3,4-dihydro-1-isoquinolinamines: A novel class of nitric oxide synthase inhibitors with a range of isoform selectivity and potency

Article abstract of DOI:10.1016/S0960-894X(01)00119-6

3-Phenyl-3,4-dihydro-1-isoquinolinamine is a weak inhibitor of iNOS and nNOS. Structural variation of 5a results in inhibitors with a range of potency and selectivity for the NOS enzymes, including a potent and very selective iNOS inhibitor 5j.

Full text of DOI:10.1016/S0960-894X(01)00119-6

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1023–1026
3,4-Dihydro-1-isoquinolinamines: A Novel Class of Nitric Oxide
Synthase Inhibitors with a Range of Isoform Selectivity and
Potency
Haydn Beaton,a Peter Hamley,a,* David J. Nicholls,b Alan C. Tinkera,* and
Alan V. Wallaceb
aDepartment of Medicinal Chemistry, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, LE11 5RH, UK
bDepartment of Bioscience, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, LE11 5RH, UK
Received 19 December 2000; revised 5 February 2001; accepted 16 February 2001
Abstract—3-Phenyl-3,4-dihydro-1-isoquinolinamine is a weak inhibitor of iNOS and nNOS. Structural variation of 5a results in
inhibitors with a range of potency and selectivity for the NOS enzymes, including a potent and very selective iNOS inhibitor 5j.
# 2001 Elsevier Science Ltd. All rights reserved.
The nitric oxide synthases (NOS) are a family of haem-
containing oxygenases which convert the natural amino-
acid l-arginine into nitric oxide.1 Three enzyme iso-
forms have been characterised. Two of these are con-
stitutive and calcium dependent: neuronal NOS (nNOS
or NOS-I) found in nervous tissue and endothelial NOS
(eNOS or NOS-III) found in the vascular endothelium.
eNOS has a pivotal role in maintaining vascular home-
ostasis. The third enzyme isoform, inducible NOS
(iNOS or NOS-II) is synthesised de novo in response to
various pathological challenges. It is not calcium-
dependent and produces nitric oxide at much higher,
cytotoxic concentrations. Overexpression of iNOS has
been implicated in a number of inflammatory dis-
orders,2 in particular rheumatoid arthritis and septic
shock. Overstimulation of nNOS (by excessive calcium
concentrations) is thought to contribute to neurological
diseases, for example, stroke.3
inhibitor that is not particularly selective. More struc-
turally diverse inhibitors including guanidine deriva-
tives,6 isothioureas7 and amidines8 have also been
described but so far there have been few reports of
selective and potent iNOS inhibitors.8b
We have discovered that the 3,4-dihydro-1-iso-
quinolinamine derivative 5a is an inhibitor of the
human iNOS and nNOS isoforms of modest potency
(IC50 9 and 12 mM, respectively) but some selectivity
against eNOS. The aromatised analogue of 5, 3-phenyl-
1-isoquinolinamine, has no significant inhibitory activity
against any of the isoforms. This paper describes the
effects of structural variation in this novel class of com-
pounds which produces inhibitors showing a wide range
of potency and isoform selectivity, including compound
5j, which is one of the most selective iNOS inhibitors
reported to date.
Many inhibitors of NOS have been described in the lit-
erature. The best known are analogues of the substrate
l-arginine such as N-monomethyl arginine (l-NMMA,
1), N-nitroarginine (l-NA, 2a) and its methyl ester (l-
NAME, 2b) and N-iminoethyllysine (l-NIL, 3).4 How-
ever, their utility is limited by low potency and undesir-
able cardiovascular side effects arising from inhibition
of eNOS. Aminoguanidine (AA, 4)5 is a frequently used
Chemistry
Most compounds were made by a new approach that we
have recently described9 based on the cycloaddition
reaction between o-tolunitrile derivatives and silyl-
aldimines (Scheme 1). This affords amino dihydro-
isoquinolines directly in moderate to good yields. In the
case of 5-fluoro-2-methylbenzonitrile, deprotonation
occurred preferentially at the 6-position. This problem was
overcome by addition of one equivalent of trimethylsilyl
*Corresponding author. Tel.: +44-509-644541; fax: +44-509-645507;
e-mail: peter.hamley@astrazeneca.com; alan.tinker@astrazeneca.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00119-6

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H. Beaton et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1023–1026
chloride to the reaction mixture to quench the unwanted
anion, then adding a second equivalent of base to
generate the required lithiated species. The silyl group is
removed quantitatively after cyclisation by fluoride
treatment.
Biology
Compounds were assayed for their ability to inhibit the
conversion of [3H]-l-Arg to [3H]-l-citrulline catalysed
by: (i) iNOS from human DLD-1 cells; (ii) nNOS
from rat cerebellum; and (iii) eNOS frum HUVEC
cells using minor modifications11 of methods already
described.12,13
A few simple 3,4-dihydroisoquinolinamines (i.e., sub-
stituted by H or methyl at position 3) were prepared by
reaction of a phenethyl halide derivative with ethyl
thiocyanate and a Lewis acid10 to give a cyclic thiomi-
date which in turn was reacted with ammonia to give
isoquinolinamines 6b and 6e. The benzazepine homo-
logue 6j could be prepared similarly from 3-bromo-1-
phenylpropane (Scheme 2).
Results and Discussion
The effects of substitution of the 3-phenyl compound 5a
were first investigated. Surprisingly, substitution at any
of the unsubstituted aryl positions on either the iso-
quinoline ring, the phenyl substituent, or the amidine
group by even a single methyl group led to loss of
activity against iNOS relative to the parent 5a (data not
shown). This suggests that 5a is close to the maximum
size acceptable for binding to the enzyme active site for
this series: indeed only fluorine was tolerated in some
positions (Table 1). Whilst fluorine substitution meta or
para in the phenyl substituent or at positions 5 or 6 in
the isoquinoline ring can be made with little or no effect
on either the potency or selectivity profile of the com-
pounds, dramatic effects occur on substitution at the
isoquinoline 7 or 8 position. While the former is almost
inactive, the latter results in a 34-fold increase in iNOS
activity without significantly affecting the activity
against the other isoforms (cf. compound 5j vs 5d). Thus,
compound 5j has good potency combined with 100-fold
selectivity for iNOS against nNOS and about 1000-fold
Scheme 1. (i) LDA, DMPU, THF, ꢀ78 ꢁC; (ii) R0CH¼NSiMe3 (pre-
pared from R0CHO and LiHMDS, THF, 0 ꢁC); (iii) HCl; (iv) Me3SiCl;
(v) TBAF, THF.
Table 1. Inhibition of NOS isoforms by 3-phenylisoquinolinamines
R
R0
iNOS IC50 (mM)
nNOS IC50 (mM)
eNOS IC50 (mM)
5a
5b
5c
5d
5e
5f
5g
5h
5i
H
H
H
H
H
8-F
5-F
6-F
7-F
8-F
8-Cl
8-Cl
H
2-F
3-F
4-F
4-Cl
H
4-F
4-F
4-F
4-F
H
9
33
12
13
10
9
30
2
5
20
44
16
n.s.@100a
44
n.s.@100a
40
86
80
44
100
9
5.5
46%@100a
0.55
4.7
9
43%@100a
55
5j
5k
5l
0.16
6
5
n.s.@100a
n.s.@100a
39%@100a
25%@100a
100
4-F
Standards :
l-NMMA
l-NAME
l-NIL
AG
1
2b
3
0.3
14
0.13
4.5
0.1
0.15
1.6
6
0.3
2.7
2.4
72
4
aPercent inhibition at test concentration n.s.=no significant effect (<25% inhibition).

H. Beaton et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1023–1026
1025
Table 2. Inhibition of NOS isoforms by alkyl and heteroaryl substituted isoquinolinamines
R
R0
n
iNOS IC50 (mM)
nNOS IC50 (mM)
eNOS IC50 (mM)
6a
6b
6c
6d
6e
6f
6g
6h
6i
H
CH3
H
H
H
H
CH3
H
H
H
H
H
1
1
1
1
1
1
1
1
1
2
32
18
2.7
1.3
0.4
3
0.4
1.5
10
2
0.9
8
2
4
0.9
CꢃCH
c-Pr
CH3
3.6
n.s.@100a
n.s@100a
2.7
1.2
4.2
47%@100a
n.s.@100a
2-Thienyl
2-Furanyl
2-Thiazolyl
2-Imidazolyl
H
8
5
19
n.s.@100a
2
n.s.@100a
12
6j
aSee footnote to Table 1.
selectivity against eNOS. The suspicion that compound
5a represented a close to maximum size fit for the active
site of NOS led us to examine the preparation of smaller
analogues (Table 2).
cells) the compounds show a similar rank order of
potency. Several show potency in the cell comparable
or superior to that of the standard inhibitors such as
l-NMMA 1, l-NAME 2b or aminoguanidine 4.
The 3-unsubstituted compound 6a has a reversed selec-
tivity compared to that of 5 resulting from a reduction
in iNOS potency combined with an increase in activity
against nNOS and eNOS. Small alkyl substituents or
five-membered heterocycles at position 3 give com-
pounds with little selectivity. Disubstitution at the 3-
position as in 6e and increase in amidine ring size to
seven (6j) reduces potency.
In conclusion, we have described a series of dihydro-
isoquinolines which led to the discovery of a highly
selective iNOS inhibitor. The levels of selectivity
achieved demonstrate that good isoform selectivity is
indeed an achievable goal and points the way to com-
pounds which could not only more closely define the
role of inducible nitric oxide synthase, but also offer a
novel therapy for inflammatory diseases.
Some of the more potent and selective compounds were
examined for their ability to inhibit nitric oxide synth-
esis in intact DLD-1 cells (Table 3). Although the
absolute value of the IC50 in these experiments is much
higher than in the enzyme assays (largely due to the
much higher arginine concentration of ꢂ1 mM in the
References and Notes
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