Pyridine containing M1 positive allosteric modulators with reduced plasma protein binding

Article abstract of DOI:10.1016/j.bmcl.2009.11.059

Incorporation of pyridines and diazines into the biphenyl region of quinolone carboxylic acid derived M₁ positive allosteric modulators was investigated as a means of lowering plasma protein binding to enhance CNS exposure.

Full text of DOI:10.1016/j.bmcl.2009.11.059

Bioorganic & Medicinal Chemistry Letters 20 (2010) 657–661
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyridine containing M1 positive allosteric modulators with reduced plasma
protein binding
a
a
a
b
Scott D. Kuduk a, , Christina N. Di Marco , Victoria Cofre , Daniel R. Pitts , William J. Ray ,
Lei Ma b, Marion Wittmann b, Matthew Seager b, Kenneth Koeplinger c, Chuck 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:
Incorporation of pyridines and diazines into the biphenyl region of quinolone carboxylic acid derived M1
positive allosteric modulators was investigated as a means of lowering plasma protein binding to
enhance CNS exposure.
Received 8 October 2009
Revised 9 November 2009
Accepted 16 November 2009
Available online 2 December 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
M1
Muscarinic
Allosteric modulator
PAM
Alzheimer’s
Cholinergic neurons serve critical functions in both the periph-
eral and central nervous systems (CNS). Acetylcholine is the key
neurotransmitter in these systems, targeting nicotinic and metab-
otropic (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 implying it may play a central role in memory and
higher brain function.
One of the hallmarks of Alzheimer’s disease (AD) is the progres-
sive degeneration of cholinergic neurons in the basal forebrain
leading to cognitive decline.4 Accordingly, direct activation of the
M1 receptor represents an approach to treat the symptoms of
AD.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
due to activation of other muscarinic subtypes via binding to the
highly conserved orthosteric acetylcholine binding site.6,7
lator of the M1 receptor with exquisite selectivity for the M1 sub-
type.10 Recent efforts to improve the potency of 1a led to the iden-
tification of biphenyl replacements for the para-methoxybenzyl
group.11 While these compounds were improved in terms of
in vitro activity, higher plasma protein binding led to decreased
CNS exposure impeding further in vivo evaluation. It has been
demonstrated in an unrelated series of compounds that insertion
of a pyridine into a biphenyl motif can effectively reduce protein
binding and enhance physical properties.12 This Letter describes ef-
forts to replace the proximal B-ring phenyl with a pyridine or other
diazine and subsequent SAR observed on the distal C-ring phenyl
(Fig. 1).
O
N
O
O
N
O
OH
OH
A
One avenue 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. recently reported the
quinolone carboxylic acid 1 as a selective positive allosteric modu-
B
OMe
C
1a: M1 IP = 660 nM
1b: -Increased potency
-Higher protein binding
-Reduced CNS exposure
PB = 96-98%
* Corresponding author.
E-mail address: scott_d_kuduk@merck.com (S.D. Kuduk).
Figure 1.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.059

658
S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 20 (2010) 657–661
Table 1
The chemistry employed to prepare target compounds 6–11 is
M1 FLIPR and protein binding data for select compounds
shown in Scheme 1. The 8-fluoroquinolone ethyl ester 2 could be
prepared by a Gould–Jacobs cyclization. Alkylation of the appropri-
ate alkyl halide or mesylate 3a–f was accomplished using potas-
sium carbonate in DMF. Subsequent base mediated hydrolysis of
4a–f followed by Suzuki coupling with the appropriate boronic
acid afforded analogs 6–11. This late stage cross-coupling allowed
for greater diversity in a rapid analog fashion.
O
O
OH
N
F
N
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.13 The percent max represents the maximum
potentiated EC20 response generated. Plasma protein binding was
determined using the equilibrium dialysis method in the presence
of rat and human serum.
Incorporation of the pyridine nitrogen at the 2-position of the B-
ring (6a) enhanced functional activity relative to phenyl compound
1b with an M1 inflection point (IP) = 153 nM with an 80% maximal
response, but had little effect on the rat or human plasma protein
binding (Table 1). Location of the nitrogen at the 3-position (7a)
lowered potency ꢀ2-fold with 97% maximal response, but did
not affect protein binding similar to the 2-pyridine 6a. Pyrazine 8
did lower protein binding significantly, but with a notable loss
(ꢀ4-fold) in M1 potency. Much of this activity could be reclaimed
in the form of pyridazine 9a, which was similar to the phenyl ana-
log 1b, with a modest reduction in rat protein binding. Pyrimidines
10 and 11 lost significant M1 activity. Due to the satisfying boost in
M1 potency seen with 2-pyridyl 6a, variation of the distal phenyl C-
ring was investigated in this series for further enhancements to M1
functional activity and protein binding.
Compds R1
M1 Pot IPa (nM) % Max Rat PB Human PB
1b
378
153
84
80
98.0
97.3
98.4
98.4
6a
7a
8
N
604
1300
390
97
85
97
98.0
95.1
96.1
98.2
97.1
98.4
N
N
N
N
N
N
9a
10
11
4000
2324
88
90
nd
nd
nd
nd
N
N
N
A library of boronic acids was employed to survey the SAR for
analogs of 6a. Overall the effects on potency were minimal when
the C-ring was varied relative to phenyl comparator 6a. Selected
examples are shown in Table 2. For brevity, the % max responses
are not shown, but were generally >90% unless otherwise
indicated.
A variety of substituents such as fluorine (6b) or methoxy (6c)
on the distal phenyl C-ring was found to modestly enhance M1
activity with little effect on protein binding. Lipophilic heterocy-
cles such as thiophene (6d) also reduced plasma free fractions as
well. One exception was methyl sulfones 6e–g that exhibited good
M1 activity (except the para-analog 6g) and reduced protein bind-
ing, particularly for the ortho position. An amino group at the meta
position (6h) was the most potent amongst the series with an M1
IP = 21 nM and had an ꢀ4% free fraction. Methylation of the group
(6i) increased protein binding, but acetylation (6j) was similar to
6h, albeit with a loss of M1 activity.
a
Values represent the numerical average of at least two experiments. Interassay
variability was 30% (IP, nM), unless otherwise noted.
ity similar to that of 6a, and did provide an enhanced free fraction.
A range of substituents (6m–q) were found to vary these proper-
ties with 6o showing a good balance of potency and free fraction
for further study. Other nitrogen containing heterocycles were also
examined. Amongst them, N-methyl pyrazole 6r, exhibited M1 po-
tency similar to the phenyl group and appeared to show a large
species dependent drop in rat protein binding. The highly active
potentiator, indole 6s, was extensively bound in both human and
rat plasma.
Select compounds from Table 2 were evaluated for their ability
to potentiate a dose–response of acetylcholine with a fixed concen-
tration of potentiator.10 As can be seen from Table 3, in the pres-
ence of 30 lM of potentiator, a left-shift between 29- and
81-fold was observed in the acetylcholine dose–response showing
they are potent positive allosteric modulators of the human M1
receptor.
Pyridines were also examined in lieu of the distal phenyl C-ring
to see if additional gains in free fraction could be obtained. The
3-pyridine (6l) looked the most promising in maintaining M1 activ-
Cl/Br/OMs
O
N
O
O
N
O
W
O
O
Z
OH
Z
Jacobs-
Gould
OEt
Z
X
b, c
OEt
CO2Et
CO2Et
Y
R
N
H
a
N
H
F
F
W
Y
F
W
Y
F
X
X
2
R1
3a: X = N; W,Y, Z = CH; R = Br
R
3b: Y = N, W; X, Z = CH; R = Cl
3c: X, Z = N; Y, W = CH; R = Cl
3d: X, Y = N; W, Z = CH; R = Cl
3e: Y, Z = N; X, W = CH; R = Cl
3f: X, W, = N; Y, Z = CH; R = Br
6: X = N; W,Y, Z = CH
7: Y = N, W; X, Z = CH
8: X, Z = N; Y, W = CH
9: X, Y = N; W, Z = CH
10: Y, Z = N; X, W = CH
11: X, W, = N; Y, Z = CH
4a-f
Scheme 1. Reagents and conditions: (a) K2CO3, KI, DMF, rt–50 °C; (b) LiOH, dioxane; (c) Pd(t-Bu3P)2, R1B(OH)2, DMSO, 1 N Cs2CO3, uwave-160 °C, 10 min.

S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 20 (2010) 657–661
659
Table 2
Table 2 (continued)
M1 FLIPR and protein binding data for select compounds
Compds
R1
M1 Pot IPa (nM)
Rat PB
99.4
Human PB
O
O
OH
6s
57
99.8
N
H
N
F
a
Values represent the numerical average of at least two experiments. Interassay
N
variability was 30% (IP, nM), unless otherwise noted.
R
Compds
R1
M1 Pot IPa (nM)
67
Rat PB
98.7
Human PB
98.1
Table 3
Fold potentiation data for select compounds
6b
Compds
Fold shift
F
6a
6b
6g
6h
6o
6r
37
29
33
45
81
37
6c
89
98.4
99.4
97.8
98.1
OMe
S
6d
150
Having identified a number of interesting C-rings from Table 2,
combinations of these were prepared and evaluated with the 3-
pyridyl (7) and pyridazine (9) B-rings to assess whether further
improvements could be gained. Results for select compounds are
shown in Table 4. Overall, the SAR for the 3-pyridyl compounds
(7b–e) translated very well from the 2-pyridine series, while the
pyridazine analogs 9b–e were all less potent across the board.
For example, meta-methylsulfone 7e maintained good M1 activity,
6e
97
91.7
85.8
MeO2S
6f
97
260
21
96.8
nd
95.5
nd
SO2Me
while the pyridazine version 9e gave an M1 IP = 1.5 lM. This find-
6g
6h
ing was unfortunate as the pyridazines showed very good free
fractions as exemplified by 9c, which had protein binding in the
58–68% range. Amongst the 3-pyridines (7b–i), meta-amino analog
7f possessed the most interesting potency and protein binding
profile.
SO2Me
96.8
95.5
NH2
With a number of potent compounds available with good free
fractions, evaluation of brain exposure was required. P-glycopro-
tein (P-gp) is an ATP-driven efflux transporter at the blood–brain
barrier (BBB), responsible for the efflux of a number of xenobiotic
substances from the brain. Accordingly, P-gp efflux potential for
human (MDR1) and rat (MDR1a), as well as passive permeability,
were evaluated to triage potential candidates.
6i
6j
110
131
99.3
95.8
98.7
95.9
NMe2
NHAc
As can be seen in Table 5, with the exception of pyrazole 6r
(MDR1a = 6.3), the majority of compounds exhibited efflux ratios
less than 3, indicating that they were not substrates for human
or rat P-gp. However, four compounds possessed sub-adequate
passive permeability (Papp <15), another important component
necessary to achieve permeation across the BBB. These include
all three methylsulfones 6e–g as well as aminopyridine 6n.
The four remaining compounds (6l, 6o, 6r, and 7e) were evalu-
ated for brain exposure in rat, utilizing oral dosing (10 mpk) and
sampling at a 2 h time point. With the exception of 7e, which also
had the lowest plasma exposure, all compounds gave very low to-
tal brain levels (5% or less). Bi-pyridyl 6l and pyrazole 6r, also have
quite poor CSF levels relative to the free plasma concentrations
(<10%). While 6l did have the highest protein binding of the four
compounds, which may explain the low brain exposure observed,
it is less clear why 6r gave such poor results despite the very high
plasma levels. Only fluoro-pyridine 6o gave a suitable CSF/Uplamsa
ratio of 0.32, and also exhibited a very high plasma concentra-
tion.14 It appears that more than P-gp efflux potential and free frac-
tion are involved in the CNS disposition of these quinolone
carboxylic acids.15
6k
6l
78
370
82
92.3
82.5
98.1
97.0
95.1
99.0
NHAc
N
N
6m
NMe2
6n
6o
200
100
83.9
86.3
90.9
97.6
N
N
NH2
F
N
6p
169
97.5
96.5
Cl
F
6q
6r
680
160
90.9
89.0
92.9
98.4
The SAR of the substitution pattern on the quinolone A-ring was
also examined utilizing compound 6r as the scaffold (Table 6).
Prior SAR has revealed that only the 5- and 8-positions on the quin-
olinone tolerate substitution, ideally with a small replacement
N
N
N

660
S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 20 (2010) 657–661
Table 4
M1 FLIPR and protein binding data for select compounds
O
N
O
OH
F
R1
R2
Compds
R1
R2
M1 Pot IPa (nM)
Rat PB
99.6
Human PB
100
7b
65
N
H
N
N
9b
8662
nd
nd
N
N
H
7c
9c
7d
9d
951
778
190
466
86.4
58.6
98.6
nd
90.5
68.0
99.1
nd
N
N
N
N
N
N
N
N
N
F
F
7e
9e
7f
28
94.7
nd
93.5
nd
SO2Me
SO2Me
NH2
N
1543
21
N
N
N
94.7
98.9
93.5
97.8
7g
130
NMe2
7h
7i
169
202
97.5
86.3
95.5
97.6
N
N
N
N
Cl
F
a
Values represent the numerical average of at least two experiments. Interassay variability was 30% (IP, nM), unless otherwise noted.
Table 5
Permeability, P-gp, and bioanalysis of plasma, brain, and CSF levels for selected compounds
Pappa
MDR1b
MDR1ab
Plasma concnc (nM)
Brain concnc (nM)
CSF concnc (nM)
B/P
CSF/Uplamsa
d
Compds
6e
6f
6g
6l
6n
6o
6r
7e
9
6
7
30
11
28
28
19
1.2
1.1
0.9
0.7
1.1
0.5
1.6
2.1
1.8
1.5
1.0
2.5
1.5
1.0
6.3
4.5
nd
nd
nd
2794
nd
12,837
33,274
382
nd
nd
nd
154
nd
311
430
154
nd
nd
nd
25
nd
97
40
0
nd
nd
nd
0.05
nd
0.05
0.03
0.4
nd
nd
nd
0.05
nd
0.32
0.08
—
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 4.
d

S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 20 (2010) 657–661
661
Table 6
pyridyls generally retained the most potency and gave analogs
with the best free fraction. However, good permeability and not
being substrates for P-gp did not necessarily translate into higher
CNS exposure, indicating other factors may be in play, such as
the potential for the involvement of other CNS transporters. Stud-
ies are underway to investigate these possibilities.
SAR for fluorine substitution on the A-ring
R1
O
N
O
F reduces PB
and permeability
OH
5
8
F increase PB
and permeability
R2
N
References and notes
N
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.
Compds R1 R2 M1 IPa Rat PB Human PB Pappb MDR1c MDR1ac
4. Geula, C. Neurology 1998, 51, 18.
5. Langmead, C. J. Pharmacol. Ther. 2008, 117, 232.
6r
6t
6u
H
F
F
F
H
F
118
176
114
88.9
90.6
92
98.4
78.6
77.0
28
6.6
11
1.6
2.6
1.4
6.3
6.8
8.1
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.;
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.
a
Values represent the numerical average of at least two experiments. Interassay
variability was 30% (IP, nM), unless otherwise noted.
Passive permeability (10À6 cm/s).
b
c
MDR1 Directional Transport Ratio (B to A)/(A to B). Values represent the average
of three experiments and interassay variability was 20%.
such as fluorine.16 While moving the fluorine to the 5-position (6t)
or the 5,8-di-fluoro variant (6u) has little effect on M1 activity, sub-
stantial effects are noted on permeability and protein binding.
Interestingly, the 5-fluoro group decreased protein binding, but
has the undesired effect of eroding permeability. The 8-fluoro 6r
had much higher protein binding (in human), but good permeabil-
ity, while the combination 6u has attenuated properties that ap-
pear largely modulated by the 5-fluorine. As a result the 8-fluoro
is a preferred A-ring scaffold, at least for this particular pyridine
containing class of quinolone carboxylic acid allosteric modulators.
In summary, the synthesis and SAR of pyridine containing quin-
olone carboxylic acid M1 positive allosteric modulators has been
detailed in an effort to identify potent compounds with reduced
plasma protein binding. It was initially found that pyridine and
pyridazines were acceptable replacements for the phenyl B-ring,
but further analogs showed the pyridines presented to be the most
flexible with respect to SAR via modification of the C-ring. The bi-
11. 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. 2009, 19.
12. Kuduk, S. D.; DiPardo, R. M.; Chang, R. K.; Di Marco, C. N.; Murphy, K. L.;
Ransom, R.; Tang, C.; Prueksaritanont, T.; Pettibone, D. J.; Bock, M. G. Bioorg.
Med. Chem. Lett. 2007, 17, 3608.
13. Selected compounds were examined in functional assays at other muscarinic
receptors and showed no activity at M2, M3, or M4 receptors.
14. As a typical example of other pharmacokinetic properties, acid 6o gave low
clearance (Cl = 6.0 ml/min/kg), Vdss = 0.7, and a half-life of 5.1 h in dog.
15. A number of isosteres have been prepared in an effort to alter transport
affinity, but the carboxylic acid group is required for M1 functional activity.
16. Shipe, W.D. et al., in preparation.