This invention relates to compounds of formula (1), wherein substituents are defined in the description, that inhibit farnesylation of mutant ras gene products tough inhibition of the enzyme farnesyl-protein transferase (FPTase). The invention also relates to methods of manufacturing the compounds, pharmaceutical compositions and methods of treating diseases, especially cancer, which are mediated through farnesylation of ras.

This is a 371 of International Application PCT/GB99/04308 filed Dec. 17, 1999.
This invention relates to compounds that inhibit farnesylation of mutant ras gene products through inhibition of the enzyme farnesyl-protein transferase (FPTase). The invention also relates to methods of manufacturing the compounds, pharmaceutical compositions and methods of treating diseases, especially cancer, which are mediated through farnesylation of ras.
Cancer is believed to involve alteration in expression or function of genes controlling cell growth and differentiation. Whilst not wishing to be bound by theoretical considerations the following text sets out the scientific background to ras in cancer. Ras genes are frequently mutated in tumours. Ras genes encode guanosine triphosphate (GTP) binding proteins which are believed to be involved in signal transduction, proliferation and malignant transformation. H-, K- and N-ras genes have been identified as mutant forms of ras (Barbacid M, Ann. Rev. Biochem. 1987, 56: 779-827). Post translational modification of ras protein is required for biological activity. Farnesylation of ras catalysed by FPTase is believed to be an essential step in ras processing. It occurs by transfer of the farnesyl group of farnesyl pyrophosphate (FPP) to a cysteine at the C-terminal tetrapeptide of ras in a structural motif called the CAAX box. After further post-translational modifications, including proteolytic cleavage at the cysteine residue of the CAAX box and methylation of the cysteine carboxyl, ras is able to attach to the cell membrane for relay of growth signals to the cell interior. In normal cells activated ras is believed to act in conjunction with growth factors to stimulate cell growth. In tumour cells it is believed that mutations in ras cause it to stimulate cell division even in the absence of growth factors (Travis J, Science 1993, 260: 1877-1878), possibly through being permanently in GTP activated form rather than cycled back to GDP inactivated form. Inhibition of farnesylation of mutant ras gene products will stop or reduce activation.
One class of known inhibitors of farnesyl transferase is based on farnesyl pyrophosphate analogues; see for example European patent application EP 534546 from Merck. Inhibitors of farnesyl transferase based on mimicry of the CAAX box have been reported. Reiss (1990) in Cell 62, 81-8 disclosed tetrapeptides such as CVIM (Cys-Val-Ile-Met). James (1993) in Science 260, 1937-1942 disclosed benzodiazepine based peptidomimetic compounds. Lerner (1995) in J. Biol. Chem. 270, 26802 and Eisai in International Patent Application WO 95/25086 disclosed further peptidomimetic compounds based on Cys as the first residue. Bristol-Myers Squibb in European Patent Application EP 696593 disclosed farnesyl transferase inhibitors having a 4-sulfanylpyrrolidine residue in the first position.
International Patent Application WO 97/17070 discloses a broad range of compounds which can contain an imidazole group and a methionine substituent. We have now discovered a narrow class of imidazole compounds which have improved properties.
According to one aspect of the present invention there is provided a compound of Formula (1):
wherein Ar1 represents:
R5 is hydrogen, C1-4alkyl, phenylC1-4alkyl;
R6 is hydrogen, C1-4alkyl, hydroxyC1-4alkyl, haloC1-4alkyl, dihaloC1-4alkyl, C1-4alkoxy, C1-4alkoxyC1-4alkyl, sulfanylC1-4alkyl, aminoC1-4alkyl, Nxe2x80x94(C1-4alkyl)aminoC1-4alkyl, N,N-di(C1-4alkyl)aminoC1-4alkyl or phenylC1-4alkyl; m is 0,1 or 2;
R12 and R13 are independently hydrogen or C1-4alkyl;
Ar1 is phenyl, pyridyl, pyridazinyl, pyrimidyl, pyrazinyl, thienyl, thiazolyl, furyl or oxazolyl, the ring being substituted on ring carbon atoms by R2 and xe2x80x94(CH2)nR3, wherein Ar2 is attached to Ar1C(R12)R13CHxe2x95x90CHxe2x80x94 by a ring carbon atom; or Ar1 is pyrrolyl, pyrazolyl or imidazolyl, substituted by R2 and xe2x80x94(CH2)nR3 (the pyrrolyl, pyrazolyl or imidazolyl rings can bear a substituent on the sp3 hybridised ring nitrogen or Ar2 can be attached to Ar1(R12)R13CHxe2x95x90CHxe2x80x94 by the sp3 hybridised ring nitrogen);
R2 is a group of the Formula (2):
wherein R7 is hydrogen or C1-4alkyl, R8 is xe2x80x94(CH2)qxe2x80x94R10 wherein q is 0-4 and R10 is C1-4alkylsulfanyl, C1-4alkylsulfinyl, C1-4alkylsulfonyl, hydroxy, C1-4alkoxy, carbamoyl, Nxe2x80x94C1-4alkyl carbamoyl, N,N-(diC1-4alkyl)carbamoyl, C1-4alkyl, phenyl, thienyl, or C1-4alkanoylamino, R9 is hydroxy, C1-6alkoxy, C3-9cycloalkyloxy, heterocyclyloxy, heterocyclylC1-4alkoxy or xe2x80x94NHxe2x80x94SO2xe2x80x94R11 wherein R11 represents, trifluoromethyl, C1-4alkyl, phenyl, heteroaryl, arylC1-4alkyl or heteroarylC1-4alkyl;
or R2 represents a lactone of Formula (3)
xe2x80x83the group of Formula (2) or (3) having L or D configuration at the chiral alpha carbon in the corresponding free amino acid;
n is 0, 1, or 2;
R3 is phenyl or heteroaryl;
or R2 is a group of the Formula (4):
xe2x80x94CONHCH(R14)R15xe2x80x83xe2x80x83Formula (4)
xe2x80x83wherein R14 is xe2x80x94(CH2)qxe2x80x94R15 wherein q is 0-4 and R15 is C1-4alkylsulfanyl, C1-4alkylsulfinyl, C1-4alkylsulfonyl, hydroxy, C1-4alkoxy, carbamoyl, Nxe2x80x94C1-4alkyl carbamoyl, N,N-(diC1-4alkyl)carbamoyl, C1-4alkyl, phenyl, thienyl, or C1-4alkanoylamino; R14 is of the formula xe2x80x94CH2OR17 (wherein R17 is hydrogen, C1-4alkyl, phenyl, heteroaryl, C2-5alkanoyl, C1-4alkoxymethyl, phenoxymethyl or heteroaryloxymethyl), of the formula xe2x80x94COR18 or of the formula xe2x80x94CH2COR15 (wherein R18 is C1-4alkyl (optionally substituted by halo, cyano, C2-5alkanoyloxy, hydroxy, C1-4alkoxy or C1-4alkanoyl), phenyl, phenylC1-4alkyl, heteroaryl, heteroarylC1-4alkyl, C5-7cycloalkyl, C5-7cycloalkylC1-3alkyl, 2-(phenyl)ethenyl, 2-(heteroaryl)ethenyl or N-methoxy-N-methylamino); or R13 is morpholinoC1-4alkyl, pyrrolidin-1-ylC1-4alkyl or piperidin-1-ylC1-4alkyl wherein the morpholine, pyrrolidine and piperidine rings are optionally substituted by C1-4alkyl or C5-7cycloalkyl; or R13 is phenyl-1-hydroxyC1-4alkyl or heteroaryl-1-hydroxyC1-4alkyl;
phenyl and heteroaryl rings in R3, R5, R6, R11 and R15 (including R17 and R18) are independently optionally substituted on ring carbon atoms by up to three substituents selected from C1-4alkyl, halogen, hydroxy, C1-4alkoxy, C1-4alkoxycarbonyl, C1-4alkanoyl, C1-4alkanoyloxy, amino, C1-4alkylamino, di(C1-4alkyl)amino, C1-4alkanoylamino, nitro, cyano, carboxy, thiol, C1-4alkylsulfanyl, C1-4alkylsulfinyl, C1-4alkylsulfonyl, C1-4alkanesulphonamido, Nxe2x80x94(C1-4alkylsulphonyl)xe2x80x94Nxe2x80x94C1-4alkylamino, aminosulfonyl, Nxe2x80x94(C1-4alkyl)aminosulfonyl, N,N-di(C1-4alkyl)aminosulfonyl, carbamoyl, Nxe2x80x94(C1-4alkyl)carbamoyl, N,N-(diC1-4alkyl)carbamoyl, carbamoylC1-4alkyl, Nxe2x80x94(C1-4alkyl)carbamoylC1-4alkyl, N,N-(diC1-4alkyl)carbamoylC1-4alkyl, hydroxyC1-4alkyl and C1-4alkoxyC1-4alkyl and on ring NH groups (replacing hydrogen) by C1-4alkyl, C1-4alkanoyl, C1-4alkylsulfonyl, haloC1-4alkyl, difluoromethyl or trifluoromethyl;
or a pharmaceutically-acceptable salt, prodrug or solvate thereof
In this specification the generic term xe2x80x9calkylxe2x80x9d includes both straight-chain and branched-chain alkyl groups. However references to individual alkyl groups such as xe2x80x9cpropylxe2x80x9d are specific for the straight-chain version only and references to individual branched-chain alkyl groups such as xe2x80x9cisopropylxe2x80x9d are specific for the branched-chain version only. An analogous convention applies to other generic terms.
It is to be understood that, insofar as certain of the compounds of Formula (1) defined above may exist in optically active or racemic forms by virtue of one or more asymmetric carbon atoms, the invention includes in its definition any such optically active or racemic form which possesses the property of inhibiting FTPase. The synthesis of optically active forms may be carried out by standard techniques of organic chemistry well known in the art, for example by synthesis from optically active starting materials or by resolution of a racemic form. Similarly, inhibitory properties against FTPase may be evaluated using the standard laboratory techniques referred to hereinafter.
The term xe2x80x9cheterocyclylxe2x80x9d refers to a 5- or 6-membered monocyclic ring containing 1 to 3 heteroatoms selected from nitrogen, oxygen and sulfur.
The term xe2x80x9cheteroarylxe2x80x9d refers to a 5-10 membered monocyclic heteroaryl ring containing 1 to 3 heteroatoms selected from nitrogen, oxygen and sulphur.
The ring sp3 hybridised ring nitrogen in the pyrrolyl, pyrazolyl or imidazolyl rings is the ring nitrogen which can be substituted without becoming quaternised i.e. the ring  greater than NH nitrogen.
The term xe2x80x9chalogen xe2x80x9d refers to fluorine, chlorine, bromine and iodine. The term xe2x80x9ccarbamoylxe2x80x9d refers to xe2x80x94C(O)NH2. The term xe2x80x9cBOC xe2x80x9d refers to tert-butoxycarbonyl.
Examples of C1-4alkyl include methyl, ethyl, propyl, isopropyl, sec-butyl and tert-butyl; examples of C1-4alkoxy include methoxy, ethoxy and propoxy; examples of C1-4alkanoyl include formyl, acetyl and propionyl; examples of C1-4alkanoyloxy include acetyloxy and propionyloxy; examples of C1-4alkylamino include methylamino, ethylamino, propylamino, isopropylamino, sec-butylamino and tert-butylamino; examples of di-(C1-4alkyl)amino include di-methylamino, di-ethylamino and N-ethyl-N-methylamino; examples of C1-4alkanoylamino include acetamido and propionylamino; examples of C1-4alkoxycarbonyl include methoxycarbonyl, ethoxycarbonyl and propoxycarbonyl; examples of C1-4alkylsulfanyl include methylsulfanyl, ethylsulfanyl, propylsulfanyl, isopropylsulfanyl, sec-butylsulfanyl and tert-butylsulfanyl; examples of C1-4alkylsulfinyl include methylsulfinyl, ethylsulfinyl, propylsulfinyl, isopropylsulfinyl, sec-butylsulfinyl and tert-butylsulfinyl; examples of C1-4alkylsulfonyl include methylsulfonyl, ethylsulfonyl, propylsulfonyl, isopropylsulfonyl, sec-butylsulfonyl and tert-butylsulfonyl; examples of Nxe2x80x94(C1-4alkyl)carbamoyl include N-methylcarbamoyl and N-ethylcarbamoyl; examples of N,N-(diC1-4alkyl)carbamoyl include N,N-dimethylcarbamoyl and N-methyl-N-ethylcarbamoyl; examples of C1-4alkanesulfonamido include methanesulfonamido, ethanesulphonamido and propanesulfonamido; examples of C1-4alkylsulfonyl-Nxe2x80x94C1-4alkylamino include methylsulfonyl-N-methylamino, ethylsulfonyl-N-methylamino and propylsulfonyl-N-methylamino; examples of fluoroC1-4alkyl include fluoromethyl, 2-fluoroethyl and 3-fluoropropyl; examples of difluoroC1-4alkyl include difluoromethyl, 2,2-difluoroethyl and 3,3-difluoropropyl; examples of carbamoylC1-4alkyl include carbamoylmethyl, carbamoylethyl and carbamoylpropyl; examples of Nxe2x80x94(C1-4alkyl)carbamoylC1-4alkyl include N-methyl-carbamoylmethyl and N-ethyl-carbamoylethyl; examples of N,N-(diC1-4alkyl)carbamoylC1-4alkyl include N,N-dimethylcarbamoylethyl and N-methyl-N-ethylcarbamoylethyl; examples of hydroxyC1-4alkyl include hydroxymethyl, hydroxyethyl, hydroxypropyl, 2-hydroxypropyl, 2-(hydroxymethyl)propyl and hydroxybutyl; examples of C1-4alkoxyC1-4alkyl include methoxyethyl, ethoxyethyl and methoxybutyl; examples of sulfanylC1-4alkyl include sulfanylmethyl, sulfanylethyl, sulfanylpropyl; and examples of Nxe2x80x94(C1-4alkyl)aminoC1-4alkyl include N-methyl-aminomethyl and N-ethyl-aminoethyl.
Examples of 5- or 6-membered heteroaryl ring systems include imidazole, triazole, pyrazine, pyrimidine, pyridazine, pyridine, isoxazole, oxazole, isothiazole, thiazole and thiophene.
Preferably the NH group in imidazole is unsubstituted or substituted by C1-4alkyl. Examples of heterocyclyl rings include pyrrolidinyl, morpholinyl, piperidinyl, dihydropyridinyl and dihydropyrimidinyl.
Preferred heteroatoms are N and S, especially N. In general, attachment of heterocyclic rings to other groups is via carbon atoms.
Examples of values for R8 in Formula (2) are side chains of lipophilic amino acids including such as for example methionine, phenylglycine, phenylalanine, serine, leucine, isoleucine or valine. The L configuration in the corresponding free amino acid is preferred. Examples of amino acid side chains are set out below.
The lactone in Formula (3) can be formed from a group of Formula (2) when R9 is OH to give a carboxyl and R8 is xe2x80x94CH2xe2x80x94CH2xe2x80x94OH where R8 and R9 together lose a water molecule to form part of a dihydrofuran-2-one heterocyclic ring.
Preferably, phenyl and heteroaryl rings in R3, R5, R6, R11 and R15 (including R17 and R18) are independently optionally substituted on ring carbon atoms by up to two substituents selected from C1-4alkyl, halogen, hydroxy, C1-4alkoxy, C1-4alkoxycarbonyl, C1-4alkanoyl, C1-4alkanoyloxy, amino, C1-4alkylamino, di(C1-4alkyl)amino, C1-4alkanoylamino, cyano, carboxy, C1-4alkylsufanyl, C1-4alkylsulfinyl, C1-4alkylsulfonyl, C1-4alkanesulphonamido, carbamoyl, Nxe2x80x94(C1-4alkyl)carbomoyl, N,N-(diC1-4alkyl)carbamoyl and on ring NH groups (replacing hydrogen) by C1-4alkyl or C1-4alkanoyl.
Preferably R12 and R13 are independently hydrogen or methyl.
Most preferably R12 and R13 are hydrogen. Preferably R6 is hydrogen, C1-4alkyl, hydroxyC1-4alkyl, aminoC1-4alkyl, fluoroC1-4alkyl, difluoroC1-4alkyl, C1-4alkoxy or C1-4alkoxyC1-4alkyl.
More preferably R6 is hydrogen, methyl, fluoromethyl, difluoromethyl, methoxy or methoxymethyl.
Most preferably R6 is hydrogen or methyl.
Preferably m is 0 or 1.
Preferably R5 is hydrogen or methyl. More preferably R5 is hydrogen.
In a particular aspect Ar1 is 1 -methylimidazol-5-yl.
Preferably Ar2 is phenyl, pyridyl, pyridazinyl, pyrimidyl, pyrazinyl, thienyl, thiazolyl, or oxazolyl, the ring being substituted on ring carbon atoms by R2 and xe2x80x94(CH2)nR3. Most preferred is where Ar2 is phenyl, pyridinyl or thienyl, the ring being substituted on ring carbon atoms by R2 and xe2x80x94(H2)nR3.
More preferably Ar2 is phenyl, pyridinyl or thienyl, the ring being substituted on ring carbon atoms by R2 and xe2x80x94(CH2)nR3.
Yet more preferably Ar2 is phenyl or pyridyl, the ring being substituted on ring carbon atoms by R2 and xe2x80x94(CH2)nR3.
Most preferably Ar2 is phenyl, the ring being substituted on ring carbon atoms by R2 and xe2x80x94(CH2)nR3.
Preferably, when n is 0, Ar2 is substituted by R2 in the 4-position and xe2x80x94(CH2)nR3 in the 3- or 5-position and when n is 1 or 2, Ar2 is substituted by R2 in the 3- or 5-position and xe2x80x94(CH2)nR2 in the 4-position. The positions indicated are relative to the point of attachment of Ar2 to Ar1C(R12)R13CHxe2x95x90CHxe2x80x94.
Preferably n is 0 or 2. In a particular aspect n is 0.
R2 is preferably a group of formula:
R7 is preferably hydrogen or methyl, especially hydrogen. In R8, q is preferably 1-4, more preferably 1 or 2, especially 2.
Within R8, R10 is preferably C1-4alkylsulfanyl, C1-4alkylsulfinyl, C1-4alkylsulfonyl, hydroxy or C1-4alkoxy. More preferably R10 is methylsulfanyl or methylsulfonyl.
R9 is preferably hydroxy, C1-4alkoxy, C3-9cycloalkyloxy, heterocyclyloxy or heterocyclylC1-4alkoxy. More preferably R9 is hydroxy, methoxy, propoxy, butoxy, tert-butoxy, cyclopentyloxy, piperidin-4-yloxy or morpholinoC1-4alkyl. Most preferably, R9 is methoxy, prop-2-oxy, n-butoxy, tert-butoxy or cyclopentyloxy.
Preferably R11 in R9 is phenyl.
Preferred substituents for NH groups in heterocyclic groups in R9 include methyl, ethyl, acetyl, propionyl, fluoromethyl, difluoromethyl and trifluoromethyl.
More preferred substituents for NH groups in heterocyclic groups in R9 include methyl and acetyl.
Preferred substituents for ring carbon atoms in phenyl or heteroaryl groups in R11 include methyl, halo, C1-4alkanoyl, nitro, cyano, C1-4alkylsulfinyl, C1-4alkylsulfonyl, carbamoyl, C1-4alkylcarbamoyl and diC1-4alkylcarbamoyl.
Formula (4) is preferably a group of formula:
In R14, q is preferably 1-4, more preferably 1 or 2, especially 2.
Within R14, R14 is preferably C1-4alkylsulfanyl, C1-4alkylsulfinyl, C1-4alkylsulfonyl, hydroxy or C1-4alkoxy. More preferably R16 in R14 is methylsulfanyl or methylsulfonyl. Preferably R17 in R15 is hydrogen or phenyl.
Most preferably R17 in R15 is hydrogen.
Preferably R18 in R15 is C1-4alkyl, phenyl, phenylC1-3alkyl, heteroaryl, heteroarylC1-3alkyl or C5-7cycloalkylC1-3alkyl.
More preferably R18 in R15 is C1-4alkyl, phenyl, phenylC1-3alkyl or heteroaryl.
Most preferably R18 in R15 is C1-4alkyl, phenyl or benzyl.
Preferably when R18 is C1-4alkyl, it is optionally substituted by halo, cyano or C2-6alkanoyloxy.
Preferably morpholinoC1-4alkyl is morpholinomethyl, pyrrolidin-1-ylC1-4alkyl is pyrrolidin-1-ylmethyl and piperidin-1-ylC1-4alkyl is piperidin-1-ylmethyl.
In one aspect R15 is morpholinomethyl.
More preferably R15 is hydroxymethyl, benzylcarbonyl, 3-(pyridyl)propionyl or morpholinomethyl.
Most preferably R15 is hydroxymethyl or benzylcarbonyl. R14 is xe2x80x94(CH2)qR16 wherein q is 0-4 and R16 is C1-4alkylsulfanyl, C1-4alkylsulfinyl, C1-4alkylsulfonyl, hydroxy or C1-4alkoxy; or R15 is morpholinomethyl, pyrrolidin-1-ylmethyl or piperidin-1-ylmethyl; or R15 is of the formula xe2x80x94CH2OR17 wherein R17 is hydrogen or phenyl; or R15 is of the formula xe2x80x94COR18 or xe2x80x94CH2COR18 wherein R18 is C1-4alkyl, phenyl, phenylC1-3alkyl, heteroaryl, heteroarylC1-3alkyl or C5-7cycloalkylC1-3alkyl; Preferably R3 is phenyl, pyridyl or thiazolyl.
Most preferably R3 is phenyl.
Preferred substituents for ring carbon atoms in R3 include C1-4alkyl, halo, trifluoromethyl, C1-4alkoxy, nitro, cyano and C1-4alkoxy C1-4alkyl.
More preferred substituents for ring carbon atoms in R3 include methyl, fluoro, chloro, trifluoromethyl, methoxy, nitro, cyano and methoxymethyl.
When R3 is phenyl it is preferably mono-substituted by fluoro, chloro or cyano or di-substituted by fluoro and triflouromethyl, chloro and trifluoromethyl, fluoro and fluoro or chloro and chloro.
A preferred substituent for a ring NH group in a heteroaryl group in R3 is C1-4alkyl, particularly methyl.
When R3 is phenyl it is preferably substituted in the 4-position, and when di-substituted it is preferably substituted in the 2- and 4-positions.
Preferably n is 0 or 2.
Preferably R7 is hydrogen.
Preferably R8 is xe2x80x94(CH2)xe2x80x94R10 is C1-4alkylsulfonyl or C1-4alkylsulfonyl.
Preferably R9 is hydroxy, C1-4alkoxy, C3-9cycloalkyloxy, heterocyclyloxy or heterocyclylC1-4alkoxy.
A preferred compound of the invention is a compound of the Formula (I) wherein:
Ar1 is of the formula (A), (B) or (C);
R5 is hydrogen or methyl;
R6 is hydrogen, C1-4alkyl, fluoroC1-4alkyl, difluoroC1-4alkyl, C1-4alkoxy or C1-4alkoxyC1-4alkyl;
m is 0 or 1;
R12 and R13 are independently hydrogen or methyl;
Ar2 is phenyl or pyridyl, the ring being substituted on ring carbon atoms by R2 and xe2x80x94(CH2)nR10 and wherein Ar2 is attached to Ar1C(R12)R13CHxe2x95x90CHxe2x80x94 by a ring carbon atom; and
n is 0, 1 or 2;
R2 is of the formula (2) wherein R7 is hydrogen or methyl;
R5 is xe2x80x94(CH2)qR10 wherein q is 0-4 and R10 is C1-4alkylsulfanyl, C1-4alkylsulfinyl, C1-4alkylsulfonyl, hydroxy or C1-4alkoxy;
R9 is hydroxy, C1-4alkoxy, C3-9cycloalkyloxy, heterocycloxy or heterocyclylC1-4alkoxy;
or R2 is of the formula (3);
R3 is phenyl, pyridyl or thiazolyl; and phenyl, heteroaryl and heterocyclyl rings in R3 and R9 are independently optionally substituted on ring carbon atoms by one or two substituents selected from C1-4alkyl, halo, C1-4alkoxy, C1-4alkanoyl, nitro, cyano, C1-4alkylsulfinyl, C1-4alkylsulfonyl, carbamoyl, C1-4alkylcarbamoyl and diC1-4alkylcarbamoyl; and optionally substituted on ring NH groups by C1-4alkyl, C1-4alkanoyl, fluoromethyl, difluoromethyl or trifluoromethyl;
or a pharmaceutically-acceptable salt, prodrug or solvate thereof.
A more preferred compound of the invention is a compound of the formula (I) wherein:
Ar1 is of the formula (A), (B) or (C);
R5 is hydrogen or methyl;
R6 is hydrogen, methyl, fluoromethyl, difluoromethyl, methoxy or methoxymethyl;
m is 0 or 1;
R12 and R13 are independently hydrogen or methyl;
Ar2 is phenyl or pyridyl; the ring being substituted on ring carbon atoms by R2 and xe2x80x94(CH2)nR3 and wherein Ar2 is attached to Ar1C(R12)R13CHxe2x95x90CHxe2x80x94 by a ring carbon atom; and n is 0, 1 or 2;
R2 is of formula (2) wherein R7 is hydrogen or methyl;
R8 is (CH2)qR10 wherein q is 1 or 2, and
R10 is methylsulfanyl or methylsulfonyl;
R9 is hydroxy, methoxy, prop-2-oxy, butoxy, tert-butoxy, cyclopentyloxy, piperidin -4 -yloxy, or morpholinoC1-4alkyl; or R2 is of the formula (3);
R3 is phenyl optionally substituted by one or two substituents selected from C1-4alkyl, halo, C1-4alkoxy, nitro, cyano, C1-4alkoxyC1-4alkyl and trifluoromethyl;
or a pharmaceutically-acceptable salt, prodrug or solvate thereof.
An even more preferred compound of the invention is a compound of the formula (I) wherein:
Ar1 is of the formula (A), (B) or (C);
R5 is hydrogen or methyl;
R6 is hydrogen or methyl;
m is 0 or 1;
R11 and R12 are hydrogen;
Ar2 is phenyl; the ring being substituted on ring carbon atoms by R2 and xe2x80x94(CH2)nR3 and wherein
Ar2 is attached to Ar1(R12)R13CHxe2x95x90CHxe2x80x94 by a ring carbon atom;
and n is 0, 1 or 2;
R2 is of the Formula (2) wherein R7 is hydrogen;
R8 is xe2x80x94(CH2)8R10 wherein q is 2 and within Formula (2)
R10 is methylsulfanyl or methylsulfonyl;
R9 is hydroxy, methoxy, prop-2-oxy, butoxy, tert-butoxy, cyclopentyloxy, piperidin-4-yloxy, or 2-morpholinoprop-2-yl; or within Formula (4)
R15 is of the fomula xe2x80x94CH2OR17 wherein R17 is hydrogen; or R15 is of the formula xe2x80x94COR18 or xe2x80x94CH2COR15 wherein R18 is C1-4alkyl, phenyl, phenylC1-3alkyl or heteroaryl; or
R15 is morpholinomethyl, pyrrolidin-1-ylmethyl or piperidin-1-ylmethyl;
R3 is phenyl optionally substituted by fluoro, chloro, cyano or trifluoromethyl;
or a pharmaceutically-acceptable salt, prodrug or solvate thereof.
Preferably Ar1C(R12)R13xe2x80x94 and Ar2 are on opposite sides of the double bond (this gives the E isomeric configuration).
Particular compounds of the present invention include those compounds specifically described in the Examples; or a pharmaceutically-acceptable salt, prodrug or solvate thereof.
Compounds of Formula (1) may form salts which are within the ambit of the invention. Pharmaceutically acceptable salts are preferred although other salts may be useful in, for example, isolating or purifying compounds.
When the compound contains a basic moiety it may form pharmaceutically-acceptable salts with a variety of inorganic or organic acids, for example hydrochloric, hydrobromic, sulphuric, phosphoric, trifluoroacetic, citric or maleic acid. A suitable pharmaceutically-acceptable salt of the invention when the compound contains an acidic moiety is an alkali metal salt, for example a sodium or potassium salt, an alkaline earth metal salt, for example a calcium or magnesium salt, an ammonium salt or a salt with an organic base which affords a pharmaceutically-acceptable cation, for example a salt with methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine.
Solvates, for example hydrates, are also within the ambit of the invention and may be prepared by generally known methods.
Various forms of prodrugs are well known in the art. For examples of such prodrug derivatives, see:
a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and Methods in Enzymology, Vol. 42, p. 309-396, edited by K. Widder, et al. (Academic Press, 1985);
b) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and H. Bundgaard, Chapter 5 xe2x80x9cDesign and Application of Prodrugsxe2x80x9d, by H. Bundgaard p. 113-191 (1991);
c) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992);
d) H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77, 285 (1988); and
e) N. Kakeya, et al., Chem Pharm Bull, 32, 692 (1984).
Examples of pro-drugs include in vivo hydrolysable esters of a compound of the Formula I. Suitable pharmaceutically-acceptable esters for carboxy include C1-4alkyl esters, C5-8cycloalkyl esters, cyclic amine esters, C1-6alkoxymethyl esters for example methoxymethyl, C1-6alkanoyloxymethyl esters for example pivaloyloxymethyl, phthalidyl esters, C3-8cycloalkoxycarbonyloxyC1-6alkyl esters for example 1-cyclohexylcarbonyloxyethyl; 1,3-dioxolen-2-onylmethyl esters for example 5-methyl-1,3-dioxolen-2-onylmethyl; and C1-6alkoxycarbonyloxyethyl esters for example 1-methoxycarbonyloxyethyl wherein alkyl, cycloalkyl and cyclicamino groups are optionally substituted by, for example, phenyl, heterocyclcyl, alkyl, amino, alkylamino, dialkylamino, hydroxy, alkoxy, aryloxy or benzyloxy, and may be formed at any carboxy group in the compounds of this invention.
According to another aspect of the invention there is provided a pharmaceutical composition comprising a compound as defined in Formula (1) or an individual compound listed above together with a pharmaceutically-acceptable diluent or carrier. A preferred pharmaceutical composition is in the form of a tablet.
The compositions of the invention may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular dosing or as a suppository for rectal dosing).
The compositions of the invention may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art. Thus, compositions intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and/or preservative agents.
Suitable pharmaceutically-acceptable excipients for a tablet formulation include, for example, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate, granulating and disintegrating agents such as corn starch or algenic acid; binding agents such as starch; lubricating agents such as magnesium stearate, stearic acid or talc; preservative agents such as ethyl or propyl p-hydroxybenzoate, and anti-oxidants, such as ascorbic acid. Tablet formulations may be uncoated or coated either to modify their disintegration and the subsequent absorption of the active ingredient within the gastrointestinal tract, or to improve their stability and/or appearance, in either case, using conventional coating agents and procedures well known in the art.
Compositions for oral use may be in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin, or olive oil.
Aqueous suspensions generally contain the active ingredient in finely powdered form together with one or more suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as lecithin or condensation products of an alkylene oxide with fatty acids (for example polyoxethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, 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 long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, 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 monooleate. The aqueous suspensions may also contain one or more preservatives (such as ethyl or propyl p-hydroxybenzoate, anti-oxidants (such as ascorbic acid), colouring agents, flavouring agents, and/or sweetening agents (such as sucrose, saccharine or aspartame).
Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil (such as arachis oil, olive oil, sesame oil or coconut oil) or in a mineral oil (such as liquid paraffin). The oily suspensions may also contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set out above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water generally contain the active ingredient together 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 such as sweetening, flavouring and colouring agents, may also be present.
The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, or a mineral oil, such as for example liquid paraffin or a mixture of any of these. Suitable emulsifying agents may be, for example, naturally-occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soya bean, lecithin, an 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 such as polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavouring and preservative agents.
Syrups and elixirs may be formulated with sweetening agents such as glycerol, propylene glycol, sorbitol, aspartame or sucrose, and may also contain a demulcent, preservative, flavouring and/or colouring agent.
The pharmaceutical compositions may also be in the form of a sterile injectable aqueous or oily suspension, which may be formulated according to known procedures using one or more of the appropriate dispersing or wetting agents and suspending agents, which have been mentioned above. A sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example a solution in 1,3-butanediol.
Suppository formulations may be prepared by mixing the active ingredient 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 excipients include, for example, cocoa butter and polyethylene glycols.
Topical formulations, such as creams, ointments, gels and aqueous or oily solutions or suspensions, may generally be obtained by formulating an active ingredient with a conventional, topically acceptable, vehicle or diluent using conventional procedure well known in the art.
Compositions for administration by insufflation may be in the form of a finely divided powder containing particles of average diameter of, for example, 30xcexc or much less, the powder itself comprising either active ingredient alone or diluted with one or more physiologically acceptable carriers such as lactose. The powder for insufflation is then conveniently retained in a capsule containing, for example, 1 to 50 mg of active ingredient for use with a turbo-inhaler device, such as is used for insufflation of the known agent sodium cromoglycate.
Compositions for administration by inhalation may be in the form of a conventional pressurised aerosol arranged to dispense the active ingredient either as an aerosol containing finely divided solid or liquid droplets. Conventional aerosol propellants such as volatile fluorinated hydrocarbons or hydrocarbons may be used and the aerosol device is conveniently arranged to dispense a metered quantity of active ingredient.
For further information on Formulation the reader is referred to Chapter 25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.
The amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the host treated and the particular route of administration. For example, a formulation intended for oral administration to humans will generally contain, for example, from 0.5 mg to 2 g of active agent compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition. Dosage unit forms will generally contain about 1 mg to about 500 mg of an active ingredient. For further information on Routes of Administration and Dosage Regimes the reader is referred to Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.
The size of the dose for therapeutic or prophylactic purposes of a compound of the Formula (1) will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well known principles of medicine. As mentioned above, compounds of the Formula (1) are useful in treating diseases or medical conditions which are due alone or in part to the effects of farnesylation of ras.
In using a compound of the Formula (1) for therapeutic or prophylactic purposes it will generally be administered so that a daily dose in the range, for example, 0.5 mg to 75 mg per kg body weight is received, given if required in divided doses. In general lower doses will be administered when a parenteral route is employed. Thus, for example, for intravenous administration, a dose in the range, for example, 0.5 mg to 30 mg per kg body weight will generally be used. Similarly, for administration by inhalation, a dose in the range, for example, 0.5 mg to 25 mg per kg body weight will be used. Oral administration is however preferred.
Compounds of this invention may be useful in combination with known anti-cancer and cytotoxic agents. If formulated as a fixed dose such combination products employ the compounds of this invention within the dosage range described herein and the other pharmaceutically active agent within its approved dosage range. Sequential use is contemplated when a combination formulation is inappropriate. According to another aspect of the invention there is provided a compound of Formula (1) or a pharmaceutically-acceptable salt thereof, for use as a medicament.
According to another aspect of the invention there is provided a compound of Formula (1) or a pharmaceutically-acceptable salt thereof, for use in preparation of a medicament for treatment of a disease mediated through farnesylation of ras.
According to another aspect of the present invention there is provided a method of treating ras mediated diseases, especially cancer, by administering an effective amount of a compound of Formula (1) or a pharmaceutically-acceptable salt thereof, to a mammal in need of such treatment.
Diseases or medical conditions may be mediated alone or in part by farnesylated ras. A particular disease of interest is cancer. Specific cancers of interest include:
carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin;
hematopoietic tumors of lymphoid lineage, including acute lymphocytic leukemia, B-cell lymphoma and Burketts lymphoma;
hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia;
tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; and
other tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma and glioma.
The compounds of Formula (1) are especially useful in treatment of tumors having a high incidence of ras mutation, such as colon, lung, and pancreatic tumors. By the administration of a composition having one (or a combination) of the compounds of this invention, development of tumors in a mammalian host is reduced.
Compounds of Formula (1) may also be useful in the treatment of diseases other than cancer that may be associated with signal transduction pathways operating through Ras, e.g., neuro-fibromatosis.
Compounds of Formula (1) may also be useful in the treatment of diseases associated with CAAX-containing proteins other than Ras (e.g., nuclear lamins and transducin) that are also post-translationally modified by the enzyme farnesyl protein transferase.
Although the compounds of the Formula (1) are primarily of value as therapeutic agents for use in warm-blooded animals (including man), they are also useful whenever it is required to inhibit the effects of activation of ras by farnesylation. Thus, they are useful as pharmacological standards for use in the development of new biological tests and in the search for new pharmacological agents.
In another aspect the present invention provides a process for preparing a compound of the Formula (1) or a pharmaceutically-acceptable salt prodrug or solvate thereof which process comprises:
deprotecting a compound of the formula (5)
wherein Ar1xe2x80x2 is Ar1 or protected Ar1 and Ar2xe2x80x2 is Ar2 or protected Ar2 and R12 and R13 are as hereinabove defined; wherein at least one protecting group is present; and thereafter if necessary:
(i) forming a pharmaceutically-acceptable salt,
(ii) forming a prodrug,
(iii) forming a solvate.
Protecting groups may in general be chosen from any of the groups described in the literature or known to the skilled chemist as appropriate for the protection of the group in question, and may be introduced by conventional methods.
Protecting groups may be removed by any convenient method as described in the literature or known to the skilled chemist as appropriate for the removal of the protecting group in question, such methods being chosen so as to effect removal of the protecting group with minimum disturbance of groups elsewhere in the molecule.
Specific examples of protecting groups are given below for the sake of convenience, in which xe2x80x9clowerxe2x80x9d signifies that the group to which it is applied preferably has 1-4 carbon atoms. It will be understood that these examples are not exhaustive. Where specific examples of methods for the removal of protecting groups are given below these are similarly not exhaustive. The use of protecting groups and methods of deprotection not specifically mentioned is of course within the scope of the invention.
A carboxy protecting group may be the residue of an ester-forming aliphatic or araliphatic alcohol or of an ester-forming silanol (the said alcohol or silanol preferably containing 1-20 carbon atoms).
Examples of carboxy protecting groups include straight or branched chain C1-12alkyl groups (for example isopropyl, t-butyl); lower alkoxy lower alkyl groups (for example methoxymethyl, ethoxymethyl, isobutoxymethyl); lower aliphatic acyloxy lower alkyl groups, (for example acetoxymethyl, propionyloxymethyl, butyryloxymethyl, pivaloyloxymethyl); lower alkoxycarbonyloxy lower alkyl groups (for example 1-methoxycarbonyloxyethyl, 1-ethoxycarbonyloxyethyl); phenyl lower alkyl groups (for example benzyl, p-methoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, benzhydryl and phthalidyl); tri(lower alkyl)silyl groups (for example trimethylsilyl and t-butyldimethylsilyl); tri(lower alkyl)silyl lower alkyl groups (for example trimethylsilylethyl); and C1-6alkenyl groups (for example allyl and vinylethyl).
Methods particularly appropriate for the removal of carboxy protecting groups include for example acid-, base-, metal- or enzymically-catalysed hydrolysis.
Examples of hydroxy protecting groups include lower alkyl groups (for example t-butyl), lower alkenyl groups (for example allyl); lower alkanoyl groups (for example acetyl); lower alkoxycarbonyl groups (for example t-butoxycarbonyl); lower alkenyloxycarbonyl groups (for example allyloxycarbonyl); phenyl lower alkoxycarbonyl groups (for example benzoyloxycarbonyl, p-methoxybenzyloxycarbonyl, o-nitrobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl); tri lower alkylsilyl (for example trimethylsilyl, t-butyldimethylsilyl) and phenyl lower alkyl (for example benzyl) groups.
Examples of amino protecting groups include formyl, aralkyl groups (for example benzyl and substituted benzyl, p-methoxybenzyl, nitrobenzyl and 2,4-dimethoxybenzyl, and triphenylmethyl); di-p-anisylmethyl and furylmethyl groups; lower alkoxycarbonyl (for example t-butoxycarbonyl); lower alkenyloxycarbonyl (for example allyloxycarbonyl); phenyl lower alkoxycarbonyl groups (for example benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, o-nitrobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl; trialkylsilyl (for example trimethylsilyl and t-butyldimethylsilyl); alkylidene (for example methylidene); benzylidene and substituted benzylidene groups.
Methods appropriate for removal of hydroxy and amino protecting groups include, for example, acid-, base-, metal- or enzymically-catalysed hydrolysis, for groups such as p-nitrobenzyloxycarbonyl, hydrogenation and for groups such as o-nitrobenzyloxycarbonyl, photolytically.
The reader is referred to Advanced Organic Chemistry, 4th Edition, by Jerry March, published by John Wiley and Sons 1992, for general guidance on reaction conditions and reagents. The reader is referred to Protective Groups in Organic Synthesis, 2nd Edition, by Green et al., published by John Wiley and Sons for general guidance on protecting groups.
Compounds of the formula (1) and (5) can be formed by:
(i) reacting a compound of the formula (6) with a compound of the formula (7):
or (ii) converting one value of R9 in R2 into another value of R9;
or (iii), where the compound of formula (I) includes alternatives of formula (2) and (3), reacting a compound in which R2 in Ar2xe2x80x2 is carboxy with a compound of the formula (8):
or (iv), where the compound of formula (I) includes alternative of formula (4), reacting a compound in which R2 in Ar2 is carboxy with a compound of formula (8a).
or (v) when Ar1xe2x80x2 is of the formula (B) or (C) (optionally protected), reacting a compound of the formula (9) or (9a) with a compound of the formula (10):
xe2x80x83wherein p, Ar1xe2x80x2, Ar2xe2x80x2, Ar3xe2x80x2 R7 and R8 are as hereinabove defined, R21 is R9 or protected R9, L1 and L2 are leaving groups and Pxe2x80x2 is an amino-protecting group or R5 providing it is not hydrogen; and thereafter if necessary:
(i) removing any protecting groups;
(ii) forming a pharmaceutically-acceptable salt, prodrug or solvate thereof.
The reaction between compounds of the formula (6) and (7) is conveniently carried out under conditions know for the Wittig reaction.
Suitable Wittig reaction conditions include using a polar aprotic organic solvent such as THF or methylene chloride in the presence of a crown ether and an alkali metal carbonate, such as potassium carbonate, preferably at xe2x88x9210xc2x0 C. to ambient temperature. C18 HPLC may be used to separate E and Z isomers if desired.
The Wittig reaction can be used to prepare a compound wherein R2 in Ar2xe2x80x2 is an alkoxycarbonyl group which may be hydrolysed to a carboxy group. If Ar1xe2x80x2 is of the formula (A), and the latter route is chosen and basic conditions are used, the E and Z isomers produced by the Wittig reaction are generally not separated if the E isomer is wanted, because the base-hydrolysis step causes isomerisation of the Z isomer to the E isomer.
A compound of the formula (6) is generally prepared by reducing the corresponding ester to the aldehyde. A suitable reducing agent is for example DIBAL which is used in an inert non-polar solvent such as diethyl ether at low temperature, for example in the range xe2x88x9278xc2x0 C. to 0xc2x0 C. Other suitable reducing agents are known in the art.
Alternatively, the ester could be reduced to the corresponding alcohol with a reducing agent such as lithium aluminium hydride or sodium borohydride and the alcohol oxidised to the aldehyde with an oxidising agent such as pyridinium chlorochromate.
The ester used to form a compound of the formula (6) can be prepared by introducing Are into a compound of the formula L4CH2COOR, wherein L4 is leaving group such as bromo, in the presence of a base such as sodium hydride, sodium hydroxide or potassium carbonate.
A compound of the formula (7) may be known in the art (for example see International Patent Application publication no. WO 98/32741) or may be prepared by reacting a compound of the formula Ar2xe2x80x2CH2Br with triphenylphosphine.
A compound of the formula Ar2xe2x80x2CH2Br may be formed by brominating a compound of the formula Ar2xe2x80x2CH3 with a suitable brominating agent such as N-bromosuccinimide.
A compound of the formula Ar2xe2x80x2CH3 wherein the Ar2xe2x80x2 group is substituted by R2 and xe2x80x94(CH2)nR3 group may be prepared from a compound of the formula Ar2xe2x80x2CH3 wherein the Ar2 group is substituted by xe2x80x94(CH2)nR3 and xe2x80x94COOHxe2x80x2 using methods described above for the conversion of xe2x80x94COOH to R2. This latter Ar2xe2x80x2CH3 compound could be prepared from a compound of the formula Ar2xe2x80x2CH3 wherein the Ar2xe2x80x2 group is substituted by a protected carboxy group (xe2x80x94COOP2) and a leaving group (L3). When n is 0, the CH3Ar2xe2x80x2(xe2x80x94COOP2)xe2x80x94L3 compound is conveniently reacted with aryl (or heteroaryl) boronic acid in the presence of a palladium catalyst such as palladium tetrakis (triphenylphosphine) palladium (0) under conditions known for the Suzuki reaction (Synth. Commun. 11, 513 (1981)). An aprotic organic solvent such as dimethyl ether (DME), toluene, dimethylsulphoxide (DMSO) or THF is generally used and a base such as sodium bicarbonate, sodium carbonate and sometimes sodium hydroxide. A fluoride such as caesium fluoride could be used instead of the base (J. Org. Chem. 1994, 59, 6095-6097). Preferably L3 is bromo or triflate.
When n is 1, the CH3Ar2xe2x80x2(xe2x80x94COOP2)xe2x80x94L3 compound wherein L3 is bromo or chloro, is conveniently reacted with a benzyl (or heteroarylmethyl) zinc chloride or a benzyl (or heteroarylmethyl)-magnesium bromide in the presence of a nickel or palladium catalyst, such as bis(triphenylphosphine)palladium (II) chloride or Pd2(dba)3, in an inert organic solvent such as tetrahydrofuran (THF). For example see the conditions used for the xe2x80x98Negishixe2x80x99 reaction (J. Org. Chem. 42 (10), 1821-1822, 1977).
When n is 2, the CH3Ar2xe2x80x2(xe2x80x94COOP2)xe2x80x94L3 compound is conveniently reacted with a styrene under conditions known for the Heck reaction. Briefly this involves an inorganic or organic base such as triethylamine, a palladium catalyst such as bis (o-tolylphosphine)palladium (II) chloride in water. (Acc. Chem. Res. 12, 146-151 (1979), J. Organometallic Chem. 486, 259-262 (1995)).
The resulting alkene can then be reduced using standard methods known in the art, for example, catalytic hydrogenation.
Alternatively the alkyne could be formed by reacting a CH3Ar2xe2x80x2(xe2x80x94COOP2)xe2x80x94L3 compound, wherein L3 is triflate or bromo, with a phenyl acetylene in the presence of an organic base such as triethylamine and a palladium catalyst such as palladium tetrakis (triphenylphosphine). For example see the conditions used for the Sonogashira reaction (J. Org. Chem. 1993,58, 6614-6619).
The resultant alkyne can be reduced using standard methods known in the art, for example, catalytic hydrogenation.
The carboxy-protecting group may then be removed.
A compound of the formula (1) in which R9 in R2 is alkoxy can conveniently be hydrolysed to another compound of the formula (1) in which R9 is hydroxy using standard methods known in the art. For example, the alkoxy group could be subjected to acid or base hydrolysis with, for example, in the case of base hydrolysis, aqueous sodium hydroxide solution in an organic solvent such as an alcohol in a temperature range of ambient temperature to 60xc2x0 C. When R9 is a hydroxy group the carboxy group in a compound of the formula (1) can be converted to an acylsulphonamide by reacting the carboxy group with the appropriate sulphonamido group in the presence of an organic base such as triethylamine or dimethylaminopyridine, in an inert organic solvent such as dimethylformamide (DMF), in temperature range of xe2x88x9220xc2x0 C. to ambient temperature.
The reaction between a compound in which R2 in Ar2xe2x80x2 is carboxy and a compound of the formula (8) and (8a) is generally carried out in the presence of a reagent that converts the carboxy group into a reactive ester, for example a carbodiimide such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) or pentafluorophenyl, and in the presence of an organic base such as N-methylmorpholine. The reaction is usually carried out in the temperature range of xe2x88x9220xc2x0 C. to ambient temperature. The reagents, 1-hydroxybenzotriazole and dimethylaminopyridine (DMAP), are often added to assist the reaction (see Chem. Ber. 103, 788, 2024 (1970), J. Am. Chem. Soc. 93, 6318 (1971), Helv. Chim. Acta. 56, 717, (1973)). Suitable solvents include DMF and dichloromethane.
For examples of suitable conditions for amide bond forming reactions see International Patent Application No. WO 98/07692.
A compound of the formula (1) in which R2 in Ar2xe2x80x2 is carboxy can be prepared by reacting a compound of the formula (6) with a compound of the formula (7), or a compound of the formula (9) or (9a) with a compound of the formula (10) wherein R2 in Ar2xe2x80x2 is protected carboxy and subsequently removing the carboxy-protecting group.
A compound of the formula (8a) wherein R22 is of the formula xe2x80x94COR18 can be formed via the intermediate NH2CH(R21)CON(OMe)Me which itself is formed by reacting together NH2CH(R21)COOH with N,O-dimethylhydroxylamine under standard amide bond forming conditions. A compound of the formula NH2CH(R21)CON(OMe)Me is then conveniently reacted with a Gringard reagent (such as PhCH2MgCl) to form a compound of the formula (8).
Alternatively, when R18 contains an alkyl chain linked to the carbonyl group, a compound of the formula NH2CH(R21)CON(OMe)Me can be converted to the corresponding dimethylphosphono compound (NH2CH(R21)COP(O)(OMe)2) by reacting the former compound with dimethylmethylphosphonate in the presence of a strong base such as n-butyl lithium. A compound of the formula (8) can be formed by reacting the dimethylphosphono compound with the appropriate aldehyde or ketone under conditions known for the Wittig or Emmons-Horner reactions.
A compound of the formula (8) wherein R22 is morpholinomethyl, pyrrolidin-1-ylmethyl or piperidin-1-ylmethyl is conveniently prepared by reacting NH2CH(R21)COOH with the appropriate heterocyclic ring under standard amide bond forming conditions to form a compound of the formula (8), wherein R15 is heterocyclylcarbonyl, and subsequently reducing the carbonyl group to a methyl group with a reducing agent such as lithium aluminium hydride.
A compound of the formula NH2CH(R21)COOH can be extended by one carbon length to produce a compound of the formula NH2CH(R21)CH2COOH using the Arndt-Eistert homologation method. For example by converting the carboxy group to an acid chloride, converting the latter to the diazoketone and hydrolysing this to the carboxylic acid. This homologation method could be used to produce subsequent homologues. A compound of the formula NH2CH(R21)CH2COOH and homologues may be used to prepare a compound of the formula NH2CH(R21)R22 wherein R22 is of the formula xe2x80x94CH2COR15, morpholino C1-4alkyl, pyrrolidin-1-ylC1-4alkyl or piperidin-1-ylC1-4alkyl.
An anion of the formula (9) is conveniently formed with a strong base, such as n-butyl lithium. This generally forms an anion at the 2-position of the imidazole compound. The anion formation is generally carried out at low temperature e.g. xe2x88x9278xc2x0 C., in an inert or organic solvent such as diethyl ether.
The anion (usually the lithium anion) of a compound of the formula (9) is normally converted to an organozinc reagent in the presence of lithium chloride, zinc iodide and copper (I) cyanide and this complex is reacted with a compound of the formula (10) in an inert organic solvent such as tetrahydrofuran in a temperature range of xe2x88x9278xc2x0 C. to ambient temperature (see the review of organozinc reagents by Paul Knochel and Robert D Singer in Chem. Rev. 1993, 93, 2117-2188).
The anion of a compound of the formula (9a) is conveniently formed as a Grignard reagent by reacting the latter compound with an alkyl magnesium bromide in an ether at low temperature. The resulting Grignard reagent is typically reacted with a compound of the formula (10) in the presence of copper (I) cyanide and lithium chloride, allowing the temperature to rise from xe2x88x9278xc2x0 C. to ambient temperature. Preferably L1 in the compound of the formula (9a) is halo, for example iodo or bromo.
Preferably L2 in the compound of the formula (10) is halo, for example bromo.
A compound of the formula (10) may be prepared by brominating the corresponding hydroxy compound with a brominating agent such as tetrabromomethane/triphenylphosphine in a suitable solvent such as dichloromethane. The hydroxy compound is typically prepared by reacting a compound of the formula (7) and glycoaldehyde under conditions known for the Wittig reaction such as those described above for the reaction between compounds of the formulae (6) and (7).
The order in which the various groups are introduced can be varied depending upon the nature of the desired final product. In general, the preferred last step is the formation or modification of R2 in Ar2xe2x80x2. For example, when R9 is hydroxy, the hydrolysis of the compound wherein R9 is alkoxy, or when R9 is other than hydroxy, the conversion of a carboxy group to R2 using the amide bond-forming conditions described above.
When Ar1xe2x80x2 of the formula (A), it is preferable to form the alkene which contains a protected carboxy group later to be converted to R2, by reacting together compounds of the formulae (6) and (7).
When Ar2xe2x80x2 is of the formula (B) or (C) the alkene which contains a protected carboxy group later to be converted to R2, is preferably formed by reacting together a compound of the formula Ar2xe2x80x2CH2P+Ph3Br (wherein Ar2xe2x80x2 is substituted by xe2x80x94COOP2 in place of R2) and glycoaldehyde, converting the resultant hydroxy compound to a compound of the formula (9) (wherein Ar2xe2x80x2 is substituted by xe2x80x94COOP2 in place of R2) and reacting the latter compound with a compound of the formula (8) or (8a).
Optionally substituents in a compound of the formula (1) and (5) or intermediates in their preparation may be converted into other optional substituents. For example an alkylthio group may be oxidised to an alkylsulphinyl or alkysulphonyl group, a nitro group reduced to an amino group, a hydroxy group alkylated to a methoxy group, or a bromo group converted to an alkylthio group.
Various substituents may be introduced into compounds of the formulae (1) and (5) and intermediates in this preparation, when appropriate, using standard methods known in the art. For example, an acyl group or alkyl group may be introduced into an activated benzene ring using Friedel-Crafts reactions, a formyl group by formylation with titanium tetrachloride and dichloromethyl ethyl ester, a nitro group by nitration with concentrated nitric acid concentrated sulphuric acid and bromination with bromine or tetra(n-butyl)ammonium tribromide.
It will be appreciated that, in certain steps in the reaction sequence to compounds of the formula (1), it will be necessary to protect certain functional groups in intermediates in order to prevent side reactions. Deprotection may be carried out at a convenient stage in the reaction sequence once protection is no longer required.
Biological activity was tested as follows:
(i) In-vitro Assay
The following stock solutions were used and the assays were conducted in 96 well plates: TRIS Buffer (500 mM TRIS, 5 mM MgCl2.6H2O, pH=8.0); Farnesyl pyrophosphate (6.4 mg/ml); Aprotinin (1.9 mg/ml); Ki-ras (0.5 mg/ml, stored at xe2x88x9280xc2x0 C.); Acid ethanol (850 ml absolute ethanol+150 ml concentrated HCl).
Farnesyl protein transferase (FPT) was partially purified from human placenta by ammonium sulphate fractionation followed by a single Q-Sepharose(trademark) (Pharmacia, Inc) anion exchange chromatography essentially as described by Ray and Lopez-Belmonte (Ray K P and Lopez-Belmonte J (1992) Biochemical Society Transactions 20 494-497). The substrate for FPT was Kras (CVIM C-terminal sequence). The cDNA for oncogenic val 12 variant of human c-Ki-ras-2 4B was obtained from the plasmid pSW11-1 (ATCC). This was then subcloned into the polylinker of a suitable expression vector e.g. pIC147. The Kras was obtained after expression in the E. coli strain, BL21. The expression and purification of c-KI-ras-2 4B and the val12 variant in E. coli has also been reported by Lowe et al (Lowe P N et al. J. Biol. Chem. (1991) 266 1672-1678). The farnesyl protein transferase enzyme preparation was stored at xe2x88x9280xc2x0 C.
The farnesyl transferase solution for the assay contained the following: dithiothreitol (DTT)(0.6 ml of 7.7 mg/ml), TRIS buffer (0.6 ml), aprotinin (0.48 ml), distilled water (1.2 ml), farnesyl transferase (0.6 ml of the crude enzyme preparation prepared as described above), zinc chloride (12 xcexcl of 5 mM). This was left at ambienttemperature for 30 minutes. After this incubation 60 xcexcl Ki-ras solution was added and the whole left to incubate for a further 60 minutes prior to use in the assay.
Assays were performed in 96 well plates as follows: 10 xcexcl of test compound solution was added to each well. Then 30 xcexcl farnesyl transferase solution (above) was added and the reaction started by addition of 10 xcexcl radiolabelled farnesyl pyrophosphate solution. After 20 minutes at 37xc2x0 C. the reaction was stopped with 100 xcexcl acid ethanol (as described in Pompliano D L et al (1992) 31 3800-3807). The plate was then kept for 1 hour at 4xc2x0 C. Precipitated protein was then collected onto glass fibre filter mats (B) using a Tomtec(trademark) cell harvester and tritiated label was measured in a Wallac(trademark) 1204 Betaplate scintillation counter. Test compounds were added at appropriate concentrations in DMSO (3% final concentration in test and vehicle control).
(ii) Intracellular Farnesylation Assay
HER313A cells (Grand et al, 1987 Oncogene 3, 305-314) were routinely cultured in Dulbecos Modified Essential Medium (DMEM) plus 10% foetal calf serum (FCS). For the assay HER313A cells were seeded at 200,000 cells/well in a volume of 2.5 ml in a 6 well tissue culture plate. After an overnight incubation at 37xc2x0 C. in 10% CO2 the medium was removed and replaced with methionine-free minimal essential medium (MEM) and the cells incubated as above for 2 hours. After this time the medium was removed and replaced by methionine-free MEM (1 ml) and test compound (1-3 xcexcl). The plates were then incubated for a further 2 hours as described above and then 30 xcexcCi of 35S-methionine added to each well. The plate was then incubated overnight as described above. The medium was then removed and the cells were lysed with lysis buffer (1 ml) (composed of 1000 ml phosphate buffered saline, 10 ml trition X-100, 5 g sodium deoxycholate, 1 g sodium dodecylsulphate) containing aprotinin (10 xcexcl/ml), the plate scrapped and then left for 10 minutes at 4xc2x0 C. The lysate was then clarified by centrifugation. To 0.8 ml of the clarified lysate 80 xcexcl of Y13-259 pan-Ras antibody (isolated from the hybridomaxe2x80x94American Tissue Culture Collection Accession Number CRL-1742) (final concentration approximately 1 xcexc/ml, the exact working concentration was optimised for each batch of antibody isolated) and protein G beads (30 xcexcof 0.5 xcexcg/ml) were added and the mixture incubated overnight with constant agitation. The pellet was then collected by centrifugation, washed and separated by SDS PAGE using a 15% gel. Radioactive bands were detected using a phosphorimager system.
(iii) Morphology and Proliferation Assay
MIA PaCa 2 cells (American Tissue Culture Collection Accession Number: CRL-1420) were routinely cultured in Dulbecos Modified Essential Medium (DMEM) plus 10% FCS in a 162 cm2 tissue culture flask. For the assay the cells were seeded at 16,000 cells/well, in 12 well plates, in DMEM containing 5% charcoal dextran treated stripped FCS (1 ml) (obtained from Pierce and Warriner). The cells were then incubated overnight at 37xc2x0 C. in 10% CO2. Test compound was then added (10 xcexcl) and the cells incubated for 6 days as described above. On days 1, 2, 3 and 6 the cells were monitored for signs of morphological change and toxicity. On day 6 the cells were removed from the plate using trypsin/EDTA and counted to determine the proliferation rate.
Although the pharmacological properties of the compounds of the Formula (1) vary with structural change as expected, in general compounds of the Formula (1) possess an IC50 in test (i) above in the range, for example, 0.00005 to 50 xcexcM in test (i). Thus by way of example the compound of Example 29 herein has an IC50 of approximately 0.0004 xcexcM in test unacceptable toxicity was observed at the effective dose for compounds tested of the present invention.


An emission reduction device which may be removably affixed to a diesel engine's exhaust system. The device comprises an outer casing which may be divided into a lower portion and an upper portion, the lower portion of which is removably attachable to the exhaust system of a diesel engine; a carcass for holding a bobbin wherein the carcass is attached to the lower portion of the outer casing at the point where the exhaust enters the outer casing and the carcass has a beveled opening in a diagonal line in its proximal part and a bobbin positioned in its distal part; one or more fibrous blanket cylinders; and a guide for arranging and securing the one or more fibrous blanket cylinders within the outer casing. The fibrous blanket cylinders may be wrapped in a wire mesh. In an alternative embodiment, a second fibrous blanket formed into a cone with the larger diameter of the cone positioned proximally may be removably inserted in the carcass.

There is a need for a method and system capable of efficiently and effectively filtering pollutants from exhaust gases. Although there are a number of devices available which are useful for filtering exhaust gases from diesel engines, each of these devices is incapable of providing an effective method for reducing pollutants cost effectively for the reasons described herein.
In a diesel engine, air is drawn into the cylinders and is compressed by the pistons at compression ratios as high as 25:1, much higher than used for spark-ignited combustion engines. Near the end of the compression stroke, diesel fuel is injected into the combustion chamber through an injector (or atomizer). The fuel ignites from contact with the air that due to compression has been heated to a temperature of about 700-900° C. The resulting combustion causes increased heat and expansion in the cylinder which increases pressure and moves the piston downward. A connecting rod transmits this motion to a crankshaft to convert linear motion to rotary motion for use as power in a variety of applications. Intake air to the engine is usually controlled by mechanical valves in the cylinder head. For increased power output, most modern diesel engines are equipped with a turbocharger, and in some derivatives, a supercharger to increase intake air volume. Use of an aftercooler to cool intake air that has been compressed, and thus heated, by the turbocharger increases the density of the air and typically leads to power and efficiency improvements.
In general, diesel emissions are bi-products of diesel combustion. This can be a function of injection within the engine. For example, advancing the start of injection (injecting before the piston reaches top of dead center) results in higher in-cylinder pressure and temperature, and higher efficiency, but also results in higher emissions of oxides of nitrogen oxides through higher combustion temperatures. At the other extreme, delayed start of injection causes incomplete combustion and emits visible black smoke made of particulate matter and unburned hydrocarbon. While many diesel emissions are problematic, the most highly regulated diesel emissions are:    1. Diesel Particuiate Material ("PM", or "DPM") (also referred to as "Diesel Particulate Matter", "Particulate Material", or "Particulate Matter"): Particulate matter is an aerosol comprised of complex physical and chemical structures. Particulate matter contributes to the greenhouse effect, it causes grave environmental damage, and it seriously affects human health. Particulate matter is primarily responsible for the black smoke normally associated with diesel exhaust. It is also a primary source of urban smog.    2. Nitrogen Oxides (NOx): Nitrogen Oxides are highly active ozone precursors and account for a large component of visible smog. Besides particulate matter, nitrogen oxides are one of the most pollutive diesel emissions.    3. Hydrocarbons (HC): The production of hydrocarbons is often a result of the inefficient combustion of fuel and engine lube oils. In the atmosphere, hydrocarbons undergo photochemical reactions with nitrogen oxides leading to formation of smog and ground level ozone.    4. Carbon Monoxide (CO): This is a highly toxic greenhouse gas that is poisonous to humans and is a contributor to global warming.
Examples of non-regulated bi-products of diesel combustion include polynuclear aromatic hydrocarbons, aldehydes, sulfur dioxide, nitrous oxide, and metal oxide.
Inefficient combustion of diesel fuel produces emissions that pollute the environment and harm human health. The environmental consequences of particulate material emissions include air pollution, water pollution, acid rain, acidification of waterways, deforestation, smog, reduced atmospheric visibility, crop degradation, global warming, and climate forcing. In addition, the human health consequences of particulate material emissions include cardiovascular disease, respiratory disease, cancer, fibrosis, allergic responses, reduced pulmonary function, worsening of asthmatic symptoms and occurrences, increased morbidity, and premature death. Moreover, a number of internationally publicized studies demonstrate a high correlation between ambient particulate material and increases in adverse health outcomes such as respiratory hospital admissions, emergency room visits, restricted activity days, respiratory symptoms for adults, lower respiratory tract illnesses for children, asthmatic attacks, chronic diseases, and mortality.
Although conventional diesel emission filtration technologies are numerous, there are essentially two categories into which all such technologies fall:    1. Catalyzed Diesel Particulate Filters ("CDPFs"): catalyzed diesel particulate filters are referred to by many different names. Some of the most commonly used—and misused—are: "catalytic converters," "catalytic Reactors," "catalytic purifiers," "exhaust purifiers," "trap filters," "diesel traps," "exhaust scrubbers," "catalyst filters," "catalyzed wall-flow filters," "wall-flow filters," and "catalytic mufflers."    2. Diesel Oxidation Catalysts ("DOCs"): diesel oxidation catalysts are also commonly referred to as "oxidation catalysts," "flow-through catalysts," and "flow-through devices."
Both catalyzed diesel particulate filters and diesel oxidation catalysts employ the same basic method to achieve the reduction of particulate materials; they utilize heat to "oxidize" or bun the particulate material. In most cases, the heat from the engine's exhaust system is used to achieve oxidation. The reoccurring process of oxidation is also often referred to "regeneration" because the process of oxidation not only reduces particulate material emissions, it also regenerates the catalytic device's filtration capacity.
In order for the process of regenerative oxidation to occur, high temperatures, normally between 250° and 350° C., must be attained and preferably sustained during operation. In many operating conditions, attaining sufficiently high temperatures can prove difficult or unattainable. Catalytic devices (CDPF's and DOC's) employ precious metals such as platinum, palladium and rhodium as catalysts to lower the minimum temperatures necessary to achieve "light off", the point at which oxidation of the particulate material is initiated. Manufactures use these highly conductive, and very expensive, metals to coat or impregnate the substrate surfaces of their catalytic devices.
The catalytic devices discussed above can generally be described as either active or passive. Catalytic technologies which rely on heat from an engine's exhaust system in order to achieve oxidation are frequently referred to as "passive" catalytic devises. Other systems may incorporate fuel burners, electric heating elements, and fuel-borne additives which aid in attaining the temperatures at which oxidation occurs. Technologies which employ these types of components are often referred to as "active" catalytic devices.
For purposes of eliminating potential confusion, it should be noted that some manufacturers define catalyzed diesel particulate filters which only contain precious metal catalysts as "active" devices, even though these devices rely solely upon the heat contained in an engine's exhaust to achieve oxidation. This classification usually occurs when the manufacturer also produces a diesel particulate filter which contains no catalyst, i.e. a device which is in all other ways similar to a catalyzed diesel particulate filter, however; the device relies solely upon the heating of its component base metal to achieve temperatures sufficient to initiate oxidation. Because exhaust temperatures are commonly required to exceed 500° C. for these non-catalyzed devices to affect oxidation, their widespread use is significantly restricted.
The primary difference between catalyzed diesel particulate filters and diesel oxidation catalyst technologies is that catalyzed diesel particulate filter technologies physically trap and store particulate material—usually by using catalyzed ceramic, cordierite or silicon carbide wall flow monoliths, or ceramic fiber or ceramic cartridge filters. Once the particulate material becomes trapped, it is oxidized and particulate material emissions are reduced.
Conversely, diesel oxidation catalyst technologies do not trap particulate material emissions. Rather, particulate materials "pass-through" the internal structures of these devices. When exhaust gases traverse the catalyst, carbon monoxide, gaseous hydrocarbons and liquid hydrocarbon particles are oxidized, thereby reducing total particulate material emissions.
There are a number of other differences between catalyzed diesel particulate filters and diesel oxidation catalyst technologies as well. For example, catalyzed diesel particulate filters can achieve particulate material filtration rates of ≧90% given specific, controlled operating conditions. Moreover, catalyzed diesel particulate filters reduce each sub-category of particulate material (i.e. solid inorganic fractions, solid organic fraction and sulfate particulates). It is necessary to note however, the application and effectiveness of catalyzed diesel particulate filters technology is significantly constrained by the following limitations:                Catalyzed diesel particulate filters are very expensive. The California Air Resources Board provides cost-range information for DPF's corresponding to the following engine capacitates:                    100 horsepower: US$5,000-US$7,000            275 horsepower: US$6,900-US$9,000            400 horsepower: US$10,000 average            1,400 horsepower: US$32,000+                        Catalyzed diesel particulate filters are incapable of affecting particulate material emissions reductions when using fuels that exceed 150 ppm Sulfur.        Catalyzed diesel particulate filters performance is adversely affected by insufficient operating temperatures.        In less-than-optimal conditions, catalyzed diesel particulate filters are prone to clogging and failure. When failure occurs, the potential for engine damage or destruction is significant.        Because catalyzed diesel particulate filters can create significant engine back pressure, expensive engine recalibrations are often required upon their installation.        catalyzed diesel particulate filters often need to be equipped with expensive electronic back pressure monitoring devices, such as data loggers.        Because passive catalyzed diesel particulate filters regeneration is entirely dependent on operating temperature, passive catalyzed diesel particulate filters do not work under "low load" conditions.        "Active" components in catalyzed diesel particulate filter technologies significantly increase catalyzed diesel particulate filters unit price and complexity.        Catalyzed diesel particulate filters do not work well on older engines.        Catalyzed diesel particulate filters can become a source of hazardous zinc, sulfuric, calcium, and phosphorus ash particulate.        Catalyzed diesel particulate filters can reduce engine performance.        Catalyzed diesel particulate filters often produce fuel economy penalties.
According to the United States Department of Energy (USDOE), fuel sulfur has significant effects on post-filter total particulate material emissions, and, as fuel sulfur levels increase, catalyzed diesel particulate filter reduction efficiencies decreases to a point where they actually becomes a source of particulate emissions when using fuels with sulfur concentrations ≧150 PPM.
Tests conducted by the USDOE report that catalyzed diesel particulate filters that achieved 95% reductions of particulate material emissions when using fuels with 3 ppm sulfur concentrations had their filtration efficiencies reduced to only 74% when using fuels with 30 ppm sulfur concentrations. Further, these same devices were reduced to particulate material filtration rates of 0% to −3% when using fuels with 150 ppm sulfur concentrations, and they experienced total particulate material emissions increases of 122% to 155% when using fuels with sulfur concentrations ≧350 ppm.
Moreover, the Natural Resources Defense Council (NRDC) has stated that catalytic technologies can not work properly if there is sulfur in the fuel—and in some cases, sulfur in the fuel will render the catalytic filtration equipment and even the vehicle inoperable.
By comparison, diesel oxidation catalyst technologies are generally less expensive than catalyzed diesel particulate filter technologies, and because diesel oxidation catalysts are "flow through", instead of "wall flow" devises, they do not have the same propensity to create engine back pressure, clog and/or cause potential engine damage like their catalyzed diesel particulate filter counterparts. Diesel oxidation catalysts can achieve particulate material filtration rates between 19% and 50%. However, the application of diesel oxidation catalyst technology is constrained by the following:                Diesel oxidation catalysts are too expensive for wide-spread application. The California Air Resources Board provides cost average information for diesel oxidation catalysts corresponding to the following engine capacitates:                    275 horsepower: US$2,100            400 horsepower: US$20,000+            The Everett School District in Washington state reported an average per-unit-cost of US$2,500 per DOC for each bus in its fleet                        Diesel oxidation catalyst reduction of total particulate material is significantly reduced when using fuels with high sulfur fuels.        Diesel oxidation catalysts do not filter solid organic fraction sometimes called "dry") particulate and dry particulates typically comprise the majority of total particulate material.        Diesel oxidation catalysts do not work well on older engines.        Diesel oxidation catalyst effectiveness is extremely dependent upon operating temperatures.        When operating at higher temperatures, diesel oxidation catalysts oxidize sulfur oxides, and in doing so become generators of sulfuric acid. When this occurs, diesel oxidation catalysts create a net increase total particulate material emissions by increasing production of sulfate particulates at rates that offset soluble organic fraction reductions
The University of Washington's Extension Energy Program has stated that diesel oxidation catalysts can oxidize sulfur dioxide to form sulfate particulates (sulfuric acid (H2SO4)). Therefore, high sulfur content fuels can increase total particulate emissions via the production of sulfuric acid, which can offset soluble organic fraction (sometimes called "wet" particulate material) reductions."
The United States Department of Energy has found statistically significant increases in particulate material with high sulfur fuel due almost exclusively to the increase in the SO4 fraction of the total particulate material. At this high exhaust temperature (405° C. at catalyst inlet), the diesel oxidation catalyst accelerates the conversion of SO2 to SO3, thereby increasing the SO4 fraction of the particulate material. As expected, the effect is seen only with the higher sulfur (150 ppm and 350 ppm sulfur content) fuels. With the 350 ppm sulfur content fuel, post catalyst particulate material emissions were approximately 200% higher than those measured without an active catalyst.
Despite the promoted efficiency of the methods and systems of the prior art, many are impracticable from the commercial point of view for the reasons set forth above. Moreover, the use of fuel with low concentration of sulphur (below 130 ppm) is an essential factor in the employment of catalytic regeneration filters. In Brazil and in the majority of the countries, the diesel is sold with 2000 ppm of sulphur. Therefore using the catalytic regeneration filters in diesel that contains more than 300 ppm of sulphur, turn the filters into a source of pollution.


The present invention is directed to isolated cDNA which codes for spider silk protein or a fragment or variant thereof, a replicable vector containing cDNA which codes for spider silk protein and which is capable of expressing spider silk protein, a transformed cell or microorganism containing cDNA which codes for spider silk protein or a fragment thereof which is capable of expressing spider silk protein and products, such as fibers, which may be manufactured utilizing the recombinant protein of the present invention.

Major ampullate (dragline) silk of orb web spiders possesses unique physical properties, combining high tensile strength and substantial elasticity, Denny, M. W. J. Exp. Biol., 65, 483-506 (1976); Lucas, F. Discovery, 25, 20-26 (1964). Previous investigations suggest that spider silk is composed of a single large protein, primarily containing pseudo-crystalline regions of stack .beta.-pleated sheet alternating with amorphous domains, Warwicker, J. O., J. Mol. Biol., 2, 350-362 (1960); Lucase, F. et al, J. Text Inst., 46, T440-T452 (1985); Hepburn, H. R. et al., Insect Biochem., 9, 69-71 (1979). The molecular basis for spider silk elasticity is presently unknown, although it has been suggested that an entropy driven process like that found in rubber is involved, Gosline, J. M., et al., Nature, 309, 551-552 (1984). It has also been speculated that the amorphous regions contribute substantially to the elastic properties of the fiber, Hepburn, H. R., et al., Insect Biochem., 9, 69-77 (1979).

Isolated DNA coding for spider silk protein, a replicable vector and a transformed cell containing the DNA


A layered construction of busbars and insulating plates wherein the lower surfaces of the busbars on the lower surface of the upper insulating plate are opposed to and spaced apart from the upper surfaces of the busbars on the upper surface of the lower insulating plate by a specified distance T. Clearance providing ribs project from the lower surface of the upper insulating plate and the upper surface of the lower insulating plate and are in contact with the surfaces of the busbars.

An electrical connection box used to connect wiring harnesses of an automotive vehicle is, as shown in FIG. 3, provided with upper casing 1 and lower casing 2. Layered element 10, including busbars 3 to 6 for forming an internal circuit, and insulating plates 7 to 9, is accommodated in a space defined by casings 1 and 2. Busbars 3 to 6 are connected via connectors 13 to relays 11 and fuses 12 mounted in receptacles formed on the upper surface of upper casing 1.
As shown in detail in FIG. 4, in layered element 10, first tier busbars 3 are arranged on the upper surface of upper insulating plate 7, second tier busbars 4 are located on the upper surface of intermediate insulating plate 8, third tier busbars 5 are on the upper surface of lower insulating plate 9, and fourth tier busbars 6 are on the lower surface of lower insulating plate 9.
The lower surface of upper insulating plate 7 and the lower surface of intermediate insulating plate 8 are in contact with the upper surfaces of ribs 8a in intermediate insulating plate 8 and the upper surfaces of ribs 9a in the lower insulating plate 9, respectively. Further, the lower surface of upper insulating plate 7 and the lower surface of intermediate plate 8 are substantially in contact with the upper surfaces of second tier busbars 4 and the upper surfaces of third tier busbars 5, respectively.
However, the layered element 10 requires three insulating plates 7 to 9 to arrange four layers of busbars 3 to 6. This leads to an increased number of parts, increased weight of the electrical connection box, and consequently to increased production cost for layered element 10.

Layered construction of busbars and insulating plates with ribs


A drumhead and a method for coating a drumhead membrane comprising applying a coating having cross linkable polymeric material to the membrane and applying energy to cross link the polymeric material and thereby permanently adhere the material to the membrane as a play surface. The cross-linking not only hardens the coating to improve durability, but also improves the adhesion of the coating to the base film. Actinically cured coatings can completely harden in a matter of 3 to 5 seconds, with UV cured inks being especially desirable.

The present invention relates to drumheads and in particular to the membrane and associated play surface of the drumhead.
Conventionally, drumheads are manufactured by processing a plastic film or membrane for desirable acoustic properties when the membrane is tensioned on the bearing edge of a drum shell. Sheets of processed film are cut and shaped with a collar region which is secured into a circular hoop that is engaged by tensioning components on the drum shell. A circular area within the hoop defines a play surface on which the percussionist can produce various sounds with, for example, sticks or brushes.
It is common to coat a plastic film such as Mylar with ink, using a mesh screen, to achieve coloration as well as subtle variations in the acoustic response of the drumhead. Typically, the ink is dried by evaporation in an oven or under a heating lamp. The drying time generally ranges from about 3 hours to 10 minutes. When a large area of coated film or many drumheads in progress are to be dried in one relatively small oven or arrangement of lamps, the drying step becomes a bottleneck in the overall drumhead manufacturing process.


There is provided a semiconductor light emitting device 100 including a substructure 101, 120, 130 including at least one light emitting region R1 including a plurality of three-dimensional (3-D) light emitting nanostructures 140 and at least one electrode region R2, R3 including a plurality of locations CP2A, 17A, 17B, 18A, 18B wherein an arrangement of the plurality of three-dimensional (3-D) light emitting nanostructures 140 and the plurality of locations CP2A, 17A, 17B, 18A, 18B are identical.

The present disclosure relates to a semiconductor light emitting device, a method for manufacturing a semiconductor light emitting device, and a method for manufacturing a semiconductor light emitting device package.
Light Emitting Diodes (LEDs) have many advantages as compared to related art light sources, such as relatively long lifespans, low degrees of power consumption, rapid response speeds, environmental friendliness, and the like, and have thus been widely seen as next generation lighting sources and have increased in prominence as a type of light source for use in various products, such as in general lighting devices and in the backlights of display devices. For example, LEDs based on Group III nitrides, such as gallium nitride (GaN), aluminum gallium nitride (AlGaN), indium gallium nitride (InGaN), indium aluminum gallium nitride (InAlGaN), and the like, may play a role as semiconductor light emitting devices outputting blue or ultraviolet light.
Recently, as LEDs have come into more widespread use, the utilization thereof has extended to the light sources of devices for use in high current and high output applications. Demand for LEDs in devices for use in high current and high output applications has spurred ongoing research in the art to which the present disclosure is directed into improvements in light emitting characteristics. For example, in order to increase luminous efficiency through enhancements in crystallinity and increases in light emitting areas, semiconductor light emitting devices having light emitting nanostructures and manufacturing techniques therefor have been proposed.

Semiconductor light emitting device, method for manufacturing semiconductor light emitting device, and method for manufacturing semiconductor light emitting device package


A first computing device running a particular program is used to identify a second computing device also running the particular program and substantially co-located with the first computing device in a particular physical location. The first computing device and the second computing device are joined in a use session of the particular program. It is determined that the first computing device displays a first user interface of the particular program at a first instance, the first user interface showing a first context in a plurality of contexts. The second computing device displays a second user interface of the particular program at the first instance based at least in part on the first user interface showing the first context, the second user interface showing a second context in the plurality of contexts.

Advances in computing have allowed people and systems to communicate in new and revolutionary ways. Graphical user interfaces are being developed that assist in the communication of concepts to and performance of tasks by users of software programs and applications. Some graphical user interfaces have been developed that allow users to control other computing devices from a remote computing device. User interfaces can be employed to assist and direct users in their manipulations of other computing devices, including remote display devices. Further, recent developments in video gaming have introduced the use of content-rich controllers that can be utilized by users to interact with a second user interface provided at least in part by a second computing device. In such systems, a first user interface is displayed on the controller device itself thereby supplementing a second, main user interface device, such as a television connected to or integrated with a video game console. Examples of such systems include Nintendo Co., Ltd.'s Wii™ U™ prototype and Apple Inc.'s Apple TV™'s airplay game mirroring technology allowing a user to utilize Apple™ iPad™ devices as controllers in video games displayed in part on an Apple TV and in part on an Apple iPad.
Like reference numbers and designations in the various drawings indicate like elements.


A pressure sensor includes a body made of semiconductor material having a first type of conductivity and a pressure-sensitive structure having the first type of conductivity defining a suspended membrane. One or more piezoresistive elements having a second type of conductivity (P) are formed in the suspended membrane. The piezoresistive elements form, with the pressure-sensitive structure, respective junction diodes. A temperature sensing method includes: generating a first current between conduction terminals common to the junction diodes; detecting a first voltage value between the common conduction terminals when the first current is supplied; and correlating the detected first voltage value to a value of temperature of the diodes. The temperature value thus calculated can be used for correcting the voltage signal generated at output by the pressure sensor when the latter is operated for sensing an applied outside pressure which deforms the suspended membrane.

Known to the art are micromachining techniques for providing integrated pressure sensors made of semiconductor material. Said sensors present numerous advantages in terms of low cost, high degree of functionality and reliability, good signal-to-noise ratio, integrability with memory circuits to obtain smart sensors, and high reproducibility. Semiconductor pressure microsensors present on the market are essentially based upon two physical effects: the piezoresistive effect, whereby the deflection of a silicon membrane caused by the pressure unbalances a Wheatstone bridge provided with resistances diffused in the membrane; and the capacitive effect, whereby the pressure induces displacement of a membrane that constitutes the mobile electrode of a capacitor (thus determining variation of capacitance thereof). In what follows only pressure sensors that use the first effect, i.e., piezoresistive sensors, will be considered.
A method of manufacture of a piezoresistive pressure sensor of a known type is described, for example, in European Patent No. 822398 or in European Patent No. 1577656, the disclosures of which are incorporated by reference. The membranes of said sensors, in order to guarantee proper operation, must have a well-controlled homogeneous thickness and moreover must not present intrinsic mechanical stresses (of a tensile and compressive nature). A method of fabrication of a membrane designed for use in piezoresistive pressure sensors is, for example, described in U.S. Pat. No. 7,871,894, the disclosure of which is incorporated by reference.
One of the main disadvantages of piezoresistive pressure sensors is the high thermal drift, that they undergo as the temperature varies. In the absence of compensation, a variation temperature of approximately 10° C. in a piezoresistive pressure sensor can cause a non-negligible drift of the output signal, in particular for applications that require high sensitivity (e.g., medical applications such as artificial breathers, spirometers, altimeters, barometers, etc.). For this reason, it is necessary to equip these sensors with a system for compensation of thermal drift. One of the known methods comprises inserting said sensors in a transduction circuit based upon the Wheatstone bridge. This modality envisages inserting on the opposite branch of the bridge piezoresistive elements that are substantially the same as those mounted on the sensor element, but arranged so as not to undergo deformations linked to the pressure applied to the membrane. As the temperature varies, all the piezoresistors undergo approximately the same thermal drifts. In this way, the elements used for compensation rebalance the Wheatstone bridge, reducing the dependence of the output pressure signal upon the temperature of the transducer.
However, on account of variations of layout of the sensor, variations of morphology of the piezoresistors linked to spreads of the manufacturing process, local concentrations of impurities, and in general other conditions of physical mismatch, also in the case of Wheatstone-bridge connection of the piezoresistors, variations of the pressure signal at output from the bridge are not completely independent of temperature variations of the sensor. A further step of compensation of the variations of the output signal caused by the temperature is consequently necessary. For this purpose, it is necessary to acquire a signal correlated to the temperature to which the piezoresistors are subjected in use. There has consequently been proposed temperature sensors located in the proximity of the pressure sensor, adapted to be used for thermal compensation of the pressure sensor. Said double-sensor systems show a temperature gradient between the temperature sensor and the membrane of the sensor, on account of the different physical location. The time that elapses for stabilization of the temperature gradient is known as "warm-up drift".
A solution to this problem has, for example, be proposed by Kuo Huan Peng, C. M. Tiang, and Yih Min Chang. "The temperature compensation of the silicon piezoresistive pressure sensor using the half-bridge technique", Proc. SPIE 5343, 292 (2004), the disclosure of which is incorporated by reference. In this document, the output drift of the bridge due to the variation in temperature is minimized by means of auto-gain circuits (AGCs) for adjusting automatically the voltage supplied to the bridge. This solution presents, however, certain disadvantages. In particular, the size of the pressure sensor is considerably increased for housing the temperature-sensing circuit. In addition, a high number of pads are used for biasing correctly both the piezoresistors and the temperature-sensing circuit.
There exists a need in the art to provide a transducer equipped with a temperature sensor, and a method for sensing a temperature of said transducer.

Integrated transducer provided with a temperature sensor and method for sensing a temperature of the transducer


The invention provides for compositions and methods for producing isoprene using isoprene synthase variants with improved properties.

Isoprene (2-methyl-1,3-butadiene) is a volatile hydrocarbon that is insoluble in water and soluble in alcohol. Commercially viable quantities of isoprene can be obtained by direct isolation from petroleum C5 cracking fractions or by dehydrogenation of C5 isoalkanes or isoalkenes (Weissermel and Arpe, Industrial Organic Chemistry, 4th ed., Wiley-VCH, pp. 117-122, 2003). The C5 skeleton can also be synthesized from smaller subunits. It would be desirable, however, to have a commercially viable method of producing isoprene that was independent of nonrenewable resources.
Biosynthetic production of isoprene occurs by two distinct metabolic pathways (Julsing et al., Appl Microbiol Biotechnol, 75:1377-1384, 2007). In eukaryotes and archae, isoprene is formed via the mevalonate (MVA) pathway, while some eubacteria and higher plants produce isoprene via the methylerythritol phosphate (MEP) pathway. Isoprene emissions from plants are light and temperature-dependent with increases linked to leaf development. An isoprene-producing enzyme, isoprene synthase, has been identified in Aspen trees (Silver and Fall, Plant Physiol, 97:1588-1591, (1991); and Silver and Fall, J Biol Chem, 270:13010-13016, (1995)) and is believed to be responsible for the in vivo production of isoprene from whole leaves. Bacterial production of isoprene has also been described (Kuzma et al., Curr Microbiol, 30:97-103, (1995); and Wilkins, Chemosphere, 32:1427-1434, (1996)), and varies in amount with the phase of bacterial growth and the nutrient content of the culture medium (U.S. Pat. No. 5,849,970 to Fall et al.; and Wagner et al., J Bacteriol, 181:4700-4703, (1999)). The levels of isoprene obtainable through bacterial systems of the prior art, however, are insufficient for commercial uses. Thus what the art needs is an efficient, large scale isoprene biological production process to produce isoprene. The invention described herein addresses these problems and provides additional benefits as well.

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Identification of isoprene synthase variants with improved properties for the production of isoprene