Antitrypanosomal and Antiplasmodial Activity of Bis(2-aminoimidazoline) and Bisguanidine Derivatives

Christophe Dardonvillea (dardonville@iqm.csic.es), Lidia Nietoa, Fernando Rodríguezb, Isabel Rozasb, Marcel Kaiserc, Reto Brunc, Binh Nguyend, W. David Wilsond, Rory Nelson Garcíae

a Instituto de Química Médica, CSIC, Juan de la Cierva 3, E–28006 Madrid, Spain; b Centre for Synthesis and Chemical Biology, School of Chemistry, Trinity College Dublin, Dublin 2, Ireland; c Swiss Tropical Institute, Socinstrasse, 57, CH-4002 Basel, Switzerland; d Department of Chemistry, Georgia State University, Atlanta, GA

30303-3083, USA; e Departamento de Parasitología, Facultad de Farmacia, Universidad Complutense de Madrid, Av. complutense s/n, E-28040 Madrid, Spain.

Antecedents: Recent findings by our group have shown that bisguanidine and especially bis(2-aminoimidazoline)diphenyl compounds displayed potent antitrypanosomal activity in vitro1 and vivo2 against T. b. rhodesiense, the causative agent of acute human African trypanosomiasis (HAT). In addition, a correlation between antitrypanosomal activity and DNA binding affinity was observed, suggesting a possible mechanism of action for these compounds.2 We also showed that this kind of molecules entered into trypanosomes via different transporters in addition to P2, indicating that parasites that have lost the P2 transporter in selection of resistance to other drugs will not show cross-resistance to this class of molecules.

Others have described excellent antiplasmodial activity of related aromatic dicationic structures such as pentamidine or DB289.3 This prompted us to test our compounds against P. falciparum as well.

Acknowledgements: This work was supported by the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases (RB), PIE grant 200680I121 from the CSIC (CD, LN), the “Programa Nacional de Biomedicina” SAF2006-04698 grant from the Spanish “Ministerio de Educación y Ciencia” (RNG), Cycle III HEA PRTLI grant by means of the CSCB in Ireland,  and by a grant of the Consejeria de Educacion Cultura y Deporte de la Comunidad Autonoma de La Rioja, Spain (F.R.).

References: [1] Dardonville, C.; Brun, R. J Med Chem 2004, 47, 2296. [2] Dardonville, C.; Barrett, M. P.; Brun, R.; Kaiser, M.; Tanious, F.; Wilson, W. D. J Med Chem 2006, 49, 3748. [3] Soeiro, M. N. C.; De Souza, E. M.; Stephens, C. E.; Boykin, D. W. Expert Opin Inv Drug 2005, 14, 957. [4] Rodriguez, F.; Rozas, I.; Ortega, J.E.; Meana, J.J.; Callado, L.F. J. Med. Chem., 2007 (submitted). [5] Deharo, E.; Garcia, R. N.; Oporto, P.; Gimenez, A.; Sauvain, M.; Jullian, V.; Ginsburg, H. Exp Parasitol 2002, 100, 252.

R

X

R

X 1

23

4

56

Y

R

4'R

1-12 13-14

1) In vitro antiprotozoal activity [Table 1]:

Antitrypanosomal activity: 9 new compounds with IC50 < 100 nM. Best anti-T. brucei agent in vitro: bis(2-aminoimidazoline)fluorene 13b. Antiplasmodial activity: 20 dicationic compounds with IC50 < 50 nM. Best antimalarial compound in vitro: 1b and 13b. Two cations are essential for good activity (data not shown) Boc-protected derivatives (1d, 1e, 6e) show excellent in vitro antimalarial activity.

Conclusions: Bis(2-aminoimidazoline) compounds are promising anti-T. brucei and antimalarial agents with better safety profile vs bisguanidine counterparts. Binding to DNA minor groove possibly plays a role in the antiprotozoal activity. Inhibition of heme biosynthesis may be responsible to some extent for theobserved antiplasmodial activity. In vivo assays are ongoing.

Table 1. In vitro antitrypanosomal, antiplasmodial and DNA binding activity of diphenyl dicationic compounds.

a Gua = guanidinium, Imi = 2-aminoimidazolinium, (Boc)Gua = N,N’-di(tert-butoxycarbonyl)guanidine, (Boc)Imi = N,N’-di(tert-butoxycarbonyl)imidazolidin-2-yl; b T. brucei rhodesiense STIB900 strain. Control: melarsoprol, IC50 = 0.0055 M; c P. falciparum K1 strain. Control: chloroquine, IC50 = 0.278 M; d Data taken from ref. 1; e Data taken from: Arafa et al. J. Med. Chem. 2005, 48, 5480.

IC50 (M) IC50 (M) Tm (ºC) Cpd Ra X Y T.b.r.b SI P.f. c SI poly(dA•dT)2

1a Gua - 0.022d 30 0.018 36 1b Imi - 0.069d 3072 0.0088 24091 1c (EtO)2CHCH2-Gua NH - 0.228d >767 0.113 > 1548 1d (Boc)Gua - 0.470d 8 0.077 48 not soluble

1e (Boc)Imi - 0.048d 204 0.059 166 not soluble

2a Gua - 0.161d 17 0.032 88 2b Imi CH2 - 0.897d 71 0.0157 4051 2c (EtO)2CHCH2-Gua - 0.316d >553 0.036 > 4861 3a Gua O - 0.196 8 0.046 36 2.0 3b Imi - 0.467 88 0.038 1079 4.3 4a Gua S - 0.102 26 0.035 76 2.2 4b Imi - 0.386 60 0.025 932 3.3 5a Gua - 0.206d 13 0.068 40 5b Imi - 2.05d >104 0.129 > 1658 5f CH2P

+(n-pentyl)3

O

- 0.414d 29 0.053 223 6a Gua - 4.3d > 51 0.444 > 500 6b Imi - 32.4d - 5.6 - 6e (Boc)Imi

SO O

- 2.6d 22 0.055 1025 7a Gua - 0.187 > 1256 0.096 > 2547 1.5 7b Imi N

HNH

O

- 0.122 852 0.028 3714 4.1

8a Gua NH

NH

S

- 0.538 29 0.607 26 1.4

9a Gua - 0.036 319 0.055 209 15.0 9b Imi N

H

O

- 0.025 7720 0.028 6893 18.2 10a Gua - 0.045 24 0.019 57 4.1 10b Imi - 0.054 646 0.016 2181 6.1 11a Gua - 0.217 111 0.041 585 3.1 11b Imi

N - 0.038 3474 0.011 12000 4.8

12a Gua - 0.270d 172 0.0152 3053 12b 4,4’-Imi

N N - 0.118d 881 0.0123 8455

13a 4,4’-Gua nil CH2 0.024e 196 0.0023e 2043 13b 4,4’-Imi nil CH2 0.0049 17020 0.0115 7252 18.1 14a 5,4’-Gua CH2 CH2 0.050 15 0.0088 83 4.1 14b 5,4’-Imi CH2 CH2 0.060 620 0.0186 2000 5.2

3) Possible mechanism of action [Table 2]: DNA binding: -Significant binding to A-T sites for several compounds: 1a, 1b, 5a,

12a, 12b, 9a, 9b, 13b.-Tm (4,4’-Imi) > Tm (4,4’-Gua) in all cases.

FBIT: - Some compounds show FPIX biomineralization inhibition similar to quinine- There is no correlation between FPIX inhibition and antimalarial activity.

2) Selectivity index (safety profile) [Table 1]: Higher selectivity for the 2-aminoimidazolinium cation vs guanidinium.

Aim of the study:

1) In vitro evaluation of new series of bis(2-aminoimidazolines) and their bisguanidine counterparts4 against T. b. rhodesiense (STIB900) and P. falciparum (K1). 2) Evaluation of the selectivity index (SI = IC50 mammalian L6-cells/ IC50 parasite). 3) Study of the mechanism of action:

- DNA binding at AT-sites (Tm). - Ferriprotoporphyrin IX binding inhibition test (FBIT).5

Table 2. Inhibition of FPIX biomineralisation

a The percentage of inhibition at 1 mg/mL (highest concentration tested)is given when the IC50 could not be determined. b Taken from ref. 4.

Inhibition FPIX Compound IC50 (M)a

Quinine 324b Chloroquine 17.9

1a 0% 1b 429.2 1c 161.8 1d 45% 1e 170.7 2a 0% 2c 15% 5b 117.2 5f 219.2 6a 42% 6e 27%

12b 127.5