Design, synthesis, and activity of 2-imidazol-1-ylpyrimidine derived inducible nitric oxide synthase dimerization inhibitors

Article abstract of DOI:10.1021/jm061319i

By the screening of a combinatorial library for inhibitors of nitric oxide (NO) formation by the inducible isoform of nitric oxide synthase (iNOS) using a whole-cell assay, 2-(imidazol-1-yl)pyrimidines were identified. Compounds were found to inhibit the dimerization of iNOS monomers, thus preventing the formation of the dimeric, active form of the enzyme. Optimization led to the selection of the potent, selective, and orally available iNOS dimerization inhibitor, 21b, which significantly ameliorated adjuvant-induced arthritis in a rat model. Analysis of the crystal structure of the 21b-iNOS monomer complex provided a rationalization for both the SAR and the mechanism by which 21b blocks the formation of the protein-protein interaction present in the dimeric form of iNOS.

Full text of DOI:10.1021/jm061319i

1146
J. Med. Chem. 2007, 50, 1146-1157
Design, Synthesis, and Activity of 2-Imidazol-1-ylpyrimidine Derived Inducible Nitric Oxide
Synthase Dimerization Inhibitors
David D. Davey,† Marc Adler,† Damian Arnaiz,† Keith Eagen,† Shawn Erickson,† William Guilford,† Margaret Kenrick,†
Michael M. Morrissey,† Mike Ohlmeyer,‡ Gonghua Pan,† Vidyadhar M. Paradkar,‡ John Parkinson,† Mark Polokoff,†
Kurt Saionz,‡ Cecile Santos,† Babu Subramanyam,† Ron Vergona,† Robert G. Wei,† Marc Whitlow,† Bin Ye,†
Zuchun (Spring) Zhao,† James J. Devlin,† and Gary Phillips*,†
Berlex Biosciences, 2600 Hilltop DriVe, P.O. Box 4099, Richmond, California 94804-0099, and Pharmacopeia Drug DiscoVery, Inc.,
P.O. Box 5350, Princeton, New Jersey 08543-5350
ReceiVed NoVember 14, 2006
By the screening of a combinatorial library for inhibitors of nitric oxide (NO) formation by the inducible
isoform of nitric oxide synthase (iNOS) using a whole-cell assay, 2-(imidazol-1-yl)pyrimidines were identified.
Compounds were found to inhibit the dimerization of iNOS monomers, thus preventing the formation of
the dimeric, active form of the enzyme. Optimization led to the selection of the potent, selective, and orally
available iNOS dimerization inhibitor, 21b, which significantly ameliorated adjuvant-induced arthritis in a
rat model. Analysis of the crystal structure of the 21b-iNOS monomer complex provided a rationalization
for both the SAR and the mechanism by which 21b blocks the formation of the protein-protein interaction
present in the dimeric form of iNOS.
Introduction
describe the optimization of the initial hits, identified by
screening the library, into orally available, potent, selective iNOS
dimerization inhibitors.7,12
Nitric oxide synthase (NOSa) is a dimeric enzyme that
catalyzes the production of NO from arginine using heme,
biopterin, and NADPH as cofactors. The three isoforms of NOS,
iNOS, endothelial (ecNOS), and neuronal (bcNOS) differ in their
tissue distribution and biological role but are similar in that they
are only active in the dimeric form. iNOS is up-regulated during
an immune response and has been implicated in the pathogenesis
of numerous inflammatory diseases, including multiple sclerosis
and rheumatoid arthritis. In contrast, bcNOS and ecNOS are
constitutively expressed in mammals and are required for normal
function. Therefore, a potent inhibitor of iNOS could ameliorate
disease; however, high selectivity would be needed to avoid
inhibiting the essential functions of the bcNOS and ecNOS
isoforms. The potential therapeutic benefit of an iNOS inhibitor
has prompted a major effort in the pharmaceutical industry to
discover a potent, selective iNOS inhibitor.1-3
Our initial search for iNOS inhibitors focused on direct
enzyme inhibition, specifically compounds that could mimic the
substrate, arginine, or those that could directly interact with the
heme. The heme binding approach led to the finding that
1-phenylimidazoles were selective but were only moderately
potent inhibitors of iNOS.4-6 To further explore the phenylimid-
dazole template, a library was prepared using ECLIPS7 technol-
ogy (Figure 1) and screened for direct enzyme inhibitors, but
no compounds were identified. The library was screened a
second time using a whole-cell assay with NO production as
the readout, and several library compounds, exemplified by 1,
significantly decreased levels of NO production.8 On further
evaluation, these compounds were found to permeate the cell
and to inhibit formation of the enzymatically active dimer. Other
groups have identified imidazole-containing compounds that also
block NO formation by this mechanism.9-11 In this paper, we
Chemistry
Compounds were originally prepared by an efficient method,
which required a difficult separation of regioisomeric pyri-
midines (Scheme 1). Reaction of 2,4-dichloropyrimidine with
imidazole gave a mixture of chloro(imidazol-1-yl)pyrimidines,
2 and 3, which were separated. The monochloropyrimidines
were subsequently reacted with secondary amines to obtain the
desired analogues. Because of problems in obtaining sufficient
quantities of the desired regioisomers, an alternative synthetic
strategy using 2-sulfonopyrimidines as an intermediate was
developed.
The 2-sulfonopyrimidines were prepared under standard
conditions (Scheme 2). Alkylation of the pyrimidinethione, 4,
with methyl iodide in the presence of base gave methylmer-
captan, 5. Reaction of the resulting hydroxypyrimidine with
POCl3 resulted in the formation of the corresponding chloro-
pyrimidine, 6, which was oxidized with mCPBA to give the
sulfone, 7. The sulfones are not stable and were best used soon
after preparation or stored under anhydrous conditions.
The pyrimidinesulfone was reacted with the protected amines,
and the intermediate sulfone, whose isolation is unnecessary
but possible, was subsequently reacted with imidazole (Scheme
3). In general, by addition of the secondary amine to the
chloropyrimidinesulfone and subsequently reaction of the
intermediate with imidazole, the desired regioisomer was
obtained with >98% regioselectivity. Under identical conditions,
reaction of primary amines with the chloropyrimidinesulfone
gave regioisomeric mixtures with addition of the amine at the
4 position predominating (>6:1). Derivatization of the ester or
piperazine nitrogen was performed sequentially under standard
conditions by initial deprotection of the ester or amine,
depending on the group to be modified, to give the desired
compounds. The regioisomeric piperazines, 19, were prepared
in a similar manner by starting with the alternatively protected
starting piperazine.
* To whom correspondence should be addressed. Phone: (925) 372-
7882. E-mail: garybphillips@comcast.net.
† Berlex Biosciences.
‡ Pharmacopeia Drug Discovery, Inc.
a Abbreviations: NOS, nitric oxide synthase; NO, nitric oxide; iNOS,
inducible NOS; ecNOS, endothelial constitutive NOS; bcNOS, brain
constitutive NOS.
10.1021/jm061319i CCC: $37.00 © 2007 American Chemical Society
Published on Web 02/23/2007

NOS Dimerization Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1147
Figure 1. Discovery of dimerization inhibitors.
Scheme 1. Original Synthesis of Disubstituted Pyrimidinesa
Scheme 3. Synthesis of Piperazine Analoguesa
a Reagents and conditions: (a) imidazole; (b) amine, K2CO3, DMSO,
heat.
Scheme 2. Synthesis of 2-Pyrimidinesulfonea
a (a) K2CO3, heat; (b) imidazole, heat; (c) LiOH, THF; (d) carbonyldi-
imidazole, piperonylamine; (e) TFA, CH2Cl2; (f) standard methods.
metrically with the Griess reagent.15 All selected compounds
were inactive in a standard noncellular iNOS enzyme assay up
to 1 µM in which the enzyme had been isolated.16 Selectivity
between the NOS isoforms was determined using BSC-1 cells
transfected with a vaccinia virus system or SF9 cells transfected
with a tetracycline-induced system that expressed one of the
three NOS isoforms. In these assays, NOS activity was
monitored by measuring the production of radiolabeled citrulline
from radiolabeled arginine. The two selectivity assays (vaccinia
virus and tetracycline) gave similar results for iNOS potency
but different results for ecNOS and bcNOS potency. The
relevance of these differences is unknown. ecNOS was found
to be more potent in the tetracycline induced system, while
bcNOS was found to be more potent in the vaccinia system.
Empirically, the tetracycline induced system gives us the ability
to monitor ecNOS selectivity while the vaccinia system indicated
the inhibitors to be very selective against ecNOS. The IC50
values for iNOS in the A172 cells and the two selectivity assays
differed, possibly because of differences in NO production
between the different cell types. It was difficult to make a good
correlation concerning the selectivity with the less active
compounds because the maximum compound concentrations
were 10 µM.
a Reagents and conditions: (a) MeI, K2CO3, DMSO; (b) POCl3, heat;
(c) mCPBA, CH2Cl2.
The regioselective piperazine synthesis has been described.13
Reaction of the ethyl ester of piperazine-2-acetic acid with
BOCON places a BOC group on the less hindered piperazine
nitrogen. A synthesis of the benzyl protected piperazine has also
been described.14 BOC protection of the benzyl protected
piperazine gives the diprotected compound. Selective removal
of either protecting group gives the selected regioisomer that
can be used in the synthesis of any of the piperazine substituted
pyrimidines.
The synthesis of optically active 11d and 19f was ac-
complished by separating a precursor on a chiral column. The
syntheses were completed by standard methods. Absolute
stereochemistry was determined by an X-ray crystal structure
of 11d and an analogue of 19f bound to the enzyme, and all
other stereochemistry is inferred. The absolute stereochemistry
of 11d corresponds to that of the D-amino acid used as starting
material in the syntheses of the more active compounds shown
in the pyrrolidine series illustrated in Table 7.
Our optimization strategy was to first modify the different
parts of the template independently and then to combine the
best substituents into a single compound that would have an
improved overall profile. The template was partitioned into the
amide (R1), piperazine substitution (R2), and pyrimidine sub-
stitution (R3) (Figure 2). In addition, different attachment loci
of the piperazine side chain, chain lengths, and alternatives to
the piperazine were tested.
Results and Discussion
Compounds were initially tested for inhibition of active iNOS
formation in a whole-cell assay using human A172 cells induced
with cytokines. NO formation was monitored spectrophoto-
Modification of the group on the piperazine nitrogen resulted
in a series that had only modest differences in potency and NOS

1148 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
DaVey et al.
Figure 2. General optimization strategy.
Table 1. Substitution at the 4-Position of the Piperazine15
Figure 3. Comparison of piperazine alternatives in the A172 assay.
Table 2. Data Illustrating Differences of the Central Aromatic Ring15
vaccinia
vaccinia
selectivity
IC50 a (nM)
selectivity ratioa
IC50 a (nM)
A172 vaccinia
ratioa
compd
X
Y
Z
A172
0.5
52
10
77
vaccinia
b/i
e/i
520
>500
>1000
NT
compd
R2
b/i
e/i
12a
15
16
N
N
CH
CH
N
CH
N
CH
N
N
7
220
100
NT
5
6
19
11a
11b
11c
11d
11e
11f
11g
11h
11i
COCH3
CH2Ph
COPh
1
1
320
13
85
24
450
74
70
11
120
25
72
34
13
28
48
16
18
10
24
10
3
7
4
5
4
12
7
2
7
3
4
9
2
11
6
5
7
6
4
>10
770
220
1700
>250
>1000
1200
1300
40
0.5
0.38
1
0.7
0.55
0.48
17
CH
CH
NT
COOCH3
CONHCH3
SO2CH3
a NT: not tested.
H
CH3
or carbon to give morpholine (13) or piperidine (12a) based
systems gives highly potent compounds with activity similar
to the activity of the piperazines (11). For faster synthetic access,
the piperidines were utilized to explore subsequent modifica-
tions. The perhydroazepine (14) was also a highly potent
compound.
CH2-1-naphthalene
CONHPh
CONHBn
SO2Ph
CH2-furan
COCH2OBn
CO-2-furan
CO2Ph
69
11j
11k
11l
0.87
0.96
2.7
0.58
0.50
0.67
1.4
0.8
1.0
1.0
0.28
0.93
350
490
1100
1100
1220
1000
700
660
1000
800
11m
11n
11o
11p
11q
11r
11s
11t
Early in the optimization, we looked at the difference in
activity between the three possible pyrimidine regioisomers
(Table 2). Previous results from libraries had indicated a potency
preference for the compounds with the imidazole in the 2
position of the pyrimidine, and this was borne out. Comparison
of the three regioisomers clearly shows that the 2-imidazolepy-
rimidine, 12a, is the most potent. A possible explanation for
this is that the imidazole needs to be nearly planar with the
pyrimidine in the active site, and this is the only regioisomer
that has no hydrogens flanking the imidazole on the pyrimidine,
allowing the imidazole to be more planar. This hypothesis was
further supported by the relatively weak iNOS activity shown
by 17, where the pyrimidine is replaced by a carbocyclic
aromatic ring. In addition to trends in the SAR, the planar
relationship of the imidazole and pyrimidine is demonstrated
in the crystal structure (Figure 6). In the vaccinia virus induced
systems, these compounds demonstrated similar overall selectiv-
ity, which can be described as good selectivity against ecNOS
and as poor selectivity against bcNOS.
To examine the effect of side chain modifications, the
corresponding piperidines, 12, were prepared (Table 3). Unlike
the modifications on the piperazine, different side chains gave
compounds with very different activity. The initial hits from
the library showed a clear preference for oxygen-containing
substitution on an aromatic moiety and a specific chain length
between the pyrimidine and the aromatic moiety. Placement of
the methoxy group was important with a rank order of para,
meta, and ortho (12b > 12c > 12d). The 3,4-disubstituted
compound 12e has a similar potency compared to the 4-sub-
stituted compound, 12b, but the 3,4,5-trisubstituted analogue,
COOBn
(CH2)2CHMe2
COOiPr
6-F-2-pyridine
CH2COOEt
C(dNH)Me
dansyl
6
5
>10
NT
850
1700
350
11u
11v
11w
4.9
33
1.1
370
NT
NT
a NT: not tested.
selectivity (Table 1). Amides, amines, carbamates, ureas, and
sulfonamides were well tolerated (e.g., 11a, 11b, 11d, 11e, and
11f, respectively), with the unsubstituted compound, 11g, as
potent as any other compound. Steric bulk may have some effect
on potency, with the large group (seen in 11i) being the least
potent compound, but other compounds with large substituents,
such as benzoyl (11q) and dansyl (11w), gave compounds with
good activity. The basicity of the piperazine nitrogen did not
seem to have much of an effect on activity in comparing 11b
with 11c, 11m with 11o, or 11r with 11s. Although the highly
basic amidine 11v was less potent than the isosteric 11a, the
aminopyridine 11t was highly active. Isosteric carbamates and
ureas had similar activity (11d vs 11e, 11j vs 11p, and 11k vs
11q), as well as amides and sulfonamides (11a vs 11f and 11c
vs 11l). In the vaccinia virus induced systems, these compounds
demonstrated similar selectivity, good selectivity against ecNOS,
and little selectivity against bcNOS.
Since we saw little effect of substituents on the piperazine
nitrogen, we explored the effect of modulating ring size and
replacement of the nitrogen (Figure 3). Incorporation of oxygen

NOS Dimerization Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1149
Table 4. Variation of the 6-substituent of the Pyrimidine15
Table 3. Variation of the Amide Moiety15
vaccinia
selectivity
ratioa
vaccinia
selectivity ratio
IC50 (nM)
IC50 a (nM)
A172 vaccinia
compd
R3
A172
vaccinia
b/i
e/i
compd
R1
b/i
e/i
12a
18a
18b
18c
18d
18e
H
0.5
0.49
7
9
5
5
4
520
1100
36
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
12o
12p
12q
12r
12s
CH2-benzodioxolane
CH2Ph-4-OCH3
CH2Ph-3-OCH3
CH2Ph-2-OCH3
CH2Ph-3,4-OCH3
CH2Ph-3,4,5-OCH3
CH2Ph
0.5
0.8
3.7
7
23
230
5
2
4
56
1
NT
6
520
Cl
Ph
Me
200
>430
>200
900
NT
67
4.1
180
35
450
16
320
515
5
>31
13
1200
>310
130
283
1.5
>100
25
5.7
3.0
19
500
110
NT
440
160
210
140
CF3
imidazole
90
12z or 12e with 12aa. Lengthening the chain caused a modest
decrease in activity; compare 12e with 12bb or 12b with 12cc.
Substitution of the pyrimidine 6 position indicated a prefer-
ence for smaller groups when comparing potency, although the
trend between hydrogen and methyl substitution was inconsistent
(Table 4; additional compounds were prepared in this series,
but the trends are consistent with those shown). The phenyl
(18b) and imidazole (18e) substituted pyrimidines were less
active than the corresponding unsubstituted compound, 12a. The
trifluoromethyl substituted analogue (18d) had the worst activity,
but the methyl analogue (18c) was highly active. The differences
in activity between the methyl and hydrogen substituted
analogues were dependent on the amide substituent. When the
benzodioxolane was the amide substituent, the hydrogen-
containing compound 12a was more potent than the methyl-
containing compound 18c, but with other substituted benzyla-
mides, the hydrogen-containing compound was the same or was
less active than the methyl-containing compound (data not
shown). While the activity of 6-chloropyrimidines was consis-
tently high, as exemplified by 18a, the potential for displacement
of the chlorine from these analogues was a concern and the
analogues were not pursued. The selectivity of the compounds
was similar to those already discussed.
By screening secondary libraries, we found that the alterna-
tively substituted piperazines, 19, had improved bcNOS selec-
tivity but somewhat decreased activity. Further studies bore out
this observation. Analogues were prepared (Table 5), and
although most of the SAR was similar to that of the initially
prepared piperazines, differences did appear. Of the few amide
analogues prepared, the benzodioxalanemethyl substituent was
found to be the most active. Substitution of the 6 position of
the pyrimidine showed a clear preference for either methyl or
chloro (data not shown). Unlike the initially substituted pip-
erazine, substitution of the 4 position of these analogues resulted
in large differences in activity, depending on the substituent.
The unsubstituted analogue, 19a, was the least active among
the analogues. The lower activity did not seem to be due to the
basicity of the compound, because the methyl and benzyl
substituted compounds (19b and 19c, respectively) were more
active than the unsubstituted analogue, but may be due to the
ability of the side chain to have freedom to rotate over the
piperazine. The isosteric sulfonamide 19d and acetamide 19e
have the same potency as 19b. In general, the different
placement of the side chain had an effect on bcNOS selectivity.
The isobutyramide 19f was the most active compound prepared
in this series, and the bcNOS selectivity in the vaccinia system
CH2Ph-4-Cl
CH2Ph-4-F
8
5
5
3
270
490
220
48
CH2Ph-4-OCF3
CH2Ph-4-CH3
CH2Ph-4-NH2
CH2Ph-4-NHSO2CH3
CH2Ph-4-N(CH3)2
CH2Ph-4-CF3
CH2Ph-4-CN
CH2Ph-4-NO2
CH2Ph-4-SO2NH2
CH2Ph-4-SO2CH3
CH2-2-furan
23
270
274
850
>12
120
>100
>1000
66
245
29
3
86
70
150
260
>100
168
>1000
940
>10
74
>10000
17
>330
940
>13
13
NT
NT
NT
110
140
NT
NT
NT
12t
63
260
12u
12v
12w
12x
12y
12z
12aa
12bb
12cc
CH2-2-pyridine
(NCH3)CH2Ph
CHPh2
539
NT
885
NT
1805
>100
11
NT
CH3
2-benzimidazole
benzodioxolane
Ph-3,4-OCH3
(CH2)2Ph-3,4-OCH3
(CH2)2Ph-4-OCH3
1100
NT
1800
680
42
47
NT
1
16
NT
>55
120
>100
66
12
3.5
1
8
>1000
a NT: not tested.
12f, lost significant potency. The potency difference between
the isoelectronic 12a and 12e possibly indicates a steric
interaction that is borne out in the crystal structure (Figure 6).
Since the unsubstituted 12g lost a significant amount of activity
and the 4-substituted compound 12b was the most potent
monosubstituted compound, we further explored substituents
at the 4 position but found no better substituent. The sterically
similar 12h and 12i are potent, having activity less than 10 nM,
but 12j-n were all much less active. Compounds with electron-
withdrawing groups, 12o-r, were much less potent, but the
sulfonamide 12s has an IC50 of 17 nM. Some of the differences
may be explained by a preference for a more electron-rich
aromatic moiety (12b vs 12j and 12k vs 12o), but the anilines
(12l-n) do not fit this pattern. The monocyclic heterocycles
tested, 12t and 12u, were less active than the unsubstituted 12g.
Substitution of the nitrogen (12v vs 12g) and benzylic position
(12w vs 12g) both decreased activity. Loss of the aromatic
moiety as in the methylamide, 12x, gave a compound with little
activity. In the vaccinia virus selectivity assays, the more active
compounds (IC50 < 20 nM) demonstrated similar selectivity,
good selectivity against ecNOS, and little selectivity against
bcNOS.
Potency was very sensitive to the distance between the
piperidine and the aromatic moiety (Table 3). Shortening the
chain caused a dramatic decrease in activity; compare 12a with

1150 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Table 5. Piperazine Regioisomers15
DaVey et al.
Table 7. Pyrrolidine Analogues Arising from D-Proline15
tet
vaccinia
selectivity
ratioa
a
a
IC50
(nM)
selectivity
IC50
(nm)
ratioa
vaccinia
IC50 (nM)
selectivity ratio
compd R3
A172
0.28
0.12
0.13
0.48
0.29
tet
5.5
4.5
7.1
b/i
e/i
47
91
65
vaccinia b/i
e/i
compd
R2
A172
vaccinia
b/i
e/i
20a
20b
20c
21a
21b
H
Me
Et
H
Me
6
27
18
5.6
5.9
7.5
5
8
15
2900
1000
7800
19a
19b
19c
19d
19e
19f
H
CH3
Bn
SO2CH3
Ac
COCH(CH3)2
CO2CH3
100
12
25
>1000
340
72
130
>350
350
160
11
74 181
327 540
NT
280 170
25
19 >2000
12
>1000
14
NT
16
20
NT
25
62 >5000
21bb Me 6400
NT
NT
67 >3800
NT
8.5
340
>32
80
50
>320
>300
>200
1.9
5.4
80
21c
Et
0.52
19g
420
a NT: not tested. b Arose from L-proline.
Table 6. Comparison of Enantiomers15
vaccinia
tet
absolute stereochemistry of 11d was determined by X-ray
crystallography of the compound in complex with the protein
and is consistent with the stereochemistry of compounds
prepared from D-proline (Table 7). The absolute stereochemistry
of 19d is inferred from a related analogue, for which a structure
was obtained.17 Conveniently, the active isomers of 11d and
19f have different stereochemistry because they each arose from
different isomers of the starting material.
a
IC50
(nM)
selectivity
ratio
IC50
selectivity
(nm)
ratioa
compd
A172
vaccinia
b/i
e/i
tet
b/i
e/i
82
32
NT
240
74
11d
0.38
0.24
34
1.9
0.75
300
24
18
5
6
1700
1100
12
10
NT
46
130
NT
25
16
NT
120
80
(S)-11d
(R)-11d
19f
(R)-19f
(S)-19f
>1000
80
80
29
>300
>400
350
>1000
NT
NT
In order to avoid separating isomers, we tested commercially
available starting materials that contained the desired stereo-
center. Analogues of proline were prepared and found to be
highly active, some showing unexpectedly high bcNOS selectiv-
ity (Table 7). Again, keeping the distance between the pyrimi-
dine and benzodioxolane group to six atoms was important, as
changes caused a significant decrease in activity (compounds
not shown). Moving the amide function in the side chain had
no effect on potency but resulted in a change in selectivity for
both isoforms (20 vs 21). Having the amide adjacent to the
pyrrolidine, 21, gave the most selective compounds identified
in this series. The analogues arising from D-proline were
significantly more potent than the analogues arising from
L-proline and may better reflect the difference in activity of the
stereoisomers (21b vs 21b*) than the earlier described pairs.
a NT: not tested.
was improved compared to the alternatively substituted pipera-
zines, while the ecNOS selectivity remained the same.
Two compounds were chosen for synthesis of their respective
stereoisomers, 11d and 19f. The compounds were prepared by
separating a common intermediate using chromatography on a
chiral support and continuing on to the final compound. A large
difference was found in the activity of the two isomers (Table
6), with, as expected, the more active isomer having activity
and selectivity similar to those of the mixture. The activity of
the less active isomer should be interpreted with caution, as
the analytical methods would not discern a small impurity
(<2%), which could easily explain all of the activity. The
Figure 4. (A) Pharmacokinetics profile in rats. Error bars represent (SEM; n ) 3 rats per route. (B) Pharmacokinetics profile in dogs. Error bars
represent (SEM; n ) 3 dogs per route.

NOS Dimerization Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1151
Figure 5. Effects of 21b in a rat adjuvant-induced arthritis model. (A) Clinical scoring. Each limb was graded on a scale of 0-4 for (with
increasing degrees of redness) gross swelling and distortion of the paw and joint fusion. The sum of the scores for each limb was totaled and
designated as the clinical score. (B) Radiological scoring. Both hind limbs were graded on a scale of 0-3 for each of the following parameters:
soft tissue swelling, cartilage loss, erosion, and heterotopic ossification. The sum of these scores for each limb was totaled and was designated as
the radiological score.
Figure 6. Compound 21b (green carbon atoms) bound to murine iNOS monomer. The porphyrin is shown in brown and the iron in magenta: (A)
a ribbon diagram with the residues that make contact with the inhibitor (<4.0 Å); (B) the solvent accessible surface (PDB entry 2ORO).
Figure 7. (A) Complex formed with L-Arg (green) and the iNOS dimer. The purple tube denotes the residues that become disordered in the
iNOS-21b complex. (B) 21b superimposed on the iNOS dimer complex (1NSI). In 1NSI, Glu377 forms a salt bridge to the guanidine of the
arginine substrate. The benzodioxolane of 21b occupies the same space as Glu377. This presumably disrupts the folding of helix 7a.
We selected 21b for in vivo characterization because of its
high potency and high selectivity versus the two other isoforms.
The compound had good oral availability in both rats and dogs.
In rats, the compound was dosed both intravenously and orally
at 10 mg/kg, and although the half-life after iv dosing was short
(0.7 h), the clearance and oral availability were reasonable (10
mL min-1 kg-1 and 21%, respectively, Figure 4A). The
compound was administered to dogs at 3 mg/kg. The half-life
after iv dosing was better (2.5 h), and the clearance and oral
availability were improved (4.5 mL min-1 kg-1 and 71%,
respectively; Figure 4B).
We then tested the ability of 21b to inhibit adjuvant-induced
arthritis in a rat model. The compound was dosed subcutane-
ously at 3, 10, and 30 mg/kg twice a day, and the rats were
monitored and scored for arthritis using two methods, clinical
signs and radiological scores (Figure 5). Compared to vehicle,
both the 10 and 30 mg/kg dose resulted in a significantly lower
clinical score. The 30 mg/kg dose also resulted in significantly

1152 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
DaVey et al.
filtered and washed with water, acetonitrile, and ether to afford 79
g (72% yield) of a white solid.
lower radiological scores. For rats dosed with 30 mg/kg, neither
the clinical scores nor the radiological scores were significantly
different from naive animals by analysis of both methods and
at least 6 of the 10 animals in this high-dose group were free
of disease. No adverse effects were observed for any of the
doses tested.
4-Chloro-6-methyl-2-(methylthio)pyrimidine (6). To phos-
phorus oxychloride (300 mL) under nitrogen was added 6-methyl-
2-methylthiopyrimidin-4-one (79 g, 0.51 mol), and the mixture was
heated at 80 °C for 5 h. The mixture was concentrated under
vacuum, and the residue was poured onto 1 kg of ice and extracted
with dichloromethane. The extract was washed with saturated
potassium carbonate, dried over basic alumina, charcoal-treated,
filtered, and concentrated under vacuum to give 81 g (91% yield)
Structural Analysis
An analysis of the structure of 21b bound to monomeric iNOS
oxygenase domain explains much of the SAR previously
described (Figure 6).18 The compound occupies the arginine
binding pocket. The imidazole coordinates to the Fe, setting
the inhibitors into the protein. The distal group of the piperazine
(R2) would point into the solvent and consequently does not
have much effect on the activity of the resulting compounds.
The substituent off the amide points back toward the porphyrin,
and the benzodioxolane fills the pocket. The linker between the
benzodioxolane and imidazole is critical because sufficient
flexibility and distance between the two groups are needed to
accommodate the bend between the two aromatics and to
accommodate the ability of the compound to bind to the enzyme.
The saturated nitrogen heterocycle probably aides the bend by
predisposing the molecule to a â turn. We attempted to
crystallize the less active enantiomer bound to the protein to
better understand the binding, but the resulting crystals all
contained the more potent isomer, which was presumably
present as an undetected impurity (up to 2% would not be
detected by our analytical methods).
A comparison of the inhibitor-monomer structure with a
structure of the dimer helped rationalize the mechanism of
inhibiting dimer formation seen with the compounds (Figure
7). The protein-protein interactions necessary for dimer forma-
tion are shown in Figure 7A. Compound 21b displaces helix
7a (red) side chains from the Arg binding site and disrupts part
of the dimer interface (Figure 7B), a disruption resulting in the
disordering of the residues starting at helix 7a and up to the
proximal half of helix 8.
1
of a pale-yellow liquid. H NMR (CDCl3) δ 6.8 (s, 1H), 2.5 (s,
3H), 2.4 (s, 3H).
4-Chloro-6-methyl-2-(methylsulfonyl)pyrimidine (7). A solu-
tion of 3-chloroperoxybenzoic acid (240 g, 77%, 0.97 mol) in
dichloromethane (2.5 L) was washed with saturated NaCl (500 mL),
dried over magnesium sulfate, transferred to a 3 L flask with
mechanical stirring, and cooled to -5 °C. A solution of 4-chloro-
6-methyl-2-(methylsulfonyl)pyrimidine (81 g, 0.46 mol) in CH2-
Cl2 (100 mL) was added over 30 min. The mixture was allowed to
warm to room temperature overnight. The solids were filtered and
washed with cold CH2Cl2 (500 mL). The filtrate was washed with
10% K2CO3 (2 L), dried over basic alumina, charcoal-treated,
filtered, and concentrated under vacuum. The residue was crystal-
lized from hexanes (1 L) to afford 80 g (83% yield) of a white
1
solid, which was stored under N2 at 0 °C. H NMR (CDCl3) δ
7.45 (s, 1H), 3.35 (s, 3H), 2.65 (s, 3H).
Methyl 4-[(1,1-Dimethylethoxy)carbonyl]-2-piperazineacetate
(8). Methyl 1,4-dibenzylpiperazine-2-acetate14 (26.8 g, 79 mmol)
was dissolved in MeOH (500 mL) and 0.5 N HCl (aqueous, 400
mL, 0.2 mol). Pd/C (10%, 9.5 g) was added, and the mixture was
hydrogenated with H2 (50 psi) for 24 h. The mixture was filtered
through Celite, and the filtrate was concentrated in vacuo to remove
most of the MeOH. The residue was made basic with K2CO3 (∼16
g) to pH 9-10, and THF (300 mL) was added. The mixture was
cooled to 0 °C, and BOC-ON (19.5 g, 79 mmol) dissolved in THF
(100 mL) was added dropwise. The mixture was stirred at 0 °C for
1 h, allowed to warm to room temperature, and stirred for 2 h.
Some solvent was removed in vacuo, and the residue was extracted
with ethyl acetate. The combined extracts were treated with 100
mL of 1 M HCl (aqueous). The aqueous layer was separated,
washed with ethyl acetate, basified with K2CO3, and extracted with
ethyl acetate. The combined organic layers were washed with brine,
dried (Na2SO4), and concentrated in vacuo to give 16.6 g (81%) of
Conclusion
1
A series of substituted pyrimidines containing an imidazole
have been discovered that interrupts protein-protein interac-
tions, displayed by inhibiting the dimerization of the iNOS
monomers. This series has been optimized for their potency,
selectivity against other NOS isoforms, and oral availability.
Compound 21b is a potent selective compound that shows
efficacy in a model of rheumatoid arthritis. Future publications
will illustrate the utilization of this template and the crystal
structure in the design of novel templates.
8. H NMR (CDCl3) δ 3.88 (b, 2H), 3.75 (s, 3H), 2.69-3.02 (m,
4H), 2.55 (b, 1H), 2.37 (dd, 1H), 2.31 (dd, 1H), 1.4 (s, 9H).
Methyl 1-[2-(1H-Imidazol-1-yl)pyrimidin-4-yl]-4-[(1,1-dim-
ethylethoxy)carbonyl]-2-piperazineacetate (9). Methyl 4-[(1,1-
dimethylethoxy)carbonyl]piperazine-2-acetate (8, 11.7 g, 45 mmol),
4-chloro-2-methylsulfonylpyrimidine (8.5 g, 44 mmol), and K2CO3
(13 g, 94 mmol) were dissolved in CH3CN (250 mL) and heated
to 35 °C for 18 h. Imidazole (15 g, 220 mmol) was added, and the
mixture was heated to 65 °C for 24 h. The solvent was removed in
vacuo, and the residue was slurried in CH2Cl2 and washed with
water. The organic layer was separated, dried (MgSO4), and
concentrated in vacuo. The residue was chromatographed on silica
(3% MeOH/CH2Cl2) to give 14.5 g (82%) of 9.
Experimental Section
All starting materials not described below were purchased from
commercial sources. All reagents and solvents were used as received
from commercial sources without additional purification. Elemental
analyses were performed by Robertson Microlit Laboratories
(Madison, NJ), and results were within (0.4% of the calculated
values. NMR spectra were recorded with a Varian XL-300
spectrometer and were consistent with the assigned structures.
Reverse-phase HPLC was performed with a Rainin SD-1 Dynamax
system and a C-18 reverse-phase Dynamax 60A column using a
gradient of acetonitrile (0.1% TFA) in water (0.1% TFA).
6-Methyl-2-(methylthio)pyrimidin-4-one (5). To a solution of
4-hydroxy-2-mercapto-6-methylpyrimidine (100 g, 0.70 mol) in
DMSO (1 L) was added potassium carbonate (106 g, 0.77 mol),
followed by iodomethane (48 mL, 0.77 mol), and the mixture was
stirred at room temperature for 24 h. The reaction mixture was
poured into 4 L of water with stirring. The resulting solids were
N-[(1,3-Benzodioxol-5-yl)methyl]-1-[2-(1H-imidazol-1-yl)py-
rimidin-4-yl]-4-[(1,1-dimethylethoxy)carbonyl]-2-piperazineac-
etamide (10). To methyl 1-[2-(1H-imidazol-1-yl)pyrimidin-4-yl]-
4-[(1,1-dimethylethoxy)carbonyl]piperazine-2-acetate (14.5 g, 36
mmol) dissolved in THF (250 mL) and water (70 mL) was added
lithium hydroxide hydrate (7 g, 0.16 mol). The mixture was stirred
for 18 h, and some of the solvent was removed in vacuo. Water
(200 mL) was added, and the material was washed with ether. The
aqueous solution was acidified with acetic acid, and the resulting
precipitate was collected by filtration and washed with water, CH3-
CN, and ether to give 10 g of a white solid. The solid was suspended
in DMF (100 mL), and carbonyldiimidazole (4.4 g, 27 mmol) was
added. The mixture was stirred for 3 h and became homogeneous.
Piperonylamine (3.5 mL, 28 mmol) was added, and the mixture
was stirred for another 1 h. The mixture was poured into 0.1 N

NOS Dimerization Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1153
KOH and extracted with CH2Cl2. The extract was washed with
water and dried (MgSO4), and the solvent was removed in vacuo
to give 13 g (69%) of 10 as a colorless oil.
N-[(1,3-Benzodioxol-5-yl)methyl]-1-[2-(1H-imidazol-1-yl)py-
rimidin-4-yl]-4-[(1-naphthylenyl)methyl]-2-piperazineaceta-
1
mide (11i). H NMR (CDCl3) δ 8.55 (s, 1 H), 8.45 (s, 1 H), 8.15
(d, 1 H), 7.8 (m, 3 H), 7.4 (m, 4 H), 7.1 (s, 1 H), 6.65 (d, 1 H),
6.55 (m, 1H), 6.4 (m, 2 H), 5.9 (s, 2 H), 4.6 (m, 1H), 4.3 (m, 2 H),
4.0 (dd, 1H), 3.65 (m, 1 H), 3.1 (m, 3 H), 2.65 (m, 2 H), 2.4 (t, 1
H), 2.1 (m, 2 H). Anal. (C32H31N7O3‚HCl‚0.3H2O) C, H, N.
N-(1,3-Benzodioxol-5-ylmethyl)-1-[2-(1H-imidazol-yl)-4-pyri-
midinyl]-4-(phenylamino)carbonyl]-2-piperazineacetamide (11j).
1H NMR (CDCl3) δ 8.25 (m, 1H), 8.20 (m,1H), 7.80 (m, 1H), 7.50
(m, 2H), 7.30 (m, 2H), 7.00 (m, 1H), 6.80 (m, 4H), 6.40 (d, 1H),
5.80 (s, 2H), 5.0 (m, 1H), 4.35-4.65 (m, 4H), 3.60 (m, 1H),3.30
(m, 1H), 3.20 (m, 1H), 3.00 (m, 1H), 2.80 (m, 1H), 2.30 (d, 1H),
1.80 (m, 2H). Anal. (C28H28N8O4‚0.5H2O) C, H, N.
4-(Acetyl)-N-[(1,3-benzodioxol-5-yl)methyl]-1-[2-(1H-imida-
zol-1-yl)pyrimidin-4-yl]-2-piperazineacetamide (11a). 1H NMR
(DMSO-d6) δ 8.5 (s, 1 H), 8.35 (br, 1 H), 8.2 (m, 1 H), 7.85 (s, 1
H), 7.03 (s, 1 H), 6.6 (m, 4 H), 5.9 (s, 2 H), 5.3 (br, 1 H), 4.6 (br,
1 H), 4.25 (m, 1 H), 4.1 (m, 2 H), 3.8 (m, 1 H), 3.4 (m, 2 H), 3.0
(m, 1 H), 2.5 (m, 2 H), 1.95 and 2.05 (2 s, 3 H). Anal. (C23H25N7O4‚
0.3H2O) C, H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]-1-[2-(1H-imidazol-1-yl)py-
rimidin-4-yl]-4-(phenylmethyl)-2-piperazineacetamide (11b). 1H
NMR (DMSO-d6) δ 8.5 (s, 1 H), 8.4 (br, 1 H), 8.2 (d, 1 H), 7.85
(s, 1 H), 7.3 (m, 5 H), 7.03 (s, 1 H), 6.8 (m, 3 H), 6.5 (d, 1 H), 5.9
(s, 2 H), 5.3 (br, 1 H), 4.6 (br, 1 H), 4.1 (m, 2 H), 3.95 (dd, 1 H),
355 (d, 1 H), 3.45 (d, 1 H), 2.9 (d, 1 H), 2.75 (d, 1 H), 2.7 (br, 1
H), 2.1 (m, 2 H). Anal. (C28H29N7O3) C, H, N.
N-(1,3-Benzodioxol-5-ylmethyl)-1-[2-(1H-imidazol-yl)-4-pyri-
midinyl]-4-[[(phenylmethyl)amino]carbonyl]-2-piperazineaceta-
1
mide (11k). H NMR (CDCl3) δ 8.40 (s, 1H), 8.20 (m,1H), 7.70
(m, 1H), 7.30 (m, 5H), 6.95 (s, 1H), 6.80 (m, 3H), 6.45 (m, 1H),
5.90 (s, 2H), 4.20-4.50 (m, 8H), 3.20 (m, 3H), 2.70 (m, 1H), 2.20
(m, 1H), 1.90 (m, 2H). Anal. (C29H30N8O4‚0.8H2O) C, H, N.
N-(1,3-Benzodioxol-5-ylmethyl)-1-[2-(1H-imidazol-yl)-4-pyri-
midinyl]-4-(phenylsulfonyl)-2-piperazineacetamide (11l). 1H NMR
(CDCl3) δ 8.50 (s, 1H), (8.20 d, 1H), 7.50-7.80 (m, 6H), 7.00 (m,
1H),6.70 (m, 3H), 6.50 (m, 2H), 5.90 (s, 2H), 4.30 (m, 2H), 3.90
(m, 1H), 3.70 (m, 1H), 3.20 (m, 1H), 2.95 (m, 1H), 2.50 (m, 3H),
2.0 (m, 2H). Anal. (C27H27N7O5S‚0.7H2O) C, H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]-1-[2-(1H-imidazol-1-yl)py-
rimidin-4-yl]-4-(2-furylmethyl)-2-piperazineacetamide (11m). 1H
NMR (CDCl3) δ 8.45 (s, 1 H), 8.1 (d, 1 H), 7.78 (s, 1 H), 7.35 (s,
1 H), 7.03 (s, 1 H), 6.6 (m, 4 H), 6.3 (s, 1 H), 6.2 (s, 1 H), 5.85 (s,
2 H), 4.6 (br, 1 H), 4.25 (dd, 1 H), 4.15 (dd, 1 H), 4.0 (m, 2 H),
3.55 (AB, 2 H), 3.15 (m, 1 H), 2.8 (m, 3 H), 2.55 (dd, 1H), 2.2 (m,
2 H). Anal. (C26H27N7O4‚0.7H2O) C, H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]-1-[2-(1H-imidazol-1-yl)py-
rimidin-4-yl]-4-(methylsulfonyl)-2-piperazineacetamide (11n). 1H
NMR (DMSO-d6) δ 8.45 (s, 1 H), 8.2 (d, 1 H), 7.9 (br, 1 H), 7.83
(s, 1 H), 7.35 (m, 4 H), 7.3 (m, 1 H), 7.03 (s, 1 H), 6.7 (m, 4 H),
5.9 (s, 2 H), 4.95 (m, 1 H), 4.6 (s, 2 H), 4.25 (m, 4 H), 4.15 (m, 3
H), 3.35 (m, 2 H), 3.2 (m, 1 H), 2.55 (m, 2 H). Anal. (C30H31N7O5‚
0.5H2O) C, H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]-1-[2-(1H-imidazol-1-yl)py-
rimidin-4-yl]-4-(phenylcarbonyl)-2-piperazineacetamide (11c).
1H NMR (DMSO-d6) δ 8.45 (s, 1 H), 8.2 (d, 1 H), 7.95 (br, 1 H),
7.85 (s, 1 H), 7.45 (s, 5 H), 7.03 (s, 1 H), 6.7 (m, 4 H), 5.9 (s, 2
H), 5.0 (m, 1 H), 4.3 (d, 1 H), 4.1 (m, 4 H), 3.4 (m, 2 H), 3.3 (m,
1 H), 2.6 (m, 2 H). Anal. (C28H27N7O4) C, H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]-1-[2-(1H-imidazol-1-yl)py-
rimidin-4-yl]-4-[(methoxy)carbonyl]-2-piperazineacetamide (11d).
Compound 11g (5.04 g, 12 mmol) was slurried in CH2Cl2 (100
mL). Methyl chloroformate (1.03 mL, 13 mmol) and 1 N NaOH
(12.5 mL) was added, and the mixture was stirred vigorously
overnight. The clear solutions were separated, and the organic layer
was dried (Na2SO4) and the solvent removed in vacuo. The residue
was chromatographed (5% MeOH in CH2Cl2) to give 5.19 g (90%).
1H NMR (DMSO-d6) δ 8.45 (s, 1 H), 8.2 (d, 1 H), 8.0 (br, 1 H),
7.85 (s, 1 H), 7.03 (s, 1 H), 6.7 (m, 4 H), 5.9 (s, 2 H), 4.95 (br, 1
H), 4.15 (d, 1 H), 4.15 (d, 2 H), 3.95 (d, 2 H), 3.65 (s, 3 H), 3.3
(m, 2 H), 3.15 (dd, 1H), 2.5 (m, 2 H). Anal. (C23H25N7O5) C,
H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]-1-[2-(1H-imidazol-1-yl)py-
rimidin-4-yl]-4-[(methylamino)carbonyl]-2-piperazineaceta-
mide (11e). 1H NMR (DMSO-d6) δ 8.45 (s, 1 H), 8.2 (d, 1 H), 8.0
(br, 1 H), 7.8 (s, 1 H), 7.03 (s, 1 H), 6.7 (m, 4 H), 6.15 (br, 1 H),
5.9 (s, 2 H), 4.9 (br, 1 H), 4.2 (m, 3 H), 3.9 (d, 2 H), 3.25 (dt, 1
H), 3.1 (dd, 1H), 3.0 (m, 1 H), 2.6 (s, 3 H), 2.5 (m, 2 H). Anal.
(C23H26N8O4‚0.5H2O) C, H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]-1-[2-(1H-imidazol-1-yl)py-
rimidin-4-yl]-4-(methylsulfonyl)-2-piperazineacetamide (11f). 1H
NMR (DMSO-d6) δ 8.45 (s, 1 H), 8.2 (d, 1 H), 8.0 (br, 1 H), 7.8
(s, 1 H), 7.03 (s, 1 H), 6.7 (m, 4 H), 5.85 (s, 2 H), 5.1 (br, 1 H),
4.45 (d, 1 H), 4.1 (m, 2 H), 3.6 (m, 2 H), 3.3 (dt, 1 H), 3.05 (dd,
1H),2.9(m,1H),2.9(s,3H),2.65(d,2H).Anal.(C22H25N7O5S‚0.25CH2-
Cl2) C, H, N.
N-(1,3-Benzodioxol-5-ylmethyl)-4-(2-furanylcarbonyl)-1-[2-
(1H-imidazolyl)-4-pyrimidinyl]-2-piperazineacetamide (11o). 1H
NMR (CDCl3) δ 8.50 (m, 1 H), 8.20 (m, 1H), 7.80 (m, 1H), 7.50
(m, 1H), 7.00 (m, 2H), 6.70 (m, 4H), 6.50 (m, 1H), 5.90 (s, 2H),
4.50 (m, 2H), 4.30 (m, 2H), 3.20 (m, 4H), 2.70 (m, 1H), 2.40 (m,
1H), 2.05 (m, 2H). Anal. (C28H25N7O5‚0.2CH2Cl2) C, H, N.
3-[2-[(1,3-Benzodioxol-5-ylmethyl)amino]-2-oxoethyl]-4-[2-
(1H-imidazolyl)-4-pyrimidinyl]-1-piperazinecarboxylic Acid, Phen-
1
yl Ester (11p). H NMR (CDCl3) δ 8.50 (m, 1 H), 8.20 (m, 1H),
7.80 (m, 1H), 7.35 (m, 1H), 7.20 (m, 1H), 7.10 (m, 4H), 6.60 (m,
5H), 5.85 (s, 2H), 4.30 (m, 4H), 3.30 (m, 3H), 2.50 (m, 2H), 2.00
(m, 2H). Anal. (C28H27N7O5‚0.1CH2Cl2) C, H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]-1-[2-(1H-imidazol-1-yl)py-
rimidin-4-yl]-2-piperazineacetamide (11g). N-[(1,3-Benzodioxol-
5-yl)methyl]-1-[2-(1H-imidazol-1-yl)pyrimidin-4-yl]-4-[(1,1-di-
methylethoxy)carbonyl]piperazine-2-acetamide (13 g, 24 mmol) was
dissolved in CH2Cl2 (200 mL) and cooled to 0 °C. Trifluoroacetic
acid (100 mL) was added, and the mixture was allowed to warm
to room temperature. The solvent was removed in vacuo and the
residue dissolved in EtOAc (200 mL). The organic layer was
washed with saturated K2CO3 and dried (MgSO4), and the solvent
was removed in vacuo. Crystallization from ether gave 6 g (57%)
N-[(1,3-Benzodioxol-5-yl)methyl]-1-[2-(1H-imidazol-1-yl)py-
rimidin-4-yl]-4-[(phenylmethoxy)carbonyl]piperazine-2-aceta-
mide (11q). 1H NMR (CDCl3) δ 8.45 (s, 1 H), 8.2 (d, 1 H), 7.8 (s,
1 H), 7.35 (m, 5 H), 7.05 (s, 1 H), 6.6 (m, 4 H), 5.85 (s, 2 H), 5.15
(m, 2 H), 4.2 (m, 5 H), 3.1 (m, 3 H), 2.5 (m, 2 H), 2.0 (m, 1 H).
Anal. (C29H29N7O5‚0.5H2O) C, H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]-1-[2-(1H-imidazol-1-yl)py-
rimidin-4-yl]-4-(3-methylbutyl)-2-piperazineacetamide (11r). 1H
NMR (CDCl3) δ 8.5 (s, 1 H), 8.15 (d, 1 H), 7.8 (s, 1 H), 7.05 (s,
1 H), 6.65 (m, 3 H), 6.5 (m, 1 H), 6.25 (m, 1 H), 5.9 (s, 2 H), 4.7
(br, 1 H), 4.25 (m, 2 H), 3.15 (m, 1 H), 2.85 (m, 3 H), 2.5 (dd, 1
H), 2.3 (m, 2 H), 2.1 (m, 3 H), 1.6 (m, 1 H), 1.3 (m, 2 H), 0.9 (m,
6 H). Anal. (C26H33N7O3‚0.5H2O) C, H, N.
1
of a white solid, mp 160-162 °C. H NMR (CDCl3) δ 8.5 (s, 1
H), 8.18 (d, 1 H), 7.8 (s, 1 H), 7.1 (s, 1 H), 6.7 (m, 3 H), 6.55 (m,
1H), 6.2 (t, 1H), 5.9 (s, 2 H), 4.8 (br, 1 H), 4.3 (m, 2 H), 2.8-3.2
(m, 7 H), 2.55 (dd, 1 H). Anal. (C21H23N7O3‚0.3H2O) C, H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]-1-[2-(1H-imidazol-1-yl)py-
1
rimidin-4-yl]-4-methyl-2-piperazineacetamide (11h). H NMR
3-[2-[(1,3-Benzodioxol-5-ylmethyl)amino]-2-oxoethyl]-4-[2-
(CDCl3) δ 8.45 (s, 1 H), 8.15 (d, 1 H), 7.78 (s, 1 H), 7.03 (s, 1 H),
6.4-6.7 (m, 4 H), 5.85 (s, 2 H), 4.25 (m, 2 H), 3.1 (m, 1 H), 2.8
(m, 4 H), 2.5 (dd, 1H), 2.25 (s, 3 H), 2.2 (m, 1 H), 2.05 (t, 1 H).
Anal. (C22H26N7O3‚0.2CH2Cl2) C, H, N.
(1H-imidazolyl)-4-pyrimidinyl]-1-piperazinecarboxylic Acid, 1-
Methyl Ethyl Ester (11s). H NMR (CDCl3) δ 8.5 (s, 1 H), 8.2
(m,1H), 7.80 (m, 1H), 7.05 (m, 1H), 6.90 (m, 1H), 6.60 (m, 4H),
5.90 (s, 2H), 5.00 (m, 2H), 4.40 (m, 1H), 4.20 (m, 3H), 3.10 (m,
1

1154 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
DaVey et al.
2H), 2.60 (m, 1H), 2.40 (m, 1H), 1.95 (s, 2H), 1.30 (m, 6H). Anal.
(C25H29N7O5‚0.5H2O) C, H, N.
H), 5.15 (br, 1 H), 4.3 (m, 1 H), 2.95 (m, 1 H), 2.65 (dd, 1 H),
2.55 (dd, 1 H), 1.3-1.8 (m, 6 H). Anal. (C24H24N6O‚0.2H2O) C,
H, N.
N-[(4-Chlorophenyl)methyl]-1-[2-(1H-imidazol-1-yl)pyrimi-
din-4-yl]-2-piperidineacetamide (12h). 1H NMR (CDCl3) δ 8.65
(br, 1 H), 8.05 (d, 1 H), 7.8 (s, 1 H), 7.4 (br, 1 H), 7.0 (m, 5 H),
6.5 (m, 1 H), 5.2 (br, 1 H), 4.2 (m, 2 H), 3.65 (m, 1H), 3.05 (m, 1
H), 2.75 (dd, 1 H), 2.6 (dd, 1 H), 1.3-1.8 (m, 6 H). Anal. (C21H23N6-
OCl‚0.8H2O) C, H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]-1-[2-(1H-imidazol-1-yl)py-
rimidin-4-yl]-4-(6-fluoropyridin-2-yl)-2-piperazineacetamide (11t).
1H NMR (DMSO-d6) δ 8.5 (s, 1 H), 8.4 (m, 1 H), 8.2 (d, 1 H), 7.9
(s, 1 H), 7.65 (m, 1H), 7.05 (s, 1 H), 6.75 (d, 1 H), 6.7 (m, 2 H),
6.6 (m, 1 H), 6.3 (m, 1 H), 5.9 (s, 2 H), 4.0 (m, 3 H), 3.3 (m, 4 H).
Anal. (C26H25N8O3F‚0.5H2O) C, H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]-1-[2-(1H-imidazol-1-yl)py-
rimidin-4-yl]-4-(2-furylmethyl)-2-piperazineacetamide (11u). 1H
NMR (DMSO-d6) δ 8.45 (s, 1 H), 8.35 (br, 1 H), 8.15 (d, 1 H),
7.8 (s, 1 H), 7.0 (s, 1 H), 6.6 (m, 4 H), 5.85 (s, 2 H), 4.0 (m, 5 H),
3.3 (m, 2 H), 3.2 (q, 2 H), 2.9 (d, 1 H), 2.8 (d, 1 H), 2.6 (m, 2H),
2.4 (d, 1 H), 2.25 (m, 1 H), 1.15 (t, 3 H). Anal. (C25H29N7O5‚
0.4H2O) C, H, N.
N-[(4-Fluorophenyl)methyl]-1-[2-(1H-imidazol-1-yl)pyrimidin-
1
4-yl]-2-piperidineacetamide (12i). H NMR (CDCl3) δ 8.4 (s, 1
H), 8.05 (d, 1 H), 7.75 (s, 1 H), 7.0 (m, 3 H), 6.8 (m, 2 H), 6.7 (m,
1 H), 6.5 (m, 1 H), 5.1 (br, 1 H), 4.25 (m, 3 H), 3.0 (m, 1 H), 2.7
(dd, 1 H), 2.55 (dd, 1 H), 1.3-1.8 (m, 6 H). Anal. (C21H23N6OF‚
0.8H2O) C, H, N.
1-[2-(1H-Imidazol-1-yl)-4-pyrimidinyl]-N-[[4-(trifluorometh-
oxy)phenyl]methyl]-2-piperidineacetamide (12j). 1H NMR (CDCl3)
δ 8.5 (s, 1 H), 8.1 (d, 1 H), 7.8 (s, 1 H), 7.1 (m, 4 H), 6.5 (d, 1 H),
6.4 (br s, 1 H), 5.2 (br s, 1 H), 4.3 (m, 2 H), 3.0 (m, 1 H), 2.6 (m,
2 H), 1.8 (m, 6 H, partially obscured by water), 1.6 (m, 1 H). Anal.
(C22H23F3N6O2‚H2O) C, H, N.
1-[2-(1H-Imidazol-1-yl)pyrimidin-4-yl]-N-[(4-methylphenyl)-
methyl]-2-piperidineacetamide (12k). 1H NMR (CDCl3) δ 8.4 (s,
1 H), 8.03 (d, 1 H), 7.75 (s, 1 H), 6.95 (m, 5 H), 6.8 (s, 1 H), 6.45
(s, 1 H), 5.0 (br, 1 H), 4.25 (m, 3 H), 2.9 (m, 1 H), 2.6 (m, 1 H),
2.5 (m, 1 H), 2.2 (s, 3 H), 1.3-1.8 (m, 6 H). Anal. (C23H28N6O3‚
H2O‚0.6H2O) C, H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]-1-[2-(1H-imidazol-1-yl)py-
rimidin-4-yl]-4-(1-iminoethyl)-2-piperazineacetamide, Hydro-
chloride (11v). 1H NMR (DMSO-d6) δ 9.5 (m, 1 H), 8.9 (s, 1 H),
8.6 (m, 2 H), 8.25 (m, 1 H), 7.9 (m, 1 H), 7.05 (s, 1 H), 6.7 (m, 4
H), 5.9 (s, 2 H), 4.1 (m, 2 H), 4.0 (m, 3 H), 3.1-3.8 (m, 4 H), 2.6
(m, 2 H), 2.35 and 2.25 (2s, 3 H). MS m/z 463 (M + 1). Anal.
(C23H26N8O3‚H2O) C, H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]-4-[(5-dimethylaminonaph-
thyl)sulfonyl]-1-[2-(1H-imidazol-1-yl)pyrimidin-4-yl]-2-pipera-
1
zineacetamide (11w). H NMR (CDCl3) δ 8.6 (d, 1 H), 8.5 (s, 1
H), 8.3 (d, 1 H), 8.2 (d, 1 H), 8.1 (d, 1 H), 7.8 (s, 1 H), 7.6 (m, 2
H), 7.2 (d, 1 H), 7.1 (s, 1 H), 6.8 (m, 3 H), 6.6 (br s, 1 H), 6.0 (s,
2 H), 4.3 (m, 1 H), 4.2 (m, 1 H), 4.1 (d, 1 H), 3.7 (d, 1 H), 3.2 (br
s, 1 H), 2.9 (s, 6 H), 2.8 (m, 4 H), 2.3 (dd, 1 H). Anal. (C33H34N8O5
S‚CH2Cl2) C, H, N.
N-[(4-Aminophenyl)methyl]-1-[2-(1H-imidazol-1-yl)-4-pyrim-
1
idinyl]-2-piperidineacetamide (12l). H NMR (CDCl3) δ 8.4 (s,
1 H), 8.1 (d, 1 H), 7.7 (s, 1 H), 7.0 (s, 1 H), 6.8 (d, 2 H), 6.5 (m,
3 H), 6.0 (br s, 1 H), 5.1 (s, 1 H), 4.2 (m, 2 H), 2.9 (m, 1 H), 2.6
(m, 2 H), 1.7 (m, 6 H), 1.5 (m, 1 H). Anal. (C21H25N7O‚CH2Cl2)
C, H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]-1-[2-(1H-imidazol-1-yl)-4-
1
pyrimidinyl]-2-piperidineacetamide (12a). H NMR (CDCl3) δ
8.45 (s, 1 H), 8.1 (d, 1 H), 7.85 (s, 1 H), 7.05 (s, 1 H), 6.7 (s, 1 H),
6.5 (m, 4 H), 5.85 (m, 2 H), 5.1 (br, 1 H), 4.2 (m, 3 H), 3.0 (m, 1
H), 2.7 (dd, 1 H), 2.55 (dd, 1 H), 1.6-1.8 (m, 5 H), 1.5 (m, 1 H).
Anal. (C22H24N6O3‚0.7CH2Cl2) C, H, N.
1-[2-(1H-Imidazol-1-yl)-4-pyrimidinyl]-N-[[4-(methylsulfonyl-
1
amino)phenyl]methyl]-2-piperidineacetamide (12m). H NMR
(DMSO-d6) δ 9.6 (s, 1 H), 8.5 (s, 1 H), 8.4 (m, 1 H), 8.1 (d, 1 H),
7.8 (s, 1 H), 7.1 (m, 5 H), 6.7 (m, 1 H), 4.2 (m, 1 H), 4.1 (m, 1 H),
3.0 (br s, 1 H), 2.9 (s, 3 H), 2.5 (m, 4 H, partially obscured by
DMSO), 1.7 (m, 5 H), 1.4 (m, 1 H). Anal. (C22H27N7O3S‚CH2Cl2)
C, H, N.
N-[(4-Dimethylaminophenyl)methyl]-1-[2-(1H-imidazol-1-yl)-
pyrimidin-4-yl]-2-piperidineacetamide (12n). 1H NMR (CDCl3)
δ 8.5 (s, 1 H), 8.1 (d, 1 H), 7.8 (s, 1 H), 7.1 (s, 1 H), 7.0 (d, 2 H),
6.5 (d, 2 H), 6.1 (s, 1 H), 5.1 (br s, 1 H), 4.2 (m, 2 H), 2.9 (m, 1
H), 2.8 (s, 6 H), 2.6 (m, 1 H), 2.5 (m, 1 H), 2.0 (s, 1 H), 1.8 (m,
5 H), 1.5 (m, 1 H). Anal. (C23H29N7O‚CH2Cl2) C, H, N.
1-[2-(1H-Imidazol-1-yl)-4-pyrimidinyl]-N-[[4-(trifluorometh-
yl)phenyl]methyl]-2-piperidineacetamide (12o). 1H NMR (CDCl3)
δ 8.4 (s, 1 H), 8.1 (d, 1 H), 7.8 (s, 1 H), 7.5 (br s, 1 H), 7.4 (d, 2
H), 7.2 (d, 2 H), 7.0 (s, 1 H), 6.5 (s, 1 H), 5.1 (br s, 1 H), 4.4 (m,
2 H), 3.0 (m, 1 H), 2.6 (m, 2 H), 2.5 (br s, 1 H), 1.7 (m, 5 H), 1.5
(m, 1 H). Anal. (C22H23F3N6O‚CH2Cl2) C, H, N.
1-[2-(1H-Imidazol-1-yl)-4-pyrimidinyl]-N-[(4-methoxyphenyl-
)methyl]-2-piperidineacetamide (12b). 1H NMR (CDCl3) δ 8.45
(s, 1 H), 8.1 (d, 1 H), 7.75 (s, 1 H), 7.1 (s, 1 H), 7.0 (d, 2 H), 6.7
(d, 2 H), 6.5 (s, 1 H), 6.25 (m, 1 H), 5.1 (br, 1 H), 4.25 (m, 3 H),
3.75 (s, 3 H), 2.59 (m, 1 H), 2.65 (dd, 1 H), 2.55 (dd, 1 H), 1.3-
1.8 (m, 6 H). Anal. (C22H26N6O2‚0.8H2O) C, H, N.
1-[2-(1H-Imidazol-1-yl)pyrimidin-4-yl]-N-[(3-methoxyphenyl-
1
)methyl]-2-piperidineacetamide (12c). H NMR (CDCl3) δ 8.4
(s, 1 H), 8.0 (m, 1 H), 7.75 (s, 1 H), 7.3 (m, 1 H), 7.05 (t, 1 H),
6.95 (s, 1 H), 6.6 (m, 3 H), 6.6 (m, 1 H), 5.0 (br, 1 H), 4.25 (m, 3
H), 3.65 (s, 3 H), 2.9 (m, 3 H), 2.6 (m, 2 H), 1.3-1.8 (m, 6 H).
Anal. (C22H26N6O2‚0.3H2O) C, H, N.
1-[2-(1H-Imidazol-1-yl)pyrimidin-4-yl]-N-[(2-methoxyphenyl-
1
)methyl]-2-piperidineacetamide (12d). H NMR (CDCl3) δ 8.4
(s, 1 H), 8.0 (d, 1 H), 7.8 (s, 1 H), 7.1 (m, 2 H), 6.7 (m, 2 H), 6.4
(d, 1 H), 6.3 (m, 1 H), 5.0 (br s, 1H), 4.3 (m, 2 H), 3.8 (s, 3 H), 2.9
(m, 1 H), 2.8 (m, 2 H), 2.0 (m, 1 H), 1.7 (m, 5 H), 1.5 (m, 1 H).
Anal. (C22H26N6O2‚H2O) C, H, N.
N-[(4-Cyanophenyl)methyl]-1-[2-(1H-imidazol-1-yl)pyrimidin-
1
4-yl]-2-piperidineacetamide (12p). H NMR (CDCl3) δ 8.4 (s, 1
H), 8.1 (d, 1 H), 7.8 (s, 1 H), 7.4 (d, 2 H), 7.2 (d, 2 H), 7.0 (s, 1
H), 6.8 (m, 1 H), 6.5 (m, 1 H), 5.2 (br s, 1 H), 4.4 (m, 2 H), 3.0
(m, 1 H), 2.8 (dd, 1 H), 2.6 (dd, 1 H), 1.8 (m, 5 H), 1.6 (m, 1 H).
Anal. (C22H23N7O‚CH2Cl2) C, H, N.
1-[2-(1H-Imidazol-1-yl)pyrimidin-4-yl]-N-[(3,4-dimethoxyphe-
1
nyl)methyl]-2-piperidineacetamide (12e). H NMR (DMSO-d6)
δ 8.5 (s, 1 H), 8.4 (m, 1 H), 8.15 (d, 1 H), 7.85 (s, 1 H), 7.05 (s,
1 H), 6.7 (m, 3 H), 6.6 (d, 1 H), 4.1 (m, 2 H), 3.6 (2 s, 6 H), 3.1
(m, 1 H), 3.0 (m, 2 H), 1.3-1.8 (m, 6 H). Anal. (C23H28N6O3‚
H2O) C, H, N.
1-[2-(1H-Imidazol-1-yl)pyrimidin-4-yl]-N-[(4-nitrophenyl)-
methyl]-2-piperidineacetamide (12q). 1H NMR (CDCl3) δ 8.4 (s,
1 H), 8.1 (d, 1 H), 8.0 (d, 2 H), 7.8 (s, 1 H), 7.2 (d, 2H), 7.1 (s, 1
H), 6.5 (d, 1 H), 5.2 (br s, 1H), 4.5 (dd, 1 H), 4.3 (dd, 1 H), 3.1
(m, 1 H), 2.8 (dd, 1 H), 2.6 (dd, 1 H), 1.8 (m, 4 H), 1.6 (m, 3 H,
partially obscured by water). Anal. (C21H23N7O3‚CH2Cl2) C, H, N.
N-[(4-Aminosulfonylphenyl)methyl]-1-[2-(1H-imidazol-1-yl)-
pyrimidin-4-yl]-2-piperidineacetamide (12r). 1H NMR (DMSO-
d6) δ 8.6 (s, 2 H), 8.2 (d, 1 H), 7.8 (s, 1 H), 7.7 (d, 2 H), 7.3 (m,
4 H), 7.1 (s, 1 H), 6.7 (d, 1 H), 4.2 (m, 2 H), 3.0 (m, 1 H), 2.5 (m,
3 H, partially obscured by DMSO), 2.3 (m, 1 H), 1.6 (m, 5 H), 1.4
(m, 1 H). Anal. (C21H25N7O3) C, H, N.
1-[2-(1H-Imidazol-1-yl)pyrimidin-4-yl]-N-[(3,4,5-trimethoxy-
1
phenyl)methyl]-2-piperidineacetamide (12f). H NMR (CDCl3)
δ 8.5 (s, 1 H), 8.1 (d, 1 H), 7.8 (s, 1 H), 7.1 (s, 1 H), 6.5 (m, 1 H),
6.3 (s, 2 H), 6.2 (br, 1 H), 5.15 (br, 1 H), 4.4 (dd, 1 H), 4.2 (m, 1
H), 3.82 (s, 3 H), 3.77 (s, 6 H), 3.0 (m, 1 H), 2.7 (dd, 1 H), 2.55
(dd, 1 H), 1.3-1.8 (m, 6 H). Anal. (C24H30N6O4) C, H, N.
1-[2-(1H-Imidazol-1-yl)pyrimidin-4-yl]-N-(phenylmethyl)-2-
piperidineacetamide (12g). 1H NMR (CDCl3) δ 8.4 (s, 1 H), 8.05
(d, 1 H), 7.75 (s, 1 H), 7.0-7.3 (m, 6 H), 6.7 (br, 1 H), 6.5 (m, 1

NOS Dimerization Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1155
1-[2-(1H-Imidazol-1-yl)-4-pyrimidinyl]-N-[[4-(methylsulfonyl)-
phenyl]methyl]-2-piperidineacetamide (12s). H NMR (CDCl3)
δ 8.4 (s, 1 H), 8.1 (d, 1 H), 7.8 (s, 1 H), 7.6 (d, 2 H), 7.4 (s, 1 H),
7.2 (d, 2 H), 7.0 (s, 1 H), 6.5 (s, 1 H), 5.1 (br s, 1 H), 4.4 (m, 2 H),
3.0 (s, 4 H), 2.7 (m, 2 H), 2.4 (s, 1 H), 1.8 (m, 5 H), 1.5 (m, 1 H).
Anal. (C22H26N6O3S‚CH2Cl2) C, H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]hexahydro-1-[2-(1H-imida-
zol-1-yl)-4-pyrimidinyl]-1H-azepine-2-acetamide (14). 14 was
prepared as described above from 1-(dimethylethoxycarbonyl)-
hexahydro-1H-azepine-2-acetic acid.19 1H NMR (CDCl3, 300 MHz)
δ 1.10-3.00 (m, 11H), 3.20-3.62 (m, 1H), 4.05-4.50 (m, 3H),
5.00-5.30 (m, 1H), 5.80-6.10 (m, 2H), 6.25-6.90 (m, 4H), 7.05
(d, 1H), 7.80 (d, 1H), 8.10 (d, 1H), 8.50 (d, 1H). MS m/z 435.3
(M + H)+.
1
1-[2-(1H-Imidazol-1-yl)-4-pyrimidinyl]-N-[(2-furanyl)methyl]-
1
2-piperidineacetamide (12t). H NMR (CDCl3) δ 8.45 (s, 1 H),
N-[(1,3-Benzodioxol-5-yl)methyl]-1-[4-(1H-imidazol-1-yl)-2-
pyrimidinyl]-2-piperidineacetamide (15). 1H NMR (CDCl3) δ 8.3
(s, 1 H), 8.15 (m, 1 H), 7.6 (s, 1 H), 7.15 (s, 1 H), 6.95 (br, 1 H),
6.6 (m, 3 H), 6.4 (d, 1 H), 5.9 (m, 2 H), 5.25 (m, 1 H), 4.7 (d, 1
H), 4.3 (dd, 1 H), 4.15 (dd, 1 H), 3.0 (t, 1 H), 2.85 (dd, 1 H), 2.55
(dd, 1 H), 1.6-1.8 (m, 5 H), 1.5 (m, 1 H). Anal. (C22H24N6O3) C,
H, N.
8.05 (d, 1 H), 7.8 (s, 1 H), 7.2 (s, 1 H), 7.05 (s, 1 H), 6.7 (m, 1 H),
6.5 (m, 1H), 6.2 (s, 1H), 6.05 (s, 1H), 5.1 (br s, 1 H), 4.3 (m, 3 H),
2.95 (m, 1 H), 2.6 (m, 2 H), 1.7 (m, 5 H), 1.5 (m, 1 H). Anal.
(C19H22N6O2‚0.5H2O) C, H, N.
1-[2-(1H-Imidazol-1-yl)-4-pyrimidinyl]-N-[(2-pyridinyl)methyl]-
1
2-piperidineacetamide (12u). H NMR (CDCl3) δ 8.45 (s, 1 H),
8.4 (m, 1H), 8.05 (d, 1 H), 7.8 (s, 1 H), 7.55 (t, 1 H), 7.0-7.2 (m,
4 H), 6.5 (m, 1 H), 5.1 (br s, 1 H), 4.4 (m, 2 H), 4.4 (br, 1H), 3.0
(m, 1 H), 2.6 (m, 2 H), 1.7 (m, 5 H), 1.5 (m, 1 H). Anal.
(C20H23N7O‚0.4H2O) C, H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]-1-[4-(1H-imidazol-1-yl)-6-
1
pyrimidinyl]-2-piperidineacetamide (16). H NMR (DMSO-d6)
δ 8.5 (s, 1 H), 8.4 (m, 2 H), 7.95 (s, 1 H), 7.1 (s, 1 H), 7.0 (s, 1 H),
6.7 (m, 2 H), 6.6 (d, 1 H), 5.9 (s, 2 H), 5.1 (br, 1 H), 4.4 (br, 1 H),
4.05 (m, 2 H), 3.03 (t, 1 H), 2.5 (m, 2 H), 1.5-1.8 (m, 5 H), 1.3
(m, 1 H). Anal. (C22H24N6O3‚0.5H2O) C, H, N.
1-[2-(1H-Imidazol-1-yl)-4-pyrimidinyl]-N-methyl-N-phenyl-
1
2-piperidineacetamide (12v). H NMR (DMSO-d6) δ 8.45 (m, 1
H), 8.1 (m, 1H), 7.8 (m, 1 H), 7.2 (m, 3 H), 7.05 (m, 3 H), 6.7 (dd,
1 H), 4.6 (m, 1 H), 4.45 (m, 2 H), 2.85 (m, 3 H), 2.9 and 2.75 (2
s, 3 H), 1.4-1.8 (m, 6 H). Anal. (C20H23N7O‚0.4H2O) C, H, N.
1-[2-(1H-Imidazol-1-yl)-4-pyrimidinyl]-N-[(diphenyl)methyl]-
N-(1,3-Benzodioxol-5-ylmethyl)-1-[3-(1H-imidazol-1-yl)phe-
nyl]-2-piperidineacetamide (17). A mixture of 1-(3-aminophenyl)-
imidazole20 (755 mg, 4.7 mmol), ethyl 7-chloro-3-oxoheptanoate21
(982 mg, 4.7 mmol), Na2HPO4 (667 mg, 4.7 mmol), iodine (60
mg, 0.23 mmol), and 500 mg of powdered 4 Å molecular sieves in
20 mL of dry benzene is refluxed for 5 h. An additional 60 mg of
iodine is added, and the reaction mixture is refluxed overnight. After
filtration of the reaction mixture, the filtrate is evaporated and the
residue is purified by chromatography (silica gel, methylene
chloride/methanol, 98:2) to yield 120 mg (8%) of [1-[3-(1H-
imidazol-1-yl)phenyl]-2-piperedinylidine]ethanoic acid, ethyl ester.
1H NMR (CDCl3) δ 7.85 (s, 1H), 7.05-7.50 (m, 6H), 4.80 (s, 1H),
4.15 (q, 2H), 3.45 (t, 2H), 2.4 (t, 2H), 1.50-1.80 (m, 6H), 1.30 (t,
3H). The ester (120 mg, 0.38 mmol) is dissolved in methanol (20
mL), and 10% Pd/C (60 mg) is added. The mixture is hydrogenated
at 1 atm for 60 h. The catalyst is filtered, and the filtrate is
evaporated. The residue is partially purified (silica gel, hexane/
ethyl acetate, 1:1), and the slightly impure product (24 mg, 0.076
mmol) is stirred in 5% methanolic NaOH overnight. After acidifica-
tion with methanolic HCl, the contents are evaporated to dryness
and the residue is coupled with piperonylamine using 2 equiv of
Hunig’s base and 1 equiv of HATU in dry DMF. After aqueous
workup the residue is purified by chromatography (silica gel, ethyl
acetate/methanol, 98:2) to yield 6 mg (19%) of the desired product.
1H NMR (CDCl3) δ 7.80 (s, 1H), 7.10-7.30 (m, 4H), 6.50-6.70
(m, 5H), 5.90 (s, 2H), 4.20-4.35 (m, 2H), 3.85 (bs, 1H), 3.50 (t,
2H), 2.95 (d, 1 H), 2.35-2.55 (m, 2H), 1.40-1.90 (m, 6H). MS
m/z 419 (M + H)+.
1
2-piperidineacetamide (12w). H NMR (CDCl3) δ 8.45 (s, 1 H),
8.05 (d, 1 H), 7.75 (s, 1 H), 6.9-7.4 (m, 12 H), 6.45 (m, 1 H), 6.2
(d, 1H), 5.1 (br s, 1 H), 4.4 (br, 1H), 3.0 (m, 1 H), 2.8 (dd, 1 H),
2.55 (dd, 1H), 1.7 (m, 5 H), 1.5 (m, 1 H). Anal. (C27H28N6O‚
0.5H2O) C, H, N.
1-[2-(1H-Imidazol-1-yl)pyrimidin-4-yl]-N-methyl-2-piper-
idineacetamide (12x). 1H NMR (DMSO-d6) δ 8.45 (s, 1 H), 8.15
(d, 1 H), 7.85 (s, 1 H), 7.55 (br, 1 H), 7.05 (s, 1 H), 6.65 (d, 1 H),
5.0 (br, 1 H), 4.25 (m, 1 H), 2.4 (m, 2 H), 1.3-1.8 (m, 6 H). Anal.
(C15H20N6O‚0.1CH2Cl2‚0.9H2O) C, H, N.
N-[(2-Benzimidazolyl)methyl]-1-[2-(1H-imidazol-1-yl)pyrimi-
din-4-yl]-2-piperidineacetamide (12y). 1H NMR (DMSO-d6) δ 8.7
(m, 1 H), 8.5 (s, 1 H), 8.15 (d, 1 H), 7.85 (s, 1 H), 7.45 (m, 2 H),
7.15 (m, 2 H), 7.05 (s, 1 H), 6.7 (d, 2 H), 5.3 (br, 2 H), 4.6 (br 1
H), 4.4 (m, 2 H), 3.0 (m, 1 H), 2.6 (m, 1 H), 2.5 (m, 1 H), 1.7 (m,
5 H), 1.4 (m, 1 H). Anal. (C22H24N8O) C, H, N.
N-(1,3-Benzodioxol-5-yl)-1-[2-(1H-imidazol-1-yl)pyrimidin-4-
1
yl]-2-piperidineacetamide (12z). H NMR (DMSO-d6) δ 9.9 (s,
1 H), 8.5 (s, 1 H), 8.15 (d, 1 H), 7.85 (s, 1 H), 7.15 (s, 1 H), 7.05
(m, 1 H), 6.75 (m, 3 H), 5.9 (s, 2 H), 3.1 (m, 1 H), 2.65 (m, 2 H),
1.7 (m, 5 H), 1.4 (m, 1 H). Anal. (C21H28N6O3‚H2O) C, H, N.
N-(3,4-Dimethoxyphenyl)-1-[2-(1H-imidazol-1-yl)pyrimidin-
4-yl]-2-piperidineacetamide (12aa). 1H NMR (DMSO-d6, 100 °C)
δ 9.3 (s, 1 H), 8.5 (s, 1 H), 8.15 (d, 1 H), 7.85 (s, 1 H), 7.1 (s, 1
H), 7.0 (m, 2 H), 6.85 (d, 1 H), 6.7 (d, 1 H), 5.2 (s, 1 H), 4.3 (d,
1 H), 3.8 (s, 3 H), 3.75 (s, 3H), 3.1 (m, 1 H), 2.7 (m, 2 H), 1.3-
1.8 (m, 6 H). Anal. (C22H26N6O3‚0.2CH2Cl2) C, H, N.
N-(1,3-Benzodioxol-5-ylmethyl)-1-[6-chloro-2-(1H-imidazol-
1-yl)-4-pyrimidinyl]-2-piperidineacetamide (18a). 1H NMR (DM-
SO-d6) δ 8.45 (s, 1 H), 8.4 (br, 1H), 7.85 (s, 1 H), 7.05 (s, 1 H),
6.85 (br, 1 H), 6.6 (m, 3 H), 5.9 (s, 2 H), 4-4.6 (m, 4H), 3.1 (m,
1 H), 2.56 (m, 2 H), 1.3-1.7 (m, 6 H). Anal. (C22H23N6O3Cl) C,
H, N, Cl.
1-[2-(1H-Imidazol-1-yl)-4-pyrimidinyl]-N-[2-(3,4-dimethoxy-
1
phenyl)ethyl]-2-piperidineacetamide (12bb). H NMR (CDCl3)
δ 8.5 (s, 1 H), 8.1 (d, 1 H), 7.8 (s, 1 H), 7.1 (s, 1 H), 6.8 (s, 1 H),
6.6 (m, 2 H), 6.5 (m, 1 H), 5.95 (br, 1H), 5.0 (br, 1 H), 3.8 (2 s, 6
H), 3.4 (m, 2 H), 2.9 (m, 1 H), 2.65 (m, 2 H), 2.45 (m, 2 H), 1.7
(m, 6 H). Anal. (C24H30N6O3‚0.3H2O‚0.15C4H10O) C, H, N.
1-[2-(1H-Imidazol-1-yl)-4-pyrimidinyl]-N-[2-(4-methoxyphe-
N-(1,3-Benzodioxol-5-ylmethyl)-1-[2-(1H-imidazol-1-yl)-6-
phenyl-4-pyrimidinyl]-2-piperidineacetamide (18b). 1H NMR
(DMSO-d6) δ 8.5 (s, 1 H), 8.4 (s, 1H), 8.2 (d, 1 H), 7.95 (d, 2 H),
7.47 (m, 2 H), 7.2 (s, 1 H), 7.05 (s, 1 H), 6.5-6.8 (m, 3 H), 5.9 (s,
2 H), 4-4.2 (m, 4H), 3.1 (m, 1 H), 2.56 (m, 2 H), 1.3-1.7 (m, 6
H). Anal. (C28H28N6O3‚0.5C3H7NO‚0.05C3H4N2) C, H, N.
N-(1,3-Benzodioxol-5-ylmethyl)-1-[2-(1H-imidazol-1-yl)-6-
methyl-4-pyrimidinyl]-2-piperidineacetamide (18c). 1H NMR
(DMSO-d6) δ 9.6 (brd, 1 H), 8.5 (s, 1H), 8.25 (d, 1 H), 7.7 (m, 2
H), 6.5-6.8 (m, 3 H), 5.9 (s, 2 H), 4-4.2 (m, 4H), 3.1 (m, 1 H),
2.6 (m, 2 H), 2.2 (s, 3H), 1.3-1.7 (m, 6 H). Anal. (C23H26N6O3‚
0.8H2O‚2.4C2HF3O2) C, H, N.
1
nyl)ethyl]-2-piperidineacetamide (12cc). H NMR (CDCl3) δ 8.5
(s, 1 H), 8.1 (d, 1 H), 7.8 (s, 1 H), 7.1 (s, 1 H), 7.0 (d, 2 H), 6.8
(d, 2 H), 6.5 (br s, 1 H), 6.3 (m, 1 H), 5.0 (br s, 1 H), 3.8 (s, 3 H),
3.4 (m, 2 H), 2.9 (m, 1 H), 2.7 (m, 2 H), 2.4 (m, 3 H), 1.7 (m, 6
H). Anal. (C23H28N6O2‚CH2Cl2) C, H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]-4-[2-(1H-imidazol-1-yl)-4-
pyrimidinyl]-3-morpholineacetamide (13). 1H NMR (CDCl3, 300
MHz) δ 2.45-2.55 (dd, 1H), 2.75-2.90 (dd, 1H), 3.20 (bt, 1H),
3.55-3.80 (m, 2H), 3.85-4.20 (m, 4H), 4.20-4.40 (dd, 2H), 5.90
(d, 2H), 6.10 (bs, 1H), 6.25-6.75 (m, 4H), 7.10 (m, 1H), 7.80 (m,
1H), 8.20 (d, 1H), 8.50 and 8.75 combined (d, 1H). MS m/z 423.5
(M + H)+.
N-(1,3-Benzodioxol-5-ylmethyl)-1-[2-(1H-imidazol-1-yl)-6-(tri-
1
fluoromethyl)-4-pyrimidinyl]-2-piperidineacetamide (18d). H
NMR (DMSO-d6) δ 9.4 (brd, 1 H), 8.5 (brd, 1H), 8.3 (s, 1 H), 7.5
(m, 2 H), 6.5-6.8 (m, 3 H), 5.9 (s, 2 H), 5.36 (s, 1H), 4.61 (m,

1156 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
DaVey et al.
1H), 4.02 (m, 1H), 3.1 (m, 1H), 1.3-1.7 (m, 6 H). Anal.
(C23H23F3N6O3‚0.5H2O‚1.5C2HF3O2) C, H, N, F.
4.3 (m, 1 H), 3.4 (m, 2 H), 2.8 (br s, 1 H), 2.6 (q, 2H), 2.4 (br s,
1 H), 2.1 (m, 4 H), 1.3 (t, 3H). Anal. (C23H26N6O3‚CH2Cl2) C,
H, N.
N-[2-(1,3-Benzodioxol-5-yl)ethyl]-1-[2-(1H-imidazol-1-yl)-4-
pyrimidinyl]-2-pyrrolidinecarboxamide (21a). 1H NMR (CDCl3)
δ 8.4 (s, 1 H), 8.2 (d, 1 H), 7.7 (br s, 1 H), 7.1 (s, 1 H), 6.4 (m, 5
H), 5.8 (m, 2 H), 4.6 (br s, 1 H), 3.4 (m, 4 H), 2.8 (m, 2 H), 2.1
(m, 4 H). Anal. (C21H22N6O3) C, H, N.
N-(1,3-Benzodioxol-5-ylmethyl)-1-[2,6-di(1H-imidazol-1-yl)-
1
4-pyrimidinyl]-2-piperidineacetamide (18e). H NMR (DMSO-
d6) δ 10.3 (br, 1 H), 9.9 (s, 1H), 8.78 (s, 1 H), 8.63 (m, 3 H), 7.95
(m, 2H), 7.41 (d, 1H), 6.5 (m, 2 H), 5.8 (s, 2H), 4.9 (m, 1H), 4.03
(m, 3H), 3.5 (t, 1H), 3.1 (m, 1H), 1.3-1.7 (m, 6 H). Anal.
(C25H26N8O3‚1.2H2O‚3C2HF3O2) C, H, N, F.
N-[2-(1,3-Benzodioxol-5-yl)ethyl]-1-[2-(1H-imidazol-1-yl)-6-
methyl-4-pyrimidinyl]-2-pyrrolidinecarboxamide (21b). 1H NMR
(CDCl3) δ 8.5 (s, 1 H), 7.7 (s, 1 H), 7.1 (s, 1 H), 6.5 (m, 4 H), 6.1
(br s, 1 H), 5.8 (m, 2 H), 4.6 (br s, 1 H), 3.5 (m, 4 H), 2.6 (m, 2
H), 2.4 (m, 4H), 2.1 (br s, 2 H), 1.3 (s, 1 H). Anal. (C22H24N6O3)
C, H, N.
N-[(1,3-Benzodioxol-5-yl)ethyl]-4-[2-(1H-imidazol-1-yl)-6-eth-
ylpyrimidin-4-yl]-2-pyrrolidinecarboxamide (21c). 1H NMR
(CDCl3) δ 8.5 (s, 1 H), 7.8 (s, 1 H), 7.1 (s, 1 H), 6.5 (m, 4 H), 6.1
(br s, 1 H), 5.8 (m, 2 H), 4.6 (br s, 1 H), 3.5 (m, 4 H), 2.6 (m, 4
H), 2.4 (br s, 1H), 2.1 (br s, 3 H), 1.3 (t, 3H). Anal. (C23H26N6O3)
C, H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]-4-[2-(1H-imidazol-1-yl)-6-
methylpyrimidin-4-yl]-2-piperazineacetamide (19a). 1H NMR
(CDCl3) δ 8.55 (s, 1 H), 7.8 (s, 1 H), 7.1 (s, 1 H), 6.75 (m, 4 H),
6.25 (s, 1 H), 5.95 (s, 2 H), 4.4 (m, 2 H), 4.35 (m, 2 H), 3.2 (m, 3
H),2.85(m,2H),2.4(m,3H),2.4(s,3H).Anal.(C22H25N7O3‚0.1CH2-
Cl2‚0.8H2O) C, H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]-4-[2-(1H-imidazol-1-yl)-6-
methylpyrimidin-4-yl]-1-methyl-2-piperazineacetamide (19b). 1H
NMR (CDCl3) δ 8.5 (s, 1 H), 7.8 (s, 1 H), 7.7 (m, 1 H), 7.05 (s,
1 H), 6.75 (m, 3 H), 6.2 (s, 1 H), 5.9 (s, 2 H), 4.35 (m, 2 H),
3.9-4.2 (m, 2 H), 3.25 (m, 2 H), 2.85 (d, 1 H), 2.4-2.7 (m, 4 H),
2.4 (2 s, 6 H). Anal. (C23H27N7O3‚0.2H2O) C, H, N.
Optically Active N-[(1,3-Benzodioxol-5-yl)methyl]-1-[2-(1H-
imidazol-1-yl)pyrimidin-4-yl]-4-[(methoxy)carbonyl]-2-pipera-
zineacetamide (11d*). N-[(1,3-Benzodioxol-5-yl)methyl-4-[(meth-
oxy)carbonyl]-1-[(dimethylethoxy)carbonyl)]piperazine-2-
acetamide was separated on a Chiralpak AD column (Chiral
Technologies Inc.) using hexanes/isopropanol (4:1). After separa-
tion, the two enantiomers were treated identically by manners
previously described to give the final compounds. Initial treatment
with trifluoroacetic acid deprotected the piperazine. The piperazine
was added to 4-chloro-2-methylsulfonylpyrimidine, and subsequent
reaction with imidazole gave the title compound. The earlier eluting
enantiomer resulted in the more potent final product, which was
identified as the S-enantiomer by analysis of the crystal structure.
Assay To Determine Potency Inhibiting NO Formation. A172
cells were obtained from the American Type Culture Collection
and were cultured routinely in DMEM without phenol red or sodium
pyruvate but containing high glucose (Gibco BRL), supplemented
with 10% (v/v) fetal bovine serum (Gibco BRL), in a humidified
atmosphere of 5% CO2 in air at 37 °C. Cells were harvested and
plated at 100 000 cells/well into 96-well tissue culture dishes in a
total of 100 µL of culture medium. After 18-24 h, inducible nitric
oxide synthase (iNOS) activity was induced by the addition of 222
U/mL human interferon-γ, 22 ng/mL of human tumor necrosis
factor-R, and 2.2 ng/mL of human interleukin 1-â. All cytokines
were purchased from Boehringer Mannheim. Concomitant with
cytokine addition, the appropriate concentration of the compound
was also added. Compound stock solutions were prepared in
DMSO, and vehicle was added to control wells. The final
concentration of DMSO in the incubations was less than 0.2% and
had no influence on iNOS induction or activity measurements.
Incubations were continued for 18-24 h, at which time an aliquot
of the culture medium was removed and tested for nitrite concentra-
tion. A 100 µL aliquot of the culture medium was removed and
mixed with 150 µL of the Griess reagent (5% v/v phosphoric acid
containing 2% w/v sulfanilamide plus 0.2% w/v naphthylethylene-
diamine) in a separate 96-well plate. The plates were read within
15 min at 550 nm in a SpectraMax spectrophotometer. The
inhibition of NO formation by compound resulted in a decrease in
the OD550 of the medium. IC50 values were calculated from a log-
logit analysis of the data. Inhibition curves with Hill slopes of less
than 0.5 or greater than 1.5 were rejected. Control experiments
showed that no significant conversion of nitrite to nitrate occurred
over the course of an experiment. Cells incubated in the absence
of cytokines produced no measurable nitrite.
N-[(1,3-Benzodioxol-5-yl)methyl]-4-[2-(1H-imidazol-1-yl)-6-
methylpyrimidin-4-yl]-1-(phenylmethyl)-2-piperazineaceta-
mide (19c). 1H NMR (CDCl3) δ 8.5 (s, 1 H), 7.8 (s, 1 H), 7.2 (m,
6 H), 7.1 (s, 1 H), 6.75 (m, 3 H), 6.22 (s, 1 H), 5.97 (s, 2 H), 4.4
(dd, 1 H), 4.3 (dd, 1 H), 3.95 (d, 1 H), 3.8 (m, 3 H), 3.6 (m, 1 H),
3.45 (d, 1 H), 3.15 (m, 1 H), 2.75 (m, 1 H), 2.4-2.7 (m, 3 H), 2.4
(s, 3 H). Anal. (C29H31N7O3‚0.1CH2Cl2‚0.4H2O) C, H, N.
N-(1,3-Benzodioxol-5-ylmethyl)-4-[2-(1H-imidazol-1-yl)-6-
methyl-4-pyrimidinyl]-1-(methylsulfonyl)-2-piperazineaceta-
mide (19d). 1H NMR (DMSO-d6) δ 9.7 (s, 1H), 8.4 (m, 2 H), 7.8
(s, 1H), 6.6 (m, 3H), 5.95 (s, 2H), 4.45 (m, 4H), 4.3 (m, 3H), 3.62
(m, 2H), 3.1 (m, 1H), 3 (s, 3H), 2.45 (m, 3H), 2.1 (s, 3H). Anal.
(C23H27N7O5S‚0.5H2O‚2.6C2HF3O2) C, H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]-4-[2-(1H-imidazol-1-yl)-6-
methylpyrimidin-4-yl]-1-(methylcarbonyl)-2-piperazineaceta-
1
mide (19e). H NMR (DMSO-d6, 100 °C) δ 8.45 (s, 1 H), 7.85
(m, 1 H), 7.8 (s, 1 H), 7.05 (s, 1 H), 6.7 (m, 3 H), 6.55 (s, 1 H),
5.95 (s, 2 H), 4.7 (m, 1H), 4.0-4.5 (m, 5 H), 3.35 (m, 1 H), 3.15
(m, 1 H), 2.4 (s, 3 H), 2.1 (s, 3 H). Anal. (C26H31N7O4‚0.2CH2-
Cl2‚0.1H2O) C, H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]-4-[2-(1H-imidazol-1-yl)-6-
methylpyrimidin-4-yl]-1-[(1-methylethyl)carbonyl]-2-pipera-
1
zineacetamide (19f). H NMR (DMSO-d6, 100 °C) δ 8.45 (s, 1
H), 7.85 (m, 2 H), 7.05 (s, 1 H), 6.7 (m, 3 H), 6.55 (s, 1 H), 5.95
(s, 2 H), 4.8 (m, 1H), 4.45 (d, 1 H), 4.3 (d, 1H), 4.15 (m, 3 H),
3.35 (dd, 1 H), 3.2 (m, 2 H), 2.9 (m, 1 H), 2.55 (m, 1 H), 2.4 (m,
1 H), 2.35 (s, 3 H), 1.05 (2 s, 6 H). Anal. (C26H31N7O4) C, H, N.
2-[2-[(1,3-Benzodioxol-5-ylmethyl)amino]-2-oxoethyl]-4-[2-
(1H-imidazol-1-yl)-6-methyl-4-pyrimidinyl]-1-piperazinecar-
boxylic Acid, Methyl Ester (19g). 1H NMR (DMSO-d6) δ 9.4 (s,
1H), 8.2 (m, 2 H), 7.6 (s, 1H), 6.7 (m, 3H), 5.95 (s, 2H), 4.6 (m,
2H), 3.9-4.41 (m, 4H), 3.62 (s, 3H), 3.1-3.4 (m, 3H), 2.6 (m,
4H), 2.3 (s, 3H). Anal. (C24H27N7O5‚0.5H2O‚1.4C2HF3O2) C,
H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]-1-[2-(1H-imidazol-1-yl)-4-
pyrimidinyl]-2-pyrrolidineacetamide (20a). 1H NMR (CDCl3) δ
8.5 (s, 1 H), 8.1 (d, 1 H), 7.8 (s, 1 H), 7.0 (s, 1 H), 6.7 (m, 3 H),
6.1 (m, 4 H), 4.7 (br s, 1 H), 4.3 (m, 2 H), 3.4 (m, 2 H), 2.8 (br s,
1 H), 2.3 (br s, 1 H), 2.1 (m, 4 H). Anal. (C21H22N6O3‚CH2Cl2) C,
H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]-1-[2-(1H-imidazol-1-yl)-6-
1
methyl-4-pyrimidinyl]-2-pyrrolidineacetamide (20b). H NMR
(CDCl3) δ 8.4 (m, 1 H), 7.7 (m, 1 H), 7.0 (s, 1 H), 6.7 (m, 3 H),
6.0 (m, 4 H), 4.6 (br s, 1 H), 4.3 (m, 2 H), 3.4 (m, 2 H), 2.8 (br s,
1 H), 2.2 (m, 8 H). Anal. (C22H24N6O3‚CH2Cl2) C, H, N.
N-[(1,3-Benzodioxol-5-yl)methyl]-1-[6-ethyl-2-(1H-imidazol-
1-yl)-4-pyrimidinyl]-2-pyrrolidineacetamide (20c). 1H NMR
(CDCl3) δ 8.6 (s, 1 H), 7.8 (s, 1 H), 7.0 (s, 1 H), 6.7 (m, 3 H), 6.1
(m, 1 H), 5.9 (s, 2 H), 5.8 (m, 1 H), 4.7 (br s, 1 H), 4.4 (dd, 1 H),
Assays To Determine Selectivity. BSC-1 cells (American Type
Culture Collection) were maintained in DMEM containing 10%
fetal bovine serum. Cells were plated into 96-well tissue culture
plates at 30 000 cells per well. After 6 h, recombinant vaccinia
virus strains encoding human inducible, endothelial, and neuronal
NOS isoforms were added, along with inhibitor or vehicle. After
17 h, the medium was aspirated and 20 µL of 40 mM Tris, pH 7.5,

NOS Dimerization Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1157
G. B.; Polokoff, M. A.; Sigal, N. A.; Vergona, R.; Whitlow, M.;
Young, T.; Devlin, J. J. Allosteric inhibitors of inducible nitric oxide
synthase dimerization discovered via combinatorial chemistry. Proc.
Natl. Acad. Sci. U.S.A. 2000, 97, 1506-1511.
containing 0.1% NP-40, 5 mg/mL aproitinin, 1 µg/mL leupeptin,
1 µg/mL pepstatin, and 24 µg/mL of Pefabloc SC was added. The
plates were then frozen at -80 °C. NOS activity in cell lysates
was determined by measuring the conversion of arginine into
citrulline. The assay mixture contained 40 mM Tris, pH 7.5, 3 mM
dithiothreitol (DTT), 4 µM each of H4B, FAD, and FMN, 0.5 µM
calmodulin, 15 mM calcium chloride, 1 mM NADPH, and 3 µM
[14C]arginine (300 Ci/mol). NOS activity in Superdex 200 fractions
was measured using the same protocol except with 4 µM [14C]-
arginine and 16 µM unlabeled arginine in the assay mixture.
Rat Adjuvant Arthritis Model.22,23 Adult male Lewis rats were
injected with 0.1 mL of Mycobacterium butyricum in incomplete
Freund’s adjuvant (10 mg/mL, intradermally) into the proximal
quarter of the tail. Drug treatment started on the day following
immunization. Compound (30, 10, and 3 mg/kg) or vehicle was
administered subcutaneously twice daily (12 h intervals). Naive rats
received no treatment. Rats were weighed and observed for clinical
signs of arthritis 3 times per week. All rats were sacrificed 34-35
days after immunization. Each dosing group consisted of 10
animals. Clinical scoring was done on a scale of 0-4 for each limb,
with increasing degrees of redness, gross swelling and distortion
of the paw, and joint fusion. The sum of these scores for each limb
was totaled and was designated as the clinical score. Radiological
(X-ray) scoring was done by grading both hind limbs of the
sacrificed animals on a scale of 0-3 for each of the following
parameters: soft tissue swelling, cartilage loss, erosion, and
heterotopic ossification. The sum of these scores for each limb was
totaled and was designated as the radiological score.
(9) Sennequier, N.; Wolan, D.; Stuehr, D. J. Antifungal imidazoles block
assembly of inducible NO synthase into an active dimer J. Biol.
Chem. 1999, 274, 930-938.
(10) Ohtsuka, M.; Konno, F.; Honda, H.; Oikawa, T.; Ishikawa, M.; Iwase,
N.; Isomae, K.; Ishii, F.; Hemmi, H.; Sato, S. PPA250 [3-(2,4-
Difluorophenyl)-6-{2-[4-(1H-imidazol-1-ylmethyl)phenoxy]ethoxy}-
2-phenylpyridine], a novel orally effective inhibitor of the dimer-
ization of inducible nitric-oxide synthase, exhibits an anti-inflammatory
effect in animal models of chronic arthritis. J. Pharmacol. Exp. Ther.
2002, 303, 52-57.
(11) Chida, N.; Hirasawa, Y.; Ohkawa, T.; Ishii, Y.; Sudo, U.; Tamura,
K.; Mutoh, S. Pharmacological profile of FR260330, a novel orally
active inducible nitric oxide synthase inhibitor. Eur. J. Pharmacol.
2005, 509, 71-6.
(12) 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. J.; Parkinson, J. F. Mechanistic studies
with potent and selective inducible nitric-oxide synthase dimerization
inhibitors. J. Biol. Chem. 2002, 277, 295-302.
(13) Afonso, A.; Baldwin, J. J.; Doll, R. J.; Li, G.; Mallams, A. K.;
Njoroge, F. G.; Rane, D. F.; Reader, J. C.; Rossman, R. R. Preparation
of Heterocyclic Tricyclic Compounds Useful for Inhibition of
g-Protein Function and for Treatment of Cell Proliferative Diseases.
WO9631478, 1996.
(14) Van den Branden, S.; Compernolle, F.; Hoorneaert, G. J. Synthesis
of lactam and ketone precursors of 2,7-substituted octahydropyrrolo-
[1,2-a]pyrazines and octahydro-2H-pyrido[1,2-a]pyrazines. J. Chem.
Soc., Perkin Trans. 1 1992, 1035-1042.
(15) The IC50 values reported for the assays in Tables 1-7 are based on
an average of at least two separate experiments. The values
determined in the vaccinia assays are referred to as vaccinia in the
tables. The values determined in the tetracycline assay are referred
to as tet in the tables. The selectivity ratios were calculated by
dividing the IC50 from either the ecNOS or bcNOS assay by dividing
the IC50 from the iNOS assay in the same system and is referred to
as e/i or b/i, respectively. For all compounds that had IC50 > 10 µM
in one of the selectivity assays, 10 µM was used in the ratio
calculation.
(16) Bredt, D. S.; Snyder, S. H. Isolation of NOS, a calmodulin-requiring
enzyme. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 682-685.
(17) The structure of the 4-methoxyphenethylamide analogue (instead of
the piperonylamide of 19d) was determined by X-ray crystallography,
and the corresponding stereochemistry of 19d is inferred (PDB entry
2ORP).
(18) Li, H.; Raman, C. S.; Glaser, C. B.; Blasko, E.; Young, T. A.;
Parkinson, J. F.; Whitlow, M.; Poulos, T. L. Crystal structures of
zinc-free and -bound heme domain of human iNOS. J. Biol. Chem.
1999, 274, 21276-21284.
Supporting Information Available: Results from elemental
analysis. This material is available free of charge via the Internet
at http://pubs.acs.org.
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