Synthesis of indolizidinone analogues of cytotoxic alkaloids: Monocyclic precursors are also active

Article abstract of DOI:10.1016/j.bmcl.2012.03.109

Readily available proline derivatives can be transformed in just two steps into analogues of cytotoxic phenanthroindolizidine alkaloids. The key step uses a sequential radical scission-oxidation-alkylation process, which yields 2-substituted pyrrolidine amides. A second process effects the cyclization to give the desired alkaloid analogues, which possess an indolizidine core. The major and minor isomers (dr 3:2 to 3:1) can be easily separated, allowing their use to study structure-activity relationships (SAR). The process is versatile and allows the introduction of aryl and heteroaryl groups (including biphenyl, halogenated phenyl, and pyrrole rings). Some of these alkaloid analogues displayed a selective cytotoxic activity against tumorogenic human neuronal and mammary cancer cells, and one derivative caused around 80% cell death in both tumor lines at micromolar doses. The cytotoxicity of some monocyclic precursors was also studied, being comparable or superior to the bicyclic derivatives.

Full text of DOI:10.1016/j.bmcl.2012.03.109

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3402–3407
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of indolizidinone analogues of cytotoxic alkaloids: Monocyclic
precursors are also active
Alicia Boto a, , Javier Miguélez a, Raquel Marín b,c,e, Mario Díaz c,d,e
⇑
a Instituto de Productos Naturales y Agrobiología del CSIC, Avda. Astrofísico Francisco Sánchez 3, 38206-La Laguna, Tenerife, Spain
b Laboratorio de Neurobiología Celular, Departamento de Fisiología, Facultad de Medicina, Campus de Ofra s/n, Universidad de La Laguna, 38071-La Laguna, Tenerife, Spain
c Instituto Canario de Investigación del Cáncer (ICIC), Canary Islands, Spain
d Laboratorio de Fisiología y Biofísica de Membranas, Departamento de Biología Animal, Facultad de Biología, Universidad de La Laguna, Avda. Astrofísico Francisco Sánchez s/n,
38206-La Laguna, Tenerife, Spain
e Instituto de Tecnologías Biomédicas, Universidad de La Laguna, 38071-La Laguna, Tenerife, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Readily available proline derivatives can be transformed in just two steps into analogues of cytotoxic
phenanthroindolizidine alkaloids. The key step uses a sequential radical scissionꢀoxidationꢀalkylation
process, which yields 2-substituted pyrrolidine amides. A second process effects the cyclization to give
the desired alkaloid analogues, which possess an indolizidine core. The major and minor isomers (dr
3:2 to 3:1) can be easily separated, allowing their use to study structure–activity relationships (SAR).
The process is versatile and allows the introduction of aryl and heteroaryl groups (including biphenyl,
halogenated phenyl, and pyrrole rings). Some of these alkaloid analogues displayed a selective cytotoxic
activity against tumorogenic human neuronal and mammary cancer cells, and one derivative caused
around 80% cell death in both tumor lines at micromolar doses. The cytotoxicity of some monocyclic pre-
cursors was also studied, being comparable or superior to the bicyclic derivatives.
Received 25 February 2012
Revised 30 March 2012
Accepted 31 March 2012
Available online 7 April 2012
Keywords:
Cytotoxicity
Indolizidine alkaloids
Alkaloid analogues
Synthetic methodology
Sequential processes
Ó 2012 Elsevier Ltd. All rights reserved.
Many alkaloids with a phenanthroindolizidine core,1 such as
tylophorine (1, Fig. 1) have displayed a potent cytotoxic activity.
(ꢀ)-Tylophorine (1) is active against several cancer cell lines,
including multidrug-resistant lines such as KB-V1 and HEPG.2 This
alkaloid presents nanomolar IC50, comparable to drugs in clinic use.
In order to modulate the selectivity and bioavailability of the
alkaloids,3 and also to study their mechanisms of action and tumor
targets,4 different analogues have been synthesized.1,5 For instance,
Sharma6a and Georg6b reported that several tetrahydroindolizin-
5(1H)-ones, such as compound 2 (Fig. 1),6 displayed a potent and
selective cytotoxic activity. Thus, indolizidinone 2 (registered as
NSC 707904),6b,c presents submicromolar IC50 (0.39
M against
l
HO
AcO
AcO
1
2
8a
N
COOH
N
1
N
Ar
O
O
Ar
O
O
One-Pot
One-Pot
Scission−
Hydrolysis−
Alkylation
Cyclization
4
5
6
X
1
H
8a
N
Scheme 1. Strategy towards the synthesis of indolizidinones 6.
8
N
OMe
OMe
O
Nu−
OMe
MeO
Radical
scission−
oxidation−
Ar
H
H
OMe
CO2H
O
+
H
2
2 X = H NSC 707904
3 X = OH
N
1
O
H
3
Z
O
N
(−)-tylophorine
N
alkylation
process
AcO
AcO
AcO
Nu−
Ph
Ph
cis-5
Figure 1. Cytotoxic alkaloids and synthetic analogues.
4
7 Z = COCH2Ph
major isomer
⇑
Corresponding author.
E-mail address: alicia@ipna.csic.es (A. Boto).
Scheme 2. Stereoselective synthesis of 2-substituted pyrrolidines 5.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.109

A. Boto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3402–3407
3403
Table 1
MCF7
Stereoselective decarboxylationꢀalkylation process to prepare 2-substituted pyrrol-
(30 μM / 48 hours)
idines 5a–e
100
80
60
40
20
0
AcO
O
AcO
DIB, I2, hν, CH2Cl2,
4
O
a
2
°
°
26 C, 4 h; then 0 C,
1'
Ar
CO2H
Ph
N
N
CH2=C(OTMS)Ar,
Ph
O
.
BF3 OEt2, 3h
ab
4
5a−e
ab
b
Entry
Ar
Ph
Products (%)
Global yielda (%)
b
bc
b
b
1
2
3
cis-5a (77), trans-5a (17)
cis-5b (79), trans-5b (14)
cis-5c (81), trans-5c (12)
94
93
93
bc
C6H4-p-OMe
C6H4-p-Br
c
c
4
5
cis-5d (74), trans-5d (17)
cis-5e (70), trans-5e (13)
91
83
Ph
N
a
is-6c
c
d
cis-6e
csi-6b
Yields after purification by chromatography.
Ctnrol
tarns-6c
t
d
trans-6e
tarns-6b
SHSY-5Y
(
30 μM / 48 hours)
5% KOH
a
AcO
AcO
in MeOH,
100
2
65 oC, 4 h
N
N
Ar
Ar
-O
O
O
O
80
60
40
20
0
a
Ph
Ph
7a
cis-5a−e
b
b
b
AcO
HO
H
N−
N
Ar
c
c
cd
c
cd
Ar
O
O
O
d
Ph
Ph
7b
6a−e
Scheme 3. Mechanism for the partial epimerization at 8a-C during the conversion
cis-5a-e?6a-e.
i-s6c
c
d
cis-6e
csi-6b
Ctnrol
tarns-6c
t
d
trans-6e
tarns-6b
Figure 2. Cell toxicity of compounds 6a–e in MCF-7 (above) and SHSY-5Y cultures
(below) following exposure at 30 M for 48 h to the different compounds. Data
l
expressed as average value SEM for, at least, three different experiments. Different
letters indicate significant statistical differences with p <0.05. Trans-6 corresponds
to (8aR)-6 while cis-6 corresponds to (8aS)-6.
Table 2
Cyclization and isolation of the indolizidinones 6a–e
AcO
HO
HO
2
1
5% KOH
in MeOH,
8a
2
3
8
+
N
N5
N
7 Ar
Ar
Ar
HCT-116) and is selective against the colon and leukemia cancer
lines.
°
65 C, 4 h
O
O
O
O
Ph
Ph
Ph
cis-5a−e
The indolizidinone 2 was initially obtained as a racemic mix-
ture. In order to evaluate the antitumoral activity of the pure enan-
tiomers, Georg synthesized the 8aR- and 8aS-hydroxyproline
precursors 3 as a separable mixture, and then converted them into
the (8aS)-2 or (8aR)-2 enantiomers.6c Interestingly, the most active
one was the minor (8aR)-2 isomer.6c
(8a
S
)-6a−e
(8a
R)-6a−e
Entry Substrate Ar
Products (%)
Global yielda
(%)
1
2
3
cis-5a
cis-5b
cis-5c
Ph
(8aR)-6a (59), (8aS)-6a
84
81
80
(25)
C6H4-p-OMe
C6H4-p-Br
(8aR)-6b (55), (8aS)-6b
The bicyclic products 3 were prepared from an unusual b-amino
acid derivative, N-Boc-4-hydroxy-L-b-homoproline, by transforma-
(26)
(8aR)-6c (48), (8aS)-6c
tion of its carboxyl group into an aryl ketone, followed by several
(32)
steps.6c We report herein an alternative strategy which starts from
(8aR)-6d (53), (8aS)-6d
(33)
4
5
cis-5d
cis-5e
86
86
readily available 4-hydroxy-L-a-proline amides 4 (Scheme 1),
whose carboxyl group is removed7,8 and replaced in situ by an al-
kyl chain to afford the aryl ketones 5 with the desired (2R) stereo-
chemistry. A one-pot saponificationꢀcyclization reaction followed
to give the bicyclic derivatives 6, whose cytotoxic activity was
evaluated.
Ph
(8aR)-6e (66), (8aS)-6e
(20)
N
a
Yields after purification by chromatography.

3404
A. Boto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3402–3407
Figure 3. Morphological aspects of MCF-7 (left column) and SHSY-5Y (right column) cell cultures in the presence of compound (8aS)-6d (lower panels) as compared with
vehicle-treated cultures (upper panels). Scale bars: 50 m.
l
products.10 The cis-isomers presented the desired (2R) configura-
tion; it must be noticed that most reported methods to prepare re-
lated 2-substituted pyrrolidine amides afford the (2S) isomers.5,6c
The procedure allowed the introduction of aryl rings with either
electron-donor or electron-withdrawing substituents, and differ-
ent sizes. The halogenated aryl groups are particularly useful, since
the halogen can be replaced by alkyl, allyl and aryl groups by
radical or sp2–spn couplings (allowing the conversion of compound
5c into libraries of biaryl derivatives such as compound 5d). The
introduction of heteroaryl substituents (compound 5e) is also
interesting,11 since the bioavailability and interaction with the bio-
logical targets can be modified.
RO
RO
N
N
O
O
O
O
Ph
Ph
trans-5 R = Ac
trans-8 R = H
cis-5 R = Ac
cis-8 R = H
K2CO3, MeOH,
26 oC, 18 h
94%
90%
It must be stressed that the major diastereomers, cis-5aꢀe, may
also display cytotoxic activity, since many flexible analogues of ri-
gid drugs are also able to interact with the biological receptors and
induce physiological responses, as will be commented on later.
The conversion of the substituted pyrrolidines cis-5aꢀe into the
indolizidinones 6 (Table 2) was carried out with 5% methanolic
KOH,6b,6c,11 which allowed not only the cyclization but also the
saponification of the acetate group.
The reaction proceeded in good yields (80ꢀ86%) to afford the
bicyclic products 6aꢀe. However, in all the cases, a separable mix-
ture of diastereomers was obtained (dr from 3:2 to 3:1). To our sur-
prise, the major products were the 8a,2-trans isomers, according to
the 1H NMR coupling constants and NOESY experiments.12 The
cyclization was then studied with trans-5a, which also afforded
the 8a,2-trans derivative as the major product [(8aR)-5a:(8aS)-5a,
3:2, 91% overall yield].
Scheme 4. Synthesis of the monocyclic analogues cis-8 and trans-8.
The first step is a sequential process initiated by the radical
decarboxylation of compound 4 (Scheme 2) by treatment with
(diacetoxyiodo)benzene (DIB) and iodine under irradiation with
visible light.7,8 The resulting C-radical is oxidized in situ to an acyli-
minium ion 7, which reacts with silyl enol ethers to give 2-substi-
tuted pyrrolidines 5.
The addition of the nucleophile was stereoselective, and affor-
ded the 2,4-cis products as the major ones. This result is due to a
stereoelectronic effect observed for substituted cyclic iminium or
oxocarbenium ions.9 Thus, when intermediate 7 adopts an enve-
lope conformation with a pseudoaxial OR substituent, the electro-
static interactions between the oxygen lone electron pairs and the
iminium ion are maximized. In this favored conformation, the
nucleophile adds mainly from the inner face, to avoid eclipsing
interactions on the formation of the product, resulting in com-
pounds cis-5.
The isomerization of the 8a-C can be explained considering that
for methyl aryl acetamides, the pKa of the
while for aryl ketones, the pKa of the
a
-protons is about 25ꢀ27,
a
-protons is about 20ꢀ25.13
The decarboxylationꢀalkylation process was studied under dif-
ferent conditions, changing the amount of the reagents, the order
of addition, the temperature of the addition step, the nucleophile,
the addition of different Lewis acids to increase the reactivity of
the intermediate, etc. The best conditions are summarized in Table
1, andyieldedcompounds5a–e ingoodtoexcellentyields(83ꢀ94%).
The diastereoselectivities ranged from 9:2 to 7:1 (cis:trans ratio)
and the major cis-isomers could be readily separated from the trans
Therefore, under the reaction conditions, both the enolate from
the amide and the enolate from the ketone are formed. In the first
case, the cyclization takes place with no epimerization. However,
in the second case, the enolate 7a (Scheme 3) undergoes a retro-Mi-
chael reaction to give the intermediate 7b. Then a recyclization
takes place,11 yielding a mixture of the (8aR)- and (8aS)-diastereo-
mers 6aꢀe, with the thermodynamically favored 8a,2-trans isomer
as the major product.

A. Boto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3402–3407
3405
As observed in Figure 2, the results show that, in the case of
MCF-7 cells, compounds (8aS)-6a, (8aR)- and (8aS)-6b, (8aR)-
and (8aS)-6c, (8aR)- and (8aS)-6d, (8aR)- and (8aS)-6e have a
significant level of toxicity at these experimental conditions.
Furthermore, compounds (8aR)-6b, (8aR)- and (8aS)-6d, (8aR)-
and (8aS)-6e were toxic in SHSY-5Y cells. Interestingly, derivatives
(8aR)-6b, (8aR)-6c, (8aR)- and (8aS)-6d, and (8aS)-6e were toxic at
different levels in both cell lines.
a
ab
The highest toxicity (>70%, p <0.005) was obtained with the
biphenyl derivative (8aS)-6d, and also its isomer, (8aR)-6d. The
other para-substituted phenyl derivatives, such as the methoxy-
phenyl compounds (8aR)- and (8aS)-6b, and the p-bromophenyl
derivatives (8aR)- and (8aS)-6c, also produced an important cell
mortality (particularly in MCF-7 cells). The lowest levels of toxicity
were observed with the phenyl derivatives (8aR)- and (8aS)-6a.
With respect to the heterocyclic compounds 6e, the (8aR)- and
(8aS)-6e isomers displayed differential effects in both cell lines.
Figure 3 illustrates cultures of MCF-7 (left column) and SHSY-5Y
(right column) cell lines after exposure to compound (8aS)-6d, at
b
b
c
30 lM for 48 h (lower panels), as compared to these cells under
non-toxic conditions in the presence of the vehicle (upper panels).
As observed in the figure, the morphology of the cells was dramat-
ically changed in the presence of this derivative, observing, at first
glance, not only a change in cell shape, perimeter and volume, but
also a high degree of cell mortality.
These results suggest that many of these derivatives may have
antitumorigenic properties. In order to evaluate their therapeutic
utility, investigation of these compounds in other tumor cell lines,
and lower-dose longer-time treatments, are currently under
investigation.
a
a
b
b
The cytotoxicity of the monocyclic precursors was studied next.
As commented before, the 2-substituted pyrrolidines 5aꢀe are
flexible analogues of the indolizidinones 6aꢀe, and may also
interact with the biological receptors and display some cytotoxic
activity. Since the biphenyl indolizidinones 6d were the most cyto-
toxic bicyclic compounds, their monocyclic analogues were pre-
pared (Scheme 4). Thus, the substituted pyrrolidines cis-5d and
trans-5d were saponified in excellent yields to give the hydroxy
derivatives cis-8 and trans-8, respectively.
c
The cytotoxicity of these derivatives was also studied in the
SHSY-5Y and MCF-7 tumor cell lines (Fig. 4). The conditions of
the previous experiments (30
lM, 48 h) were repeated for compar-
ison, but new conditions were also tried (10
l
M, 24 h).
In the first case, it was observed that the activity of the mono-
cyclic compounds 8 was comparable or superior to their bicyclic
derivatives 6d. At lower doses and exposure time the cell death
was lower but still significant with respect to the control. Remark-
ably, although the activity of cis-8 was superior to the activity of
trans-8, both compounds displayed considerable cytotoxicity.
The cytotoxic activity of these compounds is accompanied by
dramatic changes in cell morphology, as shown in Figure 5. Thus,
treatment of MCF-7 cells with compound cis-8 at a dose of
Figure 4. Cell toxicity of compounds cis-8 and trans-8 in SHSY-5Y (above) and
MCF-7 cultures (below) following exposure at 10 M (for 24 h) or 30 M (for 48 h)
to the different compounds, with respect to the vehicle. Data expressed as average
value SEM for at least three different experiments. Different letters indicate
significant differences with respect to the control with p <0.05.
l
l
The reversal of configuration in the cyclization step suggests
that, in order to obtain the desired (8aS)-diastereomers, the start-
ing material should be a (4S)-hydroxyproline derivative. Since a
variety of methodologies (such as Mitsunobu reaction)14 are avail-
able to convert inexpensive 4R-hydroxyproline into its 4S epimer,
the opposite series of alkaloid enantiomers could also be prepared.
The cytotoxicity of the new indolizidinones was studied in the
human breast cancer MCF-7 and human neuroblastoma SHSY-5Y
cell lines (Fig. 2).
30 lM and after only 6 h causes an increase in the volume of the
nucleus and the refringency of the nucleoli, as well as cytoplasm
vacuolization and cell swelling. These early changes in morpholog-
ical features indicate that compound cis-8 provokes cell death
through cell necrosis rather than apoptosis. After 24 h, a high cell
death is evident, together with morphological changes in the
remaining cells. These alterations were also observed with the
SHSY-5Y tumor line. These interesting results suggest that synthet-
ically simpler compounds such as 8 can be valuable as cytotoxic
agents.
In summary, this Letter reports the shortest route available (two
steps) for the transformation of readily available and inexpensive
hydroxyproline N-amides into indolizidinones. The first step is a
sequential process which allows the scission of the hydroxyproline
Cells were exposed to the different alkaloid derivatives (30 lM
for 48 h) and then were trypsinized. The cell viability was quanti-
fied, observing a highly significant specific toxicity with some of
these compounds. Other doses and preincubation times were also
tried (10
lM for 24 h; 20 lM for 24 h, etc.) but the results were not
statistically significant, although cell death was also observed.

3406
A. Boto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3402–3407
Figure 5. Effect of compound cis-8 on breast cancer cells MCF-7 at a dose of 30
The lower pannel amplifies the view from the medium pannel, showing the increases in cellular and nuclear volumes (arrow 1), cytoplasm vacuolization (arrow 2) and an
important increase in nucleoli refringency (arrow 3). Scale bars: 50 m.
lM after 6 h treatment (medium and lower pannels) with respect to control (upper pannel).
l
carboxylic group and its replacement by an alkyl chain. The resul-
tant 2-substituted pyrrolidines are obtained in good overall yield,
and the two possible diastereomers are readily separated. Remark-
ably, the major isomers are the 2,4-cis diastereomers with the de-
sired (2R) configuration, while most of the alternative reported
methods afford the (2S) pyrrolidines. Besides, the reported meth-
odology allows the introduction of a variety of aryl and heteroaryl
groups of different volumes and lipophilicities, in order to modu-
late the potency or bioavailability of the alkaloid analogues.
The second step was a one pot saponification–cyclization. A
partial epimerization took place, to give a separable mixture of
trans- and cis-indolizidinones in good global yield.
indolizidinone derivatives. The use of these simpler monocyclic
compounds as cytotoxic agents is currently being studied and will
be reported in a future.
Acknowledgments
This work was supported by the Research Projects CTQ2009-
07109 (AB) and SAF2010-22114-C02-01 (MD)/02 (RM) (Plan Nac-
ional de I+D, Ministerio de Ciencia e Innovación, MICINN, Spain),
and Proyectos Intramurales 2004-8-0E211 and 2004-80E642 (CSIC,
Spanish Research Council, Spain). We also acknowledge financial
support from FEDER funds. J. M. thanks CSIC for a JAE predoctoral
fellowship. We also thank technician Irene Guerra-Palma for labo-
ratory support. This article is dedicated to the memory of an inspir-
ing man, D. Juan Marín, who gave one of the authors (RM) the
unique chance to enjoy and be devoted to science.
The cytotoxic activity of the bicyclic alkaloid analogues was
studied against the human neuroblastoma SHSY-5Y and human
breast cancer MCF-7 cell lines, revealing that most of the alkaloids
displayed cytotoxic activities at 30 lM for 48 h, particularly for the
bulky biphenyl derivatives (8aS)-6d and (8aR)-6d. Thus, (8aS)-6d
caused around 80% cell death in both tumor lines at micromolar
doses.
Supplementary data
The cytotoxicity of the 2-substituted pyrrolidines was also
studied with the biphenyl compounds cis- and trans-8, which dis-
played a potent cytotoxic activity, comparable or superior to their
Supplementary data (synthesis and spectroscopic data of com-
pounds cis-5d, trans-5d, (8aS)-6d, (8aR)-6d. cis-8, and trans-8.
Experimental protocols for the biological screenings. Additional

A. Boto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3402–3407
3407
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10. The stereochemistries were assigned according to their spectroscopic data; for
instance, product cis-5a presented J2,3a ꢁ J3a,4 ꢁ 0 Hz, while J2,3b = 8.9 Hz and
J3b,4 = 6.0 Hz. In the case of trans-5a the 3-Ha was a double doublet (J2,3a = 5.5,
J3a,4 = 7.0 Hz), while 3-Hb appeared as a multiplet. The NOESY experiments
supported these stereochemistries (10-H/3-H correlations for the cis isomers).
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12. (a) The 8a,2-trans isomer was the major product. The indolizidine (8aR)-6a
presented J8a,8 = 5.0, 13.7 Hz; J8a,1 = 5.2, 12.0 Hz; J1,2 = 0, 4.2 Hz and J2,3 = 0,
4.3 Hz. On the other side, the indolizidine (8aS)-6a presented J8a,8 = 4.4,
14.1 Hz; J8a,1 = 6.3, 6.8 Hz; J1,2 = 6.5, 7.8 Hz and J2,3 = 5.2, 6.1 Hz. (b) The NOESY
experiment of the major product (8aR)-6a showed a strong spatial interaction
between 1-Ha and the two protons on 8-C. There was also an interaction
between 1-Hb and 3-Ha, since both are trans with respect to 2-H. The NOESY
cytotoxicity studies for compounds (8aS)-6d, (8aR)-6d, cis-8 and
trans-8) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmcl.2012.03.109.
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