Imidazole-containing amino acids as selective inhibitors of nitric oxide synthases

Article abstract of DOI:10.1016/S0968-0896(99)00117-0

Two series of imidazole-containing amino acids (1a-e and 2a-c), all larger homologues and analogues of L-histidine, were prepared. Since imidazole and phenyl substituted imidazoles have been reported to be inhibitors of NOS and the mode of action of these compounds as heme ligands is a potential mechanism of inhibitory action, we designed imidazole-containing amino acids as combined inhibitors at both the amino acid as well as heme binding sites. To study the influence of the distance between the amino acid moiety and the imidazole moiety on inhibitory potency, the number of carbons between these two functional groups was varied from two to six. The structure-activity relationships of this class of inhibitors can be correlated with the distance between the heme and the amino acid binding sites of the enzyme. Two of the compounds (1b and 1d) with three and five methylenes between the imidazole and amino acid functional groups, respectively, were found to be potent and selective inhibitors for nNOS and iNOS over eNOS. When phenyl was substituted on the nitrogen of the imidazole, both the potency and isoform selectivity diminished.

Full text of DOI:10.1016/S0968-0896(99)00117-0

Bioorganic & Medicinal Chemistry 7 (1999) 1941±1951
Imidazole-Containing Amino Acids as Selective Inhibitors of
Nitric Oxide Synthases
Younghee Lee, a,1 Pavel Martasek, b,2 Linda J. Roman, b,2
Bettie Sue Siler Masters b,2 and Richard B. Silverman a,*
aDepartment of Chemistry and Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston,
IL 60208-3113, USA
bDepartment of Biochemistry, The University of Texas Health Science Center, San Antonio, TX 78284-7760, USA
Received 8 December 1998; accepted 5 April 1999
AbstractÐTwo series of imidazole-containing amino acids (1a±e and 2a±c), all larger homologues and analogues of l-histidine,
were prepared. Since imidazole and phenyl substituted imidazoles have been reported to be inhibitors of NOS and the mode of
action of these compounds as heme ligands is a potential mechanism of inhibitory action, we designed imidazole-containing amino
acids as combined inhibitors at both the amino acid as well as heme binding sites. To study the in¯uence of the distance between the
amino acid moiety and the imidazole moiety on inhibitory potency, the number of carbons between these two functional groups
was varied from two to six. The structure±activity relationships of this class of inhibitors can be correlated with the distance
between the heme and the amino acid binding sites of the enzyme. Two of the compounds (1b and 1d) with three and ®ve methyl-
enes between the imidazole and amino acid functional groups, respectively, were found to be potent and selective inhibitors for
nNOS and iNOS over eNOS. When phenyl was substituted on the nitrogen of the imidazole, both the potency and isoform selec-
tivity diminished. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
The normal biological functions regulated by NO are
attributed to the di€erent NOS isoforms. However,
overproduction of NO has been implicated in numerous
disease states,2 such as septic shock,3 in¯ammatory
arthritis,4,5 seizures,6 schizophrenia,7 Alzheimer's dis-
ease,8 impotence,9 and susceptibility to infection.10
Therefore, potent and isoform selective inhibitors of
NOS are desired for medicinal purposes as well as to
de®ne the roles of each NOS isoform in biological sys-
tems. Methylarginine11 and nitroarginine12 are two of
the ®rst inhibitors of NOS, and they are derived from a
modi®cation of the terminal amino group of the guani-
dine of l-arginine. Numerous other arginine analogues
have been made,2 although, except for nitroarginine,
No-propyl-l-arginine13 and N5-(imino-3-butenyl)-l-
ornithine,14 most of these inhibitors lack selectivity
among the isozymes. Non-amino acid NOS inhibitors
also have been reported, including isothioureas,15,16
amidines,17±21 guanidines,22,23 imidazole,24,25 and inda-
zoles.26 Because NOS contains heme, one of the strategies
for its inhibition has been to design compounds that ligate
to the heme. Imidazole24,25 and several phenyl-substituted
imidazoles27 have been known to be inhibitors of NOS
with a characteristic binding spectrum indicating that
Nitric oxide (NO) has diverse roles both in normal
and pathological processes including the regulation of
blood pressure, in neurotransmission, and in the
macrophage defense systems.1 NO is synthesized by
three isoforms of nitric oxide synthase (NOS; EC
1.14.13.39), two of which, one in endothelial cells
(eNOS) and one in neuronal cells (nNOS), are con-
stitutive, and the one in macrophage cells is inducible
(iNOS). All NOS isoforms are heme-containing and
require NADPH, FAD, FMN, and tetrahydrobiopterin
for the endogenous synthesis of NO and l-citrulline
from l-arginine. The two constitutive isozymes are
Ca2+ and calmodulin-dependent, whereas the inducible
form is independent of added calmodulin because its
calmodulin is tightly bound.
Key words: Nitric oxide synthase; inhibitors; imidazole-containing
amino acids; histidine analogues.
* Corresponding author. Tel.: +1-847-491-5653; fax: +1-847-491-
7713; e-mail: agman@chem.nwu.edu
Designed and carried out all of the experiments in this study.
Provided the recombinant eNOS and the Escherichia coli cells
expressed with bovine neuronal nNOS used in this study.
1
2
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00117-0

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Y. Lee et al. / Bioorg. Med. Chem. 7 (1999) 1941±1951
they interact at the heme binding site. Also, some of the
sulfur-containing citrulline analogues, such as l-thioci-
trulline, l-homothiocitrulline, and S-methyl-l-thiocitrul-
line, were synthesized and shown to be potent inhibitors of
NOS.28,29 These compounds were designed so that the
sulfur atom of the bound inhibitor would be in close
proximity to the heme iron, thereby encouraging the
interaction between the sulfur ligand and the heme iron
and increasing the binding anity of the inhibitors for
NOS. Further exploitation of the design of arginine ana-
logues that have the potential to bind to the active-site
heme is a series of S-2-amino-5-azolylpentanoic acids
(including 1b below) which was reported as nonselective
inhibitors of rat iNOS and nNOS.30
Results and Discussion
Syntheses of imidazole-containing amino acids (1a±e)
Among the compounds that we studied, l-homo-
histidine (1a) had been synthesized previously in nine
steps starting from N-benzyloxycarbonyl-l-glutamic
acid.33 However, this method is not applicable to the
preparation of a series of imidazole-containing amino acid
derivatives. Consequently, a new enantioselective gen-
eral synthesis of 1a±e was designed utilizing the reaction
of various imidazolyl alkyl bromides (5b±e) with chiral
lithiated bislactim ether 634 as the key step (Scheme 1).
1-(N,N-Dimethylsulfamoyl)imidazole (3)35 was pro-
tected with a tert-butyldimethylsilyl group (TBDMS) by
sequential treatment with n-butyllithium followed by
tert-butyldimethylsilyl chloride. The lithium salt of the
1,2-diprotected imidazole (4)36 was allowed to react
with a series of excess dibromoalkanes to a€ord bro-
moalkyl substituted imidazoles (5b±e). Carbon±carbon
bond formation between these alkyl bromides and lithi-
ated bislactim ether (6) proceeded in good yields. The
homologated bis-lactim ether products (7a±e) were
hydrolyzed with 0.25 N HCl to the amino acid ethyl
esters (8a±e). Under these reaction conditions the
TBDMS protecting group of the imidazole was cleaved.
Removal of the sulfamoyl group requires relatively vig-
orous conditions (re¯uxing 30% HBr for 4 h); conse-
quently, the ethyl esters also were hydrolyzed to a€ord
the desired amino acids (1b±e). The bromoethyl imida-
zole analogue 5a was not prepared by the route used for
the other analogues, because the dibromoethane under-
went elimination of HBr in the presence of n-butyl-
lithium, and not a trace of the substitution product 7a
was detected. Therefore 5a was obtained by a two-step
procedure employing a two-carbon homologation of
the 1,2-diprotected imidazole 4 with ethylene oxide
followed by conversion of the resulting alcohol (9) to
the bromide (5a) under standard conditions. Treatment
In view of the well-established anity of heme iron for
imidazole,31,32 we designed compounds containing an
imidazole positioned at various distances from the
amino acid part of the molecules to determine if there
was a di€erence in the distances between these two
potential pharmacophores at the active site of the three
isoforms of NOS. The imidazole and amino acid parts
of the molecules were separated by an alkyl chain con-
taining from two to six methylenes. Since phenyl-sub-
stituted imidazoles also bind to NOS,27 another related
series of compounds with a 1-phenyl-substituted imida-
zole also was synthesized. The syntheses of compounds
1a±e and 2a±c and the study of their inhibition of the
three isozymes of NOS are reported here.
Scheme 1.

Y. Lee et al. / Bioorg. Med. Chem. 7 (1999) 1941±1951
1943
of 5a with the lithiated bislactim ether 6 ( 70ꢀC in
THF) yielded the desired compound 7a as a major
component of the reaction.
The syntheses of the N-phenyl substituted imidazole
derivatives 2a±c were carried out by the same route as
the imidazole analogues, starting from 1-phenylimida-
zole (10, Scheme 2). Surprisingly, the TBDMS group of
13a±c survived the hydrolysis reaction conditions (0.25
N HCl), so the deprotection of the TBDMS group and
the hydrolysis of the ester group were carried out
simultaneously in a basic solution (2 N NaOH) to
a€ord compounds 2a±c.
Absorption spectra changes during interaction of 1a±e
with NOS
Figure 1. (a) Spectrum of the puri®ed recombinant iNOS (3 mM) in
HEPES bu€er (100 mM, pH 7.5). (b) Spectrum after addition of 1b (to
a ®nal concentration of 1.3 mM). Virtually the same absorbance max-
imum shifts to 425 nm were observed when each of the other analo-
gues (1a, 1c, 1d, and 1e) was added to (a).(c) Spectrum after addition
of 1 mM of l-arginine to (b). Maximum absorbance shift to 395 nm
indicates the conversion of the heme to the high spin state.
One of the characteristics observed with the heme con-
taining enzymes when they are exposed to heme binding
inhibitors is a spectral change of the heme chromo-
phore. The known heme binding inhibitors of NOS,
such as imidazole24,25,35 and l-thiocitrulline,28,29 have
been shown to elicit a type II di€erence spectrum, a
transition of the high spin state of the unperturbed
heme to the low spin state when bound with the inhibi-
tors. As a ®rst step for our study of the imidazole-con-
taining amino acids, we examined whether 1a±e interact
as an axial sixth ligand of the heme cofactor of NOS.
Upon addition of 1a±e to iNOS, the initial maximum
absorbance of the enzyme at 398 nm shifted to 425 nm,
indicating that the imidazole moiety of these com-
pounds contributes as a sixth ligand to the cofactor
(Fig. 1). However, no spectral change was detected
when 20 mM l-histidine was added, suggesting that the
donation of the imidazole ligand to the heme is related
to the distance between the imidazole ring and the
amino acid. Although the degree of maximum absor-
bance shifts varied depending on the NOS isozyme and
carbon chain length of the compounds used, 1a±e
appear to be interacting with the heme based on the
spectral shift.
inhibitory potency. The ability of imidazole containing
amino acids to inhibit NOS was determined by the
hemoglobin capture assay as previously described.37 All
of the compounds tested are competitive inhibitors of
three recombinant isoforms of NOS, rat nNOS, murine
iNOS, and bovine eNOS. Since the homology for each
isozyme in rat, mouse, and bovine are very high, the use
of enzymes from di€erent species should not a€ect the
signi®cance of the results. None of the kinetic plots of
enzyme activity showed curvature, suggesting that there
was no time dependency to the inhibition. As shown in
Table 1, 1b and 1d are the two most potent inhibitors,
especially of nNOS, with values of Ki=2 mM for both
of the compounds. The isoform selectivity of 1b for
nNOS over iNOS and eNOS is 5 and 17, respectively.
The highest isoform selectivity for nNOS over eNOS
among these analogues was observed with 1d, which has
®ve methylenes between the imidazole and the amino
acid. This compound shows selectivities of 4 and 25 in
favor of nNOS over iNOS and eNOS, respectively.
Both the selectivity and the inhibitory potency were
E€ect of chain length on inhibitory potency
The in¯uence of the carbon chain length between the
imidazole and the amino acid then was measured on
Scheme 2.

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Y. Lee et al. / Bioorg. Med. Chem. 7 (1999) 1941±1951
Table 1. Inhibition of NOS by imidazole-containing amino acids
Selectivitya
All of the NOS isoforms have high speci®city for bind-
ing of l-amino acids at the active site, supported by the
fact that d-arginine is neither a substrate nor an inhi-
bitor of the enzyme. In addition to that, most of the
inhibitors classi®ed as substrate arginine analogues are
active only when the a-carbon is in the l-con®guration.
As demonstrated above, 1a±e are recognized by the
heme cofactor of the enzyme and could interact with the
amino acid binding site of the enzyme if appropriate
conditions, such as orientation and/or distance of the
imidazole and amino acid moieties are satis®ed. The
SAR of these analogues, derived from the kinetic data,
may be correlated to the distance of the heme from the
amino acid binding site. Inhibition data in Table 1 and
Figure 2 indicate that the potencies of 1a and 1e are
signi®cantly smaller than that of the other analogues.
This suggests that the distance between the imidazole
ring and the amino acid is either too short in the case of
1a or too long in the case of 1e for optimal binding at
the active site. Both elongation of 1a to a propylene
chain (1b) and shortening of 1e to a pentylene chain (1d)
results in dramatic increases in potency, suggesting that
the anity of these compounds for both the imidazole
and the amino acid binding sites are the strongest.
Interestingly, the potency of 1c decreases compared to
that of 1b and 1d. It also should be noted that the trend
described above is the same for all three isoforms of
NOS, suggesting that the general topology of the heme
cofactor and the residues responsible for recognition of
these molecules is similar.
Ki (mM)
Compound iNOS nNOS eNOS nNOS/iNOS nNOS/eNOS
1a
1b
1c
1d
1e
2a
2b
2c
950
10
35
8
40
100
50
120
50
140
170
2
65
2
150
80
100
70
500
33
150
50
250
50
17
350
65
6000
5.6
5.0
0.5
4.0
0.3
1.3
0.5
1.7
0.5
0.1
2.9
16.5
2.3
25.0
1.7
0.6
0.2
5.0
0.6
5
Imidazole
1-Phenyl
imidazole
110
1200
a
The ratio of the inverse of the Ki values; a value >1 indicates
better nNOS inhibition.
diminished when the distance between the imidazole
and the amino acid was either decreased to two methy-
lenes 1a or elongated to ®ve methylenes 1e. Compound
1c resulted in reduced inhibition compared to 1a and 1b,
but showed higher inhibition than 1a and 1e. Therefore,
the analogues with an odd number of methylenes
between the two potential pharmacophores are both
more potent and selective than the analogues with an
even number of methylenes (Fig. 2). It is clear that the
variation in the methylene chain length has a drastic
e€ect on the potencies of these compounds with all three
forms of NOS. The observed order of potency is as fol-
lows: 1b and 1d>1c>1a and 1e. Another compound in
the literature that was designed to be bifunctional is l-
thiocitrulline, which has three methylenes in between
the amino acid groups and the thiourea, which binds to
the heme.29 Its e€ect on nNOS and iNOS was tested; the
Ki values are 0.06 and 3.6 mM, respectively, or a 60-fold
selectivity for nNOS. When one more methylene is
added to the chain to give l-homothiocitrulline, the
potency was found to drop dramatically to 3 and
45 mM, respectively.29 These two compounds are similar
in binding distances to 1b and 1c and show a similar
trend.
One possible explanation for the observation that the
homologues with an odd number of methylene groups
are more potent inhibitors is the expected change in
geometry that results as methylene groups are added to
the chain (Fig. 3). If the imidazole is held rigid, bound
to the heme, then with each addition of a methylene,
assuming an all anti-coplanar arrangement of the back-
bone, the orientations of the amino and carboxylate
groups oscillate. This should have an e€ect on the
binding eciency.
Another possibility is that there is more than one amino
acid binding site at the active site of the enzyme, one
closer to the heme into which 1b ®ts well and the other a
little farther away into which 1d binds better. If there is
only one amino acid binding site at the active site, pos-
sibly two di€erent tautomeric forms of the mono-
substituted imidazole may interact with the heme
depending on the chain length of the methylenes. As
illustrated in Figure 4, the amino acid portion of 1b is
tightly bound to the amino acid binding site. To reach
the heme for favorable binding, the rather short methyl-
ene spacer of 1b needs to stretch out and also utilize
the nitrogen atom of the imidazole that is two carbons
away from alkyl chain. On the other hand, 1d, which
has a long enough alkyl chain, may be folded for optimal
coordination to the heme through the nitrogen one carbon
away from the alkyl chain (i.e. the other tautomer of the
imidazole). It is known that the monocationic form of
histamine exists as a mixture of two tautomers.38 At phy-
siological pH (7.4), the Nt-H tautomer is more prevalent
than the Nt-H form in a relative concentration ratio of 4:1.
Figure 2. Plot of inhibitory potency versus the carbon chain length in
1a±e.

Y. Lee et al. / Bioorg. Med. Chem. 7 (1999) 1941±1951
1945
selectivity for nNOS and iNOS over eNOS for 1b±e was
reversed with the phenyl substituted analogues 2a and
2b which are more selective for eNOS over the other
isoforms of NOS, although the selectivity is poor or
nonexistent. Contrary to our initial expectations, there
was no clear order of potency among 2a±c, and the
inhibitory potencies were not improved compared to
those of 1b±d. When 1-phenylimidazole was tested in
our laboratory, it was observed that it is a much poorer
inhibitor of all three isoforms (Table 1). It is not clear
why this di€erence in inhibitory potencies was observed.
Conclusion
A series of novel imidazole-containing amino acids was
synthesized using Schollkopf's chiral bislactim ether.34
Further derivatization of the imidazole ring demon-
strated that this synthetic approach can be used as a
general method for this class of compounds. Several of
these analogues were found to be inhibitors of NOS, but
selectivity among the isozymes is modest. The SAR
study revealed that the inhibitory potencies of the imi-
dazole-containing amino acids 1a±e are related to the
distance between the imidazole ring and the amino acid.
Since it was found that all three isoforms of NOS exhi-
bit similar interactions with 1a±e, we conclude that the
topologies of the active sites of all of the NOS isozymes,
especially the distance between the heme cofactor and
residues responsible for recognition of l-amino acid, are
quite similar.
Figure 3. The e€ect of increasing the number of methylene groups in
the side chain of 1a±e on the orientation of the amino acid moieties.
In an attempt to provide support for the explanation
that both tautomers of the imidazole ring may be
involved in binding, another series of analogues was
synthesized. Introduction of a substituent on one of the
nitrogens of the imidazole ring would give rise to only
one tautomeric form. This should lead to a new inhibi-
tory potency order among the analogues of varying
chain lengths. Phenyl was chosen as the substituent to
attach to the nitrogen atom because it was reported that
the inhibitory potency of 1-phenylimidazole for NOS is
several times higher than that of imidazole.24,25 Fur-
thermore, if the increased potency of 1-phenylimidazole
compared to imidazole results from a hydrophobic
interaction of the phenyl ring with the ceiling of the
heme pocket, then 1-phenylimidazole-containing amino
acids might have improved anity for the enzyme
compared to the analogues without this substitution.
As shown in Table 1, however, the general isoform
Experimental
Materials
Acids, bases, and conventional organic solvents were
purchased from Fisher. NADPH, HEPES, calmodulin,
ferrous hemoglobin, and l-arginine were purchased
from Sigma Chemical Co. Tetrahydrobiopterin (H4B)
was obtained from B. Schircks Laboratories (Jona,
Switzerland). Ion exchange resins were bought from
Bio-Rad Laboratories. All of the chemicals were pur-
chased from Aldrich Chemical Co, unless stated other-
wise. Flash chromatography was performed with Merck
silica gel (230±400 mesh). TLC plates (silica gel 60-
F254) were purchased from VWR Scienti®c. Compounds
were visualized with a ninhydrin spray reagent or a UV±
vis lamp.
Figure 4. Possible explanation for why 1b and 1d are good inhibitors of NOS. The hypothetical binding sites for the imidazole-containing amino
acid analogues.

1946
Y. Lee et al. / Bioorg. Med. Chem. 7 (1999) 1941±1951
Analytical methods
5-(2-Bromoethyl)-2-(tert-butyldimethylsilyl)-1-(N,N-di-
methylsulfamoyl) imidazole (5a). A solution containing
2-(tert-butyldimethylsilyl)-1-(N,N-dimethylsulfamoyl)-5-
(2-hydroxyethyl) imidazole (9, 200 mg, 0.60 mmol) and
triphenylphosphine (173 mg, 0.66 mmol) in dry DMF
(2 mL) and CH2Cl2 (1 mL) under N2 was stirred at room
temperature for 10 min and then to 70ꢀC. A solution
of N-bromosuccinimide (117 mg, 0.66 mmol) in DMF
(1.5 mL) was added over a period of 20min. The reaction
mixture was allowed to warm to room temperature and
was stirred for an additional 1 h. The solvent was removed
under reduced pressure, and the residue was treated with
water (2 mL) and saturated NaHCO3 (1 mL), then
extracted with ethyl acetate (2Â5 mL). The combined
organic extract was concentrated, and the residue was
puri®ed by ¯ash chromatography on a silica gel column
(2Â7 cm). Elution with 1/4 ethyl acetate/hexane gave a
white solid (130 mg, 55%); 1H NMR (300 MHz, CDCl3)
d 0.39 (s, 6H), 1.00 (s, 9H), 2.85 (s, 6H), 3.31 (t,
J=7.41 Hz, 2H), 3.59 (t, J=7.41 Hz, 2H), 7.08 (s, 1H);
13C NMR (75 MHz, CDCl3) 3.6 (2C), 18.4, 27.3 (3C),
28.9, 29.4, 37.8 (2C), 130.6, 131.4, 156.1. HRMS calcd
for C13H26BrN3O2SSi 396.0718, found 396.0768.
Optical rotations were determined with a AA-100
polarimeter (Optical Activity Ltd., England). All 1H
NMR spectra were recorded on a Varian Gemini
300 MHz (75 MHz for 13C NMR spectra). Chemical
shifts (d) are reported down®eld from tetramethylsilane
(Me4Si) in parts per million (ppm). Enzyme assays were
recorded on a Perkin±Elmer Lamda 10 UV±vis spec-
trophotometer. Mass spectra (EI and FAB) were recor-
ded on a VG Instrument VG70-250SE high-resolution
mass spectrometer. Combustion analyses were per-
formed by the Department of Geological Sciences at
Northwestern University. Melting points were obtained
with a Fisher±Johns melting point apparatus and are
not corrected.
2-(tert-Butyldimethylsilyl)-1-(N,N-dimethylsulfamoyl)-
imidazole (4). To a solution of 1-(N,N-dimethylsulfa-
moyl)imidazole (20 g, 114 mmol) in dry THF (500 mL)
at 70ꢀC under an atmosphere of nitrogen was added
n-butyllithium (46 mL of 2.5 M in hexane, 114 mmol)
over a period of 20 min. After 20 min of stirring, a
solution of tert-butyldimethylsilyl chloride (17.1 g,
114 mmol) in dry THF (100 mL) was added over a per-
iod of 30 min (internal temperature did not exceed
60ꢀC), then the solution was allowed to rise to room
temperature and was stirred for an additional hour. The
reaction mixture was poured into water (100 mL), and
the THF was removed under reduced pressure. The
product was extracted with ethyl acetate (3Â150 mL),
dried (Na2SO4), and concentrated. The crude residue
was puri®ed by ¯ash chromatography with ethyl ace-
tate/hexane (1/4) as eluent to obtain a colorless solid
(25 g, 76%); 1H NMR (300 MHz, CDCl3) d 0.39 (s, 6H).
0.95 (s, 9H), 2.84 (s, 6H), 7.22 (d, J=1.26 Hz, 1H), 7.31
(d, J=1.26 Hz, 1H). 13C NMR (75 MHz, CDCl3) 3.9
(2C), 18.1, 27.1 (3C), 38.3 (2C), 120.2, 130.8. HRMS
(M-CH3) calcd for C11H20N3O2SSi 274.1045, found
274.1047.
5-(4-Bromobutyl)-2-(tert-butyldimethylsilyl)-1-(N,N-di-
methylsulfamoyl) imidazole (5c). n-Butyllithium (9.36 mL
of 2.5 M in hexane, 23.4 mmol) was added to a stirred
solution of 2-(tert-butyldimethylsilyl)-1-(N,N-dimethyl-
sulfamoyl) imidazole (4, 6.75 g, 23.4 mmol) in THF
(200 mL) at 70ꢀC. The mixture was stirred at this
temperature for 30 min then was transferred to the
solution of 1,4-dibromobutane (8.38 mL, 70.2 mmol) in
150 mL of THF at 70ꢀC. After 1 h the temperature of
the mixture was allowed to rise to room temperature
and was stirred for 3 h. The solvent was removed, and
the residue was puri®ed by ¯ash chromatography on
silica eluting with ethyl acetate/hexane (1/4) to give a
white solid (6.84 g, 69%) which was recrystallized
from ethyl acetate/hexane (1/6); mp 73ꢀC; 1H NMR
(300 MHz, CDCl3) d 0.38 (s, 6H), 1.00 (s, 9H), 1.84 (m,
2H), 1.97 (m, 2H), 2.75 (t, J=7.53 Hz, 2H), 2.84 (s, 6H),
3.45 (t, J=6.42 Hz, 2H), 6.96 (s, 1H); 13C NMR
(75 MHz, CDCl3) 3.5 (2C), 18.4, 24.2, 26.6, 27.3 (3C),
32.3, 33.4 37.7 (2C), 129.5, 134.2, 155.4. HRMS (M-CH3)
calcd for C14H27BrN3O2SSi 410.0756, found 410.0737.
2-(tert-Butyldimethylsilyl)-1-(N,N-dimethylsulfamoyl)-5-
(2-hydroxyethyl)imidazole (9). n-Butyllithium (7.9 mL of
2.5 M in hexane, 19.9 mmol) was added to a stirred
solution of 2-(tert-butyldimethylsilyl)-1-(N,N-dimethyl-
sulfamoyl)imidazole (4, 5.2 g, 18.1 mmol) in THF
(100 mL) at 70ꢀC. The mixture was stirred at this
temperature for 30 min then transferred to a solution of
ethylene oxide (2.1 g, 47.6 mmol) in 200 mL THF at
70ꢀC. The reaction mixture was allowed to rise to
room temperature and was stirred for 1 h. Saturated
ammonium chloride (4 mL) was added to the mixture,
and the solvent was removed under reduced pressure.
The residue was extracted with ethyl acetate (2Â40 mL),
dried (Na2SO4), and recrystallized from ethyl acetate/
hexane (1/2) to give a white solid (5.2 g, 15.6 mmol,
Using the same procedure as described above for 5c, the
following compounds were synthesized:
5-(3-Bromopropyl)-2-(tert-butyldimethylsilyl)-1-(N,N-di-
methylsulfamoyl)imidazole (5b). Mp 44ꢀC; 1H NMR
(300 MHz, CDCl3) d 0.37 (s, 6H), 0.99 (s, 9H), 2.22 (m,
2H), 2.85 (s, 6H), 2.91 (t, J=7.41 Hz, 2H), 3.50 (t,
J=6.17 Hz, 2H), 6.95 (s, 1H); 13C NMR (75 MHz,
CDCl3) 3.6 (2C), 18.4, 23.6, 27.3 (3C), 30.7 33.2, 37.8
(2C), 129.6, 133.1, 155.7. HRMS (M-CH3) calcd for
C13H25BrN3O2SSi 396.0599, found 396.0600.
55%); mp 123±125ꢀC; H NMR (300 MHz, CDCl3) d
1
0.35 (s, 6H), 0.96 (s, 9H), 2.81 (s, 6H), 2.98 (td, J=6.62,
1.02 Hz, 2H), 3.87 (t, J=6.62 Hz, 2H), 7.01 (s, 1H); 13C
NMR (75 MHz, CDCl3) 3.5 (2C), 18.4, 27.3 (3C),
28.4, 37.7 (2C), 60.7, 130.0, 131.5, 155.5. HRMS
(M+H) calcd for C13H28N3O3SSi 334.1620, found
334.1625.
5-(5-Bromopentyl)-2-(tert-butyldimethylsilyl)-1-(N,N-di-
methylsulfamoyl)imidazole(5d). Recrystallized from pet-
roleum ether; mp 43±45ꢀC; 1H NMR (300 MHz,
CDCl3) d 0.38 (s, 6H), 1.00 (s, 9H), 1.56 (m, 2H), 1.71 (m,
2H), 1.91 (m, 2H), 2.72 (t, J=6.63 Hz, 2H), 2.83 (s, 6H),

Y. Lee et al. / Bioorg. Med. Chem. 7 (1999) 1941±1951
1947
3.43 (t, J=6.63 Hz, 2H), 6.94 (s, 1H); 13C NMR (75 MHz,
CDCl3) 3.5 (2C), 18.4, 24.9, 27.2, 27.3 (3C), 28.0, 32.5,
33.7, 37.7 (2C), 129.3, 134.6, 155.2. HRMS (M-CH3)
calcd for C15H29BrN3O2SSi 424.0913, found 424.0904.
2H), 2.82 (s, 6H), 3.90 (t, J=3.44 Hz, 1H), 4.00 (m, 1H),
4.04±4.24 (4H), 6.94 (s, 1H); 13C NMR (75 MHz,
CDCl3) 3.5 (2C) 14.4, 14.5, 16.7, 18.4, 19.1, 23.1, 24.9,
27.3 (3C), 31.9, 34.0, 37.6 (2C), 55.1, 60.6, 60.8, 129.3,
134.8, 155.1, 163.1, 163.4. HRMS (M-CH3) calcd for
C24H44N5O4SSi 526.2883, found 526.2884.
5-(6-Bromohexyl)-2-(tert-butyldimethylsilyl)-1-(N,N-di-
methylsulfamoyl)imidazole (5e). Obtained as a colorless
1
oil, H NMR (300 MHz, CDCl3) d 0.38 (s, 6H), 0.99 (s,
(3R,6S)-6-{5-[2-(tert-Butyldimethylsilyl)-1-(N,N-dimethyl-
sulfamoyl)imidazol-4-yl]-pentyl}-3-isopropyl-2,5-diethoxy-
3,6-dihydropyrazine (7d). 1H NMR (300 MHz, CDCl3) d
0.30 (s, 6H), 0.62 (d, J=6.81 Hz, 3H), 0.92 (s, 9H), 0.96
(d, J=6.81 Hz, 3H), 1.19 (t, J=7.00 Hz, 6H), 1.20 (m,
2H), 1.32 (m, 2H), 1.60 (m, 2H), 1.68 (m, 2H), 2.29 (m,
2H), 2.61 (t, J=7.65 Hz, 2H), 2.74 (s, 6H), 3.81 (t,
J=3.44 Hz, 1H), 3.91 (m, 1H), 3.95±4.15 (4H), 6.84 (s,
1H); 13C NMR (75 MHz, CDCl3) 3.6 (2C), 14.3, 14.4,
16.6, 18.3, 19.1, 24.3, 24.9, 27.3 (3C), 27.8, 29.3, 31.7,
34.0, 37.5 (2C), 55.3, 60.4, 60.6, 129.2, 134.9, 154.8,
162.9, 163.2. HRMS (M-C4H9) calcd for C23H42N5
O4SSi 512.2727, found 512.2728.
9H), 1.40±1.57 (4H), 1.70 (m, 2H), 1.87 (m, 2H), 2.70 (t,
J=7.70 Hz, 2H), 2.83 (s, 6H), 3.42 (t, J=6.62 Hz, 2H),
6.93 (s, 1H); 13C NMR (75 MHz, CDCl3) 3.5 (2C),
18.4, 24.9, 27.3 (3C), 27.7, 27.9, 28.6, 32.6, 34.0, 37.7
(2C), 129.3, 134.8, 155.2. HRMS (M-CH3) calcd for
C16H31BrN3O2SSi 438.1069, found 438.1062.
(3R,6S)-6-{4-[2-(tert-Butyldimethylsilyl)-1-(N,N-dimethyl-
sulfamoyl)imidazol-4-yl]-butyl}-3-isopropyl-2,5-diethoxy-
3,6-dihydropyrazine (7c). n-Butyllithium (2 mL of 2.1 M
solution in hexane, 4.18 mmol) was injected into the
solution of (3R)-3-isopropyl-2,5-diethoxy-3,6-dihydro-
pyrazine (6, 800 mg, 3.8 mmol) in THF (20 mL) and
stirring continued for 30 min at 70ꢀC. Then a solution
of 5-(4-bromobutyl)-2-(tert-butyldimethylsilyl)-1-(N,N-
dimethylsulfamoyl)imidazole (5c, 1.6 g, 3.8 mmol) in
THF (15 mL) was added. After 1 h stirring at 70ꢀC
and 2 h at room temperature, saturated NH4Cl (1 mL)
was added, and the solvent was removed in vacuo. The
residue was extracted with ethyl acetate/H2O, and the
organic layer was concentrated to give an oil that was
puri®ed by ¯ash chromatography on silica, eluting with
ethyl acetate/hexane (1/4) to give a colorless oil (1.66 g,
(3R,6S)-6-{6-[2-(tert-Butyldimethylsilyl)-1-(N,N-dimethyl-
sulfamoyl)imidazol-4-yl]-hexyl}-3-isopropyl-2,5-diethoxy-
3,6-dihydropyrazine (7e). 1H NMR (300 MHz, CDCl3) d
0.38 (s, 6H), 0.70 (d, J=6.87 Hz, 3H), 1.00 (s, 9H), 1.03
(d, J=6.87 Hz, 3H), 1.27 (t, J=7.03 Hz, 6H), 1.26±1.43
(6H), 1.65 (m, 2H), 1.73 (m, 2H), 2.28 (m, 1H), 2.67 (t,
J=7.81 Hz, 2H), 2.82 (s, 6H), 3.88 (t, J=3.30 Hz, 1H),
3.98 (m, 1H), 4.04±4.23 (m, 4H), 6.93 (s, 1H); 13C NMR
(75 MHz, CDCl3) 3.5 (2C), 14.4, 14.5, 16.7, 18.4, 19.2,
24.4, 25.1, 27.4 (3C), 27.8, 29.4, 29.5, 31.8, 34.2, 37.7
(2C), 55.4, 60.5, 60.7, 129.2, 135.1, 155.0, 163.0, 163.4.
HRMS (M-C4H9) calcd for C24H44N5O4SSi 526.2883,
found 526.2889.
1
3 mmol, 78%); H NMR (300 MHz, CDCl3) d 0.37 (s,
6H), 0.69 (d, J=6.81 Hz, 3H), 0.99 (s, 9H), 1.03 (d,
J=6.81 Hz, 3H), 1.27 (app t, J=7.01 Hz, 6H), 1.33 (m,
2H), 1.68 (m, 2H), 1.82 (m, 2H), 2.26 (m, 1H), 2.68 (app
t, J=7.57 Hz, 2H), 2.82 (s, 6H), 3.89 (m, 1H), 3.95 (m,
1H), 3.99±4.20 (4H), 6.92 (s, 1H); 13C NMR (75 MHz,
CDCl3) 3.5 (2C), 14.4, 14.5, 16.6, 18.4, 19.1, 24.5,
25.0, 27.3 (3C), 27.8, 31.8, 33.9, 37.6 (2C), 55.3, 60.5,
60.7, 129.2, 134.9, 154.9, 163.1, 163.2. HRMS (M-C4H9)
calcd for C22H40N5O4SSi 498.2571, found 498.2564.
(S)-Ethyl-2-amino-6-[1-(N,N-dimethylsulfamoyl)imidazol-
4-yl]hexanoate (8c). A suspension of (3R,6S)-6-{4-[tert-
butyldimethyl)-1-(N,N-dimethylsulfamoyl)imidazol-4-
yl]-butyl}-3-isopropyl-2,5-diethoxy-3,6-dihydropyrazine
(7c, 1.6 g, 2.9 mmol) in 0.25 N HCl (35 mL) was stirred
for 16 h at room temperature. The precipitate was
removed by ®ltration, and the ®ltrate was concentrated
in vacuo. The residue was dissolved in water, covered
with ether and vigorously shaken with concd NH4OH
until the pH was 8±10. The ether was separated, the
aqueous phase was extracted with ether and the com-
bined ether phases dried over Na2SO4. The ether was
removed and the residue was puri®ed by ¯ash chroma-
tography on silica gel eluting with ethyl acetate/ethanol/
NH4OH (40/4/1) to yield a colorless oil (414 mg,
Using the same procedure as described above for 7c, the
following compounds were synthesized:
(3R,6S)-6-{2-[2-(tert-Butyldimethylsilyl)-1-(N,N-dimethyl-
sulfamoyl)imidazol-4-yl]-ethyl}-3-isopropyl-2,5-diethoxy-
1
3,6-dihydropyrazine (7a). H NMR (300 MHz, CDCl3)
d 0.37 (s, 6H), 0.70 (d, J=6.85 Hz, 3H), 1.00 (s, 9H),
1.02 (d, J=6.85 Hz, 3H), 1.26 (app t, J=7.08 Hz, 6H),
2.02 (m, 1H), 2.23 (m, 2H), 2.70 (m, 2H), 2.82 (s, 6H),
3.91 (t, J=3.45 Hz, 1H), 4.02±4.22 (5H), 6.97 (s, 1H);
13C NMR (75 MHz, CDCl3) 3.5 (2C), 14.4, 16.7, 18.4,
19.1, 20.5, 27.3 (3C), 32.0, 32.3, 37.6 (2C), 54.7, 60.6,
60.9, 129.1, 134.6, 155.0, 162.8, 163.4. HRMS calcd for
C24H45N5O4SSi 527.2961, found 527.2956.
1
1.24 mmol, 43%). H NMR (300 MHz, CDCl3) d 1.08
(t, J=6.93 Hz, 3H), 1.25±1.64 (6H), 2.56 (t, J=7.41 Hz,
2H), 2.70 (s, 6H), 3.24 (t, J=6.21 Hz, 1H), 3.97 (q, J=
6.93 Hz, 2H), 6.64 (s, 1H), 7.67 (s, 1H); 13C NMR
(75 MHz, CDCl3) 14.2, 24.7, 25.2, 27.7, 34.4, 37.9 (2C),
54.2, 60.7, 128.1, 132.3, 138.0, 175.9. HRMS calcd for
C13H24N4O4S 332.1520, found 332.1527.
(3R,6S)-6-{3-[2-(tert-Butyldimethylsilyl)-1-(N,N-dimethyl-
sulfamoyl)imidazol-4-yl]-propyl}-3-isopropyl-2,5-diethoxy-
3,6-dihydropyrazine (7b). 1H NMR (300 MHz, CDCl3) d
0.37 (s, 6H), 0.70 (d, J=6.81 Hz, 3H), 1.00 (s, 9H), 1.03
(d, J=6.81 Hz, 3H), 1.27 (app t, J=7.11 Hz, 6H), 1.70
(m, 2H), 1.86 (m, 2H), 2.26 (m, 1H), 2.70 (t, J=7.41 Hz,
Using the same procedure as described above for 8c, the
following compounds were synthesized:
(S)-Ethyl-2-amino-4-[1-(N,N-dimethylsulfamoyl)imidazol-
1
4-yl]butanoate (8a). H NMR (300 MHz, CDCl3) d 1.20

1948
Y. Lee et al. / Bioorg. Med. Chem. 7 (1999) 1941±1951
(t, J=7.20 Hz, 3H), 1.78 (bs, 2H), 1.82 (m, 2H), 2.03 (m,
1H), 2.81 (s, 6H), 2.83 (m, 2H), 3.41 (dd, J=8.10,
5.10 Hz, 1H), 4.10 (q, J=7.20 Hz, 2H), 6.80 (s, 1H), 7.79
(s, 1H); 13C NMR (75 MHz, CDCl3) 14.1, 21.2, 33.3,
38.0 (2C), 53.7, 61.0, 128.5, 131.5, 138.1, 175.5. HRMS
calcd for C11H20N4O4S 304.1205, found 304.1201.
7.94 Hz, 2H), 3.59 (t, J=5.64 Hz, 1H), 6.77 (s, 1H), 7.56
(s, 1H); 13C NMR (75 MHz, D2O) 22.1, 30.4, 54.3,
115.9, 135.5, 135.8, 174.8. HRMS (M+H) calcd for
C7H12N3O2 170.0929, found 170.0925.
(S)-2-Amino-5-[1(3)H-imidazol-4-yl]pentanoic acid (1b).
Mp 229±231ꢀC, [a]d22+13.2ꢀ (c 1, H2O); 1H NMR
(300 MHz, D2O) d 1.62 (m, 2H), 1.76 (m, 2H), 2.55 (t,
J=7.04 Hz, 2H), 3.60 (t, J=6.03 Hz, 1H), 6.81 (s, 1H),
7.60 (s, 1H); 13C NMR (75 MHz, D2O) 24.3, 25.4, 30.4,
54.8, 116.6, 135.5, 136.7, 175.5. HRMS calcd for
C8H13N3O2 183.1009, found 183.1017. Anal. calcd for
C8H13N3O20.1H2O: C 51.93; H 7.13; N 22.71. Found: C
52.09; H 7.29; N 22.64.
(S)-Ethyl-2-amino-5-[1-(N,N-dimethylsulfamoyl)imidazol-
4-yl]pentanoate (8b). 1H NMR (300 MHz, CDCl3) d
1.25 (t, J=7.14 Hz, 3H), 1.73 (bs, 2H), 1.78 (m, 2H),
2.73 (app t, J=6.85 Hz, 2H), 2.85 (s, 6H), 3.42 (m, 1H),
4.15 (q, J=7.14 Hz, 2H), 6.83 (s, 1H), 7.83 (s, 1H); 13C
NMR (75 MHz, CDCl3) 14.3, 24.3, 24.8, 34.4, 38.1 (2C),
54.2, 61.0, 128.4, 132.1, 138.2, 175.8. HRMS calcd for
C12H22N4O4S 318.1364, found 318.1351.
(S)-2-Amino-7-[1(3)H-imidazol-4-yl)heptanoic acid (1d).
Mp 207±209ꢀC, [a]d22+12ꢀ (c 1, H2O); 1H NMR
(300MHz, D2O) d 1.15±2.10 (4H), 1.47 (m, 2H), 1.71 (m,
2H), 2.43 (t, J=7.41 Hz, 2H), 3.58 (t, J=6.08 Hz, 1H),
6.72 (s, 1H). 7.59 (s, 1H); 13C NMR (75 MHz, D2O)
24.0, 25.2, 27.8, 28.1, 30.5, 54.8, 116.8, 135.0, 136.9,
175.4. HRMS calcd for C10H15N3O2 211.1321, found
211.1323. Anal. calcd for C10H17N3O20.1H2O: C 55.90;
H 8.06; N 19.55. Found: C 55.77; H 8.03; N 19.39.
(S)-Ethyl-2-amino-7-[1-(N,N-dimethylsulfamoyl)imidazol-
4-yl]heptanoate (8d). 1H NMR (300 MHz, CDCl3) d
1.26 (t, J=7.11 Hz, 3H), 1.36±1.78 (8H), 2.71 (t, J=
7.41 Hz, 2H), 2.87 (s, 6H), 3.40 (dd, J=7.26, 5.40 Hz,
1H), 4.16 (q, J=7.11 Hz, 2H), 6.81 (s, 1H), 7.84 (s, 1H);
13C NMR (75 MHz, CDCl3) 14.3, 24.9, 25.4, 28.0, 29.1,
34.8, 38.1 (2C), 54.4, 60.9, 128.2, 132.6, 138.1, 176.2.
HRMS calcd for C14H26N4O4S 346.1676, found
346.1671.
(S)-2-Amino-8-[1(3)H-imidazol-4-yl]octanoic acid (1e).
Mp 201±202ꢀC, [a]d22+10.4ꢀ (c 1, H2O); 1H NMR
(300 MHz, D2O) d 1.21±1.36 (6H), 1.56 (m, 2H), 1.76
(m, 2H), 2.53 (t, J=7.42 Hz, 2H), 3.63 (t, J=6.09 Hz,
1H), 6.81 (s, 1H), 7.65 (s, 1H); 13C NMR (75 MHz,
D2O) 24.2, 25.4, 27.8, 28.1, 28.3, 30.6, 54.9, 116.9, 135.2,
137.2, 175.6. HRMS calcd for C11H19N3O2 225.1477,
found 225.1480. Anal. calcd for C11H19N3O2: C 58.64;
H 58.50; N 18.65. Found, C 58.26; H 8.30; N 18.38.
(S)-Ethyl-2-amino-8-[1-(N,N-dimethylsulfamoyl)imidazol-
1
4-yl]octanoate (8e). H NMR (300 MHz, CDCl3) d 1.24
(t, J=7.13 Hz, 3H), 1.30±1.74 (10H), 2.68 (t, J=
7.73 Hz, 2H), 2.85 (s, 6H), 3.38 (dd, J=7.34, 5.45 Hz,
1H), 4.13 (q, J=7.13 Hz, 2H), 6.78 (s, 1H), 7.81 (s, 1H);
13C NMR (75 MHz, CDCl3) 14.3, 24.9, 25.5, 28.0, 29.1,
29.2, 34.9, 38.1 (2C), 54.5, 60.8, 128.2, 132.7, 138.1,
176.2. HRMS calcd for C15H28N4O4S 360.1832, found
360.1837.
2-(tert-Butyldimethylsilyl)-1-phenylimidazole (11). To a
solution of 1-phenylimidazole (10, 5.0 g, 34.6 mmol) in
dry THF (200 mL) at 70ꢀC under an atmosphere of
nitrogen was added n-butyllithium (13.8 mL of 2.5 M in
hexane, 34.6 mmol). After 20 min of stirring, a solution
of tert-butyldimethylsilyl chloride (5.2 g, 34.6 mmol) in
dry THF (50 mL) was added over a period of 10 min,
and the solution was allowed to warm to room tem-
perature. Stirring continued for an additional hour. The
solvent was removed under reduced pressure and the
remaining crude product was extracted with ethyl ace-
tate (2Â50 mL), dried (Na2SO4), and concentrated. The
residue was puri®ed by ¯ash chromatography with ethyl
acetate/hexane (1/4) as eluent to obtain a white solid
which crystallized upon standing (6.8 g, 76%); 1H NMR
(300 MHz, CDCl3) d 0.00 (s, 6H), 0.89 (s, 9H), 7.11 (d,
J=1.05 Hz, 1H), 7.35±7.42 (3H), 7.49±7.55 (3H); 13C
NMR (75 MHz, CDCl3) 4.5 (2C), 17.6, 26.8 (3C),
124.1, 127.5 (2C), 128.8 (2C), 128.9, 130.0, 139.5,
150.6. HRMS calcd for C15H22N2Si 258.1552, found
258.1557.
(S)-2-Amino-6-[1(3)H-imidazol-4-yl]hexanoic acid (1c).
(S)-Ethyl-2-amino-6-[1-(N,N-dimethylsulfamoyl)imidazol-
4-yl]hexanoate (8c, 350 mg, 1.05 mmol) was dissolved in
30% HBr (20 mL) and heated under re¯ux. After 4 h the
mixture was cooled and concentrated in vacuo. The
residue was dissolved in water (5 mL) and was applied
to a column of Dowex 50 W (hydrogen form), washed
with 50 mL of H2O, then eluted with 2 N NH4OH
(100 mL). The combined fractions reactive to ninhydrin
solution were concentrated to 2 mL, then were lyophi-
lized to give a white solid (150 mg, 73%). Recrystallized
from ethanol/H2O (20/1); mp 217±219ꢀC, [a]d22+12.1ꢀ
1
(c 1, H2O); H NMR (300 MHz, D2O) d 1.19 (m, 2H),
1.40 (app quintet, J=7.53 Hz, 2H), 1.64 (m, 2H), 2.34
(t, J=7.41 Hz, 2H), 3.49 (t, J=6.15 Hz, 1H), 6.62 (s,
1H), 7.48 (s, 1H); 13C NMR (75 MHz, D2O) 23.8, 25.1,
28.1, 30.3, 54.7, 116.6, 135.0, 136.6, 175.3. HRMS calcd
for C9H15N3O2 197.1165, found 197.1164. Anal. calcd
for C9H15N3O20.2H2O: C 53.82; H 7.72; N 20.92.
Found: C 53.43; H 7.70; N 20.72.
Using the same procedure as described above for 1c, the
following compounds were synthesized:
5-(3-Bromopropyl)-2-(tert-butyldimethylsilyl)-1-phenyl-
imidazole (12a). n-Butyllithium (2.48 mL of 2.5 M in
hexane, 6.2 mmol) was added to a stirred solution of 2-
(tert-butyldimethylsilyl)-1-phenylimidazole (11, 1.6 g,
6.2 mmol) in THF (70 mL) at 70ꢀC. The mixture
was stirred at this temperature for 30 min and then was
(S)-2-Amino-4-[1(3)H-imidazol-4-yl]butanoic acid (1a).
Mp 219±222ꢀC, [a]d22+9.8ꢀ (c 0.5, H2O); 1H NMR
(300 MHz, D2O) d 2.00 (m, 2H), 2.55 (app t, J=

Y. Lee et al. / Bioorg. Med. Chem. 7 (1999) 1941±1951
1949
transferred to the solution of 1,3-dibromopropane
(2.5 mL, 24.8 mmol) in 50 mL of THF at 70ꢀC. After
1 h the temperature of the mixture was allowed to rise to
room temperature and was stirred further for 1 h. The
solvent was removed, and the residue was puri®ed by
¯ash chromatography on silica gel eluting with ethyl
163.1. HRMS calcd for C29H46N4O2Si 510.3389, found
510.3398.
Using the same procedure as described above for 13a,
the following compounds were synthesized:
1
acetate/hexane (1/4) to give a viscous oil (1 g, 43%); H
(3R,6S)-6-{4-[2-(tert-Butyldimethylsilyl)-1-phenylimidazol-
4-yl]-butyl}-3-isopropyl-2,5-diethoxy-3,6-dihydropyrazine
(13b). 1H NMR (300 MHz, CDCl3) d 0.10 (s, 6H), 0.66
(d, J=6.81 Hz, 3H), 0.86 (s, 9H), 1.00 (d, J=6.81 Hz,
3H), 1.21 (q, J=7.10 Hz, 3H), 1.23 (q, J=7.10 Hz, 3H),
1.47 (m, 2H), 1.63 (m, 2H), 2.21 (m, 1H), 2.26 (m, 2H),
3.83 (t, J=3.42 Hz, 1H), 3.78±4.25 (4H), 7.02 (s, 1H),
7.16±7.22 (2H), 7.40±7.45 (3H); 13C NMR (75 MHz,
CDCl3) 4.9 (2C), 14.3, 14.4, 16.6, 17.6, 19.1, 24.3,
24.5, 26.8 (3C), 28.3, 31.8, 33.8, 55.3, 60.4, 60.7, 127.1,
128.8 (2C), 128.9 (2C), 129.0, 135.9, 138.2, 150.0, 163.0,
163.2. HRMS calcd for C30H48N4O2Si 524.3546, found
524.3538.
NMR (300 MHz, CDCl3) d 0.09 (s, 6H), 0.87 (s, 9H),
1.98 (m, 2H), 2.47 (app t, J=7.29 Hz, 2H), 3.32 (t,
J=6.40 Hz, 2H), 7.07 (s, 1H), 7.18±7.50 (5H). 13C NMR
(75 MHz, CDCl3) 4.9 (2C), 17.6, 22.8, 26.8 (3C), 31.3,
32.8, 127.5, 128.7 (2C), 129.1 (2C), 129.2, 133.8, 137.7,
150.7. HRMS calcd for C18H27BrN2Si 378.1126 and
380.1108, found 378.1118 and 380.1127.
Using the same procedure as described above for 12a,
the following compounds were synthesized:
5-(4-Bromobutyl)-2-(tert-butyldimethylsilyl)-1-phenyl-
1
imidazole (12b). H NMR (300 MHz, CDCl3) d 0.09
(s, 6H), 0.87 (s, 9H), 1.61 (m, 2H), 1.79 (m, 2H), 2.31
(app t, J=7.28 Hz, 2H), 3.30 (t, J=6.62 Hz, 2H), 7.06
(s, 1H), 7.18±7.26 (2H), 7.43±7.48 (3H); 13C NMR
(75 MHz, CDCl3) 4.9 (2C), 17.6, 23.6, 26.7, 26.8 (3C),
32.1, 33.3, 127.4, 128.8 (2C), 129.0 (2C), 129.2, 135.0,
138.0, 150.4. HRMS calcd for C19H29BrN2Si 392.1283
and 394.1264, found 392.1281 and 394.1264.
(3R,6S)-6-{4-[2-(tert-Butyldimethylsilyl)-1-phenylimidazol-
4-yl]-butyl}-3-isopropyl-2,5-diethoxy-3,6-dihydropyrazine
(13b). 1H NMR (300 MHz, CDCl3) d 0.12 (s, 6H), 0.65
(d, J=6.75 Hz, 3H), 0.84 (s, 9H), 0.99 (d, J=3.40 Hz,
3H), 1.20 (q, J=7.10 Hz, 3H), 1.21 (q, J=7.10 Hz, 3H),
1.23 (m, 2H), 1.43 (m, 2H), 1.62 (m, 2H), 2.21 (app, t,
J=7.43 Hz, 2H), 2.22 (m, 1H), 3.83 (t, J=3.40 Hz, 1H),
3.86±4.14 (4H), 7.00 (s, 1H), 7.16±7.19 (2H), 7.38±7.43
(3H); 13C NMR (75 MHz, CDCl3) 4.8 (2C), 14.3, 14.4,
16.6, 17.6, 19.1, 24.1, 24.3, 26.8 (3C), 28.3, 29.1, 31.7,
33.9, 55.3, 60.4, 60.6, 127.1, 128.8 (2C), 128.9 (2C),
129.0, 135.9, 138.2, 140.9, 162.9, 163.3. HRMS calcd for
C31H50N4O2Si 538.3703, found 538.3701.
5-(5-Bromopentyl)-2-(tert-butyldimethylsilyl)-1-phenyl-
1
imidazole (12c). H NMR (300 MHz, CDCl3) d 0.08
(s, 6H), 0.88 (s, 9H), 1.38 (m, 2H), 1.50 (m, 2H), 1.78 (q,
J=7.20 Hz, 2H), 2.30 (app t, J=7.56 Hz, 2H), 3.33 (t,
J=6.75 Hz, 2H), 7.06 (s, 1H), 7.20±7.25 (2H), 7.42±7.49
(3H); 13C NMR (75 MHz, CDCl3) 4.8 (2C), 17.6, 24.2,
27.5, 27.7, 32.3, 33.6, 127.2, 128.8 (2C), 128.9 (2C),
129.1, 135.4, 138.0, 150.2. HRMS calcd for C18H27
BrN2Si 406.1439 and 408.1421, found 406.1428 and
408.1424.
(S)-Ethyl-2-amino-5-(1-phenylimidazol-4-yl)pentanoate
(14a). A suspension of (3R,6S)-6-{3-[2-(tert-butyldi-
methylsilyl)-1-phenylimidazol-4-yl]-propyl}-3-isopropyl-
2,5-diethoxy-3,6-dihydropyrazine (13a, 600 mg, 1.17 mmol)
in 0.25 N HCl (20 mL) was stirred for 3 h at room tem-
perature. The reaction mixture was concentrated under
reduced pressure and the residue was puri®ed by ¯ash
chromatography on silica gel eluting with ethyl acetate/
(3R,6S)-6-{3-[2-(tert-Butyldimethylsilyl)-1-phenylimidazol-
4-yl]-propyl}-3-isopropyl-2,5-diethoxy-3,6-dihydropyr-
azine (13a). n-Butyllithium (672 mL of 2.5 M solution in
hexane, 1.68 mmol) was injected into a solution of (3R)-
ethanol/NH4OH (80/4/1) to yield
a colorless oil
3-isopropyl-2,5-diethoxy-3,6-dihydropyrazine
(6,
(263 mg, 56%). 1H NMR (300 MHz, CDCl3) d 0.23 (s,
6H), 0.73 (s, 9H), 1.09 (t, J=7.16 Hz, 3H), 1.30±1.60
(4H), 2.17 (m, 2H), 3.19 (m, 1H), 3.97 (q, J=7.16 Hz,
2H), 6.91 (s, 1H), 7.05±7.10 (2H), 7.28±7.37 (3H). 13C
NMR (75 MHz, CDCl3) 4.9 (2C), 14.2, 17.4, 24.1,
24.4, 26.7 (3C), 34.3, 54.1, 60.7, 127.2, 128.7 (2C), 128.9
(2C), 129.0, 135.1, 137.9, 150.1, 175.8. HRMS calcd for
C22H35N3O2Si 401.2498, found 401.2507.
357 mg, 1.68 mmol) in THF (20 mL) and stirring con-
tinued for 30 min at 70ꢀC. Then a solution of 5-(3-
bromopropyl)-2-(tert-butyldimethylsilyl)-1-phenylimid-
azole (12a, 637 mg, 1.68 mmol) in THF (10 mL) was
added. After being stirred for 1 h at 70ꢀC and 2 h at
room temperature, saturated NH4Cl (100 mL) was
added and the solvent was removed in vacuo. The resi-
due was extracted with ethyl acetate/H2O, and the
organic layer was concentrated to give an oil which was
puri®ed by ¯ash chromatography on silica gel eluting
with ethyl acetate/hexane (1/4) to give a colorless oil
(608 mg, 71%). 1H NMR (300 MHz, CDCl3) d 0.16 (s,
6H), 0.61 (d, J=6.60 Hz, 3H), 0.81 (s, 9H), 0.94 (d,
J=6.60 Hz, 3H), 1.13±1.21 (6H), 1.38 (m, 2H), 1.63 (m,
2H), 2.18 (m, 1H), 2.22 (m, 2H), 3.77 (m, 1H), 3.81±4.12
(4H), 6.99 (s, 1H), 7.12±7.17 (2H), 7.35±7.39 (3H); 13C
NMR (75 MHz, CDCl3) 4.9 (2C), 14.4 (2C), 16.6,
17.5, 19.1, 23.5, 24.4, 26.7 (3C), 31.8, 33.7, 55.0, 60.5,
60.6, 127.2, 128.8 (5C), 135.7, 138.1, 150.0, 163.0,
Using the same procedure as described above for 14a,
the following compounds were synthesized:
(S)-Ethyl-2-amino-6-(1-phenylimidazol-4-yl)hexanoate
(14b). 1H NMR (300 MHz, CDCl3) d 0.14 (s, 6H),
0.83 (s, 9H), 1.19 (t, J=7.16 Hz, 3H), 1.29 (m, 1H), 1.45
(m, 2H), 1.67 (m, 1H), 2.24 (app t, J=7.56 Hz, 2H), 3.29
(dd, J=7.17, 5.49 Hz, 1H), 4.08 (q, J=7.16 H, 2H), 7.00
(s, 1H), 7.14±7.18 (2H), 7.39±7.44 (3H); 13C NMR
(75 MHz, CDCl3) 4.9 (2C), 14.3, 17.5, 24.3, 25.3, 26.8
(3C), 28.2, 34.5, 54.3, 60.8, 127.2, 128.8 (2C), 128.9

1950
Y. Lee et al. / Bioorg. Med. Chem. 7 (1999) 1941±1951
(2C), 129.0, 135.6, 138.0, 150.1, 176.1. HRMS calcd for
C23H37N3O2Si 415.2655, found 415.2659.
macrophage iNOS was expressed39 and isolated40 as
reported; the bovine endothelial eNOS was prepared as
previously described,41 and the rat neuronal nNOS was
expressed and puri®ed as described.42
(S)-Ethyl-2-amino-7-(1-phenylimidazol-4-yl)heptanoate
(14c). 1H NMR (300 MHz, CDCl3) d 0.13 (s, 6H),
0.83 (s, 9H), 1.21 (t, J=7.13 Hz, 3H), 1.26 (m, 1H),
1.35±1.48 (4H), 1.58 (m, 1H), 2.23 (app t, J=7.70 Hz,
2H), 3.31 (dd, J=7.41, 5.49 Hz, 1H), 4.10 (q, J=
7.13 Hz, 2H), 7.00 (s, 1H), 7.15±7.19 (2H), 7.39±7.44
(3H); 13C NMR (75 MHz, CDCl3) 4.8 (2C), 14.3, 17.5,
24.3, 25.3, 26.8 (3C), 28.2, 29.0, 34.7, 54.4, 60.8, 127.2,
128.8 (2C), 128.9 (2C), 129.0, 135.8, 138.1, 150.1, 176.2,
177.1. HRMS calcd for C24H37N3O2Si 429.2811, found
429.2817.
Initial velocity measurement via the hemoglobin assay
The generation of nitric oxide by NOS was measured
using the hemoglobin capture assay.37 A typical assay
mixture for nNOS and eNOS contained 10 mM l-argi-
nine, 1.6 mM CaCl2, 11.6 mg/mL calmodulin, 100 mM
NADPH, 6.5 mM tetrahydrobiopterin, and 3 mM oxy-
hemoglobin in 100 mM HEPES (pH 7.5). The reaction
mixture for iNOS contained 10 mM of l-arginine,
100 mM NADPH, 6.5 mM tetrahydrobiopterin, and
3 mM oxyhemoglobin in 100 mM HEPES (pH 7.5). All
assays were in a ®nal volume of 600 mL and were initi-
ated with enzyme. Nitric oxide reacts with oxy-
(S)-2-Amino-5-(1-phenylimidazol-4-yl)pentanoic acid (2a).
(S)-Ethyl-2-amino-5-(1-phenylimidazol-4-yl)pentanoate
(14a, 250 mg, 0.62 mmol) was dissolved in 2 N NaOH
(20 mL) and was heated to re¯ux overnight. The mix-
ture was cooled, acidi®ed with concd HCl and con-
centrated in vacuo. The residue was dissolved in water
(5 mL) and was loaded on a column of Dowex 50 W
(hydrogen form), washed with H2O, then eluted with 2
N NH4OH. The combined fractions reactive to nin-
hydrin solution was concentrated to give a colorless
hemoglobin to yield methemoglobin which is detected at
1
401 nM (e=19,700 M
Lamda 10 UV±vis spectrophotometer.
cm 1) on a Perkin±Elmer
Reversible inhibition kinetics. The reversible inhibition
of NOS by imidazole-containing amino acids was stu-
died under initial velocity measurement conditions by
the hemoglobin capture assay described above. The Ki
values were determined by the methods of Dixon.43
22
ꢀ
1
solid (140 mg, 86%); [a]d +12.2 (c 1, H2O); H NMR
(300 MHz, D2O) d 1.59 (m, 2H), 1.77 (m, 2H), 2.53 (app
t, J=7.28 Hz, 2H), 3.69 (t, J=6.05 Hz, 1H), 7.19 (s,
1H), 7.31±7.36 (2H), 7.52±7.56 (3H), 8.17 (s, 1H); 13C
NMR (75 MHz, D2O) 23.0, 23.1, 29.9, 54.6, 120.9, 126.0
(2C), 130 (3C), 133.8, 134.3, 136.3, 174.5. HRMS (FAB)
calcd for C14H18N3O2 260.1399, found 260.1423. Anal.
calcd for C14H17N3O2: C 64.85; H 6.59; N 16.20.
Found: C 64.69; H 6.72; N 16.12.
Acknowledgments
We are grateful to the National Institutes of Health for
®nancial support of this research to R.B.S. (GM49725)
and B.S.S.M. (GM52419) and to the Robert A. Welch
Foundation (AQ-1192) for ®nancial support to
B.S.S.M. Sincere thanks go to Professor Michael A.
Marletta (University of Michigan) for providing the
E. coli cells with an overexpression system for murine
macrophage iNOS.
Using the same procedure as described above for 2a, the
following compounds were synthesized:
(S)-2-Amino-5-(1-phenylimidazol-4-yl)hexanoic acid (2b).
[a]2d2+12ꢀ (c 1, H2O); 1H NMR (300 MHz, D2O) d
1.06 (m, 2H), 1.23 (m, 2H), 1.28 (m, 1H), 1.39 (m, 1H),
2.26 (t, J=7.30 Hz, 2H), 3.03 (t, J=6.34 Hz, 1H), 6.76
(s, 1H), 7.00±7.03 (2H), 7.30±7.33 (3H), 7.35 (s, 1H); 13C
NMR (75 MHz, D2O) 23.3, 24.7, 27.5, 34.6, 56.0, 125.3,
125.6 (2C) 128.8, 129.7 (2C), 133.5, 135.7, 137.4, 183.4.
HRMS (FAB) calcd for C15H20N3O2 274.1555, found
274.1600. Anal. calcd for C15H19N3O20.1H2O: C 65.48;
H 7.03; N 15.27. Found: C 65.29; H 7.11; N 15.21.
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