N-Heterocyclic derived M1 positive allosteric modulators

Article abstract of DOI:10.1016/j.bmcl.2010.01.013

Replacement of a phenyl ring with N-linked heterocycles in a series of quinolone carboxylic acid M₁ positive allosteric modulators was investigated. In particular, a pyrazole derivative exhibited improvements in potency, free fraction, and CNS exposure.

Full text of DOI:10.1016/j.bmcl.2010.01.013

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1334–1337
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-Heterocyclic derived M1 positive allosteric modulators
a
a
a
b
b
Scott D. Kuduk a, , Christina N. Di Marco , Victoria Cofre , Daniel R. Pitts , William J. Ray , Lei Ma ,
Marion Wittmann b, Lone Veng b, Matthew A. Seager b, Kenneth Koeplinger c, Charles D. Thompson c,
George D. Hartman a, Mark T. Bilodeau a
*
a Department of Medicinal Chemistry, Merck Research Laboratories, Sumneytown Pike, PO Box 4, West Point, PA 19486, USA
b Department of Alzheimer’s Research, Merck Research Laboratories, Sumneytown Pike, PO Box 4, West Point, PA 19486, USA
c Department of Drug Metabolism, Merck Research Laboratories, Sumneytown Pike, PO Box 4, West Point, PA 19486, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Replacement of a phenyl ring with N-linked heterocycles in a series of quinolone carboxylic acid M1 posi-
tive allosteric modulators was investigated. In particular, a pyrazole derivative exhibited improvements
in potency, free fraction, and CNS exposure.
Received 3 December 2009
Revised 4 January 2010
Accepted 5 January 2010
Available online 11 January 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
M1
Muscarinic
Allosteric
Modulator
GPCR
Alzheimer’s
Quinolone
Neurotransmission via cholinergic pathways plays a critical role
in both the peripheral and central nervous systems (CNS). In these
systems, acetylcholine is the key neurotransmitter targeting nico-
tinic and metabotropic (muscarinic) receptors. Muscarinic recep-
tors are class A G-protein coupled receptors (GPCR) with five
muscarinic subtypes, designated M1–M5,1,2 of which M1 is most
highly expressed in the hippocampus, striatum, and cortex,3 imply-
ing it may play a central role in memory and higher brain function.
In Alzheimer’s disease (AD) there is a progressive degeneration
of cholinergic neurons in the basal forebrain leading to cognitive
decline.4 One approach to treat the symptoms of AD is the direct
activation of the M1 receptor.5 Toward this goal, a number of
non-selective M1 agonists have shown potential to improve cogni-
tive performance in AD patients, but were clinically inadequate
due to cholinergic side effects thought to be due to activation of
the other muscarinic sub-types via the highly conserved orthoster-
ic acetylcholine binding site.6,7
the potency of 1 led to the identification of biphenyl replacements
such as 2 for the para-methoxybenzyl group.12 While these com-
pounds were improved in terms of in vitro activity, higher plasma
protein binding led to decreased CNS exposure impeding further
in vivo evaluation. This Letter describes efforts to replace the phe-
nyl C-ring via N-linked heterocycles in order to improve the po-
tency, free fraction, and CNS exposure for this class of M1
allosteric modulators.
The chemistry employed to prepare the requisite test com-
pounds is shown in Scheme 1. The quinolone esters 3a–c were pre-
O
N
O
O
N
O
OH
OH
A
A potential strategy to generate selectively for M1 over the other
sub-types is to identify allosteric sites on M1 that are less highly
conserved than the orthosteric site.8,9 Ma et al.10 recently reported
the quinolone carboxylic acid 1 as a selective positive allosteric
modulator of the M1 receptor (Fig. 1).11 Recent efforts to improve
B
OMe
C
1: M1 IP = 660 nM
2: -Increased potency
PB = 96-98%
-Higher protein binding
-Reduced CNS exposure
* Corresponding author.
E-mail address: scott_d_kuduk@merck.com (S.D. Kuduk).
Figure 1.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.013

S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1334–1337
1335
Cl/Br
R1
O
N
O
Y
R1
O
N
O
R1
O
O
OH
X
OEt
OEt
b, c
Y
Br
a
N
H
R2
R2
R2
4a: X, Y = CH
4b: Y = N, Y = CH
4c: X = CH, Y = N
4d: X= N, Y = N
X
X
X
3a: R1 = F, R2 = H
3b: R1 = H, R2 = F
3c: R1 = F, R2 = F
N
Y
Br
Y
Z
6a-r
5a-f
Scheme 1. Reagents and conditions: (a) K2CO3, KI, DMF, rt to 50 °C. (b) Amine, Cs2CO3, CuI, phenanthroline, DMSO, 120 °C. (c) LiOH, dioxane.
pared via a Gould–Jacobs cyclization13 as previously described.
Alkylation of 3a–c with the appropriate halide 4a–d afforded 5a–
Table 1 (continued)
Compd
R1
M1 Pot IPa (nM)
Rat PB
nd
Human PB
f. Subsequent copper iodide mediated N-arylation of the resultant
bromide with the appropriate N–H containing heterocycles fol-
lowed by ester hydrolysis afforded analogs 6a–r.
The SAR data for select analogs is shown in Table 1. Compound
potencies were determined in the presence of an EC20 concentra-
tion of acetylcholine at human M1 expressing CHO cells using cal-
cium mobilization readout on a FLIPR384 fluorometric imaging
plate reader. Plasma protein binding was determined using the
N
N
N
N
N
N
6k
620
nd
N
N
6l
96
96.1
nd
91.6
nd
Table 1
N
M1 Potentiation, rat and human protein binding for compounds 6a–r
6m
6n
228
90
F
O
N
O
OH
98.8
99.0
N
R1
6o
6p
160
150
98.2
96.1
96.9
93.0
Compd
R1
M1 Pot IPa (nM)
Rat PB
99.5
Human PB
99.7
N
N
N
N
N
6a
181
N
N
N
N
6b
6c
94
36
89.8
92.3
88.4
94.3
N
O
6q
6r
2500
500
nd
nd
N
O
6d
6e
6f
2900
290
nd
nd
77.6
88.4
N
O
N
N
N
N
nd
nd
CF3
a
Values represent the numerical average of at least two experiments. Interassay
variability was 30% (IP, nM), unless otherwise noted.
110
88.1
83.8
N
N
equilibrium dialysis method in the presence of rat and human
serum.
6g
6h
140
920
89.8
nd
88.9
nd
Pyrrole derivative 6a provided enhanced M1 potency relative to
1, but was very highly protein bound. A very nice result was noted
with pyrazole 6b however, which gave an M1 IP = 94 nM and a high
free fraction (ꢀ10%) in rat and human plasma. Further SAR on the
pyrazole indicated a strong preference for the 3-position (6c) over
the 5-position (6d) with a methyl group. Larger groups were toler-
ated, but electron withdrawing ones tended to decrease potency as
exemplified by 6e. Imidazole 6f was not as good an M1 potentiator
(M1 IP = 110 nM) as the pyrazole, but did provide a substantial free
fraction. The SAR on the imidazole showed a phenyl group (6i) was
the most active, but lost all benefits in terms of decreased protein
binding.
N
N
N
N
6i
6j
39
99.1
nd
98.8
nd
Ph
N
250

1336
S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1334–1337
Table 2
A number of benzo-fused heterocycles were also examined (6j–
Passive permeability and P-gp effects for selected compounds
p). Among them, benzimidole 6l provided a nice balance of M1
activity (IP = 96 nM) and free fraction (ꢀ9% in human). Aza-indoles
6n–p were all more potent than the parent indole 6j (M1
IP = 250 nM), but the protein binding was greatly affected by the
relative position of nitrogen atom. Lactam 6q and oxazolidinone
6r were less potent than the other simple heterocycles, although
substantial free fraction was observed with 6r.
In order to further evaluate compounds for their CNS exposure
potential, key compounds were evaluated for their passive perme-
ability (Table 2). In addition, since P-gp is considered the major ef-
flux transporter at the blood–brain barrier (BBB) responsible for
the efflux of a number of xenobiotic substances from the CNS, com-
pounds were examined for their potential as substrates for this
transporter.
Pyrazole 6b was not a substrate for human P-gp (2.0), but was
for rat (5.1). In fact, this was a trend observed for all of these N-het-
erocyclic derivatives which presented higher rat efflux ratios rela-
tive to human. The 3-methyl pyrazole 6c had improved
permeability, but higher efflux. Interestingly, imidazoles 6f–g had
poor permeability, but this could be modulated by incorporating
a highly lipophilic phenyl group (6i). Azaindole 6o was a P-gp sub-
strate with poor permeability, but regioisomer 6p, improved these
properties, with the exception of rat P-gp. Oxazolidinone 6r was
also a substrate with poor Papp.
All compounds in Tables 1 and 2 possessed a fluorine at the 5-
position on the A-ring. Variable effects have been noted on po-
tency, permeability, P-gp, and CNS availability depending on both
the number of fluorines and their relative positions on the A-ring.14
Accordingly, the 5,8-di-fluoro (7) and 8-F (8) variants of pyrazole
6b were prepared and evaluated (Table 3). Pyrazole 6b had limited
brain exposure in rat as measured by the CSF to unbound plasma
ratio of 0.05, which may be in part due to the susceptibility of this
compound as a rat P-gp substrate. CSF is viewed as a surrogate for
free brain levels compared to total drug level in the brain homog-
enate. The addition of the 8-fluorine in 7 led to a modest reduction
in M1 activity and free fraction, but did improve permeability and
brain exposure despite still having some degree of rat P-gp suscep-
tibility. The single fluorine containing analog 8 was similarly po-
tent to 6b, but with markedly higher protein binding. Thus, the
5-fluorine enhanced free fraction while the 8-fluorine has a role
in improving the permeability.
Compd
R1
Pappa
MDR1b
MDR1ab
5.1
N
N
N
N
N
N
6b
17
2.0
6c
6f
30
5
4.4
4.6
16.7
9.8
N
N
6g
6i
9
1.0
0.8
3.9
5.7
N
N
N
24
Ph
6o
6
15.9
17
N
N
6p
6r
23
7
1.7
7.8
11.8
8.7
N
O
N
O
a
Passive permeability (10À6 cm/s).
MDR1 (human) and and MDR1a (rat) Directional Transport Ratio (B to A)/(A to
b
B). Values represent the average of three experiments and interassay variability was
20%.
tiator. As can be seen from Figure 2, in the presence of 1 lM of
potentiator, a left-shift of 46-fold was observed in the acetylcho-
line dose response showing 7 is a potent positive allosteric modu-
lators of the human M1 receptor. It should be noted that some
degree of agonism is observed at 10 lM and higher concentrations.
We have previously shown that incorporation of a pyridine or
pyridazine ring in place of the B-ring phenyl in the biaryl series
maintains good M1 receptor activity and augments the free frac-
Compound 7 was evaluated for the ability to fold potentiate a
dose response of acetylcholine with a fixed concentration of poten-
Table 3
Evaluation of substituted A-rings
R2
O
N
O
OH
5
8
R1
N
N
R1
R2
M1 IP (nM)
Rat PB
Human PB
Pappa
MDR1b
MDR1ab
B/Pc
CSF/Uplasma
d
Compd
6b
7
8
H
F
F
F
F
H
94
171
96
89.8
94.9
98.3
88.4
95.7
98
17
29
—
2.0
1.4
—
5.1
4.1
—
0.04
0.03
—
0.05
0.1
—
a
b
c
Passive permeability (10À6 cm/s).
MDR1 Directional Transport Ratio (B to A)/(A to B). Values represent the average of three experiments and interassay variability was 20%.
Sprague–Dawley rats. Oral dose 10 mg/kg in 0.5% methocel, Interanimal variability was less than 20% for all values.
Determined using rat plasma protein binding from Tables 2 and 3.
d

S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1334–1337
1337
concentration was required for efficacy. Additional in vivo evalua-
tion of pyrazole 7 will be the subject of a pending manuscript.
In summary, the synthesis and SAR of N-heterocyclic containing
quinolone carboxylic acid M1 positive allosteric modulators has
been detailed in an effort to identify potent compounds with re-
duced plasma protein binding. A number of N-linked heterocycles
were found to be acceptable replacements for the phenyl C-ring,
but many of them presented lower free fractions or were sub-
strates for P-gp. A pyrazole in the form of compound 7 retained
good potency, showed improved free fraction, and was not a hu-
man P-gp substrate. Limited brain exposure was observed as 7
was a rat P-gp substrate. In spite of this, pyrazole 7 showed efficacy
in a mouse model of cognition at lower plasma levels relative to
previously reported quinolone carboxylic acids.
125
100
75
Vehicle
0.01µM of 7
0.1µM of 7
1µM of 7
10µM of 7
100µM of 7
50
25
0
-12 -11 -10 -9 -8 -7 -6 -5 -4
Log[M], Acetylcholine
-25
Figure 2. Fold potentiation plot for compound 7.
Table 4
Evaluation of B-ring pyridine or pyridazine incorporation
Supplementary data
O
O
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.01.013.
OH
N
F
References and notes
X
N
Y
N
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Compd
X
Y
M1 Pot IPa (nM)
5. Langmead, C. J. Pharmacol. Ther. 2008, 117, 232.
6b
9
10
11
CH
N
CH
N
CH
CH
N
96
1400
864
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N
24,000
a
Values represent the numerical average of at least two experiments. Interassay
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tion.12 Accordingly, this strategy was investigated utilizing a C-ring
pyrazole as shown in Table 4. Interestingly, incorporation of a 2-
pyridine (9), 3-pyridine (10), and pyridazine (11) B-rings led to
dramatic decreases, 9–240-fold, in functional activity relative to
6b showing the previously reported biaryl SAR did not translate
to this N-heterocyclic series.
Due to the high potency, good free fraction, and relative degree
of brain penetration, pyrazole 7 was chosen for further evaluation
for performance in a mouse contextual fear conditioning assay,
which serves as a model of episodic memory. In this model, mice
were treated with scopolamine before introduction to a novel envi-
ronment to block association with a novel environment. Mice trea-
ted with 10 mpk of 7 (dosed ip) exhibited a significant reversal
compared to mice treated with scopolamine alone (see Supple-
mentary data). The corresponding plasma levels were ꢀ9
lM, a
significant improvement over compound 1, where ꢀ33 M plasma
l