Potent, highly selective, and orally bioavailable gem-difluorinated monocationic inhibitors of neuronal nitric oxide synthase

Article abstract of DOI:10.1021/ja106175q

In our efforts to discover neuronal isoform selective nitric oxide synthase (NOS) inhibitors, we have developed a series of compounds containing a pyrrolidine ring with two stereogenic centers. The enantiomerically pure compounds, (S,S) versus (R,R), exhibited two different binding orientations, with (R,R) inhibitors showing much better potency and selectivity. To improve the bioavailability of these inhibitors, we have introduced a CF₂ moiety geminal to an amino group in the long tail of one of these inhibitors, which reduced its basicity, resulting in compounds with monocationic character under physiological pH conditions. Biological evaluations have led to a nNOS inhibitor with a K_i of 36 nM and high selectivity for nNOS over eNOS (3800-fold) and iNOS (1400-fold). MM-PBSA calculations indicated that the low pK_a NH is, at least, partially protonated when bound to the active site. A comparison of rat oral bioavailability of the difluorinated compound to the parent molecule shows 22% for the difluorinated compound versus essentially no oral bioavailability for the parent compound. This indicates that the goal of this research to make compounds with only one protonated nitrogen atom at physiological pH to allow for membrane permeability, but which can become protonated when bound to NOS, has been accomplished.

Full text of DOI:10.1021/ja106175q

Published on Web 09/15/2010
Potent, Highly Selective, and Orally Bioavailable
Gem-Difluorinated Monocationic Inhibitors of Neuronal Nitric
Oxide Synthase
Fengtian Xue,†,⊥ Huiying Li,‡ Silvia L. Delker,‡ Jianguo Fang,† Pavel Marta´sek,§,|
Linda J. Roman,§ Thomas L. Poulos,*,‡ and Richard B. Silverman*,†
Departments of Chemistry and Biochemistry, Molecular Biology, and Cell Biology, Center for
Molecular InnoVation and Drug DiscoVery, Chemistry of Life Processes Institute, Northwestern
UniVersity, 2145 Sheridan Road, EVanston, Illinois 60208-3113, Departments of Molecular
Biology and Biochemistry, Pharmaceutical Chemistry, and Chemistry, UniVersity of California,
IrVine, California 92697-3900, Department of Biochemistry, UniVersity of Texas Health Science
Center, San Antonio, Texas 78229, and Department of Pediatrics and Center for Applied
Genomics, First School of Medicine, Charles UniVersity, Prague, Czech Republic
Received July 12, 2010; E-mail: Agman@chem.northwestern.edu; poulos@uci.edu
Abstract: In our efforts to discover neuronal isoform selective nitric oxide synthase (NOS) inhibitors, we
have developed a series of compounds containing a pyrrolidine ring with two stereogenic centers. The
enantiomerically pure compounds, (S,S) versus (R,R), exhibited two different binding orientations, with
(R,R) inhibitors showing much better potency and selectivity. To improve the bioavailability of these inhibitors,
we have introduced a CF2 moiety geminal to an amino group in the long tail of one of these inhibitors,
which reduced its basicity, resulting in compounds with monocationic character under physiological pH
conditions. Biological evaluations have led to a nNOS inhibitor with a Ki of 36 nM and high selectivity for
nNOS over eNOS (3800-fold) and iNOS (1400-fold). MM-PBSA calculations indicated that the low pKa NH
is, at least, partially protonated when bound to the active site. A comparison of rat oral bioavailability of the
difluorinated compound to the parent molecule shows 22% for the difluorinated compound versus essentially
no oral bioavailability for the parent compound. This indicates that the goal of this research to make
compounds with only one protonated nitrogen atom at physiological pH to allow for membrane permeability,
but which can become protonated when bound to NOS, has been accomplished.
mitter.6,7 Although normal activity of nNOS is important for
neurotransmission, NO overproduction by nNOS has been
Introduction
Since its first discovery as the endothelium-derived relaxing
factor (EDRF) in 1987,1 nitric oxide (NO) has emerged as an
important biological messenger involved in a wide variety of
physiological functions as well as pathophysiological states. In
living organisms, NO is produced by nitric oxide synthase
(NOS), a homodimeric flavohemoprotein, through the oxidation
ofL-argininetoL-citrullinewithNADPHandO2 ascosubstrates.2-4
shown to be associated with chronic neurodegenerative patholo-
gies such as Parkinson’s,8 Alzheimer’s,9 and Huntington’s
diseases,10 also with chronic headaches,11 as well as neuronal
damage in stroke.12 iNOS, the isozyme of NOS that produces
cytotoxic NO, is responsible for maintaining normal function
of the immune system.13 eNOS, on the other hand, regulates
blood pressure. It has been shown experimentally that the
reduced NO production by eNOS causes hypertension and
atherosclerosis.14 Therefore, selective inhibition of nNOS activ-
To date, three distinct isoforms of mammalian NOS, namely,
neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial
NOS (eNOS), have been identified with high homology
(>55%).5 nNOS catalyzes the oxidation of L-arginine in the
central nervous system, generating NO as a critical neurotrans-
(6) Hall, A. V.; Antoniou, H.; Wang, Y.; Cheung, A. H.; Arbus, A. M.;
Olson, S. L.; Lu, W. C.; Kau, C. L.; Marsden, P. A. J. Biol. Chem.
1994, 269, 33082.
(7) Wang, Y.; Newton, D. C.; Marsden, P. A. Crit. ReV. Neurobiol. 1999,
13, 21.
† Northwestern University.
(8) Zhang, L.; Dawson, V. L.; Dawson, T. M. Pharmacol. Ther. 2006,
109, 33.
⊥ Present address: Department of Chemistry, University of Louisiana at
Lafayette, P.O. Box 44370, Lafayette, Louisiana 70504, USA.
‡ University of California.
(9) Dorheim, M.-A.; Tracey, W. R.; Pollock, J. S.; Grammas, P. Biochem.
Biophys. Res. Commun. 1994, 205, 659.
§ University of Texas Health Science Center.
| Charles University.
(10) Norris, P. J.; Waldvogel, H. J.; Faull, R. L. M.; Love, D. R.; Emson,
P. C. Neuronsicence 1996, 72, 1037.
(1) Palmer, R. M. J.; Ferrige, A. G.; Moncada, S. Nature 1987, 327, 524.
(2) Marletta, M. A. J. Biol. Chem. 1993, 268, 12231.
(3) Marletta, M. A. Cell 1994, 78, 927.
(11) Ashina, M. Exp. Opin. Pharmacother. 2002, 3, 395.
(12) Sims, N. R.; Anderson, M. F. Neurochem. Int. 2002, 40, 511.
(13) Hobbs, A. J.; Higgs, A.; Moncada, S. Annu. ReV. Pharmacol. 1999,
39, 191.
(4) Griffith, O. W.; Stuehr, D. J. Annu. ReV. Physiol. 1995, 57, 707.
(5) Alderton, W. K.; Cooper, C. E.; Knowles, R. G. Biochem. J. 2001,
357, 593.
(14) Dawson, V. L.; Dawson, T. M.; London, E. D.; Bredt, D. S.; Snyner,
S. H. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 6368.
9
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A R T I C L E S
Xue et al.
derived from inhibitor 2, in an attempt to further improve the
pharmacodynamic and pharmacokinetic properties of these
inhibitors.
Results and Discussion
Chemistry. An improved synthesis of enantiomerically pure
pyrrolidine core (4a,b) is shown in Scheme 1. First, racemic
trans-alcohol 5 underwent a Mitsunobu reaction with (S)-(-)-
camphanic acid as the nucleophile to produce two separable
diastereomers (6a and 6b) in excellent yields. Next, the ester
in 6a and 6b was hydrolyzed in aqueous Na2CO3 to generate
4a and 4b in high yields.
Figure 1. Chemical structures of 1 and 2.
As shown in Scheme 2, single enantiomer 4a or 4b was
treated with NaH, and the resulting anion was allowed to react
with allyl bromide to generate 7a and 7b in excellent yields.
Ozonolysis of 7a and 7b using Zn dust as the reducing reagent
yielded 8a and 8b in good yields. Aldehydes 8a and 8b were
subjected to reductive amination reactions with different etha-
namines in the presence of NaHB(OAc)3 to generate secondary
amines, which were further protected by another Boc-protecting
group to produce fully protected inhibitors 9a-d in good yields.
Next, the benzyl-protecting group was removed by catalytic
hydrogenation using Pd(OH)2 at 60 °C to provide 10a-d in
modest yields. Finally, the three Boc-protecting groups were
removed at the same time in HCl to generate the final inhibitors
(3a-d) in high yields.
The synthesis of inhibitor 3e began with 9d (Scheme 3).
Catalytic hydrogenation of 9d using Pd(OH)2 at elevated
temperature removed the benzyl-protecting group; when run for
48 h, the pyridinyl ring also was reduced to generate 10e in
modest yields. Then, the three Boc-protecting groups were
removed in HCl to generate final inhibitor 3e in high yields.
The synthesis of inhibitor 3f is shown in Scheme 4. Reductive
amination between aldehyde 8b and 2,2-difluoro-2-(pyridin-2-
yl)ethanamine generated a secondary amine, which was further
protected by another Boc-protecting group to provide 9f in good
yields. Next, catalytic hydrogenation of 9f removed the benzyl
protecting group and also reduced the pyridinyl group adjacent
to the CF2 group to generate 10f in modest yields. Finally, the
three Boc-protecting groups were removed at the same time in
HCl to generate inhibitor 3f in high yields.
Crystal Structures of nNOS with 3a-3f Bound. Consistent
with the binding preference of enantiomerically pure parental
compound 1,23 the binding mode of these difluorinated deriva-
tives is also dependent on the configuration around the two chiral
centers, the 3′ and 4′ positions, of the pyrrolidine ring. While
(S,S) inhibitor 3a was found to bind with its aminopyridine
moiety hydrogen bonded to the side chain of Glu592 in nNOS
(Figure 2A), (R,R) inhibitor 3b adopted a 180° flipped binding
mode with its aminopyridine making bifurcated hydrogen bonds
with the heme propionate of pyrrole ring D (Figure 2B). To
make room for these new hydrogen bonds, the Tyr706 side chain
rotates away and π-stacks against the aminopyridine ring of
the inhibitor. The pyrrolidine nitrogen in 3b also makes
favorable hydrogen bonds with propionate A as well as the O4
atom of H4B. In comparison, the pyrrolidine nitrogen in 3a is
only loosely hydrogen bonded to Glu592. The long, flexible
linker extending from the pyrrolidine allows the fluorophenyl
group in 3b to reach the vicinity of Glu592 and to π-stack with
the heme. The amino group vicinal to the CF2 moiety points
ity over its closely related isoforms, eNOS and iNOS, represents
an exciting drug approach for the development of new thera-
peutic agents to treat neurodegenerative diseases.5,15,16
In our continuing efforts to develop nNOS selective inhibitors,
we have discovered a pyrrolidine-based compound (1, Figure
1), which showed great potency (Ki ) 15 nM) and excellent
selectivity for nNOS over eNOS (2100-fold) and iNOS (630-
fold).17 However, further application of this compound as a drug
candidate for neurodegenerative diseases is limited by its poor
bioavailability.18 The two positive charges of 1 at physiological
pH, derived from the two basic amino (NH) groups, dramatically
impair the ability of 1 to penetrate the blood brain barrier (BBB)
by passive diffusion.18 In addition, the benzylic position of the
m-fluorophenylethyl substituent is susceptible to metabolic
oxidation.19
To circumvent these problems, different strategies have been
employed to improve the bioavailability of inhibitors by limiting
the number of basic functional groups in 1.18,20,21 For example,
by incorporating an electron-withdrawing group adjacent to the
amino group in the hydrophobic tail, a series of low pKa
inhibitors were synthesized with monocationic or pseudo-
monocationic characters at physiological pH.20 Evaluation of
these inhibitors led to the discovery of inhibitor 2, with two
fluorines substituted at the benzylic position of the m-fluorophe-
nylethyl tail of 1. The racemic mixture of 2 indicated good
potency (Ki ) 80 nM) and high selectivity for nNOS over both
eNOS (1500-fold) and iNOS (650-fold). More importantly, it
showed improved cell-membrane permeability compared to the
parent compound 1.20 The CF2 group is widely used in drug
development because of its unique pharmacological properties.22
For inhibitor 2, the additional CF2 group not only decreases
the basicity of the adjacent amino group dramatically (pKa ≈
5.5), but also effectively blocks the potential metabolic oxidation
at the benzylic position of the m-fluorophenyl ring.19 We report
here the structure-based design, synthesis, and biological
evaluation of a series of enantiomerically pure inhibitors (3a-f)
(15) Southan, G. J.; Szabo, C. Biochem. Pharmacol. 1996, 51, 383.
(16) Babu, B. R.; Griffith, O. W. Curr. Opin. Chem. Biol. 1998, 2, 491.
(17) Ji, H.; Li, H.; Marta´sek, P.; Roman, L. J.; Poulos, T. L.; Silverman,
R. B. J. Med. Chem. 2009, 52 (3), 779.
(18) Lawton, G. R.; Ranaivo, H. R.; Wing, L. K.; Ji, H.; Xue, F.; Marta´sek,
P.; Roman, L. J.; Watterson, D. M.; Silverman, R. B. Bioorg. Med.
Chem. 2009, 17, 2371.
(19) Silverman, R. B. The Organic Chemistry of Drug Design and Drug
Action, 2nd ed.: Elsevier: Boston, MA, 2004.
(20) Xue, F.; Fang, J.; Lewis, W. W.; Marta´sek, P.; Roman, L. J.; Silverman,
R. B. Bioorg. Med. Chem. Lett. 2010, 20, 554.
(21) Xue, F.; Huang, J.; Ji, H.; Fang, J.; Li, H.; Marta´sek, P.; Roman, L. J.;
Poulos, T. P.; Silverman, R. B. Bioorg. Med. Chem. 2010, 18, 6526.
(22) Smart, B. E. J. Fluorine Chem. 2001, 109, 3.
(23) Delker, D. L.; Ji, H.; Li, H.; Jamal, J.; Fang, J.; Xue, X.; Silverman,
R. B.; Poulos, T. L. J. Am. Chem. Soc. 2010, 132, 5437.
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Monocationic Inhibitors of Neuronal NOS
A R T I C L E S
Scheme 1. Synthesis of 4a,ba
a Reagents and conditions: (a) (S)-(-)-camphanic acid, PPh3, DIAD, rt, 16 h, 95%; (d) Na2CO3, rt, 4 h, 95%.
Scheme 2. Synthesis of 3a-da
a Reagents and conditions: (a) (i) NaH, (ii) allyl bromide, rt, 1 h, 96%; (b) O3, -78 °C, (ii) Zn, -78 °C to rt, 2 h, 81%; (c) (i) ethanamines, THF, rt, 5
min, (ii) NaHB(OAc)3, rt, 3 h; (d) (Boc)2O, Et3N, MeOH, rt, 3 h, 48-60% for two steps; (e) Pd(OH)2/C, H2, EtOH, 60 °C, 30 h, 45-60%; (f) 6 N HCl/
MeOH (2:1), rt, 16 h, 95-100%.
Scheme 3. Synthesis of 3ea
a Reagents and conditions: (a) Pd(OH)2/C, H2, EtOH, 60 °C, 48 h, 55%; (d) 6 N HCl/MeOH (2:1), rt, 16 h, 96%.
toward the Glu592 side chain, resulting in an alternate confor-
mation of the carboxylate enabling a hydrogen bond to form
between the amino nitrogen and the carboxylate oxygen (Figure
2B). This alternate conformation of Glu592 was not observed
in the structure of nNOS bound with 3a because the tight
bifurcated hydrogen bonds from the inhibitor aminopyridine to
the carboxylate of Glu592 made an excellent match to the
original conformation of the Glu side chain. The fit of the
fluorophenyl group in 3b near Glu592 is not ideal, and to avoid
close van der Waals contacts with the inhibitor, Glu592 adopts
the alternate conformation. In addition, the fluorophenyl tail of
3b is disordered, as indicated by the weak electron density for
this group. When only one conformation of the tail was modeled
with the two fluorine atoms pointing away from the heme plane,
strong negative difference density clustered around the fluorine
atoms. Also, the electron density of the fluorophenyl ring could
not be accounted for with only one ring orientation. The tail
portion of 3b was, therefore, modeled with two different
conformations (0.6 and 0.4 occupancy) as shown in Figure 2B.
There also is a partially occupied site for a water molecule
bridging between the heme propionate and the amino group in
the tail portion of 3b in the minor conformation. In contrast,
the fluorophenyl ring in 3a fits into a pocket formed by Met336,
Leu337, and Tyr706 (Figure 2A), but the density for 3a is clear
only up to the position of the two fluorine atoms, thus,
precluding determination of the fluorophenyl ring orientation.
Inhibitor 3c has its fluorine position in the phenyl ring
changed from meta in 3b to para in 3c. Because the C-F bond
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Scheme 4. Synthesis of 3fa
a Reagents and conditions: (a) (i) 2,2-difluoro-2-(pyridin-2-yl)ethanamine, THF, rt, 5 min, (ii) NaHB(OAc)3, rt, 3 h; (b) (Boc)2O, Et3N, MeOH, rt, 3 h,
55% for two steps; (c) Pd(OH)2/C, H2, EtOH, 60 °C, 30 h, 60%; (d) 2 N HCl/MeOH (1:1), rt, 16 h, 100%.
Figure 2. The nNOS active site with inhibitor 3a (A) or 3b (B) bound. The sigmaA weighted 2Fo - Fc density for inhibitor is also shown at contour level
of 1 σ. Hydrogen bonds are depicted with dashed lines. The atomic color schemes are: N, blue; O, red; S, yellow; F, light cyan. Alternate conformations for
3b (yellow and green) and E592 were observed. Figures 2-4 were prepared with PyMol (http://www.pymol.org).
is 1.34 Å, 3c is at least 1.3 Å longer than 3b. As a result, the
para-F in 3c makes a H-bond directly to the protein backbone
amide (Gly586), while the meta-F of 3b makes only a weak
van der Waals contact with the protein (Figure 3A). Because
of the tighter contact of 3c, the CF2 moiety is forced into a
downward conformation. The major conformation in 3b,
however, has the CF2 moiety pointing upward. The downward
conformation causes clashes between the CF2 and heme pro-
pionate A, so the upward CF2 conformation of 3b represents a
more relaxed fit in the active site, leading to higher potency.
Inhibitor 3d is a derivative of 3b and differs only by the
absence of the fluorine on the phenyl ring. Its binding mode to
nNOS is, therefore, almost identical to that of 3b (Figure 3B).
Without the meta-fluorine, the phenyl ring makes a looser
contact with the hydrophobic pocket defined by Pro565, Val567,
and Phe584. The phenyl tail portion in 3d exhibits two alternate
conformations similar to what is observed for 3b.
Inhibitor 3e has its aminopyridine ring partially reduced from
that in 3d, which has a negligible impact on the binding mode
of the inhibitor compared to 3d (Figure 3C). The amino and
the ring nitrogens remain planar and are still tightly hydrogen
bonded to the heme propionate of pyrrole D, and the pyrrolidine
nitrogen is hydrogen bonded to propionate A and H4B. As in
3b and 3d, the phenyl tail in 3e shows two conformations above
the heme plane, and the Glu592 side chain also has two
corresponding conformations.
into the pocket of Met336, Leu337, and Tyr706 (Figure 3D).
The density for the tail portion is good only up to the CF2 moiety
with the piperidine partially disordered. Therefore, the orienta-
tion of the ring (position of nitrogen) is not well-defined.
Crystal Structures of eNOS with 3b and 3f Bound. The eNOS
structure bound with (R,R) inhibitor 3b (Figure 4A) shows that
it has adopted a ‘flipped’ binding mode, the same orientation
as in nNOS. The Tyr477 side chain rotates farther out than in
nNOS and, therefore, does not experience optimized π-stacking
interactions with the aminopyridine of the inhibitor, as was
observed in nNOS. Similar to what was seen in nNOS, the
aminopyridine in 3b makes bifurcated hydrogen bonds with the
heme propionate of pyrrole ring D, while the pyrrolidine
nitrogen makes favorable hydrogen bonds with propionate A
as well as the O4 atom of H4B (Figure 4A). A hydrogen bond
between the inhibitor amino group with alternate conformations
for Glu363 also is observed. With the available density at the
limited resolution, only one conformation of the fluorophenyl
tail portion can be modeled.
The binding of the (S,S) inhibitor 3f in eNOS is similar to
that in nNOS, with its aminopyridine hydrogen bonded to the
Glu363 side chain (Figure 4B) in the normal binding mode.
The pyrrolidine nitrogen also hydrogen bonds with the conserved
glutamate residue, but the density for the tail portion is good
only up to the CF2 moiety with the piperidine partially
disordered. Therefore, the orientation of the ring (position of
nitrogen) and the exact configuration of the puckered ring are
not clear.
Another (S,S) inhibitor, 3f, is similar to 3a except the
fluorophenyl tail has been replaced by a piperidine ring. The
binding of 3f is similar to 3a with its aminopyridine hydrogen
bonded to the Glu592 side chain while the piperidine ring fits
Inhibitory Assays and Structure-Based Evaluation. Inhibitors
3a-f were evaluated for in Vitro inhibition activities against
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Monocationic Inhibitors of Neuronal NOS
A R T I C L E S
Figure 3. The nNOS active site with 3c (A), 3d (B), 3e (C), or 3f (D) bound. Around each inhibitor is the sigmaA weighted 2Fo - Fc density contoured
at 1 σ. Major hydrogen bonds are depicted with dashed lines. The same atomic color schemes as in Figure 1 are used. Note the alternate conformations for
E592 occur when the inhibitor shows multiple conformations in the nNOS-3d or nNOS-3e structure.
Figure 4. The eNOS active site with 3b (A) and 3f (B) bound. Around each inhibitor is the sigmaA weighted 2Fo - Fc density contoured at 1 σ. Major
hydrogen bonds are depicted with dashed lines. The same atomic color schemes as that in Figure 2 are used. As observed with nNOS, alternate conformations
for Glu363 occur in the eNOS-3b structure.
three isozymes of NOSs including rat nNOS, bovine eNOS,
and murine iNOS,24 as summarized in Table 1. Compared to
racemic lead compound 2, (S,S) enantiomer 3a is a weak
inhibitor of nNOS with a Ki value of 390 nM, which is 5-fold
less potent than 2. In addition, the selectivity of this inhibitor
for nNOS over eNOS and iNOS also decreases by 5-fold and
2-fold, respectively. The (R,R) enantiomer (3b), however, shows
excellent potency for nNOS (Ki ) 36 nM) and remarkable
selectivity over eNOS (3800-fold) and iNOS (1400-fold). These
results indicate that the chirality around the cis-chiral pyrrolidine
core plays a key role in determining the potency and selectivity
of inhibitors, as we have observed for another series of trans-
or cis-chiral pyrrolidine inhibitors.23 The potency and selectivity
shown with racemic compound 2 can be attributed mainly to
(R,R)-component 3b. We now know that a large difference in
binding affinity to nNOS between 3a and 3b originates from
two different binding modes.23 The flipped binding mode of
3b relative to 3a allows both the aminopyridine and pyrrolidine
(24) Hevel, J. M.; Marletta, M. A. Methods Enzymol. 1994, 233, 250.
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Table 1. Kia Values of Inhibitors for Rat nNOS, Bovine eNOS, and
Murine iNOS
selectivityb
nNOS
(µM)
eNOS
(µM)
iNOS
(µM)
compound
n/e
n/i
2
0.080
0.390
0.036
0.160
0.085
0.170
2.70
120
110
140
31
130
130
64
52
130
51
190
85
1500
280
3800
190
1500
770
24
650
330
1400
1200
1000
150
3a
3b
3c
3d
3e
3f
26
450
170
a The Ki values were calculated based on the directly measured IC50
values, which represent at least duplicate measurements with standard
deviations of (10%. b The ratio of Ki (eNOS or iNOS) to Ki (nNOS).
Figure 5. Plot of the computed free energy (PB) vs experimental free
energy (∆Gexp) obtained from Ki values.
nNOS than does the polar piperidinyl ring in 3f, and the
π-stacking with the heme is lost in the case of 3f. However,
both 3a and 3f bind to eNOS with similar potency, possibly
because of the smaller Val106 side chain in the pocket in eNOS
compared to Met336 in nNOS.
nitrogen atoms to make extensive hydrogen bonds with the heme
and H4B (Figure 2B). In addition, as we have argued else-
where,23 the conformation of 3a when bound to NOS places
the pyrrolidine nitrogen atom very close to the aminopyridine,
and because of electrostatic repulsion, this aminopyridine is only
partially protonated. However, in the 3b flipped orientation, the
aminopyridine is farther from the pyrrolidine nitrogen atom and
remains fully protonated. Thus, the 3b conformation provides
greater electrostatic stabilization than the 3a conformation,
which accounts for the 10-fold lower Ki for 3b than 3a.
As a p-fluorophenyl derivative of 3b, inhibitor 3c shows a
significant drop in potency for nNOS (Ki ) 160 nM), which is
4.5-fold less potent than 3b. More interestingly, 3c loses 20-
fold of selectivity over eNOS observed with 3b by moving one
single F atom from the m-position to the p-position of the phenyl
tail. The new fluorine position leads to a ‘difluoromethylene-
down’ binding mode of the inhibitor’s phenyl tail. As a result,
the hydrogen bond between the Glu592 side chain and the amino
nitrogen in the tail seen in 3b has been eliminated, which may
explain the weaker potency and selectivity of 3c.
Inhibitor 3d, with the F atom removed from the phenyl tail,
has restored good potency for nNOS (Ki ) 85 nM) and high
isozyme-selectivity (1500-fold over eNOS and 1000-fold over
iNOS). This is because 3d has retained the binding mode of
3b. The only difference is that without a fluorine atom on the
phenyl ring the van der Waals contacts to the protein (Pro565,
Val567, and Phe584) are less optimal compared to that for 3b.
This results in a bit less potency and selectivity for 3d than 3b.
The amidino inhibitor 3e, with the aromatic system of the
aminopyridine fragment partially reduced, exhibits only a 2-fold
drop in potency against nNOS compared to 3d. Although π-π
stacking of 3e with Tyr706 should be greatly diminished relative
to that with 3d, the increased basicity of the amidine of 3e over
the aminopyridine of 3d may compensate by producing a tighter
electrostatic interaction with the heme propionate, resulting in
only a factor of 2 difference for the Ki values of the these
inhibitors. The removal of the aromatic system of the aminopy-
ridine ring from inhibitor 3d allows 3e to bind more than 3-fold
better to iNOS, thus, exhibiting a much lower selectivity.
Finally, inhibitor 3f, with an (S,S) configuration of the
pyrrolidine core and a piperidinyl tail, showed poor inhibition
activity and isozyme-selectivity. Both 3a and 3f have similar
binding orientations and rather poor potency among the inhibi-
tors reported in this work. This result emphasizes again that
the chirality of the pyrrolidine core is the key to higher inhibitory
activity with (R,R) inhibitors. In addition, a less polar aromatic
ring such as the fluorophenyl group in 3a seems to fit better
into the pocket surrounded by Met336, Leu337, and Tyr706 in
The isoform selection shown for this series of inhibitors in
Table 1 is not that straightforward to interpret based only on
the structures presented here. For any one particular inhibitor
among those reported in Table 1, the binding mode in eNOS is
no different from that seen in nNOS. Similar to what we have
discussed for another series of chiral pyrrolidine containing
inhibitors,23 one of the potential reasons for the isoform
selectivity shown by 3b is the difference in π-stacking interac-
tion between the aminopyridine and Tyr706 in nNOS (or Tyr
477 in eNOS). We have consistently observed tighter interac-
tions between this Tyr and the aminopyridine with nNOS
complexed with inhibitors that adopt the 3b flipped orientation.23
Calculations To Determine the Protonation State of Bound
Inhibitors. The principal aim of this research was to lower the
pKa of one of the NH groups in the inhibitors, so that, at
physiological pH, there would be effectively only one positive
charge on the molecules, which should be acceptable for CNS
penetration.25 However, on the basis of past results, two positive
charges are required for potency and isozyme selectivity. The
aim was to find the appropriate pKa so that, once bound to NOS,
the low pKa NH would become protonated and could undergo
the appropriate electrostatic interaction with a carboxylate.
In our initial work on flipped inhibitors,23 we employed the
Molecular Mechanics Poisson-Boltzmann/Surface Area (MM-
PBSA)26 computational method to calculate the free energy of
binding of a series of aminopyridine inhibitors to nNOS and
eNOS. We employed the empirical equation derived from Figure
5, ∆Gcalc ) (PB - 5.2874)/6.029, to obtain computed free
energies. In our earlier work23 we found that the best fits with
∆Gexp resulted when the orientation of the inhibitor with the
aminopyridine stacked over the heme (normal orientation) was
assumed to be 50% protonated, while in the flipped orientation
in which the aminopyridine interacts with the heme propionate,
a +1 charge was assigned to the aminopyridine. The way partial
charges were handled was to carry out the MM-PBSA calcula-
tions with a 0 or +1 charge and average the results. We carried
out the same types of calculations with the difluorinated
inhibitors used in this study, but the goal was to probe the state
of protonation of the NH group whose pKa has been lowered to
≈5.5 as a result of the fluorine atoms. The results of these
calculations are shown in Table 2. With inhibitor 3b, additional
(25) Kerns, E. H.; Di, L. Drug-like Properties: Concepts, Structure Design
and Methods; Academic Press: Amsterdam, 2008; p 130.
(26) Massova, I.; Kollman, P. A. J. Am. Chem. Soc. 1999, 8133.
9
14234 J. AM. CHEM. SOC. VOL. 132, NO. 40, 2010

Monocationic Inhibitors of Neuronal NOS
A R T I C L E S
Table 2. Calculated Free Energies for Three Inhibitors Used in This Study Assuming Different Chargesa
inhibitor
charge on pyrrolidine
charge on NH
charge on amino pyridine
PB
∆Gcalc
∆Gexp
∆∆G
3a
3a
3a
1
1
1
1
1
0
1
0
0
-77.3
-47.66
-18.75
-13.70
-8.78
-3.99
-8.82
-4.86
-10.15
-7.50
-11.83
-14.59
-8.34
-6.89
-10.41
-8.8
-8.8
4.90
0.018
4.81
0.022
3.06
2.23
0.42
1.63
4.39
1.86
3.31
0.21
-8.8
average
1
1
average
1
1
1
-8.8
3c
3c
0
1
1
1
-24.02
-55.9
-7.92
-7.92
-7.92
-10.2
-10.2
-10.2
-10.2
-10.2
3b A
3b B
3b A
3b B
1
1
0
0
1
1
1
1
-66.03
-82.7
-45.02
-36.25
1
average
a For 3b, two different conformations of the inhibitor were observed in the crystal structure referred to here as conformations A and B.
calculations were carried out because we observed two orienta-
tions in the nNOS active site. The best fit to ∆Gexp results
from assigning a full +1 charge to the aminopyridine in the
“flipped” orientation and a +0.5 charge in the “normal”
orientation and a +0.5 charge for the NH group in all the
inhibitors independent of orientation. Our strategy for lowering
the pKa of the NH group was to decrease the total charge of the
inhibitors in solution for better bioavailability but with the hope
that they would become at least partially protonated once bound
in the active site, where the nearby heme propionates and
Glu592 might promote protonation. The calculations support
this scenario and indicate that the NH group is partially
protonated. This is reasonable because the NH group in these
inhibitors, no matter which binding orientation, always H-bonds
with one carboxylate and is within 4 Å from another carboxylate
(either the Glu592 side chain or the heme propionate).
with therapeutic potential in the treatment of diseases caused
by unregulated NO generation from nNOS.
Experimental Section
1. General Methods. All experiments were conducted under
anhydrous conditions in an atmosphere of argon, using flame-dried
apparatus and employing standard techniques in handling air-
sensitive materials. All solvents were distilled and stored under an
argon or nitrogen atmosphere before use. All reagents were used
as received. Aqueous solutions of sodium bicarbonate, sodium
chloride (brine), and ammonium chloride were saturated. Analytical
thin layer chromatography was visualized by ultraviolet, ninhydrin,
or phosphomolybdic acid (PMA). Flash column chromatography
1
was carried out under a positive pressure of nitrogen. H NMR
spectra were recorded on 500 MHz spectrometers. Data are
presented as follows: chemical shift (in ppm on the δ scale relative
to δ ) 0.00 ppm for the protons in TMS), integration, multiplicity
(s ) singlet, d ) doublet, t ) triplet, q ) quartet, m ) multiplet,
br ) broad), coupling constant (J/Hz). Coupling constants were
taken directly from the spectra and are uncorrected. 13C NMR
spectra were recorded at 125 MHz, and all chemical shift values
are reported in ppm on the δ scale, with an internal reference of δ
77.0 or 49.0 for CDCl3 or MeOD, respectively. High-resolution
mass spectra were measured on liquid chromatography/time-of-
flight mass spectrometry (LC-TOF).
2. Preparation and Characterization of New Compounds.
2.1. General Method A: Reductive Amination. To a solution of
aldehyde 8a or 8b (0.1 mmol) in THF (3 mL) was added the
ethanamine (0.2 mmol), followed by NaHB(OAc)3 (0.12 mmol).
The mixture was stirred at room temperature for an additional 3 h
and then concentrated. The crude product was purified by flash
column chromatography (EtOAc/hexanes, 2:1-4:1) to yield the
corresponding secondary amines as colorless oils, which were used
without further purification.
2.2. General Method B: Boc-Protection. To a solution of the
secondary amine (0.5 mmol) in MeOH (10 mL) was added (Boc)2O
(164 mg, 0.75 mmol) and TEA (140 µL, 1.0 mmol). The reaction
mixture was allowed to stir at room temperature for 30 min. The
solvent was removed by rotary evaporation, and the resulting
material was purified by flash column chromatography (EtOAc/
hexanes, 1:4-1:2) to yield 9a-f as colorless oils.
2.3. General Method C: Catalytic Hydrogenation. To a
solution of 9a-f (0.2 mmol) in EtOH (20 mL) was added
Pd(OH)2/C (100 mg). The reaction vessel was charged with H2,
heated at 60 °C for 24-48 h, then cooled to room temperature.
The catalyst was removed by filtration, and the resulting solution
was concentrated by rotary evaporation. The crude material was
purified by flash column chromatography (EtOAc/hexanes, 1:4-1:
2) to yield 10a-f as white foamy solids.
Pharmacokinetic Data for 3b and (R,R)-1. The goal for
insertion of two gem-fluorines into 1 was to lower the pKa of
one of the two basic nitrogen atoms to enhance metabolic
stability and oral bioavailability but allow for potential repro-
tonation of that nitrogen atom once in the active site of nNOS.
Pharmacokinetic properties of compound 3b (the (R,R) isomer
of the gem-difluorinated analogue corresponding to (R,R)-1)
were compared to those of (R,R)-1 to determine the effect of
the difluorine substitution on pharmacokinetics. The in vivo
compound half-life (t1/2) for iv dosing at 1 mg/kg in rats (average
of 3 rats) was 0.33 h for (R,R)-1 and 7.5 h for 3b; the t1/2 for
oral dosing at 5 mg/kg was too low to measure for (R,R)-1 and
3.7 h for 3b; oral bioavailability for (R,R)-1 was essentially 0
and 22.2% for 3b. It is apparent that the addition of the two
fluorines has a remarkable effect on in vivo stability and oral
bioavailability.
In summary, we have designed and synthesized a new series
of selective nNOS inhibitors (3a-f) with monocationic character
and, therefore, potentially possessing better bioavailability.
Biological evaluation of these new inhibitors based on crystal
structures led to the discovery of inhibitor 3b, which not only
retains most of the inhibitory activity of lead compound 1, but
also shows remarkable selectivity for nNOS over both eNOS
and iNOS. MM-PBSA calculations demonstrate that despite the
pKa of one of the nitrogen atoms in 3b being 5.5, when bound
to the active site, it becomes partially protonated for good
binding. Whereas the in vivo half-life (t1/2) and oral bioavail-
ability in rats for the parent compound [(R,R)-1] are nil, when
the two fluorines are added (3b), the t1/2 rises to 10 h and oral
bioavailability increases to 22%. This work represents a
significant step forward toward the goal of developing drugs
2.4. General Method D: Boc-Deprotection. To a solution of
10a-f (50 µmol) in MeOH (0.5 mL) was added 6 N HCl (1.0 mL).
The reaction mixture was allowed to stand at room temperature
9
J. AM. CHEM. SOC. VOL. 132, NO. 40, 2010 14235

A R T I C L E S
Xue et al.
for 12 h. The solvent was removed by rotary evaporation. The crude
product was recrystallized with cold diethyl ether to provide 3a-f
as pale yellow solids.
7.75-7.85 (m, 1H), 8.60-8.71 (m, 1H); 13C NMR (125 MHz,
CDCl3) δ 14.2, 21.11, 21.13, 24.7, 28.0, 28.1, 28.2, 28.3, 28.5,
29.7, 34.4, 34.5, 36.6, 42.2, 42.6, 42.7, 47.6, 47.7, 48.0, 48.8, 49.2,
49.89, 49.92, 50.1, 50.2, 50.8, 60.4, 67.5, 67.6, 67.7, 78.7, 79.1,
79.6, 80.2, 81.1, 81.2, 117.0, 117.1, 120.0, 120.4, 120.5, 124.76,
124.84, 126.4, 126.5, 126.6, 126.9, 127.0, 128.06, 128.11, 136.9,
137.0, 139.8, 139.9, 148.5, 149.3, 149.5, 153.8, 154.3, 154.4, 154.5,
154.7, 155.0, 155.4, 157.7; LC-TOF (M + H+) calcd for
C42H58F2N5O7 782.4304, found 782.4299.
(9a). 9a was synthesized by general methods A and B using
1
aldehyde 8b as the starting material (60%): H NMR (500 MHz,
CDCl3) δ 1.35-1.55 (m, 27H), 2.20-2.40 (s, 3H), 2.50-2.80 (m,
2H), 2.80-2.95 (m, 1H), 3.00-3.21 (m, 2H), 3.30-3.79 (m, 6H),
3.80-4.00 (m, 2H), 5.10-5.25 (s, 2H), 6.60-6.70 (br s, 1H),
7.00-7.30 (m, 8H), 7.31-7.60 (m, 2H); 13C NMR (125 MHz,
CDCl3) δ 14.4, 21.2, 21.3, 27.8, 28.1, 28.4, 28.5, 28.7, 29.9, 34.8,
42.3, 42.9, 47.5, 47.7, 49.0, 49.4, 50.1, 50.4, 50.9, 60.6, 67.9, 76.9,
78.9, 79.3, 79.7, 79.8, 80.6, 81.4, 113.0, 113.1, 117.2, 117.5, 120.1,
121.4, 126.7, 126.8, 126.9, 127.0, 127.1, 127.2, 127.5, 127.6, 128.3,
128.5, 128.6, 130.4, 140.0 148.8, 154.1, 154.5, 154.9, 157.8, 161.5,
163.4; LCQ-MS (M + H+) calcd for C43H58F3N4O7 799, found 799;
LC-TOF (M + H+) calcd for C43H58F3N4O7 799.4252, found
799.4258.
(3a). 3a was synthesized by general methods C and D using 9a
1
as the starting material (95%): H NMR (500 MHz, D2O) δ 2.29
(s, 3H), 2.78-2.81 (m, 2H), 2.95-3.05 (dd, J ) 8.0, 15.0 Hz, 1H),
3.15-3.20 (t, J ) 6.0, 1H), 3.31-3.35 (dd, J ) 3.0, 13.0 Hz, 1H),
3.40-3.55 (m, 3H), 3.63-3.66 (d, J ) 13.0 Hz, 1H), 3.71-3.79
(m, 1H), 3.87-3.95 (m, 3H), 4.24-4.26 (t, J ) 3.0 Hz, 1H), 6.55
(s, 1H), 6.64 (s, 1H), 7.25-7.29 (dt, J ) 2.5, 8.5 Hz, 1H),
7.34-7.36 (dd, J ) 2.5, 14.0 Hz, 1H), 7.38-7.40 (dd, J ) 2.5, 8.0
Hz, 1H), 7.49-7.52 (dd, J ) 6.0, 8.0 Hz, 1H); 13C NMR (125
MHz, D2O) δ 21.0, 29.1, 41.3, 47.0, 47.5, 49.2, 51.5, 51.7, 51.9,
63.6, 78.3, 110.4, 112.3, 112.5, 114.0, 118.2, 118.4, 118.6, 121.0,
131.2, 131.3, 134.2, 145.5, 153.9, 158.1, 161.4, 163.3; LC-TOF
(M + H+) calcd for C21H28F3N4O 409.2215, found 409.2226.
(9b). 9b was synthesized by general methods A and B using
1
aldehyde 8a as the starting material (58%): H NMR (500 MHz,
CDCl3) δ 1.35-1.55 (m, 27H), 2.20-2.40 (s, 3H), 2.50-2.80 (m,
2H), 2.80-2.95 (m, 1H), 3.00-3.21 (m, 2H), 3.30-3.79 (m, 6H),
3.80-4.00 (m, 2H), 5.10-5.25 (s, 2H), 6.60-6.70 (br s, 1H),
7.00-7.30 (m, 8H), 7.31-7.60 (m, 2H); 13C NMR (125 MHz,
CDCl3) δ 14.4, 21.2, 21.3, 27.8, 28.1, 28.4, 28.5, 28.7, 29.9, 34.8,
42.3, 42.9, 47.5, 47.7, 49.0, 49.4, 50.1, 50.4, 50.9, 60.6, 67.9, 76.9,
78.9, 79.3, 79.7, 79.8, 80.6, 81.4, 113.0, 113.1, 117.2, 117.5, 120.1,
121.4, 126.7, 126.8, 126.9, 127.0, 127.1, 127.2, 127.5, 127.6, 128.3,
128.5, 128.6, 130.4, 140.0 148.8, 154.1, 154.5, 154.9, 157.8, 161.5,
163.4; LCQ-MS (M + H+) calcd for C43H58F3N4O7 799, found 799;
LC-TOF (M + H+) calcd for C43H58F3N4O7 799.4252, found
799.4248.
(3b). 3b was synthesized by general methods C and D using 9b
as the starting material (100%): 1H NMR (500 MHz, D2O) δ 2.29
(s, 3H), 2.78-2.81 (m, 2H), 2.95-3.05 (dd, J ) 8.0, 15.0 Hz, 1H),
3.15-3.20 (t, J ) 6.0, 1H), 3.31-3.35 (dd, J ) 3.0, 13.0 Hz, 1H),
3.40-3.55 (m, 3H), 3.63-3.66 (d, J ) 13.0 Hz, 1H), 3.71-3.79
(m, 1H), 3.87-3.95 (m, 3H), 4.24-4.26 (t, J ) 3.0 Hz, 1H), 6.55
(s, 1H), 6.64 (s, 1H), 7.25-7.29 (dt, J ) 2.5, 8.5 Hz, 1H),
7.34-7.36 (dd, J ) 2.5, 14.0 Hz, 1H), 7.38-7.40 (dd, J ) 2.5, 8.0
Hz, 1H), 7.49-7.52 (dd, J ) 6.0, 8.0 Hz, 1H); 13C NMR (125
MHz, D2O) δ 21.0, 29.1, 41.3, 47.0, 47.5, 49.2, 51.5, 51.7, 51.9,
63.6, 78.3, 110.4, 112.3, 112.5, 114.0, 118.2, 118.4, 118.6, 121.0,
131.2, 131.3, 134.2, 145.5, 153.9, 158.1, 161.4, 163.3; LC-TOF
(M + H+) calcd for C21H28F3N4O 409.2215, found 409.2223.
(9c). 9c was synthesized by general methods A and B using
1
aldehyde 8a as the starting material (48%): H NMR (500 MHz,
CDCl3) δ 1.20-1.50 (m, 27H), 2.25-2.35 (m, 3H), 2.45-2.65 (m,
1H), 2.66-2.70 (m, 1H), 2.80-2.95 (m, 1H), 3.00-3.10 (m, 1H),
3.10-3.20 (m, 1H), 3.25-3.70 (m, 7H), 3.80-4.00 (m, 2H),
5.10-5.20 (m, 2H), 6.60-6.70 (m, 1H), 7.00-7.15 (m, 2H),
7.16-7.20 (m, 1H), 7.21-7.27 (m, 4H), 7.35-7.60 (m, 3H); 13C
NMR (125 MHz, CDCl3) δ 13.7, 14.2, 19.1, 21.0, 21.1, 24.7, 27.91,
27.96, 28.01, 28.2, 28.3, 28.5, 30.6, 34.4, 34.5, 34.6, 42.0, 42.1,
42.6, 42.7, 47.4, 47.5, 47.6, 47.8, 48.8, 49.3, 49.9, 50.1, 50.3, 50.8,
53.4, 53.8, 54.0, 60.4, 64.4, 67.7, 67.8, 68.0, 78.7, 78.8, 79.1, 79.2,
79.3, 79.6, 80.3, 81.17, 81.22, 115.2, 115.4, 115.6, 115.7, 117.0,
117.1, 119.9, 126.5, 126.6, 126.9, 127.0, 127.5, 128.1, 131.5, 139.7,
139.8, 148.6, 153.9, 154.3, 154.4, 154.5, 154.6, 154.7, 154.8, 155.1,
157.4, 157.5, 157.6, 162.7, 164.6, 171.2; LC-TOF (M + H+) calcd
for C43H58F3N4O7 799.4258, found 799.4237.
(3c). 3c was synthesized by general methods C and D using 9c
1
as the starting material (95%): H NMR (500 MHz, D2O) δ 2.30
(s, 3H), 2.78-2.81 (m, 2H), 2.95-3.05 (dd, J ) 8.0, 15.0 Hz, 1H),
3.15-3.20 (t, J ) 6.0, 1H), 3.31-3.35 (dd, J ) 3.0, 13.0 Hz, 1H),
3.40-3.55 (m, 3H), 3.63-3.66 (d, J ) 13.0 Hz, 1H), 3.71-3.79
(m, 1H), 3.87-3.95 (m, 3H), 4.24-4.26 (t, J ) 3.0 Hz, 1H), 6.55
(s, 1H), 6.64 (s, 1H), 7.21-7.25 (dd, J ) 8.5, 8.5 Hz, 2H),
7.59-7.62 (dd, J ) 5.0, 8.5 Hz, 2H); 13C NMR (125 MHz, D2O)
δ 21.0, 29.0, 41.3, 47.0, 47.4, 49.2, 51.7, 51.9, 51.9, 63.6, 78.3,
110.4, 113.9, 116.0, 116.1, 118.7, 127.42, 127.47, 127.55, 127.59,
145.5, 153.9, 158.1; LC-TOF (M + H+) calcd for C21H28F3N4O
409.2215, found 409.2230.
(9d). 9d was synthesized by general methods A and B using
1
(3d). 3d was synthesized by general methods C and D using 9d
aldehyde 8a as the starting material (55%): H NMR (500 MHz,
1
as the starting material (95%): H NMR (500 MHz, D2O) δ 2.30
CDCl3) δ 1.20-1.50 (m, 27H), 2.25-2.35 (m, 3H), 2.45-2.65 (m,
1H), 2.66-2.71 (m, 1H), 2.80-2.95 (m, 1H), 3.00-3.10 (m, 1H),
3.10-3.20 (m, 1H), 3.25-3.70 (m, 7H), 3.80-4.00 (m, 2H),
(s, 3H), 2.73-2.84 (m, 2H), 2.95-3.05 (dd, J ) 8.5, 15.0 Hz, 1H),
3.10-3.20 (t, J ) 6.0, 1H), 3.31-3.35 (dd, J ) 3.0, 13.5 Hz, 1H),
3.40-3.55 (m, 3H), 3.63-3.66 (d, J ) 13.5 Hz, 1H), 3.71-3.79
(m, 1H), 3.87-3.95 (m, 3H), 4.24-4.26 (t, J ) 3.0 Hz, 1H), 6.55
(s, 1H), 6.64 (s, 1H), 7.45-7.65 (m, 5H); 13C NMR (125 MHz,
D2O) δ 21.0, 29.0, 41.3, 47.0, 47.4, 49.2, 51.6, 51.8, 52.0, 63.6,
78.2, 110.4, 113.9, 119.0, 120.9, 124.81, 124.86, 124.91, 129.1,
131.6, 131.9, 132.1, 145.5, 153.9, 158.1; LC-TOF (M + H+) calcd
for C21H29F2N4O 391.2309, found 391.2337.
5.10-5.20 (m, 2H), 6.60-6.70 (m, 1H), 7.00-7.60 (m, 12H); 13
C
NMR (125 MHz, CDCl3) δ 13.7, 14.2, 19.1, 21.0, 21.1, 24.7, 27.91,
27.96, 28.01, 28.2, 28.3, 28.5, 30.6, 34.4, 34.5, 34.6, 42.0, 42.1,
42.6, 42.7, 47.4, 47.5, 47.6, 47.8, 48.8, 49.3, 49.9, 50.1, 50.3, 50.8,
53.4, 53.8, 54.0, 60.4, 64.4, 67.7, 67.8, 68.0, 78.7, 78.8, 79.1, 79.2,
79.3, 79.6, 80.3, 81.17, 81.22, 115.2, 115.4, 115.6, 115.7, 117.0,
117.1, 119.9, 126.5, 126.6, 126.9, 127.0, 127.5, 128.1, 131.5, 139.7,
139.8, 148.6, 153.9, 154.3, 154.4, 154.5, 154.6, 154.7, 154.8, 155.1,
157.4, 157.5, 157.6, 162.7, 164.6, 171.2; LC-TOF (M + H+) calcd
for C43H59F2N4O7 781.4352, found 781.4366.
(3e). 3e was synthesized by general methods C and D using 9e
as the starting material (96%): 1H NMR (500 MHz, D2O) δ
0.85-0.95 (d, J ) 2.5, 3H), 1.45-1.55 (m, 1H), 1.61-1.70 (m,
1H), 1.71-1.97 (m, 3H), 2.00-2.10 (m, 1H), 2.35-2.45 (m, 1H),
2.46-2.57 (m, 1H), 2.90-3.00 (t, J ) 6.0, 1H), 3.16-3.21 (m,
1H), 3.30-3.50 (m, 4H), 3.51-3.60 (d, J ) 13.5 Hz, 1H),
3.61-3.70 (m, 1H), 3.75-3.90 (m, 3H), 4.14s-4.16 (t, J ) 3.0
Hz, 1H), 7.45-7.65 (m, 5H); 13C NMR (125 MHz, D2O) δ 17.5,
17.6, 18.2, 26.8, 27.2, 27.3, 32.6, 32.9, 40.0, 40.2, 48.3, 48.9, 49.1,
49.6, 50.6, 52.2, 58.8, 65.2, 79.9, 80.0, 115.4, 115.6, 116.8, 117.0,
(9f). 9f was synthesized by general methods A and B using
1
aldehyde 8b as the starting material (55%): H NMR (500 MHz,
CDCl3) δ 1.40-1.55 (m, 27H), 2.27-2.29 (m, 3H), 2.45-2.67 (m,
1H), 2.68-2.75 (m, 1H), 2.85-2.95 (m, 1H), 3.00-3.11 (m, 1H),
3.12-3.20 (m, 1H), 3.30-3.45 (m, 3H), 3.46-3.65 (m, 3H),
4.05-4.20 (m, 2H), 5.16 (s, 2H), 6.67 (s, 1H), 7.17-7.20 (m, 1H),
7.21-7.26 (m, 4H), 7.30-7.45 (m, 2H), 7.50-7.70 (m, 1H),
9
14236 J. AM. CHEM. SOC. VOL. 132, NO. 40, 2010

Monocationic Inhibitors of Neuronal NOS
A R T I C L E S
Table 3. Crystallographic Data Collection and Refinement Statistics
data seta
nNOS-3a
nNOS-3b
nNOS-3c
nNOS-3d
Data collection
PDB code
Space group
Cell dimensions
a, b, c (Å)
Resolution (Å)
Rsym or Rmerge
I/σI
No. unique reflections
Completeness (%)
Redundancy
Refinement
3NLV
P212121
3NLX
P212121
3NLY
P212121
3NLZ
P212121
52.3, 111.7, 164.4
2.10 (2.14-2.10)
0.080 (0.59)
9.1 (2.2)
56857
99.4 (99.5)
5.6 (2.9)
52.3, 111.5, 164.4
1.87 (1.90-1.87)
0.053 (0.36)
13.5 (3.9)
79054
98.2 (96.5)
4.1 (4.1)
51.8, 111.2, 164.1
2.00 (2.03-2.00)
0.078 (0.40)
7.6 (1.8)
63439
96.7 (80.9)
3.9 (3.1)
51.9, 110.4, 164.0
1.92 (1.95-1.92)
0.050 (0.32)
11.5 (2.6)
73017
99.4 (89.8)
4.1 (3.6)
Resolution (Å)
No. reflections used
Rwork/Rfree
2.10
54011
0.175/0.211
1.87
75069
0.178/0.209
2.00
60245
0.201/0.249
1.92
69320
0.173/0.210
No. atoms
Protein
Ligand/ion
6671
187
6689
223
6676
190
6707
217
Water
434
479
190
417
Rms deviations
Bond lengths (Å)
Bond angles (deg)
0.013
1.325
0.012
1.390
0.015
1.554
0.013
1.456
data seta
nNOS-3e
nNOS-3f
eNOS -3b
eNOS -3f
Data collection
PDB code
3NM0
3NLW
3NLU
3NLT
Space group
P212121
P212121
P212121
P212121
Cell dimensions
a, b, c (Å)
Resolution (Å)
Rsym or Rmerge
I/σI
No. unique reflections
Completeness (%)
Redundancy
52.1, 110.9, 164.2
1.81 (1.84-1.81)
0.048 (0.38)
12.1 (2.7)
86908
99.0 (92.5)
4.0 (3.8)
52.2, 111.4, 164.7
2.10 (2.14-2.10)
0.069 (0.55)
9.2 (2.4)
56364
99.2 (100.0)
4.1 (4.1)
58.0, 107.0, 156.9
2.65 (2.70-2.65)
0.121(0.58)
9.7 (1.9)
28366
97.3 (99.1)
3.7 (3.7)
57.9, 106.9, 157.0
2.75 (2.80-2.75)
0.103 (0.65)
12.4 (1.9)
25670
95.6 (94.4)
3.9 (4.0)
Refinement
Resolution (Å)
No. reflections used
1.81
82532
0.178/0.209
2.10
53523
0.171/0.207
2.65
26957
0.185/0.254
2.74
24379
0.186/0.262
b
Rwork/Rfree
No. atoms
Protein
Ligand/ion
Water
6716
217
459
6676
185
319
6451
197
111
6419
195
60
Rms deviations
Bond lengths (Å)
Bond angles (deg)
0.013
1.360
0.013
1.347
0.014
1.502
0.014
1.519
b
a See Schemes 2-4 for nomenclature and chemical formula of inhibitors. Rfree was calculated with the 5% of reflections set aside throughout the
refinement. For each NOS isoform, the set of reflections for the Rfree calculation were kept the same for all data sets according to those used in the data
of the starting model.
126.0, 132.1, 132.2, 140.2, 163.1, 165.0, 168.4; LC-TOF (M +
overexpressed (in Escherichia coli) and isolated as reported.27 IC50
values for inhibitors 3a-f were measured for the three different
isoforms of NOS using L-arginine as a substrate. The formation of
nitric oxide was measured using a hemoglobin capture assay
described previously.24 All NOS isozymes were assayed at room
temperature in a 100 mM Hepes buffer (pH 7.4) containing 10 µM
L-arginine, 1.6 mM CaCl2, 11.6 µg/mL calmodulin, 100 µM DTT,
100 µM NADPH, 6.5 µM H4B, 3.0 µM oxyhemoglobin (for iNOS
assays, no Ca2+ and calmodulin were 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 corresponding
H+) calcd for C21H33F2N4O 395.2622, found 395.2633.
(3f). 3f was synthesized by general methods C and D using 9f
as the starting material (100%): 1H NMR (500 MHz, D2O) δ
1.40-1.50 (m, 1H), 1.51-1.60 (m, 2H), 1.79-1.90 (m, 2H),
1.91-1.98 (d, J ) 13.5 Hz, 1H), 2.20 (s, 3H), 2.65-2.75 (m, 1H),
2.79-2.85 (m, 1H), 2.94-2.96 (d, J ) 7.5 Hz, 1H), 2.97-3.00
(m, 1H), 3.07-3.12 (dd, J ) 11.5, 11.5 Hz, 1H), 3.20-3.26 (dd,
J ) 0.5, 13.0 Hz, 1H), 3.31-3.47 (m, 4H), 3.52-3.55 (d, J )
13.5 Hz, 1H), 3.61-3.64 (m, 1H), 3.74-3.82 (m, 5H), 4.11 (s,
1H), 6.52 (s, 1H), 6.57 (s, 1H); 13C NMR (125 MHz, D2O) δ 20.4,
21.05, 21.06, 22.4, 28.95, 29.01, 41.48, 41.51, 45.1, 47.0, 48.08,
48.14, 49.4, 57.7, 63.7, 63.9, 78.3, 78.4, 110.4, 114.09, 114.12,
145.7, 153.9, 158.1; LC-TOF (M + H+) calcd for C20H34F2N5O
398.2742, found 398.2726.
(27) (a) Hevel, J. M.; White, K. A.; Marletta, M. J. Biol. Chem. 1991, 266
(34), 22789. (b) Roman, L. J.; Sheta, E. A.; Marta´sek, P.; Gross, S. S.;
Liu, Q.; Masters, B. S. S. Proc. Natl. Acad. Sci. U.S.A. 1995, 92 (18),
8428. (c) Martasek, P.; Liu, Q.; Roman, L. J.; Gross, S. S.; Sessa,
W. C.; Masters, B. S. S. Biochem. Biophys. Res. Commun. 1996, 219
(2), 359.
3. Enzyme Assays. The three isozymes, murine macrophage
iNOS, rat nNOS, and bovine eNOS, were recombinant enzymes,
9
J. AM. CHEM. SOC. VOL. 132, NO. 40, 2010 14237

A R T I C L E S
Xue et al.
Ki values of inhibitors were calculated from the IC50 values using
eq 1 with known Km values (rat nNOS, 1.3 µM; iNOS, 8.3 µM;
eNOS, 1.7 µM).
were then modeled in O34 or COOT35 and refined using REFMAC.
Water molecules were added in REFMAC and checked by COOT.
The TLS36 protocol was implemented in the final stage of
refinements with each subunit as one TLS group. The refined
structures were validated in COOT before deposition to RCSB
protein data bank. The crystallographic data collection and structure
refinement statistics are summarized in Table 3 with PDB accession
codes included.
6. Molecular Mechanics Poisson-Boltzmann/SurfaceA (MM-
PBSA) Method for Free Energy of Binding of Inhibitors. The MM-
PBSA method26 as implemented in Amber 9.0 was used to compute
the free energy of binding of inhibitors to NOS and was previously
described in detail.23 To briefly summarize, the free energies of 10
different aminopyridine inhibitors-NOS complexes were calculated
from a single energy minimized structure starting from the known
crystal structures described in our previous work.23 A plot of the
resulting computed free energy (PB) versus ∆Gexp was obtained
from experimental Ki values. The empirical equation derived from
Figure 5, ∆Gcalc ) (PB - 5.2874)/6.029, was used to calculate
free energies of binding for the inhibitors used in the present study.
7. Pharmacokinetic Data. All pharmacokinetic results were
obtained by LC-MS-MS analysis and calculation of PK param-
eters using WinNonlin software at BioDuro, Inc., Shanghai, China.
Ki ) IC50/(1 + [S]/Km)
(1)
4. 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.28 The
enzyme-inhibitor complex crystals were obtained by soaking rather
than co-crystallization as reported in the earlier structural work.29
The nNOS heme domain at 7-9 mg/mL containing 20 mM
histidine or the eNOS heme domain at 20 mg/mL with 2 mM
imidazole was used for the sitting drop vapor diffusion crystalliza-
tion setup under the conditions reported before.28,30 Fresh crystals
(1-2 day old) were first passed stepwise through cryo-protectant
solutions described28,30 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. Crystals were stored in
liquid nitrogen until data collection.
5. 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-Ice31 and the crystal mounting robot. Raw
data frames were indexed, integrated, and scaled using HKL2000.32
Typically, each data set consisted of 90-100° of data with a 0.5°
frame width for both nNOS and eNOS crystals because of their
identical orthorhombic P212121 space group symmetry.
Acknowledgment. The authors are grateful for financial support
from the National Institutes of Health (GM49725 to R.B.S. and
GM57353 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 is grateful to 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 staff at SSRL for their
assistance during the remote X-ray diffraction data collections.
The binding of inhibitors was detected by the initial difference
Fourier maps calculated with REFMAC.33 The inhibitor molecules
(28) Li, H.; Shimizu, H.; Flinspach, M.; Jamal, J.; Yang, W.; Xian, M.;
Cai, T.; Wen, E. Z.; Jia, Q.; Wang, P. G.; Poulos, T. L. Biochemistry
2002, 41, 13868.
Supporting Information Available: Copies of complete
spectroscopic data of compounds 9a-d, 9f, and 3a-f. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(29) Flinspach, M. L.; Li, H.; Jamal, J.; Yang, W.; Huang, H.; Hah, J. M.;
Gomez-Vidal, J. A.; Litzinger, E. A.; Silverman, R. B.; Poulos, T. L.
Nat. Struct. Mol. Biol. 2004, 11, 54.
(30) Raman, C. S.; Li, H.; Martasek, P.; Kral, V.; Masters, B. S.; Poulos,
T. L. Cell 1998, 95, 939.
JA106175Q
(31) McPhillips, T. M.; McPhillips, S. E.; Chiu, H. J.; Cohen, A. E.; Deacon,
A. M.; Ellis, P. J.; Garman, E.; Gonzalez, A.; Sauter, N. K.;
Phizackerley, R. P.; Soltis, S. M.; Kuhn, P. J. Synchrotron Radiat.
2002, 9, 401.
(32) Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307.
(33) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Acta Crystallogr. 1997,
D53, 240.
(34) Jones, T. A.; Zou, J.-Y.; Cowan, S. W.; Kjeldgaarrd, M. Acta
Crystallogr. 1991, A47, 110.
(35) Emsley, P.; Cowtan, K. Acta Crystallogr. 2004, D60, 2126.
(36) Winn, M. D.; Isupov, M. N.; Murshudov, G. N. Acta Crystallogr.
2001, D57, 122.
9
14238 J. AM. CHEM. SOC. VOL. 132, NO. 40, 2010