Mechanistic investigation and implications of a sacrificial aniline for the tandem cascade synthesis of 4-aza-podophyllotoxin analogues

Article abstract of DOI:10.1016/j.tetlet.2013.08.071

Mechanistic investigations were pursued to determine a plausible mechanism in the cascade reaction involving tetronic acid with aldehydes and anilines to generate the 4-aza-2,3-dehydropodophyllotoxin core structure. The mechanistic driven hypothesis paved

Full text of DOI:10.1016/j.tetlet.2013.08.071

Tetrahedron Letters xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Mechanistic investigation and implications of a sacrificial aniline
for the tandem cascade synthesis of 4-aza-podophyllotoxin
analogues
⇑
Nagalakshmi Jeedimalla, Jennifer Johns, Stéphane P. Roche
Department of Chemistry and Biochemistry, Florida Atlantic University, Physical Science Building, 777 Glades Road, Boca Raton, FL 33431, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Mechanistic investigations were pursued to determine a plausible mechanism in the cascade reaction
involving tetronic acid with aldehydes and anilines to generate the 4-aza-2,3-dehydropodophyllotoxin
core structure. The mechanistic driven hypothesis paved the way for a new reaction design using for
the first time a sacrificial electron deficient aniline leading to an optimized cascade protocol (up to three-
fold yield improvement) for the synthesis of novel 4-aza-2,3-dehydropodophyllotoxin analogues.
Published by Elsevier Ltd.
Received 12 July 2013
Revised 9 August 2013
Accepted 19 August 2013
Available online xxxx
Keywords:
Natural product-mimetic scaffolds
Heterolignans
4-Aza-podophyllotoxin
Cascade reaction
Mechanistic investigation
The heterolignan podophyllotoxin (1) is known to inhibit the
assembly of tubulin into microtubules through tubulin binding,1
but high toxicity has limited its therapeutic application making
the development of structural analogues highly desirable.2 While
these efforts have resulted in the development of the commercially
marketed chemotherapeutic agent etoposide (VP-16), the lack of
chemical reactivity of certain building blocks has significantly im-
peded the preparation of diverse structural analogues. Similarly,
the 4-aza-structural analogues 2 (Fig. 1) have also shown impor-
tant biological activity as tubulin assembly inhibitors, insecticides
and vascular-disrupting agents (VDAs).3 Starting a new medicinal
chemistry program, we decided to revisit some structural varia-
tions of the 4-aza-2,3-dehydropodophyllotoxins chemotype 2,
aiming to quickly build up a library. Herein, we report the develop-
ment of an aniline mediated cascade reaction leading to improved
access of 4-aza-2,3-dehydropodophyllotoxins 2, podophyllotoxin
analogues, significantly improving diversity and chemical yield rel-
ative to previous reports (up to threefold yields improvement).
This new process was developed based on a rational mechanism,
supported by control experiments.
activity.4 Since the seminal work from Giorgi–Renault and
Husson,4b several reports have examined the one pot synthesis of
4-aza-2,3-dehydropodophyllotoxins compounds, screening for
solvents effect, temperature and potential catalysts in this reaction.
Recently, scientists at Bayer Cropscience prepared a large library of
4-aza-2,3-dehydropodophyllotoxins (ꢀ140 compounds), reporting
a lack of synthetic efficiency using either the typical or improved
R2
N
OH
H
O
4
3
B
A
C
O
O
2
1
O
R1
H
O
O
D
1
2
MeO
OMe
R3
OMe
4-aza-podophyllotoxins
R2
N
R2
N
O
H
O
H
O
One-pot
Mechanistic studies
R1
R1
O
H
O
3
5
2
OH
In the last decade numerous 4-aza-analogues of podophyllo-
toxin (1) have been prepared due to their important biological
R3
4
R3
Figure 1. General approach to 4-aza-podophyllotoxin analogues.
⇑
Corresponding author.
E-mail address: sroche2@fau.edu (S.P. Roche).
0040-4039/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.tetlet.2013.08.071
Please cite this article in press as: Jeedimalla, N.; et al. Tetrahedron Lett. (2013), http://dx.doi.org/10.1016/j.tetlet.2013.08.071

2
N. Jeedimalla et al. / Tetrahedron Letters xxx (2013) xxx–xxx
O
H
2-pentanol,
120 o
(eq. 1)
HO
C
unproductive pathway
no major product observed af ter 1h
O
MeO
4a
OMe
O
5
OMe
H
N
2-pentanol,
120 o
4a (1.0 equiv.)
HO
HO
HO
NH2
3a
C
(eq. 2)
O
O
O
unproductive
pathway
5
O
6a
5 mins
full conv.
H
OMe
N
H
HO
OMe
OMe
2-pentanol,
120 o
O
5 (1.0 equiv.)
C
(eq. 3)
3a
4a
HO
N
O
MeO
MeO
31% yield
5 mins
f ull conv.
2a
7a
OMe
Scheme 1. Control experiments between the three partners 3–5 involved in the cascade transformation.
one-pot procedures.5 The perfect orchestration in the reaction
between the three components 3–5 remains unclear and the harsh
conditions required in the reaction generate large amounts of
decomposition in a number of cases. While facing similar difficul-
ties in the synthesis of several novel 4-aza-2,3-dehydropodo-
phyllotoxins modified on the A and B rings (most likely due to
electronic effects and/or substitution patterns), we decided to
investigate the reaction mechanism in an attempt to improve the
previous synthetic protocols. To date, several hypothetical mecha-
nisms have been reported in the literature;4b,c,e,f,h,l however, none
have established the importance in the order of addition of the
three partners (aniline 3, benzaldehyde 4 and tetronic acid 5)
engaged in the cascade transformation. While several reaction
pathways may advance simultaneously to the desired 4-aza-2,3-
dehydropodophyllotoxin core,4e,h we began our studies by a thor-
ough examination of the condensation of each reaction component
by sets of two independently (Scheme 1). While the Knoevenagel
condensation product of tetronic acid 5 and 3,4,5-trimethoxy benz-
aldehyde 4a was not formed efficiently (Scheme 1, Eq. 1), both con-
densation between aniline 3a and tetronic acid 5 (Eq. 2), or
aldehyde 4a (Eq. 3) proceed rapidly to generate respectively enam-
ine 6a and imine 7a in a quantitative manner.6 Surprisingly, in situ
addition of aldehyde 4a to enamine 6a did not afford the desired
cyclized product 2a, demonstrating an unproductive reaction path-
way. On the other hand, imine 7a reacts rapidly with tetronic acid
5 to deliver the desired 4-aza-2,3-dehydropodophyllotoxin 2a in
31% overall yield. These control experiments not only showed that
only one pathway (eq. 3) leads to the desired product 2a, but the
order of addition of each component is also crucial for the success
of the cascade reaction.
Dual role played by the aniline component in the mechanism
We hypothesized that aniline 3a was acting in a dual role, first
activating the aldehyde component 4a (LUMO lowering effect of
the resulting iminium from 7a) for the addition of tetronic acid 5
R2
N
O
H
NH2
one-pot
H
O
R1
O
R1
3
R3
4
2
R3
R3
6-π
H
N
electrocyclization
6-exo-trig
R2
O
7
R2
R1
X
N
N
O
O
O
O
O
5
R1
R1
O
O
H
H
O
R3
X
N
MeO
iminium 12
quinolinium methide 11
O
R1
8
O
R3
R2
X
N
O
H
NH2
O
O
O
R1
9
R1
regenerated
10
3
R3
R3
Figure 2. Proposed mechanism for the formation of 4-azapodophyllotoxin 2 based on the established control experiment results.
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N. Jeedimalla et al. / Tetrahedron Letters xxx (2013) xxx–xxx
3
OMe
H
N
OMe
5 (1.0 equiv.)
H
O
H
O
10 mins,
pentanol, 120 o
2 hours
C
NH2
N
Cl
MeO
2a
OMe
7b
O
(31% yield)
then
3a
(1.0 equiv.)
30 mins
Cl
(1.0 equiv.)
Cl
MeO
2b
3b
MeO
OMe
OMe
not observed
4a
(1.0 equiv.)
OMe
Scheme 2. Control experiment highlighting the dual role of aniline 3 in the cascade transformation producing 4-aza-podophyllotoxin 2a.
O
H
N
O
method A:
CHO
typical one pot
NH2
H
O
2-pentanol, 120 oC
5
R1
OH
O
2c-g
R1
3c-f
, R1 = 3-SMe
4a-c
method B:
R2
NH2
sequential addition using
R2
3c
3d
3e
4a, R2 = H
4b
4c
the sacrificial aniline 3b
1
, R = 3,4-C4H8
2
, R = 3,4-OCH2O-
Cl
, R1 = 3-OH
, R2 = 3,4,5-OMe
3f, R1 = 3-OCHF2
H
N
H
N
H
N
H
N
H
F
MeS
N
O
HO
MeS
H
O
H
H
O
O
H
H
O
O
F
O
O
O
O
O
2d
2e
2f
2g
MeO
2c
O
O
O
OMe
O
O
MeO
O
method A: 12% yield
method B: 39% yield
method A: 18% yield
method B: 49% yield
method A: <2% yield
method B: 7% yield
method A: 15% yield
method B: 25% yield
method A: 7% yield
method B: 21% yield
Scheme 3. Comparative synthesis of novel 4-aza-podophyllotoxins 2c–2g between the typical and the new one-pot protocol references.
(Knoevenagel condensation) before being regenerated and con-
densed to form the product B ring (proposed mechanism in
Fig. 2). To test this hypothesis, a competitive reactivity study of
anilines was designed (Scheme 2). A sacrificial electron deficient
p-chloroaniline 3b was condensed with the same benzaldehyde
derivative 4a generating imine 7b over the course of 2 hours at
120 °C. In the same reaction vessel was then added successively
tetronic acid 5 and aniline 3a in equimolar ratio which provided
to our delight 4-aza-2,3-dehydropodophyllotoxin 2a exclusively
in 31% yield (the other possible product 2b was not detected in
the crude reaction mixture by 1H NMR or mass spectrometry anal-
yses). This result was encouraging and provides a strong support
for the dual role played by the aniline substrate 3 in the cascade
reaction.7 Incorporating this information, we therefore propose
the mechanism outlined in Figure 2. The first condensation most
likely occurs between aniline 3 and benzaldehyde 4 to deliver acti-
vated imine 7 which further endures the addition of tetronic acid 5.
Through the addition of 5, aniline 3 is then regenerated and may
then condense either on the b-position of the Michael acceptor 9
or on the carbonyl (1,4 vs 1,2 addition). To better understand this
information (even small ee) may be retained in the final product
2a, which was not observed.
Sacrificial aniline 3b in the tandem cascade reaction
After further confirmation of our mechanistic hypothesis for the
cascade reaction, we decided to examine the effect of the sacrificial
electron deficient aniline 3b on the cascade transformation scope
for several other substrates. We proposed that the successful for-
mation of the Knoevenagel intermediate 9 may also result in the
overall reaction improvement. After considerable experimental ef-
forts to optimize the stepwise cascade process (Scheme 3), we
were pleased that the more reluctant anilines 3c–f tested in the
reaction were successively condensed to yield the new 4-aza-2,3-
dehydropodophyllotoxin derivatives 2c–g in 39%, 49%, 25%, 21%
and 7% yields respectively (method B).6 For the comparative study,
all the new 4-aza-2,3-dehydropodophyllotoxins 2c–g were iso-
lated in average with threefold higher yields than using the typical
one pot procedure (isolated yields after the same purification by
method A versus method B) or a straightforward stepwise ap-
proach without sacrificial aniline 3b.9,10 Most of the compounds
2c–g synthesized via the method B were simply recrystallized from
ethanol for optimum purity affording the recovered ‘sacrificial’ ani-
line 3b as major by-product from the mother liquors.6
The cascade transformation discussed herein, highlights a novel
stepwise condensation of anilines with aldehydes and tetronic acid
resulting in 4-aza-2,3-dehydropodophyllotoxins. Several control
experiments shed light on the proposed mechanism and revealed
the order of reactivity between the three components. Our mecha-
nistic investigation resulted in the design of a new process incor-
porating an electron deficient, ‘sacrificial aniline’, enabling a
simple and versatile protocol for the synthesis of problematic
aza-analogues of podophyllotoxin (1). Using this strategy, a series
step we repeated experiments reported by Shi,4l using
a catalyst. In this reaction -proline should react in the similar fash-
L-proline as
L
ion as the aniline 3 by a LUMO lowering effect to facilitate the
nucleophilic addition on a chiral Michael acceptor (similar to 9).
For reasons that we do not explain yet, the proline catalysis pro-
ceeded poorly for our substrates leading to low isolated yields.8
As a result and in total agreement with Shi’s report, product 2a
was isolated in a racemic form (no enantiomeric excess (ee) was
detected)8 persuading us to propose that the condensation occurs
at the carbonyl carbon-centre to form 10, leading after elimination
to either quinolinium methide 11 or iminium 12 which do not car-
ry any chiral information towards the final product 2. If the con-
densation arises at the b-position, we believe that some chiral
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4
N. Jeedimalla et al. / Tetrahedron Letters xxx (2013) xxx–xxx
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of 4-aza-podophyllotoxin derivatives and the expansion of the
new method to synthesize 4-oxo-podophyllotoxin analogues.
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Acknowledgments
The authors thank the Department of Chemistry and Biochem-
istry from Florida Atlantic University, for providing financial sup-
port. The authors would like to thank Ms. M. Flint and Dr. A.
Haces for preliminary results in the present manuscript, as well
as the Mass Spectrometry Facility at the University of Florida
(National Science Foundation for the MS instruments) for analytical
data. The authors would also like to thank Professor S. Lepore at
FAU and Dr. A. Kleinke at Dart Neuroscience for helpful scientific
discussions and editing this manuscript.
5. As reported in Refs. 3b,f, depending on substrate patterns, isolated yields for
various 4-aza-2,3-dehydropodophyllotoxins varied from 3% to 95%.
6. For full experimental details and 1H NMR study, please refer to the Supporting
information.
7. p-Nitroaniline was also tested as ‘sacrificial aniline’ providing also product 2a
in a lower yield (25% isolated yield) with more decomposition as observed on
the TLC profile of the reaction.
Supplementary data
Supplementary data (NMR overlays for the proposed mecha-
nism (Fig. 2) and copies of spectra associated with this communi-
cation is furnished) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.tetlet.2013.08.071.
8. Similar conditions to the general protocol (Method A; Ref. 4l) were used with
the addition of L-Proline (10 mol %) as chiral catalyst. The reaction produced 4-
aza-2,3-dehydropodophyllotoxin 2a in 25% overall yield. Aliquot of the
reaction mixture was taken overtime at 10, 30 and 60 mins, further purified
by preparative TLC to isolate compound 2a in a pure form for analysis by
optical rotation in DMSO (c = 0.1). No light deviation was noticeable in the
three different samples suggesting that product 2a was achiral therefore no
chiral information was transferred from the catalyst to the final product 2a. The
results suggest that compound 10 (Fig. 2) may endure several transformations
before the final cyclization leading to 4-aza-podophyllotoxins, loosing then the
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Please cite this article in press as: Jeedimalla, N.; et al. Tetrahedron Lett. (2013), http://dx.doi.org/10.1016/j.tetlet.2013.08.071