First Contact: 7-Phenyl-2-Aminoquinolines, Potent and Selective Neuronal Nitric Oxide Synthase Inhibitors That Target an Isoform-Specific Aspartate

Article abstract of DOI:10.1021/acs.jmedchem.9b01573

Inhibition of neuronal nitric oxide synthase (nNOS), an enzyme implicated in neurodegenerative disorders, is an attractive strategy for treating or preventing these diseases. We previously developed several classes of 2-aminoquinoline-based nNOS inhibitor

Full text of DOI:10.1021/acs.jmedchem.9b01573

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First Contact: 7‑Phenyl-2-Aminoquinolines, Potent and Selective
Neuronal Nitric Oxide Synthase Inhibitors That Target an Isoform-
Specific Aspartate
Maris A. Cinelli,§ Cory T. Reidl,§ Huiying Li, Georges Chreifi, Thomas L. Poulos,
and Richard B. Silverman*
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ABSTRACT: Inhibition of neuronal nitric oxide synthase (nNOS), an enzyme implicated
in neurodegenerative disorders, is an attractive strategy for treating or preventing these
diseases. We previously developed several classes of 2-aminoquinoline-based nNOS
inhibitors, but these compounds had drawbacks including off-target promiscuity, low
activity against human nNOS, and only modest selectivity for nNOS over related enzymes.
In this study, we synthesized new nNOS inhibitors based on 7-phenyl-2-aminoquinoline and
assayed them against rat and human nNOS, human eNOS, and murine and (in some cases)
human iNOS. Compounds with a meta-relationship between the aminoquinoline and a positively charged tail moiety were potent
and had up to nearly 900-fold selectivity for human nNOS over human eNOS. X-ray crystallography indicates that the amino groups
of some compounds occupy a water-filled pocket surrounding an nNOS-specific aspartate residue (absent in eNOS). This
interaction was confirmed by mutagenesis studies, making 7-phenyl-2-aminoquinolines the first aminoquinolines to interact with this
residue.
flavin mononucleotide (FMN), and reduced nicotinamide
adenine dinucleotide phosphate (NADPH), whereas the
oxygenase domain contains binding sites for (6R)-5,6,7,8-
tetrahydrobiopterin (H4B), the metallocofactor heme, and the
substrate L-arginine. Electron flow proceeds from one
monomer’s reductase domain, sequentially through NADPH,
FAD, and FMN, to the opposite monomer’s oxygenase
domain, where the electron is transferred between FMN and
heme, by which L-arginine is oxidized to L-citrulline and NO.4
Most compounds initially investigated for nNOS inhibition
were designed as competitive mimics of L-arginine. These
inhibitors have high basicity and polarity, a large total polar
surface area (tPSA), and an overabundance of hydrogen bond
donors and, as a result, suffer from poor bioavailability and
blood−brain barrier (BBB) penetrability. Furthermore, many
promising L-arginine-mimetic inhibitors are not nNOS-
selective, owing to the high sequence similarity and nearly
identical active-site architecture between the three NOS
isoforms. High nNOS selectivity is crucial; nonselective
inhibitors have the potential for dangerous side effects. For
example, iNOS inhibition could impair immune system
INTRODUCTION
■
Neurodegenerative diseases such as Alzheimer’s, Huntington’s,
Parkinson’s, and amyotrophic lateral sclerosis (ALS) are
characterized by the gradual loss of neuronal integrity and
are responsible for a wide range of neurological deficiencies.
Neuronal damage or death associated with stroke, ischemic
events, and cerebral palsy (as well as acute or chronic brain
injuries) has also been linked to similarly debilitating motor,
cognitive, and psychological impairments. The overproduction
of the vital secondary messenger nitric oxide (NO), produced
by neuronal nitric oxide synthase (nNOS) in tissues of the
central (CNS) and peripheral nervous system (PNS), is
directly implicated in these disorders.1,2 Because NO plays a
key role in these diseases, rational control of NO levels in
neuronal tissues via nNOS-specific inhibition is therapeutically
desirable.
Nitric oxide synthases (NOSs) are a family of homodimeric
enzymes that are responsible for the biosynthesis of NO.
Functional regulation of NO is differentiated by subcellular
localization, tissue distribution, and regulatory gene expression
of three isoforms of NOS: endothelial NOS (eNOS), inducible
NOS (iNOS), and neuronal NOS (nNOS), which are
responsible for regulating blood pressure and vascular tone,
immune activation, and normal neuronal communication,
respectively.3 Functional NOS is a homodimer. Each NOS
monomer contains a reductase domain and an oxygenase
domain, separated by a flexible region where calmodulin binds
when activated by calcium ions. The reductase domain
contains binding sites for flavin adenine dinucleotide (FAD),
Received: September 20, 2019
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activation, while eNOS inhibition can lead to severe hyper-
tension or other cardiovascular complications.5
We have been investigating 2-aminoquinoline-based scaf-
folds as isoform-selective arginine bioisosteres with more
favorable pharmacokinetic properties. Since 2014, we have
reported several generations of aminoquinolines that are
modestly potent and selective toward nNOS (Figure 1). The
and 4, improved upon their respective parent series by
incorporating elements such as the quinoline 4-methyl group
and cyano-containing tail moieties.8,9 These compounds have
greatly enhanced hnNOS potency, hn/heNOS selectivity, and
improved cellular permeability and off-target profiles.
Because of some of the drawbacks associated with previous
inhibitor generations, we have been investigating alternative
aminoquinoline-containing scaffolds. Interestingly, the 7-
phenylquinoline compound 5 appears in the literature as part
of a Glaxo-SmithKline screening library and was recently
employed in several high-throughput screening studies.10,11
However, there are no articles, patents, or other reports of
what research program this compound may have belonged to
originally, but it appears to now be part of an “open source”
drug discovery program.
Because of its distinctively nNOS inhibitor-like structure,
but with fewer rotatable bonds than earlier series, a docking
study with 5 in a nNOS crystal structure was conducted (see
Supplemental Information for protocol). Consequently, 5 was
predicted to bind in an nNOS inhibitor-like mode, in which
the aminoquinoline forms a salt-bridge with Glu592/Glu597
(rnNOS/hnNOS) and the phenethylamine tail portion faces
out toward the regions of the active site. To this end, lead
compound 5 and related compounds 6−9 (modified at the
amine portion (Figure 2)) were designed, synthesized, and
assayed against purified NOS isoforms to test the hypothesis
that 5 and analogues could act as nNOS inhibitors.
Satisfyingly, these compounds possessed encouraging nNOS
inhibitory activity and good isoform selectivity, and we chose
to undertake a more thorough structure−activity relationship
(SAR) study. First, we investigated whether meta- or ortho-
substitution of the central phenyl ring (compounds 10−13)
might be more effective than the para-substitution of the
parent compound. This early optimization revealed that meta-
substituted analogue 12 displayed good inhibitory potency
against rat and human nNOS, excellent hn/heNOS selectivity
and n/i selectivity, as well as good solubility and desirable
properties (few rotatable bonds and low tPSA).
Figure 1. Representative aminoquinoline nNOS inhibitors.
first generation of aminoquinolines (such as 1)6 was found to
be potent and selective nNOS inhibitors with improved
pharmacokinetics. Unfortunately, 1 was found to have high
selectivity for rat nNOS (rnNOS) over human nNOS
(hnNOS), low selectivity for hnNOS over human eNOS
(heNOS), and caused toxic side effects, possibly because of its
off-target promiscuity.4 The second generation of NOS
inhibitors, (e.g., phenyl ether 2)7 reduced off-target binding
while preserving potency and selectivity against rnNOS.
However, these compounds suffered from decreased Caco-2
permeability, low hnNOS activity, and similarly low hn/
heNOS selectivity. Newer generations of inhibitors, such as 3
Figure 2. Initial derivatives of 5 prepared in this study.
B
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Figure 3. Optimization of 12 by 5-substitution, amino group constraint, and 4-substitution.
a
Scheme 1. Synthesis of Lead 5
a
Reagents and conditions: (a) (i) B2Pin2, KOAc, Pd(dppf)Cl2, dioxane, 75 °C; (ii) KHF2, tetrahydrofuran (THF)/H2O, room temperature (r.t.);
(b) Boc2O, THF, r.t.; (c) Pd(dppf)Cl2, NaHCO3, 1,2-dimethoxyethane (DME)/H2O, microwave, 120 °C; (d) (i) K2CO3, MeOH, reflux; (ii)
HCl/MeOH, ether, r.t. (after isolation).
Encouraged by both the inhibitory constants and the
agreement between X-ray crystallography and our docking
results (Supplemental Information, Figure S1), 12 was used as
a launching point for further optimization. We developed a set
of compounds with modifications made at the 5-position of the
central phenyl ring (e.g., nitrile 14 and pyridine 15) to
investigate whether additional interactions could be made with
the heme propionate or another active-site residue.
Because of 12’s high n/eNOS selectivity, we hypothesized
that the flexible tail amino group might be contacting (directly
or otherwise) a specific aspartate residue (Asp597/Asp602 in
rnNOS/hnNOS, respectively). This residue is missing in
eNOS isoforms, replaced by asparagine. Consequently, contact
(H-bonding or electrostatic) between an inhibitor and this
residue can impart very high n/eNOS selectivity (1000-fold or
more). We hypothesized that a second set of compounds could
be designed to solidify any existing contacts with Asp597/
Asp602 by incorporating the tail amino group functionality
into a rigid ring system, thereby reducing its overall flexibility
and locking the interaction in place. As the amino group of 5 is
quite flexible, both meta-/para- and ortho-/meta-constrained
derivatives (isoindoline 16 and the two isomeric racemic
indanylamines 17 and 18) were prepared.
Additional docking studies indicated that a variety of groups
might be accommodated at the 4-position of the central phenyl
ring of 12, which could form van der Waals interactions with
Met336/Met341 (rnNOS/hnNOS), a residue that was
previously implicated in high n/eNOS selectivity for 2-
aminoquinoline-based inhibitors7 as it is absent in eNOS
isoforms (replaced by a smaller valine).12 To this end, 3,4-
substituted compounds 19−37 (Figure 3), possessing a variety
of steric, electronic, and H-bonding substituents at the 4-
position, were investigated to determine if this substitution
pattern could make extra contacts with the isoform-specific
residues Met336/Met341 and/or the hnNOS-specific residue
His342.
All synthesized compounds were assayed against rnNOS,
and selected compounds were also assayed against hnNOS.
Murine iNOS and human eNOS were used to determine
selectivity, and selected compounds were also assayed against
human iNOS.
RESULTS AND DISCUSSION
■
Chemistry. To prepare compound 5, we envisioned that
the quinoline−aryl bond could be constructed via Suzuki
C
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coupling. To this end, we sought to install the boron-
containing moiety on the quinoline, taking advantage of a large
and diverse set of available aryl halides (which are less
expensive and easier to synthesize than an analogous series of
boronates or boronic esters). Using versatile 7-bromoquinoline
38,8 a 7-BPin moiety was first installed via Miyaura borylation
(Scheme 1). This intermediate was not isolated but rather
converted to trifluoroborate 39, which was readily purified
because of its insolubility in most organic solvents.
To prepare the halide precursor, commercially available 4-
bromophenethylamine 40 was Boc-protected to yield 41.
Many Suzuki conditions were screened for the coupling of 39
and 41, but the strong protic bases usually required for
reductive elimination and activation of 39 often led to
deacetylation of the quinoline and decomposition. The use
of NaHCO3 as the base13 in a mixed aqueous solvent was more
successful, and microwave irradiation of this mixture yielded
phenylquinoline 42 within 25 min at 120 °C without
substantial deacetylation. The intermediate protected phenyl-
quinolines were not extensively characterized but were isolated
and immediately deprotected. Deprotection was accomplished
stepwise, first with K2CO3 in refluxing methanol to cleave the
acetyl group, followed by treatment of the free aminoquinoline
with methanolic HCl to remove the Boc group and provide 5
as its water-soluble dihydrochloride salt.6
Ketone 47 was condensed with (S)-tert-butylsulfinamide, and
the intermediate sulfinyl imine was reduced at low temperature
to afford (S,S)-48 in a good d.r. of ∼7:1.
Desulfinylation under acidic conditions and Boc-protection
subsequently afforded derivative 49. For ortho- and meta-
substituted derivatives 10/11 and 12/13, respectively, the
commercially available benzylamines (50, 54) and phenethyl-
amines (51, 55) were Boc-protected to yield o-substituted (52,
53) and m-substituted (56, 57) bromides, respectively
(Scheme 2D,E). Suzuki coupling between 39 and these halides
under the conditions described above (Scheme 3) was facile
and displayed a high substrate tolerance, affording protected
phenylquinolines 58−65 in good yields. Generally, the only
impurity isolated was a small amount (<10%) of proto-
deborylated acetamidoquinoline. Deprotection of 58−66
(Scheme 3) afforded analogues 6−13.
To synthesize 5-cyano derivative 14, bromobenzene 66 was
prepared as previously described. Treatment of 66 with the
anion derived from Boc2NH (Scheme 4A) yielded, surpris-
ingly, mono-Boc protected amine 67, indicating that one Boc
group was cleaved during the reaction or workup. Suzuki
coupling with 39 yielded 68, which was then deprotected to
provide amine 14. In contrast, heating Boc2NH and pyridine
69 under basic conditions (Scheme 4B) afforded the N,N-di-
Boc compound (70).15 Likewise, the major product (71)
isolated upon coupling of 70 and 39 contained both Boc
groups intact. During deacetylation, a longer period of heating
(4.5 h) was employed to remove both the acetyl group and one
Boc group, and the second Boc group was then removed with
HCl to yield 15.
Synthesis of isoindoline derivative 16 (Scheme 5A)
commenced with commercially available 4-bromoisoindoline
salt 72, which was converted to the free base and Boc-
protected to yield 73. Coupling with 39 afforded 74, which
then yielded 16 upon deprotection. Indanylamine derivatives
17 and 18 (Scheme 5B) were prepared from the 5- and 6-
bromoindanones (75 and 76), respectively. The Ellman
method (as in Scheme 2C)14 was used to install the amino
group as its racemate. However, yields of sulfinamides 77 and
78 were fairly low, and large amounts of insoluble ketone
condensation byproducts were obtained. Nonetheless, 77 and
78 were desulfinylated and Boc-protected, and carbamates 79
and 80 were readily amenable to Suzuki coupling with 39,
affording 81 and 82, which were deprotected to yield,
respectively, 17 and 18.
To prepare the initial set of derivatives with different para-
aminoalkyl tail portions (6−9), the halides were first prepared.
Compound 41 was methylated to yield 43 (Scheme 2A).
Commercially available iodobenzylamine 44 was Boc-pro-
tected to yield 45, which was also methylated to yield 46
(Scheme 2B). For the (S)-α-methyl-phenethylamine group of
9, the Ellman auxiliary method14 was used (Scheme 2C).
Scheme 2. Preparation of Precursor p-, o-, and m-
a
Substituted Halides
We envisioned that 4-substituted derivatives 19−24 could
be accessed from commercially available benzaldehydes or
toluene derivatives via conversion to the benzyl halides. To this
end, fluorotoluene 83 and chlorotoluene 84 (for 19 and 20)
́
were converted to benzyl bromides 85 and 86. A Delepine
reaction, involving treatment with hexamethylenetetramine
and acidic hydrolysis of the resulting hexaminium adduct, and
subsequent Boc-protection of the amine yielded 87 and 88
(Scheme 6A). For the trifluoromethyl derivative en route to
21, commercially available nitrile 90 was reduced with BH3-
DMS,16 and the isolated amine was protected to yield 91
(Scheme 6B). 2-Ethybenzaldehyde (91, for 22) was complexed
17
with AlCl3 and brominated to yield the major regioisomer
(92b) as an inseparable 3:1 mixture with 92a. Following
borohydride reduction, the isomers were separated, and the
a
Reagents and conditions: (a) (i) NaH, THF, 0 °C to r.t.; (ii) Mel,
́
major isomer (93) was chlorinated to yield 94. Delepine
r.t. or reflux; (b) Boc2O, THF, r.t.; (c) (i) (S)-t-butylsulfinamide,
Ti(OEt)4, THF, 70 °C; (ii) NaBH4, THF, −40 °C; (d) (i) HCl/
MeOH, ether, r.t.; (e) Boc2O, Et3N, THF/MeOH, r.t.
reaction and Boc protection provided bromobenzene deriva-
tive 95 (Scheme 6C).
D
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a
Scheme 3. Assembly and Deprotection of p-, o-, and m-Substituted Phenylquinolines
a
Reagents and conditions: (a) halides 43, 45−46, 49, 52−53, 56−57, Pd(dppf)Cl2, NaHCO3, 1,2-dimethoxyethane (DME)/H2O, microwave, 120
°C; (b) (i) K2CO3, MeOH, reflux; (ii) HCl/MeOH, ether, r.t. (after isolation).
a
Scheme 4. Synthesis of 14 and 15
a
Reagents and conditions: (a) (i) NaH, Boc2NH, THF, r.t.; (ii) halide, r.t. or r.t. −50 °C (for 69); (b) 39, Pd(dppf)Cl2, NaHCO3, 1,2-
dimethoxyethane (DME)/H2O, microwave, 120 °C; (c) (i) K2CO3, MeOH, reflux; (ii) HCl/MeOH, ether, r.t. (after isolation).
Methoxybenzyl alcohol 96 (for 23) was prepared as
previously described (Scheme 6D) and elaborated via 97 as
described above to yield 98 (see Supporting Information for
details). Finally, ethoxylated toluene 99 was brominated to
are only commercially available as the HCl and HBr salts,
respectively, these salts were converted to their free bases
immediately prior to the formation of the 2-pyridylmethyl
(111), 3-pyridylmethyl (112), and thiazol-5-methyl (116)
ethers. All of these ether-containing halides were subjected to
Suzuki coupling with 39 to yield protected phenylquinolines
117−135 in moderate to excellent yields, and stepwise
deprotection as described above yielded final analogues 19−
37.
nNOS Inhibitory Assay and Crystallography. The
hemoglobin capture assay (see the Experimental Section)
was used to determine the inhibition constants (Ki) of
synthesized compounds 5−37.18,19 All compounds were
assayed against purified rnNOS as a prescreen, and eighteen
of the most potent compounds against rnNOS were further
assayed against purified human nNOS (hnNOS), murine
iNOS (miNOS), and human eNOS (heNOS) to determine
isoform selectivity. Table 1 summarizes the apparent Ki values
and isoform selectivities for compounds 5−37. Values for
compounds 1−4 are included for comparison. The rnNOS and
́
yield 100 (Scheme 6E). Delepine reaction and Boc protection
afforded carbamate 101.
As the multiple steps of this route would make the
preparation of many similar analogues time consuming, and
the Boc2NH method of Scheme 4 was unpredictable, a slightly
different strategy was used to prepare 4-ether halide derivatives
25−37 (Scheme 7). In this route, protected amine 103 was
first prepared via reduction of 102, and then, the ether
functionality was installed by deprotonation of the phenol and
treatment with alkyl or benzyl halides.
By this method, the n-propyl (104), isopropyl (105),
isobutyl (106), methylcyclobutyl (107), methylcyclopropyl
(108), 3-fluorobenzyl (109), 4-cyanobenzyl (110), 5-methyl-
isoxazol-3-methyl (113), 4- and 5-methyl thiazoles (114/116),
and oxazol-4-methyl (115) ethers were prepared. As the
thiazol-5-methyl chloride and 2- and 3-pyridylmethyl bromides
E
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a
Scheme 5. Synthesis of Conformationally Constrained Derivatives 16−18
a
Reagents and conditions: (a) Boc2O, Et3N, THF/MeOH, r.t.; (b) 39, Pd(dppf)CI2, NaHCO3, 1,2-dimethoxyethane (DME)/H2O, microwave,
120 °C; (c) (i) K2CO3, MeOH, reflux; (ii) HCI/MeOH, ether, r.t. (after isolation); (d) (i) (RS)-t-butylsulfinamide, Ti(OEt)4, THF, 70 °C; (ii)
NaBH4, THF, −40 °C; (e) (i) HCI/MeOH, ether, r.t.
miNOS isoforms were used to approximate n/i isoform
selectivity because they are the easiest to express and purify,
and those are the species used for crystallography.
Furthermore, for preclinical purposes, it is essential to prove
efficacy and selectivity in lower animals prior to advancement
to clinical trials. Recent advances have made it possible to
obtain and crystallize both hnNOS and heNOS, which were
used to support our human isoform SAR development.
Because high-resolution structures of murine or human
iNOS with inhibitors bound are still not readily available, the
majority of the structural discussion will focus on nNOS and
eNOS; discussion of a comparison between murine and human
iNOS inhibition follows that.
Initial Inhibitory and Structural Analysis of Modified
Amine Tail Analogues. The initial lead 7-phenyl-2-amino-
quinoline (5) has good rat and human nNOS inhibitory
activity (105 and 122 nM, respectively) with moderately high
n/iNOS and n/eNOS selectivities of 207-fold and 191-fold,
respectively. The X-ray crystal structure of 5 bound to rnNOS,
hnNOS, and heNOS (Figure 4A−C) revealed the structural
basis for inhibitory potency. The quinoline portion of 5 mimics
arginine and forms a bifurcated hydrogen bond system with
the main-chain carbonyl of Trp587/Trp592 (rnNOS/hnNOS)
and the side-chain carboxylate of Glu592/Glu602 (rnNOS/
hnNOS). This is identical to the structural details observed for
other nNOS inhibitors containing a 2-aminoquinoline.6−9 All
three crystal structures clearly reveal that the central phenyl
ring resides between heme propionates A and D. In the
hnNOS-5 structure, the tail phenethylamine moiety makes a
direct H-bond interaction with the H4B and propionate A,
displacing the water there, while the rnNOS-5 structure only
makes H-bonding contacts with the carbonyl of the H4B and is
unable to displace the water molecule bridging propionate A
and H4B.
direction opposite to what we observed in the nNOS structures
and instead interacts with propionate D. Ligand B is stabilized
by aromatic stacking interactions with both Trp447 as well as
Trp74 and Phe460 from the opposite chain at the dimer
interface. Two-site binding is not unique to 5 and has been
observed in several NOS-inhibitor structures.20,21 In all four
molecules of the asymmetric unit of heNOS, the aminoquino-
line and phenyl rings of 5 are clearly defined. The electron
density is weaker for the tail ethylamines, with slightly more
density observed near propionate D, indicating a potential
interaction with the heme propionate. Interestingly, the ability
of 5 and other compounds to bind in both sites has little
correlation with inhibitory potency, indicating that active-site
binding (and not H4B displacement) determines potency.
With the goal of improving hnNOS activity and isoform
selectivity, we made efforts to optimize the conformational
positioning of the tail amine of 5. To this end, homologation,
chain shortening, and isomerization of lead molecule 5 resulted
in compounds 6−13. Methylation of the tail nitrogen atom of
5 (compound 6) resulted in over a 2-fold loss in rnNOS
activity, while shortening the ethylene linker between the
central ring and the terminal nitrogen atom by one carbon
(compound 7) resulted in a 2.3-fold increase in rnNOS
activity. Moreover, 7 showed increased hnNOS inhibitory
activity, as well as improvements in both rn/miNOS and hn/
heNOS selectivity ratios, relative to the parent molecule 5. The
X-ray crystal structures of 7 bound to rnNOS and hnNOS
(Figure 5) reveal well-defined densities at the aminoquinoline
and central ring regions that closely overlap with lead 5.
However, the orientation of the tail aminomethyl group in
both structures could not be determined because of poor
density, even at a low contour levels, suggesting that free
rotation of the aminomethyl occurs either toward propionate A
and the H4B site water, as modeled in Figure 5A,B, or toward
propionate D and Tyr706/Tyr711 (rnNOS/hnNOS). In both
nNOS structures, the position of the central phenyl ring of 7
forces heme propionate D into a downward conformation. As
observed for 5 and 6, methylation of the tail nitrogen atom of 7
to yield 8 resulted in a loss of potency against both rat and
In heNOS, there are two molecules of 5 bound. Ligand A
binds in the active site in a manner very similar to the nNOS
structures, while Ligand B displaces H4B, with the amino-
quinoline positioned in the pterin binding pocket. As a result,
the tail ethylamine of 5 bound to heNOS is oriented in the
F
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was generally observed for benzyl and phenethyl ether
compounds.
Scheme 6. Synthesis of Intermediate Carbamates for the
Preparation of 4-Substituted Derivatives
a
The o-substituted isomers 11 and 10 possess less inhibitory
potency (11, Ki (rnNOS) = 935 nM; 10, Ki (rnNOS) = 119
nM) than their corresponding para-substituted derivatives 5
and 7, respectively. Furthermore, 10 displays a similar loss in
hnNOS potency and a sharp decrease in rn/miNOS selectivity.
However, poor binding affinity to heNOS (10, Ki (heNOS) =
41 900 nM) results in a gain in hnNOS selectivity over heNOS
compared to 7. In contrast to o-substitution, m-substituted
isomers 13 and 12 exhibited comparable potencies to 5 and 7,
respectively [13, Ki (rnNOS) = 107 nM; 12, Ki (rnNOS) = 55
nM]. Moreover, 12 also has very good nNOS selectivity over
iNOS (rn/miNOS = 440) and outstanding selectivity over
eNOS (hn/heNOS = 877). The assay data for 12 suggest that
placing the aminoalkyl tail portion meta- to the quinoline
favors binding to the human nNOS isoform, which prompted
examination of the crystal structures of both rnNOS-12 and
hnNOS-12. As shown in Figure 6A,B, the tail amino group in
the meta-position of the benzene ring may participate in
maximal binding interactions with a water-filled polar pocket
composed of the carboxylate of propionate A, the side chains
of Gln478/Gln483 and Arg481/Arg486, and, most notably,
Asp597/Asp602 (rnNOS/hnNOS, respectively), resulting in
reduced flexibility in the tail aminomethyl group.
In sharp contrast, the tail aminomethyl group in the heNOS-
12 structure (Figure 6C) shows a different orientation, making
H-bonds with heme propionate A. It is important to note that
there is a major difference in the active site of human eNOS,
namely, Asn366 replaces Asp597/Asp602 (rnNOS/hnNOS) in
the polar pocket noted for nNOS. In earlier studies, we found
that this Asp/Asn difference makes substantial contributions to
isoform selectivity.23 This is likely the basis for the high
selectivity observed for 12 because the aminomethyl group is
oriented toward Asp597/Asp602 in the rnNOS/hnNOS
structures.
We proposed that homologated analogue 13 could have
improved activity because the longer chain might allow it to
potentially displace one of the structural waters near the
nNOS-specific Asp residue. However, the nNOS inhibitory
activity of 13 is weaker, comparable to the para-substituted
derivative 5 (and about 2-fold lower than 12), indicating that
either (a) this water molecule is not displaced or (b) any
energy gained from water displacement does not offset
entropic costs of chain elongation, internal torsion, or other
negative interactions between the enzyme and inhibitor. The
crystal structures of rnNOS-13 and hnNOS-13 (Figure 7)
support the former hypothesis; the tail amino group does not
occupy this polar pocket but rather only displaces the water
molecule bridging propionate A and the H4B. Consequently,
the aminoethyl group can only reach the closer heme
propionate A for an electrostatic interaction rather than the
farther Asp597/Asp602 site (rnNOS/hnNOS).
Previously, installation of a nitrile at the 5-position of phenyl
ether-linked aminoquinolines greatly improved potency, as the
nitrile fit into a small, previously undiscovered auxiliary pocket,
where it formed a H-bond with a deep structural water and
stabilized binding;8 likewise, 1,3,5-trisubstituted nitrile-con-
taining aminopyridine derivatives24 exert augmented potency
and selectivity via differential interactions with the Asp site (vs
the Asn site in eNOS). For the phenylquinoline scaffold,
however, the nitrile is a very deleterious modification [14, Ki
(rnNOS) = 1,030 nM]. Examination of the crystal structure of
a
Reagents and conditions: (a) NBS, (PhCO2)2, CCl4, reflux; (b) (i)
HMTA, NaI, EtOH, r.t.; (ii) HCl, reflux; (iii) Boc2O, THF (after
isolation); (c) (i) BH3-Me2S, THF, 0 °C to reflux; (ii) HCl/MeOH,
reflux; (iii) Boc2O, THF, r.t. (after isolation); (d) Br2, AlCl3, CH2Cl2,
0 °C to r.t.; (e) NaBH4, MeOH, r.t.; (f) SOCl2, CH2Cl2, 0 °C to r.t.
human nNOS, suggesting that a primary amino group at this
position is important for achieving maximal inhibitory activity
against nNOS isoforms.
It appears that some parts of the phenylaminoquinoline SAR
overlap with that of the previously reported phenyl ether
compounds.7 For example, phenyl ether compounds with one
methylene between the amino group and the aryl ring (benzyl)
are more potent and selective than those with two (phenethyl)
or more methylenes, and the same trend is observed here (cf. 7
and 8 vs 5 and 6); compound 9 (Ki (rnNOS) = 140 nM) also
has slightly less potency than 7. Nonetheless, other parts of the
SAR are distinct from the previous aminoquinoline SARs. For
instance, N-methylated compounds (6, 8) are less potent than
the desmethyl analogues (5, 7), which is the reverse of what
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a
Scheme 7. Preparation of Ether-Substituted Carbamates and Final Assembly of 4-Substituted Analogues 19−37
a
Reagents and conditions: (a) (i) BH3-Me2S, THF, 0 °C to reflux; (ii) HCl-MeOH, reflux; (iii) Boc2O, MeOH/THF, r.t. (after isolation); (b) (i)
NaH, N,N-dimethylformamide (DMF), 0 °C; (ii) alkyl or benzyl iodide or bromide, 0 °C to r.t.; (c) alkyl bromide, K2CO3, acetone, reflux; (d)
heterocyclic halides, Cs2CO3, DMF, r.t.; (e) 39, Pd(dppf)Cl2, NaHCO3, 1,2-dimethoxyethane (DME)/H2O, pwave, 120 °C; (f)b (i) B2(OH)2,
KOAc, XPhos-Pd-G3, XPhos, EtOH, 80 °C, 2 h; (ii) 38, K2CO3, H2O, 80 °C, 15 h; (g) (i) K2CO3, MeOH, reflux; (ii) HCl/MeOH, ether, r.t.
(after isolation).
12 (Figure 6A) indicates that the 5-position nitrile of the more
compact and rigid phenylquinoline (compared to the more
flexible phenyl ether molecule) cannot reach this auxiliary
pocket. Instead, the position of the phenyl ring of 14 (just like
12) would force heme propionate D into a downward
conformation; thus, the 5-position nitrile would cause serious
clashes with nearby residues. This is another area where the
SARs of phenyl ether-linked aminoquinolines and phenyl-
quinolines drastically diverge.
On the basis of the 12-bound NOS crystal structures (Figure
6), pyridine 15 was expected to be a potent inhibitor. In
addition to having an identical binding mode to 12, the
pyridine nitrogen atom forms a hydrogen bond with heme
propionate D in all three NOS structures (Figure 8). The
aminomethyl group of 15 approaches Gln478 in rnNOS and is
disordered but may point to Asp602 in hnNOS and heads to
the water molecule near heme propionate A in heNOS, where
a second molecule of 15 is bound in the pterin site.
Nonetheless, the pyridine analogue is approximately 5-fold
less potent than 12 in rnNOS, contradicting both the
crystallographic observations and previous results for pyr-
idine-containing 2-aminopyridine and 2-aminoquinoline com-
pounds.7 The unpredicted effect may be electronic, or the
pyridine may influence interactions of the aminomethyl group
with nearby water molecules.
bonding properties that could provide crucial SAR informa-
tion, were prepared and assayed for NOS inhibitory potency.
In general, small substituent modifications at the 4-position of
lead 12, such as fluorine (19), chlorine (20), ethyl (22), and
methoxyl (23), do not add additional good contacts in the rat
nNOS active site, resulting in a loss of potency. Compound 21
is also a less potent inhibitor given that the trifluoromethyl
group of 21 is likely too bulky to fit. However, changing the 4-
position methoxyl group (23) to an ethoxyl group (24)
restored the potency in rnNOS. The crystal structure of 24
bound to rnNOS (Figure 9A) shows strong density for the
aminoquinoline and central phenyl ring as seen in the parent
compound 12. While the aminomethyl moiety occupies the
water site between H4B and propionate A, the ethoxyl group is
large enough to establish some contacts with Met336. It seems
that as a minimum a 3-atom, nonpolar moiety at the 4-position
fits better into rnNOS.
In the hnNOS-24 structure, the central phenyl ring of 24
flips 180° relative to the orientation seen in rnNOS. The
aminomethyl moiety interacts with Asn574 (Figure 9B), and
the ethoxyl group still can reach Met341 for contact. It might
be that the polar nature of His342 pushes the ethoxyl group
away and causes the central phenyl ring to flip. The binding
mode of 24 in heNOS (Figure 9C) is almost identical to that
observed in rnNOS (Figure 9A). The better nonbonded
contacts between the ethoxyl moiety of 24 and the bulky
Phe105 side chain makes the inhibitor more ordered in
structure and leads to rather low n/e selectivity (164).
Discussion of the small-4-alkoxy-3-aminomethyl and 4-
cycloalkoxy analogues (25−28) is provided in the Supporting
Information.
Constrained Amine Analogues 16−18. See the
Supporting Information (SI) for details.
4-Alkoxy-3-aminomethyl Analogues 19−29. Docking
studies with 12 also indicated that a variety of groups might be
accommodated at position 4 of the phenyl ring (Supporting
Information Figure S1). Substituents at this position could
form favorable van der Waals interactions with Met336/
Met341 or Leu337/His342 (rnNOS/hnNOS), residues that
have previously been implicated in high n/eNOS selectivity for
2-aminoquinoline-based inhibitors,7 and which are replaced by
Val104 and Phe105 in heNOS, respectively.12 To this end,
19−29, which have a variety of steric, electronic, and H-
A further improvement for rnNOS inhibition was observed
by the addition of a small cycloalkyl group (28 and 29). The
addition of a cyclobutyl tail moiety allows 28 to form van der
Waals interactions in the Met336-Leu337-Tyr706 pocket (see
Supporting Information, Figure S5), resulting in good potency
with rnNOS (Ki (rnNOS) = 49 nM). The slightly less bulky
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Table 1. Inhibition of NOS Enzymes by Synthesized
a
Compounds 5−37
a
Ki (μM)
selectivity
compd
rnNOS
hnNOS
miNOS
heNOS
rn/mi
hn/he
1
2
3
4
5
6
7
8
0.066
0.058
0.033
0.025
0.105
0.246
0.045
0.090
0.140
0.119
0.935
0.055
0.107
1.03
0.285
0.254
0.159
0.180
0.108
0.235
1.42
0.390
0.106
0.058
0.072
0.071
0.062
0.049
0.039
0.083
0.055
0.052
0.044
0.056
0.043
0.043
0.053
0.440
0.295
0.031
0.030
0.122
28.4
27.7
6.7
4.83
21.7
11.8
7.41
5.63
5.76
23.3
431
478
203
193
207
27
25
181
192
191
0.088
0.149
16.3
18.7
362
212
9
27.5
2.86
197
24
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
0.151
0.060
41.9
52.6
277
877
24.2
440
0.111
0.058
0.066
0.072
0.096
0.046
24.2
23.9
25.5
9.66
10.4
32.8
18.2
12.9
20.2
5.65
22.8
21.0
418
333
360
156
212
841
164
223
307
78
238
457
0.114
0.076
0.045
0.106
0.031
0.063
0.046
13.5
45.7
8.18
8.31
23.7
7.73
246
879
186
73
312
172
4.32
16.4
21.0
6.20
27.3
20.0
100
382
397
200
433
435
a
The compounds were assayed for in vitro inhibition against four
Figure 4. X-ray crystal structures of 5 bound in the active sites of (A)
rnNOS, (B) hnNOS, and (C) heNOS at 1.80, 1.95, and 2.19 Å
resolution, respectively. In this and all of the following figures, the
Polder Fo − Fc omit maps are contoured at 3.5 σ for the bound
inhibitors. Major hydrogen bonds are depicted with dashed lines. Two
heme propionates are labeled as A and D. Figures were made with
PyMol.22
purified NOS isoforms: rat nNOS (rnNOS), human nNOS (hnNOS),
murine iNOS (miNOS), and human eNOS (heNOS) using known
literature methods (see the Experimental Section for details), and Ki
values are calculated directly from IC50 values. IC50 values are the
average of at least two replicates from 6−9 data points; all
experimental standard error values (for the log IC50) are less than
10%, and all correlation coefficients are good (r2 > 0.87). Selectivity
values are ratios of respective Ki values.
2-fold increase in rn/miNOS selectivity compared to 12 (29,
rn/miNOS = 841; 12, rn/miNOS = 441).
cyclopropane analogue 29 was found to have even greater
rnNOS potency [Ki (rnNOS) = 39 nM], making it the most
potent rnNOS inhibitor in the entire series. The X-ray crystal
structure of 29 bound to rnNOS (Figure 10A) reveals that the
tail cyclopropane ring fits nicely into the hydrophobic pocket
surrounded by Met336, Leu337, and Tyr706, with the side
chain of Tyr706 occupying two alternate conformations.
Propionate D is pushed by 29 into the downward
conformation. However, the orientation of the aminomethyl
group shows two alternate directions that form H-bonds with
either the H4B site water (as shown in Figure 10A) or the side
chain of Arg481 via water bridging. Compound 29 has a nearly
Not only does 29 have high inhibitory potency against
rnNOS, but it also maintains equally high potency against
hnNOS, making it a potent dual rnNOS and hnNOS inhibitor.
Similar to the binding mode found in the rnNOS-29 structure,
the central phenyl ring of 29 bound to hnNOS presses into
heme propionate D. The polar nature of His342 forces the tail
cyclopropyl group to move away from the imidazole side chain,
making contacts with Met341 instead. The binding con-
formation of the tail cyclopropyl group closely overlaps with
the tail cyclobutyl group of 28 in the hnNOS-28 structure,
suggesting that the placement of the aminomethyl substituent
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Figure 5. X-ray crystal structures of 7 bound in the active sites of (A) rnNOS and (B) hnNOS at 1.75 and 2.05 Å resolution, respectively.
weak in one chain but visible in the other, which indicates that
the central phenyl ring is flipped compared to that seen in
hnNOS-28 (see Supporting Information, Figure S5), so the
aminomethyl group of 29 points to a water near Gln483, which
is very similar to the binding conformation of the aminomethyl
moiety of 12 bound to hnNOS. The positive ammonium group
of 29 faces the water-filled pocket noted above for 12 that is
influenced by the negative charge of Asp602. In heNOS, again,
the bulky Phe105 pushes the cyclopropyl group toward heme
propionate A, allowing the aminomethyl moiety to make H-
bonds with both the propionate and H4B. The aminomethyl
group would not point to the water-filled pocket because there
is no negatively charged residue lining the pocket in heNOS.
Rather, Asn366 is part of this pocket and is very likely the
origin of the 457-fold hn/heNOS selectivity for this
compound.
4-Phenyloxymethylaryl Analogues 30−37. Thus far, it
appears that considerably larger alkoxyl substituents can be
accommodated at the 4-position. Aryl substituents are also
tolerated; for example, compound 30, with its bulky 3-
fluorobenzyl group at position 4, is only slightly less potent
than 24, although it is considerably less potent than an
aminopyridine-pyrrolidine compound (18 nM) after which it is
modeled.25 This trend is also observed with the benzonitrile
ring of 31; previously, 4-cyanoaryl compounds had good
hnNOS activity but lower hn/heNOS selectivity (generally
∼30-fold),9 but in this phenylquinoline scaffold, the hn/
heNOS selectivity is higher with the 4-cyanoaryl group present.
However, the hn/heNOS selectivity of 31 remains inferior to
leads 5, 12, and 24 because of increased binding to heNOS
(8300 nM). The unusual crystallographic binding behavior of
31 is discussed in the Supporting Information.
Given the lower hn/heNOS selectivity for 31, as well as the
disfavored binding to the human nNOS active site, we decided
to reduce the overall length of the inhibitors to better fit into
the hnNOS active site while maintaining the H-bond accepting
capability in the tail ring. The 2- and 3-pyridinylmethyl ether
groups of 32 and 33, respectively, were introduced to provide
contact with either Leu337 in rnNOS or His342 in hnNOS.
Relative to 12, both pyridinyl modifications provided slight
improvements in rnNOS potency (32, Ki = 52 nM; 33, Ki = 44
nM). The crystal structure of rnNOS-32 with compound 33
overlaid (Figure 11A) shows nearly identical binding positions
for the aminoquinoline and tail pyridine, with the pyridinyl
nitrogen facing away from Leu337 in both cases (to allow
hydrophobic interactions). The difference lies in the
orientation of the central phenyl ring. As a result, the
central-ring aminomethyl group of 33 displaces the H4B site
Figure 6. X-ray crystal structures of 12 bound in the active sites of
(A) rnNOS, (B) hnNOS, and (C) heNOS at 2.05 Å, 2.15 Å, and 2.05
Å resolution, respectively.
gives rise to the difference in hnNOS activity between 28 and
29. In hnNOS-29, the density of the aminomethyl group is
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Figure 7. X-ray crystal structures of 13 bound in the active sites of (A) rnNOS and (B) hnNOS at 1.95 and 2.40 Å resolution, respectively.
Figure 9. X-ray crystal structures of 24 bound in the active sites of
(A) rnNOS, (B) hnNOS, and (C) heNOS at 2.23, 2.10, and 2.20 Å
resolution, respectively.
Figure 8. X-ray crystal structures of 15 bound in the active sites of
(A) rnNOS, (B) hnNOS, and (C) heNOS at 2.10, 2.10, and 2.15 Å
resolution, respectively.
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Figure 11. X-ray crystal structures of 32 (yellow) bound in the active
sites of (A) rnNOS with 33 (magenta) overlaid, (B) hnNOS, and (C)
heNOS at 1.70, 1.81, and 1.95 Å resolution, respectively.
Figure 10. X-ray crystal structures of 29 bound in the active sites of
(A) rnNOS, (B) hnNOS, and (C) heNOS at 1.90, 1.95, and 1.76 Å
resolution, respectively.
water, whereas this moiety points away from the existing water
in 32.
interaction with Asn574. These deleterious interactions lead
to a decreased binding affinity of 32 to hnNOS (Ki = 76 nM).
However, by virtue of the substantially diminished binding
potency toward miNOS and heNOS, the rn/miNOS and hn/
heNOS selectivities of 32 remain quite high (rn/miNOS =
879, hn/heNOS = 312).
Compound 32 is unique in that previously reported
aminoquinolines substituted with pyridines3 generally had
only 10−20-fold n/eNOS selectivity. However, 32 exhibits
fairly good hn/heNOS selectivity (312-fold). The main
difference is that in the heNOS-32 crystal structure (Figure
11C), the orientation of the central phenyl ring is
perpendicular to the aminoquinoline ring rather than parallel
as in hnNOS-32. As a result, the amino group of 32 in heNOS
is about 2 Å farther from heme propionate D than it is in
hnNOS-32. In addition, the amino group in hnNOS-32 H-
In the hnNOS-32 structure (Figure 11B), the overall
binding remains largely the same, although the orientation of
the middle phenyl ring is different in the two nNOS cases.
Here, the ring does not press sufficiently against heme
propionate D, and the latter is not distorted as seen in the
rnNOS-32 structure. The differences might be the result of the
Leu337 (rat) and His342 (human) variation, where the bulkier
His342 side chain in human nNOS pushes the tail pyridine of
32 away so that the central phenyl ring does not make such
close contacts with heme propionate D. Because of the 2-
position of the pyridine nitrogen, a H-bond with the His342
side chain is not possible. Additionally, the position of the
aminomethyl group on the phenyl ring has some uncertainty,
although some weak electron densities support a likely
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bonds with Asn574. These interactions are possible reasons for
the good hn/heNOS selectivity.
bound to. The aminomethyl moiety of 33 still interacts with
Asn574 of hnNOS (Figure 12B), while in rnNOS-33, it makes
an electrostatic interaction with heme propionate A (Figure
12A). Both interactions are favorable, and the results taken
together may suggest a reason for equal potency against rat and
human enzymes.
In heNOS-33, the aminomethyl moiety H-bonds heme
propionate A (Figure 12C) rather than pointing out toward
Gln247 as in the case of 32 (Figure 11C). How could the
pyridinyl nitrogen atom position make such a large difference?
The pyridine ring nitrogen atom of 33 can make an extra H-
bond with a water molecule next to the H4B, which pulls the
inhibitor closer to heme propionate A and results in better
interactions. This may explain the 3-fold improvement in
binding affinity of 33 (7700 nM) versus 32 (23 700 nM)
toward heNOS, which leads to the poorer n/eNOS selectivity
observed for 33 (172-fold).
The 3-pyridylmethyl ether of 33 moderately increases
hnNOS potency (Ki = 45 nM for 33 vs 60 nM for 12). This
is not the first instance where the 3-pyridyl moiety has
improved binding to hnNOS,9,26 although it is the first
example where such a compound has equal potency for both
the rat and human enzymes. The hnNOS-33 X-ray structure
(Figure 12B) shows good density for the aminoquinoline and
The incorporation of heterocyclic H-bond acceptors leads to
favorable increases in both potency and selectivity. Utilizing
heterocycles with the dual ability to form hydrophobic
interactions with Leu337 in rnNOS while also forming H-
bonding interactions with isoform-specific His342 in hnNOS
may be advantageous for in vivo studies (where activity in rats
is necessary). Considering the importance of the heterocycle,
we also sought to exchange the bulky pyridine for smaller
heterocycles (as in analogues 34−37). These compounds all
demonstrated high potencies against rnNOS, with Ki values all
below 60 nM.
The use of isoxazole (as in 34) resulted in lost activity
against hnNOS. However, 4-(methyloxy)-1,3-thiazole (35)
resulted in a Ki of 31 nM against hnNOS, making 35 the most
potent hnNOS inhibitor in this series. Comparing the X-ray
crystal structures of rnNOS-35 (Figure 13A) and hnNOS-35
(Figure 13B) reveals a common binding orientation of the
aminoquinoline ring but with differences in the central phenyl
ring resulting from different interactions between the thiazole
ring and the enzyme. In the rnNOS structure, the thiazole is
positioned near Leu337 with its carbon atom making the
closest contact. In hnNOS-35, the thiazole is near His342, with
its S atom facing toward His342 and its N atom H-bonding
with a water molecule. The bulkier His342 in hnNOS pushes
35 slightly farther away from the heme than its position in
rnNOS. Therefore, the aminomethyl group on the central
phenyl ring in hnNOS H-bonds with Asn574, whereas the
same moiety points toward Gln478 in rnNOS, and the phenyl
ring flips almost 180°. Unfortunately, single-digit micromolar
inhibitory activity of 35 against miNOS and heNOS results in
low rn/mi and hn/heNOS selectivities relative to 32.
The use of oxazole (36) results in a slight loss in hnNOS
potency (Ki = 63 nM). However, reduced inhibitory activity
against miNOS and heNOS led to excellent selectivities (rn/
miNOS = 382, hn/heNOS = 433). Crystallographic details of
36 and 37 are discussed in the Supporting Information
(Figures S7 and S8, respectively).
Human iNOS Inhibition Study. Recently, we successfully
expressed and purified human iNOS protein21 that showed
robust activity. Human iNOS (hiNOS) assay data were
collected for eight compounds, including the three simple
aminomethyl compounds 7, 10, and 12, as well as a sampling
of the more potent compounds from the latter series (29, 32,
36, and 37) to determine the SAR for the human system. Ki
values obtained from rat and human nNOS, as well as murine
and human iNOS, were used to approximate the cross-species
Figure 12. X-ray crystal structures of 33 bound in the active sites of
(A) rnNOS, (B) hnNOS, and (C) heNOS at 1.84, 1.81, and 2.20 Å
resolution, respectively.
central phenyl rings. The tail pyridine portion shows signs of
disorder, but a potential H-bond between the pyridine and
His342 is more feasible for 33 than for 32. In the rnNOS-33
structure, however, the hydrophobic portions of the pyridine
reach the nonpolar residues Leu337 and Met336 (Figure 12A),
suggesting that the pyridine can be either a hydrophobic or a
H-bonding moiety depending on the nNOS isoform it is
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Figure 13. X-ray crystal structures of 35 bound in the active sites of (A) rnNOS and (B) hnNOS at 1.88 and 1.95 Å resolution, respectively.
selectivity between lower- (rn/mi) and higher-order species
(hn/hi) (vide infra). The major difference between murine and
human iNOS that might affect inhibitor binding is that Asn115
in miNOS is Thr121 in hiNOS. The tail part of many
inhibitors might reach the site based on observations in the
available nNOS and eNOS structures. Without an iNOS-
inhibitor structure, we can only speculate on potential
interactions by superimposing the iNOS structures onto the
known nNOS-inhibitor structures.
The general inhibition trends in Table 2 are more or less
consistent (<2-fold) between murine and human iNOS, with
Table 2. Inhibition of Rat and Human nNOS Compared to
a
Murine and Human iNOS by Selected Compounds
Ki (μM)
selectivity
compd
rnNOS
hnNOS
miNOS
hiNOS
rn/mi
hn/hi
Figure 14. Structure of hnNOS-33 (yellow) with the variant residues
of miNOS (Asn115 and Ser256, gray, PDB code 1NOD) and hiNOS
(Thr121 and Ala262, magenta, PDB code 4NOS) overlaid.
7
0.045
0.119
0.055
0.039
0.052
0.044
0.043
0.053
0.088
0.151
0.060
0.046
0.076
0.045
0.063
0.046
16.3
2.86
24.2
32.8
45.7
8.18
16.4
21.0
6.94
3.55
29.9
21.1
43.9
29.4
24.6
34.8
362
24
79
24
10
12
29
32
33
36
37
441
841
879
186
382
397
498
459
578
653
390
757
the aminomethyl moiety of 33, likely too far away to influence
inhibitor binding.
Directed Mutagenesis Supports Water-Mediated
Interaction of Inhibitors with Asp597. Previous studies
indicated that Asp597 in rnNOS electrostatically stabilizes
cationic inhibitors and can account for much of the selectivity
for nNOS over eNOW (as this residue is Asn in heNOS). A
second difference, Met336 in rnNOS (Val in heNOS), can
provide better nonpolar contacts with inhibitors and impart
selectivity. To test the importance of these differences further,
we determined the Ki values for certain compounds against
various rnNOS mutants (Table 3).
a
Compounds 7, 10, 12, 29, 32, 33, 36, and 37 were assayed for in
vitro inhibition against purified human iNOS (hiNOS) using known
literature methods, and Ki values were calculated directly from IC50
values using the Cheng−Prusoff equation. IC50 values are the average
of at least two replicates from 6−9 data points; all experimental
standard error values (for the log IC50) are less than 0.10. Ki values
for isoforms: rat nNOS (rnNOS), human nNOS (hnNOS), and
murine iNOS (miNOS) were included for comparison. Selectivity
values are ratios of respective Ki values.
All tested compounds exhibited decreased potency against
the single-mutant enzyme (D597N) (perhaps, as expected),
but against the double mutant, all compounds had potency
comparable to the single mutant (except for 12, where it was
greatly reduced). Compound 36 actually displayed 5-fold
increased potency against the double mutant (versus the single
mutant). The rnNOS-36 structure shows that the tail end of
the inhibitor contacts Met336 and Leu337. These two residues
are Val104 and Phe105 in heNOS, and contacts made with the
tail end of the inhibitor may involve more than the Met/Val
difference. It is possible that the local environment near the
Val336 and Leu337 residues in the M336V/D597N double
mutant enables better contacts with the tail end of the inhibitor
than the same area in the D597N single mutant does, thus
improving the potency against the double mutant versus the
only 7 and 33 as the principal exceptions. Compound 7 is a
compact inhibitor that would make only weak contacts with
the Asn115/Thr121 (miNOS/hiNOS) site. The twofold
difference in binding affinity might result from the variation
in a water-mediated H-bonding network or another unknown
reason. Contrarily, overlaying the structure of miNOS or
hiNOS on hnNOS-33 (Figure 14) reveals that the pyridine
nitrogen atom of 33 is capable of making a direct H-bond with
either Asn115 or Thr121. The three-fold weaker binding
affinity to hiNOS might indicate a weaker interaction with
Thr121 in hiNOS (than with Asn115 in miNOS). In the same
overlay, another variation site, Ser256/Ala262 (miNOS/
hiNOS), is more than 5.0 Å from the potential position of
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a
Table 3. Inhibition Data for Wild-Type and Mutant NOS
Enzymes by Selected Compounds (7, 10, 12, 29, 32, and
36)
Table 4. PDSP Binding Summary for Selected Compounds
compd
concerning
moderate
weak
insignificant
total
a
1
4
12
29
32
33
8
3
1
1
4
3
7
6
0
4
7
5
22
17
21
27
22
17
8
22
23
13
12
20
45
45
45
45
45
45
Ki (μM)
selectivity
WT-
D597N
rnNOS
M336V/D597N WT/ WT/
compd rnNOS heNOS
rnNOS
SM
DM
7
0.045
0.119
0.055
0.039
0.052
0.043
18.7
41.9
52.6
21.0
23.7
27.3
0.273
2.11
0.416
0.246
0.384
0.575
0.215
1.57
1.60
0.189
0.159
0.114
6
18
8
6
7
5
13
29
5
3
3
10
12
29
32
36
a
Off-target binding is classified into four categories: concerning (Ki <
100 nM, or < ∼2× nNOS Ki value), moderate (100−300 nM, or ∼2−
5× nNOS Kivalue), weak (>300 nM, or > ∼5× nNOSKi 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 28). See Supporting Information, Table S1 for PDSP
secondary binding affinities (Ki).
13
a
The compounds were assayed for in vitro inhibition against purified
NOS isoforms: rat nNOS (rnNOS), human eNOS (heNOS), as well
as single mutant (SM) (D597N) and double mutant (DM) (M336V/
D597N) of rat nNOS using known literature methods, and Ki values
were calculated directly from IC50 values using the Cheng−Prusoff
equation. IC50 values are the average of at least two replicates from 6−
9 data points; all experimental standard error values (for the log IC50)
are less than 0.10. Selectivity values are ratios of respective Ki values.
of the flagged targets for 32 and 33 are serotonin receptors,
suggesting that the heteroaryl-alkyl tails may resemble a
GPCR-ligand-like pharmacophore.29 Conversely, the off-target
profiles for 12 and 29 reveal the cleanest CNS counter-
screening observed for 2-aminoquinolines to date, flagging
only the H2 and H3 receptors as concerning for 12 and 29,
respectively, which is unsurprising as these receptors are
known to bind cationic amidine groups (e.g., ranitidine), while
29 only flagged an additional 4/45 targets for moderate
binding.30 For 12, 21/45 targets were classified as weak (27/45
for 29), while 23/45 (12) and 13/45 (29) were classified as
insignificant. These results indicate that reducing the tail
group’s size (as in 29) or eliminating it completely (as in 12)
are both effective strategies to reduce off-target CNS binding,
which may translate to improved safety in vivo.
Membrane Permeability and Microsome Stability. As
our ultimate goal is to utilize these compounds as CNS drugs,
membrane permeability for compounds 7, 12, 29, 33, and 37
was determined using the parallel artificial membrane
permeability for the blood−brain barrier (PAMPA-BBB)
assay. In this assay, an artificial membrane containing BBB
phospholipids is used to assess permeability. A compound is
predicted to have good BBB penetration and is classified as a
“CNS (+)” molecule if its effective permeability (Pe) in this
assay is larger than 4.0 × 10−6 cm s−1.31−34 Additionally, the
web tool SwissADME was used to make in silico passive-BBB
penetrability predictions based on their validated BOILED-Egg
(Brain Or IntestinaL EstimateD permeation) predictive
model.35,36 Table 5 contains Pe values of three commercial
drug standards and selected nNOS inhibitors 7, 12, 29, 33, and
37. All of the selected nNOS inhibitors are predicted “CNS
(+)” with Pe values of up to 15.5 × 10−6 cm s−1. Compounds
33 and 37 display the lowest permeability among the selected
compounds (Pe = 8.09 0.67 × 10−6 and 7.04 2.43 × 10−6
cm s−1, respectively), indicating that the presence of the
heterocyclic tail portion may reduce the permeability of these
compounds. However, while the Pe values for 33 and 37 were
high enough to score as “CNS (+)”, the BOILED-Egg
permeant model predicted that they would not have BBB
penetration because of their higher tPSA values of 87.05 and
115.29 Å2, respectively, although these two compounds, along
with all of the tested compounds, have predicted log D and
log P values that are favorable for BBB penetration. Cyclo-
propyl compound 29, which lacks polar heterocycles of 33 and
37, has a lower tPSA (74.16 Å2), a twofold higher Pe, and is
predicted to be BBB (+). Eliminating the substitution at the 4-
single mutant. This trend is reflected in the behaviors of 29,
32, and 36, all of which have a long tail moiety that fits in this
region of the enzyme. On the other hand, more compact
inhibitors might be less sensitive to the Met/Val mutation site.
We conclude by focusing on 12, since this inhibitor exhibits
the best hn/heNOS selectivity (877-fold). The D597N mutant
decreases potency 8-fold, which further drops to 29-fold
against the M336V/D597N mutant, even though this inhibitor
does not appear to make contact with the Met/Val site. It is
perhaps more instructive to consider the change in ΔGbind
obtained from the Ki values, where ΔGbind = −RT ln Ki. ΔGbind
for WT hnNOS, WT heNOS, and the hnNOS D597N mutant
are −9.9, −4.5, and −7.3 kcal/mol, respectively. Thus, the
Asp/Asn difference accounts for about half of ΔΔGbind
between heNOS and hnNOS, leaving about −2.6 kcal/mol
of binding affinity unexplained. This underscores the limitation
of quantitatively explaining selectivity (against isoforms or
mutants) by a few simple amino acid differences and a
comparison of static X-ray structures. There are clear examples
of differences in active-site dynamics and the ability of even
conserved side chains to adjust to inhibitor binding that could
also contribute to selectivity (anchored plasticity) in cases like
this.27
Off-Target Profiling. Four structurally diverse compounds
from this series (12, 29, 32, and 33) were screened by the
National Institute of Mental Health’s Psychoactive Drug
Screening Program (PDSP, Table 4). In this assay,28
compounds were screened against a panel of 45 pharmaco-
logically relevant CNS targets and receptors using a radioligand
displacement assay. Initially, the assay used a primary high
dose (10 μM) and then a secondary Ki determination was
performed for compounds showing >50% binding in the
primary assay. We classify off-target binding using the
following rubric: concerning (Ki < 100 nM, or <∼2 × nNOS
Ki value), moderate (100−300 nM, or ∼2−5 × nNOS Ki
value), weak (>300 nM, or> ∼5 × nNOS Ki value, typically ∼1
μM), and insignificant (<50% at 10 μM). The off-target
profiles of previous aminoquinolines 1 and 4 have been
included for comparison. Although not as effective as 4, a slight
decrease in the fraction of concerning or moderate hits for 32
is observed (11/45 for 32 compared to 15/45 for 1), while this
fraction decreases further to 8/45 for 33. Unfortunately, most
O
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a
Table 5. Effective Permeability (Pe) of Five Commercial Drugs and nNOS Inhibitors in the PAMPA−BBB Assay
b
b
c
d
e
c
compd
log D
log P
TPSA (Å2)
63.95
reported Pe (10−6 cm s−1
)
determined Pe (10−6 cm s−1
)
BBB permeant prediction
BBB (+)
prediction
CNS (+)
f
verapamil
2.29
2.76
4.55
4.56
0.03
16
21.3 1.5
18.5 1.9
8.04 0.41
f
chlorpromazine
dopamine
31.78
66.48
6.5
0.2
BBB (+)
CNS (+)
8.90 0.68
0.12 0.41
f
−1.50
BBB (−)
CNS (−)
0.125 0.14
11.3 1.64
15.5 2.32
14.6 0.97
8.09 0.67
7.04 2.43
7
1.36
1.27
2.37
2.13
1.94
3.32
3.18
3.78
3.53
3.35
64.93
64.93
74.16
87.05
115.29
BBB (+)
BBB (+)
BBB (+)
BBB (−)
BBB (−)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
CNS (+)
12
29
33
37
a
b
All assays were performed over 17 h at a concentration of 200 μM; see the Experimental Section for details. log D (pH = 7.4) and log P values of
c
the free-base species were predicted using ChemAxon software. TPSA calculations and BBB permeation were predicted using the free web tool
SwissADME. Effective permeability values from the literature.32 Effective permeability values obtained in-house. Experimental Pe values reported
d
e
f
previously by Do et al.37
position gave mixed results. Although not as high as 29,
compound 7 maintained a high Pe value (11.3 1.64 × 10−6
cm s−1) relative to 33 and 37, although compound 12 was
found to have the highest Pe out of all compounds assayed.
Additional cellular pharmacokinetic assays are in progress.
Additionally, stability in the presence of human liver
microsomes (HLMs) was determined for 12 and positive
control terfenadine. The results of this study (Table 6) show
specific residues on the far end of the substrate access channel,
namely, Leu337/His342 (rnNOS/hnNOS). Crystal structures
indicate that these compounds act as competitive arginine
mimics, where the aminoquinoline forms hydrogen bonds with
the active-site glutamate residue, but also that substitutions at
the 4-position can reduce phenyl ring rotation to favor
interactions with the Asp (Asp597/Asp602)/H4B/propionate
A site over the propionate D site. Mutagenesis studies
confirmed the influence of the Asp site on inhibitor potency,
making this class of aminoquinolines the first with the ability to
target this site in nNOS, although preference for this site varies
from compound to compound. Finally, with these inhibitors,
the hn/heNOS selectivity derives more from a weakening of
affinity to heNOS rather than from an increase in affinity for
hnNOS; inhibitors more complex than 12 tend to be less
selective because of an increased affinity for heNOS.
Apparently, more complex substituents provide additional
contacts that improve binding to heNOS relative to hnNOS.
On the basis of both their good potency and selectivity, clean
CNS counterscreening (PDSP) profiles, and excellent
permeability in our PAMPA-BBB assay, the most promising
compounds, 12 and 29, are being advanced into preclinical
studies.
Table 6. Metabolic Stability of 12 and Positive Control in
a
Human Liver Microsome
% remaining at % remaining
60 min
at 60 min
t1/2
CLint
b
c
compd
(−NADPH)
(buffer)
(min) (mL/(min kg)
12
terfenadine
87
101
67
100
>60
23
8
108
d
a
All assays were performed in 50 mM Kphos buffer (pH 7.4)
containing HLM (0.714 mg/mL) over 60 min at 1.428 μM drug
concentration. Parent compound peak disappearance was monitored
by liquid chromatography−mass spectrometry (LC−MS)/MS; see
b
c
Experimental Section for details.
t1/2: half-life. CLint:: in vitro
d
intrinsic clearance. Positive control.
that 12 displays high stability relative to the positive control, as
indicated by a half-life (t1/2) > 60 min. However, 12 did show a
sign of degradation in buffer control samples (Supporting
Information, Table S3).
EXPERIMENTAL SECTION
■
General Procedures. Anhydrous solvents (THF, CH2Cl2,
MeOH, Et3N, and DMF) were distilled prior to use. All other
solvents, reactants, and reagents were purchased from commercial
vendors and were used without further purification. Methanolic HCl
(3 M, for ammonium hydrochloride salt formation and Boc-
deprotection) was prepared fresh by the reaction of acetyl chloride
and anhydrous MeOH at 0 °C. Melting points were determined in
capillary tubes using a Buchi melting point B-540 apparatus and are
CONCLUSIONS
■
In summary, we prepared a series of novel 7-phenyl-2-
aminoquinolines possessing tail amines designed to target
nNOS-specific aspartate residues Asp597/Asp602 (rnNOS/
hnNOS) and thereby result in higher n/eNOS selectivity.
Initially, screening compounds 5−13 revealed a preference for
meta-substituted benzylamines, such as 12, which shows
excellent potency and outstanding selectivity for nNOS over
the other isozymes. A number of modifications to 12 were
made, which included reducing or constraining amino group
flexibility, substituting the 4-position of the phenyl ring, and
using a 1,3,5-tri-substituted phenyl core. While the amine
cannot be constrained effectively and 1,3,5-tri-substitutions
clash deleteriously with heme propionates in the enzyme, some
4-position additions enhance potency and maintain high
isoform selectivity via favorable interactions with isoform-
1
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. High-resolution
mass spectral (HRMS) data were obtained at the Integrated
Molecular Structure Education and Research Center (IMSERC,
Northwestern University) on an Agilent 6210A TOF mass
spectrometer in the positive ion mode coupled to an Agilent 1200
series high-performance liquid chromatography (HPLC) system. Data
were processed using MassHunter software version B.04.00. Flash
column chromatography was performed using an Agilent 971-FP
automated flash purification system with a Varian column station and
SiliCycle cartridges (12−80 g, both normal and high performance).
Analytical HPLC was performed using an Agilent Infinity 1260 HPLC
P
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system with injection volumes of 5−10 μL. A Phenomenex Luna 5
μm C-8(2) 100 Å column, 50 mm × 4.60 mm, was used for all HPLC
experiments, using a 10 min gradient of 95% H2O/5% acetonitrile +
0.05% trifluoroacetic acid (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. Analytical thin-layer chromatog-
raphy was performed on Silicycle extra-hard 250 μm TLC plates.
Compounds were visualized with short-wavelength UV light, and with
ninhydrin and CAM stains, where appropriate. The preparation of
quinoline precursors and assembly of final compounds have been
described below, while the preparation of other precursors has been
discussed in the Supporting Information. Compounds 38,8 41,38 43,39
45,40 52,41 53,42 56,43 57,8 66,44 69,45 70,15 86,46 89,47 90,40 and
9648 were prepared by literature procedures or are known, and their
spectral or analytical data are identical to those reported.
General Procedure 1: Suzuki Coupling between 39 and Aryl
Halides. Compound 39 (1 equiv), the requisite aryl halide (1−1.2
equiv), Pd(dppf)Cl2 (5 mol %), and NaHCO3 (3.5−4 equiv) were
combined in dimethoxyethane/H2O (3:1, 4 mL of solvent per 0.25
mmol 39 is sufficient) in a 20 mL sealable microwave vial. The
mixture was briefly sparged with argon, sealed, and heated to 120 °C
under microwave irradiation with stirring (720 rpm) for 20−25 min.
After cooling, the mixture was partitioned between EtOAc and H2O
(10 mL each), and the layers were separated, and the aqueous layer
was extracted with EtOAc (3 × 30 mL), and the organic layer was
washed with 5% aq. NaCl (2 × 50 mL) and sat. aq. NaCl (50 mL),
dried over anhydrous sodium sulfate, and concentrated. The crude
residue was purified as listed below under subheadings for individual
compounds.
General Procedure 2: Deprotection of Aminoquinolines.
The protected intermediate was immediately diluted with MeOH (9−
10 mL/0.2 mmol of protected quinoline), and K2CO3 (2 equiv) was
added. The mixture was heated at reflux for 2−2.5 h, cooled, and
concentrated, and the residue was partitioned between EtOAc (10
mL) and H2O/sat. aq. NaCl (1:1, 10 mL). The layers were separated,
the aqueous phase was extracted with EtOAc (3 × 20 mL), and the
organic layers were combined, washed with sat. aq. NaCl (20 mL),
dried over anhydrous sodium sulfate, and concentrated. Purification, if
necessary, is described below under subheadings for individual
compounds. The resulting free 2-aminoquinoline was dissolved in
MeOH or ether/MeOH (10−15 mL), filtered to remove particulate
matter, and treated with methanolic HCl (∼3 M, ∼1.5−2 mL). The
mixture was stirred overnight at r.t., and workup (as described below)
afforded the deprotected compounds as hydrochloride salts.
was immediately deprotected using General Procedure 2. After
workup, the free aminoquinoline was passed through a short SiO2
plug, eluting with 1% MeOH in EtOAc. The filtrate was concentrated,
washed with hexanes, and diluted in ether/MeOH (8:1, 20 mL). After
treatment with methanolic HCl, 6 was obtained as a flocculent cream-
colored solid (0.057 g, 76% from 58) after filtering, precipitating from
MeOH (1 mL) with ether (5 mL), triturating with ether, and drying
in vacuo: mp 307−308.3 °C (dec). 1H NMR (500 MHz; DMSO-d6):
δ 14.09 (s, 1H), 8.86−8.85 (m, 2H), 8.06 (d, J = 8.5 Hz, 1H), 7.94 (d,
J = 1.7 Hz, 1H), 7.82 (dd, J = 8.5, 1.7 Hz, 1H), 7.77−7.75 (m, 2H),
7.46 (d, J = 8.3 Hz, 2H), 6.93 (d, J = 1.0 Hz, 1H), 3.22−3.16 (m,
2H), 3.02 (t, J = 8.0 Hz, 2H), 2.66 (d, J = 0.9 Hz, 3H), 2.59 (t, J = 5.1
Hz, 3H); the aminoquinoline −NH protons are mostly broadened
into the baseline at 8.1 and 8.9 ppm; 13C NMR (126 MHz; DMSO-
d6): δ 154.33, 152.50, 143.75, 138.36, 137.40, 136.98, 130.14 (2C),
127.79 (2C), 126.66, 123.84, 120.89, 115.42, 112.93, 49.36, 32.93,
+
31.57, 19.41; HRMS calcd for C19H22N3 : 292.1808; found, 292.1822.
7-(4-(Aminomethyl)phenyl)-4-methylquinolin-2-amine Dihydro-
chloride (7). Compounds 39 (0.075 g, 0.245 mmol) and 45 (0.082 g,
0.245 mmol) were coupled using General Procedure 1. Purification by
flash column chromatography, eluting with a gradient of 5% EtOAc in
CH2Cl2 to 40% EtOAc in CH2Cl2, afforded protected phenylquino-
line 59 as a yellow solid (0.062 g, 63%). This compound was
immediately deprotected using General Procedure 2. After workup,
the free aminoquinoline was precipitated from EtOAc (1 mL) with
hexanes (9 mL), triturated with hexanes, and diluted in ether/MeOH
(5:1). After treatment with methanolic HCl, 7 was obtained as a
cream-colored solid (0.043 g, 84% from 59) after filtering,
precipitating twice from hot MeOH (2 mL) with ether (10 mL),
washing with ether, and drying in vacuo: mp > 300 °C (darkens
slowly), >360 °C (melts or decomposes). 1H NMR (500 MHz;
DMSO-d6): δ 14.10 (s, 1H), 9.06 (br s, 1H), 8.43 (s, 3H), 8.30 (br s,
1H), 8.09 (d, J = 8.5 Hz, 1H), 7.98 (d, J = 1.7 Hz, 1H), 7.86−7.84
(m, 3H), 7.67 (d, J = 8.4 Hz, 2H), 6.95 (d, J = 1.0 Hz, 1H), 4.13−
4.10 (m, 2H), 2.67 (d, J = 0.9 Hz, 3H); 13C NMR (126 MHz;
DMSO-d6): δ 154.3, 152.6, 143.5, 138.9, 136.7, 135.1, 130.3 (2C),
127.7 (2C), 126.7, 124.0, 121.0, 115.5, 113.1, 42.3, 19.4; HRMS calcd
+
for C17H18N3 : 264.1495; found, 264.1503.
4-Methyl-7-(4-((methylamino)methyl)phenyl)quinolin-2-amine
Dihydrochloride (8). Compounds 39 (0.075 g, 0.245 mmol) and 46
(0.085 g, 0.245 mmol) were coupled using General Procedure 1.
Purification by flash column chromatography, eluting with a gradient
of 5% EtOAc in CH2Cl2 to 40% EtOAc in CH2Cl2, afforded protected
phenylquinoline 60 as a yellow solid (0.072 g, 70%). This compound
was immediately deprotected using General Procedure 2. After
workup, the free aminoquinoline was precipitated from EtOAc (1
mL) with hexanes (9 mL). The solid was collected and diluted in
ether/MeOH (3:1). After treatment with methanolic HCl, ether (10
mL) was added, and 8 was obtained as a cream-colored solid (0.047 g,
78% from 60) after filtering, precipitating from hot MeOH (2 mL)
7-(4-(2-Aminoethyl)phenyl)-4-methylquinolin-2-amine Dihydro-
chloride (5). Compounds 39 (0.050 g, 0.163 mmol) and 41 (0.050 g,
0.167 mmol) were coupled using General Procedure 1. Purification by
flash column chromatography, eluting with a gradient of 5% EtOAc in
CH2Cl2 to 30% EtOAc in CH2Cl2, afforded protected phenylquino-
line 42 as a white solid (0.052 g, 76%). This compound was
immediately deprotected using General Procedure 2. After workup,
the free aminoquinoline was diluted in ether/MeOH (1:1, 20 mL),
heated gently to affect solution, filtered, and cooled. After treatment
with methanolic HCl, 5 was obtained as a cream-colored solid (0.037
1
with ether (10 mL), washing with ether, and drying in vacuo. H
NMR (500 MHz; DMSO-d6): δ 14.06 (s, 1H), 9.20 (s, 2H), 9.00 (br
s, 1H), 8.20 (br s, 1H), 8.08 (d, J = 8.5 Hz, 1H), 7.97 (d, J = 1.7 Hz,
1H), 7.86−7.82 (m, 2.0 Hz, 3H), 7.70 (d, J = 8.3 Hz, 2H), 6.94 (d, J
= 1.0 Hz, 1H), 4.21−4.19 (m 2H), 2.66 (d, J = 0.9 Hz, 3H), 2.58−
2.56 (m, 3H); 13C NMR (126 MHz; DMSO-d6): δ 154.3, 152.6,
143.3, 139.4, 136.8, 133.1, 131.3 (2C), 127.8 (2C), 126.7, 124.0,
1
g, 86% from 42) after triturating with ether and drying in vacuo: H
NMR (500 MHz; DMSO-d6): δ 14.09 (s, 1H), 8.07−7.94 (m, 5H),
7.81 (dd, J = 8.5, 1.6 Hz, 1H), 7.75 (d, J = 8.2 Hz, 2H), 7.46 (d, J =
8.2 Hz, 2H), 6.93 (s, 1H), 3.12−3.08 (m, 2H), 2.96 (t, J = 7.8 Hz,
2H), 2.65 (s, 3H); the aminoquinoline −NH protons are mostly
broadened into the baseline at 8.1 and 8.9 ppm; 13C NMR (126
MHz; DMSO-d6): δ 154.3, 152.6, 143.8, 138.6, 137.4, 136.9, 130.2
(2C), 127.8 (2C), 126.7, 123.9, 120.9, 115.4, 112.9, 33.1, 19.4; one of
the aliphatic carbon signals is obscured by the solvent peak; HRMS
+
121.1, 115.6, 113.1, 51.2, 32.6, 19.4; HRMS calcd for C18H20N3 :
278.1652; found, 278.1664.
(S)-7-(4-(2-Aminopropyl)phenyl)-4-methylquinolin-2-amine Di-
hydrochloride (9). Compounds 39 (0.075 g, 0.245 mmol) and 49
(0.077 g, 0.245 mmol) were coupled using General Procedure 1.
Purification by flash column chromatography, eluting with a gradient
of 5% EtOAc in CH2Cl2 to 35% EtOAc in CH2Cl2, afforded protected
phenylquinoline 61 as a pale-yellow foam (0.092 g, 87%). This
compound was immediately deprotected using General Procedure 2.
After workup, the free aminoquinoline was washed with 10:1 hexane/
CH2Cl2 (10 mL). The solid was collected and diluted in ether/
MeOH (3:1). After treatment with methanolic HCl, the mixture was
concentrated, and the residue was azeotroped with toluene twice.
+
calcd for C18H20N3 : 278.1652; found, 278.1661.
4-Methyl-7-(4-(2-(methylamino)ethyl)phenyl)quinolin-2-amine
Dihydrochloride (6). Compounds 39 (0.075 g, 0.245 mmol) and 43
(0.079 g, 0.251 mmol) were coupled using General Procedure 1.
Purification by flash column chromatography, eluting with a gradient
of 5% EtOAc in CH2Cl2 to 30% EtOAc in CH2Cl2, afforded protected
phenylquinoline 58 as a yellow foam (0.070 g, 70%). This compound
Q
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After precipitation of the residue from MeOH (1 mL) with ether (10
mL), 9 was obtained as a tan hygroscopic powder (0.058, 75% from
61) after washing with ether and drying in vacuo: mp > 260 °C
8.11 (d, J = 8.5 Hz, 1H), 7.95−7.93 (m, 2H), 7.84 (dd, J = 8.4, 1.2
Hz, 1H), 7.78 (d, J = 7.6 Hz, 1H), 7.62−7.59 (m, 2H), 6.94 (s, 1H),
4.15 (q, J = 5.3 Hz, 2H), 2.67 (s, 3H); 13C NMR (126 MHz; DMSO-
d6): δ 154.4, 143.7, 139.2, 135.6, 130.1, 129.7, 128.3, 127.6, 126.8,
124.0, 121.1, 115.5, 113.2, 42.7, 19.4; two of the quinoline carbons are
not visible due to baseline broadening; HRMS calcd for C17H18N3 :
264.1495; found, 264.1504.
1
(darkens slowly), 281−282 °C (dec). H NMR (500 MHz; DMSO-
d6): δ 14.27 (s, 1H), 9.00 (br s, 1H), 8.20 (br s, 1H), 8.14−8.13 (m,
3H), 8.06 (d, J = 8.5 Hz, 1H), 7.96 (d, J = 1.5 Hz, 1H), 7.82 (dd, J =
8.5, 1.5 Hz, 1H), 7.76 (d, J = 8.2 Hz, 2H), 7.45 (d, J = 8.2 Hz, 2H),
6.94 (s, 1H), 3.50−3.45 (m, 1H), 3.10 (dd, J = 13.4, 5.3 Hz, 1H),
2.78 (dd, J = 13.4, 8.8 Hz, 1H), 2.66 (s, 3H), 1.17 (d, J = 6.5 Hz, 3H).
13C NMR (126 MHz; DMSO-d6): δ 154.3, 152.5, 143.8, 138.1, 137.3,
136.8, 130.7 (2C), 127.7 (2C), 126.7, 123.9, 120.8, 115.3, 112.9, 48.4,
19.4, 18.2; one of the aliphatic carbons is obscured by the solvent
+
7-(3-(2-Aminoethyl)phenyl)-4-methylquinolin-2-amine Dihydro-
chloride (13). Compounds 39 (0.075 g, 0.245 mmol) and 57 (0.077
g, 0.257 mmol) were coupled using General Procedure 1. Purification
by flash column chromatography, eluting with a gradient of 5% EtOAc
in CH2Cl2 to 45% EtOAc in CH2Cl2, afforded protected phenyl-
quinoline 65 as a yellow syrup (0.090 g, 88%). This compound was
immediately deprotected using General Procedure 2. After workup,
the free aminoquinoline was diluted in ether/MeOH (5:1), and after
treatment with methanolic HCl, ether (5 mL) was added and 13 was
obtained as a tan hygroscopic semisolid (0.053 g, 71% from 65) after
filtering, precipitating twice from hot MeOH (2 mL) with ether (10
+
peak; HRMS calcd for C19H22N3 : 292.1808; found, 292.1818.
7-(2-(Aminomethyl)phenyl)-4-methylquinolin-2-amine Dihydro-
chloride (10). Compounds 39 (0.075 g, 0.245 mmol) and 52 (0.070
g, 0.245 mmol) were coupled using General Procedure 1. Purification
by flash column chromatography, eluting with a gradient of 5% EtOAc
in CH2Cl2 to 35% EtOAc in CH2Cl2, afforded protected phenyl-
quinoline 62 as pale-yellow crystals (0.098 g, 99%). This compound
was immediately deprotected using General Procedure 2. After
workup, the free aminoquinoline was diluted in ether/MeOH (5:1).
After treatment with methanolic HCl, the mixture was concentrated,
and 10 was obtained as a white solid (0.070 g, 86% from 62) after
precipitating twice from hot MeOH (1 mL) with ether (10 mL),
washing with ether, and drying in vacuo: mp 290−291.5 °C. 1H NMR
(500 MHz; DMSO-d6): δ 14.21 (s, 1H), 9.00 (br s, 1H), 8.42 (s, 3H),
8.30 (br s, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.74 (d, J = 7.6 Hz, 1H),
7.67 (s, 1H), 7.59−7.51 (m, 3H), 7.39−7.38 (m, 1H), 6.97 (s, 1H),
3.97 (br d, J = 4.0 Hz, 2H), 2.67 (s, 3H); 13C NMR (126 MHz;
DMSO-d6): δ 154.4, 143.7, 140.4, 132.0, 130.5, 129.29, 129.18,
129.09, 126.4, 126.0, 120.9, 118.7, 113.3, 19.5; two of the quinoline
carbons are not visible due to baseline broadening; one of the
aliphatic carbon signals is obscured by the solvent peak; HRMS calcd
1
mL), washing with ether, and drying in vacuo: H NMR (500 MHz;
DMSO-d6): δ 14.17 (s, 1H), 9.00 (br s, 1H), 8.08−8.06 (m, 5H),
7.95 (d, J = 1.4 Hz, 1H), 7.84 (dd, J = 8.5, 1.5 Hz, 1H), 7.66−7.64
(m, 2H), 7.53 (t, J = 7.8 Hz, 1H), 7.39 (d, J = 7.6 Hz, 1H), 6.94 (s,
1H), 3.17−3.09 (m, 2H), 3.01 (t, J = 7.8 Hz, 2H), 2.66 (s, 3H); 13C
NMR (126 MHz; DMSO-d6): δ 154.3, 152.6, 144.1, 139.2, 139.0,
136.7, 130.1, 129.6, 128.0, 126.6, 126.1, 124.1, 120.9, 115.5, 113.0,
+
33.5, 19.4; HRMS calcd for C18H20N3 : 278.1652; found, 278.1662.
3-(2-Amino-4-methylquinolin-7-yl)-5-(aminomethyl)benzonitrile
Dihydrochloride (14). Compounds 39 (0.075 g, 0.245 mmol) and 67
(0.083 g, 0.269 mmol) were coupled using General Procedure 1.
Purification by flash column chromatography, eluting with a gradient
of 5% EtOAc in CH2Cl2 to 35% EtOAc in CH2Cl2, afforded protected
phenylquinoline 68 as a yellow foam (0.082 g, 78%). This compound
was immediately deprotected using General Procedure 2. After
workup, the free aminoquinoline was purified by flash column
chromatography, eluting with a gradient of EtOAc to 5% MeOH in
EtOAc to yield a white semisolid that was diluted in ether/MeOH
(10:1). After treatment with methanolic HCl, ether (5 mL) was
added, and 14 was obtained as a cream-colored solid (0.069 g, 68%
from 68) after filtering, precipitating from hot MeOH (2 mL) with
ether (10 mL), washing with ether, and drying in vacuo: mp > 320 °C
+
for C17H18N3 : 264.1495; found, 264.1503.
7-(2-(2-Aminoethyl)phenyl)-4-methylquinolin-2-amine Dihydro-
chloride (11). Compounds 39 (0.075 g, 0.245 mmol) and 53 (0.077
g, 0.257 mmol) were coupled using General Procedure 1. Purification
by flash column chromatography, eluting with a gradient of 5% EtOAc
in CH2Cl2 to 35% EtOAc in CH2Cl2, afforded protected phenyl-
quinoline 63 as pale-yellow crystals (0.093 g, 90%). This compound
was immediately deprotected using General Procedure 2. After
workup, the free aminoquinoline was sonicated with hexanes, and the
solid was collected and diluted in ether/MeOH (2:1). After treatment
with methanolic HCl, the mixture was concentrated, and 11 was
obtained as an off-white powder (0.064 g, 83% from 63) after
precipitating twice from hot MeOH (2 mL) with ether (10 mL),
washing with ether, and drying in vacuo: mp > 300 °C (darkens
slowly), 323−325 °C (dec). 1H NMR (500 MHz; DMSO-d6): δ
14.37 (s, 1H), 8.10 (br s, 1H), 8.10 (br s, 1H), 8.07 (d, J = 8.4 Hz,
1H), 7.92 (s, 3H), 7.68 (d, J = 1.0 Hz, 1H), 7.49−7.39 (m, 4H), 7.30
(d, J = 7.4 Hz, 1H), 6.98 (s, 1H), 2.89−2.86 (m, 4H), 2.67 (s, 3H);
13C NMR (126 MHz; DMSO-d6): δ 154.2, 152.6, 144.9, 140.5, 136.1,
1
(darkens) >360 °C (melts or decomposes). H NMR (500 MHz;
DMSO-d6): δ 14.15 (s, 1H), 9.10 (br s, 1H), 8.63 (s, 3H), 8.40 (br s,
1H), 8.34 (s, 1H), 8.29 (s, 1H), 8.14 (d, J = 8.5 Hz, 1H), 8.08 (s,
1H), 7.99 (d, J = 1.1 Hz, 1H), 7.91 (dd, J = 8.5, 1.2 Hz, 1H), 6.99 (s,
1H), 4.21 (s, 2H), 2.67 (s, 3H). 13C NMR (126 MHz; DMSO-d6): δ
154.6, 152.5, 141.6, 140.2, 137.2, 136.6, 133.36, 133.19, 131.1, 127.0,
124.1, 121.6, 118.8, 115.9, 113.6, 112.8, 41.9, 19.5; HRMS calcd for
+
C18H17N4 : 289.1448; found, 289.1459.
7-(5-(Aminomethyl)pyridin-3-yl)-4-methylquinolin-2-amine Tri-
hydrochloride (15). Compounds 39 (0.075 g, 0.245 mmol) and 70
(0.114 g, 0.294 mmol) were coupled using General Procedure 1.
Purification by flash column chromatography, eluting with a gradient
of 5% EtOAc in CH2Cl2 to 70% EtOAc in CH2Cl2, afforded protected
phenylquinoline 71 as a white solid (0.080 g, 65%). 1H NMR
confirmed the presence of two Boc groups. This compound was
immediately deprotected using General Procedure 2, and after 3 h, an
additional equivalent of K2CO3 was added to remove one of the Boc
groups. The mixture was heated for a total of 4.5 h, and after workup,
the free aminoquinoline was precipitated from EtOAc (0.5 mL) with
hexanes (5 mL) and diluted in ether/MeOH (1:1). After treatment
with methanolic HCl, ether (20 mL) was added and 15 was obtained
as a tan solid (0.053 g, 90% from 71) after filtering, precipitating from
hot MeOH (5 mL) with ether (10 mL), washing with ether, and
drying in vacuo: mp 290−292 °C. 1H NMR (500 MHz; DMSO-d6): δ
14.18 (s, 1H), 9.03 (d, J = 2.0 Hz, 1H), 8.81 (d, J = 1.6 Hz, 1H), 8.62
(s, 3H), 8.52 (s, 1H), 8.17 (d, J = 8.5 Hz, 1H), 8.01 (d, J = 1.5 Hz,
1H), 7.91 (dd, J = 8.5, 1.7 Hz, 1H), 6.99 (s, 1H), 4.22 (q, J = 5.7 Hz,
2H), 2.68 (s, 3H); the pyridinium −NH proton is broadened into
residual water around 4 ppm; 13C NMR (126 MHz; DMSO-d6): δ
154.5, 152.7, 149.8, 147.5, 140.4, 137.0, 136.5, 134.4, 131.1, 127.2,
135.1, 130.40, 130.22, 129.1, 127.6, 126.4, 126.1, 120.7, 118.1, 113.1,
30.7, 19.5; one of the aliphatic carbon signals is obscured by the
+
solvent peak; HRMS calcd for C18H20N3 : 278.1652; found,
278.1664.
7-(3-(2-Aminoethyl)phenyl)-4-methylquinolin-2-amine Dihydro-
chloride (12). Compounds 39 (0.075 g, 0.245 mmol) and 56 (0.070
g, 0.245 mmol) were coupled using General Procedure 1. Purification
by flash column chromatography, eluting with a gradient of 5% EtOAc
in CH2Cl2 to 40% EtOAc in CH2Cl2, afforded protected phenyl-
quinoline 64 as a yellow foam (0.089 g, 90%). This compound was
immediately deprotected using General Procedure 2. After workup,
the free aminoquinoline was precipitated from EtOAc (2 mL) with
hexanes (20 mL) and diluted in ether/MeOH (5:1). After treatment
with methanolic HCl, 12 was obtained as a cream-colored flocculent
solid (0.053 g, 72% from 64) after filtering, precipitating twice from
hot MeOH (2 mL) with ether (10 mL), washing with ether, and
1
drying in vacuo: mp 305.5−306.5 °C. H NMR (500 MHz; DMSO-
d6): δ 14.01 (s, 1H), 9.00 (br s, 1H), 8.41 (s, 3H), 8.25 (br s, 1H),
R
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124.1, 121.5, 115.7, 113.6, 19.5; one of the aliphatic carbon signals is
obscured by the solvent peak; HRMS calcd for C16H17N4 : 265.1459;
126.9, 126.2, 124.3, 123.9, 120.9, 115.2, 113.0, 55.1, 31.0, 30.2, 19.4;
HRMS calcd for C19H20N3 : 290.1652; found, 290.1664.
+
+
7-(3-(Aminomethyl)-4-fluorophenyl)-4-methylquinolin-2-amine
Dihydrochloride (19). Compounds 39 (0.075 g, 0.245 mmol) and 87
(0.099 g, 0.282 mmol) were coupled using General Procedure 1.
Purification by flash column chromatography, eluting with a gradient
of 5% EtOAc in CH2Cl2 to 35% EtOAc in CH2Cl2, afforded protected
phenylquinoline 117 as a dark-yellow foam (0.088 g, 85%). This
compound was immediately deprotected using General Procedure 2.
After workup, the free aminoquinoline was diluted in ether/MeOH
(5:1). After treatment with methanolic HCl, 19 was obtained as a
cream-colored flocculent solid (0.060 g, 81% from 117) after
precipitating from hot MeOH (1 mL) with ether (10 mL), washing
found, 265.1464.
7-(Isoindolin-4-yl)-4-methylquinolin-2-amine Dihydrochloride
(16). Compounds 39 (0.075 g, 0.245 mmol) and 73 (0.077 g,
0.257 mmol) were coupled using General Procedure 1. Purification by
flash column chromatography, eluting with a gradient of 5% EtOAc in
CH2Cl2 to 35% EtOAc in CH2Cl2, afforded protected phenylquino-
line 74 as a yellow gum (0.093 g, 91%). This compound was
immediately deprotected using General Procedure 2. After workup,
the free aminoquinoline was purified by flash column chromatog-
raphy, eluting with EtOAc to yield a pale-yellow solid. The solid was
diluted in ether/MeOH (2:1). After treatment with methanolic HCl,
the mixture was concentrated, and the residue was diluted in MeOH
(3 mL) and filtered through a small pad of Celite. Reconcentration
afforded a residue that was then precipitated three times from hot
MeOH (3 mL) with ether (10 mL) to yield 16 as a pale-tan solid
(0.040 g, 52% from 74) after washing with ether and drying in vacuo:
mp 220−224 °C (softens), 233−235 °C (dec). 1H NMR (500 MHz;
DMSO-d6): δ 13.98 (s, 1H), 9.87 (s, 2H), 9.00 (br s, 1H), 8.20 (br s,
1H), 8.09 (d, J = 8.5 Hz, 1H), 7.78 (s, 1H), 7.63 (d, J = 8.3 Hz, 1H),
7.59−7.53 (m, 3H), 6.95 (s, 1H), 4.64 (s, 2H), 4.61 (s, 2H), 2.66 (s,
3H); 13C NMR (126 MHz; DMSO-d6): δ 154.4, 152.3, 142.5, 137.1,
136.8, 135.5, 133.5, 129.9, 128.9, 126.6, 124.9, 123.7, 121.1, 117.2,
1
with ether, and drying in vacuo: mp 300−302 °C (dec). H NMR
(500 MHz; DMSO-d6): δ 14.08 (s, 1H), 9.05 (br s, 1H), 8.52 (s, 3H),
8.25 (br s, 1H), 8.12−8.08 (m, 2H), 7.92 (d, J = 1.4 Hz, 1H), 7.86−
7.82 (m, 2H), 7.49 (t, J = 9.2 Hz, 1H), 6.95 (s, 1H), 4.18 (br d, J =
3.2 Hz, 2H), 2.67 (s, 3H); 13C NMR (126 MHz; DMSO-d6): δ
(162.1 + 160.1, 1C), 154.5, 152.6, 142.8, 136.6, (135.34 + 135.31,
1C), (130.90 + 130.87, 1C), (129.99 + 129.92, 1C), 126.9, 124.0,
(122.47 + 122.35, 1C), (117.05 + 116.87, 1C), 115.3, 113.2, (36.17 +
+
36.14, 1C), 19.5; HRMS calcd for C17H17FN3 : 282.1401; found,
282.1408.
7-(3-(Aminomethyl)-4-chlorophenyl)-4-methylquinolin-2-amine
Dihydrochloride (20). Compounds 39 (0.075 g, 0.245 mmol) and 88
(0.105 g, 0.282 mmol) were coupled using General Procedure 1.
Purification by flash column chromatography, eluting with a gradient
of 5% EtOAc in CH2Cl2 to 40% EtOAc in CH2Cl2, afforded protected
phenylquinoline 118 as a yellow foam (0.095 g, 88%). This
compound was immediately deprotected using General Procedure
2. After workup, the free aminoquinoline was diluted in ether/MeOH
(5:1). After treatment with methanolic HCl, 20 was obtained as a
cream-colored powder (0.057 g, 71% from 118) after precipitating
four times from hot MeOH (2 mL) with ether (15 mL), washing with
ether, and drying in vacuo: mp 249−250 °C (softens slightly), 255−
+
113.4, 50.5, 50.2, 19.4; HRMS calcd for C18H18N3 : 276.1495; found,
276.1508.
7-(1-Amino-2,3-dihydro-1H-inden-5-yl)-4-methylquinolin-2-
amine Dihydrochloride (17). Compounds 39 (0.075 g, 0.245 mmol)
and 79 (0.076 g, 0.245 mmol) were coupled using General Procedure
1. Purification by flash column chromatography, eluting with a
gradient of 5% EtOAc in CH2Cl2 to 35% EtOAc in CH2Cl2, afforded
protected phenylquinoline 81 as a yellow foam (0.091 g, 86%). This
compound was immediately deprotected using General Procedure 2.
After deprotection, the free aminoquinoline was precipitated from
EtOAc (1 mL) with hexanes (20 mL), and the gel-like solid was
filtered and diluted in ether/MeOH (2:1). After treatment with
methanolic HCl, ether (8 mL) was added, and 17 was obtained as a
white solid (0.059 g, 78% from 81) after precipitating from hot
MeOH (5 mL) with ether (10 mL), washing with ether, and drying in
1
257 °C (melts). H NMR (500 MHz; DMSO-d6): δ 14.13 (s, 1H),
8.65 (s, 3H), 8.13−8.11 (m, 2H), 7.95 (d, J = 1.5 Hz, 1H), 7.87 (dd, J
= 8.5, 1.1 Hz, 1H), 7.81 (dd, J = 8.4, 2.2 Hz, 1H), 7.72 (d, J = 8.4 Hz,
1H), 6.95 (s, 1H), 4.24 (s, 2H), 3.39−3.30 (m, 3H), 2.66 (s, 3H); the
aminoquinoline −NH protons are not visible as discrete peaks but are
broadened into the baseline around 8.4 ppm; 13C NMR (126 MHz;
DMSO-d6): δ 154.7, 152.5, 142.3, 138.1, 133.8, 132.9, 130.8, 130.0,
129.1, 126.9, 123.8, 121.4, 115.8, 113.3, 19.4; one of the quinoline
carbons is not visible due to baseline broadening; one of the aliphatic
carbon signals is obscured by the solvent peak; HRMS calcd for
1
vacuo: mp >300 °C (darkens slowly), >340 °C (chars). H NMR
(500 MHz; DMSO-d6): δ 13.98 (s, 1H), 9.00 (br s, 1H), 8.49 (s, 3H),
8.10 (br s, 1H), 8.07 (d, J = 8.5 Hz, 1H), 7.94 (s, 1H), 7.82 (dd, J =
8.5, 1.3 Hz, 1H), 7.76−7.69 (m, 3H), 3.20−3.14 (m, 1H), 3.02−2.95
(m, 1H), 2.57−2.54 (m, 1H), 2.10−2.03 (m, 1H); 13C NMR (126
MHz; DMSO-d6): δ 154.3, 145.8, 143.9, 140.5, 139.9, 126.7, 126.32,
126.18, 124.10, 124.01, 121.0, 115.8, 113.1, 54.9, 31.0, 30.4, 19.4; two
of the quinoline carbons are not visible due to baseline broadening;
+
C17H17ClN3 : 298.1106; found, 298.1114.
7-(3-(Aminomethyl)-4-trifluoromethylphenyl)-4-methylquinolin-
2-amine Dihydrochloride (21). Compounds 39 (0.075 g, 0.245
mmol) and 90 (0.100 g, 0.282 mmol) were coupled using General
Procedure 1. Purification by flash column chromatography, eluting
with a gradient of 5% EtOAc in CH2Cl2 to 35% EtOAc in CH2Cl2,
afforded protected phenylquinoline 119 as a yellow solid (0.102 g,
88%). This compound was immediately deprotected using General
Procedure 2. After workup, the free aminoquinoline was purified by
flash column chromatography, eluting with EtOAc to yield a pale-
yellow gum that was diluted in ether/MeOH (10:1). After treatment
with methanolic HCl, ether (5 mL) was added, and 21 was obtained
as a cream-colored powder (0.054 g, 62% from 119) after
precipitating from hot MeOH (2 mL) with ether (10 mL), washing
+
HRMS calcd for C19H20N3 : 290.1652; found, 290.1663.
7-(3-Amino-2,3-dihydro-1H-inden-5-yl)-4-methylquinolin-2-
amine Dihydrochloride (18). Compounds 39 (0.075 g, 0.245 mmol)
and 80 (0.076 g, 0.245 mmol) were coupled using General Procedure
1. Purification by flash column chromatography, eluting with a
gradient of 2% EtOAc in CH2Cl2 to 35% EtOAc in CH2Cl2, afforded
protected phenylquinoline 82 as a yellow semisolid (0.093 g, 88%).
This compound was immediately deprotected using General
Procedure 2. After deprotection, the free aminoquinoline was purified
by flash column chromatography, eluting with EtOAc. Concentration
afforded a residue that was diluted in MeOH. After treatment with
methanolic HCl, the mixture was concentrated, azeotroped with
toluene twice, and 18 was obtained as a white solid (0.064 g, 82%
from 82) after precipitating twice from hot MeOH (2 mL) with ether
(10 mL), washing with ether, and drying in vacuo: mp 250−256 °C
1
with ether, and drying in vacuo: mp 327−329 °C. H NMR (500
MHz; DMSO-d6): δ 14.09 (s, 1H), 9.00 (br s, 1H), 8.30 (br s, 1H),
8.76 (s, 3H), 8.31 (s, 1H), 8.17 (d, J = 8.5 Hz, 1H), 8.03 (d, J = 1.5
Hz, 1H), 7.99−7.95 (m, 3H), 6.98 (s, 1H), 4.29 (s, 2H), 2.68 (s,
3H); 13C NMR (126 MHz; DMSO-d6): δ 154.6, 152.4, 143.0, 141.9,
136.8, 133.6, 130.2, 127.82, (127.77 + 127.73 + 127.68 + 127.64 +
127.59, 1C), (127.49 + 127.3, 1C), (125.6 + 123.4, 1C), 127.0, 124.2,
121.7, 116.1, 113.7, 39.14, 39.13, 19.5; the splitting of the signals for
the trifluoromethyl group and nearby carbons is partially obscured by
1
(darkens, then chars). H NMR (500 MHz; DMSO-d6): δ 14.15 (s,
1H), 9.00 (br s, 1H), 8.61 (s, 3H), 8.20 (br s, 1H), 8.11 (t, J = 4.2 Hz,
2H), 7.90 (d, J = 1.5 Hz, 1H), 7.79 (dd, J = 8.5, 1.6 Hz, 1H), 7.74
(dd, J = 7.9, 1.5 Hz, 1H), 7.51 (d, J = 7.9 Hz, 1H), 6.94 (s, 1H),
4.83−4.80 (m, 1H), 3.14 (ddd, J = 19.1, 7.1, 4.3 Hz, 1H), 2.98−2.92
(m, 1H), 2.58−2.52 (m, 1H), 2.10−2.03 (m, 1H); 13C NMR (126
MHz; DMSO-d6): δ 154.5, 145.3, 144.1, 141.1, 137.6, 136.8, 128.4,
+
other signals; HRMS calcd for C18H17F3N3 : 332.1369; found,
332.1383.
S
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Article
7-(3-(Aminomethyl)-4-ethylphenyl)-4-methylquinolin-2-amine
Dihydrochloride (22). Compounds 39 (0.060 g, 0.196 mmol) and 95
(0.065 g, 0.206 mmol) were coupled using General Procedure 1.
Purification by flash column chromatography, eluting with a gradient
of 5% EtOAc in CH2Cl2 to 35% EtOAc in CH2Cl2, afforded protected
phenylquinoline 120 as a cream-colored solid (0.074 g, 87%). This
compound was immediately deprotected using General Procedure 2.
After workup, the free aminoquinoline was triturated with 5% EtOAc
in hexanes and the solid was collected, washed with hexanes, diluted
in EtOAc, filtered, and concentrated. The residue was diluted in
ether/MeOH (6:1). After treatment with methanolic HCl, the
mixture was concentrated, and 22 was obtained as an off-white
solid (0.035 g, 56% from 120) after precipitating three times from hot
MeOH (1.5 mL) with ether (10 mL), washing with ether, and drying
in vacuo: mp 307−309 °C. 1H NMR (500 MHz; DMSO-d6): δ 14.06
(s, 1H), 9.05 (br s, 1H), 8.48 (s, 3H), 8.25 (br s, 1H), 8.10 (d, J = 8.5
Hz, 1H), 7.94 (t, J = 2.2 Hz, 2H), 7.88 (dd, J = 8.5, 1.6 Hz, 1H), 7.73
(dd, J = 8.0, 1.8 Hz, 1H), 7.46 (d, J = 8.1 Hz, 1H), 6.94 (d, J = 0.6 Hz,
1H), 4.16−4.13 (m, 2H), 2.76 (q, J = 7.5 Hz, 2H), 2.65 (s, 3H), 1.22
(t, J = 7.5 Hz, 3H); 13C NMR (126 MHz; DMSO-d6): δ 154.4, 152.7,
143.71, 143.55, 136.65, 136.54, 133.1, 130.0, 128.6, 127.6, 126.7,
124.0, 120.9, 115.0, 113.0, 39.5, 25.1, 19.5, 15.4; HRMS calcd for
afforded protected phenylquinoline 123 as an off-white solid (0.083 g,
73%). This compound was immediately deprotected using General
Procedure 2. After deprotection, the free aminoquinoline was
triturated with 5% EtOAc in hexanes (15 mL), and the obtained
solid was diluted in ether/MeOH (2:1). After treatment with
methanolic HCl, the mixture was concentrated, the residue was
azeotroped twice with toluene, and 25 was obtained as a yellow glassy
solid (0.044 g, 62% from 123) after precipitating twice from hot
MeOH (2 mL) with ether (10 mL) and drying in vacuo: mp 220 °C
(softens), 264−266 °C (bubbles, melts). 1H NMR (500 MHz;
DMSO-d6): δ 14.06 (s, 1H), 9.00 (br s, 1H), 8.32 (s, 4H), 8.07 (d, J =
8.5 Hz, 1H), 7.91 (d, J = 2.2 Hz, 1H), 7.89 (d, J = 1.4 Hz, 1H), 7.82
(dd, J = 8.6, 1.2 Hz, 1H), 7.78 (dd, J = 8.6, 2.3 Hz, 1H), 7.25 (d, J =
8.7 Hz, 1H), 6.91 (s, 1H), 4.09 (br t, J = 6.5 Hz, 4H), 2.63 (t, J = 1.8
Hz, 3H), 1.83 (sextet, J = 7.0 Hz, 2H), 1.03 (t, J = 7.4 Hz, 3H); 13C
NMR (126 MHz; DMSO-d6): δ 157.6, 154.4, 143.51, 130.7, 129.4,
129.2, 126.7, 123.6, 123.2, 120.6, 114.6, 113.1, 112.7, 70.2, 37.8, 22.4,
19.4, 10.9; two of the quinoline carbons are not visible due to baseline
broadening; HRMS calcd for C20H24N3O+: 322.1914; found,
322.1927.
7-(3-(Aminomethyl)-4-isopropoxyphenyl)-4-methylquinolin-2-
amine Dihydrochloride (26). Compounds 39 (0.075 g, 0.245 mmol)
and 105 (0.093 g, 0.269 mmol) were coupled using General
Procedure 1. Purification by flash column chromatography, eluting
with a gradient of 5% EtOAc in CH2Cl2 to 35% EtOAc in CH2Cl2,
afforded protected phenylquinoline 124 as an off-white solid (0.083 g,
73%). This compound was immediately deprotected using General
Procedure 2. After deprotection, the free aminoquinoline was
precipitated from EtOAc (1 mL) with hexanes (10 mL) and the
obtained solid was diluted in ether/MeOH (4:1). After treatment
with methanolic HCl, the mixture was concentrated and azeotroped
twice with toluene, and 26 was obtained as an off-white solid (0.0097
g, 14% from 124) after precipitating twice from hot MeOH (1 mL)
with ether (10 mL), washing with ether, and drying in vacuo: mp
+
C19H22N3 : 292.1808; found, 292.1815.
7-(3-(Aminomethyl)-4-methoxyphenyl)-4-methylquinolin-2-
amine Dihydrochloride (23). Compounds 39 (0.075 g, 0.245 mmol)
and 98 (0.085 g, 0.269 mmol) were coupled using General Procedure
1. Purification by flash column chromatography, eluting with a
gradient of 5% EtOAc in CH2Cl2 to 40% EtOAc in CH2Cl2, afforded
protected phenylquinoline 121 as a yellow foam (0.077 g, 72%). This
compound was immediately deprotected using General Procedure 2.
After workup, the free aminoquinoline was triturated with hexanes.
The solid was collected and diluted in ether/MeOH (8:1). After
treatment with methanolic HCl, 23 was obtained as a yellow
amorphous solid (0.057 g, 88% from 121) after precipitating from hot
MeOH (5 mL) with ether (10 mL), washing with ether, and drying in
vacuo: mp 232−234 °C. 1H NMR (500 MHz; DMSO-d6): δ 14.16 (s,
1H), 9.00 (br s, 1H), 8.34−8.30 (2 br s, 4H), 8.07 (d, J = 8.6 Hz,
1H), 7.91 (d, J = 2.2 Hz, 1H), 7.90 (d, J = 1.4 Hz, 1H), 7.82 (ddd, J =
8.5, 4.7, 2.0 Hz, 2H), 7.26 (d, J = 8.7 Hz, 1H), 6.93 (s, 1H), 4.08 (q, J
= 5.3 Hz, 2H), 3.92 (s, 3H), 2.65 (s, 3H); 13C NMR (126 MHz;
DMSO-d6): δ 158.3, 154.4, 152.7, 143.5, 136.7, 130.8, 129.7, 129.3,
126.7, 123.7, 123.1, 120.5, 114.5, 112.7, 112.4, 56.4, 38.1, 19.4;
HRMS calcd for C18H20N3O+: 294.1601; found, 294.1611.
1
237−239 °C (softens), 245−246 °C (melts). H NMR (500 MHz;
DMSO-d6): δ 13.97 (s, 1H), 9.00 (br s, 1H), 8.26 (br s, 4H), 8.07 (d,
J = 8.6 Hz, 1H), 7.89 (d, J = 9.5 Hz, 2H), 7.81 (d, J = 8.3 Hz, 1H),
7.77 (d, J = 8.7 Hz, 1H), 7.28 (d, J = 8.4 Hz, 1H), 6.91 (s, 1H), 4.81−
4.75 (m, 1H), 4.06 (d, J = 4.9 Hz, 2H), 2.66 (s, 3H), 1.37 (d, J = 6.0
Hz, 6H); 13C NMR (126 MHz; DMSO-d6): δ 156.5, 154.5, 152.5,
143.5, 136.9, 130.4, 129.5, 129.0, 126.7, 123.80, 123.61, 120.5, 114.5,
114.2, 112.7, 71.0, 37.9, 22.2 (2C), 19.4; HRMS calcd for
C20H24N3O+: 322.1914; found, 322.1933.
7-(3-(Aminomethyl)-4-ethoxyphenyl)-4-methylquinolin-2-amine
Dihydrochloride (24). Compounds 39 (0.075 g, 0.245 mmol) and
101 (0.089 g, 0.269 mmol) were coupled using General Procedure 1.
Purification by flash column chromatography, eluting with a gradient
of 5% EtOAc in CH2Cl2 to 40% EtOAc in CH2Cl2, afforded protected
phenylquinoline 122 as a cream-colored solid (0.072 g, 65%). This
compound was immediately deprotected using General Procedure 2.
After workup, the free aminoquinoline was triturated with hexanes.
The solid was collected and diluted in ether/MeOH (2:1). After
treatment with methanolic HCl, 24 was obtained as a yellow glassy
solid (0.060 g, 99% from 122) after washing with 2% MeOH in ether,
diluting in MeOH and reconcentrating, washing with ether, and
drying in vacuo: mp 250 °C (softens), 265−267 °C (bubbles, melts).
1H NMR (500 MHz; DMSO-d6): δ 14.23 (s, 1H), 9.07 (br s, 1H),
8.39 (s, 3H), 8.25 (br s, 1H), 8.07 (d, J = 8.6 Hz, 1H), 7.92 (d, J = 2.1
Hz, 1H), 7.89 (d, J = 1.2 Hz, 1H), 7.82 (dd, J = 8.5, 1.4 Hz, 1H), 7.78
(dd, J = 8.6, 2.2 Hz, 1H), 7.24 (d, J = 8.7 Hz, 1H), 6.93 (s, 1H), 4.18
(br q, J = 6.9 Hz, 2H), 4.07 (d, J = 5.4 Hz, 2H), 2.65 (s, 3H), 1.42 (t,
J = 6.9 Hz, 3H); 13C NMR (126 MHz; DMSO-d6): δ 157.5, 154.4,
152.6, 143.5, 136.7, 130.6, 129.6, 129.2, 126.7, 123.7, 123.1, 120.5,
114.4, 113.0, 112.7, 64.5, 37.9, 19.4, 15.0; HRMS calcd for
C19H22N3O+: 308.1757; found, 308.1772.
7-(3-(Aminomethyl)-4-isobutoxyphenyl)-4-methylquinolin-2-
amine Dihydrochloride (27). Compounds 39 (0.050 g, 0.163 mmol)
and 106 (0.058 g, 0.160 mmol) were coupled using General
Procedure 1. Purification by flash column chromatography, eluting
with a gradient of 5% EtOAc in CH2Cl2 to 35% EtOAc in CH2Cl2,
afforded protected phenylquinoline 125 as a yellow solid (0.056 g,
72%). This compound was immediately deprotected using General
Procedure 2. After deprotection, the free aminoquinoline was diluted
in MeOH (10 mL), filtered, and diluted with 4 mL of ether. After
treatment with methanolic HCl, the mixture was concentrated and
azeotroped with toluene twice to yield 27 as a cream-colored solid
(0.043 g, 90% from 125) after precipitating from MeOH (2 mL) with
ether (15 mL), washing with ether, and drying in vacuo: mp 283−285
°C (softens, bubbles), 308 °C (chars). 1H NMR (500 MHz; DMSO-
d6): δ 13.85 (s, 1H), 8.26 (s, 3H), 8.08 (d, J = 8.5 Hz, 1H), 7.89 (dd,
J = 8.8, 1.6 Hz, 2H), 7.82−7.78 (m, 2H), 7.26 (d, J = 8.7 Hz, 1H),
6.90 (s, 1H), 4.11 (q, J = 5.4 Hz, 2H), 3.91 (d, J = 6.5 Hz, 2H), 2.66
(s, 3H), 2.16−2.10 (m, 1H), 1.04 (d, J = 6.7 Hz, 6H); the
aminoquinoline −NH protons are broadened into the baseline around
8.5 ppm; 13C NMR (126 MHz; DMSO-d6): δ 157.5, 154.4, 152.7,
143.5, 130.7, 129.23, 129.12, 126.7, 123.6, 123.2, 120.6, 114.5, 113.1,
112.7, 74.8, 37.7, 28.1, 19.60, 19.43; one of the aminoquinoline
carbons is not visible due to baseline broadening; HRMS calcd for
C21H26N3O+: 336.2070; found, 336.2080.
7-(3-(Aminomethyl)-4-propoxyphenyl)-4-methylquinolin-2-
amine Dihydrochloride (25). Compounds 39 (0.075 g, 0.245 mmol)
and 104 (0.093 g, 0.269 mmol) were coupled using General
Procedure 1. Purification by flash column chromatography, eluting
with a gradient of 5% EtOAc in CH2Cl2 to 35% EtOAc in CH2Cl2,
7-(3-(Aminomethyl)-4-(cyclobutylmethoxy)phenyl)-4-methylqui-
nolin-2-amine Dihydrochloride (28). Compounds 39 (0.082 g, 0.270
mmol) and 107 (0.110 g, 0.300 mmol) were coupled using General
T
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Procedure 1. Purification by flash column chromatography, eluting
with a gradient of 5% EtOAc in CH2Cl2 to 80% EtOAc in CH2Cl2,
afforded protected phenylquinoline 126 as a tan foam (0.052 g, 40%).
A portion of this compound (0.04 g) was immediately deprotected
using General Procedure 2. After workup, the free aminoquinoline
was diluted in MeOH (1.0 mL) and treated with methanolic HCl for
18 h. The mixture was concentrated to minimal solvent and triturated
with ether (10 mL × 3) to afford 28 as an off-white solid (0.031 g,
88% from 126) after filtering, washing with ether, and drying in
vacuo: mp 83−84 °C (softens) > 209.5 °C (darkens) > 241.2 °C
General Procedure 2. After deprotection, the free aminoquinoline was
purified by passing through a short plug of SiO2 and washing with
EtOAc and then 10% MeOH in EtOAc. Concentration afforded a
residue that was diluted in ether/MeOH (2:1). After treatment with
methanolic HCl, the mixture was concentrated, and 30 was obtained
as a white solid (0.057 g, 84% from 128) after precipitating twice
from MeOH (2 mL) with ether (10 mL) and drying in vacuo: mp
1
242−243.5 °C. H NMR (500 MHz; DMSO-d6): δ 14.00 (s, 1H),
9.00 (br s, 1H), 8.35 (br s, 4H), 8.07 (d, J = 8.5 Hz, 1H), 7.94 (d, J =
2.2 Hz, 1H), 7.89 (d, J = 1.4 Hz, 1H), 7.81 (ddd, J = 12.4, 8.8, 1.8 Hz,
2H), 7.50−7.46 (m, 1H), 7.43−7.38 (m, 2H), 7.29 (d, J = 8.7 Hz,
1H), 7.19 (td, J = 8.7, 2.2 Hz, 1H), 6.91 (s, 1H), 4.16 (br q, J = 5.5
Hz, 2H), 2.65 (s, 3H); 13C NMR (126 MHz; DMSO-d6): δ (163.8
+161.8, 1C) 157.0, 154.4, 152.8, 143.4, (140.16 + 140.10, 1C),
131.19, (131.06 + 130.99, 1C), 129.7, 129.2, 126.7, 123.88, (123.86 +
123.67, 1C), 123.5, 120.6, (115.31 + 115.15, 1C), (114.74 + 114.57,
1C), 113.6, 112.8, 69.4, 37.9, 19.4; three of the quinoline and aryl ring
carbons are not visible because of baseline broadening; HRMS calcd
for C24H23FN3O+: 388.1820; found, 388.1828.
1
(dec) H NMR (500 MHz, DMSO-d6) δ 14.38 (s, 1H), 9.20 (bs,
1H), 8.58 (s, 3H), 8.28 (bs, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.97 (d, J =
2.3 Hz, 1H), 7.88 (d, J = 1.7 Hz, 1H), 7.84 (dd, J = 8.6, 1.7 Hz, 1H),
7.75 (dd, J = 8.6, 2.4 Hz, 1H), 7.24 (d, J = 8.6 Hz, 1H), 6.96 (s, 1H),
4.09 (d, J = 6.4 Hz, 2H), 4.06 (d, J = 5.3 Hz, 2H), 2.81 (hept, J = 7.0
Hz, 1H), 2.64 (s, 3H), 2.18−2.05 (m, 2H), 1.99−1.82 (m, 4H). 13C
NMR (126 MHz, DMSO-d6) δ 156.97, 153.88, 151.95, 143.00,
135.99, 130.05, 128.66, 128.37, 126.08, 123.12, 122.73, 119.86,
113.68, 112.59, 112.08, 71.86, 36.84, 33.78, 24.15, 18.85, 18.07; one
of the aminoquinoline carbons is not visible due to baseline
broadening; HRMS calcd for C22H26N3O+: 348.2076; found,
348.2073.
4-((4-(2-Amino-4-methylquinolin-7-yl)-2-(aminomethyl)-
phenoxy)methyl)benzonitrile Dihydrochloride (31). Compounds 39
(0.075 g, 0.245 mmol) and 110 (0.102 g, 0.245 mmol) were coupled
using General Procedure 1. After workup, the crude residue was
suspended in EtOAc/ether (1:1) and filtered through a small pad of
Celite overlaid with SiO2. The filtrate was concentrated to yield a
residue that was washed with 5% hexane/EtOAc (30 mL) to afford
129 as a white solid (0.073 g, 56%) after washing with hexanes. This
compound was immediately deprotected using General Procedure 2.
After deprotection, the free aminoquinoline was purified by flash
column chromatography, eluting with a gradient of EtOAc to 2%
MeOH in EtOAc to yield a white solid that was diluted in ether/
MeOH (2:1). After treatment with methanolic HCl, the mixture was
concentrated, the residue was azeotroped with toluene, and 31 was
obtained as a white solid (0.054 g, 84% from 129) after precipitating
twice from MeOH (3 mL) with ether (15 mL) and drying in vacuo:
7-(3-(Aminomethyl)-4-(cyclopropylmethoxy)phenyl)-4-methyl-
quinolin-2-amine Dihydrochloride (29). Compound 108 (0.078 g,
0.219 mmol), tetrahydroxydiboron (0.059 g, 0.77 mmol), KOAc
(0.064 g, 0.66 mmol), XPhos-Pd-G3 (2.4 mg, 2.8 μmol), and XPhos
(2.6 mg, 5.5 μmol) were added to an oven-dried glass vial equipped
with a Teflon-lined cap.49 EtOH (2.2 mL, degassed) was then added
to the reaction vessel, and a nitrogen stream was allowed to bubble
through the solution at room temperature for 15 min with stirring.
The vessel was then sealed, and the reaction mixture was heated to 80
°C for 2 h. The reaction was then allowed to cool to ambient
temperature before 3 equiv (0.364 mL, 0.66 mmol) of 1.8 M degassed
aqueous K2CO3 was added via a syringe followed by compound 38
(0.055 g, 0.20 mmol). The reaction vial was degassed with argon,
resealed with a Teflon-lined cap, and left to stir at 80 °C for an
additional 15 h. The reaction mixture was cooled to room
temperature and partitioned between EtOAc and H2O (20 mL
each). The layers were separated, the aqueous layer was extracted with
EtOAc (3 × 20 mL), and the organic layer was washed with 5% aq.
NaCl (2 × 20 mL) and sat. aq. NaCl (20 mL), dried over anhydrous
sodium sulfate, and concentrated. Purification by flash column
chromatography, eluting with a gradient of 5% EtOAc in CH2Cl2 to
90% EtOAc in CH2Cl2, afforded protected phenylquinoline 127 as a
colorless solid (0.073 g, 78%). A portion of this compound (0.069 g)
was immediately deprotected using General Procedure 2. After
workup, the free aminoquinoline was diluted in MeOH (3.0 mL),
treated with methanolic HCl for 18 h, and concentrated to dryness.
The title compound (29) was obtained as a yellow-colored solid
(0.029 g, 49% from 127) after reverse phase flash column
chromatography, eluting through a 15.5 g C18 RediSep RF Gold
cartridge column with a gradient of 100% water to 100% acetonitrile,
azeotropic removal of water with toluene, and drying in vacuo: mp >
1
mp 280−282 °C. H NMR (500 MHz; DMSO-d6): δ 14.03 (s, 1H),
8.37 (s, 3H), 8.06 (d, J = 8.4 Hz, 1H), 7.94−7.88 (m, 4H), 7.80−7.75
(m, 4H), 7.27 (d, J = 8.5 Hz, 1H), 6.90 (s, 1H), 5.40 (s, 2H), 4.17 (s,
2H), 2.64 (s, 3H); the aminoquinoline −NH protons are not visible
as discrete peaks but are broadened into the baseline around 8.4 and
7.9 ppm; 13C NMR (126 MHz; DMSO-d6): δ 19.38, 37.79, 69.30,
111.09, 112.77, 113.52, 115.19, 119.23, 120.72, 123.49, 126.60,
128.52, 129.11, 129.80, 131.43, 132.93, 143.01, 143.16, 154.74,
156.78; three of the aminoquinoline and aryl carbons are not visible
because of baseline broadening; HRMS calcd for C25H23N4O+:
395.1866; found, 395.1872.
7-(3-(Aminomethyl)-4-(pyridin-2-ylmethoxy)phenyl)-4-methyl-
quinolin-2-amine Trihydrochloride (32). Compounds 39 (0.075 g,
0.245 mmol) and 111 (0.096 g, 0.245 mmol) were coupled using
General Procedure 1. Purification by flash column chromatography,
eluting with a gradient of 10% EtOAc in CH2Cl2 to 85% EtOAc in
CH2Cl2, afforded protected phenylquinoline 130 as a white powder
(0.091 g, 72%) after precipitating from EtOAc (1 mL) with hexanes
(20 mL). This compound was immediately deprotected using General
Procedure 2. After deprotection, the free aminoquinoline was purified
by flash column chromatography, eluting with a gradient of EtOAc to
15% MeOH in EtOAc. Concentration afforded a residue that was
diluted in MeOH. After treatment with methanolic HCl, the mixture
was concentrated, the residue was azeotroped three times with
toluene, and 32 was obtained as a white powder (0.063 g, 74% from
130) after precipitating three times from MeOH (5 mL) with ether
(10 mL), washing with ether, and drying in vacuo: mp 231−233 °C
1
193.5 °C (foams and decomposes) H NMR (500 MHz, DMSO-d6)
δ 14.30 (s, 1H), 8.50 (s, 3H), 8.06 (d, J = 8.5 Hz, 1H), 7.95 (d, J = 2.4
Hz, 1H), 7.89 (d, J = 1.7 Hz, 1H), 7.83 (dd, J = 8.6, 1.8 Hz, 1H), 7.75
(dd, J = 8.7, 2.4 Hz, 1H), 7.23 (d, J = 8.7 Hz, 1H), 6.99−6.87 (m,
1H), 4.09 (d, J = 5.0 Hz, 2H), 3.99 (d, J = 6.9 Hz, 2H), 2.65 (s, 3H),
1.33 (tt, J = 7.3, 5.0 Hz, 1H), 0.61 (td, J = 7.7, 6.1, 4.5 Hz, 2H), 0.41
(q, J = 4.9 Hz, 2H); 13C NMR (126 MHz, DMSO-d6) δ 156.93,
153.87, 151.99, 142.97, 136.11, 130.08, 128.75, 128.43, 126.09,
123.08, 122.71, 119.91, 113.79, 112.80, 112.10, 72.70, 37.08, 18.84,
9.93, 3.01; HRMS calcd for C21H24N3O+: 334.1914; found, 334.1910.
7-(3-(Aminomethyl)-4-((3-fluorobenzyl)oxy)phenyl)-4-methyl-
quinolin-2-amine Dihydrochloride (30). Compounds 39 (0.060 g,
0.196 mmol) and 109 (0.090 g, 0.216 mmol) were coupled using
General Procedure 1. Purification by flash column chromatography,
eluting with a gradient of 5% EtOAc in CH2Cl2 to 35% EtOAc in
CH2Cl2, afforded protected phenylquinoline 128 as a brown foam
(0.078 g, 76%). This compound was immediately deprotected using
1
(dec). H NMR (500 MHz; DMSO-d6): δ 14.08 (s, 1H), 9.04 (br s,
1H), 8.66 (d, J = 4.8 Hz, 1H), 8.46 (s, 3H), 8.22 (br s, 1H), 8.08 (d, J
= 8.6 Hz, 1H), 7.99 (t, J = 7.3 Hz, 1H), 7.94 (d, J = 2.3 Hz, 1H), 7.89
(d, J = 1.6 Hz, 1H), 7.82 (dd, J = 8.6, 1.7 Hz, 1H), 7.79 (dd, J = 8.6,
2.3 Hz, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.49 (t, J = 6.1 Hz, 1H), 7.33
(d, J = 8.7 Hz, 1H), 6.92 (s, 1H), 4.21 (d, J = 5.7 Hz, 2H); the
pyridinium −NH proton is broadened into residual water around 4.3
ppm; 13C NMR (126 MHz; DMSO-d6): δ 157.1, 156.0, 154.4, 152.7,
U
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148.7, 143.4, 139.0, 136.6, 131.5, 130.0, 129.3, 126.8, 124.2, 123.73,
123.67, 122.8, 120.6, 114.6, 113.8, 112.8, 70.4, 38.3, 19.4; HRMS
calcd for C23H23N4O+: 371.1866; found, 371.1880.
(0.044 g, 99% from 133) after filtering, washing with ether, and drying
in vacuo. Because of the NMR spectral overlap of differing HCl
species of 35, the precipitate was further purified using reverse phase
chromatography, eluting through a 15.5 g C18 RediSep RF Gold
cartridge column with a gradient of 100% water to 100% acetonitrile
to afford 35 as a yellow solid (0.009 g, 38%), which was used for
7-(3-(Aminomethyl)-4-(pyridin-3-ylmethoxy)phenyl)-4-methyl-
quinolin-2-amine Trihydrochloride (33). Compounds 39 (0.075 g,
0.245 mmol) and 112 (0.096 g, 0.245 mmol) were coupled using
General Procedure 1. As the product was poorly soluble in EtOAc, the
aqueous layer was extracted with 200 mL of EtOAc during the
workup. After concentration, the residue was washed with 50%
hexane/EtOAc (30 mL) to afford 131 as an off-white solid (0.100 g,
80%) after washing with hexanes. This compound was immediately
deprotected using General Procedure 2. After deprotection, the free
aminoquinoline was purified by flash column chromatography, eluting
with a gradient of 3% MeOH in EtOAc to 15% MeOH in EtOAc to
yield a colorless foam. This was diluted with ether/MeOH (1:1).
After treatment with methanolic HCl, the mixture was concentrated,
and 33 was obtained as a pale-orange solid (0.062 g, 66% from 131)
after precipitating twice from hot MeOH (1 mL) with ether (10 mL),
washing with ether, and drying in vacuo: mp 238−240 °C (softens)
1
characterization: mp 210.8−212 °C (softens) > 315 °C (dec); H
NMR (500 MHz, DMSO-d6) δ 14.20 (s, 1H), 9.18 (d, J = 1.9 Hz,
1H), 8.43 (s, 4H), 8.05 (d, J = 8.5 Hz, 1H), 7.94 (d, J = 2.4 Hz, 1H),
7.92 (d, J = 2.0 Hz, 1H), 7.89 (d, J = 1.8 Hz, 1H), 7.84−7.75 (m,
2H), 7.42 (d, J = 8.6 Hz, 1H), 6.91 (s, 1H), 5.42 (s, 2H), 4.12 (s,
2H), 2.64 (s, 3H); 13C NMR (126 MHz, 500 MHz, DMSO-d6) δ
156.45, 154.96, 154.03, 152.12, 151.95, 142.98, 136.00, 130.73,
129.20, 128.44, 126.18, 123.27, 123.18, 119.99, 118.35, 113.82,
113.36, 112.24, 65.99, 37.17, 18.97. HRMS calcd for C21H21N4OS+:
377.1436; found, 377.1429.
7-(3-(Aminomethyl)-4-(oxazol-4-ylmethoxy)phenyl)-4-methyl-
quinolin-2-amine Trihydrochloride (36). Compounds 39 (0.089 g,
0.289 mmol) and 115 (0.133 g, 0.347 mmol) were coupled using
General Procedure 1. Purification by flash column chromatography,
eluting with a gradient of 5% EtOAc in CH2Cl2 to 80% EtOAc in
CH2Cl2, afforded protected phenylquinoline 134 as a tan powder
(0.027 g, 19%). A portion of this compound (0.024 g) was
immediately deprotected using General Procedure 2. After workup,
the free aminoquinoline was diluted in MeOH (1.0 mL), treated with
methanolic HCl (1.5 mL) for 18 h, and concentrated to dryness. The
title compound (36) was obtained as a tan solid (0.014 g, 65% from
134) in 90% purity after recrystallizing once from 1:3 methanol/
ethanol followed by an additional overnight recrystallization from
methanol that was slowly diffused with ether, followed by removal of
mother liquor, washing with 1:20 methanol/ether, and drying in
vacuo. Additional purification of the crude product was achieved after
reverse phase flash column chromatography, eluting through a 15.5 g
C18 RediSep RF Gold cartridge column with a gradient of 100%
water to 100% acetonitrile to afford pure 36 as a cream-colored solid
(0.011 g, 47% from 135) after azeotropic removal of water with
toluene and drying in vacuo: mp > 151 °C (slowly browns) 253−254
1
248−250 °C (melts). H NMR (500 MHz; DMSO-d6): δ 14.07 (s,
1H), 9.00 (br s, 1H), 8.94 (s, 1H), 8.72 (d, J = 4.7 Hz, 1H), 8.39 (s,
3H), 8.30 (d, J = 6.9 Hz, 1H), 8.25 (br s, 1H), 8.08 (d, J = 8.6 Hz,
1H), 7.95 (d, J = 2.1 Hz, 1H), 7.90 (d, J = 1.3 Hz, 1H), 7.84−7.81
(m, 2H), 7.74−7.72 (m, 1H), 7.35 (d, J = 8.7 Hz, 1H), 6.93 (s, 1H),
5.40 (s, 2H), 4.15 (q, J = 5.5 Hz, 3H), 2.66 (s, 3H); the pyrdinium
−NH proton is broadened into residual water around 4.0 ppm; 13C
NMR (126 MHz; DMSO-d6): δ 156.9, 154.4, 152.8, 146.9, 143.4,
136.6, 134.4, 131.4, 130.0, 129.3, 126.8, 125.4, 123.74, 123.55, 120.6,
114.5, 113.6, 112.8, 67.5, 37.9, 19.4; one of the quinoline carbons is
not visible due to baseline broadening; 13C NMR (126 MHz; DMSO-
d6): δ 157.1, 156.0, 154.4, 152.7, 148.6, 143.4, 139.0, 136.6, 131.5,
130.0, 129.3, 126.8, 124.2, 123.73, 123.67, 122.8, 120.6, 114.6, 113.8,
112.8, 70.4, 38.3, 19.4; HRMS calcd for C23H23N4O+: 371.1866;
found, 371.1878.
7-(3-(Aminomethyl)-4-((5-methylisoxazol-3-yl)methoxy)phenyl)-
4-methylquinolin-2-amine Dihydrochloride (34). Compounds 39
(0.085 g, 0.276 mmol) and 113 (0.132 g, 0.33 mmol) were coupled
using General Procedure 1. Purification by flash column chromatog-
raphy, eluting with a gradient of 5% EtOAc in CH2Cl2 to 70% EtOAc
in CH2Cl2, afforded protected phenylquinoline 133 as a white solid
(0.032 g, 22%). This compound was immediately deprotected using
General Procedure 2. After workup, the free aminoquinoline was
diluted in MeOH (1.0 mL) and treated with methanolic HCl (1 mL)
for 18 h, resulting in the formation of a white precipitate, which was
concentrated to dryness. The title compound (34) was obtained as a
tan solid (0.012 g, 43% from 132) after recrystallization overnight
from methanol that was slowly diffused with ether, followed by
removal of mother liquor, washing with 1:20 methanol/ether, and
drying in vacuo: mp > 267 °C (browns slowly) 272−274 °C (melts/
1
°C. H NMR (500 MHz, DMSO-d6) δ 8.45 (s, 1H), 8.30 (s, 1H),
8.22−7.91 (m, 1H), 7.88 (d, J = 2.5 Hz, 1H), 7.82 (d, J = 8.4 Hz,
1H), 7.75 (dd, J = 8.6, 2.5 Hz, 1H), 7.72 (d, J = 1.9 Hz, 1H), 7.50
(dd, J = 8.5, 2.1 Hz, 1H), 7.35 (dd, J = 8.7, 2.8 Hz, 1H), 6.61 (s, 1H),
6.33 (s, 2H), 5.18 (s, 2H), 4.05 (s, 2H); the aminoquinoline -NH
proton is broadened into baseline and the benzylic CH3 protons are
obscured by the solvent peak around 2.5; 13C NMR (126 MHz,
DMSO-d6) δ 158.23, 155.33, 152.46, 148.39, 143.80, 139.52, 137.92,
135.55, 132.62, 128.25, 127.67, 124.30, 123.67, 122.30, 121.93,
119.46, 113.05, 112.08, 62.21, 37.52, 18.08. HRMS calcd for
+
C21H21N4O2 : 361.1659; found, 361.1655.
7-(3-(Aminomethyl)-4-(thiazol-5-ylmethoxy)phenyl)-4-methyl-
quinolin-2-amine Trihydrochloride (37). Compounds 39 (0.081 g,
0.265 mmol) and 116 (0.127 g, 0.32 mmol) were coupled using
General Procedure 1. Purification by flash column chromatography,
eluting with a gradient of 5% EtOAc in CH2Cl2 to 70% EtOAc in
CH2Cl2, afforded protected phenylquinoline 135 as a tan solid (0.036
g, 26%). A portion of this compound (0.032 g) was immediately
deprotected using General Procedure 2. After workup, the free
aminoquinoline was diluted in MeOH (1.0 mL) and treated with
methanolic HCl (1 mL) for 18 h, resulting in the formation of a white
precipitate, which was concentrated to dryness. The pure title
compound (37) was obtained as a brown crystalline solid (0.018 g,
60% from 135) after recrystallizing once from 1:5 methanol/ethanol
and then from methanol, washing with 1:20 methanol/ether, and
drying in vacuo: mp > 238 °C (darkens) 252−255 °C (slowly
1
gels) > 277 °C (foams and decomp.). H NMR (500 MHz, DMSO-
d6) δ 14.29 (s, 1H), 8.52 (s, 3H), 8.10−8.01 (m, 1H), 8.01−7.93 (m,
1H), 7.90 (s, 1H), 7.83 (dd, J = 8.5, 2.0 Hz, 1H), 7.79 (dt, J = 8.8, 2.4
Hz, 1H), 7.36 (d, J = 8.6 Hz, 1H), 6.94 (d, J = 4.6 Hz, 1H), 6.49 (d, J
= 4.4 Hz, 1H), 5.32 (s, 2H), 4.10 (d, J = 5.6 Hz, 2H), 2.65 (s, 3H),
2.44 (s, 3H); 13C NMR (126 MHz, DMSO-d6) δ 169.98, 164.79,
160.32, 156.15, 153.88, 142.80, 130.94, 129.19, 128.56, 126.44,
126.14, 123.17, 122.96, 120.01, 114.03, 112.93, 112.20, 101.47, 61.85,
+
37.13, 18.84, 11.76; HRMS calcd for C22H23N4O2 : 375.1821; found,
375.1816.
7-(3-(Aminomethyl)-4-(thiazol-4-ylmethoxy)phenyl)-4-methyl-
quinolin-2-amine Trihydrochloride (35). Compounds 39 (0.087 g,
0.282 mmol) and 114 (0.122 g, 0.304 mmol) were coupled using
General Procedure 1. Purification by flash column chromatography,
eluting with a gradient of 5% EtOAc in CH2Cl2 to 80% EtOAc in
CH2Cl2, afforded protected phenylquinoline 133 as a cream-colored
solid (0.056 g, 38%). A portion of this compound (0.047 g) was
immediately deprotected using General Procedure 2. After workup,
the free aminoquinoline was treated with methanolic HCl (2 mL) and
sonicated for 10 min before stirring at room temperature for 18 h.
The resulting gray precipitate was collected to afford 35 as a gray solid
1
softens) > 256 °C (decomposes into brown foam). H NMR (500
MHz, DMSO-d6) δ 14.33 (s, 1H), 9.18 (s, 1H), 8.54 (d, J = 6.1 Hz,
3H), 8.12 (s, 1H), 8.06 (d, J = 8.5 Hz, 1H), 7.98 (d, J = 2.4 Hz, 1H),
7.90 (d, J = 1.8 Hz, 1H), 7.84 (dd, J = 8.6, 1.8 Hz, 1H), 7.79 (dd, J =
8.6, 2.3 Hz, 1H), 7.42 (d, J = 8.7 Hz, 1H), 6.96 (s, 1H), 5.57 (s, 2H),
4.06 (q, J = 5.8 Hz, 3H), 2.65 (s, 4H); 13C NMR (126 MHz, DMSO)
δ 155.94, 155.51, 153.86, 152.02, 142.93, 142.82, 135.97, 133.64,
V
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130.85, 129.06, 128.42, 126.13, 123.18, 123.04, 119.97, 115.67,
113.86, 113.19, 112.18, 62.40, 37.04, 18.85; HRMS calcd for
C21H21N4OS+: 377.1436 found, 377.1431.
was carefully placed on top of the acceptor plate to make a
“sandwich”. The plate was incubated at 25 °C for 17 h in a saturated
humidity atmosphere with orbital agitation at 100 rpm. Verapamil,
chlorpromazine, and dopamine were used as a positive, intermediate,
and negative control, respectively. After incubation, 150 μL of the test
solution was taken from each well from both sides (donor and
acceptor) and transferred to the UV plate for measurement. The
effective permeability (Pe) was calculated using the following
equation57
Potassium 2-Acetamido-4-methylquinoline-7-trifluoroborate
(39). Compound 38 (0.368 g, 1.32 mmol), B2Pin2 (0.503 g, 1.98
mmol), anhydrous KOAc (0.388 g, 3.96 mmol), and Pd(dppf)Cl2 (5
mol %, 0.048 g) were combined in a sealable vial and diluted with
anhydrous dioxane (5 mL). The mixture was purged with argon,
sealed, and heated at 75 °C for 19 h. The mixture was then cooled,
diluted with EtOAc (20 mL), and filtered through a pad of Celite
overlaid with a thin layer of SiO2. The pad was washed with EtOAc
(200 mL), and the combined filtrate was washed with 5% aq. NaCl
and sat. aq. NaCl (100 mL each). The organic layer was dried over
anhydrous sodium sulfate and concentrated to yield a brown foam
containing the crude 7-BPin quinoline, which was immediately diluted
in THF/H2O (5/1.5 mL). KHF2 (0.412 g, 5.28 mmol) was added,
and the mixture was stirred at r.t. for 3 h. THF (2 mL) and H2O (1
mL) were then added, and the mixture was sonicated vigorously for 5
min and concentrated. The residue was azeotroped three times with
toluene, diluted with acetone, and filtered. The filter cake was
extracted with boiling acetone (6 × 60 mL), and the combined
acetone filtrates were concentrated. The residue was washed with 10%
hexanes in CH2Cl2 to yield 39 as a light-tan powder (0.371 g, 92%):
VA × VD
(VA + VD)
2.303
P =
×
e
A × (t−τ )
ss
Ä
Å
Å
Å
Å
Å
É
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
i
j
j
y
i
j
j
y
z
z
z
z
z
VA + VD
(1 − R) × VD
C (t)
CD(0)
z
z
A
× lg 1 − j
z × j
Å
Å
Å
Å
Å
j
j
z
z
j
j
k
{
k
{
Å
Ç
Ñ
Ö
where Pe is the effective permeability (cm/s); VA and VD are the
volumes of the acceptor and donor wells (0.25 cm3), respectively; CA
(t) is the concentration of the acceptor well at time t; CD (0) and CD
(t) are the concentrations of the donor well at t0 and t, respectively; A
is the filter well area (0.21 cm2); t is the incubation time (s); and τss is
the time to reach a steady state (usually very short compared to the
incubation time). R is the retention membrane factor and was
calculated using the following equation
1
mp > 260 °C (darkens), 290−292 °C (dec). H NMR (500 MHz;
Ä
É
acetone-d6): δ 9.33 (s, 1H), 8.12 (s, 1H), 7.91 (s, 1H), 7.71 (d, J = 8.2
Hz, 1H), 7.66 (d, J = 8.2 Hz, 1H), 2.63 (d, J = 0.8 Hz, 3H), 2.23 (s,
3H); 13C NMR (126 MHz; DMSO-d6): δ 169.4, 150.2, 146.0, 145.1,
129.46, 129.45, 129.42, 123.9, 121.0, 112.7, 24.0, 18.7; this compound
does not ionize well by ESI-MS or liquid chromatography-time-of-
flight (LC-TOF).
Å
Ñ
Å
Ñ
Å
CD(t)
CD(0)
VA
VD
CA(t) Ñ
Å
Ñ
Ñ
Å
R = 1 −
−
×
Å
Ñ
Å
Å
Å
Ñ
Ñ
Ñ
C (0)
D
Å
Ç
Ñ
Ö
Pe was reported as an average of triplicate measurements with a
standard deviation.
Human Liver Microsome Stability Assay. A 1120 μL aliquot of
potassium phosphate buffer (50 mM, pH 7.4) containing liver
microsomes (0.714 mg/mL) was added to individual 2 mL tubes
(final concentration 0.5 mg/mL). Positive control (terfenadine) and
12 (1 mM DMSO stocks) were directly spiked into respective tubes
to prepare a concentration of 1.428 μM (final concentration 1 μM).
From the above mixture, 70 μL was added to individual wells of 96-
well reaction plates and preincubated at 37 °C for 5 min. All reactions
were initiated by adding 30 μL of 3.33 mM NADPH (1 mM final
concentration). Reactions without NADPH and buffer controls
(minus NADPH) at 0 min and 60 min were also incubated to rule
out non-NADPH metabolism or chemical instability in the incubation
buffer. All reactions were terminated using 100 μL of ice-cold
acetonitrile containing an internal standard (glipizide) at 0, 5, 15, 30,
and 60 min. The plates were centrifuged at 4000 rpm for 15 min, and
100 μL aliquots were analyzed for parent compound disappearance in
the multiple-reaction monitoring (MRM) mode using liquid
chromatography−mass spectrometry (LC−MS)/MS. The percent
remaining of test compounds and positive controls in each sample was
determined by considering the peak area ratio in the 0 minute sample
as 100%. The half-life (t1/2 in min) and the in vitro intrinsic clearance
(CLint units in mL/min/kg) were calculated according to the
following equations, where k is the gradient of line determined
from a plot of the peak area ratio (compound peak area/internal
standard peak area; plot can be found in Supporting Information,
Table S3) against time:
Purified NOS Enzyme Assays. Rat and human nNOS, rat nNOS
mutants, murine macrophage iNOS, human iNOS, and human eNOS
were recombinant enzymes (expressed in Escherichia coli and purified
as reported previously).21,50−52 To test for NOS inhibition, the
hemoglobin capture assay was used to measure nitric oxide
production. The assay was performed at 37 °C in 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES, 100
mM with 10% glycerol, pH 7.4) in the presence of 10 μM L-arginine;
this concentration was used because (a) it is close to the Km values of
all three isoforms, where detection of competitive inhibitors is the
most sensitive, and (b) significant NOS uncoupling does not occur at
this concentration. Also included were 100 μM NADPH, 0.83 mM
CaCl2, approximately 320 units/mL of calmodulin, 10 μM H4B, and
human oxyhemoglobin (3 μM). For iNOS, the CaCl2 and calmodulin
were omitted and replaced with HEPES buffer (as neither are
required for activation of iNOS). The assay was performed in 96-well
plates using a Synergy 4 BioTek hybrid reader. The dispensing of
NOS enzyme and hemoglobin was automated, and after 30 s
(maximum delay), NO production was read by monitoring the
absorbance at 401 nm (resulting from the conversion of oxy-
hemoglobin to methemoglobin). Kinetic readouts were performed for
5 min. Each compound was assayed at least in duplicate, and six to
nine concentrations (500 μM−50 nM or 100 μM−10 nM for eNOS
and iNOS; 50 μM−5 nM for rat and human nNOS) were used to
construct concentration−response curves. IC50 values were calculated
by nonlinear regression (variable slope, four parameters) using
GraphPad Prism software (standard error is reported for the
log IC50), and Ki values were obtained from IC50 values using the
Cheng−Prusoff53 equation [Ki = IC50/(1 + [S]/Km)] with the
following Km values: 1.3 μM (rat nNOS), 1.6 μM (human nNOS), 8.2
μM (murine macrophage iNOS), 8 μM (human iNOS),54 3.9 μM
(human eNOS),55 1.7 μM (rat nNOS D597N single mutant),56 and
1.9 μM (rat nNOS D597N/M336V double mutant).48
0.693
k
0.693
in vitro t1/2
t1/2
=
=
; CLint
45 mg microsomes
gm liver
mL incubation
mg microsomes
×
×
20 mg liver weight
kg b.w
×
PAMPA-BBB Assay. The PAMPA-BBB assay was performed
following a protocol described previously.21 Briefly, the assay was
performed in 10 mM phosphate-buffered saline (PBS) buffer (pH
7.5), and compounds were tested at a concentration of 200 μM. The
donor plate was first coated with 4 μL of porcine brain lipid (20 mg/
mL in dodecane), followed by addition of 250 μL of a test compound.
The acceptor plate was filled with 250 μL of PBS, and the donor plate
For liver microsomes, the scaling factor used was 45 mg of
microsomal protein per gram liver.
Inhibitor Complex Crystal Preparation. The sitting drop vapor
diffusion method was used to grow crystals at 4 °C for the heme
domains of rnNOS (8 mg/mL containing 20 mM histidine), the
hnNOS K301R/R354A/G357D mutant (10 mg/mL containing 20
W
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Article
mM histidine), and heNOS (6 mg/mL). The crystal growth
conditions were as described previously.8 The only exception is that
the pH for the heNOS crystal growth is 7.5 rather than 6.5 as
mistakenly reported there. Fresh crystals were first passed stepwise
through cryoprotectant solutions. The pH of the final soaking solution
for rnNOS was adjusted from 5.8 through 6.5 to 7.0 (in MES) to 7.5
(in HEPES) and that for hnNOS from pH 7.2 to 7.5 (in HEPES); for
heNOS, the BIS-TRIS buffer at pH 7.5 was unchanged. At pH 7.5,
crystals were soaked with 5−10 mM inhibitor for 2−4 h at 4 °C
before being flash-cooled with liquid nitrogen and stored until data
collection.
6PMV, 6PMW, 6PMX, 6PMY, 6PMZ, 6PN0, 6PN1, 6PN2,
6PN3, 6PN4, 6PN5, 6PN6, 6PN7, 6PN8, 6PN9, 6PNA,
6PNB, 6PNC, 6PND, 6PNE, 6PNF, 6PNG, 6PNH, 6PO5,
6PO7, 6PO8, 6PO9, 6POA, 6POB, 6POC, 6POT, 6POU,
6POV, 6POW, 6POX, 6POY, 6POZ, 6PP0, 6PP1, 6PP2, 6PP3,
6PP4.
AUTHOR INFORMATION
■
Corresponding Author
Richard B. Silverman − Department of Chemistry, Department
of Molecular Biosciences, Chemistry of Life Processes Institute,
Center for Molecular Innovation and Drug Discovery,
Northwestern University, Evanston, Illinois 60208-3113, United
States; orcid.org/0000-0001-9034-1084; Phone: +1 847
491 5653; Email: Agman@chem.northwestern.edu; Fax: +1
847 491 7713
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 data
collection control software Blu-Ice58 and a crystal-mounting robot.
When a CCD detector was used, 100−125° of data were typically
collected with 0.5° per frame. If a Pilatus pixel array detector was
used, 140−200° of fine-sliced data were collected with 0.2° per frame.
More data frames were collected for heNOS, which has a low
symmetry (P21). Raw CCD data frames were indexed, integrated, and
scaled using iMOSFLM,59 but the pixel array data were preferably
processed with XDS60 and scaled with Aimless.61 The binding of
inhibitors was detected by initial difference Fourier maps calculated
with REFMAC.62 The inhibitor molecules were then modeled in
Coot63 and refined using REFMAC or PHENIX.64 The symmetry of
heNOS crystals was changed from the orthorhombic P212121 reported
previously65 to monoclinic P21, with a β angle only 0.6−0.7° off
compared to the original 90°. Therefore, a molecular replacement
calculation with PHASER-MR66 was needed to solve the structure. In
the P21 space group, there are two heNOS dimers in the asymmetric
unit. Disordering in portions of inhibitors bound in the NOS active
sites was often observed, sometimes resulting in poor density quality.
However, partial structural features were usually still visible if the
contour level of the sigmaA weighted 2m|Fo| − D|Fc| map was
dropped to 0.5 σ, which afforded the building of reasonable models
into the disordered regions. Water molecules were added in PHENIX
and visually checked by Coot. The Transport/Libration/Screw
(TLS)67 protocol was implemented in the PHENIX refinements
with each subunit as one TLS group. The omit Fo − Fc density maps
were calculated by the Polder map facility in PHENIX for the bound
inhibitors.68 The refined structures were validated in Coot before
deposition in the Protein Data Bank. The X-ray diffraction data
collection and structure refinement statistics are summarized in Table
S1 in the Supporting Information.
Authors
Maris A. Cinelli − Department of Chemistry, Department of
Molecular Biosciences, Chemistry of Life Processes Institute,
Center for Molecular Innovation and Drug Discovery,
Northwestern University, Evanston, Illinois 60208-3113, United
States
Cory T. Reidl − Department of Chemistry, Department of
Molecular Biosciences, Chemistry of Life Processes Institute,
Center for Molecular Innovation and Drug Discovery,
Northwestern University, Evanston, Illinois 60208-3113, United
States
Huiying Li − Department of Molecular Biology and
Biochemistry, University of California, California 92697-3900,
United States
Georges Chreifi − Department of Molecular Biology and
Biochemistry, University of California, California 92697-3900,
United States
Thomas L. Poulos − Department of Molecular Biology and
Biochemistry, Pharmaceutical Sciences, and Chemistry,
University of California, California 92697-3900, United States;
orcid.org/0000-0002-5648-3510
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.9b01573
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.9b01573.
■
Author Contributions
sı
§M.A.C. and C.T.R. contributed equally to this work.
Notes
The authors declare no competing financial interest.
Crystallographic data collection and refinement statistics
for rat and human nNOS and human eNOS structures;
results and discussion pertaining to constrained amine
analogues (16−18) and small 4-alkyloxy-3-aminomethyl
analogues 25−28, and binding behaviors of 31, 36, and
37; relevant X-ray structures for 25, 26, 28, 31, 36, 37,
and protein secondary structure displacement (Figures
S1−S6); molecular docking protocol, synthesis, and
analytical data for relevant precursor compounds 46−49,
67, 73, 77−80, 85, 87−88, 90, 92−95, 98, 100−116;
full PDSP secondary binding assay results and
informaton on liver microsome stability (PDF)
ACKNOWLEDGMENTS
■
The authors thank the National Institutes of Health (R01
GM049725 and R35 GM131788 to R.B.S.; GM057353 and
GM131920 to T.L.P.) for their support of this work. We thank
the beamline staff at SSRL and ALS for their assistance during
the remote X-ray diffraction data collections. H.L. would like
to thank Carla Plaza for her efforts in protein preparations,
which provided samples for both the structure determinations
and the inhibitory assays. M.A.C. would like to thank Saman
Shafaie and Dr. S. Habibi Goudarzi for assistance with HRMS
experiments. This work made use of IMSERC at Northwestern
University, which has received support from the Soft and
Hybrid Nanotechnology Experimental (SHyNE) Resource
(NSF NNCI-1542205), the State of Illinois, and the
International Institute for Nanotechnology (IIN).
Molecular formula strings table (CSV)
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 (see Table S2 in SI for details):
X
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(Tyr706, Leu337, and Met336) for potent and selective inhibition of
neuronal nitric oxide synthase. Bioorg. Med. Chem. Lett. 2010, 20,
6258−6261.
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1999, 40, 6709−6712.
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T.; Erlanson, D. A.; Guckian, K.; Simmons, R. C.; Lee, W-C.; Sun, L.;
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WO2008005457, January 10, 2008.
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ABBREVIATIONS USED
■
NO, nitric oxide; nNOS, neuronal nitric oxide synthase; iNOS,
inducible nitric oxide synthase; eNOS, endothelial nitric oxide
synthase; rnNOS, rat neuronal nitric oxide synthase; hnNOS,
human neuronal nitric oxide synthase; heNOS, human
endothelial nitric oxide synthase; miNOS, murine inducible
nitric oxide synthase; hiNOS, human inducible nitric oxide
synthase; FMN, flavin mononucleotide; H4B, (6R)-5,6,7,8-
tetrahydrobiopterin; tPSA, total polar surface area; BIS-TRIS,
bis(2-hydroxyethyl)amino-tris(hydroxymethyl)-methane;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
PAMPA-BBB, parallel artificial membrane permeability for
the blood−brain barrier; PDSP, Psychoactive Drug Screening
Program; WT, wild-type
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