In search of potent and selective inhibitors of neuronal nitric oxide synthase with more simple structures

Article abstract of DOI:10.1016/j.bmc.2013.06.014

In certain neurodegenerative diseases damaging levels of nitric oxide (NO) are produced by neuronal nitric oxide synthase (nNOS). It, therefore, is important to develop inhibitors selective for nNOS that do not interfere with other NOS isoforms, especially endothelial NOS (eNOS), which is critical for proper functioning of the cardiovascular system. While we have been successful in developing potent and isoform-selective inhibitors, such as lead compounds 1 and 2, the ease of synthesis and bioavailability have been problematic. Here we describe a new series of compounds including crystal structures of NOS-inhibitor complexes that integrate the advantages of easy synthesis and good biological properties compared to the lead compounds. These results provide the basis for additional structure-activity relationship (SAR) studies to guide further improvement of isozyme selective inhibitors.

Full text of DOI:10.1016/j.bmc.2013.06.014

Bioorganic & Medicinal Chemistry 21 (2013) 5323–5331
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
In search of potent and selective inhibitors of neuronal nitric oxide
synthase with more simple structures
Qing Jing a,b, Huiying Li c,d,e, Jianguo Fang a,b, Linda J. Roman f, Pavel Martásek f, , Thomas L. Poulos c,d,e,
,
⇑
Richard B. Silverman a,b,
⇑
a Department of Chemistry, Chemistry of Life Processes Institute, Center for Molecular Innovation and Drug Discovery, Northwestern University, Evanston, IL 60208-3113, USA
b Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA
c Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900, USA
d Department of Pharmaceutical Sciences, University of California, Irvine, CA 92697, USA
e Department of Chemistry, University of California, Irvine, CA 92697-2025, USA
f Department of Biochemistry, The University of Texas Health Science Center, San Antonio, TX 78384-7760, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In certain neurodegenerative diseases damaging levels of nitric oxide (NO) are produced by neuronal
nitric oxide synthase (nNOS). It, therefore, is important to develop inhibitors selective for nNOS that
do not interfere with other NOS isoforms, especially endothelial NOS (eNOS), which is critical for proper
functioning of the cardiovascular system. While we have been successful in developing potent and iso-
form-selective inhibitors, such as lead compounds 1 and 2, the ease of synthesis and bioavailability have
been problematic. Here we describe a new series of compounds including crystal structures of NOS-inhib-
itor complexes that integrate the advantages of easy synthesis and good biological properties compared
to the lead compounds. These results provide the basis for additional structure–activity relationship
(SAR) studies to guide further improvement of isozyme selective inhibitors.
Received 26 April 2013
Revised 30 May 2013
Accepted 6 June 2013
Available online 15 June 2013
Keywords:
Nitric oxide
Neuronal nitric oxide synthase
Inhibition
Aminopyridines
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
tive nNOS inhibitors,10 combining ease of synthesis, bioavailability,
and selectivity remains a challenging task.
Nitric oxide (NO) is an important second-messenger molecule
that plays many fundamental physiological roles. It is produced
from L-arginine by the nitric oxide synthase (NOS) family of en-
We have previously developed two lead compounds (1 and 2)
that exhibit excellent potency against nNOS. Compound 1 provides
an excellent dual-selectivity of nNOS over the other two isoforms
(Ki = 7 nM, e/n = 2667, i/n = 806),11 but, unfortunately, the tedious
synthesis limits its structure/activity optimization to improve bio-
availability. The synthesis of 2 (three synthetic steps) is much eas-
ier than 1 (15 synthetic steps plus a chiral resolution), but its
selectivity needs to be improved considerably.12 On the basis of
the current results, we attempted a new strategy to integrate both
the advantages of easy synthesis and good activity for the next
generation of inhibitors. The new compounds were designed with
short synthetic routes, and their structures are ready for further
optimization.
zymes, which includes neuronal NOS (nNOS), endothelial NOS
(eNOS), and inducible NOS (iNOS). However, overproduction of
NO by the nNOS in the brain is closely associated with many neu-
rodegenerative diseases, including chronic pathologies such as Par-
kinson’s,1 Alzheimer’s,2 Huntington’s,3 headaches,4 and neuronal
damage in stroke.5 Therefore, it has become a promising strategy
to inhibit nNOS to block the excess generation of NO for the treat-
ment of neurodegeneration.6–8 A large number of nNOS inhibitors
have been reported9 but none of these inhibitors has entered clin-
ical trials because of low potency or poor isoform selectivity.9 The
isozymes of NOS share ꢀ50% sequence homology and show a
highly similar heme active site structure, which is why most inhib-
itors directed at the substrate binding site show limited isoform
selectivity. While we have succeeded in developing highly selec-
2. Chemistry
The synthesis of inhibitors 5 began with a condensation reac-
tion to protect 4,6-dimethylpyridine, and the product, compound
3, was deprotonated with n-BuLi then treated with a dibromide
to obtain compound 4. After removal of the protecting groups in
the presence NH2OH–HCl, inhibitors 5a–c were obtained, as de-
scribed in Scheme 1.
⇑
Corresponding authors. Tel.: +1 949 824 7020 (T.L.P.), +1 847 491 5653 (R.B.S.).
E-mail addresses: poulos@uci.edu (T.L. Poulos), Agman@chem.northwestern.edu
(R.B. Silverman).
Present address: Department of Pediatrics and Center for Applied Genomics, 1st
School of Medicine, Charles University, Prague, Czech Republic.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.06.014

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Q. Jing et al. / Bioorg. Med. Chem. 21 (2013) 5323–5331
O
a
b
+
n
(
)
N
N
N
N
N
N
H2N
N
c
O
Br
nBr
(
)
3
4a: n = 3
4b: n = 4
4c: n = 5
n = 3, 4, 5
n
(
)
N
NH2
H2N
N
5a: n = 3
5b: n = 4
5c: n = 5
Scheme 1. Synthesis of 5a–c. Reagents and conditions: (a) p-TsOH, toluene, reflux, 89%; (b) (i) n-BuLi, THF, ꢁ78 to 0 °C; (ii) dibromide, 51–55%; (c) NH2OH–HCl, EtOH/H2O (2/
1), 100 °C, 20 h, 79–82%.
HO
OH
a
HO
O
N
N
Br
N
+
7
6
d
b
c
TMS
Br
N
N
N
N
N
8
3
9
e
O
O
NH2
O
O
N
H2N
N
N
N
11
10
Scheme 2. Synthesis of 11. Reagents and conditions: (a) K2CO3, acetone, reflux, 71%; (b) (i) n-BuLi, THF, ꢁ78 to 0 °C; (ii) TMSCl, 0 °C, 95%; (c) BrF2CCF2Br, CsF, DMF, 91%; (d)
K2CO3, acetone, reflux, 79%; (e) NH2OH–HCl, EtOH/H2O (2/1), 20 h, 100 °C.
Compound 6 was prepared according to the general method for
amine protection (see details in Supplementary Data). Addition of a
three-fold excess of 1,3-dihydroxybenzene over 6 provided mono-
substituted 7. Compound 9 was prepared according to a literature
report13 as shown in Scheme 2. A standard ether synthesis linked
fragments 7 and 9 together to produce 10 followed by deprotection
of the amine to afford inhibitors 11.
Thesynthesisof14and15followeda similarmethodasthat for11
but using NaH to deprotect 1,3-dihydroxybenzene without a large
excess over 4-(bromomethyl)pyridine (Scheme 3). A second ether
synthesis connected the 4-methyl aminopyridine head to generate
13 followed by deprotection of the amino group to yield 14. Com-
pound 15 followed the same pathway starting from 2,20-biphenol.
In the synthesis of 18 and 19 (Scheme 4), a Mitsunobu reaction
was used to add the N-Boc-piperidinemethyl fragment to dihy-
droxybenzene, and mono-substituted 16 was isolated in a 53%
yield. An ether synthesis connected the 4-methylaminopyridine
to generate 17, followed by deprotection of the amino and Boc
groups to yield 18. Compound 19 followed the same pathway
but started with 2,20-biphenol.
and 3-fluorophenethyl aldehyde to provide 23. After deprotection
of the amino group, 24 was isolated in a 92% yield.
The synthesis of inhibitor 27 began with 3-hydroxybenzalde-
hyde, and the 4-methylpyridine head was added in the presence
of 9 and K2CO3 in acetone (Scheme 6). Reductive amination con-
nected 25 and 3-fluorophenethyl amine together to provide 26.
Compound 27 was obtained after deprotection of the amino group
with NH2OH–HCl.
2,6-Di(hydroxymethyl)pyridine was used as the starting mate-
rial for the synthesis of 32 (Scheme 7). It was oxidized to mono
aldehyde 28 with Dess–Martin periodinane (DMP) followed by
reductive amination with 3-fluorophenethyl amine. The resulting
secondary amine (29) was Boc-protected and then treated with 9
in the presence of NaH in DMF to construct 31. Inhibitor 32 was ob-
tained after deprotection of dimethylpyrrole and Boc, respectively,
in a yield of 71% for the two steps.
The synthesis of 36 is illustrated in Scheme 8. First, 5-hydroxy-
3-pyridinecarboxylic acid methyl ester was linked to the 4-methyl-
2-aminopyridine head, and then the methyl ester was reduced to
aldehyde 34 with Dibal-H at ꢁ78 °C. Reductive amination success-
fully connected the second head, 3-fluorophenethyl amine. Com-
pound 36 was afforded after removing the protecting group.
The synthesis of inhibitor 24 started from 3-hydroxyaniline; the
first step was protection of the amino group with Boc (Scheme 5),.
A standard ether synthesis linked the 4-methylpyridine head suc-
cessfully in the presence of K2CO3 in acetone. The Boc group was
removed in 1.5 M HCl methanol after reaction for four hours. The
3-fluorophenethyl aldehyde was prepared from 3-fluorophenetha-
nol via Dess–Martin oxidation. Reductive amination connected 22
3. Results and discussion
All of the inhibitors were assayed against three different iso-
forms of NOS, rat nNOS, bovine eNOS, and murine macrophage

Q. Jing et al. / Bioorg. Med. Chem. 21 (2013) 5323–5331
5325
H2N
N
N
O
O
15
Scheme 3. Synthesis of 14 and 15. Reagents and conditions: (a) (i) NaH, DMF, 0 °C; (ii) 4-(bromomethyl)pyridine, 46%; (b) (i) NaH, DMF, 0 °C; (ii) compound 9, 59%; (c)
BrF2CCF2Br, CsF, DMF, 91%; (d) K2CO3, acetone, reflux, 79%; (e) NH2OH–HCl, EtOH/H2O (2/1), 100 °C, 20 h.
NH
H2N
N
O
O
19
Scheme 4. Synthesis of 18 and 19. Reagents and conditions: (a) DIAD, Ph3P, N-Boc-piperidinemethanol, THF, 53%; (b) K2CO3, acetone, compound 9, reflux, 73%; (c) (i) NH2OH–
HCl, EtOH/H2O, 100 °C, 20 h; (ii) 2 M HCl in dioxane/CH3OH (1/1), 80%.
HO
NHBoc
HO
NH2
NH2
O
a
c
b
O
NHBoc
N
N
N
N
20
22
21
H
N
d
H
N
F
O
e
F
O
N
N
H2N
N
23
24
Scheme 5. Synthesis of 24. Reagents and conditions: (a) (Boc)2O, THF, reflux, 93%; (b) K2CO3, acetone, compound 9, reflux, 90%; (c) 1.5 M HCl in CH3OH, 98%; (d) NaBH(OAc)3,
3-fluorophenethyl aldehyde, DCE, 3 Å MS, 67%; (e) NH2OH–HCl, EtOH/H2O (2/1), 100 °C, 20 h, 92%.
iNOS, using
L
-arginine as a substrate. Although inhibitors 5a–c
aminopyridine next to the heme propionate from pyrrole ring D
(referred to as propionate D). The orientation of this second amino-
pyridine group in nNOS is clearly different from that seen in eNOS,
even though the electron density for the second aminopyridine is
less certain compared to that for the first aminopyridine. This ami-
nopyridine bends over forming H-bonds with both Asn569 and
heme propionate D in nNOS, while it forms only one H-bond with
Asn340 in eNOS. The Met336 side chain in nNOS has two unusual
rotamer conformations (Fig. 2A), which might prevent 5a from
have good potency with nNOS, selectivity over the other two iso-
forms remains at the same level as compound 2 (<100).11 The crys-
tal structures of these compounds in complex with nNOS or eNOS
were determined to test the effects of the linker length between
the two aminopyridine head groups. As shown in Figure 1, 5a binds
to nNOS and eNOS in a similar way with its first aminopyridine
moiety H-bonded with active site glutamate residue Glu592 in
nNOS and Glu363 in eNOS. The 5-carbon linker brings the second

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Q. Jing et al. / Bioorg. Med. Chem. 21 (2013) 5323–5331
HO
CHO
a
b
O
CHO
O
N
N
N
N
N
H
F
25
26
c
O
H2N
N
N
H
F
27
Scheme 6. Synthesis of 27. Reagents and conditions: (a) K2CO3, acetone, compound 9, reflux, 72%; (b) NaBH(OAc)3, 3-fluorophenethyl amine, DCE, 3 Å MS, 64%; (c) NH2OH–
HCl, EtOH/H2O (2/1), 100 °C, 20 h, 91%.
F
F
N
CHO
N
c
a
b
HO
N
N
HO
OH
N
H
N
Boc
HO
HO
28
30
29
e
d
H2N
N
N
N
N
N
O
N
H
F
O
N
Boc
F
32
31
Scheme 7. Synthesis of 32. Reagents and conditions: (a) Dess–Martin periodinane (DMP), CH2Cl2, 46%; (b) NaBH(OAc)3, 3-fluorophenethyl amine, DCE, 3 Å MS, 78%; (c)
(Boc)2O, NaHCO3, CH3OH, 76%; (d) NaH, compound 9, DMF, 0 °C, 51%; (e) (i) NH2OH–HCl, EtOH/H2O (2/1), 100 °C, 20 h; (ii) 2 M HCl in dioxane/CH3OH (1/1), 71%.
O
O
O
c
HO
a
b
O
O
O
O
N
N
N
N
N
N
d
N
33
34
O
O
N
N
N
H
F
H2N
N
N
F
H
N
N
35
36
Scheme 8. Synthesis of 36. Reagents and conditions: (a) NaH, compound 9, DMF, 0 °C, 70%; (b) Dibal-H, ꢁ78 °C, 56%; (c) NaBH(OAc)3, 3-fluorophenethyl amine, DCE, 3 Å MS,
90%; (d) NH2OH–HCl, EtOH/H2O (2/1), 100 °C, 20 h, 79%.
Figure 1. Structures of lead compounds 1 and 2.
adopting the more extended conformation seen in eNOS (Fig. 2B),
where a smaller and more rigid Val106 residue is in the same
location.
Since working with the double-headed aminopyridine inhibi-
tors,13 we have noticed that inhibitors with a 7- or 8-atom spacer
between the two aminopyridine groups are capable of having one
aminopyridine form tight bifurcated H-bonds with the other ami-
nopyridine bound to heme propionate D, in addition to the H-
bonds between the first aminopyridine and the NOS active site glu-
tamate (Fig. 3A). This is referred to as the double-headed binding
mode. Consistent with the earlier observations, 5c, with a 7-carbon
linker, binds to both nNOS and eNOS in the double-headed binding

Q. Jing et al. / Bioorg. Med. Chem. 21 (2013) 5323–5331
5327
Figure 2. Inhibitor 5a bound to nNOS (A) or eNOS (B). The omit Fo–Fc density for inhibitor is shown at a 2.5r contour level. Relevant H-bonds are depicted as dashed-lines. In
nNOS-5a structure a partially occupied (50%) zinc site is detected similar to what was observed in some other nNOS inhibitor complex structures14 Compound 5a is small and
flexible enough even to bind in a groove on the molecular surface of nNOS next to Glu705 (data not shown). Structure figures were made with PyMol (http://www.pymol.org).
Figure 3. The active site of nNOS (A) or eNOS (B) in complex with inhibitor 5c. The omit Fo–Fc density for inhibitor is shown at a 2.5
depicted as dashed-lines. Two alternate conformations are observed in nNOS for inhibitor 5c and Tyr706.
r contour level. Relevant H-bonds are
Figure 4. The active site of nNOS complexed with inhibitor 11 (A) and 18 (B). The omit Fo–Fc density for inhibitor is shown at a 2.5r contour level. Relevant H-bonds are
depicted as dashed-lines.
mode, as shown in Figure 3. However, one binding conformation of
5c cannot fully account for the electron density features in nNOS.
The second conformation of 5c also exists that makes a H-bond
with Asn569. To match the alternate binding of 5c, the Tyr706 side
chain shows two alternate rotamers as well (Fig. 3A). Inhibitor 5b
has a 6-carbon linker, which is more flexible than 5a, but the linker
is not long enough to allow its second aminopyridine to H-bond
with the heme propionate D carboxylic acid. For the nNOS–5b
complex, only the first aminopyridine that interacts with the active
site Glu is ordered (data not shown).
Compound 11 has a potency of 60 nM against nNOS as well as
selectivity of 298 and 140 over eNOS and iNOS, respectively, the
highest among the inhibitors tested in this work. Unfortunately,
we could only obtain partial information from the crystal structure

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Q. Jing et al. / Bioorg. Med. Chem. 21 (2013) 5323–5331
of the nNOS–11 complex because the aminopropyl tail is disor-
dered. As shown in Figure 4, while the aminopyridine forms tight
H-bonds with Glu592, the orientation of the phenyl ring is less cer-
tain. The ether oxygen can indirectly interact with the heme and
H4B through a bridging water molecule (Fig. 4A). It is reasonable
to assume that the aminopropyl N atom does not have any favor-
able contact with the protein; otherwise, it would have been visi-
ble in the structure. No eNOS structure was attempted because of
the poor binding of 11 to eNOS. The introduction of a rigid pyridine
group in 14 did not work, but having a non-planar piperidine in
inhibitor 18 led to better potency and selectivity. As seen in Figure
4B, although the density for the piperidine is not as good as that for
other part of 18, it does show the potential for the ring N atom to
interact with both heme and H4B, replacing a conserved water
molecule that normally H-bonds to both the heme and H4B (see
Fig. 4A). The planar pyridine ring in 14 is not expected to establish
similar interactions with the protein observed for piperidine in 18.
Inhibitors 11 and 18 share high structural similarity, thus exhibit-
ing similar inhibitory activity. When the phenyl linker was re-
placed with a biphenyl linker, such as inhibitors 15 and 19, the
potency dropped dramatically. Their bulky and rigid structures
might be the cause of the loss of activity, which is consistent with
what we learned about the inhibitor binding pocket in the struc-
tures shown in Figure 4.
Compound 27, which resulted by merging functional groups in
lead compounds 1 and 2, is the best inhibitor in this series,
affording a potency of 40 nM with a dual selectivity of 267 (e/
n) and 147 (i/n). A very similar inhibitor, 24, with one carbon
atom less than 27 in the right arm, exhibits a 54-fold decrease
in potency. The structure of nNOS-27, shown in Figure 5, provides
some clues as to why the benzylamine of 27 binds to the active
site of nNOS much better than does the aniline fragment in 24.
Even though the density for the fluorophenyl tail of 27 is weaker
than that for the other part of the inhibitor, the benzylamine N
atom is likely involved in an H-bonding network with heme
and H4B through one bridging water molecule (Fig. 5A). The ani-
line N atom in 24 is co-planer with the ring and, therefore, is
much less flexible. A rotation of the entire aniline ring would
be required for the N atom to H-bond with the heme propionate.
We have also tested inhibitors 32 and 36, where the phenyl linker
was replaced with a more hydrophilic pyridine ring. In addition,
one more carbon was introduced between the aminopyridine
and the middle ring in 32. However, those changes decrease the
potency and selectivity relative to 27 (Table 1). For the nNOS-
32 structure only the aminopyridine ring that H-bonds to
Glu592 is visible. The partial density indicates that the N atom
of the middle pyridine might interact with the heme propionates,
but the orientation of the ring is not well defined, which leads to
total disordering of the tail (data not shown). Fortunately, struc-
tures of 36 with both nNOS and eNOS are available, as shown
in Figure 5B and C, respectively. It is clear that the N atom in
the middle pyridine ring of 36 does not interact with heme. The
ring N atom is at the meta-position relative to the other substit-
uents so that if the ring N atom had H-bonded with the heme by
flipping 180° from what is shown in Figure 5B and C, the fluoro-
phenyl tail would be forced to reposition, thereby losing impor-
tant protein-inhibitor contacts. In the observed binding
conformation, the amine N atom adjacent to the middle pyridine
ring can join the H-bonding network, through two water mole-
cules, with heme and H4B. This two-water bridged H-bonding
network in 36 is likely weaker than the one-water bridged net-
work seen in 27 in terms of the binding energy gained by the
inhibitor. The orientation of the fluorophenyl ring is different in
nNOS vs. eNOS, which may be influenced by the nearby amino
acid variant, Met336 in nNOS vs. Val106 in eNOS; this may con-
tribute to the observed isoform selectivity.
Figure 5. The active site of nNOS complexed with inhibitor 27 (A) and 36 (B), and
that of eNOS complexed with 36 (C). The omit Fo–Fc density for inhibitor is shown at
a 2.5r contour level. Relevant H-bonds are depicted as dashed-lines.
In conclusion, we designed and synthesized a series of inhibi-
tors by using the functional groups from lead compounds 1 and
2. The new series of inhibitors feature both the advantages of easy
synthesis and improved isoform selectivity properties (up to

Q. Jing et al. / Bioorg. Med. Chem. 21 (2013) 5323–5331
5329
Table 1
Kia values of inhibitors for rat nNOS, bovine eNOS and murine iNOS
Inhibitors
Kia (nM)
Selectivity
e/n
nNOS
eNOS
iNOS
i/n
5a
5b
5c
11
14
15
18
19
24
27
32
36
48
33
86
60
616
3847
117
4235
2156
40
979
375
20
86
25
77
54
25
140
44
2842
2154
18018
62839
NDb
1798
2193
8472
27123
NDb
298
102
NDb
239
NDb
NDb
261
21
NDb
146
NDb
NDb
147
31
27963
17124
NDb
NDb
NDb
NDb
10429
5623
10366
5886
8427
2862
270
140
74
21
a
The IC50 values were measured for three different isoforms of NOS including rat nNOS, bovine eNOS, and murine macrophage iNOS using
L-arginine as a substrate. Nine
data points (concentrations) were collected, and the IC50 values were calculated using a nonlinear regression method. Each experiment was repeated three times, and the
average numbers are presented in Table 1. The experimental standard deviations were less than 10%. The corresponding Ki values were calculated from the IC50 values using
the equation Ki = IC50/(1 + [S]/Km) with known Km values (rat nNOS, 1.3
lM; iNOS, 8.3
lM; eNOS, 1.7
lM).
b
Not determined.
298-fold) compared to 2. However, the high isoform selectivity of
lead 1 has not yet been matched. Utilizing a planar aromatic ring
as in 2, whether polar or non-polar, as the middle bridging group
increases rigidity, thereby restraining the possible positions for
the remaining tail groups. Disadvantages of this approach seem
to outweigh the advantages, as this greatly limits the variety of
tails that can be used. The most critical, but challenging, task is
to introduce more specific enzyme-inhibitor contacts into nNOS
relative to the other isoforms in order to overcome the disordering
of the tail group, which extends out of the active site. Certainly the
structure–activity-relationship studies here will form a basis for
further improvement. The main advantage of our current com-
pounds is that the ether bond and heteroatom connecting the
two heads simplify the chemistry required to probe a variety of
tails for optimization of isoform selectivity.
4. Experimental section
4.1. General methods
All experiments were conducted under anhydrous conditions in
an atmosphere of argon, using flame-dried apparatus and employ-
ing standard techniques in handling air-sensitive materials. All sol-
vents were distilled and stored under an argon or nitrogen
atmosphere before using. Non-synthesized reagents were pur-
chased from Sigma–Aldrich Co., LLC. and used as received. Aqueous
solutions of sodium bicarbonate, sodium chloride (brine), and
ammonium chloride were saturated. Analytical thin layer
chromatography was visualized by ultraviolet light, potassium
permanganate or phosphomolybdic acid (PMA). Flash column
chromatography was carried out under a positive pressure of air.

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Q. Jing et al. / Bioorg. Med. Chem. 21 (2013) 5323–5331
Table 2
Crystallographic data collection and refinement statistics
Data seta
nNOS-5a
nNOS-5c
nNOS-11
nNOS-18
Data collection
PDB code
4JSE
4JSF
4JSG
4JSH
Space group
P212121
P212121
P212121
P212121
Cell dimensions a, b, c (Å)
Resolution (Å)
Rmerge
51.7 110.2 163.9
1.97 (2.00–1.97)
0.060 (0.553)
25.7 (2.9)
66,633
99.3 (99.7)
4.0 (3.9)
51.8 110.5 164.3
2.05 (2.09–2.05)
0.072 (0.664)
22.7 (2.5)
59,677
99.1 (99.9)
4.0 (4.0)
52.2 111.2 164.2
1.95 (1.98–1.95)
0.066 (0.530)
27.9 (2.3)
71,051
51.7 111.6 164.3
2.35 (2.39–2.35)
0.076 (0.775)
18.9 (1.8)
40,870
I/rI
No. unique reflections
Completeness (%)
Redundancy
99.6 (99.3)
4.0 (3.9)
99.0 (99.9)
3.7 (3.3)
Refinement
Resolution (Å)
No. reflections used
Rwork/Rfree
1.97
63,104
0.170/0.205
2.05
56,506
0.175/0.217
1.95
67,268
0.189/0.226
2.35
38,227
0.193/0.253
b
No. atoms
Protein
Ligand/ion
Water
6728
219
431
6711
197
386
6665
172
357
6832
177
74
R.m.s. deviations
Bond lengths (Å)
Bond angles (°)
0.014
1.42
0.014
1.42
0.010
1.36
0.011
1.52
Data seta
nNOS-27
nNOS-36
eNOS-5a
eNOS-5c
eNOS-36
Data collection
PDB code
4JSI
4JSJ
4JSK
4JSL
4JSM
Space group
P212121
P212121
P212121
P212121
P212121
Cell dimensions a, b, c (Å)
Resolution (Å)
Rmerge
51.6, 110.8, 164.6
2.09 (2.13–2.09)
0.075 (0.656)
22.6 (2.3)
56,724
51.7, 111.3, 164.4
1.92 (1.95–1.92)
0.066 (0.620)
31.3 (2.8)
73,068
99.3 (100.0)
4.0 (4.0)
58.4, 106.6, 157.0
2.28 (2.32–2.28)
0.050 (0.585)
27.8 (2.3)
45,192
98.9 (100.0)
3.3 (3.3)
57.8, 106.6, 157.0
58.3, 106.4, 157.1
2.25 (2.29–2.25)
0.073 (0.674)
18.7 (1.8)
47,060
99.7 (100.0)
3.6 (3.6)
2.04 (2.08–2.04)
0.066 (0.669)
22.6 (2.0)
62,475
99.6 (99.7)
3.4 (3.4)
I/rI
No. unique reflections
Completeness (%)
Redundancy
99.5 (99.9)
4.0 (4.1)
Refinement
Resolution (Å)
No. reflections used
Rwork/Rfree
2.09
53,714
0.193/0.241
1.92
69,161
0.193/0.225
2.28
42,763
0.205/0.258
2.04
59,144
0.167/0.208
2.25
44,635
0.186/0.244
b
No. atoms
Protein
Ligand/ion
Water
6668
183
222
6689
183
366
6427
197
145
6446
205
457
6455
201
244
R.m.s. deviations
Bond lengths (Å)
Bond angles (deg)
0.013
1.56
0.015
1.46
0.016
1.61
0.014
1.47
0.016
1.61
a
See Table 1 for inhibitor chemical formulae.
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
b
isoform according to those used in the data of the starting model.
1H NMR spectra were recorded on 500 MHz Varian or Bruker
AVANCE spectrometers. Data are presented as follows: chemical
shift (in ppm on the d scale relative to d = 0.00 ppm for the protons
in TMS), integration, multiplicity (s = singlet, d = doublet, t = trip-
let, q = quartet, m = multiplet, br = broad), coupling constant (J/
Hz). Coupling constants were taken directly from the spectra and
are uncorrected. 13C NMR spectra were recorded at 125 MHz, and
all chemical shift values are reported in ppm on the d scale with
an internal reference of d 77.0 or 49.0 for CDCl3 or CD3OD, respec-
tively. LC–MS (ESI) was conducted on Agilent LCQ mass spectrom-
eter. High resolution mass spectra (HRMS) were measured with an
Agilent 6210 LC-TOF (ESI) mass spectrometer. The enzyme assay
was monitored on a BioTek Synergy 4 microplate reader.
coli and isolated as reported.15 The formation of nitric oxide was
measured using a hemoglobin capture assay, as described previ-
ously.11 All NOS isozymes were assayed at room temperature in
a 100 mM Hepes buffer (pH 7.4) containing 10
1.6 mM CaCl2, 11.6 g/mL calmodulin, 100
(DTT), 100 M NADPH, 6.5 M H4B, and 3.0
l
M
L
-arginine,
dithiothreitol
lM oxyhemoglobin
l
lM
l
l
(for iNOS assays, no CaCl2 and calmodulin were added). The assay
was initiated by the addition of enzyme, and the initial rates of the
enzymatic reactions were determined on a UV–vis spectrometer by
monitoring the formation of methemoglobin at 401 nm from 0 to
60 s after mixing. The corresponding Ki values of inhibitors were
calculated from the IC50 values using Eq. (1) with known Km values
(rat nNOS, 1.3
lM; iNOS, 8.3 lM; eNOS, 1.7 lM).
Ki ¼ IC50=ð1 þ ½Sꢂ=Km
Þ
ð1Þ
4.2. NOS inhibition assays
4.3. Inhibitor complex crystal preparation
IC50 values for inhibitors 5a–36 were measured for three differ-
ent isoforms of NOS, rat nNOS, bovine eNOS, and murine macro-
The nNOS or eNOS heme domain proteins used for crystallo-
graphic studies were produced by limited trypsin digest from the
phage iNOS using
isoforms were recombinant enzymes overexpressed in Escherichia
L-arginine as a substrate. The three enzyme

Q. Jing et al. / Bioorg. Med. Chem. 21 (2013) 5323–5331
5331
corresponding full length enzymes and further purified through a
Superdex 200 gel filtration column (GE Healthcare) as described
previously.16 The nNOS heme domain at 7–9 mg/mL containing
20 mM histidine or the eNOS heme domain at 12 mg/mL contain-
ing 2 mM imidazole were used for the sitting drop vapor diffusion
crystallization setup under the conditions reported before.16 Fresh
crystals (1–2 day old) were first passed stepwise through cryo-pro-
tectant solutions as described16 and then soaked with 10 mM
inhibitor for 4–6 h at 4 °C before being flash cooled with liquid
nitrogen.
lic. We also thank the beamline staff at SSRL and ALS for their assis-
tance during the remote X-ray diffraction data collections.
Supplementary data
Supplementary data associated (Detailed synthetic procedures
and full characterization (1H NMR, 13C NMR) of compounds 3–
36) with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bmc.2013.06.014.
References and notes
4.4. X-ray diffraction data collection, processing, and structure
refinement
1. Zhang, L.; Dawson, V. L.; Dawson, T. M. Pharmacol. Ther. 2006, 109, 33.
2. Dorheim, M.-A.; Tracey, W. R.; Pollock, J. S.; Grammas, P. Biochem. Biophys. Res.
Commun. 1994, 205, 659.
3. Norris, P. J.; Waldvogel, H. J.; Faull, R. L. M.; Love, D. R.; Emson, P. C.
Neuroscience 1996, 1037, 72.
4. Ashina, M. Exp. Opin. Pharmacother. 2002, 3, 395.
The cryogenic (100 K) X-ray diffraction data were collected re-
motely at various beamlines at Stanford Synchrotron Radiation
Lightsource (SSRL) or Advanced Light Source (ALS) through the
data collection control software Blu-Ice17 and a crystal mounting
robot. Raw data frames were indexed, integrated, and scaled using
HKL2000.18 The binding of inhibitors was detected by the initial
difference Fourier maps calculated with REFMAC.19 The inhibitor
molecules were then modeled in COOT20 and refined using REF-
MAC. Water molecules were added in REFMAC and checked by
COOT. The TLS21 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 inhibitor coordinate removed from the input
PDB file to generate the map coefficients DELFWT and SIGDELFWT.
The refined structures were validated in COOT before deposition in
the RCSB protein data bank. The crystallographic data collection
and structure refinement statistics are summarized in Table 2 with
PDB accession codes included.
5. Sims, N. R.; Anderson, M. F. Neurochem. Int. 2002, 40, 511.
6. Alderton, W. K.; Cooper, C. E.; Knowles, R. G. Biochem. J. 2001, 357, 593.
7. Southan, G. J.; Szabo, C. Biochem. Pharmacol. 1996, 51, 383.
8. Babu, B. R.; Griffith, O. W. Curr. Opin. Chem. Biol. 1998, 2, 491.
9. Ji, H.; Erdal, E. P.; Litzinger, E. A.; Seo, J.; Zhu, Y.; Xue, F.; Fang, J.; Huang, J.;
Silverman, R. B. In Frontiers in Medicinal Chemistry; Reitz, A. B., Choudhary, M. I.,
Atta-ur-Rahman, Eds.; Bentham Science, 2009; Vol. 54, p 842.
10. (a) Silverman, R. B. Acc. Chem. Res. 2009, 42, 439; (b) Poulos, T. P.; Li, H. Acc.
Chem. Res. 2012, 46, 390.
11. Ji, H.; Li, H.; Martásek, P.; Roman, L. J.; Poulos, T. S. L.; Silverman, R. B. J. Med.
Chem. 2009, 52, 779.
12. Xue, F.; Delker, S. L.; Li, H.; Fang, J.; Martásek, P.; Roman, L. J.; Poulos, T. P.;
Silverman, R. B. J. Med. Chem. 2011, 54, 2039.
13. Xue, F.; Gu, W.; Silverman, R. B. Org. Lett. 2009, 11, 5194.
14. Delker, S. L.; Ji, H.; Li, H.; Jamal, J.; Fang, J.; Xue, F.; Silverman, R. B.; Poulos, T. L.
J. Am. Chem. Soc. 2010, 132, 5437.
15. (a) Hevel, J. M.; White, K. A.; Marletta, M. A. J. Biol. Chem. 1991, 266, 22789; (b)
Roman, L. J.; Sheta, E. A.; Martasek, P.; Gross, S. S.; Liu, Q.; Masters, B. S. S. Proc.
Natl. Acad. Sci. U.S.A. 1995, 92, 8428; (c) Martasek, P.; Liu, Q.; Liu, J. W.; Roman,
L. J.; Gross, S. S.; Sessa, W. C.; Masters, B. S. S. Biochem. Biophys. Res. Commun.
1996, 219, 359.
16. (a) Li, H.; Shimizu, H.; Flinspach, M.; Jamal, J.; Yang, W.; Xian, M.; Cai, T.; Wen,
E. Z.; Jia, Q.; Wang, P. G.; Poulos, T. L. Biochemistry 2002, 41, 13868; (b) Raman,
C. S.; Li, H.; Martásek, P.; Král, V.; Masters, B. B. S.; Poulos, T. L. Cell 1998, 95,
939.
Acknowledgments
17. McPhillips, T. M.; McPhillips, S. E.; Chiu, H. J.; Cohen, A. E.; Deacon, A. M.; Ellis,
P. J.; Garman, E.; Gonzalez, A.; Sauter, N. K.; Phizackerley, R. P.; Soltis, S. M.;
Kuhn, P. J. Synchrotron Radiat. 2002, 9, 401.
18. Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307.
19. Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Acta Crystallogr., Sect. D 1997, 53,
240.
20. Emsley, P.; Cowtan, K. Acta Crystallogr., Sect. D 2004, 60, 2126.
21. Winn, M. D.; Isupov, M. N.; Murshudov, G. N. Acta Crystallogr., Sect. D 2001, 57,
122.
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 Distin-
guished Professorship in Chemistry (AQ0012). P.M. is supported by
grants 0021620806 and 1M0520 from MSMT of the Czech Repub-