Selective l-nitroargininylaminopyrrolidine and l-nitroargininylaminopiperidine neuronal nitric oxide synthase inhibitors

Article abstract of DOI:10.1016/j.bmc.2007.01.001

Selective inhibition of the localized excess production of NO by neuronal nitric oxide synthase (nNOS) has been targeted as a potential means of treating various neurological disorders. Based on observations from the X-ray crystal structures of complexes of nNOS with two nNOS-selective inhibitors, (4S)-N-{4-amino-5-[(2-amino)ethylamino]pentyl}-N′-nitroguanidine (l-Arg(NO₂)-l-Dbu-NH₂ (1) and 4-N-(N^ω-nitro-l-argininyl)-trans-4-amino-l-proline amide (2), a series of descarboxamide analogues was designed and synthesized (3-7). The most potent compound was aminopyrrolidine analogue 3, which exhibited better potency and selectivity for nNOS than parent compound 2. In addition, 3 provided higher lipophilicity and a lower molecular weight than 2, therefore having better physicochemical properties. N^α-Methylated analogues (8-11) also were prepared for increased lipophilicity of the inhibitors, but they had 4- to 5-fold weaker binding affinity compared to their parent compounds.

Full text of DOI:10.1016/j.bmc.2007.01.001

Bioorganic & Medicinal Chemistry 15 (2007) 1928–1938
Selective L-nitroargininylaminopyrrolidine
and L-nitroargininylaminopiperidine neuronal
nitric oxide synthase inhibitors
Jiwon Seo, Pavel Mart a´ sek,b,ꢀ Linda J. Roman and Richard B. Silverman
a
b,ꢁ
a,
*
a
Department of Chemistry, Department of Biochemistry, Molecular Biology, and Cell Biology, and the Center for Drug Discovery
and Chemical Biology, Northwestern University, Evanston, IL 60208-3113, USA
b
Department of Biochemistry, The University of Texas Health Science Center, San Antonio, TX 78384-7760, USA
Received 7 December 2006; accepted 1 January 2007
Available online 4 January 2007
Abstract—Selective inhibition of the localized excess production of NO by neuronal nitric oxide synthase (nNOS) has been targeted
as a potential means of treating various neurological disorders. Based on observations from the X-ray crystal structures of complex-
0
es of nNOS with two nNOS-selective inhibitors, (4S)-N-{4-amino-5-[(2-amino)ethylamino]pentyl}-N -nitroguanidine (L-Arg(NO
2
)-
x
2
L-Dbu-NH (1) and 4-N-(N -nitro-L-argininyl)-trans-4-amino-L-proline amide (2), a series of descarboxamide analogues was
designed and synthesized (3–7). The most potent compound was aminopyrrolidine analogue 3, which exhibited better potency
and selectivity for nNOS than parent compound 2. In addition, 3 provided higher lipophilicity and a lower molecular weight than
a
2
, therefore having better physicochemical properties. N -Methylated analogues (8–11) also were prepared for increased lipophilicity
of the inhibitors, but they had 4- to 5-fold weaker binding affinity compared to their parent compounds.
2007 Elsevier Ltd. All rights reserved.
ꢀ
1
. Introduction
Because NO generated from eNOS is crucial to vascular
regulation, selective inhibition of nNOS is a prerequisite
10,11
for the inhibitor to be therapeutically useful.
1
Nitric oxide (NO), an essential cellular signaling mole-
cule, is produced in mammalian cells by a family of
enzymes called nitric oxide synthases (NOS, EC
.14.13.39). Three isoforms of NOS have been isolated
so far: neuronal NOS (nNOS) in the central nervous sys-
tem, endothelial NOS (eNOS) in the cardiovascular sys-
x
Previously, N -nitro-L-arginine-containing dipeptide
2
12
13
1
amide inhibitors 1 and 2 (Fig. 1) were found to be
highly selective for neuronal nitric oxide synthase. To
further the understanding of the inhibitor–active site
interactions, Flinspach et al. resolved the X-ray crystal
structures for nNOS complexed with 1 and 2 (Support-
3
tem, and inducible NOS (iNOS) in the immune system.
Each isozyme catalyzes the conversion of L-arginine to
NO and L-citrulline. NO is involved with diverse physi-
ological events, which include neurotransmission
1
4
ing Information). Compound 2 is a conformationally
restricted analogue of 1, but the binding modes for both
inhibitors in the nNOS active site are somewhat differ-
ent. The C-terminal carboxamide of 1 faces toward the
ceiling of the active site and is surrounded by a hydrogen
bond network of Arg481, Gln478, and Ser477; it also
appears to have a weak interaction with Asn569. The
carboxamide of 2, however, is placed in the substrate ac-
cess channel and does not form hydrogen bonds with
Arg481, Gln478, and Ser477. Instead, the carboxamide
appears to have a weak hydrogen bond interaction with
the heme propionate. The strength of each hydrogen
bond cannot be quantitatively evaluated, but it is pre-
sumed that the carboxamide of 1 contributes more sig-
nificantly to the enzyme–inhibitor binding than does
the carboxamide of 2 based on the number and the
4
5
(
(
nNOS), vasodilation (eNOS), and cytoprotection
6
iNOS). Despite the importance of this molecule,
uncontrolled production of NO is associated with poten-
tially harmful effects; an overproduction of NO in neu-
ronal cells has been implicated in strokes, migraine
8
headaches, and various neurodegenerative diseases.
7
9
Keywords: Neuronal nitric oxide synthase; Enzyme inhibitors; Selective
inhibition; Aminopyrrolidines; Aminopiperidines.
*
Corresponding author. Tel.: +1 847 491 5653; fax: +1 847 491
713; e-mail: Agman@chem.northwestern.edu
7
ꢀ
Developed the eNOS overexpression system in E. coli and isolated
and purified the eNOS.
Developed the nNOS overexpression system in E. coli.
ꢁ
0
968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.01.001

J. Seo et al. / Bioorg. Med. Chem. 15 (2007) 1928–1938
1929
HN
HN
NHNO2
HN
HN
NHNO2
O
O
H
N
H
N
R
H N
NH2
N
NH2
2
H
O
O
NH
NH2
1
2, R = H
8
, R = CH
3
HN
NHNO2
NH
HN
NHNO2
NH
HN
NHNO2
NH
HN
NHNO2
NH
HN
NHNO2
NH
H
H
N
H
H
H
N
R
N
R
N
N
R
N
H
H N
2
N
NH
H N
2
N
H
H
O
NH
NH
O
NH
O
O
NH
O
3
9
, R = H
, R = CH3
4
5, R = H
10, R = CH3
6
7, R = H
11, R = CH3
Figure 1. Structures of neuronal nitric oxide synthase-selective inhibitors.
distances of the hydrogen bonds. Indeed, Huang et al.
demonstrated the importance of the carboxamide moie-
ty in 1 by evaluating a series of descarboxamide ana-
by a peptide coupling reaction using HBTU/HOBt/
DIEA and subsequent deprotection (Scheme 1).
1
5
a
logues.
2
These descarboxamide derivatives showed
.5–4 times lower nNOS binding affinity than 1 depend-
2.2. N -Methylated inhibitors
a
a
ing on the side-chain carbon length. However, as ob-
served in the X-ray crystal structures, the carboxamide
moiety of 2 may not be as important as that in 1. Based
on this hypothesis, we designed a series of descarbox-
amide analogues of 2 with different ring sizes and stereo-
chemistry (3–7, Fig. 1). These aminopyrrolidine and
aminopiperidine analogues represent three possible
advantages: first, removal of the polar carboxamide
functionality from 2 should afford a less polar com-
pound for potential improved pharmacokinetic proper-
ties; second, the lower molecular weight is a desirable
property; and third, there is one less stereogenic center.
For the synthesis of 8–11, N -Boc-N -methyl-L-nit-
roarginine (21) was prepared as shown in Scheme 2.
N -Boc-N -Methyl-L-ornithine (19) was synthesized
a
a
1
8
according to a literature procedure using a modifica-
tion of the nitrile reduction method. The synthesis
started with Boc-L-Gln-OH; the c-carboxamide of
Boc-L-Gln-OH was dehydrated to a nitrile using acetic
anhydride and pyridine. The resulting nitrile-containing
compound was selectively alkylated with iodomethane,
a
providing N -Boc-2-methylamino-4-cyanobutyric acid
(18). As originally reported, the reduction of the nitrile
to an amine was first tried with PtO -catalyzed hydroge-
2
1
8
nation in isopropanol in the presence of HCl. Later we
found that the nitrile reduction could be performed in a
more facile and consistent manner by hydrogenation at
In a continued effort to modify inhibitors for enhanced
lipophilicity, we also designed simple N -methylated ni-
a
tro-L-argininamide analogues (8–11, Fig. 1). Recently,
we reported analogues of 2 in which the pyrrolidine ring
nitrogen was alkylated; this led to a decrease in both
1 atmosphere pressure with Pd(OH) /C as the catalyst in
2
methanol/acetic acid (1:1) solution. The Pd(OH) cata-
2
lyst also is much cheaper than PtO , propitious for a
2
1
6
a
potency and selectivity. The N -methylated analogues
described here will be assessed for effective hydrogen
bonding interactions between active site residues and
the a-amino moiety as either a primary amine or a second-
ary amine. Provided that the additional methyl group
does not disrupt existing hydrogen bonding interactions,
large scale reaction. After filtration of the catalyst and
evaporation of the solvent, Boc-L-MeOrn-OH (19)
directly underwent nitroguanidination using 2-methyl-
1-nitro-2-thiopseudourea (20), which was prepared by
nitration of commercially available S-methylisothiourea
1
9
hemisulfate salt (Scheme 2).
a
it is expected that the N -methylated derivatives would
7
have more favorable physicochemical properties.
1
Boc-L-MeArg(NO )-OH (21) was then coupled to (R)-
2
(+)-1-Boc-3-aminopyrrolidine, (R)-(+)-1-Boc-3-amino-
piperidine, and 4-amino-1-Boc-piperidine, and the
a
2
. Chemistry
resulting N -methylated L-nitroargininamides were
deprotected with TFA, purified by preparative HPLC,
and lyophilized (9–11, Scheme 1).
2
.1. Descarboxamide inhibitors
Five descarboxamide analogues were designed using
-aminopyrrolidines (3 and 4) and 3 or 4-aminopiperi-
dines (5–7 in Fig. 1). Compounds 3–7 were synthesized
Compound 8 was prepared by the synthetic route shown
in Scheme 3. Key intermediate 30 was synthesized from
the starting material, N-Boc-L-trans-4-hydroxyproline.
3

1930
J. Seo et al. / Bioorg. Med. Chem. 15 (2007) 1928–1938
HN
HN
NHNO2
HN
HN
NHNO2
Boc-aminopyrrolidines
or Boc-aminopiperidines
TFA
3 - 7 (R = H)
1
9
- 11 (R = CH )
HBTU, HOBt
CH Cl , ~quant.
1 3
2
2
H
DIEA, CH Cl /DMF
2
2
Boc
OH
O
Boc
N
N
68 - 99%
N
R2
R1
R1
O
1
2 - 16 (R = H)
1
2
2 - 24 (R = CH )
1
3
R =
2
N
N
N
N
N
Boc
Boc
Boc
Boc
Boc
1
2, R = H
13, R = H 14, R = H
15, R = H 16, R = H
1
1
1
1
1
2
2, R = CH
23, R = CH
24, R = CH
1
3
1
3
1
3
Scheme 1.
N
O
N
O
NH2
H N
2
a
b
c
OH
OH
OH
OH
Boc
N
Boc
N
H
Boc
N
Boc N
H
O
O
O
CH3
CH3
Boc-Gln-OH
17
1
8
19
HN
HN
NHNO2
d
NH
NH2
NH
NHNO2
HNO3
H SO
2
4
CH3S
CH S
H SO
3
2
4
OH
O
2
2
0
Boc N
CH3
21
Scheme 2. Reagents and conditions: (a) acetic anhydride, pyridine, 60%; (b) CH
quantitative; (d) 20, Et
N, EtOH, 40 ꢁC, 43%.
3
I, NaH, THF, 83%; (c) H
2
2
/Pd(OH) , MeOH/AcOH (1:1),
3
Methyl ester protection and hydroxyl group inversion
were achieved by an intramolecular Mitsunobu reac-
compound 2, by approximately two orders of magni-
tude, and the molecular weights are lower. N -Methyla-
tion (8–11) also resulted in increased lipophilicity of the
compounds.
a
2
0
21
tion and a sodium azide-catalyzed methanolysis to
provide 26. As precursor of a primary amine, an azide
was incorporated by another Mitsunobu reaction using
diphenylphosphoryl azide (DPPA). Methyl ester 27
was then hydrolyzed and converted to amide 29 by a
mixed anhydride coupling reaction using 29% aqueous
ammonia. Palladium on carbon-catalyzed hydrogena-
tion provided 30. Peptide coupling of 21 and 30 followed
by Boc deprotection provided 8.
The in vitro enzyme inhibitory activity was evaluated
using a hemoglobin capture assay with descarboxamides
3–7 as well as parent compound 2; the assay results are
summarized in Table 2. These results indicate that 3 is a
more potent inhibitor of nNOS than is 2.
Two inferences can be made from this result: (1) the car-
boxamide moiety of 2 is not crucial for enzyme binding
provided that other changes resulting from the removal
of the carboxamide moiety, such as solubility or confor-
mation, have minimal effects; (2) removal of the carbox-
amide moiety affords better interactions between the
a-amino group of 3 and the active site Glu592 (Figs. 2
and 3). From computer modeling, the excision of the
carboxamide group results in the loss of a weak interac-
tion between the heme propionate and the carboxamide
3
. Results and discussion
Physicochemical properties (M , logP, and logD7.4) for
W
the inhibitors were calculated using Advanced Chemis-
try Development, Inc. software (Toronto, Canada),
and the results are summarized in Table 1. Descarboxa-
mide compounds 3, 5, and 7, although still poorly lipo-
philic, have increased lipophilicity compared with parent

J. Seo et al. / Bioorg. Med. Chem. 15 (2007) 1928–1938
1931
O
O
O
O
O
HO
a
b
HO
c
N3
OH
OCH3
OCH3
N
N
N
N
Boc
Boc
Boc
Boc
27
25
26
O
O
O
N3
N3
H2N
d
e
f
g
OH
NH2
NH2
N
N
N
Boc
Boc
Boc
28
29
30
HN
NHNO2
HN
NHNO2
NH
NH
h
O
O
H
H
N
Boc
N
H C
3
N
NH2
N
H
NH2
O
N
CH3
O
NH
Boc
31
8
Scheme 3. Reagents and conditions: (a) DIAD, PPh
THF, 91%; (e) iso-butylchloroformate, NMM, NH
DMF, 30%; (h) 50% TFA, CH Cl , quant.
3
, THF, 97%; (b) MeOH, NaN
3
3
, 40 ꢁC, 98%; (c) DPPA, DEAD, PPh , THF, 94%; (d) 5% LiOH,
4
OH, THF, 83%; (f) H , Pd/C, MeOH, 94%; (g) Boc-MeArg(NO
2
2
)-OH, HBTU, HOBt, DCM/
2
2
a
Table 1. Physicochemical properties of nNOS inhibitors
Table 2. In vitro NOS inhibition by descarboxamide and N -methyl-
a
ated analogues
a,b
a
Compound
M
W
logP
logD7.4
b
i
d
Compound
K
(lM)
Selectivity
n/i n/e
1.2
218
2
3
5
7
8
9
330.34
287.32
301.35
301.34
344.37
301.35
315.37
315.37
ꢀ3.81
ꢀ2.22
ꢀ1.79
ꢀ1.84
ꢀ3.58
ꢀ1.99
ꢀ1.56
ꢀ1.60
ꢀ6.57
ꢀ5.20
ꢀ4.77
ꢀ4.83
ꢀ6.33
ꢀ4.96
ꢀ4.52
ꢀ4.58
c
nNOS
iNOS
eNOS
L-NNA
0.61 ± 0.005
0.33 ± 0.019
0.22 ± 0.021
1.76 ± 0.065
0.48 ± 0.018
7.62 ± 0.587
0.47 ± 0.011
1.09 ± 0.106
1.08 ± 0.025
2.26 ± 0.038
2.38 ± 0.110
4.28
17
55
0.72
72
68
7
52
2
3
4
5
6
7
8
9
250
32
309
44
57
90
77
1
0
1
225
221
262
290
160
174
114
188
70
469
29
1
535
202
65
a
Lipophilicity data were calculated with ACD/LogD version 7.0,
Advanced Chemistry Development, Inc., Toronto, Canada.
Octanol–water partition coefficient calculated for neutral compounds.
430
60
557
266
148
77
b
124
242
515
115
107
216
1
1
0
1
48
a
–
NH, but the loss of this interaction allows the a-amino
i
The enzymes used for the K determinations are rat brain nNOS,
group of 3 to move slightly closer to Glu592 and results
in a stronger hydrogen bond. The slightly higher nNOS
potency of 3 relative to 2 indicates that the latter effect
probably outweighs the former. The key hydrogen bond
between the secondary amine of the pyrrolidine ring and
the heme propionate group through structural water
molecule 2 remains similar (Fig. 3b). Compound 3 also
shows enhanced selectivity for nNOS over the other iso-
forms. The opposite stereochemistry at the 3-position of
the pyrrolidine ring (S-stereochemistry) has little effect
on the affinity for iNOS and eNOS, but is detrimental
to nNOS binding (compare 3 and 4).
recombinant murine iNOS, and recombinant bovine eNOS. See
Section 5.
b
c
In most cases, the K
data points and correlation coefficients 0.948–0.996.
± SEM. Results are given as a mean of two or more independent
i
values represent single measurements with 5–6
K
i
experiments.
d
i i
The ratio of K (iNOS or eNOS) to K (nNOS).
of the ring. Piperidines (5–7) have about three times low-
er activity against eNOS than do the pyrrolidines (2–4).
This reflects the difference between the active site sizes of
nNOS and eNOS, and is consistent with the fact that
1
1,22
nNOS has a slightly larger active site than eNOS.
Piperidine-containing compounds 5 and 7 exhibited a
twofold reduced potency for nNOS relative to 3, indicat-
ing that a five-membered ring fits better than a six-mem-
bered ring at that position in the nNOS active site. As in
the case of the pyrrolidine-containing compounds, the
S-stereochemistry at the piperidine ring (6) was disad-
vantageous, relative to R-stereochemistry (5), for bind-
ing with the neuronal and inducible isoforms. The
binding affinity for eNOS is highly dependent on the size
In this series of descarboxamide derivatives, 3 showed
the best potency for nNOS, and 7 showed the best selec-
tivity for nNOS over iNOS and eNOS.
An additional structure–activity relationship study was
carried out by N -methylation of 2, 3, 5, and 7
a
(Fig. 1). We rationalized that if methylation did not dis-
rupt the hydrogen bonds of the a-amino group, we
could generate inhibitors with higher lipophilicities.

1932
J. Seo et al. / Bioorg. Med. Chem. 15 (2007) 1928–1938
˚
Figure 2. Two perspectives of X-ray crystal structure of 2 bound in nNOS active site. The distances of the hydrogen bonds are marked in A.
a
In addition, the effect of N -methylation could be eval-
uated for future amine-prodrug design, because a pro-
moiety on a secondary amine produces a more stable
ondary amino group on the pyrrolidine ring and water
molecule 2 (Fig. 2).
2
3
prodrug than that on a primary amine.
4. Conclusion
Entries 8–11 in Table 2 give the in vitro NOS inhibitory
activities of the N -methylated derivatives. All of the
a
x
We designed and synthesized N -nitro-L-argininamides
containing aminopyrrolidine or aminopiperidine sub-
stituents as descarboxamide analogues of 2. In accor-
dance with our hypothesis, the carboxamide group of
2 is not essential for enzyme–inhibitor binding; 3 pro-
vided higher in vitro potency and selectivity with nNOS
than did 2. The descarboxamides also offer the potential
benefit of lower polarity. We also corroborated the
importance of the hydrogen bonding interaction be-
tween the a-amino group on the nitroargininamide
methylated compounds had approximately 4- to 5-fold
decreased nNOS binding affinity, which suggests that
the methyl moiety on the a-amino group causes an unfa-
vorable effect on the hydrogen bond network. Apparent-
ly, the methyl group cannot be accommodated tightly in
the space around Glu592 and water molecule 3 (Fig. 2);
rather, the methyl group might cause the inhibitor to
move away from Glu592, water molecule 2, and water
molecule 3, weakening the hydrogen bonding interac-
tions with them. This shift of the inhibitor (moving to-
ward the top in Fig. 2a or the right in Fig. 2b) does
a
and Glu592/water 3 by preparing and testing N -meth-
ylated analogues 8–11, which exhibited lower affinities
for nNOS than 2. These results broaden our under-
standing of nNOS-inhibitor interactions and provide
strategies for the design of more effective nNOS-selec-
tive inhibitors.
a
not seem to be beneficial. The assay results for the N -
methylated derivatives support the significance of the
interaction between the a-amino group and Glu592/
water molecule 3, and the interaction between the sec-

J. Seo et al. / Bioorg. Med. Chem. 15 (2007) 1928–1938
1933
absorbance at 254 nm with a Waters 2487 Dual lambda
absorbance detector. Generally, product peaks appeared
at 10–15 min elution times. Fractions containing the
pure products were concentrated in vacuo, and the res-
idues were lyophilized.
All reagents were purchased from Aldrich, Advanced
ChemTech, and Nova Biochem, and were used without
further purification unless stated otherwise. NADPH,
calmodulin, and human ferrous hemoglobin were ob-
tained from Sigma–Aldrich Co. Tetrahydrobiopterin
(H B) was purchased from Alexis Biochemicals Co.,
4
San Diego, CA, USA. HEPES, DTT, and conventional
organic solvents were purchased from Fisher Scientific.
Tetrahydrofuran was distilled under nitrogen from sodi-
um/benzophenone. Diethyl azodicarboxylate (DEAD)
was purchased from Lancaster Synthesis, Inc., Pelham,
NH, USA. (R) and (S) isomers of both 1-Boc-3-amino-
pyrrolidine and 1-Boc-3-aminopiperidine were pur-
chased from CNH Technologies, Inc., Woburn, MA,
USA.
5.2. General method for the syntheses of 3–7
Figure 3. Superimposition of the crystallographic conformation of 2
gray) and the docked conformation of 3 (orange) in the nNOS active
site. Distances of the hydrogen bonds are marked in angstrom.
To a solution of Boc-L-Arg(NO )-OH (2 mmol), HOBt
2
(
(2 mmol), and HBTU (2 mmol) in dry DMF (3 mL)
and dry CH Cl (6 mL) was added the appropriate
2
2
amine (1 mmol) in dry DMF (3 mL) dropwise at 0 ꢁC.
5. Experimental
DIEA (2 mmol) in dry CH Cl (4 mL) was added slow-
2
2
ly, and stirring continued overnight at room tempera-
ture under argon. The solvent was removed under
reduced pressure, and the residue was treated with
EtOAc (10 mL). The organic mixture was washed suc-
cessively with 5% NaHCO3 (10 mL), water (10 mL),
0.5 N HCl (10 mL), and brine (10 mL), and dried over
Na SO . The solution was concentrated in vacuo, and
5
.1. General methods
Fisher silica gel 60 (230–400 mesh) was used for flash
column chromatography with >10% methanol eluent
systems; otherwise Sorbent Technologies silica gel 60
(
200–400 mesh) was used. Thin-layer chromatography
2
4
was carried out on E. Merck precoated silica gel 60
F254 plates. Compounds were visualized with a ninhy-
drin spray reagent or a UV/vis lamp. Combustion anal-
yses were performed by Atlantic Microlab, Inc.,
Norcross, Georgia, USA. Electrospray mass spectra
were obtained on a VG70-250SE mass spectrometer in
the Analytical Service Laboratory at Northwestern Uni-
versity. High resolution mass spectrometric analyses
were carried out at the University of Illinois, Urbana-
Champaign, using a Micromass Quattro II spectrome-
the residue was purified by flash column chromatogra-
phy (EtOAc/MeOH = 19:1, R = 0.20) to afford the
f
product as a white foam. The Boc-protected analogues
were treated with trifluoroacetic acid/CH Cl (1:1) at
2
2
0
ꢁC under argon, the reaction temperature was allowed
to rise to room temperature, and stirring was continued
for 1 h. Excess TFA and solvent were removed by evap-
oration in vacuo. The residue was repeatedly dissolved
in CH Cl and the solvents evaporated to remove traces
2
2
of TFA. The residue was dissolved in a small amount of
water, and the solution was washed with CH Cl and
lyophilized to give a white solid in a quantitative yield.
1
ter. H NMR spectra were recorded on a Varian
2
2
Mercury 400 or Varian Inova 500 NMR spectrometer.
Chemical shifts are reported as d values in parts per mil-
lion downfield from TMS (d 0.0) as the internal standard
5.2.1. (3R)-3-((2S)-2-tert-Butoxycarbonylamino-5-nitro-
guanidinopentanoylamino)-pyrrolidine-1-carboxylic acid
in CDCl . Melting points were measured on a Buchi
3
melting point B-540 apparatus and are uncorrected.
An Orion Research model 701 pH meter with a general
combination electrode was used for pH measurements.
tert-butyl ester (12). Boc-L-Arg(NO )-OH (342 mg,
2
1.07 mmol), HOBt (145 mg, 1.07 mmol), HBTU (406 mg,
1.07 mmol), DIEA (138 mg, 1.07 mmol), and (R)-(+)-1-
Boc-3-aminopyrrolidine (100 mg, 0.54 mmol) were used
as described above to provide 12 (240 mg, 0.49 mmol,
Final compounds (1.0 mL injection of a solution of
8
1
mg/mL sample) were purified by preparative HPLC
Waters Prep LC 4000 system) using a Phenomenex
9
1%) as a white solid; mp 102.9–103.7 ꢁC. H NMR
(
(500 MHz, CDCl ) d 8.70 (s, 1H), 7.50 and 7.44 (2 · s,
3
Gemini 5 l C18 (250 · 21.20 mm) column. Samples were
eluted using a mobile phase of 4% solvent A (60%
CH CN + 40% water + 0.08% TFA) and 96% solvent
3
B (water + 0.1% TFA) for 30 min isocratically at a flow
rate of 18 mL/min. Product peaks were detected by
1H), 5.76 and 5.67 (2 · s, 1H), 4.38 (m, 1H), 4.26 (m, 1H),
3.56 (dd, J = 6.0 Hz, 4.5 Hz, 1H), 3.29–3.39 (m, 4H), 3.17
(m, 1H), 2.06 (m, 2H), 1.82 (s, 1H), 1.75 (s, 1H), 1.67
1
3
(s, 2H), 1.41 (s, 9H), 1.39 (s, 9H), rotamers; C NMR
125 MHz, CDCl ) d 172.2, 159.6, 156.6, 154.8, 80.4, 79.9,
(
3

1934
J. Seo et al. / Bioorg. Med. Chem. 15 (2007) 1928–1938
6
0.7, 51.6, 50.8, 49.7, 48.9, 44.5, 44.0, 40.7, 38.9, 31.7, 31.2,
0.9, 28.7, 28.6, 24.8, 21.3, 14.4, rotamers; HRMS (ES)
5.2.6. (2S)-2-Amino-5-nitroguanidinopentanoic acid ((3R)-
piperidin-3-yl)amide (5). A white solid was obtained after
3
(
+
m/z): M+H calcd for C H N O 488.2833, found
1
Boc deprotection; mp 58.5–59.5 ꢁC.
H
NMR
2
0
38
7 7
488.2841.
(400 MHz, D O) d 3.88 (t, J = 10.4 Hz, 1H), 3.80 (t,
2
J = 6.0 Hz, 1H), 3.30 (m, 1H), 3.12–3.19 (m, 3H), 2.78
(t, J = 12.0 Hz, 1H), 2.70 (t, J = 11.6 Hz, 1H), 1.83 (d,
J = 11.6 Hz, 2H), 1.75 (s, 2H), 1.61 (m, 1H), 1.49 (s,
5.2.2. (2S)-2-Amino-5-nitroguanidinopentanoic acid ((3R)-
pyrrolidin-3-yl)amide (3). A white solid was obtained
1
13
after Boc deprotection; mp 58.0–59.8 ꢁC. H NMR
2H), 1.40 (m, 1H); C NMR (100 MHz, D O) d
2
(
J = 6.0 Hz, 1H), 3.43 (dd, J = 8.0 Hz, 6.5 Hz, 1H), 3.26
400 MHz, D O) d 4.13 (t, J = 6.0 Hz, 1H), 3.82 (t,
169.1, 158.9, 114.9, 53.0, 46.1, 44.2, 43.6, 40.3, 28.0,
27.5, 20.7; Anal. Calcd for C H N O Æ2.5TFAÆH O:
2
1
1
23
7
3
2
(
(
m, 2H), 3.15 (s, 2H), 3.11 (m, 1H), 2.19 (m, 1H), 1.85
m, 1H), 1.76 (s, 2H), 1.49 (s, 2H); C NMR (100 MHz,
C, 31.79, H, 4.59, N, 16.22; Found: C, 31.79, H, 4.36,
N, 16.39.
1
3
D O)d169.6,158.9,114.9,52.9,49.5,49.1,44.6,40.3,29.6,
2
2
H,4.14,N,17.13;Found:C,31.11,H,4.35,N,17.22.
8.0; Anal. Calcd for C H N O Æ2.5TFA: C, 31.48,
5.2.7. (3S)-3-((2S)-2-tert-Butoxycarbonylamino-5-nitro-
guanidinopentanoylamino)-piperidine-1-carboxylic acid
1
0
21
7
3
tert-butyl ester (15). Boc-L-Arg(NO )-OH (224 mg,
2
5.2.3. (3S)-3-((2S)-2-tert-Butoxycarbonylamino-5-nitro-
guanidinopentanoylamino)-pyrrolidine-1-carboxylic acid
tert-butyl ester (13). Boc-L-Arg(NO )-OH (342 mg,
2
0.70 mmol), HOBt (95 mg, 0.70 mmol), HBTU
(265 mg, 0.70 mmol), DIEA (90 mg, 0.70 mmol), and
(S)-1-Boc-3-aminopiperidine (70 mg, 0.35 mmol) were
used as described above to provide 15 (152 mg,
1
1
.07 mmol),HOBt(145 mg,1.07 mmol),HBTU(406 mg,
.07 mmol), DIEA (138 mg, 1.07 mmol), and (S)-(ꢀ)-1-
0.30 mmol, 87%) as a white solid; mp 107.2–
1
Boc-3-aminopyrrolidine (100 mg, 0.54 mmol) were used
as described above to provide 13 (261 mg, 5.35 mmol,
9
108.0 ꢁC. H NMR (500 MHz, CDCl ) d 8.60 (s,
3
1H), 6.86 (s, 1H), 5.62 and 5.61 (2 · s, 1H), 4.28 (s,
1H), 3.90 (s, 1H), 3.75 (t, J = 6.0 Hz, 1H), 3.60 (s,
1H), 3.33 (m, 3H), 3.25 (s, 1H), 1.85 (s, 1H), 1.79
(m, 1H), 1.68 (br s, 4H), 1.53 (m, 2H), 1.45 (s, 9H),
1
9%) as a white solid; mp 100.9–101.7 ꢁC. H NMR
(
500 MHz,CDCl )d8.61(s,1H),7.47(s,1H),5.73(s,1H),
3
4
3
9
.34 (s, 1H), 4.24 (s, 1H), 3.53 (s, 1H), 3.20–3.36 (m, 4H),
.12(m,1H),2.05(s,2H),1.82(s,1H),1.63(m,3H),1.39(s,
H),1.36(s,9H),rotamers; CNMR(125 MHz,CDCl )
1
3
1.42 (s, 9H), rotamers; C NMR (125 MHz, CDCl3)
d 171.4, 159.7, 156.5, 155.3, 80.3, 68.2, 60.6, 45.7,
40.8, 28.6, 28.5, 25.8, 21.3, 14.4, rotamers; HRMS
1
3
3
d 172.4, 159.6, 156.4, 154.8, 80.2, 79.9, 60.6, 51.6, 50.9,
+
4
9.6,49.0,44.4,44.0,40.7,38.8,31.6,31.2,30.7,28.7,28.5,
4.8,21.3,14.4,rotamers;HRMS(ES)(m/z):M+H calcd
(ES) (m/z): M+H calcd for C H N O 502.2989,
2
1
40
7
7
+
2
forC H N O 488.2833,found488.2847.
found 502.2987.
2
0
38
7
7
5.2.8. (2S)-2-Amino-5-nitroguanidinopentanoic acid ((3S)-
piperidin-3-yl)amide (6). A white solid was obtained
5.2.4. (2S)-2-Amino-5-nitroguanidinopentanoic acid ((3S)-
pyrrolidin-3-yl)amide (4). A white solid was obtained after
1
after Boc deprotection; mp 89.5–91.3 ꢁC. H NMR
1
Boc deprotection; mp 63.6–65.0 ꢁC. H NMR (500 MHz,
(400 MHz, D O) d 3.87 (t, J = 10.0 Hz, 1H), 3.80 (t,
2
D O) d 4.29 (t, J = 6.0 Hz, 1H), 3.79 (t, J = 6.5 Hz, 1H),
J = 5.6 Hz, 1H), 3.27 (d, J = 11.6 Hz, 1H), 3.16 (m,
1H), 3.11 (s, 2H), 2.75 (t, J = 12.8 Hz, 1H), 2.64 (t,
J = 11.6 Hz, 1H), 1.85 (m, 2H), 1.73 (s, 2H), 1.61
2
3
.41 (dd, J = 7.5 Hz, 5.0 Hz, 1H), 3.27 (m, 1H), 3.19
(
(
1
1
(
m,1H),3.10(s,2H),3.01(dd,J = 7.5 Hz,5.0 Hz,1H),2.15
q, J = 7.0 Hz, 1H), 1.84 (q, J = 7.0 Hz, 1H), 1.72 (s, 2H),
1
3
(m, 1H), 1.47 (s, 2H), 1,41 (m, 1H);
C NMR
1
3
.47 (s, 2H); C NMR (100 MHz, D O) d 169.7, 158.9,
(100 MHz, D O) d 169.1, 158.9, 114.9, 52.9, 46.1,
2
2
14.8, 52.8, 49.4, 49.0, 44.6, 40.3, 29.6, 28.0. HRMS (ES)
calcd for C H N O
44.3, 43.7, 40.3, 28.1, 27.4, 20.8. HRMS (ES) (m/z):
M+H
+
+
m/z): M+H
288.1784,
calcd for C H N O 302.1941, found
11 24 7 3
1
0
22
7
3
found288.1783;Anal. Calcdfor C H N O Æ3TFAÆ0.75-
302.1940; Anal. Calcd for C H N O Æ2.5TFAÆH O:
C, 31.79, H, 4.59, N, 16.22; Found: C, 31.64, H,
4.54, N, 16.10.
1
0
21
7
3
11 23 7 3 2
H O:C, 29.89, H, 4.00, N, 15.25; Found: C, 29.89, H, 4.13,
N,15.35.
2
5.2.5. (3R)-3-((2S)-2-tert-Butoxycarbonylamino-5-nitro-
guanidinopentanoylamino)-piperidine-1-carboxylic acid
5.2.9. 4-((2S)-2-tert-Butoxycarbonylamino-5-nitroguanid-
inopentanoylamino)-piperidine-1-carboxylic acid tert-butyl
tert-butyl ester (14). Boc-L-Arg(NO )-OH (224 mg,
2
ester (16). Boc-L-Arg(NO )-OH (319 mg, 1.0 mmol),
2
0
.70 mmol), HOBt (95 mg, 0.70 mmol), HBTU
265 mg, 0.70 mmol), DIEA (90 mg, 0.70 mmol), and
HOBt (135 mg, 1.0 mmol), HBTU (379 mg, 1.0 mmol),
DIEA (129 mg, 1.0 mmol), and 1-Boc-4-aminopiperidine
(100 mg, 0.50 mmol) were used as described above to pro-
(
(
R)-1-Boc-3-aminopiperidine (70 mg, 0.35 mmol) were
used as described above to provide 14 (162 mg,
0
vide 16 (219 mg, 0.44 mmol, 87%) as a white solid; mp
1
.32 mmol, 92%) as a white solid; mp 111.6–113.0 ꢁC.
119.4–120.1 ꢁC. H NMR (500 MHz, CDCl ) d 8.83
3
1
H NMR (500 MHz, CDCl ) d 8.69 (s, 1H), 7.10 (s,
(s, 1H), 7.25 (s, 1H), 5.68 (s, 1H), 4.31 (s, 1H), 3.97
(s, 2H), 3.86 (t, J = 3.5 Hz, 1H), 3.50 (s, 1H), 3.24
(s, 1H), 2.84 (m, 2H), 1.82 (m, 3H), 1.70 (br s, 5H), 1.43
3
1
(
1
H), 5.70 (s, 1H), 3.78 (s, 2H), 3.54 (s, 1H), 3.28–3.42
m, 3H), 3.03 (s, 2H), 1.83 (s, 1H), 1.75 (s, 1H), 1.63–
1
3
13
.66 (m, 6H), 1.40 (s, 9H), 1.38 (s, 9H), rotamers;
C
(s, 9H), 1.41 (s, 9H), rotamers; C NMR (125 MHz,
CDCl ) d 171.5, 159.6, 156.8, 155.0, 80.5, 80.0, 60.7,
NMR (125 MHz, CDCl ) d 171.8, 159.6, 156.5, 155.2,
3
3
8
0.4, 80.1, 60.6, 54.1, 45.8, 40.8, 38.9, 31.2, 30.1, 28.6,
8.5, rotamers; HRMS (ES) (m/z): M+H calcd for
47.0, 40.7, 31.8, 28.7, 28.6, 21.3, 14.4, rotamers; HRMS
(ES) (m/z): M+H calcd for C H N O 502.2989, found
502.2978.
+
+
2
C H N O 502.2989, found 502.2994.
2
1
40
7
7
2
1
40
7
7

J. Seo et al. / Bioorg. Med. Chem. 15 (2007) 1928–1938
1935
5.2.10. (2S)-2-Amino-5-nitroguanidinopentanoic acid pip-
eridin-4-ylamide (7). A white solid was obtained after Boc
36.4, 35.5, 28.5, 28.3, rotamers; MS (ESI, CH OH)
3
+
[M+Na ] = 293.3.
1
deprotection; mp 78.5–81.0 ꢁC. H NMR (400 MHz,
D O) d 3.78 (t, J = 5.6 Hz, 2H), 3.25 (s, 2H), 3.11
5.2.14. (N-Boc-trans-4-azido-L-proline (28). A mixture of
27 (0.10 g, 0.37 mmol) in THF (3 mL) and 5% aq LiOH
(3 mL) was stirred at 0 ꢁC. The reaction temperature
was allowed to rise to room temperature, and stirring
continued for 17 h. After evaporation of THF under re-
duced pressure, the aqueous solution was washed twice
with EtOAc (5 mL), acidified with 1 N HCl to pH 3,
and extracted twice with EtOAc (10 mL). The organic
extract was washed with brine, dried over Na SO , fil-
2
(
2
s, 2H), 2.92 (t, J = 12.0 Hz, 2H), 1.93 (t, J = 14.0 Hz,
1
3
H), 1.73 (s, 2H), 1.52 (m, 4H); C NMR (100 MHz,
D O) d 168.8, 158.8, 114.8, 52.9, 44.8, 42.9, 40.3, 28.1,
2
2
3
1
7.8, 27.7; Anal. Calcd for C H N O Æ2.5TFA: C,
2.77, H, 4.38, N, 16.72; Found: C, 32.63, H, 4.60, N,
7.00.
11 23 7 3
5
.2.11. (N-Boc-4-hydroxy-L-proline lactone (25). To a
2
4
stirred ice-cold solution of Boc-Hyp-OH (4.48 g,
9.4 mmol) and triphenylphosphine (6.26 g, 23.2 mmol)
tered, and evaporated. The residue was further dried
under reduced pressure and used in the next reaction
1
1
in freshly distilled THF (100 mL) under nitrogen was
added DIAD (4.70 g, 23.2 mmol) dropwise. The reac-
tion temperature was allowed to rise to room tempera-
ture, and stirring continued for 20 h. The solvent was
evaporated under reduced pressure, and the residue
was directly purified by flash column chromatography
without further purification (28, 0.86 mg, 91%).
H
NMR (500 MHz, CDCl ) d 10.4 (s, 1H), 4.40 and 4.30
3
(2 · t, J = 6.5 Hz and J = 7.0 Hz, 1H), 4.18 (s, 1H),
3.61–3.66 (m, 1H), 3.55 and 3.44 (2 · d, J = 12.0 Hz,
1H), 2.18–2.36 (m, 2H), 1.38 and 1.43 (2 · s, 9H), rota-
1
3
mers; C NMR (125 MHz, CDCl ) d 177.1, 175.9,
3
(
EtOAc/hexane = 1:1, R = 0.40), to give 4.05 g (97%)
f
171.9, 155.1, 153.9, 81.7, 81.5, 59.2, 58.9, 57.9, 57.7,
51.7, 51.5, 36.4, 35.2, 28.5, 28.3, 21.2, 14.3, rotamers;
1
of 25 as a white foamy solid; mp 89.9–91.2 ꢁC.
NMR (400 MHz, CDCl ) d 4.95 (s, 1H), 4.38 (br s,
H
+
MS (ESI, CH CN) [3M+Na ] = 790.5.
3
3
1H), 3.37 (d, J = 10.4 Hz, 1H), 3.24 (d, J = 10.4 Hz,
1H), 2.04 (d, J = 10.8 Hz, 1H), 1.87 (d, J = 6.4 Hz,
1H), 1.28 (s, 9H), rotamers.
5.2.15. (N-Boc-trans-4-azido-L-prolinamide (29). Com-
pound 28 (0.14 g, 0.55 mmol) was dissolved in THF
(5 mL) at ambient temperature, and the solution was
5
.2.12. (N-Boc-cis-4-hydroxy-L-proline methyl ester (26).
cooled to ꢀ10 ꢁC. To this solution were added N-
methylmorpholine (0.09 g, 0.83 mmol) and iso-butyl
chloroformate (0.11 g, 0.83 mmol) successively, and
stirring continued for 30 min at ꢀ10 ꢁC. Aqueous
ammonia (29%, 0.15 mL) was added, and the mixture
was stirred at ꢀ10 ꢁC for 30 min then at room temper-
ature for 2.5 h. After addition of water (10 mL), the
solution was extracted twice with EtOAc (10 mL),
washed with brine, dried over Na SO , filtered, and
A solution of 25 (0.11 g, 0.50 mmol) and sodium azide
0.06 g, 1.00 mmol) in dry MeOH (15 mL) was stirred
(
for 5 h at 40 ꢁC under argon. The solvent was evaporat-
ed under reduced pressure, and the residue was parti-
tioned between water and EtOAc. The organic layer
was washed with brine, dried over Na SO , and concen-
trated. The residue was purified by flash column chro-
matography (EtOAc/hexane = 2:1, R = 0.3) to give 26
2
4
f
2
4
1
(
0.12 g, 98%) as a white foamy solid; 81.6–82.0 ꢁC. H
evaporated. The residue was purified by flash column
chromatography (EtOAc, 100%, R = 0.30) to afford
NMR (500 MHz, CDCl ) d 4.30 (d, J = 7.0 Hz, 1H),
3
f
1
29 (0.12 g, 83%) as an oil. H NMR (500 MHz,
3
2
.71 and 3.70 (2 · s, 3H), 3.66 (m, 1H), 3.47–3.56 (m,
H), 2.25–2.33 (m, 1H), 2.03 (t, J = 6.5 Hz, 1H), 1.35
CDCl ) d 6.99 and 6.54 (2 · s, 1H), 6.38 and 6.28
3
1
3
and 1.39 (2 · s, 9H), rotamers; C NMR (125 MHz,
CDCl ) d 175.4, 175.1, 154.7, 154.0, 80.5, 71.1, 70.0,
58.1, 57.8, 55.8, 55.2, 52.9, 52.6, 38.7, 38.0, 28.5, 28.4,
rotamers.
(2 · s, 1H), 4.36 and 4.21 (2 · s, 1H), 4.16 (d,
J = 4.5 Hz, 1H), 3.41–3.57 (m, 2H), 2.43 and 2.30
(2 · m, 1H), 2.21 and 2.07 (2 · m, 1H), 1.41 (s, 9H),
3
1
3
rotamers;
C NMR (125 MHz, CDCl ) d 175.3,
3
174.2, 171.4, 155.4, 154.3, 81.3, 60.6, 59.5, 59.2, 58.3,
51.8, 36.9, 34.3, 28.5, 21.2, 14.4, rotamers; MS (ESI,
5
.2.13. (N-Boc-trans-4-azido-L-proline methyl ester (27).
+
CH CN/H O) [M+H ] = 256.0.
DEAD (0.09 g, 0.53 mmol) was slowly added dropwise
via syringe to an ice-cold stirred solution of 26 and tri-
phenylphosphine (0.13 g, 0.51 mmol) in dry THF
3
2
5.2.16. (N-Boc-trans-4-amino-L-prolinamide (30). Com-
pound 29 (33 mg, 0.13 mmol) was dissolved in MeOH
(3 mL) and subjected to catalytic hydrogenation with
10% Pd/C (8 mg) at 1 atm. After 2 h, the reaction mix-
ture was filtered on Celite, and the filtrate was concen-
(
5 mL) under nitrogen. DPPA (0.14 g, 0.51 mmol) was
then added dropwise via syringe to the solution, and
the temperature was allowed to rise to room tempera-
ture. After 21 h of stirring, the solvent was evaporated
under reduced pressure, and the residue was purified
by flash column chromatography (EtOAc/hexane = 1:4,
trated to dryness to afford an oily residue 30 (28 mg,
94%). R = 0.40 (EtOAc/MeOH/Et N = 1:1:0.1);
1
H
f
3
R = 0.30), to give 0.10 g (94%) of 27 as a colorless oil.
f
NMR (500 MHz, acetone-d ) d 7.21 and 7.16 (2 · s,
6
1
H NMR (500 MHz, CDCl ) d 4.33 and 4.26 (2 · t,
1H), 6.82 and 6.76 (2 · s, 1H), 4.35 and 4.32 (2 · s,
1H), 4.20 (s, 1H), 3.65 (m, 2H), 3.26 (m, 1H), 3.10 (br
s, 2H), 2.07–2.14 (m, 2H), 1.47 and 1.43 (2 · s, 9H),
3
J = 6.0 Hz and J = 7.0 Hz, 1H), 4.14 (s, 1H), 3.67 (s,
3H), 3.61–3.65 (m, 1H), 3.51 and 3.41 (2 · d,
J = 11.5 Hz and J = 11.0 Hz, 1H), 2.23–2.28 (m, 1H),
1
3
rotamers; C NMR (125 MHz, acetone-d ) d 175.8,
6
2
.08–2.13 (m, 1H), 1.34 and 1.40 (2 · s, 9H), rotamers;
175.1, 167.9, 154.9, 154.1, 79.3, 79.2, 60.1, 59.8, 58.0,
57.3, 53.1, 38.5, 36.7, 28.1, 28.0, rotamers; MS (ESI,
1
3
C NMR (125 MHz, CDCl ) d 173.1, 172.9, 154.1,
3
+
CH OH) [M+H ] = 230.1.
1
53.5, 80.7, 59.4, 58.9, 57.9, 57.5, 52.5, 52.4, 51.6, 51.4,
3

1936
J. Seo et al. / Bioorg. Med. Chem. 15 (2007) 1928–1938
5
.2.17. (N-Boc-4-trans-[(2S)-5-nitroguanidino-2-(tert-but-
5.2.20. (2S)-2-(tert-Butoxycarbonylmethylamino)-4-cya-
nobutyric acid (18). To an ice-cold solution of 17
(4.15 g, 18.2 mmol) in THF (40 mL) was added iodom-
ethane (20.6 g, 145.4 mmol) and NaH (1.31 g,
54.5 mmol), and the mixture was stirred at room tem-
perature for 3 days. EtOAc (10 mL) were added fol-
lowed by water (20 mL). The pH of the solution was
adjusted to 9 by addition of 0.1 N HCl, and the solution
was concentrated to a small volume. Sodium thiosulfate
(0.1 N, 5 mL) was added followed by water (20 mL).
The solution was extracted twice with ether, and the
water layer was acidified to pH 3 with 5% citric acid with
stirring. The solution was extracted three times with
EtOAc, and the combined ethyl acetate solution was
washed with brine four times, dried over Na SO , and
oxycarbonylmethylamino)-pentanoylamino]-L-proli-
namide (31). To a solution of 21 (124 mg, 0.37 mmol),
HOBt (62 mg, 0.46 mmol), and HBTU (174 mg,
0
.46 mmol) in dry DMF (1 mL) and dry CH Cl2
2
(
4 mL) was added 30 (70 mg, 0.31 mmol) in dry DMF
(3 mL) dropwise at 0 ꢁC. DIEA (80 mg, 0.62 mmol) in
dry CH Cl (1 mL) was added slowly, and stirring con-
2
2
tinued overnight at room temperature under argon. The
solvent was removed under reduced pressure, and the
residue was treated with EtOAc (10 mL). The organic
mixture was washed successively with 5% NaHCO3,
water, 0.5 N HCl, and brine, and dried over Na SO .
The solution was concentrated in vacuo, and the residue
was purified by flash column chromatography (CH Cl /
MeOH = 10:1, R = 0.15) to afford 31 (46 mg, 30%) as a
2
4
2
2
2
4
concentrated. The residue was purified by flash column
chromatography (CH Cl /MeOH/AcOH = 20:1:0.1, R =
0.25) to afford 18 (3.66 g, 83%) as a yellowish crystalline
f
1
white foam. H NMR (500 MHz, acetone-d ) d 7.43 (s,
6
2
2
f
1
4
3
2
1
H), 7.11 and 7.04 (2 · s, 1H), 6.55 (s, 1H), 4.64 and
.50 (2 · s, 1H), 4.31 and 4.28 (2 · s, 1H), 3.72 (s, 1H),
.40 (s, 2H), 3.29 (m, 2H), 2.97 and 2.81 (2 · s, 3H),
.26 (s, 1H), 2.21 (m, 1H), 1.96–2.03 (m, 2H), 1.54–
1
solid; mp 86.2–87.6 ꢁC. H NMR (500 MHz, CD OD) d
3
4.63 and 4.43 (2 · m, 1H), 2.87 and 2.85 (2 · s, 3H),
2.47–2.59 (m, 2H), 2.30 (m, 1H), 2.16 (m, 1H), 1.47
and 1.49 (2 · s, 9H), cis & trans; C NMR (125 MHz,
CD OD) d 172.3, 156.8, 156.1, 122.3, 119.5, 81.2, 80.6,
1
3
.77 (m, 4H), 1.45 (s, 9H), 1.41 (s, 9H), rotamers;
+
HRMS (ES) (m/z): M+H calcd for C H N O
2
2
41
8
8
3
5
45.3047, found 545.3046.
59.2, 58.2, 32.3, 31.5, 27.6, 27.5, 25.5, 24.8, 13.8, cis &
trans; MS (ESI, CH Cl ) [M+Na ] = 265.1.
+
2
2
5.2.18. 4-trans-((2S)-5-Nitroguanidino-2-methylamino-
pentanoylamino)-L-prolinamide (8). Compound 31
5.2.21.
(2S)-2-(tert-Butoxycarbonylmethylamino)-5-
(
(
33 mg, 0.066 mmol) was treated with TFA/CH Cl2
2
3 mL/3 mL) at 0 ꢁC under nitrogen. The reaction tem-
nitroguanidinopentanoic acid (21). Compound 18
(0.69 g, 2.83 mmol) was dissolved in a mixture of
MeOH/AcOH (10 mL/10 mL) and subjected to catalytic
hydrogenation with Pd(OH)2 on carbon (20 wt %,
93 mg) at 1 atm. After overnight stirring, the reaction
mixture was filtered through Celite, and the filtrate
was concentrated to dryness to afford 19 (0.69 g, 99%)
as a solid. Without further purification, 19 (0.69 g,
2.83 mmol) was dissolved in absolute EtOH (20 mL).
To this stirred solution were added 2-methyl-1-nitro-2-
perature was then allowed to rise to room temperature,
and stirring continued for 1 h. Excess TFA and solvent
were removed by evaporation in vacuo. The residue
was repeatedly dissolved in CH Cl (10 mL) and the
solvents evaporated to remove traces of TFA. The res-
idue was dissolved in a small amount of water, and the
solution was washed with CH Cl and lyophilized to
give 8 as a white solid (47 mg, 96%); mp 108.7–
1
J = 8.0 Hz, 1H), 4.43 (t, J = 6.0 Hz, 1H), 3.75 (t,
J = 6.5 Hz, 1H), 3.67 (dd, J = 7.0 Hz and J = 5.5 Hz,
2
2
2
2
1
16
10.0 ꢁC. H NMR (500 MHz, D O) d 4.48 (t,
thiopseudourea (1.9 g, 14.2 mmol) and triethylamine
2
(2.9 g, 28.3 mmol), successively. The reaction tempera-
ture was then raised to 40 ꢁC, and stirring continued
overnight under nitrogen. The solvent was evaporated
under reduced pressure, and the residue was purified
by flash column chromatography (CH Cl /MeOH/
1
(
(
H), 3.25 (dd, J = 7.5 Hz and J = 5.5 Hz, 1H), 3.17
s, 2H), 2.55 (s, 3H), 2.35 (m, 2H), 1.83 (s, 2H), 1.51
s, 2H); Anal. Calcd for C H N O Æ2.75TFAÆ1.5H O:
1
2
24
8
4
2
2
2
C, 30.69, H, 4.38, N, 16.36; Found: C, 30.49, H, 4.27,
N, 16.58.
AcOH = 10:1:0.1, R = 0.25) to give 21 (0.41 g, 43%) as
f
1
a clear syrup. H NMR (500 MHz, D O) d 4.42 and
2
4
.12 (m, 1H), 3.20 (m, 2H), 2.74 and 2.68 (2 · s, 3H),
5
.2.19. (2S)-2-tert-Butoxycarbonylamino-4-cyanobutyric
acid (17). A solution of Boc-L-Gln-OH (100 mg,
.41 mmol) in dry pyridine (3 mL) and acetic anhy-
1.82 (s, 2H), 1.51 (s, 2H), 1.30 and 1.24 (2 · s, 9H), rota-
+
mers; MS (ESI, CH OH) [2M+H ] = 666.9.
3
0
dride (50 mg, 0.49 mmol) was stirred at room temper-
ature for 6 h and concentrated. The residue was taken
up in EtOAc, and the solution was washed three times
with 5% citric acid (10 mL) and brine (10 mL), and
dried over Na SO . The solution was concentrated in
5.3. General method for the syntheses of 9–11
Compounds 9–11 were prepared by the same procedure
described above for the syntheses of 3–7 except Boc-L-
MeArg(NO )-OH (21) was used instead of Boc-L-
2
4
2
vacuo, and the residue was purified by flash column
chromatography (CH Cl /MeOH = 10:1, R = 0.15) to
Arg(NO )-OH.
2
2
2
f
1
afford 17 (56 mg, 60%) as a syrup. H NMR
5.3.1. (3R)-3-[(2S)-2-(tert-Butoxycarbonylmethylamino)-
5-nitroguanidinopentanoylamino]-pyrrolidine-1-carboxylic
(
500 MHz, acetone-d ) d 6.33 (d, J = 7.0 Hz, 1H),
6
4
2
.27 (d, J = 4.0 Hz, 1H), 2.62 (t, J = 7.0 Hz, 2H),
acid tert-butyl ester (22). Boc-L-MeArg(NO )-OH
2
1
.25 (m, 1H), 2.05 (m, 1H), 1.42 (s, 9H); C NMR
3
(100 mg, 0.30 mmol), HOBt (45 mg, 0.33 mmol), HBTU
(125 mg, 0.33 mmol), DIEA (49 mg, 0.38 mmol), and
(R)-(+)-1-Boc-3-aminopyrrolidine (47 mg, 0.25 mmol)
were used as described above to provide 22 (85 mg,
(
125 MHz, acetone-d ) d 172.6, 156.0, 119.3, 79.0,
6
5
[
2.7, 28.8, 27.9, 13.8; MS (ESI, CH OH)
3
+
2M+H ] = 457.3.

J. Seo et al. / Bioorg. Med. Chem. 15 (2007) 1928–1938
1937
1
0
1
3
1
9
.17 mmol, 68%). H NMR (500 MHz, CDCl ) d 4.59 (s,
C, 32.01, H, 4.48, N, 14.52; Found: C, 31.96, H, 4.37, N,
14.20.
3
H), 4.37 (t, J = 5.0 Hz, 1H), 3.57 (d, J = 5.5 Hz, 1H),
.38 (m, 4H), 3.13 (m, 1H), 2.74 (s, 3H), 2.12 (m, 1H),
.93 (m, 1H), 1.81 (s, 1H), 1.66–1.74 (m, 3H), 1.45 (s,
5.4. Enzyme and assay
+
H), 1.43 (s, 9H), rotamers; HRMS (ES) (m/z): M+H
calcd for C H N O 502.2989, found 502.2972.
2
All of the NOS isoforms used were recombinant en-
zymes overexpressed in E. coli from different sources.
1
40
7
7
2
4
25
5
acid ((3R)-pyrrolidin-3-yl)amide (9). A white foamy solid
.3.2.
(2S)-2-Methylamino-5-nitroguanidino-pentanoic
Murine macrophage iNOS, rat nNOS, and bovine
2
6
eNOS were expressed and isolated as reported. Nitric
oxide formation from NOS was monitored by the hemo-
1
was obtained after deprotection; mp 59.6–59.9 ꢁC. H
2
7
NMR (500 MHz, D O) d 4.31 (t, J = 5.0 Hz, 1H), 3.67
globin capture assay at 30 ꢁC as described previously.
Briefly, nitric oxide transforms oxyhemoglobin (Fe ) to
2
2+
(
t, J = 6.5 Hz, 1H), 3.43 (dd, J = 7.5 Hz and
J = 5.0 Hz, 1H), 3.24 (m, 2H), 3.08 (m, 3H), 2.47 (s,
H), 2.17 (m, 1H), 1.76–1.81 (m, 3H), 1.43 (s, 2H); Anal.
Calcd for C H N O Æ3TFAÆH O: C, 30.87, H, 4.27, N,
3
+
methemoglobin (Fe ), which is monitored at the maxi-
mum absorbance (401 nm) upon the addition of the en-
zyme into a mixture of substrate L-Arg, inhibitor,
cofactors, and Hepes buffer. A solution of nNOS or
eNOS contained 10 lM L-arginine, 1.6 mM CaCl2,
11.6 lg/mL calmodulin, 100 lM DTT, 100 lM NADPH,
3
1
1
23
7
3
2
1
4.82; Found: C, 30.71, H, 4.20, N, 15.01.
5.3.3. (3R)-3-[(2S)-2-(tert-Butoxycarbonyl-methyl-ami-
no)-5-nitroguanidinopentanoyl-amino]-piperidine-1-car-
6.5 lM H B, 6 lM oxyhemoglobin, and specified inhibi-
4
boxylic acid tert-butyl ester (23). Boc-L-MeArg(NO )-
2
OH (120 mg, 0.36 mmol), HOBt (49 mg, 0.36 mmol),
tor concentrations in 100 mM Hepes (pH 7.5) in 600 lL
total volume; iNOS contained the same concentrations
HBTU
(137 mg,
0.36 mmol),
DIEA
(70 mg,
.54 mmol), and (R)-1-Boc-3-aminopiperidine (34 mg,
of cofactors, except CaCl and calmodulin were not
2
0
0
2
added. The assays were initiated by addition of enzyme
and recorded on a Perkin-Elmer Lambda 10 UV/vis
spectrophotometer.
.18 mmol) were used as described above to provide
3 (67 mg, 0.13 mmol, 71%). H NMR (500 MHz,
1
CDCl ) d 4.52 (s, 1H), 3.88 (s, 1H), 3.57 (s, 1H),
3
3
2
2
.36 (m, 3H), 3.27 (s, 1H), 3.15 (s, 1H), 2.76 (s, 3H),
.00 (m, 1H), 1.84 (s, 1H), 1.70 (s, 2H), 1.62 (m,
H), 1.55 (m, 2H), 1.48 (s, 9H), 1.46 (s, 9H), rotamers;
5.5. Determination of K values
i
The reversible inhibition of NOS by the L-nitroarginin-
amide compounds was studied under initial rate condi-
tions with the hemoglobin assay as described above.
The parameters of the following inhibition equation
were fitted to the initial velocity data: % inhibi-
tion = 100[I]/{[I] + K (1 + [S]/K )}. K values for L-ar-
i
ginine were 1.3 lM (nNOS), 8.3 lM (iNOS), and
1.7 lM (eNOS). The selectivity of an inhibitor was
+
HRMS (ES) (m/z): M+H calcd for C H N O
7
2
2
42
7
5
16.3146, found 516.3137.
2
8
5.3.4. (2S)-2-Methylamino-5-nitroguanidino-pentanoic
acid ((3R)-piperidin-3-yl)amide (10). A white foamy solid
m
m
1
was obtained after deprotection; mp 61.0–61.6 ꢁC. H
NMR(500 MHz,D O)d3.94(s,1H),3.71(s,1H),3.36(d,
2
J = 12.0 Hz, 1H), 3.21 (m, 1H), 3.16 (s, 2H), 2.78 (dt,
J = 12.0 Hz, 6.0 Hz, 2H), 2.54 (s, 3H), 1.82–1.88 (m, 4H),
defined as the ratio of the respective K values.
i
1
.66 (d, J = 12.0 Hz, 1H), 1.40–1.49 (m, 3H); Anal. Calcd
5.6. Docking analysis
forC H N O Æ3TFA:C,32.88,H,4.29,N,14.91;Found:
C,32.58,H,4.60,N,14.96.
1
2
25
7
3
Molecular modeling calculations were performed using
the software packages SYBYL 6.8 and AutoDock 3.0.5
running on a Silicon Graphics Octane 2 workstation.
The protein structure used in the docking study was
5.3.5. 4-[(2S)-2-(tert-Butoxycarbonyl-methyl-amino)-5-
nitroguanidino-pentanoylamino]-piperidine-1-carboxylic
acid tert-butyl ester (24). Boc-L-MeArg(NO )-OH
2
2
9
prepared as described previously. The 3-D structure
of the ligand was built in SYBYL 6.8 by modifying
the molecular structure of 2, which was extracted from
the crystal structure (pdb id: 1p6j). Using the SYBYL
program, correct atom types were assigned assuming
physiological pH. Energy minimizations were per-
formed following both the addition of polar hydrogen
atoms and partial atom charge calculations by the
(
(
56 mg, 0.17 mmol), HOBt (24 mg, 0.18 mmol), HBTU
68 mg, 0.18 mmol), DIEA (27 mg, 0.21 mmol), and 1-
Boc-4-aminopiperidine (28 mg, 0.14 mmol) were used
as described above to provide 24 (63 mg, 0.12 mmol,
1
8
3
2
1
7%). H NMR (500 MHz, CDCl ) d 4.57 (s, 1H),
3
.97 (s, 2H), 3.87 (m, 1H), 3.34 (s, 2H), 2.86 (m, 2H),
.74 (s, 3H), 1.94 (m, 1H), 1.83 (m, 2H), 1.71 (m, 2H),
.44 (m, 18H), 1.31 (m, 3H), rotamers; HRMS (ES)
3
0
Gasteiger–Marsilli method. The torsion and rotatable
bonds in the ligand were defined by AutoTors, an aux-
iliary program of AutoDock 3.0.5, which also united
the nonpolar hydrogens and partial atomic charges to
the bonded carbon atoms. Parameters for the docking
experiment were used as described in detail previous-
+
(
m/z): M+H calcd for C H N O 516.3146, found
7
2
2
42
7
5
16.3151.
5.3.6. (2S)-2-Methylamino-5-nitroguanidino-pentanoic acid
piperidin-4-ylamide (11). A white foamy solid was
1
27
obtained after deprotection; mp 58.9–59.3 ꢁC. H NMR
ly. A flexible docking calculation yielded 100 docked
(
500 MHz, D O) d 3.85 (m, 1H), 3.68 (t, J = 6.5 Hz,
conformations, and appropriate conformations were
chosen by visual comparison of the superimposition
of the guanidino moiety of the predicted conformations
and that of 2.
2
1
3
1
H), 3.30 (m, 2H), 3.14 (s, 2H), 2.96 (m, 2H), 2.53 (s,
H), 1.94–2.02 (m, 2H), 1.79 (s, 2H), 1.55–1.63 (m, 2H),
.47 (m, 2H); Anal. Calcd for C H N O Æ3TFAÆH O:
1
2
25
7
3
2

1938
J. Seo et al. / Bioorg. Med. Chem. 15 (2007) 1928–1938
13. G o´ mez-Vidal, J. A.; Mart a´ sek, P.; Roman, L. J.; Silver-
man, R. B. J. Med. Chem. 2004, 47, 703–710.
Acknowledgments
1
4. Flinspach, M. L.; Li, H.; Jamal, J.; Yang, W.; Huang, H.;
Hah, J.; G o´ mez-Vidal, J. A.; Litzinger, E. A.; Silverman,
R. B.; Poulos, T. L. Nat. Struct. Mol. Biol. 2004, 11,
We are grateful to the National Institutes of Health
GM 49725 to R.B.S., and GM52419 and HL30050 to
Prof. Bettie Sue Masters, in whose laboratory P.M.
and L.J.R. work) for financial support of this work.
(
5
4–59.
1
1
1
5. Huang, H.; Mart a´ sek, P.; Roman, L. J.; Silverman, R. B.
J. Med. Chem. 2000, 43, 2938–2945.
6. Ji, H.; G o´ mez-Vidal, J. A.; Mart a´ sek, P.; Roman, L. J. J.;
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7. Rodgers, J. D.; Johnson, B. L.; Wang, H.; Erickson-
Viitanen, S.; Klabe, R. M.; Bacheler, L.; Cordova, B. C.;
Chang, C.-H. Bioorg. Med. Chem. Lett. 1998, 8, 715–
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Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.
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1