First-in-class, dual-action, 3,5-disubstituted indole derivatives having human nitric oxide synthase (nNOS) and norepinephrine reuptake inhibitory (NERI) activity for the treatment of neuropathic pain

Article abstract of DOI:10.1021/jm300138g

A family of different 3,5-disubstituted indole derivatives having 6-membered rings were designed, synthesized, and demonstrated inhibition of human nitric oxide synthase (NOS) with norepinephrine reuptake inhibitory activity (NERI). The structure-activity

Full text of DOI:10.1021/jm300138g

Article
pubs.acs.org/jmc
First-in-Class, Dual-Action, 3,5-Disubstituted Indole Derivatives
Having Human Nitric Oxide Synthase (nNOS) and Norepinephrine
Reuptake Inhibitory (NERI) Activity for the Treatment of Neuropathic
Pain
Gabriela Mladenova,*,† Subhash C. Annedi,† Jailall Ramnauth,† Shawn P. Maddaford,† Suman Rakhit,†
John S. Andrews,† Dongqin Zhang,‡ 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 family of different 3,5-disubstituted indole derivatives having 6-membered rings
were designed, synthesized, and demonstrated inhibition of human nitric oxide synthase (NOS)
with norepinephrine reuptake inhibitory activity (NERI). The structure−activity relationship
(SAR) within the cyclohexane ring showed the cis-isomers to be more potent for neuronal NOS
and selective over endothelial NOS compared to their trans-counterparts. Compounds, such as
cis-(+)-37, exhibited dual nNOS and NET inhibition (IC50 of 0.56 and 1.0 μM, respectively) and
excellent selectivity (88-fold and 12-fold) over eNOS and iNOS, respectively. The lead compound (cis-(+)-37) showed lack of
any direct vasoconstriction or inhibition of ACh-mediated vasorelaxation in isolated human coronary arteries. Additionally, cis-
(+)-37 was effective at reversing both allodynia and thermal hyperalgesia in a standard Chung (spinal nerve ligation) rat
neuropathic pain model. Overall, the data suggest that cis-(+)-37 is a promising dual action development candidate having
therapeutic potential for the treatment of neuropathic pain.
alertness. It is believed that by inhibiting norepinephrine
transporter (NET), pain is attenuated by blocking reuptake of
NE, leading to increased postsynaptic NE levels and sustained
activation of the descending pain inhibitory pathway. In
addition, animal studies and clinical observations suggest that
drugs with NERI activity are more effective for the treatment of
pain.11−13
INTRODUCTION
■
Nitric oxide (NO) is a biological signaling molecule, and since
its discovery it has been under investigation as a potential
therapeutic for the treatment of neurodegenerative disorders
such as inflammation and pain. Nitric oxide synthase (NOS)
inhibitors have repeatedly been shown to be active in multiple
models of neuropathic pain.1 NO is synthesized by catalysis
from three isoforms of nitric oxide synthase (NOS), a
constitutive form in endothelial cells (eNOS), a constitutive
form in neuronal cells (nNOS), and an inducible form found in
macrophage cells (iNOS). These enzymes are homodimeric
proteins that catalyze a five-electron oxidation of L-arginine,
yielding NO and L-citrulline. The role of NO produced by each
of the NOS isoforms is quite unique, and its functions in
normal and pathological processes are well established.2 For
example, NO produced from overstimulation of individual
NOS isoforms, especially nNOS and iNOS, can lead to several
disorders such as septic shock, arthritis,3 pain,4 and various
neurodegenerative disorders.5 On the other hand, stimulation
of eNOS produces NO which has mainly a physiological role in
maintaining normal blood pressure and flow, while it is
inhibition (for example N-methyl-L-arginine (L-NMMA)) can
lead to enhanced white cell, platelet activation, and hyper-
tension.6−9
Neuropathic pain is a complex phenomenon characterized by
burning pain coupled with hyperalgesia and allodynia that
involves several mechanisms in both the peripheral and central
nervous system. At present, first-line treatment options for this
pathology are represented by the so-called “analgesic adjuvants”
such as antidepressants, anticonvulsivants, and local anesthetics
(gabapentin, lidocaine, tramadol, nortriptyline, doxepine).14−17
In recent years, an increased understanding of the
mechanism of action has led to the use of antidepressant
medication for the treatment of neuropathic pain.18−21 Further,
there has been a growing interest in the design of drugs that act
specifically on multiple targets (“targeted polypharmacology”)
with the aim of improving drug efficacy and safety. These types
of compounds, with defined multitarget profiles, have been
classified as designed multiple ligands (DMLs) to distinguish
them from nonselective drugs that often possess activities
irrelevant to disease management, with the potential to elicit
undesirable side effects. Success with the DML approach has
Norepinephrine (NE) is a monoamine neurotransmitter
found in the CNS and plays an important role in human
physiology and pathology.10 It is involved in mood and sleep
regulation, expression of behavior, and the general degree of
Received: January 31, 2012
Published: March 15, 2012
© 2012 American Chemical Society
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been achieved in the development of dual inhibitors of
serotonin and norepinephrine reuptake for the treatment of
depression or pain22 such as Duloxetine (1)23,24 Venlafaxine
(2),25 and Milnacipran (3).26 In general, these new dual action
antidepressants show superior efficacy22 via the action of both
ascending and descending noradrenergic and serotonergic
pathways.
acid hydrolysis of the ketal (Scheme 1). The resulting ketone
was subjected to reductive amination with the appropriate
amine to give di- and trisubstituted amines as racemic mixtures
in 60−70% yield. The separation of the enatiomers was not
attempted at this time. After protection of the secondary
amines (( )-8 and ( )-10), compounds ( )-7, ( )-9, and
( )-11 underwent two separate transformations. In the first
pathway, only the nitro group was reduced using hydrazine in
the presence of Raney nickel as catalyst to obtain anilines
( )-12−( )-14, which were then coupled with methyl
thiophene-2-carbimidothioate 15. After deprotection of
( )-17 and ( )-19 under acid conditions, the first set of
target compounds, ( )-16, ( )-18, and ( )-20, was obtained.
Only compound ( )-16 was separated into its enantiomers in
high optical purity using preparative chiral HPLC. The absolute
configurations were not assigned.
To obtain the saturated analogues, compounds ( )-7, ( )-9,
and ( )-11 were subjected to hydrogenation in the presence of
palladium on carbon at 1 atm, where the nitro groups were
reduced as well. Mixtures of inseparable diastereomers were
obtained. The anilines were further coupled with 15 as
previously mentioned, and after removal of the Boc-protecting
group a new set of compounds 24, 26, and 28 was obtained. At
this time the final compounds were separated into their
corresponding cis and trans isomers using HPLC. Although
NOE data were obtained for all isomers, the 2D data proved
difficult for stereochemical assignment owing to the fact that
the cyclohexane ring would undergo interconversion between
the boat and chair conformations on the NMR time scale. This
interconversion would tend to average the chemical shift values
of the axial and equatorial protons and decrease the resolution
of the couplings. Therefore, in all three cases 1D spectra were
used to assign the cis and trans stereochemistry (see Supporting
Information). The argument is based on the fact that there is
good chemical shift dispersion for the protons of the trans
isomers as well as good resolution of the coupling constants
and similar observations have been previously reported.31,32
The cis isomers did not have the same chemical shift
differences, and the coupling constants were not as well
resolved. Using the above argument, the proton chemical shift
range for the cis-configurations in compounds 24, 26, and 28 is
0.3−0.5 and 0.8−0.93 ppm for the trans-configurations.
Compound ( )-29, the second key intermediate, was
obtained from the Michael addition of 4 to cyclohex-2-enone
with Bi(NO3)3 as the catalyst (Scheme 2). Reductive amination
of the ketone with the appropriate amine yielded compounds
( )-30 (cis/trans 1:3) and ( )-32 (cis/trans 1:3) as separable
isomers. The enantiomers were not separated at this time. The
cis/trans stereochemistry was assigned by using COSY and
NOESY spectroscopic techniques and proved much easier in
the stereochemical assignment compared to the 1,4-disub-
stituted analogues (see Supporting Information). Each of the
isomers underwent further transformation separately, first
protection, followed by reduction of the nitro group with
hydrazine hydrate and Raney nickel and coupling with 15 as
previously. After removal of the Boc- group, the final set of
target compounds cis/trans-( )-37 and cis/trans-( )-39 was
obtained as racemates. Because cis-( )-37 showed a better in
vitro profile, it was further separated into its enatiomers with
high optical purity using chiral supercritical fluid chromatog-
raphy, however, the absolute configuration was not established
at this time.
Considering that both norepinephrine and nitric oxide are
involved in the progression of pain, the plan was to design and
synthesize dual action selective neuronal nitric oxide synthase
(nNOS) inhibitors with NERI activity. Both nNOS and NET,
an enzyme and membrane transporter, respectively, are
completely distinct biological targets. Given that a sufficient
overlap of pharmacophores must exist between the two targets
of interest in order for a drug to interact sufficiently, it may be
more challenging to find suitable dual action compounds. The
goal of this project was to establish the SAR based on our NOS
pharmacophore model and to optimize the potency and
selectivity for nNOS versus eNOS as well as the relative balance
of activity for nNOS versus NET (Figure 1).
Figure 1. (a) NET pharmacophore model: basic amine linked (flexible
or constrained) to two aromatic systems. Generally N-methyl
compounds are more active; (b) nNOS pharmacophore scaffold is
usually an aromatic ring. Main interaction is usually achieved through a
2° or 3° amine, and the anchoring point is made with an amidine
group.
On the basis of our previous findings,27−30 bulkier groups
showed better potency and selectivity for the nNOS inhibitors
and therefore a number of different 3,5-disubstituted indole
derivatives having 1,4-disubstituted or 1,3-disubstituted cyclo-
hexane rings were designed, synthesized, and tested for their
potencies at these two targets. The synthesis would also provide
an insight into the SAR within the series. Specifically, it would
enable an examination of the effect of stereochemistry within
the cyclohexane ring (cis versus trans) and the relative potencies
of secondary versus tertiary amines.
RESULTS AND DISCUSSION
■
Chemistry. The syntheses started with commercially
available 5-nitroindole (4) and are outlined in Schemes 1 and
2. Compounds 6 and ( )-29 are the key intermediates in the
preparation of our final targets consisting of 3,5-disubstituted
indoles with 1,4-disubstituted cyclohexanes or 1,3-disubstituted
cyclohexanes, respectively. Compound 6 was prepared from the
condensation of 4 and 5 under basic conditions followed by the
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a
Scheme 1. 3,5-Disubstituted Indoles with 1,4-Disubstituted Cyclohexane Rings
a
Reagents and Conditions: (a) 5, KOH, MeOH, reflux; (b) 10% HCl, acetone, rt; (c) amine, AcOH, NaBH(OAC)3, 1,2-DCE; (d) (Boc)2O, Et3N,
1,4-dioxane; (e) hydrazine hydrate, Raney nickel, MeOH, reflux; (f) 15, EtOH, rt; (g) 1N HCl, reflux or TFA/CH2Cl2 rt; (h) chiral HPLC
separation; (i) EtOH, Pd/C, H2, 1 atm; (j) preparative HPLC.
All final compounds were converted into their hydrochloride
salts prior to in vitro and in vivo testing.
An apparent difference in both potency and selectivity was
observed for the cis/trans pairs of compounds 24−28. The cis
analogues are 5−15 times more potent for the nNOS isoform
as well as for NET compared to their trans counterparts. For
example, cis-26 exhibited an IC50 value of 0.39 μM for nNOS,
while the corresponding trans isomer was 5-fold less potent.
Similar difference in activity was observed for NET, with IC50
value of 0.4 μM for cis-26 and IC50 value of 2.0 μM for trans-26.
The cis analogues also showed better selectivity for nNOS over
eNOS. This is consistent with our previous observations where
the cis isomers were found to be more potent and selective for
the nNOS over eNOS isoform,30 and the conformational
difference between these stereoisomers appears to be just as
important on NET activity. This provides additional support
for the suggested pharmacophore model, where a bulky or
cyclic side chain with a basic amine is necessary to obtain the
submicromolar potency for nNOS33 and selectivity over eNOS
isoform and to achieve potency for NET. Also, translocation of
the basic amine out of the cyclic ring can make the functional
Structure−Activity Relationship (SAR) Studies. The
synthesized compounds were screened for their in vitro human
NOS and human NET inhibitory activities by measuring the
conversion of [3H]L-arginine to [3H]L-citrulline using a
radiometric method for NOS inhibition and the inhibition of
[3H] nisoxetine binding to CHO cells in the presence of the
test compound for NET inhibition (Table 1). Initial look into
the SAR of 1,4-disubstituted cyclohexane analogues for both
the unsaturated and their saturated counterparts of all
compounds showed very good potency for nNOS and NET
and excellent selectivity for nNOS over eNOS and iNOS.
It is interesting to note that compared to the racemate
( )-16, the separated enantiomers (+)-16 and (−)-16 became
more potent for nNOS (i.e., from IC50 = 3.0 to IC50 ∼ 0.8 μM)
while the potencies for NET were reduced by half (i.e., IC50
1 from IC50 = 0.55 μM).
∼
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Scheme 2. 3,5-Disubstituted Indoles with 1,3-Disubstituted Cyclohexane Rings
Reagents and Conditions: (a) cyclohex-2-enone, Bi(NO3)3, MeCN, rt; (b) amine, AcOH, NaBH(OAC)3, 1,2-DCE; (c) (Boc)2O, Et3N, 1,4-dioxane;
(d) hydrazine hydrate, Raney nickel, MeOH, reflux; (e) 15, EtOH, rt; (f) TFA, CH2Cl2, rt; (g) chiral HPLC
group less rigid and orient its position to the active site of the
NOS enzyme.
The selected compounds also showed a low potential for
drug−drug interaction with low cytochrome P450 (CYP450)
inhibition against multiple isozymes (Table 2). This rules out
the inhibitory activity with CYP450 enzymes, where com-
pounds that bind to the heme iron (for example, imidazole-
containing compounds)35 have shown to be potent inhibitors
of CYP450.
Because cis-(+)-37 exhibited excellent potency for nNOS and
NET and excellent selectivity over eNOS, the compound was
considered for further evaluation in a variety of in vitro and in
vivo assays. In addition to the low hERG inhibition and low
potential for drug−drug interaction, cis-(+)-37 also possess
drug-like molecular properties such as polar surface area (PSA)
less than 7036 and cLogP less than 4, which are expected to be
beneficial to the PK and the in vivo efficacy of this drug
candidate.37,38
Mediated Vasoconstriction Effect on Isolated Human
Resistance Arteries. To investigate potentially undesirable
cardiovascular effects associated with the inhibition of eNOS,
cis-(+)-37 was assessed for the contractile response (inhibition
of acetylcholine-mediated vasorelaxation) on isolated human
resistance arteries. Acetylcholine (Ach), an endothelium and
nitric oxide dependent vasodilator, slows the heart rate when
functioning as an inhibitory neurotransmitter by inhibiting
contraction of cardiac muscle fibers.39,40 The test was
performed in the absence and presence of L-arginine (substrate
for eNOS) with positive (L-NMMA) and negative (vehicle)
controls (Figure 3). The arteries were preconstricted with
U46619, a thromboxane A2 (TxA2) mimetic agent, and then
exposed to acetylcholine. The response to ACh provides
information on the activity of eNOS in an active human
biological tissue and thereby any inhibitory effect of cis-(+)-37
In the 1,3-disubstituted cyclohexane series, the cis and trans
isomers did not show drastic differences in potency for nNOS
and NET, however the selectivity over eNOS was better for the
cis isomers. In contrast, the NET potency of the trans isomers
increased over their cis analogues (ex. cis-( )-37 IC50 = 2.4 μM
vs trans-( )-37 IC50 = 0.88 μM). With separated enantiomers
cis-(+)-37 and cis-(−)-37 and comparison to their correspond-
ing racemate, cis-(+)-37 demonstrated an increase in potency
over its antipode for both nNOS and NET (2- and 5-fold,
respectively) and better selectivity over eNOS.
It is interesting to note that the size of the alkyl group on the
basic amine did not play a major role in the potency and
selectivity of these compounds. For example, cis-26 and cis-
( )-37 (R2 = Me), cis-28 and cis-( )-39 (R2 = Et) all had
comparable potencies for nNOS (IC50 = 0.38, 0.49, 0.54, and
0.73 μM, respectively) and comparable selectivity over eNOS
(IC50 = 25.4, 77.6, 19.0, and 19.2 μM, respectively). Similarly,
comparing secondary versus tertiary amines the overall
selectivity for nNOS and potency over eNOS also remained
unchanged as in cis-24 (R1 = R2 = Me) and cis-26 (R2 = Me, R1
= H) (IC50 = 0.34 and 0.39 μM for nNOS and 62.8 and 35.2
μM for eNOS, respectively).
Several compounds cis-26, cis-28, cis-(+)-37, and cis-( )-39
exhibited the expected in vitro profile and were subject to
further investigation. Compounds cis-26 and cis-(+)-37
demonstrated low hERG inhibition (IC50 ≥ 30 μM), which
suggests a low potential for QTc prolongation.34 However cis-
28 and cis-( )-39 were more selective at hERG with IC50 of 23
and 16.3 μM (Table 2).
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Table 1. Human NOS and NET Inhibitory Activity by 3,5-Disubstituted Indole Derivatives
a
Values reported in parentheses are 95% confidence intervals. In a radiometric method, inhibitory activities were measured by the conversion of
b
[3H]-L-arginine into [3H]-L-citrulline. Inhibition of [3H] nisoxetine binding to CHO cells. Protriptyline was used as a reference compound (Ki = 2.0
c
0.5 nM). Each NET value is within accepted limits of the historic average 0.5 log units. L-NMMA is a known nonselective NOS inhibitor; tested
d
e
for comparison. NT not tested. Not calculable
on eNOS. Furthermore, if inhibition of relaxation is due to the
inhibition of eNOS, the addition of L-arginine (substrate for
eNOS) would recover the relaxation due to ACh. As seen in
Figure 3a, no significant inhibition with cis-(+)-37 on the ability
of the blood vessels to respond to ACh was observed at 100
μM concentration. Also, the addition of L-arginine did not
significantly affect the response to ACh in the presence of the
compound, providing further evidence that cis-(+)-37 does not
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Table 2. Inhibition of Human Cytochrome P450 Enzymes and hERG Inhibition Activity with Selected 3,5-Disubstituted Indole
Derivatives
CYP450 (IC50 μM)
compd
cis-26
hERG (IC50 μM)
CYP1A2
CYP2C9
CYP2C19
CYP2D6
CYP3A4
CYP3A4
39
23
>100
>100
>100
26.4
23.4
>100
NT
3.48
0.927
5.14
NT
∼57.3
23.1
>100
>100
cis-28
∼21.5
a
a
a
cis-(+)-37
cis-( )-39
33
n/a
n/a
∼100
NT
n/a
b
16.3
NT
NT
NT
a
b
n/a: no inhibition observed over the concentration range tested. NT: not tested.
Figure 2. (a) Effect of cis-(+)-37 on the responses to ACh in absence and presence of L-arginine. (b) Effect of L-NMMA on the responses to ACh in
absence and presence of L-arginine.
Figure 3. Neuropathic pain model. (a) Antiallodynic effects of cis-(+)-37 in the L5/L6 SNL SD rats. (b) Antihyperalgesia effects of cis-(+)-37 in the
L5/L6 SNL SD rats.
have an effect on agonist-mediated activation of human eNOS.
The in vitro data of cis-(+)-37 is therefore consistent with the
weak inhibitory potency in human eNOS with IC50 of 49.3 μM.
On the other hand, L-NMMA, a nonselective NOS inhibitor,
causes vasoconstriction at 100 μM concentration, and this
effect is reversed with the addition of L-arginine (100 μM)
(Figure 2b).
Chung or Spinal Nerve Ligation (SNL) Model Studies
with cis-(+)-37. The activity of cis-(+)-37 was evaluated in a
rat spinal nerve ligation (SNL) model of neuropathic pain.
Animal models of disease serve as a useful tool in assessing
efficacy in a disease state as it relates to drug potency and PK
properties of the molecule. The Chung model of neuropathic
pain is an animal model that involves spinal nerve ligation and
the development of mechanical or tactile allodynia and thermal
hyperalgesia in the paws of the animal. This model is widely
used for various investigative studies on pain mechanisms and
for assessing the potential of a drug analgesic effectiveness.
Allodynia is pain due to a stimulus that does not normally
provoke pain and are distinct from analgesia, which is defined
as reduction in the intensity of pain that occurs in response to a
normally painful stimulus, and hyperalgesia is defined as an
increased response to a stimulus that is normally painful.
Allodynia and hyperalgesia can be assessed by measuring the
paw withdrawal times from a thermal stimulus on the paws of
the animals (hyperalgesia) or by application of a mechanical
force using a calibrated filament applied to the paws
(allodynia).
In this model,41 surgery is performed to tightly ligate the L5
and L6 spinal nerves on one side of the rat. This produces
mechanical allodynia and thermal hyperalgesia of the affected
foot. Withdrawal threshold to application of mechanical force
and withdrawal latency to application of radiant heat to the foot
was tested. Intraperitoneal administration of cis-(+)-37 at 30
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mg/kg resulted in both reversal of allodynia and thermal
hyperalgesia. Maximum reversal effect of 90% for allodynia and
96% for hyperalgesia was observed at 30 min. (Figure 3). The
results clearly show that cis-(+)-37 is effective at reversing both
hyperalgesia and allodynia, suggesting it may be efficacious in
treating neuropathic pain. This is also a strong evidence for the
dual action activity of cis-(+)-37 in comparison to our previous
published pure nNOS inhibitors, where only reversal of thermal
hyperalgesia was observed.27,30
Encouraged by the later results, the metabolic profile of cis-
(+)-37 was investigated and its stability tested in liver
hepatocytes of several species (human, primate, dog, and rat).
No turnover was observed over 120 min, indicating a great
metabolic stability of this compound.
High Throughput Profile of cis-(+)-37. The high
throughput profile is useful tool for identifying off-target
activities of compounds with potential drawbacks associated
with the drug development process. This drawback can include
the interaction of the lead candidate with a specific protein or a
biological pathway and thus contribute to undesirable side
effects once administered in an individual. This process is also a
rapid and cost-effective way of prioritizing the most promising
compound such as cis-(+)-37 for further evaluation in various
preclinical toxicology studies during the selection process. To
identify off-target activities, cis-(+)-37 was tested in several
validated in vitro pharmacological assays at a single
concentration of 10 μM (40 nonpeptidic receptors, 30 peptidic
receptors, two nuclear receptors, five ion channels, and three
transporters).30 The compound was found to be selective for
most of the targets tested (<50% inhibition at 68 out of 80
receptors) with weak inhibition at: opioid receptors μ (53%)
and sigma receptor (σ, 61%). The compound showed strong
inhibition at: human muscarinic M1 (80%), M2 (87%), M3
(88%), M4 (93%), and M5 (87%). Overall cis-(+)-37 possesses
an excellent safety profile.
EXPERIMENTAL SECTION
■
General. All the 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
and 600 MHz (for 2D data) spectrometers. 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 reverse phase column, and
the purity was determined to be >95% for all final compounds, unless
otherwise indicated (see Supporting Information for complete
analytical methods and retention times). No attempts were made to
optimize the yields.
4-(5-Nitro-1H-indol-3-yl)cyclohex-3-enone (6). (a) A solution of 4
(3.0 g, 18.50 mmol) in dry MeOH (50 mL) was treated with KOH
(5.6 g, 100 mmol) at rt. After stirring for 10 min, 1,4-cyclohexanedione
monoethylene acetal (7.22 g, 46.25 mmol) was added and the
resulting solution was refluxed for 36 h. The reaction was brought to
rt, and solvent was evaporated. Crude was diluted with water (50 mL),
and precipitated solid was filtered off and washed with water (2 × 10
mL). The precipitate was dried under vacuum to obtain 5-nitro-3-(1,4-
1
dioxaspiro[4.5]dec-7-en-8-yl)-1H-indole (4.7 g, 85%) as a solid. H
NMR (CDCl3) δ 8.78 (d, 1H, J = 2.1 Hz), 8.36 (brs, 1H), 8.05 (dd,
1H, J = 2.1, 9.0 Hz), 7.32 (d, 1H, J = 8.7 Hz), 7.22 (d, 1H, J = 2.4 Hz),
6.12 (t, 1H, J = 3.9 Hz), 2.66−2.49 (m, 2H), 4.00−3.96 (m, 4H), 2.49
(brs, 2H), 1.91 (t, 2H, J = 6.6 Hz). ESI-MS (m/z, %) 301 (MH+, 100).
(b) A solution of 5-nitro-3-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)-1H-
indole (4.7 g, 15.65 mmol) in acetone (50 mL) was treated with 10%
aq HCl (50 mL) at rt and stirred overnight (14 h). Acetone was
evaporated, and crude was basified using 10% aq NH4OH solution
(100 mL). The precipitate was filtered off and washed with 10%
NH4OH solution (2 × 10 mL) and water (2 × 10 mL). The product
was dried under vacuum to obtain compound 6 (4.0 g, quantitative) as
a solid. 1H NMR (CDCl3) δ, 6.24 (t, 1H, J = 3.6 Hz), 7.57 (d, 1H, J =
9.0 Hz), 7.76 (d, 1H, J = 2.1 Hz), 8.03 (dd, 1H, J = 2.1, 9.0 Hz), 8.71
(d, 1H, J = 2.1 Hz), 3.19−3.18 (m, 2H), 2.97−2.88 (m, 2H), 2.71 (t, J
= 6.9 Hz, 2H). ESI-MS (m/z, %) 279 (M + Na, 36), 257 (MH+, 100).
N,N-Dimethyl-4-(5-nitro-1H-indol-3-yl)cyclohex-3-enamine
(( )-7). A solution of compound 6 (1.0 g, 3.90 mmol) in dry 1,2-
dichloroethane (10 mL) was treated with N,N-dimethyl amine
hydrochloride (0.31 g, 3.902 mmol), AcOH (0.22 mL, 3.90 mmol),
and NaBH(OAc)3 (1.24 g, 5.85 mmol) at rt, and the resulting mixture
was stirred overnight (14 h). The reaction was diluted with 1 N NaOH
(30 mL), and product was extracted into ethyl acetate (2 × 50 mL).
The combined ethyl acetate layer was washed with brine (20 mL) and
dried (Na2SO4). Solvent was evaporated, and crude was purified by
column chromatography (2 M NH3 in MeOH:CH2Cl2 1:9) to obtain
CONCLUSION
■
Novel 3,5-disubstituted indole derivatives with various 1,3- and
1,4-disubstituted cyclohexane side chains were synthesized and
shown to be selective nNOS inhibitors with NERI activity.
Further, with respect to potential cardiovascular effects,
excellent selectivity for nNOS over eNOS was also achieved.
In terms of potency, the cis-isomers were found to be more
potent for nNOS and selective over eNOS compared to the
trans-isomers. In the case of 1,4-disubstitution, cis-isomers were
found to have better NERI activity than the trans-isomer,
whereas the reverse was seen in the 1,3-disubstituted
cyclohexane rings.
1
compound ( )-7 (0.73 g, 66%) as a brown solid. H NMR (DMSO-
d6) δ 11.82 (s, 1H), 8.67 (d, 1H, J = 2.1 Hz), 8.00 (dd, 1H, J = 2.1, 9.0
Hz), 7.62 (s, 1H), 7.54 (d, 1H, J = 9.0 Hz), 6.15 (t, 1H, J = 1.5 Hz),
2.62−2.39 (m, 4H), 2.23−2.12 (m, 7H), 2.06−1.98 (m, 1H), 1.57−
1.43 (m, 1H). ESI-MS (m/z, %) 286 (MH+, 100).
As seen in the broad chemical series, the lead compound, cis-
(+)-37, showed lack of any direct vasoconstriction or inhibition
of ACh-mediated vasorelaxation in isolated human coronary
arteries, suggesting that a cardiovascular effect associated with
the inhibition of human eNOS would be greatly diminished. cis-
(+)-37 does not have any off-target activities (80 receptors),
suggesting that clinically effective concentrations would
engender a minimal side effect profile. The compound was
active in the neuropathic pain model. Intraperitoneal
administration of cis-(+)-37 was shown to reverse the thermal
hyperalgesia and mechanical allodynia in the SNL model,
indicating the potential of cis-(+)-37 to treat neuropathic pain.
Overall, the chemistry, safety pharmacology, and animal efficacy
data support the continued advancement of cis-(+)-37 as a
novel pain therapeutic.
N-Methyl-4-(5-nitro-1H-indol-3-yl)cyclohex-3-enamine (( )-8). A
solution of compound 6 (1.44 g, 5.62 mmol) in 1,2-dichloroethane
(20 mL) was treated with AcOH (0.31 mL, 5.62 mmol), methylamine
hydrochloride (0.37 g, 5.62 mmol), and NaBH(OAC)3 (1.78 g, 8.43
mmol) at rt and stirred overnight (14 h). Same workup as in ( )-7.
The crude was purified by column chromatography (2 M NH3 in
MeOH:CH2Cl2 1:9) to obtain compound ( )-8 (0.82 g, 54%) as a
1
solid. H NMR (DMSO-d6) δ 11.85 (brs, 1H), 8.67 (s, 1H), 8.00 (d,
1H, J = 7.5 Hz), 7.63 (s, 1H), 7.54 (d, 1H, J = 9.0 Hz), 6.13 (brs, 1H),
2.70−2.60 (m, 1H), 2.57−2.40 (m, 3H), 2.35 (s, 3H), 2.01−1.97 (m,
2H), 1.53−1.44 (m, 1H). ESI-MS (m/z, %) 272 (MH+, 100).
tert-Butyl Methyl(4-(5-nitro-1H-indol-3-yl)cyclohex-3-enyl)-
carbamate (( )-9). A solution of compound ( )-8 (0.8 g, 2.95
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mmol) in dry 1,4- dioxane (20 mL) was treated with Et3N (0.82 mL,
5.90 mmol) followed by (Boc)2O (0.67 g, 3.10 mmol) at rt, and the
resulting solution was stirred overnight (16 h). Solvent was
evaporated, and crude was purified by column chromatography
(EtOAc:hexanes 1:1) to obtain compound ( )-9 (1.0 g, 91%) as a
solid. 1H NMR (DMSO-d6) δ 11.87 (s, 1H), 8.68 (d, 1H, J = 2.1 Hz),
8.01 (dd, 1H, J = 2.4, 9.0 Hz), 7.66 (s, 1H), 7.55 (d, 1H, J = 9.0 Hz),
6.17 (brs, 1H), 4.16−4.10 (m, 1H), 2.74 (s, 3H), 2.70−2.60 (m, 2H),
2.45−2.29 (m, 2H), 1.87−1.81 (m, 2H), 1.42 (s, 9H). ESI-MS (m/z,
%) 394 (M.Na+, 100), 316 (44), 272 (82).
N-Ethyl-4-(5-nitro-1H-indol-3-yl)cyclohex-3-enamine (( )-10). A
solution of compound 6 (1.0 g, 3.902 mmol) in dry 1,2-dichloroethane
(10 mL) was treated with ethyl amine hydrochloride (0.31 g, 3.902
mmol), AcOH (0.22 mL, 3.902 mmol), and NaBH(OAc)3 (1.24 g,
5.853 mmol) at room temperature, and the resulting mixture was
stirred for overnight (14 h). Same workup as in ( )-7. The crude was
purified by column chromatography (2 M NH3 in MeOH:CH2Cl2
1:9) to obtain compound ( )-10 (1.08 g, 97%) as a dark-yellow solid.
1H NMR (DMSO-d6) δ 11.83 (brs, 1H), 8.67 (d, 1H, J = 2.4 Hz), 8.00
d6) δ 10.58 (s, 1H), 7.15 (d, 1H, J = 2.7 Hz), 7.04 (d, 1H, J = 8.4 Hz),
6.99 (d, 1H, J = 1.5 Hz), 6.48 (dd, 1H, J = 2.1, 8.5 Hz), 6.00−5.98 (m,
1H), 4.48 (s, 2H), 4.06 (brs, 1H), 3.18−3.12 (m, 2H), 2.60−2.20 (m,
4H), 1.91- 1.80 (m, 2H), 1.41 (s, 9H), 1.08 (t, 3H, J = 6.9 Hz). ESI-
MS (m/z, %) 356 (MH+, 10), 300 (100).
N-(3-(4-(Dimethylamino)cyclohex-1-enyl)-1H-indol-5-yl)-
thiophene-2-carboximidamide (( )-16). A solution of compound
( )-12 (0.18 g, 0.70 mmol) in dry EtOH (10 mL) was treated with
compound 15 (0.4 g, 1.41 mmol) at rt and stirred for 24 h. Solvent
was evaporated, and crude was diluted with satd NaHCO3 solution (20
mL), and product was extracted into CH2Cl2 (2 × 25 mL). The
combined CH2Cl2 layer was washed with brine (20 mL) and dried
(Na2SO4). Solvent was evaporated, and crude was purified by column
chromatography (2 M NH3 in MeOH:CH2Cl2 1:9) to obtain
1
compound ( )-16 (0.24 g, 90%) as a solid. H NMR (DMSO-d6) δ
10.88 (s, 1H), 7.71 (d, 1H, J = 2.7 Hz), 7.58 (d, 1H, J = 4.5 Hz), 7.31−
7.28 (m, 2H), 7.20 (s, 1H), 7.09 (t, 1H, J = 4.2 Hz), 6.65 (dd, 1H, J =
1.2, 8.4 Hz), 6.21 (brs, 2H), 6.03 (s, 1H), 2.60−2.31 (m, 3H), 2.22−
2.08 (m, 8H), 2.02−1.97 (m, 1H), 1.53−1.42 (m, 1H). ESI-MS (m/z,
%) 365 (MH+, 39), 320 (38), 183 (76), 160 (100). ESI-HRMS calcd
for C21H25N4S (MH+), 365.1813; obsd 365.1794.
(dd, 1H, J = 2.4, 9.0 Hz), 7.62 (s, 1H), 7.54 (d, 1H, J = 9.0 Hz), 6.13
(s, 1H), 4.07 (brs, 1H), 3.16 (s, 2H), 2.80−2.40 (m, 3H), 2.00−1.94
(m, 2H), 1.52 −1.39 (m, 2H), 1.03 (t, 3H, J = 6.9 Hz). ESI-MS (m/z,
%) 286 (MH+, 100).
The enantiomeric mixture was separated by using preparative chiral
HPLC column chromatography to obtain (−)-16 and (+)-16.
First Enantiomer (−)-16). ESI-MS (m/z, %) 365 (MH+, 35), 320
(43), 160 (82), 119 (100). ESI-HRMS calcd for C21H25N4S (MH+),
365.1794; obsd, 365.1794. Chiral purity: 98.87%. Chemical purity:
95.3%.
tert-Butyl Ethyl(4-(5-nitro-1H-indol-3-yl)cyclohex-3-enyl)-
carbamate (( )-11). A solution of compound ( )-10 (1.05 g, 3.68
mmol) in dry 1,4-dioxane (20 mL) was treated with Et3N (1.02 mL,
7.36 mmol) followed by (Boc)2O (0.84 g, 3.863 mmol) at rt, and the
resulting solution was stirred for overnight (14 h). Solvent was
evaporated, and crude was purified by column chromatography (2 M
NH3 in MeOH:CH2Cl2 1:1) to obtain compound ( )-11 (1.1 g, 78%)
as a yellow solid. 1H NMR (DMSO-d6) δ 11.85 (s, 1H), 8.67 (d, 1H, J
= 2.1 Hz), 8.01 (dd, 1H, J = 2.1, 8.7 Hz), 7.64 (s, 1H), 7.55 (d, 1H, J =
9.0 Hz), 6.16 (s, 1H), 4.05 (brs, 1H), 3.18−3.14 (m, 2H), 2.62−2.56
(m, 2H), 2.43 −2.27 (m, 2H), 1.96−1.83 (m, 2H), 1.42 (s, 9H), 1.09
(t, 3H, J = 6.9 Hz). ESI-MS (m/z, %) 408 (M + Na, 95), 386 (MH+,
9), 330 (73), 286 (100).
3-(4-(Dimethylamino)cyclohex-1-enyl)-1H-indol-5-amine
(( )-12). A solution of compound ( )-7 (0.21 g, 0.74 mmol) in dry
MeOH (5 mL) was treated with Raney nickel (0.05 g) followed by
hydrazine hydrate (0.22 mL, 7.36 mmol) at rt. The reaction was placed
in a preheated oil bath and refluxed for 5 min. The reaction brought to
room temperature, filtered through a Celite bed, and washed with
methanol (2 × 10 mL). The solvent was evaporated, and crude was
purified by column chromatography (2 M NH3 in MeOH:CH2Cl2
1:9) to obtain compound ( )-12 (0.185 g, quantitative) as a foam. 1H
NMR (DMSO-d6) δ 10.55 (s, 1H), 7.13 (d, 1H, J = 2.4 Hz), 7.04 (d,
1H, J = 8.7 Hz), 6.99 (d, 1H, J = 0.9 Hz), 6.47 (dd, 1H, J = 1.8, 8.4
Hz), 5.99 (brs, 1H), 4.47 (s, 2H), 2.57−2.08 (m, 11H), 2.02−1.97 (m,
1H), 1.52−1.40 (m, 1H). ESI-MS (m/z, %) 256 (MH+, 100), 211
(41).
Second Enantimer (+)-16). ESI-MS (m/z, %) 365 (MH+, 34), 320
(46), 160 (89), 119 (100). ESI-HRMS calcd for C21H25N4S (MH+),
365.1794; obsd, 365.1795. Chiral purity: 99.38%. Chemical purity:
95%.
tert-Butyl Methyl(4-(5-(thiophene-2-carboximidamido)-1H-indol-
3-yl)cyclohex-3-enyl)carbamate (( )-17). A solution of compound
( )-13 (0.42 g, 1.22 mmol) in dry EtOH (20 mL) was treated with
compound 15 (0.69 g, 2.43 mmol) at room temperature, and the
resulting solution was stirred for 24 h. Same workup as in ( )-16. The
crude was purified by column chromatography (2 M NH3 in
MeOH:CH2Cl2 5:95) to obtain compound ( )-17 (0.37 g, 68%) as
foam. 1H NMR (DMSO-d6) δ 10.94 (s, 1H), 7.72 (d, 1H, J = 3.3 Hz),
7.60 (d, 1H, J = 4.8 Hz), 7.32−7.25 (m, 2H), 7.22 (s, 1H), 7.10 (t, 1H,
J = 4.2 Hz), 6.66 (d, 1H, J = 8.4 Hz), 6.28 (brs, 1H), 6.06 (s, 1H),
4.16−4.06 (m, 1H), 2.72 (s, 3H), 2.40−2.22 (m, 2H), 1.87−1.77 (m,
2H), 1.40 (s, 9H), 1.26−1.20 (m, 1H), 0.85 (t, 1H, J = 7.2 Hz). ESI-
MS (m/z, %) 451 (MH+, 100).
N-(3-(4-(Methylamino)cyclohex-1-enyl)-1H-indol-5-yl)thiophene-
2-carboximidamide (( )-18). A solution of compound ( )-17 (0.35
g, 0.78 mmol) was treated with 20% TFA in CH2Cl2 (20 mL) at 0 °C,
and stirring was continued for 1 h at same temperature. Solvent was
evaporated, crude was diluted with 10% aq NH3 (15 mL), and product
was extracted into CH2Cl2 (3 × 20 mL). The combined CH2Cl2 layer
was washed with brine (10 mL) and dried (Na2SO4). Solvent was
evaporated, and crude was purified by column chromatography (2 M
NH3 in MeOH:CH2Cl2 1:9) to obtain compound ( )-18 (0.2 g, 74%)
as a solid. 1H NMR (DMSO-d6) δ 10.87 (s, 1H), 7.71 (d, 1H, J = 3.3
Hz), 7.59 (d, 1H, J = 4.2 Hz), 7.31−7.28 (m, 2H), 7.20 (s, 1H), 7.09
(dd, 1H, J = 4.2, 4.9 Hz), 6.65 (dd, 1H, J = 1.5, 8.2 Hz), 6.19 (brs,
2H), 6.01 (s, 1H), 2.61−2.57 (m, 1H), 2.46−2.40 (m, 1H), 2.33 (s,
3H), 1.96−1.88 (m, 3H), 1.47−1.39 (m, 2H). ESI-MS (m/z, %) 351
(MH+, 66), 320 (54), 160 (63), 119 (100). ESI-HRMS calcd for
C20H23N4S (MH+), 351.1654; obsd, 351.1637. HPLC purity 84.6%.
tert-Butyl Ethyl(4-(5-(thiophene-2-carboximidamido)-1H-indol-3-
yl)cyclohex-3-enyl)carbamate (( )-19). A solution of compound
( )-14 (0.44 g, 1.24 mmol) in dry EtOH (20 mL) was treated with
compound 15 (0.7 g, 2.48 mmol) at rt and stirred for 24 h. Same
workup as in ( )-16. The crude was purified by column
chromatography (2 M NH3 in MeOH:CH2Cl2 5:95) to obtain
compound ( )-19 (0.49 g, 85%) as a yellow solid. 1H NMR (DMSO-
d6) δ 10.92 (s, 1H), 7.72−7.70 (m, 1H), 7.59 (d, 1H, J = 5.1 Hz), 7.
36−7.27 (m, 2H), 7.20 (s, 1H), 7.09 (t, 1H, J = 4.2 Hz), 6.66 (d, 1H, J
= 7.8 Hz), 6.22 (s, 2H), 6.04 (brs, 1H), 4.02 (brs, 1H), 3.17−3.13 (m,
tert-Butyl 4-(5-Amino-1H-indol-3-yl)cyclohex-3-enyl(methyl)-
carbamate (( )-13). A solution of compound ( )-9 (0.5 g, 1.35
mmol) in dry MeOH (20 mL) was treated with hydrazine hydrate
(0.41 mL, 13.46 mmol) followed by Raney nickel (0.1 g), and the
resulting mixture was refluxed for 30 min. Same workup as in ( )-12.
The crude was purified by column chromatography (EtOAc:hexanes
1
1:1) to obtain compound ( )-13 (0.43 g, 94%) as a foam. H NMR
(DMSO-d6) δ 10.60 (s, 1H), 7.16 (d, 1H, J = 2.7 Hz), 7.05 (d, 1H, J =
8.4 Hz), 6.99 (d, 1H, J = 1.5 Hz), 6.48 (dd, 1H, J = 1.8, 8.2 Hz), 6.00
(brs, 1H), 4.49 (s, 2H), 4.15−4.05 (m, 1H), 2.73 (s, 3H), 2.42−2.14
(m, 2H), 1.86−1.76 (m, 2H), 1.41−1.38 (m, 11H). ESI-MS (m/z, %)
364 (MNa+, 7), 342 (MH+, 11), 286 (100).
tert-Butyl 4-(5-Amino-1H-indol-3-yl)cyclohex-3-enyl(ethyl)-
carbamate (( )-14). A solution of compound ( )-11 (0.5 g, 1.30
mmol) in dry MeOH (10 mL) was treated with Raney nickel (0.05 g)
followed by hydrazine hydrate (0.4 mL, 12.971 mmol) at rt. The
reaction was placed in a preheated oil bath and refluxed for 5 min.
Same workup as in ( )-12. The crude was purified by column
chromatography (2 M NH3 in MeOH:CH2Cl2 5:95) to obtain
compound ( )-14 (0.46 g, quantitative) as a foam. 1H NMR (DMSO-
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2H), 2.58 −2.26 (m, 4H), 1.92−1.82 (m, 2H), 1.41 (s, 9H), 1.07 (t,
3H, J = 6.9 Hz). ESI-MS (m/z, %) 465 (MH+, 100).
2H). ESI-MS (m/z, %) 367 (MH+, 100) 322 (70). ESI-HRMS
(MH+) calcd for C21H27N4S, 367.1950; found, 367.1940. HPLC
purity 99.3%.
N-(3-(4-(Ethylamino)cyclohex-1-enyl)-1H-indol-5-yl)thiophene-2-
carboximidamide (( )-20). A solution of compound ( )-19 (0.3 g,
0.645 mmol) was treated with 20% TFA in CH2Cl2 (20 mL) at 0 °C,
and stirring was continued for 2 h at same temperature. Same workup
as in ( )-18. The crude was purified by column chromatography (2 M
NH3 in MeOH:CH2Cl2 1:9) to obtain compound ( )-20 (0.125 g,
53%) as a solid. 1H NMR (DMSO-d6) δ 10.87 (s, 1H), 7.70 (d, 1H, J
= 2.7 Hz), 7.58 (d, 1H, J = 5.1 Hz), 7.31−7.26 (m, 2H), 7.19 (s, 1H),
7.09 (dd, 1H, J = 3.9, 4.9 Hz), 6.64 (d, 1H, J = 8.4 Hz), 6.19 (s, 2H),
6.01 (s, 1H), 2.74−2.40 (m, 5H), 1.96 −1.88 (m, 2H), 1.48−1.37 (m,
2H), 1.02 (t, 3H, J = 7.2 Hz). ESI-MS (m/z, %) 365 (MH+, 22), 320
(44), 160 (66), 127 (41), 119 (100). ESI-HRMS calcd for C21H25N4S
(MH+), 365.1794; obsd, 365.1811. HPLC purity 94.2%.
tert-Butyl 4-(5-Amino-1H-indol-3-yl)cyclohex-3-enyl(methyl)-
carbamate (22). A solution of compound ( )-9 (0.5 g, 1.36 mmol)
in 2 M NH3 in MeOH (20 mL) was treated with Pd−C (0.05 g) and
flushed with hydrogen gas. The reaction was stirred at rt for overnight
(16 h) under hydrogen atm (balloon pressure). The solution was
filtered using Celite bed and washed with CH2Cl2:MeOH (1:1, 3 × 20
mL). The solvent was evaporated, and crude was purified by column
chromatography (EtOAc:hexanes 1:1) to obtain compound 22 (0.46
g, quantitative) as a solid in 1:2 ratio of diastereomers. 1H NMR
(DMSO-d6) δ 10.28, 10.23 (2s, 1H), 6.87−6.85, 7.06−6.99 (2 m, 2H),
6.68−6.66 (m, 1H), 6.50−6.42 (m, 1H), 4.41 (brs, 2H), 3.85−3.82
(m, 1H), 2.72−2.60 (2s, 3H), 2.57−2.53 (m, 1H), 2.17−2.02 (m, 2H),
1.84−1.46 (m, 6H), 1.41, 1.38 (2s, 9H). ESI-MS (m/z, %) 366 (M +
Na+, 8), 344 (MH+, 10), 288 (100).
trans-24. 1H NMR (CDCl3) δ 8.00 (brs, 1H), 7.46−7.43 (m, 2H),
7.34 (d, 1H, J = 12.7 Hz), 7.28−7.26 (m, 1H), 7.11 (dd, 1H, J = 5.8,
7.3 Hz), 6.96 (d, 1H, J = 3.3 Hz), 6.89 (dd, 1H, J = 2.3, 12.7 Hz), 4.88
(brs, 1H), 2.79−2.72 (m, 1H), 2.35 (s, 6H), 2.33−2.27 (m, 1H),
2.22−2.19 (m, 2H), 2.05−2.02 (m, 2H), 1.60−1.58 (m, 2H), 1.48−
1.38 (m, 2H). ESI-MS (m/z, %) 367 (MH+, 100) 322 (75). ESI-
HRMS (MH+) calcd for C21H27N4S, 367.1950; found, 367.1951.
HPLC purity 95.3%.
tert-Butyl Methyl(4-(5-(thiophene-2-carboximidamido)-1H-indol-
3-yl)cyclohexyl)carbamate (25). A solution of compound 22 (0.44 g,
1.28 mmol) in dry EtOH (20 mL) was treated with compound 15
(0.73 g, 2.56 mmol) at rt and stirred for 24 h. Same workup as in
( )-16. The crude was purified by column chromatography (2 M NH3
in MeOH:CH2Cl2 5:95) to obtain compound 25 (0.425 g, 73%) as a
1
foam in 1:2 ratio of diastereomers. H NMR (DMSO-d6) δ 10.63,
10.59 (2s, 1H), 7.71 (d, 1H, J = 3.6 Hz), 7.59 (d, 1H, J = 5.1 Hz),
7.29−7.22 (m, 1H), 7.11−6.95 (m, 3H), 6.66−6.62 (m, 1H), 6.27
(brs, 1H), 3.90−3.80 (m, 1H), 2.70−2.62 (m, 4H), 2.18−2.06 (m,
2H), 1.82−1.64 (m, 4H), 1.56−1.38 (m, 11H). ESI-MS (m/z, %) 453
(MH+, 100).
N-(3-(4-(Methylamino)cyclohexyl)-1H-indol-5-yl)thiophene-2-
carboximidamide (26). Compound 25 (0.2 g, 0.44 mmol) was treated
with 1 N HCl solution at room temperature, and the resulting solution
was refluxed for 2 h. The reaction was brought to room temperature,
filtered, and washed with water (5 mL). The solvent was evaporated,
and crude was recrystallized from ethanol/ether to obtain compound
26 (0.18 g, 94%) as HCl salt in 1:2 ratio of diastereomers.
Compound 26 was separated (as a free base) using normal phase
HPLC semipreparative column chromatography.
tert-Butyl 4-(5-Amino-1H-indol-3-yl)cyclohexyl(ethyl)carbamate
(23). A solution of compound ( )-11 (0.55 g, 1.43 mmol) in 2 M
NH3 in MeOH (10 mL) was treated with Pd−C (0.05 g) and flushed
with hydrogen gas. The reaction was stirred at rt for overnight (16 h)
under hydrogen atm (balloon pressure). Same workup as in 22. The
crude was purified by column chromatography (2 M NH3 in
MeOH:CH2Cl2 2.5:97.5) to obtain compound 23 (0.43 g, 84%) as
1
cis-26 (Less Polar Isomer). H NMR (DMSO-d6) δ 10.52 (s, 1H),
7.70 (d, 1H, J = 2.7 Hz), 7.58 (d, 1H, J = 5.4 Hz), 7.25 (d, 1H, J = 8.4
Hz), 7.09 (dd, 1H, J = 3.9, 5.1 Hz), 6.96−7.00 (m, 2H), 6.61 (d, 1H, J
= 8.4 Hz), 6.18 (brs, 2H), 2.80−2.73 (m, 1H), 2.64−2.59 (m, 1H),
2.27 (s, 3H), 1.96−1.53 (m, 6H), 1.13−1.08 (m, 1H), 1.01−0.94 (m,
1H), 0.91−0.81 (m, 1H). ESI-MS (m/z, %) 353 (MH+ for free base,
30) 322 (100), 119 (51). ESI-HRMS calcd for C20H25N4S (MH+ for
free base), 353.1794; obsd, 353.1777. HPLC purity 97%.
1
a solid in 2:3 ratio of diastereomers. H NMR (DMSO-d6) δ 10.27,
10.23 (2s, 1H), 6.88−6.86, 7.06−6.99 (2 m, 2H), 6.70−6.66 (m, 1H),
6.47−6.44 (m, 1H), 4.52 (brs, 2H), 3.80−3.68 (m, 1H), 3.17−3.11
(m, 2H), 3.04−2.98 (m, 1H), 2.18−2.01 (m, 2H), 1.78−1.63 (m, 4H),
1.51−1.37 (m, 11H), 1.07, 0.99 (2t, 3H, J = 7.2, 6.6 Hz). ESI-MS (m/
z, %) 380 (M + Na, 6), 358 (MH+, 5), 302 (100), 258 (54). ESI-
HRMS calcd for C21H32N3O2 (MH+), 358.2507; obsd, 358.2489.
N-(3-(4-(Dimethylamino)cyclohexyl)-1H-indol-5-yl)thiophene-2-
carboximidamide (24). A solution of compound ( )-7 (0.43 g, 1.51
mmol) in dry EtOH (5 mL) was treated with Pd−C (0.04 g) and
purged with hydrogen gas at rt. The reaction was stirred at same
temperature under hydrogen atm (balloon pressure) for overnight (14
h). The reaction was filtered using Celite bed and washed with dry
EtOH (2 × 5 mL). The combined EtOH layer was treated with
compound 15 (0.85 g, 3.01 mmol) at room temperature and stirred
for 24 h. Solvent was evaporated, crude was diluted with satd NaHCO3
solution (20 mL), and product was extracted into CH2Cl2 (2 × 25
mL). The combined CH2Cl2 layer was washed with brine (20 mL) and
dried (Na2SO4). Solvent was evaporated, and crude was purified by
column chromatography (2 M NH3 in MeOH:CH2Cl2 1:9) to obtain
compound 24 (0.4 g, 72%, over two steps) as a yellow solid. 1H NMR
(DMSO-d6) δ 10.57 (s, 1H), 7.71 (d, 1H, J = 3.3 Hz), 7.60 (d, 1H, J =
5.4 Hz), 7.27 (d, 1H, J = 8.4 Hz), 7.10 (t, 1H, J = 4.2 Hz), 7.05−6.99
(m, 2H), 6.64 (dd, 1H, J = 1.5, 8.4 Hz), 6.48 (brs, 1H), 2.96−2.91 (m,
1H), 2.71−2.64 (m, 1H), 2.34 (s, 3H), 2.23 (s, 3H), 2.08−2.05 (m,
1H), 1.94−1.82 (m, 3H), 1.72−1.66 (m, 1H), 1.60−1.39 (m, 3H).
ESI-MS (m/z, %) 367 (MH+, 31), 322 (18), 184 (100). ESI-HRMS
calcd for C21H27N4S (MH+), 367.1965; obsd, 367.1950.
1
trans-26 (More Polar Isomer). H NMR (DMSO-d6) δ 10.54 (s,
1H), 7.70 (d, 1H, J = 2.7 Hz), 7.58 (d, 1H, J = 5.4 Hz), 7.25 (d, 1H, J
= 8.4 Hz), 7.09 (t, 1H, J = 4.5 Hz), 6.99−6.95 (m, 2H), 6.61 (dd, 1H, J
= 1.2, 8.2 Hz), 6.18 (brs, 2H), 2.71−2.61 (m, 1H), 2.35−2.24 (m,
4H), 2.02−1.90 (m, 3H), 1.52−1.40 (m, 1H), 1.28−1.08 (m, 3H),
1.01−0.94 (m, 1H), 0.91−0.81 (m, 1H). ESI-MS (m/z, %) 353 (MH+
for free base, 28) 322 (100), 119 (47). ESI-HRMS calcd for
C20H25N4S (MH+ for free base), 353.1794; obsd, 353.1799. HPLC
purity 92%.
tert-Butyl Ethyl(4-(5-(thiophene-2-carboximidamido)-1H-indol-3-
yl)cyclohexyl)carbamate (27). A solution of compound 23 (0.4 g,
1.12 mmol) in dry EtOH (20 mL) was treated with compound 15
(0.63 g, 2.24 mmol) at rt and stirred for 24 h. Same workup as in
( )-16. The crude was purified by column chromatography (2 M NH3
in MeOH:CH2Cl2 5:95) to obtain compound 27 (0.4 g, 60%) as a
1
yellow solid in 2:3 ratio of diastereomers. H NMR (DMSO-d6) δ
10.62, 10.59 (2s, 1H), 7.72−7.70 (m, 1H), 1.08−0.98 (m, 3H), 7.60
(d, 1H, J = 5.1 Hz), 7.30−7.22 (m, 2H), 7.11−7.09 (m, 1H), 7.01−
6.96 (m, 1H), 6.67−6.62 (m, 1H), 6.31 (brs, 2H), 3.76−3.70 (m, 1H),
3.17 −3. 02 (m, 3H), 2.18−2.05 (m, 2H), 1.85−1.68 (m, 4H), 1.56
−1.38 (m, 11H). ESI-MS (m/z, %) 467 (MH+, 100).
N-(3-(4-(Ethylamino)cyclohexyl)-1H-indol-5-yl)thiophene-2-car-
boximidamide (28). Compound 27 (0.26 g, 0.557 mmol) was treated
with 1 N aqueous HCl solution at rt, and the resulting solution was
refluxed for 2 h. Same procedure as for compound 26. The crude was
recrystallized from ethanol/ether to obtain compound 28 (0.23 g,
94%) as a solid in 2:3 ratio of diastereomers. Compound 28 was
separated (as a free base) using reverse phase semipreparative column
on HPLC.
Compound 24 Was Separated by Preparative SFC. cis-24. 1H
NMR (CDCl3) δ 8.29 (brs, 1H), 7.45−743 (m, 2H), 7.33 (d,
1H, J = 12.6 Hz), 7.26 (s, 1H), 7.10 (dd, 1H, J = 5.8, 7.3 Hz),
7.06 (d, 1H, J = 2.8 Hz), 6.88 (dd, 1H, J = 2.4, 12.6 Hz), 4.88
(brs, 1H), 3.11−3.06 (m, 1H), 2.31 (s, 6H), 2.19−2.16 (m,
1H), 2.07−1.98 (m, 2H), 1.89−1.81 (m, 4H), 1.71−1.64 (m,
3496
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1
cis-28. H NMR (CD3OD) δ 7.61 (dd, 1H, J = 1.2, 3.9 Hz), 7.54
was added AcOH (0.28 mL, 4.65 mmol), EtNH2·HCl (0.38 g, 4.65
mmol), and NaBH(OAc)3 (1.50 g, 7.00 mmol) and the mixture left to
stir overnight at rt. Same workup as in ( )-7. The crude was purified
by column chromatography (2 M NH3 in MeOH:CH2Cl2 1:9) to
obtain two diastereomers as yellow solids.
(d, 1H, J = 5.4 Hz), 7.34 (d, 1H, J = 8.4 Hz), 7.16 (d, 1H, J = 1.5 Hz),
7.12 (d, 1H, J = 3.9 Hz), 7.10 (brs, 1H), 6.78 (dd, 1H, J = 1.8, 8.4 Hz),
2.79−2.74 (m, 1H), 3.06−3.00 (m, 1H), 1.98−1.81 (m, 4H), 2.65 (q,
2H), 1.75−1.71 (m, 4H), 1.13 (t, 3H, J = 7.5 Hz). ESI-MS (m/z, %)
367 (MH+ for free base, 18), 322 (100), 184 (19), 119 (39). ESI-
HRMS calcd for C21H27N4S (MH+, free base), 367.1959; obsd,
367.1950. HPLC purity 93.5%.
1
trans-( )-32 (Less Polar Diastereomer): (0.70 g, 52%). H NMR
(CDCl3) δ 8.64 (d, 1H, J = 2.1 Hz), 8.34 (s, 1H, NH), 8.09 (dd, 1H, J
= 2.1, 9.0 Hz), 7.37 (d, 1H, J = 9.0 Hz), 7.12 (d, 1H, J = 2.1 Hz),
3.42−3.24 (m, 1H), 3.06−3.01 (m, 1H), 2.70 (q, 2H, J = 7.2, 7.2 Hz),
2.07−2.01 (m, 2H), 1.82−1.74 (m, 2H), 1.70−1.55 (m, 4H), 1.17 (t,
3H, J = 8.4 Hz). EI-MS (m/z, %) 287 (M+, 10), 242 (100).
1
trans-28. H NMR (CD3OD) δ 7.62 (d, 1H, J = 3.6 Hz), 7.54 (d,
1H, J = 5.4 Hz), 7.34 (d, 1H, J = 8.4 Hz), 7.15 (d, 1H, J = 1.2 Hz),
7.12 (t, 1H, J = 4.2 Hz), 6.98 (s, 1H), 6.77 (dd, 1H, J = 1.5, 8.5 Hz),
2.81−2.64 (m, 3H), 2.59−2.52 (m, 1H), 2.10 (t, 4H, J = 13.8 Hz),
1.63−1.51 (m, 2H), 1.38−1.28 (m, 2H), 1.13 (t, 3H, J = 7.2 Hz). ESI-
MS (m/z, %) 367 (MH+ for free base, 18), 322 (100), 184 (19), 119
(39). ESI-HRMS calcd for C21H27N4S (MH+, free base), 367.1959;
obsd, 367.1950. HPLC purity 98.5%.
3-(5-Nitro-1H-indol-3-yl)cyclohexanone (( )-29). To a solution of
4 (4.00 g, 25.61 mmol) in dry MeCN (5.00 mL) was added cyclohex-
2-enone (7.40 mL, 76.83 mmol) and Bi(NO3)3 (0.12 g, 0.26 mmol)
and the mixture stirred overnight at rt The solvent then was
evaporated, and the crude was purified by column chromatography
(hexane:EtOAc 1:1) to obtain compound ( )-29 (2.70 g, 41%) as a
1
cis-( )-32 (More Polar Diastereomer): (0.21 g, 16%). H NMR
(CDCl3) δ 8.61 (d, 1H, J = 2.1 Hz), 8.37 (s, 1H, NH), 8.10 (dd, 1H, J
= 2.1, 9.0 Hz), 7.37 (d, 1H, J = 9.0 Hz), 7.10 (d, 1H, J = 1.8 Hz),
3.00−2.89 (m, 1H), 2.75 (q, 2H, J = 7.2 Hz), 2.32−2.28 (m, 1H),
2.11−2.04 (m, 2H), 1.97−1.84 (m, 1H), 1.63−1.47 (m, 2H), 1.44−
1.29 (m, 3H), 1.14 (t, 3H, J = 7.1 Hz). EI-MS (m/z, %) 287 (M+, 15),
244 (100).
The stereochemistry of both diastereomers was determined using
COSY and NOESY spectroscopic techniques (see Supporting
Information).
tert-Butyl Ethyl(3-(5-nitro-1H-indol-3-yl)cyclohexyl)carbamate
(trans-( )-33). To a solution of trans-( )-32 (0.67 g, 2.36 mmol)
in 1,4-dioxane (10 mL) was added (Boc)2O (0.57 g, 2.60 mmol) and
triethyl amine (0.66 mL, 4.74 mmol) and the resulting mixture left to
stir overnight at rt. The solvent was evaporated and the crude purified
on column chromatography (EtOAc:hexane 1:1) to obtain trans-
1
yellow solid. H NMR (CDCl3) δ 8.59 (d, 1H, J = 2.1 Hz), 8.51 (s,
1H, NH), 8.12 (dd, 1H, J = 2.1, 9.0 Hz), 7.41 (d, 1H, J = 9.0 Hz), 7.15
(d, 1H, J = 2.1 Hz), 3.56 −3.47 (m, 1H), 2.85−2.77 (m, 1H), 2.65 (t,
1H, J = 9.9 Hz), 2.55−2.37 (m, 2H), 2.34−2.26 (m, 1H), 2.09−1.81
(m, 3H). EI-MS (m/z, %) 258 (M+, 100).
N-Methyl-3-(5-nitro-1H-indol-3-yl)cyclohexanamine (( )-30). To
a solution of ( )-29 (1.20 g, 4.65 mmol) in 1,2-dichloroethane (50
mL) was added AcOH (0.28 mL, 4.65 mmol), MeNH2·HCl (0.38 g,
4.65 mmol), and NaBH(OAc)3 (1.50 g, 7.00 mmol) and the mixture
left to stir overnight at rt. Same workup as in ( )-7. The crude was
purified by column chromatography (2 M NH3 in MeOH:CH2Cl2
1:9) to obtain two diastereomers as yellow solids.
1
( )-33 as a yellow solid (0.72 g, 78%). H NMR (CDCl3) δ 8.57 (d,
1H, J = 2.1 Hz), 8.08 (dd, 1H, J = 2.1, 9.0 Hz), 7.63 (s, 1H, NH), 7.35
(d, 1H, J = 9.0 Hz), 7.26 (s, 1H), 3.61−3.57 (m, 1H), 3.28−3.07 (m,
2H), 2.17−2.07 (m, 2H), 1.96−1.86 (m, 1H), 1.79−1.62 (m, 3H),
1.49−1.45 (s, m, 9H, 3H), 1.14 (t, 3H, J = 6.9 Hz). ESI-MS (m/z, %)
410 (NaM+, 50), 288 (100).
tert-Butyl Ethyl(3-(5-nitro-1H-indol-3-yl)cyclohexyl)carbamate
(cis-( )-33). Same procedure was used as for trans-( )-33 (0.26 g,
97%). 1H NMR (DMSO-d6) δ 8.55 (d, 1H, J = 2.1 Hz), 7.97 (dd, 1H,
J = 2.1, 9.0 Hz), 7.50 (d, 1H, J = 9.0 Hz), 7.39 (s, 1H), 3.14 (q, 2H, J =
6.9 Hz), 3.04−2.96 (m, 1H), 1.95−1.86 (m, 2H), 1.75−1.64 (m, 2H),
1.57−1.51 (m, 2H), 1.42 (s, 9H), 1.49−1.23 (m, 2H), 1.04 (t, 3H, J =
6.9 Hz). EI-MS (m/z, %) 387 (M+, 20), 270 (100).
tert-Butyl 3-(5-Amino-1H-indol-3-yl)cyclohexyl(methyl)-
carbamate (trans-( )-34). To a solution of trans-( )-31 (0.70, g
1.87 mmol) in dry MeOH (15 mL) was added Raney nickel (0.1 g
50% as a slurry in water) and hydrazine hydrate (1.00 mL, 18.70
mmol). The resulting mixture was immersed in a preheated oil bath
and refluxed for 15 min or until the solution became clear. Same
workup as in ( )-12. The crude was purified on column
chromatography (2 M NH3 in MeOH:CH2Cl2 2:98) to give trans-
( )-34 as a light-brown solid (0.60 g, 92%). 1H NMR (CDCl3) δ 7.76
(s, 1H, NH), 7.28 (s, 1H), 7.16 (d, 1H, J = 8.4 Hz), 6.89 (d, 1H, J =
2.1 Hz), 6.64 (dd, 1H, J = 2.1, 8.4 Hz), 4.51−4.36 (m, 1H), 3.50 (s,
2H, NH), 2.76 (s, 3H), 2.16−2.05 (m, 2H), 1.88 (ddd, 1H, J = 5.4,
12.4, 25.1 Hz), 1.72−1.46 (m, 6H), 1.42 (s, 9H). EI-MS (m/z, %) 343
(M+, 70), 212 (100).
1
trans-( )-30 (Less Polar Diastereomer): (0.58 g, 46%). H NMR
(CDCl3) δ 8.63 (d, 1H, J = 2.1 Hz), 8.44 (s, 1H, NH), 8.09 (dd, 1H, J
= 2.1, 9.0 Hz), 7.36 (d, 1H, J = 9.0 Hz), 7.12 (s, 1H), 3.37−3.26 (m,
1H), 2.97−2.87 (m, 1H), 2.41 (s, 3H), 2.08−2.04 (m, 2H), 1.88−1.69
(m, 3H), 1.65−1.49 (m, 3H). EI-MS (m/z, %) 273 (M+, 10), 242
(100).
1
cis-( )-30 (More Polar Diastereomer): (0.21 g, 16%). H NMR
(CDCl3) δ 8.93 (s, 1H, NH), 8.54 (d, 1H, J = 2.4 Hz), 8.06 (dd, 1H, J
= 2.1, 9.0 Hz), 7.35 (d, 1H, J = 9.0 Hz), 7.06 (s, 1H), 2.93−2.75 (m,
2H), 2.56 (s, 3H), 2.44−2.33 (m, 1H), 2.17−2.13 (m, 1H), 2.08−2.01
(m, 1H), 1.99−1.85 (m, 1H), 1.57−1.45 (m, 2H), 1.38−1.26 (m, 2H).
EI-MS (m/z, %) 273 (M+, 30), 230 (100).
The stereochemistry of both diastereomers was determined using
COSY and NOESY spectroscopic techniques (see supporting info).
tert-Butyl Methyl(3-(5-nitro-1H-indol-3-yl)cyclohexyl)carbamate
(trans-( )-31). To a solution of trans-( )-30 (0.55 g, 2.0 mmol) in
1,4-dioxane (10 mL) was added (Boc)2O (0.48 g, 2.21 mmol) and
triethyl amine (0.56 mL, 4.10 mmol) and the resulting mixture left to
stir overnight at rt. The solvent was evaporated and the crude purified
on column chromatography (hexane:EtOAc 1:1) to give trans-( )-31
1
as a yellow solid (0.73 g, quantitative). H NMR (CDCl3) δ 8.57 (d,
tert-Butyl 3-(5-Amino-1H-indol-3-yl)cyclohexyl(methyl)-
carbamate (cis-( )-34). Same procedure as for trans-( )-34 (0.34
1H, J = 2.1 Hz), 8.50 (s, 1H, NH), 8.08 (dd, 1H, J = 2.1, 9.0 Hz), 7.35
(d, 1H, J = 9.0 Hz), 7.26 (s, 1H), 4.52−4.35 (m, 1H), 3.63−3.57 (m,
1H), 2.78 (s, 3H), 2.18−2.09 (m, 2H), 1.98−1.86 (m, 1H), 1.81−1.64
(m, 3H), 1.57−1.49 (m, 2H), 1.43 (s, 9H). EI-MS (m/z, %) 299 (M+,
100).
1
g, 97%). H NMR (CDCl3) δ 7.72 (s, 1H, NH), 7.15 (d, 1H, J = 8.4
Hz), 6.95 (s, 1H), 6.88 (d, 1H, J = 2.4 Hz), 6.65 (dd, 1H, J = 2.1, 8.4
Hz), 4.26−4.13 (m, 1H), 3.52 (s, 2H, NH), 2.93−2.84 (m, 1H), 2.74
(s, 3H), 2.11−2.03 (m, 2H), 1.96−1.89 (m, 1H), 1.80−1.75 (m, 1H),
1.66−1.31 (m, 4H), 1.48 (s, 9H). EI-MS (m/z, %), 343 (M+, 100).
tert-Butyl 3-(5-Amino-1H-indol-3-yl)cyclohexyl(ethyl)carbamate
(trans-( )-35). To a solution of trans-( )-33 (0.70, g 1.81 mmol)
in dry MeOH (15 mL) was added Raney nickel (0.1 g 50% as a slurry
in water) and hydrazine hydrate (0.90 mL, 18.10 mmol). The resulting
mixture was immersed in a preheated oil bath and refluxed for 15 min
or until the solution became clear. Same workup as for ( )-12 . The
crude was purified on column chromatography (2 M NH3 in
MeOH:CH2Cl2 2:98) to give trans-( )-35 as a brownish solid (0.64
tert-Butyl Methyl(3-(5-nitro-1H-indol-3-yl)cyclohexyl)carbamate
(cis-( )-31). Same procedure as for trans-( )-31. (0.40 g, 73%). H
1
NMR (CDCl3) δ8.61 (d, 1H, J = 2.1 Hz), 8.37 (s, 1H, NH), 8.10 (dd,
1H, J = 2.1, 9.0 Hz), 7.38 (d, 1H, J = 9.0 Hz), 7.11 (d, 1H, J = 1.8 Hz),
4.27−3.96 (m, 1H), 3.06−2.95 (m, 1H), 2.78 (s, 3H), 2.10−2.03 (m,
2H), 2.00−1.92 (m, 1H), 1.86−1.78 (m, 1H), 1.69−1.57 (m, 3H),
1.49 (s, 9H), 1.44−1.34 (m, 1H). EI-MS (m/z, %), 373 (M+, 20), 242
(100).
N-Ethyl-3-(5-nitro-1H-indol-3-yl)cyclohexanamine (( )-32). To a
solution of ( )-29 (1.20 g, 4.65 mmol) in 1,2-dichloroethane (50 mL)
1
g, quantitative). H NMR (CDCl3) δ 7.82 (s, 1H, NH), 7.33 (s, 1H),
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7.26 (s, 1H), 7.15 (d, 1H, J = 8.4 Hz), 6.89 (d, 1H, J = 2.1 Hz), 6.64
(dd, 1H, J = 2.1, 8.4 Hz), 4.43 (s, 1H), 3.56−3.43 (m, 1H), 3.24−3.06
(m, 2H), 2.17−2.07 (m, 2H), 1.92−1.82 (m, 1H), 1.79−1.71 (m 1H),
1.69−1.53 (m, 3H), 1.45 (s, 9H), 1.12 (t, 3H, J = 6.8 Hz). EI-MS (m/
z, %) 357 (M+, 70), 212 (100).
( )-35 (0.62 g, 1.73 mmol) in dry EtOH (25 mL) was added 15 (1.00
g, 3.47 mmol) and the reaction left to stir at rt for 48 h. Same workup
as for ( )-16. The crude was purified on column chromatography (2
M NH3 in MeOH:CH2Cl2 2:98 then 5:95) to give trans-( )-38 as a
1
yellow solid (0.80 g, quantitative). H NMR (DMSO-d6) δ 10.67 (s,
tert-Butyl 3-(5-Amino-1H-indol-3-yl)cyclohexyl(ethyl)carbamate
1H, NH), 7.70 (d, 1H, J = 3.9 Hz), 7.58 (d, 1H, J = 5.1 Hz), 7.29 (s,
1H), 7.26 (s, 1H), 7.09 (dd, 1H, J = 3.6, 5.1 Hz), 6.92 (s, 1H), 6.64
(dd, 1H, J = 1.8, 8.4 Hz), 6.21 (s, 2H), 4.26−4.19 (m, 1H), 3.53−3.42
(m, 1H), 3.20−3.05 (m, 2H), 2.04−1.84 (m, 3H), 1.68−1.44 (m, 5H),
1.36 (s, 9H), 1.04 (t, 3H, J = 6.9 Hz). ESI-MS (m/z, %) 467 (MH+,
100).
1
(cis-( )-35). Same procedure as for trans-( )-35 (0.21 g, 96%). H
NMR (CDCl3) δ7.725 (s, 1H), 7.15 (d, 1H, J = 8.7 Hz), 6.96 (s, 1H),
6.87 (d, 1H, J = 2.1 Hz), 6.65 (dd, 1H, J = 2.1, 8.7 Hz), 4.19−4.12 (m,
1H), 3.22−3.05 (m, 2H), 2.90−2.80 (m, 1H), 2.15−2.11 (m, 1H),
2.04−1.98 (m, 1H), 1.94−1.90 (m, 1H), 1.83−1.80 (m, 1H), 1.48 (s,
9H), 1.66−1.30 (m, 3H), 1.09 (t, 3H, J = 6.9 Hz). EI-MS (m/z, %)
357 (M+, 100).
tert-Butyl Ethyl(3-(5-(thiophene-2-carboximidamido)-1H-indol-3-
yl)cyclohexyl)carbamate (cis-( )-38). The same procedure as for
1
trans-( )-38 (0.19 g, 78%). H NMR (DMSO-d6) δ 10.83 (s, 1H,
tert-Butyl Methyl(3-(5-(thiophene-2-carboximidamido)-1H-indol-
3-yl)cyclohexyl)carbamate (trans-( )-36). To a solution of trans-
( )-34 (0.57 g, 1.66 mmol) in dry EtOH (25 mL) was added 15 (0.75
g, 3.32 mmol) and the reaction left to stir at rt for 48 h. Same workup
as for ( )-16. The crude was purified on column chromatography (2
M NH3 in MeOH:CH2Cl2 2:98 then 5:95) to give trans-( )-36 as a
NH), 7.88 (d, 1H, J = 2.1 Hz), 7.84 (d, 1H, J = 3.3 Hz), 7.37 (d, 1H, J
= 8.7 Hz), 7.29 (s, 1H), 7.22 (dd, 1H, J = 4.5, 8.7 Hz), 7.13 (s, 1H),
6.83 (d, 1H, J = 8.4 Hz), 4.03−3.89 (m, 1H), 3.13 (q, 2H, J = 6.0 Hz),
2.89−2.77 (m, 1H), 1.94−1.80 (m, 3H), 1.74−1.57 (m, 2H), 1.56−
1.46 (m, 3H), 1.39 (s, 9H), 1.04 (t, 3H, J = 6.9 Hz). ESI-MS (m/z, %)
467 (MH+, 100).
1
yellow solid (0.62 g, 81%). H NMR (DMSO-d6) δ 10.68 (s, 1H,
NH), 7.70 (d, 1H, J = 3.6 Hz), 7.58 (d, 1H, J = 4.5 Hz), 7.28 (d, 2H, J
= 8.4 Hz), 7.09 (dd, 1H, J = 3.6, 5.1 Hz), 6.93 (s, 1H), 6.64 (dd, 1H, J
= 1.8, 8.4 Hz), 6.22 (s, 2H, NH), 4.27−4.24 (m, 1H), 3.53−3.40 (m,
1H), 2.69 (s, 3H), 2.04−1.98 (m, 1H), 1.93−1.88 (m, 2H), 1.71−1.42
(m, 5H), 1.35 (s, 9H). ESI-MS (m/z, %) 453 (NaM+, 100).
N-(3-(3-(Ethylamino)cyclohexyl)-1H-indol-5-yl)thiophene-2-car-
boximidamide (trans-( )-39). Compound trans-( )-38 (0.75 g, 1.61
mmol) was treated with 20% TFA solution (31 mL) in dichloro-
methane at 0 °C and the mixture left to stir for 2 h at 0 °C. Same
workup as for trans-( )-37. The crude was purified by column
chromatography (2 M NH3 in MeOH:CH2Cl2 1:4) to give trans-
( )-39 as a yellow solid (0.50 g, 85%). 1H NMR (DMSO-d6) δ 10.54
(s, 1H, NH), 7.70 (d, 1H, J = 3.0 Hz), 7.58 (d, 1H, J = 5.1 Hz), 7.26
(d, 1H, J = 8.4 Hz), 7.09 (dd, 1H, J = 3.9, 5.1 Hz), 7.00−6.98 (m, 2H),
6.62 (d, 1H, J = 8.4 Hz), 6.19 (s, 2H, NH), 3.23−3.08 (m, 1H), 2.99−
2.85 (m, 1H), 2.58 (q, 2H, J = 7.2 Hz), 1.97−1.89 (m, 2H), 1.82−1.58
(m, 3H), 1.51−1.44 (m, 3H), 1.05 (t, 3H, J = 6.9 Hz). ESI-MS (m/z,
%) 367 (MH+, 50%), 322 (100). ESI-HRMS (MH+) calcd for
C21H27N4S, 367.1950; found, 367.1956. HPLC purity 91.02%
N-(3-(3-(Ethylamino)cyclohexyl)-1H-indol-5-yl)thiophene-2-car-
boximidamide (cis-( )-39). Same procedure as for trans-( )-39 (0.50
tert-Butyl Methyl(3-(5-(thiophene-2-carboximidamido)-1H-indol-
3-yl)cyclohexyl)carbamate (cis-( )-36). Same procedure as for trans-
1
( )-36 (0.32 g, 75%). H NMR (DMSO-d6) δ 10.59 (s, 1H, NH),
7.70 (d, 1H, J = 3.3 Hz), 7.58 (d, 1H, J = 4.8 Hz), 7.26 (d, 1H, J = 8.4
Hz), 7.09 (dd, 1H, J = 3.6, 4.8 Hz), 7.04 (s, 1H), 6.98 (s, 1H), 6.62
(dd, 1H, J = 1.8, 8.4 Hz), 6.20 (s, 2H, NH), 4.09−3.78 (m, 1H), 2.87−
2.79 (m, 1H), 2.69 (s, 3H), 2.00−1.84 (m, 5H), 1.68−1.46 (m, 5H),
1.38 (s, 9H). ESI-MS (m/z, %) 453 (NaM+, 100).
N-(3-(3-(Methylamino)cyclohexyl)-1H-indol-5-yl)thiophene-2-
carboximidamide (trans-( )-37). Compound trans-( )-36 (0.60 g,
0.13 mmol) was treated with 20% TFA solution (31 mL) in
dichloromethane at 0 °C and the mixture left to stir for 2 h at 0 °C.
The solution then was neutralized with 10% NH4OH and the organic
layer separated and evaporated. The crude was purified by column
chromatography (2 M NH3 in MeOH:CH2Cl2 1:4) to give trans-
( )-37 as a yellow solid (0.45 g, quantitative). 1H NMR (DMSO-d6) δ
10.59 (s, 1H, NH), 7.71 (d, 1H, J = 3.3 Hz), 7.58 (d, 1H, J = 5.1 Hz),
7.09 (dd, 1H, J = 3.6, 4.8 Hz), 7.02 (d, 2H, J = 10.4 Hz), 6.63 (d, 1H, J
= 10.2 Hz), 6.20 (s, br, 2H, NH), 3.51−3.24 (m, 3H), 2.40 (s, 3H),
2.07−1.96 (m, 1H), 1.91−1.83 (m, 1H), 1.77−1.69 (m, 3H), 1.60−
1.51 (m, 3H). ESI-MS (m/z, %) 353 (MH+, 80), 322 (100). ESI-
HRMS (MH+) calcd for C20H25N4S, 353.1794; found, 353.1812.
HPLC purity 98.46%.
1
g, 85%). H NMR (DMSO-d6) δ 10.62 (s, 1H, NH), 7.71 (d, 1H, J =
3.0 Hz), 7.59 (d, 1H, J = 5.1 Hz), 7.28 (d, 1H, J = 8.7 Hz), 7.10 (dd,
1H, J = 3.6, 5.1 Hz), 7.03−7.01 (m, 2H), 6.64 (d, 1H, J = 8.4 Hz), 6.22
(s, 2H, NH), 3.07−2.99 (m, 1H), 2.88−2.82 (m, 3H), 2.37−2.27 (m,
1H), 2.11−1.81 (m, 3H), 1.53−1.21 (m, 4H), 1.11 (t, 3H, J = 6.9 Hz).
ESI-MS (m/z, %) 367 (MH+, 50), 322 (100). ESI-HRMS calcd for
C21H27N4S (MH+), 367.1950; found, 367.1968. HPLC purity 96.69%
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.
N-(3-(3-(Methylamino)cyclohexyl)-1H-indol-5-yl)thiophene-2-
carboximidamide (cis-( )-37). Same procedure as for trans-( )-37
1
(0.22 g, quantitative). H NMR (DMSO-d6) δ, 9.61 (s, 1H), 8.58 (s,
NOS Enzyme Assays. Recombinant human nNOS, eNOS, and
iNOS were produced in Baculovirus-infected Sf9 cells. In a radiometric
method, NOS activity was 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.
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 were carried out at 37 °C for 45 min. The reaction
was stopped by adding 20 μL of ice-cold buffer containing 100 mM
2H, NH), 8.16 (d, 1H, J = 4.5 Hz), 8.12 (d, 1H, J = 3.6 Hz), 7.65 (s,
1H), 7.52 (d, 1H, J = 8.4 Hz), 7.39 (pseudo t, 1H, J = 4.5 Hz), 7.28 (d,
1H, J = 2.1 Hz), 7.10 (d, 1H, J = 8.4 Hz), 3.25−3.08 (m, 1H), 2.94−
2.86 (m, 1H), 2.58 (s, 3H), 2.35−2.27 (m, 1H), 2.11−2.08 (m, 1H),
2.01−1.84 (m, 2H), 1.61−1.28 (m, 4H). ESI-MS (m/z, %) 353 (MH+,
100). ESI-HRMS (MH+) calcd for C20H25N4S, 353.1794; found,
353.1792.
Compound cis-( )-37 was separated into its enantiomers on a
chiral HPLC.
cis-(+)-37 (less polar enantiomer). [α]D = +23.77 (4.50 mg in 2 mL
MeOH). ESI-MS (m/z, %) 353 (MH+, 50), 322 (100). ESI-HRMS
calcd for C16H25N4O5 (MH+), 353.1819; found, 353.1807. ee 100% by
HPLC. HPLC purity 99.2%.
trans-(−)-37 (more polar enantiomer). [α]D = −28.64 (4.80 mg in
2 mL MeOH). ESI-MS (m/z, %) 353 (MH+, 50), 322 (100). ESI-
HRMS (MH+) calcd for C16H25N4O5, 353.1819; found, 353.1809. ee
by 99% HPLC. HPLC purity 99.1%
tert-Butyl Ethyl(3-(5-(thiophene-2-carboximidamido)-1H-indol-3-
yl)cyclohexyl)carbamate (trans-( )-38). To a solution of trans-
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Journal of Medicinal Chemistry
Article
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 radioactivity 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 L-NMMA. All assays were
performed in duplicate.
vasoconstricted with U46619 prior to CCRCs to ACh (1 ×
10−10 M to 1 × 10−5 M). L-NMMA was studied in each artery
ring (100 μM) in the presence and absence of L-arginine (10
mM) as positive control.
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.
Efficacy in the Chung Model of Neuropathic Pain. Nerve
ligation injury was performed according to the literature procedure.
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 recovery 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 Binding Assay. The assay was carried with
human recombinant HEK-293 cells using [3H]astemizole as a ligand
(2 nM) with incubation at 22 °C for 75 min with reference to
astemizole according to the literature procedure. 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) obtained in the presence of the compounds (i.e., cis-
( )-37). The IC50 values (concentration causing a half-maximal
inhibition of control specific binding) and Hill coefficients (nH) were
determined by nonlinear regression analysis of the competition curves
generated with mean replicate values using Hill equation curve fitting
(Y = D + [(A − D)/(1 + (C/C50)nH)], where Y = specific binding, D =
minimum specific binding, A = maximum specific binding, C =
compound concentration, C50 = IC50, and nH = slope factor).
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
KPSS (62.5 mM) three times to provide a reference means of
contractility.
NE Transporter (Antagonist Radioligand) Assay.42 Evaluation
of the affinity of compounds for the human norepinephrine
transporter in transfected CHO cells determined in a radioligand
binding assay.
Experimental protocol: Cell membrane homogenates (20 μg
protein) were incubated for 120 min at 4 °C with 1 nM
[3H]nisoxetine in the absence or presence of the test compound in
a buffer containing 50 mM Tris-HCl (pH 7.4), 120 mM NaCl, and 5
mM KCl.
Nonspecific binding was determined in the presence of 1 μM
desipramine. Following incubation, the samples were filtered rapidly
under vacuum through glass fiber filters (GF/B, Packard) presoaked
with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl
using a 96-sample cell harvester (Unifilter, Packard). The filters were
dried and then counted for radioactivity in a scintillation counter
(Topcount, Packard) using a scintillation cocktail (Microscint 0,
Packard). The results are expressed as a percent inhibition of the
control radioligand specific binding.
The standard reference compound was protriptyline, which was
tested in each experiment at several concentrations to obtain a
competition curve from which its IC50 is calculated.
High Throughput Broad Screen. In each experiment, the
respective reference compound was tested concurrently with cis-
(+)-37, and the data were compared with historical values determined.
Results showing an inhibition (or stimulation for assays run in basal
conditions) higher than 50% are considered to represent significant
effect of cis-(+)-37. 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.
ASSOCIATED CONTENT
■
S
* Supporting Information
Analytical and preparative HPLC methods including chiral
methods for all final compounds. 1D 1H NMR for cis-24/trans-
1
24, cis-26/trans-26, and cis-28/trans-28. 1D and 2D H NMR
for cis-( )-30, trans-( )-30, cis-( )-32, and trans-( )-32. This
material is available free of charge via the Internet at http://
pubs.acs.org.
The pharmacology was 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.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 1-416-479-4116. Fax: 1-905-823-5836. E-mail:
gmladenova@neuraxon.com.
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.
Notes
The authors declare no competing financial interest.
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 (10−3 M). cis-( )-37 ( L-arginine) was added at the
selected concentration for a period of 50 min. In the presence
of cis-( )-37 ( L-arginine), all vessels were then submaximally
ACKNOWLEDGMENTS
■
We are grateful to NoAb BioDiscoveries Inc. (Mississauga, ON,
Canada) and Asinex Ltd (Moscow, Russia) for performing the
human NOS inhibition assay. We would also like to thank
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ABBREVIATIONS USED
■
NE, norepinephrine; NERI, norepinephrine reuptake inhibitor;
NET, norepinephrine transporter; CNS, central nervous
system; NO, nitric oxide; NOS, nitric oxide synthase; eNOS,
endothelial nitric oxide synthase; nNOS, neuronal nitric oxide
synthase; iNOS, inducible nitric oxide synthase; L-NMMA, N-
methyl-L-arginine; SAR, structure−activity relationship;
CYP450, cytochrome P450; hERG, human ether-a-go-go
related gene; QTc, heartrate-corrected QT interval; SNL,
spinal nerve ligation; PSA, polar surface area; PK, pharmaco-
kinetics; ACh, acetylcholine
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