Analogues of 2-aminopyridine-based selective inhibitors of neuronal nitric oxide synthase with increased bioavailability

Article abstract of DOI:10.1016/j.bmc.2009.02.017

Overproduction of nitric oxide by neuronal nitric oxide synthase (nNOS) has been linked to several neurodegenerative diseases. We have recently designed potent and isoform selective inhibitors of nNOS, but the lead compound contains several basic function

Full text of DOI:10.1016/j.bmc.2009.02.017

Bioorganic & Medicinal Chemistry 17 (2009) 2371–2380
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Analogues of 2-aminopyridine-based selective inhibitors of neuronal nitric
oxide synthase with increased bioavailability
Graham R. Lawton a,b, Hantamalala Ralay Ranaivo b, Laura K. Chico b, Haitao Ji a,b, Fengtian Xue a,b
,
Pavel Martásek c,d, Linda J. Roman c, D. Martin Watterson b, Richard B. Silverman a,b,
*
a Department of Chemistry and Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208-3113, United States
b Center for Drug Discovery and Chemical Biology, Northwestern University, Evanston, IL 60208-3113, United States
c Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX, United States
d 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:
Overproduction of nitric oxide by neuronal nitric oxide synthase (nNOS) has been linked to several neu-
rodegenerative diseases. We have recently designed potent and isoform selective inhibitors of nNOS, but
the lead compound contains several basic functional groups. A large number of charges and hydrogen
bond donors can impede the ability of molecules to cross the blood brain barrier and thereby limit the
effectiveness of potential neurological therapeutics. Replacement of secondary amines in our lead com-
pound with neutral ether and amide groups was made to increase bioavailability and to determine if the
potency and selectivity of the inhibitor would be impacted. An ether analogue has been identified that
retains a similar potency and selectivity to that of the lead compound, and shows increased ability to pen-
etrate the blood brain barrier.
Received 3 February 2009
Accepted 7 February 2009
Available online 14 February 2009
Keywords:
Neuronal nitric oxide synthase
Neuronal nitric oxide synthase inhibitors
Ether
Bioavailability
Blood-brain barrier
Aminopyridine
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
ecules that inhibited nNOS with a potency and selectivity compa-
rable to 1, but which carried less charge, or at least had fewer
Neuronal nitric oxide synthase (nNOS) has been implicated in
various neurodegenerative diseases, including Parkinson’s disease1
and neuronal damage resulting from stroke.2 Inhibition of nNOS
could have therapeutic benefit in these and other diseases, but this
inhibition must be achieved without effect on the other isoforms of
NOS, endothelial (eNOS) and inducible (iNOS); inhibition of eNOS
could lead to side effects such as hypertension,3 and inhibition of
iNOS could result in a higher probability of Alzheimer’s disease.4
We have recently shown that a highly potent and isoform selec-
tive nNOS inhibitor (1) (Fig. 1) affects the extent of neurological
damage suffered by rabbit fetuses under hypoxic conditions when
administered to the dam intravenously.5 Although this in vivo effi-
cacy was encouraging, we were concerned that the efficacy of the
compound in adult disease models may be limited by unfavorable
pharmacokinetics.6 Specifically, the high number of basic groups
leads to significant positive charge at physiological pH, which
could impede the ability of the compound to cross a fully formed
blood brain barrier (BBB).7
hydrogen bond donors, and therefore could show increased uptake
into the CNS.
The secondary amine of the pyrrolidine ring of 1 interacts with
a key aspartate residue in nNOS.8 This is critical for nNOS over
eNOS selectivity, so this group was not altered. The secondary
amine adjacent to the pyrrolidine was replaced with an ether link-
age to form 2 and an amide linkage to form 3. Replacing the sec-
ondary amine in the chain with an ether linkage gave 4. These
three compounds were tested for activity against the NOS iso-
forms, and it was discovered that having an ether next to the pyr-
rolidine was a beneficial modification, but that having an ether in
the chain resulted in a significant decrease in potency. The ether
modification was incorporated into the design of 5 and 6, which
had amides in the chain, with the carbonyls on either side of the
nitrogen.
2. Chemistry
Systematic replacement of basic functional groups in the lead
with non-basic ones was carried out. The goal was to identify mol-
The synthesis of alcohol 7 has been described previously. The
carbamate proton adjacent to the pyridine ring is more acidic than
the hydroxyl group proton, allowing selective alkylation of the
nitrogen preferentially to the oxygen (Scheme 1). Protection of this
nitrogen with a benzyl group is necessary for both the Mitsunobu
* Corresponding author. Tel.: +1 847 491 5653; fax: +1 847 491 7713.
E-mail address: Agman@chem.northwestern.edu (R.B. Silver.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.017

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G. R. Lawton et al. / Bioorg. Med. Chem. 17 (2009) 2371–2380
H
N
H
N
H
N
H
N
H2N
H2N
H2N
N
N
N
H2N
N
F
F
F
F
N
O
H
1 ( )
2 ( )
H
N
H
N
O
H
N
H2N
N
O
F
N
H
N
H
3 ( )
4 ( )
H
N
H
N
H
N
H
N
H2N
N
F
O
O
O
O
5 ( )
6 ( )
Figure 1. nNOS-selective analogue inhibitors synthesized.
reaction9 and ether formation. A Mitsunobu reaction using acetic
acid followed by hydrolysis of the resulting ester converted the
trans-alcohol (8) to the desired cis-alcohol (10). Allyl bromide
proved to be the best alkylating agent for ether formation. Alkylat-
ing reagents such as bromoacetonitrile and other alkylbromides
and tosylates gave poor yields, most likely because of the high ba-
sicity of the secondary alkoxide. The allyl ether could be converted
to aldehyde 12 under ozonolysis conditions.
3-Fluorophenethylamine was alkylated with ethyl bromoace-
tate, and the resulting secondary amine was Boc protected (Scheme
3). The ester was hydrolyzed to give acid 16. 3-Fluorophenethanol
was alkylated with allyl bromide and converted to aldehyde 18.
Compound 12 and 3-fluorophenethylamine were mixed to form
the imine, which was then reduced to the secondary amine using
sodium triacetoxyborohydride (Scheme 4). Simultaneous removal
of the benzyl group and the Boc groups was achieved using high-
pressure hydrogenation and strong acid to give 2 in a high yield.
Amine 14 was coupled to 16 using standard peptide coupling
reagents to give the amide. This was treated with acid to remove
Performing the Mitsunobu reaction on 8 using DPPA as a source
of azide gave 13, which could be reduced to amine 14 with con-
comitant removal of the benzyl group (Scheme 2).
Boc
N
Boc
N
Boc
N
iii
ii
i
BocN
N
BocN
N
BocHN
BocN
N
OH
OAc
OH
Ph
N
Ph
7 ( )
8 ( )
9 ( )
Boc
Boc
Boc
N
N
N
iv
v
BocN
N
N
BocN
O
O
OH
O
Ph
Ph
Ph
H
11 ( )
10 ( )
12 ( )
Scheme 1. Reagents and conditions: (i) NaH, DMF, 0 °C, 30 min, then BnBr, rt, 16 h; (ii) PPh3, DIAD, AcOH, THF, rt, 16 h; (iii) 1 N NaOH (aq), MeOH, rt, 16 h; (iv) NaH, DMF,
0 °C, 30 min, then allyl bromide; (v) O3, CH2Cl2, ꢀ78 °C, then Zn, AcOH (aq), rt, 1 h.
Boc
N
Boc
N
Boc
N
ii
i
BocN
N
BocN
N
BocHN
N
OH
N3
NH2
Ph
Ph
8 ( )
13 ( )
14 ( )
Scheme 2. Reagents and conditions: PPh3, DIAD, DPPA, THF, rt, 16 h; (ii) H2, Pd(OH)2/C, MeOH, rt, 1–2 d.

G. R. Lawton et al. / Bioorg. Med. Chem. 17 (2009) 2371–2380
2373
O
O
H
Boc
N
Boc
N
i
F
NH2
OH
a
b
ii
F
F
F
F
OEt
OH
O
15
16
18
F
i
O
ii
O
17
Scheme 3. Reagents and conditions: (a) (i) ethyl bromoacetate, MeOH, rt, 4 h, then DIEA, Boc2O, rt, 2 h; (ii) 1 N NaOH (aq), MeOH, rt, 16 h; (b) (i) NaH, THF, 0 °C, then allyl
bromide, rt, 16 h; (ii) O3, CH2Cl2, ꢀ78 °C, 1 h, then Zn, AcOH (aq), rt, 3 h.
H N
Boc
N
2
i
ii
12
2
Boc
N
+
BocN
Bn
N
F
O
F
19 ( )
Boc
N
iii
iv
iv
O
14
+
H
N
16
3
4
BocHN
BocHN
N
F
N
H
20 ( )
Boc
N
v
+
18
14
N
O
F
N
H
21 ( )
Scheme 4. Reagents and conditions: (i) NaHB(OAc)3, MeOH, rt, 1 h, then Boc2O, TEA, 3 h; (ii) H2, Pd(OH)2/C, EtOH/HCl, rt, 575 psi, 2 d; (iii) EDC, HOBt, DIEA, CH2Cl2, rt, 16 h;
(iv) 4 N HCl, dioxanes; (v) NaHB(OAc)3, CH2Cl2, rt, 1 h.
the Boc groups, producing 3. A second portion of amine 14 was
mixed with 18 then reduced with sodium triacetoxyborohydride.
The Boc groups were removed with acid to give 4.
secondary amine adjacent to the pyrrolidine ring in 1 is either
not contributing to binding to the active site, or that the ether link-
age contributes equally as an amine to binding, possibly as a
hydrogen bond acceptor. Crystal structures of analogues of 1 in
the active site of nNOS indicate that this amine interacts with a
heme propionate group via an active site water molecule. It is pos-
sible that an ether could also hydrogen bond to the same water
molecule. Replacing the amine with an amide to give 3 resulted
in a slight loss of binding affinity (3–4 fold), perhaps the result of
increased steric hindrance. However, replacing the amine in the
chain with an ether linkage as in 4, or with an amide as in 5 and
6 resulted in a significant loss in potency with nNOS. In the case
of 4, the ether linkage is not only neutral, but a hydrogen bond do-
nor has been replaced with a hydrogen bond acceptor. In the case
of the amides, the NH is a potential H-bond donor, but the potency
is not recovered. This suggests that this amine is involved in a
charge–charge interaction that is important for binding, not a sim-
ple hydrogen bonding interaction.
Aldehyde 12 was oxidized with oxone,10 and the resulting acid
was coupled to 3-fluorophenethylamine (Scheme 5). Deprotection
gave 5. Aldehyde 12 and benzylamine were mixed and reduced to
the secondary amine. The two benzyl protecting groups were re-
moved under hydrogenation conditions. The primary amine was
coupled to 3-fluorophenylacetic acid, and the Boc groups were re-
moved to give 6.
3. Results and discussion
3.1. In vitro NOS inhibition assay
The compounds were tested for their ability to inhibit the three
NOS isoforms using an established in vitro assay.11 The results are
shown in Table 1.
Racemic lead compound 1 is a competitive inhibitor of nNOS
with low nanomolar potency. It shows 2000-fold selectivity for
nNOS over eNOS, which is critical for this class of compounds to
be therapeutically useful. Ether 2 has the same potency with nNOS
as the analogous amine compound 1. The potencies for 2 with
eNOS and iNOS are also comparable with those for 1, so the selec-
tivity for nNOS over the other isoforms is excellent (ꢁ2000-fold
over eNOS, ꢁ600-fold over iNOS). These results indicate that the
3.2. In vivo brain uptake assay
To determine the extent to which 1 and 2 cross the blood brain
barrier, the compounds were administered to mice at 3.7 mg/kg
(10 nmol/g) via intraperitoneal injection. At 5, 10, 20, and 40 min
after administration, five mice were sacrificed, and blood and brain
samples were taken. The amount of compound in each sample was

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G. R. Lawton et al. / Bioorg. Med. Chem. 17 (2009) 2371–2380
Boc
N
Boc
N
ii
i
BocN
N
BocN
N
O
OH
O
O
Ph
Ph
H
O
O
12 ( )
22 ( )
Boc
N
iv
5
H
N
BocN
R
N
F
O
23 ( ), R = Bn
24 ( ), R = H
iii
Boc
Boc
N
N
v
iii
12 ( )
BocN
N
NHBn
BocHN
N
O
NH
2
O
Ph
25 ( )
26 ( )
Boc
N
v
vi
H
N
6
BocHN
N
F
O
O
27 ( )
Scheme 5. Reagents and conditions: (i) oxone, DMF, rt, 16 h; (ii) 3-fluorophenethylamine, EDC, HOBt, DIEA, CH2Cl2, rt, 16 h; (iii) H2, Pd(OH)2/C, MeOH, rt, 1–2 d; (iv) 4 N HCl,
dioxanes, 16 h; (v) benzylamine, NaHB(OAc)3, MeOH, rt, 1 h; (vi) 3-fluorophenylacetic acid, EDC, HOBt, DIEA, CH2Cl2, rt, 16 h.
Table 1
and separate administrations, suggesting that the two compounds
do not interfere with one another. The values were initially quan-
Inhibition of the three isozymes of NOS by 1–6
tified in
concentrations were converted to
brain tissue is approximately 1 g/mL.
A comparison of the plasma concentrations gives a surprising
result. The plasma concentration of 1 is significantly higher than
that of 2 throughout the 40 min experiment. The brain level of 1
is also slightly higher than that of 2. Figure 4 shows the ratio of
brain to blood concentration for the two compounds.
l
mol/mL of plasma and
l
l
mol/g of brain tissue. The brain
M by assuming the density of
1
2
3
4
5
6
Ki (nNOS) (
Ki (eNOS) (
Ki (iNOS) (
l
M)
0.014
28
4
0.015
31
9.5
0.053
27
5.4
0.4
33
4
2.3
145
52
5.0
107
77
lM)
l
M)
determined by LCMS and quantified by comparison to a standard
curve. The assay was performed on 1 and 2 separately and as a
co-administration experiment. The plasma and brain concentra-
tions of 1 and 2 calculated in the co-administration experiment
are shown in Figures 2 and 3. The shapes of the curves and relative
values did not differ significantly between the co-administration
The brain/blood ratio for 2 is consistently higher than that of 1,
indicating that it does indeed penetrate the BBB more efficiently.
Brain Concentrations of 1b and 4 after co-
administration
Plasma Levels of 1b and 4 after co-administration
6
0.09
2
1
5
4
0.08
2
1
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
3
2
1
0
0
10
20
30
40
50
0
10
20
30
40
50
Time after administration (min)
Time after administration (min)
Figure 2. Comparison of the plasma concentrations of 1 and 2 after ip adminis-
tration at 3.7 mg/kg. Error bars show the standard error mean.
Figure 3. Comparison of the brain concentrations of 1 and 2 after ip administration
at 3.7 mg/kg. Error bars show the standard error mean.

G. R. Lawton et al. / Bioorg. Med. Chem. 17 (2009) 2371–2380
Ratio of brain to plasma concentration
2375
5. Experimental procedures
5.1. General methods
0.3
0.25
0.2
2
1
Proton nuclear magnetic resonances (1H NMR) were recorded
in deuterated solvents on a Varian Inova 500 (500 MHz) spec-
trometer. Chemical shifts are reported in parts per million
(ppm, d) relative to tetramethylsilane (d 0.00). 1H NMR splitting
patterns are designated as singlet (s), doublet (d), triplet (t), or
quartet (q). Splitting patterns that could not be interpreted or
easily visualized were recorded as multiplet (m) or broad (br).
Coupling constants are reported in Hertz (Hz). Proton-decoupled
carbon (13C NMR) spectra were recorded on a Varian Inova 500
(125 MHz) spectrometer and are reported in ppm using the sol-
vent as an internal standard (CDCl3, d 77.23). NMR spectra re-
corded in D2O were not normalized. In many cases, the
presence of rotamers made the NMR spectra complex. In the
case of two peaks that are clearly a pair of rotamers, but are
too far apart for an average to accurately represent the spec-
trum, the pair is written enclosed in parentheses, or the pres-
ence of rotamers is indicated. Electrospray mass spectra (ESMS)
were obtained using an LCQ-Advantage with methanol as the
solvent in positive ion mode, unless otherwise stated. For most
compounds, 1H and 13C NMR and ESMS data are presented. Final
compounds were analyzed for purity using multiple HPLC condi-
tions (see Supplementary data). Analysis of biological samples
was carried out using an API 3000 liquid chromatography-tan-
dem mass spectrometry system (Applied Biosystems, Foster City,
CA) equipped with an Agilent 1100 series HPLC system (Agilent
Technologies, Wilmington, DE).
All chemical reagents were purchased from Aldrich and were
used without further purification. NADPH, calmodulin, and human
ferrous hemoglobin were obtained from Sigma–Aldrich. Tetrahy-
drobiopterin (H4B) was purchased from Alexis Biochemicals.
HEPES, DTT, and some conventional organic solvents were pur-
chased from Fisher Scientific.
Tetrahydrofuran (THF) was distilled from sodium and benzo-
phenone prior to use. Methylene chloride (CH2Cl2) was distilled
from calcium hydride prior to use, if dry solvent were required.
Dimethylformamide (DMF) was purchased as an anhydrous sol-
vent and was used directly.
0.15
0.1
0.05
0
0
10
20
30
40
50
Time after administration (min)
Figure 4. Comparison of the ratio of brain to blood concentrations of 1 and 2 after
ip administration at 3.7 mg/kg. Error bars show the standard error mean.
The secondary amine in 1 that is adjacent to the pyrrolidine ring
is probably not protonated at physiological pH, as the presence of
two secondary amines two carbons away will lower the pKa signif-
icantly.12 This may explain why there is little loss in binding affin-
ity when this amine is replaced with an ether linkage. The ether
modification in 2 is therefore not useful in reducing the overall
charge on the molecule, which may explain why there is only a
small difference in BBB penetration between 1 and 2. However,
the ether modification does reduce the number of H-bond donors.
Furthermore, according to molecular properties calculations, 2 has
a slightly higher calculated logD (0.11) and lower polar surface
area (81 Å2) than 1 (ꢀ0.09 and 84 Å2, respectively).13 All of these
subtle changes may be responsible for the modest increase in the
ability of the compound to cross the BBB.
A cursory inspection of the in vivo results suggests that
although 2 crosses the BBB better than 1, there has been no
improvement as there is no positive increase in final brain concen-
tration. However, the concentration of 2 is above 60 nM through-
out the 40 min experiment. This is four times the Ki with nNOS,
and could be enough to produce a therapeutic response. In con-
trast, the plasma levels are less than 2 lM, which is significantly
less than the Ki with eNOS. Although the brain to blood ratio is
low, when administered at this dose, the compound should reach
sufficient levels in the brain to have a therapeutic effect while
not affecting eNOS, and therefore not causing a hypertensive side
effect. The plasma concentration of 1 is considerably higher than
that of 2, although still insufficient to effectively inhibit eNOS. In
contrast, the brain concentrations of the two compounds are
approximately the same. Since the two compounds have the same
ability to inhibit nNOS, compound 2 could potentially have the
same therapeutic effect as 1 while at a lower systemic level, and
therefore have a lower potential for side effects. Compound 1
was found to be highly effective in a cerebral palsy rabbit model
and exhibited no cardiovascular effects.5
5.2. General alkylation procedure
To a solution of the compound in anhydrous DMF (3–10 mL) at
0 °C was added NaH (1.1 equiv). The mixture was allowed to warm
to room temperature and stirred for 30 min. The alkylating agent
(1.1 equiv) was added dropwise, and the solution was stirred for
16 h. The solvent was removed in vacuo, and the residue was dis-
solved in EtOAc, washed with brine, dried over anhydrous Na2SO4,
and concentrated in vacuo.
5.3. General Mitsunobu procedure
4. Conclusions
To a solution of the alcohol in anhydrous THF (5 mL) were
added PPh3, DIAD and the nucleophile. The mixture was stirred
for 16 h. The mixture was poured into brine and extracted with
EtOAc (3 ꢂ 10 mL), dried over anhydrous Na2SO4, and concentrated
in vacuo.
The replacement of secondary amino groups in a potent and
selective nNOS inhibitor with ether or amide linkages had varied
effects on potency. In one case, replacement with an ether resulted
in a negligible difference in potency and selectivity. In other cases,
the replacement resulted in dramatic losses in potency. The ether
analogue that was as potent as the lead compound showed a mod-
est increase in brain uptake over the lead compound. These results
are consistent with the notion that hydrogen bond donor groups
are more detrimental to BBB penetration than hydrogen bond
acceptor groups.
5.4. General reductive amination procedure
A solution of the aldehyde and the amine in CH2Cl2 (5 mL) was
stirred for 10 min. NaHB(OAc)3 was added, and the solution was
stirred for 1 h.

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G. R. Lawton et al. / Bioorg. Med. Chem. 17 (2009) 2371–2380
5.5. General procedure for amide formation
5.10. cis-tert-Butyl 3-((6-(benzyl(tert-butoxycarbonyl)amino)-
4-methylpyridin-2-yl)methyl)-4-hydroxypyrrolidine-1-
carboxylate (10)
To a solution of the acid and the amine in CH2Cl2 (3 mL) were
added TEA, EDC, and HOBt. The mixture was stirred for 16 h. The
solution was diluted with CH2Cl2 (20 mL) and washed with 1 N
HCl (2 ꢂ 15 mL), satd NaHCO3 (15 mL), and brine (15 mL). The or-
ganic layer was dried over anhydrous Na2SO4 and concentrated in
vacuo.
To a solution of 9 (216 mg, 0.40 mmol) in MeOH (3 mL) was
added aqueous NaOH (1 N, 3 mL). The mixture was stirred at room
temperature overnight. The pH of the solution was adjusted to ꢁ8,
and the product was extracted with EtOAc (3 ꢂ 15 mL). The organic
layers were combined, dried over anhydrous Na2SO4, and concen-
trated in vacuo. The crude product was purified using flash column
chromatography (silica gel, EtOAc/hexanes, 2:3) to afford 10 as a
white solid (184 mg, 0.37 mmol, 93%). 1H NMR (500 MHz, CDCl3)
d 7.36 (d, 1H), 7.28–7.20 (m, 5H), 6.74 (d, 1H), 5.09 (s, 2H), 3.95
(m, 1H), 3.59–3.36 (m, 4H), 3.14 (m, 1H), 2.86 (m, 1H), 2.78 (m,
1H), 2.31 (s, 3H), 1.44 (s, 9H), 1.41 (s, 9H); 13C NMR (125 MHz,
CDCl3) d 157.8, 154.5, 154.2, 149.8, 139.2, 128.5, 127.0, 120.7,
118.7, 81.7, 79.3, (71.2 + 70.4), (53.9 + 53.6), 50.6, (49.6 + 49.2),
(45.2 + 44.6), 35.3, 28.8, 28.4, 21.4; ESMS m/z = 498 (M+H)+, 520
(M+Na)+.
5.6. General procedure for benzyl group removal
To a solution of the compound in EtOH (5 mL) was added
Pd(OH)2/C (ꢁ5 mg). The mixture was stirred under a hydrogen
atmosphere at 50 °C for 48 h. The mixture was filtered through Cel-
ite, and the solvent was removed in vacuo.
5.7. General procedure for Boc group removal
A solution of the compound in HCl in dioxanes (4 N, 3 mL)
was stirred for 16 h. The solvent was removed under a stream
of nitrogen, and the residue was dissolved in water (10 mL).
The solution was washed with EtOAc, and the solvent was re-
moved in vacuo. The resulting salt was dissolved in the mini-
mum amount of MeOH, and anhydrous ether was added
causing the salt to precipitate. The ether layer was decanted to
leave the salt as a white solid.
5.11. tert-Butyl 3-(allyloxy)-4-((6-(benzyl(tert-butoxy-
carbonyl)amino)-4-methylpyridin-2-yl)methyl)pyrrolidine-1-
carboxylate (11)
To a solution of 10 (994 mg, 2.0 mmol) in DMF (10 mL) was
added NaH (60% in mineral oil, 120 mg, 3.0 mmol) at 0 °C. After
being stirred for 15 min, allyl bromide (360 lL, 3.0 mmol) was
5.8. trans-tert-Butyl 3-((6-(benzyl(tert-butoxycarbonyl)
amino)-4-methylpyridin-2-yl)methyl)-4-hydroxypyrrolidine-1-
carboxylate (8)
added. The reaction mixture was allowed to stir at room tempera-
ture for 30 min, then quenched by H2O (1.0 mL). The solvent was
removed in vacuo, and the resulting material was purified by flash
column chromatography (silica gel, EtOAc/hexanes, 1:9–1:4) to
yield 11 (1.07 g, 99%) as a colorless oil. 1H NMR (500 MHz, CDCl3)
d 7.40 (m, 1H), 7.27–7.17 (m, 5H), 6.69 (s, 1H), 5.80 (m, 1H),
5.22–5.11 (m, 4H), 3.96 (m, 1H), 3.72–3.64 (m, 2H), 3.55–3.37
(m, 2H), 3.21–3.09 (m, 2H), 2.93 (m, 1H), 2.72 (m, 1H), 2.59 (m,
1H), 2.29 (m, 3H), 1.45–1.41 (m, 18H); 13C NMR (125 MHz, CDCl3)
d 158.0, 154.9, 154.1, 148.7, 140.1, 134.9, 128.3, 127.2, 126.7,
120.4, 117.3, 116.8, 81.4, 79.3, 78.0, 70.4, (51.2 + 50.7), 50.1,
(49.6 + 49.2), (43.1 + 42.4), 34.8, 28.8, 28.4, 21.4; ESMS m/z = 538
(M+H)+, 560 (M+Na)+.
The general alkylation procedure was carried out on
7
(491 mg, 1.21 mmol) using benzyl bromide (148 L, 1.25 mmol)
l
as the alkylating agent. The crude product was purified using
flash column chromatography (silica gel, EtOAc/hexanes, 2:3)
to afford
NMR (500 MHz, CDCl3)
8 as a
white solid (402 mg, 0.81 mmol, 67%). 1H
d
7.34–7.18 (m, 6H), 6.70 (m, 1H),
5.12 (s, 2H), 4.05 (m, 1H), 3.74–3.55 (m, 2H), 3.19 (m, 1H),
3.08 (m, 1H), 2.81–2.63 (m, 2H), 2.40 (m, 1H), 2.29 (s, 3H),
1.45 (s, 9H), 1.42 (s, 9H); 13C NMR (125 MHz, CDCl3)
d
157.5, 154.8, 154.5, 154.1, 149.4, 139.6, 128.4, 127.3, 127.0,
120.8, 118.4, 81.6, 79.5, (75.3 + 74.6), (52.9 + 52.5), 50.6,
(49.9 + 49.5), (45.6 + 44.8), 39.4, 28.7, 28.4, 21.3; ESMS m/
z = 498 (M+H)+.
5.12. tert-Butyl 3-((6-(benzyl(tert-butoxycarbonyl)amino)-4-
methylpyridin-2-yl)methyl)-4-(2-oxoethoxy)-pyrrolidine-1-
carboxylate (12)
5.9. tert-Butyl 3-acetoxy-4-((6-(benzyl(tert-butoxycarbonyl)-
amino)-4-methylpyridin-2-yl)methyl)pyrrolidine-1-
carboxylate (9)
A solution of 11 (535 mg, 1.0 mmol) in CH2Cl2 (20 mL) was
cooled to -78 °C, to which O3 was charged until the reaction solu-
tion turned purple (ꢁ20 min). The O3 flow was stopped, and the
reaction was allowed to stir at the same temperature for 1.5 h.
To the resulting solution was added Zn (195 mg, 3.0 mmol) and
50% aqueous AcOH (20 mL). The reaction mixture was then
warmed to room temperature over 45 min. The solvent was re-
moved by rotary evaporation and the resulting material was puri-
fied by flash column chromatography (silica gel, ethyl acetate/
hexanes, 1:4–1:1) to yield 12 (405 mg, 75%) as an oily white solid.
1H NMR (500 MHz, CDCl3) d 9.56 (s, 1H), 7.44 (m, 1H), 7.28–7.16
(m, 5H), 6.69 (s, 1H), 5.17 (s, 2H), 4.06–3.94 (m, 1H), 3.81–3.63
(m, 2H), 3.54–3.41 (m, 2H), 3.22–3.11 (m, 2H), 2.98 (m, 1H), 2.77
(m, 1H), 2.71–2.53 (m, 1H), 2.30 (s, 3H), 1.46 (s, 9H), 1.42 (s, 9H);
13C NMR (125 MHz, CDCl3) d 202.4, 157.6, 154.7, 154.1, 148.9,
140.0, 128.4, 127.1, 120.2, 117.2, 81.5, 80.7, 79.7, 74.7,
(51.1 + 50.5), 50.1, (49.4 + 49.0), (42.9 + 42.3), 34.4, 28.7, 28.4,
21.3; ESMS m/z = 572 (M+MeOH)+.
The general Mitsunobu procedure was carried out on
(402 mg, 0.81 mmol) using PPh3 (262 mg, 1 mmol), DIAD
(188 L, 1 mmol) and acetic acid (86 L, 1.5 mmol) as the nucle-
8
l
l
ophile. The crude product was purified using flash column chro-
matography (silica gel, EtOAc/hexanes, 1:3) to afford 9 as a
white solid (216 mg, 0.40 mmol, 49%). Unreacted 8 was also
recovered. 1H NMR (500 MHz, CDCl3) d 7.44 (m, 1H), 7.29–7.18
(m, 5H), 6.62 (s, 1H), 5.16 (s, 2H), 4.11 (m, 1H), 3.43 (m, 3H),
3.10 (m, 1H), 2.85 (m, 1H), 2.66 (m, 2H), 2.29 (s, 3H), 2.05 (s,
3H), 1.45 (s, 9H), 1.41 (s, 9H); 13C NMR (125 MHz, CDCl3) d
170.7, 156.9, 154.5, 154.2, 148.9, 140.1, 128.3, 127.2, 126.8,
126.7, 119.9, 117.5, 81.4, 79.6, (75.0 + 74.1), 60.6, (53.0 + 52.6),
50.2, (49.6 + 49.1), (42.0 + 41.4), 35.0, 28.7, 28.4, 21.3; ESMS m/
z = 540 (M+H)+, 562 (M+Na)+.

G. R. Lawton et al. / Bioorg. Med. Chem. 17 (2009) 2371–2380
2377
5.13. tert-Butyl 3-azido-4-((6-(benzyl(tert-butoxycarbonyl)-
5.17. 1-(2-(Allyloxy)ethyl)-3-fluorobenzene (17)
amino)-4-methylpyridin-2-yl)methyl)pyrrolidine-1-
carboxylate (13)
The general alkylating procedure was carried out on 3-fluorophe-
nethanol (127 L, 1 mmol) using allyl bromide (130 L, 1.5 mmol)
l
l
The general Mitsunobu procedure was carried out on
8
as the alkylating agent and THF as the solvent. The crude product
was purified using flash column chromatography (silica gel,
EtOAc/hexanes, 1:4) to afford 17 (143 mg, 0.79 mmol, 79%) as a col-
orless oil. 1H NMR (500 MHz, CDCl3) d 7.23 (m, 1H), 7.00 (d,
J = 7.5 Hz, 1H), 6.91 (m, 2H), 5.89 (m, 1H), 5.25 (dd, J = 1, 17 Hz,
1H), 5.17 (d, J = 10 Hz, 1H), 3.98 (d, J = 5.5 Hz, 2H), 3.64 (t, J = 7 Hz,
2H), 2.89 (t, J = 7 Hz, 2H); 13C NMR (125 MHz, CDCl3) d 162.1,
141.9, 134.9, 129.9, 124.8, 117.2, 116.0, 113.3, 72.2, 70.9, 36.3.
(519 mg, 1.04 mmol) using PPh3 (342 mg, 1.3 mmol), DIAD
(255 L, 1.35 mmol) and DPPA (281 L, 1.30 mmol) as the nucle-
l
l
ophile source. The crude product was purified using flash col-
umn chromatography (silica gel, EtOAc/hexanes, 1:4) to afford
13 as
a
white solid (461 mg, 0.88 mmol, 85%) 1H NMR
(500 MHz, CDCl3) d 7.47 (m, 1H), 7.37 (m, 1H), 7.24 (m, 4H),
6.69 (s, 1H), 5.17 (s, 2H), 3.76 (m, 1H), 3.58–3.37 (m, 2H), 3.31
(m, 1H), 3.00 (q, J = 11 Hz, 1H), 2.85 (m, 1H), 2.72 (m, 1H),
2.62 (m, 1H), 2.30 (m, 3H), 1.45 (s, 9H), 1.42 (s, 9H); 13C NMR
(125 MHz, CDCl3) d 156.8, 154.6, 154.2, 149.0, 140.0, 130.3,
128.3, 127.0, 126.8, 126.4, 120.5, 117.4, 81.5, 79.8, (63.5 + 62.7),
51.6, 50.2, 48.9, (42.8 + 42.2), 35.2, 28.7, 28.4, 21.4; ESMS m/
z = 523 (M+H)+, 545 (M+Na)+.
5.18. 2-(3-Fluorophenethoxy)acetaldehyde (18)
A solution of 17 (143 mg, 0.79 mmol) in CH2Cl2 (5 mL) was cooled
to ꢀ78 °C. Ozone was passed through the solution for 1 h. Zn powder
(104 mg, 1.6 mmol) and 50% aqueous acetic acid (5 mL) were added,
and the mixture was allowed to warm to room temperature. The
mixture was stirred a further 1 h. The mixture was poured into NaH-
CO3 (aq), and the product was extracted with CH2Cl2 (3 ꢂ 20 mL).
The organic layers were combined, dried over anhydrous Na2SO4
and concentrated in vacuo. The crude product was purified using
flash column chromatography (silica gel, EtOAc/hexanes, 1:3) to af-
ford 18 as a white solid (110 mg, 0.61 mmol, 80%). 1H NMR
(500 MHz, CDCl3) d 9.69 (s, 1H), 7.25 (m, 1H), 6.94 (m, 3H), 4.07–
3.43 (m, 4H), 2.90 (m, 2H); 13C NMR (125 MHz, CDCl3) d 200.1,
163.0, 141.2, 130.1, 124.8, 116.1, 113.6, 73.3, 72.6, 36.1.
5.14. tert-Butyl 3-amino-4-((6-(tert-butoxycarbonylamino)-4-
methylpyridin-2-yl)methyl)pyrrolidine-1-carboxylate (14)
The general procedure for the removal of benzyl groups was
carried out on 13 (461 mg, 0.88 mmol). The crude product was
purified using flash column chromatography (silica gel, EtOAc/
methanol, 9:1) to afford 14 as a white solid (170 mg, 0.42 mmol,
48%). 1H NMR (500 MHz, CDCl3) d 7.62 (s, 1H), 6.64 (s, 1H), 3.56–
3.14 (m, 4H), 2.78–2.61 (m, 3H), 2.46 (m, 1H), 2.30 (m, 3H), 1.52
(s, 9H), 1.44 (s, 9H); 13C NMR (125 MHz, CDCl3) d 161.4, 158.1,
155.0, 152.6, 151.6, 150.4, 119.4, 110.5, 81.0, 79.4, (71.8 + 70.7),
(61.3 + 60.4), (50.0 + 49.6), (49.0 + 48.5), (35.3 + 34.7), 28.7, 28.4,
21.5; ESMS m/z = 407 (M+H)+.
5.19. tert-Butyl 3-((6-(benzyl(tert-butoxycarbonyl)amino)-4-
methylpyridin-2-yl)methyl)-4-(2-(tert-butoxycarbonyl(3-fluor-
ophenethyl)-amino)ethoxy) pyrrolidine-1-carboxylate (19)
5.15. Ethyl 2-(tert-butoxycarbonyl(3-fluorophenethyl)-amino)-
The general reductive amination procedure was carried out using
acetate (15)
aldehyde 12 (463 mg, 0.86 mmol), 3-fluorophenethylamine (224
1.72 mmol),andNaHB(OAc)3 (212 mg, 1 mmol).Tothecrudemixture
was added DIEA (200 L, 1.39 mmol) and Boc2O (218 mg, 1 mmol),
lL,
A solution of ethyl bromoacetate (119
lL, 1 mmol) and 3-flu-
l
orophenethylamine (130 L, 1.5 mmol) in ethanol was stirred for
l
and the solution was stirred for 2 h. The mixture was poured into
brine (15 mL) and extracted with EtOAc (3 ꢂ 15 mL). The organic lay-
erswere combined, driedoveranhydrousNa2SO4 andconcentratedin
vacuo.Thecrudeproductwaspurifiedusingflashcolumnchromatog-
raphy (silica gel, EtOAc/hexanes, 1:4) to afford 19 as a white solid
(249 mg, 0.33 mmol, 38%). 1H NMR (500 MHz, CDCl3) d 7.42 (m,
1H), 7.24–7.17 (m, 6H), 6.94–6.86 (m, 3H), 6.62 (m, 1H), 5.17 (s,
2H), 3.60 (m, 1H), 3.52–3.17 (m, 9H), 3.06 (q, J = 11 Hz, 1H), 2.88–
2.79 (m, 3H), 2.68 (m, 1H), 2.58 (m, 1H), 2.28 (m, 3H), 1.43 (m,
27H); 13C NMR (125 MHz, CDCl3) d 164.1, 162.1, 157.9, 155.5, 154.8,
154.6, 154.1, 148.8, 142.2, 140.1, 130.1, 128.3, 127.2, 126.7, 124.8,
120.2, 117.3, 115.9, 113.5, 81.4, 79.8, 79.3, 68.3, 64.5, 60.6, 50.3,
50.2, 49.5, 47.6, 43.0, 35.0, 34.5, 28.7, 28.4, 21.4; ESMS m/z = 763
(M+H)+.
16 h. Boc2O (327 mg, 1.5 mmol) was added, and the solution was
stirred a further 2 h. The mixture was poured into brine and ex-
tracted with EtOAc (3 ꢂ 15 mL). The organic layers were combined,
dried over Na2SO4, and concentrated in vacuo. The crude product
was purified using flash column chromatography (silica gel,
EtOAc/hexanes, 1:4) to afford 15 as a colorless oil (146 mg,
0.45 mmol, 45%). 1H NMR (500 MHz, CDCl3) d 7.24 (m, 1H), 6.99–
6.88 (m, 3H), 4.18 (q, J = 7 Hz, 2H), 3.88 (s, 1H), 3.76 (s, 1H), 3.49
(m, 2H), 2.84 (m, 2H), 1.44 (s, 9H), 1.27 (t, J = 6.5 Hz, 3H); 13C
NMR (125 MHz, CDCl3) d 170.2, (164.7 + 162.8), 155.8, 141.9,
130.2, 124.8, 115.9, 113.5, 80.6, 61.3, 50.4, 49.4, 35.0, 28.5, 14.5;
ESMS m/z = 326 (M+H)+.
5.16. 2-(tert-Butoxycarbonyl(3-fluorophenethyl)amino)acetic
acid (16)
5.20. 6-((4-(2-(3-Fluorophenethylamino)-ethoxy)-pyrrolidin-3-
yl)methyl)-4-methyl-pyridin-2-amine (2)
To a solution of 15 (146 mg, 0.45 mmol) in MeOH (3 mL) was
added aqueous NaOH (1 N, 3 mL) dropwise. The solution was stir-
red for 16 h. Aqueous HCl (1 N, 4 mL) was added dropwise. The
mixture was diluted with brine (5 mL) and extracted with ethyl
acetate (3 ꢂ 15 mL). The organic layers were combined, dried over
Na2SO4, and concentrated in vacuo to afford 16 as a white solid
(133 mg, 0.45 mmol, quant). 1H NMR (500 MHz, CDCl3) d 10.22
(br, 1H), 7.24 (m, 1H), 6.98–6.86 (m, 3H), (3.93 + 3.81) (s, rotamers,
2H), 3.49 (m, 2H), 2.84 (m, 2H), 1.43 (s, 9H); 13C NMR (125 MHz,
To a solution of 19 (190 mg, 0.25 mmol) in EtOH (5.0 mL) was
added a 1:1 mixture of EtOH/concentrated HCl (10 mL) and
Pd(OH)2/C (20%, 150 mg). The mixture was charged with H2 at 575
psi. The reaction was allowed to stir at room temperature for 48 h.
Thecatalystwas removedby filtrationthroughCelite, and theresult-
ingCelitecake was washedwithEtOH (4 ꢂ 3 mL)and 2 N HCl(3 mL).
The combined solution was concentrated in vacuo to yield 2 as a
white trihydrochloride salt (84 mg, 0.23 mmol, 90%). 1H NMR
(500 MHz, D2O) d 7.24 (q, J = 7 Hz, 1H), 7.02 (d, J = 8 Hz, 1H), 6.97
(d, J = 10 Hz, 1H), 6.90 (t, J = 9 Hz), 6.57 (s, 1H), 6.45 (s, 1H), 4.10
(m, 1H), 3.75 (m, 1H), 3.70 (m, 1H), 3.63–3.53 (m, 4H), 3.38 (m,
CDCl3)
d (175.9 + 175.4), (164.1 + 162.2), 156.1, 141.7, 130.3,
124.8, 116.0, 113.5, 81.2, 50.5, 49.3, 34.8, 28.5; ESMS (negative
ion mode) m/z = 296 (MꢀH)ꢀ.

2378
G. R. Lawton et al. / Bioorg. Med. Chem. 17 (2009) 2371–2380
1H), 3.29–3.20 (m, 4H), 3.04 (m, 1H), 2.96 (m, 1H), 2.84 (m, 1H), 2.68
(m, 1H), 2.21 (s, 3H); 13C NMR (125 MHz, D2O) d 163.8, 161.8, 158.4,
154.0, 145.9, 138.9, 130.8, 124.9, 115.6, 114.4, 110.6, 78.4, 64.0, 60.4,
49.5, 48.4, 47.2, 47.1, 41.7, 31.4, 29.3, 21.3; ESMS m/z = 373 (M+H)+.
HRMS calcd 373.24036, found: 373.24036 (M+H)+.
3.10–2.99 (m, 2H), 2.82–2.69 (m, 2H), 2.54 (m, 1H), 2.20 (s, 3H);
ESMS m/z = 373 (M+H)+. HRMS calcd 373.24036, found:
373.24082 (M+H)+.
5.25. 2-(4-((6-(Benzyl(tert-butoxycarbonyl)amino)-4-methyl-
pyridin-2-yl)methyl)-1-(tert-butoxycarbonyl)-pyrrolidin-3-
yloxy)acetic acid (22)
5.21. tert-Butyl 3-(2-(tert-butoxycarbonyl-(3-fluorophenethyl)
amino)acetamido)-4-((6-(tert-butoxycarbonylamino)-4-
methylpyridin-2-yl)methyl)pyrrol-idine-1-carboxylate (20)
To a solution of 12 (166 mg, 0.31 mmol) in anhydrous DMF
(3 mL) was added oxone (190 mg, 0.31 mmol). The mixture was
stirred at room temperature for 3 h, and the solvent was removed
in vacuo. The residue was dissolved in 1 N HCl (aq) and extracted
with EtOAc (5 ꢂ 15 mL). The organic layers were combined, dried
over anhydrous Na2SO4, and concentrated in vacuo to afford 22
as a white solid (143 mg, 0.258 mmol, 83%). 1H NMR (500 MHz,
CDCl3) d 7.25 (m, 6H), 6.92–6.70 (m, 1H), 5.18 (m, 2H), 4.12 (m,
1H), 3.89 (m, 2H), 3.48 (m, 2H), 3.17 (m, 2H), 2.92 (m, 2H), 2.59
(m, 1H), 2.32 (m, 3H), 1.44 (m, 18H); 13C NMR (125 MHz, CDCl3)
d 172.4, 158.4, 157.0, 155.1, 154.4, 151.5, 139.3, 128.5, 127.2,
120.7, 87.9, 82.1, 79.8, 74.8, 66.6, 51.1, 50.4, 49.4, 42.9, 34.8,
28.7, 28.4, 21.6; ESMS m/z = 556 (M+H)+, ESMS (negative ion mode)
m/z = 554 (MꢀH)+.
The general procedure for amide formation was carried out on 16
(76 mg, 0.26 mmol) and 14 (106 mg, 0.25 mmol) using EDC (54 mg,
0.28 mmol), HOBt(38 mg, 0.28 mmol), and DIEA(52 lL, 0.28 mmol).
The crude product was purified using flash column chromatography
(silica gel, EtOAc/hexanes, 2:1) to afford 20 (151 mg, 0.22 mmol,
88%) as a colorless oil. 1H NMR (500 MHz, CDCl3) d 7.62 (s, 1H),
7.24 (m, 1H), 6.91 (m, 3H), 6.60 (m, 1H), 4.57 (m, 1H), 3.81 (m, 2H),
3.56–3.39 (m, 4H), 3.25–3.06 (m, 2H), 2.86–2.60 (m, 5H), 2.27 (s,
3H), 1.52 (s, 9H), 1.42 (s, 9H), 1.38 (s, 9H); 13C NMR (125 MHz, CDCl3)
d 171.3, 164.1, 162.1, 157.2, 156.5, 154.4, 152.7, 151.6, 150.3, 141.2,
130.3, 124.8, 119.4, 116.0, 113.7, 110.7, 81.4, 80.9, 79.6, 52.4, 51.1,
50.5, 49.2, 49.0, 40.9, 36.3, 34.7, 28.6, 21.5; ESMS m/z = 686
(M+H)+, 708 (M+Na)+.
5.26. tert-Butyl 3-((6-(benzyl(tert-butoxycarbonyl)-amino)-4-
methylpyridin-2-yl)methyl)-4-(2-(3-fluorophenethylamino)-2-
oxoethoxy)pyrrolidine-1-carboxylate (23)
5.22. N-(4-((6-Amino-4-methylpyridin-2-yl)-methyl)-
pyrrolidin-3-yl)-2-(3-fluorophenethylamino)-acetamide (3)
The general procedure for the removal of Boc groups was car-
ried out on 20 (151 mg, 0.22 mmol) to give 3 (94 mg, 0.19 mmol,
86%) as a pale yellow trihydrochloride salt. 1H NMR (500 MHz,
D2O) d 7.24 (q, J = 7.5 Hz, 1H), 6.99 (d, J = 7.5 Hz, 1H), 6.92 (m,
2H), 6.50 (s, 2H), 4.55 (m, 1H), 3.89 (s, 2H), 3.59 (m, 1H), 3.46
(m, 1H), 3.32 (m, 1H), 3.22 (m, 2H), 3.10 (m, 1H), 2.93 (t, J = 8 Hz,
2H), 2.83 (m, 1H), 2.73 (t, J = 7.5 Hz, 2H), 2.15 (s, 3H); 13C NMR
(125 MHz, D2O) d 166.3, 163.8, 161.9, 158.3, 154.0, 145.2, 138.8,
130.9, 124.9, 115.8, 114.4, 110.7, 50.5, 50.4, 48.6, 48.1, 48.0, 40.4,
31.5, 29.4, 21.4; ESMS m/z = 386 (M+H)+. HRMS calcd 386.23561,
found: 386.23616 (M+H)+.
The general procedure for amide formation was carried out on 22
(143 mg, 0.258 mmol) and 3-fluorophenethylamine (40
lL,
0.3 mmol) using TEA (43 L, 0.3 mmol, EDC (50 mg, 0.26 mmol),
l
and HOBt (35 mg, 0.26 mmol). The crude product was purified using
flash column chromatography (silica gel, EtOAc/hexanes, 2:1) to af-
ford 23 as a white solid (35 mg, 0.05 mmol, 20%). 1H NMR (500 MHz,
CDCl3) d 7.44 (m, 1H), 7.23 (m, 6H), 6.95 (m, 1H), 6.89 (m, 2H), 6.52
(m, 1H), 5.14 (s, 2H), 3.87 (m, 1H), 3.67–3.36 (m, 6H), 3.13 (m, 1H),
2.99 (m, 1H), 2.79 (m, 2H), 2.69 (m, 1H), 2.54 (m, 2H), 2.30 (m, 3H),
1.45 (s, 9H), 1.40 (s, 9H); 13C NMR (125 MHz, CDCl3) d 169.5, 164.2,
157.2, 154.3, 149.0, 140.0, 130.4, 128.4, 127.0, 126.9, 124.6, 120.0,
117.5, 115.9, 113.8, 83.7, 81.6, 80.4, 79.7, 68.9, (50.8 + 50.5), 50.2,
(49.3 + 49.0), (42.7 + 42.2), 39.8, 35.6, 34.5, 28.7, 28.4, 21.4; ESMS
m/z = 677 (M+H)+.
5.23. tert-Butyl 3-(tert-butoxycarbonyl(2-(3-fluorophe
nethoxy)ethyl)amino)-4-((6-(tert-butoxycarbonylamino)-4-
methylpyridin-2-yl)methyl)pyrrolidine-1-carboxylate (21)
5.27. tert-Butyl 3-((6-(tert-butoxycarbonylamino)-4-
methylpyridin-2-yl)methyl)-4-(2-(3-fluorophenethylamino)-2-
oxoethoxy)pyrrolidine-1-carboxylate (24)
The general reductive amination procedure was carried out using
amine 14 (69 mg, 0.17 mmol) and aldehyde 18 (27 mg, 0.15 mmol)
with NaHB(OAc)3 (42 mg, 0.2 mmol). DIEA (100 lL, 0.7 mmol) and
Boc2O (50 mg, 0.23 mmol) were added to the crude product, and
the solution was stirred another 2 h. The mixture was poured into
NaHCO3 (aq), and the product was extracted with CH2Cl2
(3 ꢂ 20 mL). The organic layers were combined, dried over anhy-
drous Na2SO4, and concentrated in vacuo. The crude product was
purified using flash column chromatography (silica gel, EtOAc/hex-
anes, 1:3) to afford 21 as a white solid (81 mg, 0.12 mmol, 80%). 1H
NMR (500 MHz, CDCl3) d 7.60 (m, 1H), 7.31–7.23 (m, 2H), 6.93 (m,
2H), 6.61 (m, 1H), 3.64–3.48 (m, 5H), 3.42–3.04 (m, 5H), 2.87 (m,
4H), 2.62 (m, 1H), 2.44 (m, 1H), 2.29 (m, 3H), 1.52 (s, 9H), 1.44 (s,
18H); ESMS m/z = 673 (M+H)+, 695 (M+Na)+.
The general procedure for the removal of benzyl groups was car-
ried out on 23 (34 mg, 0.05 mmol). The crude product was purified
using flash column chromatography (silica gel, EtOAc/hexanes, 2:1)
to afford 24 as a yellow oil (21 mg, 0.036 mmol, 72%). 1H NMR
(500 MHz, CDCl3) d 7.61 (m, 1H), 7.05 (s, 1H), 6.98 (m, 1H), 6.89 (m,
2H), 6.71 (m, 1H), 6.53 (s, 1H), 4.01 (m, 1H), 3.84 (m, 2H), 3.65–3.46
(m, 4H), 3.29 (m, 1H), 3.07 (m, H), 2.84 (m, 2H), 2.71 (m, 1H), 2.57
(m, 2H), 2.30 (s, 3H), 1.52 (s, 9H), 1.46 (s, 9H); ESMS m/z = 587 (M+H)+.
5.28. 2-(4-((6-Amino-4-methylpyridin-2-yl)-methyl)-
pyrrolidin-3-yloxy)-N-(3-fluorophenethyl)-acetamide (5)
5.24. 6-((4-(2-(3-Fluorophenethoxy)ethylamino)pyrrolidin-3-
yl)methyl)-4-methyl-pyridin-2-amine (4)
The general procedure for the removal of Boc groups was car-
ried out on 24 (0.036 mmol) to give 5 as a greasy solid (5.1 mg,
0.011 mmol, 31%). 1H NMR (500 MHz, D2O) d 7.14 (q, J = 7 Hz,
1H), 6.95 (d, J = 7.5 Hz, 1H), 6.90 (d, J = 10 Hz, 1H), 6.75 (m, 1H),
6.55 (s, 1H), 6.26 (s, 1H), 4.00 (m, 1H), 3.86 (m, 1H), 3.70 (m,
1H), 3.42 (m, 6H), 3.13 (m, 1H), 3.01 (m, 1H), 2.75–2.58 (m, 3H),
2.16 (s, 3H); ESMS m/z = 387 (M+H)+. HRMS calcd 387.21963,
found: 387.22094 (M+H)+.
The general procedure for the removal of Boc groups was car-
ried out on 21 (81 mg, 0.12 mmol) to give 4 (54 mg, 0.11 mmol,
92%) as a white trihydrochloride salt. 1H NMR (500 MHz, D2O) d
7.12 (m, 1H), 6.97–6.91 (m, 2H), 6.73 (m, 1H), 6.61–6.53 (m, 2H),
4.24 (m, 1H), 4.12 (m, 1H), 3.85–3.45 (m, 6H), 3.34–3.17 (m, 3H),

G. R. Lawton et al. / Bioorg. Med. Chem. 17 (2009) 2371–2380
2379
5.29. tert-Butyl 3-((6-(benzyl(tert-butoxycarbonyl)-amino)-4-
methylpyridin-2-yl)methyl)-4-(2-(benzylamino)ethoxy)-
pyrrolidine-1-carboxylate (25)
mationofnitricoxidewasmonitoredusinga hemoglobin captureas-
say as described previously. Briefly, a solution of nNOS or eNOS
containing 10
lM
L
-arginine, 1.6 mM CaCl2, 11.6 g /mL calmodulin,
l
100 M DTT, 100
l
l
M NADPH, 6.5 M H4B, 125 lg/mL oxyhemoglo-
l
The general reductive amination procedure was carried out
using aldehyde 12 (59 mg, 0.11 mmol), benzylamine (36 lL,
bin, and varying concentrations of inhibitor in 100 mM Hepes (pH
7.4) was monitored at 30 °C. For the determination of inhibition of
iNOS, no additional Ca2+ or calmodulin were added. The assay was
initiated by the addition of enzyme, and the absorption of UV light
at 400 nm was recorded over 1 min. As NO was evolved and coordi-
nated to the hemoglobin, the absorption at 400 nm increased, pro-
ducing a value for the enzyme velocity under these conditions. A
value for the initial rate was obtained when no inhibitor was added
0.33 mmol), and NaHB(OAc)3 (32 mg, 0.15 mmol). The crude prod-
uct was purified using flash column chromatography (silica gel,
EtOAc) to afford 25 as a white solid (63 mg, 0.1 mmol, 91%). 1H
NMR (500 MHz, CDCl3) d 7.41–7.18 (m, 11H), 6.64 (s, 1H), 5.17
(s, 2H), 3.79 (s, 2H), 3.65–3.52 (m, 3H), 3.44–3.35 (m, 2H), 3.28
(m, 1H), 3.19 (m, 1H), 3.09 (m, 1H), 2.89 (m, 1H), 2.74–2.67 (m,
2H), 2.57 (m, 1H), 2.25 (s, 3H), 1.45 (s, 9H), 1.41 (s, 9H); 13C NMR
(125 MHz, CDCl3) d 157.9, 155.0, 154.7, 154.1, 148.8, 140.6,
140.1, 128.6, 128.3, 127.3, 127.2, 126.8, 126.7, 120.2, 117.3, 81.4,
79.7, 79.4, 78.8, (68.9 + 68.8), 54.0, (51.1 + 50.6), 50.2, 49.6, (49.0,
(43.0 + 42.4), 34.7, 28.8, 28.5, 21.4; ESMS m/z = 631 (M+H)+.
(v0). The velocity of the enzyme (v) was then determined in the pres-
ence of varying concentrations of inhibitor, until a concentration of
inhibitor that reduced the enzyme velocity to half its initial value
(
v
/
v0 ꢁ 0.5) was discovered. Concentrations of inhibitor above and
belowthis value were tested and a graph of v/ 0 versus inhibitorcon-
v
centration ([I]) was plotted. Extrapolation of this graph allowed the
determination of an IC50 value. Experiments were repeated at least
three times or until graphs had R2 > 0.95. The Ki value can be esti-
mated from the IC50 value if the Km for the substrate is known, using
5.30. tert-Butyl 3-(2-aminoethoxy)-4-((6-(tert-butoxycarbonyl-
amino)-4-methylpyridin-2-yl)methyl)-pyrrolidine-1-
carboxylate (26)
the equations below. The Km values used were: 1.3
8.3 M (iNOS) and 1.7 M (eNOS):
lM (nNOS),
The general procedure for the removal of benzyl groups was
carried out on 25 (63 mg, 0.10 mmol). The crude product was puri-
fied using flash column chromatography (silica gel, EtOAc/MeOH,
9:1) to afford 26 as a white solid (13 mg, 0.03 mmol, 30%). 1H
NMR (500 MHz, CDCl3) d 7.55 (s, 1H), 7.20 (s, 1H), 6.58 (s, 1H),
3.73 (m, 1H), 3.58–3.20 (m, 6H), 3.08 (m, 1H), 2.82 (m, 2H), 2.61
(m, 1H), 2.46 (m, 1H), 2.22 (s, 3H), 1.45 (s, 9H), 1.39 (s, 9H); ESMS
m/z = 451 (M+H)+.
l
l
% inhibition ¼ 100½Iꢃ=ð½Iꢃ þ Kif1 þ ½Sꢃ=KmgÞ
Ki ¼ IC50=ð1 þ ½Sꢃ=Km
Þ
5.34. In vivo brain uptake assay
1 and 2 were administered at 3.7 mg/kg to naive mice via ip
injection as a 1 mM solution in phosphate buffered saline (PBS).
At 5, 10, and 20 min after administration, mice were sacrificed,
and blood was drawn by intracardiac puncture. The mice were per-
fused with PBS, and the brains were harvested.
Plasma and brain concentrations of 1 and 2 were quantified by
liquid chromatography-tandem mass spectrometry after sample
preparation by solid phase extraction. In brief, to an aliquot of plas-
5.31. N-(2-(4-((6-Amino-4-methylpyridin-2-
yl)methyl)pyrrolidin-3-yloxy)ethyl)-2-(3-
fluorophenyl)acetamide (27)
The general procedure for amide formation was carried out on
26 (13 mg, 0.03 mmol) and 3-fluorophenylacetic acid (5 mg,
0.03 mmol) using EDC (6 mg, 0.03 mmol), HOBt (5 mg, 0.03 mmol),
ma (100
standard. The mixture was diluted with 2% acetic acid (500
Brains were weighed and homogenized in 2% formic acid/acetoni-
trile (2:1, 700 L) and 3 or 4 (10 L of 10 M) was added. The
lL) was added 3 or 4 (10
lL of 10
lM stock) as an internal
l
L).
and TEA (3 lL, 0.03 mmol). The crude product was purified using
flash column chromatography (silica gel, EtOAc/hexanes, 3:1) to af-
ford 27 as a white solid (9 mg, 0.015 mmol, 51%). 1H NMR
(500 MHz, CDCl3) d 7.63 (m, 1H), 7.23 (m, 2H), 7.01–6.88 (m,
3H), 6.58 (s, 1H), 6.42 (s, 0.5H), 6.30 (s, 0.5H), 3.71 (m, 1H), 3.66–
3.52 (m, 4H), 3.50–3.34 (m, 4H), 3.24 (m, 1H), 3.06 (m, 1H), 2.70
(m, 1H), 2.49 (m, 2H), 2.31 (s, 3H), 1.53 (s, 9H), 1.46 (s, 9H); ESMS
m/z = 587 (M+H)+, 609 (M+Na)+.
l
l
l
homogenate was transferred to a centrifuge tube, and the homog-
enizer was washed with 2% formic acid in water/acetonitrile (2:1,
300 lL), which was also added to the centrifuge tube. The samples
were centrifuged (10 krpm, 12 min), and the supernatant was
transferred to a separate tube. Plasma and brain samples were
loaded onto a preconditioned Oasis MCX micro-elution plate. The
well was washed with 2% formic acid (500
(500 L), and the compound was eluted with 5% NH4OH in meth-
anol (2 ꢂ 400 L). The solvent was removed under a stream of
nitrogen, and the residue was reconstituted in LCMS/MS mobile
phase (200 L). An aliquot (20 L) was analyzed by LCMS and com-
pared to standard curves. Standard curves for each compound in
each matrix were linear between 0.005 and 5
g/mL with R2 > 0.99.
Samples containing 2 and 4 were eluted isocratically from a
Phenomenex MAX column (50 ꢂ 2.0 mm, Phenomenex, Torrance,
CA) with a mobile phase consisting of water and methanol
lL) and methanol
5.32. N-(2-(4-((6-Amino-4-methylpyridin-2-yl)-
methyl)pyrrolidin-3-yloxy)ethyl)-2-(3-fluorophenyl)acetamide
(6)
l
l
l
l
The general procedure for the removal of Boc groups was car-
ried out on 27 (9 mg, 0.015 mmol) to give 6 as a greasy solid
(3 mg, 0.0065 mmol, 44%). 1H NMR (500 MHz, D2O) d 7.27 (s,
1H), 7.10 (q, J = 7.5 Hz, 1H), 6.97 (d, J = 7.5, 1H), 6.92 (d, J = 8Hz,
1H), 6.72 (m, 1H), 6.52 (s, 1H), 6.16 (s, 1H), 3.86 (m, 1H), 3.67
(m, 1H), 3.59 (m, 1H), 3.52 (m, 1H), 3.50–3.39 (m, 2H), 3.29 (m,
3H), 3.08 (dd, J = 3, 13 Hz, 1H), 2.89 (t, J = 11.5 Hz, 1H), 2.50–2.37
(m, 3H), 2.13 (s, 3H); ESMS m/z = 387 (M+H)+. HRMS calcd
387.21963, found: 387.22097 (M+H)+.
l
(80:20) containing 0.1% TFA at a flow rate of 250 lL/ min. The tan-
dem mass spectrometer was operated with its electrospray source
in the positive ionization mode. The mass to charge ratios of the
precursor-to-product ion reactions monitored were 373.3?123.1
for 2 and 4. The retention time of 2 was 2.38 min; that of 4 was
4.74 min. Samples containing 1 and 4 were eluted isocratically
from a Phenomenex MAX column (50 ꢂ 2.0 mm, Phenomenex, Tor-
rance, CA) with a mobile phase consisting of water and methanol
5.33. In vitro enzyme assay
The NOS isoforms used were recombinant enzymes overexpres-
sed in Escherichia coli. Murine macrophage iNOS,14 rat nNOS,15 and
bovineeNOS16 were overexpressed and isolated as reported. Thefor-
(75:25) containing 0.1% TFA at a flow rate of 125 lL/min. The tan-

2380
G. R. Lawton et al. / Bioorg. Med. Chem. 17 (2009) 2371–2380
dem mass spectrometer was operated with its electrospray source
in the positive ionization mode. The mass to charge ratios of the
precursor-to-product ion reactions monitored were 372.3?123.2
for 1. The retention time of 1 was 2.46 min; that of 4 was 4.74 min.
References and notes
1. Zhang, L.; Dawson, V. L.; Dawson, T. M. Pharmacol. Ther. 2006, 109, 33.
2. Sims, N. R.; Anderson, M. F. Neurochem. Int. 2002, 40, 511.
3. Zicha, J.; Pechanova, O.; Dobesova, Z.; Kunes, J. Clin. Sci. 2003, 105, 483.
4. Wilcock, D. M.; Lewis, M. R.; Van Nostrand, W. E.; Davis, J.; Previti, M.
L.; Gharkholonarehe, N.; Vitek, M. P.; Colton, C. A. J. Neurosci. 2008, 28,
1537.
Acknowledgments
5. 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., in press.
6. Adinolfi, M.; Beck, Susan E.; Haddad, Susan A.; Seller, Mary J. Nature 1976, 259,
140.
7. Seelig, A. J. Mol. Neurosci. 2007, 33, 32.
8. Ji, H.; Stanton, B. Z.; Igarashi, J.; Li, H.; Martásek, P.; Roman, L. J.; Poulos, T. L.;
Silverman, R. B. J. Am. Chem. Soc. 2008, 130, 3900.
9. Lawton, G. R.; Ji, H.; Silverman, R. B. Tetrahedron Lett. 2006, 47, 6113.
10. Travis, B. R.; Sivakumar, M.; Hollist, G. O.; Borhan, B. Org. Lett. 2003, 5, 1031.
11. Hevel, J. M.; Marletta, M. A. Method Enzymol. 1994, 233, 250.
12. Dagnall, S. P.; Hague, D. N.; McAdam, M. E. J. Chem. Soc., Perkin Trans. 2 1984,
1111.
13. Molecular properties were calculated using Molinspiration software
(www.molinspiration.com) on the expected form at pH 7.4.
14. Hevel, J. M.; White, K. A.; Marletta, M. A. J. Biol. Chem. 1991, 266, 22789.
15. 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.
The authors are grateful to the National Institutes of Health for
financial support to R.B.S. (GM49725) and Dr. Bettie Sue Masters
(GM52419, with whose laboratory P.M. and L.J.R. are affiliated),
as well as the Robert A. Welch Foundation to Dr. Bettie Sue Masters
(AQ1192). P.M. is supported by Grants 0021620806 and 1M0520
from MSMT of the Czech Republic. The authors wish to thank Dr.
Michael Avram and Lynn Luong of the Northwestern Clinical Phar-
macology Core Facility and the Pharmaceutical Chemistry Transla-
tional Resource of the Center for Drug Discovery and Chemical
Biology for carrying out the SPE and LCMS experiments.
Supplementary data
16. Martasek, P.; Liu, Q.; Liu, J. W.; Roman, L. J.; Gross, S. S.; Sessa, W. C.; Masters, B.
S. S. Biochem. Biophys. Res. Commun. 1996, 219, 359.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.02.017.