Potent and selective neuronal nitric oxide synthase inhibitors with improved cellular permeability

Article abstract of DOI:10.1016/j.bmcl.2009.11.086

Recently, a series of potent and selective neuronal nitric oxide synthase inhibitors containing two basic nitrogen atoms was reported (Ji, H.; Stanton, B. Z.; Igarashi, J.; Li, H.; Martasek, P.; Roman, L. J.; Poulos, T. L.; Silverman, R. B. J. Am. Chem. Soc. 2008, 130, 3900-3914). In an effort to improve their bioavailability, three compounds (2a-c) were designed with electron-withdrawing groups near one of the basic nitrogen atoms to lower its pK_a. Inhibition studies with these compounds showed that two of them not only retained most of the potency and selectivity of the best analogue of the earlier series, but also showed improved membrane permeability based on data from a cell-based assay.

Full text of DOI:10.1016/j.bmcl.2009.11.086

Bioorganic & Medicinal Chemistry Letters 20 (2010) 554–557
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Potent and selective neuronal nitric oxide synthase inhibitors
with improved cellular permeability
Fengtian Xue a,b, Jianguo Fang a,b, William W. Lewis a,b, Pavel Martásek c,d, Linda J. Roman c,
Richard B. Silverman a,b,
*
a Department of Chemistry, Center for Molecular Innovation and Drug Discovery, Chemistry of Life Processes Institute, Northwestern University, 2145 Sheridan Road, Evanston,
IL 60208-3113, USA
b Department of Biochemistry, Molecular Biology, and Cell Biology, Center for Molecular Innovation and Drug Discovery, Chemistry of Life Processes Institute, Northwestern
University, 2145 Sheridan Road, Evanston, IL 60208-3113, USA
c Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX, USA
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:
Recently, a series of potent and selective neuronal nitric oxide synthase inhibitors containing two basic
nitrogen atoms was reported (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, 3900–3914). In an effort to improve their bioavailability,
three compounds (2a–c) were designed with electron-withdrawing groups near one of the basic nitrogen
atoms to lower its pKa. Inhibition studies with these compounds showed that two of them not only
retained most of the potency and selectivity of the best analogue of the earlier series, but also showed
improved membrane permeability based on data from a cell-based assay.
Received 31 October 2009
Revised 17 November 2009
Accepted 18 November 2009
Available online 22 November 2009
Keywords:
Neuronal nitric oxide synthase
Selective inhibitors
Increased membrane permeability
pKa
Ó 2009 Elsevier Ltd. All rights reserved.
Neuronal nitric oxide synthase (nNOS) catalyzes the oxidation
-arginine to -argi-
-citrulline via the intermediate NG-hydroxy-
of nNOS, designed inhibitors should not interfere with the activity of
either eNOS or iNOS.15 The second challenge for drugs that target
nNOS, which primarily resides in the CNS, is the ability of the de-
signed compounds to penetrate the blood brain barrier (BBB). The
BBB is a unique barrier formed by brain capillary endothelial cells
that are linked via tight junctions. Accordingly, drug molecules need
to cross the endothelial cell monolayer via the transcellular pathway
to exhibit a pharmaceutical effect in the CNS.
Our laboratory has previously reported the structure-based de-
sign, synthesis, and biological evaluation of a series of pyrrolidine-
based nNOS selective inhibitors.17–19 Among them, 1 demonstrates
great potency (Ki = 15 nM) and very high selectivity for nNOS over
eNOS (2100-fold) and iNOS (630-fold). These data indicated that 1
is a promising candidate to be considered as a lead compound for
neurodegenerative drugs.19
of
L
L
L
nine in the central nervous system (CNS), generating the highly
reactive free radical neurotransmitter nitric oxide (NO).1–3 Signifi-
cant research has shown that overproduction of NO from nNOS is
implicated in various neurodegenerative diseases,4,5 including Par-
kinson’s,6–8 Alzheimer’s,9 Huntington’s10 diseases, and stroke.11
Since nNOS plays a critical role in the production of neuronal NO,
it is considered to be a promising neurodegenerative therapeutic
target.12–14
Although intense research efforts have been devoted to the de-
sign and development of small molecules to inhibit the activity of
nNOS,15 none has been reported to enter clinical trials for neurode-
generative disease. There are two major challenges, in addition to
high potency, involved in developing novel nNOS inhibitors. The first
is the selectivity of inhibitors for nNOS over its closely related iso-
zymes, endothelial NOS (eNOS) and inducible NOS (iNOS). eNOS is
a key regulator of blood pressure and vascular tissues. It has been
shown that inhibition of eNOS activity leads to high blood pressure
and vascular effects.16 On the other hand, iNOS is the enzyme
responsible for the generation of cytotoxic NO, playing an important
role in the immune system.16 Therefore, when blocking the activity
H
N
H
N
F
H2N
N
O
( )-1
Although 1 exhibits high in vitro potency and selectivity, its
therapeutic utility is limited by poor BBB permeability.19 We spec-
ulated that the two positive charges of 1 at physiological pH, de-
* Corresponding author.
E-mail address: r-silverman@northwestern.edu (R.B. Silverman).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.086

F. Xue et al. / Bioorg. Med. Chem. Lett. 20 (2010) 554–557
555
H
N
H
N
H2N
F
N
O
O
N
F
( )-2a
H
N
H
N
F
F
H
N
H
N
F
F
H2N
N
O
H2N
O
( )-2b
( )-2c
Figure 1. Chemical structures of 2a–c.
nitrogen atom by induction, would decrease the cationic character
of inhibitors 2a–c compared to 1, and therefore, improve mem-
brane permeability of the inhibitors.
Table 1
Physiochemical properties of inhibitors 1 and 2a–c
a
Compound
Mw
pKa
As shown in Scheme 1, the synthesis of 3b began with 3-fluoro-
styrene (4). Bromofluorination of 4 using NBS and Et3Nꢀ3HF gener-
ated 1-(2-bromo-1-fluoroethyl)-3-fluorobenzene (5) in high
yields.22 Next, 5 was converted to azide (6) using NaN3 in DMSO
at 65 °C in good yields.23 Finally, catalytic hydrogenation of 6 in
a mixture of EtOH and 1 N HCl (2:1) provided 3b as a HCl salt in
excellent yields.
1
372
388
390
408
8.94
8.12
7.32
5.56
2a
2b
2c
a
Lipophilicity data were calculated with ACD/Log D version 7.0, Advanced
Chemistry Development, Inc., Toronto, Canada.
2,2-Difluoro-2-(3-fluorophenyl)ethanamine (3c) was synthe-
sized as shown in Scheme 2. 2-Bromo-30-fluoroacetophenone (7)
was treated with diethylaminosulfur trifluoride (DAST) to give 1-
(2-bromo-1,1-difluoroethyl)-3-fluorobenzene (8) in good yields.24
Next, 8 was allowed to react with NaN3 in DMSO at 110 °C to give
azide (9) in good yields. Finally, 9 was subjected to catalytic hydro-
genation under acidic conditions to give 3c as an HCl salt in excel-
lent yields.
With 3a–c in hand (3a is commercially available), we finished
the syntheses of inhibitors 2a–c using a three-step procedure
(Scheme 3). First, reductive amination between 1019 and 3a–c
using NaHB(OAc)3 gave 11a–c in modest yields. To simplify the
purification process, the resulting secondary amines (11a–c) were
protected with (Boc)2O to give 12a–c. Finally, the Bn-protecting
group and the three Boc-protecting groups were removed at the
same time in a mixture of EtOH and 12 N HCl (2:1) under high
pressure catalytic hydrogenation conditions to provide 2a–c as
HCl salts in good yields.
rived from the two secondary amino groups, dramatically impaired
the ability of 1 to penetrate the highly lipophilic BBB by passive
diffusion.
The low membrane permeability of 1 represents a major chal-
lenge for further investigation of this compound. To circumvent this
problem, different strategies have been applied in an attempt to im-
prove the bioavailability of 1.19–21 In this Letter, we describe the de-
sign, synthesis, andbiologicalevaluationof anewseriesofanalogues
of 1 with electron-withdrawing groups (Fig. 1) to lower the pKa of
one of the basic nitrogens (2a–c), and demonstrate the possibility
of using them as inhibitors with improved bioavailability.
In the current design, different electron-withdrawing groups,
including ether (2a), monofluoro methylene (2b), and difluoro
methylene (2c), were introduced at a vicinal position to the amino
group in the lipophilic tail of 1. Chemical structures of these new
inhibitors are closely related to that of 1, while the predicted pKa
values of the protonated vicinal nitrogen atoms are significantly
lower than the corresponding protonated nitrogen atom in 1
(Table 1). We anticipate that the additional electron-withdrawing
groups, which partially remove the positive charge from the vicinal
Inhibitors 2a–c were evaluated for in vitro inhibition against
three isozymes of NOS: rat nNOS, bovine eNOS, and murine iNOS
using known methods (Table 2).25 Inhibitor 2a, with an electroneg-
a
c
b
F
H2N
F
Br
F
N3
F
F
F
F
HCl
4
5
6
3b
Scheme 1. Reagents and conditions: (a) NBS, Et3Nꢀ3HF, CH2Cl2, 0 °C to rt, 12 h, 85%; (b) NaN3, DMSO, 65 °C, 4 h, 85%; (c) Pd(OH)2/C, H2, EtOH/1 N HCl (2:1), rt, 24 h, 98%.
b
a
c
Br
F
N3
F
H2N
F
Br
F
HCl
F
F
F
F
F
F
O
7
8
9
3c
Scheme 2. Reagents and conditions: (a) DAST (neat), rt, 7 days, 70%; (b) NaN3, DMSO, 110 °C, 1 h, 88%; (c) Pd(OH)2/C, H2, EtOH/1 N HCl (2:1), rt, 24 h, 91%.

556
F. Xue et al. / Bioorg. Med. Chem. Lett. 20 (2010) 554–557
Boc
N
Boc
N
a
b
H
N
Boc
O
Boc
Boc
N
Bn
N
N
Bn
N
O
O
O
R
( )-10
( )-11a-c
Boc
N
H
N
Boc
N
H
N
c
3 HCl
H2N
N
N
Bn
N
O
R
R
( )-2a-c
( )-12a-c
R =
F
F
O
F
F
F
F
a
b
c
Scheme 3. Reagents and conditions: (a) (i) 3a–c, THF, rt, 5 min; (ii) NaHB(OAc)3, rt, 3 h; (b) (Boc)2O, Et3N, MeOH, rt, 12 h, 48–60% for two steps; (c) Pd(OH)2/C, H2, 2:1 EtOH/
HCl (12 N), rt, 500 psi, 40 h, 85–91%.
ative oxygen atom inserted into the lipophilic tail of 1, is almost
sixfold less potent for nNOS compared to the lead compound (1),
while the selectivity of this inhibitor for nNOS over eNOS and iNOS
decreases by 10-fold and threefold, respectively. There are two
likely reasons for these results. First, compound 2a is one atom
longer than 1, which probably makes it too long to fit snugly into
the active site of nNOS. Moreover, it has been shown previously
that the positively charged amine functionality in the lipophilic tail
plays an important role for tight binding of 1 to nNOS;19 therefore,
partial removal of the positive charge from this group may impair
the activity of inhibitors.19 Inhibitor 2b, with monofluoromethyl-
ene vicinal to one of the basic amino groups, shows very good po-
tency for nNOS and excellent selectivity for nNOS over eNOS and
iNOS. Given that the pKa of the vicinal amino group in 2b is lower
than that in 2a, partial removal of the positive charge on that ami-
no group cannot explain the lower potency of 2a. Finally, inhibitor
2c, with a difluoromethylene in the lipophilic tail of 1, exhibited a
2.2-fold drop in potency relative to 2b. The introduction of the
strongly electron-withdrawing difluoromethylene group essen-
tially completely removes the positive charge on the amino group
in the lipophilic tail at neutral pH, which decreases its ability for an
electrostatic interaction with the heme carboxylate. However,
inhibitor 2c still showed excellent selectivity for nNOS over eNOS
and iNOS.
Table 3
IC50 values of inhibitors in purified enzyme assay and cell-based assay
a
a
Compound IC50
M)
IC50(cell)
(lM)
IC50(1)
IC50(x)
/
IC50(1)(cell)
IC50(x)(cell)
/
(
l
1
2b
2c
0.13
0.31
0.70
9
10
19
—
0.42
0.19
—
0.9
0.47
a
The IC50 values represent at least duplicate measurements with standard
deviations of 10%.
the first ratio column. Compound 2b is 0.42 times as potent as 1;
compound 2c is 0.19 times as potent as 1. The second ratio column
compares IC50(cell) values for 2b and 2c relative to 1 in the cell-
based assay, which should relate to the relative ability of the com-
pounds to penetrate the cell membrane. Compound 2b is only 0.9
as potent as 1, which indicates that 2b crosses the cell membrane
2.1 times (0.9/0.42) better than 1. Compound 2c is 0.47 times as
potent as 1 in the cell-based assay, indicating that 2c crosses the
cell membrane 2.5 times (0.47/0.19) better than 1. This suggests
that the difluoromethylene electron-withdrawing group of 2c,
which decreases the pKa of the vicinal amino group compared to
the monofluoromethylene group of 2b and no electron-withdraw-
ing group of 1, improves cell permeability. On the other hand, the
absolute potency of inhibitors may be sacrificed because of the
importance of the positively charged amino group in binding to
nNOS.
The two best inhibitors (2b and 2c), together with the lead com-
pound (1), were tested for their potency in a cell-based assay.26
This assay can provide information about their membrane perme-
ability. The results are summarized in Table 3. The IC50 values for
inhibitors 2b and 2c are compared to the lead compound (1) in
In conclusion, a new series of potent and selective nNOS inhib-
itors (2a–2c) were designed and synthesized. Purified enzyme and
cell-based evaluation of these new inhibitors led to the discovery
of inhibitors 2b and 2c, which not only retain most of the activity
of the lead compound (1), but also have improved membrane
permeability.
Table 2
Kia values of inhibitors for rat nNOS, bovine eNOS, and murine iNOS
Compound
nNOS (lM)
eNOS (lM)
iNOS (lM)
Selectivityb
n/e
n/i
Acknowledgments
119
2a
2b
2c
0.015
0.086
0.036
0.080
31
21
36
62
9.5
18
13
2100
240
1000
780
630
210
360
650
The authors are grateful to the National Institutes of Health
(GM49725 to R.B.S. and GM52419 to Professor Bettie Sue Siler Mas-
ters, with whose laboratory P.M. and L.J.R. are associated) for finan-
cial support of this research. B.S.S.M. also is grateful to the Welch
Foundation for a Robert A. Welch Distinguished Professorship in
52
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).

F. Xue et al. / Bioorg. Med. Chem. Lett. 20 (2010) 554–557
557
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