Reduced amide bond peptidomimetics. (4S)-N-(4-amino-5-[aminoalkyl]aminopentyl)-N′-nitroguanidines, potent and highly selective inhibitors of neuronal nitric oxide synthase

Article abstract of DOI:10.1021/jm0101491

Selective inhibition of the isoforms of nitric oxide synthase (NOS) could be therapeutically useful in the treatment of certain disease states arising from the overproduction of nitric oxide. Recently, we reported nitroarginine-containing dipeptide amides (Huang, H; Martasek, P.; Roman, L. J.; Masters, B. S. S.; Silverman, R. B. J. Med. Chem. 1999, 42, 3147.) and some peptidomimetic analogues (Huang, H; Martasek, P.; Roman, L. J.; Silverman, R.B. J. Med Chem. 2000, 43, 2938.) as potent and selective inhibitors of neuronal NOS (nNOS). Here, reduced, amide bond pseudodipeptide analogues are synthesized and evaluated for their activity. The deletion of the carbonyl group from the amide bond either preserves or improves the potency for nNOS. Significantly, the selectivities for nNOS over eNOS (endothelial NOS); and iNOS (inducible NOS) are greatly increased in these series. The most potent nNOS inhibitor among these compounds is (4S)-N-(4-amino-5-[aminoethyl]aminopentyl)-N′-nitroguanidine (7) (K_i = 120 nM), which also shows the highest selectivity over eNOS (greater than 2500-fold) and 320-fold selectivity over iNOS. The reduced amide bond is an excellent surrogate of the amide bond, and it will facilitate the design of new potent and selective inhibitors of nNOS.

Full text of DOI:10.1021/jm0101491

J . Med. Chem. 2001, 44, 2667-2670
2667
Brief Articles
Red u ced Am id e Bon d P ep tid om im etics.
(4S)-N-(4-Am in o-5-[a m in oa lk yl]a m in op en tyl)-N′-n itr ogu a n id in es, P oten t a n d
High ly Selective In h ibitor s of Neu r on a l Nitr ic Oxid e Syn th a se
J ung-Mi Hah,†,‡ Linda J . Roman,§,| Pavel Marta´sek,§, and Richard B. Silverman*,†
Department of Chemistry and Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University,
Evanston, Illinois 60208-3113, and Department of Biochemistry, The University of Texas Health Science Center,
San Antonio, Texas 78284-7760
Received April 2, 2001
Selective inhibition of the isoforms of nitric oxide synthase (NOS) could be therapeutically
useful in the treatment of certain disease states arising from the overproduction of nitric oxide.
Recently, we reported nitroarginine-containing dipeptide amides (Huang, H; Martasek, P.;
Roman, L. J .; Masters, B. S. S.; Silverman, R. B. J . Med. Chem. 1999, 42, 3147.) and some
peptidomimetic analogues (Huang, H; Martasek, P.; Roman, L. J .; Silverman, R.B. J . Med Chem.
2000, 43, 2938.) as potent and selective inhibitors of neuronal NOS (nNOS). Here, reduced
amide bond pseudodipeptide analogues are synthesized and evaluated for their activity. The
deletion of the carbonyl group from the amide bond either preserves or improves the potency
for nNOS. Significantly, the selectivities for nNOS over eNOS (endothelial NOS), and iNOS
(inducible NOS) are greatly increased in these series. The most potent nNOS inhibitor among
these compounds is (4S)-N-(4-amino-5-[aminoethyl]aminopentyl)-N′-nitroguanidine (7) (Ki )
120 nM), which also shows the highest selectivity over eNOS (greater than 2500-fold) and
320-fold selectivity over iNOS. The reduced amide bond is an excellent surrogate of the amide
bond, and it will facilitate the design of new potent and selective inhibitors of nNOS.
In tr od u ction
disorders;14 however, because of the importance of nitric
oxide to physiological functioning, potent as well as
isoform-selective inhibitors are essential. nNOS inhibi-
tion has been targeted for treatment of strokes15 and
iNOS inhibition for the treatment of septic shock16 and
arthritis.17 Although there may be pathologies associ-
ated with overactivity of eNOS, blood pressure homeo-
stasis is so critical that most investigators believe that
therapeutically useful NOS inhibitors must not inhibit
eNOS.
Nitric oxide (NO)1 is synthesized enzymatically from
arginine in numerous tissues and cell types by a family
of enzymes collectively known as nitric oxide synthase
(NOS, EC 1.14.13.39).2 Three principal isoforms of this
enzyme have been isolated and characterized,3 each
associated with different physiological functions: the
immune response (inducible NOS or iNOS),4 smooth
muscle relaxation (endothelial NOS or eNOS),5 and
neuronal signaling (neuronal NOS or nNOS).6 All of
these isoforms utilize NADPH, FAD, FMN, (6R)-5,6,7,8-
tetrahydrobiopterin, and heme as cofactors.
Overproduction of NO has been a factor in numerous
disease states. NO overproduction by nNOS has been
implicated in strokes,7 migrane headaches,8 and Alzhe-
imer’s disease9 and with tolerance to and dependence
on morphine.10 iNOS-mediated overproduction of NO
has been associated with development of colitis,11 tissue
damage and inflammation,12 and rheumatoid arthritis.13
Animal studies and early clinical trials suggest that
NOS inhibitors could be therapeutic in many of these
In our labs, excellent inhibitory potency and selectiv-
ity for nNOS over eNOS and iNOS have been achieved
with certain nitroarginine dipeptide amides that have
an amine-containing side chain (1-3).18 The most potent
nNOS inhibitor among these compounds is L-ArgNO2-L-
Dbu-NH2 (1) (Ki ) 130 nM), which also shows excellent
selectivity over eNOS (greater than 1500-fold) and 192-
fold selectivity over iNOS. The change in carbon length
of the side chain produced only a small effect on the
potency of all of the isoforms of NOS. Peptidomimetic
modifications19 were made on compounds 1-3. Incor-
poration of protecting groups at the N terminus of the
dipeptide and masking of the NH- group of the peptide
bond resulted in a dramatic loss in potency of nNOS,
which demonstrates the importance of the R-amino
group of the dipeptide and NH moiety of the peptide
bond for binding at the active site. Surprisingly, removal
of the carboxamide group (4-6) had only a minor effect
on both potency and selectivity.
* To whom correspondence should be addressed. Phone: (847) 491-
5653. Fax: (847) 491-7713. E-mail: Agman@chem.northwestern.edu.
Address correspondence to this author at the Department of Chemistry.
† Northwestern University.
‡ Carried out all experiments in this study.
§ The University of Texas Health Science Center.
| Developed the overexpression system for nNOS in E. coli and the
purification of the enzyme.
Developed the eNOS overexpression system in E. coli and the
purification of eNOS and isolated the eNOS.
10.1021/jm0101491 CCC: $20.00 © 2001 American Chemical Society
Published on Web 06/28/2001

2668 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16
Brief Articles
Ta ble 1. NOS Inhibition by the Reduced-Amide Bond
Analogues 7-9 and Nω-Nitroarginine-Containing Dipeptides
(1-6)a
Ki (µM)b
selectivityc
compound nNOS iNOS eNOS eNOS/nNOS iNOS/nNOS
1d
2d
3d
4e
5e
6e
0.13
0.33
0.45
0.54
0.46
0.35
25
97
104
100
118
108
200
245
141
199
213
70
1538
742
313
368
463
200
192
294
231
185
256
308
7
8
9
0.12
0.29
0.46
39
73
123
314
524
411
2617
1807
893
325
252
267
a
The enzymes used for the Ki measurements are recombinant
rat nNOS, recombinant murine iNOS, and recombinant bovine
b
eNOS. The Ki values represent at least duplicate measurements;
standard deviations of (8-12% were observed. c The ratio of Ki
d
(eNOS or iNOS) to Ki(nNOS); all are nNOS-selective. Data taken
from ref 18. e Data taken from ref 19.
(12)23 were reductively coupled using sodium triacetoxy-
borohydride in dry methanol,24 providing the reduced
dipeptides 13. After purification of these compounds,
cleavage of the Boc groups was achieved with TFA.
Compounds 7-9 were isolated as pale-yellow powders
after lyophilization, and the elemental analyses showed
that all of these compounds were triple trifluoroacetic
acid dihydrate salts.
Further peptidomometic modifications are necessary
for therapeutically useful compounds especially because
peptides are generally not good drug candidates as a
result of their poor bioavailability. Many bioisosteric
replacements of the amide bond were considered; how-
ever, considering the results from the first peptidomi-
metic approach mentioned above,19 the reduced amide
bond (-CH2-NH-) was chosen as the first target.
Compounds 7-9 contain features important to good
inhibitors of NOS. First, they contain several amine
nitrogens, which have been found to be very important
for interactions with the active site of the enzyme, and
second, they do not contain amide bonds, so they should
be stable toward endogenous peptidases in vivo.
Resu lts a n d Discu ssion
The Ki data for the reduced amide bond analogues
(7-9) are given in Table 1 along with the data for the
corresponding dipeptides (1-3)18 and descarboxamide
analogues (4-6).19 Deletion of the carbonyl group of the
amide bond either preserves or improves the potency
toward nNOS. Compound 7 shows the best potency over
nNOS (Ki ) 120 nM) as well as the highest selectivity
over eNOS (greater than 2500-fold) and iNOS (320-fold)
in these series of compounds. The length of the amine
side chain seems to have only a minor effect on the
potency for all isoforms of NOS; 8 and 9 inhibit nNOS
with Ki values of 290 and 460 nM, respectively. How-
ever, the shorter chain has better potency as well as
selectivity.
Ch em istr y
The three reduced amide bond analogues (7-9) were
synthesized according to the method in Scheme 1, which
employs Weinreb amide 1020 as a key intermediate. The
Weinreb amide was reduced to an aldehyde using
lithium aluminum hydride according to a modified
procedure of Goel et al.21 The resulting N-Boc-nitro-L-
argininal (11)22 and mono-Boc-protected alkanediamines
Sch em e 1

Brief Articles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16 2669
methanol, NR-tert-butoxycarbonyl-1,2-ethanediamine (12, n )
1; 184 µL, 1 mmol) and 3 Å molecular sieves were added, and
the mixture was stirred at room temperature. After being
stirred for 1 h, the reaction mixture was treated with sodium
triacetoxyborohydride (334.6 mg, 1.5 mmol) and stirred over-
night. The reaction mixture was filtered, and the filtrate was
concentrated in vacuo. The residue was purified by flash
column chromatography on silica gel (EtOAc/MeOH ) 1:1) to
afford 13a (201 mg, 45%) as a pale-yellow solid: 1H NMR (500
MHz, CD3OD) δ 3.67 (m, 1H), 3.33 (m, 2H), 3.04-3.20 (m, 2H),
2.62-2.73 (m, 4H), 1.52-1.75 (m, 4H), 1.45 (brs, 18H).
(4S)-4-N-ter t-Bu t oxyca r b on yla m in o-5-(2-[N-ter t-b u -
toxyca r bon yla m in op r op yl]a m in op en tyl]-N′-n itr ogu a n i-
d in e (13b). This compound (234 mg, 51%) was prepared as
described above using NR-tert-butoxycarbonyl-1,3-propanedi-
amine (12b, n ) 2): 1H NMR (500 MHz, CDCl3) δ 4.90 (m,
1H), 3.69 (m, 1H), 3.18-3.21 (m, 4H), 2.77(m, 2H), 2.64 (m,
2H), 1.50-1.65 (m, 6H), 1.44 (brs, 18H).
(4S)-4-N-ter t-Bu t oxyca r b on yla m in o-5-(2-[N-ter t-b u -
t oxyca r b on yla m in ob u t yl]a m in op en t yl]-N′-n it r ogu a n i-
d in e (13c). This compound (265 mg, 56%) was prepared as
described above using NR-tert-butoxycarbonyl-1,4-butanedi-
amine: 1H NMR (500 MHz, CDCl3) δ 4.67 (m, 1H), 3.71 (m,
1H), 3.14 (m, 4H), 2.72 (t, 2H, J ) 6.0), 2.64 (m, 2H), 1.71 (m,
2H), 1.49-1.54 (m, 8H), 1.45 (brs, 18H).
(4S)-N-(4-Am in o-5-[a m in oet h yl]a m in op en t yl)-N′-n i-
tr ogu a n id in e (7). Compound 13a (201 mg, 0.45 mmol) was
treated with 10 mL of trifluoroacetic acid/CH2Cl2 (1:1 v/v) for
30 min. Excess TFA and solvent were removed by evaporation.
The residue was dissolved in a small amount of water, and
the mixture was washed with ether and lyophilized to give a
pale-yellow foam (110 mg, 99%): 1H NMR (500 MHz, D2O) δ
3.63 (m, 1H), 3.32-3.39 (m, 6H), 3.24 (m, 2H), 1.69-1.78 (m,
4H). HRMS (M + 1) calcd for C8H21N7O2 248.183, found
248.180. Anal. (C8H21N7O2‚3TFA‚2H2O) C, H, N.
(4S)-N-(4-Am in o-5-[a m in op r op yl]a m in op en t yl)-N′-n i-
tr ogu a n id in e (8). This compound was prepared as described
above using compound 13b: 1H NMR (500 MHz, D2O) δ 3.62
(m, 1H), 3.32 (m, 2H), 3.24 (m, 2H), 3.13 (m, 2H), 3.01 (m,
2H), 2.03 (quin, 2H, J ) 7.0), 1.69-1.78 (m, 4H). HRMS (M
+ 1) calcd for C9H23N7O2 262.199, found 262.195. Anal.
(C9H23N7O2‚3TFA‚2H2O) C, H, N.
(4S)-N-(4-Am in o-5-[a m in ob u t yl]a m in op en t yl)-N′-n i-
tr ogu a n id in e (9). This compound was prepared as described
above using compound 13c: 1H NMR (500 MHz, D2O) δ 3.75
(m, 1H), 3.62 (m, 2H), 3.32 (m, 2H), 3.24 (m, 2H), 2.94 (m,
2H), 1.67-1.82 (m, 8H). HRMS (M + 1) calcd for C10H25N7O2:
276.214, found 276.214. Anal. (C10H25N7O2‚3TFA‚2H2O) C,
H, N.
En zym e a n d Assa y. All of the NOS isoforms used were
recombinant enzymes overexpressed in E. coli from different
sources; there is very high sequence identity for the isoforms
from different sources. The murine macrophage iNOS was
expressed and isolated according to the procedure of Hevel et
al.25 The rat nNOS was expressed26 and purified as described.27
The bovine eNOS was isolated as reported.28 Nitric oxide
formation from NOS was monitored by the hemoglobin capture
assay as described.29
Deter m in a tion of Ki Va lu es. The apparent Ki values were
obtained by measuring percent inhibition in the presence of
10 µM L-arginine with at least three concentrations of inhibi-
tor. The parameters of the following inhibition equation30 were
fitted to the initial velocity data: % inhibition ) 100[I]/{[I] +
Ki (1 +[S]/Km)}. Km values for L-arginine were 1.3 µM (nNOS),
8.2 µM (iNOS), and 1.7 µM (eNOS). The selectivity of an
inhibitor was defined as the ratio of the respective Ki values.
When the data for 7-9 are compared with those for
4-6 to see the intrinsic effect of deletion of the amide
carbonyl group, it is apparent that the potency on nNOS
and iNOS is about the same or increased a little, but
the potency with eNOS has greatly decreased. In
particular, the large increase in selectivity for nNOS
over eNOS by 7 comes from a 4.5-fold increase in
potency for nNOS and a 1.5-fold decrease in potency for
eNOS. For 8 and 9 this selectivity increase is driven
more by large decreases in potency for eNOS (2.5-fold
and almost 6-fold, respectively). Therefore, the selectiv-
ity for nNOS over eNOS is significantly increased for
7-9, which implies that the carbonyl moiety of the
amide bond might not be necessary for its activity
toward nNOS and iNOS but that the rigid -CO-NH-
group interacts better with the active site of eNOS than
the reduced, flexible -CH2-NH- group. The difference
may also be the result of the nonbasic amide nitrogen
becoming basic when reduced to the corresponding
amine, which may not bind as well to eNOS.
In conclusion, the reduced amide bond analogues 7-9
show potencies similar to that of nNOS but greatly
increased selectivities over eNOS and iNOS. Therefore,
the reduced amide bond peptidomimetics are significant
surrogates for the dipeptide inhibitors of nNOS, and this
should facilitate further development of potent and
selective inhibitors for NOS.
Exp er im en ta l Section
Gen er a l Meth od s. NOS assays were recorded on a Perkin-
Elmer Lambda 10 UV/vis spectrophotometer. 1H NMR spectra
were recorded on a Varian Inova 500 MHz NMR spectrometer.
Chemical shifts are reported as δ values in parts per million
downfield from TMS (δ 0.00) as the internal standard in
CDCl3. For samples run in D2O, the HOD resonance was
arbitrarily set at 4.80 ppm. An Orion Research model 701 pH
meter with a general combination electrode was used for pH
measurements. Electrospray mass spectra were obtained on
a Micromass Quattro II spectrometer. Elemental analyses
were obtained by Oneida Research Services, Inc., Whiteboro,
NY. Thin-layer chromatography was carried out on E. Merck
precoated silica gel 60 F254 plates. Amino acids were visualized
with a ninhydrin spray reagent or a UV/vis lamp. E. Merck
silica gel 60 (230-400 mesh) was used for flash column
chromatography.
Rea gen ts a n d Ma ter ia ls. Amino acids were purchased
from Advanced ChemTech, Inc. NADPH, calmodulin, and
human ferrous hemoglobin were obtained from Sigma Chemi-
cal Co. Tetrahydrobiopterin (H4B) was purchased from Alexis
Biochemicals. HEPES, DTT, and conventional organic solvents
were purchased from Fisher Scientific. All other chemicals
were purchased from Aldrich unless otherwise stated.
Nr-(ter t-Bu toxyca r bon yl)-L-n itr oa r gin in e N-Meth yl-O-
m eth ylca r boxa m id e (10). This compound was prepared from
12.8 g (40.1 mmol) of NR-(tert-butoxycarbonyl)-L-nitroarginine
as described in ref 21 except that isobutyl chloroformate was
used instead of methyl chloroformate. The residue was further
evacuated on an oil pump to give a white solid product (12.9
g, 89%): 1H NMR δ 5.65 (d, 1H, N-H, J ) 9.0), 4.69 (t, 1H, J
) 9.0), 3.79 and 3.73 (s, 3H), 3.25 and 3.10 (s, 3H), 3.64 (m,
1H), 3.32 (m, 1H), 1.79 (m, 2H), 1.63 (m, 2H), 1.47 (s, 9H).
Nr-(ter t-Bu toxyca r bon yl)-L-n itr oa r gin in a l (11). This
compound was prepared according to the method in ref 22.
From 3.62 g of 10 (10 mmol), the white powder product 2.17 g
(72%) was obtained, and it was stored in a deep freezer (-80
Ack n ow led gm en t. The authors are grateful to the
National Institutes of Health (Grant GM49725) for
financial support of this research.
1
°C) prior to use. H NMR showed that 11 is a mixture of the
free aldehyde and cyclized hemiaminal.22
Refer en ces
(4S)-4-N-ter t-Bu t oxyca r b on yla m in o-5-(2-[N-ter t-b u -
t oxyca r b on yla m in oet h yl]a m in op en t yl]-N′-n it r ogu a n i-
d in e (13a ). To a solution of 11 (303 mg, 1 mmol) in dry
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