Improved synthesis of chiral pyrrolidine inhibitors and their binding properties to neuronal nitric oxide synthase

Article abstract of DOI:10.1021/jm200411j

We report an efficient synthetic route to chiral pyrrolidine inhibitors of neuronal nitric oxide synthase (nNOS) and crystal structures of the inhibitors bound to nNOS and to endothelial NOS. The new route enables versatile structure-activity relationship studies on the pyrrolidine-based scaffold, which can be beneficial for further development of nNOS inhibitors. The X-ray crystal structures of five new fluorine-containing inhibitors bound to nNOS provide insights into the effect of the fluorine atoms on binding.

Full text of DOI:10.1021/jm200411j

BRIEF ARTICLE
pubs.acs.org/jmc
Improved Synthesis of Chiral Pyrrolidine Inhibitors and Their Binding
Properties to Neuronal Nitric Oxide Synthase†
Fengtian Xue,‡,#,∞ James M. Kraus,‡,∞ Kristin Jansen Labby,‡ Haitao Ji,‡ Jan Mataka,‡ Guoyao Xia,‡ Huiying Li,§
Silvia L. Delker,§ Linda J. Roman,|| Pavel Martꢀasek,||,^ Thomas L. Poulos,*,§ and Richard B. Silverman*,‡
‡Departments of Chemistry and 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 Chemistry, 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 and Center for Applied Genomics, 1st School of Medicine, Charles University, Prague, Czech Republic
S Supporting Information
b
ABSTRACT: We report an efficient synthetic route to chiral pyrrolidine inhibitors of neuronal nitric oxide synthase (nNOS) and crystal
structures of the inhibitors bound to nNOS and to endothelial NOS. The new route enables versatile structureÀactivity relationship
studies on the pyrrolidine-based scaffold, which can be beneficial for further development of nNOS inhibitors. The X-ray crystal structures
of five new fluorine-containing inhibitors bound to nNOS provide insights into the effect of the fluorine atoms on binding.
’ INTRODUCTION
catalytic hydrogenation of an N-Boc-N-benzyl-protected intermediate,
proceeded poorly. As a result, the utility of this route was dramatically
compromised by the lack of scalability. More importantly, the strong
reducing conditions used to remove the benzyl protecting group pro-
hibited the possibility of incorporating reduction sensitive functional
groups (e.g., nitriles, ketones, alkenes, cyclopropanes, and halophenyls)
into the inhibitors, which significantly impaired structureÀ
activity optimization based on this scaffold. For instance, attempts to
remove the benzyl protecting group from a compound containing a
chlorophenyl substituent led to dechlorination. Removal of the benzyl
protecting group by catalytic hydrogenation from a compound contain-
ing a cyclopropane led to a partial reduction of the cyclopropyl ring.9
We report here a synthesis with improved overall yield and
functional group compatibility. In the current method, the proble-
matic benzyl protecting group in the previous synthesis is replaced
by a Boc-protecting group. The new route not only produces 1b in
an enhanced overall yield but also provides a way to synthesize
inhibitors with reduction-sensitive chlorophenyl groups.
Selective inhibition of neuronal nitric oxide synthase (nNOS)
over its closely related isozymes, inducible nitric oxide synthase
(iNOS) and endothelial nitric oxide synthase (eNOS), has attracted
tremendous drug discovery efforts for neurotoxicity and certain
neurodegenerative conditions such as Parkinson’s, Alzheimer’s, and
Huntington’s diseases and cerebral palsy.1À5 As part of our research
program directed toward developing novel nNOS inhibitors, we
recently disclosed a series of gem-difluorinated monocationic in-
hibitors including 1a and 1b, which are potent and highly selective
inhibitors of nNOS.6,7 Moreover, according to the results of detailed
pharmacokinetic studies,7,8 1b has demonstrated intriguing cell
membrane permeability and oral bioavailability, which makes it a
promising drug candidate for neurodegenerative diseases.
’ CHEMISTRY
As shown in Scheme 1, the synthesis of key precursor 9 began
with Boc-protected 4,6-dimethylaminopyridine 2. Compound 2
was treated with 2 equiv of n-butyllithium (n-BuLi), and the
resulting dianion was allowed to react with tert-butyl 6-oxa-3-
azabicyclo[3.1.0]hexane-3-carboxylate6 to generate the trans-
alcohol (3) in modest yields. Attempts to convert 3 directly to
6 failed because the free hydroxyl group of 3 is more reactive
toward (Boc)2O than the carbamate NH group, and the hydro-
lytic stability of the formed carbonate is very similar to that of the
di-Boc-protected aminopyridine. Therefore, we had to use a
Although they possess desirable potency, selectivity, and
pharmacokinetic properties, it is difficult to prepare gram-scale
quantities of 1b for a comprehensive preclinical study.7,8 The
previously reported route takes 16 steps starting from 4,6-dimeth-
yl-2-aminopyridine. Several of the steps suffered from unsatisfactory
yields, resulting in an overall yield of less than 1%.7,8 In particular, the
late stage benzyl deprotection step, employing a high temperature
Received: April 7, 2011
Published: August 02, 2011
r
2011 American Chemical Society
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|

Journal of Medicinal Chemistry
BRIEF ARTICLE
Scheme 1. Synthesis of 9a
a Reagents and conditions: (a) (i) n-BuLi (2 equiv), À78 ꢀC to room temp, (ii) epoxide, À78 ꢀC to room temp, 41%; (b) TBSCl, imidazole, DMF, room
temp, 30 h, 93%; (c) (Boc)2O, DMAP, room temp, 24 h, 95%; (d) TBAF, room temp, 15 min, 99%; (e) (S)-(À)-camphanic acid, DIAD, room temp,
16 h, 94%; (f) Na2CO3, H2O/MeOH, 35 ꢀC, 30 min, 97%.
Scheme 2. Synthesis of 1b and 8aÀea
a Reagents and conditions: (a) allyl methyl carbonate, Pd(PPh3)4, 45 ꢀC, 5 h, 66%; (b) O3, À78 ꢀC, 30 min, (ii) Me2S, À78 ꢀC to room temp, 2 h, 87%;
(c) amine hydrochloride, triethylamine, NaHB(OAc)3, room temp, 3 h, 86À91%; (d) 6 N HCl in MeOH (2:1), room temp, 12 h, 90À99%.
three-step procedure to convert 3 to 6. Di-Boc protection of
2 also was fruitless because one of the Boc groups is transferred to
the oxyanion of 3 upon condensation with diBoc 2 with tert-butyl
6-oxa-3-azabicyclo[3.1.0]hexane-3-carboxylate, as described earlier.10
The hydroxyl group of 3 was first protected with tert-butyldimethyl-
silyl chloride (TBSCl) in the presence of imidazole to give the silyl
ether (4) in excellent yields. Next, the NH of the carbamate group on
the pyridine ring was further protected with another Boc protecting
group using (Boc)2O in the presence of 4-dimethylaminopyridine
(DMAP) to yield 5 in high yields. Then the silyl ether was cleaved
using tetrabutylammonium fluoride (TBAF) to provide the tri-Boc
protected alcohol (6) in very high yields. Finally, the two enantiomers
of 6 were resolved through their camphanic ester derivatives (7a and
7b) employing a Mitsunobu reaction using (S)-(À)-camphanic acid
as the nucleophile. The ester linkage of 7b was hydrolyzed carefully
using Na2CO3 in MeOH/H2O to provide chiral precursor 9 in
excellent yields.
Allylation of chiral cis-alcohol 9 in the presence of allyl methyl
carbonate using Pd(PPh3)4 as a catalyst provided 10 in good
yields (Scheme 2).10,11 Alkene 10 was treated with ozone,
followed by dimethyl sulfide to generate 11 in excellent yields.
Aldehyde 11 underwent a reductive amination reaction with the
corresponding amines in the presence of NaHB(OAc)3 to give
12, 13aÀe in high yields. Finally, the three Boc-protecting groups
were removed simultaneously in HCl to provide the final inhibitors
in excellent yields.
’ RESULTS AND DISCUSSION
We have determined the crystal structures of rat nNOS with
inhibitors 8aÀe bound and that of bovine eNOS in complex with
8c. The PDB codes are given in Supporting Information Table 1.
As expected from the chirality of their pyrrolidine moiety,
inhibitors 8aÀe adopt the same flipped binding mode as leads
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BRIEF ARTICLE
1a,b (Figure 1).7 The aminopyridine motif extends to the surface
hydrophobic pocket (Tyr706, Leu337, and Met336) in nNOS,
forming a chargeÀcharge interaction with the heme propionate
D and a πÀπ stacking interaction with the aromatic side chain of
Tyr706 in its newly adopted conformation. Substitution of the
m-fluoro atom in the phenyl tail of 1b with a larger chlorine atom
slightly increases the potency (1.3-fold) of the inhibitor (8a vs
1b, Table 1). A comparison between the structures of nNOS-8a
(Figure 1A) and nNOS-1b provides some clues of why nNOS
prefers the m-chloro over m-fluoro atom on the phenyl group.
Similar to what was observed for nNOS-1b7 the 2,2-difluoro-
2-(m-chlorophenyl)ethyl moiety of 8a has adopted two confor-
mations, with the CF2 group pointing away from (major) and
toward (minor) the heme. Residual Fo À Fc difference density is
present especially around the fluorine atoms if only one conforma-
tion is modeled, clearly indicating two conformations. The two
different conformations also result in two slightly different orienta-
tions of the chlorophenyl group, which is consistent with two
conformations. However, because of the bulkier chlorine atom in 8a,
there is increased van der Waals contact and the phenyl ring has
been pushed closer to the Glu592 side chain but is still well tolerated.
The Glu592 side chain shows two rotamers in the nNOS-8a
structure, which were seen in nNOS-1b as well. The new Glu592
rotamer forms a new hydrogen bond to the amino nitrogen of the
inhibitor (Figure 1A). The active site of eNOS might also accom-
modate the m-chlorophenyl ring better; therefore, the selectivity of
8a for nNOS over eNOS has dropped 6-fold, which makes this
inhibitor comparable to inhibitor 1a.
Table 1. Ki of Inhibitors for Rat nNOS, Bovine eNOS, and
Murine iNOSa
selectivityb
compd nNOS (μM) eNOS (μM) iNOS (μM)
n/e
n/i
1a
1bc
8a
8b
8c
8d
8e
0.16
31
130
16
190
25
194
1182
186
159
731
221
272
1188
227
907
535
1000
221
722
0.11
0.086
0.17
78
27
91
0.026
0.077
0.18
19
26
17
17
49
130
a The Ki values were calculated based on the directly measured IC50
values, which represent at least duplicate measurements with standard
deviations of (10%. There is high homology among these isozymes
from different species.5 b The ratio of Ki (eNOS or iNOS) to Ki (nNOS).
c The Ki (nNOS) for inhibitor 1b is different from that previously
reported because of the use of a new high throughput assay improving
both speed and reproducibility.
Inhibitor 8b, the corresponding o-chloro isomer of 8a, has
2-fold lower potency for rat nNOS (8b vs 8a, Table 1). However,
eNOS does not show a preference for inhibitors 8b and 8a. The
structure of nNOS-8b (Figure 1B) reveals only one conforma-
tion for the inhibitor where the CF2 group points toward the
Figure 1. Active site structures in enzymeÀinhibitor complexes: (A) nNOS-8a, (B) nNOS-8b, (C) nNOS-8c, (D) eNOS-8c, (E) nNOS-8d, and
(F) nNOS-8e. The σ weighted 2Fo À Fc electron density map for each bound inhibitor at 1σ contour level is shown for parts AÀD and the omit Fo À Fc
density map at 2.5σ contour level shown for parts E and F. Major hydrogen bonds are drawn with dashed lines. Alternative side chain rotamers are
observed for Glu592 in nNOS or Glu363 in eNOS in parts AÀD. The inhibitor 8a also shows two conformations in its lipophilic tail (colored as gray and
magenta in part A). The density for part of inhibitor 8e is weak as shown, but the model presented in part F is supported by the 2Fo À Fc map at lowered
contour level (0.5σ). Figures were prepared with PyMol (www.pymol.org). The PDB codes are as follows: nNOS-8a, 3PNE; nNOS-8b, 3PNF; nNOS-
8c, 3PNG; eNOS-8c, 3PNH; nNOS-8d, 3SVP; nNOS-8e, 3SVQ.
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BRIEF ARTICLE
heme. In this conformation one of the fluorine atoms makes an
unfavorable intramolecular contact with the chlorine atom on the
phenyl ring. However, there is no electron density indicating an
altered orientation for the CF2 group that avoids this unfavorable
contact. This steric hindrance may partially account for the 2-fold
drop in potency for 8b compared to 8a. When both meta positions of
the phenyl ring are substituted (8d), the conformation of the phenyl
ring is determined by the bulkier chlorine atom and the conformation
is more like 8a than 1b. This ring is still well tolerated in the active site,
and of the CF2 analogues reported here (1a,b, 8a,b, 8d,e), 8d is the
most potent of the series. Interestingly, the side chain of 8d shows
only one conformation because the meta disubstituted ring is too
large to be accommodated in the alternative conformation. Inhibitor
8e is the least potent analogue of the series. Similar to 8b, ortho
substitution in 8e leads to an intramolecular clash between fluorine of
the ring and the CF2 group, and again, only one side chain
conformation is observed with CF2 pointing downward to the heme
(Figure 1B,F).
Inhibitor 8c, a mixture of diastereomers, is the monofluoro
methylene derivative of 1b with a chiral pyrrolidine core. The mixture
shows increased potency for rat nNOS (8c vs 1b, Table 1). This may
result from the higher basicity of the amino group in the lipophilic tail
of 8c compared to that of 1b.8 However, the nNOS selectivity of 8c
over eNOS is somewhat diminished compared with that for 1b
because of its relatively higher potency toward eNOS. The structures
of 8c bound to nNOS and eNOS were obtained (Figure 1C,D). In
both structures, the same binding mode for 8c is observed, with its
single fluorine atom pointing down toward the heme. Apparently,
despite being a mixture of diastereomers, the (R,R,R)-diastereomer
has greater binding affinity to the isozymes than the (R,R,S)-diaster-
eomer because the observed electron density for the fluorine atom only
supports one position (R). This may be because there is no unfavorable
contact between this fluorine atom and heme propionate A in con-
trast to the clashes observed for the CF2 group in its “downward”
conformation in the nNOS-8a structure (Figure 1A). The amino group
in the lipophilic tail of the inhibitor lures Glu592 of nNOS (Glu363 of
eNOS) into an alternative rotamer by hydrogen bonding.
’ EXPERIMENTAL SECTION
The purity of the final compounds was determined by HPLC analysis
to be g95%. For experimental details, see the Supporting Information.
6-(((3R,4R)-4-(2-((2,2-Difluoro-2-(3-fluorophenyl)ethyl)-
amino)ethoxy)pyrrolidin-3-yl)methyl)-4-methylpyridin-2-
amine (1b). To a solution of 12a (60 mg, 85 umol) in MeOH (2mL) was
added 6 N HCl (4 mL) at room temperature. The mixture was stirred for
12 h and then concentrated. The crude product was purified by recrystalli-
zation (EtOH/H2O) to give inhibitor 1b (40 mg, 99%) as a tri-HCl salt;
[R]20 +6.25 (c 4, MeOH).
6-(((3R,4R)-4-(2-((2,2-Difluoro-2-(3-chlorophenyl)ethyl)-
amino)ethoxy)pyrrolidin-3-yl)methyl)-4-methylpyridin-2-
amine (8a). To a solution of 12b (70 mg, 0.10 mmol) in MeOH
(2 mL) was added 6 N HCl (4 mL) at room temperature. The mixture
was stirred for 12 h and then concentrated. The crude product was
purified by recrystallization (EtOH/H2O) to give inhibitor 8a (50 mg,
1
95%) as a tri-HCl salt: H NMR (500 MHz, D2O) δ 2.19 (s, 3H),
2.60À2.75 (m, 1H), 2.85À2.95 (m, 1H), 3.00À3.10 (m, 2H),
3.20À3.30 (m, 1H), 3.30À3.45 (m, 3H), 3.55À3.60 (d, J = 13.0 Hz,
1H), 3.65À3.70 (m, 1H), 3.75À3.90 (m, 3H), 4.15 (d, J = 3.0 Hz, 1H),
6.41 (s, 1H), 6.55 (s, 1H), 7.30À7.45 (m, 3H), 7.52 (s, 1H); 13C NMR
(125 MHz, D2O) δ 20.0, 29.2, 41.3, 41.4, 47.0, 47.4, 49.1, 51.7, 63.6,
78.3, 110.4, 114.0, 118.2, 123.39, 123.42, 123.47, 125.07, 125.12,
130.7, 131.5, 133.9, 134.4, 145.4, 153.9, 158.1; LC-TOF (M + H+)
calcd for C21H28ClF2N4O 425.1920, found 425.1919.
6-(((3R,4R)-4-(2-((2,2-Difluoro-2-(2-chlorophenyl)ethyl)-
amino)ethoxy)pyrrolidin-3-yl)methyl)-4-methylpyridin-2-
amine (8b). Inhibitor 8b was synthesized using a procedure similar
to that for 8a (50 mg, 95%) as a tri-HCl salt: 1H NMR (500 MHz, D2O) δ
2.19 (s, 3H), 2.60À2.75 (m, 1H), 2.85À2.95 (m, 1H), 3.03À3.08 (t, J =
11.5 Hz, 1H), 3.19 (s, 1H), 3.21À3.25 (dd, J = 3.0, 13.0 Hz, 1H),
3.35À3.42 (m, 3H), 3.52À3.58 (d, J = 13.0 Hz, 1H), 3.63À3.66 (m,
1H), 3.82À3.88 (m, 1H), 3.90À4.00 (m, 2H), 4.14À4.16 (t, J = 3.5 Hz,
1H), 6.42 (s, 1H), 6.54 (s, 1H), 7.30À7.35 (m, 1H), 7.40À7.45 (m, 2H),
7.55À7.60 (m, 1H); 13C NMR (125 MHz, D2O) δ 21.0, 29.0, 41.2, 47.0,
47.4, 48.8, 49.2, 50.3, 50.5, 63.4, 78.2, 110.4, 113.9, 118.1, 127.57, 127.63,
127.70, 129.1, 130.8, 131.4, 131.5, 133.1, 145.5, 153.9, 158.1; LC-TOF
(M + H+) calcd for C21H28ClF2N4O 425.1920, found 425.1919.
6-(((3R,4R)-4-(2-((2-Fluoro-2-(3-fluorophenyl)ethyl)amino)-
ethoxy)pyrrolidin-3-yl)methyl)-4-methylpyridin-2-amine (8c).
Inhibitor 8c was synthesized as a mixture of two diastereomers using a
procedure similar to that for 8a (32 mg, 90%) as a tri-HCl salt: 1H NMR
(500 MHz, D2O) δ 2.19 (s, 3H), 2.65À2.75 (m, 1H), 2.85À2.95 (m, 1H),
3.03À3.11 (m, 1H), 3.20 (s, 1H), 3.21À3.25 (dd, J = 3.0, 13.0 Hz, 1H),
3.30À3.45 (m, 4H), 3.50À3.58 (m, 2H), 3.60À3.66 (m, 1H), 3.80À3.85
(m, 1H), 4.14À4.16 (m, 1H), 5.80À6.00 (m, 1H), 6.46 (s, 1H),
6.50À6.55 (m, 1H), 7.00À7.15 (m, 3H), 7.30À7.41 (m, 1H);
13C NMR (125 MHz, D2O) δ 21.0, 29.0, 29.1, 41.3, 41.4, 43.7, 43.9,
47.0, 47.1, 47.3, 48.7, 49.2, 49.3, 51.3, 51.5, 51.7, 51.8, 63.6, 63.9, 78.2, 88.4,
89.8, 110.4, 112.48, 112.54, 112.56, 112.61, 112.69, 112.72, 112.74, 114.0,
114.1, 116.3, 116.5, 116.6, 121.35, 121.40, 121.46, 121.49, 121.51, 130.85,
130.92, 131.0, 136.8, 136.99, 137.02, 145.60, 145.64, 153.85, 153.86,
158.11, 158.12, 161.6, 163.5; LC-TOF (M + H+) calcd for C21H29F2N4O
391.2309, found 391.2288.
6-(((3R,4R)-4-(2-((2,2-Difluoro-2-(3-chloro-5-fluorophenyl)-
ethyl)amino)ethoxy)pyrrolidin-3-yl)methyl)-4-methylpyridin-
2-amine (8d). Inhibitor 8d was synthesized as a mixture of two dia-
stereomers using a procedure similar to that for 8a (7 mg, 80%) as a tri-HCl
salt: 1H NMR (500 MHz, MeOD) δ 2.35 (s, 3H), 2.80À2.87 (m, 1H),
2.93À2.98 (m, 1H), 3.07À3.13 (m, 1H), 3.23À3.27 (m, 1H), 3.38À3.45
(m, 1H), 3.51À3.56 (m, 2H), 3.50À3.58 (m, 2H), 3.65À3.68 (d, J = 13.0
Hz, 1H), 3.73À3.79 (m, 2H), 3.94À3.99 (m, 1H), 4.00À4.07 (m, 1H),
4.19À4.21 (m, 1H), 6.66 (s, 1H), 6.67 (s, 1H), 7.42À7.46 (m, 2H), 7.56
(s, 1H); 13C NMR (125 MHz, MeOD) δ 22.0, 30.4, 43.5, 52.5, 52.7, 64.9,
StructureÀactivity relationship (SAR) studies demonstrate
the key role of the meta substituent in 1b, 8c, and 8d for retaining
high inhibitory activity for rat nNOS. A bulkier m-chloro group in 8a
increases potency relative to the m-fluoro substituent but leads to a
loss in selectivity over the other two isoforms. Although m-chloro
and m-fluoro disubstitution (8d) achieves another small boost in
potency, it also results in an additional drop in selectivity. The
o-chloro group in 8b or o-fluoro group in 8e leads to tight intra-
molecular contacts with its CF2 group, which is accompanied by an
unfavorable side chain conformation and a drop in potency. The
p-fluoro in 1a forces its CF2 group into a downward conformation,
weakening the hydrogen bond from the amino group to the Glu592
side chain. It seems then that only meta substituents place the
phenyl ring in the right position to optimize van der Waals contact
with the hydrophobic pocket surrounded by Val567 and Phe584 in
nNOS,7 and m-fluoro inhibitor 1b achieves the optimum balance
between potency and selectivity.
’ CONCLUSION
An improved synthesis of chiral pyrrolidine inhibitors of nNOS
has been developed. Compared with the reported synthesis, it is
three steps shorter with an overall yield of ∼10% (>10-fold larger). It
also enables expanded SAR studies on the pyrrolidine-based scaffold,
which can be beneficial for further development of nNOS inhibitors.
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BRIEF ARTICLE
80.0, 111.5, 112.8, 112.9, 115.1, 120.2, 120.4, 123.1, 137.47, 137.48, 147.95,
156.0, 159.2, 163.2, 165.2; LC-TOF (M + H+) calcd for C21H26ClF3N4O
443.1747, found 443.3306.
chloride; DMAP, 4-dimethylaminopyridine; TBAF, tetrabutyl-
ammonium fluoride; SAR, structureÀactivity relationship
6-(((3R,4R)-4-(2-((2,2-Difluoro-2-(2,3-difluorophenyl)ethyl)-
amino)ethoxy)pyrrolidin-3-yl)methyl)-4-methylpyridin-2-
amine (8e). Inhibitor 8e was synthesized as a mixture of two diastere-
omers using a procedure similar to that for 8a (12 mg, 75%) as a tri-HCl salt:
1H NMR (500 MHz, MeOD) δ 2.35 (s, 3H), 2.82À2.88 (m, 1H),
2.95À3.00 (m, 1H), 3.08À3.14 (m, 1H), 3.23À3.30 (m, 1H), 3.40À
3.44 (m, 1H), 3.53À3.55 (m, 2H), 3.60 (d, J = 13 Hz, 1H), 3.77À3.79 (m,
1H), 3.95À3.97 (m, 1H), 4.04À4.10 (m, 2H), 4.19À4.20 (m, 1H), 6.66 (s,
1H), 6.67 (s, 1H), 7.32À7.55 (m, 3H); 13C NMR (125 MHz, MeOD) δ
22.0, 30.35, 30.40, 43.5, 49.9, 50.4, 52.5, 65.0, 79.9, 111.5, 115.1, 122.2,
122.3, 123.5, 126.7, 137.47, 137.48, 147.9, 148.0, 156.0, 159.2; LC-TOF
(M + H+) calcd for C21H26F4N4O 427.2043, found 427.4021.
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S
Supporting Information. Procedure for the syntheses of
b
4, 5, 6, 7aÀb, 9, 10, 11, 12, and 13aÀe, enzyme assays of 8aÀe,
and crystal structures of 8aÀe in the active site of enzyme and
crystallographic statistics table. This material is available free of
charge via the Internet at http://pubs.acs.org.
(6) Lawton, G. R.; Ranaivo, H. R.; Wing, L. K.; Ji, H.; Xue, F.;
Martesek, P.; Roman, L. J.; Watterson, D. M.; Silverman, R. B. Analogues
of 2-aminopyridine-based selective inhibitors of neuronal nitric oxide
synthase with increased bioavailability. Bioorg. Med. Chem. 2009,
17, 2371–2380.
Accession Codes
†The PDB codes for compounds in Figure 1 are as follows:
nNOS-8a, 3PNE; nNOS-8b, 3PNF; nNOS-8c, 3PNG; eNOS-
8c, 3PNH; nNOS-8d, 3SVP; nNOS-8e, 3SVQ.
(7) Xue, F.; Li, H.; Delker, S.; Fang, J.; Martꢀasek, P.; Roman, L. J.;
Poulos, T. L.; Silverman, R. B. Potent, highly selective, and orally
bioavailable gem-difluorinated monocationic inhibitors of neuronal
nitric oxide synthase. J. Am. Chem. Soc. 2010, 132, 14229–14238.
(8) Xue, F.; Fang, J.; Lewis, W. W.; Martasek, P.; Roman, L. J.;
Silverman, R. B. Potent and selective neuronal nitric oxide synthase
inhibitors with improved cellular permeability. Bioorg. Med. Chem. Lett.
2010, 20, 554–557.
(9) Xue, F.; Li, H.; Kraus, J. M.; Ji, H.; Mataka, J.; Labby, K. J.;
Poulos, T. L.; Silverman, R. B. Unpublished results.
(10) Xue, F.; Silverman, R. B. An alkoxide anion triggered tert-
butyloxycarbonyl group migration. Mechanism and application. Tetra-
hedron Lett. 2010, 51, 2536–2538.
’ AUTHOR INFORMATION
Corresponding Author
*For T.L.P.: phone, 949-824-7020; e-mail, poulos@uci.edu. For
R.B.S.: phone, 847-491-5653; fax, 847-491-7713; e-mail, Agman@
chem.northwestern.edu.
Present Addresses
#Department of Pharmaceutical Sciences, University of Maryland
School of Pharmacy, 20 N. Pine Street, Room N719, Baltimore,
Maryland 21201, United States.
(11) Haight, A. R.; Stoner, E. J.; Peterson, M. J.; Grover, V. K.
General method for the palladium-catalyzed allylation of aliphatic
alcohols. J. Org. Chem. 2003, 68, 8092–8096.
Author Contributions
∞These authors contributed equally to this paper.
’ ACKNOWLEDGMENT
We are grateful to the National Institutes of Health Grants
GM049725 to R.B.S., GM057353 to T.L.P., and GM052419 to
Dr. Bettie Sue Siler Masters (B.S.S.M.), with whose lab P.M. and
L.J.R. are affiliated, and Grant AQ1192 from The Robert A.
Welch Foundation to B.S.S.M. B.S.S.M. is also grateful to the
Welch Foundation for a Robert A. Welch Foundation Distin-
guished Professorship in Chemistry (Grant AQ0012). P.M. is
supported by Grants 0021620806 and 1M0520 from MSMT of
the Czech Republic for financial support of this research. We
thank the SSRL beamline staff for their support during remote
X-ray diffraction data collection.
’ ABBREVIATIONS USED
nNOS, neuronal nitric oxide synthase; NO, nitric oxide; eNOS,
endothelial nitric oxide synthase; iNOS, inducible nitric oxide
synthase; n-BuLi, n-butyllithium; TBSCl, tert-butyldimethylsilyl
6403
dx.doi.org/10.1021/jm200411j |J. Med. Chem. 2011, 54, 6399–6403