Structure-Activity Relationships of 7-Deaza-6-benzylthioinosine Analogues as Ligands of Toxoplasma gondii Adenosine Kinase Young Ah Kim, † Ashoke Sharon, † Chung K. Chu,* ,† Reem H. Rais, ‡ Omar N. Al Safarjalani, ‡ Fardos N. M. Naguib, ‡ and Mahmoud H. el Kouni ‡ Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, UniVersity of Georgia, Athens, Georgia 30602, and Department of Pharmacology and Toxicology, Center for AIDS Research, UniVersity of Alabama School of Medicine, Birmingham, Alabama 35294 ReceiVed February 25, 2008 Several 7-deaza-6-benzylthioinosine analogues with varied substituents on aromatic ring were synthesized and evaluated against Toxoplasma gondii adenosine kinase (EC.2.7.1.20). Structure-activity relationships indicated that the nitrogen atom at the 7-position does not appear to be a critical structural requirement. Molecular modeling reveals that the 7-deazapurine motif provided flexibility to the 6-benzylthio group as a result of the absence of H-bonding between N7 and Thr140. This flexibility allowed better fitting of the 6-benzylthio group into the hydrophobic pocket of the enzyme at the 6-position. In general, single substitutions at the para or meta position enhanced binding. On the other hand, single substitutions at the ortho position led to the loss of binding affinity. The most potent compounds, 7-deaza-p-cyano-6-benzylthioinosine (IC 50 ) 5.3 µM) and 7-deaza-p-methoxy-6-benzylthioinosine (IC 50 ) 4.6 µM), were evaluated in cell culture to delineate their selective toxicity. Introduction Toxoplasma gondii is an obligate intracellular protozoan parasite belonging to the phylum Apicomplexa. T. gondii infects a wide range of hosts, including human and warm-blooded animals. 1–3 Toxoplasmic infection in human is estimated to affect more than a billion individuals worldwide, resulting in an important public health problem. 4,5 T. gondii infection causes morbidity and mortality in immunocompromised individuals such as organ transplant, leukemia, and AIDS patients, as well as congenitally infected children. In immunocompromised patients, reactivation of chronic T. gondii infection can cause a severe central nervous system disease, and thus, toxoplasmic encephalitis and focal neurological lesions are the most common clinical manifestations. 6,7 Infection acquired during pregnancy can lead to congenital toxoplasmosis of the fetus, which results in abortion, neonatal death, or fetal abnormalities that may be present at birth or may develop later in life. 8,9 At the present time, there is no vaccine available to prevent human infection with this class of pathogens. 10,11 Antifolate drugs, such as the combination of pyrimethamine and sulfadi- azine, are currently used for the treatment of toxoplasmic infection. 12–15 This therapy, however, is ineffective against toxoplasma tissue cysts 6,16 and causes side effects such as bone marrow depression and skin rashes. 15,17 In order to reduce undesirable side effects, folic acid and spiramycin are used as alternative regimen of sulfa drugs. 11,18 However, the emergence of drug resistance and complications associated with long-term treatment of toxoplasmosis warrant the need for the development of new and effective chemotherapeutic agents. Recently, T. gondii adenosine kinase (EC.2.7.1.20) has been investigated as an attractive chemotherapeutic target for the development of antitoxoplasmic agents. 19,20 Several differences between T. gondii and their host are found in purine metabolism. Unlike their host, T. gondii lack de novo purine synthesis and thus depend on the salvage pathways for their purine require- ments. Specifically, the activity of adenosine kinase in T. gondii is 10-fold higher than those of other enzymes in the purine salvage pathways. Therefore, T. gondii adenosine kinase contributes significantly to the survival of the parasite. 20 However, deficiency of adenosine kinase was shown not to be lethal to the parasites, indicating that inhibition of the enzyme will not lead to toxicity in toxoplasma. Structure-activity relationships, comparative enzymatic, metabolic, and X-ray structural studies of T. gondii adenosine kinase have identified subversive substrates that are selectively metabolized to cyto- toxic nucleotides by the T. gondii but not human adenosine kinase. 19–30 We have previously reported that 6-benzylthioinosine analogues 19,20,22,24–26 are selective subversive substrates for the T. gondii, but not host, adenosine kinase. Certain 6-benzylth- ioinosine analogues showed potent antitoxoplasma activity in cell culture as well as in mouse infection model, 19,22,25 demonstrating the potential usefulness of these classes of compounds in the treatment of toxoplasmosis. Furthermore, the 6-benzylthioinosine analogues act as inhibitors of the mam- malian nucleoside transporter ENT1 (es) in host cells, 26 thus elucidating the basis for the lack of uptake of these compounds by uninfected host cells. 23 Several N 6 -benzyladenosine ana- logues also showed appreciable antiparasitic activity, however; they exhibited host toxicity. 27 In light of these findings, 19,22,24–27 we have chosen the 6-benzylthio group as a more suitable scaffold for further development of purine nucleoside analogues as antitoxoplasmic agents. In our continuing effort to develop potent and selective antitoxoplasmic agents, our interest has focused on base- modified 7-deazapurine (pyrrolo[2,3-d]pyrimidine) nucleosides in view of their significant biological properties, including antiparasitic as well as antiviral, antimicrobial, and anticancer activities. 31–34 The base-modified nucleosides are synthetically challenging and can generate new classes of chemotherapeutic agents. 7-Deazapurine is an ideal mimic of purine in which the nitrogen atom at the 7-position is replaced by a carbon atom. * To whom correspondence should be addressed. Phone: (706) 542-5379. Fax: (706) 542-5381. E-mail: dchu@rx.uga.edu. † University of Georgia. ‡ University of Alabama School of Medicine. J. Med. Chem. 2008, 51, 3934–3945 3934 10.1021/jm800201s CCC: $40.75  2008 American Chemical Society Published on Web 06/19/2008