1,2,3,4-tetrahydroquinoline-based selective human neuronal nitric oxide synthase (nNOS) inhibitors: Lead optimization studies resulting in the identification of N-(1-(2-(methylamino)ethyl)-1,2,3,4-tetrahydroquinolin-6-yl) thiophene-2-carboximidamide as a

Article abstract of DOI:10.1021/jm3000449

Numerous studies have shown that selective nNOS inhibitors could be therapeutic in many neurological disorders. Previously, we reported a series of 1,2,3,4-tetrahydroquinoline-based potent and selective nNOS inhibitors, highlighted by 1 (J. Med. Chem. 201

Full text of DOI:10.1021/jm3000449

Article
pubs.acs.org/jmc
1,2,3,4-Tetrahydroquinoline-Based Selective Human Neuronal Nitric
Oxide Synthase (nNOS) Inhibitors: Lead Optimization Studies
Resulting in the Identification of N-(1-(2-(Methylamino)ethyl)-1,2,3,4-
tetrahydroquinolin-6-yl)thiophene-2-carboximidamide as a
Preclinical Development Candidate
Jailall Ramnauth,*,† Paul Renton,† Peter Dove,† Subhash C. Annedi,† Joanne Speed,† Sarah Silverman,†
Gabriela Mladenova,† Shawn P. Maddaford,† Salvatore Zinghini,† Suman Rakhit,† John Andrews,†
David K. H. Lee,‡ Dongqin Zhang,§ and Frank Porreca§
†NeurAxon Inc., 2395 Speakman Drive, Suite 1001, Mississauga, Ontario, L5K 1B3, Canada
‡NoAb BioDiscoveries Inc., 2820 Argentia Road, Mississauga, Ontario, L5N 8G4, Canada
§Department of Pharmacology, University of Arizona, 1501 N. Campbell Avenue, Tucson, Arizona 85724, United States
ABSTRACT: Numerous studies have shown that selective nNOS
inhibitors could be therapeutic in many neurological disorders.
Previously, we reported a series of 1,2,3,4-tetrahydroquinoline-based
potent and selective nNOS inhibitors, highlighted by 1 (J. Med. Chem.
2011, 54, 5562−5575). Despite showing activity in two rodent pain
models, 1 suffered from low oral bioavailability (18%) and moderate
hERG channel inhibition (IC50 = 4.7 μM). To optimize the properties of
1, we synthesized a small focused library containing various alkylamino groups on the 1-position of the 1,2,3,4-
tetrahydroquinoline scaffold. The compounds were triaged based on their activity in the NOS and hERG manual patch
clamp assays and their calculated physicochemical parameters. From these studies, we identified 47 as a potent and selective
nNOS inhibitor with improved oral bioavailability (60%) and no hERG channel inhibition (IC50 > 30 μM). Furthermore, 47 was
efficacious in the Chung model of neuropathic pain and has an excellent safety profile, making it a promising preclinical
development candidate.
nNOS has the potential to be therapeutic in many disease
states, but in order to maintain the important roles of eNOS in
regulating blood pressure and iNOS in immune responses, the
selective inhibition of nNOS is crucial.11
INTRODUCTION
■
The discovery of nitric oxide (NO) as a mediator of vascular
tone in the early 1980s1,2 has led to the unraveling of the
diverse biological functions of NO.3,4 This small, short-lived
molecule is synthesized from L-arginine by a family of three
enzymes called nitric oxide synthase (NOS).5 Two isoforms,
endothelial NOS (eNOS) and neuronal NOS (nNOS), are
constitutively expressed and depend on increases in external
calcium and binding of a calcium/calmodulin complex for
activation. The third isoform is inducible NOS (iNOS),
requiring an external stimulus for production and independent
of calcium activation due to the tight binding of a calcium/
calmodulin complex at the dimer interface. Under normal
conditions, these enzymes produce NO in a delicate balance to
control diverse physiological functions such as blood pressure
regulation, platelet aggregation, inflammation, and neuro-
transmission.6 However, overproduction of NO by the different
isoforms can lead to many pathological conditions. In
particular, a large body of evidence indicates that over-
production of NO by nNOS can lead to many neurological
disease states including neurodegeneration during Alzheimer’s
and Parkinson’s diseases,7 altered spinal transmission of
neuropathic pain,8,9 and progression of migraine and chronic
tension-type headaches.10 Consequently, the inhibition of
The design and development of selective nNOS inhibitors
have challenged both academic and industrial researchers alike
for over 2 decades.12−14 The early inhibitors of NOS were
nonselective L-arginine-based compounds such as N-mono-
methyl-L-arginine (L-NMMA) and N-nitro-L-arginine methyl
ester (L-NAME). The lack of selectivity prevented these
compounds from being therapeutically useful, primarily because
of the effects of eNOS inhibition;15 for example, L-NMMA has
been shown to increase blood pressure in human clinical
trials.16 Using traditional medicinal chemistry efforts and
structure-guided drug discovery techniques, researchers have
identified many structurally diverse compounds that inhibit
nNOS potently and selectively (structures 1−4 are some recent
examples, Figure 1).17−20 Despite this progress, a major
challenge still exists in designing CNS druglike molecules
with favorable physicochemical properties to target the
relatively polar active site of nNOS.21
Received: January 11, 2012
Published: February 15, 2012
© 2012 American Chemical Society
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12 via a Finkelstein reaction. Reaction of iodide 12 with a series
of amines gave compounds 13, 14, and 16. The last two
compounds were then Boc protected to give bromo
intermediates 15 and 17. The synthesis of intermediates 23
and 24 followed synthetic paths from 10 and 19, respectively
(Scheme 3). Nucleophilic displacement of the chloride 10 with
methylethylamine gave 20, while displacement of chloride 19
with ethanolamine gave 21, which was Boc protected to give
compound 22. Compounds 20 and 22 were reduced with
borane in THF to give 23 and 24, respectively. Compared to
Scheme 2, this route is much more efficient and provided a
a
Scheme 2. Synthesis of Intermediates 16, 17, and 18
Figure 1. Examples of recently published nNOS inhibitors.
Our research group has been interested in designing and
developing druglike selective nNOS inhibitors for treating CNS
disorders, and we have reported a number of structurally
diverse nNOS inhibitors.17,22−25 Recently, we identified 1 as a
druglike potent and selective human nNOS inhibitor that was
efficacious in the rat Chung model of neuropathic pain and also
in a rodent model of dural inflammation relevant to migraine
pain.17 However, because of the low oral bioavailability of this
compound (18%), we could not advance it further into
preclinical development. Later, we discovered that compound 1
was moderately active at the hERG ion channel (vide infra),
which further hampered its development. To address these
drawbacks, we now describe the lead optimization studies of 1
that led to the identification of N-(1-(2-(methylamino)ethyl)-
1,2,3,4-tetrahydroquinolin-6-yl)thiophene-2-carboximidamide
(47), which is a potent and selective human nNOS inhibitor
with the desirable features of a preclinical development
candidate.
a
Reagents and conditions: (a) chloroacetyl chloride, toluene, 95 °C,
30 min; (b) 1 M BH3·THF, THF, rt, 17 h; (c) NaI, acetone, reflux, 2
days; (d) amine, K2CO3, ACN, reflux, 17 h; (e) Boc2O, Et3N, dioxane,
rt, 17 h.
a
Scheme 3. Synthesis of Intermediates 23 and 24
RESULTS AND DISCUSSION
■
Chemistry. We developed a general method for synthesiz-
ing the target compounds in this study from either a bromo or
nitro intermediate (Scheme 5). The synthesis of intermediates
7, 8, 13, 15, 17, 23, 24, and 28 are outlined in Schemes 1−4,
a
a
Reagents and conditions: (a) chloroacetyl chloride, toluene, 105 °C,
Scheme 1. Synthesis of Intermediates 7 and 8
15 min; (b) Boc2O, Et3N, dioxane, rt, 17 h; (c) 1 M BH3·THF, THF,
rt, 17 h.
simpler route for synthesizing the intermediates. The final
intermediate 28 was synthesized according to Scheme 4 from
25.17 Nucleophilic displacement of the chloride 25 with
ethanolamine gave 26, and this compound was Boc protected
to give 27, which was converted to 28 after reduction with
borane in THF.
The intermediates were converted to the target compounds
according to Scheme 5. The nitro intermediates 7, 8, 24, and
28 were reduced under catalytic hydrogenation conditions to
give the corresponding anilines 29, 36, 32, and 37, respectively,
and the bromo intermediates 13, 15, 17, 18, and 23 were
converted to the anilines 35, 31, 30, 33, and 34, respectively, by
a Buchwald−Hartwig amination reaction using LiHMDS as an
ammonia surrogate. These anilines were coupled with
thiophene-2-carbimidothioate hydroiodide at room temper-
ature in ethanol to give either the target compounds 43 and 44
or the protected compounds 38−42, 45, and 46. The
carbamate protected compounds 38 and 45 were converted
a
Reagents and conditions: (a) PhOCOCl, CH2Cl2, rt, 17 h.
while the synthesis of intermediate 18 was described
previously.17 The nitro intermediates 7 and 8 were prepared
according to Scheme 1 from compounds 5 and 6, respectively,
by N-demethylation with phenyl chloroformate and concom-
itant protection as the phenyl carbamate. To synthesize
compounds 13, 15, and 17, compound 9 was treated with
chloroacetyl chloride to give the chloroamide 10, which was
reduced with borane in THF to give the alkyl chloride 11. In
order to efficiently couple a series of amines to this scaffold, it
was necessary to convert 11 to the corresponding alkyl iodide
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a
Scheme 4. Synthesis of Intermediate 28
a
Reagents and conditions: (a) ethanolamine, K2CO3, ACN, reflux, 17 h; (b) Boc2O, Et3N, dioxane, rt, 17 h; (c) 1 M BH3·THF, THF, rt, 17 h.
a
Scheme 5. Synthesis of Target Compounds 43, 44, 47−53
a
Reagents and conditions: (a) Pd/C, H2, EtOH, rt (X = NO2) or LiHMDS, Pd2(dba)3, PtBu3(X = Br), THF, reflux, 2 h; (b) thiophene-2-
carbimidothioate·HI, EtOH, rt; (c) 1 N NaOH, ethylene glycol, 100 °C; (d) 3 N HCl in MeOH.
to target compounds 47 and 48 by base hydrolysis, whereas the
Boc protected compounds 39−42 and 46 were converted to
target compounds 49−53 under standard acidic conditions.
Human NOS Inhibition Studies. Our design strategy was
based on targeting the L-arginine binding site of nNOS using a
pharmacophore model described by us and others.14,17 In brief,
this model featured a guanidine isosteric group and a basic
amine side chain attached to a central scaffold. In our previous
publication, we prepared a number of analogues based on the
1,2,3,4-tetrahydroquinoline core with a thiophene amidine
group attached at the 6-position and a variety of acyclic and
cyclic alkylamino side chains attached at the 1-position of this
scaffold.17 This study led to the identification of a series of
potent and selective inhibitors of nNOS. From this series, the
most potent and selective nNOS inhibitor was compound 1.
Although compound 1 was active in two rodent pain models, it
was not an optimal compound to advance further into
preclinical development. As mentioned in the Introduction,
we sought to improve the oral bioavailability and decrease the
hERG inhibition properties of 1 while retaining or improving
the nNOS inhibitory potency and selectivity. To achieve this
objective, a small focused library was prepared by varying the
nature of the aminoalkyl side chain while keeping the 1,2,3,4-
tetrahydroquinoline core and thiophene amidine group
constant. From our previous work using the 1,2,3,4-
tetrahydroquinoline scaffold, the nature of the aminoalkyl
side chain did not play a major role in the nNOS inhibitory
potency and selectivity.17 Consequently, we felt that this would
be an ideal moiety to modify in order to optimize the ADME
properties of the compounds.
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Table 1. In Vitro NOS Inhibitory and hERG Manual Patch-Clamp Data along with Calculated Ligand Efficiencies for nNOS
a
Inhibition
a
Inhibitory activities were measured by following the conversion of [3H]L-arginine into [3H]L-citrulline. All assays were performed at least in
duplicate. Values in parentheses are the 95% confidence intervals. NT: not tested. Selectivity ratios for nNOS are defined as e/n = IC50(eNOS)/
IC50(nNOS) and i/n = IC50(iNOS)/IC50(nNOS).
The compounds prepared were tested as the dihydrochloride
salts for inhibitory activity against all three human NOS
isoforms. The inhibitory activities of these compounds were
measured by following the conversion of [3H]L-arginine into
[3H]L-citrulline in the presence of the requisite cofactors.5,26
The enzymatic reaction was carried out in the presence or
absence of varying concentrations of the compound. Following
that, the negatively charged [3H]L-citrulline was separated from
the positively charged [3H]L-arginine using resin beads.
Inhibition of enzyme activity by the compound is measured
by dividing the enzymatic conversion in the presence of
compound by the enzymatic conversion in the absence of
compound. The IC50 is the concentration of compound that
gives rise to 50% inhibition. The observed NOS IC50 values and
the selectivity ratios for nNOS, defined as IC50(eNOS)/
IC50(nNOS) and IC50(iNOS)/IC50(nNOS), are shown in
Table 1.
All of the compounds prepared inhibited nNOS in the
submicromolar range and were highly selective over both
eNOS and iNOS. The most potent nNOS inhibitor was
compound 43 (IC50 = 97 nM), while the most selective
compound against eNOS was compound 44, with over 400-fold
selectivity. As observed previously,17 despite the diverse nature
of the aminoalkyl side chains, the differences in inhibitory
potencies against nNOS for all compounds are within 0.5 log
unit, demonstrating that the aminoalkyl substituents on the 1-
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a
Table 2. Calculated Physicochemical Properties and CNS MPO Scores of the Compounds
compd
43
clogP
PSA
clogD (pH 7.4)
pKa (most basic)
Hb-A
Hb-D
MW
FRB
CNS MPO
3.59
2.71
2.77
3.06
3.28
3.63
2.07
2.83
2.36
3.97
−0.08
70.6
90.83
79.4
79.4
79.4
79.4
98.5
79.4
99.6
70.6
111.2
0.54
0.46
9.1
9.1
4
5
4
4
4
4
5
4
5
4
6
2
3
3
3
3
3
4
3
4
2
6
342.5
6
8
5
6
6
6
8
3
9
5
6
4.7
4.6
4.4
4.1
4.3
4.1
4.2
4.4
4.0
4.0
3.3
44
358.5
47
−0.7
9.52
10.28
9.52
9.52
9.1
314.45
328.48
328.48
342.5
48
−0.82
−0.26
0.17
49
50
51
−0.72
−0.66
−1.06
0.42
344.47
326.46
358.5
( )-52
9.46
9.3
53
1
10.26
14.06
368.54
188.23
L-NMMA
−3.55
a
Hb-A: sum of H-bond acceptors. Hb-D: sum of H-bond donors. MW: molecular weight. FRB: number of freely rotating bonds. PSA: polar surface
area. CNS MPO: central nervous system multiple parameter optimization.
position of the 1,2,3,4-tetrahydroquinoline core occupy a fairly
tolerant region in the nNOS enzyme. Furthermore, the ligand
efficiencies27 (nNOS potency/non-H atoms) of all compounds
prepared were within a narrow range of 0.42−0.34. The
compounds do not inhibit iNOS to any appreciable extent, and
inhibitory activity against eNOS was fairly weak for these
compounds. A number of compounds showed similar or better
nNOS/eNOS selectivity than compound 1, and all compounds
were more nNOS/iNOS selective than 1. On the basis of this
primary screen, we advanced nNOS inhibitors that were highly
selective over eNOS (>200-fold) and iNOS (>500-fold).
Accordingly, compounds 48, 50, and ( )-52 were given
lower priority.
hERG Channel Inhibition Studies. Inhibition of the
human ether-a-go-go-related gene (hERG) channel is known to
cause torsade de pointes cardiac arrhythmias via a prolongation
of cardiac QT action potential, and it is a major reason for
compound attrition and withdrawal from the market.28 We
evaluated these compounds in the hERG manual patch clamp
functional assay, and the results are presented in Table 1. It is
clear that compounds containing a tertiary amine (1, 43, and
44) inhibit the channel moderately in the single digit
micromolar range. The corresponding secondary amines for
43 (47 and 49) and 44 (47 and 51) do not inhibit the channel
to any extent. The other secondary amines are devoid of hERG
activity as well, with only compound 50 showing weak activity
(IC50 = 27.5 μM). The relationship between physicochemical
properties, particularly lipophilicity, and hERG channel activity
is well established and explains the current trend we observe.29
The calculated log D (Table 2) for compounds 1, 43, and 44
are 0.54, 0.45, and 0.42, respectively, which are significantly
higher than the calculated log D for all other compounds. It is
noteworthy that the secondary amine 50, which has a positive
calculated log D (0.17), showed some weak inhibition of the
hERG ion channel, further supporting the key role lipophilicity
plays in hERG channel blockage. By use of these results,
compounds 43 and 44 were not progressed further because of
their inhibition of the hERG channel. These two compounds
along with 1 have over 40-fold selectivity of nNOS inhibition
over hERG channel inhibition. Nevertheless, the other
compounds are much weaker (>30 μM) hERG channel
blockers.
challenge when designing nNOS inhibitors to treat CNS
disorders. In the past decade, several seminal papers have
established the importance of physicochemical properties on
the in vivo behavior of drugs.30,31 More recently, Wager et al.
examined and utilized the interplay among six of these
physicochemical properties to create a druglikeness central
nervous system multiparameter optimization (CNS MPO)
algorithm.32 Using this prospective design tool, which is based
on a scale of 0−6, they evaluated a large number of drugs for
CNS disorders and showed that most marketed CNS drugs
(74%) have a CNS MPO score of ≥4. To assess the
druglikeness of these new selective nNOS inhibitors, the
physicochemical properties and CNS MPO scores were
calculated (Table 2).33 All new compounds follow Lipinski’s
rule of five30 (log P, hydrogen bond donor/acceptor properties,
and MW) and had better CNS MPO scores than 1.
Interestingly, the tertiary amines 43 and 44 had the highest
CNS MPO scores while the nonselective amino acid based L-
NMMA, not surprisingly, had the lowest CNS MPO scores.
Using the CNS MPO as a final selection criterion for the
remaining five compounds, we selected compound 47 for
further evaluation.
L5/L6 Spinal Nerve Ligation (SNL) or Chung Model of
Neuropathic Pain in Rats. To evaluate the in vivo behavior
of 47, its efficacy was assessed in the rat Chung model of
neuropathic pain (Figure 2).34 This model serves as a
preliminary in vivo screen to assess the druglikeness of our
selective nNOS inhibitors. The inhibitory activity of 47 against
Physicochemical Properties and CNS Druglikeness of
the nNOS Inhibitors. Because of the polar and acidic nature
of the nNOS active site, compounds that mimic the substrate L-
arginine are likely to be polar and basic, which poses a major
Figure 2. Compound 47 reverses thermal hyperalgesia in the L5/L6
spinal nerve ligation (Chung) model of neuropathic pain in rats.
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a
Table 3. Comparing Rat Pharmacokinetics of Compounds 1 and 47
b
compd
Cmax (μM)
Tmax (h)
t1/2 (h)
AUC (μg·h/mL)
Vdss (L/kg)
Clp (L h−1 kg−1
)
Fpo (%)
1
0.24
0.88
1.7
2.0
9.4
8.1
0.55
1.16
94.7
83.9
4.02
5.26
18.4
59.9
47
a
b
3 mg/kg iv; 10 mg/kg po. po t1/2
.
Table 4. Experimental Conditions for hERG K+ Channel Conventional Patch-Clamp Assay
cells
solution
incubation
detection
HEK-293 cell line extracellular solution: 137 mM NaCl, 4 mM KCl, 1.8 mM 5−10 min for concentration at rt (22−24 °C) conventional whole-cell patch-
stably expressing
hERG
CaCl2, 1 mM MgCl2, 10 mM D-(+)-glucose, 10 mM
HEPES (pH 7.4 by NaOH)
cumulatively
clamp (by Axopatch 200 B or
HEKA EPC9)
intracellular solution: 130 mM KCl, 10 mM NaCl, 1 mM
MgCl2, 10 mM EGTA, 5 mM MgATP, 10 mM HEPES
(pH 7.2 by KOH)
rat nNOS was similar to that of human nNOS (192 nM versus
176 nM). As shown in Figure 2, this compound reverses
thermal hyperalgesia when given to rats at a dose of 30 mg/kg
intraperitoneally with a maximum effect at 30 min after
administration. The activity of 47 in this in vivo model
demonstrated that this compound possesses druglike proper-
ties.
Comparison of Rat Pharmokinetics of Compounds 47
and 1. Rat pharmacokinetics was carried out on compound 47,
and the results were compared to those obtained with
compound 1 (Table 3).17 Compound 47 showed a significant
improvement in oral exposure over 1 with an oral bioavailability
of 60%. Also, AUC (10 mg/kg po) was more than twice as high
for 47, and compound 47 showed a 3-fold increase in Cmax. The
AUC and Cmax are higher than the nNOS inhibitory activity
(0.2 μM).
Off-Target Activitiy of Compound 47. Having identified
a compound with improved oral bioavailability and decreased
hERG inhibition over 1, we evaluated 47 for off-target activity.
First, compound 47 did not exhibit any inhibitory activity
against the five major human CYP P450 isoforms, making it
less likely to have any drug−drug interactions. In addition,
compound 47 had a relatively low human plasma protein
binding of only 40%, further reducing the likelihood of drug−
drug interactions. Second, the selectivity profile of 47 was
determined against a panel of 80 targets comprising G-protein-
coupled receptors, ion channels, transporters of biogenic
amines, and enzymes (CEREP Screen) at a test concentration
of 10 μM. Compound 47 only showed significant inhibition
(>70%) at the following targets: rat α-2 adrenergic receptor
(80%) and human muscarinic receptor (M1, 72%; M2, 74%; M3,
89%; M4, 94%). As a follow-up to the high throughput profile,
the cellular functional assays of these targets were evaluated and
the EC50 values were greater than 40 μM. Overall, 47 possesses
an excellent safety profile and has been advanced for further in
vivo preclinical studies.
compound that addressed these drawbacks. Using a previously
described pharmacophore model, we prepared a small focused
library centered around the 1-position alkylamino side chain of
the 1,2,3,4-tetrahydroquinoline scaffold. This region was
tolerant to modifications, since all compounds displayed
nNOS inhibitory potencies in the submicromolar range. The
compounds were triaged based on a number of experimental
studies and calculated physicochemical parameters. In addition
to the NOS inhibition assay, hERG channel inhibition was
assessed using a manual patch clamp functional assay, which
revealed that tertiary amines in this series were active with
inhibitory potencies in the low micromolar range while
secondary amines were essentially inactive. Further analysis
revealed that the clogD value might be a key driver in the
hERG channel blockage activity. The recently described CNS
MPO parameter was used to further triage the compounds. On
the basis of this parameter, all new compounds scored equal or
better than the lead compound 1, and by use of this parameter
as a final selection criterion, compound 47 was selected for
further evaluation. A preliminary screen in the Chung model of
neuropathic pain showed that 47 was efficacious, demonstrating
its druglikeness. Furthermore, the oral bioavailability of 47
(60%) was much better than that for 1 (18%). The off-target
activity of 47 as assessed in a high throughput screen, CYP
P450 inhibition studies, and plasma protein binding studies was
minimal, making 47 a promising preclinical development
selective nNOS inhibitor candidate.
EXPERIMENTAL SECTION
■
Chemistry. General Procedures. All reactions were conducted
under an atmosphere of argon, and the mixtures were stirred
magnetically unless otherwise noted. Commercial reagents and
anhydrous solvents were used as received without further purification.
When necessary, Sure/Seal anhydrous solvents were utilized.
Reactions were monitored by analytical TLC using precoated silica
gel aluminum plates (Sigma-Aldrich, 0.2 mm, 60 Å) and were
visualized with UV light or stained where appropriate. Flash column
chromatography was performed using Silicycle Siliaflash F60 (40−63
μm) silica gel. The 1H NMR spectra were performed at York
University, Canada, on a Bruker 300 spectrometer. Chemical shifts (δ)
are reported in parts per million (ppm) relative to CDCl3 (7.26 ppm),
CD3OD (4.87 ppm), or DMSO-d6 (2.50 ppm). Coupling constants
(J) are given in hertz (Hz). Low and high resolution MS were
performed at the University of Toronto AIMS (Mass Spectrometry
Laboratory), Canada, on an Applied Biosystems/MDS Sciex QstarXL
hybrid quadrupole/TOF instrument using electrospray ionization
except where indicated. Analytical HPLC spectra were collected on an
Agilent 1100 HPLC system using a reverse phase column. All final
CONCLUSIONS
■
In a previous study, we identified a series of potent and
selective nNOS inhibitors based on the 1,2,3,4-tetrahydroqui-
noline core. The most potent and nNOS selective compound
(1) was selected for further evaluation to demonstrate the
usefulness of selective nNOS inhibitors. Although this
compound was efficacious in two in vivo rodent pain models,
we could not advance this compound further because of its low
oral bioavailability and low micromolar inhibition of the hERG
ion channel. We undertook this current study to identify a
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2.79 (t, J = 6.3 Hz, 2H), 1.99−1.96 (m, 2H). ESI-MS (m/z, %): 356
(MH+).
Table 5. HPLC Purity of Final Compounds
compd
retention time (min)
purity (%)
HPLC method
Phenyl Methyl(3-(6-nitro-3,4-dihydroquinolin-1(2H)-yl)-
propyl)carbamate (8). 8 was prepared from N,N-dimethyl-3-(6-
nitro-3,4-dihydroquinolin-1(2H)-yl)propan-1-amine (6) (1 g, 3.80
mmol) and phenyl carbonochloridate (0.71 mL, 5.70 mmol) as
described for 7 to obtain the title compound (1.02 g, 72.7%). 1H NMR
(DMSO-d6) δ 7.88 (t, J = 7 Hz, 1H), 7.78 (s, 1H), 7.37 (t, J = 7 Hz,
2H), 7.20 (t, J = 7 Hz, 1H), 7.12−7.06 (m, 2H), 6.70 (d, J = 9 Hz,
1H), 3.50−3.33 (m, 6H), 3.06, 2.93 (2s, 3H), 2.76−2.71 (m, 2H),
1.92−1.81 (m, 4H). ESI-MS (m/z, %): 370 (MH+, 100).
43
11.94
11.36
8.65
96.7
95.0
96.0
96.3
96.3
96.5
95.5
98.1
98.4
C
C
A
B
C
C
B
A
B
44
47
48
7.92
49
15.20
17.02
7.66
50
51
( )-52
53
6.82
1-(6-Bromo-3,4-dihydroquinolin-1(2H)-yl)-2-chloroetha-
none (10). A solution of 6-bromo-1,2,3,4-tetrahydroquinoline (9)
(2.09 g, 9.85 mmol) in toluene (55 mL) was treated with 2-
chloroacetyl chloride (0.86 mL, 10.84 mmol) dropwise over 10 min.
The resulting suspension was stirred at room temperature for 1.5 h
and then heated at 95 °C for 30 min. After cooling to room
temperature, the yellow solution was diluted with EtOAc and sat.
bicarbonate solution. The mixture was poured into a separatory funnel
and extracted. The organic layer was separated, dried (Na2SO4),
filtered, and concentrated. The crude product was purified by flash
column chromatography (EtOAc/hexanes, 3:7) to obtain the title
7.95
a
Method A
time (min)
solvent A (%)
solvent B (%)
0
20
25
30
35
20
80
80
20
20
80
20
20
80
80
b
Method B
1
compound (2.4 g, 84%) as a white solid. H NMR (CDCl3) δ 7.35−
time (min)
solvent A (%)
solvent B (%)
7.33 (m, 3H), 4.19 (s, 2H), 3.80 (t, J = 6.3 Hz, 2H), 2.74 (t, J = 6.6
Hz, 2H), 2.04−1.98 (m, 2H).
0
5
0
30
30
50
50
30
0
100
70
6-Bromo-1-(2-chloroethyl)-1,2,3,4-tetrahydroquinoline (11).
A round-bottom flask was charged with 1-(6-bromo-3,4-dihydroqui-
nolin-1(2H)-yl)-2-chloroethanone (10) (0.546 g, 1.89 mmol) and
treated with borane−THF complex (1 M in THF, 18.9 mL, 18.9
mmol), and the resulting mixture was stirred overnight at room
temperature. After the mixture was cooled to 0 °C, the reaction was
quenched by dropwise addition of MeOH (5 mL) and the mixture was
stirred for 10 min at 0 °C. The mixture was concentrated under
reduced pressure and purified by column chromatography (EtOAc/
hexanes, 1:9) to obtain the title compound (0.519 g, 100%) as a
7
70
7.5
10
12
14
15
50
50
70
100
100
0
c
Method C
time (min)
solvent A (%)
solvent B (%)
1
viscous residue. H NMR (CDCl3) δ 7.11 (dd, 1H, J = 8.7, 2.4 Hz),
0
7
18
24
37
65
18
18
82
76
63
35
82
82
7.08−7.04 (m, 1H), 6.42 (d, 1H, J = 8.7 Hz), 3.64−3.56 (m, 4H), 3.35
(t, 2H, J = 5.6 Hz), 2.73 (t, 2H, J = 6.3 Hz), 1.97−1.89 (m, 2H). ESI-
MS (m/z, %): 274/276 (MH+, 100).
15
25
26
35
6-Bromo-1-(2-iodoethyl)-1,2,3,4-tetrahydroquinoline (12). A
suspension of 6-bromo-1-(2-chloroethyl)-1,2,3,4-tetrahydroquinoline
(11) (1.80 g, 6.56 mmol) in acetone (50 mL) was heated to reflux for
2 days. A further portion of sodium iodide (19.66 g, 132.0 mmol) was
added and the suspension refluxed for 5 more days. The mixture was
cooled to room temperature, filtered through a pad of Celite, and
concentrated to obtain the title compound (2.19 g, 91%) as a yellow
oil. 1H NMR (CDCl3) δ 7.12 (dd, 1H, J = 8.7, 2.5 Hz), 7.09−7.04 (m,
1H), 6.42 (d, 1H, J = 8.7 Hz), 3.65−3.60 (m, 2H), 3.33 (t, 2H, J = 5.6
Hz), 3.25−3.19 (m, 2H), 2.72 (t, 2H, J = 6.3 Hz), 1.98−1.90 (m, 2H).
ESI-MS (m/z, %): 366/368 (MH+, 100).
a
Method A: solvent A, acetonitrile; solvent B, aqueous buffer, pH 10.6
(ammonium carbonate/NaOH); column, Waters XTerra RP8, 5 μm,
4.6 mm × 150 mm; flow rate, 1 mL/min; concentration, 1 mg/mL in
MeOH; injection volume, 10 μL; wavelength, 254 nm. Method B:
b
solvent A, acetonitrile/TFA (100:0.1); solvent B, water/TFA
(100:0.1); column, Phenomenex Luna C18 2.5 μm, 100 mm × 3.0
mm; flow rate, 0.5 mL/min; concentration, 1 mg/mL in MeOH;
c
injection volume, 5 μL; wavelength, 254 nm. Method C: solvent A,
acetonitrile; solvent B, buffer, pH 10.6 (water/triethylamine); column,
Waters XTerra RP8, 5 μm, 4.6 mm × 150 mm; flow rate, 1.5 mL/min;
concentration, 1 mg/mL in MeOH; injection volume, 5 μL;
wavelength, 254 nm.
2-((2-(6-Bromo-3,4-dihydroquinolin-1(2H)-yl)ethyl)(methyl)-
amino)ethanol (13). A solution of 6-bromo-1-(2-iodoethyl)-1,2,3,4-
tetrahydroquinoline 12 (0.475 g, 1.298 mmol) in acetonitrile (19 mL)
and water (1 mL) in a 50 mL pressure vessel fitted with a stir bar was
treated with potassium carbonate (1.793 g, 12.98 mmol) and 2-
(methylamino)ethanol (0.975 g, 12.98 mmol), and the sealed vessel
was stirred at 80 °C overnight. After 18 h the mixture was partitioned
between CH2Cl2 (100 mL) and water (20 mL) and transferred to a
separatory funnel. The organic layer was separated and the aqueous
layer (pH 12) extracted further with CH2Cl2. The combined organic
layer was washed with brine, dried over Na2SO4, filtered, and
concentrated to a brown residue. Purification on silica gel, eluting with
5% 2 M NH3 in methanol/95% dichloromethane, yielded a pale yellow
compounds were >95% purity. No attempts were made to optimize
yields.
Phenyl Methyl(2-(6-nitro-3,4-dihydroquinolin-1(2H)-yl)-
ethyl)carbamate (7). A solution of N,N-dimethyl-2-(6-nitro-3,4-
dihydroquinolin-1(2H)-yl)ethanamine (5) (0.5 g, 2.01 mmol) in
CH2Cl2 (10 mL) was treated dropwise with phenyl chloroformate
(0.37 mL, 3.01 mmol). The mixture was stirred at room temperature
for 17 h and then partitioned between CH2Cl2 and 1 N NaOH
solution. After extraction, the organic layer was separated, dried
(Na2SO4), filtered, and concentrated to give a yellow residue. This
residue was subjected to flash column chromatography (EtOAc/
1
oil, 2 (341 mg, 84%). H NMR (DMSO-d6) δ 7.06 (dd, 1H, J = 8.8,
2.5 Hz), 6.99 (d, 1H, J = 2.4 Hz), 6.48 (d, 1H, J = 8.8 Hz), 4.34 (t, 1H,
J = 5.3 Hz), 3.44 (q, 2H, J = 6.2 Hz), 3.32−3.25 (m, 4H), 2.64 (t, 2H,
J = 6.2 Hz), 2.47−2.41 (m, 4H), 2.23 (s, 3H), 1.83−1.76 (m, 2H).
ESI-MS (m/z, %): 313/315 (MH+, 100).
N-(2-(6-Bromo-3,4-dihydroquinolin-1(2H)-yl)ethyl)propan-
2-amine (14). 14 was prepared from 6-bromo-1-(2-iodoethyl)-
1
CH2Cl2, 1:4) to obtain the title compound (0.61 g, 85.4%). H NMR
(CDCl3) δ 7.99−7.92 (m, 1H), 7.87 (d, J = 2.1 Hz, 1H), 7.39−7.33
(m, 2H), 7.23−7.19 (m, 1H), 7.06−7.01 (m, 2H), 6.69 (m, 1H),
3.67−3.52 (m, 4H), 3.48 (t, J = 5.7 Hz, 2H), 3.14 and 3.07 (2s, 3H),
2888
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Journal of Medicinal Chemistry
Article
1,2,3,4-tetrahydroquinoline (12) (0.4 g, 1.09 mmol), potassium
carbonate (0.75 g, 5.46 mmol), and isopropylamine (0.64 g, 10.93
mmol) as described for 13 to obtain the title compound (0.27 g, 83%)
as a pale yellow oil. 1H NMR (DMSO-d6) δ 7.06 (dd, 1H, J = 8.8, 2.5
Hz), 6.99 (d, 1H, J = 2.5 Hz), 6.52 (d, 1H, J = 8.8 Hz), 3.31−3.20 (m,
4H), 2.78−2.67 (m, 1H), 2.67−2.59 (m, 4H), 1.84−1.76 (m, 2H),
0.96 (d, 6H, J = 6.2 Hz). ESI-MS (m/z, %): 297/299 (MH+, 10), 238/
240 (20), 159 (100).
tert-Butyl 2-Hydroxyethyl(2-(6-nitro-3,4-dihydroquinolin-
1(2H)-yl)-2-oxoethyl)carbamate (22). 22 was prepared from 2-
(2-hydroxyethylamino)-1-(6-nitro-3,4-dihydroquinolin-1(2H)-yl)-
ethanone (21) (3.1 g, 11.10 mmol), triethylamine (3.12 mL, 22.20
mmol), and di-tert-butyl dicarbonate (2.71 mL, 11.65 mmol) as
described for 15 to obtain the title compound (4.2 g, 100%). 1H NMR
(DMSO-d6) δ 8.09−7.90 (m, 3H), 4.65−4.59 (m, 1H), 3.76−3.71 (m,
2H), 3.56 (s, 2H), 3.52−3.47 (m, 2H), 3.31−3.25 (m, 2H), 2.88−2.83
(m, 2H), 1.95−1.89 (m, 2H), 1.39, 1.31 (2s, 9H). ESI-MS (m/z, %):
380 (MH+, 17), 280 (100).
tert-Butyl 2-(6-Bromo-3,4-dihydroquinolin-1(2H)-yl)ethyl-
(isopropyl)carbamate (15). A solution of N-(2-(6-bromo-3,4-
dihydroquinolin-1(2H)-yl)ethyl)propan-2-amine 14 (0.140 g, 0.471
mmol) in anhydrous dioxane (10 mL) was treated with triethylamine
(0.132 mL, 0.942 mmol) and di-tert-butyl dicarbonate (0.108 g, 0.495
mmol) and the resulting mixture stirred overnight at room
temperature. The mixture was concentrated to residue and purified
directly on silica gel, eluting with 10% EtOAc/90% hexanes to yield a
2-(6-Bromo-3,4-dihydroquinolin-1(2H)-yl)-N-ethyl-N-meth-
ylethanamine (23). 23 was prepared from 1-(6-bromo-3,4-
dihydroquinolin-1(2H)-yl)-2-(ethyl(methyl)amino)ethanone (20)
(1.05 g, 3.37 mmol) and 1 M borane in THF (33.7 mL, 33.7
mmol) as described for 11 to obtain the title compound (0.89 g, 89%)
1
as a colorless residue. H NMR (CDCl3) δ 7.09 (dd, J = 1.8, 6.6 Hz,
1
1H), 7.02−7.01 (m, 1H), 6.44 (d, J = 6.6 Hz, 1H), 3.36 (t, J = 5.7 Hz,
2H), 3.29 (t, J = 4.2 Hz, 2H), 2.70 (t, J = 4.8 Hz, 2H), 2.53−2.43 (m,
4H), 2.28 (s, 3H), 1.94−1.88 (m, 2H), 1.06 (t, J = 5.4 Hz, 3H). ESI-
MS (m/z, %): 397 and 399 (MH+, 100%).
tert-Butyl 2-Hydroxyethyl(2-(6-nitro-3,4-dihydroquinolin-
1(2H)-yl)ethyl)carbamate (24). 24 was prepared from tert-butyl 2-
hydroxyethyl(2-(6-nitro-3,4-dihydroquinolin-1(2H)-yl)-2-oxoethyl)-
carbamate (22) (4.2 g, 11.07 mmol) and 1 M borane in THF (33.2
mL, 33.2 mmol) as described for 11 to obtain the title compound
(3.39 g, 84%). 1H NMR (DMSO-d6) δ 7.84 (d, J = 9.0 Hz, 1H), 7.78
(d, J = 9.9 Hz, 1H), 6.74 (m, 1H), 4.73 (t, J = 4.8 Hz, 1H), 3.58−3.53
(m, 2H), 3.49−3.39 (m, 6H), 3.27−3.17 (m, 2H), 2.75−2.68 (m, 2H),
1.88−1.81 (m, 2H), 1.35 (s, 9H). ESI-MS (m/z, %): 366 (MH+, 19),
310 (100).
colorless oil (0.150 g, 80%). H NMR (CDCl3) δ 7.09 (dd, 1H, J =
8.7, 2.0 Hz), 7.05−7.00 (m, 1H), 6.55 (d, 1H, J = 8.7 Hz), 4.50−4.00
(m, 1H), 3.42−3.27 (m, 4H), 3.24−3.11 (m, 2H), 2.70 (t, 2H, J = 6.2
Hz), 1.97−1.86 (m, 2H), 1.53−1.51 (2s, 9H), 1.13 (d, 6H, J = 6.8
Hz). ESI-MS (m/z, %): 397/399 (MH+, 80), 341/343 (100).
2-(6-Bromo-3,4-dihydroquinolin-1(2H)-yl)-N-ethylethan-
amine (16). 16 was prepared from 6-bromo-1-(2-iodoethyl)-1,2,3,4-
tetrahydroquinoline (12) (0.1 g, 0.27 mmol), potassium carbonate
(0.37 g, 2.73 mmol), and ethanamine hydrochloride (0.22 g, 2.73
mmol) as described for 13 to obtain the title compound (55 mg,
71.1%) as a yellow oil. 1H NMR (CDCl3) δ 7.09 (dd, 1H, J = 8.7, 2.4
Hz), 7.06−7.01 (m, 1H), 6.51 (d, 1H, J = 8.7 Hz), 3.36 (t, 2H, J = 6.8
Hz), 3.29 (t, 2H, J = 5.5 Hz), 2.82 (t, 2H, J = 6.8 Hz), 2.80−2.65 (m,
4H), 1.96−1.88 (m, 2H), 1.11 (t, 3H, J = 7.1 Hz).
tert-Butyl 2-(6-Bromo-3,4-dihydroquinolin-1(2H)-yl)ethyl-
(ethyl)carbamate (17). 17 was prepared from 2-(6-bromo-3,4-
dihydroquinolin-1(2H)-yl)-N-ethylethanamine (16) (0.25 g, 0.88
mmol), triethylamine (0.24 mL, 1.76 mmol), and di-tert-butyl
dicarbonate (0.20 g, 0.92 mmol) as described for 15 to give the title
1-(3-(2-Hydroxyethylamino)propyl)-6-nitro-3,4-dihydroqui-
nolin-2(1H)-one (26). 26 was prepared from 1-(3-chloropropyl)-6-
nitro-3,4-dihydroquinolin-2(1H)-one (25) (0.5 g, 1.86 mmol) and 2-
aminoethanol (1.12 mL, 18.61 mmol) as described for 13 to obtain the
1
title compound (0.27 g, 49.5%). H NMR (DMSO-d6) δ 8.10−8.05
1
compound (0.28 g, 83%) as a colorless oil. H NMR (CDCl3) δ 7.09
(m, 2H), 7.37 (d, J = 8.7 Hz, 1H), 4.42 (brs, 1H), 3.93 (t, J = 7.4 Hz,
2H), 3.39 (t, J = 5.7 Hz, 2H), 2.96 (t, J = 7.35 Hz, 2H), 2.61−2.57 (m,
2H), 2.52−2.45 (m, 4H), 1.66−1.57 (m, 2H). ESI-MS (m/z, %): 294
(MH+, 100), 233 (19).
(dd, 1H, J = 8.7, 2.3 Hz), 7.03 (brs, 1H), 6.50 (d, 1H, J = 8.7 Hz),
3.48−3.11 (m, 8H), 2.70 (t, 2H, J = 6.3 Hz), 2.05−1.87 (m, 2H), 1.47
(s, 9H), 1.10 (brs, 3H). ESI-MS (m/z, %): 383/385 (MH+, 38), 327/
329 (100).
tert-Butyl 2-Hydroxyethyl(3-(6-nitro-2-oxo-3,4-dihydroqui-
nolin-1(2H)-yl)propyl)carbamate (27). 27 was prepared from 1-
(3-(2-hydroxyethylamino)propyl)-6-nitro-3,4-dihydroquinolin-2(1H)-
one (26) (0.27 g, 0.92 mmol), triethylamine (0.25 mL, 1.84 mmol),
and di-tert-butyl dicarbonate (0.21 g, 0.96 mmol) as described for 15
to obtain the title compound (0.359 g, 99%). 1H NMR (DMSO-d6) δ
8.16−8.10 (m, 2H), 7.34 (d, J = 9.0 Hz, 1H), 4.67−4.64 (m, 1H),
3.98−3.91 (m, 2H), 3.47−3.41 (m, 2H), 3.28−3.16 (m, 4H), 3.01 (t, J
= 7.4 Hz, 2H), 2.62 (t, J = 7.4 Hz, 2H), 1.81−1.72 (m, 2H), 1.39−1.30
(m, 9H). ESI-MS (m/z, %): 416 (100), 294 (77).
tert-Butyl 2-Hydroxyethyl(3-(6-nitro-3,4-dihydroquinolin-
1(2H)-yl)propyl)carbamate (28). 28 was prepared from tert-butyl
2-hydroxyethyl(3-(6-nitro-2-oxo-3,4-dihydroquinolin-1(2H)-yl)-
propyl)carbamate (27) (0.35 g, 0.90 mmol) and 1 M borane in THF
(2.69 mL, 2.70 mmol) as described for 11 to obtain the title
compound (0.25 g, 73.8%). 1H NMR (DMSO-d6) δ 7.87 (dd, J = 9.3,
2.7 Hz, 1H), 7.78 (d, J = 2.7 Hz, 1H), 6.65 (d, J = 9.3 Hz, 1H), 4.68 (t,
J = 5.0 Hz, 1H), 3.48−3.32 (m, 6H), 3.26−3.18 (m, 4H), 2.75 (t, J =
6.0 Hz, 1H), 1.88−1.76 (m, 4H), 1.36 (brs, 9H). ESI-MS (m/z, %):
402 (100), 380 (MH+, 14), 280 (95).
1-(6-Bromo-3,4-dihydroquinolin-1(2H)-yl)-2-(ethyl(methyl)-
amino)ethanone (20). A suspension of 1-(6-bromo-3,4-dihydroqui-
nolin-1(2H)-yl)-2-chloroethanone (10) (1.0 g, 3.47 mmol) and
potassium iodide (0.11 g, 0.69 mmol) in THF (10 mL) was treated
with N-methylethanamine (1.78 mL, 20.79 mmol). The mixture was
stirred at room temperature for 2 h and then heated at 65 °C for 1 h.
The mixture was concentrated and partitioned between CH2Cl2 and
saturated NaHCO3 solution. After extraction, the organic layer was
separated, dried (Na2SO4), filtered, and concentrated to give a light
brown residue. This residue was purified by flash column
chromatography (2 M NH3 in MeOH/CH2Cl2, 5:95) to obtain the
1
title compound (1.06 g, 98%) as a light brown residue. H NMR
(CDCl3) δ 7.52−7.40 (m, 1H), 7.30−7.27 (m, 2H), 3.80 (t, J = 6.0
Hz, 2H), 3.28 (s, 2H), 2.72 (t, J = 6.9 Hz, 2H), 2.50 (q, J = 7.2 Hz,
2H), 2.30 (s, 3H), 1.96 (quint, J = 6.6 Hz, 2H), 1.03 (t, J = 7.2 Hz,
3H). ESI-MS (m/z, %): 311 and 313 (MH+, 100%).
2-(2-Hydroxyethylamino)-1-(6-nitro-3,4-dihydroquinolin-
1(2H)-yl)ethanone (21). To a solution of 2-chloro-1-(6-nitro-3,4-
dihydroquinolin-1(2H)-yl)ethanone (19) (5.9 g, 23.17 mmol) in
acetonitrile (75 mL) was added potassium carbonate (9.61 g, 69.5
mmol), followed by ethanolamine (14.01 mL, 232 mmol). The
mixture was stirred at room temperature for 30 min. The mixture was
diluted with water and extracted into CH2Cl2. The combined organic
layer was dried (Na2SO4), filtered, concentrated, and purified by
column chromatography (EtOAc, followed by 2 M NH3 in MeOH/
CH2Cl2, 1:99 to 3:97) to obtain the title compound (3.1 g, 47.9%). 1H
NMR (DMSO-d6) δ 8.07 (d, J = 2.4 Hz, 1H), 8.03 (dd, J = 9.0, 2.4 Hz,
1H), 7.96 (d, J = 9.3 Hz, 1H), 3.73 (t, J = 6.15 Hz, 2H), 3.45 (t, J = 5.7
Hz, 2H), 2.84 (t, J = 6.4 Hz, 2H), 2.61 (t, J = 5.5 Hz, 2H), 1.90 (quint,
J = 6.2 Hz, 2H). ESI-MS (m/z, %): 280 (MH+, 100).
Phenyl 2-(6-Amino-3,4-dihydroquinolin-1(2H)-yl)ethyl-
(methyl)carbamate (29). A suspension of phenyl methyl(2-(6-
nitro-3,4-dihydroquinolin-1(2H)-yl)ethyl)carbamate (7) (0.5 g, 1.41
mmol) and palladium on activated carbon (10%, 75 mg, 0.07 mmol) in
a 1:1 mixture of THF/EtOH (20 mL) was stirred under a balloon of
hydrogen for 4.5 h. The suspension was filtered through a pad of
Celite. The pad was rinsed with methanol, and the filtrate was
concentrated to obtain the title compound (0.46 g, quantitative) as a
1
viscous oil. H NMR (CDCl3) δ 7.39−7.34 (m, 2H), 7.22−7.17 (m,
1H), 7.12−7.08 (m, 2H), 6.61−6.42 (m, 3H), 3.61−3.45 (m, 4H),
3.26 (t, J = 6.0 Hz, 2H), 3.20 (brs, 2H), 3.13 and 3.06 (2s, 3H), 2.70
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Journal of Medicinal Chemistry
Article
(t, J = 6.3 Hz, 2H), 1.96−1.87 (m, 2H). ESI-MS (m/z, %): 326
(MH+).
butylphosphine in hexane (10 wt %) (0.32 mL, 0.10 mmol), 2-((2-(6-
bromo-3,4-dihydroquinolin-1(2H)-yl)ethyl)(methyl)amino)ethanol
(13) (0.16 g, 0.52 mmol), and lithium bis(trimethylsilyl)amide (1 M
in THF, 1.58 mL, 1.58 mmol) as described for 30 to obtain the title
compound (57 mg, 43.4%) as an orange-red residue. 1H NMR
(DMSO-d6) δ 6.36−6.27 (m, 2H), 6.21 (brs, 1H), 4.31 (t, 1H, J = 5.4
Hz), 4.18 (brs, 2H), 3.44 (q, 2H, J = 6.3 Hz), 3.18 (t, 2H, J = 7.3 Hz),
3.10 (t, 2H, J = 5.3 Hz), 2.56−2.40 (m, 6H), 2.22 (s, 3H), 1.84−1.72
(m, 2H). ESI-MS (m/z, %): 250 (MH+, 100).
Phenyl 3-(6-Amino-3,4-dihydroquinolin-1(2H)-yl)propyl-
(methyl)carbamate (36). 36 was prepared from phenyl methyl(3-
(6-nitro-3,4-dihydroquinolin-1(2H)-yl)propyl)carbamate (8) (1.02 g,
2.76 mmol) as described for 29 to obtain the title compound (0.83 g,
89%) as a dark oil. ESI-MS (m/z, %): 340 (MH+, 100).
tert-Butyl 2-(6-Amino-3,4-dihydroquinolin-1(2H)-yl)ethyl-
(ethyl)carbamate (30). A suspension of tris(dibenzylideneacetone)-
dipalladium(0) (66 mg, 0.07 mmol) in anhydrous THF (3 mL) was
treated with tri-tert-butylphosphine in hexane (10 wt %) (0.43 mL,
0.14 mmol) and the mixture stirred for 5 min at room temperature. A
solution of tert-butyl 2-(6-bromo-3,4-dihydroquinolin-1(2H)-yl)ethyl-
(ethyl)carbamate (17) (0.27 g, 0.71 mmol) in THF (7 mL) was added
followed by lithium bis(trimethylsilyl)amide (1 M in THF, 1.43 mL,
1.43 mmol) and the mixture heated in a sealed reaction vial at 90 °C
for 3 h. The mixture was cooled to room temperature and treated with
TBAF (1 M in THF, 4 mL, 4 mmol) and stirred for 30 min. The
mixture was partitioned between water and EtOAc, transferred to a
separatory funnel and the organic layer separated. The aqueous layer
(pH 10) was further extracted with EtOAc and the combined organic
layers were washed with brine, dried (Na2SO4), filtered, and
concentrated to give a dark brown residue. The crude was purified
by column chromatography (2 M NH3 in MeOH/CH2Cl2, 2.5:97.5)
to obtain the title compound (171 mg, 74.6%) as a brown residue. 1H
NMR ((DMSO-d6) δ 6.42 (d, 1H, J = 8.5 Hz), 6.31−6.26 (m, 1H),
6.22 (brs, 1H), 4.20 (brs, 2H), 3.26−3.08 (m, 8H), 2.55 (t, 2H, J = 6.3
Hz), 1.82−1.74 (m, 2H), 1.41 (s, 9H), 1.02 (t, 3H, J = 7.0 Hz). ESI-
MS (m/z, %): 320 (MH+, 90), 264 (100).
tert-Butyl 2-(6-Amino-3,4-dihydroquinolin-1(2H)-yl)ethyl-
(isopropyl)carbamate (31). 31 was prepared from tris-
(dibenzylideneacetone)dipalladium(0) (35 mg, 0.03 mmol), tri-tert-
butylphosphine in hexane (10 wt %) (0.22 mL, 0.07 mmol), tert-butyl
2-(6-bromo-3,4-dihydroquinolin-1(2H)-yl)ethyl(isopropyl)carbamate
(15) (0.15 g, 0.37 mmol), and lithium bis(trimethylsilyl)amide (1 M
in THF, 0.75 mL, 0.75 mmol) as described for 30 to obtain the title
compound (85 mg, 67.5%) as a dark brown residue. 1H NMR
(DMSO-d6) δ 6.44 (d, 1H, J = 8.0 Hz), 6.28 (dd, 1H, J = 8.3, 2.1 Hz),
6.24−6.18 (m, 1H), 4.33−3.91 (m, 3H), 3.24−3.04 (m, 6H), 2.60−
2.50 (m, 2H), 1.85−1.74 (m, 2H), 1.44 (s, 9H), 1.07 (d, 6H, J = 6.8
Hz). ESI-MS (m/z, %): 334 (MH+, 100).
tert-Butyl 3-(6-Amino-3,4-dihydroquinolin-1(2H)-yl)propyl-
(2-hydroxyethyl)carbamate (37). 37 was prepared from tert-butyl
2-hydroxyethyl(3-(6-nitro-3,4-dihydroquinolin-1(2H)-yl)propyl)-
carbamate (28) (0.23 g, 0.60 mmol) as described for 29 to obtain the
1
title compound (0.21 g, 99%) as a dark oil. H NMR (DMSO-d6) δ
6.34−6.25 (m, 2H), 6.22 (m, 1H), 4.65−4.60 (m, 1H), 4.20 (brs, 2H),
3.48−3.41 (m, 2H), 3.22−3.15 (m, 4H), 3.06−3.01 (m, 4H), 2.59−
2.50 (m, 2H), 1.38−1.78 (m, 2H), 1.72−1.62 (m, 2H), 1.38 (s, 9H).
ESI-MS (m/z, %): 350 (MH+, 100).
Phenyl Methyl(2-(6-(thiophene-2-carboximidamido)-3,4-di-
hydroquinolin-1(2H)-yl)ethyl)carbamate (38). A solution of
phenyl 2-(6-amino-3,4-dihydroquinolin-1(2H)-yl)ethyl(methyl)-
carbamate (29) (0.445 g, 1.37 mmol) in EtOH (20 mL) was treated
with methyl thiophene-2-carbimidothioate hydroiodide (0.78 g, 2.73
mmol) and stirred overnight at room temperature. Argon was bubbled
through the mixture for 20 min. Then it was partitioned between
CH2Cl2 and saturated NaHCO3 solution. After extraction, the organic
layer was separated and the aqueous layer was extracted with an
additional CH2Cl2. The combined organic layer was dried (Na2SO4),
filtered, and concentrated to give a dark oil which was purified by flash
column chromatography (MeOH/CH2Cl2, 2.5:97.5, then 2 M NH3 in
MeOH/CH2Cl2, 2.5:97.5 to 7.5:92.5) to obtain the title compound
1
(0.4 g, 67.2%) as a yellow-brown solid. H NMR (DMSO-d6) δ 7.68
tert-Butyl 2-(6-Amino-3,4-dihydroquinolin-1(2H)-yl)ethyl(2-
hydroxyethyl)carbamate (32). 32 was prepared from tert-butyl 2-
hydroxyethyl(2-(6-nitro-3,4-dihydroquinolin-1(2H)-yl)ethyl)-
carbamate (24) (3.3 g, 9.03 mmol) as described for 29 to obtain the
(brs, 1H), 7.58 (d, J = 5.1 Hz, 1H), 7.42−7.34 (m, 2H), 7.24−7.18 (m,
1H), 7.11−7.03 (m, 3H), 6.68 (d, J = 9.0 Hz, 1H), 6.57−6.51 (m,
2H), 6.29 (brs, 2H), 3.58−3.44 (m, 4H), 3.32−3.27 (m, 2H), 3.09,
2.97 (2s, 3H), 2.73−2.65 (m, 2H), 1.90−1.83 (m, 2H). ESI-MS (m/z,
%): 435 (MH+, 100%).
tert-Butyl Ethyl(2-(6-(thiophene-2-carboximidamido)-3,4-di-
hydroquinolin-1(2H)-yl)ethyl)carbamate (39). 39 was prepared
from tert-butyl 2-(6-amino-3,4-dihydroquinolin-1(2H)-yl)ethyl(ethyl)-
carbamate (30) (0.165 g, 0.51 mmol) and methyl thiophene-2-
carbimidothioate hydroiodide (0.295 g, 1.03 mmol) as described for
38 to obtain the title compound (0.12 g, 54.2%) as a yellow-orange
solid. 1H NMR (DMSO-d6) δ 7.69 (d, 1H, J = 3.2 Hz), 7.58 (d, 1H, J
= 5.0 Hz), 7.08 (dd, 1H, J = 5.0, 3.7 Hz), 6.68−6.59 (m, 1H), 6.59−
6.47 (m, 2H), 6.50−6.20 (brs, 2H), 3.33−3.16 (m, 8H), 2.66 (t, 2H, J
= 6.2 Hz), 1.91−1.79 (m, 2H), 1.41 (s, 9H), 1.04 (t, 3H, J = 7.0 Hz).
ESI-MS (m/z, %): 429 (MH+, 100).
tert-Butyl Isopropyl(2-(6-(thiophene-2-carboximidamido)-
3,4-dihydroquinolin-1(2H)-yl)ethyl)carbamate (40). 40 was
prepared from tert-butyl-2-(6-amino-3,4-dihydroquinolin-1(2H)-yl)-
ethyl(isopropyl) carbamate (31) (0.085 g, 0.25 mmol) and methyl
thiophene-2-carbimidothioate hydroiodide (0.127 g, 0.44 mmol) as
described for 38 to obtain the title compound (0.054 g, 47.9%) as a
yellow solid. 1H NMR (DMSO-d6) δ 7.70 (d, 1H, J = 3.4 Hz), 7.59 (d,
1H, J = 5.0 Hz), 7.09 (dd, 1H, J = 5.0, 3.6 Hz), 6.71−6.51 (m, 3H),
6.51−6.28 (brs, 2H), 4.29−3.94 (m, 1H), 3.43−3.18 (m, 6H), 2.66 (t,
2H, J = 5.8 Hz), 1.91−1.80 (m, 2H), 1.46 (s, 9H), 1.11 (d, 6H, J = 6.8
Hz). ESI-MS (m/z, %): 443 (MH+, 100).
tert-Butyl 2-Hydroxyethyl(2-(6-(thiophene-2-carboximida-
mido)-3,4-dihydroquinolin-1(2H)-yl)ethyl)carbamate (41). 41
was prepared from tert-butyl 2-(6-amino-3,4-dihydroquinolin-1(2H)-
yl)ethyl(2-hydroxyethyl)carbamate (32) (3.61 g, 10.76 mmol) and
methyl thiophene-2-carbimidothioate hydroiodide (6.14 g, 21.52
mmol) as described for 38 to obtain the title compound (3.13 g,
1
title compound as a dark oil. H NMR (DMSO-d6) δ 6.43 (d, J = 8.4
Hz, 1H), 6.28 (d, J = 8.7 Hz, 1H), 6.22 (s, 1H), 4.35 (t, J = 5.1 Hz,
1H), 4.18 (s, 2H), 3.48−3.31 (m, 2H), 3.28−3.16 (m, 6H), 3.13−3.10
(m, 2H), 2.55 (t, J = 6.4 Hz, 2H), 1.80−1.76 (m, 2H), 1.41 (s, 9H).
ESI-MS (m/z, %): 336 (MH+, 100), 280 (100).
tert-Butyl 3-(6-Amino-3,4-dihydroquinolin-1(2H)-yl)-
pyrrolidine-1-carboxylate (33). 33 was prepared from Pd2(dba)3
(46 mg, 0.05 mmol), PtBu3 (0.6 mL of a 10 wt % in hexanes solution,
0.2 mmol), tert-butyl 3-(6-bromo-3,4-dihydroquinolin-1(2H)-yl)-
pyrrolidine-1-carboxylate (18) (0.38 g, 1.00 mmol), and lithium
hexamethyldisilizane (2.0 mL, 1 M solution in THF, 2.0 mmol) as
described for 30 to obtain the title compound (0.295 g, 93.1%) as a
viscous dark brown residue. 1H NMR (CDCl3) δ 6.59−6.44 (m, 3H),
4.35−4.23 (m, 1H), 3.59−3.11 (m, 8H), 2.69 (t, J = 6.3 Hz, 2H),
2.09−2.04 (m, 2H), 1.93−1.87 (m, 2H), 1.47 (s, 9H). ESI-MS (m/z,
%): 318 (MH+, 100%).
1-(2-(Ethyl(methyl)amino)ethyl)-1,2,3,4-tetrahydroquinolin-
6-amine (34). 34 was prepared from 2-(6-bromo-3,4-dihydroquino-
lin-1(2H)-yl)-N-ethyl-N-methylethanamine (23) (0.5 g, 1.68 mmol),
tris(dibenzylideneacetone)dipalladium(0) (77 mg, 0.08 mmol), tri-
tert-butylphosphine in hexane (10 wt %) (0.61 mL, 0.20 mmol), and
lithium hexamethyldisilazide (1 M in THF, 5.05 mL, 5.05 mmol) as
described for 30 to obtain the title compound (0.35 g, 89%) as a dark
1
brown residue. H NMR (CDCl3) δ 6.49−6.48 (2H), 6.41 (brs, 1H),
3.36−3.31 (m, 2H), 3.21 (t, J = 5.7 Hz, 2H), 3.20 (brs, 2H), 2.68 (t, J
= 6.3 Hz, 2H), 2.56−2.43 (m, 4H), 2.28 (s, 3H), 1.95−1.87 (m, 2H),
1.07 (t, J = 7.2 Hz, 3H). ESI-MS (m/z, %): 234 (MH+).
2-((2-(6-Amino-3,4-dihydroquinolin-1(2H)-yl)ethyl)(methyl)-
amino)ethanol (35). 35 was prepared from tris-
(dibenzylideneacetone)dipalladium(0) (48 mg, 0.05 mmol), tri-tert-
2890
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65.4%). 1H NMR (DMSO-d6) δ 7.68 (d, J = 3.3 Hz, 1H), 7.56 (d, J =
4.8 Hz, 1H), 7.07 (dd, J = 4.8, 3.3 Hz, 1H), 6.66−6.62 (m, 1H), 6.55−
6.46 (m, 2H), 6.27 (brs, 2H), 4.71 (t, J = 4.8 Hz, 1H), 3.49−3.45 (m,
2H), 3.36−3.32 (m, 4H), 3.25−3.21 (m, 4H), 2.66 (t, J = 6.2 Hz, 2H),
1.86−1.82 (m, 2H), 1.42 (s, 9H). ESI-MS (m/z, %): 445 (MH+, 100).
tert-Butyl 3-(6-(Thiophene-2-carboximidamido)-3,4-dihy-
droquinolin-1(2H)-yl)pyrrolidine-1-carboxylate (42). 42 was
prepared from tert-butyl 3-(6-amino-3,4-dihydroquinolin-1(2H)-yl)-
pyrrolidine-1-carboxylate (33) (0.21 g, 0.66 mmol) and methyl
thiophene-2-carbimidothioate hydroiodide (0.37 g, 1.32 mmol) as
described for 38 to obtain the title compound (0.168 g, 59.6%) as a
yellow solid. 1H NMR (DMSO-d6) δ 7.68 (d, J = 3.3 Hz, 1H), 7.57 (d,
J = 5.1 Hz, 1H), 7.07 (dd, J = 3.9, 5.1 Hz, 1H), 6.73 (d, J = 8.7 Hz,
1H), 6.59−6.52 (m, 2H), 6.34 (brs, 2H), 4.46−4.34 (m, 1H), 3.51−
3.10 (m, 6H), 2.69 (t, J = 6.3 Hz, 2H), 2.08−1.99 (m, 2H), 1.85−1.78
(m, 2H), 1.41 (s, 9H). ESI-MS (m/z, %): 427 (MH+, 100%).
N-(1-(2-(Ethyl(methyl)amino)ethyl)-1,2,3,4-tetrahydroquino-
lin-6-yl)thiophene-2-carboximidamide (43). 43 was prepared
from 1-(2-(ethyl(methyl)amino)ethyl)-1,2,3,4-tetrahydroquinolin-6-
amine (34) (0.335 g, 1.43 mmol) and methyl thiophene-2-
carbimidothioate hydroiodide (0.81 g, 2.87 mmol) as described for
38 to obtain the title compound (0.36 g, 73.2%) as a dark yellow
residue. 1H NMR (DMSO-d6) δ 7.67 (d, J = 3.6 Hz, 1H), 7.55 (dd, J =
0.9, 5.1 Hz, 1H), 7.07 (dd, J = 3.6, 4.8 Hz, 1H), 6.58−6.48 (3H), 6.30
(brs, 2H), 3.30−3.22 (m, 4H), 2.65 (t, J = 6.0 Hz, 2H), 2.46−2.36 (m,
4H), 2.20 (s, 3H), 1.87−1.80 (m, 2H), 0.97 (t, J = 7.2 Hz, 3H). ESI-
MS (m/z, %): 343 (MH+). ESI-HRMS calculated for C19H27N4S-
(MH+), 343.1950; observed, 343.1946.
extraction, the organic layer was separated, and the aqueous layer
was extracted once again with CH2Cl2. The combined organic layer
was washed with brine, dried (Na2SO4), filtered, and concentrated to
give a dark residue. This residue was purified by flash column
chromatography (2 M NH3 in MeOH/CH2Cl2, 5:95) to obtain the
title compound (0.155 g, 56.8%) as a yellow solid. 1H NMR (CD3OD)
δ 7.98−7.95 (m, 2H), 7.31−7.28 (m, 1H), 7.05 (dd, J = 2.4, 8.7 Hz,
1H), 6.97 (d, J = 2.4 Hz, 1H), 6.81 (d, J = 8.7 Hz, 1H), 3.62 (t, J = 6.6
Hz, 2H), 3.34 (t, J = 5.4 Hz, 2H), 3.20 (t, J = 6.9 Hz, 2H), 2.78 (t, J =
6.0 Hz, 2H), 2.72 (s, 3H), 1.99−1.92 (m, 2H); 13C NMR (DMSO-d6)
δ 156.39, 144.55, 134.30, 133.92, 129.17, 128.55, 126.13, 124.46,
123.38, 122.35, 111.24, 48.86, 46.88, 44.40, 32.57, 27.32, 21.17. ESI-
MS (m/z, %): 315 (MH+). ESI-HRMS calculated for C17H23N4S-
(MH+), 315.1637; observed, 315.1629.
N-(1-(3-(Methylamino)propyl)-1,2,3,4-tetrahydroquinolin-6-
yl)thiophene-2-carboximidamide (48). 48 was prepared from
phenyl methyl(3-(6-(thiophene-2-carboximidamido)-3,4-dihydroqui-
nolin-1(2H)-yl)propyl)carbamate (45) (0.63 g, 1.40 mmol) as
1
described for 47 to obtain the title compound (0.13 g, 28.2%). H
NMR (DMSO-d6) δ 7.66 (d, J = 3.3 Hz, 1H), 7.54 (d, J = 5.1 Hz, 1H),
7.06 (dd, J = 5.1, 3.6 Hz, 1H), 6.54 (brs, 2H), 6.47 (s, 1H), 6.21 (brs,
2H), 3.26−3.16 (m, 4H), 2.66 (t, J = 6.4 Hz, 2H), 2.53−2.48 (m, 2H),
2.28 (s, 3H), 1.85 (quint, J = 5.5 Hz, 2H), 1.64 (quint, J = 7.2 Hz, 2H).
N-(1-(2-(Ethylamino)ethyl)-1,2,3,4-tetrahydroquinolin-6-yl)-
thiophene-2-carboximidamide (49). A solution of tert-butyl
ethyl(2-(6-(thiophene-2-carboximidamido)-3,4-dihydroquinolin-
1(2H)-yl)ethyl)carbamate (39) (0.115 g, 0.26 mmol) in dry MeOH
(10 mL) was treated with 2 M HCl (1.35 mL, 2.7 mmol) and the
mixture heated to reflux for 90 min. After cooling to room
temperature, the solution was concentrated and dried briefly on a
high-vacuum pump. The residue was purified by column chromatog-
raphy (2 M NH3 in MeOH/CH2Cl2, 1:9) to obtain the title
N-(1-(2-((2-Hydroxyethyl)(methyl)amino)ethyl)-1,2,3,4-tetra-
hydroquinolin-6-yl)thiophene-2-carboximidamide (44). 44 was
prepared from 2-((2-(6-amino-3,4-dihydroquinolin-1(2H)-yl)ethyl)-
(methyl)amino)ethanol (35) (52 mg, 0.209 mmol) and methyl
thiophene-2-carbimidothioate hydroiodide (0.119 g, 0.41 mmol) as
described for 38 to obtain the title compound (44 mg, 58.9%) as a
1
compound (78 mg, 88%) as a yellow solid. H NMR (DMSO-d6) δ
7.66 (d, 1H, J = 3.0 Hz), 7.54 (dd, 1H, J = 5.0, 0.9 Hz), 7.06 (dd, 1H, J
= 5.0, 3.7 Hz), 6.59−6.52 (m, 2H), 6.47 (brs, 1H), 6.21 (brs, 2H),
3.32−3.20 (m, 4H), 2.70−2.64 (m, 4H), 2.56 (q, 2H, J = 7.1 Hz),
1.88−1.80 (m, 2H), 1.04 (t, 3H, J = 7.0 Hz). ESI-MS (m/z, %): 329
(MH+, 100), 258 (100). ESI-HRMS calculated for C18H25N4S (MH+),
329.1794; observed, 329.1798.
N-(1-(2-(Isopropylamino)ethyl)-1,2,3,4-tetrahydroquinolin-
6-yl)thiophene-2-carboximidamide (50). 50 was prepared from
tert-butyl isopropyl(2-(6-(thiophene-2-carboximidamido)-3,4-dihydro-
quinolin-1(2H)-yl)ethyl)carbamate (40) (50 mg, 0.11 mmol) as
described for 49 to obtain the title compound (40 mg, quantitative) as
a yellow solid. 1H NMR (DMSO-d6) δ 7.66 (d, 1H, J = 3.1 Hz), 7.54
(d, 1H, J = 5.1 Hz), 7.06 (dd, 1H, J = 5.0, 3.7 Hz), 6.61−6.50 (m, 2H),
6.47 (brs, 1H), 6.21 (brs, 2H), 3.30−3.18 (m, 4H), 2.79−2.70 (m,
1H), 2.70−2.61 (m, 4H), 1.88−1.80 (m, 2H), 0.97 (d, 6H, J = 6.2
Hz). ESI-MS (m/z, %): 343 (MH+, 100). ESI-HRMS calculated for
C19H27N4S (MH+), 343.1950; observed, 343.1953.
N-(1-(2-(2-Hydroxyethylamino)ethyl)-1,2,3,4-tetrahydroqui-
nolin-6-yl)thiophene-2-carboximidamide (51). 51 was prepared
from tert-butyl 2-hydroxyethyl(2-(6-(thiophene-2-carboximidamido)-
3,4-dihydroquinolin-1(2H)-yl)ethyl)carbamate (41) (3.13 g, 7.04
mmol) as described for 49 to obtain the title compound (1.65 g,
68.0%). 1H NMR (DMSO-d6) δ 7.67 (d, J = 3.6 Hz, 1H), 7.54 (d, J =
4.8 Hz, 1H), 7.06 (dd, J = 4.8, 3.6 Hz, 1H), 6.60−6.52 (m, 2H), 6.48
(s, 1H), 3.45 (t, J = 5.7 Hz, 2H), 3.30−3.21 (m, 4H), 2.72−2.59 (m,
6H), 1.88−1.80 (m, 2H). ESI-MS (m/z, %): 345 (MH+, 100). ESI-
HRMS calculated for C18H25N4OS (MH+), 344.1741; observed,
345.1743.
1
yellow solid. H NMR (DMSO-d6) δ 7.67 (d, 1H, J = 3.0 Hz), 7.55
(dd, 1H, J = 5.0, 0.9 Hz), 7.07 (dd, 1H, J = 5.0, 3.7 Hz), 6.60−6.51 (m,
2H), 6.48 (brs, 1H), 6.29 (brs, 2H), 4.34 (t, 1H, J = 5.3 Hz), 3.46 (q,
2H, J = 6.2 Hz), 3.32−3.22 (m, 4H), 2.65 (t, 2H, J = 6.2 Hz), 2.50−
2.44 (m, 4H), 2.25 (s, 3H), 1.88−1.80 (m, 2H). ESI-MS (m/z, %):
359 (MH+, 100). ESI-HRMS calculated for C19H27N4OS (MH+),
359.1900; observed, 359.1908.
Phenyl Methyl(3-(6-(thiophene-2-carboximidamido)-3,4-di-
hydroquinolin-1(2H)-yl)propyl)carbamate (45). 45 was prepared
from phenyl 3-(6-amino-3,4-dihydroquinolin-1(2H)-yl)propyl-
(methyl)carbamate (36) (0.83 g, 2.44 mmol) and methyl thiophene-
2-carbimidothioate hydroiodide (1.39 g, 4.89 mmol) as described for
1
38 to obtain the title compound (0.63 g, 57.4%). H NMR (DMSO-
d6) δ 7.67 (d, J = 3.0 Hz, 1H), 7.55 (d, J = 4.8 Hz, 1H), 7.41−7.35 (m,
2H), 7.23−7.21 (m, 1H), 7.13−7.05 (m, 3H), 6.56 (m, 2H), 6.49 (brs,
1H), 6.25 (brs, 2H), 3.50−3.45 (m, 2H), 3.38−3.32 (m, 2H), 3.23−
3.19 (m, 2H), 3.06, 2.93 (2s, 3H), 2.69−2.64 (m, 2H), 1.89−1.81 (m,
4H).
tert-Butyl 2-Hydroxyethyl(3-(6-(thiophene-2-carboximida-
mido)-3,4-dihydroquinolin-1(2H)-yl)propyl)carbamate (46). 46
was prepared from tert-butyl 3-(6-amino-3,4-dihydroquinolin-1(2H)-
yl)propyl(2-hydroxyethyl)carbamate (37) (0.2 g, 0.57 mmol) and
methyl thiophene-2-carbimidothioate hydroiodide (0.32 g, 1.14 mmol)
as described for 38 to obtain the title compound (0.112 g, 42.7%). 1H
NMR (DMSO-d6) δ 7.67 (d, J = 3.6 Hz, 1H), 7.55 (d, J = 4.2 Hz, 1H),
7.07 (dd, J = 4.2, 3.6 Hz, 1H), 6.54−6.47 (m, 3H), 6.29−6.21 (brs,
2H), 4.68−4.62 (m, 1H), 3.50−3.42 (m, 2H), 3.27−3.12 (m, 8H),
2.69−2.63 (m, 2H), 1.89−1.81 (m, 2H), 1.78−1.69 (m, 2H), 1.39 (s,
9H). ESI-MS (m/z, %): 459 (MH+, 100).
N-(1-(2-(Methylamino)ethyl)-1,2,3,4-tetrahydroquinolin-6-
yl)thiophene-2-carboximidamide (47). A solution of phenyl
methyl(2-(6-(thiophene-2-carboximidamido)-3,4-dihydroquinolin-
1(2H)-yl)ethyl)carbamate (38) (0.38 g, 0.87 mmol) in ethanol (15
mL) was treated with NaOH (0.35 g, 8.70 mmol) followed by H2O (8
mL). The mixture was heated at reflux for 6 h. The solution was
concentrated and partitioned between CH2Cl2 and brine. After
N-(1-(Pyrrolidin-3-yl)-1,2,3,4-tetrahydroquinolin-6-yl)-
thiophene-2-carboximidamide (52). A solution of tert-butyl-3-(6-
(thiophene-2-carboximidamido)-3,4-dihydroquinolin-1(2H)-yl)-
pyrrolidine-1-carboxylate (42) (150 mg, 0.35 mmol) in methanol (5
mL) was treated with 1 N HCl (10 mL) and heated at 70 °C for 30
min. The solution was concentrated and dried under reduced pressure
to give a yellow solid. The solid was triturated with MeOH/Et2O
(5:95) and dried under reduced pressure to obtain the dihydrochloride
1
salt of the title compound (0.125 g 89.3%). H NMR (DMSO-d6) δ
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11.23 (s, 1H), 9.78 (brs, 1H), 9.65 (brs, 1H, 9.59 (brs, 1H), 8.61 (s,
1H), 8.15−8.14 (m, 2H), 7.36 (pseudo t, J = 4.5 Hz, 1H), 7.09−6.88
(m, 3H), 4.76−4.66 (m, 1H), 3.39−3.06 (m, 6H), 2.72 (t, J = 5.4 Hz,
2H), 2.16−2.01 (m, 2H), 1.87−1.83 (m, 2H). ESI-MS (m/z, %): 327
(MH+). ESI-HRMS calculated for C18H23N4S (MH+), 327.1637;
observed, 327.1649.
N-(1-(3-(2-Hydroxyethylamino)propyl)-1,2,3,4-tetrahydro-
quinolin-6-yl)thiophene-2-carboximidamide (53). 53 was pre-
pared from tert-butyl 2-hydroxyethyl(3-(6-(thiophene-2-carboximida-
mido)-3,4-dihydroquinolin-1(2H)-yl)propyl)carbamate (46) (0.1 g,
0.21 mmol) as described for 49 to obtain the title compound (0.042 g,
53.7%). 1H NMR (DMSO-d6) δ 7.67 (d, J = 3.6 Hz, 1H), 7.54 (d, J =
4.8 Hz, 1H), 7.06 (dd, J = 4.8, 3.6 Hz, 1H), 6.55 (m, 2H), 6.47 (m,
1H), 6.22 (brs, 2H), 4.45 (t, J = 4.9 Hz, 1H), 3.45 (t, J = 5.2 Hz, 2H),
3.26−3.17 (m, 4H), 2.68−2.63 (m, 2H), 2.59−2.54 (m, 4H), 1.89−
1.82 (m, 2H), 1.69−1.60 (m, 2H). ESI-HRMS calculated for
C19H27N4OS (MH+), 359.1907; observed, 359.19.
General Procedure for the Conversion of the Free Base to
the Dihydrochloride Salt. To a solution of the free base (1.0 equiv)
in methanol was added 1 M HCl in diethyl ether (3.0 equiv). The
solution was stirred at room temperature for 10 min and then
concentrated to dryness. The residue was dried under reduced
pressure for 2 days to give a solid. In all cases, the HPLC purity of the
salt is similar to that of the free base.
Biology. NOS Inhibition Assay. Recombinant human inducible
NOS (iNOS), human endothelial constitutive NOS (eNOS), or
human neuronal constitutive NOS (nNOS) was produced in
baculovirus-infected Sf9 cells (ALEXIS). In a radiometric method,
NO synthase activity is determined by measuring the conversion of
[3H]L-arginine to [3H]L-citrulline. To measure iNOS, 10 μL of enzyme
is 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 eNOS or nNOS, 10 μL of enzyme is 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 enzyme inhibition, a 15 μL solution of a test substance
is added to the enzyme assay solution, followed by a preincubation
time of 15 min at room temperature. The reaction is 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 is
150 μL in every well. The reactions are carried out at 37 °C for 45
min. The reaction is 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 is separated by DOWEX (ion-exchange resin
DOWEX 50WX 8-400, Sigma), and the DOWEX is removed by
spinning at 12000g for 10 min in the centrifuge. An 70 μL aliquot of
the supernatant is added to 100 μL of scintillation fluid, and the
samples are 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 inhibitor L-NMMA.
All assays are performed at least in duplicate. Standard deviations are
10% or less.
Chung Model of Injury-Induced Neuropathic-like Pain.
Nerve ligation injury was performed according to the method
described by Kim and Chung.29 This technique produces signs of
neuropathic dysesthesias, including tactile allodynia, thermal hyper-
algesia, and guarding of the affected paw which begins on day 1 of the
surgery and peaks on day 16. Rats were anesthetized with halothane,
and the vertebrae over the L4 to S2 region were exposed. The L5 and
L6 spinal nerves were exposed, carefully isolated, and tightly ligated
with 4-0 silk suture distal to the DRG. After homeostatic stability was
ensured, the wounds were sutured and the animals allowed to recover
in individual cages. Sham-operated rats were prepared in an identical
fashion except that the L5/L6 spinal nerves were not ligated. Rats
exhibiting signs of motor deficiency were euthanized. 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. 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 was stabilized, the
compound 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 4.
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 the compound was con-
structed from the percentage reductions of current amplitude by
sequential concentrations.
HPLC Purity. Analytical HPLC spectra were collected on an
Agilent 1100 HPLC system using a reverse phase column. All final
compounds were >95% purity. Results are shown in Table 5.
AUTHOR INFORMATION
■
Corresponding Author
*Telephone: 1-416- 673-8461. Fax: 1-905-823-5836. E-mail:
jramnauth@neuraxon.com.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to CEREP (U.S. and France) for performing
the hERG manual 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; L-NMMA, N-mono-
methyl-L-arginine; CNS MPO, central nervous system multi-
parameter optimization; hERG, human ether-a-go-go-related
gene; SAR, structure−activity relationship; SNL, spinal nerve
ligation
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