Investigation of Indolglyoxamide and Indolacetamide Analogues of Polyamines as Antimalarial and Antitrypanosomal Agents

Pure compound screening has previously identified the indolglyoxylamidospermidine ascidian metabolites didemnidine A and B (2 and 3) to be weak growth inhibitors of Trypanosoma brucei rhodesiense (IC50 59 and 44 μM, respectively) and Plasmodium falciparum (K1 dual drug resistant strain) (IC50 41 and 15 μM, respectively), but lacking in selectivity (L6 rat myoblast, IC50 24 μM and 25 μM, respectively). To expand the structure–activity relationship of this compound class towards both parasites, we have prepared and biologically tested a library of analogues that includes indoleglyoxyl and indoleacetic “capping acids”, and polyamines including spermine (PA3-4-3) and extended analogues PA3-8-3 and PA3-12-3. 7-Methoxy substituted indoleglyoxylamides were typically found to exhibit the most potent antimalarial activity (IC50 10–92 nM) but with varying degrees of selectivity versus the L6 rat myoblast cell line. A 6-methoxyindolglyoxylamide analogue was the most potent growth inhibitor of T. brucei (IC50 0.18 μM) identified in the study: it, however, also exhibited poor selectivity (L6 IC50 6.0 μM). There was no apparent correlation between antimalarial and anti-T. brucei activity in the series. In vivo evaluation of one analogue against Plasmodium berghei was undertaken, demonstrating a modest 20.9% reduction in parasitaemia.


Introduction
Alkyl amines belonging to the polyamine family [1] are widely distributed in nature, being isolated from a diverse range of terrestrial and marine sources. From the simple diamines putresine and cadaverine through to more complex examples of spermidine and spermine, polyamines have been reported to exhibit biological activities towards a large number of cellular targets and processes. While N-alkyl derivatives are generally cytotoxic or act synergistically with cytotoxins [2][3][4], examples have been reported to act as potent epigenetic modulators [5][6][7], to act as antioxidants [8], and to exhibit anti-trypanosomal [9,10] and anti-malarial properties [11][12][13][14][15][16].
As part of our own continuing search for new natural product leads for the development of treatments for neglected human diseases [17][18][19][20][21], we recently reported the discovery of polyamine alkaloids orthidine F (1) [22,23] and didemnidines A (2) and B (3) [24] as in vitro growth inhibitors of Plasmodium falciparum (K1 dual drug-resistant strain) ( Figure 1). In the case of orthidine F, the antimalarial potency of the natural product (IC 50 0.89 μM) [23] was increased substantially (IC 50 1.3 nM) by undertaking a structure-activity relationship study [25], which also identified optimal structural attributes for antimalarial activity to be either a polyamine PA3-8-3 or PA3-12-3 [1] scaffold, and bearing 1, ω-disubstitution. Didemnidines A and B were found to be more modest growth inhibitors of both P. falciparum (IC 50 41 and 15 μM, respectively) and Trypanosoma brucei rhodesiense (IC 50 59 and 44 μM, respectively) [24]. Analogue 4, prepared during the synthesis of 3, was identified as the most active anti-protozoal compound in the limited series (Pf IC 50 8.4 μM, Tbr IC 50 9.9 μM), again suggesting that 1, ω-disubstitution of this alkaloid family might lead to the identification of more active examples. Herein we report the results of a structure-activity relationship study investigating the influence of indole substitution, the requirement for the side chain keto group and nature of the polyamine core to the observed anti-protozoal activity of didemnidines A and B. The library was evaluated for antimalarial activity against the NF54 drug sensitive strain of P. falciparum, for anti-trypanosomal activity against Trypanosoma brucei rhodesiense and for cytotoxicity towards the non-malignant L6 rat myoblast cell line. One analogue was also tested for in vivo antimalarial activity against Plasmodium berghei in mice.

In Vivo Anti-Malarial Evaluation
Analogue 20 was selected for in vivo evaluation in Plasmodium berghei infected mice. Using a standard test protocol [30], a repeated ip dose of 50 (mg/kg)/day for four days led to a 20.9% reduction in parasitaemia. No increase in mean survival time was observed.

General Procedure A: Amide Bond Formation
To a solution of carboxylic acid (2.05 equiv.), diamine (1 equiv.), and PyBOP (2.05 equiv.) in DMF (1 mL) was added Et 3 N (3 equiv.). The reaction mixture was allowed to stir under N 2 at room temperature for 23 h. The solution was dried in vacuo and the crude reaction product purified by C 8 reversed-phase column chromatography (20%-30% MeOH/H 2 O (+0.05%TFA)) to afford the target diamide as the bis-trifluoroacetate salt or by silica gel column chromatography (0%-1% MeOH in CH 2 Cl 2 ) to afford the target diamide as the free base.

2-(5-Methoxy-1H-indol-3-yl)-2-oxoacetic Acid
Mp   (11) The target compound 11 was prepared using a previously published method [26]. To a solution of 6-methoxyindole (0.13 g, 0.866 mmol) in anhydrous diethyl ether (10 mL) was added oxalyl chloride (0.11 mL, 1.30 mmol) dropwise at 0 °C. The reaction mixture was allowed to stir at 0 °C for 3 h before it was warmed to r.t. Saturated aq. NaHCO 3 (10 mL) was then added, and the reaction mixture heated at reflux for 1 h. After cooling to r.t., the pH of the reaction mixture was adjusted to 1 using 10% HCl. The resulting green precipitate was filtered, washed with cold diethyl ether (30 mL) and dried under vacuum to yield 11 as a green powder (0.18 g, 97% yield) which was used in the next step without further purification.

In Vivo Anti-Malarial Efficacy Studies
In vivo anti-malarial activity was assessed as previously described [30]. Groups of three female NMRI mice (20-22 g) were intravenously infected with 2 × 10 7 parasitized erythrocytes on day 0 with GFP-transfected P. berghei strain ANKA [32]. Compounds were formulated in 100% DMSO, diluted 10-fold in distilled water and administered intraperitoneally in a volume of 10 mL· kg −1 on four consecutive days (4, 24, 48 and 72 h post infection). Parasitemia was determined on day 4 post infection (24 h after last treatment) by FACS analysis. Activity was calculated as the difference between the mean per cent parasitaemia for the control (n = 5 mice) and treated groups expressed as a per cent relative to the control group. The survival of the animals was usually monitored up to 30 days: a compound was considered curative if the animal survived to day 30 after infection with no detectable parasites. In vivo efficacy studies in mice were conducted according to the rules and regulations for the protection of animal rights ("Tierschutzverordnung") of the Swiss "Bundesamt für Veterinä rwesen". They were approved by the veterinary office of Canton Basel-Stadt, Switzerland.

Conclusions
The polyamine marine natural products didemnidine A (2) and B (3) have been previously identified as weak in vitro growth inhibitors of Trypanosoma brucei rhodesiense and Plasmodium falciparum. A series of 1, ω-substituted polyamine analogues were prepared that explored the influence of "capping acids" indole-3-glyoxylic acid and indole-3-acetic acid, length of polyamine chain and the presence or absence of mid-chain nitrogen substitution on antiprotozoal activity. Three analogues, one containing a PA3-8-3 core (20) and two containing PA3-12-3 cores (29, 32) were identified as particularly potent antimalarials, with the former example also exhibiting good selectivity. Several analogues were identified that exhibit more enhanced anti-Trypanosoma brucei activity than the original natural product hits, but these same analogues also exhibited cytotoxicity, making them poorly selective. PA3-8-3 analogue 20 was only mildly active against P. berghei infection in a mouse model.