1048 Journal of Medicina stry. 1994,Vol.37,No.8 Figure 12. The of R-2 within active f HCA I as determined by x-rav when e 18 nt d The that the alkylatic this initial igne 4 conver ted o an midi d th In order to fill the remaining space with A 169.and se d donor tothe Ala263carb solubi ing group (in a9 ondingom distan th er azine ld sho able to mo ve back to itsori I position n and b he that the n ce left y the d wat r in the nan TS ith the 2 mple (Table of .the la np tions to be correct. The water4 the Ala 263 onded tspieiouspositioi 263 s a hyd displaced the he and Asp 169 as predicte eerae del beca d23 f m of der botto part the activ eotantechg water 430. e indole ring. d to fill th of the nd is 2 nM a Replacement in1048 Journal of Medicinal Chemistry, 1994, Vol. 37, No. 8 Perspective Figure 12. The bound conformation of R-2 within the active site of HCA I1 as determined by X-ray crystallographic studies. when substituted, and changing its substituents for subsequent designs could be accomplished by simple alkylation chemistry. The fit of this initially designed fragment is shown in Figure 16 superimposed on the methyl probe from GRID. In order to fill the remaining space at the opening of the active site, a benzyl group was used as one of the nitrogen alkyl substituents. To complete the molecule, a solubilizing group (in this case a basic piperazine ring) was placed at the 4-position of the benzene ring. Modeling indicated that the piperazine ring would sit at the interface of protein and bulk solvent. Shown in Table 8 is the initial lead compound 22 along with its inhibition constants against human TS. Solution of the crystal structure of compound 22 complexed with E. coli TS was accomplished and is shown in Figure 17. Com￾pound 22 was found to bind in a similar fashion to the original model with one major exception. In the actual fitted structure, the lactam carbonyl oxygen makes an unfavorable interaction with the Ala 263 carbonyl oxygen and as a result moves the Ala 263 oxygen about 1 A from its previous position. This movement breaks a hydrogen￾bond between water 430 and Ala 263 carbonyl oxygen and the water is displaced by the lactam oxygen of the benzindole. It appears that the naphthalene ring wedges itself deeper in the pocket than the model because of more favorable hydrophobic interactions at the top and bottom of the active site. It accomplishes this at the expense of water 430. Optimization of the New Inhibitor Series. Analysis of this structure resulted in the design of a second generation compound 23 shown in Table 8. Replacement of the lactam carbonyl oxygen with a nitrogen atom was intended to accomplish two goals. The first was that the lactam would be converted to an amidine functionality which should be protonated while bound and therefore make a charged hydrogen bond with Asp 169, and second, the exocyclic NH2 of the amidine should be an excellent hydrogen-bond donor to the Ala 263 carbonyl oxygen. Since hydrogen-bonding atom distances are inside their respec￾tive van der Waals radius, the Ala 263 carbonyl oxygen should be able to move back to its original position and restore the hydrogen bond to water 430. The result would be that the empty space left by the displaced water in the compound 22 complex would be filled. This single atom change resulted in an improvement in binding of roughly a factor of 50 (see Table 8). The structure of compound 23 complexed with E. coli TS is shown in Figure 18. Analysis of this structure revealed the assumptions to be correct. The water 430 had returned and was hydrogen bonded to Ala 263 carbonyl oxygen. The NH2 of the amidine was hydrogen bonded to both Ala 263 carbonyl oxygen and water 430. In addition, there was a hydrogen bond between the ami￾dine and Asp 169 as predicted. Using the structure of compound 23 as a guide, a number of derivatives intended to fill remaining space in the deep part of the active site and at the protein solvent interface were designed. Compound 25 has two additional methyl groups on the benzindole ring, both intended to fill the remaining hydrophobic space, and the inhibition constant of this compound is 2 nM against the human enzyme. Compound 24 has replaced the original piperazine ring