Improving the oral efficacy of CNS drug candidates: Discovery of highly orally efficacious piperidinyl piperidine M2 muscarinic receptor antagonists

Article abstract of DOI:10.1021/jm0255163

In search of a backup M₂ muscarinic receptor antagonist to the previously reported compound 1, we discovered compound (+)-14, which showed superior oral efficacy in animal models. The improvement of oral efficacy was achieved by modulating both

Full text of DOI:10.1021/jm0255163

© Copyright 2002 by the American Chemical Society
Volume 45, Number 25
December 5, 2002
Letters
centrally and peripherally.3 Muscarinic autoreceptors
(M2), located presynaptically on cholinergic nerve ter-
minals, are associated with the inhibitory feedback
regulation of ACh release. Muscarinic M2 receptor
antagonists such as himbacine and AF-DX116 have
been demonstrated to increase the levels of ACh release
in rat striatum.4a Since increased levels of ACh are
beneficial for the improvement of cognition,5 we have
embarked on the discovery of potent and selective
muscarinic M2 receptor antagonists for the treatment
of neurodegenerative diseases such as Alzheimer’s
disease (AD). Our criteria for selection of a muscarinic
M2 receptor antagonist as a drug candidate required
high M2 receptor binding affinity (Ki < 5 nM), 100-fold
selectivity versus M1 and M3 receptor subtypes, as well
as oral efficacy in animal models.6
Im p r ovin g th e Or a l Effica cy of CNS Dr u g
Ca n d id a tes: Discover y of High ly Or a lly
Effica ciou s P ip er id in yl P ip er id in e M2
Mu sca r in ic Recep tor An ta gon ists
Yuguang Wang,* Samuel Chackalamannil,
Zhiyong Hu, William J . Greenlee, J ohn Clader,
Craig D. Boyle, J ames J . Kaminski, William Billard,
Herbert Binch, III, Gordon Crosby, Vilma Ruperto,
Ruth A. Duffy, Mary Cohen-Williams, Vicki L. Coffin,
Kathleen A. Cox, Diane E. Grotz, and
J ean E. Lachowicz
Schering-Plough Research Institute, 2015 Galloping Hill
Road, Kenilworth, New J ersey 07033
Received February 25, 2002
To screen the synthesized muscarinic M2 receptor
antagonists for oral efficacy, we used the rat micro-
dialysis and the young rat passive avoidance response
(PAR) assays. In the microdialysis assay, the level of
ACh released from rat striatum is measured after oral
administration of a drug, thereby determining the
ability of the drug to reach brain M2 receptors and block
the ACh negative feedback mechanism.4 The PAR assay
is a cognitive paradigm which measures whether refer-
ence memory is improved upon oral administration of
a drug.7
Abstr a ct: In search of a backup M2 muscarinic receptor
antagonist to the previously reported compound 1, we discov-
ered compound (+)-14, which showed superior oral efficacy in
animal models. The improvement of oral efficacy was achieved
by modulating both the molecular weight and lipophilicity of
the lead compounds.
In tr od u ction . The design of drug candidates with
good oral efficacy is a major challenge in modern drug
discovery. The oral efficacy of a CNS drug can be
affected by many factors such as oral bioavailability and
blood-brain barrier (BBB) permeability. Numerous
publications have addressed the relationship between
oral activity and the physicochemical properties of drug
candidates.1,2 For example, the rule-of-52 can be em-
ployed to predict the oral behavior of a drug by the
physicochemical properties of molecular weight (MW),
lipophilicity (logP), and hydrogen bonding. In addition,
metabolic stability must be taken into account when
designing orally active drugs. Herein, we would like to
report the positive outcome of our efforts to optimize
the oral efficacy of selective muscarinic M2 receptor
antagonists.
Ra tion a le. Compounds 1 and 2 are representatives
of our first generation of muscarinic M2 receptor an-
tagonists (Figure 1).8 These compounds were highly
potent with the required subtype selectivity (Table 1).
In addition, both compounds were orally active in rat
microdialysis and rat PAR assays. Compound 1 was
selected for phase I clinical trials based on its plasma
concentration and efficacy upon oral dosing.8a
A common characteristic of compounds 1 and 2 was
a high molecular weight (MW > 550). In the develop-
ment of a backup candidate for compound 1, we syn-
thesized lower molecular weight analogues by truncat-
ing the left-hand portion of compound 1. Extensive
structure-activity relationship (SAR) studies led to the
discovery of the lower MW analogue 3 (Figure 1).9
Muscarinic receptors have been shown to mediate the
actions of the neurotransmitter acetylcholine (ACh) both
* To whom correspondence should be addressed. Phone: 908-740-
3508. Fax: 908-740-7152. E-mail: yuguang.wang@spcorp.com.
10.1021/jm0255163 CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/05/2002

5416 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25
Letters
Ta ble 2. Profile of Tetrasubstituted Olefin Analogs
F igu r e 1. Structures of earlier M2 receptor antagonists.
Ta ble 1. Properties of the Earlier M2 Receptor Antagonists
a
compd M2 M1/M2
MDb
PARc MW ClogPd rat PKe
18a
28b
39
0.89 734 180 ( 18
0.51 125 180 ( 25
0.001 566
0.1 659
205 110 ( 15(po) inact 487
210 ( 21 (iv)
4.6
5.0
6.3
908
603
nd
0.9
a
Ki, nM; all determinations were performed three times (SEM
b
< 15%). Microdialysis: %ACh release compared with baseline
in rat striatum, 0 f 2 h, 10 mg/kg, po, 20% HPâCD (baseline )
100%). c Passive avoidance response: orally active dose (mg/kg,
significant change in latency over vehicle, n ) 36, SEM < 10%).
a
Ki, nM; all determinations were performed three times (SEM
b
< 15%). Human microsomal stability: % parent compound
remaining after 20 min incubation with human liver microsomes.
c ClogP values were calculated using SYBYL version 6.6 accessed
via the ClogP column type and various expression generators with
a special license (Biobyte) available from Tripos, Inc.
d
ClogP values were calculated using SYBYL version 6.6 accessed
via the ClogP column type and various expression generators with
a special license (Biobyte) available from Tripos, Inc. e AUC (high
throughput single measurement): h•ng/mL, 0 f 6 h, 10 mg/kg,
po, 20% HPâCD.10
Compound 3 met our binding criteria and was sub-
jected to the rat microdialysis assay. While it was highly
active after intravenous (iv) dosing, compound 3 did not
show the desired level of oral efficacy in this assay
(Table 1). In addition to its low metabolic stability,11 the
high lipophilicity (ClogP ) 6.3) of compound 3 also
contributed to its low oral activity in the microdialysis
assay. To improve the oral efficacy, we focused on
modulating the lipophilicity and metabolic stability in
this series of compounds. We oxidized the sulfide atom
of compound 3 to a more polar and robust sulfone
moiety. At the right-hand side of compound 3, the
naphthyl group was replaced with either a metabolically
more stable 4-fluoronaphthyl group (9) or a polar
2-amino-3-methylphenyl group (10). However, com-
pounds 9 and 10 showed undesirable M2 receptor Ki
values (Table 2). Our challenge was to improve the M2
receptor binding affinity of compounds 9 and 10.
Among the known strategies to increase receptor
binding affinity, we chose conformational restriction. A
double bond was introduced at the benzylic position as
shown to limit the degrees of freedom in the truncated
analogues (Figure 2).
F igu r e 2. Conformational restriction by a double bond.
oxidation transformed amine 6 to sulfoxide 7 or sulfone
8. Moderate selectivity in the formation of sulfoxide 7
or sulfone 8 was achieved by adjusting the number of
equivalents of sodium perborate during the oxidation
step.12 Both compounds 7 and 8 were treated with TFA
to remove the N-BOC protecting group, and subsequent
coupling with different aromatic acids provided the final
targets.
Resu lts a n d Discu ssion . The binding affinity and
selectivity of the synthesized targets against cloned
human muscarinic receptors were assayed according to
the reported protocol.13 To help select the right drug
candidate for clinical trials, the stability of our M2
antagonists in human liver microsomes after a 20 min
incubation time was assessed.14
A comparison of the muscarinic M2 receptor binding
affinities of the compounds in Table 2 indicated that the
introduction of a double bond improved the binding
affinity. The muscarinic M2 receptor binding affinity
was increased by more than 20-fold from 9 to 11 and
3-fold from 10 to 12. More importantly, the M2 receptor
Ki values of 13 and 14 coupled with their microsomal
stability results warranted further studies of these two
sulfoxides.
Ch em istr y. The synthesis of the tetrasubstituted
olefins is outlined in Scheme 1.
Protection of commercially available compound 4 was
followed by the displacement of fluorine atom with
isopropylthiol to form sulfide/ketone 5. Addition of
methyllithium to ketone 5 followed by dehydration in
refluxing TFA/CH2Cl2 (1:1) afforded amine 6 as the only
olefin regioisomer. Reductive amination and sulfide

Letters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25 5417
Sch em e 1a
a
Conditions: (a) (BOC)2O, 10% NaOH/Et2O, 95%; (b) NaH, DMF, isopropylthiol, 65 °C, 6 h, 92%; (c) MeLi, THF, 90%; (d) 50% TFA/
CH2Cl2, reflux, 92%; (e) NaBH(AcO)3, 1,2-dichloroethane, 1-tert-butoxycarbonyl-4-piperidone, 75%; (f) NaBO3, HOAc, 65%; (g) 30% TFA/
CH2Cl2, 95%; (h) ArCOOH, EDCI, DMAP, CH2Cl2, 85-96%.
Ta ble 3. Binding Affinity of Chiral Sulfoxides
a
compd
M2
M1/M2
M3/M2
M4/M2
M5/M2
(-)-13
(+)-13
(-)-14
(+)-14
1
2.4
1.3
12
0.89
0.89
245
166
6
101
734
376
122
nd
170
787
36
7
nd
22
69
35
6
nd
36
96
a
Ki, nM; all determinations were performed three times (SEM
< 15%).
Ta ble 4. Efficacy and Pharmacological Profile of Chiral
Sulfoxides
compd
MDa
PARb
nd
nd
nd
0.0001
0.001
rat PKc
MW
ClogPd
F igu r e 3. PAR data of (+)-14.7 *Significant changes in latency
over vehicle (4% MC) (p < 0.05, Dunnet’s “T” test, n ) 36).
(-)-13
(+)-13
(-)-14
(+)-14
1
155 ( 18
179 ( 18
nd
225 ( 35
180 ( 21
nd
nd
nd
5149
908
533
533
494
494
566
4.5
4.5
2.8
2.8
4.6
a
Microdialysis: %ACh release compared with baseline in rat
b
striatum, 0 f 2 h, 10 mg/kg, po, 20% HPâCD. Passive avoidance
response: orally active dose (mg/kg, significant change in latency
over vehicle, n ) 36, SEM < 10%). c AUC (high throughput single
measurement): h•ng/mL, 0 f 6 h, 10 mg/kg, po, 20% HPâCD.10
d
ClogP values were calculated using SYBYL version 6.6 accessed
via the ClogP column type and various expression generators with
a special license (Biobyte) available from Tripos, Inc.
Next, we prepared the enantiomers of sulfoxides 13
and 14. These targets were derived from (+)-7 and (-)-7
which were separated by chiral HPLC.15 Three of the
enantiomerically pure compounds met our binding
criteria ((-)-13, (+)-13, and (+)-14; Table 3) and were
tested further in vivo.
F igu r e 4. Comparison of microdialysis data of (+)-14 and 1.
Significant stimulation over preinjection baseline (p ) 0.05,
Duncan’s Multiple Range Statistic). Drug concn ) 10 mg/kg,
po; vehicle HP âCD.
14 in human microsomal incubation studies.16 There
was no indication of metabolism of the tetrasubstituted
double bond.
Among the resolved enantiomers, compound (+)-14
demonstrated superior oral efficacy in both the micro-
dialysis and PAR assays (Table 4). Compared with
compound 1, the new lead (+)-14 increased ACh release
in the microdialysis assay by 45%, and showed equiva-
lent oral efficacy in the rat PAR assay at a 10-fold lower
dose. It was not surprising that the rat plasma level of
compound (+)-14 was approximately five times that of
compound 1 upon oral administration. Figures 3 and 4
summarize the PAR and microdialysis4b data of (+)-14.
We attributed the excellent oral efficacy of (+)-14 to
its relatively low molecular weight (MW ) 494) and
optimized lipophilicity (ClogP ) 2.8). We believed that
the data in Table 4 provided a convincing example of
the effect that modulating the physical chemical proper-
ties of a compound can have on oral efficacy. In addition,
for further development of this series of compounds, we
specifically examined the metabolites of (+)-13 and (+)-
Con clu sion . We have successfully improved oral
efficacy of the first generation muscarinic M2 receptor
antagonists. The discovery of (+)-14 demonstrated the
effects of molecular weight and lipophilicity on the oral
efficacy of this class of CNS drug candidates. We also
applied the strategy of conformational restriction to
increase the muscarinic M2 receptor binding affinity
significantly. The excellent muscarinic M2 receptor
binding affinity of the tetrasubstituted double bond
series made the modulation of molecular weight and
lipophilicity possible.
Ack n ow led gm en t. The authors would like to thank
Mr. Yao H. Ing and Dr. P. Das for mass spectral data;
Dr. Blythin for chiral HPLC separation condition.

5418 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25
Letters
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G.; Billard, W.; Binch, H., III; Crosby, G.; Cohen-Williams, M.;
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Su p p or tin g In for m a tion Ava ila ble: Analytical data and
spectral information available for the key compounds dis-
cussed. This material is available free of charge via the
Internet at http://pubs.acs.org.
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equiv of NaBO3; sulfone was obtained as a major product (80-
95%) with 2.5-3 equiv of NaBO3. Sulfoxide and sulfone can be
easily separated with flash column chromatography. For separa-
tion details, please see the Supporting Information.
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paradigm than in the PAR assay.
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(14) Compounds (10 µg/mL concentration) were incubated in human
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percent of the parent compound remaining after 20 min. The
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2592), which was used as a reference incubated at the same time
under the same conditions. A difference of 30% was considered
significant, and compounds that showed stabilities 30% more
than SCH 72788 were considered further. SCH 72788 typically
had 15-30% parent remaining after 20 min. See the following
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(15) For details of chiral HPLC separation of 7, please see the
Supporting Information.
(16) Compounds were subjected to human liver microsomal incuba-
tion for 30 min. The metabolite characterization was done by
LC MS/MS of the microsomal incubation. The details will be
reported by Cox, K. A., et al.
J M0255163