First total synthesis of (±)-3-hydroxy-11-norcytisine: Structure confirmation and biological characterization

Article abstract of DOI:10.1021/ol802145b

(Chemical Equation Presented) The first total synthesis of the natural product 3-hydroxy-11-norcytisine (1), structurally related to cytisine (2), a benchmark ligand at neuronal nicotlnic acetylcholine receptors (NNRs), has been achieved. The synthesis permits the unambiguous confirmation of the structure originally proposed for 1 and has enabled initial biological characterization of 1 and its related compounds against NNRs.

Full text of DOI:10.1021/ol802145b

ORGANIC
LETTERS
2
008
Vol. 10, No. 23
353-5356
First Total Synthesis of
5
(()-3-Hydroxy-11-norcytisine: Structure
Confirmation and Biological
Characterization
,
†
‡
Daniel Yohannes,* Camilla P. Hansen, Srinivasa Rao Akireddy,
†
†
†
†
Terry A. Hauser, Melanie N. Kiser, Nicholas J. Gurnon, Cynthia S. Day,
§
,†
†
Balwinder Bhatti,* and William S. Caldwell
Targacept, Inc., 200 East First Street, Suite 300, Winston-Salem, North Carolina 27101,
Faculty of Pharmaceutical Sciences, UniVersity of Copenhagen, UniVersitetsparken 2,
DK-2100 Copenhagen, Denmark, and Chemistry Department, Wake Forest UniVersity,
Salem Hall, Box 7486, Winston-Salem, North Carolina 27109
daniel.yohannes@targacept.com
Received September 15, 2008
ABSTRACT
The first total synthesis of the natural product 3-hydroxy-11-norcytisine (1), structurally related to cytisine (2), a benchmark ligand at neuronal
nicotinic acetylcholine receptors (NNRs), has been achieved. The synthesis permits the unambiguous confirmation of the structure originally
proposed for 1 and has enabled initial biological characterization of 1 and its related compounds against NNRs.
Hydroxynorcytisine (1), an alkaloid from the legume family
Laburnum anagyroides), was isolated in 1989 by Hayman
heteromeric nicotinic acetylcholine receptor subtype in the
brain. Given our focus on the discovery of therapeutic agents
3
(
1
and Gray. It is a naturally occurring skeletal congener of
-)-cytisine (2), which is prodigiously found in the Legu-
acting at neuronal nicotinic receptors (NNRs), we were
curious as to whether 1 would also display affinity at NNRs
and, if so, what its selectivity profile would be across central
and peripheral nicotinic acetylcholine receptor subtypes.
Herein, we disclose the first total synthesis of hydroxynor-
cytisine (1), its unambiguous structure confirmation, and
biological data at NNR subtypes for both 1 and its parent
scaffold 3 (Figure 1).
(
2
minosae family. Despite its obvious structural and potential
biosynthetic relationship to (-)-cytisine, hydroxynorcytisine
has received little, if any, attention in the literature since the
original isolation disclosure.
(
-)-Cytisine (2) binds with high affinity to the R4ꢀ2
neuronal nicotinic acetylcholine receptor, the predominant
†
Targacept, Inc.
University of Copenhagen.
Wake Forest University.
‡
§
(3) (a) Whiting, P.; Lindstrom, J. Proc. Natl. Acad. Sci. U.S.A. 1987, 8,
595. (b) Flores, C. M.; Rogers, S. W.; Pabreza, L. A.; Wolfe, B. B.; Kellar,
K. J. Mol. Pharmacol. 1992, 41, 31. (c) Hall, M.; Zerbe, L.; Leonard, S.;
Freedman, R. Brain Res. 1993, 600, 127. (d) Happe, H. K.; Peters, J. L.;
Bergman, D. A.; Murrin, L. C. Neuroscience 1994, 62, 929.
(
1) Hayman, A. R.; Gray, D. O. Phytochemistry 1989, 28, 673–5.
(
2) (a) Partheil, A. Arch. Pharm. (Weinheim, Ger.) 1894, 232, 161. (b)
Ing, H. R. J. Chem. Soc. 1932, 2778. (c) Leonard, N. J. The Alkaloids;
Academic: New York, NY, 1953; Vol. III, p 120.
10.1021/ol802145b CCC: $40.75
Published on Web 11/13/2008
 2008 American Chemical Society

Pd-C) of which cleanly afforded the pyrrolidine. The free
nitrogen of the crude pyrrolidine was immediately protected
2 2 3
(Boc O, Na CO ) to afford 7 in high overall yield (74% over
3
steps). Reduction of the ester to the primary alcohol (LAH)
gave 8 in nearly quantitative yield, providing a compound
well suited to participate in the modified Van Tamelen
4
cyclization utilized in our earlier synthesis of cytisine.
Indeed, mesylation of the primary alcohol and subsequent
heating (toluene reflux) afforded 9 in 84% yield. The intact
scaffold of the title compound (sans hydroxyl) was revealed
when 9 was deprotected (TFA) to give 3.
Figure 1. Structures of (()-3-hydroxy-11-norcytisine (1), (-)-
cytisine (2), and (()-11-norcytisine (3).
Cytisine has been extensively derivatized in efforts to
5
delineate its structure-activity relationship. Of relevance
Our preferred synthetic approach was to first access
intermediate 9 for multiple reasons. Structure-function
precedents on the pyridone of cytisine suggested to us that
such an intermediate might be one on which we could rapidly
elaborate to the hydroxypyridone, as well as other analogues
substituted on the pyridone moiety. Additionally, we were
interested in the nicotinic potential of 3 (derived from 9,
Scheme 1) as a direct comparator to natural cytisine, whose
pyridone ring is similarly placed and unsubstituted. Finally,
for the construction of the pyridone ring, we were relying
on the precedent of our earlier total synthesis of cytisine
which utilized a methoxypyridine f pyridone cyclization,
a dependable method for construction of fused and bridged
to our synthesis was the knowledge that cytisine could be
readily nitrated or brominated. Our expectation was that
6
either of these approaches, applied to 9, could provide the
penultimate transformation that would give us access to the
natural scaffold of 1. The introduction of the hydroxyl
substituent on intermediate 9, containing the preformed
pyridone ring, is described below (Scheme 2).
Our initial attempts at functionalization next to the
pyridone carbonyl involved nitration of 9 (HNO
3
, H
2
SO
4
;
/
(Boc) O, 22%) followed by reduction to the amine 11 (H
2
2
Pd-C; 40%). However, preliminary attempts to convert the
7
amine into the hydroxyl were unsuccessful. Ultimately, we
were most successful using the bromination of 9 (NBS,
4
bicyclic pyridones. As such, we were disinclined to
CCl
4
), which provided the desired 3-substituted isomer (12)
complicate that cyclization with substituents on the pyridine
ring (such as hydroxyl or an equivalent synthon) which may
alter its reactivity toward cyclization.
in 54% yield, along with a substantial amount of the
(4) (a) O’Neill, B. T.; Yohannes, D.; Bundesmann, M. W.; Arnold, E. P.
Org. Lett. 2000, 2, 4201. (b) Yohannes, D.; Procko, K.; Lebel, L. A.; Fox,
C. B.; O’Neill, B. T. Bioorg. Med. Chem. 2008, 18, 2316.
In the event, we embarked on the synthesis utilizing the
commercially available racemic 4, hoping to position our-
selves for a future asymmetric synthesis by using a starting
material which possessed a stereogenic center. Diazomethane
esterification of the carboxylic acid was followed by activa-
tion of the nitrogen with Boc anhydride (73% for the two
steps). Addition of the 6-lithio-2-methoxypyridine to the
lactam carbonyl was compromised by concomitant addition
to the methyl ester, but a 55% yield of 6 was nevertheless
obtained. Liberation of the free amine of 6 (excess TFA,
(
5) (a) O’Neill, B. T. U.S. Patent WO 98/18798, 1998. (b) Rouden, J.;
Seitz, T.; Lemoucheux, L.; Lasne, M.-C. J. Org. Chem. 2004, 69, 3787.
c) Carbonnelle, E.; Sparatore, F.; Canu-Boido, C.; Salvagno, C.; Baldani-
(
Guerra, B.; Terstappen, G.; Zwart, R.; Vijverberg, H.; Clementi, F.; Gotti,
C. Eur. J. Pharmacol. 2003, 471, 85. (d) Slater, Y. E.; Houlihan, L. M.;
Maskell, P. D.; Exley, R.; Bermudez, I.; Lukas, R. J.; Valdivia, A. C.;
Cassels, B. K. Neuropharmacology 2003, 44, 503. (e) Boido, C.; Tasso,
B.; Boido, V.; Sparatore, F. Farmaco 2003, 58, 265. (f) Roger, G.; Lagnel,
B.; Rouden, J.; Besret, L.; Valette, H.; Demphel, S.; Gopisetti, J. M.; Coulon,
C.; Ottaviani, M.; Wrenn, L. A.; Letchworth, S. R.; Bohme, G. A.;
Benavides, J.; Lasne, M.-C.; Bottlaender, M.; Dolle, F. Bioorg. Med. Chem.
2
003, 11, 5333. (g) Imming, P.; Klaperski, P.; Stubbs, M. T.; Seitz, G.;
Gundisch, D. Eur. J. Med. Chem. 2001, 36, 375. (h) Marriere, E.; Rouden,
J.; Tadino, V.; Lasne, M.-C. Org. Lett. 2000, 2, 1121. (i) Boido, C.;
Sparatore, F. Farmaco 1999, 54, 438–451. (j) Lin, N. H.; Meyer, M. D.
Exp. Opin. Ther. Pat. 1998, 8, 991. (k) Chellappan, S. K.; Xiao, Y.;
Tueckmantel, W.; Kellar, K. J.; Kozikowski, A. P. J. Med. Chem. 2006,
3
then NaHCO ) spontaneously closed the amino-ketone
intermediate to the cyclic imine, the rapid hydrogenation (H /
2
4
9, 2673. (l) Blackall, K. J.; Hendry, D.; Pryce, R. J.; Roberts, S. M.
J. Chem. Soc., Perkin Trans. 1 1995, 2767. (m) Fitch, R. W.; Kaneko, Y.;
Klaperski, P.; Daly, J. W.; Seitz, G.; Guendisch, D. Bioorg. Med. Chem.
Lett. 2005, 15, 1221.
Scheme 1
(
6) (a) Marriere, E.; Rouden, J.; Tadino, V.; Lasne, M. C. Org. Lett.
2
000, 2, 1121–1124. (b) O’Neill, B. T. PCT Int. Appl. WO9818798, 1998;
Chem. Abstr. 1998, 129, 4774. (c) Houlihan, L. M.; Slater, Y; Guerra, D. L.;
Jian-Hong, P.; Y.-P., K.; Lukas, R. J.; Cassels, B. K.; Bermudez, I.
J. Neurochem. 2001, 78, 1029–1043. (d) Imming, P.; Klaperski, P.; Stubbs,
M. T.; Seitz, G.; Gundisch, D. Eur. J. Med. Chem. 2001, 36, 375. (e)
Nicolotti, O.; Canu Boido, C.; Sparatore, F.; Carotti, A. Farmaco 2002,
5
2
7, 469. (f) Boido, C. C.; Tasso, B.; Boido, V.; Sparatore, F. Farmaco
003, 58, 265. (g) Slater, Y. E.; Houlihan, L. M.; Maskell, P. D.; Exley,
R.; Bermudez, I.; Lukas, R. J.; Valdivia, A. C.; Cassels, B. K. Neurophar-
macology 2003, 44, 503. (h) Fitch, R. W.; Kaneko, Y.; Klaperski, P.; Daly,
J. W.; Seitz, G.; Guendisch, D. Bioorg. Med. Chem. Lett. 2005, 15, 1221.
(
i) Abin-Carriquiry, J. A.; Voutilainen, M. H.; Barik, J.; Cassels, B. K.;
Iturriaga-Vasquez, P.; Bermudez, I.; Durand, C.; Dajas, F; Wonnacott, S.
Eur. J. Pharmacol. 2006, 536, 1.
(
7) The yields were very low for conversion of 11 to 14 through
diazotization followed by copper-mediated displacement of diazonium by
water.
5354
Org. Lett., Vol. 10, No. 23, 2008

undesired 5-substituted bromide (29% yield) and trace
amounts of the 3,5-dibromopyridone (2-3% yield). Conver-
sion of bromide 12 to the 3-acyl compound under Stille
conditions (tributyl(1-ethoxyvinyl)stannane, cat. Pd(0), tolu-
ene, reflux) provided 13 in good isolated yield (70%),
containing functionality that could be converted into the
hydroxyl. The Bayer-Villager oxidation of ketone 13 (m-
CPBA, CHCl ) and subsequent saponification (KOH-MeOH)
3
thus afforded 14 containing the hydroxyl functionality in the
desired position (40%). Deprotection of the Boc group (TFA,
2 2
CH Cl ) cleanly gave what we believed to be the natural
product (1).
Scheme 2
Figure 2. Displacement ellipsoids (50% probability) for the X-ray
crystal structure of 15, the bisdansyl derivative of 3-hydroxy-11-
9
norcytisine (1).
To assess the value of hydroxynorcytisine (1) as a ligand
at the nicotinic acetylcholine receptor, we evaluated the
natural product 1 and scaffold 3 in neuronal nicotinic
acetylcholine receptor binding assays, along with cytisine
(
2) for comparison (Table 1). The NNR subtypes chosen
were the rat R4ꢀ2 and R7 receptor subtypes. The R4ꢀ2
subtype is thought to be involved in important clinical
indications such as addiction and dementia, while the R7
subtype is believed to be implicated in the neuroprotective
10
effects of nicotine. To our great surprise, the natural product
and its parent scaffold 3 displayed more than 4 orders of
1
magnitude less affinity than cytisine (2) at R4ꢀ2 NNRs and
at least an order of magnitude less affinity at R7 nicotinic
11
receptors. The disparate affinities at R4ꢀ2 nicotinic recep-
tors of hydroxynorcytisine (1) and cytisine (2) illustrate the
profound effect that minor differences in structure can exert
at this receptor. Our current efforts are directed toward the
molecular and structure-activity understanding of why such
ostensibly minor structural variability between natural prod-
ucts 1 and 2 translates to vastly divergent affinities at the
R4ꢀ2 nicotinic acetylcholine receptor.
To our knowledge, this disclosure represents the first total
synthesis of 3-hydroxy-11-norcytisine (1). We have addition-
ally provided unambiguous confirmation of the structure
originally proposed for the natural product. Finally, we
present herein the first characterization of 1 in biological
screens, in particular the R4ꢀ2 nicotinic acetylcholine
receptor, at which its well-known congener cytisine (2) is
considered a benchmark ligand. Additional studies, including
Gray et al. had characterized the natural product as its
bisdansyl (DNS) adduct 15, by NMR. We therefore prepared
1
a sample of 15 from 1 for direct spectral comparison and
1
were pleased to find that our H NMR of 15 was identical
to that which was reported. As we did not have a sample of
the isolated natural product compound with which to draw
a direct comparison, we endeavored to generate high-quality
crystals for X-ray determination of the structure. Our efforts
were rewarded with an X-ray structure confirmation of the
natural product characterized as its bisdansylated derivative
8
1
5 (Figure 2).
(
8) The CIF for 15 has been deposited with the Cambridge Crystal-
lographic Data Centre database, deposition code: 701795.
9) Pale yellow single crystals of C34H34N4O6S2 (15) are, at 193(2) K,
(
monoclinic, space group P21/n (an alternate setting of P21/c - C25h (No.
1
9
4)) with a ) 6.933(2) Å, b ) 22.473(5) Å, c ) 20.118(4) Å, ꢀ )
3
-
3
6.409(4)°, V ) 3115.1(12) Å , and Z ) 4 {dcalcd ) 1.405 g·cm
;
-1
µa(MoK Rj ) ) 0.225 mm }. A full hemisphere of diffracted intensities (1868
0-second frames with a ω scan width of 0.30°) was measured for a single-
domain specimen using graphite-monochromated MoK Rj radiation (λ )
.71073 Å) on a Bruker SMART APEX CCD Single Crystal Diffraction
3
Table 1. Affinities of Hydroxynorcytisine (1), Cytisine (2), and
0
1
2
System. X-rays were provided by a fine-focus sealed X-ray tube operated
at 50 kV and 30 mA. For more information, see Supporting Information.
Analogue (3) at Neuronal Nicotinic Receptors (NNRs)
Ki (nM)
(
10) (a) Romanelli, M. N.; Gratteri, P.; Guandalini, L.; Martini, E.;
Bonaccini, C.; Gualtieri, F. ChemMedChem 2007, 2, 746. (b) Mazurov,
A. A.; Hauser, T.; Miller, C. H. Curr. Med. Chem. 2006, 13, 1567. (c)
Breining, S. R.; Mazurov, A. A.; Miller, C. H. Annu. Rep. Med. Chem.
compound
R4ꢀ2
R7
1
2
3
14000 ( 4000
0.4 ( 0.1
41000 ( 7000
260000 ( 31000
1700 ( 300
>100000
2
005, 40, 3. (d) Breining, S. R. Curr. Top. Med. Chem. (Sharjah, United
Arab Emirates) 2004, 4, 609.
(
11) The estimate of >100000 is based on the failure of the compound
to pass single concentration determination at 100000 nM.
Org. Lett., Vol. 10, No. 23, 2008
5355

molecular modeling and the full structure-activity work
which addresses the disparate NNR profiles of 1 and 2, will
be delineated in a subsequent disclosure.
Supporting Information Available: Experimental pro-
cedures and spectroscopic data for compounds 1, 3, 5, 7-9,
and 12-15 and X-ray crystallographic information file (CIF)
and detailed report for 15. This material is available free of
charge via the Internet at http://pubs.acs.org.
(
12) Experimental details for the determination of affinities at R4ꢀ2 and
R7 NNRs can be found in the following reference: Marrero, M. B.; Papke,
R. L.; Bhatti, B. S.; Shaw, S; Bencherif, M. J. Pharmacol. Exp. Ther. 2004,
3
09, 16–27.
OL802145B
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Org. Lett., Vol. 10, No. 23, 2008