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Azithromycin, a first member of the azalide family of macrolides, while having substantial antimalarial activity, failed as a single agent for malaria prophylaxis. In this paper we present the first analogue campaign to identify more potent compounds from this class. Ureas and thioureas of 15-membered azalides, N′′-substituted 9a-(N′-carbamoyl-β-aminoethyl), 9a-(N′- thiocarbamoyl-β-aminoethyl), 9a-[N′-(β- cyanoethyl)-N′-(carbamoyl-β-aminoethyl)], 9a-[N′- (β-cyanoethyl)-N′-(thiocarbamoyl-β-aminoethyl)], 9a-{; ; ; ; N′-[β-(ethoxycarbonyl)ethyl]-N′(carbamoyl-β- aminoethyl)}; ; ; ; , and 9a-[N′-(β-amidoethyl)-N′- (carbamoyl-β-aminoethyl)] of 9-deoxo-9-dihydro-9a- aza-9a-homoerythromycin A, were synthesized and their biological properties evaluated. The results obtained indicate a substantial improvement of the in vitro activity against P. falciparum (up to 88 times over azithromycin), particularly for compounds containing both sugars on the macrocyclic ring and aromatic moiety on 9a- position. The improved in vitro activity was not confirmed in the mouse model, likely due to an increase in lipophilicity of these analogues leading to a higher volume of distribution. Overall, with increased in vitro activity, promising PK properties, and modest in vivo efficacy, this series of molecules represents a good starting platform for the design of novel antimalarial azalides.

A series of 15-membered azalide urea and thiourea derivatives has been synthesized and evaluated for their in vitro antimalarial activity against chloroquine-sensitive (D6), chloroquine/pyremethamine resistant (W2) and multidrug resistant (TM91C235) strains of Plasmodium falciparum. We have developed an effective automated synthetic strategy for the rapid synthesis of urea/thiourea libraries of a macrolide scaffold. Compounds have been synthesized using a solution phase strategy with overall yields of 50-80%. Most of the synthesized compounds had inhibitory effects. The top 10 compounds were 30-65 times more potent than azithromycin, an azalide with antimalarial activity, against all three strains.

The synthesis of the chimeric molecules consisting of two pharmacophores, tetraoxane and 7-chloro-4-aminoquinoline, is reported. The tetraoxanes 2, 4, and 8 show relatively potent in vitro antimalarial activities, with IC90 values for the Plasmodium falciparum strain W2 of 2.26, 12.44, and 10.74 nM, respectively. In addition, two compounds, 2 and 4, cured mice in a modified Thompson test for antimalarial blood stage activity, with a minimum curative dose of 80 mg/kg, a minimum active dose of 20 mg/kg/day, and a maximum tolerated dose of gt 960 mg/kg.

The development of widespread drug resistance to chloroquine (CQ(a)) has resulted in severe health issues for countries in malaria endemic regions. The antimalarial properties of artemisinin 2 and of other peroxides, such as 1,2,4,5-tetraoxacy-cloalkanes (tetraoxanes), have recently begun to be exploited in the development of new approaches to fighting CQ-resistant strains of malaria. New tetraoxanes employing a steroidal backbone have now been prepared that are highly active, are inexpensive, and demonstrate low toXieity.(5,6) A part of our research in this field is focused on the development of a new type of tetraoxane with nonidentical substituents(6) that utilize a steroid and small cyclohexylidene carriers possessing secondary amide bonds. Also, during our work in this field we discovered that tetraoxanes are unusually stable, even. at pH 1.6,(6c) a characteristic that subsequently allowed the synthesis of many interesting derivatives. This communication encompasses the synthesis of various amino-functionalized antimalarials based on the appreciable stability of the tetraoxane moiety to reaction conditions such as reductive amination and LiAlH4 reduction. Their respective antimalarial activities and the pronounced antiproliferative activity of certain products are reported along with in vitro metabolism studies.

Several dicyclohexylidene tetraoxanes were prepared in order to gain a further insight into structure-activity relationship of this kind of antimalarials. The tetraoxanes 2-5, obtained as a cis/trans mixture, showed pronounced antimalarial activity against Plasmodium falciparum chloroquine susceptible D6, chloroquine resistant W2 and multidrug-resistant TM91C235 (Thailand) strains. They have better than or similar activity to the corresponding desmethyl dicyclohexylidene derivatives. Two chimeric endoperoxides with superior antimalarial activity to the natural product ascaridole were also synthesized.

A series of acid-stable carboxamide derivatives of 2-guanidinoimidazolidinedione (5a-c and 6a-c) were prepared as potential malaria prophylactic and radical cure agents. The new compounds showed moderate to good causal prophylactic activity in mice infected with Plasmodium yoelii sporozoites. Three compounds were further tested for causal prophylactic activity in Rhesus monkeys infected with Plasmodium cynomolgi sporozoites, and all showed a delay in patency from 13 to 40 days at 30 mg/kg/day x 3 days by IM dosing. Two out of four compounds tested for radical curative activity in Rhesus showed cure at 30 mg/kg/day x 3 days. The other two compounds showed delay in relapse from 16 to 68 days. Conversion of new carboxamides (5 and 6) to s-triazine derivatives (7) was demonstrated in mouse and human microsomal preparations and in rat plasma. The results suggest the metabolites, s-triazine derivatives 7, may be the active species of the new carboxamides 5a-c and 6a-c prepared in this study.

The synthesis of deoxycholic acid (DCA)- and cholic acid (CA)-derived mixed tetraoxanes revealed that N-(2-dimethylamino)ethyl derivatives are potent antimalarials in vitro and in vivo. The tetraoxanes presented in this paper are dual inhibitors: besides curing mice in vivo without observed toxic effects, they kill cancer cell lines at very low concentrations. For example, DCA and CA derivatives 16 and 25 cured 3/5 (160 mg/kg/day) and 2/5 (40 mg/kg/day, MTD960 mg/kg), respectively, and they were extremely active against melanoma LOX IMVI cancer, LC50 = 22 nM and 69 nM, respectively.

The high level of attrition of drugs in clinical development has led pharmaceutical companies to increase the efficiency of their lead identification and development through techniques such as combinatorial chemistry and high-throughput (HTP) screening. Since the major reasons for clinical drug candidate failure other than efficacy are pharmacokinetics and toxicity, attention has been focused on assessing properties such as metabolic stability, drug-drug interactions (DDI), and absorption earlier in the drug discovery process. Animal studies are simply too labor-intensive and expensive to use for evaluating every hit, so it has been necessary to develop and implement higher throughput in vitro ADME screens to manage the large number of compounds of interest. The antimalarial drug development program at the Walter Reed Army Institute of Research, Division of Experimental Therapeutics (WRAIR / ET) has adopted this paradigm in its search for a long-term prophylactic for the prevention of malaria. The overarching goal of this program is to develop new, long half-life, orally bioavailable compounds with potent intrinsic activity against liver- and blood-stage parasites. From the WRAIR HTP antimalarial screen, numerous compounds are regularly identified with potent activity. These hits are now immediately evaluated using a panel of in vitro ADME screens to identify and predict compounds that will meet our specific treatment criteria. In this review, the WRAIR ADME screening program for antimalarial drugs is described as well as how we have implemented it to predict the ADME properties of small molecule for the identification of promising drug candidates.