Structure-based design, synthesis, and biological evaluation of lipophilic-tailed monocationic inhibitors of neuronal nitric oxide synthase

Article abstract of DOI:10.1016/j.bmc.2010.06.074

Selective inhibitors of neuronal nitric oxide synthase (nNOS) have the potential to develop into new neurodegenerative therapeutics. Recently, we described the discovery of novel nNOS inhibitors (1a and 1b) based on a cis-pyrrolidine pharmacophore. These compounds and related ones were found to have poor blood-brain barrier permeability, presumably because of the basic nitrogens in the molecule. Here, a series of monocationic compounds was designed on the basis of docking experiments using the crystal structures of 1a,b bound to nNOS. These compounds were synthesized and evaluated for their ability to inhibit neuronal nitric oxide synthase. Despite the excellent overlap of these compounds with 1a,b bound to nNOS, they exhibited low potency. This is because they bound in the nNOS active site in the normal orientation rather than the expected flipped orientation used in the computer modeling. The biphenyl or phenoxyphenyl tail is disordered and does not form good protein-ligand interactions. These studies demonstrate the importance of the size and rigidity of the side chain tail and the second basic amino group for nNOS binding efficiency and the importance of the hydrophobic tail for conformational orientation in the active site of nNOS.

Full text of DOI:10.1016/j.bmc.2010.06.074

Bioorganic & Medicinal Chemistry 18 (2010) 6526–6537
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure-based design, synthesis, and biological evaluation of lipophilic-tailed
monocationic inhibitors of neuronal nitric oxide synthase
Fengtian Xue a,b, ,à, Jinwen Huang a,b, , Haitao Ji a,b, Jianguo Fang a,b, Huiying Li c,d, Pavel Martásek e,f
,
a,b,
Linda J. Roman e, Thomas L. Poulos c,d, , Richard B. Silverman
*
*
a Department of Chemistry, Center for Molecular Innovation and Drug Discovery, and Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208-3113, USA
b Department of Biochemistry, Molecular Biology, and Cell Biology, Center for Molecular Innovation and Drug Discovery, and Chemistry of Life Processes Institute,
Northwestern University, Evanston, IL 60208-3113, USA
c Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900, USA
d Department of Pharmaceutical Chemistry, and Chemistry, University of California, Irvine, CA 92697-3900, USA
e Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX, USA
f Department of Pediatrics and Center for Applied Genomics, 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 inhibitors of neuronal nitric oxide synthase (nNOS) have the potential to develop into new neu-
rodegenerative therapeutics. Recently, we described the discovery of novel nNOS inhibitors (1a and 1b)
based on a cis-pyrrolidine pharmacophore. These compounds and related ones were found to have poor
blood–brain barrier permeability, presumably because of the basic nitrogens in the molecule. Here, a ser-
ies of monocationic compounds was designed on the basis of docking experiments using the crystal
structures of 1a,b bound to nNOS. These compounds were synthesized and evaluated for their ability
to inhibit neuronal nitric oxide synthase. Despite the excellent overlap of these compounds with 1a,b
bound to nNOS, they exhibited low potency. This is because they bound in the nNOS active site in the
normal orientation rather than the expected flipped orientation used in the computer modeling. The
biphenyl or phenoxyphenyl tail is disordered and does not form good protein–ligand interactions. These
studies demonstrate the importance of the size and rigidity of the side chain tail and the second basic
amino group for nNOS binding efficiency and the importance of the hydrophobic tail for conformational
orientation in the active site of nNOS.
Received 15 May 2010
Revised 18 June 2010
Accepted 21 June 2010
Available online 1 July 2010
Keywords:
Neuronal nitric oxide synthase
cis-Pyrrolidine pharmacophore
Neurodegenerative therapeutics
Enzyme inhibitors
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
the discovery of the novel nNOS inhibitors 1a,b (Fig. 1)12 based
on a cis-pyrrolidine pharmacophore.13,14 Inhibitor 1b has excellent
Many lines of biological evidence have shown that overproduc-
tion of nitric oxide (NO) in the central nervous system (CNS) is
implicated in various types of neurodegenerative diseases, includ-
ing Parkinson’s,1–3 Alzheimer’s,4 and Huntington’s disease.5 Neuro-
nal nitric oxide synthase (nNOS), the enzyme responsible for the
generation of neuronal NO, is, therefore, a promising target for
the development of new neurodegenerative therapeutics.6–9 Over
the past two decades, significant research has been devoted to
developing nNOS selective inhibitors.10,11 Recently, we described
potency (Ki = 15 nM), as well as high isozyme selectivity for nNOS
(2100-fold over endothelial NOS; 630-fold over inducible NOS),
which makes this compound a promising lead for neurodegenera-
tive therapeutics.12
Inhibition of an enzyme that is implicated in the CNS, such
as nNOS, however, requires the designed molecules to cross
the blood–brain barrier (BBB).15 Generally, there are three criteria
for a small molecule to cross the BBB: (1) molecular weight
<450 Da;16 (2) reasonably good lipid solubility;16 and (3) no affinity
for one of BBB active efflux transporters (e.g., p-glycoprotein).17–21
Recent animal tests demonstrated that 1a,b have low BBB
permeability, which strongly impedes further application of this
compound as a neurodegenerative therapeutic.12 Among the mech-
anisms that could limit 1b from crossing the BBB, the dicationic
character of this molecule under physiological conditions, as a result
of the two high pKa secondary amino groups, is a crucial factor
prohibiting passive diffusion.16 To improve the BBB permeability,
prodrug approaches have been carried out to partially decrease
Abbreviations: nNOS, neuronal nitric oxide synthase; CNS, central nervous
system; BBB, blood–brain barrier; TEA, triethylamine; TBAF, tetrabutylammonium
fluoride; TBS, t-butyldimethylsilyl.
* Corresponding authors. Tel.: +1 847 491 5653; fax: +1 847 491 7713 (R.B.S.).
E-mail addresses: poulos@uci.edu (T.L. Poulos), r-silverman@northwestern.edu,
agman@chem.northwestern.edu (R.B. Silverman).
These authors contributed equally to the research.
Present address: Department of Chemistry, University of Louisiana at Lafayette,
à
PO Box 44370, Lafayette, LA 70504, USA.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.074

F. Xue et al. / Bioorg. Med. Chem. 18 (2010) 6526–6537
6527
R
H
N
H
N
H
H2N
H2N
H2N
N
R
O
O
O
N
F
H2N
N
N
H
2a
( )-
( )-1a
H
N
R1
N
R
O
H
N
2b-f
( )-
H
H
N
F
H2N
N
O
N
( )-1b
N
( )-2g-k
R2
Figure 1. Design of monocationic inhibitors 2 based on 1b.
the cationic character of 1b.22 Herein, we describe the structure-
based design and synthesis of a new series of nNOS inhibitors
(2, Fig. 1) with the aminoalkyl tail of 1b replaced by a lipophilic
biphenyl fragment or a phenoxyphenyl fragment. We reasoned that
these compounds would be effective, given that they were designed
based on a recent unexpected finding on how the (3R,4R) enantio-
mer of 1a binds to nNOS.23 It was anticipated that the (3R,4R)-1a
would bind such that the aminopyridine ring stacks over the heme
and H-bonds with the active site Glu592. Instead, we found that it
binds in a flipped orientation with the aminopyridine extending
out of the active site, where it H-bonds with a heme propionate.
We also found that the (3R,4R)-1b binds the same as its counterpart
of 1a (to be published).23,24 To make room for the aminopyridine,
a monocationic compound, which should have a better chance to
cross the BBB by passive diffusion.25 Second, using a phenoxyphenyl
or biphenyl fragment, new inhibitors 2 possess a more rigid struc-
ture compared to 1b, which can potentially improve the potency
of inhibitors and be less susceptible to metabolic degradation. The
4-methyl group on the aminopyridine ring of 1b was removed in
compounds 2a–f because the methyl group of the eutomer of 1b is
stabilized at the hydrophobic pocket of rat nNOS lined with
Met336, Leu337, and Trp306 (chain B).23,24 In human NOS, the resi-
due that corresponds to rat nNOS Leu337 is His342, and it is the only
residue in the active site that differs between rat nNOS and human
nNOS.26 It was hypothesized that demethylated compound 2a–f
might fit better in the active site of human nNOS, which does not
have this hydrophobic pocket.
Tyr706 must swing out of the way, where it forms a p-stacking inter-
action with the aminopyridine (Fig. 2).23,24 Because of this new bind-
ing orientation, compounds with fewer basic amino groups could
overlay nicely on the flipped binding mode of 1b. There are two po-
tential advantages to this new design. First, one of the two high pKa
amino groups is removed from the lead compound (1b), resulting in
2. Chemistry
The synthesis of key intermediate 9 began with 2-amino-6-
methylpyridine (3) (Scheme 1). Protection of the amino group in
3 with (Boc)2O in the presence of triethylamine (TEA) gave 4 in
an excellent yield. Boc-protected aminopyridine 4 was treated
with 2 equiv of n-BuLi, and the resulting dianion was allowed to re-
act with epoxide 513 to give secondary alcohol 6 in good yields.
Next, 6 was converted to a bis-Boc-protected alcohol using a
three-step procedure.27 First, the hydroxyl group was protected
as the t-butyldimethylsilyl (TBS)-ether (7) using TBSCl in the pres-
ence of imidazole. Then, 7 was treated with (Boc)2O in the presence
of N,N-dimethylpyridine (DMAP) to provide bis-Boc-protected silyl
ether 8. Finally, the TBS-protecting group was removed using tetra-
butylammonium fluoride (TBAF) to generate 9 in high yields.
The substituted phenoxyphenols (10b–f) were synthesized
using a two-step procedure (Scheme 2). First, Ullmann couplings
of m-methoxyphenol (11) with iodobenzene derivatives using
Cs2CO3 in the presence of a catalytic amount of CuBr provided
12b–f.28 Then, the methyl ethers were cleaved using BBr3 to give
10b–f in high yields.
With both 9 and 10a–f in hand, the syntheses of inhibitors 2a–f
were completed (Scheme 3). After screening a series of reaction
conditions, the Mitsunobu reaction using 9 and 10a–f as starting
materials went smoothly at room temperature to generate 12a–f
in modest to good yields. Then, the three Boc-protecting groups
of 12a–f were removed in TFA to generate inhibitors 2a–f.
The synthesis of single enantiomer 14a and 14b is shown in
Scheme 4. The free NH group on the pyridine ring of 15 was pro-
tected with a SEM-protecting group using SEM-Cl using NaH as a
Figure 2. Docking conformation of 2h (green) in the active site of nNOS. The potent
and selective inhibitor N1-[(3R,4R)-40-((600-amino-400-methylpyridin-200-yl)methyl)-
pyrrolidin-30-yl]-N2-(30-fluorophenethyl)ethane-1,2-diamine (blue), the (3R,4R)
enantiomer of 1a in Figure 1 in the crystal structure of rat nNOS (PDB code:
3JWT)23 is overlaid for comparison.

6528
F. Xue et al. / Bioorg. Med. Chem. 18 (2010) 6526–6537
Boc
N
i
ii
H2N
N
BocHN
N
BocHN
N
OH
3
6
4
Boc
N
Boc
N
7 R = TBS, R' = H
8 R = TBS, R' = Boc
9 R = H, R' = Boc
iv
v
iii
Boc
N
R'
N
OR
5
O
Scheme 1. Synthesis of 9. Reagents and conditions: (i) (Boc)2O, TEA, rt, 12 h, 97%; (ii) (1) n-BuLi, ꢀ78 °C to 0 °C; (2) 5, ꢀ78 °C to rt, 16 h, 70%; (iii) TBSCl, imidazole, rt, 16 h,
78%; (iv) (Boc)2O, DMAP, rt, 30 h, 92%; (v) TBAF, rt, 30 min, 97%.
R
R
i
ii
MeO
OH
MeO
F
O
12b-f
HO
O
10b-f
11
Cl
Cl
F
F
b
f
c
d
e
Scheme 2. Synthesis of 10b–f. Reagents and conditions: (i) iodobenzene, Cs2CO3, CuBr, 60 °C, 48 h, 87–95%; (ii) BBr3, ꢀ78 °C to rt, 85–92%.
Boc
N
H
N
i
ii
9
H2N
N
OR
(Boc)2N
N
OR
2a-f
13a-f
F
F
O
b
O
c
a
Cl
Cl
F
O
O
O
d
e
f
Scheme 3. Syntheses of inhibitors 2a–f. Reagents and conditions: (i) phenol, PPh3, DEAD, 0 °C to rt, 72 h, 35–51%; (ii) TFA/CH2Cl2 (1:2), rt, 4 h, 91–95%.
base to yield 16 in good yields. The two enantiomers were resolved
through camphanic ester derivatives using a Mitsunobu reaction to
generate two separable diastereomers 17a and 17b in reasonable
yields. Finally, the ester linkage was hydrolyzed using Na2CO3 to
provide chiral precursor 14a and 14b in excellent yields.
The bi-aryl fragment 19a–e was synthesized using a two-step
procedure (Scheme 5). Suzuki coupling of 4-(bromomethyl)phenol
with a series of boronic acid generated to 18a–e. Then, the hydro-
xyl group of 18a–e was converted to bromide (19a–e) by refluxing
in HBr in high yields.
Then, global deprotection of SEM-protecting group and Boc-pro-
tecting groups generated inhibitors 2g–k in high yields.
3. Results and discussion
Inhibitors 2a–k were evaluated for in vitro inhibition activity
against three isozymes of NOS: rat nNOS, bovine eNOS, and murine
iNOS using known methods.22 The results are summarized in
Table 1. Inhibitor 2a, with a biphenyl group in place of the aminoal-
kyl tail of 1b, had a Ki value of 10
phenyl analog 2b had slightly better inhibitory activity (Ki = 3.0
for nNOS). Inhibitors 2c and 2d, with halogen-substituents at the
lM for nNOS, while the phenoxy-
The syntheses of inhibitors 2g–k were finished as shown in
Scheme 6. Compound 14b was treated with NaH, and the resulting
anion was reacted with 19a–e to provide 13g–k in excellent yields.
lM

F. Xue et al. / Bioorg. Med. Chem. 18 (2010) 6526–6537
6529
Boc
N
Boc
N
Boc
N
O
O
O
O
O
b
O
Boc
Boc
Boc
N
N
O
N
N
O
N
R
N
OH
SEM
SEM
17a
17b
15
16
(+/-)-
(+/-)-
R = H
R = SEM
a
c
c
Boc
N
Boc
N
Boc
Boc
N
N
OH
N
N
OH
14a
14b
SEM
SEM
Scheme 4. Synthesis of 14a and 14b. Reagents and conditions: (a) NaH, SEM-Cl, rt, 16 h, 68%; (b) (S)-(ꢀ) camphanic acid, PPh3, DEAD, rt, 16 h, 88%; (d) Na2CO3, rt, 4 h, 95%.
b
a
OH
OH
18a-e
Br
19a-e
Br
Ar
Ar
Cl
F3C
Cl
F
F
F
a
b
c
d
e
Scheme 5. Synthesis of 19a–e. Reagents and conditions: (a) ArB(OH)2, Pd(Ph3P)4, 2 N Na2CO3, DME, 80 °C, 48 h; (b) HBr, reflux, 3 h.
Boc
N
H
N
a
F
b
Boc
14b
N
SEM
N
O
H2N
N
O
13g-k
2g-k
Ar
Ar
Cl
Cl
F3C
F
F
g
h
i
j
k
Scheme 6. Synthesis of 2g–k. Reagents and conditions: (a) NaH, 4-arylbenzyl bromide, rt, 6 h; (b) 4 N HCl in MeOH, rt, 16 h, 90%.
para position of the terminal phenyl ring, are weaker inhibitors than
the non-substituted analog (2b). However, the additional substitu-
ent at the meta position on the ring, inhibitors 2e and 2f, showed
slightly enhanced potency relative to 2a against nNOS. We believe
that the additional fluorine or methyl substituent fits into a small
hydrophobic pocket at Met336, providing additional binding en-
ergy. Inhibitors 2g–k, with the installation of a 4-methyl group on
the aminopyridine ring, indicated some improved inhibitory activ-
ity. All new inhibitors are significantly less potent (300–600-fold)
than lead compound 1b, with Ki values in the low micromolar range,
although the shape and size of the active site of nNOS can fit this ser-
ies of compounds according to the docking calculation. These results
were initially disappointing, given that this series overlaid very well
with the crystal structure of the (3R,4R)-1b bound to nNOS in the
flipped mode (Fig. 2). However, the crystal structure of a representa-
tive phenoxyphenyl analogue (2b) bound to nNOS showed that
although the racemic mixture of 2b was used in crystal preparation,
Table 1
Kia values of inhibitors for rat nNOS, bovine eNOS, and murine iNOS
Compound
nNOS (
l
M)
eNOS (
l
M)
iNOS (
l
M)
Selectivityb
n/e
n/i
2a
2b
2c
2d
2e
2f
2g
2h
2i
10.0
3.0
7.8
8.4
2.2
4.3
0.75
1.2
0.82
0.62
0.76
>200
>200
18
16
8.3
>200
>200
221
>20
>67
2.3
1.9
1.8
1.7
18
22
17
13
18
>20
>67
28
10
18
9
53
46
45
36
66
79.5
81.8
79.1
39.5
55.6
37.3
22.1
50.0
15
13.3
26.0
13.6
8.0
2j
2k
13.3
a
The Ki values were calculated based on the directly measured IC50 values, which
represent at least duplicate measurements with standard deviations of 10%.
b
The ratio of Ki (eNOS or iNOS) to Ki (nNOS).

6530
F. Xue et al. / Bioorg. Med. Chem. 18 (2010) 6526–6537
only the (3R,4R) enantiomer was observed in the structure, and it did
not bind in the flipped orientation (Fig. 3). Instead, the aminopyri-
dine was observed to bind in the normal orientation in which the
aminopyridine interacts with Glu592, and Tyr706 remains in its na-
tive position. This is the first case in the pyrrolidine series of com-
pounds in which the (3R,4R) stereochemistry does not bind in the
flipped orientation. It appears that the configuration around the
(3,4) positions of the pyrrolidine, therefore, is not the sole determi-
nant controlling the binding orientation of these inhibitors; the size
and rigidity of the tail also are very important. When the tail is bulky
and rigid, as that in 2b, the inhibitor cannot adopt the flipped mode
because its tail no longer fits into the active site over the heme distal
surface. Instead, the aminopyridine ring interacts with Glu592, and
the tailend ofthe inhibitor extendsout of theactive site. The absence
of electron density for the phenoxyphenyl tail indicates poor inter-
actions with protein groups, which may account, in part, for the rel-
atively weak affinity seen for these compounds. It is interesting that
the (3S,4S)-1b24 was bound in the same orientation as was observed
for (3R,4R)-2b in this structure. The pyrrolidine nitrogen of (3S,4S)-
2b would have H-bonded directly to Glu592, which is not observed
here (Fig. 3). It is likely that the bulky phenoxyphenyl tail in (3S,4S)-
2b would have an even poorer fit than what was seen for the tail in
(3R,4R)-2b. The bulkiness of this hydrophobic tail, apparently, does
not permit the molecule from assuming the flipped binding mode.
ally prefer the normal binding mode with a disordered tail, and,
therefore, these compounds are only weak inhibitors of nNOS.
The secondary amino group in the lipophilic tail also might be crit-
ical for tight binding of 1 to nNOS in the flipped mode, as com-
pounds without this amino group, to date, bind poorly to nNOS.
5. Experimental procedures
5.1. General methods
All syntheses were conducted under anhydrous conditions in an
atmosphere of argon, using flame-dried apparatus and employing
standard techniques in handling air-sensitive materials. All sol-
vents were distilled and stored under an argon or nitrogen atmo-
sphere before use. 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 TMS), integration, multiplicity
(s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br =
broad), coupling constant (J/Hz). Coupling constants were taken di-
rectly 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 CD3OD, respectively. High-resolution mass spectra were
measured on liquid chromatography/time-of-flight mass spec-
trometry (LC–TOF).
4. Conclusion
A new series of nNOS inhibitors (2a–k) has been designed and
synthesized based on docking experiments with the crystal struc-
ture of the lead compound, (3R,4R)-1b, which bound to nNOS in a
flipped binding mode. Because of the bulky tail of 2a–k, they actu-
5.2. General procedure A (Ullmann reaction)
To a mixture of Cs2CO3 (1.37 g, 4.2 mmol), CuBr (29 mg, 0.2 mmol),
and ethyl 2-oxocyclohexanecarboxylate28 (64
lL, 0.4 mmol) was
added DMSO (1.0 mL). The mixture was allowed to stir at room
temperature for 30 min, followed by addition of a mixture of 3-
methoxyphenol (2.0 mmol) and iodobenzene (2.0 mmol) as a solu-
tion in DMSO (1.0 mL) through a cannula. The flask was sealed and
heated at 60 °C for 24 h and then cooled to room temperature. The
reaction was filtered through Celite, and the resulting cake was
washed with EtOAc (4 ꢁ 25 mL). The combined organic layers were
washed with brine (50 mL), dried over Na2SO4, and concentrated.
The crude product was purified by flash chromatography (EtOAc/
hexanes, 1:9) to yield 12b–f (87–95%) as pale yellow oils.
5.3. General procedure B (demethylation)
To a solution of a methyl ether (12b–f, 1.0 mmol) in dichloro-
methane at ꢀ78 °C was added BBr3 (1 M solution, 1.2 mL, 1.2 mmol)
dropwise. The mixture was warmed to room temperature over a
period of 2 h and kept stirring overnight. The solvent was removed
by rotary evaporation, and the resulting material was purified by
flash chromatography (EtOAc/hexanes, 1:9) to yield 10b–f (85–
92%) as colorless oils.
5.4. General procedure C (Mitsunobu reaction)
Figure 3. The active site crystal structure of nNOS with 2b bound (PDB code: 3N2R)
showing some relevant hydrogen bonds as dashed lines. The 2Fo–Fc (gray) and
A mixture of 9 (100 mg, 0.2 mmol) and PPh3 (100 mg, 0.3 mmol)
was dissolved in THF (5 mL). To the resulting solution was added a
phenol (10a–f, 0.3 mmol) as a solution in THF (1.0 mL), followed by
Fo–Fc (red) electron density map of 2b are contoured at 1.0
r and ꢀ3.0 r,
respectively. Although crystals were soaked in a racemic mix of 2b, the electron
density is consistent with only the (3R,4R) enantiomer. The pyrrolidine ring
nitrogen of the (3S,4S) enantiomer would have directly hydrogen bonded to Glu592,
replacing the position of Wat1. The electron density for the aminopyridine group is
very clear and partially so for the pyrrolidine. However, there is no electron density
for the phenoxyphenyl tail, indicating disordering and, therefore, the absence of any
tight protein–phenoxyphenyl interactions.
DEAD (60 lL, 0.3 mmol) dropwise. The mixture was allowed to stir
at room temperature for 40 h and then concentrated. The crude
product was purified by flash chromatography to yield 13a–f
(35–51%) as white solids.

F. Xue et al. / Bioorg. Med. Chem. 18 (2010) 6526–6537
6531
5.5. General procedure D (Boc-deprotection)
To a solution of 13a–f (50 mol) in dichloromethane (1.0 mL)
was added TFA (1.0 mL). The reaction mixture was allowed to sit
at room temperature for 4 h and then concentrated. The crude
product was purified by flash chromatography (5–10% methanol
in dichloromethane) to yield 2a–f (91–95%) as white solids.
ture for an additional 1 h, and then the reaction flask was trans-
ferred to an ice-bath for another 30 min. The reaction was cooled
to ꢀ78 °C, to which a solution of epoxide 5 (890 mg, 4.81 mmol)
in THF (10 mL) was added dropwise over a period of 30 min along
the side of the reaction flask. After addition, the Dry Ice-acetone
bath was removed, and the reaction was kept stirring for an addi-
tional 2.5 h. The reaction was quenched with H2O (1.0 mL) and
concentrated by rotary evaporation. The crude product was puri-
fied using flash column chromatography (EtOAc/hexanes, 2:3–
1:1) to provide 6 (1.32 g, 3.37 mmol, 70%) as a white solid: 1H
NMR (500 MHz, CDCl3) d 1.45 (s, 9H), 1.49 (s, 9H), 2.30–2.50 (m,
1H), 2.60–2.80 (m, 2H), 3.00–3.30 (m, 2H), 3.50–3.80 (m, 2H),
4.00–4.20 (m, 1H), 5.04 (br s, 1H), 6.80–6.90 (m, 1H), 7.50–7.80
(m, 3H); 13C NMR (125 MHz, CDCl3) d 28.3, 28.6, 39.2, 44.8, 45.2,
49.2, 49.7, 52.3, 52.7, 60.5, 64.5, 74.5, 75.2, 79.5, 81.1, 110.3,
118.2, 139.2, 151.6, 152.4, 154.6, 157.9; LCQ-MS (M+H+) calcd for
C20H32N3O5 394, found 394.
l
5.6. General procedure E (Suzuki coupling)
To a solution of 3-fluorophenylboronic acid (462 mg, 3.3 mmol)
and 4-bromophenylmethanol (561 mg, 3 mmol) in DME (10 mL)
was added Pd(Ph3P)4 (173 mg, 0.15 mmol) and 2 N Na2CO3
(1.5 mL). The mixture was stirred at 80 °C for 48 h. After cooling
to room temperature, the reaction mixture was filtered through
Celite. The filtrate was concentrate, and the resulting residue was
purified by flash column chromatography (EtOAc/hexane, 1:4) to
generate 18a–e as white solids (80–84%).
5.9.3. tert-Butyl 3-((6-(tert-butoxycarbonylamino)pyridin-2-yl)-
tert-butyldimethylsilyloxy)pyrrolidine-1-carboxylate (7)
5.7. General procedure F
To a solution of 6 (850 mg, 2.16 mmol) in DMF (5.0 mL) were
added TBSCl (408 mg, 2.7 mmol) and imidazole (367 mg, 5.4 mmol).
The reaction mixture was stirred at 40 °C for 24 h and then parti-
tioned between EtOAc (150 mL) and aqueous NH4Cl (100 mL). The
organic layer was washed with brine (100 mL), dried over Na2SO4,
and concentrated. The crude product was purified using flash col-
To 2.0 mmol of 18a–e was added HBr solution (48%, 5 mL). The
reaction was heated under reflux for 3 h, and then cooled to room
temperature. The mixture was partitioned between EtOAc
(100 mL) and H2O (30 mL). The organic layer was washed with
H2O (2 ꢁ 30 mL), dried over Na2SO4, and concentrated. The crude
product was purified by flash column chromatography on silica
gel to provide the product 19a–e (84–90%).
umn chromatography (EtOAc/hexanes, 1:9–1:6) to provide
7
(855 mg, 1.68 mmol, 78%) as a white solid: 1H NMR (500 MHz,
CDCl3) d ꢀ0.03 (s, 6H), 0.81 (s, 9H), 1.40 (s, 9H), 1.47 (s, 9H), 2.40–
2.50 (m, 2H), 2.75–2.85 (m, 1H), 2.98–3.14 (m, 2H), 3.38–3.62 (m,
2H), 3.90–3.98 (m, 1H), 6.73 (d, J = 7.5 Hz, 1H), 7.38–7.54 (m, 2H),
7.71 (m, 1H); 13C NMR (125 MHz, CDCl3) d ꢀ4.6, 18.2, 25.9, 28.4,
28.7, 39.3, 46.1, 46.7, 48.8, 49.1, 52.7, 53.0, 74.5, 75.3, 79.4, 81.0,
109.9, 117.9, 138.7, 151.6, 152.5, 154.9, 158.3; LCQ-MS (M+H+) calcd
for C26H46N3O5Si 508, found 508.
5.8. General procedure G
To a solution of 14b (0.1 mmol) in DMF (2 mL) was added NaH
(60% in mineral oil, 10 mg, 0.25 mmol) at 0 °C. After 10 min, 19a–e
(0.105 mmol) was added as a solution DMF (1 mL). The reaction
was stirred at room temperature for 16 h, and concentratedin vacuo.
The resulting residue was partitioned between EtOAc (100 mL) and
H2O (10 mL). The organic layer was washed with H2O (2 ꢁ 10 mL),
dried over Na2SO4, and concentrated. The crude product was puri-
fied by flash column chromatography on silica gel (EtOAc/hexanes,
5:2) to generate 13g–k as colorless oils (60–66%).
5.9.4. tert-Butyl 3-((6-(bis(tert-butoxycarbonyl)amino)pyridin-
2-yl)-methyl)-4-(tert-butyldimethylsilyloxy)pyrrolidine-1-
carboxylate (8)
To a solution of 7 (507 mg, 1.0 mmol) in THF (10 mL) was added
(Boc)2O (327 mg, 1.5 mmol) and DMAP (30 mg, 0.25 mmol). The
solution was stirred at room temperature for 16 h. The solvent
was removed by rotary evaporation, and the crude product was
purified using flash column chromatography (EtOAc/hexanes,
1:9–1:6) to generate 8 (558 mg, 0.92 mmol, 92%) as a white solid:
1H NMR (500 MHz, CDCl3) d ꢀ0.01 (s, 6H), 0.84 (s, 9H), 1.40–1.45
(m, 27H), 2.45–2.55 (m, 2H), 2.83–3.14 (m, 3H), 3.40–3.64 (m,
2H), 3.98–4.04 (m, 1H), 6.98–7.02 (m, 1H), 7.06–7.14 (m, 1H),
7.55–7.65 (m, 1H); 13C NMR (125 MHz, CDCl3) d ꢀ4.5, 18.2, 25.9,
28.1, 28.7, 39.1, 46.1, 46.7, 48.5, 48.9, 52.6, 53.2, 74.5, 75.1, 79.4,
83.0, 118.9, 121.5, 138.3, 151.5, 152.0, 154.9, 159.2; LCQ-MS
(M+H+) calcd for C31H54N3O7Si 608, found 608.
5.9. General procedure H
A solution of 13g–k (0.1 mmol) in 3 N HCl in methanol (3 mL)
was stirred at room temperature for 24 h. The mixture was concen-
trated, and the resulting crude material was purified by Sephdex
LH-20 to give final inhibitors 2g–k (95–99%) as hydrochloride salts.
5.9.1. tert-Butyl 6-methylpyridin-2-ylcarbamate (4)
To a solution of 2-amino-6-methylpyridine (4.32 g, 40 mmol) in
t-butanol (40 mL) was added (Boc)2O (10.9 g, 50 mmol) and trieth-
ylamine (7.0 mL, 50 mmol). The solution was heated at 50 °C for
24 h. The solvent was removed by rotary evaporation, and the
crude product was purified using flash column chromatography
(EtOAc/hexanes, 1:18–1:9) to generate 4 (7.5 g, 36 mmol, 97%) as
a white solid: 1H NMR (500 MHz, CDCl3) d 1.42 (s, 9H), 2.40 (s,
3H), 7.46–7.50 (dd, J = 7.5, 8.5 Hz, 1H), 6.72–6.74 (d, J = 8.0 Hz,
1H), 7.71–7.73 (d, J = 8.5 Hz, 1H), 8.79 (s, 1H); 13C NMR
(125 MHz, CDCl3) d 24.0, 28.4, 80.7, 109.6, 118.0, 138.6, 152.0,
153.0, 156.9; LCQ-MS (M+H+) calcd for C11H17N2O2 209, found 209.
5.9.5. tert-Butyl 3-((6-(bis(tert-butoxycarbonyl)amino)pyridin-
2-yl)-methyl)-4-hydroxypyrrolidine-1-carboxylate (9)
To a solution of 8 (607 mg, 1.0 mmol) in THF (10 mL) was added
TBAF (1 M in THF, 1.25 mL, 1.25 mmol). The solution was stirred at
room temperature for 16 h. The solvent was removed by rotary
evaporation, and the resulting crude product was purified using
flash column chromatography (EtOAc/hexanes, 1:1) to generate 9
(476 mg, 0.97 mmol, 97%) as a white solid: 1H NMR (500 MHz,
CDCl3) d 1.42–1.47 (m, 27H), 2.43–2.50 (m, 1H), 2.85–2.95 (m,
2H), 3.03–3.12 (m, 1H), 3.15–3.24 (m, 1H), 3.61–3.80 (m, 2H),
4.14–4.18 (m, 1H), 7.06–7.14 (m, 2H), 7.70 (dd, J = 7.5, 8.5 Hz,
1H); 13C NMR (125 MHz, CDCl3) d 28.1, 28.7, 39.5, 39.7, 44.9,
45.7, 49.7, 50.0, 52.5, 53.0, 74.6, 75.3, 79.5, 83.7, 119.8, 120.0,
5.9.2. tert-Butyl 3-((6-(tert-butoxycarbonylamino)pyridin-2-yl)-
methyl)-4-hydroxypyrrolidine-1-carboxylate (6)
To a solution of 4 (1.0 g, 4.81 mmol) in THF (30 mL) at ꢀ78 °C
was added n-BuLi (1.6 M in hexanes, 6.3 mL, 10.0 mmol) dropwise.
The resulting dark red solution was stirred at the same tempera-

6532
F. Xue et al. / Bioorg. Med. Chem. 18 (2010) 6526–6537
122.2, 122.4, 139.1, 151.5, 151.8, 154.7, 159.4; LCQ-MS (M+H+)
calcd for C25H40N3O7 494, found 494.
13C NMR (125 MHz, CDCl3) d 14.9, 150.0, 105.6, 110.2, 110.5,
116.0, 116.2, 118.46, 118.52, 122.67, 122.71, 126.5, 126.7, 130.7,
152.09, 152.11, 157.0, 158.9, 159.5; LC–MS (M+H+) calcd for
5.9.6. 1-(4-Fluorophenoxy)-3-methoxybenzene (12c)
Compound 12c was synthesized using general procedure A
(95%): 1H NMR (500 MHz, CDCl3) d 3.76 (s, 3H), 6.52–6.54 (d,
J = 8.5 Hz, 2H), 6.62–6.64 (d, J = 10.0 Hz, 1H), 6.90–7.10 (m, 4H),
7.18–7.22 (m, 1H); 13C NMR (125 MHz, CDCl3) d 55.5, 104.5,
108.9, 110.5, 116.4, 116.6, 120.9, 121.0, 130.4; LC–MS (M+H+) calcd
for C13H12FO2 219, found 219.
C13H12FO2 219, found 219.
5.9.14. (3R,4R/3S,4S)-tert-Butyl 3-(biphenyl-4-yloxy)-4-((6-(bis-
(tert-butoxycarbonyl)amino)pyridin-2-yl)methyl)pyrrolidine-
1-carboxylate (13a)
Compound 13a was synthesized using general procedure C
(40%): 1H NMR (500 MHz, CDCl3) d 1.40–1.60 (m, 27H), 2.90–3.10
(m, 2H), 3.15–3.25 (m, 1H), 3.35–3.45 (m, 1H), 3.50–3.65 (m,
2H), 3.66–3.80 (m, 1H), 4.68–4.71 (m, 1H), 6.90–6.92 (d,
J = 8.0 Hz, 2H), 7.02–7.06 (m, 1H), 7.07–7.12 (m, 1H), 7.30–7.35
(m, 1H), 7.40–7.65 (m, 4H); 13C NMR (125 MHz, CDCl3) d 24.9,
28.2, 28.7, 28.8, 30.0, 34.9, 42.8, 76.7, 79.7, 79.8, 83.2, 100.0,
116.1, 116.2, 119.3, 120.0, 126.9, 127.0, 128.5, 128.9, 129.0,
134.6, 138.6, 140.9, 151.5, 152.1, 154.6, 154.8, 157.0, 159.3; LCQ-
MS (M+H+) calcd for C37H48N3O7 646, found 646.
5.9.7. 1-(4-Chlorophenoxy)-3-methoxybenzene (12d)
Compound 12d was synthesized using general procedure A
(87%): 1H NMR (500 MHz, CDCl3) d 3.76 (s, 3H), 6.56–6.67 (m,
2H), 6.66–6.68 (d, J = 10.0 Hz, 1H), 6.94–6.96 (d, J = 11.0 Hz, 2H),
7.15–7.35 (m, 3H); 13C NMR (125 MHz, CDCl3) d 55.6, 105.2,
109.5, 111.2, 120.5, 129.9, 130.5; LC–MS (M+H+) calcd for
C13H12ClO2 235, found 235.
5.9.8. 1-Chloro-2-fluoro-4-(3-methoxyphenoxy)benzene (12e)
Compound 12e was synthesized using general procedure A
(88%): 1H NMR (500 MHz, CDCl3) d 3.76 (s, 3H), 6.56–6.67 (m,
2H), 6.66–6.68 (d, J = 10.0 Hz, 1H), 6.94–6.96 (d, J = 11.0 Hz, 2H),
7.15–7.35 (m, 3H); 13C NMR (125 MHz, CDCl3) d 55.6, 105.2,
109.5, 111.2, 120.5, 129.9, 130.5; LC–MS (M+H+) calcd for
5.9.15. (3R,4R/3S,4S)-tert-Butyl 3-((6-(bis(tert-butoxycarbonyl)-
amino)pyridin-2-yl)methyl)-4-(3-phenoxyphenoxy)pyrrol-
idine-1-carboxylate (13b)
Compound 13b was synthesized using general procedure C
(35%): 1H NMR (500 MHz, CDCl3) d 1.40–1.60 (m, 27H), 2.80–3.00
(m, 2H), 3.10–3.20 (dd, J = 7.5, 16.5 Hz, 1H), 3.25–3.35 (m, 1H),
3.45–3.50 (m, 1H), 3.55–3.60 (m, 1H), 3.61–3.70 (m, 1H), 4.57–
4.60 (d, J = 10.5 Hz, 1H), 7.00–7.60 (m, 12H); 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; LC–MS (M+H+)
calcd for C37H48N3O8 662, found 662.
C13H11ClFO2 253, found 253.
5.9.9. 1-Fluoro-4-(3-methoxyphenoxy)-2-methylbenzene (12f)
Compound 12f was synthesized using general procedure A
(87%): 1H NMR (500 MHz, CDCl3) d 3.76 (s, 3H), 6.56–6.67 (m,
2H), 6.66–6.68 (d, J = 10.0 Hz, 1H), 6.94–6.96 (d, J = 11.0 Hz, 2H),
7.15–7.35 (m, 3H); 13C NMR (125 MHz, CDCl3) d 55.6, 105.2,
109.5, 111.2, 120.5, 129.9, 130.5; LC–MS (M+H+) calcd for
5.9.16. (3R,4R/3S,4S)-tert-Butyl 3-((6-(bis(tert-butoxycarbonyl)-
amino)pyridin-2-yl)methyl)-4-(3-(4-fluorophenoxy)phenoxy)-
pyrrolidine-1-carboxylate (13c)
C14H14FO2 233, found 233.
Compound 13c was synthesized using general procedure C
(35%): 1H NMR (500 MHz, CDCl3) d 1.40–1.60 (m, 27H), 2.80–3.00
(m, 2H), 3.10–3.20 (dd, J = 7.5, 16.5 Hz, 1H), 3.25–3.35 (m, 1H),
3.45–3.50 (m, 1H), 3.55–3.60 (m, 1H), 3.61–3.70 (m, 1H), 4.57–
4.60 (d, J = 10.5 Hz, 1H), 7.00–7.60 (m, 12H); 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; LC–MS (M+H+)
calcd for C37H48N3O8 662, found 662.
5.9.10. 3-(4-Fluorophenoxy)phenol (10c)
Compound 10c was synthesized using general procedure B
(85%): 1H NMR (500 MHz, CDCl3) d 5.51 (br s, 1H), 6.44 (s, 1H),
6.49–6.55 (m, 2H), 6.90–7.10 (m, 4H), 7.12–7.16 (dd, J = 10.0,
10.0 Hz, 1H); 13C NMR (125 MHz, CDCl3) d 105.6, 110.3, 110.6,
116.4, 116.7, 121.15, 121.24, 130.7; LC–MS (M+H+) calcd for
C12H10FO2 205, found 205.
5.9.11. 3-(4-Chlorophenoxy)phenol (10d)
5.9.17. (3R,4R/3S,4S)-tert-Butyl 3-((6-(bis(tert-butoxycarbonyl)-
amino)pyridin-2-yl)methyl)-4-(3-(4-chlorophenoxy)phenoxy)-
pyrrolidine-1-carboxylate (13d)
Compound 10d was synthesized using general procedure B
(85%): 1H NMR (500 MHz, CDCl3) d 5.79 (br s, 1H), 6.47 (s, 1H),
6.52–6.58 (dd, J = 11.0, 14.5, 2H), 6.91–6.94 (d, J = 11.0, 2H),
7.13–7.17 (dd, J = 10.0, 11.0 Hz, 1H), 7.23–7.25 (d, J = 11.0, 2H);
13C NMR (125 MHz, CDCl3) d 106.4, 110.9, 111.2, 120.7, 128.8,
130.0, 130.8, 155.6, 157.1, 158.5; LC–MS (M+H+) calcd for
Compound 13d was synthesized using general procedure C
(46%): 1H NMR (500 MHz, CDCl3) d 1.40–1.60 (m, 27H), 2.80–3.00
(m, 2H), 3.10–3.20 (dd, J = 7.5, 16.5 Hz, 1H), 3.25–3.35 (m, 1H),
3.45–3.50 (m, 1H), 3.55–3.60 (m, 1H), 3.61–3.70 (m, 1H), 4.57–
4.60 (d, J = 10.5 Hz, 1H), 7.00–7.60 (m, 12H); 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; LC–MS (M+H+)
calcd for C37H48N3O8 662, found 662.
C12H10ClO2 221, found 221.
5.9.12. 3-(4-Chloro-3-fluorophenoxy)phenol (10e)
Compound 10e was synthesized using general procedure B
(92%): 1H NMR (500 MHz, CDCl3) d 5.79 (br s, 1H), 6.47 (s, 1H),
6.52–6.58 (dd, J = 11.0, 14.5, 2H), 6.91–6.94 (d, J = 11.0, 2H),
7.13–7.17 (dd, J = 10.0, 11.0 Hz, 1H), 7.23–7.25 (d, J = 11.0, 2H);
13C NMR (125 MHz, CDCl3) d 107.1, 107.6, 107.8, 110.7, 110.8,
115.27, 115.29, 131.0, 131.1, 157.2, 157.4; LC–MS (M+H+) calcd
for C12H9ClFO2 239, found 239.
5.9.18. (3R,4R/3S,4S)-tert-Butyl 3-((6-(bis(tert-butoxycarbonyl)-
amino)pyridin-2-yl)methyl)-4-(3-(4-chloro-3-fluorophenoxy)-
phenoxy)pyrrolidine-1-carboxylate (13e)
Compound 13e was synthesized using general procedure C
(48%): 1H NMR (500 MHz, CDCl3) d 1.40–1.60 (m, 27H), 2.80–3.00
(m, 2H), 3.10–3.20 (dd, J = 7.5, 16.5 Hz, 1H), 3.25–3.35 (m, 1H),
3.45–3.50 (m, 1H), 3.55–3.60 (m, 1H), 3.61–3.70 (m, 1H), 4.57–
4.60 (d, J = 10.5 Hz, 1H), 7.00–7.60 (m, 12H); 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; LC–MS (M+H+)
calcd for C37H48N3O8 662, found 662.
5.9.13. 3-(4-Fluoro-3-methylphenoxy)phenol (10f)
Compound 10f was synthesized using general procedure B
(86%): 1H NMR (500 MHz, CDCl3) d 5.79 (br s, 1H), 6.47 (s, 1H),
6.52–6.58 (dd, J = 11.0, 14.5, 2H), 6.91–6.94 (d, J = 11.0, 2H),
7.13–7.17 (dd, J = 10.0, 11.0 Hz, 1H), 7.23–7.25 (d, J = 11.0, 2H);

F. Xue et al. / Bioorg. Med. Chem. 18 (2010) 6526–6537
6533
5.9.19. (3R,4R/3S,4S)-tert-Butyl 3-((6-(bis(tert-butoxycarbonyl)-
5.9.24. 6-(((3R,4R/3S,4S)-4-(3-(4-Chloro-3-fluorophenoxy)-
amino)pyridin-2-yl)methyl)-4-(3-(4-fluoro-3-methylphenoxy)-
phenoxy)pyrrolidine-1-carboxylate (13f)
phenoxy)pyrrolidin-3-yl)methyl)pyridin-2-amine (2e)
Compound 2e was synthesized using general procedure D
(92%): 1H NMR (500 MHz, CD3OD) d 3.00–3.20 (m, 3H), 3.30–3.35
(m, 1H), 3.50–3.75 (m, 3H), 5.02 (s, 1H), 6.64–6.75 (m, 3H), 6.80–
6.90 (m, 3H), 6.88–6.91 (m, 2H), 7.34–7.37 (dd, J = 8.0, 8.5 Hz,
1H), 7.75–7.79 (dd, J = 7.0, 8.0 Hz, 1H); 13C NMR (125 MHz, CD3OD)
d 28.5, 30.5, 43.4, 51.5, 77.2, 108.3, 108.4, 108.5, 112.4, 112.7,
112.8, 113.0, 114.2, 116.0, 116.1, 116.4, 116.7, 119.0, 132.3,
132.4, 145.5, 145.6, 147.9, 148.7, 156.8, 156.9, 158.5, 158.6,
158.7, 158.9, 159.1, 160.8, 162.6, 162.9; LC–MS (M+H+) calcd for
Compound 13f was synthesized using general procedure C
(51%): 1H NMR (500 MHz, CDCl3) d 1.40–1.60 (m, 27H), 2.80–3.00
(m, 2H), 3.10–3.20 (dd, J = 7.5, 16.5 Hz, 1H), 3.25–3.35 (m, 1H),
3.45–3.50 (m, 1H), 3.55–3.60 (m, 1H), 3.61–3.70 (m, 1H), 4.57–
4.60 (d, J = 10.5 Hz, 1H), 7.00–7.60 (m, 12H); 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; LC–MS (M+H+)
calcd for C37H48N3O8 662, found 662.
C22H22ClFN3O2 414.1385, found 414.1397.
5.9.20. 6-(((3R,4R/3S,4S)-4-(Biphenyl-4-yloxy)pyrrolidin-3-yl)-
methyl)pyridin-2-amine (2a)
5.9.25. 6-(((3R,4R/3S,4S)-4-(3-(4-Fluoro-3-methylphenoxy)-
phenoxy)pyrrolidin-3-yl)methyl)pyridin-2-amine (2f)
Compound 2a was synthesized using general procedure D
(95%): 1H NMR (500 MHz, CD3OD) d 3.00–3.20 (m, 2H), 3.21–3.30
(dd, J = 9.0, 14.0 Hz, 1H), 3.61–3.72 (m, 3H), 5.01 (s, 1H), 6.69–
6.71 (d, J = 7.5 Hz, 1H), 6.81–6.83 (d, J = 9.0 Hz, 1H), 7.05–7.07 (d,
J = 7.5 Hz, 2H), 7.29–7.32 (dd, J = 7.0, 7.5 Hz, 1H), 7.40–7.43 (dd,
J = 7.5, 8.0 Hz, 2H), 7.55–7.59 (m, 3H), 7.73–7.77 (dd, J = 7.5,
9.0 Hz, 1H); 13C NMR (125 MHz, CD3OD) d 21.3, 41.5, 47.7, 50.4,
75.6, 99.9, 116.2, 119.3, 122.0, 127.0, 129.0, 134.6, 138.6, 140.9,
151.5, 152.1, 154.6, 154.8, 157.0, 158.3; LC–MS (M+H+) calcd for
Compound 2f was synthesized using general procedure D
(95%): 1H NMR (500 MHz, CD3OD) d 2.24 (s, 3H), 2.95–3.18 (m,
3H), 3.30–3.35 (m, 1H), 3.50–3.75 (m, 3H), 4.95–4.96 (t,
J = 2.0 Hz, 1H), 6.56–6.58 (m, 1H), 6.67–6.70 (m, 1H), 6.80–6.85
(m, 3H), 6.86–6.90 (m, 3H), 7.01–7.07 (m, 1H), 7.72–7.74 (dd,
J = 7.0, 8.0 Hz, 1H); 13C NMR (125 MHz, CD3OD) d 14.6, 27.7, 30.5,
43.4, 51.5, 77.1, 107.1, 110.8, 111.8, 112.7, 112.8, 113.0, 116.6,
116.8, 117.0, 118.9, 119.4, 119.5, 123.5, 127.5, 127.6, 131.9,
145.5, 147.9, 148.7, 153.5, 156.8, 156.9, 158.1, 158.9, 161.0,
162.4, 162.6, 162.9; LC–MS (M+H+) calcd for C23H25FN3O2
394.1931, found 394.1920.
C22H24N3O 346.1919, found 346.1911.
5.9.21. 6-(((3R,4R/3S,4S)-4-(3-Phenoxyphenoxy)pyrrolidin-3-yl)-
methyl)pyridin-2-amine (2b)
Compound 2b was synthesized using general procedure D
(91%): 1H NMR (500 MHz, D2O) d 2.70–3.00 (m, 3H), 3.14–3.19
(dd, J = 11.0, 11.0 Hz, 1H), 3.25–3.40 (m, 2H), 3.45–3.55 (dd,
J = 7.5, 11.5 Hz, 1H), 6.29 (s, 1H), 6.41–6.50 (m, 2H), 6.51–6.55
(dd, J = 1.5, 8.0 Hz, 1H), 6.56–6.60 (d, J = 8.5 Hz, 1H), 6.79–6.81 (d,
J = 8.0 Hz, 2H), 6.98–7.01 (dd, J = 7.0, 7.0 Hz, 1H), 7.06–7.10
(dd, J = 8.0, 8.0 Hz, 1H), 7.17–7.21 (d, J = 8.0 Hz, 2H), 7.45–7.55
(dd, J = 7.5, 8.5 Hz, 1H); 13C NMR (125 MHz, D2O) d 21.3, 41.5,
47.7, 50.4, 75.6, 106.1, 110.9, 111.5, 112.4, 119.4, 124.4, 130.3,
131.1, 144.7, 146.7, 154.2, 156.2, 157.5, 158.4; LC–MS (M+H+) calcd
for C22H24N3O2 362.18685, found 362.18597.
5.9.26. ( )-tert-Butyl-{{6-{tert-butoxycarbonyl{[2-(trimethyl-
silyl)ethoxy]methyl}amino}-4-methylpyridine-2-yl}methyl}-4-
hydroxypyrrolidine-1-carboxylate (16)
To an ice-cooled solution of 1512 (1.328 g, 3.26 mmol) in DMF
(10 mL) was added sodium hydride (0.157 g, 60% in mineral oil,
3.92 mmol). After 15 min, SEM-Cl (0.59 g, 0.64 mL, 3.59 mmol)
was added dropwise. The mixture was stirred at room temperature
for 16 h, and then quenched with. H2O (5 mL). The solvent was re-
moved by rotary evaporation, and the resulting material was par-
titioned between EtOAc (30 mL) and H2O (20 mL). The organic
layer was extracted with EtOAc (2 ꢁ 30 mL). The combined organic
layers were washed with brine (10 mL), dried over Na2SO4, and
concentrated in vacuo. The residue was purified by flash column
chromatography (silica gel, EtOAc/hexanes, 2:3) to yield a white
foamy solid (1.25 g, 68%): 1H NMR (400 MHz, CDCl3) d 0.0 (s, 9H),
0.93 (t, J = 8.0 Hz, 2H), 1.47 (s, 9H), 1.52 (s, 9H), 2.35 (s, 3H),
2.38–2.58 (m, 1H), 2.78–2.98 (m, 2H), 3.01–3.30 (m, 2H), 3.63 (t,
J = 8.0 Hz, 2H), 3.64–3.90 (m, 2H), 4.10–4.25 (m, 1H), 4.85 + 5.12
(brs, OH), 5.29 (s, 2H), 6.82 and 6.84 (s, 1H), 7.17 (s, 1H); LC–TOF
(M+H+) calcd for C27H47N3O6Si 537.3234, found 537.3244.
5.9.22. 6-(((3R,4R/3S,4S)-4-(3-(4-Fluorophenoxy)phenoxy)-
pyrrolidin-3-yl)methyl)pyridin-2-amine (2c)
Compound 2c was synthesized using general procedure D
(95%): 1H NMR (500 MHz, CD3OD) d 2.99–3.08 (m, 2H), 3.09–3.16
(dd, J = 8.5, 14.5 Hz, 1H), 3.30–3.35 (m, 1H), 3.50–3.52 (m, 2H),
3.60–3.72 (dd, J = 8.0, 11.5 Hz, 1H), 4.94–4.95 (t, J = 2.0 Hz, 1H),
6.56 (s, 1H), 6.64–6.66 (d, J = 7.0 Hz, 1H), 6.67–6.70 (m, 1H),
6.80–6.82 (d, J = 8.5 Hz, 1H), 6.98–7.00 (m, 2H), 7.07–7.11 (m,
2H), 7.23–7.27 (dd, J = 8.0, 8.5 Hz, 1H), 7.72–7.74 (dd, J = 7.0,
8.0 Hz, 1H); 13C NMR (125 MHz, CD3OD) d 28.5, 30.5, 43.4, 51.5,
77.2, 107.2, 111.0, 112.7, 112.8, 112.9, 117.4, 117.5, 122.15,
122.22, 132.0, 145.5, 148.7, 153.91, 153.93, 156.8, 159.0, 159.6,
160.8, 161.5, 162.4, 162.6; LC–MS (M+H+) calcd for C22H23FN3O2
380.1774, found 380.1777.
5.9.27. (3S,4S)-tert-Butyl 3-((6-(tert-butoxycarbonyl((2-(tri-
methylsilyl)ethoxy)methyl)amino)-4-methylpyridin-2-yl)-
methyl)-4-((4S)-4,7,7-trimethyl-3-oxo-2-oxabicyclo[2.2.1]-
heptane-1-carbonyloxy)pyrrolidine-1-carboxylate (17a) and
(3R,4R)-tert-butyl 3-((6-(tert-butoxycarbonyl((2-(trimethyl-
silyl)ethoxy)methyl)amino)-4-methylpyridin-2-yl)methyl)-4-
((4S)-4,7,7-trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-
carbonyloxy)pyrrolidine-1-carboxylate (17b)
5.9.23. 6-(((3R,4R/3S,4S)-4-(3-(4-Chlorophenoxy)phenoxy)-
pyrrolidin-3-yl)methyl)pyridin-2-amine (2d)
Compound 2d was synthesized using general procedure D
(95%): 1H NMR (500 MHz, CD3OD) d 2.99–3.16 (m, 3H), 3.30–3.35
(m, 1H), 3.50–3.52 (m, 2H), 3.60–3.72 (m, 1H), 4.96 (s, 1H), 6.60–
6.95 (m, 6H), 6.96–6.97 (d, J = 2.5 Hz, 2H), 6.97–6.98 (d,
J = 2.5 Hz, 2H), 7.27–7.29 (m, 1H), 7.73–7.76 (dd, J = 7.5, 9.0 Hz,
1H); 13C NMR (125 MHz, CD3OD) d 27.7, 30.5, 43.4, 51.5, 77.2,
107.8, 111.6, 112.7, 112.9, 113.0, 116.5, 118.8, 121.6, 130.9,
132.1, 145.5, 145.6, 147.9, 148.7, 156.8, 156.9, 157.0, 159.0,
159.9, 162.3, 162.6; LC–MS (M+H+) calcd for C22H23ClN3O2
396.1479, found 396.1465.
To an ice-cooled solution of 16 (0.935 g, 1.74 mmol), (1S)-(ꢀ)-
camphanic acid (0.4 g, 2.02 mmol) and Ph3P (0.75 g, 2.86 mmol)
in THF (10 mL) was added DEAD (0.9 mL, 3.0 mmol) dropwise.
The mixture was stirred at room temperature for 6 h, and then con-
centrated in vacuo. The crude product was purified by flash column
chromatography (silica gel, EtOAc/hexanes, 1:5) to obtain the two
diastereomers in 88% overall yield.
Compound 17a (Foamy solid, 44%; 0.55 g): 1H NMR (400 MHz,
CDCl3) d 0 (s, 9H), 0.92 (t, J = 8.0 Hz, 2H), 0.97 + 0.99 (s, 3H),
1.07 + 1.09 (s, 3H), 1.16 (s, 3H), 1.25–1.4 (m, 1H), 1.46 (s, 9H),

6534
F. Xue et al. / Bioorg. Med. Chem. 18 (2010) 6526–6537
1.54 (s, 9H), 1.65–1.80 (m, 1H), 1.90–2.10 (m, 2H), 2.326 + 2.336 (s,
3H), 2.40–2.55 (m, 1H), 2.75–3.05 (m, 2H), 3.18–3.30 (m, 1H),
3.50–3.70 (m, 3H), 4.10–4.44 (m, 2H), 5.36 (s, 2H), 6.79 (s, 1H),
7.18 (s, 1H); LC–MS (M+H+) calcd for C37H59N3O9Si 718, found 718.
Compound 17b (Foamy solid, 0.55 g, 44%): 1H NMR (400 MHz,
CDCl3) d 0 (s, 9H), 0.92 (t, J = 8.8 Hz, 2H), 0.99 + 1.01 (s, 3H), 1.08
(s, 3H), 1.34 + 1.60 (s, 3H), 1.25–1.40 (m, 1H), 1.70–1.80 (m, 1H),
1.90–2.04 (m, 1H), 2.06–2.19 (m, 1H), 2.33 (s, 3H), 2.39–2.54 (m,
1H), 2.70–3.04 (m, 2H), 3.25 (t, J = 10.4 Hz, 1H), 3.50–3.70 (m,
3H), 4.22–4.42 (m, 3H), 5.36 (s, 1H), 6.74 (s, 1H), 7.17 (s, 1H);
LC–MS (M+H+) calcd for C37H59N3O9Si 718, found 718.
5.9.35. 40-(Bromomethyl)-3-fluorobiphenyl (19b)
Compound 19b was synthesized using general procedure F
(87%): 1H NMR (400 MHz, CDCl3) d 4.55 (s, 2H), 7.0–7.10 (m, 1H),
7.22–7.3 (m, 1H, overlapped by CDCl3), 7.35–7.44 (m, 2H), 7.50
(d, J = 8.0 Hz, 2H), 7.55 (d, J = 8.0 Hz, 2H).
5.9.36. 40-(Bromomethyl)-3-(trifluoromethyl)biphenyl (19c)
Compound 19c was synthesized using general procedure F
(87%): 1H NMR (400 MHz, CDCl3)
d 4.55 (s, 2H), 7.50 (d,
J = 7.6 Hz, 2H), 7.57 (d, J = 7.6 Hz, 2H), 7.62 (d, J = 8.0 Hz, 1H),
7.75 (d, J = 8.0 Hz, 1H), 7.82 (s, 1H).
5.9.37. 40-(Bromomethyl)-3-chloro-4-fluorobiphenyl (19d)
Compound 19d was synthesized using general procedure F (87%):
1H NMR (400 MHz, CDCl3) d 4.52 (s, 2H), 7.18 (t, J = 8.8 Hz, 1H), 7.38–
7.47 (m, 5H), 7.57 (dd, J = 8.8, 2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3):
33.0, 116.8 (d, JC–F = 84 Hz), 121.2, 126.6 (d, JC–F = 29.2 Hz), 127.3,
129.1, 129.6, 137.3, 137.5 (d, JC–F = 12 Hz), 138.9, 156.4, 158.9; 19F
NMR (376 MHz, CDCl3): ꢀ119.5.
5.9.28. (3R,4R)-tert-Butyl-3-((6-(tert-butoxycarbonyl((2-(tri-
methylsilyl)ethoxy)methyl) amino)-4-methylpyridin-2-yl)-
methyl)-4-hydroxypyrrolidine-1-carboxylate (14b)
To a solution of 17b (397 mg, 0.55 mmol) in methanol (5 mL)
and water (0.5 mL) was added Na2CO3 (0. 11 g, 1 mmol). The mix-
ture was stirred at room temperature overnight, and concentrated.
The resulting residue was partitioned between EtOAc (20 mL) and
H2O (20 mL). The organic layer was dried over Na2SO4, and concen-
trated in vacuo. The crude product was purified by flash column
chromatography (silica gel, EtOAc/hexanes, 2:3) to yield com-
pound 17b as a foamy solid (0.291 g, 98%): 1H NMR (400 MHz,
CDCl3) d 0 (s, 9H), 0.95 (t, J = 8.0 Hz, 2H), 1.46 (s, 9H), 2.35 (s,
3H), 2.80 (t, J = 12.0 Hz, 1H), 2.96 (t, 1H), 3.22 (t, 1H), 3.40–3.70
(m, 4H), 4.05–4.25 (m, 2H), 4.53 + 4.63 (br s, OH), 5.25 (s, 2H),
6.85 (s, 1H), 7.21 (s, 1H). 13C NMR (100 MHz, CDCl3): 17.7, 20.8,
28.0, 28.3, 34.8, 44.3, 45.0, 48.8, 49.2, 53.3, 53.6, 61.6, 65.6, 69.8,
70.6, 76.6, 78.8, 81.4, 118.8, 120.8, 120.9, 149.5, 149.6, 153.1,
153.7, 154.2, 157.8; ESI-MS: calcd for C27H47N3O6Si 537.3234,
found 537.32437.
5.9.38. 40-(Bromomethyl)-4-fluoro-3-methylbiphenyl (19e)
Compound 19e was synthesized using general procedure F
(87%): 1H NMR (400 MHz, CDCl3) d 2.32 (s, 3H), 4.53 (s, 2H), 7.05
(t, J = 8.8 Hz, 1H), 7.30–7.40 (m, 2H), 7.44 (d, J = 8.8 Hz, 2H), 7.49
(d, J = 8.8 Hz, 2H); 13C NMR (100 MHz, CDCl3): 314.5, 32.2, 115.2
(d, JC–F = 90.8 Hz), 125.0 (d, JC–F = 69.2 Hz), 125.8 (d, JC–F = 29.2 Hz),
127.2, 129.4, 130.0 (d, JC–F = 18.4 Hz), 136.0 (d, JC–F = 14.4 Hz),
136.5, 140.3, 159.8, 162.3; 19F NMR (376 MHz, CDCl3): ꢀ117.8.
5.9.39. (3S,4S)-tert-Butyl 3-((6-(tert-butoxycarbonyl((2-(tri-
methylsilyl)ethoxy)methyl)amino)-4-methylpyridin-2-yl)-
methyl)-4-((30-chlorobiphenyl-4-yl)methoxy)pyrrolidine-1-
carboxylate (13g)
5.9.29. (30-Chlorobiphenyl-4-yl)methanol (18a)
Compound 13g was synthesized using general procedure G
(87%): 1H NMR (400 MHz, D2O) d 0 (s, 9H), 0.94 (t, J = 8.0 Hz, 2H),
1.49 (s, 9H), 1.53 (s, 9H), 2.31 + 2.32 (s, 3H), 2.66–2.82 (m, 1H),
2.83–2.94 (m, 1H), 3.08 (dd, J = 14.0, 6.8 Hz, 1H), 3.22–3.33 (m,
1H), 3.35–3.42 (m, 1H), 3.48–3.60 (m, 1H), 3.63 (t, J = 8.0 Hz, 2H),
3.78 (d, J = 12.0 Hz, 1H), 3.97–4.03 (m, 1H), 4.38–4.48 (m, 1H),
4.68 (t, J = 11.6 Hz, 1H), 5.37 (s, 2H), 6.79 (s, 1H), 7.16 (d,
J = 9.6 Hz, 1H), 7.33–7.46 (m, 4H), 7.50 (d, J = 7.2 Hz, 1H), 7.55–
7.63 (m, 3H); 13C NMR (100 MHz, CDCl3) d ꢀ1.48, 18.0, 21.0,
28.2, 28.5, 28.9, 34.9, 42.6, 43.3, 49.0, 49.3, 50.2, 50.8, 65.7,
70.6 + 70.7, 76.5, 78.0, 79.0, 79.16 + 79.21, 81.3, 119.2, 121.3,
125.2, 127.0, 127.1, 127.2, 127.9, 128.2, 130.0, 134.6, 137.8,
138.0, 139.1, 142.7, 148.8, 153.4, 154.1, 154.5, 154.8, 158.4; ESI-
MS: m/z = 738 [M+H]+; HR-ESI-MS: 737.3627 calcd for
Compound 18a was synthesized using general procedure E
(87%): 1H NMR (400 MHz, CDCl3) d 4.75 (s, 2H), 7.30–7.34 (m,
1H), 7.37 (t, J = 8.0 Hz, 1H), 7.42–7.48 (m, 3H), 7.54–7.58 (m, 3H).
5.9.30. (30-Fluorobiphenyl-4-yl)methanol (18b)
Compound 18b was synthesized using general procedure E
(87%): 1H NMR (400 MHz, CDCl3) d 4.75 (s, 2H), 7.00–7.08 (m,
1H), 7.22–7.32 (m, overlapped by CDCl3, 1H), 7.34–7.42 (m, 2H),
7.45 (d, J = 8.0 Hz, 2H), 7.57 (d, J = 8.0 Hz, 2H).
5.9.31. (30-(Trifluoromethyl)biphenyl-4-yl)methanol (18c)
Compound 18c was synthesized using general procedure E
(87%): 1H NMR (400 MHz, CDCl3)
d 4.75 (s, 2H), 7.46 (d,
J = 8.0 Hz, 2H), 7.56 (d, J = 7.2 Hz, 1H), 7.59 (d, J = 8.0 Hz, 2H),
7.76 (d, J = 7.2 Hz, 1H), 7.83 (s, 1H).
C40H56ClN3O6Si, found: 737.3634.
5.9.40. (3S,4S)-tert-Butyl 3-((6-(tert-butoxycarbonyl((2-(tri-
methylsilyl)ethoxy)methyl)amino)-4-methylpyridin-2-yl)-
methyl)-4-((30-fluorobiphenyl-4-yl)methoxy)pyrrolidine-1-
carboxylate (13h)
5.9.32. (30-Chloro-40-fluorobiphenyl-4-yl)methanol (18d)
Compound 18d was synthesized using general procedure E
(87%): 1H NMR (400 MHz, CDCl3) d 4.75 (s, 2H), 7.20 (t, J = 8.8 Hz,
1H), 7.40–7.47 (m, 3H), 7.49–7.55 (m, 2H), 7.61 (dd, J = 7.8,
2.4 Hz, 1H).
Compound 13h was synthesized using general procedure G
(87%): 1H NMR (400 MHz, D2O) d 0 (s, 9H), 0.92 (t, J = 8.0 Hz, 2H),
1.49 (s, 9H), 1.54 (s, 9H), 2.31 + 2.33 (s, 3H), 2.68–2.83 (m, 1H),
2.84–2.93 (m, 1H), 3.04–3.13 (m, 1H), 3.22–3.34 (m, 1H), 3.35–
3.43 (m,1H), 3.48–3.60 (m,1H), 3.63 (t, J = 8.0 Hz, 2H), 3.78 (d,
J = 12.0 Hz, 1H), 3.96–4.04 (m, 1H), 4.38–4.49 (m, 1H), 3.68 (t,
J = 11.2 Hz, 1H), 5.37 (s, 2H), 6.80 (s, 1H), 7.04–7.12 (m, 1H), 7.16
(d, J = 8.8 Hz, 1H), 7.32–7.35 (m, 1H), 7.38–7.50 (m, 4H), 7.55–
7.62 (m, 2H); 13C NMR (100 MHz, CDCl3) d ꢀ1.46, 18.0, 21.0,
28.2, 28.5, 29.7, 34.9 + 35.0, 42.6, 43.3, 49.0, 49.3, 50.2, 50.8, 64.8,
65.7, 70.6 + 70.7, 76.5, 78.0, 78.9, 79.17 + 79.22, 81.23 + 81.28,
113.8, 113.9, 114.0, 114.1, 119.2, 121.3, 122.6 + 122.7, 127.0,
127.2, 127.4, 127.9, 128.2, 130.16 + 130.24, 137.9, 138.0, 139.2,
5.9.33. (40-Fluoro-30-methylbiphenyl-4-yl)methanol (18e)
Compound 18e was synthesized using general procedure E
(87%): 1H NMR (400 MHz, CDCl3) d 2.33 (s, 3H), 4.73 (s, 2H), 7.06
(t, J = 9.2 Hz, 1H), 7.25–7.54 (m, 6H).
5.9.34. 40-(Bromomethyl)-3-chlorobiphenyl (19a)
Compound 19a was synthesized using general procedure F
(87%): 1H NMR (400 MHz, CDCl3)
d 4.54 (s, 2H), 7.29–7.33
(m,1H), 7.35 (t, J = 8.0 Hz, 1H), 7.41–7.43 (m, 1H), 7.45 (d,
J = 8.0 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H), 7.53–7.55 (m, 1H).

F. Xue et al. / Bioorg. Med. Chem. 18 (2010) 6526–6537
6535
143.0, 148.8, 153.4, 154.2, 154.5, 154.8, 158.4, 161.9, 164.4; ESI-
5.9.44. 6-(((3S,4S)-4-((30-Chlorobiphenyl-4-yl)methoxy)pyrr-
olidin-3-yl)methyl)-4-methylpyridin-2-amine hydrochloride
salt (2g)
MS: m/z = 700 [M+H]+; HR-ESI-MS: 699.4119 calcd for C41H57F3
N3O6Si, found: 699.4113.
Compound 2g was synthesized using general procedure H
(100%): 1H NMR (400 MHz, D2O) d 1.80 (s, 3H), 2.50–2.72 (m,
3H), 3.04–3.10 (m, 1H), 2.32–2.34 (m, 1H), 3.48–3.59 (m, 1H),
3.71 (d, J = 12.8 Hz, 1H), 4.01 (s, 1H), 4.15 (d, J = 12 Hz, 1H), 4.62
(d, J = 12 Hz, 1H), 5.86 (s, 1H), 6.24 (s, 1H), 7.09 (d, J = 7.2 Hz,
1H), 7.17–7.35 (m, 7H); 13C NMR (100 MHz, D2O) d 21.1, 28.4,
40.7, 47.4, 49.8, 70.1, 75.5, 110.0, 113.3, 124.9, 126.2, 126.4,
127.4, 130.5, 134.2, 136.8, 138.2, 141.4, 145.4, 153.4, 157.1; ESI-
MS: m/z = 408 [M+H]+; HR-ESI-MS: 407.1764 calcd for
5.9.41. (3S,4S)-tert-Butyl 3-((6-(tert-butoxycarbonyl((2-(tri-
methylsilyl)ethoxy)methyl)amino)-4-methylpyridin-2-yl)-
methyl)-4-((30-(trifluoromethyl)biphenyl-4-yl)methoxy)-
pyrrolidine-1-carboxylate (13i)
Compound 13i was synthesized using general procedure G
(87%): 1H NMR (400 MHz, D2O) d 0 (s, 9H), 0.92 (t, J = 8.0 Hz, 2H),
1.49 (s, 9H), 1.54 (s, 9H), 2.31 + 2.33 (s, 3H), 2.65–2.83 (m, 1H),
2.84–2.93 (m, 1H), 3.02–3.13 (m, 1H), 3.22–3.34 (m, 1H), 3.35–
3.43 (m, 1H), 3.47–3.60 (m, 1H), 3.63 (t, J = 8.0 Hz, 2H), 3.79 (d,
J = 12.8 Hz, 1H), 4.00 (s, 1H), 4.40–4.50 (m, 1H), 4.69 (t,
J = 10.8 Hz, 1H), 5.37 (s, 2H), 6.80 (s, 1H), 7.16 (d, J = 8.8 Hz, 1H),
7.46 (d, J = 7.2 Hz, 2H), 7.55–7.67 (m, 4H), 7.80 (d, J = 7.2 Hz, 1H),
7.86 (s, 1H); 13C NMR (100 MHz, CDCl3) d ꢀ1.47, 18.0, 21.0, 28.2,
28.5, 29.7, 34.85 + 34.95, 42.6, 43.3, 49.0, 49.3, 50.2, 50.8, 65.7,
70.6 + 70.7, 76.5, 78.1, 79.0, 79.16 + 79.23, 81.23 + 81.27, 119.2,
121.3, 123.8 + 123.9, 127.1, 128.0, 128.3, 129.2, 130.3, 138.1,
138.2, 139.0, 141.6, 148.8, 153.4, 154.2, 154.8, 158.4; 19F NMR
(100 MHz, CDCl3): ꢀ63.0; ESI-MS: m/z = 772 [M+H]+; HR-ESI-MS:
771.3891 calcd for C41H57F3N3O6Si, found: 771.3898.
C24H26ClN3OS, found: 407.1773.
5.9.45. 6-(((3S,4S)-4-((30-Fluorobiphenyl-4-yl)methoxy)pyrr-
olidin-3-yl)methyl)-4-methylpyridin-2-amine hydrochloride
salt (2h)
Compound 2h was synthesized using general procedure H
(100%): 1H NMR (400 MHz, D2O) d 1.86 (s, 3H), 2.55–2.80 (m,
3H), 3.04–3.15 (m, 1H), 3.30–3.40 (m, 1H), 3.50–3.61 (m, 1H),
3.68–3.75 (m, 1H), 4.06 (s, 1H), 4.17 (d, J = 12 Hz, 1H), 4.62 (d,
J = 12 Hz, 1H), 6.97 (s, 1H), 6.20 (s, 1H), 6.95 (s, 1H), 7.11–7.44
(m, 7H); 13C NMR (100 MHz, D2O) d 21.0, 28.1, 40.0, 47.5, 49.9,
70.0, 75.0, 109.8, 113.0, 114.2, 122.3, 126.4, 129.6, 130.8, 136.6,
138.4, 141.8, 145.5, 153.4, 157.2, 161.8, 164.3; 19F NMR
(100 MHz, D2O): ꢀ113.7; ESI-MS: m/z = 392 [M+H]+; HR-ESI-MS:
391.2071 calcd for C24H26FN3OS, found: 391.2072.
5.9.42. (3S,4S)-tert-Butyl 3-((6-(tert-butoxycarbonyl((2-(tri-
methylsilyl)ethoxy)methyl)amino)-4-methylpyridin-2-yl)-
methyl)-4-((40-fluoro-30-methylbiphenyl-4-yl)methoxy)-
pyrrolidine-1-carboxylate (13j)
Compound 13j was synthesized using general procedure G
(87%): 1H NMR (400 MHz, D2O) d 0 (s, 9H), 0.92 (t, J = 8.0 Hz, 2H),
1.49 (s, 9H), 1.54 (s, 9H), (2.31 + 2.33) (s, 3H), 2.37 (s, 3H), 2.67–
2.83 (m, 1H), 2.84–2.94 (m,1H), 3.08 (dd, J = 14.0, 6.8 Hz, 1H),
3.22–3.34 (m, 1H), 3.35–3.42 (m, 1H), 3.48–3.59 (m, 1H), 3.63 (t,
J = 8.0 Hz, 2H), 3.78 (d, J = 12.0 Hz, 1H), 3.96–4.04 (m, 1H), 4.38–
4.46 (m, 1H), 4.67 (t, J = 11.2, 10.8 Hz, 1H), 5.37 (s, 2H), 6.79 (s,
1H), 7.10 (t, J = 8.8 Hz, 1H), 7.16 (d, t, J = 9.2 Hz, 1H), 7.36–7.45
(m, 4H), 7.51–7.58 (m, 2H); 13C NMR (100 MHz, CDCl3) d ꢀ1.46,
14.669 + 14.696, 18.0, 20.983 + 20.965, 28.2, 28.5, 29.7,
34.9 + 35.0, 42.6, 43.3, 49.0, 49.3, 50.2, 50.8, 65.7, 70.7 + 70.8,
76.5, 77.9, 78.9, 79.1, 79.2, 81.22 + 81.26, 115.1, 115.3, 119.2,
121.3, 125.80 + 125.88, 126.9, 127.9, 128.1, 130.09 + 130.14,
136.6, 137.0, 137.1, 139.8, 148.8, 153.4, 154.2, 154.5, 154.8,
158.4, 159.8, 162.2; ESI-MS: m/z = 736 [M+H]+; HR-ESI-MS:
735.4079 calcd for C24H26ClN3OS, found: 735.4082.
5.9.46. 4-Methyl-6-(((3S,4S)-4-((30-(trifluoromethyl)biphenyl-4-
yl)methoxy)pyrrolidin-3-yl)methyl)pyridin-2-amine hydro-
chloride salt (2i)
Compound 2i was synthesized using general procedure H
(100%): 1H NMR (400 MHz, D2O) d 1.67 (s, 3H), 2.42–2.68 (m, 3H),
2.94–3.08 (m, 1H), 3.24–3.32 (m, 1H), 3.42–3.54 (m, 1H), 3.69 (d,
J = 12.4 Hz, 1H), 3.91 (s, 1H), 4.07 (d, J = 11.2 Hz, 1H), 4.57 (d,
J = 11.2 Hz, 1H), 5.70 (s, 1H), 6.23 (s, 1H), 7.08–7.28 (m, 6H), 7.37–
7.50 (m, 2H); 13C NMR (100 MHz, D2O) d 20.8, 28.7, 41.4, 47.3,
49.5, 70.1, 76.2, 110.1, 113.5, 122.6, 122.8, 123.7, 126.4, 129.1,
129.5, 129.9, 130.3, 130.6, 137.3, 137.9, 140.4, 145.6, 153.6, 156.8;
19F NMR (100 MHz, D2O): ꢀ63.2; ESI-MS: m/z = 442 [M+H]+; HR-
ESI-MS: 441.2028 calcd for C25H26F3N3O, found: 441.2032.
5.9.47. 6-(((3S,4S)-4-((40-Fluoro-30-methylbiphenyl-4-yl)methoxy)-
pyrrolidin-3-yl)methyl)-4-methylpyridin-2-amine hydrochloride
salt (2j)
5.9.43. (3S,4S)-tert-Butyl 3-((6-(tert-butoxycarbonyl((2-(tri-
methylsilyl)ethoxy)methyl)amino)-4-methylpyridin-2-yl)-
methyl)-4-((30-chloro-40-fluorobiphenyl-4-yl)methoxy)-
pyrrolidine-1-carboxylate (13k)
Compound 2j was synthesized using general procedure H
(100%): 1H NMR (400 MHz, D2O) d 1.84 (s, 3H), 2.06 (s, 3H),
2.50–2.78 (m, 3H), 3.05–3.12 (m, 1H), 3.32–3.34 (m, 1H), 3.51–
3.60 (m, 1H), 3.72 (d, J = 12.8 Hz, 1H), 4.04 (s, 1H), 4.17 (d,
J = 12.0 Hz, 1H), 4.63 (d, J = 12.0 Hz, 1H), 5.92 (s, 1H), 6.25 (s, 1H),
6.86 (t, J = 8.8 Hz, 1H), 7.14–7.29 (m, 6H); 13C NMR (100 MHz,
D2O) d 13.9, 21.1, 28.4, 40.6, 47.4, 49.8, 70.1, 75.3, 110.0, 113.3,
115.1, 115.3, 125.0, 125.2, (125.34 + 125.42), 126.2, 129.4, 135.5,
135.9, 138.9, 145.5, 153.4, 157.2, 159.6, 162.1; ESI-MS: m/z = 406
[M+H]+; HR-ESI-MS: 405.2216 calcd for C25H28FN3O, found:
405.2285.
Compound 13k was synthesized using general procedure G
(87%): 1H NMR (400 MHz, D2O) d 0 (s, 9H), 0.92 (t, J = 8.0 Hz, 2H),
1.49 (s, 9H), 1.54 (s, 9H), 2.31 + 2.33 (s, 3H), 2.65–2.82 (m,1H),
2.83–2.94 (m, 1H), 3.08 (dd, J = 14.0, 7.2 Hz, 1H), 3.21–3.34 (m,
1H), 3.34–3.43 (m, 1H), 3.48–3.60 (m, 1H), 3.63 (t, J = 8.0 Hz, 2H),
3.78 (d, J = 12.0 Hz, 1H), 3.96–4.05 (m, 1H), 4.38–4.49 (m, 1H),
4.67 (t, J = 12.0, 11.6, 9.2 Hz, 1H), 5.38 (s, 2H), 6.79 (s, 1H), 7.16
(d, J = 9.2 Hz, 1H), 7.24 (t, J = 8.8 Hz, 1H), 7.40–7.49 (m, 3H),
7.50–7.56 (m, 2H), 7.64 (dd, J = 6.8, 2.1 Hz, 1H); 13C NMR
(100 MHz, CDCl3) d ꢀ1.47, 17.9, 21.0, 28.2, 28.5, 29.6, 34.9, 42.6,
43.3, 48.9, 49.3, 50.2, 50.8, 65.7, 70.5 + 70.6, 76.5, 78.0, 79.0,
79.1 + 79.2, 81.3, 116.7, 116.9, 119.2, 121.2, 126.6, 126.7, 126.9,
128.0, 128.2, 129.1, 137.8, 138.0, 138.1, 138.3, 148.8, 153.4,
154.1, 154.8, 156.3, 158.4, 158.8; 19F NMR (100 MHz, CDCl3):
ꢀ120.4; ESI-MS: m/z = 756 [M+H]+; HR-ESI-MS: 755.3533 calcd
for C24H26ClN3OS, found: 755.3540.
5.9.48. 6-(((3S,4S)-4-((30-Chloro-40-fluorobiphenyl-4-yl)methoxy)-
pyrrolidin-3-yl)methyl)-4-methylpyridin-2-amine hydrochloride
salt (2k)
Compound 2k was synthesized using general procedure H
(100%): 1H NMR (400 MHz, D2O) d 1.86 (s, 3H), 2.55–2.74 (m,
3H), 3.06–3.12 (m, 1H), 3.32 (d, J = 12.8 Hz, 1H), 3.50–3.57 (m,
1H), 4.05 (s, 1H), 4.17 (d, J = 12.0 Hz, 1H), 4.62 (d, J = 12.0 Hz,
1H), 5.95 (s, 1H), 6.24 (s, 1H), 7.06 (t, J = 8.8 Hz, 1H), 7.20–7.40

6536
F. Xue et al. / Bioorg. Med. Chem. 18 (2010) 6526–6537
(m, 6H); 13C NMR (100 MHz, D2O) d 21.0, 28.6, 40.7, 47.4, 49.8,
70.1, 75.5, 109.8, 113.3, 116.8, 117.0, (120.5 + 120.7), 126.3,
126.5, 128.2, 129.4, 136.6, 137.0, 137.5, 146.1, 153.7, 156.1,
156.8, 158.5; ESI-MS: m/z = 426 [M+H]+; HR-ESI-MS: 425.1670
calcd for C24H25ClFN3O, found: 425.1674.
Table 2
Crystallographic data collection and refinement statistics
nNOS-2b
P212121
Data collection
Space group
Cell dimensions
a, b, c (Å)
Resolution (Å)
Rsym
52.28, 111.56, 164.85
1.90 (1.93–1.90)
0.075 (0.572)
24.2 (2.0)
75,896
99.3 (99.7)
5.10. Enzyme assays
I/rI
IC50 valuesfor inhibitors2a–d weremeasuredfor thethreediffer-
ent isoforms of NOS, including rat nNOS, bovine eNOS, and murine
macrophage iNOS using L-arginine as the substrate. The three iso-
No. unique reflections
Completeness (%)
Redundancy
4.0 (4.0)
zymes were recombinant enzymes, which were overexpressed (in
E. coli) and isolated as reported.23 The formation of nitric oxide was
measured using a hemoglobin capture assay described previously.29
All NOS isozymes were assayed at room temperature in a 100 mM
Refinement
Resolution (Å)
No. reflections used
1.90
71,875
0.182/0.215
44.53
a
Rwork/Rfree
Mean B value
No. atoms
Protein
Hepes buffer (pH 7.4) containing 10
11.6 g/mL calmodulin, 100 M DTT, 100
3.0
M oxyhemoglobin (for iNOS assays, no Ca2+ and calmodulin
lM
L-arginine, 1.6 mM CaCl2,
l
l
l
M NADPH, 6.5 M H4B,
l
6,671
186
l
Ligand/ion
was 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 from 0 to 60 s after. The corresponding Ki values of inhibitors
were calculated from the IC50 values using Eq. 1 with known Km val-
Water
380
Rms deviations
Bond lengths (Å)
Bond angles (°)
0.014
1.350
a
The 5% of reflections were set aside for the free R calculation throughout the
refinement according to the free R flags used in the refinement of the starting model
(1OM4).
ues (rat nNOS, 1.3 lM; iNOS, 8.3 lM; eNOS, 1.7 lM).
Ki ¼ IC50=ð1 þ ½Sꢂ=KmÞ:
ð1Þ
mounting robot. Raw data frames were indexed, integrated, and
scaled using HKL2000.36
5.11. Molecular docking
The binding of inhibitors was detected by the initial difference
Fourier maps calculated with REFMAC.37 The inhibitor molecules
were then modeled in O38 or COOT39 and refined using REFMAC.
Water molecules were added in REFMAC and checked by COOT.
The TLS40 protocol was implemented in the final stage of refine-
ments with each subunit as one TLS group. The refined structures
were validated in COOT before deposition to RCSB protein data
bank with PDB accession code 1OM4. The crystallographic data
collection and structure refinement statistics are summarized in
Table 2.
Docking simulations were carried out as described by Ji et al.
with AutoDock 3.0.5.30 Polar hydrogen atoms were added to the
nNOS crystal structure (PDB code: 3JWT),24 and Kollman united
atom charges31 were assigned. Hydrogens were also added to the
heme and H4B, and charges were calculated by the Gasteiger–Mar-
sili method.32 The charge of the Fe atom bound to heme was as-
signed +3. The nonpolar hydrogen atoms of heme and H4B were
removed manually, and their charges were united with the bonded
carbon atoms. Atomic solvation parameters and fragmental vol-
umes were assigned using the AddSol utility. The 3D structures
of the ligands were built and partial atomic charges were also cal-
culated using the Gasteiger–Marsili method. The rotatable bonds in
the ligands were defined using AutoTors, which also unites the
nonpolar hydrogens and partial atomic charges to the bonded car-
bon atoms. The grid maps were calculated using AutoGrid. The
dimensions of the grid box was 31 ꢁ 28 ꢁ 31 Å, and the grid spac-
ing was set to 0.375 Å. Docking was performed using the Lamarck-
ian genetic algorithm (LGA), and the pseudo-Solis and Wets
method were applied for the local search.
Acknowledgments
The authors are grateful to the National Institutes of Health for
financial support to R.B.S. (GM49725), T.L.P (GM57353), and Dr.
Bettie Sue Masters (GM52419, with whose laboratory P.M. and
L.J.R. are affiliated). B.S.S.M. also is grateful to the Welch Founda-
tion for a Robert A. Welch Distinguished Professorship in Chemis-
try (AQ0012). P.M. is supported by grants 0021620806 and
1M0520 from MSMT of the Czech Republic.
Supplementary data
5.12. Crystal preparation, X-ray diffraction data collection, and
structure refinement
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.06.074. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
The nNOS heme domain protein used for crystallographic stud-
ies was produced by limited trypsin digest from the corresponding
full length enzyme and further purified through a Superdex 200 gel
filtration column (GE Healthcare) as described previously.33,34 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 reported before.33 Fresh crystals (1–2 day old) were
first passed stepwise through cryo-protectant solutions described
and then soaked with 10 mM inhibitor for 4–6 h at 4 °C before
being flash cooled by plunging into liquid nitrogen.
The cryogenic (100 K) X-ray diffraction data were collected re-
motely at the Stanford Synchrotron Radiation Lightsource through
the data collection control software Blu-Ice35 and the crystal
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