Synthesis and cytotoxicity evaluation of olivacine-indole hybrids tethered by alkyl linkers

Article abstract of DOI:10.1080/14786419.2021.1880401

In this work, eleven new derivatives were prepared of the alkaloid olivacine (1), which was isolated from the bark of Aspidosperma australe. These compounds (7a–k) are hybrids of olivacine and indoles or carbazole, tethered by alkyl chains of variable lengths (C-4, C-5 or C-6). Compounds 7a–k showed increased cytotoxicity towards a panel of four cell lines. The subcellular localization of olivacine and of the synthetic derivatives was studied by fluorescence microscopy. The cycles of K562 cells exposed to olivacine or compounds 7a–k were analysed by flow cytometry, and showed, for some of the new derivatives, a different profile of cell distribution among the phases of the cycle when compared to olivacine, which is indicative of lysosomal apoptosis.

Full text of DOI:10.1080/14786419.2021.1880401

Natural Product Research
Formerly Natural Product Letters
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Synthesis and cytotoxicity evaluation of olivacine-
indole hybrids tethered by alkyl linkers
Cristian M. Pis Diez, Mariela Céspedes, Gabriela M. Di Venosa, Gustavo
Calvo, Esteban Avigliano, Adriana G. Casas & Jorge A. Palermo
To cite this article: Cristian M. Pis Diez, Mariela Céspedes, Gabriela M. Di Venosa, Gustavo
Calvo, Esteban Avigliano, Adriana G. Casas & Jorge A. Palermo (2021): Synthesis and cytotoxicity
evaluation of olivacine-indole hybrids tethered by alkyl linkers, Natural Product Research, DOI:
10.1080/14786419.2021.1880401
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2021.1880401
Synthesis and cytotoxicity evaluation of olivacine-indole
hybrids tethered by alkyl linkers
c
c
Cristian M. Pis Dieza,b, Mariela Cespedes , Gabriela M. Di Venosa , Gustavo
ꢀ
Calvoc, Esteban Aviglianod,e, Adriana G. Casasc and Jorge A. Palermoa,b
a
ꢀ
ꢀ
Universidad de Buenos Aires, Departamento de Quımica Organica, Facultad de Ciencias Exactas y
b
Naturales, Ciudad Universitaria, Buenos Aires, Argentina; Universidad de Buenos Aires, Unidad de
Microanalisis y Metodos Fısicos en Quımica Organica (UMYMFOR), Buenos Aires, Argentina; Centro
de Investigaciones sobre Porfirinas y Porfirias (CIPYP) CONICET and Hospital de Clınicas Jose de San
Martın, Universidad de Buenos Aires, Buenos Aires, Argentina; Centro de Investigaciones Antonia
Ramos (CIAR), Fundacion Bosques Nativos Argentinos. Camino Balneario s/n, Villa Bonita, Misiones,
Argentina; Instituto de Investigaciones en Produccion Animal (INPA-CONICET-UBA), Universidad de
c
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
d
ꢀ
ꢀ
e
ꢀ
Buenos Aires, Buenos Aires, Argentina
ABSTRACT
ARTICLE HISTORY
Received 22 January 2020
Accepted 17 January 2021
In this work, eleven new derivatives were prepared of the alkaloid
olivacine (1), which was isolated from the bark of Aspidosperma
australe. These compounds (7a–k) are hybrids of olivacine and
indoles or carbazole, tethered by alkyl chains of variable lengths
(C-4, C-5 or C-6). Compounds 7a–k showed increased cytotoxicity
towards a panel of four cell lines. The subcellular localization of
olivacine and of the synthetic derivatives was studied by fluores-
cence microscopy. The cycles of K562 cells exposed to olivacine
or compounds 7a–k were analysed by flow cytometry, and
showed, for some of the new derivatives, a different profile of cell
distribution among the phases of the cycle when compared to
olivacine, which is indicative of lysosomal apoptosis.
KEYWORDS
Olivacine; hybrid
compounds; cytotoxic
activity;
Aspidosperma australe
CONTACT Jorge A. Palermo
palermo@qo.fcen.uba.ar
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2021.1880401.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

2
C. M. PIS DIEZ ET AL.
1. Introduction
In the course of a collaborative project aimed at the discovery and sustainable use of
natural resources of the Atlantic rainforest of Misiones province in north-eastern
Argentina, the bark tissues of several trees of this region were investigated, in the
search for abundant and easy to isolate secondary metabolites that may be amenable
for chemical diversification. Olivacine (1) was isolated as one of the main alkaloids of
the bark of the “yellow guatambu” tree (Aspidosperma australe) together with uleine
ꢀ
(3), N-methyltetrahydroellipticine (4) and apparicine (5) (Perez et al. 2019) (Figure 1).
From this biological source, and due to its simple purification protocol (see
Supplementary Material), a steady and sustainable supply of olivacine became avail-
able (typically 0.8 g/1 kg of bark) for a chemical diversification project, with the aim to
explore structural modifications that had not been thoroughly investigated in previ-
ous works.
Alkaloids with a pyridocarbazole nucleus have been intensely studied due to their
planar, highly conjugated structures, and their reported antitumor activity (Asche and
Demeunynck 2007; Roesch 2016; Tsutsumi et al. 2016 ). Olivacine (1), and its isomer
ellipticine (2), are the best known alkaloids of this family. Since the first reports of their
antitumor activity, these compounds have been the subject of numerous studies
focused on their biological activity, modes of action and structure-activity relationships
ꢀ
(OSullivan et al. 2013). Initially, ellipticine and olivacine were regarded as DNA interca-
lators and inhibitors of topoisomerase II (Garbett and Graves 2004). Later studies
showed that these alkaloids, as well as some synthetic derivatives, have a multimodal

NATURAL PRODUCT RESEARCH
3
Figure 1. Olivacine (1), ellipticine (2) and other alkaloids isolated from the bark of
Aspidosperma australe.
type of action. This includes the interaction with several proteins of the cell-cycle con-
trol system, such as p53, p21, p73 and several kinases such as AKT and c-Kit kinase
^
(Shi et al. 1998; Jin et al. 2004; Vendome et al. 2005). The development of several syn-
ꢀ
thetic routes to olivacine triggered the preparation of a series of derivatives (Tylinska
et al. 2018; Schmidt et al. 2018; (see Table S1 for a selection of structural types and
additional references in Supplementary Material). In particular, one of these derivatives,
S-16020-2 (1-diethylaminoethylolivacine), reached Phase II clinical trials as an anti-
ꢀ
cancer drug (Leonce et al. 1996; additional references in Supplementary Material).
However, olivacine still offers several unexplored possibilities of structural diversifica-
tion, such as the chemistry of the indole nitrogen.
In this work, eleven olivacine derivatives (compounds 7a–k) were prepared, which
are hybrid compounds based on olivacine and a series of simpler substituted indole or
carbazole structures, tethered by hydrocarbon chains of variable lengths. The rationale
for the design of these hybrids was that the presence of a second planar hetero-
aromatic moiety connected by a linker may modify the intercalating capacity of the
compounds. Several substituted indoles, as well as carbazole, were selected as the
second heterocyclic moiety. All the new compounds were tested for cytotoxic activity
against a panel of four cell lines, and their effect on the cell cycle was also analysed
by flow cytometry. In addition, due to the intrinsic fluorescence of olivacine and com-
pounds 7a–k, their subcellular location was also evaluated. The derivatives of olivacine
with the three N-bromoalkyl chains (8a-c) were also prepared in order to compare
their bioactivity profiles with those of the hybrid compounds.
2. Results and discussion
2.1. Chemistry
Initial attempts to obtain compounds with two units of olivacine tethered by N-alkyl
chains by typical SN2 reactions of the alkaloid with the corresponding alkyl dihalides
and base, gave only the monosubstituted N-alkylated product, in which a bromine
substituent was still present at the end of the alkyl chain. Although different condi-
tions were tried for this transformation (use of different solvents, bases, temperatures,
microwave heating, ionic liquids), the steric hindrance of the olivacine core (especially
C5-Me) prevented the formation of the disubstituted compounds in reasonable yields.
However, in a previous work with simpler indoles, geminal disubstituted compounds
could be readily prepared from alkyl dihalides and an excess of indole under the same
conditions (Bertinetti et al. 2011). In view of this, we decided to prepare hybrid com-
pounds with an olivacine core and simpler, sterically less challenged indoles, by two
consecutive SN2 reactions as outlined in Figure 2.

4
C. M. PIS DIEZ ET AL.
Figure 2. Synthesis of compounds 6a-k, 7a-k and 8a-c.
Although the order of the two SN2 N-alkylations could be interchanged with
comparable yields, a conservative approach was adopted regarding the natural
product component, trying to minimize the purification steps of the olivacine-con-
taining products. In this way, different substituted indoles, as well as carbazole,
were first treated with an excess of NaOH in DMSO, and after the formation of the
corresponding anions, N-alkylation with the alkyl dihalide (1,4-dibromobutane, 1,5-
dibromopentane or 1,6-dibromohexane) yielded the N-bromoalkylindoles 6a–k.
These intermediates, after quick purification by flash column chromatography, were
used as electrophyles in a second SN2 step, this time with the anion of olivacine in
DMSO as nucleophyle, to yield the final hybrids 7a–k. Full experimental procedures
can be found in the Supplementary Material. Derivatives of olivacine with the N-bro-
moalkyl chains (8a-c) were also prepared by the same technique, in order to study
the influence of the tether or the second heterocyclic moiety on the bioactiv-
ity profile.
2.2. Biological assays
2.2.1. Cytotoxicity Evaluation
The cytotoxicity of olivacine and compounds 7a–k and 8a-c was tested against a
panel of four cell lines: LM2 (murine mammary adenocarcinoma), K562 (human leuke-
mia), IGROV-1 (human ovarian cancer) and HaCaT (human normal keratinocytes) (Van
Merloo et al. 2011). Doxorubicin and cisplatin were used as positive controls. All IC50
values are listed in Table 1. From these results, it is clear that all the derivatives show
an increase in cytotoxic activity (two or three-fold) compared to olivacine. The
observed IC50 are in the same range as those of the clinical drugs doxorubicin and cis-
platin. However, there are no marked differences in the bioactivity of the hybrids with
different indole moieties or tethers of different length. There is also a lack of selectivity
against the different cell lines, either normal or tumoral.
2.2.2. Fluorescence microscopy
Since olivacine and compounds 7a–k are all fluorescent, their subcellular location was
studied by fluorescence microscopy. LM2 cells grown on coverslips were analysed by
fluorescence microscopy after 10 min and 48 h exposures to the compounds. A fast

NATURAL PRODUCT RESEARCH
5
Table 1. Cytotoxicity evaluation for compounds 1,7a-k and 8a-c.
IC50 (mM)a
Compound
LM2
HaCaT
K562
IGROV-1
1
7a
7 b
7c
7d
7e
7f
7 g
7 h
7i
7j
7k
8a
8 b
8c
11.2 2.7
4.2 0.5
3.6 0.4
5.0 0.3
6.0 0.7
3.8 0.5
4.9 0.3
4.0 0.5
3.6 0.4
3.2 0.6
4.6 0.4
5.2 0.7
3.4 0.5
5.5 0.4
5.3 0.6
1.12 0.02
6.9 0.8
11.8 2.5
4.8 0.4
4.1 0.6
5.6 0.3
7.1 0.8
4.2 0.5
5.0 0.4
4.5 0.5
4.1 0.3
3.6 0.7
5.8 0.6
6.1 0.7
3.8 0.5
5.9 0.6
4.9 0.5
1.83 0.18
10.9 1.3
5.9 0.7
2.3 0.2
1.9 0.1
2.5 0.4
3.1 0.5
1.7 0.2
2.8 0.3
2.0 0.5
1.8 0.2
1.4 0.3
2.3 0.2
3.5 0.3
1.8 0.2
3.2 0.4
2.5 0.3
0.44 0.04
3.5 0.6
9.8 1.1
2.9 0.3
2.7 0.3
3.0 0.4
2.6 0.2
2.9 0.4
3.7 0.5
3.2 0.3
3.3 0.2
3.9 0.7
4.5 0.5
4.0 0.4
2.8 0.3
5.5 0.4
5.4 0.2
Doxorubicin
Cisplatin
1.60 0.02
7.8 0.9
aMTT method after 48 h. incubation.
incorporation of olivacine and its derivatives was detected by the observation of an
intense blue/green fluorescence. Figure S15 (Supplementary Material) shows the
micrographs of LM2 cells exposed to olivacine or compound 7c as a representative
hybrid. The micrographs corresponding to the rest of the compounds are shown in
Figures S16 and S17 in the Supplementary Material section. In the case of olivacine,
the fluorescence is mostly blue, and is located mainly at the cytoplasmatic organules
together with some weaker nuclear staining. However, in the case of the hybrids, the
fluorescence is greener, and located mainly at the lysosomes with almost no fluores-
cence at the nuclei.
Previous studies of the fluorescence emission spectrum of the related alkaloid ellip-
ticine showed that it is composed of two bands: a blue band (440 nm) which is
favored at basic pH, characterizes the neutral form. Another intense green band
(520 nm), is favoured in more acidic conditions and characterizes the protonated form
(Schwaller et al. 1991). This fact suggests that a green fluorescence may be associated
to a more acidic lysosomal location. This preferential lysosomal location of the hybrids
at short incubation times, together with their weaker nuclear fluorescence when com-
pared to olivacine may indicate a diminished nuclear uptake and DNA intercalation,
and these facts point to a different mechanism of action. The cytotoxic action of the
compounds is evidenced by the presence of a lower cell density in the micrographs
obtained after an incubation of 48 h (see Supplementary Material).
2.2.3. Flow cytometry
The effect of olivacine and the synthesised hybrids on the cycle of K562 cells was eval-
uated by flow cytometry. The analysis of the histogram of olivacine at two different
concentrations: IC25 and IC50 (Figure S18, Supplementary Material), allowed the follow-
ing conclusions: at lower cytotoxic doses of olivacine, the cell cycle arrest is probably
due to DNA topoisomerase failure (Sharma et al. 2012), whereas at higher doses, an
apoptotic pathway is likely to take place. The cytoplasmatic (probably lysosomal) and

6
C. M. PIS DIEZ ET AL.
nuclear localization of the compound, are in accordance with this dual apoptotic and
cycle arrest cell death.
Surprisingly, the histograms of some of the hybrid derivatives were strikingly differ-
ent. Figure S19 (SupplementaryMaterial) shows a comparison of the cell-cycle profiles
of olivacine and compounds 7a–k. Most of the derivatives (especially 7a, 7e-h) show a
dramatic increase in sub G1 cells, which more than doubles the corresponding per-
centage observed in the case of olivacine, together with a decrease of the number of
cells in the G2/M phase. These results suggest that at high concentrations (IC75), oliva-
cine may be acting preferentially as an inducer of cell cycle G2/M arrest, whereas the
derivatives trigger cell death mainly by apoptosis. These observations are in line with
the subcellular localization pattern of the derivatives, which is mainly lysosomal even
at long incubation periods, suggesting that a lysosomal apoptotic pathway is likely to
be involved. However, the contribution of apoptotic death mediated by cell cycle
arrest due to DNA topoisomerase failure cannot be fully discarded. It is interesting to
note that in the case of compounds 8a-c, in which olivacine has an N-bromoalkyl
chain bound to the indole nitrogen but no second heteroaromatic ring, although their
IC50 are in the same range as those of the hybrids, their cell cycle profiles are very
similar to that of olivacine. These results indicate that the introduction of an alkyl-teth-
ered additional heterocyclic moiety induced a different mode of action in deriva-
tives 7a–k.
3. Conclusions
Almost all of the previously reported derivatives of olivacine were obtained by total
synthesis, however, in the present study, the derivatives were prepared from the nat-
ural product itself, as a way to add value to the natural resource. A literature search
showed that there are no reported derivatives of olivacine with indole N-alkyl groups
larger than a methyl group. In this sense compounds 7a–k represent a new class of
olivacine derivatives, which are nearly three times more cytotoxic than the parent
compound, and, at the same time, display a different mode of action. In the case of
olivacine, inspection of the subcellular location by fluorescence microscopy revealed
that, although there is some fluorescence at the nuclei, most of the compound
remains in the cytoplasmic organules. This dispersion of the compound at the subcel-
lular level is in accordance to the previously reported multimodal action of olivacine.
On the other hand, there is very little uptake of compounds 7a–k into the nuclei, and
consequently these compounds do not interact with DNA. The green fluorescence
observed in the cytoplasm may be indicative of a lysosomal location of compounds
7a–k, taking into account that lysosomal pH is particularly acidic. A striking difference
in the mode of action of olivacine and 7a–k is evident by comparison of the cell-cycle
profiles obtained by flow cytometry. Most of these derivatives show a marked increase
of sub G1 cells, which is indicative of apoptosis. All these results put together point
towards a possible mechanism of lysosomal apoptosis for compounds 7a–k, which in
turn may explain the lack of selectivity of the compounds against the different cell
lines. The fact that compounds 8a-c show cell-cycle profiles comparable to olivacine
proves that the hybridization strategy of tethering a second heteroaromatic moiety

NATURAL PRODUCT RESEARCH
7
causes this change in the mechanism of action. Finally, it must be noted that the
structural modifications performed in this work can be easily combined with the previ-
ously reported synthetic strategies, in the search for more active olivacine derivatives.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
We are grateful to Dr. Nahuel Schenone, Mr. Javier Unisony and all the staff at the C.I.A.R. for
their help and the kind permission to use their facilities (transportation, housing and laboratory)
during our field trips to Misiones. Research at UMYMFOR-FCEN-UBA was supported by grants
from CONICET to JAP (PIP 2014-2016 Nꢀ 11220130100523CO), UBA (UBACyT 2014-2017 Nꢀ
20020130100457BA) and ANPCyT (PICT 2014-2063). Work at CIPYP was supported by a grant
from CONICET (PIP 0237, to AGC). GMDV, EA, AGC and JAP are researchers of CONICET. CMPD
and GC thank CONICET for doctoral fellowships, while MC thanks INC for a student fellowship.
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