A simple synthesis of bannucine and 5′-epibannucine from (-)-vindoline

Article abstract of DOI:10.1016/j.tet.2015.10.020

Bannucine is an Aspidosperma alkaloid isolated from the dried leaves of Catharanthus roseus. The molecule is a derivative of vindoline bearing a C10 substituent, a pattern common to the antineoplastic dimeric indole alkaloids of C. roseus. In bannucine, a 2-pyrrolidone moiety is attached at C5′ to the aromatic ring of the vindoline core at C10. In the present work we report the synthesis of bannucine and its 5′-epimer from natural (-)-vindoline using a cyclic N-acyliminium ion intermediate whose N-acylaminocarbinol precursor is synthesized by the partial reduction of succinimide. We also describe the separation and the structural analysis of the two epimers, using among others, single crystal X-ray diffraction methods, in order to clarify the orientation of the proton attached to the C5′ carbon. The in vitro antineoplastic activity of the pure epimers was also investigated, but none of the two substances showed significant activity on the examined tumour cell lines.

Full text of DOI:10.1016/j.tet.2015.10.020

Accepted Manuscript
A simple synthesis of bannucine and 5’-epibannucine from (−)-vindoline
Viktor Ilkei, Péter Bana, Flórián Tóth, Anna Palló, Tamás Holczbauer, Mátyás
Czugler, Zsuzsanna Sánta, Miklós Dékány, Áron Szigetvári, László Hazai, Csaba
Szántay, Jr., Csaba Szántay, György Kalaus
PII:
S0040-4020(15)30120-4
10.1016/j.tet.2015.10.020
TET 27192
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 29 June 2015
Revised Date: 24 September 2015
Accepted Date: 6 October 2015
Please cite this article as: Ilkei V, Bana P, Tóth F, Palló A, Holczbauer T, Czugler M, Sánta Z, Dékány
M, Szigetvári Á, Hazai L, Szántay Jr. C, Szántay C, Kalaus G, A simple synthesis of bannucine and 5’-
epibannucine from (−)-vindoline, Tetrahedron (2015), doi: 10.1016/j.tet.2015.10.020.
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A simple synthesis of bannucine and 5’-
epibannucine from (−)-vindoline
Leave this area blank for abstract info.
Viktor Ilkeia, *, Péter Banaa, Flórián Tóthb, Anna Pallóc, Tamás Holczbauerc, Mátyás Czuglerc, Zsuzsanna
Sántad, Miklós Dékányd, Áron Szigetvária, László Hazaia, Csaba Szántay Jr.d, Csaba Szántaya and György
Kalausa (†)
a Department of Organic Chemistry and Technology, Budapest University of Technology and Economics,
Gellért tér 4. Budapest, H-1111, Hungary
bXiMo Hungary Ltd., Záhony utca 7. Budapest, H-1031, Hungary
c Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences,
Magyar tudósok körútja 2. Budapest, H-1117, Hungary
d Spectroscopic Research Division, Gedeon Richter Plc., Gyömrői út 19-21. Budapest, H-1103, Hungary

1
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Tetrahedron
journal homepage: www.elsevier.com
A simple synthesis of bannucine and 5’-epibannucine from (−)-vindoline
Viktor Ilkeia, *, Péter Banaa, Flórián Tóthb, Anna Pallóc, Tamás Holczbauerc, Mátyás Czuglerc, Zsuzsanna
Sántad, Miklós Dékányd, Áron Szigetvária, László Hazaia, Csaba Szántay Jr.d, Csaba Szántaya and György
Kalausa (†)
a Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Gellért tér 4. Budapest, H-1111, Hungary
bXiMo Hungary Ltd., Záhony utca 7. Budapest, H-1031, Hungary
c Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok körútja 2. Budapest, H-1117, Hungary
d Spectroscopic Research Division, Gedeon Richter Plc., Gyömrői út 19-21. Budapest, H-1103, Hungary
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Bannucine is an Aspidosperma alkaloid isolated from the dried leaves of Catharanthus roseus.
The molecule is a derivative of vindoline bearing a C10 substituent, a pattern common to the
antineoplastic dimeric indole alkaloids of Catharanthus roseus. In bannucine, a 2-pyrrolidone
moiety is attached at C5’ to the aromatic ring of the vindoline core at C10. In the present work
we report the synthesis of bannucine and its 5’-epimer from natural (−)-vindoline using a cyclic
N-acyliminium ion intermediate whose N-acylaminocarbinol precursor is synthesized by the
partial reduction of succinimide. We also describe the separation and the structural analysis of
the two epimers, using among others, single crystal X-ray diffraction methods, in order to clarify
the orientation of the proton attached to the C5’ carbon. The in vitro antineoplastic activity of
the pure epimers was also investigated, but none of the two substances showed significant
activity on the examined tumor cell lines.
Available online
Keywords:
Bannucine
Aspidosperma alkaloids
Alkaloid synthesis
N-acylaminocarbinol
Iminium chemistry
2015 Elsevier Ltd. All rights reserved
.
1. Introduction
Catharanthus roseus (L.) G. Don is a herbaceous evergreen
plant endemic to Madagascar. It is grown around the globe as an
ornamental plant and also as a medical plant used for the
treatment of a wide range of diseases including hypertension,
malaria, diabetes and Hodgkin’s lymphoma.1 During the past
decades numerous alkaloids have been isolated from
Catharanthus roseus, among which the most important ones are
vinblastine and vincristine. These two antineoplastic dimeric
indole alkaloids are used in the clinical treatment of certain types
of cancer including leukemias, non-small cell lung cancer and
Hodgkin’s lymphoma. The isolation and structural analysis of
potentially antineoplastic alkaloids derived from Catharanthus
roseus is still a relevant field of research today. The structure
elucidation, rational synthesis and chemical modification of the
isolated compounds provide a constant challenge for organic
chemists.2
Figure 1. The structure of bannucine ((−)-1)
the antineoplastic dimeric indole alkaloids. In bannucine, a 2-
pyrrolidone moiety is attached at C5’ to the aromatic ring of the
vindoline core at C10. Apart from spectroscopic structure
elucidation the authors also described the new compound by
giving its melting point (152-154 °C) and specific rotation
([α]D = −33 (c = 0.26; chloroform)). Based on their NMR
measurements the authors assigned a β-orientation to the proton
attached to C5’.
The Aspidosperma alkaloid bannucine ((−)-1, Figure 1.) was
isolated by Atta-ur-Rahman and co-workers from the dried leaves
of Catharanthus roseus in 1986.3 The molecule is a derivative of
vindoline ((−)-2) bearing a C10 substituent, a pattern common to
During our work we set the aim of synthesizing bannucine
from natural (−)-vindoline.
Corresponding author. Tel.: +36-1-463-1277; e-mail: vilkei@mail.bme.hu

2
Tetrahedron
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2. Results and discussion
N-acylaminocarbinol derivatives, as well as the N-
the methoxy and the dialkylamino group.5-7 Considering the
statements above we proposed a simple retrosynthetic scheme for
the synthesis of bannucine ((−)-1) (Scheme 1.). Disconnection of
the C10-C5’ bond leads to the cyclic N-acylaminocarbinol
reagent of type I, and vindoline ((−)-2).
acyliminium ions that arise from them, are often used as
electrophilic reagents and preferred intermediates during the
synthesis of alkaloids, especially for the construction of
heterocyclic units.4 The reactivity of vindoline ((−)-2) and the use
of these reagents provide an opportunity for the synthesis of
bannucine ((−)-1). The electron-rich aromatic ring of vindoline
((−)-2) is highly activated toward electrophilic substitution; its
most reactive position is C10, owing to the directing effects of
Scheme 1. Retrosynthetic analysis of bannucine ((−)-1)
We first synthesized 5-acetoxypyrrolidine-2-one (5) according
to the procedure described in the literature.8-9 Compound 5 can be
prepared from succinimide (3) in two steps (Scheme 2.). The
partial reduction of 3 yields 5-ethoxypyrrolidine-2-one (4),8
which is converted to acetate 5 by stirring in acetic acid at
ambient temperature.9 Considering the sensitivity of N-
acylaminocarbinol acetate 5, no attempts were made at its
purification, instead it was used immediately as an oil in the next
reaction. Vindoline ((−)-2) was allowed to react with an excess of
acetate 5 at 130 °C under neat conditions for 2 hours, according
to the procedure described by Nagasaka et al.9 The reaction gave
a mixture of bannucine and 5’-epibannucine in an acceptable
yield (Scheme 2.). Purification of the epimeric mixture was
carried out by column chromatography. The pure epimeric
mixture was crystallized by trituration in ether and characterized
by its melting point (151-153 °C) and specific rotation ([α]D =
−52.3 (c = 0.26; CHCl3)).
Scheme 2. Synthesis of bannucine epimers under neat conditions
Optimization of the excess of reagent 5 used is included in Table
1. (entries 1-5). Since raising the amount of the N-
acylaminocarbinol reagent to more than 5 equivalents relative to
vindoline did not result in a higher yield, experiments hereafter
were conducted using 5 equivalents of acetate 5 relative to (−)-2.
gave oily reaction mixtures (Scheme 3.) (Table 1., entries 6-8).
A shorter reaction time resulted in a clear reaction mixture, while
longer reaction times gave lower yields presumably due to the
thermal degradation of the product, which resulted in the
formation of tarry impurities in the reaction mixture. The highest
yield (62%) was reached after a 30 minute run at 130 °C (Table
1., entry 8), which was virtually equal to that of the conventional
heating method (Table 1., entry 3). Compared to conventional
heating, the advantage of using microwave heating was a
significantly shorter reaction time and a reduced amount of tarry
Next, we investigated the possibility of preparing the epimeric
mixture using different methods. Applying microwave heating to
neat mixtures of a five-fold excess of the N-acylaminocarbinol
acetate 5 and vindoline ((−)-2) at 130 °C for different durations

3
side products, owing to the direct and uniform heating of the
reaction mixture.
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Scheme 3. Synthesis of bannucine epimers using microwave irradiation
Next, we investigated the acid catalysed solution-phase synthesis
of the bannucine epimers. Small-scale model experiments were
conducted to establish the most suitable solvent for the reaction
(Table 1., entries 9-15). Reactions were run in boiling solvents
for 4 hours, with catalytic amounts of p-toluenesulfonic acid. The
best solvent was found to be nitromethane, with the isolated yield
77%. The suitability of nitromethane can probably be attributed
to its ability to solvate the ionic intermediates of the reaction due
to its high polarity. A larger scale (100 mg) experiment in
nitromethane gave the title compounds in an improved 90%
overall yield (Table 1., entry 16), likely due to decreased loss of
material during isolation (Scheme 5.). Under the same
conditions, amidocarbinol 6, which was prepared by hot water
hydrolysis of 4 (Scheme 4.),10 gave virtually the same yield
(Table 1., entry 17), which comes as no surprise, considering that
the same N-acyliminium intermediate (7) is involved in the
reaction (Scheme 6.).
Scheme 4. Synthesis of 5-hydroxypyrrolidine-2-one (6)
Scheme 5. Acid catalysed solution-phase synthesis of bannucine epimers
Table 1. Yield of the bannucine epimers under different conditions
Excess of
Temp.
(°C)
Time
(h)
Yield*, †
(%)
Entry
Reagent
Solvent
Catalyst
Heating
reagent
(mol. equiv.)
2
1
2
5
5
-
-
-
-
130
130
2
2
oil bath
oil bath
31
39
3
5
3
4
5
5
5
5
5
5
-
-
-
-
-
-
-
-
-
-
-
-
130
130
130
130
130
130
2
2
oil bath
oil bath
oil bath
MW
63
66
64
44
50
62
10
20
5
5
2
6‡
7‡
8‡
2
5
1
MW
5
0.5
MW

4
Tetrahedron
9
4
4
4
4
4
4
4
4
6
EtOAc
pTsOH
4
4
4
4
4
4
4
4
4
oil bath
oil bath
oil bath
oil bath
oil bath
oil bath
oil bath
oil bath
oil bath
22
56
25
66
36
77
58
90
86
5ACCEPTED MANUS7C7 R(∆)IPT
10
11
12
13
14
15
16#
17#
5
5
5
5
5
5
5
5
THF
pTsOH
pTsOH
pTsOH
pTsOH
pTsOH
pTsOH
pTsOH
pTsOH
66 (∆)
DMF
120
Dioxane
Toluene
CH3NO2
CH3CN
CH3NO2
CH3NO2
101 (∆)
111 (∆)
101 (∆)
81 (∆)
101 (∆)
101 (∆)
*Experiments were run with 71 mg vindoline ((−)-2), except where otherwise noted.
† Isolated yield for the epimeric mixture of bannucine ((−)-1) and 5’-epibannucine ((−)-5’-epi-1). The ratio of
bannucine and 5’-epibannucine in the product was identical (2:3) in all of the experiments.
‡ Microwave reactions were run with 212 mg vindoline ((−)-2) (for details see the Experimental section).
# Larger-scale experiments were run with 100 mg vindoline ((−)-2), accounting for a higher isolated yield.
According to the NMR measurements of the product the
experimental section, as deduced from higher resolution 13C
mixture contained the two epimers in an approximate ratio of 2:3.
Separation of the epimers was accomplished by multiple
consecutive chromatographic purifications on preparative TLC
plates. The structures of the pure epimers were elucidated by
spectroscopic methods including MS and NMR measurements. In
comparing our NMR results with those of Atta-ur-Rahman et al.,3
we found that the synthetic epimers (–)-1 and (–)-5’-epi-1 and
the bannucine isolated by them had very similar 13C NMR
spectra. However, some of our 13C NMR assignments differ from
those given by Atta-ur-Rahman et al.; in particular, we concluded
that the chemical shift values for the OCOCH3 and C4’ carbons
had been misassigned in the original work, presumably because
of the lower spectral resolution available at that time. For this
reason we herein provide our own assignments in the
NMR and 13C–1H correlation spectra (HSQC and HMBC).
The configuration of the stereogenic carbon at C5’ could not
be assigned unambiguously solely on the basis of the NMR
spectra. In order to be able to assign the configuration of the
carbon at C5’ in each epimer, we grew a single crystal of the
epimer with the lower Rf value, the component which is more
abundant in the epimeric mixture, and could also be isolated with
a higher yield. The unknown configuration was elucidated by X-
ray crystallography (XRD) (c.f. Figure 2.). The absolute
configuration of C5’ turned out to be S, which translates to the α-
orientation of the hydrogen at C5’. Therefore the examined
isomer is 5’-epibannucin ((−)-5’-epi-1), and the ratio of epimers
in the original mixture was (−)-1 : (−)-5’-epi-1 = 2:3. Further
reactions yielded the epimers in approximately the same ratio.
Figure 2. The XRD structure of 5’-epibannucine shown in 30% atomic displacement ellipsoids
The absolute configuration of the crystal could be ascertained
by both the deduction of the configuration based on the known
stereocenters and by the use of the anomalous dispersion effect
mainly due to the dichloromethane solvent molecule. This latter
proved to be successful even in this case when only a slightly
populated solvent was retained in the crystal. The site
occupancies were modelled as 50% population of the
dichloromethane atomic positions. Both the Flack11 and the Hooft
parameters12 show that the X-ray structure is the correct hand of
the crystal. Analysis of the absolute structure using Bayesian
likelihood methods was done using PLATON.13 The results
indicated that the absolute structure had been correctly assigned.
The calculated probability that the structure is inverted is given
as 0 (Figure 3.). As only a part of the original solvent quantity
was retained in the crystal it might still be useful like a “heavy
atom” derivative in defining the absolute stereochemistry.

5
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Figure 3. Bijvoet pair analysis12 of the 5’-epibannucin ((−)-5’-epi-1) : dichloromethane 1 : 0.5 crystal
Based on the literature, we proposed a plausible mechanism
for the formation of the bannucine epimers. Depending on the N-
acylaminocarbinol reagent used, either the protonation of 4 or 6,
or the thermal heterolysis of 5 leads to the formation of the N-
acyliminium salt 7, which is an electrophile reactive enough to
attack the aromatic ring of vindoline ((−)-2) at C10. The reaction
follows the general mechanism of electrophilic aromatic
substitutions: the σ-complex (8) formed in the reaction is
deprotonated by the acetate or tosylate anion yielding a mixture
of bannucine ((−)-1) and 5’-epibannucine ((−)-5’-epi-1) (Scheme
6.).
pTsOH
O
O
H
R
O
OR
N
N
H
H
- ROH
X
R = Et
R = H
4
6
∆
O
O
OAc
N
H
N
H
X- = AcO- or pTsO-
5
7
X
N
N
H
H
H
O
N
H
H3CO
N
H
OCOCH3
COOCH3
H3CO
N
OCOCH3
H
COOCH3
HO
N
CH3
HO
CH3
8
(-)-2
- HX
N
H
H
H
H
O
O
N
H
N
H
H3CO
N
OCOCH3
COOCH3
H3CO
N
OCOCH3
COOCH3
H
H
HO
CH3
HO
CH3
(-)-1
(-)-5'-epi-1
Scheme 6. Proposed reaction mechanism for the formation of the bannucine epimers

6
Tetrahedron
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The pure epimers of bannucine were characterized by their
rotations we measured for bannucine ((−)-1) ([α]D = −12.3 (c =
melting points and specific rotations. Bannucine ((−)-1) melted at
284-286 °C with a specific rotation [α]D = −12.3 (c = 0.26;
CHCl3), while 5’-epibannucine ((−)-5’-epi-1) melted at 191-193
°C and had specific rotation [α]D = −72.3 (c = 0.26; CHCl3).
Comparison of the melting points and specific rotations we
measured for the pure epimers to those from the literature3 gave
no leads in the identification of the epimers (Figure 4.). Based on
the fact that the specific rotation given by Atta-ur-Rahman et al.
for the substance they regarded as pure bannucine
([α]D = −33 (c = 0.26; CHCl3))3 significantly differs from the
value measured by us, and that it falls between the specific
0.26; CHCl3)) and 5’-epibannucine ((−)-5’-epi-1) ([α]D = −72.3
(c = 0.26; CHCl3)), it is likely that the substance Atta-ur-Rahman
et al. isolated from Catharanthus roseus was not pure bannucine,
but in fact a mixture of the two epimers. This assumption is
corroborated by the fact that the melting point given by Atta-ur-
Rahman et al. for their substance (152-154 °C),3 is very close to
the one we measured for the solid epimeric mixture that we
synthesized (151-153 °C), and it is much lower than the melting
point we have measured for each pure epimer (bannucine ((−)-1)
mp.: 284-286 °C; 5’-epibannucine ((−)-5’-epi-1) mp.: 191-193
°C).
[α]D = −12.3 (c = 0.26; CHCl3)
[α]D = −72.3 (c = 0.26; CHCl3)
m.p.: 284-286 °C
m.p.: 191-193 °C
Substance isolated by Atta-ur-Rahman et al.,
regarded as pure bannucine ((−)-1)3
[α]D = −52.3 (c = 0.26; CHCl3)
[α]D = −33 (c = 0.26; CHCl3)
m.p.: 151-153 °C
m.p.: 152-154 °C
Figure 4. Comparison of own and literature data: melting points and specific rotations of the epimeric mixture and of the pure epimers
3. Biology
The in vitro antineoplastic activity of the pure epimers ((−)-1)
ovarian cancer (OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8,
NCI/ADR-RES, SK-OV-3), renal cancer (786-0, A498, ACHN,
CAKI-1, RXF 393, SN12C, TK-10, UO-31), prostate cancer
(PC-3, DU-145), breast cancer (MCF7, HS 578T, BT-549, T-
47D, MDA-MB-468).
and ((−)-5’-epi-1) was investigated at the National Institutes of
Health (NIH), where the proliferation inhibitory effect of the two
samples was measured against different tumor cell lines. The
experiments were performed on 57 different tumor cell lines
embracing 9 frequently occurring tumor types. The tumor types
and cell lines were the following: leukemia (CCRF-CEM, HL-
60(TB), K-562, MOLT-4, SR), non-small cell lung cancer
(A549/ATCC, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23,
NCI-H322M, NCI-H460, NCI-H522), colon cancer (COLO 205,
HCC-2998, HCT-116, HCT-15, HT29, KM12, SW-620), CNS
cancer (SF-268, SF-295, SF-539, SNB-19, SNB-75, U251),
melanoma (LOX IMVI, MALME-3M, M14, MDA-MB-435, SK-
MEL-2, SK-MEL-28, SK-MEL-5, UACC-257, UACC-62),
The NCI screening procedures were conducted as described in
the literature,14 and the origins and processing of the cell lines
were also described.14-16 The inhibitory effect of (−)-1 and (−)-5’-
epi-1 samples were measured against the listed tumor cell lines at
the concentration of 2.5×10-6 M and 10-5 M respectively, but both
epimers were ineffective on the examined tumor cell lines (no
negative growth percentage value was greater than 20%).
4. Conclusion
In the present work we successfully elaborated the synthesis
of the Aspidosperma alkaloid bannucine ((–)-1) and its 5’-epimer
((–)-5’-epi-1) from natural (–)-vindoline ((–)-2). In our synthesis
we applied a cyclic N-acyliminium intermediate, whose N-

7
acylaminocarbinol precursor was synthesized by the partial
reduction of succinimide. The epimers were separated by
preparative TLC and fully characterised, including X-ray
crystallography, which allowed the unambiguous assignment of
the configuration of the C5’ carbon in each epimer, allowing
clarification of the data previously reported for bannucine ((–)-1).
The in vitro antineoplastic activity of the pure epimers was tested
against 57 different tumor cell lines, but none of the two
substances showed significant antitumor activity.
and mean shift/esd is 0.004 and 0.001). The maximum and
ACCEPTED MANUSCRIPT
minimum residual electron density in the final difference map
was 0.52 and -0.28 e·Å-3. Hydrogen atomic positions were
calculated from assumed geometries. Hydrogen atoms were
included in structure factor calculations but they were not
refined. The isotropic displacement parameters of the hydrogen
atoms were approximated from the U(eq) value of the atom they
were bonded to. Crystal structure data are deposited with the
Cambridge Crystallographic Data Centre under CCDC 1063928
((−)-5’-epi-1) and can be obtained free of charge upon
application.
5. Experimental section
5.1. General
5-Ethoxypyrrolidine-2-one (4)
To a solution of succinimide (3) (7.156 g, 72.22 mmol) in ethanol
(300 mL) at 0 °C was added sodium borohydride (4.00 g, 105.74
mmol) in one portion. The reaction mixture was stirred at 0 °C
for 4 h, during which time every 15 min 5 drops of 2 M ethanolic
hydrogen chloride solution were added. Then the reaction
mixture was acidified to pH=3 with 2 M ethanolic hydrogen
chloride solution over 30 min, after which it was stirred at 5 °C
for 45 min. Then the reaction mixture was neutralised (pH=7)
with 5% ethanolic potassium hydroxide solution and evaporated
in vacuo to give a syrupy solid, which was suspended in
chloroform (80 mL), filtered, and the precipitate washed with
chloroform (3×20 mL). The filtrate was evaporated in vacuo to
give a colourless oil, which was dissolved in dichloromethane
(80 mL) and washed with water (3×10 mL). The aqueous phase
was extracted with dichloromethane (6×20 mL), then the organic
phases were unified, dried (MgSO4) and the solvent evaporated
in vacuo to give the title compound 4 (4.327 g, 46%) as a
colourless oil, which crystallized on standing, giving white
crystals, m.p. 51-53 °C (lit.: 48-53 °C);8 Rf (acetone) 0.72;
νmax(KBr) 3200, 2978, 1707, 1689, 1668, 1457, 1282, 1250,
1067, 986 cm-1; δH (400 MHz, DMSO-d6) 8.62 (1 H, s, NH),
4.89-4.83 (1 H, m, H-5), 3.54-3.44 (1 H, m, OCH2y), 3.35-3.27 (1
H, m, OCH2x), 2.30-2.11 (1 H, m, Hy-4), 2.30-2.11 (1 H, m, Hy-
3), 2.05-1.95 (1 H, m, Hx-3), 1.89-1.78 (1 H, m, Hx-4), 1.10 (3 H,
t, J 7.0 Hz, CH3); δC (100.6 MHz, DMSO-d6) 177.4 (CON), 85.0
(C-5), 61.6 (OCH2), 28.1 (C-3), 27.7 (C-4), 15.1 (CH3);
MS(ESI): 130 (C6H12NO2); ESI-MS-MS (cid=35) (rel. int. %):
84(100).
Melting points were measured on a SANYO Gallenkamp
apparatus and are uncorrected. IR spectra were recorded on a
Bruker FT-IR instrument. Optical rotations were measured on a
PerkinElmer 241 polarimeter at ambient temperature, at the D-
line of sodium (λ = 589.3 nm). 1H NMR and 13C NMR
measurements were performed on Varian 400 MHz, Varian 500
MHz and Varian 800 MHz spectrometers. Chemical shifts are
given on the delta scale as parts per million (ppm) with
tetramethylsilane (TMS) (1H) or dimethylsulfoxide-d6 (13C) as the
internal standard (0.00 ppm and 39.5 ppm, respectively). MS
spectra were recorded on VG-Trio-2 and Finnigan MAT 95SQ
instrument using EI or ESI techniques. HRMS analyses were
performed on an LTQ FT Ultra (Thermo Fischer Scientific,
Bremen, Germany) system. Preparative TLC was carried out
using Kieselgel 60 F254 (Merck) coated glass plates. Column
chromatography was performed using Geduran Si 60 (Merck)
silica. Investigations using microwave heating were conducted in
a single-mode Discover System (CEM Corporation) using sealed
reaction vessels (borosilicate, 10 mL volume). Experiments were
performed in temperature control mode (using the built-in
calibrated IR sensor) with 200 W initial power. The reaction
mixtures were stirred („low” setting), the specified time is equal
to the time at set point. (–)-Vindoline (isolated from
Catharanthus roseus) was provided by Gedeon Richter Plc.
5.2. X-ray diffraction
This experiment was carried out at ambient temperature on an
apparently intact crystal selected from a batch vial. All data
collections were run on a RIGAKU R-AXIS RAPID II image
plate diffractometer using Cu-Kα radiation. Crystal data:
C29.5H38ClN3O7, Fwt.: 582.08, white, prism, size: 0.03 × 0.02 ×
0.01 mm, space group P21, a = 11.4810(10) Å, b = 12.1940(10)
Å, c = 12.7760(10) Å, β = 115.174(4)°, V = 1618.7(2) Å3, T =
293(2) K, Z = 2, F(000) = 618, Dx = 1.194 Mg/m3, ꢀ = 1.429
mm-1. A crystal of 5’-epibannucin was mounted on a loop. Cell
parameters were determined by least-squares of the setting angles
of 5730 (6.84 ≤ θ ≤ 54.23) reflections. Intensity data were
5-Hydroxypyrrolidine-2-one (6)
A solution of 5-ethoxypyrrolidin-2-one (4) (2.0 g, 15.5 mmol) in
water (25 mL) was refluxed for 3 h. The solvent was evaporated
in vacuo to give a colourless oil, which was triturated with ethyl
acetate to give a white solid. The solid was filtered and
recrystallized from acetone to give the title compound 6 (0.89 g,
60%) as a white solid, m.p. 94-96 °C (lit.: 90 °C);13 Rf (33%
hexane/acetone) 0.19; νmax(KBr) 3254, 2996, 2962, 1668, 1475,
1415, 1323, 1271, 1166, 1101, 1070, 1016cm-1; δH (400 MHz,
DMSO-d6) 8.18 (1 H, s, NH), 5.70 (1 H, d, J 6.9 Hz, OH), 5.06
(1 H, m, H-5), 2.33-2.23 (1 H, m, Hy-3), 2.24-2.14 (1 H, m, Hy-
4), 2.03-1.94 (1 H, m, Hx-3), 1.75-1.66 (1 H, m, Hx-4); δC (100.6
MHz, DMSO-d6) 176.7 (CON),78.5 (C-5), 30.3 (C-4), 28.4 (C-
3); HRMS: 102.05491 (C4H8NO2; calc. 102.05496); ESI-MS-MS
(cid=55) (rel. int. %): 85(100).
collected on
a R-AXIS RAPID diffractometer (graphite
monochromator; Cu-Kα radiation, λ = 1.54178 Å) at 293(2) K in
the range 6.83 ≤ θ ≤ 54.22 using θ/2θ scans. A total of 8724
reflections were collected of which 3778 were unique [R(int)
=
0.0396, R(σ) = 0.0861]; intensities of 2824 reflections were
greater than 2(σI). Completeness to 2θ = 0.984. A numerical
absorption correction was applied to the data (the minimum and
maximum transmission factors were 0.952 and 0.994). The
structure was solved by direct methods17 (and subsequent
difference syntheses). Anisotropic full-matrix least-squares
Bannucine ((−)-1) and 5’-epibannucine ((−)-5’-epi-1)
method a): neat mixture, conventional heating
refinement17 on F2 for all non-hydrogen atoms yielded R1
=
A solution of 5-ethoxypyrrolidine-2-one (4) (300 mg, 2.32 mmol)
in acetic acid (10 mL) was stirred at room temperature for 24 h.
The solvent was evaporated in vacuo in a water bath of 35-40 °C,
then vindoline ((−)-2) (212 mg, 0.46 mmol) was added and the
neat mixture was stirred at 130 °C for 2 h. The arising dark
brown oil, which solidified on standing at room temperature, was
0.0893 and wR2 = 0.2111 for 2824 [I > 2(σI)] and R1 = 0.1172
and wR2 = 0.2290 for all (3778) intensity data, (number of
parameters = 394, goodness-of-fit = 1.083, the Flack absolute
structure parameter x = 0.02(7), Hooft y = 0.01(2), the maximum

8
Tetrahedron
ACCEPTED MANUSCRIPT
dissolved in dichloromethane (20 mL), the solution was washed
1047, 813, 759 cm-1; δH (799.7 MHz, DMSO-d6) 8.78 (1 H,
with saturated sodium bicarbonate solution (10 mL), dried
(MgSO4) and the solvent evaporated in vacuo. Purification of the
crude product by column chromatography (acetone) gave the
epimeric mixture of the title compounds (−)-1 and (−)-5’-epi-1
(158 mg, 63%) as an off-white amorphous solid, which was
crystallized by trituration in ether to give white crystals, m.p.
151-153 °C (lit. (bannucine): 152-154 °C);3 Rf (9%
MeOH/CH2Cl2) 0.68 and 0.63; [α]ꢁꢀꢂ −52.3 (c 0.26, CHCl3) (lit.
(bannucine): [α]ꢀꢁꢂ −33 (c 0.26, CHCl3));3 νmax(KBr) 3573, 3311,
3028, 2965, 2946, 2877, 1740, 1694, 1612, 1599, 1500, 1465,
s,C(16)OH), 7.70(1 H, s, H-1’), 6.97 (1 H, s, H-9), 6.33 (1 H, s,
H-12), 5.82 (1 H, ddd, J 10.1, 4.9, 1.7 Hz, H-14), 5.19 (1 H, s, H-
17), 5.09 (1 H, dt, J 10.1, 2.0 Hz, H-15), 4.80 (1 H, dd, J 7.7, 5.8
Hz, H-5’), 3.78 (3 H, s,C(11)OCH3), 3.66 (3 H,
s,C(16)COOCH3), 3.53 (1 H, s, H-2), 3.43 (1 H, dd, J 16.4, 4.9
Hz, Hβ-3), 3.29 (1 H, td, J 9.0, 4.1 Hz, Hβ-5), 2.82 (1 H, brd, J
16.4 Hz, Hα-3), 2.66 (1 H, s, H-21), 2.60 (3 H, s, H3-1), 2.58-2.54
(1 H, m, Hα-5), 2.38-2.33 (1 H, m, Hy-4’), 2.24-2.18 (2 H, buried
m, H2-6), 2.21-2.12 (2 H, buried m, H2-3’), 1.94 (3 H, s,
C(17)OCOCH3), 1.69-1.65 (1 H, m,Hx-4’), 1.47-1.43 (1 H, m,
Hy-19), 0.95-0.91 (1 H, m, Hx-19), 0.43 (3 H, t, J 7.5 Hz, H3-18);
δC (201.1 MHz DMSO-d6) 177.1 (C-2’), 171.6 (C(16)COOCH3),
170.0 (C(17)OCOCH3), 157.3 (C-11), 152.5 (C-13), 130.0 (C-
15), 124.31 (C-14), 124.28 (C-8), 122.1 (C-10), 119.7 (C-9), 93.7
(C-12), 82.9 (C-2), 78.7 (C-16), 75.9 (C-17), 66.1 (C-21), 55.6
(C(11)OCH3), 52.3 (C-7), 51.7 (C(16)COOCH3), 51.5 (C-5’),
51.2 (C-5), 50.5 (C-3), 43.7 (C-6), 42.5 (C-20), 38.6 (C-1), 30.6
(C-19), 30.0 (C-3’), 29.1 (C-4’), 20.7 (C(17)OCOCH3), 7.6 (C-
18); MS(ESI): 540 (C29H38N3O7); ESI-MS-MS (cid=35) (rel. int.
%): 522(10); 480(100); 448(5); 271(26).
1431, 1374, 1242, 1047 cm-1. Comparison of the H and 13C
1
NMR spectra of the epimeric mixture to literature data3 showed
that the substance we synthesized was indeed a mixture of
bannucine and its C5’-epimer. Epimeric ratios can be calculated
from the integrated intensities of the following protons: H-
C(16)OH, H-1', H-17, Ha-3, H-21, Hx-4' (see the spectra of the
pure epimers in the Supporting Information). MS(ESI): 540
(C29H38N3O7); ESI-MS-MS (cid=35) (rel. int. %): 522(10);
480(100); 448(5); 271(28).
method b): neat mixture, microwave heating
5’-Epibannucine ((−)-5’-epi-1) (75 mg, 47%) was obtained as
a white solid, m.p. 191-193 °C (lit. (bannucine): 152-154 °C);3 Rf
(9% MeOH/CH2Cl2) 0.63; [α]ꢁꢀꢂ −72.3 (c 0.26, CHCl3) (lit.
(bannucine): [α]ꢀꢁꢂ −33 (c 0.26, CHCl3));3 νmax(KBr) 3573, 3321,
2960, 2877, 1741, 1694, 1615, 1600, 1500, 1466, 1433, 1373,
1241, 1047, 740 cm-1; δH (799.7 MHz, DMSO-d6) 8.89 (1 H,
s,C(16)OH),7.78 (1 H, s, H-1’), 6.98 (1 H, s, H-9), 6.33 (1 H, s,
H-12), 5.82 (1 H, ddd, J 10.1, 4.9, 1.4 Hz, H-14), 5.16 (1 H, s, H-
17), 5.09 (1 H, dt, J 10.1, 1.4 Hz, H-15), 4.82 (1 H, dd, J 7.5, 6.0
Hz, H-5’), 3.78 (3 H, s, C(11)OCH3), 3.65 (3 H, s,
C(16)COOCH3), 3.54 (1 H, s, H-2), 3.40 (1 H, ddd, J 16.4, 4.9,
1.4 Hz, Hβ-3), 3.29 (1 H, td, J 9.1, 4.3 Hz, Hβ-5), 2.88 (1 H, dt, J
16.4, 1.4 Hz, Hα-3), 2.70 (1 H, s, H-21), 2.62-2.59 (1 H, buried
m, Hα-5), 2.60 (3 H, s, H3-1), 2.38-2.36 (1 H, m, Hy-4’), 2.24-
2.19 (2 H, m, H2-6), 2.18-2.14 (1 H, m, Hy-3’), 2.12-2.08 (1 H,
m, Hx-3’), 1.93 (3 H, s,C(17)OCOCH3),1.59-1.56 (1 H, m, Hx-
4’), 1.48-1.45 (1 H, m, Hy-19), 0.89-0.86 (1 H, m,Hx-19), 0.41 (3
H, t, J 7.4 Hz,H3-18); δC (201.1 MHz DMSO-d6) 177.0 (C-2’),
171.6 (C(16)COOCH3), 170.1 (C(17)OCOCH3), 157.2 (C-11),
152.4 (C-13), 129.8 (C-15), 124.6 (C-14), 124.1 (C-8), 122.0 (C-
10), 119.5 (C-9), 93.7 (C-12), 82.9 (C-2), 78.7 (C-16), 75.9 (C-
17), 65.8 (C-21), 55.6 (C-C(11)OCH3), 52.4 (C-7), 51.7
(C(16)COOCH3), 51.1 (C-5’), 50.9 (C-5), 50.3 (C-3), 43.4 (C-6),
42.4 (C-20), 38.6 (C-1), 30.4 (C-19), 30.0 (C-3’), 29.9 (C-4’),
20.7 (C(17)OCOCH3), 7.4 (C-18); MS(ESI): 540 (C29H38N3O7);
ESI-MS-MS (cid=35) (rel. int. %): 522(10); 480(100); 448(5);
271(26).
A solution of 5-ethoxypyrrolidine-2-one (4) (300 mg, 2.32 mmol)
in acetic acid (10 mL) was stirred at room temperature for 24 h.
The solution was transferred to a standard 10 mL MW vessel and
the solvent was evaporated in vacuo in a water bath of 35-40 °C.
Vindoline ((−)-2) (212 mg, 0.46 mmol) was added, the vessel
was sealed and the neat mixture was heated at 130 °C in a
microwave reactor for 30 min. The arising colourless oil was
dissolved in dichloromethane (20 mL), the solution was washed
with saturated sodium bicarbonate solution (10 mL), dried
(MgSO4) and the solvent evaporated in vacuo. Purification of the
crude product by column chromatography (acetone) gave the
epimeric mixture of the title compounds (−)-1 and (−)-5’-epi-1
(156 mg, 62%) as an off-white amorphous solid, which was
crystallized by trituration in ether to give white crystals. The
physical and spectral characteristics of the product are the same
as in method a).
method c): acid catalysed solution-phase reaction
To a mixture of vindoline ((−)-2) (100 mg, 0.22 mmol) and 5-
ethoxypyrrolidine-2-one (4) (141 mg, 1.09 mmol) in
nitromethane (5 mL) a catalytic amount of p-toluenesulfonic acid
was added and the mixture refluxed for 4 h. The solvent was
evaporated in vacuo, the residue was dissolved in
dichloromethane (20 mL), the solution was washed with
saturated sodium bicarbonate solution (10 mL), dried (MgSO4)
and the solvent evaporated in vacuo. Purification of the crude
product by column chromatography (acetone) gave the epimeric
mixture of the title compounds (−)-1 and (−)-5’-epi-1 (106 mg,
90%) as an off-white amorphous solid, which was crystallized by
trituration in ether to give white crystals. The physical and
spectral characteristics of the product are the same as in method
a).
Acknowledgments
The authors are grateful to Gedeon Richter Plc. for providing
(–)-vindoline and for the financial assistance. The authors are
also grateful to the National Institutes of Health (NIH) for the
biological testing of the samples.
Separation of the epimers of bannucine
Supplementary data
Purification of an epimeric mixture of bannucine ((−)-1) and 5’-
epibannucine ((−)-5’-epi-1) (158 mg) by preparative TLC (9%
MeOH/CH2Cl2) gave the title compounds as white amorphous
solids, which were crystallized by trituration in ether. Bannucine
((−)-1) (40 mg, 25%) was obtained as a white solid, m.p. 284-286
°C (lit. (bannucine): 152-154 °C);3 Rf (9% MeOH/CH2Cl2) 0.68;
[α]ꢁꢀꢂ −12.3 (c 0.26, CHCl3) (lit. (bannucine): [α]ꢁꢀꢂ −33 (c 0.26,
CHCl3));3 νmax(KBr) 3315, 2945, 2876, 2834, 1735, 1710, 1612,
1599, 1497, 1466, 1448, 1430, 1377, 1342, 1259, 1243, 1224,
Supplementary data (1H, 13C, HSQC and HMBC NMR spectra of
bannucine and 5’-epibannucine) associated with this article can
be found in the online version, at http://xxxxxxxxxxxxxxxxxxx.

9
References and notes
ACCEPTED MANUSCRIPT
1. Flora of China; Zhengyi, W.; Raven, P. H.; Deyuan, H. (Ed.)
Science Press (Beijing) & Missouri Botanical Garden Press (St.
Louis), 1994; Vol. 16, pp 157.
2. For recent advances in the field see: (a) Keglevich, P.; Hazai, L.;
Dubrovay, Z.; Dékány, M.; Szántay Jr., C.; Kalaus, G.; Szántay,
C. Heterocycles 2014, 89, 653-668.; (b) Keglevich, P.; Hazai, L.;
Kalaus, G.; Szántay, C. Molecules 2012, 17, 5893-5914.; (c)
Sears, J. E; Boger, D. L. Acc. Chem. Res. 2015, 48, 653-662.; (d)
Gherbovet, O.; Coderch, C.; Alvarez, M. C. G.; Bignon, J.;
Thoret, S.; Guéritte, F.; Gago, F.; Roussi, F. J. Med. Chem. 2014,
57, 5470-5476.; (e) Schleicher, K. D.; Sasaki, Y.; Tam, A.; Kato,
D.; Duncan, K. K.; Boger, D. L. J. Med. Chem. 2013, 56, 483-
495.; (f) Leggans, E. K.; Duncan, K. K.; Barker, T. J.; Schleicher,
K. D.; Boger, D. L. J. Med. Chem. 2013, 56, 628-639; (g)
Leamon, C. P.; Vlahov, I. R.; Reddy, J. A.; Vetzel, M.;
Santhapuram, H. K. R.; You, F.; Bloomfield, A.; Dorton, R.;
Nelson, M.; Kleindl, P.; Vaughn, J. F.; Westrick, E. Bioconjugate
Chem. 2014, 25, 560-568.; (h) Barker, T. J.; Duncan, K. K.;
Otrubova, K.; Boger, D. L. ACS Med. Chem. Lett. 2013, 4, 985-
988.; (i) Giovanelli, E.; Moisan, L.; Comesse, S.; Leroux, S.;
Rousseau, B.; Hellier, P.; Nicolas, M.; Doris, E. Org. Biomol.
Chem. 2013, 11, 5885-5891.; (j) Turner, T. C.; Shibayama, K.;
Boger, D. L. Org. Lett. 2013, 15, 1100-1103.
3. Atta-ur-Rahman; Ali, I.; Chaudhary, M. I. J. Chem. Soc., Perkin
Trans. 1 1986, 923-926.
4. For reviews on the chemistry of N-acyliminium ions and the
synthesis of their precursors see: (a) Speckamp, W. N.; Hiemstra,
H. Tetrahedron 1985, 41, 4367-4416.; (b) Speckamp, W. N.;
Moolenaar, M. J. Tetrahedron 2000, 56, 3817-3856.; (c) Marson,
C. M. ARKIVOC (Gainesville, FL, U.S.) 2001, 1-16.; (d)
Maryanoff, B. E.; Zhang, H.; Cohen, J. H.; Turchi, I. J.;
Maryanoff, C. A. Chem. Rev. (Washington, DC, U.S.) 2004, 104,
1431-1628.; (e) Royer, J.; Bonin, M.; Micouin, L. Chem. Rev.
(Washington, DC, U.S.) 2004, 104, 2311-2352.; (f) Yazici, A.;
Pyne, S. G. Synthesis 2009, 339-368.; (g) Yazici, A.; Pyne, S. G.
Synthesis 2009, 513-541.
5. Johnson, P.D.; Sohn, J-H.; Rawal, V. H. J. Org. Chem. 2006, 71,
7899-7902.
6. Gorka-Kereskényi, Á.; Szabó, L.; Hazai, L.; Lengyel, M.; Szántay
Jr., C.; Sánta, Z.; Kalaus, G.; Szántay, C. Heterocycles 2007, 71,
1553-1563.
7. Honty, K.; Szántay Jr., C.; Kolonits, P.; Demeter, A.; Szántay, C.
Tetrahedron 1993, 49, 10421-10426.
8. Hubert, J. C.; Wijnberg, J. B. P. A.; Speckamp, W. N.
Tetrahedron 1975, 31, 1437-1441.
9. Nagasaka, T.; Abe, M.; Ozawa, N.; Kosugi, Y.; Hamaguchi, F.
Heterocycles 1983, 20, 985-990.
10. Cue, B. W.; Chamberlain, N. Org. Prep. Proc. Int. 1979, 11,
285-286.
11. (a) Flack, H. D. Acta Crystallogr., Sect. A: Found. Adv. 1983, 39,
876-881.; (b) Flack, H. D.; Bernardinelli, G. J. Appl. Crystallogr.
2000, 33, 1143-1148.
12. Hooft, R. W. W.; Straver, L. H.; Spek, A. L. J. Appl. Crystallogr.
2008, 41, 96-103.
13. Spek, A. L. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2009, 65,
148-155.
14. Monks, A.; Scudiero, D. A.; Skehan, P.; Shoemaker, R. H.; Paull,
K.; Vistica, D.; Hose, C.; Lamgley, J.; Cronise, P.; Waigro-Wolff,
A.; Grog-Goodrich, M.; Campbell, H.; Mayo, J. G.; Boyd, M. R.
J. Natl. Cancer Inst. 1991, 83, 757-766.
15. Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.;
Czerwinski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.;
Shoemaker, R. H.; Boyd, M. R. Cancer Res. 1988, 48, 589-601.
16. Shomaker, R. H.; Monks, A.; Alley, M. C.; Scudiero, D. A.; Fine,
D. L.; McLemore, T. L.; Abbott, B. J.; Paull, K. D.; Mayo, J. G.;
Boyd, M. R. Prog. Clin. Biol. Res. 1988, 276, 265-286.
17. Sheldrick, G. M. Acta Crystallogr., Sect. A: Found. Adv. 2008, 64,
112-122.