Design of Novel N-(2,4-Dioxo-1,2,3,4-tetrahydro-thieno[3,2- d]pyrimidin-7-yl)-guanidines as Thymidine Phosphorylase Inhibitors, and Flexible Docking to a Homology Model Melissa L. P. Price, a, * Wayne C. Guida, b,c Tara E. Jackson, b Jason A. Nydick, b Patricia L. Gladstone, a Jose´ C.Juarez, a Fernando Don˜ate a and Robert J. Ternansky a a Attenuon, L.L.C., San Diego, CA 92121, USA b Department of Chemistry, Eckerd College, St. Petersburg, FL 33711, USA c Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute at the University of South Florida, Tampa, FL 33612, USA Received 22 May 2002; accepted 24 September 2002 Abstract—A novel class of thymidine phosphorylase (TP) inhibitors has been designed based on analogy to the enzyme substrate as well as known inhibitors. Flexible docking studies, using a homology model of human TP, of the designed N-(2,4-dioxo-1,2,3,4- tetrahydro-thieno[3,2-d]pyrimidin-7-yl)-guanidines as well as their synthetic precursors provide insight into the observed experi- mental trends in binding affinity. # 2002 Published by Elsevier Science Ltd. Thymidine phosphorylase (TP) catalyzes the reversible phosphorolysis of thymidine (1) to thymine and 2-deoxyribose-1-phosphate (Scheme 1). 1,2 By analogy to purine nucleoside phosphorylase, 3 the reaction likely proceeds through an oxocarbenium ion-like transition state. The phosphorylated sugar product is further con- verted to 2-deoxy-d-ribose which has been shown to have chemotactic activity in vitro and angiogenic activ- ity in vivo. 4 Therefore, inhibitors of TP may find utility as suppressors of tumor growth. 5 Taiho Pharmaceutical Co. has reported the most potent TP inhibitor to date, 5-chloro-6-(2-iminopyrrolidin-1- yl)methyl-2,4(1H,3H)-pyrimidinedione hydrochloride (2) which inhibits human TP with an IC 50 value of 35 nM. 6 Our designed thienopyrimidinyl guanidines, 3 and 4, preserve the interactions of the pyrimidine ring in 1 and 2, substitute an isosteric sulfur atom for the chlor- ine atom in 2, and maintain the positive-charge charac- ter of the iminopyrrolidine in 2,whichlike 2, may mimic the oxocarbenium ion-like transition state in thymidine phosphorolysis. Pyrimidine nucleoside phosphorylase (PyNP) (42% sequence identity using Clustalw 7 for alignment against the human sequence) catalyzes the same reaction as TP, as well as the conversion of uridine to uracil. Unlike other structures of TP, 8,9 the high-resolution 0960-894X/03/$ - see front matter # 2002 Published by Elsevier Science Ltd. PII: S0960-894X(02)00828-4 Bioorganic & Medicinal Chemistry Letters 13 (2003) 107–110 Scheme 1. *Corresponding author. Fax: +1-858-622-0517; e-mail: price@ attenuon.com