Intramolecular hydrogen bonding: A potential strategy for more bioavailable inhibitors of neuronal nitric oxide synthase

Article abstract of DOI:10.1016/j.bmc.2012.01.037

Selective neuronal nitric oxide synthase (nNOS) inhibitors have therapeutic applications in the treatment of numerous neurodegenerative diseases. Here we report the synthesis and evaluation of a series of inhibitors designed to have increased cell membran

Full text of DOI:10.1016/j.bmc.2012.01.037

Bioorganic & Medicinal Chemistry 20 (2012) 2435–2443
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Intramolecular hydrogen bonding: A potential strategy for more
bioavailable inhibitors of neuronal nitric oxide synthase
Kristin Jansen Labby a, Fengtian Xue a, , James M. Kraus a, Haitao Ji a,à, Jan Mataka a, Huiying Li b,
a,
Pavel Martásek c,d, Linda J. Roman c, Thomas L. Poulos b, , Richard B. Silverman
⇑
⇑
a Department of Chemistry, Department of Molecular Biosciences, Chemistry of Life Processes Institute, Center for Molecular Innovation and Drug Discovery, Northwestern
University, 2145 Sheridan Road, Evanston, IL 60208-3113, USA
b Departments of Molecular Biology and Biochemistry, Chemistry, and Pharmaceutical Sciences, University of California, Irvine, Irvine, CA 92697-3900, USA
c Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX, USA
d Department of Pediatrics, 1st School of Medicine, Charles University, Prague, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Selective neuronal nitric oxide synthase (nNOS) inhibitors have therapeutic applications in the treatment
of numerous neurodegenerative diseases. Here we report the synthesis and evaluation of a series of
inhibitors designed to have increased cell membrane permeability via intramolecular hydrogen bonding.
Their potencies were examined in both purified enzyme and cell-based assays; a comparison of these
results demonstrates that two of the new inhibitors display significantly increased membrane permeabil-
ity over previous analogs. NMR spectroscopy provides evidence of intramolecular hydrogen bonding
under physiological conditions in two of the inhibitors. Crystal structures of the inhibitors in the nNOS
active site confirm the predicted non-intramolecular hydrogen bonded binding mode. Intramolecular
hydrogen bonding may be an effective approach for increasing cell membrane permeability without
affecting target protein binding.
Received 9 December 2011
Revised 18 January 2012
Accepted 22 January 2012
Available online 7 February 2012
Keywords:
Neuronal nitric oxide synthase
Enzyme inhibitors
Intramolecular hydrogen bond
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
migraine headaches,4 as well as neuronal damage in stroke.5 High
NO concentrations provide a rationale for the therapeutic use of
Neurodegeneration, characterized by progressive loss of neu-
rons in isolated areas of the central nervous system (CNS), has
become a rapidly spreading health crisis in the world. It affects
the lives of millions of people, causing problems with motor skills
and memory. Although significant research advances have been
made, effective treatments for neurodegenerative disorders have
remained elusive. An abnormally high concentration of cerebral
nitric oxide (NO), the metabolite of the neuronal isoform of nitric
nNOS inhibitors as a potential simultaneous treatment for different
types of neurodegeneration. In the past two decades, a large num-
ber of nNOS inhibitors have been reported.6–9 However, none of
these compounds has been approved for medical use. Recently,
we reported a family of syn-3,4-substituted pyrrolidine inhibitors
(e.g., 1–3, Fig. 1) that were discovered using structure-based drug
design.10–13 These inhibitors showed remarkable potency for nNOS
and excellent isoform selectivity over endothelial NOS (eNOS) and
inducible NOS (iNOS). We have previously found that the (3S,4S)-
isomers of 1 and 3 bind to nNOS in the predicted mode with the
3-fluorophenyl group bound in the hydrophobic cleft comprised
of M336 and L337 and the aminopyridine interacting with E592,
but unexpectedly the (3R,4R)-isomers bind with 180o rotation with
oxide synthases (nNOS), has been observed as
a common
pathogenic phenomenon shared by various neurodegenerative
conditions, including Alzheimer’s,1 Parkinson’s,2 Huntington’s,3
Abbreviations: CNS, central nervous system; NO, nitric oxide; nNOS, neuronal
nitric oxide synthase; eNOS, endothelial nitric oxide synthase; iNOS, inducible
nitric oxide synthase; BBB, blood–brain barrier; H-bond, hydrogen bond; L-NNA,
-N-nitroarginine; H4B, tetrahydrobiopterin.
Corresponding authors. Tel.:+1 847 491 5653; fax: +1 847 491 7713 (R.B.S.);
tel.:+1 949 824 7020. (T.L.P.).
E-mail addresses: poulos@uci.edu (T.L. Poulos), Agman@chem.northwestern.edu
(R.B. Silverman).
Present address: Department of Pharmaceutical Sciences, University of Maryland
School of Pharmacy, 20 North Pine Street, Baltimore, MD 21201, USA
the 3-fluorophenyl group p-stacked over the active site heme and
the aminopyridine electrostatically interacting with a carboxylate
of heme propionate.11Also surprising, the (3R,4R)-isomers are more
potent than the (3S,4S)-isomers. However, results from animal
tests indicated that racemic mixtures of 1 and 3 (and their
corresponding 3R,4R stereoisomers) show minimal blood–brain
barrier (BBB) penetration;13 the BBB is a unique barrier formed
by brain capillary endothelial cells that molecules must be able
to penetrate to be effective in the CNS. This result limits further
investigation of these lead compounds as oral therapeutics. Given
L
⇑
à
Present address: Department of Chemistry, University of Utah, Salt Lake City, UT
84112, USA
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.01.037

2436
K. J. Labby et al. / Bioorg. Med. Chem. 20 (2012) 2435–2443
and binds to nNOS, it may encounter various intermolecular
interactions and stabilize an ‘open’ (not intramolecular hydrogen
bonded) conformation. In this conformation, the pharmacologically
critical amino group may be released from the intramolecular H-
bond. Compounds 4a–d have benzyl-like aryl substituents like 225
rather than phenylethyl-like aryl substituents like 126 and 3,26so
that 6-membered instead of 7-membered intramolecular hydrogen
bonds would form. Therefore, we chose to make the (3S,4S)-isomers
that bind in the normal mode, despite the generally lower potency
in the phenylethyl series; computer modeling suggested that with
a one-carbon shorter side chain compounds would have better
binding, although the (3R,4R) compounds have not been made to
confirm. Six-membered intramolecular H-bonds are most common,
the majority of which are linked by sp2-hybridized atoms and are
therefore part of planar conjugated systems; five-membered intra-
molecular hydrogen bonds also have precedence.18
Figure 1. Chemical structures and inhibitory activities of inhibitors (3S,4S)-1 and
(3S,4S)-3 and ( )-2.
2. Chemistry
The synthesis of inhibitors 4a–d began with chiral precursor 5
(Scheme 1).27 Allylation of 5 using allyl methyl carbonate in the
presence of Pd(PPh3)4 gave alkene 6 in good yields.28 Ozonolysis
of 6 yielded aldehyde 7, which was subjected to reductive amina-
tion with amines to provide 8a–d. Global deprotection of the three
Boc-protecting groups with TFA generated inhibitors 4a–d in quan-
titative yields.
the chemical structures of the inhibitors, we reasoned that the
multiple positive charges and hydrogen bond donor properties of
1–3 at physiological pH, derived from the amino groups, limit them
from penetrating the BBB by passive diffusion.13 Lead compound 3
was designed to eliminate one of the hydrogen bond donors of 1,
the amino group attached to the pyrrolidine ring, by replacing it
with a hydrogen bond acceptor ether linkage; hydrogen bond do-
nors are believed to lower the ability to cross the BBB more than
hydrogen bond acceptors. Supporting our rationale, BBB penetra-
tion was slightly improved from the design of 1 to 3, but there is
still much room for improvement in the bioavailability of these
selective inhibitors.13
3. Results and discussion
3.1. Assay with purified nNOS
The potencies of these compounds have been tested both in a
purified enzyme assay and in a cell-based assay. The purified en-
zyme assay results demonstrate that 4a–d have lower potencies
for nNOS and lower selectivities over eNOS and iNOS in compari-
son with our previous inhibitors 1–3 (Table 1),25,26 but compounds
4a–d are proof-of-principle compounds to test their ability to per-
meate a cell membrane.
In previous efforts we pursued different strategies to modify the
structure of compounds in this class.13–17 For example, the high
pKa amino groups have been replaced by neutral functionalities
such as ethers and amides.13 We have also inserted electron-with-
drawing groups, such as ether, monofluoromethylene, difluoro-
methylene, and cyclopropyl, to inductively decrease the pKa of
the amino group.14–17 Despite the improved membrane permeabil-
ity of the new inhibitors, the successes of these modified structures
were compromised by a significant drop in the in vitro potency and
isoform selectivity.
3.2. Assay in HEK293t cells
Here we describe the design and synthesis of a new series of
nNOS inhibitors (4a–d, Fig. 2) to diffuse the overall charge of 3 by
incorporation of an intramolecular hydrogen bond, a known strat-
egy in the design of novel inhibitors for a variety of enzymes,18–23
sometimes used to improve BBB penetration.24 In the highly hydro-
phobic environment of the BBB, the inhibitor is hypothesized to
adopt a ‘closed’ conformation by forming an intramolecular H-bond,
which decreases the overall polarity of the compound to improve
BBB permeability. On the other hand, when the inhibitor reaches
We have employed a simple, colorimetric, cell-based assay
using HEK293t cells stably transfected with and expressing nNOS
to assess the potencies of these compounds in a cellular environ-
ment.29 Although other processes may be influencing nNOS inhibi-
tion in these assays, cell permeability can be predicted from the
cellular potencies of these compounds as measured by the inhibi-
tion of the production of nitric oxide in the HEK293t cells (Table 2).
Comparing the IC50 values of these compounds in the purified
enzyme assay versus the cellular assay clearly indicates that
Figure 2. Chemical structures of (S.S)-4a–d. The desired hydrogen bond is shown in red. Inhibitor 4c may take on a conformation in which the –OH acts as both a H-bond
acceptor and donor, delocalizing the positively charged nitrogen.

K. J. Labby et al. / Bioorg. Med. Chem. 20 (2012) 2435–2443
2437
Scheme 1. Synthesis of 4a–d. Reagents and conditions: (a) allyl methyl carbonate, Pd(PPh3)4, 45 °C, 5 h, 66%; (b) O3, ꢀ78 °C, 30 min, (ii) Me2S, ꢀ78 °C to rt, 2 h, 87%; (c)
amines, NaHB(OAc)3, rt, 3 h, 91%; (d) 6 N HCl in MeOH (2:1), rt, 12 h, 99%.
same (cell based IC50/purified enzyme IC50 = 1.0). The lower the
RPI, the more cell membrane-penetrable is the compound. There-
fore, 4a and 4b have the highest cell penetrability compared to
1–3, and 4c is comparable in penetrability relative to 3, despite
the poorer inhibitory properties of these compounds relative to
1–3. As depicted in Figure 2, however, 4c had the greatest theoret-
ical potential to neutralize the charge on the ammonium ion, yet it
did not exhibit high cell permeability. That may suggest that the
phenolic hydroxyl group does not act as a hydrogen bond donor.
Compound 4d, with the highest IC50 of all compounds in the table,
is nonetheless more cell-penetrable than 1 and 2. We also tested
just the aminopyridine fragment of inhibitors 1–4 (2-amino-4,6-
dimethylpyridine hydrochloride), which is a potent, nonselective
inhibitor of purified nNOS (50 nM); however, its RPI is much higher
than any of our inhibitors, indicating that it poorly crosses the cell
Table 1
Kiavalues of 1–3 and 4a–d with all three NOS isoforms
Compound
nNOS (
l
M)
eNOS (
l
M)
iNOS (
lM)
Selectivityb
n/e n/i
505
1c
0.052
0.085
0.12
0.41
0.82
0.39
0.40
26
85
26
40
77
37
66
3.9
9.4
7.5
44
114
57
74
110
65
2d
1000
226
98
3c
4a
4b
4c
4d
105
139
143
92
93
93
36
169
a
The Ki values were calculated based on the directly measured IC50 values (see
methods), which represent at least triplicate measurements with standard devia-
tions of 10%.
b
The ratio of Ki (eNOS or iNOS) to Ki (nNOS).
Data from Ref. 26
Data from Ref. 25
c
d
membrane despite having a neutral pKa. Although
L-N-nitroargi-
nine ( -NNA) is a relatively poor inhibitor of purified nNOS, it ap-
L
pears to penetrate the cell membrane freely, having the same
IC50 value in the cell-based assay as the isolated enzyme assay
and a RPI of 1.
It is interesting to note that the Relative Permeability Index has
little correlation with partition coefficients for the inhibitors. As
calculated by Chem Axon,30 the cLogP values are as follows: 2.18
(1); 2.35 (2); 2.49 (3); 2.2 (4a); 1.9 (4b); ꢀ0.09 (4c); 1.12 (4d).
membrane permeability is a major factor that limits the effective
concentration of these inhibitors within cells. Therefore, the Rela-
tive Permeability Index (RPI) is a measure of how similar the IC50
value is for a compound as measured in the cell-based assay rela-
tive to the purified enzyme assay. If a compound freely passed
through the cell membrane, then those IC50 values should be the
Table 2
IC50 valuesa of NOS inhibitors in purified enzyme assay and cell-based assay. See methods for assay details
Compound
Purified IC50
(lM)
Cell-based IC50
(l
M)
Relative Permeability Index (RPI)b
(cell based IC50/purified IC50
)
1
2
3
4a
4b
4c
4d
0.45
0.74
1.0
3.6
7.1
3.5
3.4
0.05
5.0
19
42
13
11
22
43
100
25
42
57
13
3.1
3.1
12
29
500
1
Aminopyridinec
-NNAd
5.0
L
a
See methods. IC50 values represent at least triplicate measurements with standard deviations of less than10%.
The smaller the number, the closer the difference in IC50 for the cell-based assay and purified enzyme assay, which suggests improved permeability
b
into cells.
c
aminopyridine = 2-amino-4,6-dimethylpyridine hydrochloride.
d
L
-NNA = L-N-nitroarginine.

2438
K. J. Labby et al. / Bioorg. Med. Chem. 20 (2012) 2435–2443
Figure 3. (A) Binding conformations of 4a–d overlaid: 4a (green), 4b (cyan), 4c (yellow), 4d (magenta); heme is shown in light pink; H4B (tetrahydrobiopterin) is blue. The
enzyme conformation is highly conserved between all four structures; therefore only the active site residues from the crystal structure of 4a are shown. Met336 of the 4d
crystal structure is also shown (magenta) because of the difference in its configuration. (B). Binding conformations of 1 (3JWS, cyan), 3 (3NLK, magenta) and 4a (green),
overlaid. Residues overlay almost exactly; therefore only those corresponding to the crystal structure of 4a are shown. Hydrogen bonds are depicted with dashed lines. PDB
codes: nNOS-4a, 3TYL; nNOS-4b, 3TYM; nNOS-4c, 3TYN; nNOS-4d, 3TYO.
For this series, cLogP seems to be a poor predictor of permeability
as measured by the NOS cell-based assay.
3.3. Crystallography of 4a–d bound to nNOS
Crystal structures of all four compounds bound to nNOS were
obtained, showing that when bound to nNOS, they all adopt the
open, non-intramolecular hydrogen bonded form (Fig. 3). Inhibi-
tors 4a–d (like 1–3), bear a (3S,4S) configuration at the pyrrolidine
ring, and all bind to the nNOS active site in the normal binding
mode, with the 2-amino-4-methylpyridine stacked over the heme
plane and hydrogen bonding with Glu592.11 The pyrrolidine nitro-
gen hydrogen bonds with Glu592 and interacts through a water-
bridged hydrogen bond with Asp597, the residue that can be
exploited for selectivity between nNOS and eNOS (Asn368 in
eNOS). The amino group that was designed to form an intramolec-
ular hydrogen bond with the substituent from the tail aromatic
ring is exposed in the crystal structures and hydrogen bonds with
heme propionate D in all four inhibitors. Compounds 4a–c have a
substituted phenyl tail, but 4d contains a furan in place of the phe-
nyl ring. The substituted phenyls and the furan ring occupy the
pocket lined with hydrophobic residues Met336, Leu337, and
Tyr706. The furan is smaller than the phenyl substituents, so it is
able to accommodate a different rotamer of Met336 in which it ro-
tates in toward the inhibitor molecule. The slightly higher Ki of 4b,
the methoxyl-substituted inhibitor, may result from a slight steric
clash with Trp306 (subunit B) in this pocket.
Figure 3B shows a comparison of the binding mode of 4a–d with
that of previously designed (3S,4S) inhibitors 1–3 using an overlay
of 1 and 3 with 4a. Inhibitor 1 (2 as well) contains a nitrogen atom
in place of the oxygen atom adjacent to the pyrrolidine in 3 and 4a–
d. It is this amino nitrogen in 1 (and 2) that makes a hydrogen bond
with a different heme propionate (of pyrrole A). The ether oxygen in
3 and 4a–d does not have a proton to make this hydrogen bond. In-
stead, 3 and 4a–d all curl so that the other amino group next to the
aromatic tail group can hydrogen bond with heme propionate D.
The inhibitors with an amine next to the pyrrolidine (1 and 2), gen-
erally bind tighter to nNOS (Fig. 1) than the ones bearing an ether
oxygen at the same position (3 and 4a–d, Table 1). However, the
Figure 4. 1H NMR spectra of 4a–d taken in D2O at 25 °C. Doublets appear in place of
the singlet usually seen with compounds of this scaffold for the methylene protons
adjacent to the H-bonded amine in 4b and 4c; this is evidence for the H-bond. In
both cases, the doublet further upfield overlaps with the triplet from one of the
pyrrolidine hydrogens; a coupling value cannot be reported for the doublet further
upfield but is presumed to be the same.
ether inhibitors show better bioavailability.14,17 Another noticeable
difference in the crystal structure between inhibitors 1 and 3 and

K. J. Labby et al. / Bioorg. Med. Chem. 20 (2012) 2435–2443
2439
series 4a–d occurs because of oneless carbon unit in the chain to the
phenyl substituent in the latter. The hydrophobic pocket is large en-
ough to accommodate either chain length. However, the inhibitors
with a longer chain length make tighter van der Waals contacts
with Met336, Leu337, and Tyr706, thus showing better binding
affinity than the shorter tailed 4a–d. Inhibitor 2 has the same chain
length as 4a–d but 2 has better binding affinity than 4a–d. This is
because the amino group next to the pyrrolidine in 2 hydrogen
bonds to heme propionate A, so that the rest of its fluorophenyl tail
adopts an extended conformation rather the curled one seen in 4a–
d, allowing tighter hydrophobic interactions with the surrounding
protein.
In contrast, the observed signal in the NMR spectra for the
methylene protons of 4a and 4d (Fig. 4) is the expected singlet at
approximately d 4.2 ppm. In these two compounds the methylene
protons are also diastereotopic (as in 4b and 4c), but the lack of a
stable interaction to differentiate the protons of the ammonium
gives rise to a singlet for the methylene protons. Therefore, there
is likely no significant intramolecular hydrogen bonding interac-
tion occurring in these compounds at room temperature in an
aqueous environment, and more importantly, not under physiolog-
ical conditions (37 °C). Fluorine-mediated hydrogen bonds are
weak (if observable at all), and these results (4a) are consistent
with this observation. A five-membered intramolecular hydrogen
bond (4d) is known to be the least likely to form.18
3.4. NMR spectroscopy to determine extent of hydrogen
bonding
An examination of the NMR spectra of 4b and 4c in D2O at var-
ious temperatures provides further evidence of the hydrogen bond
character. The two doublets shift at higher temperatures, as seen in
Fig. 5. Unfortunately the doublets of 4b are slightly obscured by the
shift of the pyrrolidine proton (at 25 °C, d 4.12 ppm) and spectral
resolution decreases at 95 °C; nonetheless, Fig. 5 illustrates that
the chemical shifts of the doublets are moving closer together as
the temperature increases. In both inhibitors, hydrogen bonding
is observed in water at physiological pH, and the temperature at
which doublets would be expected to collapse into a singlet is well
above the physiological temperature of 37 °C. This suggests that
the intramolecular hydrogen bonds of 4b and 4c are stable in solu-
tion at high temperatures and that this approach would be amena-
ble to enhancing membrane permeability in vivo.
Inspection of the 1H NMR spectra of these compounds in D2O at
room temperature shows a striking difference in the splitting pat-
tern of the methylene group (d 4.2 ppm) in the arylalkylside chain
(Fig. 4). When the intramolecular H-bond forms, the interaction of
the phenyl ortho-substituent differentiates the two protons of the
ammonium and makes the ammonium nitrogen chiral.31 This
anisotropy32–34 is evident as a second-order effect in the 1H NMR
spectrum because the side chain methylene protons are diastereo-
topic. In the 1H NMR spectra of 4b and 4c, these protons exhibit an
AB pattern, two separate but coupled doublets, instead of the sin-
glet expected for these identical protons. The average shift of the
AB quartet is centered at the chemical shift of the expected singlet,
and the doublets for the individual protons are found shifted up-
field or downfield.
Interestingly, 4a and 4b demonstrate the highest relative
permeability (Table 2), although it is 4b and 4c that display NMR
spectral evidence of a H-bond. The o-fluorophenyl group of
Figure 5. 1H NMR spectra of 4b and 4c in D2O at 95, 85, 55, 25, and 5 °C. The AB doublets shift toward one another at higher temperatures. In the spectra of 4b, the doublet
further upfield is obscured by the shift of a pyrrolidine proton (d 4.12 at 25 °C). The J value within the doublets remains the same, J = ꢁ13 Hz. The difference between the
chemical shifts (D) is reported to better illustrate the changes in the chemical shifts, as instrument resolution is lost at higher temperatures.

2440
K. J. Labby et al. / Bioorg. Med. Chem. 20 (2012) 2435–2443
permeable inhibitor 4a is the most lipophilic side chain group of
the series and also the most electron withdrawing; the increased
electron withdrawing character lowers the pKa of the ammonium
group, making it less basic. Both of these properties might account
for the enhanced permeability of 4a. This is consistent with the
findings of Ballet et al. who concluded that lipophilicity rather than
side chain flexibility is the key determinant for BBB penetration.35
Compound 4c, despite its ability to form an intramolecular hydro-
gen bond has a polar phenolic hydroxyl group, which would hinder
cell permeability.
mum 5 volumes. The compounds were eluted with a gradient from
solvent A (water, 0.1% TFA) to solvent B (acetonitrile, 0.1% TFA), 0–
5 min, 0–25% B, 5–10, 25–35% B, 10–20 35% isocratic. The com-
pounds had the following retention times, 4a, 11.2 min, 4b,
11.6 min, 4c, 11.4 min, 4d, 10.5 min.
5.2. Preparation and characterization of new compounds
5.2.1. (3S,4S)-tert-Butyl-3-(allyloxy)-4-((6-(bis(tert-butoxy-
carbonyl)amino)-4-methylpyridin-2-yl)methyl)pyrrolidine-
1-carboxylate (6)
To a solution of 5 (1.15 g, 2.0 mmol) and Pd(Ph3P)4 (235 mg,
0.2 mmol) in dry THF (50 mL) was added allyl methyl carbonate
4. Conclusion
(700 lL, 6.0 mmol). The reaction mixture was allowed to stir at
New nNOS inhibitors 4a–d have been synthesized derived from
chiral pyrrolidine lead 2. Crystal structures confirm that these com-
pounds bind to nNOS in the ‘open’ conformation, while 1H NMR
spectra provide evidence of intramolecular hydrogen bonding, the
‘closed’ conformation, in two of the compounds (4b and 4c). It ap-
pears, however, that incorporation of an intramolecular hydrogen
bond is not sufficient to enhance cell membrane permeability; lipo-
philicity seems to play a larger role, as evidenced by the good per-
meability of 4a and the poor permeability of 4c. Inhibitors 4a and
4b demonstrate greatly improved cell permeability when com-
pared to previous inhibitors 1–3. For inhibitor 4b, its improved per-
meability likely results from intramolecular hydrogen bonding to
diffuse the positive charge on the vicinal ammonium ion. Once
the compounds enter the active site of nNOS, however, intermolec-
ular binding interactions overcome the intramolecular hydrogen
bonds, resulting in the predicted open-conformation binding mode.
This suggests that intramolecular hydrogen bonding can accom-
plish the desired decreased polarity for enhanced cell membrane
permeability, but not interfere with desired binding interactions
in the target protein. The purpose of these studies was to demon-
strate the effect of hydrogen bonding on cell permeability; the next
step in the process will be to modify the compounds for better bind-
ing potency without loss of cell membrane permeability.
45 °C for 5 h and then concentrated. The resulting material was
purified by flash column chromatography (silica gel, EtOAc/hex-
anes, 1:2, Rf = 0.40) to yield 6 (675 mg, 66%) as a colorless oil: 1H
NMR (500 MHz, CDCl3) d 1.40–1.50 (m, 27H), 2.25–2.27 (m, 3H),
2.60–2.75 (m, 1H), 2.78–2.85 (dd, J = 9.0, 13.5 Hz, 1H), 2.98–3.05
(dd, J = 9.0, 13.5 Hz, 1H), 3.10–3.21 (m, 1H), 3.25–3.29 (dd, J = 4.0,
12.5 Hz, 1H), 3.40–3.62 (m, 2H), 3.75–3.85 (m, 2H), 4.00–4.10 (td,
J = 5.5, 13.0 Hz, 1H), 5.15–5.17 (d, J = 10.5 Hz, 1H), 5.25–5.29 (d,
J = 17.0 Hz, 1H), 5.84–5.91 (ddd, J = 5.0, 10.5, 17.0 Hz, 1H), 6.85–
6.95 (m, 2H); 13C NMR (125 MHz, CDCl3) d 20.9, 27.9, 28.4, 28.5,
34.7, 34.8, 42.7, 43.3, 48.9, 49.2, 50.4, 51.0, 70.2, 70.3, 77.8, 78.6,
79.1, 79.2, 82.8, 116.7, 116.9, 119.6, 122.9, 134.6, 134.7, 149.50,
149.52, 151.4, 151.5, 151.8, 154.5, 154.8, 159.2, 159.3; LC-TOF
(M+H+) calcd for C29H46N3O7 548.3336, found 548.3339.
5.2.2. (3S,4S)-tert-Butyl-3-((6-(bis(tert-butoxycarbonyl)amino)-
4-methylpyridin-2-yl)methyl)-4-(2-oxoethoxy)pyrrolidine-1-
carboxylate (7)
A solution of 6 (100 mg, 0.19 mmol) in CH2Cl2 (10 mL) was
cooled to ꢀ78 °C, to which O3 was charged until the reaction solu-
tion turned purple (ꢁ10 min). The O3 flow was stopped, and the
reaction was allowed to stir at the same temperature for 30 min.
To the resulting solution was added Me2S (150 lL). The reaction
mixture was then warmed to room temperature and kept stirring
at room temperature for an additional 2 h. The solvent was removed
by rotary evaporation and the resulting crude product was purified
by flash column chromatography (EtOAc/hexanes, 1:1, Rf = 0.2) to
yield 6 (87 mg, 87%) as a colorless oil: 1H NMR (500 MHz, CDCl3) d
1.45 (s, 27H), 2.22 (s, 3H), 2.70–2.85 (m, 1H), 2.85–2.95 (m, 1H),
3.05–3.15 (m, 1H), 3.16–3.25 (m, 1H), 3.30–3.37 (m, 1H), 3.45–
3.70 (m, 2H), 3.85–3.95 (m, 2H), 4.05–4.20 (t, J = 10.0 Hz, 1H),
6.90–6.93 (m, 2H), 9.66 (s, 1H); 13C NMR (125 MHz, CDCl3) d 20.9,
24.7, 27.9, 28.5, 29.7, 34.4, 42.5, 43.2, 48.8, 49.1, 50.3, 51.0, 74.6,
74.9, 79.4, 79.5, 80.4, 83.0, 119.66, 119.73, 122.8, 149.7, 149.8,
151.57, 151.60, 151.8, 154.4, 154.8, 159.87, 158.94, 200.2, 200.6;
LC-TOF (M+H+) calcd for C28H44N3O8 550.3128, found 550.3130.
5. Experimental section
5.1. General methods
All experiments were conducted under anhydrous conditions in
an atmosphere of argon, using flame-dried apparatus and employ-
ing standard techniques in handling air-sensitive materials. All
solvents were distilled and stored under an argon or nitrogen atmo-
sphere before using. All reagents were used as received. Aqueous
solutions of sodium bicarbonate, sodium chloride (brine), and
ammonium chloride were saturated. Analytical thin layer chroma-
tography was visualized by ultraviolet light. Flash column chroma-
tography was carried out under a positive pressure of nitrogen. 1H
NMR spectra were recorded on 500 MHz spectrometers. Data are
presented as follows: chemical shift (in ppm on the d scale relative
to d = 0.00 ppm for the protons in tetramethylsilane (TMS)), integra-
tion, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet,
m = multiplet, br = broad), coupling constant (J/Hz). Coupling con-
stants 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 d scale with an internal reference
of d 77.0 or 49.0 for CDCl3 or MeOD, respectively. High-resolution
mass spectra were measured by liquid chromatography/time-of-
flight mass spectrometry (LC-TOF). The purity of the final com-
pounds was determined by HPLC analysis to be P95%. The column
5.3.3. (3S,4S)-tert-Butyl-3-((6-(bis(tert-butoxycarbonyl)amino)-
4-methylpyridin-2-yl)methyl)-4-(2-(2-fluorobenzylamino)etho
xy)pyrrolidine-1-carboxylate (8a)
To a solution of aldehyde 7 (100 mg, 0.18 mmol) in DCM (4 mL)
was added 2,2-difluoro-2-(3-fluorophenyl)ethanamine hydrochlo-
ride (83 mg, 0.36 mmol), triethylamine (11 lL, 0.36 mmol), fol-
lowed by NaHB(OAc)3 (100 mg, 0.45 mmol). The reaction mixture
was stirred for an additional 3 h and then concentrated. The crude
product was purified by flash column chromatography (EtOAc/
hexanes, 1:1, Rf = 0.15) to yield 8a (115 mg, 91%) as a colorless
oil: 1H NMR (500 MHz, CDCl3) d 1.43–1.44 (s, 27H), 2.29–2.30 (m,
3H), 2.60–2.75 (m, 1H), 2.76–2.85 (m, 2H), 2.92–3.00 (m, 1H),
3.05–3.14 (m, 1H), 3.20–3.50 (m, 5H), 3.52–3.70 (m, 2H), 3.72–
3.80 (m, 1H), 3.91 (s, 2H), 6.84–6.85 (d, J = 8.0 Hz, 1H), 6.89–6.91
used was a Higgins Analytical, TARGAÒ C18, 5
n: TS-2546-C185, The Nest Group, Inc., http://www.nestgrp.com).
The column was thoroughly equilibrated at 100% solvent A, mini-
l
m, 4.6 ꢂ 250 mm (p/

K. J. Labby et al. / Bioorg. Med. Chem. 20 (2012) 2435–2443
2441
(d, J = 10.0 Hz, 1H), 7.02–7.06 (dd, J = 9.0, 9.5 Hz, 1H), 7.10–7.13
(dd, J = 7.0, 7.5 Hz, 1H), 7.20–7.30 (m, 1H), 7.35–7.40 (m, 1H); 13C
NMR (125 MHz, CDCl3) d 20.9, 24.7, 27.9, 28.47, 28.50, 29.7, 34.6,
34.7, 36.6, 42.6, 43.3, 46.8, 48.2, 48.8, 49.1, 50.3, 50.8, 68.0, 68.2,
78.7, 79.2, 79.3, 79.4, 82.8, 115.2, 115.3, 115.4, 115.5, 119.5,
119.6, 122.8, 124.17, 124.19, 128.89, 128.92, 128.95, 128.99,
130.46, 130.51, 130.55, 149.6, 151.4, 151.5, 151.8, 154.5, 154.8,
159.08, 159.14, 160.3, 162.2; LC-TOF (M+H+) calcd for C35H52FN4O7
659.3820, found 659.3818.
stirred for 12 h and then concentrated. The crude product was
purified by recrystallization (EtOH/H2O) to give inhibitor 4a
(38 mg, 97%): 1H NMR (500 MHz, D2O) d 2.18 (s, 3H), 2.68–2.73
(m, 1H), 2.76–2.82 (dd, J = 7.0, 15.5 Hz, 1H), 2.85–2.90 (dd, J = 8.0,
15.0 Hz, 1H), 3.04–3.09 (t, J = 11.5 Hz, 1H), 3.15–3.25 (m, 2H),
3.39–3.43 (dd, J = 8.5, 11.5 Hz, 1H), 3.50–3.53 (d, J = 13.5 Hz, 1H),
3.53–3.60 (m, 1H), 3.73–3.80 (m, 1H), 4.05–4.10 (m, 1H), 4.22 (s,
2H), 6.47 (s, 1H), 6.53 (s, 1H), 7.12–7.14 (dd, J = 1.0, 8.5 Hz, 1H),
7.16–7.18 (dd, J = 1.0, 8.0 Hz, 1H), 7.35–7.40 (m, 1H); 13C NMR
(125 MHz, D2O) d 21.0, 28.8, 41.3, 44.67, 44.70, 46.4, 47.1, 49.3,
63.9, 78.0, 110.3, 114.0, 115.8, 115.9, 117.4, 117.5, 125.0, 125.1,
132.1, 132.2, 132.3, 132.4, 145.7, 153.8, 158.1, 160.1, 162.0; LC-
TOF (M+H+) calcd for C20H28FN4O 359.2247, found 359.2253.
5.2.4. (3S,4S)-tert-Butyl 3-((6-(bis(tert-butoxycarbonyl)amino)-
4-methylpyridin-2-yl)methyl)-4-(2-(2-methoxybenzylamino)
ethoxy)pyrrolidine-1-carboxylate (8b)
Compound 8b was synthesized using a similar procedure to
that of 8a (88%): 1H NMR (500 MHz, CDCl3) d 1.42–1.43 (s, 27H),
2.31–2.33 (m, 3H), 2.60–2.70 (m, 1H), 2.70–2.80 (m, 1H), 2.90–
3.10 (m, 4H), 3.28–3.32 (m, 1H), 3.35–3.52 (m, 3H), 3.65–3.75
(m, 1H), 3.79–3.81 (m, 2H), 3.85–3.87 (m, 3H), 4.01–4.20 (m,
2H), 6.75–7.00 (m, 4H), 7.26–7.30 (m, 1H), 7.35–7.40 (m, 1H);
13C NMR (125 MHz, CDCl3) d 20.9, 21.9, 27.9, 28.3, 28.4, 28.5,
29.7, 34.4, 34.5, 42.5, 43.2, 46.1, 47.5, 47.7, 48.7, 48.9, 50.3, 50.8,
55.4, 55.5, 55.6, 60.4, 65.2, 65.3, 79.3, 79.4, 79.8, 82.92, 82.94,
110.5, 110.6, 119.7, 119.8, 121.0, 121.2, 121.3, 122.7, 122.8,
130.50, 130.52, 131.4, 149.9, 151.47, 151.51, 151.77, 151.79,
154.5, 154.7, 157.7, 158.89, 158.91, 176.2; LC-TOF (M+H+) calcd
for C36H55N4O8 671.4020, found 671.4016.
5.2.8. 6-(((3S,4S)-4-(2-(2-Methoxybenzylamino)ethoxy)-
pyrrolidin-3-yl)methyl)-4-methylpyridin-2-amine (4b)
Compound 4b was synthesized using a similar procedure to
that of 4a (96%): 1H NMR (500 MHz, CDCl3) d 2.18 (s, 3H), 2.65–
2.85 (m, 3H), 3.03–3.10 (t, J = 11.5 Hz, 1H), 3.17–3.32 (m, 3H),
3.40–3.47 (dd, J = 8.5, 12.0 Hz, 1H), 3.50–3.57 (m, 2H), 3.72–3.80
(m, 4H), 4.05–4.17 (m, 3H), 6.43 (s, 1H), 6.49 (s, 1H), 6.91–6.94
(dd, J = 7.0, 7.5 Hz, 1H), 6.97–6.99 (d, J = 8.5 Hz, 1H), 7.23–7.25
(dd, J = 1.0, 7.5 Hz, 1H), 7.34–7.38 (ddd, J = 1.5, 8.5, 9.0 Hz, 1H);
13C NMR (125 MHz, CDCl3) d 21.0, 38.7, 41.0, 46.2, 46.9, 47.1,
49.1, 55.4, 64.0, 77.8, 110.3, 111.2, 113.8, 118.2, 121.0, 131.6,
131.8, 145.6, 153.8, 157.6, 158.0; LC-TOF (M+H+) calcd for
C21H31N4O2 371.2447, found 371.2450.
5.2.5. (3S,4S)-tert-Butyl 3-((6-(bis(tert-butoxycarbonyl)amino)-
4-methylpyridin-2-yl)methyl)-4-(2-(2-hydroxybenzylamino)eth
oxy) pyrrolidine-1-carboxylate (8c)
5.2.9. 2-((2-((3S,4S)-4-((6-Amino-4-methylpyridin-2-yl)methyl)-
pyrrolidin-3-yloxy)ethylamino)methyl)phenol (4c)
Compound 8c was synthesized using a similar procedure to that
of 8a (55%): 1H NMR (500 MHz, CDCl3) d 1.45–1.46 (s, 27H), 2.34 (s,
3H), 2.60–2.75 (m, 2H), 2.75–2.85 (m, 1H), 2.85–3.00 (m, 2H),
3.00–3.07 (m, 1H), 3.07–3.20 (m, 1H), 3.20–3.33 (m, 1H), 3.33–
3.51 (m, 3H), 3.51–3.65 (m, 1H), 3.65–3.74 (m, 2H), 3.74–3.90
(m, 1H), 4.00–4.18 (m, 2H), 6.75–6.85 (m, 2H), 6.93 (s, 1H), 7.02–
7.10 (m, 1H), 7.15–7.20 (m, 1H); 13C NMR (125 MHz, CDCl3) d
20.9, 21.1, 27.9, 28.5, 29.7, 34.1, 42.4, 43.1, 46.8, 47.0, 48.4, 48.9,
50.1, 50.3, 50.7, 50.8, 53.4, 60.3, 66.3, 66.4, 78.6, 79.3, 79.5, 79.6,
82.9, 83.2, 83.3, 116.1, 116.3, 118.9, 119.4, 119.7, 120.3, 120.5,
120.9, 122.9, 123.0, 123.2, 128.8, 128.9, 129.4, 129.6, 129.7,
150.3, 150.6, 151.4, 151.6, 151.7, 154.7, 154.9, 157.2, 157.3,
159.1, 159.2; LC-TOF (M+H+) calcd for C35H53N4O8 657.3863, found
657.3874.
Compound 4c was synthesized using a similar procedure to that
of 4a (92%): 1H NMR (500 MHz, CDCl3) d 2.15 (s, 3H), 2.60–2.70 (m,
1H), 2.77–2.82 (m, 2H), 3.00–3.10 (t, J = 11.5 Hz, 1H), 3.18–3.22 (m,
3H), 3.42–3.51 (dd, J = 8.5, 11.5 Hz, 1H), 3.51–3.56 (m, 2H), 3.73–
3.80 (m, 1H), 4.00–4.25 (m, 3H), 6.38 (s, 1H), 6.50 (s, 1H), 6.83–
6.88 (m, 2H), 7.21–7.25 (m, 2H); 13C NMR (125 MHz, CDCl3) d
21.0, 28.8, 41.2, 46.0, 47.0, 47.1, 49.2, 63.7, 77.8, 110.3, 114.0,
115.4, 117.0, 120.6, 131.6, 131.7, 145.6, 153.8, 155.0, 158.0; LC-
TOF (M+H+) calcd for C20H29N4O2 357.2291, found 357.2277.
5.2.10. 6-(((3S,4S)-4-(2-(Furan-2-ylmethylamino)ethoxy)pyrroli-
din-3-yl)methyl)-4-methylpyridin-2-amine (4d)
Compound 4b was synthesized using a similar procedure to
that of 4a (96%): 1H NMR (500 MHz, CDCl3) d 2.32 (s, 3H),
2.65–2.73 (m, 1H), 2.76–2.83 (dd, J = 7.5, 15.0 Hz, 1H), 2.88–
2.93 (dd, J = 7.5, 14.5 Hz, 1H), 3.04–3.09 (t, J = 11.5 Hz, 1H),
3.17–3.24 (m, 3H), 3.39–3.43 (dd, J = 9.0, 11.5 Hz, 1H), 3.48–
3.51 (d, J = 13.5 Hz, 2H), 3.51–3.56 (m, 1H), 3.70–3.75 (m, 1H),
4.05–4.10 (m, 1H), 4.22 (s, 2H), 6.38–6.40 (dd, J = 2.0, 5.0 Hz,
1H), 5.48 (s, 1H), 6.52–6.53 (d, J = 3.5 Hz, 1H), 6.55 (s, 1H),; 13C
NMR (125 MHz, CDCl3) d 21.0, 28.8, 41.4, 43.1, 46.0, 47.0, 49.4,
64.0, 78.1, 110.3, 110.8, 111.0, 113.0, 114.0, 144.1, 144.3, 144.9,
145.8, 153.9, 158.1; LC-TOF (M+H+) calcd for C18H27N4O2
331.2134, found 331.2136.
5.2.6. (3S,4S)-tert-Butyl 3-((6-(bis(tert-butoxycarbonyl)amino)-
4-methylpyridin-2-yl)methyl)-4-(2-(furan-2-ylmethylamino)
ethoxy)pyrrolidine-1-carboxylate(8d)
Compound 8b was synthesized using a similar procedure to
that of 8a (90%): 1H NMR (500 MHz, CDCl3) d 1.43–1.44 (s, 27H),
2.30–2.32 (m, 3H), 2.50–2.60 (m, 1H), 2.75–2.83 (m, 2H), 2.92–
3.17 (m, 3H), 3.20–3.50 (m, 5H), 3.52–3.70 (m, 2H), 3.77–3.80
(m, 1H), 3.81 (s, 2H), 6.20–6.21 (d, J = 3.0 Hz, 1H), 6.32 (s, 1H),
6.86–6.92 (m, 2H), 7.37 (s, 1H); 13C NMR (125 MHz, CDCl3) d
19.1, 20.9, 21.0, 23.4, 24.7, 27.9, 28.39, 28.48, 28.51, 29.7, 30.6,
34.6, 34.7, 36.6, 42.6, 43.2, 45.8, 45.9, 48.2, 48.3, 48.8, 49.1, 50.3,
50.8, 64.4, 68.2, 68.4, 78.7, 79.2, 79.3, 79.4, 82.85, 82.86, 107.0,
107.2, 110.16, 110.24, 119.57, 119.61, 122.8, 141.90, 141.94,
141.97, 149.6, 151.45, 151.50, 151.8, 153.4, 154.5, 154.7, 159.1,
159.2, 171.3; LC-TOF (M+H+) calcd for C33H51N4O8 631.3703, found
631.3703.
5.3. NOS purified enzyme assays
The three NOS isoforms, rat nNOS, murine iNOS, and bovine
eNOS were obtained as recombinant enzymes overexpressed in
and purified from E. coli as previously reported.36–39 The hemo-
globin capture assay was used to measure nitric oxide produc-
tion.40 Briefly, the assay was run at 37 °C in 100 mM HEPES
5.2.7. 6-(((3S,4S)-4-(2-(2-Fluorobenzylamino)ethoxy)pyrrolidin-
3-yl)methyl)-4-methylpyridin-2-amine (4a)
To a solution of 8a (70 mg, 0.10 mmol) in MeOH (2 mL) was
added 6 N HCl (4 mL) at room temperature. The mixture was
buffer (10% glycerol; pH 7.4) in the presence of 10
The following were also included in the assay: 100
10 M tetrahydrobiopterin, 1 mM CaCl2, 11.6 g/mL calmodulin
and 3.0 M oxyhemoglobin. For iNOS, calmodulin and CaCl2 were
l
M
l
L
-arginine.
M NADPH,
l
l
l

2442
K. J. Labby et al. / Bioorg. Med. Chem. 20 (2012) 2435–2443
Table 3
Crystallographic data collection and refinement statistics
Data seta
nNOS-4a
nNOS-4b
nNOS-4c
nNOS-4d
PDB code
3TYL
3TYM
3TYN
3TYO
Data collection
Space group
P212121
P212121
P212121
P212121
Cell dimensions
a, b, c (Å)
Resolution (Å)
Rsym or Rmerge
51.9,110.8,164.3
1.90 (1.93–1.90)
0.055 (0.629)
29.2 (2.1)
51.8,110.7, 164.2
2.00 (2.03–2.00)
0.056 (0.590)
15.6 (2.0)
51.8,110.8,164.2
1.97 (2.00–1.97)
0.046 (0.564)
31.0 (2.5)
51.9,110.9,164.3
1.93 (1.96–1.93)
0.047 (0.605)
32.1 (2.5)
I/
r
No. unique reflections
Completeness (%)
Redundancy
75,516
99.9 (100.0)
4.1 (4.1)
64,550
99.9 (97.8)
4.1 (4.1)
67,186
98.8 (98.0)
4.1 (4.1)
72,491
99.8 (99.9)
4.1 (4.0)
Refinement
Resolution (Å)
No. reflections
Rwork/Rfree
1.90
71,520
0.179/0.212
2.00
61,119
0.186/0.227
1.97
63,641
0.185/0.223
1.93
68,646
0.180/0.215
2
No. atoms
Protein
Ligand/ion
Water
6709
185
377
6671
183
283
6679
181
321
6671
177
366
Mean B-factor
45.16
49.50
48.17
47.50
R.m.s. deviations
Bond lengths (Å)
Bond angles (°)
0.014
1.411
0.013
1.386
0.013
1.403
0.012
1.324
a
See Scheme 1 for chemical formula of inhibitors.
omitted because iNOS is calcium independent; CaM is bound
tightly. All NOS isozymes were used at a concentration of approx-
imately 100 nM. The assay was run in a 96 well plate, using the
5.5. Crystal preparation, X-ray diffraction data collection, and
structure refinement
Synergy
Throughput Analysis Facility. The assay was run in triplicate;
Forty-eight 100 L reactions were performed at once. The addi-
tion of hemoglobin and NOS were automated with a maximum
of 30 s delay before the reactions could be recorded at
401 nm. The absorbance increase at 401 nm is due to the
formation of NO via the conversion of oxyhemoglobin to
methemoglobin.
4
by BioTek, at the Northwestern University High
The nNOS heme domain protein used for crystallization was
generated by limited trypsin digest from the full length nNOS
and further purified through a Superdex 200 gel filtration column
(GE Healthcare) as described previously.42 The nNOS heme domain
at 7–9 mg/mL containing 20 mM histidine was used for the sitting
drop vapor diffusion crystallization setup under the conditions re-
ported42with minor modification by addition of 10% (v/v) ethylene
glycol in well solution. Fresh crystals were first passed stepwise
through cryo-protectant solutions as described42and then soaked
with 10 mM inhibitor for 4–6 h at 4 °C before being flash cooled
by plunging into liquid nitrogen.
l
a
The IC50 values were obtained using non-linear regression in
GraphPad Prism5 software. Subsequent Ki values were determined
using the Cheng-Prusoff relationship:41
The cryogenic (100 K) X-ray diffraction data were collected re-
motely at beamline 7–1 at Stanford Synchrotron Radiation Light
source through the data collection control software Blu-Ice43 and
the crystal mounting robot. Raw data frames were indexed, inte-
grated, and scaled using HKL2000.44 The binding of inhibitors
was detected by the initial difference Fourier maps calculated with
REFMAC.45 The inhibitor molecules were then modeled in COOT46
and refined using REFMAC. Water molecules were added in REF-
MAC and checked by COOT. The TLS47 protocol was implemented
in the final stage of refinements with each subunit as one TLS
group. The refined structures were validated in COOT before depo-
sition to RCSB protein data bank. The crystallographic data collec-
tion and structure refinement statistics are summarized in Table 3
with PDB accession codes included.
Ki ¼ IC50=ð1 þ ½Sꢃ=KmÞ
The following known Km values were used: rat nNOS 1.3
murine iNOS 8.3 M; bovine eNOS 1.7 M.
lM;
l
l
5.4. nNOS cell-based assay
HEK293t cells stably transfected with rat nNOS were cultured
as previously described.29 The nNOS inhibition assay was per-
formed as previously reported,29 with the following modifications:
assays were performed in 96-well plates with a total volume of
100
l
L, and 10
l
M A23187 (Sigma Aldrich, St Louis, MO, USA)
M.
(added as 100ꢂ stock in 50% DMSO) was used in place of 5
l
A23187 is a calcium ionophore; it transports calcium into the cells
and activates nNOS. Nine concentrations of each inhibitor were
tested, in at least triplicate wells. All inhibitors were assayed with-
in the same experiment to assure consistencies in cell concentra-
tion and passage number. The entire assay was repeated at least
three times for each inhibitor, and the IC50 values averaged. Cells
were plated in the 96-well plates 24 h before activation. After 6 h
of activation (in the presence or absence of inhibitor, which was
Acknowledgments
We are grateful to the National Institutes of Health, Grants
GM049725 to RBS and GM057353 to TLP. 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 grant 0021620849from
MSMT of the Czech Republic. We thank the SSRL beamline staff
for their support during remote X-ray diffraction data collection.
added 30 min before activation) 50 lL aliquots of the media were
removed and nitrite production was quantified using Griess
reagent.40

K. J. Labby et al. / Bioorg. Med. Chem. 20 (2012) 2435–2443
2443
21. McDonagh, A. F.; Lightner, D. A. J. Med. Chem. 2007, 50, 480.
22. Sasaki, S.; Cho, N.; Nara, Y.; Harada, M.; Endo, S.; Suzuki, N.; Furuya, S.; Fujino,
M. J. Med. Chem. 2003, 46, 113.
23. Alex, A.; Millan, D. S.; Perez, M.; Wakenhut, F.; Whitlock, G. A. Med. Chem.
Commun. 2011, 2, 669.
We thank Drs. Josh Kurutz and Yuyang Wu of the Northwestern
University IMSERC facility for valuable assistance and discussions
concerning the NMR experiments.
24. Ashwood, V. A.; Field, M. J.; Horwell, D. C.; Julien-Larose, C.; Lewthwaite, R. A.;
McCleary, S.; Pritchard, M. C.; Raphy, J.; Singh, L. J. Med. Chem. 2001, 44, 2276.
25. Ji, H.; Li, H.; Martásek, P.; Roman, L. J.; Poulos, T. L.; Silverman, R. B. J. Med.
Chem. 2009, 52, 779.
26. Ji, H.; Delker, S. L.; Li, H.; Martásek, P.; Roman, L. J.; Poulos, T. L.; Silverman, R. B.
J. Med. Chem. 2010, 53, 7804.
27. Xue, F.; Kraus, J. M.; Labby, K. J.; Ji, H.; Mataka, J.; Xia, G.; Li, H.; Delker, S. L.;
Roman, L. J.; Martásek, P.; Poulos, T. L.; Silverman, R. B. J. Med. Chem. 2011, 54,
6399.
28. Haight, A. R.; Stoner, E. J.; Peterson, M. J.; Grover, V. K. J. Org. Chem. 2003, 68,
8092.
29. Fang, J.; Silverman, R. B. Anal. Biochem. 2009, 390, 74.
30. Viswanadhan, V. N.; Ghose, A. K.; Revankar, G. R.; Robins, R. K.; Che, J. Inf.
Comput. Sci 1989, 29, 163.
31. Wilson, J. C.; Munro, S. L. A.; Craik, D. J. Magn. Reson. Chem. 1995, 33, 367.
32. Oki, M. Top. Stereochem. 1983, 14, 1.
33. Clayden, J.; Moran, W. J.; Edwards, P. J.; LaPlante, S. R. Angew. Chem., Int. Ed.
2009, 48, 6398.
34. Kraus, J. M.; Tatipaka, H. B.; McGuffin, S. A.; Chennamaneni, N. K.; Karimi, M.;
Arif, J.; Verlinde, C. L. M. J.; Buckner, F. S.; Gelb, M. H. J. Med. Chem. 2010, 53,
3887.
35. Ballet, S.; Misicka, A.; Kosson, P.; Lemieux, C.; Chung, N. N.; Schiller, P. W.;
Lipkowski, A. W.; Tourwé, D. J. Med. Chem. 2008, 51, 2571.
36. Hevel, J. M.; White, K. A.; Marletta, M. A. J. Biol. Chem. 1991, 266, 22789.
37. Roman, L. J.; Sheta, E. A.; Martasek, P.; Gross, S. S.; Liu, Q.; Masters, B. S. Proc.
Natl. Acad. Sci. U.S.A. 1995, 92, 8428–8432.
38. Gerber, N. C.; Ortiz de Montellano, P. R. J. Biol. Chem. 1995, 270, 17791.
39. Martasek, P.; Liu, Q.; Liu, J.; Roman, L. J.; Gross, S. S.; Sessa, W. C.; Masters, B. S.
S. Biochem. Biophys. Res. Commun. 1996, 219, 359.
40. Hevel, J. M.; Marletta, M. A. Methods Enzymol. 1994, 233, 250.
41. Cheng, Y.-C.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.
42. 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.
43. 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.
44. Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307.
45. Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Acta Crystallogr., Sect. D: Biol.
Crystallogr 1997, 53, 240.
Supplementary data
Supplementary data (data include full 1H and 13C NMR spectra
of 4a–d.) associated with this article can be found, in the online
version, at doi:10.1016/j.bmc.2012.01.037.
References and notes
1. Dorheim, M.-A.; Tracey, W. R.; Pollock, J. S.; Grammas, P. Biochem. Biophys. Res.
Commun. 1994, 205, 659.
2. Zhang, L.; Dawson, V. L.; Dawson, T. M. Pharmacol. Ther. 2006, 109, 33.
3. Norris, P. J.; Waldvogel, H. J.; Faull, R. L. M.; Love, D. R.; Emson, P. C.
Neuroscience 1996, 72, 1037.
4. Ashina, M. Expert Opin. Pharmacother. 2002, 3, 395.
5. Sims, N. R.; Anderson, M. F. Neurochem. Int. 2002, 40, 511.
6. Southan, G. J.; Szabó, C. Biochem. Pharmacol. 1996, 51, 383.
7. Babu, B. R.; Griffith, O. W. Curr. Opin. Chem. Biol. 1998, 2, 491.
8. Alderton, W. K.; Cooper, C. E.; Knowles, R. G. Biochem. J. 2001, 357, 593.
9. Ji, H.; Erdal, E.; Litzinger, E. A.; Seo, J.; Zhu, Y.; Xue, F.; Fang, J.; Huang, J.;
Silverman, R. B. In Frontiers in Medicinal Chemistry; Reitz, A. B., Choudhary, M. I.,
Atta-ur-Rahman, Eds., fifth ed.; Bentham Science Publishers, 2009; pp 842–
882.
10. Silverman, R. B. Acc. Chem. Res. 2009, 42, 439.
11. Delker, S. L.; Ji, H.; Li, H.; Jamal, J.; Fang, J.; Xue, F.; Silverman, R. B.; Poulos, T. L.
J. Am. Chem. Soc. 2010, 132, 5437.
12. Ji, H.; Tan, S.; Igarashi, J.; Li, H.; Derrick, M.; Martásek, P.; Roman, L. J.; Vásquez-
Vivar, J.; Poulos, T. L.; Silverman, R. B. Ann. Neurol. 2009, 65, 209.
13. Lawton, G. R.; Ralay Ranaivo, H.; Chico, L. K.; Ji, H.; Xue, F.; Martásek, P.; Roman,
L. J.; Watterson, D. M.; Silverman, R. B. Bioorg. Med. Chem. 2009, 17, 2371.
14. Xue, F.; Fang, J.; Lewis, W. W.; Martásek, P.; Roman, L. J.; Silverman, R. B. Bioorg.
Med. Chem. Lett. 2010, 20, 554.
15. Xue, F.; Huang, J.; Ji, H.; Fang, J.; Li, H.; Martásek, P.; Roman, L. J.; Poulos, T. L.;
Silverman, R. B. Bioorg. Med. Chem. 2010, 18, 6526.
16. Xue, F.; Li, H.; Fang, J.; Roman, L. J.; Martásek, P.; Poulos, T. L.; Silverman, R. B.
Bioorg. Med. Chem. Lett. 2010, 20, 6258.
17. Xue, F.; Li, H.; Delker, S. L.; Fang, J.; Martásek, P.; Roman, L. J.; Poulos, T. L.;
Silverman, R. B. J. Am. Chem. Soc. 2010, 132, 14229.
18. Kuhn, B.; Mohr, P.; Stahl, M. J. Med. Chem. 2010, 53, 2601.
19. Laurence, C.; Brameld, K. A.; Graton, J.; Le Questel, J.-Y.; Renault, E. J. Med. Chem.
2009, 52, 4073.
46. Emsley, P.; Cowtan, K. Acta Crystallogr., Sect. D: Biol. Crystallogr 2004, D60, 2126.
47. Winn, M. D.; Isupov, M. N.; Murshudov, G. N. Acta Crystallogr., Sect. D: Biol.
Crystallogr 2001, D57, 122.
20. Jansma, A.; Zhang, Q.; Li, B.; Ding, Q.; Uno, T.; Bursulaya, B.; Liu, Y.; Furet, P.;
Gray, N. S.; Geierstanger, B. H. J. Med. Chem. 2007, 50, 5875.