Nitric oxide synthase inhibitors that interact with both heme propionate and tetrahydrobiopterin show high isoform selectivity

Article abstract of DOI:10.1021/jm5004182

Overproduction of NO by nNOS is implicated in the pathogenesis of diverse neuronal disorders. Since NO signaling is involved in diverse physiological functions, selective inhibition of nNOS over other isoforms is essential to minimize side effects. A series of α-amino functionalized aminopyridine derivatives (3-8) were designed to probe the structure-activity relationship between ligand, heme propionate, and H₄B. Compound 8R was identified as the most potent and selective molecule of this study, exhibiting a K _i of 24 nM for nNOS, with 273-fold and 2822-fold selectivity against iNOS and eNOS, respectively. Although crystal structures of 8R complexed with nNOS and eNOS revealed a similar binding mode, the selectivity stems from the distinct electrostatic environments in two isoforms that result in much lower inhibitor binding free energy in nNOS than in eNOS. These findings provide a basis for further development of simple, but even more selective and potent, nNOS inhibitors.

Full text of DOI:10.1021/jm5004182

Article
pubs.acs.org/jmc
Open Access on 04/23/2015
Nitric Oxide Synthase Inhibitors That Interact with Both Heme
Propionate and Tetrahydrobiopterin Show High Isoform Selectivity
Soosung Kang,† Wei Tang,† Huiying Li,‡ Georges Chreifi,‡ Pavel Martasek,§ Linda J. Roman,§
́
Thomas L. Poulos,*,‡ and Richard B. Silverman*,†
†Department of Chemistry, Department of Molecular Biosciences, Chemistry of Life Processes Institute, Center for Molecular
Innovation and Drug Discovery, Northwestern University, Evanston, Illinois 60208-3113, United States
‡Departments of Molecular Biology and Biochemistry, Pharmaceutical Sciences, and Chemistry, University of California, Irvine,
California 92697-3900, United States
§Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78384-7760, United States
S
* Supporting Information
ABSTRACT: Overproduction of NO by nNOS is implicated in the pathogenesis of diverse neuronal disorders. Since NO
signaling is involved in diverse physiological functions, selective inhibition of nNOS over other isoforms is essential to minimize
side effects. A series of α-amino functionalized aminopyridine derivatives (3−8) were designed to probe the structure−activity
relationship between ligand, heme propionate, and H4B. Compound 8R was identified as the most potent and selective molecule
of this study, exhibiting a Ki of 24 nM for nNOS, with 273-fold and 2822-fold selectivity against iNOS and eNOS, respectively.
Although crystal structures of 8R complexed with nNOS and eNOS revealed a similar binding mode, the selectivity stems from
the distinct electrostatic environments in two isoforms that result in much lower inhibitor binding free energy in nNOS than in
eNOS. These findings provide a basis for further development of simple, but even more selective and potent, nNOS inhibitors.
various cancers.13 In addition, NO synthesis from bacterial
INTRODUCTION
The free radical nitric oxide (NO) is an important signaling
■
NOS was reported to play a critical role in antibiotic resistance
and pathogenicity.14,15 This suggests that the inhibition of
NOSs can be effective for the control of these diverse diseases,
but because NO signaling is involved in various physiological
functions, selective inhibition is essential to minimize any
unwanted side effects.16
molecule,1 controlling diverse physiological and pathological
processes in various species.2 In mammals, NO is endogenously
produced using L-arginine and molecular oxygen with NADPH
by three principal nitric oxide synthases (NOSs): neuronal
NOS (nNOS), endothelial NOS (eNOS), and inducible NOS
(iNOS).3 Selective inhibition of each NOS can regulate
different biological functions of NO signaling because each
NOS isoform is localized differently in the neuron,
endothelium, and immune system, and is activated by a specific
pathway.4 Overproduction of NO by nNOS in the central
nervous system has been implicated in the pathogenesis of
diverse neuronal disorders such as strokes,5 septic shock,6
seizures,7 migraine headaches,8 Alzheimer’s disease,9 Parkin-
son’s disease,10 and ALS.11 Recently, nNOS has also been
implicated to play a critical role in melanoma tumor
development and growth.12 In the immune system, excess
NO production from iNOS is also linked to inflammation and
NOSs are homodimeric enzymes; each monomer consists of
a reductase domain and an oxygenase domain. A C-terminal
reductase domain contains NADPH, FAD, and FMN
cofactors,17 and an N-terminal oxygenase domain contains
iron protoporphyrin IX (heme), where the substrate L-Arg
binds, and tetrahydrobiopterin (H4B) cofactors.18 H4B forms
tight H-bonds with the propionate of the heme A-ring and
provides an electron that is crucial for activating the heme-
bound dioxygen during the catalytic reaction.19 Although H4B
Received: March 17, 2014
Published: April 23, 2014
© 2014 American Chemical Society
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Figure 1. (a) Chemical structures of leads 1 and 2 and inhibitory activities; (b) overlay of inhibitors 1 (cyan) and 2 (orange) complexed with nNOS,
showing the heme (pink), H4B (yellow), and key residues (green) in the active site (PDB: 3NLM and 3N5W); and (c) the proposed scaffold derived
from compounds 1 and 2.
Figure 2. Prepared and tested molecules in this study.
binding is not required for dimerization, it interacts with both
subunits of the dimer by forming part of the dimerization
interface to enrich the structural stability of the dimer.20,21
For over a decade, our research groups have been interested
in the development of selective inhibitors of nNOS for the
treatment of neurodegenerative disease. Among diverse NOS
inhibitors, compounds 122 and 223 (Figure 1A) are the most
potent inhibitors for nNOS. They are spotlighted by excellent
isoform selectivity for 1 and easy synthesis for 2. Compound 1
has >700-fold selectivity against iNOS and >3800-fold
selectivity against eNOS. The X-ray crystal structures of 1
complexed with nNOS and eNOS24 reveal features of enzyme−
inhibitor interactions that form the basis for high potency and
selectivity (Figure 1B): the aminopyridine of 1 interacts with a
heme D-ring propionate via two H-bonds, as well as with
Tyr706 in a π−π stacking interaction. The pyrrolidine nitrogen
of 1 is located within hydrogen-bonding distances to both H4B
and the heme A-ring propionate, replacing a water molecule,
while the fluorophenyl ring stacks with the heme plane. Despite
the excellent isoform selectivity of this molecule, the
construction of the two unnaturally occurring chiral centers
of 1 is not efficient and requires multiple steps with a low
overall yield. This limits the opportunities for optimizing the
pharmacokinetic properties of the inhibitor and for carrying out
in vivo studies. Compound 2, the other potent nNOS inhibitor
(Ki = 25 nM), is only moderately selective (i/n = 58, e/n =
107) but can be prepared from commercial starting materials in
four chemical steps, in an excellent overall yield. The common
feature of 1 and 2 is that both utilize one aminopyridine to
make H-bonds with the heme D-ring propionate and to stack
with Tyr706 (Figure 1B). The additional H-bonds between the
other aminopyridine of 2 and Glu592 of nNOS anchor the
inhibitor to the substrate binding site above the heme in a
double-headed mode. However, 2, in contrast to 1, does not
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a
Scheme 1. Synthesis of 3R and 3S
a
Reagents and conditions: (a) 9 (2.5 equiv), BuLi (2.5 equiv), THF, 0 °C to −78 °C; (b) DEAD, DPPA, PPh3, THF, room temp., 12 h; (c) LiAlH4;
(d) (i) (S)-camphamic chloride, TEA, CH2Cl2, room temp., (ii) chiral resolution on a silica gel column; (e) conc HCl, AcOH, microwave, 150 °C, 5
h.
a
Scheme 2. Synthesis of 5R and 5S
a
Reagents and conditions; (a) BuLi, THF; (b) DIBAL; (c) (S)-t-butylsulfinamide, Ti(OEt)4; (d) 9, BuLi; (e) conc HCl, EtOH, microwave, 120 °C,
20 min.
directly interact with the H4B or with the propionate of the
heme A-ring. The lack of these interactions may explain the
moderate selectivity of 2 against iNOS and eNOS.
A possible strategy to confirm this structure-selectivity issue
is to install a new functional group on molecule 2 to create an
interaction with H4B and the heme A-ring propionate. This
approach will allow us to perform a SAR study in this area for a
new chemotype design in which the molecules will be easy to
prepare while still being highly isoform selective. From the
structure overlay of 1 and 2 (Figure 1B), we found that the
pyrrolidine amine of 1 could be replaced by the addition of an
amino group on the side chain of 2 (Figure 1C). The alignment
of an amino group with an adapting position of the middle
aromatic ring of 2, especially maintaining the structural
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a
Scheme 3. Synthesis of 5
a
Reagents and conditions: (a) (i) 9, BuLi, THF; (ii) 15; (b) DIBAL-H; (c) (i) MeNO2, TEA, (ii) AcCl; (d) 9, BuLi; (e) LAH; (f) conc HCl, EtOH,
microwave, 120 °C, 20 min.
a
Scheme 4. Synthesis of 7 and 8
a
Reagents and conditions: (a) (i) MeNO2, TEA, (ii) AcCl; (b) 9, BuLi; (c) 28, Pd(PPh3)2Cl2, CuI, PPh3, DEA, DMF; (d) Raney-Ni, H2, MeOH/
EtOH; (e) conc HCl/EtOH (1/2), microwave, 120 °C, 20 min.
similarity to the pyrrolidine amine of 1, was virtually performed
using Surflex-Sim in the Sybyl-X program. Although the R-
enantiomer of the α-amino derivative was predicted to interact
with H4B and a propionate of the heme, the preparation of the
other enantiomer was also desirable to confirm the stereo-
activity relationship. After 2-amino-4-methylpyridine was
selected as a head near the α-amino group, three different
meta-substituted aromatic rings were adapted as linkers, and
another 2-amino-4-methylpyridine or a 4-methylpyridine ring
was chosen as the second head in the hopes that it would stay
above the heme with a proposed interaction with Glu592. The
crystal structural information gathered with compounds bearing
an α-amino group (3−5) led to the introduction of an
aminomethyl group (6−8) to further improve the binding
affinity and selectivity. Figure 2 summarizes the prepared and
assayed compounds in this study.
CHEMISTRY
■
The synthesis of compounds 3R and 3S is shown in Scheme 1.
Benzyl alcohol 11 was prepared by coupling of 3-
bromomethylbenzaldehyde (9) with two equivalents of
lithiated pyrrolyl-4,6-lutidine (10). The hydroxyl group of 11
was then converted to benzyl azide 12 via a Mitsunobu reaction
with DPPA. Reduction of the azide with LiAlH4 gave the free
amine, which subsequently underwent amidation with (S)-
camphanic chloride to give a separable diastereomeric mixture.
Each pure diastereomer, 13a and 13b, was successfully isolated
using general silica gel column chromatography. An asymmetric
approach toward the target compounds using Ellman’s chiral
sulfinamide in the synthesis of 4R and 4S (Scheme 2) was not
successful; only inseparable diastereomeric mixtures were
produced. The (S)-camphanyl auxiliary and the two protecting
groups on the aminopyridine rings were removed together by
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Table 1. List of Ki Values and Selectivity of 3−8
a
Reference molecules that were reported previously.23
microwave-aided hydrolysis to give optically active 3R and 3S
(Scheme 1).
and 19b were removed by microwave-aided acidic hydrolysis to
give 5R and 5S in high yields.
Compounds 5R and 5S were prepared from 2,4-dimethyllu-
tidine and 15 using a five-step procedure (Scheme 2). Lithiated
2,4-dimethyllutidine was coupled with benzyl bromide 15 to
give nitrile 16. The cyano group of 16 was reduced to an
aldehyde (17) using DIBAL, which then underwent con-
densation with Ellman’s chiral sulfinamide to give (S)-N-tert-
butanesulfinyl aldimine 18 in a moderate yield. This
intermediate was coupled with lithiated pyrrolyl-4,6-lutidine 9
to give diastereomeric mixture 19a−b. Minor diasteromeric
product 19b eluted first, and the major product 19a eluted
second during silica gel column chromatography. Protecting
groups on the aminopyridine and t-butyl sulfinamide of 19a
Compound 6 was also prepared from benzyl bromide 15
using a six-step procedure (Scheme 3). Coupling of lithiated 9
with 15 gave nitrile 20, which was then reduced to aldehyde 21
by treatment with DIBAL. Condensation of 21 with nitro-
methane gave nitrovinyl compound 22 in good yield. Michael
addition of 22 with lithiated 9 produced nitro intermediate 23,
which was reduced to amine 24 using Raney-Ni under a
hydrogen atmosphere. Both aminopyridine protecting groups
were removed by microwave-aided hydrolysis to give 6 in good
yields.
Compounds 7 and 8 were prepared in five steps from
commercially available brominated pyridinylaldehydes 25a−b
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Table 2. Ki Values and Selectivity of 8S and 8R
Figure 3. Active site structures of rat nNOS in complex with (A) 3S (PDB: 4CTP), (B) 3R (PDB: 4CTQ), (C) 4 (PDB: 4CTR), and (D) 5 or 5S
(PDB: 4CTT). The Fo − Fc omit electron density for the inhibitor is displayed at the 2.5 σ contour level. The weaker density in the tail part of 4 and
5 indicates disordering. The major H-bonds are depicted with dashed lines. All crystal structure figures were prepared with PyMol (www.pymol.org).
(Scheme 4). Condensation of 25a−b with nitromethane in the
presence of TEA and acetyl chloride, followed by Michael
reaction with lithiated 4,6-dimethylpyridine afforded 27a−b.
Sonogashira coupling between 27a−b and alkyne 28, which
was prepared from 6-bromo-2-aminopyridine and trimethylsi-
lylacetylene, produced intermediate 29a−b. Raney nickel-
mediated hydrogenation of 29a−b yielded reduced product
30a−b. Finally, the protecting groups on the aminopyridine
rings were removed using microwave conditions to yield
products 7 and 8. A portion of intermediate 30b was protected
with Boc and then injected onto an OD-H chiral HPLC
column to separate the enantiomers. Each enantomerically pure
compound was deprotected via microwave-assisted acidic
hydrolysis to give optically active enantiomers 8S and 8R.
Our diverse approach for the chiral resolution of the final
deprotected compound (8) using CrownPak CR-(+), Chiralcel
OD-RH, and Whelk-O 1 chiral reverse phase HPLC columns,
and a Chiralcel OD-H chiral normal phase column with DEA,
were not successful. Chiral derivatization of the final compound
using Mosher’s acid chloride, (S)-camphanyl chloride, and (S)-
mandelic chloride also were not successful because the
molecules have multiple reactive amines.
RESULTS AND DISCUSSION
■
In Vitro Inhibitory Assays. The NOS isoform assays
involved subjecting 3−8 to an oxyhemoglobin NO capture
assay using a Biotek Gen5 microplate reader. IC50 values for
each compound were determined in duplicate or triplicate
using dose−response curves with nine concentration points (1
pM−3 mM). The standard deviation of the assays were less
than 15% with nNOS or iNOS and less than 25% with eNOS.
The inhibition constants (Ki) of these compounds were
determined from the IC50 and Km values (rat nNOS = 1.3
μM; murine iNOS = 8.2 μM; and bovine eNOS = 1.7 μM) for
all three NOS isoforms using the following relationship: Ki =
IC50/(1 + [S]/KM)
The selectivity of antagonism of nNOS relative to iNOS or
eNOS was determined by calculating the ratios of the Ki values
with iNOS or eNOS to those with nNOS. Compounds 3−8,
having various amino functional groups, were found to have
moderate to excellent selectivity (50−2822 of e/n, 36−273 of
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i/n) and moderate to good binding affinity (24−4370 nM) to
nNOS. All of the synthesized molecules as well as three
reference molecules (31, 32, and 2; for comparative purpose)
and their associated activities are shown in Tables 1 and 2.
The compounds with an α-amino (or a hydroxyl) group and
symmetric double heads, 3S, 3R, and 4, exhibited about a 100
nM binding affinity to nNOS and modest selectivity against
eNOS and iNOS, whereas 5R and 5S, having α-amino tailed
asymmetric double heads, showed poor potency. Compounds
6−8, having an α-aminomethyl tail with two aminopyridine
head groups, showed improved potency by 1- to 5-fold. The
orientation of the nitrogen on the middle aromatic ring was
important; compound 7 was a relatively poor inhibitor of
nNOS when the nitrogen was located in the narrow arc of the
middle ring. Although the α-aminomethyl derivatives 6, 7, and
8 did not display improved binding affinity for nNOS
compared with that of their parent molecules 31, 32, and 2,
it is noteworthy that all of those α-aminomethyl derivatives
have better selectivity (Table 1) against iNOS and eNOS. In
general, the α-aminomethyl derivatives are 2- to 3-fold (i/n)
and 3- to 10-fold (e/n) more selective than their parent
molecules. To further explore the inhibition potency and
selectivity of racemic 8, each enantiomer was prepared and
assayed with the three NOS isoforms. Compound 8R, the (R)-
enantiomer of 8, showed excellent potency (Ki = 24 nM) for
nNOS with a 273-fold selectivity over iNOS and a 2822-fold
selectivity over eNOS, the best in the series.
We also assayed compound 8 against human nNOS to
explore whether the interactions are acceptable for the human
isoform as well. Human nNOS is very similar to rat nNOS,
except that the hydrophobic pocket surrounded by Met341,
Leu337, and Tyr706 in rat nNOS is replaced by Met340,
His342, and Tyr711. This pocket is where the second
headgroup of this series of double-headed inhibitors fits. The
inhibitory potency of 8 for human nNOS is 90 nM, similar to
that (70 nM) of compound 1,25 which is our most potent
human nNOS inhibitor with potential selectivity over human
iNOS and eNOS.
Structure−Activity Relationship Studies. To aid in the
structure−activity relationship studies for the series of double-
headed aminopyridine compounds, we first determined the
crystal structures of nNOS in complex with 3S, 3R, 4, and 5
that bear an α-amino group (Table 1). Both 3S and 3R are able
to bind to nNOS with both aminopyridine heads involved in H-
bonds, one with Glu592 and the other with the propionate of
the heme D-ring, respectively (Figure 3). In contrast, the parent
compound (31) showed only one aminopyridine H-bonded
with Glu592, while the rest of the inhibitor was badly
disordered in structure (unpublished data). Introducing an α-
amino group next to the center phenyl ring in 3S and 3R helps
to stabilize the double-headed binding. Interestingly, the
binding orientation of 3 is dependent on the chirality leading
to different positions for the α-amine. The α-amino group of 3S
is next to the aminopyridine that H-bonds with Glu592 and
points downward to the heme (Figure 3A), while the α-amino
group of 3R is on the side of the aminopyridine that H-bonds
with the propionate of the heme D-ring (Figure 3B). This
amino group can H-bond with a conserved water molecule that
is bridging between the H4B and the propionate of heme A-ring
rather than replace the water as expected from modeling.
When the α-amino group is replaced with a hydroxyl group
in 4, the compound can no longer achieve a double-headed
binding (Figure 3C). Instead, the aminopyridine next to the
hydroxyl group forms H-bonds with Glu592 so that the
hydroxyl points toward the heme, similar to the position of the
amino group in 3S, but the second aminopyridine is partially
disordered, hanging in the active site access channel and
making only van der Waals contacts with the protein.
The structure of nNOS complexed with 5 was also checked
to explore the binding mode for the compound bearing
asymmetric double head groups. As expected, only the
aminopyridine is able to anchor the inhibitor above the heme
via H-bonds with Glu592, while the 4-methylpyridine head is
poorly defined in the active site access channel (Figure 3D).
Although a racemic mixture of 5S and 5R was used in crystal
soaking, only the 5S enantiomer was picked up by the nNOS in
the crystal, which agrees with the better affinity of 5S compared
to that of 5R (Table 1). The α-amino nitrogen is also able to
H-bond with Glu592, which forces the center phenyl ring to
bend toward the heme, causing a distortion of the propionate of
the heme D-ring. Both the α-amino group and the amino-
pyridine ring in 5S wrap around the Glu592 side chain, which
resembles the binding mode of the substrate, L-Arg, where the
α-amino and guanidinium groups embrace Glu592. Note that
the α-amino group from the same S-chiral centers of 3S and 5S
ends up with two totally different positions, H-bonding either
with a water molecule (3S) or with the heme D-ring propionate
(5S), because the binding orientation of 3S and 5S is flipped by
180° relative to each other.
Both 3S and 5S showed modest isoform selectivity for nNOS
over eNOS. We, therefore, also determined the crystal
structures of 3S, 3R, and 5S bound to eNOS. In the eNOS-
3S structure (data not shown), only one aminopyridine was
visible in the electron density that H-bonds with Glu363; the
rest of 3S was disordered, which prevented us from obtaining a
fully refined structure. Nevertheless, double-headed binding, as
seen in the nNOS-3S structure, can be ruled out because the
Tyr477 side chain still H-bonds with the propionate of the
heme D-ring, thus blocking the access of the second
aminopyridine to the site. The one-headed binding of 3S
results in its poorer affinity to eNOS, leading to ∼500-fold
selection for nNOS over eNOS (Table 1). In contrast, 3R binds
to eNOS in a bouble-headed mode, as seen in Figure 4A, which
is almost no different from what is observed in the nNOS-3R
structure (Figure 3B), thus giving 5-fold improved affinity to
eNOS compared with that of 3S (Table 1). Although having a
similar binding mode to both nNOS and eNOS, 3R still shows
118-fold better affinity to nNOS than eNOS. Below, we discuss
in more detail what gives rise to the isoform selection when the
binding mode for an inhibitor is identical in the two isoforms.
The structure of eNOS-5S shows some intriguing features
(Figure 4B). While the aminopyridine makes H-bonds with
Glu363, which is the same as that seen in the nNOS-5S
structure, the α-amino group does not directly H-bond with
Glu363. Instead, a water molecule is bridging in between. This
binding preference resembles what we have observed for some
nNOS selective dipeptide amide inhibitors, where the α-amino
group of the dipeptide inhibitor made a direct H-bond with
Glu592 in nNOS but was bridged by a water molecule in
between the α-amino of the inhibitor and Glu363 in eNOS.26
This different binding preference results from the better
electrostatic stabilization that the α-amino group of the
inhibitor experiences by its proximity next to two negatively
charged residues, Glu592 and Asp597 in nNOS, compared with
just one negatively charged residue, Glu363 and Asn368 in
eNOS. Therefore, the loss of the electrostatic interactions in
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Figure 4. Active site structures of bovine eNOS in complex with (A)
3R (PDB: 4CTY) and (B) 5 or 5S (PDB: 4CTZ). The Fo − Fc omit
electron density for the inhibitor is displayed at the 2.5 σ contour level.
The major H-bonds are depicted with dashed lines.
eNOS explains the 363-fold selectivity observed for 5S (Table
1).
The double-headed compounds with only an α-amino group
on the side chain (3S, 3R, 4, and 5S) do not quite reach the
position of the pyrroline amine of parent compound 1 that can
replace a water molecule (Figure 1B). This might be why the
low nanomolar affinity and good isoform selectivity of 1 had
not been achieved with 3−5. We, therefore, introduced an
aminomethyl group to racemic compounds 6, 7, and 8 (Figure
2). We also designed three different center aromatic rings to
further explore the influence of the polarity of this ring to the
potency and selectivity of inhibitors. The inhibitory assays
indicated that 6 and 8 indeed have improved potency (Table
1). The crystal structures of 6 and 8 bound to nNOS showed
that they share a similar double-headed binding mode (Figure
5). Although the racemic samples were used for crystal
preparation, the resulting structures were dominated by the
R-enantiomer in both cases. The structure of nNOS-6R (Figure
5A) overlays well with that of nNOS-3R (Figure 3B) except
that the aminomethyl group of 6 replaces a water molecule,
thereby allowing it to make H-bonds with both the H4B and the
propionate of heme A-ring, as was expected from the design.
The better interactions from the aminomethyl group of 6 to
both the H4B and the propionate afford a 2-fold improvement
in potency compared with that of 3 (Table 1).
Figure 5. Active site structures of rat nNOS in complex with (A) 6
(PDB: 4CTU), (B) 8 or 8R (PDB: 4CTW), and (C) 7 or 7S (PDB:
4CTV). The Fo − Fc omit electron density for the inhibitor is
displayed at the 2.5 σ contour level. The major H-bonds are depicted
with dashed lines. The weak H-bond in nNOS-8R is labeled with a
distance in Å.
at an ortho-position relative to the other two substituents in
contrast to the meta-position in 8. To our surprise, 7 binds to
nNOS (Figure 5C) in an orientation that is 180° flipped from
that of 8 (Figure 5B). Moreover, it is the S-enantiomer of 7 that
populates the structure, even though a racemic mixture of 7 was
used in crystal preparation. In this binding orientation, the
aminomethyl group of 7 makes a tight H-bond with Glu592.
The positions of the neighboring aminopyridine, H-bonding to
Glu592, and the center phenyl ring, bending toward the heme,
overlay well with their counterparts in 5S (Figure 3D). In
addition, the second aminopyridine of 7 can also reach the site
of the heme D-ring propionate in a double-headed mode
(Figure 5C). Although in this flipped binding orientation, 7
shows poorer potency than 6 or 8, it does have an impressive
isoform selectivity (Table 1). We have tried, but failed, to get
Compound 8 exhibits even better potency with nNOS than
does 6 (Table 1), which may result from the extra nitrogen on
the center pyridine ring of 8 (Figure 5B) versus the phenyl ring
in 6 (Figure 5A). This pyridine ring in 8 is pulled a bit further
up toward Asp597 in nNOS than is the phenyl ring of 6,
making water bridged H-bonds with the acidic residue and
another weak H-bond (3.4 Å) with Gln478 (Figure 5B). We
have also designed compound 7, having the pyridine nitrogen
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an eNOS-7 structure because the inhibitor was badly
disordered except for the aminopyridine that H-bonds with
Glu363 (data not shown). From the known structure of eNOS-
5S (Figure 4B), we reasoned that the poor binding affinity of 7
toward eNOS must result from its inability to establish direct
H-bonds with Glu363 by its aminomethyl group because of the
more electropositive environment of Glu363 and Asn368 in
eNOS, which would not allow a positively charged amino-
methyl group to enter the pocket.
Compound 8 exhibits the best potency toward nNOS in the
series, which prompted us to further explore the effects of
chirality on the inhibitory potency and selectivity. Enantiopure
8S and 8R were synthesized, and crystal structures of both
enantiomers bound to nNOS and eNOS were determined.
Indeed, 8R gives the best potency (24 nM) with nNOS and
2822-fold selectivity for nNOS over eNOS (Table 2). Both 8S
and 8R show very similar double-headed binding to nNOS; the
nNOS-8R structure is the same as that of nNOS-8 (Figure 5B),
while the nNOS-8S structure (Figure 6A) can be, more or less,
superimposed on nNOS-8R, with the only exception being
around the chiral center. Also, the center pyridine of 8S can
make a better H-bond (2.9−3.0 Å) with Gln478. Regardless of
the chirality, the aminomethyl group in both 8S and 8R can
replace a water molecule equally well, thus making direct H-
bonds with both the H4B and the propionate of the heme D-
ring. The similarity in structure seems to be reasonable
considering the ∼3-fold difference in potency between 8S and
8R. The eNOS-8S structure (Figure 6B) shows a one-headed
binding mode since the access to the heme D-ring propionate
from the second aminopyridine of 8S is blocked by the Tyr477
side chain, which remains in its position, H-bonding with the
same propionate. Therefore, the second aminopyridine group is
poorly defined in structure, although the first aminopyridine
and aminomethyl groups maintain the H-bonding interactions
that are also observed in nNOS-8S (Figure 6A). To our
surprise, we found no significant difference in the binding mode
of 8R to eNOS (Figure 6C) versus that to nNOS (Figure 5B).
With a double-headed binding mode, the center pyridine of 8R
in eNOS points toward the direction of Asn368, just like its
counterpart in nNOS, which points toward Asp597. In contrast,
the central pyridine ring of 8S in eNOS, in a one-headed
binding, bends away from Asn368 and makes a better H-bond
with Gln249 (Figure 6B). This may be part of the reason that
8S binds better to eNOS than does 8R (Table 2).
The structural binding mode alone cannot explain the 2822-
fold isoform selectivity of 8R for nNOS over eNOS. This is not
the first time that a highly selective NOS inhibitor shows an
almost identical binding mode to nNOS and eNOS. The
previous examples were parent compound 122 and its amino-
analogue.24 To better understand the basis for isoform
selectivity, we turned to a computational approach that proved
useful in previous studies with NOS inhibitors.24 Using the
MM-PBSA approach, we first calculated the ΔG for 8R bound
to nNOS. The value from this calculation is called PBtotal and
includes the solvation and enthalpic terms but not the decrease
in entropy upon inhibitor moving from solution to the active
site. Since we are examining exactly the same inhibitor binding
to nNOS and eNOS, ignoring this entropic contribution
introduces little error. Table 3 provides the results of the MM-
PBSA calculations.
Figure 6. Active site structures of rat nNOS in complex with (A) 8S
(PDB: 4CTX) and of bovine eNOS with (B) 8S (PDB: 4CU1) and
(C) 8R (PDB: 4CU0). The Fo − Fc omit electron density for inhibitor
is displayed at 2.5 σ contour level. The major H-bonds are depicted
with dashed lines. The weak H-bond in eNOS-8R is labeled with a
distance in Å.
Table 3. Results of the MM-PBSA Calculations (kcal/mol)
protein
ELE
−1018.7 −44.92 −41.1 −92.2
−818.7 −41.82 −9.57 −57.51
VDW
PBele
PBtotal
ΔGcalc
ΔGexp
a
nNOS
eNOS
−10.4
−6.5
−10.4
−5.7
a
ΔGcalc for nNOS was normalized to match ΔGexp by dividing PBtotal
by ΔGexp and then using this conversion factor to compute ΔGclac for
eNOS. ELE and VDW are the electrostatic and van der Waals
contributions, respectively, to inhibitor binding. PBele is the sum of
ELE and the reaction field energy calculated with the Poisson−
Boltzmann equation and thus represents the total electrostatic
component of inhibitor binding. PBtotal is the sum of all energies
and represents the total free energy of binding uncorrected for the
entropic contribution of the inhibitor moving from solution to the
active site.
This approach comes to within 0.8 kcal/mol of estimating
the ΔG of binding to eNOS and also shows that the primary
difference between eNOS and nNOS is the more favorable
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the compounds was evaluated on an Beckman Gold reverse phase
analytical HPLC system using an Phenonemex Gemini C-18 (4.6 ×
250 mm, 5 μm) or Luna C-8 (4.6 × 250 mm, 5 μm) reverse phase
columns with UV absorbance and evaporative light scattering
detection. Purities of all compounds that were subjected to the
biological assay were >95%. The direct chiral resolutions of racemic 8R
and 8S were performed on a Beckman Gold HPLC system using a
Chiralcel OD-H HPLC column (Daicel, 250 × 4.6 mm i.d., 5 mm).
Hexanes and isopropanol (85−92% hexanes) were used as the mobile
phases. The operation temperature was 25 °C, and the flow rate was
0.8 mL/min with 254 nm UV detection. Optical rotations were
measured on a PerkinElmer Model 341 digital readout polarimeter.
General Procedure for the Deprotection of 2-(2,5-Dimethyl-
1H-pyrrol-1-yl)pyridine Derivatives Using Microwave Irradi-
ation:27 Method A. To a 5 mL microwave vial equipped with a
magnetic stir bar was added the protected aminopyridine (1.0 mmol),
ethanol (2.5 mL), and concentrated hydrochloric acid (0.5 mL). After
being capped, the vial was shaken vigorously and then heated in the
microwave irradiator for 20 min at 120 °C (as recorded via the IR
sensor of the microwave instrument). After being cooled to room
temperature, the reaction mixture was concentrated in vacuo and
purified by flash column chromatography using a SiliaSep C18 flash
cartridge (25g, 40−63 μm/230−400 mesh, pore size 60 Å) with 5 to
80% MeOH in water as the mobile phase.
electrostatic interactions between the inhibitor and nNOS than
those in eNOS. We attribute this primarily to Asp597 in nNOS
vs Asn368 in eNOS, even though Asp597 does not directly
contact the inhibitor. If the active site pocket exhibits a
relatively low dielectric milieu, then long-range electrostatic
stabilization by Asp597 on both the aminomethyl group and
the central pyridine of 8R could be quite substantial. However,
when the binding of 8R to eNOS is forced into the same
double-headed mode as that with nNOS, the penalty toward
electrostatic interactions leads to poorer binding and, therefore,
higher isoform selectivity.
CONCLUSIONS
■
A series of α-amino functionalized aminopyridine derivatives
(3−8) were designed based on the comparison and rationale
that the pyrrolidine of lead 1, interacting with a heme
propionate and H4B, is the key to isoform selectivity.
Therefore, an α-amino or aminomethyl group has been
installed in the other lead (2) to probe the structure−selectivity
relationship, while providing a substantially simple scaffold that
retains selectivity (Figure 1). In general, the symmetric double-
headed aminopyridine compounds (3 and 4) showed better
potency than the asymmetric one (5) because the former can
establish H-bonds through both head groups. Although a
simple α-amino group installed on the side chain can stabilize
the double-headed inhibitor binding mode, only the amino-
methyl group is long enough to reach a water site. By replacing
the water molecule, the inhibitor is able to make H-bonds with
both the H4B and the propionate of the heme A-ring. These
interactions seem to be crucial to gain the isoform selectivity for
the inhibitors. Compound 8R, the best inhibitor discovered in
this study, exhibits excellent nNOS potency (24 nM) and
isoform selectivity (273-fold for i/n and 2822-fold for e/n). It
also showed <100 nM potency for human nNOS. However, the
crystal structures of 8R complexed with nNOS and eNOS share
an almost identical binding mode, which is similar to what was
observed with parent compound 1.22 A free energy calculation
indicated that the different electrostatic environments in the
active site of the two NOS isoforms give rise to the isoform
distinct binding affinity, even for inhibitors that exhibit the
same binding mode. The knowledge gained by this study will
provide the basis for the design of further novel and isoform-
selective inhibitors.
(S)-6-(2-Amino-2-(3-(2-(6-amino-4-methylpyridin-2-yl)ethyl)-
phenyl)ethyl)-4-methylpyridin-2-amine (3S) and (R)-6-(2-Amino-2-
(3-(2-(6-amino-4-methylpyridin-2-yl)ethyl)phenyl)ethyl)-4-methyl-
pyridin-2-amine (3R). The title compounds were synthesized using
general method A. 3S (55 mg, 58%) was prepared as a pale yellow gel
from 13a (180 mg, 0.26 mmol), and 3R (50 mg, 60%) was prepared as
a pale yellow gel from 13b (160 mg, 0.23 mmol). 3S, [α]20D 1= +72.1°
(c 3 g/L, MeOH); 3R, [α]20 = −79.2° (c 2 g/L, MeOH). H NMR
(500 MHz, MeOD) δ 7.22−D7.17 (m, 1H), 7.14 (qt, J = 2.9, 1.9, 1.4
Hz, 2H), 7.05 (dt, J = 7.4, 1.6 Hz, 1H), 6.25 (s, 1H), 6.24 (ss, 2H),
6.18 (s, 1H), 4.21 (t, J = 7.1 Hz, 1H), 2.89 (dd, J = 9.6, 6.4 Hz, 2H),
2.81 (d, J = 7.1 Hz, 2H), 2.79−2.71 (m, 2H), 2.13 (s, 3H), 2.11 (s,
3H); 13C NMR (126 MHz, MeOD) δ 160.86, 160.70, 159.73, 157.35,
151.03, 150.86, 145.85, 143.10, 129.46, 128.35, 127.82, 125.18, 115.89,
114.67, 108.22, 107.92, 57.15, 48.09, 40.64, 37.32, 21.08, 21.05. HRMS
(ESI): calcd for C22H28N5 [M + H]+, 362.2339; found, 362.2338.
(R)-6-(2-Amino-2-(3-(2-(4-methylpyridin-2-yl)ethyl)phenyl)ethyl)-
4-methylpyridin-2-amine (5R) and (S)-6-(2-Amino-2-(3-(2-(4-meth-
ylpyridin-2-yl)ethyl)phenyl)ethyl)-4-methylpyridin-2-amine (5S).
Method A was used to prepare 5R (153 mg, 65%, colorless gel)
from 19a (360 mg, 0.682 mmol) and 5S (22 mg, 60%, colorless gel)
from 19b (55 mg, 0.104 mmol). 5S, [α]20 = −20.0° (c 2 g/L,
D
MeOH); 5R, [α]20D = +19.2° (c 2 g/L, MeOH). 1H NMR (500 MHz,
MeOD) δ 8.54 (d, J = 6.0 Hz, 1H), 7.79 (s, 1H), 7.68 (d, J = 5.9 Hz,
1H), 7.63 (s, 1H), 7.51−7.37 (m, 2H), 7.36−7.29 (m, 1H), 6.68 (s,
2H), 4.87 (d, J = 7.8 Hz, 1H), 3.51 (dd, J = 14.7, 7.9 Hz, 1H), 3.42−
3.34 (m, 3H), 3.21−3.14 (m, 2H), 2.62 (s, 3H), 2.34 (s, 3H); 13C
NMR (126 MHz, MeOD) δ 162.50, 158.89, 156.51, 156.12, 143.95,
142.17, 141.24, 137.24, 131.02, 130.88, 129.16, 128.68, 127.00, 126.98,
116.76, 112.22, 55.32, 38.40, 35.76, 22.36, 21.96. HRMS (ESI): calcd
for C22H27N4 [M + H]+, 347.2230; found, 347.2229.
EXPERIMENTAL SECTION
■
Materials, Synthetic Methods, and Molecular Character-
ization. All starting reagents and solvents were purchased from
Sigma-Aldrich, TCI America, and Matrix Scientific and were used
without further purification. Solvents were purified by passage through
a solvent column composed of activated alumina and a supported
copper redox catalyst. Moisture or oxygen-sensitive reactions were
performed under an atmosphere of dry N2 or argon. A Biotage
Initiator microwave system was used for microwave-assisted reactions.
Thin-layer chromatography was carried out on E. Merck precoated
silica gel 60 F254 plates. An Agilent 971-FP flash purification system
with various SiliaSep (Silicycle, 40−63 μm, 60 Å) prepacked silica gel
6-(3-Amino-2-(3-(2-(6-amino-4-methylpyridin-2-yl)ethyl)phenyl)-
propyl)-4-methylpyridin-2-amine (6). Method A was used to prepare
1
6 (144 mg, 43%, pale yellow gel) from 24 (475 mg, 0.89 mmol). H
NMR (500 MHz, MeOD) δ 7.43 (s, 1H), 7.32 (t, J = 7.6 Hz, 1H),
7.21 (dt, J = 7.5, 1.8 Hz, 2H), 6.68 (s, 2H), 6.62 (s, 1H), 6.50 (s, 1H),
3.60−3.51 (m, 1H), 3.40−3.34 (m, 2H), 3.29 (dd, J = 14.3, 6.1 Hz,
1H), 3.07−3.00 (m, 5H), 2.38 (s, 3H), 2.28 (s, 3H); 13C NMR (126
MHz, MeOD) δ 159.08, 158.70, 155.78, 150.00, 147.22, 142.47,
139.40, 130.61, 129.59, 129.31, 127.58, 116.07, 114.88, 111.16, 110.83,
44.87, 44.69, 37.99, 35.93, 35.61, 22.01, 21.91. HRMS (ESI): calcd for
C23H30N5 [M + H]+, 376.2496; found, 376.2502.
1
cartridges was used for flash column chromatography. H NMR and
13C NMR spectra were recorded in the indicated solvent on a Bruker
Avance-III (500 and 126 MHz for 1H and 13C, respectively)
spectrometer. Chemical shifts are reported as δ values in parts per
million downfield from TMS (δ 0.0) as the internal standard in
CDCl3. MS was performed on a system consisting of an electrospray
ionization (ESI) source in a Thermo Finnigan LCQ and Bruker
amaZon SL mass spectrometer. High-resolution mass spectra were
obtained using an Agilent 6210 LC-TOF spectrometer. The purity of
6-(3-Amino-2-(6-(2-(6-amino-4-methylpyridin-2-yl)ethyl)pyridin-
2-yl)propyl)-4-methylpyridin-2-amine (7). Method A was used to
prepare 7 (87 mg, two step 43%, pale yellow gel) from crude 30a (300
1
mg). H NMR (500 MHz, MeOD) δ 7.83 (m, 1H), 7.38 (m, 1H),
7.33 (m, 1H), 6.73 (s, 1H), 6.70 (s, 1H), 6.65 (s, 1H), 6.45 (s, 1H),
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3.80 (s, 1H), 3.61 (dd, J = 12.9, 9.0 Hz, 1H), 3.37 (t, J = 4.7 Hz, 1H),
3.23 (m, 6H), 2.39 (s, 3H), 2.29 (s, 3H); 13C NMR (126 MHz,
MeOD) δ 160.66, 159.10, 158.71, 157.66, 155.90, 155.79, 149.94,
146.66, 124.54, 124.02, 115.82, 114.76, 111.48, 110.92, 44.82, 43.32,
37.01, 36.55, 32.91, 21.99, 21.89. HRMS (ESI): calcd for C22H29N6 [M
+ H]+, 377.2448; found, 377.2455.
was quenched by sequential addition of i-PrOH (1 mL), water (15
mL), and then 1 M NaOH aq (10 mL). The organic materials were
extracted with ethyl acetate (25 mL) three times, and the combined
organic layers were washed with brine, dried over anhydrous MgSO4,
and then concentrated in vacuo to yield a crude amine (2-(6-(2,5-
dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)-1-(3-(2-(6-(2,5-di-
methyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)phenyl)ethan-1-
amine). To the solution of the produced amine in dichloromethane
(25 mL) was added (S)-camphamic chloride (325 mg, 1.5 mmol) and
triethylamine (0.279 mL, 2.0 mmol) at 0 °C. After being stirred for 12
h at room temp, the reaction mixture was quenched with H2O (50
mL). After the addition of dichloromethane (25 mL), the organic layer
was partitioned, dried with MgSO4, concentrated by rotary
evaporation, and purified by flash chromatography (EtOAc/hexanes)
to yield diastereomeric pure compounds 13a (180 mg, 28%) and 13b
(161 mg, 25%) as a pale yellow oil. 13a: 1H NMR (500 MHz, CDCl3)
δ 7.19 (t, J = 7.6 Hz, 1H), 7.14 (t, J = 1.8 Hz, 1H), 7.11 (dt, J = 7.6, 1.5
Hz, 1H), 7.06 (dt, J = 7.5, 1.4 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 6.93
(s, 1H), 6.92 (s, 1H), 6.84 (ss, 2H), 5.87 (s, 2H), 5.86 (s, 2H), 5.51
(td, J = 8.7, 5.8 Hz, 1H), 3.26 (dd, J = 14.0, 5.9 Hz, 1H), 3.17 (dd, J =
14.0, 9.0 Hz, 1H), 3.01 (q, J = 2.5, 1.8 Hz, 4H), 2.40 (ddd, J = 13.4,
10.4, 4.1 Hz, 1H), 2.36 (s, 3H), 2.31 (s, 3H), 2.11 (s, 6H), 2.06 (s,
6H), 1.90−1.73 (m, 2H), 1.61 (ddd, J = 12.7, 8.9, 4.2 Hz, 1H), 1.04 (s,
3H), 0.97 (s, 3H), 0.57 (s, 3H); 13C NMR (126 MHz, CDCl3) δ
178.24, 165.99, 160.78, 157.36, 151.82, 151.54, 149.74, 142.05, 141.27,
128.80, 128.51, 128.49, 127.75, 126.68, 124.04, 123.29, 122.58, 120.64,
120.12, 106.85, 106.73, 92.49, 55.28, 53.72, 53.23, 44.37, 39.63, 35.88,
30.27, 29.03, 21.04, 20.95, 16.61, 16.18, 13.36, 13.30, 9.72; MS (ESI)
m/z 720.36 [M + Na]+. 13b: 1H NMR (500 MHz, CDCl3) δ 7.16 (t, J
= 7.6 Hz, 1H), 7.14−7.09 (m, 2H), 7.06 (dd, J = 7.7, 1.6 Hz, 1H), 7.03
(dt, J = 7.6, 1.4 Hz, 1H), 6.89 (s, 2H), 6.84 (s, 2H), 5.87 (s, 2H), 5.84
(s, 2H), 5.44 (td, J = 8.2, 6.1 Hz, 1H), 3.27 (dd, J = 14.0, 6.1 Hz, 1H),
3.20 (dd, J = 14.0, 8.3 Hz, 1H), 2.98 (d, J = 2.0 Hz, 4H), 2.40−2.34
(m, 4H), 2.32 (s, 3H), 2.11 (s, 6H), 2.04 (s, 6H), 1.85 (ddd, J = 13.0,
10.8, 4.2 Hz, 1H), 1.67 (ddd, J = 13.0, 9.3, 4.1 Hz, 1H), 1.59 (ddd, J =
13.2, 9.3, 3.8 Hz, 1H), 1.04 (s, 3H), 0.98 (s, 3H), 0.74 (s, 3H); 13C
NMR (126 MHz, CDCl3) δ 178.22, 166.28, 160.74, 157.33, 151.67,
149.73, 141.92, 141.45, 128.66, 128.49, 127.63, 126.64, 124.05, 123.42,
122.51, 120.77, 120.12, 106.75, 106.70, 92.30, 55.25, 54.01, 53.37,
43.95, 39.64, 35.87, 30.23, 29.06, 21.04, 21.01, 16.69, 16.43, 13.30,
13.24, 9.69; MS (ESI) m/z 720.37 [M + Na]+.
3-(2-(4-Methylpyridin-2-yl)ethyl)benzonitrile (16). To a solution of
14 (0.804 g, 7.5 mmol) in dry THF (30 mL) was added n-BuLi (1.6 M
solution in hexanes, 4.68 mL, 7.5 mmol), and the reaction was stirred
for 30 min at 0 °C. This solution (red color) was added dropwise to a
solution of 3-(bromomethyl)benzonitrile (15, 1.16 g, 6.0 mmol) in
THF (20 mL) at −78 °C using a cannula, until the solution became
pale red. The reaction mixture was allowed to stir for an additional 20
min and then quenched with H2O (50 mL). After the addition of ethyl
acetate (100 mL), the organic layer was partitioned, dried with
MgSO4, and concentrated in vacuo. The resulting yellow oil was
purified by flash chromatography (EtOAc/hexanes) to yield the title
compound as a yellow oil (680 mg, 51%). 1H NMR (500 MHz,
CDCl3) δ 8.40 (d, J = 5.0 Hz, 1H), 7.52−7.39 (m, 3H), 7.36 (t, J = 7.6
Hz, 1H), 6.96 (dd, J = 5.1, 1.5 Hz, 1H), 6.89 (d, J = 1.6 Hz, 1H),
3.19−2.94 (m, 4H), 2.30 (s, 3H); 13C NMR (126 MHz, CDCl3) δ
159.85, 149.15, 147.57, 143.05, 133.15, 132.02, 129.78, 129.12, 123.96,
122.49, 119.04, 112.24, 39.44, 35.32, 20.97; MS (ESI) m/z 222.97 [M
+ H]+.
(S)-6-(3-Amino-2-(5-(2-(6-amino-4-methylpyridin-2-yl)ethyl)-
pyridin-3-yl)propyl)-4-methylpyridin-2-amine (8S) and (R)-6-(3-
Amino-2-(5-(2-(6-amino-4-methylpyridin-2-yl)ethyl)pyridin-3-yl)-
propyl)-4-methylpyridin-2-amine (8R). Method A was used to
prepare 8 (45 mg, 41%) from racemic 30b (180 mg), 8S (21 mg,
69%) from enantiomer 30c-1 (43 mg), and 8R (15 mg, 79%) from
enantiomer 30c-2 (32 mg). 8S, [α]20 = −85.3° (c 4 g/L, MeOH);
D
8R, [α]20D = +84.9° (c 3 g/L, MeOH). 1H NMR (500 MHz, MeOD)
δ 8.44 (s, 2H), 8.20 (d, J = 2.2 Hz, 1H), 6.74 (s, 1H), 6.71 (s, 1H),
6.65 (s, 1H), 6.58 (s, 1H), 3.75−3.66 (m, 1H), 3.50 (dd, J = 13.1, 9.9
Hz, 1H), 3.42 (dd, J = 13.1, 5.3 Hz, 1H), 3.39−3.34 (m, 1H), 3.13 (td,
J = 9.8, 9.4, 5.6 Hz, 5H), 2.39 (s, 3H), 2.31 (s, 3H); 13C NMR (126
MHz, MeOD) δ 159.12, 158.75, 155.95, 155.84, 149.33, 147.89,
146.49, 138.99, 138.68, 136.11, 116.10, 114.96, 111.51, 111.08, 44.14,
42.24, 37.57, 34.83, 32.89, 21.99, 21.89. HRMS (ESI): calcd for
C22H29N6 [M + H]+, 377.2448; found, 377.2454.
2-(6-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)-1-(3-(2-
(6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)phenyl)-
ethanol (11). To a solution of 9 (1.0 g, 5.0 mmol) in THF (25 mL)
was added n-BuLi (1.6 M solution in hexanes, 3.12 mL, 5.0 mmol),
and the reaction was stirred for 30 min at 0 °C. This mixture was
transferred to a solution of 3-(bromomethyl)benzaldehyde (10, 396
mg, 2.0 mmol) in THF (25 mL) at −78 °C via a cannula. The reaction
mixture was allowed to stir for an additional 20 min and then
quenched with H2O (50 mL). After the addition of ethyl acetate (50
mL), the organic layer was partitioned, dried with MgSO4, and
concentrated by rotary evaporation. The resulting yellow oil was
purified by flash chromatography (EtOAc/hexanes) to yield 2,5-
dimethylpyrrole-protected product 11 as a yellow oil (663 mg, 64%).
1H NMR (500 MHz, CDCl3) δ 7.33−7.25 (m, 3H), 7.14−7.09 (m,
1H), 6.99 (s, 1H), 6.95 (s, 2H), 6.89 (s, 1H), 5.93 (s, 2H), 5.91 (s,
2H), 5.15 (m, 1H), 3.17 (m, 2H), 3.10 (m, 4H), 2.42 (s, 3H), 2.40 (s,
3H), 2.18 (s, 6H), 2.16 (s, 6H); 13C NMR (126 MHz, CDCl3) δ
161.01, 159.29, 151.62, 151.13, 150.28, 149.53, 143.99, 141.63, 128.56,
128.50, 128.40, 127.51, 125.97, 123.52, 123.46, 122.67, 120.77, 120.10,
106.93, 106.68, 73.25, 45.73, 39.78, 36.64, 35.98, 24.72, 21.10, 13.31;
MS (ESI) m/z 519.32 [M + H]+.
2-(2-Azido-2-(3-(2-(6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyri-
din-2-yl)ethyl)phenyl)ethyl)-6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-meth-
ylpyridine (12). To a stirred solution of triphenylphosphine (314 mg,
1.2 mmol) and diethylazodicarboxylate (210 mg, 1.2 mmol) in dry
THF (15 mL), diphenylphosphonyl azide (331 mg, 1.2 mmol) was
added dropwise followed by a THF solution of 11 (518 mg, 1.0
mmol). After stirring for 1 h at room temperature, the reaction mixture
was concentrated in vacuo and purified by flash column chromatog-
raphy to yield the title compound (522 mg, 96%) as a colorless oil. 1H
NMR (500 MHz, CDCl3) δ 7.30 (t, J = 7.9 Hz, 2H), 7.22−7.15 (m,
3H), 6.99 (s, 1H), 6.94 (s, 1H), 6.92 (s, 1H), 6.89 (s, 1H), 5.94 (s,
2H), 5.92 (s, 2H), 5.12 (t, J = 7.3 Hz, 1H), 3.16 (d, J = 7.4 Hz, 2H),
3.10 (s, 4H), 2.38 (s, 3H), 2.16 (ss, 12H); 13C NMR (126 MHz,
CDCl3) δ 160.66, 157.25, 151.78, 151.67, 149.86, 149.52, 142.22,
139.42, 128.84, 128.52, 128.48, 126.94, 124.55, 123.86, 122.67, 120.85,
120.13, 106.80, 106.70, 65.75, 44.75, 39.66, 35.79, 21.05, 20.99, 13.29,
13.25; MS (ESI) m/z 544.21 [M + H]+.
(1S,4S)-N-((S)-2-(6-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-methylpyri-
din-2-yl)-1-(3-(2-(6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-
yl)ethyl)phenyl)ethyl)-4,7,7-trimethyl-3-oxo-2-oxabicyclo[2.2.1]-
heptane-1-carboxamide (13a) and (1S,4S)-N-((R)-2-(6-(2,5-Dimeth-
yl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)-1-(3-(2-(6-(2,5-dimethyl-1H-
pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)phenyl)ethyl)-4,7,7-trimeth-
yl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-carboxamide (13b). To the
solution of 12 (500 mg, 0.92 mmol) in THF (25 mL) was added
dropwise a 1.0 M solution of LiAlH4 in THF (1.4 mL, 1.4 mmol) at 0
°C. After being stirred for 3 h at the same temperature, the reaction
mixture was allowed to warm to room temperature, and the reaction
3-(2-(4-Methylpyridin-2-yl)ethyl)benzaldehyde (17). To a solution
of 16 (660 mg, 2.94 mmol) in dichloromethane (30 mL) was added
1.0 M solution of DIBAL in toluene (8.8 mL, 8.8 mmol) at 0 °C, and it
was stirred for 3 h. The reaction mixture was then quenched with
MeOH (3 mL) and water (25 mL). The mixture was warmed to
ambient temperature, stirred for 30 min, and diluted with CH2Cl2 (50
mL). The organic layer was washed with Rochelle’s solution and brine,
dried over MgSO4, and concentrated in vacuo. The resulting residue
was purified by flash chromatography (EtOAc/hexanes) to yield the
1
title compound as a yellow oil (350 mg, 53%). H NMR (500 MHz,
CDCl3) δ 10.01 (s, 1H), 8.44 (d, J = 5.0 Hz, 1H), 7.77−7.68 (m, 2H),
4392
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7.52−7.42 (m, 2H), 6.98 (dd, J = 5.2, 1.6 Hz, 1H), 6.93 (s, 1H), 3.21−
3.13 (m, 2H), 3.13−3.05 (m, 2H), 2.32 (s, 3H); 13C NMR (126 MHz,
CDCl3) δ 192.58, 160.31, 149.17, 147.49, 142.80, 136.55, 134.81,
129.59, 129.03, 127.69, 123.94, 122.39, 39.79, 35.61, 21.00.
(m, 2H), 7.52−7.42 (m, 2H), 6.94 (s, 1H), 6.90 (s, 1H), 5.92 (s, 2H),
3.19 (ddd, J = 8.4, 5.9, 2.1 Hz, 2H), 3.14 (ddd, J = 8.9, 6.0, 2.1 Hz,
2H), 2.39 (s, 3H), 2.14 (s, 6H); 13C NMR (126 MHz, CDCl3) δ
192.52, 160.27, 151.71, 149.62, 142.62, 136.56, 134.81, 129.50, 129.07,
128.46, 127.79, 122.68, 120.28, 106.73, 39.34, 35.33, 21.01, 13.27.
2-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-methyl-6-(3-(2-nitrovinyl)-
phenethyl)pyridine (22). Compound 21 (600 mg, 1.88 mmol),
nitromethane (0.151 mL, 2.83 mmol), and triethylamine (0.524 mL,
3.76 mmol) were dissolved in dichloromethane and stirred at room
temperature for 2 h. After the solvent was removed under reduced
pressure, the organic residue was redissolved in dichloromethane and
then mixed with acetyl chloride (0.134 mL, 3.76 mmol) and
triethylamine (0.655 mL, 4.7 mmol). After being stirred at room
temperature for 1 h, the mixture was concentrated in vacuo and
purified by column chromatography to give the title compound (577
mg, 85%) as a yellow solid. 1H NMR (500 MHz, CDCl3) δ 8.00 (d, J =
13.7 Hz, 1H), 7.58 (d, J = 13.6 Hz, 1H), 7.45−7.34 (m, 4H), 6.97 (s,
1H), 6.91 (s, 1H), 5.92 (s, 2H), 3.14 (h, J = 3.0 Hz, 4H), 2.41 (s, 3H),
2.15 (s, 6H); 13C NMR (126 MHz, CDCl3) δ 151.74, 149.66, 142.97,
139.23, 137.00, 132.50, 130.11, 129.45, 129.28, 128.47, 128.44, 126.97,
122.67, 120.31, 120.07, 106.79, 106.63, 39.36, 35.36, 21.03, 13.28,
13.25; MS (ESI) m/z 362.45 [M + H]+.
2-(2,5-Dimethyl-1H-pyrrol-1-yl)-6-(3-(1-(6-(2,5-dimethyl-1H-pyr-
rol-1-yl)-4-methylpyridin-2-yl)-3-nitropropan-2-yl)phenethyl)-4-
methylpyridine (23). The title compound (531 mg, 61%) was
prepared according to similar procedures described for the synthesis of
19a−b using n-BuLi (1.6 M, 1.21 mL, 1.94 mmol), 9 (387 mg, 1.94
mmol), and 22 (560 mg, 1.55 mmol). Yellow oil; 1H NMR (500 MHz,
CDCl3) δ 7.21 (t, J = 7.84 Hz, 1H), 7.09−7.02 (m, 3H), 6.90 (s, 1H),
6.89 (s, 1H), 6.88 (s, 2H), 5.92 (s, 4H), 4.74−4.57 (m, 2H), 4.12−
4.02 (m, 1H), 3.15 (d, J = 7.69 Hz, 2H), 3.04 (s, 4H), 2.38 (s, 3H),
2.37 (s, 3H), 2.16 (s, 6H), 2.11 (s, 6H); 13C NMR (126 MHz, CDCl3)
δ 160.60, 157.76, 151.80, 151.57, 149.94, 149.68, 142.17, 139.19,
128.90, 128.53, 128.47, 127.89, 127.66, 125.05, 123.37, 122.71, 120.84,
120.15, 106.83, 106.71, 79.79, 44.04, 41.34, 39.62, 35.77, 21.00, 20.99,
13.28, 13.24; MS (ESI) m/z 562.29 [M + H]+.
3-(6-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)-2-(3-(2-
(6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)phenyl)-
propan-1-amine (24). A solution of 23 (500 mg, 0.89 mmol) in
EtOH (5 mL) and MeOH (5 mL) was stirred with Raney-Ni (50% in
water, 0.4 mL) for 1 h at ambient temperature under a hydrogen
atmosphere. The reaction mixture was filtered through Celite and
concentrated in vacuo to yield the title compound (475 mg, 99%). 1H
NMR (500 MHz, CDCl3) δ 7.22 (t, J = 7.9 Hz, 1H), 7.08−7.01 (m,
3H), 6.93 (s, 1H), 6.89 (s, 1H), 6.83 (ss, 2H), 5.91 (s, 2H), 5.90 (s,
2H), 3.21 (td, J = 7.9, 5.0 Hz, 1H), 3.13 (dd, J = 13.5, 7.4 Hz, 1H),
3.07−2.98 (m, 5H), 2.92 (qd, J = 12.9, 6.9 Hz, 2H), 2.39 (s, 3H), 2.33
(s, 3H), 2.15 (s, 6H), 2.10 (s, 6H). 13C NMR (126 MHz, CDCl3) δ
160.93, 159.89, 151.63, 151.49, 149.51, 149.32, 142.58, 141.77, 128.60,
128.47, 128.43, 128.18, 126.78, 125.66, 123.35, 122.56, 120.20, 120.07,
106.71, 106.58, 49.54, 47.14, 42.21, 39.83, 35.94, 21.01, 20.96, 13.28,
13.20; MS (ESI) m/z 532.27 [M + H]+.
2-Bromo-6-(2-nitrovinyl)pyridine (26a). The title compound (295
mg, 65%) was prepared according to a similar procedure described for
the synthesis of 22 using MeNO2 (0.160 mL, 3.0 mmol) and 25a (372
mg, 2.0 mmol). Yellow solid; 1H NMR (500 MHz, CDCl3) δ 8.05 (d, J
= 13.07 Hz, 1H), 7.85 (d, J = 13.10 Hz, 1H), 7.67 (t, J = 7.71 Hz, 1H),
7.59 (dd, J = 0.92, 7.98 Hz, 1H), 7.45 (dd, J = 0.88, 7.45 Hz, 1H); 13C
NMR (126 MHz, CDCl3) δ 150.37, 143.04, 141.72, 139.27, 135.26,
130.34, 125.09; MS (ESI) m/z 455.77 [2 M + H]+.
(S,E)-2-Methyl-N-(3-(2-(4-methylpyridin-2-yl)ethyl)benzylidene)-
propane-2-sulfinamide (18). To a solution of (S)-tert-butanesulfina-
mide (0.210 g, 1.7 mmol) in THF (5 mL) was added 17 (350 mg, 1.55
mmol) followed by Ti(OEt)4 (0.70 g, 3 mmol). The reaction solution
was stirred overnight at room temperature (16 h), and then the
reaction was quenched by the slow addition of saturated aqueous
NaHCO3 (10 mL). The resulting mixture was diluted with EtOAc (20
mL) and filtered through Celite, and the Celite pad was washed with
EtOAc (20 mL). The organic layers were partitioned, dried over
MgSO4, and concentrated in vacuo. The residue was purified by silica
gel chromatography (EtOAc/hexanes) to give the title compound
1
(361 mg, 71% yield) as a pale yellow solid. H NMR (500 MHz,
CDCl3) δ 8.56 (s, 1H), 8.43 (d, J = 5.0 Hz, 1H), 7.70 (d, J = 1.6 Hz,
1H), 7.67 (dt, J = 6.9, 1.8 Hz, 1H), 7.43−7.34 (m, 2H), 6.96 (dd, J =
5.0, 1.6 Hz, 1H), 6.94 (s, 1H), 3.16−3.00 (m, 4H), 2.31 (s, 3H), 1.28
(s, 9H); 13C NMR (126 MHz, CDCl3) δ 162.85, 160.44, 149.12,
147.45, 142.62, 134.13, 132.75, 129.08, 128.98, 127.46, 123.95, 122.36,
57.77, 39.87, 35.75, 22.63, 20.99; MS (ESI) m/z 329.35 [M + H]+.
(S)-N-((R)-2-(6-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-
yl)-1-(3-(2-(4-methylpyridin-2-yl)ethyl)phenyl)ethyl)-2-methylpro-
pane-2-sulfinamide (19a down, major) and (S)-N-((S)-2-(6-(2,5-
Dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)-1-(3-(2-(4-methyl-
pyridin-2-yl)ethyl)phenyl)ethyl)-2-methylpropane-2-sulfinamide
(19b). To a solution of 9 (250 mg, 1.25 mmol) in dry THF (15 mL)
was added n-BuLi (1.6 M solution in hexanes, 0.781 mL, 1.25 mmol),
and the reaction was stirred for 30 min at 0 °C. This solution was
added dropwise to a solution of sulfonamide (18, 350 mg, 1.07 mmol)
in THF (15 mL) at −78 °C using a cannula. After being stirred for an
additional 20 min, the reaction mixture was quenched with H2O (20
mL) and diluted with EtOAc (25 mL). The organic layer was
partitioned, dried with MgSO4, and concentrated in vacuo. The
resulting yellow oil was purified by flash chromatography (EtOAc/
hexanes) to yield title compounds 19a (367 mg, 65%) and 19b (62
mg, 11%). The minor product (19b) eluted first. 19a: pale brown oil;
1H NMR (500 MHz, CDCl3) δ 8.43 (dd, J = 4.9, 0.9 Hz, 1H), 7.25−
7.17 (m, 3H), 7.14−7.09 (m, 1H), 6.97−6.94 (m, 2H), 6.88 (s, 1H),
6.87 (s, 1H), 5.89 (s, 2H), 4.92−4.82 (m, 1H), 4.33 (d, J = 4.8 Hz,
1H), 3.47−3.34 (m, 1H), 3.20 (dd, J = 13.9, 6.1 Hz, 1H), 3.02 (s, 3H),
2.35 (s, 3H), 2.32 (s, 3H), 2.08 (s, 6H), 1.10 (s, 9H); 13C NMR (126
MHz, CDCl3) δ 160.87, 158.06, 151.49, 149.57, 149.03, 147.43,
142.08, 141.84, 128.64, 128.44, 127.94, 127.34, 124.90, 123.91, 123.87,
122.24, 120.59, 106.67, 59.49, 56.09, 45.43, 40.14, 36.09, 22.47, 22.13,
21.01, 20.97, 13.26; MS (ESI) m/z 551.15 [M + H]+. 19b: pale brown
1
oil; H NMR (500 MHz, CDCl3) δ 8.44 (d, J = 5.1 Hz, 1H), 7.30−
7.19 (m, 3H), 7.15 (dt, J = 7.5, 1.6 Hz, 1H), 6.97 (dd, J = 5.1, 1.5 Hz,
1H), 6.95 (s, 1H), 6.94 (s, 2H), 5.88 (s, 2H), 4.76 (ddd, J = 9.3, 4.1,
1.9 Hz, 1H), 3.21−3.09 (m, 2H), 3.04 (p, J = 2.8 Hz, 4H), 2.40 (s,
3H), 2.33 (s, 3H), 2.14 (s, 6H), 1.09 (s, 9H); 13C NMR (126 MHz,
CDCl3) δ 160.89, 158.28, 151.28, 150.35, 149.07, 147.41, 142.17,
141.93, 128.58, 128.48, 127.83, 127.62, 125.13, 123.89, 123.57, 122.25,
120.91, 106.50, 57.95, 55.45, 45.58, 40.14, 36.12, 22.59, 21.08, 21.03,
13.27; MS (ESI) m/z 551.22 [M + Na]+.
3-(2-(6-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)-
benzonitrile (20). The title compound (785 mg, 83%) was prepared
according to a similar procedure described for the synthesis of 16
using n-BuLi (1.6 M, 2.34 mL, 3.75 mmol), 9 (750 mg, 3.75 mmol),
and 15 (0.582 g, 3.0 mmol). Colorless oil; 1H NMR (500 MHz,
CDCl3) δ 7.54−7.43 (m, 3H), 7.39 (d, J = 15.3 Hz, 1H), 6.93 (s, 1H),
6.91 (s, 1H), 5.92 (s, 2H), 3.22−3.00 (m, 4H), 2.40 (s, 3H), 2.14 (s,
6H); 13C NMR (126 MHz, CDCl3) δ 159.86, 151.76, 149.72, 142.92,
133.17, 132.00, 129.85, 129.18, 128.45, 122.69, 120.41, 119.00, 112.32,
106.77, 39.05, 35.05, 21.02, 13.26; MS (ESI) m/z 632.34 [2 M + H]+.
3-(2-(6-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)-
benzaldehyde (21). The title compound (615 mg, 78%) was prepared
according to similar procedures described for the synthesis of 17 using
DIBAL (1M, 7.5 mL, 7.5 mmol) and 20 (780 mg, 2.48 mmol). Pale
3-Bromo-5-(2-nitrovinyl)pyridine (26b). The title compound (390
mg, 86%) was prepared according to a similar procedure described for
the synthesis of 22 using MeNO2 (0.160 mL, 3.0 mmol) and 25b (372
mg, 2.0 mmol). Yellow solid; 1H NMR (500 MHz, CDCl3) δ 8.80 (d, J
= 2.2 Hz, 1H), 8.74 (t, J = 2.7 Hz, 1H), 8.05 (t, J = 2.1 Hz, 1H), 7.97
(d, J = 13.8 Hz, 1H), 7.64 (d, J = 13.8 Hz, 1H); 13C NMR (126 MHz,
CDCl3) δ 153.48, 148.10, 139.35, 137.53, 133.83, 128.15, 121.42; MS
(ESI) m/z 455.98 [2 M + H]+.
2-(2-(6-Bromopyridin-2-yl)-3-nitropropyl)-6-(2,5-dimethyl-1H-
pyrrol-1-yl)-4-methylpyridine (27a). The title compound (329 mg,
1
yellow oil; H NMR (500 MHz, CDCl3) δ 10.00 (s, 1H), 7.79−7.69
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60%) was prepared according to a similar procedure described for the
NMR (126 MHz, MeOD) δ 162.44, 162.33, 161.77, 161.33, 152.85,
152.74, 152.53, 152.24, 138.13, 129.41, 129.39, 125.28, 124.53, 122.59,
122.34, 122.17, 122.12, 107.65, 107.57, 46.94, 41.38, 38.69, 38.12,
25.32, 20.97, 20.87, 13.25, 13.21; MS (ESI) m/z 533.19 [M + H]+.
3-(6-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)-2-(6-(2-
(6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)pyridin-
2-yl)propan-1-amine (30b). The crude title compound (330 mg) was
prepared using the same procedure described for the synthesis of 30a
from 27b (350 mg, 0.63 mmol). 1H NMR (500 MHz, CD3Cl) δ 8.26
(s, 1H), 8.17 (s, 1H), 7.62 (s, 1H), 7.03 (s, 1H), 6.89 (s, 1H), 6.88 (s,
1H), 6.79 (s, 1H), 5.87 (s, 2H), 5.80 (s, 2H), 3.80−3.71 (m, 1H),
3.54−3.44 (m, 1H), 3.24−3.14 (m, 1H), 3.10−2.95 (m, 4H), 2.96−
2.77 (m, 2H), 2.39 (s, 3H), 2.27 (s, 3H), 2.09 (s, 6H), 1.99 (s, 6H);
13C NMR (126 MHz, CDCl3) δ 160.26, 157.89, 151.45, 151.31,
synthesis of 19a−b using n-BuLi (1.6 M, 1.00 mL, 1.60 mmol), 9 (320
1
mg, 1.60 mmol), and 26a (290 mg, 1.28 mmol). Pale brown oil; H
NMR (500 MHz, CDCl3) δ 7.43 (t, J = 7.67 Hz, 1H), 7.36 (d, J = 7.92
Hz, 1H), 7.10 (d, J = 7.40 Hz, 1H), 6.90 (s, 1H), 6.88 (s, 1H), 5.93 (s,
2H), 5.04 (dd, J = 9.68, 13.55 Hz, 1H), 4.66 (dd, J = 4.52, 13.58 Hz,
1H), 4.35−4.24 (m, 1H), 3.24 (dd, J = 7.27, 14.09 Hz, 1H), 3.19−3.07
(m, 1H), 2.38 (s, 3H), 2.13 (s, 6H); 13C NMR (126 MHz, CDCl3) δ
160.51, 157.17, 151.93, 150.09, 142.07, 138.94, 128.43, 126.95, 123.59,
122.75, 120.94, 106.91, 106.84, 77.55, 44.24, 40.14, 21.00, 13.27; MS
(ESI) m/z 450.96 [M + Na]+.
2-(2-(5-Bromopyridin-3-yl)-3-nitropropyl)-6-(2,5-dimethyl-1H-
pyrrol-1-yl)-4-methylpyridine (27b). The title compound (375 mg,
51%) was prepared according to a similar procedure described for the
synthesis of 19a−b using n-BuLi (1.6 M, 1.34 mL, 2.15 mmol), 9 (430
mg, 2.15 mmol), and 26b (390 mg, 1.72 mmol). 1H NMR (500 MHz,
CDCl3) δ 8.59 (s, 1H), 8.42 (s, 1H), 6.92 (ss, 2H), 5.93 (s, 2H), 4.79
(dd, J = 13.1, 5.6 Hz, 1H), 4.70 (dd, J = 13.1, 9.2 Hz, 1H), 4.22 (dtd, J
= 9.3, 7.6, 5.5 Hz, 1H), 3.23 (dd, J = 14.2, 7.7 Hz, 1H), 3.16 (dd, J =
14.2, 7.6 Hz, 1H), 2.39 (s, 3H), 2.10 (s, 6H); 13C NMR (126 MHz,
CDCl3) δ 156.39, 152.04, 150.42, 150.35, 147.36, 137.72, 136.59,
128.44, 123.47, 121.32, 120.94, 106.98, 78.82, 40.86, 40.55, 21.02,
13.25; MS (ESI) m/z 450.98 [M + Na]+.
150.51, 150.27, 148.37, 147.03, 137.22, 136.23, 134.99, 128.55, 128.53,
124.19, 122.87, 121.01, 120.55, 106.91, 106.74, 44.00, 41.31, 40.72,
38.72, 32.72, 21.06, 20.96, 13.21, 13.11; MS (ESI) m/z 533.30 [M +
H]+.
N-Boc-3-(6-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)-
2-(6-(2-(6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)-
pyridin-2-yl)propan-1-amine (30c). To a portion of the crude
product of 30b (∼0.27 mmol, 150 mg) in 10 mL of dichloromethane
was added Boc2O (109 mg, 0.5 mmol) and triethylamine (70 μL, 0.5
mmol), and it was stirred for 12 h. The reaction mixture was
concentrated in vacuo and then purified by flash chromatography
(EtOAc/hexanes) to give the title compound (110 mg, 62%) as a
colorless oil. The chiral resolution of racemic 30c was performed using
an OD-H chiral column with an autocollector-equipped HPLC
system; 30c (100 mg) was dissolved in 2 mL of EtOH, and then
0.1 mL of the solution per time was injected until the parent solution
was all consumed. The separated enantiomers were collected and
concentrated in vacuo to give 30c-1 (43 mg, Rt = 16.1 min, ee = 98%)
2-(2,5-Dimethyl-1H-pyrrol-1-yl)-6-((6-(1-(6-(2,5-dimethyl-1H-pyr-
rol-1-yl)-4-methylpyridin-2-yl)-3-nitropropan-2-yl)pyridin-2-yl)-
ethynyl)-4-methylpyridine (29a). The reaction mixture of 27a (300
mg, 0.70 mmol), Pd(PPh3)2Cl2 (23 mg, 0.035 mmol), CuI (6 mg,
0.035 mmol), PPh3 (37 mg, 0.14 mmol), 28 (167 mg, 0.80 mmol),
diethylamine (1.5 mL), and DMF (1.5 mL) were heated at 120 °C for
20 min in the microwave cavity. Then, the reaction mixture was
treated with diethyl ether (20 mL), filtered, and concentrated in vacuo.
The residue was purified by flash chromatography (EtOAc/hexanes)
1
and 30c-2 (32 mg, Rt = 23.1 min, ee = 97%). H NMR (500 MHz,
1
to give the title compound (306 mg, 78%) as a pale yellow oil. H
CDCl3) δ 8.27 (s, 1H), 8.22 (s, 1H), 7.42 (s, 1H), 6.93 (s, 1H), 6.90
(s, 1H), 6.84 (s, 2H), 5.91 (s, 2H), 5.89 (s, 2H), 4.64 (d, J = 6.7 Hz,
1H), 3.55−3.37 (m, 3H), 3.35−3.24 (m, 1H), 3.24−3.14 (m, 1H),
3.06−2.93 (m, 3H), 2.39 (s, 3H), 2.33 (s, 3H), 2.15 (s, 6H), 2.07 (s,
6H), 1.40 (s, 9H); 13C NMR (126 MHz, CDCl3) δ 160.02, 158.72,
155.74, 151.75, 151.64, 149.74, 149.69, 148.28, 147.32, 137.04, 136.73,
135.32, 128.42, 123.41, 122.64, 120.54, 120.33, 106.79, 106.67, 79.38,
45.18, 43.46, 41.36, 39.13, 32.69, 28.35, 21.02, 20.98, 13.30, 13.21; MS
(ESI) m/z 654.56 [M + Na]+.
Computational Methods. The MM-PBSA method, as imple-
mented in Amber 9.028 used in our previous work, was used to
calculate free energies of binding. In this method, the total free energy
of the NOS−inhibitor complex is taken as the sum of the following
energy terms:
NMR (500 MHz, CDCl3) δ 7.58 (t, J = 7.77 Hz, 1H), 7.53 (s, 1H),
7.47 (dd, J = 1.03, 7.76 Hz, 1H), 7.11 (d, J = 7.74 Hz, 1H), 7.08 (s,
1H), 6.90 (s, 1H), 6.85 (s, 1H), 5.93 (s, 2H), 5.91 (s, 2H), 5.16 (dd, J
= 9.67, 13.66 Hz, 1H), 4.70 (dd, J = 4.66, 13.62 Hz, 1H), 4.33 (m,
1H), 3.26 (dd, J = 7.45, 14.02 Hz, 1H), 3.19 (dd, J = 7.75, 14.02 Hz,
1H), 2.49 (s, 3H), 2.36 (s, 3H), 2.18 (s, 6H), 2.13 (s, 6H); 13C NMR
(126 MHz, CDCl3) δ 159.78, 157.45, 152.39, 151.91, 150.01, 142.40,
141.88, 136.77, 128.57, 128.45, 127.37, 126.61, 123.82, 123.57, 123.00,
120.87, 106.95, 106.87, 88.03, 87.44, 44.65, 40.38, 20.98, 20.96, 13.28,
13.22; MS (ESI) m/z 559.24 [M + H]+.
2-(2,5-Dimethyl-1H-pyrrol-1-yl)-6-((5-(1-(6-(2,5-dimethyl-1H-pyr-
rol-1-yl)-4-methylpyridin-2-yl)-3-nitropropan-2-yl)pyridin-3-yl)-
ethynyl)-4-methylpyridine (29b). The title compound (355 mg, 74%)
was prepared using the same procedure described for the synthesis of
29a from 27b (370 mg, 0.86 mmol) and 28 (207 mg, 0.98 mmol). 1H
NMR (500 MHz, CDCl3) δ 8.71 (s, 1H), 8.45 (s, 1H), 7.43 (s, 1H),
7.07 (s, 1H), 6.91 (ss, 2H), 5.92 (ss, 4H), 4.80 (dd, J = 13.0, 5.6 Hz,
1H), 4.71 (dd, J = 13.0, 9.2 Hz, 1H), 4.25 (m, 1H), 3.25 (dd, J = 14.2,
7.6 Hz, 2H), 3.16 (dd, J = 14.2, 7.7 Hz, 1H), 2.49 (s, 3H), 2.38 (s,
3H), 2.17 (s, 6H), 2.10 (s, 6H); 13C NMR (126 MHz, CDCl3) δ
156.52, 152.43, 152.01, 151.80, 150.37, 150.09, 148.99, 141.78, 137.74,
134.50, 128.55, 128.46, 126.97, 123.47, 122.94, 121.27, 107.01, 106.93,
91.87, 85.09, 78.96, 40.96, 40.56, 21.02, 20.98, 14.23, 13.25, 13.22; MS
(ESI) m/z 559.17 [M + H]+.
G = EMM + Gsolv + Gnp − TSsolute
where EMM = the total molecular mechanics energy computed with the
Sander module in Amber 9.0, Gsolv is the solvation free energy
estimated from the Poisson−Boltzmann equation, Gnp = the nonpolar
solvation energy estimated from the solvent accessible surface area,
and TSsolute = the solute entropy. From a single energy-minimized
structure, the free energy is computed for the NOS−inhibitor
complex, NOS alone with the inhibitor removed, and the inhibitor
alone. The overall free energy of binding is computed from the
following equation:
3-(6-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)-2-(6-(2-
(6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)pyridin-
2-yl)propan-1-amine (30a). A solution of 29a (300 mg, 0.54 mmol)
in EtOH (5 mL) and MeOH (5 mL) was stirred with Raney-Ni (50%
in water, 0.5 mL) for 7 h at ambient temperature under a hydrogen
atmosphere. The reaction mixture was filtered through a PTFE
membrane filter (diameter 25 mm, pore size 0.2 μm) and concentrated
ΔGbind = (Gcomplex − Greceptor − Ginhibitor
)
As others have done, the solute entropy is ignored.29 Given that the
inhibitors used for these calculations are exactly the same, ignoring
entropy introduces little error. Parameters for the inhibitor and heme
were the same as described previously.24
Enzyme Assay Methods. All of the NOS isoforms were
overexpressed and purified,30−32 and enzyme kinetics data were
determined using the hemoglobin capture assay (HCA) at 37 °C in a
high-throughput method using 96-well plates. A typical assay mixture
for nNOS and eNOS contained various concentrations of the test
compound, 10 μM L-Arg, 1.0 mM CaCl2, 300 units/mL calmodulin
1
in vacuo to give the crude title compound (300 mg). H NMR (500
MHz, MeOD) δ 7.50 (t, J = 7.7 Hz, 1H), 7.14 (s, 1H), 7.02 (d, J = 7.4
Hz, 1H), 6.99 (s, 1H), 6.97 (s, 1H), 6.95 (d, J = 7.5 Hz, 1H), 6.92 (s,
1H), 5.82 (s, 3H), 5.81 (s, 2H), 3.60−3.43 (m, 1H), 3.37 (s, 2H),
3.27−3.14 (m, 7H), 3.07 (dd, J = 12.9, 8.0 Hz, 1H), 2.93 (dd, J = 12.9,
5.4 Hz, 1H), 2.40 (s, 3H), 2.31 (s, 3H), 2.05 (s, 7H), 2.00 (s, 6H); 13C
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Article
(Sigma, P-2277), 100 μM NADPH, 0.125 mg/mL hemoglobin-A0
(ferrous form, Sigma, H0267), and 10 μM H4B in 100 mM HEPES
(pH 7.5). A typical assay mixture for iNOS contained various
concentrations of the test compound, 100 μM NADPH, 0.125 mg/mL
hemoglobin-A0 (ferrous form), and 10 μM H4B in 100 mM HEPES
(pH 7.5). All assays were in a final volume of 100 μL and were
initiated by the addition of enzyme (approximately 100 nM final
concentration). Nitric oxide-mediated oxidation of hemoglobin-A0 was
monitored at 401 nm for 1 min on a Synergy H1 reader by Biotek.
Curves were fitted using the Michaelis−Menten equation in GraphPad
Prism 5.0 (GraphPad Software, Inc.). For Ki determinations, IC50
values were calculated using nonlinear regressions (dose−response
inhibition, four-parameter variable slope). Subsequent Ki values were
calculated using the Cheng-Prusoff relationship: Ki = IC50/(1 + [S]/
Km) (Km values used for rat nNOS, murine iNOS, bovine eNOS, and
human nNOS were 1.3, 8.3, 1.7, and 1.6 μM respectively).
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rat cardiac myocytes. Pharmacol. Res. 2005, 51, 409−417.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis of 28, inhibitor complex crystal preparation, X-ray
crystallographic data collection and refinement statistics, and
1H and 13C spectra of 3, 4, 5, 6, 7, and 8. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*(T.L.P.) Phone: 949-824-7020. Fax: 949-824-3280. E-mail:
poulos@uci.edu.
■
(12) Yang, Z.; Misner, B.; Ji, H.; Poulos, T. L.; Silverman, R. B.;
Meyskens, F. L.; Yang, S. Targeting nitric oxide signaling with nNOS
inhibitors as a novel strategy for the therapy and prevention of human
melanoma. Antioxid. Redox Signaling 2013, 19, 433−447.
(13) Lian, K.; Murad, F. Nitric oxide (NO)-biogeneration, regulation,
and relevance to human disease. Front. Biosci. 2003, 8, d264−d278.
(14) Holden, J. K.; Li, H.; Jing, Q.; Kang, S.; Richo, J.; Silverman, R.
B.; Poulos, T. L. Structural and biological studies on bacterial nitric
oxide synthase inhibitors. Proc. Natl. Acad. Sci. U.S.A. 2013, 110,
18127−18131.
*(R.B.S.) Phone: 847-491-5653. Fax: 847-491-7713. E-mail:
Agman@chem.northwestern.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
(15) van Sorge, N. M.; Beasley, F. C.; Gusarov, I.; Gonzalez, D. J.;
■
von Kockritz-Blickwede, M.; Anik, S.; Borkowski, A. W.; Dorrestein, P.
̈
We are grateful for financial support from the National
Institutes of Health (GM049725 to R.B.S. and GM057353 to
T.L.P.). We thank Dr. Bettie Sue Siler Masters (NIH grant
GM52419, with whose laboratory P.M. and L.J.R. are affiliated).
B.S.S.M. also acknowledges the Welch Foundation for a Robert
A. Welch Distinguished Professorship in Chemistry (AQ0012).
P.M. is supported by grants 0021620806 and 1M0520 from
MSMT of the Czech Republic. We also thank the beamline staff
at SSRL and ALS for their assistance during the remote X-ray
diffraction data collection.
C.; Nudler, E.; Nizet, V. Methicillin-resistant Staphylococcus aureus
bacterial nitric-oxide synthase affects antibiotic sensitivity and skin
abscess development. J. Biol. Chem. 2013, 288, 6417−6426.
(16) Mukherjee, P.; Cinelli, M. A.; Kang, S.; Silverman, R. B.
Development of nitric oxide synthase inhibitors for neurodegeneration
and neuropathic pain. Chem. Soc. Rev. 2014, DOI: 10.1039/
C3CS60467E.
(17) Ghosh, D. K.; Stuehr, D. J. Macrophage NO synthase:
characterization of isolated oxygenase and reductase domains reveals
a head-to-head subunit interaction. Biochemistry 1995, 34, 801−807.
́ ́
(18) Raman, C. S.; Li, H.; Martasek, P.; Kral, V.; Masters, B. S.;
ABBREVIATIONS USED
Poulos, T. L. Crystal structure of constitutive endothelial nitric oxide
synthase: a paradigm for pterin function involving a novel metal
center. Cell 1998, 95, 939−950.
■
nNOS, neuronal nitric oxide synthase; iNOS, inducible nitric
oxide synthase; eNOS, endothelial nitric oxide synthase; ALS,
amyotrophic lateral sclerosis; FMN, flavin mononucleotide;
MM-PBSA, molecular mechanics−Poisson−Boltzmann surface
area
(19) Woodward, J. J.; Nejatyjahromy, Y.; Britt, R. D.; Marletta, M. A.
Pterin-centered radical as a mechanistic probe of the second step of
nitric oxide synthase. J. Am. Chem. Soc. 2010, 132, 5105−5113.
(20) Li, H.; Poulos, T. L. Structure-function studies on nitric oxide
synthases. J. Inorg. Biochem. 2005, 99, 293−305.
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