WO2001022963A1 - Procede de prevention de l'osteoporose - Google Patents

Procede de prevention de l'osteoporose Download PDF

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Publication number
WO2001022963A1
WO2001022963A1 PCT/US2000/026357 US0026357W WO0122963A1 WO 2001022963 A1 WO2001022963 A1 WO 2001022963A1 US 0026357 W US0026357 W US 0026357W WO 0122963 A1 WO0122963 A1 WO 0122963A1
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Prior art keywords
cyanobenzyl
piperazine
ylmethyl
substituted
imidazol
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PCT/US2000/026357
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English (en)
Inventor
Alfred A. Reszka
Gideon A. Rodan
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Merck & Co., Inc.
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Priority to AU77153/00A priority Critical patent/AU7715300A/en
Publication of WO2001022963A1 publication Critical patent/WO2001022963A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings

Definitions

  • disorders in humans and other mammals involve or are associated with abnormal bone resorption.
  • Such disorders include, but are not limited to, osteoporosis, glucocorticoid induced osteoporosis, Paget's disease, abnormally increased bone turnover, periodontal disease, tooth loss, bone fractures, rheumatoid arthritis, periprosthetic osteolysis, osteogenesis imperfecta, metastatic bone disease, hypercalcemia of malignancy, and multiple myeloma.
  • osteoporosis which in its most frequent manifestation occurs in postmenopausal women.
  • Osteoporosis is a systemic skeletal disease characterized by a low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. Osteoporotic fractures are a major cause of morbidity and mortality in the elderly population. As many as 50% of women and about 20% of men will experience an osteoporotic fracture. A large segment of the older population already has low bone density and a high risk of fractures. There is a significant need to both prevent and treat osteoporosis and other conditions associated with bone reso ⁇ tion. Because osteoporosis, as well as other disorders associated with bone loss, are generally chronic conditions, it is believed that appropriate therapy will typically require chronic treatment.
  • Normal bone physiology involves a process wherein bone tissue is continuously being turned over by the processes of modeling and remodeling. In other words, there is normally an appropriate balance between reso ⁇ tion of existing bone tissue and the formation of new bone tissue. The exact mechanism underlying the coupling between bone reso ⁇ tion and formation is still unknown. However, an imbalance in these processes is manifested in various disease states and conditions of the skeleton.
  • osteoblasts Two different types of cells called osteoblasts and osteoclasts are involved in the bone formation and reso ⁇ tion processes, respectively. See H. Fleisch, Bisphosphonates In Bone Disease, From The Laboratory To The Patient, 3rd Edition, Parthenon Publishing (1997), which is inco ⁇ orated by reference herein in its entirety.
  • Osteoblasts are cells that are located on the bone surface. These cells secrete an osseous organic matrix, which then calcifies. Substances such as fluoride, parathyroid hormone, and certain cytokines such as protaglandins are known to provide a stimulatory effect on osteoblast cells. However, an aim of current research is to develop therapeutic agents that will selectively increase or stimulate the bone formation activity of the osteoblasts.
  • Osteoclasts also known as Oc
  • Oc Osteoclasts
  • the osteoclasts resorb bone in a closed, sealed-off microenvironment located between the cell and the bone.
  • the recruitment and activity of osteoclasts is known to be influenced by a series of cytokines and hormones. It is well known that bisphosphonates are selective inhibitors of osteoclastic bone reso ⁇ tion, making these compounds important therapeutic agents in the treatment or prevention of a variety of systemic or localized bone disorders caused by or associated with abnormal bone reso ⁇ tion.
  • FPTase farnesyl-protein transferase
  • GGPTase-I geranylgeranyl-protein transferase type I
  • Rab GGPTase geranylgeranyl-protein transferase type-II
  • Each of these enzymes selectively uses farnesyl diphosphate or geranyl-geranyl diphosphate as the isoprenoid donor and selectively recognizes the protein substrate.
  • FPTase farnesylates CaaX-containing proteins that end with Ser, Met, Cys, Gin or Ala.
  • CaaX tetrapeptides comprise the minimum region required for interaction of the protein substrate with the enzyme.
  • the enzymological characterization of these three enzymes has demonstrated that it is possible to selectively inhibit one with little inhibitory effect on the others (Moores, S. L., Schaber, M. D., Mosser, S. D., Rands, E., O'Hara, M. B., Garsky, V. M., Marshall, M. S., Pompliano, D. L., and Gibbs, J. B., J. Biol. Chem., 266:17438 (1991), U.S. Pat. No. 5,470,832).
  • the prenylation reactions have been shown genetically to be essential for the function of a variety of proteins (Clarke, 1992; Cox and Der, 1992a; Gibbs, J. B. (1991); Cell 65: 1-4; Newman and Magee, 1993; Schafer and Rine, 1992). This requirement often is demonstrated by mutating the CaaX Cys acceptors so that the proteins can no longer be prenylated. The resulting proteins are devoid of their central biological activity. These studies provide a genetic "proof of principle" indicating that inhibitors of prenylation can alter the physiological responses regulated by prenylated proteins.
  • the Ras protein is part of a signaling pathway that links cell surface growth factor receptors to nuclear signals initiating cellular proliferation.
  • Ras functions like a G-regulatory protein.
  • Ras In the inactive state, Ras is bound to GDP.
  • Ras Upon growth factor receptor activation, Ras is induced to exchange GDP for GTP and undergoes a conformational change.
  • the GTP-bound form of Ras propagates the growth stimulatory signal until the signal is terminated by the intrinsic GTPase activity of Ras, which returns the protein to its inactive GDP bound form (D.R. Lowy and D.M. Willumsen, Ann. Rev. Biochem. 62:851-891 (1993)).
  • Activation of Ras leads to activation of multiple intracellular signal transduction pathways, including the MAP Kinase pathway and the Rho Rac pathway (Joneson et al., Science 271 :810-812).
  • Ras protein is one of several proteins that are known to undergo post-translational modification.
  • Farnesyl-protein transferase utilizes farnesyl pyrophosphate to covalently modify the Cys thiol group of the Ras CAAX box with a farnesyl group (Reiss et al., Cell, 62:81-88 (1990); Schaber et al., J. Biol.
  • R2 and R3 are attached to the same C atom and are combined to form -(CH2)u- wherein one of the carbon atoms is optionally replaced by a moiety selected from: O, S(O) m , -NC(O)-, and -N(CORlO)- ;
  • R4 is selected from H and CH3;
  • R2, R3 and R4 are optionally attached to the same carbon atom;
  • R6, R7 and R? are independently selected from: H; Ci-4 alkyl, C3-6 cycloalkyl, heterocycle, aryl, aroyl, heteroaroyl, arylsulfonyl, heteroarylsulfonyl, unsubstituted or substituted with: a) Ci-4 alkoxy, b) aryl or hete ⁇ c) halogen, d) HO,
  • R is independently selected from C1-C6 alkyl and aryl
  • A3 is selected from: -C(O)-, -C(O)NRlO-, -C(O)O-, S(O) m or a bond;
  • G is H2 or O, provided that G is not H2, if A3 is a bond;
  • n 0, 1 or 2
  • n 0, 1, 2, 3 or 4
  • p 0, 1, 2, 3 or 4
  • q is 1 or 2
  • r is 0 to 5, provided that r is 0 when V is hydrogen
  • s is 0 or 1
  • t is O or l
  • u is 4 or 5
  • v is 0, 1, 2 or 3; provided that v is not 0 if A3 is a bond;
  • R2 and R3 are independently selected from: H; unsubstituted or substituted Ci-8 alkyl, unsubstituted or substituted C2-8 alkenyl, unsubstituted or substituted C2-8 alkynyl, unsubstituted or substituted aryl, unsubstituted or substituted heterocycle,
  • substituted group is substituted with one or more of: 1) aryl or heterocycle, unsubstituted or substituted with: a) Ci-4 alkyl,
  • halogen e) CN, f) aryl or heteroaryl, g) perfluoro-Ci.4 alkyl, h) SR6a S(O)R6a, SO2R 6a ;
  • R2 and R3 are attached to the same C atom and are combined to form -(CH2)u- wherein one of the carbon atoms is optionally replaced by a moiety selected from: O, S(O)m, -NC(O)-, and -N(CORlO)- ;
  • R2, R3 and R4 are optionally attached to the same carbon atom;
  • R6a i s selected from: Cl-4 alkyl, C3-6 cycloalkyl, heterocycle, aryl, unsubstituted or substituted with: a) Ci-4 alkoxy, b) aryl or heterocycle, c) halogen, d) HO,
  • R O is independently selected from hydrogen, Ci-Cg alkyl, benzyl and aryl;
  • Rl is independently selected from Ci-C ⁇ alkyl and aryl
  • V is selected from: a) hydrogen, b) heterocycle, c) aryl, d) C1-C20 alkyl wherein from 0 to 4 carbon atoms are replaced with a heteroatom selected from O, S, and N, and e) C2-C20 alkenyl, provided that V is not hydrogen if Al is S(O) m and V is not hydrogen if Al is a bond, n is 0 and A is S(O) m ;
  • Rl lOC(O)NRl0- c) unsubstituted or substituted C1-C6 alkyl wherein the substituent on the substituted C1-C6 alkyl is selected from unsubstituted or substituted aryl, heterocyclic, C3-C10 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, RlOO-, Rl lS(O) m -, Rl0C(O)NRl0-, (RlO) 2 NC(O)-, Rl0 2 N-
  • R2 and R3 are attached to the same C atom and are combined to form -(CH2)u- wherein one of the carbon atoms is optionally replaced by a moiety selected from: O, S(O) m , -NC(O)-, and -N(CORlO)- ;
  • R2, R3 and R4 are optionally attached to the same carbon atom;
  • R6 and R7 may be joined in a ring
  • R7 and R7a may be joined in a ring
  • R6 is selected from: Ci-4 alkyl, C3-6 cycloalkyl, heterocycle, aryl, unsubstituted or substituted with: a) C 1-4 alkoxy, b) aryl or heterocycle, c) halogen, d) HO, e > Y O R
  • R9 is selected from: a) hydrogen, b) alkenyl, alkynyl, perfluoroalkyl, F, Cl, Br, Rl OO-, Rl 1 S(O) m -, Rl0C(O)NRl0-, (RlO) 2 NC(O)-, R10 2 N-C(NR10)-, CN, NO2,
  • RlO is independently selected from hydrogen, C1-C6 alkyl, benzyl and aryl;
  • Rl 1 is independently selected from Cl-Cg alkyl and aryl
  • A3 is selected from: -C(O)-, -C(O)NRl0-, -C(O)O-, and S(O) m ;
  • A4 is selected from: a bond, O, and NRlO;
  • V is selected from: a) hydrogen, b) heterocycle, c) aryl, d) C1-C20 alkyl wherein from 0 to 4 carbon atoms are replaced with a heteroatom selected from O, S, and N, and e) C2-C20 alkenyl, provided that V is not hydrogen if A is S(O) m and V is not hydrogen if Al is a bond, n is 0 and A2 is S(O) m ;
  • Z is unsubstituted or substituted aryl or unsubstituted or substituted heteroaryl
  • n 0, 1 or 2
  • n 0, 1, 2, 3 or 4
  • p 0, 1, 2, 3 or 4
  • q is 1 or 2
  • r is 0 to 5, provided that r is 0 when V is hydrogen
  • s is 0 or 1
  • t is O or l
  • u is 4 or 5
  • v is 0, 1, 2 or 3; provided that v is not 0 if A3 is -C(O)- or S(O) m ;
  • R8 is independently selected from: a) hydrogen, b) unsubstituted or substituted aryl, unsubstituted or substituted heterocycle, unsubstituted or substituted C3-C10 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, perfluoroalkyl, F, Cl, Br, RlOO-, Rl lS(O) m -, Rl0C(O)NRl0-, (RlO) 2 NC(O)-, Rl0 2 N-C(NRlO)-, CN, NO2, RlOC(O)-, N3, -N(RlO) 2 , or Rl lOC(O)NRl0-, -C(O)ORl0 and c) C 1 -C ⁇ alkyl unsubstituted or substituted by aryl, cyanophenyl, heterocycle, C3-C10 cycloalkyl, C2-C6 alkenyl
  • RlO is independently selected from hydrogen, C1-C6 alkyl, benzyl and aryl;
  • Rl 1 is independently selected from C1-C6 alkyl and aryl
  • A3 is selected from: -C(O)- or S(O) m ;
  • W is a heterocycle
  • n 0, 1, 2, 3 or 4
  • p 0, 1, 2, 3 or 4
  • q is 1 or 2
  • r is 0 to 5, provided that r is 0 when V is hydrogen; and s is 0 or 1 ,
  • Rib is independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, RlOO-, -N(Rl ) 2 or C2-C6 alkenyl, c) C1-C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, RlOO-, or -N(RlO) 2 ;
  • Rlc is independently selected from: a) hydrogen, b) RlOO-, RlOC(O)NRlO-, (RlO) 2 NC(O)-, Rl0 2 N-C(NRlO)-, CN, NO2,
  • ⁇ .NR 6 R 7 R is selected from H; T O ; or Cl-5 alkyl, unbranched or branched, unsubstituted or substituted with one or more of:
  • R6 is selected from: Ci-4 alkyl or C3-6 cycloalkyl, unsubstituted or substituted with: a) C 1-4 alkoxy, b) halogen, or c) aryl or heterocycle;
  • R3 is selected from H and CH3; ;
  • R6 and R are independently selected from: H; Ci-4 alkyl, C3-6 cycloalkyl, aryl, heterocycle, unsubstituted or substituted with: a) C 1-4 alkoxy, b) halogen, or c) aryl or heterocycle;
  • A3 is selected from: -C(O)-, -C(O)NRl0-, -C(O)O- and S(O) m ;
  • Z is unsubstituted or substituted phenyl, unsubstituted or substituted napthyl, unsubstituted or substituted pyridyl, unsubstituted or substituted quinoline or unsubstituted or substituted 1,2 methylenedioxybenzene;
  • Rib is independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, R OO-, -N(RlO) 2 or C2-C6 alkenyl, c) C1-C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, RlOO-, or -N(RlO) ;
  • R9a is hydrogen, C l -C6 alkyl or chloro
  • RlO is independently selected from hydrogen, C1-C6 alkyl, benzyl and aryl;
  • Rl 1 is independently selected from C1-C6 alkyl and aryl
  • A3 is -C(O)-
  • the compounds useful in the methods of the present invention may have asymmetric centers and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers, including optical isomers, being included in the present invention.
  • any variable e.g. aryl, heterocycle, Rl, R2 etc.
  • its definition on each occmrence is independent at every other occurrence.
  • combinations of substituents/or variables described for the compounds hereinabove are permissible only if such combinations result in stable compounds.
  • heterocycle or heterocyclic represents a stable 5- to 7-membered monocyclic or stable 8- to 11 -membered bicyclic heterocyclic ring which is either saturated or unsaturated, and which consists of carbon atoms and from one to four heteroatoms selected from the group consisting of N, O, and S, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring.
  • heterocycle or heterocyclic includes heteroaryl moieties.
  • the heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure.
  • the substituted group is intended to mean a substituted Ci-8 alkyl, substituted C2-8 alkenyl, substituted C2-8 alkynyl, substituted aryl or substituted heterocycle from which the substituent(s) R2 and R3 are selected.
  • C6-C10 multicyclic alkyl ring in the definition of R c in formula C wherein two Rlcs are combined is intended to include polycyclic saturated and unsaturated aliphatic hydrocarbon groups having the specified number of carbon atoms. Examples of such cycloalkyl groups includes, but are not limited to:
  • cyclic moieties When R and R3 are combined to form -(CH2) ⁇ r, and when two Rl s on the same carbon are combined with that carbon to form a C4-C6 cycloalkyl, cyclic moieties are formed. Examples of such cyclic moieties include, but are not limited to:
  • Lines drawn into the ring systems from substituents indicate that the indicated bond may be attached to any of the substitutable ring carbon atoms.
  • R is selected from: hydrogen,
  • Al and A are independently selected from: a bond, -C(O)NRl0-, -NRlOC(O)-, O, -N(RlO)-, -S(O)2N(RlO)- and -N(RlO)S(O)2-. Most preferably, Al and A are a bond.
  • Z is selected from methylcyclohexyl, dimethylcyclohexyl and trimethylcyclohexyl, optionally substituted with one or two moieties selected from the following: a) Ci-4 alkoxy, b) NR6R7, c) C3-6 cycloalkyl, d) -NR6C(O)R7, e) HO, f) -S(O) m R6a g) halogen, or h) perfluoroalkyl.
  • Z is selected from phenyl and pyridyl, optionally substituted with one or two moieties selected from the following: a) Ci-4 alkoxy, b) NR6R7, c) C3-6 cycloalkyl, d) -NR6C(O)RV, e) HO, f) -S(O) m R6a g) halogen, or h) perfluoroalkyl.
  • p is 1, 2 or 3.
  • s is 0.
  • t is 1.
  • v 0, 1 or 2.
  • the moiety Al(CRl a 2) n A (CRla2) n is not a bond. It is intended that the definition of any substituent or variable (e.g.,
  • Rl , R9 5 n, etc. at a particular location in a molecule be independent of its definitions elsewhere in that molecule.
  • -N(RlO)2 represents -NHH, -NHCH3, -NHC2H5, etc. It is understood that substituents and substitution patterns on the compounds of the instant invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials.
  • the pharmaceutically acceptable salts of the compounds useful in the methods of this invention include the conventional non-toxic salts of the compounds of this invention as formed, e.g., from non-toxic inorganic or organic acids.
  • such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like: and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic and the like.
  • the pharmaceutically acceptable salts of the compounds useful in the methods of this invention can be synthesized from the compounds of this invention which contain a basic moiety by conventional chemical methods.
  • the salts are prepared either by ion exchange chromatography or by reacting the free base with stoichiometric amounts or with an excess of the desired salt- forming inorganic or organic acid in a suitable solvent or various combinations of solvents.
  • Reactions used to generate the compounds useful in the methods of this invention are prepared by employing reactions as shown in the Schemes 1-23, in addition to other standard manipulations such as ester hydrolysis, cleavage of protecting groups, etc., as may be known in the literature or exemplified in the experimental procedures.
  • the product VI can be reacted with a suitably substituted carboxylic acid to provide the piperazine VII; a final acid deprotection as previously described gives the intermediate VIII (Scheme 2).
  • the intermediate VIII can itself be reductively alkylated with a variety of aldehydes, such as IX.
  • the aldehydes can be prepared by standard procedures, such as that described by O. P. Goel, U. Krolls, M. Stier and S. Kesten in Organic Syntheses, 1988, 67, 69-75, from the appropriate amino acid (Scheme 3).
  • XV can be accomplished by literature procedures.
  • Scheme 9 illustrates synthesis of an instant compound wherein a non-hydrogen R9b is inco ⁇ orated in the instant compound.
  • a readily available 4-substituted imidazole XXVI may be selectively iodinated to provide the 5-iodoimidazole XXVII. That imidazole may then be protected and coupled to a suitably substituted benzyl moiety to provide intermediate XXVffl. Attachment of the imidazolyl nitrogen via an ethyl linker to the piperazine nitrogen of intermediate XXI, described above, provides the instant compound XXIX.
  • the piperazine Vm is reductively alkylated with an aldehyde which also has a protected hydroxyl group, such as XXXIII in Scheme 11 , the protecting groups can be subsequently removed to unmask the hydroxyl group.
  • the Boc protected amino alcohol XXX1N can then be utilized to synthesize 2- aziridinylmethylpiperazines such as XXXV.
  • 5-Substituted piperazin-2-ones can be prepared as shown in Scheme 12. Reductive amination of Boc-protected amino aldehydes XXXVI (prepared from I as illustrated) gives rise to compound XXXVH This is then reacted with bromoacetyl bromide under Schotten-Baumann conditions; ring closure is effected with a base such as sodium hydride in a polar aprotic solvent such as dimethylformamide to give XXX Vm The carbamate protecting group is removed under acidic conditions such as trifluoroacetic acid in methylene chloride, or hydrogen chloride gas in methanol or ethyl acetate, and the resulting piperazine can then be carried on to final products as described in above.
  • acidic conditions such as trifluoroacetic acid in methylene chloride, or hydrogen chloride gas in methanol or ethyl acetate
  • the isomeric 3-substituted piperazin-2-ones can be prepared as described in Scheme 13.
  • the imine formed from arylcarboxamides IXL and 2- aminoglycinal di ethyl acetal (XL) can be reduced under a variety of conditions, including sodium triacetoxyborohydride in dichloroethane, to give the amine XLI.
  • a suitably substituted amino acid I can be coupled to amine XLI under standard conditions, and the resulting amide XLII when treated with aqueous acid in tetrahydrofuran can cyclize to the unsaturated XLHI.
  • Catalytic hydrogenation under standard conditions gives the requisite intermediate XL1N, which is elaborated to final products as described in above.
  • Reaction Scheme 14 provides an illustrative example of the synthesis of compounds of the instant invention wherein the substituents R and R3 are combined to form -(CH2)u-- For example, 1-aminocyclohexane-l-carboxylic acid
  • Intermediate XLVm may be deprotected and reductively alkylated or acylated as illustrated in the previous Schemes.
  • the hydroxyl moiety of intermediate XLVHI may be mesylated and displaced by a suitable nucleophile, such as the sodium salt of ethane thiol, to provide an intermediate IL.
  • Intermediate XL VIII may also be oxidized to provide the carboxylic acid on intermediate L, which can be utilized form an ester or amide moiety.
  • Amino acids of the general formula LI which have a sidechain not found in natural amino acids may be prepared by the reactions illustrated in Scheme 16 starting with the readily prepared imine LIT.
  • Schemes 17-20 illustrate syntheses of suitably substituted aldehydes useful in the syntheses of the instant compounds wherein the variable W is present as a pyridyl moiety. Similar synthetic strategies for preparing alkanols that inco ⁇ orate other heterocyclic moieties for variable W are also well known in the art. For example, Scheme 21 illustrates the preparation of the corresponding quinoline aldehyde.
  • therapeutically effective amount means that amount of the compound that inhibits GGTase-I, and other active ingredients useful in the instant method invention, that will elicit the desired therapeutic effect or response or provide the desired benefit when administered in accordance with the desired treatment regimen.
  • a preferred therapeutically effective amount is a bone reso ⁇ tion inhibiting amount.
  • “Pharmaceutically acceptable” as used herein means generally suitable for administration to a mammal, including humans, from a toxicity or safety standpoint.
  • the compound that inhibits GGTase-I is typically administered for a sufficient period of time until the desired therapeutic effect is achieved.
  • the term "until the desired therapeutic effect is achieved”, as used herein, means that the therapeutic agent or agents are continuously administered, according to the dosing schedule chosen, up to the time that the clinical or medical effect sought for the disease or condition being mediated is observed by the clinician or researcher.
  • the compounds are continuously administered until the desired change in bone mass or structure is observed. In such instances, achieving an increase in bone mass or a replacement of abnormal bone structure with normal bone structure are the desired objectives.
  • the compounds are continuously administered for as long as necessary to prevent the undesired condition. In such instances, maintenance of bone mass density is often the objective.
  • Nonlimiting examples of administration periods can range from about 2 weeks to the remaining lifespan of the mammal.
  • administration periods can range from about 2 weeks to the remaining lifespan of the human, preferably from about 2 weeks to about 20 years, more preferably from about 1 month to about 20 years, more preferably from about 6 months to about 10 years, and most preferably from about 1 year to about 10 years.
  • the compounds useful in the methods of this invention may be administered to mammals, preferably humans, either alone or, preferably, in combination with pharmaceutically acceptable carriers, excipients or diluents, in a pharmaceutical composition, according to standard pharmaceutical practice.
  • the compounds can be administered orally or parenterally, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of administration.
  • compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.
  • Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, microcrystalline cellulose, sodium crosscarmellose, corn starch, or alginic acid; binding agents, for example starch, gelatin, polyvinyl-pyrrolidone or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc.
  • the tablets may be uncoated or they may be coated by known techniques to mask the unpleasant taste of the drug or delay disintegration and abso ⁇ tion in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a water soluble taste masking material such as hydroxypropylmethyl-cellulose or hydroxypropylcellulose, or a time delay material such as ethyl cellulose, cellulose acetate buryrate may be employed.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water soluble carrier such as polyethyleneglycol or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
  • Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene- oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan
  • the aqueous suspensions may also contain one or more preservatives, for example ethyl, or n- propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame.
  • Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as butylated hydroxyanisol or alpha-tocopherol.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • the pharmaceutical compositions useful in the methods of the invention may also be in the form of an oil-in-water emulsions.
  • the oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these.
  • Suitable emulsifying agents may be naturally- occurring phosphatides, for example soy bean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions may also contain sweetening, flavoring agents, preservatives and antioxidants.
  • Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, flavoring and coloring agents and antioxidant.
  • sweetening agents for example glycerol, propylene glycol, sorbitol or sucrose.
  • Such formulations may also contain a demulcent, a preservative, flavoring and coloring agents and antioxidant.
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous solutions.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • the sterile injectable preparation may also be a sterile injectable oil-in- water microemulsion where the active ingredient is dissolved in the oily phase.
  • the active ingredient may be first dissolved in a mixture of soybean oil and lecithin. The oil solution then introduced into a water and glycerol mixture and processed to form a microemulation.
  • the injectable solutions or microemulsions may be introduced into a patient's blood-stream by local bolus injection.
  • a continuous intravenous delivery device may be utilized.
  • An example of such a device is the Deltec CADD-PLUSTM model 5400 intravenous pump.
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension for intramuscular and subcutaneous administration.
  • This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this pu ⁇ ose any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • compositions useful in the instant methods may also be administered in the form of a suppositories for rectal administration of the drug.
  • These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol.
  • suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • Such materials include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol.
  • creams, ointments, jellies, solutions or suspensions, etc. containing the compound useful
  • compositions are intended to encompass a product comprising the specified ingredients in the specific amounts, as well as any product which results, directly or indirectly, from combination of the specific ingredients in the specified amounts.
  • the daily dosage will normally be determined by the prescribing physician with the dosage generally varying according to the age, weight, sex and response of the individual patient, as well as the severity of the patient's symptoms. Appropriate amounts can be determined by routine experimentation from animal models and human clinical studies. Generally, an appropriate amount of the compound that inhibits GGTase-I is chosen to obtain a bone reso ⁇ tion inhibiting effect, i.e. a bone reso ⁇ tion inhibiting amount of the compound that inhibits GGTase- I is administered.
  • a suitable amount of compound is administered to a mammal for the prevention or inhibition of bone reso ⁇ tion.
  • Administration occurs in an amount between about 0.1 mg/kg of body weight to about 60 mg/kg of body weight per day, preferably of between 0.5 mg/kg of body weight to about 40 mg/kg of body weight per day.
  • the compounds described above may also be co-administered with other well known therapeutic agents that are selected for their particular usefulness against the condition that is being treated.
  • the compounds of the instant invention may be co-administered with other well known compounds useful in the treatment and prevention of bone reso ⁇ tion and osteoporosis. Included in such combinations of therapeutic agents are combinations of the instant geranylgeranyl- protein transferase type I inhibitors and a bisphosphonate active or a pharmaceutically acceptable salt thereof.
  • bisphosphonate actives are defined herein to be distinct from and not to include the geranylgeranyl-protein transferase type I inhibitors of the present invention.
  • the geranylgeranyl-protein transferase type I inhibitor may itself be a bisphosphonate.
  • nitrogen-containing means that the bisphosphonate compound or pharmaceutically acceptable salt thereof comprises at least one nitrogen atom in the bisphosphonate portion of the molecule.
  • any nitrogen atom contained in the positive counter ion of such a salt e.g., the nitrogen atom of an ammonium counter ion, would not be considered in meeting the "nitrogen- containing" definition.
  • alendronic acid i.e. 4-amino-l- hydroxybutylidene-l,l-bisphosphonic acid is an example of a nitrogen-containing bisphosphonate.
  • ammonium salt of the unsubstituted 1- hydroxybutylidene- 1 , 1 -bisphosphonic acid would not be a nitrogen-containing bisphosphonate as defined herein.
  • n is an integer from 0 to 7 and wherein A and X are independently selected from the group consisting of H, OH, halogen, NH2, SH, phenyl, C1-C30 alkyl, C3- C30 branched or cycloalkyl, Ci -C30 substituted alkyl, C1-C10 alkyl substituted NH2, C3-C10 branched or cycloalkyl substituted NH2, Ci -Ci 0 dialkyl substituted NH2, C3-C10 branched or cycloalkyl disubstituted NH2, C1 -C10 alkoxy, C1-C10 alkyl substituted thio, thiophenyl, halophenylthio, C1-C10 alkyl substituted phenyl, pyridyl, furanyl, pyrrolidinyl, imidazolyl, imidazopyridinyl, and benzyl, such that both A and
  • the alkyl groups can be straight, branched, or cyclic, provided that sufficient atoms are selected for the chemical formula.
  • the Ci -C30 substituted alkyl can include a wide variety of substituents, nonlimiting examples which include those selected from the group consisting of phenyl, pyridyl, furanyl, pyrrolidinyl, imidazonyl, N ⁇ 2, Cl -Cl 0 alkyl or dialkyl substituted NH2, OH, SH, and Cl-Cio alkoxy.
  • the foregoing chemical formula Bp is also intended to encompass complex carbocyclic, aromatic and hetero atom structures for the A and/or X substituents, nonlimiting examples of which include naphthyl, quinolyl, isoquinolyl, adamantyl, and chlorophenylthio.
  • a non-limiting class of structures of bisphosphonate compounds useful in the instant invention are those in which A is selected from the group consisting of H, OH, and halogen, X is selected from the group consisting of C1-C30 alkyl, Ci -C30 substituted alkyl, halogen, and Cl -C10 alkyl or phenyl substituted thio, and n is 0.
  • a non-limiting subclass of structures of bisphosphonate compounds useful in the instant invention are those in which A is selected from the group consisting of H, OH, and Cl, X is selected from the group consisting of C1 -C30 alkyl, C1-C30 substituted alkyl, Cl, and chlorophenylthio, and n is 0.
  • a non-limiting example of the subclass of structures of bisphosphonate compounds useful in the instant invention is when A is OH and X is a 3-aminopropyl moiety, and n is 0, so that the resulting compound is a 4-amino-l,-hydroxybutylidene- 1,1 -bisphosphonate, i.e. alendronate.
  • salts include those selected from the group consisting alkali metal, alkaline metal, ammonium, and mono-, di, tri-, or tetra-C ⁇ -C30-alkyl-substituted ammonium.
  • Preferred salts are those selected from the group consisting of sodium, potassium, calcium, magnesium, and ammonium salts.
  • derivatives include those selected from the group consisting of esters, hydrates, and amides.
  • bisphosphonate and “bisphosphonates”, as used herein in referring to the therapeutic agents of the present invention are meant to also encompass diphosphonates, biphosphonic acids, and diphosphonic acids, as well as salts and derivatives of these materials.
  • the use of a specific nomenclature in referring to the bisphosphonate or bisphosphonates is not meant to limit the scope of the present invention, unless specifically indicated. Because of the mixed nomenclature currently in use by those or ordinary skill in the art, reference to a specific weight or percentage of a bisphosphonate compound in the present invention is on an acid active weight basis, unless indicated otherwise herein.
  • the phrase "about 5 mg of a bisphosphonate selected from the group consisting of alendronate, pharmaceutically acceptable salts thereof, and mixtures thereof, on an alendronic acid active weight basis” means that the amount of the bisphosphonate compound selected is calculated based on 5 mg of alendronic acid. For other bisphosphonates, the amount of bisphosphonate is calculated based on the corresponding bisphosphonic acid.
  • Nonlimiting examples of bisphosphonates useful herein include the following:
  • Alendronate also known as alendronate sodium or alendronate monosodium trihydrate
  • 4-amino-l-hydroxybutylidene- 1,1 -bisphosphonic acid monosodium trihydrate 4-amino-l-hydroxybutylidene- 1,1 -bisphosphonic acid monosodium trihydrate.
  • 1,1 -dichloromethylene- 1 , 1 -diphosphonic acid (clodronic acid), and the disodium salt (clodronate, Procter and Gamble) are described in Belgium Patent 672,205 (1966) and J. Org. Chem 32, 4111 (1967), both of which are inco ⁇ orated by reference herein in their entirety.
  • a non-limiting class of bisphosphonates useful in the instant invention are selected from the group consisting of alendronate, cimadronate, clodronate, tiludronate, etidronate, ibandronate, neridronate, olpandronate, risedronate, piridronate, pamidronate, zolendronate, pharmaceutically acceptable salts thereof, and mixtures thereof.
  • a non-limiting subclass of the above-mentioned class in the instant case is selected from the group consisting of alendronate, pharmaceutically acceptable salts thereof, and mixtures thereof.
  • a non-limiting example of the subclass is alendronate monosodium trihydrate.
  • a bisphosphonate that may be utilized in the instant method will vary with the dosing schedule, the particular bisphosphonate chosen, the age, size, sex and condition of the mammal or human, the nature and severity of the disorder to be treated, and other relevant medical and physical factors. Thus, a precise therapeutically effective amount cannot be specified in advance and can be readily determined by the caregiver or clinician. Appropriate amounts can be determined by routine experimentation from animal models and human clinical studies. Generally, an appropriate amount of bisphosphonate is chosen to obtain a bone reso ⁇ tion inhibiting effect, i.e. a bone reso ⁇ tion inhibiting amount of the nitrogen-containing bisphosphonate is administered. For humans, an effective oral dose of nitrogen-containing bisphosphonate is typically from about 1.5 to about 6000 ⁇ g/kg body weight and preferably about 10 to about 2000 ⁇ g/kg of body weight.
  • alendronate monosodium trihydrate common human doses which are administered are generally in the range of about 2 mg/day to about 40 mg/day, preferably about 5 mg/day to about 40 mg/day.
  • presently approved dosages for alendronate monosodium trihydrate are 5 mg/day for preventing osteoporosis, 10 mg/day for treating osteoporosis, and 40 mg/day for treating Paget's disease.
  • the bisphosphonate can be administered at intervals other than daily, for example once-weekly dosing, twice-weekly dosing, biweekly dosing, and twice-monthly dosing.
  • appropriate multiples of the bisphosphonate dosage would be administered.
  • alendronate monosodium trihydrate would be administered at dosages of 35 mg/week or 70 mg/week in lieu of seven consecutive daily dosages of 5 mg or 10 mg.
  • the pharmaceutical compositions herein comprise from about 1 mg to about 100 mg of bisphosphonate, preferably from about 2 mg to 70 mg, and more preferably from about 5 mg to about 70, on a bisphosphonic acid basis.
  • the pharmaceutical compositions useful herein comprise about 2.5 mg, 5 mg, 10 mg, 35, mg, 40 mg, or 70 mg of the active on an alendronic acid active weight basis.
  • Such agents include those selected from the group consisting of calcitonin, estrogens, progesterone, androgens, calcium supplements, fluoride, growth hormone secretagogues, vitamin D analogues, and selective estrogen receptor modulators.
  • the calcitonins useful herein can be from human or nonhuman sources, e.g. salmon calcitonin.
  • Nonlimiting examples of estrogens include estradiol.
  • Nonlimiting examples of selective estrogen receptor modulators include raloxifene, iodoxifene, and tamoxifene. Growth horomone secretagogues are described in U.S. Patent No. 5,536,716, to Chen et al., issued July 16, 1996, which is inco ⁇ orated by reference herein in its entirety.
  • the inhibitor of geranylgeranyl-protein transferase may also be useful in combination with an integrin antagonist for the inhibition or prevention of bone reso ⁇ tion.
  • an integrin antagonist refers to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to an integrin(s) that is involved in the regulation of angiogenisis, or in the growth and invasiveness of tumor cells.
  • the term refers to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the ⁇ v ⁇ 3 integrin, which selectively antagonize, inhibit or counteract binding of a physiological ligand to the ⁇ v ⁇ 5 integrin, which antagonize, inhibit or counteract binding of a physiological ligand to both the ⁇ v ⁇ 3 integrin and the ⁇ v ⁇ 5 integrin, or which antagonize, inhibit or counteract the activity of the particular integrin(s) expressed on capillary endothelial cells.
  • the term also refers to antagonists of the ⁇ v ⁇ , ⁇ v ⁇ 8, ⁇ l ⁇ l, ⁇ 2 ⁇ l, ⁇ 5 ⁇ l, ⁇ 6 ⁇ l and ⁇ 6 ⁇ 4 integrins.
  • the term also refers to antagonists of any combination of ⁇ v ⁇ 3, ⁇ v ⁇ 5, ⁇ v ⁇ , ⁇ v ⁇ 8, ⁇ l ⁇ l, ⁇ 2 ⁇ l, ⁇ 5 ⁇ l, ⁇ l and ⁇ 6 ⁇ 4 integrins.
  • the instant compounds may also be useful with other agents that inhibit angiogenisis and thereby inhibit the growth and invasiveness of tumor cells, including, but not limited to angiostatin and endostatin.
  • combination products employ the combinations of this invention within the dosage range described below and the other pharmaceutically active agent(s) within its approved dosage range.
  • Combinations of the instant invention may alternatively be used sequentially with known pharmaceutically acceptable agent(s) when a multiple combination formulation is inappropriate.
  • the inhibitor of geranylgeranyl-protein transferase may also be useful in combination with an inhibitor of a cysteine cathepsin for the inhibition or prevention of bone reso ⁇ tion.
  • a combination of the instant inhibitors of geranylgeranyl protein transferase and an inhibitor(s) of cathepsin K and L may be useful in the treatment of osteoporosis and the prevention of bone reso ⁇ tion.
  • Cathepsin K formerly known as cathepsin 02, is a cysteine protease and is described in PCT International Application Publication No. WO 96/13523, published May 9, 1996; U.S. Patent No. 5,501,969, issued March 3, 1996; and U.S. Patent No.
  • compositions further comprising an active ingredient selected from the group consisting of a) an anabolic agent, b) an estrogen receptor modulator, c) an androgen receptor modulator, d) a cytotoxic/antiproliferative agent, e) a matrix metalloproteinase inhibitor, f) an inhibitor of epidermal-derived, fibroblast-derived, or platelet- derived growth factors, g) an inhibitor of VEGF, h) an antibody to a growth factor or to a growth factor receptor, i) an inhibitor ofFlk-l/KDR, Flt-l, Tck/Tie-2, or Tie-1, j) a sex steroid, k) a growth hormone secretagogue, 1) an inhibitor of osteoclast proton ATPase, and m) parathyroid hormone n) a bone mo ⁇ hogenetic protein and mixtures thereof.
  • an active ingredient selected from the group consisting of a) an anabolic agent, b) an estrogen receptor
  • the second active ingredient is selected from the group consisting of: a) an organic bisphosphonate or a pharmaceutically acceptable salt or ester thereof, b) an estrogen receptor modulator, c) an androgen receptor modulator, d) an inhibitor of osteoclast proton ATPase, and e) a cathepsin K inhibitor; and mixtures thereof.
  • Nonlimiting examples of estrogen receptor modulators include estrogen, progestin, estradiol, droloxifene, raloxifene, and tamoxifene.
  • Nonlimiting examples of cytotoxic/antiproliferative agents are taxol, vincristine, vinblastine, and doxorubicin.
  • composition is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
  • Method 1 (Hydrochloride salt): A 72 liter vessel was charged with 190 proof ethanol (14.4 L) followed by the addition of 4-cyanobenzylbromide (2.98 kg) and HMTA (2.18 kg) at ambient temperature. The mixture was heated to about 72- 75 °C over about 60 min. On warming, the solution thickens and additional ethanol (1.0 liter) was added to facilitate stirring. The batch was aged at about 72-75°C for about 30 min.
  • the mixture was allowed to cool to about 20°C over about 60 min, and HCl gas (2.20 kg) was sparged into the slurry over about 4 hours during which time the temperature rose to about 65°C.
  • the mixture was heated to about 70-72°C and aged for about 1 hour.
  • the slurry was cooled to about 30°C and ethyl acetate (22.3 L) added over about 30 min.
  • the slurry was cooled to about -5°C over about 40 min and aged at about -3 to about -5°C for about 30 min.
  • the mixture was filtered and the crystalline solid was washed with chilled ethyl acetate (3 x 3 L).
  • the solid was dried under a N2 stream for about 1 hour before charging to a 50 liter vessel containing water (5.5 L).
  • the pH was adjusted to about 10-10.5 with 50% NaOH (4.0 kg) maintaining the internal temperature below about 30°C.
  • methylene chloride 2.8 L was added and stirring continued for about 15 min.
  • the layers were allowed to settle and the lower organic layer was removed.
  • the aqueous layer was extracted with methylene chloride (2 x 2.2 L).
  • the combined organic layers were dried over potassium carbonate (650 g). The carbonate was removed via filtration and the filtrate concentrated in vacuo at about 25°C to give a free base as a yellow oil.
  • the oil was transferred to a 50 liter vessel with the aid of ethanol (1.8 L). Ethyl acetate (4.1 L) was added at about 25°C. The solution was cooled to about 15°C and HCl gas (600 g) was sparged in over about 3 hours, while keeping batch temperature below about 40°C. At about 20-25°C, ethyl acetate (5.8 L) was added to the slurry, followed by cooling to about -5°C over about 1 hour. The slurry was aged at about -5°C for about 1 hour and the solids isolated via filtration.
  • Method 2 (phosphate salt): A slurry of HMTA in 2.5 L EtOH was added gradually over about 30 min to about 60 min to a stirred slurry of cyanobenzyl- bromide in 3.5 L EtOH and maintained at about 48-53°C with heating & cooling in a 22L neck flask (small exotherm). Then the transfer of HMTA to the reaction mixture was completed with the use of 1.0 L EtOH. The reaction mixture was heated to about 68-73°C and aged at about 68-73°C for about 90 min. The reaction mixture was a slurry containing a granular precipitate which quickly settled when stirring stopped.
  • the mixture was cooled to a temperature of about 50°C to about 55°C.
  • Propionic acid was added to the mixture and the mixture was heated and maintained at a temperature of about 50°C to about 55°C.
  • Phosphoric acid was gradually added over about 5 min to about 10 min, maintaining the reaction mixture below about 65 °C to form a precipitate-containing mixture.
  • the mixture was gradually warmed to about 65°C to about 70°C over about 30 min and aged at about 65°C to about 70°C for about 30 min.
  • the mixture was then gradually cooled to about 20-25°C over about 1 hour and aged at about 20-25°C for about 1 hour.
  • the reaction slurry was then filtered.
  • the filter cake was washed four times with EtOH, using the following sequence, 2.5 L each time.
  • the filter cake was then washed with water five times, using 300 mL each time.
  • the filter cake was washed twice with MeCN (1.0 L each time) and the above identified compound was obtained.
  • Step 2 Preparation of 1 -(4-Cyanobenzyl)-2-mercapto-5- hydroxymethylimidazole
  • the mixture was heated to 70°C, and aged for 2 hours. The temperature of the mixture was then cooled to room temperature and was aged overnight.
  • the thioimidazole product was obtained by vacuum filtration.
  • the filter cake was washed four times acetonitrile (25 mL each time) until the filtrates became nearly colorless. Then the filter cake was washed three times with water (approximately 25-50 mL each time) and dried in vacuo to obtain the above-identified compound.
  • Step 3 Preparation of l-(4-Cvanobenzyl)-5-Hvdroxymethylimidazole
  • the solution was cooled to 20°C and quenched by slowly adding 20% aqueous Na2SO3 (25 mL) maintaining the temperature at less than 25°C.
  • the solution was filtered through a bed of DARCO G-60 (9.0 g) over a bed of SolkaFlok (1.9 g) in a sintered glass funnel. The bed was washed with 25 mL of 10% acetic acid in water.
  • the combined filtrates were cooled to 15°C and a 25% aqueous ammonia was added over a 30 minute period, maintaining the temperature below 25°C, to a pH of 9.3.
  • the yellowish slurry was aged overnight at 23°C (room temperature).
  • the solids were isolated via vacuum filtration.
  • the cake (100 mL wet volume) was washed with 2 x 250 mL 5% ammonia (25%) in water, followed by 100 mL of ethyl acetate.
  • the wet cake was dried with vacuum/N2 flow and the above- titled compound was obtained.
  • Step 4 Preparation of l-(4-cyanobenzyl)-5-chloromethyl imidazole HCl salt
  • Method 1 l-(4-Cyanobenzyl)-5-hydroxymethylimidazole (1.0 kg), as described in above in Step 3, was slu ⁇ ed with DMF (4.8 L) at 22°C and then cooled to -5°C. Thionyl chloride (390 mL) was added dropwise over 60 min during which time the reaction temperature rose to a maximum of 9°C. The solution became nearly homogeneous before the product began to precipitate from solution. The slurry was warmed to 26°C and aged for 1 h.
  • Method 2 To an ice cold solution of dry acetonitrile (3.2 L, 15 L/Kg hydroxymethyhmidazole) was added 99% oxalyl chloride (101 mL, 1.15 mol, 1.15 equiv.), followed by dry DMF (178 mL, 2.30 mol, 2.30 equiv.), at which time vigorous evolution of gas was observed. After stirring for about 5 to 10 min following the addition of DMF, solid hydroxymethyhmidazole (213 g, 1.00 mol), as described above in Step 3, was added gradually. After the addition, the internal temperature was allowed to warm to a temperature of about 23°C to about 25°C and stirred for about 1 to 3 hours.
  • the mixture was filtered, then washed with dry acetonitrile (400 mL displacement wash, 550 mL slurry wash, and a 400 mL displacement wash).
  • the solid was maintained under a N2 atmosphere during the filtration and washing to prevent hydrolysis of the chloride by adventitious H2O. This yielded the crystalline form of the chloromethylimidazole hydrochloride.
  • Method 3 To an ice cold solution of dry DMF (178 mL, 2.30 mol, 2.30 equiv.) in dry acetonitrile (2.56 L, 12 L/Kg Hydroxymethyhmidazole) was added oxalyl chloride (101 mL, 1.15 mol, 1.15 equiv). The heterogeneous mixture in the reagent vessel was then transferred to a mixture of hydroxymethyhmidazole (213 g, 1.00 mol), as described above in Step 3, in dry acetonitrile (1.7 L, 8 L/Kg hydroxymethyhmidazole).
  • Step 1 l-(4'-Cyanobenzyl) imidazol-5 -ylmethyl piperazine-4-carboxylic acid benzyl ester
  • Step 1 The product from Step 1 (6.17 mmol) was dissolved in absolute ethanol followed by the introduction of 10% Pd/C catalyst then hydrogen under atmospheric pressure. The catalyst was removed via filtration through filter-aid and the product was isolated by removing the solvent under reduced pressure.
  • Step 1 Preparation of l-(4'-Cyanobenzyl) imidazol-5-ylmethyl piperazine-4- carboxylic acid tgrt-butyl ester
  • Step 2 Preparation of 1 -(4 '-Cyanobenzyl) imidazol-5-ylmethyl piperazine trishydrochloride
  • Step 1 2,6-Dimethylcvclohexane methyl-(4-nitropheny)carbonate
  • a THF:acetonitrile (7:1, lOmL) solution of 4-nitrophenyl chloroformate (7.96 mmol) was added to a THF: acetonitrile (7:1, 20mL) solution of 2,6-dimethylcyclohexane methanol (7.24 mmol) at 25°C and then stined for 0.25 hour. Pyridine (7.96mmol) was then added dropwise over 4 minutes. Stirring was continued for 2 hours at 25°C and then the reaction was diluted with ethyl acetate and washed with water, a saturated sodium chloride solution, dried with sodium sulfate and then evaporated.
  • Step 2 l-(4'-Cyanobenzyl) imidazol-5-ylmethyl piperazine-4-carboxylic acid-
  • Step 1 (3,3,5,5-Tetramethyl)- 1 -cyclohexyl-(4'-nitrophenyl)carbonate
  • the title compound was prepared as described above in Example 3, Step 1 except starting with 3,3,5,5-tetramethyl cyclohexanol (6.40 mmol), 4- nitrophenyl chloroformate (7.04 mmol) and pyridine (7.04 mmol).
  • Step 2 l-(4'-Cyanobenzyl) imidazol-5-ylmethyl piperazine-4-carboxylic acid- (3,3,5,5 -tetramethvDcyclohexyl ester 1 -(4 ' -Cyanobenzyl) imidazol-5 -ylmethyl piperazine prepared as described in Example 2, Step 2 (0.35 mmol) and the product from Step 1 (0.42 mmol) were dissolved in dichloromethane and diisopropylethyl amine (0.84 mmol) and stirred for 18 hours at 25°C. The product was isolated via preperative hplc followed by lyophilization. FAB-MS: calc: 463.5 found: 464.3.
  • Step 1 (3,3-Dimethyl)cvclohexane-(4-nitrophenyl)carbonate
  • Example 5 The product from Example 5, Step 1 was prepared as described above in Example 3, Step 1 starting with 3,3-dimethyl cyclohexanol (1.07 mmol), 4- nitrophenyl chloroformate (1.18 mmol) and pyridine (1.18 mmol).
  • Step 2 1 -(4 '-Cyanobenzyl) imidazol-5-ylmethyl piperazine-4-carboxylic acid- (3,3-dimethyl)cvclohexyl ester l-(4'-Cyanobenzyl) imidazol-5 -ylmethyl piperazine prepared as described in Example 2, Step 2 (0.32 mmol) and the product from Example 4, Step 1 (0.32 mmol) were dissolved in dichloromethane and diisopropylethyl amine (0.84 mmol) and stined for 18 hours at 25°C. The product was isolated via preperative hplc followed by lyophilization. FAB-MS: calc: 435.6 found: 436.3.
  • Step 2 2-( 1.3.3-Trimethyl- 1 -cvclohexyDacetamide
  • the product from Step 1 (6.19 mmol) was refluxed in toluene and thionyl chloride (18.6 mmol) for 0.25 hour, concentrated to dryness and then retreated for an additional 0.25 hour.
  • the resulting 2-(l,3,3-Trimethyl-l-cyclohexyl)acetyl chloride was then dissolved in dioxane and added to an ammonia saturated dioxane solution at 0°C and stirred for 0.5 hour.
  • the dioxane was removed under reduced pressure and partitioned with water and ethyl acetate.
  • the organic layer was removed, dried with sodium sulfate and removed under reduced pressure.
  • FAB-MS calc: 183.4 found: 184.2.
  • Step 3 1 -Amino-2-(l ,3,3-trimethyl- 1 -cyclohexyDethane
  • Step 4 N-(tert-Butyloxy carbonyl)-N'-[2-(l,3,3-trimethyl-l-cyclohexyl)ethyl] diaminoethane
  • N-boc glycine aldehyde (12.42 mmol) as described in Org. Prep. Proc, Int., 25(4), 457-461, 1993.
  • l-amino-2-(l,3,3-trimethyl-l-cyclohexyl)ethane, N-boc glycine aldehyde and sodium acetate (8.28 mmol) were dissolved in 30 mL of MeOH.
  • Step 5 1 - ⁇ 2-( 1 ,3 ,3 -trimethyl- 1 -cyclohexyl)ethyl]piperazin- 1 -one
  • the reaction was partitioned between water and ethyl acetate and then the organic was washed successively with KHSO4, NaHCO3, NaCl and then dried with sodium sulfate and concentrated. The residue was then treated with 4N HCl in dioxane for 2.0 hours.
  • Step 6 (R,S) 1 -(4 '-Cyanobenzyl) imidazol-5-ylmethyl piperazin-3-one- ⁇ 4-[2-
  • the oil was purified by reversed phase prep LC to give the pure racemic product as a fluffy white amo ⁇ hous powder after concentration and lyophilization.
  • a 70 mg sample of the racemate was further purified by chiral prep LC (CHIRALPAK-AD) to give the earlier eluting enantiomer as a glass.
  • the glass was dissolved in a mixture of acetonitrile and trifluoroacetic acid, and was reconcentrated.
  • the concentrate was lyophilized form dioxane to give the bis trifluoroacetate salt of the earlier eluting enantiomer as an amo ⁇ hous white powder.
  • Step 1 S-Hexahydromandelic acid tert-butyldimethyl silyl ether
  • Step 2 1 -(4 '-Cyanobenzyl) imidazol-5-ylmethyl piperazine-4-(2-hydroxy-2- cyclohexypacetamide l-(4'-cyanobenzyl) imidazol-5-ylmethyl piperazine (0.25 mmol), l-(4'- Cyanobenzyl) imidazol-5-ylmethyl piperazine prepared as described in Example 2, Step 2 (0.26 mmol), HOBt (0.3 mmol), EDC (0.325 mmol) and N-methymo ⁇ holine (0.75 mmol) were dissolved in DMF and stirred at 25°C for 18 hours. The reaction was purified on a Ci 8 preperative hplc column and the title compound was isolated via lyophilization. FAB-MS: calc: 421.5 found: 422.3.
  • Step 2 1 -(4 '-Cyanobenzyl) imidazol-5-ylmethyl piperazine-4-(2-hydroxy-2- cyclohexyDcarboxamide
  • Step 1 Preparation of 2,6-Dimethoxybenzyloxy-(4-nitropheny)carbonate A THF: acetonitrile (7:1, 2 mL) solution of 4-nitrophenyl chloroformate
  • Step 1 4-Benzyloxycarbonyl-[l-(2-oxo-2-(adamant-l-yl))ethyl]piperazin-2- one
  • Step 2 1 - ⁇ 1 -(2-oxo-2-(adamant- 1 -yl))ethyl]piperazin-2-one
  • Step 3 1 -(4-Cyanobenzyl)-5-[ 1 -(2-oxo-2-(adamant- 1 -yl)ethyl)-2-oxo- piperazin-4-yl-methyl]imidazole l-[l-(2-Oxo-2-(adamant-l-yl))ethyl]piperazin-2-one (253.4 mg, 0.81 mmol) was placed into a round bottomed flask and diisopropylethylamine (0.71ml), acetonitrile (20.0 ml) and l-(4-cyanobenzyl-5-chloromethyl)imidazole hydrochloride (prepared as described in Example 1) (217.2 mg, 0.81 mmol).
  • step 2 To 1 -(4 '-cyanobenzyl) imidazol-5-ylmethyl piperazine Example 2, step 2 (0.445 mmol) in ethyl acetate and methanol was added the commercially available 1-adamantyl isocyanate (0.423 mmol) and diisopropylethyl amine (0.89 mmol). After stirring for 18 hours the reaction was purified via preparative hplc and the title compound was then isolated via lyophilization.
  • Step 1 1 -(4 '-Cyanobenzyl) imidazol-5-ylmethyl piperazine-4-carboxylic acid-
  • the reaction was stirred overnight and then injected onto a C18 preparative HPLC column and purified with a mixed gradient of 5%-95% acetonitrile/0.1% TFA; 95%-5%/0.1% aqueous TFA over 15 min.
  • the title compound was isolated after conversion to the hydrochloride salt.
  • the reaction was stined overnight and then injected onto a Cl 8 preparative HPLC column and purified with a mixed gradient of 5%-95% acetonitrile/0.1% TFA; 95%-5%/0.1% aqueous TFA over 15 min.
  • the title compound was isolated after conversion to the hydrochloride salt.
  • the reaction was stined overnight and then injected onto a C18 preparative HPLC column and purified with a mixed gradient of 5%-95% acetonitrile/0.1% TFA; 95%-5% 0.1% aqueous TFA over 15 min.
  • the title compound was isolated after conversion to the hydrochloride salt.
  • the reaction was heated for 2 hours, filtered, and then injected onto a Cl 8 preparative HPLC column and purified with a mixed gradient of 5%-95% acetonitrile/0.1% TFA; 95%-5%/0.1% aqueous TFA over 15 min.
  • the title compound was isolated after conversion to the hydrochloride salt.
  • Step 2 Preparation of 6-diethylaminopyridine-2-carboxylic acid
  • the ester from step 1 (2g, 9.0 mmol) and NaOH (IM, 50ml) were stined in MeOH (50ml) at reflux for 3hrs.
  • the reaction was concentrated in vacuo.
  • the residue was dissolved in methylene chloride (15ml) and HCl (IM in ether, 5ml) was added.
  • the solvent was removed in vacuo.
  • the crude product was purified by a C18 preparative HPLC column with a mixed gradient of 5%-95% acetonitrile/0.1% TFA; 95%-5%/0.1% aqueous TFA over 15 min and the title compound was isolated.
  • Step 3 Preparation of 4-[ 1 -(4-Cyanobenzyl)imidazol-5-ylmethyl]- 1 -(6- diethylamino-pyrid-2-oyl)piperazine trihydrochloride
  • the reaction was concentrated in vacuo and then worked up with ethyl acetate and H 2 O.
  • the crude product was purified by a C18 preparative HPLC column with a mixed gradient of 5%-95% acetonitrile/0.1% TFA; 95%-5%/0.1% aqueous TFA over 15 min.
  • the title compound was isolated after conversion to the hydrochloride salt.
  • Compounds useful in the instant methods of treatment may also be identified by assays that determine the extent of inhibition by the compound of the activity of a prenyl-protein transferase, in particular geranylgeranyl-protein transferase type I (GGTase-I). While any inhibitor of GGTase-I is useful in the instant methods of treatment, preferably the compounds useful in the instant methods inhibit GGTase- I in vitro (Example 29) at an IC50 of less than 1000 nM. More preferably, the compounds useful in the instant methods inhibit GGTase-I in vitro at an IC50 of less than 50 nM. Such compounds that are useful in the instant methods may be generally described as inhibitors of prenyl-protein transferase.
  • the compounds useful in the instant methods inhibit the cellular processing of the Rapl protein at a MIC of less than about 20 nM (Example 31). More preferably, the compounds useful in the instant methods inhibit the cellular processing of the Rapl protein at an EC50 of less than about 50 nM (Example 31).
  • the inhibitors of prenyl- protein transferase are dual inhibitors of farnesyl-protein transferase and geranylgeranyl-protein transferase type I (GGTase-I).
  • the compounds are further characterized in that the inhibitory activity of the compounds against GGTase-I is greater than the inhibitory activity against FPTase.
  • the compounds of this subembodiment of the instant invention inhibit FPTase in vitro (Example 30) at an ICsoof less than 1 ⁇ M and inhibit GGTase-I in vitro (Example 29) at an IC50 of less than 50 nM.
  • the compounds of this subembodiment of the instant invention inhibit the cellular processing of the Rapl protein (Example 31) at an EC50 of less than 50 nM. Also more preferably, the ratio of the IC50 of the compounds of this subembodiment of the instant invention for in vitro inhibition of FPTase to the IC50 of the compounds of the instant invention for in vitro inhibition of GGTase type I is greater than 25.
  • the compounds are further characterized in that the inhibitory activity of the compounds against FPTase is greater than or equal to the inhibitory activity against GGTase-I.
  • the compounds of this second subembodiment of the instant invention inhibit FPTase in vitro (Example 30) at an IC50 of less than 100 nM and inhibit GGTase-I in vitro (Example 29) at an IC50 of less than 1 ⁇ M.
  • the compounds of this second subembodiment of the instant invention inhibit the cellular processing of the Rapl protein (Example 31) at an EC50 of less than 200 nM.
  • test procedures to employ to measure prenyl-protein transferase activity and the bone reso ⁇ tion inhibiting activity of the compounds of the present invention are described below.
  • the modified geranylgeranyl-protein transferase inhibition assay is carried out at room temperature.
  • a typical reaction contains (in a final volume of 50 ⁇ L): [3H]geranylgeranyl diphosphate, biotinylated Ras peptide, 50 mM HEPES, pH 7.5, a modulating anion (for example 10 mM glycerophosphate or 5 mM ATP), 5 mM MgCl2, 10 ⁇ M ZnCl2, 0.1% PEG (15,000-20,000 mw), 2 mM dithiothreitol, and geranylgeranyl-protein transferase type I (GGTase-I).
  • the GGTase-type I enzyme employed in the assay is prepared as described in U.S. Pat. No. 5,470,832, inco ⁇ orated by reference.
  • the Ras peptide is derived from the K4B-Ras protein and has the following sequence: biotinyl-GKKKKKKSKTKCVIM (single amino acid code). Reactions are initiated by the addition of GGTase and stopped at timed intervals (typically 15 min) by the addition of 200 ⁇ L of a 3 mg/mL suspension of streptavidin SPA beads (Scintillation Proximity Assay beads, Amersham) in 0.2 M sodium phosphate, pH 4, containing 50 mM EDTA, and 0.5% BSA. The quenched reactions are allowed to stand for 2 hours before analysis on a Packard TopCount scintillation counter.
  • IC50 values are determined with Ras peptide near KM concentrations. Enzyme and substrate concentrations for inhibitor IC50 determinations are as follows: 75 pM GGTase-I, 1.6 ⁇ M Ras peptide, 100 nM geranylgeranyl diphosphate.
  • Isoprenyl-protein transferase activity assays are canied out at 30°C unless noted otherwise.
  • a typical reaction contains (in a final volume of 50 ⁇ L): [3H]farnesyl diphosphate, Ras protein, 50 mM HEPES, pH 7.5, 5 mM MgCl2, 5 mM dithiothreitol, 10 ⁇ M ZnCl2, 0.1% polyethyleneglycol (PEG) (15,000-20,000 mw) and isoprenyl-protein transferase.
  • the FPTase employed in the assay is prepared by recombinant expression as described in Omer, C.A., Krai, A.M., Diehl, R.E., Prendergast, G.C., Powers, S., Allen, CM., Gibbs, J.B. and Kohl, N.E. (1993) Biochemistry 32:5167-5176. After thermally pre-equilibrating the assay mixture in the absence of enzyme, reactions are initiated by the addition of isoprenyl- protein transferase and stopped at timed intervals (typically 15 min) by the addition of 1 M HCl in ethanol (1 mL). The quenched reactions are allowed to stand for 15 m (to complete the precipitation process).
  • inhibitors are prepared as concentrated solutions in 100% dimethyl sulfoxide and then diluted 20-fold into the enzyme assay mixture.
  • Substrate concentrations for inhibitor IC50 determinations are as follows: FTase, 650 nM Ras-CVLS, 100 nM farnesyl diphosphate.
  • Protocol A PSN-1 (human pancreatic carcinoma) or viral-K4B-ras-transformed
  • Ratl cells are used for analysis of protein processing. Subconfluent cells in 100 mm dishes are fed with 3.5 ml of media (methionine-free RPMI supplemented with 2% fetal bovine serum or cysteine-free/methionine-free DMEM supplemented with 0.035 ml of 200 mM glutamine (Gibco), 2% fetal bovine serum, respectively) containing the desired concentration of test compound, lovastatin or solvent alone. Cells treated with lovastatin (5-10 ⁇ M), a compound that blocks Ras processing in cells by inhibiting a rate-limiting step in the isoprenoid biosynthetic pathway, serve as a positive control.
  • media methionine-free RPMI supplemented with 2% fetal bovine serum or cysteine-free/methionine-free DMEM supplemented with 0.035 ml of 200 mM glutamine (Gibco), 2% fetal bovine serum, respectively.
  • Test compounds are prepared as lOOOx concentrated solutions in DMSO to yield a final solvent concentration of 0.1%. Following incubation at 37°C for two hours 204 ⁇ Ci/ml [35s]Pro-Mix (Amersham, cell labeling grade) is added. After introducing the label amino acid mixture, the cells are incubated at 37°C for an additional period of time (typically 6 to 24 hours). The media is then removed and the cells are washed once with cold PBS.
  • the cells are scraped into 1 ml of cold PBS, collected by centrifugation (10,000 x g for 10 sec at room temperature), and lysed by vortexing in 1 ml of lysis buffer (1% Nonidet P-40, 20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% deoxycholate, 0.1% SDS, 1 mM DTT, 10 ⁇ g/ml AEBSF, 10 ⁇ g/ml aprotinin, 2 ⁇ g/ml leupeptin and 2 ⁇ g/ml antipain). The lysate is then centrifuged at 15,000 x g for 10 min at 4°C and the supernatant saved.
  • lysis buffer 1% Nonidet P-40, 20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% deoxycholate, 0.1% SDS, 1 mM DTT, 10 ⁇ g/m
  • Rap 1 For immunoprecipitation of Rap 1 , samples of lysate supernatant containing equal amounts of protein are utilized. Protein concentration is determined by the bradford method utilizing bovine serum albumin as a standard. The appropriate volume of lysate is brought to 1 ml with lysis buffer lacking DTT and 2 ⁇ g of the Rapl antibody, Rapl/Krevl (121) (Santa Cruz Biotech), is added. The protein antibody mixture is incubated on ice at 4°C for 1 hour. The immune complex is collected on pansorbin (Calbiochem) by tumbling at 4°C for 45 minutes.
  • the pellet is washed 3 times with 1 ml of lysis buffer lacking DTT and protease inhibitors and resuspended in 100 ⁇ l elution buffer (10 mM Tris pH 7.4, 1% SDS).
  • the Rapl is eluted from the beads by heating at 95 °C for 5 minutes, after which the beads are pelleted by brief centrifugation (15,000 x g for 30 sec. at room temperature).
  • the supernatant is added to 1 ml of Dilution Buffer (0.1% Triton X- 100, 5 mM EDTA, 50 mM NaCl, 10 mM Tris pH 7.4) with 2 ⁇ g Rapl antibody, Rapl/Krevl (121) (Santa Cruz Biotech).
  • the second protein/antibody mixture is incubated on ice at 4°C for 1-2 hours.
  • the immune complex is collected on pansorbin (Calbiochem) by tumbling at 4°C for 45 minutes.
  • the pellet is washed 3 times with 1 ml of lysis buffer lacking DTT and protease inhibitors and resuspended in Laemmli sample buffer.
  • Rapl is eluted from the beads by heating at 95°C for 5 minutes, after which the beads are pelleted by brief centrifugation. The supernatant is subjected to SDS-PAGE on a 12% acrylamide gel (bis-acrylamide:acrylamide, 1 :100), and the Rapl visualized by fluorography.
  • PSN-1 cells are passaged every 3-4 days in 10cm plates, splitting near- confluent plates 1 :20 and 1 :40. The day before the assay is set up, 5x 106 cells are plated on 15 cm plates to ensure the same stage of confluency in each assay. The media for these cells is RPMI 1640 (Gibco), with 15% fetal bovine serum and lx
  • cells are collected from the 15 cm plates by trypsinization and diluted to 400,000 cells/ml in media. 0.5 ml of these diluted cells are added to each well of 24- well plates, for a final cell number of 200,000 per well.
  • the cells are then grown at 37°C overnight.
  • the compounds to be assayed are diluted in DMSO in 1/2-log dilutions.
  • the range of final concentrations to be assayed is generally 0.1-100 ⁇ M.
  • concentrations per compound is typical.
  • the compounds are diluted so that each concentration is lOOOx of the final concentration (i.e., for a 10 ⁇ M data point, a 10 mM stock of the compound is needed).
  • each lOOOx compound stock is diluted into 1 ml media to produce a 2X stock of compound.
  • a vehicle control solution (2 ⁇ L DMSO to 1 ml media), is utilized.
  • 0.5 ml of the 2X stocks of compound are added to the cells.
  • the media is aspirated from the assay plates.
  • Each well is rinsed with 1 ml PBS, and the PBS is aspirated. 180 ⁇ L SDS-PAGE sample buffer
  • RNAse/DNase mix is added per well. This mix is 1 mg/ml DNasel (Worthington Enzymes), 0.25 mg/ml Rnase A (Worthington Enzymes), 0.5 M Tris-HCl pH 8.0 and 50 mM MgCl2-
  • the plate is left on ice for 10 minutes. Samples are then either loaded on the gel, or stored at -70°C until use.
  • Each assay plate (usually 3 compounds, each in 4-point titrations, plus controls) requires one 15-well 14% Novex gel. 25 ⁇ l of each sample is loaded onto the gel. The gel is run at 15mA for about 3.5 hours. It is important to run the gel far enough so that there will be adequate separation between 21kd (Rapl) and 29kd (Rab6).
  • the gels are then transferred to Novex pre-cut PVDF membranes for 1.5 hours at 30V (constant voltage). Immediately after transferring, the membranes are blocked overnight in 20 ml Western blocking buffer (2% nonfat dry milk in Western wash buffer (PBS + 0.1% Tween-20). If blocked over the weekend, 0.02% sodium azide is added. The membranes are blocked at 4°C with slow rocking.
  • the blocking solution is discarded and 20 ml fresh blocking solution containing the anti Rapla antibody (Santa Cruz Biochemical SC1482) at 1 :1000 (diluted in Western blocking buffer) and the anti Rab6 antibody (Santa Cruz Biochemical SC310) at 1 :5000 (diluted in Western blocking buffer) are added.
  • the membranes are incubated at room temperature for 1 hour with mild rocking.
  • the blocking solution is then discarded and the membrane is washed 3 times with Western wash buffer for 15 minutes per wash.
  • the developed transparency sheet is scanned on a phosphorimager and the Rap la Minimum Inhibitory Concentration is determined from the lowest concentration of compound that produces a detectable Rap la Western signal.
  • the Rap la antibody used recognizes only unprenylate ⁇ Vunprocessed Rap la, so that the precence of a detectable Rap la Western signal is indicative of inhibition of Rap la prenylation.
  • This protocol allows the determination of an EC50 for inhibition of processing of Rap la.
  • the assay is run as described in Protocol B with the following modifications. 20 ⁇ l of sample is run on pre-cast 10-20% gradient acrylamide mini gels (Novex Inc.) at 15 mA/gel for 2.5-3 hours. Prenylated and unprenylated forms of Rap la are detected by blotting with a polyclonal antibody (Rapl/Krev-1 Ab#121;Santa Cruz Research Products #sc-65), followed by an alkaline phosphatase- conjugated anti-rabbit IgG antibody. The percentage of unprenylated Rap la relative to the total amount of Rap la is determined by peak integration using Imagequant® software (Molecular Dynamics).
  • Unprenylated Rap la is distinguished from prenylated protein by virtue of the greater apparent molecular weight of the prenylated protein. Dose-response curves and EC50 values are generated using 4-parameter curve fits in SigmaPlot software. EXAMPLE 32
  • osteoclasts When osteoclasts engage in bone reso ⁇ tion, they can cause the formation of pits in the surface of bone that they are acting upon. Therefore, when testing compounds for their ability to inhibit osteoclasts, it is useful to measure the ability of osteoclasts to excavate these reso ⁇ tion pits when the inhibiting compound is present.
  • Consecutive 200 micron thick cross sections from a 6 mm cylinder of bovine femur diaphysis are cut with a low speed diamond saw (Isomet, Beuler, Ltd., Lake Bluff, II). Bone slices are pooled, placed in a 10% ethanol solution and refrigerated until further use.
  • bovine bone slices Prior to experimentation, bovine bone slices are ultrasonicated twice, 20 minutes each in H2O. Cleaned slices are placed in 96 well plates such that two control lanes and one lane for each drug dosage are available. Each lane represents either triplicate or quadruplicate cultures.
  • the bone slices in 96 well plates are sterilized by UV inadiation. Prior to incubation with osteoclasts, the bone slices are hydrated by the addition of 0.1 ml ⁇ MEM, pH 6.9 containing 5% fetal bovine serum and 1% penicillin/streptomycin.

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Abstract

L'invention concerne un procédé de prévention ou d'inhibition de la résorption osseuse chez un mammifère, ce procédé consistant à administrer à un mammifère nécessitant un tel traitement une dose efficace sur le plan thérapeutique d'un inhibiteur de la géranylgéranyl-transférase de type I.
PCT/US2000/026357 1999-09-27 2000-09-25 Procede de prevention de l'osteoporose WO2001022963A1 (fr)

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US8993577B2 (en) 2009-02-20 2015-03-31 Astrazeneca Ab Cyclopropyl amide derivatives
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US8063215B2 (en) 2007-08-22 2011-11-22 Astrazeneca Ab Cyclopropyl amide derivatives
US9029381B2 (en) 2007-08-22 2015-05-12 Astrazeneca Ab Cyclopropyl amide derivatives
WO2009106640A2 (fr) * 2008-02-28 2009-09-03 Maastricht University Dérivés de n-benzyl-imidazole
WO2009106640A3 (fr) * 2008-02-28 2009-10-29 Maastricht University Dérivés de n-benzyl-imidazole
US8993577B2 (en) 2009-02-20 2015-03-31 Astrazeneca Ab Cyclopropyl amide derivatives
US9012452B2 (en) 2010-02-18 2015-04-21 Astrazeneca Ab Processes for making cyclopropyl amide derivatives and intermediates associated therewith
US9045445B2 (en) 2010-06-04 2015-06-02 Albany Molecular Research, Inc. Glycine transporter-1 inhibitors, methods of making them, and uses thereof
US9290458B2 (en) 2012-05-17 2016-03-22 Genentech, Inc. Amorphous form of an AKT inhibiting pyrimidinyl-cyclopentane compound, compositions and methods thereof
US9278917B2 (en) 2012-05-17 2016-03-08 Genentech, Inc. Process for making amino acid compounds
US9309204B2 (en) 2012-05-17 2016-04-12 Array Biopharma Inc. Process for making hydroxylated cyclopentylpyrimidine compounds
US9315471B2 (en) 2012-05-17 2016-04-19 Genetech, Inc. Process of making hydroxylated cyclopentapyrimidine compounds and salts thereof
US9416110B2 (en) 2012-05-17 2016-08-16 Array Biopharma Inc. Process for making hydroxylated cyclopentylpyrimidine compounds
US9505725B2 (en) 2012-05-17 2016-11-29 Genentech, Inc. Crystalline and mesomorphous forms of an AKT inhibiting pyrimidinyl-cyclopentane compound, compositions and methods thereof
US9676730B2 (en) 2012-05-17 2017-06-13 Array Biopharma Inc. Process for making hydroxylated cyclopentylpyrimidine compounds
US9790190B2 (en) 2012-05-17 2017-10-17 Array Biopharma Inc. Process for making hydroxylated cyclopentylpyrimidine compounds
US20160297792A1 (en) * 2013-11-12 2016-10-13 Merck Sharp & Dohme Corp. Piperidine or piperazine linked imidazole and triazole derivatives and methods of use thereof for improving the pharmacokinetics of a drug
US10745377B2 (en) * 2013-11-12 2020-08-18 Merck Sharp & Dohme Corp. Piperidine or piperazine linked imidazole and triazole derivatives and methods of use

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