Quinolizidinone carboxylic acid selective M1 allosteric modulators: SAR in the piperidine series

Article abstract of DOI:10.1016/j.bmcl.2011.01.094

SAR study of the piperidine moiety in a series of quinolizidinone carboxylic acid M₁ positive allosteric modulators was examined. While the SAR was generally flat, compounds were identified with high CNS exposure to warrant additional in vivo evaluation.

Full text of DOI:10.1016/j.bmcl.2011.01.094

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1710–1715
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Quinolizidinone carboxylic acid selective M1 allosteric modulators:
SAR in the piperidine series
Scott D. Kuduk a, , Ronald K. Chang a, Christina N. Di Marco a, William J. Ray b, Lei Ma b,
⇑
Marion Wittmann b, 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:
SAR study of the piperidine moiety in a series of quinolizidinone carboxylic acid M1 positive allosteric
modulators was examined. While the SAR was generally flat, compounds were identified with high
CNS exposure to warrant additional in vivo evaluation.
Received 4 January 2011
Revised 19 January 2011
Accepted 19 January 2011
Available online 28 January 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Allosteric
Modulator
Muscarinic
Quinolizidinone
Cholinergic neurons serve critical functions in both the periph-
eral and central nervous systems (CNS). Acetylcholine serves as the
key neurotransmitter in these systems, targeting both nicotinic
and metabotropic (muscarinic) receptors. Muscarinic receptors
are class A G-protein coupled receptors (GPCR) widely expressed
in the CNS. There are five muscarinic subtypes, designated M1 to
M5,1,2 of which M1 is most highly expressed in the hippocampus,
striatum, and cortex,3 suggesting a central role in memory and
higher brain function.
During the course of Alzheimer’s disease (AD) there is a pro-
gressive degeneration of cholinergic neurons in the basal forebrain
leading to cognitive decline.4 One potential tactic to treat the
symptoms of AD is the direct activation of the M1 receptor.5 As a
result, a number of non-selective M1 agonists have shown poten-
tial to improve cognitive performance in AD patients, but were
clinically limited due to cholinergic side effects thought to be med-
iated via activation of the other muscarinic sub-types thru the
highly conserved orthosteric acetylcholine binding site.6,7
O
N
O
O
N
O
OH
OH
A
B
OMe
C
1: M1 IP = 660 nM
2: M1 IP = 212 nM
-Higher protein binding
-Reduced CNS exposure
PB = 96-98%
O
O
O
O
N
OH
N
N
OH
One approach to engender 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 Ma et al.10 recently reported
the quinolone carboxylic acid 1 as a selective positive allosteric
modulator of the M1 receptor (Fig. 1).11 Attempts to improve the
N
N
CN
CF3
3: M1 IP = 520 nM
4h: M1 IP = 460 nM
-Lower protein binding and increased CNS exposure
⇑
Corresponding author.
E-mail address: scott_d_kuduk@merck.com (S.D. Kuduk).
Figure 1. Quinolone and quinolizidinone leads.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.094

S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1710–1715
1711
Table 1
potency and physicochemical properties of 1 led to a number of
advancements such as potent biphenyl 2.12 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.
M1 FLIPR data for select piperidines
O
O
N
OH
We recently described the identification of quinolizidinone car-
boxylic acids as replacements for the quinolone ring system.13
Inclusion of this motif allowed for the incorporation of basic
amines such as piperazine 3 and piperidine 4 in lieu of a benzylic
group, which led to dramatic improvement of physicochemical
properties and free fraction, leading to enhanced CNS exposure.
This communication describes efforts to optimize the potency
and CNS exposure of piperidine containing M1 positive allosteric
modulators as represented by 4h.
The synthesis of requisite quinolizidinones is shown in Scheme
1. The chemistry commenced with lithiation of 2-picoline 5 with
LDA followed by addition of ethoxymethylene diethylmalonate 6
and subsequent thermal cyclization in refluxing o-xylene to pro-
vide quinolizidinone 7. Vilsmeier formylation at the 1-position
provided the requisite aldehyde 8. Reductive amination of 8 with
the appropriate amines was followed by saponification of the ethyl
ester to afford target N-linked quinolizidinone carboxylic acid
piperidines 4a–z0, and tetrahydroquinolines 9a–y.
N
R1
Compd
R1
M1 Pot IP (
l
M)a
4a
1.3
4b
4c
4d
4e
4f
1.7
2.1
0.77
1.2
1.3
1.7
F
N
F
Cl
Br
CN
A library of amines was employed in the reductive amination
protocol. The SAR data for a representative group of piperidine con-
taining quinolizidinone carboxylic acids is shown in Table 1. Com-
pound 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.
4g
CH3
CF3
Phenyl piperidine 4a was a modest potentiator with an M1
IP = 1.3 l
M.14 Substitution at the para position of the phenyl ring
4h
0.46
(compounds 4b–i) generally presented flat SAR with the aforemen-
tioned 4h proving to be the most potent analog (M1 IP = 460 nM).
The position of the substituent on the ring also had little effect
as movement of fluorine or chlorine to the meta- (4k, 4m) and
ortho-positions (4t) did not provide any significant improvements.
Replacement of phenyl with un-substituted (4n) or substituted
pyridines (4c, j, s) provided similarly flat SAR. Diazines 4o–r were
a notable exception where a significant decrease was noted
relative to the phenyl or pyridine counterparts. N-Linked azoles
(4u–y) proved to be acceptable phenyl alternatives, with benz-
imidazole 4x the best among the group. Moreover, the 2-substi-
tuted benzimidazole 4z was the most potent piperidine identified
4i
4j
4.3
2.2
OCH3
F
N
N
F
4k
4l
1.5
1.5
NH
Cl
4m
0.91
O
O
O
4n
4o
4p
4q
4r
0.95
11.0
4.3
CO2Et
N
N
a, b
N
+
EtO
OEt
N
OEt
N
N
5
6
7
c
N
N
O
O
O
7.0
CO2H
CO2H
CO2Et
N
N
d, e
N
N
N
or
R
13.0
0.84
N
N
O
N
X
Y
X
4s
8
R
Z
F
F
N
9a-y
4a-z'
4t
1.7
Scheme 1. Reagents and conditions: (a) LDA, THF, ꢀ80 to ꢀ20 °C; (b) o-xylene,
reflux; (c) POCl3, DMF, 0 to 20 °C; (d) NaBH(OAc)3, DCE, AcOH, 4 Å molecular sieves;
(e) NaOH, THF, EtOH, 50 °C.
(continued on next page)

1712
S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1710–1715
Addition of a chlorine (9b–d) on the phenyl moiety of 9a led to
Table 1 (continued)
an improvement of functional activity with 6-chloro 9d showing
an ꢁ5-fold increase (M1 IP = 330 nM) relative to 9a. Incorporation
of heteroatoms provided equipotent (9e–f) or improved activity
(9g–h) relative to the phenyl counterpart. A number of variations
of 9d were made substituting for the chlorine (9i–p). This position
Compd
R1
M1 Pot IP (
0.78
l
M)a
N
N
N
N
N
N
4u
4v
1.9
1.7
N
N
N
Table 2
M1 FLIPR data for select tetrahydroisoquinolines
4w
O
O
N
OH
4x
0.55
R1
N
N
Compd
R1
M1 Pot IP (
l
M)a
N
4y
4z
4z0
6.0
0.12
30.0
N
N
9a
1.7
H
N
N
N
9b
0.85
Me
N
Cl
Cl
Cl
N
N
9c
0.63
0.33
a Values represent the numerical average of at least two experiments. Interassay
variability was 30% (IP, nM), unless otherwise noted.
9d
N
N
N
9e
1.8
(M1 IP = 120 nM), but capping the N–H with a methyl (4z0) led to a
>200-fold decrease in activity.
N
N
N
N
9f
1.7
In the quinolone carboxylic acid series, we have previously ob-
served that the B- and C-rings of the biphenyl could be tied back,
followed by C-ring excision to provide naphthyl 10 leading to an
ꢁ3-fold improvement in functional activity (Fig. 2).15 In order to
drive potency beyond what was observed in Table 1, this strategy
was employed for piperidine 4a leading to tetrahydroisoquinoline
9a. While 9a did not show an improvement over 4a,16 a series of
analogs was examined to see if substitution would lead to
improvements in this tetrahydroisoquinoline context. Results are
shown in Table 2.
9g
0.90
N
9h
9i
0.83
1.4
N
N
OCH3
SCH3
CH3
N
N
9j
>10
O
N
O
O
N
O
OH
OH
9k
9l
1.1
N
N
N
N
B
0.78
0.63
0.61
F
C
9m
9n
2: M1 IP = 212 nM
10: M1 IP = 80 nM
Br
CN
O
O
O
O
N
OH
N
OH
9o
9p
0.56
0.39
N
N
N
N
4a: M1 IP = 1300 nM
Figure 2. Design of tetrahydroisoquinolines.
9a: M1 IP = 1700 nM
a Values represent the numerical average of at least two experiments. Interassay
variability was 30% (IP, nM), unless otherwise noted.

S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1710–1715
1713
Table 4
proved highly tolerant of substitution, with a flat SAR similar to
Protein binding data for select analogs
that for the piperidines in Table 1, albeit with a slight preference
for small, lipophilic groups such as fluorine (9l) or cyano (9n).
Aromatics were also tolerated at this position of the tetrahydroiso-
quinoline, with 4-pyridyl 9p showing very similar potency (M1
IP = 390 nM) to chloro 9d.
O
O
N
OH
In addition to substitution of the phenyl sector of 9a, a scan of
the tetrahydroisoquinoline alkyl skeleton of 9a was also conducted
as shown in Table 3. Ethyl esters 9q–s showed the 3-position was
completely intolerant of substitution, while the 1-position led to
significant increase in activity (M1 IP = 340 nM). Additional substi-
tutions were subsequently investigated at the 1-position (9u–y).
Aliphatic groups were inactive as shown by cyclohexyl 9u, but
more favorable results were obtained with aromatic groups. Phe-
nyl 9v and thiophene 9y gave similar data, but ortho-fluro phenyl
9w showed a marked improvement. Moreover, 4-pyridyl 9x
proved to be the most potent compound reported here, with an
M1 IP = 53 nM.
R1
Compd‘
R1
Rat PB (%)
Human PB (%)
91
Log D
4g
89
2.97
0.92
CF3
H
N
4x
76
79
N
N
N
4z
77
96
82
98
nd
Table 3
M1 FLIPR data for select tetrahydroisquinolines
N
N
9d
2.37
Compd
R1
M1 Pot IP (
l
M)a
Cl
N
N
9a
1.7
9x
94
96
nd
N
9q
9r
>100
3.6
EtO2C
N
Select compounds from Tables 1–3 were evaluated for their hu-
man and rat plasma protein binding (Table 4). The piperidines gave
high free fractions, particularly for the two benzimidazole regioi-
somers 4x,z with ꢁ20% free in each species. The tetrahydroisoquin-
olines 9d,x were more highly bound, but still have between 2% and
6% free fraction. In addition, the log D’s were all in a desirable
range, similar to what was observed for piperazines such as 3.
Based on the acceptable levels of protein binding, the com-
pounds in Table 4 were evaluated further for their CNS exposure
potential. Accordingly, they were evaluated for passive permeabil-
ity and potential as substrates for the CNS efflux transporter P-gp,
as well as their CNS exposure in rat utilizing oral dosing at 10 mpk
(Table 5).
All compounds except for benzimidazoles 4x and 4z gave
acceptable permeability (>15). With the exception of 9x, all other
compounds were not P-gp substrates with good efflux ratios
(<2.5). The 4-trifluoromethylphenyl piperidine gave a very high
CSF/Uplamsa ratio of 0.7 and a surprisingly high brain to plasma
(B/P) ratio of 6. Benzimidazole 4z showed poor oral absorption
and undetectable CNS levels consistent with the low passive per-
meability.17 Chloro-tetrahydroisoquinoline 9d presented higher
plasma levels than piperidine 4g, with a similar CSF/Uplamsa ratio.
Lastly, the pyridyl-tetrahydroisoquinoline 9x provided the best
plasma levels among the group, but CNS ratios were lower, consis-
tent with 9x being a substrate for P-gp.
CO2Et
CO2Et
9s
9t
0.34
1.7
N
N
CO2Me
9u
9v
>100
0.56
0.15
N
N
N
F
9w
N
Based on their overall profiles18 and high CSF levels, piperidine
4g and tetrahydroisoquinoline 9d were examined in a contextual
fear conditioning model of episodic memory using B6SJL mice.
Compounds were tested at 3, 10, and 30 mpk, but no significant
reversal of scopolamine deficit was observed at the doses exam-
ined (data not shown). High plasma levels were obtained at
9x
9y
0.053
0.6
N
N
S
30 mpk with both 4g (31 lM) and 9d (16 lM) suggesting the lack
of efficacy in this model was not due to insufficient drug levels.19
This lack of activity in this mouse model20 with the piperidines
was particularly surprising as the related piperazine analog 3,
which had similar in vitro activity (M1 IP = 520 nM) and brain
a Values represent the numerical average of at least two experiments. Interassay
variability was 30% (IP, nM), unless otherwise noted.

1714
S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1710–1715
Table 5
Permeability, P-gp, and bioanalysis of plasma, brain, and CSF levels for selected compounds
a
b
b
d
Compd
Papp
MDR1
MDR1a
Plasma concnc
(nM)
Brain concnc
(nM)
CSF concnc
(nM)
B/P
CSF/Uplamsa
4g
4x
4z
9d
9x
35
<5
6.6
42
27
0.9
—
0.4
1.4
2.8
0.9
—
1.9
2.9
9.7
915
—
132
4245
9777
5779
—
28
—
0
86
230
6.0
—
—
0.86
0.14
0.70
—
—
0.63
0.37
0
3692
1345
a
b
c
Passive permeability (10ꢀ6 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 Table 4.
d
Vehicle
0.1µM 4g
1 µM 4g
Vehicle
0.1µM 9d
1 µM 9d
120
110
100
90
80
70
60
50
40
30
20
10
0
120
110
100
90
80
70
60
50
40
30
20
10
0
-10
-10
-13 -12 -11 -10 -9 -8 -7 -6 -5 -4
Log[M], Acetylcholine
-13 -12 -11 -10 -9 -8 -7 -6 -5 -4
Log[M], Acetylcholine
Figure 3. Fold potentiation curves for 4g and 9d.
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.
exposure (CSF:Uplasma = 0.42), worked in this model at 7.4
plasma.13
lM
To confirm that 4g and 9d were behaving similarly with respect
to allosteric modulator 3, their effects on the affinity of acetylcho-
line for the M1 receptor in a functional assay utilizing calcium
mobilization as the readout were evaluated. In CHO cells express-
ing the human M1 receptor, increasing concentrations of both 4g
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. USA 2009, 106, 15950.
11. For additional characterization of 1, see: Shirey, J. K.; Brady, A. E.; Jones, P. J.;
Davis, A. A.; Bridges, T. M.; Kennedy, J. P.; Jadhav, S. B.; Menon, U. N.; Xiang, Z.;
Watson, M. L.; Christian, E. P.; Doherty, J. J.; Quirk, M. C.; Snyder, D. H.; Lah, J. J.;
Nicolle, M. M.; Lindsley, C. W.; Conn, P. J. J. Neurosci. 2009, 45, 14271.
12. Yang, F. V.; Shipe, W. D.; Bunda, J. L.; Wisnoski, D. D.; Zhao, Z.; Lindsley, C. W.;
Ray, W. J.; Ma, L.; Wittmann, M.; Seager, M. W.; Koeplinger, K.; Thompson, C.
D.; Hartman, G. D. Bioorg. Med. Chem. Lett. 2010, 19, 651.
and 9d from 0.1 to 1 lM potentiates the effect of acetylcholine
leading to a leftward shift in the acetylcholine M1 dose–response
curves (Fig. 3). Thus it appears the lack of efficacy observed in
the mouse CFC assay is not due to inability to potentiate the acetyl-
choline dose response.21
13. Kuduk, S. D.; Chang, R. K.; Di Marco, C. N.; Pitts, D. R.; Ray, W. J.; Ma, L.;
Wittmann, M.; Seager, M.; Koeplinger, K. A.; Thompson, C. D.; Hartman, G. D.;
Bilodeau, M. T. A. C. S. Med. Chem. Lett. 2010, 1, 263.
In summary, a series of piperidine and tetrahydroisoquinoline
quinolizidinone carboxylic acids were prepared and evaluated.
The SAR was thoroughly investigated and found that substitution
on the piperidine was generally flat. Potentiators 4g and 9d were
identified as having reasonable potency and both showed high
CNS exposure. However, unlike the case for piperazine derived
quinolizidione modulators such as 3, these piperidine containing
analogs did not show efficacy in a mouse model of episodic mem-
ory. Additional analog work along with more advanced in vitro
studies is ongoing to explain this lack of activity.
14. The tetrahydropyridine variant of 4a was ꢁ2 fold less potent than the saturated
variant and was not investigated further.
O
O
N
OH
N
References and notes
M1 IP = 2.7 µM
1. Bonner, T. I. Trends Neurosci. 1989, 12, 148.
2. Bonner, T. I. Trends Pharmacol. Sci. 1989, 11.
3. Levey, A. I. Proc. Natl. Acad. Sci. 1996, 93, 13451.
4. Geula, C. Neurology 1998, 51, 18.
15. Kuduk, S.D.; Di Marco, C.N.; Cofre, V.; Pitts, D.R.; Ray, W.J.; Ma, L.; Wittmann,
M.; Seager, M.; Koeplinger, KA..; Thompson, C.D.; Hartman, G.D.; Bilodeau, M.T.
Bioorg. Med. Chem. Lett. 2010, 19, accepted.
16. The tetrahydroquinoline variant of 9a was ꢁ10 fold less potent than the
5. Langmead, C. J. Pharmacol. Ther. 2008, 117, 232.
isoquinoline.
6. Bodick, N. C.; Offen, W. W.; Levey, A. I.; Cutler, N. R.; Gauthier, S. G.; Satlin, A.;
Shannon, H. E.; Tollefson, G. D.; Rasumussen, K.; Bymaster, F. P.; Hurley, D. J.;
Potter, W. Z.; Paul, S. M. Arch. Neurol. 1997, 54, 465.
7. Greenlee, W.; Clader, J.; Asbersom, T.; McCombie, S.; Ford, J.; Guzik, H.;
Kozlowski, J.; Li, S.; Liu, C.; Lowe, D.; Vice, S.; Zhao, H.; Zhou, G.; Billard, W.;
Binch, H.; Crosby, R.; Duffy, R.; Lachowicz, J.; Coffin, V.; Watkins, R.; Ruperto,
V.; Strader, C.; Taylor, L.; Cox, K. Il Farmaco 2001, 56, 247.
8. Conn, P. J.; Christopulos, A.; Lindsley, C. W. Nat. Rev. Drug Disc. 2009, 8, 41.
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.;
O
O
N
OH
N
M1 IP = 17 µM

S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1710–1715
1715
17. It is worth noting that although 4z does not appear to be a P-gp substrate,
those values are not likely to reflect the true efflux ratio due the low passive
permeability of the molecule in the parent cell line.
18. Compound 9x was removed from further consideration due to its high P-gp
efflux ratio and as it had significant binding to the hERG cardiac channel
similar CSF/Uplasma ratio of ꢁ0.42, showing that P-gp efflux in mouse was not a
factor.
20. Potentiator 4g was active at rat (M1 IP = 760 nM) receptors, within twofold of
human (M1 IP = 460 nM).
21. Potentiators 4g and 9d were also evaluated for activity in the b-arrestin
pathway, which recruits different signaling proteins than the Ga
s-coupled Ca2+
(<1 lM).
19. To confirm CNS exposure in mouse and that P-gp was not playing a role in CNS
disposition in this species, CF-1 wild type and P-gp knock out mice were
treated with 4g, and plasma and CSF were taken after 30 minutes. The wild
type mice gave a CSF/Uplasma ratio of ꢁ0.37, and mice lacking P-gp gave a
release measured by the FLIPR assay, in the presence and absence of an EC15
concentration of ACh. Both compounds perform comparably in both FLIPR and
b-arrestin assays relative to 3, indicating that possible different signaling
pathways are not responsible for the lack of in vivo activity.