Diamine and aminoalcohol derivatives active against Trypanosoma brucei

Bioorganic & Medicinal Chemistry Letters 22 (2012) 440–443

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Diamine and aminoalcohol derivatives active against Trypanosoma brucei Esther del Olmo a,⇑, Rosario Diaz-González b, Ricardo Escarcena a, Luis Carvalho b, Luis A. Bustos a, Miguel Navarro b,⇑, Arturo San Feliciano a a b

Departamento de Química Farmacéutica, Facultad de Farmacia – CIETUS, Universidad de Salamanca, Campus Unamuno, E-37007 Salamanca, Spain Instituto de Parasitología y Biomedicina ‘‘Lopez Neyra’’, CSIC, Avda. del Conocimiento, E-18100 Granada, Spain

a r t i c l e

i n f o

Article history: Received 22 September 2011 Revised 28 October 2011 Accepted 30 October 2011 Available online 6 November 2011 Keywords: Diamines Aminoalcohols Trypanocide Sleeping sickness

a b s t r a c t Twenty compounds selected as representative members of three series of long-chain 1,2-diamines, 2-amino-1-alkanols and 1-amino-2-alkanols structurally related to dihydrosphingosin, were synthesized and tested in vitro for their ability to inhibit the sleeping sickness parasites Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Eight compounds showed EC50 values in the submicromolar range, with selectivity indexes up to 39 related to the respective cytotoxicity values for Vero cells. The parasite phenotype detected after treatment with the most potent compounds showed irreversible cell morphology alterations of the flagellar pocket that lead to inhibition of cell growth and parasite death. Ó 2011 Elsevier Ltd. All rights reserved.

Trypanosoma brucei is the causal agent of Human African Trypanosomiasis (HAT), a vector-borne neglected disease transmitted by insects of Glossina genus, the tsetse fly. Also known as sleeping sickness, the disease is distributed among 36 sub-Saharan countries, with more than 7000 new cases reported in 2009 and more than 60 million people at risk.1 The clinical manifestation of HAT depends on the sub-species involved. T. b. rhodesiense is responsible of the acute form of the disease, mainly distributed in Eastern and Southern Africa, whereas in West and Central Africa, T. b. gambiense, is responsible of chromic infections that may develop symptoms years later. Wild animals, as well as livestock, are reservoirs of the HAT for both species, being an obstacle to eradicate this disease. Evolution of infection begin with an early stage or haemolymphatic phase, where the parasite replicates in blood and body fluids, provoking lymphadenophathy and fever, followed by a second stage where the infectious cells cross the blood–brain barrier causing neurologic symptoms and if untreated, patients would fall into coma and die.2 Drugs used in the present time exhibit shortage in healing and potent toxicity.3 Vaccination is hampered by antigenic variation, a process developed by the parasite that changes the glycoprotein surface eluding the host immune response. Thus, it is evident the need of developing new drugs and therapeutic strategies to improve efficacy and reduce toxicity in treatments. Success in the treatment of HAT is conditioned to an early diagnosis, since current pharmacological formulations that get through ⇑ Corresponding authors. E-mail address: [email protected] (E.del Olmo). 0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2011.10.108

the blood–brain barrier have very high toxic side effects and complicated administration. The therapeutic arsenal to treat HAT is scarce, mainly based on a few old drugs, which were donated to the WHO by the manufacturers Sanofi-Aventis and Bayer. Pentamidine isethionate and suramin are used, respectively, against the first stage of T. b. gambiense and T. b. rhodesiense disease. The very toxic melarsoprol is used against the second stage HAT caused by T. b. gambiense. Eflornithine is safer than melarsoprol and was recommended as first line treatment for second stage HAT caused by T. b. gambiense, but recently clinical trials showed that a nifurtimox–eflornithine combination therapy presents advantages over the eflornithine monotherapy. Unfortunately, T. b. rhodesiense is resistant to eflornithine so actually the only choice against the second stage is melarsoprol. Due to the lack of really safe and efficacious drugs, the discovery and development of better and cheaper new compounds against HAT is needed.2,4 In an attempt to discover new types of therapeutic agents against T. brucei, along with several series of fused heterocyclic compounds, we prospectively evaluated a number of aliphatic 1,2-diamine, 1-amino-2-alkanol and 2-amino-1-alkanol derivatives (Fig. 1), structurally considered as simplified sphingosin pseudo-analogues. Preliminarily, a small library of 20 compounds representative of the three series were selected for evaluation on the basis of previous bioactivity results found for this type of compounds against Mycobacterium tuberculosis5 and Leishmania spp. parasites6 as well as, those found for a collection of cyclic analogues tested against Leishmania spp. and Trypanosoma cruzi.7 The preparation of the 1,2-diamine and 2-aminoalkan-1-ol derivatives being assayed was previously reported by us.5,6,8,9

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series, all the compounds were assayed in vitro for 72 h against the human-infective strain T. b. rhodesiense EATRO3, grown and tested as bloodstream form,12 with various concentrations of the different test compounds, according to a reported procedure based on the Alamar Blue assay13 and covering a range from 0.05 to 2.5 lM.14 The 50% effective concentration (EC50) value of each compound was determined and the results are shown in Table 1. Cells cultured without any compound, incubated with the calculated proportion of DMSO used as solvent of the samples served as control and pentamidine was used as reference drug showing an EC50 value of 3.7 nM against T. b. rhodesiense, a value significantly lower than that previously reported by Bodley and Shapiro.15 As it can be seen in Table 1 the three series of compounds tested contain representative elements displaying anti-trypanosomal effects with submicromolar EC50 values, though no one attained the potency of the reference drug. In the case of those 1,2alkanediamine derivatives 1 to 5, two compounds attained the submicromolar level for their EC50 values, the ethylamino/Boc (t-butoxycarbonyl) derivative 2 resulting as the most potent trypanocide within those compounds evaluated of this series. It can be observed, though being not enough sure for this short group, that the presence of the Boc substituent on N2 (compounds 1 and 2) seems comparatively useful for enhancing the activity, while the increase of size or polarity of the substituent on N1 (compounds 3, 4 and 5) fairly lead to lowering the antiparasitic potency. Five members of the series of 2-amino-1-alkanol derivatives (compounds 6–13), also attained micromolar or submicromolar levels in their respective EC50 values. For this series the preferred substituent on N2 was also the ethyl group and the comparison

Y-R2 2

H3C

1

(CH2)n

X-R1

X, Y = O, NH, N, n = 9, 13, 15. R1,R2 = H, Et, n-Bu, n-Pent, n-Hex, cyclohex, Bn, 4-Me-piperidin-1-yl, 4-Bu-piperidin-1-yl, morpholin-1-yl, Boc, HOOC-(CH2)2(3)CO-. Figure 1. Global structure and variants of the evaluated compounds.

R1R2NH

H3C (CH2)13

R1

OH

O

MeOH, reflux

H3C

N R2

(CH2)13

Scheme 1. The synthesis of 1-amino-2-alkanol derivatives.

Compounds of the new series, with inverted positions for the amino and the hydroxyl functions to mimic positions 2 and 3 of dihydrosphingosin, were easily prepared by nucleophilic attack of 1,2-epoxyhexadecane with the appropriate alkyl-, dialkyl- or heterocyclic amine derivative (Scheme 1). Examples of preparation and characterisation by physical and spectral data for compounds of the new series are given in the references and notes section.10,11 After a prospective preliminary assay of parasite growth inhibition, at 1 and 100 lM concentrations of the compounds, that served to reveal the anti-trypanosomal potential of the three

Table 1 Activity of long-chain diamine and aminoalcohol derivatives against Trypanosoma brucei rhodesiense EATRO3

Y-R2 2

H3C

1

X-R1

(CH2)n

Compda

X

Y

R1

R2

n

T. brucei rhodesiense EC50 (nM) ± SD

1 2 3 4

NH NH NH NH

NH NH NH NH

H Et n-Hex HO2CCH2–

Boc Boc Boc H

13 13 13 13

961 ± 67 704 ± 55 >2500 >2500

5 6 7 8 9 10 11 12

NH O O O O O O O

NH NH NH NH NH NH NH N

HO2C(CH2)2CO– H H H H H H H

Boc H Et Et Et n-Bu HO2C(CH2)3CO– Et2

13 13 9 13 15 13 13 13

>2500 >2500 1087 ± 88 1139 ± 26 584 ± 14 817 ± 57 >2500 722 ± 58

13 14 15 16 17 18 19 20

O NH NH NH N NH NH NH

NH O O O O O O O

Bn n-Hex Cyclohexyl 2-Propyl Et2 4-Me-piperazin-1-yl 4-Bu-piperazin-1-yl Morpholin-1-yl

HO2C(CH2)2CO– H H H H H H H

13 13 13 13 13 13 13 13

>2500 >2500 790 ± 36 436 ± 4 2001 ± 95 524 ± 12 1781 ± 194 >2500

Pentamidine

3.7 ± 0.4

Suramin

185.3 ± 8.5

EC50 values under 1 lM are bolded. a All the compounds were obtained as racemic mixtures and evaluated per triplicate.

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of potencies corresponding to three different aliphatic chain lengths denoted a potency maximum for the compound with a C18-chain total size, that could correlate well with the fact that this series of compounds is structurally closer to dihydrosphingosin than the first series of 1,2-diamines. Indeed, the most potent trypanocidal agent of this group, compound 9, corresponds to 2-methylaminopalmitol, which can also be recognised as a dihydrosphingosin analogue, namely N-ethyl-3-deoxy-17,18-dinor-dihydrosphingosin. Dialkylation of N2 tends to reduce the activity, as it does the increase of size from ethyl to n-butyl in the N2-substituent. Related to the series of 1-amino-2-alkanol derivatives, the inverted dihydrosphingosin analogues, three other compounds attained the submicromolar level in their EC50 values. No structure–activity comparison can be done within this group due to the scarce number of compounds and the varied substituents present. Nevertheless, certain homogeneity in size and/or volume can be detected for the N1-substituents, isopropyl, 4-methylpiperazinyl or cyclohexyl groups, respectively, present in the three most potent compounds 16, 18 and 15 of this series. In spite of the difference in the measurement units (EC50 vs MIC100), it is interesting to note the relative trypanocidal potency showed by compounds 1, 2, 9, 10 and 15 against T. b. rhodesiense (EC50 = 0.96, 0.70, 0.58, 0.82 and 0.79 lM, respectively) in comparison with their previously reported effects5 against Mycobacterium tuberculosis (H37Rv strain: MIC = 30, 32, 16.6, 30 and 33 lM, respectively). This finding would point towards the existence of an appreciable selectivity of these compounds against the parasite. In order to establish the intrinsic toxicity of these compounds a selection of them, representative of the three series, was assayed on Vero cells, normal kidney cells of an African green monkey, and the results were used to calculate the respective selectivity inTable 2 Calculated selectivity indexes and comparative activity of compounds 9, 16 and 18 against T. b. gambiense ELIANE. Compd

1 2 8 9 16 17 18

Selectivity Index (SI)a

EC50 (nM) T. b. rhodesiense

T. b. gambiense

T. b. rhodesiense

T. b. gambiense

961 ± 67 704 ± 25 1139 ± 26 584 ± 14 436 ± 04 2001 ± 95 524 ± 12

nt nt nt 451 ± 27 329 ± 13 nt 431 ± 25

17.0 38.8 39.1 27.7 27.1 19.9 23.7

— — — 35.9 35.8 — 28.8

EC50 values under 1 lM are bolded. a Calculated with the equation: SI = EC50 (Vero cells)/EC50 (T. brucei). nt, not tested; —, not calculated.

dexes, which ranged within the 17–39 interval (Table 2). Previous work described a much higher SI value of 1945 for melarsoprol,16 thus indicating that compounds reported here deserve further structure optimisation, though they can be considered as valuable new hits according to DNDI (Drugs for Neglected Disease Initiative) recommendations (EC50 < 1 lM and SI > 10). Additionally, aiming to further prospect on the usefulness and the real spectrum against HAT, the three most potent compounds against T. b. rodhesiense, 9, 16 and 18 were also tested against T. b. gambiense, showing 20–30% better activity results than against T. b. rhodesiense (Table 2). This fact could be of interest due to the fair lipidic nature of these compounds, with log P values higher than 5 (calculated for 18:5.3, for 16:5.8 and for 9:6.3) that ensure their ability to cross the blood/brain barrier and would further support their potential use during the neurological phase of chronic HAT induced by T. b. gambiense. In order to attain some insight into the mechanism and mode of action of the compounds, images of parasites after 24 h treatment were obtained. The marked differences observed with respect to untreated parasites are shown in Figure 2. As it is seen in the picture on the right, the 2-ethylaminostearyl alcohol (9) induced a substantial morphological alteration of the flagellar pocket structure, in the bloodstream form of the parasite. Similar results were obtained with the inverted 1-aminoalkan-2-ol derivatives 16 [1-(2-propylamino)hexadecan-2-ol] and 18 [1-(4methylpiperazin-1-yl)hexadecan-2-ol]. The alteration known as the ‘Big Eye’ phenotype, denotes a considerable enlargement of the flagellar pocket, a small invagination of the plasma membrane, where important processes take place. 17Thus, membrane trafficking occurs exclusively toward this region and inhibition of endocytosis leads to excessive growth of the flagellar-pocket membrane resulting in this characteristic phenotype.18 This is probably the best understood morphological defect described in trypanosomes. Endocytosis in T. brucei is an essential process for the infective bloodstream form, therefore the big eye phenotype generated by treatment with these compounds invariably leads to cell death.19 In summary, the spectrum amplitude against both types of HAT and the modest to good selectivity indexes shown by these series and their most representative compounds, have provided several promising lead compounds to continue the research. Future investigations will be focused on expanding the number of molecules to be evaluated and on establishing the actual mechanism of action, including target molecules and the affected metabolic pathways in the parasite. The morphological results shown in the pictures (Fig. 2) will probably help us for the target characterisation. In addition, the new series will include structural optimisation to improve substantially the selectivity index and to make further

Figure 2. Alteration of the flagellar pocket structure of Trypanosoma brucei rhodesiense by compound 9 visualised in live cells. Left: untreated parasite culture; right: treated with 9 (2 M, 24 h, images captured using Normarski optics).

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considerations about formulation, acute toxicity and in vivo preclinical assays and pharmacokinetic aspects. Acknowledgements Collaborative research performed under the auspices of Network RICET (ISCIII) (RD06/0021, Groups: 0022 and 0010). RE and RD thank their respective research contract financed by ISCIII-RICET. LAB thanks the PhD fellowships received from USAL-B. Santander and Becas-Chile. Financial support from FIS-PI-060782, MEC-AGL 2005-02168 and SAF2009-07587, is also acknowledged. References and notes 1. WHO. Fact sheet N 259. October 2010. African trypanosomiasis (sleeping sickness). 2. Malvy, D.; Chappuis, F. Clin. Microbiol. Infect. 2011, 17, 986. 3. Barrett, M. P.; Boykin, D. W.; Brun, R.; Tidwell, R. R. British J. Pharmacol. 2007, 152, 1155. 4. Burri, C. Parasitology 2010, 137, 1987. 5. Del Olmo, E.; Molina-Salinas, G. M.; Escarcena, R.; Alves, M.; Hernández-Pando, R.; López-Pérez, J. L.; Said-Fernández, S.; San Feliciano, A. Bioorg. Med. Chem. Lett. 2009, 19, 5764. 6. Del Olmo, E.; Alves, M.; López, J. L.; Inchaustti, A.; Yaluff, G.; Rojas de Arias, A.; San Feliciano, A. Bioorg. Med. Chem. Lett. 2002, 12, 659. 7. Rebollo, O.; del Olmo, E.; Ruiz, G.; López-Pérez, J. L.; Giménez, A.; San Feliciano, A. Bioorg. Med. Chem. Lett. 2008, 18, 184. 8. Lucas, R.; Úbeda, A.; Payá, M.; Alves, M.; del Olmo, E.; López, J. L.; San Feliciano, A. Bioorg. Med. Chem. Lett. 2000, 10, 285. 9. Del Olmo, E.; Plaza, A.; Muro, A.; Martínez-Fernández, A. R.; Nogal-Ruiz, J. J.; López-Pérez, J. L.; San Feliciano, A. Bioorg. Med. Chem. Lett. 2006, 16, 6091. 10. Preparation of compound 14: To a stirring solution of 1,2-epoxyhexadecane [500 mg, 2.0 mmol] in 10 mL of dry methanol, 221 lL (2.0 mmol) of hexylamine was added. The mixture was refluxed for 4 h followed by 20 h a room temperature. Reaction evolution was controlled by TLC. The methanol was removed under vaccum, and the residue dissolved in ethyl acetate (30 mL), washed with 2 N HCl (2  25 mL), and then with sat. NaHCO3(aq) (3  25 mL), and the organic layer dried over Na2SO4 to provide a solid reaction crude that was purified by column chromatography (9:1 CH2Cl2/CH3OH) to give 466 mg (65%) of a white solid mp: 67–69 °C; HRMS: Calcd: 342.3736; found: 342.3721 (M+H+); IR: 3268, 2953, 2919, 2853, 1468, 1121, 923 cm 1. Compounds 15, 16, 17, 18, 19 and 20 were obtained under similar conditions in 71, 69, 46, 65, 70 and 60% yield, respectively.

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11. Compound 15: 1-cyclohexylaminohexadecan-2-ol. Mp = 68 °C; EM: m/z 338 (M+ H); 296 (M+ C2H3O); 266(M+ C7H13N); 256 (M+ C6H11); 196 (C14H28); 142 (C8H16ON); 112 (M+ C16H31O); 83 (C6H4). IR (NaCl):mmax 3382, 2918, 2850, 1455, 1215,1010 cm 1. 1H NMR (200 MHz, CDCl3, d): 0.89 (3H, t, J = 6.6 Hz, H16), 1.26 (26H, b s), 1.43 (2H, m), 1.61 (2H, m), 1.93 (2H, m), 2.41 (1H, dd, J = 12.0, 9.1 Hz, H-1a), 2.42 (1H, m, H-1´), 2.81 (1H, dd, J = 12.0, 2.9 Hz, H-1b), 3.58 (1H, m, H-2). 13C NMR (50 MHz, CDCl3, d ppm) 14.2; 22.7; 25.1; 25.8; 26.1; 29.4; 29.7; 32.0; 33.3; 33.8; 35.2; 52.3; 56.7; 69.7. Compound 18: 1-(4-methylpiperazin-1-yl)hexadecan-2-ol. Mp = 38–39 °C; HRMS: Calcd: 341.3454; found: 341.3524 (M+H+); IR (NaCl): mmax: 3462, 2921, 2840, 2797, 1459, 1288, 1161, 1113, 1077, 812 cm 1. 1H NMR (200 MHz, CDCl3, d): 0.86 (3H, t, J = 7.0 Hz), 1.23 (26H, b s); 2.23 (1H, dd, J = 12.2, 3.2 Hz); 2.28 (3H, s); 2.32 (1H, dd, J = 12.2, 2.0 Hz); 2.44 (4H, m); 2.69 (4H, m); 3.63 (1H, m) ppm. 13C NMR (50 MHz, CDCl3, d): 14.0; 22.6; 25.5; 29.3; 29.5; 29.6; 29.7; 31.9; 34.9; 45.9; 53.1; 55.1; 64.1; 66.1 ppm. 12. Cell cultures: Human-infective strains Trypanosoma brucei rhodesiense EATRO3 and T.b. gambiense ELIANE were grown in HMI-9 medium (Invitrogen) supplemented with 20% heat-inactivated fetal bovine serum (HIFBS) (Invitrogen) at 37 °C with 5% CO2. Both Trypanosoma strains were grown and tested as bloodstream forms. Vero cells, derived from the kidney of an African green monkey, were similarly grown in Dulbecco’s modification of Eagle medium (DMEM) (Invitrogen) supplemented with 10% HIFBS at 37 °C with 5% CO2. 13. Räz, B.; Iten, M.; Grether-Bühler, Y.; Kaminsky, R.; Brun, R. Acta Trop. 1997, 68, 139. 14. In vitro antitrypanosomal and cytotoxicity assays: T. b. rhodesiense EATRO3 (2  103 cells/ml) and T. b. gambiense ELIANE (2  104 cells/ml) were incubated in 96-well plates with concentrations covering a range from 0.05 to 2.5 lM of the compounds, for 72 h at 37 °C with 5% CO2 in culture medium. Vero cells (5  104 cells/ml) were incubated in 96-well plates with various concentrations (covering a range from 4 to 35 lM) of the different compounds for 72 h at 37 °C with 5% CO2 in culture medium. The 50% effective/cytotoxic concentration (EC50/CC50) value of the different compounds was determined by the Alamar Blue assay.12 Fluorescence was recorded with an Infinite F200 microplate reader (Tecan Austria GmbH, Austria) equipped with 550 and 590 nm filters for excitation and emission wavelengths, respectively. 15. Bodley, A. L.; Shapiro, T. A. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 3726. 16. Bakunova, S. M.; Bakunov, S. A.; Patrick, D. A.; Kumar, E. V.; Ohemeng, K. A.; Bridges, A. S.; Wenzler, T.; Barszcz, T.; Jones, S. K.; Werbovetz, K. A.; Brun, R.; Tidwell, R. R. J. Med. Chem. 2009, 52, 2016. 17. Field, M. C.; Carrington, M. Nat. Rev. Microbiol. 2009, 7, 775. 18. Morgan, G. W.; Hall, B. S.; Denny, P. W.; Field, M. C.; Carrington, M. Trends Parasitol. 2002, 18, 540. 19. Allen, C. L.; Goulding, D.; Field, M. C. Embo J. 2003, 22, 4991.