Potent and selective double-headed thiophene-2-carboximidamide inhibitors of neuronal nitric oxide synthase for the treatment of melanoma

Article abstract of DOI:10.1021/jm401252e

Selective inhibitors of neuronal nitric oxide synthase (nNOS) are regarded as valuable and powerful agents with therapeutic potential for the treatment of chronic neurodegenerative pathologies and human melanoma. Here, we describe a novel hybrid strategy

Full text of DOI:10.1021/jm401252e

Article
pubs.acs.org/jmc
Potent and Selective Double-Headed Thiophene-2-carboximidamide
Inhibitors of Neuronal Nitric Oxide Synthase for the Treatment of
Melanoma
He Huang,† Huiying Li,‡ Sun Yang,§ Georges Chreifi,‡ Pavel Martasek,∥,⊥ Linda J. Roman,∥
́
Frank L. Meyskens,§ Thomas L. Poulos,*,‡ and Richard B. Silverman*,†
†Department of Chemistry, Department of Molecular Biosciences, Chemistry of Life Processes Institute, Center for Molecular
Innovation and Drug Discovery, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113, United States
‡Departments of Molecular Biology and Biochemistry, Pharmaceutical Sciences, and Chemistry, University of California, Irvine,
California 92697-3900, United States
§Chao Family Comprehensive Cancer Center, University of California, Irvine, California 92697-3900, United States
∥Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78384-7760, United States
⊥Department of Pediatrics and Center for Applied Genomics, First School of Medicine, Charles University, Prague, Czech Republic
S
* Supporting Information
ABSTRACT: Selective inhibitors of neuronal nitric oxide
synthase (nNOS) are regarded as valuable and powerful agents
with therapeutic potential for the treatment of chronic
neurodegenerative pathologies and human melanoma. Here,
we describe a novel hybrid strategy that combines the
pharmacokinetically promising thiophene-2-carboximidamide
fragment and structural features of our previously reported
potent and selective aminopyridine inhibitors. Two inhibitors,
13 and 14, show low nanomolar inhibitory potency (Ki = 5 nM
for nNOS) and good isoform selectivities (nNOS over eNOS [440- and 540-fold, respectively] and over iNOS [260- and 340-
fold, respectively]). The crystal structures of these nNOS−inhibitor complexes reveal a new hot spot that explains the selectivity
of 14 and why converting the secondary to tertiary amine leads to enhanced selectivity. More importantly, these compounds are
the first highly potent and selective nNOS inhibitory agents that exhibit excellent in vitro efficacy in melanoma cell lines.
the treatment of such diseases, inhibition of iNOS and eNOS,
especially the latter, may cause undesired side effects.8,9
Therefore, to precisely control biological processes and
improve the safety profile, selective inhibition of nNOS over
the other two isoforms is important for any potential
therapeutic agent. The close similarity of active site structures
in all three isoforms10,11 presents a critical barrier to the design
of selective nNOS inhibitors.
In our continuous efforts to develop nNOS selective
inhibitors, we discovered a series of highly potent and selective
nNOS small molecule inhibitors with a 2-aminopyridinomethyl
pyrrolidine scaffold (Figure 1, 1 and 2).12−16 Some showed
excellent potency (Ki < 10 nM) and excellent selectivity for
nNOS over eNOS (>2500-fold) and iNOS (>700-fold).
Moreover, we have found that the 2-aminopyridine moiety
not only can make hydrogen bonds (H-bonds) with the active
site glutamate (Glu592 in nNOS or Glu363 in eNOS) but also
can hydrogen bond with the heme propionate in a
conformation flipped 180°, depending on the chirality of the
INTRODUCTION
■
Nitric oxide synthases (NOSs) are responsible for the biological
production of nitric oxide (NO), an important second
messenger that regulates many biological processes such as
neurotransmission, vasodilation, and immune response.1 NOSs
catalyze two sequential reactions that convert L-arginine to L-
citrulline and the free radical NO in the presence of oxygen and
reduced nicotinamide adenine dinucleotide phosphate
(NADPH) with Nω-hydroxy-L-arginine (NHA) as an inter-
mediate.2−4
There are three mammalian NOS isoforms: neuronal NOS
(nNOS), inducible NOS (iNOS), and endothelial NOS
(eNOS). All three isoforms are homodimers and share catalytic
properties. However, according to various tissue distributions,
NO generated from different NOS isoforms can play a wide
range of physiological functions.5,6 Overproduction of NO by
nNOS has been implicated in many chronic neurodegenerative
pathologies. Our previous studies have shown that NO from
nNOS plays an important role in increasing the invasion and
proliferation of human melanoma cells, suggesting that
targeting NO signaling may facilitate therapy and prevention.7
Although inhibition of nNOS activity is a promising strategy for
Received: August 13, 2013
© XXXX American Chemical Society
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Figure 1. Chemical structures of 1−3.
Figure 2. Chemical structures of 4−6.
pyrrolidine. To achieve the two possible enzyme−inhibitor H-
bonding interactions with a single compound and to simplify
the synthetic routes, we developed a series of symmetric
double-headed aminopyridines (e.g., 3 in Figure 1).17,18
However, all of the aminopyridine-based inhibitors suffered
from some limitations such as lack of oral bioavailability and/or
poor membrane permeability. A fragment-based comparison of
the in vitro enzyme assay versus the cellular assay clearly
indicates that the aminopyridine moiety in these molecules may
be an important factor that limits their membrane perme-
ability.19
In addition to the aminopyridine-based inhibitors, thiophene-
2-carboximidamides (Figure 2, 4−6) represent another
important class of nNOS inhibitors. Recently, some of these
compounds were reported with promising pharmacological
profiles.20−25 Despite their favorable pharmacokinetics, they
have significant challenges to overcome with respect to their
lower potency and isozyme selectivities in vitro compared with
the aminopyridine-containing inhibitors. In addition, few crystal
structures depicting the binding mode of these molecules to the
NOS isoforms have been reported; therefore, more crystallo-
graphic studies of thiophene-2-carboximidamides would be
highly desirable.
Both the 2-aminopyridine and thiophene-2-carboximidamide
moieties exhibit similar H-bond interactions with the enzyme,
acting as mimics of the guanidinium group of the natural
substrate L-Arg. Taken together, the thiophene-2-carboximida-
mide fragment would serve as a bioisostere of the 2-
aminopyridine fragment with the potential for improved
pharmacological profiles. Here we adopted a hybrid strategy
and designed and synthesized a series of compounds that
combines the thiophene-2-carboximidamide fragment and
structural features of our potent and selective double-headed
aminopyridine inhibitors (Figure 3). Furthermore, we con-
ducted crystallographic studies to identify the inhibitor−
enzyme interactions that promote nNOS-specific inhibition.
CHEMISTRY
■
The synthesis of 7 began with 1-iodo-3-nitrobenzene (Scheme
1). Homocoupling of 1-iodo-3-nitrobenzene gave 18 in a
moderate yield. The nitro groups of 18 were reduced to amino
groups in a quantitative yield, and then coupling with methyl
thiophene-2-carbimidothioate hydroiodide salt was used to
generate final product 7. The coupling reaction of 3,3′-
methylenedianiline with methyl thiophene-2-carbimidothioate
hydroiodide salt gave final product 8.
A synthetic route for target molecule 9 is shown in Scheme 2.
Wittig reaction of 3-nitrobenzaldehyde with the corresponding
phosphorus ylide of 1-(bromomethyl)-3-nitrobenzene allowed
the isolation of the intermediate alkene in an 87% yield.
Compound 23 was obtained by reduction of 21, followed by
treatment with methyl thiophene-2-carbimidothioate hydro-
iodide salt. Catalytic hydrogenation of 23 reduced the double
bond, giving final product 9 in a high yield.
Nucleophilic addition of 3-nitrophenol with 2-bromo-1-(3-
nitrophenyl)ethanone yielded 24 (Scheme 3). The carbonyl
group of 24 was reduced to a hydroxyl group. Reduction of 25
gave 26, which was allowed to react with methyl thiophene-2-
carbimidothioate hydroiodide salt to give 10.
Inhibitors 11 and 12 were synthesized as illustrated in
Scheme 4. Nucleophilic addition of 3-nitrophenol with the
corresponding alkyl halides generated 27 and 28. Their nitro
groups were reduced to give intermediates 29 and 30, which
were subsequently coupled with methyl thiophene-2-carbimi-
dothioate hydroiodide salt to give final products 11 and 12,
respectively.
To synthesize inhibitors 13−17 (Scheme 5), reductive
amination of 3-nitrobenzaldehyde with the corresponding
amines generated 34−36. The amino groups of 34−36 were
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Figure 3. Target molecules designed and synthesized in this study.
protected with a Boc group. Reductive amination of 35 and 36
with acetaldehyde gave 40 and 41, respectively. Amino group
reduction and coupling with methyl thiophene-2-carbimido-
thioate hydroiodide salt were performed under conditions
similar to those we adopted for the synthesis of 7, giving target
molecules 15, 17, and intermediates 47−49. Deprotection of
the Boc groups of 47−49 generated final products 13, 14, and
16.
To gain more insight into the structural basis for relative
potencies and selectivities, we determined the crystal structure
of several inhibitors bound to nNOS. The crystal structures
revealed that 7 and 9 bind to nNOS as designed with one
thiophene-2-carboximidamide head interacting with Glu592,
while the other head occupies a hydrophobic pocket
surrounded by Met336, Leu337, and Tyr706 near the active
site entrance (Figure 4). The two phenyl rings in 7 are in van
der Waals contacts with both heme propionates (Figure 4A). In
contrast, having a 2-carbon linker between the two phenyl rings
in 9 pushes the second phenyl and thiophene-2-carboximida-
mide head farther away from the heme (Figure 4B). The
Leu337 side chain must adopt an alternate rotamer to
accommodate lengthy compound 9. The second carboximida-
mide in 9 does not make any direct contacts with the protein,
while that in 7 makes only a weak H-bond with heme
propionate D. Therefore, the submicromolar binding affinity is
mainly attributed to the first phenyl ring and the thiophene-2-
carboximidamide head that tightly anchors the inhibitor to the
NOS active site. The structure of 10 bound to nNOS (data not
shown) also supports this conclusion. While the first phenyl
ring and thiophene-2-carboximidamide head is clearly shown in
the electron density, the remainder of the compound is
RESULTS AND DISCUSSION
■
Compounds 7−12 were designed with double thiophene-2-
carboximidamide heads having diverse linkers. These flexible
linkers mainly vary in length and, therefore, can provide
precursor structural features that fit the enzyme active site
appropriately. Table 1 shows the results of inhibition assays
using purified NOS enzymes with 7−12.
For this series, 7−10 display moderate inhibitory activity,
while 11 and 12 are the best nNOS inhibitors. Compound 11
also shows outstanding nNOS selectivity over iNOS and good
selectivity over eNOS. Compared to 11, addition of one
methylene group leads to a compound (12), with half the
potency and a sharp decrease in selectivity for nNOS over both
eNOS and iNOS.
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a
Scheme 1. Synthesis of 7 and 8
a
Reagents and conditions: (a) Pd2(dba)3, tri(o-tolyl)phosphine, DIEA, 100 °C, 8 h, 61%; (b) Raney Ni, hydrazine hydrate, room temp, 30 min,
quantitative; (c) EtOH, room temp, 24 h, 54−58%.
a
Scheme 2. Synthesis of 9
a
Reagents and conditions: (a) PPh3, toluene, 90 °C, 12 h, 97%; (b) LHMDS, THF, −78 °C to room temp, 4 h, 87%; (c) Raney Ni, hydrazine
hydrate, room temp, 30 min, quantitative; (d) EtOH, room temp, 24 h, 46%; (e) H2, Pd/C, EtOH, room temp, 24 h, 91%.
disordered. Nevertheless, 10 shares a similar affinity to nNOS
as 7 and 9 (Table 1).
bonding network involving Arg596, Asp600, and Arg603 via a
water molecule. Interestingly, this thiophene-2-carboximida-
mide head reaches to a pocket surrounded by Trp306 (in the
other subunit of the dimer), Asp600, Ser602, and Arg603
(Figure 5B). This is the first time we have found an inhibitor
reaching into this pocket. Sequence alignment reveals that
Ser602 in nNOS is a His or a Gln at the corresponding
positions in eNOS and iNOS, respectively. It is likely that
interactions between the thiophene-2-carboximidamide and this
unique hot spot affects the selectivity of 11 while the extensive
aforementioned enzyme−inhibitor interactions account for the
better potency. Linker length also is quite important because
increasing the linker by one atom in going from 11 to 12 results
in a 2-fold lower potency. The 12-nNOS structure (data not
Compounds with a longer (4- or 5-atom) linker between the
two head groups, 11 and 12, exhibit much improved potency
(Table 1). The 11-nNOS structure (Figure 5A) provides an
explanation. Similar to the cases of 7 and 9, one phenyl ring and
the thiophene-2-carboximidamide head of 11 recognizes the
nNOS active site and H-bonds to Glu592 with the
carboximidamide nitrogens. The O atom in the central linker
distal to the active site forms a weak H-bond (3.4 Å) with
Gln478. This brings the second phenyl ring next to Arg481.
The Arg481 guanidinium plane has to rotate to make a better
cation−aromatic stacking interaction with the phenyl ring of
11. In addition, the N atom in the second head joins a H-
D
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a
Scheme 3. Synthesis of 10
a
Reagents and conditions: (a) K2CO3, DMF, room temp, 6 h, 73%; (b) NaBH4, THF, room temp, 8 h, 80%; (c) Raney Ni, hydrazine hydrate, room
temp, 30 min, quantitative; (d) EtOH, room temp, 24 h, 41%.
a
Scheme 4. Synthesis of 11 and 12
a
Reagents and conditions: (a) K2CO3, DMF, 60 °C, 6 h, 71−80%; (b) Raney Ni, hydrazine hydrate, room temp, 30 min, quantitative; (c) EtOH,
room temp, 24 h, 59−58%.
shown) shows that while the first phenyl ring and thiophene-2-
carboximidamide head clearly bind to the nNOS active site
similarly to that of 11, the other head is too flexible to be
modeled with certainty.
group in the linker is situated between the two heme
propionates. These electrostatic interactions are very likely
the main source of binding affinity gained by the two
compounds. However, 13 and 14 have a different phenyl ring
in the second head. While 13 bears a meta-bisubstituted phenyl
ring, the one in 14 is para-bisubstituted. The structural
differences around the phenyl ring influence the way the
second thiophene-2-carboximidamide headgroup interacts with
the protein. The head in 13 reaches the same pocket next to
Ser602 described for 11 but through a different “route”. This is
because the new inhibitor−heme electrostatic interactions
direct the second phenyl ring of 13 to pack against Met336
(Figure 6A) rather than Arg481, as in the case of 11 (Figure
5A). The second thiophene-2-carboximidamide head in 14 is
partially disordered because the para-bisubstituted phenyl
forces the thiophene tail toward the position of Trp306 (in
the other subunit). The tail has to make a sharp turn to avoid
clashes (Figure 6B).
nNOS has two negative charges in the active site (Glu592
and Asp597) compared to one in eNOS (Glu363). As a result,
nNOS provides better electrostatic stabilization for positively
charged inhibitors. Inspired by our previously reported highly
selective and potent pyrrolidine-containing nNOS inhibitors (1
and 2), we introduced a basic nitrogen atom mimicking the
pyrrolidine N atom in the linker part of 11, leading to 13 and
14, to enhance the binding affinity and selectivity (Table 2).
The results are impressive because both Ki values of 13 and 14
for nNOS are 5 nM, which is 25-fold more potent than that of
11. Moreover, they display good selectivities of nNOS over
iNOS and eNOS. The crystal structures of 13 and 14 bound to
nNOS show that, as expected, both use the first phenyl ring and
thiophene-2-carboximidamide head to anchor the inhibitor to
the nNOS active site (Figure 6). The newly introduced amino
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a
Scheme 5. Synthesis of 13−17
a
Reagents and conditions: (a) Et3N, NaBH3CN, THF, room temp, 12 h, 69−72%; (b) (Boc)2O, DMAP, MeOH, room temp, 12 h, 92−94%; (c)
NaBH3CN, THF, room temp, 12 h, 58−64%; (d) Raney Ni, hydrazine hydrate, room temp, 30 min, quantitative; (e) EtOH, room temp, 24 h, 48−
53%; (f) 3N HCl/MeOH, room temp, 24 h, quantitative.
The basicity of the amine in the linker is important for
potency, but this secondary amine is also a potential metabolic
site that can be easily converted to a secondary hydroxylamine
or serve as a substrate for flavin monooxygenases.26 Therefore,
we introduced a small ethyl group at the basic N atom of 14 to
block this potential metabolic site. Such a substitution (15)
decreases the potency (leading to an almost 10-fold drop in
nNOS inhibition) but affords an increase in selectivity of nNOS
over eNOS (845-fold). To our surprise, the 15-nNOS structure
reveals that 15 (Figure 6C) binds to nNOS in an orientation
180° flipped from that of 14 (Figure 6B). With 15, it is the
para-bisubstituted phenyl ring and its adjacent thiophene-2-
carboximidamide that binds to Glu592. This orientation still
allows a H-bond between the ethylated tertiary amine and
F
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nNOS structure (Figure 6C). That is, the para-bisubstituted
phenyl ring brings the thiophene-2-carboximidamide next to
Glu363 on one end and the tertiary amine next to heme
propionate D on the other end, for H-bonds. This means that
the 845-fold selectivity of 15 for nNOS over eNOS (Table 2) is
associated with how the tail end of the inhibitor extending out
of the active site interacts with the protein. Unfortunately, the
position of this thiophene ring is less certain because of poorer
density in this region in both the 15-eNOS and 15-nNOS
structures. Even so, there are sufficient sequence differences in
this area to provide a possible explanation for selectivity. The
main difference is that Met336 in nNOS is Val106 in eNOS.
The second phenyl ring of 15 in eNOS makes van der Waals
contacts with Val106 and Leu107, while the same group in
nNOS contacts Met336 and Leu337. As a result, there is the
potential for greater nonpolar interactions in nNOS. Another
factor is the contacts between the thiophene ring and a Trp side
chain (Trp306 in nNOS or Trp76 in eNOS). These contacts
are closer in nNOS than those in eNOS.
Changing the secondary amine in 14 to a tertiary amino in
15 improves selectivity. To explore whether such substitution is
effective in other cases, especially in mono-thiophene-2-
carboximidamide nNOS inhibitors, we designed and synthe-
sized 16 and 17 (Table 3). One thiophene-2-carboximidamide
head was replaced by a fluorobenzene fragment because
fluorine has been widely used to alter the pharmacokinetic
properties and overall drug-like properties of compounds.27
Compound 16 drops about half of the nNOS inhibitory activity
compared to parent compound 14, and selectivity also
decreases sharply, especially over eNOS. However, addition
of an ethyl group at the basic nitrogen of 16 further enhances
the nNOS over eNOS selectivity although it decreases the
potency (Table 3), which shows the same trend as 14 and 15.
The reason for decreased potency may be the shorter length,
thereby preventing additional van der Waals contacts with the
protein that are afforded by the longer thiophene-2-
carboximidamide head in 14. Detailed interactions of 16 and
17 are shown in Figure 8.
Table 1. Inhibition of NOS Isozymes by Synthetic Inhibitors
7−12
a
Ki (μM)
selectivity
n/i n/e
compd
no.
nNOS
iNOS
eNOS
7
0.787 0.061
0.776 0.069
0.739 0.053
0.819 0.067
0.130 0.062
0.237 0.019
66.4 5.2
79.0 4.0
103.8 8.7
10.1 0.9
66.8 2.7
12.6 0.8
177.3 10.9
133.0 12.4
66.0 5.8
5.2 0.4
84
102
141
12
225
171
89
8
9
10
11
12
6
13.7 0.7
4.0 0.3
513
53
105
17
a
The compounds were evaluated for in vitro inhibition against three
NOS isozymes: rat nNOS, bovine eNOS, and murine iNOS.
Considering that three NOS isozymes are isolated from three different
species, the activity or selectivity of these compounds may differ in
human NOS isozymes.39
heme propionate D. Inhibitor 14 is close to both heme
propionates while 15 is close to only one, which may be the
structural basis for why 14 is a more potent inhibitor.
Nevertheless, the second phenyl ring and thiophene-2-
carboximidamide head in 15 are quite well-defined, making
van der Waals contacts with Met336, Leu337, and Trp306 (in
the other subunit).
To understand isoform selectivity, we also determined the
structures of 14-eNOS and 15-eNOS. As shown in Figure 7A,
14 anchors its meta-bisubstituted phenyl ring and the
connected thiophene-2-carboximidamide head to the eNOS
active site next to Glu363, which is the same interaction seen in
the 14-nNOS structure. The salt bridges from the secondary
amine in the linker of 14 to both heme propionates also exist in
eNOS, which affords a low micromolar binding affinity of 14 to
eNOS (Table 2). However, the second phenyl and thiophene-
2-carboximidamide head of 14 are less well-defined in eNOS.
The residual electron density is nevertheless clear enough to
indicate that the thiophene head steers away from the pocket
where the same thiophene in 14-nNOS establishes more
favorable inhibitor−protein interactions. We hypothesize that
the reason for this different binding preference stems from an
amino acid variant, Ser602 in nNOS but His373 in eNOS. The
bulkier His373 side chain in eNOS makes the pocket too
shallow to accommodate the thiophene head of 14.
Indeed, the structure of 16-nNOS (Figure 8A) reveals that
apart from the common features of NOS active site recognition
through the thiophene-2-carboximidamide head seen for other
analogues, the fluorobenzene head of 16 interacts only with
Trp306 (other subunit). Compound 17 shows ∼7-fold lower
potency than 16, mainly because it maintains only one H-bond
The inhibitor binding mode observed in the 15-eNOS
structure (Figure 7B) is almost the same as that seen in the 15-
Figure 4. Crystallographic binding conformation of 7 (A) and 9 (B) in rat nNOS. The omit Fo − Fc electron density maps for the inhibitors are
shown at the 2.5 σ contour level. Relevant H-bonds are depicted with dashed lines. Key distances are labeled in Å. All structural figures were
prepared with PyMol (www.pymol.org).
G
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Figure 5. Crystallographic binding conformation of 11 with rat nNOS (A) and the new hot spot around the second thiophene head (B). The omit
Fo − Fc electron density maps for inhibitors are shown at the 2.5 σ contour level. Relevant H-bonds are depicted with dashed lines.
proliferation of melanoma and increased invasion, leading to
subsequent development of metastases. nNOS inhibitors were
able to efficiently inhibit melanoma proliferation and invasion
with significant reduction of intracellular NO levels. Inhibitors
13 and 14 exhibited potent antimelanoma activity in vitro
(Table 4). Their EC50 values in metastic melanoma A375 cells
are 1.3 and 3.4 μM (Table 4), which is better than that of the
chemotherapeutic drug cisplatin (4.2 and 14.3 μM in A375 and
Sk-Mel28 cells, respectively).7 Notably, the inhibition by 13
and 14 is more predominant in metastatic melanoma A375 cells
compared to primary early stage Wm3211 cells, which supports
our hypothesis that nNOS/NO signaling is more critical to
melanoma progression than to the initiation phase. However,
unexpectedly, these compounds also exhibited apparent
inhibition of cell proliferation in primary normal cells, including
melanocytes and fibroblast cells. Taken together, these results
suggest that targeting nNOS provides a potential approach to
block overactivated NO signaling in human melanoma.
Table 2. Inhibition of NOS Isozymes by Synthetic Inhibitors
13−15
a
Ki (μM)
selectivity
compd no.
nNOS
iNOS
eNOS
n/i
n/e
13
14
15
0.005 0.0005
0.005 0.0003
0.049 0.002
1.3 0.2
2.2 0.1
2.7 0.2
41.4 2.2
260
340
290
440
540
845
1.7 0.1
14.2 0.8
a
The compounds were evaluated for in vitro inhibition against three
NOS isozymes: rat nNOS, bovine eNOS, and murine iNOS.
with heme propionate A via its tertiary amine (Figure 8B),
while the secondary amine in 16 makes H-bonds/salt bridges
with both heme propionates (Figure 8A). Moreover, the
fluorobenzene in the 17-nNOS complex has higher flexibility
with weaker electron density, making a new hydrophobic
interaction with Tyr706. This interaction forces the phenyl
group of Tyr706 to rotate about 70° from what is seen in the
16-nNOS structure. Although 17 is a weaker binder than 16,
the former has better selectivity (Table 3). The crystal structure
shows that 16 (Figure 8C) binds to eNOS in an almost
identical manner as that found in the 16-nNOS structure
(Figure 8A), and 17 also binds to eNOS (Figure 8D) similar to
nNOS (Figure 8B). The only difference for 16 is the
orientation of the F atom from fluorobenzene. In eNOS the
F atom points toward Val106 and Leu107 for van der Waals
contacts. To avoid clashes with the fluorobenzene of 16 (or
17), the Tyr477 side chain rotates, as we observed with 17-
nNOS (Figure 8B). The subtle difference between 17-eNOS
(Figure 8D) and 17-nNOS (Figure 8B) is also only in the
position of fluorobenzene, which might be attributed to the
amino acid variation in the vicinity. Met336 in nNOS is
corresponds to Val106 in eNOS. Hydrophobic contacts (∼3.5−
3.9 Å) found between the fluorobenzene and Val106/Leu107
in 17-eNOS are closer than those contacts (∼4.3−4.8 Å) to
Met336/Leu337 seen with 17-nNOS. Although it is not clear
that the differences near the tail end of the inhibitors is the
explanation for isoform selectivity, it is clear that attaching an
ethyl group to the secondary amine is a useful method to
improve the nNOS/eNOS selectivity of these thiophene
compounds.
CONCLUSIONS
■
In summary, we report the rational design of selective nNOS
inhibitors using a hybrid strategy. Two compounds, 13 and 14,
show excellent potency (5 nM) and good selectivities for
nNOS over the other isozymes. The crystal structures of these
inhibitors bound to nNOS or eNOS have identified a new hot
spot near Ser602 in nNOS (Figure 5B). The equivalent residue
in eNOS is His373, which may be a contributing structural
factor for the selectivity observed for 14 (Figure 6). A small
ethyl substituent attached to the amine of the inhibitor acts as
an important component for improving isoform selectivity.
Importantly, 13 and 14 achieve good cellular activity in two
melanoma cell lines. Altogether, these inhibitors are promising
candidates for further development of improved therapeutics,
and their structural features provide new clues for the future
design and development of new selective nNOS inhibitors.
EXPERIMENTAL SECTION
■
All reagents were purchased from Sigma-Aldrich, Alfa Aesar, or TCI
and were used without further purification unless stated otherwise.
Analytical thin layer chromatography was visualized by ultraviolet light.
Flash column chromatography was carried out under a positive
1
pressure of air. H NMR spectra were recorded on 500 or 400 MHz
Given the enzyme potency of 13 and 14, we next tested
whether these inhibitors were effective in melanoma cell lines.
Detailed in our previous study,7 nNOS is associated with the
spectrometers. Data are presented as follows: chemical shift (in ppm
on the δ scale, and the reference resonance peaks set at 0 ppm
[TMS(CDCl3)] and 3.31 ppm (CD2HOD), multiplicity (s = singlet, d
H
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Figure 6. Crystallographic binding conformation of 13 (A), 14 (B), and 15 (C) with rat nNOS. The omit Fo − Fc electron density maps for
inhibitors are shown at the 2.5 σ contour level. Relevant H-bonds are depicted with dashed lines. Key distances are drawn with dashed lines and
labeled in Å.
Figure 7. Crystallographic binding conformation of 14 (A) and 15 (B) with bovine eNOS. The omit Fo − Fc electron density maps for inhibitors are
shown at the 2.5 σ contour level. Relevant H-bonds are depicted with dashed lines. Key distances are drawn with dashed lines and labeled in Å.
= doublet, t = triplet, q = quartet, p = quintet, m = multiplet), coupling
constant (J/Hz), and integration. 13C NMR spectra were recorded at
125 or 100 MHz, and all chemical shift values are reported in ppm on
the δ scale, with an internal reference of δ 49.0 for CD3OD. High-
resolution mass spectra were measured on liquid chromatography/
time-of-flight mass spectrometry (LC-TOF). The purity of the tested
compounds was determined by high-performance liquid chromatog-
raphy (HPLC) analysis and was >95%.
at room temperature for 30 min. Then the reaction mixture was
filtered through Celite. The filtration was diluted with water, extracted
with EtOAc, and washed with saturated NaHCO3 and brine. The
organic layer was concentrated in vacuo. The residue was purified by
column chromatography to yield the product.
General Procedure B: Reductive Amination. To a solution of
the aldehyde (1.0 mmol) and amine (1.0 mmol) in THF (20 mL) at 0
°C was added NaBH3CN (1.05 mmol) in three portions carefully. The
reaction mixture was stirred at 0 °C for 30 min and then stirred
overnight at room temperature. The reaction mixture was quenched
with MeOH in an ice bath, extracted with EtOAc, and washed with
General Procedure A: Nitro Group Reduction. To a solution of
the reagent (1 mmol) in MeOH (10.0 mL) was added hydrazine
hydrate (0.2 mL) and Raney nickel. The reaction mixture was stirred
I
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Journal of Medicinal Chemistry
Article
crude product was recrystallized with MeOH and cold diethyl ether to
provide the product.
Table 3. Inhibition of NOS Isozymes by Synthetic Inhibitors
16 and 17
a
N,N′-([1,1′-Biphenyl]-3,3′-diyl)bis(thiophene-2-carboximida-
mide) (7). To a solution of 1-iodo-3-nitrobenzene (3.0 mmol) in dry
DIEA (20 mL) was added Pd2(dba)3 (0.05 equiv) and tri(o-
tolyl)phosphine (0.1 equiv). The reaction mixture was stirred under
argon at 100 °C for 8 h and then diluted with H2O, extracted with
EtOAc, and washed with water and brine. The organic layer was dried
over Na2SO4 and concentrated in vacuo. The residue was purified by
column chromatography to yield 18 (yield 61%). 7 was synthesized by
general procedures A and D using 18 as the starting material (yield
54%). 1H NMR (500 MHz, CD3OD): δ 7.68 (d, J = 3.5 Hz, 2H), 7.60
(d, J = 5.0 Hz, 2H), 7.46 (t, J = 7.5 Hz, 2H), 7.40 (d, J = 7.5 Hz, 2H),
7.26 (s, 2H), 7.15 (dd, J = 5.0, 3.5 Hz, 2H), 7.00 (d, J = 7.5 Hz, 2H).
13C NMR (125 MHz, CD3OD): δ 156.61, 140.59, 135.37, 134.96,
Ki (μM)
selectivity
compd
no.
nNOS
iNOS
eNOS
n/i
n/e
16
17
0.011 0.001
0.073 0.003
1.6 0.07
0.9 0.05
145
166
82
12.1 0.74
21.7 1.57
297
a
The compounds were evaluated for in vitro inhibition against three
NOS isozymes: rat nNOS, bovine eNOS and murine iNOS.
saturated NaHCO3 and brine. The organic layer was concentrated in
vacuo. The residue was purified by column chromatography to yield
product.
General Procedure C: Boc-Protection. To a solution of the
amine (1 mmol) in MeOH (15 mL) was added di-tert-butyl
dicarbonate (1.2 equiv) and DMAP (cat.). The reaction mixture was
stirred at room temperature for 12 h. Then the reaction mixture was
concentrated in vacuo. The residue was purified by column
chromatography to yield the product.
General Procedure D: Coupling Reaction of Amine with
Methyl Thiophene-2-carbimidothioate Hydroiodide Salt. To a
solution of the amine (1 mmol) in EtOH (15.0 mL) was added
thiophene-2-carbimidothioate hydroiodide salt (1.5 equiv for mono-
amine and 3.0 equiv for diamine). The reaction mixture was stirred at
room temperature for 36 h. Then the reaction mixture was
concentrated in vacuo. The residue was purified by column
chromatography to yield product.
General Procedure E: Boc-Deprotection. To a solution of the
Boc-protected reagent (0.2 mmol) in MeOH (1.0 mL) was added 3N
HCl (10.0 mL). The reaction mixture was stirred at room temperature
for 24 h. Then the reaction mixture was concentrated in vacuo. The
134.59, 130.65, 129.07, 128.62, 126.63, 125.25, 124.12. LC-TOF (M +
H+) calcd for C22H19N4S2 403.1046, found 403.1046.
N,N′-(Methylenebis(3,1-phenylene))bis(thiophene-2-carbox-
imidamide) (8). Compound 8 was synthesized by general procedure
D using 3,3′-methylenediamiline as the starting material (yield 58%).
1H NMR (500 MHz, CD3OD): δ 8.10−8.00 (m, 4H), 7.53 (t, J = 7.5
Hz, 2H), 7.41−7.36 (m, 6H), 7.34 (d, J = 7.5 Hz, 2H), 4.18 (s, 2H).
13C NMR (125 MHz, CD3OD): δ 159.39, 144.70, 135.82, 135.50,
135.15, 131.76, 130.92, 130.30, 130.02, 127.25, 124.71, 41.99. LC-
TOF (M + H+) calcd for C23H21N4S2 417.1202, found 417.1195.
N,N′-(Ethane-1,2-diylbis(3,1-phenylene))bis(thiophene-2-
carboximidamide) (9). LHMDS (1.0 M in THF, 2.4 mmol) was
added to a suspension of 20 (2 mmol) in anhydrous THF (15 mL) at
−78 °C. The reaction mixture was stirred under argon for 30 min,
then warmed to 0 °C over 30 min. To this reaction mixture was added
3-nitrobenzaldehye (2.2 mmol) in anhydrous THF (3 mL) at −78 °C.
The reaction mixture was warmed to room temperature and stirred for
4 h. The reaction mixture was then quenched with saturated NH4Cl,
Figure 8. Crystallographic binding conformation of 16 (A) and 17 (B) with rat nNOS, as well as 16 (C) and 17 (D) with bovine eNOS. The omit
Fo − Fc electron density maps for inhibitors are shown at the 2.5 σ contour level. Relevant H-bonds are depicted with dashed lines. Key distances are
drawn with dashed lines and labeled in Å.
J
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Table 4. EC50 Values (μM) of 13 and 14 with Human Melanoma Cells
compd primary melanoma wm3211 cell line metastatic melanoma A375 cell line primary fibroblast cells primary keratinocytes immortalized melanocytes
a
b
13
14
5.6
2.7
3.4
1.3
1.4
7.9
ND
cannot calculate
cannot calculate
cannot calculate
a
b
Not done. Toxic even at lower concentration and no dose−activity curve occurred.
extracted with EtOAc, and washed with water and brine. The organic
layer was concentrated in vacuo. The residue was purified by column
chromatography to yield 21 (yield 87%). Intermediate 24 was
synthesized by general procedures A and D using 21 as the starting
material (yield 46%). A solution of 24 (0.5 mmol) in MeOH (20 mL)
was treated with 10% Pd/C (15 mg). The reaction mixture was stirred
at room temperature under a hydrogen atmosphere for 24 h. The
catalyst was removed by filtration through Celite, and the resulting
solution was concentrated in vacuo. The crude material was purified by
2H), 4.28 (t, J = 6.0 Hz, 4H), 2.33 (p, J = 6.0 Hz, 2H). 13C NMR (125
MHz, CD3OD): δ 161.77, 158.79, 146.61, 134.93, 134.51, 132.32,
129.83, 118.43, 115.89, 112.62, 65.88, 30.21. LC-TOF (M + H+) calcd
for C25H25N4O2S2 477.1413, found 477.1411.
N-(3-(2-((3-(Thiophene-2-carboximidamido)benzyl)amino)-
ethyl)phenyl)thiophene-2-carboximidamide (13). 13 was syn-
thesized by general procedures B, C, A, D, and E using 3-
nitrobenzaldehyde and 2-(3-nitrophenyl)ethanamine as the starting
1
materials. H NMR (500 MHz, CD3OD): δ 7.65 (t, J = 5.0 Hz, 2H),
1
7.56 (dt, J = 5.0, 2.0 Hz, 2H), 7.38−7.27 (m, 2H), 7.17−7.09 (m, 2H),
7.06 (dt, J = 7.5, 1.5 Hz, 1H), 7.01−6.94 (m, 2H), 6.91 (dt, J = 7.5, 1.5
Hz, 1H), 6.88−6.82 (m, 2H), 3.79 (s, 2H), 2.96−2.81 (m, 4H). 13C
NMR (125 MHz, CD3OD): δ 161.52, 153.94, 150.23, 142.53, 141.90,
141.06, 130.73, 130.67, 129.87, 129.83, 128.49, 128.39, 125.03, 124.76,
123.95, 123.69, 122.60, 121.58, 54.30, 51.28, 36.59. LC-TOF (M +
H+) calcd for C25H26N5S2 460.1624, found 460.1618.
column chromatography to yield 9 (yield 91%). H NMR (400 MHz,
CD3OD): δ 8.17−8.02 (m, 4H), 7.56−7.51 (t, J = 7.5 Hz, 2H), 7.45−
7.38 (m, 6H), 7.36−7.30 (d, J = 7.5 Hz, 2H), 3.10 (s, 4H). 13C NMR
(100 MHz, CD3OD): δ 156.90, 143.97, 136.57, 133.15, 132.73,
130.64, 129.92, 128.32, 128.26, 125.00, 122.43, 36.92. LC-TOF (M +
H+) calcd for C24H23N4S2 431.1359, found 431.1359.
N-(3-(1-Hydroxy-2-(3-(thiophene-2-carboximidamido)-
phenoxy)ethyl)phenyl)thiophene-2-carboximidamide (10). To
a solution of 3-nitrophenol (2 mmol) in DMF (20 mL) was added
K2CO3 (2.2 mmol) and 2-bromo-1-(3-nitrophenyl)ethanone (2.2
mmol). The suspension was stirred for 6 h at room temperature. The
reaction mixture was then diluted with H2O (50 mL), extracted with
EtOAc, and washed with water and brine. The organic layer was dried
over Na2SO4 and concentrated in vacuo. The residue was purified by
column chromatography to yield 24 (yield 73%). To a solution of 24
(1 mmol) in THF (10 mL) was added NaBH4 (1.2 mmol) in three
portions. The reaction mixture was stirred at room temperature for 8 h
under a N2 atmosphere. The reaction mixture was quenched with H2O
(20 mL), extracted with CH2Cl2, and washed with water and brine.
The organic layer was dried over Na2SO4 and concentrated in vacuo.
The residue was purified by column chromatography to yield 25 (yield
80%). 10 was synthesized by general procedures A and D using 25 as
N-(4-(2-((3-(Thiophene-2-carboximidamido)benzyl)amino)-
ethyl)phenyl)thiophene-2-carboximidamide (14). 14 was syn-
thesized by the same procedures as those to prepare 13 using 2-(4-
nitrophenyl)ethanamine as the starting material. 1H NMR (500 MHz,
CD3OD): δ 7.65−7.58 (m, 2H), 7.58−7.51 (m, 2H), 7.33 (t, J = 7.5
Hz, 1H), 7.24−7.17 (m, 2H), 7.13−7.07 (m, 2H), 7.04 (dt, J = 7.5, 2.0
Hz, 1H), 6.95 (t, J = 2.0 Hz, 1H), 6.93−6.85 (m, 3H), 3.84−3.70 (m,
2H), 2.93−2.74 (m, 4H). 13C NMR (125 MHz, CD3OD): δ 153.93,
141.90, 141.03, 136.14, 130.99, 130.86, 130.66, 130.30, 129.86, 129.80,
128.50, 128.44, 128.37, 124.76, 123.80, 123.66, 123.60, 122.61, 54.27,
51.49, 36.09. LC-TOF (M + H+) calcd for C25H26N5S2 460.1624,
found 460.1620.
N-(4-(2-(Ethyl(3-(thiophene-2-carboximidamido)benzyl)-
amino)ethyl)phenyl)thiophene-2-carboximidamide (15). 35
was synthesized by general procedure B using 3-nitrobenzaldehyde
and 2-(4-nitrophenyl)ethanamine as the starting materials. To a
solution of 35 (1 mmol) and acetaldehyde (2 equiv) in THF (10 mL)
was added NaBH4 (1.2 mmol) in three portions. The reaction mixture
was stirred at room temperature for 8 h under a N2 atmosphere. The
reaction mixture was then quenched with H2O (20 mL), extracted
with CH2Cl2, and washed with water and brine. The organic layer was
dried over Na2SO4 and concentrated in vacuo. The residue was
purified by column chromatography to yield 39 (yield 64%). 15 was
synthesized by general procedures A and D using 39 as the starting
material. 1H NMR (500 MHz, CD3OD): δ 7.69−7.60 (m, 2H), 7.60−
7.54 (m, 2H), 7.36 (t, J = 7.5 Hz, 1H), 7.24−7.17 (m, 2H), 7.14−7.09
(m, 3H), 7.05−6.99 (m, 1H), 6.96−6.87 (m, 3H), 3.72 (s, 2H), 2.88−
2.73 (m, 4H), 2.72−2.64 (q, J = 7.0 Hz, 2H), 1.16 (t, J = 7.0 Hz, 3H).
13C NMR (125 MHz, CD3OD): δ 150.04, 141.15, 141.01, 136.83,
1
the starting material (yield 41%). H NMR (500 MHz, CD3OD): δ
7.62 (dt, J = 7.5, 3.5 Hz, 2H), 7.58−7.51 (m, 2H), 7.37 (t, J = 7.5 Hz,
1H), 7.25 (t, J = 7.5 Hz, 1H), 7.20 (d, J = 7.5 Hz, 1H), 7.13−7.07 (m,
3H), 6.93 (dt, J = 7.5, 2.5 Hz, 1H), 6.69 (dd, J = 7.5, 2.5 Hz, 1H),
6.60−6.54 (m, 2H), 5.05−5.00 (m, 1H), 4.15−4.00 (m, 2H). 13C
NMR (125 MHz, CD3OD): δ 161.37, 144.06, 131.34, 130.62, 129.88,
128.51, 128.38, 123.20, 122.77, 121.79, 116.23, 110.97, 110.09, 74.18,
73.46. LC-TOF (M + H+) calcd for C24H23N4O2S2 463.1257, found
463.1256.
N,N′-((Ethane-1,2-diylbis(oxy))bis(3,1-phenylene))bis-
(thiophene-2-carboximidamide) (11). To a solution of 3-nitro-
phenol (2 mmol) in DMF (20 mL) was added K2CO3 (2.2 mmol) and
1,3-dibromopropane (1 mmol). The suspension was then stirred for 6
h at 60 °C. The reaction mixture was then diluted with H2O (50 mL),
extracted with EtOAc, and washed with water and brine. The organic
layer was dried over Na2SO4 and concentrated in vacuo. The residue
was purified by column chromatography to yield 27 (yield 80%). 11
was synthesized by general procedures A and D using 27 as the
130.90, 130.43, 129.86, 129.79, 128.43, 128.37, 128.35, 125.79, 124.76,
123.65, 122.58, 58.73, 55.97, 33.01, 11.75. LC-TOF (M + H+) calcd
for C27H30N5S2 488.1937, found 488.1932.
N-(3-(((3-Fluorophenethyl)amino)methyl)phenyl)thiophene-
2-carboximidamide (16). 16 was synthesized by the same
procedures as those to prepare 13 using 3-fluorobenzaldehyde as the
1
starting material (yield 59%). H NMR (500 MHz, CD3OD): δ 8.01
1
(t, J = 4.0 Hz, 4H), 7.47 (t, J = 8.0 Hz, 2H), 7.33 (t, J = 4.0 Hz, 2H),
7.03 (dd, J = 8.0, 2.0 Hz, 2H), 6.97 (t, J = 2.0 Hz, 2H), 6.94 (dd, J =
8.0, 2.0 Hz, 2H), 4.43 (s, 4H). 13C NMR (125 MHz, CD3OD): δ
161.52, 157.72, 133.75, 133.12, 132.15, 129.64, 129.49, 118.15, 114.83,
112.12, 68.12. LC-TOF (M + H+) calcd for C24H23N4O2S2 463.1257,
found 463.1257.
N,N′-((Propane-1,3-diylbis(oxy))bis(3,1-phenylene))bis-
(thiophene-2-carboximidamide) (12). 12 was synthesized by the
same procedures as those to prepare 11 using 1,4-dibromobutane as
the starting material. 1H NMR (500 MHz, CD3OD): δ 8.09−7.99 (m,
4H), 7.49 (t, J = 8.0 Hz, 2H), 7.41−7.33 (m, 2H), 7.11−7.05 (dd, J =
8.0, 2.5 Hz, 2H), 7.03 (t, J = 2.5 Hz, 2H), 7.00 (dd, J = 8.0, 2.5 Hz,
starting material. H NMR (400 MHz, CD3OD): δ 8.18−8.04 (m,
2H), 7.81−7.69 (m, 2H), 7.65 (t, J = 7.5 Hz, 1H), 7.58−7.50 (m, 1H),
7.41−7.28 (m, 2H), 7.18−7.06 (m, 2H), 7.03−6.93 (m, 1H), 4.37 (s,
2H), 3.42−3.31 (m, 2H), 3.20−3.10 (m, 2H). 13C NMR (100 MHz,
CD3OD): δ 164.17, 161.74 (s), 139.31 (d, J = 7.5 Hz), 134.72, 134.45,
134.22, 133.50, 130.87, 130.55, 130.34 (d, J = 7.5 Hz), 128.66, 127.22,
126.47, 124.49, 124.46, 115.30 (d, J = 20 Hz), 113.56 (d, J = 20 Hz),
109.99, 50.38, 48.27, 31.49 (d, J = 2.0 Hz). LC-TOF (M + H+) calcd
for C20H21FN3S 354.1435, found 354.1441.
N-(3-((Ethyl(3-fluorophenethyl)amino)methyl)phenyl)-
thiophene-2-carboximidamide (17). 17 was synthesized by the
same procedures as those to prepare 15 using 3-fluorobenzaldehyde
K
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Table 5. Crystallographic Data Collection and Refinement Statistics
a
data set
nNOS-7
nNOS-9
nNOS-11
4KCJ
nNOS-13
Data Collection
PDB code
4KCH
4KCI
4KCK
P212121
space group
P212121
P212121
P212121
cell dimensions a, b, c (Å)
resolution (Å)
Rmerge
51.7, 111.6, 165.0
2.15 (2.19−2.15)
0.066 (0.559)
20.0 (1.3)
51.8, 111.9, 165.0
2.27 (2.31−2.27)
0.055 (0.677)
29.7 (1.9)
51.8, 110.0, 164.7
2.05 (2.09−2.05)
0.055 (0.799)
25.1 (2.1)
51.9, 111.0, 165.3
2.10 (2.14−2.10)
0.069 (0.720)
20.7 (1.5)
I/σI
no. unique reflns
completeness (%)
redundancy
52573
44477
61070
52371
99.4 (98.1)
3.6 (3.6)
98.3 (97.8)
4.0 (4.2)
99.6 (97.8)
3.6 (3.4)
92.6 (100.0)
3.5 (3.7)
Refinement
resolution (Å)
2.15
2.27
2.05
2.10
no. reflns used
49887
42147
57366
49419
b
R
work/Rfree
no. atoms
protein
0.194/0.250
0.181/0.238
0.195/0.242
0.216/0.264
6659
185
6659
189
6665
193
6681
179
ligand/ion
water
167
122
259
379
RMS deviations
bond lengths (Å)
0.011
0.016
0.013
0.012
bond angles (deg)
data set
2.02
2.27
2.11
2.10
a
nNOS-14
nNOS-15
nNOS-16
nNOS-17
Data Collection
PDB code
4KCL
4KCM
4KCN
4KCO
space group
P212121
P212121
P212121
P212121
cell dimensions a, b, c (Å)
resolution (Å)
Rmerge
52.1, 111.1, 165.1
1.93 (1.96−1.93)
0.057 (0.479)
26.3 (2.1)
71898
51.7, 110.8, 165.2
2.08 (2.12−2.08)
0.068 (0.762)
23.1 (1.7)
52.1, 111.0, 165.0
1.85 (1.88−1.85)
0.050 (0.660)
29.2 (2.6)
82346
52.0, 110.9, 165.0
1.86 (1.89−1.86)
0.057 (0.680)
28.2 (2.1)
I/σI
no. unique reflns
completeness (%)
redundancy
58123
81114
98.0 (97.8)
3.3 (3.2)
99.6 (100.0)
3.8 (3.8)
99.3 (99.9)
4.0 (4.1)
99.4 (100.0)
3.9 (3.8)
Refinement
resolution (Å)
2.10
2.07
1.85
1.86
no. reflns used
68,060
55,146
77,959
76,678
b
R
work/Rfree
no. atoms
protein
0.182/0.213
0.191/0.236
0.185/0.217
0.187/0.220
6666
195
6668
188
6677
219
6685
177
ligand/ion
water
341
224
397
380
RMS deviations
bond lengths (Å)
0.011
0.011
0.012
0.014
bond angles (deg)
data set
1.45
2.05
1.52
1.62
a
eNOS-14
eNOS-15
eNOS-16
eNOS-17
Data Collection
PDB code
4KCP
4KCQ
4KCR
4KCS
space group
P212121
P212121
P212121
P212121
cell dimensions a, b, c (Å)
resolution (Å)
Rmerge
57.7, 106.2, 156.8
2.07 (2.11−2.07)
0.060 (0.712)
24.2 (1.9)
59332
57.7, 106.4, 156.7
2.03 (2.07−2.03)
0.064 (0.494)
26.9 (2.6)
62254
57.6, 106.2, 156.5
2.09 (2.13−2.09)
0.059 (0.576)
27.3 (2.6)
57.7, 106.4, 157.1
2.05 (2.09−2.05)
0.051 (0.528)
31.0 (2.9)
61246
I/σI
no. unique reflns
Completeness (%)
Redundancy
57743
99.1 (99.9)
4.0 (4.0)
98.4 (99.7)
3.9 (3.9)
99.7 (100.0)
4.0 (4.1)
99.2 (100.0)
4.0 (4.0)
Refinement
Resolution (Å)
2.07
2.03
2.09
2.05
No. reflns used
56198
59095
54719
58050
b
R
work/Rfree
0.166/0.213
0.179/0.224
0.164/0.205
0.168/0.213
No. atoms
protein
6446
6429
6436
6446
L
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Journal of Medicinal Chemistry
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Table 5. continued
a
data set
eNOS-14
eNOS-15
eNOS-16
eNOS-17
Refinement
ligand/ion
216
305
223
359
206
354
201
350
water
RMS deviations
bond lengths (Å)
0.020
0.013
1.63
0.011
1.45
0.020
2.05
bond angles (deg)
2.02
a
b
See Figure 3 for the inhibitor chemical structure. Rfree was calculated with the 5% of reflections set aside throughout the refinement. The set of
reflections for the Rfree calculation were kept the same for all data sets of each isoform according to those used in the data of the starting model.
1
and 2-(3-nitrophenyl)ethanamine as the starting materials. H NMR
(400 MHz, CD3OD): δ 7.85−7.72 (m, 2H), 7.43 (t, J = 7.5 Hz, 1H),
7.31−7.17 (m, 4H), 7.12 (dt, J = 7.5, 1.0 Hz, 1H), 7.02 (dt, J = 7.5, 1.0
Hz, 1H), 6.97 (dt, J = 9.0, 2.5 Hz, 1H), 6.88 (td, J = 9.0, 2.5 Hz, 1H),
3.96 (s, 2H), 2.99−2.82 (m, 6H), 1.18 (t, J = 7.0 Hz, 3H). 13C NMR
(100 MHz, CD3OD): δ 164.05, 161.62, 141.73 (d, J = 7.5 Hz), 135.05,
130.92, 130.00, 129.91, 129.82, 129.80, 127.70, 126.86, 124.80, 124.44,
124.41, 123.27, 115.19 (d, J = 20 Hz), 112.72 (d, J = 20 Hz), 109.99,
56.82, 53.51, 47.10, 31.10, 9.67. LC-TOF (M + H+) calcd for
C22H25FN3S 382.1748, found 382.1742.
model into the disordered regions. Water molecules were added in
REFMAC and checked by COOT. The TLS38 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 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 5 with PDB accession codes included.
Enzyme Assays. The three isozymes, rat nNOS, murine
macrophage iNOS, and bovine eNOS, were recombinant enzymes,
overexpressed (in Escherichia coli) and isolated as reported.28−30 Ki
values for inhibitors 7−17 were measured for the three different
isoforms of NOS using L-arginine as a substrate. The formation of
nitric oxide was measured using the hemoglobin capture assay
described previously.31 All NOS isozymes were assayed using the
Synergy H1 hybrid multimode microplate reader (BioTek Instru-
ments, Inc.) at 37 °C in a 100 mM Hepes buffer (pH 7.4) containing
10 μM L-arginine, 0.83 mM CaCl2, 320 units/mL calmodulin, 100 μM
NADPH, 10 μM H4B, and 3.0 μM oxyhemoglobin (for iNOS assays,
no Ca2+ and calmodulin was added). The assay was initiated by the
addition of enzyme, and the initial rates of the enzymatic reactions
were determined by monitoring the formation of NO−hemoglobin
complex at 401 nm for 60 s. The apparent Ki values were obtained by
measuring the percent enzyme inhibition in the presence of 10 μM L-
arginine with at least five concentrations of inhibitor. The parameters
of the following inhibition equation were fitted to the initial velocity
data: % inhibition = 100[I]/{[I] + Ki(1 + [S]/Km)}. Km values for L-
arginine were 1.3 μM (nNOS), 8.2 μM (iNOS), and 1.7 μM (eNOS).
The selectivity of an inhibitor was defined as the ratio of the respective
Ki values.
Inhibitor Complex Crystal Preparation. The nNOS or eNOS
heme domain proteins used for crystallographic studies were produced
by limited trypsin digest from the corresponding full length enzymes
and further purified through a Superdex 200 gel filtration column (GE
Healthcare) as described previously.32,33 The nNOS heme domain at
7−9 mg/mL containing 20 mM histidine or the eNOS heme domain
at 12 mg/mL containing 2 mM imidazole were used for the sitting
drop vapor diffusion crystallization setup under the conditions
reported before.32,33 Fresh crystals (1−2 day old) were first passed
stepwise through cryoprotectant solutions as described32,33 and then
soaked with 10 mM inhibitor for 4−6 h at 4 °C before being mounted
on nylon loops and flash cooled by plunging into liquid nitrogen.
X-ray Diffraction Data Collection, Processing, and Structure
Refinement. The cryogenic (100 K) X-ray diffraction data were
collected remotely at various beamlines at Stanford Synchrotron
Radiation Lightsource through the data collection control software
Blu-Ice34 and a crystal mounting robot. Raw data frames were indexed,
integrated, and scaled using HKL2000.35 The binding of inhibitors was
detected by the initial difference Fourier maps calculated with
REFMAC.36 The inhibitor molecules were then modeled in
COOT37 and refined using REFMAC. Disordering in portions of
inhibitor bound in the NOS active site was often observed, resulting in
poor density quality. However, partial structural features usually could
still be visible if the contour level of the sigmaA weighted 2m|Fo| − D|
Fc| map dropped to 0.5 σ, which afforded building of a reasonable
ASSOCIATED CONTENT
■
S
* Supporting Information
Details of HPLC purity and copies of complete spectroscopic
data of compounds 7−17. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*For R.B.S.: phone, +1 847 491 5653; fax, +1 847 491 7713; E-
mail, Agman@chem.northwestern.edu.
*For T.L.P.: phone, +1 949 824 7020; E-mail, poulos@uci.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful for financial support from the National
Institutes of Health (GM049725 to R.B.S. and GM057353 to
T.L.P.). We thank Dr. Bettie Sue Siler Masters (NIH grant
GM52419, with whose laboratory P.M. and L.J.R. are affiliated).
B.S.S.M. also acknowledges the Welch Foundation for a Robert
A. Welch Distinguished Professorship in Chemistry (AQ0012).
P.M. is supported by grants 0021620806 and 1M0520 from
MSMT of the Czech Republic. We also thank the beamline staff
at SSRL and ALS for their assistance during the remote X-ray
diffraction data collections.
ABBREVIATIONS USED
■
NOS, nitric oxide synthase; NO, nitric oxide; NADPH, reduced
nicotinamide adenine dinucleotide phosphate; NHA, Nω-
hydroxy-L-arginine; nNOS, neuronal nitric oxide synthase;
eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric
oxide synthase; H-bond, hydrogen bond
REFERENCES
■
(1) Calabrese, V.; Mancuso, C.; Calvani, M.; Rizzarelli, E.;
Butterfield, D. A.; Stella, A. M. G. Nitric oxide in the central nervous
system: neuroprotection versus neurotoxicity. Nature Rev. Neurosci.
2007, 8, 766−775.
(2) Rosen, G. M.; Tsai, P.; Pou, S. Mechanism of free-radical
generation by nitric oxide synthase. Chem. Rev. 2002, 102, 1191−1199.
M
dx.doi.org/10.1021/jm401252e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(3) Griffith, O. W.; Stuehr, D. J. Nitric Oxide Synthases: Properties
and Catalytic Mechanism. Annu. Rev. Physiol. 1995, 57, 707−734.
(4) Granik, V. G.; Grigor’ev, N. A. Nitric oxide synthase inhibitors:
biology and chemistry. Russ. Chem. Bull. 2002, 51, 1973−1995.
(5) Alderton, W. K.; Cooper, C. E.; Knowles, R. G. Nitric oxide
synthases: structure, function and inhibition. Biochem. J. 2001, 357,
593−615.
Tetrahydroquinoline-based selective human neuronal nitric oxide
synthase (nNOS) inhibitors: lead optimization studies resulting in the
identification of N-(1-(2-(methylamino)ethyl)-1,2,3,4-tetrahydroqui-
nolin-6-yl)thiophene-2-carboximi damide as a preclinical development
candidate. J. Med. Chem. 2012, 55, 2882−2893.
(22) Annedi, S. C.; Ramnauth, J.; Cossette, M.; Maddaford, S. P.;
Dove, P.; Rakhit, S.; Andrews, J. S.; Porreca, F. Novel, druglike 1,7-
disubstituted 2,3,4,5-tetrahydro-1H-benzo[b]azepine-based selective
inhibitors of human neuronal nitric oxide synthase (nNOS). Bioorg.
Med. Chem. Lett. 2012, 22, 2510−2513.
(23) Renton, P.; Speed, J.; Maddaford, S.; Annedi, S. C.; Ramnauth,
J.; Rakhit, S.; Andrews, J. 1,5-Disubstituted indole derivatives as
selective human neuronal nitric oxide synthase inhibitors. Bioorg. Med.
Chem. Lett. 2011, 21, 5301−5304.
(24) Annedi, S. C.; Maddaford, S. P.; Mladenova, G.; Ramnauth, J.;
Rakhit, S.; Andrews, J. S.; Lee, D. K. H.; Zhang, D. Q.; Porreca, F.;
Bunton, D.; Christie, L. Discovery of N-(3-(1-methyl-1,2,3,6-
tetrahydropyridin-4-yl)-1H-indol-6-yl) thiophene-2-carboximidamide
as a selective inhibitor of human neuronal nitric oxide synthase
(nNOS) for the treatment of pain. J. Med. Chem. 2011, 54, 7408−
7416.
(25) Mladenova, G.; Annedi, S. C.; Ramnauth, J.; Maddaford, S. P.;
Rakhit, S.; Andrews, J. S.; Zhang, D. Q.; Porreca, F. First-in-class, dual-
action, 3,5-disubstituted indole derivatives having human nitric oxide
synthase (nNOS) and norepinephrine reuptake inhibitory (NERI)
activity for the treatment of neuropathic pain. J. Med. Chem. 2012, 55,
3488−3501.
(26) Ziegler, D. M.; Ansher, S. S.; Nagata, T.; Kadlubar, F. F.; Jakoby,
W. B. N-Methylation: potential mechanism for metabolic activation of
carcinogenic primary arylamines. Proc. Natl. Acad. Sci. U. S. A. 1988,
85, 2514−2517.
(27) Hodgetts, K. J.; Combs, K. J.; Elder, A. M.; Harriman, G. C. The
Role of Fluorine in the Discovery and Optimization of CNS Agents:
Modulation of Drug-Like Properties. Annu. Rep. Med. Chem. 2010, 45,
429−448.
(28) Hevel, J. M.; White, K. A.; Marletta, M. A. Purification of the
inducible murine macrophage nitric oxide synthase. Identification as a
flavoprotein. J. Biol. Chem. 1991, 266, 22789−22791.
(6) Vallance, P.; Leiper, J. Blocking NO synthesis: How, where and
why? Nature Rev. Drug Discovery 2002, 1, 939−950.
(7) Yang, Z.; Misner, B.; Ji, H.; Poulos, T. L.; Silverman, R. B.;
Meyskens, F. L.; Yang, S. Targeting Nitric Oxide Signaling with nNOS
Inhibitors As a Novel Strategy for the Therapy and Prevention of
Human Melanoma. Antioxid. Redox Signaling 2013, 19, 433−447.
(8) Villanueva, C.; Giulivi, C. Subcellular and cellular locations of
nitric oxide synthase isoforms as determinants of health and disease.
Free Radical Biol. Med. 2010, 49, 307−316.
(9) Olesen, J. Nitric oxide-related drug targets in headache.
Neurotherapeutics 2010, 7, 183−190.
(10) 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. The Novel
Binding Mode of N-Alkyl-N′-hydroxyguanidine to Neuronal Nitric
Oxide Synthase Provides Mechanistic Insights into NO Biosynthesis.
Biochemistry 2002, 41, 13868−13875.
(11) Masic, L. P. Arginine mimetic structures in biologically active
antagonists and inhibitors. Curr. Med. Chem. 2006, 13, 3627−3648.
(12) Silverman, R. B. Design of selective neuronal nitric oxide
synthase inhibitors for the prevention and treatment of neuro-
degenerative diseases. Acc. Chem. Res. 2009, 42, 439−451.
(13) Delker, S. L.; Ji, H.; Li, H..; Jamal, J.; Fang, J.; Xue, F.;
Silverman, R. B.; Poulos, T. L. Unexpected binding modes of nitric
oxide synthase inhibitors effective in the prevention of a cerebral palsy
phenotype in an animal model. J. Am. Chem. Soc. 2010, 132, 5437−
5442.
(14) Ji, H.; Li, H.; Martasek, P.; Roman, L. J.; Poulos, T. L.;
Silverman, R. B. Discovery of highly potent and selective inhibitors of
neuronal nitric oxide synthase by fragment hopping. J. Med. Chem.
2009, 52, 779−797.
(15) Ji, H.; Delker, S. L.; Li, H.; Martasek, P.; Roman, L. J.; Poulos,
T. L.; Silverman, R. B. Exploration of the active site of neuronal nitric
oxide synthase by the design and synthesis of pyrrolidinomethyl 2-
aminopyridine derivatives. J. Med. Chem. 2010, 53, 7804−7824.
(16) Huang, H.; Ji, H.; Li, H.; Jing, Q.; Jansen Labby, K.; 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,
11559−11572.
(29) Roman, L. J.; Sheta, E. A.; Martasek, P.; Gross, S. S.; Liu, Q.;
Masters, B. S. S. High-level expression of functional rat neuronal nitric
oxide synthase in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 1995,
92, 8428−8432.
(30) Martasek, P.; Liu, Q.; Liu, J. W.; Roman, L. J.; Gross, S. S.;
Sessa, W. C.; Masters, B. S. S. Characterization of Bovine Endothelial
Nitric Oxide Synthase Expressed in E. coli. Biochem. Biophys. Res.
Commun. 1996, 219, 359−365.
(17) Xue, F.; Fang, J.; Delker, S. L.; Li, H.; Martasek, P.; Roman, L. J.;
Poulos, T. L.; Silverman, R. B. Symmetric double-headed amino-
pyridines, a novel strategy for potent and membrane-permeable
inhibitors of neuronal nitric oxide synthase. J. Med. Chem. 2011, 54,
2039−2048.
(31) Hevel, J. M.; Marletta, M. A. Nitric-oxide synthase assays. In
Oxygen Radicals in Biological Systems, Part C; Methods in Enzymology;
Academic Press: San Diego, CA, 1994; Vol. 233, pp 250−258.
(32) 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. The Novel
Binding Mode of N-Alkyl-N′-hydroxyguanidine to Neuronal Nitric
Oxide Synthase Provides Mechanistic Insights into NO Biosynthesis.
Biochemistry 2002, 41, 13868−13875.
́
(18) Huang, H.; Li, H.; Martasek, P.; Roman, L. J.; Poulos, T. L.;
Silverman, R. B. Structure-guided design of selective inhibitors of
neuronal nitric oxide synthase. J. Med. Chem. 2013, 56, 3024−3032.
(19) Jansen Labby, K.; Xue, F.; Kraus, J. M.; Ji, H.; Mataka, J.; Li, H.;
Martasek, P.; Roman, L. J.; Poulos, T. L.; Silverman, R. B.
Intramolecular hydrogen bonding: a potential strategy for more
bioavailable inhibitors of neuronal nitric oxide synthase. Bioorg. Med.
Chem. 2012, 20, 2435−2443.
(20) Ramnauth, J.; Speed, J.; Maddaford, S. P.; Dove, P.; Annedi, S.
C.; Renton, P.; Rakhit, S.; Andrews, J.; Silverman, S.; Mladenova, G.;
Zinghini, S.; Nair, S.; Catalano, C.; Lee, D. K.; De Felice, M.; Porreca,
F. Design, synthesis, and biological evaluation of 3,4-dihydroquinolin-
2(1H)-one and 1,2,3,4-tetrahydroquinoline-based selective human
neuronal nitric oxide synthase (nNOS) inhibitors. J. Med. Chem.
2011, 54, 5562−5575.
(21) Ramnauth, J.; Renton, P.; Dove, P.; Annedi, S. C.; Speed, J.;
Silverman, S.; Mladenova, G.; Maddaford, S. P.; Zinghini, S.; Rakhit,
S.; Andrews, J.; Lee, D. K.; Zhang, D.; Porreca, F. 1,2,3,4-
́ ́
(33) Raman, C. S.; Li, H.; Martasek, P.; Kral, V.; Masters, B. B. S.;
Poulos, T. L. Crystal structure of constitutive endothelial nitric oxide
synthase: a paradigm for pterin function involving a novel metal
center. Cell 1998, 95, 939−950.
(34) 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. Blu-Ice and the Distributed
Control System: Software for Data Acquisition and Instrument
Control at Macromolecular Crystallography Beamlines. J. Synchrotron
Radiat. 2002, 9, 401−406.
(35) Otwinowski, Z.; Minor, W. Processing of X-ray Diffraction Data
Collected in Oscillation Mode. Methods Enzymol. 1997, 276, 307−326.
(36) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Refinement of
Macromolecular Structures by the Maximum-Likelihood Method. Acta
Crystallogr., Sect. D: Biol. Crystallogr. 1997, D53, 240−255.
N
dx.doi.org/10.1021/jm401252e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(37) Emsley, P.; Cowtan, K. Coot: model-building tools for
molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004,
D60, 2126−2132.
(38) Winn, M. D.; Isupov, M. N.; Murshudov, G. N. Use of TLS
Parameters to Model Anisotropic Displacements in Macromolecular
Refinement. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2001, D57,
122−133.
(39) Patman, J.; Bhardwaj, N.; Ramnauth, J.; Annedi, S. C.; Renton,
P.; Maddaford, S. P.; Rakhit, S.; Andrews, J. S. Novel 2-amino-
benzothiazoles as selective neuronal nitric oxide synthase inhibitors.
Bioorg. Med. Chem. Lett. 2007, 17, 2540−2544.
O
dx.doi.org/10.1021/jm401252e | J. Med. Chem. XXXX, XXX, XXX−XXX