Substituted 6-phenyl-pyridin-2-ylamines: Selective and potent inhibitors of neuronal nitric oxide synthase

Article abstract of DOI:10.1016/j.bmcl.2004.06.043

The synthesis and nNOS and eNOS activity of 6-(4-(dimethylaminoalkyl)-/6- (4-(dimethylaminoalkoxy)-5-ethyl-2-methoxyphenyl)-pyridin-2-ylamines and 6-(4-(dimethylaminoalkyl)-/6-(4-(dimethylaminoalkoxy)-2,5-dimethoxyphenyl) -pyridin-2-ylamines 1-8 are described. These compounds are potent inhibitors of the human nNOS isoform.

Full text of DOI:10.1016/j.bmcl.2004.06.043

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4511–4514
Substituted 6-phenyl-pyridin-2-ylamines: selective and
potent inhibitors of neuronal nitric oxide synthase
Deane M. Nason, Steven D. Heck, Mathew S. Bodenstein, John A. Lowe, III,
Robert B. Nelson, Dane R. Liston, Charles E. Nolan, Lorraine F. Lanyon,
Karen M. Ward and Robert A. Volkmann*
Pfizer Global Research and Development, Groton, CT 06340, USA
Received 16 April 2004; revised 14 June 2004; accepted 14 June 2004
Available online 3 July 2004
Abstract—The synthesis and nNOS and eNOS activity of 6-(4-(dimethylaminoalkyl)-/6-(4-(dimethylaminoalkoxy)-5-ethyl-2-meth-
oxyphenyl)-pyridin-2-ylamines and 6-(4-(dimethylaminoalkyl)-/6-(4-(dimethylaminoalkoxy)-2,5-dimethoxyphenyl)-pyridin-2-yl-
amines 1–8 are described. These compounds are potent inhibitors of the human nNOS isoform.
Ó 2004 Elsevier Ltd. All rights reserved.
Nitric oxide (NO), which is generated oxidatively from
L
-arginine by nitric oxide synthase (NOS), is an
N
NH2
important neuromodulator in the CNS. All the NOS
isoforms (nNOS, eNOS, and iNOS) are present in the
brain. Given the role NO plays in a variety of normal
and pathological states, selective inhibitors of NOS
isozymes are of considerable interest. Inhibition of
nNOS, for example, has been proposed as a neuropro-
tective strategy in stroke, Parkinson’s disease, and
trauma.1;2 The role of NO in the pathophysiology of
stroke has revealed complex and contradictory results.
With 7-nitroindazole (7-NI), an nNOS/eNOS inhibitor,
reduction in infarction volumes in animals is dependant
on serum glucose concentration and ischemic intracel-
lular pH.3 Answering the question of the viability of
selective nNOS inhibition as a therapeutic approach for
neuroprotection will require the identification of not
only potent but selective nNOS inhibitors.4 Herein we
report the preparation of potent and selective nNOS
inhibitors.
O
N
N
A
Early efforts in our laboratory were focused on a series
of 6-(4-(substituted)phenyl)-2-aminopyridines, the best
of which were potent nNOS inhibitors5 (IC50 human
nNOS ¼ 100–250 nM) with modest selectivity (sevenfold
selective against eNOS) for the nNOS isozyme (see
structure A).6 Efforts to increase potency and selectivity
were successful when substituents were placed on addi-
tional positions of the phenyl ring. In particular, analogs
bearing 2-methoxy and 5-ethyl (or 5-methoxy) substit-
uents on the phenyl ring and having aminoalkyl and
aminoalkoxy substitution in the 4-position provided
potent and selective nNOS inhibitors. Herein we
describe the synthesis and NOS activity of eight potent
6-(4-(dimethylaminoalkyl)-/6-(4-(dimethylaminoalkoxy)-
5-substituted-2-methoxyphenyl)-pyridin-2-ylamines 1–8
in which we have evaluated 5-methoxy and 5-ethyl
derivatives and have systematically varied the length
and structure of the linker between the dimethylamino
moiety and the phenyl ring.
Keywords: nNOS inhibitors.
* Corresponding author. Tel.: +1-860-441-4662; fax: +1-860-686-1060;
e-mail: robert_a_volkmann@groton.pfizer.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.043

4512
D. M. Nason et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4511–4514
removal of the 2,5-dimethylpyrrole protecting group,
OMe
XMe
OMe
aminopyridine 1. 4-Dimethylaminomethyl-2-methoxy-5-
ethyl-2-aminopyridine 2 was similarly prepared by
coupling of boronic acid 21 with Boc-protected amino-
6-bromopyridine 22 (see Fig. 3). Boronic acid 21, in
turn, was generated in a four-step sequence from bro-
mide 17 via a Stille coupling followed by olefin reduc-
tion, bromination, and metallation and treatment with
triethyl borate to generate 21.
N
NH2
N
NH2
Me2N(CH2)n
Me2N(CH2)2O
XMe
7 (X=O)
8 (X=CH2)
1 (X=O, n=1) 2 (X=CH2, n=1)
3 (X=O, n=2) 4 (X=CH2, n=2)
5 (X=O, n=3) 6 (X=CH2, n=3)
Preparation of the dimethylaminoethyl derivatives 3 and
4 bearing the carbon atom linker is shown in Figure 4
and followed a strategy similar to that used for the
preparation of 1 and 2. Alcohol 26 was converted in
three steps to boronic acid 28, which was coupled with
bromopyridine 22 to afford pyridine ether 29. Removal
of the TBDMS protecting group followed by a Dess–
Martin oxidation of alcohol 30 generated aldehyde 31,
which was allowed to react with dimethylamine under
standard reductive amination conditions to afford 40.
Boc removal yielded aminopyridine 3. Aminopyridine 4
was generated using the identical strategy starting with
3-methoxy-6-ethyl-phenethyl alcohol 33, which in turn
was prepared in a two-step sequence from 32 involving a
Friedel–Crafts acylation/ketone reduction protocol.
Figure 1. nNOS targets.
Multistep syntheses were required for preparing the
eight targeted 2-aminopyridine derivatives shown in
Figure 1. The general strategy for accessing each of the
compounds involved convergent routes utilizing a Su-
zuki coupling of appropriately substituted phenylbo-
ronic acids and protected 6-bromo-2-aminopyridines to
build up the biaryl framework. The targeted amino-
pyridines 1–8 all contain a terminal dimethylamino
group. Other substituted distal alkyl and aryl amines
were prepared. In general, the dimethylamino group
provides potent nNOS activity and was therefore used in
this investigation.
Benzaldehydes 14 and 42, which provided 1 and 2, were
also employed to generate aminopyridines with a three
carbon atom linker (see Fig. 5).
6-(4-Dimethylaminomethyl-2,5-dimethoxy-2-aminopyr-
idine 1 was prepared by coupling boronic acid 11 with
bromopyridine 12 as described in Figure 2. Standard
acetal hydrolysis of 13, followed by reductive amination
involving aldehyde 14, and dimethylamine in the pres-
ence of sodium triacetoxyborohydride provided after
Accordingly, a Wittig condensation involving aldehyde
14 provided allylic amine 43. Catalytic hydrogenation of
43 provided propylamine 45. Removal of the pyrrole
OMe
OMe
OMe
OMe
1) Pd(PPh3)4/Na2CO3/
90oC/ EtOH/ H2O
1) (CH3)2NH/
Na(OAc)3BH
/AcOH/ THF
2) NH2OH/6:1
EtOH/H2O/ 90oC
1) HO(CH2)2OH/TsOH
benzene 100%
Br
R
N
NH2
2) BuLi/ THF/ -78oC
N
N
H
H
B(OEt)3 85%
Br
N
12
N
Me2NCH2
R
O
O
O
OMe
OMe
OMe
2) HOAc/ THF/ H2O 95%
OMe
9
1
13 (R =CH(OCH2CH2O))
14 (R =CHO)
10 (R =Br)
11 (R =B(OH)2)
Figure 2.
OMe
Br
OMe
Br
OMe
1) Bu3SnCHCH2
Pd(PPh3)4/ toluene
heat 85%
2) H2/ PtO2/EtOAc
30 PSI 100%
1) B2H6/ THF/ 0oC 96%
2) TBDMSCl/
imidazole/ THF/ 96%
1) NBS/ silica gel/
CCl4 62%
HO
RO
TBDMSO
2) BuLi/ THF/
-78oC/ B(OEt)3
O
R
16 (R =H)
15
OMe
17 (R =TBDMS)
18 (R =CHCH2)
19 (R =CH2CH3)
OMe
OMe
1) (CH3)2NH/Na(OAc)3BH/
AcOH/ THF
R
1) Pd(PPh3)4/Na2CO3/
90oC/ EtOH/ H2O
N
NHBOC
N
NH2
2) 6N HCl/ dioxane/
TBDMSO
R
115oC
Me2NCH2
Br
N
NHBOC
CH2CH3
CH2CH3
CH2CH3
20 (R =Br)
21 (R =B(OH)2)
22
23 (R =CH2OTBDMS)
24 (R =CH2OH)
25 (R=CHO)
2) TBAF/ THF/ 45% (2 steps)
3) MnO2/ toluene/ reflux 83%
2
Figure 3.

D. M. Nason et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4511–4514
4513
OMe
OMe
OMe
OMe
1) TBDMSCl/ imidazole/
1) Pd(PPh3)4/Na2CO3/
90oC/ EtOH/ H2O
R
THF/ 23oC
N
NHBOC
2) NBS/ silica gel/
CCl4
3) BuLi/ THF/
-78oC/ B(OEt)3
HOCH2CH2
TBDMSOCH2CH2
R
Br
N
NHBOC
22
OMe
OMe
OMe
2) TBAF/ THF
29 (R =CH2CH2OTBDMS)
30 (R =CH2CH2OH)
31 (R=CH2CHO)
27 (R =Br)
28 (R =B(OH)2)
3) Dess Martin/ CH2Cl2
26
OMe
OMe
1) AcCl/ AlCl3/ CH2Cl2
2) LiAlH4/ AlCl3
3) TBDMSCl/
1) NBS/ silica gel/
CCl4
2) BuLi/ THF/
-78oC/ B(OEt)3
1) Pd(PPh3)4/Na2CO3/
90oC/ EtOH/ H2O
R
imidazole/
THF 23oC
Br
N
22
NHBOC
HOCH2CH2
TBDMSOCH2CH2
ROCH2CH2
CH2CH3
2) TBAF/ THF
CH2CH3
3) Dess Martin/ CH2Cl2
35 (R =Br)
36 (R =B(OH)2)
32
33 (R =H)
34 (R =TBDMS)
OMe
OMe
OMe
1) (CH3)2NH/Na(OAc)3BH/
AcOH/ THF
N
NHBOC
N
NHR
N
NHBOC
2) NaOH/ dioxane/
115oC
OCHCH2
XCH3
Me2N(CH2)2
R
XCH3
CH2CH3
31 (X =O)
39 (X =CH2)
40 (X=O; R= BOC)
41 (X=CH2; R=BOC)
3 (X=O; R= H)
37 (R =CH2CH2OTBDMS)
38 (R =CH2CH2OH)
39 (R=CH2CHO)
4 (X=CH2; R=H)
Figure 4.
Finally, 2-hydroxy-4-methoxy acetophenone 47 served
as a common starting material for the preparation of
dimethylaminoethoxy derivatives 7 and 8 having an
ethoxy linker (Fig. 6). Ketone reduction followed by
phenol protection yielded 48, which was converted to
boronic acid 49 following bromination and subsequent
lithiation and triethylborate addition. Suzuki Coupling
of 49 provided pyridine 50. Removal of the benzyl
moiety via transfer hydrogenolysis and the pyrrole
moiety with hydroxylamine hydrochloride provided
phenol 55. Treatment of 55 with N-(2-chloroethyl)di-
methylamine in the presence of cesium carbonate gener-
ated aminopyridine 8. To access aminopyridine 7, 47
was converted to 51 following phenol protection and
ring bromination. Baeyer–Villiger oxidation followed by
phenol protection provided bromide 52. Metal halogen
exchange followed by boronic acid formation and sub-
jection to standard Suzuki coupling conditions with
bromopyridine 12 afforded pyridine 53. Removal of the
TBDMS protecting group and alkylation of the resul-
tant phenol with iodomethane provided pyridine 54.
Removal of the phenol and amino protecting groups
using standard conditions (transfer hydrogenolysis/pyr-
role hydrolysis) afforded phenol 56, which was alkylated
to provide aminopyridine 7 as described for the con-
version of 55 to 8 (Table 1).
OMe
N
N
LiN(Si(CH3)3)2
Ph3PCH2CH2N(CH3)2I
14 (X =O)
42 (X =CH2)
OCH
XCH3
OMe
N
10% Pd/C/ EtOH
50 PSI H2 28%
N
43 (X =O)
44 (X =CH2)
Me2NCH2CHCH
XCH3
OMe
N
N
NH2OH HCl
90oC
Me2N(CH2)3
45 (X =O)
46 (X =CH2)
XCH3
OMe
N
NH2
5 (X =O)
6 (X =CH2)
Me2N(CH2)3
XCH3
Figure 5.
The nNOS and eNOS inhibitory activity of the com-
pounds reported in this manuscript were measured in
vitro using human NOS isoforms derived from the
cloned genes expressed baculovirus in Sf9 cells.7 All
eight targeted aminopyridines have attractive in vitro
protecting group with hydroxylamine provided amino-
pyridine 5. Using the same methodology, aldehyde 42
was converted to aminopyridine 6.

4514
D. M. Nason et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4511–4514
OMe
OMe
OMe
OMe
1) NBS/ silica gel/
CCl4
2) BuLi/ THF/
-78oC/ B(OEt)3
B(OH)2
1) EtO2CCl/ TEA
NaBH4
2) BnBr/ K2CO3
Chem. Pharm. Bull.
1979, 27, 1490.
1) Pd(PPh3)4/Na2CO3/
90oC/ EtOH/ H2O
N
N
BnO
HO
BnO
BnO
Br
N
12
N
CH2CH3
COCH3
CH2CH3
CH2CH3
50
49
48
47
1) KOH/ BnBr/ dibenzo-18-
crown-6/ CH3CN
2) NBS/ silica gel/ CCl4
OMe
OMe
OMe
1) BuLi/ THF/ -78oC/
B(OEt)3
OMe
OMe
Br
Br
1) KF/ 40% HBr
2) KOH/ MeI/
dibenzo-18-crown-
1) MCPBA/ CH2Cl2
N
N
N
N
2) Pd(PPh3)4/Na2CO3/
90oC/ EtOH/ H2O
2) TBDMSCl/
imidazole/ THF
BnO
BnO
BnO
6/ CH3CN
BnO
COCH3
OTBDMS
OTBDMS
Br
N
12
N
51
53
52
54
OMe
OMe
XMe
OMe
1) HCO2NH4/
20% Pd(OH)2/
MeOH/ heat
2) NH2OH HCl/
EtOH/ H2O/ heat
N
NH2
N
N
N
NH2
Cs2CO3/ acetone/ heat
(CH3)2NCH2CH2Cl HCl
HO
BnO
Me2N(CH2)2O
XCH3
XCH3
50 (X=CH2)
54 (X=O)
7 (X=O)
8 (X=CH2)
55 (X=CH2)
56 (X=O)
Figure 6.
Table 1. Nitric oxide synthase inhibition for compounds A and 1–8
References and notes
Compound Human nNOS
a
Human eNOS
a
(IC50)
nNOS/
eNOS
1. Panahian, N.; Yoshida, T.; Huang, P. L.; Hedley-Whyte, E.
T.; Dalkara, T.; Fishman, M. C.; Moskowitz, M. A.
Neuroscience 1996, 72, 343.
2. Eskott, K. J.; Beech, J. S.; Haga, K. K.; Williams, S. C. R.;
Meldrum, B. S.; Bath, P. M. W. J. Cereb. Blood Flow
Metab. 1998, 18, 281.
(IC50
)
A
1
2
3
4
5
6
7
8
140
810
54
887
>32,000
8833
6.3
40
164
136
220
160
103
196
64
235
75
>32,000
16,500
>32,000
7700
3. Coert, B. A.; Anderson, R. E.; Meyer, F. B. Am. J. Physiol.
Heart Circ. Physiol. 2003, 284, H151.
200
75
4. Salerno, L.; Sorrenti, V.; DiGiacomo, C.; Romeo, G.;
Siracusa, M. A. Curr. Pharm. Des. 2002, 8, 177.
5. NO synthase activity was measured by modification of the
procedure described in: Bredt, D. S.; Snyder, S. H. Proc.
Natl. Acad. Sci. U.S.A. 1990, 87, 682, 10 lL of enzyme
solution and 10 lL of 3[H]-arginine were added to 80 lL of
buffer (contents of which varied depending on the NOS
isoform being tested). After incubation for 50 min at 30 °C,
the reaction was terminated by application to a 0.15 mL
column containing Biorex-60 cation exchange resin, sodium
form, and washed with 90 lL water. 3[H]-Citrulline was
quantified by liquid scintillation spectroscopy of the eluant.
6. Lowe, J. A., III; Qian, W.; Volkmann, R. A.; Heck, S.;
Nowakowski, J.; Nelson, R.; Nolan, C.; Liston, D.; Ward,
K.; Zorn, S.; Johnson, C.; Vanase, M.; Faraci, W. S.;
Verdries, K. A.; Baxter, J.; Doran, S.; Sanders, M.; Ashton,
M.; Whittle, P.; Stefaniak, M. Biorg. Med. Chem. Lett.
1999, 9, 2569.
112
58
21,943
3710
a Values are means of three experiments. Inhibition of human neuronal
and human endothelial nitric oxide synthase activity given as an IC50
value in nM units.
nNOS activity. All are selective for nNOS with a range
of 40- to 220-fold. In general, the 2,5-dimethoxy analogs
(1, 3, 5, and 7) are devoid of eNOS activity. The 2-
methoxy-5-ethyl analogs (2, 4, 6, and 8), on the other
hand, in which the 5-methoxy group has been replaced
with a 5-ethyl group are more potent for human nNOS
and still have significant nNOS selectivity (nNOS/eNOS:
64–220). Of the eight analogs, dimethylaminomethyl
analog 2 and dimethylaminoethoxy analog 8 are
the most potent nNOS inhibitors. Compounds
described in this manuscript have been profiled in vivo.
Efficacy in animal models will be described in due
course.
7. Lowe, J. A., III; Qian, W.; Drozda, S. E.; Volkmann, R. A.;
Nason, D.; Nelson, R. B.; Nolan, C.; Liston, D.; Ward, K.;
Faraci, S.; Verdries, K.; Seymour, P.; Majchrzak, M.;
Villalobos, A.; White, W. F. J. Med. Chem. 2004, 47,
1575.