Nitrile in the Hole: Discovery of a Small Auxiliary Pocket in Neuronal Nitric Oxide Synthase Leading to the Development of Potent and Selective 2-Aminoquinoline Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.7b00259

Neuronal nitric oxide synthase (nNOS) inhibition is a promising strategy to treat neurodegenerative disorders, but the development of nNOS inhibitors is often hindered by poor pharmacokinetics. We previously developed a class of membrane-permeable 2-amino

Full text of DOI:10.1021/acs.jmedchem.7b00259

Article
pubs.acs.org/jmc
Nitrile in the Hole: Discovery of a Small Auxiliary Pocket in Neuronal
Nitric Oxide Synthase Leading to the Development of Potent and
Selective 2‑Aminoquinoline Inhibitors
Maris A. Cinelli,† Huiying Li,‡ Georges Chreifi,‡ Thomas L. Poulos,*,‡ and Richard B. Silverman*,†
†Department of Chemistry, Department of Molecular Biosciences, Chemistry of Life Processes Institute, Center for Molecular
Innovation and Drug Discovery, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113, United States
‡Departments of Molecular Biology and Biochemistry, Pharmaceutical Sciences, and Chemistry, University of California, Irvine,
Irvine, California 92697-3900, United States
S
* Supporting Information
ABSTRACT: Neuronal nitric oxide synthase (nNOS) inhibition is a promising strategy
to treat neurodegenerative disorders, but the development of nNOS inhibitors is often
hindered by poor pharmacokinetics. We previously developed a class of membrane-
permeable 2-aminoquinoline inhibitors and later rearranged the scaffold to decrease off-
target binding. However, the resulting compounds had decreased permeability, low
human nNOS activity, and low selectivity versus human eNOS. In this study, 5-
substituted phenyl ether-linked aminoquinolines and derivatives were synthesized and
assayed against purified NOS isoforms. 5-Cyano compounds are especially potent and
selective rat and human nNOS inhibitors. Activity and selectivity are mediated by the
binding of the cyano group to a new auxiliary pocket in nNOS. Potency was enhanced
by methylation of the quinoline and by introduction of simple chiral moieties, resulting
in a combination of hydrophobic and auxiliary pocket effects that yielded high (∼500-
fold) n/e selectivity. Importantly, the Caco-2 assay also revealed improved membrane permeability over previous compounds.
reserves,5,6 damage to various cellular structures, and the
eventual apoptosis or necrosis of neurons, leading progressively
to the symptoms characteristic of neurodegeneration. Studies
INTRODUCTION
■
Neurodegenerative disorders (Alzheimer’s and Parkinson’s
diseases, amyotrophic lateral sclerosis, Huntington’s disease,
have shown that hyperactive nNOS and dysfunctional nitrergic
and others) are characterized by the gradual loss of neuronal
signaling are affiliated with or directly implicated in the
function and structure. The resulting symptoms cause great
suffering not only to patients but also to their caretakers, the
economy, and to global health in general. Effective treatments
for neurodegenerative diseases are limited, and the develop-
pathology of many neurodegenerative disorders7−10 making
nNOS a desirable target for therapeutic intervention.9,11,12
nNOS functions by converting L-arginine to L-citrulline and
NO via an electron relay proceeding through five cofactors.
ment of novel therapeutics to treat neurodegeneration is a
nNOS is only functional as a homodimer with each monomer
highly desirable unmet medical need.
containing an oxygenase domain and a reductase domain that
Neuronal nitric oxide synthase (nNOS) is an enzymatic
are joined by a linker domain where calmodulin, in response to
target under investigation for the treatment of neurodegener-
elevated calcium levels, binds and activates the enzyme. Once
ative disorders (as well as other conditions characterized by
activated, electron flow proceeds from the reductase domain-
bound reduced nicotinamide adenine dinucleotide phosphate
(NADPH), to flavin adenine dinucleotide (FAD), to flavin
neuronal damage, such as stroke, ischemic events, cerebral
palsy, and neuropathic pain).1 Three NOS isoenzymes produce
nitric oxide (NO), a free-radical second-messenger molecule, in
the human body: endothelial NOS (eNOS) produces the NO
employed in blood pressure regulation and smooth muscle
tone, inducible NOS (iNOS) plays a role in immune activation,
mononucleotide (FMN), and then from the FMN subdomain
of one monomer to the other monomer’s oxygenase domain,13
through (6R)-5,6,7,8-tetrahydrobiopterin (H4B), and finally to
the heme active center, where the bound L-arginine is oxidized
and in the CNS, the NO produced by nNOS is required for
in the presence of molecular oxygen.14
normal neuronal signaling.2
Most nNOS inhibitors are competitive with the substrate and
resemble L-arginine in their physicochemical properties.
Unfortunately, these polar, high-pKa, ionizable molecules
Under neuroinflammatory or neurodegenerative phenotypes,
however, nNOS can become overactive or overexpressed, and
NO levels surge several orders of magnitude, where NO can
cause damage or combine to form other damaging species like
peroxynitrite.3 These species can cause protein nitration and
aggregation,4 depletion of cellular energy and glutathione
Received: February 17, 2017
© XXXX American Chemical Society
A
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often suffer from low bioavailability and low CNS permeation,
which severely limits their therapeutic use. An additional
challenge is that an inhibitor must be selective for nNOS over
eNOS and iNOS, as inhibition of eNOS could cause
cardiovascular liabilities,15 whereas iNOS inhibition could
disrupt immune system activation. This is a daunting task,
however, as all three NOS isoforms share very similar
sequences and structure.16
rats, and was extremely promiscuous in CNS counterscreens.
The second-generation,18 rearranged phenyl ether 4 (optimized
from lead 3), preserved the potency and selectivity of 1 and 2
while drastically decreasing the off-target binding, but this
compound had significantly decreased Caco-2 permeability, low
human nNOS activity, and similarly low selectivity for hnNOS
over heNOS.
We chose to continue investigating this cleaner-binding
phenyl ether scaffold in an attempt to improve n/e selectivity,
hnNOS inhibitory potency, and possibly cellular permeability.
First, the 5-position of the phenyl ring (Figure 2) was
substituted with a variety of groups, leading to analogues 5−
9. Previously, the 1,3,5-trisubstituted phenyl or pyridyl moieties
of 2-aminopyridine inhibitors19−21 were able to access nNOS-
specific residues such as Asp597 (Asp602 in hnNOS) or other
nNOS-specific regions and lead to high n/e selectivity. It was
proposed that analogous substituents on the phenyl ether
scaffold could reach potentially similar nNOS-specific regions
that could improve hnNOS potency, such as the hnNOS-
specific residue His342.
Previously, we reported several classes of nNOS inhibitors
based on a 2-aminoquinoline scaffold.17,18 Our first generation
of inhibitors, such as 1 and 2 (Figure 1), were potent, selective,
Second, it was previously reported that for 2-aminopyridines,
installation of a methyl group at the 4-position of the pyridine
could drastically improve potency and, in some cases,
selectivity.22 A fragment screen then showed that 2-amino-4-
methylquinoline bound nearly 7-fold tighter (Ki = 94 nM) to
rat nNOS than the unmethylated 2-aminoquinoline (630 nM).
X-ray crystallography indicated that the 4-methyl compound
acted as a competitive L-arginine antagonist. To this end, the
methylated analogues of compounds 8 and 9 (11 and 13,
respectively) and of the original phenyl ether leads 3 and 4 (10
and 12, respectively) were prepared.
We also investigated the removal of the oxygen from the
ether linkage entirely (as seen in many previous amino-
pyridines),19,21 as the methylene adjacent to the phenyl ether
could serve as a potential site of metabolism, and removal of the
oxygen would lower the tPSA of the molecule (it is 50.41 Å2 for
14). Therefore, analogues 14 and 16, and desmethyl analogue
15 (deoxygenated analogues of active compounds 10, 11, and
8, respectively) were synthesized.
Figure 1. Previous use of 2-aminoquinolines as nNOS inhibitors.
and possessed excellent cellular and in vivo pharmacokinetics.17
Unfortunately, 2 was selective for rat nNOS (rnNOS) over
human nNOS (hnNOS), displayed low selectivity for human
nNOS over human eNOS (heNOS), caused toxic side effects in
Figure 2. Design strategy utilized and compounds synthesized in this study. All molecules have a CLogP between 2.5 and 4 (lower for cyano
compounds and higher for deoxy compounds), and tPSA (total polar surface area) of 50−83 Å2 (higher for cyano compounds and lower for deoxy
compounds).
B
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a
Finally, more specific efforts to optimize the most potent and
selective scaffold (the 5-cyanophenyl ether, as in 8 and 11)
were made, all centered on improving n/e selectivity and
overall hydrophobicity. The short methylamine tail of 11 was
replaced with the ethylamine of 18 and the chiral α-methyl
ethylamine of 19, to direct an extra alkyl group in the area of
the nNOS-specific hydrophobic residue, Met336/Met341
(rnNOS/hnNOS).23 van der Waals contact between inhibitors
and this residue has been implicated in improved n/e
selectivity, as this residue is replaced by a smaller valine in
eNOS isoforms.18 Similarly, compound 17 combines a 4-
substitution pattern (as in 4) with the 5-cyano group.
Scheme 2
All compounds were assayed against rnNOS, and select
compounds were also assayed against hnNOS. To shift our
structure−activity relationship (SAR) work toward increasingly
more human systems, human eNOS (heNOS) was employed
instead of previously used bovine eNOS. Murine iNOS
(miNOS) was used, and n/i selectivity is reported as the
ratio of miNOS/rnNOS. Finally, as most of these molecules
have excellent chemical properties (Figure 1), potent and
selective compounds were assayed in a Caco-2 assay to
approximate their cellular permeability.
a
CHEMISTRY
Reagents and conditions: (a) (i) phenols 23−26, 29, NaH, DMF, 0
°C, ii. 30 (in DMF), 0 °C; (b) (i) K2CO3, MeOH, reflux; (ii) HCl/
MeOH, ether, r.t., or TFA/DCM (for 35), after isolation.
■
To prepare the initial set of 5-substituted quinoline analogues
(5−9), a series of 3,5-disubstituted phenols was first
synthesized (Scheme 1). Commercially available aldehydes
35). Compounds 31−35 were first deacetylated using K2CO3
in hot methanol, and the Boc groups were then cleaved using
HCl (31, 32, and 34) or TFA (33 and 35) to yield pure final
compounds 5−9 as water-soluble hydrochloride or trifluor-
oacetate salts.
a
Scheme 1
Because of the reactivity and acidity of 4-methylquinolines,
the analogous bromide (43) could not be prepared by our
previous synthetic route (which utilized both free-radical
bromination of a 7-methylquinoline and basic conditions that
are incompatible with the 4-methyl group). A new synthetic
route had to be devised,25 and we envisioned that installing a
readily derivatizable handle at position 7 and then functionaliz-
ing the 2-position would be the most viable strategy. To this
end, 7-bromoquinoline 37 was prepared by the Doebner-Miller
condensation of 7-bromoaniline (36) with methyl vinyl
ketone26 (Scheme 3). Treatment with m-CPBA afforded N-
oxide 38, which readily underwent deoxygenative amination
upon treatment with Ts2O and t-BuNH2;27,28 heating with TFA
removed the t-butyl group to yield the free aminoquinoline
(39) in good yields (even on a multigram scale) following
neutralization and column chromatography. The obtained
aminoquinoline was then protected as acetamidoquinoline 40
as previously described. To further functionalize 40, the
bromide was converted into aldehyde 41 using Ueda et al.’s
palladium-catalyzed hydrocarbonylation,29 which employs N-
formylsaccharin as the CO donor and Et3SiH as the reductant.
Reduction of the aldehyde afforded 42, which could be
brominated (Appel conditions18 or chlorinated with SOCl2) to
yield bromide 43 or chloride 44. Bromide 43 was treated with
phenols 26 and 29, as well as with 45 and 46 (prepared by
literature procedures;18 not in Scheme 1), to yield assembled
cores 47−50, which were deprotected as described above to
yield 4-methylated analogues 10−13.
a
Reagents and conditions: (a) (i) MeNH2 in THF, cat. AcOH,
CHCl3/MeOH, Na2SO4, r.t.; (ii) NaBH4, MeOH, 0 °C−r.t.; (iii)
Boc2O, THF, r.t.; (b) K4Fe(CN)6 × 3 H2O, t-BuXPhos, t-BuXPhos
G3, KOAc, H2O/dioxane, 100 °C; (c) [(PPh3)2PdCl2], CuI, Et3N, 70
°C; (d) TBAF in THF, THF, 0 °C; (e) H2, Pd/C, MeOH, r.t.
20−22 were reductively aminated and Boc-protected18 to
afford phenols 23−25, respectively. Brominated compound 24
was converted, via palladium-catalyzed cyanation, into
cyanophenol 26.24 Sonogashira coupling between 24 and
ethynyltrimethylsilane yielded 27, which was desilylated (to
give 28) and reduced to give ethylphenol 29. The requisite
phenols were then treated with quinolinemethyl bromide 30
(prepared by previously reported procedures18 (Scheme 2))
under basic conditions to afford the phenyl ether cores (31−
To prepare the deoxygenated analogues 14−16, bromide 40
was employed (to prepare 4-methyl analogues), and compound
51 (to prepare compounds with no substituent at the 4-
C
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a
Scheme 3
a
Reagents and conditions: (a) 2-buten-1-one, FeCl3·6H2O, AcOH, reflux; (b) m-CBPA, CH2Cl2, r.t.; (c) (i) t-BuNH2, Ts2O, PhCF3/CH2Cl2, 0 °C;
(ii) TFA, 75 °C; (d) N-acetylimidazole, DMAP, THF, reflux; (e) N-formylsaccharin, Pd(OAc)2, dppb, Et3SiH, Na2CO3, DMF, 75 °C; (f) NaBH4,
MeOH, 50 °C−r.t.; (g) PPh3, CBr4, THF, 0 °C−r.t. (for 43), SOCl2, 0 °C (for 44); (h) (i) phenols 26, 29, 45, 46, NaH, DMF, 0 °C; (ii) 43 (in
DMF), 0 °C; (i) (i) K2CO3, MeOH, reflux; (ii) HCl/MeOH, ether, r.t., or TFA/DCM (for 48), after isolation.
position), via aminoquinoline 52,30 was converted into
desmethyl 7-bromoquinoline 53 (Scheme 4A). Next, suitable
Sonogashira coupling partners were prepared. To prepare 14,
3-iodobenzyl bromide (54, Scheme 4B) was converted to
carbamate 55, and coupling with ethynyltrimethylsilane
afforded 56 in excellent yields, which was then desilylated to
yield 57. Synthesis of cyanated analogues 15 and 16 began with
bromination of commercially available cyanotoluene 58
(Scheme 4C); bromide 59 was subsequently aminated and
protected to yield 60. As described above, Sonogashira coupling
and desilylation of 61 yielded 62. Phenylacetylene 62 is
sensitive and polymerizes at room temperature, so it must be
kept cold until use.
a
Scheme 4
With the quinolines and phenylacetylenes in hand, copper-
free Sonogashira conditions31 were used to join the halves
(Scheme 5). The quinolinyl-acetylenes 63−65 were readily
identifiable by TLC because of their bright blue fluorescence.
After purification, the triple bonds were hydrogenated to
alkanes 66−68. For 63, this was readily accomplished with
palladium on carbon (to yield 66), but these conditions also
reduced the nitriles of 64 and 65. For these compounds, a Pd/
C-ethylenediamine complex32,33 was used to reduce the alkyne,
which showed excellent chemoselectivity, yielding 67 and 68
despite requiring extended reaction times or higher pressures.
Finally, 66−68 were deprotected to afford 14−16.
Preparing the phenols required for analogues 17−19 proved
more challenging, as phenolic (or other) precursors with these
particular 1,3,5-substitution patterns are not commercially
available or readily synthesized. Nonetheless, the meta-
borylation/oxidation strategy has been employed by Smith,
Maleczka, and others34,35 to prepare similar 3,5-disubstituted
phenols. In our case, to prepare a borylation substrate,
commercially available toluene 69 (Scheme 6) was brominated
(to give 70) and converted into protected amine 71. Cyanation
afforded 72. To prepare the phenol, iridium-catalyzed
borylation with Pin2B2 afforded an intermediate boronic ester
(not isolated), which was then treated with Oxone in aqueous
acetone to yield phenol 73 in moderate yield. The phenoxide of
73 was then treated with chloride 44. Because of 44’s lower
reactivity, the etherification reaction was performed at 50 °C
a
Reagents and conditions: (a) AcNH2, K2CO3, reflux (230 °C); (b)
N-acetylimidazole, DMAP, THF, reflux; (c) (i) MeNH2 in THF,
CH2Cl2, r.t.; (ii) Boc2O, CH2Cl2, r.t.; (d) [(PPh3)2PdCl2], CuI, Et3N,
70 °C; (e) TBAF in THF, THF, 0 °C; (f) NBS, (PhCO2)2, CCl4,
reflux.
D
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a
(instead of 0 °C as for 43), and deprotection of 74 afforded
analogue 17.
Scheme 5
Similarly, phenethylamine analogues 18 and (R,S)-19 were
also prepared via meta-borylation (Scheme 7). The substrates
(unsubstituted 79 and α-methylated (R,S)-80) were prepared
by Boc-protection and methylation of 75 (to yield 77) or
reductive amination and Boc-protection of 76 (to yield (R,S)-
78). Cyanation (as described above) afforded benzonitriles 79
and (R,S)-80, and the borylation−oxidation strategy produced,
respectively, 81 and (R,S)-82. These phenols were treated with
44, and deprotection of 83 and (R,S)-84 afforded final
compounds 18 and (R,S)-19, respectively.
As derivatives of 78 proved difficult to resolve into their
enantiomers, an asymmetric synthesis of the two enantiomers
of 19 (Scheme 8) was developed, based on the Ellman
auxiliary.36,37 Commercially available (R)- or (S)-t-butylsulfina-
mides (R- and S-85) were condensed with 76. Low-temper-
ature reduction of the imine with NaBH4 afforded the expected
(R,R)-diastereomer (R,R)-86 (when (R)-85 was used), and
(S,S)-diastereomer (S,S)-86 (when (S)-85 was used) in
satisfactory (>7:1) diastereomeric ratios. After purification,
cleavage of the sulfinamide auxiliaries and Boc-protection of the
free amines yielded (R)- and (S)-87, which were both
methylated to yield (R)- and (S)-78. The remainder of the
synthesis proceeded as with the racemic material: cyanation
afforded (R)- and (S)-80, which were borylated and treated
with Oxone to yield the phenols (R)- and (S)-82. Enantomeric
purity of these phenols (as assessed by chiral HPLC) was very
high (>99%). Subsequent phenyl ether formation and
deprotection of (R)- and (S)-84 afforded (R)- and (S)-19,
which were confirmed as single enantiomers via derivatization
(at the secondary amine) with (S)-camphanic chloride.
a
Reagents and conditions: (a) XPhos, Pd(MeCN)2Cl2, Cs2CO3,
MeCN, 80 °C; (b) H2, Pd/C, MeOH, r.t. (for 66), H2, Pd/C(en),
THF, atmospheric pressure or 50 PSI (for 67 and 68); (c) (i) K2CO3,
MeOH, reflux; (ii) HCl/MeOH, ether, r.t., after isolation.
a
Scheme 6
RESULTS AND DISCUSSION
■
Compounds 5−19 were assayed against purified rat nNOS
(rnNOS). Select compounds were then assayed against murine
iNOS (miNOS) and also against hnNOS and heNOS. With
recent advances,38 it is now possible to obtain and crystallize
both hnNOS and heNOS, so these enzymes were used as part
of a shift to obtain more human SAR data. RnNOS and miNOS
were still used, as historically, they are the easiest to express and
purify, and for clinical purposes, it is desirable to prove efficacy
and selectivity in lower animals. As miNOS is often difficult to
crystallize, the majority of the structural discussion will
concentrate on nNOS and eNOS. Inhibition data are
summarized in Table 1, where iNOS (n/i) selectivity is
reported as the ratio of the Ki values obtained for miNOS/
rnNOS, whereas eNOS (n/e) selectivity is for heNOS/hnNOS.
Values for compounds 1−4 are included for comparison.
Compared to unsubstituted compound 3, the 5-substituted
phenyl ethers (5−9) all have considerably greater potencies
against rnNOS. The standout among this series is the 5-cyano
compound 8, which is approximately 4 times more potent than
3 and 1.6 times more potent than previous phenyl ether lead 4.
The X-ray crystal structure of 8 bound to rnNOS (Figure 3A)
was examined to determine what causes this dramatic effect. As
observed for similar aminoquinoline-containing com-
pounds,17,18 the 2-aminoquinoline moiety of 8 acts as a
competitive arginine mimic by hydrogen bonding with the
conserved glutamate residue (Glu592). As also previously
observed, the phenyl ring extends beyond the heme-binding
pocket, where the methylamine tail H-bonds the water bridging
a heme propionate and the cofactor H4B. In most nNOS-
a
Reagents and conditions: (a) NBS, (PhCO2)2, CCl4, reflux; (b) (i)
MeNH2 in THF, CH2Cl2, r.t.; (ii) Boc2O, CH2Cl2, r.t.; (c) K4Fe(CN)6
× 3 H2O, t-BuXPhos, t-BuXPhos G3, KOAc, H2O/dioxane, 100 °C;
(d) (i) [Ir(OCH3) (COD)]2, dtbpy, Pin2B2, hexanes, 50 °C; (ii)
Oxone, H2O/acetone, 0 °C; (e) (i) phenol, NaH, DMF, 0 °C; (ii) 44
(in DMF), 0 °C−50 °C; (f) (i) K2CO3, MeOH, reflux; (ii) HCl/
MeOH, ether, r.t., after isolation.
E
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a
Scheme 7
a
Reagents and conditions: (a) (i) Boc2O, THF, r.t.; (ii) NaH, DMF, then MeI, 0 °C−60 °C; (b) (i) MeNH2 in THF, Na(OAc)3BH, AcOH,
CH2Cl2; (ii) Boc2O, THF, r.t.; (c) K4Fe(CN)6 × 3 H2O, t-BuXPhos G3, t-BuXPhos, 1:1 KOAc in H2O/dioxane, 100 °C; (d) (i) [Ir(OCH3)
(COD)]2, dtbpy, Pin2B2, hexanes, r.t. or 50 °C; (ii) Oxone, H2O/acetone, 0 °C−r.t. or r.t.; (e) (i) NaH, DMF, 0 °C; (ii) 44 (in DMF), 0 °C−50 °C;
(f) (i) K2CO3, MeOH, reflux; (ii) HCl/MeOH, ether, r.t.
a
Scheme 8
a
Reagents and conditions: (a) (i) Ti(OEt)4, THF, reflux; (ii) NaBH4, THF, −48 °C; (b) (i) HCl/MeOH, ether, r.t.; (ii) Boc2O, Et3N, MeOH/
THF, r.t.; (c) NaH, THF, °C−r.t., then MeI, 0 °C−r.t.; (d) K4Fe(CN)6 × 3 H2O, t-BuXPhos G3, t-BuXPhos, 1:1 KOAc in H2O/dioxane, 100 °C;
(e) i. [Ir(OCH3) (COD)]2, dtbpy, Pin2B2, hexanes, 50 °C, ii. Oxone, H2O/acetone, 0 °C-r.t.; (e) i. NaH, DMF, 0 °C; ii. 44 (in DMF), 0 °C-50 °C;
(f) (i) K2CO3, MeOH, reflux; (ii) HCl/MeOH, ether, r.t.
phenyl ether-linked structures (such as with 3), the phenyl ring
region is flexible, as evidenced by poor electron density in this
region in many crystal structures.18 The electron density of 8,
by contrast, is intact throughout the inhibitor, indicating greatly
reduced flexibility. This stabilization comes from the cyano
group of 8, which fits into a narrow pocket formed by the side
chains of Tyr706, Met570, and Asn569. The backbone carbonyl
of Asn569, the amide of Gln707, and the side-chain of Asn569
also surround 8’s cyano group. While other nNOS inhibitors
previously studied contain a 1,3,5-trisubstituted cyanophenyl
motif,19,20 those portions of the inhibitors occupy entirely
different spaces within the enzyme; this newly revealed
“auxiliary pocket” has never been previously reported to
interact with any nNOS inhibitor. It is possible that the
increased stabilization caused by the nitrile (and as a result,
improved potency) is partially electrostatic in nature. One
outcome of the nitrile fitting into this pocket is that the
electron-poor benzonitrile ring is now anchored in close
proximity (<4 Å) to the electron-rich Tyr706, enhancing π-
stacking interactions with this residue.
This “nitrile effect” also extends to human nNOS. In the
hnNOS-8 structure (Figure 3B), Leu337 of rat nNOS is
replaced by His342,38 but the small pocket is still present
(bounded by Tyr711, Met575, and Asn574), which similarly
accommodates the nitrile of 8. The binding modes of 8
between the two isozymes are virtually identical, and as
expected, 8 is equipotent against rat and human nNOS. This
represents a remarkable improvement in hnNOS potency over
leads 1−4, all of which are highly selective (5−7-fold) for
rnNOS over hnNOS. With the improved data quality, a water
molecule now is visible inside the auxiliary pocket in the
hnNOS-8 structure, forming a bridging H-bond between the
nitrile and both the carbonyl of Asn574 and amide of Gln712
(Asn569 and Gln707 in rnNOS). There is no doubt that this
water molecule is structural, as it has previously been observed
in nNOS crystal structures with L-arginine, as well as many
other inhibitor molecules.39,40 The solvent structure is highly
sensitive to crystal quality, however, and this is likely why the
water is not present in the rnNOS-8 structure. This suggests
that the effects of the nitrile may be multifaceted by providing
enhanced electrostatic and H-bond acceptor stabilization.
F
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Table 1. Inhibition of NOS Enzymes by Synthesized
Compounds
a
Ki (μM)
selectivity
compd
rnNOS
hnNOS
miNOS
heNOS
rn/mi
hn/he
1
0.049
0.066
0.142
0.058
0.079
0.071
0.082
0.035
0.054
0.039
0.033
0.028
0.037
0.033
0.063
0.036
0.032
0.042
0.050
0.061
0.050
0.318
0.440
0.911
0.295
NT
44.0
28.4
33.2
27.7
16.8
15.2
32.7
12.5
40.7
5.75
6.7
9.49
11.8
17.3
7.41
NT
899
431
237
478
213
214
399
357
754
147
203
266
351
138
316
95
30
2
27
3
19
4
25
5
ND
ND
ND
342
344
202
181
81
6
NT
NT
7
0.136
0.036
0.125
0.033
0.031
0.052
0.078
0.051
NT
NT
8
12.3
43.0
6.66
5.63
4.19
21.0
6.09
NT
9
10
11
12
7.44
13.0
4.54
19.9
3.42
3.35
25.7
10.8
7.34
25.6
13
269
119
ND
90
14
15
16
0.056
0.054
0.061
0.074
0.065
0.046
5.04
6.08
18.9
15.0
16.1
22.2
17
105
612
216
120
512
113
310
203
248
483
18
(R,S)-19
(R)-19
(S)-19
a
The compounds were assayed for in vitro inhibition against four
purified NOS isoforms: rat nNOS (rnNOS), human nNOS (hnNOS),
murine iNOS (miNOS), and human eNOS (heNOS) using known
literature methods (see Experimental Section for details), and Ki values
are calculated directly from IC50 values. IC50 values are the average of
at least two replicates from 6 to 9 data points; all experimental
standard error values (for the LogIC50) are less than 0.012, and all
correlation coefficients are good (R2 > 0.85). Specific LogIC50 and SE
data can be found in the Supporting Information, SI Table 2.
Selectivity values are ratios of respective Ki values. NT = not tested;
ND = not determined.
In addition to the improvements in potency, the nitrile−
auxiliary pocket interaction also enhances n/e selectivity.
Compound 8 is over 300-fold selective for hnNOS over
heNOS, high selectivity in a human-based system. We recently
obtained some of our first heNOS-inhibitor crystal structures,
belonging to a new P21 space group (see Experimental
Section). The chief differences between hnNOS and heNOS
are that the Asp602, His342, and Met341 residues of the former
isoform are replaced with Asn366, Phe105, and Val104 in the
latter.16 As previously reported, the Asp/Asn difference does
not appear to play any role in n/e selectivity for these phenyl
ether-linked compounds,18 whereas the Met/Val difference can
cause dramatic changes in Ki values. In the heNOS-8 structure
(Figure 3C), the smaller Val104 residue causes the methyl-
amine tail to assume an alternate conformation, where it is
accommodated next to Phe105, breaking the H-bond present
between the amine and H4B-site water molecule in hnNOS.
Although the small auxiliary pocket (into which the nitrile fits)
is conserved between hnNOS and heNOS, the stabilization
from the nitrile appears to be less pronounced in heNOS, as
evidenced by the incomplete electron density of the
benzonitrile ring. The bulky Phe105 residue in heNOS pushes
Tyr475 down toward O1D of the heme propionate (away from
the inhibitor), whereas in hnNOS, Tyr711 H-bonds O2D of
the same propionate and can thus interact more tightly with the
benzonitrile ring. Tyrosine residues in nNOS isoforms are also
Figure 3. Active site structures of 8 bound to rat nNOS (A), human
nNOS (B), and human eNOS (C). In this and all the following figures
for crystal structures, the omit Fo − Fc density map for the inhibitor is
shown at the 2.5 σ contour level. Major hydrogen bonds are shown as
dashed lines with distances in Ångstroms. Figures were prepared with
PyMol (www.pymol.org).
reported to be more flexible than those in eNOS isoforms,41
suggesting a greater possibility for induced fit in nNOS.
While the nitrile-containing molecule confers the highest
potency, there is a general trend of 5-substituted compounds
having greater potency than 3; the 5-ethylated analogue (9)
shows the second-highest potency after 8. Although the density
near the ethyl group indicates some flexibility, the rnNOS-9
structure (Figure 4A) suggests that the 5-ethyl group does not
bind in the small auxiliary pocket, likely due to its different
shape from the linear nitrile and the lack of a H-bonding
partner. Instead, the phenyl ring swings over toward the H4B
site, and the ethyl group reaches the nNOS-specific hydro-
phobic pocket, where it likely makes favorable van der Waals
contact with Met336, Leu337, and Tyr706, although the
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interaction is lacking in the heNOS-9 structure (Figure 4C);
the combination of the missing H4B-amine interaction, along
with different contact between the ethyl group and Val104 (vs
Met341 of hnNOS), may be responsible for the high n/e
selectivity for 9. Interestingly, 9 (and to a lesser degree, the
structurally similar compound 7) is highly selective for rnNOS
over miNOS. Murine iNOS has a polar asparagine residue
(Asn115, instead of Leu337 in rat nNOS) next to the
conserved substrate access channel tyrosine (Tyr485 in
miNOS or Tyr706 in rnNOS),42 and it is likely that large,
hydrophobic groups (such as methoxyl or ethyl) would disfavor
binding near Asn115.
In our second series of compounds (10−13), a methyl group
was installed at position 4 of the 2-aminoquinoline. Previous
studies with 2-aminopyridines indicated that 4-methylation
improved nNOS inhibitory potency (and in some cases, n/e
selectivity). Computer modeling predicted (and X-ray
crystallography confirmed) that the 4-methyl groups fit into a
sterically small, hydrophobic region (termed the “S-pock-
et”)22,43 located along the “back wall” of the heme-binding site.
Originally, we believed that the 2-aminoquinoline group may
have been too bulky for this positive modification to be
successfully translated to this scaffold, but this hypothesis
appears disproven, both from fragment screening and crystallo-
graphic results (vide supra) and from the observation that the
4-methyl group in these phenyl ether analogues has positive
effects on potency for both rat nNOS and human nNOS when
compared to those of unmethylated congeners (cf. 3 vs 10 and
4 vs 12). As expected, the rnNOS-10 crystal structure (Figure
5) shows that, as with the aminopyridines, the methyl group fits
Figure 4. Active site structures of 9 bound to rat nNOS (A), human
nNOS (B), and human eNOS (C). Note the alternate rotamer
assumed by His342 as a result of a possible clash with the ethyl group
in hnNOS (B).
Figure 5. Active site structure of 10 bound to rat nNOS. The phenyl
ring shows weaker density.
phenyl-Tyr706 π-stack is broken, and the amine-H4B site
interaction is much weaker (compared to that in the nNOS-8
structure) with less defined density. It is likely that the other 5-
substituents of 5, 6, and 7 (with lower potency than 8) might
also fit into this region of the enzyme as 9 does, which reflects
the requirement for a linear and polar substituent for auxiliary
pocket access.
Compared to 8, compound 9 is also a poorer human nNOS
inhibitor. In the hnNOS-9 structure (Figure 4B), the nitrile
pocket is again unoccupied, but there is a possible clash
between the hydrophobic ethyl group and the side chain of
His342. Indeed, an alternate rotamer of His342 can be modeled
in this structure, where the bulky, polar imidazole ring faces
away from the ethyl group. The bulk of the histidine forces the
amine of 9 to displace the H4B-site water molecule. This
into the S-pocket, bound by Phe584, Val567, and the backbone
of Ser585. The bulk of the aminoquinoline is still well
accommodated in the binding mode: 4-methylation forces the
2-aminoquinoline into a more parallel orientation above the
heme, instead of the tilted conformation assumed by
unmethylated compounds to avoid steric clashes with Phe584
and Val567.17
The enhancing effects that the 4-methyl group have on the 5-
substituted compounds are much less pronounced (cf. 8 and
11, where there is little effect, vs 3 and 10), which indicates that
the combination of the 4-methyl and 5-substituent is not
additive. Nonetheless, compound 11, with its combination of S-
pocket and nitrile−auxiliary pocket interactions, is a very potent
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dual rnNOS/hnNOS inhibitor. As with 8, the binding mode is
identical in the rnNOS-11 (Figure 6A) and hnNOS-11 (Figure
6B) crystal structures, with clear electron density throughout
and the bridging structural water molecule present in both the
rnNOS and hnNOS structures.
methylated analogue is lower than the desmethyl one. This
(along with the universal decrease in n/i selectivity) could
reflect a nonspecific increase in inhibitor binding, as the S-
pocket is conserved among all isoforms. The X-ray crystal
structure of heNOS-11 (Figure 6C), however, is surprising: two
molecules of 11 are present in the binding site. One binds in
the “usual” mode (L-arginine mimicry with nitrile−auxiliary
pocket interactions), while the other displaces the H4B cofactor
and π-stacks with Trp477. Arg365 (not shown in the figure),
which normally H-bonds with H4B, swings away as H4B is
displaced, and a Zn2+ ion is coordinated by Asp369, His461
(from the other monomer), and two water molecules. The H4B
displacement occurs even when micromolar amounts of H4B
are added to the crystal soaking solution, suggesting that 11
outcompetes H4B binding in the pterin pocket. Supporting this
observation is the fact that the Ki value for 11 for heNOS does
not change significantly when the concentration of H4B in the
assay is increased from 10 μM to 50 μM (5.6 μM for the former
vs 4.8 μM for the latter). The 4-methyl group on the quinoline
of 11 likely enhances this stacking with Trp477, which explains
why H4B displacement is not observed with 8 or other
desmethyl compounds. Although this is the first report of an
aminoquinoline-based inhibitor binding a NOS enzyme in this
way, dual inhibitor binding, H4B displacement, and extra Zn2+-
coordination were previously observed in rat nNOS with
double-headed aminopyridine inhibitors44 but has not been
observed in bovine eNOS owing to its more rigid dimer
interface45 and thus was proposed to be exploitable for selective
inhibitor design. Indeed, the bovine eNOS-11 structure (Figure
S1 ) only shows one inhibitor bound to the substrate site (even
when Zn2+ is added to the crystal soaking solution), with a
binding mode similar to that of the heNOS-8 structure (where
the methylamine can face away from the H4B site). It is possible
that some global structural difference between bovine eNOS
and human eNOS simply causes a decrease in H4B affinity (or
it may only occur under certain conditions) and that an extra
bound inhibitor could cause the decrease in Ki value obtained
upon methylation (cf. 8 and 11). However, the poor electron
density of 11 in the substrate site in the heNOS-11 structure
(Figure 6C), especially in the amine and benzonitrile portions
of the substrate-site molecule that are crucial for binding,
suggests that this is an unstable complex. This could occur
because binding of the second 11 in the H4B site may weaken
the binding of the first 11 in the substrate site (i.e., negative
cooperativity) and, as such, result in a much higher heNOS Ki
value than that observed for hnNOS.
To examine these different binding modes (and the
requirements for the nitrile to reach the auxiliary pocket)
further, deoxygenated derivatives 14−16 were prepared. These
compounds are also desirable because of their high cLogP value
and lack of the polar oxygen, which increases tPSA and could
be a potential site of metabolism in vivo. Interestingly, 14
behaves similarly to compound 10; the Ki values for rnNOS,
miNOS, or heNOS for 14 are very close to those for 10,
suggesting that the oxygen is not a crucial requirement for
selectivity for this compound, although the hnNOS activity has
decreased slightly. The rnNOS-14 structure (Figure 7A),
however, indicates that removal of the oxygen changes the
binding mode. While the 4-methylaminoquinoline is fixed in
the same position as it is in 10, the larger ethylene linker of 14,
compared to the oxaethylene linker of 10, prefers an “upward”
position away from Met570 and Val567. However, the phenyl
ether linker snugly abuts these two residues, and the phenyl
Figure 6. Active site structures of 11 bound to rat nNOS (A), human
nNOS (B), and human eNOS (C). There are two molecules of 11
bound in heNOS (C), the first one has a disordered benzonitrile
portion while the second one displaces H4B and creates a new Zn site.
The effects of methylation on nNOS/eNOS selectivity are
more complicated. It was previously reported that 4-
methylation of 2-aminopyridine inhibitors can considerably
improve n/e selectivity,22 but this is not consistently observed
with 2-aminoquinolines. Compounds 10 and 12 have higher
selectivity because of the greatly improved hnNOS activities
relative to 3 and 4 (respectively), but for 5-substituted
compounds 11 and 13, the n/e selectivity decreases upon
methylation compared to that of unmethylated compounds 8
and 9, respectively. In all cases, the heNOS Ki value for the
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its Ki value 8-fold to 42 nM. The rnNOS-18 structure (Figure
8A) shows that while the nitrile-auxiliary pocket interaction is
Figure 7. Active site structures of 14 (A) and 16 (B) bound to rat
nNOS. Note that unlike 11 (Figure 6A), the benzonitrile of 16 is not
bound in the auxiliary pocket because of the repulsion between the
bulkier ethylene linker and Val567.
Figure 8. Active site structures of 18 bound to rat nNOS (A) and
human nNOS (B). The structural water is missing in rnNOS likely due
to the diffraction data quality.
ring swings over toward the H4B site where the methylamine
tail displaces the H4B-site water instead of H-bonding it. As the
rnNOS Ki value of 14 is close to that of 10, this is obviously a
favorable conformation.
retained, the longer phenethylamino group displaces the H4B-
site water, whereas the hnNOS-18 structure (Figure 8B)
indicates that the amine H-bonds the existing H4B site water.
Compound 19, assayed initially as its racemate (R,S)-19, is
also very close in rnNOS and hnNOS potency to those of 18.
Interestingly, when rnNOS and hnNOS crystals were soaked
with (R,S)-19, the electron density of the resulting crystal
structures (hnNOS and rnNOS, Figure S2A and B) was
consistent with both enantiomers being bound, suggesting that
the two are similar in potency, which was confirmed by assaying
them separately. The Ki value for (R)-19 is 61 nM against
rnNOS and 65 nM against hnNOS, whereas those for (S)-19
are 50 nM and 46 nM, respectively. Compounds 18 and 19
(and isomers) were designed to place longer tail groups in the
vicinity of Met336/Met341 (rnNOS/hnNOS). In the hnNOS-
18 structure, the hnNOS-(R)-19, and hnNOS-(S)-19 structures
(Figure 9A and B), and the rnNOS-(S)-19 and rnNOS-(R)-19
structures (Figure S3A and B, respectively) large, flexible
portions of the alkylamine tails are all <5 Å away from and
could contact Met336 or Met341 with the rest of the molecules
anchored by aminoquinoline-glutamate, nitrile-auxiliary pocket,
and H4B-site-amine interactions. No obvious differences in
binding, other than the positioning of the α-methyl group, are
observable between (R)-19, and (S)-19.
As in the phenyl ether scaffold, methylation of the quinoline
in the alkyl scaffold (15 to 16) also improves potency.
However, the rnNOS-16 (Figure 7B) structure indicates that
16 is less stable when bound than is 11. The nitrile is not
bound in the auxiliary pocket, and the electron density indicates
greatly increased flexibility. This suggests that the geometric
and steric requirements to place a substituent into the auxiliary
pocket are quite strict. The methylene bridge again assumes the
“upward” position observed in the rnNOS-14 structure to avoid
steric clashes with Val567, breaking the Tyr706-aryl interaction
and pointing the nitrile toward Leu337 instead.
Despite the hypothesis that a 4-substituent, such as fluorine,
could improve potency, compound 17 does not have improved
rnNOS and hnNOS activity compared to that of 11, and the
selectivity values are lower. Previously, it was also shown that
fluorine pointing roughly toward the region of the nNOS-
specific hydrophobic pocket was deleterious to n/i selectivity.18
This same decrease is also observed here; the fluorine-Asn115
interaction may be favorable in miNOS, as indicated by a lower
Ki of 3.34 μM for 17 (compared to 6.7 μM for 11). Compound
18 represents an interesting example where cyanation and
methylation can be used to “rescue” a substitution pattern with
low activity. Previously, an uncyanated, unmethylated version of
this compound18 was reported to have a rat nNOS Ki value of
332 nM; the methyl and cyano substituents together decrease
As these methionine residues are not present in eNOS
enzymes, extra van der Waals contact with these methionine
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Figure 9. Active site structures of (R)-19 (A) and (S)-19 (B) bound to
human nNOS. The only difference between the two enantiomers is the
α-methyl position. Alternate rotamers for His342 were observed.
Figure 10. Active site structures of (S)-19 (A) and (R)-19 (B) bound
to human eNOS. There are two molecules of (R)-19 bound in human
eNOS. The first one in the active site shows a disordered tail portion,
while the second one displaces H4B and creates a new Zn site.
residues should improve n/e selectivity, as the analogous eNOS
valines have a smaller surface area and thus make less contact
with inhibitors. As predicted, the hnNOS/heNOS selectivities
for these bulkier compounds (18 and the enantiomers of 19)
exceed that of 11. Despite the similar potencies, there is a clear
difference in selectivity between (R)- and (S)-19; the n/e
selectivity for (S)-19 is nearly twice that of (R)-19.
Approaching 500-fold, (S)-19 has the highest n/e selectivity
ever observed for a 2-aminoquinoline-based inhibitor.
We examined the heNOS crystal structures with the two
enantiomers bound for insights into this disparity. In the (S)-
19-heNOS structure (Figure 10A), only one molecule of (S)-19
is bound, with the density in the H4B site largely consistent
with H4B being bound, although there is some ambiguity about
the occupancy of the new Zn2+-site in several chains (not
shown in the figure). As observed for 8, Tyr475 in heNOS is
pushed by the bulky Phe105 from O2D toward O1D of the
heme propionate and appears to form a weaker electrostatic/
van der Waals interaction with the benzonitrile ring than the
analogous Tyr711 in hnNOS. The amine tail of (S)-19 is
disordered in this structure compared to the relatively intact
density of the tail in the hnNOS-(S)-19 structure, suggesting
destabilization of the amine binding, possibly the result of
reduced interactions between Val104 and the α-methyl group,
compared to those stronger interactions with nNOS’s
methionine. By contrast, the (R)-19-heNOS structure (Figure
10B) resembles that of 11, with two molecules of inhibitor
present in the binding site. The heNOS Ki value for (R)-19
(16.1 μM) is not substantially increased from (S)-19 (22.1
μM), suggesting that the heNOS-dual-inhibitor complex, like
that of 11, may be relatively weak or unstable. As much of the
binding of these phenyl ether-linked compounds is dependent
on tail amine-H4B site interactions, any complex that lacks
these interactions, as a result of displacement of H4B and its
attendant water molecule, may destabilize inhibitor binding. A
similar dual-inhibitor complex is observed for heNOS-18
(Figure S4), suggesting that the ability to displace H4B is
ligand-dependent but may be common among certain classes of
these benzonitrile compounds. Although far from the first
record of stereochemical influence on the potency or selectivity
of nNOS inhibitors,46,47 the case of 19 reveals that even very
simple chiral groups, such as the tail of 19, can impart excellent
n/e selectivity for 2-aminoquinolines.
Finally, two potent dual rnNOS/hnNOS inhibitors with
different structural motifs and high n/e selectivity (8 and 10)
were assayed for membrane permeability in a Caco-2 assay. In
this assay, the permeability of a compound through a
monolayer of cells resembling the intestinal epithelium is
measured. Caco-2 assays have been used to approximate the
potential for both oral bioavailability and blood−brain barrier
permeation,48,49 although the latter is generally less accurate.
Although compounds 1 and 2 were unfavorably promiscuous
binders, they were highly permeable in this assay. Compound 4,
despite its significantly cleaner off-target profile, was much less
permeable and had higher efflux, indicating that the structural
rearrangement had detrimental effects on cellular pharmacoki-
netics. Compared to 4, both 8 and 10 have modestly increased
permeability, lower efflux, and higher recovery values (Table 2;
values for compounds 2 and 4 are given for comparison),
K
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modification of the cyanoaryl-containing tail (elongation to 18,
or introduction of a stereocenter, such as 19) could also be
used to disfavor binding to heNOS, and one compound, (S)-
19, had about 500-fold selectivity over both eNOS and iNOS,
exerted through a mixture of differential hydrophobic pocket
and tyrosine−benzonitrile interactions, although the role of
H4B antagonism in heNOS cannot entirely be excluded.
Additionally, both cyanation and 4-methylation showed some
ability to improve the Caco-2 permeability of the phenyl ether
scaffold. These results, taken together, highlight the promise of
this new auxiliary pocket in nNOS as a novel “hot spot” for
further structure-based design of nNOS inhibitors.
Table 2. Caco-2 Permeability Summary for Select
Compounds
a
apparent permeability (Papp
,
b
10−6 cm s−1
)
recovery
efflux
f
compd
mean A → B mean B → A
ratio
A → B B → A
2
4
8
16.9
2.3
41.9
12.6
32.1
34.4
17.7
2.4
2.5
63%
37%
63%
70%
103%
79%
5.5
9.2
3.5
89%
10
7.1
4.8
100%
c
Warfarin
26.0
0.33
0.12
0.68
7.3
d
Ranitidine
e
Talinolol
8.2
68.3
a
EXPERIMENTAL SECTION
All assays were performed over 2 h at a concentration of 10 μM. See
■
b
c
Experimental Section for details. Apparent permeability value. High
General Procedures. Anhydrous solvents (THF, CH2Cl2, MeOH,
Et3N, MeCN, and DMF) were distilled prior to use. All other solvents,
reactants, and reagents were purchased from commercial vendors and
were used without further purification. Methanolic HCl (3 M, for
ammonium hydrochloride salt formation and Boc-deprotection) was
freshly prepared by the reaction of acetyl chloride and anhydrous
MeOH at 0 °C. Melting points were determined in capillary tubes
d
e
permeability control. Low permeability control. High efflux control.
f
Efflux ratio is defined as Papp (B → A)/Papp (A → B); efflux ratio
values >3 indicate that a compound may be a substrate for P-gp or
other active transport systems.
although their permeability is still considerably lower than that
of 2. Interestingly, the cyano compound 8 is more permeable
than 10, despite having a higher tPSA (83.4 Å2, increased
because of the nitrile, vs 59.6 Å2) and lower cLogP (2.5 vs 3.2),
which suggests that these parameters may not be entirely
predictive of cellular permeability. Regardless, 4-methylation
and 5-cyanation might be favored modifications for improving
the bioavailability of future compounds.
1
using a Buchi melting point B-540 apparatus and are uncorrected. H
NMR spectra were recorded at 500 MHz, using a Bruker Avance III
500 (direct cryoprobe), and 13C NMR spectra were obtained at 126
MHz using the same instrument. Low-resolution ESIMS were
obtained on a Bruker AmaZon SL Ion Trap mass spectrometer
system. High-resolution mass spectral data were obtained at the
Integrated Molecular Structure Education and Research Center
(IMSERC, Northwestern University) on an Agilent 6210A TOF
mass spectrometer in positive ion mode using electrospray ionization
with an Agilent G1312A HPLC pump and an Agilent G1367B
autoinjector. Data were processed using MassHunter software version
B.04.00. Flash column chromatography was performed using an
Agilent 971-FP automated flash purification system with a Varian
column station and SiliCycle cartridges (12−80 g, both normal and
high performance). Analytical HPLC was performed using an Agilent
Infinity 1260 HPLC system and injection volumes of 5−10 μL. A
Phenomenex Luna 5 μm C-8(2) 100 Å column, 50 × 4.60 mm, was
used for all HPLC experiments, using a 10 min gradient of 95% H2O/
5% acetonitrile + 0.05% TFA to 95% acetonitrile/5% H2O + 0.05%
TFA, at 1.5 mL/min. Chiral analytical HPLC was performed using a
Chiralpak AD-H 5 μm column, 250 × 4.60 mm, using 25 min isocratic
elution at 5% isopropanol in hexanes, at 1 mL/min. The purity of all
final target compounds was found to be ≥95% by HPLC. Analytical
thin-layer chromatography was performed on SiliCycle extra-hard 250
μm TLC plates. Compounds were visualized with short-wavelength
UV light and with ninhydrin, FeCl3, CAM, and KMnO4 stains, where
appropriate. Compounds 30,17,18 45,18 46,18 and 5150 were prepared
by known literature procedures, and their spectral data are consistent
with those data reported for them. The optical rotation of chiral
compounds was measured using a Rudolph Research Analytical
Autopol IV automatic polarimeter, using a 50 mm cell and the sodium
D-line (589 nm). The preparation of quinoline precursors and
assembly of final compounds is described below, while the preparation
of phenols and precursors 23−25, 26−29, 55−57, 59−62, 70−73,
and 77−82 is discussed in the Supporting Information.
CONCLUSIONS
■
To summarize, we undertook further modification of our
“cleaner-binding” phenyl ether-linked aminoquinoline scaffold
with hopes that we could improve human nNOS inhibition,
hnNOS/heNOS selectivity, and possibly the cellular perme-
ability of this promising class of compounds. We discovered
that 5-substitution of the phenyl ring results in greatly
improved hnNOS activity and hnNOS/heNOS selectivity,
especially when the substituent is cyano. X-ray crystallographic
studies revealed that the 5-cyano group fits into a small,
previously unreported, auxiliary pocket located next to the
heme-binding sites of both rnNOS and hnNOS, resulting in
good potency against both isoforms. The nitrile H-bonds to a
structural water within this pocket and anchors an electron-
deficient aryl ring next to an electron-rich tyrosine, an
interaction that is weaker in heNOS. Additionally, the
methylamine tails of 8 and 11 favor a water-mediated H-
bond to H4B and a heme propionate in nNOS, an interaction
that is missing in heNOS. These combined interactions resulted
in the first 2-aminoquinolines with high selectivity for human
nNOS over human eNOS. Although this new auxiliary pocket is
a promising site for further optimization, the structural
requirements to fit a substituent into this region may be fairly
strict; replacement of the nitrile with an ethyl group resulted in
the 5-substituent being bound elsewhere, and replacement of
the phenyl ether oxygen of 11 with a slightly larger methylene
(16) resulted in increased flexibility because of the increased
steric bulk, as observed by crystallography. Additionally, we
found that methylation of the 4-position of the aminoquinoline
could greatly improve rnNOS and hnNOS potency further,
although it decreased hnNOS/heNOS selectivity in some cases.
4-Methylation also caused some inhibitors to displace H4B in
heNOS in addition to mimicking L-arginine, although this
appears ligand-dependent. In spite of the decrease, further
General Procedure for the Synthesis and Deprotection of Phenyl
Ether-Linked Aminoquinolines. The procedure is similar to that
recently reported:18 sodium hydride (60% suspension in mineral oil, 1
equiv) was diluted with anhydrous DMF (1−2 mL) and cooled to 0
°C under argon. A solution of the required phenol (1 equiv) in
anhydrous DMF (1−2 mL) was added slowly to the suspension and
stirred at 0 °C for 10−30 min (typically ∼25 min), following which
bromides 30 or 43 (1 equiv) or chloride 44 (0.90-equiv) was added as
a solution in anhydrous DMF. If 30 or 43 were employed, the reaction
mixture was stirred at 0 °C for 40 min−1 h (typically ∼50 min), and if
44 was employed, the reaction mixture was warmed to r.t. and then
heated to 50 °C for 45 min−1 h. In both cases, the reaction mixture
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was then quenched at r.t. by the addition of a 1:1 sat. aq. NaCl/H2O
mixture or a sat. aq. NaHCO3 solution (∼15 mL). The mixture was
extracted with EtOAc (usually 3 × 20 mL was sufficient for these
lipophilic carbamates), and the organic phase was washed with 5% aq.
NaCl ((3−4) × 30−80 mL) and sat aq. NaCl (30−50 mL). The
organic layer was dried over anhydrous sodium sulfate, concentrated,
and purified by flash column chromatography (12 g SiO2 cartridge),
using gradients as described for individual compounds below. The
resulting intermediate acetamides were not characterized or purified
further, but were diluted with anhydrous MeOH (5−10 mL), and
anhydrous K2CO3 (∼2 equiv) was added. The mixture was heated at
reflux for 2−2.5 h, cooled, and concentrated. The resulting residue was
partitioned between EtOAc and 1:1 H2O/sat. aq. NaCl, and the
aqueous layer was extracted with EtOAc ((2−3) × 5−20 mL). The
organic layers were washed with sat. aq. NaCl and dried over
anhydrous sodium sulfate. Purification is detailed under subheadings
for individual compounds. The free aminoquinoline was diluted in dry
ether (or 10:1−4:1 ether/MeOH) or dichloromethane (for 31, 35,
and 48) and filtered to remove any particulate matter. To the filtered
solution, methanolic HCl (3 M, 1−2 mL) was added (except for 31,
35, and 48, where 150−300 μL trifluoroacetic acid was added instead),
and the mixture was stirred at room temperature either overnight or
for 20−30 min (for 31, 35, and 48). The salts were isolated by
filtration or precipitation, and the final purification was performed as
described below for individual compounds.
7-[(3-Chloro-5-((methylamino)methyl)phenoxy)methyl]quinolin-
2-amine Dihydrochloride (5). This compound was prepared from 30
(0.100 g, 0.358 mmol) and phenol 23 (0.097 g, 0.358 mmol). Workup
and purification by flash column chromatography, eluting with a
gradient of 2% EtOAc in CH2Cl2 to 35% EtOAc in CH2Cl2, afforded
intermediate acetamide 31 as a colorless foam (0.138 g, 82%), which
was immediately deprotected using K2CO3 (0.081 g, 0.587 mmol), as
described in the General Procedure. After workup, the resulting gum
was purified by flash column chromatography, eluting with EtOAc to
yield the free aminoquinoline as a semisolid residue that was diluted in
10:1 ether/MeOH (∼12 mL) and treated with methanolic HCl (2
mL). After being stirred overnight, filtration afforded 5 (0.081 g, 69%
from 31) as a white flocculent solid after precipitation from hot
MeOH (1 mL) with ether (5 mL) and washing with ether: mp 298−
299 °C. 1H NMR (500 MHz; DMSO-d6): δ 14.36 (br s, 1 H), 9.31 (br
s, 2 H), 9.20 (br s, 1 H) 8.37 (d, J = 9.3 Hz, 1 H), 8.30 (br s, 1 H),
7.96 (d, J = 8.2 Hz, 1 H), 7.76 (s, 1 H), 7.53 (dd, J = 8.2, 1.2 Hz, 1 H),
7.30 (t, J = 1.7 Hz, 1 H), 7.25 (t, J = 1.4 Hz, 1 H), 7.22 (t, J = 2.0 Hz, 1
H), 7.10 (d, J = 9.3 Hz, 1 H), 5.38 (s, 2 H), 4.09 (s, 2 H), 2.55−2.50
(m, 3 H, partially obscured by solvent peak). 13C NMR (126 MHz;
DMSO-d6): δ 159.2, 154.8, 143.1, 141.7, 135.6, 134.3, 129.4, 124.2,
122.7, 120.8, 116.3, 115.7, 115.4, 114.2, 69.3, 50.7, 32.3; one of the
quinoline carbons is not visible due to baseline broadening; ESIMS m/
z (rel. intensity) 328/330 (MH+, 100/33). HRMS calcd for
C18H19ClN3O+: 328.1211; found, 328.1215.
123.77, 122.22, 120.49, 117.81, 116.34, 115.51, 113.83, 68.97, 50.27,
32.00; one of the quinoline carbons is not visible due to baseline
broadening; ESIMS m/z (rel. intensity) 372/374 (MH+, 100/100).
HRMS calcd for C18H19BrN3O+: 372.0706; found, 372.0709.
7-[(3-Methoxy-5-((methylamino)methyl)phenoxy)methyl]-
quinolin-2-amine Ditrifluoroacetate (7). This compound was
prepared from 30 (0.054 g, 0.193 mmol) and phenol 25 (0.052 g,
0.193 mmol). Workup and purification by flash column chromatog-
raphy, eluting with a gradient of 3% EtOAc in CH2Cl2 to 43% EtOAc
in CH2Cl2, afforded intermediate acetamide 33 as a pale-yellow foam
(0.064 g, 71%), which was immediately deprotected using K2CO3
(0.038 g, 0.273 mmol) as described in the General Procedure. After
workup, the free aminoquinoline was diluted with CH2Cl2, filtered,
and concentrated. The residue was diluted with anhydrous CH2Cl2 (3
mL), and trifluoroacetic acid (200 μL) was added. The mixture was
stirred for 25 min and concentrated, and ether (10 mL) was added to
the residue. The mixture was sonicated until a solid formed, which was
collected to yield 7 (0.061 g, 81% from 33) as a white solid after
precipitation from hot MeOH (1 mL) with ether (10 mL) and
1
washing with ether: mp 130 °C (softens), 156−158 °C (melts). H
NMR (500 MHz; DMSO-d6): δ 14.16 (br s, 1 H), 8.79 (s, 2 H), 8.23
(s, 1 H), 7.87−7.82 (m, 1 H), 7.64 (s, 1 H), 7.43 (s, 1 H), 6.98−6.97
(m, 1 H), 6.76 (t, J = 1.5 Hz, 1 H), 6.69 (s, 1 H), 6.68 (t, J = 2.1 Hz, 1
H), 5.29 (s, 2 H), 4.05 (s, 2 H), 3.76 (s, 3 H), 2.55 (s, 3 H); the
aminoquinoline protons are not visible but are broadened into the
baseline and cause an overall broadening of the signals from 7 to 8
ppm. 13C NMR (126 MHz; DMSO-d6): δ 160.6, 159.4, (158.5 + 158.3
+ 158.0 + 157.8, 1 C), 134.1, 128.6, 121.0, (118.5 + 116.1, 1 C), 113.5,
108.7, 107.9, 101.3, 68.9, 55.4, 51.3, 32.1; several of the aminoquino-
line and trifloroacetate carbon signals are not visible due to baseline
broadening; ESIMS m/z (rel. intensity) 324 (MH+,100). HRMS calcd
+
for C19H22N3O2 : 324.1707; found, 324.1711.
3-[(2-Aminoquinolin-7-yl)methoxy]-5-((methylamino)methyl)-
benzonitrile Dihydrochloride (8). This compound was prepared from
30 (0.085 g, 0.305 mmol) and phenol 26 (0.080 g, 0.305 mmol).
Workup and purification by flash column chromatography, eluting
with a gradient of 5% EtOAc in CH2Cl2 to 40% EtOAc in CH2Cl2,
afforded intermediate acetamide 34 as a yellow foam (0.127 g, 90%),
which was immediately deprotected using K2CO3 (0.076 g, 0.551
mmol) as described in the General Procedure. After workup, the free
aminoquinoline was purified by flash column chromatography (eluting
with a gradient of EtOAc to 2% MeOH in EtOAc), and the resultant
white solid was diluted in 4:1 ether/MeOH and treated with
methanolic HCl (1.5 mL). After being stirred overnight, filtration
afforded 8 (0.073 g, 68% from 34) as a white flocculent solid after
precipitation from hot MeOH (2 mL) with ether (10 mL) and
1
washing with ether: mp 304−305 °C (dec). H NMR (500 MHz;
DMSO-d6): δ 14.24 (br s, 1 H), 9.30 (br s, 2 H), 9.20 (br s, 1 H), 8.37
(d, J = 9.2 Hz, 1 H), 8.21 (br s, 1 H), 7.96 (d, J = 8.2 Hz, 1 H), 7.76 (s,
1 H), 7.67 (t, J = 1.8 Hz, 1 H), 7.64 (t, J = 1.8 Hz, 1 H), 7.61 (s, 1 H),
7.54 (d, J = 7.9 Hz, 1 H), 7.09 (d, J = 9.3 Hz, 1 H), 5.42 (s, 2 H), 4.15
(s, 2 H), 2.53−2.50 (m, 3 H). 13C NMR (126 MHz; DMSO-d6): δ
158.2, 154.5, 142.7, 141.0, 135.3, 129.1, 126.3, 125.1, 123.9, 122.4,
120.6, 118.19, 118.00, 115.6, 113.9, 112.4, 69.2, 50.1, 32.0; ESIMS m/z
(rel. intensity) 319 (MH+, 100). HRMS calcd for C19H19N4O+:
319.1553; found, 319.1559.
7-[(3-Bromo-5-((methylamino)methyl)phenoxy)methyl]quinolin-
2-amine Dihydrochloride (6). This compound was prepared from 30
(0.085 g, 0.305 mmol) and phenol 24 (0.096 g, 0.305 mmol). Workup
and purification by flash column chromatography, eluting with a
gradient of 2% EtOAc in CH2Cl2 to 35% EtOAc in CH2Cl2, afforded
intermediate acetamide 32 as an off-white foam (0.110 g, 70%), which
was immediately deprotected using K2CO3 (0.059 g, 0.427 mmol) as
described in the General Procedure. After workup, the free
aminoquinoline was obtained as a gummy residue that was diluted
in 4:1 ether/MeOH and treated with methanolic HCl (2 mL). After
being stirred overnight, filtration afforded 6 (0.081 g, 84% from 32) as
a white flocculent solid after precipitation from hot MeOH (2 mL)
7-[(3-Ethyl-5-((methylamino)methyl)phenoxy)methyl]quinolin-2-
amine Ditrifluoroacetate (9). This compound was prepared from 30
(0.042 g, 0.152 mmol) and phenol 29 (0.041 g, 0.152 mmol). Workup
and purification by flash column chromatography, eluting with a
gradient of 5% EtOAc in CH2Cl2 to 30% EtOAc in CH2Cl2, afforded
intermediate acetamide 35 as a translucent, colorless gum (0.052 g,
74%), which was immediately deprotected using K2CO3 (0.031 g,
0.224 mmol) as described in the General Procedure. After workup, the
free aminoquinoline was diluted with anhydrous CH2Cl2 (3 mL), and
trifluoroacetic acid (150 μL) was added. The mixture was stirred for 15
min and concentrated, and ether (10 mL) was added to the residue.
The mixture was stirred until a solid formed, which was collected to
yield 9 (0.027 g, 67% from 35) as a white solid after being washed with
ether: mp 154−156 °C. 1H NMR (500 MHz; DMSO-d6): δ 14.23 (br
1
with ether (10 mL) and washing with ether: mp 286−287.5 °C. H
NMR (500 MHz; DMSO-d6): δ 14.32 (br s, 1 H), 9.26 (br s, 2 H),
9.20 (br s, 1 H), 8.37 (d, J = 9.3 Hz, 1 H), 8.21 (br s, 1 H), 7.96 (d, J =
8.2 Hz, 1 H), 7.76 (s, 1 H), 7.53 (dd, J = 8.2, 1.0 Hz, 1 H), 7.37 (t, J =
1.3 Hz, 1 H), 7.35 (t, J = 2.0 Hz, 1 H), 7.34 (t J = 1.3 Hz, 1 H), 7.10
(d, J = 9.3 Hz, 1 H), 5.38 (s, 2 H), 4.09 (t, J = 4.7 Hz, 2 H), 2.52−2.50
(m, 3 H, partially obscured by solvent peak). 13C NMR (126 MHz;
DMSO-d6): δ 158.88, 154.51, 142.59, 141.30, 135.56, 129.03, 125.19,
M
DOI: 10.1021/acs.jmedchem.7b00259
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
s, 1 H), 8.81−8.70 (m, 4 H), 8.36 (d, J = 9.3 Hz, 1 H), 7.95 (d, J = 8.2
Hz, 1 H), 7.70 (s, 1 H), 7.53 (d, J = 8.1 Hz, 1 H), 7.06 (d, J = 9.3 Hz, 1
H), 7.00 (d, J = 1.2 Hz, 1 H), 6.98 (s, 1 H), 6.94 (s, 1 H), 5.31 (s, 2
H), 4.07 (s, 2 H), 2.61 (q, J = 7.6 Hz, 2 H), 2.56 (s, 3 H), 1.19 (t, J =
7.6 Hz, 3 H). 13C NMR (126 MHz; DMSO-d6): δ (158.8 + 158.5 +
158.30 + 158.0, 1 C), 158.27, 154.7, 146.1, 142.6, 142.0, 133.3, 129.0,
123.7, 121.8, 120.5, (118.3 + 115.9), 114.8, 113.8, 113.5, 68.5, 51.3,
32.2, 28.1, 15.3; the trifluoroacetate carbon signals are only partially
visible; two of the aminoquinoline carbons are not visible due to
baseline broadening; ESIMS m/z (rel. intensity) 322 (MH+, 100).
HRMS calcd for C20H24N3O+: 322.1914; found, 322.1916.
MeOH added) and treated with methanolic HCl (1 mL). After being
stirred overnight, filtration afforded 12 (0.058 g, 81% from 49) as a
white flocculent solid after precipitation from hot MeOH (1 mL) with
1
ether (10 mL) and being washed with ether: mp 280−282 °C. H
1
NMR (500 MHz; DMSO- d6): δ H NMR (500 MHz; DMSO-d6), δ
14.13 (s, 1 H), 8.84−8.70 (s, 4 H), 8.02 (d, J = 8.4 Hz, 1 H), 7.70 (s, 1
H), 7.55 (d, J = 8.3 Hz, 1 H), 7.00 (s, 1 H), 6.98 (s, 1 H), 6.94 (s, 1
H), 6.91 (s, 1 H), 5.38 (s, 2 H), 4.21 (s, 2 H), 2.64−2.58 (m, 5 H),
2.55 (s, 3 H), 1.19 (t, J = 7.5 Hz, 3 H). 13C NMR (126 MHz; DMSO-
d6): δ 156.9, 153.8, 152.3, 141.3, 135.7, 130.9, 130.5, 125.8, 124.9,
123.7, 120.7, 118.3, 117.0, 115.6, 112.6, 68.9, 48.3, 32.4, 19.0; ESIMS
m/z (rel. intensity) 342/344 (MH+, 100/30). HRMS calcd for
C19H21ClN3O+: 342.1368; found, 342.1371.
4-Methyl-7-[(3-((methylamino)methyl)phenoxy)methyl]quinolin-
2-amine Dihydrochloride (10). This compound was prepared from 43
(0.065 g, 0.221 mmol) and phenol 46 (0.052 g, 0.221 mmol). Workup
and purification by flash column chromatography, eluting with a
gradient of 5% EtOAc in CH2Cl2 to 30% EtOAc in CH2Cl2, afforded
intermediate acetamide 50 as a white solid (0.076 g, 77%), which was
immediately deprotected using K2CO3 (0.047 g, 0.338 mmol) as
described in the General Procedure. After workup, the free
aminoquinoline was triturated with hexanes and filtered, and the
resultant white solid was diluted in 10:1 ether/MeOH (8 mL) and
treated with methanolic HCl (1.5 mL). After being stirred overnight,
filtration afforded 10 (0.051 g, 80% from 50) as a white flocculent
solid, after precipitation from hot MeOH (0.5 mL) with ether (5 mL)
7-((3-Ethyl-5-((methylamino)methyl)phenoxy)methyl)-4-methyl-
quinolin-2-amine Ditrifluoroacetate (13). This compound was
prepared from 43 (0.065 g, 0.22 mmol) and phenol 29 (0.058 g,
0.22 mmol). Workup and purification by flash column chromatog-
raphy, eluting with a gradient of 5% EtOAc in CH2Cl2 to 37% EtOAc
in CH2Cl2, afforded intermediate acetamide 48 as a white solid (0.076
g, 72%), which was immediately deprotected using K2CO3 (0.044 g,
0.318 mmol) as described in the General Procedure. After workup, the
free aminoquinoline was diluted with anhydrous CH2Cl2 (6 mL), and
trifluoroacetic acid (300 μL) was added. The mixture was stirred for 30
min and concentrated, and ether (10 mL) was added to the residue.
The mixture was sonicated until a solid formed and filtered to yield 13
(0.079 g, 89% from 48) as a white solid after being washed with ether:
1
and being washed with ether: mp 249−251 °C. H NMR (500 MHz;
DMSO-d6): δ 14.12 (br s, 1 H), 9.19 (br s, 2 H), 9.00 (br s, 1 H), 8.10
(br s, 1 H), 8.03 (d, J = 8.4 Hz, 1 H), 7.78 (s, 1 H), 7.57 (dd, J = 8.4,
1.3 Hz, 1 H), 7.39 (t, J = 7.9 Hz, 1 H), 7.32 (t, J = 1.8 Hz, 1 H), 7.12−
7.09 (m, 2 H), 6.94 (d, J = 0.8 Hz, 1 H), 5.37 (s, 2 H), 4.09 (t, J = 4.8
Hz, 2 H), 2.65 (d, J = 0.9 Hz, 3 H), 2.54−2.50 (m, 3 H, partially
obscured by solvent peak). 13C NMR (126 MHz; DMSO-d6): δ 158.1,
153.8, 141.8, 133.6, 130.0, 125.8, 123.6, 122.4, 120.6, 116.6, 115.5,
115.1, 112.6, 68.4, 51.0, 32.0, 19.0; two of the aminoquinoline carbons
are not visible due to baseline broadening; ESIMS m/z (rel. intensity)
308 (MH+, 100). HRMS calcd for C19H22N3O+: 308.1757; found,
308.1757.
1
mp 199.5−201 °C. H NMR (500 MHz; DMSO-d6): 14.13 (br s, 1
H), 8.02 (d, J = 8.4 Hz, 1 H), 7.70 (s, 1 H), 7.55 (d, J = 8.3 Hz, 1 H),
7.00 (s, 1 H), 6.98 (s, 1 H), 6.94 (s, 1 H), 6.91 (s, 1 H), 5.31 (s, 2 H),
4.06 (S, 2 H), 2.64−2.58 (m, 5 H), 2.55 (s, 3 H), 1.19 (t, J = 7.5 Hz, 3
H). 13C NMR (126 MHz; DMSO-d6): δ (158.9 + 158.7 + 158.43 +
158.19, 1 C), 158.25, 154.1, 152.2, 146.0, 141.7, 136.2, 133.3, 125.7,
123.6, 121.9, 120.7, (118.3 + 115.9, 1 C), 115.6, 114.8, 113.5, 112.7,
68.4, 51.3, 32.1, 28.1, 18.9, 15.3; the trifluoroacetate carbon signals are
only partially visible; ESIMS m/z (rel. intensity) 336 (MH+, 100).
HRMS calcd for C21H25N3O+: 336.2070; found, 336.2075.
3-[(2-Amino-4-methylquinolin-7-yl)methoxy]-5-((methylamino)-
methyl)benzonitrile Dihydrochloride (11). This compound was
prepared from 43 (0.065 g, 0.221 mmol) and phenol 26 (0.058 g,
0.221 mmol). Workup and purification by flash column chromatog-
raphy, eluting with a gradient of 5% EtOAc in CH2Cl2 to 38% EtOAc
in CH2Cl2, afforded intermediate acetamide 47 as a yellow foam
(0.043 g, 41%), which was immediately deprotected using K2CO3
(0.025 g, 0.181 mmol) as described in the General Procedure. After
workup, the free aminoquinoline was purified by flash column
chromatography (eluting with a gradient of EtOAc to 5% MeOH in
EtOAc), and the resultant residue was diluted in 10:1 ether/MeOH
(10 mL) and treated with methanolic HCl (1.5 mL). After being
stirred overnight, filtration afforded 8 (0.023 g, 68% from 47) as a pale
yellow flocculent solid after precipitation twice from hot MeOH (0.4
mL) with ether (10 mL) and being washed with ether: mp 185−188
°C. 1H NMR (500 MHz; DMSO-d6): δ 14.14 (br s, 1 H), 9.38 (br s, 2
H), 8.99 (br s, 1 H), 8.10 (br s, 1 H), 8.03 (d, J = 8.4 Hz, 1 H), 7.76 (s,
1 H), 7.69 (d, J = 1.4 Hz, 1 H), 7.64−7.63 (m, 1 H), 7.62 (s, 1 H),
7.56 (dd, J = 8.4, 1.2 Hz, 1 H), 6.94 (s, 1 H), 5.43 (s, 2 H), 4.15 (s, 2
H), 2.63 (d, J = 0.7 Hz, 3 H), 2.55−2.50 (m, 3 H, partially obscured by
solvent peak). 13C NMR (126 MHz; DMSO-d6): δ 158.2, 153.8, 152.2,
140.8, 135.8, 135.3, 126.3, 125.9, 123.8, 122.5, 120.8, 118.2, 118.0,
115.8, 112.7, 112.4, 69.0, 50.1, 32.0, 19.0; ESIMS m/z (rel. intensity)
333 (MH+, 100). HRMS calcd for C20H21N4O+: 333.1710; found,
333.1716.
4-Methyl-7-[3-((methylamino)methyl)phenethyl]quinolin-2-
amine Dihydrochloride (14). Compound 66 (0.058 g, 0.129 mmol)
was deprotected using K2CO3 (0.036 g, 0.259 mmol) as described in
the General Procedure. After workup, the free aminoquinoline was
purified by flash column chromatography, eluting with a gradient of
EtOAc to 7% MeOH in EtOAc, to yield a colorless gum, which was
diluted in 20:1 ether/MeOH (10 mL) and treated with methanolic
HCl (0.7 mL). After being stirred overnight, filtration afforded 14
(0.026 g, 54% from 66) as a white solid, after precipitation from hot
MeOH (0.5 mL) with ether (3 mL) and being washed with ether: mp
1
135−137 °C. H NMR (500 MHz; DMSO-d6): δ 14.05 (br s, 1 H),
9.13−9.10 (br m, 3 H), 9.00 (br s, 1 H), 8.10 (br s, 1 H), 7.90 (d, J =
8.4 Hz, 1 H), 7.50 (s, 1 H), 7.47 (s, 1 H), 7.41 (dd, J = 8.4, 1.1 Hz, 1
H), 7.35−7.31 (m, 2 H), 7.28−7.26 (m, 1 H), 6.87 (d, J = 0.4 Hz, 1
H), 4.08 (s, 2 H), 3.10 (t, J = 7.8 Hz, 2 H), 2.98 (t, J = 7.8 Hz, 2 H),
2.60 (s, 3 H), 2.55−2.50 (m, 3 H, obscured by solvent peak); 13C
NMR (126 MHz; DMSO-d6): δ 153.6, 152.3, 146.4, 141.3, 135.6,
132.0, 130.0, 129.0, 128.7, 127.5, 125.8, 125.4, 119.5, 116.6, 111.8,
51.2, 36.5, 36.2, 31.9, 18.9; ESIMS m/z (rel. intensity) 306 (MH+,
+
100). HRMS calcd for C20H24N3 : 306.1965; found, 306.1969.
3-[2-(2-Aminoquinolin-7-yl)ethyl]-5-((methylamino)methyl)-
benzonitrile Dihydrochloride (15). Compound 67 (0.026 g, 0.057
mmol) was deprotected using K2CO3 (0.016 g, 0.114 mmol) as
described in the General Procedure. After workup, the free
aminoquinoline was passed through a short SiO2 plug (eluting with
EtOAc) to yield a colorless gum that was diluted in ether (10 mL) and
treated with methanolic HCl (0.5 mL). After being stirred for 18 h,
filtration afforded 15. The filtrate was concentrated, and the residue
was resuspended in ether and treated with methanolic HCl (0.5 mL);
after 24 h, additional 15 was obtained. The total obtained solid was
precipitated from hot MeOH (0.5 mL) with ether (3 mL) and washed
with ether to afford 15 as a cream-colored solid (0.014 g, 62% from
67): mp 260−261.5 °C. 1H NMR (500 MHz; DMSO-d6): δ 14.01 (br
s, 1 H), 9.14−9.10 (br m, 3 H), 8.32 (d, J = 9.3 Hz, 1 H), 8.10 (br s, 1
7-[(4-Chloro-3-((methylamino)methyl)phenoxy)methyl]-4-meth-
ylquinolin-2-amine Dihydrochloride (12). This compound was
prepared from 43 (0.065 g, 0.220 mmol) and phenol 45 (0.060 g,
0.220 mmol). Workup and purification by flash column chromatog-
raphy, eluting with a gradient of 5% EtOAc in CH2Cl2 to 37% EtOAc
in CH2Cl2, afforded intermediate acetamide 49 as a white solid (0.084
g, 79%), which was immediately deprotected using K2CO3 (0.048 g,
0.347 mmol) as described in the General Procedure. After workup, the
free aminoquinoline was diluted in ether (10 mL, with a few drops of
N
DOI: 10.1021/acs.jmedchem.7b00259
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Journal of Medicinal Chemistry
Article
H), 7.85−7.81 (m, 4 H), 7.49 (s, 1 H), 7.38 (d, J = 8.3 Hz, 1 H), 7.01
(d, J = 9.3 Hz, 1 H), 4.14 (s, 2 H), 3.12 (dd, J = 9.2, 6.2 Hz, 2 H), 3.03
(dd, J = 9.3, 6.3 Hz, 2 H), 2.55−2.50 (m, 3 H, obscured by solvent
peak). 13C NMR (126 MHz; DMSO-d6): δ 154.30. 146.24, 143.00,
142.84, 135.89, 135.22, 133.57, 132.46, 131.33, 128.75, 125.86, 119.33,
118.47, 116.36, 112.92, 111.38, 50.18, 36.13, 35.61, 31.93; ESIMS m/z
white flocculent solid after precipitation from hot MeOH (1 mL) with
ether (10 mL) and washing with ether: mp 213−215 °C. H NMR
1
(500 MHz; DMSO-d6): δ 14.07 (br s, 1 H), 9.10−8.79 (br m, 3 H),
8.10 (br m, 1 H), 8.03 (d, J = 8.4 Hz, 1 H), 7.75 (s, 1 H), 7.56 (dd, J =
8.4, 1.1 Hz, 1 H), 7.47 (dd, J = 2.3, 1.3 Hz, 1 H), 7.41−7.49 (m, 1 H),
7.37 (t, J = 1.8 Hz, 1 H), 6.93 (s, 1 H), 5.41 (s, 2 H), 3.22−3.17 (m, 2
H), 2.98 (t, J = 7.7 Hz, 2 H), 2.63 (d, J = 0.8 Hz, 3 H), 2.56 (t, J = 5.3
Hz, 3 H). 13C NMR (126 MHz; DMSO-d6): δ 158.3, 153.8, 141.1,
140.6, 125.8, 125.4, 123.7, 121.4, 120.8, 118.5, 116.0, 115.7, 112.7,
112.4, 68.8, 48.3, 32.5, 30.8, 19.0; ESIMS m/z (rel. intensity) 347
(MH+, 100). HRMS calcd for C21H23N4O+: 347.1866; found,
347.1872.
+
(rel. intensity) 317 (MH+, 100). HRMS calcd for C20H21N4 :
317.1761; found, 317.1764.
3-[2-(2-Amino-4-methylquinolin-7-yl)ethyl]-5-((methylamino)-
methyl)benzonitrile Dihydrochloride (16). Compound 68 (0.045 g,
0.095 mmol) was deprotected using K2CO3 (0.026 g, 0.190 mmol) as
described in the General Procedure. After workup, the free
aminoquinoline was purified by flash column chromatography, eluting
with a gradient of EtOAc to 3% MeOH in EtOAc, to yield a colorless
gum that was diluted in 5:1 ether/MeOH (10 mL) and treated with
methanolic HCl (1 mL). After being stirred overnight, filtration
afforded 16 (0.019 g, 50% from 68) as a white solid after precipitation
from hot MeOH (0.75 mL) with ether (13 mL) and washed with 30%
(R,S)-3-[(2-Amino-4-methylquinolin-7-yl)methoxy]-5-(2-
(methylamino)propyl)benzonitrile Dihydrochloride (R,S-19). This
compound was prepared from 44 (0.054 g, 0.217 mmol) and phenol
82 (0.060 g, 0.206 mmol) at 50 °C. Workup and purification by flash
column chromatography, eluting with a gradient of 5% EtOAc in
CH2Cl2 to 40% EtOAc in CH2Cl2, afforded intermediate acetamide 84
as a yellow foam (0.063 g, 61%), which was immediately deprotected
using K2CO3 (0.035 g, 0.251 mmol) as described in the General
Procedure. After workup, the free aminoquinoline was purified by flash
column chromatography, eluting with a gradient of EtOAc to 2%
MeOH in EtOAc, and the obtained residue was diluted in 4:1 ether/
MeOH (10 mL) and treated with methanolic HCl (0.5 mL). After
being stirred overnight, filtration afforded (R,S)-19 (0.041 g, 76% from
84) as a white flocculent solid after precipitation from hot MeOH (1
mL) with ether (10 mL) and washing with ether: mp 190−200 °C
1
MeOH in ether, with ether, and dried: mp 213−215 °C. H NMR
(500 MHz; DMSO-d6): δ 13.99 (s, 1 H), 9.29 (s, 2 H), 8.91 (br s, 1
H), 7.91 (d, J = 8.4 Hz, 1 H), 7.85−7.82 (m, 2 H), 7.83−7.81 (m, 1
H), 7.49 (d, J = 1.2 Hz, 1 H), 7.41 (dd, J = 8.4, 1.5 Hz, 1 H), 6.87 (d, J
= 1.0 Hz, 1 H), 4.14 (s, 2 H), 3.13 (dd, J = 9.1, 6.4 Hz, 2 H), 3.03 (dd,
J = 9.2, 6.5 Hz, 2 H), 2.61 (d, J = 0.8 Hz, 3 H), 2.55−2.50 (m, 3 H,
obscured by solvent peak). One of the aminoquinoline−NH protons is
not visible due to baseline broadening. 13C NMR (126 MHz; DMSO-
d6): δ 153.7, 152.3, 146.0, 143.0, 135.7, 135.2, 133.6, 132.4, 131.3,
125.7, 125.5, 119.6, 118.5, 116.6, 111.8, 111.4, 50.2, 35.9, 35.6, 31.9,
18.9; ESIMS m/z (rel. intensity) 331 (MH+, 100). HRMS calcd for
1
(softens); 246−250 °C (melts). H NMR (500 MHz; DMSO-d6): δ
14.10 (s, 1 H), 9.00−8.90 (m, 3 H), 8.10 (br s, 1 H), 8.03 (d, J = 8.4
Hz, 1 H), 7.75 (d, J = 0.8 Hz, 1 H), 7.56 (dd, J = 8.4, 1.4 Hz, 1 H),
7.47 (dd, J = 2.4, 1.3 Hz, 1 H), 7.41 (s, 1 H), 7.38 (dd, J = 2.1, 1.6 Hz,
1 H), 6.93 (d, J = 0.8 Hz, 1 H), 5.40 (s, 2 H), 3.48−3.44 (m, 1 H),
3.17 (dd, J = 13.6, 4.5 Hz, 1 H), 2.73 (dd, J = 13.4, 9.4 Hz, 1 H), 2.63
(d, J = 0.9 Hz, 3 H), 2.57 (t, J = 5.4 Hz, 3 H), 1.10 (d, J = 6.5 Hz, 3
H). 13C NMR (126 MHz; DMSO-d6): δ 158.3, 153.8, 141.1, 140.1,
125.88, 125.84, 123.8, 121.8, 120.7, 118.5, 116.2, 115.7, 112.7, 112.4,
68.8, 54.7, 37.6, 29.7, 19.0, 15.1; two of the aminoquinoline carbons
are not visible due to baseline broadening; ESIMS m/z (rel. intensity)
361 (MH+, 100). HRMS calcd for C22H25N4O+: 361.2023; found,
361.2028.
+
C21H23N4 : 331.1917; found, 331.1922.
5-[(2-Amino-4-methylquinolin-7-yl)methoxy]-2-fluoro-3-
((methylamino)methyl)benzonitrile Dihydrochloride (17). This
compound was prepared from 44 (0.050 g, 0.202 mmol) and phenol
73 (0.054 g, 0.192 mmol) at 50 °C. Workup and purification by flash
column chromatography, eluting with a gradient of 5% EtOAc in
CH2Cl2 to 40% EtOAc in CH2Cl2, afforded intermediate acetamide 74
as a colorless glass (0.072 g, 73%), which was immediately deprotected
using K2CO3 (0.040 g, 0.291 mmol) as described in the General
Procedure. After workup, the free aminoquinoline was purified by flash
column chromatography, eluting with a gradient of EtOAc to 3%
MeOH in EtOAc, and the obtained residue was diluted in 5:1 ether/
MeOH (10 mL) and treated with methanolic HCl (1 mL). After being
stirred overnight, filtration afforded 17 (0.047 g, 77% from 74) as a
white flocculent solid after precipitation from hot MeOH (0.75 mL)
with ether (10 mL) and the product was washed with 30% MeOH in
(R)-3-[(2-Amino-4-methylquinolin-7-yl)methoxy]-5-(2-
(methylamino)propyl)benzonitrile Dihydrochloride ((R)-19). This
compound was prepared from 44 (0.057 g, 0.228 mmol) and phenol
(R)-82 (0.070 g, 0.241 mmol) at 50 °C. Workup and purification by
flash column chromatography, eluting with a gradient of 5% EtOAc in
CH2Cl2 to 40% EtOAc in CH2Cl2, afforded intermediate acetamide
(R)-84 as a white foam (0.079 g, 69%), which was immediately
deprotected using K2CO3 (0.043 g, 0.312 mmol) as described in the
General Procedure. After workup, the free aminoquinoline was
purified by flash column chromatography, eluting with a gradient of
EtOAc to 2% MeOH in EtOAc, and the obtained residue was diluted
in 4:1 ether/MeOH (10 mL) and treated with methanolic HCl (0.75
mL). The mixture was stirred for 20 h at r.t. and concentrated. The
residue was triturated with 5% MeOH in ether (10 mL) and collected
by filtration. The precipitate was recrystallized from MeOH/ether
(5:1, 15 mL) to yield (R)-19 as a white solid ((0.049 g, 72% from (R)-
84)) after washing with ether: mp 146−148 °C (softens), 170−173 °C
(melts). 1H NMR (500 MHz; DMSO-d6): δ 14.03 (s, 1 H), 8.90−8.80
(m, 3 H), 8.10 (br s, 1 H), 8.03 (d, J = 8.4 Hz, 2 H), 7.75 (s, 1 H),
7.56 (dd, J = 8.4, 0.7 Hz, 1 H), 7.48 (dd, J = 2.3, 1.3 Hz, 1 H), 7.41 (s,
1 H), 7.38 (dd, J = 2.0, 1.6 Hz, 1 H), 6.93 (s, 1 H), 5.40 (s, 2 H),
3.48−3.44 (m, 1 H), 3.17 (dd, J = 13.4, 4.5 Hz, 1 H), 2.73 (dd, J =
13.4, 9.4 Hz, 1 H), 2.63 (d, J = 0.8 Hz, 3 H), 2.57 (t, J = 5.2 Hz, 3 H),
1.10 (d, J = 6.5 Hz, 3 H). 13C NMR (126 MHz; DMSO-d6): δ 158.3,
153.8, 141.0, 140.1, 125.88, 125.81, 123.7, 121.8, 120.8, 118.5, 116.2,
115.8, 112.7, 112.4, 68.8, 54.7, 37.6, 29.7, 19.0, 15.1; two of the
aminoquinoline carbons are not visible due to baseline broadening;
ESIMS m/z (rel. intensity) 361 (MH+, 100). HRMS calcd for
C22H25N4O+: 361.2023; found, 361.2030. The compound displays
1
ether and then with ether: mp 279.5−281 °C (dec). H NMR (500
MHz; DMSO-d6): δ 14.15 (br s, 1 H), 9.44 (br s, 2 H), 9.00 (br s, 1
H), 8.10 (br s, 1 H), 8.03 (d, J = 8.4 Hz, 1 H), 7.85−7.84 (m, 1 H),
7.76−7.74 (m, 2 H), 7.56 (dd, J = 8.4, 1.4 Hz, 1 H), 6.94 (d, J = 0.9
Hz, 1 H), 5.40 (s, 2 H), 4.21 (s, 2 H), 2.63 (d, J = 0.9 Hz, 3 H), 2.59
(s, 3 H). 13C NMR (126 MHz; DMSO-d6): δ (156.7 + 154.7, 1 C),
(153.92 + 153.90, 1 C), 153.80, 152.2, 140.7, 135.7, 125.9, (124.92 +
124.89, 1C), 123.8, (121.92 + 121.80, 1 C), 120.8, 119.2, 115.9, 113.6,
112.7, (100.83 + 100.69, 1 C) 69.5, (43.84 + 43.82, 1 C), 32.2, 19.0;
ESIMS m/z (rel. intensity) 351 (MH+, 100). HRMS calcd for
C20H20FN4O+: 351.1616; found, 351.1623.
3-[(2-Amino-4-methylquinolin-7-yl)methoxy]-5-(2-
(methylamino)ethyl)benzonitrile Dihydrochloride (18). This com-
pound was prepared from 44 (0.024 g, 0.0949 mmol) and phenol 81
(0.025 g, 0.0904 mmol). The reaction mixture was stirred at r.t. for 4
days. Workup and purification by flash column chromatography,
eluting with a gradient of 5% EtOAc in CH2Cl2 to 35% EtOAc in
CH2Cl2, afforded intermediate acetamide 83 as a white semisolid
(0.025 g, 57%), which was immediately deprotected using K2CO3
(0.014 g, 0.102 mmol) as described in the General Procedure. After
workup, the free aminoquinoline was purified by flash column
chromatography, eluting with a gradient of EtOAc to 7% MeOH in
EtOAc, and the obtained clear gum was diluted in 10:1 ether/MeOH
(10 mL) and treated with methanolic HCl (0.5 mL). After being
stirred overnight, filtration afforded 18 (0.010 g, 49% from 83) as a
O
DOI: 10.1021/acs.jmedchem.7b00259
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Journal of Medicinal Chemistry
Article
little optical rotation at a concentration of 10 mg/mL but was
confirmed as a single enantiomer via chiral derivatization.
7-Bromo-4-methylquinolin-2-amine (39). N-Oxide 38 (3.81 g, 16
mmol) was diluted in 2:1 trifluorotoluene/CH2Cl2 (120 mL) and
cooled to 0 °C. t-Butylamine (8.41 mL, 80 mmol) was added, followed
by p-toluenesulfonic anhydride (10.4 g, 32 mmol) in small portions.
After 10 min, additional t-BuNH2 (1 mL) and Ts2O (1.0 g) were
added, and after a total of 20 min, TLC indicated the presence of a less
polar product (intermediate t-butylaminoquinoline). The suspension
was concentrated until the CH2Cl2 was removed, trifluoroacetic acid
(34 mL) was added, and the mixture was heated until the internal
temperature reached 80 °C, where it was held for 5 h. The mixture was
then cooled and stirred 17 h at r.t. The dark red mixture was
concentrated and neutralized with 1 N NaOH until the pH was ∼12.
The suspension was extracted with EtOAc (3 × 150 mL), and the
organic layers were washed with H2O and sat. aq. NaCl (100 mL
each). The organic layers were dried over anhydrous sodium sulfate
and concentrated to yield a residue that was adsorbed onto silica gel
and purified by flash column chromatography, eluting with a gradient
of 50% EtOAc in CH2Cl2 to EtOAc, to yield 39 as a tan crystalline
(S)-3-[(2-Amino-4-methylquinolin-7-yl)methoxy]-5-(2-
(methylamino)propyl)benzonitrile Dihydrochloride ((S)-19). This
compound was prepared from 44 (0.047 g, 0.190 mmol) and phenol
(S)-82 (0.058 g, 0.200 mmol) at 50 °C. Workup and purification by
flash column chromatography, eluting with a gradient of 5% EtOAc in
CH2Cl2 to 40% EtOAc in CH2Cl2, afforded intermediate acetamide
(S)-84 as a white foam (0.059 g, 62%), which was immediately
deprotected using K2CO3 (0.033 g, 0.239 mmol) as described in the
General Procedure. After workup, the free aminoquinoline was
purified by flash column chromatography, eluting with a gradient of
EtOAc to 2% MeOH in EtOAc, and the obtained residue was diluted
in 6:1 ether/MeOH (7 mL) and treated with methanolic HCl (0.75
mL). The mixture was stirred for 20 h at r.t., ether (6 mL) was added,
and the mixture was filtered. The precipitate was recrystallized from
MeOH/ether (5:1, 10 mL) to yield (S)-19 as a white solid ((0.042 g,
83% from (S)-84)) after washing with ether: mp 146−148 °C
1
1
(softens), 170−173 °C (melts). H NMR (500 MHz; DMSO-d6): δ
solid (2.26 g, 60%) after washing with hexanes: mp 180−182 °C. H
14.00 (br s, 1 H), 8.90−8.80 (m, 3 H), 8.10 (br s, 1 H), 8.03 (d, J = 8.4
Hz, 2 H), 7.75 (s, 1 H), 7.56 (dd, J = 8.4, 0.6 Hz, 1 H), 7.48 (dd, J =
2.4, 1.3 Hz, 1 H), 7.41 (s, 1 H), 7.38 (t, J = 1.8 Hz, 1 H), 6.93 (s, 1 H),
5.41 (s, 2 H), 3.48−3.44 (m, 1 H), 3.17 (dd, J = 13.4, 4.6 Hz, 1 H),
2.73 (dd, J = 13.4, 9.4 Hz, 1 H), 2.64 (d, J = 0.8 Hz, 3 H), 2.58 (t, J =
5.3 Hz, 3 H), 1.10 (d, J = 6.5 Hz, 3 H). 13C NMR (126 MHz; DMSO-
d6): δ 158.8, 154.3, 141.5, 140.6, 126.37, 126.32, 124.3, 122.3, 121.3,
119.0, 116.6, 116.3, 113.2, 112.9, 69.3, 55.2, 38.0, 30.2, 19.4, 15.6; two
of the aminoquinoline carbons are not visible due to baseline
broadening; ESIMS m/z (rel. intensity) 361 (MH+, 100); ESIMS m/z
(rel. intensity) 361 (MH+, 100). HRMS calcd for C22H25N4O+:
361.2023; found, 361.2033. The compound displays little optical
rotation at a concentration of 10 mg/mL but was confirmed as a single
enantiomer via chiral derivatization.
NMR (500 MHz; DMSO-d6): δ 7.68 (d, J = 8.7 Hz, 1 H), 7.57 (d, J =
2.0 Hz, 1 H), 7.27 (dd, J = 8.7, 2.1 Hz, 1 H), 6.62 (d, J = 1.0 Hz, 1 H),
6.53 (s, 2 H), 2.46 (d, J = 1.0 Hz, 3 H). 13C NMR (126 MHz; CDCl3):
δ 157.6, 148.5, 145.9, 128.5, 125.6, 125.1, 123.5, 122.6, 112.3, 18.7;
ESIMS m/z (rel. intensity) 237/239 (MNa+, 100/94).
N-(7-Bromo-4-methylquinolin-2-yl)acetamide (40). Compound
39 (2.20 g, 9.28 mmol) was diluted in anhydrous THF (50 mL),
and N-acetylimidazole (1.53 g, 13.9 mmol) and a catalytic amount of
DMAP (∼20 mg) were added. The mixture was heated at reflux for 17
h. The mixture was cooled and concentrated, and the residue was
dissolved in EtOAc (200 mL) and washed with H2O (100 mL). The
aqueous layer was extracted with EtOAc (2 × 200 mL), and the
organic layers were washed with H2O (200 mL) and sat. aq. NaCl
(200 mL). The combined organic layers were dried over anhydrous
sodium sulfate and concentrated, and the residue was suspended in
EtOAc (5 mL) and precipitated by the addition of hexanes (150 mL).
The precipitate was collected and washed with hexanes to yield 40 as
7-Bromo-4-methylquinoline (37).26 3-Bromoaniline (36, 13.8 g, 80
mmol) and FeCl3·6 H2O (22.8 g, 84 mmol) were diluted with glacial
acetic acid (200 mL). The mixture was heated to 60 °C to ensure
dissolution of the solids (ca. 15 min), and then methyl vinyl ketone (8
mL, 88 mmol) was added slowly. The mixture was heated at reflux
(∼140 °C) for 3 h, during which time a yellow precipitate formed. The
mixture was then cooled to r.t., and the precipitate was filtered from
the solution and washed with EtOAc until the filtrate was colorless
(this filtrate was discarded). The filter cake was then diluted with
EtOAc (400 mL), and 1 M NaOH was added until the pH of the
resulting suspension was >9. The resulting emulsion was filtered
through a coarse fritted funnel to remove iron salts, and the organic
and aqueous layers were separated. The aqueous layer was extracted
with EtOAc (4 × 400 mL). The combined organic layers (∼1.5 L)
were washed with H2O (2 × 400 mL) and sat. aq. NaCl (400 mL),
dried over anhydrous sodium sulfate, and concentrated to 1/10th of
the original volume. The solution was filtered through a pad of Celite
and reconcentrated to afford 37 as a yellow-green solid (7.71 g, 44%).
The analytical data for this compound are consistent with those
previously reported.26
7-Bromo-4-methylquinoline N-Oxide (38). Compound 37 (7.71 g,
34.7 mmol) was diluted in anhydrous CH2Cl2, and m-chloroperox-
ybenzoic acid (8.39 g of 70% m-CPBA, 48.6 mmol) was added. The
mixture was stirred at r.t. for 19 h, and 1 M NaOH (∼100 mL) was
added. The layers were separated, and the aqueous layer was extracted
exhaustively with CH2Cl2 (3 × 100 mL), following which the
combined organic layers were washed with sat. aq. NaHCO3 (100 mL)
and sat. aq. NaCl (100 mL) and dried over anhydrous sodium sulfate.
Concentration afforded a residue that was washed with 5% CH2Cl2 in
hexanes to afford the desired compound (7.58 g, 92%) as a yellow
crystalline solid after washing with hexanes and drying: mp 179−181
°C. 1H NMR (500 MHz; DMSO-d6): δ 9.01 (d, J = 2.0 Hz, 1 H), 8.48
(d, J = 6.0 Hz, 1 H), 7.86 (d, J = 9.0 Hz, 1 H), 7.79 (dd, J = 9.0, 2.0
Hz, 1 H), 7.18 (d, J = 6.0 Hz, 1 H), 2.69 (s, 3 H). 13C NMR (126
MHz; DMSO-d6): δ 140.8, 135.4, 133.5, 131.6, 128.4, 127.8, 123.7,
122.7, 121.7, 17.5; ESIMS m/z (rel. intensity) 260/262 (MNa+, 13/
13).
1
an off-white iridescent solid (2.32 g, 90%): mp 246−248.5 °C. H
NMR (500 MHz; CDCl3): δ 8.75 (br s, 1 H), 8.33 (s, 1 H), 8.01 (d, J
= 1.9 Hz, 1 H), 7.81 (d, J = 8.8 Hz, 1 H), 7.59 (dd, J = 8.8, 1.5 Hz, 1
H), 2.73 (s, 3 H), 2.31 (s, 3 H). 13C NMR (126 MHz; CDCl3): δ
169.9, 151.7, 147.9, 146.4, 129.4, 128.4, 125.4, 124.8, 124.0, 114.9,
24.8, 19.2; ESIMS m/z (rel. intensity) 301/303 (MNa+, 20/19).
N-(7-Formyl-4-methylquinolin-2-yl)acetamide (41). Compound
40 (1.08 g, 3.6 mmol), Na2CO3 (0.576 g, 5.38 mmol), dppb (0.070
g, 0.164 mmol), Pd(OAc)2 (0.024 g, 0.107 mmol), and N-
formylsaccharin (1.15 g, 5.44 mmol) were placed in a sealable,
heavy-walled glass tube, which was evacuated and backfilled with argon
several times. To the tube was added anhydrous, degassed DMF (20
mL) containing Et3SiH (0.744 mL, 4.66 mmol). The tube was sealed,
and the reaction mixture was allowed to stir at r.t. for 10 min before
being heated to 70 °C for 22 h. The reaction mixture was cooled and
quenched with 1:1 H2O/sat. aq. NaCl (50 mL) and extracted with
EtOAc (3 × 70 mL). The organic layer was washed with 5% aq. NaCl
(3 × 100 mL) and sat. aq. NaCl (100 mL), dried over anhydrous
sodium sulfate, and concentrated to yield a residue that was adsorbed
onto silica gel and purified by flash column chromatography, eluting
with a gradient of 5% EtOAc in CH2Cl2 to 40% EtOAc in CH2Cl2 to
yield 41 as a white flocculent solid (0.514 g, 63%); mp 209−211 °C
(softens), 218−221 °C (melts). 1H NMR (500 MHz; CDCl3): δ 10.20
(d, J = 0.5 Hz, 1 H), 8.40 (s, 1 H), 8.25 (d, J = 1.3 Hz, 1 H), 8.05 (d, J
= 8.6 Hz, 2 H), 8.00 (br s, 1 H), 7.95 (dd, J = 8.6, 1.6 Hz, 1 H), 2.76
(d, J = 1.0 Hz, 3 H), 2.29 (s, 3 H). 13C NMR (126 MHz; CDCl3): δ
192.2, 169.3, 151.7, 147.7, 146.2, 137.3, 133.3, 130.1, 125.3, 122.1,
116.8, 25.2, 19.5; ESIMS m/z (rel. intensity) 229 (MH+, 5).
N-(7-(Hydroxymethyl)-4-methylquinolin-2-yl)acetamide (42). Al-
dehyde 41 (0.514 g, 2.25 mmol) was diluted in MeOH (30 mL) and
heated gently to 50 °C to effect solution. When the mixture was just
dissolved, NaBH4 (0.111 g, 2.92 mmol) was added in one portion, and
the mixture was stirred for 20 min and concentrated. The residue was
partitioned between EtOAc and 1:1 sat. aq. NaHCO3/H2O (30 mL
P
DOI: 10.1021/acs.jmedchem.7b00259
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Journal of Medicinal Chemistry
Article
each). The aqueous layer was extracted with EtOAc (3 × 30 mL), and
the organic layers were washed with sat. aq. NaCl and dried over
anhydrous sodium sulfate. Concentration afforded a residue that was
partially dissolved in EtOAc (5 mL) and precipitated with hexanes
(100 mL). Filtration afforded 42 as a white crystalline powder (0.493
5% EtOAc in CH2Cl2 to 25% EtOAc in CH2Cl2 to yield 53 as a
flocculent, off-white solid (0.262 g, 46%) after washing with hexanes;
mp 220−222 °C. H NMR (500 MHz; CDCl3): δ 8.42 (br d, J = 8.1
Hz, 1 H), 8.20 (br s, 1 H), 8.14 (d, J = 9.1 Hz, 1 H), 7.99 (s, 1 H),
7.64 (d, J = 8.6 Hz, 1 H), 7.54 (d, J = 8.7 Hz, 1 H), 2.29 (s, 3 H). 13C
NMR (126 MHz; CDCl3): δ 169.3, 151.6, 147.0, 138.9, 129.6, 128.96,
128.94, 125.0, 124.5, 114.5, 25.1; 287/289 (MNa+, 21/20).
1
1
g, 95%) that was used without further purification. H NMR (500
MHz; CDCl3): δ 9.75 (br s, 1 H), 8.22 (s, 1 H), 7.89 (d, J = 8.5 Hz, 1
H), 7.78 (s, 1 H), 7.49 (dd, J = 8.5, 1.2 Hz, 1 H), 4.83 (s, 2 H), 3.50
(br s, 1 H) 2.69 (s, 3 H), 2.28 (s, 3 H).
General Procedure for Sonogashira Coupling between 7-
Bromoquinolines and Phenylacetylenes.31 A sealable vial or tube
was charged with the requisite 7-bromoquinoline (1 equiv) and
phenylacetylene (1.1−1.5 equiv), XPhos (6 mol %), Pd(MeCN)2Cl2
(3 mol %), and Cs2CO3 (1.5 equiv). The vial was evacuated and
backfilled with argon several times, and then anhydrous MeCN (2
mL/100 mg bromoquinoline) was added, and the mixture was heated
to 75−80 °C overnight (typically 16−18 h). The mixture was then
cooled, diluted with water, and extracted with EtOAc (typically 3 × 30
mL is sufficient), and the organic layers were washed with 5% sat. aq.
NaCl (3 × 50 mL) and sat. aq. NaCl (50 mL). The organic phases
were dried over anhydrous sodium sulfate and concentrated to yield
residues that were purified using flash column chromatography (SiO2)
with gradients as described for the individual compounds below.
tert-Butyl (3-((2-Acetamido-4-methylquinolin-7-yl)ethynyl)-
benzyl)-(methyl)carbamate (63). This compound was prepared
from 40 (0.059 g, 0.179 mmol) and 57 (0.048 g, 0.197 mmol) at
75 °C. Workup and purification by flash column chromatography,
eluting with a gradient of 5% EtOAc in CH2Cl2 to 30% EtOAc in
CH2Cl2 gave the desired product as a yellow powder (0.058 g, 73%).
The identity of the product was confirmed by NMR spectroscopy, and
the product was carried on to the next step without further purification
or characterization.
tert-Butyl (3-((2-Acetamidoquinolin-7-yl)ethynyl)-5-cyanoben-
zyl)-(methyl)carbamate (64). This compound was prepared from
53 (0.071 g, 0.268 mmol) and 62 (0.083 g, 0.307 mmol) at 80 °C.
Workup and purification by flash column chromatography, eluting
with a gradient of 5% EtOAc in CH2Cl2 to 30% EtOAc in CH2Cl2,
gave the desired product as a tan powder (0.031 g, 28%). The product
was carried on to the next step without further purification or
characterization.
tert-Butyl (3-((2-Acetamido-4-methylquinolin-7-yl)ethynyl)-5-cy-
anobenzyl)-(methyl)carbamate (65). This compound was prepared
from 40 (0.120 g, 0.430 mmol) and 62 (0.180 g, 0.665 mmol) at 80
°C. Workup and purification by flash column chromatography, eluting
with a gradient of 5% EtOAc in CH2Cl2 to 30% EtOAc in CH2Cl2 gave
the desired product as a brownish-yellow semisolid (0.191 g, 95%).
The product was carried on to the next step without further
purification or characterization.
N-(7-(Bromomethyl)-4-methylquinolin-2-yl)acetamide (43). Alco-
hol 42 (0.147 g, 0.64 mmol) was diluted in anhydrous THF (5 mL)
and cooled to 0 °C. CBr4 (0.255 g, 0.768 mmol) and PPh3 (0.201 g,
0.768 mmol) were added. The mixture was stirred at 0 °C for 15 min,
warmed to r.t., and stirred for 3.5 h. The mixture was then
concentrated and diluted in CH2Cl2 and filtered to remove insoluble
materials. The filtrate was concentrated and purified by flash column
chromatography, eluting with a gradient of 3% EtOAc in CH2Cl2 to
30% EtOAc in CH2Cl2 to yield 43 as a white solid (0.135 g, 72%).
This crude product (containing some byproducts arising from
overbromination of the acetamide) was suitable for the formation of
phenyl ethers and was used without further purification or character-
ization. 1H NMR (500 MHz; CDCl3): δ 8.38 (s, 1 H), 7.98 (d, J = 8.6
Hz, 1 H), 7.85 (d, J = 1.4 Hz, 1 H), 7.60 (dd, J = 8.6, 1.4 Hz, 1 H),
4.63 (s, 2 H), 2.77 (d, J = 0.4 Hz, 3 H), 2.36 (s, 3 H).
N-(7-(Chloromethyl)-4-methylquinolin-2-yl)acetamide (44). Alco-
hol 42 (0.500 g, 2.17 mmol) was cooled to 0 °C under argon. Chilled
SOCl2 (2.1 mL) was added, and the mixture was stirred at 0 °C for 1
h. The mixture was then concentrated, and the residue was azeotroped
with toluene (2 × 5 mL) to remove traces of SOCl2. The residue was
suspended in H2O, and sat. aq. NaHCO3 was added until a white solid
precipitated (at pH ∼8). The suspension was extracted with EtOAc (3
× 50 mL). Sat. aq. NaHCO3 (50 mL) was added to the organic layer,
and the mixture was sonicated vigorously until the EtOAc became
cloudy, and the mixture was allowed to sit overnight for 17 h to ensure
hydrolysis of any byproducts arising from overchlorination at the
acetyl group. The layers were then separated, and the organic layer was
washed with sat. aq. NaCl (100 mL), dried over anhydrous sodium
sulfate, and concentrated. The resulting residue was washed with 1%
EtOAc in hexanes to yield 51 as a white iridescent solid (0.508 g,
1
94%); mp 211−212.5 °C. H NMR (500 MHz; CDCl3): δ 8.28 (s, 1
H), 8.15 (br s, 1 H), 7.94 (d, J = 8.6 Hz, 1 H), 7.79 (d, J = 1.4 Hz, 1
H), 7.51 (dd, J = 8.6, 1.8 Hz, 1 H), 4.75 (s, 2 H), 2.72 (d, J = 0.9 Hz, 3
H), 2.27 (s, 3 H). 13C NMR (126 MHz; CDCl3): δ 169.4, 151.1,
148.1, 139.5, 127.0, 126.1, 125.5, 124.9, 114.9, 46.0, 25.2, 19.4; one of
the quinoline carbons is not visible due to baseline broadening; ESIMS
m/z (rel. intensity) 271/273 (MNa+, 41/12).
7-Bromoquinolin-2-amine (52). Compound 51 (0.700 g, 2.89
mmol), anhydrous K2CO3 (1.99 g, 14.4 mmol), and dry acetamide
(13.7 g, 231 mmol) were heated under argon to reflux (∼230 °C) for
18 h. The mixture was cooled and diluted with H2O (60 mL), and the
resulting suspension was extracted with EtOAc (3 × 70 mL). The
organic layers were washed with H2O and sat. aq. NaCl (70 mL each),
dried over anhydrous sodium sulfate, and concentrated. The resulting
solid was dissolved in hot EtOAc (5 mL) and precipitated by the
addition of hexanes (100 mL). The yellow solid was collected and
dried to yield crude compound 52 (0.481 g, 75%), which was used
without any further purification; analytical data for this compound are
consistent with those previously reported.28
N-(7-Bromoquinolin-2-yl)acetamide (53). Compound 52 (0.481 g,
2.15 mmol) was diluted in anhydrous THF (25 mL). N-
Acetylimidazole (0.284 g, 2.58 mmol) was added, and the mixture
was heated to reflux under argon for 20 h, after which additional N-
acetylimidazole (0.284 g, 2.58 mmol) and a catalytic amount of DMAP
(∼20 mg) were added. After a total of 23 h at reflux, the mixture was
cooled and concentrated, and the residue was partitioned between
H2O and EtOAc (30 mL each). The aqueous layer was then extracted
with EtOAc (3 × 30 mL), and the organic layers were washed with
H2O and sat. aq. NaCl (100 mL each). The organic phase was dried
over anhydrous sodium sulfate and concentrated, and the residue was
purified by flash column chromatography, eluting with a gradient of
tert-Butyl (3-(2-(2-Acetamido-4-methylquinolin-7-yl)ethyl)-
benzyl)-(methyl)carbamate (66). Compound 63 (0.385 g, 0.868
mmol) was diluted with MeOH (40 mL) and heated gently to affect
solution. A catalytic amount of 10% Pd/C (∼20 mg) was added, and
the mixture was hydrogenated (using a balloon) for 20 h at r.t. The
Pd/C was then removed via syringe filter, and the filtrate was
concentrated. The residue was diluted in minimal CH2Cl2 (2 mL) and
precipitated by the addition of hexanes (50 mL). The suspension was
filtered to yield 66 as a white solid (0.362 g, 93%) after washing with
hexanes and drying in vacuo. This compound was deprotected
immediately without further characterization or purification.
tert-Butyl (3-(2-(2-Acetamidoquinolin-7-yl)ethyl)-5-cyanoben-
zyl)-(methyl)carbamate (67). Compound 64 (0.035 g, 0.077 mmol)
was diluted in THF (5 mL), and a catalytic amount of Pd/C(en) was
added. The mixture was hydrogenated (using a balloon) for 66 h, with
another equivalent of catalyst being added after 18 h. The Pd/C(en)
was removed via filtration, and the filtrate was concentrated. The
residue was purified by flash column chromatography, eluting with a
gradient of 5% EtOAc in CH2Cl2 to 45% EtOAc in CH2Cl2, to yield
67 as a yellow gum (0.026 g, 76%), which was deprotected
immediately without further characterization or purification.
tert-Butyl (3-(2-(2-Acetamido-4-methylquinolin-7-yl)ethyl)-5-cya-
nobenzyl)-(methyl)carbamate (68). Compound 65 (0.100 g, 0.213
mmol) was diluted in THF (12 mL), and a catalytic amount of Pd/
Q
DOI: 10.1021/acs.jmedchem.7b00259
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
C(en) was added. The mixture was hydrogenated at 50 PSI on a Parr
shaker for 48 h. The Pd/C(en) was removed via filtration, and the
filtrate was concentrated. The residue was purified by flash column
chromatography, eluting with a gradient of 5% EtOAc in CH2Cl2 to
40% EtOAc in CH2Cl2, to yield 68 as a yellow gum (0.045 g, 45%),
which was immediately deprotected without further characterization or
purification.
using REFMAC or PHENIX.62 Replacement of magnesium acetate in
the cryoprotectant with MgSO4 or MgCl2 for heNOS crystals caused a
shrinkage of cell dimensions (MgSO4) or a distortion of the symmetry
(MgCl2) compared to that in the heNOS crystal structures reported
previously.38 Therefore, a molecular replacement calculation with
PHASER-MR63was needed to solve the structure. The crystal packing
of the MgCl2 soaked heNOS crystals was changed slightly, resulting in
a symmetry change from orthorhombic P212121 to monoclinic P21,
with a β angle only 0.6−0.7° off compared to the original 90°. In the
P21 space group, there are two heNOS dimers in the asymmetric unit.
Disordering in portions of inhibitors bound in the NOS active sites
was often observed, sometimes resulting in poor density quality.
However, partial structural features were usually still visible if the
contour level of the sigmaA weighted 2m|Fo| − D|Fc| map was dropped
to 0.5 σ, which afforded the building of reasonable models into the
disordered regions. Water molecules were added in PHENIX and
checked by Coot. The TLS64 protocol was implemented in the final
stage of refinements with each subunit as one TLS group. The omit Fo
− Fc density maps were calculated by removing inhibitor coordinates
from the input PDB file before running one more round of TLS
refinement in PHENIX (simulated annealing protocol with a 2000 K
initial temperature). The resulting map coefficients DELFWT and
PHDELWT were used to generate maps. The refined structures were
validated in Coot before deposition in the Protein Data Bank.
Caco-2 Permeability Assay. Caco-2 monolayer assays were
performed by Cyprotex US, LLC (Watertown, MA), using standard
procedures, as previously reported for compound 4.18
Purified NOS Enzyme Assays. Rat and human nNOS, murine
macrophage iNOS, and human eNOS were recombinant enzymes
(expressed in E. coli and purified as reported previously).38,51−53 To
test for NOS inhibition, the hemoglobin capture assay was used to
measure nitric oxide production. The assay was performed at 37 °C in
HEPES buffer (100 mM with 10% glycerol, pH 7.4) in the presence of
10 μM L-arginine (used because (a) it is close to the Km values all three
isoforms, where detection of competitive inhibitors is most sensitive,
and (b) significant NOS uncoupling does not occur at this
concentration). Also included were 100 μM NADPH, 0.83 mM
CaCl2, approximately 320 units/mL of calmodulin, 10 μM H4B, and
human oxyhemoglobin (3 μM). For iNOS, the CaCl2 and calmodulin
were omitted and replaced with HEPES buffer (neither is required for
activation of iNOS). The assay was performed in 96-well plates using a
Synergy 4 BioTek hybrid reader. The dispensing of NOS enzyme and
hemoglobin were automated, and after 30 s (maximum delay), NO
production was read by monitoring the absorbance at 401 nm
(resulting from conversion of oxyhemoglobin to methemoglobin).
Kinetic readouts were performed for 5 min. Each compound was
assayed at least in duplicate, and six to nine concentrations (500 μM−
50 nM or 100 μM−10 nM for eNOS and iNOS; 50 μM to 5 nM for
rat and human nNOS) were used to construct dose−response curves.
IC50 values were calculated by nonlinear regression (variable slope,
four parameters) using GraphPad Prism software (standard error is
reported for the LogIC50), and Ki values were obtained from IC50
values using the Cheng−Prusoff54 equation [Ki = IC50/(1 + [S]/Km)]
with the following Km values: 1.3 μM (rat nNOS), 1.6 μM (human
nNOS), 8.2 μM (murine macrophage iNOS), and 3.9 μM (human
eNOS).55
Inhibitor Complex Crystal Preparation. The sitting drop vapor
diffusion methods were used to grow crystals at 4 °C for the heme
domains of rat nNOS (8 mg/mL containing 20 mM histidine), the
human nNOS K301R/R354A/G357D mutant (10 mg/mL), and
human eNOS (7 mg/mL). The well solutions are for rat nNOS, 20−
24% PEG3350, 0.1 M MES, pH 5.8, 140−200 mM ammonium
acetate, 10% ethylene glycol, 5 mM GSH, and 30 μM SDS; for human
nNOS, 8% PEG3350, 35 mM citric acid, 65 mM Bis-Tris-propane, pH
7.2, 10% glycerol, and 5 mM TCEP; for human eNOS, 12−15%
PEG3350, 0.1 M Bis-Tris, pH 6.5, 200−250 mM magnesium acetate,
100 mM GdCl3, 10% glycerol, and 5 mM TCEP. Fresh crystals were
first passed stepwise through cryoprotectant solutions and then soaked
with 5−10 mM inhibitor for 3−4 h at 4 °C before being flash cooled
with liquid nitrogen and stored until data collection. The presence of
an acetate ion near the heme active site in bovine eNOS had caused
interference in the binding mode of some phenyl ether-linked
aminoquinoline compounds.18 The high concentration of magnesium
acetate in the heNOS growth conditions may also introduce an acetate
near the active site that can influence the binding mode of inhibitors.
To avoid having this acetate in the structure, the magnesium acetate in
the cryoprotectant solution was replaced with either MgSO4 or MgCl2.
X-ray Diffraction Data Collection, Data Processing, and
Structural Refinement. The cryogenic (100 K) X-ray diffraction
data were collected remotely at the Stanford Synchrotron Radiation
Lightsource (SSRL) or Advanced Light Source (ALS) through the
data collection control software Blu-Ice56 and a crystal-mounting
robot. When a Q315r CCD detector was used, 100−125° of data were
typically collected with 0.5° per frame. If a Pilatus pixel array detector
was used, 140−160° of fine-sliced data were collected with a 0.2° per
frame. Raw CCD data frames were indexed, integrated, and scaled
using iMOSFLM,57 but the pixel array data were processed with
XDS58 and scaled with Aimless.59 The binding of inhibitors was
detected by initial difference Fourier maps calculated with REFMAC.60
The inhibitor molecules were then modeled in Coot61 and refined
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.7b00259.
Crystallographic data collection and refinement statistics
for rat and human nNOS, human eNOS, and bovine
eNOS; bovine eNOS-11, rnNOS and hnNOS-(R,S)-19,
rnNOS (R)- and (S)-19, and heNOS-18 Table S1)
crystal structures, and NOS inhibition LogIC50 values
and standard errors for new compounds (PDF)
Synthesis and analytical data for compounds 23−25, 26−
29, 55−57, 59−62, 70−73, and 77−82 (CSV)
Accession Codes
PDB codes for X-ray crystal structures described in this study
have been deposited in the Protein Data Bank. We will release
the atomic coordinates and experimental data upon article
publication under the following accession codes: rnNOS-8,
5UNR; rnNOS-9, 5UNS; rnNOS-10, 5UNT; rnNOS-11,
5UNU; rnNOS-14, 5UNV; rnNOS-16, 5UNW; rnNOS-18,
5UNX; rnNOS-(R,S)-19, 5UNY; rnNOS-(R)-19, 5UNZ;
rnNOS-(S)-19, 5UO0; hnNOS-8, 5UO1; hnNOS-9, 5UO2;
hnNOS-11, 5UO3; hnNOS-18, 5UO4; hnNOS-(R,S)-19,
5UO5; hnNOS-(R)-19, 5UO6; hnNOS-(S)-19, 5UO7;
heNOS-8, 5UO8; heNOS-9, 5UO9; heNOS-11, 5UOA;
heNOS-(R)-19, 5UOB; heNOS-(S)-19, 5UOC; beNOS-11,
5UOD.
AUTHOR INFORMATION
Corresponding Authors
■
*(T.L.P.) Tel:+1 949 824 7020. E-mail: poulos@uci.edu.
*(R.B.S.) Tel:+1 847 491 5653. Fax: +1 847 491 7713. E-mail:
Agman@chem.northwestern.edu.
ORCID
Thomas L. Poulos: 0000-0002-5648-3510
Richard B. Silverman: 0000-0001-9034-1084
R
DOI: 10.1021/acs.jmedchem.7b00259
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the National Institutes of Health (R01GM049725, to
R.B.S., GM057353 to T.L.P., and F32GM109667 to M.A.C.),
for generous support of this work. M.A.C. wishes to thank Mr.
Saman Shafaie and Dr. S. Habibi Goudarzi for assistance with
HRMS experiments, Mr. Ben McDonald for his help with chiral
HPLC, and Dr. Gashaw Goshu for his help with polarimetry
measurements. This work made use of IMSERC at North-
western University, which has received support from the Soft
and Hybrid Nanotechnology Experimental (SHyNE) Resource
(NSF NNCI-1542205), the State of Illinois, and the Interna-
tional Institute for Nanotechnology (IIN). H.L. thanks Carla
Plaza for her assistance in protein purifications that provided
samples for both enzyme activity assays and crystallization. We
also wish to thank the SSRL and ALS beamline staff for their
support during remote X-ray diffraction data collection.
(16) Mukherjee, P.; Cinelli, M. A.; Kang, S.; Silverman, R. B.
Development of nitric oxide synthase (NOS) inhibitors for neuro-
degenerative diseases and neuropathic pain. Chem. Soc. Rev. 2014, 43,
6814−6838.
ABBREVIATIONS USED
■
́
(17) Cinelli, M. A.; Li, H.; Chreifi, G.; Martasek, P.; Roman, L. J.;
NO, nitric oxide; nNOS, neuronal nitric oxide synthase; iNOS,
inducible nitric oxide synthase; eNOS, endothelial nitric oxide
synthase; rnNOS, rat neuronal nitric oxide synthase; hnNOS,
human neuronal nitric oxide synthase; heNOS, human
endothelial nitric oxide synthase; miNOS, murine inducible
nitric oxide synthase; FMN, flavin mononucleotide; H4B, (6R)-
Poulos, T. L.; Silverman, R. B. Simplified 2-aminoquinoline-based
scaffold for potent and selective neuronal nitric oxide synthase
inhibition. J. Med. Chem. 2014, 57, 1513−1530.
́
(18) Cinelli, M. A.; Li, H.; Pensa, A. V.; Kang, S.; Martasek, P.;
Roman, L. J.; Poulos, T. L.; Silverman, R. B. Phenyl ether- and aniline-
containing 2-aminoquinolines as potent and selective inhibitors of
neuronal nitric oxide synthase. J. Med. Chem. 2015, 58, 8694−8712.
(19) Huang, H.; Li, H.; Martasek, P.; Roman, L. J.; Poulos, T. J.;
Silverman, R. B. Structure-guided design of selective inhibitors of
neuronal nitric oxide synthase. J. Med. Chem. 2013, 56, 3024−3032.
5,6,7,8-tetrahydrobiopterin; tPSA, total polar surface area; Papp
,
apparent permeability; Bis-Tris, bis(2-hydroxyethyl)amino-tris-
(hydroxymethyl)-methane; HEPES, 4-(2-hydroxyethyl)-1-pi-
perazineethanesulfonic acid; GSH, glutathione, reduced;
TCEP, tris(2-carboxyethyl)phosphine; dppb, 1,4-bis-
(diphenylphosphino)butane
́
(20) Kang, S.; Li, H.; Tang, W.; Martasek, P.; Roman, L. J.; Poulos,
T. L.; Silverman, R. B. 2-Aminopyridines with a truncated side chain to
improve human neuronal nitric oxide synthase inhibitory potency and
selectivity. J. Med. Chem. 2015, 58, 5548−5560.
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