Oncogenic forms of Ras proteins are associated with a broad range of human cancers including an estimated 90% of all colon cancers (1). Ras proteins undergo a complex series of posttranslational processing events, which have been defined over the past several years (2, 3). The initial post-translational event is the transfer of the 15-carbon isoprene farnesyl from farnesyl pyrophosphate to a Cys residue (Cys 186 in Ha-Ras) in the conserved carboxyl-terminal "CAAX" motif (where "A" is an aliphatic residue) present in all Ras proteins (4, 5). Studies employing site-directed mutagenesis (6, 7) or inhibitors of hydroxymethylglutaryl-CoA reductase (8), the rate-limiting enzyme in isoprenoid biosynthesis, demonstrated that isoprenylation is required for Ras proteins to become membraneassociated and to induce cellular transformation. The farnesyl protein transferase (FPT) 1 that catalyzes this reaction has been purified (9) and cDNA clones for its ␣ and ␤ subunits isolated (10 -12).A number of other cellular proteins are also isoprenylated on a Cys residue near their COOH terminus (13,14). These include other substrates for FPT, such as the nuclear lamins (15). However, the majority of cellular isoprenylated proteins are modified with geranylgeranyl, a 20-carbon isoprene. Two distinct geranylgeranyl protein transferases (GGPT I and II) have been identified (16,17) and cDNA clones for their ␣ and ␤ subunits isolated (18,19). GGPT I and FPT share a common ␣ subunit (18,20).The primary determinant for recognition of protein substrates by the isoprenyl transferases is the substrate's carboxyl-terminal amino acid sequence. Proteins ending in Cys-X-XSer (or Met) are preferred substrates for FPT, while proteins terminating in Cys-X-X-Leu are preferred substrates for GGPT I (21, 22). Substitution of leucine for serine at the COOH terminus of the Ha-Ras CAAX box (Ser 189 3 Leu) makes this protein a substrate for geranylgeranylation (rather than farnesylation) both in vitro and in cells (23). The different substrate specificities of FPT and GGPT-1 are likely mediated by their distinct ␤ subunits. GGPT II utilizes protein substrates terminating in Cys-Cys or Cys-X-Cys (17,24).A number of inhibitors of FPT have been reported over the past several years (25). The design of CAAX peptidomimetics (26 -29) has resulted in potent and selective FPT inhibitors capable of blocking Ras processing in cells. These compounds have shown considerable promise as antitumor agents based on their ability to inhibit cellular transformation induced by oncogenic Ras proteins (26,27) and the growth of Ras-dependent

Novel tricyclic Ras farnesyl-protein transferase (FPT) inhibitors are described. A comprehensive structure-activity relationship (SAR) study of compounds arising from substitution at the 3-position of the tricyclic pyridine ring system has been explored. In the case of halogens, the chloro, bromo, and iodo analogues 19, 22, and 28 were found to be equipotent. However, the fluoro analogue 17 was an order of magnitude less active. Whereas a small alkyl substituent such as a methyl group resulted in a very potent FPT inhibitor (SCH 56580), introduction of bulky substituents such as tert-butyl, compound 33, or a phenyl group, compound 29, resulted in inactive FPT inhibitors. Polar groups at the 3-position such as amino 5, alkylamino 6, and hydroxyl 12 were less active. Whereas compound SCH 44342 did not show appreciable in vivo antitumor activity, the 3-bromo-substituted pyridyl N-oxide amide analogue 38 was a potent FPT inhibitor that reduced tumor growth by 81% when administered q.i.d. at 50 mpk and 52% at 10 mpk. These compounds are nonpeptidic and do not contain sulfhydryl groups. They selectively inhibit FPT and not geranylgeranyl-protein transferase-1 (GGPT-1). They also inhibit H-Ras processing in COS monkey kidney cells and soft agar growth of Ras-transformed cells.

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The synthesis of a variety of novel 4-amido, 4-carbamoyl and 4-carboxamido derivatives of 1-(8-chloro-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl) piperazine to explore the SAR of this series of FPT inhibitors is described. This resulted in the synthesis of the 4- and 3-pyridylacetyl analogues 45a and 50a, respectively, both of which were orally active but were found to be rapidly metabolized in vivo. Identification of the principal metabolites led to the synthesis of a variety of new compounds that would be less readily metabolized, the most interesting of which were the 3- and 4-pyridylacetyl N-oxides 80a and 83a. Novel replacements for the pyridylacetyl moiety were also sought, and this resulted in the discovery of the 4-N-methyl and 4-N-carboxamidopiperidinylacetyl derivatives 135a and 160a, respectively. All of these derivatives exhibited greatly improved pharmacokinetics. The synthesis of the corresponding 3-bromo analogues resulted in the discovery of the 4-pyridylacetyl N-oxides 83b (+/-) and 85b [11S(-)] and the 4-carboxamidopiperidinylacetamido derivative 160b (+/-), all of which exhibited potent FPT inhibition in vitro. All three showed excellent oral bioavailability in vivo in nude mice and cynomolgus monkeys and exhibited excellent antitumor efficacy against a series of tumor cell lines when dosed orally in nude mice.

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