Synthesis and muscarinic M(2) subtype antagonistic activity of enantiomeric pairs of 3-demethylhimbacine (3-norhimbacine) and its C(4)-epimer.

Article abstract of DOI:10.1016/S0960-894X(02)00695-9

In the course of our studies of the structure-activity relationships of himbacine 1, a potent antagonist of the M(2) subtype of muscarinic receptor, the four title compounds, 2, ent-2, 3, and ent-3, were synthesized with a highly stereoselective intermole

Full text of DOI:10.1016/S0960-894X(02)00695-9

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3271–3273
Synthesis and Muscarinic M2 Subtype Antagonistic Activity of
Enantiomeric Pairs of 3-Demethylhimbacine (3-Norhimbacine)
and Its C4-Epimer
Masanori Takadoi,a,* Kentaro Yamaguchib and Shiro Terashimac
aDiscovery Research Laboratories, Kyorin Pharmaceutical Co. Ltd., 2399-1 Nogi, Nogi-machi, Tochigi 329-0114, Japan
bChemical Analysis Center, Chiba University, 1-33 Yayoicho, Inage-ku, Chiba 263-8522, Japan
cSagami Chemical Research Center, 2743-1 Hayakawa, Ayase, Kanagawa 252-1193, Japan
Received 1 July 2002;accepted 20 August 2002
Abstract—In the course of our studies of the structure–activity relationships of himbacine 1, a potent antagonist of the M2 subtype
of muscarinic receptor, the four title compounds, 2, ent-2, 3, and ent-3, were synthesized with a highly stereoselective intermolecular
Diels–Alder reaction of tetrahydroisobenzofuran 4 with achiral furan-2(5H)-one 5 as a key step, followed by simultaneous optical
resolution and epimer separation of the racemic intermediates. Among these compounds, 3-demethylhimbacine (3-norhimbacine) 2,
bearing an absolute configuration corresponding to that of 1, was found to show more potent muscarinic M2 subtype receptor
binding activity than natural 1.
# 2002 Elsevier Science Ltd. All rights reserved.
Himbacine (1) is a piperidine alkaloid isolated from the
bark of Galbulimima Baccata of the magnolia family1
and shows potent antagonistic activity against muscari-
nic M2 subtype receptors, with 10–20-fold selectivity
toward the M1 subtype (Fig. 1).2 Accordingly, it
appears evident that 1 is the reasonable and attractive
lead compound of a drug for the treatment of Alzheimer’s
disease.3
ent-3) for the purpose of further evaluating the struc-
ture–activity relationships, and in order to explore the
advantages of our explored synthetic scheme. We wish
to report here the successful total synthesis of 2, ent-2, 3,
and ent-3 and their muscarinic M2 subtype receptor
binding activity. Among these compounds, the synthetic
himbacine 2, which bears an absolute configuration
corresponding to that of 1, was found to show more
potent receptor binding activity than natural 1.
Recently, we reported the novel total synthesis of 1 by a
method featuring a highly stereoselective intermolecular
Diels–Alder reaction of the furan derivative with chiral
furan-2(5H)-one as a key step.4 The efficiency and
directness of our explored synthetic route were demon-
strated by the successful synthesis of various structural
types of himbacine congeners and by the evaluation of
their structure–activity relationships.5 In the course of
our study of 1 from the viewpoint of medicinal chem-
istry, we next focused on the effects of the lactone ring
moiety, especially that of the C-3 methyl group, of 1 on
the M2 receptor subtype antagonistic activity. Thus, we
designed enantiomeric pairs of 3-demethylhimbacine (3-
norhimbacine) (2 and ent-2) and its C4-epimer (3 and
According to our reported procedure,4 racemic exo-
methylene compound 7 was prepared from the adduct 6,
which was prepared by a highly stereoselective inter-
molecular
hydroisobenzofuran
Diels–Alder
reaction
4 and commercially available
of
tetra-
*Corresponding author. Tel.: +81-280-56-2201;fax: +81-280-57-
1293;e-mail: masanori.takadoi@mb2.kyorin-pharm.co.jp
Figure 1.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00695-9

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M. Takadoi et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3271–3273
achiral furan-2(5H)-one 5 (Scheme 1).6 Hydroboration–
oxidation of 7 afforded the carbinol 8 as an inseparable
mixture of 4a, 4b-epimers in a ratio of 2:1, as demon-
strated by 1H NMR analysis. In our previous total
synthesis of 1, the stereoselectivity of the hydrobora-
tion–oxidation reaction was 1:8 (4a:4b).4 This difference
in stereoselectivity may have been due to the steric hin-
drance effects of the C-3 methyl group. Without
separation, the racemic epimeric mixture was directly
converted to the O-4-bromobenzoates 9 in a 99% yield.
The compounds were subjected to simultaneous optical
resolution and epimer separation by means of HPLC,
using CHIRALCEL OD, giving four stereoisomers, 9a–
9d, each of which possessed high optical purity.7 In
order to determine the absolute configurations of these
molecules, X-ray crystal structure analysis of 9a and 9c
was performed.8 Based on the results obtained by X-ray
analysis, it was clearly established that 9a bears an
absolute configuration corresponding to that of 1, and
that 9c is the 4a-epimer of 9a. The absolute configur-
ations of 9a–9d were unambiguously determined, and
are shown in Scheme 1. Accordingly, 9a was trans-
formed into the key intermediate sulfone 11a by a three-
step sequence involving alkaline hydrolysis, conversion
to the phenyl sulfide 10a using (cyanomethyl)-
trimethylphosphonium iodide,9 and mCPBA oxidation
of 10a (Scheme 2). A Julia–Lythgoe coupling reaction of
11a with 124 was quenched with excess benzoyl chloride,
followed by treatment of the resulting diastereomeric
mixture of O-benzoates with 5% Na–Hg in the presence
of Na2HPO4, which gave rise to (E)-olefin 13a in a 61%
combined yield. Then, 13a was sequentially subjected to
oxidation of the hemiacetal moiety, deprotection of the
N-Boc group, and reductive N-methylation, furnishing
210,11 in a 40% combined yield. By the same sequences,
9b (ent-9a), 9c, and 9d (ent-9c) were successfully con-
verted to the corresponding target compounds ent-2, 3,
and ent-3, respectively.10,11 To avoid confusion, the com-
pounds carrying the natural configurations corresponding
to that of 1 are only depicted in Scheme 2.
Scheme 2. (a) (Cyanomethyl)trimethylphosphonium iodide, thiophe-
nol, N,N-diisopropylethylamine, MeCN, 80 ꢀC, 2.5 h, 92%;(b)
mCPBA, NaHCO3, CH2Cl2, rt, 1.5 h, 82%;(c) (i) nBuLi, 12, 1,2-
dimethoxyethane, ꢁ78ꢂ0 ꢀC, 3 h;(ii) benzoyl chloride, ꢁ78 ꢀCꢂrt,
1 h;(iii) 3-(dimethylamino)propylamine, rt, 97% (a mixture of dia-
stereomers);(iv) 5% Na–Hg, Na 2HPO4, MeOH, rt, 1 h, 63%;(d)
Jones reagent, acetone, rt, 1.5 h, 58%;(e) trifluoroacetic acid, CH 2Cl2,
rt, 0.5 h, 87%;(f) 37% HCHO aq NaBH 3CN, CH3CN, rt, 1 h, 80%.
The four 3-demethylhimbacine (3-norhimbacine) deri-
vatives, 2, ent-2, 3, and ent-3, were then subjected to
receptor binding activity assay against M1 and M2 sub-
type muscarinic receptors (Table 1).12 As was hoped, it
was found that 2 bore an absolute configuration corre-
sponding to that of 1, and showed superior affinity to 1.
However, the other three stereoisomers, ent-2, 3, and
ent-3, exhibited only weak receptor binding activity
compared with that of 1 and 2. These results clearly
demonstrated that the C-3 methyl group on the lactone
ring moiety of 1 is not important for its strong mus-
carinic M2 subtype antagonistic activity. In addition,
the stereochemistry at the C-4 position was shown to
play an important role in eliciting M2 antagonistic
activity, in a manner similar to that played by the
stereochemistry of 1 and its 4-epimer.5
ꢀ
Scheme 1. (a) 5 M LiClO4–Et2O, 4,40-thiobis(6-tert-butyl-m-cresol), rt, 48 h, 49%;(b) ref 4, 18% (eight steps);(c) (i) BH 3 THF, THF, 0 Cꢂrt, 7 h;
(ii) 10% H2O2, 10% NaOH, 0 ꢀC, 0.5 h, 82%;(d) (i) 4-bromobenzoic acid, SOCl 2, 80 ꢀC, 1 h;(ii) 8, Et3N, CH2Cl2, 0 ꢀCꢂrt, 2 h, 99%;(e) CHIR-
ALCEL OD, C6H14/2-propanol=98:2, 9a: 19%, 9b: 18%, 9c: 30%, 9d: 32%;(f) 10% NaOH, EtOH, rt, 0.5 h, 8a: 100%, ent-8a: 100%, 8c: 100%,
ent-8c: 91%.
.

M. Takadoi et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3271–3273
3273
Table 1. In vitro binding activity of 3-norhimbacines
5. Takadoi, M.;Katoh, T.;Ishiwata, A.;Terashima, S.
rahedron. Submitted for publication.
Tet-
Entry
Compd
ꢁlogKi
M2 (brainstem)
6. The Diels–Alder cycloadduct 6 was also synthesized by
reacting 4 with maleic anhydride followed by NaBH4 reduc-
tion of one of the carbonyl groups. See: Tochtermann, W.;
Bruhn, S.;Wolff, C. Tetrahedron Lett. 1994, 35, 1165.
7. HPLC conditions were as follows: CHIRALCEL OD col-
umn (DAICEL Chemical Industries, Ltd.) (1 Â 25 cm), mobile
phase [hexane/2-propanol=10:1 (v/v)], flow rate (1.0 mL/min),
and temperature (40 ꢀC). The effluent was monitored at
254 nm. The retention times and ee values were as follows: 9c,
21.2 min, 99% ee; 9b (ent-9a), 22.7 min, 99% ee; 9a, 23.5 min,
94% ee; 9d (ent-9c), 33.6 min, 98% ee.
8. The crystallographic data (excluding structure factors) for
structures 9a and 9c reported in this paper have been depos-
ited to the Cambridge Crystallographic Data Centre as sup-
plementary publication numbers CCDC 183427 and 183426,
respectively. Copies of the data can be obtained, free of
charge, on application to CCDC, 12 Union Road, Cambridge
CB2 1EZ, UK (fax: +44–1223–336033;e-mail: http://
www.deposit@ccdc.cam.ac.uk).
M1 (cortex)
1
2
3
4
5
1
2
7.1
7.4
6.0
6.2
6.3
7.9
8.1
6.1
6.4
6.4
ent-2
3
ent-3
In conclusion, we have succeeded in the synthesis of
four novel himbacine congeners, 3-demethylhimbacines
(3-norhimbacines) (2, ent-2, 3, and ent-3) lacking a
methyl group at the C-3 position on the lactone ring of
1. Among them, 2, which bears an absolute configur-
ation corresponding to that of 1, exhibited more potent
binding activity against the M2 subtype receptor than
did natural himbacine 1. Further investigation of the
pharmacological profile of 2 is therefore in progress.
9. (a) Zaragona, F. Tetrahedron 2001, 57, 5451. (b) Zaragona,
F.;Stephensen, H. J. Org. Chem. 2001, 66, 2518.
1
10. All compounds were characterized by H and 13C NMR,
IR, and mass spectroscopic methods. Yields of each reaction
are as follows: (a) thiophenylation; ent-10a: 88%, 10c: 79%,
ent-10c: 76%;(b) mCPBA oxidation, ent-11a: 99%, 11c: 86%,
ent-11c: 82%;(c) (i) Julia–Lythgoe coupling reaction (isolated
as O-benzoates), (ii) 5% Na–Hg, ent-13a: 52% from ent-11a,
13c: 77% from 11c, ent-13c: 59% from ent-11c;(d) Jones oxi-
dation, ent-14a: 62%, 14c: 60%, ent-14c: 70%;(e) removal of
the N-Boc group, ent-15a: 94%, 15c: 98%, ent-15c: 99%;(f)
reductive methylation, ent-2: 59%, 3: 78%, ent-3: 63%. Details
will be reported in a separate paper.
Acknowledgements
We are grateful to Dr. H. Ohkubo and Dr. T. Ishizaki,
Kyorin Pharmaceutical Co. Ltd., for their encourage-
ment and many valuable suggestions. We would also
like to thank Dr. Y. Fukuda, Kyorin Pharmaceutical
Co. Ltd., for his suggestions and helpful input in dis-
cussions of this study. The in vitro binding activity
assay was carried out by Dr. T. Kawai, Kyorin Phar-
maceutical Co. Ltd., to whom our thanks are also due.
24
11. 2: Colorless oil, ½ꢀꢃD +69ꢀ (c 0.44, CHCl3), HRMS (FAB)
(m/z): calcd for C21H34NO2 (M++H): 332.2590. Found,
24
332.2609, ent-2: colorless oil, ½ꢀꢃD ꢁ72ꢀ (c 0.30, CHCl3),
HRMS (FAB) (m/z): calcd for C21H34NO2 (M++H):
24
332.2590. Found, 332.2579, 3: mp 86–88 ꢀC, ½ꢀꢃD ꢁ9.1ꢀ (c
References and Notes
0.42, CHCl3), HRMS (FAB) (m/z): calcd for C21H34NO2
(M++H): 332.2590. Found, 332.2573, ent-3: mp 85–87 ꢀC,
24
½ꢀꢃD +9.7ꢀ (c 0.47, CHCl3), HRMS (FAB) (m/z): calcd for
C21H34NO2 (M++H): 332.2590. Found, 332.2569.
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12. Binding assay: the receptor binding analysis of the M1 and
M2 subtypes of muscarinic receptors was performed using
homogenates of the cerebral cortex and the brainstem of the
rat, respectively. The radioligands used were [3H]-pirenzepine
for the cerebral cortex and [3H]-quinuclidinyl benzilate (QNB)
for the brainstem, respectively. The homogenates were incu-
bated in a 50 mM Tris–buffer (pH 7.4) at 25 ꢀC for 90 min, and
rapidly filtrated on Whatman GF-B filters. The radioactivities
were counted using a liquid scintillation counter. Non-specific
binding was defined in the presence of 2 mM atropine. Test
compounds were dissolved in DMSO and diluted with buffer
to the final concentrations. The competition binding experi-
ments were performed in the presence of less than 0.1%
DMSO, which did not affect the specific binding. The equili-
brium dissociation constants (Ki) were calculated using the
Cheng–Prusoff equation, Ki=IC50/(1+L/Kd), where L and Kd
were the concentration and the dissociation constant of the
radioligand, respectively. The Kd values were determined by a
Scatchard analysis.
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Tet-