Discovery of cis - N -(1-(4-(methylamino)cyclohexyl)indolin-6-yl)thiophene- 2-carboximidamide: A 1,6-disubstituted indoline derivative as a highly selective inhibitor of human neuronal nitric oxide synthase (nNOS) without any cardiovascular liabilities

Article abstract of DOI:10.1021/jm201564u

A series of 1,6-disubstituted indoline derivatives were synthesized and evaluated as inhibitors of human nitric oxide synthase (NOS) designed to mitigate the cardiovascular liabilities associated with previously reported tetrahydroquinoline-based selectiv

Full text of DOI:10.1021/jm201564u

Article
pubs.acs.org/jmc
Discovery of cis-N-(1-(4-(Methylamino)cyclohexyl)indolin-6-
yl)thiophene-2-carboximidamide: A 1,6-Disubstituted Indoline
Derivative as a Highly Selective Inhibitor of Human Neuronal Nitric
Oxide Synthase (nNOS) without Any Cardiovascular Liabilities
Subhash C. Annedi,*,† Jailall Ramnauth,† Shawn P. Maddaford,† Paul Renton,† Suman Rakhit,†
Gabriela Mladenova,† Peter Dove,† Sarah Silverman,† John S. Andrews,† Milena D. Felice,‡
and Frank Porreca‡
†NeurAxon Inc., 2395 Speakman Drive, Suite 1001, Mississauga, Ontario L5K 1B3, Canada
‡Department of Pharmacology, University of Arizona, 1501 North Campbell Avenue, Tucson, Arizona 85724, United States
S
* Supporting Information
ABSTRACT: A series of 1,6-disubstituted indoline derivatives were
synthesized and evaluated as inhibitors of human nitric oxide synthase
(NOS) designed to mitigate the cardiovascular liabilities associated with
previously reported tetrahydroquinoline-based selective neuronal NOS
inhibitors due to higher lipophilicity (J. Med. Chem. 2011, 54,
5562−5575). This new series produced similar potency and selectivity
among the NOS isoforms and was devoid of any cardiovascular liabilities
associated with QT prolongation due to hERG activity or endothelial
NOS mediated vasoconstriction effect. The SAR studies led to the
identification of cis-45, which was shown to reverse thermal hyperalgesia
in vivo in the spinal nerve ligation model of neuropathic pain with
excellent safety profile (off-target activities at 80 CNS related receptors/
ion channels/transporters). The results presented in this report make cis-
45 as an ideal tool for evaluating the potential role of selective nNOS inhibitors in CNS related disorders where excess NO
produced by nNOS is thought to play a crucial role.
(heme). Both the nNOS and eNOS isoforms are regulated
by their dependence on Ca2+/calmodulin concentration, while
the iNOS isoform is not dependent on Ca2+/calmodulin
concentration because it already has a tightly bound calm-
odulin. The reduction of BH4 and heme iron allows the
activation of O2, which is followed by the hydroxylation of L-
arginine to Nω-hydroxy-L-arginine and then conversion to L-
citrulline, ultimately releasing NO.
When it is highly regulated, the production of NO is
considered beneficial to the host organism; however, excess
NO or overproduction has been implicated in numerous
disease states.5 In particular, excess NO produced by nNOS has
been shown to play an important role in the development of
several disorders including septic shock, stroke, pain (migraine,
chronic tension-type headache, visceral, and neuropathic) and
neurodegenerative disorders (Parkinson’s disease, MS, and
Alzheimer’s disease).6,7 To gain the potential therapeutic
benefits associated with nNOS inhibition, it is critical that the
selective inhibition of nNOS is achieved to preserve the
important functions of the other NOS isoforms such as eNOS.
INTRODUCTION
■
Nitric oxide (NO) is an inorganic free radical gas that is
involved in a wide range of physiological functions and
pathophysiological states.1,2 NO is produced by a family of
three heme binding enzymes called nitric oxide synthase
(NOS) via the five electron oxidation of L-arginine to L-
citrulline.3,4 Although the three NOS isoforms are similar in
their mechanism, they are produced by different genes, with
different functions. Neuronal or brain NOS (nNOS or NOS1)
and endothelial NOS (eNOS or NOS3) are constitutively
expressed and thought to play a role in neurotransmission and
smooth muscle relaxation, respectively. The third isoform is an
inducible NOS (iNOS or NOS2) that is expressed during
bacterial infection, tumor cell cytolysis and inflammation.
The NOS enzymes are homodimeric proteins consisting of a
C-terminal reductase domain and an N-terminal catalytic
oxygenase domain connected by a calmodulin-binding linker.
The C-terminal reductase domain transfers electrons from
NADPH through two prosthetic groups, flavinadenine
dinucleotide (FAD) and flavine mononucleotide (FMN), to
the N-terminal oxygenase domain, which binds L-arginine,
(6R)-2-amino-6-[(1R,2S)-1,2-dihydroxypropyl]-5,6,7,8-tetrahy-
dropteridin-4(1H)-one (BH4) and iron protoporphyrin
Received: November 21, 2011
Published: December 16, 2011
© 2011 American Chemical Society
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a
For example, eNOS plays a crucial role in the regulation of
blood pressure, and the inhibition of eNOS by a nonselective
NOS inhibitor such as 1 (Figure 1) has been shown to cause
Scheme 1
a
Reagents and conditions: (a) 7−9, NaBH(OAc)3, AcOH, DCE, rt;
(b) (i) Raney-Ni, hydrazine hydrate, MeOH, reflux, (ii) 13, EtOH, rt.
transfer hydrogenation conditions, followed by coupling with
thiophene-2-carbimidothioate 13,23 gave the target compounds
14−16. The N-alkylation of 6 with 2-chloroacetyl chloride24
provided chloro intermediate 17, which was substituted with
two different amines under basic conditions to obtain 18 and
19, respectively (Scheme 2). The secondary amine in 19 was
protected as tert-butyl carbamate to obtain 20. The amide
group in 18 and 20 was reduced into the corresponding amine
using borane in THF to obtain 21 and 22, respectively.
Reduction of nitro group in 21 and 22 into the corresponding
amine followed by the coupling to thiophene-2-carbimido-
thioate 13 gave 23 and 24, respectively. The deprotection of
Boc- protecting group in 24 was carried out under strong acidic
conditions to obtain the target compound 25.
The reductive amination of 6-bromoindoline (26), 6-
nitroindoline (6), and 5-fluoro-6-nitroindoline (27)25 with
tert-butyl 3-oxopyrrolidine-1-carboxylate (28) and tert-butyl 4-
oxopiperidine-1-carboxylate (29) gave the coupled compounds
30−32, respectively (Scheme 3). The bromo- group in 30 was
converted into the corresponding amine using standard
Buchwald−Hartwig amination conditions,26 followed by
coupling with thiophene-2-carbimidothioate 13 to give 33.
The reduction of the nitro- group in 31 and 32 into the
corresponding amine was carried under transfer hydrogenation
conditions, followed by coupling with thiophene-2-carbimido-
thioate 13 to give 34 and 35, respectively. The Boc-protecting
group in 33−35 was removed under strong acidic conditions to
obtain the target compounds 36−38, respectively. At this time
no attempts were made to separate ( )-36 into its
enantiomers, and the compound was tested as a racemic
mixture in the human NOS assay.
The reductive amination of 6-nitro indoline (6) with 1,4-
dioxaspiro[4.5]decan-8-one (39) as described above provided
40 (Scheme 4). The deprotection of the acetal group was
carried out under acidic conditions to obtain 41, followed by
the reductive amination with methyl amine hydrochloride to
provide two diastereomers cis-42 and trans-42 in 1:1 ratio,
which were partially separated by regular silica gel column
chromatography (the stereochemistry was conformed with final
compounds after the sequence was completed). The protection
of the secondary amine in cis-42 and trans-42 was achieved
under basic conditions to obtain cis-43 and trans-43,
respectively. The nitro- group reduction in cis-43 and trans-
43 to the corresponding amine followed by coupling with
thiophene-2-carbimidothioate 13 gave cis-44 and trans-44,
respectively. Finally, the Boc-deprotection was carried out
Figure 1. Compound 1 is one of the most studied early nonselective
NOS inhibitor. Literature examples of selective nNOS inhibitors with
basic amine side chain (2−5).
vasoconstriction in humans.8,9 Investigation into the synthesis
and chemistry of novel isoform selective inhibitors of NOS has
been an ongoing challenge over the past two decades even
though the general pharmacophore requirements are well
established.10 Early NOS inhibitors designed by mimicking the
natural substrate, L-arginine (the most common mode),
produced only nonselective NOS inhibitors. However, later
generations of peptidic and nonpeptidic NOS inhibitors
targeting the arginine binding site or BH4 site as well as
dimerization inhibitors have been shown to be more potent and
selective among NOS isoforms.11−13
The simplified pharmacophore model adapted by our group
for competitively inhibiting NOS at the substrate binding site
contains a guanidine isosteric group and a basic amine group
both attached to a central aryl scaffold as described earlier.14 By
varying the guanidine isostere, the central linker, or the basic
amine group, potent and selective nNOS inhibitors have been
obtained (for example: 3−5, Figure 1).14−19 We recently
reported a series of tetrahydroquinoline-based selective nNOS
inhibitors with druglike properties and was shown to be active
in two different pain models with reference compound 5
(Figure 1).18 However 5 was associated with cardiovascular
liabilities due to moderate inhibition at human ether-a-go-go-
related gene (hERG K+) and vasoconstrictive effect through an
unknown mechanism, both attributable to its higher lip-
ophilicity (vide infra).20,21 To mitigate the cardiovascular
liabilities associated with 5, indoline-based compounds with
smaller ring size with lower lipophilicity were designed,
synthesized, and evaluated as NOS inhibitors. Herein, we
now describe the synthesis and structure−activity relationship
(SAR) of 1,6-disubstituted indoline-based selective nNOS
inhibitors that led to the identification of cis-45, which has
lower lipophilicity with better side effect profile when
compared to reference compound 5.
RESULTS AND DISCUSSION
■
Chemistry. Synthesis of all target compounds was carried
out as described in Schemes 1 through 5. Reductive amination
of 6-nitroindoline (6) with various ketones (7−9) was carried
out with sodium triacetoxy borohydride to obtain compounds
10−12 (Scheme 1).22 Reduction of the nitro group under
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a
Scheme 2
a
Reagents and conditions: (a) 2-chloroacetyl chloride, toluene, 110 °C; (b) amine, K2CO3, acetonitrile:water (9:1), 85−100 °C; (c) Boc2O, Et3N,
dioxane, rt; (d) 1 M BH3 in THF, rt; (e) (i) Raney-Ni, hydrazine hydrate, MeOH, reflux, (ii) 13, EtOH, rt; (f) 3 N HCl, MeOH, 90 °C.
a
a
Scheme 3
Scheme 4
a
Reagents and conditions: (a) 28, 29, NaBH(OAc)3, AcOH, DCE, rt;
(b) (i) Pd2(dba)3, PtBu3, LiHMDS, THF, reflux or Raney-Ni,
hydrazine hydrate, MeOH, reflux, (ii) 13, EtOH, rt; (c) 3 N HCl,
MeOH, reflux.
under mild acidic conditions using TFA to obtain the final
compounds cis-45 and trans-45. Both cis-45 and trans-45 differ
by the cis- and trans- substitution at the 1,4 positions of the
cyclohexyl ring. It is difficult to observe a clear NOE between
these positions due to a certain degree of interconversion
between boat and chair conformations, although it was
expected that the trans- conformation would permit the 1,4
substituents to be in a more stable, equatorial position to
stabilize the chair conformation (Supporting Information). In
this conformation, the protons in the 2,3 (and 5,6) positions
would be predominantly in equatorial and axial positions and
experiencing the maximum affect of chemical shift anisotropy
from the carbon−carbon bonds (Supporting Information).
Therefore the trans- conformation have the largest chemical
shift range (0.7 ppm) for proton 2,3 (and 5,6) as opposed to
the cis- compound (0.4 ppm).27 To avoid the sequential
transformations as described in Scheme 4, the tert-butyl
methyl(4-oxocyclohexyl)carbamate (46) side chain was
coupled to 5-fluoro-6-nitro indoline (27) as described above
to provide 47 as a nonseparable mixture of diastereomers
(trans:cis, 1.5:1) (Scheme 5). Nitro- group reduction followed
a
Reagents and conditions: (a) 39, NaBH(OAc)3, AcOH, DCE, rt; (b)
HCl, acetone; (c) CH3NH2·HCl, NaBH(OAc)3, AcOH, DCE, rt; (d)
Boc2O, Et3N, dioxane, rt; (e) (i) Raney-Ni, hydrazine hydrate, MeOH,
reflux, (ii) 13, EtOH, rt; (f) TFA, CH2Cl2, rt.
by coupling to thiophene-2-carbimidothioate 13 provided 48 as
a nonseparable mixture of diastereomers. The deprotection of
the Boc- group under acidic conditions provided the final
compounds cis-49 and trans-49 (cis:trans, 1:1.5), which were
separated by regular silica gel column chromatography. The
stereochemistry of cis-49 and trans-49 was determined as
described for cis-45 and trans-45.
Structure−Activity Relationship (SAR) Studies. All
compounds were converted into their corresponding dihydro-
chloride salts and inhibitory activities were measured against all
three human NOS isoforms (Table 1). Recombinant human
nNOS, eNOS, and iNOS were produced in baculovirus-260
infected Sf9 cells. In a radiometric method, inhibitory activities
were measured by the conversion of [3H]-L-arginine into [3H]-
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a
Scheme 5
a
Reagents and conditions: (a) 46, NaBH(OAc)3, AcOH, DCE, rt; (b) (i) Pd−C/H2, EtOH, rt, (ii) 13, EtOH, rt; (c) 3 M HCl, MeOH, reflux.
L-citrulline. The enzymatic reaction was carried out in the
presence or absence of varying concentrations of the inhibitor
in water. Inhibition of enzyme activity by the inhibitor was
measured by dividing the enzymatic conversion in the presence
of inhibitor divided by the enzymatic conversion in the absence
of inhibitor. IC50 value is the concentration of compound that
gives rise to 50% inhibition.
In general, in vivo toxicity and in vitro receptor promiscuity
are both associated with lipophilicity and are most likely to arise
when clogP is greater than 3, particularly compounds with a
basic amine.28 The current design strategy of selecting the
indoline core as a central aromatic linker is based on their lower
lipophilicity (cis-45: clogP = 3.14), when compared to
previously reported tetrahydroquinoline-based selective nNOS
inhibitors represented by reference compound 5 (clogP =
4.44).18 Attaching a bulky or cyclic basic amine to the 1-
position of the indoline ring is adapted from our previous
results with substituted indole and tetrahydroquinoline
compounds.15−18 To test this hypothesis with the new indoline
core as a central aromatic linker, 14−16 were prepared without
a basic amine. Even though 14−16 contain the bulky cyclic side
chains, they showed 4−23-fold weaker potency for the nNOS
isoform when compared to the compounds that contain basic
amine side chains such as ( )-36 and 37.
Compounds 23 and 25 with flexible side chains were
substituted with hydroxy- and hydroxy ethyl- groups,
respectively, to increase the steric bulk to maximize the
selectivity for nNOS over eNOS and to reduce the Log P values
(vide infra). Compounds 23 and 25 showed increased potency
across all NOS isoforms, particularly for eNOS (23: IC50 = 7.12
μM; 25: IC50 = 3.36 μM, most potent compound for nNOS
and eNOS isoforms) among the current series with good
selectivity for nNOS over eNOS (eNOS/nNOS = 71 for 23, 48
for 25). The cyclic amine side chains showed very good
potency for nNOS and selectivity over eNOS (eNOS/nNOS =
40 for ( )-36 and 183 for 37). Moving the basic amine out of
the cyclic ring (cis-45 and trans-45) not only increase the side
chain length by an extra carbon but also change the orientation
and position of the basic amine, which is also considered as an
important contributing factor for the human nNOS selectivity.
cis-45 and trans-45 showed very good selectivity for nNOS over
eNOS (527, 86 fold for cis-45 and trans-45, respectively). There
is a clear difference between the diastereomers as both the
potency for nNOS and the selectivity for nNOS over eNOS
reside in the cis- isomer.
The polar and acidic residues in the nNOS active site require
the inhibitor to contain polar and positively charged functional
group such as a basic amine (prevailing as protonated at
physiological pH), which is not an ideal combination due to
blood−brain barrier and cell membrane permeability issues. In
an effort to increase the nonprotonated species, it was
envisioned that a fluorine substitution at the ortho- position
(to the amidine group) should be able to lower the basicity of
the amidine group while retaining the nNOS activity and
selectivity over eNOS.29 Accordingly, 38, cis-49, and trans-49
were prepared with a fluorine substitution ortho- to the
amidine group in order to compare their nNOS activity and
selectivity over eNOS with that of 37, cis-45, and trans-45. The
fluoro derivative 38 showed weaker potency across all isoforms
(35-fold for nNOS, 2.5-fold for eNOS, and 201-fold for iNOS)
when compared to 37 without a fluorine substitution. This may
be due to restricted flexibility with the fluorine substitution,
which may be preventing a favorable binding orientation as in
case of 37 without fluorine substitution. Similar results were
observed with cis-49 and trans-49, where both the compounds
showed weaker potencies across all NOS isoforms when
compared to the nonfluorinated compounds cis-45 and trans-45
and is consistent with our previous observation with
tetrahydroquinoline-based nNOS inhibitors.18 Compound 25
was the most potent compound (nNOS IC50 = 0.07 μM)
among the current series with good selectivity (∼50-fold) over
both eNOS and iNOS isoforms, whereas cis-45 was the most
selective for nNOS isoform over eNOS (eNOS/nNOS = 527-
fold) and iNOS (iNOS/nNOS = 224-fold) isoforms. On the
basis of the potency at the nNOS isoform and selectivity over
eNOS, cis-45 was considered for further evaluation in a variety
of in vitro and in vivo assays.
The Chung or Spinal Nerve Ligation (SNL) Model
Study with cis-45. To investigate the potential therapeutic
use of these selective nNOS inhibitors, cis-45 was evaluated in
an in vivo model of neuropathic pain (Figure 2).30 The Chung
or spinal nerve ligation (SNL) model involves ligation of both
the L5 and L6 spinal nerves of one side of the rat, which
produces thermal and mechanical allodynia of the affected paw.
Withdrawal latency following the application of radiant heat to
the paw was tested. Intraperitoneal administration of cis-45 at
30 mg/kg resulted in a partial reversal of thermal hyperalgesia
with a maximum reversal effect of 66% observed at 30 min. The
poor in vivo activity of cis-45 may be attributed to its 5-fold
weaker inhibitory activity in rat nNOS isoform (IC50 = 1.93
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g
Table 1. Inhibition of Human NOS Enzymes by 1, 6-Disubstituted Indoline Derivatives
a
b
In a radiometric method, inhibitory activities were measured by the conversion of [3H]-L-arginine into [3H]-L-citrulline. >100, Not active at the
c
d
e
maximum tested concentration of 100 μM. NC, Not calculable. Inhibitory values are for rat NOS isoforms. Compound 1 is a known nonselective
NOS inhibitor; tested for comparison. Compound 5 is a recently reported tetrahydroquinoline-based selective nNOS inhibitor; included for
comparison. Values reported in parentheses are 95% confidence intervals.
f
g
μM) compared to its human nNOS isoform (IC50 = 0.37 μM).
In addition cis-45 was shown to be nonselective among the rat
NOS isoforms (eNOS/nNOS = 2, iNOS/nNOS = 1),
indicating a significant inhibitory activity difference between
human and rat NOS enzymes, which is consistent with our
previous observation with 2-aminobenzothiazole compounds.14
hERG K+ Channel Inhibition Studies with cis-45.
Antiarrhythmic drugs that induce QT prolongation by
inhibiting the hERG K+ channel has been linked to drug
induced sudden cardiac death and are now the second leading
cause for withdrawing approved drugs from the market.31
Reference compound 5, when tested in human recombinant
Figure 2. Compound cis-45 attenuates thermal hyperalgesia in the L5/
HEK-293 cell lines for its ability to inhibit the hERG K+
L6 SNL model of neuropathic pain. The mean values reported are
channel, showed moderate inhibitory activity (IC50 = 4.7
from six animals.
μM),32 which poses a significant hurdle for the rapid drug
development process and also receive an increased regulatory
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attention.33 The hERG activity of 5 was assumed to be
associated with the higher lipophilicity (clogP = 4.44) of the
molecule. Reducing the lipophilicity of the molecule to control
the hERG activity is one of the well-known phenomenon
reported in the literature.34 On the basis of the literature, a
discrete structural modification from tetrahydroquinoline to
indoline was designed, which will not only change the
physicochemical properties but also reduce the lipophilicity
(cis-45: clogP = 3.15 vs 4.44 for 5) by one log unit due to its
smaller ring size. To compare the hERG activity (conventional
patch-clamp assay) of the lead candidate cis-45 with reference
5, cis-45 was tested in human recombinant HEK-293 cell lines
for its ability to inhibit the hERG K+ channel. Compound cis-45
showed ∼3-fold weaker functional activity against hERG K+
channel with IC50 of 13 μM.32
Figure 5. Effect of 5 on the responses to ACh in absence and presence
of L-arginine. Significant vasoconstriction effect was observed with 5
(due to an unknown mechanism), which is not reversed with the
addition of L-arginine (substrate for eNOS), indicating that the effect is
not due to the eNOS inhibition.
eNOS Mediated Vasoconstriction Effect on Isolated
Human Resistance Arteries with cis-45. cis-45 was assessed
for the contractile response (inhibition of acetylcholine (ACh)-
mediated vasorelaxation) on isolated human resistance arteries
to assess the undesirable cardiovascular effect associated with
the inhibition of eNOS in absence and presence of L-arginine
(substrate for eNOS) with positive (1), negative (vehicle), and
reference compound 5 (Figures 3, 4, and 5). The arteries were
eNOS) would recover the relaxation due to ACh. No significant
inhibition with cis-45 on the ability of the blood vessels to
respond to ACh was observed at 100 μM concentration (Figure
3). Furthermore, the addition of L-arginine did not significantly
affect the response to ACh in the presence of cis-45, providing
further evidence that cis-45 did not have an effect on agonist-
mediated activation of human eNOS. The data suggests that cis-
45 would be devoid of any vasoconstrictive effect at
concentrations that are physiologically relevant based on its
human eNOS potency (IC50 = 195 μM).
In contrast, 1 and 5 both caused vasoconstriction at 100 μM
concentration (Figure 4 and 5). The vasoconstriction effect
caused by a nonselective NOS inhibitor 1 (due to eNOS
inhibition) was reversed with the addition of L-arginine (100
μM) (Figure 4), whereas vasoconstriction effect caused by
selective nNOS inhibitor 5 was not reversed with the addition
of L-arginine (100 μM) (Figure 5), which indicates that the
vasoconstriction effect is not due to the eNOS inhibition, but
through an unknown mechanism, once again attributable to its
higher lipophilicity (vide supra). Even though 5 is very selective
for nNOS over eNOS (465) with weak inhibitory activity at
eNOS isoform (IC50 = 45.6 μM), it still possess a
cardiovascular liability and was hindered from further develop-
ment. Where as cis-45, with its lower lipophilicity mitigates the
cardiovascular liabilities and further evaluated in various in vitro
assays to determine the side activity.
Figure 3. Effect of cis-45 on the responses to ACh in absence and
presence of L-arginine. No significant vasoconstriction effect was
observed with cis-45.
High Throughput Profile of cis-45. To identify off-target
activities, cis-45 was tested in 80 validated in vitro
pharmacological assays (40 nonpeptidic receptors, 30 peptidic
receptors, 2 nuclear receptors, 5 ion channels, and 3
transporters) that cover a broad range of CNS related targets.35
This high throughput profile is used to identify the potential
limitations and liabilities of the selection candidate associated
with off-target activities to minimize the risks and overall cost
associated with the drug development process. This process is
also a rapid and cost-effective way of prioritizing the most
promising compounds such as cis-45 for further evaluation in
various preclinical toxicology studies during the selection
process. Accordingly, cis-45 was tested at a single concentration
of 10 μM in 80 different pharmacological assays to identify the
off-target activities (Table 1 from Supporting Information). cis-
45 was found to be selective for most of the targets tested
(<50% inhibition at 68 out of 80 receptors). cis-45 showed
weak inhibition at: opioid receptors μ (52%) and κ (69%), σ
receptor (σ, 72%), α-2 adrenergic receptor (α2, 58%), and
serotonin transporters 5-HT1B (60%) and 5-HT5a (59%). cis-45
showed strong inhibition at human muscarinic receptors M1
Figure 4. Effect of 1 on the responses to ACh in absence and presence
of L-arginine. Significant vasoconstriction effect was observed with 1
(due to eNOS inhibition), which is reversed with the addition of L-
arginine (substrate for eNOS), indicating that the effect is due to the
eNOS inhibition.
preconstricted with U46619, a thromboxane A2 (TxA2)
mimetic agent, and then exposed to ACh, an endothelium
and nitric oxide dependent vasodilator. The response to ACh
provides information on the activity of eNOS in an active
human biological tissue and thereby any inhibitory effect of cis-
45 on eNOS. Furthermore, if inhibition of relaxation is due to
the inhibition of eNOS, the addition of L-arginine (substrate for
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was extracted into CH2Cl2. The combined CH2Cl2 layer was washed
with brine and dried (Na2SO4). Solvent was evaporated and crude was
purified by column chromatography (EtOAc:hexanes, 1:9) on silica gel
(80%), M2 (80%), M3 (85%), M4 (95%), and M5 (90%) and
human norepinephrine transporter (86%). As a follow-up to
the high throughput profile, the IC50 for norepinephrine
transporter was calculated as 4.4 μM, which is considered to be
very weak to have any effect at concentrations that are
physiologically relevant. Overall, cis-45 possesses an excellent in
vitro safety profile.
1
to obtain the title compound (0.72 g, 96%) as a dark-brown solid. H
NMR (CDCl3) δ 7.46 (dd, 1H, J = 2.1, 8.1 Hz), 7.09−7.04 (m, 2H),
3.52 (t, 2H, J = 8.4 Hz), 3.42−3.34 (m, 1H), 3.01 (t, 2H, J = 8.4 Hz),
1.85−1.70 (m, 5H), 1.45−1.30 (m, 4H), 1.19−1.11 (m, 1H). ESI-MS
(m/z, %): 247 (MH+, 100), 165 (61), 119 (39).
6-Nitro-1-(tetrahydro-2H-pyran-4-yl)indoline (11). Prepared
from 6-nitroindoline (6) (0.5 g, 3.05 mmol) and dihydro-2H-pyran-
4(3H)-one (8) (0.562 mL, 6.09 mmol) as described for 10 to obtain
the title compound (0.75 g, 99%) as a dark-red solid. 1H NMR
(CDCl3) δ 7.49 (dd, 1H, J = 1.8, 7.9 Hz), 7.12−7.07 (m, 2H), 4.12−
4.07 (m, 2H), 3.68−3.60 (m, 1H), 3.56−3.48 (m, 4H), 3.04 (t, 2H, J =
8.4 Hz), 1.81−1.73 (m, 4H). ESI-MS (m/z, %): 271 (M + Na, 100),
249 (MH+, 76).
6-Nitro-1-(tetrahydro-2H-thiopyran-4-yl)indoline (12). Pre-
pared from 6-nitroindoline (6) (0.6 g, 3.65 mmol) and dihydro-2H-
thiopyran-4(3H)-one (9) (0.84 g, 7.31 mmol) as described for 10 to
obtain the title compound (0.95 g, 98%) as an orange solid. 1H NMR
(CDCl3) δ 7.48 (dd, 1H, J = 2.1, 7.8 Hz), 7.09−7.05 (m, 2H), 3.53 (t,
2H, J = 8.7 Hz), 3.43−3.34 (m, 1H), 3.03 (t, 2H, J = 8.7 Hz), 2.91−
2.71 (m, 4H), 2.20−2.04 (m, 2H), 1.89−1.68 (m, 2H). ESI-MS (m/z,
%): 287 (M + Na, 95), 265 (MH+, 100).
N-(1-Cyclohexylindolin-6-yl)thiophene-2-carboximidamide
(14). A suspension of 1-cyclohexyl-6-nitroindoline (10) (0.6 g, 2.43
mmol) in dry methanol (10 mL) was treated with hydrazine hydrate
(0.88 mL, 24.36 mmol) followed by Raney-Ni (0.1 g, 2.43 mmol) at
room temperature. The resulting suspension was refluxed for 5 min in
a preheated oil bath. The reaction was brought to room temperature,
filtered through Celite bed, and washed with methanol. The combined
methanol layer was evaporated and crude was purified by flash column
chromatography (EtOAc:hexanes, 1:3) to obtain 1-cyclohexylindolin-
6-amine as a solid.
A solution of above 1-cyclohexylindolin-6-amine in dry ethanol (10
mL) was treated with methyl thiophene-2-carbimidothioate hydro-
iodide (13) (1.37 g, 4.86 mmol) at room temperature, and the
resulting mixture was stirred at same temperature for overnight (18 h).
The reaction was diluted with satd NaHCO3 solution, and product was
extracted into CH2Cl2. The combined CH2Cl2 layer was washed with
brine and dried (Na2SO4). Solvent was evaporated, and crude was
purified by column chromatography (EtOAc:hexanes, 1:2) to obtain
the title compound (0.5 g, 73.8%, over two steps) as a solid. 1H NMR
(DMSO-d6) δ 7.69 (d, 1H, J = 2.7 Hz), 7.58 (d, 1H, J = 5.1 Hz), 7.07
(dd, 1H, J = 3.9, 4.9 Hz), 6.90 (d, 1H, J = 7.5 Hz), 6.23 (s, 2H), 5.98
(d, 1H, J = 7.5 Hz), 5.90 (s, 1H), 3.35−3.29 (m, 3H, merged with
water peak), 2.80 (t, 2H, J = 8.4 Hz), 1.73−1.59 (m, 5H), 1.36−1.26
(m, 4H), 1.17−1.07 (m, 1H). ESI-MS (m/z, %): 326 (MH+, 100).
ESI-HRMS calculated for C19H24N3S (MH+), 326.1685; observed,
326.1678. HPLC purity: 97.82%.
N-(1-(Tetrahydro-2H-pyran-4-yl)indolin-6-yl)thiophene-2-
carboximidamide (15). Prepared from 6-nitro-1-(tetrahydro-2H-
pyran-4-yl)indoline (11) (0.5 g, 2.01 mmol) as described for 14 to
obtain the title compound (0.55 g, 92%) as a solid. 1H NMR (DMSO-
d6) δ 7.69 (d, 1H, J = 3.3 Hz), 7.58 (d, 1H, J = 5.1 Hz), 7.07 (t, 1H, J =
3.9 Hz), 6.92 (d, 1H, J = 7.8 Hz), 6.23 (brs, 2H), 6.02−6.00 (m, 2H),
3.92−3.88 (m, 2H), 3.64−3.57 (m, 1H), 3.45−3.38 (m, 4H), 2.81 (t,
2H, J = 8.1 Hz), 1.64−1.60 (m, 4H). ESI-MS (m/z, %): 328 (MH+,
100). ESI-HRMS calculated for C18H21N3OS (MH+), 328.1478;
observed, 328.1471. HPLC purity: 96.66%.
N-(1-(Tetrahydro-2H-thiopyran-4-yl)indolin-6-yl)thiophene-
2-carboximidamide (16). Prepared from 6-nitro-1-(tetrahydro-2H-
thiopyran-4-yl)indoline (12) (0.5 g, 1.89 mmol) as described for 14 to
obtain the title compound (0.54 g, 92%) as a solid. 1H NMR (DMSO-
d6) δ 7.69 (d, 1H, J = 2.7 Hz), 7.58 (d, 1H, J = 5.1 Hz), 7.08 (t, 1H, J =
4.8 Hz), 6.91 (d, 1H, J = 7.5 Hz), 6.23 (brs, 2H), 6.00 (dd, 1H, J = 1.5,
7.5 Hz), 5.96 (s, 1H), 3.40−3.29 (m, 3H), 2.84−2.77 (m, 4H), 2.66−
2.61 (m, 2H), 2.03−1.98 (m, 2H), 1.69−1.64 (m, 2H). ESI-MS (m/z,
%): 344 (MH+, 100). ESI-HRMS calculated for C18H22N3S2 (MH+),
344.1249; observed, 344.1234. HPLC purity: 98.26%.
CONCLUSIONS
■
In conclusion, a series of 1,6-disubstituted indoline derivatives
were synthesized and shown to be selective inhibitors of human
nNOS over eNOS and iNOS isoforms. Compounds with bulky
cyclic side chain (14−16) without a basic amine showed up to
20-fold weaker potency when compared to a similar compound
with basic amine side chain (37). This indicates that the
presence of a basic amine is an important contributing factor to
obtain submicromolar inhibitory potency for the nNOS
isoform. A bulky cyclic amino- substituent at the 1-position
of the indoline ring such as 4-(methylamino)cyclohexyl- group
(cis-45 and trans-45) provided better selectivity over
pyrrolidine (( )-36) or piperidine (37) based cyclic amine
substitutions. Substitution of fluorine ortho- to the thiophene
amidine group (5-position of the indoline ring) dramatically
reduced the potency across all NOS isoforms, indicating an
unfavorable orientation in the enzyme active site in this
particular 1,6-disubstituted indoline derivatives. Intraperitoneal
administration of cis-45 was shown to reverse the thermal
hyperalgesia in the SNL model of neuropathic pain. At the
same time, cis-45 is devoid of any cardiovascular liabilities
associated with QT prolongation with hERG K+ channel
binding or vasoconstriction on human resistance arteries
associated with the inhibition of human eNOS when compared
to reference compound 5. cis-45 does not have any off-target
activities (80 receptors) associated with the side effect profile,
making it an ideal candidate to carry forward through the
preclinical development process such as toxicological evalua-
tions. The significant in vitro safety results presented in this
communication with cis-45 provide an opportunity to
investigate the role that the nNOS enzyme plays in CNS
related disorders such as migraine and chronic tension type
headache (CTTH).
EXPERIMENTAL SECTION
■
General. All reactions were performed under an atmosphere of
argon and stirred magnetically unless otherwise noted. Commercial
reagents and anhydrous solvents were used as received without further
purification. Reactions were monitored by analytical TLC using
precoated silica gel aluminum plates (0.2 mm, 60 Å) and were
visualized with UV light or stained appropriately. Flash column
chromatography was performed using Silicycle Siliaflash F60 (40−63
μm) silica gel. The 1H NMR spectra were performed on a Bruker 300
MHz spectrometer. Low and high resolution mass spectra were
performed on an applied Biosystems/MDS Sciex QstarXL hybrid
quadrupole/TOF instrument using electrospray ionization. The
chemical purity of all final compounds was determined by Agilent
1100 series HPLC system using Agilent Zorbax, SB-C18, and Waters
XTerra RP8.5 μM reverse phase columns, and the purity was
determined to be ≥95% for all final compounds. No attempts were
made to optimize the yields.
1-Cyclohexyl-6-nitroindoline (10). A solution of 6-nitroindoline
(6) (0.5 g, 3.05 mmol) in dry 1,2-dichloroethane (10 mL) was treated
with cyclohexanone (7) (0.63 mL, 6.09 mmol), followed by acetic acid
(0.43 mL, 7.61 mmol) and sodium triacetoxyborohydride (0.96 g, 4.57
mmol) at room temperature, and the resulting mixture was stirred for
3 h. The reaction was basified with 1 N NaOH solution, and product
949
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Article
2-Chloro-1-(6-nitroindolin-1-yl)ethanone (17). A suspension
of 6-nitroindoline (6) (1.0 g, 6.09 mmol) in toluene (20 mL) was
treated with chloroacetyl chloride (0.97 mL, 12.18 mmol) dropwise.
The mixture was heated at 110 °C for 15 min, cooled to room
temperature, and treated with saturated sodium bicarbonate solution.
The product was extracted into ethyl acetate, washed with brine, and
dried (Na2SO4). The crude was purified by flash column
chromatography (EtOAc:hexanes, 5:95 to 1:1, then 2 M NH3
MeOH: CH2Cl2, 1:9) on silica gel to obtain the title compound
tert-Butyl 2-Hydroxyethyl(2-(6-nitroindolin-1-yl)ethyl)-
carbamate (22). Prepared from tert-butyl 2-hydroxyethyl(2-(6-
nitroindolin-1-yl)-2-oxoethyl)carbamate (20) (0.97 g, 2.65 mmol) as
described for 21 to obtain the title compound (0.9 g, 96%) as an
orange−red residue. 1H NMR (DMSO-d6) δ 7.41−7.37 (m, 1H),
7.21−7.16 (m, 1H), 7.12−7.10 (m, 1H), 4.71 (t, 1H, J = 5.4 Hz), 3.58
(t, 2H, J = 8.4 Hz), 3.49−3.46 (m, 2H), 3.40−3.35 (m, 4H), 3.29−
3.23 (m, 2H), 3.04−2.95 (m, 2H), 1.35 and 1.31 (2s, 9H). ESI-MS
(m/z, %): 374 (MNa+, 75%), 352 (MH+, 25%), 296 (95%), 252
(100%).
1
(1.22 g, 83%) as a light-brown foam. H NMR (CDCl3) δ 9.00 (s,
(R)-N-(1-(2-(3-Hydroxypyrrolidin-1-yl)ethyl)indolin-6-yl)-
thiophene-2-carboximidamide (23). Prepared from (R)-1-(2-(6-
nitroindolin-1-yl)ethyl)pyrrolidin-3-ol (21) (1.24 g, 4.47 mmol) as
described for 14 to obtain the title compound (0.113 g, 7%) as a solid.
1H NMR (DMSO-d6) δ 7.70 (dd, 1H, J = 0.9, 3.6 Hz), 7.58 (dd, 1H, J
= 0.6, 5.1 Hz), 7.07 (dd, 1H, J = 3.6, 5.1 Hz), 6.93 (d, 1H, J = 7.5 Hz),
6.26 (brs, 2H), 6.03 (brd, 1H, J = 8.7 Hz), 5.97 (brs, 1H), 4.67 (d, 1H,
J = 4.8 Hz), 4.19−4.13 (m, 1H), 3.42−3.28 (m, 2H), 3.11 (t, 2H, J =
6.9 Hz), 2.83 (brt, 2H, J = 7.8 Hz), 2.73 (dd, 1H, J = 6.0, 9.3 Hz),
2.64−2.53 (m, 3H), 2.47−2.42 (m, 1H), 2.34 (dd, 1H, J = 3.9, 9.6
Hz), 2.01−1.89 (m, 1H), 1.56−1.46 (m, 1H). ESI-MS (m/z, %): 357
(MH+, 100%). ESI-HRMS calculated for C19H25N4OS (MH+),
357.1743; observed, 357.1742. HPLC purity: 97.88%.
1H), 7.96 (dd, 1H, J = 2.1, 8.1 Hz), 7.32 (d, 1H, J = 8.1 Hz), 4.31 (t,
2H, J = 8.4 Hz), 4.18 (s, 2H), 3.35 (t, 2H, J = 8.7 Hz). ESI-MS (m/z,
%): 263 (44, M + Na), 241 (MH+, 100).
(R)-2-(3-Hydroxypyrrolidin-1-yl)-1-(6-nitroindolin-1-yl)-
ethanone (18). A mixture of 2-chloro-1-(6-nitroindolin-1-yl)-
ethanone (17) (1.1 g, 4.57 mmol), potassium carbonate (1.89 g,
13.71 mmol), and (R)-pyrrolidin-3-ol (0.55 mL, 6.86 mmol) in
acetonitrile (20 mL) and H2O (2 mL) was heated at 85 °C for 2 h.
The dark mixture was concentrated and treated with H2O under rapid
stirring. The solid was filtered, washed with H2O, and dried under
reduced pressure to obtain the title compound (1.25 g, 94%) as a
1
brown solid. H NMR (DMSO-d6) δ 8.80 (d, 1H, J = 1.5 Hz), 7.91
(dd, 1H, J = 2.1, 8.1 Hz), 7.48 (d, 1H, J = 8.4 Hz), 4.73 (brd, 1H, J =
4.5 Hz), 4.28−4.15 (m, 3H), 3.42 (brs, 2H), 3.25 (t, 2H, J = 8.7 Hz),
2.85 (dd, 1H, J = 6.0, 9.3 Hz), 2.72 (dd, 1H, J = 7.8, 15.6 Hz), 2.62−
2.55 (m, 1H), 2.50−2.44 (m, 1H, overlap with DMSO peak), 2.05−
1.93 (m, 1H), 1.60−1.52 (m, 1H). ESI-MS (m/z, %): 292 (MH+,
100%).
tert-Butyl 2-Hydroxyethyl(2-(6-(thiophene-2-
carboximidamido)indolin-1-yl)ethyl)carbamate (24). Prepared
from tert-butyl 2-hydroxyethyl(2-(6-nitroindolin-1-yl)ethyl)carbamate
(22) (0.89 g, 2.53 mmol) as described for 14 to obtain the title
1
compound (0.57 g, 53%) as a yellow solid. H NMR (DMSO-d6) δ
7.69 (d, 1H, J = 3.3 Hz), 7.58 (d, 1H, J = 4.8 Hz), 7.08 (pseudo t, 1H,
J = 3.9 Hz), 6.92 (d, 1H, J = 7.8 Hz), 6.25 (brs, 2H), 6.06−5.99 (m,
2H), 4.68 (t, 1H, J = 5.4 Hz), 3.47−3.32 (m, 6H, overlap with H2O
peak), 3.25−3.12 (m, 4H), 2.85 (t, 2H, J = 7.5 Hz), 1.37 (s, 9H). ESI-
MS (m/z, %): 431 (MH+, 100%).
2-(2-Hydroxyethylamino)-1-(6-nitroindolin-1-yl)ethanone
(19). A suspension of 2-chloro-1-(6-nitroindolin-1-yl)ethanone (17)
(1.5 g, 6.23 mmol), potassium carbonate (4.31 g, 31.2 mmol), and 2-
aminoethanol (1.5 mL, 24.93 mmol) in a mixture of acetonitrile (20
mL) and water (4.00 mL) was heated at 55 °C for 2 h and then 100
°C for 30 min. The mixture was allowed to cool to room temperature,
transferred to a separatory funnel containing EtOAc and H2O, and
then extracted. The organic layer was separated, dried (Na2SO4),
filtered, and concentrated to give a brown residue. This residue was
purified by flash column chromatography (MeOH:CH2Cl2, 5:95 to 2
M NH3 in MeOH:CH2Cl2, 1:9) on silica gel to obtain the title
N-(1-(2-(2-Hydroxyethylamino)ethyl)indolin-6-yl)thiophene-
2-carboximidamide (25). A solution of tert-butyl 2-hydroxyethyl(2-
(6-(thiophene-2-carboximidamido)indolin-1-yl)ethyl)carbamate (24)
(0.55 g, 1.27 mmol) in MeOH (10 mL) was treated with 3 N HCl
(4.26 mL, 12.77 mmol) and heated at 90 °C for 30 min. The yellow
solution was concentrated, diluted with H2O (20 mL), and washed
with CH2Cl2. The aqueous layer was concentrated and dried to obtain
dihydrochloride salt of the title compound (0.45 g, 87%) as a brown
solid. 1H NMR (DMSO-d6) δ 11.50 (brs, 1H), 9.81 (s, 1H), 9.15 (brs,
2H), 8.76 (brs, 1H), 8.16 (d, 2H, J = 4.8 Hz), 7.37 (pseudo t, 1H, J =
4.2 Hz), 7.18 (d, 1H, J = 7.5 Hz), 6.69 (s, 1H), 6.62 (d, 1H, J = 7.5
Hz), 3.70 (t, 2H, J = 5.1 Hz), 3.52−3.46 (m, 4H), 3.16 (brs, 2H),
3.02−2.95 (m, 4H). ESI-MS (m/z, %): 331 (MH+, free base, 100%).
ESI-HRMS calculated for C17H23N4OS (MH+, free base), 331.1587;
observed, 331.1587. HPLC purity: 96.43%.
tert-Butyl 3-(6-Bromoindolin-1-yl)pyrrolidine-1-carboxylate
(30). Prepared from 6-bromoindiline (26) (5.0 g, 25.2 mmol) and tert-
butyl 3-oxopyrrolidine-1-carboxylate (28) (5.84 g, 31.6 mmol) as
described for 10 to obtain the title compound (7.91 g, 85%) as a syrup.
1H NMR (DMSO-d6) δ 6.93 (d, 1H, J = 7.5 Hz), 6.69−6.65 (m, 2H),
4.28−4.16 (m, 1H), 3.49−3.16 (m, 6H), 2.83 (t, 2H, J = 8.4 Hz),
2.07−1.09 (m, 2H), 1.40 (s, 9H). ESI-MS (m/z, %): 391 and 389
(MNa+, 65%), 369 and 367 (MH+, 2%), 313 and 311 (100%).
tert-Butyl 4-(6-Nitroindolin-1-yl)piperidine-1-carboxylate
(31). Prepared from 6-nitroindoline (6) (5.0 g, 30.5 mmol) and tert-
butyl 4-oxopiperidine-1-carboxylate (29) (12.14 g, 60.9 mmol) as
described for 10 to obtain the title compound (8.88 g, 84%). 1H NMR
(DMSO-d6) δ 7.40 (dd, 1H, J = 8.1, 2.1 Hz), 7.19 (d, 1H, J = 8.1 Hz),
7.17 (d, 1H, J = 2.1 Hz), 4.04 (brd, 2H, J = 11.7 Hz), 3.81−3.71 (m,
1H), 3.47 (t, 2H, J = 8.6 Hz), 3.00 (t, 2H, J = 8.6 Hz), 2.95−2.75 (m,
2H), 1.64 (brd, 2H, J = 11.1 Hz), 1.52−1.42 (m, 2H), 1.40 (s, 9H). EI-
MS (m/z, %): 347 (M+, 100), 274 (78).
1
compound (0.75 g, 45.4%) as a light-brown solid. H NMR (DMSO-
d6) δ 8.81 (d, 1H, J = 2.1 Hz), 7.91 (dd, 1H, J = 2.1, 8.1 Hz), 7.49 (d,
1H, J = 8.1 Hz), 4.59−4.48 (m, 1H), 4.18 (t, 2H, J = 8.4 Hz), 3.53 (s,
2H), 3.49−3.47 (m, 2H), 3.27 (t, 2H, J = 8.4 Hz), 2.64 (t, 2H, J = 5.7
Hz), 2.16 (brs, 1H). ESI-MS (m/z, %): 266 (MH+, 100%).
tert-Butyl 2-Hydroxyethyl(2-(6-nitroindolin-1-yl)-2-
oxoethyl)carbamate (20). A mixture of 2-(2-hydroxyethylamino)-
1-(6-nitroindolin-1-yl)ethanone (19) (0.72 g, 2.71 mmol), di-tert-butyl
dicarbonate (0.62 g, 2.85 mmol), and triethylamine (0.75 mL, 5.43
mmol) in dioxane (30 mL) was stirred at room temperature for 17 h.
The solution was concentrated and subjected to flash chromatography
(2 M NH3 in MeOH:CH2Cl2, 5:95) on silica gel to obtain the title
compound (0.99 g, 100%) as a yellow residue. 1H NMR (DMSO-d6) δ
8.79 (brs, 1H), 7.93 (d, 1H, J = 7.8 Hz), 7.51 (d, 1H, J = 8.1 Hz),
4.69−4.64 (m, 1H), 4.21−4.15 (m, 4H), 3.57 (s, 2H), 3.55−3.49 (m,
2H), 3.35−3.23 (m, 2H, overlap with H2O peak), 1.42 and 1.32 (2s,
9H). ESI-MS (m/z, %): 388 (MNa+, 50%), 366 (MH+, 35%), 266
(100%).
(R)-1-(2-(6-Nitroindolin-1-yl)ethyl)pyrrolidin-3-ol (21). A sus-
pension of (R)-2-(3-hydroxypyrrolidin-1-yl)-1-(6-nitroindolin-1-yl)-
ethanone (18) (1.22 g, 4.19 mmol) in THF (20 mL) at 0 °C was
treated with 1 M borane in THF (20.94 mL, 20.94 mmol). The
resulting yellow solution was allowed to warm to room temperature
and stirred for 17 h. The yellow solution was cooled to 0 °C and
quenched with MeOH (20 mL) dropwise with caution (very slowly at
first). The resulting orange solution was concentrated, redissolved in
MeOH (30 mL), and concentrated to dryness. This residue was
purified by flash column chromatography (2 M NH3 in
MeOH:CH2Cl2, 5:95) on silica gel to obtain the title compound
(1.16 g, quantitative) as an orange−red residue.
tert-Butyl 4-(5-Fluoro-6-nitroindolin-1-yl)piperidine-1-car-
boxylate (32). Prepared from 5-fluoro-6-nitroindoline (27) (0.115
g, 0.63 mmol) and tert-butyl 4-oxo-1-piperidinecarboxylate (29) (0.25
g, 1.26 mmol) as described for 10 to obtain the title compound (0.206
g, 89%) as an orange oil. 1H NMR (DMSO-d6) δ 7.23 (d, 1H, J = 11.0
950
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Journal of Medicinal Chemistry
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Hz), 7.02 (d, 1H, J = 5.9 Hz), 4.03 (app d, 2H, J = 12.0 Hz), 3.77−
3.67 (m, 1H), 3.42 (app t, 2H, J = 8.5 Hz), 2.99 (app t, 2H, J = 8.3
Hz), 2.93−2.70 (m, 2H), 1.73−1.58 (m, 2H), 1.51−1.40 (m, 2H),
1.40 (s, 9H).
(MH+, 100%). ESI-HRMS calculated for C17H21N4S (MH+),
313.1481; observed, 313.1471. HPLC purity: 99.57%.
N-(1-(Piperidin-4-yl)indolin-6-yl)thiophene-2-carboximida-
mide (37). Prepared from tert-butyl 4-(6-(thiophene-2-
carboximidamido)indolin-1-yl)piperidine-1-carboxylate (34) (1.05 g,
2.46 mmol) as described for 36 to obtain the title compound (0.214 g,
27%). 1H NMR (DMSO-d6) δ 7.69 (d, 1H, J = 3.0 Hz), 7.58 (d, 1H, J
= 5.1 Hz), 7.08 (dd, 1H, J = 5.1, 3.9 Hz), 6.91 (d, 1H, J = 7.5 Hz), 6.20
(brs, 2H), 6.00 (d, 1H, J = 7.5 Hz), 5.94 (s, 1H), 3.45−3.29 (m, 3H),
2.98 (m, 2H), 2.81 (t, 2H, J = 8.2 Hz), 2.57−2.49 (m, 2H), 1.64−1.60
(m, 2H), 1.52−1.38 (m, 2H). EI-MS (m/z, %): 326 (M+, 100). EI-
HRMS calculated for C18H22N4S (M+), 326.1564; observed, 326.1565.
HPLC purity: 97.99%.
N-(5-Fluoro-1-(piperidin-4-yl)indolin-6-yl)thiophene-2-car-
boximidamide (38). Prepared from tert-butyl 4-(5-fluoro-6-(thio-
phene-2-carboximidamido)indolin-1-yl)piperidine-1-carboxylate (35)
(0.125 g, 0.28 mmol) as described for 36 to obtain the title compound
(0.067 g, 69%) as a yellow solid. 1H NMR (DMSO-d6) δ 7.71 (d, 1H,
J = 3.5 Hz), 7.60 (d, 1H, J = 5.0 Hz), 7.12−7.04 (m, 1H), 6.84 (d, 1H,
J = 10.1 Hz), 6.43 (brs, 2H), 5.97 (d, 1H, J = 6.8 Hz), 3.44−3.19 (m,
3H, merged with H2O peak), 2.98 (app d, 2H, J = 11.9 Hz), 2.81 (t,
2H, J = 8.0 Hz), 2.61−2.50 (m, 2H), 1.69−1.52 (m, 2H), 1.46−1.33
(m, 2H). 19F NMR (DMSO-d6) δ −138.130 to −138.190 (m). ESI-
MS (m/z, %): 345 (MH+, 100). ESI-HRMS calculated for
C18H22N4FS (MH+), 345.1543; observed, 345.1541. HPLC purity:
98.21%.
tert-Butyl 3-(6-(Thiophene-2-carboximidamido)indolin-1-
yl)pyrrolidine-1-carboxylate (33). A suspension of tris-
(dibenzylideneacetone)dipalladium(0) (0.125 g, 0.13 mmol) in THF
(3 mL) was treated with tritert-butylphosphine in hexanes, 10 wt %
(1.65 mL, 0.54 mmol) and then stirred for 5 min at room temperature.
To this mixture was added lithium hexamethyldisilizane (1 M in THF)
(5.45 mL, 5.45 mmol) and tert-butyl 3-(6-bromoindolin-1-yl)-
pyrrolidine-1-carboxylate (30) (1.0 g, 2.72 mmol) in THF (20 mL).
The reaction vessel was sealed and the dark mixture was heated at 100
°C for 3 h and then allowed to cool to room temperature, treated with
tetrabutylammonium fluoride (1 M in THF) (13.61 mL, 13.61 mmol),
and stirred for 30 min. The mixture was concentrated and partitioned
between EtOAc and H2O in a separatory funnel and extracted. The
organic layer was separated and rinsed with brine, dried (Na2SO4),
filtered, and concentrated to give a dark residue. This residue was
purified by silica gel column chromatography using the Biotage
purification system (column:, Silicycle 80 g; EtOAc:hexanes, 1:3
gradient to EtOAc over 10 column volumes; flow rate, 30 mL/min;
collection wavelength, 254 nm) to obtain tert-butyl 3-(6-aminoindolin-
1-yl)pyrrolidine-1-carboxylate as a light-brown semisolid.
A solution of above tert-butyl 3-(6-aminoindolin-1-yl)pyrrolidine-1-
carboxylate in dry EtOH (20 mL) was treated with methyl thiophene-
2-carbimidothioate hydroiodide (13) (1.53 g, 5.44 mmol) and stirred
at room temperature for 17 h. The reaction was worked up and
purified as described for 14 to obtain the title compound (0.7 g, 77%,
6-Nitro-1-(1,4-dioxaspiro[4.5]decan-8-yl)indoline (40). Pre-
pared from 6-nitroindoline (6) (3.0 g, 18.27 mmol) and 1,4-
dioxaspiro[4.5]decan-8-one (39) (4.28 g, 27.4 mmol) as described
for 10 to obtain the title compound (5.02 g, 90%) as a dark-orange
1
over two steps) as a light-yellow solid. H NMR (DMSO-d6) δ 7.72
1
solid. H NMR (CDCl3) δ 7.46 (dd, 1H, J = 2.1, 7.8 Hz), 7.08−7.04
(d, 1H, J = 8.7 Hz), 7.59 (d, 1H, J = 4.8 Hz), 7.08 (dd, 1H, J = 3.9, 4.8
Hz), 6.94 (d, 1H, J = 7.5 Hz), 6.32 (brs, 2H), 6.09−6.07 (m, 2H),
4.28−4.08 (m, 1H), 3.49−3.14 (m, 6H, overlap with H2O peak), 2.82
(t, 2H, J = 7.8 Hz), 2.08−1.91 (m, 2H), 1.39 (s, 9H). ESI-MS (m/z,
%): 413 (MH+, 100%).
tert-Butyl 4-(6-(Thiophene-2-carboximidamido)indolin-1-
yl)piperidine-1-carboxylate (34). Prepared from tert-butyl 4-(6-
nitroindolin-1-yl)piperidine-1-carboxylate (31) (1.0 g, 2.88 mmol) as
described for 14 to obtain the title compound (1.08 g, 90%). 1H NMR
(DMSO-d6) δ 7.74−7.69 (m, 1H), 7.61−7.57 (m, 1H), 7.12−7.06 (m,
1H), 7.93−6.90 (m, 1H), 6.23 (brs, 2H), 6.03−6.00 (m, 2H), 4.12−
3.99 (m, 3H), 3.61−3.52 (m, 1H), 3.33 (m, 2H), 2.84−2.78 (m, 3H),
1.72−1.62 (m, 2H), 1.49−1.39 (m, 11H). EI-MS (m/z, %): 426 (M+,
100).
(m, 2H), 3.96 (s, 4H), 3.55−3.43 (m, 3H), 3.01 (t, 2H, J = 8.4 Hz),
1.88−1.67 (m, 8H). ESI-MS (m/z, %): 305 (MH+, 100).
4-(6-Nitroindolin-1-yl)cyclohexanone (41). To a solution of 6-
nitro-1-(1,4-dioxaspiro[4.5]decan-8-yl)indoline (40) (4.8 g, 15.77
mmol) in acetone (30 mL) was added hydrogen chloride (20 mL,
20.00 mmol) and the mixture left to stir at room temperature
overnight. The solvent was removed and diluted with dichloromethane
and 1 N NaOH solution. The organic layer was extracted, washed with
brine, dried (Na2SO4), and solvent was evaporated to obtain the crude
1
title compound (4.2 g, quantitative). H NMR (CDCl3) δ 7.53 (dd,
1H, J = 1.8, 7.8 Hz), 7.17 (d, 1H, J = 1.8 Hz), 7.10 (d, 1H, J = 7.8 Hz),
3.99−3.89 (m, 1H), 3.52 (t, 2H, J = 8.7 Hz), 3.05 (t, 2H, J = 8.7 Hz),
2.57−2.51 (m, 4H), 2.20−2.14 (m, 2H), 1.97−1.83 (m, 2H). ESI-MS
(m/z, %): 261 (MH+, 40).
tert-Butyl 4-(5-Fluoro-6-(thiophene-2-carboximidamido)-
indolin-1-yl)piperidine-1-carboxylate (35). Prepared from tert-
butyl 4-(5-fluoro-6-nitroindolin-1-yl)piperidine-1-carboxylate (32)
(0.20 g, 0.54 mmol) as described for 14 to obtain the title compound
(0.15 g, 56%) as a sticky yellow residue. 1H NMR (DMSO-d6) δ 7.74
(d, 1H, J = 4.2 Hz), 7.63 (d, 1H, J = 4.9 Hz), 7.16−7.05 (m, 1H), 6.68
(d, 1H, J = 10.0 Hz), 6.53 (brs, 2H), 6.06 (d, 1H, J = 6.8 Hz), 4.10−
3.92 (m, 2H), 3.60−3.45 (m, 1H), 3.30−3.19 (m, 2H), 2.92−2.65 (m,
4H), 1.73−1.57 (m, 2H), 1.49−1.30 (m, 11H). 19F NMR (DMSO-d6)
δ −137.856 to −137.924 (m). ESI-MS (m/z, %): 445 (MH+, 100).
N-(1-(Pyrrolidin-3-yl)indolin-6-yl)thiophene-2-carboximida-
N-Methyl-4-(6-nitroindolin-1-yl)cyclohexanamine (cis-42
and trans-42). Prepared from 4-(6-nitroindolin-1-yl)cyclohexanone
(41) (2.0 g, 7.68 mmol) and methylamine hydrochloride (0.62 g, 9.22
mmol) as described for 10 to obtain two partially separable mixture of
diastereomers (1.21 g, 57%) in 1:1 ratio. cis-42: dark-orange solid; 1H
NMR (CDCl3) δ 7.45 (dd, 1H, J = 2.1, 8.1 Hz), 7.06−7.04 (m, 2H),
3.56 (t, 2H, J = 8.7 Hz), 3.47−3.36 (m, 1H), 3.00 (t, 2H, J = 8.7 Hz),
2.83−2.81 (m, 1H), 2.44 (s, 3H), 1.93−1.87 (m, 2H), 1.83−1.74 (m,
2H), 1.68−1.57 (m, 4H); ESI-MS (m/z, %) 276 (MH+, 100). trans-
1
42: dark-orange solid; H NMR (CDCl3) δ 7.46 (dd, 1H, J = 1.8, 7.8
mide (36).
A solution of tert-butyl 3-(6-(thiophene-2-
Hz), 7.07−7.04 (m, 2H), 3.51 (t, 2H, J = 8.7 Hz), 3.47−3.37 (m, 1H),
3.01 (t, 2H, J = 8.7 Hz), 2.45 (s, 3H), 2.41−2.33 (m, 1H),2.15−2.04
(m, 2H), 1.93−1.83 (m, 2H), 1.76−1.55 (m, 4H); ESI-MS (m/z, %):
276 (MH+, 100), 245 (60).
carboximidamido)indolin-1-yl)pyrrolidine-1-carboxylate (33) (0.65 g,
1.57 mmol) in MeOH (15 mL) was treated with 3 N HCl (5.25 mL,
15.76 mmol) and then heated at 100 °C for 30 min. The mixture was
concentrated to remove most of the methanol, diluted with H2O (20
mL), and washed with CH2Cl2. The aqueous layer was basified with 3
N NaOH solution (∼15 mL) and extracted into CH2Cl2. The organic
layer was separated, dried (Na2SO4), filtered, and concentrated to
tert-Butyl Methyl(4-(6-nitroindolin-1-yl)cyclohexyl)-
carbamate (cis-43). Prepared from N-methyl-4-(6-nitroindolin-1-
yl)cyclohexanamine (cis-42) (0.15 g, 0.54 mmol) and di-tert-butyl
dicarbonate (0.13 g, 0.59 mmol) as described for 20 to obtain the title
1
1
obtain the title compound (0.435 g, 88%) as a yellow solid. H NMR
compound as an orange solid (0.2 g, 98%). H NMR (CDCl3) δ 7.51
(DMSO-d6) δ 7.69 (d, 1H, J = 3.0 Hz), 7.58 (dd, 1H, J = 0.9, 5.1 Hz),
7.08 (dd, 1H, J = 3.6, 4.8 Hz), 6.92 (d, 1H, J = 7.2 Hz), 6.26 (brs, 2H),
6.06−6.02 (m, 2H), 4.03−3.91 (m, 1H), 3.34 (t, 2H, J = 8.4 Hz,
overlap with H2O peak), 2.97 (dd, 1H, J = 4.5, 8.1 Hz), 2.87−2.69 (m,
5H), 1.94−1.83 (m, 1H), 1.77−1.65 (m, 1H). ESI-MS (m/z, %): 313
(dd, 1H, J = 2.1, 7.8 Hz), 7.12 (d, 1H, J = 2.1 Hz), 7.09 (d, 1H, J = 7.8
Hz), 4.03−3.95 (m, 1H), 3.70 (t, 2H, J = 8.1 Hz), 3.49−3.44 (m, 1H),
3.05 (t, 2H, J = 8.1 Hz), 2.78 (s, 3H), 2.17−2.10 (m, 2H), 1.79−1.62
(m, 6H), 1.47 (s, 9H). ESI-MS (m/z, %): 376 (MH+, 10), 276 (95),
245 (100).
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tert-Butyl Methyl(4-(6-nitroindolin-1-yl)cyclohexyl)-
carbamate (trans-43). Prepared from N-methyl-4-(6-nitroindolin-
1-yl)cyclohexanamine (trans-42) (0.195 g, 0.70 mmol) and di-tert-
butyl dicarbonate (0.17 g, 0.77 mmol) as described for 20 to obtain
the title compound (0.26 g, 98%) as an orange solid. 1H NMR
(CDCl3) δ 7.46 (dd, 1H, J = 2.1, 8.1 Hz), 7.07−7.05 (m, 2H), 4.07−
3.95 (m, 1H), 3.50 (t, 2H, J = 8.7 Hz), 3.42−3.34 (m, 1H), 3.02 (t,
2H, J = 8.7 Hz), 2.76 (s, 3H), 1.92−1.81 (m, 4H), 1.68−1.55 (m, 4H),
1.47 (s, 9H). ESI-MS (m/z, %): 376 (MH+, 10), 276 (90), 245 (100).
tert-Butyl Methyl(4-(6-(thiophene-2-carboximidamido)-
indolin-1-yl)cyclohexyl)carbamate (cis-44). Prepared from tert-
butyl methyl(4-(6-nitroindolin-1-yl)cyclohexyl)carbamate (cis-43)
(0.19 g, 0.50 mmol) as described for 14 to obtain the title compound
tert-Butyl 4-(5-Fluoro-6-(thiophene-2-carboximidamido)-
indolin-1-yl)cyclo-hexyl(methyl)carbamate (48). A solution of
tert-butyl 4-(5-fluoro-6-nitroindolin-1-yl)cyclohexyl(methyl)carbamate
(47) (0.106 g, 0.26 mmol) in dry EtOH (7 mL) was treated with 10%
palladium on carbon (0.029 g, 0.02 mmol) and stirred for 1 h under
hydrogen atm. The mixture was filtered through a pad of Celite and
washed with EtOH to obtain crude tert-butyl 4-(6-amino-5-
fluoroindolin-1-yl)cyclohexyl(methyl)carbamate.
To the above ethanolic solution of tert-butyl 4-(6-amino-5-
fluoroindolin-1-yl)cyclohexyl(methyl)carbamate was added methyl
thiophene-2-carbimidothioate hydroiodide (13) (0.154 g, 0.539
mmol) and the resulting mixture stirred at room temperature for 66
h. The reaction was worked up and purified as described for 14 to
obtain the title compound (70 mg, 55% over two steps) as a mixture of
diastereomers (trans:cis, 1.5:1). 1H NMR (DMSO-d6) δ 7.72 (d, 1H, J
= 3.8 Hz), 7.60 (d, 1H, J = 5.0 Hz), 7.12−7.05 (m, 1H), 6.90−6.80
(m, 1H), 6.44 (brs, 2H), 5.98 (d, 0.6H, J = 6.9 Hz), 5.93 (d, 0.4H, J =
6.9 Hz), 3.91−3.69 (m, 1H), 3.54−3.45 (m, 1H), 3.30−3.16 (m, 2H),
2.89−2.75 (m, 2H), 2.72 and 2.65 (2s, 3H), 2.09−1.93 (m, 1H),
1.85−1.44 (m, 7H), 1.39 (s, 9H). 19F NMR (DMSO-d6) δ −137.973
to −138.033 (m), −138.197 to −138.256 (m).
N-(5-Fluoro-1-(4-(methylamino)cyclohexyl)indolin-6-yl)-
thiophene-2-carbox -imidamide (cis-49 and trans-49). Prepared
from tert-butyl 4-(5-fluoro-6-(thiophene-2-carboximidamido)indolin-
1-yl)cyclohexyl(methyl)carbamate (48) (0.105 g, 0.22 mmol) as
described for ( )-36 to obtain the title compound as a separable
mixture of diastereomers. cis-49: yellow solid (20 mg, 24%); 1H NMR
(DMSO-d6) δ 7.72 (d, 1H, J = 3.0 Hz), 7.60 (d, 1H, J = 5.0 Hz), 7.09
(dd, 1H, J = 5.0, 3.8 Hz), 6.83 (d, 1H, J = 10.1 Hz), 6.42 (brs, 2H),
5.93 (d, 1H, J = 6.8 Hz), 3.33−3.19 (m, 4H, merged with H2O peak),
2.80 (t, 2H, J = 8.1 Hz), 2.62−2.53 (m, 1H), 2.23 (s, 3H), 1.83−1.61
(m, 4H), 1.56−1.30 (m, 4H); 19F NMR (DMSO-d6) δ −138.388 to
−138.448 (m); ESI-MS (m/z, %), 373 (MH+, 69), 342 (100); ESI-
HRMS calculated for C20H26N4FS (MH+), 373.1856; observed:,
373.1869; HPLC purity:, 97.74%. trans-49: yellow solid (30 mg, 36%);
1H NMR (DMSO-d6) δ 7.72 (d, 1H, J = 2.9 Hz), 7.60 (d, 1H, J = 5.0
Hz), 7.09 (dd, 1H, J = 5.0, 3.8 Hz), 6.83 (d, 1H, J = 10.1 Hz), 6.42
(brs, 2H), 5.93 (d, 1H, J = 6.8 Hz), 3.33−3.15 (m, 4H, merged with
H2O peak), 2.80 (t, 2H, J = 8.0 Hz), 2.32−2.15 (m, 1H), 2.27 (s, 3H),
1.99−1.86 (m, 2H), 1.77−1.63 (m, 2H), 1.49−1.28 (m, 2H), 1.20−
1.00 (m, 2H); 19F NMR (DMSO-d6) δ −138.231 to −138.290 (m);
ESI-MS (m/z, %), 373 (MH+, 46), 342 (100); ESI-HRMS calculated
for C20H26N4FS (MH+), 373.1856; observed, 373.1865; HPLC purity,
97.96%.
General Procedure for Conversion of the Free Base to the
Corresponding Dihydrochloride Salt. A solution of the free base
(1.0 equiv) in methanol was treated with 1 M HCl solution in diethyl
ether (3.0 equiv) dropwise at room temperature. The resulting mixture
was stirred for 10 min and concentrated to dryness. The product was
dried under reduced pressure to obtain the dihydrochloride salt as a
solid. The chemical purity of the dihydrochloride salts was similar to
their corresponding free bases.
NOS Enzyme Assays. Recombinant human nNOS, eNOS, and
iNOS were produced in baculovirus-infected Sf9 cells. In a radiometric
method, NOS activity is determined by measuring the conversion of
[3H]L-arginine to [3H]L-citrulline. To measure eNOS and nNOS, 10
μL of enzyme was added to 100 μL of 40 mM HEPES, pH = 7.4,
containing 2.4 mM CaCl2, 1 mM MgCl2, 1 mg/mL BSA, 1 mM
EDTA, 1 mM dithiothreitol, 1 μM FMN, 1 μM FAD, 10 μM
tetrahydrobiopterin, 1 mM NADPH, and 1.2 μM CaM. To measure
iNOS, 10 μL of enzyme was added to 100 μL of 100 mM HEPES, pH
= 7.4, containing 1 mM CaCl2, 1 mM EDTA, 1 mM dithiotheitol, 1
μM FMN, 1 μM FAD, 10 μM tetrahydrobiopterin, 120 μM NADPH,
and 100 nM CaM.
1
(0.19 g, 90%) as a yellow solid. H NMR (CDCl3) δ 7.42−7.38 (m,
2H), 7.11−7.05 (m, 1H), 7.01 (d, 1H, J = 7.5 Hz), 6.24 (dd, 1H, J =
1.8, 7.8 Hz), 6.12 (s, 1H), 5.71 (brs, 2H), 4.02−3.92 (m, 1H), 3.56 (t,
2H, J = 8.1 Hz), 3.38−3.31 (m, 1H), 2.93 (t, 2H, J = 8.1 Hz), 2.77 (s,
3H), 2.17−2.11 (m, 2H), 1.81−1.56 (m, 6H), 1.46 (s, 9H). ESI-MS
(m/z, %): 455 (MH+, 100).
tert-Butyl Methyl(4-(6-(thiophene-2-carboximidamido)-
indolin-1-yl)cyclohexyl)carbamate (trans-44). Prepared from
tert-butyl methyl(4-(6-nitroindolin-1-yl)cyclohexyl)carbamate (trans-
43) (0.25 g, 0.66 mmol) as described for 14 to obtain the title
compound (0.22 g, 84%) as a yellow solid. 1H NMR (CDCl3) δ 7.42−
7.37 (m, 2H), 7.11−7.05 (m, 1H), 6.95 (d, 1H, J = 7.8 Hz), 6.21 (d,
1H, J = 7.2 Hz), 6.08 (s, 1H), 4.87 (brs, 2H), 4.02−3.90 (m, 1H),
3.40−3.24 (m, 3H), 2.91 (t, 2H, J = 7.8 Hz), 2.72 (s, 3H), 1.92−1.76
(m, 4H), 1.67−1.55 (m, 4H), 1.46 (s, 9H). ESI-MS (m/z, %): 455
(MH+, 100).
N-(1-(4-(Methylamino)cyclohexyl)indolin-6-yl)thiophene-2-
carboximidamide (cis-45). To a solution of tert-butyl methyl(4-(6-
(thiophene-2-carboximidamido)indolin-1-yl)cyclohexyl)carbamate
(cis-44) (0.18 g, 0.39 mmol) in CH2Cl2 (10 mL) was added TFA (2
mL) and the mixture left to stir for 2 h at room temperature. The
contents were transferred to a separatory funnel, 3 N NaOH was
added, and the organic layer was extracted and evaporated. The crude
was purified on a silica gel column chromatography (2 M NH3 in
MeOH:CH2Cl2 1:9) to obtain the title compound (0.075 g, 53%) as a
yellow solid. 1H NMR (CDCl3) δ 7.41−7.39 (m, 2H), 7.06 (dd, 1H, J
= 3.9, 4.8 Hz), 6.98 (d, 1H, J = 7.8 Hz), 6.19 (dd, 1H, J = 1.5, 7.5 Hz),
6.07 (s, 1H), 4.86 (brs, 2H), 3.45 (t, 2H, J = 8.4 Hz), 3.38−3.31 (m,
1H), 2.90 (t, 2H, J = 8.4 Hz), 2.81 (brs, 1H), 2.45 (s, 3H), 1.96−1.92
(m, 2H), 1.88−1.72 (m, 2H), 1.63−1.54 (m, 4H). ESI-MS (m/z, %):
355 (MH+, 100), 324 (50), 178 (60). ESI-HRMS calculated for
C20H27N4S (MH+), 355.1950; observed, 355.1950. HPLC purity:
99.17%.
N-(1-(4-(Methylamino)cyclohexyl)indolin-6-yl)thiophene-2-
carboximidamide (trans-45). Prepared from tert-butyl methyl(4-(6-
(thiophene-2-carboximidamido)indolin-1-yl)cyclohexyl)carbamate
(trans-44) (0.20 g, 0.44 mmol) as described for cis-44 to obtain the
1
title compound (0.13 g, 83%) as a yellow solid. H NMR (CDCl3) δ
7.41−7.38 (m, 2H), 7.06 (dd, 1H, J = 3.9, 4.8 Hz), 6.98 (d, 1H, J = 7.5
Hz), 6.20 (dd, 1H, J = 1.5, 7.5 Hz), 6.08 (s, 1H), 4.86 (brs, 2H), 3.41−
3.29 (m, 3H), 2.91 (t, 2H, J = 8.1 Hz), 2.45 (s, 3H), 2.40−2.25 (m,
1H), 2.07−1.99 (m, 2H), 1.1.91−1.86 (m, 2H), 1.53−1.36 (m, 2H),
1.28−1.10 (m, 2H). ESI-MS (m/z, %): 355 (MH+, 100), 324 (50),
178 (60). ESI-HRMS calculated for C20H27N4S (MH+), 355.1950;
observed, 355.1942. HPLC purity: 98.65%.
tert-Butyl 4-(5-Fluoro-6-nitroindolin-1-yl)cyclohexyl-
(methyl)carbamate (47). Prepared from 5-fluoro-6-nitroindoline
(27) (0.21 g, 1.15 mmol) and tert-butyl methyl(4-oxocyclohexyl)-
carbamate (46) (0.524 g, 2.30 mmol) as described for 10 to obtain the
title compound (0.215 g, 47%) as a mixture of diastereomers (trans:cis,
1.5:1). 1H NMR (DMSO-d6) δ 7.27−7.20 (m, 1H), 6.96 (d, 0.6H, J =
5.9 Hz), 6.82 (d, 0.4H, J = 5.9 Hz), 3.91−3.78 (m, 1H), 3.68 (app t,
1H, J = 8.3 Hz), 3.55−3.39 (m, 2H), 3.08−2.93 (m, 2H), 2.72 and
2.69 (2s, 3H), 2.06−1.94 (m, 1H), 1.81−1.44 (m, 7H), 1.40 (s, 9H).
19F NMR (DMSO-d6) δ −132.311 to −132.370 (m), −132.645 to
To measure enzyme inhibition, a 15 μL solution of a test substance
was added to the enzyme assay solution, followed by a preincubation
time of 15 min at RT. The reaction was initiated by addition of 20 μL
of L-arginine containing 0.25 μCi of [3H] arginine/mL and 24 μM L-
arginine. The total volume of the reaction mixture was 150 μL in every
well. The reactions are carried out at 37 °C for 45 min. The reaction
−132.705 (m). ESI-MS (m/z, %): 416 (MNa+, 48%), 394 (MH+, 30),
338 (100).
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Table 2. Experimental Conditions for hERG K+ Channel Conventional Patch-Clamp Assay
cells
solutions
incubation
detection
HEK-293 cell line
stably expressing
hERG
extracellular solutions: 137 NaCl, 4 KCl, 1.8
CaCl2, 1 MgCl2, 10 D(+)-glucose, 10 HEPES (pH
7.4 by NaOH).
5−10 min for concentration at rt (22−24 °C)
conventional whole-cell patch
clamp (by Axopatch 200 B or
HEKA EPC9).
cumulatively.
intracellular solutions: 130 KCl, 10 NaCl, 1
MgCl2, 10 EGTA, 5 MgATP, 10 HEPES (pH 7.2
by KOH).
was stopped by adding 20 μL of ice-cold buffer containing 100 mM
HEPES, 3 mM EGTA, 3 mM EDTA, pH = 5.5. [3H]L-citrulline was
separated by DOWEX (ion-exchange resin DOWEX 50 W X 8−400,
SIGMA), and the DOWEX was removed by spinning at 12000g for 10
min in the centrifuge. Then a 70 μL aliquot of the supernatant was
added to 100 μL of scintillation fluid and the radio activity was
counted in a liquid scintillation counter (1450 Microbeta Jet, Wallac).
Specific NOS activity is reported as the difference between the activity
recovered from the test solution and that observed in a control sample
containing 240 mM of the inhibitor (1). All assays were performed in
duplicate.
Efficacy in the Chung Model of Neuropathic Pain. Nerve
ligation injury was performed according to the literature procedure.30
Rats were anesthetized with halothane and the L5 and L6 spinal nerves
were exposed, carefully isolated, and tightly ligated with 4−0 silk
suture distal to the DRG. After ensuring homeostatic stability, the
wounds were sutured and the animals allowed to recover in individual
cages. This technique produces signs of neuropathic dysesthesias,
including tactile allodynia, thermal hyperalgesia, and guarding of the
affected paw which begins on day 1 of the surgery and peaks on day
16. After a period of recovery following the surgical intervention, rats
show enhanced sensitivity to painful and normally nonpainful stimuli.
hERG K+ Channel Conventional Patch-Clamp Assay.32
Cultured cells (1−7 days) were used for patch clamp assay. The
cells were cultured in DMEM/GlutaMax-1 + 10% FBS and were
planted on collagen-coated dishes at low density (∼2 × 104 cells/
dish). The cell was held at −80 mV. A 50 ms pulse to −40 mV was
delivered to measure the leaking currents, which were subtracted from
the tail currents online. Then the cell was depolarized to +20 mV for 2
s, followed by a second pulse to −40 mV for 1 s to reveal the tail
currents. This paradigm was delivered once every 5 s to monitor the
current amplitude. After the current amplitude stabilized, cis-45 was
delivered to the extracellular medium by a rapid solution changer
perfusion system. During superfusion, the cell was repetitively
stimulated with the protocol described above, and the current
amplitude was continuously monitored. The experimental conditions
are described in Table 2.
KPSS (62.5 mM) three times to provide a reference means of
contractility.
The pharmacology conducted in the following order.
(1) Test tissue was first challenged to provide a measure of
maximum contractility.
(2) Test tissue was washed with PSS and allowed to return to
baseline.
(3) Test tissue was then tested for endothelial integrity by
precontracting the tissue with thromboxane mimetic
U46619 (1 × 10−7 M) and then adding cumulative
concentrations of a known endothelium-dependent
dilator agonist (ACh; 1 × 10−10 M to 1 × 10−5 M). If
the endothelium was intact, ACh produced relaxations.
(4) Test tissue was rinsed and allowed to return to baseline.
(5) The test article was tested in the presence and absence of
L-arginine (100 μM). cis-45 ( L-arginine) was added at
the selected concentration for a period of 50 min. In the
presence of cis-45 ( L-arginine), all vessels were then
submaximally vasoconstricted with U46619 prior to
CCRCs to ACh (1 × 10−10 M to 1 × 10−5 M).
Compound 1 was studied in each artery ring (100 μM)
in the presence and absence of L-arginine (100 μM) as a
positive control and reference compound 5 was studied
for comparison.
Responses were expressed as a % of the maximal contractile
response to U46619 (as a negative % change for a vasodilatory
response). The % relaxation (reversal) of U46619-mediated
contractions in response to ACh was plotted as concentration versus
response. Direct contractile effects were expressed as a % of the
maximum contractile response to KPSS (62.5 mM). Best-fit curves
were constructed using nonlinear regression.
High Throughput Broad Screen. In each experiment, the
respective reference compound was tested concurrently with cis-45,
and the data were compared with historical values (Table 1 from
Supporting Information). Results showing an inhibition (or
stimulation for assays run in basal conditions) higher than 50% are
considered to represent significant effect of cis-45. The specific ligand
binding to the receptor is defined as the difference between the total
binding and the nonspecific binding determined in the presence of an
excess of unlabeled ligand. The results are expressed as a percent of
control specific binding ((measured specific binding/control specific
binding) × 100) and as a percent inhibition of control specific binding
(100 − ((measured specific binding/control specific binding) × 100))
obtained in the presence of the test compound.
The degree of inhibition (%) was obtained by measuring the tail
current amplitude before and after drug superfusion (the difference
current was normalized to control and multiplied by 100 to obtain the
percent of inhibition). Concentration (log) response curves were fitted
to a logistic equation to generate estimates of IC50. The
concentration−response relationship of cis-45 was constructed from
the percentage reductions of current amplitude by sequential
concentrations.
Contractile Effects on Human Resistance Arteries. Fresh
specimens of human resistance arteries were obtained from surgical
explant tissue with full informed consent and ethical permission from
the donor. All test tissues, having been cut into ring segments of
approximately 2 mm length, were attached by 40 μm diameter wire
running through the lumen of the vessel to stainless steel heads in 10
mL myograph baths containing Krebs-bicarbonate physiological saline
solution (PSS), aerated with 95% O2 and 5% CO2, and maintained at a
temperature of 37 °C. Changes in tension were recorded using a
Danish Myotech isometric transducer. The segments were allowed to
equilibrate for at least 30 min and were washed with PSS every 15 min
during the equilibration period. Segments were processed through a
standardization procedure to reduce signal variability prior to
pharmacological intervention. All segments were then exposed to
ASSOCIATED CONTENT
* Supporting Information
Full details on the HPLC methods for all final compounds, 2D
spectral data for cis-45 and trans-45 and the broad screen
results for cis-45 with 80 receptors. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: 1-416-673-8457. Fax: 1-905-823-5836. E-mail:
sannedi@neuraxon.com.
■
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ACKNOWLEDGMENTS
■
We are grateful to NoAb BioDiscoveries Inc. (Mississauga, ON,
Canada) and Asinex Ltd. (Moscow, Russia) for performing the
human NOS inhibition assays, Biopta Ltd. (Glasgow, UK) for
studying the contractile effect on human resistance arteries, and
Cerep (USA and France) for hERG binding assay, hERG
patch-clamp assay, and broad screen profile.
ABBREVIATIONS USED
■
NO, nitric oxide; NOS, nitric oxide synthase; nNOS, neuronal
nitric oxide synthase; eNOS, endothelial nitric oxide synthase;
iNOS, inducible nitric oxide synthase; CTTH, chronic tension
type headache; FAD, flavinadenine dinucleotide; FMN, flavine
mononucleotide; BH4, (6R)-2-amino-6-[(1R,2S)-1,2-dihydrox-
ypropyl]-5,6,7,8-tetrahydropteridin-4(1H)-one; L-NMMA, N-
monomethyl L-arginine; hERG, human ether-a-go-go-related
gene; SAR, structure−activity relationship; SNL, spinal nerve
ligation; ACh, acetylcholine
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