Organic &

Biomolecular
Chemistry
View Article Online
COMMUNICATION View Journal | View Issue

An unexpected formation of the novel 7-oxa-2-

Cite this: Org. Biomol. Chem., 2013, 11, azabicyclo[2.2.1]hept-5-ene skeleton during the
407

Received 29th August 2012,

reaction of furfurylamine with maleimides and their
Published on 22 October 2012 on http://pubs.rsc.org | doi:10.1039/C2OB26699G

Accepted 19th October 2012 bioprospection using a zebrafish embryo model†

DOI: 10.1039/c2ob26699g

www.rsc.org/obc Carlos E. Puerto Galvis and Vladimir V. Kouznetsov*

Downloaded by University of Arizona on 15 December 2012

An unexpected intramolecular cyclization during the reaction of maleimides 4 and furfurylamine 5, according to the retro-
furfurylamine with maleimides is reported as a novel strategy for synthetic analysis depicted in Scheme 1. Followed by our retro-
the efficient green synthesis of the 7-oxa-2-azabicyclo[2.2.1]hept- synthetic analysis of functionalized DNC A, our study began
5-ene skeleton. Under the same reaction conditions, 7-oxabicyclo with the three-step synthesis of the commercially unavailable
[2.2.1]hept-5-enes were synthesized when furfurylamine was N-arylmethylmaleimides 4a–g derived from benzylamines 2
N-protected by the acetyl group. Both types of bicycloheptenes and maleic anhydride 3 using the respective substituted ben-
were screened using the zebrafish model system for genetics and zaldehydes 1 as the main starting materials7 (see ESI† for
developmental biology. details).
Having prepared the required diverse maleimides 4a–g, we
focused our study on the desired DNCs A. The first experiment
Introduction was carried out at room temperature under an inert atmos-
phere, N-benzylmaleimide 4a and furfurylamine 5 were chosen
Among the oxygen and nitrogen-containing heterocycles, 7-oxa as model substrates, acetonitrile was employed as the solvent.
and 7-azabicycles are the common core components of some After 3 hours the reaction was complete (TLC) and once the
biologically active natural alkaloids as well as potent pharma- main product was purified, the structural elucidation of
ceutical drugs like cantharidines1 and (−)-epibatidine ana- the isolated substance revealed surprisingly that instead of the
logues.2 However, the known natural and synthetic examples desired molecule A, a new compound 6a with the 7-oxa-2-aza-
of oxa and/or aza bicyclic skeletons are scarce and the syn- bicyclo[2.2.1]hept-5-ene skeleton was obtained as a single
thetic methods to prepare them remain the main objective of product and in moderate yield (Scheme 2).
many current investigations.3 To date the most fundamental The reaction conditions were varied (Table 1), in order to
and common strategies for the synthesis of these oxa-azabi- improve the yield and to establish a green method for the
cyclo rings are based on the Diels–Alder reaction (DAR) selective preparation of product 6a, finding that (i) all the
between nitroso derivatives and cyclopentadiene to afford experiments gave in moderate to excellent yields the same
2-oxa-3-azabicyclo[2.2.1]hept-5-enes4 or via an intramolecular product 6a as a stable oil, (ii) this reaction can be carried out
1,3-dipolar cycloaddition using nitrones to give 7-oxa-1-aza- smoothly in CH3CN or polyethylene glycol 400 (PEG-400)
bicyclo[2.2.1]heptanes.5 Nevertheless, both methods are in without any catalyst at room temperature (entries 1,2) and at
general expensive, laborious and not eco-friendly. 90 °C (entries 5,6), (iii) 10 mol% H3BO3 catalyses this process
With our current interest in the development of new syn- reducing the reaction times in both polar solvents, CH3CN and
thetic routes for the preparation of diverse heterocycles using PEG-400 at rt (entries 3,4), and (iv) heating the reaction at
the DARs,6 we directed our efforts to complement the back-
grounds and improve the drawbacks in the synthesis of de-
hydronorcantharimides (DNC), designing a logic route for the
selective synthesis of functionalized DNC A from diverse

Laboratorio de Química Orgánica y Biomolecular, Universidad Industrial de

Santander, Cra 27 calle 9, Bucaramanga A.A. 678, Colombia.
E-mail: kouznet@uis.edu.co; Fax: +57 76 349069
† Electronic supplementary information (ESI) available: Detailed synthetic pro- Scheme 1 Synthetic design for the preparation of N-substituted
cedures and spectral data. See DOI: 10.1039/c2ob26699g dehydronorcantharimides.

This journal is © The Royal Society of Chemistry 2013 Org. Biomol. Chem., 2013, 11, 407–411 | 407
 View Article Online

Communication Organic & Biomolecular Chemistry

Table 2 H3BO3-Catalyzed reaction of furfurylamine 5 with various male-

imides 4a–i in PEG-400

Scheme 2 Formation of the unexpected product 6a during the model reaction

between maleimide 4a and furfurylamine 5.
Published on 22 October 2012 on http://pubs.rsc.org | doi:10.1039/C2OB26699G

Table 1 Criteria for the selection of the best conditions for the reaction of the
N-benzylmaleimides 4a with furfurylamine 5
Downloaded by University of Arizona on 15 December 2012

Catalyst Conversiona Time Yield of

Entry Solvent (10 mol%) (%) (h) 6ab (%)

1 CH3CN None 57c 3 48

2 PEG-400 None 52c 2.3 43
3 CH3CN H3BO3 63c 1 52
4 PEG-400 H3BO3 60c 1 57
5 CH3CN None 100d 4 80
6 PEG-400 None 100d 3 79
7 CH3CN H3BO3 100d 1 84
8 PEG-400 H3BO3 100d 1 82
a
Selecting maleimide 4a as a limit reactant. b Isolated yield. c Reaction
performed at room temperature. d Reaction performed at 90 °C.

a
Isolated yields. b Commercial (R)- and (S)-1-phenylethanamines were
90 °C enhances the catalytic activity of boric acid, accelerating used to prepare the corresponding maleimides 4h and 4i.
considerably the formation of product 6a (entries 7,8).
Next, having optimized the reaction conditions, a small
library of novel and diverse 7-oxa-2-azabicyclo[2.2.1]hept-5- According to Corey’s work, where the possible interactions
enes 6a–i (Table 2) were easily prepared from the respective of the boron atom (oxazaborolidines) with the maleimide core
maleimides 4a–i, selecting boric acid (10 mol%) and PEG-400 are mentioned,9 we proposed a reasonable mechanistic
as a solvent (Table 1, entry 8) as standard conditions, in agree- hypothesis, in which in a first step the maleimides lose their
ment with the current environmental concerns, designing and properties of dienophiles when they interact with boric acid to
developing economically and environmentally benign syn- rapidly form the intermediate I1, this species has a positive
thesis.8 We examined the generality of the reaction by varying charge on one of the olefinic carbons of maleimides that is
the steric and electronic properties of the substituents on the stabilized by the boronic diacid ion H2BO3−. Due to the trigo-
maleimide ring finding that (i) the course of this reaction is nal geometry of this anion and the symmetry of the pyrrolidine
not affected by the chemical nature of the N-benzyl male- ring, the boronic diacid ion induces the stereoselective attack of
imides and (ii) the novel series of the unexpected products 6a– nucleophilic species to the positively-charged carbon (Scheme 3).
i were obtained in good yields instead of the desired DNC A, The second step involves the selective attack of furfuryl-
without the observation of any collateral product or isomers. amine 5 to intermediate I1, which adopts one of its possible
1
H NMR, 13C NMR, DEPT-135 and HSQC experiments of resonance structures, promoted by the electron-donor nature
products 6a–i revealed the presence of the 2,5-dioxopyrrolidine of the furan oxygen,10 to form the intermediate I2 through the
and the 7-oxa-2-azabicyclo[2.2.1]hept-5-ene rings, and a new possible pre-transition state TS. Thus, we suggested a con-
methylene group (C-4′) confirmed the saturation of the five- certed rearrangement, promoted by the regeneration of H3BO3,
membered ring. The COSY experiment confirmed the for- in which the CvO function is restored and promoted the
mation of the 7-oxa-2-azabicyclo[2.2.1]hept-5-ene ring as a abstraction of one of the H bonded to furfurylamine nitrogen.
rigid system, while the HMBC experiment, through the corre- This fact generates a nucleophilic centre on that atom that
lations observed between H-3 to C-3′ and H-3′ to C-3, indicated induces the attack on the electrophilic centre on the oxocarbe-
the connection between C-4 (the bicyclic moiety) and C-3′ nium ion and leads to the formation of the 7-oxa-2-azabicyclo-
( pyrrolidine ring) (see ESI† for details). [2.2.1]hept-5-ene ring.

408 | Org. Biomol. Chem., 2013, 11, 407–411 This journal is © The Royal Society of Chemistry 2013
 View Article Online

Organic & Biomolecular Chemistry Communication

Table 4 Synthesis of N-substituted DNCs 7a–c using boric acid as a catalyst in

PEG-400
Published on 22 October 2012 on http://pubs.rsc.org | doi:10.1039/C2OB26699G
Downloaded by University of Arizona on 15 December 2012

a
Isolated yield. b Reaction times (hours) monitored by TLC.
Scheme 3 Possible mechanistic hypothesis for the reaction of maleimides and
furfurylamine under H3BO3 catalysis.

Table 3 Reaction between maleimides 4j and 4k with furfurylamine 5a giving

products 6j and 6k

a
Isolated yield.

Finally, according to the reaction mechanism, there is no

evidence that the pre-existing chirality of maleimides 4h and
4i generates an asymmetric induction that affects the selective
formation of the enantiomers 6h and 6i (see ESI† for details). Fig. 1 A: Zebrafish embryos treated with compound 6g at 72 hpf. The
embryos treated at 100, 150 and 200 μM died after chemical exposure at this
Next, we turned our attention to other functionalized male-
time. The main visual phenotype, to measure the development delay at this
imides in order to explore the reactivity and versatility of our stage, was when the eggs hatch and the alevins could be photographed. At
reaction. Thus, making furfurylamine 5a react with male- 80 μM after 72 hpf, the eggs have not hatched at the same rate than the
imides 4j and 4k, two new molecules 6j and 6k were obtained control did and the digestive damage (DD) could be observed at 60 μM. B:
with both 7-oxa-2-azabicyclo[2.2.1]hept-5-ene and 2,5-dioxo- Zebrafish embryos treated with compound 6g at 96 hpf. Embryos treated at
80 μM finally died after 96 hpf. Head–trunk angle (red dotted line) 134.4°
pyrrolidine rings as exclusive products (Table 3).
(control angle: 148°) at 60 μM indicating several development delays. The
At the end of our investigation and with the need to accom- yellow dotted line indicates that the embryos, treated with 60 μM and below,
plish our initial objective, we focused our efforts on the syn- did not consume their yolk at the same rate to the control fish, putting in evi-
thesis of the desired DNC A. Based on the possible reaction dence the DD induced by the 7-oxa-2-azabicyclo[2.2.1]hept-5-ene 6g.

This journal is © The Royal Society of Chemistry 2013 Org. Biomol. Chem., 2013, 11, 407–411 | 409
 View Article Online

Communication Organic & Biomolecular Chemistry

Table 5 Criteria for the selection of the best conditions for the reaction of the in vitro AChE inhibitory activity, highlighting compound 6b,
N-benzylmaleimides 4a with furfurylamine 5 with an IC50 = 0.2 mM, as the most potent inhibitor of this
enzyme (Table 5).
Compound IC50 a (mM)

6a 0.233 ± 0.003
6b 0.200 ± 0.004
6c 0.267 ± 0.008 Conclusions
6d 0.315 ± 0.014
6e 0.293 ± 0.004 In summary, the present work is the first example of an easy,
6f 0.235 ± 0.011 efficient and green protocol for the synthesis of both novel
6g 0.279 ± 0.015
6h 0.249 ± 0.012 7-oxa-2-azabicyclo[2.2.1]hept-5-enes and 4-aminomethyl-7-oxa-
6i 0.262 ± 0.012 bicyclo [2.2.1]hept-5-enes, further investigations of the scope
6j 0.302 ± 0.008 and the reaction mechanism of this synthetic method could
6k 0.419 ± 0.026
extend its application to the synthesis of some natural and bio-
Published on 22 October 2012 on http://pubs.rsc.org | doi:10.1039/C2OB26699G

7a 0.236 ± 0.006
7b 0.287 ± 0.009 logically active molecules.
7c 0.212 ± 0.008 Through zebrafish embryo in vivo screening it was discov-
Physostigmine 0.173 ± 0.009b
ered that 6g is a novel inhibitor of early-stage zebrafish embryo
a
IC50 values are the mean ± SEM of at least three different experiments development and an extremely toxic agent.
Downloaded by University of Arizona on 15 December 2012

in duplicate. b IC50 values are expressed in μM.

Possessing several degrees of structural diversity, both types
of bicycloheptenes 6a–k and 7a–c are novel, interesting models
for zebrafish embryo in vivo screening that encourage our syn-
mechanism proposed in Scheme 3, the nucleophilicity of the thetic and biological investigations on phenotypic characteriz-
furfurylamine nitrogen plays a key role in the ring closure ation of a whole vertebrate organism in order to exploit this
during the formation of the bicyclic system, so the acetylation platform for medium to high throughput compound testing.
of this nitrogen probably would avoid the ring closure and
allow the DAR between the reactants.
From this hypothesis we performed the reaction between
Acknowledgements
N-acetylfurfurylamine 5b and maleimides 4b, 4j and 4k under
the same conditions studied above. We were pleased that We acknowledge the financial support given by Universidad
N-substituted DNCs 7a–c were obtained in excellent yields Industrial de Santander during the development of this study.
(Table 4).
Finally and as part of our current chemical-biology
program, preliminary studies were carried out to establish the
Notes and references
possible biological activity of molecules 6a–k and 7a–c.
We addressed the zebrafish (Danio rerio, Cyprinidae) as a 1 For reports on cantharidins biological activities, see:
system for biomedical research. Through zebrafish embryo (a) I. Bertini, V. Calderone, M. Fragai, C. Luchinat and
in vivo screening11 we discovered that compound 6g was a E. Talluri, J. Med. Chem., 2009, 52, 4838;
novel inhibitor of early-stage zebrafish embryo development (b) M. J. Robertson, C. P. Gordon, C. Gilbert, A. McCluskey
and an extremely toxic agent at diverse concentrations. The and J. A. Sakoff, Bioorg. Med. Chem., 2011, 19, 5734.
embryos treated with 100, 150 and 200 μM died after 72 hours 2 (a) Z.-L. Wei, P. A. Petukhov, Y. Xiao, W. Tückmantel,
after chemical exposure. The head–trunk angle, used as a C. George, K. J. Kellar and A. P. Kozikowski, J. Med. Chem.,
measure of developmental rate (normal angle from the middle 2003, 46, 921; (b) Y. Huang, Z. Zhu, Y. Xiao and
of the ear and eye to the notochord: 148° at 96 hpf ), indicates M. Laurelle, Bioorg. Med. Chem. Lett., 2005, 15, 4385.
that the embryos treated with 60 μM, and below, are slightly 3 (a) E. Gómez-Sánchez, E. Soriano and J. Marco-Contelles,
delayed in development, head–trunk angle 134.4° (red dotted J. Org. Chem., 2007, 72, 8656; (b) P. D. Crose and C. L. Rosa,
line). The yellow dotted line indicates that the embryos, Tetrahedron: Asymmetry, 2002, 13, 197.
treated with 60 μM and below, did not consume their yolk at 4 S. Yan, M. J. Miller, T. A. Wencewicz and U. Möllmann,
the same rate to the control fish, putting in evidence the diges- Bioorg. Med. Chem. Lett., 2010, 20, 1302.
tive damage (DD) (Fig. 1B). 5 A. Budzińska and W. Sas, Tetrahedron, 2001, 57, 2021.
Additionally, we followed the method described by 6 For recent reviews on the Diels–Alder reaction, see:
Ellman12 and adapted in our laboratory with some modifi- (a) V. V. Kouznetsov, Tetrahedron, 2009, 65, 2721;
cations.13 Compounds 6a–k and 7a–c were evaluated as inhibi- (b) D. R. Merchán Arenas, F. A. Rojas Ruiz and
tors of the enzyme acetylcholinesterase (AChE) from V. V. Kouznetsov, Tetrahedron Lett., 2011, 52, 1388;
Electrophorus electricus (type V-S). Comparing the AChE inhibi- (c) V. V. Kouznetsov, D. R. Merchán Arenas, C. J. Ortiz
tory activity for the evaluated compounds with the obtained Areniz and C. M. Meléndez Gómez, Synthesis, 2011, 4011;
inhibitory activity for the reference compound ( physostig- (d) V. V. Kouznetsov, C. M. Meléndez Gómez, F. A. Rojas
mine), we found that these compounds possess moderate Ruiz and E. del Olmo, Tetrahedron Lett., 2012, 53, 3115.

410 | Org. Biomol. Chem., 2013, 11, 407–411 This journal is © The Royal Society of Chemistry 2013
 View Article Online

Organic & Biomolecular Chemistry Communication

7 (a) L. V. B. Hoelz, B. T. Gonçalves, J. C. Barros and 10 C. G. Francisco, A. J. Herrera and E. Suárez, J. Org. Chem.,
J. F. Mendes da Silva, Molecules, 2010, 15, 94; 2003, 68, 1012.
(b) C. S. J. Walpole, R. Wrigglesworth, S. Bevan, 11 For small molecule screening in zebrafish, see:
E. A. Campbell, A. Dray, I. F. James, M. N. Perkins, (a) L. R. Donaldson, S. Wallace, D. Haigh, E. E. Patton and
D. J. Reid and J. Winter, J. Med. Chem., 1993, 36, 2362; A. N. Hulme, Org. Biomol. Chem., 2011, 9, 2233;
(c) N. Matuszak, G. G. Muccioli, G. Labar and (b) K. L. Taylor, N. J. Grant, N. D. Temperley and
D. M. Lambert, J. Med. Chem., 2009, 52, 7410. E. E. Patton, Cell Commun. Signaling, 2010, 8, 11;
8 For a review of the Green Chemistry and their principles, (c) J. Berger and P. Currie, Curr. Med. Chem., 2007, 14,
see: (a) S. L. Y. Tang, R. L. Smith and M. Poliakoff, Green 2413.
Chem., 2005, 7, 761–762; (b) P. T. Anastas and N. Eghbali, 12 G. L. Ellman, K. D. Courtney, V. Andres Jr. and
Chem. Soc. Rev., 2010, 39, 301–312; (c) C.-J. Li and R. M. Feather-Stone, Biochem. Pharmacol., 1961, 7, 88.
P. T. Anastas, Chem. Soc. Rev., 2012, 41, 1413–1414. 13 V. V. Kouznetsov, L. Y. Vargas Méndez and A. Muñoz
9 S. Mukherjee and E. J. Corey, Org. Lett., 2010, 12, 632. Acevedo, Lett. Drug Des. Discov., 2010, 7, 710.
Published on 22 October 2012 on http://pubs.rsc.org | doi:10.1039/C2OB26699G
Downloaded by University of Arizona on 15 December 2012

This journal is © The Royal Society of Chemistry 2013 Org. Biomol. Chem., 2013, 11, 407–411 | 411