Antiparasitic activities of new lawsone Mannich bases

A series of new lawsone Mannich bases derived from salicylaldehydes or nitrofurfural were prepared and tested for their activities against Leishmania major, Toxoplasma gondii, and Trypanosoma brucei brucei parasites. The hydrochloride salts 5a and 6a of the Mannich bases 2a and 3a, derived from unsubstituted salicylaldehyde and long‐chained alkyl amines, were selectively and strongly active against T. gondii cells and appear to be new promising drug candidates against this parasite. Compound 6a showed an even higher activity against T. gondii than the known lawsone Mannich base 1b. Compound 4a, derived from salicylaldehyde and 2‐methylaminopyridine, was also distinctly active against T. gondii cells. The derivatives 3a (salicyl derivative), 3b (3,5‐dichloro‐2‐hydroxyphenyl derivative), and 3d (5‐nitrofuranyl derivative) as well as the hydrochlorides 6a and 6b were also efficacious against T. b. brucei cells with compounds 3a and 3b being more selective for T. b. brucei over Vero cells when compared with the known control compound 1b. The derivatives 5a, 5c, 6a, and 6c proved to be up to five times more active than 1b against L. major promastigotes and up to four times more efficacious against L. major amastigotes.

the current study, we prepared new lawsone Mannich bases with long-chain appendages and tested their activities against the cutaneous leishmaniasis causing parasite L. major, against the Nagana cattle-disease causing parasite T. b. brucei (both kinetoplastid parasites), and against toxoplasmosis causing T. gondii parasites (an apicomplexan parasite).

| RESULTS AND DISCUSSION
Compounds 2a-d, 3a-d, and 4a were prepared in moderate yields by Mannich reactions of lawsone, the respective aryl aldehyde and the respective amine (Scheme 1). [19] They were obtained as racemic orange to orange-red solids. Compounds 2a-c and 3a-c were converted to the hydrochloride salts 5a-c and 6a-c by reaction with acetyl chloride in ethyl alcohol (EtOH; Scheme 2).
The new lawsone Mannich bases were tested on three protozoal parasites including T. gondii against which the related lawsone derivative atovaquone had already shown reasonable activity. [20] Our hydrochlorides 5a and 6a proved highly active against T. gondii tachyzoites, surpassing even the effect of the known positive control 1b (Table 1). Both 5a and 6a also displayed some selectivity for the T.
gondii parasite when compared with nonmalignant Vero cells with selectivity index (SI) values of 2.38 for 5a and 3.12 for 6a. The new compounds 4a, 5c, and 6c also exhibited good activities against T.
gondii in the range of that of 1b with some selectivity in the case of 5c (SI = 1.91) and 6c (SI = 3.38).
The new compounds were also tested for trypanocidal activity against the bloodstream-form T. b. brucei parasites using the Alamar Blue (AB) assay (Table 2). Compounds 3a, 3b, and 3d, carrying a long hexadecyl chain, reached IC 50 values in the low one-digit micromolar concentration range (IC 50 = 1.17-1.39 µM) and so performed better than their dodecyl analogs 2a, 2b, and 2d. It is possible that 3a, 3b, and 3d share similar modes of action with the approved antileishmanial drug miltefosine, which is a hexadecylphosphocholine derivative. [21,22] Interestingly, 3a, 3b, and 3d showed only a weak activity against T. gondii and Vero cells and the selectivity of 3a and 3b for T. b. brucei was distinctly higher when compared with that of the known 1b. Finally, the new compounds were tested against L. major promastigotes and amastigotes ( Table 3). The hydrochlorides 5c, 6a, and 6c were active against the promastigotes with IC 50 values ranging from 5.04 to 6.54 µM, and against the amastigotes with IC 50 values between 4.06 and 4.71 µM. The dibromo derivative 6c showed SI values of 3.19 for promastigotes and 3.97 for amastigotes, each over Vero cells. The highest activity against the amastigotes was found for 5a (IC 50 = 3.62 µM), which showed a distinctly weaker activity against the promastigotes. With the exception of the dichloro derivatives 2b and 3b, all new compounds were more active against the amastigotes than the promastigotes. The combination of the lawsone pharmacophore with hydrophobic side-chains (as in atovaquone) generates amphiphilic conjugates with potentially enhanced cell membrane penetration to the effect of an increased cytotoxic activity. Such a hybrid molecule strategy can indeed lead to compounds with high activity against L. major. [23] Atovaquone, for instance, showed a reasonable activity against visceral leishmaniasis. [24] The observation of a higher activity of most of the new lawsone Mannich bases against L. major amastigotes compared to promastigotes supports earlier studies with similar drugs and drug candidates. The approved antileishmaniasis drug miltefosine proved toxic to L. major amastigotes without negatively affecting the host immune system. [25] Ketotifen and cromolyn sodium also performed better against L. major amastigotes than promastigotes. [26] Another study described the potent activity of the CM11 peptide against L. major amastigotes. [27] 3 | CONCLUSIONS A series of new lawsone Mannich bases derived from (halo-) salicylaldehydes and long-chained alkyl amines (C 12 and C 16 chains) were prepared in reasonable yields and were tested against the pathogenic parasites L. major, T. gondii, and T. b. brucei. New compounds with inhibitory activities against T. gondii (5a and 6a) exceeding that of the known 2-pyridyl lawsone Mannich base 1b were identified. Compound 6a is a particularly promising new drug candidate for the treatment of toxoplasmosis, warranting further indepth tests. In addition, we observed a distinctly higher selectivity of compounds 3a and 3b for T. b. brucei cells when compared to the known Mannich base 1b. Improved selectivity of 3a and 3b might lead to less severe side effects in future tests with Trypanosoma animal models. The most striking data were obtained from tests against L. major with distinctly improved activities of the new compounds 5a, 5c, 6a, and 6c when compared with the known compound 1b. A topical application of these new lawsone Mannich Value is taken from Ahmed et al. [11] AL NASR ET AL. All starting compounds were purchased from Aldrich. The known compound 1b was prepared according to a literature procedure. [13] The following instruments were used: melting points (uncorrected),    −1 2,922, 2,852, 1,679, 1,645, 1,590, 1,516, 1,459,  1,364, 1,275, 1,218, 1,143, 1,093, 1,046, 865, 794, 723, 689, 657,   625, 610, and 601; 1 H NMR (300 MHz, CDCl 3 ); δ 0.7-0.9 (3H, m),

| Alamar blue (AB) assay
The AB assay was used to identify viable cells after treatment with drug candidates. [29][30][31][32] This assay is based on the irreversible reaction of the blue dye resazurin and NADH to pink resofurin in intact cells.