Phenyl ether- and aniline-containing 2-aminoquinolines as potent and selective inhibitors of neuronal nitric oxide synthase

Article abstract of DOI:10.1021/acs.jmedchem.5b01330

Excess nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) is implicated in neurodegenerative disorders. As a result, inhibition of nNOS and reduction of NO levels is desirable therapeutically, but many nNOS inhibitors are poorly bioavailable. Promising members of our previously reported 2-aminoquinoline class of nNOS inhibitors, although orally bioavailable and brain-penetrant, suffer from unfavorable off-target binding to other CNS receptors, and they resemble known promiscuous binders. Rearranged phenyl ether- and aniline-linked 2-aminoquinoline derivatives were therefore designed to (a) disrupt the promiscuous binding pharmacophore and diminish off-target interactions and (b) preserve potency, isoform selectivity, and cell permeability. A series of these compounds was synthesized and tested against purified nNOS, endothelial NOS (eNOS), and inducible NOS (iNOS) enzymes. One compound, 20, displayed high potency, selectivity, and good human nNOS inhibition, and retained some permeability in a Caco-2 assay. Most promisingly, CNS receptor counterscreening revealed that this rearranged scaffold significantly reduces off-target binding.

Full text of DOI:10.1021/acs.jmedchem.5b01330

Article
pubs.acs.org/jmc
Phenyl Ether- and Aniline-Containing 2‑Aminoquinolines as Potent
and Selective Inhibitors of Neuronal Nitric Oxide Synthase
Maris A. Cinelli,† Huiying Li,‡ Anthony V. Pensa,† Soosung Kang,† Linda J. Roman,§ Pavel Martasek,§,∥,⊥
́
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,
California 92697-3900, United States
§Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78384-7760, United States
∥Department of Pediatrics, First Faculty of Medicine, Charles University, Prague, Czech Republic
⊥BIOCEV, Prague, Czech Republic
S
* Supporting Information
ABSTRACT: Excess nitric oxide (NO) produced by neuronal
nitric oxide synthase (nNOS) is implicated in neurodegener-
ative disorders. As a result, inhibition of nNOS and reduction
of NO levels is desirable therapeutically, but many nNOS
inhibitors are poorly bioavailable. Promising members of our
previously reported 2-aminoquinoline class of nNOS inhib-
itors, although orally bioavailable and brain-penetrant, suffer
from unfavorable off-target binding to other CNS receptors,
and they resemble known promiscuous binders. Rearranged
phenyl ether- and aniline-linked 2-aminoquinoline derivatives
were therefore designed to (a) disrupt the promiscuous
binding pharmacophore and diminish off-target interactions
and (b) preserve potency, isoform selectivity, and cell
permeability. A series of these compounds was synthesized and tested against purified nNOS, endothelial NOS (eNOS), and
inducible NOS (iNOS) enzymes. One compound, 20, displayed high potency, selectivity, and good human nNOS inhibition, and
retained some permeability in a Caco-2 assay. Most promisingly, CNS receptor counterscreening revealed that this rearranged
scaffold significantly reduces off-target binding.
trite, can cause free radical damage to cellular structures and
protein nitration, which can lead to misfolding and
aggregation.3,4 Abnormally high levels of nNOS and NO have
therefore been associated with or implicated in numerous
neurodegenerative disorders,5−8 making nNOS inhibition a
desirable therapeutic avenue for the treatment or prevention of
these conditions.9−11
Nitric oxide synthases are unique among mammalian
enzymes in that they are homodimers requiring five cofactors
to function: flavin adenine dinucleotide (FAD), flavin
mononucleotide (FMN), and reduced nicotinamide adenine
dinucleotide phosphate (NADPH), found in each monomer’s
reductase domain, and (6R)-5,6,7,8-tetrahydrobiopterin (H4B)
and the final electron acceptor, heme, which bind in each
monomer’s oxygenase domain along with the substrate, L-
arginine. Between the two domains lies a flexible region where
calmodulin binds when activated by calcium ion, and electron
INTRODUCTION
■
Neurodegenerative disorders, such as Alzheimer’s, Parkinson’s,
and Huntington’s diseases, amyotrophic lateral sclerosis (ALS),
and others, are diseases where the structure and function of
neurons gradually deteriorate, leading to the motor, cognitive,
and psychological difficulties associated with these disorders.
Neuronal damage also occurs in head trauma, cerebral palsy,
stroke, and ischemic events. With the world’s increasingly aged
population, the incidence of neurodegenerative disorders is also
increasing, and, with most treatments limited to palliative care,
the development of new therapeutics is of utmost importance.
A target receiving considerable attention is neuronal nitric
oxide synthase (nNOS).1 Nitric oxide (NO), an important
second messenger, is produced by three nitric oxide synthase
(NOS) enzymes: inducible nitric oxide synthase (iNOS),
required for immune response activation, endothelial nitric
oxide synthase (eNOS), which regulates blood pressure and
vascular tone, and nNOS, necessary for neuronal signaling.2 If
nNOS is overexpressed or overactive in neuronal tissue,
however, the excess NO, especially if converted to peroxyni-
Received: August 27, 2015
© XXXX American Chemical Society
A
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flow proceeds from one monomer’s reductase domain to the
other’s oxygenase domain,12 where NO is produced as L-
arginine is oxidized to L-citrulline.13
permeation in mice. Although only modestly potent in vivo, 2
also inhibited nNOS in rat brain homogenates and in the brains
of live rats. Despite their promise, 1 and 2 have major
disadvantages. Compound 2 has a poor safety profile in rats,
displaying toxic side effects at the doses required for effective
nNOS inhibition. Furthermore, a counterscreen against a panel
of relevant CNS targets and receptors performed by the
Psychoactive Drug Screening Program (PDSP)19 revealed that
1 and especially 2 are promiscuous binders with strong (<100
nM) affinities for serotonin, opioid, and histamine receptors, as
well as for the dopamine and norepinephrine transporters,
which could cause unfavorable side effects. Additionally,
compounds 1 and 2 are quite time-consuming to synthesize
(12 steps). Because of these problems, we chose to investigate
alternative aminoquinoline-containing scaffolds for nNOS
inhibition.
Interestingly, the literature revealed that compounds such as
ketanserin, haloperidol, and risperidone, which also bind to
multiple, similar CNS targets, have overall structural motifs in
common with 1 and 2: two hydrophobic or aromatic exteriors
(one, often halogenated), joined by an interior alkylamine
linker, a pharmacophore (Figure 2A) that appears to “stick” to
GPCRs and other CNS targets.20
One important challenge in the design of nNOS inhibitors is
that eNOS and iNOS, in sequence and overall structure, are
nearly identical to nNOS, especially in their active site
residues.14,15 Nonselective NOS inhibition is potentially
dangerous: iNOS inhibition could interfere with immune
activation, while eNOS inhibition can cause severe hyper-
tension and other cardiovascular liabilities.16 Additionally, many
nNOS inhibitors are mimics of L-arginine and thus have high
basicity and polarity, large total polar surface area (tPSA), and
many hydrogen bond donors, all of which hinder the inhibitors’
blood−brain barrier (BBB) penetration and diminish ther-
apeutic use.17
In 2014, we reported a series of 2-aminoquinolines as
competitive nNOS inhibitors.18 Two compounds, 1 and 2
(Figure 1), were considered promising candidates for further
Lowe and colleagues at Pfizer21 reported the structure of 3, a
potent nNOS inhibitor with very high affinity for dopamine and
serotonin receptors. Removal of the exterior hydrophobic
group diminished off-target binding, and when an exterior polar
amine was attached to an interior hydrophobic group (such as
in 4 and 5), higher potency and isoform selectivity resulted.22,23
Similar exterior polar amines (especially N-methyl and N,N-
dimethylamines) were also effective in conferring potency and
selectivity of NeurAxon’s thiophenecarboximidamides.24,25
Design of an analogous “inverted” aminoquinoline-contain-
ing compound (Figure 2B) resulted in 6, with the halves of the
Figure 1. Aminquinoline-containing nNOS inhibitors 1 and 2.
development because of their high potency and selectivity over
iNOS and eNOS. These compounds are permeable in a Caco-2
assay, and 1 displayed good oral bioavailability and brain
Figure 2. (A) Similarity of 1 and 2 to known “promiscuous” CNS binders; (B) truncation−inversion strategy (3 to 4 and 5) and design of
compound 6.
B
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Figure 3. Molecules investigated in this study and overall design strategy.
molecule joined by a phenyl ether, the same as in 4 and 5. We
hypothesized that in addition to reducing off-target binding,
this structural rearrangement could have additional benefits.
First, the external amine of 6 could potentially form
interactions in various spots peripheral to the nNOS active
site such as Asp597, the H4B site, or residues of the substrate
access channel hydrophobic pocket, all of which have been
shown to play significant roles in conferring potency or isoform
selectivity.26−28 Second, the predicted physicochemical proper-
ties of 6 are similar to those of 1 and 2 (CLogP: 2−4, <7
rotatable bonds, TPSA < 80 Å2, and <4 hydrogen-bond
donors), making it plausible that the cellular permeability of 1
and 2 could be preserved.
In addition to 6, we designed and synthesized a small library
of related compounds (7−12, Figure 3); all were assayed
against purified rat nNOS, murine iNOS, and bovine eNOS.
Encouraged by the potency and isoform selectivity of 8 and 9,
different modifications were performed to optimize the
structure and examine the SAR. First, 13 and 14 were
synthesized to investigate if longer tail lengths could provide
additional or enhanced beneficial contacts with the enzyme.
Second, although 8 and 9 have the amine-containing moiety
located meta to the phenyl ether, para-substituted compounds
15 and 16 were also prepared to investigate the role that
different ring substitution patterns may play. Third, we
reasoned that bioisosteric replacement of the phenyl ether
with an aniline (17 and 18) might introduce another contact in
the region near the heme propionates, resulting in greater
affinity for nNOS.
Finally, we performed specific, structure-based optimizations
to improve the potency and selectivity of lead 9. X-ray
crystallography indicated that the center aryl rings of 8 and 9
abut two residues, Tyr706 and Asn569. We hypothesized that
introduction of halogens para to the phenyl ether linkage, as in
19 and 20, could provide additional van der Waals interactions
with Tyr706 or the surrounding hydrophobic structures.
Likewise, by converting the phenyl ring to a pyridine (21), a
hydrogen bond or polar contact could potentially form between
the pyridine nitrogen and the side chain of Asn569. We also
proposed that contacts might be made with other residues of
the hydrophobic pocket (such as Met336 and Leu337) by
placing a methyl group alpha to the exterior amine group of 9
(to yield 22). All compounds were assayed against purified rat
nNOS, and select compounds were assayed against eNOS,
iNOS, and human nNOS, for cellular permeability in a Caco-2
model, and against a panel of CNS targets in the PDSP to
assess their off-target binding.
Chemistry. Previously, 7-substituted-2-aminoquinolines
were prepared via readily accessible chloroquinoline 2318,29
́ ́
(Scheme 1) by performing a Korod
i amidation30 to produce 2-
acetamidoquinoline 25. Unfortunately, this procedure gives
irreproducible yields and is not readily amenable to scale-up.
Smith et al.31 reported the palladium-catalyzed amination of 2-
chloroquinolines using LHMDS as both an ammonia surrogate
C
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a
of 31 and immediate Boc-protection (to aid in purification and
prevent later interference by the free secondary amine). For the
benzylic amine of 8, phenol 34 (Scheme 2C) was prepared by
reductive amination of commercially available aldehyde 33 with
N,N-dimethylamine. Similarly, exchanging N,N-dimethylamine
for methylamine (needed for 9) yielded an amine, which was
protected as 35. Phenol 37 (Scheme 2D) was prepared for the
synthesis of 10 by Boc-protecting commercially available 36.
The longer linker of 13, via phenol 40, was prepared by a
Sonogashira coupling of 3-iodophenol 38 with N,N-dimethyl-
propargylamine, followed by reduction of the triple bond of 39
to yield 40 (Scheme 3A). For compound 14, 42 was prepared
Scheme 1
a
Reagents and conditions: (a) LHMDS, Pd2(dba)3, DavePhos, THF/
dioxane, 100 °C; (b) N-acetylimidazole, THF, reflux; (c) NBS,
(PhCO2)2, benzene, reflux.
a
Scheme 3
and base; applying this procedure to 23 afforded 2-amino-
quinoline 24 in nearly quantitative yield on a multigram scale.
Treatment with N-acetylimidazole in refluxing THF afforded
25, and free-radical bromination (as previously reported)
yielded versatile bromide 26.
The phenol and aniline halves were then prepared. To
prepare 6 (Scheme 2A), 3-methoxyphenethylamine (27) was
dimethylated, and the O-methyl group of 28 was removed to
yield phenol 29. Monomethyl phenol 32 (needed for
compound 7) was prepared from 3-methoxyphenethyl bromide
(30, Scheme 2B) and methylamine, followed by demethylation
a
a
Scheme 2
Reagents and conditions: (a) N,N-dimethylpropargylamine, CuI (10
mol %), Pd(PPh3)4 (5 mol %), THF/Et3N, r.t.; (b) H2, Pd/C, MeOH,
r.t.; (c) N,N-dimethylethanolamine, PPh3, DEAD, THF, 0 °C, r.t.
by a Mitsunobu reaction between resorcinol 41 and N,N-
dimethylethanolamine32 (Scheme 3B). As performed for the
meta-analogues, phenols 44 and 45 (for analogues 15 and 16,
respectively, Scheme 4) were prepared from 4-hydroxybenzal-
dehyde (43) and either N,N-dimethylamine (for 44) or
methylamine (for 45).
a
Scheme 4
a
Reagents and conditions: (a) (i) Me2NH-HCl, Et3N, Na2SO4,
CHCl3/MeOH, r.t.; (ii) Na(OAc)3BH, r.t., (b) (i) MeNH2 in THF,
CHCl3/MeOH, Na2SO4, AcOH, r.t., (ii) NaBH4, MeOH, 0 °C−r.t.,
(iii) Boc2O, THF, r.t.
The anilines required for 17 and 18 were prepared from 3-
nitrobenzyl bromide (46, Scheme 5) upon treatment with
either dimethylamine (47) or methylamine (followed by Boc-
protection to yield 49). Reduction of the nitro group with
Raney nickel afforded 48 (for 17) and 50 (for 18).
Finally, the substituted phenols (for 19−22, Scheme 6A)
could be prepared by employing the reductive amination/Boc
protection protocol to commercially available aldehydes (51,
52) or acetophenone 53, to yield the protected amines 54−56,
a
Reagents and conditions: (a) formalin, formic acid, DMF, 60 °C
−120 °C, 5 h; (b) 48% HBr, AcOH, reflux; (c) 40% MeNH2 in H2O,
THF/H2O, r.t.; (d) (i) 48% HBr, HOAc, reflux, (ii) Boc2O, Et3N,
THF, r.t.; (e) (i) Me2NH-HCl, Et3N, CHCl3/MeOH, Na2SO4 r.t., (ii)
Na(OAc)3BH, r.t., (f) (i) MeNH2 in THF, cat. AcOH, CHCl3/
MeOH, Na2SO4, r.t.; (ii) NaBH4 MeOH, 0 °C−r.t., (iii) Boc2O, THF,
r.t.; (g) Boc2O, THF, 0 °C−r.t.
D
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a
75, which was deprotected as described above. Unfortunately,
because of its nucleophilic amine, 48 only produced water-
soluble quaternization products upon reaction with 26. It was
proposed that the two halves of N-linked compound 17 could
be joined via reductive amination, beginning with the
quinolinecarboxaldehyde 81. After many unsuccessful attempts
to prepare 81 from 25 and 26, 81 was prepared in five steps34
(Scheme 8), starting with a Wittig cyanovinylation of
commercially available aldehyde 76; the desired trans-isomer
77 was obtained in good yield. Iron-catalyzed reductive
cyclization yielded aminoquinoline 78, which was acetylated
in good yield using N-acetylimidazole. Ester 79 was reduced to
alcohol 80, and oxidation to 81 was performed using Dess-
Martin periodinane. An indirect reductive amination with 48
was effective at elevated temperatures, and the crude acetamide
was deprotected to yield 18.
Scheme 5
a
Reagents and conditions: (a) Me2NH-HCl, Et3N, CH2Cl2/MeOH,
r.t.; (b) H2, Raney Ni, MeOH, r.t.; (c) (ii) MeNH2 in THF, CH2Cl2,
r.t., (ii) Boc2O, CH2Cl2, r.t.
a
Scheme 6
RESULTS AND DISCUSSION
■
Compounds 6−22 were first assayed against purified rat nNOS,
murine macrophage iNOS, and bovine eNOS using the
hemoglobin capture assay.35,36 These species are used because
(a) it is easiest to purify and readily obtain crystallographic data
with these isozymes, and (b) there is a large active-site
homology between mammalian species and therefore, while not
100% accurate, an adequate approximation of isoform
selectivity can be obtained. Table 1 summarizes the apparent
Ki values and isoform selectivity values for 6−22; for the sake of
comparison, values for 1 and 2 are included.
The first series of compounds, 6−12, is less potent than 1 or
2. At first glance, it appears that compounds with a basic amino
group (6−10) are more potent against nNOS than those with
less basic groups such as pyridine (11) and N,N-dimethylani-
line, which rendered compound 12 essentially inactive. The
positioning of the tail amino group plays a role in enzyme−
inhibitor interactions. The crystal structures of nNOS-6 and
nNOS-7 are compared side by side in Figure 4. The strong 2-
aminoquinoline-Glu592 (arginine mimic) interaction character-
istic of this class is well supported by the strong electron
density. In contrast, the central aryl ring and the tail regions
show rather weak density, indicative of disordering. The
methylamine of 7 reaches the H4B site because the tail is long
enough to displace the water molecule normally present in this
site, and H-bond either with H4B or with the nearby heme
propionate (Figure 4B). This interaction pushes the central aryl
ring into nonbonded contact with the Tyr706 side chain.
However, the tertiary amino group of 6 does not interact with
H4B or heme, but hangs adjacent to Met336 because the central
aryl ring presses against the heme propionate (Figure 4A). The
lack of both the H4B site and Tyr706 contacts likely explains
why 6 is less potent than 7.
When the tail is one methylene shorter, (8 and 9 vs 6 and 7,
respectively) the potency is also increased. As shown in Figure
5, the tertiary amino group of 8 can form an H-bond with
Asn569, while the secondary amino group of 9 is involved in a
water-mediated H-bonding network with H4B and heme. The
phenyl ring of 9 forms a pi-stacking interaction with Tyr706
(these rings are >4 Å apart in the nNOS-8 structure). These
same interactions are observed in the nNOS-10 structure,
which has a primary amino group, (see Supporting Information,
Figure S1).
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) (i) HBr/H2O, AcOH, 130 °C, (ii) Et3N, Boc2O,
THF/MeOH, r.t.
respectively. Lastly, the pyridinol 59 (for 21, Scheme 6B) was
prepared by reductive amination of nicotinaldehyde 57 and
cleavage of the methyl group of 58; Boc-protection furnished
59.
With the halves of the phenyl ether-substituted quinolines in
hand, assembly of the final cores (Scheme 7) was completed by
first treating the desired phenol [29, 32, 34, 35, 37, 40, 42, 44,
45, 54−56, 59, or commercially available 3-hydroxypyridine or
3-(N,N-dimethylamino)phenol (for compounds 11 and 12,
respectively)] with sodium hydride in DMF at 0 °C. A solution
of 26 was then added; the reaction was typically complete
within 1 h. Yields were good but were lower for the tertiary
amines because of partial solubility in the aqueous DMF
solutions produced in the workup. The intermediate
acetamides (60−74) were not characterized and were
deprotected immediately after purification: the acetyl group
was first removed by K2CO3 in refluxing methanol, and after
isolation, the free aminoquinolines were treated with
methanolic HCl in ether to produce water-soluble hydro-
chloride salts. Compounds without a Boc group were isolated
after 5−15 min; those with a Boc group were stirred overnight
for deprotection. In the case of 72, HCl induced unfavorable
side reactions, so TFA was used instead.
The microwave alkylation procedure of Romero et al.33 was
employed to synthesize aniline 18 (Scheme 7). Compound 26,
excess 50, and catalytic potassium iodide were heated in
acetonitrile under microwave irradiation to furnish intermediate
Two trends are clear. First, potency is increased slightly upon
removal of one methyl group from the tertiary amines; 9 and
10 are more potent than 8, and 7 is more potent than 6. The
E
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a
Scheme 7
a
Reagents and conditions: (a) (i) phenols 29, 32, 34, 35, 37, 40, 42, 44, 45, 54−56, 59, 3-(dimethylamino)phenol, or 3-hydroxypyridine, NaH,
DMF, 0 °C, (ii) 26 (in DMF), 0 °C; (b) (i) K2CO3, MeOH, reflux, (ii) HCl/MeOH, ether, r.t., or TFA/DCM (for 72), after isolation, 5 min-
overnight; (c) 50 (2.5 equiv), cat. KI, μwave, MeCN, 110 °C.
a
Scheme 8
a
Reagents and conditions: (a) (triphenylphosphoranylidene)acetonitrile (slow addition), CH2Cl2, −10 °C; (b) Fe powder, DMF/AcOH, 100 °C;
(c) N-acetylimidazole, cat. DMAP, dioxane, 100 °C; (d) LiAlH4 (1.5 equiv), THF, −10 °C−0 °C; (e) PPh3, CBr4 THF, 0 °C−r.t.; (e) Dess-Martin
periodinane, CH2Cl2, r.t.; (f) (i) 48, EtOH, AcOH, Na2SO4, 60 °C, (ii) NaBH4, r.t., (g) (i) K2CO3, MeOH, reflux; (ii) HCl/MeOH, ether, r.t. (after
isolation).
protonated secondary amino group bears two hydrogens and is
a stronger H-bond donor than the tertiary amino group, as
demonstrated by 7 and 9. Second, the longer chains of 6 and 7
show higher flexibility (structure disordering) in the phenyl
ether and tail region when compared to that of 8 and 9 and
thus could result in weaker or partial interactions as a result of
entropy, leading to lower potency than that of 8 and 9.
Interactions with Asn569 or the H4B site are crucial for the
activity of these compounds; the short amino group of 12 is
incapable of forming these interactions, and this could be why
12 has little activity.
Interestingly, the n/e selectivity for 8 and 9 is similar to that
observed for 1 or 2. It is known that contact between the
halophenyl rings of 1 and 2 and the hydrophobic pocket
residues Tyr706, Leu337, and Met336 improves potency and
n/e selectivity.18 What is responsible for the n/e selectivity for
these compounds lacking obvious hydrophobic contact? X-ray
crystallography indicates that in eNOS, a common binding
mode is observed for most phenyl ether-linked aminoquino-
lines, characterized by an “upward” position of the ether bond,
where the phenyl ring sits perpendicular to the plane of the
aminoquinoline. In the eNOS-9 structure (Figure 6A), the
F
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nNOS and eNOS resulted in these binding modes. Further
complicating the interpretation of the eNOS structural data is
the presence of both glycerol and acetate (crystallization buffer
components) in all of the eNOS structures. While the glycerol
is located peripherally relative to the inhibitor, the acetate
(found in eNOS because of the alternate rotamer assumed by
Arg252) abuts the perpendicular phenyl rings of the inhibitors
and in several cases results in clearer electron density in the
crystal structures, suggesting that the perpendicular binding
modes may be stabilized by nonbonded contacts with the
acetate and are thus likely a crystallographic artifact. By
removing acetate from the cryo-soaking buffer, we were able to
confirm that acetate is, indeed, the reason for some of these
inhibitors to adopt a different binding mode in eNOS. This will
be considered in more detail when the structures of 20 bound
to eNOS and nNOS are discussed below.
To confirm this hypothesis further, the rat nNOS double
mutant D597N/M336V was employed, where Asp597 and
Met336 are mutated to, respectively, the corresponding
asparagine and valine found in bovine eNOS. This enzyme
has previously been used as an eNOS “surrogate” to elucidate
the effects that amino acid differences have on inhibitor
binding.37,38 While the active-site sequence resembles eNOS,
the overall local environment and domain architecture still
resemble wild-type rat nNOS; Arg481 (the equivalent of
Arg252) is retained in the WT nNOS rotameric state, and
acetate no longer binds. In the nNOS-D597N/M336V-9-
structure (Figure 6B) the phenyl ether portion of 9 reverts to
the coplanar binding mode observed in WT rat nNOS and not
the perpendicular mode observed in the eNOS-9 structure,
indicating that the upward position of the phenyl ring is likely
stabilized or induced by the acetate. Nonetheless, a difference in
the positioning of the methylamine tail in the nNOS-D597N/
M336V-9 structure (Figure 6B) could explain the observed
selectivity for nNOS over eNOS. In the WT nNOS structure,
the methylamine tail rotates away from bulky Met336, and the
amino group faces toward the bridging water in the H4B site
(Figure 5B). In the double mutant structure, the smaller Val336
(analogous to eNOS’ Val106) allows for the amine to instead
face toward the hydrophobic pocket, breaking the H4B site
interaction. This is not the first reported case where subtle
differences in Met/Val contacts have led to large effects on n/e
selectivity18,39 and also serves as a cautionary note against over-
reliance on X-ray structures in interpretation of SAR data. As
no acetate is used in the eNOS purification or assay, the
Table 1. Inhibition of NOS Enzymes by Phenyl Ether and
Aniline Compounds
a
Ki (μM)
selectivity
compd
nNOS
iNOS
eNOS
n/i
n/e
1
0.049
0.066
0.468
0.332
0.179
0.142
0.160
0.712
>5.75
0.652
0.475
0.283
0.332
0.375
0.569
0.147
0.058
0.043
0.360
44.0
28.4
150
43.6
60.2
33.2
33.6
NT
11.16
7.24
17.7
15.1
18.0
25.3
12.9
NT
899
431
320
131
338
237
210
ND
ND
NT
379
413
147
89
228
110
38
2
6
7
45
8
101
178
80
9
10
11
12
13
14
15
16
17
18
19
20
21
22
ND
ND
ND
44
NT
NT
NT
NT
178
117
48.7
33.2
NT
31
31.4
NT
111
ND
31
11.5
NT
ND
78
ND
ND
216
11.4
27.7
13.5
67.1
NT
12.5
3.31
NT
478
313
186
b
77
ND
a
The compounds were assayed for in vitro inhibition against three
purified NOS isoforms: rat nNOS, bovine eNOS, and murine iNOS,
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 7 to 9 data points; all
b
experimental standard error values are less than 12%, except for the
eNOS value for 21, which is 19%. All correlation coefficients are good
(R2 > 0.82). Selectivity values are ratios of respective Ki values. NT =
not tested; ND = not determined.
perpendicular orientation of the phenyl ring breaks the pi-
stacking interactions with Tyr706 (distance >6 Å), and the
phenyl ring and methylamine portion can be modeled in
alternate conformations where the amine can either face toward
or away from the H4B site. Similar upward phenyl ring
positions are also observed for 7 and 8 bound to eNOS (see
Figure S2), indicating that this position occurs regardless of tail
length or amine alkylation pattern.
While the perpendicular orientations found in these eNOS
structures would diminish many of the interactions that confer
binding to nNOS (and thus result in the observed n/e
selectivity), it was unclear what structural difference between
Figure 4. Active site structure of 6 (A) or 7 (B) bound to rat nNOS. The omit Fo − Fc density map for the inhibitor is shown at the 2.5 σ contour
level. The tail of 6 shows weaker density indicative of disordering. The alkylamine of 7 displaces a water molecule, thus making H-bonds with both
H4B and the heme. Major hydrogen bonds are shown as dashed lines. Figures prepared using PyMol (www.pymol.org).
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Figure 5. Active site structure of 8 (A) or 9 (B) bound to rat nNOS. The omit Fo − Fc density map for the inhibitor is shown at the 2.5 σ contour
level. The tail of 8 shows weaker density, indicative of disordering. The dimethylamine of 9 makes a H-bond with the water molecule bridging both
H4B and heme. Major hydrogen bonds are shown as dashed lines.
Figure 6. Active site structure of 9 bound to bovine eNOS (A) and rat nNOS D597N/M336V double mutant (B). The omit Fo − Fc density map for
the inhibitor is shown at the 2.5 σ contour level. An acetate ion (Act) is stabilized by Arg252 in eNOS, which in turn makes contacts with the phenyl
ring of 9. Major hydrogen bonds are shown as dashed lines.
Figure 7. Active site structure of 15 (A) or 17 (B) bound to rat nNOS. 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.
observed eNOS selectivity for 9 was assumed to result from
actual structural changes by comparing WT nNOS-9 and
nNOS-D597N/M336V-9 structures. Assaying 9 against nNOS-
D597N/M336V yielded a Ki of 585 nM. This value is 4-fold
higher than the WT nNOS Ki, indicating that the Met-to-Val
switch indeed disfavors binding to the double mutant. This
value is also considerably lower than the eNOS Ki value (25.3
μM), demonstrating that different amino acids are also not the
full determinant of selectivity; subtle differences in sterics,
electronics, or the local active site environment could also be
responsible.
chain by one methylene unit (to phenylpropyl, such as in 13
and 14) might either force the N-methyl groups toward the
nNOS-specific hydrophobic pocket or place the basic amine
near Asp597. Asn368 replaces Asp597 in eNOS (vide supra),
and electrostatic or water-mediated contact with the latter
residue has been previously implicated in high n/e
selectivity.26,40 Disappointingly, this modification was ineffec-
tive: 13 and 14 are less potent than 6. Loss of activity upon
extensive homologation was seen previously for 2-aminoquino-
line compounds,18 hypothesized to result from sterically
disfavored, suboptimal, or partial interactions, or internal
torsional strain.
Although 6 and 7 are less potent than the shorter
compounds (8−10), we reasoned that elongation of the alkyl
H
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Figure 8. Active site structure of 21 (A) or 20 (B) bound to rat nNOS, 20 bound to acetate-free eNOS (C), or 20 bound to rat nNOS D597N/
M336V double mutant (D). The omit Fo − Fc density map for the inhibitor is shown at the 2.5 σ contour level. The dimethylamine of 20 displaces
the water molecule bridging H4B and heme in the WT nNOS but not in the acetate-free eNOS or the nNOS double mutant structures. Major
hydrogen bonds are shown as dashed lines.
We also synthesized para-substituted analogues 15 and 16 to
investigate whether different ring substitution could improve
potency or selectivity. Compound 15 has lower potency than
meta-substituted analogue 9 (Ki 283 nM vs 142 nM), and
desmethyl compound 16 is slightly less potent than 15,
indicating that the binding mode of these compounds must be
different from the meta-substituted analogues. The nNOS-15
crystal structure (Figure 7A) indicates that the phenyl ring of
15 is oriented quite differently from that of 9. The alkylamine
tail of 15, as a para-substituent, extends out farther with high
flexibility. The methyl group makes possible contact with
Met336, but the amine loses its effective H-bond interaction
with the H4B site water. The flexible nature of the tail may
allow it to make both the hydrophobic pocket and H4B site
contacts transiently.
We also employed the secondary aniline (17 and 18) as a
bioisosteric replacement for the ether oxygen. Crystal structures
of the phenyl ether-linked compounds indicated a possible
placement for the NH group between the heme propionates,
where it could make another electrostatic or H-bonding
interaction. Surprisingly, the potency for 17 was lower than
that of 8, and the loss of additional potency upon
demethylation (to 18) indicated that 17 and 18 do not bind
like 8 and 9. The nNOS-17 structure (Figure 7B) reveals
instead that the aminoquinoline and central aryl rings sit ∼120°
relative to each other in a “butterfly” position, reflecting the
geometry required for the aniline nitrogen to interact with the
nearby heme propionate. As a result, the aryl ring moves
upward, and the dimethylamine H-bonds to Asn569. It is not
evident from this structure as to why 17 should be a worse
nNOS inhibitor than 8 (or why demethylation should further
attenuate the activity), but both steric crowding around the
Asn569-dimethylamine area (highly disordered in the crystal
structure) and the lack of the inhibitor-Tyr706 pi-stacking
interaction observed in 8 and 9 could play a role. Additionally,
the eNOS-17 structure (see Figure S3) reveals a nearly
identical binding mode to that assumed in the nNOS-17
structure, which could be consistent with the low selectivity of
17.
While the addition of a fluorine or α-methyl group to 9 (to
give 19 or 22, respectively) was neutral (19) or a deleterious
(22) modification with regard to both potency and selectivity
(relative to 9, and thus were not investigated further or
examined crystallographically), the replacement of the phenyl
ring of 9 with pyridyl (21) resulted in a substantial increase in
potency (Ki 142 nM to 43 nM; more potent than 1 and 2). The
nNOS-9 structure indicated the potential for the ring nitrogen
to make an additional contact with Asn569, a prediction proven
correct by the nNOS-21 structure (Figure 8A). Compound 21
assumes a well-defined, 9-like binding mode, where the external
amine binds the H4B-site water and the pyridine ring
simultaneously forms an edge-to-face pi-stacking interaction
with Tyr706 and a polar contact with Asn569 (although at 3.7
Å, it is likely too far for a true H-bond). The high potency of 21
also highlights the absolute necessity of the external polar
amine; compound 11, the unsubstituted 3-pyridine, binds
nearly 17-fold weaker to nNOS than 21. While potent, 21 does
not possess improved n/e selectivity relative to that of 9: this
asparagine residue is conserved across all NOS isoforms, and
interaction with the pyridine likely nonspecifically increases
binding affinity to other isoforms, as manifested by the
decreased Ki values for eNOS and iNOS as well.
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In contrast to para-fluoro compound 19, the para-chloro
analogue 20 is much more potent than compound 9, with high
isoform selectivity comparable to that of 1 and 2. The nNOS-
20 structure (Figure 8B) shows that the phenyl ring is flexible,
having two possible orientations in the two nNOS monomers,
where the amine can either displace the H4B-site water or point
toward the hydrophobic pocket by rotation of the phenyl ring,
while the chlorine can make van der Waals contacts with
Met336. The fluorine atom of 19 could be too small to make
the latter contact, whereas the α-methyl group of 22 may be
sterically disfavored in this region.
Table 2. Inhibition of Rat and Human nNOS by Select
Compounds
a
a
Ki (μM)
compd
rat nNOS
human nNOS
selectivity (rat/human)
1
0.049
0.066
0.468
0.179
0.142
0.283
0.375
0.058
0.043
0.318
0.440
1.86
6.5
6.7
4.0
4.8
6.4
3.8
1.8
5.1
11.8
2
6
8
0.855
0.911
1.08
9
15
17
20
0.657
0.295
0.507
The eNOS-20 crystal structure (see Figure S4) also displays
the “upward” binding mode and the stabilizing acetate ion.
However, if acetate is removed from the cryo-soaking buffer,
the phenyl ring of 20 no longer adopts the upward orientation
(Figure 8C) but points toward the pocket bound by Val106,
Leu107, and Tyr477, similar to the binding mode observed in
the rat nNOS double mutant D597N/M336V-20 structure
(Figure 8D). In addition, the positions of the phenyl ring and
its two substituents (chlorine and methylamine) are better
resolved than in the double mutant nNOS structure. The
methylamine tail contacts Val106 but does not H-bond with the
H4B or heme propionate A as observed in the wild type nNOS
structure (Figure 8B). The chlorine makes no contact with
Val106. As this binding mode is quite similar to that found in
the double mutant structure (Figure 8D), we can confirm that
acetate strongly influences the binding mode of inhibitors in
eNOS structures and that the nNOS double mutant is indeed a
good mimic of eNOS. In the nNOS double mutant-20
structure (Figure 8D), the Met-to-Val switch causes a
significant change in binding mode: the phenyl ring has a
single orientation rather than the two different ones observed in
WT nNOS. The amine-H4B site interaction is broken; instead,
the methylamine tail fits in the hydrophobic pocket, larger
because of the smaller Val336 residue. In addition, there is a
lack of van der Waals contact between 20’s chlorine atom and
Val336. Assaying 20 against nNOS D597N/M336V gave a Ki
value (863 nM) that is in between that of the WT nNOS (58
nM) and eNOS (12.5 μM) Ki values, indicating, like for 9, that
differences in amino acid sequences (such as Met to Val) do
not fully account for the observed selectivity. Nonetheless, the
double mutant nNOS/wild-type nNOS selectivity ratio (15) is
higher than 9’s (4) showing, as the crystal structure does, that
the inhibitor-Met336 contact plays a greater role in n/e
selectivity for 20 than it does for 9.
In addition to the good n/e selectivity, the majority of the
phenyl ether compounds are also poor iNOS inhibitors (20−
150 μM). We had previously reported this incompatibility
between iNOS and 2-aminoquinolines.17 The high n/i
selectivity was attributed in part to the mismatch between the
smaller and more rigid heme-binding pocket of iNOS41 and the
bulky aminoquinoline, and in part to hydrophobic pocket
contacts (as murine iNOS contains a polar residue, Asn115, in
place of Leu337 of rat nNOS). Indeed, a bulky and
hydrophobic group, such as chlorophenyl, may clash in the
region of Asn115 and possibly lead to the high n/i selectivity
observed for 20.
Several compounds of interest (6, 8, 9, 15, 17, 20, and 21)
were also assayed against purified human nNOS (Table 2). The
human nNOS active site differs from rat nNOS by one residue:
Leu337 is replaced by His342.42,43 The peripheral pocket of
human nNOS, therefore, prefers binding smaller and more
hydrophilic fragments, as evident from the high rat nNOS/
21
a
The compounds were assayed for in vitro inhibition against human
nNOS 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 7 to 9 data points;
all experimental standard error values are less than 12%, and all
correlation coefficients are good (R2 > 0.87). Selectivity values are
ratios of respective Ki values; rat nNOS Ki values are from Table 1.
human nNOS selectivity for the hydrophobic-tailed compounds
1 and 2. If the bound inhibitor cannot reach the Leu/His site,
an identical binding mode should be found in both rat and
human nNOS structures.42 Aniline 17 is a typical example. In
the human nNOS-17 structure (see Figure S5), the “butterfly”
binding mode is identical to that found in the rat nNOS-17
structure, and the bound inhibitor is far from the Leu/His site.
Therefore, there should be little selectivity (r/h = 1.8 only)
between these two nNOS enzymes. Most of the ether-linked
compounds are poor human nNOS inhibitors (Ki = 1−2 μM).
The most potent human nNOS inhibitor, 20, is still selective
for rat nNOS over human nNOS, even though it is
approximately as potent as 1. The human nNOS-20 structure
(Figure 9A) shows a consistent phenyl ring orientation where
the dimethylamine makes an H-bond with the H4B site water;
whereas in the rat nNOS-20 structure (Figure 8B), the phenyl
ring rotates and allows the dimethylamine to displace the H4B
site water molecule in one orientation but extends the amine to
the hydrophobic pocket in the other orientation (not shown).
Apparently, the bulkier His342 in human nNOS influences the
phenyl position of 20 differently than Leu337 in rat nNOS.
Pyridine analogue 21 strongly prefers to bind to rat nNOS
(r/h = 11.8), although the binding mode shows almost no
difference in the two nNOS structures (Figure 9B for human).
The only subtle difference observed in the structures is the side
chain orientation of Tyr706 in rat compared to that of Tyr711
in human nNOS (because of the closer contacts from His342 in
the human enzyme). A slight change in the edge-to-face pi-
stacking interaction between the tyrosine and the inhibitor’s
pyridyl ring, however, would likely not fully account for the
great difference in inhibitory potency.
Encouraged by the potency and selectivity of 20, this
compound was tested in a Caco-2 assay (Table 3), in which a
compound’s ability to cross a monolayer of cells (resembling
the intestinal lumen) is measured. This assay can be used to
approximate the permeability of the gut epithelium, and, to a
lesser extent, the BBB.44,45 Compound 1, an orally bioavailable,
brain-penetrant nNOS inhibitor, has good membrane perme-
ability in the apical to basolateral (A → B) direction (a mean
Papp of 16.9 × 10−6 cm s−1), high compound recovery values,
and a low efflux ratio [ratio of membrane permeability (A → B)
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Figure 9. Active site structure of 20 (A) or 21 (B) bound to human nNOS. 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.
Table 3. Caco-2 Permeability Summary for Select
Compounds
binding fraction has decreased to 6 targets, and the insignificant
binding fraction has increased considerably, from 8/45 to 22/
45, with the majority of the decrease coming from serotonin
receptors. These results indicate that truncation and structural
rearrangement, as observed for 4 and 5, are effective at reducing
off-target CNS binding and could potentially translate to
improved safety in vivo, making the phenyl ether-linked core an
exciting scaffold for further optimization.
a
apparent permeability (Papp
,
b
10−6 cm s−1
mean A → B mean B → A efflux ratio A → B B → A
)
recovery
compd
20
warfarin
2.3
12.6
8.2
5.5
37%
79%
c
21.1
0.084
0.061
0.39
22.6
125
d
ranitidine
1.9
e
talinolol
7.6
CONCLUSIONS
■
a
All assays were performed over 2 h at a concentration of 10 μM. See
To summarize, we have developed a novel series of 2-
aminoquinoline-based nNOS inhibitors, using the rationale that
this particular “rearranged” scaffold could maintain or enhance
the potency and isoform selectivity of our previous leads (1 and
2) while diminishing the off-target binding of these
compounds. Assaying this new generation of inhibitors
(containing an exterior polar amine and an interior hydro-
phobic portion linked to the aminoquinoline via a phenyl ether
or aniline) against purified NOS enzymes indicated that 9,
while having lower potency than 1 or 2, maintained the triple-
digit isoform selectivity of the leads. X-ray crystallography
(using rat nNOS and a rat nNOS double mutant) indicated
that the n/e selectivity might arise from enhanced hydrophobic
contacts between the tail of the inhibitor and the nNOS-specific
hydrophobic pocket. Elongating the chain between the aryl ring
and the amine, changing the ring substitution pattern, and
replacing the phenyl ether linkage with an aniline failed to
improve 9’s potency and selectivity, but one compound (20),
chlorinated on the central aryl ring, was found to be a potent
and selective inhibitor of both rat and human nNOS. Although
lower than compound 1, 20 still displayed some cellular
permeability in a Caco-2 assay despite the significant structural
rearrangement. Most promisingly, when assayed against a panel
of 45 relevant CNS targets and receptors in the PDSP, 20
showed diminished off-target interactions relative to those of 2
(a promiscuous binder) indicating that potent and selective
compounds based on the inverted phenyl ether core may have
the potential for improved safety in vivo.
b
c
Experimental Section for details. Apparent permeability value. High
permeability control. Low permeability control. High efflux control.
d
e
to efflux (B → A), < 3 is considered favorable]. Unfortunately,
compound 20 is less cell-penetrant, has a slightly higher efflux
ratio than 1, and has a lower A → B recovery value, potentially
indicative of poor solubility or metabolic lability in these cells.
Nonetheless, although suboptimal, some permeability is
retained despite the drastic structural rearrangement.
Finally, 20 was screened by the PDSP.19 In this assay,
compounds are assayed against a panel of 45 pharmacologically
relevant CNS targets via radioligand binding assays. We have
classified off-target binding (Table 4) into four categories:
a
Table 4. PDSP Binding Summary for Select Compounds
compd
concerning
moderate
weak
insignificant
total
2
8
3
7
3
22
17
8
45
45
20
22
a
Off-target binding is classified into four categories: concerning (Ki <
100 nM, or < ∼2× nNOS Ki value), moderate (100−300 nM, ∼ 2−5×
nNOS Ki value), weak (>300 nM, or > ∼5× nNOS Ki value, typically
∼1 μM), and insignificant (<50% bound at 10 μM), for a total of 45
receptors as assayed by the PDSP’s “comprehensive screen”. See ref
18. The three concerning targets for compound 20 are the alpha-2C
adrenergic receptor, histamine H2 receptor, and the serotonin
transporter (SERT).
concerning (Ki < 100 nM, or < ∼2× nNOS Ki value), moderate
(100−300 nM, ∼2−5× nNOS Ki value), weak (>300 nM, or >
∼5× nNOS Ki value, typically ∼1 μM), and insignificant (<50%
at 10 μM). For compound 2, a promiscuous scaffold with low
therapeutic index, binding was considered concerning or
moderate at 15/45 targets (mostly serotonin and histamine
receptors), with 22/45 targets classified as weak, and 8/45 as
insignificant. For compound 20, the concerning/moderate
EXPERIMENTAL SECTION
■
General Procedures. Anhydrous solvents (THF, CH2Cl2, MeOH,
and DMF) were distilled prior to use. The remaining solvents,
reactants, and reagents were purchased from commercial vendors and
were used without further purification. Methanolic HCl (3 M, for
ammonium salt formation and Boc-deprotection) was prepared fresh
by the reaction of acetyl chloride and anhydrous MeOH at 0 °C.
Melting points were determined in capillary tubes using a Buchi
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1
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 was performed using 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 (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). 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. The purity of all final target compounds was found to
be ≥95% by HPLC. Preparative HPLC was performed at the
Northwestern University Center for Molecular Innovation and Drug
Discovery ChemCore laboratory, using an Agilent 1200 Series HPLC
and Agilent 6120 Quadrupole Mass Spectrometer (API-MS mode)
and 150 × 21.2 mm preparative HPLC columns (described under
subheadings for individual compounds). Microwave chemistry was
performed using a Biotage Initiator Sixty research microwave in
Biotage vials. 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, and
KMnO4 stain, where relevant. Compounds 23,18 26,31,46 31,47 37,48
and 4232 were prepared by known literature procedures, and their
spectral data are consistent with those data reported for them. The
preparations of phenols, anilines, and precursors 28−59 are discussed
in the Supporting Information.
aq. NaCl/H2O mixture (∼15 mL) or a sat. aq. NaHCO3 solution. The
mixture was extracted with EtOAc (usually 3 × 20 mL was sufficient;
for tertiary amines, more exhaustive extraction was required), 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 (2−2.5 equiv)
was added. The mixture was heated at reflux for 2−2.5, 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 ether/MeOH) and
filtered to remove any particulate matter. To the filtered solution,
methanolic HCl (3 M, 1.5 mL) was added, and the mixture was stirred
either 5 min (when a Boc group was not present) or overnight (when
a Boc group was present). The hydrochloride salts were isolated by
filtration, and the final purification was performed as described below
for individual compounds.
7-[(3-(2-(Dimethylamino)ethyl)phenoxy)methyl]quinolin-2-
amine Dihydrochloride (6). This was prepared from 26 (0.150 g, 0.54
mmol) and phenol 29 (0.089 g, 0.54 mmol). Workup and purification
by flash column chromatography, eluting with a gradient of 2% MeOH
in EtOAc to 30% MeOH in EtOAc, afforded the intermediate
acetamide 60 as a white semisolid (0.081 g, 41%) that was immediately
deprotected using K2CO3 (0.062 g, 0.446 mmol). After workup, the
resulting gum was dissolved in minimal EtOAc, and hexanes were
added to precipitate a white solid. This procedure was repeated twice,
and the solid was diluted with 10% MeOH in ether (10 mL), filtered,
and concentrated. The resulting residue was diluted in ether (∼10
mL) and treated with methanolic HCl for 5 min. Filtration afforded 6
(0.050 g, 57% from 64) as a cream-colored powder after washing with
ether (3 × 2 mL): mp 115−120 °C (softens), 240 °C (dec). 1H NMR
(500 MHz) DMSO-d6: δ 14.37 (s, 1 H), 10.46 (s, 1 H), 9.21 (br s, 1
H), 8.38 (d, J = 9.3 Hz, 1 H), 8.22 (br s, 1 H), 7.95 (d, J = 8.2 Hz, 1
H), 7.78 (s, 1 H), 7.54 (dd, J = 8.2, 1.4 Hz, 1 H), 7.28 (t, J = 7.9 Hz, 1
H), 7.11 (d, J = 9.3 Hz, 1 H), 7.02 (d, J = 1.7 Hz, 1 H), 6.95 (dd, J =
8.0, 2.3 Hz, 1 H), 6.89 (d, J = 7.6 Hz, 1 H), 5.32 (s, 2 H), 3.29 (dt, J =
12.1, 4.6 Hz, 2 H), 3.01−2.98 (m, 2 H), 2.80 (s, 3 H), 2.79 (s, 3 H).
13C- NMR (126 MHz; DMSO-d6): δ 158.2, 154.4, 142.8, 142.3, 138.6,
2-Amino-7-methylquinoline (24). Compound 23 (1.00 g, 5.63
mmol), DavePhos (0.051 g, 2 mol %), and Pd2(dba)3 (0.051 g, ∼1
mol %) in a sealed tube or Biotage 20 mL microwave vial were diluted
with anhydrous dioxane (6 mL), and LHMDS (1 M in THF, 12 mL,
12 mmol) was added slowly. The reaction was purged with argon for 5
min, sealed, and heated to 100 °C for 20 h. The mixture was cooled
and poured into aqueous HCl (1 M, 20 mL) and stirred for 10 min
before EtOAc (20 mL) was added. The layers were separated, and the
aqueous layer was extracted with 1 M HCl (3 × 50 mL); the aqueous
phase was washed repeatedly with EtOAc (3 × 30 mL), and the
combined organic phase was discarded. The aqueous phase was
basified to pH 9 with NaOH and extracted with EtOAc (2 × 150 mL).
The organic phase was washed with sat. aq. NaCl (100 mL), dried, and
concentrated to yield the crude aminoquinoline as an off-white
iridescent solid (0.900 g, 100% with minor impurities); analytical data
for this compound are as previously described.29
2-(Acetamido)-7-methylquinoline (25). Compound 24 (3.02 g,
19.1 mmol) was diluted with anhydrous THF (100 mL), N-
acetylimidazole (2.52 g, 23.0 mmol) was added, and the mixture was
heated at reflux for 17 h. The mixture was cooled, concentrated, and
the residue was diluted with EtOAc (200 mL) and washed with H2O
(∼500 mL) and sat. aq. NaCl (100 mL). The organic layer was dried
over anhydrous sodium sulfate and concentrated. The residue was
dissolved in a minimal amount of hot EtOAc and precipitated with
hexanes (∼200 mL) to yield 25 as a pale-tan iridescent solid (3.23 g,
85%) after washing with hexanes. Analytical data for this compound
are consistent with those previously reported.18,29
135.8, 129.8, 129.0, 123.8, 121.5, 120.3, 115.5, 115.1, 113.7, 113.0,
68.4, 57.1, 42.1, 29.8; ESIMS m/z (rel. intensity) 322 (MH+, 100).
HRMS calcd for C20H24N3O: 322.1919; found, 322.1912.
7-[(3-(2-(Methylamino)ethyl)phenoxy)methyl]quinolin-2-amine
Dihydrochloride (7). This was prepared from 26 (0.150 g, 0.54 mmol)
and phenol 32 (0.138 g, 0.54 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 61
as a white semisolid (0.181 g, 74%), which was immediately
deprotected using K2CO3 (0.111g, 0.806 mmol). After workup, the
resulting gum was triturated with hexanes and 1:1 ether/hexanes (5
mL each) and filtered to yield a white solid that was suspended in
ether (8 mL) and treated with methanolic HCl (1.5 mL) for 20 h.
Filtration afforded 7 (0.105 g, 69% from 61) as a chalky white solid
after trituration with 10% MeOH in ether (3 × 1 mL): mp 211.5−213
1
°C. H NMR (500 MHz; DMSO-d6): δ 14.48 (s, 1 H), 9.26 (br s, 1
H), 9.01 (s, 2 H), 8.37 (d, J = 9.3 Hz, 1 H), 8.27 (br s, 1 H), 7.95 (d, J
= 8.2 Hz, 1 H), 7.78 (s, 1 H), 7.54 (dd, J = 8.2, 1.0 Hz, 1 H), 7.27 (t, J
= 7.9 Hz, 1 H), 7.12 (d, J = 9.3 Hz, 1 H), 6.99 (s, 1 H), 6.94 (dd, J =
8.2, 2.4 Hz, 1 H), 6.87 (d, J = 7.6 Hz, 1 H), 5.32 (s, 2 H), 3.16−3.11
(m, 2 H), 2.93 (t, J = 8.0 Hz, 2 H), 2.55 (t, J = 5.4 Hz, 3 H). 13C NMR
(126 MHz; DMSO-d6): δ 158.2, 154.4, 142.8, 142.3, 138.9, 135.8,
129.8, 129.0, 123.8, 121.4, 120.3, 115.4, 115.1, 113.7, 113.0, 68.4, 48.9,
32.3, 31.4; ESIMS m/z (rel. intensity) 308 (MH+, 100). HRMS calcd
for C19H22N3O: 308.1763; found, 308.1760.
General Procedure for the Synthesis and Deprotection of
Phenyl Ether-Linked Aminoquinolines. 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 bromide 26 (1 equiv) was added as a solution in
anhydrous DMF. The reaction mixture was stirred at 0 °C for 40 min−
1 h (typically ∼50 min) and quenched at 0 °C by addition of a 1:1 sat.
L
DOI: 10.1021/acs.jmedchem.5b01330
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
7-[(3-(Dimethylamino)methyl)phenoxy)methyl]quinolin-2-amine
Dihydrochloride (8). This was prepared from 26 (0.120 g, 0.42 mmol)
and phenol 34 (0.065 g, 0.43 mmol). Workup and purification by flash
column chromatography, eluting with a gradient of 2% MeOH in
EtOAc to 30% MeOH in EtOAc, afforded intermediate acetamide 62
as a white gum (0.091 g, 61%), which was deprotected using K2CO3
(0.089g, 0.65 mmol). After workup, the resulting syrup was triturated
with 5:1 DCM/hexanes (5 mL) to yield a white solid. This was diluted
in EtOAc (3 mL), filtered to remove particulates, and concentrated.
The residue was diluted in MeOH (1 mL) and treated with
methanolic HCl (1.5 mL). Ether was added to precipitate a gum
that was washed with 5% MeOH in ether and ether (1 mL each) to
yield 8 (0.063 g, 64% from 62) as a pale-yellow foam: mp 76 °C
which was deprotected using K2CO3 (0.064 g, 0.466 mmol). After
workup, the resulting residue was triturated with DCM/hexanes (∼4
mL), and the pale-yellow iridescent solid was collected, dissolved in
MeOH, filtered to remove particulates, and concentrated. The residue
was resuspended in MeOH (1 mL), and methanolic HCl (1.5 mL)
was added. After 5 min, the addition of ether (5 mL) precipitated 11 as
an off-white solid (0.067 g, 88% from 74) after washing with ether (2
mL): mp 188 °C (softens), 211−214 °C (melts). 1H NMR (500
MHz; DMSO-d6): δ14.35 (s, 1 H), 9.23 (br s, 1 H), 8.65 (s, 1 H), 8.43
(s, 1 H), 8.38 (d, J = 9.3 Hz, 1 H), 8.27 (br s, 1 H), 7.98−7.97 (m, 2
H), 7.79 (s, 1 H), 7.76−7.74 (m, 1 H), 7.57 (d, J = 8.1 Hz, 1 H),
7.13−7.11 (m, 1 H), 5.51 (s, 2 H); the pyridinium N−H proton is
broadened into the baseline and is not visible; 13C NMR (126 MHz;
DMSO-d6): δ 155.7, 154.6, 143.0, 140.9, 138.2, 135.9, 133.6, 129.3,
127.7, 126.5, 124.2, 120.7, 115.7, 114.2, 69.7; ESIMS m/z (rel.
intensity) 252 (MH+, 100). HRMS calcd for C15H14N3O: 252.1137;
found, 252.1130.
1
(softens), 91−94 °C (melts), 240 °C (dec). H NMR (500 MHz;
DMSO-d6): δ 14.37 (s, 1 H), 10.77 (s, 1 H), 9.21 (br s, 1 H), 8.38 (d, J
= 9.3 Hz, 1 H), 8.24 (s, 1 H), 7.95 (d, J = 8.2 Hz, 1 H), 7.78 (s, 1 H),
7.55 (dd, J = 8.2, 1.4 Hz, 1 H), 7.41−7.38 (m, 2 H), 7.15−7.10 (m, 3
H), 5.36 (s, 2 H), 4.24 (d, J = 5.3 Hz, 2 H), 2.68 (s, 3 H), 2.67 (s, 3
H). 13C NMR (126 MHz; DMSO-d6): δ 158.2, 154.4, 142.9, 142.1,
132.1, 130.1, 129.1, 124.0, 123.6, 120.4, 117.5, 115.9, 115.2, 113.9,
68.6, 59.4, 48.7, 41.6; ESIMS m/z (rel. intensity) 308 (MH+, 100).
HRMS calcd for C19H22N3O: 308.1763; found, 308.1757.
7-((3-(Dimethylamino)phenoxy)methyl)quinolin-2-amine Dihy-
drochloride (12). This was prepared from 26 (0.120 g, 0.43 mmol)
and 3-(dimethylamino)phenol (0.041 g, 0.43 mmol). After workup,
the resulting residue was purified by flash column chromatography,
eluting with a gradient of 5% EtOAc in CH2Cl2 to 25% EtOAc in
CH2Cl2, to yield intermediate acetamide 65 (0.070 g, 49%) as a clear
syrup, which was immediately deprotected using K2CO3 (0.058 g, 0.42
mmol). After workup, the resulting oil was treated with methanolic
HCl (4 mL), and ether (12 mL) was added to afford an off-white
solid; an analytically pure sample for assay was prepared by preparative
LC-MS, using the instrument described in the General Procedures
section and a Phenomenex Luna 5 μm C8(2) 100 Å, 150 × 21.2 mm
preparative HPLC column, eluting with a gradient of 95% H2O + 0.1%
formic acid/5% MeCN + 0.1% formic acid to 81% H2O + 0.1% formic
acid/19% MeCN + 0.1% formic acid over 20 min. Evaporation of the
desired mass fraction and retreatment with methanolic HCl (1 mL)
and ether (1 mL) afforded 12 as a white solid (0.023 g, 30% from 65):
7-[(3-(Methylamino)methyl)phenoxy)methyl]quinolin-2-amine
Dihydrochloride (9). This was prepared from 26 (0.150 g, 0.54 mmol)
and phenol 35 (0.128 g, 0.54 mmol). Workup and purification by flash
column chromatography, eluting with 5% EtOAc in CH2Cl2 to 33%
EtOAc in CH2Cl2, afforded intermediate acetamide 63 as a colorless
foam (0.184 g, 78%), which was deprotected using K2CO3 (0.117 g,
0.844 mmol). After workup, the resulting residue was diluted in ether
(5 mL), filtered to remove particulates, and treated with methanolic
HCl (1.5 mL) for 20 h. Ether (3 mL) was added, and the precipitate
was filtered to yield 9 as a white amorphous solid (0.102 g, 66% from
63) after trituration with 2:1 ether/MeOH (2 mL) and washing with
1
ether (2 mL): mp 257−259 °C. H NMR (500 MHz; DMSO-d6): δ
14.41 (s, 1 H), 9.27 (s, 3 H), 8.37 (d, J = 9.3 Hz, 1 H), 8.30 (br s, 1
H), 7.95 (d, J = 8.2 Hz, 1 H), 7.78 (s, 1 H), 7.55−7.53 (m, 1 H), 7.37
(t, J = 7.9 Hz, 1 H), 7.33 (s, 1 H), 7.12−7.08 (m, 3 H), 5.35 (s, 2 H),
4.08 (t, J = 5.5 Hz, 2 H), 2.52 (d, J = 5.2 Hz, 3 H, partially obscured by
solvent signal). 13C NMR (126 MHz; DMSO-d6): δ 158.1, 154.4,
142.8, 142.1, 135.8, 133.6, 129.9, 129.0, 123.8, 122.5, 120.3, 116.6,
115.14, 115.10, 113.8, 68.5, 51.0, 31.9; ESIMS m/z (rel. intensity) 294
(MH+, 100). HRMS calcd for C18H20N3O: 294.1606; found, 294.1603.
7-[(3-Aminomethyl)phenoxy)methyl]quinolin-2-amine Dihydro-
chloride (10). This was prepared from 26 (0.100 g, 0.358 mmol)
and phenol 37 (0.080 g, 0.358 mmol). Workup and purification by
flash column chromatography, eluting with 5% EtOAc in CH2Cl2 to
50% EtOAc in CH2Cl2, afforded intermediate acetamide 64 as a
colorless foam (0.121 g, 80%), which was deprotected using K2CO3
(0.079 g, 0.569 mmol). After workup, the resulting gum was triturated
with 5:1 ether/hexanes and 2:1 hexanes/EtOAc (5 mL each) to yield
the product as a white solid, which was diluted in MeOH, filtered to
remove particulates, and concentrated. The residue was diluted with
ether (8 mL) and treated with methanolic HCl (1.5 mL) for 20 h. The
precipitate was filtered to yield 10 as a white powder (0.050 g, 50%
from 64) after trituration with 15% MeOH in ether (2 × 2 mL) and
2:1 ether/MeOH (2 mL) and washing with ether (2 mL): mp 254−
255 °C. 1H NMR (500 MHz; DMSO-d6): δ 14.49 (s, 1 H), 9.24 (br s,
1 H), 8.45 (s, 3 H), 8.37 (d, J = 9.3 Hz, 1 H), 8.28 (br s, 1 H), 7.95 (d,
J = 8.2 Hz, 1 H), 7.78 (s, 1 H), 7.54 (dd, J = 8.2, 1.4 Hz, 1 H), 7.35 (t,
J = 7.9 Hz, 1 H), 7.28 (d, J = 1.8 Hz, 1 H), 7.13−7.05 (m, 3 H), 5.34
(s, 2 H), 3.99 (q, J = 5.7 Hz, 2 H). 13C NMR (126 MHz; DMSO-d6):
δ 158.1, 154.4, 142.8, 142.1, 135.82, 135.69, 129.8, 129.0, 123.8, 121.5,
120.3, 115.7, 115.1, 114.6, 113.8, 68.5, 42.1; ESIMS m/z (rel.
intensity) 280 (MH+, 100). HRMS calcd for C17H18N3O: 280.1450;
found, 280.1442.
1
mp 195−197 °C. H NMR (500 MHz; DMSO-d6): δ 14.30 (s, 1 H),
9.22 (br s, 1 H), 8.38 (d, J = 9.5 Hz, 1 H), 8.22 (br s, 1 H), 7.95 (d, J =
9.0 Hz, 1 H), 7.77 (s, 1 H), 7.55 (d, J = 9.0 Hz, 1 H), 7.27 (s, 1 H),
7.11 (d, J = 9.0 Hz, 1 H), 6.68 (br s, 3 H), 5.32 (s, 2 H), 2.99 (s, 6 H);
one proton is not visible due to baseline broadening; 13C NMR (126
MHz; DMSO-d6): δ 159.4, 154.8, 143.4, 142.9, 136.3, 130.6, 129.4,
124.4, 120.8, 115.6, 114.2, 66.9; four of the aryl carbons are not visible
due to baseline broadening, and the methyl carbon signals are
obscured by the solvent peak; ESIMS m/z (rel. intensity) 294 (MH+,
100). HRMS calcd for C18H20N3O, 294.1606; found, 294.1599.
7-[(3-(3-(Dimethylamino)propyl)phenoxy)methyl]quinolin-2-
amine Dihydrochloride (13). This was prepared from 26 (0.150 g,
0.54 mmol) and phenol 49 (0.097 g, 0.54 mmol). Workup and
purification by flash column chromatography, eluting with a gradient
of 2% MeOH in EtOAc to 40% MeOH in EtOAc, afforded
intermediate acetamide 66 as a pale yellow gum (0.105 g, 51%)
after filtering an EtOAc solution to remove particulates and
reconcentrating. The intermediate acetamide was deprotected using
K2CO3 (0.077 g, 0.556 mmol). After workup, the resulting residue was
diluted in CH2Cl2 (1 mL) and precipitated with hexanes (5 mL) to
yield the free aminoquinoline as a white solid, which was diluted with
CH2Cl2, filtered again, and concentrated. The residue was diluted in
ether (5 mL) and treated with methanolic HCl (1 mL) for 5 min. The
mixture was then concentrated, and the residue was triturated with
ether (2 × 5 mL), 10% MeOH in ether (4 × 5 mL), and EtOAc (5
mL), to yield 13 as an off-white foam (0.045 g, 40% from 66): mp 72−
1
75 °C (softens), 260 °C (dec). H NMR (500 MHz; DMSO-d6): δ
14.34 (s, 1 H), 10.41 (s, 1 H), 9.17 (br s, 1 H), 8.37 (d, J = 9.3 Hz, 1
H), 8.21 (br s, 1 H), 7.95 (d, J = 8.2 Hz, 1 H), 7.77 (s, 1 H), 7.54 (dd,
J = 8.2, 1.3 Hz, 1 H), 7.24 (t, J = 7.9 Hz, 1 H), 7.10 (d, J = 9.3 Hz,
1H), 6.96 (t, J = 1.8 Hz, 1 H), 6.90−6.88 (m, 1 H), 6.85 (d, J = 7.7 Hz,
1 H), 5.31 (s, 2 H), 3.02−2.99 (m, 2 H), 2.72 (s, 3 H), 2.72 (s, 3 H),
2.61 (t, J = 7.7 Hz, 2 H), 2.00−1.94 (m, 2 H). 13C NMR (126 MHz;
DMSO-d6): δ 158.1, 154.4, 142.8, 142.36, 142.27, 135.9, 129.6, 129.0,
123.8, 121.1, 120.3, 115.12, 115.01, 113.7, 112.3, 68.3, 56.1, 42.0, 31.9,
7-[(Pyridin-3-yloxy)methyl]quinolin-2-amine Dihydrochloride
(11). This was prepared from 26 (0.120 g, 0.43 mmol) and 3-
hydroxypyridine (0.041 g, 0.43 mmol). Workup and purification by
trituration with 50% CH2Cl2 in hexanes (4 mL) afforded intermediate
acetamide 74 as a flocculent, peach-colored solid (0.068 g, 54%),
M
DOI: 10.1021/acs.jmedchem.5b01330
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
25.1; ESIMS m/z (rel. intensity) 336 (MH+, 100). HRMS calcd for
C21H26N3O, 336.2076; found, 336.2073.
16 as a white chalky solid (0.131 g, 85% from 69) after washing with
20% MeOH in ether (5 mL) and drying: mp 283−285 °C. H NMR
1
7-((3-(2-(Dimethylamino)ethoxy)phenoxy)methyl)quinolin-2-
amine Dihydrochloride (14). This was prepared from 26 (0.100 g,
0.36 mmol) and phenol 42 (0.065 g, 0.36 mmol). After workup, the
resulting residue was purified by flash column chromatography, eluting
with 50% EtOAc in hexanes + 5% Et3N, to yield intermediate
acetamide 67 (0.050 g, 37%) as an opaque syrup, which was
immediately deprotected using K2CO3 (0.036 g, 0.26 mmol). After
workup, the resulting oil was treated with methanolic HCl (3 M, 3
mL), and ether (9 mL) was added, affording an off-white solid; an
analytically pure sample for assay was prepared by preparative LC-MS,
using the instrument described in the General Procedures section and
a Phenomenex Luna 5 μm C8(2) 100 Å, 150 × 21.2 mm preparative
HPLC column, eluting with a gradient of 99% H2O + 0.1% formic
acid/1% MeCN + 0.1% formic acid to 98% H2O + 0.1% formic acid
2% MeCN + 0.1% formic acid over 20 min. Evaporation of the desired
mass fraction and retreatment with methanolic HCl (1 mL) and ether
(1 mL) afforded 14 as a white solid (0.0065 g, 12%, from 67): mp
(500 MHz; DMSO-d6): δ 14.31 (s, 1 H), 9.07 (br s, 3 H), 8.36 (d, J =
9.3 Hz, 1 H), 8.29 (br s, 1 H), 7.94 (d, J = 8.2 Hz, 1 H), 7.75 (s, 1 H),
7.53 (dd, J = 8.2, 1.3 Hz, 1 H), 7.47−7.45 (m, 2 H), 7.10−7.09 (m, 3
H), 5.35 (s, 2 H), 4.03 (t, J = 5.6 Hz, 2 H), 2.48 (m, 3 H, partially
obscured by solvent peak). 13C NMR (126 MHz; DMSO-d6): δ 158.4,
154.4, 142.7, 142.0, 136.0, 131.6, 129.0, 124.5, 123.7, 120.4, 115.2,
114.9, 113.7, 68.5, 50.6, 31.7; ESIMS m/z (rel. intensity) 294 (MH+,
100). HRMS calcd for C18H20N3O, 294.1606; found, 294.1597.
7-[((3-((Dimethylamino)methyl)phenyl)amino)methyl]quinolin-
2-amine Trihydrochloride (17). Aldehyde 81 (0.080 g, 0.374 mmol)
and aniline 48 (0.067 g, 0.449 mmol) were diluted in abs. EtOH (12
mL), and anhydrous Na2SO4 (∼0.5 g) was added. The mixture was
heated to 60 °C for 1 h, glacial AcOH (8 μL) was added, and heating
was continued for 22 h. The mixture was cooled to room temperature,
and NaBH4 (0.018 g, 0.486 mmol) was added, and after 20 min, the
mixture was filtered. The filtrate was evaporated and partitioned
between EtOAc and sat. aq. NaHCO3 (20 mL each). The aqueous
layer was extracted with EtOAc (2 × 30 mL), and the organic layer
was washed with H2O and sat. aq. NaCl (20 mL each), dried over
anhydrous sodium sulfate, and concentrated. The resulting residue was
purified by flash column chromatography, eluting with a gradient of
CH2Cl2 to 25% MeOH in CH2Cl2, to yield the crude acetamide as a
white solid (0.032 g, 25%) after trituration with 10% CH2Cl2 in
hexanes. The intermediate acetamide was deprotected using K2CO3
(0.026 g, 0.188 mmol) and worked up using the general procedure;
the obtained yellow gum was triturated with 10% EtOAc in hexanes
and hexanes (3 mL each), diluted with 20% MeOH in ether (∼5 mL),
and treated with methanolic HCl (0.5 mL) for 5 min. The precipitate
was collected, diluted in MeOH (0.5 mL), and precipitated with ether
(3 mL) to afford 17 (0.019 g, 12% from the acetamide) as a pale-
yellow powder: mp 219−221 °C. 1H NMR (500 MHz; DMSO-d6): δ
14.18 (s, 1 H), 10.35 (s, 1 H), 9.11 (br s, 1 H), 8.33 (d, J = 9.3 Hz, 1
H), 8.12 (br s, 1 H), 7.87 (d, J = 8.2 Hz, 1 H), 7.64 (s, 1 H), 7.48 (dd,
J = 8.2, 1.3 Hz, 1 H), 7.11 (t, J = 7.8 Hz, 1 H), 7.04 (d, J = 9.3 Hz, 1
H), 6.75 (s, 1 H), 6.68 (d, J = 7.5 Hz, 1 H), 6.63 (dd, J = 8.2, 1.6 Hz, 1
H), 4.49 (s, 2 H), 4.07 (d, J = 5.5 Hz, 2 H), 2.63 (s, 3 H), 2.62 (s, 3
H); the anilinium N−H protons appear as a broad singlet, exchanging
with residual water at 4.2 ppm. 13C NMR (126 MHz; DMSO-d6): δ
154.3, 148.5, 145.9, 142.9, 135.8, 131.1, 129.4, 128.8, 124.3, 119.8,
118.3, 114.7, 114.1, 113.5, 113.1, 60.0, 46.1, 41.5; ESIMS m/z (rel.
intensity) 307 (MH+, 100). HRMS calcd for C19H23N4, 307.1923;
found, 307.1916.
1
181−183 °C. H NMR (500 MHz; DMSO-d6): δ 14.43 (s, 1 H),
10.57 (s, 1 H), 9.24 (br s, 1 H), 8.37 (d, J = 9.0 Hz, 1 H), 8.23 (br s, 1
H), 7.95 (d, J = 8.0 Hz, 1 H), 7.77 (s, 1 H), 7.53 (d, J = 8.0 Hz, 1 H),
7.24 (dd, J = 8.0, 8.0 Hz, 1 H), 7.11 (d, J = 9.0 Hz, 1 H), 6.72−6.67
(m, 2 H), 6.62 (dd, J = 8.0, 2.5 Hz, 1 H), 5.32 (s, 2 H), 4.36 (t, J = 5.0
Hz, 2 H), 3.48 (dt, J = 5.0, 5.0 Hz, 2 H), 2.83 (s, 6 H). 13C NMR (126
MHz; DMSO-d6): δ 159.7, 159.2, 154.9. 143.2, 142.6, 136.3, 130.7,
129.5, 124.2, 120.8, 115.8, 114.2, 108.2, 108.0, 102.5, 69.0, 62.8, 55.7,
48.2. ESIMS m/z (rel. intensity) 338 (MH+, 100). HRMS calcd for
C20H24N3O2, 338.1868; found, 338.1864.
7-[(4-(Dimethylamino)methyl)phenoxy)methyl]quinolin-2-amine
Dihydrochloride (15). This was prepared from 26 (0.120 g, 0.43
mmol) and phenol 44 (0.065 g, 0.43 mmol). Workup and purification
by flash column chromatography, eluting with a gradient of 3% MeOH
in EtOAc to 20% MeOH in EtOAc, afforded crude acetamide 68 as a
foamy pale-yellow semisolid (0.099 g, 66%), which was deprotected
using K2CO3 (0.078 g, 0.57 mmol). After workup, the resulting
yellowish gum was triturated with 10% EtOAc in hexanes (10 mL),
diluted with MeOH, filtered, and reconcentrated. The resulting residue
was diluted with ether (5 mL) and treated with methanolic HCl (1
mL) for 5 min. Concentration afforded an off-white foamy solid; an
analytically pure sample for assay was prepared by preparative LC-MS,
using the instrument described in the General Procedures section and
a Phenomenex Luna 5 μm C8(2) 100 Å, 150 × 21.2 mm preparative
HPLC column, eluting with a gradient of 95% H2O + 0.1% formic
acid/5% MeCN + 0.1% formic acid to 80% H2O + 0.1% formic acid
20% MeCN + 0.1% formic acid over 20 min. Evaporation of the
desired mass fraction yielded a syrup that was suspended in MeOH (2
mL), filtered, concentrated, and treated with methanolic HCl (0.7
mL). Ether (5 mL) was added to precipitate 15, which was obtained as
a white hygroscopic solid (0.022 g, 21% from 68) after washing with
7-[((3-((Methylamino)methyl)phenyl)amino)methyl]quinolin-2-
amine Trihydrochloride (18). Bromide 26 (0.092 g, 0.330 mmol),
aniline 50 (0.195 g, 0.826 mmol), and KI (0.006 g, 15 mol %) were
combined in anhydrous MeCN (2.5 mL), sealed, and heated under
microwave irradiation at 110 °C for 20 min. The mixture was diluted
with CH2Cl2 (30 mL) and washed with sat. aq. NaHCO3 (20 mL).
The aqueous layer was extracted with CH2Cl2 (20 mL), and the
organic layers were washed with H2O and sat. aq. NaCl (30 mL), dried
over anhydrous sodium sulfate, and 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 intermediate
acetamide 75 as a yellow foam (0.103 g, 72%), which was deprotected
using K2CO3 (0.065 g, 0.474 mmol) and worked up according to the
general procedure. The resulting residue was purified by flash column
chromatography, eluting with a gradient of EtOAc to 2% MeOH in
EtOAc, to yield a pale-yellow foam. The free aminoquinoline was
diluted with 30% MeOH in ether (11 mL) and was treated with
methanolic HCl (1.5 mL) for 20 h. The precipitate was collected and
precipitated from hot MeOH (0.5 mL) with ether (5 mL) to yield 18
as a cream-colored solid (0.050 g, 53% from 75): mp 230−232 °C. 1H
NMR (500 MHz; DMSO-d6): δ14.30 (s, 1 H), 9.10 (br s, 3 H), 8.34
(d, J = 9.3 Hz, 1 H), 8.16 (br s, 1 H), 7.88 (d, J = 8.2 Hz, 1 H), 7.65 (s,
1 H), 7.49 (dd, J = 8.2, 1.3 Hz, 1 H), 7.09 (t, J = 7.8 Hz, 1 H), 7.06 (d,
J = 9.3 Hz, 1 H), 6.76 (s, 1 H), 6.68 (d, J = 7.5 Hz, 1 H), 6.60 (dd, J =
8.1, 1.7 Hz, 1 H), 4.49 (s, 2 H), 3.92 (t, J = 5.9 Hz, 2 H), 2.47 (t, J =
5.4 Hz, 3 H, partially obscured by solvent peak); the anilinium N−H
1
ether and drying: mp 251−253.5 °C. H NMR (500 MHz; DMSO-
d6): δ 14.27 (s, 1 H), 10.50 (s, 1 H), 9.15 (br s, 1 H), 8.37 (d, J = 9.3
Hz, 1 H), 8.21 (br s, 1 H), 7.95 (d, J = 8.2 Hz, 1 H), 7.76 (s, 1 H),
7.55−7.54 (m, 1 H), 7.50 (d, J = 8.7 Hz, 2 H), 7.12 (d, J = 8.7 Hz, 2
H), 7.10 (d, J = 9.4 Hz, 1 H), 5.35 (s, 2 H), 4.19 (d, J = 4.7 Hz, 2 H),
2.66 (s, 3 H), 2.65 (s, 3 H). 13C NMR (126 MHz; DMSO-d6): δ
158.8, 154.4, 142.8, 142.0, 135.9, 132.7, 129.0, 123.8, 122.8, 120.4,
115.22, 115.02, 113.8, 68.5, 58.9, 41.3; ESIMS m/z (rel. intensity) 308
(MH+, 100). HRMS calcd for C19H22N3O, 308.1763; found, 308.1756.
7-[(4-(Methylamino)methyl)phenoxy)methyl]quinolin-2-amine
Dihydrochloride (16). This was prepared from 26 (0.150 g, 0.54
mmol) and phenol 45 (0.128 g, 0.54 mmol). Workup and purification
by flash column chromatography, eluting with a gradient of 5% EtOAc
in CH2Cl2 to 33% EtOAc in CH2Cl2, afforded intermediate acetamide
69 as a pale yellow foam (0.184 g, 78%), which was deprotected using
K2CO3 (0.117 g, 0.844 mmol). After workup, the resulting residue was
purified by flash column chromatography, eluting with a gradient of
EtOAc to 2% MeOH in EtOAc, to yield a colorless foam. The free
aminoquinoline was diluted with ether (7 mL) and treated with
methanolic HCl (1.5 mL) for 18 h. The precipitate was filtered to yield
N
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protons appear as a broad singlet, exchanging with residual water at 4.4
ppm. 13C NMR (126 MHz; DMSO-d6): δ 154.5, 148.5, 146.1, 143.0,
136.0, 132.9, 129.5, 129.0, 124.4, 120.0, 117.6, 114.9, 113.6, 113.24,
113.19, 51.7, 46.4, 32.1; ESIMS m/z (rel. intensity) 293 (MH+, 100).
HRMS calcd for C18H21N4, 293.1766; found, 293.1760.
0.36 mmol) and phenol 56 (0.090 g, 0.36 mmol). After workup, the
resulting residue was purified by flash column chromatography, eluting
with a gradient of 7% EtOAc in CH2Cl2, to 30% EtOAc in CH2Cl2 to
yield intermediate acetamide 72 as a white solid (0.119 g, 74%), which
was immediately deprotected using K2CO3 (0.073 g, 0.53 mmol).
After workup, the resulting oil was treated with trifluoroacetic acid
(∼0.3 mL) in ether (5 mL) and stirred at room temperature 18 h to
afford 22 as a white solid (0.077 g, 50% from 72): mp 120−123 °C.
1H NMR (500 MHz; DMSO-d6): δ 14.37 (s, 1 H), 9.15 (s, 1 H), 8.92
(br s, 2 H), 8.39 (d, J = 9.5 Hz, 1 H), 7.97 (d, J = 8.5 Hz, 1 H), 7.72 (s,
1 H), 7.56 (d, J = 9.5 Hz, 1 H), 7.42 (dd, J = 8.0, 8.0 Hz, 1 H), 7.23
(dd, J = 2.0, 1.5 Hz, 1 H), 7.14−7.05 (m, 3 H), 5.34 (s, 2 H), 4.30 (q, J
= 6.0 Hz, 1 H), 2.44 (t, J = 5.0 Hz, 3 H), 1.54 (d, J = 6.0 Hz, 3 H); 13C
NMR (126 MHz, DMSO-d6) δ (159.5 + 159.2 + 159.0 + 158.7, 1 C),
158.8, 155.1, 143.3, 142.4, 139.1, 136.7, 130.8, 129.5, 124.3, 120.9,
120.7, 115.7, 115.6, 114.8, 114.4, 103.5, 69.1, 58.3, 30.9, 19.3; ESIMS
m/z (rel. intensity) 308 (MH+, 100). HRMS calcd for C19H22N3O,
308.1763; found, 308.1755.
7-[(4-Fluoro-3-((methylamino)methyl)phenoxy)methyl]quinolin-
2-amine Dihydrochloride (19). This was prepared from 26 (0.100 g,
0.358 mmol) and phenol 54 (0.091 g, 0.358 mmol). Workup and
purification by flash column chromatography, eluting with 2% EtOAc
in CH2Cl2 to 30% EtOAc in CH2Cl2, afforded intermediate acetamide
70 as a colorless foam (0.128 g, 79%), which was immediately
deprotected using K2CO3 (0.078 g, 0.564 mmol). After workup, the
resulting gum was triturated with hexanes (2 × 5 mL), diluted in 10%
MeOH in ether (9 mL), and treated with methanolic HCl (1.5 mL)
for 20 h. The precipitate was collected and precipitated 3 times from
hot MeOH (0.5 mL) with ether (2.5 mL) to yield 19 as a white
amorphous solid (0.053 g, 49% from 70): mp 293−294 °C. 1H NMR
(500 MHz; DMSO-d6): δ 14.35 (s, 1 H), 9.29 (br s, 3 H), 8.37 (d, J =
9.3 Hz, 1 H), 8.29 (br s, 1 H), 7.95 (d, J = 8.2 Hz, 1 H), 7.78 (s, 1 H),
7.54 (dd, J = 8.2, 1.3 Hz, 1 H), 7.44 (dd, J = 6.0, 3.1 Hz, 1 H), 7.27 (t,
J = 9.2 Hz, 1 H), 7.14 (dt, J = 8.9, 3.6 Hz, 1 H), 7.10 (d, J = 9.3 Hz, 1
H), 5.33 (s, 2 H), 4.14 (s, 2 H), 2.57 (s, 3 H). 13C NMR (126 MHz;
DMSO-d6): δ (156.0 + 154.04, 1 C), 154.4, (154.11 + 154.10, 1 C),
142.8, 141.8, 136.0, 129.0, 123.8, 120.4, (119.89 + 119.76, 1 C),
(118.18 + 118.15, 1 C), (117.10 + 117.03, 1C), (116.53 + 116.35, 1
C), 115.3, 113.8, 69.2, (44.44 + 44.41, 1 C), 32.2; ESIMS m/z (rel.
intensity) 312 (MH+, 100). HRMS calcd for C18H19FN3O, 312.1512;
found, 312.1502.
(E)-Methyl 4-(2-Cyanovinyl)-3-nitrobenzoate (77).23 Compound
76 (2.00 g, 9.56 mmol) was diluted in anhydrous CH2Cl2 (24 mL) and
cooled to −15 °C. (Triphenylphosphoranylidene)acetonitrile (3.08 g,
10.2 mmol) was diluted in anhydrous CH2Cl2 (30 mL) and added to
the reaction mixture dropwise over 50 min. The mixture was stirred a
total of 90 min, warmed to room temperature, and concentrated. The
residue was purified by flash column chromatography, eluting with a
gradient of 10% EtOAc in hexanes to 35% EtOAc in hexanes.
Fractions containing pure 77 were saved, and the remaining eluate was
concentrated and repurified using the same column gradient to yield
7-[(4-Chloro-3-((methylamino)methyl)phenoxy)methyl]quinolin-
2-amine Dihydrochloride (20). This was prepared from 26 (0.100 g,
0.358 mmol) and phenol 55 (0.097 g, 0.358 mmol). Workup and
purification by flash column chromatography, eluting with 2% EtOAc
in CH2Cl2 to 30% EtOAc in CH2Cl2, afforded intermediate acetamide
71 as a colorless foam (0.136 g, 81%), which was deprotected using
K2CO3 (0.080 g, 0.578 mmol). After workup, the resulting gum was
triturated with hexanes (2 × 5 mL), diluted in 10% MeOH in ether (9
mL), and treated with methanolic HCl (1.5 mL) for 20 h. The
precipitate was collected and precipitated 4 times from hot MeOH (1
mL) with ether (3 mL) to yield 20 as a white amorphous solid (0.059
1
77 as a white flocculent solid (1.76 g, 79%); H NMR (500 MHz,
CDCl3) δ 8.72 (d, J = 1.7 Hz, 1 H), 8.32 (dd, J = 8.1, 1.7 Hz, 1 H),
7.87 (d, J = 8.1 Hz, 1 H), 7.69 (d, J = 11.8 Hz, 1 H), 5.77 (d, J = 11.8
Hz, 1 H), 3.94 (s, 3 H); 13C NMR (126 MHz, CDCl3) δ 164.2, 147.1,
145.7, 134.7, 133.3, 132.8, 131.1, 126.2, 115.6, 101.5, 53.0; ESIMS m/z
(rel. intensity) 233 (MH+, 100).
Methyl 2-Aminoquinoline-7-carboxylate (78).23 Compound 77
(2.2 g, 9.47 mmol) was diluted in DMF/glacial AcOH (1:1, 20 mL),
and one portion of iron powder (1.85 g, 33.2 mmol, divided into two
roughly equal amounts) was added in smaller portions. The mixture
was heated to 100 °C for 90 min, after which the second portion of
iron was added in smaller portions. After a total of 2 h 50 min of
heating, the mixture was cooled, concentrated to dryness, and the
mixture was treated with sat. aq. NaHCO3 (100 mL). This mixture was
exhaustively extracted with EtOAc (total, 700 mL), and the organic
phase was washed with sat. aq. NaCl (200 mL), dried over anhydrous
sodium sulfate, and concentrated. The resulting yellow residue was
diluted with EtOAc (10 mL) and heated until dissolution occurred.
Hexanes (100 mL) were then added, and the mixture was sonicated
vigorously until a precipitate formed. The precipitate was collected and
washed with hexanes to yield 78 as a pale-yellow crystalline solid (1.59
g, 83%). 1H NMR (500 MHz, CDCl3) δ 8.40 (s, 1 H), 7.96 (d, J = 8.8
Hz, 1 H), 7.91 (dd, J = 8.3, 1.6 Hz, 1 H), 7.70 (d, J = 8.3 Hz, 1 H),
6.87 (d, J = 8.8 Hz, 1 H), 5.19 (s, 2 H), 3.99 (s, 3 H); 13C NMR (126
MHz, CDCl3) δ 167.0, 157.1, 146.0, 138.2, 131.4, 127.8, 127.6, 126.1,
122.8, 113.8, 52.4; ESIMS m/z (rel. intensity) 203 (MH+, 100).
Methyl 2-Acetamidoquinoline-7-carboxylate (79). Compound 78
(1.89 g, 9.37 mmol) was diluted with anhydrous dioxane (60 mL), and
N-acetylimidazole (1.24 g, 11.2 mmol) and a catalytic amount (∼0.050
g) of DMAP were added. The mixture was heated at 100 °C for 19 h,
cooled, and concentrated, and the resulting residue was suspended in
CH2Cl2 (300 mL). The organic layer was washed with H2O (200 mL),
and the aqueous layer was extracted with CH2Cl2 (2 × 200 mL). The
organic layers were washed with H2O (400 mL) and sat. aq. NaCl
(300 mL), dried over anhydrous sodium sulfate, and concentrated.
The residue was diluted in CH2Cl2 (10 mL), and hexanes (150 mL)
were added until a precipitate formed. This was collected and dried to
yield 79 as a tan iridescent solid (2.08 g, 91%): mp 218−219.5 °C. 1H
NMR (500 MHz, CDCl3) δ 8.68 (br s, 1 H), 8.56−8.49 (m, 2 H), 8.22
(d, J = 9.0 Hz, 1 H), 8.06 (dd, J = 8.4, 1.7 Hz, 1 H), 7.85 (d, J = 8.4
Hz, 1 H), 4.00 (s, 3 H), 2.28 (s, 3 H); 13C NMR (126 MHz, CDCl3) δ
1
g, 51% from 71): mp 290−291.5 °C (dec). H NMR (500 MHz;
DMSO-d6): δ 14.36 (s, 1 H), 9.21 (br s, 3 H), 8.37 (d, J = 9.3 Hz, 1
H), 8.27 (br s, 1 H), 7.95 (d, J = 8.2 Hz, 1 H), 7.77 (s, 1 H), 7.54 (dd,
J = 5.2, 2.2 Hz, 2 H), 7.49 (d, J = 8.9 Hz, 1 H), 7.14 (dd, J = 8.9, 3.0
Hz, 1 H), 7.10 (d, J = 9.3 Hz, 1 H), 5.37 (s, 2 H), 4.21 (s, 2 H), 2.60
(s, 3 H). 13C NMR (126 MHz; DMSO-d6): δ 156.9, 154.4, 142.7,
141.5, 135.9, 130.9, 130.5, 129.1, 124.9, 123.8, 120.4, 118.3, 117.0,
115.4, 113.8, 69.0, 48.3, 32.4; ESIMS m/z (rel. intensity) 328 (MH+,
100). HRMS calcd for C18H19ClN3O, 328.1217; found, 328.1212.
7-[((5-((Methylamino)methyl)pyridin-3-yl)oxy)methyl]quinolin-2-
amine Trihydrochloride (21). This was prepared from 26 (0.100 g,
0.358 mmol) and phenol 59 (0.085 g, 0.358 mmol). Workup and
purification by flash column chromatography, eluting with 15% EtOAc
in CH2Cl2 to 90% EtOAc in CH2Cl2, afforded intermediate acetamide
73 as a white amorphous solid (0.098 g, 63%), which was deprotected
using K2CO3 (0.062 g, 0.449 mmol). After workup, the resulting gum
was triturated with hexanes (2 × 5 mL), diluted in 10% MeOH in
ether (9 mL), and treated with methanolic HCl (1.5 mL) for 20 h. The
precipitate was collected and precipitated from hot MeOH (0.5 mL)
with ether (2.5 mL) to yield 21 as a flocculent, cream-colored solid
1
(0.056 g, 62% from 73): mp 224−226 °C. H NMR (500 MHz;
DMSO-d6): δ 14.36 (s, 1 H), 9.47 (s, 2 H), 9.23 (br s, 1 H), 8.51 (d, J
= 2.7 Hz, 1 H), 8.38 (d, J = 9.4 Hz, 2 H), 8.26 (br s, 1 H), 7.97 (d, J =
8.2 Hz, 2 H), 7.80 (s, 1 H), 7.56 (dd, J = 8.2, 1.4 Hz, 1 H), 7.11 (d, J =
9.3 Hz, 1 H), 5.46 (s, 2 H), 4.18 (t, J = 5.8 Hz, 2 H), 2.54 (t, J = 5.4
Hz, 3 H). 13C NMR (126 MHz; DMSO-d6): δ 154.54, 154.42, 143.0,
142.3, 141.2, 137.1, 136.0, 129.51, 129.32, 125.2, 124.2, 120.7, 115.6,
114.1, 69.4, 48.3, 32.1; ESIMS m/z (rel. intensity) 295 (MH+, 100).
HRMS calcd for C17H19N4O, 295.1559; found, 295.1553.
(RS)-7-[(3-(1-(Methylamino)ethyl)phenoxy)methyl]quinolin-2-
amine Ditrifluoroacetate (22). This was prepared from 26 (0.100 g,
O
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169.3, 166.8, 151.8, 145.8, 138.4, 131.4, 129.8, 128.7, 127.9, 124.7,
116.2, 52.5, 24.9; ESIMS m/z (rel. intensity) 245 (MH+, 100).
N-(7-(Hydroxymethyl)quinolin-2-yl)acetamide (80). Compound
79 (0.732 g, 3.0 mmol) was diluted in anhydrous THF (10 mL)
and cooled to −15 °C. LiAlH4 (1 M in THF, 4.5 mL, 4.5 mmol) was
added dropwise over 5 min, and the mixture was stirred for 1 h. The
reaction was quenched by the addition of EtOAc (10 mL) and sat. aq.
sodium potassium tartrate solution (20 mL) and stirred until two
layers formed. The layers were separated, and the aqueous layer was
extracted with EtOAc (4 × 50 mL). The organic phase was washed
with sat. aq. NH4Cl (50 mL), the aqueous phase was re-extracted with
20 mL of EtOAc following this wash, and the combined organic layers
were washed with sat. aq. NaCl (50 mL), and dried over anhydrous
sodium sulfate. Concentration afforded a white residue, which was
precipitated from minimal EtOAc with hexanes to afford crude 80 as a
white solid (0.433 g, 67%), which was used in the following step
without further purification; 1H NMR (500 MHz, CDCl3) δ 8.41 (d, J
= 8.9 Hz, 1 H), 8.32 (s, 1 H), 8.17 (d, J = 8.9 Hz, 1 H), 7.81 (s, 1 H),
7.79 (d, J = 8.3 Hz, 1 H), 7.47 (d, J = 8.3 Hz, 1 H), 4.91 (s, 2 H), 2.28
(s, 3 H); 13C NMR (126 MHz, CDCl3) δ 169.1, 151.1, 146.6, 143.1,
138.5, 127.9, 125.6, 124.5, 124.3, 114.0, 65.0, 25.0; ESIMS m/z (rel.
intensity) 217 (MH+, 100)
and human nNOS heme domains used for crystallographic studies
were carried out using the procedures described previously.42,53 The
rat nNOS heme domain (WT or double mutant, at 9 mg/mL
containing 20 mM histidine) or the bovine eNOS heme domain (at 10
mg/mL containing 2 mM imidazole) were used for the sitting drop
vapor diffusion crystallization setup under conditions previously
reported.54,55 Human nNOS crystals were obtained with the triple
K301R/R354A/G357D mutant heme domain sample at 10 mg/mL.
By slightly modifying the original conditions42 where the monoclinic
C2 crystals grew, a new crystal form belonging to the P212121 space
group was obtained.42 Fresh crystals (1−2 days old) were first passed
stepwise through cryoprotectant solutions and then soaked with 10
mM inhibitor for 4−6 h at 4 °C before being flash cooled with liquid
nitrogen.
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 0.2° per
frame. Raw CCD data frames were indexed, integrated, and scaled
using HKL2000,57 but the pixel array data were processed with XDS58
and scaled with Scala (or 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
using REFMAC or PHENIX.62 Disordering in portions of inhibitors
bound in the NOS active sites was often observed, sometimes resulting
in poor density quality. However, partial structural features usually
could still be visible if the contour level of the σA weighted 2m|Fo| −
D|Fc| map dropped to 0.5 σ, which afforded the building of reasonable
models into the disordered regions. Water molecules were added in
REFMAC and checked by COOT. The TLS63 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 REFMAC or 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 RCSB protein data bank. The crystallographic data
collection and structure refinement statistics are summarized in Table
S1, with the PDB accession codes included.
Caco-2 Permeability Assay. Caco-2 monolayer assays were
performed by Cyprotex US, LLC (Watertown, MA) using the
following standard procedure: Caco-2 cells, grown in tissue culture
flasks, were trypsinized, resuspended, and grown and differentiated in
96-well plates for 3 weeks; monolayer formation was determined by
measuring the transport of Lucifer yellow, an impermeable dye. All
assays were performed at a concentration of 10 μM for 2 h. For apical
to basolateral (A → B) permeability, compounds were added on the
apical side (A), with permeation determined at the receiving
(basolateral, B) side, where the receiving buffer was removed for
analysis by LC/MS/MS using an Agilent 6410 mass spectrometer
(ESI, MRM mode) coupled with an Agilent 1200 HPLC, using
propranolol as an analytical internal standard. Buffers used were 100
μM Lucifer yellow in transport buffer (1.98 g/L glucose in 10 mM
HEPES, 1× Hank’s Balanced Salt Solution, pH 6.5) (apical side) and
transport buffer at pH 7.4 (basolateral side). Apparent permeability
(Papp) is expressed using the following equation: Papp = (dQ/dt)/C0A,
where the numerator is the rate of permeation, C0 is initial
concentration, and A is the monolayer area (0.11 cm2). For
bidirectional permeability, the efflux ratio was defined as Papp (B →
A)/Papp (A → B); high efflux ratio values (>3) indicate that a
compound may be a substrate for P-gp or other active transport
systems.
N-(7-Formylquinolin-2-yl)acetamide (81). Dess-Martin period-
inane (0.869 g, 2.05 mmol) was diluted in anhydrous CH2Cl2 (25
mL), and alcohol 80 (0.364 g, 1.71 mmol) was added in one portion.
The mixture was stirred at room temperature for 70 min and then
quenched by the addition of sat. aq. Na2S2O3 (15 mL). After being
stirred for 15 min, the layers were separated, and the aqueous layer was
extracted with CH2Cl2 (2 × 20 mL). The organic layers were washed
with sat. aq. NaHCO3 and sat. aq. NaCl (20 mL each). The organic
layer was dried over anhydrous sodium sulfate and concentrated, and
the residue was purified by flash column chromatography, eluting with
a gradient of CH2Cl2 to 40% EtOAc in CH2Cl2, to yield 81 as a yellow
1
amorphous solid (0.300 g, 82%); mp 199−200 °C. H NMR (500
MHz; CDCl3): δ 10.21 (d, J = 0.5 Hz, 1 H), 8.57 (br d, J = 9.0 Hz, 1
H), 8.29 (t, J = 0.7 Hz, 1 H), 8.25 (d, J = 9.0 Hz, 1 H), 7.96 (dd, J =
8.3, 1.4 Hz, 1 H), 7.92 (d, J = 8.4 Hz, 1 H), 2.32 (s, 3 H); the
acetamidoquinoline N−H proton is broadened into the baseline ∼8.3
ppm. 13C NMR (126 MHz; CDCl3): δ 192.0, 151.9, 139.2, 137.7,
132.3, 129.8, 129.0, 122.6, 116.7, 26.1; one of the quinoline carbons is
broadened into the baseline and is not visible; ESIMS m/z (rel.
intensity) 215 (MH+, 14).
Purified NOS Enzyme Assays. Rat and human nNOS, murine
macrophage iNOS, bovine eNOS, and rat nNOS double mutant
D597N/M336V were recombinant enzymes, expressed in E. coli and
purified as previously reported.49−51 To test for enzyme 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. 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, CaCl2 and calmodulin were
omitted and replaced with HEPES buffer (as neither are required for
activation of iNOS). The assay was performed in 96-well plates using a
Synergy 4 BioTek hybrid reader, and the dispensing of NOS enzyme
and hemoglobin were automated; after 30 s (maximum delay), NO
production was read by monitoring the absorbance at 401 nm
(resulting from the conversion of oxyhemoglobin to methemoglobin).
Kinetic readouts were performed for 3 or 5 min. Each compound was
assayed at least in duplicate, and seven to nine concentrations (500
μM−50 nM or 100 μM−10 nM for eNOS and iNOS; 50 μM to 5 nM
for nNOS) were used to construct dose−response curves. IC50 values
were calculated by nonlinear regression using GraphPad Prism
software, and Ki values were obtained using the Cheng−Prusoff
equation52 [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), 1.7 μM (bovine eNOS), and 1.9 μM (rat
nNOS D597N/M336V double mutant).38
Inhibitor Complex Crystal Preparation. The preparations of rat
nNOS (wild type or D597N/M336V double mutant), bovine eNOS,
P
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01330.
■
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Accession Codes
PDB codes for X-ray crystal structures described in this study
have been deposited in the Protein Data Bank under the
following accession codes: 5AD4, 5AD5, 5AD6, 5AD6, 5AD8,
5AD9, 5ADA, 5ADB, 5ADC, 5ADD, 5ADE, 5ADF, 5ADG,
5ADI, 5ADJ, 5ADK, 5ADL, 5ADN, 5FJ2, and 5FJ3.
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.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to the National Institutes of Health
(GM049725, to R.B.S., GM057353 to T.L.P., and
F32GM109667 to M.A.C.), for support of this work. L.J.R. is
currently supported on NIH GM081568 and NSF grant 13-
573. P.M. is supported by grants UNCE 204011 and PRVOUK
P24/LF1/3 from Charles University, Prague, Czech Republic,
and A.P. is supported by a Lambert Fellowship (Chemistry of
Life Processes Institute, Northwestern University) and by the
Katherine L. Kreighbaum Scholarship (Northwestern Univer-
sity). M.A.C. and A.P. wish to thank Drs. Arsen Gaisin and
Neha Malik of the Center for Molecular Innovation and Drug
Discovery (Northwestern University) for valuable assistance
with preparative HPLC, and Mr. Saman Shafaie and Dr. S.
Habibi Goudarzi for assistance with HRMS experiments. We
also wish to thank the SSRL and ALS beamline staff for their
support during X-ray diffraction and data collection. Off-target
Ki determinations were generously provided by the National
Institute of Mental Health’s Psychoactive Drug Screening
Program, contract #HHSN-271-2013-00017-C (NIMH PDSP),
directed by Dr. Bryan L. Roth (University of North Carolina at
Chapel Hill) and project officer Jamie Driscoll (NIH).
ABBREVIATIONS USED
■
NOS, neuronal nitric oxide synthase; iNOS, inducible nitric
oxide synthase; eNOS, endothelial nitric oxide synthase; FMN,
flavin mononucleotide; H4B, (6R)-5,6,7,8-tetrahydrobiopterin;
tPSA, total polar surface area; Papp, apparent permeability;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
PDSP, psychoactive drug screening program; WT, wild-type
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