1H-Pyrazole-1-carboxamidines: New inhibitors of nitric oxide synthase

Article abstract of DOI:10.1016/S0960-894X(00)00573-4

1H-Pyrazole-1-carboxamidines were prepared as potential inhibitors of the three isozymes of nitric oxide synthase. All of the compounds were found to be competitive inhibitors of all three isoforms. The most selective compound prepared was 1H-pyrazole-N-(3-aminomethylanilino)-1-carboxamidine (14), which is 100-fold selective for nNOS over eNOS with a K(i) value of 2μM. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00573-4

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2771±2774
1H-Pyrazole-1-carboxamidines:
New Inhibitors of Nitric Oxide Synthase
Younghee Lee,a Pavel Martasek,b Linda J. Romanb
and Richard B. Silvermana,*
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 2 August 2000; accepted 2 October 2000
AbstractÐ1H-Pyrazole-1-carboxamidines were prepared as potential inhibitors of the three isozymes of nitric oxide synthase. All of the
compounds were found to be competitive inhibitors of all three isoforms. The most selective compound prepared was 1H-pyrazole-N-(3-
aminomethylanilino)-1-carboxamidine (14), which is 100-fold selective for nNOS over eNOS with a Ki value of 2 mM. # 2000
Elsevier Science Ltd. All rights reserved.
Introduction
We recently reported that No-propyl-l-arginine was a
competitive inhibitor and also an inactivator of nNOS.11
This compound was found to have about a 3000-fold
selectivity for nNOS over iNOS and 150-fold selectivity
of nNOS versus eNOS, unusually large selectivities
compared to other analogues of l-arginine.12 In an e€ort
to design selective inhibitors of NOS based on the other
known l-arginine-derived inhibitiors of NOS, a library
of No-nitroarginine-containing dipeptides (or dipeptide
analogues) was synthesized by varying the chirality and
the order of amino acids. Several of the compounds
from this library showed selectivities higher than 1000-
fold in favor of one or two isozymes over the others.13
These studies suggested that selective inhibition of NOS
via substrate modi®cation is an achieveable goal.
A family of enzymes known as nitric oxide synthase
(NOS) catalyze the oxidation of l-arginine to l-citrulline
and nitric oxide (NO), a molecule that plays an important
role in the regulation of blood pressure, neurotransmis-
sion, and the immune response.1 Three isoforms of nitric
oxide synthase have been identi®ed in di€erent tissues.
Neuronal (nNOS) and endothelial (eNOS) nitric oxide
synthase are expressed constitutively and are Ca++ ion-
and calmodulin-dependent for activity; inducible mac-
rophage (iNOS) nitric oxide synthase is expressed by
in¯ammatory stimuli and is independent of calmodulin.
The normal biological functions regulated by NO are
attributed to those NOS isoforms. However, an excess
or de®ciency of nitric oxide produced by NOS isoforms
has been implicated in a variety of diseases, such as
septic shock,2 in¯ammatory arthritis,3 schizophrenia,4
Alzheimer's disease,5 impotence,6 and susceptibility to
infection.7 Therefore, selective inhibition of NOS iso-
forms would be bene®cial in cases when an excess of
NO is produced as well as to de®ne the roles of each
NOS isoform in biological systems.
In our continued e€orts to examine other compounds
for inhibition of NOS, a series of 1H-pyrazole-1-car-
boxamidines was synthesized, and the inhibition by each
of these compounds with the three isozymes of NOS
was investigated.
Chemistry
A wide variety of inhibitors of NOS are known,8 and
some of the earliest inhibitors are No-substituted-l-
arginines, such as methylarginine9 and nitroarginine.10
Treatment of alkylcyanamides 1a±g with pyrazole in 4
N HCl/dioxane produced a series of 1H-pyrazole-1-car-
boxamidines 2a±g,14 as shown in Scheme 1. To determine
the e€ect of methylation of the pyrazole ring in 2 on
enzyme binding, 3-methyl-, 4-methyl-, and 3,5-dimethyl-
pyrazole (3a±c, respectively) were treated with cyanamide
*Corresponding author. Tel.: +1-847-491-5653; fax: +1-847-491-
7713; e-mail: agman@chem.nwu.edu
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00573-4

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Y. Lee et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2771±2774
in acidic media to a€ord 4a±c, respectively (Scheme 2).
N-Butyl-N0-propylguanidine (5) was prepared in a 67%
yield by condensation of guanylating agent 2b with
excess n-butylamine, as described in Scheme 3; this
compound was made to determine if the pyrazole ring
was important to binding.
1H-Pyrazole-N-(4-aminobutyl)-1-carboxamidine (9) was
synthesized from mono N-Cbz-protected 1,4-diamino-
butane (6) in three steps as outlined in Scheme 4.
Treatment of 6 with cyanogen bromide and sodium
acetate in methanol a€orded cyanamide 7 in 73% yield.
Subsequent condensation of 7 with pyrazole in 4 N
HCl/dioxane gave 8 followed by hydrogenolysis of the
N-Cbz protecting group provided the desired 9 in 82%
yield for the last two steps.
Scheme 1.
Scheme 5 illustrates the synthetic route to 1H-pyrazole-
N-(3-aminomethylanilino)-1-carboxamidine (14). Com-
mercially available 3-nitrobenzylamine (10) was treated
with acetyl chloride in the presence of triethylamine to
a€ord N-acetyl-3-nitrobenzylamine in an 83% yield;
reduction of the nitro group under catalytic hydrogena-
tion conditions (H2, 10% Pd/C) provided the desired ani-
line analogue 13 (92%). In a similar manner as described
above, 11 was converted to cyanamide 12 (78%), which
was subsequently condensed with pyrazole in 4 N HCl/
dioxane to give rise to carboxamidine 13 (61%). Depro-
tection of the N-acetyl group of 13 was achieved in
re¯uxing 6 N HCl for 30min to give 14 in a 72% yield.
Scheme 2.
Results and Discussion
Compounds 2, 4, 5, 9, and 14 were evaluated as inhibi-
tors of the three isoforms of NOS using the hemoglobin
capture assay.15 All of the NOS isoforms used were
Scheme 3.
Scheme 4.
Scheme 5.

Y. Lee et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2771±2774
2773
recombinant enzymes overexpressed in Escherichia coli
from di€erence sources; there is very high sequence
identity for the isoforms from di€erent sources. The
murine macrophage iNOS was expressed and isolated
according to the procedure of Hevel et al.16 The rat
neuronal NOS was expressed17 and puri®ed as descri-
bed.18 The bovine endothelial NOS was isolated as
reported.19 The 1H-pyrazole-1-carboxamidines (2) exhi-
bit inhibitory activity against NOS. As summarized in
Table 1, 1H-pyrazole-1-carboxamidine (2a) was found
to be the most potent inhibitor of NOS among the series
of compounds tested in this study. However, the isoform
selectivity of 2a was insigni®cant. Substitution of
straight-chain alkyl groups (2b, 2c, and 2d) on the nitro-
gen resulted in decreased potency compared to 2a. The
decrease in potency was higher as the chain length of the
alkyl group increased from propyl to pentyl, suggesting
a steric hindrance to binding. There is a slight decrease
in potency for the compounds with cyclo- or branched
alkyl groups such as cyclopropyl (2e) (compared with
2b) and cyclopropylmethyl (2f) and isobutyl (2g) (com-
pared with the corresponding butyl analogue 2c), again
indicating a steric hindrance to binding.
on the pyrazole ring. Replacement of the pyrazole ring of
2b with butylamine su€ered about a 100-fold decrease in
potency.
We found earlier that nitroarginine-containing dipep-
tides in which the other amino acid has an amino-con-
taining side chain are quite potent and selective nNOS
inhibitors.13b To mimic the potential binding of a side-
chain amino group, 9 was synthesized, which was found
to be 10 times more potent at inhibiting nNOS (Ki=
6 mM) than its methyl isostere (i.e., 2d, Ki=60 mM),
again suggesting an important interaction of the side-
chain amino group with nNOS. It also exhibited one of
the highest selectivities (77-fold) of the compounds tes-
ted in this study. The conformationally-restricted ana-
logue of 9, namely 14, also was synthesized. This
compound was one of the more potent inhibitors of
nNOS of the compounds studied here and was the most
selective, 100-fold selective, for nNOS versus eNOS. A
similar observation to our result was found in a report
by the Glaxo Wellcome group in which the aryl amidine
analogues 15 and 16 were selective for nNOS over iNOS
and eNOS, although they were much more potent than
14 (Table 1).20
Because of the reactivity of 1H-pyrazole-1-carbox-
amidines as guanylating agents, it was suspected that
they may be inactivators as well as inhibitors. As a
representative example, 2b was shown to exhibit time-
dependent inhibition of nNOS with a Ki=33 mM and a
kinact=0.024 min 1. The site of reactivity is under
investigation.
Conclusion
To investigate steric e€ects on the pyrazole ring, several
methylated pyrazole analogues were made (4a±c).
Appending methyl groups to the 3-, 4-, or 5-position of
the pyrazole ring results in signi®cant loss of inhibitory
activity for all of the isoforms of NOS compared to the
unsubstituted counterpart 2a. The disubstituted analogue
4c showed a dramatic decrease in potency (ca. 1000-
fold) compared to its parent compound 2a. This drop in
potency for all isoforms is presumed to be steric in nat-
ure; eNOS is the most sensitive to methyl substitution
1H-Pyrazole-1-carboxamidines exhibit only moderate
potency and no selectivity for the NOS isozymes. Mod-
i®cation of the side chain by the addition of an amino
group increases potency and selectivity for nNOS over
iNOS and eNOS. These compounds show time-depen-
dent inhibition, presumably the result of their guany-
lating reactivity.
Acknowledgements
We are grateful to the National Institutes of Health for
®nancial support of this research to R.B.S. (GM49725)
and Bettie Sue Siler Masters (GM52419) and to the
Robert A. Welch Foundation (AQ-1192) for ®nancial
support to B.S.S.M, in whose lab P.M. and L.J.R.
work. Sincere thanks go to Professor Michael A. Mar-
letta (University of Michigan) for the E. coli cells which
express murine macrophage iNOS.
Table 1. Inhibition of NOS by 1H-pyrazole-1-carboxamidines
Ki (mM)
Selectivity
Compound nNOS
iNOS eNOS nNOS/iNOS nNOS/eNOS
2a
2b
2c
2d
2e
2f
2g
4a
4b
4c
5
0.4
3
14
0.7
5
14
50
80
22
150
9
0.5
9
25
1.8
1.7
1
1.3
3
1.8
14
2.4
8
60
50
10
45
8
12
350
700
6
700
120
80
180
100
190
1500
650
460
200
0.8
1.6
2.2
3.3
1.1
0.3
2.1
1.1
8.3
6
4
References and Notes
13
16
4.3
0.9
77
100
56
46
3
750
780
50
12
0.16
0.12
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