Hydroxy cycloalkyl fused pyridone carboxylic acid M1 positive allosteric modulators

Article abstract of DOI:10.1016/j.bmcl.2010.02.095

Incorporation of hydroxycycloalkyl fused pyridone carboxylic acids in lieu of quinolone carboxylic acids enhance free fraction without increased susceptibility to P-glycoprotein transport.

Full text of DOI:10.1016/j.bmcl.2010.02.095

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2538–2541
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Hydroxy cycloalkyl fused pyridone carboxylic acid M1 positive
allosteric modulators
a
a
b
b
b
Scott D. Kuduk a, , Robert M. DiPardo , Douglas C. Beshore , 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:
Incorporation of hydroxycycloalkyl fused pyridone carboxylic acids in lieu of quinolone carboxylic acids
enhance free fraction without increased susceptibility to P-glycoprotein transport.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 4 February 2010
Revised 24 February 2010
Accepted 25 February 2010
Available online 10 March 2010
Keywords:
M1
Muscarinic
Alzheimer’s
Quinolone
Allosteric
The central cholinergic nervous system serves essential func-
tions and is activated by acetylcholine as the endogenous ligand,
targeting nicotinic and metabotropic muscarinic receptors. The lat-
ter are class A G-protein coupled receptors (GPCR) of which there
are five muscarinic subtypes, designated M1–M5.1,2
The progressive degeneration of cholinergic neurons in Alzhei-
mer’s disease (AD) is proposed as a leading cause of the resultant
cognitive decline.3 A therapeutic approach would be the direct
activation of the M1 receptor, which is highly expressed in the
affected brain regions,4 implying it may play a central role in mem-
ory and higher brain function.5 Non-selective M1 agonists exhib-
ited improved cognitive performance in AD patients, but
exhibited intolerable side effects attributed to activation of the
highly conserved orthosteric acetylcholine binding site of other
muscarinic sub-types.6,7
The activation of a less-highly conserved allosteric binding site
in preference to the orthosteric domain, is one pathway to produce
selectivity for M1 over the other sub-types.8,9 Quinolone carboxylic
acid 1 has been described as a selective positive allosteric modula-
tor of the M1 receptor with excellent specificity for the M1 sub-
type.10,11 SAR evaluation of 1 led to the identification of biaryl
replacements for the para-methoxybenzyl group such as biphenyl
2 (Fig. 1),12 but higher plasma protein binding led to decreased free
CNS exposure impeding further in vivo evaluation. Previous SAR ef-
forts on the A-ring showed substitution was not tolerated, with the
exception of fluorination at the 5 and 8 positions. The preceding
paper described efforts to identify heterocyclic replacements for
the phenyl A-ring.13 This communication describes efforts to iden-
tify non-aromatic, cycloalkyl A-rings that would retain M1 potency
and show reduced protein binding leading to improved in vivo
activity.
The synthesis of requisite A-ring modified biphenyls is shown
in Scheme 1. Condensation of 3-aminocyclohexenone 3 with
O
N
O
O
N
O
5
A
8
OH
OH
B
OMe
C
1: M1 IP = 660 nM
2: M1 IP = 212 nM
-Higher protein binding
PB = 96-98%
-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.2010.02.095

S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2538–2541
2539
EtO2C
HN
CO2Et
O
O
NH2
EtO2C
EtO
CO2Et
a
CO2Et
d, e
f
b
10e
10f
10a
10b
+
N
H
O
O
9a
3
4
8a
EtO2C
HN
O
CO2Et
EtO2C
H2N
CO2Et
O
d, e
c
b
CO2Et
f
O
+
N
H
O
O
5
6
9b
8b
O
EtO2C
HN
CO2Et
EtO2C
H2N
CO2Et
d, e
O
b
CO2Et
c
10c,d
+
( )n
N
H
( )n
7a,b
( )n
6
9c-d
8c-d
Scheme 1. Reagents and conditions: (a) 130 °C, neat; (b) diphenylether, 230 °C; (c) 4-toluene sulfonic acid, toluene, reflux; (d) 4-(bromomethyl)biphenyl, DMF, K2CO3; (e) 1 N
NaOH, THF, EtOH; (f) NaBH4, THF, MeOH.
ethoxymethylene malonate 4 produced 8a. Similarly, condensation
of di-ketone 5 or ketones 7a,b with aminomethylene malonate 6
provided 8b and 8c. Cyclization of 8a–d was carried out using a
modified Gould–Jacobs cyclization14 to afford heterocycles 9a–
d.15 Alkylation of 9a–d with 4-(bromomethyl)biphenyl followed
by subsequent ethyl ester hydrolysis afforded carboxylic acids
10a–d. The ketones present in 10a,b were converted to alcohols
10e,f via reduction with sodium borohydride.
The preparation of analogs bearing modified B/C-ring combina-
tions is shown in Scheme 2. Alkylation of 10a with appropriate ha-
lide followed by sodium borohydride reduction affords alcohols
11a (X = N) or 11b (X = CH). Subsequent Suzuki cross-coupling fol-
lowed by ester hydrolysis affords acids 10h–y.
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 represents the effect of compound and
EC20 of acetylcholine relative to the maximal possible acetylcholine
effect. Plasma protein binding was determined using the equilib-
rium dialysis method in the presence of rat and human serum.
Data for modified A-ring compounds is shown in Table 1. Cyclo-
hexanone 10a lost ꢀfourfold in terms of M1 activity relative to
quinolone 2, but exhibited a markedly reduced plasma protein
binding profile. The corresponding 8-isomer 10b was substantially
less active (M1 IP = 4.8 lM), while the cyclohexyl 10c and cyclo-
pentyl 10d were comparable with minimal improvement in free
fraction compared to 2. Reduction of 10a to secondary alcohol
10e provided the most potent compound amongst the series (M1
IP = 440 nM) with high maximal acetylcholine activity, represent-
ing only a two fold potency decrease with respect to 2. The 8-hy-
droxy isomer 10f and the methyl ether of 10e were substantially
less potent.17 Based upon the good potency and higher free fraction
(ꢀ5%) of 10e relative to 2, additional SAR evaluation of the B and C
rings with the 5-hydroxycyclohexane in place was investigated
with selected compounds as shown in Table 2.
Previously, it was observed in the quinolone series that incorpo-
ration of a 2-pyridyl (10h,i) in place of the phenyl B-ring reduced
protein binding and was neutral in terms of potency.18 Compound
10h did possess enhanced free fraction, but both analogs lost M1
activity implying a poor SAR translation from the quinolone series.
In addition, a range of substitutions at the three positions of the
phenyl C-ring were evaluated (10j–o), and the SAR was flat, with
the meta isomers proving to be the most well tolerated.
OH
O
O
O
CO2Et
CO2Et
a, b
N
N
H
Lipophilic heterocycles such as thiophene 10p and furan 10q
showed no potency advantages. In earlier quinolone SAR, N-linked
heterocycles exhibited good properties over their C-linked coun-
terparts,19 but pyrazole 10r and imidazole 10s had reduced M1
activity. Substituted pyridines (10t–w) also were not particularly
advantageous, with only methoxypyridine 10u showing improved
M1 potency. Lastly, substituted pyrazoles were investigated with
isobutyl 10y exhibiting the best potency of all B/C-ring analogs
examined with a similar free fraction relative to biphenyl 10e.
Overall, the SAR for B/C-ring combinations was flat20 and did not
translate well from the quinolone SAR to this hydroxycyclohexane
class of compounds.
10a
X
Br
OH
O
N
11a, b
CO2H
c, d
X
R
10h-y
Scheme 2. Reagents and conditions: (a) ArCH2X, DMF, K2CO3; (b) NaBH4, THF,
MeOH; (c) Pd(OAc)2, X-PHOS, K2CO3, CH3CN, H2O, 80 °C; (d) 1 N NaOH, THF, EtOH.

2540
S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2538–2541
Table 1
Table 2 (continued)
M1 FLIPR and protein binding data for select compounds
Compd
R1
M1 Pot IPa (nM)
Rat PB
—
Human PB
—
O
O
OH
R1
10k
410
N
OMe
10l
1386
1400
—
—
—
—
OMe
Compd
R1
M1 Pot IPa (nM)
212
% Max
92
Rat PB
98.7
Human PB
99.5
10m
Cl
2
10n
10o
340
340
—
—
—
—
O
Cl
10a
10b
800
94
70
86.7
—
91.4
—
Cl
4890
10p
10q
10r
10s
2640
750
—
—
S
O
—
—
10c
10d
751
810
91
99
98.5
97.9
98.8
98.5
O
N
N
N
1369
2500
62.3
21.2
56.1
40.9
OH
N
10e
10f
10g
440
8190
1100
99
59
90
93.6
—
94.4
—
10t
10u
10v
14790
320
—
—
—
—
—
—
N
N
N
N
F
OH
OMe
OMe
NH2
N
—
—
2425
a
Values represent the numerical average of at least two experiments. Interassay
variability was 30% (IP, nM), unless otherwise noted.
10w
1095
—
—
Table 2
10x
10y
908
200
59.4
84.4
82.1
94.9
N
N
M1 FLIPR and protein binding data for select compounds
OH
O
O
N
N
OH
N
a
Values represent the numerical average of at least two experiments. Interassay
variability was 30% (IP, nM), unless otherwise noted.
X
R1
Compd
R1
M1 Pot IPa (nM)
Rat PB
78.6
Human PB
75.4
Selected compounds were examined for CNS exposure as shown
in Table 3. Since P-glycoprotein (P-gp) is a major efflux transporter
of xenobiotics at the blood–brain barrier (BBB), P-gp efflux in hu-
man (MDR1) and rat (MDR1a) P-gp, as well as passive permeabil-
ity, were evaluated to triage potential candidates. Biphenyl 10e
and phenylpyrazole 10x exhibited good permeability (Papp >15),
but N-linked phenylpyrazole 10r was low removing it from further
consideration.
The two remaining compounds (10e, and 10x) were evaluated
for brain exposure in rat, utilizing oral dosing (10 mpk) and sam-
pling at a 2 h time point. Biphenyl 10e gave a modest CSF/Uplasma
ratio of 0.22 and a suitably high plasma concentration. Phenylpyr-
10h (X = N)
1100
F
10i (X = N)
420
—
—
—
—
NH2
10j
3100
MeO

S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2538–2541
2541
Table 3
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/Uplasma
d
Compd
10e
10r
10x
20
2.8
19
1.5
3.5
2.2
1.9
8.1
7.3
6145
—
26677
146
—
368
87
—
110
0.03
—
0.01
0.22
—
0.10
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 1 and 2.
d
improvement over compound 1, where ꢀ33
l
M plasma was re-
120
quired for in vivo efficacy.
110
100
90
80
70
60
50
40
30
20
10
0
Vehicle
In summary, a series of substituted cycloalkyl fused pyridone car-
boxylic acids in lieu of quinolone carboxylic acids were prepared and
evaluated. Optimal A-rings were the cyclohexane and 5-hydroxy
cyclohexane 10e. The SAR for B/C-ring combinations of 10e was flat
relative to previous data with the quinolones. Potentiator 10e
showed adequate CNS exposure and performed very well in a mouse
model of episodic memory. Additional structural types employing
the hydroxycyclohexane A-ring motif are undergoing evaluation.
0.01µM 10e
0.1µM 10e
1µM 10e
10µM 10e
100µM 10e
-10
-13 -12 -11 -10 -9 -8 -7 -6 -5 -4
References and notes
Log[M], Acetylcholine
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Scop (0.3 mpk)
Scop/10e (3 mpk)
Scop/10e (10 mpk)
Scop/10e (30 mpk)
#
#
#
*
Baseline
24 Hr
Figure 3.
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azole 10x exhibited robust plasma levels, but a lower CSF/Uplasma
ratio of 0.10, which could be attributed to the result of 10x being
a substrate for rat P-gp.
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D.; Hartman, G. D. Bioorg. Med. Chem. Lett. 2009, 19, 531.
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18. Kuduk, S. D.; Di Marco, C. N.; Cofre, V.; Pitts, D. R.; Ray, W. J.; Ma, L.; Wittmann,
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Compound 10e was evaluated for the ability to fold potentiate a
dose response of acetylcholine (Fig. 2). In the presence of 1 lM or
greater concentration of potentiator 10e, a leftward-shift was ob-
served in the acetylcholine dose response curve showing it is a po-
tent positive allosteric modulator of the human M1 receptor. No
effects were seen at concentrations that were below the inflection
point (440 nM) of 10e.
Based on the observed M1 potency and reasonable CSF/Uplasma
ratio, compound 10e was evaluated in a mouse contextual fear
conditioning assay, which serves as a model of episodic memory
(Fig. 3). In this experiment, mice were treated with scopolamine
before introduction to a novel environment to block this new asso-
ciation. Mice dosed ip with all three doses of 10e exhibited a full
reversal compared to mice treated with scopolamine alone. The
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21. CSF levels were not examined.
corresponding plasma levels at 3 mpk were 6 l
M,21 a notable