Antitumor activity of SCH 66336, an orally bioavailable tricyclic inhibitor of farnesyl protein transferase, in human tumor xenograft models and wap-ras transgenic mice.

We have been developing a series of nonpeptidic, small molecule farnesyl protein transferase inhibitors that share a common tricyclic nucleus and compete with peptide/protein substrates for binding to farnesyl protein transferase. Here, we report on pharmacological and in vivo studies with SCH 66336, a lead compound in this structural class. SCH 66336 potently inhibits Ha-Ras processing in whole cells and blocks the transformed growth properties of fibroblasts and human tumor cell lines expressing activated Ki-Ras proteins. The anchorage-independent growth of many human tumor lines that lack an activated ras oncogene is also blocked by treatment with SCH 66336. In mouse, rat, and monkey systems, SCH 66336 has excellent oral bioavailability and pharmacokinetic properties. In the nude mouse, SCH 66336 demonstrated potent oral activity in a wide array of human tumor xenograft models including tumors of colon, lung, pancreas, prostate, and urinary bladder origin. Enhanced in vivo efficacy was observed when SCH 66336 was combined with various cytotoxic agents (cyclophosphamide, 5-fluorouracil, and vincristine). In a Ha-Ras transgenic mouse model, prophylactic treatment with SCH 66336 delayed tumor onset, reduced the average number of tumors/mouse, and reduced the average tumor weight/animal. In a therapeutic mode in which gavage treatment was initiated after the transgenic mice had developed palpable tumors, significant tumor regression was induced by SCH 66336 in a dose-dependent fashion. This was associated with increased apoptosis and decreased DNA synthesis in tumors of animals treated with SCH 66336. Enhanced efficacy was also observed in this model when SCH 66336 was combined with cyclophosphamide. SCH 66336 is presently being evaluated in Phase I clinical trials.

Farnesyl protein transferase inhibition: a novel approach to anti-tumor therapy. the discovery and development of SCH 66336.

Farnesyl protein transferase (FPT) inhibition is an interesting and promising approach to non-cytotoxic anticancer therapy. Research in this area has resulted in several orally active compounds that are currently in clinical evaluation. This review focuses on FPT inhibitors in clinical trials and concentrates on the benzocycloheptapyridine class, with details on the discovery and development of SCH 66336, currently in Phase II clinical trials.

Isobenzofurans as conformationally constrained miconazole analogues with improved antifungal potency.

A series of halogen-substituted isobenzofuran analogues was synthesized, which represented conformationally constrained analogues of miconazole (1). In vitro and in vivo topical antifungal activity against both dermatophytes and Candida species varied widely, but 13c proved to be significantly superior to both 1 and clotrimazole against a vaginal Candida infection in hamsters, while 13b was significantly more active than 1 against a a topical Trichophyton infection in guinea pigs. None of the compounds were orally active. When the most direct analogue of 1 proved to be among the least active, a molecular modeling study was done using 1, the two active analogues 13b and 13c, and the inactive analogue 13a. All four compounds possessed skeletally similar conformations either at or energetically readily accessible from the global minimum energy conformations. This common conformation of the inactive analogue 13a, however, occupies unique molecular volume space associated with two chlorine atoms, which must also present unique electrostatic properties at the receptor. The conformation-activity relationships discussed may contribute toward deduction of additional structural requirements for pharmacophore optimization and more efficacious antifungal drugs.

Identification of pharmacokinetically stable 3, 10-dibromo-8-chlorobenzocycloheptapyridine farnesyl protein transferase inhibitors with potent enzyme and cellular activities.

Farnesyl protein transferase (FPT) is a promising target for the development of cancer chemotherapeutics because it is responsible for the farnesylation of oncogenic p21 Ras proteins which are found in nearly 30% of all human cancers and necessary for cellular development and growth. The recent discovery and progression to phase II clinical trials of trihalobenzocycloheptapyridine Sch-66336 as a potent inhibitor of FPT with oral, in vivo efficacy in mice have spawned extensive structure-activity relationship studies (SAR) of this class of compounds. Of the many trihalobenzocycloheptapyridine analogues prepared, we have identified several which inhibit FPT and cellular proliferation at single-digit nanomolar concentrations and which have good pharmacokinetic properties in mice.

Tricyclic farnesyl protein transferase inhibitors: crystallographic and calorimetric studies of structure-activity relationships.

Crystallographic and thermodynamic studies of farnesyl protein transferase (FPT) complexed with novel tricyclic inhibitors provide insights into the observed SAR for this unique class of nonpeptidic FPT inhibitors. The crystallographic structures reveal a binding pattern conserved across the mono-, di-, and trihalogen series. In the complexes, the tricycle spans the FPT active site cavity and interacts with both protein atoms and the isoprenoid portion of bound farnesyl diphosphate. An amide carbonyl, common to the tricyclic compounds described here, participates in a water-mediated hydrogen bond to the protein backbone. Ten high-resolution crystal structures of inhibitors complexed with FPT are reported. Included are crystallographic data for FPT complexed with SCH 66336, a compound currently undergoing clinical trials as an anticancer agent (SCH 66336, 4-[2-[4-(3,10-dibromo-8-chloro-6,11-dihydro-5H-benzo[5, 6]cyclohepta[1, 2-b]pyridin-11-yl)-1-piperidinyl]-2-oxoethyl]-1-piperidinecarbo xamide ). Thermodynamic binding parameters show favorable enthalpies of complex formation and small net entropic contributions as observed for 4-[2-[4-(3,10-dibromo-8-chloro-6,11-dihydro-11H-benzo[5, 6]cyclohepta[1, 2-b]pyridin-11-ylidene)-1-piperidinyl]-2-oxoethyl]pyridine N-oxide where DeltaH degrees bind = -12.5 kcal/mol and TDeltaS degrees bind = -1.5 kcal/mol.

Inhibitors of farnesyl protein transferase. 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 and 1-(3-bromo-8-chloro-6,11- dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl)piperazine.

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.

Effects of SCH 59228, an orally bioavailable farnesyl protein transferase inhibitor, on the growth of oncogene-transformed fibroblasts and a human colon carcinoma xenograft in nude mice.

The products of the Ha-, Ki-, and N-ras proto-oncogenes comprise a family of 21 kDa guanine nucleotide-binding proteins which play a crucial role in growth factor signal transduction and in the control of cellular proliferation and differentiation. Activating mutations in the ras oncogenes occur in a wide variety of human tumors. Ras proteins undergo a series of posttranslational processing events. The first modification is addition of the 15-carbon isoprene, farnesyl, to a Cys residue near the carboxy-terminus of Ras. Prenylation allows the Ras oncoprotein to localize to the plasma membrane where it can initiate downstream signalling events leading to cellular transformation. Inhibitors of the enzyme which catalyzes this step, farnesyl protein transferase (FPT), are a potential class of novel anticancer drugs which interfere with Ras function. SCH 59228 is a tricyclic FPT inhibitor which inhibits the farnesylation of purified Ha-Ras with an IC50 of 95 nM and blocks the processing of Ha-Ras in Cos cells with an IC50 of 0.6 microM. SCH 59228 has favorable pharmacokinetic properties upon oral dosing in nude mice. The in vivo efficacy of SCH 59228 was evaluated using a panel of tumor models grown in nude mice. These included several rodent fibroblast lines expressing mutationally-activated (val12) forms of the Ha-Ras oncogene. In some cases, these proteins contain their native C-terminal sequence (CVLS) which directs farnesylation. In one model, the C-terminal sequence was altered to CVLL, making the expressed protein a substrate for a distinct prenyl transferase, geranylgeranyl protein transferase-1. When dosed orally at 10 and 50 mg/kg (four times a day, 7 days a week) SCH 59228 significantly inhibited tumor growth of cells expressing farnesylated Ha-Ras in a dose-dependent manner; over 90% growth inhibition was observed at the 50 mg/kg dose. Tumor growth of cells expressing the geranylgeranylated form of Ha-Ras was less potently inhibited. Growth of tumors derived from a rodent fibroblast line expressing activated Ki-Ras containing its native C-terminal sequence (CVIM), which preferentially directs farnesylation, was also inhibited by SCH 59228. Inhibition in the Ki-Ras model was less than that observed in the Ha-Ras model. In contrast, tumors derived from cells transformed with the mos oncogene were not significantly inhibited even at the highest dose level. SCH 59228 also significantly and dose-dependently inhibited the growth of human colon adenocarcinoma DLD-1 xenografts (which express activated Ki-ras). These results indicate that SCH 59228 possesses in vivo antitumor activity upon oral dosing in tumor models expressing activated ras oncogenes. This is the first report of oral antitumor activity with an FPT inhibitor. These results are discussed in light of recent observations on alternative prenylation of some Ras isoforms.

Chemical modification of everninomicins.

Novel antibiotic everninomicin D is chemically transformed into new biologically active derivatives. Reactions of a nitro group attached to a tertiary carbon center have been investigated. Synthesis and reactions of hydroxylaminoeverninomicin D, aminoeverninomicin D and their derivatives have been discussed.

Novel HIV-protease inhibitors containing beta-hydroxyether and -thioether dipeptide isostere surrogates: modification of the P3 ligand.

Studies involving modifications to the P3 position of previously described HIV-protease inhibitors containing beta-hydroxyether and thioether dipeptide isostere replacements led to the discovery of pseudopeptides 8o and 8p with improved antiviral activities.

Synthesis and preliminary in-vitro profile of Sch 34343--a new penem antibacterial agent.

Sch 34343, a new penem antibiotic, has been synthesized by several routes and found to be highly active against both aerobic and anaerobic Gram-positive and Gram-negative organisms (except Pseudomonas). It has excellent beta-lactamase stability and is bactericidal. The synthetic routes to Sch 34343 and its in-vitro activity compared with other structurally related penems are discussed.

The synthesis of novel HIV-protease inhibitors.

The syntheses, enzyme inhibition and antiviral activity of potent HIV-protease inhibitors containing novel beta-hydroxy ether and thioethers based on the transition state mimetic concept are discussed.

Enzymatic kinetic resolution of piperidine atropisomers: synthesis of a key intermediate of the farnesyl protein transferase inhibitor, SCH66336.

The resolution of secondary amines via enzyme-catalyzed acylation is a relatively rare process. The kinetic resolution of a series of intermediates of SCH66336 (1), by either enzymatic acylation of the pendant piperidine (4, 5) or hydrolysis of the corresponding carbamate 3, was investigated. In the case of 4, the molecule exists as a pair of enantiomers due to atropisomerism about the exocyclic double bond. The enzymatic acylation of (+/-)-4 was optimized in terms of acylating agent, solvent, and moisture content. The use of lipase, Toyobo LIP-300, and trifluoroethyl isobutyrate as acylating agent resulted in isobutyrylation of the (+)-enantiomer, which is easily separated from the unwanted (-)-4. Hydrolysis of the isobutyramide 6c yielded the desired (+)-4 in high enantiomeric excess. (-)-4 may be recovered from the resolution step, racemized, and resubjected to enzymatic acylation to increase material throughput.

Atropisomeric trihalobenzocycloheptapyridine analogues provide stereoselective FPT inhibitors with antitumor activity.

Introduction of bromine at the 10-position of 3-bromo-8-chloro-benzocycloheptapyridine analogues of type 3 results in formation of atropisomeric compounds of type (+/-)-1 and (+/-)-2 that are easily separable at room temperature on a ChiralPak AD column providing pure atropisomers, (+)-1, (-)-1, and (+)-2, (-)-2, respectively. Evaluation of the FPT activity of these atropisomers revealed that compounds (+)-1 and (+)-2 were more potent in the FPT enzyme and cellular assay than their (-)-isomer counterparts. Compounds (+)-1 and (+)-2 were found to inhibit FPT processing in COS cells at low micro molar range. They were also found to have excellent cellular antitumor activity. Evaluation of compound (+)-1 and (+)-2 in DLD-tumor model in nude mice revealed that they were efficacious, inhibiting tumor growth by 55 and 63% at 50 mpk, respectively.

Interaction of a novel GDP exchange inhibitor with the Ras protein.

Mutated, tumorigenic Ras is present in a variety of human tumors. Compounds that inhibit tumorigenic Ras function may be useful in the treatment of Ras-related tumors. The interaction of a novel GDP exchange inhibitor (SCH-54292) with the Ras-GDP protein was studied by NMR spectroscopy. The binding of the inhibitor to the Ras protein was enhanced at low Mg2+ concentrations, which enabled the preparation of a stable complex for NMR study. To understand the enhanced inhibitor binding and the increased GDP dissociation rates of the Ras protein, the conformational changes of the Ras protein at low Mg2+ concentrations was investigated using two-dimensional 1H-15N HSQC experiments. The Ras protein existed in two conformations in slow exchange on the NMR time scale under such conditions. The conformational changes mainly occurred in the GDP binding pocket, in the switch I and the switch II regions, and were reversible. The Ras protein resumed its regular conformation after an excess amount of Mg2+ was added. A model of the inhibitor in complex with the Ras-GDP protein was derived from intra- and intermolecular NOE distance constraints, and revealed that the inhibitor bound to the critical switch II region of the Ras protein.