Identification of a methoxynaphthalene scaffold as a core replacement in quinolizidinone amide M1 positive allosteric modulators

Article abstract of DOI:10.1016/j.bmcl.2014.01.012

A series of methoxynaphthalene amides were prepared and evaluated as alternatives to quinolizidinone amide M₁ positive allosteric modulators. A methoxy group was optimal for M₁ activity and addressed key P-gp issues present in the aforementioned quinolizidinone amide series.

Full text of DOI:10.1016/j.bmcl.2014.01.012

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1417–1420
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of a methoxynaphthalene scaffold as a core
replacement in quinolizidinone amide M1 positive allosteric
modulators
a
a
b
b
Scott D. Kuduk a, , Christina N. Di Marco , Jonathan R. Saffold , William J. Ray , Lei Ma ,
Marion Wittmann b, Kenneth A. Koeplinger c, Charles D. Thompson c, George D. Hartman a,
Mark T. Bilodeau a, Douglas C. Beshore a
⇑
a Department of Medicinal Chemistry, Sumneytown Pike, PO Box 4, West Point, PA 19486, USA
b Department of Alzheimer’s Research, 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:
A series of methoxynaphthalene amides were prepared and evaluated as alternatives to quinolizidinone
amide M1 positive allosteric modulators. A methoxy group was optimal for M1 activity and addressed key
P-gp issues present in the aforementioned quinolizidinone amide series.
Received 12 December 2013
Revised 2 January 2014
Accepted 6 January 2014
Available online 13 January 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
M1
Allosteric modulator
Muscarinic
Alzheimer’s disease
P-glycoprotein
Neurons expressing cholinergic receptors operate critical func-
tions in both the peripheral and central nervous systems (CNS).
The key neurotransmitter in these systems, acetylcholine, targets
both nicotinic and metabotropic (muscarinic) receptors. The mus-
carinic receptors are members of class A G-protein coupled recep-
tors (GPCR) that are extensively expressed in the CNS. Within the
family, there are five muscarinic subtypes, designated M1 to
M5.1,2 The M1 receptor is most highly expressed in brain regions
of the hippocampus, striatum, and cortex,3 signifying a fundamen-
tal role in memory and cognition.
As Alzheimer’s disease (AD) advances, there is a gradual degen-
eration of these cholinergic neurons in the basal forebrain leading
to increasing cognitive deficits.4 An approach to address the symp-
toms of AD is via the direct stimulation of the M1 receptor.5Toward
this end, a number of non-selective M1 agonists have previously
shown promising clinical results on cognitive performance in pa-
tients with AD. However, none of these could advance beyond
proof-of-concept studies as a result of untoward cholinergic side
effects thought to be mediated via pan activation of other
muscarinic sub-types as a consequence of the highly conserved
orthosteric acetylcholine binding site.6,7
In order to preferentially activate the M1 receptor over the other
muscarinic sub-types, the strategy chosen was to target an alloste-
ric site on M1 that would be expected to be less well conserved
than the orthosteric site.8,9 Toward this end, lead quinolone car-
boxylic acid BQCA10 was identified via screening of the MRL sam-
ple repository as a highly selective positive allosteric modulator
(PAM) of the M1 receptor and has been the subject of extensive
lead optimization.11 For example, we have described the evolution
of BQCA into quinolizidinone carboxylic acids such as 1 as potent,
selective, and CNS penetrant M1 positive allosteric modulators.12,13
Further analog work in this quinolizidinone context allowed fur-
ther advancement identifying replacement of the carboxylic acid
with amides such as 2,14 which were highly potent in functional
assays, but were substrates for the CNS efflux transporter P-glyco-
protein (P-gp). This communication describes efforts to identify an
alternate core template for the quinolizidinone nucleus that would
be bereft of P-gp mediated transport (Fig. 1).
One of the key features targeted in the selection of a core
replacement would be the ability to form a putative intra-molecu-
lar hydrogen bond between the amidic N–H and an acceptor on the
template.15 This was a requirement for M1 functional activity
⇑
Corresponding author.
E-mail address: scott_d_kuduk@merck.com (S.D. Kuduk).
0960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2014.01.012

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S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1417–1420
H
N
Putative H-bond acceptor
needed for potency
X
H
O
O
O
N
H
O
H
OH
N
O
N
O
X = O, NHR
O
N
CN
N
CN
N
H
O
N
O
MeO
O
1: M1 IP = 136 nM
PB = 60-70%
2: M1 IP = 31 nM
PB = 52-86%
O
O
Figure 1.
OMe
OMe
4: M1 IP = 3330 nM
Figure 2.
during the quinolizidinone carboxamide SAR analysis as both ter-
tiary amides and esters lose substantial activity. During the course
of SAR efforts on HTS lead BQCA 3, it was found that the quinolone
ring system could be replaced with a methoxynaphthalene, in the
context of the carboxylic acid, at the expense of ꢀ5-6-fold loss of
functional potency.16 It was proposed to investigate the
methoxynaphthalene in the framework of amides, to see if the
methoxy group could serve to mimic the proposed hydrogen bond
motif thought to be necessary for M1 activity (Fig. 2).
The synthesis of requisite compounds is shown in Scheme 1.
Starting with commercially available methyl 1-methoxy-2-nap-
thoate, bromination provided intermediate 8. Suzuki cross-cou-
pling followed by hydrolysis afforded acid 9. Amide formation
with (1S,2S)-2-aminocyclohexanol was implemented using Bop
reagent followed by oxidative cleavage of the olefin produced
aldehyde 10. Reductive amination of 10 delivered target amines
5a–c. Alternatively, bromide 8 underwent Negishi coupling with
the appropriate zinc reagent followed by cleavage of the methyl
ester and methyl ether to provide acid 11. Aforementioned Bop
coupling of 11 was followed by alkylation with the appropriate
alkyl halide to lead to targets 6a–f. Lastly, the chloropyridine pres-
ent in 6a was further functionalized to prepare analogs 6g–n.
The initial SAR data for a representative group of methoxynaph-
thalene amides that possess different groups at the 4-position of
the naphthalene ring is shown in Table 1. Compound potencies
were determined in the presence of an EC20 concentration of
3: M1 IP = 660 nM
acetylcholine at human M1 expressing CHO cells using calcium
mobilization readout on a FLIPR384 fluorometric imaging plate
reader. The percent max represents the maximum potentiated
EC20 response generated. Both N- and C-linked analogs were exam-
ined. The N-linked analog (5a) of amide 2 lost ꢀ10-fold in func-
tional potency, but the piperazines 5b and 5c were substantially
less active. C-linked analog 6a possessed good potency for M1,
and the chloropyridine was selected to keep in place for additional
SAR work on the naphthalene scaffold. All compounds possessed
relatively similar percent max.
To test the hypothesis that the methoxy group was a potential
hydrogen bond acceptor, a number of alternative groups were
examined (Table 2). The des-methyl compound, naphthol 6b, lost
ꢀ35 fold relative to methoxy. Increasing the steric size of the group
to ethyl (6c, M1 IP = 980 nM) and allyl (6d, M1 IP = 1100 nM) also
reduced potency while acetate 6e was completely inactive. To
match the steric size without the potential for hydrogen bonding,
ethyl analog 6f was made and was found to be a very weak M1
PAM with an IP = 7400 nM. While this SAR does not prove the exis-
tence and bioactive role of a hydrogen bond between the amidic
MeO HN
MeO HN
MeO
O
OH
OH
O
g
d, e
O
OH
N
N
N Me
N
N
6m
6l
O
NR1R2
5a-c
6n
9
10
k
l
m
b, c
n
OH
O
MeO HN
RO HN
MeO
R
O
6g-i
OH
R
OH
Cl
d, j
h, i
OH
N
O
N
o
O
N
OMe
6j: R = OMe
6k: R = SMe
p
7: R = H
8: R = Br
Cl
a
11
6a: R = Cl
6a-f
Scheme 1. (a) Br2, AcOH, (b) vinylpotassium fluoroborate, K2CO3, PdCl2(dppf), THF, H2O, 80 °C, (c) NaOH, THF, H2O, (d) (1S,2S)-2-aminocyclohexanol, Bop reagent, TEA, DCM,
(e) OsO4, NMO, THF, acetone, H2O, (f) Amine, AcOH, NaBH(OAc)3, DCE, (g) NaBH(OAc)3, CH2Cl2, (h) Pd(PtBu3)2, ((6-chloropyridin-3-yl)methyl)zinc(II) chloride, THF, 100 °C, (i)
33% HBr, AcOH, (j) Alkylhalide, K2CO3, DMF, (k) pyrazole, CuI, trans-N,N-dimethylcyclohexane-1,2-diamine, Cs2CO3, DMSO, 120 °C, (l) (1-methyl-1H-pyrazol-4-yl)boronic
acid, Pd(PtBu3)2, THF, 100 °C, (m) 3-pyridine boronic acid, Pd(PtBu3)2, THF, 100 °C, (n) alkylB(OH)2, Pd(PtBu3)2, Cs2CO3, THF, 100 °C, (o) CuI, MeOH, 160°C, (p) NaSMe, DMSO,
80°C.

S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1417–1420
1419
Table 1
Table 2
M1 FLIPR and protein binding data for select compounds
M1 FLIPR and protein binding data for select compounds
MeO HN
R
HN
OH
OH
O
O
N
R
Compd
R1
—
M1 Pot IPa (nM)
% Max
62
Cl
2
31
Compd
R1
M1 Pot IPa (nM)
% Max
N
N
N
6a
6b
6c
6d
6e
6f
OMe
OH
OEt
OAllyl
OAc
Et
260
9100
980
1100
>10,000
7400
74
35
37
68
18
47
CN
N
5a
5b
303
62
57
N
N
4000
a
Values represent the numerical average of at least two experiments. Interassay
variability was 30% (IP, nM), unless otherwise noted.
N
5c
6a
3900
260
83
74
N
Me
Table 3
N
M1 FLIPR and protein binding data for select compounds
N
Cl
MeO HN
OH
O
a
Values represent the numerical average of at least two experiments. Interassay
variability was 30% (IP, nM), unless otherwise noted.
N
R
NH and the methoxy group, it is clear that having said functionality
in place is important with this core.
Compd
R1
M1 Pot IP (nM)a
% Max
Rat PPB
Having identified the methoxy as the preferred group, addi-
tional SAR was examined at the 4-position on the naphthalene ring
using chloride 6a as the starting point. The chloro group could be
replaced with a variety of alkyl substituents (6g–n) that increased
potency with the methyl 6g (M1 IP = 121 nM) and vinyl 6h (M1
IP = 74 nM) standing out. Similarly, methyl ether (6j; M1
IP = 140 nM) and thiomethyl (6k, M1 IP = 170) were also well toler-
ated. However, the most substantial gains in modulator potency
were obtained with the inclusion of heterocycles (6l–n). Pyrazoles
6l and 6m were excellent alternatives to a smaller substituent,
especially the C-linked methyl pyrazole 6m that gave an M1
IP = 17 nM. Lastly, a simple 3-pyridyl ring (6n) afforded potency
very similar to pyrazole 6m, but both compounds are very highly
protein bound in rat relative to the smaller groups.
Since quinolizidinone amides such as 2 were P-gp substrates,
naphthalene derivatives were evaluated for P-gp efflux potential
in human (MDR1) and rat (MDR1a) expressing cell lines, as well
as passive permeability, and select examples for CNS exposure in
rat. As can be seen in Table 4, all naphthalene amides examined
had good permeability (>15 cm/s) and were not substrates for
6a
6g
6h
6i
6j
6k
Cl
Me
Vinyl
Et
OMe
SMe
260
121
74
190
140
170
74
93
88
73
85
76
90.0
98.4
Nd
Nd
Nd
Nd
N
N
6l
6m
6n
87
17
24
83
77
73
98.6
99.9
99.4
N
Me
N
N
a
Values represent the numerical average of at least two experiments. Interassay
variability was 30% (IP, nM), unless otherwise noted.
P-gp with low efflux ratios (ER); notably N-linked naphthalene
amide 5a differentiates from the direct comparator quinolizidi-
none amide 2. One exception was N-linked analog 6l that was a
Table 4
Permeability, P-gp, and bioanalysis of plasma, brain, and CSF levels for selected compounds
a
d
Compd
Papp
MDR1b
MDR1ab
Plasma Conc.c (nM)
Brain Conc.c (nM)
CSF Conc.c (nM)
B/P
CSF/Uplasma
2
5a
6a
6g
6l
29
31
32
35
31
27
28
11.1
1.7
1.3
1.6
1.7
1.1
0.9
21.1
2.3
1.9
2.1
3.2
1.4
1.5
—
—
—
—
—
—
5
91
8.7
—
—
—
—
—
<0.1
0.9
0.34
—
2060
6020
2100
29,000
4900
420
1200
370
1600
860
0.22
0.21
0.17
0.06
0.2
6m
6n
11
0.38
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

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S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1417–1420
Vehicle
Vehicle
110
110
100
90
80
70
60
50
40
30
20
10
0
0.12µM 6g
1.2 µM 6g
12 µM 6g
0.024µM 6n
0.24 µM 6n
2.4 µM 6n
100
90
80
70
60
50
40
30
20
10
0
-10
-10
-13 -12 -11 -10 -9 -8 -7 -6 -5 -4
-13 -12 -11 -10 -9 -8 -7 -6 -5 -4
Log[M], Acetylcholine
Log[M], Acetylcholine
Figure 3. Fold potentiation curves for 6n and 6g.
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.
11. 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. 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. ACS Med. Chem. Lett. 2010, 1, 263.
13. Kuduk, S. D.; Chang, R. K.; Di Marco, C. N.; Pitts, D. R.; Greshock, T. J.; Ma, L.;
Wittmann, M.; Seager, M.; Koeplinger, K. A.; Thompson, C. D.; Hartman, G. D.;
Bilodeau, M. T.; Ray, W. J. J. Med. Chem. 2011, 13, 4773.
14. Kuduk, S. D.; Chang, R. K.; Greshock, T. J.; Ray, W. J.; Ma, L.; Wittmann, M.;
Seager, M.; Koeplinger, K. A.; Thompson, C. D.; Hartman, G. D.; Bilodeau, M. T.
ACS Med. Chem. Lett. 2012, 3, 1070.
15. Beshore, D. C. Presented at the 33rd National Medicinal Chemistry Symposium;
Tuscon: AZ, May 2012.
16. N-linked methoxynaphthalene carboxylic acids such as 12 below were also
made and retained similar potency to C-linked acid 4.
substrate for rodent P-gp (ER > 2.5). In terms of central exposure,
methyl analog 6g gave a CSF/Uplasma ratio of 0.9, substantially high-
er than Cl analog 6a.17 N-linked pyrazole 6l was somewhat lower,
which could be a reflection of being a rat P-gp substrate. While the
highly potent pyrazole analog 6m did not show any detectable CSF
exposure, pyridine 6n was measurable with a CSF/Uplasma ratio of
0.38.
Finally, to confirm that the new methoxynaphthalenes behave
similarly with respect to allosteric modulator 2, the effects of 6n
and 6g on the affinity of acetylcholine for the M1 receptor in a func-
tional assay utilizing calcium mobilization as the readout was eval-
uated. In CHO cells expressing the human M1 receptor, increasing
concentrations of 6n and 6g from their respective IP’s, and 10
and 100Â over, potentiates the effect of acetylcholine leading to
a leftward shift in the acetylcholine M1 dose-response curves
(Fig. 3).
In summary, a series of methoxynaphthalene amides were pre-
pared and evaluated as alternatives to quinolizidinone amide M1
positive allosteric modulators. The methoxy group was optimal
for M1 activity and addressed the key P-gp issues present in the
aforementioned quinolizidinone amide series. Accordingly, addi-
tional analog work is ongoing to leverage this and further optimize
methoxynaphthalene analogs for improvements for additional
characterization.
MeO
OH
O
References and notes
N
CN
N
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.
5. Langmead, C. J. Pharmacol. Ther. 2008, 117, 232.
12: M1 IP = 5400 nM
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.
17. With respect to methyl analog 6g exhibiting a higher CSF ratio than Cl analog
6a, the high-throughput solubility of 6g (90
lM at pH 7) was much better than
Cl 6a (insoluble at pH 7) which may help explain the difference.