Design and Synthesis of Potent Antileishmanial Cycloalkylidene-Substituted Ether Phospholipid Derivatives Theodora Calogeropoulou,* ,† Panagiotis Angelou, † Anastasia Detsi, †,‡ Irene Fragiadaki, § and Effie Scoulica* ,§ Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, 48 Vassileos Constantinou AVenue, 11635 Athens, Greece, and Department of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, School of Medicine, UniVersity of Crete, 71409 Heraklion, Greece ReceiVed September 18, 2007 Two series of novel ether phospholipids (EPs) have been synthesized. The first includes cyclodecylidene- or cyclopentadecylidene-substituted EPs carrying N,N,N-trimethylammonium or N-methylpiperidino or N-methylmorpholino head groups. The second series encompasses more rigid head groups in combination with cycloalkylidene moieties in the lipid portion. In addition, hydrogenated derivatives were obtained. All the new analogues, except 33, were 1.5- to 62-fold more potent than miltefosine against the intracellular L. infantum, and the most active ones were also less cytotoxic against the human monocytic cell line THP1 and less hemolytic than miltefosine. The analogues that combine high potency with low cytotoxicity and hemolytic activity were 19, 37, 21 23, 38, 39, and 40. Cyclopentadecylpentylphosphocholine (38) possesses an IC 50 of 0.7 µM against L. infantum amastigotes and is the least cytotoxic analogue, since it does not present toxicity against THP1 macrophages, even at a concentration that is 800-fold the antiparasitic IC 50 value, and does not present significant hemolytic activity. Introduction Leishmaniasis, one of the most neglected tropical diseases, is a parasitic disease that is transmitted by the bite of the infected phlebotomine sandfly and constitutes a major public health problem especially in the tropical and subtropical regions of the world. It is currently endemic in 88 countries on five continents (Africa, Asia, Europe, and North and South America), and the population at risk reaches 350 million people. The parasite exists in two different forms: the flagellated one in the gut of the sandfly vector and the amastigote in the mammalian host that is the cause of the acute desease. Visceral leishmaniasis (VL a , or “kala-azar”), mucocutaneous leishmaniasis (MCL, ulceration of the skin and hyperdevelop- ment of the mucous membranes), and cutaneous and diffuse cutaneous leishmaniasis (CL and diffuse CL) are the distin- guishable forms of the disease, which includes several clinical syndromes and, if untreated, can have devastating consequences. Establishment of the infection and progression of the disease are favored by the compromised immune system of patients, like those infected with HIV, and as a result, leishmania/HIV coinfection is now considered an extremely serious new disease with severe clinical, diagnostic, chemotherapeutic, epidemio- logical, and economic implications. 1 Even though chemotherapy is currently the only way to treat the various forms of leishmaniasis, since no vaccine is yet available, the arsenal of drugs against the disease is still limited. Today, first line antileishmanial drugs include pentavalent antimonials (sodium stibogluconate and meglumine antimonate), pentamidine, and amphotericin B. 2 The high resistance developed to pentavalent antimonials, high-dose regimens, and long treatment courses using parenteral administration, as well as, renal and cardiac toxicity, are major drawbacks. On the other hand, the high toxicity and declining efficacy of pentamidine have restricted its use. Amphotericin B and its lipid formulation proved to be very effective in the treatment of leishmaniasis, but the cost is still prohibitively high. Paromomycin and sitamaquine are two compounds currently undergoing clinical trials. 3,4 The serious problems associated with the treatment of leishmania infections, such as development of drug resistance, the need for low cost drugs, and the limited funding available for parasitic diseases, have recently led many research groups to design and synthesize novel antileishmanial compounds as well as screen natural products for their antileishmanial activity. Most recent examples of synthetic compounds include nitrogen heterocycles, such as quinoline, 5,6 dihydropyridopyrimidine, 7 and pyrazolopyridine 8 derivatives as well as nitrogen-containing biphosphonates 9 and arylanthranilodinitriles. 10 Moreover, screen- ing of natural products has identified several promising leads and has provided new scaffolds for chemical derivatization. 11–15 In 1987, the “serendipitous” discovery of the antileishmanial activity of hexadecylphosphocholine (miltefosine, HePC), 16 an alkyllysophospholipid initially developed as an antitumor agent, constituted a major breakthrough in antileishmanial chemo- therapy. This compound is effective against both visceral and cutaneous leishmaniasis, displays good bioavailability, and is currently registered as an oral drug for the treatment of the disease in India (in 2002) and Colombia (in 2005). 17 According to the latest clinical studies in Colombia, cure rates of 91-100% were obtained with a dose of 2.5 (mg/kg)/day for 28 days, with mild side effects (mostly gastrointestinal) in 35–60% of the patients. 18 Despite its advantages, miltefosine has a long half-life (100–200 h) in humans and a low therapeutic ratio, character- istics that could encourage development of resistance, especially * To whom correspondence should be addressed. For T.C.: phone, +3010 7273833; fax, +3010 7273818; e-mail, tcalog@eie.gr. For E.S.: phone/ fax, +302810 394576; e-mail, scoulica@med.uoc.gr. † National Hellenic Research Foundation. ‡ Current Address: Laboratory of Organic Chemistry, School of Chemical Engineering, National Technical University of Athens, Heroon Polytechniou 9, Zografou Campus, 15773 Athens, Greece. § University of Crete. a Abbreviations: HePC, hexadecylphosphocholine; HIV, human immu- nodeficiency virus; VL, visceral leishmaniasis; CL, cutaneous leishmaniasis; GPI, glycosylphosphatidylinositol; PI, propidium iodide. J. Med. Chem. 2008, 51, 897–908 897 10.1021/jm701166b CCC: $40.75  2008 American Chemical Society Published on Web 01/26/2008