Novel 2-aminobenzothiazoles as selective neuronal nitric oxide synthase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.02.011

A series of substituted 2-aminobenzothiazole compounds have been synthesized and evaluated as nitric oxide synthase (NOS) inhibitors. Compound 14 shows activity in the nM range and is selective for the human neuronal NOS isoform. We have also evaluated the compounds against the rat NOS isoforms. For some of the compounds, there are significant differences in NOS inhibitory activities between the human and rat enzymes. For example, compound 10b has nM activity against the rat nNOS while low μM activity against the human nNOS.

Full text of DOI:10.1016/j.bmcl.2007.02.011

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2540–2544
Novel 2-aminobenzothiazoles as selective neuronal nitric oxide
synthase inhibitors
Joanne Patman, Namrta Bhardwaj, Jailall Ramnauth,* Subhash C. Annedi, Paul Renton,
Shawn P. Maddaford, Suman Rakhit and John S. Andrews
NeurAxon Inc. Suite 318, 16-1375 Southdown Road, Mississauga, Ont., Canada L5J 2Z1
Received 14 December 2006; revised 2 February 2007; accepted 6 February 2007
Available online 8 February 2007
Abstract—A series of substituted 2-aminobenzothiazole compounds have been synthesized and evaluated as nitric oxide synthase
(NOS) inhibitors. Compound 14 shows activity in the nM range and is selective for the human neuronal NOS isoform. We have
also evaluated the compounds against the rat NOS isoforms. For some of the compounds, there are significant differences in
NOS inhibitory activities between the human and rat enzymes. For example, compound 10b has nM activity against the rat nNOS
while low lM activity against the human nNOS.
Ó 2007 Elsevier Ltd. All rights reserved.
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), two of which, one in the
endothelial cells (eNOS) and one in the neuronal cells
(nNOS), are constitutive while the one in macrophage
cells is inducible (iNOS).2 These enzymes are homodimer-
ic proteins that catalyze a five-electron oxidation of L-ar-
ginine, yielding NO and citrulline. The role of NO
produced by each of the NOS isoforms is quite unique.
Overstimulation or overproduction of individual NOS
isoforms plays a role in several disorders including septic
shock, arthritis, diabetes, ischemia-reperfusion injury,
pain, and various neurodegenerative diseases.3–5 NOS
inhibitors can be therapeutic in many disorders, but
preservation of physiologically important nitric oxide
synthase function requires the development of isoform-
selective inhibitors. In particular, when designing nNOS
inhibitors, selectivity for nNOS relative to eNOS is of
paramount importance in order to avoid adverse cardio-
vascular effects.6
were structural analogs of the natural substrate L-argi-
nine, which lack isoform selectivity.7 However, some lat-
er generations of L-arginine analog NOS inhibitors are
potent and selective.8,9 In addition, potent and selective
non-amino acid NOS inhibitors have been reported.10–14
The general pharmacophore model for a NOS inhibitor
that targets the arginine binding site is a guanidine isos-
teric group and a basic site attached to a central linker
(Fig. 1). The minimum requirement is an isostere of
the substrate’s guanidine group to make a bidentate
interaction with a conserved glutamic acid residue of
the enzyme.
In our research on designing selective nNOS inhibitors
for treating CNS disorders, we envisioned using a bicy-
clic heterocyclic scaffold as a central linker. In this re-
port, we present novel substituted 2-aminobenzothiazole
inhibitors of NOS that are potent and selective for the
neuronal isoform.15
Our initial focus was to determine if the 2-aminobenzo-
thiazole scaffold is tolerated in the NOS active site. We
A number of different classes of NOS inhibitors have
been reported in the literature. Early NOS inhibitors
Keywords: Nitric oxide synthase; Nitric oxide; Inhibitor; 2-Amino-
benzothiazole.
*
Corresponding author. Tel.: +1 416 673 8461; fax: +1 416 595
8249; e-mail: jramnauth@nrxn.com
Figure 1. NOS inhibitor pharmacophore model.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.02.011

J. Patman et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2540–2544
2541
synthesized a few analogs containing guanidine isosteric
groups with no other substitution on the bicyclic hetero-
cyclic ring. The syntheses of 5- and 6- substituted
2-aminobenzothiazoles are outlined in Scheme 1.
Reduction of the commercially available nitro com-
pounds furnished the anilines. These anilines provided
a handle for attaching the guanidine isostere. Two com-
mon guanidine isosteric groups were employed: a thio-
phene amidine and an S-ethyl isothiourea.
to Scheme 2. The 2-hydrazinylnitrobenzothiazoles 6a,b
were obtained from the corresponding nitro-
aminobenzothiazole compounds by treating with hydra-
zine at elevated temperature.17 These compounds were
then converted to the 2-chloronitrobenzothiazoles 7a,b
in refluxing thionyl chloride.18 Treatment of the 2-chlo-
ronitrobenzothiazoles with a series of amines in H2O or
DMF with K2CO3 at 90 °C gave the corresponding
2-amino substituted nitrobenzothiazoles of general
structure 8. These compounds were reduced with SnCl2
in refluxing ethanol to furnish the anilines with the gen-
eral structure 9. Since the thiophene amidine proved to
be more potent and selective for nNOS than the corre-
sponding isothioureas, it was used as a guanidine isoste-
re for all subsequent compounds. Thus, this group was
coupled to the anilines to give the final compounds
10–21.
The inhibitory activities of these compounds16 against
all three human isoforms of NOS were measured by fol-
lowing the conversion of [3H]L-arginine into [3H]L-cit-
rulline. The observed NOS IC50 values and the
selectivity for nNOS ratios, defined as IC50 (eNOS)/
IC50 (nNOS) and IC50 (iNOS)/IC50 (nNOS), are shown
in Table 1.
The data indicated that compounds with the thiophene
amidines (4a and 4b) displayed better NOS inhibitory
activities over the S-ethyl isothioureas (5a and 5b). Also,
compounds 4a and 4b showed excellent selectivities for
nNOS over iNOS (400- and 90-fold, respectively). Sim-
ilar inhibitory activities against nNOS and eNOS were
obtained regardless of whether the guanidine isostere
is attached at the 5- or 6-position of the 2-aminobenzo-
thiazole scaffold. Compounds with the 6-substituted
guanidine isostere are slightly more active against iNOS
than their 5-substituted counterpart, however, all of the
compounds displayed poor inhibitory activity against
this isoform.
The inhibitory activities and selectivities of the 2,5- and
2,6-di-substituted aminobenzothiazoles against the hu-
man NOS isozymes are presented in Table 2. In general,
introduction of the sidechains did not have any detri-
mental effects on the inhibitory activity against all three
NOS isoforms. Similar inhibitory activities against
nNOS were obtained regardless of whether the thio-
phene amidine is attached at the 5- or 6-position of
the 2-substituted aminobenzothiazole scaffold. Howev-
er, for eNOS and iNOS, the 6-substituted thiophene
amidines showed an increase in activity when compared
to their corresponding 5-substituted analogs. In a simi-
lar fashion to the mono-substituted analogs, all com-
pounds displayed poor inhibitory activity against iNOS.
In an effort to improve nNOS potency and selectivity for
nNOS over eNOS, we explored substitution of the amin-
obenzothiazole by tethering different groups from the
2-position. These analogs were synthesized according
Scheme 1. Reagents and conditions: (a) SnCl2Æ2H2O, EtOH, reflux; (b)
EtOH, rt; (c) (i) benzoyl isothiocyanate, THF; (ii) 1 N NaOH, THF;
(iii) EtI, K2CO3, THF.
Scheme 2. Reagents and conditions: (a) NH2NH2HCl, NaOH, ethyl-
ene glycol, 145 °C; (b) SOCl2, reflux; (c) RNH2, H2O, 90 °C or DMF,
K2CO3, 90 °C; (d) SnCl2Æ2H2O, EtOH, reflux; (e) EtOH, rt.
Table 1. Inhibition of human NOS isozymes by 2-aminobenzothiazoles
eNOS/nNOSb
iNOS/nNOSc
a
a
a
Compound
Subst.
nNOS IC50 (lM)
eNOS IC50 (lM)
iNOS IC50 (lM)
4a
4b
5a
5b
5
6
5
6
0.2
0.2
4.6
1.4
2.3
2.9
12
79
25
97
46
13
18
3
395
90
21
32
9.2
7
a Inhibitory activity for human NOS was measured by monitoring the conversion of [14C]-Arg to [14C]-Cit. For each compound, the percent
inhibition was determined in duplicate at 8–12 different concentrations.
b Selectivity against nNOS over eNOS is the ratio of eNOS IC50/nNOS IC50
c Selectivity against nNOS over iNOS is the ratio of iNOS IC50/nNOS IC50
.
.

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J. Patman et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2540–2544
Table 2. Inhibition of human NOS isozymes by di-substituted aminobenzothiazoles
6
S
HN
S
NHR
5
N
HN
a
a
a
Compound
Subst.
R
nNOS IC50
(lM)
eNOS IC50
(lM)
iNOS IC50
(lM)
eNOS/nNOSb
iNOS/nNOSc
10a
11a
12a
13a
14
5
5
5
5
5
6
6
6
6
6
6
6
2-(Pyridin-2-yl)ethyl
2-Morpholinoethyl
1.1
1.3
0.4
0.8
0.03
1.3
2.0
0.5
0.2
0.3
4.4
2
6
12
16
13
1.6
0.9
1.5
6
139
141
48
91
15
13
37
14
14
6
5
9
126
108
123
118
483
10
1-Benzylpiperidin-4-yl
1-(4-Fluorobenzyl)piperidin-4-yl
40
16
53
1
( )-2-(1-Methylpyrrolidin-2-yl)ethyl
2-(Pyridin-2-yl)ethyl
2-Morpholinoethyl
10b
11b
12b
13b
15
1
18
28
1-Benzylpiperidin-4-yl
12
40
2
1-(4-Fluorobenzyl)piperidin-4-yl
2-(1H-Imidazol-5-yl)ethyl
4-Bromophenethyl
8
70
19
0.7
2.1
16
16
17
75
64
0.5
8
17
32
Tetrahydro-2H-pyran-4-yl
a Inhibitory activity for human NOS was measured by monitoring the conversion of [14C]-Arg to [14C]-Cit. For each compound, the percent
inhibition was determined in duplicate at 8–12 different concentrations.
b Selectivity against nNOS over eNOS is the ratio of eNOS IC50/nNOS IC50
.
c Selectivity against nNOS over iNOS is the ratio of iNOS IC50/nNOS IC50
.
The most potent and selective nNOS inhibitor obtained
was with the racemic sidechain 2-(1-methylpyrrolidin-2-
yl)ethyl, compound 14 (nNOS IC50 = 0.03 lM, 53- and
483-fold selectivity against eNOS and iNOS, respective-
ly). No attempts were made to separate the enantiomers.
This compound contains a basic group with a 3-carbon
linker separating the basic nitrogen from the 2-amino
group on the heterocyclic core. In general, a 3-carbon
linker from the 2-aminobenzothiazole scaffold to a basic
site provided potent nNOS inhibitors in the sub-micro-
molar range (compounds 12a,b, 13a,b, 14, and 15).
Although compounds 10a and 10b contain this structur-
al requirement, these compounds did not give sub-mi-
cromolar activity in the nNOS inhibition assay. One
possible reason for this could be the reduced basicity
of the pyridine group as compared to an alkyl substitut-
ed nitrogen. Further evidence for the importance of the
basicity of the nitrogen comes from comparing the
nNOS inhibitory activity of the imidazole analog 15
(nNOS IC50 = 0.3 lM) with that of the pyridine analog
10b (nNOS IC50 = 1.3lM). For these analogs, one
would expect the imidazole to be more basic than the
pyridine. Also, compounds 16 and 17 do not contain ba-
sic groups in the sidechain and as a result weaker activ-
ities were observed.
As a sub-series, the N-benzylpiperidine analogs (Table 2,
compounds 12a,b and 13a,b) gave potent and selective
nNOS inhibitory activities. We focused our attention
on optimizing this series by exploring substitution on
the phenyl ring. Keeping the amidine group at the 6-po-
sition of the heterocyclic scaffold, a few analogs were
synthesized according to Scheme 2 and evaluated for
NOS inhibitory activity (Table 3). Compounds contain-
ing the 3- and 4-methoxyphenyl groups, 18 and 19,
respectively, proved to be potent nNOS inhibitors. In
addition, comparing iNOS inhibitory activities for these
two compounds revealed that the position of the meth-
oxy group is crucial for activity. This could be attributed
to a steric clash between 18 and the iNOS enzyme. For
nNOS and eNOS, it is interesting that an ester 20 is tol-
erated at the 4-position of the phenyl ring but not an
Table 3. Inhibition of human NOS isozymes by substituted benzylpiperidine aminobenzothiazoles
R
N
H
N
S
N
a
S
NH
NH
eNOS/nNOSb
iNOS/ nNOSc
a
a
Compound
R
nNOS IC50 (lM)
eNOS IC50 (lM)
iNOS IC50 (lM)
18
19
20
21
3-OMe
4-OMe
0.1
0.2
0.3
5.1
3.8
5.7
5.2
59
>100d
9
>100d
>100d
27
28
18
12
>1000
45
4-CO2Me
4-CO2H
>300
>20
a Inhibitory activity for human NOS was measured by monitoring the conversion of [14C]-Arg to [14C]-Cit. For each compound, the percent
inhibition was determined at 8–12 different concentrations.
b Selectivity against nNOS over eNOS is the ratio of eNOS IC50/nNOS IC50
.
c Selectivity against nNOS over iNOS is the ratio of iNOS IC50/nNOS IC50
.
d No inhibition at the highest test concentration of 100 lM.

J. Patman et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2540–2544
Table 4. Inhibition of rat NOS isozymes by di-substituted aminobenzothiazoles
2543
eNOS/nNOSb
iNOS/ nNOSc
a
a
a
Compound
nNOS IC50
(lM)
eNOS IC50
(lM)
iNOS IC50
(lM)
10b
11b
12b
15
0.05
0.8
3.4
9.1
25
44
36
26
NT
19
63
12
139
1
500
55
0.09
4.8
12.5
4.6
400
5
16
18
4.7
6.7
5.2
1.3
1
0.2
ND
3
NT, not tested.
ND, not determined.
a Inhibitory activity for rat NOS was measured by monitoring the conversion of [14C]-Arg to [14C]-Cit. For each compound, the percent inhibition
was determined in duplicate at 8–12 different concentrations.
b Selectivity against nNOS over eNOS is the ratio of eNOS IC50/nNOS IC50
.
c Selectivity against nNOS over iNOS is the ratio of iNOS IC50/nNOS IC50
.
acid 21 which is probably due to electrostatic repulsion.
Excellent selectivity for nNOS against iNOS was ob-
tained for compounds 18 and 20.
in rat models of disease states in which nNOS is thought
to play a role and will present the results in due course.
Since there are species differences among the NOS en-
zymes and we would like to evaluate our compounds
in rat models of disease states, a few compounds have
been tested for inhibitory activity in the rat NOS assays
(Table 4). In a similar fashion to the human enzymes,
inhibition was determined by measuring their effect on
the conversion by NOS of [3H]L-arginine into [3H]L-cit-
rulline. While there are differences between the rat and
human NOS enzymes, we were somewhat surprised by
the dramatic differences in nNOS activities and selectiv-
ities of some of the compounds. For example, com-
pound 10b (human nNOS IC50 = 1.3 lM vs rat nNOS
IC50 = 0.05 lM) and compound 12b (human nNOS
IC50 = 0.5 lM vs rat nNOS IC50 = 0.09 lM) showed
the largest differences in nNOS inhibitory activities be-
tween the two species. In the case of 10b, there was no
selectivity for the human nNOS over eNOS while there
was a 60-fold selectivity for the rat nNOS over eNOS.
Similarly, compound 12b was much more selective
(140-fold) for the rat nNOS over eNOS. In addition
compounds 10b and 12b showed excellent selectivity
(500-fold and 400-fold, respectively) for rat nNOS over
iNOS. It should be noted that while the inhibitory activ-
ities can vary by as much as 2 orders of magnitude for
nNOS (compound 10b) in the two species, such is not
the case for eNOS and iNOS inhibition. Interestingly,
18 showed a dramatic loss of nNOS potency but an in-
crease in its eNOS activity.
Acknowledgments
We are grateful to NoAb BioDiscoveries Inc. (Mississa-
uga, ON, Canada) and Asinex Ltd (Moscow, Russia)
for performing the NOS inhibition assays. We thank
the Natural Sciences and Engineering Council (NSERC)
of Canada for Industrial Research Fellowships to two of
us (N.B. and J.R.).
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In summary, we have designed and synthesized a series
of 2-aminobenzothiazole inhibitors that are selective
for human and rat nNOS. Compound 14 was the most
potent and selective for human nNOS over eNOS and
iNOS, while compound 12b was the most selective for
rat nNOS over eNOS. We have shown that by introduc-
ing substituents at the 2-position of the heterocyclic
scaffold we were able to design nNOS selective com-
pounds by as much as 50-fold over eNOS for the human
enzymes. We have also shown that for a few compounds
significant differences in NOS inhibitory activities
among the rat and human enzymes can occur. We are
currently evaluating some of our selective compounds
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2544
J. Patman et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2540–2544
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15. Part of this work has been presented: Patman, J.;
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Garcin-Hosfield, E.; Bhardwaj, N.; Ramnauth, J.
Novel Aminobenzothiazole Nitric Oxide Synthase
Inhibitors. Applications in the treatment of central
nervous system disorders, 89th Canadian Chemistry
Conference Meeting, Halifax, Nova Scotia, Canada,
May 27–31, 2006.