For Ras oncoproteins to transform mammalian cells, they must be post-translationally modified with a farnesyl group in a reaction catalysed by the enzyme farnesyl-protein transferase (FPTase). Inhibitors of FPTase have therefore been proposed as anti-cancer agents. We show that L-744,832, which mimics the CaaX motif to which the farnesyl group is added, is a potent and selective inhibitor of FPTase. In MMTV-v-Ha-ras mice bearing palpable tumours, daily administration of L-744,832 caused tumour regression. Following cessation of treatment, tumours reappeared, the majority of which regressed upon retreatment. No systemic toxicity was found upon necropsy of L-744,832-treated mice. This first demonstration of anti-FPTase-mediated tumour regression suggests that FPTase inhibitors may be safe and effective anti-tumour agents in some cancers.
The farnesyltransferase inhibitor L-744,832 selectively blocks the transformed phenotype of cultured cells expressing a mutated H-ras gene and induces dramatic regression of mammary and salivary carcinomas in mouse mammary tumor virus (MMTV)-v-Ha-ras transgenic mice. To better understand how the farnesyltransferase inhibitors might be used in the treatment of human tumors, we have further explored the mechanisms by which L-744,832 induces tumor regression in a variety of transgenic mouse tumor models. We assessed whether L-744,832 induces apoptosis or alterations in cell cycle distribution and found that the tumor regression in MMTV-v-Ha-ras mice could be attributed entirely to elevation of apoptosis levels. In contrast, treatment with doxorubicin, which induces apoptosis in many tumor types, had a minimal effect on apoptosis in these tumors and resulted in a less dramatic tumor response. To determine whether functional p53 is required for L-744,832-induced apoptosis and the resultant tumor regression, MMTV-v-Ha-ras mice were interbred with p53 ؊/؊ mice. Tumors in ras/p53 ؊/؊ mice treated with L-744,832 regressed as efficiently as MMTV-v-Haras tumors, although this response was found to be mediated by both the induction of apoptosis and an increase in G 1 with a corresponding decrease in the S-phase fraction. MMTV-v-Ha-ras mice were also interbred with MMTV-c-myc mice to determine whether ras/myc tumors, which possess high levels of spontaneous apoptosis, have the potential to regress through a further increase in apoptosis levels. The ras/myc tumors were found to respond nearly as efficiently to L-744,832 treatment as the MMTV-v-Ha-ras tumors, although no induction of apoptosis was observed. Rather, the tumor regression in the ras/myc mice was found to be mediated by a large reduction in the S-phase fraction. In contrast, treatment of transgenic mice harboring an activated MMTVc-neu gene did not result in tumor regression. These results demonstrate that a farnesyltransferase inhibitor can induce regression of v-Ha-ras-bearing tumors by multiple mechanisms, including the activation of a suppressed apoptotic pathway, which is largely p53 independent, or by cell cycle alterations, depending upon the presence of various other oncogenic genetic alterations.
Inhibition of kinesin spindle protein (KSP) is a novel mechanism for treatment of cancer with the potential to overcome limitations associated with currently employed cytotoxic agents. Herein, we describe a C2-hydroxymethyl dihydropyrrole KSP inhibitor ( 11) that circumvents hERG channel binding and poor in vivo potency, issues that limited earlier compounds from our program. However, introduction of the C2-hydroxymethyl group caused 11 to be a substrate for cellular efflux by P-glycoprotein (Pgp). Utilizing knowledge garnered from previous KSP inhibitors, we found that beta-fluorination modulated the p K a of the piperidine nitrogen and reduced Pgp efflux, but the resulting compound ( 14) generated a toxic metabolite in vivo. Incorporation of fluorine in a strategic, metabolically benign position by synthesis of an N-methyl-3-fluoro-4-(aminomethyl)piperidine urea led to compound 30 that has an optimal in vitro and metabolic profile. Compound 30 (MK-0731) was recently studied in a phase I clinical trial in patients with taxane-refractory solid tumors.
A series of macrocyclic 3-aminopyrrolidinone farnesyltransferase inhibitors (FTIs) has been synthesized. Compared with previously described linear 3-aminopyrrolidinone FTIs such as compound 1, macrocycles such as 49 combined improved pharmacokinetic properties with a reduced potential for side effects. In dogs, oral bioavailability was good to excellent, and increases in plasma half-life were due to attenuated clearance. It was observed that in vivo clearance correlated with the flexibility of the molecules and this concept proved useful in the design of FTIs that exhibited low clearance, such as FTI 78. X-ray crystal structures of compounds 49 and 66 complexed with farnesyltransferase (FTase)-farnesyl diphosphate (FPP) were determined, and they provide details of the key interactions in such ternary complexes. Optimization of this 3-aminopyrrolidinone series of compounds led to significant increases in potency, providing 83 and 85, the most potent inhibitors of FTase in cells described to date.
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