Discovery, synthesis, and characterization of an orally bioavailable, brain penetrant inhibitor of mixed lineage kinase 3

Article abstract of DOI:10.1021/jm401094t

Inhibition of mixed lineage kinase 3 (MLK3) is a potential strategy for treatment of Parkinson's disease and HIV-1 associated neurocognitive disorders (HAND), requiring an inhibitor that can achieve significant brain concentration levels. We report here URMC-099 (1) an orally bioavailable (F = 41%), potent (IC₅₀ = 14 nM) MLK3 inhibitor with excellent brain exposure in mouse PK models and minimal interference with key human CYP450 enzymes or hERG channels. The compound inhibits LPS-induced TNFα release in microglial cells, HIV-1 Tat-induced release of cytokines in human monocytes and up-regulation of phospho-JNK in Tat-injected brains of mice. Compound 1 likely functions in HAND preclinical models by inhibiting multiple kinase pathways, including MLK3 and LRRK2 (IC₅₀ = 11 nM). We compare the kinase specificity and BBB penetration of 1 with CEP-1347 (2). Compound 1 is well tolerated, with excellent in vivo activity in HAND models, and is under investigation for further development.

Full text of DOI:10.1021/jm401094t

Article
pubs.acs.org/jmc
Discovery, Synthesis, and Characterization of an Orally Bioavailable,
Brain Penetrant Inhibitor of Mixed Lineage Kinase 3
Val S. Goodfellow,*,† Colin J. Loweth,†,⊥ Satheesh B. Ravula,†,# Torsten Wiemann,† Thong Nguyen,†
Yang Xu,∥,¶ Daniel E. Todd,§ David Sheppard,§ Scott Pollack,§ Oksana Polesskaya,‡ Daniel F. Marker,‡
Stephen Dewhurst,‡ and Harris A. Gelbard‡
†Califia Bio Inc., 11575 Sorrento Valley Road, San Diego 92121, California, United States
‡University of Rochester Medical Center, School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, New York 14642,
United States
§BioFocus, Chesterford Research Park, Saffron Walden, Essex CB10 1XL, U.K.
∥Biofocus DPI, 9640 Towne Centre Drive, San Diego, California 92121, United States
S
* Supporting Information
ABSTRACT: Inhibition of mixed lineage kinase 3 (MLK3) is a potential strategy
for treatment of Parkinson’s disease and HIV-1 associated neurocognitive disorders
(HAND), requiring an inhibitor that can achieve significant brain concentration levels.
We report here URMC-099 (1) an orally bioavailable (F = 41%), potent (IC50
=
14 nM) MLK3 inhibitor with excellent brain exposure in mouse PK models and
minimal interference with key human CYP450 enzymes or hERG channels. The
compound inhibits LPS-induced TNFα release in microglial cells, HIV-1 Tat-induced
release of cytokines in human monocytes and up-regulation of phospho-JNK in Tat-
injected brains of mice. Compound 1 likely functions in HAND preclinical models by inhibiting multiple kinase pathways,
including MLK3 and LRRK2 (IC50 = 11 nM). We compare the kinase specificity and BBB penetration of 1 with CEP-1347 (2).
Compound 1 is well tolerated, with excellent in vivo activity in HAND models, and is under investigation for further
development.
monocytes to the CNS, (3) viral infection of microglia leading
to interruption of their normal function, and (4) extensive
synaptodendritic damage, which ultimately impacts polysynap-
tic pathways that are the substrate for HAND in affected
regions of the brain. A host of inflammatory mediators have
been implicated in cellular models of HAND, where TNF-α
release and signaling likely play a major central role. A more
limited subset of mediators has been identified as being up-
regulated in the cerebrospinal fluid (CSF) and post-mortem
brain tissues of HAND patients. These mediators/effectors
include TNFα, the chemokine monocyte chemoattractant
protein (MCP-1), and from preclinical models, mixed-lineage
kinase 3 (MLK3), an important control point in MAPK kinase
regulated inflammation pathways.3
Mixed lineage kinases are mitogen activated protein kinase
kinase kinases (MAPKKKs) with features of both serine−
threonine and tyrosine kinases that regulate the c-Jun N-terminal
kinase (JNK) mitogen activated protein kinase (MAPK) sig-
naling cascade and also regulate p38 and extracellular signal-
regulated kinase (ERK).4−6 MLK3 (MAP3K11) is the most
widely expressed MLK family member4−6 and is expressed in
neurons7 (as well as other cell types).8,9 At the cellular level,
MLK3 is activated by stress, including reactive oxygen species,
INTRODUCTION
■
We report here the discovery, synthesis, and characterization of
URMC-099 (1), a new inhibitor of mixed lineage kinase type 3
(MLK3) with excellent blood−brain barrier penetration
properties, which has shown neuroprotective and antineuroin-
flammatory properties in in vitro and in vivo models of HIV-1
associated neurocognitive disorders (HAND).1 Combination
antiretroviral therapy (cART) has greatly increased both the life
expectancy and quality of life for HIV-1 seropositive individuals
and is one of the greatest success stories of modern drug
development. However, as the population of AIDS patients has
aged, it has become apparent that neurological impairments
resulting from HIV infection are not controlled by cART and
may indeed be exacerbated by some CNS penetrating
antiretroviral agents used in HIV therapy.2 HAND encom-
passes a broad range of neurologic deficits that range from mild
cognitive impairment to frank dementia and is the result of
damage to normal synaptic architecture that is likely mediated
by dysregulation of immune cells in the CNS. In the U.S.,
greater than 50% of AIDS patients experience some symptoms
of HAND, with a significant percentage (15%) exhibiting neu-
rologic morbidity severe enough to preclude normal activities
of daily living with substantial economic impact for their health-
care.2
The hallmarks of HAND include: (1) a dysregulation of
inflammatory cytokines and chemokines, (2) the recruitment of
Received: July 18, 2013
© XXXX American Chemical Society
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ceramide, and TNFα.10,11 At the molecular level, it is activated
by Cdc42 and Rac, which interact with MLK3, and can cause it
to dimerize via a leucine zipper interface, resulting in
autophosphorylation at Thr277 and Ser281 within the protein
activation loop and enzyme activation.12,13 HIV-1 Tat also leads
to phosphorylation at these same residues in primary rat neu-
rons14 and to activation of glycogen synthase kinase (GSK-3ß)
in neurons.15,16 This is important because MLK3 can be acti-
vated as a result of direct phosphorylation by GSK-3β.17
Previously published MLK3 inhibitors, CEP-134718 (2), K252a6
(3), CEP-70119 (4), CEP-1100420 (5), and compound 621
(Figure 1), have been based largely on the protein kinase-
significant CNS penetration, which might serve as a first-in-
class treatment, adjunctive to cART, for HAND.
RESULTS AND DISCUSSION
■
We have identified a 7-azaindole based MLK3 inhibitor 1, with
potent in vitro activity against HIV-1 Tat mediated release of
cytokines in human and mouse immune cells, including cul-
tured mouse microglial BV-2 cells; it is also neuroprotective in
mice exposed to HIV-1 Tat.1 In mouse brain imaging
experiments, compound 1 has demonstrated its in vivo efficacy
for preservation of normal synaptic architecture and reversal of
neuroinflammation following CNS exposure to HIV-1 Tat.1
Because of a compelling profile of central anti-inflammatory
effects and neuroprotection, compound 1 is being evaluated for
adjunctive therapy with or without cART in humanized mice
engrafted with human CD34 cells that can sustain peripheral
and central infection with HIV-1.31
A proprietary focused library of kinase inhibitors from
BioFocus Ltd. containing approximately 15000 unique com-
pounds was screened against MLK3 in a radiometric filter plate
assay format. A large number of active compounds with con-
sistent structure activity themes were identified from
structurally distinct chemical libraries. We focused our initial
hit-to-lead development on the imidazopyrazinone series based
around screening hit (7) (see Figure 3). This compound gave
variable potency in enzyme inhibition assays (MLK3 IC50
ranged between 40−120 nM); we believed this variability was
due to solubility issues. Cellular assays also suggested poor
ability to penetrate cells (LPS-stimulated TNF-α release in
BV-2 mouse microglial cells, IC50 = 23.6 μM). However, the
simple structure, well-developed SAR from screening hits, ease
of synthesis, and potential for scaffold hopping from this series
of compounds more than compensated for its initial drawbacks.
During the modification of this scaffold, the design of
compounds for optimization focused on low molecular weight
compounds (<450 MW) with small numbers of hydrogen bond
donors (<3) and low polar surface area to maximize chances of
CNS penetration. Analysis of virtual libraries of potential
synthetic targets with Stardrop (Optibrium Ltd.) guided us
toward the 7-azaindole type scaffold and compounds related to
the indole containing (8), a direct analogue of 7. Compound 8
(see figures 2 and 3) was much more potent against MLK3 and
showed significant inhibition of the release of TNFα in BV-2
cells stimulated with LPS. We rapidly synthesized a number of
potent analogues around this structure type with the goal of
simultaneously optimizing (1) high potency in a biochemical
MLK3 inhibition assay, (2) inhibitory activity in macrophages/
microglia to prevent HIV-1 Tat-induced stimulation of a
relevant panel of cytokines of validated importance in HAND,
(3) metabolic stability, (4) CNS penetration, and (5) high
brain and plasma concentrations upon oral dosing. Ultimately,
compound 1 emerged as an attractive candidate in a small
focused library of very active compounds and was designed
through a strategy to introduce solubilizing groups known to be
effective in marketed kinase inhibitors. Our goal was to replace
the trimethoxy aryl residue, with a group that could exhibit very
low polar surface area in order to increase the potential for
CNS penetration. The benzyl-linked N-methyl piperazine
moiety met these criteria and yielded compound 1, both highly
potent against MLK3 and with very low polar surface area.
Inhibition of LPS-Induced TNFα Release in Murine
BV-2 Microglial Cells. After obtaining potent MLK3 inhi-
bition data, we used an ELISA-based assay to gauge if our
Figure 1. Examples of known potent MLK3 inhibitors.
promiscuous staurosporine scaffold. Compound 2 has been
used as a tool compound to explore the effects of MLK3
inhibition for HAND and Parkinson’s disease22−24 in cellular
and animal models, although the compound is in no way
specific for MLK3. Compound 2 has also been a central player
in the target validation of mixed lineage kinases for HAND.
Compound 2 protected primary rat hippocampal neurons as
well as dorsal root ganglion neurons from the otherwise lethal
effects of exposure to HIV-1 coat protein gp120.25,26 Tat and
gp120 induce autophosphorylation of MLK3 in primary rat
neurons, which was abolished by the addition of 2. Compound
2 also enhanced survival of both rat and human neurons and
inhibited the activation of human monocytes after exposure to
Tat and gp120.14 Compound 2 is neuroprotective in an in vivo
model of HIV-1 infection, reversing microglial activation and
restoring normal synaptic architecture, as well as restoring
macrophage secretory profiles to a trophic vs toxic phenotype
in response to HIV-1 infection.27
Unfortunately, 2 failed to show efficacy in a large phase II
clinical trial for early stage Parkinson’s disease.28 Compound 2
has a high molecular weight (MW = 615) with a large polar
surface area (PSA = 95 Å2), properties which are not conducive
to blood−brain barrier (BBB) penetration. There are additional
reasons to suspect that 2 likely did not maintain therapeutic
levels in the brains of significant numbers of patients. No
published data for CNS penetration is available for this
compound, however, 2 is known to interact with and inhibit
CYP450 enzymes.29 Plasma PK concentration data for human
subjects receiving 2 and antiviral therapy showed very large
patient to patient variation in blood levels of 2, which may be
indicative of induction of metabolic liver enzymes. In silico
assessment using Optibrium30 software suggested poor CNS
penetration of 2, confirmed by blood−brain barrier studies
reported here, as well as the likelihood of interaction with liver
enzymes, which could lead to further degradation of CNS
exposure over time. Therefore our goal was to identify a new
potent MLK3 inhibitor with improved pharmacokinetics and
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Figure 2. Potent and drug-like 7-azaindole MLK3 inhibitors selected for initial profiling.
Figure 3. Optimization design path to compound 1.
potent MLK3 inhibitor compounds were affecting the MAPK
kinase pathway, which regulates TNFα release in microglial
cells, one of the key cytokines responsible for inflammatory
effects in HAND.32−35 This secondary assay allowed us to
identify chemotypes that readily penetrated relevant immune
cells and elicited functional activity in reduction of this key pro-
inflamatory cytokine. We profiled 132 potent compounds for
activity in MLK3 inhibition, TNFα release inhibition assays, as
well as drug-like properties (see Figure 2) and selected six
compounds (1, 8, 9, 10, 11, 12) for preliminary pharmaco-
kinetic screening in mice (see Figure 2).
Screening Phamacokinetics Studies and Blood−Brain
Barrier Penetration. It is a widely held belief that few potent
protein kinase inhibitors penetrate the CNS, although pub-
lished data are scant regarding CNS exposure for these mostly
anticancer drugs. Recent strategies in kinase drug development
have skewed the propensity for these drugs to have properties
unfavorable for BBB penetration.36
possible, with nonlinear shapes, few hydrogen bond donors,
Log D between 2 and 5, and low polar surface area. We tested
the most promising compounds in screening PK studies for
BBB penetration in mice. Test compounds were dosed iv in
C57BL/6 male mice, through the tail vein, for a total dose of
10 mg/kg per animal, and standard pharmacokinetic parame-
ters were determined (Table 1). Both 1 and 8 exhibited
significant brain concentrations (brain AUC (μg/kg·h) = 5130
and 1535, respectively) over the time course of the experiment
with excellent brain/plasma ratios. The brain concentrations of
1 remained well above the in vitro MLK3 inhibition IC50 for
over 6 h after injection. The volume of distribution for 1 is
within acceptable bounds and does not indicate that the
compound will accumulate in tissues. The brain and plasma
concentrations for 8 were very closely matched, giving B/P
ratios of ∼1 throughout the entire experiment. A B/P ratio of 1
suggests that the compound partitions fairly freely across the
BBB, indicating few issues with PGP efflux pumps or accu-
mulation of compound in brain tissue. However, compound 8
was rapidly metabolized in mice. In our experience with the
Our strategy for finding CNS penetrant compounds for
proof-of-concept studies was to keep structures as simple as
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Table 1. Screening PK Study (iv, 10 mg/kg, C57 BL/6 Mice)
parameter
AUC (μg/L·h)
1
8
9
10
11
12
3410
2.72
11.5
2.9
1560
0.69
6.43
6.42
2744
1535
3736
0.99
1400
1.13
11.7
7.1
1630
1.21
10.7
6.2
3150
1.95
8.92
3.2
1840
0.29
2.2
T1/2 (h)
b
Vz (L/kg)
CLz (L/h/kg)
5.4
Cmax (μg/L)
1848
5130
4685
1.6
4742
213
530
1.2
4823
83
9788
693
4250
136
283
0.1
brain AUC (μg/kg·h)
brain Cmax (μg/kg)
314.5
834
a
brain/plasma ratio @ 1 h
BQL
1.02
a
b
Below quantification level. Apparent volume of distribution during terminal phase.
7-azaindole based inhibitors, polar surface area, molecular
weight, and number of hydrogen bond donors were most
important in determining whether a compound would exhibit
good CNS exposure. Thus compounds such as 12 with high
polar surface area or 10 with large number of hydrogen bond
donors, exhibited poor BBB penetration in this preliminary
study (see Table 1.)
Oral PK Study with Compound 1. To gauge the potential of
compound 1 to function as a drug and to establish that oral
dosing would be allowed in animal models, we initiated an oral
PK study where compound 1 was dosed at 10 mg/kg in
C57BL/6 mice (see Tables 2 and 3). To determine %F, iv
BBB penetration assay, mice were dosed iv at 10 mg/kg and
brain and plasma levels are measured at three time points
utilizing averages from three mice per time point (see Table 4).
In this assay format, 1 again achieved significant brain levels
(e.g., at 3 h, the whole brain concentration was 950 ng/g, with a
B/P ratio of 0.81). We utilized this rapid assessment of BBB
penetration to compare 2 with our compounds. In this iv dos-
ing assay, 2 showed significantly lower plasma and brain levels
at all time points, with poor B/P ratios. For example, by three
hours after iv dosing at the same starting dose (10 mg/kg), the
brain concentration of compound 1 (ng/g) was measured as
14 times higher than that observed for 2. While these numbers
may not necessarily translate into high free fraction concen-
trations in human brain, taken together with human micro-
somal and hepatocyte stability data, they suggest that
compound 1 is much more likely to achieve significant CNS
levels. Our method does not determine free brain/free plasma
fraction concentration ratios. This determination may be
important when drugs are very tightly protein bound in the
brain (>99%). With very high protein binding, the drug may
be present in the brain but may not engage the therapeutic
target. Compounds showing weaker protein binding (<99%)
obviously are in equilibrium with an apparent binding constant
that is usually much weaker than the highly potent Kd or Km of
drugs with significantly increased activity for the target kinase
MLK3. It is well-known that for such drugs, the protein bound
fraction represents a reservoir of displaceable drug in equilib-
rium with free drug. We have provided data (see Figure 2) to
rank order compounds for protein binding in human plasma.
This data shows that protein binding for compound 1 is
approximately 98%, and we expect the reservoir of drug in
the brain to be in free equilibrium and available to bind to the
MLK 3 target enzyme with high affinity (Ki ∼ 10 nM). This
conclusion is supported by the fact that the terminal half-life for
brain concentrations for compound 1 has been measured in
mice at between 1.5 and 2 h, very close to the plasma half-life,
suggesting that compound 1 in mice is not highly protein
bound in the brain and is available to be metabolized by sys-
temic CYP450 enzymes with modest Km affinity and is also
available for target engagement with high affinity for MLK3.
A Direct Control Analogue of Compound 1 with Poor CNS
Penetration. We have also identified a very closely related ana-
logue (13) of compound 1 with very limited CNS penetration.
While compound 13 had similar MLK3 potency and activity
compared to compound 1 in simple kinase specificity screens, it
had good systemic exposure but much lower brain levels when
administered iv to C57Bl/6 mice, with B/P ratios of 0.04, 0.07,
and 0.13 at 0.5, 1, and 3 h, respectively. This result was not fully
anticipated based on in silico predictions or results with similar
analogues. The formal replacement of carbon by nitrogen
Table 2. Low Dose iv Administration of Compound 1 in C57
BL/6 Mice (2.5 mg/kg)
parameter
1
AUC (μg/L·h)
T1/2 (h)
1400
2.14
5.6
Vz (L/kg)
CLz (L/h/kg)
Cmax (μg/L)
1.8
570
Table 3. Oral Dosing of Compound 1 in C57 BL/6 Mice
(10 mg/kg)
parameter
1
AUC (μg/L·h)
T1/2 (h)
2300
1.92
2670
4.0
Cmax (μg/L)
Tmax (h)
%F
41
dosing was repeated at a lower concentration (2.5 mg/kg) to
ensure that metabolic mechanisms would not be saturated by a
high dose of test article dissolved in DMSO/PEG400. Using iv
(2.5 mg/kg) and PO (10 mg/kg) dosing in C57Bl/6 mice,
analysis of plasma concentrations of compound 1 yielded
standard pharmacokinetic parameters, confirming proportion-
ally similar exposures as measured by AUC levels at lower doses
and similar half-life (terminal half-life iv = 2.14 h, oral = 1.92 h)
and good oral bioavailability (%F = 41) using a simple standard
oral dosing vehicle containing 0.4% Tween-80 and 0.5%
hydroxypropylmethyl cellulose in pH 7.4 buffered saline.
Blood−Brain Barrier Penetration of 2. Our stated goal was
to identify a compound with significantly improved CNS pen-
etration over 2. To rapidly assess and compare the ability of a
large number of compounds to penetrate the CNS, we adopted
a simple screening model utilizing C57 BL/6 mice which were
also employed in our imaging efficacy assays.1 In the screening
D
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Table 4. Blood−Brain Barrier Penetration Comparison (iv Dosing, 10 mg/kg)
B/P ratio
30 min
B/P ratio
60 min
B/P ratio
180 min
compd
sample
30 min
60 min
180 min
a
1
plasma
4163
2833
2920
2249
1167
950
b
brain
0.68
0.77
0.07
0.27
0.81
a
a
13
2
plasma
3613
164
2566
179
936
130
b
brain
0.04
0.25
0.138
0.21
plasma
2403
594
1716
463
326
67
b
brain
a
b
ng/mL. ng/g.
provides both a new basic center and a site for hydrogen bon-
ding. It may be that the side chain azaindole pyridine nitrogen
alters the preferred solution conformation compared to com-
pound 1, allowing better exposure to hydrogen bond inter-
actions with that side chain with proteins or water. Such
increased H-bonding could impede BBB penetration.
to Tat-only treatment, was achieved at the lowest drug
concentration tested (100 nM) for TNFα and IL-6 and at
300 nM for MCP1; in the case of IL-8, more than 50% inhi-
bition of chemokine release was attained by 300 nM of drug,
although this did not achieve statistical significance due to high
variation in the Tat-only control samples (Figure 4). Similar
Metabolic Stability, CYP450 Inhibition, hERG Inhibition.
When 1 was tested in vivo in mice, the terminal half-life after
oral and iv administration was measured as 2.1 and 1.9 h,
respectively. Data were obtained for the stability of 1 in the
presence of human microsomes (45%) as well as human (55%),
monkey (55%), and mouse hepatocytes (73%), where the data
is expressed as “% remaining after 1 h”. The human microsome
and hepatocyte stability data are similar to several marketed
drugs used as standards in the assays, suggesting that while the
half-life is not ideal for once a day dosing, it likely would be
acceptable for a first-in-class drug for HAND where no known
treatment currently exists. There were no significant CYP450
inhibition issues for 1 identified from the three most common
isoforms of interest for drug/drug interactions (see Table 5).
Table 5. CYP450 Inhibition (IC50, μM)
CYP450
1
8
9
10
11
12
Figure 4. Inhibition of HIV Tat stimulated cytokine release in human
monocytes with compound 1 at 100, 300, and 1000 nM
concentrations (NT = no treatment).
2D6
3A4
2C9
>30
>30
>30
>30
3.7
4.2
>30
>30
>30
13.2
21.8
>30
>30
>30
16.2
>30
13.7
>30
findings were observed in human monocyte-derived macro-
phages (MDM, data not shown) with the most potent inhibition
of our inhibitors against MCP-1 and TNF-α release from MDM.
In Vivo Inhibition of HIV-1 Tat Induced JNK Signaling with
Compound 1. HIV-1 Tat activates JNK in neurons and other
cell types, via an MLK-dependent pathway.14,41−44 JNKs are
major regulators of mammalian apoptotic cell death pathways
and have also been proposed to have an important role in
neurodegeneration.45 However, direct inhibition of JNK may
not be an acceptable strategy for disease modification because
the role of JNK in cell death is so ubiquitous in different cell
types, tissues, and organs. Further, there is significant JNK
phosphorylation and activity in neurons from healthy brain
tissue, indicating that JNK plays multiple roles in the healthy
CNS. A selective strategy of interfering with HIV-induced
signaling upstream of JNK, such as MLK signaling, may pro-
vide an opportunity to develop a more selective treatment
for HAND with less risk of side effects and undesired toxicity
as well as a potential treatment for other neurodegenerative
diseases.
Kinase inhibitors possessing amine-solubilizing groups such as
the piperazine present potential risk for inhibition of hERG
channels, with associated risks of QT prolongation. The auto-
mated Cerep whole cell patch-clamp technique37 (Qpatch 16,
Sophion Biosciences) was used to record outward potassium
currents from single cells transfected with the hERG channel.
The IC50 for 1 was determined as 21 μM and was rated as
low cardiac toxicity risk by the CEREP compound evaluation
criteria.
Inhibition of Tat-Induced Pro-inflammatory Cytokine
Release by Primary Human Monocytes. We tested whether
1 could regulate the HIV-1 Tat-induced release of a set of
pro-inflammatory cytokines from primary human monocytes.
We focused on TNF-α, MCP-1, IL-6, and IL-8 because these
are believed to be of importance of the pathogenesis of
HAND.32−40
Compound 1 potently inhibited all of the mediators in a dose
dependent fashion. Statistically significant inhibition, compared
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To verify in vivo inhibition of the HIV-1 Tat-induced JNK
pathway activation by compound 1, C57BL/6 mice were
injected intracerebrally with saline or HIV Tat1−72, 700 μm
deep in somatosensory cortex by stereotactic injection. This
paradigm reproducibly results in sustained in vivo neuro-
inflammation after Tat injection.1 Half of the mice received ip
pretreatment/treatment with 10 mg/kg of compound 1, every
12 h, which continued until sacrifice, with a total of three doses
before Tat injection and two doses post injection while the
other half were untreated (n = 3 for each of the four groups).
Mice received the Tat injection at 4−6 h after the third ip
injection of compound 1. In separate single dose PK exper-
iments, plasma levels measured at 4 h post injection of drug
were 500 nM and brain levels were also at 500 nM. At 6 h post
drug injection, plasma levels were at 275 nM and brain levels
were at 233 nM. Mice were anesthetized and sacrificed 24 h
after Tat injection. Levels of phosphorylated JNK (pJNK) were
measured by Western blotting from whole brain lysates
collected directly adjacent to the Tat injection site (the caudal
medial quadrant of the left hemisphere). To control for vari-
ations in protein loading between experimental replicates, levels
of pJNK were normalized to the expression of total α-tubulin
(see Figure 5A). The untreated Tat exposed animals had a 75%
based inhibitors cocrystallized in kinase active sites. Docking
studies of compound 1 in the MLK1 ATP binding site were
performed using the automated docking utilities contained
within the molecular modeling suite MOE46 as well as manual
dockings and minimizations, allowing complete flexibility of the
protein and ligand (refer to Figure 6). MLK1 is an excellent
Figure 6. Potential binding site interactions analysis for MLK1 and
compound 1 based on manual docking and minimization studies.
model for MLK3 binding, and 1 potently inhibits MLK1 (IC50 =
19 nM). In aggregate, these analyses suggested that the com-
pound is likely a type 1 kinase inhibitor forming two hydrogen
bonds to the hinge. The indole is likely positioned so that it
may make potential key interactions with neighboring lysine,
aspartic acid, asparagine, or phenylalanine residues. In these
models, the piperazine moiety interacts with a critical arginine
residue and is also partially solvent exposed.
To the best of our knowledge, no X-ray crystal structure for
MLK3 has yet been published. Cephalon has released an X-ray
crystal structure (3DTC) of one of their inhibitors 6 co-
crystallized in MLK1.22 All but one of the significant residues in
the MLK1 ATP binding site are identical to MLK3 (Figure 6).
Phe222, present in the hinge region of MLK1, is a tyrosine in
MLK3, however, the side chain is outwardly facing and its
impact is minimal on ligand binding. In our SAR studies, we
observed that the pyrrolo NH and the pyridine nitrogen of the
scaffold were required for potency. In addition, for the side
chains, having an aromatic group such as an indole, aniline, or
phenol was associated with high in vitro enzyme inhibition
potency as they provided a hydrogen bond donor from the para
position of an aromatic ring.
Figure 5. Intraperitoneal (ip) compound 1 treatment prevents HIV-1
Tat mediated increase in JNK phosphorylation in vivo in brain tissue
ipsilateral to stereotactically injected Tat. (A) Immunoblots of the
expression of pJNK and α-tubulin in brain tissue of mice stereotacti-
cally injected with saline (Sa) vehicle and no treatment or Sa+
systemic administration of 1 or stereotaxic injection of HIV-1 Tat and
no treatment or stereotaxic injection of Tat with systemic
administration of 1. (B) Densitometric analyses of the optical density
of bands in (A), normalized to the expression of α tubulin in each
experimental replicate. * = p < 0.05.
increase in the levels of phosphorylated JNK compared to the
saline control animals (Figure 5B). Compound 1 treatment in
the Tat exposed animals normalized phosphorylated JNK levels
to those observed in saline controls (Figure 5B) (*, p < 0.05).
We analyzed data depicted in Figure 5A,B by one-way ANOVA
with Newman−Keuls post hoc test. Error bars indicate SEM,
but the compound had no effect on pJNK levels in saline-
treated animals.
In studying the sequences of related kinases, only MLK1,
MLK3, and ZAP70 contained the Gly225-Gly226-Pro227 motif,
positioned at the top of the hinge, however 10 kinases con-
tained a proline residue at the position equivalent to Pro227
.
Molecular Modeling Studies with Compound 1. Analysis of
the potential binding modes of compound 1 were based on
known SAR as well as crystal structures of similar 7-azaindole
One such kinase was spleen tyrosine kinase, SYK, which had
been crystallized with the Rigel compound, R406.47 In this
structure, the 3,4,5-trimethoxyphenyl group extended into the
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solvent exposed region where it was observed to buttress the
proline and also interact with a lysine residue in the equivalent
position to Arg203. (MLK3 numbering) This group was recog-
nized within our SAR in early compounds such as 7 and 8, and
it was proposed that a similar interaction could take place with
this group extending into the solvent exposed region.
The 7-azaindole scaffold, in recent years, has been demon-
strated to be a privileged structure for forming interactions with
the hinge region of ATP binding sites of several kinases, and
these compounds are the subject of several patents and studies
of binding fragments for several protein kinases including
AKT1, IKKB, JAK3, SGK1, and ABL1 mutants.48 A binding
mode analysis performed against kinase X-ray structures con-
taining ligands with an azaindole scaffold revealed consistency
with our binding mode hypothesis identified through docking
studies with homology models and PDB 3DTC.
Of particular interest to us were 7-azaindole ligands bound in
PDB 3ETA (insulin-like growth factor-1 receptor (IGF-1R))
tyrosine kinase and 2Z60 (T315I mutant of ABL1), which
exhibited a binding mode we had originally focused on from
docking studies with homology models. In these structures, the
pyrrolo hydrogen of the inhibitor donates a hydrogen bond to
the carbonyl of the Hinge +1 residue, and the pyridine nitrogen
accepts a hydrogen bond from the hinge +3 N−H moiety.
3-Position substituents would likely interact with Phe155 in
MLK1 and Asn273. 5-Position substituents on the 7-azaindole
scaffold may interact with Arg230 and may be largely solvent
exposed, allowing the incorporation of solubilizing groups. This
model is consistent with all structure−activity data.
P38a, SRK, SYK, JNK1, and ZAP70; see Table 6). In this panel
of important diverse kinases from TK and AGC families, only
LCK, a fairly promiscuous kinase, yielded an IC50 potency
below 500 nM. As interest in this compound progressed with
uniformly superior activity in cellular and in vivo models of
HAND, we began to explore reasons for the unique activity.
Using a bioinformatics approach, we identified six kinases which
have known high affinity for closely related azaindole structures
for further screening of the inhibition profile of 1. Two of these
kinases had crystal structures of bound 7-azaindole inhibitors
available to allow us to explore modeling of kinase specificity
based on the observed subsite interactions.
Although compound 1 had initially appeared fairly selective
on the original screening panel, on these six selected kinases
compound 1 exhibited significant potency for several of the
kinases in these biochemical assays for inhibition of phosphor-
ylation of substrates (Table 7). We expanded our investigation
a
Table 7. IC50 Values for Kinase Panel Selected by
Bioinformatics Analysis of Kinase Ligands
compounds
kinase/assay
1
13
ABL1 (T315I)
3
285
720
CDK2
1180
4
FLT3
9.5
IKKa
591
257
200
560
330
288
IKKb
IR
1290
730
FLT3(MEF Cells)
Protein Kinase Specificity. Previously, little protein kinase
specificity data has been published for MLK3 inhibitors. Com-
pound 3 is a fairly promiscuous inhibitor.49 Compound 4 was
originally identified as a potent inhibitor of FLT3 or JAK iso-
forms but is also a fairly potent inhibitor of MLK kinases and a
close analogue of 2.49 Compound 6 was reported to be a potent
inhibitor of MLK1 and a weak inhibitor of TrkA and Fyn.21
Compound 1 (MLK3 IC50 = 14 nM) inhibits both MLK1
(IC50 = 19 nM) as well as MLK2 (IC50 = 42 nM) and the
related MLK family member DLK (IC50 = 150 nM), as ex-
pected from the analysis of the active sites of these enzymes.
We initially utilized a screen of nine protein kinases to profile
potential compounds (AMPK, CDK1, ERK2, GSK3B, LCK,
a
Average of two determinations (nanomolar). IC50s for various kinases
in specificity screens were conducted by Reaction Biology, Inc.
(Malvern, Pennsylvania) as described in ref 51. MEF cell experiments
described in Experimental Section.
of specificity using the binding kinome scan ScanMax assay from
DiscoverRx (originally conducted at Ambit Biosciences), so com-
parison could be made with compounds described in the literature.
In this assay, compound 1 was screened against a panel of
442 kinases and showed greater than 90% inhibition against 111
wild-type human protein kinases at 1 μM (data in Supporting
Information). The ScanMax assay from DiscoverRx49,50 is
widely used to compare kinase inhibitor specificity based on
extrapolated kinase/inhibitor binding and to produce thermo-
dynamic binding constants, Kd. The assay measures the ability
of significantly modified kinases to bind to a solid support
immobilized ligand and utilizes conditions not present in living
cells, such as the absence of ATP. To confirm the results
obtained with this assay, we therefore also conducted a scan of
342 human wild-type kinases for inhibition of substrate
phosphorylation using a high throughput ATP-P33 radiolabeled
assay from Reaction Biology Corp. at a dose of 1 μM in the
presence of 10 μM ATP (data in Supporting Information).51
We followed up with individual radioligand binding data for
ATP uptake to determine IC50 values for some of the more
interesting kinases (see Tables 6 and 7). In the ScanMax bind-
ing format assay, at a concentration of 1 μM, compound 1
exhibited greater than 50% inhibition of binding for 265 kinases
and showed greater than 99% inhibition of binding for 36
kinases. In the Reaction Biology format in a screen of 342
human wild-type kinases, compound 1 exhibited 50% or greater
inhibition of 202 kinases, with 15 showing greater than 99%
inhibition (see Supporting Information).
a
Table 6. IC50s (nM) Determined for Selected Protein
Kinases for Compound 1
kinase
IC50
1512
kinase
IC50
AMPK
AurA
LCK
333
661
108
MEKK2
P38a
AurB
123
12050
1030
111
AurC
CDK1
CDK2
c-MET
ERK2
FLT1
GSK3B
IGF1R
IR
290
ROCK1
ROCK2
SGK
1125
1180
177
67
SGK1
SRC
201
6290
39
4330
731
SYK
>10,000
307
TRKA
TRKB
ZAP70
ABL1
85
217
200
5050
JNK1
3280
6.8
a
Average of two determinations (nanomolar). IC50s for various kinases
in specificity screens were conducted by Reaction Biology, Inc.
(Malvern, Pennsylvania) as described in ref 51.
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Figure 7. Percent inhibition profiles in the DiscoverRx ScanMax kinome scan for compounds 1 and 2.
A large number of kinases showed very significant affinity for
compound 1 in the ScanMax binding assay. These included
the following important kinases that showed >90% inhibition at
1 μM: ABL1, CDK11, CDK4, CDKL2, CLK1, CLK2, CLK4,
DYRK1B, FLT3, KIT, MELK, PDGFRB, SRPK2, ALK, ARK5,
AXL, IKKalpha, IKKbeta, ROCK1, TYK2. Additionally, several
kinases that are potential targets for antineurodegenerative
kinase inhibitor programs showed substantial inhibition: DLK
In the ScanMax binding format assay, at a concentration of
1 μM, 2 exhibited greater than 50% inhibition of binding of
185 kinases and showed greater than 99% inhibition of binding
of 28 kinases including LRRK2, FLT3, CAMK1D, KIT, MLK1,
and Aurora kinases A and C (see Figure 7 and Supporting
Information). Overall, both compound 1 and 2 showed only
modest kinase specificity with notable differences in selectivity.
Compound 1 showed potent activity on the ABL1 family and
important mutants, whereas 2 lacked potent activity in this
family. In contrast, 2 showed high activity against certain CAMK
family kinases, unlike compound 1. Activity against CAMK2A
(84% inhibition for 2) and CAMK2D (87% inhibition for 2) is
highly correlated with positive activity in micronucleus
screening models for kinase induced chromosome damage, as
are BRSK2 (94% inhibition) and AMPK-alpha2 (98% inhi-
bition).53 Unlike 1, compound 2 also strongly interacted with a
subset of AGC family kinases, inhibiting RSK1 and RSK4 and
related kinases.
Both compounds 1 and 2, as well as other MLK3 inhibitors
not reported here, strongly interact with members of the STE
family kinases (MAP4K4, TNIK, and MINK) involved in MAPK
pathways. Our working hypothesis is that the remarkable effects
observed in in vivo efficacy studies1 of 1 may be the result of
compound 1 being a “selectively non-selective kinase inhibitor.”
Indeed, recent strategies in kinase drug discovery now rec-
ognize that the most efficacious drugs modulate multiple kinase
pathways, but a key issue remains in finding safe compounds
with multiple activities.54 Two kinases, FLT3 and LRRK2, are
both very potently inhibited by both compounds 1 and 2 and
seem potentially interesting with regard to off-target and neu-
roprotective effects.
(88%, Kd = 70 nM, IC50 = 150 nM), LRRK2 (92%, IC50
=
11 nM), LRRK2 (G2019S mutant, 96%), MARK1 (92%),
MARK2 (49%), IGF1R (48%), and SGK1 (70%). Compound
1 is essentially inactive against two kinases known to interact
with MLK3 (GSK3β IC50 > 10 μM, as well as AKT1) and
several other pathway-related or interesting kinases including
AKT2, AKT3, ERK1, ERK2, p38 (all isoforms), BRAF, and
EGFR. It is also noteworthy that 1 inhibited binding of JNK
kinases potently in the ScanMax format, but that high JNK1, -2,
and -3 activity was not confirmed in the biochemical assay
measuring inhibition of substrate phosphorylation. For
example, JNK1 showed 99% binding inhibition at 1 μM in
the ScanMax assay, but the IC50 for phosphorylation inhibition
was 3.2 μM.
Comparison Kinome Scan of Compound 2. To define the
specificity of 2 and to compare and contrast activity at potential
control nodes for anti-inflammatory and neuroprotective
pathways, we synthesized 252 as a standard and conducted ki-
nase inhibition scans using the ScanMax assay to examine
activity on 456 kinases, including most known human kinases
and important mutants thereof. Compound 2 was characterized
1
by H NMR, LC-MS, and high-resolution mass spectrometry,
and the activity was confirmed in enzyme inhibition assays.
As expected, 2 was a very potent inhibitor of the three major
MLK enzymes with IC50s (average of two determinations) for
MLK3 = 6 nM, MLK2 = 2 nM, MLK1 < 1 nM (refer to
Experimental Section, method B).
FLT3 Inhibition. To date, every compound showing potent
activity in our human monocyte assay for inhibition of cytokine
induction by HIV-1 Tat have been potent inhibitors of FLT3
and MLK3. Moreover, a literature survey revealed that most
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Parkinson’s disease (which has been associated with the
G2019S mutation in LRRK2) and Crohn’s disease.60 LRRK2
is potently inhibited by compound 1 (IC50 = 11 nM), and 2
also showed 100% inhibition of LRRK2 binding and 98%
inhibition of the LRRK2 (G2019S) mutant binding in the
ScanMax assay at a concentration of 1 μM.
Interestingly, exposure of murine BV2 microglial cells to
HIV-1 Tat results in phosphorylation of serine 935 on LRRK2
as well as release of pro-inflammatory cytokines and an increase
in phagocytic activity. LRRK2 kinase inhibition attenuated Tat-
induced cytokine release and phagocytosis, suggesting that
LRRK2 may be a novel regulator of microglial inflammation in
HAND.61
The role that kinases other than MLK3 play in the potent in
vivo and in vitro activity of compound 1 in models of HAND is
the subject of current investigation. Our hypothesis is that
strong neuroprotective and antineuroinflammatory efficacy of
compound 1 in our in vivo models for HAND are attributable
to its ability to safely interfere with multiple kinase pathways
that act cooperatively (or synergistically) to drive the path-
ogenesis of HAND. This has important implications for our
understanding of HAND and suggests that cooperative kinase-
regulated gene networks may play a critical and previously
underappreciated role in this disease.
The knowledge we have gained from the structure−activity
relationships and requirements for metabolic stability and
blood−brain barrier penetration have allowed us to design
second-generation compounds not based on the 7-azaindole
core that are much more selective for MLK3 and are potently
neuroprotective in rodent cells, but the details of biological
activity deviate surprisingly from those exhibited by 2 and
compound 1; the details of these studies will be published in
due course.
Table 8. Kinase Inhibitors with High Affinity for MLK3 and
FLT357
a
a
kinase inhibitor
FLT3
MLK3
BIBF-1120
CEP-701
EXEL-2880
KW-2449
Pazopanib
PD-173955
PKC-412
R406
3.8
8.5
0.9
700
18
340
230
740
550
17
15
1100
150
11
0.71
2.9
15
6.5
11
Staurosporine
TAE-684
VX-680
20
420
680
a
ScanMax Binding Kd, nM.
previously described MLK3 inhibitors also potently inhibit
FLT3 (Table 8).
FLT3 is a cytokine/growth factor receptor tyrosine kinase
and is important for survival, proliferation, and differentiation of
hematopoietic cells in bone marrow. Inhibitors of FLT3 are
being developed for the treatment of acute myeloid leukemia
(AML)55 and are also of interest for chronic neuroinflammatory
and neurodegenerative conditions such as multiple sclerosis.56
It is therefore notable that all of the azaindole MLK3 inhibitors
disclosed here exhibited significant FLT3 activity (Table 9).
Table 9. IC50 (Nanomolar) or Percent Inhibition at 1 μmol
a
for FLT3 and LRRK2
compd
FLT3
LRRK2
7
88%
4.0
2970
11
1
SUMMARY
■
8
98%
98%
99%
97%
98%
9.5
35
Using potent and drug-like screening hits as a starting point, we
employed a strategy to find CNS penetrant compounds based
on low molecular weight, low polar surface area, limited
number of hydrogen bond donors, and well-defined log D. By
doing so, we identified a potent, orally bioavailable MLK3 in-
hibitor with excellent pharmacokinetic properties and improved
CNS penetration over previously developed MLK3 inhibitors.
This compound has excellent activity in preclinical models for
HIV associated neurocognitive disease1 and is undergoing
safety testing for potential development. Compound 1 potently
inhibits several key kinases involved in multiple inflammatory
and neurodegenerative pathways, including MLK3 and LRRK2,
and is likely a ”selectively nonselective” kinase inhibitor which
modulates cooperative kinase-regulated gene networks involved
in the pathogenesis of HAND.
9
46
10
11
12
13
5
ND
ND
ND
a
Average of two determinations.
Global suppression of FLT3 is not expected to be benign,
and potent FLT3 inhibitors such as compound 4 have caused
suppression of platelet production.58,59 However, in practice it
is difficult to achieve cellular and in vivo inhibition of FLT3. We
explored the cellular activity of our inhibitors in a cell-based
FLT3 phosphorylation assay, using a murine embryonal fibro-
blast (MEF) cell line that expresses a high level of exogenous
full-length human FLT3-wt. Stimulation of these cells with
human FLT3-ligand results in FLT3 autophosphorylation. The
IC50 for compound 1 against purified FLT3 was 4 nM, but in
this cell-based assay, compound 1’s IC50 against FLT3 was 140-
fold less (560 nM). The activity of compound 13 was similar
(730 nM). These findings suggest that our azaindole MLK3
inhibitors exhibit only modest levels of activity against FLT3 in
cells and that FLT3 inhibition may not make a dominant
contribution to the in vivo efficacy of compound 1.
CHEMISTRY
■
Compounds were efficiently synthesized using sequential
Suzuki couplings utilizing key intermediate 16. For routine syn-
thesis of diverse analogues, best yields and purity were obtained
by protecting the azaindole nitrogen as a tosyl group. The
synthesis of compound 1 is illustrated in Scheme 1. 5-Bromo-
1H-pyrrolo[2,3-b]pyridine 14 was iodinated with NIS to afford
15. The N−H was protected using TsCl, and the resulting
tosylate 16 was subjected to regioselective Suzuki coupling reac-
tion using indole-5-boronic acid at room temperature to yield
intermediate 17. The second Suzuki coupling with 4-formyl
LRRK2 Inhibition. Leucine-rich repeat kinase 2 (LRRK2)
represents another interesting kinase implicated in neuro-
degenerative and inflammatory diseases, including familial
I
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a
Scheme 1. Synthesis of Compound 1
a
Reagents and conditions: (a) NIS, acetone; (b) NaH, p-TSA, THF; (c) indole-5-boronic acid, PdCl2(PPh3)2, MeCN, aq 2 M Na2CO3; (d)
4-formylphenyl boronic acid, PdCl2(PPh3)2, MeCN, aq 2 M Na2CO3; (e) 1-methylpiperazine, Na(OAc)3BH, CH2Cl2; (f) aq 5N NaOH, CH2Cl2,
acetone.
a
Scheme 2. Synthesis of Compound 13
a
Reagents and conditions: (a) 7-azaindole-5-boronic acid pinacol ester, PdCl2(PPh3)2, MeCN, aq 1 M Na2CO3; (b) NaH, p-toluene sulfonyl
chloride, DMF; (c) 4-formylphenyl boronic acid, PdCl2(PPh3)2, MeCN, aq 1 M Na2CO3; (d) 1-methyl piperazine, Na(OAc)3BH, CH2Cl2; (e)
NaOH, MeOH.
boronic acid with intermediate 17 gave the benzaldehyde 18.
Reductive amination reaction with 1-methyl piperazine fol-
lowed by hydrolysis of the tosyl group resulted in the desired
product 1 in an overall efficient synthesis. Compounds 8, 9, 10,
11, and 12 were produced using the same synthetic approach.
Synthesis of compound 13 proceeded by similar sequential
Suzuki couplings but required the protection of the side chain aza-
indole nitrogen to achieve best yields and purity (Scheme 2).
Suzuki coupling intermediate 16 with 7-aza-1H-indol-5-
ylboronic acid provided 20, which was then tosylated to yield
21. The second Suzuki coupling produced intermediate
aldehyde 22, which was reductively aminated and deprotected
to yield compound 13.
diimidazole. Suzuki coupling of 27, using standard conditions
with the 3,4,5-trimethoxy substituted boronic acid, provided
compound 7 (Scheme 3).
EXPERIMENTAL SECTION
■
The structures of compounds synthesized in the examples below were
confirmed using the following procedures. LC-MS/UV/ELS analysis
was performed on instrumentation consisting of Shimadzu LC-10AD
vp series HPLC pumps and dual wavelength UV detector, a Gilson
215 autosampler, a Sedex 75c evaporative light scattering (ELS)
detector, and a PE/Sciex API 150EX mass spectrometer. Then 5.0 μL
injections were performed for each sample on a Phenomenex Gemini
5 μm C18 column. Mobile phases consisted of 0.05% formic acid in
both HPLC grade water (A) and HPLC grade acetonitrile (B). Then
5.0 μL injections were performed for each sample, using gradient
elution from 5% B to 100% B in 4 min at a flow rate of 2.0 mL/min
with a final hold at 100% B of 1.8 min. UV (220 and 254 nm) and ELS
data was collected for 4.5 min. All final compounds exhibited >95%
purity. Routine one-dimensional NMR spectroscopy was performed
Resynthesis of Screening Hit 7. Aminopyrazine 24 was
brominated with NBS, and the resulting dibromo compound
25 underwent regioselective substitution with 5-aminoindole.
Cyclization of 26 was accomplished by heating with carbonyl
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a
Scheme 3. Synthesis of Screening Hit 7
a
Reagents and conditions: (a) NBS, CH2Cl2, 0 °C; (b) EtOH, DIEA, 80 °C; (c) CDI, THF, 65 °C; (d) PdCl2(PPh3)2, CH3CN, H2O, 150 °C.
on a 300 MHz Varian Mercury-Plus spectrometer. The samples were
dissolved in deuterated solvents obtained from Cambridge Isotope
Laboratories, Inc. and transferred to 5 mm ID NMR tubes. The spec-
tra were acquired at 293 K. C, H, N, Pd combustion analysis provided
by Robertson Mircrolit Laboratories, and high resolution mass spectra
were obtained by the UCSD Department of Chemistry Agilent 6230
ESI-TOF mass spectrometer service.
bis(triphenylphosphine)-palladium(II) dichloride (6.0 mg, 0.008 mmol),
and 1 M Na2CO3 (1 mL). The resulting mixture was degassed with Ar
for 10 min, after which it was heated at 150 °C for 10 min in a
Personal Chemistry Optimizer. The organic layer was separated,
filtered, and concentrated in vacuo. The residue was purified by
1
preparatory HPLC to yield 7 (6.5 mg, 19%). H NMR (DMSO-d6,
300 MHz): δ 12.18 (s, 1H), 11.28 (s, 1H), 8.57 (s, 1H), 7.83 (d, J =
1.8 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.42 (m, 1H), 7.37 (dd, J = 1.8,
8.4 Hz, 1H), 7.20 (s, 2H), 6.51 (m, 1H), 3.78 (s, 6H), 3.66 (s, 3H).
HPLC retention time: 2.30 min. HR-MS (ESI-TOFMS) 418.1511
(M + H), calcd 418.1511.
3-(1H-Indol-5-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)-
phenyl)-1H-pyrrolo[2,3-b]pyridine (1). In a round-bottom flask,
19 (12.4 g, 21.5 mmol) was dissolved in CH2Cl2 (105 mL) and
MeOH (105 mL), then 5N NaOH (210 mL) added. The resulting
mixture was heated at 50 °C for 60 min. After completion of the
reaction, the product was extracted using EtOAc and purified on silica
gel chromatography. After the purification, all combined batches
were again dissolved in DCM (250 mL), MeOH (250 mL), and THF
(250 mL) and added quadra pure TU thiourea resin (10 g) and stirred
overnight on rotary evaporator. After filtration, the solvents were re-
moved and redissolved in EtOH (450 mL) then heated to 55 °C. After
2 h, HPLC grade water was added and the resulting mixture kept in a
freezer to afford white solid was collected by filtration to afford 3-(1H-
indol-5-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo-
3-(1H-Indol-5-yl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrrolo-
1
[2,3-b]pyridine (8). H NMR (DMSO-d6, 300 MHz): δ 11.78 (s,
1H), 11.03 (s, 1 H), 8.51 (d, J = 2.1 Hz, 1H), 8.36 (d, J = 1.8 Hz, 1H),
7.86 (s, 1H), 7.72 (d, J = 2.4 Hz, 1H), 7.45 (s, 2H), 7.32 (m, 1H), 6.92
(s, 2H), 6.45 (m, 1 H), 3.85 (s, 6H), 3.70 (s, 3H). MS ESI (m/z):
400.4 (M + H)+, calcd 399. HRMS (EI) m/z calcd for C24H21N3O3Na
(M + Na)+ 422.1475, found 422.1476.
5-(3-(1H-Indol-5-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)pyridin-2-
1
amine (9). H NMR (DMSO-d6, 300 MHz): δ 11.73 (d, J = 1.8 Hz,
1H), 11.05 (s, 1 H), 8.43 (d, J = 2.4 Hz, 1H), 8.29 (d, J = 1.8 Hz, 1H),
8.27 (d, J = 2.1 Hz, 1H), 7.88 (s, 1H), 7.76 (dd, J = 2.4, 8.4 Hz, 1H),
7.46 (s, 2H), 7.33 (m, 1H), 6.55 (dd, J = 0.6, 8.7 Hz, 1H), 6.46 (m,
1 H), 5.99 (s, 2 H). HPLC retention time: 1.10 min. MS ESI (m/z):
326.2 (M + H)+, calcd 325. HRMS (EI) m/z calcd for C20H16N5 (M +
H)+ 326.1400, found 326.1401.
1
[2,3-b]pyridine 1 (6.4 g, 70%). H NMR (DMSO-d6, 300 MHz): δ
11.82 (bs, 1H), 11.07 (bs, 1H), 8.54 (d, J = 1.2 Hz, 1H), 8.41 (d, J =
1.2 Hz, 1H), 7.89 (s, 1H), 7.76 (d, J = 1.5 Hz, 1H), 7.69 (d, J = 5.1 Hz,
2H), 7.48 (m, 2H), 7.39 (d, J = 4.8 Hz, 2H), 7.35 (m, 1H), 6.49 (dd,
J = 2.7, 1.2 Hz, 1H), 3.48 (s, 2H), 2.50 (m, 3H), 2.38 (m, 5H). ESI
(m/z) 422 (M + H), calcd 421. HRMS (EI) m/z calcd for C27H28N5
(M + H)+ 422.2341, found 423.2329. CHN.
5-(5-(3,4,5-Trimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-3-
1
yl)pyridin-2-amine (10). H NMR (DMSO-d6, 300 MHz): δ 11.82
(s, 1H), 8.53 (d, J = 1.8 Hz, 1H), 8.31 (d, J = 1.8, 1 H), 8.28 (d, J =
1.5 Hz), 7.76 (dd, J = 2.1, 8.4 Hz, 1 H), 7.70 (d, J = 2.4 Hz, 1H), 6.95
(s, 2H), 6.54 (d, J = 8.4 Hz, 1 H), 5.87 (s, 2H), 3.86 (s, 6H), 3.68 (s,
3H). MS ESI (m/z): 377.4 (M + H), calcd 376. HRMS (EI) m/z calcd
for C21H21N4O3 (M + H)+ 377.1608, found 377.1607.
5-(3-(1H-Indol-5-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrimidin-
2-amine (11). MS ESI (m/z): 327.2 (M + H), calcd 326. HRMS (EI)
m/z calcd for C19H15N6 (M + H)+ 327.1353, found 327.1354.
5-(5-(3,4,5-Trimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-3-
yl)pyrimidin-2-amine (12). MS ESI (m/z): 378.4 (M + H), calcd
377. HRMS (EI) m/z calcd for C20H20N5O3 (M + H)+ 378.1561,
found 378.1563.
5-(5-(4-((4-Methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo-
[2,3-b]pyridin-3-yl)-1H-pyrrolo[2,3-b]pyridine (13). To a solution
of 23 (4.00 g, 5.46 mmol) in MeOH (20 mL) was added NaOH
(723 mg, 16.4 mmol). The resulting mixture was heated at 50 °C for
60 min. After completion of the reaction, the product was extracted
(9S,10R,12R)-9,12-Epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]-
pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic Acid, 5,16-Bis-
[(ethylthio)methyl]-2,3,9,10,11,12-hexahydro-10-hydroxy-9-
methyl-1-oxo Methyl Ester (2). 2 was synthesized from K252a (LC
Laboratories, Woburn, MA) according to the method of Kaneko.52 1H
NMR (DMSO-d6, 300 MHz): δ 9.13 (s, 1H), 8.63 (s, 1H), 7.95 (s,
1H), 7.88 (d, J = 5.1 Hz, 1H), 7.84 (d, J = 5.1 Hz, 1H), 7.46−7.44 (m,
2H), 7.10 (dd, J = 3.0, 4.2 Hz, 1H), 6.33 (s, 1H), 5.02 (d, J = 10.2 Hz,
1H), 4.95 (d, J = 10.2 Hz, 1H), 3.98 (s, 2H), 3.94 (s, 2H), 3.92 (s,
3H), 3.38−3.35 (m, 1H), 2.50−2.46 (m, 4H), 2.13 (s, 3H), 1.99 (dd,
J = 3.0, 8.4 Hz, 1H), 1.23 (t, J = 2.4 Hz, 6H). MS ESI (m/z): 616.3
(M + H)+, calcd 616.2. HRMS (EI) m/z calcd for C33H33N3O5Na
(M + Na)+ 638.1754, found 638.1755.
1-(1H-Indol-5-yl)-6-(3,4,5-trimethoxyphenyl)-1H-imidazo-
[4,5-b]pyrazin-2(3H)-one (7). To a solution of 24 (27 mg, 0.08 mmol)
in CH3CN (1 mL) in a Personal Chemistry microwave reaction vial
was added 3,4,5-trimethoxyphenylboronic acid (17 mg, 0.08 mmol),
K
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using 250 mL of IPA:CHCl3 (1:3) and water. The organic portions
were dried and concentrated in vacuo. The crude product was puri-
fied on a silica gel column to afford 13 (1.57 g, 68%). 1H NMR
(DMSO-d6, 300 MHz): δ 11.96 (s, 1H), 11.63 (s, 1H), 8.61 (d, J =
1.5 Hz, 1H), 8.56 (d, J = 1.2 Hz, 1H), 8.41 (d, J = 0.9 Hz, 1H), 8.31
(d, J = 1.2 Hz, 1H), 7.89 (d, J = 1.5 Hz, 1H), 7.73 (d, J = 4.8 Hz, 2H),
7.49−7.48 (m, 1H), 7.40 (d, J = 4.8 Hz, 2H), 6.51−6.49 (m, 1H), 3.49
(s, 2H), 2.38−2.36 (m, 4H), 2.21 (s, 3H). ESI (m/z) 423.4 (M + H)+,
calcd 422. HRMS (EI) m/z calcd for C26H27N6 (M + H)+ 423.2292,
found 423.2295.
(8.9 g, 89 mmol) and sodium triacetoxyborohydride (14.2 g, 67.2 mmol).
The reaction mixture was stirred for 1 h at room temperature, after
which it was partitioned between CH2Cl2 and brine. The organic layer
was separated, dried over MgSO4, and concentrated in vacuo. The
crude product was purified on silica gel column to give 15 (17 g, 68%
yield). 1H NMR (DMSO-d6, 300 MHz): δ 11.23 (bs, 1H), 8.70 (d, J =
1.2 Hz, 1H), 8.40 (d, J = 1.5 Hz, 1H), 8.09 (d, J = 0.9 Hz, 2H), 8.07 (s,
1H), 7. 98 (s, 1H), 7.71 (d, J = 5.1 Hz, 2H), 7.52 (s, 2H), 7.41 (m,
5H), 7.24 (d, J = 4.5 Hz, 1H), 7.16 (dd, J = 10.8, 4.5 Hz, 2H), 6.52
(dd, J = 2.7, 1.2 Hz, 1H), 3.53 (s, 2H), 2.57 (m, 3H), 2.34 (m, 5H)
2.29 (s, 3H). MS ESI (m/z): 576 (M + H)+, calcd 575.
5-Bromo-3-iodo-1H-pyrrolo[2,3-b]pyridine (15). In a 3 L
round-bottom flask, 5-bromo-1H-pyrrolo[2,3-b]pyridine (63g,
319 mmol) was dissolved in 1500 mL of acetone. To the stirred
mixture was added NIS (79.1g, 351 mmol), and the resulting mixture
was stirred for 1.5 h; the precipitated solid was collected by filtration
and washed with cold acetone (400 mL) to afford 15 (89.8 g, 88%
5-(5-Bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-
pyrrolo[2,3-b]pyridine (20). To a stirred suspension of 16 (60 g,
125 mmol) and 7-aza-1H-indol-5-ylboronic acid (22.2 g, 138 mmol) in
CH3CN (625 mL) was added 1 M Na2CO3 (312 mL) followed by
bis(triphenylphosphine)palladium(II) dichloride (4.4 g, 6.2 mmol).
The resulting mixture was stirred at room temperature for 1 h. After
complete consumption of starting materials, the mixture was extracted
with EtOAc and evaporated to dryness in vacuo and it was dissolved in
CH2Cl2 (50 mL), absorbed onto Celite, and dried. The residue was
purified via silica gel chromatography using CH2Cl2 as the eluent to
1
yield) as white solid. H NMR (DMSO-d6, 300 MHz) δ 12.35 (bs,
1H), 8.31 (d, J = 1.5 Hz, 1H), 7.86 (dd, J = 1.2 Hz, 0.3 Hz, 1 H), 7.80
(d, J = 1.5 Hz, 1H). MS ESI (m/z): 322/324 (M + H)+, calcd 323.
5-Bromo-3-iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (16). To a
stirred solution of 15 (45.0g, 139 mmol) in 700 mL of anhydrous
THF cooled to 0 °C with an ice bath was added NaH [60% dispersion
in mineral oil] (8.3 g, 208 mmol). The reaction mixture was stirred for
20 min at 0 °C, after which p-toluenesulfonyl chloride (29.1 g,
153 mmol) was added. The resulting mixture was stirred at 0 °C for
1.5 h, after confirmation of completion by LCMS and TLC, the reac-
tion mixture was warmed to room temperature, evaporated to dryness,
and quenched with water. The crude product was mixed with EtOAc
(1000 mL) refluxed for 1 h, and then hexane (500 mL) was added to
precipitate the product 16 (60 g, 90% yield) as a light-yellow powder.
1H NMR (DMSO-d6, 300 MHz) δ 8.50 (d, J = 1.5 Hz, 1H), 8.21 (s,
1
obtain 20 (38.6 g, 65% yield). H NMR (DMSO-d6, 300 MHz): δ
11.21 (bs, 1H), 8.52 (d, J = 1.2 Hz, 1H), 8.47 (d, J = 1.5 Hz, 1H), 8.13
(s, 1H), 8.05 (d, J = 5.1 Hz, 2H), 7.92 (s, 1H), 7.51 (d, J = 4.8 Hz,
1H), 7.46 (dd, J = 5.1, 1.2 Hz, 1H), 7.43 (d, J = 5.1 Hz, 1H), 7.40 (dd,
J = 3.9, 1.8 Hz, 2H), 6.52 (dd, J = 2.7, 1.2 Hz,1 H), 2.33 (s, 3H). MS
ESI (m/z): 466.2/468.2 (M + H)+, calcd 466.
5-(5-Bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-1-tosyl-
1H-pyrrolo[2,3-b]pyridine (21). To a stirred solution of 20 (1.01 g,
2.17 mmol) in 20 mL of anhydrous DMF was added NaH [60%
dispersion in mineral oil] (130 mg, 3.25 mmol). The reaction mixture
was stirred for 20 min at room temperature, after which p-toluene
sulfonyl chloride (538 mg, 2.82 mmol) was added. The resulting mix-
ture was stirred at room temperature for 2 h. The mixture was
evaporated to dryness and quenched with water. The crude product
was mixed with EtOAc (100 mL) and extracted. The organic solution
was dried and evaporated in vacuo. The residue was purified via silica
gel chromatography eluting with 20% EtOAc in hexanes to obtain 21
as a white solid (1.21 g, 90%). 1H NMR (DMSO-d6, 300 MHz) δ 8.76
(d, J = 1.2 Hz, 1H), 8.56 (d, J = 1.2 Hz, 1H), 8.54 (d, J = 1.2 Hz, 1H),
8.45 (d, J = 1.5 Hz, 1H), 8.39 (s, 1H), 8.04 − 8.01 (m, 4H), 7.96 (d,
J = 2.4 Hz, 1H), 7.43 (d, J = 4.5 Hz, 4H), 6.87 (d, J = 2.4 Hz, 1H),
2.34 (s, 6H). MS ESI (m/z): 622.4/624.1 (M + 1)+, calcd 621.
4-(1-Tosyl-3-(1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-
pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (22),. To a solution of
21 (1.32 g, 2.12 mmol) in CH3CN (20 mL) in a round-bottom flask
was added 4-formylphenylboronic acid (349 mg, 2.33 mmol), bis-
(triphenylphosphine)-palladium(II) dichloride (149 mg, 0.212 mmol),
and 1 M Na2CO3 (20 mL). The resulting mixture was heated to reflux
for 3 h and then cooled to room temperature. The precipitated
product was filtered and dried. The organic layer was extracted with
EtOAc and washed with brine and evaporated to dryness to afford
more crude material. The filtered solid and crude material from evap-
oration were redissolved in CH2Cl2, absorbed on Celite, and purified
on silica gel column chromatography to yield 22 as a white solid
(822 mg, 60% yield). MS ESI (m/z): 647.2 (M + H)+, calcd 646.
5-(5-(4-((4-Methylpiperazin-1-yl)methyl)phenyl)-1-tosyl-1H-
pyrrolo[2,3-b]pyridin-3-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine
(23). To a solution of 22 (3.35 g, 5.18 mmol) in CH2Cl2 (50 mL) was
added 1-methylpiperazine (1.15 mL, 10.4 mmol) and sodium triace-
toxyborohydride (2.19 g, 10.4 mmol). The reaction mixture was stirred
for 1 h at room temperature, after which it was partitioned between
CH2Cl2 and brine. The organic layer was separated, dried over MgSO4,
and concentrated in vacuo. The crude product was purified on silica
1H), 8.00 (d, J = 4.8 Hz, 0.3 Hz, 2H), 7.98 (d, J = 1.5 Hz, 1H), 7.44
(dd, J = 4.8 Hz, 0.3 Hz, 2H), 2.34 (s, 3H). MS ESI (m/z): 477.0/479.0
(M + 1)+, calcd 477.
5-Bromo-3-(1H-indol-5-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine
(17). To a stirred suspension of 16 (60 g, 125 mmol) and 1H-indol-5-
ylboronic acid (22.2 g, 138 mmol) in CH3CN (625 mL) was added
1 M Na2CO3 (312 mL) followed by bis(triphenylphosphine)-
palladium(II) dichloride (4.4 g, 6.2 mmol). The resulting mixture
was stirred at room temperature for 1 h. After complete consumption
of starting materials, the mixture was extracted with EtOAc and
evaporated to dryness in vacuo, it was dissolved in CH2Cl2 (50 mL),
absorbed onto Celite, and dried. The residue was purified via silica gel
chromatography using CH2Cl2 as the eluent to obtain 17 (38.6 g, 65%
yield). 1H NMR (DMSO-d6, 300 MHz): δ 11.21 (bs, 1H), 8.52 (d, J =
1.2 Hz, 1H), 8.47 (d, J = 1.5 Hz, 1H), 8.13 (s, 1H), 8.05 (d, J = 5.1 Hz,
2H), 7.92 (s, 1H), 7.51 (d, J = 4.8 Hz, 1H), 7.46 (dd, J = 5.1, 1.2 Hz,
1H), 7.43 (d, J = 5.1 Hz, 1H), 7.40 (dd, J = 3.9, 1.8 Hz, 2H), 6.52
(dd, J = 2.7, 1.2 Hz,1 H), 2.33 (s, 3H). MS ESI (m/z): 466.2/468.2
(M + H)+, calcd 466.
4-(3-(1H-Indol-5-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)-
benzaldehyde (18). To a solution of 17 (29.5 g, 63.3 mmol) in CH3CN
(315 mL) in a round-bottom flask was added 4-formylphenylboronic
acid (11.4 g, 76 mmol), bis(triphenylphosphine)-palladium(II)
dichloride (4.4 g, 6.3 mmol), and 1 M Na2CO3 (160 mL). The
resulting mixture was heated to reflux for 2.5 h. The reaction was
cooled to room temperature; the precipitated product was filtered and
dried. The organic layer was extracted with EtOAc and washed with
brine and evaporated to dryness to afford more crude material. The
filtered solid and crude and material from evaporation were re-
dissolved in CH2Cl2, absorbed on Celite, and purified via silica gel
chromatography using CH2Cl2 as the eluent to afford 18 (38.6 g, 65%
1
yield). H NMR (DMSO-d6, 300 MHz): δ 11.21 (bs, 1H), 10.07 (s,
1H), 8.81 (d, J = 1.2 Hz, 1H), 8.53 (d, J = 1.2 Hz, 1H), 8.13 (s, 1H),
8.08 (d, J = 5.1 Hz, 2H), 8.02 (m, 5H), 7.53 (dd, J = 5.1 Hz, 2H), 7.45
(d, J = 5.1 Hz, 2H), 7.46 (dd, J = 3.0, 1.5 Hz, 1H), 6.52 (dd, J = 2.7,
1.2 Hz,1 H), 2.34 (s, 3H). MS ESI (m/z): 492 (M + H)+, calcd 491.
3-(1-Tosyl-1H-indol-5-yl)-5-(4-((4-methylpiperazin-1-yl)-
methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (19). To a solution of
18 (22 g, 44.8 mmol) in CH2Cl2 (448 mL) was added 1-methylpiperazine
1
gel column to afford 23 (2.65 g, 70% yield). H NMR (DMSO-d6,
300 MHz): δ 8.83 (d, J = 1.2 Hz, 1H), 8.71 (d, J = 1.2 Hz, 1H), 8.52
(d, J = 1.5 Hz, 1H), 8.44 (d, J = 1.2 Hz, 1H), 8.36 (s, 1H), 8.07 (d, J =
5.1 Hz, 2H), 8.02 (d, J = 5.1 Hz, 2H), 7.96 (d, J = 2.4 Hz, 1H), 7.71
(d, J = 4.8 Hz, 2H), 7.43 (d, J = 5.1 Hz, 4H), 7.38 (d, J = 4.8 Hz, 2H),
L
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6.87 (d, J = 2.4 Hz, 1H), 3.48 (s, 2H), 2.40 − 2.25 (m, 8H), 2.14 (s,
3H). MS ESI (m/z): 731.4 (M + H)+, calcd 730.
weighed, and stored at −80 °C until analysis. For compound quan-
titation, mouse brains were homogenized in 5 volumes of water. The
homogenates were extracted by protein precipitation with acetonitrile.
LC-MS/MS analysis was conducted as described for the plasma. Brain
homogenate concentrations were converted to brain concentrations
for the calculations of brain to plasma ratios.
Brain Penetration Comparison for Compounds 1, 2, and 13.
Compounds were dissolved in 5% DMSO, 40% PEG-400, and 55%
saline to yield a nominal concentration of 2 mg/mL (pH = 8) and
were dosed at 10 mg/kg in C57BL/6 mice by tail vein injection.
Samples were collected as described above at 30, 60, and 180 min
postdose. Mice were anesthetized, sacrificed and blood collected by
retro-orbital bleeds, and brains collected (3 time points over 24 h, 3
mice per time point). Concentration of compounds was determined in
plasma and brain samples as described above.
Detailed Oral PK Study for Compound 1 in C57/Bl/6 Mice.
Intravenous dosing for %F determination: Male C57/BL/6 mice were
dosed iv (2.5 mg/kg) by tail vein injection of a solution of 2 mg/mL of
compound 1 dissolved in 5% DMSO, 40% PEG-400, and 55% saline.
Blood samples of approximately 0.30 μL were collected from each
mouse (n = 3 mice per time point) by retro-orbital bleed while the
animals were anesthetized with isoflurane. Blood samples were col-
lected in tubes containing sodium heparin as the anticoagulant,
predose and at 0.083, 0.25, 0.5, 1, 2, 4, 7, and 24 h postdose. Samples
were centrifuged within 1 h of collection and plasma was collected and
stored at −80 °C until analysis.
Oral Dosing for %F determination: Compound was dosed by oral
gavage (10 mg/kg) as a suspension of 1 mg/mL of 1 in a solution of
0.5% hydroxypropyl methylcellulose and 0.4% Tween-80 in pH 7.4
PBS buffer. Blood samples were collected in tubes containing sodium
heparin as the anticoagulant, predose and at 0.25, 0.5, 1, 2, 4 6, 8, and
24 h post dose. Total concentrations of the compound were
determined by liquid chromatography−tandem mass spectrometry
(LC-MS/MS) as described above.
Stability in Human Liver Microsomes. Stability of the test com-
pound was determined in human liver microsomes by Cerep Inc.,
using standard assay conditions of 1 μM concentration of the test
compound. Compound concentration was determined by HPLC, and
reported values are the average of duplicate values.
Stability in Hepatocytes. Stability of the test compound was
determined in cryopreserved human, cynomolgus monkey, and CD-1
mice hepatocytes, by Cerep Inc., using standard assay conditions of
1 μM concentration of the test compound. Compound concentration
was determined by HPLC, and reported values are the average of
tripicate values.
CYP2C9, 2D6, and 3A4 Inhibition. Test compound was incubated
with pooled human liver microsomes at 37 °C in 0.1 M Tris buffer, pH
7.4, and its effect on the metabolism of probe substrates for CYP
enzymes determined (2D6, diclofenac; 3A4, dextromethorphan; 2C9,
midazolam). The compound was tested at six concentrations ranging
from 0.12 to 30 μM. Conditions of incubation in this assay have been
optimized to maintain first-order reaction conditions and to minimize
the potential for nonspecific binding of probe or study compound.
Reactions were terminated with acetonitrile containing analytical
internal standard (carbamazepine), and samples were centrifuged to
remove microsomal protein and analyzed using optimized HPLC and
MS conditions. The MS responses for the solvent control samples
were taken as the 100% reference values against which the inhibition of
metabolism was measured. IC50 values were calculated using a sig-
moidal dose−response equation within GraphPad Prism.
Inhibition of HIV Tat-Induced Cytokine Release in Primary
Human Monocytes. This assay was performed essentially as
described.14,63 Briefly, human monocytes were isolated from freshly
collected whole blood using CD14 immunomagnetic beads (Miltenyi-
Biotec), plated in 24-well plates and incubated with the specified
compounds at the indicated concentrations (100, 3000, 1000 nM); in
control wells, no compound was added. Then 30 min later, HIV-1 Tat
was added to a final concentration of 50 nM; the cells were incubated
for 8 h (in control wells, nothing was added [NT]). Cell supernatants
were then collected, centrifuged to remove debris, transferred to new
3,5-Dibromopyrazin-2-amine (25). To a stirred solution of
aminopyrazine 24 (8.21 g, 86.4 mmol) in anhydrous methylene chlo-
ride (215 mL) cooled to 0 °C was added N-bromosuccinimide (32.3 g,
181 mmol) in portions over a 6 h period, during which time the tem-
perature of the reaction was kept below 0 °C. The resulting mixture
was stored at 4 °C overnight, after which it was stirred vigorously and
quenched with H2O (100 mL). The organic layer was separated, after
which it was washed with saturated aqueous NaHCO3, washed with
brine, dried over MgSO4, filtered, and evaporated in vacuo to yield
a residue that was triturated with 20% EtOAc in hexanes to yield
25 (10.3 g, 47%) as a yellow−brown powder. 1H NMR (CDCl3,
300 MHz) δ 8.02 (s, 1H), 5.05 (bs, 2H). HPLC retention time:
1.99 min. MS ESI (m/z): 252.0/254.0/256.2 (M + 1)+, calcd 251.
6-Bromo-N2-(1H-indol-5-yl)pyrazine-2,3-diamine (26). To a
stirred suspension of 25 (3.48 g, 13.7 mmol) and 1H-indol-5-amine
(2.00 g, 15.0 mmol) in EtOH (3.5 mL) was added diisopropylethyl-
amine (2.60 mL, 15.0 mmol). The resulting mixture was stirred for
48 h at 80 °C, after which it was partitioned between EtOAc and H2O.
The organic layer was separated, after which it was washed with brine,
dried over Na2SO4, filtered, and evaporated in vacuo to yield a residue
that was purified via silica gel chromatography eluting with 1:1
1
EtOAc:hexanes to yield 26 (1.75 g, 42%) as a red−brown solid. H
NMR (DMSO-d6, 300 MHz): δ 10.98 (s, 1H), 8.22 (s, 1H), 7.83 (s,
1H), 7.31−7.28 (m, 3H), 7.19 (d, J = 8.7 Hz, 1H), 6.43 (s, 2H), 6.36
(s, 1H). HPLC retention time: 2.07 min. MS ESI (m/z): 304.2/306.2
(M + H)+, calcd 303.
6-Bromo-1-(1H-indol-5-yl)-1H-imidazo[4,5-b]pyrazin-2(3H)-
one (27). To a solution of 26 (0.450 g, 1.48 mmol) in THF (5 mL)
was added carbonyldiimidazole (1.20 g, 7.40 mmol). The resulting
mixture was heated at 65 °C for 48 h, after which it was concentrated
in vacuo and partitioned between EtOAc and H2O. The organic layer
was separated, dried over MgSO4, filtered, and concentrated in vacuo
to yield a residue that was purified via silica gel chromatography
eluting with EtOAc to yield 27 (0.20 g, 41%). HPLC retention time:
+
2.07 min. MS ESI (m/z): 330.2/332.2 (M + 1) , calcd 329.
LPS-Induced TNFα Release from Microglial BV2 Cells. A
TNFα release assay in LPS-stimulated BV-2 cells was performed essen-
tially as described.62 Briefly, mouse microglial BV-2 cells were treated
with test compounds followed by LPS (100 ng/mL final concen-
tration), and culture supernatants were harvested 8 h thereafter for
TNFα ELISA.
Screening PK (iv Dosing) in Mice. Three mice were used for
each time point. Male C57/BL/6 mice were dosed iv (10 mg/mL) by
tail vein injection of a solution of 2 mg/mL in solutions containing the
indicated compound and vehicles (compounds 1, 9, 10, 11 and 13, 5%
DMSO, 40% PEG-400, 55% saline; compounds 8 and 12, 5% DMSO,
40% PEG-400, 55% H2O containing 20% HP-βCD). Blood samples of
approximately 0.30 μL were collected from each mouse (n = 3 mice
per time point) by retro-orbital bleed while the animals were
anesthetized with isoflurane. Blood samples were collected in tubes
containing sodium heparin as the anticoagulant, predose and at 0.083,
0.25, 0.5, 1, 2, 4, 6, 8, and 24 h postdose. Samples were centrifuged
within 1 h of collection and plasma was collected and stored at −20 °C
until analysis. Total concentrations of the compound were determined
by liquid chromatography−tandem mass spectrometry (LC-MS/MS),
following plasma protein precipitation with acetonitrile and injection
of the supernatant onto the column (XTerraMS C18, 5 μm, 4.6 mm ×
50 mm). The LC system comprised an Agilent (Agilent Technologies
Inc., USA) 1100 series liquid chromatography equipped with G1379A
degasser, G1311A Quantpump, G1313A autosampler, and G1316A
column oven. Mass spectrometric analysis was performed using an
API4000 (triple-quadrupole) instrument from AB Inc. (Canada) with
an ESI interface.
The aqueous mobile phase was water with 0.1% formic acid, and the
organic mobile phase was methanol with 0.1% formic acid. The lower
and upper limits of quantitation of the assay were 2.5 and 5000 ng/mL
based on known standards, respectively. Brains were collected from
three different animals at each time point, rinsed with ice-cold saline,
M
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microcentrifuge tubes, and frozen at −20 °C. A Luminex bead array
assay was then performed to quantitate the indicated chemokines and
cytokines. Results were measured in triplicate or quadruplicate, and
data are presented as mean values; error bars denote the standard
deviation. Note that similar results were obtained with monocytes
derived from multiple (n > 5) different donors, as well as in terminally
differentiated monocyte-derived macrophages (data not shown).
* = p < 0.05; one-way ANOVA with Bonferroni’s correction (when
compared to Tat only control).
Inhibition of JNK Phosphorylation in HIV-Tat Treated Mice. Wild-
type C57BL/6 mice were either pretreated with three doses of
10 mg/kg Compound 1 spaced 12 h apart (n = 6) or were left un-
treated (n = 6). Treatment with compound 1 continued every 12 h
throughout the duration of the experiment. Half of the mice (three
pretreated with compound 1, three untreated) received a stereotactic
injection of 3 μL of 3 μg/μL HIV-1 Tat1−72 in PBS into the somato-
sensory cortex at the coordinates 1.0 mm posterior to Bregma, 1.0 mm
lateral left of Bregma, and 0.7 mm ventral to the pial surface. The
remaining mice received an injection of 3 μL of sterile PBS at the same
coordinates. A 35 gauge needle with a 10 μL Hamilton syringe
controlled by a microsyringe pump (Micro4 World Precision
Instruments) was used to perform the injections, which were delivered
at a flow rate of 80 nL/min to minimize brain injury occurring as a
result of injection pressure. The needle and syringes were coated with
Sigmacote (Sigma SL-2) to prevent the Tat from sticking to the inside
of the syringe. Then 24 hours after injection, the mice were sacrificed
by pentobarbital overdose; animals were then briefly transcardially
perfused with ice-cold saline, and the brains were removed, sectioned,
and flash frozen on dry ice.
of 442 known protein kinases. Data are presented as percent of
control activity remaining. (0% indicates very tight binders, 100%
indicates no binding). Complete data are presented in the
Supporting Information.
Reaction Biology Wild-Type Human Kinome Inhibition Scan.
Compound 1 was tested against 342 wild-type human kinases for
inhibition of protein phosphorylation using the Reaction Biology Inc.
Hot Spot P33 radio binding assay.51 The compound was tested in
single dose duplicate mode at a concentration of 1 μM . Control
Compound was tested in 10 dose IC50 mode with 3-fold serial dilution
starting at 20 μM. Reactions were carried out at 10 μM ATP. Com-
plete data are available in the Supporting Information.
Kinase Specificity Inhibition Data. Kinase inhibition IC50 values
were determined from 10 point curves. IC50s for various kinases in
specificity screens were conducted by Reaction Biology, Inc. (Malvern,
Pennsylvania) as described.51
Radiometric Filter Plate MLK3 Assay (Method A). MLK3
(200 ng (130 nM), Dundee, DU8313) was incubated with 1 μM
inactive MKK7b (Dundee, DU703) in the presence of 2 μM cold ATP
(Km) and 0.5 μCi/assay 33P ATP and appropriate concentrations of
compounds. After a 20 min incubation, the reactions were washed
through filter plates and read on a scintillation counter.
Biochemical Assay for the Inhibition of Kinase Activity for
MLK3 (Method B). Myelin basic protein (20 μM final concentration)
was dissolved in 20 mM Hepes (pH 7.5) containing 10 μM MgCl2,
1 μM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 μM Na3VO4,
2 mM DTT, and 1% DMSO. Activated MLK3 was added and mixed
(20 nM final concentration), and inhibitors were added in DMSO.
33P-ATP (specific activity 500 μCi/μL) was delivered into the reaction
The brain tissue of interest was homogenized in a glass
homogenizer in a solution of 1X Tris-buffered saline pH 7.4 with
0.05% Tween and protease and phosphatase inhibitors (no. 161280;
Thermo Fisher Scientific). The homogenate was spun down at 13000
rcf for 15 min to remove insoluble debris, and the lysis supernatant
was collected. The protein concentrations were measured and
normalized using a Bradford assay. Then 12 μg of protein sample
was then mixed with loading dye, boiled for 5 min, and run on a 4% to
15% SDS-PAGE gel (BioRad 456−1086) at 100 V. The gel was
transferred onto a nitrocellulose membrane at 100 V for 66 min on ice.
The membranes were blocked in 5% milk in 1× Tris-buffered saline
with 0.05% Tween (TBS-T) for 1 h at room temperature with shaking.
The membranes were washed three times in TBS-T for 10 min a wash.
The primary antibody against phosphorylated JNK (Cell Signaling
4668P) was applied overnight at 4 °C at a concentration of 1:2000 in
TBS-T with 5% milk. The next day, the membranes were washed
three times in TBS-T for 10 min a wash. The horseradish peroxidase
(HRP)-conjugated secondary antibody (BioRad 170−6515) was
applied at a concentration of 1:11000 in 5% milk TBS-T for 45 min
at room temperature with shaking. The membranes were washed three
times in TBS-T and enhanced chemiluminescence (ECL) substrate
(Thermo 34076, Thermo 34080) was applied for 3 min. The mem-
branes were developed on film (Thermo 34091). To control for
variations in protein loading, the membranes were stripped (Millipore
2504) and then blocked in 5% milk TBS-T for 30 min. The mouse
anti-α-tubulin (Sigma T5168) was applied in 5% milk TBS-T over-
night with shaking. The process of washing, secondary application, and
developing was repeated in order to obtain the α-tubulin loading
control blot. The optical density measurements used for quantification
were obtained using ImageJ. * = p < 0.05; one-way ANOVA with
Bonferroni’s correction.
mixture to initiate the reaction (ATP concentration: 10 μM), and the
mixture was incubated at room temperature for 20 min. Percent
Activity was determined using a proprietary HOTSPOT microfluidic
filter binding technology.51
FLT3 Cellular Assay. This was performed by Proqinase GMBH,
Freiburg, Germany. Briefly, this assay uses a murine embryonal
fibroblast (MEF) cell line, which expresses a high level of exogenously
introduced full-length human, wild-type FLT3. Stimulation of these
cells with human FLT3-ligand results in receptor tyrosine auto-
phosphorylation. MEF-FLT3-wt cells were plated in DMEM
supplemented with 10% FCS in multiwell cell culture plates. After
serum-starvation overnight, cells were incubated with compounds in
serum-free medium. After 90 min incubation at 37 °C, cells were
stimulated with FLT3-L at 250 ng/mL for 5 min. Quantification of
substrate phosphorylation was assessed in 96 well plates via sandwich
ELISA using a substrate specific capture antibody and an anti-
phosphotyrosine detection antibody. Raw data were converted into
percent substrate phosphorylation relative to controls (incubated with
FLT3L alone), which were set to 100%. IC50 values were determined
using GraphPad Prism 5.01 software using a nonlinear regression
curve fit with variable hill slope. The equation is a four-parameter
logistic equation.
Plasma Protein Binding. Protein binding in human plasma was
assessed by performing equilibrium dialysis with plasma containing
test compounds (10 μM) against 0.1 M PBS (pH 7.4). Following
incubation (6 h at 37 °C), the parent compound was measured in both
plasma and buffer compartments by LC-MS and the percentage of
compound bound to plasma proteins determined. Test compounds
(10 μM) were added to plasma (n = 2) and dialyzed against 0.1 M
phosphate buffered saline (pH 7.4) for 6 h at 37 °C. After incubation,
the contents of each plasma and buffer compartment were removed
and mixed with equal volumes of control buffer or plasma as appro-
priate to maintain matrix equivalence for analysis. Plasma proteins
were then precipitated by the addition of acetonitrile containing
carbamazepine as analytical internal standard, centrifuged, and
the supernatant removed for analysis by mass spectrometry
(LC-MS/MS).
Discover RX ScanMax Kinome Binding Scan. Compounds that
bind the kinase active site directly (sterically) or indirectly (allosteri-
cally) prevent kinase binding to the immobilized ligand, and will
reduce the amount of kinase captured on a solid support. Conversely,
test molecules that do not bind the kinase have no effect on the
amount of kinase captured on the solid support. Screening “hits” are
identified by measuring the amount of kinase captured in test versus
control samples by using a quantitative qPCR method that detects
the associated DNA label to determine a Kd for ligand binding.
Compounds 1 and 2 were tested at 1 μM concentration against a panel
Molecular Modeling and Ligand Docking. The 3DTC.PDB
structure of MLK1 was used as a surrogate for MLK3 to evaluate the
binding mode for the series. The compounds contained a conserved
scaffold that was hand docked into MLK1 and protein and ligand were
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Article
energy minimized using the molecular modeling suite MOE. It was
hypothesized that the compounds acted as type 1 kinase inhibitor so
the scaffold was positioned to ensure that the hydrogen bond donor
and acceptor of the 7-azaindole core interacted with the corresponding
contacts on the hinge. This provided two binding mode hypotheses for
evaluation. The binding mode that fit the SAR data allowed the
scaffold to donate and accept a hydrogen bond to and from Glu221 and
Ala223, respectively. This placed the side-chain indole group to the rear
of the ATP site with the N-methyl-piperazine group positioned toward
the solubilizing pocket.
kinase 3 inhibitor URMC-099 is neuroprotective and anti-inflamma-
tory in models of human immunodeficiency virus-associated neuro-
cognitive disorders. J. Neurosci. 2013, 33, 9998−10010.
(2) Heaton, R. K.; Franklin, D. R.; Ellis, R. J.; McCutchan, J. A.;
Letendre, S. L.; Leblanc, S.; Corkran, S. H.; Duarte, N. A; Clifford, D.
B.; Woods, S. P.; Collier, A. C.; Marra, C. M.; Morgello, S.; Mindt, M.
R.; Taylor, M. J; Marcotte, T. D.; Atkinson, J. H.; Wolfson, T.;
Gelman, B. B.; McArthur, J. C.; Simpson, D. M.; Abramson, I.; Gamst,
A.; Fennema-Notestine, C.; Jernigan, T. L.; Wong, J.; Grant, I. HIV-
associated neurocognitive disorders before and during the era of
combination antiretroviral therapy: differences in rates, nature, and
predictors. J NeuroVirol. 2011, 17, 3−16.
(3) Gelbard, H. A.; Dewhurst, S.; Maggirwar, S. B.; Kiebala, M.;
Polesskaya, O.; Gendelman, H. E. Rebuilding synaptic architecture in
HIV-1 associated neurocognitive disease: a therapeutic strategy based
on modulation of mixed lineage kinase. Neurotherapeutics 2010, 7,
392−398.
(4) Gallo, K. A.; Johnson, G. L. Mixed-lineage kinase control of JNK
and p38 pathways. Nature Rev. Mol. Cell. Biol. 2002, 9, 663−672.
(5) Silva, R. M.; Kuan, C. Y.; Rakic, P.; Burke, R. E. Mixed lineage
kinase-c-jun N-terminal kinase signaling pathway: a new therapeutic
target in Parkinson’s disease. Mov. Disord. 2005, 20, 653−664.
(6) Wang, L. H.; Besirli, C. G.; Johnson, E. M., Jr. Mixed-lineage
kinases: a target for the prevention of neurodegeneration. Annu. Rev.
Pharmacol. Toxicol. 2004, 44, 451−474.
(7) Maroney, A. C.; Finn, J. P.; Connors, T. J.; Durkin, J. T.; Angeles,
T.; Gessner, G.; Xu, Z.; Meyer, S. L.; Savage, M. J.; Greene, L. A.;
Scott, R. W.; Vaught, J. L. Cep-1347 (KT7515), a semisynthetic
inhibitor of the mixed lineage kinase family. J. Biol. Chem. 2001, 276,
25302−25308.
ASSOCIATED CONTENT
* Supporting Information
■
S
Kinase inhibition profiles for 442 kinases for compound 1 and 2
performed by DiscoverRx; inhibition profile using radio ligand
binding assay for 342 wild-type human kinases performed by
Reaction Biology Corp for compound 1. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 858-720-1948. E-mail: vsgoodfellow@califiabio.com.
■
Present Addresses
⊥C.J.L.: Dart NeuroScience LLC, 7473 Lusk Boulevard, San
Diego, California 92121, United States.
#S.B.R.: Epigen Biosciences 10225 Barnes Canyon Road, San
Diego, California 92121, United States.
¶Y.X.: Ropes and Gray LLP, 1900 University Avenue, East Palo
Alto, California 94303, United States.
(8) Handley, M. E.; Rasaiyaah, J.; Barnett, J.; Thakker, M.; Pollara,
G.; Katz, D. R.; Chain, B. M. Expression and function of mixed lineage
kinases in dendritic cells. Int. Immunol. 2007, 19, 923−933.
(9) Handley, M. E.; Rasaiyaah, J.; Chain, B. M.; Katz, D. R. Mixed
lineage kinases (MLKs): a role in dendritic cells, inflammation and
immunity? Int. J. Exp. Pathol. 2007, 88, 111−126.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
(10) Jaeschke, A.; Davis, R. J. Metabolic stress signaling mediated by
mixed-lineage kinases. Mol. Cell 2007, 27, 498−508.
The authors declare no competing financial interest.
(11) Sathyanarayana, P.; Barthwal, M. K.; Kundu, C. N.; Lane, M. E.;
Bergmann, A.; Tzivion, G.; Rana, A. Activation of the drosophila MLK
by ceramide reveals TNF-alpha and ceramide as agonists of
mammalian MLK3. Mol. Cell 2002, 10, 1527−1533.
(12) Leung, I. W.; Lassam, N. Dimerization via tandem leucine
zippers is essential for the activation of the mitogen-activated protein
kinase kinase kinase, MLK-3. J. Biol. Chem. 1998, 273, 32408−32415.
(13) Leung, I. W.; Lassam, N. The kinase activation loop is the key to
mixed lineage kinase-3 activation via both autophosphorylation and
hematopoietic progenitor kinase 1 phosphorylation. J. Biol. Chem.
2001, 276, 1961−1967.
(14) Sui, Z.; Fan, S.; Sniderhan, L.; Reisinger, E.; Litzburg, A.;
Schifitto, G.; Gelbard, H. A.; Dewhurst, S.; Maggirwar, S. B. Inhibition
of mixed lineage kinase 3 prevents HIV-1 Tat-mediated neurotoxicity
and monocyte activation. J. Immunol. 2006, 177, 702−711.
(15) New, D. R.; Maggirwar, S. B.; Epstein, L. G.; Dewhurst, S.;
Gelbard, H. A. HIV-1 Tat induces neuronal death via tumor necrosis
factor-alpha and activation of non-N-methyl-D-aspartate receptors by a
NFkappaB-independent mechanism. J. Biol. Chem. 1998, 273, 17852−
17858.
(16) Dewhurst, S.; Maggirwar, S. B.; Schifitto, G.; Gendelman, H. E.;
Gelbard, H. A. Glycogen synthase kinase 3 beta (GSK-3 beta) as a
therapeutic target in neuroAIDS. J. Neuroimmune Pharmacol. 2007, 2,
93−96.
ACKNOWLEDGMENTS
■
We thank John Harris, John McCall, Angus MacLeod, and
Sanjay B. Maggirwar for helpful guidance for this project and
the National Institutes of Health for continuing funding of
these efforts. This work was funded in part by grants from the
National Institutes of Health, Division of Mental Health, NIH
grant P01MH064570 (H.A.G., PI), and a Qualifying Ther-
apeutic Discovery Project Grant to Califia Bio, Inc.
ABBREVIATIONS USED
■
HAND, HIV-1-associated neurocognitive disorders; HIV-1,
human immunodeficiency virus type 1; LPS, lipopolysaccha-
ride; TNF-α, tumor necrosis factor α; MLK, mixed lineage
kinase; CNS, central nervous system; BBB, blood−brain
̀
barrier; CSF, cerebrospinal fluid; hERG, human ether-a-go-
go-related gene; JNK, c-Jun N-terminal kinase; FLT3, fms-
related tyrosine kinase 3; LRRK2, leucine-rich repeat kinase 2;
MOE, Molecular Operating Environment; NIS, N-iodosuccini-
mide; MeCN, CH3CN, acetonitrile; DIEA, di-isopropylethyl-
amine; THF, tetrahydrofuran; CDI, 1,1′-carbonyldiimidazole;
EtOAC, ethyl acetate; DMSO, dimethylsulfoxide; CH2CL2,
DCM, dichloromethane; PK, pharmacokinetic
(17) Mishra, R.; Barthwal, M. K.; Sondarva, G.; Rana, B.; Wong, L.;
Chatterjee, M.; Woodgett, J. R.; Rana, A. Glycogen synthase kinase-
3beta induces neuronal cell death via direct phosphorylation of mixed
lineage kinase 3. J. Biol. Chem. 2007, 282, 30393−30405.
(18) Sweeney, Z. K.; Lewcock, J. W. ACS Chemical Neuroscience
Spotlight on CEP-1347. ACS Chem. Neurosci. 2011, 2, 3−4.
REFERENCES
■
(1) Marker, D. F.; Tremblay, M. E.; Puccini, J. M.; Barbieri, J.; Gantz
Marker, M. A.; Loweth, C. J.; Muly, E. C.; Lu, S. M.; Goodfellow, V. S.;
Dewhurst, S.; Gelbard, H. A. The new small-molecule mixed-lineage
O
dx.doi.org/10.1021/jm401094t | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(19) Davis, M. I.; Hunt, J. P.; Herrgard, S.; Ciceri, P.; Wodicka, L. M.;
Pallares, G.; Hocker, M.; Treiber, D. K.; Zarrinkar, P. P.
Comprehensive analysis of kinase inhibitor selectivity. Nature
Biotechnol. 2011, 29, 1046−1051.
(35) Wesselingh, S. L.; Glass, J.; McArthur, J. C.; Griffin, J. W.;
Griffin, D. E. Cytokine dysregulation in HIV-associated neurological
disease. Adv. Neuroimmunol. 1994, 4, 199−206.
(36) Chico, L. K.; Van Eldik, L. J.; Watterson, D. M. Targeting
protein kinases in central nervous system disorders. Nature Rev. Drug
Discovery 2009, 8, 892−909.
(37) Cardiac Toxicity hERG; Cerep: Celle L’Evescault, France, 2012;
http://www.cerep.fr/cerep/users/pages/downloads/Documents/
Marketing/Pharmacology%20&%20ADME/Application%20notes/
Cardiac%20toxicity%20hERG-2012.pdf.
(38) Weiss, J. M.; Nath, A.; Major, E. O.; Berman, J. W. HIV-1 Tat
induces monocyte chemoattractant protein-1-mediated monocyte
transmigration across a model of the human blood−brain barrier
and up-regulates CCR5 expression on human monocytes. J. Immunol.
1999, 163, 2953−2959.
(39) Gonzalez, E.; Rovin, B. H.; Sen, L.; Cooke, G.; Dhanda, R.;
Mummidi, S.; Kulkarni, H.; Bamshad, M. J.; Telles, V.; Anderson, S.
A.; Walter, E. A.; Stephan, K. T.; Deucher, M.; Mangano, A.;
SBologna, R.; Ahuja, S. S.; Dolan, M. J.; Ahuja, S. K. HIV-1 infection
and AIDS dementia are influenced by a mutant MCP-1 allele linked to
increased monocyte infiltration of tissues and MCP-1 levels. Proc. Natl.
Acad. Sci. U. S. A. 2002, 99, 13795−13800.
(40) Mankowski, J. L.; Queen, S. E.; Clements, J. E.; Zink, M. C.
Cerebrospinal fluid markers that predict SIV CNS disease. J
Neuroimmunol. 2004, 157, 66−70.
(41) Li, C. J.; Ueda, Y.; Shi, B.; Borodyansky, L.; Huang, L.; Li, Y. Z.;
Pardee, A. B. Tat protein induces self-perpetuating permissivity for
productive HIV-1 infection. Proc. Natl. Acad. Sci. U. S. A. 1997, 94,
8116−8120.
(42) Ganju, R. K.; Munshi, N.; Nair, B. C.; Liu, Z. Y.; Gill, P.;
Groopman, J. E. Human immunodeficiency virus tat modulates the
Flk-1/KDR receptor, mitogen-activated protein kinases, and compo-
nents of focal adhesion in Kaposi’s sarcoma cells. J. Virol. 1998, 72,
6131−6137.
(20) Falsig, J.; Porzgen, P.; Lotharius, J.; Leist, M. Specific
̈
modulation of astrocyte inflammation by inhibition of mixed lineage
kinases with CEP-1347. J. Immunol. 2004, 173, 2762−2770.
(21) Hudkins, R. L.; Diebold, J. L.; Tao, M.; Josef, K. A.; Park, C. H.;
Angeles, T. S.; Aimone, L. D.; Husten, J.; Ator, M. A.; Meyer, S. L.;
Holskin, B. P.; Durkin, J. T.; Fedorov, A. A.; Fedorov, E. V.; Almo, S.
C.; Mathiasen, J. R.; Bozyczko-Coyne, D.; Saporito, M. S.; Scott, R.
W.; Mallamo, J. P. Mixed-lineage kinase 1 and mixed-lineage kinase 3
subtype-selective dihydronaphthyl [3,4-a]pyrrolo[3,4-c]carbazole-5-
ones: optimization, mixed-lineage kinase 1 crystallography, and oral
in vivo activity in 1-methyl-4-phenyltetraphydropyridine models. J.
Med. Chem. 2008, 51, 5680−5689.
(22) Lotharius, J.; Falsig, J.; van Beek, J.; Payne, S.; Dringen, R.;
Brundin, P.; Leist, M. Progressive degeneration of human mesen-
cephalic neuron-derived cells triggered by dopamine-dependent
oxidative stress is dependent on the mixed-lineage kinase pathway. J.
Neurosci. 2005, 25, 6329−6342.
(23) Saporito, M. S.; Brown, E. M.; Miller, M. S.; Carswell, S. CEP-
1347/KT-7515, an inhibitor of c-jun N-terminal kinase activation,
attenuates the 1-methyl-4-phenyl tetrahydropyridine-mediated loss of
nigrostriatal dopaminergic neurons in vivo. J. Pharmacol. Exp. Ther.
1999, 288, 421−427.
(24) Mathiasen, J. R.; McKenna, B. A.; Saporito, M. S.; Ghadge, G.
D.; Roos, R. P.; Holskin, B. P.; Wu, Z. L.; Trusko, S. P.; Connors, T.
C.; Maroney, A. C.; Thomas, B. A.; Thomas, J. C.; Bozyczko-Coyne,
D. Inhibition of mixed lineage kinase 3 attenuates MPP+-induced
neurotoxicity in SH-SY5Y cells. Brain Res. 2004, 1003, 86−97.
(25) Bodner, A.; Toth, P. T.; Miller, R. J. Activation of c-Jun N-
terminal kinase mediates gp120IIIB- and nucleoside analogue-induced
sensory neuron toxicity. Exp. Neurol. 2004, 188, 246−253.
(26) Bodner, A.; Maroney, A. C.; Finn, J. P.; Ghadge, G.; Roos, R.;
Miller, R. J. Mixed lineage kinase 3 mediates gp120IIIB-induced
neurotoxicity. J. Neurochem. 2002, 82, 1424−1434.
(43) Kumar, A.; Manna, S. K.; Khawan, S.; Aggarwal, B. B. HIV-Tat
protein activates c-Jun N-terminal kinase and activator protein-1. J.
Immunol. 1998, 161, 776−781.
(27) Eggert, D.; Dash, P. K.; Gorantla, S.; Dou, H.; Schifitto, G.;
Maggirwar, S. B.; Dewhurst, S.; Poluektova, L.; Gelbard, H. A.;
Gendelman, H. E. Neuroprotective activities of CEP-1347 in models
of neuroAIDS. J. Immunol. 2010, 184, 746−756.
(44) Singh, I. N.; El-Hage, N.; Campbell, M. E.; Lutz, S. E.; Knapp, P.
E.; Nath, A.; Hauser, K. F. Differential involvement of p38 and JNK
MAP kinases in HIV-1 Tat and gp120-induced apoptosis and neurite
degeneration in striatal neurons. Neuroscience 2005, 135, 781−790.
(45) Davies, C.; Tournier, C. Exploring the function of the JNK (c-
Jun N-terminal kinase) signaling pathway in physiological and
pathological processes to design novel therapeutic strategies. Biochem.
Soc. Trans. 2012, 40, 85−89.
(46) Santiago, V.; Giorgio, C.; Stefano, M. Medicinal Chemistry and
the Molecular Operating Environment (MOE): application of QSAR
and molecular docking to drug discovery. Curr. Top. Med. Chem. 2008,
8, 1555−1572.
(47) Braselmann, S.; Taylor, V.; Zhao, H.; Wang, S.; Sylvain, C.;
Baluom, M.; Qu, K.; Herlaar, E.; Lau, A.; Young, C.; Wong, B. R.;
Lovel, S.; Sun, T.; Park, G.; Argade, A.; Jurcevic, S.; Pine, P.; Singh, R.;
Grossbard, E. B.; Payan, D. G.; Masuda, E. S. R406, an orally available
spleen tyrosine kinase inhibitor blocks fc receptor signaling and
reduces immune complex-mediated inflammation. J. Pharmacol. Exp.
Ther. 2006, 319, 998−1008.
(48) Bamborough, P.; Brown, M. J.; Christopher, J. A.; Chung, C.;
Mellow, G. W. Selectivity of kinase inhibitor fragments. J. Med. Chem.
2011, 54, 5131−5143.
(49) Karaman, M. W.; Herrgard, S.; Treiber, D. K.; Gallant, P.;
Atteridge, C. E.; Campbell, B. T.; Chan, K. W.; Ciceri, P.; Davis, M. I.;
Edeen, P. T.; Faraoni, R.; Floyd, M.; Hunt, J. P.; Lockhard, D. J.;
Milanov, Z. V.; Morrison, M. J.; Pallares, G.; Patel, H. K.; Pritchard, S.;
Wodicka, L. M.; Zarrinkar, P. P. A quantitative analysis of kinase
inhibitor selectivity. Nature Biotechnol. 2008, 26, 127−132.
(50) Fabian, M. A.; Biggs, W. H., 3rd; Treiber, D. K.; Atteridge, C. E.;
Azimioara, M. D.; Benedetti, M. G.; Carter, T. A.; Ciceri, P.; Edeen, P.
T.; Floyd, M.; Ford, J. M.; Galvin, M.; Gerlach, J. L.; Grotzfeld, R. M.;
Herrgard, S.; Insko, D. E.; Insko, M. A.; Lai, A. G.; Lelias, J. M.; Mehta,
(28) Parkinson Study Group PRECEPT Investigators. Mixed lineage
kinase inhibitor CEP-1347 fails to delay disability in early Parkinson
disease. Neurology 2007, 69, 1480-1490.
(29) Ma, Q.; Gelbard, H. A.; Maggirwar, S. B.; Dewhurst, S.;
Gendelman, H. E.; Peterson, D. R.; Difrancesco, R.; Hochreiter, J. S.;
Morse, G. D.; Schifitto, G. Pharmacokinetic interaction of CEP-1347
and atazanavir in HIV-infected patients. J. Neurovirol. 2013, 3, 254−60.
(30) StarDrop: Guiding Decisions in Drug Discovery; Optibrium Ltd.:
Cambridge, UK, 2013; http://www.optibrium.com/stardrop/.
(31) Dash, P. K.; Gorantla, S.; Gendelman, H. E.; Knibbe, J.; Casale,
G. P.; Makarov, E.; Epstein, A. A.; Gelbard, H. A.; Boska, M. D.;
Poluektova, L. Y. Loss of neuronal integrity during progressive HIV-1
infection of humanized mice. J. Neurosci. 2011, 31, 3148−3157.
(32) Genis, P.; Jett, M.; Bernton, E. W.; Boyle, T.; Gelbard, H. A.;
Dzenko, K.; Keane, R. W.; Resnick, L.; Mizrachi, Y.; Volsky, D. J.;
Epstein, L. G.; Gendelman, H. E. Cytokins and arachidonic
metabolites produced during human immunodeficiency virus (HIV)-
infected macrophage−astroglia interactions: implications for the
neuropathogenesis of HIV disease. J. Exp. Med. 1992, 176, 1703−
1718.
(33) Wesselingh, S. L.; Power, C.; Glass, J. D.; Tyor, W. R.;
McArthur, J. C.; Farber, J. M.; Griffin, J. W.; Griffin, D. E. Intracerebral
cytokine messenger RNA expression in acquired immunodeficiency
syndrome dementia. Ann. Neurol. 1993, 33, 576−582.
(34) Tyor, W. R.; Glass, J. D.; Griffin, J. W.; Becker, P. S.; McArthur,
J. C.; Bezman, L.; Griffin, D. E. Cytokine expression in the brain
during the acquired immunodeficiency syndrome. Ann. Neurol. 1992,
31, 349−360.
P
dx.doi.org/10.1021/jm401094t | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
S. A.; Milanov, Z. V.; Velasco, A. M.; Wodicka, L. M.; Patel, H. K.;
Zarrinkar, P. P.; Lockhart, D. J. A small molecule-kinase interaction
map for clinical kinase inhibitors. Nature Biotechnol. 2005, 23, 329−
336.
(51) Ma, H.; Deacon, S.; Horiuchi, K. The challenge of selecting
protein kinase assays for lead discovery optimization. Expert Opin.
Drug Discovery 2008, 3, 607−621.
(52) Kaneko, M.; Saito, Y.; Saito, H.; Matsumoto, T.; Matsuda, Y.;
Vaught, J. L.; Dionne, C. A.; Angeles, T. S.; Glicksman, M. A.; Neff, N.
T.; Rotella, D. P.; Kauer, J. C.; Mallamo, J. P.; Hudkins, R. L.;
Murakata, C. Neurotrophic 3,9-bis[(alkylthio)methyl]-and-bis-
(alkoxymethyl)-K-252a derivatives. J. Med. Chem. 1997, 40, 1863−
1869.
(53) Olaharski, A. J.; Gonzaludo, N.; Bitter, H.; Goldstein, D.;
Kirchner, S.; Uppal, H.; Kolaja, K. Identification of a kinase profile that
predicts chromosome damage induced by small molecule kinase
inhibitors. PLoS Comput. Biol. 2009, 5, e1000446.
(54) Morphy, R. Selectively nonselective kinase inhibition: striking
the right balance. J. Med. Chem. 2010, 53, 1413−1437.
(55) Grunwald, M. R.; Levis, M. J. FLT3 inhibitors for acute myeloid
leukemia: a review of their efficacy and mechanisms of resistance. Int. J.
Hematol. 2013, 97, 683−694.
(56) DeBoy, C. A.; Rus, H.; Tegla, C.; Cudrici, C.; Jones, M. V.;
Pardo, C. A.; Small, D.; Whartenby, K. A.; Calabresi, P. A. FLT-3
expression and function on microglia in multiple sclerosis. Exp. Mol.
Pathol. 2010, 89, 109−116.
(57) Davis, M. I.; Hunt, J. P.; Herrgard, S.; Ciceri, P.; Wodicka, L. M.;
Pallares, G.; Hocker, M.; Treiber, D. K.; Zarrinkar, P. P.
Comprehensive analysis of kinase inhibitor selectivity. Nature
Biotechnol. 2011, 29, 1046−1051.
(58) Santos, F. P.; Kantarjian, H. M.; Jain, N.; Manshouri, T.;
Thomas, D. A.; Garcia-Manero, G.; Kennedy, D.; Extrov, Z.; Cortes, J.;
Verstovsek, S. Phase 2 study of CEP-701, an orally available JAK2
inhibitor, in patients with primary or post-polycythemia vera/essential
thrombocythemia myelofibrosis. Blood 2010, 115, 1131−1136.
(59) Olaharski, A. J.; Bitter, H.; Gonzaludo, N.; Kondru, R.;
Goldstein, D. M.; Zabka, T. S.; Lin, H.; Singer, T.; Kolaja, K. Modeling
bone marrow toxicity using kinase structural motifs and the inhibition
profiles of small molecular kinase inhibitors. Toxicol. Sci. 2010, 118,
226−275.
(60) Tsika, E.; Moore, D. J. Mechanisms of LRRK2-mediated
neurodegeneration. Curr. Neurol. Neurosci. Rep. 2012, 12, 251−260.
(61) Marker, D. F.; Puccini, J. M.; Mockus, T. E.; Barbieri, J.; Lu, S.
M.; Gelbard, H. A. LRRK2 kinase inhibition prevents pathological
microglial phagocytosis in response to HIV-1 Tat protein. J.
Neuroinflammation 2012, 9, 261.
(62) Kiebala, M.; Maggirwar, S. B. Ibudilast, a pharmacologic
phosphodiesterase inhibitor, prevents human immunodeficiency virus-
1 Tat-mediated activation of microglial cells. PLoS One 2011, 6,
e18633.
(63) Sui, Z.; Sniderhan, L. F.; Schifitto, G.; Phipps, R. P.; Gelbard, H.
A.; Dewhurst, S.; Maggirwar, S. B. Functional synergy between CD40
ligand and HIV-1 Tat contributes to inflammation: implication in HIV
type 1 dementia. J. Immunol. 2007, 178, 3226−3236.
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