Discovery of a neuroprotective chemical, (S)- N -(3-(3,6-Dibromo-9 H -carbazol-9-yl)-2-fluoropropyl)-6-methoxypyridin-2-amine [(-)-P7C3-S243], with improved druglike properties

Article abstract of DOI:10.1021/jm401919s

(-)-P7C3-S243 is a neuroprotective aminopropyl carbazole with improved druglike properties compared with previously reported compounds in the P7C3 class. It protects developing neurons in a mouse model of hippocampal neurogenesis and protects mature neurons within the substantia nigra in a mouse model of Parkinsons disease. A short, enantioselective synthesis provides the neuroprotective agent in optically pure form. It is nontoxic, orally bioavailable, metabolically stable, and able to cross the blood-brain barrier. As such, it represents a valuable lead compound for the development of drugs to treat neurodegenerative diseases and traumatic brain injury.

Full text of DOI:10.1021/jm401919s

Article
pubs.acs.org/jmc
Discovery of a Neuroprotective Chemical, (S)‑N‑(3-(3,6-
Dibromo‑9H‑carbazol-9-yl)-2-fluoropropyl)-6-methoxypyridin-2-
amine [(−)-P7C3-S243], with Improved Druglike Properties
Jacinth Naidoo,†,⊥ Hector De Jesus-Cortes,‡,§,⊥ Paula Huntington,† Sandi Estill,† Lorraine K. Morlock,†
Ruth Starwalt,† Thomas J. Mangano,∥ Noelle S. Williams,† Andrew A. Pieper,*,§ and Joseph M. Ready*,†
‡
Departments of †Biochemistry and Neuroscience, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard,
Dallas, Texas 75390-9038, United States
§Departments of Psychiatry, Neurology, and Veterans Affairs, University of Iowa Carver College of Medicine, 200 Hawkins Avenue,
Iowa City, Iowa 52242, United States
∥National Institute of Mental Health Psychoactive Drug Screening Program, University of North Carolina at Chapel Hill, Chapel Hill,
North Carolina 27599, United States
S
* Supporting Information
ABSTRACT: (−)-P7C3-S243 is a neuroprotective amino-
propyl carbazole with improved druglike properties compared
with previously reported compounds in the P7C3 class. It
protects developing neurons in a mouse model of hippocampal
neurogenesis and protects mature neurons within the
substantia nigra in a mouse model of Parkinson’s disease. A
short, enantioselective synthesis provides the neuroprotective
agent in optically pure form. It is nontoxic, orally bioavailable, metabolically stable, and able to cross the blood−brain barrier. As
such, it represents a valuable lead compound for the development of drugs to treat neurodegenerative diseases and traumatic
brain injury.
eurodegenerative diseases currently affect millions of
untreated or vehicle-treated mice underwent apoptotic cell
Npeople worldwide, and the incidence of disease is rapidly death within 1 week of their birth. Our screen revealed an
aminopropyl carbazole, which we named P7C3 (1),2 that
increasing as the aging population expands. The magnitude and
approximately doubled the number of surviving newborn
hippocampal cells at this time point. Additional experiments
demonstrated that 1 and derivatives thereof increased the
number of new neurons by blocking apoptotic cell death rather
than by increasing neural stem cell proliferation. Subsequent
chemical optimization yielded P7C3-A20 (2),3 a derivative with
improved potency and toxicity profile that is available on a large
scale.4 Recently, we and others have shown that the P7C3 class
of compounds are broadly protective of mature neurons, with
potent efficacy in multiple models of neurodegenerative disease.
For instance, we discovered that 1 and 2 protect neurons in the
substantia nigra (SNc) in a mouse model of PD involving the
neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP).5 Similarly, we found that they minimize motor
neuron cell death in the spinal cord of G93A SOD1 transgenic
mice, a common model of ALS.6 More recently, 2 was found to
protect rat cerebral cortical neurons in the moderate fluid
percussion model of TBI and to exert antidepressant effects by
increasing hippocampal neurogenesis.7,8 Finally, 1 demonstra-
ted neuroprotective effects in a rat model of age-related
cognitive decline2 and a zebrafish model of retinal degener-
trend of this problem places a growing human and financial
strain on healthcare systems, which is exacerbated by the
absence of effective treatments for many of the most common
afflictions.1 Neurodegenerative diseases, such as amyotrophic
lateral sclerosis (ALS) and Parkinson’s disease (PD), traumatic
brain injury (TBI), and normal age-related cognitive decline
feature, by definition, neuronal cell death. Accordingly, we have
searched for small molecules that could prevent the death of
neurons in a variety of in vivo contexts. We hypothesize that
such neuroprotective agents could possess general utility for
treating disorders associated with neuron cell death.
Rather than focusing on predefined molecular targets
thought to be related to specific neurodegenerative diseases,
we have searched for small molecules with in vivo neuro-
protective properties. In 2010 we reported the results of an
unbiased in vivo screen designed to identify such agents. This
assay involved administration of compounds concurrent with
exposure to the thymidine analogue bromodeoxyuridine
(BrdU), which marked newly born neurons through incorpo-
ration into newly synthesized DNA. The neuroprotective
efficacy of compounds was gauged by evaluating their
protective effect on newborn neurons within the hippocampus
over a 1 week period. Under the assay conditions, around 40%
of newborn neural precursor cells in the hippocampus of
Received: December 14, 2013
Published: April 3, 2014
© 2014 American Chemical Society
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ation.9 While the antidepressant activity of 1 and 2 is correlated
with protection of immature neurons, in all other cases, 1 and/
or 2 protected mature neurons from cell death. Moreover, in
cases where neuron death was associated with behavioral or
learning deficits, neuroprotective efficacy was reflected in an
improvement in motor function and/or learning and memory.
campal neurons in mice because this assay gives a simultaneous
readout of efficacy, acute toxicity, and cell permeability. For this
reason, all analogues of 1 were evaluated initially by infusing
them directly into the left lateral ventricle of environmentally
deprived10 live mice using a subcutaneously implanted osmotic
minipump. Compounds were delivered intracerebroventricu-
larly (ICV) over a 7 day period at a concentration of 10 μM (12
μL/day).11 Mice were additionally dosed intraperitoneally (IP)
daily with BrdU (50 mg/kg) to mark newly born hippocampal
neural precursor cells. At the end of the 1 week dosing, animals
were sacrificed and transcardially perfused. Brain slices were
stained with antibodies to BrdU, and the number of surviving
neural precursor cells was quantified by counting the number of
BrdU+ cells in the hippocampal dentate gyrus. To avoid
complications arising from the surgical implantation of the
pump, we quantified neurogenesis in the hemisphere opposite
to compound infusion. Finally, to obtain consistent data across
many animals, the number of BrdU+ cells was normalized to
the volume of the dentate gyrus.
Our initial objective was to replace the aniline ring with a
heterocyclic ring. Previous studies had shown that the N-phenyl
ring of 1 could be replaced with a 2-pyridyl group (giving 3),
but not a 3- or 4-pyridyl group, while maintaining activity in the
in vivo hippocampal neuroprotection assay.3 Moreover, we had
discovered that fluorination of the central methylene and
substitution of the N-aryl ring improved the activity, but neither
change alone had substantial impact. For example, 4 (i.e.,
methoxy-1) and 5 (i.e., fluoro-1) showed similar activity as 1.
However, fluorinating the anisidine congener 4 to provide 2
substantially improved the activity in the hippocampal
neuroprotection assay. In this context, we targeted 15, a
fluorinated compound containing a methoxy-substituted 2-
aminopyridine. Following the approach we described for the
large-scale synthesis of 2,4 terminal epoxide 7 was opened with
While 2 is active, orally available, brain-penetrant, and
nontoxic, even at doses in excess of the minimally efficacious
dose, we recognized the opportunity to design a molecule with
improved druglike properties. In particular, we sought to
replace the aniline moiety of 2 with an alternative heterocycle,
increase the polarity of the compound, and synthesize the drug
lead as a single enantiomer. Few marketed drugs contain aniline
moieties, and most chiral drugs are produced in optically active
form. Here we describe the discovery and evaluation of
(−)-P7C3-S243 (15), a neuroprotective agent with improved
physicochemical properties. It appears to be nontoxic and more
efficacious than 1 and 2 in animal models of PD. It therefore
represents an optimized neuroprotective agent emerging from
the P7C3 class of compounds.
RESULTS
■
Prior and ongoing work in our groups indicates that the P7C3
class of compounds exert their neuroprotective effects by
stabilizing mitrochondria against the depolarization events that
are known to trigger apoptosis.2 Nonetheless, our most robust
assay involves monitoring neuroprotection of newborn hippo-
Scheme 1
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4-nitrobenzenesulfonyl (4-nosyl, 4-Ns)-protected 2-methoxy-6-
aminopyridine (Scheme 1). Surprisingly, when the resultant
amino alcohol 8 was subjected to fluorination conditions with
penetration. Nonetheless, ICV is clearly not an ideal method
of dosing. Fortunately, the optimized lead 15 showed a smooth
dose−response curve upon intraperitoneal (IP) administration.
It was active at a dose of 1 mg kg−1 day−1 and reached its ceiling
of efficacy at 10 mg kg−1 day−1, results which parallel those
found in the MPTP model of Parkinson’s disease (see below).
Control experiments demonstrated that the increase in BrdU+
neurons within the hippocampus was due to neuroprotection
rather than increased neural stem cell proliferation (Figure S1
in the Supporting Information).
Previous work had demonstrated that the preponderance of
neuroprotective activity resided in a single enantiomer of the
P7C3 class of compounds. The enantiomers of 18, 19, and 20
that are shown in Scheme 2 were the more active of each pair,
1
MorphoDast, HPLC/MS and H NMR analyses indicated the
formal loss of methanol. We speculated that the pyridine
nitrogen displaced an activated alcohol and the cyclized
pyridinium salt (not shown) was demethylated to yield
aminopyridone 9.
We next considered an approach involving amination of a
primary amine. To this end, epoxide 7 was opened with sodium
azide. The resulting secondary alcohol 10 was fluorinated to
provide azido fluoride 11, and the azide was reduced with
triphenylphosphine to yield fluoroamine 12. Reaction of this
amine with iodopyridine 1312 in the presence of CuI and
diketone ligand 14 provided the desired analogue 15 in high
yield on a multigram scale.13 Alternative conditions featuring
Pd catalysts or lacking the diketone ligand provided less than
20% yield of the intended product. In a similar manner, amino
alcohol 16 was arylated to provide 17, the hydroxy congener of
15. Amino alcohol 16 proved to be much less reactive than
fluoroamine 12, resulting in incomplete conversion. Moreover,
in this case adding the diketone ligand improved the yield by
only around 10%, suggesting that the amino alcohol itself can
serve as a ligand for Cu.14
Scheme 2. Stereoselective Synthesis of (−)-15
As described above, compounds were administered to mice
ICV over a 1 week period concurrent with dosing of BrdU IP.
Figure 1 shows that aminopyridine 15 approximately doubled
Figure 1. Efficacy of 15 in a mouse model of neurogenesis.
Compounds were administered using the indicated dose and method
for 7 days, during which time mice were dosed IP daily with BrdU (50
mg kg−1 day−1) to label newborn hippocampal neurons. Data are
shown as mean standard error of the mean (SEM).
suggesting a specific protein-binding partner. We also
determined that tertiary alcohol 21 was more active than the
initial hit 1, suggesting the presence of a hydrophobic binding
site into which the methyl group could project.3 These
observations prompted the question of which, if either,
enantiomer of fluorinated analogues such as 2 and 15 would
display higher potency. In particular, we sought to determine
whether fluorine would act as a polar group and occupy the
binding site favored by the hydroxyl group of 18, 19, and 20 or
act as a hydrophobic group and bind similarly to 21.
Unfortunately, previous attempts to fluorinate an optically
active precursor to 2 provided material with 0−20% ee, so
developing an enantioselective synthesis remained a high
priority.
the number of surviving newborn hippocampal neurons over
this time period and displayed an activity superior to that of 1
and comparable to that of 2. Several of the synthetic
intermediates also displayed neuroprotective activity in this
assay. While azide 11 was inactive under the conditions of this
experiment, the primary amines 12 and 16 as well as the N-
pyridyl amino alcohol 17 showed an increase in the number of
BrdU+ cells compared with vehicle. However, these inter-
mediates were less potent than 15 and even slightly less active
than our initial hit 1. We originally adopted intracerebroven-
tricular delivery to avoid complications related to brain
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Condensation of 3,6-dibromocarbazole (6) with the 3-Ns-
protected glycidol derivative 22 provided the epoxide (−)-7 in
97% yield with 96% ee.15 Epoxide ring opening with azide was
quantitative and provided a substrate for fluorination, (+)-10.
Comparison with the material formed from a Mitsunobu
reaction with (S)-glycidol demonstrated that, as described by
Sharpless and co-workers,16 sulfonate 22 reacted by direct
displacement of the leaving group rather than by epoxide
opening/epoxide closure. Consistent with previous results, we
found that reaction with MorphoDast was not stereospecific, as
azidofluoride 11 was recovered with only 63% ee. We speculate
that the common invertive mechanistic pathway was compro-
mised by an intramolecular displacement with the carbazole
nitrogen, yielding an aziridinium ion intermediate (not shown).
This pathway would occur with overall retention and lower the
observed optical purity. In contrast, we discovered that the
reagent combination perfluorobutanesulfonyl fluoride and
tetrabutylammonium difluorotriphenylsilicate (TBAT) yielded
the desired fluoride with clean inversion.17 Subsequent
processing as before provided optically active (−)-15 with
95% ee. Recrystallization from isopropanol enriched the optical
purity to 99% ee. The (+) enantiomer was similarly prepared
starting from ent-22.
this assay. The more active enantiomer shows roughly the same
activity as the racemic mixture at identical doses. We interpret
this observation to mean that this in vivo assay cannot resolve
the 2-fold difference in concentration between, for example, a
single enantiomer at 10 μM and a racemic mixture at 10 μM (5
μM each enantiomer). X-ray crystallographic analysis of a single
crystal of (−)-15 revealed it to be the S enantiomer (Figure S2
in the Supporting Information). Interestingly, the more active
fluoride is enantiomeric to the more active enantiomer in the
hydroxyl series (18−20). This observation implies that the
binding partner for the P7C3 class of neuroprotective chemicals
contains both a hydrophobic pocket, which can be occupied by
the fluoride or methyl group, and a polar pocket, which
preferentially accommodates a hydroxyl group.
The mouse model of neurogenesis involves protecting
newborn hippocampal neurons from premature apoptotic cell
death. We also sought to determine whether 15 could protect
mature neurons from cell death. In this context, we had
previously demonstrated that 1 and 2 could protect mature
dopaminergic neurons within the SNc from toxicity associated
with MPTP.5 This mouse model of PD entails treating mice for
5 days with a toxic dose of MPTP (30 mg kg−1 day−1) to
initiate cell death within the SNc. In the brain, MPTP is
oxidized to 1-methyl-4-phenylpyridinium cation (MPP+), which
is selectively transported into dopaminergic neurons within the
SNc.18 Within these cells, MPP+ acts as a mitochondrial poison,
generating a pathology that resembles that of Parkinsonian
patients.19 Under our assay conditions, treatment with drug was
initiated a full 24 h after the final dose of MPTP. This dosing
regime ensured that any effect of the P7C3 class of compounds
could be attributed to neuroprotective activity rather than to
simply blocking the uptake or metabolism of MPTP.20 Mice
were administered drug twice daily for 21 days, after which time
they were sacrificed by transcardial perfusion. Fixed brains were
sectioned through the SNc at 30 μM intervals. Every fourth
section was stained with antibodies specific to the enzyme
tyrosine hydroxylase (TH). TH catalyzes the oxidation of L-
tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), which is
the first step in the biosynthesis of dopamine. Accordingly, the
number of TH+ cells provides a quantification of the
neuroprotective efficacy of test compounds following MPTP
exposure. Importantly, all microscopic analyses were performed
blinded to the treatment group.
The pure enantiomers of 15 were first evaluated in the
mouse model of hippocampal neuroprotection after they were
dosed ICV. As shown in Figure 2, (−)-15 is highly active,
whereas its enantiomer shows no neuroprotective activity in
As shown in Figure 3, MPTP effects an approximately 50%
reduction in the number of TH+ neurons under the chronic
dosing protocol described above. Consistent with our earlier
reports, 2 exhibits marked neuroprotective activity, showing a
significant neuroprotective effect when administered at 5 mg
kg−1 day−1 and protection resulting in around 75% rescue at a
dose of 10 mg kg−1 day−1.5 Encouragingly, 15 demonstrated
even higher neuroprotective activity than 2, with the former
showing significant activity at a dose of 1 mg kg−1 day−1 and
rescuing over 85% of the TH+ neurons at the highest dose
tested (10 mg kg−1 day−1). Moreover, the enantiomeric
specificity observed in the MPTP model of PD mirrored that
observed in the mouse model of neurogenesis. Specifically,
(−)-(S)-15 was strongly protective, while the (+)-R enantiomer
was indistinguishable from vehicle treatment under the
conditions of the assay. In this side-by-side experiment, 15
appeared to be at least as effective as 2, a compound that has
proven efficacy in several animal models of neurodegenerative
disease.
Figure 2. Enantiomer-specific efficacy of 15 in a mouse model of
neurogenesis. Compound was administered ICV at the indicated
concentration (0.5 μL/h) for 7 days, during which time mice were
dosed IP daily with BrdU (50 mg kg−1 day−1) to label newborn
hippocampal neurons. The graph shows the mean SEM for each
group (N = 4 or 5). Images are of representative stained sections. Scale
bars = 0.3 mm.
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Figure 3. Efficacy of 15 in the MPTP model of Parkinson’s disease. Mice were administered MPTP (30 mg kg−1 day−1) for 5 days. On the sixth day,
treatment with drug was initiated at the indicated dose (IP, BID, 21 days) . Bars indicate numbers of tyrosine hydroxylase-positive (TH+) cells
detected by immunohistochemical staining of the substantia nigra (mean SEM). Asterisks indicate p < 0.01 (*), p < 0.001 (**), or p < 0.0001
(***) relative to the vehicle (Veh) + MPTP group. The number of mice in each group is shown within the corresponding bar. Representative
immunohistochemical pictures of TH staining in the SNc are shown for 5 mg kg−1 day−1 treatment groups. Scale bars = 300 μM.
Table 1. Pharmacokinetic Properties of ( )-15
a
administration
dose (mg/kg)
AUC(brain) (μg·min/g)
%F (plasma/brain)
AUCbrain/AUCplasma
t1/2 (h)
IV
IP
10
10
20
20
671 14
189 35
319 68
388 29
−
1.1
7.4
>24
15
66/28
90/48
38/29
0.45
0.28
0.81
IP
PO
9.6
a
Presented as mean SEM.
The pharmacokinetic and toxicity profiles of 15 were
of the hERG channel at 50 μM using a patch clamp assay.
Several cytochrome P450s (CYP3A4, -2C9, -3D6, -2C8, -2B6)
were insensitive to (−)-15, while CYP1A2 and CYP2C19
showed modest IC50 values of 20 and 1.9 μM, respectively. On
the basis of the high protein binding of 15 (>99%21), free
concentrations of the drug are unlikely to reach those
concentrations. Finally, (−)-15 was screened through the
NIH’s Psychoactive Drug Screening Program, and no
compelling binding partners were identified. Of 47 neuronal
receptors and channels tested, only the μ-opioid receptor (IC50
= 8.2 μM) and the peripheral benzodiazepine receptor [also
known as translocator protein (TSPO)] (IC50 = 0.35 μM)
showed any ligand displacement. The binding affinity of the
former is clearly too weak to be causative for the biological
activity we observed, provided that the in vitro data are
predictive of binding in vivo. The relevance of binding to TSPO
is less clear, however. While the total brain concentration
remains in the low micromolar range for several hours after
dosing, the free concentration is much lower. Moreover, while
we observed enantiomeric specificity in the neurogenesis
(Figure 2) and MPTP assays (Figure 3), both enantiomers of
15 bind TSPO with similarly weak affinities (0.35 and 0.68
μM). Finally, 2, which shows similar in vivo activity as (−)-15,
binds TSPO more weakly (IC50 = 2.0 μM). A wide structural
range of chemicals have been found to bind TSPO, from the
eponymous benzodiazapines to cholesterol to porphyrins,22
evaluated using both in vivo and in vitro methods. We
observed nearly complete metabolic stability in the presence of
murine hepatocytes (t1/2 > 240 min) and good stability in the
presence of liver microsomes (t1/2 values: mouse, 49 min; rat,
>240 min; dog, 45 min; monkey, 81 min; human, 117 min).
We then monitored plasma and brain levels of the compound
following intravenous, oral, or intraperitoneal dosing. As shown
in Table 1, 15 exhibited a dose-proportional increase in brain
levels when dosed IP. Plasma bioavailability was higher when
the compound was dosed IP as opposed to per os (PO) by oral
gavage, but partitioning into the brain was superior when the
compound was administered by oral gavage. We noted the
same effect with 2, and thus, comparable brain levels are
achieved either IP or by oral gavage. By all routes of
administration, 15 was metabolically stable. While full
pharmacokinetic profiling was performed with ( )-15, we
additionally observed indistinguishable plasma and brain
concentrations for racemic and (−)-15 at a single time point
following intraperitoneal injection of 1, 3, 10, or 30 mg/kg
doses (Figure S3 in the Supporting Information). These data
are consistent with similar ADME properties for the racemic
and optically active forms of 15.
(−)-15 did not show significant toxicity toward the three
human cancer cell lines tested (IC50 > 10 μM vs H2122,
H2009, and HCC44). Likewise, we observed <15% inhibition
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124.5, 141.1, 145.4, 147.6, 163.7. ESI-MS (m/z): 309.9 [M + 1]+;
C12H11N3O5S requires 309.04.
suggesting that it might represent a nonspecific binding partner
for small molecules. Moreover, a tight-binding inhibitor of this
protein showed no toxicity in phase I and II clinical trials for
ALS and is currently in phase II trials for spinal muscular
atrophy.23 Overall, the clean receptor binding results, which are
shown in full in Table S1 in the Supporting Information, are
consistent with our observation that long-term treatment with 2
(up to 40 mg kg−1 day−1 for 21 days) or 15 (up to 10 mg kg−1
day−1 for 21 days) has no negative impact on the weight,
behavior, or appearance of the treated animals.
(−)-(S)-3,6-Dibromo-9-(oxiran-2-ylmethyl)-9H-carbazole
[(−)-(S)-7]. In a modification of a published method,17 a stirred
solution of 3,6-dibromocarbazole (6.27 g, 19.3 mmol) and powdered
potassium hydroxide (1.30 g, 23.2 mmol) in dimethylformamide
(DMF) (100 mL) was cooled in ice before the dropwise addition of
(R)-glycidyl-3-nitrobenzenesulfonate (22)18 (5.01 g, 19.3 mmol). The
ice bath was removed, and the reaction mixture was stirred overnight
at ambient temperature, diluted with water, and washed several times
with water and then brine. The organic layer was dried over Na2SO4,
filtered, and concentrated to give epoxide (−)-7 in quantitative yield,
which was used without additional purification. 1H NMR (CDCl3, 400
MHz): δ 8.13 (d, J = 2.0 Hz, 2H), 7.57 (dd, J = 8.7, 1.8 Hz, 2H),
7.39−7.31 (m, 2H), 4.65 (dd, J = 16.0, 2.5 Hz, 1H), 4.28 (dd, J = 16.1,
5.0 Hz, 1H), 3.32 (ddd, J = 5.2, 2.6, 1.3 Hz, 1H), 2.82 (t, J = 4.3 Hz,
1H), 2.50 (dd, J = 4.7, 2.6 Hz, 1H). 13C NMR (CDCl3, 100 MHz): δ
45.0, 45.1, 50.6, 110.7, 112.8, 123.4, 123.8, 129.5, 139.8. Poor
ionization precluded analysis by mass spectrometry. HPLC (ChiralPak
AD-H, 1.0 mL/min 1% iPrOH/hexanes): tR(major) = 24.3−34.0 min;
tR(minor) = 48.4−58.6 min; ee = 95.9%. [α]2D0 = −5.74 (c = 0.244, THF).
N-(3-(3,6-Dibromo-9H-carbazol-9-yl)-2-hydroxypropyl)-N-
(6-methoxypyridin-2-yl)-4-nitrobenzenesulfonamide (8). n-
BuLi (0.65 mL of a 2.5 M solution in hexanes) was added dropwise
to a solution of N-(6-methoxypyridin-2-yl)-4-nitrobenzenesulfonamide
(278.7 mg, 0.90 mmol) in DMF (3.0 mL) at 0 °C. The solution was
stirred for 15 min before the addition of 3,6-dibromo-9-(oxiran-2-
ylmethyl)-9H-carbazole (7) (265.1 mg, 0.70 mmol). The reaction
mixture was heated to 80 °C for 4 h, after which more epoxide 7
(113.0 mg, 0.3 mmol) was added. The reaction mixture was stirred
overnight at 80 °C, cooled, diluted with EtOAc, and washed twice with
water, twice with 1 N NaOH, twice more with water, and finally brine.
The crude mixture was purified on an automated flash chromatog-
raphy system in 0−10% MeOH/DCM to provide a light-orange solid
CONCLUSIONS
■
We have discovered a novel class of potent neuroprotective
agents known as the P7C3 series of aminopropyl carbazoles.
We have now synthesized and characterized an optimized
member of this series that lacks an aniline moiety. Moreover, it
exhibits improved polarity and is readily available as a single
enantiomer. These characteristics improve its druglike proper-
ties compared with previously reported analogues. Here we
have demonstrated that (−)-15 potently blocks neuron cell
death under two conditions: apoptosis of newborn hippo-
campal neurons and MPTP-mediated killing of dopaminergic
neurons in the substantia nigra. The latter effect is notable for
almost complete protection in this model of Parkinson’s
disease. Future reports will describe the effect of this
neuroprotective chemical in a rat model of PD and mouse
models of TBI. Likewise, we are actively seeking to validate the
molecular target of the P7C3 class of compounds and will
report the results of those studies in due course. Taken
together, the data suggest that (−)-15 is a promising candidate
for treating some of the most devastating neurological
disordersdisorders that are likely to increase in prevalence
and currently lack effective therapies.
1
in 12.5% yield. H NMR (CDCl3, 400 MHz): δ 8.17 (d, J = 1.9 Hz,
2H), 8.04 (d, J = 9.2 Hz, 2H), 7.56 (dd, J = 8.7, 2.0 Hz, 2H), 7.44 (t, J
= 7.9 Hz, 1H), 7.38 (d, J = 8.7 Hz, 2H), 7.05−6.77 (m, 2H), 6.48 (d, J
= 7.7 Hz, 1H), 6.40 (d, J = 8.1 Hz, 1H), 4.43 (ddd, J = 15.5, 13.8, 7.5
Hz, 3H), 4.22−4.03 (m, 1H), 3.92 (dd, J = 15.3, 8.9 Hz, 1H), 3.49 (s,
3H). 13C NMR (CDCl3, 100 MHz): δ 163.9, 155.5, 140.9, 140.0,
129.4, 126.1, 125.7, 123.7, 123.4, 120.2, 114.6, 112.8, 111.2, 107.4,
105.0, 69.1, 68.1, 53.7, 25.8. ESI-MS (m/z): 732.6 [M + HCOO]−;
C27H22Br2N4O6S requires 688.0.
3-((3,6-Dibromo-9H-carbazol-9-yl)methyl)-1-((4-
nitrophenyl)sulfonyl)-2,3-dihydroimidazo[1,2-a]pyridin-5(1H)-
one (9). MorphoDast (100 μL, 0.82 mmol) was added to a solution of
alcohol 8 (60.2 mg, 0.087 mmol) in methylene chloride (2.0 mL). The
reaction mixture was stirred overnight, and the reaction was quenched
by the addition by an equivalent volume of saturated aqueous sodium
bicarbonate. The mixture was extracted three times with methylene
chloride. The combined organic layers were dried over Na2SO4,
filtered, concentrated, and purified by preparative TLC in 5% MeOH/
DCM to provide the pyridone as a yellow solid in 21% yield. 1H NMR
(CDCl3, 400 MHz): δ 8.26−8.13 (m, 2H), 8.06 (d, J = 1.8 Hz, 2H),
7.48 (dd, J = 8.7, 1.9 Hz, 2H), 7.39 (d, J = 8.7 Hz, 2H), 7.35 (dd, J =
9.1, 7.5 Hz, 1H), 7.01−6.80 (m, 2H), 6.22 (dd, J = 9.1, 0.8 Hz, 1H),
5.87 (dd, J = 7.5, 0.8 Hz, 1H), 5.31 (s, 1H), 5.28 (dd, J = 7.4, 3.2 Hz,
1H), 4.88 (dd, J = 15.2, 3.1 Hz, 1H), 4.70 (dd, J = 15.1, 7.5 Hz, 1H),
4.15 (dd, J = 10.2, 8.3 Hz, 1H), 3.87 (dd, J = 10.2, 1.3 Hz, 1H). 13C
NMR (CDCl3, 100 MHz): δ 161.3, 146.3, 144.9, 142.4, 139.4, 129.8,
125.7, 123.9, 123.6, 117.8, 113.2, 111.4, 110.3, 95.2, 86.3, 53.7, 52.0.
ESI-MS (m/z): 700.6 [M + HCOO]−; C26H18Br2N4O5S requires
655.9.
EXPERIMENTAL SECTION
■
General Methods. Unless otherwise stated, commercially available
materials were used without further purification, and reactions were
performed under a nitrogen atmosphere using freshly purified solvents.
Solvents were purified using solvent purification columns purchased
from Glass Contour (Laguna Beach, CA). All of the reactions were
monitored by thin-layer chromatography (TLC) using E. Merck silica
gel 60 F254 precoated plates (0.25 mm). Flash chromatography was
performed with the indicated solvents using silica gel (particle size
0.032−0.063 μm) purchased from Sorbent Technologies. H and 13C
1
NMR spectra were recorded on a Varian Inova 400 or 500
spectrometer. Chemical shifts are reported relative to internal solvent.
Electrospray ionization mass spectrometry (ESI-MS) was performed
on an Agilent 2010 LC−MS instrument. Optical rotations were
measured at 20 °C on a Rudolph Research Analytical Autopol IV
polarimeter. Compounds ( )-7, 10, and 16 were synthesized as
previously described.3,4 All of the synthetic compounds were
1
confirmed to be ≥95% pure by reversed-phase HPLC and H NMR
analyses.
N-(6-Methoxypyridin-2-yl)-4-nitrobenzenesulfonamide. A
solution of 6-methoxypyridin-2-ylamine24 (206.8 mg, 1.66 mmol)
and pyridine (0.2 mL) in methylene chloride (8.0 mL) was stirred for
30 min before being cooled in an ice bath. 4-Nitrobenzenesulfonyl
chloride (383.9 mg, 1.73 mmol) was added slowly to the cold stirring
solution, and the ice bath was removed 10 min later. The reaction
mixture was stirred overnight and then condensed, and the dark-
colored mixture was purified on an automated flash chromatography
system in 0−10% MeOH/dichloromethane (DCM) to provide the
nosylate as a yellow liquid in 55% yield. 1H NMR (CDCl3, 400 MHz):
δ 8.34 (d, J = 8.9 Hz, 2H), 8.14 (d, J = 8.9 Hz, 2H), 7.53 (t, J = 8.0 Hz,
1H), 7.35−7.29 (m, 1H), 6.81 (d, J = 7.7 Hz, 1H), 6.46 (d, J = 8.2 Hz,
1H), 3.74 (s, 3H). 13C NMR (CDCl3, 100 MHz): δ 53.9, 103.7, 106.7,
(+)-(S)-1-Azido-3-(3,6-dibromo-9H-carbazol-9-yl)propan-2-
ol [(+)-(S)-10]. A 20% aqueous solution of sodium azide (75 mL) was
added to a mixture of (S)-(−)-7 (7.79 g, 20.4 mmol) and
tetrahydrofuran (THF) (65 mL), and the mixture was heated to 75
°C. A further 20 mL of a 20% aqueous solution of sodium azide was
added after 24 h to ensure complete conversion of the epoxide. The
biphasic mixture was condensed under reduced pressure, diluted with
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EtOAc, and washed twice with water and then brine. The organic layer
filtered, and concentrated. The well-dried solid was dissolved in 80%
DCM/hexanes, passed through a short silica plug, and washed with
DCM to remove unreacted amine. The filtrate was condensed to give a
residue in 30% yield. 1H NMR (CDCl3, 500 MHz): δ 8.15 (d, J = 2.0
Hz, 2H), 7.57 (dd, J = 8.6, 2.0 Hz, 2H), 7.36 (m, 3H), 6.09 (d, J = 7.9
Hz, 1H), 6.02 (d, J = 7.8 Hz, 1H), 4.55 (bs, 1H), 4.45−4.22 (m, 3H),
3.76 (s, 3H), 3.50 (d, J = 14.7 Hz, 1H), 3.26 (dt, J = 14.1, 5.6 Hz, 1H).
13C NMR (CDCl3, 100 MHz): δ 163.3, 157.4, 140.2, 139.2, 128.8,
was dried over Na2SO4, filtered, and concentrated to give the azido
1
alcohol in 91% yield, which was used without further purification. H
NMR (CDCl3, 400 MHz): δ 8.06 (d, J = 1.9 Hz, 2H), 7.53 (dd, J =
8.7, 2.0 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 4.26 (dd, J = 6.1, 1.6 Hz,
2H), 4.23−4.14 (m, 1H), 3.44 (dd, J = 12.6, 4.3 Hz, 1H), 3.30 (dd, J =
12.6, 5.6 Hz, 1H), 2.48 (s, 1H). 13C NMR (CDCl3, 100 MHz): δ 46.4,
54.1, 69.5, 110.6, 110.7, 112.7, 123.3, 123.3, 123.6, 129.4, 139.5. ESI-
MS (m/z): 466.6 [M + HCOO]−; C15H12Br2N4O requires 421.9.
[α]2D0 = +3.18 (c = 0.26, THF).
123.2, 122.9, 112.0, 110.5, 99.9, 97.4, 70.2, 53.3, 46.5, 46.3. ESI-MS
(m/z): 503.7 [M + H]+; C21H19Br2N3O2 requires 503.0.
(−)-(R)-9-(3-Azido-2-fluoropropyl)-3,6-dibromo-9H-carba-
zole [(−)-(R)-11]. Following a published procedure, diisopropylethyl-
amine (7.75 mL, 44.5 mmol) and toluene (140 mL) were added
alternatingly (ca. 20 portions each) to a mixture of tetrabutylammo-
nium difluorotriphenylsilicate (TBAT) (9.50 g, 17.6 mmol) and (S)-1-
azido-3-(3,6-dibromo-9H-carbazol-9-yl)propan-2-ol (7.41 g, 17.5
mmol) at room temperature.19 Perfluoro-1-butanesulfonyl fluoride
(PBSF) (6.91 mL, 38.5 mmol) was added dropwise, and the reaction
mixture was stirred overnight at ambient temperature. The mixture
was concentrated and purified by automated chromatography (SiO2,
50% DCM/hexanes) to provide the fluoro azide in 70% yield as a
white solid. 1H NMR (CDCl3, 400 MHz): δ 8.14 (t, J = 2.3 Hz, 2H),
7.64−7.52 (m, 2H), 7.32 (dd, J = 8.7, 1.8 Hz, 2H), 5.12−4.85 (m,
1H), 4.54 (ddt, J = 17.9, 5.4, 2.4 Hz, 2H), 3.58 (ddd, J = 17.7, 13.5, 4.5
Hz, 1H), 3.40 (ddd, J = 24.1, 13.5, 4.7 Hz, 1H). 13C NMR (CDCl3,
100 MHz): δ 44.2 (d, JC−F = 23.9 Hz), 51.6 (d, JC−F = 23.9 Hz), 90.0
(S)-N-(3-(3,6-Dibromo-9H-carbazol-9-yl)-2-fluoropropyl)-6-
methoxypyridin-2-amine [(−)-(S)-15. First, (S)-12 (72.2 mg, 0.18
mmol), 2-iodo-6-methoxypyridine14 (45.4 mg, 0.19 mmol), copper(I)
iodide (2.9 mg, 0.015 mmol), and cesium carbonate (119.4 mg, 0.36
mmol) were added to an oven-dried vial, which was then purged with
nitrogen for 10 min. Then DMSO (0.36 mL) and 14 (4 μL, 0.019
mmol) were added. The reaction mixture was heated at 40 °C for 3 h.
The cooled mixture was diluted with EtOAc and washed three times
with a 9:1 saturated NH4Cl/NH4OH solution, three times with water,
and then brine. The organic layer was dried over Na2SO4, filtered, and
concentrated. The well-dried solid was dissolved in methylene chloride
and passed through a short silica plug, which removed the small
amount of unreacted amine. The filtrate was condensed to give a white
solid, which was purified on an automated flash chromatography
system in 50−80% DCM/hexanes to provide (−)-(S)-15 as a white
solid in 61% yield with 96% ee, which was stored under nitrogen.
Recrystallization to enhance the enantiomeric excess was carried out
by heating a solution of 15.8 mg of the purified product in 1.0 mL of
isopropanol. Thin needles formed over 24 h at room temperature. The
overall yield from (S)-12 was 59% with 98.5% ee. Single crystals
suitable for X-ray diffraction were grown by diffusion of hexanes into a
concentrated solution of (−)-15 in dichloroethane (see Figure S2 in
the Supporting Information). 1H NMR (CDCl3, 400 MHz): δ 8.16 (d,
J = 1.9 Hz, 2H), 7.56 (d, J = 1.9 Hz, 2H), 7.35 (t, J = 7.8 Hz, 1H), 7.30
(d, J = 8.7 Hz, 1H), 6.04 (dd, J = 32.7, 8.0 Hz, 2H), 5.29−5.02 (dm,
1H), 4.65−4.46 (m, 3H), 3.87−3.74 (m, 1H), 3.70 (s, 3H), 3.66−3.49
(m, 1H). 13C NMR (CDCl3, 100 MHz): δ 163.7, 156.9, 140.3, 139.7,
129.5, 123.9, 123.4, 112.8, 110.7 (d, JC−F = 1.8 Hz), 99.6, 98.2, 91.4 (d,
JC−F = 174.9 Hz), 45.31 (d, JC−F = 23.9 Hz), 43.6 (d, JC−F = 22.0 Hz).
ESI-MS (m/z): 505.8 [M + 1]+; C22H19Br2FN2O requires 505.0.
HPLC (ChiralPak IA, 1.0 mL/min 100% MeOH): tR(major) = 8.37−
10.02 min; tR(minor) = 10.26−11.26 min. [α]2D0 = −9.5 (c = 0.46, THF).
Animal Studies. All of the animal studies were performed in
accordance with the ethical guidelines established by UT Southwestern
and the University of Iowa.
In Vivo Neuroprotection Assay. Test compounds were
evaluated as described previously.2 Compounds were infused ICV
into the left lateral ventricle of four adult (12 week old) wild-type
C57BL/J6 mice by means of surgically implanted Alzet osmotic
minipumps that delivered solution into the animals at a constant rate
of 0.5 μL/h for 7 days. Alternatively, compounds were administered IP
twice daily. Bromodeoxyuridine (BrdU) was injected IP at 50 mg kg−1
day−1 for 6 days during pump infusion. Twenty-four hours after the
final BrdU administration, mice were sacrificed by transcardial
perfusion with 4% paraformaldehyde at pH 7.4, and their brains
were processed for immunohistochemical detection of incorporated
BrdU in the subgranular zone (SGZ). Dissected brains were immersed
in 4% paraformaldehyde overnight at 4 °C and then cryoprotected in
sucrose before being sectioned into 40 μm thick free-floating sections.
Unmasking of BrdU antigen was achieved by incubation of tissue
sections for 2 h in 50% formamide/2× saline sodium citrate (SSC) at
65 °C followed by a 5 min wash in 2× SSC and subsequent incubation
for 30 min in 2 M HCl at 37 °C. Sections were processed for
immunohistochemical staining with mouse monoclonal anti-BrdU
(1:100). The number of BrdU+ cells in the entire dentate gyrus SGZ
in the contralateral hemisphere (opposite side of the surgically
implanted pump) was quantified by counting BrdU+ cells within the
SGZ and dentate gyrus in every fifth section throughout the entire
hippocampus and then normalizing for dentate gyrus volume.
(d, JC−F = 177.6 Hz), 110.5 (d, JC−F = 1.3 Hz), 113.1, 123.5 (d, JC−F
=
2.2 Hz), 123.9, 129.6, 139.5. ESI-MS (m/z): 468.7 [M + HCOO]−;
C15H11Br2FN4 requires 423.9. HPLC (ChiralPak AD-H, 1.0 mL/min
0.5% iPrOH/hexanes): tR(minor) = 25.0−29.1 min; tR(major) = 29.4−40.7
20
min; ee = 96.2%. [α] = −5.90 (c = 0.217, THF).
(+)-(S)-3-(3,6-DibrDomo-9H-carbazol-9-yl)-2-fluoropropan-1-
amine [(S)-(+)-12]. Triphenylphosphine (3.24, 12.3 mmol) was
added to a solution of azide (−)-(R)-11 (5.10 g, 12.0 mmol) in THF
(80 mL). The cloudy mixture was heated at 65 °C overnight. Water
was added to the cooled reaction mixture, and the resulting mixture
was stirred for 6 h before volatile solvents were removed under
reduced pressure. The viscous mixture was diluted with EtOAc and
washed twice with water and then brine. The organic layer was dried
over Na2SO4, filtered, and concentrated to give a yellow waxy residue.
Sufficient cold methylene chloride was added to dissolve the waxy
residue (ca. 100 mL), and then at 0 °C 1 M HCl was added dropwise
to precipitate the hydrochloride salt. The slurry was stirred for about
20 min before it was filtered and then washed with cold DCM several
times and finally with hexanes. The powdery solid (5.29 g,
quantitative) was dried on a fritted funnel and contained less than
1% PPh3O byproduct. The salt was freebased by adding a saturated
solution of sodium bicarbonate (90.0 mL) to a milky mixture of the
amine HCl salt (5.29 g) in methylene chloride (90.0 mL) with Et3N
(3.0 mL). The solution was stirred for 1 h until the organic layer was
translucent. The organic layer was separated, and the aqueous phase
was extracted with methylene chloride. The combined organic layers
were dried over Na2SO4, filtered, and concentrated to give the free
amine as an off-white solid. 1H NMR (CDCl3, 400 MHz): δ 8.15 (d, J
= 2.0 Hz, 2H), 7.57 (dd, J = 8.8, 1.9 Hz, 2H), 7.36 (d, J = 8.7 Hz, 2H),
4.85 (dtt, J = 47.9, 6.1, 4.4 Hz, 1H), 4.67−4.39 (m, 2H), 3.22−2.78
(m, 2H). 13C NMR (CD3OD, 100 MHz): δ 141.1, 130.5, 125.1, 124.3,
113.8, 112.4, 112.4, 91.2 (d, JC−F = 172.4 Hz), 45.5 (d, JC−F = 20.4
Hz), 42.1 (d, JC−F = 20.4 Hz). [α]2D0 = +8.21 (c = 0.268, MeOH). ESI-
MS (m/z): 398.7 [M + 1]+; C15H13Br2FN2 requires 397.94.
1-(3,6-Dibromo-9H-carbazol-9-yl)-3-((6-methoxypyridin-2-
yl)amino)propan-2-ol (17). 1-Amino-3-(3,6-dibromo-9H-carbazol-
9-yl)propan-2-ol (16) (60.1 mg, 0.15 mmol), 2-iodo-6-methoxypyr-
idine (13) (35.9 mg, 0.15 mmol), copper(I) iodide (2.3 mg, 0.012
mmol), and cesium carbonate (100.9 mg, 0.31 mmol) were combined
in an oven-dried vial under nitrogen. Dimethyl sulfoxide (DMSO) (0.3
mL) and 2,2,6,6-tetramethyl-3,5-heptanedione (14) (3.5 μL, 0.017
mmol) were then added. The reaction mixture was stirred overnight at
ambient temperature, diluted with EtOAc, and washed several times
with water and then brine. The organic layer was dried over Na2SO4,
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MPTP Assay. As previously reported,5 adult male C57Bl/6 mice
were individually housed for 1 week and then injected IP daily for 5
days with 30 mg kg−1 day−1 free-base MPTP (Sigma). On day 6, 24 h
after the mice received the fifth and final dose of MPTP, daily
treatment with compound was initiated. Mice received twice-daily
doses of each compound (or vehicle) for the following 21 days, after
which mice were sacrificed by transcardial perfusion with 4%
paraformaldehyde. The brains were dissected, fixed overnight in 4%
paraformaldehyde, and cryoprotected in sucrose for freezing by
standard procedures. Frozen brains were sectioned through the
striatum and SNc at 30 μM intervals, and every fourth section (spaced
120 μM apart) was stained with antibodies directed against tyrosine
hydroxylase (TH) (Abcam, rabbit anti-TH, 1:2500). Diaminobenzi-
dine was used as a chromagen, and tissue was counter-stained with
hematoxylin to aid in visualization of the neuroanatomy. Images were
analyzed with a Nikon Eclipse 90i motorized research microscope with
Plan Apo lenses coupled with Metamorph Image Acquisition software
(Nikon). TH+ neurons were counted with ImageJ software (NIH) in
every section by two blinded investigators, and the results were
averaged and multiplied by the sectioning interval to determine the
total number of TH+ neurons per SNc.
Pharmacokinetic Analysis. 15 was formulated in 5% DMSO/
10% cremophor EL (Sigma, St. Louis, MO)/85% D5W (5% dextrose
in water, pH 7.2). Adult mice were dosed IV, IP or via oral gavage
(PO) in a total volume of 0.2 mL. Animals were sacrificed at varying
times after dosing by inhalation overdose of CO2. Whole blood was
collected with a syringe and needle coated with anticoagulant citrate
dextrose (ACD) solution (sodium citrate). The blood was
subsequently centrifuged at 9300g for 10 min to isolate plasma.
Plasma was stored at −80 °C until analysis. Brains were isolated from
mice immediately after sacrifice, rinsed extensively with phosphate-
buffered saline (PBS), blotted dry, weighed, and snap-frozen in liquid
nitrogen. Lysates were prepared by homogenizing the brain tissue in a
3-fold volume of PBS (weight of brain in g × 3 = volume in mL of
added PBS). The total lysate volume was estimated as the volume of
added PBS plus the volume of brain. Either plasma or brain (100 μL)
was processed by addition of 200 μL of acetonitrile containing 0.15%
formic acid to precipitate plasma or tissue protein and release bound
drug. This mixture was vortexed for 15 s and then centrifuged at
13120g for 5 min, and the supernatant was analyzed directly by
HPLC/MS/MS. Standard curves were prepared by addition of
compound to blank plasma or blank brain lysate. A value 3-fold
above the signal obtained from blank plasma or brain lysate was
designated as the limit of detection (LOD). The limit of quantitation
(LOQ) was defined as the lowest concentration at which back-
calculation yielded a concentration within 20% of the theoretical value.
Pharmacokinetic parameters were calculated using the noncompart-
mental analysis tool of WinNonLin (Pharsight). Bioavailability was
calculated as (AUCoral/AUCIV) × (DoseIV/Doseoral) × 100%, where
AUC denotes the area under the concentration−time curve). The
brain:blood ratio was calculated using AUC values.
Author Contributions
⊥J.N. and H.D.J.-C. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Prof. Jef De Brabander (UT Southwestern) for
helpful discussions regarding Cu-catalyzed amination and the
NIH’s Psychoactive Drug Screening Program at the University
of North Carolina. Funding was provided by the Edward N.
and Della C. Thome Memorial Foundation and the Welch
Foundation (I-1612) (to J.M.R.) and the NSF (2012140236-
02) (to H.D.J.-C.). Additional support was received from the
NIH (R01 MH087986) to A.A.P. and an unrestricted
endowment provided to Steven L. McKnight by an anonymous
donor.
ABBREVIATIONS USED
■
ALS, amyotrophic lateral sclerosis; PD, Parkinson’s disease;
TBI, traumatic brain injury; BrdU, bromodeoxyuridine; MPTP,
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; SOD, superoxide
dismutase; ICV, intracerebroventricularly; IP, intraperitoneally;
PO, per os (by mouth); TH, tyrosine hydroxylase; SNc,
substantia nigra; AUC, area under the curve.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Table of screening results from the Psychoactive Drug
Screening Program, X-ray crystal structure of (−)-15,
comparison of plasma and brain levels for rac-15 and (−)-15,
and spectra of synthetic materials. This material is available free
of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: andrew-pieper@uiowa.edu (A.A.P.).
*E-mail: joseph.ready@utsouthwestern.edu (J.M.R.).
■
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