Substituted 2-(R)-Methyl piperazines as muscarinic M2 selective ligands

Article abstract of DOI:10.1016/S0960-894X(02)00023-9

A novel series of 2-(R)-methyl-substituted piperazines (e.g., 2) is described. They are potent M₂ selective ligands that have > 100-fold selectivity versus the M₁ receptor. In the rat microdialysis assay, compound 14 showed significantly enchanced levels of acetylcholine after oral administration.

Full text of DOI:10.1016/S0960-894X(02)00023-9

Bioorganic & Medicinal Chemistry Letters 12 (2002) 791–794
Substituted 2-(R)-Methyl Piperazines as Muscarinic
M2 Selective Ligands
Joseph A. Kozlowski,* Guowei Zhou, Jayaram R. Tagat, Sue-Ing Lin,
Stuart W. McCombie, Vilma B. Ruperto, Ruth A. Duffy, Robert A. McQuade,
Gordon Crosby, Jr., Lisa A. Taylor, William Billard, Herbert Binch, III and
Jean E. Lachowicz
Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033-0539, USA
Received 19 October 2001; accepted 21 December 2001
Abstract—A novel series of 2-(R)-methyl-substituted piperazines (e.g., 2) is described. They are potent M2 selective ligands that
have >100-fold selectivity versus the M1 receptor. In the rat microdialysis assay, compound 14 showed significantly enchanced
levels of acetylcholine after oral administration. # 2002 Elsevier Science Ltd. All rights reserved.
People suffering from Alzheimer’s Disease (AD) show a
progressive loss of memory and cognitive function,
which has been associated with impairment of the cho-
linergic system.1 One method of treatment for AD is to
increase acetylcholine levels through administration of
acetylcholinesterase inhibitors.2 An alternate approach
is to develop drugs that enhance the release of acetyl-
choline.1,3 The approach we have chosen is to enhance
acetylcholine levels through antagonism of presynaptic
M2 muscarinic receptors.4À6 The M1/M2 selectivity is
crucial since antagonism of post-synaptic M1 receptors,
with agents such as scopolamine, is known to produce
cognitive deficits.7 Recently we reported on a series of
M2 selective antagonists 1, which increased acetylcho-
line levels in a microdialysis paradigm in rats.8
Although the chemical series described showed desirable
activity in vitro and in vivo, we sought to find an alter-
native to the chiral sulfoxide, and a-cyano amine moi-
eties. We wish to report our progress in this area as
represented by structure 2, wherein the sulfoxide and
cyano moieties were replaced with sulfone, and (S)-
methyl, respectively (Fig. 1).
p-fluorobenzaldehyde in DMF, gave the coupled
diphenylsulfide. Reaction with Ti(i-OPr)4, 4-cyclohexyl-
2-(R)-methylpiperazine, and either methylmagnesium
bromide or diethylaluminum cyanide or sodium cyano-
borohydride gave the product with R equal to methyl,
cyano, or hydrogen, respectively. When R is methyl, the
mixture of diastereomers was separated by flash chro-
matography. The cyclohexylpiperazine was prepared
from the reaction of (R)-methyl piperazine with cyclo-
hexanone, Ti(i-OPr)4, and NaCNBH3. Oxidation with
m-chloroperoxybenzoic acid (m-CPBA) gave the sul-
fone. Stereochemical assignment of the benzylic methyl
was done via NOE studies, following flash chromato-
graphic separation of the diastereomers.
A non-racemic route to compounds containing an
(S)-methyl group at the benzylic carbon is described
in Scheme 2. Sodium hydrogensulfide was added
to p-fluoroacetophenone, and BHT in hot DMF to
Compounds containing a cyclohexylpiperazine, with R2
hydrogen (Table 1), were prepared as shown in Scheme
1. Reaction of the sodium salt of benzenethiol and
*Corresponding author. Tel.: +1-908-740-3488; fax: +1-908-740-
7152; e-mail: joseph.kozlowski@spcorp.com
Figure 1.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00023-9

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J. A. Kozlowski et al. / Bioorg. Med. Chem. Lett. 12 (2002) 791–794
generate sodium-4-mercapto acetophenone, which was
coupled in the same pot with 3,4-methylenedioxy-
iodobenzene under Suzuki’s conditions.9 After work up
and chromatography, the ketone was isolated in ꢀ80%
yield. Oxidation of the diphenylsulfide to the sulfone
was accomplished with m-CPBA. Corey’s enantio-
selective reduction gave the chiral (R)-alcohol in >98%
yield and 94% ee.10 Mesylation and displacement with
4-N-Boc-2-(R)-methylpiperazine gave the (S)-benzylic
methyl. Removal of the BOC with acid and reductive
amination N-BOC-4-piperidinone proceeded in good
yield.11 Deprotection of the BOC gave the free piper-
idine, which was converted to amides, sulfonamides,
and ureas with acid chlorides, sulfonyl chlorides, or
isocyanates, respectively.
similar to 1 observed. In looking for alternates to the
Strecker amine moiety, we focused on methyl as a non-
racemizable replacement for the cyano group. Thus,
substituting the cyano of compound 3 with methyl gave
the diastereomeric compounds 6 and 7. A slight pref-
erence for (S)-methyl at the benzylic site was observed,
with 6 was apparent. The importance of the combina-
tion of the benzylic and piperazine methyl together was
Scheme 1. (a) NaH, 0 ꢁC, DMF, add p-fluorobenzaldehyde, warm to
rt, 12 h; (b) 4-cyclohexyl-2-(R)-methylpiperazine, rt, Ti(i-OPr)4,
CH3MgBr or Et2AlCN or NaCNBH3, for R=CH3, CN, H, respec-
tively, flash chromatography; (c) m-CPBA, CH2Cl2, rt.
Compounds with R2 equal to methyl (Table 1) were
prepared from either (R)- or (S)-methyl lactate (Scheme
3). 2-Nitrophenylsulfonyl chloride was treated with
aminoethanol, and the product was acylated with acetic
anhydride in pyridine. This sulfonamide was treated
with the triflate of either (R)- or (S)-methyl lactate, fol-
lowed by reduction of the esters with borane-methyl
sulfide complex. The piperazine was formed from reac-
tion of the diol, activated with triflic anhydride, with the
diphenylsulfonyl-a-methylbenzylamine, prepared as
shown from a-methylbenzylamine. Removal of the sul-
fonamide protecting group, under Fukayama’s condi-
tions,12 gave the free piperazine, which was converted to
9 or 10.
The binding affinities for synthesized compounds
described here were determined using cloned human
muscarinic receptors, as previously described.13 Our
initial objective was to find a compound with affinity
and selectivity comparable to or better than 1, but
without the chiral sulfoxide and a-cyano amine moi-
eties. First we focused on switching the chiral sulfoxide
of 1 to a sulfone, thereby simplifying the synthetic
scheme and avoiding the possibility of metabolic race-
mization of the sulfoxide in vivo. The chirality lost in
changing sulfoxide to sulfone, was introduced again by
addition of a methyl to the piperazine ring (e.g., 3 and 4,
Table 1). A preference for the (R)-configuration for the
R1 methyl was apparent (e.g., 3) with M1/M2 selectivity
Scheme 2. (a) NaHS, DMF, 95 ꢁC, 2 h; (b) 3,4-methylenedioxy-
iodobenzene, CuI, K2CO3, DMF, 100 ꢁC, 6 h; (c) m-CPBA, NaHCO3,
1,2-dichloroethane, rt, 24 h; (d) 0.6 equiv BH3(CH3)2S, THF, rt, 0.2
equiv Corey’s oxoborolidine catalyst; (e) CH3SO2Cl, CH2Cl2, 0 ꢁC,
Et3N; (f) tetramethylpiperidine, 2-(R)-methyl-4-BOC-piperazine,
CH3CN, reflux, 12 h; (g) 6 N HCl, reflux 7 h; (h) NaHB(OAc)3, N-
BOC-4-piperidinone; (i) 6 N HCl, rt 3 h; (j) EtSO2Cl, Et3N, rt.
Table 1.
Compd
R
R1
R2
Ki (nM)
M2
M1/M2
3
4
5
6
7
8
9
10
CN
CN
CH3
(S)-CH3
(R)-CH3
H
(R)-CH3
(R)-CH3
(R)-CH3
(S)-CH3
H
(R)-CH3
(R)-CH3
(R)-CH3
H
H
H
H
H
H
0.16
16
0.6
0.3
0.15
0.3
36
5
Scheme 3. (a) 2-Hydroxyethyl amine, rt, CH2Cl2; (b) acetic anhydride,
pyridine, rt; (c) (R)-CH3CH(OSO2CF3)CO2Et; (d) BH3(CH3)2S, THF;
(e) Tf2O, Collidine, CH2Cl2, 0 ꢁC; (f) 2 equiv Na2CO3, CH3CN, rt
24 h; (g) HSCH2CO2H, LiOH–H2O; (h) Ti(i-OPr)4, cyclohexanone,
NaCNBH3; (i) 1,3-dibromo-5,5-dimethylhydantoin, rt, MeSO3H,
CH2Cl2; (j) n-BuLi, THF, À70 ꢁC, then ArSO2F, warm to rt; (k)
KOH, MeOH, rt.
10
44
30
23
10
10
H
(S)-CH3
(R)-CH3
0.04
0.14
H

J. A. Kozlowski et al. / Bioorg. Med. Chem. Lett. 12 (2002) 791–794
793
demonstrated by removing each separately (e.g., 5 and
8); a significant loss of selectivity resulted when either
the benzylic or piperazine methyl was replaced with
hydrogen.
improved M2 selectivity. Although this strategy was
successful with piperidines,14 it was untested with the
methylated-piperazines described here. The results of
these modifications are summarized in Table 2. When
the 4-methoxyphenyl was exchanged with 3,4-methyl-
enedioxy phenyl, an increase in M2 affinity and selec-
tivity was observed (e.g., 11 vs 6). In general, piperidine
derivatives such as sulfonamides and amides were more
selective than cyclohexyl piperazine, but the free piper-
idine, 13, had weaker affinity and lower selectivity. Aryl
sulfonamides maintained M2 affinity, but were less
selective than the corresponding alkyl sulfonamides.
Both amides and urea’s lost affinity and selectivity
relative to the equivalent alkyl sulfonamide.
The 3-methyl piperazines (R2=methyl and R1=hydro-
gen) indicate that the position of the methyl in the
piperazine ring is important for optimizing M2 selectiv-
ity, as these compounds showed good affinity but not
selective.
To further optimize the binding profile of 6, we chose to
add more functionality to the structure by conversion of
the cyclohexyl to a 4-piperidine, and replacing the
4-methoxyphenyl with 3,4-methylenedioxy phenyl. It
was anticipated that additional steric and hydrogen-
bonding sites could provide an opportunity for
The in vivo effect of 14 was measured using a micro-
dialysis paradigm,5,15 in which the acetylcholine level
was measured as a function of time through a dialysis
probe inserted into the striatum of conscious rats.
Compound 14 produced a sustained increase in acetyl-
choline levels (Fig. 2), which was not observed with the
more selective 15 (data not shown). The affinity of 14
and 15 are similar, and since acetylcholine release is
determined by M2 potency, not M1/M2 selectivity, the
differences observed in the microdialysis paradigm may
reflect differences in the pharmacokinetic profiles of 14
and 15.
Table 2.
Compd
R
X
R3
Ki (nM)
M1/M2
M2
We have demonstrated that both the chiral sulfoxide
and Strecker amine moieties of 1 can be replaced with
sulfone and methyl respectively, when a methyl group is
added to the piperazine ring. The chirality of the
benzylic and piperazine methyl groups is critical for
maintaining good M2 affinity and selectivity. The posi-
tion of the piperazine methyl is important for M2 selec-
tivity. Further enhancements in M2 affinity and
selectivity were found through modification of the
4-methoxy phenyl and the cyclohexyl moieties. Alkyl
11
12
(S)-CH3
(R)-CH3
CH2
CH2
—
—
0.03
0.03
61
30
13
14
15
16
17
18
19
20
(S)-CH3
(S)-CH3
(S)-CH3
(S)-CH3
(S)-CH3
(S)-CH3
(S)-CH3
(S)-CH3
N
N
N
N
N
N
N
N
H
9
4
109
281
198
40
32
15
10
SO2–C2H5
SO2–n-C3H7
SO2–n-C4H9
SO2–Ph
COPh
CO–n-C4H9
CONHC2H5
0.7
0.2
0.2
0.8
0.9
1.3
2.5
Figure 2. Acetylcholine release from striatum of conscious rat.

794
J. A. Kozlowski et al. / Bioorg. Med. Chem. Lett. 12 (2002) 791–794
sulfonamides were the preferred capping groups for the
piperidine nitrogen. Compound 14 displays a M1/M2
selectivity of 109-fold with a M2 Ki=0.7 nM. It pro-
duced a sustained increase in acetylcholine levels when
administered orally in the microdialysis paradigm.
These initial results are promising for the development
of M2 selective muscarinic antagonists for the treatment
of Alzheimer’s Disease.
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