Cyclopropyl- and methyl-containing inhibitors of neuronal nitric oxide synthase

Article abstract of DOI:10.1016/j.bmc.2012.12.019

Inhibitors of neuronal nitric oxide synthase have been proposed as therapeutics for the treatment of different types of neurological disorders. On the basis of a cis-3,4-pyrrolidine scaffold, a series of trans-cyclopropyl- and methyl-containing nNOS inhib

Full text of DOI:10.1016/j.bmc.2012.12.019

Bioorganic & Medicinal Chemistry 21 (2013) 1333–1343
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Cyclopropyl- and methyl-containing inhibitors of neuronal nitric
oxide synthase
Huiying Li a,b,c, Fengtian Xue d,e, , James M. Kraus II d,e, Haitao Ji d,e,à, Kristin Jansen Labby d,e
,
Jan Mataka d,e,§, Silvia L. Delker a,b,c,–, Pavel Martásek f,g, Linda J. Roman f, Thomas L. Poulos a,b,c,
,
⇑
Richard B. Silverman d,e,
⇑
a Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900, United States
b Department of Pharmaceutical Chemistry, University of California, Irvine, CA 92697-3900, United States
c Department of Chemistry, University of California, Irvine, CA 92697-3900, United States
d Department of Chemistry, Chemistry of Life Processes Institute, Center for Molecular Innovation and Drug Discovery, Northwestern University, 2145 Sheridan Road, Evanston,
IL 60208-3113, United States
e 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, United States
f Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX, United States
g 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:
Inhibitors of neuronal nitric oxide synthase have been proposed as therapeutics for the treatment of
different types of neurological disorders. On the basis of a cis-3,4-pyrrolidine scaffold, a series of trans-
cyclopropyl- and methyl-containing nNOS inhibitors have been synthesized. The insertion of a rigid elec-
tron-withdrawing cyclopropyl ring decreases the basicity of the adjacent amino group, which resulted in
decreased inhibitory activity of these inhibitors compared to the parent compound. Nonetheless, three of
them exhibited double-digit nanomolar inhibition with high nNOS selectivity on the basis of in vitro
enzyme assays. Crystal structures of nNOS and eNOS with these inhibitors bound provide a basis for
detailed structure–activity relationship (SAR) studies. The conclusions from these studies will be used
as a guide in the future development of selective NOS inhibitors.
Received 31 October 2012
Revised 4 December 2012
Accepted 12 December 2012
Available online 22 December 2012
Keywords:
Neuronal nitric oxide synthase
Inhibition
Isozyme selectivity
Cyclopropyl analogues
X-ray crystallography
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
and O2 to L
-citrulline and NO.4 A considerable body of research
has shown that overproduction of cerebral NO by the neuronal iso-
form of NOS (nNOS) is a general pathological phenomenon for var-
ious neurological disorders such as Parkinson’s,5 Alzheimer’s,6
Huntington’s,7 headaches,8 and cerebral palsy.5–10 This has led to
the search for and development of selective inhibitors of nNOS
over endothelial NOS (eNOS), the isozyme that is responsible for
the regulation of blood pressure, and inducible NOS (iNOS), the iso-
zyme that is critical for immune responses, as therapeutic agents
for the treatment of neurological disorders.11
In our on-going effort to develop novel nNOS inhibitors, we re-
cently reported a series of cis-3,4-pyrrolidine-based inhibitors.12–14
In this family, several inhibitors have shown remarkable neuropro-
tective properties in a preclinical rabbit model for cerebral palsy.13
Compound 1, as an example, not only is neuroprotective but is the
most selective nNOS inhibitor over eNOS and iNOS yet reported.14
Despite its promising inhibitory activity, further application of 1
has been impeded by several of its structural characteristics. First,
the flexible m-fluorophenyl ethanamino tail produces multiple
rotatable bonds in the inhibitor. In addition, the benzylic position
Nitric oxide (NO) has a wide variety of functions in the body, the
most well-studied of which are its regulatory action on smooth
muscle relaxation, cytotoxic activity in the immune system, neuro-
transmission, brain development and protection, and maintenance
of synaptic plasticity.1 The production of NO is mediated via the
activity of nitric oxide synthase (NOS),2,3 a family of homodimeric
heme-containing monooxygenases, which metabolizes L-arginine
⇑
Corresponding authors. Tel.: +1 949 824 7020 (T.L.P.); tel.: +1 847 491 5653
(R.B.S.).
E-mail addresses: poulos@uci.edu (T.L. Poulos), Agman@chem.northwestern.edu
(R.B. Silverman).
Current address: Department of Pharmaceutical Sciences, University of Maryland
School of Pharmacy, 20 North Pine Street, Baltimore, MD 21201, United States.
à
Current address: Department of Chemistry, University of Utah, Salt Lake City, UT
84112-0850, United States.
§
Current address: Department of Chemistry, Northeastern Illinois University,
Chicago, IL 60625-4699, United States.
–
Current address: Celgene, 4550 Towne Centre Court, San Diego, CA 92121, United
States.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.12.019

1334
H. Li et al. / Bioorg. Med. Chem. 21 (2013) 1333–1343
of the m-fluorophenyl ring is highly susceptible to metabolic oxi-
dation.15 Also, the two positive charges of 1 at physiological pH,
derived from the two secondary amino groups, decreases the
chance of 1 to penetrate the blood-brain barrier (BBB).13 These
considerations prompted the development of new pyrrolidine
nNOS inhibitors with a potentially more desirable pharmacokinetic
and pharmacodynamic profile.
benzenes 4a–c, Rh(II)-catalyzed cyclopropanation produced 5a–c
as cis/trans mixtures in good yields.27 Ethyl esters 5a–c were trea-
ted with NaOCH3 in refluxing EtOH to induce epimerization, gener-
ating the thermodynamically more stable trans isomers, which
were hydrolyzed in aqueous LiOH to yield 6a–c in good yields.27
Carboxylic acids 6a–c were converted to Boc-protected amines
(7a–c) through Curtius rearrangement reactions in reasonable
H
N
H
N
H
H
N
F
N
R
H2N
N
O
H2N
N
O
1
2
Ki-nNOS = 7 nM
R = F, Cl, Me
Ki-eNOS = 19,000 nM
K
i-iNOS = 5,800 nM
Different strategies have been applied to modify the chemical
structure of 1.16–20 Herein we describe the design and synthesis
of a new series of inhibitors (2), with a structurally constrained
cyclopropyl ring inserted in the position adjacent to the amino
group of the ethanamino tail. Conformational restriction using
cyclopropyl fragments is a strategy that has been widely used in
modern drug design to create novel inhibitors for a variety of en-
zymes.21–23 Introduction of the cyclopropyl group (2) can poten-
tially enhance inhibitory activity by stabilizing and thereby
reducing the energetic penalty in binding to the enzyme active site
and thus improve selectivity.24,25 In addition, the insertion of a
cyclopropyl fragment can block the potential metabolic oxidation
at the benzylic position of the m-fluorophenyl ring.15 Furthermore,
the electron-withdrawing character of the cyclopropyl ring de-
creases the basicity of the adjacent amino group. The calculated
pKa value of the amino group in the lipophilic tail of 2 is ꢀ7.4,
which is significantly lower than 8.9 in 1.26 As a result, pseudo-
monocationic molecule 2 may have improved BBB permeability.15
Here we report structure–activity relationship studies on these
cyclopropyl containing inhibitors to determine if enhanced po-
tency and selectivity can be attained prior to consideration of phar-
macokinetic property assessment.
yields.28 Finally, the Boc-protecting groups of 7a–c were removed
in trifluoroacetic acid (TFA) to provide ( )-trans-3a–c as TFA salts
in high yields (see Scheme 3)
The two enantiomers of 3a were resolved in two steps. First, 3a
was treated with (S)-(ꢁ) camphanic chloride in the presence of tri-
ethylamine (TEA) to produce two separable diastereomers 8a and
8b. Then the amide bonds of 8a and 8b were hydrolyzed in concen-
trated HCl to generate single enantiomers 3d and 3e in good yields.
The syntheses of inhibitors 2a–d began with (R,R)-9a.29 Com-
pound 9a underwent reductive amination with amines 3a–c and
3e using NaBH(OAc)3 as a reducing reagent to generate 10a–d in
good yields. Next, the three Boc-protecting groups of 10a–d were
removed in HCl to yield inhibitors 2a–d in high yields.
The synthesis of inhibitors 2e,f began with aldehyde 9b
(Scheme 4).13 Reductive amination of 9b with amine 3a or 3e pro-
vided the corresponding secondary amine, which was further pro-
tected with a Boc-protecting group to give 10e,f in reasonable
yields. Next, the cyclopropyl group was reduced, along with depro-
tection of the Bn-protecting group, by catalytic hydrogenation
using Pd(OH)2 as a catalyst at 60 °C to generate 11e,f. Finally, the
three Boc-protecting groups were removed in HCl to provide inhib-
itors 2e,f as tris–HCl salts in good yields.
2. Chemistry
3. Structure–activity studies
The key amine building blocks (3a–e) were synthesized as
Nitric oxide hemoglobin capture assays were performed with
shown in Schemes 1 and 2. Starting from 1-substituted-3-vinyl-
2a through 2f against three NOS isoforms to obtain the in vitro
R
EtO2C
R
HO2C
R
b
a
6a
R = F
4a
5a
(+/-)-
R = F
(+/-)-
R = F
(+/-)-6b R = CH3
6c
4b R = CH3
(+/-)-5b R = CH3
4c
5c
(+/-)-
R = Cl
R = Cl
(+/-)-
R = Cl
c
d
BocHN
R
H2N
R
3a
R = F
7a
(+/-)-
(+/-)-
(+/-)-7b R = CH3
7c
R = F
(+/-)-3b R = CH3
3c
(+/-)-
R = Cl
(+/-)-
R = Cl
Scheme 1. Synthesis of 3a–c. Reagents and conditions: (a) EtO2CCHN2, Rh2(OAc)4, toluene, 80–85 °C, 2 h; (b) (i) NaOCH3 in EtOH (1 M), reflux, 40 h, (ii) LiOH, MeOH/H2O,
70 °C, 16 h, 75–80% for two steps; (c) diphenyl phosphorazidate, triethylamine, t-BuOH, 85 °C, 48 h, 75–82%; (d) TFA/CH2Cl2 (1:2), room temp, 45 min.

H. Li et al. / Bioorg. Med. Chem. 21 (2013) 1333–1343
1335
H
H
H
H
H2N
H2N
F
N
F
O
O
O
O
O
O
H
H
8a
a
H2N
F
b
3d
H
H
N
(+/-)-3a
F
F
H
H
8b
3e
Scheme 2. Resolution of enantiomers 3d and 3e. Reagents and conditions: (a) (S)-(ꢁ)-camphanic chloride, CH2Cl2, TEA, room temp, 30 min, 91% for two diastereomers; (b)
12 N HCl, EtOH (2:1), reflux, 72 h, 67–70%.
Boc
N
Boc
N
a
H
N
*
O
R
(Boc)2N
N
O
(Boc)2N
N
O
*
9a
10a (R,S/S,R), R = F
10b (R,S), R = F
10c (R,S/S,R), R = CH3
10d (R,S/S,R), R = Cl
H
N
H
b
*
N
R
H2N
N
O
*
3 HCl
2a (R,S/S,R), R = F
2b (R,S), R = F
2c (R,S/S,R), R = CH3
2d (R,S/S,R), R = Cl
Scheme 3. Synthesis of 2a–d. Reagents and conditions: (a) amine hydrochloride, TEA, NaHB(OAc)3, room temp, 3 h, 81–87%; (d) 6 N HCl in MeOH (2:1), room temp, 16 h, 90–
99%. The R/S notations shown indicate the chirality of the two chiral centers of the cyclopropyl ring; the pyrrolidine ring has (R,R) stereochemistry in all of the compounds.
Boc
N
Boc
N
Boc
a
b
*
O
Boc
Boc
Boc
N
R
N
Bn
N
N
Bn
N
N
O
O
*
9b
10e
(R,S/S,R)
10f (R,S)
Boc
N
H
N
Boc
c
H
N
N
F
F
*
*
N
H
H2N
N
O
O
3HCl
11e
(R/S)
11f (R)
2e
(R/S)
2f (R)
Scheme 4. Synthesis of inhibitors 2e–f. Reagents and conditions: (a) (i) 3a or 3e, TEA, NaHB(OAc)3, room temp, 3 h, (ii) (Boc)2O, TEA, MeOH, room temp, 6 h, 60% for two
steps; (b) H2, Pd(OH)2/C, 60 °C, 30 h; (c) 6 N HCl/MeOH (2:1), room temp, 16 h, 25% for two steps. The R/S notations shown indicate the chirality of the two chiral centers of the
cyclopropyl ring; the pyrrolidine ring has (R,R) stereochemistry in all of the compounds.
inhibitory potency and isoform selectivity for this series of com-
pounds (Table 1). Crystal structures of nNOS and eNOS with these
inhibitors bound were also determined, which provides the basis
for structure–activity relationship (SAR) studies.
The introduction of a cyclopropyl ring adjacent to the amine in
the tail end of the inhibitor was designed to lower the pKa
(pKa = 7.4, calculated by ACD/Labs 7.0) of this otherwise basic
group (1, pKa = 8.9, calculated by ACD/Labs 7.0) to improve mem-
brane permeability of the inhibitor. The rigidity along the inhibitor
tail results in the cyclopropyl ring forcing the amino group to face
more exclusively toward the active site Glu residue (Glu592 in
nNOS or Glu363 in eNOS). This amino group of the inhibitor re-
cruits the Glu residue as its hydrogen-bonding partner, forcing
the Glu side chain into an alternate rotamer position (Fig. 1). The
alternate Glu side chain rotamer also was observed in the eNOS–
1 complex structure but not in the nNOS–1 structure.14 The more
flexible tail in 1 allows the amino nitrogen to point in various
Consistent with the binding mode of (3R,4R)-1,14 all six inhibitors
in this study bind to both nNOS and eNOS in a flipped orientation. As
shown in Figure 1, the two nitrogen atoms of aminopyridine donate
two hydrogen bonds to heme propionate D. The pyrrolidine ring
nitrogen is positioned between heme propionate A and the C@O
group of the pterin, thereby forming hydrogen bonds to both. This
binding mode leaves the cyclopropyl and phenyl rings of the inhibi-
tor tail right above the heme and provides the primary differences
between the 1 and 2 interactions with the local surroundings.

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H. Li et al. / Bioorg. Med. Chem. 21 (2013) 1333–1343
Table 1
Kia Values of inhibitors for rat nNOS, bovine eNOS, and murine iNOS
H
N
H
N
H
H
N
H
N
F
F
H2N
N
O
H2N
N
O
H
2a
2b
H
N
H
N
H
N
H
N
Cl
H2N
H2N
N
N
O
O
H2N
H2N
N
N
O
O
2c
2d
H
N
H
N
H
N
H
N
F
F
2e
2f
Compound
nNOS (lM)
eNOS (lM)
iNOS (
lM)
Selectivityb
n/e
n/i
( )2a
2b
( )2c
( )2d
( )2e
2f
0.170
0.175
0.140
0.071
0.046
0.070
51
31
45
26
68
40
16
12
10
0.86
10.2
7.6
300
177
320
370
1500
570
94
69
72
12
220
110
a
The Ki values were calculated based on the directly measured IC50 values, which represent at least duplicate measurements with standard deviations of 10%.
The ratio of Ki (eNOS or iNOS) to Ki (nNOS).
b
directions. And, therefore, there is not enough driving force to
bring the Glu side chain into an alternate rotamer in nNOS.
We also have tested the impact of chirality at the cyclopropyl
ring on inhibitor binding potency. The nNOS complex structures
show that when a racemic compound mixture is used in crystal
soaking, the ‘ring up’ (R,S) enantiomer binds exclusively (Fig. 1B
and C), although the ‘ring down’ (S,R) enantiomer (2b) can still bind
if it is the only isomer used for crystal soaking (Fig. 1A). The differ-
ent stereochemistry around the cyclopropyl ring results in slightly
different orientations (more than 12°) for the tail phenyl ring,
which is more parallel to the heme plane in the ‘ring down’ (S,R)
enantiomer than in the ‘ring up’ (R,S) isomer (Fig. 2A). Moreover,
in the ‘ring down’ (S,R) enantiomer both the cyclopropyl (3.1 Å)
and phenyl (2.7 Å) rings are farther away from the native Glu592
side chain position and, thus, can tolerate both the native and
alternate Glu592 rotamer positions. In contrast, the cyclopropyl
or phenyl ring in the ‘ring up’ (R,S) enantiomer, as in 2c or 2d, is
so close to the native Glu592 side chain, being 2.5 Å to cyclopropyl
and 2.3 Å to phenyl ring in 2c assuming the Glu592 is in its native
position, that the alternate Glu592 rotamer is favored (Fig. 1B and
C). Therefore, two factors can potentially influence the Glu592 side
chain conformation: (1) a H-bond (or salt bridge) with the inhibitor
amino group and (2) a steric repulsion between the phenyl ring
and Glu592. The cyclopropyl-containing inhibitors reported here
either force the amino group to face Glu592 or bring the tail phenyl
ring closer to Glu592, resulting in alternate Glu592 side chain
conformations.
phenyl ring about 0.5 Å closer to Glu592, which forces Glu592 to
adopt primarily the alternate rotamer conformation. As noted ear-
lier, this alternate rotamer makes a better hydrogen bond with the
inhibitor. Therefore, it is both the size of the substituent attached
to the phenyl ring and the chirality of the inhibitor that contribute
to differences in binding affinity. It is probably the resulting differ-
ence in hydrogen bond strength from the tail amino group to the
alternate Glu592 side chain position that is the main reason why
2d shows a two-fold better binding affinity to nNOS than that of
either 2a or 2c (Table 1).
Introduction of a cyclopropyl ring next to the amino group in
the tail decreases the charge, which results in decreased potency;
the analogous compounds with a methyl group at this position,
such as 2e and 2f, show slightly better binding affinity to nNOS
(Table 1). The methyl group should increase the basicity of the
amino group in these two compounds compared to the cyclopro-
pyl-containing counterparts. Although the tail amino groups in
both the methyl and cyclopropyl inhibitors superimpose well,
and both are in position to H-bond with Glu592 (Fig. 2), the higher
basicity of the tail amino group (pKa = 8.0, calculated by ACD/Labs
12.0), resulting from the methyl group compared to the cyclopro-
pyl group, very likely contributes to the tighter binding of the
methyl inhibitors. The higher affinity of 2e over 2f also is readily
understood on the basis of amine-Glu592 interactions (Fig. 3). In
2f the amino nitrogen is pointing away from the Glu592 side chain
because of the R-chirality at the methyl position in this inhibitor.
The S-stereochemistry seen in the nNOS-2e structure must be
the dominant one preferred by the enzyme because it is the only
enantiomer observed in the structure, even though 2e used for
the crystal soak is a racemic (R/S) mixture. This is consistent with
the lower Ki value towards nNOS measured for 2e versus 2f (Ta-
ble 1). The R-configuration can only be observed when the enantio-
mer pure compound (2f) is used.
Overall the ‘ring up’ (R,S) enantiomer fits better into the nNOS
active site. The alternate rotamer of Glu592 plays an important role
here since in the better binding ‘ring up’ (R,S) mode Glu592 is in a
better position to H-bond with the tail amine group of inhibitors 2c
and 2d (Fig. 1). Another factor to consider is how the halogen/
methyl group of the phenyl ring fits into the hydrophobic pocket
defined by Val567 and Phe584 (Fig. 1). The larger methyl group
and chloro atom relative to the smaller fluoro atom pushes the
To explore the structural basis for isoform selectivity of these
cyclopropyl-containing inhibitors, crystal structures of eNOS with

H. Li et al. / Bioorg. Med. Chem. 21 (2013) 1333–1343
1337
chlorophenyl ring also forces the Glu363 side chain into its alter-
nate rotamer conformation to avoid the potential clashes with
Glu363 in its native rotamer conformation. For eNOS-2e, the smal-
ler fluorophenyl ring and the absence of the cyclopropyl ring allow
both Glu363 rotamer conformations to exist. In both cases, the en-
zyme-inhibitor interactions right above the heme in eNOS replicate
what we have seen in nNOS. However, the binding affinity of these
inhibitors to eNOS is significantly lower than that to nNOS. Isoform
selection has been attributed primarily to two amino acid differ-
ences in the active site of NOS, Asp597 in nNOS versus Asn368 in
eNOS and Met336 in nNOS versus Val106 in eNOS. Binding free en-
ergy calculations19 indicated that although Asp597 (or Asn368) is
not directly involved in binding of these pyrrolidine inhibitors, it
does impose a significant electrostatic influence on how tight these
inhibitors fit into the NOS active site. On the other side of the bind-
ing site, the Met336 (Val106) residue does contact the aminopyri-
dine ring of the inhibitors. One of the valine methyl groups actually
makes close contact with the inhibitor, thereby pushing the amino-
pyridine ring of inhibitor farther away from the Val106 position in
eNOS compared to the aminopyridine position seen in nNOS. This
different position of the aminopyridine ring in eNOS also pushes
the Tyr477 side chain away relative to the Tyr706 position in
nNOS, and as a result, Tyr706 in nNOS makes better stacking inter-
actions with the aminopyridine ring of the inhibitor than does
Tyr477 in eNOS. Taken together these differences observed be-
tween eNOS and nNOS provide a structural basis for the relatively
poor binding of these inhibitors to eNOS.
In summary, the cyclopropyl containing pyrrolidine inhibitors
reported in this study show a decrease in potency with both nNOS
and eNOS compared with parental inhibitor 1. This may result in
part from the lowered basicity of the tail amino group. The struc-
tures here demonstrate the importance of the hydrogen bond
strength of this amino group to the alternate side chain rotamer
of the active site Glu residue. Weakening of this hydrogen bond
results in a loss in potency toward nNOS, leading to poorer
isoform selectivity of these more rigid cyclopropyl-containing
inhibitors. These observations indicate that other chemical modi-
fications are needed to retain the inhibitory potency in future
inhibitor design.
4. Experimental section
4.1. General method A: Rh(II)-catalyzed cyclopropanation
To a solution of styrene derivative 4a–c (20 mmol) in dry tolu-
ene (40 mL) was added Rh2(OAc)4 (0.4 mmol) catalyst. The result-
ing mixture was heated at 80 °C for 30 min, then ethyl diazoacetate
(10 mmol) was added dropwise at the same temperature over a
period of 1 h. The reaction mixture was allowed to stir at 85 °C
for an additional 2 h and then cooled to room temperature. The sol-
vent was removed by rotary evaporation, and the resulting oil was
purified by flash chromatography (1–10% ethyl acetate in hexanes)
to generate 5a–c as mixtures of cis/trans isomers.
4.2. General method B: epimerization and hydrolysis
Figure 1. The nNOS active site structure with (A) 2b (1.85 Å, PDB code 3RQJ), (B) 2c
(2.21 Å, 3RQK), or (C) 2d (1.93 Å, 3RQL) bound. The omit Fo ꢁ Fc difference density
To a solution of 5a–c (10 mmol) in EtOH (10 mL) was added
NaOCH3 (40 mL) portionwise. The reaction solution was heated un-
der reflux for 40 h and then concentrated by rotary evaporation.
The resulting residue was partitioned between CH2Cl2 (200 mL)
and H2O (100 mL). The aqueous layer was extracted with CH2Cl2
(2 ꢂ 100 mL). The combined organic layers were dried over Na2SO4
and concentrated. The crude ethyl ester was taken up in MeOH
(70 mL), to which was added LiOH (345 mg, 15 mmol) and H2O
contoured at 3
r level is shown around the bound inhibitor. Note that the two
alternate Glu592 side chain rotamers are observed for 2b, but only the new rotamer
is seen for 2c and 2d. Significant hydrogen bonds are depicted with dashed lines.
The heme pyrrole ring positions are labeled in panel A. All structural figures were
made with PyMol (www.pymol.org).
2d or 2e bound were also determined as shown in Figure 4. Both
inhibitors show the same binding mode in eNOS as that observed
in nNOS structures (see Figs 1C and 3A). For eNOS-2d the bulky

1338
H. Li et al. / Bioorg. Med. Chem. 21 (2013) 1333–1343
Figure 2. (A) The active site structure of nNOS-2b (green, 3RQJ) with the molecule of 2d (gray, 3RQL) overlaid in order to illustrate the chirality difference at the cyclopropyl
ring. Two alternate Glu592 side chain rotamers are shown. (B) The active site structure of nNOS-2e (gray, 3RQM) with the molecule of 2f (green, 3RQN) overlaid. The two
compounds in the structures show different stereochemistry (S or R) at the position of the methyl group.
Figure 3. The active site of nNOS with (A) 2e (1.95 Å, 3RQM) or (B) 2f (1.95 Å, 3RQN) bound. The omit Fo ꢁ Fc difference density contoured at 3
r level is shown around the
bound inhibitor. Significant hydrogen bonds are depicted with dashed lines. In panel A, two partially occupied water molecules are also shown which are complimentary to
the two alternate Glu592 side chain positions.
(70 mL). The reaction was heated at 70 °C for 16 h. After cooling to
room temperature, MeOH was removed by rotary evaporation. The
resulting aqueous solution was acidified with concentrated HCl to
pH 1 and then extracted with ethyl acetate (3 ꢂ 150 mL). The com-
bined organic layers were dried over Na2SO4, and concentrated.
The crude product was purified by flash chromatography to yield
6a–c (75–80%) as white solids.
concentrated. The crude product was purified by flash chromatog-
raphy to yield 7a–c (75–82%) as white solids.
4.4. General method D: Boc-deprotection of 7a–c
To a solution of 7a–c (1.0 mmol) in CH2Cl2 (5 mL) was added
TFA (5 mL). The reaction mixture was stirred at room temperature
for 30 min. The solvent was removed by rotary evaporation. The
yellow oil was dried under vacuum for 24 h to give crude amines
3a–c as yellow oils, which were used in the next step without fur-
ther purification.
4.3. General method C: Curtius rearrangement
To a solution of 6a–c (2.0 mmol) in dry t-BuOH (0.3 M) was
added diphenyl phosphorazidate (DPPA, 2.2 mmol) and TEA
(3.0 mmol). The reaction solution was heated at 85 °C for two days
then cooled to room temperature and concentrated. The resulting
solution was partitioned between ether (50 mL) and NaHCO3
(50 mL). The aqueous layer was extracted with ether (2 ꢂ 50 mL).
The combined organic layers were dried over Na2SO4 and
4.5. General method E: reductive amination
To a solution of aldehyde (0.1 mmol) in CH2Cl2 (3 mL) was
added amine (0.11 mmol), followed by TEA (0.2 mmol), and
NaBH(OAc)3 (0.12 mmol). The mixture was stirred at room

H. Li et al. / Bioorg. Med. Chem. 21 (2013) 1333–1343
1339
Figure 4. The eNOS active site with (A) 2d (2.08 Å, 3RQO) or (B) 2e (2.35 Å, 3RQP) bound. The omit Fo ꢁ Fc difference density contoured at 3
r
level is shown around the bound
inhibitor. Significant hydrogen bonds are depicted with dashed lines.
temperature for an additional 3 h and then concentrated. The
crude product was purified by flash column chromatography (ethyl
acetate/hexanes, 2:1–4:1) to give the product as a colorless oil.
130.2, 130.3, 142.4, 142.5, 162.2, 164.2, 179.8; LC-MS (MꢁH+) calcd
for C10H10ClO2 197, found 197.
4.10. tert-Butyl 2-(3-fluorophenyl)cyclopropylcarbamate (7a)
4.6. General method F: Boc-deprotection of 10a–d and 11e–f
Compound 7a was synthesized using general method C (75%):
1H NMR (500 MHz, CDCl3) d 1.10–1.25 (m, 1H), 1.40–1.55 (m,
10H), 2.00–2.10 (br s, 1H), 2.73 (br s, 1H), 5.04 (br s, 1H), 6.82–
6.88 (m, 2H), 6.92–6.94 (d, J = 7.5 Hz, 1H), 7.20–7.25 (dd, J = 7.5,
14.0 Hz, 1H); 13C NMR (125 MHz, CDCl3) d 16.6, 25.1, 28.6, 32.9,
113.0, 113.2, 113.4, 113.6, 122.4, 129.9, 130.0, 130.1, 143.77,
143.83, 162.2, 164.1; LCQ-MS (M+H+) calcd for C14H19FNO2 252,
found 252.
To a solution of tri-Boc-protected inhibitor (0.2 mmol) in MeOH
(0.5 mL) was added 6 N HCl (1.0 mL). The reaction mixture was al-
lowed to stand at room temperature for 16 h and then concen-
trated. The resulting pale yellow oil was evacuated for 30 h to
give the inhibitors (95–99%).
4.7. 2-(3-Fluorophenyl)cyclopropanecarboxylic acid (6a)
Compound 6a was synthesized using general method A and B
(75%): 1H NMR (500 MHz, CDCl3) d 0.87–0.91 (dd, J = 6.0, 13.5 Hz,
1H), 1.21–1.29 (m, 1H), 1.39–1.43 (ddd, J = 5.0, 6.5, 8.0 Hz, 1H),
1.67–1.72 (m, 1H), 1.90–1.94 (m, 1H), 2.58–2.63 (m, 1H), 6.79–
6.82 (dd, J = 2.0, 5.5 Hz, 1H), 6.90–6.95 (m, 2H), 7.24–7.28 (m,
1H), 8.90–11.00 (br s, 1H); 13C NMR (125 MHz, CDCl3) d 17.8,
24.4, 26.9, 31.8, 113.3, 113.5, 113.8, 114.0, 122.30, 122.32, 130.2,
130.3, 142.4, 142.5, 162.2, 164.2, 179.8; LCQ-MS (MꢁH+) calcd
for C10H8FO2 179, found 179.
4.11. tert-Butyl 2-m-tolylcyclopropylcarbamate (7b)
Compound 7b was synthesized using general method C (82%):
1H NMR (500 MHz, CDCl3) d 1.00–1.20 (m, 1H), 1.46 (s, 9H),
1.95–2.05 (ddd, J = 3.0, 6.5, 9.5 Hz, 1H), 2.31 (s, 3H), 2.74 (br s,
1H), 4.85 (br s, 1H), 6.91–6.93 (m, 2H), 6.97–6.99 (d, J = 7.5 Hz,
1H), 7.13–7.16 (dd, J = 7.5, 8.0 Hz, 1H); 13C NMR (125 MHz, CDCl3)
d 16.3, 21.4, 24.5, 28.4, 32.6, 120.2, 120.3, 123.4, 126.1, 126.8,
127.2, 128.2, 130.1, 137.9, 140.6; LCQ-MS (M+H+) calcd for
C15H21NO2 248, found 248.
4.8. 2-m-Tolylcyclopropanecarboxylic acid (6b)
4.12. tert-Butyl 2-(3-chloroluorophenyl)cyclopropylcarbamate
Compound 6b was synthesized using general method A and B
(77%): 1H NMR (500 MHz, CDCl3) d 1.30–1.40 (ddd, J = 4.5, 7.0,
7.5 Hz, 1H), 1.60–1.65 (dd, J = 5.0, 9.0 Hz, 1H), 1.85–1.90 (ddd,
J = 4.5, 5.0, 7.5 Hz, 1H), 2.30 (s, 3H), 2.50–2.60 (ddd, J = 4.5, 7.0,
9.0 Hz, 1H), 6.85–6.95 (m, 1H), 7.00–7.05 (m, 1H), 7.15–7.22 (m,
2H), 9.00–11.00 (br s, 1H); 13C NMR (125 MHz, CDCl3) d 17.5,
21.4, 24.0, 27.1, 123.2, 127.0, 127.4, 128.4, 138.2, 139.4, 180.1;
LCQ-MS (MꢁH+) calcd for C11H13O2 177, found 177.
(7c)
Compound 7c was synthesized using general method C (77%):
1H NMR (500 MHz, CDCl3) d 1.14–1.17 (dd, J = 6.5, 7.0 Hz, 2H),
1.45 (s, 9H), 1.99–2.03 (ddd, J = 2.5, 7.5, 10.5 Hz, 1H), 2.72 (br s,
1H), 4.88 (br s, 1H), 6.95–7.02 (d, J = 7.0 Hz, 1H), 7.10–7.25 (m,
3H); 13C NMR (125 MHz, CDCl3) d 16.3, 24.5, 28.4, 32.6, 120.1,
120.3, 124.8, 126.2, 126.6, 129.5, 129.9, 134.1, 142.9; LCQ-MS
(M+H+) calcd for C14H19ClNO2 268, found 268.
4.9. 2-(3-Chlorophenyl)cyclopropanecarboxylic acid (6c)
4.13. Compounds 8a and 8b
Compound 6c was synthesized using general method A and B
(80%): 1H NMR (500 MHz, CDCl3) d 1.30–1.40 (ddd, J = 2.0, 3.5,
7.0 Hz, 1H), 1.60–1.65 (dd, J = 5.0, 9.0 Hz, 1H), 1.85–1.91 (m, 1H),
2.50–2.60 (m, 1H), 6.85–7.02 (m, 1H), 7.05–7.10 (m, 1H), 7.15–
7.22 (m, 2H), 9.00–11.00 (br s, 1H); 13C NMR (125 MHz, CDCl3) d
17.8, 24.4, 26.9, 31.8, 113.3, 113.5, 113.8, 114.0, 122.30, 122.32,
To a solution of amine 3a (450 mg, 3.0 mmol) in CH2Cl2 was
added camphanic chloride (650 mg, 3.0 mmol) followed by TEA
(510 lL, 3.75 mmol). The reaction was allowed to stir at room tem-
perature for 30 min and then concentrated. The resulting oil was
purified by flash chromatography (ethyl acetate/hexanes, 1:4) to

1340
H. Li et al. / Bioorg. Med. Chem. 21 (2013) 1333–1343
generate 8a (445 mg, 45%): 1H NMR (500 MHz, CDCl3) d 0.92 (s,
3H), 1.12 (s, 6H), 1.19–1.22 (m, 1H), 1.28–1.30 (m, 2H), 1.65–
1.75 (m, 1H), 1.85–2.00 (m, 2H), 2.05–2.15 (m, 1H), 2.50–2.60
(m, 1H), 2.90–2.98 (m, 1H), 6.68 (br s, 1H), 6.84–6.90 (m, 2H),
6.95–6.97 (d, J = 7.5 Hz, 1H), 7.21–7.25 (m, 1H); 13C NMR
(125 MHz, CDCl3) d 9.7, 15.8, 16.5, 16.7, 24.67, 24.78, 29.0, 30.3,
31.6, 54.0, 55.3, 92.4, 113.1, 113.27, 113.32, 113.5, 122.30,
122.32, 129.8, 129.9, 142.6, 142.7, 162.0, 163.9, 168.3, 178.2; LC-
TOF (M+H+) calcd for C19H23FNO3 332.1662, found 332.1673.
Compound 8b (360 mg, 36%): 1H NMR (500 MHz, CDCl3) d 0.91
(s, 3H), 1.11 (s, 6H), 1.20–1.35 (m, 3H), 1.65–1.75 (m, 1H), 1.85–
2.00 (m, 2H), 2.00–2.10 (m, 1H), 2.50–2.58 (m, 1H), 2.89–2.95
(m, 1H), 6.67 (br s, 1H), 6.84–6.90 (m, 2H), 6.95–6.97 (d,
J = 8.0 Hz, 1H), 7.21–7.24 (m, 1H); 13C NMR (125 MHz, CDCl3) d
9.7, 16.0, 16.5, 16.7, 24.61, 24.63, 29.0, 30.3, 31.5, 54.0, 55.3,
92.38, 92.40, 113.1, 113.29, 113.32, 113.4 113.5, 113.6, 122.30,
122.32, 129.8, 129.9, 142.66, 142.72, 162.0, 163.9, 168.2, 168.3,
178.2; LC-TOF (M+H+) calcd for C19H23FNO3 332.1662, found
332.1677.
159.7; LC-TOF (M+H+) calcd for C43H60F3N4O8 685.3977, found
685.3991.
4.17. (3R,4R)-tert-Butyl 3-((6-(bis(tert-butoxycarbonyl)amino)-
4-methylpyridin-2-yl)methyl)-4-(2-((1S,2R)-2-(3-fluorophenyl)-
cyclopropylamino)ethoxy)pyrrolidine-1-carboxylate (10b)
Compound 10b was synthesized using general method D (82%):
1H NMR (500 MHz, CDCl3) d 0.95–1.01 (m, 1H), 1.10–1.15 (m, 1H),
1.40–1.45 (m, 27H), 1.89–1.92 (m, 1H), 2.28–2.32 (m, 3H), 2.33–
2.37 (m, 1H), 2.60–2.75 (m, 1H), 2.76–2.83 (m, 1H), 2.85–2.93
(m, 2H), 2.95–3.00 (m, 1H), 3.05–3.15 (m, 1H), 3.27–3.31 (m,
1H), 3.32–3.34 (m, 1H), 3.35–3.55 (m, 1H), 3.57–3.74 (m, 1H),
3.75–3.85 (m, 1H), 6.65–6.72 (m, 1H), 6.80–6.89 (m, 2H), 6.90–
6.95 (m, 2H), 7.15–7.20 (m, 1H); 13C NMR (100 MHz, CDCl3) d
14.2, 17.4, 17.5, 21.0, 22.7, 24.7, 24.8, 27.9, 28.5, 29.7, 31.6, 34.6,
34.7, 36.6, 41.4, 41.5, 42.5, 43.1, 44.7, 48.7, 48.8, 49.1, 50.3, 50.9,
60.4, 68.3, 78.6, 79.2, 79.3, 79.4, 82.8, 82.9, 112.2, 112.4, 112.6,
119.5, 119.6, 121.6, 122.7, 128.5, 128.6, 129.6, 129.7, 131.9,
132.0, 132.1, 132.2, 149.6, 151.5, 151.8, 154.6, 154.8, 159.0,
159.1, 162.0, 163.9; LC-TOF (M+H+) calcd for C37H54FN4O7
685.3977, found 685.3979.
4.14. (1S,2R)-2-(3-Fluorophenyl)cyclopropanamine (3d)
To a solution of 8a (330 mg, 1.0 mmol) in EtOH (5 mL) was
slowly added 12 N HCl (10 mL). The resulting mixture was heated
under reflux for 72 h and then cooled to room temperature. The
solvent was removed by rotary evaporation, and the resulting
crude material was purified with flash chromatography (2–5%
MeOH in CH2Cl2) to give 3d as a yellow oil (105 mg, 70%): 1H
NMR (500 MHz, CDCl3) d 1.10–1.30 (br s, 1H), 1.40–1.60 (br s,
1H), 2.30–2.50 (br s, 1H), 2.70–2.90 (br s, 1H), 6.70–6.72 (m, 1H),
6.72–6.77 (m, 1H), 6.89–7.00 (m, 1H), 7.10–7.30 (m, 1H), 7.80–
8.20 (br s, 2H); 13C NMR (125 MHz, CDCl3) d 13.2, 21.5, 31.6,
113.5, 113.7, 114.2, 114.4, 122.29, 122.31, 130.4, 130.5, 140.1,
140.2; LC-TOF (M+H+) calcd for C9H11FN 152.0876, found
152.0870.
4.18. (3R,4R)-tert-Butyl 3-((6-(bis(tert-butoxycarbonyl)amino)-
4-methylpyridin-2-yl)methyl)-4-(2-((1S,2R/1R,2S)-2-m-
tolylcyclopropylamino)ethoxy)pyrrolidine-1-carboxylate (10c)
Compound 10c was synthesized using general method D (87%):
1H NMR (500 MHz, CDCl3) d 0.90–0.97 (m, 1H), 1.00–1.05 (m, 1H),
1.40–1.45 (m, 27H), 1.84–1.91 (m, 1H), 2.27–2.32 (m, 6H), 2.33–
2.40 (m, 1H), 2.60–2.75 (m, 1H), 2.75–2.83 (m, 1H), 2.85–2.93
(m, 2H), 2.95–3.00 (m, 1H), 3.05–3.15 (m, 1H), 3.25–3.30 (m, 1H),
3.31–3.34 (m, 1H), 3.35–3.55 (m, 1H), 3.57–3.74 (m, 1H), 3.75–
3.85 (m, 1H), 6.85–6.95 (m, 4H), 6.92–7.01 (m, 2H), 7.10–7.20
(m, 2H); 13C NMR (100 MHz, CDCl3) d 13.7, 14.1, 14.2, 17.2, 17.3,
17.4, 19.1, 20.9, 21.0, 21.1, 21.4, 22.2, 24.96, 24.99, 25.0, 25.5,
27.9, 28.5, 29.7, 30.6, 34.6, 34.7, 41.0, 41.1, 42.6, 43.2, 48.8, 48.9,
49.0, 49.1, 50.3, 60.4, 64.4, 68.4, 78.6, 79.2, 79.3, 79.4, 82.8, 82.9,
119.5, 119.6, 122.7, 122.8, 122.9, 126.3, 126.5, 126.6, 126.7,
126.8, 128.17, 128.24, 128.3, 128.5, 128.6, 132.0, 132.1, 137.8,
137.9, 141.3, 142.3, 149.6, 151.4, 151.5, 151.8, 154.8, 159.1,
159.2, 171.2; LC-TOF (M+H+) calcd for C38H57N4O7 681.4227, found
681.4224.
4.15. (1R,2S)-2-(3-Fluorophenyl)cyclopropanamine (3e)
Compound 3e was synthesized using a similar procedure to that
of 3d using 8b as a starting material (67%): 1H NMR (500 MHz,
CDCl3) d 1.10–1.30 (br s, 1H), 1.40–1.60 (br s, 1H), 2.30–2.50 (br
s, 1H), 2.70–2.90 (br s, 1H), 6.70–6.72 (m, 1H), 6.72–6.77 (m, 1H),
6.89–7.00 (m, 1H), 7.10–7.30 (m, 1H), 7.80–8.20 (br s, 2H); 13C
NMR (125 MHz, CDCl3) d 13.2, 21.5, 31.6, 113.5, 113.7, 114.2,
114.4, 122.29, 122.31, 130.4, 130.5, 140.1, 140.2; LC-TOF (M+H+)
calcd for C9H11FN 152.0876, found 152.0870.
4.19. (3R,4R)-tert-Butyl 3-((6-(bis(tert-butoxycarbonyl)amino)-
4-methylpyridin-2-yl)methyl)-4-(2-((1S,2R/1R,2S)-2-(3-
clorophenyl)cyclopropylamino)ethoxy)pyrrolidine-1-
carboxylate (10d)
4.16. (3R,4R)-tert-Butyl 3-((6-(bis(tert-butoxycarbonyl)-
amino)-4-methylpyridin-2-yl)methyl)-4-(2-((1S,2R/1R,2S)-
2-(3-fluorophenyl)cyclopropylamino)ethoxy)pyrrolidine-1-
carboxylate (10a)
Compound 10d was synthesized using general method D (87%):
1H NMR (500 MHz, CDCl3) d 0.92–0.99 (m, 1H), 1.07–1.11 (m, 1H),
1.40–1.45 (m, 27H), 1.85–1.90 (m, 1H), 2.29–2.33 (m, 3H), 2.34–
2.38 (m, 1H), 2.60–2.75 (m, 1H), 2.76–2.83 (m, 1H), 2.85–2.93
(m, 2H), 2.95–3.00 (m, 1H), 3.05–3.15 (m, 1H), 3.25–3.30 (m, 1H),
3.31–3.34 (m, 1H), 3.35–3.52 (m, 1H), 3.57–3.74 (m, 1H), 3.75–
3.85 (m, 1H), 6.85–6.92 (m, 2H), 6.93–7.05 (m, 2H), 7.08–7.20
(m, 4H); 13C NMR (100 MHz, CDCl3) d 17.4, 17.5, 17.6, 18.7, 20.9,
21.0, 22.2, 24.9, 25.0, 25.3, 26.2, 27.9, 28.5, 34.6, 34.7, 35.6, 41.3,
41.4, 41.5, 41.6, 42.5, 43.1, 48.7, 48.8, 49.1, 50.3, 52.0, 68.4, 68.5,
76.7, 78.6, 79.2, 79.3, 79.4, 82.8, 82.9, 119.5, 119.6, 122.7, 124.0,
124.07, 124.10, 124.12, 124.3, 125.5, 125.6, 125.7, 125.81, 125.83,
125.86, 125.94, 128.5, 128.6, 129.4, 129.5, 129.6, 132.0, 132.1,
134.1, 134.2, 143.6, 144.6, 144.7, 149.6, 151.47, 151.53, 151.8,
154.8, 159.7; LC-TOF (M+H+) calcd for C37H53ClN4O7 701.3681,
found 701.3884.
Compound 10a was synthesized using general method D (81%):
1H NMR (500 MHz, CDCl3) d 0.95–1.00 (m, 1H), 1.06–1.10 (m, 1H),
1.40–1.46 (m, 27H), 1.86–1.90 (m, 1H), 2.26–2.33 (m, 3H), 2.35–
2.40 (m, 1H), 2.60–2.75 (m, 1H), 2.76–2.85 (m, 1H), 2.86–2.92
(m, 2H), 2.95–2.98 (m, 1H), 3.05–3.13 (m, 1H), 3.24–3.30 (m,
1H), 3.30–3.35 (m, 1H), 3.36–3.51 (m, 1H), 3.57–3.75 (m, 1H),
3.77–3.85 (m, 1H), 6.85–6.92 (m, 2H), 6.93–7.20 (m, 4H); 13C
NMR (100 MHz, CDCl3) d 17.4, 17.5, 17.6, 18.7, 20.9, 21.0, 22.2,
24.9, 25.0, 25.3, 26.2, 27.9, 28.5, 34.6, 34.7, 35.6, 41.3, 41.4, 41.5,
41.6, 42.5, 43.1, 48.7, 48.8, 49.1, 50.3, 52.0, 68.4, 68.5, 76.7, 78.6,
79.2, 79.3, 79.4, 82.8, 82.9, 119.5, 119.6, 122.7, 124.0, 124.07,
124.10, 124.12, 124.3, 125.5, 125.6, 125.7, 125.81, 125.83, 125.86,
125.94, 128.5, 128.6, 129.4, 129.5, 129.6, 132.0, 132.1, 134.1,
134.2, 143.6, 144.6, 144.7, 149.6, 151.47, 151.53, 151.8, 154.8,

H. Li et al. / Bioorg. Med. Chem. 21 (2013) 1333–1343
1341
4.20. 6-(((3R,4R)-4-(2-((1S,2R/1R,2S)-2-(3-Fluorophenyl)-
cyclopropylamino)ethoxy)pyrrolidin-3-yl)methyl)-4-
methylpyridin-2-amine (2a)
s, 0.5H), 4.12 (br s, 0.5H), 6.34 (s, 0.5H), 6.39 (s, 0.5H), 6.51 (s,
0.5H), 6.53 (s, 0.5H), 6.95–7.01 (m, 1H), 7.05–7.20 (m, 3H); 13C
NMR (125 MHz, D2O) d 12.3, 12.4, 12.6, 20.3, 20.4, 20.5, 21.1,
28.8, 28.9, 30.6, 37.4, 38.6, 41.2, 41.3, 47.0, 47.1, 47.2, 47.5, 49.2,
49.3, 63.5, 64.5, 78.2, 78.4, 110.31, 110.34, 113.9, 114.0, 124.7,
124.75, 124.82, 126.1, 126.16, 126.23, 126.7, 126.9, 127.0, 130.0,
130.1, 130.2, 133.8, 133.9, 134.0, 140.2, 140.7, 145.52, 145.54,
153.8, 158.1; LC-TOF (M+H+) calcd for C22H30ClN4O 401.2108,
found 401.2093.
Compound 2a was synthesized using general method E as a
mixture of two diastereomers (99%): 1H NMR (500 MHz, D2O) d
1.30–1.40 (m, 1H), 1.41–1.48 (m, 1H), 2.10–2.20 (m, 3H), 2.30–
2.50 (m, 1H), 2.51–2.77 (m, 2H), 2.80–2.90 (m, 1H), 2.91–3.03
(m, 1H), 3.04–3.40 (m, 3H), 3.41–3.72 (m, 2H), 3.73–3.88 (m,
1H), 3.90–4.11 (m, 1H), 4.40–4.50 (m, 1H), 6.30–6.60 (m, 2H),
6.70–6.90 (m, 2H), 7.00–7.20 (m, 2H); 13C NMR (125 MHz, D2O) d
12.6, 12.9, 20.7, 20.9, 21.2, 21.4, 29.1, 37.7, 37.8, 38.6, 41.5, 45.3,
47.3, 47.6, 47.7, 49.6, 63.8, 64.5, 78.3, 78.5, 109.0, 110.5, 113.2,
113.4, 113.8, 114.0, 114.3, 122.4, 127.2, 128.1, 129.2, 130.1,
136.4, 140.7, 140.9, 145.9, 146.6, 153.0, 154.0, 158.3, 161.8,
163.8; LC-TOF (M+H+) calcd for C21H30FN4O2 385.2404, found
385.2393.
4.24. (3R,4R)-tert-Butyl 3-((6-(benzyl(tert-butoxycarbonyl)-
amino)-4-methylpyridin-2-yl)methyl)-4-(2-((tert-butoxycarbonyl)
((1S,2R/1R,2S)-2-(3-fluorophenyl)cyclopropyl)amino)ethoxy)-
pyrrolidine-1-carboxylate (10e)
To a solution of aldehyde 9b (100 mg, 0.18 mmol) in CH2Cl2
(2 mL) was added 3a (60 mg, 0.37 mmol) followed by TEA
(125 lL, 0.9 mmol). The mixture was allowed to stir at room tem-
perature for 5 min before NaBH(OAc)3 (50 mg, 0.23 mmol) was
added. The reaction mixture was stirred for an additional 3 h then
was partitioned between ethyl acetate (50 mL) and brine (25 mL).
The organic layer was dried over Na2SO4 and concentrated to yield
crude secondary amine. To a solution of the resulting crude amine
in MeOH (1.5 mL) was added (Boc)2O (120 mg, 0.56 mmol) and
4.21. 6-(((3R,4R)-4-(2-((1S,2R)-2-(3-Fluorophenyl)-
cyclopropylamino)ethoxy)pyrrolidin-3-yl)methyl)-4-
methylpyridin-2-amine (2b)
Compound 2b was synthesized using general method E (95%):
1H NMR (500 MHz, D2O) d 1.30–1.40 (m, 1H), 1.41–1.46 (m, 1H),
2.18 (s, 3H), 2.19–2.20 (m, 1H), 2.40–2.50 (m, 1H), 2.55–2.70 (m,
2H), 2.81–3.00 (m, 3H), 3.19–3.23 (dd, J = 3.5, 13.5 Hz, 1H), 3.30–
3.40 (m, 2H), 3.49–3.52 (m, 1H), 3.55–3.61 (m, 1H), 3.75–3.85
(m, 1H), 4.06 (br s, 1H), 6.38 (s, 1H), 6.54 (s, 1H), 6.80–6.95 (m,
3H), 7.15–7.25 (dd, J = 5.5, 10.5 Hz, 1H); 13C NMR (125 MHz, D2O)
d 12.6, 20.4, 21.0, 28.8, 30.1, 38.4, 41.3, 47.1, 47.5, 49.3, 64.3,
78.3, 110.3, 112.9, 113.1, 113.6, 113.8, 114.0, 122.1, 128.9, 129.0,
130.4, 130.5, 131.8, 131.9, 133.1, 140.6, 140.7, 145.6, 153.9,
158.1; LC-TOF (M+H+) calcd for C21H30FN4O2 385.2404, found
385.2384.
Et3N (75 lL, 0.56 mmol). The reaction mixture was stirred at room
temp for 6 h and was partitioned between ethyl acetate (50 mL)
and brine (20 mL). The organic layer was dried over Na2SO4, and
the solvents were removed by rotary evaporation. The resulting
material was purified by flash column chromatography (ethyl ace-
tate/hexanes, 1:4–1:2) to yield 10e (70 mg, 60%) as a colorless oil
(71%): 1H NMR (500 MHz, CDCl3) d 1.15–1.21 (m, 2H), 1.35–1.50
(m, 28H), 1.65–1.80 (br s, 1H), 2.10–2.20 (br s, 1H), 2.25–2.33
(m, 3H), 2.50–2.80 (m, 3H), 2.80–2.97 (m, 1H), 3.00–3.10 (m, 1H),
3.15–3.20 (m, 1H), 3.25–3.50 (m, 3H), 3.55–3.70 (m, 3H), 4.05–
4.10 (m, 1H), 5.18 (s, 2H), 6.55–6.65 (m, 1H), 6.70–6.90 (m, 3H),
7.10–7.30 (m, 6H), 7.40–7.45 (m, 1H); 13C NMR (125 MHz, CDCl3)
d 14.1, 14.3, 14.5, 19.5, 21.2, 21.4, 23.0, 28.5, 28.6, 28.9, 30.0,
30.7, 31.7, 34.7, 34.7, 40.2, 40.3, 42.9, 49.1, 49.6, 50.3, 50.4, 51.1,
60.6, 64.6, 78.1, 79.4, 79.7, 80.2, 81.3, 81.5, 112.9, 113.0, 117.2,
117.3, 120.2, 122.2, 126.7, 127.0, 127.2, 127.3, 128.3, 130.1,
140.2, 148.8, 148.8, 154.2, 154.7, 154.7, 155.1, 157.9, 162.1,
164.2, 171.4; LC-TOF (M+H+) calcd for C44H60FN4O7 775.4441,
found 775.4432.
4.22. 4-Methyl-6-(((3R,4R)-4-(2-((1S,2R/1R,2S)-2-m-
tolylcyclopropylamino)ethoxy)pyrrolidin-3-yl)methyl)pyridin-
2-amine (2c)
Inhibitor 2c was synthesized using general method E as a mix-
ture of two diastereomers (90%): 1H NMR (500 MHz, D2O) d 1.10–
1.30 (m, 1H), 1.35–1.45 (m, 1H), 2.10–2.15 (m, 3H), 2.15–2.20 (m,
3H), 2.20–2.50 (m, 1H), 2.50–2.80 (m, 2H), 2.81–3.00 (m, 2H),
3.19–3.25 (m, 1H), 3.30–3.40 (m, 2H), 3.47–3.52 (m, 1H), 3.55–
3.70 (m, 1H), 3.71–3.85 (m, 1H), 4.00–4.15 (m, 1H), 6.35–6.60
(m, 2H), 6.85–6.90 (m, 2H), 6.91–7.15 (m, 2H); 13C NMR
(125 MHz, D2O) d 12.2, 12.3, 15.1, 20.2, 20.4, 20.5, 20.6, 20.7,
21.0, 21.6, 23.2, 28.8, 28.9, 30.1, 30.5, 31.8, 37.4, 38.5, 41.2, 41.3,
47.0, 47.2, 47.5, 49.2, 49.3, 63.6, 64.5, 78.1, 78.3, 110.2, 110.3,
113.9, 114.0, 122.9, 123.1, 123.3, 126.3, 126.5, 126.6, 126.7,
126.8, 127.5, 127.6, 127.7, 128.6, 128.7, 128.78, 128.82, 128.9,
129.0, 131.8, 131.9, 137.9, 138.1, 138.7, 138.9, 139.0, 145.59,
145.62, 153.8, 158.1; LC-TOF (M+H+) calcd for C21H30FN4O2
381.2654, found 381.2653.
4.25. (3R,4R)-tert-Butyl 3-((6-(benzyl(tert-butoxycarbonyl)-
amino)-4-methylpyridin-2-yl)methyl)-4-(2-((tert-
butoxycarbonyl)((1S,2R)-2-(3-
fluorophenyl)cyclopropyl)amino)ethoxy)pyrrolidine-1-
carboxylate (10f)
Compound 10f was synthesized, using a procedure similar to
that for 10e (60%), as a colorless oil (71%): 1H NMR (500 MHz,
CDCl3) d 1.10–1.22 (m, 2H), 1.35–1.50 (m, 28H), 1.65–1.80 (br s,
1H), 2.10–2.20 (br s, 1H), 2.20–2.35 (m, 3H), 2.40–2.80 (m, 3H),
2.80–2.95 (m, 1H), 2.97–3.10 (m, 1H), 3.16–3.21 (m, 1H), 3.22–
3.44 (m, 3H), 3.45–3.70 (m, 3H), 4.04–4.10 (m, 1H), 5.10–5.25
(br s, 2H), 6.55–6.65 (m, 1H), 6.65–6.95 (m, 3H), 7.10–7.27 (m,
6H), 7.31–7.45 (m, 1H); 13C NMR (125 MHz, CDCl3) d 14.0, 14.4,
14.5, 19.4, 21.3, 21.4, 22.9, 28.4, 28.7, 28.8, 30.0, 30.9, 31.8,
34.8, 34.9, 40.0, 40.1, 42.9, 49.1, 49.5, 50.2, 50.5, 51.1, 60.7,
64.6, 78.2, 79.4, 79.7, 80.2, 81.4, 81.5, 112.9, 113.0, 117.2, 117.3,
120.2, 122.1, 126.8, 127.1, 127.2, 127.3, 128.3, 130.0, 140.1,
148.7, 148.8, 154.1, 154.6, 154.7, 155.0, 157.9, 162.2, 164.1,
171.4, 171.5; LC-TOF (M+H+) calcd for C44H60FN4O7 775.4441,
found 775.4441.
4.23. 6-(((3R,4R)-4-(2-((1S,2R/1R,2S)-2-(3-Clorophenyl)-
cyclopropylamino)ethoxy)pyrrolidin-3-yl)methyl)-4-
methylpyridin-2-amine (2d)
Compound 2d was synthesized using general method E as a
mixture of two diastereomers (91%): 1H NMR (500 MHz, D2O) d
1.10–1.30 (m, 1H), 1.35–1.50 (m, 1H), 2.15–2.20 (m, 3H), 2.25–
2.30 (m, 1H), 2.35–2.50 (m, 1H), 2.55–2.70 (m, 2H), 2.70–2.77
(m, 1H), 2.81–3.00 (m, 2H), 3.19–3.25 (m, 1H), 3.30–3.40 (m, 1H),
3.49–3.52 (m, 1H), 3.55–3.70 (m, 1H), 3.75–3.85 (m, 1H), 4.05 (br

1342
H. Li et al. / Bioorg. Med. Chem. 21 (2013) 1333–1343
4.26. 6-(((3R,4R)-4-(2-(((R/S)-1-(3-Fluorophenyl)propan-2-
yl)amino)ethoxy)pyrrolidin-3-yl)methyl)-4-methylpyridin-2-
amine (2e)
44.2, 44.4, 47.3, 49.5, 55.6, 64.0, 64.4, 78.4, 110.6, 114.3, 114.4,
114.5, 116.2, 116.4, 125.4, 125.6, 130.8, 130.9, 138.3, 145.9,
146.0, 154.1, 158.4, 161.9, 163.9; LC-TOF (M+H+) calcd for
C22H32FN4O 387.2560, found 385.2556.
To a solution of 10e (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 30 h, then cooled to room temperature. The cat-
alyst was removed by filtration, and the resulting solution was
concentrated by rotary evaporation. The crude material was puri-
fied by flash column chromatography (ethyl acetate/hexanes,
1:4–1:2) to yield 11e as a white foamy solid. To a solution of the
resulting 11e in MeOH (0.5 mL) was added 6 N HCl (1.0 mL). The
reaction mixture was allowed to stand at room temperature for
16 h. The solvent was removed by rotary evaporation. The crude
product was recrystallized using cold diethyl ether to provide 2e
as a pale yellow solid (20 mg, 25%): 1H NMR (500 MHz, D2O) d
1.18–1.20 (m, 3H), 2.20 (s, 3H), 2.60–2.72 (m, 2H), 2.75–2.90 (m,
2H), 3.00–3.10 (m, 2H), 3.15–3.33 (m, 3H), 3.34–3.42 (m, 1H),
3.44–3.60 (m, 3H), 3.70–3.80 (m, 1H), 4.09 (s, 1H), 6.46–4.67 (m,
1H), 6.56 (s, 1H), 6.90–7.10 (m, 3H), 7.20–7.25 (m, 1H); 13C NMR
(125 MHz, D2O) d 15.2, 15.5, 21.3, 29.2, 38.4, 38.5, 41.6, 41.7,
4.27. 6-(((3R,4R)-4-(2-(((S)-1-(3-Fluorophenyl)propan-2-
yl)amino)ethoxy)pyrrolidin-3-yl)methyl)-4-methylpyridin-2-
amine (2f)
Compound 2f was synthesized using a procedure similar to that
for 2e (25%): 1H NMR (500 MHz, D2O) d 1.28–1.30 (d, J = 6.5 Hz,
3H), 2.31 (s, 3H), 2.75–2.85 (m, 2H), 2.85–3.00 (m, 2H), 3.10–3.20
(m, 2H), 3.30–3.37 (m, 2H), 3.37–3.46 (m, 1H), 3.46–3.55 (m,
1H), 3.55–3.70 (m, 3H), 3.80–3.90 (m, 1H), 4.20 (s, 1H), 6.57 (s,
1H), 6.67 (s, 1H), 7.00–7.05 (m, 1H), 7.06–7.09 (m, 1H), 7.09–7.13
(d, J = 7.5 Hz, 1H), 7.33–7.40 (dd, J = 7.5, 14.5 Hz, 1H); 13C NMR
(125 MHz, D2O) d 15.0, 21.0, 29.0, 38.2, 41.4, 43.9, 47.0, 49.3,
55.4, 64.2, 78.1, 110.4, 114.0, 114.1, 114.3, 115.9, 116.1, 125.18,
125.20, 130.6, 130.7, 130.07, 130.13, 145.7, 153.9, 158.1, 161.7,
163.6; LC-TOF (M+H+) calcd for C22H32FN4O 387.2560, found
385.2547.
Table 2
Crystallographic data collection and refinement statistics
Data set
nNOS-2b
nNOS-2c
nNOS-2d
nNOS-2e
Data collection
PDB code
3RQJ
3RQK
3RQL
3RQM
Space group
Cell dimensions
a, b, c (Å)
Resolution (Å)
Rsym or Rmerge
P212121
P212121
P212121
P212121
51.8, 110.7, 164.3
1.85 (1.88–1.85)
0.044 (0.63)
36.2 (2.5)
52.0, 111.5, 164.4
2.20 (2.24–2.20)
0.076 (0.54)
25.6 (2.1)
51.7, 110.8, 163.9
1.93 (1.96–1.93)
0.052 (0.59)
29.8 (2.3)
51.9, 110.4, 164.0
1.95 (1.98–1.95)
0.061 (0.59)
22.9 (2.0)
I/rI
No. unique reflections
Completeness (%)
Redundancy
82,378
99.6 (99.7)
4.1 (4.1)
47,658
97.9 (97.5)
3.8 (3.7)
71,869
99.7 (100.0)
4.1 (4.0)
69,867
99.7 (99.5)
3.8 (3.6)
Refinement
Resolution (Å)
No. reflections used
Rwork/Rfree
1.85
78,043
0.189/0.221
2.21
45,045
0.200/0.258
1.93
68,065
0.190/0.235
1.95
66,358
0.173/0.208
No. atoms
Protein
Ligand/ion
6,677
185
6,662
185
6,697
188
6,672
179
Water
328
170
312
538
R.m.s. deviations
Bond lengths (Å)
Bond angles (°)
0.015
1.508
0.015
1.535
0.017
1.637
0.013
1.372
Data seta
nNOS-2f
eNOS-2d
eNOS -2e
Data collection
PDB code
3RQN
3RQO
3RQP
Space group
Cell dimensions
a, b, c (Å)
Resolution (Å)
Rsym or Rmerge
P212121
P212121
P212121
52.1, 110.9, 164.2
1.95 (1.98–1.95)
0.056 (0.55)
27.0 (2.6)
58.1, 106.6, 156.5
2.08 (2.12–2.08)
0.072 (0.66)
22.8 (2.1)
57.9, 106.6, 156.9
2.35 (2.39–2.35)
0.086 (0.67)
13.8 (1.5)
I/rI
No. unique reflections
Completeness (%)
Redundancy
70,044
99.9 (100.0)
4.1 (4.1)
59,039
99.6 (99.9)
3.7 (3.8)
41,301
99.5 (99.2)
3.6 (3.5)
Refinement
Resolution (Å)
1.95
2.08
2.35
No. reflections used
Rwork/Rfree
66,498
0.173/0.209
55,805
0.174/0.221
39,184
0.178/0.228
b
No. atoms
Protein
Ligand/ion
6,698
185
6,416
193
6,462
203
Water
396
403
255
R.m.s. deviations
Bond lengths (Å)
Bond angles (°)
0.013
1.352
0.015
1.557
0.014
1.522
a
See Table 1 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
b
for all data sets according to those used in the data of the starting model.

H. Li et al. / Bioorg. Med. Chem. 21 (2013) 1333–1343
1343
thank the staff at SSRL for their assistance during the remote X-
ray diffraction data collections.
5. Inhibitory activity assay
The three isozymes, murine macrophage iNOS, rat nNOS, and
bovine eNOS, were recombinant enzymes, overexpressed in
Escherichia coli and isolated as reported.30 IC50 values for inhibitors
2a–f were measured for the three different isoforms of NOS using
L-arginine as a substrate. The formation of nitric oxide was mea-
sured using a hemoglobin capture assay described previously.31
References and notes
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Commun. 1994, 205, 659.
8. Norris, P. J.; Waldvogel, H. J.; Faull, R. L. M.; Love, D. R.; Emson, P. C.
Neuronscience 1996, 1037, 72.
9. Sims, N. R.; Anderson, M. F. Neurochem. Int. 2002, 40, 511.
10. Calabrese, V.; Mancuso, C.; Calvani, M.; Rizzarelli, E.; Butterfield, D. A.; Stella, A.
M. Nat. Rev. 2007, 8, 766.
11. Hobbs, A. J.; Higgs, A.; Moncada, S. Annu. Rev. Pharmacol. Toxicol. 1999, 39, 191.
12. 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.
13. 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.
14. (a) Lawton, G. R.; Ranaivo, H. R.; Wing, L. K.; Ji, H.; Xue, F.; Martesek, P.; Roman,
L. J.; Watterson, D. M.; Silverman, R. B. Bioorg. Med. Chem. 2009, 17, 2371; (b) Ji,
H.; Delker, S. L.; Li, H.; Martasek, P.; Roman, L. J.; Poulos, T. L.; Silverman, R. B. J.
Med. Chem. 2010, 53, 7804.
All NOS isozymes were assayed at room temperature in
100 mM Hepes buffer (pH 7.4) containing 10 -arginine,
1.6 mM CaCl2, 11.6 g/mL calmodulin, 100 DTT, 100
NADPH, 6.5 M H4B, 3.0 M oxyhemoglobin (for iNOS assays, no
a
lM L
l
lM
lM
l
l
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 Ki values of
inhibitors were calculated from the IC50 values using Equation 1
with known Km values (rat nNOS, 1.3
1.7 M).
l
M; iNOS, 8.3
l
M; eNOS,
l
Ki ¼ IC50=ð1 þ ½Sꢃ=Km
Þ
ð1Þ
15. Silverman, R. B. The Organic Chemistry of Drug Design and Drug Action, 2nd
edition; Academic Press: San Diego, 2004.
6. Crystal structure determination
16. Xue, F.; Fang, J.; Lewis, W. W.; Martasek, P.; Roman, L. J.; Silverman, R. B. Bioorg.
Med. Chem. Lett. 2010, 20, 554.
17. Xue, F.; Huang, H.; Li, H.; Ji, H.; Fang, J.; Poulos, T. L.; Silverman, R. B. Bioorg.
Med. Chem. 2010, 18, 6526.
18. Delker, S. L.; Xue, F.; Li, H.; Jamal, J.; Silverman, R. B.; Poulos, T. L. Biochemistry
2010, 49, 10803.
19. Xue, F.; Li, H.; Fang, J.; Poulos, T. L.; Silverman, R. B. J. Am. Chem. Soc. 2010, 132,
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20. Xue, F.; Delker, S. L.; Li, H.; Fang, J.; Jamal, J.; Silverman, R. B.; Poulos, T. L. J. Med.
Chem. 2011, 54, 2039.
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Lajoiea, G. A. Bioorg. Med. Chem. 2003, 11, 869.
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4167.
The preparation of protein samples of nNOS and eNOS heme do-
main and the procedures for crystal growth and inhibitor soaks
were the same as reported.19 The cryogenic (100 K) X-ray diffrac-
tion data were collected remotely at various beamlines at Stanford
Synchrotron Radiation Light Source (SSRL) through the data collec-
tion control software Blu-Ice32 and crystal mounting robot.
Raw data frames were indexed, integrated, and scaled using
HKL2000.33 The reflection files were then concerted to the mtz for-
mat using routines Scalepack2mtz and Truncate in the CCP4
suite.34 The binding of each inhibitor was revealed by the initial
difference Fourier maps calculated with REFMAC.35 The inhibitor
molecules were then modeled in COOT36 and refined using REF-
MAC. Water molecules were added in REFMAC and inspected visu-
ally through COOT. The TLS37 protocol was implemented in the
final stage of refinements with each subunit as one TLS group.
The omit Fo ꢁ Fc electron density map was calculated at the end
of the refinement with the coefficient DELFWT in a mtz file gener-
ated by a round of TLS refinement fed with an inhibitor-free coor-
dinate file. The refined structures were validated in COOT before
deposition in the RCSB protein data bank. The crystallographic data
collection and structure refinement statistics are summarized in
Table 2 with PDB accession codes included.
24. Kozikowski, A.; Raven Press: New York, 1993.
25. Wermuth, C. G.; The Practice of Medicinal Chemistry; Academic Press: San
Diego, 1996.
26. Perrin, C. L.; Fabian, M. A.; Rivero, I. A. Tetrahedron 1999, 55, 5773.
27. Pryde, D. C.; Cook, A. S.; Burring, D. J.; Jones, L. H.; Foll, S.; Platts, M. Y.;
Sanderson, V.; Corless, M.; Stobie, A.; Middleton, D. S.; Foster, L.; Barker, L.;
Graaf, P. V. D.; Stacey, P.; Kohl, C.; Coggon, S.; Beaumont, K. Bioorg. Med. Chem.
2007, 15, 142.
28. Tchilibon, S.; Kim, S.-K.; Gao, Z.-G.; Harris, B. A.; Blaustein, J. B.; Gross, A. S.;
Duong, H. T.; Melman, N.; Jacobson, K. A. Bioorg. Med. Chem. 2004, 12, 2021.
29. Xue, F.; Kraus, J. M.; Jansen Labby, K.; 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.
30. (a) Hevel, J. M.; White, K. A.; Marletta, M. A. J. Biol. Chem. 1991, 266, 22789; (b)
Roman, L. J.; Sheta, E. A.; Martásek, P.; Gross, S. S.; Liu, Q.; Masters, B. S. S. Proc.
Natl. Acad. Sci. U.S.A. 1995, 92, 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,
359.
Acknowledgments
31. Hevel, J. M.; Marletta, M. A. Method Enzymol. 1994, 233, 250.
32. 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.
33. Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307.
34. Collaborative Computational Project Number 4, the CCP4 Suite: Programs for
Protein Crystallography. Acta Cryst. 1994, D50, 760.
35. Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Acta Cryst. 1997, D53, 240.
36. Emsley, P.; Cowtan, K. Acta Cryst. 2004, D60, 2126.
37. Winn, M. D.; Isupov, M. N.; Murshudov, G. N. Acta Cryst. 2001, D57, 122.
The authors are grateful for financial support from the National
Institutes of Health (GM49725 to RBS and GM57353 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 Distin-
guished Professorship in Chemistry (AQ0012). P.M. is supported
by Grant 0021620849 from MSMT of the Czech Republic. We also