Potent and Selective Conformationally Restricted Neuronal Nitric Oxide Synthase Inhibitors

Article abstract of DOI:10.1021/jm030297m

Selective inhibition of the isoforms of nitric oxide synthase (NOS) in pathologically elevated synthesis of nitric oxide has great therapeutic potential. We previously reported nitroarginine-containing dipeptide amides (Huang, H.; Martasek, P.; Roman, L.

Full text of DOI:10.1021/jm030297m

J . Med. Chem. 2004, 47, 703-710
703
P oten t a n d Selective Con for m a tion a lly Restr icted Neu r on a l Nitr ic Oxid e
Syn th a se In h ibitor s
J os e´ A. G o´ mez-Vidal,§,⊥ Pavel Mart a´ sek,‡,† Linda J . Roman,‡,| and Richard B. Silverman*,§
Department of Chemistry, Department of Biochemistry, Molecular Biology, and Cell Biology, and the Drug Discovery Program,
Northwestern University, Evanston, Illinois 60208-3113, and Department of Biochemistry, The University of Texas Health
Science Center, San Antonio, Texas 78284-7760
Received J une 18, 2003
Selective inhibition of the isoforms of nitric oxide synthase (NOS) in pathologically elevated
synthesis of nitric oxide has great therapeutic potential. We previously reported nitroarginine-
containing dipeptide amides (Huang, H.; Martasek, P.; Roman, L. J .; Masters, B. S. S.;
Silverman, R. B. J . Med. Chem. 1999, 42, 3147) and some peptidomimetic analogues (Huang,
H.; Martasek, P.; Roman, L. J .; Silverman, R. B. J . Med. Chem. 2000, 43, 2938) as potent and
selective inhibitors of neuronal NOS (nNOS). Here we report conformationally restricted
NO
dipeptides derived from the dipeptide L-Arg
2
2
-L-Dbu-NH (8). The selectivities for nNOS over
endothelial NOS and inducible NOS of the most potent nNOS inhibitor (10a ) among these
compounds are comparable to that of the parent compound. An unsubstituted amide bond is
necessary for potency against nNOS. The stereochemistry of compound 10a was optimum for
potency and selectivity and thus provides the binding conformation of the parent compound
with nNOS.
In tr od u ction
cofactors. The constitutive forms do not bind Ca2+/
2
+
calmodulin at basal levels of Ca , while the inducible
form does. All three NOS isoforms are functional only
as tight homodimers through interactions between the
heme domains. NOS monomers consist of a reductase
domain that contains binding sites for NADPH, FAD,
and FMN and an oxygenase domain that has binding
sites for L-Arg, the heme prosthetic group, and tetrahy-
Nitric oxide (NO), a ubiquitous biological messenger
involved in a variety of physiological processes, acts as
a signal transducer but also exerts a variety of regula-
tory and cytostatic functions. In most instances, NO
mediates its biological effects by activating guanylate
cyclase and increasing cyclic GMP synthesis. However,
effects of nitric oxide that are independent of cyclic GMP
have also been described.1
drobiopterin (BH ). A zinc tetrathiolate center at the
4
Nitric oxide synthase (NOS; EC 1.14.13.39) isoforms2
dimer interface aids in dimer stabilization and forma-
4
-6
tion of the pterin binding site.
Dimerization of NOS
are homodimers that catalyze the oxidation of L-arginine
is required for fully coupled enzyme activity because the
flow of electrons during catalysis occurs from the
reductase domain of one monomer subunit to the
to L-citrulline and nitric oxide in an NADPH- and O2-
dependent process (reaction I).3
7
+
oxygenase domain of the other monomer.
L-Arg + 2O + 1.5NADPH + 2H f
2
The crystal structures of the oxygenase domains of
+
L-citrulline + •NO + 2H O + 1.5NADP (I)
8
2
the murine iNOS monomer, murine and human iNOS
9
-12
dimers,
and human and bovine endothelial NOS
1
3
There are at least three distinct isoforms of NOS. The
constitutive endothelial isoform (eNOS) is involved in
the regulation of smooth muscle relaxation and blood
pressure and in the inhibition of platelet aggregation.
A second constitutive isoform is the neuronal NOS
dimers indicate a high degree of structural similarity
within the catalytic center and dimer interface regions
between the NOS isoforms. They share only approxi-
mately 50% of the primary sequence homology, sug-
gesting that they may differ from each other in regu-
latory aspects. The N-terminal segment exhibits the
largest variation in sequence. While the N-terminal
segments are not essential for catalytic activity, they
are involved in cellular targeting and protein-protein
interactions. The ceiling heights directly above the iron
atom of the heme sites differ, suggesting the size of the
active sites decrease in the order nNOS > iNOS >
(nNOS), which is important for neurotransmission. A
third isozyme, inducible NOS (iNOS), is located in
activated macrophage cells and acts as a cytotoxic agent
in normal immune responses. All of the isoforms utilize
NADPH, FAD, FMN, tetrahydrobiopterin, and heme as
*
To whom correspondence should be addressed at the Department
of Chemistry. Phone: (847) 491-5653. Fax: (847) 491-7713. E-mail:
Agman@chem.northwestern.edu.
1
4
eNOS. The subtle differences among the substrate
sites of the NOS isozymes can be used to design a second
generation of selective NOS inhibitors with therapeutic
potential.
§
Northwestern University.
Carried out all experiments in this study.
The University of Texas Health Science Center.
Developed the eNOS overexpression system in E. coli and isolated
⊥
‡
†
and purified the eNOS.
|
The use of NOS inhibitors against pathologically
elevated synthesis of NO has great therapeutic poten-
Developed the nNOS overexpression system in E. coli and the
purification of the enzyme.
1
0.1021/jm030297m CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/31/2003

7
04 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3
G o´ mez-Vidal et al.
F igu r e 2. Compounds 9 and 10 are conformationally re-
stricted analogues of 8.
F igu r e 1. NOS inhibitors.
tial.15 NO overproduction by nNOS has been associated
Sch em e 1
1
6
17
with neurodegeneration during stroke, chronic head-
aches,18 and Alzheimer’s disease. Thus, nNOS repre-
19
2
0
sents a therapeutic target for inhibitors. Enhanced
formation of NO following the induction of iNOS ap-
2
1
pears to be important in arthritis, development of
colitis,22 and tissue damage and inflammation.23 Selec-
tive inhibition of one isoform over the others is essential
because the three isoforms of NOS have unique roles
in separate tissues. Selective inhibitors may also be
useful tools for investigating other biological functions
of NO.24
of 152 dipeptide amides containing nitroarginine was
screened for activity. Excellent inhibitory potency and
selectivity for nNOS over eNOS and iNOS was achieved
with dipeptide amides containing a basic amine side
chain. The most potent nNOS inhibitor among these
initial compounds was L-Arg
NO
2
-L-Dbu-NH (8; K ) 130
2
i
Two broad categories of NOS inhibitors have been
described: (a) substrate analogues and heme ligands
and (b) pterin antagonists. Analogues of the substrate
nM, Figure 2), which also displayed selectivity over
eNOS (>1500-fold) and iNOS (192-fold).
The side chain amino group of 8 binds to the enzyme
via a hydrogen bond to a water molecule, which inter-
ω
L-arginine include N-alkyl-L-arginines (1), N -nitro-L-
5
37
arginine (2), and N -(imino-3-butenyl)-L-ornithine (3)
acts with the active-site tetrahydrobiopterin. Because
(
Figure 1). Most of these have minimal selectivity among
of free rotation in the aminoethyl side chain of L-Dbu,
there are many conformations that are not ideally suited
for this interaction. If the ideal conformation were
incorporated, greater potency and selectivity would be
attained.
For these reasons, 8 has been used as a lead com-
pound for the development of more potent and selective
conformationally restricted dipeptide analogues. By this
approach, a more optimal orientation of the amino group
may be determined for better activity and selectivity.
To that end, we have synthesized 4-aminoproline ana-
logues 9 and 10. The synthesis of these compounds was
implemented on a solid phase, and the compounds were
screened for activity against nNOS and iNOS. The most
potent compounds were purified by HPLC, and the Ki
values were determined against all three isozymes.
2
5
26
the isozymes, except for N-propyl-L-arginine and 3,
which are highly selective inhibitors for nNOS over
iNOS and eNOS. There also are numerous non amino
acid based NOS inhibitors, for example, (+)-cis-5-pentyl-
4
-(trifluoromethyl)pyrrolidin-2-imine (5), N-(3-(amino-
methyl)phenyl)acetamidine (6), and N-(3-(aminomethyl)-
benzyl)acetamidine (7) (Figure 1). The iminopyrrolidine
5
belongs to a large group of cyclic amidine type
2
7
inhibitors of NOS. These non amino acid inhibitors
have significantly improved selectivity. Arylamidine 6
is a highly potent and selective nNOS inhibitor (nNOS,
IC50 ) 11 nM; eNOS, IC50 ) 1100 nM; iNOS, IC50 )
2
8
4
80 nM), and arylamidine 7 has been characterized
2
9
as a selective iNOS inhibitor. In some cases, the
selectivity is as high as 5000-fold (5) and 900-fold (7),
in favor of iNOS over eNOS. However, such high
selectivity has not normally been observed for the
inhibition of nNOS over eNOS (the above few examples
are exceptions). Recently, allosteric inhibitors of iNOS
dimerization have been described.30
Ch em istr y
cis- and trans-4-Amino-L- and -D-proline derivatives
were designed as constrained mimics to obtain confor-
mationally restricted dipeptide amides derived from the
dipeptide amide 8. The 4-substituted proline precursors
used in solid-phase synthesis were synthesized in 80-
95% yield from the corresponding esters using LiOH in
2
(S)-Alkyl-L-isothiocitrulline-containing dipeptides have
been reported as selective iNOS competitive inhibi-
tors.3
1,32
(S)-Methyl-L-isothiocitrulline (4) was identified
3
3
as a potent nNOS inhibitor (IC50 ) 60 nM). Thio-
imidates generally confer more NOS inhibitory potency
than their corresponding amidines. The combination of
a hydrophobic L-amino acid with the isothiourea deriva-
tive altered the inhibition pattern to give selective iNOS
inhibitors.
THF/H O (Scheme 1). The synthesis of the methyl esters
3
8
has already been reported.
The general solid-phase synthesis of L-ArgNO2-L-Dbu-
NH derivatives with cis- and trans-4-amino-L- and -D-
2
proline at the N-terminus is depicted in Scheme 2. The
3
9
Burgess methodology to deprotect Boc-amines on a
Our interest has been in the selective inhibition of
nNOS. Because 2 was reported to have more than 250-
fold selectivity in favor of nNOS over iNOS, we
synthesized and evaluated nitroarginine-containing
dipeptides to see if the incorporation of nitroarginine
Rink resin was found not to be selective for N-Boc-L-
NO
and -D-Arg
2
(15). This synthetic procedure, which uses
3
4
an excess of (TMS)OTf and 2,6-lutidine, afforded a pure
1
dipeptide derivative but with different H NMR and
ESMS data; the nitroarginine moiety was affected under
these conditions. It has been reported that nitroalkanes
are silylated by an excess of (TMS)OTf and lead to a
into a dipeptide could increase the inhibitory potency
and selectivity of L-nitroarginine.3
5,36
Hence, a library

Neuronal Nitric Oxide Synthase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3 705
Sch em e 2
migration of the trialkylsilyloxy group to give oximes.40
These compounds were not inhibitors of nNOS, and
their complete structure determination was not pur-
sued. The addition of only 2 equiv of (TMS)OTf or
TBSOTf (Scheme 2) instead of an excess of (TMS)OTf
is critical to avoid a rearrangement of the N-nitroguani-
dino fragment during the deprotection reaction. TBAF
is needed to cleave the intermediate silyl carbamate and
to obtain the free amine if TBSOTf is used.
applied to the N-Boc-L-Pro-loaded Rink resin, it allowed
for the synthesis of L-Pro-containing dipeptides in high
purity. This represents a new use of (TMS)I for the
removal of N-Boc on the TFA-sensitive Rink resin.
However, when (TMS)I was used to deprotect azide 19,
no product was obtained, showing the lack of selectivity
of this reagent. These results demonstrate that the azide
substituent in 19 is unstable during the deprotection
step. This reaction with the azide is not general because
it has been described previously with N-Boc deprotection
4
1
Analysis of the conditions of the reaction also showed
that a nucleophilic hydroxide is needed to cleave the
adduct intermediate during the Staudinger azide reduc-
4
4
in the presence of a secondary azide. A possible
explanation for this unusual reactivity has been offered
4
2
45
tion on a solid phase (16, Scheme 2). The use of lithium
hydroxide in THF afforded the final products (18a -h )
in high purity after resin cleavage. Azido compounds of
type 17 also were synthesized for SAR studies.
recently.
The reduction of azide 19 was initially accomplished
under Staudinger conditions using triphenylphosphine.
The dipeptide was obtained in 80% purity as shown in
1
1
The synthesis of dipeptide amides containing C-
terminal 4-aminoprolinamide isomers with the proline
nitrogen as part of the peptide bond also was carried
out (9 and 24, Scheme 3). After azide reduction the
resultant amine was protected as the trifluoroacetamide
its H NMR spectrum. Analysis of the H NMR spec-
trum of the final compound showed that the main
impurity was a derivative of the adduct intermediate
during the Staudinger reaction. Consequently, trimeth-
ylphosphine was used in place of triphenylphosphine,
but the purity did not improve. Triethylammonium tris-
(phenylthio)stannate has been described as a very
efficient reducing agent with a high chemoselectivity
2
1. This additional step was found to be necessary
because of the instability of azide 19 during N-Boc
deprotection using (TMS)OTf. A variety of conditions
with (TMS)OTf and other reagents were tried, but the
purity of the products obtained was always lower than
expected.
4
6
toward azides. This reagent has been applied success-
4
7
fully on a solid phase using Rink resin and other
4
8
resins, and it afforded amine 20 in high purity
(Scheme 3). Therefore, the azide group was reduced, and
the resulting amine 20 was protected as its trifluoro-
(
TMS)I has been used for N-Boc deprotection,43 but
it had not been applied to a solid-phase synthesis. When

7
06 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3
G o´ mez-Vidal et al.
Sch em e 3
acetamide49 using (trifluoroacetyl)benzotriazole50 prior
Biologica l Resu lts
to N-Boc deprotection (Scheme 3).
None of the 32 analogues containing the 4-aminopro-
line isomers at either the N- or C-terminus (17, 18, 9,
and 24) exhibited significant activity at 100 µM against
iNOS (IC50 > 100 µM). Some compounds have an IC50
value of 100 µM or less against nNOS. Only three
dipeptide amide analogues that contain a C-terminal
N-Boc deprotection of intermediate 21 was studied to
find the most mild conditions using (TMS)OTf. The
results showed that an excess of 2,6-lutidine is neces-
sary. The reaction did not work, and/or the resin was
affected using a 1:1 ratio of (TMS)OTf and 2,6-lutidine.
The best conditions were 4 equiv of (TMS)OTf and a
ratio of 1:1.5 of the reagents.
Following the coupling reaction to give 23, deprotec-
tion of 23 using lithium hydroxide in a THF/H2O
mixture and resin cleavage afforded 9a (and its isomers
4
-aminoprolinamide (9a , 9h , 24a ) exhibited some nNOS
inhibition at 100 µM (IC50 ≈ 100 µM). This supports the
importance of a nonsubstituted peptide bond for the
5
1
interaction with the enzyme.
The potency and selectivity of nitroarginine-contain-
ing dipeptides diminish dramatically when the peptide
bond hydrogen is substituted by a methyl group.51 This
suggests a possible hydrogen bond at that position,
which would be lost in compounds such as 9 and 24. It
is now known that, in fact, there is an important
hydrogen bond between the peptide NH and a water
9
b-h ) in high purity (Scheme 3). The corresponding
trifluoroacetamido-protected analogues 24a -h were
also obtained for SAR studies.
The synthesis of nonnatural dipeptide amides 10a -h
NO
containing L- or D-Arg
2
and isomers of 4-aminopro-
linamide with the 4-amino group in the peptide bond
was conducted as shown in Scheme 4. This third family
of dipeptide amides was synthesized using the 4-amino
group of resin-bound Boc-4-aminoproline (20) in the
coupling step. These structures are rigid analogues of
the lead compound but without a substituted peptide
bond nitrogen. Thirty-two isomers were synthesized
from Schemes 2 and 3, and eight isomers were synthe-
sized from Scheme 4.
3
7
molecule at the active site. The fact that dipeptide
analogue 24a , having a protected 4-amino substituent,
has an activity similar to that of the nonprotected 9a
suggests that this amino group is not interacting with
the enzyme in this series of compounds.
Eleven dipeptide analogues (17a ,c,e, 18a -h ) that
contain 4-amino- or 4-azidoproline at the N-terminus
showed some nNOS inhibition at 100 µM (IC50 ) 50-

Neuronal Nitric Oxide Synthase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3 707
Sch em e 4
Ta ble 1. nNOS and iNOS Inhibition by the C-Terminal
Ta ble 2. IC50 and Ki Values for 10a and 10c
4
4
-Aminoprolinamide-Containing Dipeptides in Which the
-Amino Nitrogen Is in the Peptide Bond
IC50 (µM)
Ki (µM)
compd
nNOS
iNOS
eNOS
nNOS
iNOS
eNOS
activity at 100 µMa
% enzyme activity)
activity at 100 µMa
(% enzyme activity)
(
10a
0.64
9.6
65
441
880
760
0.10a
1.10
29
128
110
200
1
8
0c
199
25
compd
nNOS
iNOS
compd
nNOS
iNOS
0.13b
b
b
1
1
1
1
0a
0b
0c
0d
54b
50
49
10
95
78
94
10e
10f
10g
10h
80
78
52
83
97
96
96
97
a
Experimentally determined. b From ref 27.
c
65
nitro-L-argininyl-4-N-(4(R)-amino-L-prolinamide, 10a ) is
the most potent compound, having an IC50 value of 0.64
µM (Table 2). This compound represents the first
conformationally restricted analogue of 8 with a similar
Ki and selectivity over iNOS and eNOS. L-Arg
a
Enzyme residual activity at 100 µM inhibitor. b Enzyme
residual activity at 0.5 µM inhibitor. Enzyme residual activity
at 1 µM inhibitor.
c
NO
2
-
1
00 µM). L-trans-Pro4-NH
2
-L-ArgNO
-NH2 (18a ) is the most
2
containing dipeptide amides were more potent than the
potent compound of this second series with an IC50 value
slightly lower than 50 µM. The azido analogues showed
a decrease in inhibition, indicating either a steric
problem with the azido group or a loss in hydrogen
NO
NO
2
D-Arg
2
analogues. The diastereomer L-Arg
-4-N-(D-
4-NH
trans-Pro
2
-NH2 (also, nitro-L-argininyl-4-N-(4(S)-
amino-D-prolinamide, 10c) showed a greater selectivity
over iNOS although it was 15-fold less potent against
nNOS than 10a . No time dependence of inhibition was
observed, suggesting that these compounds do not act
as slow-binding inhibitors or irreversible inhibitors. In
general, L-trans-4-aminoproline-containing dipeptide
amides are the most potent compounds in each of the
three series.
bonding of the amino group. The stereochemistry of 18a
NO
is important for its activity. The introduction of D-Arg
2
instead of L-ArgNO
and the inversion of configuration
2
at the 4-aminoproline CR position in 18a caused a
decrease in potency.
A third family of dipeptides has been synthesized
(
Scheme 4) using the 4-amino group of the 4-aminopro-
line to form the dipeptide bond. These structures are
rigid analogues of the lead compound and without a
substituted peptide bond. Some of these compounds
were the most potent compounds of the three series
Con clu sion s
It is apparent from the results described here that
the stereochemistry at each of the three stereocenters
in 10a strongly affects the potency and selectivity
displayed with nNOS over iNOS (290-fold) and eNOS
(1280-fold). The stereochemistry of 10a suggests a
(Table 1).
Trans derivatives are more potent than cis derivatives
NO
4-NH
2
in this series. L-Arg
2
-4-N-(L-trans-Pro
-NH2 (or

7
08 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3
G o´ mez-Vidal et al.
conformation of 8 that binds effectively with nNOS.
From the crystal structures of 8 and 10a bound to both
nNOS and eNOS, it is apparent that these two com-
pounds bind essentially identically in each of the
isozymes; however, L-nitroarginine binds differently to
these isozymes than does either 8 or 10a . This could
account for the increased selectivity of the dipeptide
amide analogues relative to L-nitroarginine.
2 h. A portion of the azidodipeptide-loaded resin (0.05 mmol)
was cleaved to obtain the corresponding azidodipeptides. The
rest of the resin (0.05 mmol) was suspended in DCM (3 mL),
and triphenylphosphine (0.15 mmol) was added. The suspen-
sion was shaken at room temperature overnight. The resin
was washed (5 mL each time) with MeOH (3×) and DCM (3×),
3
7
2
and a solution of LiOH (0.5 mmol) in THF/H O (5:1) was
added. The resin was shaken for 4 h at room temperature.
The resin was washed (5 mL each time) with THF (3×), H
2
O
(
3×), and MeOH (3×) and dried under vacuum for 2 h. The
resulting Rink amide resin loaded with the dipeptide (0.05
mmol) was swollen in DCM and filtered. A solution of TFA
Exp er im en ta l Section
Gen er a l Meth od s, Rea gen ts, a n d Ma ter ia ls. All re-
agents were purchased from Aldrich or Advanced ChemTech
and were used without further purification unless stated
otherwise. NADPH, calmodulin, and human ferrous hemoglo-
bin were obtained from Sigma Chemical Co. Tetrahydrobio-
3
and DCM (50:50, 3 mL) containing 1% Et SiH was added, and
the resulting mixture was shaken for 30 min at room temper-
ature. The suspension was filtered, and the resin was washed
(5 mL each time) with DCM/TFA (8:2, 3×). The solvent was
removed on the rotary evaporator, and the residue was treated
with DCM and concentrated to dryness. This last step was
repeated twice. The dipeptide was prepurified by solid-phase
extraction using a C-18 preconditioned column in water. The
dipeptide amide (100 µL injections of a solution of 5 mg of
dipeptide) was purified further by HPLC using a semiprepara-
tive column [Phenomenex, Luna, 250 × 10 mm, C18(2)] with
a precolumn [Phenomenex, Luna 50 × 10 mm, C18(2)] at a
flow rate of 4 mL/min at 214 nm. The elution program was as
follows: (A, water + 0.1% TFA; B, MeCN + 0.1% TFA) 5 min
using 1% B, then a gradient to 10% B over 15 min followed by
4
pterin (H B) was purchased from Alexis Biochemicals. HEPES,
DTT, and conventional organic solvents were purchased from
Fisher Scientific. Tetrahydrofuran was distilled under nitrogen
from sodium/benzophenone. Sodium sulfate was used to dry
organic layers during workup. Thin-layer chromatography was
carried out on E. Merck precoated silica gel 60 F254 plates. E.
Merck silica gel 60 (230-400 mesh) was used for flash
chromatography. An Orion Research model 701 pH meter with
a general combination electrode was used for pH measure-
ments. Combustion analyses were performed by Oneida Re-
1
search Laboratories, New York. H NMR spectra were recorded
5
min with 10% B, and then a gradient to 1% B over 5 min.
on a Gemini 2000, Mercury 400, or Inova 500 spectrometer
The total run time was 30 min in a Beckman HPLC instru-
ment (System Gold 125, UV detector 166). Fractions containing
the pure dipeptide were concentrated to dryness, and the
residue was lyophilized.
1
3
(
75.4, 100.6, or 125.7 MHz, for C NMR spectra) in CDCl
Chemical shifts are reported as values in parts per million
relative to the peak for TMS in CDCl . For samples run in
O, the HOD resonance was arbitrarily set at 4.80 ppm. Mass
spectra were obtained on a Micromass Quattro II spectrometer
ESI) or on a VG Instrument (VG70-250SE) high-resolution
3
.
3
D
2
Gen er a l Meth od for th e Syn th esis of D- a n d L-Ni-
tr oa r gin in yl-2-N-(cis- a n d tr a n s-4-a m in o-D- a n d -L-p r o-
lin a m id es) (Sch em e 3). Deprotected Rink amide resin
(
mass spectrometer. NOS assays were recorded on a Perkin-
Elmer Lambda 10 UV/vis spectrophotometer.
(prepared as above, 0.1 mmol) was suspended in DMF (1 mL).
DIEA (0.2 mmol) and a solution of the N-Boc-4-azidoproline
isomer (0.2 mmol) and HBTU (0.2 mmol) in DMF (4 mL) were
added sequentially. The resulting mixture was shaken at room
temperature for 6 h and filtered. The resin was washed (5 mL
each time) with DMF (3×), DCM (2×), and MeOH (3×). The
resulting resin-bound amino acid was dried under vacuum for
Tr iet h yla m m on iu m Tr is(p h en ylt h io)st a n n a t e. Cau-
tion! Stench. SnCl
THF by sonicating the suspension under nitrogen. Thiophenol
0.8 mmol, 8 equiv) and TEA (1 mmol, 10 equiv) were
2
(0.2 mmol, 2 equiv) was solubilized in dry
(
sequentially injected under nitrogen. The obtained yellow
solution containing particulate matter was drawn into a dry
syringe using a 0.45 µM filter on line. This solution was used
immediately.
2
h. Rink amide resin loaded with the azidoproline isomer (0.1
mmol) was suspended in dry THF under nitrogen. A yellow
+
-
solution of preformed [Et NH] [Sn(PhS) ] (0.2 mmol) was
3
3
Gen er a l Meth od for th e Syn th esis of cis- a n d tr a n s-
injected, and the mixture was stirred for 1 h at room temper-
ature. The reaction was quenched with a saturated solution
4
(
-Am in o-D- a n d -L-p r olin yl-D- a n d -L-n itr oa r gin in a m id es
Sch em e 2). Rink amide resin (0.1 mmol) was treated twice
with 20% piperidine in DMF (5 mL) at room temperature for
and 20 min, respectively. The resin was washed (5 mL each
time) with DMF (3×), DCM (2×), and MeOH (3×). The
deprotected Rink amide resin (0.1 mmol) was suspended in
DMF (1 mL). DIEA (0.3 mmol) and a solution of Boc-D- or
L-ArgNO (0.3 mmol) and HBTU (0.3 mmol) in DMF (4 mL)
2
were added sequentially. The resulting mixture was shaken
at room temperature for 6 h and filtered. The resin was washed
of K
each time) with water (3×) and THF (3×). Then the resin was
treated twice with a mixture of THF/saturated aqueous K CO
2:1) for 15 min. The resin was filtered and washed (5 mL each
time) with water (3×), MeOH (3×), DCM (3×), and methanol
3×). Rink amide resin loaded with the 4-aminoproline isomer
0 (0.1 mmol) was preswollen with dry THF, and a solution
2 3
CO in water. The resin was filtered and washed (5 mL
5
2
3
(
(
2
of (trifluoroacetyl)benzotriazole (1 mmol) in dry THF was
added. The suspension was shaken at room temperature for 2
h, and then the resin was washed (5 mL each time) with THF
(3×), MeOH (3×), DCM (3×), and MeOH (3×). The resulting
resin-bound bisprotected amino acid was dried under vacuum
for 2 h. The bisprotected-4-aminoproline-loaded resin was dried
overnight under nitrogen and then suspended in dry DCM (0.1
mmol in 3 mL of dry DCM). The suspension was cooled in an
ice bath, and 2,6-lutidine (0.6 mmol) and (TMS)OTf (0.4 mmol)
were sequentially injected dropwise under argon. The suspen-
sion was warmed to room temperature after 10 min and stirred
for 2 h under argon. The reaction was quenched using a
saturated solution of ammonium chloride in water. The resin
was washed (5 mL each time) with DCM (3×), MeOH (3×),
water (3×), MeOH (3×), and DCM (3×). Rink amide resin
loaded with the Boc-deprotected aminoproline derivative (0.1
(
5 mL each time) with DMF (3×), DCM (2×), and MeOH (3×).
The resulting resin-bound amino acid was dried under vacuum
for 2 h. The Boc-L-ArgNO -loaded resin was dried overnight
2
under nitrogen and then was suspended in dry DCM (0.1 mmol
in 3 mL of dry DCM). The suspension was cooled in an ice
bath, and 2,6-lutidine (0.4 mmol) and (TMS)OTf (0.2 mmol)
were sequentially dropwise injected under argon. The suspen-
sion was warmed to room temperature after 10 min and was
allowed to stir for 2 h under argon. The reaction was quenched
using a saturated solution of ammonium chloride in water.
The resin was washed (5 mL each time) with DCM (3×), MeOH
(
3×), water (3×), MeOH (3×), and DCM (3×). Rink amide resin
loaded with the deprotected L-ArgNO isomer (0.1 mmol) was
2
suspended in DMF (1 mL). A solution of the N-Boc-4-azidopro-
line isomer (0.2 mmol), HBTU (0.2 mmol), and DIEA (0.3
mmol) in 4 mL of DMF was added. The resulting mixture was
shaken for 6 h at room temperature and washed (5 mL each
time) with DMF (3×), DCM (2×), and MeOH (3×). The
resulting resin-bound dipeptide was dried under vacuum for
mmol) was suspended in DMF (1 mL). DIEA (0.4 mmol) and
NO
a solution of Boc-D- or -L-Arg
2
(0.4 mmol) and HBTU (0.4
mmol) in DMF (4 mL) were added sequentially. The resulting
mixture was shaken at room temperature for 3 h and filtered.

Neuronal Nitric Oxide Synthase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3 709
The resin was washed (5 mL each time) with DMF (3×), DCM
(2) Moncada, S.; Higgs, A.; Furchgott, R. XIV. International union
of pharmacology nomenclature in nitric oxide research. Phar-
macol. Rev. 1997, 49, 137-142.
3) Hobbs, A. J .; Higgs, A.; Moncada, S. Inhibition of nitric oxide
synthase as a potential therapeutic target. Annu. Rev. Pharma-
col. Toxicol. 1999, 39, 191-220.
(4) Li, H.; Raman, C. S.; Mart a´ sek, P.; Masters, B. S. S.; Poulos, T.
L. Crystallographic studies on endothelial nitric oxide synthase
complexed with nitric oxide and mechanism-based inhibitors.
Biochemistry 2001, 40, 5399-5406.
(2×), and MeOH (3×). This coupling reaction was repeated
for a second time. The resulting resin-bound protected dipep-
tide was dried under vacuum for 2 h. A portion of the
protected-dipeptide-loaded Rink resin (0.05 mmol) was cleaved
(
(see below) to obtain the corresponding trifluoroacetamide
derivative. The rest of the protected-dipeptide-loaded resin
(
(
0.05 mmol) was preswollen with THF, and a solution of LiOH
0.5 mmol) in THF/H O (5:1) was added. The resin was shaken
2
(
5) Hillier, B. J .; Christopherson, K. S.; Prehoda, K. E.; Bredt, D.
S.; Lim, W. A. Unexpected modes of PDZ domain scaffolding
revealed by structure of nNOS-syntrophin complex. Science
at room temperature for 3 h and washed (5 mL each time)
with THF (3×), MeOH (3×), H O (3×), and MeOH (3×). The
2
resulting 4-amino-deprotected-dipeptide-loaded resin was dried
under vacuum for 2 h. The resulting Rink amide resin loaded
with the dipeptide (0.05 mmol) was cleaved, and the dipeptides
were purified as described for Scheme 2.
1
999, 284, 812-815.
(6) Nishida, C. R.; Ortiz de Montellano, P. R. Electron transfer and
catalytic activity of nitric oxide synthases. Chimeric constructs
of the neuronal, inducible, and endothelial isoforms. J . Biol.
Chem. 1998, 273, 5566-5571.
Gen er a l Meth od for th e Syn th esis of D- a n d L-Nitr o-
ar gin in yl-4-N-(4-am in opr olin am ide) Dipeptides (Sch em e
(
7) Siddhanta, U.; Presta, A.; Fan, B.; Wolan, D.; Rousseau, D. L.;
Stuehr, D. J . Domain swapping in inducible nitric-oxide syn-
thase. Electron transfer occurs between flavin and heme groups
located on adjacent subunits in the dimer. J . Biol. Chem. 1998,
273, 18950-18958.
4
). The resin loaded with the aminoproline isomer 20 (Scheme
3
, 0.1 mmol) was suspended in DMF (1 mL). DIEA (0.4 mmol)
NO
and a solution of Boc-D- or -L-Arg
2
(0.4 mmol) and HBTU
(0.4 mmol) in DMF (4 mL) were added sequentially. The
(8) Crane, B. R.; Arvai, A. S.; Gachhui, R.; Wu. C.; Ghosh, D. K.;
Getzoff, E. D.; Stuehr, D. J .; Tainer, J . A. The structure of nitric
oxide synthase oxygenase domain and inhibitor complexes.
Science 1997, 278, 425-431.
resulting mixture was shaken at room temperature for 3 h
and then filtered. The resin was washed (5 mL each time) with
DMF (3×), DCM (2×), and MeOH (3×). This coupling reaction
was repeated a second time, and then the resulting resin-
bound dipeptide was dried under vacuum for 2 h. The resulting
resin loaded with the protected dipeptide (0.1 mmol) was
cleaved, and the dipeptides were purified as described for
Scheme 2.
(
9) Crane, B. R.; Arvai, A. S.; Ghosh, S.; Getzoff, E. D.; Stuehr, D.
ω
J .; Tainer, J . A. Structures of the N -hydroxy-L-arginine complex
of inducible nitric oxide synthase oxygenase dimer with active
and inactive pterins. Biochemistry 2000, 39, 4608-4621.
(10) Crane, B. R.; Arvai, A. S.; Ghosh, D. K.; Wu. C.; Getzoff, E. D.;
Stuehr, D. J .; Tainer, J . A. Structure of nitric oxide synthase
oxygenase dimer with pterin and substrate. Science 1998, 279,
4
-N-(Nitr o-L-a r gin in yl)-4R-a m in o-L-p r olin a m id e (10a ).
H NMR (D O): δ 4.66 (t, 1H), 4.58 (q, 1H), 4.05 (t, 1H), 3.83
dd, 1H), 3.41 (dd, 1H), 3.35 (br s, 2H), 2.56-2.47 (m, 2H), 1.98
2
121-2126.
1
2
(
11) Fischmann, T. O.; Hruza, A.; Niu, X. D.; Fossetta, J . D.; Lunn,
C. A.; Dolphin, E.; Prongay, A. J .; Reichert, P.; Lundell, D. J .;
Narula, S. K.; Weber, P. Structural characterization of nitric
oxide synthase isoforms reveals striking active-site conservation.
Nat. Struct. Biol. 1999, 6, 233-242.
(
(
1
3
br s, 2H), 1.72 (br s, 2H). C NMR (D
2
O): δ 170.8, 169.8,
1
4
62.8 (c, TFA), 159.2, 116.3 (c, TFA), 58.9, 53.0, 50.0, 49.1,
+
0.3, 34.6, 28.0. MS (ES): [C11
22 8 4
H N O ] m/z 331.1 (MH ). Anal.
(
12) Li, H.; Raman, C. S.; Glaser, C. B.; Blasko, E.; Young, T. A.;
Parkinson, J . F.; Whitlow, M.; Poulos, T. L. Crystal structures
of zinc-free and -bound heme domain of human inducible nitric-
oxide synthase. J . Biol. Chem. 1999, 274, 21276-21284.
13) Raman, C. S.; Li, H.; Mart a´ sek, P.; Kr a´ l, V.; Masters, B. S. S.;
Poulos, T. L. Crystal structure of constitutive endothelial nitric
oxide synthase: A paradigm for pterin function involving a novel
metal center. Cell 1998, 95, 939-950.
Calcd: C, 31.23; H, 4.01; N, 18.21. Found: C, 31.17; H, 3.95;
N, 17.98 (+2.5TFA).
4
-N-(Nitr o-L-a r gin in yl)-4S-a m in o-D-p r olin a m id e (10c).
1
H NMR (D
2
O): δ 4.58 (t, 1H), 4.52 (q, 1H), 3.98 (t, 1H), 3.77
(
(
dd, 1H), 3.32 (dd, 1H), 3.27 (br s, 2H), 2.46 (dd, 2H), 1.90 (br
s, 2H), 1.65 (br s, 2H). C NMR (D
c, TFA), 159.0, 116.3 (c, TFA), 59.0, 52.9, 49.9, 49.1, 40.4, 34.7,
22 8 4
8.0. MS (ES): [C11H N O ] m/z 331.1 (MH ). Anal. Calcd:
1
3
2
O): δ 170.8, 169.8, 162.8
(
2
+
(14) Gerber, N. C.; Rodriguez-Crespo, I.; Nishida, C. R.; Ortiz de
Montellano, P. R. Active site topologies and cofactor-mediated
conformational changes of nitric-oxide synthases. J . Biol. Chem.
C, 31.72; H, 4.16; N, 19.09. Found: C, 32.03; H, 4.12; N, 18.86
(+2.25TFA).
1
997, 272, 6285-6290.
En zym e a n d Assa y. All of the NOS isoforms used were
(
15) Alcaraz, M. J .; Guillen, M. I. Nitric oxide related therapeutic
phenomenon: A challenging task. Curr. Pharm. Des. 2002, 8,
215-231.
recombinant enzymes overexpressed in E. coli from different
sources; there is very high sequence identity for the isoforms
from different sources. The murine macrophage iNOS was
expressed and isolated according to the procedure of Hevel et
(
16) (a) Vallance, P.; Leiper, J . Blocking NO synthesis: How, where
and why? Nat. Rev. Drug Discuss. 2002, 1, 939-950. (b)
Grunewald, T.; Beal, M. F. NOS knockouts and neuroprotection.
Nat. Med. 1999, 5, 1354-1355.
17) Huang, Z.; Effects of cerebral ischaemia in mice deficient in
neuronal nitric oxide synthase. Science 1994, 265, 1883-1885.
(18) Ashina, M. Nitric oxide synthase inhibitors for the treatment of
5
2
53
al. The rat nNOS was expressed and purified as described.
5
4
The bovine eNOS was isolated as reported. Nitric oxide
formation from NOS was monitored by the hemoglobin capture
assay as described.
Deter m in a tion of K
obtained by measuring percent inhibition in the presence of
(
5
5
Va lu es. The apparent K
i
values were
chronic tension-type headache. Expert Opin. Pharmacother.
i
2
002, 3, 395-399.
(
19) Schulz, J . B.; Matthews, R. T.; Klockgether, T.; Dichgans, J .;
Flint B., M. The role of mitochondrial dysfunction and neuronal
nitric oxide in animal models of neurodegenerative diseases. Mol.
Cell. Biochem. 1997, 174, 193-197.
1
0 µM L-arginine with at least three concentrations of inhibi-
tor. The deviation from the mean of the three measurements
was (5%. The parameters of the following inhibition equa-
5
6
tion were fitted to the initial velocity data: % inhibition )
(20) Lowe, J . A., III. Nitric oxide synthase inhibitors: Recent patent
activity. IDrugs 2000, 3, 63-72.
(21) Boughton-Smith, N. K.; Tinker, A. C. Inhibitors of nitric oxide
synthase in inflammatory arthritis. IDrugs 1998, 1, 321-333.
1
00[I]/{[I] + K
i
m m
(1 + [S]/K )}. K values for L-arginine were 1.3
µM (nNOS), 8.2 µM (iNOS), and 1.7 µM (eNOS). The selectivity
of an inhibitor was defined as the ratio of the respective K
i
(
22) Kankuri, E.; Vaali, K.; Knowles, R. G.; Lahde, M.; Korpela, R.;
Vapaatalo, H.; Moilanen, E. Suppression of acute experimental
colitis by a highly selective inducible nitric-oxide synthase
inhibitor, N-[3-(Aminomethyl)benzyl]acetamidine. J . Pharmacol.
Exp. Ther. 2001, 298, 1128-1132.
(23) Tinker, A. C.; Beaton, H. G.; Boughton-Smith, N.; Cook, T. R.;
Cooper, S. L.; Fraser-Rae, L.; Hallam, K.; Hamley, P.; McInally,
T.; Nicholls, D. J .; Pimm, A. D.; Wallace, A. V. 1,2-Dihydro-4-
quinazolinamines: Potent, highly selective inhibitors of inducible
nitric oxide synthase which show antiinflammatory activity in
vivo. J . Med. Chem. 2003, 46, 913-916.
values.
Ack n ow led gm en t. Grants from NATO and the
Spanish Ministry of Science and Technology to J .A.G.-
V. and from the National Institutes of Health (GM49725)
to R.B.S. are gratefully acknowledged.
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