Heterocyclic fused pyridone carboxylic acid M1 positive allosteric modulators

Article abstract of DOI:10.1016/j.bmcl.2010.02.096

The phenyl ring in a series of quinolone carboxylic acid M₁ positive allosteric modulators was replaced with a variety of heterocycles in order to reduce protein plasma binding and enhance CNS exposure.

Full text of DOI:10.1016/j.bmcl.2010.02.096

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2533–2537
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Heterocyclic fused pyridone carboxylic acid M1 positive allosteric modulators
a
a
b
b
b
Scott D. Kuduk a, , Christina N. Di Marco , Ronald K. Chang , William J. Ray , Lei Ma , Marion Wittmann ,
Matthew A. Seager b, Kenneth A. 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:
The phenyl ring in a series of quinolone carboxylic acid M1 positive allosteric modulators was replaced
with a variety of heterocycles in order to reduce protein plasma binding and enhance CNS exposure.
Ó 2010 Published by Elsevier Ltd.
Received 4 February 2010
Revised 24 February 2010
Accepted 25 February 2010
Available online 3 March 2010
Keywords:
M1
Muscarinic
Quinolone
Allosteric
Alzheimer’s
Cholinergic neurons serve essential functions in both the
peripheral and central nervous systems (CNS). Acetylcholine is
the principal neurotransmitter targeting nicotinic and muscarinic
metabotropic receptors. Muscarinic receptors are class A G-protein
coupled receptors (GPCR) widely expressed in the CNS. There are
five muscarinic sub-types, designated M1–M5,1,2 of which M1 is
most highly expressed in the hippocampus, striatum, and cortex,3
implying it may play a central role in memory and higher brain
function.
A common observation in Alzheimer’s disease (AD) is the pro-
gressive degeneration of cholinergic neurons in the basal forebrain
leading to cognitive decline.4 One approach to treat these cognitive
symptoms of AD would be the direct activation of the M1 receptor.5
In this regard, a number of non-selective M1 agonists have shown
potential to improve cognitive performance in AD patients, but
were clinically limited by cholinergic side effects thought to be
the result of activation of other muscarinic sub-types via binding
to the highly conserved orthosteric acetylcholine binding site.6,7
One conduit to stimulate selectively for M1 over the other sub-
types is to target allosteric sites on M1 that are less highly con-
served than the orthosteric site.8,9 It was recently reported that
quinolone carboxylic acid 1 is a selective positive allosteric modu-
lator of the M1 receptor with excellent selectivity for this sub-
type.10,11 Endeavors to improve the potency of 1 led to the
identification of biaryl replacements for the para-methoxybenzyl
group such as biphenyl 2 (Fig. 1).12 While these compounds were
improved in terms of in vitro activity, higher plasma protein bind-
ing led to decreased CNS exposure impeding further in vivo evalu-
ation. Previous SAR efforts on the A-ring showed substitution was
not tolerated, with the exception of fluorination at the 5- and 8-
positions. This Letter, describes efforts to identify heterocyclic
fused A-rings that would retain M1 potency, reduce protein bind-
ing, and improve CNS exposure.
The chemistry used to prepare test compounds is shown in
Scheme 1. The appropriate heterocyclic acid chloride 3 is treated
with potassium 3-(ethoxy)-3-oxopropanoate and MgCl2 to pro-
O
N
O
O
N
O
5
A
8
OH
OH
B
OMe
C
1: M1 IP = 660 nM
2: M1 IP = 212 nM
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 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.02.096

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S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2533–2537
O
O
O
N
O
O
F
O
O
d, e
OEt
NMe2
OH
Cl
OEt
a
b
N
N
N
N
F
F
3
4
5
c
9-15
O
O
O
O
X
X
Z
EtO2C
CO2Et
OEt
X
Z
f, g
OH
h, e
OEt
+
Y
Y
Y
Z
NH2
N
N
H
6
7
8
16-19
Scheme 1. Reagents: (a) Potassium 3-(ethoxy)-3-oxopropanoate, TEA, MgCl2, CH3CN; (b) dimethylformamide diethylacetal, neat, 50 °C; (c) dimethylamino ethyl acrylate,
TEA, toluene, 70 °C; (d) 1-(biphenyl-4-yl)methanamine, K3PO4, DMA, 50 °C; (e) 1 N NaOH, THF, EtOH; (f) 130 °C, neat; (g) diphenylether, 230 °C; (h) 4-(bromomethyl)biphe-
nyl, DMF, K2CO3.
Table 1
duce beta keto ester 4. Subsequent treatment with dimethylform-
amide diethylacetal afforded 5.13 Alternatively, 3 may be converted
directly to 5 in a single pot process using dimethylamino ethyl
acrylate and TEA. Treatment of 5 with 1-(biphenyl-4-yl)methan-
amine followed by ester hydrolysis afforded 9–15.
M1 FLIPR and protein binding data for select compounds
O
O
OH
R1
N
A second approach initiated with the condensation of the
appropriate heterocyclic amine 6 with diethyl ethoxymethylene
malonate 7, and subsequent thermal cyclization afforded 8.14
Alkylation of 8 with 4-(bromomethyl)biphenyl followed by ester
hydrolysis provided 16–19. Analogous compounds were made by
similar methods using modifications previously described.15
Compound potencies were determined in the presence of an
EC20 concentration of acetylcholine at human M1 expressing CHO
cells using calcium mobilization readout on a FLIPR384 fluorometric
imaging plate reader and are presented as the inflection point
(IP).16 The percent max is the response of compound + EC20 acetyl-
choline relative to maximal acetylcholine effect. Plasma protein
binding was determined using the equilibrium dialysis method in
the presence of rat and human serum.
Data for A-ring modified compounds is shown in Table 1. Incor-
poration of a pyridine led to variable effects compared to 2, in
which the 6-pyridyl 10 exhibited a complete loss of activity and
the 5-pyridyl 9 an ꢀ7-fold decrease in activity. The 7- and 8-pyri-
dyl analogs (11–12) lost functional activity; the latter did not exhi-
bit any benefit in terms of reduced protein binding. Diazines 13–15
were even less active with only pyrazine 13 having potency under
Compd R1
M1 Pot IPa
0.21
(lM) % Max Rat PB (%) Human PB (%)
2
92
92
4
98.8
n.d.
n.d.
n.d.
99.5
n.d.
n.d.
n.d.
N
9
10
1.38
>100
0.43
N
N
11a
62
12a
13
0.58
0.78
87.2
5.95
56
96
48
34
99.7
97.7
n.d.
n.d.
99.2
97.7
n.d.
n.d.
N
N
N
1 lM. Thiophenes 16–17 exhibited good M1 functional activity,
N
N
with 16a representing the most potent replacement (M1
IP = 150 nM) with similar protein binding relative to the phenyl
in 2. However, further insertion of a nitrogen into these scaffolds
in the form of thiadiazole 18 or isothiazole 19 lost an additional
ꢀ10-fold activity.
14
N
15
N
S
In light of the relatively good potency observed for the 7- and 8-
pyridyl isomers, and previous experience with N-heterocyclic C-
ring quinolone carboxylic acids,17 hybrid analogs of 11a and 12a
were evaluated as shown in Table 2. Replacement of the C-ring
phenyl with a pyrazole did not improve potency (11b–12b), but
a 3-methylpyrazole was better, particularly in the context of 7-
aza isomer 11c (M1 IP = 250 nM). This compound also had lower
free fraction relative to 8-aza isomer 12c, although a very signifi-
cant species difference was observed. The related 5-methyl analogs
11d–12d were substantially less potent, and imidazoles 11e–12e
were also less active than had been anticipated from experience
with N-aryl derivatives in the quinolone carboxylic acid series.
16a
17
0.15
0.37
3.50
3.35
90
78
69
76
99.2
99.6
n.d.
n.d.
98.7
99.3
n.d.
n.d.
S
N
S
S
18
N
N
19
a
Values represent the numerical average of at least two experiments. Interassay
variability was 30% (IP, M), unless otherwise noted.
l

S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2533–2537
2535
Table 2
M1 FLIPR and protein binding data for select compounds
O
O
O
N
O
OH
OH
N
N
N
R1
R1
Compd
R1
M1 Pot IPa
(l
M)
Rat PB
—
Human PB
—
Compd
R1
M1 Pot IPa
(lM)
Rat PB
99.7
Human PB
11a
0.43
12a
0.58
99.2
11b
11c
1.14
0.25
—
—
12b
12c
0.81
0.56
95.2
100
93.9
98.8
N
N
N
N
N
N
N
N
N
N
N
N
98.9
87.5
11d
11e
13.0
1.03
—
—
—
—
12d
12e
17.9
0.34
—
—
94.8
76.7
N
N
N
N
a
Values represent the numerical average of at least two experiments. Interassay variability was 30% (IP, lM), unless otherwise noted.
Due to the improved potency of thiophene 16a, a number of
imidazole ring. Passive permeability of aniline 16h was the highest
of the group, but was a substrate for P-gp. Thus incorporation of
heteroatoms in the A-ring tended toward compounds with de-
creased permeability and increased P-gp efflux. However, thio-
phene 16d bearing an N-linked pyrazole, was an exception with
a CSF/Uplasma ratio of 0.21.
Based on the high CSF levels of 16d, this compound was exam-
ined in a mouse contextual fear conditioning model of episodic
memory, but had no effect at the doses examined (data not
shown). This was surprising as the homologous quinolone analog
worked in this model,16 and 16d also exhibited good pharmacoki-
netic properties.18
It was found that 16d was a mouse P-gp substrate with a
large efflux ratio of 18. To confirm P-gp affected CNS exposure,
CF-1 wild type and P-gp knock out mice were treated with
16d, and plasma and CSF were taken after 30 min. The wild type
mice gave a low CSF/Uplasma ratio of ꢀ0.05, while the mice lack-
ing P-gp possessed three fold higher CSF/Uplasma ratio of ꢀ0.16,
approaching that observed in rat.19,20 Consequently, P-gp may
contribute to the lack of efficacy observed with 16d in the mouse
CFC model.
In summary, the SAR for A-ring heterocycles in place of the
phenyl ring in quinolone carboxylic acid based M1 positive allo-
steric modulators was examined. Thiophene and aza versions at
the 7- and 8-position were tolerated, while diazines were not.
Incorporation of many of the A-ring heterocycles negatively im-
pacted the passive permeability and conferred increased P-gp
susceptibility. Moreover, for thiophene 16d, P-gp efflux in mouse
likely contributed to a lack of in vivo efficacy. Evaluation of non-
aromatic A-rings in lieu of the phenyl is the subject of the follow-
ing paper.
both N- and C-linked C-ring heterocycles were evaluated and select
examples are shown in Table 3. Pyridine C-rings (16b,c) and N-
linked heterocycles did not confer any potency improvements rel-
ative to 16a, although pyrazole 16d and imidazole 16e did have
very high free fractions. The C-linked pyrazole 16g had similar
activity relative to 16a, but species dependent plasma protein
binding was also noted.
Previous SAR in the quinolone series identified that a pyridine
in the B-ring was an acceptable replacement that could lower pro-
tein binding. Accordingly, analogs 16h–n incorporated a 2-pyridyl
B-ring with a variety of different C-rings. Amongst them, meta-ani-
line 16h (M1 IP = 38 nM) and C-linked pyrazole 16n (M1
IP = 340 nM), looked most promising from a potency and free frac-
tion standpoint. Bi-pyridyls (16i–l) were all less potent than their
arylpyridine counterparts as were other C-ring heterocycles such
as thiophene (16m).
Select compounds with good potency and free fractions were
evaluated for P-glycoprotein transport and brain exposure in
rat. P-glycoprotein (P-gp) is an ATP-driven efflux transporter at
the blood brain barrier (BBB), responsible for the efflux of a num-
ber of xenobiotic substances from the brain. Accordingly, P-gp ef-
flux potential for human (MDR1) and rat (MDR1a), as well as
passive permeability, were evaluated to triage potential
candidates.
As can be seen in Table 4, 4 out of 5 analogs examined had sub-
adequate permeability (<15) and appeared to be substrates for P-
gp, with the exception of thiophene 16d. These poor properties
were consistent with low CNS exposure in rat for 7-pyridyl 11c
and thiophene 16n. Imidazole 16e exhibited particularly low per-
meability (Papp <2) presumably due to the strong basicity of the

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S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2533–2537
Table 3
M1 FLIPR and protein binding data for select compounds
O
N
O
S
OH
R1
Compd
R1
M1 Pot IPa
0.15
(l
M)
Rat PB
99.2
Human PB
98.7
Compd
R1
M1 Pot IPa
0.038
(lM)
Rat PB
90.1
Human PB
88.3
N
N
N
NH2
16a
16h
16b
16c
0.57
0.60
—
—
—
—
16i
16j
1.05
2.0
—
—
—
—
N
N
N
N
OMe
N
N
N
N
N
16d
0.52
86.8
86.0
16k
0.5
—
—
N
Cl
16e
16f
16g
0.24
0.51
0.15
74.1
—
80.1
—
16l
0.81
1.66
0.34
—
—
N
N
N
N
S
16m
16n
—
—
N
N
N
99.1
93.4
90.6
72.1
N
N
N
N
a
Values represent the numerical average of at least two experiments. Interassay variability was 30% (IP, lM), unless otherwise noted.
Table 4
Permeability, P-gp, and bioanalysis of plasma, brain, and CSF levels in rat for selected compounds
Pappa
MDR1b
MDR1ab
Plasma Conc.c (nM)
Brain Conc.c (nM)
CSF Conc.c (nM)
Brain/plasma
CSF/Uplasma
d
Compd
11c
16d
16e
16h
16n
9.0
31
<2
12
5.6
2.4
1.9
—
7.5
4.3
5.3
5.0
—
18.6
7.4
78511
4240
—
—
9415
1201
316
—
—
159
24
123
—
—
42
0.15
0.07
—
—
0.02
0.03
0.21
—
—
0.05
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.
CSF to unbound plasma ratio determined using rat plasma protein binding from Tables 2 and 3.
d
9. For an example of an allosteric activator of the M1 receptor, see: Jones, C. K.;
Brady, A. E.; Davis, A. A.; Xiang, Z.; Bubser, M.; Noor-Wantawy, M.; Kane, A. S.;
Bridges, T. M.; Kennedy, J. P.; Bradley, S. R.; Peterson, T. E.; Ansari, M. S.;
Baldwin, R. M.; Kessler, R. M.; Deutch, A. Y.; Lah, J. J.; Levey, A. I.; Lindsley, C.
W.; Conn, P. J. J. Neurosci. 2008, 41, 10422.
10. Ma, L.; Seager, M.; Wittmann, M.; Bickel, D.; Burno, M.; Jones, K.;
Kuzmick-Graufelds, V.; Xu, G.; Pearson, M.; McCampbell, A.; Gaspar, R.;
Shughrue, P.; Danziger, A.; Regan, C.; Garson, S.; Doran, S.; Kreatsoulas,
C.; Veng, L.; Lindsley, C.; Shipe, W.; Kuduk, S. D.; Jacobsen, M.; Sur, C.;
Kinney, G.; Seabrook, G.; Ray, W. J. Proc. Natl. Acad. Sci. U.S.A. 2009, 106,
15950.
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16. Select compounds were examined in functional assays at other muscarinic
receptors and showed no activity at M2, M3, or M4 receptors.