Anti-tumoral activity of imidazoquines, a new class of antimalarials derived from primaquine

Article abstract of DOI:10.1016/j.bmcl.2009.10.081

The growth inhibitory activity of imidazoquines, antimalarial imidazolidin-4-ones derived from primaquine, on human cancer cell lines HT-29, Caco-2, and MCF-7 has been evaluated. Primaquine, N-dipeptidyl-primaquine derivatives, and other quinolines have b

Full text of DOI:10.1016/j.bmcl.2009.10.081

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6914–6917
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Anti-tumoral activity of imidazoquines, a new class of antimalarials
derived from primaquine
Iva Fernandes a, Nuno Vale a, Victor de Freitas a, Rui Moreira b, Nuno Mateus a, Paula Gomes a,
*
a CIQUP, Departamento de Química, Faculdade de Ciências, Universidade do Porto, P-4169-007 Porto, Portugal
b iMed.UL, CECF, Faculdade de Farmácia de Lisboa, P-1600-083 Lisboa, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
The growth inhibitory activity of imidazoquines, antimalarial imidazolidin-4-ones derived from prima-
quine, on human cancer cell lines HT-29, Caco-2, and MCF-7 has been evaluated. Primaquine, N-dipepti-
dyl-primaquine derivatives, and other quinolines have been included in the study for comparison
purposes. Primaquine and some of its derivatives were significantly active against the MCF-7 human
breast cancer cell line, so these compounds might represent useful leads targeted at the development
of novel specific agents against breast cancer. Conversely, all compounds were generally inactive against
Received 3 September 2009
Revised 16 October 2009
Accepted 17 October 2009
Available online 22 October 2009
Keywords:
HT-29, with only one of the imidazoquines having IC50 below 50
line were modest and did not follow any defined trend.
lM. Activities against the Caco-2 cell
Anti-tumoral
Breast cancer
Imidazolidin-4-ones
Malaria
Ó 2009 Elsevier Ltd. All rights reserved.
Primaquine
Quinolines
Quinolines and derivatives have been classically known for
their antimalarial properties.1–3 Quinolines may have other rele-
vant biological effects, such as the potent and selective antagonism
At the light of the above findings and of our former promising
results with imidazoquines, derivatives of the 8-aminoquinoline
antimalarial primaquine, 1, as a new class of antimalarials not sus-
ceptible to oxidative deamination,12–19 we decided to investigate
their activity as anti-proliferative agents against the human tumoral
cell lines HT-29 (human colon adenocarcinoma), Caco-2 (human
epithelial colo-rectal adenocarcinoma), and MCF-7 (breast cancer).
The study was focused on imidazoquines 2–4, but also included the
parent drug (1), its N-acetyl (5) and two N-dipeptidyl (6 and 7)
derivatives, as well as other three quinolines (8–10) for compari-
son and establishment of preliminary SAR.
Primaquine (1) and quinolines 8–10 were commercially avail-
able from Sigma–Aldrich, whereas primaquine derivatives (2–7)
were prepared in good yields and with correct spectral data, by
previously reported methods12–19 described in Supplementary
data.
Cytotoxicity assays were performed according to the procedure
adopted by the National Cancer Institute (NCI, USA),20,21 as detailed
in Supplementary data, where inhibition curves obtained for all
compounds against all three cell lines are also given.
Figure 1 displays the inhibition curve obtained for 3a on MCF-7
cells, as an example. IC50 values were deduced from these curves
and are given in Table 1. From these data, it can be inferred that
compound activity against the cell lines assayed depends on both
compound structure and specific cell line. The clearest observation
is that compounds have highest growth inhibitory effects (lower
IC50 values) on the breast cancer cell line (MCF-7), while are
to a2C-adrenoreceptors of some 4-aminoquinolines with potential
applications in the therapy of CNS disorders.4 Recently, Strobl
and co-workers found that the cinchona tree bark antimalarial
quinidine inhibited growth of human breast tumor cells,5 which
prompted them to engage in the study of other anti-proliferative
quinolines and to the proposal of four of such compounds as breast
tumor cell differentiation agents.6 Mahajan et al. have also de-
scribed new 7-chloroquinolyl thioureas structurally related to
chloroquine as potential antimalarial and anticancer agents.7 The
mechanism of anti-tumoral action of quinolines is not fully estab-
lished,8 but reported increase of caspase 3 levels in breast cancer
cells suggests involvement in apoptosis.9 Reports on new drug can-
didates with dual antimalarial and anticancer activity are not lim-
ited to quinoline derivatives, as shown by most recent works by
O’Neill’s and Posner’s groups with trioxanes.10,11 While the former
authors have found artemisinin–acridine hybrids that displayed
in vitro antimalarial and anti-tumor activity,10 the latter have con-
ceived new dimeric trioxane sulfones able to cure malaria-infected
mice with a single oral dose and to display potent and selective
cytotoxicity against several cancer cell lines.11
* Corresponding author.
E-mail address: pgomes@fc.up.pt (P. Gomes).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.081

I. Fernandes et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6914–6917
6915
Interestingly, while quinolines 8–10 were generally inactive
against any of the three cell lines, primaquine (1) had the highest
activity against MCF-7 and Caco-2 cells and the second highest
activity against HT-29 cells, suggesting a key role of the aliphatic
chain of primaquine (the 4-amino-1-methylbutyl substituent at
the quinolinic’s 8-amino group) for its anti-tumoral activity. This
becomes quite clear when comparing 8-amino-6-methoxyquino-
line (8) with primaquine (1).
3a
150.0
100.0
50.0
**
**
***
***
***
***
0.0
O
Modification of the primaquine’s terminal primary amine, by
insertion of either imidazolidin-4-one (3, 4) or dipeptide (6, 7)
moieties generally had a detrimental effect on anti-tumoral activ-
ities, which was more pronounced for imidazolidin-4-one pepti-
domimetic structures (3, 4). Exception is made for imidazoquine
3a that was the best against HT-29 cells and one of the best against
MCF-7 cells. For these two cell lines, comparison of data for 3a with
those for 3b show that replacement of the N1-aminoacyl substitu-
ent of the imidazoldin-4-one ring by an N1-acetyl group leads to
activity loss, thus indicating that the primary amine from the N-
terminal amino acid has a relevant role for the anti-tumoral activ-
ity of peptidomimetic imidazoquines as 3a. This is possibly a key
structural requirement for activity against MCF-7 cells, as the com-
pounds displaying lowest IC50 values on this cell line, that is, pri-
maquine (1), imidazoquine 3a and N-dipeptidylprimaquines 6, 7
all have an aliphatic primary amine whose acetylation leads to sig-
nificant loss of activity (5 vs 1, 3b vs 3a). We believe that it is the
presence of such a primary amine rather than the existence of an
amino acid or dipeptide moiety that is relevant, otherwise prima-
quine would not rank the best. Interestingly, the primary amine
-50.0
-100.0
***
100.0
0.0
1.23
3.70
11.1
33.3
Concentration (µM)
HT-29
Caco-2
MCF-7
Figure 1. Effect of imidazoquine 3a on the growth of different human tumoral cell
lines evaluated by Sulforhodamine B assay. Cells, seeded in 96-well plates, were
treated with a broad concentration range (6.3–100.0 lM) of each compound for
48 h. Each value represents the mean SEM (n = 3–6). **p <0.001, ***p <0.0001
(significant decrease vs control).
practically inactive against the colon adenocarcinoma cell line (HT-
29). In what concerns growth inhibition of Caco-2 cells, results are
more disperse and no clear structure–activity relationships could
be drawn in this case.
Table 1
In vitro activity of test compounds against HT-29, Caco-2, and MCF-7 human tumoral cell lines (average SEM from 3 to 6 independent experiments)
O
Me
O
H
N
4, X =
6, X =
N
X = Me
Me
O
N
H
NH2
N
H
O
N
N
N
R3
R4
O
O
R2
R1
O
HN
NH2
NH
7, X =
O
N
Me
N
H
4a, R3 = R4 = -Me
2, R1 = -Me, R2 = -H
Me
Me
4b, R3 and R4 = -(CH2)5
Me
-
Me
3a, R1 = -H, R2
3b, R1 = -H, R2
=
=
R5,R6
NH2
O
O
O
8, R5 = 6-OMe, R6 = 8-NH2
9, R5 = 2-Cl, R6 = 3-COOH
10, R5 = 4-Cl, R6 = 7-CF3
X
1, X = -NH2
Me
5, X =
N
H
Me
N
Estimated values for log P, log S and overall drug scores are also given (see text).
a
Estimated by the ALOGPS 2.1 algorithm.22
b
Estimated by the Osiris Property Explorer.23

6916
I. Fernandes et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6914–6917
group of primaquine has been claimed to also have a key role on
the drug’s antimalarial activity.1,2 Possibly, the aliphatic primary
amine has the appropriate pKa (ca. 10.6 and 9.7 for, respectively,
butylamine and alanine24,25) for cellular internalization, whereas
molecules devoid of primary amino groups or where such groups
are aromatic (pKa for aniline is ca. 4.226) have not. Apparently, spe-
cific amino acid or oligopeptide transporters like hPept127,28 do not
have a role here, or else better results would expected for 3a, 6, and
7 on Caco-2 cells, given their high expression of such transporters
and their consequent use in intestinal absorption studies of com-
pounds bearing oligopeptide moieties.27,28
At this preliminary stage, we can say that results are promising
concerning compounds’ specific anti-proliferative action against
the MCF-7 cell line model of breast cancer. Though primaquine
(1) and its N-alanylprolyl derivative (7) were better than imidazoq-
uine 3a against MCF-7 cells, the latter has the advantage of being
both resistant against proteases (that promptly degrade the pep-
tide moiety of 7) and oxidative deamination (the main metabolic
process behind premature deactivation and low oral bioavailability
of primaquine).12–19,29 Known algorithms22,23 were used to calcu-
late parameters relevant to predict drug oral bioavailability, like
log P or log S and to estimate the overall drug score of the test com-
pounds (Table 1). Data show that imidazoquine 3a has not only the
highest drug score of the set, but is also considerably soluble
(120 mg/L) and has a log P value that falls within the optimal inter-
val for gastric absorption (2 1).30
treatment is known to cause oxidative stress in liver, kidneys,
and blood by generating reactive oxygen species (ROS).2,37,38
ROS have a crucial role both on differentiation and suppression
of hypoxic (e.g., breast) tumors39–44 and on lipid peroxidation,38,45
whose enhancement is known to be protective against breast can-
cer.45 In view of this, the anti-proliferative action of primaquine, 1,
and derived imidazoquine, 3a, herein reported might be related to
the intracellular generation of drug-derived ROS.
Ongoing in vitro redox and radical-scavenging properties of
primaquine and imidazoquines 3, as well as in vivo anti-tumoral
activity and oral bioavailability studies on the same compounds
are in course and will be timely reported.
Acknowledgments
Thanks are due to the Portuguese Foundation for Science and
Technology (FCT) for financial support through Project Grants
PTDC/QUI/65142/2006, PTDC/QUI/65501/2006, CONC-REEQ/275/
QUI and REDE/1517/RMN/2005. N.V. and I.F. thank FCT for, respec-
tively, Post-Doc Grant SFRH/BPD/48345/2008 and Ph.D. Grant
SFRH/BD/38883/2007.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.10.081.
Imidazoquines like 3a had already been found to display inter-
esting antimalarial and anti-pneumocystic activities.16,17,19 Rele-
vantly, such activities were consistently higher for those
compounds derived from smaller amino acids (Gly or Ala, i.e., R1
and/or R2 = H or Me), as is the case of 3a. Moreover, the in vitro
References and notes
1. Rosenthal, P. J.. In Antimalarial Chemotherapy: Mechanisms of Action, Resistance
and New Directions in Drug Discovery; Humana Press: Totowa, NJ, 2001.
2. Vale, N.; Moreira, R.; Gomes, P. Eur. J. Med. Chem. 2009, 44, 937.
3. Suryanaryana, V.; Meenakshi, J.; Kirandeep, K.; Premanand, P.; Sanjay, R. P.;
Rahul, J. Med. Res. Rev. 2007, 27, 65.
4. Hoglund, I. P.; Silver, S.; Engstrom, M. T.; Salo, H.; Tauber, A.; Kyyronen, H. K.;
Saarenketo, P.; Hoffren, A. M.; Kokko, K.; Pohjanoksa, K.; Sallinen, J.; Savola, J.
M.; Wurster, S.; Kallatsa, O. A. J. Med. Chem. 2006, 49, 6351.
5. Zhou, Q.; Melkoumian, Z. K.; Lucktong, A.; Moniwa, M.; Davie, J. R.; Strobl, J. S. J.
Biol. Chem. 2000, 275, 35256.
6. Martirosyan, A. R.; Rahim-Bata, R.; Freeman, A. B.; Clarke, C. D.; Howard, R. L.;
Strobl, J. S. Biochem. Pharmacol. 2004, 68, 1729.
7. Mahajan, A.; Yeh, S.; Nell, M.; van Rensburg, C. E. J.; Chibale, K. Bioorg. Med.
Chem. Lett. 2007, 17, 5683.
8. Shi, A.; Nguyen, T. A.; Battina, S. K.; Rana, S.; Takemoto, D. J.; Chiang, P. K.; Hua,
D. H. Bioorg. Med. Chem. Lett. 2008, 18, 3364.
9. Gunjan, G.; Takahiro, O.; Aibin, S.; Duy, H. H.; Thu Annelise, N. Drug Dev. Res.
2008, 69, 526.
10. Jones, M.; Mercer, A. E.; Stocks, P. A.; La Pensée, L. J. I.; Cosstick, R.; Park, B. K.;
Kennedy, M. E.; Piantanida, I.; Ward, S. A.; Davies, J.; Bray, P. G.; Rawe, S. L.; Baird,
J.; Charidza, T.; Janneh, O.; O’Neill, P. M. Bioorg. Med. Chem. Lett. 2009, 19, 2033.
11. Rosenthal, A. S.; Chen, X.; Liu, J. O.; West, D. C.; Hergenrother, P. J.; Shapiro, T.
A.; Posner, G. H. J. Med. Chem. 2009, 52, 1198.
12. Araújo, M. J.; Bom, J.; Capela, R.; Casimiro, C.; Chambel, P.; Gomes, P.; Iley, J.; Lopes, F.;
Morais, J.; Moreira, R.; de Oliveira, E.; do Rosario, V.; Vale, N. J. Med. Chem. 2005, 48,
888.
13. Chambel, P.; Capela, R.; Lopes, F.; Iley, J.; Morais, J.; Gouveia, L.; Gomes, J. R. B.;
Gomes, P.; Moreira, R. Tetrahedron 2006, 62, 9883.
14. Ferraz, R.; Gomes, J. R. B.; de Oliveira, E.; Moreira, R.; Gomes, P. J. Org. Chem.
2007, 72, 4189.
15. Gomes, P.; Araújo, M. J.; Rodrigues, M.; Vale, N.; Azevedo, Z.; Iley, J.; Chambel,
P.; Morais, J.; Moreira, R. Tetrahedron 2004, 60, 5551.
16. Vale, N.; Collins, M. S.; Gut, J.; Ferraz, R.; Rosenthal, P. J.; Cushion, M. T.;
Moreira, R.; Gomes, P. Bioorg. Med. Chem. Lett. 2008, 18, 485.
17. Vale, N.; Matos, J.; Gut, J.; Nogueira, F.; do Rosario, V.; Rosenthal, P. J.; Moreira,
R.; Gomes, P. Bioorg. Med. Chem. Lett. 2008, 18, 4150.
activity of imidazoquines
3 may become more pronounced
in vivo due to their expectedly higher oral bioavailability as com-
pared to 1, 6, and 7.
In view of the above, we believe that imidazoquines like 3a may
constitute useful leads as multi-drugs against malaria, pneumocy-
stic pneumonia (PCP), and breast cancer. Obviously, the develop-
ment of novel anticancer agents is of undeniable value worldwide.
Though breast cancer prevails in North-Western and temperate
regions of the World, with relatively low incidence on African,
South-American, and East-Asian regions where malaria is
endemic,31 recent reports account for a significant increase of
incidence in young Asian women32 and to alarming mortality rates
among African women.33 So, we believe that discovery of multi-tar-
get drugs effective against cancer, malaria, and opportunistic infec-
tions like PCP, which may be deadly for HIV-infected persons, is
especially relevant for those regions of the globe.
Finally, we must again outline that the mechanisms of both
antimalarial and anti-proliferative action of quinolines are not fully
understood. 4-Aminoquinolines, like chloroquine, are likely to
exert their antimalarial action by impairment of hemozoin crystal-
lization (the mechanism through which blood-stage parasites get
rid of heme after digestion of host’s hemoglobin), which explains
why is chloroquine active only against intraerythrocytic para-
sites.34 In what concerns chloroquine’s anti-tumoral activity,
DNA intercalation-induced apoptosis has been proposed35 but is
still controversial.36
18. Vale, N.; Nogueira, F.; do Rosário, V. E.; Gomes, P.; Moreira, R. Eur. J. Med. Chem.
2009, 44, 2506.
In the case of 8-aminoquinolines like primaquine (and, proba-
bly, imidazoquines 3), which are not active against intraerythro-
cytic parasites and exert an antimalarial role complementary to
that of 4-aminoquinolines, the mechanism of action is certainly
quite different. So far, studies on primaquine’s mode of action
suggests a key role for active quinone metabolites behind inter-
ference with the mitochondrial respiratory chain, as swelling of
parasite’s mitochondria and collapse of the mitochondrial mem-
brane potential have been observed.2,34 Further, primaquine
19. Vale, N.; Prudêncio, M.; Marques, C. A.; Collins, M. S.; Gut, J.; Nogueira, F.;
Matos, J.; Rosenthal, P. J.; Cushion, M. T.; do Rosário, V. E.; Mota, M. M.;
Moreira, R.; Gomes, P. J. Med. Chem. ahead-of-print (doi:10.1021/jm900738c).
20. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.;
Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82,
1107.
21. Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paul, K.; Vistica, D.; Hose, C.;
Langley, J.; Cronise, P.; Vaigro-Wolff, A., et al J. Natl. Cancer Inst. 1991, 83, 757.
22. http://www.vcclab.org/lab/alogps.
23. http://www.organic-chemistry.org/prog/peo.
24. Hall, H. K. J. Am. Chem. Soc. 2002, 79, 5441.

I. Fernandes et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6914–6917
6917
25. Dawson, R. M. C.; Elliott, D. C.; Elliott, W. H.; Jones, K. M. Data for Biochemical
34. Foley, M.; Tilley, L. Pharmacol. Ther. 1998, 79, 55.
Research; Oxford University Press, 1959.
35. Zhou, Q.; McCracken, M. A.; Strobl, J. S. Breast Cancer 2002, 75, 107.
26. Elliott, J. J.; Mason, S. F. J. Chem. Soc. (Resumed) 1959, 2352.
27. Santos, C.; Morais, J.; Gouveia, L.; De Clercq, E.; Pannecouque, C.; Nielsen, C. U.;
Steffansen, B.; Moreira, R.; Gomes, P. ChemMedChem 2008, 3, 970.
28. Santos, C. R.; Capela, R.; Pereira, C. S. G. P.; Valente, E.; Gouveia, L.;
Pannecouque, C.; De Clercq, E.; Moreira, R.; Gomes, P. Eur. J. Med. Chem.
2009, 44, 2339.
29. Portela, M. J.; Moreira, R.; Valente, E.; Constantino, L.; Iley, J.; Pinto, J.; Rosa, R.;
Cravo, P.; do Rosario, V. E. Pharm. Res. 1999, 16, 949.
30. Handbook of Preformulation: Chemical, Biological, and Botanical Drugs; Niazi, S.
K., Ed.; CRC Press, 2006.
36. Savarino, A.; Lucia, M. B.; Giordano, F.; Cauda, R. Lancet Oncol. 2006, 7, 792.
37. Vazquez-Vivar, J.; Augusto, O. Free Radical Res. 1990, 7, 383.
38. Šimunovic´, M.; Perkovic´, I.; Zorc, B.; Ester, K.; Kralj, M.; Hadjipavlou-Litina, D.;
Pontiki, E. Bioorg. Med. Chem. 2009, 17, 5605.
39. Pugh, C. W.; Gleadle, J. I.; Maxwell, P. H. Breast Cancer Res. 2001, 3, 313.
40. Knowles, H. J.; Harris, A. L. Breast Cancer Res. 2001, 3, 318.
41. Williams, K. J.; Cowen, R. L.; Stratford, I. J. Breast Cancer Res. 2001, 3, 328.
42. Van den Eynden, G. G.; Van der Auwera, I.; Van Laere, S. J.; Colpaert, C. G.;
Turley, H.; Harris, A. L.; Van Dam, P.; Dirix, L. Y.; Vermeulen, P. B.; Van Marck, E.
A. Br. J. Cancer 2005, 93, 1128.
31. Parkin, D. M. Lancet Oncol. 2001, 2, 533.
32. Yip, A. Y.; Chow, L. W. Breast Cancer 2006, 13, 192.
33. Parkin, D. M.; Leticia, M. G. F. Breast J. 2006, 12, S70.
43. Kim, J.-W.; Gao, P.; Dang, C. V. Cancer Metast. Rev. 2007, 26, 291.
44. Milani, M.; Harris, A. L. Eur. J. Cancer 2008, 44, 2766.
45. Gago-Domíngeuz, M.; Jiang, X.; Castelao, J. E. Breast Cancer J. 2007, 9, 201.