Accessible chiral linker to enhance potency and selectivity of neuronal nitric oxide synthase inhibitors

Article abstract of DOI:10.1021/ml400381s

The three important mammalian isozymes of nitric oxide synthase (NOS) are neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Inhibitors of nNOS show promise as treatments for neurodegenerative diseases. Eight easily synthesized compounds containing either one (20a,b) or two (9a-d; 15a,b) 2-amino-4-methylpyridine groups with a chiral pyrrolidine linker were designed as selective nNOS inhibitors. Inhibitor 9c is the best of these compounds, having a potency of 9.7 nM and dual selectivity of 693 and 295 against eNOS and iNOS, respectively. Crystal structures of nNOS complexed with either 9a or 9c show a double-headed binding mode, where each 2-aminopyridine headgroup interacts with either a nNOS active site Glu residue or a heme propionate. In addition, the pyrrolidine nitrogen of 9c contributes additional hydrogen bonds to the heme propionate, resulting in a unique binding orientation. In contrast, the lack of hydrogen bonds from the pyrrolidine of 9a to the heme propionate allows the inhibitor to adopt two different binding orientations. Both 9a and 9c bind to eNOS in a single-headed mode, which is the structural basis for the isozyme selectivity.

Full text of DOI:10.1021/ml400381s

Letter
pubs.acs.org/acsmedchemlett
Accessible Chiral Linker to Enhance Potency and Selectivity of
Neuronal Nitric Oxide Synthase Inhibitors
Qing Jing,† Huiying Li,‡ Linda J. Roman,§ Pavel Martasek,§,∥ 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, Evanston, Illinois 60208-3113, United States
‡Departments of Molecular Biology and Biochemistry, Pharmaceutical Sciences, and Chemistry, University of California, Irvine,
California 92697-3900, United States
§Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78384-7760, United States
∥Department of Pediatrics, First Faculty of Medicine, Charles University, Prague, Czech Republic
S
* Supporting Information
ABSTRACT: The three important mammalian isozymes of nitric oxide synthase (NOS) are neuronal NOS (nNOS),
endothelial NOS (eNOS), and inducible NOS (iNOS). Inhibitors of nNOS show promise as treatments for neurodegenerative
diseases. Eight easily synthesized compounds containing either one (20a,b) or two (9a−d; 15a,b) 2-amino-4-methylpyridine
groups with a chiral pyrrolidine linker were designed as selective nNOS inhibitors. Inhibitor 9c is the best of these compounds,
having a potency of 9.7 nM and dual selectivity of 693 and 295 against eNOS and iNOS, respectively. Crystal structures of nNOS
complexed with either 9a or 9c show a double-headed binding mode, where each 2-aminopyridine headgroup interacts with
either a nNOS active site Glu residue or a heme propionate. In addition, the pyrrolidine nitrogen of 9c contributes additional
hydrogen bonds to the heme propionate, resulting in a unique binding orientation. In contrast, the lack of hydrogen bonds from
the pyrrolidine of 9a to the heme propionate allows the inhibitor to adopt two different binding orientations. Both 9a and 9c
bind to eNOS in a single-headed mode, which is the structural basis for the isozyme selectivity.
KEYWORDS: Nitric oxide, nitric oxide synthase, enzyme inhibition, neurodegenerative diseases
itric oxide (NO), which is synthesized from L-arginine by
the nitric oxide synthase (NOS) family of enzymes, plays
our group,14 the integration of easy synthesis, bioavailability,
and selectivity remains a challenge.
N
many fundamental physiological roles in mammals. Although all
three isoforms of NOS, neuronal NOS (nNOS), endothelial
NOS (eNOS), and inducible NOS (iNOS), produce NO as an
important second messenger molecule, overexpression of NO
by nNOS in brain is associated with many neurodegenerative
diseases, including Parkinson’s,1 Alzheimer’s,2 Huntington’s,3
migraine headaches,4 and neuronal damage from stroke.5
Inhibition of nNOS is a potential treatment for neuro-
degeneration;6−8 however, it is challenging to make a selective
inhibitor of nNOS over eNOS and iNOS in order to prevent
side effects, such as hypertension, and not to interfere with
normal immunological functions. The high similarity of NOS
isozymes, particularly the heme active site structure, is the
reason that most of the reported inhibitors provide limited
isozyme selectivity,9−13 even though those molecules bind at
the active site and afford excellent potency. While some highly
selective nNOS inhibitors have been successfully developed in
We have previously developed two lead compounds (1 and
2, Figure 1) that exhibit excellent potency against nNOS.
Despite the high selectivity of compound 1,15 the tedious
synthesis limits its structure/activity optimization to improve
bioavailability. The synthesis of 2 is much easier than that of 1,
but its selectivity needs to be improved.16 Compound 3
features advantages of synthetic ease and good activity for the
next generation of inhibitors. In this work, we design a new
class of inhibitors, using a straightforward synthesis, having
excellent inhibition properties.17 These compounds, which
exhibit unique binding features with NOS, provide a basis for
developing this integrative strategy of ease of synthesis and
Received: September 8, 2013
Accepted: November 5, 2013
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
Figure 1. Structures of lead compounds 1, 2, and 3.
a
inhibitor structure for improved inhibition potency and
isozyme selectivity. Because the inhibitors derive from chiral
natural scaffolds that are commercially available, there is no
need for chiral synthesis or resolution, which makes these new
compounds easily accessible and ready for further optimization.
Compounds 9a−d were synthesized via the route shown in
Scheme 1. Starting from the corresponding chiral proline
Scheme 2. Chemical Synthesis of 15a,b
a
Scheme 1. Chemical Synthesis of 9a−d
a
Reagents and conditions: (a) NaH, DMF, 7, 0 °C, 86−87%. (b)
LiBH4, THF, 87−91%. (c) PPh3, CBr4, THF, 81%. (d) NaH, DMF,
NaI, 0 °C, 45−50%. (e) (i) NH2OH−HCl, EtOH/H2O (2/1); (ii) 2
M HCl in CH3OH/dioxane (1/1), two steps, 70−73%.
a
Reagents and conditions: (a) (Boc)2O, CH3OH, 86−91%. (b)
a
Scheme 3. Chemical Synthesis of 20a,b
LiBH4, THF, 94−98%. (c) NaH, DMF, compound 7, 0 °C, 80−86%.
(d) (i) NH2OH−HCl, EtOH/H2O (2/1); (ii) 2 M HCl in CH3OH/
dioxane (1/1), two steps, 68−81%. Note: The synthesis of 7 follows
the literature report.18
analogues 4, the secondary amine was protected as a Boc
carbamate in methanol, and then the methyl ester was reduced
to an alcohol with LiBH4 in excellent yields. Diol 6 was allowed
to react with 7 in the presence of NaH, providing doubly
substituted 8 in one step. After deprotection of the amino
groups of the 2-aminopyridines with NH2OH−HCl and the
Boc protecting group under acidic conditions, 9 was obtained
successfully.
a
The synthesis of 15 requires two key intermediates, 11 and
12, as shown in Scheme 2. Starting from 5, ether formation
with 7 afforded 10, followed by reduction with LiBH4, gave 11.
Under similar conditions, ethylene glycol reacted with 7,
yielding 12, which was brominated with PPh3 and CBr4 in THF
to give 13. Base-promoted reaction of 11 and 13 produced 14;
deprotection gave 15.
As described in Scheme 3, compound 18, a key intermediate
to 20, was prepared from 3-fluorophenylmethanol by allylation,
oxidation to the aldehyde,19 reduction to the alcohol, and
bromination. Coupling of 18 with 11 gave 19, which was
deprotected, as described above, to generate 20.
Reagents and conditions: (a) NaH, DMF, allyl bromide, 0 °C, 84%.
(b) (i) RuCl3, NaIO4, BnEt3NCl, EtOAc; (ii) TMSCl, LiBH4, two
steps, 76%. (c) PPh3, CBr4, CH2Cl2, 82%. (d) NaH, DMF, NaI, 0 °C,
51−53%. (e) (i) NH2OH−HCl, EtOH/H2O (2/1); (ii) 2 M HCl in
CH3OH/dioxane (1/1), two steps, 74−78%.
selective (693- and 295-fold for nNOS over eNOS and iNOS,
respectively). We extended the left arm of the (2S, 4R) and
(2R, 4R) isomer to give 15a,b. However, both were less potent
and selective than 9. Replacement of one of the 2-amino-4-
methylpyridine groups with a 3-fluorophenyl moiety (20a,b)
decreased the potency further.
All inhibitors were assayed against three different isoforms of
NOS, including rat nNOS, bovine eNOS, and murine
macrophage iNOS using L-arginine as a substrate. Ki values
and selectivities are summarized in Table 1. The new
compounds contain a similar five-membered ring linker as
does 1, which derives from proline analogues. Reference
compound 3 can be regarded as having a bioisosteric open-
chain pyrrolidine. Four isomers of 9 (9a−d) exhibit excellent
potency against nNOS, but 9c is most potent (9.7 nM) and
Not surprisingly, the stereochemistry of inhibitors signifi-
cantly affects binding. To understand the structure−activity
relationship of these inhibitors, we have determined the crystal
structures of 9a and 9c complexed with rat nNOS (Figure 2).
While the 2-aminopyridine with a 3-atom ether linker to the
center pyrrolidine ring hydrogen bonds to Glu592, the 2-
aminopyridine with a 2-atom ether linker hydrogen bonds to
the heme propionate D. In order for the 2-aminopyridine group
to make two hydrogen bonds with the heme propionate D,
B
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ACS Medicinal Chemistry Letters
Letter
a
shown in Figure 2A, can coexist with the flipped one as an
alternate conformation, but because of the limited data
resolution, and for simplicity, only the flipped orientation was
modeled into subunit B.
Table 1. Ki Values of Inhibitors for nNOS, eNOS, and
iNOS
Inspired by compounds 1−3, we designed 15a,b and 20a,b,
except without a basic nitrogen atom in the linker, to match a
similar size and/or structure of 1. Unfortunately, none of these
compounds afforded improved potency or selectivity. These
results suggest that a long ether bonded linker arm in this series
of compounds is not a promising strategy.
It is interesting to compare the binding mode differences
between the best inhibitor reported here, 9c, with the lead
compounds in Figure 1. The aminopyridine in 1 makes
bifurcated H-bonds with the heme propionate D, while the
pyrrolidine nitrogen directly H-bonds to both the heme
propionate A and H4B,15 which affords the inhibitor low
nanomolar potency and more than 2500-fold selectivity for
nNOS over eNOS. Although the interactions from the
aminopyridine to the heme propionate D are preserved with
one of the two head groups in 9c, the longer 2-atom spacer
only allows the pyrrolidine nitrogen of 9c to H-bond with heme
propionate A and not with H4B (Figure 2B). The double-
headed binding mode initially observed with lead compound
216 is retained with 9c. The additional H-bonding interaction
introduced by the pyrrolidine nitrogen to the heme makes 9c a
more potent inhibitor than 2. Being able to establish both
double-headed binding, as well as a H-bond from the
pyrrolidine N atom to heme in 9c, provides an improvement
compared to 3, which has lost its H-bonds from the second
aminopyridine to the heme propionate D because of the short
2-atom linker between the chiral center and the headgroup.17
To interpret the isozyme selectivity reported in Table 1, we
also determined the crystal structures of 9a and 9c complexed
with bovine eNOS. As shown in Figure 3, for both 9a and 9c,
a
b
Ki (nM)
selectivity
e/n
inhibitors
nNOS
eNOS
iNOS
i/n
9a
26
2179
3473
6727
2356
6248
9300
7963
11726
2271
5588
2866
1902
2316
13564
5858
8472
84
87
9b
34
102
693
76
164
295
61
9c
9.7
31
9d
15a
15b
20a
20b
70
89
33
199
153
330
47
68
52
38
35
26
a
The IC50 values were measured for three different isoforms of NOS,
rat nNOS, bovine eNOS, and murine macrophage iNOS, using L-
arginine as a substrate with standard deviation of 10%. The
corresponding Ki values were calculated from the IC50 values using
the equation Ki = IC50/(1 + [S]/Km) with known Km values (nNOS,
b
1.3 μM; iNOS, 8.3 μM; eNOS, 1.7 μM). The ratio of Ki (eNOS or
iNOS) to nNOS.
Figure 2. View of the active site of nNOS in complex with inhibitor 9a
(A) and 9c (B). Shown also are the omit Fo − Fc density maps for
inhibitor contoured at the 2.5σ level. Relevant hydrogen bonds are
depicted as dashed lines. All structural figures were prepared with
PyMol (www.pymol.org).
Trp706, which normally H-bonds to the heme propionate,
must move out of the way and adopt a new rotamer
conformation. In addition, because of the different chirality,
the ring N atom from the center pyrrolidine in 9c points
toward the heme where it can H-bond (2.7−3.0 Å) with both
propionates (Figure 2B). In contrast, the pyrrolidine ring N
atom in 9a makes no hydrogen bonds with the heme (Figure
2A). This difference in the pyrrolidine ring orientation results
in better binding affinity for 9c (9.7 nM) compared to that for
9a (26 nM), which is consistent with the much better electron
density quality shown in the crystal structure of former than
that of the latter. The lack of hydrogen bonding from the
pyrrolidine ring of 9a to the heme propionates allows a
different inhibitor binding orientation in subunit B, where 9a
flips 180°. This places the 2-aminopyridine with a 2-atom ether
linker to pyrrolidine in the active site, where it H-bonds with
Glu592, as illustrated in Figure S1 (see Supporting
Information). Note that the normal binding orientation, as
Figure 3. View of the active site of eNOS in complex with inhibitor 9a
(A) and 9c (B). Shown also are the omit Fo − Fc density maps for the
inhibitors contoured at the 2.5σ level. Relevant hydrogen bonds are
depicted as dashed lines.
only a single 2-aminopyridine, the one with a 3-atom ether
linker to the pyrrolidine, makes hydrogen bonds with Glu363.
The other 2-aminopyridines cannot interact with the heme
propionate D because Tyr477 is still in position to hydrogen
bond with the same propionate. As a result, the second 2-
aminopyridine is disordered in both eNOS structures. The
disordering is more severe for 9a than for 9c, possibly because
the hydrogen bonding geometry between the pyrrolidine and
the heme propionates is much poorer in the former (Figure
3A) than that seen in the latter (Figure 3B). The exact position
of the second 2-aminopyridine in 9a is poorly defined.
On the basis of what we know about the binding features of
9a and 9c to nNOS and eNOS, we expect that compounds
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ACS Medicinal Chemistry Letters
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15a,b and 20a,b can still bind to NOS through hydrogen bonds
from the active site Glu to the 2-aminopyridine with a 2-atom
ether linker to the pyrrolidine. This would allow the pyrrolidine
ring N atom to interact with the heme propionate(s). However,
the extended ether linkers to the second 2-aminopyridine
(15a,b) or to the fluorophenyl moiety (20a,b), most likely,
result in steric clashes between the long tail of the inhibitors
and the surrounding protein, which is reflected in their poor
affinity to both nNOS and eNOS (Table 1).
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 grant 0021620849 from MSMT of the
Czech Republic. We thank the beamline staff at SSRL and ALS
for their assistance during the remote X-ray diffraction data
collections. We also thank the ChemCore from the Center for
Molecular Innovation and Drug Discovery (CMIDD), North-
western University for the help of inhibitor purity assays.
The binding of 9a and 9c to nNOS and eNOS teaches us
again the importance of inhibitor chirality and NOS active site
dynamics. We have a number of examples of double-headed
aminopyridine inhibitors that bind to nNOS in multiple
orientations but to eNOS only in a single orientation.20 The
conserved Tyr residue (Tyr706 in nNOS or Tyr477 in eNOS)
that normally hydrogen bonds to heme propionate D can adopt
a different rotamer conformation, leaving space for the
aminopyridine of the inhibitor to make hydrogen bonds to
the heme propionate. This difference between nNOS and
eNOS results from the ability of Tyr706 to more readily adopt
alternate rotamer conformations than can Tyr477 in eNOS.
This enhanced dynamics and adaptability of nNOS is no doubt
an important structural basis that we can utilize in designing
isozyme selective NOS inhibitors.
In conclusion, we have designed and synthesized double-
headed compounds with a chiral linker derived from proline,
which has substantially simplified the synthesis of these
inhibitors relative to that for 1 and increased selectivity relative
to 2. Inhibitor (2R, 4S)-9c affords excellent potency (9.7 nM)
as well as dual selectivity (693- and 295-fold for nNOS over
eNOS and iNOS, respectively). With the application of our
integrative strategy, easy accessibility and good inhibitory
properties are taken into account during inhibitor design.
Combined with our earlier work on double-headed inhibitors,
we not only developed a series of potent and selective
inhibitors but also provided an efficient methodology for the
design of small molecules and accelerated the structure
optimization process.
ABBREVIATIONS
■
NO, nitric oxide; NOS, nitric oxide synthase; nNOS, neuronal
NOS; eNOS, endothelial NOS; iNOS, inducible NOS;
(Boc)2O, di-tert-butyl dicarbonate
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ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic procedures, characterization of compounds, including
1H and 13C NMR spectra, assay details, and crystallographic
procedures. This material is available free of charge via the
Internet at http://pubs.acs.org.
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C.; Renton, P.; Rakhit, S.; Andrews, J.; Silverman, S.; Mladenova, G.;
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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.
AUTHOR INFORMATION
Corresponding Authors
*(R.B.S.) E-mail: agman@chem.northwestern.edu. Tel: +1-
847-491-5653.
*(T.L.P.) E-mail: poulos@uci.edu. Tel: +1-949-824-7020.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
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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
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