Pre-clinical characterization of aryloxypyridine amides as histamine H3 receptor antagonists: Identification of candidates for clinical development

Article abstract of DOI:10.1016/j.bmcl.2010.05.041

The pre-clinical characterization of novel aryloxypyridine amides that are histamine H₃ receptor antagonists is described. These compounds are high affinity histamine H₃ ligands that penetrate the CNS and occupy the histamine H₃ receptor in rat brain. Several compounds were extensively profiled pre-clinically leading to the identification of two compounds suitable for nomination as development candidates.

Full text of DOI:10.1016/j.bmcl.2010.05.041

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4210–4214
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pre-clinical characterization of aryloxypyridine amides as histamine H3
receptor antagonists: Identification of candidates for clinical development
*
Michael A. Letavic , Leah Aluisio, John R. Atack, Pascal Bonaventure, Nicholas I. Carruthers,
Christine Dugovic, Anita Everson, Mark A. Feinstein, Ian C. Fraser, Kenway Hoey, Xiaohui Jiang,
John M. Keith, Tatiana Koudriakova, Perry Leung, Brian Lord, Timothy W. Lovenberg, Kiev S. Ly,
Kirsten L. Morton, S. Timothy Motley, Diane Nepomuceno, Michele Rizzolio, Raymond Rynberg,
Kia Sepassi, Jonathan Shelton
Johnson & Johnson Pharmaceutical Research & Development L.L.C., 3210 Merryfield Row, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The pre-clinical characterization of novel aryloxypyridine amides that are histamine H3 receptor antag-
onists is described. These compounds are high affinity histamine H3 ligands that penetrate the CNS and
occupy the histamine H3 receptor in rat brain. Several compounds were extensively profiled pre-clinically
leading to the identification of two compounds suitable for nomination as development candidates.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 26 March 2010
Revised 11 May 2010
Accepted 12 May 2010
Available online 16 May 2010
Keywords:
Histamine
Histamine H3
There have been many reports over the past few years demon-
strating that histamine H3 receptors play a pivotal role in regula-
tion of the sleep-wake cycle and that histamine H3 antagonists
should have therapeutic utility for the treatment of a variety of
conditions, including ADHD, narcolepsy and potentially Alzhei-
mer’s disease, among other conditions. In fact, several pharmaceu-
tical companies have reported initiation of clinical trials for these
indications and numerous reviews covering the topic have ap-
peared in the recent literature.1–6
Some of the structures of histamine H3 antagonists that are in
clinical trials, or that were in the clinic at one time, are shown in
Figure 1. These include the Bioprojet compound BF2.649 (1, tiproli-
sant), a benzoazepine from GSK (2) and the aminopyrrolidine 3 (A-
331440) from Abbott. Tiprolisant is reported to be in Phase II trials
for the treatment of narcolepsy, epilepsy and Parkinson’s disease
and compound 2 is also in Phase II trials for narcolepsy.2–6
As part of our efforts to discover novel histamine H3 antagonists
we now report on a series of high affinity aryloxypyridine amides
that have drug-like properties and pre-clinical profiles amenable
for the selection of potential clinical candidates.
yl)benzylamines9 as represented by structures 4–6, among others
(Fig. 2). We have also described our efforts towards the identifica-
tion of benzyl amine-based compounds, such as 7, that are dual
histamine H3 antagonists/serotonin transporter (SERT) inhibitors
with in vivo efficacy at both targets.10 During the course of these
latter studies, we prepared numerous pyridine-linked compounds
as represented by 8 (Fig. 3).11
We immediately recognized that compound 8 and related ana-
logs represented a possible new chemotype for optimization of his-
tamine H3 affinity as 8 had moderate affinity for the H3 receptor
and no affinity for SERT.
Also, based on our work in the benzyl amine series, we realized
that these compounds were likely not optimized for H3 affinity. For
instance, in the benzyl amine series diazepane is preferred over
O
H
N
N
O
N
N
O
2
NC
Over the past several years we have reported on a number of
histamine H3 antagonist chemotypes including propyloxypiperi-
dines,7 2-aryloxymethyl-morpholines,8 and (4-aminobutyn-1-
1
Cl
O
N
N
3
* Corresponding author.
E-mail address: mletavic@prdus.jnj.com (M.A. Letavic).
Figure 1. Literature histamine H3 antagonists.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.041

M. A. Letavic et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4210–4214
4211
R2
O
N
X
N
O
N
(a)
(b)
n
O
N
O
N
N
O
N
O
N
R1
Br
R1
Br
O
X=Br or F
N
16
9
Scheme 1. Synthesis of compounds 9. Reagents and conditions: (a) phenol, K2CO3,
DMF, 90 °C, 18 h, 90–97%; (b) amine, THF, DBU, Hermann’s catalyst12, t-Bu3PHBF4,
Mo(CO)6, 125 °C, microwave, 6 min, ꢀ50%.
4, hH3 Ki=0.97 nM
5, hH3 Ki=1.9 nM
Cl
Cl
N
R2
Br R2
(b)
O
Br
O
N
N
N
(a)
N
n
n
H
N
N
N
N
N
HO
HO
O
R1
N
N
N
N
N
O
O
O
O
10
6, hH3 pKi=8.93
7, hH3 Ki=2.4 nM
hSERT Ki=6.3 nM
R2
R2
N
N
(a)
(b)
Br
Figure 2. Literature histamine H3 antagonists and H3/SERT ligands.
R1
N
O
N
Br
O
O
O
11
Cl
Cl
Scheme 2. Synthesis of compounds 10–11. Reagents and conditions: (a) amine,
EDCI, HOBt, (i-Pr)2NEt, DCM, 23 °C, 18 h, 25–90%; (b) phenol, Cs2CO3, DMA, 120 °C,
18 h, 23–60%.
R1
R2
O
N
N
n
N
O
N
N
N
CN
R2
N
R2
N
R1= halo, cyano, SMe, H
R2= alkyl, cycloalky, H
O
O
9-15
N
N
N
8, hH3 Ki=49 nM
rSERT Ki >10000 nM
(a)
(c)
(b)
(d)
N
N
N
N
O
O
R1
Br
Br
O
O
Br
Br
O
O
O
O
OH
Figure 3. H3/SERT ligands and novel histamine H3 ligands.
14
R2
N
R2
N
piperazine.10 In addition, the cyano substituent may not be re-
quired for H3 affinity since both the cyano and benzyl amine sub-
stituents are tolerated. Thus, we proposed several series of
isomeric aryloxy-pyridine amides (9–15) as potential histamine
H3 antagonists. These compounds utilize the amide that was pre-
ferred for H3 affinity in 7 and lack the cyano moiety in 8 while
retaining the pyridine linker present in 8. We now report our pro-
gress towards the identification of compounds from these series
that are suitable for progression into early development.
The procedures used to prepare the desired compounds are
shown in Schemes 1–3. Compounds 9 (Scheme 1) were easily pre-
pared by addition of a phenol to either 5-bromo-2-fluoro-pyridine
or 2,5-dibromopyridine which produced the 5-bromo-2-aryloxy-
pyridines 16 in acceptable yields. The 5-bromo-2-aryloxypyridines
16 were then converted directly to compounds 9 via an aminocarb-
onylation reaction.11,12
N
N
N
R1
OH
15
Scheme 3. Synthesis of compounds 14–15. Reagents and conditions: (a) oxalyl
chloride, DMF, DCM, 23 °C, 1 h, then amine, Et3N, 18 h, 26–58%; (b) phenol, Cs2CO3,
DMA, 200 °C, 2 h, microwave, 63–91%; (c) amine-HCl, bromotripyrro-lidinophos-
phonium hexaflurophosphate, (i-Pr)2NEt, DCM, 18 h, 26-58%; (d) phenol, Cs2CO3,
DMA, 200 °C, 3 h, microwave, 63–91%.
ally tolerated, however the position of the aryloxy ring relative to
the amide, and the position of nitrogen in the pyridine ring do have
a significant effect on H3 affinity. It appears that compounds 11
have substantially decreased affinity as compared to compounds
9–10, and 12–14.
Compounds 10 and 11 were prepared as shown in Scheme 2 uti-
lizing a simple amino acid coupling followed by addition of a phe-
nol to the resulting 5- or 6-bromopyridine-2-carboxylic acid
amide. The phenylsulfanylpyridines 12 and 13 were made using
the same methods, substituting the appropriate benzenethiol for
the phenol.
Several compounds were chosen for an abbreviated in vivo
screen based on their affinity for the H3 receptor and/or prelimin-
ary microsomal stability data. Typically, the compounds were
screened for affinity at the rat histamine H3 receptor13 and for sta-
bility in the presence of human and rat liver microsomes either
prior to, or in some cases concurrent with, dosing in a rat.
Our strategy was designed to very quickly identify compounds
with acceptable brain exposures following oral administration. In
order to do this, compounds were dosed po in rats then plasma
and brain concentrations, as well as ex vivo receptor occupancy,
were obtained at 1, 3 and 6 h post dose. This allowed for a determi-
nation of relative exposure in the plasma following a po dose, thus
giving a crude estimate of %F. The results of these studies are
shown in Table 3. Some, but not all, of the compounds have lower
affinity for the rat histamine H3 receptor than the human receptor
and typically the compounds are less stable in rat liver microsomes
than in human liver microsomes. However, the rat brain and plas-
ma concentrations (shown at 1 h) do not necessarily correlate with
microsomal stability in rat. Even so, these experiments allowed us
to identify compounds that efficiently penetrate the CNS, occupy
R2
S
R2
S
N
N
n
n
N
N
N
R1
R1
N
O
O
12
13
Scheme 3 details the synthesis of the 5-phenoxy-nicotinamides 14
and the 4-phenoxy-pyridine-2-carboxylic acid amides 15. The pro-
cedures are similar to those described previously.
Tables 1 and 2 show representative human histamine H3 bind-
ing affinities and pA2’s for the compounds that were prepared in
this study.13 These data clearly show that diazepane (9n–9ab) is
preferred over piperazine (9a–9j) and that cyclic or branched alkyl
groups are preferred (i.e., 9k–9m vs 9n and 9q) as substituents on
the diazepane. Substituents on the aryloxy phenyl ring are gener-

4212
M. A. Letavic et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4210–4214
Table 1
Table 3
Binding and functional data for the human H3 receptor for compounds 9–10 and 12–13
Binding and functional data for the rat H3 receptor, RLM and HLM stability, and
in vivo data for select compounds 9–10, 12
b
Compds
R1
R2
n
Human H3 Ki (nM)a
Human H3 pA2
Compds
r.b. H3 Kia
Rat H3 pA2
RLMc
(%)
HLMc
(%)
R.O.d
Concne
b
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
9q
9r
9s
9t
9u
9v
9w
9x
9y
3,4-Cl
4-MeS
H
3-CN
4-F
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
c-Pr
i-Pr
i-Pr
i-Pr
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
78 28
230 23
260 76
110 39
79 10
(nM)
9p
9q
9s
9u
9w
9x
9y
9aa
10e
12b
21
23
11
15 (n = 2)
10 (n = 1)
75 (n = 2)
15
23
24 (n = 2)
37 (n = 1)
2
8
9
8.38
8.45
8.02
8.43
8.16
7.94
8.30
8.40
7.58
86
63
87
90
4
41
58
63
56
7
99
87
99
99
30
70
98
95
95
82
88
94
87
n.d.
100
n.d.
96
95
87
1.96/0.36
2.65/1.48
0.96/6.02
0.30/0.21
4.79/1.52
0.30/0.31
1.72/0.63
2.62/4.47
2.37/1.02
0.12/0.03
4-F
430 86
4-Cl
3-Cl
2-Cl
2-Cl
4-F
4-F
4-F
4-F
3-F
3-F
4-F
3-CN
3-CN
H
73
100 48
31
40 10
9000
860 210
9
8
4
9
c-Pent
H
Me
n.d.
Et
34
25
16
3
3
6
a
Except where indicated values are the means of at least three experiments in
c-Pr
c-Pr
c-Bu
c-Bu
c-Pr
c-Bu
c-Pr
c-Bu
i-Pr
c-Bu
c-Pr
c-Bu
c-Pr
c-Bu
c-Bu
i-Pr
c-Pr
c-Bu
c-Bu
c-Bu
c-Bu
c-Bu
c-Bu
c-Bu
c-Bu
c-Bu
8.80
triplicate. r.b. is rat brain. Ki SD is reported.
b
The result of a single experiment.
Percent remaining after 30 min in rat liver microsomes (RLM) or human liver
4.1 0.8
1.4 0.1
9.40
9.42 0.10
8.53
c
microsomes (HLM).
12
1.8 0.7
10
4.4 0.5
18
7.6 2.7
17
1.0 0.4
8
d
Receptor occupancy (R.O.) at 1 h after a 10 mpk po dose.
Brain/plasma concentrations (lM) at 1 h after a 10 mpk po dose.
9.39 0.10
e
5
H
9.57
9.26
9.06
9.22
9.51
3,4-Cl
3,4-Cl
3-Cl
3-Cl
4-Cl
4-Cl
2-Cl
3,4-Cl
3,4-Cl
4-Cl
3-Cl
4-F
3-F
2-F
H
4-F
H
4-F
5
Table 4
Pre-clinical PK data for select compounds 9 and 10ea
4
9z
13
59
17
5
4
3
Compds
Cl (mL/min/kg)
Vss (L/kg)
t1/2 (h)
%F
9aa
9ab
10a
10b
10c
10d
10e
10f
10g
12a
12b
13a
13b
Rat
9q
9s
49
41
3.5
1.4
4.2
5.0
3.0
4.5
1.2
0.7
0.8
1.0
1.3
1.0
50
89
11
19
62
23
1.5 0.3
10
2
9p
9y
9aa
10e
Canine
9q
118
104
36
5.5 3.5
4.7 6.6
2.9 0.9
5.5 1.0
4.3 3.7
8.61
8.72
8.85 0.55
80
9.27
35
21
35
7.2
1.6
3.6
2.6
0.9
1.9
28
29
14
3.7
1
9s
4.6 0.7
2.3 0.2
10e
Mouse
9q
9.48
9.48
29
8
94
97
8.7
4.4
1.1
0.8
28
21
a
9s
Ki’s are the mean of at least three experiments in triplicate. Ki SD is reported.
b
The result of a single experiment, unless SD is shown, then it is the mean of
a
For rat (Sprague–Dawley) and mouse (Balb-C), the compounds were dosed
1 mg/kg i.v. and 10 mg/kg po, for dog (beagle) the doses were 1 mg/kg i.v. and 5 mg/
kg po.
three experiments.
Table 2
PK for these compounds are shown in Table 4. All three compounds
have high clearance and moderate bioavailability in canine. In vitro
cardiovascular safety assessment (Table 5) revealed that although
none of the compounds bound efficiently to the hERG channel, as
measured by displacement of astemizole, the compounds tested
variably had significant activity in a functional patch clamp assay.
Based on the patch clamp data 9aa, 9y, and 10e were not pursued
further.
Binding and functional data for the human H3 receptor for compounds 11 and 14–15
R1
R2
Human H3 Ki (nM)a
Human H3 pA2
b
Compds
11a
11b
11c
14a
14b
15a
15b
3,4-Cl
3,4-Cl
3,4-Cl
H
4-F
H
i-Pr
430 130
1040 180
160 55
11 10
c-Pr
c-Bu
c-Bu
c-Bu
c-Bu
c-Bu
17 17
8.87
41
5.7
3
3
All of the compounds shown in Table 5 were also screened in a
commercial panel of 50 receptor, ion channel and transporter as-
says (CEREP, www.cerep.com) and none had any significant affinity
4-F
a
Ki’s are the means of at least three experiments in triplicate. Ki SD is reported.
The result of a single experiment.
b
(at 1 lM) for any of the targets that were screened.
After evaluating all the data we had at this time compounds 9q
and 9s were chosen for additional pre-clinical evaluation.
the histamine H3 receptor in a rat brain, and that are relatively sta-
ble to microsomes, thus allowing us to choose compounds for more
thorough in vivo evaluation.
Table 5
The more interesting compounds shown in Table 3 were then
evaluated in more detailed rat PK studies. The results of these
experiments are shown in Table 4.
In vitro CV safety assessment for select compounds 9 and 10e
Compds
hERG binding IC50 (%)
M/%inhib@10 M)
Patch clamp IC50 (lM)
(
l
l
Typically, the compounds have rather high clearance in rat and
moderate to high volumes of distribution. Compounds 9q, 9s and
9aa have high bioavailability in rat.
Balancing microsomal stability as well as rat plasma and brain
concentrations and receptor occupancy, several compounds were
chosen for further profiling. These included 9q, 9s and 10e. Canine
9q
9s
9p
9y
9aa
10e
>10/0
>10/23
>10/0
>10/28
>10/23
8.4/53
3.5
>10
5.4
1.6
1.4
0.9

M. A. Letavic et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4210–4214
4213
Table 6
Permeability, solubility and physical properties of 9q and 9s
110
100
90
80
70
60
50
40
30
20
10
0
Compds
9q
9s
Caco-2 A to B
Caco-2 B to A
Solubility
SGFa,b
78 Â 10À6 cm/s
77 Â 10À6 cm/s
36 Â 10À6 cm/s
25 Â 10À6 cm/s
>5 mg/mL
>5 mg/mL, pH 3.82
369.44
8.15
2.08
>1 mg/mL
>100 mg/mL, pH 5.49
376.46
8.01( 0.04)
1.38
Waterb
9q
9s
MW
c
pKa
c
Log D7.4
a
b
c
For 9q aqueous HCl at pH 1, for 9s 0.2% NaCl in 0.1 N HCl.
For the HCl salt.
Measured by Analiza (www.analiza.com).
0
1
10
100
1000
10000
Plasma Concentration (ng/mL)
Both compounds were found to be permeable, did not show the
Figure 4. Ex vivo H3 receptor occupancy with 9q or 9s in rat striatum: dose
dependency after oral administration (t = 30 min for 9q or 60 min for 9s). Results
are presented as% receptor occupancy versus vehicle treated rats (each data point
represents an individual animal). Ex vivo autoradiography was performed as
potential for efflux (Caco-2 data, Table 6) and both were suffi-
ciently soluble in simulated gastric fluid and in water as the HCl
salts. Neither compound showed any CYP inhibition (in vitro
previously described using [3H]-R- -methylhistamine.13
a
microsome data for 3A4, 2D6 and 2C9 at 10 lM) and both com-
pounds met pre-clinical developability criteria with regards to
physical properties including log D7.4 and molecular weight. It is
notable that the brain to plasma ratios (Table 2) for 9q and 9s
are quite different (1.8 vs 0.16). This difference is possibly attrib-
uted to differences in Vss, (3.5 L/kg vs 1.4 L/kg in rat, Table 4) and
correlates with log D7.4 (2.08 vs 1.38).
Figure 4 shows the plasma drug concentration-response curves
for receptor occupancy in a rat for 9q and 9s.13 These data demon-
strate that the plateau for maximal receptor occupancy (ꢀ90%) for
9q was reached at 3 mg/kg po corresponding to a plasma concen-
tration of 150 ng/mL. Likewise, 9s had maximal receptor occu-
Vehicle
9q 3 mg/kg SC
9q 1 mg/kg SC
A
B
600
*
30000
20000
10000
0
9q 0.3 mg/kg SC
500
400
300
200
100
0
*
-120 -60
0
60
120 180 240 300
Time Post Dose (min)
Vehicle
C
D
9s 3 mg/kg PO
9s 10 mg/kg PO
9s 30 mg/kg PO
600
500
400
300
200
100
0
**
50000
40000
30000
20000
10000
0
*
*
-90 -60 -30
0
30 60 90 120 150 180
Time Post Dose (min)
Figure 5. (A and B) Effects of 9q (0.3, 1.0 and 3.0 mg/kg s.c.) and 9s (3, 10 and 30 mg/kg po) on in vivo extracellular levels of histamine in the frontal cortex of freely moving
rats (AP = À3.2 mm, ML = 0.8 mm, V = À5.0 mm from bregma and dura). All rats were administered the drugs after measuring stable baseline. Histamine levels were
measured using high performance liquid chromatography with mass spectroscopy (HPLC MS/MS).14 Data represents the means SEM, n = 4–6 animals. (A and C) Area under
the curve values of the data presented in panel B and D. p* < 0.05, **p < 0.01 by one way ANOVA and Newman–Keuls multiple comparison post hoc test versus vehicle treated
rats.

4214
M. A. Letavic et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4210–4214
Wake Minutes / 2 Hours
Wake Minutes / 2 Hours
A
B
100
80
60
40
20
0
100
80
60
40
20
0
Vehicle
9q
Vehicle
9s
***
***
1-2
3-4
5-6
1-2
3-4
5-6
Time Interval (2hrs.)
Time Interval (2hrs.)
Figure 6. (A and B) Effects of 9q (10 mg/kg s.c., n = 7) and 9s (30 mg/kg s.c., n = 6) on wake duration (min., mean SEM) during the first 6 h following the administration at the
beginning of the light phase. ***p < 0.01 v. vehicle as determined by two way ANOVA followed by Bonferroni post hoc analysis.
pancy (again, ꢀ90%) at 1 mg/kg po corresponding to a plasma con-
centration of 160 ng/mL. It is interesting to note that, because of
the differences in brain to plasma ratios, this calculation implies
that much lower brain concentrations are required for occupancy
with 9s as compared to 9q. This may be explained in part by the
differences in plasma protein binding for the two compounds.
Compound 9s has a free fraction of 79% in rat whereas compound
9q has a free fraction of only 15% in rat.
Considering that both compounds have 10-fold lower affinity
for the rat receptor as compared to the human receptor (pA2’s,
Tables 1 and 3), the expected plasma concentration required for
human receptor 90% occupancy is estimated at ꢀ15 ng/mL, assum-
ing similar brain to plasma ratios in rat and human. Next, microdi-
alysis experiments were performed on 9q and 9s in rat.
we have demonstrated acceptable PK in three pre-clinical species
for both compounds. Based on this work we identified 9q and 9s
as potential clinical candidates. Additional toleration studies and
baboon PET studies are ongoing in order to support future human
clinical trials.
References and notes
1. Gemkow, M. J.; Davenport, A. J.; Harich, S.; Ellenbroek, B. A.; Cesura, A.; Hallett,
D. Drug Discovery Today 2009, 14, 509.
2. Esbenshade, T. A.; Browman, K. E.; Bitner, R. S.; Strakhova, M.; Cowart, M. D.;
Brioni, J. D. Br. J. Pharmacol. 2008, 154(6), 1166.
3. Sander, K.; Kottke, T.; Stark, H. Biol. Pharm. Bull. 2008, 31(12), 2163.
4. Stocking, E. M.; Letavic, M. A. Curr. Top. Med. Chem. 2008, 8(11), 988.
5. Letavic, M. A.; Barbier, A. J.; Dvorak, C. A.; Carruthers, N. I. Prog. Med. Chem.
2006, 44, 181.
6. Celanire, S.; Wijtmans, M.; Talaga, P.; Leurs, R.; de Esch, I. J. P. Drug Discovery
Today 2005, 10, 1613.
7. Apodaca, R.; Dvorak, C. A.; Xiao, W.; Barbier, A. J.; Boggs, J. D.; Wilson, S. J.;
Lovenberg, T. W.; Carruthers, N. I. J. Med. Chem. 2003, 46, 3938.
8. Letavic, M. A.; Keith, J. M.; Ly, K. S.; Bonaventure, P.; Feinstein, M. A.; Lord, B.;
Miller, K. L.; Motley, S. T.; Nepomuceno, D.; Sutton, S. W.; Carruthers, N. I.
Bioorg. Med. Chem. Lett. 2008, 18, 5796.
Figure 5 shows the increase in histamine release in the rat frontal
cortex following s.c. administration of 9q and following po adminis-
tration of 9s versus time. The concentrations of 9q obtained in a sim-
ilar experiment at 3 mpk s.c. in the brain and plasma were 0.956
lM
and 0.257 M, respectively and the concentration of 9s in the rat
l
brain and plasma at 10 mpk is listed on Table 3. Both compounds
are linear with dose. Both compounds significantly increased the
levels of histamine in the frontal cortex, a result which is consistent
with antagonism of the histamine H3 receptor in rat. Finally, EEG
studies were also performed on 9q and 9s in rat in order to confirm
wake promoting effects.9 Figure 6 shows the total wake duration
for each compound. Both compounds showed a significant increase
in wake duration following s.c. administration of doses that resulted
in maximal receptor occupancy.
In conclusion, we have designed and prepared a series of aryl-
oxypyridine amides and demonstrated that select members of this
series are high affinity histamine H3 antagonists. We have also
demonstrated that these compounds are highly selective for the
histamine H3 receptor and that several compounds readily pene-
trate the rat brain and occupy the histamine H3 receptor following
oral administration. Finally, compounds 9q and 9s occupy the H3
receptor at low plasma concentrations, increase histamine release
in rat frontal cortex and promote wake in rat. Because these
compounds have higher affinity for the human H3 receptor, it is ex-
pected that around 10-fold lower plasma concentrations will be re-
quired in human brain H3 receptor occupancy compared to rat. We
have also demonstrated that compounds 9q and 9s have physical
properties consistent with good absorption and distribution and
9. Dvorak, C. A.; Apodaca, R.; Xiao, W.; Jablonowski, J. A.; Bonaventure, P.;
Dugovic, C.; Shelton, J.; Lord, B.; Miller, K.; Dvorak, L. K.; Lovenberg, T. W.;
Carruthers, N. I. Eur. J. Med. Chem. 2009, 44, 4098.
10. Ly, K. S.; Letavic, M. A.; Keith, J. M.; Miller, J. M.; Stocking, E. M.; Barbier, A. J.;
Bonaventure, P.; Lord, B.; Jiang, X.; Boggs, J. D.; Dvorak, L.; Miller, K. L.;
Nepomuceno, D.; Wilson, S. J.; Carruthers, N. I. Bioorg. Med. Chem. Lett. 2008, 18,
39.
11. Keith, J. M.; Letavic, M. A.; Ly, K. S.: Mani, N.; Mills, J.; Villani, F.; Zhong, H. M.
U.S. 20070281923.
12. Letavic, M. A.; Ly, K. S. Tetrahedron Lett. 2007, 48, 2339.
13. The affinity of test compounds for the human recombinant H3 receptor was
determined as described in Ref. 8. Functional data (human pA2 data) and all rat
data was determined as described in Barbier, A. J.; Berridge, C.; Dugovic, C.;
Laposky, A. D.; Wilson, S. J.; Boggs, J.; Aluisio, L.; Lord, B.; Mazur, C.; Pudiak, C.
M.; Langlois, X.; Xiao, W.; Apodaca, R.; Carruthers, N. I.; Lovenberg, T. W. Br. J.
Pharmacol. 2004, 143, 649. Under the assay conditions used (no constitutive
histamine H3 activity) we are unable to distinguish between antagonists versus
inverse agonists.
14. A Supelco Discovery HS F5, 2.1 Â 100 mm, 3
lm column was used with a
mobile phase containing a 0.1% HCO2H in H2O (solvent A) and 0.1% HCO2H in
H3CCN (solvent B). The mobile phase was held on the B:A ratio of 100:0 for
1.5 min. The linear gradient was changed over the next 0.5 min to B:A ratio of
5:95 and held for 2.5 min, then returned to the starting conditions. The total
run time was 6.5 min and flow rate was 0.6 mL/min (Shimadzu LC-10AD VP
with SCL-10A VP system controller). Tandem mass spectrometric (MS/MS)
detection was carried out on a PE Sciex API4000 in the positive ion mode (ESI)
by multiple reaction monitoring (MH+/daughter was 112.09 95.1 m/z). The
concentration for each sample was calculated from the peak area of the
chromatographic signal and the slope from the corresponding standard curve.