Novel inhibitors of neurotropic alphavirus replication that improve host survival in a mouse model of acute viral encephalitis

Article abstract of DOI:10.1021/jm300214e

Arboviral encephalitis is a potentially devastating human disease with no approved therapies that target virus replication. We previously discovered a novel class of thieno[3,2-b]pyrrole-based inhibitors active against neurotropic alphaviruses such as western equine encephalitis virus (WEEV) in cultured cells. In this report, we describe initial development of these novel antiviral compounds, including bioisosteric replacement of the 4H-thieno[3,2-b]pyrrole core with indole to improve metabolic stability and the introduction of chirality to assess target enantioselectivity. Selected modifications enhanced antiviral activity while maintaining low cytotoxicity, increased stability to microsomal metabolism, and also revealed striking enantiospecific activity in cultured cells. Furthermore, we demonstrate improved outcomes (both symptoms and survival) following treatment with indole analogue 9h (CCG-203926) in an in vivo mouse model of alphaviral encephalitis that closely correlate with the enantiospecific in vitro antiviral activity. These results represent a substantial advancement in the early preclinical development of a promising class of novel antiviral drugs against virulent neurotropic alphaviruses.

Full text of DOI:10.1021/jm300214e

Article
pubs.acs.org/jmc
Novel Inhibitors of Neurotropic Alphavirus Replication That Improve
Host Survival in a Mouse Model of Acute Viral Encephalitis
Janice A. Sindac,‡ Bryan D. Yestrepsky,‡ Scott J. Barraza,‡ Kyle L. Bolduc,‡ Pennelope K. Blakely,∥
Richard F. Keep,⊥ David N. Irani,∥ David J. Miller,*,§,# and Scott D. Larsen*,†,‡,#
‡
§
†Vahlteich Medicinal Chemistry Core and Department of Medicinal Chemistry, College of Pharmacy, Departments of Internal
∥
⊥
Medicine and Microbiology and Immunology, Department of Neurology, Departments of Neurosurgery and Neuroanatomy,
University of Michigan, Ann Arbor, Michigan 48109, United States
ABSTRACT: Arboviral encephalitis is a potentially devastating
human disease with no approved therapies that target virus
replication. We previously discovered a novel class of
thieno[3,2-b]pyrrole-based inhibitors active against neurotropic
alphaviruses such as western equine encephalitis virus (WEEV)
in cultured cells. In this report, we describe initial development
of these novel antiviral compounds, including bioisosteric
replacement of the 4H-thieno[3,2-b]pyrrole core with indole to
improve metabolic stability and the introduction of chirality to assess target enantioselectivity. Selected modifications enhanced
antiviral activity while maintaining low cytotoxicity, increased stability to microsomal metabolism, and also revealed striking
enantiospecific activity in cultured cells. Furthermore, we demonstrate improved outcomes (both symptoms and survival)
following treatment with indole analogue 9h (CCG-203926) in an in vivo mouse model of alphaviral encephalitis that closely
correlate with the enantiospecific in vitro antiviral activity. These results represent a substantial advancement in the early preclinical
development of a promising class of novel antiviral drugs against virulent neurotropic alphaviruses.
alphavirus western equine encephalitis virus (WEEV).12 The
lead compound 1 (CCG-32091, Figure 1), discovered via
INTRODUCTION
■
Infections caused by insect-borne viruses (arboviruses)
represent some of the most dramatic examples of disease re-
emergence throughout the world.1 This is due in part to the
significant growth in urban centers in the latter half of the 20th
century, which has produced societal conditions that can greatly
facilitate arbovirus epidemics. One particularly frightening public
health scenario is the recent expansion of specific arboviral
diseases outside of their historical geographic boundaries.2−4
Furthermore, there is the potential for an intentional
introduction of a virulent arbovirus into the environment or
select communities. This has prompted public health authorities
to list numerous arboviruses as high priority biodefense
pathogens, particularly those that infect the central nervous
system (CNS) and cause acute encephalitis.5 This is due in part
to numerous characteristics that make them potential biological
weapons: (i) high clinical morbidity and mortality, (ii) potential
for aerosol transmission, (iii) lack of effective countermeasures
for disease prevention or control, (iv) public anxiety elicited by
CNS infections, (v) ease with which large volumes of infectious
materials can be produced, and (vi) potential for malicious
introduction of foreign genes designed to increase virulence.6,7
The near complete absence of effective drugs to treat
neurotropic arbovirus infections highlights the need for novel
approaches to identify and develop antiviral agents for these
potentially devastating diseases, but research to date in this area
has been sparse.8−11
Figure 1. Structure of HTS lead 1.
high-throughput screening (HTS) in the University of Michigan
Center for Chemical Genomics (CCG), possessed modest
potency with low cytotoxicity. Furthermore, initial structure−
activity relationship (SAR) analysis elucidated with available
library and commercial analogues was encouraging, with small
changes in structure resulting in large changes in activity.
We initiated a medicinal chemistry program around 1 with
two goals in mind: (1) to identify the macromolecular target
and (2) to achieve proof-of-concept activity in an animal model
of alphaviral encephalitis. With regard to the first goal, we
We recently reported the identification of a class of small
molecules that inhibit RNA replication of the neurotropic
Received: February 16, 2012
Published: March 19, 2012
© 2012 American Chemical Society
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designed initial analogues of 1 bearing chiral centers to
determine if eudismic ratios were detectable, which would
establish confidence that our lead was acting through binding
to a chiral macromolecule. With regard to in vivo efficacy,
RESULTS AND DISCUSSION
■
WEEV Replicon SAR. All new analogues were evaluated
with the cell-based replicon assay initially developed for the
HTS, in which the majority of the WEEV structural genes are
replaced with the firefly luciferase gene as a reporter for viral
RNA replication12 and with an MTT assay to evaluate their
effects on cell viability (Table 1). We limited our initial SAR
investigation to thieno[3,2-b]pyrroles and indoles that incorpo-
rated R1 groups that were derivatives of benzyl, based on the
favorable activity exhibited by such analogues in our previous
work.12 After replacing the 2-furanylmethyl amide of 1 with
equipotent benzyl amide (5a) to improve predicted stability to
oxidative metabolism, an initial increase in potency was realized
by replacing the 4-fluorobenzyl at R1 with 4-chlorobenzyl (5b).
The impact of adding chirality at two different positions was
evaluated with analogues 5c−5f. Interestingly, a eudismic ratio
was established at each position but was apparently larger on the
benzyl amide (5c vs 5d) than the pyrrole N-substituent (5e vs
5f). In the absence of a known molecular target, these results are
highly significant in that they are strong evidence that our series is
binding to a chiral macromolecule.
we needed to improve the modest potency of the lead (IC50
=
24 μM) while maintaining low cytotoxicity, and we were con-
cerned about the potential metabolic instability of the electron-
rich 4H-thieno[3,2-b]pyrrole ring.13 Thienopyrrole is a classic
bioisostere for indole,14,15 so the feasibility of replacing the
central bicyclic template of 1 with indole was also investigated.
CHEMISTRY
■
The preparation of new thieno[3,2-b]pyrrole analogues is
summarized in Scheme 1. Methyl ester 2 was N-alkylated
Scheme 1. Preparation of Thieno[3,2-b]pyrrole Analogues
a
of 1
Remaining SAR was elucidated on the bioisosteric indole
template 9. Importantly, the prototype analogue 9a showed no
loss in activity in the replicon assay versus the corresponding
thieno[3,2-b]pyrrole 5a (Table 1). Interestingly, replacing the 4-
fluorobenzyl with 4-chlorobenzyl (9b) did not improve activity
as in the thieno[3,2-b]pyrrole series. Moving the benzylamide
moiety from the 4-position of the piperidine to the 3-position
(9c) was detrimental to activity, as we observed previously in
the thienopyrrole series.12 Shortening the benzylamide by one
carbon (9d) resulted in total loss of activity, while lengthening it
to 2-phenylethylamide (9e) maintained activity but introduced
some cytotoxicity. Appending a para-methyl group to the
phenylethylamide (9f), however, provided a significant boost
in activity, improving the therapeutic index (CC50/IC50) to 14.
As anticipated, incorporating chirality into the benzylamide
established the same eudismic ratio as observed with the thieno-
[3,2-b]pyrroles, with the (R)-enantiomer 9h having superior
activity over (S)-enantiomer 9g. These results strongly suggest
that the indoles are binding in a very similar manner to the
thieno[3,2-b]pyrroles. Final SAR in the benzyl amide series
consisted of 4-methoxy (9i) and 3-chloro (9j) substitution, the
former leading to complete loss of activity. One simple alkyl
amide (9k) evaluated in this initial SAR survey maintained
activity but at the cost of increased cytotoxicity. A preliminary
examination of heterocycles was more encouraging, with
4-pyridylmethyl amide 9l demonstrating a significant boost in
activity over the benzyl amide 9b with no apparent increase in
cytotoxicity. The closely related 3-pyridylmethyl amide 9m was
somewhat less active.
a
Reagents and conditions: (a) K2CO3, DMF, R1Cl or Ph3P,
DIAD, R1OH, THF; (b) KOH, aq EtOH, 40 °C; (c) ethyl
isonipecotate, EDC, HOBt, DIEA, DCM, RT; (d) HNR2R3, EDC,
HOBt, DIEA, RT.
under basic conditions with various benzyl chlorides or under
Mitsunobu conditions16 with benzyl alcohols to afford thieno-
[3,2-b]pyrroles 3. Following saponification of the ester, EDC-
mediated amidation with ethyl isonipecotate provided esters 4.
Final analogues 5 were obtained after a second saponification
and EDC-mediated amidation with various amines. Indole
analogues 9 were prepared in an analogous fashion starting with
ethyl indole-2-carboxylate (Scheme 2).
a
Scheme 2. Preparation of Indole Analogues of 1
Included in Table 1 are calculated partition coefficients (ClogP),
a common estimator of lipophilicity. It is quite possible that some
of the observed differences in replicon inhibition were due to
simple changes in this parameter, as it is generally accepted
that higher lipophilicity results in greater cell permeability.17
For example, some or all of the improved activity of phenethyl
amide 9f vs benzyl amide 9b could simply be due to its higher
lipophilicity and thus expected higher passive permeability.
However, it is conversely very likely that the improved potency
of 4-pyridylmethyl amide 9l vs 9b is a reflection of better intrinsic
activity rather than passive permeability due to its significantly
lower ClogP. Similarly, the eudismic ratios observed for the three
a
Reagents and conditions: (a) K2CO3, DMF, R1Cl, 60 °C; (b) LiOH,
aq THF, 60 °C; (c) ethyl isonipecotate, EDC, HOBt, DIEA, DMF,
RT; (d) HNR2R3, EDC, HOBt, DIEA, RT.
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Table 1. WEEV Replicon and Physical Property Data for New Analogues (Schemes 1 and 2)
a
b
c
d
e
compd
R1
NR2R3
IC50 (μM)
CC50 (μM)
CC50/IC50 ClogP
Log Peff
MLM T1/2 (min)
1
4-F-Ph-CH2
NHCH2furan-2-ylNH
NHCH2Ph
24.4 6.9
25.4 7.3
10.1 2.6
>100
>100
>100
>100
>100
93.4
>4.1
>3.9
>9.9
3.05
3.88
4.45
4.76
4.76
4.04
4.04
3.98
4.55
5.71
4.92
4.62
5.12
4.86
4.86
4.47
5.26
3.58
3.05
3.05
5a
5b
5c
5d
5e
5f
4-F-Ph-CH2
4-Cl-Ph-CH2
4-F-Ph-CH2
NHCH2Ph
−3.6 0. 01
1.7 1.7
(S)-NHCH(CH3)Ph
(R)-NHCH(CH3)Ph
NHCH2Ph
4-F-Ph-CH2
8.3 1.3
18.4 4.7
>100
11
(R)-4-F-PhCH(CH3)
(S)-4-F-PhCH(CH3)
4-F-Ph-CH2
>100
>100
>100
>100
>100
>5.4
NHCH2Ph
9a
9b
9c
9d
9e
9f
NHCH2Ph
14.8 5.8
15.5 5.6
30.6 7.3
>50
>6.7
>6.5
>3.3
4-Cl-Ph-CH2
4-Cl-Ph-CH2
4-Cl-Ph-CH2
4-Cl-Ph-CH2
4-Cl-Ph-CH2
4-Cl-Ph-CH2
4-Cl-Ph-CH2
4-Cl-Ph-CH2
4-Cl-Ph-CH2
4-Cl-Ph-CH2
4-Cl-Ph-CH2
4-Cl-Ph-CH2
NHCH2Ph
−3.6 0.03
7.3 3.9
f
NHCH2Ph
g
NHPh
ND
NHCH2CH2Ph
15.2 9.3
5.5 2.2
>100
62.1
75.2
>100
89.9
ND
4.1
14
NHCH2CH2(4-Me-Ph)
(S)-NHCH(CH3)Ph
(R)-NHCH(CH3)Ph
NHCH2(4-MeO-Ph)
NHCH2(3-Cl-Ph)
N-piperidinyl
−3.9 0.11
−3.6 0.03
9g
9h
9i
6.5 1.5
>50
14
31 7.1
9j
15.3 9.0
9.2 2.8
6.8 1.7
11.4 0.4
>100
51.7
>100
>100
>6.5
5.6
9k
9l
NHCH2pyridin-4-yl
NHCH2pyridin-3-yl
>14
>8.7
−3.7 0.17
9m
a
b
Inhibition of luciferase expression in the WEEV replicon assay. Ribavirin (positive control) has an IC50 in this assay of 16 μM. Cell viability as
c
d
determined by inhibition of mitochondrial reduction of MTT. Calculated logP (ChemBioDraw Ultra 11.0). Log of effective permeability (cm/s) as
determined by PAMPA Explorer (pION). Half-life in mouse liver microsome incubations. The amide CONHCH2Ph is bonded to the 3-position of
the piperidine instead of the 4-position. ND: not determined.
e
f
g
pairs of enantiomers likely reflect actual differences in intrinsic
activity because of their identical ClogPs.
In this regard, we also evaluated selected analogues in the
parallel artificial membrane permeability assay (PAMPA) as a
more direct estimate of passive permeability (Table 1).18 The
most important conclusion from these studies is that as a class
the analogues are predicted to have medium to high passive
cell permeability (Peff ≥ 10−4 cm/s), a critical requirement
for compounds to be active in the CNS. It is also clear that
among the analogues tested there was not a wide variation in
permeability, even when lipophilicity was reduced (9l), which
suggests that the series will have some flexibility for structural
modification without loss of permeability.
In Vitro Antiviral Assays. Four analogues were selected
for advancement to in vitro infection studies: the prototype
thieno[3,2-b]pyrrole 5b and indole 9b analogues, along with
the indole enantiomers 9g and 9h. We wished to directly
compare the effectiveness of the two heterocyclic templates
against live virus and to assess whether the indole enantiomers
had antiviral activity that correlated with activity in the replicon
assay.
We measured antiviral activity using two complementary
assays in cultured neuronal cells: reduction in cytopathic effect
(CPE) and extracellular virus titers (Figure 2). Alphaviruses
such as WEEV and the related neuroadapted Sindbis virus
(NSV) are highly cytolytic to cultured cells, due in part to
vigorous replication and virion production, such that antiviral
compounds are predicted to increase cell viability and decrease
virus titers after infection. Both the thieno[3,2-b]pyrrole 5b
and indole 9b analogues, as well as the indole enantiomer 9h
that was active in the replicon inhibition assay (see Table 1),
Figure 2. Neuronal cell infection studies. Cultured human BE(2)-C
neuronal cells were simultaneously infected with WEEV or NSV and
incubated with the indicated compounds at a final concentration of
25 μM. Cell viability (A) and virus titers (B) were determined 24 h
post-infection by MTT and plaque assays, respectively. Ribavirin (R)
was used as the positive control for both assays. *p < 0.05 compared to
DMSO () controls.
increased cell viability by 1.5−2-fold after either WEEV or NSV
infection (Figure 2A). In contrast, the indole enantiomer 9g,
which was inactive in the replicon inhibition assay, was unable
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Figure 3. Clinical effects of the indole enantiomers, 9g and 9h, in mice with acute NSV encephalomyelitis. Cohorts of infected mice (n = 13/group)
were otherwise not manipulated or were treated with 9g or 9h (30 mg/kg/dose) or with a vehicle control by intraperitoneal injection every 12 h
beginning 12 h after virus challenge and continuing for the next 7 days. (A) Survival differences between drug- and vehicle-treated animals were
measured using a log-rank (Mantel−Cox) test. (B) Similarly, the proportion of mice that either developed mild or no hind limb paralysis following
NSV challenge was determined in each group, and differences between drug- and vehicle-treated animals determined by a log-rank (Mantel−Cox) test.
to rescue virus-induced CPE. We obtained consistent results
when we measured virion production via plaque assays, where
the thieno[3,2-b]pyrrole 5b, the indole 9b analogue, and the
indole enantiomer 9h all significantly reduced both WEEV and
NSV titers, whereas the indole enantiomer 9g was inactive in
this assay (Figure 2B). These antiviral assay results correlated
with the replicon inhibition studies (Table 1) and support the
conclusion that chirality at the benzylamide position is a major
determinant of compound activity. Moreover, the antiviral
effects of 5b, 9b, and 9h were comparable to those of ribavirin,
an established broad spectrum antiviral agent used as a positive
control.
benefit against the development of severe hind limb paralysis
prior to death (Figure 3B), a characteristic feature of NSV-
induced disease that follows intracerebral challenge.19,20
To investigate the effects of 9g and 9h on virus replication in
vivo, the amount of infectious NSV present directly in the CNS
was measured early (1−4 days) after infection, when titers peak
and then begin to wane as antiviral host immunity is activated.
Plaque titration assays showed that viral titers were lower in the
brains of mice receiving 9h compared to 9g or a vehicle control,
achieving statistical significance on day 3 (Figure 4A). A similar
Metabolic Stability. Because the new indole template 9 was
selected in part based on its expected higher resistance to
oxidative metabolism, we also undertook a preliminary evaluation
of the stability of prototypes 5b and 9b to metabolism by
mouse liver microsomes (MLMs). Indole 9b was found to have a
longer half-life than the corresponding thieno[3,2-b]pyrrole 5b
(7.3 min vs 1.7 min, Table 1), suggesting that the new indole
series should have greater potential for in vivo efficacy. On
the basis of its promising antiviral activity, we also evaluated
α-methyl benzylamide analogue 9h. Interestingly, the stability of
9h to metabolism by MLMs is significantly higher than
prototype indole benzylamide 9b (T1/2 = 31 min vs 7.3 min,
Table 1), suggesting that the benzyl amide CH2 may be a major
site of oxidative metabolism that is being sterically shielded by
the methyl group.
In Vivo Infection Studies. We selected the indole
enantiomers 9g and 9h for initial in vivo testing. Compound
9h offered favorable antiviral activity, relatively low cytotoxicity,
and good stability to microsomal metabolism, while 9g provided
an ideal negative control as a closely related but inactive
enantiomer. We chose NSV as our initial in vivo model due to
the lower biosafety requirements for this pathogen (BSL2
containment) compared to highly virulent WEEV (BSL3
containment). Without treatment, direct intracerebral injection
of NSV causes hind limb paralysis and death in weanling mice.
Pilot survival assays using 9h at doses of 10 mg/kg and 30 mg/kg
suggested that the higher concentration was more effective
(data not shown). In larger experimental cohorts, mice treated
with 9h at a dose of 30 mg/kg twice daily beginning 12 h after
viral challenge and continuing for a 7-day period (that reflects
the interval of peak viral replication and clearance) were
significantly protected from lethal NSV infection compared to
animals that were otherwise untreated, that received a vehicle
control, or that were given 9g at the identical concentration
(Figure 3A). Importantly, treatment with 9h also conferred
Figure 4. Virological effects of the indole enantiomers, 9g and 9h, in
mice with acute NSV encephalomyelitis. Cohorts of NSV-infected
mice were treated with 9g or 9h (30 mg/kg/dose) or with a vehicle
control by intraperitoneal injection every 12 h beginning 12 h after
virus challenge. At 24 h intervals, viral titers were measured in
quadruplicate brain (A) and spinal cord (B) tissue samples from each
group by plaque titration assay. Statistical differences (*p < 0.05) in
tissue viral loads were determined by an unpaired Student’s t-test
compared to vehicle-treated controls.
extent of viral inhibition was also observed in the spinal cords
of NSV-infected mice (Figure 4B). Prior studies have shown
that reducing CNS viral titers by a log are associated with
improved disease outcome after NSV infection.19,20
To determine the extent to which clinical protection
correlated with enhanced neuronal survival, an established
fluorojade labeling method was employed. In the hippocampus,
where many neurons are infected by NSV, treatment with 9h
led to reduced neuronal injury compared to mice that were
given either 9g or a vehicle control (Figure 5B). In these assays,
the Nissl substance was stained to enumerate hippocampal
neurons and the proportion of these cells that were also
fluorojade-positive was counted by fluorescence microscopy
(example shown in Figure 5A). In the spinal cord, silver staining
was used to label motor neuron axons in lumbar ventral nerve
roots (Figure 6A), and the density of preserved axons following
treatment with 9h showed enhanced cell survival compared to
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Figure 5. Effects of the indole enantiomers, 9g and 9h, on neuronal survival in the brains of NSV-infected mice. The anatomically defined
hippocampus, a site of heavy NSV infection, was chosen for further study. (A) Representative fluorojade staining of damaged hippocampal neurons
in the brains of NSV-infected mice. NisslRed was first used to label neurons in sections through the hippocampus, while fluorojade staining (arrows)
was then performed to identify those cells undergoing active degeneration. (B) The proportion of fluorojade-positive hippocampal neurons was
determined in quadruplicate slides prepared from 3 mice in each group. Statistical differences (*p < 0.05) in the number of labeled (injured) neurons
were determined by an unpaired Student’s t-test compared to vehicle-treated control animals.
Figure 6. Effects of the indole enantiomers, 9g and 9h, on neuronal survival in the spinal cords of NSV-infected mice. Silver staining of tissue
sections prepared from the lumbar spinal column was used to identify the ventral nerve roots and the motor neuron axons they carry. (A) A
representative specimen from an uninfected animal is shown, with the L4 and L5 ventral nerve roots identified (marked with *). (B) Quantification
of axonal density in these lumbar ventral nerve roots shows relative neuronal sparing in 9h-treated animals compared to vehicle- or 9g-treated
controls. Statistical differences (*p < 0.05) were determined using unpaired Student’s t-tests.
mice given 9g or a vehicle control (Figure 6B). Taken together,
these data show that 9h exerts an antiviral effect within the CNS
of NSV-infected mice, causing enhanced neuronal survival that
leads to improved disease outcome.
suggesting that it is a chiral macromolecule. Importantly,
we have established that an analogue (9h, CCG-203926) with
only modest potency is capable of improving outcomes, both
symptoms and survival, in a mouse model of alphavirus
infection, and that the observed protective effects correlate with
both in vitro and in vivo antiviral activity, and with the extent of
neuronal protection from viral damage. Current studies in our
laboratories are aimed at further improving the potency of this
new class of indole-based antiviral agents. We also intend to
evaluate their efficacy in mouse models of WEEV infection,
with results to be reported in due course.
Successful antiviral therapy in animal models of acute
alphavirus encephalitis has only been accomplished in a few
settings. In newborn mice, seco-pregnane steroids delayed
mortality when given within 4 h of viral challenge but did not
alter overall survival.21 In weanling mice infected intranasally
with a vaccine strain of Venezuelan equine encephalitis virus
(VEEV), (−)-carbodine improved outcome when initiated
up to 4 days after viral challenge while lowering peak CNS
viral titers by a half-log.22 The same drug showed no benefit
following wild-type VEEV challenge. While of relatively modest
clinical utility, these studies highlight the fact that successful
compounds do not have to exert dramatic antiviral effects
within the CNS to exert protective effects. Future studies
will be aimed at broadening the window of effectiveness from
the time of viral challenge and synergizing with unrelated
treatments known to exert more direct neuroprotective effects.
EXPERIMENTAL SECTION
■
General Synthetic Procedures. All reagents were used as
received from commercial sources unless otherwise noted. 1H and
13C spectra were obtained in DMSO-d6 or CDCl3 at room temperature,
unless otherwise noted, on a Varian Inova 400 MHz, Varian Inova
500 MHz, or Bruker Avance DRX 500 instruments. Chemical shifts for
the 1H NMR and 13C NMR spectra were recorded in parts per million
(ppm) on the δ scale from an internal standard of residual
tetramethylsilane (0 ppm). Mass spectroscopy data were obtained on
a Waters Corporation LCT. Purities of final compounds used for
biological testing were determined by HPLC and were ≥95% unless
reported otherwise. HPLC retention times were recorded in minutes
(min) using an Agilent 1100 series with an Agilent Zorbax Eclipse
Plus−C18 column and a 10−90% acetonitrile/water over 13 min
gradient (gradient A). Combustion analyses (CHN) were performed
by Robertson Microlit Laboratories (Madison, NJ). Solvent abbrevia-
tions used: MeOH (methanol), DCM (dichloromethane), EA
CONCLUSIONS
■
In summary, our initial medicinal chemistry efforts have been
successful at improving both the potency and metabolic
stability of lead alphavirus RNA replication inhibitor 1.
Furthermore, we have demonstrated that the as yet unidentified
molecular target exhibits a significant level of enantioselectivity,
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(ethyl acetate), hex (hexanes), DMSO (dimethylsulfoxide), DMF
(dimethylformamide), H2O (water), THF (tetrahydrofuran). Reagent
abbreviations used: DIAD (diisopropyl diazodicarboxylate), HOBt
(1-hydroxy-1,2,3-benzotriazole), EDC (N-(3-dimethylaminopropyl)-
N′-ethylcarbodiimide hydrochloride), DIEA (diisopropylethylamine),
PPh3 (triphenylphosphine), MgSO4 (magnesium sulfate), NaHCO3
(sodium bicarbonate), NH4Cl (ammonium chloride), K2HCO3
(potassium bicarbonate), K2CO3 (potassium carbonate), KOH
(potassium hydroxide), and HCl (hydrogen chloride). Assay abbrevia-
tions: LUC(luciferase), MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide.
and washed in a separatory funnel sequentially with satd aq NH4Cl,
H2O, and brine. The organic layer was dried over MgSO4 and purified
via Biotage silica column (10 g, 100% hexane to 33% EA:hex elution),
yielding 4a as a light-brown oil (49 mg, 80% yield). TLC Rf (1:1
hex:EA): 0.60. 1H NMR (CDCl3, 500 MHz): δ (ppm) 7.21 (d, J = 5.3
Hz, 1H), 7.15 (dd, J = 5.5, 8.5 Hz, 2H), 6.97 (t, J = 8.5 Hz, 2H), 6.91
(d, J = 5.4 Hz, 1H), 6.58 (s, 1H), 5.45 (s, 2H), 4.36−4.26 (m, 2H),
4.17 (quart, J = 7.2 Hz, 2H), 3.08−2.97 (m, 2H), 2.50 (quint, J = 3.7
Hz, 1H), 1.92−1.80 (bm, 2H), 1.54−1.35 (bm, 2H), 1.29 (t, J = 7.2
Hz, 3H).
(R)-Ethyl 1-(4-(1-(4-Fluorophenyl)ethyl)-4H-thieno[3,2-b]pyrrole-
5-carbonyl)piperidine-4-carboxylate (4e). Synthesized in the same
manner as compound 4a. TLC Rf (33% EA:hex): 0.45. 1H NMR
(CDCl3, 500 MHz): δ (ppm) 7.21 (d, J = 5.3 Hz, 1H), 7.15 (dd, J =
5.5, 8.5 Hz, 2H), 6.97 (t, J = 8.5 Hz, 2H), 6.91 (d, J = 5.4 Hz, 1H),
6.58 (s, 1H), 6.22 (q, 6.4 Hz, 1H), 4.36−4.26 (m, 2H), 4.17 (quart, J =
7.2 Hz, 2H), 3.08−2.97 (m, 2H), 2.50 (quint, J = 3.7 Hz, 1H), 1.93 (d,
6.4 Hz, 3H), 1.92−1.80 (bm, 2H), 1.54−1.35 (bm, 2H), 1.29 (t, J =
7.2 Hz, 3H).
(S)-Ethyl 1-(4-(1-(4-Fluorophenyl)ethyl)-4H-thieno[3,2-b]pyrrole-
5-carbonyl)piperidine-4-carboxylate (4f). Synthesized in the same
manner as compound 4a. TLC Rf (33% EA:hex): 0.45. 1H NMR
(CDCl3, 500 MHz): δ (ppm) 7.21 (d, J = 5.3 Hz, 1H), 7.15 (dd, J =
5.5, 8.5 Hz, 2H), 6.97 (t, J = 8.5 Hz, 2H), 6.91 (d, J = 5.4 Hz, 1H),
6.58 (s, 1H), 6.22 (q, 6.4 Hz, 1H), 4.36−4.26 (m, 2H), 4.17 (quart, J =
7.2 Hz, 2H), 3.08−2.97 (m, 2H), 2.50 (quint, J = 3.7 Hz, 1H), 1.93 (d,
6.4 Hz, 3H), 1.92−1.80 (bm, 2H), 1.54−1.35 (bm, 2H), 1.29 (t, J =
7.2 Hz, 3H).
General Procedure A for Generating Analogues 5a−f from
4. Compound 4a (333 mg, 0.804 mmol) and KOH (158 mg,
2.82 mmol) were dissolved in 3:1 EtOH:H2O solution (8 mL). The
mixture was heated to 40 °C and allowed to stir for 16 h. The reaction
was judged complete by TLC monitoring and was quenched by the
addition of 1N HCl (40 mL), causing immediate precipitation of a
white solid. The aqueous suspension was extracted 3× with ethyl
acetate (3× 15 mL) and then washed with H2O and brine (20 mL).
The organic layer was dried over MgSO4 and concentrated in vacuo to
a light-brown oily solid (287 mg, 91% yield).
A portion of the isolated carboxylic acid (50 mg, 0.13 mmol) was
dissolved in dichloromethane (1.3 mL), followed by EDC (30 mg,
0.16 mmol), hydroxybenzotriazole (21 mg, 0.16 mmol), and DIEA
(338 μL, 0.194 mmol). The solution was allowed to stir at RT 30 min,
at which point amine (0.194 mmol) was added. The solution was
allowed to stir 16 h under nitrogen atmosphere. The reaction was
diluted with additional dichloromethane (20 mL) and then washed
with saturated aqueous NH4Cl solution, H2O, and brine. The organic
layer was dried over MgSO4 and then concentrated in vacuo. Further
purification via Biotage silica column (10 g, 100% DCM to 1%
MeOH:DCM) afforded the desired compound as a white solid.
N-Benzyl-1-(4-(4-fluorobenzyl)-4H-thieno[3,2-b]pyrrole-5-
carbonyl)piperidine-4-carboxamide (5a). Synthesized from 4a by
procedure A, using benzylamine as the amide coupling partner. TLC
Rf (10% MeOH:DCM): 0.45. 1H NMR (CDCl3, 500 MHz): δ (ppm)
7.36 (t, J = 6.0 Hz, 2H), 7.33−7.24 (m, 3H), 7.21 (d, J = 5.4 Hz, 1H),
7.15 (t, J = 5.4 Hz, 2H), 6.97 (t, J = 8.7 Hz, 2H), 6.88 (d, J = 5.4 Hz,
1H), 6.58 (s, 1H), 5.68 (bs, 1H), 5.44 (s, 2H), 4.54−4.35 (m, 4H),
2.93 (t, J = 11.7 Hz, 2H), 2.36 (m, 1H), 1.87−1.79 (m, 2H), 1.61−
1.50 (m, 2H). TOF ES+ MS: 476.2 (M + H). HPLC (gradient A):
retention time = 7.23, purity >95%.
N-Benzyl-1-(4-(4-chlorobenzyl)-4H-thieno[3,2-b]pyrrole-5-
carbonyl)piperidine-4-carboxamide (5b). Synthesized from 4b
(prepared from 2 as described for 4a using 4-chlorobenzyl chloride)
by procedure A, using benzylamine as the amide coupling partner.
TLC Rf (70% EA/hex): 0.2. 1H NMR (400 MHz, DMSO-d6) δ 8.34(t,
J = 5.8 Hz,1H), 7.42−7.12 (m, 13H), 6.71 (s, 1H), 5.45 (s, 2H), 4.27
(d, J = 5.8 Hz, 4H), 2.93 (m, 1H), 2.42 (m, 1H), 1.71 (d, J = 11.2 Hz,
2H), 1.44 (d, J = 11.3 Hz, 1H). 13C NMR (400 MHz, DMSO-d6)
173.5, 161.7, 142.1, 139.5, 137.2, 132.0, 129.5, 129.0, 128.4, 128.1,
127.0, 126.7, 126.6, 121.2, 111.3, 103.9, 48.3, 41.8, 41.5, 28.4. TOF ES
+ MS: 492.0 (M + H). HPLC (gradient A): retention time = 7.73.
Methyl 4H-Thieno[3,2-b]pyrrole-5-carboxylate (2). 4H-Thieno-
[3,2-b]pyrrole-5-carboxylic acid (200 mg, 1.20 mmol) was dissolved in
DMF (2.4 mL) and cooled to 0 °C in an ice bath. To this solution
were added KHCO3 (132 mg, 1.32 mmol) and iodomethane (150 μL,
2.40 mmol). The reaction was allowed to warm to RT and stirrred
for 16 h. The reaction was diluted with H2O (∼15 mL), causing the
precipitation of a light-tan solid. The precipitate was isolated via
vacuum filtration and washed with H2O, yielding compound 2 (178
1
mg, 78% yield). TLC Rf (10% MeOH:DCM): 0.9. H NMR (CDCl3,
500 MHz): δ (ppm) 9.15 (bs, 1H), 7.36 (d, J = 5.4 Hz, 1H), 7.16 (d,
J = 1.4 Hz, 1H), 6.99 (d, J = 5.4 Hz, 1H), 3.93 (s, 3H).
Methyl N-(4-Fluoro)benzyl-4H-thieno[3,2-b]pyrrole Carboxylate
(3a). To a stirred solution of 2 (200 mg, 1.10 mmol) in 18 mL of
DMF at 60 °C were added finely powdered K2CO3 (304 mg,
2.20 mmol) and 4-fluorobenzyl chloride (264 uL, 2.20 mmol). The
reaction mixture was allowed to stir 18 h and monitored by TLC. The
reaction mixture was diluted with ∼30 mL of H2O and extracted with
ethyl acetate (3 × 10 mL). The organic layer was washed with satd
aq NaHCO3, satd aq NH4Cl, and brine. The organic layer was dried
over MgSO4, concentrated in vacuo, and purified via 40 g Biotage
CombiFlash silica column (2:1 hexane:ethyl acetate), affording 3a
(221 mg, 69% yield) as a viscous brown oil. TLC Rf (33% EA:hexane):
0.81. 1H NMR (CDCl3, 500 MHz): δ (ppm) 7.40 (d, J = 5.4 Hz, 2H),
7.14 (m, 2H), 6.99 (t, J = 8.7 Hz, 2H), 6.88 (d, J = 5.4 Hz, 1H), 5.74
(s, 2H), 3.86 (s, 3H).
(R)-Methyl 4-(1-(4-Fluorophenyl)ethyl)-4H-thieno[3,2-b]pyrrole-
5-carboxylate (3e). Compound 2 (100 mg, 0.551 mmol), PPh3
(289 mg, 1.10 mmol), and (S)-1-(4-fluorophenyl)ethanol (104 μL,
1.10 mmol) were combined in 1.8 mL of anhydrous THF at 0 °C.
DIAD (212 μL, 1.10 mmol) was then added dropwise. The reaction
mixture was stirred at 0 °C for 1 h, then allowed to warm to RT for an
additional 12 h. The solvent was removed in vacuo, leaving a viscous
black liquid that was purified via Biotage (10 g silica column, 5:1
hexanes:ethyl acetate) to yield compound 3e (104 mg, 62% yield) as a
clear oil. TLC Rf (20% EA:hex): 0.65. 1H NMR (CDCl3, 500 MHz): δ
(ppm) 7.25 (m, 3H), 7.19 (d, J = 5.4 Hz, 1H), 7.02 (t, J = 8.7 Hz, 2H),
6.97 (q, J = 6.9 Hz, 1H), 6.45 (d, J = 5.4 Hz, 1H), 3.90 (s, 3H), 1.91
(d, J = 6.9 Hz, 3H).
(S)-Methyl 4-(1-(4-Fluorophenyl)ethyl)-4H-thieno[3,2-b]pyrrole-
5-carboxylate (3f). Prepared in the same manner as compound 3e
starting with (R)-1-(4-fluorophenyl)ethanol. TLC Rf (20% EA:hex):
1
0.65. H NMR (CDCl3, 500 MHz): δ (ppm) 7.25 (m, 3H), 7.19 (d,
J = 5.4 Hz, 1H), 7.02 (t, J = 8.7 Hz, 2H), 6.97 (quart, J = 6.9 Hz, 1H),
6.45 (d, J = 5.4 Hz, 1H), 3.90 (s, 3H), 1.91 (d, J = 6.9 Hz, 3H).
Ethyl 1-(4-(4-Fluorobenzyl)-4H-thieno[3,2-b]pyrrole-5-carbonyl)-
piperidine-4-carboxylate (4a). Compound 3a (179 mg, 0.619 mmol)
and KOH (118 mg, 2.11 mmol) were dissolved in a 3:1 EtOH:H2O
solution (6 mL). The mixture was heated to 40 °C and allowed to stir
for 16 h. The reaction was judged complete by TLC. Reaction was
quenched by the addition of 1N HCl (30 mL), causing immediate
precipitation of a white solid. Solid was collected via vacuum filtration
and washed with H2O. Drying in vacuo yielded the carboxylic acid
as a white powder (147 mg, 86% yield). The isolated carboxylic acid
(391 mg, 1.42 mmol) was dissolved in DCM (14 mL), to which EDC
(326 mg, 1.70 mmol), 1-hydroxybenzotriazole (230 mg, 1.70 mmol),
and DIEA (371 μL, 2.13 mmol) were added. The solution was allowed
to stir at RT for 30 min, at which point ethyl isonipecotate (328 μL,
2.13 mmol) was added. The reaction was allowed to stir at RT for
16 h. The reaction was then diluted with additional DCM (25 mL)
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(S)-1-(4-(4-Fluorobenzyl)-4H-thieno[3,2-b]pyrrole-5-carbonyl)-N-
(1-phenylethyl)piperidine-4-carboxamide (5c). Synthesized from 4a
by Procedure A, using (S)-α-methylbenzylamine as the amide coupling
5.87 (s, 2H). TOF ES+ MS: 286.0 (M + H). HPLC (gradient A):
retention time = 7.88 min.
Ethyl 1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)piperidine-4-
carboxylate (8b). 1-(4-Chlorobenzyl)-1H-indole-2-carboxylic acid
(1.00 g, 3.50 mmol), EDC (1.34 g, 6.99 mmol), and 1-hydro-
xybenzotriazole (0.946 g, 7.00 mmol) were added to an oven-dried
RBF. The solids were dissolved in 4.0 mL of DCM and allowed to
stir at room temperature for 15 min. Ethyl piperidine-4-carboxylate
(1.08 mL, 7.01 mmol) and DIEA (1.22 mL, 7.00 mmol) were added
sequentially. The reaction was allowed to stir overnight at room
temperature. The reaction was diluted with water and ethyl acetate.
The layers were separated. The ethyl acetate layer was washed with
saturated sodium chloride. The organic layer was dried over magnesium
sulfate, filtered through a silica gel plug, and concentrated to obtain an
oily product. The crude material was triturated in ethyl acetate to
obtain white solid as the product (1.29 g, 87%). TLC Rf (60% EA/hex):
0.5. 1H NMR (400 MHz, DMSO-d6) δ 7.63−7.55 (m, 2H), 7.33−7.28
(m, 2H), 7.22 (t, J = 7.1 Hz, 1H), 7.12−7.02 (m, 3H), 6.72 (s, 1H),
5.49 (s, 2H), 4.25 (s, 1H), 4.06 (q, J = 7.1, 4.2 Hz, 2H), 3.88 (s, 2H),
2.60−2.52 (m, 1H), 1.75 (s, 2H), 1.44−1.01 (m, 6H). TOF ES+ MS:
286.0 (M + H). HPLC (gradient A): retention time = 7.88 min.
1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)piperidine-4-carbox-
ylic Acid. Compound 8b (4.2 g, 9.8 mmol) and lithium hydroxide
hydrate (4.1 g, 98 mmol) were dissolved in 10 mL of THF and 40 mL
of water. The reaction was allowed to stir overnight. The reaction was
diluted with water and diethyl ether. The aqueous layer was washed
with another aliquot of diethyl ether before it was acidified with 2N
HCl to pH ∼ 2. The aqueous layer solution was washed with ethyl
acetate (3 × 200 mL). The ethyl acetate layers were combined and
washed with saturated aqueous solution of sodium chloride, dried over
magnesium sulfate, filtered, and concentrated in vacuo to obtain the
product as a white solid (3.33 g, 86%). TLC Rf (60% EA/hex): 0.25.
1H NMR (400 MHz, DMSO-d6) δ 12.30 (s, 1H), 7.66 (d, J = 7.9 Hz,
1H), 7.59 (d, J = 8.4 Hz, 1H), 7.35 (d, J = 8.4 Hz, 2H), 7.25 (t, J =
11.4, 3.9 Hz, 1H), 7.16−7.09 (m, 3H), 6.75 (s, 1H), 5.52 (s, 2H), 4.29
(s, 1H), 4.04 (s, 1H), 3.06 (s, 2H), 2.52−2.47 (m, 1H), 1.80 (s, 2H),
1.22 (s, 2H). TOF ES+ MS: (M + H). HPLC (gradient A): retention
time = 6.98 min.
General Procedure B for Preparing Compounds 9a−m. 1-(1-
(4-Chlorobenzyl)-1H-indole-2-carbonyl)piperidine-4-carboxylic acid
(1.0 equiv), EDC (2.0 equiv), and 1-hydroxybenzotriazole (2.0 equiv)
were dissolved in DCM (3.0 mL) and allowed to stir at room temp-
erature for 15 min. DIEA (2.0 equiv) and desired amine (2.0 equiv)
were added. The reaction was allowed to stir at room temperature
overnight. The reaction was diluted with water and ethyl acetate.
The organic layer was washed with water, saturated sodium bicarbonate,
1N HCl, and saturated sodium chloride. The organic layer was dried
over magnesium sulfate, filtered, and concentrated in vacuo. The crude
material was recrystallized in diethyl ether/ethyl acetate to afford desired
product.
1
partner. TLC Rf (1:1 hex:EA): 0.20. H NMR (CDCl3, 500 MHz): δ
(ppm) 7.37 (t, J = 6.0 Hz, 2H), 7.31 (m, 3H), 7.20 (d, J = 5.3 Hz, 1H),
7.14 (dd, J = 5.3, 8.4 Hz, 2H), 6.96 (t, J = 8.7 Hz, 2H), 6.87 (d, J = 5.3
Hz, 1H) 6.58 (s, 1H), 5.63 (d, J = 7.5 Hz, 1H), 5.43 (s, 2H), 5.15
(quint, J = 7.3 Hz, 1H), 4.44 (m, 2H), 2.91 (m, 2H), 2.31 (m, 1H),
1.81 (m, 2H), 1.58 (m, 2H), 1.52 (d, J = 6.9 Hz, 3H). TOF ES+ MS:
490.1 (M + H), 512.0 (M + Na). HPLC (gradient A): retention
time = 7.47.
(R)-1-(4-(4-Fluorobenzyl)-4H-thieno[3,2-b]pyrrole-5-carbonyl)-N-
(1-phenylethyl)piperidine-4-carboxamide (5d). Synthesized from 4a
by procedure A, using (R)-α-methylbenzylamine as the amide coupling
1
partner. TLC Rf (1:1 hex:EA): 0.20. H NMR (CDCl3, 500 MHz): δ
(ppm) 7.37 (t, J = 6.0 Hz, 2H), 7.31 (m, 3H), 7.20 (d, J = 5.3 Hz, 1H),
7.14 (dd, J = 5.3, 8.4 Hz, 2H), 6.96 (t, J = 8.7 Hz, 2H), 6.87 (d, J = 5.3
Hz, 1H) 6.58 (s, 1H), 5.63 (d, J = 7.5 Hz, 1H), 5.43 (s, 2H), 5.15
(quint, J = 7.3 Hz, 1H), 4.44 (m, 2H), 2.91 (m, 2H), 2.31 (m, 1H),
1.81 (m, 2H), 1.58 (m, 2H), 1.52 (d, J = 6.9 Hz, 3H). TOF ES+ MS:
490.1 (M + H), 512.1 (M + Na). HPLC (gradient A): retention
time = 7.47.
(R)-N-Benzyl-1-(4-(1-(4-fluorophenyl)ethyl)-4H-thieno[3,2-b]-
pyrrole-5-carbonyl)piperidine-4-carboxamide (5e). Synthesized from
4e by procedure A, using benzylamine as the amide coupling partner.
1
TLC Rf (1:1 hex:EA): 0.17. H NMR (CDCl3, 500 MHz): δ (ppm)
7.36 (d, J = 6.5 Hz, 3H), 7.30 (bm, 4H), 7.09 (d, J = 5.2 Hz, 1H), 6.99
(t, J = 8.4 Hz, 2H), 6.63 (d, J = 5.3 Hz, 1H), 6.55 (s, 1H), 6.02 (quart,
J = 6.8 Hz, 1H), 5.86 (bs, 1H), 4.52 (m, 2H), 4.47 (s, 2H), 2.96 (m,
2H), 2.38 (quint, J = 11.0 Hz, 1H), 1.94 (d, J = 6.5 Hz, 3H), 1.89 (m,
2H), 1.63 (m, 2H). TOF ES+ MS: 490.1 (M + H), 512.1 (M + Na).
HPLC (gradient A): retention time = 7.50.
(S)-N-Benzyl-1-(4-(1-(4-fluorophenyl)ethyl)-4H-thieno[3,2-b]-
pyrrole-5-carbonyl)piperidine-4-carboxamide (5f). Synthesized from
4f by procedure A, using benzylamine as the amide coupling partner.
1
TLC Rf (1:1 hex:EA): 0.17. H NMR (CDCl3, 500 MHz): δ (ppm)
7.36 (d, J = 6.5 Hz, 3H), 7.30 (bm, 4H), 7.09 (d, J = 5.2 Hz, 1H), 6.99
(t, J = 8.4 Hz, 2H), 6.63 (d, J = 5.3 Hz, 1H), 6.55 (s, 1H), 6.02 (quart,
J = 6.8 Hz, 1H), 5.86 (bs, 1H), 4.52 (m, 2H), 4.47 (s, 2H), 2.96 (m,
2H), 2.38 (quint, J = 11.0 Hz, 1H), 1.94 (d, J = 6.5 Hz, 3H), 1.89 (m,
2H), 1.63 (m, 2H). TOF ES+ MS: 490.1 (M + H), 512.1 (M + Na).
HPLC (gradient A): retention time = 7.50.
Ethyl 1-(4-Chlorobenzyl)-1H-indole-2-carboxylate (7b). Ethyl 1H-
indole-2-carboxylate 6 (1.50 g, 7.93 mmol) and potassium carbonate
(1.42 g, 10.3 mmol) were dissolved in 10 mL of DMF. A 5.0 mL DMF
solution of 1-chloro-4-(chloromethyl)benzene (1.66 g, 10.3 mmol)
was added. The reaction was placed under nitrogen and heated at
60 °C for 24 h. The reaction was cooled to room temperature and
diluted with water (50 mL) and extracted with ethyl acetate (80 mL).
The organic layers were combined, washed with saturated sodium
chloride solution (100 mL × 3), dried over magnesium sulfate, and
concentrated in vacuo to afford a tan solid. Trituration with methanol
afforded 7b as a white solid (1.8 g, 72%). TLC Rf (50% EA/Hex): 0.5.
1H NMR (400 MHz, DMSO-d6) δ = 7.74 (d, J = 8.0 Hz, 1H), 7.59 (d,
J = 8.5 Hz, 1H), 7.41−7.30 (m, 4H), 7.16 (t, J = 7.5 Hz, 1H), 7.04 (d,
J = 8.3 Hz, 2H), 5.85 (s, 2H), 4.29 (q, J = 7.1 Hz, 2H), 1.29 (t, J = 7.1
Hz, 3H). TOF ES+ MS: 314.0 (M + H), 336.0 (M + Na). HPLC
(gradient A): retention time = 7.88 min.
1-(4-Chlorobenzyl)-1H-indole-2-carboxylic Acid. Compound 7b
and lithium hydroxide (1.53 g, 63.9 mmol) were dissolved in 10 mL of
THF and 20 mL of water. The reaction was heated at 60 °C overnight.
The reaction was cooled and diluted with water and washed with
diethyl ether. The aqueous solution was acidified using 2N HCl acid
to pH 2. The resulting suspension was extracted with ethyl acetate.
The organic layer was dried over magnesium sulfate, filtered, and
concentrated in vacuo to obtain pure product as a white solid (1.47 g,
81%). TLC Rf (50% EA/hex): 0.2. 1H NMR (400 MHz, DMSO-d6) δ
= 13.02 (s, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.54 (d, J = 8.5 Hz, 1H),
7.37−7.26 (m, 4H), 7.14 (t, J = 7.5 Hz, 1H), 7.05 (d, J = 5.6 Hz, 2H),
N-Benzyl-1-(1-(4-fluorobenzyl)-1H-indole-2-carbonyl)piperidine-
4-carboxamide (9a). Synthesized from 6 as described for 9b using
4-fluorobenzyl chloride instead of 4-chlorobenzyl chloride. Flash
chromatography with 0−5% methanolic ammonia: DCM afforded
CCG 203880 as a white solid (60 mg, 48%). TLC Rf (5% methanolic
1
ammonia/DCM): 0.2. H NMR (400 MHz, DMSO-d6) δ 8.33 (t, J =
5.8 Hz, 1H), 7.60 (dd, J = 16.0, 8.2 Hz, 2H), 7.35−7.06 (m, 11H),
6.72 (s, 1H), 5.48 (s, 2H), 4.44 (bs, 1H), 4.27 (d, J = 5.7 Hz, 2H), 4.03
(bs, 1H), 2.91 (bs, 2H), 2.47−2.40 (m, 1H), 1.78 (bs, 2H), 1.45 (bs,
2H). TOF ES+ MS: 470.1 (M + H), 492.1 (M + Na). HPLC (gradient
A): retention time = 7.77 min.
N-Benzyl-1-(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)piperidine-
4-carboxamide (9b). Prepared by procedure B using benzylamine.
1
TLC Rf (70% EA/hex): 0.2. H NMR (400 MHz, DMSO-d6) δ 8.35
(t, J = 5.9 Hz, 1H0, 7.63 (d, J = 7.9 Hz, 1H), 7.55 (d, J = 8.3 Hz, 1H),
7.38−7.29 (m, 4H), 7.27−7.29 (m, 4H), 7.27−7.18 (m, 4H), 7.14−
7.08 (m, 3H), 6.74 (s, 1H), 5.50(s, 2H), 4.43 (bs, 1H), 4.28 (d, J = 5.9
Hz, 2H), 4.04 (bs, 1H), 2.95 (bs, 2H), 2.48−2.42 (m, 2H), 1.74 (bs,
2H), 1.47 (bs, 2H). 13C NMR (500 MHz, DMSO-d6) 173.4, 161.8,
139.5, 137.1, 136.7, 131.7, 126.1, 123.0, 121.3, 120.1, 110.6, 103.30.
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TOF ES+ MS: 486.1 (M + H), 508.0 (M + Na). HPLC (gradient A):
retention time = 7.73 min.
N-(3-Chlorobenzyl)-1-(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)-
piperidine-4-carboxamide (9j). Synthesized according to procedure B
using 3-chlorobenzylamine. Trituration in methanol afforded afforded
1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-phenylpiperidine-
1
4-carboxamide (9d). Synthesized according to procedure B using
9j as a white solid (179 mg, 49%). TLC Rf (70% EA/hex): 0.3. H
1
aniline. White solid (77 mg, 65%). TLC Rf (70% EA/hex): 0.2. H
NMR (400 MHz, DMSO-d6). δ 8.40 (t, J = 6.0 Hz, 1H), 7.63 (d, J =
8.0 Hz, 1H), 7.54 (d, J = 8.3 Hz, 1H), 7.38−7.06 (m, 14H), 6.73 (1H),
5.49 (s, 2H), 4.45 (bs, 1H) 4.27 (d, J = 5.6 Hz, 2H), 4.08 (bs, 1H), 2.94
(bs, 2H), 2.48−2.43 (m, 1H), 1.77 (bs, 2H), 1.40 (bs, 2H). TOF ES+
MS: 520.0 (M + H). HPLC (gradient A): retention time = 8.06 min.
(1-(4-Chlorobenzyl)-1H-indol-2-yl)(4-(piperidine-1-carbonyl)-
piperidin-1-yl)methanone (9k). Synthesized according to procedure B
using piperidine. Flash chromatography with 0−5% methanolic
ammonia/DCM afforded 9k as a white solid (48 mg, 41%). TLC Rf
(0−5% methanolic ammonia/DCM): 0.2. 1H NMR (400 MHz,
DMSO-d6) δ 7.62 (d, J = 7.8 Hz, 1H), 7.54 (d, J = 8.3 Hz, 1H), 7.33
(d, J = 8.5 Hz, 2H), 7.21 (t, J = 11.8, 4.6 Hz, 1H), 7.13−7.07 (m, 3H),
6.73 (s, 1H), 5.49 (s, 2H), 4.44 (bs, 1H), 4.02 (bs, 1H), 3.43 (s, 4H),
3.20−3.83 (m, 3H), 1.79−1.20 (m, 10H). TOF ES+ MS: 464.1 (M +
H), 486.1 (M + Na). HPLC (gradient A): retention time = 7.95 min.
1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(pyridin-4-
ylmethyl)piperidine-4-carboxamide (9l). Synthesized according to
procedure B using 4-aminomethyl pyridine to afford 9l as a white solid
(61 mg, 50%). TLC Rf (100% EA): 0.1. 1H NMR (400 MHz, DMSO-
d6). δ 8.68−8.59 (m, 4H), 7.63 (d, J = 7.8 Hz, 1H), 7.54 (d, J = 8.4 Hz,
1H), 7.49 (d, J = 5.9 Hz, 4H), 7.36−7.30 (m, 3H), 7.22 (t, J = 7.6 Hz,
1H), 7.14−7.07 (m, 2H), 6.74 (s, 1H), 5.49 (s, 2H), 4.56−4.14 (m,
4H), 2.97 (bs, 2H), 1.76 (bs, 2H), 1.43 (bs, 2H). TOF ES+ MS: 487.1
(M + H). HPLC (gradient A): retention time = 8.00 min.
NMR (400 MHz, DMSO-d6) δ 9.89 (s, 1H), 7.66−7.52 (m, 4H),
7.37−7.18 (m, 5H), 7.13−7.07 (m, 3H), 7.02 (t, J = 7.4 Hz, 1H), 6.75
(s, 1H), 5.51(s, 2H), 4.45 (bs, 1H), 4.03 (bs, 1H), 2.95 (bs, 2H), 2.64−
2.55 (m, 1H), 1.79 (bs, 2H), 1.47 (bs, 2H). TOF ES+ MS: 472.1 (M +
H), 494.1 (M + Na). HPLC (gradient A): retention time = 8.01 min.
1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-phenethylpiperi-
dine-4-carboxamide (9e). Synthesized according to procedure B using
2-phenethylamine. White solid (76 mg, 60%). TLC Rf (70% EA/hex):
1
0.2. H NMR (400 MHz, DMSO-d6) δ 7.85 (t, J = 5.4 Hz, 1H), 7.62
(d, J = 7.8 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H), 7.36−7.05 (m, 10H), 6.71
(s, 1H), 5.48 (s, 2H), 4.38 (bs, 1H), 4.01 (bs, 1H), 3.26 (q, J = 13.6, 6.7
Hz, 2H), 2.90 (bs, 2H), 2.69 (t, J = 13.6, 6.5 Hz, 2H), 2.39−2,28 (m,
2H), 1.61 (bs, 2H), 1.36 (bs, 2H). TOF ES+ MS: 500.2 (M + H),
522.1 (M + Na). HPLC (gradient A): retention time = 7.90 min.
1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(4-
methylphenethyl)piperidine-4-carboxamide (9f). Synthesized ac-
cording to procedure B using p-methyl phenethylamine. Flash
chromatography with 0−5% methanolic ammonia/DCM afforded 9f
as a white solid (24 mg, 19%). TLC Rf (0−5% methanolic ammonia/
1
DCM): 0.25. H NMR (400 MHz, DMSO-d6). δ 7.86 (t, J = 5.5 Hz,
1H), 7.64 (d, J = 8.0 Hz, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.35 (d, J = 8.4
Hz, 2H), 7.23 (t, J = 7.6 Hz, 1H), 7.14−7.03 (m, 7H), 6.73 (s, 1H),
5.50 (s, 2H), 4.40 (bs, 1H), 3.91 (bs, 1H), 3.24 (q, J = 12.0, 6.3 Hz,
2H), 2.90 (bs, 2H), 2.66 (t, J = 7.3 Hz, 2H), 2.40−2.30 (m, 1H), 2.27
(s, 3H), 1.65 (bs, 2H), 1.36 (bs, 2H). TOF ES+ MS: 514.1 (M + H),
536.1 (M + Na). HPLC (gradient A): retention time = 8.15 min.
(S)-1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(1-
phenylethyl)piperidine-4-carboxamide (9g). Synthesized according
to procedure B using (S)-1-phenylethylamine. White solid (32.9 mg,
26%). TLC Rf (70% EA/hex): 0.2. 1H NMR (400 MHz, DMSO-d6) δ
8.25 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 7.9 Hz, 1H), 7.53 (d, J = 8.4 Hz,
1H), 7.35−7.25 (m, 5H), 7.21 (t, J = 7.3 Hz, 2H), 7.09 (d, J = 7.7 Hz,
3H), 6.72 (s, 1H), 5.48 (s, 2H), 4.97−4.82 (m, 1H), 4.42 (bs, 1H),
4.03 (bs, 1H), 2.89 (bs, 2H), 2.46−2.39 (m, 1H), 1.68 (bs, 2H), 1.52−
1.26 (m, 5H). 13C NMR (400 MHz, DMSO-d6) 172.6, 161.8, 144.8,
137.1, 136.7, 131.8, 131.7, 128.7, 128.3, 128.1, 126.4, 126.1, 125.7,
123.0, 121.3, 120.1, 110.6, 103.3, 47.4, 46.1, 41.4, 22.4. TOF ES+ MS:
500.0 (M + H), 522.1 (M + Na). HPLC (gradient A): retention time
= 7.88 min. [CHN] C = 71.8%, H = 6.07%, N = 8.37%. Theoretical
[CHN]: C = 72.06%, 6.05%, 8.4%.
(R)-1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(1-
phenylethyl)piperidine-4-carboxamide (9h). Synthesized according
to procedure B using (R)-1-phenylethylamine. White solid (282 mg,
45%). TLC Rf (70% EA/hex): 0.2. 1H NMR (400 MHz, DMSO-d6) δ
8.25 (d, J = 8.1 Hz, 1H), 7.62 (d, J = 7.9 Hz, 1H), 7.53 (d, J = 8.3 Hz,
1H), 7.36−7.26 (m, 6H), 7.21 (t, J = 7.4 Hz, 2H), 7.10 (d, J = 8.2 Hz,
3H), 6.72 (s, 1H), 5.48 (s, 2H), 4.90 (p, J = 7.1 Hz, 1H), 4.41 (bs,
1H), 4.03 (bs, 1H), 2.89 (bs, 2H), 2.47−2.38 (m, 1H), 1.69 (bs, 2H),
1.52−1.25 (m, 5H). 13C NMR (400 MHz, DMSO-d6) 172.6, 161.8,
144.7, 137.1, 136.7, 131.8, 131.7, 128.7, 128.3, 128.1, 126.4, 126.1,
125.7, 123.0, 121.3, 120.1, 110.6, 103.3, 47.4, 46.1, 41.4, 22.4. TOF ES
+ MS: 500 (M + H), 522.1 (M + Na). HPLC (gradient A): retention
time = 7.87 min. [CHN] C = 71.93%, H = 6.06%, N = 8.39%.
Theoretical [CHN]: C = 72.06%, 6.05%, 8.4%.
1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(pyridin-3-
ylmethyl)piperidine-4-carboxamide (9m). Synthesized according to
procedure B using 3-aminomethyl pyridine to afford 9m as a white
1
solid (315 mg, 70%). TLC Rf (100% EA): 0.1. H NMR (400 MHz,
DMSO-d6). δ 8.46 (d, J = 8.3 Hz, 1H), 8.40(t, J = 5.7 Hz, 1H), 7.70−
7.60 (m, 1H), 7.54 (d, J = 8.3 Hz, 1H), 7.40−7.29 (m, 1H), 7.21 (t, J =
7.6 Hz, 1H), 7.13−7.05 (m, 1H), 6.73 (s, 1H), 5.49 (s, 1H), 4.38 (bs,
1H), 4.30 (d, J = 5.8 Hz, 2H), 4.04 (bs, 1H), 2.95 (bs, 2H), 2.47−2.41
(m, 1H), 1.72 (bs, 2H), 1.41 (bs, 2H). TOF ES+ MS: 487.1 (M + H).
HPLC (gradient A): retention time = 5.62 min.
Ethyl 1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)piperidine-3-
carboxylate. Compound 7b (100 mg, 0.319 mmol), EDC (134 mg,
0.699 mmol) and 1-hydroxybenzotriazole (95.0 mg, 0.703 mmol) were
dissolved in DCM (volume: 3.0 mL). The reaction was allowed to stir
for 10 min before the addition of DIEA (0.122 mL, 0.700 mmol) and
ethyl piperidine-3-carboxylate (0.109 mL, 0.707 mmol). The reaction
was allowed to stir overnight at room temperature. The reaction was
diluted with water and ethyl acetate. The organic phase was washed
with water, 1N HCl, saturated sodium bicarbonate, and saturated
sodium chloride. The organic layer was dried over magnesium sulfate,
filtered, and concentrated in vacuo. The crude material was purified
using Biotage SP1 system with 0−45% ethyl acetate/hexanes to afford
1
white solid. (100 mg, 67%) TLC Rf (60% EA/hex): 0.5. H NMR
(400 MHz, DMSO-d6). δ 7.63 (d, J = 7.9 Hz, 1H), 7.55 (d, J = 8.3 Hz,
1H), 7.33 (d, J = 8.4 Hz, 2H), 7.22 (t, J = 7.3 Hz, 1H), 7.16−7.06 (m,
3H), 6.73 (s, 1H), 5.46 (s, 2H), 4.47−3.63 (m, 5H), 3.29−2.70 (m,
2H), 1.94−1.03 (m, 4H). TOF ES+ MS: 425.1(M + H), 447.1 (M +
Na). HPLC (gradient A): retention time = 8.36 min.
1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)piperidine-3-carbox-
ylic Acid. Ethyl 1-(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)-
piperidine-3-carboxylate (100 mg, 0.235 mmol) and solid lithium
hydroxide (99 mg, 2.4 mmol) were dissolved in 1.5 mL of THF and
3.0 mL of water. The reaction was allowed to stir overnight. After 24 h
at room temperature, the reaction was diluted with water and extracted
twice with diethyl ether. The aqueous layer was acidified with 2N HCl
aqueous solution to pH 2 and extracted with three times with ethyl
acetate. The organic layers were combined and washed with saturated
sodium chloride solution, dried over magnesium sulfate, filtered, and
concentrated in vacuo to obtain a white solid. The crude material was
taken directly to the next step without further purification. (50 mg,
53%) TLC Rf (60% EA/hex): 0.2. 1H NMR (400 MHz, DMSO-d6) δ
= 12.26 (s, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H),
1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(4-
methoxybenzyl)piperidine-4-carboxamide (9i). Synthesized accord-
ing to procedure B using 4-methoxybenzylamine. Trituration in
methanol afforded 9i as white solid (104 mg, 80%). TLC Rf (70% EA/
hex): 0.2. 1H NMR (400 MHz, DMSO-d6). δ 8.25 (t, J = 5.8 Hz, 1H),
7.57 (dd, J = 34.9, 8.1 Hz, 2H), 7.33 (d, J = 8.4 Hz, 2H), 7.20 (t, J =
7.7 Hz, 1H), 7.11 (m, 4H), 6.86 (d, J = 8.6 Hz, 2H), 6.72 (s, 1H), 5.48
(s, 2H), 4.43 (s, 1H), 4.18 (d, J = 5.8 Hz, 2H), 4.01 (s, 1H), 3.71 (s,
3H), 2.93 (s, 2H), 2.41 (d, J = 11.7 Hz, 1H), 1.70 (s, 2H), 1.42 (s,
2H). TOF ES+ MS: 516.1 (M + H), 538.1 (M + Na). HPLC (gradient
A): retention time = 8.27 min; purity = 91%.
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7.33 (d, J = 8.4 Hz, 2H), 7.22 (t, J = 7.6 Hz, 1H), 7.16−7.04 (m, 3H)
6.73 (s, 1H), 5.46 (s, 2H), 4.21 (s, 1H), 3.94 (s, 1H), 3.02 (s, 2H),
1.99 (s, 1H), 1.96−1.52 (m, 2H), 1.40−0.97 (m, 2H). TOF E+ MS:
397.1 (M + H), 419.1 (M + Na). HPLC (gradient A): retention time
= 6.98 min.
of infection of 0.1 or 10, respectively, to obtain approximately 20−25%
residual cell viability at 24 h post-infection.
Induction of Experimental Viral Encephalitis. Female C57BL/
6 mice were purchased from The Jackson Laboratory (Bar Harbor,
ME). All animals were housed and used on-site under specific
pathogen-free conditions in strict accordance with guidelines set by the
National Institutes of Health and protocols approved by the University
Committee on the Use and Care of Animals. Mice were housed on a
10/14 h light/dark cycle in ventilated cages containing no more than
five animals per cage. Food and water were available ad libitum.
To induce encephalomyelitis, 5−6-week-old mice were anesthetized
with isoflurane (Abbott Laboratories, Chicago, IL) and 1000 plaque-
forming units (PFU) of the prototype alphavirus, neuroadapted
Sindbis virus (NSV), suspended in 10 μL of phosphate-buffered saline
(PBS) were inoculated directly into the right cerebral hemisphere of
each animal. Experimental antiviral compounds were solubilized in
dimethyl sulfoxide (DMSO) as stock solutions (100 μM) and then
diluted in PBS to generate working solutions for intraperitoneal
injection into infected mice on a twice-daily dosing schedule. For those
experiments where clinical outcome was the primary end point, each
infected mouse was scored daily into one of the following categories:
(1) normal, (2) mild paralysis (some weakness of one or both hind
limbs), (3) moderate paralysis (weakness of one hind limb, paralysis of
the other hind limb), (4) severe paralysis (complete paralysis of both
hind limbs), or (5) dead. Other groups of animals were sacrificed at
defined intervals post-infection in order to collect brain and spinal
cord tissue for ex vivo analysis. Following intracardiac perfusion with
ice-cold PBS, the left cerebral hemisphere and lower spinal cord was
isolated from some animals, snap-frozen on dry ice, and stored at
N-Benzyl-1-(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)piperidine-
3-carboxamide (9c). Synthesized according to procedure B using 1-
(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)piperidine-3-carboxylic acid
and benzylamine. Flash chromatography with 0−5% methanolic
ammonia: DCM afforded 9c as a white solid (38 mg, 62%). TLC Rf
1
(70% EA/hex): 0.2. H NMR (400 MHz, DMSO-d6). δ 8.44 (s, 1H),
7.60 (d, J = 7.9 Hz, 1H), 7.49 (s, 1H), 7.36−7.03 (m, 12H), 6.72 (s,
1H), 5.46 (s, 2H), 4.58−3.74 (m, 4H), 2.93 (bs, 2H), 2.30 (s, 1H),
1.93−1.48 (m, 3H). TOF ES+ MS: 486.1 (M + H), 508.1 (M + Na).
HPLC (gradient A): retention time = 7.99 min.
Mouse Liver Microsome (MLM) Stability Assay. Balb-C mouse
liver microsomes (MLM) were purchased from Invitrogen. MgCl2 and
NADPH were obtained from Sigma. Solvents were of HPLC grade or
better. The HPLC-MS/MS system consisted of a ThermoElectron
Finnigan TSQ Quantum Ultra AM.
Microsomal incubations were done in triplicate. Incubation mixtures
contained 2 μL of 20 mg/mL microsomes (approximately 0.04 mg
microsomal protein), 4 μL of 100 mM DMSO-dissolved substrate
(1.0 μM final), in 475 μL potassium phosphate buffer (0.1 M, pH 7.5,
containing 3.3 mM MgCl2.) The incubation mixture was allowed to
shake at 37 °C for 5 min, and a T = 0 aliquot was removed. Reaction
was initiated by the addition of 20 μL of NADPH (22 mM under the
same buffer conditions), and the mixture was allowed to shake at
37 °C until the final aliquot was removed. Then 30 μL aliquots were
taken after 0, 5, and 15 min. All aliquots were quenched by dilution in
90 μL of MeCN containing an internal standard, and the precipitate
was pelleted via centrifugation. Then 20 μL of supernatant was
injected onto the HPLC-MS/MS. Mobile phase A was 95:5 H2O/
MeCN with 0.1% formic acid, and mobile phase B was MeCN with
0.1% formic acid. Column used was a Luna C18(2) 4.6 mm × 30 mm
column with 3 μM particle size. The flow rate was 2 mL/min, and a
gradient mobile phase composition was used: isocratic hold for 1 min
at 90% A (10% B), 1 min gradient to 10% A (90% B), 1 min gradient
back to 90% A (10% B), and 1 min isocratic hold at 90% A (10% B).
Ionization method consisted of positive electrospray. Source
parameters were optimized for each individual substrate. Substrates
and internal standards were followed by selected reaction monitoring.
Substrate/internal standard area ratios were determined and converted
into percent substrate remaining. The natural log of the percent
remaining was plotted against time, the slope of the linear regression
was determined, and the equation T1/2 = −ln(2)/k was used to
calculate half-life. T1/2 values cited in the manuscript are means of
N ≥ 3 experiments.
̈
−80 °C for virus titration assays (see below). Alternatively, naive or
NSV-infected mice were sequentially perfused with ice-cold PBS and
then chilled 4% paraformaldehyde (PFA) in PBS so that the brains and
spinal cords could be removed intact for histopathological analyses
(see below).
Virus Titration Assays. Homogenates (10% w/v) of each tissue
sample were prepared in PBS, and serial 10-fold dilutions of each
homogenate were assayed for plaque formation on monolayers of
BHK-21 cells, as previously described.24 Results are presented as the
geometric mean
standard error of the mean (SEM) of the log10
number of PFU per gram of tissue derived from four animals at each
time point.
Histopathology Analyses. Nervous system tissues were post-
fixed overnight at 4 °C in 4% PFA. The axonal processes of motor
neurons (MN) in the lumbar spinal cord that innervate the hind limb
musculature were quantified in cross sections of ventral spinal nerve
roots as a correlate of hind limb paralysis according to published
methods.25,26 All experimental samples were collected from mice
14 days post-infection because the loss of MN axons is delayed
following destruction of the cell body within the spinal cord itself.
Sections of the lumbar spinal column at the L4−L5 level were
decalcified (Immunocal, Decal Corporation, Tallman, NY) and
embedded in paraffin. Sections were then stained using a modified
Bielchowsky silver staining method to label the neurofilament proteins
of each nerve axon.25−29 Axonal density (the number of intact axons
per cross-sectional area of each nerve root) was determined for the
right and left L4 and L5 ventral nerve roots from a minimum of four
animals in each experimental group.
Parallel Artificial Membrane Permeability Assay (PAMPA).
Compounds were dissolved in DMSO to generate a 10 mM compound
solution. The experiment was performed using the Double-Sink
protocol provided by pION, Inc. with the PAMPA Explorer system.
A cosolvent system solution was used for the experiment.
In Vitro Antiviral and Cytotoxicity Assays. The WEEV replicon
assay was done as previously described12 with the following
modifications. We obtained a clonal derivative of the original BSR-
T7 cell line by limiting dilution and used this clonal cell line for all
replicon assays. The BSR-T7/C3 clone was cultured in Dulbecco’s
Modified Eagle Medium containing 5% heat inactivated fetal bovine
serum, 1% sodium pyruvate, 0.1 mM non-essential amino acids,
10 U/mL penicillin, and 10 μg/mL streptomycin. Cells were cultured
in the above media with 0.5 mg/mL G418 every third passage to
maintain selection. Cells were transfected in 10 cm tissue culture plates
for 2 h, detached by trypsinization, and transferred to 96-well plates
preloaded with compound dilutions. Final cell concentrations were
∼2 × 106 cells/mL, and plates were harvested 18−20 h later for
luciferase and MTT assays as previously described.12 BE(2)-C cell
culture, virus infections, and plaque assays were done as previously
described.23 Cells were infected with WEEV or NSV at a multiplicities
Neuronal damage in the brain was assessed in cryosections through
̈
the hippocampal formations of naive and day 14 NSV-infected mice.
Virus consistently and prominently infects this brain region.20 Before
staining, each section was incubated in 0.1% Triton X-100 for 15 min to
expose intracellular antigens. Slides were then incubated with NisslRed
(NeuroTrace 530/615 fluorescent Nissl stain, Invitrogen, Grand Island,
NY) diluted 1:100 for 20 min, washed, incubated in a 0.06% potassium
permanganate solution, washed again, and stained in 0.0001% Fluoro-
Jade C compound (Millipore, Billerica, MA) in 1% acetic acid for 10 min.
After further washing, slides were dried, dehydrated in xylene, and
coverslipped using VectaMount permanent mounting media (Vector
Laboratories, Burlingame, CA). The right and left hippocampi from
each animal were imaged at 20× magnification using a Nikon Ti−U
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Journal of Medicinal Chemistry
Article
inverted fluorescence microscope supported by the NIS-Elements Basic
Research acquisition and analysis software (Nikon Instruments Inc.,
Melville, NY). The total number of Fluoro-Jade-positive/NisslRed-
positive cells (degenerating neurons) and NisslRed-positive cells
(all neurons) was counted in duplicate slides from each hippocampus
of triplicate mice for each experimental condition to determine the
proportion of Fluoro-Jade-positive neurons.
Statistical Analyses. The Prism 5.0 software package (GraphPad
Software, La Jolla, CA) was used for all statistical analyses. Differences in
severity of paralysis and survival among cohorts of infected mice were
measured using a log-rank (Mantel−Cox) test. Unpaired Student’s t test
was used to assess differences between tissue viral titers or neuronal
counts between two experimental groups at single time points. In all
cases, differences at a p < 0.05 level were considered significant.
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AUTHOR INFORMATION
■
Corresponding Author
*For S.D.L.: phone, 734 615 0454; E-mail, sdlarsen@umich.
edu. For D.J.M.: phone, 734 763 0565; E-mail, milldavi@
umich.edu.
Author Contributions
#S.D.L. and D.J.M. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by an NIH Partnerships for
Biodefense Viral Pathogens grant (R01 AI089417), an NIH
Pharmacological Sciences Training Program fellowship for
B.D.Y. (T32 GM007767), and a UM Rackham Merit
Scholarship for S.J.B. We thank Dr. Brian Shay for assistance
with the LC/MS/MS analysis of samples from the MLM
stability assays.
ABBREVIATIONS USED
■
CNS, central nervous system; CPE, cytopathic effect; HTS,
high-throughput screen; EDC, N-(3-dimethylaminopropyl)-N′-
ethylcarbodiimide hydrochloride; MTT, 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide; NSV, neuroadapted
Sindbis virus; VEEV, Venezuelan equine encephalitis virus;
WEEV, western equine encephalitis virus; PAMPA, parallel
artificial membrane permeability assay; MLM, mouse liver
microsome
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