Synthetic analogues of the montanine-type alkaloids with activity against apoptosis-resistant cancer cells

Article abstract of DOI:10.1016/j.bmcl.2018.01.041

In a search of small molecules active against apoptosis-resistant cancer cells, a skeletal rearrangement of alkaloid haemanthamine was utilized to generate a series of compounds possessing the alkaloid montanine ring system. The synthesized compounds were found to inhibit proliferation of cancer cells resistant to apoptosis at micromolar concentrations. Selected compounds were also active against patient-derived glioblastoma cells expressing stem-cell markers. This is the first report describing the preparation of synthetic analogues of the montanine-type alkaloids with antiproliferative activity. The compounds prepared in the current investigation appear to be a useful starting point for the development of agents to fight cancers with apoptosis resistance, and thus, associated with poor prognoses.

Full text of DOI:10.1016/j.bmcl.2018.01.041

Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthetic analogues of the montanine-type alkaloids with activity
against apoptosis-resistant cancer cells
Karthik Govindaraju a,e, Aude Ingels b,e, Md Nabiul Hasan c, Dandan Sun c, Veronique Mathieu b,
Marco Masi d, Antonio Evidente d, Alexander Kornienko a,
⇑
a Department of Chemistry and Biochemistry, Texas State University, San Marcos, TX 78666, United States
b Laboratoire de Cancérologie et de Toxicologie Expérimentale, Faculté de Pharmacie, Université Libre de Bruxelles (ULB), Brussels, Belgium
c Department of Neurology, University of Pittsburgh, Pittsburgh, PA 15213, United States
d Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Complesso Universitario Monte Sant’Angelo, Via Cintia 4, 80126 Napoli, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In a search of small molecules active against apoptosis-resistant cancer cells, a skeletal rearrangement of
alkaloid haemanthamine was utilized to generate a series of compounds possessing the alkaloid monta-
nine ring system. The synthesized compounds were found to inhibit proliferation of cancer cells resistant
to apoptosis at micromolar concentrations. Selected compounds were also active against patient-derived
glioblastoma cells expressing stem-cell markers. This is the first report describing the preparation of
synthetic analogues of the montanine-type alkaloids with antiproliferative activity. The compounds
prepared in the current investigation appear to be a useful starting point for the development of agents
to fight cancers with apoptosis resistance, and thus, associated with poor prognoses.
Received 26 December 2017
Revised 18 January 2018
Accepted 21 January 2018
Available online xxxx
Keywords:
Antiproliferative activity
Apoptosis resistance
Montanine
Ó 2018 Elsevier Ltd. All rights reserved.
Haemanthamine
Manthine
Cancers with intrinsic resistance to apoptosis are characterized
by the lack of responsiveness to current chemotherapeutic agents
that generally work by the induction of apoptosis in cancer cells.
These cancers include tumors of the lung, melanoma and glioblas-
toma and they represent a major challenge in the clinic.1 For exam-
ple, patients afflicted by glioblastoma multiforme,2,3 have a median
survival expectancy of less than 14 months when treated with the
best available protocol that involves surgery, radiation and
chemotherapy with temozolomide.4 Further, the main cause of
death of cancer patients are tumor metastases. Metastatic cells
are resistant to anoikis, a type of apoptotic cell death triggered
by the loss of contact with extracellular matrix or neighboring
cells.5 Resistance to anoikis, and thus apoptosis, renders metastatic
cells unresponsive to the large majority of proapoptotic agents as
well.3,6 In addition, it is believed that a population of cancer cells,
referred to as cancer stem cells, is particularly unresponsive to
pro-apoptotic chemotherapy. Glioblastoma cells exhibiting stem
cell markers (GSCs) can be grown in culture as neurospheres. They
possess the ability to self-renew, differentiate into multiple neural
lineages and faithfully recapitulate human glioblastoma on both
histological and genetic levels when injected into the brains of
mice.7–9 Therefore, a search for novel anticancer agents that can
overcome cancer cell resistance, including resistance to apoptosis,
is an important pursuit.
In this connection, natural products represent a valuable source
of not only cytotoxic compounds, but also those capable of over-
coming the intrinsic resistance of cancer cells to apoptosis.10–13
Our recent studies have focused on isocarbostyrils and alkaloids
belonging to the Amaryllidaceae plant family. The medicinal value
of the Amaryllidaceae plants was already recognized in the fourth
century BC, when Hippocrates of Cos used oil from the daffodil
Narciclasus poeticus L. for the treatment of cancer.14 In more recent
times, over 100 structurally diverse alkaloids, possessing a wide
spectrum of biological activities have been isolated from the
Amaryllidaceae species.15 Narciclasine and lycorine (Fig. 1) have
been actively investigated for their potent anti-tumor effects, both
in vitro and in vivo, in various pre-clinical models of human cancers
by us16–21 and others.22–25 Our interest in these natural products
primarily stems from their ability to overcome apoptosis resistance
and display promising activity in orthotopic mouse models of
glioblastoma and metastatic melanoma.16,19,21 Although, not eval-
uated in vivo, crinine-type alkaloids, such as haemanthamine
(Fig. 1)17 and bulbispermine,26 also show promising activity
against apoptosis-resistant cancer cells. To investigate whether
⇑
Corresponding author.
E-mail address: a_k76@txstate.edu (A. Kornienko).
e
These authors contributed equally.
https://doi.org/10.1016/j.bmcl.2018.01.041
0960-894X/Ó 2018 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Govindaraju K., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.01.041

2
K. Govindaraju et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
practical multigram isolation procedures for narciclasine, lycorine
and haemanthamine.28 We thus drew our attention to an old
report of a semisynthetic transformation of haemanthamine-type
skeleton to the montanine-type scaffold (Fig. 2).29 This elegant
work, which was reproduced in a later study,30 described the
intramolecular nucleophilic attack of the electron rich aromatic
moiety on the activated C11-hydroxyl (A in Fig. 2) to form the
C10a-C11 bond presumably leading to the arenium ion B. The
C10a–C10b bond then breaks upon the SN20 attack of a nucleophile
at C2 with the translocation of the C1–C2 alkene to the C1–C10b
position resulting in the montanine-type skeleton C (Fig. 2).
The reaction can be conducted without the addition of an exter-
nal nucleophile for 48 h at rt, in which case it will result in chlo-
rine-containing derivative 1, where the chloride liberated upon
mesylation serves as the nucleophile (Fig. 3).29 Alternatively, the
reaction can be run at 0 °C for 8 h with the subsequent addition
of a nucleophilic species. In this case a wide variety of C2-substi-
tuted montanine-type synthetic derivatives can be prepared. Thus,
the addition of water results in 2.29 The addition of methoxide, pre-
pared by the reaction of MeOH with NaH leads to another monta-
nine-type alkaloid manthine, isolated by Wildman from South
African plant genus Haemanthus.29,31 Depending on the species
the yield varied from 1 to 44 mg per kg of wet bulbs, clearly insuf-
ficient for its supply for biological studies.31 This alkaloid was also
obtained by a total synthesis by Sha and coworkers, which
involved a 24-step procedure from commercially available quinic
acid.32 The one step synthesis of manthine from haemanthamine,
readily available in multigram quantities, is thus significant.
Fig. 1. Structures of Amaryllidaceae constituents with promising anticancer
activities.
promising antiproliferative properties are also associated with
other representative Amaryllidaceae alkaloid groups, we noted that
montanine (Fig. 1) was shown to be cytotoxic to human cancer
cells.27 The montanine group of alkaloids is characterized by a
5,11-methanomorphanthridine ring system and some examples
of the montanine-type alkaloids are shown in Fig. 1. The work
reported in the current manuscript describes the first systematic
attempt to assess the anticancer potential of compounds based
on the montanine-type alkaloid structural system.
Synthetic derivatization of the montanine-type alkaloids to
generate a library of analogues is challenging because their stable
and practical supply from the plant sources has not been estab-
lished to our knowledge. In contrast, we and others have developed
Fig. 2. Hypothetical mechanism for haemanthamine-montanine skeletal rearrangement.
Fig. 3. Synthesis of C2-substituted montanine-type compounds.
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K. Govindaraju et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
3
Fig. 4. Derivatization of 1 (A), 2 (B) and a similar rearrangement of alkaloid haemanthidine (C).
We found that other nucleophiles that can be utilized in this
transformation are ethanol, allylic and propargylic alcohols, lead-
ing to compounds 3, 4 and 5. In addition, a primary amine, indole
and pyrrole can be added to the C2-position quite readily following
their deprotonation with NaH, resulting in 6, 7 and 8 (Fig. 3). In the
case of indole, only C3-alkylation was observed, although both N1-
and C3-alkylations are possible.
Because chloride 1 could be readily prepared on the preparative
scale, it was utilized in further transformations, involving N-oxida-
tion, N-propargylation, and single or double iodination of the aro-
matic ring, resulting in derivatives 9, 10, 11 and 12, respectively
(Fig. 4A). In addition, 2 can be converted to bromide 13 (Fig. 3B).
Finally, we found that the transformation of the crinine to monta-
nine skeleton can be also achieved using a related alkaloid, hae-
manthidine (Fig. 4C). Here, the C6-mesylated product 14 was
isolated in a reasonable yield. The stability of the mesylate is
accounted for by the bridgehead nature of the tertiary nitrogen
and thus its inability to stabilize the adjacent positive charge that
would develop in the transition state of the hydrolysis
reaction.33,34
panel of six cancer cell lines.35 This included cells resistant to a
number pro-apoptotic stimuli, such as human A549 non-small cell
lung cancer (NSCLC), human glioblastoma U373, and human
SKMEL-28 melanoma, as well as tumor models, which are largely
susceptible to apoptosis-inducing stimuli, such as human Hs683
anaplastic oligodendroglioma, human MCF-7 breast adenocarci-
noma and mouse B16F10 melanoma (Table 1). Analysis of these
data reveals that the active derivatives displayed comparable
potencies in both cell types, irrespective to cell sensitivities to
apoptosis induction and suggesting that they are capable of over-
coming apoptosis resistance (Table 1).19
Despite the large variations in cancer cell line sensitivities, the
data indicate that a small C2-substituent is preferred as observed
in chloride 1, alcohol 2 and bromide 13. Activity of chloride 1
and bromide 13, in which the halogen atom has the inverted con-
figuration, appears to indicate that the
a-C2 configuration is not
critical. The variations in cell line sensitivities to both chloride 1
and bromide 13 suggest that they are not indiscriminate alkylating
agents, but have distinct intracellular targets. Of the C2-ethers,
alkaloid manthine, containing the small C2-methoxy group, stands
out by affording single digit GI50 values across the entire panel,
including the glioblastoma U373 cells for which 1 and 2 are less
The synthesized compounds were evaluated for in vitro antipro-
liferative properties using the MTT colorimetric assay against a
Table 1
In vitro growth inhibitory effects of compounds 1–14 and alkaloid manthine.
Compound
GI50 in vitro values (mM)a
Resistant to apoptosis
Sensitive to apoptosis
A549
SKMEL-28
U373
MCF7
Hs683
B16F10
1
6
26
51
17
6
7
2
5
8
31
13
4
8
Manthine
3
4
5
4
3
3
3
4
59
10
>100
14
>100
20
82
20
>100
7
40
7
5
6
7
23
59
18
28
65
9
42
72
9
28
44
23
24
67
24
10
10
4
8
9
86
67
>100
>100
>100
>100
>100
25
68
95
11
>100
>100
>100
39
7
>100
>100
>100
78
9
>100
>100
>100
>100
>100
18
>100
>100
>100
78
19
>100
>100
>100
>100
71
5
>100
10
11
12
13
14
>100
>100
72
a
Mean concentration required to reduce the viability of cells by 50% after a 72 h treatment relative to a control, each experiment performed in sextuplicates, as determined
by MTT assay.
Please cite this article in press as: Govindaraju K., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.01.041

4
K. Govindaraju et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
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33. Selected procedure for the preparation of alcohol 2:
A solution of
proliferation at concentrations as low as 1
pounds inhibited proliferation by 80–95% at concentrations of 10
and 30 M.
lM. All tested com-
haemanthamine (100 mg, 0.332 mmol) in dry pyridine (3 mL) was treated
with methanesulfonyl chloride (0.1 mL, 1.33 mmol) and allowed to stand at 0
°C for 8 h. The mixture was poured into of water (10 mL) containing sodium
bicarbonate (100 mg). The solution was again allowed to stand overnight and
then extracted with chloroform. The solvent was removed under reduced
pressure. The resulting residue was purified by preparative TLC using CH2Cl2/
MeOH (9:1) as an eluent to yield 74 mg of 2 (74%) as an amorphous white solid.
1H NMR (400 MHz, CDCl3) d 6.54 (s, 1H), 6.49–6.42 (m, 1H), 5.87 (dd, J = 10.6,
1.4 Hz, 2H), 5.53 (d, J = 14.9 Hz, 1H), 4.35 (dd, J = 16.6, 7.6 Hz, 1H), 4.06 (s, 1H),
3.86 (dd, J = 18.8, 9.8 Hz, 1H), 3.53 (d, J = 11.4 Hz, 1H), 3.41–3.34 (m, 4H), 3.31
(t, J = 3.0 Hz, 1H), 3.19–3.02 (m, 2H), 2.85 (s, 1H), 2.32 (ddd, J = 12.9, 5.1, 3.5 Hz,
1H), 1.56 (td, J = 12.5, 3.1 Hz, 1H); 13C NMR (100 MHz, CDCl3) d 152.8, 146.9,
146.1, 131.9, 123.8, 115.5, 107.4, 106.78, 100.8, 81.1, 67.1, 60.6, 58.9, 56.9, 55.4,
45.4, 28.3; HRMS (ESI) calcd for C17H20NO+4, 302.1392 (M+H)+; found, 302.1388.
l
In conclusion, utilizing a previously described transformation of
the crinine to montanine-type skeletons, we prepared the first ser-
ies of synthetic analogues of the montanine alkaloids differing by
the substituents at C2, N5, C6, C7 and C10 positions. It was found
that the reaction readily proceeded not only with oxygen or halo-
gen-based nucleophiles, but also with primary amines and nucle-
ophilic heterocycles. The synthesized compounds were found to
inhibit proliferation of cancer cells resistant to apoptosis at micro-
molar concentrations, with alkaloid manthine, C2-OH and C2-
indole-substituted compounds appearing to be most potent.
Selected compounds also were active against patient-derived
glioblastoma cells expressing stem-cell markers. These preliminary
results indicate that compounds based on the montanine skeleton
should be investigated as potential anticancer agents active against
drug-resistant cancer cells, including cancer stem cells.
34. Selected procedure for the preparation of indole 7:
haemanthamine (30 mg, 0.1 mmol) in dry pyridine (2 mL) was treated with
methanesulfonyl chloride (33 L, 0.4 mmol) and allowed to stand at 0 °C for 8
A solution of
l
h. After another hour at 25 °C the reaction mixture was added via a cannula to
an ice-cold THF (3 mL) solution of indole (23 mg, 0.15 mmol), pretreated with
NaH (100 mg, 0.45 mmol). After stirring for 30 min, the reaction mixture was
poured into water (10 mL) and extracted with chloroform (3 ꢀ 5 mL). The
solvent was removed under reduced pressure and the residue was purified by
preparative TLC using CH2Cl2:MeOH (20:1) mixture as the eluent to yield 7 (25
mg, 62%) as a semi-solid substance. 1H NMR (400 MHz, CDCl3) d 8.19 (s, 1H),
7.66 (d, J = 7.8 Hz, 1H), 7.46–7.34 (m, 1H), 7.27–7.22 (m, 1H), 7.21–7.14 (m,
1H), 6.90 (d, J = 1.8 Hz, 1H), 6.65 (s, 1H), 6.56 (s, 1H), 5.95 (dt, J = 8.9, 4.5 Hz,
2H), 5.77 (s, 1H), 4.58 (d, J = 16.4 Hz, 1H), 4.16–4.01 (m, 1H), 3.90–3.85 (s, 1H),
3.80–3.71 (m, 2H), 3.64–3.56 (m, 1H), 3.49 (s, 3H), 3.43–3.27 (m, 2H), 2.53 (s,
1H), 1.75–1.65 (m, 1H); 13C NMR (100 MHz, CDCl3) d 135.8, 126.2, 122.5, 121.9,
119.7, 118.3, 117.1, 111.5, 107.7, 106.9, 101.0, 79.1, 77.2, 56.9, 56.1, 44.0, 36.9,
30.9; HRMS (ESI) calcd for C25H25N2O3 (M+H)+: 401.1865, found, 401.1870.
35. The human cell lines: breast carcinoma MCF-7 (DSMZ ACC107),
oligodendroglioma Hs683 (ATCC HTB138), non-small cell lung cancer A549
(DSMZ ACC107), glioblastoma U373 (ECACC 08061901), melanoma SKMEL-28
(ATCC HTB72) and the murine melanoma B16F10 (ATCC CRL-6475) were
cultured in RPMI supplemented with 10% heat-inactivated FBS (GIBCO code
10270106), 4 mM glutamine (Lonza code BE17-605E), 100 mg/mL gentamicin
(Lonza code 17-5182), and penicillin-streptomycin (200 units/ml and 200 mg/
Acknowledgments
AK acknowledges the National Cancer Institute (CA186046-
01A1) and DS acknowledges the support from the National Insti-
tutes of Health (R01 NS075995).
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2018.01.041.
Please cite this article in press as: Govindaraju K., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.01.041

K. Govindaraju et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
5
ml) (Lonza code 17-602E). Cell lines were cultured in flasks, maintained and
grown at 37 °C, 95% humidity, 5% CO2. Antiproliferative effects of the
compounds on these cell lines were evaluated through the colorimetric assay
MTT. Briefly, cells were trypsinized and seeded in 96 well plates. Prior to
treatment compounds were dissolved in DMSO at a concentration of 10 mM.
After 24 h, cells were treated with the compounds at different concentrations
ranging from 10 nM to 100 mM or left untreated for 72 h. Cell viability was
estimated by means of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide, Sigma, Bornem, Belgium) mitochondrial reduction into
formazan in living cells. The optical density of the untreated control was
normalized as 100% of viable cells allowing determination of the concentration
that reduced their global growth by 50%.
38. Cell proliferation of GC22 glioblastoma cells was measured by quantifying BrdU
(Bromodeoxyuridine) incorporation using an immunoassay Brdu Kit (EMD
Millipore, code 2750) as described in Zhu W, Carney KE, Pigott VT, et al.
Carcinogenesis. 2016;37:839. Briefly, GC22 cells (1 ꢀ 103 cells/well) were
seeded in 96-well plates in 180 lL media. After 24 h in culture cells were
incubated for 72 h with compounds of different concentrations ranging from 1
M to 30 M and DMSO as vehicle control. BrdU was added in the last 4 h
l
l
period of 72 h incubation. The incorporation of BrdU into newly synthesized
DNA of proliferating cells was detected by using a peroxidase-conjugated
antibody which reacts with the thymidine analogue BrdU. Bound anti-BrdU-
peroxidase conjugated antibody was measured by a substrate reaction, and
then quantified calorimetrically by an ELISA plate reader (Spectra MAX 190,
Molecular Devices, Sunnyvale, CA) at dual wavelength of 450/550 nm..
36. Zorniak M, Clark PA, Leeper HE, et al. Clin Cancer Res. 2012;18:3628.
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Please cite this article in press as: Govindaraju K., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.01.041