Peripheral but crucial: A hydrophobic pocket (Tyr706, Leu 337, and Met336) for potent and selective inhibition of neuronal nitric oxide synthase

Article abstract of DOI:10.1016/j.bmcl.2010.08.096

Selective inhibition of the neuronal isoform of nitric oxide synthase (nNOS) over endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) has become a promising strategy for the discovery of new therapeutic agents for neurodege

Full text of DOI:10.1016/j.bmcl.2010.08.096

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6258–6261
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Peripheral but crucial: A hydrophobic pocket (Tyr706, Leu337, and Met336
for potent and selective inhibition of neuronal nitric oxide synthase
)
Fengtian Xue a, , Huiying Li b, Jianguo Fang a, Linda J. Roman c, Pavel Martásek c,à, Thomas L. Poulos b,
,
⇑
Richard B. Silverman a,
⇑
a Departments of Chemistry and Biochemistry, Molecular Biology, and Cell Biology, Center for Molecular Innovation and Drug Discovery, and Chemistry of Life Processes
Institute, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208-3113, USA
b Departments of Molecular Biology and Biochemistry, Pharmaceutical Chemistry, and Chemistry, University of California, Irvine, CA 92697-3900, USA
c Department of Biochemistry, The University of Texas Health Science Center, San Antonio, TX 78384-7760, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 July 2010
Revised 17 August 2010
Accepted 18 August 2010
Available online 26 August 2010
Selective inhibition of the neuronal isoform of nitric oxide synthase (nNOS) over endothelial nitric oxide
synthase (eNOS) and inducible nitric oxide synthase (iNOS) has become a promising strategy for the dis-
covery of new therapeutic agents for neurodegenerative diseases. However, because of the high sequence
homology of different isozymes in the substrate binding pocket, developing inhibitors with both potency
and excellent isoform selectivity remains a challenging problem. Herein, we report the evaluation of a
recently discovered peripheral hydrophobic pocket (Tyr706, Leu337, and Met336) that opens up upon inhib-
itor binding and its potential in designing potent and selective nNOS inhibitors using three compounds,
2a, 2b, and 3. Crystal structure results show that inhibitors 2a and 3 adopted the same binding mode as
lead compound 1. We also found that hydrophobic interactions between the 4-methyl group of the ami-
Keywords:
Nitric oxide synthase
Selective inhibitor
Crystal structure
Peripheral hydrophobic pocket
Pi-stacking
nopyridine ring of these compounds with the side chain of Met336, as well as the
p–p stacking interaction
between the pyridinyl motif and the side chain of Tyr706 are important for the high potency and selectiv-
ity of these nNOS inhibitors.
Ó 2010 Elsevier Ltd. All rights reserved.
Overproduction of the small molecule nitric oxide (NO) by the
neuronal isoform of nitric oxide synthase (NOS) in the brain is clo-
sely associated with numerous neurodegenerative diseases,
including chronic pathologies such as Parkinson’s,1 Alzheimer’s,2
Huntington’s,3 headaches,4 as well as neuronal damage in stroke.5
Inhibition of nNOS, therefore, has become a compelling strategy for
the treatment of neurodegeneration.6–8 In the past two decades, a
large number of nNOS inhibitors have been reported, including
form selectivity. At present, discovery of nNOS inhibitors with high
potency and selectivity still represents a major challenge.
To overcome this limitation, a number of selective nNOS inhib-
itors have emerged from structure-based design.10–12 One of the
more important is pyrrolidine-based inhibitor 1, which exhibits
excellent potency (Ki = 7 nM) and selectivity for rat nNOS over bo-
vine eNOS (2700-fold) and murine macrophage iNOS (830-fold).13–15
Interestingly, crystal structures of nNOS and eNOS in complex with
1 revealed that this inhibitor adopts an unexpected ‘flipped’ bind-
ing mode, shown in Figure 1, in which the 2-amino-4-methylpyri-
dine motif extended beyond the edge of the heme distal site,
hydrogen bonded to the heme propionate of pyrrole ring D, and fits
derivatives of L-arginines, guanidines, isothioureas, isothioureidos,
2-iminopiperidines, amidines, aminopyridines, thioureas, thia-
zoles, imidazoles, and indazoles.9 None of these inhibitors, how-
ever, has entered clinical trials due to drawbacks with respect to
their potency and isozyme selectivity.9 These inhibitors, when
nicely into a hydrophobic pocket (Tyr706, Leu337, and Met336
formed by an alternate rotamer of the aromatic side chain of
Tyr706 13
A close look at this pocket indicates two potential benefi-
)
binding to nNOS, target the L-arginine binding pocket where nNOS
has high sequence homology with the other two isozymes, eNOS
.
and iNOS.6 This explains why most inhibitors suffer from poor iso-
cial interactions: (1) the hydrophobic interaction between the 4-
methyl group of 1 and the side chains of Leu337 and Met336, and
(2) the p–p stacking interaction between the aminopyridine motif
⇑
Corresponding authors. Tel.: +1 949 824 7020 (T.L.P.); tel.: +1 847 491 5653
(R.B.S.).
of 1 and the aromatic side chain of Tyr706. More importantly, the
specific residues around this pocket are significantly different in
the three mammalian NOS isoforms. For example, instead of the
three amino acid residues, Tyr706, Leu337, and Met336 for rat nNOS,
for human nNOS the corresponding residues are Tyr710, His341, and
E-mail addresses: poulos@uci.edu (T.L. Poulos), Agman@chem.northwestern.edu,
r-silverman@northwestern.edu (R.B. Silverman).
Present Address: Department of Chemistry, University of Louisiana at Lafayette,
PO Box 44370, Lafayette, LA 70504, USA.
à
Department of Pediatrics and Center for Applied Genomics, 1st School of
Met340, while for bovine eNOS and murine iNOS, these are Tyr477
,
Medicine, Charles University, Prague, Czech Republic.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.096

F. Xue et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6258–6261
6259
adopt the ‘flipped’ binding mode as with 2a, and thus will provide
a direct comparison between the two different binding modes.
Inhibitor 3 removes the aromaticity of the aminopyridine from
H
N
H
N
F
H2N
N
O
2a. A comparison of 3 and 2a demonstrates the role of the
p–p
stacking interaction between the pyridine ring of 1 and the side
1
chain of Tyr706
.
H
N
H
N
F
F
F
H2N
N
N
O
O
O
2a
H
N
H
N
H2N
HN
2b
H
N
H
N
N
H
3
Figure 1. Binding of lead compound 1 to the rat nNOS active site. The major
hydrogen bonds are drawn as dashed lines. The structural figures were prepared
with PyMOL (www.pymol.org).
The syntheses of key intermediates 4a and 4b are shown in
Scheme 1, using a recently reported method.13–15 The amino group
of 2-amino-4,6-dimethylpyridine (5) was protected using (Boc)2O
in the presence of triethylamine (TEA) to provide 6 in high yields.
Compound 6 was treated with two equivalents of n-BuLi, and the
resulting dianion was allowed to react with the epoxide (7)13 to
generate the desired regioisomer (8) in modest yields. Then, the
free NH group on the pyridine ring was further protected by a ben-
zyl-protecting group using benzyl bromide to yield 9 in good
yields. Next, trans alcohol 9 underwent a Mitsunobu reaction using
acetic acid as a nucleophile to produce 10 in very high yields.
Hydrolysis of 10 yielded cis-alcohol 11 in quantitative yields. Final-
ly, the racemic mixture of 11 was successfully separated using a
two step procedure. First, 11 was treated with (1S)-(À)-camphanic
Leu107, and Val106, and Tyr487, Asn115, and Met114, respectively.16
Therefore, we speculate that this hydrophobic pocket may provide
a new ‘hot spot’ for developing isoform-selective inhibitors for
NOSs.
To further investigate the chemical environment of this periph-
eral site, and also to evaluate the possibility of utilizing it for the
development of selective nNOS inhibitors, we report herein the
synthesis and evaluation of three inhibitors, 2a, 2b, and 3. Com-
pound 2a is an analog of 1, with the 4-methyl group removed from
the 2-aminopyridine ring. The difference in potency between 1 and
2a for nNOS can demonstrate the importance of the methyl group
to inhibitory activity. Compound 2b, the enantiomer of 2a, has
opposite chirality at the pyrrolidine core. Therefore, 2b cannot
Boc
N
Boc
N
b
c
d
R
Boc
N
H
N
BocHN
N
N
Bn
N
OH
OH
(+ /-)-9
(+ /-)-8
5
6
R = H
a
R = Boc
Boc
N
Boc
N
Boc
N
f
O
O
O
O
O
O
Boc
Boc
Boc
N
Bn
N
O
N
N
N
Bn
N
O
OR
Bn
12a
12b
10
11
(+ /-)-
(+ /-)-
R = Ac
R = H
e
g
g
O
Boc
N
Boc
N
N
Boc
Boc
Boc
N
Bn
N
OH
N
Bn
N
OH
7
4a
4b
Scheme 1. Synthesis of 4a and 4b. Reagents and conditions: (a) (Boc)2O, TEA, t-BuOH, 50 °C, 24 h, 90%; (b) (i) n-BuLi (2 equiv), À78 °C to rt, 30 min; (ii) 7, À78 °C to rt, 3 h,
55%; (c) NaH, 0 °C to rt, 10 min, benzyl bromide, rt, 8 h, 92%; (d) PPh3, DIAD, acetic acid, rt, 12 h, 95%; (e) NaOH (2 N)/MeOH, 50 °C, 4 h, 90%; (f) (1S)-(À)-camphanic chloride,
TEA, DMAP, rt 4 h, 49% for each diastereomer; (g) Na2CO3, H2O/MeOH, rt, 3 h, 99%.

6260
F. Xue et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6258–6261
Boc
N
Boc
N
Boc
N
a
b
O
Boc
Boc
Boc
N
N
N
Bn
N
O
N
Bn
N
O
OH
Bn
4a (R,R)
4b (S,S)
13a (R,R)
13b (S,S)
14a (R,R)
14b (S,S)
Boc
N
Boc
N
Boc
N
c
H
e
Boc
N
F
Boc
F
N
Bn
N
O
N
R
N
O
15a
15b
(R,R)
(S,S)
16a (R,R), R = Bn
16b (S,S), R = Bn
f
17a (R,R), R = H
17b (S,S), R = H
d
3 (R,R)
g
2a (R,R)
2b (S,S)
Scheme 2. Syntheses of 2a, 2b, and 3. Reagents and conditions: (a) NaH, allyl bromide, 0 °C to rt 30 min, 99%; (b) (i) O3, À78 °C; (ii) Zn, acetic acid, À78 °C to rt, 70–75%;
(c) 2-(3-fluorophenyl)ethanamine, NaHB(OAc)3, rt, 3 h, 52–55%; (d) Pd(OH)2/C, H2, 2:1 EtOH/HCl (12 N), rt, 575 psi, 40 h, 100%; (e) (Boc)2O, Et3N, MeOH, rt, 0.5 h, 99%;
(f) Pd(OH)2/C, H2, 60 °C, 24 h, 35–50%; (g) 6 N HCl/MeOH (2:1), rt, 4 h, 80–85%.
chloride in the presence of TEA and N,N-dimethylaminopyridine
(DMAP) to yield two diastereomers (12a and 12b), which were suc-
cessfully separated using silica chromatography. Then, each ester
was subjected to aqueous Na2CO3 to produce 4a and 4b as single
enantiomers in excellent yields.
Next, allylation of 4a/b gave 13a/b in excellent yields
(Scheme 2).13–15 Alkenes 13a/b were subjected to ozonolysis using
Zn as the reducing reagent to provide aldehydes (14a/b), which
underwent reductive aminations with 2-(3-fluorophenyl)ethan-
amine to provide secondary amines 15a/b. The secondary amino
group was protected with a Boc-protecting group, and then the
benzyl-protecting groups of 16a/b were removed by catalytic
hydrogenation at 60 °C to give 17a/b. Finally, the three Boc-
protecting groups were removed in a 2:1 mixture of 6 N HCl and
MeOH to generate inhibitors 2a and 2b. Inhibitor 3 can be
synthesized from 15a using high pressure catalytic hydrogenation
conditions in very high yields.
containing Tyr706, Leu337, and Met336, forming a charge–charge
interaction with the heme propionate D, as well as a stacking
p–p
interaction with the aromatic side chain of Tyr706. However, re-
moval of the 4-methyl group from the 2-aminopyridine motif dra-
matically impaired the potency (sevenfold) of the inhibitor (2a vs
1, Table 1). This result highlights the crucial role of the 4-methyl
group for retaining the high inhibitory activity of 1 for rat nNOS.
Importantly, the selectivity of 2a for rat nNOS over bovine eNOS
also dropped significantly (2.3-fold, Table 1). This was mainly the
result of the lower sensitivity of eNOS to the presence of the 4-
methyl group, only a threefold difference when comparing 2a to
1. This methyl group might make less favorable contacts with
the smaller side chain of Val106 in eNOS. Inhibitor 2b, the corre-
sponding enantiomer of 2a, adopts the normal binding mode with
its 2-aminopyridine hydrogen bonded to the side chain of Glu592
(Fig. 2B), resulting in a fourfold lower potency for rat nNOS (2b
vs 2a, Table 1). However, eNOS has no preference for the two bind-
ing modes with 2b and 2a showing comparable affinities. Inhibitor
3, with the 2-aminopyridine of 2a reduced to a cyclic amidine,
showed diminished potency for rat nNOS (3 vs 2a, Table 1). This re-
In the crystal structure of the active site of rat nNOS, 2a adopts
the same binding mode as lead compound 1 (Fig. 2A). The amino-
pyridine motif extends to the same peripheral hydrophobic pocket
Figure 2. Active site structures of rat nNOS in complex with 2a (A, PDB code 3NNY) and 2b (B, PDB code 3NNZ). Shown also the 2Fo–Fc electron density for inhibitor at 1
r
contour level. The major hydrogen bonds are drawn as dashed lines.

F. Xue et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6258–6261
6261
Table 1
inhibitory activities were tested against various NOS isoforms.
Our results highlight the importance of the hydrophobic interac-
tion between the 4-methyl group of the 2-aminopyridine motif
and the side chain of Met336 for tight binding and high isoform-
Ki values of 1, 2a, 2b, and 3 for rat nNOS, bovine eNOS and murine iNOS
Inhibitor
Kia (nM)
Selectivity
Rat nNOS
eNOS
iNOS
5800
10,000
13,200
21,800
n/e
n/i
selectivity of the inhibitors. We also show that the
p–p stacking
1
7
50
220
110
19,000
58,000
59,000
58,000
2700
1200
270
830
200
60
interaction between the aminopyridine ring and the aromatic side
chain of Tyr706 is important for good potency. The insensitivity of
eNOS to the binding mode of pyrrolidine-based inhibitors and
2a
2b
3
530
200
the weaker p–p
stacking interaction from its Tyr477 to the 2-ami-
a
The Ki values represent at least duplicate measurements with standard devia-
nopyridine ring of the inhibitors are isoform specific properties
tions of 10%.
valuable to future inhibitor design.
Acknowledgments
Table 2
Ki values of 1, 2a, 2b, and 3 for rat nNOS and human nNOS
The authors are grateful to the National Institutes of Health for
financial support to R.B.S. (GM49725), T.L.P. (GM57353), and Dr.
Bettie Sue Masters (GM52419, with whose laboratory P.M. and
L.J.R. are affiliated). B.S.S.M. is the Robert A. Welch Distinguished
Professor in Chemistry (AQ0012). P.M. is supported by Grants
0021620806 and 1M0520 from MSMT of the Czech Republic.
Inhibitor
Kia (nM)
Human nNOS
Selectivity
Rat/human
Rat nNOS
1
7
50
220
110
50
290
1800
490
7.1
5.8
8.2
4.5
2a
2b
3
a
The Ki values represent at least duplicate measurements with standard devia-
Supplementary data
tions of 10%.
Supplementary data associated with this article can be found in
the online version at doi:10.1016/j.bmcl.2010.08.096.
sult indicates that the p–p stacking interaction between the pyri-
dine ring and Tyr706 is an important factor for tight binding of 2a
to nNOS. The stacking interaction provides less contribution to
the binding affinity of 2a to eNOS, as its Ki values are similar for
both 3 and 2a. This is probably because the Tyr477 side chain in
eNOS does not interact as closely with the 2-aminopyridine ring
of the inhibitors, as demonstrated previously in crystal structures
for other pyrrolidine inhibitors complexed to eNOS and nNOS.13
We also tested the inhibitory activity of 1, 2a, 2b, and 3 against
the human isoform of nNOS (Table 2). Human nNOS shows very
high sequence homology to rat nNOS in the active site16; the only
References and notes
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different residue in the peripheral site is His341 in place of Leu337
,
which makes the hydrophobic pocket in human nNOS smaller than
that in rat nNOS. As a result, the hydrophobic pocket of human
nNOS should prefer smaller fragments. Even though inhibitors 1,
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In summary, we demonstrate the feasibility and potential of
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as a ‘hot spot’ for designing potent and selective nNOS inhibitors.
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