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In Vitro Inhibition of Toxoplasma gondii by

Four New Derivatives of Artemisinin
Lorraine Jones-Brando, John D'Angelo, Gary H. Posner and
Robert Yolken
Antimicrob. Agents Chemother. 2006, 50(12):4206. DOI:
10.1128/AAC.00793-06.
Published Ahead of Print 23 October 2006.

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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY. Compounds were tested for in vitro efficacy against T. 2006. and 50 ␮g of streptomycin sulfate per ml. The results of the toxoplasma inhibition and cytotoxicity * Corresponding author. † These authors contributed equally to this work. Johns Hopkins University School of Medicine. 4. Maryland2 Received 30 June 2006/Returned for modification 7 September 2006/Accepted 2 October 2006 Toxoplasma gondii is an apicomplexan protozoan of worldwide medical importance. 100 mM in 100 mM HEPES (pH 7. Grand Island. 600 N. Dept. CellTiter 96 AQueous One Solution Reagent for determining cytotoxicity was purchased from Promega Corp. have the ability to inhibit toxoplasma replication in vitro (2. Department of Pediatrics. Thus. Normal human foreskin fibroblasts (American Type Culture Collection. Baltimore.2† Gary H. Fullerton. WI. they are limited in terms of absorption. a therapeutic index (TI) was calculated by the formula TI ⫽ TD50/ID50. 50 units of penicillin G per ml. We synthesized novel.. originally developed for the treatment of malaria. infrared. Fort Collins. of Pediatrics. these trioxane monomers are hydrolytically stable for at least 12 h even at 60°C in dimethyl sulfoxide-water at pH 7. In contrast to artemether. Chlorophenol red–␤-D-galactopyranoside (Roche. p. University of Michigan Medical School. 4206–4208 0066-4804/06/$08. was a gift from Vern Carruthers. gondii through contact with feces from infected cats. 6. gondii could be associated with chronic neuropsychiatric diseases or behavioral abnormalities in some populations (1. Johns Hopkins University School of Medicine. NY) containing 10% fetal bovine serum (Atlas Biologicals. Wolfe St. E-mail: lbrando @jhmi.00793-06 Copyright © 2006. Esterification of primary alcohol 2a with benzoyl chloride led to benzoate ester 2b in 92% yield. Fax: (410) 955-3723.. (St. CA. Cells were maintained in Dulbecco’s modified Eagle medium (Gibco. caused by the protozoan Toxoplasma gondii.. gondii in vitro. 50. Inc. Johns Hopkins University. or by transmission from infected mother to fetus. 䌤 Published ahead of print on 23 October 2006. While these trioxanes have a number of advantages in terms of rapid action and low levels of toxicity. we have designed and synthesized five C-10 nonacetal. First. 1. Louis.1 and Department of Chemistry and The Malaria Research Institute. is medically important and distributed worldwide.4-trioxane artemisinin and artemisinin derivatives such as artemether. Mailing address: Stanley Division of Developmental Neurovirology. Available medications for the prevention and treatment of toxoplasma infection show limited efficacy and have substantial side effects (5). Trioxane C-10 primary alcohol 2a was prepared in 74% yield by hydroboration-oxidation of the corresponding known C-10 allyl trioxane. Vol. No. 10). All Rights Reserved.. less than 5% decomposition was observed by proton nuclear magnetic resonance spectrometry. Humans are infected by T. IN). MO). The median inhibitory dose (50% inhibitory dose [ID50]) and the median cytotoxic dose (50% toxic dose [TD50]) were calculated by extrapolation of the corresponding dose-response curve on a log-linear plot employing the portions of the curve that transected the 50% response point. Indianapolis. Serological studies indicate that T. 2 mM L-glutamine. 12 In Vitro Inhibition of Toxoplasma gondii by Four New Derivatives of Artemisinin䌤 Lorraine Jones-Brando.4. For each compound. Structures of artemisinin and the derivatives are shown in Fig. 2014 by guest Toxoplasmosis. nonacetal. Published studies have indicated that the naturally occurring 1. was stored frozen at ⫺80°C. Maryland. and short half-life (i. Each of these new lipid-soluble chromatographically purified artemisinin derivatives was fully characterized spectroscopically (proton and carbon-13 nuclear magnetic resonance. Deprotonation of the primary alcohol 2a using sodium hydride and then displacement of the bromide anion from citronelyl bromide gave citronelyl ether 2d in 55% yield. American Society for Microbiology. and esterification with 2-bromobenzoyl chloride produced 2-bromobenzoate ester 2c in 86% yield.2). A culture of the tachyzoites of Toxoplasma gondii strain 2F. and high-resolution mass spectrometry). bioavailability. Currently available medications are limited in terms of efficacy and side effects..2. Under these conditions.e.2 and Robert Yolken1 Stanley Division of Developmental Neurovirology. Posner.1†* John D’Angelo. Baltimore.asm. hydrolytically stable derivatives of artemisinin (8) and measured their ability to inhibit the replication of T. by the consumption of undercooked meat from infected animals. which constitutively expresses cytoplasmic ␤-galactosidase and is derived from strain RH. 4206 Downloaded from http://aac. Infection of immunocompetent adults can result in fever and adenopathy (11). Dec. easy hydrolysis into toxic dihydroartemisinin) (9).00⫹0 doi:10. China. zinc-promoted deoxygenation of the known C-10 allyl trioxane and then hydroboration-oxidation produced dioxolane deoxy-2a in 72% yield. . University of Wuhan. MD 212874988. VA) were used to grow tachyzoites and to test compounds for activity and cytotoxicity. CO). Madison. 25 mM HEPES (Gibco).1128/AAC. Trimethoprim was purchased from Sigma Chemical Co. Artemether was generously donated by Huiling Wang. gondii and cytotoxicity by previously published methods (7). Manassas. 12). hydrolytically stable derivatives of artemisinin and showed that they inhibit the replication of Toxoplasma gondii in cell culture.org/ on April 23. Baltimore. Phone: (410) 614-0730. This parasite can cause systemic infection and widespread organ damage in immunocompromised individuals and neonates. Artemisinin was obtained from Holley Pharmaceuticals Co.edu.

J...org/ on April 23. and T. D’Angelo and G. 177:1128–1131.7 1. gondii by artemisinin and synthetic derivatives Compounda Mol wt Artemisinin Artemether 2d 2c 2b 2a Deoxy-2a Trimethoprim 282. Psychopathology 38:87–90.). nonperoxidic deoxy-2a. Yeh. assays towards T. Selection and characterization of Toxoplasma gondii mutants resistant to artemisinin. J. W.9 ⬎320b 220 160 ⬎320b ⬎320b 510 200 60 ⬎1. which inhibited toxoplasma replication at a concentration of 5. A. 2005.2 ␮g/ml. a value 1/4 log greater than the concentration tested was used to compute the TI. The five new derivatives are listed in decreasing order of efficacy according to concentrations in ␮g/ml. Artemether. 2a. D’Angelo and G. the availability of such compounds would also allow for clinical trials directed at defining the role of toxoplasma infections in human diseases. 2b.5 0. X. J. Dominguez Gerpe. 31:645–650. FlippenAnderson. artemether. Luo. In vitro inhibition of T. Arteether. Nonperoxidic deoxy-2a showed virtually no inhibitory activity against the toxoplasma. and Dongpei Sang for valuable collaboration in obtaining in vitro antimalarial data. S. Milhous. b Toxicity endpoint was not reached. R. with a TI of 1. E. which has virtually no antimalaria activity (J.VOL. and R.54 326.6 0. 1988.560 640 210 TI ⱖ243 1.100 320 ⱖ933 ⱖ933 190 1. These degrees of cytotoxicity compare favorably with that of trimethoprim. Peters. was also devoid of antitoxoplasma activity.) and NIH grant AI 34885 (to G. one of the antifolate compounds of relatively low toxicity that is used for the treatment of toxoplasma infection in humans.. Further. H. Torrey. B. Dis. We have demonstrated that four new nonacetal derivatives of artemisinin have both increased antitoxoplasma activity and decreased cytotoxicity compared to trimethoprim. C. J. H.2. B. such as pyrimethamine. These values compare favorably to that of the antifolate positive-control compound trimethoprim. This work was supported by the Theodore and Vada Stanley Foundation (L. Bucks. Infect. Krug. Brossi. F. gondii in humans would represent a major advance in the treatment of infections in immunocompromised individuals. Downloaded from http://aac. Posner. Med.2 8. Substitutions at R are as shown.H.asm. All of the compounds that showed inhibitory activity in our TABLE 1. 2c. E.3 560 17.0 0. Posner. 2b. 3. Schro ¨der. The elucidation of these shared pathways should be the subject of additional investigations. we also measured the cytotoxicity of the test compounds. which inhibited toxoplasma replication at concentrations between 2 and 3 ␮g/ml.2 12 a The parent compound is shown first followed by the known derivative.6 2. 2014 by guest FIG. C. J.J.68 509.43 310. We thank Theresa Shapiro. 1998. Chem. L. Curiel.4 8. a new antimalarial drug: synthesis and antimalarial properties. C. unpublished data). their toxicity generally precludes widespread usage. Artemisinin.43 290. D.2 0. P. Psychopathology in first-episode schizophrenia and antibodies to Toxoplasma gondii. particularly over prolonged periods of administration (5). with a TD50 of 60 ␮g/ml. Andrew Rosenthal. L.Y. Bottmer. These findings suggest that derivatives of artemisinin may affect similar pathways in toxoplasma and malarial organisms. REFERENCES 1.3 ID50 TD50 ␮g/ml ␮M ␮g/ml ␮M 2. as expected (3). and 2d all inhibited the toxoplasma at concentrations of less than 1 ␮g/ ml. Nash. gondii have been shown to inhibit the replication of chloroquine-sensitive Plasmodium falciparum (NF 54) strains of malaria with ID50s from 5 to 30 nM (J.43 430. 2006 NOTES 4207 assays are shown in Table 1. 1. Bachmann.130 740 340 ⬎630 ⬎740 1. and 2c showed little cytotoxicity at concentrations up to 510 ␮g/ml (compound 2a) or 320 ␮g/ml while the other artemisinin derivatives showed 50% cytotoxicity only at concentrations of ⬎160 ␮g/ml. P. and W.2 1. and novel derivatives.P.Somewhatlessantitoxoplasmaactivitywasnotedwithartemisinin and compound 2a. The therapeutic indices of all of the compounds except deoxy-2a were thus approximately 10⫻ to 100⫻ more favorable than that of trimethoprim. unpublished data). Berens. have increased antitoxoplasma activity.1 1. And further. Yolken. While other antifolate compounds.7 173 5. J.3 0. In order to determine therapeutic indices and thus specific antitoxoplasma activity. Venugopalan. 50.-B. L. .4 464. The availability of low-toxicity compounds capable of the prevention and treatment of T.34 298. Structures of artemisinin. and R. 2.

S. quinine sulfate. I. 1987. 1994. Antimicrob. Downloaded from http://aac. mefloquine. 6. Torrey. L. Klayman. Chem. Lillehoj. 2004. R. Drugs used in the treatment of schizophrenia and bipolar disorder inhibit the replication of Toxoplasma gondii. Yolken. and R. A. R.. 32:842–844. H. K. and R. 1994. 30:1217–1258. E. A. H. F. Torrey. Chang. Ford. 34:1961–1965. M. Clin. Drugs 48:179–188.2. J. J. Agents Chemother.4208 NOTES 4. Tenter. trifluoperazine hydrochloride. O’Neill. 2003. M. K. 1990. Milhous. Marr. thiahexatroxane.. In vitro effects of three new 1. Dis. and hexatroxanone) on Toxoplasma gondii. and W. and R. J. Int. W. Res. V. and J.. Yolken. In vitro effects of artemisinin ether. Ou-Yang. Mack. C.asm. Jefford. Krug. G. and L. Holfels. Med. J.org/ on April 23. Med.. AGENTS CHEMOTHER. McLeod. L. Ruslanova. Agents Chemother. 10. primaquine phosphate. 7. Berens. Lin. Antimicrob. Schizophr. Agents Chemother.-C. 47:2945–2964. E. 62:237–244. J. Peche`re.. J. Antimalarial activity of new water-soluble dihydroartemisinin derivatives. Toxoplasma gondii: from animals to humans. 2001. W. Management of toxoplasmosis. and G. M. P. 38:1392–1396. ANTIMICROB. Bachmann. Posner.. F. 2000. and verapamil on Toxoplasma gondii. J. C. L. D. Milhous. E. Infect. 2014 by guest . 5. 9. 1989. 30:2147–2150. Heckeroth. Antimicrob. E. R. A. Antibodies to Toxoplasma gondii in individuals with first-episode schizophrenia. Weiss.4-trioxanes (pentatroxane. Schro ¨der. McAuley. Jones-Brando. Georgiev. 12. cycloguanil hydrochloride (alone and in combination with sulfadiazine).. K. H. 8. Parasitol. E. 33:1748–1752. 11. S. and J. Chem. Inhibition of growth of Toxoplasma gondii by qinghaosu and derivatives. D.. A medicinal chemistry perspective on artemisinin and related endoperoxides.