Antiplasmodial activity of synthetic ellipticine derivatives and an isolated analog

Article abstract of DOI:10.1016/j.bmcl.2014.04.070

Ellipticine has been shown previously to exhibit excellent in vitro antiplasmodial activity and in vivo antimalarial properties that are comparable to those of the control drug chloroquine in a mouse malaria model. Ellipticine derivatives and analogs exhibit antimalarial potential however only a few have been studied to date. Herein, ellipticine and a structural analog were isolated from Aspidosperma vargasii bark. A-ring brominated and nitrated ellipticine derivatives exhibit good in vitro inhibition of Plasmodium falciparum K1 and 3D7 strains. Several of the compounds were found not to be toxic to human fetal lung fibroblasts. 9-Nitroellipticine (IC₅₀ = 0.55 μM) exhibits greater antiplasmodial activity than ellipticine. These results are further evidence of the antimalarial potential of ellipticine derivatives.

Full text of DOI:10.1016/j.bmcl.2014.04.070

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2631–2634
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antiplasmodial activity of synthetic ellipticine derivatives
and an isolated analog
Andreia Montoia a,b, Luiz F. Rocha e Silva a,b, Zelina E. Torres a, David S. Costa a, Marycleuma C. Henrique b,
Emerson S. Lima b, Marne C. Vasconcellos b, Rita C. Z. Souza c, Monica R. F. Costa d, Andriy Grafov e,
Iryna Grafova e, Marcos N. Eberlin c, Wanderli P. Tadei a, Rodrigo C. N. Amorim a, Adrian M. Pohlit a,
⇑
a National Institute for Amazon Research, Avenida André Araújo, 2936, 69067-375 Manaus, Amazonas, Brazil
b Federal University of Amazonas, Avenida General Rodrigo Otávio Jordão Ramos, 3000, 69077-000 Manaus, Amazonas, Brazil
c State University of Campinas, Campus Universitário Zeferino Vaz SN, 13083-970 Campinas, Brazil
d Heitor Vieira Dourado Tropical Medicine Foundation, Avenida Pedro Teixeira, 25, 69040-000 Manaus, Amazonas, Brazil
e University of Helsinki, A.I. Virtasen aukio 1, PL 55, FI-00014 Helsinki, Finland
a r t i c l e i n f o
a b s t r a c t
Article history:
Ellipticine has been shown previously to exhibit excellent in vitro antiplasmodial activity and in vivo
antimalarial properties that are comparable to those of the control drug chloroquine in a mouse malaria
model. Ellipticine derivatives and analogs exhibit antimalarial potential however only a few have been
studied to date. Herein, ellipticine and a structural analog were isolated from Aspidosperma vargasii bark.
A-ring brominated and nitrated ellipticine derivatives exhibit good in vitro inhibition of Plasmodium fal-
ciparum K1 and 3D7 strains. Several of the compounds were found not to be toxic to human fetal lung
Received 22 March 2014
Revised 14 April 2014
Accepted 18 April 2014
Available online 28 April 2014
Keywords:
2-Methyl-1,2,3,4-tetrahydroellipticine
9-Nitroellipticine
fibroblasts. 9-Nitroellipticine (IC50 = 0.55
These results are further evidence of the antimalarial potential of ellipticine derivatives.
Ó 2014 Elsevier Ltd. All rights reserved.
lM) exhibits greater antiplasmodial activity than ellipticine.
7-Nitroellipticine
7,9-Dibromoellipticine
Plasmodium falciparum
Despite progress made in recent years in its control, malaria
continues to be the most important parasitic disease in tropical
regions worldwide. Approximately 3.3 billion people, roughly half
the world’s population, live in regions where malaria is endemic
and are thus at risk of infection. In 2010, approximately 220 mil-
lion malaria infections were recorded worldwide leading to an esti-
mated 666,000 deaths. Approximately 80% of all malaria deaths
occur in Africa and most are among children less than 5 years
old.1 This death toll is related to the growing number of cases
involving drug-resistant malaria parasites among other factors.
The antimalarial drugs chloroquine and mefloquine (quinoline
antimalarials) are synthetic mimics of the plant-derived natural
product quinine. Today, chloroquine is the most used antimalarial
drug worldwide and reports of chloroquine-resistance (CQR) in
Plasmodium falciparum and Plasmodium vivax (together responsible
for the absolute majority of all malaria infections throughout the
world) are widespread. Artemisinin is a plant-derived antimalarial
natural product. Its derivatives are prepared on an industrial scale
from isolated artemisinin through semi-synthesis.
ACTs (artemisinin-based combined therapies) have been rec-
ommended by WHO (World Health Organization) since 2005 for
the treatment of CQR malaria. ACTs comprise a synthetic quinoline
antimalarial and an artemisinin derivative and highlight the
importance of traditionally used antimalarial plants as sources of
chemical structural classes for contemporary malaria therapy.
While ACTs are clinically effective, the history of antimalarial drug
treatment leads to concern that it is only a matter of time before
parasites resistant to ACT therapy appear. In fact, in Cambodia,
the first evidence for the resistance of malaria parasites to ACTs
has been confirmed. No new class of antimalarials has been intro-
duced into clinical practice for more than two decades. The search
for new drug leads is important for the future of malaria treat-
ment.2 Plants are not only the origin of the antimalarials used in
current clinical practice, but continue to be rich sources of natural
product drug leads for other tropical diseases.3–5
Ellipticine (1), or 5,11-dimethyl-6H-pyrido[4,3,b]carbazole, is
an indole alkaloid whose derivatives have important anticancer
properties (Fig. 1).6–10 Compound 1 can be isolated from the
⇑
Corresponding author. Tel.: +55 92 3643 3078; fax: +55 92 3643 3079.
E-mail address: ampohlit@inpa.gov.br (A. M. Pohlit).
http://dx.doi.org/10.1016/j.bmcl.2014.04.070
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

2632
A. Montoia et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2631–2634
5
6
are the only ellipticine derivatives whose antiplasmodial activity
has been described to date.
4
R
R
R
2
3
1
10
R
N
9
11
Experience with the medicinal chemistry of synthetic quinoline
alkaloids and semi-synthetic derivatives of artemisinin has shown
that small structural changes can lead to improved antimalarial
activity and better pharmacological properties.16 A number of
derivatives of 1 have been prepared previously.17–22 Among the
commonly prepared derivatives, the most frequently desired mod-
ifications have been hydroxylation at C-7 and C-9 positions, alkyl-
ation of the N-atoms and C-1, and substitution at the C-9
position.22 The aim herein was to study the antiplasmodial activity
of novel and heretofore unexplored ellipticine derivatives and a
natural, isolated analog of ellipticine.
D
3
8
2
A
C
5
R
B
4
7
N
1
H
R
R3
R1
R2
R4
Me
R5
H
R6
1
H
H
H
H
2
H
H
H
H
H
H
H
Br
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
3
H
OH
OMe
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
4
H
5
H
3,4 -dihydro
6
H
Br
7
Br
Br
H
Br
8
Br
Despite several published and patented syntheses of ellipticine
(1)23 it is available from manufacturers only in tens of milligram
quantities and is costly. In the present work, the natural com-
pounds 1 and 2-methyl-1,2,3,4-tetrahydroellipticine (11) (Fig. 1)
were isolated from Aspidosperma vargasii bark collected at the
Ducke Forest Reserve in Manaus, Amazonas State, Brazil by adapt-
ing from previously published procedures.11,12,24–27 Briefly, isola-
tion involved initial extraction of powdered bark with 1% NH4OH
in EtOH, followed by evaporation of solvents and dissolution of
the alkaloid-rich residue in EtOAc. Extraction of the EtOAc solution
with 0.1 N HCl, basification of the aqueous phase to pH 8 with
NaHCO3 and extraction with CHCl3 yielded CHCl3 extracts that
were repeatedly chromatographed on silica gel to yield 1 and 11
that were identified based on their NMR and MS data28,29 and
comparison with literature data.12 The antiplasmodial activity
9
NO2
H
10
11
NO2
H
H
Me
1,2,3,4-tetrahydro
Figure 1. Ellipticine (1), olivacine (2), ellipticine derivatives 3–515 and 6–10
(synthesized herein) and isolated alkaloid 11.
alkaline EtOH extracts of the bark of the Amazonian carapanaúba
tree Aspidosperma vargasii11,12 and this compound exhibits signifi-
cant in vitro inhibitory activity (IC50 = 0.35–0.81 lM) against Plas-
modium falciparum (Table 1).11,13,14 Recently, significant in vivo
antimalarial activity of 1 against Plasmodium berghei in mice has
been demonstrated in the Peters 4 day suppressive test. At daily
doses of 10 mg/kg/day, ellipticine suppresses parasitemia versus
controls by 70–77%. At oral doses of 50 mg/kg/day, 1 exhibits total
elimination of parasitemia 5–7 days after initial infection and
mean survival times (MST, >40 days) that are comparable to the
commercial drug standard chloroquine.14 In that same report, the
significant in vitro (Table 1) and in vivo antimalarial activity of
olivacine (2), a structural isomer of 1 exhibiting analogous ring
structure, is also demonstrated. Oral doses of 10 and 50 mg/kg/
day of 2 suppress P. berghei by 48–68% and 90–91%, respectively,
5–7 days after infection.14 Both 1 and 2 exhibit low toxicity to mur-
ine macrophages, significant selectivity indices (SI = 290 to >1200)
and greater in vivo antimalarial activity than the much studied
indole alkaloid cryptolepine and a synthetic analog of cryptolepine.
Also, 1 and 2 exhibit none of the acute toxicity associated with cry-
ptolepine and its derivatives.14
(IC50 = 4–13 l
M against P. falciparum K1 & 3D7)30 and cytotoxicity
to human fetal lung fibroblasts (IC50 >50
l
g/mL)31 of alkaloid 11
were evaluated herein (Table 1).
Reactions using ellipticine (1) as substrate were carried out on
several milligram scales (Scheme 1). Treating ellipticine in glacial
AcOH with excess Br2 at 70 °C leads to the formation of a ca. 3:1
mixture of the novel 7,9-dibromoellipticine (7) and the known 9-
bromoellipticine (6) based on UPLC–MS, HRMS and NMR analysis.
This mixture is inseparable by standard chromatographic tech-
niques.32 Interestingly, it exhibits good inhibition of both P. falcipa-
rum K1 and 3D7 strains (IC50 = 0.2–0.3 lg/mL). Attempts to fully
convert compound 1 to dibromo product 7 by first reacting as
described above and then adding a second portion of Br2 and heat-
ing at 100 °C yield an unstable product tentatively assigned the
structure 7,8,9-tribromoellipticine (8) based on LC-HRMS and
NMR spectra.33 8 could not be tested for antiplasmodial activity.
Ellipticine (1) dissolved in AcOH and then treated with 65%
HNO3 at 0 °C leads to a mixture of mononitration products (pre-
sumably, 6-nitro, 7-nitro and 9-nitroellipticines) and unreacted 1
according to LC–MS analysis. After flash chromatography, the novel
Recently, ellipticine derivatives 3–5 (Fig. 1) exhibiting in vitro
activity against P. falciparum were described among the most
active compounds in a large compound library screening.15 In that
work, significant antiplasmodial activity was described for 1
(IC50 = 1.13
IC50 = 0.30
l
l
M), 1-HCl (IC50 = 0.60
l
M), 9-hydroxyellipticine (3,
M) and 3,4-
lM). To our knowledge, these
M), 9-methoxyellipticine (4, IC50 = 1.15
l
dihydroellipticine (5, IC50 = 1.01
Table 1
In vitro inhibition of Plasmodium falciparum (Pf) strains and cytotoxicity of ellipticine derivatives and structural analogs
Compound
Classification of antiplasmodial activitya
Pf, IC50
(lg/mL)
Pf, IC50
(l
M)
Fibroblasts, IC50 (lg/mL)
K1
3D7
K1
0.81b
1.4b
—
0.55
13
3D7
1
2
Active
Active
Active
Active
0.19
0.35
0.30
0.20
3.9
0.085
0.29
0.20
nt
4.9
0.35b
ntc
ntc
>50
nt
1.2b
6 + 7 (ca. 1:3)
9-2AcOH
—
nt
17
13
10
11
Active
Active
Active/Very Active
Very Active
>50
>50
nt
1.1
3.5
4.2
Chloroquine diphosphate
Quinine sulfate
0.17
0.040
0.061
0.052
0.33
0.12
0.11
0.15
nt
nt–not tested, — = not applicable.
a
IC50 <0.1
lg/mL = very active, IC50 = 0.1–5
l
g/mL = active, IC50 = 5–10
lg/mL = moderate activity, IC50 >10 l
g/mL = inactive.36
Data reported previously.14
b
c
IC50 >1.4 ꢀ 102
l
M against murine macrophages.14

A. Montoia et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2631–2634
2633
Br
Br
O N
2
Br
N
N
N
N
Br2
HNO3
glacial AcOH
70ºC,1h
+
glacial AcOH
1h, 0ºC
N
N
H
N
H
N
then evapd
at 100ºC
7
6
8
H
9
Br
N
Br2
N
H
glacial AcOH
HNO3, AcOH
Br
70ºC,1h
excess Br2
evapd 100ºC
ellipticine (1)
1h, 0ºC
CC
N
H
N
H
O N
10
Br
2
Scheme 1. Synthesis of brominated and nitrated derivatives of ellipticine.
compound 7-nitroellipticine (10) is obtained (Scheme 1).34 This
Supplementary data
compound exhibits the lowest antiplasmodial activity of all those
tested herein (IC50 = 13–17 lM against P. falciparum K1 & 3D7). If
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
04.070. These data include MOL files and InChiKeys of the most
important compounds described in this article.
glacial AcOH is used in the above procedure, the known 9-nitroel-
lipticine (9)20 is obtained as the sole product (no chromatography
required)35 and exhibits the highest antiplasmodial activity of all
the compounds tested.
The nitration and bromination performed herein occurred by
electrophilic aromatic substitution predominantly at the C-7 and/
or C-9 positions of 1 due to ortho/para directed activation by the
indole N-atom. Deactivation of the pyridine (D) ring to electro-
philic addition, due to the electronegative sp2 N-atom, is also pre-
sumed to be an important factor in the definition of the
regiochemical outcome of these reactions.
References and notes
1. WHO (World Health Organization). 10 facts on malaria (March 2013). http://
www.who.int/features/factfiles/malaria/en/, accessed on October 22, 2013.
2. Ekland, E. H.; Fidock, D. A. Int. J. Parasitol. 2008, 38, 743.
3. Schmidt, T. J.; Khalid, S. A.; Romanha, A. J.; Alves, T. M. A.; Biavatti, M. W.; Brun,
R.; Costa, F. B.; Castro, S. L.; Ferreira, V. F.; Lacerda, M. V. G.; Lago, J. H. G.; Leon,
L. L.; Lopes, N. P.; Amorim, R. C. N.; Niehues, M.; Ogungbe, I. V.; Pohlit, A. M.;
Scotti, M. T.; Setzer, W. N.; Soeiro, M. N. C.; Steindel, M.; Tempone, A. G. Curr.
Med. Chem. 2012, 19, 2128.
4. Schmidt, T. J.; Khalid, S. A.; Romanha, A. J.; Alves, T. M. A.; Biavatti, M. W.; Brun,
R.; Andrade Neto, V. F.; Costa, F. B.; Castro, S. L.; Ferreira, V. F.; Lacerda, M. V. G.;
Lago, J. H. G.; Leon, L. L.; Lopes, N. P.; Amorim, R. C. N.; Niehues, M.; Ogungbe, I.
V.; Pohlit, A. M.; Scotti, M. T.; Setzer, W. N.; Soeiro, M. N. C.; Steindel, M.;
Tempone, A. G. Curr. Med. Chem. 2012, 19, 2176.
5. Pohlit, A. M.; Lima, R. B. S.; Frausin, G.; Rocha, L. F.; Lopes, S. C. P.; Moraes, C. B.;
Cravo, P.; Lacerda, M. V. G.; Siqueira, A. M.; Freitas-Junior, L. H.; Costa, F. T. M.
Molecules 2013, 18, 9219.
6. Acton, E. M.; Narayanan, V. L.; Risbood, P. A.; Shoemaker, R. H.; Vistica, D. T.;
Boyd, M. R. J. Med. Chem. 1994, 37, 2185.
7. Wei Xu, G.; Mawji, I. A.; Macrae, C. J.; Koch, C. A.; Datti, A.; Wrana, J. L.; Dennis,
J. W.; Shimmer, A. D. Apoptosis 2008, 13, 413.
8. Masood, F.; Chen, P.; Yasin, T.; Fatima, N.; Hasan, F.; Hameed, A. Mater. Sci. Eng.,
C 2013, 33, 1054.
9. Knölker, H.-J. Curr. Org. Synth. 2004, 1, 309.
The in vitro inhibition observed herein for 9-nitroellipticine (9)
against the K1 strain of P. falciparum is greater than that of ellipti-
cine and taken together with the result of the previous report pre-
sented in the discussion, is further evidence that A-ring
modification of 1 especially in the 9-position, may be associated
with improved antiplasmodial activity. More derivatives should
be prepared in the future using alternative synthetic approaches
that do not depend solely upon direct preparation from 1.
In conclusion, 1 exhibits promising in vivo activity against Plas-
modium berghei that is comparable to that of chloroquine, though
at doses ca. 5ꢀ those of chloroquine. Like quinine and artemisinin,
ellipticine is a potent antimalarial natural product that is isolated
from a traditionally used antimalarial plant. The nitrated and bro-
minated ellipticine derivatives prepared herein exhibit good
in vitro antiplasmodial activity. Several of the compounds studied
are not toxic to human fetal lung fibroblasts (MRC-5). The present
cost of commercial ellipticine and its availability impose limits on
the approach adopted herein (direct synthesis of derivatives from
1). Total synthesis of 1 and its derivatives using alternative meth-
ods are beyond the scope of this work. No in vivo study on the anti-
malarial activity of a derivative of 1 has been reported though
olivacine (2) exhibits in vivo activity comparable to 1.14 The deriv-
atives of ellipticine prepared herein and others should be synthe-
sized on a larger scale in future work to investigate SAR and
in vivo antimalarial activity.
10. Knölker, H.-J.; Reddy, K. R. Chem. Rev. 2002, 102, 4303.
11. Andrade-Neto, V. F.; Pohlit, A. M.; Pinto, A. C.; Silva, E. C.; Nogueira, K. L.; Melo,
M. R.; Henrique, M. C.; Amorim, R. C.; Silva, L. F.; Costa, M. R.; Nunomura, R. C.;
Nunomura, S. M.; Alecrim, W. D.; Alecrim, M. G.; Chaves, F. C.; Vieira, P. P.
Memórias do Instituto Oswaldo Cruz 2007, 102, 359.
12. Henrique, M. C.; Nunomura, S. M.; Pohlit, A. M. Quim. Nova 2010, 33, 284.
13. Pohlit, A. M.; Rocha e Silva, L. F.; Henrique, M. C.; Montoia, A.; Amorim, R. C. N.;
Nunomura, S. M.; Andrade-Neto, V. F. Phytomedicine 2012, 19, 1049.
14. Rocha e Silva, L. F.; Montoia, A.; Amorim, R. C. N.; Melo, M. R.; Henrique, M. C.;
Nunomura, S. M.; Costa, M. R. F.; Andrade-Neto, V. F.; Costa, D. S.; Danta, G.;
Lavrado, J.; Moreira, R.; Paulo, A.; Pinto, A. C.; Tadei, W. P.; Zacardi, R. S.;
Eberlin, M. N.; Pohlit, A. M. Phytomedicine 2012, 20, 71.
15. Passemar, C.; Salér, M.; Soh, M. P. N.; Linas, M. D.; Ahond, A.; Poupat, C.; Benoit-
Vical, F. Phytomedicine 2011, 18, 1118.
16. Shahabuddin, M. S.; Nambiar, M.; Advirao, G. M.; Raghava, S. C. Invest. New
Drugs 2009, 28, 36.
17. Dalton, L. K.; Demerac, S.; Elmes, B. C.; Loder, W.; Swan, M.; Teitei, T. Aust. J.
Chem. 1967, 20, 2715.
Acknowledgements
18. Boogaard, A. T.; Pandit, U. K.; Koomen, G. J. Tetrahedron 1994, 50, 2551.
19. Devraj, R.; Barrett, J. F.; Fernandez, J. A.; Katzenellenbogen, J. A.; Cushman, M. J.
Med. Chem. 1996, 39, 3367.
This research was financed through Grants from the Brazilian
National Council for Scientific and Technological Development
(CNPq, National Malaria Network, Bionorth Network), the Amazo-
nas State Foundation for the Advancement of Research (FAPEAM/
PRONEX) and the European Community FP7–Marie Curie
Actions–People–International Research Staff Exchange Scheme
(IRSES), Grant Agreement No. PIRSES-GA-2011-295262 and the
Academy of Finland and Magnus Ehrnrooth Foundation. A.M.P.
and E.S.L. are CNPq PQ fellows.
´
20. Dracínsky, M.; Sejbal, J.; Rygerova, B.; Stiborova, M. Tetrahedron Lett. 2007, 48,
6893 (Suppl. Mat.: doi:http://dx.doi.org/10.1016/j.tetlet.2007.07.160.).
21. Lee, H. Y.; Chen, G. S.; Chen, C. S.; Chern, J. W. J. Heterocycl. Chem. 2010, 47, 454.
22. Cirino, J. J. V.; Belletato, P.; Dantas, S. O.; Ribeiro, L.; Ferreira, G. R.; Santos, H. F.
Quim. Nova 2005, 28, 30.
23. Schmidt, A. W.; Reddy, K. R.; Knölker, H.-J. Chem. Rev. 2012, 112, 3193.
24. Burnell, R. H.; Della Casa, D. Can. J. Chem. 1967, 45, 89.
25. Schmutz, J.; Hunziker, F. Helv. Chim. Acta 1958, 41, 288.
26. Woodward, R. B.; Iacobucci, G. A.; Hochstein, F. A. J. Am. Chem. Soc. 1959, 81,
4434.

2634
A. Montoia et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2631–2634
27. Pereira, M. M.; Jacomé, R. L. R. P.; Alcântara, A. F. C.; Alves, R. B.; Rastan, D. S.
chromatographic peak at 8.63 min that had MS ions at m/z 325 and 327
[M]+. HRMS [M+H]+: m/z 325.0343 and 327.0443, for C17H13N2Br calcd [M+H]+
Quim. Nova 2007, 30, 970.
28. Ellipticine (1): yellow needles (56 mg), mp 311–313 °C (lit.12
)
1H NMR
m/z 325.0340 and 327.0321 (D
= 0.9–37 ppm). 7,9-Dibromoellipticine (7): 1H
(250 MHz, CD3OD): d 2.7 (s, H-13) 3.2 (other CH3 overlapped with solvent
impurity peak), 7.2 (ddd, J = 7.0, 6.8 and 1.5, H-9), 7.4 (m, H-8 and H-7), 7.9 (d,
J = 7.0 Hz, H-4), 8.3 (d, J = 7.0 Hz, H-10 and H-3), 9.5 (s, H-1). By UPLC–MS, peak
at tR 4.15 min exhibits adduct ion [M+H]+ m/z 247 HRMS [M+H]+: m/z
NMR (500 MHz, DMSO-d6) d 12.3 (s, H-6), 10.1 (s, H-1), 8.61 (d, J = 1.6 Hz, H-
10), 8.54 (d, J = 7 Hz, H-3), 8.52, (d, J = 7 Hz, H-4), 8.10 (d, J = 1.6 Hz, H-8), 3.0 (s,
H-12). UPLC–MS exhibited chromatographic peaks at tR 10.25 and MS ions at
m/z 403, 405 and 407 [M+H]+. HRMS [M+H]+: m/z 402.9480, 404.9448 and
406.9441, for C17H12N2Br2 calcd [M+H]+ m/z 402.9445, 404.9426, 406.9408
247.1222, for C17H14N2 calcd. [M+H]+ m/z 247.1229 (
D = 3 ppm).
29. 2-Methyl-1,2,3,4-tetrahydroellipticine (11): white crystals (59 mg), 218–223 °C
(D = 5–9 ppm).
1
(lit.12). H NMR (400 MHz, CD3OD): d 2.4 (s, H-13), 2.5 (s, H-20), 2.6 (s, H-12),
33. Synthesis of 7,8,9-tribromoellipticine (8): Initially, the same procedure was used
that resulted in the preparation of the above mixture of 7,9-dibromoellipticine
(7) and 9-bromoellipticine (6). After 1 h, the reaction mixture was transferred
to a watch glass, excess Br2 was added and the mixture was heated over a
boiling water bath until the reaction solvents had evaporated. The mixture was
suspended in distilled water and extracted with CHCl3/MeOH (9:1). The
organic phase was dried over Na2SO4 and evaporated. The structure of product
8 was elucidated based on LC-HRMS, 1H and 13C NMR, DEPT135 and DEPT90
spectra. This compound was unstable and decomposed in DMSO-d6 in 1 week.
7,8,9-Tribromoellipticine (8): 1H NMR (300 MHz, DMSO-d6), d 11.53 (H-6), 10.01
(H-1), 8.55–8.35 (H-3, H-4, H-10), 3.19 (CH3), 2.90 (CH3). 13C NMR/DEPT 135 &
90 (75 MHz, DMSO-d6) d 144.84 (CH), 143.98 (C), 141.29 (C), 134.86 (C), 134.62
(C), 128.76 (CH), 126.65 (CH), 126.24 (C), 124.43 (C), 123.48 (C), 120.53 (C),
120.25 (CH), 115.42 (C), 112.39 (C), 107.44 (C), 15.07 (CH3), 12.48 (CH3). HRMS
[M+H]+: m/z 480.8500 (rel. int. 1), 482.8489 (rel. int. 3), 484.8467 (rel. int. 3),
486.8451 (rel. int. 1), for C17H11N2Br3 calcd. [M+H]+ m/z 480.8551, 482.8530,
2.8 (t, J = 6 Hz , H-3), 3.0 (t, J = 6 Hz, H-4), 3.7 (s, H-1), 7.1 (t, J = 8 Hz, H-9), 7.3 (t,
J = 8 Hz, H-8), 7.4 (d, J = 8 Hz , H-7), 8.1 (d, J = 8 Hz, H-10). 13C NMR (100 MHz,
CD3OD): d 11.3 (CH3), 14.0 (CH3), 27.0 (CH2), 44.8 (CH3), 52.2 (CH2), 56.1 (CH2),
110.1 (CH), 114.5 (C), 118.0 (CH), 119.7 (C), 121.8 (C), 122.0 (CH), 123.9 (C),
124.1 (CH), 125.8 (C), 128.4 (C), 138.4 (C), 140.6 (C). Adduct ion [M+H]+ at m/z
265, tR 3.53 min., HRMS [M+H]+: m/z 265.1694, for C18H21N2 calcd [M+H]+ m/z
265.1705 (
D = 4 ppm).
30. In vitro culture of Plasmodium falciparum and assay: The antimalarial drug-
susceptible 3D7 clone of the NF54 isolate (unknown origin, MRA-102, MR4-
ATCC Manassas, Virginia, USA) and the multi-drug resistant K1 (Thailand,
MRA-159, MR4-ATCC) strains of P. falciparum were maintained in continuous
culture using the Trager and Jensen method.37 For the microtest, an initial
parasitemia of 1–2% and hematocrit of 3% were used. Substances were diluted
in DMSO to a stock concentration of 5 mg/mL. These stock solutions were each
diluted in complete culture medium to obtain sample solutions for tests in the
range 100–0.006
l
g/mL. The test was performed as described previously.11
484.8510, 486.8489 (avg.
D = 0.8 ppm).
Briefly, the diluted test samples were plated in wells containing parasitized red
blood cells. Each diluted test sample was tested in triplicate. The test plate was
incubated for 48 h at 37 °C. After incubation, the contents of the wells were
evaluated by optical microscopy. The inhibition of the growth of parasites
(IGP%) was evaluated as a percentage by comparison with controls (which did
not receive sample): IGP% = 100 ꢀ [1ꢁ(parasitemia with sample/parasitemia of
controls)].
34. Synthesis of 7-nitroellipticine (10): 1 (4.5 mg, 18.2 lmol) was dissolved in AcOH
(2 mL) with magnetic stirring and then chilled in an ice bath. Chilled 65% HNO3
(0.9 mL) was added and the resulting mixture was stirred magnetically for 1 h
then the solution was made pH 10 by addition of NaCO3 and extracted with
CHCl3/MeOH 9:1 (3 ꢀ 10 mL). The organic phase was dried over anhydrous
Na2SO4 and then evaporated to dryness on a rotary evaporator to obtain a
crude product mixture (39.5 mg). The mixture was separated by flash CC using
a gradient of i-PrOH in CHCl3 (0.2:9.8–0.5:9.5). Analytical normal-phase TLC
was performed with CHCl3/MeOH (9:1) as eluents to yield mono-nitro product
31. Cytotoxicity test using the Alamar Blue™ assay: Human fetal lung fibroblasts
(MRC-5) were grown in DMEN medium supplemented with 10% fetal bovine
serum, 2 mM glutamine, 100
l
g/mL streptomycin and 100 U/mL penicillin, and
7 (RF 0.77, yield: 1.0 mg (30%), based on recovered 1). 7-Nitroellipticine (10): 1
H
incubated at 37 °C with 5% CO2 atmosphere. For experiments the cells were
plated in 96-well plates (104 cells per well) and the Alamar Blue™ assay38 was
performed as follows. After 24 h, the compounds were dissolved in DMSO
NMR (500 MHz, DMSO-d6) d 11.25 (s, H-6), 9.83 (s, H-1), 8.86 (d, J = 8.0 Hz, H-
8), 8.55 (d, J = 6.0 Hz, H-4), 8.43 (d, J = 8.0 Hz, H-10), 8.00 (d, J = 6.0 Hz, H-3),
7.50 (t, J = 8.0 Hz, H-9), 2.95 (s, H-13). Ion adduct at m/z 292, HRMS [M+H]+: m/
(50
Doxorubicin was used as positive control. Negative controls received 0.1%
DMSO. 2 h before the end of the incubation, 10 L of Alamar Blue™ were added
lg/mL) and added to each well and the cells were incubated for 48 h.
z 292.1077, for C17H13N3O2 calcd. [M+H]+ m/z 292.1086 (
D
= 3 ppm).
35. Synthesis of 9-nitroellipticine 2AcOH (9-2AcOH): AcOH was previously
fractionally distilled and dried over molecular sieves. (2.0 mg) was
l
1
to each well. The fluorescent signal was monitored with a multiplate reader
using a 530–560 nm excitation wavelength and 590 nm emission wavelength.
dissolved in AcOH (ca. 0.75 mL) with stirring. Chilled 65% HNO3 (3 drops)
was added to the stirred solution of 1 in an ice bath and the resulting mixture
was stirred for 1 h. Then, distilled H2O was added and the mixture was basified
to pH 11 with Na2CO3. The solution was extracted with CHCl3/MeOH 9:1
(3 ꢀ 10 mL). The organic phase was dried over anhydrous Na2SO4 and then
32. Synthesis of 9-bromoelliptcine (6) and 7,9-dibromoellipticine (7):
13 mol) was dissolved in glacial AcOH (60 L, 43.1 mg). The resulting solution
was placed in an ice bath. A second solution was prepared by addition of glacial
AcOH (90 L, 65 mg) to Br2 (0.25 L, 0.5 mol) and chilled. Upon mixing of
these two chilled solutions, red brick-colored precipitate formed. The
1 (3.2 mg,
l
l
l
l
l
evaporated to dryness on a rotary evaporator. The crude product was
a
characterized by LC-HRMS and 1H NMR and found to be the 2 AcOH salt of
9-nitroellipticine (9). 9-nitroellipticine 2AcOH (9-2AcOH): 1H NMR (300 MHz,
DMSO-d6) d 9.72 (br s, H-1), 9.06 (d, J = 2.4 Hz, H-10), 8.47 (br s, H-3), 8.38 (dd,
J1 = 2.4 Hz, J2 = 9.0 Hz, H-8), 7.93 (d, J = 6.0 Hz, H-4), 7.66 (d, J = 9.0 Hz, H-7),
3.25 (s, H-12), 2.80 (s, H-13), 1.65 (s, 2CH3CO2H). HRMS [M+H]+: m/z 292.1076,
reaction mixture was heated in an H2O bath at 70 °C for 1 h. The reaction
was monitored by TLC using CHCl3/MeOH 9:1 as eluents and UV 254 and
366 nm, I2 chamber and p-anisaldehyde/heat for development of the plates.
After 1 h of reaction, the solution was transferred to a watch glass and heated
over a steam bath to total dryness to yield the crude product mixture (6.9 mg).
CC on silica gel led to the isolation of an analytical mixture of 6 and 7 by 1H
NMR, UPLC–MS and HRMS analyses. 9-Bromoellipticine (6): 1H NMR (500 MHz,
for C17H13N3O2 calcd [M+H]+ m/z 292.1086 (
D = 3 ppm).
36. Bertani, S.; Bourdy, G.; Landau, I.; Robinson, J. C.; Esterre, P.; Deharo, E. J.
Ethnopharmacol. 2005, 98, 45.
DMSO-d6)
(overlapping peaks, H-3 and H-4), 7.82 (dd, J = 1.8, 8.6 Hz, H-8), 7.66 (d,
J = 8.6 Hz, H-7), 3.17 (s, H-12), 2.89 (s, H-13). UPLC–MS exhibited
d
12.3 (s, H-6), 10.1 (s, H-1), 8.61 (d, J = 1.8 Hz, H-10), 8.50
37. Trager, W.; Jensen, J. B. Science 1976, 193, 673.
38. Ahmed, S. A.; Gogal, R. M., Jr.; Walsh, J. E. J. Immunol. Methods 1994, 15, 211.
a