Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
  • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
  • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D207/16—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• MMPs matrix metalloproteinases
• TEVIP tissue inhibitor of metalloproteinases
• MMPs Three groups of MMPs have been delineated: the collagenases which have triple helical interstitial collagen as a substrate, the gelatinases which are proteinases of denatured collagen and Type IV collagen, and the stromelysins which were originally characterized as proteoglycanases but have now been identified to have a broader proteolytic spectrum.
• specific collagenases include fibroblast collagenase (MMP-1 ), neutrophil collagenase (MMP-8), and collagenase 3 (MMP- identified to have a broader proteolytic spectrum.
• specific collagenases include fibroblast collagenase (MMP-1), neutrophil collagenase (MMP-8), and collagenase 3 (MMP- 13).
• gelatinases include 72 kDa gelatinase (gelatinase A; MMP-2) and 92 kDa gelatinase (gelatinase B; MMP-9).
• stromelysins include stromelysin 1 (MMP-3), stromelysin 2 (MMP-10) and matrilysin (MMP-7).
• MMPs which do not fit neatly into the above groups include metalloelastase (MMP-12), membrane-type MMP (MT-MMP or MMP-14) and stromelysin 3 (MMP-11). Beckett, R.P. et al., supra.
• MMPs Over-expression and activation of MMPs have been linked with a wide range of diseases such as cancer; rheumatoid arthritis; osteoarthritis; chronic inflammatory disorders, such as emphysema and smoking-induced emphysema; cardiovascular disorders, such as atherosclerosis; coraeal ulceration; dental diseases such as gingivitis and periodontal disease; and neurological disorders, such as multiple sclerosis.
• diseases such as cancer; rheumatoid arthritis; osteoarthritis; chronic inflammatory disorders, such as emphysema and smoking-induced emphysema; cardiovascular disorders, such as atherosclerosis; coraeal ulceration; dental diseases such as gingivitis and periodontal disease; and neurological disorders, such as multiple sclerosis.
• invasive proximal gastric cells express the 72 kDa form of collagenase Type IV, whereas the noninvasive cells do not. Schwartz, G.K. et al., Cancer
• Rat embryo cells transformed by the Ha-ras and v-myc oncogenes or by Ha-ras alone are metastatic in nude mice and release the 92 kDa gelatinase/collagenase (MMP-9). Bernhard, E.J. et al., Proc. Natl. Acad. Sci. 91, 4293-4597 (1994).
• the plasma concentration of MMP-9 was significantly increased (P ⁇ 0.01) in 122 patients with gastrointestinal tract cancer and breast cancer.
• a range of MMPs can hydrolyse the membrane-bound precursor of the pro-inflammatory cytokine tumor necrosis factor ⁇ (TNF- ⁇ ).
• TNF- ⁇ tumor necrosis factor ⁇
• This pharmacological action is a probable contributor to the antiarthritic action of this class of compounds seen in animal models. Beckett, R.P. et al., supra.
• Stromelysin has been observed to degrade the ⁇ i-proteinase inhibitor which regulates the activity of enzymes such as elastase, excesses of which have been linked to chronic inflammatory disorders such as emphysema and chronic bronchitis. Beeley, N.R.A. et al., supra.; Wahl, R.C. et al.. Annual Reports in Medicinal Chemistry 25. 177-184 (1990).
• MMP- 12 is required for the development of smoking- induced emphysema in mice. Science, 277, 2002 (1997). Inhibition of the appropriate MMP may thus potentiate the inhibitory activity of endogenous inhibitors of this type.
• Collagenase, stromelysin and gelatinase have been implicated in the destruction of the extracellular matrix of the cornea. This is thought to be an important mechanism of morbidity and visual loss in a number of ulcerative ocular diseases, particularly those following infection or chemical damage. Burns, F.R. et al, Invest. Opthalmol. and Visual Sci. 32, 1569-1575 (1989).
• the MMPs present in the eye during ulceration are derived either endogenously from infiltrating leucocytes or fibroblasts, or exogenously from microbes.
• Collagenase and stromelysin activities have been identified in fibroblasts isolated from inflamed gingiva and the levels of enzyme have been correlated with the severity of the gingivitis observed.
• the present invention provides novel 3-substitutedpyrrolidines of formula (1 ):
• e is an interger from 0 to 2;
• A is selected from the group consisting of -OH and -NRR'; wherein
• R and R' are independently selected from the group consisting of hydrogen and C ⁇ -C 6 alkyl or R and R' taken together with the nitrogen atom to which they are attached form a N-morpholino, N-piperidino, N-pyrrolidino, or N-isoindolyl;
• Ri is selected from the group consisting of hydrogen, C ⁇ -C 6 alkyl, -(CH 2 ) a -CO2R 5 , -(CH 2 ) a -C(O)NH 2 , -(CH 2 ) 4 NH 2 , -(CH 2 ) 3 -NH-C(NH)NH 2 , -(CH 2 ) 2 -S(O) b -CH 3 , -CH 2 -OH, -CH(OH)CH 3 , -CH 2 -SH, -(CH 2 ) d -Ar,, and -CH 2 -Ar 2 ; wherein a is 1 or 2; b is 0, 1, or 2; d is an integer from 0 to 4;
• R 5 is selected from the group consisting of hydrogen, C ⁇ _C 4 alkyl, and benzyl;
• Aj] is a radical selected from the group consisting of
• R 6 is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, C ⁇ .C alkyl, hydroxy, and C 1 -C4 alkoxy;
• R 7 is selected from the group consisting of hydrogen, halogen, C
• Ar 2 is a radical selected from the group consisting of
• R 2 is a radical selected from the group consisting of
• R 2 - is from 1 to 2 substituents selected from the group consisting of hydrogen, halogen, C ⁇ -C 4 alkyl, and C ⁇ -C 4 alkoxy;
• R 3 is selected from the group consisting of C ⁇ -C 6 alkyl, -(CH 2 ) m -W, -(CH2) P -Ar 3 , -(CH 2 ) k -CO 2 R 9 , -(CH 2 ) m -NR 8 SO 2 -Y,, and -(CH 2 ) m -Z-Q wherein m is an integer from 2 to 8; p is an integer from 0-10; k is an integer from 1 to 9;
• W is phthalimido
• Ar 3 is selected from the group consisting of
• R 23 is from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, C
• R 8 - is hydrogen or C]-C 6 alkyl;
• R is hydrogen or C ⁇ -C 6 alkyl;
• Y ⁇ is selected from the group consisting of hydrogen, -(CH 2 ) j -Ar 4 , and -N(R 24 ) 2 wherein j is 0 or 1 ;
• R 24 each time selected is independently hydrogen or C ⁇ -C 6 alkyl or are taken together with the nitrogen to which they are attached to form N-morpholino, N- piperidino, N-pyrrolidino, or N-isoindolyl;
• R 25 is from 1 to 3 substituents independently selected from the group consisting of hydrogen, halogen, C ⁇ -C 4 alkyl, and C C 4 alkoxy;
• Z is selected from the group consisting of -O-, -NR -, -C(O)NR 8 -, -NR 8 C(O)-, -NR 8 C(O)NH-, -NR 8 C(O)O -, and -OC(O)NH-;
• R 8 is hydrogen or C ⁇ -C 6 alkyl
• Q is selected from the group consisting of hydrogen, -(CH 2 ) n -Y 2 , and -(CH 2 ) -Y 3 ; wherein n is an integer from 0 to 4; x is an integer from 2 to 4;
• Y is selected from the group consisting of hydrogen, -(CH 2 ) n -Ar5 and -(CH 2 ),-C(O)OR 2 7 wherein
• Ar is selected from the group consisting of
• R 26 is from 1 to 3 substituents independently selected from the group consisting of hydrogen, halogen, C ⁇ -C 4 alkyl, and C ⁇ -C 4 alkoxy; h is an integer from 0 to 6; t is an integer from 1 to 6; R 27 is hydrogen or C ⁇ -C 6 alkyl; Y 3 is selected from the group consisting of -N(R 28 ) 2 , N-morpholino, N- piperidino, N-pyrrolidino, and N-isoindolyl; wherein
• R 2 g each time taken is independently selected from the group consisting of hydrogen and Cj-C 6 alkyl;
• R4 is selected from the group consisting of hydrogen, -C(O)R ⁇ o, -C(O)-(CH 2 ) q -K and -S-G wherein
• Rio is selected from the group consisting of hydrogen, C ⁇ _C 4 alkyl, phenyl, and benzyl; q is 0, 1, or 2; K is selected from the group consisting of
• V is selected from the group consisting of a bond, -CH 2 -, -O-, -S(O),-, -NR 21 -, and -NC(O)R 22 -; wherein r is 0, 1 , or 2;
• R 21 is selected from the group consisting of hydrogen, C ⁇ -C alkyl, and benzyl
• R 22 is selected from the group consisting of hydrogen, -CF 3 , C 1 -C 10 alkyl, phenyl , and benzyl
• R ⁇ is selected from the group consisting of hydrogen, C1-C4 alkyl, and benzyl
• Ri 1 - is selected from the group consisting of hydrogen, C ⁇ _C 4 alkyl, and benzyl;
• G is selected from the group consisting of
• w is an integer from 1 to 3;
• Ri 2 is selected from the group consisting of hydrogen, C ⁇ -C 6 alkyl,
• R 13 is selected from the group consisting of hydrogen, hydroxy, amino, C ⁇ -C 6 alkyl, N-methylamino, N,N-dimethylamino, -CO 2 R 1 7, and -OC(O)R] 8 ; wherein
• R ⁇ is hydrogen, -CH 2 O-C(O)C(CH 3 ) 3 , C,.C 4 alkyl, benzyl, or diphenylm ethyl;
• Ri 8 is hydrogen, C
• R 1 9 is hydrogen, C ⁇ _C alkyl, or benzyl
• R 2 o is hydrogen, -CF 3 , Cj-Cio alkyl, or benzyl;
• R 15 is selected from the group consisting of hydrogen, C ⁇ -C 6 alkyl and benzyl; R I O is selected from the group consisting of hydrogen and C ⁇ -C 4 alkyl; and stereoisomers, pharmaceutically acceptable salt, and hydrate thereof.
• the present invention further provides a method of inhibiting matrix metalloproteinases (MMPs) in a patient in need thereof comprising administering to the patient an effective matrix metalloproteinase inhibiting amount of a compound of formula (1).
• MMPs matrix metalloproteinases
• the present invention provides a method of treating a neoplastic disease state or cancer; rheumatoid arthritis; osteoarthritis; osteoporosis; cardiovascular disorders, such as atherosclerosis; corneal ulceration; dental diseases, such as gingivitis or periodontal disease; and neurological disorders, such as multiple sclerosis; chronic inflammatory disorders, such as emphysema and especially smoking-induced emphysema.
• the present invention provides a composition comprising an assayable amount of a compound of formula (1) in admixture or otherwise in association with an inert carrier.
• the present invention also provides a pharmaceutical composition comprising an effective MMP inhibitory amount of a compound of formula (1 ) in admixture or otherwise in association with one or more pharmaceutically acceptable carriers or excipients.
• the compounds of formula (1) exist as stereoisomers. Specifically, it is recognized that they exist as stereoisomers at the point of attachment of the substituents R,, -(CH 2 ) e -R 2 , R3, and -SR 4 , -C(O)NH-CHR,-C(O)A, R, 2 , and -NHR 1 5. Where indicated the compounds follow either the (+)- and (-)- designation for optical rotation, the (D)- and (L)- designation of relative stereochemistry, or the Cahn-Ingold- Prelog designation of (R)-and (S)- for the stereochemistry of at specific postions in the compounds represented by formula (1 ) and intermediates thereof.
• any reference in this application to one of the compounds of the formula (1) is meant to encompass either specific stereoisomers or a mixture of stereoisomers.
• the specific stereoisomers can be prepared by stereospecific synthesis using enantiomerically pure or enantiomerically enriched starting materials which are well known in the art.
• the specific stereoisomers of amino acid starting materials are commercially available or can be prepared by stereospecific synthesis as is well known in the art or analogously known in the art, such as D. A. Evans, et al. J. Am. Chem. Soc, 112, 401 1-4030 (1990); S. Ikegami et al. Tetrahedron, 44, 5333-5342 (1988); W.Oppolzer et al. Tet. Lets.
• stereoisomers of either starting materials or products can be resolved and recovered by techniques known in the art, such as chromatography on chiral stationary phases, enzymatic resolution, or fractional recrystallization of addition salts formed by reagents used for that purpose.
• Useful methods of resolving and recovering specific stereoisomers are known in the art and are described in Stereochemistry of Organic Compounds, E. L. Eliel and S. H. Wilen, Wiley (1994) and Enantiomers. Racemates, and Resolutions, J. Jacques, A. Collet, and S. H. Wilen, Wiley (1981).
• halogen refers to a fluorine atom, chlorine atom, bromine atom, or iodine atom
• C ⁇ -C 6 alkyl refers to a branched or straight chained alkyl radical containing from 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, etc.;
• C1-C4 alkyl refers to a saturated straight or branched chain alkyl group containing from 1 -4 carbon atoms and includes methyl, ethyl, propyl, isopropyl, n-butyl, s- butyl, isobutyl, and t-butyl;
• C1-C4 alkoxy refers to a straight or branched alkoxy group containing from 1 to 4 carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy. isobutoxy, t- butoxy, etc.;
• g refers to grams
• mg refers to milligrams
• ⁇ g refers to micrograms
• mol refers to moles
• mmol refers to millimoles
• nmole refers to nanomoles
• L refers to liters
• mL or “ml” refers to milliliters
• ⁇ L refers to microliters
• °C refers to degrees Celsius
• R t refers to retention factor
• mp refers to melting point
• dec refers to decomposition
• bp refers to boiling point
• mm of Hg refers to pressure in millimeters of mercury
• cm refers to centimeters
• nm refers to nanometers
• brine refers to a saturated aqueous sodium chloride solution
• M refers to molar
• mM refers to millimolar
• pharmaceutically acceptable acid addition salts is intended to apply to any non-toxic organic or inorganic acid addition salt of the base compounds represented by formula (1) or any of its intermediates.
• inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulphuric, and phosphoric acid and acid metal salts such as sodium monohydrogen orthophosphate, and potassium hydrogen sulfate.
• organic acids which form suitable salts include the mono-, di-, and tricarboxylic acids.
• Such acids are for example, acetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, salicyclic, 2-phenoxybenzoic, p-toluenesulfonic acid, and sulfonic acids such as methane sulfonic acid and 2-hydroxyethane sulfonic acid.
• Such salts can exist in either a hydrated or substantially anhydrous form.
• the acid addition salts of these compounds are soluble in water and various hydrophilic organic solvents, and which in comparison to their free base forms, generally demonstrate higher melting points.
• compositions represented by formula (1) are intended to apply to any non-toxic organic or inorganic basic addition salts of the compounds represented by formula (1) or any of its intermediates.
• Illustrative bases which form suitable salts include alkali metal or alkaline-earth metal hydroxides such as sodium, potassium, calcium, magnesium, or barium hydroxides; ammonia, and aliphatic, alicyclic, or aromatic organic amines such as methylamine, dimethylamine, trimethylamine, and picoline.
• Ri is selected from the group consisting of C-C 6 alkyl and -(CH 2 ) d -Ari are preferred;
• R is selected from the group consisting of hydrogen, -C(O)R ⁇ o and -SG are preferred;
• R 4 is selected from the group consisting of -C(O)R ⁇ o and R J O is C ⁇ _C 4 alkyl more preferred;
• Examples of compounds encompassed by the present invention include the following.
• the compounds of formula (1) can be prepared by a variety of procedures readily known to those skilled in the art. Such procedures include, peptide coupling, such as solid phase sequential procedures and solution phase sequential procedures using suitable amino acids and substituted acids and displacement, modification, and functionalization procedures, as required, utilizing suitable protecting groups and deprotection procedures.
• amino acid refers to naturally occurring amino acids as well as non-naturally occurring amino acids having substituents encompassed by R] and R 2 as described above.
• the naturally occurring amino acids included are glycine, alanine, valine, leucine, isoleucine. serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, histidine. aspartic acid, asparagine, glutamic acid, glutamine, arginine, ornithine, and lysine.
• Non-naturally occurring amino acids within the term "amino acid,” include without limitation, the D-isomers of the naturally occurring amino acids, norleucine, norvaline, alloisoleucine, t-butylglycine, methionine sulfoxide, and methionine sulfone.
• amino acid include without limitation phenylalanines, phenylglycines, homophenylalanines, 3-phenylpropylglycines, 4- phenylbutylglycines; each including those substituted by R 6 and R 6 as described above; and 1-naphthylalanines and 2-naphthylalanines; including those substituted by R 7 and R 7 - as described above.
• the compounds of formula (1) can be prepared by utilizing techniques and procedures well known and appreciated by one of ordinary skill in the art. To illustrate, general synthetic schemes for preparing intermediates and the compounds of formula (1) are set forth below. In the reaction schemes below, the reagents and starting materials are readily available to one of ordinary skill in the art and all substituents are as previously defined unless otherwise indicated.
• step 1 an appropriate protected compound of the formula (2a) is coupled with an appropriate compound of formula (3a) to give a compound of formula (2b).
• An appropriate protected compound of the formula (2a) is one in which R2 is as desired in the final compound of formula (1) or gives rise after deprotection to R 2 as desired in the final compound of formula (1), e is as desired in the final product of formula (1), and Pgi is an amine protecting group.
• an appropriate compound of formula (2a) may also be one in which the stereochemistry at the carboxy and -(CH 2 ) e -R2 bearing carbons is as desired in the final product of formula (1 ).
• the protecting group, Pgi is one in which the can be removed in the presence of the amide formed in this step.
• the use and removal of amine protecting groups is well known and appreciated in the art and described in Protective Groups in Organic Synthesis, Theodora W. Greene (Wiley-Interscience, 2nd Edition, 1991).
• the use of t-Boc and F-moc for Pgi is preferred.
• An appropriate compound of the formula (3a) is one in which Ri is as desired in the final compound of formula (1) or gives rise after deprotection to R
• a * may also be an attachment to a suitable resin. Such a protected carboxy or resin is chosen so that it does not interfere with subsequent deprotection, displacement, derivitivization, functionalization, or modification reactions, as are required. The use and removal of carboxy protecting groups is well known and appreciated in the art and described in Protective Groups in Organic Synthesis, Theodora W. Greene (Wiley- Interscience, 2nd Edition, 1991 ).
• an appropriate compound of formula (3a) may also be one in which the stereochemistry at the Ri bearing carbon is as desired in the final product of formula (1).
• Such coupling reactions are carried out by a variety of procedures readily known to those skilled in the art. Such procedures include, peptide coupling, such as solid phase sequential procedures and solution phase sequential procedures using suitable amino acids and substituted acids followed by displacement, modification, and functionalization procedures, as required, utilizing suitable protecting groups and deprotection procedures.
• amino acid refers to naturally occurring amino acids as well as non-naturally occurring amino acids having substituents encompassed by Ri and -(CH 2 ) e -R 2 as described above.
• the naturally occurring amino acids included are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, histidine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, ornithine, and lysine.
• Non-naturally occurring amino acids within the term "amino acid,” include without limitation, the D-isomers of the naturally occurring amino acids, norleucine, norvaline, alloisoleucine, t-butylglycine, methionine sulfoxide, and methionine sulfone.
• Other non-naturally occurring amino acids within the term “amino acid,” include without limitation phenylalanines substituted by R 6 as described above; phenylglycines, homophenylalanines, 3- phenylpropylglycines, 4-phenylbutylglycines; including those substituted by R 6 as described above; and 2-naphthylalanines, including those substituted by R 7 as described above.
• the preparation of amino acids bearing -(CH 2 ) e -R2 are knkown in the art and described herein.
• Solid phase sequential procedures can be performed using established methods, including automated methods such as by use of an automated peptide synthesizer.
• Automated methods such as by use of an automated peptide synthesizer.
• Solid Phase Peptide Synthesis Freeman 1969
• B. Merrifield Peptides: Synthesis, Structures, and Applications (B. Gutte, Ed., Acedemic Press 1995).
• a protected amino acid bearing Ri or protected Ri is bound to a resin support.
• the resin support employed can be any suitable resin conventionally employed in the art for the solid phase preparation of poly-peptides, preferably polystyrene which has been crossed away with about 0.5 to about 3 percent divinyl benzene, which has been either in chloromethylated or hydroxymethylated to provide sites for ester formation with the initially introduced protected amino acid.
• Suitable resins are well known and appreciated in the art, including those described in Rink, Tet. Let., 28, 3787 (1987) and Sieber, Tet. Let., 28, 2107 (1987). Included within the solid phase methods are combinatorial methods which are known in the art. K. S. Lam, Chem. Rev., 97, 41 1-448 (1997).
• the resin-bound protected amino acid bearing Ri is sequentially amino deprotected and coupled with a protected amino acids bearing -(CH 2 ) e -R 2 to give a resin-bound protected dipeptide.
• This resin bound protected dipeptide is sequentially amino deprotected and coupled with a protected amino acid bearing R 3 or protected R 3 to give a protected tripeptide.
• an appropriate protected dipeptide may be coupled by the solution method prior to coupling with the resin-bound amino acid.
• Each protected amino acids or amino acid sequence is introduced into the solid phase reactor and about a two-fold to four-fold excess.
• the coupling is carried out in a suitable medium, for example dimethylformamide, dichloromethane, or mixtures of dimethyl formamide and dichloromethane.
• a suitable medium for example dimethylformamide, dichloromethane, or mixtures of dimethyl formamide and dichloromethane.
• the compounds of formula (1) can also be prepared by solution phase sequential procedures well known and appreciated in the art. Accordingly, suitably protected amino acids, substituted acids or dipeptides are coupled by procedures requiring activation of the carbonyl group and coupling reaction with amine function of an appropriate protected amino acid or dipeptide. These procedures are well known appreciated in the art.
• Particularly suitable coupling reagents include N-((dimethylamino)-lH-l ,2,3-triazolo[4,5- b]pyridin-l-ylmethylene)-N-methylmethanaminium hexafluororphosphate N-oxide (HATU), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxy-benzotriazole or N,N'-diisopropylcarbodiimide and 1-hydroxy-benzotriazole.
• HATU hexafluororphosphate N-oxide
• l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxy-benzotriazole or N,N'-diisopropylcarbodiimide and 1-hydroxy-benzotriazole.
• coupling agents are pyridine benzotriazol-l-yloxytris(dimethylamino)phosphonium hexafluorophosphate complex , carbodiimides (e.g., N,N'-dicyclohexylcarbodiimide); cyanamides (e.g., N,N- dibenzylcyanamide); (3b) ketenimines; isoxazolium salts (e.g., N-ethyl-5-phenyl-isoxazolium- 3'-sulfonate; monocyclic nitrogen containing heterocyclic amides of aromatic character containing one through four nitrogens in the ring such as imidazolides, pyrazolides, and 1 ,2,4- triazolides.
• carbodiimides e.g., N,N'-dicyclohexylcarbodiimide
• cyanamides e.g., N,N- dibenzylcyanamide
• heterocyclic amides that are useful include N,N' -carbonyl diimidazole and N,N-carbonyl- di-l,2,4-triazole; alkoxylated acetylene (e.g., ethoxyacetylene); reagents which form a mixed anhydride with the carboxyl moiety of the amino acid (e.g., ethylchloroformate and isobutylchloro formate).
• alkoxylated acetylene e.g., ethoxyacetylene
• reagents which form a mixed anhydride with the carboxyl moiety of the amino acid e.g., ethylchloroformate and isobutylchloro formate.
• Other activating reagents and their use in peptide coupling are described by Kapoor, J. Pharm. Sci., 59, 1-27 (1970).
• Such coupling reactions to form amides are carried out in suitable solvents, such as dichloromethane, tetrahydrofuran, diethyl ether, chloroform, and the like, and using suitable bases, such as triethylamine, N-methylmorpholine, N,N-disopropylethylamine, pyridine, and the like, and coupling reagents, as required, and are well known and appreciated in the art.
• the reactions are generally carried out at -10°C to the refluxing temperature of the solvent and generally require form 1 hour to 2 days.
• the product can be isolated and purified by techniques well known in the art, such as extraction, evaporation, trituration, lyophilization, chromatography, and recrystallization.
• step 2 the amine protecting group, Pgi, of the compound of formula (2b) is selectively removed to give the compound of formula (2c).
• the product can be isolated and purified by techniques well known in the art, such as extraction, evaporation, salt formation, trituration, lyophilization. chromatogranhv. and recrvstallization.
• step 3 a compound of formula (2c) coupled with an appropriate acid derivative bearing R 3 ' and Y (compound of formula (3b)) to give a compound of formula (4).
• Such coupling reactions are well known and appreciated in the art and discussed above.
• the product can be isolated and purified by techniques well known in the art such as extraction, evaporation, salt formation, trituration, lyophilization, chromatography, and recrystallization.
• An appropriate compound of formula (3b) is one in which R 3 - is R 3 as desired in the final product of formula (1) or gives rise after deprotection to R 3 as desired in the final product of formula (1 ) and Y is a protected thio substituent or Y may be a protected hydroxy substituent or bromo which gives rise upon selective deprotection and displacement or displacement and further deprotection and/or elaboration, if required, to -SR 4 as desired in the final product of formula (1).
• an appropriate compound of formula (3b) may also be one in which R 3 - gives rise to R 3 - which, upon derivatization, gives rise R 3 as desired in the final product of formula (1) and Y is a protected thio substituent.
• an appropriate compound of formula (3b) may also be one in which the stereochemistry at the R 3 - and Y bearing carbon is as desired in the final product of formula (1 ) or gives rise after displacement to the stereochemistry as desired at that carbon in the final product of formula (1).
• the activating group (A) is one which undergoes an amidation reaction.
• an amidation reaction may proceed through an acid, X is -OH; or an acid may be first converted to an acid chloride, X is -Cl; or an activated intermediate; such as an anhydride; a mixed anhydride of aliphatic carboxylic acid, such as formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, pivalic acid, 2-ethylbutyric acid, trichloroacetic acid, trifluoroacetic acid, and the like; of aromatic carboxylic acids, such as benzoic acid and the like; of an activated ester, such as phenol ester, p-nitrophenol ester, 2,4-dinitrophenol ester, pentafluorophenol ester, pentachlorophenol ester, N-hydroxysuccinimide ester, N- hydroxyphthalimide ester, 1 -hydroxy- lH-benztriazole ester, and the like; activated amide, such as imi
• Acid chlorides and activated intermediates may be prepared but are not necessarily isolated before the addition of a compound of formula (3b).
• the use and selection of appropriate protecting groups is within the ability of those skilled in the art and will depend upon compound of formula (3b) to be protected, the presence of other protected amino acid residues, other protecting groups, and the nature of the particular R 3 and/or R 4 group(s) ultimately being introduced.
• Compounds of formula (3b) in which Y is bromo and protected thio are commercially available or can be prepared utilizing materials, techniques, and procedures well known and appreciated by one of ordinary skill in the art or described herein. See PCT Application WO 96/11209, published 18 April 1996.
• Examples commercially available compounds of formula (3b) in which Y is bromo include 2- bromopropionic acid, 2-bromobutyric acid, 2-bromovaleric acid, 2-bromohexanoic acid, 6- (benzoylamino)-2-bromohexanoic acid, 2-bromoheptanoic acid, 2-bromooctanoic acid, 2- bromo-3-methylbutyric acid, 2-bromoisocaproic acid, 2-bromo-3-(5-imidazoyl)proionic acid, (R)-(+)-2-bromopropionic acid, (S)-(-)-2-bromopropionic acid.
• Reaction Scheme B a final product of formula (1) is prepared from a compound of formula (4) (prepared as described in Reaction Scheme A) in which R 3 - is R 3 as desired in the final product of formula (1) or gives rise after deprotection to R 3 as desired in the final product of formula (1) and Y is a protected thio substituent or hydroxy or bromo.
• step 1 a compound of formula (4) in which Y is protected thio gives rise upon selective deprotection to give a compound of formula (5).
• thiol of formula (5) compounds of formula (4) in which Y is a protected thio substituents are selectively deprotected to give a thiol of formula (5).
• Protected thio substituents include thioesters, such as thioacetyl or thiobenzoyl, thioethers, such as thiobenzyl, thio-4- methoxybenzyl, thiotriphenylmethyl, or thio-t-butyl, or unsymmetrical sulfides, such as dithioethyl or dithio-t-butyl.
• thioesters such as thioacetyl or thiobenzoyl
• thioethers such as thiobenzyl, thio-4- methoxybenzyl, thiotriphenylmethyl, or thio-t-butyl
• unsymmetrical sulfides such as dithioethyl or dithio
• step 2 a compound of formula (5) undergoes modification reaction to give a compound of formula (6).
• modification reactions include, thiol esterification and disulfide formation.
• a compound of formula (5) is contacted with about an equimolar amount of an appropriate acid, such as HO-C(O)R ⁇ o or HO-C(O)-(CH 2 ) q -K in the presence of a suitable coupling agent to give a compound of formula (6) in which R 4 is -C(O)Rjo or -C(O)-(CH ) q -K.
• an appropriate acid such as HO-C(O)R ⁇ o or HO-C(O)-(CH 2 ) q -K
• the reaction is carried out in the presence of a coupling agent such as 2-fluoro-l-methylpyridinium p-toluenesulfate, l-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, carbonyldiimidazole, 1- ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline. or diethylcyanophosphonate in a suitable aprotic solvent such as methylene chloride.
• a coupling agent such as 2-fluoro-l-methylpyridinium p-toluenesulfate, l-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, carbonyldiimidazole, 1- ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline. or diethylcyanophosphonate in a suitable aprotic solvent such as
• the product can be isolated and purified by techniques well known in the art, such as extraction, evaporation, trituration, lyophilization, chromatography, and recrystallization.
• Compounds of formula (6) in which R4 is -S-G group can be synthesized according to techniques well known and appreciated by one of ordinary skill in the art, as disclosed in PCT Application No. WO 95/21839, published 17 August 1995 and U.S. Patent Nos. 5,491,143, issued February 13, 1996, and 5,731,306, issued March 24, 1998, and Roques, B.P. et al., J, Med. Chem. 33, 2473-2481 (1992).
• a compound of formula (5) is contacted with an appropriate compound of formula (7).
• An appropriate compound of formula (7) is one which gives G as desired in the final product of formula (1) or gives rise upon deprotection to G as is desired in the final product of formula (1).
• the compound of formula (7) may have stereochemistry as desired in the final product of formula (1).
• the reaction is carried out in a suitable solvent, such as ethanol, methanol, dichloromethane, or mixtures of ethanol or methanol and dichloromethane.
• the solvent is degassed by passing a stream of nitrogen gas through it for 15 minutes before the reaction is carried out.
• the reaction is carried out using from 1.0 to 4.0 molar equivalents of an appropriate compound of formula (7).
• the reaction is carried out at temperatures of from 0°C to the refluxing temperature of the solvent, with a temperature of 10°C to 30°C being preferred.
• the reaction generally requires from 1 to 48 hours.
• the product can be isolated by techniques well known in the art, such as extraction, evaporation, and precipitation and can be purified by chromatography and recrystallization.
• step 3 a compound of formula (4) in which Y is hydroxy or bromo can be displaced by an appropriate thiol, HSR 4 , to give a compound of formula (1) or a protected compound of formula (1).
• an appropriate thiol HSR 4 is one which gives R as desired in the final product of formula (1 ) or gives rise to R4 as desired in the final product of formula (1 ).
• step 3 a compound of formula (4) in which Y is hydroxy (obtained from protected hydroxy compounds of formula (4)) undergoes a displacement reaction with an appropriate thio introducing reagent by the method of Mitsunobu to give a compound of formula (4) in which Y is a protected thio substituent or -SR 4 as desired in the final compound of formula ( 1 )
• a compound of formula (4) in which Y is hydroxy reacts with thioacetic acid or thiobenzoic acid, t ⁇ phenylphosphine, and diethylazodicarboxylate in a suitable aprotic solvent, such as tetrahydrofuran to give a compound of formula (4) in which Y is thioacetyl or thiobenzoyl
• a suitable aprotic solvent such as tetrahydrofuran
• step 3 a compound of formula (4) in which Y is bromo undergo a displacement reaction with an appropriate thio introducing reagent to give a compound of formula (4) in which Y is protected thio substituent which gives ⁇ se upon deprotection and subsequent elaboration, if desired, the -SR4 as desired in the final compound of formula (1)
• An approp ⁇ ate thio introducing reagent is also one which introduces a group -SR as desired in the final compound of formula (1)
• a solution of p-methoxybenzylmercaptan a suitable organic solvent such as dimethyl formarmde is degassed and treated with a suitable base such as sodium hyd ⁇ de, sodium hydroxide, or cesium carbonate
• a suitable base such as sodium hyd ⁇ de, sodium hydroxide, or cesium carbonate
• a suitable catalyst such as tetra-n-butylammomum iodide
• the reaction mixture is carried out for 1 to 25 hours at temperatures ranging form 0°C to about 100°C
• Selective removal of the 4-methoxybenzyl moiety gives the desired compound of formula (1 )
• the product can be isolated and pu ⁇ fied by techniques well known in the art, such as extraction, evaporation, t ⁇ turation, lyophilization, chromatography, and recrystalhzation
• step 3 a compound of formula (4) m which Y is bromo can be displaced by an approp ⁇ ate thio ester, Ph 3 S-C(O)-(CH2) q -X by techniques well known and appreciated in the art. as disclosed in U S Pat No 5,424,425, issued Jun 13, 1995
• a protected compound of formula (1) is deprotected to give a compound of formula (1 ).
• Such deprotection reactions are well known appreciated in the art and may include selective deprotections.
• Reaction Scheme C a final product of formula (1 ) is prepared from a compound of formula (4) (prepared as described in Reaction Scheme A) in which R 3 - gives rise to R 3 - and Y is -SR as is desired in the final product of formula (1) or a protected thio substituent gives a compound of formula (1).
• step 1 an appropriate compound of formula (4) is deprotected, hydrolyzed, or reduced to give a compound of formula (4a).
• an approp ⁇ ate compound of formula (4) is one in which R 3 gives rise to a compound of formula (4a) in which R 3 undergoes further de ⁇ vitization (step 2) to give a compound of formula (4) in which R-, is -(CH 2 ) n. -NR 8 -SO 2 -Y ⁇ or -(CH 2 ) m -Z-Q as desired m the final product of formula ( 1 )
• step 1 an appropriate compound of formula (4) is deprotected, hydrolyzed, or reduced to give a compound of formula (4a).
• an approp ⁇ ate compound of formula (4) is one in which R 3 gives rise to a compound of formula (4a) in which R 3 undergoes further de ⁇ vitization (step 2) to give a compound of formula (4) in which R-, is -(CH 2 ) n. -NR 8 -SO 2
• an approp ⁇ ate compound of formula (4) is one in which Y is -SR as desired in the final compound of formula (1) or Y is protected thio which gives ⁇ se upon deprotection or deprotection and further functionalization to give -SR , as desired, in the final product of formula (1) as desc ⁇ bed in Reaction Scheme B, step 2, above
• a compound of formula (4) in which R is -(CH ) m -NR 8 -t-Boc is contacted with a molar excess of a suitable acid to give a compound of formula (4a) in which R 3 is -(CH ) m -NHR
• a suitable acid such as methanol, ethanol, ethyl acetate, diethyl ether, or dioxane
• Suitable acids for this reaction are well known in the art, including hydrochloric acid, hydrobromic acid, tnfluoroacetic acid, and methanesulfomc acid
• the reaction is generally carried out at room temperature for a pe ⁇ od of time ranging from 1-10 hours
• the product can be isolated by techniques well known in the art, such as extraction, evaporation, and precipitation and can be pu ⁇ fied by chromatography and recrystalhzation
• a compound of formula (4) in v ⁇ hich R 3 is -(CH 2 )m-C(O)OPg- and Pgi is methyl or ethyl is contacted with about 1 to 2 molar equivalents of lithium hydroxide, sodium hydroxide, or potassium hydroxide to give a compound of formula (4a) in which R 3 is -(CH 2 )m-CO 2 H
• a suitable solvent such as methanol.
• ethanol methanol/water mixtures, ethanol/water mixtures, or tetrahydrofuran water mixtures and generally requires 1 to 24 hours
• the reaction is carried out at temperatures of from about 0°C to the refluxing temperature of the solvent.
• the resulting acid is isolated and purified by techniques well known in the art, such as acidification, extraction, evaporation, and precipitation and can be purified by trituration, precipitation, chromatography, and recrystalhzation.
• a compound of formula (4a) in which R 3 is -(CH2)m-i-CO 2 Pg 3 in which Pg 3 is methyl or ethyl is contacted with a suitable reducing agent, such as lithium borohydride, diisobutylaluminum hydride, 9-borabicyclo[3.3.1]nonane, preferably lithium borohydride to provide a compound of formula (4a) in which Ry is - (CH 2 ) ⁇ ⁇ -CH 2 OH.
• a suitable solvent such as dichloromethane, tetrahydrofuran, or toluene, with tetrahydrofuran being preferred.
• the reaction is carried out at a temperature of from about -30°C to about 50°C and generally requires from 2 to 12 hours.
• the product can be isolated by quenching, extraction, evaporation, and precipitation and can be purified by trituration, chromatography, and recrystalhzation.
• step 2 a compound of formula (4a) undergoes a derivitization reaction to give a compound of formula (5) in which R 3 is as desired in the final product of formula (1).
• derivitization reactions include hydrolysis of esters and ester formations as are well known in the art, ether formation, amine alkylation, formation of amides, urea formation, carbamate formation, and formation of sulfonamide.
• step 2 the compound of formula (4a) is one in which Y is a protected thio group, such as thioacetyl, thiobenzoyl, 4-methoxybenzyl thiol or t-butylthiol.
• a suitable alkylating agent is one which transfers Q or protected Q as desired in the final product of formula (1), such as benzyl bromide, benzyl chloride, substituted benzyl bromide, substituted benzyl chloride, ethyl bromoacetate, t-butyl bromoaceate, ethyl 3-chloropropionate, ethyl 3- bromopropionate, ethyl 5-bromovalerate, ethyl 4-bromobutyrate, 3-chloropropionamide, 2- bromoethylbenzene, substituted 2-bromoethylbenzene, l-chloro-3-phenylpropane, l-bromo-4- phenylbutane, and the like, or nitrogen mustards, including 2-dimefhylaminoethyl chloride, 2- diethylaminoethyl chloride, and 3-dimethylaminopropyl chloride.
• the reaction is carried out in a suitable solvent, such as diethyl ether, tetrahydrofuran. dimethylformamide, dimethyl sulfoxide, or acetonitrile and using a suitable base, such as sodium hydride, potassium hydride, potassium t-butoxide, and lithium diisopropylamide.
• a suitable solvent such as diethyl ether, tetrahydrofuran. dimethylformamide, dimethyl sulfoxide, or acetonitrile
• a suitable base such as sodium hydride, potassium hydride, potassium t-butoxide, and lithium diisopropylamide.
• the reaction is generally carried out at temperatures of -70°C and room temperature and require from about 1-24 hours.
• the product can be isolated by techniques well known in the art, such as extraction, evaporation, and precipitation and can be purified by chromatography and recrystalhzation.
• an ether formation can also be carried out by a procedure similar to the one above using a compound of formula (4a) in which R 3 - is -(CH 2 ) m - ⁇ -CH 2 OH in which the hydroxy group is first converted to a leaving group, such as chloro, bromo, or mesylate and a suitable alcohol which transfers Q or protected Q as desired in the final product of formula (1 ), such as benzyl alcohol, substituted benzyl alcohol, phenol, substituted phenol, and the like.
• a leaving group such as chloro, bromo, and mesylate
• a compound of formula (4a) in which R 3 • is -(CH 2 ) m -NHR 8 is contacted with 1 to 10 molar equivalents of a suitable alkylating agent to give a compound of formula (5) in which R 3 is -(CH 2 ) m -Z-Q in which Z is -NR 8 -.
• the reaction may be carried out after protection of the amine function of R 3 - in which R 8 is hydrogen by a suitable protecting group, such as benzyl or t-Boc.
• a suitable alkylating agent is one as described above for the ether formation and also includes alkylhalides, such as methyl iodide, methyl bromide, ethyl bromide, propyl bromide, propyl chloride, butyl bromide, butyl chloride, and the like.
• the reaction is carried out in a suitable solvent, such as methanol, ethanol, dimethylformamide, or pyridine and using a suitable base, such as sodium carbonate, triethylamine, N,N-diisopropylethylamine or pyridine.
• the reaction is generally carried out at temperatures of room temperature to the refluxing temperature of the solvent and require from about 1-24 hours.
• the product can be isolated by techniques well known in the art, such as extraction, evaporation, and precipitation and can be purified by chromatography and recrystalhzation.
• a compound of formula (4a) in which R 3 - is -(CH 2 ) m -NHR 8 is contacted in a reductive alkylation with a suitable aldehyde to give a compound of formula (5) in which R 3 is -(CH 2 ) m -Z-Q in which Z is -NRg-.
• a suitable aldehyde is one which transfers Q or protected Q as desired in the final product of formula (1), such as benzaldehyde and substituted benzaldehydes.
• the reaction is carried out in a suitable solvent, such as methanol, ethanol, tetrahydrofuran, or mixtures of methanol or ethanol and tetrahydrofuran.
• a suitable solvent such as methanol, ethanol, tetrahydrofuran, or mixtures of methanol or ethanol and tetrahydrofuran.
• the reaction may be carried out in the presence of a drying agent, such as sodium sulfate or molecular sieves.
• the reaction is carried out in the presence of from 1.0 to 6.0 molar equivalents of a suitable reducing agent, such as, sodium borohydride or sodium cyanoborohydride with sodium cyanoborohydride being preferred. It may be advantageous to maintain the pH in the range of about 4 to 6.
• the reaction is generally carried out at temperatures of from 0°C to the refluxing temperature of the solvent. Generally, the reactions require 1 to 72 hours.
• the product can be isolated by techniques well known in the art, such as extraction,
• a suitable amine, HNR 8 Q gives rise to R 8 and Q as desired in the final product of formula (1), such as methylamine, ethylamine, propylamine, butylamine, N- methyl benzylamine, benzyl ⁇ -alanine, 4-(3-aminopropyl)morpholine, and the like.
• a compound of formula (4a) in which R 3 - is is -(CH 2 ) m -NHR 8 is contacted with a suitable carboxylic acid in an amide formation to give a compound of formula (5) in which R 3 is -(CH 2 ) m -Z-Q in which Z is amide.
• Suitable carboxylic acids, QC(O)-OH are ones give rise to Q as desired in the final product of formula (1), such as benzoic acid, substituted benzoic acids, phenyl acetic acids, substituted phenylacetic acids, mono-t-butyl malonate, and the like.
• An appropriate isocyanate is one which gives rise to Q as desired in the final product, such as phenyl isocyanate, substituted phenyl isocyanate. napthyl isocyanate, ethyl isocyanatoacetate, and the like.
• the reaction is carried out by adding an equivalent of, or a slight molar excess of, an appropriate isocyanate is added to a solution of a compound of formula (4a) in which R 3 - is -(CH ) m - NHR 8 in a suitable solvent, such as diethyl ether, benzene, or toluene.
• a suitable solvent such as diethyl ether, benzene, or toluene.
• the reaction is carried out at temperature of from about 0°C to the refluxing temperature of the solvent and require about 1-24 hours.
• the product can be isolated and purified by techniques well known in the art, such as filtration, extraction, evaporation, trituration, chromatography, and recrystalhzation.
• chloro formates examples include benzyl chloroformate, naphthyl chloroformate, phenyl chloroformate, and substituted phenyl chloro formates, such as 4-chlorophenyl chloroformate, 4-methylphenyl chloroformate, 4-bromophenyl chloroformate, 4-fluorophenyl chloroformate, 4-methoxyphenyl chloroformate and the like.
• the reaction is carried out by adding an equivalent of, or a slight molar excess of, an appropriate chloro formate to a solution of a compound of formula (4a) in which R 3 is -(CH 2 ) m -NHR 8 in a suitable solvent, such as toluene, tetrahydrofuran, dimethylformamide, dichloromethane, pyridine, or chloroform.
• a suitable solvent such as toluene, tetrahydrofuran, dimethylformamide, dichloromethane, pyridine, or chloroform.
• a suitable base such as triethylamine, sodium carbonate, potassium bicarbonate, pyridine or N,N-diisopropylethylamine.
• the reaction is carried out at a temperature of from -70°C to the refluxing temperature of the solvent and generally requires from 30 minutes to 24 hours.
• the product can be isolated and purified by techniques well known in the art, such as extraction, evaporation, chromat
• a compound of formula (4a) in which R 3 '- is -(CH 2 )m- ! -CH 2 OH is contacted with an appropriate isocyanate, as defined above for urea formation, to give a compound of formula (5) in which R 3 is -(CH 2 ) m -Z-Q in which Z is O- carbamoyl.
• the reaction is carried out in a suitable solvent, such as diethyl ether, tetrahydrofuran, dimethylformamide, or acetonitrile.
• the reaction may be facilitated by the use of catalytic amount of a suitable base, such as sodium hydride, potassium hydride, or potassium t-butoxide.
• the reaction is generally carried out at temperatures of from -20°C to room temperature and require from about 1-24 hours.
• the product can be isolated by techniques well known in the art. such as extraction, evaporation, and precipitation and can be purified by chromatography and recrystalhzation.
• a compound of formula (4a) in which R 3 - is -(CH2) m -NHR 8 is contacted with an appropriate sulfonamide forming reagent.
• An appropriate sulfonamide forming reagent such as a sulfonyl chloride, Y ⁇ S(O) 2 Cl, or sulfonyl anhydride, Y ⁇ (O) 2 S-O-S(O) 2 Yi, is one which gives rise to Yi as desired in the final product.
• sulfonamide forming reagents examples include, benzenesulfonyl chloride, 1-napthalenesulfonyl chloride, 2- napthalenesulfonyl chloride, dansyl chloride, N-morpholinylsulfonyl chloride, N- piperidinylsulfonyl chloride, 2,4,5-trichlorobenzenesulfonyl chloride, 2,5- dichlorobenzenesulfonyl chloride, 2,4,6-triisopropylbenzenesulfonyl chloride, 2- mesitylenesulfonyl chloride, 4-bromobenzenesulfonyl chloride, 4-fluorobenzenesulfonyl chloride, 4-chlorobenzenesulfonyl chloride, 4-methoxybenzenesulfonyl chloride, 4-t- butylbenzenesulfonyl chloride, p-to
• the reaction is carried out in a suitable solvent, such as tetrahydrofuran, dichloromethane, pyridine, or chloroform and in the presence of an excess of a suitable base, such as triethylamine, sodium carbonate, pyridine, or N,N-diisopropylethylamine.
• a suitable solvent such as tetrahydrofuran, dichloromethane, pyridine, or chloroform
• a suitable base such as triethylamine, sodium carbonate, pyridine, or N,N-diisopropylethylamine.
• the reaction is carried out at a temperature of from -50°C to the refluxing temperature of the solvent.
• the reaction generally requires from 30 minutes to 24 hours.
• the product can be isolated and purified by techniques well known in the art, such as extraction, evaporation, chromatography, and recrystalhzation.
• step 3 a compound of formula (5) in which R 3 is as desired in the final product of formula (1) undergoes a selective thiol deprotection to give a compound of formula (5).
• Such selective thiol deprotections using suitable protecting groups are well known and appreciated in the art as discussed in Reaction Scheme B. step 1, above.
• step 4 a compound of formula (5) undergoes a modification reaction to give a compound of formula (1) or protected compound of formula (1) as described in Reaction Scheme B, step 2, above.
• step 5 a compound of formula (4) in which Y is protected thio is deprotected to give a compound of formula (1) or to a protected compound of formula (1 ).
• a protected compound of formula (1) is deprotected to give a compound of formula (1).
• Such deprotection reactions are well known appreciated in the art and may include selective deprotections.
• An appropriate ⁇ -amino carboxylic acid of formula (8), and protected forms thereof, is one which is one in which R 3 - is R 3 as desired in the final product of formula (1) or gives rise after deprotection to R 3 as desired in the final product of formula (1)
• ⁇ -amino carboxylic acid of formula (8) may also be one in which the stereochemistry at the R 3 bearing carbon gives rise after displacement to the stereochemistry as desired at that carbon in the final product of formula (1).
• Such appropriate ⁇ -amino carboxylic acid of formula (8) are commercially available or may be readily prepared by techniques and procedures well known and appreciated by one of ordinary skill in the art.
• an ⁇ -amino carboxylic acid of formula (8) and a suitable bromide, such as hydrogen bromide or potassium bromide in acidic solution, such as sulfuric acid is treated with sodium nitrite.
• the reaction temperature is carried out at temperatures of from about - 25°C to about ambient temperature and require about 1 to 5 hours.
• the product can be isolated and purified by techniques well known in the art, such as acidification, extraction, evaporation, chromatography, and recrystalhzation to give the compound of formula (3b) in which Y is bromo and X is -OH.
• the product can be isolated and purified by techniques well known and appreciated in the art, such as acidification, basification, filtration, extraction, evaporation, trituration, chromatography, and recrystalhzation.
• an appropriate carboxylic acid of formula (9) is brominated to give compound of formula (3b) in which Y is bromo and X is -OH.
• An appropriate carboxylic acid of formula (9), and protected forms thereof, is one which is one in which R 3 is R 3 as desired in the final product of formula (1) or gives rise after deprotection to R 3 as desired in the final product of formula (1).
• a mixture of a carboxylic acid of formula (9) and dry red phosphorous are treated dropwise with bromine at temperature ranging from about -20° to about 10°C.
• the reaction mixture is then warmed to room temperature and then heated to about 80°C for about 2-5 hours.
• the reaction mixture is then cooled to room temperature, poured into water containing sodium bisulfite, and neutralized using solid sodium carbonate.
• the aqueous layer is extracted and acidified with a suitable acid, such as concentrated hydrochloric acid.
• the precipitate is collected by filtration and dried to give the compound of formula (3b) or formula (3b2)in which Y is bromo and X is -OH.
• the product can be isolated and purified by techniques well known and appreciated in the art, such as acidification, basification, filtration, extraction, evaporation, trituration, chromatography, and recrystalhzation.
• an appropriate ⁇ -amino carboxylic acid of formula (11) is converted to an compound of formula (9) in which R 3 - is W-(CH 2 ) m --
• An appropriate ⁇ - amino carboxylic acid of formula (11) is one in which m is as desired in the final product of formula (1) and are readily available in the art.
• the reaction is carried out in a suitable polar solvent, such as water, ethanol, diethyl ether, tetrahydrofuran, or a water/ethanol solvent mixture using a suitable base, such as sodium carbonate and N- carbethoxyphthalimide.
• the reaction mixture is typically stirred at about ambient temperature for 1-5 hours.
• the product can be isolated and purified by techniques well known in the art, such as acidification, extraction, evaporation, chromatography, and recrystalhzation to give the desired compound of formula (9) in which R 3 > is W-(CH 2 ) m -.
• step 1 an appropriate ⁇ , ⁇ -di amino acid of formula (12) undergoes a selective N- ⁇ -protection to give an N- ⁇ -protected- ⁇ -diamino acid of formula (13).
• An appropriate ⁇ , ⁇ -diamino acid of formula (12) is one in which m is as desired in the final product of formula (1).
• a selective N- ⁇ -protection of a suitable ⁇ ,cD-diamino acid is accomplished by masking the ⁇ -amino group by formation of a benzylidene imine.
• the benzylidene imine is formed by dissolving L-lysine monohydrochloride in lithium hydroxide and cooling the solution to a temperature ranging from about 0° to 10°C Freshly distilled benzaldehyde is then added and the solution is shaken. N- ⁇ -benzylidene-L-lysine is recovered by filtration and evaporation.
• N-c -benzylidene-L-lysine is added to a mixture of sodium hydroxide and ethanol, cooled to a temperature of from about -5°C to about -25°C. Then, precooled solutions of benzyloxycarbonyl chloride in a solvent, such as ethanol, is added to the reaction mixture.
• the temperature is maintained in a range of from about -10°C to about -25°C during the course of addition, and may allowed to rise afterwards.
• the reaction mixture is then acidified using a suitable acid, such as precooled hydrochloric acid, and N- ⁇ -benzyloxycarbonyl-L-lysine, which corresponds to formula (13) where m is 4, is recovered by filtration evaporate and recrystalhzation.
• step 2 N- ⁇ -benzyloxycarbonyl-L-lysine or other compounds of formula (13) is converted to ⁇ -phthalimido- ⁇ -benzyloxycarbonyl-L-lysine or other ⁇ - phthalimido- ⁇ -aminoprotected carboxylic acid of formula (14) by the method described in Reaction Scheme G.l, above.
• step 3 the ⁇ -phthalimido- ⁇ -aminoprotected carboxylic acid of formula (14) is deprotected to give compound of formula (8) in which R 3 is W-(CH 2 ) m -.
• ⁇ -phthalimido- ⁇ -benzyloxycarbonyl-L-lysine is contacted with hydrogen in the presence of a hydrogenation catalyst, such as 10% palladium/carbon.
• a hydrogenation catalyst such as 10% palladium/carbon.
• the reactants are typically contacted in a suitable solvent mixture such as ethanol, methanol, water, ethanol/water mixtures, or methanol/water mixtures.
• the reactants are typically shaken under a hydrogen atmosphere of 35-45 psi at room temperature for a period of time ranging from 5-24 hours.
• the product is typically recovered by filtration and evaporation of the solvent.
• Reaction Scheme H A route for preparing the compounds of formula (3b) and formula (3b2) in which Yi is protected thio is presented in Reaction Scheme H.
• the reagents and starting materials are readily available to one of ordinary skill in the art.
• Reaction Scheme H all substituents, unless otherwise indicated, are as previously defined.
• step 1 a bromoacetate of formula (15) is contacted with an appropriate thiol to give a protected acetic acid ester of formula (17).
• Pg 5 is a protecting group, such as methyl, ethyl, t-butyl, and benzyl.
• An appropriate thiol is one which gives rise to a protected thio group, Y, in the product of formula (3b).
• step 1 the use of 4-methoxybenzylmercaptan is preferred.
• a bromoacetate of formula (15) is contacted with an appropriate thiol in a suitable organic solvent, such as dimethylformamide.
• a suitable organic solvent such as dimethylformamide.
• the solvent is degassed.
• the reaction is carried out using a suitable base, such as sodium hydroxide, triethylamine, or N,N-diisopropylethylamine.
• the reaction is carried out at temperatures of from about -50°C to about ambient temperature and requires about 1 to 72 hours.
• the protected acetic acid ester of formula (17) can be isolated and purified by methods well known and appreciated in the art, such as extraction, evaporation, chromatography, and distillation, and recrystalhzation.
• step 2 the protected acetic acid ester of formula ( 17) is alkylated with an appropriate akylating agent to give a compound of formula (18).
• an appropriate alkylating agent is one which transfers Ry which is R 3 as desired in the final product of formula (1 ) or gives rise after deprotection to R 3 as desired in the final product of formula (1) or gives rise to R 3 as defined in Reaction Scheme C, step 1.
• alkylating agents include alkylhalides, such as methyl iodide, methyl bromide, ethyl bromide, propyl bromide, propyl chloride, butyl bromide, butyl chloride, and the like; benzyl bromide, benzyl chloride, substituted benzyl bromide, substituted benzyl chloride, ethyl bromoacetate, t-butyl bromoaceate, ethyl 3-chloropropionate, ethyl 3- bromopropionate, ethyl 5-bromovalerate, ethyl 4-bromobutyrate, 3-chloropropionamide, 2- bromoethylbenzene, substituted 2-bromoethylbenzene, l -chloro-3-phenylpropane, l-bromo-4- phenylbutane, and the like, N-(2-bromodide
• a protected acetic acid ester of formula (17) is alkylated with an appropriate alkylating agent.
• the reaction is carried out in a suitable solvent, such as diethyl ether, tetrahydrofuran, dimethylformamide, and toluene using a suitable base, such as sodium hydride, potassium hydride, potassium t-butoxide, lithium bis(trimethylsilyl)amide, sodium bis(trimethylsilyl)amide, potassium bis(trimethylsilyl)amide, or lithium diisopropylamide.
• the reaction is generally carried out at temperatures of about -70°C to about room temperature and require from about 1-24 hours.
• the product can be isolated by techniques well known in the art, such as extraction, evaporation, and precipitation and can be purified by chromatography and recrystalhzation.
• step 3 the compound of formula (18) the carboxy protecting group Pg 5 is selectively removed to give a compound of formula (3b) in which Y is protected thio.
• Y is protected thio.
• Reaction Scheme I describes the preparation of a specific diastereomer of the compounds of formula (2a).
• step 1 an appropriate aldehyde of formula (20) is converted to a compound of formula (21 ) in which Pg is a protecting group.
• Pg is a protecting group.
• An appropriate aldehyde of formula (20) is one in which e and R 2 are as desired in the final product of formula (1).
• Appropriate aldehydes of formula (20) include, benzaldehyde, substituted benzaldehydes, 1-naphthaldehyde, substitued 1- naphthaldehydes, 2-naphthaldehyde, substitued 2-naphthaldehydes, phenylacetaldehyde, substituted phenylacetaldehydes, hydrocinnamaldehyde, and substituted hydrocinnamaldehyde.
• Suitable Aldol-type condensations include the Claisen-Schmidt and Knoevenaglel reactions. Modern Synthetic Reactions, H.O. House (2 nd Ed. The Benjamin/Cummings Publishing Co. 1972). As is appreciated by one of skill in the art the Claisen-Schmidt reaction using malonic acid, or esters thereof, give compounds of formula (22) upon decarboxylation or hydrolysis and decarboxylation.
• Sutiable Wittig-type reacations include the Wittig and Wadswoth-Edmonds reactions.
• an appropriate aldehyde of formula (20) is reacted with an appropriate reagent, such as (carbethoxymethylene)triphenylphosphorane or dimethyl trimethylsilyloxycarbonylmethyl phosphonate.
• the reaction is carried out in solvent, such as ethanol, benzene, toluene, or tetrahydrofuran. Typically the reaction is carried out at temperature of from about -20° to reflux and require about 4 to 48 hours.
• the product can be isolated and purified by techniques well known and appreciated in the art, such as quenching, acidification, filtration, extraction, evaporation, trituration, chromatography, and recrystalhzation.
• an appropriate aldehyde of formula (20) is reacted with an appropriate reagent, such as dimethyl trimethylsilyloxycarbonylmethyl phosphonate.
• the reaction is carried out in solvent, such as benzene, toluene, diethyl ether, or tetrahydrofuran.
• solvent such as benzene, toluene, diethyl ether, or tetrahydrofuran.
• the reaction is carried out using a suitable base, such as potassium t-butoxide, sodium hydride, lithium diisopropylamide, or sodium or potassium bis(trimethylsilyl)amide.
• the reaction is carried out at temperature of from about -70° to ambient temperature and require about 1 to 48 hours.
• the product can be isolated and purified by techniques well known and appreciated in the art, such as quenching, acidification, filtration, extraction, evaporation, trituration, chromatography, and recrystalhzation.
• step 2 a compound of formula (21) is hydrolysed to give a compound of formula (22).
• Such hydrolysis of esters under acidic or basic conditions is well known and appreciated in the art and described in Protective Groups in Organic Synthesis, Theodora W. Greene (Wiley-Interscience, 2nd Edition, 1991).
• a compound of formula (21) is reacted with a suitable hydrolyzing agent, such as sodium hydroxide, potassium hydroxide, lithium hydroxide, or sodium carbonate to give an acid.
• a suitable hydrolyzing agent such as sodium hydroxide, potassium hydroxide, lithium hydroxide, or sodium carbonate
• the hydrolysis reaction is carried out in a suitable solvent, such as water, ethanol, methanol, or water/methanol mixtures, water/ethanol mixtures, water/tetrahydrofuran mixtures.
• the reactions are carried out at temperatures of from 0°C to the refluxing temperature of the solvent and generally require from 30 minutes to 48 hours.
• the acid produced in the hydrolysis reaction can be isolated using techniques well known in the art, such as acidification, extraction, and evaporation.
• the acid may be used after isolation without further purification or may be purified by chromatography, tritruration, and recrystalhzation as is known in the art.
• step 3 a compound of formula (22) is activated and reacted with a lithiated 4-substituted-oxazolidin-5-one to give a compound of formula (23).
• Suitable 4-substituted-oxazolidin-5-ones include 4-phenyl-2-oxazolidinone, (R)-4-phenyl-2- oxazolidinone, (S)-4-phenyl-2-oxazolidinone, 3,3-dimethyl-4-phenyl-2-oxazolidinone, (R)- 3,3-dimethyl-4-phenyl-2-oxazolidinone, and (S)-3,3-dimethyl-4-phenyl-2-oxazolidinone.
• the use of (R)-4-phenyl-2-oxazolidinone is depicted in Reaction Scheme I.
• the compound of formula (22) in a suitable organic solvent such as tetrahydrofuran diethyl ether
• a suitable tertiary organic amine such as triethylamine or N-methylmorpholine and cooled to -78°C.
• a suitable acid halide such as trimethylacetyl chloride is added and the mixture is transferred to an ice bath for 0.5 to 1.0 hours, then recooled to -78°C
• the resulting slurry is treated with lithiated (R)-4-phenyl-5- oxazolidinone, prepared by adding n-butyllithium to (S)-4-phenyl-2-oxazolidinone in tetrahydrofuran, and allowed to warm gradually to ambient temperature over a period of time ranging from about 10 to 20 hours.
• the product can be isolated by methods well known and appreciated in the art, such as extraction and evaporation.
• the product can be purified by methods well known and appreciated in the art, such as flash chromatography.
• step 4 a compound of formula (23) undergoes a 1,4-addition of a vinyl group to give a compound of formula (23a).
• a compound of formula (23) and trimethylsilyl chloride in a suitable solvent, such as tetrahydrofuran is added to a prepared solution of copper (I) iodide and N,N.N', N'-tetramethylethylenediamine and vinylmagnesium bromide in tetrhydrofuran.
• the reaction is carried out at temperatures of form about -78°C to about 0°C and requires form about 1 to 12 hours.
• the product can be isolated and purified by techniques well known and appreciated in the art, such as quenching, acidification, filtration, extraction, evaporation, trituration, chromatography, and recrystalhzation.
• step 5 a compound of formula (23a) undergoes an azide introduction reaction with a suitable azide transfer agent to give a compound of formula (23b).
• a suitable azide transfer agent for example, a compound of formula (23a)
• Such azide introductions are described in the art in J. Am. Chem. Soc, 112, 401 1-4030 (1990).
• a solution of a suitable amide such as potassium bis(trimethylsilyl)amide in a suitable organic solvent, such as tetrahydrofuran is cooled to -78°C and treated with a solution of a compound of formula (32a) in tetrahydrofuran, precooled to -78°C.
• a solution of a suitable azide transfer agent, such as trisyl azide, prepared by the method described in J. Org. Chem., 38, 1 1-16 (1973), in a suitable organic solvent, such as tetrahydrofuran, precooled to -78°C is then added. The solution is stirred, quenched with acetic acid.
• step 6 a compound of formla (23b) is hydrolyzed and esterified to give a compound of formula (24).
• a compound of formula (23b) is reacted with a suitable hydrolyzing agent, such as lithium hydroxide and hydrogen peroxide.
• a suitable hydrolyzing agent such as lithium hydroxide and hydrogen peroxide.
• the hydrolysis reaction is carried out in a suitable solvent, such as water/tetrahydrofuran mixtures.
• the reactions are carried out at temperatures of from 0°C to the refluxing temperature of the solvent and generally require from 30 minutes to 48 hours.
• the acid produced in the hydrolysis reaction can be isolated using techniques well known in the art, such as quenching of peroxides, acidification, extraction, and evaporation.
• the acid may be used after isolation without further purification or may be purified by chromatography, tritruration, and recrystalhzation as is known in the art.
• the acid is then este ⁇ fied to give a compound of formula (24).
• a ester forming reagent such as (trimethylsilyl)diazomethane.
• This reaction is carried out in a suitable solvent, such as methanol or methanol/tetrahydrofuran mixtures.
• a suitable solvent such as methanol or methanol/tetrahydrofuran mixtures.
• the reactions are carried out at temperatures of from 0°C to the refluxing temperature of the solvent and generally require from 12 to 48 hours.
• the product can be isolated and purified techniques well known in the art, such as acidification, extraction, evaporation, chromatography, tritruration, and recrystalhzation.
• the acid is contacted with methanol under acidic conditions.
• the reactions are carried out at temperatures of from 0°C to the refluxing temperature of methanol and generally require from 1 to 48 hours.
• the product can be isolated and purified techniques well known in the art, such as acidification, extraction, evaporation, chromatography, tritruration, and recrystalhzation.
• step 7 a compound of formula (24) is reduced and cyclized to give a compound of formula (25).
• a compound of formula (24) is contacted with a suitable recucing agent, such as dicyclohexylborane.
• a suitable recucing agent such as dicyclohexylborane.
• the reaction is carried out in a suitable solvent, such tetrahydrofuran.
• the reactions are carried out at temperatures of from -20°C to ambient temperature and generally require from 1 to 48 hours.
• the product can be isolated and purified techniques well known in the art, such as quenching, extraction, evaporation, chromatography, t ⁇ truration, and recrystalhzation.
• Reaction Scheme I step 8, a compound of formula (25) is protected to give a compound of formula (2a).
• the use of amine protecting groups is well known and appreciated in the art and described in Protective Groups in Organic Synthesis. Theodora W. Greene (Wiley-Interscience, 2nd Edition, 1991).
• 6-aminohexanoic acid (6-aminocaproic acid) (8.0 g, 60 mmol) and water (100 mL).
• sodium carbonate (6.84 g, 64 mmol)
• N-carbethoxyphthalimide (14.0 g, 64 mmol).
• extract the reaction mixture with ethyl acetate (100 mL).
• Collect the solid by filtration, rinse with water, and dry to give 6-phthalimidohexanoic acid (12.7 g, 80% yield).
• the reaction mixture was poured into 250 mL of a 3:2 mixture of saturated Ammonium chlroide: concentrated NH4OH.
• the layers were separated and the aqueous layer extracted with ethyl acetate (3 x 200 mL).
• the combined organic layers were washed sequentially with saturated Ammonium chlroide (1 x 100 mL) and water (1 x 100 mL).
• the organic layer was dried dry over Mg so 4. and concentrated under reduced pressure.
• the residue was purified by passage through a plug of SiO2 eluting with 4:1 hexane:ethyl acetate.
• the eluant was concentrated in vacuo to recover a white solid (3.64 g, 9.81 mmol, 87% yield).
• Potassium hexamethyldisilazide (0.5 M in toluene, 25.5 mL, 12.8 mmol) was added in one portion to anhydrous tetrahydrofuran (34 mL) at -78°C.
• Imide 4 (3.64 g, 9.81 mmol) was slurried in tetrahydrofuran (34 mL) and added via cannula, rinsing with tetrahydrofuran (2 x 11 mL) to complete the transfer.
• Trisylazide is not commercially available. Sulfonyl azides can be prepared according to J. Org. Chem. 1973, 38, 11-16. The azide transfer can be difficult. See J. Am. Chem. Soc. 1990, 1 12, 401 1-4030 for a full discussion. After the addition of the trisylazide, an intermediate that is more polar than starting material is rapidly formed. After addition of AcOH, the polar intermediate slowly disappears and the product azidoimide spot begins to form. It is only slightly less polar than the starting imide. A decomposition product of trisylazide nearly coelutes with the product.
• the residue was passed through a SiO2 plug column eluting with 1 : 1 hexane:ethyl acetate to recover, after concentration, a white solid that was presumably a mixture of the carboxylic acid and chiral auxiliary. Recrystalhzation from hexane/ethyl acetate yielded the chiral auxiliary as needles.
• the mother liquor was concentrated and carried on to the esterification step.
• the reagent was slurried in dichloromethane (36 mL) and cooled to 0°C. Vinyl azide 6 (1.23 g, 4.38 mmol) was dissolved in dichloromethane (9 mL) and added via cannula. The reaction mixture became pale yellow and gas evolution was evident. The mixture was warmed to room temperature overnight. Added MeOH (26 mL) and stirred for an additional 15 minutes. The mixture was concentrated under reduced pressure.
• N-Fmoc-trans-3-(naphth-2-yl)-L-proline (8) A solution of amino ester 7 (4.31 mmol) in 5 M hydrochloric acid (20 mL) was heated at 100°C overnight. The reaction mixture was concentrated in vacuo to recover the amino acid.
• the final target can also be recrystallized from hexane/ethyl acetate.
• the present invention provides a method of inhibiting matrix metalloproteinase
• MMP matrix metalloproteinase inhibiting amount of a compound of formula (1).
• the term "patient" refers to warm-blooded animals or mammals, including guinea pigs, dogs, cats, rats, mice, hamsters, rabbits and primates, including humans.
• a patient is in need of treatment to inhibit MMP when it would be beneficial to the patient to reduce the physiological effect of active MMP.
• a patient is in need of treatment to inhibit MMP when a patient is suffering from a disease state characterized by excessive tissue disruption or tissue degradation, such as, but not limited to, a neoplastic disease state or cancer; rheumatoid arthritis; osteoarthritis; cardiovascular disorders, such as atherosclerosis; corneal ulceration; dental diseases, such as gingivitis or periodontal disease; and neurological disorders, such as multiple sclerosis; chronic inflammatory disorders, such as emphysema and especially smoking-induced emphysema.
• a disease state characterized by excessive tissue disruption or tissue degradation
• tissue disruption or tissue degradation such as, but not limited to, a neoplastic disease state or cancer
• rheumatoid arthritis such as atherosclerosis
• corneal ulceration such as atherosclerosis
• dental diseases such as gingivitis or periodontal disease
• neurological disorders such as multiple sclerosis
• chronic inflammatory disorders such as emphysema and especially smoking-induced
• an "effective matrix metalloproteinase inhibiting amount" of a compound of formula (1) is an amount which is effective, upon single or multiple dose administration to the patient, in providing relief of symptoms associated with MMP and is thus effective in inhibiting MMP-induced tissue disruption and/or MMP-induced tissue degradation.
• "relief of symptoms" of MMP-mediated conditions refers to decrease in severity over that expected in the absence of treatment and does not necessarily indicate a total elimination or cure of the disease. Relief of symptoms is also intended to include prophylaxis.
• an effective matrix metalloproteinase inhibiting dose can be readily determined by the use of conventional techniques and by observing results obtained under analogous circumstances.
• determining the effective dose a number of factors are considered including, but not limited to: the species of the patient; its size, age, and general health; the specific disease involved; the degree of involvement or the severity of the disease; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; and the use of concomitant medication.
• An effective matrix metalloproteinase inhibiting amount of a compound of formula (1) will generally vary from about 0.1 milligram per kilogram of body weight per day (mg/kg/day) to about 300 milligrams per kilogram of body weight per day (mg/kg/day). A daily dose of from about 1 mg/kg to about 100 mg/kg is preferred.
• Neoplastic disease state refers to an abnormal state or condition characterized by rapidly proliferating cell growth or neoplasm.
• Neoplastic disease states for which treatment with a compound of formula (1) will be particularly useful include: Leukemias, such as, but not limited to, acute lymphoblastic, chronic lymphocytic, acute myeloblastic and chronic myelocytic; Carcinomas and adenocarcinomas, such as, but not limited to, those of the cervix, oesophagus, stomach, small intestines, colon, lungs (both small and large cell), breast and prostate; Sarcomas, such as, but not limited to, oesteroma, osteosarcoma, lipoma, hposarcoma, hemangioma and hemangiosarcoma; Melanomas, including amelanotic and melanotic; and mixed types of neoplasias such as, but not limited to carcinosarcoma, lymphoid tissue type, fo
• Atherosclerosis is a disease state characterized by the development and growth of atherosclerotic lesions or plaque.
• the identification of those patients who are in need of treatment for atherosclerosis is well within the ability and knowledge of one of ordinary skill in the art. For example, individuals who are either suffering from clinically significant atherosclerosis or who are at risk of developing clinically significant atherosclerosis are patients in need of treatment for atherosclerosis.
• a clinician of ordinary skill in the art can readily determine, by the use of clinical tests, physical examination and medical/family history, if an individual is a patient in need of treatment for atherosclerosis.
• chronic inflammatory disease refers to diseases or conditions characterized by persistent inflammation in the absence of an identifiable irritant or microbial pathogen.
• Inflammatory diseases for which treatment with a compound of formula (1) will be particularly useful include: emphysema, chronic bronchitis, asthma, and chronic inflammation, and especially smoking-induced emphysema.
• a compound of formula (1) can be administered in any form or mode which makes the compound bioavailable in effective amounts, including oral and parenteral routes.
• the compound can be administered orally, subcutaneously, intramuscularly, intravenously, transdermally, topically, intranasally, rectally, inhalation, and the like.
• Oral and inhalation administration is generally preferred.
• One skilled in the art of preparing formulations can readily select the proper form and mode of administration depending upon the disease state to be treated, the stage of the disease, and other relevant circumstances. Remington ' s Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (1990).
• a compound of formula (1) can be administered in the form of pharmaceutical compositions or medicaments which are made by combining a compound of formula (1) with pharmaceutically acceptable carriers or excipients, the proportion and nature of which are determined by the chosen route of administration, and standard pharmaceutical practice.
• the pharmaceutical compositions or medicaments are prepared in a manner well known in the pharmaceutical art.
• the carrier or excipient may be a solid, semi-solid, or liquid material, which can serve as a vehicle or medium for the active ingredient. Suitable carriers or excipients are well known in the art.
• the pharmaceutical composition may be adapted for oral or parenteral use and may be administered to the patient in the form of tablets, capsules, suppositories, solution, suspensions, gels, ointments, aerosol or the like.
• the pharmaceutical compositions may be administered orally, for example, with an inert diluent or with an edible ca ⁇ ier. They may be enclosed in gelatin capsules or compressed into tablets.
• a compound of formula (1 ) may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like. These preparations should contain at least 4% of a compound of formula (1), the active ingredient, but may be varied depending upon the particular form and may conveniently be between 4% to about 70%) of the weight of the unit. The amount of the active ingredient present in compositions is such that a unit dosage form suitable for administration will be obtained.
• the tablets, pills, capsules, troches and the like may also contain one or more of the following adjuvants: binders such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or lactose, disintegrating agents such as alginic acid, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; and sweetening agents such as sucrose or saccharin may be added or a flavoring agent such as peppermint, methyl salicylate or orange flavoring.
• a liquid carrier such as polyethylene glycol or a fatty oil.
• dosage unit forms may contain other various materials which modify the physical form of the dosage unit, for example, as coatings.
• tablets or pills may be coated with sugar, shellac, or other enteric coating agents.
• a syrup may contain, in addition to the present compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors. Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used.
• the compounds of the present invention may be incorporated into a solution or suspension.
• These preparations should contain at least 0.1 % of a compound of the invention, but may be varied to be between 0.1 % and about 50% of the weight thereof.
• the amount of the active ingredient present in such compositions is such that a suitable dosage will be obtained.
• Preferred compositions and preparations are able to be determined by one skilled in the art.
• the solutions or suspensions may also include one or more of the following adjuvants: sterile diluents such as water for iniection, saline solution, fixed oils, oolvethvlene glvcols. glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylene diaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of toxicity such as sodium chloride or dextrose.
• the parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic
• the compounds of the present invention may also be administered by inhalation, such as by aerosol or dry powder. Delivery may be by a liquefied or compressed gas or a suitable pump system which dispenses the compounds of the present invention or a formulation thereof.
• Formulations for administration by inhalation of compounds of formula (1 ) may be delivered in single phase, bi-phasic, or tri-phasic systems. A variety of systems are available for the administration by aerosol of the compounds of formula ( 1 ).
• Dry powder formulations are prepared by either pelletizing or milling the compound of formula (1) to a suitable particle size or by admixing the pelletized or milled compound of formula (1 ) with a suitable carrier material, such as lactose and the like. Delivery by inhalation includes the necessary container, activators, valves, subcontainers, and the like.
• Preferred aerosol and dry powder formulations for administration by inhalation can be determined by one skilled in the art.
• the MMP inhibitors of the present invention can be evaluated by the procedures that follow.
• ProMMP-1 (EC 3.4.24.7; interstitial collagenase) was purified from culture medium of human rheumatoid synovial fibroblasts stimulated with macrophage-conditioned medium according to Okada, Y. et al., J. Biol. Chem. 261, 14245-14255 (1986).
• the active MMP-1 was obtained by treatment of proMMP-1 with trypsin (5 ⁇ g/mL) at 37°C for 30 minutes, followed by addition of soybean trypsin inhibitor (50 ⁇ g/mL).
• the activated MMP-1 is assayed using a fluorogenic substrate, Mca-Pro-Leu-Gly-Leu- Dpa-Ala-Arg-NH 2 , Knight, C.G. et al., FEBS Lett. 296, 263-266 (1992), at 37°C in 2.0 mL of assay buffer containing 50 itiM Tris, pH 7.6, 0.2 M sodium chloride, 50 mM calcium chloride, and 0.02%) Brij-35.
• the enzyme (10 ⁇ L of 0.2 ⁇ M MMP-3 dilution in assay buffer) was added at the last to start the reaction.
• EXAMPLE B Source and Activation of proMMP-2 Recombinant MMP-2 was purified from the fermentation broth of yeast Pichia pastoris that carries the integrated MMP-2 gene into its chromosome.
• the full-length cDNA for MMP-2 was obtained by reverse transcription of RNA from human melanoma A375M cell line by the reverse transcriptase polymerase chain reaction (RT-PCR) using sequence specific oligonucleotides. The nucleotide sequence was confirmed by Taq cycle sequencing.
• the cDNA was ligated into the Pichia pastoris expression vector pHIL-D2 in such a way that the expression of pro-MMP-2 is under the control of the methanol inducible alcohol oxidase promoter.
• the expression construct was digested with either Sail or Nsil and used to transform the Pichia pastoris strains KM71 and SMD1 168.
• a large-scale culture of a selected clone designated 24S was performed in a high cell density fermentor and the recombinant MMP-2 was purified from the culture supernatant by gelatin-sepharose 4B (Pharmacia).
• the enzyme is sufficiently pure at this stage for routine measurement of inhibition. If desired, however, the enzyme may be further purified by Ac A 44 gel filtration (Spectra).
• the active MMP-2 was obtained by activation of proMMP-2 at 37°C for 1 h with 4- aminophenylmercuric acetate which was then removed by a Sephadex G-50 spin column.
• the enzyme is assayed using a fluorogenic substrate, Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH 2 , at 37°C in 2.0 mL of assay buffer containing 50 mM Tris, pH 7.6, 0.2 M sodium chloride, 50 mM calcium chloride, 0.02% Brij-35, and 50 ⁇ M ⁇ -mercaptoethanol.
• the increase in fluorescence is monitored ( ⁇ ex 328 nm, ⁇ em 393 nm).
• Substrate and inhibitor stock solutions are made in DMF.
• the enzyme is added at the last to start the reaction.
• ProMMP-3 (EC 3.4.24.17; Stromelysin- 1 ) was purified from culture medium of human rheumatoid synovial fibroblasts stimulated with macrophage-conditioned medium according to Okada, Y. et al., J. Biol. Chem. 261, 14245-14255 (1986).
• the active MMP-3 was obtained by treatment of proMMP-3 with trypsin (5 ⁇ g/mL) at 37°C for 30 minutes, followed by addition of soybean trypsin inhibitor (50 ⁇ g/mL). Aliquots of the activated
• the activated MMP-3 is assayed using a fluorogenic substrate, Mca-Pro-Leu-Gly-Leu- Dpa-Ala-Arg-NH 2 , Knight, C.G. et al, FEBS Lett. 296, 263-266 (1992), at 37°C in an assay buffer containing 50 mM Tris, pH 7.6, 0.2 M sodium chloride, 50 mM calcium chloride, and 0.02%) Brij-35.
• the increase in fluorescence due to cleavage of Gly-Leu peptide bond by MMP-3 was monitored with Perkin-Elmer LS50B Fluorimeter ( ⁇ e 328 nm, ⁇ e ⁇ .
• MMP- 12 macrophage metalloelastase
• MMP- 12 (EC 3.4.24.65) was cloned, expressed and purified according to Shapiro, S.D. et al., J Biol. Chem. 268, 23824-23829 (1993). Autoactivation resulted in the fully processed active form of the enzyme. Aliquots of MMP- 12 were stored at -70C.
• the potency of inhibitors of MMP- 12 was measured using either quartz cuvettes or microtiter plates.
• the activity of MMP- 12 was measured using a fluorogenic substrate, Mca- Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH 2 , Knight, C.G. et al., FEBS Lett. 296, 263-266 (1992), at 25°C in an assay buffer containing 50 mM Tris, pH 7.6, 0.2 M sodium chloride, 50 mM calcium chloride, and 0.02%o Brij-35.
• K values were determined using the cuvette method by preparing a series of intermediate inhibitors solutions in 0.1% HCl-DMF and mixing the inhibitor with substrate (final concentration 2 mM) in a quartz cuvette containing 2 ml of assay buffer. MMP- 12 was added to start the reaction at a concentration of 2 nM and progress curves were generated.
• K values were determined using the microtiter plate method in a manner similar to that described for the cuvette method with some modifications.
• Four different inhibitor concentrations (50 ml in assay buffer)of each compound were added to separate wells of a microtiter plate and substrate was added (100 ml) to get a final concentration of 4 mM.
• MMP-1 2 was added to a final concentration of 2 nM (50 ml) to start the reaction. Cleavage of substrate was recorded every 30 seconds for 30 minutes and progress curves were generated.

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