1-(1,3-Benzodioxol-5-ylmethyl)-3-[4-(1H-imidazol-1-yl)phenoxy]-piperidine analogs as potent and selective inhibitors of nitric oxide formation

Article abstract of DOI:10.1016/j.bmcl.2007.02.053

A new series of 1-(1,3-benzodioxol-5-ylmethyl)-3-[4-(1H-Imidazol-1-yl)phenoxy]-piperidine analogs were designed and identified as potent and selective inhibitors of NO formation based both on the crystal structure of a murine iNOS Δ114 monomer domain/ inhibitor complex and inhibition of the NO formation in human A172 cell assays. Compound 12S showed high potency and high iNOS selectivity versus nNOS and eNOS.

Full text of DOI:10.1016/j.bmcl.2007.02.053

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2499–2504
1-(1,3-Benzodioxol-5-ylmethyl)-3-[4-(1H-imidazol-1-yl)phenoxy]-
piperidine analogs as potent and selective inhibitors of
nitric oxide formation
Robert G. Wei,a,* Marc Adler,b David Davey,a Elena Ho,c Raju Mohan,a Mark Polokoff,b
Jih-Lie Tseng,c Marc Whitlow,b Wei Xu,a Shendong Yuana and Gary Phillipsa
aDepartment of Medicinal Chemistry, Berlex Biosciences, 2600 Hilltop Drive, Richmond, CA 94804, USA
bDepartment of Biophysics, Berlex Biosciences, 2600 Hilltop Drive, Richmond, CA 94804, USA
cDepartment of Pharmacology, Berlex Biosciences, 2600 Hilltop Drive, Richmond, CA 94804, USA
Received 18 January 2007; revised 17 February 2007; accepted 21 February 2007
Available online 27 February 2007
Abstract—A new series of 1-(1,3-benzodioxol-5-ylmethyl)-3-[4-(1H-Imidazol-1-yl)phenoxy]-piperidine analogs were designed and
identified as potent and selective inhibitors of NO formation based both on the crystal structure of a murine iNOS D114 monomer
domain/ inhibitor complex and inhibition of the NO formation in human A172 cell assays. Compound 12S showed high potency
and high iNOS selectivity versus nNOS and eNOS.
Ó 2007 Elsevier Ltd. All rights reserved.
Nitric oxide (NO) is generated from the reaction of L-
arginine with oxygen by an enzyme called nitric oxide
synthase (NOS).1,2 NOS exists in three distinct isoforms,
which fall into two categories: constitutive and induc-
ible. There are two constitutive isoforms, which are cal-
cium and calmodulin dependent, and there is an
inducible isoform, which is calcium independent. The
two constitutive isoforms are (1) a neuronal isoform,
NOS-1 or nNOS,3 which was first found in neuronal tis-
sue and (2) an endothelial isoform, NOS-3 or eNOS,
which is found in vascular endothelial cells. The induc-
ible isoform, NOS-2 or iNOS, is found in a wide range
of cells. The three isoforms differ in their location and
function, but are similar in that they are only active in
the dimeric form. eNOS and nNOS generate low, tran-
sient levels of NO in response to an increase in intracel-
lular calcium concentrations to regulate blood pressure,
platelet adhesion, and neurotransmission.4 However,
iNOS generates high, sustained levels of NO. These ele-
vated levels of NO and resulting NO-derived metabo-
lites result in cellular cytotoxicity and tissue damage
which may contribute to the pathophysiology of a num-
ber of human diseases such as multiple sclerosis (MS)
and rheumatoid arthritis (RA). The non-selective inhibi-
tion of NO formation might lead to unwanted side
effects, therefore much effort has been focused on the
discovery of novel selective iNOS inhibitors in the phar-
maceutical industry.4,5
The search for direct enzyme inhibitors of iNOS led to
the discovery of N-phenylimidazoles as selective inhibi-
tors of iNOS, albeit with moderate potency.6 In addi-
tion, N-phenylimidazoles can also promote iNOS
dimer assembly5d after binding to the heme. Because
the dimer assembly is critical for NOS activity and
NO production, prevention of iNOS dimerization is a
potential mechanism for a therapeutic agent.
We have previously disclosed a class of compounds that
block iNOS dimerization.5a,b A representative of that
class is 1 (Fig. 1). A comparison of this compound
O
H
O
N
N
N
O
N
N
Keywords: iNOS inhibitor; Dimerization.
*
Corresponding author. Tel.: +1 510 669 4742; fax: +1 510 669
4310; e-mail addresses: guo_ping_wei_@berlex.com; robertwei@
comcast.net
1
N
Figure 1. iNOS dimerization inhibitor.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.02.053

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R. G. Wei et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2499–2504
bound to the murine iNOS D114 monomer domain and
the model of the iNOS dimer suggests that these inhibi-
tors occupy the same position as Glu377 in the human
iNOS dimer structure. Our working hypothesis is that
these inhibitors bind to the iNOS monomer and prevent
Glu377 of helix 7A from occupying the position that
leads to dimer formation.
of 2-[4-(imidazol-1-yl)phenoxy]ethylamine 2 with piper-
onal gave monoalkylated compound 3 and dialkylated
compound 4. Alkylation of 3 with ethyl bromoacetate
under basic conditions, followed by hydrolysis with lith-
ium hydroxide, afforded compounds 5 and 6, respec-
tively. The acid, 6, was bound to an oxime resin using
DCC as coupling reagent, followed by reaction with
amines, affording products 7–9. The heterocyclic com-
pounds were prepared by standard methods (Scheme
2). The reaction of 3-hydroxypiperidine hydrochloride
10 with 3,4-methylenedioxybenzyl chloride using potas-
sium carbonate at 50 °C gave the alkylated intermediate
11. Mitsunobu reaction8 of 11 with 4-(imidazol-1-
yl)phenol afforded 12. The other phenoxy analogs were
prepared in a similar fashion. Oxidation of N-benzyl-3-
hydroxypiperidine hydrochloride 13 with PDC followed
by reductive amination with 4-(N-imidazol-1-yl)aniline
afforded intermediate 14. Further debenzylation of 14
with Pd/C followed by alkylation with 3-4-methylenedi-
oxybenzyl chloride gave 15. For the preparation of 17,
compound 13 was treated with sodium hydride followed
by reaction with 3-chloro-6-(imidazol-1-yl)pyridazine to
give 16. Debenzylation and alkylation of 16 afforded 17.
The original series contain an imidazol-1-ylpyrimidine
linked to a distal benzodioxolane group with a six-atom
tether including a saturated heterocycle. From the crys-
tal structure of compound 1 bound to monomeric mur-
ine iNOS D114 (Fig. 2), we knew that the imidazole
group binds to the heme and the imidazole, pyrimidine,
and pyrrolidine ring are nearly coplanar. The benzodi-
oxolane group fits snugly between residues in the iNOS
monomer active site and the pyrimidine ring resulting in
a U-shaped conformation. This binding mode is consis-
tent with the crystal structure of the fragments, 4-imi-
dazol-1-ylphenol and piperonylamine bound, to
murine iNOSD114 (not shown).7 This prompted us to
design new inhibitors with alternate linkers connecting
the benzodioxolane and imidazole moieties (Fig. 3).
Here we report the discovery of potent, selective inhibi-
tors of NO formation, which do not inhibit iNOS en-
zyme activity.
Compounds were initially tested for inhibition of NO
formation in a whole cell using human A172 cell assays
induced with cytokines.5c NO formation was monitored
with Griess reagent.9 In a standard iNOS enzyme assay,
compounds had weak activity (compound 12 had an
IC50 of 15.5 lM). Selectivity between the NOS isoforms
was determined in SF9 cells transfected with a NOS
isoform in a tetracycline-induced system.5c NOS activity
was monitored by measuring the production of radiola-
beled citrulline from radiolabeled arginine. Tet data
refer to the IC50 determined as an average of at least
two separate experiments in the iNOS transfected cells
induced by addition of tetracycline unless otherwise
stated.
Our initial work focused on compounds with a hydroxy-
ethylamine as a linker (Scheme 1). Reductive amination
We first explored straight chain tethered analogs (Table
1). The initial compound 3, with an unsubstituted linker,
had surprising activity, with an IC50 of 240 nM, but sub-
stitution of the chain was detrimental, although at-
tempts were limited. Tertiary amines were tolerated,
but compound 6 with an acetic acid substitution showed
weak activity.
Next, we explored the SAR of analogs with a heterocy-
clic linker (Table 2). Compound 18, with pyrrolidine as
a linker, exhibited low activity with an IC50 of 4 lM.
Figure 2. The crystal structure of inhibitor 1 bound to murine iNOS
monomer (PDB entry 2ORO).
N
N
N
N
O
O
N
N
O
O
O
O
O
O
N
N
R
Figure 3. iNOS dimerization inhibitor design scheme.

R. G. Wei et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2499–2504
2501
O
N
O
O
a
N
O
N
N
N
H2N
O
4
2
+
O
O
b,c
O
O
N
N
N
H
3
O
O
O
R2
=
7
8
O
O
O
N
O
N
N
d,e
O
N
N
N
CF3
R1=Et, 5
OR1
R2
O
N
9
R
1 =H, 6
N
H
O
Scheme 1. Reagents and conditions: (a) NaBH3CN, AcOH, MeOH, piperonal; (b) ethyl bromoacetate, K2CO3, MeCN, 70–80 °C, 3 h; (c) LiOH,
THF–H2O; (d) DCC, DCM, Kaiser oxime resin, 18 h; (e) NH2R2, DCM 18 h.
OH
O
OH
b
N
a
N
N
N
O
O
O
O
N
H
10
.HCl
12
11
H
N
H
N
OH
c,d
e,a
N
N
N
N
N
N
.
HCl
N
N
N
O
O
15
14
13
N
O
OH
N
N
O
N
e,a
f
N
N
N
.
HCl
N
N
O
O
17
13
16
Scheme 2. Reagents and conditions: (a) 3-4-methylenedioxybenzyl chloride, K2CO3, NaI (cat.), DMF, 50 °C, 4 h; (b) 4-1-imidazolylphenol, Ph3P,
DEAD, DMF; (c) PDC, DCM; (d) 4-(N-imidazolyl)aniline, Ti(OiPr)4, NaCNBH3, EtOH, 20 h; (e) 10% Pd/C, MeOH, NH4 CO2H, reflux, 4 h; (f) i—
NaH, DMF; ii—3-chloro-6-(imidazol-1-yl) pyridazine, rt.
Table 1. Inhibitory activity for compounds 3–9
dine, 12, a highly potent and selective compound was
obtained. Again, the activity resided in the S enantio-
mer, as 12R displayed weaker activity. Replacement of
the imidazol-1-ylphenyl group with a 6-(imidazol-1-yl)-
3-pyridazinyl group, 17, resulted in similar activity and
selectivity. Compound 15, an aniline analog, showed
5-fold higher potency than the ether 12. Extension by
one more carbon off the piperidine (20) significantly re-
duced the potency. Compound 19 with an azepine as the
linker showed similar activity to the piperidine analog
12. The 2-(4-imidazol-1-yl)phenoxy methyl substituted
analog 21 exhibited 2-fold more potency than the pyr-
rolidine analog 22R. The 3S-hydroxypiperidine was
optimal as a linker and compound 12S was optimal both
in high potency with IC50 value of 9 nM in a whole cell
assay and in iNOS high selectivity versus nNOS and
eNOS (>400 for both). It should be noted that the differ-
Compound
A172 cell IC50 (lM)
3
4
5
6
7
8
9
0.24
1.5
1.2
>10
0.64
1.8
1.6
The activity resided in the S enantiomer, as 18R dis-
played weaker activity (IC50 > 10 lM). Changing the
linker attachment site to the 2-position with one more
carbon extension gave 22R, which exhibited 5-fold higher
potency and showed some selectivity (>80-fold for
both). When the pyrrolidine was replaced with a piperi-

2502
R. G. Wei et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2499–2504
Table 2. Activity and selectivity of piperidine analogs
Compound Structure
A172 cell IC50 (nM) Tet iNOS IC50 (nM) Tet selectivity ratio n/i Tet selectivity ratio e/i
O
12
57
250
NT
72
350
NT
600
NT
NT
NT
439
>17
400
NT
420
NT
NT
NT
86a
>17
N
N
N
N
N
N
N
P
N
N
N
N
N
N
O
12R
1700
N
P
O
12S
9
N
P
O
18
4200
>10,000
1600
10
NT
NT
NT
120
590
N
P
O
18R
N
P
O
18S
N
P
H
N
15
N
N
N
P
N
N
N
O
N
17
52
N
P
O
19
78
NT
NT
NT
NT
NT
NT
N
N
P
N
N
N
O
20
>10,000
N
P
O
21
100
250
2200
1200
>46
>80
>46
N
N
P
N
22R
>80a
O
N
P
N
N
P = 3,4-methylenedioxybenzyl group. NT, not tested.
a Data were reported as for n = 1 determination.
ences in potency between the A172 cells and the tetracy-
cline-induced cell assays are unclear, although it may
be due to differences in NO production between the
different cell types.
exhibited a high clearance in the rat and extensive
distribution throughout the body. Oral bioavailability
is high partly because of the low systemic level after
intravenous administration. Metabolic stability in
rat, dog, and human liver microsomes revealed a
47%, 89%, and 31% parent remaining after 3 h,
respectively.
Further characterization of 12S was pursued due to
its activity and selectivity (Table 3). Compound 12S

R. G. Wei et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2499–2504
2503
Table 3. Rat PK data of compound 12S
Route
iv
po
10
Dose (mg/kg)
T1/2 (h)
2
2.2
128
21
2.9
na
Cl (ml/min/kg)
Vss (L/kg)
na
0.8
Cmax (lg/ml)
AUCinf (h lg/ml)
%F
na
0.28
na
2.6
ꢀ100%
na, not applicable.
These results possibly account for the high clearance.
We determined the crystal structure of 12S bound to the
murine iNOSD 114 monomer (Fig. 4). The compound
occupies the arginine binding pocket and the imidazole
directly coordinates to the iron. The benzodioxolane
and phenylimidazole adopt a favorable U-shaped con-
formation, positioning the benzodioxolane such that it
will prevent Glu377 in helix 7A from adopting a confor-
mation that is conducive to dimer formation, an essen-
tial step for activity.
Figure 5. Comparison of crystal structure of inhibitor 12S (green,
PDB entry 2ORT)-murine iNOS monomer complex with inhibitor 1
(gold, PDB entry 2ORO).
change the U-shaped conformation which accounts for
the similar potency to compound 12. However, when
the linker was changed from piperidine (12S) to pyrrol-
idine (18S) the potency drops 178-fold. A model of 18S
in iNOS monomer shows that the pyrrolidine ring needs
to adapt a strained conformation in order to position
the phenylimidazole and benzodioxolane moieties in a
productive binding conformation.
A comparison between the crystal structures of 1 and
12S bound to murine iNOS D114 monomer shows that
both compounds bind in a U-shaped conformation,
and that the 4-(imidazol-1-yl)aryl binds in the same con-
formation, with the imidazole coordinating the heme
(Fig. 5). The benzodioxolane moieties occupy the same
pocket between the arylimidazole and the iNOS active
site, but have different orientations. Compound 12S is
20-fold less potent than 1. This difference of potency
could be due to the extra interactions that the
imidazolylpyrimidine of 1 has with iNOS and the heme
(methyl group with Pro344 and Gln257 and pyrrolidine
ring with heme) and/or the different orientations of the
benzodioxolane groups.
In summary, a new series of 1-(1,3-Benzodioxol-5-ylm-
ethyl)-3-[4-(1H-Imidazol-1-yl)phenoxy]-piperi-dine ana-
logs have been discovered as inhibitors of NO
formation using structure-based design. Compound
12S potently inhibits iNOS activity in a whole cell assay
and demonstrates high iNOS selectivity versus eNOS
and nNOS. It is hypothesized that this compound pre-
vents iNOS monomer from dimerizing in a similar
fashion to compound 1.
Replacement of the phenoxy oxygen atom with a nitro-
gen atom shown in structure 15 presumably does not
References and notes
1. For recent reviews see: (a) Marletta, M. A. Cell 1994, 927;
(b) Nathan, C.; Xie, Q. J. Biol. Chem. 1994, 269, 13725; (c)
Griffith, O. W.; Stuehr, D. J. Annu. Rev. Physiol. 1995, 57,
707.
2. Luzzi, S. D.; Marletta, M. A. Bioorg. Med. Chem. Lett.
2005, 15, 3934.
3. (a) Jaroch, S.; Rehwinkel, H.; Ho¨lscher, P.; Sulzle, D.;
¨
Burton, G.; Hillmann, M.; McDonald, F. M.; Miklautz, H.
Bioorg. Med. Chem. Lett. 2004, 14, 743; (b) Jaroch, S.;
Ho¨lscher, P.; Rehwinkel, H.; Sulzle, D.; Burton, G.;
¨
Hillmann, M.; McDonald, F. M. Bioorg. Med. Chem. Lett.
2003, 13, 1981.
4. Chida, N.; Hirasawa, Y.; Ohkawa, T.; Ishii, Y.; Sudo, Y.;
Tamura, K.; Mutoh, S. Eur. J. Pharmacol. 2005, 509, 71.
5. (a) Blasko, E.; Glaser, C. B.; Devlin, J. J.; Xia, W.;
Feldman, R. I.; Polokoff, M. A.; Phillips, G. B.; Whitlow,
M.; Auld, D. S.; McMillan, K.; Ghosh, S.; Stuehr, D.;
Parkinson, J. F. J. Biol. Chem. 2002, 277, 295; (b)
McMillan, K.; Adler, M.; Auld, D. S.; Baldwin, J. J.;
Blasko, E.; Browne, L. J.; Chelsky, D.; Davey, D.; Dolle,
R. E.; Eagan, K. A.; Erickson, S.; Feldman, R. I.; Glaser,
C.; Mallari, C.; Morrissey, M. M.; Ohlmeyer, M. H. J.;
Figure 4. The crystal structure of inhibitor 12S-murine iNOS D114
monomer complex (PDB entry 2ORT).

2504
R. G. Wei et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2499–2504
Pan, G.; Parkinson, J. F.; Phillips, G. B.; Polokoff, M. A.;
Watanabe, N. Bioorg. Med. Chem. Lett. 2005, 15, 1361; (j)
Tsymbalov, S.; Hagen, T. J.; Moore, W. M.; Jerome, G.
M.; Connor, J. R.; Manning, P. T.; Pitzele, B. S.; Hallinan,
E. A. Bioorg. Med. Chem. Lett. 2002, 12, 3337; (k)
Moormann, A. E.; Metz, S.; Toth, M. V.; Moore, W. M.;
Jerome, G.; Kornmeier, C.; Manning, P.; Hansen, D. W.,
Jr.; Pitzele, B. S.; Webber, R. K. Bioorg. Med. Chem. Lett.
2001, 11, 2651; (l) Hallinan, E. A.; Hagen, T. J.; Bergmanis,
A.; Moore, W. M.; Jerome, G. M.; Spanggler, D. P.;
Stevens, A. M.; Shieh, H. S.; Manning, P. T.; Pitzele, B. S.
J. Med. Chem. 2004, 47, 900; (m) Hallinan, E. A.;
Tsymbalov, S.; Dorn, C. R.; Pitzele, B. S.; Hansen, D.
W., Jr. J. Med. Chem. 2002, 45, 1686; (n) Hamley, P.;
Tinker, A. C. Bioorg. Med. Chem. Lett. 1995, 5, 1573.
6. Wolff, D. J.; Gribin, B. J. Arch. Biochem. Biophys. 1994,
311, 293.
Sigal, N. A.; Vergona, R.; Whitlow, M.; Young, T.; Devlin,
J. J. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 1506; (c) Davey,
D. D.; Adler, M.; Arnaiz, D; Eagen, K.; Erickson, S.;
Guilford, W.; Kenrick, M.; Morrissey, M. M.; Ohlmeyer,
M.; Pan, G.; Paradkar, V. M.; Parkinson, J.; Polokoff, M.;
Santos, C.; Subramanyam, B.; Vergona, R.; Wei, R. G.;
Whitlow, M.; Ye, B.; Zhao, S.; Devlin, J.; Phillips, G.
J. Med. Chem. in press; (d) Parkinson, J. F.; Devlin, J. J.;
Phillips, G. B. Endothelial Cell Res. Ser. 1999, 505; (e)
Salerno, l.; Sorrenti, V.; Di Giacomo, C.; Romeo, G.;
Siracusa, M. A. Curr. Pharm. Des. 2002, 8, 177; (f)
Sennequier, N.; Wolan, D.; Stuehr, D. J. J. Biol. Chem.
1999, 274, 930; (g) Park, K. H.; Park, Y.-D.; Han, J.-M.;
Im, K.-R.; Lee, B. W.; Jeong, I. Y.; Jeong, T.-S.; Lee, W. S.
Bioorg. Med. Chem. Lett. 2006, 6, 5580; (h) Jackson, S. A.;
Sahni, S.; Lee, L.; Luo, Y.; Nieduzak, T. R.; Liang, G.;
Chiang, Y.; Collar, N.; Fink, D. He. W.; Laoui, A.; Merrill,
J.; Boffey, R.; Crackett, P.; Rees, B.; Wong, M.; Guilloteau,
J.-P.; Mathieu, M.; Rebello, S. S. Bioorg. Med. Chem. 2005,
13, 2723; (i) Ueda, S.; Hideo, T.; Suzuki, K.; Yano, A.;
Matsumoto, M.; Kubo, T.; Minato, H.; Arai, Y.; Tsuji, J.-I.;
7. Whitlow, M.; Adler, M.; Davey, D.; Huang Q.-L.; Pham,
E.; Polokoff, M.; Xu, W.; Yuan, S.-D.; Phillips, G. Bioorg.
Med. Chem. Lett. in press.
8. Hughes, D. L. Org. React. 1992, 42, 335.
9. Stuehr, D. J.; Marletta, M. A. Proc. Natl. Acad. Sci. U.S.A.
1985, 82, 7738.