Potent and Selective Human Neuronal Nitric Oxide Synthase Inhibition by Optimization of the 2-Aminopyridine-Based Scaffold with a Pyridine Linker

Article abstract of DOI:10.1021/acs.jmedchem.6b00273

Neuronal nitric oxide synthase (nNOS) is an important therapeutic target for the treatment of various neurodegenerative disorders. A major challenge in the design of nNOS inhibitors focuses on potency in humans and selectivity over other NOS isoforms. Here we report potent and selective human nNOS inhibitors based on the 2-aminopyridine scaffold with a central pyridine linker. Compound 14j, the most promising inhibitor in this study, exhibits excellent potency for rat nNOS (K_i = 16 nM) with 828-fold n/e and 118-fold n/i selectivity with a K_i value of 13 nM against human nNOS with 1761-fold human n/e selectivity. Compound 14j also displayed good metabolic stability in human liver microsomes, low plasma protein binding, and minimal binding to cytochromes P450 (CYPs), although it had little to no Caco-2 permeability.

Full text of DOI:10.1021/acs.jmedchem.6b00273

Subscriber access provided by NEW YORK UNIV
Article
Potent and Selective Human Neuronal Nitric Oxide Synthase Inhibition by
Optimization of the 2-Aminopyridine-based Scaffold with a Pyridine Linker
Heng-Yen Wang, Yajuan Qin, Huiying Li, Linda J. Roman,
Pavel Martásek, Thomas L. Poulos, and Richard B. Silverman
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00273 • Publication Date (Web): 06 Apr 2016
Downloaded from http://pubs.acs.org on April 7, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
6
Potent and Selective Human Neuronal Nitric Oxide Synthase Inhibition by
Optimization of the 2-Aminopyridine-based Scaffold with a Pyridine Linker
7
8
9
£ ＃
HengꢀYen Wang,†,¥ Yajuan Qin,†,‡,¥ Huiying Li,§ Linda J. Roman,¶ Pavel Martásek,¶, ,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
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 60208ꢀ3113, United States.
‡State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing
University, Nanjing 210093, People’s Republic of China.
§Departments of Molecular Biology and Biochemistry, Pharmaceutical Sciences, and
Chemistry, University of California, Irvine, California 92697ꢀ3900, United States.
¶Department of Biochemistry, University of Texas Health Science Center, San Antonio,
Texas 78384ꢀ7760, United States;
£Department of Pediatrics, First Faculty of Medicine, Charles University, Prague, Czech
Republic
＃BIOCEV, Prague, Czech Republic
¥ These author contributed equally.
Keywords：nitric oxide synthase, neurodegenerative diseases, mutant nNOS, human nNOS,
human eNOS, selective inhibition
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 46
1
2
3
4
Abstract
5
6
7
8
Neuronal nitric oxide synthase (nNOS) is an important therapeutic target for the treatment
of various neurodegenerative disorders. A major challenge in the design of nNOS inhibitors
focuses on potency in humans and selectivity over other NOS isoforms. Here we report
potent and selective human nNOS inhibitors based on the 2ꢀaminopyridine scaffold with a
central pyridine linker. Compound 14j, the most promising inhibitor in this study, exhibits
excellent potency for rat nNOS (Ki 16 nM) with 828ꢀfold n/e and 118ꢀfold n/i, selectivity,
with a Ki value of 13 nM against human nNOS with 1761ꢀfold human n/e selectivity.
Compound 14j also displayed good metabolic stability in human liver microsomes, low
plasma protein binding, and minimal binding to cytochromes P450 (CYPs), although it had
little to no Cacoꢀ2 permeability.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 3 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
6
Introduction
7
8
9
Nitric oxide (NO) is an essential second messenger in mammals,1 which regulates a variety
of biological processes, such as vasodilation,2 smooth muscle relaxation,3 neurotransmission,4
and immune response.5 NO is generated by nitric oxide synthases (NOSs), of which there are
three mammalian NOS isoforms: inducible NOS (iNOS), which activates the immune system
to destroy pathogens and microorganisms;6 endothelial NOS (eNOS), which regulates the
relaxation of smooth muscle, leading to a decrease of blood pressure;7 and neuronal NOS
(nNOS), which regulates the release of neurotransmitters8 and is involved in neuronal
communication.9 Overproduction of NO in the central nervous system (CNS) has been
reported to be associated with chronic neurodegenerative pathogenesis,10 such as Alzheimer’s
disease (AD),11 Parkinson’s disease (PD),12 and Huntington’s disease (HD),13 as well as
amyotrophic lateral sclerosis (ALS).14 Therefore, inhibition of nNOS is a viable therapeutic
strategy for treating neurodegenerative disorders.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
All of the mammalian NOSs are active only as homodimers,15 and each monomer consists
of a Cꢀterminal reductase domain, which contains the binding sites for NADPH (reduced
nicotinamide adenine dinucleotide phosphate), FAD (flavin adenine dinucleotide), and FMN
(flavin mononucleotide),16 and an Nꢀterminal oxygenase domain, which contains binding
sites for nonꢀcatalytic zinc (Zn2+), catalytic heme, cofactor H4B (tetrahydrobiopterin), and the
substrate(LꢀArg).17 NOSs catalyze the oxidation of ꢀArg to ꢀcitrulline and NO in the
L
L
presence of oxygen and NADPH, and this cascade reaction is initiated by the twoꢀelectron
donor NADPH in the reductase domain to the electron acceptor heme in the oxygenase
domain through FAD and FMN with a series of proton transfers in the process.18
Given that all NOS isoforms catalyze the same reaction, the active site structures are nearly
identical, which has made the development of isoformꢀselective inhibitors a challenging
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 46
1
2
3
4
5
6
7
8
9
problem. Nevertheless, it has been possible to exploit subtle difference in the active sites to
develop highly selective nNOS inhibitors.19 Current efforts have been focused on improving
bioavailability without sacrificing potency and selectivity. Various nNOS inhibitors that we
have developed are shown in Figure 1. Thiopheneꢀbased inhibitors with diamine and
hydroxyethylꢀdiamine structure (Figure 1, 1 and 2, respectively)20 show good potency against
human nNOS. However, compound 2 gives very poor isoform selectivity over iNOS and
eNOS (n/I = 49 and n/e = 48). Compound 3,21,22 containing 2ꢀaminopyridine, a pyrrolidine
linker, and a diamine tail, is a moderate nNOS inhibitor (Ki = 388 nM) with good isoform
selectivity (1114ꢀfold over bovine eNOS and 150ꢀfold over murine iNOS).Compound4,23 a
doubleꢀheaded inhibitor, displays good potency against nNOS (Ki= 25 nM) but lacks good
isoform selectivity (n/e=107 and n/i=58). Recently, our group reported a series of nNOS
inhibitors with a 2ꢀaminopyridine anchor, a heteroaromatic ring linker, and a diamine tail,
such as compounds5 and 6.24 Both display excellent potency for rat nNOS (Ki=17 nM and 35
nM, respectively) and good selectivity over iNOS and eNOS (5: n/i= 127, n/e= 759; 6: n/i=
138, n/e=507). However, compounds 5 and 6 exhibit decreased potency against human nNOS
(59 nM and 64 nM, respectively).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Although the human NOS isoforms are the ultimate target for these inhibitors, our efforts
have focused on rat nNOS and bovine eNOS25ꢀ30 primarily because these two NOS isoforms
generate the best crystals. However, we recently have been able to crystallize human nNOS
and eNOS and have found that potencies for our inhibitors were generally 5ꢀ10 times less
with human nNOS than with rat nNOS. This could be a problem for translation of preclinical
animal studies to clinical trials, and could, in general, account for some drug failures in
clinical trials. An overlay of human and rat nNOS crystal structures showed that the primary
sequence in the active site and peripheral pocket of the rat enzyme is almost identical to that
in the human enzyme except that Leu337 in the rat nNOS peripheral pocket is replaced by
His342 in human nNOS. Consequently, the peripheral pocket of human nNOS tends to bind
ACS Paragon Plus Environment

Page 5 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
6
smaller and more hydrophilic inhibitors, providing an insightful guide for the design of more
potent human nNOS inhibitors.24, 29b
7
8
9
On the basis of reported NOS assay results and Xꢀray crystallographic analysis, we
performed specific and structureꢀbased optimizations to improve the potency and selectivity
of lead 5. Major modifications focused on exploring the diversity of the tail portion, leaving
the aminopyridine head and middle pyridine linker unmodified. First, we adopted a
ringꢀchain transformation strategy to give compound 14b, based on the structure of 14a, to
investigate the influence of a bulker and more hydrophobic tail on the potency and selectivity
of nNOS inhibitors. Second, an O atom and N atom were employed as bioisosteric
replacements in the piperidine ring of 14b to make 14c and 14g, where the O atom or N atom
could possibly make a direct or indirect (waterꢀassisted) Hꢀbond network with the heme, H4B,
or the surrounding residues within the active site of nNOS. Next, a ringꢀchain transformation
and homologation were used to design 14d and 14e from14c, and the piperazine tail of 14g
was bioisosterically changed to a piperidine ring to obtain 14h. Lastly, 14j and 14kwere
synthesized, adopting a ringꢀchain transformation strategy from14hto investigate the
influence of flexibility on activity. The SAR of the secondary and tertiary amines on the tail
were explored to evaluate steric and polar effects.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
All compounds were assayed against purified rat nNOS, murine iNOS, and bovine eNOS
for preliminary evaluation ofthe potency and isoform selectivity for the NOSs in lower
animals. Many of the compounds were also assayed against human nNOS and eNOS to
determine human isoform selectivity.
CHEMISTRY
The synthesis of compounds 10 and 11 is shown in Scheme 1. Compound 11 was
prepared in two steps, coupling 3ꢀchloromethylpyridine (8a) with lithiated
pyrrolylꢀ4,6ꢀdimethylpyridine (7) followed by pyrrole deprotection.24 Intermediate 10 was
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 46
1
2
3
4
synthesized by the same coupling reaction described above using aryl bromide 8b.
5
6
7
Scheme 1. Synthesis of intermediate 10 and compound 11
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
b
H
R
H2N
N
N
N
N
N
(R= H)
N
N
7
11
9: R= H
10: R= Br
Cl
Br
8a=
8b=
N
N
Br
Reagents and conditions (a) 8a or 8b, nꢀBuLi, THF, ꢀ78 ºC to 0 ºC then ꢀ78 ºC; (b)
NH2OH·HCl, EtOH/H2O (2:1), 100 ºC, 20 h.
As shown in Scheme 2, compounds 14aꢀ14e were synthesized by palladiumꢀcatalyzed
BuchwaldꢀHartwig amination of intermediate 10 with amines 12aꢀ12e24,31 followed by
pyrrole deprotection.24 Compound 10 was subjected to palladiumꢀcatalyzed amination with
12f to give 13f, which was methylated (13g), and the pyrrole was deprotected to afford 14g.
Scheme 2. Synthesis of compounds 14a-14e and 14g
Reagents and conditions: (a) 12a, 12b, 12c,12d, 12e, or 12f, Pd2(dba)3, DavePhos, NaOtꢀBu,
dioxane, 100 ºC, 20 h; (b) MeI, TEA, THF, r.t., 1 h; (c) NH2OH·HCl, EtOH/H2O (2:1), 100
ACS Paragon Plus Environment

Page 7 of 46
Journal of Medicinal Chemistry
1
2
3
4
ºC, 20 h.
5
6
7
8
9
The synthesis of 14h is shown in Scheme 3. Compound 13h was obtained via Suzuki
crossꢀcoupling32 employing 10 and boronic acid 12h; 13h subsequently underwent alkene
reduction, carbamate reduction, and pyrrole deprotection to give 14h. Compound 14j and 14k
were synthesized according to Scheme 4. Compound 14j was prepared in three steps
including a Sonagashira crossꢀcoupling33 of 10 with NꢀBocꢀNꢀmethylꢀpropargylamine (12i),
Boc deprotection, and pyrrole deprotection. Sonagashiracrossꢀcoupling of 10 with
3ꢀdimethylaminoꢀ1ꢀpropyne (12k) followed by pyrrole deprotection gave 14k.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 3. Synthesis of 14h
Reagents and conditions (a) 12h, Pd(OAc)2, SPhos, K3PO4, Tol/H2O (20:1), 100 ºC, 20 h; (b)
(i) Pd/C, H2, MeOH, r.t., 20 h, (ii) LAH, THF, 0 ºC to reflux, 1 h, (iii) NH2OH·HCl,
EtOH/H2O (2:1), 100 ºC, 20 h.
Scheme 4. Synthesis of compounds 14j and 14k
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 46
1
2
3
4
5
6
Reagents and conditions: (a) 12i or 12k, Pd(PPh3)4, CuI, TEA, 90 ºC, 20 h; (b) 20 % TFA in
7
8
DCM, r.t., 1 h; (c) (i) Pd/C, H2, MeOH, r.t., 20 h, (ii) NH2OH·HCl, EtOH/H2O (2:1), 100 ºC,
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
20 h.
RESULTS AND DISCUSSION
Inhibition of nonhuman NOSs
Synthesized inhibitors were assayed against purified rat nNOS, murine macrophage iNOS,
bovine eNOS, and human nNOS and eNOS using the hemoglobin capture assay.34,35 The Ki
values and isoform selectivities for inhibitors 11, 14a-14e, 14g, 14h, 14j, and 14k against
nonhuman NOSs are shown in Table 1; values for 4,23 5,24 and 624 are included for
comparison. The excision of the terminal pyridino or amino group from 4ꢀ6 is highly
detrimental to the potency (see 11ꢀ14e). Addition of a second amino group by attachment of a
piperazine (14g) or piperidine (14h) ring begins to restore the excellent potencies and
selectivities of 4ꢀ6. The crystal structures of nNOSꢀ14g (Figure 4A) and nNOSꢀ5 (Figure 4C)
reveal
a
similar and unique inhibitor binding mode. In addition to the
2ꢀaminopyridineꢀGlu592 Hꢀbond, the pyridine linker bends upward forming a Hꢀbond to
Tyr562 (3.0 Å). To allow this upward pyridine position the Gln478 side chain has to adapt to
an alternate rotamer form. The external N atom in the piperazine ring joins a Hꢀbonding
network involving H4B and heme propionate A via a water molecule (external N to water: 3.0
Å; water to H4B: 2.7 Å; water to heme propionate A: 2.6 Å). In contrast, for 5, although the
internal N atom position in the tail is wellꢀdefined, the positions of the last three atoms are
ambiguous, having weaker density.24
The internal N atom in the tail region attached to the pyridine linker does not interact with
the surrounding residues, as shown in both the nNOSꢀ14g and nNOSꢀ5 complexes. On the
basis of the crystal structures, 14h was synthesized as a bioisostere of 14g without the
ACS Paragon Plus Environment

Page 9 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
internal N atom in the piperazine ring. As shown in the crystal structure of nNOSꢀ14h (Figure
4B), in addition to the strong 2ꢀaminopyridineꢀGlu592 salt bridge, the N atom in the pyridine
linker forms a Hꢀbond to Tyr562 (3.0 Å), and the N atom in the piperazine ring also
participates in a waterꢀassisted Hꢀbonding network with the H4B and heme propionate A (N
atom to water: 2.9 Å; water to H4B: 2.6 Å; water to heme propionate A: 2.7 Å). Therefore,
14g, 14h, and 5 bind identically to rat nNOS with the pyridine linker in an upward position,
i.e., in which the pyridine N faces away from the heme. The differences in potency among
14g, 14h, and 5 could be explained by the flexibility of the tail region and the strength
(Hꢀbond distance) of the interaction made by the central pyridine linker to Tyr562.
Compound 14g is a very weak inhibitor of bovine eNOS (Ki>30000 nM), at least 2.5ꢀfold
less potent than reference compound 5 (Ki 12910 nM); consequently, it is highly selective for
nNOS over eNOS (n/e > 698). The crystal structure of eNOSꢀ14g (Figure 6A) shows that
while the 2ꢀaminopyridine forms Hꢀbonds with Glu363, the pyridine linker is no longer in the
upward position. Rather, it forms a Hꢀbonding network involving the H4B and heme
propionate A via a water molecule (N atom to water: 2.7 Å; water to H4B: 2.6 Å; water to
heme propionate A: 2.5 Å), and the external N atom in the piperazine ring forms a weak
Hꢀbond with Asn340 (3.4 Å). In comparison, the central pyridine linker in the eNOSꢀ5
complex (Figure 6B) is also in a flat binding mode, and the tail region of 5 does not interact
with the enzyme. The pyridine linker in 14g is parallel to the 2ꢀaminopyridine anchor, while
the pyridine linker in 5 makes a 15° angle from the 2ꢀaminopyridine plane, which does not
involve the Hꢀbonding network with H4B and heme. The excellent n/e selectivities for 14g
and 5 are very likely the result of the drastically different pyridine linker positions in the two
isoforms.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The most potent and selective analogues (14j and 14k) have flexible side chains
resembling that in 5, except that the Nꢀmethylamino group adjacent to the pyridine ring has
been replaced by a methylene group. In a comparison of the crystal structures of nNOSꢀ14j
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 46
1
2
3
4
5
6
7
8
9
and nNOSꢀ14k (Figure 6) with that of nNOSꢀ5 (Figure 4C), the 2ꢀaminopyridineꢀGlu592
interaction and the identical upward pyridine linker binding mode are observed in all three of
the complexes. In the nNOSꢀ14j complex, the secondary amine N atom in the tail region
joins a waterꢀassisted Hꢀbonding network with the H4B and heme propionate A (N atom to
water: 2.9 Å; water to H4B: 2.6 Å; water to heme propionate A: 2.6 Å). In the 14kꢀnNOS
complex, the tertiary amine N atom in the tail region also participates in a waterꢀbridged
Hꢀbonding network involving the H4B and heme propionate A (N atom to water: 2.6 Å; water
to H4B: 2.6 Å; water to heme propionate A: 2.6 Å). The involvement of the inhibitor tail
region with a Hꢀbonding network seems to improve potency.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Consistent with the observed high n/e selectivity, the crystal structure of eNOSꢀ14k
(Figure 6C) shows that the upward pyridine linker binding mode of 14k in nNOS is not
observed in eNOS. Rather, its linker pyridine is parallel to the plane of the aminopyridine
anchor, making Hꢀbonds with Asn368 via a bridging water molecule (N atom to water: 2.9 Å;
water to Asn368: 2.9 Å). In addition, the tertiary amine N on the tail forms a Hꢀbond with
heme propionate D (2.9 Å).
Inhibition of human NOSs
The primary sequence of human and rat NOS is more than 93% identical,24 and the nNOS
active site in each mammalian isozyme is highly conserved. The peripheral pockets of
humanꢀ and rat nNOS differ by only one residue (His342 in human and Leu337 in rat).24, 36 If
the inhibitor is designed so it is too small to reach the His342/Leu337 site, an identical
binding mode should be observed in both the human and rat nNOSꢀinhibitor complexes,24 and
the difference in potency of an inhibitor for the enzyme in these species should be
insignificant (Ki human nNOS /rat nNOS close to 1). Compounds 5 and 6 fit into this
classification, and the human nNOS/rat nNOS ratios for these compounds were 3.5 and 1.8,
respectively.
ACS Paragon Plus Environment

Page 11 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Compounds 14a, 14c, 14g, 14h, 14j, and 14k, which also have truncated sideꢀchains, were
assayed against purified human nNOS (Table 2). Four of these analogues, 14c, 14h, 14j, 14k,
had human nNOS/rat nNOS ratios lower than 1.8; 14j is the first potent compound with a
human nNOS/rat nNOS ratio < 1. The benefit of a human/rat Ki ratio of 1 or less is that
results in a preclinical rodent model might translate to humans in clinical trials.
As shown in Figure 7A, the 2ꢀaminopyridine group of 14g forms Hꢀbonds with Glu597 in
human nNOS (Glu592 in rat); the N atom in the pyridine linker makes a Hꢀbond with Tyr567
(2.8 Å) (Tyr562 in rat); the external N atom in the piperazine ring joins a Hꢀbond network
involving H4B and heme propionate A via a water molecule (external N to water: 2.9 Å;
water to H4B: 3.0 Å; water to heme propionate A: 2.5 Å). In the human nNOSꢀ14h complex
(Figure 7B), the same Hꢀbonds exist between the 2ꢀaminopyridine group of 14h and Glu597;
the N atom of the pyridine linker makes a Hꢀbond to Tyr567 (3.0 Å), and the external N atom
in the piperazine ring participates in a waterꢀassisted Hꢀbonding network involving H4B and
heme propionate A (external N to water: 2.7 Å; water to H4B: 2.8 Å; water to heme
propionate A: 2.7 Å).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The human nNOSꢀ14g crystal structure (Figure 7A) shows an identical upward pyridine
linker binding mode as shown in Figure 4A (rat nNOSꢀ14g) because 14g is too short to reach
the His342/Leu337 site (His342 is at least 8 Å away from 14g). Similarly, an identical
binding mode is observed in both human and rat nNOSꢀ14h complexes (Figure 7B and
Figure 4B).
Compound 14j is comparable in potency and selectivity to those of compound 5; 14k has
twoꢀthirds the potency of 5 with lower selectivity. Crystal structures of these compounds
bound to human nNOS show that 14j (Figure 8A) Hꢀbonds to Glu597 through the
2ꢀaminopyridine group; the N atom in the pyridine linker forms two Hꢀbonds to Tyr567 (2.8
Å) and Tyr593 (3.4 Å) (Tyr588 in rat), and the N atom in the tail region participates in a tight
Hꢀbonding network involving the H4B and heme propionate A through a water molecule (N
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 46
1
2
3
4
5
6
7
8
9
to water: 2.6 Å; water to H4B: 2.8 Å; water to heme propionate A: 2.6 Å). Similar interactions
are involved in the 14k complex (Figure 8B). In addition to the 2ꢀaminopyridineꢀGlu597
interaction, the N atom in the pyridine linker forms two Hꢀbonds to Tyr567 (2.8 Å) and
Tyr593 (3.5 Å), and the N atom in the tail region joins a waterꢀmediated Hꢀbond network
with H4B and heme propionate A (N to water: 2.5 Å; water to H4B: 2.9 Å; water to heme
propionate A: 2.7 Å). The structures of human nNOSꢀ14j and ꢀ14k reveal a subtle but
important feature: the N atom in the pyridine linker is involved in two Hꢀbonds; the distance
to Tyr593 is within 3.5 Å. This is the consequence of a long tail region where its amine N
atom is part of a tight Hꢀbonding network with the H4B and heme via a water molecule. The
network pushes the pyridine linker closer to Tyr567 and Tyr593. This feature does not exist in
structures of human nNOSꢀ14g or ꢀ14h because the Hꢀbonding network there is established
by shorter piperidine or piperazine moieties. In all of the rat nNOS structures in complex with
14g, 14h, 14j, and 14k, the N atom in the pyridine linker is also only making one Hꢀbond
with Tyr562, but not with Tyr588. A comparison with the binding interactions in human
nNOSꢀ5 (Figure 8C) is interesting as well. While the 2ꢀaminopyridine group in 5 is also
anchored by Glu597, the N atom of the central pyridine ring makes only one hydrogen bond,
that is, with Tyr567, and the external N atom in the tail region displaces the bridging water
molecule between the H4B and heme propionate A.24 All three inhibitors, 14j, 14k, and 5,
exhibit the same upward pyridine binding mode in human nNOS; the differences in the
enzymeꢀinhibitor interactions, especially in the number and distances of the H bonds, appear
to account for the compound potency differences.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
An alignment of human nNOS and eNOS reveals four important residue differences:
Ser607/His371, His342/Phe305, Met341/Val104, and Asp602/Asn366. Among these four
amino acid pairs, the Asp602/Asn366 difference in human nNOS/eNOS makes the nNOS
active site more electronegative than eNOS, and this electronic difference can be used for
selective inhibition (nNOS tends to bind positively charged compounds).
ACS Paragon Plus Environment

Page 13 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Compounds 14a, 14c, 14g, 14h, 14j, and 14k were assayed against purified human eNOS
and compared with 5 and 6 (Table 3). Once again, 14j exhibited the best selectivity properties
of these compounds, 1761ꢀfold more potent against human nNOS than human eNOS.
Although 14g had the least affinity for human eNOS, its potency with human nNOS was 6.5
times worse than that for 14j, so the selectivity of 14g was about half that of 14j.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Effect of 14j on CYPs
The two pyridines in 14j make it a potential inhibitor of cytochromes P450 (CYPs).
Consequently, 14j was evaluated for its inhibition of seven major human liver microsomal
P450s (Table 4). At a concentration 769 times its Ki against human nNOS (10 ꢀM), it was
found to inhibit the activity of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and
CYP2D6 insignificantly; the only isozyme that showed any significant inhibition was
CYP3A4 (58%). The IC50 of compound 14j for CYP3A4 is estimated to be approximately 10
ꢀM, and the selectivity toward nNOS is more than 70ꢀfold in rat (IC50 0.142 ꢀM) and
100ꢀfold in human (IC50 0.091 ꢀM). Therefore, the inhibitory potency of 14j for CYP3A4 is
relatively weak.
Other pharmacokinetic properties of 14j
Other pharmacokinetic properties that were tested with 14j included human microsome
stability and plasma protein binding. At 1 ꢀM concentration, the halfꢀlife of 14j in human
liver microsomes was > 60 min; however, the metabolism was unrelated to CYPꢀmediated
metabolism, as 23% of the compound was lost in 60 min in the absence of NADPH or in 90
min in the presence of NADPH. At 5 ꢀM concentration, only 20% bound to human plasma
protein. Unfortunately, 14j exhibited little or no Cacoꢀ2 permeability. Detailed experimental
results and protocol can be found in the Supporting Information.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 46
1
2
3
4
CONCLUSIONS
5
6
7
8
A series of human nNOS inhibitors, based on the 2ꢀaminopyridine scaffold having a
central pyridine linker, were designed and evaluated. Compound 14j had outstanding
properties, exhibiting a potent and slightly lower Ki value for human nNOS (13 nM) than rat
nNOS (16 nM) with 118ꢀfold and 1761ꢀfold selectivity over mouse iNOS and human eNOS,
respectively. Xꢀray crystal structures of 14j bound to both rat and human nNOS showed that
the N of the pyridine linker formed two Hꢀbonds (to Tyr567 and Tyr593) in human nNOS but
only one Hꢀbond in rat nNOS. Compound 14j displayed very poor inhibition of human CYPs
(except 58% inhibition of CYP3A4 at 10 ꢀM), little or no human microsome CYP
metabolism for at least 60 min, and only 20% human plasma protein binding. However, it had
little or no Cacoꢀ2 permeability, predicting that oral absorption would be poor.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EXPERIMENTAL SECTION
Materials, Synthetic Methods, and Characterization of Molecules. Commercially
available starting materials 12a-12f were purchased from SigmaꢀAldrich and were used
without further purification. All reactions were performed under an atmosphere of dry argon.
Compounds 7, 8a, 8b, 12h, and 12i were prepared by known literature procedures using
commercially available chemicals (7-s, 8a-S, 8b-S, 12h-S, and 12i-S; see Supporting
Information for details), and their spectral data are consistent with those data reported for
them.
Anhydrous solvents (CH2Cl2 and THF) were purified prior to use by passage through a
column composed of activated alumina and a supported copper redox catalyst. Analytical
thinꢀlayer chromatography was carried out on SiliCycle preꢀcoated silica gel 60 F254 plates.
Compounds were visualized with shortꢀwavelength UV light, ninhydrin, or KMnO4 stain.
Flash column chromatography was performed using an Agilent 971ꢀFP automated flash
purification system with a Varian column station and various SiliCycle cartridges (4−80 g,
ACS Paragon Plus Environment

Page 15 of 46
Journal of Medicinal Chemistry
1
2
3
4
40ꢀ63 ꢀm, 60 Å). 1H NMR and 13C NMR spectra were recorded in the indicated solvent on a
5
6
1
Bruker AvanceꢀIII (500 MHz and 125 MHz for H and 13C, respectively) spectrometer.
7
8
9
Lowꢀresolution ESIMS was performed using a Bruker AmaZon SL Ion Trap mass
spectrometer system. Highꢀresolution mass spectra were obtained using an Agilent 6210
LCꢀTOF spectrometer in the positive ion mode using electrospray ionization with an Agilent
G1312A HPLC pump and an Agilent G1367B auto injector at the Integrated Molecular
Structure Education and Research Center (IMSERC), Northwestern University. The purity of
the compounds was evaluated on an analytical HPLC. Analytical HPLC was performed using
an Agilent Infinity 1260 HPLC system with a Phenonemex Luna Cꢀ8 (4.6 × 250 mm, 5 ꢀm)
reverse phase column, detecting with UV absorbance (254 nm). The purity of all compounds
that were subjected to biological assay was > 95%.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
General Procedure A: Pyrrolyl-lutidine and Aryl Halide Coupling. A round bottom
flask was charged with 7 (1 equiv.), and THF was added to form a 0.2 M solution. nꢀBuLi
(1.6 equiv.) was added to the solution at ꢀ78 ºC, and the reaction mixture was allowed to stir
for 30 min at 0 ºC. The mixture was transferred to a 1 M solution of aryl halide (8a or 8b) in
THF via cannula at ꢀ78 ºC, which was allowed to stir for an additional 20 min then quenched
with H2O. The crude reaction mixture was partitioned between ethyl acetate and water, and
the organic layer was washed with H2O and brine, dried with Na2SO4, and concentrated under
reduced pressure. The crude product was purified by flash column chromatography to give 9
and 10.
2-(2,5-Dimethyl-1
H
-pyrrol-1-yl)-4-methyl-6-(2-(pyridin-3-yl)ethyl)pyridine
(9).
Compound 9 was synthesized according to general procedure A using 7 (600 mg, 3 mmol)
and 8a (681 mg, 3 mmol). 9 was isolated as a yellow oil (343 mg, 39%) after flash column
1
chromatography (ethyl acetate: hexanes 1:1). H NMR (500 MHz, CDCl3): δ 8.41ꢀ8.39 (m,
2H), 7.46 (d, J = 5.0 Hz, 1H), 7.16 (dd, J = 7.0, 5.0 Hz, 1H), 6.87 (s, 1H), 6.84 (s, 1H), 5.86
(s, 2H), 3.06ꢀ3.05 (m, 4H), 2.33 (s, 3H), 2.09 (s, 6H); 13C NMR (125 MHz, CDCl3): δ 160.1,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 46
1
2
3
4
5
6
151.8, 150.0, 149.6, 147.5, 136.7, 135.9, 128.5, 123.3, 122.7, 120.3, 106.8, 39.1, 32.7, 21.0,
13.2; MS ESI: calcd. For C19H22N3 [M+H]+, 292.18; found, 292.08.
7
8
9
2-(2-(5-Bromopyridin-3-yl)ethyl)-6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridine
(10). Compound 10 was synthesized according to general procedure A using 7 (400 mg, 2
mmol) and 8b (500 mg, 2 mmol). 10 was isolated as a yellow oil (273.2 mg, 46%) after flash
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
column chromatography (ethyl acetate: hexanes 1:4). H NMR (500 MHz, CDCl3): δ 8.48 (s,
1H), 8.30 (s, 1H), 7.60 (s, 1H), 6.87 (s, 1H), 6.86 (s, 1H), 5.87 (s, 2H), 3.06ꢀ3.05 (m, 4H),
2.35 (s, 3H), 2.09 (s, 6H); 13C NMR (125 MHz, CDCl3): δ 159.4, 151.9, 149.8, 148.2, 147.6,
139.1, 138.9, 128.5, 122.8, 120.6, 120.5, 106.8, 38.7, 32.1, 21.0, 13.2; MS ESI: calcd. For
C19H21BrN3 [M+H]+, 372.09; found, 372.05.
4-Methyl-6-(2-(pyridin-3-yl)ethyl)pyridin-2-amine (11). 11 was synthesized according
to general procedure D using 9 (48.0 mg) and NH2OH·HCl (46.9 mg). 11 was isolated as a
1
brown oil (33.5 mg, 95%) after flash column chromatography (MeOH: DCM 3:7). H NMR
(500 MHz, Methanolꢀd4): δ 8.93 (s, 1H), 8.80 (d, J= 6.0 Hz, 1H), 8.66 (d, J= 8.0 Hz, 1H),
8.09 (dd, J = 8.0, 6.0 Hz, 1H), 6.72 (s, 1H), 6.70 (s, 1H), 3.36ꢀ3.33 (m, 2H), 3.18ꢀ3.15 (m,
2H), 2.37 (s, 3H); 13C NMR (125 MHz, methanolꢀd4): δ 157.7, 154.6, 147.0, 146.9, 141.2,
140.7, 139.7, 127.2, 113.7, 110.0, 32.6, 31.0, 20.6; HRMS ESI: calcd. For C13H16N3 [M+H]+,
214.1339; found, 214.1337.
General Procedure B: Preparation of 13a-13e and 13g (Buchwald-Hartwig
Palladium-Catalyzed Amination of 10). A microwave vial was charged with Pd2(dba)3(5
mol%), Davephos (10 mol%), NaOtBu (1.2 equiv.), 10 (1 equiv.) and amine 12a-12f (1.5ꢀ2
equiv.). The mixtures were diluted with dioxane to form a 0.16 M solution. The microwave
vial was then capped, and the reaction mixture was stirred at 100 ºC for 20 h. At this time, the
cap was removed, and the reaction mixture was filtered through a silica pad. The filtrate was
concentrated under reduced pressure and purified by flash column chromatography to give
13a-13e and 13g.
ACS Paragon Plus Environment

Page 17 of 46
Journal of Medicinal Chemistry
1
2
3
4
5-(2-(6-(2,5-Dimethyl-1
H
-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)-N,N-dimethylpyri
5
6
7
8
din-3-amine (13a). Compound 13a was synthesized according to general procedure B using
Pd2(dba)3 (12.1 mg), Davephos (10.2 mg), NaOtBu (29.2 mg), 10 (93.3 mg), and amine 12a
(0.25 ml, 2M in THF). 13a was isolated as a yellow oil (42.3 mg, 50%) after flash column
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
chromatography (MeOH: DCM 1:19). H NMR (500 MHz, CDCl3): δ 7.95 (s, 1H), 7.79 (s,
1H), 6.90 (s, 1H), 6.84 (s, 1H), 6.77 (s, 1H), 5.86 (s, 2H), 3.07ꢀ3.03 (m, 2H), 3.02ꢀ2.98 (m,
2H), 2.91 (s, 6H), 2.34 (s, 3H), 2.10 (s, 6H); 13C NMR (125 MHz, CDCl3): δ 160.4, 151.7,
149.6, 146.1, 137.6, 136.7, 132.6, 128.5, 122.7, 120.2, 119.2, 106.8, 40.1, 39.4, 33.1, 21.0,
13.2.
2-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-methyl-6-(2-(5-(piperidin-1-yl)pyridin-3-yl)ethyl)p
yridine (13b). Compound 13b was synthesized according to general procedure B using
Pd2(dba)3 (13.2 mg), Davephos (10.8 mg), NaOtBu (32.1 mg), 10 (102 mg, 0.28 mmol), and
amine 12b (46.8. mg, 0.55 mmol). 13b was isolated as a yellow oil (73.2 mg, 71%) after flash
1
column chromatography (ethyl acetate: hexanes 8:1). H NMR (500 MHz, CDCl3): δ 8.10 (s,
1H), 7.85 (s, 1H), 6.97 (s, 1H), 6.87 (s, 1H), 6.84 (s, 1H), 5.86 (s, 2H), 3.12 (t, J = 5.0 Hz,
4H), 3.06ꢀ2.98 (m, 4H), 2.34 (s, 3H), 2.10 (s, 6H), 1.68ꢀ1.64 (m, 4H), 1.59ꢀ1.54 (m, 2H); 13C
NMR (125 MHz, CDCl3): δ 160.3, 151.8, 149.6, 147.6, 139.4, 136.9, 136.0, 128.4, 123.2,
122.8, 120.2, 106.8, 49.8, 39.3, 32.9, 25.5, 24.1, 21.0, 13.3.
4-(5-(2-(6-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)pyridin-3-yl)mor
pholine (13c). Compound 13c was synthesized according to general procedure B using
Pd2(dba)3 (11.4 mg), Davephos (9.6 mg), NaOtBu (28.2 mg), 10 (88.1 mg), and amine 12c
(44.2 mg). 13c was isolated as a yellow oil (86.6 mg, 96%) after flash column
1
chromatography (MeOH: DCM 1:19). H NMR (500 MHz, CDCl3): δ 8.07 (s, 1H), 7.92 (s,
1H), 6.96 (s, 1H), 6.88 (s, 1H), 6.84 (s, 1H), 5.87 (s, 2H), 3.82 (t, J = 5.0 Hz, 4H), 3.11 (t, J =
5.0 Hz, 4H), 3.08ꢀ3.06 (m, 4H), 2.35 (s, 3H), 2.09 (s, 6H); 13C NMR (125 MHz, CDCl3): δ
160.2, 151.7, 149.6, 146.7, 141.2, 136.8, 136.0, 128.4, 122.7, 122.4, 120.2, 106.8, 66.7, 48.6,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 46
1
2
3
4
39.2, 32.8, 21.0, 13.3; MS ESI: calcd. For C23H29N4O [M+H]+, 377.23; found, 377.18.
5
6
5-(2-(6-(2,5-Dimethyl-1
H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)-N-(2-methoxyethyl
7
8
)-N-methylpyridin-3-amine (13d). Compound 13d was synthesized according to general
9
procedure B using Pd2(dba)3 (12.0 mg), Davephos (10.1 mg), NaOtBu (29.4 mg), 10 (93.5 mg,
0.25 mmol), and amine 12d (46.1 mg). 13d was isolated as a yellow oil (61.8 mg, 65%) after
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
flash column chromatography (MeOH: DCM 1:19). H NMR (500 MHz, CDCl3): δ 7.95 (s,
1H), 7.70 (s, 1H), 6.90 (s, 1H), 6.84 (s, 1H), 6.77 (s, 1H), 5.86 (s, 2H), 3.52ꢀ3.50 (m, 2H),
3.47ꢀ3.45 (m, 2H), 3.32 (s, 3H), 3.07ꢀ3.03 (m, 2H), 3.00ꢀ2.97 (m, 2H), 2.93 (s, 3H), 2.34 (s,
13
3H), 2.10 (s, 6H); C NMR (125 MHz, CDCl3): δ 160.5, 151.7, 149.6, 145.0, 137.7, 136.7,
132.6, 128.4, 122.7, 120.2, 118.7, 106.8, 70.1, 59.1, 52.1, 39.4, 38.6, 33.1, 21.0, 13.2.
5-(2-(6-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)-N-(3-
methoxypropyl)pyridin-3-amine (13e). Compound 13e was synthesized according to
general procedure B using Pd2(dba)3 (12.0 mg), Davephos (10.2 mg), NaOtBu (29.5 mg), 10
(94 mg), and amine 12e (46.3 mg). 13e was isolated as a yellow oil (66.5 mg, 70%) after
1
flash column chromatography (MeOH: DCM 1:19). H NMR (500 MHz, CDCl3): δ 7.82 (s,
1H), 7.73 (s, 1H), 6.89 (s, 1H), 6.83 (s, 1H), 6.66 (s, 1H), 5.85 (s, 2H), 4.06 (brs, 1H), 3.47 (t,
J = 6.0 Hz, 2H), 3.32 (s, 3H), 3.16 (t, J = 6.5 Hz, 2H), 3.04ꢀ3.01 (m, 2H), 2.96ꢀ2.93 (m, 2H),
2.33 (s, 3H), 2.09 (s, 6H), 1.86ꢀ1.81(m, 2H); 13C NMR (125 MHz, CDCl3): δ 160.5, 151.7,
149.6, 149.5, 138.5, 134.0, 133.9, 128.4, 122.7, 120.2, 118.5, 106.7, 71.2, 58.8, 41.6, 39.3,
32.9, 29.2, 21.0, 13.2; MS ESI: calcd. For C23H29N4O [M+H]+, 379.25; found, 379.20.
1-(5-(2-(6-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)pyridin-3-yl)-4-m
ethylpiperazine (13g). Compound 13g was synthesized by methylation of 13f. Compound
13f was prepared according to general procedure B using Pd2(dba)3 (28.1 mg), Davephos
(24.1 mg), NaOtBu (70.7 mg), 10 (226.2 mg), and piperazine 12f (105.4 mg). Crude product
13f was subjected to methylation without purification. A scintillation vial was charged with
13f (1 equiv.). The crude product was diluted with THF to form a 0.1 M solution followed by
ACS Paragon Plus Environment

Page 19 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
addition of triethylamine (1.5 equiv.) and MeI (1 equiv.). The reaction mixture was allowed to
stir at r.t. for 1 h. The crude product was diluted with DCM and washed with water and brine.
The organic layer was dried with Na2SO4 and concentrated under reduced pressure. The crude
product mixture was purified by flash column chromatography (MeOH: DCM 1:6) to give
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
13g (34.6 mg, 15% for the two steps). H NMR (500 MHz, CDCl3): δ 8.09 (s, 1H), 7.89 (s,
1H), 6.97 (s, 1H), 6.87 (s, 1H), 6.83 (s, 1H), 5.84 (s, 2H), 3.20 (t, J = 5.0 Hz, 4H), 3.04ꢀ2.99
(m, 4H), 2.60 (t, J = 5.0 Hz, 4H), 2.36 (s, 3H), 2.33 (s, 3H), 2.08 (s, 6H); 13C NMR (125
MHz, CDCl3): δ 160.2, 151.7, 149.6, 146.6, 140.9, 136.8, 136.3, 128.4, 122.8, 122.7, 120.3,
106.8, 54.7, 48.1, 45.9, 39.2, 32.9, 21.0, 13.2.
tert-Butyl-4-(5-(2-(6-(2,5-dimethyl-1
H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)pyridi
n-3-yl)-5,6-dihydropyridine-1(2 )-carboxylate (13h). A microwave vial was charged with
H
Pd(OAc)2 (13.4 mg), SPhos (41.1 mg), K3PO4 (849.1 mg), boronic acid 12f (459.2 mg), and
10 (364 mg, 1.0 mmol). The mixture was diluted with toluene/water (20:1) to form a 0.13 M
solution. The microwave vial was capped, and the reaction mixture was stirred at 100 ºC for
20 h. The cap was removed, and the reaction mixture was diluted with ethyl acetate. The
crude product was filtered, the filtrate was dried over Na2SO4, and concentrated under
reduced pressure. 13h was isolated as a yellow oil (316.2 mg, 68%) after flash column
1
chromatography (ethyl acetate: hexanes 1:1). H NMR (500 MHz, CDCl3): δ 8.43 (s, 1H),
8.27 (s, 1H), 7.44 (s, 1H), 6.87 (s, 1H), 6.84 (s, 1H), 6.02ꢀ6.01 (m, 1H), 5.86 (s, 2H),
4.07ꢀ4.05 (m, 2H), 3.62ꢀ3.60 (m, 2H), 3.08ꢀ3.07 (m, 4H), 2.45ꢀ2.43 (m, 2H), 2.34 (s, 3H),
2.08 (s, 6H), 1.47 (s, 9H); 13C NMR (125 MHz, CDCl3): δ 159.9, 154.8, 151.8, 149.7, 148.3,
148.2, 144.1, 136.4, 135.8, 132.7, 132.6, 128.4, 122.7, 120.3, 106.8, 79.9, 39.1, 39.0, 32.6,
32.5, 28.5, 27.1, 21.0, 13.2.
General Procedure C: Preparation of 13i and 13k (Sonagashira-Cross Coupling of
10). A microwave vial was charged with Pd(PPh3)4 (5 mol%), CuI (5 mol%), and 10 (1
equiv.). The mixtures were diluted with triethylamine to form a 0.16 M solution followed by
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 46
1
2
3
4
5
6
7
8
9
the addition of alkyne (1.5ꢀ2 equiv.). The microwave vial was capped, and the reaction
mixture was stirred at 90 ºC for 20 h. The cap was removed, and the reaction mixture was
diluted with ethyl acetate and filtered. The filtrate was washed with water, ammonium
chloride, and brine, dried with Na2SO4, and concentrated under reduced pressure. The crude
product mixture was purified by flash column chromatography to give 13i and 13k.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
tert-Butyl-3-(5-(2-(6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)pyridi
n-3-yl)prop-2-ynyl(methyl)carbamate (13i). Compound 13i was synthesized according to
general procedure C using Pd(PPh3)4 (28.8 mg), CuI (4.9 mg), alkyne 12i (126.8 mg, 0.75
mmol), and 10 (184.6 mg, 0.50 mmol). 13i was isolated as a brown oil (171.9 mg, 75%) after
1
flash column chromatography (MeOH: DCM 1:19). H NMR (500 MHz, CDCl3): δ 8.44 (s,
1H), 8.29 (s, 1H), 7.50 (s, 1H), 6.86 (s, 1H), 6.84 (s, 1H), 5.86 (s, 2H), 4.26 (brs, 2H),
3.05ꢀ3.04 (m, 4H), 2.94 (s, 3H), 2.33 (s, 3H), 2.08 (s, 6H), 1.46 (s, 9H); 13C NMR (125 MHz,
CDCl3): δ 159.7, 151.8, 150.1, 149.7, 149.0, 148.6, 148.1, 138.6, 128.4, 122.7, 120.4, 119.6,
106.8, 88.0, 80.3, 80.2, 38.9, 38.8, 33.6, 32.3, 28.4, 21.0, 13.2; MS ESI: calcd. For
C28H35N4O2 [M+H]+, 459.28; found, 459.20.
3-(5-(2-(6-(2,5-Dimethyl-1
H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)pyridin-3-yl)-N,
N
-dimethylprop-2-yn-1-amine (13k). Compound 13k was synthesized according to general
procedure C using Pd(PPh3)4 (28.8 mg), CuI (5.1 mg), alkyne 12k (62.3. mg), and 10 (185
mg). 13k was isolated as a brown oil (166.6 mg, 90%) after flash column chromatography
(MeOH: DCM 1: 9). 1H NMR (500 MHz, CDCl3): δ 8.45 (s, 1H), 8.27 (s, 1H), 7.49 (s, 1H),
6.85 (s, 1H), 6.83 (s, 1H), 5.85 (s, 2H), 3.43 (s, 2H), 3.05ꢀ3.04 (m, 4H), 2.33 (s, 6H), 2.32 (s,
13
3H), 2.08 (s, 6H); C NMR (125 MHz, CDCl3): δ 159.8, 151.8, 150.1, 149.7, 148.8, 138.6,
136.2, 128.4, 122.7, 120.4, 119.9, 106.7, 88.0, 82.1, 48.6, 44.3, 38.9, 32.3, 21.0, 13.2; MS
ESI: calcd. For C24H29N4 [M+H]+, 373.24; found, 373.19.
General Procedure D: Preparation of 11, 14a-14e, and 14g, (Pyrrole Deprotection). A
microwave vial was charged with 9, 13a-13e, or 13g (1 equiv.) and NH2OH·HCl (3ꢀ4 equiv.).
ACS Paragon Plus Environment

Page 21 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
The mixtures were diluted with EtOH/water (2:1) to form a 0.16 M solution. The microwave
vial was capped, and the reaction mixture was stirred at 100 ºC for 20 h. The cap was
removed, and the reaction mixture was concentrated under reduced pressure. The crude
product mixture was purified by flash chromatography to give 11, 14a-14e, and 14g.
6-(2-(5-(Dimethylamino)pyridin-3-yl)ethyl)-4-methylpyridin-2-amine (14a). 14a was
synthesized according to general procedure D using 13a (62.3 mg) and NH2OH·HCl (52.7
mg). 14a was isolated as a brown oil (47.2 mg, 99%) after flash column chromatography
(MeOH: DCM 4:6). 1H NMR (500 MHz, methanolꢀd4): δ 8.02 (s, 1H), 7.96 (s, 1H), 7.70 (s,
1H), 6.68 (s, 1H), 6.66 (s, 1H), 3.19ꢀ3.15 (m, 2H), 3.12ꢀ3.08 (m, 8H), 2.36 (s, 3H); 13C NMR
(125 MHz, Methanolꢀd4): δ 157.6, 154.6, 148.1, 147.5, 139.6, 127.6, 125.4, 123.6, 113.7,
109.8, 38.7, 32.2, 31.4, 20.5; HRMS ESI: calcd. For C15H21N4 [M+H]+, 257.1761; found,
251.1766.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4-Methyl-6-(2-(5-(piperidin-1-yl)pyridin-3-yl)ethyl)pyridin-2-amine (14b). 14b was
synthesized according to general procedure D using 13b (62.8 mg) and NH2OH·HCl (28.0
mg). 14b was isolated as a brown oil (32.3 mg, 65%) after flash column chromatography
(MeOH: DCM 1:9). 1H NMR (500 MHz, methanolꢀd4): δ 8.18 (s, 1H), 7.96 (s, 1H), 7.76 (s,
1H), 6.70 (s, 1H), 6.66 (s, 1H), 3.37 (t, J = 5.0 Hz, 4H), 3.16ꢀ3.12 (m, 2H), 3.10ꢀ3.06 (m, 2H),
2.35 (s, 3H), 1.71ꢀ1.68 (m, 6H); 13C NMR (125 MHz, methanolꢀd4): δ 157.5, 154.5, 148.5,
147.7, 138.7, 131.6, 128.8, 126.7, 113.7, 109.7, 48.2, 33.4, 31.4, 24.8, 23.6, 20.5; HRMS ESI:
calcd. For C18H25N4 [M+H]+, 297.2074; found, 297.2078.
4-Methyl-6-(2-(5-morpholinopyridin-3-yl)ethyl)pyridin-2-amine (14c). 14c was
synthesized according to general procedure D using 13c (82.8 mg, 0.50 mmol) and
NH2OH·HCl (62.1 mg). 14c was isolated as a brown oil (64.3 mg, 98%) after flash column
1
chromatography (MeOH: DCM 3:7). H NMR (500 MHz, methanolꢀd4): δ 8.31 (s, 1H), 8.17
(s, 1H), 8.14 (s, 1H), 6.72 (s, 1H), 6.71 (s, 1H), 3.86 (t, J = 5.0 Hz, 4H), 3.43 (t, J = 5.0 Hz,
4H), 3.25ꢀ3.22 (m, 2H), 3.15ꢀ3.12 (m, 2H), 2.37 (s, 3H); 13C NMR (125 MHz, methanolꢀd4):
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 46
1
2
3
4
5
6
δ 157.7, 154.5, 149.1, 147.2, 140.5, 129.3, 129.2, 125.2, 113.8, 109.9, 65.8, 46.6, 33.0, 31.4,
20.6; HRMS ESI: calcd. For C17H23N4O [M+H]+, 299.1866; found, 299.1872.
7
8
9
6-(2-(5-((2-Methoxyethyl)(methyl)amino)pyridin-3-yl)ethyl)-4-methylpyridin-2-amin
e (14d). 14d was synthesized according to general procedure D using 13d (61.8 mg) and
NH2OH·HCl (45.9 mg). 14d was isolated as a brown oil (48.3 mg, 98%) after flash column
chromatography (MeOH: DCM 3:7). 1H NMR (500 MHz, methanolꢀd4): δ 8.11 (s, 1H), 8.01
(s, 1H), 7.87 (s, 1H), 6.73 (s, 1H), 6.71 (s, 1H), 3.72 (t, J = 5.0 Hz, 2H), 3.63 (t, J = 5.0 Hz,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
2H), 3.32 (s, 3H), 3.23ꢀ3.20 (m, 2H), 3.15ꢀ3.12 (m, 5H), 2.36 (s, 3H); C NMR (125 MHz,
methanolꢀd4): δ 157.7, 154.5, 147.9, 147.3, 140.0, 126.6, 126.5, 122.9, 113.8, 109.9, 70.0,
58.0, 51.4, 37.6, 33.1, 31.3, 20.6; HRMS ESI: calcd. For C17H25N4O [M+H]+, 301.2023;
found, 301.2031.
6-(2-(5-(3-Methoxypropylamino)pyridin-3-yl)ethyl)-4-methylpyridin-2-amine (14e).
14e was synthesized according to general procedure D using 13e (66.5 mg) and NH2OH·HCl
(50.2 mg.). 14e was isolated as a brown oil (49.1 mg, 93%) after flash column
1
chromatography (MeOH: DCM 3:7). H NMR (500 MHz, methanolꢀd4): δ 7.95 (s, 1H), 7.93
(s, 1H), 7.65 (s, 1H), 6.71 (s, 1H), 6.68 (s, 1H), 3.51 (t, J = 6.0 Hz, 2H), 3.35 (s, 3H), 3.29 (t,
J = 7.0 Hz, 2H), 3.18ꢀ3.15 (m, 2H), 3.12ꢀ3.09 (m, 2H), 2.36 (s, 3H), 1.92ꢀ1.87 (m, 2H); 13C
NMR (125 MHz, methanolꢀd4): δ 157.7, 154.5, 148.0, 147.2, 140.4, 127.0, 126.4, 122.4,
113.8, 109.9, 69.6, 57.6, 39.6, 32.9, 31.2, 28.3, 20.6; HRMS ESI: calcd. For C17H25N4O
[M+H]+, 301.2023; found, 301.2026.
4-Methyl-6-(2-(5-(4-methylpiperazin-1-yl)pyridin-3-yl)ethyl)pyridin-2-amine (14g).
14g was synthesized according to general procedure D using 13g (34.6 mg) and NH2OH·HCl
(25.1 mg). 14g was isolated as a brown oil (18.0 mg, 65%) after flash column
chromatography (MeOH: DCM 4:6). 1H NMR (500 MHz, methanolꢀd4): δ 8.16 (s, 1H), 7.92
(s, 1H), 7.42 (s, 1H), 6.59 (s, 1H), 6.55 (s, 1H), 3.51ꢀ3.49 (m, 4H), 3.28ꢀ3.26 (m, 4H),
3.05ꢀ2.98 (m, 4H), 2.84 (s, 3H), 2.31 (s, 3H); 13C NMR (125 MHz, methanolꢀd4): δ 156.2,
ACS Paragon Plus Environment

Page 23 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
6
155.3, 149.8, 146.3, 140.1, 136.6, 135.7, 123.9, 113.6, 109.1, 53.3, 46.1, 42.8, 34.5, 31.6,
20.4; HRMS ESI: calcd. For C18H26N5 [M+H]+, 312.2183; found, 312.2186.
7
8
9
General Procedure E: Preparation of 14j and 14k (Alkyne Reduction and Pyrrole
Deprotection). A scintillation vial was charged with 10% wt. Pd/C and 13k (1 equiv.). The
mixtures were diluted with methanol to form a 0.1 M solution. The reaction mixture was
stirred at r.t. for 20 h under hydrogen gas. At this time, the crude was filtered, and the filtrate
was concentrated under reduced pressure. The crude product was subjected to
2,5ꢀdimethylpyrrole deprotection as described above (general procedure D) without
purification. A microwave vial was charged with the hydrogenation crude product (1 equiv.)
and NH2OH·HCl (3ꢀ4 equiv.). The mixtures were diluted with EtOH/water (2:1) to form a
0.16 M solution. The microwave vial was capped, and the reaction mixture was stirred at 100
ºC for 20 h. The cap was removed, and the reaction mixture was concentrated under reduced
pressure. The crude product mixture was purified by flash chromatography to give 14k.
6-(2-(5-(3-(Dimethylamino)propyl)pyridin-3-yl)ethyl)-4-methylpyridin-2-amine (14k).
14k was synthesized according to general procedure E using13k (166.6 mg), 10% wt. Pd/C
(131.2 mg), and NH2OH·HCl (79.2 mg). 14k was isolated as a brown oil (118.5 mg, 89%)
after flash column chromatography (MeOH: DCM 4:6). 1H NMR (500 MHz, methanolꢀd4): δ
8.80 (s, 2H), 8.71 (s, 1H), 6.76 (s, 2H), 3.36ꢀ3.33 (m, 2H), 3.31ꢀ3.28 (m, 2H), 3.22ꢀ3.19 (m,
2H), 3.03ꢀ3.00 (m, 2H), 2.95 (s, 6H), 2.38 (s, 3H), 2.27ꢀ2.21 (m, 2H); 13C NMR (125 MHz,
methanolꢀd4): δ 157.8, 154.5, 147.1, 147.0, 141.1, 140.4, 139.4, 139.1, 113.9, 110.0, 56.5,
42.2, 32.7, 31.1, 28.7, 24.8, 20.7; HRMS ESI: calcd. For C18H27N4 [M+H]+, 299.2230; found,
299.2236.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4-Methyl-6-(2-(5-(3-(methylamino)propyl)pyridin-3-yl)ethyl)pyridin-2-amine (14j).
14j was synthesized by Boc deprotection of 13i followed by general procedure E. Compound
13i (171.9 mg, 0.38 mmol) was diluted with DCM (3.8 ml) to form a 0.1 M solution followed
by addition of TFA (0.8 mL). The reaction mixture was allowed to stir at r.t. for 1 h. The
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 46
1
2
3
4
5
6
7
8
9
crude product was concentrated under reduced pressure, diluted with DCM, and washed with
sat. NaHCO3. The organic layer was dried over Na2SO4and concentrated to give crude
product 13j. The crude product was used for further steps without purification. 14j was
synthesized according to general procedure E using 13j (82.6 mg), 10% wt. Pd/C (56.1 mg),
and NH2OH·HCl (52.2 mg). 14j was isolated as a brown oil (63.8 mg, 60%) after flash
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
column chromatography (MeOH: DCM 4:6). H NMR (500 MHz, methanolꢀd4): δ 8.79 (s,
1H), 8.78 (s, 1H), 8.68 (s, 1H), 6.75 (s, 2H), 3.35ꢀ3.32 (m, 2H), 3.21ꢀ3.18 (m, 2H), 3.14ꢀ3.11
(m, 2H), 3.04ꢀ3.01 (m, 2H), 2.75 (s, 6H), 2.38 (s, 3H), 2.20ꢀ2.14 (m, 2H); 13C NMR (125
MHz, methanolꢀd4): δ 157.8, 154.5, 147.1, 147.0, 141.2, 140.4, 139.4, 139.2, 113.9, 110.0,
48.1, 32.7, 32.4, 31.1, 28.8, 26.3, 20.7; HRMS ESI: calcd. For C17H25N4 [M+H]+, 285.2074;
found, 285.2078.
4-Methyl-6-(2-(5-(1-methylpiperidin-4-yl)pyridin-3-yl)ethyl)pyridin-2-amine (14h).
14h was synthesized by alkyne reduction of 13h followed by Boc reduction and pyrrole
deprotection. 13h (316.2 mg) was treated with 10% wt. Pd/C (235.2 mg) and diluted with
MeOH to form a 0.1 M solution. The reaction mixture was stirred at r.t. for 20 h under
hydrogen gas. The crude product was filtered through Celite, and the filtrate was concentrated
under reduced pressure. The crude product (298.9 mg) was subjected to tertꢀbutyl carbamate
reduction without purification. A roundꢀbottom flask was charged with lithium aluminum
hydride (126.1 mg), and the hydrogenation crude product solution in THF (0.1 M) was
slowly added at 0 ºC under N2. The reaction mixture was allowed to warm up to r.t., refluxed
for 1 h, quenched, with water, filtered, and extracted with dichloromethane to give the crude
tertꢀbutyl carbamate reduction product (136.6 mg).,which was treated with NH2OH·HCl
(99.7 mg) and diluted with EtOH/water (2:1) to form a 0.16 M solution. The reaction mixture
was stirred at 100 ºC for 20 h, concentrated under reduced pressure, and purified by flash
1
column chromatography to give 14h (74.4 mg, 36%) as a brown oil. H NMR (500 MHz,
methanolꢀd4): δ 8.35 (s, 1H), 8.32 (s, 1H), 7.74 (s, 1H), 6.60 (s, 1H), 6.54 (s, 1H), 3.62ꢀ3.60
ACS Paragon Plus Environment

Page 25 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(m, 2H), 3.22ꢀ3.20 (m, 3H), 3.08ꢀ3.07 (m, 2H), 3.04ꢀ2.98 (m, 4H), 2.93 (s, 3H), 2.88ꢀ2.86 (m,
2H), 2.29 (s, 3H); 13C NMR (125 MHz, methanolꢀd4): δ 155.8, 155.6, 150.1, 147.4, 145.8,
139.7, 136.5, 135.4, 113.7, 109.0,54.1, 48.5, 42.5, 34.7, 31.6, 29.9, 20.4; HRMS ESI: calcd.
For C19H27N4 [M+H]+, 311.2230; found, 311.2231.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
NOS Enzyme Inhibition Assays. Enzyme inhibition was evaluated by measuring NO
production with the hemoglobin capture assay, which was performed with purified NOSs in
96ꢀwell plates using a Biotek Gen5TM microplate reader. Purified NOSs used in this study, rat
nNOS, human nNOS, murine macrophage iNOS, bovine eNOS, and human eNOS, are
recombinant enzymes, expressed in E. coli and purified by reported procedures.37ꢀ39 For rat
and human nNOS, bovine and human eNOS, the assays were performed at 37 ºC in 100 mM
HEPES buffer with 10% glycerol (pH 7.4) in the presence of 10 ꢀM ꢀarginine and
L
tetrahydrobiopterin, 100 ꢀM NADPH, 0.83 mM CaCl2, 320 units/mL of calmodulin, and 3
ꢀM human oxyhemoglobin.For iNOS, the assay was performed at 37 ºC in HEPES buffer in
the presence of ꢀarginine and cofactors (NADPH, H4B, and human oxyhemoglobin). For all
L
the assays, NO production was read by monitoring the absorbance at 401 nm, and kinetic
readouts were recorded for 6 min. The inhibition constants (Ki) for all NOSs were calculated
from the IC50 values and Km (human nNOS: 1.6 ꢀM; rat nNOS: 1.3 ꢀM; murine iNOS: 8.2
ꢀM; bovine eNOS: 1.7 ꢀM; human eNOS: 3.9 ꢀM)40 using the Cheng−Prusoff equation:
Ki=IC50/(1+[S]/Km).41 IC50 values were determined with seven to nine concentration points
(200 ꢀM−50 nM) to construct dose−response curves, and were calculated by nonlinear
regression using GraphPad Prism. The calculated standard deviations of the assays were less
than 10% with all NOSs using doseꢀresponse curves.
Inhibitor Complex Crystal Preparation. The preparations of rat nNOS, bovine eNOS,
human eNOS, and human nNOS heme domains used for crystallographic studies were carried
out using procedures described previously.24,42,43 The rat nNOS heme domain (at 9 mg/mL
containing 20 mM histidine) or the bovine eNOS heme domain (at 10 mg/mL containing 2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 46
1
2
3
4
5
6
7
8
9
mM imidazole) were used for the sitting drop vapor diffusion crystallization setup under
conditions previously reported.42 Human nNOS crystals were obtained with a triple
K301R/R354A/G357D mutant heme domain at 10 mg/mL. By slightly modifying the original
conditions where the monoclinic C2 crystals grew, a new crystal form belonging to the
P212121 space group was obtained. Long plate crystals were grown at 4 °C with the sitting
drop setup against a well solution of 8ꢀ9% PEG3350, 40 mM citric acid, 60 mM
BisꢀTrisꢀPropane, pH 6.2, 10% glycerol, and 5 mM TCEP. New crystals belong to the
orthorhombic P212121 space group with cell dimensions of a= 52.3 Å, b= 122.7 Å, and c=
165.0 Å with one homodimer in the asymmetric unit, which closely resembles the cell
dimensions of rat nNOS crystal. Fresh crystals were first passed stepwise through
cryoprotectant solutions and then soaked with 10 mM inhibitor for 4−6 h at 4 ºC before being
flash cooled in liquid nitrogen.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
X-ray Diffraction Data Collection, Data Processing, and Structural Refinement. The
cryogenic (100 K) Xꢀray diffraction data were collected remotely at the Stanford Synchrotron
Radiation Light source (SSRL) or Advanced Light Source (ALS) through the data collection
control software BluꢀIce44 and a crystal mounting robot. When a Q315r CCD detector was
used, 90−100° of data were typically collected with 0.5° per frame. If a Pilatus pixel array
detector was used, 120−130° of fineꢀsliced data were collected with 0.2° per frame. Raw
CCD data frames were indexed, integrated, and scaled using MOSFLM,45 but the pixel array
data were processed with XDS46 and scaled with or Aimless.47 The binding of inhibitors was
detected by the initial difference Fourier maps calculated with REFMAC.48 The inhibitor
molecules were then modeled in COOT49 and refined using REFMAC or PHENIX.50
Disordering in portions of inhibitors bound in the NOS active sites was often observed,
sometimes resulting in poor density quality. However, partial structural features usually could
still be visible if the contour level of the sigma A weighted 2m|Fo|−D|Fc| map dropped to 0.5
σ, which afforded the building of reasonable models into the disordered regions. Water
ACS Paragon Plus Environment

Page 27 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
molecules were added in PHENIX and checked by COOT. The TLS51 protocol was
implemented in the final stage of refinements with each subunit as one TLS group. The omit
Fo−Fc density maps shown in figures were calculated by running one round of simulated
annealing refinement (initial temperature 2000 K) in PHENIX with the inhibitor coordinate
removed from the input PDB file to generate map coefficients DELFWT and PHDELWT.
The refined structures were validated in COOT before deposition in the RCSB protein data
bank. The crystallographic data collection and structure refinement statistics are summarized
in Table S1 of the Supporting Information, with the PDB accession codes included.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ASSOCIATED CONTENT
Supporting Information
Preparation of compounds 7, 8a, 8b, 12h, and 12i
Crystallographic data collection and structure refinement statistics.
CYP inhibition protocol.
Biological assays for 14j (Metabolic stability in HLM, Determination of protein binding in
human plasma, and Cacoꢀ2 permeability)
This material is available free of charge via the Internet at http://pubs.acs.org.
Accession Codes
PDB codes for structures reported here: nNOSꢀ14c, 5FVP; nNOSꢀ14d, 5FW0; nNOSꢀ14e,
5FVO; nNOSꢀ14g, 5FVQ; nNOSꢀ14h, 5FVR; nNOSꢀ14j, 5FVS; nNOSꢀ14k, 5FVT;
HnNOSꢀ14g, 5FVU; HnNOSꢀ14h, 5FVV; HnNOSꢀ14j, 5FVW; HnNOSꢀ14k, 5FVX;
eNOSꢀ14g, 5FVY; eNOSꢀ14k, 5FVZ. Authors will release the atomic coordinates and
experimental data upon article publication.
AUTHOR INFORMATION
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 46
1
2
3
4
Corresponding Authors
5
6
7
8
*(T.L.P.) Tel:+1 949 824 7020. Eꢀmail: poulos@uci.edu.
*(R.B.S.) Tel:+1 847 491 5653. Fax: +1 847 491 7713.
Eꢀmail:Agman@chem.northwestern.edu.
Notes
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
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.). Y.J.Q. is funded by the China Scholarship
Council. L.J.R. is currently supported on NIH GM081568 and NSF grant 13ꢀ573. P.M. is
supported by grants UNCE 204011 and PRVOUK P24/LF1/3 from Charles University,
Prague, Czech Republic. HꢀY.W. and Y.J.Q. wish to thank Mr. Saman Shafaie and Dr. S.
Habibi Goudarzi in Northwestern’s Integrated Molecular Structure Education and Research
Center for assistance with HRMS experiments. We also wish to thank the SSRL and ALS
beamline staff for their support during Xꢀray diffraction and data collection.
ABBREVIATIONS USED
NO, nitric oxide; nNOS, neuronal nitric oxide synthase; iNOS, inducible nitric oxide
synthase; eNOS, endothelial nitric oxide synthase; HnNOS, human neuronal nitric oxide
synthase; HeNOS, human endothelial nitric oxide synthase; ꢀArg, ꢀarginine; FAD, flavin
L
L
adenine dinucleotide; FMN, flavin mononucleotide; NADPH, reduced nicotinamide adenine
dinucleotide phosphate; H4B, (6R)ꢀ5,6,7,8ꢀtetrahydrobiopterin; HEPES,
4ꢀ(2ꢀhydroxyethyl)ꢀ1ꢀpiperazineꢀethanesulfonic acid; TCEP, tris(2ꢀchloroethyl)phosphate;
WT, wild type.
ACS Paragon Plus Environment

Page 29 of 46
Journal of Medicinal Chemistry
1
2
3
4
REFERENCES
5
6
(1) Kerwin, J. F., Jr.; Lancaster, J. R., Jr.; Feldman, P. L. Nitric oxide: a new paradigm for
7
8
second messengers. J. Med. Chem. 1995, 38, 4343ꢀ4362.
9
(2) Palmer, R. M.; Ferrige, A. G.; Moncada, S. Nitric oxide release accounts for the biological
activity of endotheliumꢀderived relaxing factor. Nature 1987, 327, 524ꢀ526.
(3) Ignarro, L. J.; Lippton, H.; Edwards, J. C.; Baricos, W. H.; Hyman, A. L.; Kadowitz, P. J.;
Gruetter, C. A. Mechanism of vascular smooth muscle relaxation by organic nitrates, nitrites,
nitroprusside and nitric oxide: evidence for the involvement of Sꢀnitrosothiols as active
intermediates. J. Pharmacol. Exp. Ther. 1981, 218, 739ꢀ749.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(4) Vincent, S. R. Nitric oxide: a radical neurotransmitter in the central nervous system. Prog.
Neurobiol. 1994, 42, 129ꢀ160.
(5) Bogdan, C. Nitric oxide and the immune response. Nat. Immunol. 2001, 2, 907ꢀ916.
(6) Karpuzoglu, E.; Ahmed, S. A. Estrogen regulation of nitric oxide and inducible nitric
oxide synthase (iNOS) in immune cells: implications for immunity, autoimmune diseases,
and apoptosis. Nitric Oxide 2006, 15, 177ꢀ186.
(7) Roe, N. D.; Ren, J. Nitric oxide synthase uncoupling: a therapeutic target in
cardiovascular diseases. Vasc. Pharmacol. 2012, 57, 168ꢀ172.
(8) Baranano, D. E.; Snyder, S. H. Neural roles for heme oxygenase: contrasts to nitric oxide
synthase. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 10996ꢀ1002.
(9) Steinert, J. R.; Chernova, T.; Forsythe, I. D. Nitric oxide signaling in brain function,
dysfunction, and dementia. Neuroscientist 2010, 16, 435ꢀ452.
(10) Huang, H.; Ji, H.; Li, H.; Jing, Q.; Labby, K. J.; Martasek, P.; Roman, L. J.; Poulos, T. L.;
Silverman, R. B. Selective monocationic inhibitors of neuronal nitric oxide synthase. Binding
mode insights from molecular dynamics simulations. J. Am. Chem. Soc. 2012, 134,
11559ꢀ11572.
ACS Paragon Plus Environment