Two-step synthesis and biological evaluation of calyxamines A and B

Article abstract of DOI:10.1016/j.tetlet.2013.10.022

A two-step synthesis of naturally occurring alkaloids calyxamines A and B featuring a tandem Mannich–aldol condensation reaction under solvent free conditions, and their inhibitory activity against acetylcholinesterase (AChE) is reported.

Full text of DOI:10.1016/j.tetlet.2013.10.022

Tetrahedron Letters 54 (2013) 6852–6854
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Two-step synthesis and biological evaluation of calyxamines A and B
⇑
Rosa-L. Meza-León , Alvaro Dávila-García, Fernando Sartillo-Piscil, Leticia Quintero,
Martha Sosa Rivadeneyra, Silvano Cruz-Gregorio
Centro de Investigación de la Facultad de Ciencias Químicas, Benemérita Universidad Autónoma de Puebla (BUAP), 14 Sur Esq. San Claudio, San Manuel, 72570 Puebla, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
A two-step synthesis of naturally occurring alkaloids calyxamines A and B featuring a tandem Mannich–
aldol condensation reaction under solvent free conditions, and their inhibitory activity against acetylcho-
linesterase (AChE) is reported.
Received 1 February 2013
Revised 4 October 2013
Accepted 6 October 2013
Available online 16 October 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Organocatalysis
Piperidines
Amines
Aldol reaction
Enzymes
Calyxamines A (1) and B (2) are naturally occurring alkaloids
isolated from the marine sponge Calyx podatypa by Rodríguez
et al. in 1997.1 Their structural determination was performed by
applying NMR and X-ray methods to the corresponding trifluoro-
acetate salt of the calyxamine A. Two years later, Cóbar and Pinto
reported that calyxamines A and B possess modest antimicrobial
activity against Staphylococcus aureus and Mycobacterium smegma-
tis.2 The biosynthetic proposal for 1 and 2 envisions the formation
of piperidone 3 from the condensation of NH3 and 4 equiv of ace-
tone (or biosynthetic equivalents) via the corresponding imine
intermediate.1 Experimentally, it seems that the best way to pre-
pare calyxamines A and B is starting from the condensation of pip-
eridone 3 with acetone, especially because the preparation of 3 is
well-documented.3 In this sense, the single reported synthesis of
calyxamines A and B involves the condensation reaction between
the 2,2,6,6-tetramethyl-4-piperidone 3 and acetone under basic
conditions (Scheme 1).2
On this basis, we decided to develop a more rapid and efficient
protocol for the synthesis of calyxamines A and B, and thus to ob-
tain sufficient amount for biological assays. Our proposal involves
the preparation of the imine intermediate 4 from the condensation
reaction between NH4Cl and acetone; and then, the preparation of
piperidone 3 could be obtained from a Mannich condensation with
another equivalent of acetone followed by aldol condensation of 3
with acetone, as well. We planned to conduct both condensations
O
O
O
O
+
N
H
3
N
H
N
H
ONa
heat
Calyxamine A
Calyxamine B
(1)
(2)
Scheme 1. Synthesis of calyxamines A and B starting from piperidone 3.
in a two-step sequential process (Scheme 2). Additionally, we envi-
sioned that both condensation reactions might be catalyzed by
commercially available diamines, and as the acetone is used for
both condensations, we anticipated that the reactions could be
conducted under free-solvent conditions.
It has demonstrated the efficacy of the chiral diamines in the
enantioselective Mannich condensation;4 however, because the
calyxamines A and B are not chiral compounds, there was no need
for using optically pure amines. Thus, the imine 4 was quantita-
tively prepared from the reaction of acetone with ammonium chlo-
ride,5 followed by the use of ethyldiamine
5 (10% mol) as
organocatalyst in the Mannich reaction, wherein the acetone is
used as the enamine generator. The reaction was maintained in
vigorous stirring for 24 h expecting to obtain piperidone 3, in the
first instance; however, we only observed the formation of piperi-
done 3 as a minor by-product. Interestingly, the main observed
products were the calyxamines A (1) and B (2) in an equimolar ra-
tio (Scheme 3).6 By running a blank reaction we proved that the
⇑
Corresponding author. Tel.: +52 222 2955500x7391; fax: +52 222 2454972.
E-mail addresses: rosaluisa@yahoo.com, rosa.meza@correo.buap.mx (R.-L.
Meza-León).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.10.022

R.-L. Meza-León et al. / Tetrahedron Letters 54 (2013) 6852–6854
6853
diamine 5 is the actual organocatalyst. Apparently, diamine 5 not
only catalyzes the Mannich condensation, but also the cross-aldol
condensation of the incipient piperidone 3 with acetone.7 Dia-
mines 6 and 7 were also tested as organocatalyst for the tandem
Mannich/aldol condensation reaction obtaining lower yield for
the case of diamine 7, and only traces when cyclohexane diamine
6 was used (Scheme 3). It is worth to mention that this efficient
two-step protocol for the synthesis of calyxamines A and B was
successfully conducted under solvent free conditions.
Having established an efficient protocol for the synthesis of
calyxamines A and B, we proceed to test them on the inhibition
of acetylcholinesterase.8 The hydrolysis of the no fluorescent in-
doxyl acetate produces a highly fluorescent material for measuring
cholinesterase activity. In the presence of AChE inhibitors a de-
crease in the reaction rate of AChE is expected to quantitatively as-
sess the inhibition capacity of 1 and 2. Figures 1a and 1b show their
pronounced inhibition activities after 5 min incubation with
AChE.9
Figure 1b. Calyxamine
inhibition around 0.6 mM.
B
in
a
concentration-dependent manner reaching 50%
Table 1
The inhibitory activities on AChE
Although many natural products have been reported as inhibi-
tors of AChE (e.g., compounds in Table 1), the inhibition achieved
by calyxamine B is comparable with that exhibited by natural phe-
a
Compound
IC50
117
(lg/mL)
Calyxamine B
Ferulicacid
Quercetin
200–500
5.9
Tiliroside
13.97
29.99
32.5
O
O
Quercetrin
Artoblidxanthone
Artoninse
O
NH
O
1
2
and
NH Cl
42.5
+
4
organocatalyst
(diamine)
organocatalyst
(diamine)
N
H
Imine
(4)
a
The values indicate 50% AChE inhibitory effect and are the means of triplicate
data.
Scheme 2. Our sequential proposal route for the synthesis of calyxamines A and B.
nolic compounds such as ferulic acid,10 but less effective than trit-
erpenoidal alkaloids from Buxushyrcana: tiliroside, 3-methoxy
quercetin, quercitrin, quercetin and dehydroevodiamine,11 and
also some flavonoids from Artocarpusnobilis.12
In conclusion, we have developed a highly efficient two-step
solvent free protocol for the synthesis of calyxamines A and B.
The calyxamine B showed binding capacity to AChE comparable
to those natural inhibitors of AChE.
O
O
O
NH
O
NaHCO3
NH4Cl
2 eq
+
+
3
+
N
H
N
H
Diamine
4
1
2
Acknowledgments
NH2
NH2
H2N
H2N
5
Yield
Entry
Diamine Ratio
1:2
This work was financially supported by VIEP-BUAP No. MELL-
NAT13-I. We thank Dr. Eduardo Torres for his support in obtaining
biological studies.
%
a
5
50
1:1
NH2
NH2
6
7
6
-
b
c
-
7
1:1
35
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2013.
10.022. These data include MOL files and InChiKeys of the most
important compounds described in this article.
Scheme 3. Two step-synthesis of calyxamines A and B.
References and notes
1. Rodríguez, A. D.; Cóbar, O. M.; Padilla, O. L.; Barnes, C. L. J. Nat. Prod. 1997, 60,
1331–1333.
2. Cóbar, O. M.; Vásquez, A. A. DIGI-USAC Guatemala 2004.
3. Sosnovsky, G.; Konieczny, M. Synthesis 1976, 735–736. and references cited
therein.
4. Notz, W.; Tanaka, F.; Barbas, C. F. Acc. Chem. Res. 2004, 37, 580–591.
5. Preparation of imine 4: A 25 mL flask was charged with acetone (1.25, 17 mmol),
ammonium chloride (0.909 g, 17 mmol) and water (1 mL). Then the mixture
was heated to 60 °C maintaining the mixture under constant stirring, then
NaHCO3 was added portion wise until bubbling ceases, at that point there is
formation of a large amount of NaCl. The precipitate was filtered off and the
formed imine is used without purification in the next reaction.
Blank reaction: The imine 4 was dissolved in 2 mL of acetone and kept stirring
for 48 h, the mixture remained colorless all the time, then was concentrated
Figure 1a. Calyxamine A inhibited 50% of the AChE from 8 to 0.5 lM.

6854
R.-L. Meza-León et al. / Tetrahedron Letters 54 (2013) 6852–6854
133.0, 207.0. ESI-HRMS: m/z calcd for C12H21NO: 195.1623, found 195.1593.
under reduced pressure leaving the flask empty after evaporation. Indicating
that ethylenediamine is essential to carry out the reaction.
1-(2,2,6,6-Tetramethylpiperidin-4-ylidene)propan-2-one, calyxamine B
(2): 1H
6. Synthesis of 2,2,6,6-tetramethyl-4-piperidone (3) and calyxamines A (1) and B (2):
Imine 4 and EDA (0.1136 mL, 1.7 mmol) were dissolved in acetone (2 mL) at
room temperature. The reaction turned, form colorless to red wine, and was
maintained at room temperature for 24 h, where at this time, the major
observed product was the 2,2,6,6-tetramethyl-4-piperidone 3; however by
allowing to stir the reaction mixture for another 24 h, the major observed
products were the calyxamine A and B. The reaction mixture was concentrated
under reduced pressure to afford 1.45 g of crude reddish brown solid (approx.
55% of acetone transformed). The crude product was purified by flash
chromatography using dichloromethane (DCM) as eluent; to obtain 0.0122 g
of piperidone 3 (2%), then the mobile phase was changed to a mixture of DCM/
acetone 3:1; to yield 0.073 g of calyxamine A (12%) as a white crystalline solid
that sublimes at 225–226 °C; finally calyxamine B (0.044 g, 0.23 mmol, 7%) of a
crystalline solid mp 205–210 °C, and finally 0.522 g of a mixture of both
calyxamines that we could not separate.
NMR d (ppm) 1.62 (s, 6H), 1.63 (s, 6H), 2.2 (s, 4H), 3.1 (s, 3H), 6.2 (s, 1H). 13C
NMR d (ppm) 27.6, 32.0, 38.4, 46.7, 58.9, 59.6, 127.0, 148.4, 198.2. ESI-HRMS:
m/z calcd for C12H21NO: 195.1623, found 195.1617.
7. Similar version of this tandem Mannich–aldol condensation has been reported
by Chan and Yip: Feng, L.; Xu, L.; Lam, K.; Zhou, Z.; Yip, C.-W.; Chan, A. S. C.
Tetrahedron Lett. 2005, 46, 8685–8689.
8. Acetylcholinesterase from electrophorus electricus (electric eel), type VI-S, and
indoxyl acetate from Sigma–Aldrich.
9. Acetylcholinesterase activity: Acetylcholin esterase (AchE) activity was
determined by
a fluorescence method measuring the indoxyl acetate
hydrolysis. The 3 mL-reaction mixture contained 1.25 mM indoxyl acetate
and 2 Ache units in phosphate buffer 60 mM pH 7. The reaction progress was
followed by the increase in the fluorescence emission at 470 nm with an
excitation of 395 nm.
10. Shahwar, L. D.; Rehman, S. U.; Raza, M. A. J. Med. Plants Res. 2010, 4, 260–266.
11. (a) Ata, A.; Iverson, C. D.; Kalhari, K. S.; Akhter, S.; Betteridge, J.;
Meshkatalsadat, M. H.; Orhan, I.; Sener, B. Phytochemistry 2010, 71, 1780–
1786; (b) Jung, M.; Park, M. Molecules 2007, 12, 2130–2139.
2,2,6,6-Tetramethyl-4-piperidone (3): 1H NMR d (ppm) 1.25 (s, 12H), 2.28 (s, 4H).
13C NMR d (ppm) 30.8, 31.9, 54.05, 55.31, 203.5. ESI-HRMS: m/z calcd for
C9H17NO: 155.1310, found 155.1315.
1-(2,2,6,6-Tetramethyl-1,2,3,6-tetrahydropyridin-4-yl)propan-2-one, calixamine A
(1): 1H NMR d (ppm) 1.18 (s, 6H), 1.23 (s, 6H), 1.83 (s, 2H), 2.15 (s, 3H), 3.07 (s,
2H), 5.49 (s, 1H). 13C NMR d (ppm) 29.8, 31.0, 40.6, 49.9, 51.6, 52.9, 126.9,
12. Ata, A.; Van Den Bosch, S. A.; Harwanik, D. J.; Pidwinski, G. E. Pure Appl. Chem.
2007, 79, 2269–2276.