Structure-guided design of selective inhibitors of neuronal nitric oxide synthase

Article abstract of DOI:10.1021/jm4000984

Nitric oxide synthases (NOSs) comprise three closely related isoforms that catalyze the oxidation of l-arginine to l-citrulline and the important second messenger nitric oxide (NO). Pharmacological selective inhibition of neuronal NOS (nNOS) has the potential to be therapeutically beneficial in various neurodegenerative diseases. Here, we present a structure-guided, selective nNOS inhibitor design based on the crystal structure of lead compound 1 in nNOS. The best inhibitor, 7, exhibited low nanomolar inhibitory potency and good isoform selectivities (nNOS over eNOS and iNOS are 472-fold and 239-fold, respectively). Consistent with the good selectivity, 7 binds to nNOS and eNOS with different binding modes. The distinctly different binding modes of 7, driven by the critical residue Asp597 in nNOS, offers compelling insight to explain its isozyme selectivity, which should guide future drug design programs.

Full text of DOI:10.1021/jm4000984

Article
pubs.acs.org/jmc
Structure-Guided Design of Selective Inhibitors of Neuronal Nitric
Oxide Synthase
He Huang,† Huiying Li,‡ 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, 2145 Sheridan Road, Evanston, Illinois 60208-3113, United States
‡Departments of Molecular Biology and Biochemistry, Pharmaceutical Sciences, and Chemistry, University of California, Irvine,
California 92697-3900, United States
§Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78384-7760, United States
∥Department of Pediatrics and Center for Applied Genomics, First School of Medicine, Charles University, Prague, Czech Republic
ABSTRACT: Nitric oxide synthases (NOSs) comprise three
closely related isoforms that catalyze the oxidation of L-
arginine to L-citrulline and the important second messenger
nitric oxide (NO). Pharmacological selective inhibition of
neuronal NOS (nNOS) has the potential to be therapeutically
beneficial in various neurodegenerative diseases. Here, we
present a structure-guided, selective nNOS inhibitor design
based on the crystal structure of lead compound 1 in nNOS.
The best inhibitor, 7, exhibited low nanomolar inhibitory
potency and good isoform selectivities (nNOS over eNOS and iNOS are 472-fold and 239-fold, respectively). Consistent with
the good selectivity, 7 binds to nNOS and eNOS with different binding modes. The distinctly different binding modes of 7,
driven by the critical residue Asp597 in nNOS, offers compelling insight to explain its isozyme selectivity, which should guide
future drug design programs.
scaffold.14,15 Although some of them showed great potency and
excellent selectivity for nNOS over eNOS and iNOS, they still
suffered from serious limitations, namely, the positive charges
derived from the basic groups dramatically impair cell
INTRODUCTION
■
Nitric oxide (NO) is a widely utilized second messenger for
intracellular signaling cascades invoked by a wide variety of
biological stimuli and is of particular functional importance in the
permeability. To overcome this shortcoming, a series of
central nervous system (CNS).1,2 Nitric oxide synthases (NOSs)
symmetric double-headed aminopyridines without charged
groups were designed and synthesized.16 The best inhibitor, 1
catalyze the oxidation of L-arginine to NO and L-citrulline with
NADPH and O2 as cosubstrates.3,4 Therefore, these enzymes are
(see Figure 1 for chemical structure), shows low nanomolar
involved in a number of important biological processes and are
implicated in many chronic neurodegenerative pathologies such
as Alzheimer’s, Parkinson’s, and Huntington’s diseases as well as
neuronal damage resulting from stroke, cerebral palsy, and
migraine headaches.5−8 For this reason, there is interest in the
generation of potent small-molecule inhibitors of NOSs.9,10
NOSs comprise three closely related isoforms: neuronal NOS
(nNOS), endothelial NOS (eNOS), and inducible NOS
(iNOS).1 Each isoform is characterized by unique cellular and
Figure 1. Chemical structure of 1.
subcellular distribution, function, and catalytic properties.11
inhibitory potency and enhanced membrane permeability.
However, 1 exhibits low isoform selectivity. Therefore, we
used the crystal structure of the nNOS oxygenase domain in
complex with 1 as a template to design more selective nNOS
inhibitors. As revealed by the crystal structure (Figure 2), while
inhibitor 1 shows high affinity to nNOS by utilizing both of its
While a number of NOS inhibitors have been reported with high
affinity, the challenging task is to achieve high selectivity. Because
nNOS is abundant in neuronal cells but eNOS is vital in
maintaining vascular tone in brain, improvement in the
inhibitory selectivity of nNOS over eNOS is very important for
lowering the risk of side effects.12,13
In our continued efforts to develop nNOS selective inhibitors,
we discovered a series of highly potent and selective nNOS small
molecule inhibitors with a 2-aminopyridinomethyl pyrrolidine
Received: January 21, 2013
Published: March 1, 2013
© 2013 American Chemical Society
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CHEMISTRY
■
To synthesize inhibitors 2−5 (Scheme 1), the carboxyl groups of
bromo-substituted isophthalic acids were reduced to alcohols in
good yields, and then bromination generated intermediates 9a
and 9b. 2-(2,5-Dimethyl-1H-pyrrol-1-yl)-4,6-dimethylpyridine
was treated with n-butyllithium and was allowed to react with 9a
or 9b. Subsequently, a palladium-catalyzed amination of 10a and
10b with N-monosubstituted piperazines gave corresponding
aromatic amines 11 and 12. Deprotection of 11 and 12 gave 13
and target compound 3, respectively. Finally, the removal of the Boc-
protecting group of 13 proceeded smoothly, giving a high yield of 2.
The palladium-catalyzed Heck coupling reactions of tert-butyl
allylcarbamate with 10a and 10b gave corresponding alkenes 14a
and 14b. The 2,5-dimethylpyrrole protecting groups of 14a and
14b were removed with NH2OH·HCl at 100 °C to generate 15a
and 15b in moderate yields. Finally, catalytic hydrogenation and
the removal of the Boc-protecting group proceeded smoothly,
giving high yields of 4 and 5.
The synthesis of 6 and 7 (Scheme 2) began with 1-bromo-3,5-
dimethylbenzene. It was subjected to cyanation with CuCN and
subsequent bromination with NBS to afford 3,5-bis(bromomethyl)-
benzonitrile 18 in a 22% yield. 2-(2,5-Dimethyl-1H-pyrrol-1-yl)-4,6-
dimethylpyridine was then treated with n-butyllithium and allowed
to react with 18 to form 19 in a 48% yield. The 2,5-dimethylpyrrole
protecting groups of 19 were removed with NH2OH·HCl at 100 °C
to generate 7 in a 31% yield. Reduction of the cyano group in 7 gave
6 in a moderate yield.
Figure 2. Crystallographic binding conformation of 1 (PDB ID 3N5W)
with rat nNOS. All structural figures were prepared with PyMol (www.
pymol.org).
2-aminopyridine rings to interact with protein residues and
heme, it leaves some room near the central pyridine moiety.
The central pyridine nitrogen atom of 1 hydrogen bonds via a
bridging water molecule with negatively charged residue Asp597.
The corresponding residue in eNOS is Asn368. Our studies with
a series of dipeptide amide inhibitors had demonstrated23 that
the potency of inhibitors can be dramatically increased in eNOS
by replacing Asn368 with Asp, while the Ki rises substantially in
nNOS if Asp597 is replaced by Asn. Because of the Asp/Asn
difference in nNOS and eNOS, the nNOS active site has a more
electronegative environment compared to that of eNOS.
Therefore, an electropositive functional group is preferred in
the vicinity of Asp597 in nNOS. To target interaction with
Asp597, here, we designed and synthesized a series of nNOS
inhibitors (Figure 3) that contain a tail on the central aromatic
ring with various lengths and different substitution groups at the
terminus.
RESULTS AND DISCUSSION
■
All of the compounds tested were competitive inhibitors against
the substrate L-arginine. The first of the analogues prepared
Figure 3. Target molecules designed and synthesized in this study.
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a
Scheme 1. Synthesis of 2−5
a
Reagents and conditions: (a) LiBH4, TMSCl, THF, room temperature (rt), 12 h, 82−86%; (b) PPh3, CBr4, CH2Cl2, 0 °C, 2 h, 89−92%; (c) 9a or
9b, n-BuLi, THF, −78 °C to rt, 2 h, 48−56%; (d) Pd2(dba)3, BINAP, NaOtBu, PhMe, 80 °C, 8 h, 61−67%; (e) NH2OH·HCl, EtOH/H2O (2:1),
100 °C, 36 h, 58−69%; (f) 3N HCl/MeOH, rt, 24 h, quantitative; (g) PdCl2, DIEA, tri(o-tolyl)phosphine, DMF, 120 °C, 8 h, 58−64%;
(h) NH2OH·HCl, EtOH/H2O (2:1), 100 °C, 36 h, 49−56%; (i) H2, Pd/C, MeOH, rt, 12 h, quantitative; (j) 3N HCl/MeOH, rt, 24 h, quantitative.
(Figure 3, 2) retains the double 2-aminopyridine heads but with a
piperazino tail at the 5-position of the central phenyl core. It
showed moderate inhibition, with a Ki value of 2.1 μM for nNOS
and nNOS selectivities over eNOS and iNOS of 30-fold and
17-fold, respectively (Table 1). Blocking the terminal N atom of the
piperazino tail with a methyl group (3) slightly increases the size,
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a
Scheme 2. Synthesis of 6 and 7
a
Reagents and conditions: (a) CuCN, DMF, 180 °C, 8 h, 84%; (b) NBS, BPO, CCl4, 80 °C, 6 h, 22%; (c) n-BuLi, THF, −78 °C to rt, 2 h, 48%; (d)
NH2OH·HCl, EtOH/H2O (2:1), 100 °C, 24 h, 31%; (e) LiBH4, TMSCl, THF, rt, 12 h, 42%.
a
Table 1. Inhibition of NOS Isozymes by Synthetic Inhibitors
A similar binding mode that kicks out H4B was also observed with
another inhibitor bearing two 2-aminopyridine heads.16 As expected
from our design, the terminal N atom of the 3-aminopropyl moiety
approaches residue Asp597. However, the density for this side chain
is rather poor compared to the other part of inhibitor indicative of
flexibility of the tail, suggesting that other positions on the tail that
are somewhat distant from Asp597 also are possible sites of
attachment of the binding group. Interestingly, 4 was found not to
be competitive with H4B (data not shown).
Similar to the binding mode of 1, 4-substituted compound 5
binds to nNOS with one of its 2-aminopyridine heads interacting
with Glu592 and the other making bifurcated hydrogen bonds
with heme propionate D (Figure 4B). Again, the density for its 3-
aminopropyl tail is so poor that there is ambiguity even at a lower
contour level (0.5σ, 2Fo−Fc map) as to which way the terminal
N atom goes. Two different binding modes were modeled with
one extending out away from Asp597 in one monomer of the
heme domain dimer (Figure 4B) and the other one interacting
with Asp597 in the other monomer.
Note that 4 interacts with Asp597, which was our primary goal.
One particularly interesting finding that emerged from the crystal
structure is that the 3-aminopropyl tail on the central core is
relatively long and flexible and thus tends to be disordered. On
the basis of these observations and a desire to further improve the
potency and selectivity, we decided to pursue additional changes
to the 3-aminopropyl tail of 4. Compound 6, with a shorter tail
on the phenyl core, has a 10-fold better nNOS inhibitory potency
and 10-fold better selectivity for nNOS over eNOS than those of 4.
Crystal structures of nNOS complexed with 6 (Figure 5) show that
the aminomethyl substituent is ordered and small enough to fit
within the cavity allowing a weaker hydrogen bond (3.2−3.4 Å) with
Asp597. However, this time, the interaction between Asp597 and the
tail N atom pulls the phenyl core so that the second 2-aminopyridine
head cannot make bifurcated hydrogen bonds with the heme
propionate of pyrrole ring D, leading to partial disorder (poor density).
Replacement of the water-mediated pyridine fragment by a
cyanophenyl group is a known structure modification;17
therefore, we explored the effect of cyano substitution at the
5-position of the phenyl core. Its Ki value with nNOS is 56 nM,
and the selectivities for nNOS over eNOS and iNOS are 472-fold
and 239-fold, respectively. We have also determined the crystal
Ki (μM)
selectivity
no.
nNOS
eNOS
iNOS
n/e
n/i
2
3
4
5
6
7
2.10
5.75
0.54
0.11
0.053
0.056
63.2
97.4
12.1
33.4
11.7
26.4
36.7
138.0
32.5
21.9
6.31
13.4
30
17
17
24
22
60
303
221
472
199
119
239
a
The compounds were evaluated for in vitro inhibition against three
NOS isozymes, rat nNOS, bovine eNOS, and marine iNOS, using
known literature methods.21
which tends to decrease the potency and nNOS selectivity over
eNOS but affords a slight increase in selectivity over iNOS. The
nNOS−2 complex crystal structure (data not shown) revealed
electron density for only the 2-aminopyridine near Glu592 with
the rest of the inhibitor fully disordered. These results suggest
that the steric bulk of the piperazino group hinders interaction
between the second aminopyridine group and the enzyme.
We reasoned that only relatively small substituents would be
tolerated at the 5-position of the phenyl core. Moreover, there is
more space available between Glu592 and the 4-position of the
phenyl core. To test whether smaller and flexible substituents
could increase the potency and selectivity, we designed inhibitors
4 and 5. The 5-substituted compound (4) displays a Ki value of
0.54 μM with nNOS, which is 4- and 10-fold lower than those of
2 and 3, respectively. Compared to compound 2, 4-substituted
compound 5 exhibits about 19-fold better inhibitory potency
(Ki = 0.11 μM) and 20-fold better selectivity for nNOS over
eNOS and iNOS (n/e = 303; n/i = 199).
To better understand structure−activity relationships for 4
and 5, the crystal structures of nNOS complexed with these
inhibitors were determined. In contrast to the binding mode of 1
where both 2-aminopyridine heads are involved in hydrogen
bonds with Glu592 and heme, one of 2-aminopyridine groups of
4 interacts with Glu592 to anchor the inhibitor to the nNOS
active site, but the other 2-aminopyridine replaces the cofactor
H4B stacking with Trp678. Inhibitor binding also promotes the
binding of a new zinc ion ligated by Asp600 and His692 (chain B)
along with a chloride ion and a water molecule (Figure 4A).
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Figure 4. Crystallographic binding conformations of 4 (A, PDB ID 4IMS) and 5 (B, PDB ID 4IMT) with rat nNOS. The Fo−Fc omit density map for
inhibitor is shown at the 2.5σ contour level. Binding of 4 replaces H4B and creates a new Zn binding site. Arg596 is then salt bridged to Glu592 rather
than to Asp600 as in the Zn-free structure.
density at the lower contour level (0.5σ, 2Fo−Fc map), where
the N atom of the cyano group faces Asp597 but is a little too far
(>3.5 Å) for a strong hydrogen bond.
Consistent with the poorer binding affinity to eNOS, in the
eNOS−7 complex (Figure 6B), 7 exhibits a different binding
mode with only one 2-aminopyridine hydrogen bonded with
Glu363, while the other one is partially disordered showing poor
density. In this binding mode, the cyanophenyl part folds upward
away from Asn368 with the cyano nitrogen atom hydrogen bonded
to Ser248. In the eNOS−7 complex structure, there is an acetate ion
that is in the vicinity of Asn368 and forms salt bridges with two basic
residues, Arg252 and Arg374. This acetate has been observed in many
eNOS inhibitor complex structures. It is possible that this acetate
prevents the cyanophenyl group of 7 from interacting with Asn368.
However, the presence of a negatively charged acetate in the vicinity
of Asn368 is a consequence of the electropositive nature of the
environment at this site. This is most likely the reason for the cyano-
phenyl to steer away from Asn368 in eNOS. The binding mode with
eNOS explains why 7 has better selectivity for nNOS over eNOS than
does 1, which showed an identical 2-headed binding mode with both
isoforms.16 As noted above, the key differences we targeted between
nNOS and eNOS is the Asp597/Asn368 difference in the active site
pocket. The distinctly different binding modes of 7 driven by this
critical residue in nNOS and eNOS afford a high degree of selectivity.
From our exploration of the structure−activity relationship of
the symmetric double-headed 2-aminopyridines, 7 remains the
Figure 5. Crystallographic binding conformations of 6 (PDB ID 4IMU)
with rat nNOS. The Fo−Fc omit density map for inhibitor is shown at the
2.5σ contour level. Although binding of 6 does not disturb the H4B site, it
still creates a new Zn binding site. Arg596 is also salt bridged with Glu592.
structures of 7 complexed to both nNOS and eNOS. As expected
from the structure-guided design, the double heads of 7 bind to
the active site of nNOS in a similar pattern as those of parent
inhibitor 1: one interacts with Glu592, while the other forms
bifurcated hydrogen bonds with heme propionate D and has
aromatic π-stacking with Tyr706 (Figure 6A).
Unfortunately, the key part of the inhibitor, the cyanophenyl
ring, shows poorer density. The model built is supported by
Figure 6. Crystallographic binding conformations of 7 with rat nNOS (A, PDB ID 4IMW) and bovine eNOS (B, PDB ID 4IMX). The Fo−Fc omit
density map for inhibitor is shown at the 2.5σ contour level. For eNOS, an extra acetate ion is shown that is salt bridged between two Arg residues.
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(25 mL) and 2 N NaOH (50 mL). The aqueous layer was extracted with
Et2O (2 × 25 mL), and the combined organic layers were dried over
Na2SO4. The solvent was removed by rotary evaporation, and the
resulting yellow oil was purified by flash chromatography to yield
products 3, 7, 13, 15a, or 15b.
most selective compound we have discovered, while maintaining
good potency against nNOS.
CONCLUSIONS
■
The NOS active site is highly conserved; therefore, achieving
selectivity for nNOS inhibition is extremely difficult. Through
structure-guided design, we confirm that the nNOS inhibitor
potency and selectivity can be achieved by introducing
electropositive functional groups that can interact with Asp597
in nNOS. In this study, we demonstrate that the potent and
selective nNOS inhibitor 7 binds to nNOS and eNOS with
different binding modes because of the Asp597/Asn368
difference and thus exhibits the best selectivity in the series.
Furthermore, the active substituent in 7 is a cyano group, which is
not basic and not positively charged, as is the case with the amino
substituents. This should be favorable for bioavailability. The
results imply that inhibitors with a benzonitrile core should be
nNOS selective. Our best compound 7, which not only retains
the affinity with nNOS but also shows significantly improved
selectivity against other NOS isoforms, is a good template for the
further design of selective nNOS inhibitors.
General Procedure D: Boc Deprotection. To a solution of 13,
16a, or 16b (0.2 mmol) in MeOH (1.0 mL) was added 3 N HCl (10.0
mL). The reaction mixture was stirred at room temperature for 24 h.
Then, the reaction mixture was concentrated in vacuo. The crude
product was recrystallized with cold diethyl ether to provide 2, 4, or 5.
6,6′-((5-(Piperazin-1-yl)-1,3-phenylene)bis(ethane-2,1-diyl))-
bis(4-methylpyridin-2-amine) (2). Intermediate 8a was synthesized
by general procedure A using 5-bromoisophthalic acid as the starting
material (yield 86%). To a suspension of 8a (1.0 mmol) in dry CH2Cl2
(10 mL) was added PPh3 (2.1 mmol) and CBr4 (2.1 mmol). The
reaction mixture was stirred at room temperature for 2 h and then
quenched with H2O, extracted with CH2Cl2, and washed with water and
brine. The organic layer was dried over Na2SO4 and concentrated in
vacuo. The residue was purified by column chromatography to yield 9a
(yield 89%). Compound 10a was synthesized by general procedure B
using 9a as the starting material (yield 48%). To a suspension of 10a (1.0
mmol), 1-Boc-piperazine, and NaOtBu (1.0 mmol) in dry toluene (10
mL) was added Pd2(dba)3 (0.05 mmol) and BINAP (0.1 mmol). The
reaction mixture was stirred at 80 °C for 8 h. The reaction mixture was
then diluted with H2O (20 mL), extracted with EtOAc, and washed with
water and brine. The organic layer was dried over Na2SO4 and
concentrated in vacuo. The residue was purified by column
chromatography to yield 11 (yield 67%). Compound 2 was synthesized
by general procedures C and D using 11 as the starting material (yield
58%). 1H NMR (500 MHz, D2O): δ 6.64 (d, J = 1.5 Hz, 2H), 6.56 (s,
1H), 6.46 (s, 2H), 6.23 (d, J = 1.5 Hz, 2H), 3.29−3.25 (m, 8H), 2.82−
2.81 (m, 8H), 2.09 (s, 6H). 13C NMR (125 MHz, D2O): δ 157.75,
153.44, 148.52, 147.93, 141.52, 123.77, 116.34, 114.46, 109.38, 47.47,
42.69, 33.84, 29.49, 20.96. LC-TOF MS (M + H+) calcd for C26H35N6
431.2923, found 431.2917.
EXPERIMENTAL SECTION
■
All reagents were purchased from Sigma−Aldrich, Alfa Aesar, or TCI
and were used without further purification unless stated otherwise.
Analytical thin layer chromatography was visualized by UV light,
ninhydrin, or phosphomolybdic acid. Flash column chromatography
was carried out under a positive pressure of air. 1H NMR spectra were
recorded on 500 MHz spectrometers. Data are presented as follows:
chemical shift (in ppm on the δ scale, and the reference resonance peaks
set at 0 ppm [TMS(CDCl3)], 3.31 ppm (CD2HOD), 4.80 ppm (HOD),
and 7.26 ppm (CDCl3)), multiplicity (s = singlet, d = doublet, t = triplet,
m = multiplet), coupling constant (J/Hz), integration. 13C NMR spectra
were recorded at 125 MHz, and all chemical shift values are reported in
parts per million on the δ scale, with an internal reference of δ 77.0 or
49.0 for CDCl3 or CD3OD, respectively. High-resolution mass spectra
were measured by liquid chromatography/time-of-flight mass spec-
trometry (LC-TOF MS). The purity of the tested compounds was
determined by high-performance liquid chromatography (HPLC)
analysis and was >95%.
6,6′-((5-(4-Methylpiperazin-1-yl)-1,3-phenylene)bis(ethane-
2,1-diyl))bis(4-methylpyridin-2-amine) (3). Compound 3 was
synthesized by the same procedures as those to prepare 2 using 1-
methylpiperazine as the starting material. 1H NMR (500 MHz, CDCl3):
δ 6.63 (s, 3H), 6.348 (d, J = 1.5 Hz, 2H), 6.20 (s, 2H), 3.19 (t, J = 5.0 Hz,
4H), 2.95−2.80 (m, 8H), 2.64−2.55 (m, 4H), 2.37 (s, 3H), 2.20 (s, 6H).
13C NMR (125 MHz, CDCl3): δ 157.82, 148.81, 142.64, 141.84, 123.94,
120.45, 114.48, 114.09, 106.69, 55.15, 49.14, 46.07, 39.70, 36.44, 21.08.
LC-TOF MS (M + H+) calcd for C27H37N6 445.3080, found 445.3073.
6,6′-((5-(3-Aminopropyl)-1,3-phenylene)bis(ethane-2,1-
diyl))bis(4-methylpyridin-2-amine) (4). Intermediate 14a was
synthesized by the same procedures as those to prepare 2 using Boc-
allylamine as the starting material. Compound 15a was synthesized by
general procedure C using 14a as the starting material (yield 49%). To a
solution of 15a (0.2 mmol) in MeOH (10 mL) was added 10% Pd/C
(10 mg). The reaction mixture was stirred at room temperature under a
hydrogen atmosphere for 12 h. The catalyst was removed by filtration
through Celite, and the resulting solution was concentrated in vacuo.
The crude material was purified by column chromatography to yield
16a. Compound 4 was synthesized by general procedure D using 16a as
the starting material (quantitative). 1H NMR (500 MHz, D2O): δ 6.77
(d, J = 1.5 Hz, 2H), 6.67 (t, J = 1.5 Hz, 1H), 6.45 (s, 2H), 6.23 (d, J = 1.5
Hz, 2H), 2.79 (s, 8H), 2.76−2.70 (m, 2H), 2.48−2.44 (m, 2H), 2.08 (s,
6H), 1.75−1.69 (m, 2H). 13C NMR (125 MHz, D2O): δ 162.68, 157.75,
153.40, 148.08, 141.36, 140.23, 126.67, 114.40, 109.35, 38.95, 33.71,
33.60, 31.56, 29.44, 20.96. LC-TOF MS (M + H+) calcd for C25H34N5
404.2814, found 404.2808.
General Procedure A: LiBH4 Reduction. TMSCl (2.4 mmol) was
added to a suspension of LiBH4 (2.4 mmol) in anhydrous THF (20 mL)
at 0 °C. The reaction mixture was stirred under argon for 30 min at room
temperature. To this reaction mixture was added dropwise bromo-
substituted isophthalic acids or 7 (1.0 mmol) in anhydrous THF (2 mL).
The reaction mixture was stirred for 12 h at room temperature. The
reaction mixture was then quenched with MeOH in an ice bath,
extracted with EtOAc, and washed with water and brine. The organic
layer was concentrated in vacuo. The residue was purified by column
chromatography to yield 8a, 8b, or 6.
General Procedure B: Synthesis of 2,5-Dimethylpyrrole-
Protected Intermediates. To a solution of 2-(2,5-dimethyl-1H-
pyrrol-1-yl)-4,6-dimethyl-pyridine (1.0 mmol) in THF (20 mL) at −78
°C was added n-BuLi (1.6 M solution in hexanes, 2.5 mmol) dropwise.
The resulting dark red solution after the addition was transferred to an
ice bath. After 30 min, a solution of bromide 9a, 9b, or 18 (0.5 M in
THF) was added dropwise until the dark red color disappeared. The
reaction mixture was allowed to stir at 0 °C for an additional 10 min and
then quenched with H2O. The solvent was removed by rotary
evaporation, and the resulting yellow oil was purified by flash
chromatography to yield 2,5-dimethylpyrrole-protected intermediates
10a, 10b, or 19.
6,6′-((4-(3-Aminopropyl)-1,3-phenylene)bis(ethane-2,1-
diyl))bis(4-methylpyridin-2-amine) (5). Compound 5 was synthe-
sized by the same procedure as that to prepare 4 using 4-bromoiso-
1
phthalic acid as the starting material. H NMR (500 MHz, MeOD):
General Procedure C: 2,5-Dimethylpyrrole Deprotection. To
a solution of 11, 12, 14a, 14b, or 19 (0.5 mmol) in EtOH (20 mL)
was added hydroxylamine hydrochloride (5 mmol) followed by H2O
(10 mL). The reaction mixture was heated at 100 °C for 24 h. After
cooling to room temperature, the mixture was partitioned between Et2O
δ 6.97−6.95 (m, 1H), 6.90−6.87 (m, 2H), 6.20−6.18 (m, 4H), 2.94−
2.84 (m, 2H), 2.82−2.75 (m, 4H), 2.68−2.61 (m, 4H), 2.60−2.55
(m, 4H), 2.07 (s, 6H), 1.26−1.23 (m, 2H). 13C NMR (125 MHz,
MeOD): δ 160.33, 160.31, 160.12, 158.51, 152.01, 151.95, 151.93,
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Table 2. Crystallographic Data Collection and Refinement Statistics
a
data set
nNOS-4
nNOS-5
nNOS-6
4IMU
nNOS-7
4IMW
eNOS-7
Data Collection
PDB code
4IMS
4IMT
4IMX
P212121
space group
P212121
P212121
P212121
P212121
Cell Dimensions
a, b, c (Å)
51.8, 110.4, 164.1
2.15 (2.19−2.15)
0.093 (0.632)
18.0 (2.0)
51.7, 110.7, 163.7
2.20 (2.24−2.20)
0.073 (0.723)
19.6 (1.8)
51.9, 110.2, 163.9
2.03 (2.07−2.03)
0.067 (0.618)
21.8 (2.0)
51.7, 111.4, 164.2
2.15 (2.19−2.15)
0.075 (0.674)
23.4 (2.2)
58.1, 106.6, 156.5
2.25 (2.29−2.25)
0.063 (0.653)
21.7 (2.2)
resolution (Å)
Rsym or Rmerge
I/σI
no. unique reflections
completeness (%)
redundancy
52 114
48 484
60 761
52 331
46 588
99.5 (99.8)
3.5 (3.6)
99.6 (98.5)
4.0 (3.9)
99.0 (96.5)
3.6 (3.4)
99.6 (99.8)
4.6 (4.5)
99.6 (99.8)
3.6 (3.7)
Refinement
resolution (Å)
2.15
2.20
2.03
2.20
2.25
no. reflections used
49 363
45 951
57 686
46 313
44 205
b
Rwork/Rfree
0.183/0.235
0.199/0.248
0.184/0.225
0.213/0.273
0.176/0.222
No. Atoms
protein
6679
163
6674
189
6682
193
6659
185
6446
211
ligand/ion
water
244
162
350
158
249
RMS Deviations
bond length (Å)
bond angle (deg)
0.012
2.05
0.013
2.08
0.011
1.97
0.016
1.56
0.015
1.47
a
b
See Figure 3 for the inhibitor chemical formula. Rfree was calculated with the 5% of reflections set aside throughout the refinement. The set of
reflections for the Rfree calculation were kept the same for all data sets of each isoform according to those used in the data of the starting model.
140.29, 137.19, 130.88, 130.21, 127.64, 127.62, 114.72, 114.62, 108.26,
39.95, 39.85, 36.70, 35.30, 30.35, 29.99, 23.89, 21.16, 21.13. LC-TOF
MS (M + H+) calcd for C25H34N5 404.2814, found 404.2806.
calmodulin, 100 μM NADPH, 10 μM H4B, 3.0 μM oxyhemoglobin
(for iNOS assays, no Ca2+ and calmodulin were added). The assay was
initiated by the addition of enzyme, and the initial rates of the enzymatic
reactions were determined by monitoring the formation of the NO−
hemoglobin complex at 401 nm for 60 s. The apparent Ki values were
obtained by measuring the percent enzyme inhibition in the presence of
10 μM L-arginine with at least five concentrations of inhibitor. The
parameters of the following inhibition equation were fitted to the initial
velocity data: percent inhibition = 100[I]/{[I] + Ki(1 + [S]/Km)}. Km
values for L-arginine were 1.3 μM (nNOS), 8.2 μM (iNOS), and 1.7 μM
(eNOS). The selectivity of an inhibitor was defined as the ratio of the
respective Ki values.
Inhibitor Complex Crystal Preparation. The nNOS or eNOS
heme domain proteins used for crystallographic studies were produced
by limited trypsin digestion from the corresponding full length enzymes
and further purified through a Superdex 200 gel-filtration column (GE
Healthcare), as described previously.22,23 The nNOS heme domain at
7−9 mg/mL containing 20 mM histidine or the eNOS heme domain at
12 mg/mL containing 2 mM imidazole were used for the sitting drop
vapor diffusion crystallization setup under the conditions previously
reported.22,23 Fresh crystals (1−2 day old) were first passed stepwise
through cryo-protectant solutions as described22,23 and then soaked
with 10 mM inhibitor for 4−6 h at 4 °C before being mounted on nylon
loops and flash cooled by plunging into liquid nitrogen.
X-ray Diffraction Data Collection, Processing, and Structure
Refinement. The cryogenic (100 K) X-ray diffraction data were
collected remotely at various beamlines at the Stanford Synchrotron
Radiation Lightsource through the data collection control software Blu-
Ice24 and a crystal mounting robot. Raw data frames were indexed,
integrated, and scaled using HKL2000.25 The binding of inhibitors was
detected by the initial difference Fourier maps calculated with
REFMAC.26 The inhibitor molecules were then modeled in COOT27
and refined using REFMAC. Water molecules were added in REFMAC
and checked by COOT. The TLS28 protocol was implemented in the
final stage of refinements with each subunit as one TLS group. The omit
Fo−Fc density maps were calculated by repeating the last round of TLS
refinement with the inhibitor coordinate removed from the input PDB
file to generate the coefficients DELFWT and SIGDELFWT. The
refined structures were validated in COOT before deposition in the
6,6′-((5-(Aminomethyl)-1,3-phenylene)bis(ethane-2,1-diyl))-
bis(4-methylpyridin-2-amine) (6). Compound 6 was synthesized by
1
general procedure A using 7 as the starting material (yield 42%). H
NMR (500 MHz, MeOD): δ 7.06−6.96 (m, 3H), 6.34−6.21 (m, 4H),
2.92−2.88 (m, 4H), 2.85−2.73 (m, 6H), 2.17 (s, 6H). 13C NMR (125
MHz, MeOD): δ 161.51, 160.68, 151.03, 129.67, 129.61, 126.26, 119.69,
114.67, 107.90, 40.66, 37.29, 24.25, 21.05. LC-TOF MS (M + H+) calcd
for C23H30N5 376.2501, found 376.2495.
3,5-Bis(2-(6-amino-4-methylpyridin-2-yl)ethyl)benzonitrile
(7). To a solution of 1-bromo-3,5-dimethylbenzene (5 mmol) in dry
DMF (20 mL) was added CuCN (6 mmol). The reaction mixture was
stirred at 180 °C under N2 for 6 h. The solid was removed by filtration
through Celite, and the resulting solution was diluted by H2O, extracted
with EtOAc, and washed with water and brine. The organic layer was
dried over Na2SO4 and concentrated in vacuo. The residue was purified
by column chromatography to yield 17 (yield 84%). To a suspension of
17 (4.0 mmol) in dry CCl4 (40 mL) was added NBS (8 mmol) and BPO
(cat.). The reaction mixture was stirred at 80 °C for 6 h. The mixture was
concentrated in vacuo and then purified by column chromatography to
yield 18 (yield 22%). Compound 7 was synthesized by general
procedures B and C using 18 (0.5 mmol) as the starting material (two
steps yield 14.8%). 1H NMR (500 MHz, CDCl3): δ 7.28 (d, J = 1.5 Hz,
2H), 7.27−7.26 (m, 1H), 6.25 (s, 2H), 6.18 (s, 2H), 3.00−2.91 (m, 4H),
2.86−2.77 (m, 4H), 2.17 (s, 6H). 13C NMR (125 MHz, CDCl3): δ
158.23, 158.19, 149.51, 143.10, 133.66, 129.69, 119.33, 114.44, 111.92,
106.86, 39.16, 35.40, 21.00. LC-TOF MS (M + H+) calcd for C23H26N5
372.2188, found 372.2183.
Enzyme Assays. The three isozymes, rat nNOS, murine macro-
phage iNOS, and bovine eNOS, were recombinant enzymes, overex-
pressed (in Escherichia coli) and isolated as reported.18−20 Ki determined
for inhibitors 2−7 with the three different isoforms of NOS using
L-arginine as a substrate. The formation of nitric oxide was measured using
the hemoglobin capture assay described previously.21 All NOS isozymes
were assayed using the Synergy H1 hybrid multi-mode microplate
reader (BioTek Instruments, Inc.) at 37 °C in a 100 mM HEPES buffer
(pH 7.4) containing 10 μM L-arginine, 0.83 mM CaCl2, 320 units/mL
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Journal of Medicinal Chemistry
Article
RCSB protein data bank. The crystallographic data collection and
structure refinement statistics are summarized in Table 2 with PDB
accession codes included.
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ASSOCIATED CONTENT
Accession Codes
■
The coordinates for the five crystal structures reported in this
paper have been deposited with RCSB, which will be
immediately released upon publication. The PDB entry codes
for crystal structures reported in this work are as follows: nNOS-
4, 4IMS; nNOS-5, 4IMT; nNOS-6, 4IMU; nNOS-7, 4IMW;
eNOS-7, 4IMX.
AUTHOR INFORMATION
Corresponding Author
■
*(T.L.P.) Phone: (949) 824-7020; fax: (949) 824-3280; e-mail:
poulos@uci.edu. (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
■
The authors 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 collections.
ABBREVIATIONS USED
■
NOS, nitric oxide synthase; nNOS, neuronal nitric oxide
synthase; eNOS, endothelial nitric oxide synthase; iNOS,
inducible nitric oxide synthase; H4B, (6R)-5,6,7,8-tetrahydro-L-
biopterin; NO, nitric oxide; NADPH, reduced nicotinamide
adenine dinucleotide phosphate; CNS, central nervous system;
Ph3P, triphenylphospine; BINAP, 2,2′-bis(diphenylphosphino)-
1,1′-binaphthyl; DIAD, diisopropyl azodicarboxylate; TMSCl,
trimethylsilyl chloride; DIEA, N,N-diisopropylethylamine;
DMF, dimethylformamide
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