4) exemplifies the route to the
compounds in Table 4. An SNAr reaction between 1-fluoro-4-
nitrobenzene (25) and 4-methylpiperadin-4-ol followed by
nitro reduction gave aniline 27 in good yield. Next, aniline 27
was combined with 6,8-dibromoimidazo[1,2-a]pyrazine (12) in
the presence of N,N-diisopropylethylamine to provide the
bromide 28, which was further converted to 30 by coupling
reaction with indazole boronic ester 29. This general route was
utilized for preparation of the compounds in Table 4.
Table 2. Optimization of C-Ring Carboxamides
analogue
R1
R2
R3
[Syk] (nM) [pBLNK] (nM)
11
43
44
45
46
47
48
49
50
51
52
53
CONH2
H
H
H
27.1
17.7
50.2
244
127
619
615
248
123
268
CONH2
H
H
CONHMe
CONMe2
CONH2
CONH2
CONH2
CONH2
F
H
H
H
112
F
H
67.3
28.3
37.4
Me
H
OMe
H
H
Me
H
466
CONH2
CONH2
CONH2
CONH2
18.6
64.1
23.8
134
310
251
418
Me
H
OMe
H
H
Me
113
RESULTS AND DISCUSSION
■
Screening of our compound library identified compound 8
(Figure 2), which was found to have promising activity in a Syk
enzyme assay (IC50 = 65 nM). Early work replaced the tert-
butylphenyl group with dimethoxyphenyl (based on the
structures of 1 and Bayer’s Syk inhibitor BAY 61-3606)45 and
reversed the amide to provide our initial lead compound 9,
The initial synthetic steps of the pyran derivative 70 are
shown in Scheme 5. Friedel−Crafts alkylation of benzene with
chloropyran 31 gave phenylpyran 32, which underwent
nitration with nitric acid to provide 33. Compound 70 was
then prepared by the procedure described in Scheme 4.
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a
a
Scheme 2
Scheme 3
a
Reagents and conditions: (a) B2(pin)2, KOAc, [PdCl2(dppf)]·
CH2Cl2, DMSO, 90 °C, 16 h; (b) bromide 12, Pd(PPh3)4, Na2CO3,
1,4-dioxane, H2O, microwave, 125 °C, 20 min.
<10%)/(total number of kinases)]. Dose−response follow-up
showed a Kd for Syk binding of 4 nM. In contrast, 1 bound 215
out of 358 kinases with an S(10) = 0.60 and had a Syk Kd of 12
nM. The promising selectivity profile of 9 prompted us to
initiate a medicinal chemistry program in the imidazopyrazine
series.
a
Reagents and conditions: (a) PyBOP, NH4Cl, NMM, DMF or EDCI,
HOBt, NH4OH, NMM, THF; (b) B2(pin)2, KOAc, [PdCl2(dppf)]·
CH2Cl2, 1,4-dioxane.
To guide the initial medicinal chemistry efforts, we obtained
an X-ray crystal structure of 9 bound to the kinase domain of
Syk. 9 was found to occupy the ATP binding pocket (Figure 3),
with N-1 of the imidazopyrazine system accepting a hydrogen
bond from the backbone NH of A451, while the aniline NH of
9 forms a second H-bond with the carbonyl group of the same
residue. The aniline ring (A-ring) extends toward the solvent
and forms contacts with L377, G454, and P455. The phenyl
ring at the 6-position (C-ring) sits above the gatekeeper M448
and near the catalytic K402 side chain and also forms extensive
van der Waals contacts with V385 in the P-loop. The amide
linker is slightly twisted and forms a hydrogen bond to a water
molecule in the pocket. The terminal phenyl ring (D-ring) is
oriented up toward the top of the pocket, enabling van der
Waals contacts with the side chains of P455 and R498, and
positions the carboxylic acid to form a hydrogen bond with
N457 and a water molecule.
Table 3. C-Ring Optimization with 5,6/6,6-Heterocycles
Despite the potent inhibition of Syk enzymatic activity, 9
suffered a 200-fold loss in cellular potency as measured by
BCR-mediated phosphorylation of BLNK (pBLNK) in Ramos
cells and also exhibited poor activity in a human whole blood
assay measuring inhibition of FcεR-mediated basophil
degranulation (EC50 > 5 μM). We suspected the benzoic acid
moiety as a contributor to these observations, so our initial SAR
investigations focused on modifying this region of the molecule
(Table 1). The m-CO2H analogue 35 and phenylacetic acid
derivative 36 lost 10-fold and 3-fold enzyme potency relative to
9 but still retained single-digit nanomolar activity. Modeling
showed that it would be difficult for the carboxylic acid to
maintain an optimal interaction with N457 in both these
analogues while preserving the network of other favorable
contacts. Due to the presence of multiple attractive interactions,
the specific contact with N457 is likely to be beneficial but not
critical for potent Syk inhibition. Consistent with this, the
methyl ester analogue 34, while 20-fold less potent compared
to the acid, still retained an IC50 of 15 nM. A number of
carboxylic acid bioisosteres48−50 and alternative hydrogen bond
donating groups were investigated in an effort to improve
cellular potency. The tetrazole 39 and acylsulfonamide 38
analogues retained enzymatic potency comparable to that of 9,
but failed to improve cellular potency. The reduced enzyme−
cell shift (10−27-fold) of the primary amide 37 and the
heterocyclic derivatives 40−42 was encouraging, but we felt
that further improvements in cellular potency were required.
which possessed excellent Syk potency of 0.8 nM (Table 1).
When screened at 10 μM against 317 kinases in the
KinomeScan competition binding assay,46,47 9 bound to only
eight kinases, including Syk, with an S(10) score of 0.03 [S(10)
is defined as the (number of kinases with a control score of
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a
Scheme 4
a
Reagents and conditions: (a) 4-methylpiperadin-4-ol hydrochloride, DIEA, CH3CN, reflux, 2.5 h; (b) 10% Pd/C (wet), H2 gas (1 atm), ethanol;
(c) 6,8-dibromoimidazo[1,2-a]pyrazine, DIPEA, IPA, reflux, 2 h; (d) Pd(PPh3)4, Na2CO3, 1,4-dioxane, H2O, microwave, 135 °C, 35 min.
Table 4. A-Ring Substituent Optimization
a
optimization of cellular and whole blood potency. Subsequent
to our work, similar problems in a series of imidazopyridazine
Syk inhibitors have recently been described (10, Figure 2).51
We therefore sought to truncate the molecule to identify a
more druglike template for further chemistry efforts. We found
that removal of the benzoic acid moiety resulted in Syk IC50
values of 27 and 18 nM for the m- and p-carboxamides 11
(Figure 4) and 43, respectively. Despite the order of magnitude
loss in Syk enzyme potency, both of these compounds had
enzyme−cell shifts of less than 10-fold, suggesting that the
Scheme 5
a
Reagents and conditions: (a) AlCl3, benzene, 0 °C to rt (caution: gas
evolution!), 1 h, 92%; (b) AcOH, H2SO4, HNO3, rt, 30 min.
It became apparent that the relatively high molecular weight
and lipophilicity of this series would constrain further
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Figure 2. Initial hit 8, lead 9, and Roche extended acid 10.
Figure 3. Cocrystal structure (2.0 Å) of the Syk kinase domain with compound 9 (PDB code 4PV0). Dashed lines show hydrogen bond contacts in
the pocket between the compound and protein. Part of the P-loop is removed to make viewing the compound binding mode easier.
compound 9 (0.03). This selectivity profile coupled with the
improved molecular properties encouraged us to further
explore the truncated series, with the goal of improving
intrinsic potency.
The results of our SAR studies around the C-ring
phenylcarboxamide are summarized in Table 2. In both the
3- and 4-carboxamido series, addition of small substituents
ortho to the carboxamide (compounds 46−48 and 50−52,
respectively) had little impact on Syk enzyme potency and
failed to improve upon the parent compound 11 or 43. In
contrast, addition of a methyl group ortho to the phenyl−
imidazopyrazine linkage reduced potency significantly in both
cases (49 and 53). Energy minimizations with the Merck
molecular force field (MMFF) indicate that this methyl group
Figure 4. Results of truncation.
increases the dihedral angle between the phenyl ring and the
imidazopyrazine from 42° to 70°. It is possible that this twist
disrupts potential H-bond interactions between the carbox-
amide and the protein and also erodes productive interactions
between the phenyl ring and V385. The importance of this H-
bonding interaction is supported by the reduced potency of the
mono- and dimethyl amides 44 and 45, which suffered 2- and
6-fold reductions in Syk potency, respectively. The data in
Table 2 suggested that there was little potency advantage to be
gained by substitution of the phenyl ring and that manipulating
reduction in molecular weight (24%) and log P (1.4 units) had
indeed improved the molecular properties. Furthermore, 43
showed encouraging bioavailability (22%) in the rat after oral
dosing at 10 mg/kg. Considering the in vivo clearance (Cl =
3.05 L/h/kg), the bioavailability for 43 is indicative of good
absorption from the gastrointestinal (GI) tract. Importantly,
truncation to the primary amide did not significantly erode
kinome selectivity, as the S(10) scores for compounds 11
(0.05) and 43 (0.04) were comparable to that of the lead
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the trajectory of the NH to the enzyme could be a more
productive strategy.
compound to optimize these parameters further. To test the
role of the morpholine nitrogen, we synthesized the pyran
analogue 70 and found a significant improvement in micro-
somal stability, but this compound had surprisingly poor
cellular activity. Reduced potency was also seen with the ring-
opened 71 or ring-expanded 72 morpholine analogue. Finally,
having previously demonstrated improved enzymatic potency
of the dimethoxy relative to the monomethoxy arrangement, we
prepared the 3-methoxy-4-morpholino derivative 73. While
intrinsic and cellular potencies were improved, this did not
translate to improved activity in the whole blood assay.
Furthermore, introduction of the methoxy group reduced the
metabolic stability significantly. At this stage of our
optimization, 68 was selected for further evaluation.
Incorporating the NH into a ring system consistently
improved Syk potency relative to that of the carboxamides
(Table 3). The indole and indazole analogues 54 and 55
improved activity by 5−10-fold over the carboxamide analogues
11 and 43 (Table 2). The benzimidazole and benzotriazole
analogues 56 and 57 showed a slight reduction in potency
relative to the corresponding indole and indazole, possibly due
to the existence of tautomeric forms in the former ring systems
that are less compatible with potent Syk binding. The isomeric
5-indole and 5-indazole isomers 58 and 24 exhibited Syk IC50
values 3−4-fold weaker than their 6-substituted counterparts 54
and 55, indicating a preference for the m-NH (relative to the
imidazopyrazine core). The importance of the free NH for
potent inhibition was further confirmed by the weaker enzyme
and cellular activity of the N-methyl derivatives 59 and 60. We
also examined 6,6-semisaturated ring systems and found the
same preference for a m-NH (relative to the imidazopyrazine
core) versus the para regioisomer (61 vs 62). The lactam 61
and its reduced benzomorpholine analogue 63 had potency
comparable to that of the indazole, indicating that the NH can
be presented in a variety of contexts to form favorable
hydrogen bond interactions with the enzyme.52 Several
compounds in Table 3 demonstrated potent cell-based activity
in the pBLNK assay, but still failed to produce satisfactory
whole blood potency. Only the indazole analogue 55 had an
EC50 of less than 1 μM in whole blood, and this compound was
profiled further. When screened against 359 kinases at 10 μM,
55 produced an S(10) score of 0.08. Further analysis of the top
39 hits from the primary screen showed that 55 did not bind to
any kinases with potency within 10-fold of that of Syk (Kd = 9
nM). Thus, significant truncation of the initial lead compound
9 led to improved cellular and whole blood potency with no
appreciable loss in selectivity.
The crystal structure of 68 bound to the Syk kinase domain
(Figure 5A) shows that the aminoimidazopyrazine core
In a rat pharmacokinetic (PK) study, 55 showed moderate
clearance (2.1 L/h/kg) with encouraging oral bioavailability
(36%) when dosed at 10 mg/kg. However, the metabolic
stability in human liver microsomes was unacceptable, with a
predicted clearance of 0.72 L/h/kg. We suspected the
dimethoxyphenyl motif as a metabolic liability, so the next
phase of our investigation focused on this region of the
molecule (Table 4). Crystallographic information suggested
that the 4-position of the phenyl ring offered a trajectory
toward solvent, and we anticipated this would provide a
platform to improve the absorption, distribution, metabolism,
and excretion (ADME)/PK properties without adversely
affecting Syk binding. Compounds 55 and 64−66 confirmed
that the 3,4-dimethoxy arrangement was optimal for enzyme
potency. The monomethoxy derivatives 65 and 66, despite
retaining Syk activity within 2-fold of the dimethoxy compound
55, suffered a 7−15-fold loss in cellular potency. In contrast,
monosubstitution of the para position with a variety of aliphatic
amines (30, 67−69, 71−73) retained pBLNK potency within
1−3-fold of that of 55. Consistent with the presumed metabolic
liability of the dimethoxyphenyl group, many of these analogues
also improved metabolic stability in human liver microsomes.
In particular, the 4-methyl-4-hydroxypiperidine 30 exhibited a
predicted human clearance of 0.27 L/h/kg and excellent
enzymatic potency, although the whole blood EC50 remained
above 500 nM. However, the morpholine derivative 68
provided a good balance of whole blood potency and metabolic
stability, and we prepared several close analogues of this
Figure 5. (A) Cocrystal structure (2.1 Å) of Syk with compound 68
(PDB code 4PUZ). Along with the hinge interactions, 68 forms a new
hydrogen bond with D512. (B) Comparison of the binding modes
between 68 (green) and 1 (pink).
maintains the same hinge interactions observed with 9. The
4-morpholino group extends beyond the hinge residues toward
solvent. Surprisingly, a significant conformational difference in
the C-ring between 9 and 68 was observed. The C-ring phenyl
of 9 rotates 27° relative to the core, whereas the indazole
system of 68 is rotated 24° in the opposite direction. This
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Table 5. ADME Profile of 68 in Multiple Species
a
a
c
d
microsomal half-life
(min)
predicted hepatic Cl
(L/h/kg)
plasma protein binding
(%)
Coco-2 Papp
b
b
c
species
Cl (L/h/kg) Vss (L/kg)
F (%)
(10−6 cm/s)
rat
35
1.53
0.85
0.29
0.27 0.01
0.80 0.03
0.52 0.05
1.41 0.40
67
9
99.2
92.8
97.1
A/B 3.20, B/A 1.95
dog
23.4
98.6
53 16
human
a
b
c
d
From hepatic microsomes. Intravenous dose of 0.4 mg/kg. Oral dose of 1 mg/kg. At 5 μM concentration.
Figure 6. Kd analysis of 68 and 1. Individual kinases for which Kd values were determined are arranged in descending order of selectivity of 68 on the
x-axis. Fold selectivities for inhibition of each kinase relative to the Syk Kd for 68 (blue dots) and 1 (red dots) are shown.
Table 6. Biological Profile of GS-9973
EC50
SD (nM)
target
cell type
Ramos
stimulation
αIgM
readout
pBLNK
GS-9973
26
R406
Syk
53 32
13 1.5
46 11
Jak2
ckit
Flt3
Ret
TF-1
EPO
SCF
na
pStat5
453 335
445 100
327 175
>1000
BMMCs
MV4,11
SK-N-MC
HUVEC
B-cell
cKit
proliferation
pRet
10
36
36
5
9
KDR
Syk
VEGF
pVEGFR2
CD86
>1000
αIgM
125 78
41 27
335 36
151 109
na
B-cell
αlgM
proliferation
TNFα production
CD63
monocytes
basophils (WB)
immune complex
αlgE
147 16
387 220
3659 2116
difference in rotation brings the NH of the indazole closer to
the DFG motif. The D512 side chain rotates toward the
indazole NH and forms a hydrogen bond, which explains the
intrinsic potency of 68 even without the network of additional
interactions, including the H-bond to N457 that is evident in
the more elaborate structure 9.
The in vitro ADME and in vivo PK properties of 68 are
summarized in Table 5. 68 is highly protein bound across
species, with a human free fraction of 2.7%. 68 was relatively
stable in human liver microsomes (predicted Cl = 0.29 L/h/kg)
but was less stable in preclinical species. In vivo, the clearance
relative to hepatic blood flow was low in rat but was higher in
dog, with dog having a relatively lower extent of protein
binding. 68 showed good bidirectional permeability across
Caco-2 cell monolayers, indicating good absorption potential
and low potential for efflux at concentrations likely to be
achieved clinically. When dosed orally at 1 mg/kg in solution,
68 showed moderate to high bioavailability in rat and dog. A
comparison of the bioavailability and hepatic extraction in these
species indicates that the extent of absorption from the GI tract
is high (>75%). Since inhibition of metabolizing enzymes has
the potential to cause clinically relevant drug−drug interactions,
we evaluated the ability of 68 to inhibit CYP1A2, 2C9, 2C19,
2D6, and 3A4. IC50 values were >10 μM in all cases. The
solubility of crystalline 68 in simulated intestinal fluid under
both fed and fasted conditions was quite low (16 and 2 μM,
respectively), most likely due to its high crystallinity and
melting point (326 °C).
Maintaining a high degree of kinase selectivity was
paramount in our program, and 68 was initially profiled using
the KinomeScan platform against a panel of 359 nonmutant
kinases at a single concentration of 10 μM. The resulting S(10)
scores showed dramatically improved selectivity compared to
that of 1 [S(10) = 0.12 and 0.60, respectively]. To characterize
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Figure 7. GS-9973 was efficacious in rat established collagen-induced arthritis. Collagen-induced arthritis was initiated in rats as described in the
Experimental Section. Twice daily oral dosing with vehicle or GS-9973 was commenced at disease onset on day 10 or 11 (study day 1). Calipers
were used to measure ankle thickness daily (mean SEM; an asterisk indicates p < 0.01 vs arthritis + vehicle) (A). Blood plasma compound PK was
assessed following the final dose (B). On the basis of histological evaluation of ankle sections by hematoxylin and eosin (H&E) staining and a five-
point scale described in the Experimental Section, robust disease-modifying antirheumatic drug (DMARD) activity of GS-9973 was observed (C).
ED50 and EC50 values for GS-9973 were calculated for multiple measured disease parameters (D).
the selectivity profile in more detail, Kd values were determined
for 67 kinases, including all those that showed control scores of
<10% in the primary screen, and were compared to the
corresponding Kd values for 1 (Figure 6). Only 1 kinase, TNK1,
showed less than 10-fold selectivity versus Syk for 68, whereas
1 bound 36 kinases within 10-fold of the potency of Syk and 14
kinases more potently than Syk. Additional Kd values were
determined for 1 on the basis of the primary screen data, and
additional kinases with Kd values more potent than that of Syk
were identified (Supplementary Table 1, Supporting Informa-
tion). We selected five kinase targets for further cellular
profiling on the basis of their potential to confound efficacy
readouts or contribute to clinically observed safety issues with
1. The results showed that, in cells, 68 inhibited Flt3, Jak2, c-
Kit, KDR, and Ret activity 10−35-fold less potently than 1. 68
showed 13- to >1000-fold cellular selectivity for Syk as assessed
by target protein phosphorylation or functional response
(Table 6), whereas 1 inhibited four out of five of these kinases
more potently than Syk. Together these results demonstrated
the excellent selectivity profile of 68 and highlighted the
potential for an improved clinical safety margin.
of VEGFR2 by various conformations of the diaminopyr-
imidine scaffold has been described.54 We believe that 1
potently inhibits VEGFR2 by adopting one or more of these
active conformations, which are not accessible to the more
conformationally restricted 68.
Having demonstrated the high selectivity of 68 for Syk, we
tested its ability to inhibit multiple cellular functions that
contribute to the pathology of RA (Table 6). In vitro, 68
potently inhibited BCR-mediated activation and proliferation of
B-cells as well as immune-complex-stimulated cytokine
production in monocytes, indicating the potential for activity
in both the initiation and effector phases of inflammatory
arthritis. Furthermore, 68 demonstrated whole blood potency
that was superior to that of 1 from our own in vitro studies or
from reported clinical studies.15 In a rat collagen-induced
arthritis model where dosing is initiated after the onset of
disease, 68 significantly inhibited ankle inflammation when
dosed orally at 10, 3, and 1 mg/kg bid (Figure 7A),
demonstrating a role for Syk in the effector phase of
inflammatory arthritis. There was a direct correlation between
exposure and efficacy in this model. To build a correlation of
plasma levels, efficacy, and the degree of target inhibition, we
developed an in vitro rat whole blood phospho-Syk (pSyk)
assay and determined the EC50 for pSyk inhibition to be 1.9
μM. Plasma levels of 68 measured on the final study day (12 h
post last dose) were consistent with maintaining 50% (at 3 mg/
kg) and 90% (at 10 mg/kg) inhibition of pSyk at trough
As a potential rationale for this improved selectivity, Figure
5B shows a superposition of the crystal structures of 68 and 1
bound to Syk,53 indicating the similar binding pose of both. We
hypothesize that the flexibility provided by the extra rotatable
bond in 1 allows it to adopt conformations that can interact
with several kinases in addition to Syk. For example, inhibition
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pressure, triturated with heptane (150 mL), dissolved in methylene
chloride, and washed with saturated sodium bicarbonate. The organic
layer was dried over sodium sulfate and filtered, and the filtrate was
concentrated under reduced pressure to afford benzyl 4-amino-
benzoate (10.2 g, 57%) as an off-white solid that was used without
further purification.
Benzyl 4-(3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-
benzamido)benzoate (17). A mixture of 16 (4.39 g, 19.3 mmol), 3-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl chloride (4.30 g,
16.1 mmol), and N,N-diisopropylethylamine (7.49 g, 57.9 mmol) in
methylene chloride (50 mL) was stirred at ambient temperature for 13
h. After this time, the reaction was concentrated under reduced
pressure and purified by chromatography (silica, heptane to 1:3 ethyl
acetate/heptane) to afford benzyl 4-(3-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)benzamido)benzoate (3.07 g, 78% pure by mass,
27% yield) as a light yellow sticky foam that was used without further
purification.
(Figure 7B). 68 also showed disease-modifying activity in
multiple histological measurements, including inhibition of
pannus formation, cartilage damage, bone resorption, and
peritosteal bone formation (Figure 7C), with ED50 values
ranging from 1.2 to 3.9 mg/kg (Figure 7D). These data suggest
that 50% target coverage (pSyk EC50) at trough was sufficient
to significantly reduce disease scores.
CONCLUSION
■
The favorable in vitro and in vivo properties of 68 resulted in
its selection as a development candidate, GS-9973. Given the
excellent selectivity profile, it is hypothesized that GS-9973 will
enable high levels of Syk inhibition clinically with fewer dose-
limiting adverse effects associated with more promiscuous
inhibitors. GS-9973 is currently being evaluated in human
clinical trials.41,55
Benzyl 4-(3-(8-((3,4-Dimethoxyphenyl)amino)imidazo[1,2-a]-
pyrazin-6-yl)benzamido)benzoate (18). A mixture of 6-bromo-N-
(3,4-dimethoxyphenyl)imidazo[1,2-a]pyrazin-8-amine (13) (1.82 g,
5.22 mmol) and 17 (3.06 g, 5.22 mmol, 78% by mass) in 1 M aqueous
sodium carbonate (8 mL) and 1,4-dioxane (24 mL) was sparged with
nitrogen with stirring for 5 min. The resulting mixture was treated with
tetrakis(triphenylphosphine)palladium(0) (1.20 g, 1.04 mmol) and
stirred at reflux for 2.5 h. After this time, the reaction was cooled to
ambient temperature, diluted with ethyl acetate (100 mL), and washed
with brine (100 mL). The aqueous layer was extracted with ethyl
acetate (100 mL), the combined organic layers were dried over sodium
sulfate and filtered, and the filtrate was concentrated under reduced
pressure. The residue obtained was purified by chromatography (silica,
methylene chloride to 1:4 ethyl acetate/methylene chloride) to afford
benzyl 4-(3-(8-((3,4-dimethoxyphenyl)amino)imidazo[1,2-a]pyrazin-
6-yl)benzamido)benzoate (2.02 g, 65%) as an off-white solid: mp
EXPERIMENTAL SECTION
General Procedures. All commercial reagents were used as
provided. Flash chromatography was performed using an ISCO
Combiflash Companion purification system with RediSep Rf
■
1
prepacked silica gel cartridges supplied by Teledyne Isco. H NMR
spectra were recorded on a Varian Inova 300 MHz spectrometer.
Proton chemical shifts are reported in parts per million from an
internal standard or residual solvent. The purity of the tested
compounds was assessed to be at least 95% by HPLC analysis unless
indicated otherwise. A Gemini C18 110 Å column (50 mm × 4.6 mm,
5 μm particle size) was used with gradient elution of acetonitrile in
water, 0−30% for 5 min and then 30−98% for 5 min at a flow rate of 2
mL/min with detection at 254 nm wavelength. For all samples 0.1%
TFA was added to both eluents. High-resolution mass spectrometry
was performed on an Agilent 6210 time of flight mass spectrometer
coupled to an Agilent 1200 rapid resolution HPLC instrument. The
samples were run on a Phenomenex Luna C18 column using reversed-
phase chromatography with a gradient from 20% to 90% acetonitrile
containing 0.1% formic acid. The reference masses that were used
during data collection were 118.086255 and 922.009798. Data were
processed via Agilent Masshunter B.04 qualitative analysis.
Synthesis of 9. Benzyl 4-Nitrobenzoate (15). A solution of 4-
nitrobenzoyl chloride (14) (18.6 g, 100 mmol) in methylene chloride
(200 mL) was added dropwise to a stirred solution of benzyl alcohol
(12.9 g, 120 mmol) and triethylamine (30.3 g, 300 mmol) in
methylene chloride (200 mL), and the reaction was stirred at ambient
temperature for 22 h. After this time, the reaction was filtered, and the
filtrate was washed with brine (400 mL) and then saturated aqueous
ammonium chloride (400 mL). The organic layer was dried over
sodium sulfate and filtered, and the filtrate was concentrated under
reduced pressure. The residue obtained was solvent exchanged with
ethanol (200 mL) and then recrystallized from ethanol (150 mL) to
afford benzyl 4-nitrobenzoate (20.4 g, 79%) as a light yellow solid: 1H
NMR (300 MHz, CDCl3) δ 8.30−8.22 (m, 4H), 7.47−7.37 (m, 5H),
5.41 (s, 2H).
Benzyl 4-Aminobenzoate (16). A mixture of 15 (20.4 g, 79.3
mmol) and iron powder (66.4 g, 1190 mmol) in ethanol (600 mL, 1%
water) was treated dropwise with a solution of concentrated sulfuric
acid (52 mL) in water (204 mL), and the resulting mixture was
mechanically stirred at ambient temperature for 1 h. The reaction was
treated with additional sulfuric acid (52 mL) and stirred at ambient
temperature for 15 min and then at 60 °C for 1.5 h. After this time, the
reaction was cooled to ambient temperature and filtered through
diatomaceous earth, and the volatiles of the filtrate were removed
under reduced pressure. The resulting mixture was extracted with ethyl
acetate (500 mL and then 2 × 200 mL), and the combined organics
were washed with brine (500 mL). The organic layer was dried over
sodium sulfate and filtered, and the filtrate was concentrated under
reduced pressure until a small amount of white solid formed, which
was removed by filtration. The filtrate was concentrated under reduced
1
154−160 °C; H NMR (300 MHz, CDCl3) δ 10.72 (s, 1H), 9.57 (s,
1H), 8.74 (s, 1H), 8.57 (s, 1H), 8.25 (d, J = 7.8 Hz, 1H), 8.08−7.94
(m, 7H), 7.69−7.60 (m, 3H), 7.50−7.34 (m, 5H), 6.92 (d, J = 8.7 Hz,
1H), 5.36 (s, 2H), 3.78 (s, 3H), 3.73 (s, 3H); ESI MS m/z 600 [M +
H]+; HPLC 8.93 min, 95.9% (AUC).
4-(3-(8-((3,4-Dimethoxyphenyl)amino)imidazo[1,2-a]pyrazin-6-
yl)benzamido)benzoic Acid (9). A solution of 18 (1.75 g, 2.92 mmol)
in ethyl acetate (500 mL) and ethanol (500 mL) at 60 °C was treated
with 10% palladium on carbon (5.11 g, 50% water by weight), sparged
with hydrogen for 30 min at 60 °C, and then stirred under balloon
pressure hydrogen at 60 °C for 30 min. After this time, the reaction
was sparged with nitrogen for 10 min and filtered hot through
diatomaceous earth. The filtrate was concentrated under reduced
pressure, and the residue obtained was exhaustively triturated (boiling
acetonitrile (2 × 50 mL), then boiling methanol (50 mL), then boiling
1:1 methanol/acetonitrile (50 mL), then boiling acetonitrile (50 mL),
and then boiling 1:1 methanol/acetonitrile (50 mL)) to afford 4-(3-(8-
((3,4-dimethoxyphenyl)amino)imidazo[1,2-a]pyrazin-6-yl)-
benzamido)benzoic acid (458 mg, 31%) as an off-white solid: mp >
250 °C; 1H NMR (300 MHz, DMSO-d6) δ 12.74 (br s, 1H), 10.67 (s,
1H), 9.57 (s, 1H), 8.74 (s, 1H), 8.56 (s, 1H), 8.25 (d, J = 7.8 Hz, 1H),
8.80−7.89 (m, 7H), 7.69−7.53 (m, 3H), 6.93 (d, J = 8.7 Hz, 1H), 3.78
(s, 3H), 3.71 (s, 3H); HRMS-ESI+ m/z calcd for C28H23N5O5
510.1772 (M + H+), found 510/1770 (M + H+).
3-(8-((3,4-Dimethoxyphenyl)amino)imidazo[1,2-a]pyrazin-
6-yl)benzamide (11). The target compound was obtained following
the procedure outlined for 33 as a light green solid (40 mg, 38%): mp
226−228 °C; 1H NMR (300 MHz, DMSO-d6) δ 9.56 (s, 1H), 8.67 (s,
1H), 8.50 (s, 1H), 8.17 (d, J = 7.8 Hz, 1H), 8.10 (d, J = 2.4 Hz, 1H),
8.05 (br s, 1H), 8.03 (s, 1H), 7.87 (d, J = 7.8 Hz, 1H), 7.68 (m, 1H),
7.59−7.54 (m, 2H), 7.44 (br s, 1H), 6.95 (d, J = 8.7 Hz, 1H), 3.82 (s,
3H), 3.76 (s, 3H); ESI MS m/z 390 [M + H]+; HRMS m/z 390.1577
[M + H]+; HRMS-ESI+ m/z calcd for C21H19N5O3 390.1561 (M +
H+), found 390.1577 (M + H+).
5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole
(23). A mixture of 5-bromo-1H-indazole (22) (1.48 g, 7.50 mmol),
bis(pinacolato)diboron (2.09 g, 8.25 mmol), and potassium acetate
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(2.21 g, 22.5 mmol) in dimethyl sulfoxide (45 mL) was sparged with
nitrogen while being stirring for 10 min. Dichloro[1,1′-bis-
(diphenylphosphino)ferrocene]palladium(II)−methylene chloride ad-
duct (823 mg, 1.12 mmol) was then added, and the reaction was
stirred at 90 °C for 16 h. After this time, the reaction was cooled to
room temperature, added to a mixture of 1:1 ethyl acetate/water (400
mL), and sonicated for 5 min. The resulting biphasic mixture was
filtered through diatomaceous earth and the filter cake washed with
ethyl acetate (50 mL). The layers of the filtrate were separated, and the
aqueous phase was extracted with ethyl acetate (2 × 100 mL). The
combined organic layers were dried over sodium sulfate, filtered, and
concentrated under reduced pressure to afford 5-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)-1H-indazole (667 mg, 36%) as a dark-brown
solid that was used without further purification: ESI MS m/z 244.9 [M
+ H]+.
cake was dried to a constant weight under vacuum to afford 1-(4-((6-
bromoimidazo[1,2-a]pyrazin-8-yl)amino)phenyl)-4-methylpiperidin-
4-ol (574 mg, 87%) as a light brown solid: H NMR (300 MHz,
DMSO-d6) δ 9.71 (s, 1H), 8.16 (s, 1H), 7.90 (m, 1H), 7.75−7.72 (m,
2H), 7.59 (m, 1H), 6.94−6.91 (m, 2H), 4.25 (s, 1H), 3.30−3.23 (m,
2H), 3.14−3.05 (m, 2H), 1.58−1.54 (m, 4H), 1.51 (s, 3H).
1
1-(4-((6-(1H-Indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl)amino)-
phenyl)-4-methylpiperidin-4-ol (30). A mixture of 28 (300 mg,
0.746 mmol) and 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-
indazole (29) (270 mg, 1.11 mmol) in 1 M aqueous sodium carbonate
(1.5 mL) and 1,4-dioxane (4 mL) was sparged with nitrogen while
being stirred for 5 min. Tetrakis(triphenylphosphine)palladium(0)
(170 mg, 0.147 mmol) was then added and the reaction heated under
microwave irradiation at 135 °C for 35 min. After this time, the
reaction was cooled to room temperature, combined with a second lot
(0.496 mmol), and filtered through diatomaceous earth and the filter
cake washed with ethyl acetate (150 mL). The filtrate was washed with
water (30 mL) and then brine (30 mL) and dried over sodium sulfate.
The drying agent was removed by filtration and the filtrate
concentrated under reduced pressure. The resulting residue was
purified by trituration with methanol and then chromatography (silica,
gradient, methylene chloride to 19:1 methylene chloride/methanol) to
afford 1-(4-((6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl)amino)-
phenyl)-4-methylpiperidin-4-ol (179 mg, 36% combined) as a light
N-(3,4-Dimethoxyphenyl)-6-(1H-indazol-5-yl)imidazo[1,2-a]-
pyrazin-8-amine (24).
A mixture of 6-bromo-N-(3,4-
dimethoxyphenyl)imidazo[1,2-a]pyrazin-8-amine (200 mg, 0.570
mmol), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole
(278 mg, 1.14 mmol), and 1 M aqueous sodium carbonate (1.25
mL) in 1,4-dioxane (4.0 mL) was sparged with nitrogen while being
stirred for 5 min. Tetrakis(triphenylphosphine)palladium(0) (66 mg,
0.057 mmol) was then added and the reaction heated under
microwave irradiation at 125 °C for 20 min. After this time, the
reaction was cooled to room temperature, dissolved in ethyl acetate
(10 mL), and filtered through diatomaceous earth. The filtrate was
washed with water (20 mL) and then brine (2 × 20 mL) and dried
over sodium sulfate. The drying agent was removed by filtration, and
the filtrate was concentrated under reduced pressure. The resulting
residue was purified by chromatography (silica, gradient, methylene
chloride to 1:19 methanol/methylene chloride) to afford N-(3,4-
dimethoxyphenyl)-6-(1H-indazol-5-yl)imidazo[1,2-a]pyrazin-8-amine
1
yellow solid: mp >250 °C; H NMR (300 MHz, DMSO-d6) δ 13.17
(s, 1H), 9.45 (s, 1H), 8.65 (s, 1H), 8.19 (s, 1H), 8.08 (s, 1H), 8.00−
7.97 (m, 3H), 7.83 (d, J = 8.7 Hz, 1H), 7.72 (d, J = 8.7 Hz, 1H), 7.63
(s, 1H), 6.99 (d, J = 9.0 Hz, 2H), 4.26 (s, 1H), 3.30−3.24 (m, 2H),
3.16−3.07 (m, 2H), 1.60−1.58 (m, 4H), 1.17 (s, 3H); HRMS-ESI+ m/
z calcd for C25H25N7O 440.2193 (M + H+), found 440.2201(M + H+).
4-Phenyltetrahydro-2H-pyran (32). A solution of aluminum
chloride (2.67 g, 20.0 mmol) in anhydrous benzene (50 mL) was
cooled to 0 °C in an ice/water bath under a nitrogen atmosphere and
treated dropwise with a solution of 4-chlorotetrahydro-2H-pyran (31)
(2.11g, 17.5 mmol) in anhydrous benzene (5 mL). Caution: gas
evolution and exothermic reaction! When the addition was complete, the
cooling bath was removed and the reaction stirred at room
temperature for 1 h. After this time, the reaction was poured into a
mixture of ice/water (200 mL) and extracted with diethyl ether (200
mL). The organic phase was dried over sodium sulfate and filtered and
the filtrate concentrated under reduced pressure to afford 4-
phenyltetrahydro-2H-pyran (2.62 g, 92%) as a yellow solid which
1
(59 mg, 27%) as an off-white solid: mp > 250 °C; H NMR (300
MHz, DMSO-d6) δ 13.14 (s, 1H), 9.50 (s, 1H), 8.59 (s, 1H), 8.44 (s,
1H), 8.18 (d, J = 2.4 Hz, 1H), 8.14 (s, 1H), 8.01 (m, 2H), 7.62 (m,
3H), 6.98 (d, J = 9.0 Hz, 1H), 3.86 (s, 3H), 3.77 (s, 3H); HRMS-ESI+
m/z calcd for C21H18N6O2 387.1564 (M + H+), found 388.1584 (M +
H+).
4-Methyl-1-(4-nitrophenyl)piperidin-4-ol (26). A mixture of 1-
fluoro-4-nitrobenzene (25) (350 mg, 2.48 mmol), 4-methylpiperidin-
4-ol hydrochloride salt (400 mg, 2.64 mmol), and N,N-diisopropyle-
thylamine (670 mg, 5.18 mmol) in acetonitrile (6.3 mL) was stirred at
reflux for 2.5 h. After this time, the reaction was cooled to room
temperature and concentrated under reduced pressure. The resulting
residue was purified by chromatography (silica, methylene chloride) to
afford 4-methyl-1-(4-nitrophenyl)piperidin-4-ol (441 mg, 75%) as a
1
was used in the next step without purification: H NMR (400 MHz,
DMSO-d6) δ 7.29−7.17 (m, 5H), 3.96−3.92 (m, 2H), 3.46−3.40 (m,
2H), 2.84−2.72 (m, 1H), 1.69−1.64 (m, 4H).
4-(4-Nitrophenyl)tetrahydro-2H-pyran (33). A solution of 32
(2.60 g, 16.0 mmol) in glacial acetic acid (15 mL) was treated with a
solution of 98% sulfuric acid (0.88 mL) in glacial acetic acid (15 mL),
followed by a solution of 90% nitric acid (0.77 mL) in glacial acetic
acid (15 mL), and then diluted with 98% sulfuric acid (13.3 mL) and
the reaction stirred at room temperature for 30 min. After this time,
the mixture was poured into ice/water (50 mL) and slowly treated
with sodium bicarbonate (50 g). Caution: gas evolution and exothermic
reaction! When the addition was complete, the mixture was stirred at
40 °C until gas evolution ceased and then cooled to room
temperature. The mixture was brought to pH 14 with 5 M aqueous
sodium hydroxide and the resulting suspension filtered. The filtrate
was extracted with methylene chloride (3 × 100 mL), and the
combined organic layers were dried over sodium sulfate. The drying
agent was removed by filtration and the filtrate concentrated under
reduced pressure to afford 4-(4-nitrophenyl)tetrahydro-2H-pyran
1
yellow solid: H NMR (300 MHz, DMSO-d6) δ 8.03−7.99 (m, 2H),
7.01−6.98 (m, 2H), 4.43 (s, 1H), 3.72−3.66 (m, 2H), 3.41−3.30 (m,
2H), 1.56−1.44 (m, 4H), 1.14 (s, 3H).
1-(4-Aminophenyl)-4-methylpiperidin-4-ol (27). A solution of
26 (440 mg, 1.86 mmol) in ethanol (10 mL) and methanol (10 mL)
was degassed with nitrogen, and 10% palladium on carbon (50% wet,
170 mg dry weight) was added. The round-bottom flask was charged
with hydrogen gas to a pressure of 1 atm, and the contents were stirred
for 2.5 h. After this time, the hydrogen gas was evacuated and nitrogen
charged into the flask. The catalyst was removed by filtration through a
pad of diatomaceous earth and the filter cake washed sequentially with
ethyl acetate (30 mL), ethanol (30 mL), and ethyl acetate (30 mL).
The filtrate was concentrated under reduced pressure to afford 1-(4-
aminophenyl)-4-methylpiperidin-4-ol (373 mg, 97%) as an orange-
yellow semisolid that was used in the next step without purification or
analysis.
1
(2.05 g, 62%) as a yellow solid: H NMR (400 MHz, DMSO-d6) δ
1-(4-((6-Bromoimidazo[1,2-a]pyrazin-8-yl)amino)phenyl)-4-
methylpiperidin-4-ol (28). A mixture of 27 (370 mg, 1.79 mmol),
6,8-dibromoimidazo[1,2-a]pyrazine (450 mg, 1.63 mmol), and N,N-
diisopropylethylamine (420 mg, 3.25 mmol) in 2-propanol (5 mL)
was stirred at reflux for 2.5 h and then at room temperature for 18 h.
After this time, the mixture was poured into water (20 mL) and stirred
for 5 min. The resulting suspension was filtered and the filter cake
washed with water (20 mL) and then diethyl ether (20 mL). The filter
8.17 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 8.8 Hz, 2H), 3.98−3.94 (m, 2H),
3.48−3.42 (m, 2H), 3.00−2.92 (m, 1H), 1.74−1.67 (m, 4H).
Methyl 4-(3-(8-((3,4-Dimethoxyphenyl)amino)imidazo[1,2-
a]pyrazin-6-yl)benzamido)benzoate (34). A mixture of 3-(8-
((3,4-dimethoxyphenyl)amino)imidazo[1,2-a]pyrazin-6-yl)benzoic
acid trifluoroacetate salt (500 mg, 0.897 mmol, 70% by mass), methyl
4-aminobenzoate (136 mg, 0.897 mmol), (benzotriazol-1-yloxy)-
tripyrrolidinophosphonium hexafluorophosphate (703 mg, 1.35
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mmol), and N,N-diisopropylethylamine (1.16 g, 8.97 mmol) in DMF
(7 mL) was stirred at 60 °C for 14.5 h. After this time, the reaction was
diluted with water (100 mL) and extracted with ethyl acetate (100
mL). The organic layer was washed with water (100 mL) and then 5%
aqueous lithium chloride (100 mL), dried over sodium sulfate, and
filtered, and the filtrate was concentrated under reduced pressure. The
residue obtained was purified by chromatography (silica, methylene
chloride to 1:39 methanol/methylene chloride) followed by trituration
with methylene chloride to afford methyl 4-(3-(8-((3,4-
dimethoxyphenyl)amino)imidazo[1,2-a]pyrazin-6-yl)benzamido)-
benzoate (110 mg, 23%) as a white solid: mp 141−144 °C; 1H NMR
(300 MHz, DMSO-d6) δ 10.71 (s, 1H), 9.58 (s, 1H), 8.74 (s, 1H),
8.56 (s, 1H), 8.25 (d, J = 7.5 Hz, 1H), 8.08−7.93 (m, 7H), 7.70−7.60
(m, 3H), 6.93 (d, J = 8.7 Hz, 1H), 3.85 (s, 3H), 3.77 (s, 3H), 3.71 (s,
3H); HRMS-ESI+ m/z calcd for C29H25N5O5 524.1928 (M + H+),
found 524.1954 (M + H+).
1H), 3.77 (s, 3H), 3.69 (s, 3H); HRMS-ESI+ m/z calcd for
C28H23N9O3 534.1997 (M + H+), found 534.2019 (M + H+).
N-(4-(1H-Imidazol-2-yl)phenyl)-3-(8-((3,4-dimethoxyphenyl)-
amino)imidazo[1,2-a]pyrazin-6-yl)benzamide (40). The target
compound was obtained following the procedure outlined for 33 as a
brown solid (50 mg, 40%): mp > 250 °C; 1H NMR (300 MHz,
DMSO-d6) δ 10.52 (s, 1H), 9.58 (s, 1H), 8.74 (s, 1H), 8.57 (s, 1H),
8.24 (d, J = 6.6 Hz, 1H), 8.09−7.92 (m, 8H), 7.67−7.61 (m, 3H), 7.19
(s, 2H), 6.94 (d, J = 8.7 Hz, 1H), 3.79 (s, 3H), 3.72 (s, 3H); HRMS-
ESI+ m/z calcd for C30H25N7O3 532.2092 (M + H+), found 532.2112
(M + H+).
N-(4-(1H-Pyrazol-5-yl)phenyl)-3-(8-((3,4-dimethoxyphenyl)-
amino)imidazo[1,2-a]pyrazin-6-yl)benzamide (41). The target
compound was obtained following the procedure outlined for 33 as an
off-white solid (12.2 mg, 13%): mp 250−255 °C; 1H NMR (300 MHz,
DMSO-d6) δ 13.24−12.82 (m, 1H), 10.45 (m, 1H), 9.57 (s, 1H), 8.74
(s, 1H), 8.57 (s, 1H), 8.25 (d, J = 8.1 Hz, 1H), 8.09−7.63 (m, 11H),
6.93 (d, J = 8.7 Hz, 1H), 6.69 (s, 1H), 3.83 (s, 3H), 3.79 (s, 3H);
HRMS-ESI+ m/z calcd for C30H25N7O3 532.2092 (M + H+), found
532.2089 (M + H+).
3-(3-(8-((3,4-Dimethoxyphenyl)amino)imidazo[1,2-a]-
pyrazin-6-yl)benzamido)benzoic Acid (35). The target compound
was obtained following the procedure outlined for 33: mp > 250 °C;
1H NMR (300 MHz, DMSO-d6) δ 10.58 (s, 1H), 9.60 (s, 1H), 8.75 (s,
N-(4-(1H-Pyrazol-4-yl)phenyl)-3-(8-((3,4-dimethoxyphenyl)-
amino)imidazo[1,2-a]pyrazin-6-yl)benzamide (42). The target
compound was obtained following the procedure outlined for 33 as a
tan solid (171 mg, 46%): mp > 250 °C; 1H NMR (300 MHz, DMSO-
d6) δ 12.90 (s, 1H), 10.37 (s, 1H), 9.56 (s, 1H), 8.73 (s, 1H), 8.56 (s,
1H), 8.23 (d, J = 7.8 Hz, 1H), 8.16−8.09 (m, 2H), 8.03 (s, 1H), 7.94
(m, 2H), 7.80 (d, J = 8.4 Hz, 2H), 7.67−7.60 (m, 5H), 6.93 (d, J = 8.7
Hz, 1H), 3.79 (s, 3H), 3.72 (s, 3H); HRMS-ESI+ m/z calcd for
C30H25N7O3 532.2092 (M + H+), found 532.2087 (M + H+).
4-(8-((3,4-Dimethoxyphenyl)amino)imidazo[1,2-a]pyrazin-
6-yl)benzamide (43). A mixture of (4-(aminocarbonyl)phenyl)-
boronic acid (114 mg, 0.690 mmol), 6-bromo-N-(3,4-
dimethoxyphenyl)imidazo[1,2-a]pyrazin-8-amine (300 mg, 0.862
mmol), and 1 M aqueous sodium carbonate (1 mL) in 1,4-dioxane
(5 mL) was sparged with nitrogen with stirring for 5 min. The reaction
was treated with tetrakis(triphenylphosphine)palladium(0) (200 mg,
0.172 mmol) and reacted under microwave irradiation at 135 °C for
15 min. After this time, the reaction was cooled to ambient
temperature, and the solids that formed were collected by filtration
and triturated with acetonitrile and then methanol to afford 4-(8-((3,4-
dimethoxyphenyl)amino)imidazo[1,2-a]pyrazin-6-yl)benzamide (96
1H), 8.59 (s, 1H), 8.44 (s, 1H), 8.24 (d, J = 7.6 Hz, 1H), 8.08−7.95
(m, 4H), 7.70−7.49 (m, 5H), 6.93 (d, J = 8.7 Hz, 1H), 3.78 (s, 3H),
3.71 (s, 3H); HRMS-ESI+ m/z calcd for C28H23N5O5 510.1722 (M +
H+), found 510.1783 (M + H+).
2-(4-(3-(8-((3,4-Dimethoxyphenyl)amino)imidazo[1,2-a]-
pyrazin-6-yl)benzamido)phenyl)acetic Acid Hydrochloride
(36). The target compound was obtained following the procedure
1
outlined for 33 as a yellow solid (68 mg, 84%): mp 176−190 °C; H
NMR (300 MHz, DMSO-d6) δ 10.40 (s, 1H), 9.98 (s, 1H), 8.82 (s,
1H), 8.57 (s, 1H), 8.23 (d, J = 7.5 Hz, 1H), 8.14 (s, 1H), 8.04 (d, J =
2.1 Hz, 1H), 7.97 (d, J = 8.1 Hz, 1H), 7.91 (s, 1H), 7.75 (d, J = 8.4 Hz,
2H), 7.67 (t, J = 7.8 Hz, 1H), 7.59 (dd, J = 8.7, 2.4 Hz, 1H), 7.40 (d, J
= 8.1 Hz, 2H), 6.96 (d, J = 8.4 Hz, 1H), 3.79 (s, 3H), 3.72 (s, 3H),
3.56 (s, 2H); HRMS-ESI+ m/z calcd for C29H25N5O5 524.1928 (M +
H+), found 524.1939 (M + H+).
N-(4-Carbamoylphenyl)-3-(8-((3,4-dimethoxyphenyl)-
amino)imidazo[1,2-a]pyrazin-6-yl)benzamide (37). A mixture of
4-aminobenzamide (35 mg, 0.256 mmol), N,N-diisopropylethylamine
(265 mg, 2.05 mmol), 3-(8-((3,4-dimethoxyphenyl)amino)imidazo-
[1,2-a]pyrazin-6-yl)benzoic acid trifluoroacetate salt (145 mg, 0.256
mmol, 69% by mass), and (benzotriazol-1-yloxy)-
tripyrrolidinophosphonium hexafluorophosphate (200 mg, 0.384
mmol) in DMF (2 mL) was heated at 75 °C for 2.5 d. After this
time, the reaction was concentrated under reduced pressure. The
residue obtained was purified twice, first by chromatography (silica,
ethyl acetate to 3:7 methanol/methylene chloride) and then by
trituration with methanol, to afford N-(4-carbamoylphenyl)-3-(8-
((3,4-dimethoxyphenyl)amino)imidazo[1,2-a]pyrazin-6-yl)benzamide
1
mg, 36%) as a light-brown solid: mp 248−249 °C; H NMR (300
MHz, DMSO-d6) δ 9.55 (s, 1H), 8.71 (s, 1H), 8.16−7.92 (m, 7H),
7.66 (s, 1H), 7.61 (d, J = 8.3 Hz, 1H), 7.39 (s, 1H), 6.97 (d, J = 8.6
Hz, 1H), 3.84 (s, 3H), 3.77 (s, 3H); HRMS-ESI+ m/z calcd for
C30H25N7O3 390.1561 (M + H+), found 390.1570 (M + H+).
3-(8-((3,4-Dimethoxyphenyl)amino)imidazo[1,2-a]pyrazin-
6-yl)-N-methylbenzamide (44). The target compound was
obtained following the procedure outlined for 42: 1H NMR (300
MHz, DMSO-d6) δ 9.54 (s, 1H), 8.64 (s, 1H), 8.54−8.39 (m, 2H),
8.13 (dt, J = 7.9, 1.4 Hz, 1H), 8.10 (d, J = 2.4 Hz, 1H), 8.00 (d, J = 1.1
Hz, 1H), 7.79 (dt, J = 7.7, 1.3 Hz, 1H), 7.64 (d, J = 1.1 Hz, 1H), 7.59−
7.49 (m, 2H), 6.93 (d, J = 8.8 Hz, 1H), 3.79 (s, 3H), 3.74 (s, 3H),
3.36−3.23 (m, 1H), 2.81 (d, J = 4.5 Hz, 3H); HRMS-ESI+ m/z calcd
for C22H21N5O3 404.1717 (M + H+), found 404.1737 (M + H+).
3-(8-((3,4-Dimethoxyphenyl)amino)imidazo[1,2-a]pyrazin-
6-yl)-N,N-dimethylbenzamide (45). The target compound was
obtained following the procedure outlined for 42: 1H NMR (300
MHz, DMSO-d6) δ 9.54 (s, 1H), 8.68 (s, 1H), 8.13−8.00 (m, 3H),
7.96 (d, J = 1.1 Hz, 1H), 7.64 (d, J = 1.1 Hz, 1H), 7.60−7.44 (m, 2H),
7.38 (dt, J = 7.6, 1.3 Hz, 1H), 6.93 (d, J = 8.8 Hz, 1H), 3.77 (s, 3H),
3.74 (s, 3H), 3.37−3.23 (m, 3H), 3.01 (s, 3H), 2.93 (s, 3H); HRMS-
ESI+ m/z calcd for C23H23N5O3 418.1874 (M + H+), found 418.1882
(M + H+).
1
(15 mg, 12%) as a light brown solid: mp > 250 °C; H NMR (300
MHz, CDCl3-d6) δ 8.44 (s, 1H), 8.39−7.75 (m, 5H), 7.87−7.75 (m,
3H), 7.64−7.30 (m, 6H), 6.88 (d, J = 8.7 Hz, 1H), 3.92 (s, 3H), 3.86
(s, 3H), 1.60 (s, 9H); HRMS-ESI+ m/z calcd for C28H24N6O4
509.1932 (M + H+), found 509.1944 (M + H+).
3-(8-((3,4-Dimethoxyphenyl)amino)imidazo[1,2-a]pyrazin-
6-yl)-N-(4-((methylsulfonyl)carbamoyl)phenyl)benzamide Hy-
drochloride Salt (38). The target compound was obtained following
the procedure outlined for 33 as a yellow solid (44 mg, 18%): mp
228−235 °C; 1H NMR (300 MHz, CDCl3) δ 12.03 (br s, 1H), 10.91
(s, 1H), 10.10 (br s, 1H), 8.94 (s, 1H), 8.65 (s, 1H), 8.28−8.25 (m,
1H), 8.15−7.92 (m, 8H), 7.71−7.66 (m, 1H), 7.61−7.59 (m, 1H),
6.97 (d, J = 8.7 Hz, 1H), 3.78 (s, 3H), 3.73 (s, 3H), 3.38 (s, 3H);
HRMS-ESI+ m/z calcd for C29H26N6O6S 587.1707 (M + H+), found
587.1727 (M + H+).
N-(4-(1H-Tetrazol-5-yl)phenyl)-3-(8-((3,4-dimethoxyphenyl)-
5-(8-((3,4-Dimethoxyphenyl)amino)imidazo[1,2-a]pyrazin-
amino)imidazo[1,2-a]pyrazin-6-yl)benzamide (39). The target
6-yl)-2-fluorobenzamide (46). The target compound was obtained
1
1
compound was obtained following the procedure outlined for 33: H
following the procedure outlined for 42: mp 232−233 °C; H NMR
NMR (300 MHz, DMSO-d6) δ 10.73 (s, 1H), 9.57 (s, 1H), 8.77 (s,
1H), 8.58 (t, J = 1.8 Hz, 1H), 8.29−8.19 (m, 1H), 8.13−7.99 (m, 6H),
7.95 (dt, J = 7.9, 1.3 Hz, 1H), 7.71−7.55 (m, 3H), 6.91 (d, J = 8.8 Hz,
(300 MHz, DMSO-d6) δ 9.54 (s, 1H), 8.66 (s, 1H), 8.29 (dd, J = 6.9,
2.4 Hz, 1H), 8.15−8.09 (m, 1H), 8.06 (d, J = 2.4 Hz, 1H), 7.98 (s,
1H), 7.78 (s, 1H), 7.71 (s, 1H), 7.65 (s, 1H), 7.53 (dd, J = 8.7, 2.4 Hz,
3867
dx.doi.org/10.1021/jm500228a | J. Med. Chem. 2014, 57, 3856−3873

Journal of Medicinal Chemistry
Article
1H), 7.40 (t, J = 8.7 Hz, 1H), 6.94 (d, J = 8.7 Hz, 1H), 3.80 (s, 3H),
3.75 (s, 3H); HRMS-ESI+ m/z calcd for C21H18FN5O3 408.1466 (M +
H+), found 408.4188 (M + H+).
the procedure outlined for 23: mp 250−252 °C; 1H NMR (300 MHz,
DMSO-d6) δ 13.12 (s, 1H), 9.46 (s, 1H), 8.61 (s, 1H), 8.13 (s, 1H),
8.02−8.01 (m, 2H), 7.94 (d, J = 0.9 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H),
7.68 (dd, J = 8.4, 0.9 Hz, 1H), 7.58 (d, J = 0.9 Hz, 1H), 7.55 (dd, J =
8.4, 2.4 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 3.76 (s, 3H), 3.71 (s, 3H);
HRMS-ESI+ m/z calcd for C21H18N6O2 387.1564 (M + H+), found
387.1579 (M + H+).
6-(1H-Benzo[d]imidazol-5-yl)-N-(3,4-dimethoxyphenyl)-
imidazo[1,2-a]pyrazin-8-amine (56). The target compound was
obtained following the procedure outlined for 23: mp 135−137 °C;
1H NMR (300 MHz, DMSO-d6) δ 12.58 (s, 0.5H), 12.51 (s, 0.5H),
9.48 (s, 1H), 8.60 (s, 1H), 8.34−8.10 (m, 3H), 7.98 (s, 1H), 7.91−
7.82 (m, 1H), 7.73−7.62 (m, 3H), 6.97 (d, J = 9.0 Hz, 1H), 3.87 (s,
1.5H), 3.82 (s, 1.5H), 3.77 (s, 3H); HRMS-ESI+ m/z calcd for
C21H18N6O2 387.1564 (M + H+), found 387.1578 (M + H+).
6-(1H-Benzo[d][1,2,3]triazol-6-yl)-N-(3,4-dimethoxyphenyl)-
imidazo[1,2-a]pyrazin-8-amine (57). The target compound was
obtained following the procedure outlined for 23: mp 244−250 °C;
1H NMR (300 MHz, DMSO-d6) δ 9.54 (s, 1H), 8.71 (s, 1H), 8.49 (s,
1H), 8.10 (d, J = 2.4 Hz, 1H), 8.03−7.94 (m, 3H), 7.65 (s, 1H), 7.61
(dd, J = 8.7, 2.4 Hz, 1H), 6.98 (d, J = 8.7 Hz, 1H), 3.85 (s, 3H), 3.77
(s, 3H); HRMS-ESI+ m/z calcd for C20H17N7O2 388.1516 (M + H+),
found 388.1515 (M + H+).
N-(3,4-Dimethoxyphenyl)-6-(1H-indol-5-yl)imidazo[1,2-a]-
pyrazin-8-amine (58). The target compound was obtained following
the procedure outlined for 23: mp 235−237 °C; 1H NMR (300 MHz,
DMSO-d6) δ 11.17 (s, 1H), 9.43 (s, 1H), 8.51 (s, 1H), 8.26 (m, 2H),
7.96 (d, J = 0.9 Hz, 1H), 7.75 (dd, J = 8.7, 1.5 Hz, 1H), 7.61 (d, J = 0.9
Hz, 1H), 7.57 (dd, J = 8.7, 2.4 Hz, 1H), 7.47 (d, J = 8.7 Hz, 1H), 7.38
(t, J = 2.4 Hz, 1H), 6.97 (d, J = 8.7 Hz, 1H), 6.47 (m, 1H), 3.87 (s,
3H), 3.76 (s, 3H); HRMS-ESI+ m/z calcd for C22H19N5O2 386.1612
(M + H+), found 388.1616 (M + H+).
N-(3,4-Dimethoxyphenyl)-6-(1-methyl-1H-indazol-6-yl)-
imidazo[1,2-a]pyrazin-8-amine (59). The target compound was
obtained following the procedure outlined for 23: mp 185−187 °C;
1H NMR (300 MHz, DMSO-d6) δ 9.55 (s, 1H), 8.71 (s, 1H), 8.22 (s,
1H), 8.10−8.07 (m, 2H), 8.02 (s, 1H), 7.83 (m, 2H), 7.67 (s, 1H),
7.67−7.64 (m, 1H), 6.99 (d, J = 8.7 Hz, 1H), 4.12 (s, 3H), 3.83 (s,
3H), 3.77 (s, 3H); HRMS-ESI+ m/z calcd for C22H20N6O2 401.1721
(M + H+), found 401.1726 (M + H+).
N-(3,4-Dimethoxyphenyl)-6-(1-methyl-1H-indazol-5-yl)-
imidazo[1,2-a]pyrazin-8-amine (60). The target compound was
obtained following the procedure outlined for 23: mp 210−212 °C;
1H NMR (300 MHz, DMSO-d6) δ 9.51 (s, 1H), 8.62 (s, 1H), 8.43 (s,
5-(8-((3,4-Dimethoxyphenyl)amino)imidazo[1,2-a]pyrazin-
6-yl)-2-methylbenzamide (47). The target compound was obtained
1
following the procedure outlined for 42: mp 243−244 °C; H NMR
(300 MHz, DMSO-d6) δ 9.50 (s, 1H), 8.63 (s, 1H), 8.14 (d, J = 2.4
Hz, 1H), 8.02 (d, J = 1.5 Hz, 1H), 7.98 (s, 1H), 7.94 (dd, J = 8.1, 1.5
Hz, 1H), 7.80 (s, 1H), 7.64 (s, 1H), 7.53 (dd, J = 8.7, 2.4 Hz, 1H),
7.43 (s, 1H), 7.34 (d, J = 8.1 Hz, 1H), 6.94 (d, J = 8.7 Hz, 1H), 3.81
(s, 3H), 3.75 (s, 3H), 2.40 (s, 3H); HRMS-ESI+ m/z calcd for
C22H21N5O3 404.1717 (M + H+), found 404.1728 (M + H+).
5-(8-((3,4-Dimethoxyphenyl)amino)imidazo[1,2-a]pyrazin-
6-yl)-2-methoxybenzamide (48). The target compound was
obtained following the procedure outlined for 42: mp 228−230 °C
1
dec; H NMR (300 MHz, DMSO-d6) δ 9.58 (s, 1H), 8.60 (s, 1H),
8.44 (s, 1H), 8.11−8.06 (m, 2H), 8.01 (s, 1H), 7.70 (m, 2H), 7.61 (s,
1H), 7.55 (d, J = 8.7 Hz, 1H), 7.26 (d, J = 8.7 Hz, 1H), 6.94 (d, J = 8.7
Hz, 1H), 3.94 (s, 3H), 3.81 (s, 3H), 3.75 (s, 3H); HRMS-ESI+ m/z
calcd for C22H21N5O4 420.1666 (M + H+), found 420.1688 (M + H+).
3-(8-((3,4-Dimethoxyphenyl)amino)imidazo[1,2-a]pyrazin-
6-yl)-4-methylbenzamide (49). The target compound was obtained
following the procedure outlined for 42: mp > 250 °C; 1H NMR (300
MHz, DMSO-d6) δ 9.47 (s, 1H), 8.14 (s, 1H), 8.03−8.00 (m, 2H),
7.95−7.90 (m, 2H), 7.81 (dd, J = 8.1, 1.7 Hz, 1H), 7.66 (s, 1H), 7.55
(dd, J = 8.7, 2.3 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.30 (bs, 1H), 6.88
(d, J = 8.7 Hz, 1H), 3.70 (s, 3H), 3.68 (s, 3H), 2.47 (s, 3H); HRMS-
ESI+ m/z calcd for C22H21N5O3 404.1717 (M + H+), found 404.1735
(M + H+).
4-(8-((3,4-Dimethoxyphenyl)amino)imidazo[1,2-a]pyrazin-
6-yl)-2-fluorobenzamide (50). The target compound was obtained
following the procedure outlined for 42: mp > 250 °C; 1H NMR (300
MHz, DMSO-d6) δ 9.60 (s, 1H), 8.77 (s, 1H), 8.04 (d, J = 2.1 Hz,
1H), 7.99 (s, 1H), 7.92−7.87 (m, 2H), 7.81−7.75 (m, 1H), 7.70−7.65
(m, 3H), 7.56 (dd, J = 8.7, 2.1 Hz, 1H), 6.97 (d, J = 8.7 Hz, 1H), 3.84
(s, 3H), 3.76 (s, 3H); HRMS-ESI+ m/z calcd for C21H18FN5O3
408.1466 (M + H+), found 408.1479 (M + H+).
4-(8-((3,4-Dimethoxyphenyl)amino)imidazo[1,2-a]pyrazin-
6-yl)-2-methylbenzamide (51). The target compound was obtained
1
following the procedure outlined for 42: mp 225−237 °C; H NMR
(300 MHz, DMSO-d6) δ 9.52 (s, 1H), 8.64 (s, 1H), 8.11 (d, J = 2.3
Hz, 1H), 7.99−7.98 (m, 1H), 7.92−7.84 (m, 2H), 7.74 (bs, 1H),
7.65−7.64 (m, 1H), 7.57 (dd, J = 8.7, 2.3 Hz, 1H), 7.48 (d, J = 7.9 Hz,
1H), 7.36 (bs, 1H), 6.97 (d, J = 8.7 Hz, 1H), 3.83 (s, 3H), 3.76 (s,
3H), 2.45 (s, 3H); HRMS-ESI+ m/z calcd for C22H21N5O3 404.1717
(M + H+), found 404.1718 (M + H+).
1H), 8.18 (d, J = 2.4 Hz, 1H), 8.12 (s, 1H), 8.05 (dd, J = 9.0, 1.5 Hz,
1H), 7.98 (s, 1H), 7.74 (d, J = 9.0 Hz, 1H), 7.64 (s, 1H), 7.60 (dd, J =
9.0, 2.4 Hz, 1H), 6.99 (d, J = 9.0 Hz, 1H), 4.09 (s, 3H), 3.87 (s, 3H),
3.77 (s, 3H); HRMS-ESI+ m/z calcd for C22H20N6O2 401.1721 (M +
H+), found 401.1739 (M + H+).
4-(8-((3,4-Dimethoxyphenyl)amino)imidazo[1,2-a]pyrazin-
6-yl)-2-methoxybenzamide (52). The target compound was
obtained following the procedure outlined for 42: mp 221−222 °C
1
dec; H NMR (300 MHz, DMSO-d6) δ 9.56 (s, 1H), 8.75 (s, 1H),
6-(8-((3,4-Dimethoxyphenyl)amino)imidazo[1,2-a]pyrazin-
6-yl)-2H-benzo[b][1,4]oxazin-3(4H)-one (61). The target com-
pound was obtained following the procedure outlined for 23: mp
7.99 (d, J = 1.2 Hz, 1H), 7.92 (d, J = 8.1 Hz, 1H), 7.87 (d, J = 2.4 Hz,
1H), 7.77−7.66 (m, 5H), 7.54 (bs, 1H), 6.97 (d, J = 8.7 Hz, 1H), 4.01
(s, 3H), 3.79 (s, 3H), 3.76 (s, 3H); HRMS-ESI+ m/z calcd for
C22H21N5O4 420.1666 (M + H+), found 420.1681 (M + H+).
4-(8-((3,4-Dimethoxyphenyl)amino)imidazo[1,2-a]pyrazin-
6-yl)-3-methylbenzamide (53). The target compound was obtained
1
235−237 °C; H NMR (300 MHz, DMSO-d6) δ 10.94 (s, 1H), 9.46
(s, 1H), 8.41(s, 1H), 7.99 (s, 1H), 7.91 (d, J = 2.1 Hz, 1H), 7.74 (dd, J
= 8.7, 2.1 Hz, 1H), 7.62 (s, 1H), 7.56−7.51 (m, 2H), 7.04 (d, J = 8.7
Hz, 1H), 6.98 (d, J = 8.7 Hz, 1H), 4.62 (s, 2H), 3.78 (s, 3H), 3.77 (s,
3H); HRMS-ESI+ m/z calcd for C22H19N5O4 418.1510 (M + H+),
found 418.1512 (M + H+).
1
following the procedure outlined for 42: mp 238−241 °C; H NMR
(300 MHz, DMSO-d6) δ 9.48 (s, 1H), 8.15 (s, 1H), 8.01−7.98 (m,
2H), 7.86−7.76 (m, 3H), 7.66−7.58 (m, 3H), 7.36 (bs, 1H), 6.89 (d, J
= 8.7 Hz, 1H), 3.71 (s, 6H), 2.50 (s, 3H); HRMS-ESI+ m/z calcd for
C22H21N5O3 404.1717 (M + H+), found 404.1736 (M + H+).
N-(3,4-Dimethoxyphenyl)-6-(1H-indol-6-yl)imidazo[1,2-a]-
pyrazin-8-amine (54). The target compound was obtained following
the procedure outlined for 23: mp 215−217 °C; 1H NMR (300 MHz,
DMSO-d6) δ 11.23 (s, 1H), 9.45 (s, 1H), 8.53 (s, 1H), 8.12 (d, J = 2.4
Hz, 1H), 8.08 (s, 1H), 7.98 (s, 1H), 7.67−7.59 (m, 4H), 7.39 (t, J =
2.7 Hz, 1H), 6.97 (d, J = 8.7 Hz, 1H), 6.45 (m, 1H), 3.82 (s, 3H), 3.76
(s, 3H); HRMS-ESI+ m/z calcd for C22H19N5O2 386.1612 (M + H+),
found 386.1630 (M + H+).
7-(8-((3,4-Dimethoxyphenyl)amino)imidazo[1,2-a]pyrazin-
6-yl)-2H-benzo[b][1,4]oxazin-3(4H)-one (62). The target com-
pound was obtained following the procedure outlined for 23: mp >
1
250 °C; H NMR (300 MHz, DMSO-d6) δ 10.81 (s, 1H), 9.48 (s,
1H), 8.53 (s, 1H), 8.04 (d, J = 2.4 Hz, 1H), 7.94 (s, 1H), 7.62−7.56
(m, 4H), 6.99−6.95 (m, 2H), 4.62 (s, 2H), 3.82 (s, 3H), 3.75 (s, 3H);
HRMS-ESI+ m/z calcd for C22H19N5O4 418.1510 (M + H+), found
418.1499 (M + H+).
6-(3,4-Dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-(3,4-
dimethoxyphenyl)imidazo[1,2-a]pyrazin-8-amine (63). The
target compound was obtained following the procedure outlined for
23: mp 179−182 °C; 1H NMR (300 MHz, DMSO-d6) δ 9.39 (s, 1H),
N-(3,4-Dimethoxyphenyl)-6-(1H-indazol-6-yl)imidazo[1,2-a]-
pyrazin-8-amine (55). The target compound was obtained following
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8.32 (s, 1H), 8.01 (d, J = 2.4 Hz, 1H), 7.95 (s, 1H), 7.67 (dd, J = 8.7,
2.4 Hz, 1H), 7.59 (s, 1H), 7.23 (d, J = 2.1 Hz, 1H), 7.11 (dd, J = 8.4,
2.1 Hz, 1H), 6.96 (d, J = 8.7 Hz, 1H), 6.72 (d, J = 8.4 Hz, 1H), 5.86
(m, 1H), 4.16 (m, 2H), 3.80 (s, 3H), 3.75 (s, 3H) 3.33 (m, 2H,
merged with H2O peak); HRMS-ESI+ m/z calcd for C22H21N5O3
404.1717 (M + H+), found 404.1728 (M + H+).
(s, 1H), 8.18 (s, 1H), 8.08 (s, 1H), 7.97−7.92 (m, 3H), 7.83 (d, J = 8.4
Hz, 1H), 7.71 (dd, J = 8.4, 0.9 Hz, 1H), 7.62 (d, J = 0.9 Hz, 1H), 6.77
(d, J = 9.3 Hz, 2H), 4.64 (t, J = 5.1 Hz, 1H), 3.60−3.56 (m, 2H), 3.40
(t, J = 6.3 Hz, 2H), 2.95 (s, 3H); HRMS-ESI+ m/z calcd for
C22H21N7O 400.1880 (M + H+), found 400.1892 (M + H+).
N-(4-(1,4-Oxazepan-4-yl)phenyl)-6-(1H-indazol-6-yl)-
imidazo[1,2-a]pyrazin-8-amine (72). The target compound was
obtained following the procedure outlined for 29: mp 237−240 °C;
1H NMR (300 MHz, DMSO-d6) δ 13.17 (s, 1H), 9.35 (s, 1H), 8.61 (s,
6-(1H-Indazol-6-yl)-N-phenylimidazo[1,2-a]pyrazin-8-amine
(64). The target compound was obtained following the procedure
1
outlined for 29: mp > 250 °C; H NMR (300 MHz, DMSO-d6) δ
13.20 (bs, 1H), 9.69 (s, 1H), 8.74 (s, 1H), 8.20−8.18 (m, 3H), 8.09 (s,
1H), 8.03 (d, J = 0.9 Hz, 1H), 7.85 (d, J = 8.7 Hz, 1H), 7.73 (dd, J =
8.7, 1.2 Hz, 1H), 7.68 (d, J = 1.2 Hz, 1H), 7.41 (m, 2H), 7.08 (t, J =
6.9 Hz, 1H); HRMS-ESI+ m/z calcd for C19H14N6 327.1353 (M +
H+), found 327.1362 (M + H+).
1H), 8.17 (s, 1H), 8.08 (s, 1H), 7.97−7.93 (m, 3H), 7.82 (d, J = 8.4
Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.61 (s, 1H), 6.79 (d, J = 9.0 Hz,
2H), 3.75 (t, J = 4.5 Hz, 2H), 3.61−3.58 (m, 6H), 1.94 (t, J = 5.7 Hz,
2H); HRMS-ESI+ m/z calcd for C24H23N7O 426.2037 (M + H+),
found 426.2049 (M + H+).
6-(1H-Indazol-6-yl)-N-(3-methoxyphenyl)imidazo[1,2-a]-
pyrazin-8-amine (65). The target compound was obtained following
the procedure outlined for 29: mp 238−240 °C; 1H NMR (300 MHz,
DMSO-d6) δ 13.21 (s, 1H), 9.66 (s, 1H), 8.73 (s, 1H), 8.18 (s, 1H),
8.10 (s, 1H), 8.05−8.03 (m, 2H), 7.85 (d, J = 8.4 Hz, 1H), 7.76−7.72
(m, 2H), 7.71 (s, 1H), 7.28 (t, J = 8.4 Hz, 1H), 6.63 (dd, J = 8.4, 2.1
Hz, 1H), 3.81 (s, 3H); HRMS-ESI+ m/z calcd for C20H16N6O
357.1458 (M + H+), found 357.1477 (M + H+).
6-(1H-Indazol-6-yl)-N-(3-methoxy-4-morpholinophenyl)-
imidazo[1,2-a]pyrazin-8-amine (73). The target compound was
obtained following the procedure outlined for 29 using 3-methoxy-4-
morpholinoaniline in the coupling step: mp > 250 °C; 1H NMR (300
MHz, DMSO-d6) 13.19 (s, 1H), 9.55 (s, 1H), 8.69 (s, 1H), 8.21 (s,
1H), 8.09−8.08 (m, 2H), 8.00 (s, 1H), 7.84 (d, J = 8.7 Hz, 1H), 7.75
(d, J = 8.7 Hz, 1H), 7.67−7.65.
Kinase Assays. Full-length baculovirus-expressed Syk (Cell
Signaling Technologies, Danver, MA) kinase activity was measured
in a Lance-based assay format in a final volume of 25 μL containing 25
mM Tris−HCl, pH 7.5, 5 mM β-glycerophosphate, 2 mM DTT, 0.1
mM Na3VO4, 10 mM MgCl2, 0.5 μM Promega PTK biotinylated
peptide substrate 1, 0.01% casein, 0.01% Triton X-100, 0.25% glycerol,
and 40 mM ATP (Km for ATP) incubated at room temperature for 60
min. Reactions were stopped with the addition of 30 mM EDTA
containing 30 μL of SA-APC and 4 nM PT-66 antibody and the plates
measured on a Perkin-Elmer Envision. IC50 values for test compounds
were determined using a four-parameter linear regression algorithm.
DiscoveRx Screen. Compounds were screened at 10 μM in the
KINOMEscan (DiscoveRx, San Diego, CA) assay, and the results for
the primary screen binding interactions are reported as “percent
control”, where lower numbers indicate stronger hits in the matrix.
Values of >35% are considered “no hits”. Kd determinations were
assessed at DiscoveRx.
6-(1H-Indazol-6-yl)-N-(4-methoxyphenyl)imidazo[1,2-a]-
pyrazin-8-amine (66). The target compound was obtained following
1
the procedure outlined for 29: mp > 250 °C; H NMR (300 MHz,
DMSO-d6) δ 13.17 (s, 1H), 9.57 (s, 1H), 8.68 (s, 1H), 8.18 (s, 1H),
8.08 (s, 1H), 8.06 (d, J = 9.0 Hz, 2H), 7.99 (d, J = 0.9 Hz, 1H), 7.84
(d, J = 8.4 Hz, 1H), 7.72 (dd, J = 8.4, 1.2 Hz, 1H), 7.64 (d, J = 0.9 Hz,
1H), 6.99 (d, J = 9.0 Hz, 2H), 3.79 (s, 3H); HRMS-ESI+ m/z calcd for
C20H16N6O 357.1458 (M + H+), found 357.1477 (M + H+).
1-(4-((6-(1H-Indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl)amino)-
phenyl)-3-methylazetidin-3-ol (67). The target compound was
obtained following the procedure outlined for 29: mp 265−268 °C
1
dec; H NMR (300 MHz, DMSO-d6) δ 13.18 (s, 1H), 9.38 (s, 1H),
8.63 (s, 1H), 8.17 (s, 1H), 8.08 (s, 1H), 7.97−7.93 (m, 3H), 7.83 (d, J
= 8.4 Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.62 (s, 1H), 6.52 (d, J = 8.7
Hz, 2H), 5.47 (s, 1H), 3.77 (d, J = 7.5 Hz, 2H), 3.61 (d, J = 7.5 Hz,
2H), 1.48 (s, 3H); HRMS-ESI+ m/z calcd for C23H21N7O 412.1880
(M + H+), found 412.1884 (M + H+).
6-(1H-Indazol-6-yl)-N-(4-morpholinophenyl)imidazo[1,2-a]-
Cellular Cross-Screening Activity Assays. Bone marrow derived
mouse mast cells (BMMCs), HUVECs, or SK-N-MCs were
resuspended at (1−2) × 106 cells/wells in Tyrode’s buffer
(BMMCs) or RPMI and incubated with compound dilutions for 1 h
followed by stimulation with 50 ng/mL SCF (BMMCs), 50 ng/mL
VEGF (HUVECs), or 100 ng/mL GDNF (SK-N-MCs). Following 3−
15 min of stimulation, the cells were washed in PBS and resuspended
in cell lysis buffer (Cell Signaling) and the proteins resolved by SDS−
PAGE. Immunodetection was evaluated for phospho-cKit (Cell
Signaling) in the BMMCs and normalized to total PLCγ2 (Santa
Cruz Biotechnology, Santa Cruz, CA), phospho-KDR (Cell Signaling)
in HUVECs, and phospho-Ret (Cell Signaling) in SK-N-MCs.
Detection was enabled by the use of infared-conjugated secondary
antibodies and Odyssey software (LiCor Biosciences, Lincoln, NE).
Jak2 Activity Assay. TF1 cells were serum starved overnight in
1% fetal bovine serum (FBS) RPMI medium at 1 × 106 cells/mL.
Cells were resuspended in fresh serum-free RPMI and incubated with
compound dilutions for 1 h followed by stimulation with 5 units/mL
erythropoietin (R&D Systems, Minneapolis, MN). The cells were
lysed in 50 μL of RIPA buffer, and phospho-Stat5 was detected using
an MSD phospho-Stat5 quantitation plate (Meso Scale Discovery,
Rockville, MD).
MV-4-11 Proliferation Assays. Functional impact on cellular Flt3
activity was determined by measuring compound inhibition of MV-4-
11 (ATCC no. CRL-9591) cell proliferation. A total of 104 cells were
diluted in RPMI medium containing 10% FBS in 96-well flat-bottomed
tissue culture plates and incubated with compound dilutions for 72 h
at 37 °C. Alamar blue (10%) was added to the cells, which were
incubated for an additional 12−18 h at 37 °C, and inhibition of the
relative cell numbers was determined by spectrophotometer readings
at 570/600 nm.
pyrazin-8-amine (68). The target compound was obtained following
1
the procedure outlined for 29: mp > 250 °C; H NMR (300 MHz,
DMSO-d6) δ 13.17 (s, 1H), 9.50 (s, 1H), 8.66 (s, 1H), 8.19 (s, 1H),
8.09 (s, 1H), 8.03 (d, J = 8.7 Hz, 2H), 7.99 (d, J = 0.9 Hz, 1H), 7.84
(d, J = 8.7 Hz, 1H), 7.72 (dd, J = 8.7, 0.9 Hz, 1H), 7.63 (d, J = 0.9 Hz,
1H), 7.00 (d, J = 8.7 Hz, 2H), 3.77 (t, J = 4.6 Hz, 4H), 3.11 (t, J = 4.6
Hz, 4H); HRMS-ESI+ m/z calcd for C23H21N7O 412.1880 (M + H+),
found 412.1894 (M + H+).
6-(1H-Indazol-6-yl)-N-(4-(dioxothiomorpholino)phenyl)-
imidazo[1,2-a]pyrazin-8-amine (69). The target compound was
1
obtained following the procedure outlined for 29: mp > 250 °C; H
NMR (300 MHz, DMSO-d6) δ 13.17 (s, 1H), 9.57 (s, 1H), 8.67 (s,
1H), 8.18 (s, 1H), 8.09−8.06 (m, 3H), 7.99 (d, J = 1.2 Hz, 1H), 7.84
(d, J = 8.4 Hz, 1H), 7.72 (dd, J = 8.4, 1.2 Hz, 1H), 7.64 (d, J = 0.9 Hz,
1H), 7.08 (d, J = 9.0 Hz, 2H), 3.76−3.74 (m, 4H), 3.18−3.16 (m,
4H); HRMS-ESI+ m/z calcd for C23H21N7O2S: 460.1550 (M + H+),
found 460.1557 (M + H+).
6-(1H-Indazol-6-yl)-N-(4-(tetrahydro-2H-pyran-4-yl)phenyl)-
imidazo[1,2-a]pyrazin-8-amine (70). The target compound was
obtained following the procedure outlined for 29 using nitro reagent
32 in the second step: mp > 300 °C; 1H NMR (400 MHz, DMSO-d6)
δ 13.19 (s, 1H), 9.64 (s, 1H), 8.71 (s, 1H), 8.20 (s, 1H), 8.12 (d, J =
8.8 Hz, 2H), 8.09 (s, 1H), 8.01 (d, J = 0.8 Hz, 1H), 7.85 (d, J = 8.4 Hz,
1H), 7.73 (dd, J = 8.4, 1.2 Hz, 1H), 7.66 (d, J = 0.8 Hz, 1H), 7.28 (d, J
= 8.8 Hz, 2H), 3.99−3.96 (m, 2H), 3.49−3.43 (m, 2H), 2.81−2.76 (m,
1H), 1.75−1.65 (m, 4H); HRMS-ESI+ m/z calcd for C24H22N6O
411.1928 (M + H+), found 411.1930 (M + H+).
2-((4-((6-(1H-Indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl)amino)-
phenyl)methylamino)ethanol (71). The target compound was
obtained following the procedure outlined for 29: mp 229−231 °C;
1H NMR (300 MHz, DMSO-d6) δ 13.18 (bs, 1H), 9.33 (s, 1H), 8.62
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Ramos Assay (pBLNK). Ramos cells were serum starved at 2 × 106
cells/mL in serum-free RPMI for 1 h in an upright T175 Falcon TC
flask. The cells were centrifuged (1100 rpm for 5 min) and incubated
at a density of 5 × 106 cells/mL in the presence of 3× serial dilutions
of test compound or DMSO controls for 1 h at 37 °C. The cells were
stimulated by incubation with 3 μg/mL antihuman IgM F(ab)2
(Southern Biotech, Birmingham, AL) for 5 min at 37 °C. The cells
were pelleted and lysed in 50 μL of cell lysis buffer. Phospho-BLNK
was detected using an MSD high bind plate coated for 1 h with 30 ng/
well total BLNK capture antibody (Santa Cruz Biothechnology).
Lysate was added, and the cells were washed in TBS−1% Tween-20
and probed with an antiphospho-Blnk-Y96 antibody (Santa Cruz
Biotechnology). Inhibition of the pBLNK was quantitated versus the
control well (Meso Scale Discovery).
Human CD86 Expression. Isolated human B-cells (Stem Cell
Technologies, Vancouver, Canada) were thawed briefly in a 37 °C
water bath and plated in RPMI 1640 medium supplemented with 10%
FBS, 100 units/mL penicillin−streptomycin, 0.01 M HEPES, 2 mM
GlutaMAX, 5 mM sodium pyruvate, and 10 mM β-mercaptoethanol at
a density of 5 × 105 cells/200 μL per well of a flat-bottom 96-well
plate. The cells were incubated with compound for 1 h in a 37 °C
incubator with 5% CO2 and stimulated with 20 μg/mL mouse F(ab′)2
antihuman IgM in the continued presence of compound for 16 h. The
cells were washed two times in PBS + 4% FBS and then stained with
20 μL each of anti-CD19 and anti-CD86 and 2.5 μL 7AAD for 40 min
on ice. The cells were pelleted at 300g for 5 min, washed three times
with PBS + 4% FBS, and analyzed by flow cytometry.
Human B-Cell Proliferation. Isolated human B-cells (Stem Cell
Technologies) were thawed in a 37 °C water bath and rested in RPMI
1640 medium supplemented with 10% FBS, 100 units/mL penicillin−
streptomycin, 0.01 M HEPES, 2 mM GlutaMAX, 5 mM sodium
pyruvate, and 10 mM β-mercaptoethanol for 5 h in a 37 °C incubator
with 5% CO2 and subsequently loaded with 5 μM CFSE per the
manufacturer’s instructions (Life Technologies, Carlsbad, CA). The
cells (3 × 105 cells/200 μL per well) in a round-bottom 96-well plate
were incubated with compound for 1 h in a 37 °C incubator, then
stimulated with 20 μg/mL goat F(ab′)2 antihuman IgM and 20 μg/mL
mouse anti-CD40, and incubated for 90 h in a 37 °C incubator. The
cells were rinsed once in PBS + 4% FBS and incubated with 7AAD for
30 min on ice. The cells were pelleted at 300g for 10 min, rinsed twice,
and analyzed by flow cytometry on the 7AAD− population, and
proliferation was estimated on the basis of the reduction of fluoroscein
staining.
Immune-Complex Stimulation of TNFα Production. Frozen
human monocytes (Stem Cell Technologies) were quickly thawed in a
37 °C water bath and rested for 3 h at 37 °C in RPMI 1640 medium
supplemented with 10% heat-inactivated FBS, 2 mM Glutamax, 1×
sodium pyruvate, 0.1 M HEPES, 10 mM β-mercaptoethanol, and 100
units/mL penicillin−streptomycin prior to plating in 96-well plates at
1 × 105 cells/well in 100 μL of complete RPMI. The cells were
incubated with compound for 1 h and stimulated with 4 μL of immune
complex at 40 μg/mL (stock solution of 300 μL of a polyclonal goat
F(ab′)2 antihuman Fc + 35 μL of purified human IgG + 65 μL of
medium (final mass ratio of 3:1) incubated on ice for 1 h prior to use)
for 16 h at at 37 °C. Culture supernatants were harvested and stored at
−20 °C until they were analyzed for TNFα levels using a singleplex
Meso Scale TNFα kit (Meso Scale Discovery).
light, and cell pellets were harvested by centrifugation at 1300 rpm for
10 min at RT. The pellets were washed one time with 1.0 mL of wash
buffer A for 5 min and recentrifuged. CD63 expression on CD123+/
HLA− cells was measured by fluorescence-activated cell sorting
(FACS) analysis on a Canto FACS Calibur (BD Biosciences), and the
CD63 expresssion (%) versus DMSO controls was used to determine
the EC50 in whole blood.
Rat Collagen-Induced Arthritis (CIA) Model. Female Lewis rats
from Charles River (mean mass 178 g, eight per group for collagen
arthritis, four per group for normal controls) were anesthetized with
isoflurane and injected with 300 μL of Freund’s incomplete adjuvant
(Difco, Detroit, MI) containing 2 mg/mL bovine type II collagen
(Elastin Products, Owensville, MI) at the base of the tail and two sites
on the back on days 0 and 6. Oral dosing (bid at 12 h intervals) was
performed on arthritic days 0−7 with vehicle (Cremophor/ethanol/
saline), GS-9973 (1, 3, or 10 mg/kg), or the reference compound
dexamethasone (Dex; 0.075 mg/kg) administered daily (qd). Rats
were terminated on arthritis day 16. Efficacy evaluation was based on
animal body masses, daily ankle caliper measurements, ankle diameters
expressed as the area under the curve (AUC), terminal hind paw
masses, and histopathologic evaluation of ankles and knees. PK was
measured from plasma samples taken 0, 2, 4, 8, 12, and 24 h post last
dose. The paws were fixed in formalin and processed for hemotoxylin
(H) and eosin (E) microscopy. H and E sections were scored for bone
resorption as follows: (0) normal; (0.5) normal on low magnification
but have the earliest hint of small areas of resorption in the metaphysis
with no resorption in the tarsal bones; (1) (minimal) small definite
areas of resorption in distal tibial trabecular or cortical bone or in the
tarsal bones, not readily apparent on low magnification, rare
osteoclasts; (2) (mild) more numerous areas (<25% loss of bone in
growth plate area) of resorption in distal tibial trabecular or cortical
bone and tarsals apparent on low magnification, osteoclasts more
numerous; (3) (moderate) obvious resorption of medullary trabecular
and cortical bone without full thickness defects in both distal tibial
cortices, loss of some medullary trabeculae with 26−50% loss across
the growth plate and cortices, some loss in tarsal bones, lesion
apparent on low magnification, osteoclasts more numerous; (4)
(marked) full or nearly full thickness defects in both distal tibial
cortices, often with distortion of the profile of the remaining cortical
surface, marked loss of medullary bone of distal tibia (50−100% loss
across the growth plate area and cortices and up to 50% loss in small
tarsals if minor in tibia), numerous osteoclasts, minor to mild
resorption in smaller tarsal bones; (5) (severe) full thickness defects in
both distal tibial cortices with >75% loss across the growth plate and
both cortices and >50% loss in tarsals, often with distortion of the
profile of the remaining cortical surface, marked loss of medullary bone
of distal tibia, numerous osteoclasts. Osteoclast counts (5400× fields)
were performed on the ankles in the areas of greatest bone resorption.
For statistical analysis, the ankle thicknesses, bone erosion scores,
osteoclast counts, and c-fos expression values (mean
SE) were
analyzed for group differences using the Student’s t test. Significance
was set at p < 0.05.
Competitive Protein Binding Assay. Human plasma and cell
culture medium containing 10% fetal bovine serum (CCM) were
spiked with the test compound at a final concentration of 2 μM.
Spiked plasma (1 mL) and CCM (1 mL) were placed into opposite
sides of the assembled dialysis cells, which were separated by a
semipermeable membrane. The dialysis cells were rotated slowly in a
37 °C water bath for the time necessary to reach equilibrium.
Postdialysis plasma and CCM masses were measured, and the test
compound concentrations in plasma and CCM were determined with
LC/MS/MS.
Metabolic Stability. Metabolic stability in vitro was determined
using pooled hepatic microsomal fractions (final protein concentration
of 0.5 mg/mL) at a final test compound concentration of 3 μM. The
reaction was initiated by the addition of an NADPH-regenerating
system. An aliquot of 25 μL of the reaction mixture was transferred at
various time points to plates containing a quenching solution. The test
compound concentration in the reaction mixture was determined with
CD63 Whole Blood Assay. Fresh human whole blood was
collected in sodium heparin vacutainers (catalog no. 367874, BD
Biosciences, San Jose, CA). A 2 μL sample of compound in 2× serial
dilutions was added to 100 μL of whole blood in a 96-well microtiter
plate and incubated for 1 h at 37 °C. A 20 μL sample of antihuman IL-
3 potentiation buffer B (catalog no. 10-0500, Glycotope Biotechnology
GmbH, Heidelberg, Germany) was added for 10 min at 37 °C,
followed by goat antihuman IgE (catalog no. H15700, Invitrogen,
Chicago, IL) stimulation for 20 min at 37 °C. The reaction was placed
on ice to stop degranulation, and the cells were stained with 20 μL of
anti-CD63-FITC/anti-CD123-PE/anti-HLA-DR-PerCEP (catalog no.
341068, Becton, Dickinson and Co., Pasadena, CA). Red blood cells
were lysed with 1.6 mL of buffer G for 10 min and protected from
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Article
LC/MS/MS. Predicted clearance was calculated using the well-stirred
liver model without protein restriction.
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Metabolic stability was also determined in cryopreserved
hepatocytes using tritiated test compounds. The incubation mixture
contained 1 × 106 hepatocytes/mL and 1 μM tritiated test compound
(2.5 μCi). The incubation was carried out with gentle shaking at 37 °C
under a humid atmosphere of 95% air/5% CO2 (v/v). Aliquots of 50
μL were removed after 0, 1, 3, and 6 h and added to 100 μL of
quenching solution. The samples were analyzed on a flow scintillation
radio detector coupled to an HPLC system. The metabolites were
quantified on the basis of the peak areas from the radio detector with
the cell-free control samples used as a reference. Metabolic stabilities
in hepatocytes were determined by measuring the rate of
disappearance of the test comound as ta percentage of the total
peak area of the formed radiolabeled metabolites and the test
compound.
Pharmacokinetics. Pharmacokinetic studies were performed in
male naive Sprague−Dawley (SD) rats, non-naive beagle dogs, and
cynomolgus monkeys (three animals per dosing route) following
federal and Institutional Animal Care and Use Committee (IACUC)
guidelines. Intravenous (iv) administration was dosed via infusion over
30 min in a vehicle containing 5% ethanol, 20% PEG400, and 75%
water (pH adjusted to 3.0 with HCl). Oral dosing was administered by
gavage in a vehicle containing 5% ethanol, 45% PEG 400, and 50% 50
mM citrate buffer, pH 3. Blood samples were collected over a 24 h
period postdose into Vacutainer tubes containing EDTA-K2. Plasma
was isolated, and the concentration of the test compound in plasma
was determined with LC/MS/MS after protein precipitation with
acetonitrile.
Noncompartmental pharmacokinetic analysis was performed on
plasma concentration data to calculate pharmacokinetic parameters
using the software program WinNonLin (version 5.0.1).
ASSOCIATED CONTENT
* Supporting Information
■
S
DiscoveRx primary screen data and selectivity scores for GS-
9973. This material is available free of charge via the Internet at
http://pubs.acs.org.
Accession Codes
The PDB code for compound 9 bound to Syk is 4PV0, and that
for compound 68 bound to Syk is 4PUZ.
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AUTHOR INFORMATION
Corresponding Author
*Phone: 203-315-7459. E-mail: scotta.mitchell@gilead.com.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Kathy Brendza and Loredana Serafini for generating
HRMS data, Jayaraman Chandrasekhar for modeling input,
Will Watkins and Gregory Notte for manuscript review, and
Douglas Stafford for chemistry outsourcing management at
Albany Molecular Research Institute (AMRI).
ABBREVIATIONS USED
■
Syk, spleen tyrosine kinase; ITAM, immunoreceptor tyrosine-
based activation motif; BLNK, B-cell linker protein; RA,
rheumatoid arthritis; TNFα, tumor necrosis factor α; ITP,
idiopathic thrombocytopenia purpura; SLE, systemic lupus
erythramatosis; AHA, autoimmune hemolytic anemia; SAR,
structure−activity relationship; BCR, B-cell receptor; DTT,
dithiothreitol; BMMC, bone marrow-derived mast cell;
HUVEC, human umbilical vein endothelial cell; SCF, stem
cell factor; FACS, fluorescence-activated cell sorting
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