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Definitions

• the present invention relates to the field of agonists for the family of nuclear receptors identified as peroxisome proliferator-activated receptors.
• PPARs peroxisome proliferator-activated receptors
• PPAR ⁇ isoforms expressed at the protein level in mouse and human, ⁇ l and ⁇ 2. They differ only in that the latter has 30 additional amino acids at its N terminus due to differential promoter usage within the same gene, and subsequent alternative RNA processing.
• PPAR ⁇ 2 is expressed primarily in adipose tissue, while PPAR ⁇ l is expressed in a broad range of tissues.
• Murine PPAR ⁇ was the first member of this nuclear receptor subclass to be cloned; it has since been cloned from humans.
• PPAR ⁇ is expressed in numerous metabolically active tissues, including liver, kidney, heart, skeletal muscle, and brown fat. It is also present in monocytes, vascular endothelium, and vascular smooth muscle cells. Activation of PPAR ⁇ induces hepatic peroxisome proliferation, hepatomegaly, and hepatocarcinogenesis in rodents. These toxic effects are lost in humans, although the same compounds activate PPAR ⁇ across species.
• Human PPAR ⁇ was cloned in the early 1990s and subsequently cloned from rodents. PPAR ⁇ is expressed in a wide range of tissues and cells with the highest levels of expression found in digestive tract, heart, kidney, liver, adipose, and brain. Thus far, no PPAR ⁇ -specific gene targets have been identified.
• the PPARs are ligand-dependent transcription factors that regulate target gene expression by binding to specific peroxisome proliferator response elements PPREs) in enhancer sites of regulated genes.
• PPARs possess a modular structure composed of functional domains that include a DNA binding domain (DBD) and a ligand binding domain (LBD).
• the DBD specifically binds PPREs in the regulatory region of PPAR- responsive genes.
• the DBD located in the C-terminal half of the receptor contains the ligand-dependent activation domain, AF-2. Each receptor binds to its PPRE as a heterodimer with a retinoid X receptor (RXR).
• RXR retinoid X receptor
• a PPAR Upon binding an agonist, the conformation of a PPAR is altered and stabilized such that a binding cleft, made up in part of the AF-2 domain, is created and recruitment of transcriptional coactivators occurs. Coactivators augment the ability of nuclear receptors to initiate the transcription process.
• the result of the agonist-induced PPAR-coactivator interaction at the PPRE is an increase in gene transcription. Downregulation of gene expression by PPARs appears to occur through indirect mechanisms. (Bergen & Wagner, 2002, Diabetes Tech. & Ther., 4:163-174).
• PPAR ⁇ The first cloning of a PPAR (PPAR ⁇ ) occurred in the course of the search for the molecular target of rodent hepatic peroxisome proliferating agents. Since then, numerous fatty acids and their derivatives including a variety of eicosanoids and prostaglandins have been shown to serve as ligands of the PPARs. Thus, these receptors may play a central role in the sensing of nutrient levels and in the modulation of their metabolism, hi addition, PPARs are the primary targets of selected classes of synthetic compounds that have been used in the successful treatment of diabetes and dyslipidemia. As such, an understanding of the molecular and physiological characteristics of these receptors has become extremely important to the development and utilization of drugs used to treat metabolic disorders.
• PPAR ⁇ and PPAR ⁇ may play a role in a wide range of events involving the vasculature, including atherosclerotic plaque formation and stability, thrombosis, vascular tone, angio- genesis,and cancer.
• PPAR ⁇ and PPAR ⁇ may play a role in a wide range of events involving the vasculature, including atherosclerotic plaque formation and stability, thrombosis, vascular tone, angio- genesis,and cancer.
• TGDs Thiazolidinediones
• TZDs induced adipocyte differentiation and increased expression of adipocyte genes, including the adipocyte fatty acid-binding protein aP2.
• PPAR ⁇ interacted with a regulatory element of the aP2 gene that controlled its adipocyte- specific expression.
• experiments were performed that determined that TZDs were PPAR ⁇ ligands and agonists and demonstrate a definite correlation between their in vitro PPAR ⁇ activities and their in vivo insulin- sensitizing actions. (Bergen & Wagner, 2002, Diabetes Tech. & Ther., 4:163-174).
• TZDs include troglitazone, rosiglitazone, and pioglitazone, have insulin-sensitizing and anti-diabetic activity in humans with type 2 diabetes and impaired glucose tolerance.
• Farglitazar is a very potent non-TZD PPAR- ⁇ -selective agonist that was recently shown to have antidiabetic as well as lipid-altering efficacy in humans.
• NSAIDs non-steroidal antiinflammatory drugs
• indomethacin, fenoprofen, and ibuprofen have displayed weak PPAR ⁇ and PPAR ⁇ activities.
• Clofibrate and fenofibrate have been shown to activate PPAR ⁇ with a 10-fold selectivity over PPAR ⁇ . Bezafibrate acted as a pan-agonist that showed similar potency on all three PPAR isoforms.
• Wy-14643 the 2-arylthioacetic acid analogue of clofibrate, was a potent murine PPAR ⁇ agonist as well as a weak PPAR ⁇ agonist. In humans, all of the fibrates must be used at high doses (200-1,200 mg/day) to achieve efficacious lipid- lowering activity.
• TZDs and non-TZDs have also been identified that are dual PPAR ⁇ / ⁇ agonists.
• KRP-297 is an example of a TZD dual PPAR ⁇ / ⁇ agonist (Fajas, 1997, J. Biol. Chem., 272:18779-18789) DRF-2725 and AZ-242 are non-TZD dual PPAR ⁇ / ⁇ agonists. (Lohray, et ab, 2001, J. Med. Chem., 44:2675-2678; Cronet, et ab, 2001, Structure (Camb.) 9:699-706).
• GW501516 was a potent, highly-selective PPAR ⁇ agonist that produced beneficial changes in serum lipid parameters in obese, insulin-resistant rhesus monkeys. (Oliver et ab, 2001, Proc. Natl. Acad. Sci., 98:5306-5311).
• Yamamoto et ab U.S. Patent 3,489,767 describes "l-(phenylsulfonyl)-indolyl aliphatic acid derivatives” that are stated to have “antiphlogistic, analgesic and antipyretic actions.” (Cob 1, lines 16-19.)
• Kato et ab European patent application 94101551.3, Publication No.
• the present invention concerns molecular scaffolds of Formula I and the use of such molecular scaffolds, and the use of compounds with the structure of Formula I as modulators of the PPARs, PPAR ⁇ , PPAR ⁇ , and PPAR ⁇ , where Formula I is:
• U, V, W, X, and Y are independently substituted N or CR 8 , where there are no more than 4, and preferably no more than 3, nitrogens in the bicyclic ring structure shown in Formula I, and there are no more than 2 nitrogens in either of the rings;
• R 1 is a carboxyl group (or ester thereof) or a carboxylic acid isostere such as optionally substituted thiazolidine dione, optionally substituted hydroxamic acid, optionally substituted acyl-cyanamide, optionally substituted tetrazole, optionally substituted isoxazole, optionally substituted sulphonate, optionally substituted sulfonamide, and optionally substituted acylsulphonamide;
• R 6 and R 7 are independently hydrogen, optionally substituted lower alkyb optionally substituted cycloalkyb optionally substituted heterocycloalkyb optionally substituted aryb optionally substituted aralkyb optionally substituted heteroaryb or optionally substituted heteroaralkyb or R and R combine to form a mono-carbocyclic or mono-heterocyclic 5- or 6-membered ring system;
• R 9 is optionally substituted lower alkyb optionally substituted cycloalkyb optionally substituted heterocycloalkyb optionally substituted aryb optionally substituted aralkyb optionally substituted heteroaryb or optionally substituted heteroaralkyb
• R 10 and R 1 x are independently hydrogen, optionally substituted lower alkyb optionally substituted cycloalkyb optionally substituted heterocycloalkyb optionally substituted aryb optionally substituted aralkyb optionally substituted heteroaryb or optionally substituted heteroaralkyb or R 10 and R 11 combine to form a mono-carbocyclic or mono-heterocyclic 5- or 6-membered ring system;
• R 12 , R 13 , R 14 ' and R 15 are independently optionally substituted lower alkyb optionally substituted cycloalkyb optionally substituted monofluoroalkyb trifluoromethyb optionally substituted difiuoroalkyb optionally substituted heterocycloalkyb optionally substituted aryb optionally substituted aralkyb optionally substituted heteroaryb or optionally substituted heteroaralkyb
• Z is O or S
• n 0, l, or 2.
• Halo or "Halogen” - alone or in combination means all halogens, that is, chloro (Cl), fluoro (F), bromo (Br), iodo (I).
• Haldroxyl refers to the group -OH.
• Alkyl alone or in combination means an alkane-derived radical containing from 1 to 20, preferably 1 to 15, carbon atoms (unless specifically defined). It is a straight chain alkyb branched alkyb or cycloalkyb In many embodiments, an alkyl is a straight or branched alkyl group containing from 1-15, 1 to 8, 1-6, 1-4, or 1-2, carbon atoms, such as methyl, ethyl, propyb isopropyl, butyl, t-butyl and the like.
• lower alkyl is used herein to describe the straight chain alkyl groups of 1-6, 1-4, or 1-2 carbon atoms.
• cycloalkyl groups are monocyclic, bicyclic or tricyclic ring systems of 3-8, more preferably 3-6, ring members per ring, such as cyclopropyl, cyclopentyb cyclohexyl, and the like, but can also include larger ring structures such as adamantyl.
• Alkyl also includes a straight chain or branched alkyl group that contains or is interrupted by a cycloalkyl portion. The straight chain or branched alkyl group is attached at any available point to produce a stable compound.
• a substituted alkyl is a straight chain alkyb branched alkyb or cycloalkyl group defined previously, independently substituted with 1 to 3 groups or substituents of halo, hydroxy, alkoxy, alkylthio, alkylsulfinyb alkylsulfonyb acyloxy, aryloxy, heteroaryloxy, amino optionally mono- or di-substituted with alkyb aryl or heteroaryl groups, amidino, urea optionally substituted with alkyb aryb heteroaryl or heterocyclyl groups, aminosulfonyl optionally N-mono- or N,N-di-substituted with alkyb aryl or heteroaryl groups, alkylsulfonylamino, arylsulfonylamino, hetero
• alkenyl - alone or in combination means a straight, branched, or cyclic hydrocarbon containing 2-20, preferably 2-17, more preferably 2-10, even more preferably
• carbon to carbon double bond In the case of a cycloalkenyl group, conjugation of more than one carbon to carbon double bond is not such as to confer aromaticity to the ring. Carbon to carbon double bonds may be either contained within a cycloalkenyl portion, with the exception of cyclopropenyb or within a straight chain or branched portion. Examples of alkenyl groups include ethenyb propenyb isopropenyb butenyb cyclohexenyb cyclohexenylalkyl and the like.
• a substituted alkenyl is the straight chain alkenyb branched alkenyl or cycloalkenyl group defined previously, independently substituted with 1 to 3 groups or substituents of halo, hydroxy, alkoxy, alkylthio, alkylsulfinyb alkylsulfonyb acyloxy, aryloxy, heteroaryloxy, amino optionally mono- or di-substituted with alkyb aryl or heteroaryl groups, amidino, urea optionally substituted with alkyl, aryb heteroaryl or heterocyclyl groups, aminosulfonyl optionally N- mono- or N,N-di-substituted with alkyb aryl or heteroaryl groups, alkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, alkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino,
• Alkynyl - alone or in combination means a straight or branched hydrocarbon containing 2-20, preferably 2-17, more preferably 2-10, even more preferably 2-8, most preferably 2-4, carbon atoms containing at least one, preferably one, carbon to carbon triple bond.
• alkynyl groups include ethynyb propynyb butynyl and the like.
• a substituted alkynyl refers to the straight chain alkynyl or branched alkynyl defined previously, independently substituted with 1 to 3 groups or substituents of halo, hydroxy, alkoxy, alkylthio, alkylsulfinyb alkylsulfonyb acyloxy, aryloxy, heteroaryloxy, amino optionally mono- or di-substituted with alkyb aryl or heteroaryl groups, amidino, urea optionally substituted with alkyb aryb heteroaryl or heterocyclyl groups, aminosulfonyl optionally N-mono- or N,N-di-substituted with alkyb aryl or heteroaryl groups, alkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, alkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, or the like
• Alkyl alkynyl refers to a groups -RCCR' where R is lower alkylene or substituted lower alkylene, R' is hydrogen, lower alkyb substituted lower alkyb acyb aryb substituted aryb hetaryb or substituted hetaryl as defined below.
• Alkoxy denotes the group -OR, where R is lower alkyb substituted lower alkyb acyb aryb substituted aryl, aralkyb substituted aralkyb heteroalkyb heteroarylalkyb cycloalkyb substituted cycloalkyb cycloheteroalkyb or substituted cycloheteroalkyl as defined.
• Acyl denotes groups -C(O)R, where R is hydrogen, lower alkyl, substituted lower alkyb aryb substituted aryl and the like as defined herein.
• Aryloxy denotes groups -OAr, where Ar is an aryb substituted aryb heteroaryb or substituted heteroaryl group as defined herein.
• Amino or substituted amine denotes the group -NRR', where R and R' may independently by hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl, hetaryb or substituted heteroaryl as defined herein, acyl or sulfonyl.
• Amido denotes the group -C(O)NRR', where R and R' may independently by hydrogen, lower alkyl, substituted lower alkyb aryb substituted aryl, hetaryb substituted hetaryl as defined herein.
• Carboxyl denotes the group -C(O)OR, where R is hydrogen, lower alkyb substituted lower alkyb aryb substituted aryl, hetaryb and substituted hetaryl as defined herein.
• carboxylic acid isostere refers to a group selected from optionally substituted thiazolidine dione, optionally substituted hydroxamic acid, optionally substituted acyl-cyanamide, optionally substituted tetrazole, optionally substituted isoxazole, optionally substituted sulphonate, optionally substituted sulfonamide, and optionally substituted acylsulphonamide
• Carbocyclic refers to a saturated, unsaturated, or aromatic group having a single ring (e.g., phenyl) or multiple condensed rings where all ring atoms are carbon atoms, which can optionally be unsubstituted or substituted with, e.g., halogen, lower alkyl, lower alkoxy, alkylthio, acetylene, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryb substituted hetaryb nitro, cyano, thiob sulfarnido and the like.
• Aryl - alone or in combination means phenyl or naphthyl optionally carbocyclic fused with a cycloalkyl of preferably 5-7, more preferably 5-6, ring members and/or optionally substituted with 1 to 3 groups or substituents of halo, hydroxy, alkoxy, alkylthio, alkylsulfinyb alkylsulfonyb acyloxy, aryloxy, heteroaryloxy, amino optionally mono- or di-substituted with alkyb aryl or heteroaryl groups, amidino, urea optionally substituted with alkyl, aryl, heteroaryl or heterocyclyl groups, aminosulfonyl optionally N- mono- or N,N-di-substituted with alkyb aryl or heteroaryl groups, alkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino,
• Substituted aryl refers to aryl optionally substituted with one or more functional groups, e.g., halogen, lower alkyl, lower alkoxy, alkylthio, acetylene, amino, amido, carboxyl, hydroxyl, aryb aryloxy, heterocycle, heteroaryb substituted heteroaryb nitro, cyano, thiob sulfamido and the like.
• functional groups e.g., halogen, lower alkyl, lower alkoxy, alkylthio, acetylene, amino, amido, carboxyl, hydroxyl, aryb aryloxy, heterocycle, heteroaryb substituted heteroaryb nitro, cyano, thiob sulfamido and the like.
• Heterocycle refers to a saturated, unsaturated, or aromatic group having a single ring (e.g., morpholino, pyridyl or furyl) or multiple condensed rings (e.g., naphthpyridyb quinoxalyb quinolinyb indolizinyl orbenzo[b]thienyl) and having carbon atoms and at least one hetero atom, such as N, O or S, within the ring, which can optionally be unsubstituted or substituted with, e.g., halogen, lower alkyl, lower alkoxy, alkylthio, acetylene, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryb substituted hetaryb nitro, cyano, thiob sulfamido and the like.
• a single ring e.g., morpholino, pyridyl or furyl
• Heteroaryl alone or in combination means a monocyclic aromatic ring structure containing 5 or 6 ring atoms, or a bicyclic aromatic group having 8 to 10 atoms, containing one or more, preferably 1-4, more preferably 1-3, even more preferably 1-2, heteroatoms independently selected from the group O, S, and N, and optionally substituted with 1 to 3 groups or substituents of halo, hydroxy, alkoxy, alkylthio, alkylsulfinyb alkylsulfonyb acyloxy, aryloxy, heteroaryloxy, amino optionally mono- or di-substituted with alkyl, aryl or heteroaryl groups, amidino, urea optionally substituted with alkyb aryb heteroaryl or heterocyclyl groups, aminosulfonyl optionally N-mono- or N,N-di- substituted with alkyl, aryl or heteroaryl groups, alkyls
• Heteroaryl is also intended to include oxidized S or N, such as sulfinyl, sulfonyl and N-oxide of a tertiary ring nitrogen.
• a carbon or nitrogen atom is the point of attachment of the heteroaryl ring structure such that a stable aromatic ring is retained.
• heteroaryl groups are pyridinyb pyridazinyb pyrazinyb quinazolinyb purinyb indolyb quinolinyb pyrimidinyb pyrrolyb oxazolyb thiazolyb thienyb isoxazolyb oxathiadiazolyb isothiazolyb tetrazolyb imidazolyb triazinyb furanyb benzofuryb indolyl and the like.
• a substituted heteroaryl contains a substituent attached at an available carbon or nitrogen to produce a stable compound.
• Heterocyclyl - alone or in combination means a non-aromatic cycloalkyl group having from 5 to 10 atoms in which from 1 to 3 carbon atoms in the ring are replaced by heteroatoms of O, S or N, and are optionally benzo fused or fused heteroaryl of 5-6 ring members and/or are optionally substituted as in the case of cycloalkyb
• Heterocycyl is also intended to include oxidized S or N, such as sulfinyb sulfonyl and N-oxide of a tertiary ring nitrogen. The point of attachment is at a carbon or nitrogen atom.
• heterocyclyl groups are tetrahydrofuranyb dihydropyridinyb piperidinyb pyrrolidinyb piperazinyb dihydrobenzofuryb dihydroindolyb and the like.
• a substituted hetercyclyl group contains a substituent nitrogen attached at an available carbon or nitrogen to produce a stable compound.
• Substituted heteroaryl refers to a heterocycle optionally mono or poly substituted with one or more functional groups, e.g., halogen, lower alkyl, lower alkoxy, alkylthio, acetylene, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, substituted heterocycle, hetaryb substituted hetaryb nitro, cyano, thiob sulfamido and the like.
• functional groups e.g., halogen, lower alkyl, lower alkoxy, alkylthio, acetylene, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, substituted heterocycle, hetaryb substituted hetaryb nitro, cyano, thiob sulfamido and the like.
• Aralkyl refers to the group -R-Ar where Ar is an aryl group and R is lower alkyl or substituted lower alkyl group.
• Aryl groups can optionally be unsubstituted or substituted with, e.g., halogen, lower alkyl, alkoxy, alkylthio, acetylene, amino, amido, carboxyl, hydroxyl, aryb aryloxy, heterocycle, substituted heterocycle, hetaryb substituted hetaryb nitro, cyano, thiob sulfamido and the like.
• Heteroalkyl refers to the group -R-Het where Het is a heterocycle group and R is a lower alkylene group. Heteroalkyl groups can optionally be unsubstituted or substituted with e.g., halogen, lower alkyl, lower alkoxy, alkylthio, acetylene, amino, amido, carboxyl, aryl, aryloxy, heterocycle, substituted heterocycle, hetaryb substituted hetaryb nitro, cyano, thiob sulfamido and the like.
• Heteroarylalkyl refers to the group -R-HetAr where HetAr is an heteroaryl group and R is lower alkylene or substituted lower alkylene.
• Heteroarylalkyl groups can optionally be unsubstituted or substituted with, e.g., halogen, lower alkyl, substituted lower alkyl, alkoxy, alkylthio, acetylene, aryl, aryloxy, heterocycle, substituted heterocycle, hetaryb substituted hetaryb nitro, cyano, thiob sulfamido and the like.
• Cycloalkyl refers to a cyclic or polycyclic alkyl group containing 3 to 15 carbon atoms.
• Substituted cycloalkyl refers to a cycloalkyl group comprising one or more substituents with, e.g., halogen, lower alkyl, substituted lower alkyl, alkoxy, alkylthio, acetylene, aryl, aryloxy, heterocycle, substituted heterocycle, hetaryb substituted hetaryb nitro, cyano, thiob sulfamido and the like.
• Cycloheteroalkyl refers to a cycloalkyl group wherein one or more of the ring carbon atoms is replaced with a heteroatom (e.g., N, O, S or P).
• Substituted cycloheteroalkyl refers to a cycloheteroalkyl group as herein defined which contains one or more substituents, such as halogen, lower alkyl, lower alkoxy, alkylthio, acetylene, amino, amido, carboxyl, hydroxyl, aryb aryloxy, heterocycle, substituted heterocycle, hetaryb substituted hetaryb nitro, cyano, thiob sulfamido and the like.
• substituents such as halogen, lower alkyl, lower alkoxy, alkylthio, acetylene, amino, amido, carboxyl, hydroxyl, aryb aryloxy, heterocycle, substituted heterocycle, hetaryb substituted hetaryb nitro, cyano, thiob sulfamido and the like.
• Alkyl cycloalkyl denotes the group -R-cycloalkyl where cycloalkyl is a cycloalkyl group and R is a lower alkylene or substituted lower alkylene.
• Cycloalkyl groups can optionally be unsubstituted or substituted with e.g. halogen, lower alkyb lower alkoxy, alkylthio, acetylene, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, substituted heterocycle, hetaryb substituted hetaryb nitro, cyano, thiob sulfamido and the like.
• Alkyl cycloheteroalkyl denotes the group -R-cycloheteroalkyl where R is a lower alkylene or substituted lower alkylene.
• Cycloheteroalkyl groups can optionally be unsubstituted or substituted with e.g. halogen, lower alkyl, lower alkoxy, alkylthio, amino, amido, carboxyl, acetylene, hydroxyl, aryl, aryloxy, heterocycle, substituted heterocycle, hetaryb substituted hetaryb nitro, cyano, thiob sulfamido and the like.
• the compounds have a structure of Formula I in which the bicyclic core shown for Formula I has one of the following structures:
• the compound includes a bicyclic core as shown above.
• Such compounds can include substitutents as described for Formula I, with the understanding that ring nitrogens other than the nitrogen corresponding to position 1 of the indole structure are unsubstituted.
• the compounds have one of the bicyclic cores shown above and substitution selections as shown herein for compounds having an indolyl core; the compounds have one of the bicyclic cores above, and the substituents shown at the 5- position are instead attached at the 6-position.
• the compounds have a structure of Formula 1-1, namely
• the compounds of Formula I are compounds of Formulas la, lb, lc, Id, X, or XIV as shown in the Detailed Description.
• the aryl group is a 5- or 6-membered ring; the aryl group is a 6- membered ring; in further embodiments in which the aryl group is a 6-membered ring, the ring is substituted with one or two groups independently selected from halo, alkoxy, cycloalkyb aryb aryloxy, heteroaryb heteroaryloxy, aryl or heteroaryl substituted alkyl, and aryl or heteroaryl substituted alkoxy; in further embodiments in which a 6-membered ring is substituted with halo or alkoxy, the ring is substituted at the 3-position (met),
• the ring is substituted with one or two groups located at ring positions not adjacent to the ring atom linked to the -S(O) - group; the 5-membered ring is substituted with one or two ring substituents selected from the group consisting of halo, alkoxy, cycloalkyb aryb aryloxy, heteroaryb heteroaryloxy, aryl or heteroaryl substituted alkyl, and aryl or heteroaryl substituted alkoxy; the ring is substituted with chloro; the ring is substituted with alkoxy; the ring is substituted with alkyl; the ring is substituted with optionally substituted aryl or heteroaryl; the ring is substituted with optionally substituted aryloxy or heteroaryloxy; the 5-membered ring is fused with a second 5- or 6-membered aromatic or non-aromatic carbocyclic or heterocyclic ring.
• R 21 is optionally substituted aryl or optionally substituted heteroaryb
• R 4 is different from H and alkoxy, or R 4 is different from H and OR 9 .
• R 4 is different from H, halo, alkyl, alkoxy, alkylthio; R 4 is different from H, halo, C ⁇ - 3 alkyl, C ⁇ - 3 alkoxy, C 1 - 3 alkylthio; R 4 is different from C ⁇ _ 3 alkoxy; R 4 is not methoxy.
• n 2, R 2 is -S(O) 2 R 21 , with R 21 being optionally substituted aryl or optionally substituted heteroaryb
• R 21 being optionally substituted aryl or optionally substituted heteroaryb
• the aryl group is a 5- or 6-membered ring; the aryl group is a 6- membered ring; in further embodiments in which the aryl group is a 6-membered ring, the ring is substituted with one or two groups independently selected from halo, alkyl, cycloalkyb aryb aryloxy, heteroaryb heteroaryloxy, aryl or heteroaryl substituted alkyb and aryl or heteroaryl substituted alkoxy; in further embodiments in which a 6-membered ring is substituted with halo or alkoxy, the ring is substituted at the 3-position (meta), 4-
• the ring is substituted with one or two groups located at ring positions not adjacent to the ring atom linked to the -S(O) 2 - group; the 5-membered ring is substituted with one or two ring substituents selected from the group consisting of halo, alkoxy, cycloalkyb aryb aryloxy, heteroaryb heteroaryloxy, aryl or heteroaryl substituted alkyl, and aryl or heteroaryl substituted alkoxy; the ring is substituted with chloro; the ring is substituted with alkoxy; the ring is substituted with alkyl; the ring is substituted with optionally substituted aryl or heteroaryl; the ring is substituted with optionally substituted aryloxy or heteroaryloxy; the 5-membered ring is fused with a second 5- or 6-membered aromatic or non-aromatic carbocyclic or heterocyclic ring.
• R 21 being a substituted aryl group with a 6-membered
• the substitution on the aryl group is not methoxy
• the substitution on the aryl group is not alkoxy
• the substitution on the aryl group is not alkoxy
• R 4 and the substitution on the aryl group are not both alkoxy
• R 4 and the substitution on the aryl group are not both methoxy
• R 4 is not alkoxy
• R 4 is not methoxy.
• compounds of Formula I have a structure of Formula Ie as shown below:
• R* is hydrogen, halo, optionally substituted lower alkyl, optionally substituted cycloalkyb optionally substituted heterocycloalkyb optionally substituted aryl, optionally substituted aralkyb optionally substituted heteroaryb optionally substituted heteroaralkyb -OR 9 (e.g., optionally substituted alkoxy, for example, methoxy, ethoxy) -SR 9 , -NR 10 R ⁇ , -C(Z)NR 10 R ⁇ , -C(Z)R 20 , -S(O) 2 NR 10 R n , or-S(O) 2 R 21 ;
• R 24 is H, halo, optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted aryloxy, or optionally substituted aralkoxy (e.g., Aryl-O(CH 2 ) p O-, where p is 1-4);
• R 25 is H, halo, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted aryloxy, or R 24 and R 25 together form a fused ring with the phenyl group, e.g., benzofuran.
• R 4 is optionally substituted alkoxy (e.g., methoxy, ethoxy, propoxy, isopropoxy), optionally substituted aryloxy, optionally substituted heteroaryloxy, optionally substituted alkyl (e.g., methyl or ethyl), optionally substituted cycloalkyb optionally substituted cycloheteroalkyl, optionally substituted aryl, optionally substituted heteroaryb or halo.
• alkoxy e.g., methoxy, ethoxy, propoxy, isopropoxy
• aryloxy optionally substituted heteroaryloxy
• alkyl e.g., methyl or ethyl
• optionally substituted cycloalkyb optionally substituted cycloheteroalkyl
• optionally substituted aryl optionally substituted heteroaryb or halo.
• R 4 is optionally substituted alkoxy (e.g., methoxy, ethoxy, propoxy, isopropoxy), optionally substituted alkyl (e.g., methyl or ethyl), optionally substituted aryl, optionally substituted heteroaryb or halo.
• alkoxy e.g., methoxy, ethoxy, propoxy, isopropoxy
• alkyl e.g., methyl or ethyl
• aryl e.g., optionally substituted heteroaryb or halo.
• compounds of Formula I can be as specified for Formula Ie, but with the phenyl ring to which R 24 and R 25 are attached as a heteroaryl ring. If the heteroaryl ring is a 5-membered ring, R 24 and R 25 are attached at the ring positions that are not adjacent to the atom linking to the sulfonyl group shown in Formula Ie.
• R 4 is alkoxy and R 24 and R 25 are chloro; R 4 is alkoxy and R 24 and R 25 are fluoro; R 4 is alkoxy and R 24 is alkoxy; R 4 is alkoxy and R 24 is alkyl; R 4 is methoxy or ethoxy and R 24 and R 25 are chloro; R 4 is methoxy or ethoxy and R 24 is alkoxy; R 4 is methoxy or ethoxy and R 24 is alkyl.
• both of R 24 and R 25 are not alkyl; neither of R 24 and R 25 are alkyl; with R 24 as H, R 25 is not alkyl; with R 25 as H, R 24 is not alkyl.
• Exemplary compounds include those listed in Table 1 and in Table 4.
• Reference to compounds of Formula I herein includes specific reference to sub-groups and species of compounds of Formula I described herein (e.g., particular embodiments as described above) unless indicated to the contrary.
• any one or more of the sub-groups of compounds of Formula I or any one or more of the exemplary compounds is excluded from one of the specified compound groups or sub-groups of Formula I that would otherwise include such sub-group or sub-groups.
• the compound is specific for PPAR ⁇ ; specific for PPAR ⁇ ; specific for PPAR ⁇ ; specific for PPAR ⁇ and PPAR ⁇ ; specific for PPAR ⁇ , and PPAR ⁇ ; specific for PPAR ⁇ and PPAR ⁇ .
• specificity means that the compound has at least 5-fold greater activity (preferably at least 1-, 20-, 50-, or 100-fold or more greater activity) on the specific PPAR(s) than on the other PPAR(s), where the activity is determined using a biochemical assay suitable for determining PPAR activity, e.g., an assay as described herein.
• a first aspect of the invention concerns novel compounds of Formula I and subgroups of Formula I, e.g., as described above or otherwise described herein.
• a related aspect of this invention concerns pharmaceutical compositions that include a compound of Formula I and at least one pharmaceutically acceptable carrier, excipient, or diluent.
• the composition can include a plurality of different pharmacalogically active compounds.
• compounds of Formula I can be used in the preparation of a medicament for the treatment of a PPAR-mediated disease or condition.
• the invention concerns a method of treating or prophylaxis of a disease or condition in a mammal, by administering to the mammal a therapeutically effective amount of a compound of Formula I, a prodrug of such compound, or a pharmaceutically acceptable salt of such compound or prodrug.
• the compound can be alone or can be part of a pharmaceutical composition.
• the disease or condition is obesity, overweight condition, hyperlipidemia, dyslipidemia including associated diabetic dyslipidemia and mixed dyslipidemia, hypoalphalipoproteinemia, Syndrome X, Type II diabetes mellitus, Type I diabetes, hyperinsulinemia, impaired glucose tolerance, insulin resistance, a diabetic complication (e.g., neuropathy, nephropathy, retinopathy or cataracts), hypertension, coronary heart disease, heart failure, hypercholesterolemia, inflammation, thrombosis, congestive heart failure, cardiovascular disease (including atherosclerosis, arteriosclerosis, and hypertriglyceridemia), epithelial hyperproliferative diseases (such as eczema and psoriasis), cancer, and conditions associated with the lung and gut and regulation of appetite and food intake in subjects suffering from disorders such as obesity, anorexia bulimia and anorexia nervosa.
• disorders such as obesity, anorexia bulimia and anorexia nervosa.
• the identification of compounds of Formula I active on PPARs also provides a method for identifying or developing additional compounds active on PPARs, e.g., improved modulators, by determining whether any of a plurality of test compounds of Formula I active on at least one PPAR provides an improvement in one or more desired pharmacologic properties relative to a reference compound active on such PPAR, and selecting a compound if any, that has an improvement in the desired pharmacologic property, thereby providing an improved modulator.
• additional compounds active on PPARs e.g., improved modulators
• the desired pharmacologic property is PPAR pan-activity, PPAR selectivity for any individual PPAR (PPAR ⁇ , PPAR ⁇ , or PPAR ⁇ ), selectivity on any two PPARs (PPAR ⁇ and PPAR ⁇ , PPAR ⁇ and PPAR ⁇ , or PPAR ⁇ and PPAR ⁇ ), serum half-life longer than 2 hr or longer than 4 hr or longer than 8 hr, aqeous solubility, oral bioavailability more than 10%, oral bioavailability more than 20%.
• the reference compound is a compound of Formula I.
• the process can be repeated multiple times, i.e., multiple rounds of preparation of derivatives and/or selection of additional related compounds and evaluation of such further derivatives of related compounds, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more additional rounds.
• structural info ⁇ nation about one or more of the PPARs is utilized, e.g., in conjunction with compounds of Formula I or a molecular scaffold or scaffold core of Fo ⁇ nula I.
• the invention provides a method of designing a ligand that binds to at least one member of the PPAR protein family (PPAR ⁇ , PPAR ⁇ , and PPAR ⁇ ), by identifying as molecular scaffolds one or more compounds that bind to a binding site of a PPAR with low affinity; determining the orientation of the one or more molecular scaffolds at the binding site of the PPAR by obtaining co-crystal structures of the molecular scaffolds in the binding site; identifying one or more structures of at least one scaffold molecule that, when modified, provide a ligand having altered binding affinity or binding specificity or both for binding to the PPAR as compared to the binding of the scaffold molecule.
• the designed ligand(s) can then be provided, e.g., by synthesizing or otherwise obtaining the ligand(s).
• the molecular scaffold is a compound of Formula I, or contains a bicyclic core as shown above for Formula I.
• a plurality of distinct compounds are assayed for binding to the binding site of the PPAR; co-crystals of the molecular scaffolds bound to the PPAR are isolated, and the orientation of the molecular scaffold is determined by performing X-ray crystallography on the co-crystals; the method further involves identifying common chemical structures of the molecular scaffolds, placing the molecular scaffolds into groups based on having at least one common chemical structure, and determining the orientation of the one or more molecular scaffolds at the binding site of the PPAR for at least one representative compound from a plurality of groups; the ligand binds to the target molecule with greater binding affinity or greater binding specificity or both than the molecular scaffold; the orientation of the molecular scaffold is determined by nuclear magnetic resonance in co-crystal structure dete ⁇ nination; the plurality of distinct compounds are each assayed for binding to a plurality of members of the PPAR family.
• the compounds are grouped into classes based on common chemical structures and a representative compound from a plurality of the classes is selected for performing X-ray crystallography on co-crystals of the compound and target molecule; the distinct compounds are selected based on criteria selected from molecular weight, clogP, and the number of hydrogen bond donors and acceptors; the clog P is less than 2, and the number of hydrogen bond donors and acceptors is less than 5.
• the distinct compounds have a molecular weight of from about 100 to about 350 daltons, or more preferably from about 150 to about 350 daltons or from 150 to 300 daltons, or from 200 to 300 daltons.
• the distinct compounds can be of a variety of structures.
• the distinct compounds can have a ring structure, either a carbocyclic or heterocyclic ring, such as for example, a phenyl ring, a pyrrole, imidazole, pyridine, purine, or any ring structure.
• a compound or compounds binds with extremely low affinity, very low affinity, low affinity, moderate affinity, or high affinity; at least about 5% of the binding compounds bind with low affinity (and/or has low activity), or at least about 10%), 15%, or 20% of the compounds bind with low affinity (or very low or extremely low).
• the compounds can be grouped into classes based on common chemical structures and at least one representative compound from at least one, or preferably a plurality, of the classes selected for performing orientation determination, e.g., by X-ray crystallography and/or NMR analysis.
• the selection can be based on various criteria appropriate for the particular application, such as molecular weight, clogP (or other method of assessing lipophilicity), Polar Surface Area (PSA) (or other indicator of charge and polarity or related properties), and the number of hydrogen bond donors and acceptors.
• Compounds can also be selected using the presence of specific chemical moieties which, based on information derived from the molecular family, might be indicated as having predisposing some affinity for members of the family.
• Compounds with highly similar structures and/or properties can be identified and grouped using computational techniques to facilitate the selection of a representative subset of the group.
• the molecular weight is from about 150 to about 350 daltons, more preferably from 150 to 300 daltons.
• the clog P is preferably less than 2
• the number of hydrogen bond donors and acceptors is preferably less than 5 and the PSA less than 100.
• Compounds can be selected that include chemical structures of drugs having acceptable pharmacalogical properties and/or lacking chemical strutures that are known to result in undesirable pharmacological properties, e.g., excessive toxicity and lack of solubility.
• the assay is an enzymatic assay, and the number of groups of molecular scaffolds formed can conveniently be about 500.
• the assay is a competition assay, e.g., a binding competition assay.
• Cell-based assays can also be used. As indicated above, compounds can be used that have low, very low, or extremely low activity in a biochemical or cell-based assay.
• the modification of a molecular scaffold can be the addition, subtraction, or substitution of a chemical group.
• the modification may desirably cause the scaffold to be actively transported to or into particular cells and/or a particular organ.
• the modification of the compound includes the addition or subtraction of a chemical atom, substituent or group, such as, for example, a hydrogen, alkyl, alkoxy, phenoxy, alkenyb alkynyb phenylalkyb hydroxyalkyl, haloalkyb aryb arylalkyb alkyloxy, alkylthio, alkenylthio, phenyb phenylalkyb phenylalkylthio, hydroxyalkyl-thio, alkylthiocarbbamylthio, cyclohexyl, pyridyl, piperidinyl, alkylamino, amino, nitro, mercapto, cyano, hydroxyl, a halogen
• the information provided by performing X-ray crystallography on the co-crystals is provided to a computer program, wherein the computer program provides a measure of the interaction between the molecular scaffold and the protein and a prediction of changes in the interaction between the molecular scaffold and the protein that result from specific modifications to the molecular scaffold, and the molecular scaffold is chemically modified based on the prediction of the biochemical result.
• the computer program can provide the prediction based on a virtual assay such as, for example, virtual docking of the compound to the protein, shape-based matching, molecular dynamics simulations, free energy perturbation studies, and similarity to a three-dimensional pharmacophore.
• a virtual assay such as, for example, virtual docking of the compound to the protein, shape-based matching, molecular dynamics simulations, free energy perturbation studies, and similarity to a three-dimensional pharmacophore.
• a variety of such programs are well-known in the art.
• Chemical modification of a chemically tractable structure can result, or be selected to provide one or more physical changes, e.g., to result, in a ligand that fills a void volume in the protein-ligand complex, or in an attractive polar interaction being produced in the protein-ligand complex.
• the modification can also result in a sub-structure of the ligand being present in a binding pocket of the protein binding site when the protein- ligand complex is formed.
• the compounds can be grouped based on having a common chemical substructure and a representative compound from each group (or a plurality of groups) can be selected for co-crystallization with the protein and performance of the X-ray crystallography.
• the X-ray crystallography is preferably performed on the co-crystals under at least 20, 30, 40, or 50 distinct environmental conditions, or more preferably under about 96 distinct environmental conditions.
• the X-ray crystallography and the modification of a chemically tractable structure of the compound can each be performed a plurality of times, e.g., 2, 3, 4, or more rounds of crystallization and modification.
• one or more molecular scaffolds are selected to have binding to a plurality of members of the PPAR family.
• the method can also include the identification of conserved residues in a binding site(s) of a PPAR protein that interact with a molecular scaffold, ligand or other binding compound.
• conserved residues can, for example, be identified by sequence alignment of different members of the PPAR family, and identifying binding site residues that are the same or at least similar between multiple member of the family.
• Interacting residues can be characterized as those within a selected distance from the binding compound(s), e.g., 3, 3.5, 4, 4.5, or 5 angstroms.
• the mvention provides a method of designing a ligand that binds to at least one PPAR that is a member of the PPAR family, by identifying as molecular scaffolds one or more compounds that bind to binding sites of a plurality of members of the PPAR family, determining the orientation of one or more molecular scaffolds at the binding site of a PPAR(s) to identify chemically tractable structures of the scaffold(s) that, when modified, alter the binding affinity or binding specificity between the scaffold(s) and the PPAR(s), and synthesizing a ligand wherein one or more of the chemically tractable structures of the molecular scaffold(s) is modified to provide a ligand that binds to the PPAR with altered binding affinity or binding specificity.
• the invention also provides a method to identify properties that a likely binding compound will possess, thereby allowing, for example, more efficient selection of compounds for structure activity relationship determinations and/or for selection for screening.
• another aspect concerns a method for identifying binding characteristics of a ligand of a PPAR protein, by identifying at least one conserved interacting residue in the PPAR that interacts with at least two binding molecules; and identifying at least one common interaction property of those binding molecules with the conserved residue(s). The interaction property and location with respect to the structure of the binding compound defines the binding characteristic.
• the identification of conserved interacting residues involves comparing (e.g., by sequence alignment) a plurality of amino acid sequences in the PPAR family and identifying binding site residues conserved in that family; identification of binding site residues by determining a co-crystal structure; identifying interacting residues (preferably conserved residues) within a selected distance of the binding compounds, e.g., 3, 3.5, 4, 4.5, or 5 angstroms; the interaction property involves hydrophobic interaction, charge-charge interaction, hydrogen bonding, charge-polar interaction, polar-polar interaction, or combinations thereof.
• Another related aspect concerns a method for developing ligands for a PPAR using a set of scaffolds.
• the method involves selecting a PPAR or plurality of PPARs, selecting a molecular scaffold, or a compound from a scaffold group, from a set of at least 3 scaffolds or scaffold groups where each of the scaffolds or compounds from each scaffold group are known to bind to the target.
• the set of scaffolds or scaffold groups is at least 4, 5, 6, 7, 8, or even more scaffolds or scaffold groups.
• Another aspect concerns a method for identifying structurally and energetically allowed sites on a binding compound for attachment of an additional component(s) by analyzing the orientation of the binding compound(s) in a PPAR binding site (e.g., by analyzing co-crystal structures), thereby identifying accessible sites on the compound for attachment of the separate component.
• the binding compound is a compound of Formula I.
• the method involves calculating the change in binding energy on attachment of the separate component at one or more of the accessible sites; the orientation is determined by co-crystallography; the separate component includes a linker, a label such as a fluorophore, a solid phase material such as a gel, bead, plate, chip, or well.
• the invention provides a method for attaching a PPAR binding compound to an attachment component(s), by identifying energetically allowed sites for attachment of such an attachment component on a binding compound (e.g., as described for the preceding aspect), and attaching the compound or derivative thereof to the attachment component(s) at the energetically allowed site(s).
• the binding compound is a compound of Formula I.
• the attachment component is a linker (which can be a traceless linker) for attachment to a solid phase medium
• the method also involves attaching the compound or derivative to a solid phase medium through the linker attached at the energetically allowed site; the binding compound or derivative thereof is synthesized on a linker attached to the solid phase medium; a plurality of compounds or derivatives are synthesized in combinatorial synthesis; the attachment of the compound(s) to the solid phase medium provides an affinity medium
• a related aspect concerns a method for making an affinity matrix for a PPAR, where the method involves identifying energetically allowed sites on a PPAR binding compound for attachment to a solid phase matrix; and attaching the PPAR binding compound to the solid phase matrix through the energetically allowed site.
• the binding compound is a compound of Formula I.
• Various embodiments are as described for attachment of a separate component above; identifying energetically allowed sites for attachment to a solid phase matrix is performed for at least 5, 10, 20, 30, 50, 80, or 100 different compounds; identifying energetically allowed sites is performed for molecular scaffolds or other PPAR binding compounds having different core ring structures.
• PPAR refers to a peroxisome proliferator-activated receptor as recognized in the art.
• the PPAR family includes PPAR ⁇ (also referred to as PPARa or PPARalpha), PPAR ⁇ (also referred to as PP ARd or PPARdelta), and PPAR ⁇ (also referred to as PPARg or PPARgamma).
• the individual PPARs can be identified by their sequences, where exemplary reference sequence accession numbers are: NM_005036 (cDNA sequence for hPPARa), NP_005027 (protein sequence for hPPARa), NM_015869 (cDNA sequence for hPPARg isoform 2), NP_056953 (protein sequence for hPPARg isoform 2), NM_006238 (cDNA sequence for hPPARd), and NP_006229 (protein sequence for hPPARd).
• exemplary reference sequence accession numbers are: NM_005036 (cDNA sequence for hPPARa), NP_005027 (protein sequence for hPPARa), NM_015869 (cDNA sequence for hPPARg isoform 2), NP_056953 (protein sequence for hPPARg isoform 2), NM_006238 (cDNA sequence for hPPARd), and NP_006229 (protein sequence for hPPARd).
• sequence differences will exist due to allelic variation, and will also recognize that other animals, particularly other mammals have corresponding PPARs, which have been identified or can be readily identified using sequence alignment and confirmation of activity, can also be used.
• modifications can be introduced in a PPAR sequence without destroying PPAR activity. Such modified PPARs can also be used in the present invention, e.g., if the modifications do not alter the binding site conformation to the extent that the modified PPAR lacks substantially normal ligand binding.
• the term "bind” and “binding” and like terms refer to a non-convalent energetically favorable association between the specified molecules (i.e., the bound state has a lower free energy than the separated state, which can be measured calorimetrically).
• the binding is at least selective, that is, the compound binds preferentially to a particular target or to members of a target family at a binding site, as compared to non-specific binding to unrelated proteins not having a similar binding site.
• BSA is often used for evaluating or controlling for non-specific binding.
• the decrease in free energy going from a separated state to the bound state must be sufficient so that the association is detectable in an biochemical assay suitable for the molecules involved.
• saying is meant the creation of experimental conditions and the gathering of data regarding a particular result of the experimental conditions.
• enzymes can be assayed based on their ability to act upon a detectable substrate.
• a compound or ligand can be assayed based on its ability to bind to a particular target molecule or molecules and/or to modulate an activity of a target molecule.
• background signal in reference to a binding assay is meant the signal that is recorded under standard conditions for the particular assay in the absence of a test compound, molecular scaffold, or ligand that binds to the target molecule.
• background signal in reference to a binding assay is meant the signal that is recorded under standard conditions for the particular assay in the absence of a test compound, molecular scaffold, or ligand that binds to the target molecule.
• binding site is meant an area of a target molecule to which a ligand can bind non-covalently. Binding sites embody particular shapes and often contain multiple binding pockets present within the binding site. The particular shapes are often conserved within a class of molecules, such as a molecular family. Binding sites within a class also can contain conserved structures such as, for example, chemical moieties, the presence of a binding pocket, and/or an electrostatic charge at the binding site or some portion of the binding site, all of which can influence the shape of the binding site.
• binding pocket is meant a specific volume within a binding site.
• a binding pocket is a particular space within a binding site at least partially bounded by target molecule atoms.
• a binding pocket is a particular shape, indentation, or cavity in the binding site.
• Binding pockets can contain particular chemical groups or structures that are important in the non-covalent binding of another molecule such as, for example, groups that contribute to ionic, hydrogen bonding, van der Waals, or hydrophobic interactions between the molecules.
• chemical structure or “chemical substructure” is meant any definable atom or group of atoms that constitute a part of a molecule.
• chemical substructures of a scaffold or ligand can have a role in binding of the scaffold or ligand to a target molecule, or can influence the three-dimensional shape, electrostatic charge, and/or conformational properties of the scaffold or ligand.
• orientation in reference to a binding compound bound to a target molecule is meant the spatial relationship of the binding compound and at least some of its consitituent atoms to the binding pocket and/or atoms of the target molecule at least partially defining the binding pocket.
• crystal refers to an ordered complex of target molecule, such that the complex produces an X-ray diffraction pattern when placed in an X-ray beam.
• a crystal is distinguished from a disordered or partially ordered complex or aggregate of molecules that do not produce such a diffraction pattern.
• a crystal is of sufficient order and size to be useful for X-ray crystallography.
• a crystal may be formed only of target molecule (with solvent and ions) or may be a co-crystal of more than one molecule, for example, as a co-crystal of target molecule and binding compound, and/or of a complex of proteins (such as a holoenzyme).
• co-crystals an ordered complex of the compound, molecular scaffold, or ligand bound noncovalently to the target molecule that produces a diffraction pattern when placed in an X- ray beam.
• the co-crystal is in a form appropriate for analysis by X-ray or protein crystallography.
• the target molecule-ligand complex can be a protein-ligand complex.
• log P is meant the calculated log P of a compound, "P” referring to the partition coefficient of the compound between a lipophilic and an aqueous phase, usually between octanol and water.
• chemically tractable structures is meant chemical structures, sub-structures, or sites on a molecule that can be covalently modified to produce a ligand with a more desirable property.
• the desirable property will depend on the needs of the particular situation. The property can be, for example, that the ligand binds with greater affinity to a target molecule, binds with more specificity, or binds to a larger or smaller number of target molecules in a molecular family, or other desirable properties as needs require.
• designing a ligand By “designing a ligand,” “preparing a ligand,” “discovering a ligand,” and like phrases is meant the process of considering relevant data (especially, but not limited to, any individual or combination of binding data, X-ray co-crystallography data, molecular weight, clogP, and the number of hydrogen bond donors and acceptors) and making decisions about advantages that can be achieved with resort to specific structural modifications to a molecule, and implementing those decisions. This process of gathering data and making decisions about structural modifications that can be advantageous, implementing those decisions, and determining the result can be repeated as many times as necessary to obtain a ligand with desired properties.
• binding is meant the process of attempting to fit a three-dimensional configuration of a binding pair member into a three-dimensional configuration of the binding site or binding pocket of the partner binding pair member, which can be a protein, and determining the extent to which a fit is obtained.
• the extent to which a fit is obtained can depend on the amount of void volume in the resulting binding pair complex (or target molecule-ligand complex).
• the configuration can be physical or a representative configuration of the binding pair member, e.g., an in silico representation or other model.
• ligand In the context of development of modulators using molecular scaffolds, by "ligand” is meant a molecular scaffold that has been chemically modified at one or more chemically tractable structures to bind to the target molecule with altered or changed binding affinity or binding specificity relative to the molecular scaffold.
• the ligand can bind with a greater specificity or affinity for a member of the molecular family relative to the molecular scaffold.
• a ligand binds non-covalently to a target molecule, which can preferably be a protein or enzyme.
• binding with “low affinity” is meant binding to the target molecule with a dissociation constant (ka) of greater than 1 ⁇ M under standard conditions.
• low affinity binding is in a range of 1 ⁇ M - 10 mM, 1 ⁇ M - 1 mM, 1 ⁇ M - 500 ⁇ M, 1 ⁇ M - 200 ⁇ M, 1 ⁇ M - 100 ⁇ M.
• binding with “very low affinity” is meant binding with a kd of above about 100 ⁇ M under standard conditions, e.g., in a range of 100 ⁇ M - 1 mM, 100 ⁇ M - 500 ⁇ M, 100 ⁇ M - 200 ⁇ M.
• binding with “extremely low affinity” is meant binding at a k of above about 1 mM under standard conditions.
• moderate affinity is meant binding with a k d of from about 200 nM to about 1 ⁇ M under standard conditions.
• moderately high affinity is meant binding at a k of from about 1 nM to about 200 nM.
• binding at “high affinity” is meant binding at a k d of below about 1 nM under standard conditions.
• low affinity binding can occur because of a poorer fit into the binding site of the target molecule or because of a smaller number of non-covalent bonds, or weaker covalent bonds present to cause binding of the scaffold or ligand to the binding site of the target molecule relative to instances where higher affinity binding occurs.
• the standard conditions for binding are at pH 7.2 at 37°C for one hour.
• 100 ⁇ l/well can be used in HEPES 50 mM buffer at pH 7.2, NaCI 15 mM, ATP 2 ⁇ M, and bovine serum albumin 1 ug/welb 37°C for one hour.
• Binding compounds can also be characterized by their effect on the activity of the target molecule.
• a “low activity” compound has an inhibitory concentration (IC 50 ) (for inhibitors or antagonists) or effective concentration (EC 50 ) (applicable to agonists) of greater than 1 ⁇ M under standard conditions.
• IC 50 inhibitory concentration
• EC 50 effective concentration
• very low activity is meant an IC 50 or EC 5 o of above 100 ⁇ M under standard conditions.
• extremely low activity is meant an IC 5 o or EC 5 o of above 1 mM under standard conditions.
• moderate activity is meant an IC 5 ⁇ or EC 50 of 200 nM to 1 ⁇ M under standard conditions.
• IC 50 or EC 50 of 1 nM to 200 nM By “moderately high activity” is meant an IC 50 or EC50 of below 1 nM under standard conditions.
• the IC 5 ⁇ (or EC 50 ) is defined as the concentration of compound at which 50% of the activity of the target molecule (e.g., enzyme or other protein) activity being measured is lost (or gained) relative to activity when no compound is present.
• Activity can be measured using methods known to those of ordinary skill in the art, e.g., by measuring any detectable product or signal produced by occurrence of an enzymatic reaction, or other activity by a protein being measured.
• activities can be determined as described in the Examples, or using other such assay methods known in the art.
• molecular scaffold or "scaffold” is meant a small target binding molecule to which one or more additional chemical moieties can be covalently attached, modified, or eliminated to form a plurality of molecules with common structural elements.
• the moieties can include, but are not limited to, a halogen atom, a hydroxyl group, a methyl group, a nitro group, a carboxyl group, or any other type of molecular group including, but not limited to, those recited in this application.
• Molecular scaffolds bind to at least one target molecule with low or very low affinity and/or bind to a plurality of molecules in a target family (e.g., protein family), and the target molecule is preferably an enzyme, receptor, or other protein.
• Preferred characteristics of a scaffold include molecular weight of less than about 350 daltons; binding at a target molecule binding site such that one or more substituents on the scaffold are situated in binding pockets in the target molecule binding site; having chemically tractable structures that can be chemically modified, particularly by synthetic reactions, so that a combinatorial library can be easily constructed; having chemical positions where moieties can be attached that do not interfere with binding of the scaffold to a protein binding site, such that the scaffold or library members can be modified to form ligands, to achieve additional desirable characteristics, e.g., enabling the ligand to be actively transported into cells and/or to specific organs, or enabling the ligand to be attached to a chromatography column for additional analysis.
• a molecular scaffold is
• scaffold core refers to the core structure of a molecular scaffold onto which various substituents can be attached.
• the scaffold core is common to all the scaffold molecules.
• the scaffold core will consist of or include one or more ring structures.
• scaffold group refers to a set of compounds that share a scaffold core and thus can all be regarded as derivatives of one scaffold molecule.
• molecular family groups of molecules classed together based on structural and/or functional similarities.
• molecular families include proteins, enzymes, polypeptides, receptor molecules, oligosaccharides, nucleic acids, DNA, RNA, etc.
• a protein family is a molecular family.
• Molecules can also be classed together into a family based on, for example, homology. The person of ordinary skill in the art will realize many other molecules that can be classified as members of a molecular family based on similarities in chemical structure or biological function.
• protein-ligand complex or “co-complex” is meant a protein and ligand bound non-covalently together.
• protein is meant a polymer of amino acids.
• the amino acids can be naturally or non-naturally occurring.
• Proteins can also contain adaptations, such as being glycosylated, phosphorylated, or other common modifications.
• protein family is meant a classification of proteins based on structural and/or functional similarities.
• kinases, phosphatases, proteases, and similar groupings of proteins are protein families. Proteins can be grouped into a protein family based on having one or more protein folds in common, a substantial similarity in shape among folds of the proteins, homology, or based on having a common function. In many cases, smaller families will be specified, e.g., the PPAR family.
• Protein folds are 3-dimensional shapes exhibited by the protein and defined by the existence, number, and location in the protein of alpha helices, beta-sheets, and loops, i.e., the basic secondary structures of protein molecules. Folds can be, for example, domains or partial domains of a particular protein.
• ring structure is meant a molecule having a chemical ring or sub-structure that is a chemical ring. In most cases, ring strutures will be carbocyclic or heterocyclic rings.
• the chemical ring may be, but is not limited to, a phenyl ring, aryl ring, pyrrole ring, imidazole, pyridine, purine, or any ring structure.
• specific biochemical effect is meant a therapeutically significant biochemical change in a biological system causing a detectable result.
• This specific biochemical effect can be, for example, the inhibition or activation of an enzyme, the inhibition or activation of a protein that binds to a desired target, or similar types of changes in the body's biochemistry.
• the specific biochemical effect can cause alleviation of symptoms of a disease or condition or another desirable effect.
• the detectable result can also be detected through an intermediate step.
• standard conditions conditions under which an assay is performed to obtain scientifically meaningful data.
• Standard conditions are dependent on the particular assay, and can be generally subjective. Normally the standard conditions of an assay will be those conditions that are optimal for obtaining useful data from the particular assay. The standard conditions will generally minimize background signal and maximize the signal sought to be detected.
• standard deviation is meant the square root of the variance.
• a “set” of compounds is meant a collection of compounds.
• the compounds may or may not be structurally related.
• target molecule is meant a molecule that a compound, molecular scaffold, or ligand is being assayed for binding to.
• the target molecule has an activity that binding of the molecular scaffold or ligand to the target molecule will alter or change.
• the binding of the compound, scaffold, or ligand to the target molecule can preferably cause a specific biochemical effect when it occurs in a biological system.
• a “biological system” includes, but is not limited to, a living system such as a human, animal, plant, or insect. In most but not all cases, the target molecule will be a protein or nucleic acid molecule.
• pharmacophore is meant a representation of molecular features that are considered to be responsible for a desired activity, such as interacting or binding with a receptor.
• a pharmacophore can include 3-dimensional (hydrophobic groups, charged/ionizable groups, hydrogen bond donors/acceptors), 2D (substructures), and ID . (physical or biological) properties.
• the present invention concerns the peroxisome proliferator-activated receptors (PPARs), which have been identified in humans and other mammals.
• PPARs peroxisome proliferator-activated receptors
• a group of compounds have been identified, corresponding to Formula I, that are active on one or more of the PPARs, in particular compounds that are active one or more human PPARs.
• the PPARs have been recognized as suitable targets for a number of different disease and conditions. Some of those applications are described briefly below. Additional applications are known and the present compounds can also be used for those diseases and conditions.
• PPAR ⁇ Insulin resistance and diabetes: In connection with insulin resistance and diabetes, PPAR ⁇ is necessary and sufficient for the differentiation of adipocytes in vitro and in vivo. In adipocytes, PPAR ⁇ increases the expression of numerous genes involved in lipid metabolism and lipid uptake. In contrast, PPAR ⁇ down-regulates leptin, a secreted, adipocyte-selective protein that has been shown to inhibit feeding and augment catabolic lipid metabolism. This receptor activity could explain the increased caloric uptake and storage noted in vivo upon treatment with PPAR ⁇ agonists.
• TZDs including troglitazone, rosiglitazone, and pioglitazone
• non-TZDs including farglitazar
• PPAR ⁇ has been associated with several genes that affect insulin action.
• TNF ⁇ a proinflammatory cytokine that is expressed by adipocytes
• PPAR ⁇ agonists inhibited expression of TNF ⁇ in adipose tissue of obese rodents, and ablated the actions of TNF ⁇ in adipocytes in vitro.
• PPAR ⁇ agonists were shown to inhibit expression of 11 ⁇ -hydroxysteroid dehydrogenase 1 (1 l ⁇ -HSD-1), the enzyme that converts cortisone to the glucocorticoid agonist cortisob in adipocytes and adipose tissue of type 2 diabetes mouse models.
• Adipocyte Complement-Related Protein of 30 kDa (Acrp30 or adiponectin) is a secreted adipocyte-specific protein that decreases glucose, trigiycerides, and free fatty acids.
• adipocyte-specific protein that decreases glucose, trigiycerides, and free fatty acids.
• PPAR ⁇ agonists Treatment of diabetic mice and nondiabetic human subjects with PPAR ⁇ agonists increased plasma levels of Acrp30. Induction of Acrp30 by PPAR ⁇ agonists might therefore also play a key role in the insulin-sensitizing mechanism of PPAR ⁇ agonists in diabetes. (Berger et ab, 2002, Diabetes Tech. And Ther. 4:163-174.)
• PPAR ⁇ is expressed predominantly in adipose tissue.
• the net in vivo efficacy of PPAR ⁇ agonists involves direct actions on adipose cells with secondary effects in key insulin responsive tissues such as skeletal muscle and liver. This is supported by the lack of glucose-lowering efficacy of rosiglitazone in a mouse model of severe insulin resistance where white adipose tissue was essentially absent.
• in vivo treatment of insulin resistant rats produces acute ( ⁇ 24 h) normalization of adipose tissue insulin action whereas insulin-mediated glucose uptake in muscle was not improved until several days after the initiation of therapy.
• PPAR ⁇ agonists can produce an increase in adipose tissue insulin action after direct in vitro incubation, whereas no such effect could be demonstrated using isolated in vitro incubated skeletal muscles.
• the beneficial metabolic effects of PPAR ⁇ agonists on muscle and liver may be mediated by their ability to (a) enhance insulin-mediated adipose tissue uptake, storage (and potentially catabolism) of free fatty acids; (b) induce the production of adipose-derived factors with potential insulin sensitizing activity (e.g., Acrp30); and/or (c) suppress the circulating levels and/or actions of insulin resistance-causing adipose- derived factors such as TNF ⁇ or resistin. (Berger et ab, 2002, Diabetes Tech. And Titer. 4:163-174.)
• (b) Dyslipidemia and atherosclerosis In connection with dyslipidemia and atherosclerosis, PPAR ⁇ has been shown to play a critical role in the regulation of cellular uptake, activation, and ⁇ -oxidation of fatty acids. Activation of PPAR ⁇ induces expression of fatty acid transport proteins and enzymes in the peroxisomal ⁇ -oxidation pathway. Several mitochondrial enzymes involved in the energy-harvesting catabolism of fatty acids are robustly upregulated by PPAR ⁇ agonists.
• Peroxisome proliferators also activate expression of the CYP4As, a subclass of cytochrome P450 enzymes that catalyze the ⁇ -hydroxylation of fatty acids, a pathway that is particularly active in the fasted and diabetic states.
• CYP4As a subclass of cytochrome P450 enzymes that catalyze the ⁇ -hydroxylation of fatty acids
• PPAR ⁇ is an important lipid sensor and regulator of cellular energy-harvesting metabolism.
• Atherosclerosis is a very prevalent disease in Westernized societies.
• "dyslipidemia” characterized by elevated triglyceride-rich particles and low levels of HDL cholesterol is commonly associated with other aspects of a metabolic syndrome that includes obesity, insulin resistance, type 2 diabetes, and an increased risk of coronary artery disease.
• dyslipidemia characterized by elevated triglyceride-rich particles and low levels of HDL cholesterol is commonly associated with other aspects of a metabolic syndrome that includes obesity, insulin resistance, type 2 diabetes, and an increased risk of coronary artery disease.
• 38% were found to have low HDL ( ⁇ 35 mg dL) and 33% had elevated trigiycerides (>200 mg/dL).
• treatment with fibrates resulted in substantial triglyceride lowering and modest HDL-raising efficacy.
• PPAR ⁇ agonists can effectively improve cardiovascular risk factors and have a net benefit to improve cardiovascular outcomes.
• fenofibrate was recently approved in the United States for treatment of type IIA and IIB hyper-lipidemia.
• Mechanisms by which PPAR ⁇ activation cause triglyceride lowering are likely to include the effects of agonists to suppress hepatic apo-CIII gene expression while also stimulating lipoprotein lipase gene expression.
• PPAR ⁇ and/or PPAR ⁇ expression in vascular cell types suggests that direct vascular effects might contribute to potential antiatherosclerosis efficacy.
• PPAR ⁇ and PPAR ⁇ activation have been shown to inhibit cytokine-induced vascular cell adhesion and to suppress monocyte-macrophage migration.
• PPAR ⁇ -selective compounds have the capacity to reduce arterial lesion size and attenuate monocyte-macrophage homing to arterial lesions in animal models of atherosclerosis.
• two recent studies have suggested that either PPAR ⁇ or PPAR ⁇ activation in macrophages can induce the expression of a cholesterol efflux "pump" protein.
• (c) Inflammation Monocytes and macrophages are known to play an important part in the inflammatory process through the release of inflammatory cytokines and the production of nitric oxide by inducible nitric oxide synthase. Rosiglitazone has been shown to induce apoptosis of macrophages at concentrations that paralleled its affinity for PPAR ⁇ . This ligand has also been show to block inflammatory cytokine synthesis in colonic cell lines. This latter observation suggests a mechanistic explanation for the observed anti-inflammatory actions of TZDs in rodent models of colitis.
• Hypertension is a complex disorder of the cardiovascular system that has been shown to be associated with insulin resistance.
• Type 2 diabetes patients demonstrate a 1.5-2-fold increase in hypertension in comparison with the general population.
• Troglitazone, rosiglitazone, and pioglitazone therapy have been shown to decrease blood pressure in diabetic patients as well as troglitazone therapy in obese, insulin-resistant subjects. Since such reductions in blood pressure were shown to correlate with decreases in insulin levels, they can be mediated by an improvement in insulin sensitivity.
• TZDs also lowered blood pressure in one-kidney one-clip Sprague Dawley rats, which are not insulin resistant
• hypotensive action of PPAR ⁇ agonists is not exerted solely through their ability to improve insulin sensitivity.
• Other mechanisms that have been invoked to explain the antihypertensive effects of PPAR ⁇ agonists include their ability to (a) downregulate expression of peptides that control vascular tone such as PAI-I, endothelin, and type-c natriuretic peptide C or (b) alter calcium concentrations and the calcium sensitivity of vascular cells. (Berger et ab, 2002, Diabetes Tech. And Ther. 4:163-174.)
• isoforms of the PPAR family of nuclear receptors are clearly involved in the systemic regulation of lipid metabolism and serve as "sensors" for fatty acids, prostanoid metabolites, eicosanoids and related molecules. These receptors function to regulate a broad array of genes in a coordinate fashion. Important biochemical pathways that regulate insulin action, lipid oxidation, lipid synthesis, adipocyte differentiation, peroxisome function, cell apoptosis, and inflammation can be modulated through the individual PPAR isoforms.
• PPAR agonists such as those described herein, can be used in the prophylaxix and or therapteutic treatment of a variety of different disease and conditions, such as obesity, overweight condition, hyperlipidemia, dyslipidemia including associated diabetic dyslipidemia and mixed dyslipidemia, hypoalphalipoproteinemia, Syndrome X, Type II diabetes mellitus, Type I diabetes, hyperinsulinemia, impaired glucose tolerance, insulin resistance, a diabetic complication (e.g., neuropathy, nephropathy, retinopathy or cataracts), hypertension, coronary heart disease, heart failure, hypercholesterolemia, inflammation, thrombosis, congestive heart failure, cardiovascular disease (including atherosclerosis, arteriosclerosis, and hypertriglyceridemia), epithelial hyperproliferative diseases (such as eczema and psoriasis), and conditions associated with the lung and gut and regulation of appetite and food intake in subjects suffering from disorders such as obesity, anorexia bulimia and
• (f) Weight Control Administration of PPAR ⁇ agonists can induce satiety, and thus are useful in weight loss or maintenance.
• PPAR ⁇ agonists can act preferentially on PPAR ⁇ , or can also act on another PPAR, or can be PPAR pan-agonists.
• the satiety inducing effect of PPAR ⁇ agonists can be used for weight control or loss.
• PPAR agonist compounds described by Formula I as provided in the Summary above. Included within Formula I are sub-groups of compounds, for example, sub-groups shown by the structures la, lb, lc, Id, X, and XIV as shown in the synthetic schemes below. Included within such compounds of Formula I, are exemplary compounds provided in Table 1 below. Additional compounds within Formula I can also be prepared and tested to confirm activity using conventional methods and the guidance provided herein.
• Structure-based modulator design and identification methods are powerful techniques that can involve searches of computer databases containing a wide variety of potential modulators and chemical functional groups.
• the computerized design and identification of modulators is useful as the computer databases contain more compounds than the chemical libraries, often by an order of magnitude.
• For reviews of structure-based drug design and identification see Kuntz et al. (1994), Aec. Chem. Res. 27:117; Guida (1994) Current Opinion in Struc. Biol. 4: 777; Colman (1994) Current Opinion in Struc. Biol. 4: 868).
• the three dimensional structure of a polypeptide defined by structural coordinates can be utilized by these design methods, for example, the structural coordinates of a PPAR.
• the three dimensional structures of PPARs determined by the homology, molecular replacement, and NMR techniques can also be applied to modulator design and identification methods.
• structural information for a PPAR in particular, structural information for the active site of the PPAR can be used.
• structural information for the active site of the PPAR can be used.
• modulator identification and design can, for example, be used to identify and/or develop additional active compounds within Formula I (a sub-group thereof).
• a typical computer program used for this purpose can perform a processes comprising the following steps or functions: (a) remove the existing compound from the protein; (b) dock the structure of another compound into the active-site using the computer program (such as DOCK) or by interactively moving the compound into the active-site; (c) characterize the space between the compound and the active-site atoms; (d) search libraries for molecular fragments which (i) can fit into the empty space between the compound and the active-site, and (ii) can be linked to the compound; and (e) link the fragments found above to the compound and evaluate the new modified compound.
• Part (c) refers to characterizing the geometry and the complementary interactions formed between the atoms of the active site and the compounds. A favorable geometric fit is attained when a significant surface area is shared between the compound and active-site atoms without forming unfavorable steric interactions.
• the method can be performed by skipping parts (d) and (e) and screening a database of many compounds.
• MCSS Miranker and Karplus, (1991), Proteins: Structure, Function, and Genetics 11:29.
• AUTODOCK Goodsell and Olson, (1990), Proteins: Structure, Function, and Genetics 8:195.
• GRID Goodford, (1985), J. Med. Chem. 28:849.
• Another way of identifying compounds as potential modulators is to modify an existing modulator in the polypeptide active site.
• the computer representation of modulators can be modified within the computer representation of a PPAR active site. Detailed instructions for this technique can be found, for example, in the Accel erys User Manual, 1995 in LUDI.
• the computer representation of the modulator is typically modified by the deletion of a chemical group or groups or by the addition of a chemical group or groups.
• the atoms of the modified compound and active site can be shifted in conformation and the distance between the modulator and the active-site atoms may be scored along with any complementary interactions formed between the two molecules. Scoring can be complete when a favorable geometric fit and favorable complementary interactions are attained. Compounds that have favorable scores are potential modulators.
• a third method of structure-based modulator design is to screen compounds designed by a modulator building or modulator searching computer program. Examples of these types of programs can be found in the Molecular Simulations Package, Catalyst. Descriptions for using this program are documented in the Molecular Simulations User Guide (1995). Other computer programs used in this application are ISIS/HOST, ISIS/BASE, ISIS/DRAW) from Molecular Designs Limited and UNITY from Tripos Associates.
• a modulator construction computer program is a computer program that may be used to replace computer representations of chemical groups in a compound complexed with a PPAR or other biomolecule with groups from a computer database.
• a modulator searching computer program is a computer program that may be used to search computer representations of compounds from a computer data base that have similar three dimensional structures and similar chemical groups as compound bound to a particular biomolecule.
• a typical program can operate by using the following general steps: (a) map the compounds by chemical features such as by hydrogen bond donors or acceptors, hydrophobic/lipophilic sites, positively ionizable sites, or negatively ionizable sites; (b) add geometric constraints to the mapped features; and (c) search databases with the model generated in (b). [0200] Those skilled in the art also recognize that not all of the possible chemical features of the compound need be present in the model of (b). One can use any subset of the model to generate different models for data base searches.
• a scaffold method for identifying and developing additional PPAR active compounds.
• a scaffold method can be applied using molecular scaffolds within Formula I, or having a scaffold core of Formula I, but can also be applied to other molecular scaffolds that are identified.
• the present invention also concerns methods for designing ligands active on PPARs by using structural information about the ligand binding sites and identified PPAR binding compounds. While such methods can be implemented in many ways (e.g., as described above), highly preferably the process utilizes molecular scaffolds. Such development processes and related methods are described generally below, and can, as indicated by applied to the PPARs, individually and/or in any pair, or as a family.
• Molecular scaffolds are low molecular weight molecules that bind with low or very low affinity to the target and typically have low or very low activity on that target and or act broadly across families of target molecules.
• the ability of a scaffold or other compound to act broadly across multiple members of a target family is advantageous in developing ligands.
• a scaffold or set of scaffolds can serve as starting compounds for developing ligands with desired specificity or with desired cross-activity on a selected subset of members of a target family.
• identification of a set of scaffolds that each bind with members of a target family provides an advantageous basis for selecting a starting point for ligand development for a particular target or subset of targets.
• the ability of a scaffold to bind to and/or have activity on multiple members of a target family is related to active site or binding site homology that exists across the target family.
• a scaffold active across multiple members of the target family interacts with surfaces or residues of relatively high homology, i.e., binds to conserved regions of the binding pockets. Scaffolds that bind with multiple members can be modified to provide greater specficity or to have a particular cross-reactivity, e.g., by exploiting differences between target binding sites to provide specificity, and exploiting similarities to design in cross-reactivities. Adding substituents that provide attractive interactions with the particular target typically increases the binding affinity, often increasing the activity.
• the various parts of the ligand development process are described in more detail in following sections, but the following describes an advantageous approach for scaffold-based ligand development.
• Scaffold-based ligand development can be implemented in a variety of ways, but large scale expression of protein is useful to provide material for crystallization, co-crystallization, and biochemical screening (e.g., binding and activity assays).
• crystallization crystallization conditions can be established for apo protein and a structure determined from those crystals.
• a biased library selected for the particular target family is screening for binding and/or activity on the target. Highly preferably a plurality of members from the target family is screened. Such screening, whether on a single target or on multiple members of a target family provides screening hits. Low affinity and/or low activity hits are selected.
• Such low affinity hits can either identify a scaffold molecule, or allow identification of a scaffold molecule by analyzing common features between binding molecules. Simpler molecules containing the common features can then be tested to determine if they retain binding and/or activity, thereby allowing identification of a scaffold molecule.
• the overlap in binding and/or activity of compounds can provide a useful selection for compounds that will be subjected to crystallization. For example, for 3 target molecules from a target family, if each target has about 200-500 hits in screening of a particular library, much smaller subsets of those hits will be common to any 2 of the 3 targets, and a still smaller subset will be common to all 3 targets, e.g., 100-300. In many cases, compounds in the subset common to all 3 targets will be selected for co-crystallography, as they provide the broadest potential for ligand development.
• co-crystallography conditions for forming co- crystals are determined, allowing determination of co-crystal structure and the orientation of binding compound in the binding site of the target is determined by solving the structure (this can be highly assisted if an apo protein crystal structure has been determined or if the structure of a close homolog is available for use in a homology model.
• the co-crystals are formed by direct co-crystallization rather than by soaking the compound into crystals of apo protein.
• a binding mode filter can, for example, be based on the demonstration of a dominant binding mode. That is, a scaffold or compounds of a scaffold group bind with a consistent orientation, preferably a consistent orientation across multiple members of a target family. Filtering scaffolds for multiple sites for substitution provides greater potential for developing ligands for specific targets due to the greater capacity for appropriately modifying the structure of the scaffold. Filtering for tractable chemistry also facilitates preparation of ligands derived from a scaffold because the synthetic paths for making derivative compounds are available. Carrying out such a process of development provides scaffolds, preferably of divergent structure.
• a surrogate target from the target family can be used. It is desirable to have the surrogate be as similar as possible to the desired target, thus a family member that has high homolgy in the binding site should be used, or the binding site can be modified to be more similar to that of the desired target, or part of the sequence of the desired target can be inserted in the family member replacing the corresponding part of the sequence of the family member.
• the scaffolds can be used to develop multiple products directed at specific members of the family, or at specific subsets of family members.
• derivative compounds ligands
• ligands can be designed and tested that have increasing selectivity.
• such ligands are typically developed to have greater activity, and will also typically have greater binding affinity.
• ligands are developed that have improved selectivity and activity profiles, leading to identification of lead compounds for drug development, leading to drug candidates, and final drug products.
• scaffolds and/or compound sets or libraries for scaffold or binding compound identification
• particular types of characteristics e.g., to select compounds that are more likely to bind to a particular target and/or to select compounds that have physical and/or synthetic properties to simplify preparation of derivatives, to be drug-like, and/or to provide convenient sites and chemistry for modification or synthesis.
• Useful chemical properties of molecular scaffolds can include one or more of the following characteristics, but are not limited thereto: an average molecular weight below about 350 daltons, or between from about 150 to about 350 daltons, or from about 150 to about 300 daltons; having a clogP below 3; a number of rotatable bonds of less than 4; a number of hydrogen bond donors and acceptors below 5 or below 4; a Polar Surface Area of less than 100 A 2 .; binding at protein binding sites in an orientation so that chemical substituents from a combinatorial library that are attached to the scaffold can be projected into pockets in the protein binding site; and possessing chemically tractable structures at its substituent attachment points that can be modified, thereby enabling rapid library construction.
• PSA Molecular Polar Surface Area
• Additional useful chemical properties of distinct compounds for inclusion in a combinatorial library include the ability to attach chemical moieties to the compound that will not interfere with binding of the compound to at least one protein of interest, and that will impart desirable properties to the library members, for example, causing the library members to be actively transported to cells and/or organs of interest, or the ability to attach to a device such as a chromatography column (e.g., a streptavidin column through a molecule such as biotin) for uses such as tissue and proteomics profiling purposes.
• a chromatography column e.g., a streptavidin column through a molecule such as biotin
• the present invention provides methods of designing ligands that bind to a plurality of members of a molecular family, where the ligands contain a common molecular scaffold.
• a compound set can be assayed for binding to a plurality of members of a molecular family, e.g., a protein family.
• One or more compounds that bind to a plurality of family members can be identified as molecular scaffolds.
• a set of ligands can be synthesized starting with one or a few molecular scaffolds to arrive at a plurality of ligands, wherein each ligand binds to a separate target molecule of the molecular family with altered or changed binding affinity or binding specificity relative to the scaffold.
• a plurality of drug lead molecules can be designed to individually target members of a molecular family based on the same molecular scaffold, and act on them in a specific manner.
• the methods of the present invention can involve assays that are able to detect the binding of compounds to a target molecule at a signal of at least about three times the standard deviation of the background signal, or at least about four times the standard deviation of the background signal.
• the assays can also include assaying compounds for low affinity binding to the target molecule.
• a large variety of assays indicative of binding are known for different target types and can be used for this invention. Compounds that act broadly across protein families are not likely to have a high affinity against individual targets, due to the broad nature of their binding.
• assays e.g., as described herein
• potency is not the primary, nor even the most important, indicia of identification of a potentially useful binding compound. Rather, even those compounds that bind with low affinity, very low affinity, or extremely low affinity can be considered as molecular scaffolds that can continue to the next phase of the ligand design process.
• the assays can preferably be enzymatic or binding assays. In some embodiments it may be desirable to enhance the solubility of the compounds being screened and then analyze all compounds that show activity in the assay, including those that bind with low affinity or produce a signal with greater than about three times the standard deviation of the background signal.
• These assays can be any suitable assay such as, for example, binding assays that measure the binding affinity between two binding partners.
• Various types of screening assays that can be useful in the practice of the present invention are known in the art, such as those described in U.S. Patent Nos. 5,763,198, 5,747,276, 5,877,007, 6,243,980, 6,294,330, and 6,294,330, each of which is hereby inco ⁇ orated by reference in its entirety, including all charts and drawings.
• At least one compound at least about 5%, at least about 10%>, at least about 15%, at least about 20%, or at least about 25% of the compounds can bind with low affinity.
• up to about 20%) of the compounds can show activity in the screening assay and these compounds can then be analyzed directly with high-throughput co-crystallography, computational analysis to group the compounds into classes with common structural properties (e.g., structural core and/or shape and polarity characteristics), and the identification of common chemical structures between compounds that show activity.
• Binding parameters can be measured using surface plasmon resonance, for example, with a BIAcore ® chip (Biacore, apan) coated with immobilized binding components.
• Surface plasmon resonance is used to characterize the microscopic association and dissociation constants of reaction between an sFv or other ligand directed against target molecules.
• Such methods are generally described in the following references which are inco ⁇ orated herein by reference. Nely F. et ab, BIAcore ® analysis to test phosphopeptide-SH2 domain interactions, Methods in Molecular Biology. 121:313- 21, 2000; Liparoto et ab, Biosensor analysis of the interleukin-2 receptor complex, Journal of Molecular Recognition.
• BIAcore ® uses the optical properties of surface plasmon resonance (SPR) to detect alterations in protein concentration bound to a dextran matrix lying on the surface of a gold/glass sensor chip interface, a dextran biosensor matrix.
• SPR surface plasmon resonance
• proteins are covalently bound to the dextran matrix at a known concentration and a ligand for the protein is injected through the dextran matrix.
• HTS typically uses automated assays to search through large numbers of compounds for a desired activity.
• HTS assays are used to find new drugs by screening for chemicals that act on a particular enzyme or molecule. For example, if a chemical inactivates an enzyme it might prove to be effective in preventing a process in a cell which causes a disease.
• High throughput methods enable researchers to assay thousands of different chemicals against each target molecule very quickly using robotic handling systems and automated analysis of results.
• high throughput screening or “HTS” refers to the rapid in vitro screening of large numbers of compounds (libraries); generally tens to hundreds of thousands of compounds, using robotic screening assays.
• Ultra high-throughput Screening generally refers to the high-throughput screening accelerated to greater than 100,000 tests per day.
• a multicontainer carrier facilitates measuring reactions of a plurality of candidate compounds simultaneously.
• Multi- well microplates may be used as the carrier.
• Screening assays may include controls for pu ⁇ oses of calibration and confirmation of proper manipulation of the components of the assay. Blank wells that contain all of the reactants but no member of the chemical library are usually included.
• a known inhibitor (or activator) of an enzyme for which modulators are sought can be incubated with one sample of the assay, and the resulting decrease (or increase) in the enzyme activity used as a comparator or control.
• modulators can also be combined with the enzyme activators or inhibitors to find modulators which inhibit the enzyme activation or repression that is otherwise caused by the presence of the known the enzyme modulator.
• known ligands of the target can be present in control/calibration assay wells.
• Spectrophotometric and spectrofluorometric assays are well known in the art. Examples of such assays include the use of colorimetric assays for the detection of peroxides, as described in Gordon, A. J. and Ford, R. A., The Chemist's Companion: A Handbook Of Practical Data, Techniques, And References, John Wiley and Sons, NN., 1972, Page 437.
• Fluorescence spectrometry may be used to monitor the generation of reaction products. Fluorescence methodology is generally more sensitive than the abso ⁇ tion methodology. The use of fluorescent probes is well known to those skilled in the art. For reviews, see Bashford et ab, Spectrophotometry and Spectrofluorometry: A Practical Approach, pp. 91-114, IRL Press Ltd. (1987); and Bell, Spectroscopy In Biochemistry, Vol. I, pp. 155-194, CRC Press (1981).
• SMase activity can be detected using the Amplex ® Red reagent (Molecular Probes, Eugene, OR). In order to measure sphingomyelinase activity using Amplex ® Red, the following reactions occur. First, SMase hydrolyzes sphingomyelin to yield ceramide and phosphorylcholine. Second, alkaline phosphatase hydrolyzes phosphorylcholine to yield choline.
• choline is oxidized by choline oxidase to betaine.
• H 2 O 2 in the presence of horseradish peroxidase, reacts with Amplex ® Red to produce the fluorescent product, Resorufin, and the signal therefrom is detected using spectrofluorometry.
• Fluorescence polarization is based on a decrease in the speed of molecular rotation of a fluorophore that occurs upon binding to a larger molecule, such as a receptor protein, allowing for polarized fluorescent emission by the bound ligand.
• FP is empirically determined by measuring the vertical and horizontal components of fluorophore emission following excitation with plane polarized light. Polarized emission is increased when the molecular rotation of a fluorophore is reduced.
• a fluorophore produces a larger polarized signal when it is bound to a larger molecule (i.e. a receptor), slowing molecular rotation of the fluorophore.
• the magnitude of the polarized signal relates quantitatively to the extent of fluorescent ligand binding. Accordingly, polarization of the "bound" signal depends on maintenance of high affinity binding.
• FP is a homogeneous technology and reactions are very rapid, taking seconds to minutes to reach equilibrium.
• the reagents are stable, and large batches may be prepared, resulting in high reproducibility. Because of these properties, FP has proven to be highly automatable, often performed with a single incubation with a single, premixed, tracer- receptor reagent.
• Owickiet ab Application of Fluorescence Polarization Assays in High-Throughput Screening, Genetic Engineering News, 17:27, 1997.
• FP is particularly desirable since its readout is independent of the emission intensity (Checovich, W. J., et ab, Nature 375:254-256, 1995; Dandliker, W. B., et ab, Methods in Enzymology 74:3-28, 1981) and is thus insensitive to the presence of colored compounds that quench fluorescence emission.
• FP and FRET are well-suited for identifying compounds that block interactions between sphingolipid receptors and their ligands. See, for example, Parker et ab, Development of high throughput screening assays using fluorescence polarization: nuclear receptor-ligand-binding and kinase/phosphatase assays, J Biomol Screen 5:77-88, 2000.
• Fluorophores derived from sphingolipids that may be used in FP assays are commercially available.
• Molecular Probes (Eugene, OR) currently sells sphingomyelin and one ceramide flurophores.
• N-(4,4-difluoro- 5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene- 3-pentanoyl)sphingosyl phosphocholine BODIPY® FL C5-sphingomyelin
• N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s- indacene- 3-dodecanoyl)sphingosyl phosphocholine BODIPY® FL C12-sphingomyelin
• N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene- 3-pentanoyl)sphingosine BODIPY ® FL C5-ceramide
• U.S. Patent No. 4,150,949 discloses fluorescein-labelled gentamicins, including fluoresceinthiocarbanyl gentamicin. Additional fluorophores may be prepared using methods well known to the skilled artisan.
• Exemplary normal-and-polarized fluorescence readers include the POLARION ® fluorescence polarization. system (Tecan AG, Hombrechtikon, Switzerland). General multiwell plate readers for other assays are available, such as the VERS AMAX ® reader and the SPECTRAMAX ® multiwell plate spectrophotometer (both from Molecular Devices).
• Fluorescence resonance energy transfer is another useful assay for detecting interaction and has been described. See, e.g., Heim et ab, Curr. Biol. 6:178-182, 1996; Mitra et ab, Gene 173:13-17 1996; and Selvin et ab, Meth. Enzymol 246:300-345, 1995.
• FRET detects the transfer of energy between two fluorescent substances in close proximity, having known excitation and emission wavelengths.
• a protein can be expressed as a fusion protein with green fluorescent protein (GFP).
• GFP green fluorescent protein
• the resonance energy can be transferred from one excited molecule to the other.
• the emission spectrum of the sample shifts, which can be measured by a fluorometer, such as a fMAX multiwell fluorometer (Molecular Devices, Sunnyvale Calif).
• SPA Scintillation proximity assay
• SPA is a particularly useful assay for detecting an interaction with the target molecule.
• SPA is widely used in the pharmaceutical industry and has been described (Hanselman et ab, J. Lipid Res. 38:2365-2373 (1997); Kahl et ab, Anal. Biochem. 243:282-283 (1996); Undenfriend et al, Anal. Biochem. 161:494-500 (1987)). See also U.S. Patent Nos. 4,626,513 and 4,568,649, and European Patent No. 0,154,734.
• FLASHPLATE ® scintillant-coated plates NN Life Science Products, Boston, MA).
• the target molecule can be bound to the scintillator plates by a variety of well known means. Scintillant plates are available that are derivatized to bind to fusion proteins such as GST, His6 or Flag fusion proteins. Where the target molecule is a protein complex or a multimer, one protein or subunit can be attached to the plate first, then the other components of the complex added later under binding conditions, resulting in a bound complex.
• the gene products in the expression pool will have been radiolabeled and added to the wells, and allowed to interact with the solid phase, which is the immobilized target molecule and scintillant coating in the wells.
• the assay can be measured immediately or allowed to reach equilibrium. Either way, when a radiolabel becomes sufficiently close to the scintillant coating, it produces a signal detectable by a device such as a TOPCOUNT NXT ® microplate scintillation counter (Packard BioScience Co., Meriden Conn.). If a radiolabeled expression product binds to the target molecule, the radiolabel remains in proximity to the scintillant long enough to produce a detectable signal.
• the assay can utilize AlphaScreen (amplified /uminescent proximity homogeneous assay) format, e.g., AlphaScreening system (Packard BioScience). AlphaScreen is generally described in Seethala and Prabhavathi, Homogenous Assays: AlphaScreen, Handbook of Drug; Screening, Marcel Dekkar Pub. 2001, pp. 106-110. Applications of the technique to PPAR receptor ligand binding assays are described, for example, in Xu et ab, 2002, Nature 415:813-817.
• a scaffold include being of low molecular weight (e.g., less than 350 Da, or from about 100 to about 350 daltons, or from about 150 to about 300 daltons).
• clog P of a scaffold is from -1 to 8, more preferably less than 6, 5, or 4, most preferably less than 3.
• the clogP is in a range -1 to an upper limit of 2, 3, 4, 5, 6, or 8; or is in a range of 0 to an upper limit of 2,3, 4, 5, 6, or 8.
• the number of rotatable bonds is less than 5, more preferably less than 4.
• the number of hydrogen bond donors and acceptors is below 6, more preferably below 5.
• An additional criterion that can be useful is a Polar Surface Area of less than 100.
• Guidance that can be useful in identifying criteria for a particular application can be found in Lipinski et ab, Advanced Drug Delivery Reviews 23 (1997) 3-25, which is hereby inco ⁇ orated by reference in its entirety.
• a scaffold will preferably bind to a given protein binding site in a configuration that causes substituent moieties of the scaffold to be situated in pockets of the protein binding site. Also, possessing chemically tractable groups that can be chemically modified, particularly through synthetic reactions, to easily create a combinatorial library can be a preferred characteristic of the scaffold. Also preferred can be having positions on the scaffold to which other moieties can be attached, which do not interfere with binding of the scaffold to the protein(s) of interest but do cause the scaffold to achieve a desirable property, for example, active transport of the scaffold to cells and/or organs, enabling the scaffold to be attached to a chromatographic column to facilitate analysis, or another desirable property.
• a molecular scaffold can bind to a target molecule with any affinity, such as binding with an affinity measurable as about three times the standard deviation of the background signal, or at high affinity, moderate affinity, low affinity, very low affinity, or extremely low affinity.
• the above criteria can be utilized to select many compounds for testing that have the desired attributes.
• Many compounds having the criteria described are available in the commercial market, and may be selected for assaying depending on the specific needs to which the methods are to be applied. In some cases sufficiently large numbers of compounds may meet specific criteria that additional methods to group similar compounds may be helpful.
• a variety of methods to assess molecular similarity, such as the Tanimoto coefficient have been used, see Willett et al, Journal of Chemical Information and Computer Science 38 (1998), 983-996. These can be used to select a smaller subset of a group of highly structurally redundant compounds.
• cluster analysis based on relationships between the compounds, or structural components of the compound can also be carried out to the same end; see Lance and Williams Computer Journal 9 (1967) 373- 380, Jarvis and Patrick IEEE Transactions in Computers C-22 (1973) 1025-1034 for clustering algorithms, and Downs et ab Journal of Chemical Information and Computer Sciences 34 (1994) 1094-1102 for a review of these methods applied to chemical problems.
• One method of deriving the chemical components of a large group of potential scaffolds is to virtually break up the compound at rotatable bonds so as to yield components of no less than 10 atoms.
• the resulting components may be clustered based on some measure of similarity, e.g. the Tanimoto coefficient, to yield the common component groups in the original collection of compounds. For each component group, all compounds containing that component may be clustered, and the resulting clusters used to select a diverse set of compounds containing a common chemical core structure. In this fashion, a useful library of scaffolds may be derived even from millions of commercial compounds.
• a “compound library” or “library” is a collection of different compounds having different chemical structures.
• a compound library is screenable, that is, the compound library members therein may be subject to screening assays.
• the library members can have a molecular weight of from about 100 to about 350 daltons, or from about 150 to about 350 daltons.
• Libraries can contain at least one compound that binds to the target molecule at low affinity.
• Libraries of candidate compounds can be assayed by many different assays, such as those described above, e.g., a fluorescence polarization assay.
• Libraries may consist of chemically synthesized peptides, peptidomimetics, or arrays of combinatorial chemicals that are large or small, focused or nonfocused.
• focused it is meant that the collection of compounds is prepared using the stracture of previously characterized compounds and/or pharmacophores.
• Compound libraries may contain molecules isolated from natural sources, artificially synthesized molecules, or molecules synthesized, isolated, or otherwise prepared in such a manner so as to have one or more moieties variable, e.g., moieties that are independently isolated or randomly synthesized.
• moieties variable e.g., moieties that are independently isolated or randomly synthesized.
• Types of molecules in compound libraries include but are not limited to organic compounds, polypeptides and nucleic acids as those terms are used herein, and derivatives, conjugates and mixtures thereof.
• Compound libraries useful for the invention may be purchased on the commercial market or prepared or obtained by any means including, but not limited to, combinatorial chemistry techniques, fermentation methods, plant and cellular extraction procedures and the like (see, e.g., Cwirla et ab, Biochemistry 1990, 87, 6378-6382; Houghten et ab, Nature 1991, 354, 84-86; Lam et ab, Nature 1991, 354, 82-84; Brenner et ab, Proc. Natl Acad. Sci. USA 1992, 89, 5381-5383; R. A. Houghten, Trends Genet. 1993, 9, 235-239; E. R Felder, Chimia 1994, 48, 512-541; Gallop et al., J. Med.
• Preferred libraries can be prepared in a homogenous reaction mixture, and separation of unreacted reagents from members of the library is not required prior to screening.
• combinatorial chemistry approaches are based on solid state chemistry, liquid phase combinatorial chemistry is capable of generating libraries (Sun CM., Recent advances in liquid-phase combinatorial chemistry, Combinatorial Chemistry & High Throughput Screening. 2:299-318, 1999).
• Libraries of a variety of types of molecules are prepared in order to obtain members therefrom having one or more preselected attributes that can be prepared by a variety of techniques, including but not limited to parallel array synthesis (Houghton, Annu Rev Pharmacol Toxicol 2000 40:273-82, Parallel array and mixture-based synthetic combinatorial chemistry; solution-phase combinatorial chemistry (Merritb Comb Chem High Throughput Screen 1998 l(2):57-72, Solution phase combinatorial chemistry, Coe et ab, Mol Divers 1998-99;4(l):31-8, Solution-phase combinatorial chemistry, Sun, Comb Chem High Throughput Screen 1999 2(6):299-318, Recent advances in liquid-phase combinatorial chemistry); synthesis on soluble polymer (Gravert et ab, Curr Opin Chem Biol 1997 1(1): 107- 13, Synthesis on soluble polymers: new reactions and the construction of small molecules); and the like.
• nucleic acids are prepared by various techniques, including by way of non-limiting example the ones described herein, for the isolation of aptamers.
• Libraries that include oligonucleotides and polyaminooligonucleotides (Markiewicz et ab, Synthetic oligonucleotide combinatorial libraries and their applications, Farmaco. 55:1 '4-7, 2000) displayed on streptavidin magnetic beads are known.
• Nucleic acid libraries are known that can be coupled to parallel sampling and be deconvoluted without complex procedures such as automated mass spectrometry (Enjalbal C. Martinez J. Aubagnac JL, Mass spectrometry in combinatorial chemistry, Mass Spectrometry Reviews. 19:139-61, 2000) and parallel tagging. (Perrin DM., Nucleic acids for recognition and catalysis: landmarks, limitations, and looking to the future, Combinatorial Chemistry & High Throughput Screening 3:243-69).
• Peptidomimetics are identified using combinatorial chemistry and solid phase synthesis (Kim HO. Kahn M., A merger of rational drug design and combinatorial chemistry: development and application of peptide secondary structure mimetics, Combinatorial Chemistry 8c High Throughput Screening 3:167-83, 2000; al-Obeidi, Mol Biotechnol 1998 9(3):205-23, Peptide and peptidomimetric libraries. Molecular diversity and drug design). The synthesis may be entirely random or based in part on a known polypeptide.
• Polypeptide libraries can be prepared according to various techniques.
• phage display techniques can be used to produce polypeptide ligands (Gram H., Phage display in proteolysis and signal transduction, Combinatorial Chemistry 8c High Throughput Screening. 2:19-28, 1999) that may be used as the basis for synthesis of peptidomimetics.
• Polypeptides, constrained peptides, proteins, protein domains, antibodies, single chain antibody fragments, antibody fragments, and antibody combining regions are displayed on filamentous phage for selection.
• a group of scaffolds exhibiting binding to two or more members of a target protein family will contain scaffolds with a greater likelihood that such binding results from specific interactions with the individual target proteins. This would be expected to substantially reduce the effect of so-called "promiscuous inhibitors" which severely complicate the inte ⁇ retation of screening assays (see McGovern et al Journal of Medicinal Chemistry 45: 1712-22, 2002).
• the property of displaying binding to multiple target molecules in a protein family may be used as a selection criteria to identify molecules with desirable properties.
• groups of scaffolds binding to specific subsets of a set of potential target molecules may be selected. Such a case would include the subset of scaffolds that bind to any two of three or three of five members of a target protein family.
• Such subsets may also be used in combination or opposition to further define a group of scaffolds that have additional desirable properties. This would be of significant utility in cases where inhibiting some members of a protein family had known desirable effects, such as inhibiting tumor growth, whereas inhibiting other members of the protein family which were found to be essential for normal cell function would have undesirable effects.
• a criteria that would be useful in such a case includes selecting the subset of scaffolds binding to any two of three desirable target molecules and eliminating from this group any that bound to more than one of any three undesirable target molecules.
• the orientation of compound bound to target is determined.
• this determination involves crystallography on co-crystals of molecular scaffold compounds with target.
• Most protein crystallographic platforms can preferably be designed to analyze up to about 500 co-complexes of compounds, ligands, or molecular scaffolds bound to protein targets due to the physical parameters of the instruments and convenience of operation.
• the scaffolds can be placed into groups based on having at least one common chemical structure or other desirable characteristics, and representative compounds can be selected from one or more of the classes.
• Classes can be made with increasingly exacting criteria until a desired number of classes (e.g., 10, 20, 50, 100, 200, 300, 400, 500) is obtained.
• the classes can be based on chemical structure similarities between molecular scaffolds in the class, e.g., all possess a pyrrole ring, benzene ring, or other chemical feature.
• classes can be based on shape characteristics, e.g., space-filling characteristics.
• the co-crystallography analysis can be performed by co-complexing each scaffold with its target, e.g., at concentrations of the scaffold that showed activity in the screening assay.
• This co-complexing can, for example, be accomplished with the use of low percentage organic solvents with the target molecule and then concentrating the target with each of the scaffolds.
• these solvents are less than 5% organic solvent such as dimethyl sulfoxide (DMSO), ethanol, methanol, or ethylene glycol in water or another aqueous solvent.
• Each scaffold complexed to the target molecule can then be screened with a suitable number of crystallization screening conditions at appropriate temperature, e.g., both 4 and 20 degrees. In preferred embodiments, about 96 crystallization screening conditions can be performed in order to obtain sufficient information about the co- complexation and crystallization conditions, and the orientation of the scaffold at the binding site of the target molecule. Crystal structures can then be analyzed to determine how the bound scaffold is oriented physically within the binding site or within one or more binding pockets of the molecular family member.
• the structure of the target molecule bound to the compound may also be superimposed or aligned with other structures of members of the same protein family.
• modifications of the scaffold can be made to enhance the binding to members of the target family in general, thus enhancing the utility of the scaffold library.
• Different useful alignments may be generated, using a variety of criteria such as minimal RMSD supe ⁇ osition of alpha-carbons or backbone atoms of homologous or structurally related regions of the proteins.
• Standard X-ray protein diffraction studies such as by using a Rigaku RU-200 ® (Rigaku, Tokyo, Japan) with an X-ray imaging plate detector or a synchrotron beam-line can be performed on co-crystals and the diffraction data measured on a standard X-ray detector, such as a CCD detector or an X-ray imaging plate detector.
• Performing X-ray crystallography on about 200 co-crystals should generally lead to about 50 co-crystal structures, which should provide about 10 scaffolds for validation in chemistry, which should finally result in about 5 selective leads for target molecules.
• Additives that promote co-crystallization can of course be included in the target molecule formulation in order to enhance the formation of co-crystals.
• the scaffold to be tested can be added to the protein formulation, which is preferably present at a concentration of approximately 1 mg/ml.
• the formulation can also contain between 0%>-10% (v/v) organic solvent, e.g. DMSO, methanol, ethanol, propane diol, or 1,3 dimethyl propane diol (MPD) or some combination of those organic solvents.
• Compounds are preferably solubilized in the organic solvent at a concentration of about 10 mM and added to the protein sample at a concentration of about 100 mM.
• the protein-compound complex is then concentrated to a final concentration of protein of from about 5 to about 20 mg/ml.
• the complexation and concentration steps can conveniently be performed using a 96 well formatted concentration apparatus (e.g., Amicon Inc., Piscataway, NJ).
• Buffers and other reagents present in the formulation being crystallized can contain other components that promote crystallization or are compatible with crystallization conditions, such as DTT, propane diol, glycerol.
• the crystallization experiment can be set-up by placing small aliquots of the concentrated protein-compound complex (e.g., 1 ⁇ l) in a 96 well format and sampling under 96 crystallization conditions. (Other formats can also be used, for example, plates with fewer or more wells.) Crystals can typically be obtained using standard crystallization protocols that can involve the 96 well crystallization plate being placed at different temperatures. Co-crystallization varying factors other than temperature can also be considered for each protein-compound complex if desirable. For example, atmospheric pressure, the presence or absence of light or oxygen, a change in gravity, and many other variables can all be tested. The person of ordinary skill in the art will realize other variables that can advantageously be varied and considered. Conveniently, commercially available crystal screening plates with specified conditions in individual wells can be utilized.
• illustrations can also be used to decide where chemical groups might be added, substituted, modified, or deleted from the scaffold to enhance binding or another desirable effect, by considering where unoccupied space is located in the complex and which chemical substructures might have suitable size and/or charge characteristics to fill it.
• regions within the binding site can be flexible and its properties can change as a result of scaffold binding, and that chemical groups can be specifically targeted to those regions to achieve a desired effect.
• Specific locations on the molecular scaffold can be considered with reference to where a suitable chemical substructure can be attached and in which conformation, and which site has the most advantageous chemistry available.
• ⁇ Gbind ⁇ Gtr + ⁇ Ghb + ⁇ Gion + ⁇ Glipo + ⁇ Garom + ⁇ Grot
• ⁇ Gtr is a constant term that accounts for the overall loss of rotational and translational entropy of the lignand
• ⁇ Ghb accounts for hydrogen bonds formed between the ligand and protein
• ⁇ Gion accounts for the ionic interactions between the ligand and protein
• ⁇ Glipo accounts for the lipophilic interaction that corresponds to the protein- ligand contact surface
• ⁇ Garom accounts for interactions between aromatic rings in the protein and ligand
• ⁇ Grot accounts for the entropic penalty of restricting rotatable bonds in the ligand upon binding.
• the calculated binding energy for compounds that bind strongly to a given target will likely be lower than -25 kcal mol, while the calculated binding affinity for a good scaffold or an unoptimized compound will generally be in the range of -15 to -20.
• the penalty for restricting a linker such as the ethylene glycol or hexatriene is estimated as typically being in the range of +5 to +15.
• This method estimates the free energy of binding that a lead compound should have to a target protein for which there is a crystal structure, and it accounts for the entropic penalty of flexible linkers. It can therefore be used to estimate the penalty incurred by attaching linkers to molecules being screened and the binding energy that a lead compound must attain in order to overcome the penalty of the linker.
• the method does not account for solvation, and the entropic penalty is likely overestimated when the linkers are bound to the solid phase through an additional binding complex, e.g., a biotin: streptavidin complex.
• Another exemplary method for calculating binding energies is the MM-PBSA technique (Massova and Kollman, Journal of the American Chemical Society 121:8133- 43,1999; Chong et al, Proceedings of theNational Academy of Sciences 96:14330-5,1999; Donini and Kollman, Journal of Medicinal Chemistry 43:4180-8,2000).
• This method uses a Molecular Dynamics approach to generate many sample configurations of the compound and complexed target molecule, then calculates an interaction energy using the well- known AMBER force field (Cornell, et al Journal of the American Chemical Society 117:5179-97 1995) with corrections for desolvation and entropy of binding from the ensemble.
• binding energies highly correlated with those found experimentally The absolute binding energies calculated with this method are reasonably accurate, and the variation of binding energies is approximately linear with a slope of 1+/- 0.5.
• the binding energies of compounds interacting strongly with a given target will be lower than about -8 kcal/mob while a binding energy of a good scaffold or unoptimized compound will be in the range of -3 to -7 kcal/mob
• Computer models such as homology models (i.e., based on a known, experimentally derived stracture) can be constructed using data from the co-crystal structures.
• a computer program such as Modeller (Accelrys, San Diego CA) may be used to assign the three dimensional coordinates to a protein sequence using an alignment of sequences and a set or sets of template coordinates.
• preferred co-crystal structures for making homology models contain high sequence identity in the binding site of the protein sequence being modeled, and the proteins will preferentially also be within the same class and/or fold family.
• Knowledge of conserved residues in active sites of a protein class can be used to select homology models that accurately represent the binding site.
• Homology models can also be used to map structural information from a surrogate protein where an apo or co-crystal stracture exists to the target protein.
• Virtual screening methods such as docking, can also be used to predict the binding configuration and affinity of scaffolds, compounds, and/or combinatorial library members to homology models.
• Using this data, and carrying out "virtual experiments" using computer software can save substantial resources and allow the person of ordinary skill to make decisions about which compounds can be suitable scaffolds or ligands, without having to actually synthesize the ligand and perform co-crystallization. Decisions thus can be made about which compounds merit actual synthesis and co-crystallization.
• An understanding of such chemical interactions aids in the discovery and design of drags that interact more advantageously with target proteins and/or are more selective for one protein family member over others. Thus, applying these principles, compounds with superior properties can be discovered.
• the design and preparation of ligands can be performed with or without structural and/or co-crystallization data by considering the chemical structures in common between the active scaffolds of a set.
• structure-activity hypotheses can be formed and those chemical structures found to be present in a substantial number of the scaffolds, including those that bind with low affinity, can be presumed to have some effect on the binding of the scaffold. This binding can be presumed to induce a desired biochemical effect when it occurs in a biological system (e.g., a treated mammal).
• New or modified scaffolds or combinatorial libraries derived from scaffolds can be tested to disprove the maximum number of binding and/or structure-activity hypotheses. The remaining hypotheses can then be used to design ligands that achieve a desired binding and biochemical effect.
• co-crystallography data shows the binding pocket of the protein with the molecular scaffold bound to the binding site, and it will be apparent that a modification can be made to a chemically tractable group on the scaffold.
• a small volume of space at a protein binding site or pocket might be filled by modifying the scaffold to include a small chemical group that fills the volume. Filling the void volume can be expected to result in a greater binding affinity, or the loss of undesirable binding to another member of the protein family.
• the co-crystallography data may show that deletion of a chemical group on the scaffold may decrease a hindrance to binding and result in greater binding affinity or specificity.
• Molecular Interaction Field-computing techniques such as those implemented in the program GRID, result in energy data for particular positive and negative binding interactions of different computational chemical probes being mapped to the vertices of a matrix in the coordinate space of the target molecule. These data can then be analyzed for areas of substitution around the scaffold binding site which are predicted to have a favorable interaction for a particular target molecule.
• Compatible chemical substitution on the scaffold e.g. a methyl, ethyl or phenyl group in a favorable interaction region computed from a hydrophobic probe, would be expected to result in an improvement in affinity of the scaffold.
• a scaffold could be made more selective for a particular target molecule by making such a substitution in a region predicted to have an unfavorable hydrophobic interaction in a second, related undesirable target molecule.
• a positively charged group can be complemented with a negatively charged group introduced on the molecular scaffold. This can be expected to increase binding affinity or binding specificity, thereby resulting in a more desirable ligand.
• regions of protein binding sites or pockets are known to vary from one family member to another based on the amino acid differences in those regions.
• Chemical additions in such regions can result in the creation or elimination of certain interactions (e.g., hydrophobic, electrostatic, or entropic) that allow a compound to be more specific for one protein target over another or to bind with greater affinity, thereby enabling one to synthesize a compound with greater selectivity or affinity for a particular family member.
• certain regions can contain amino acids that are known to be more flexible than others. This often occurs in amino acids contained in loops connecting elements of the secondary stracture of the protein, such as alpha helices or beta strands. Additions of chemical moieties can also be directed to these flexible regions in order to increase the likelihood of a specific interaction occurring between the protein target of interest and the compound.
• Virtual screening methods can also be conducted in s ⁇ lico to assess the effect of chemical additions, subtractions, modifications, and/or substitutions on compounds with respect to members of a protein family or class.
• a chemical stracture or sub-structure to a scaffold can be performed with any suitable chemical moiety.
• moieties which are provided by way of example and are not intended to be limiting, can be utilized: hydrogen, alkyb alkoxy, phenoxy, alkenyb alkynyl, phenylalkyb hydroxyalkyl, haloalkyb aryb arylalkyb alkyloxy, alkylthio, alkenylthio, phenyb phenylalkyb phenylalkylthio, hydroxyalkyl-thio, alkylthiocarbbamylthio, cyclohexyl, pyridyl, piperidinyb alkylamino, amino, nitro, mercapto, cyano, hydroxyl, a halogen atom, halomethyb an oxygen atom (e.g., forming a ketone or
• Additional examples of structures or sub-structures that may be utilized are an aryl optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyb carboxylate, nitro, and ester moieties; an amine of formula -NX 2 X 3 , where X 2 and X 3 are independently selected from the group consisting of hydrogen, saturated or unsaturated alkyb and homocyclic or heterocyclic ring moieties; halogen or trihalomethyb a ketone of formula -COX , where X 4 is selected from the group consisting of alkyl and homocyclic or heterocyclic ring moieties; a carboxylic acid of formula -(X 5 ) n COOH or ester of formula (X 6 ) n COOX , where X 5 , X 6 , and X 7 and are independently selected from the group consisting of alkyl and homocyclic or heterocycl
• the binding energy with the attachment should be at least 4 kcal/mob, more preferably at least 6, 8, 10, 12, 15, or 20 kcal/mob
• the presence of the attachment at the particular site reduces binding energy by no more than 3, 4, 5, 8, 10, 12, or 15 kcal/mob
• attachment sites will be those that are exposed to solvent when the binding compound is bound in the binding site.
• attachment sites can be used that will result in small displacements of a portion of the enzyme without an excessive energetic cost.
• Exposed sites can be identified in various ways. For example, exposed sites can be identified using a graphic display or 3-dimensional model. In a grahic display, such as a computer display, an image of a compound bound in a binding site can be visually inspected to reveal atoms or groups on the compound that are exposed to solvent and oriented such that attachment at such atom or group would not preclude binding of the enzyme and binding compound. Energetic costs of attachment can be calculated based on changes or distortions that would be caused by the attachment as well as entropic changes.
• components can be attached. Persons with skill are familiar with the chemistries used for various attachments. Examples of components that can be attached include, without limitation: solid phase components such as beads, plates, chips, and wells; a direct or indirect label; a linker, which may be a traceless linker; among others. Such linkers can themselves be attached to other components, e.g., to solid phase media, labels, and/or binding moieties.
• the binding energy of a compound and the effects on binding energy for attaching the molecule to another component can be calculated approximately by manual calculation, or by using any of a variety of available computational virtual assay techniques, such as docking or molecular dynamics simulations.
• a virtual library of compounds derived from the attachment of components to a particular scaffold can be assembled using a variety of software programs (such as Afferent, MDL Information Systems, San Leandro, CA or CombiLibMaker, Tripos Associates, St. Louis, MO). This virtual library can be assigned appropriate three dimensional coordinates using software programs (such as Concord, Tripos Associates, St. Louis, MO or Omega, Openeye Scientific Software, Santa Fe, NM). These structures may then be submitted to the appropriate computational technique for evaluation of binding energy to a particular target molecule. This information can be used for purposes of prioritizing compounds for synthesis, for selecting a subset of chemically tractable compounds for synthesis, and for providing data to correlate with the experimentally determined binding energies for the synthesized compounds.
• the crystallographic determination of the orientation of the scaffold in the binding site specifically enables more productive methods of assessing the likelihood of the attachment of a particular component resulting in an improvement in binding energy.
• Such an example is shown for a docking-based strategy in Haque et al Journal of Medicinal Chemistry 42:1428-40, 1999, wherein an "Anchor and Grow" technique which relied on a crystallographically determined fragment of a larger molecule, potent and selective inhibitors were rapidly created.
• the use of a crystallographically characterized small molecule fragment in guiding the selection of productive compounds for synthesis has also been demonstrated in Boehm et al, Journal of Medicinal Chemistry 43:2664-74, 2000.
• Linkers suitable for use in the invention can be of many different types. Linkers can be selected for particular applications based on factors such as linker chemistry compatible for attachment to a binding compound and to another component utilized in the particular application. Additional factors can include, without limitation, linker length, linker stability, and ability to remove the linker at an appropriate time. Exemplary linkers include, but are not limited to, hexyl, hexatrienyl, ethylene glycob and peptide linkers. Traceless linkers can also be used, e.g., as described in Plunkett, M. J., and Ellman, J. A., 1995, /. Org. Chem., 60:6006.
• Typical functional groups, that are utilized to link binding compound(s), include, but not limited to, carboxylic acid, amine, hydroxyl, and thiol. (Examples can be found in Solid-supported combinatorial and parallel synthesis of small molecular weight compound libraries; Tetrahedron organic chemistry series Vol.17; Pergamon, 1998; p85).
• labels can also be attached to a binding compound or to a linker attached to a binding compound. Such attachment may be direct (attached directly to the binding compound) or indirect (attached to a component that is directly or indirectly attached to the binding compound). Such labels allow detection of the compound either directly or indirectly. Attachment of labels can be performed using conventional chemistries. Labels can include, for example, fluorescent labels, radiolabels, light scattering particles, light absorbent particles, magnetic particles, enzymes, and specific binding agents (e.g., biotin or an antibody target moiety).
• solid phase media Similar to attachment of linkers and labels, attachment to solid phase media can be performed using conventional chemistries.
• Such solid phase media can include, for example, small components such as beads, nanoparticles, and fibers (e.g., in suspension or in a gel or chromatographic matrix).
• solid phase media can include larger objects such as plates, chips, slides, and tubes.
• the binding compound will be attached in only a portion of such an objects, e.g., in a spot or other local element on a generally flat surface or in a well or portion of a well.
• Suitable dosage forms in part, depend upon the use or the route of administration, for example, oral, transdermal, transmucosal, or by injection (parenteral). Such dosage forms should allow the compound to reach target cells. Other factors are well known in the art, and include considerations such as toxicity and dosage forms that retard the compound or composition from exerting its effects. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, 18 th ed., Mack Publishing Co., Easton, PA, 1990 (hereby inco ⁇ orated by reference herein).
• Compounds can be formulated as pharmaceutically acceptable salts.
• Pharmaceutically acceptable salts are non-toxic salts in the amounts and concentrations at which they are administered. The preparation of such salts can facilitate the pharmacological use by altering the physical characteristics of a compound without preventing it from exerting its physiological effect. Useful alterations in physical properties include lowering the melting point to facilitate transmucosal administration and increasing the solubility to facilitate administering higher concentrations of the drag.
• Pharmaceutically acceptable salts include acid addition salts such as those containing sulfate, chloride, hydrochloride, fumarate, maleate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p- toluenesulfonate, cyclohexylsulfamate and quinate.
• acid addition salts such as those containing sulfate, chloride, hydrochloride, fumarate, maleate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p- toluenesulfonate, cyclohexylsulfamate and quinate.
• Pharmaceutically acceptable salts can be obtained from acids such as hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid,/?-toluenesulfonic acid, cyclohexylsulfamic acid, fumaric acid, and quinic acid.
• acids such as hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid,/?-toluenesulfonic acid, cyclohexylsulfamic acid, fumaric acid, and quinic acid.
• Pharmaceutically acceptable salts also include basic addition salts such as those containing benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alkylamine, and zinc, when acidic functional groups, such as carboxylic acid or phenol are present.
• basic addition salts such as those containing benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alkylamine, and zinc.
• acidic functional groups such as carboxylic acid or phenol are present.
• Such salts can be prepared using the appropriate corresponding bases.
• salts can be prepared by standard techniques. For example, the free-base form of a compound is dissolved in a suitable solvent, such as an aqueous or aqueous-alcohol in solution containing the appropriate acid and then isolated by evaporating the solution. In another example, a salt is prepared by reacting the free base and acid in an organic solvent.
• a suitable solvent such as an aqueous or aqueous-alcohol in solution containing the appropriate acid and then isolated by evaporating the solution.
• a salt is prepared by reacting the free base and acid in an organic solvent.
• the pharmaceutically acceptable salt of the different compounds may be present as a complex.
• complexes include 8-chlorotheophylline complex (analogous to, e.g., dimenhydrinate: diphenhydramine 8-chlorotheophylline (1:1) complex; Dramamine) and various cyclodextrin inclusion complexes.
• Carriers or excipients can be used to produce pharmaceutical compositions.
• the carriers or excipients can be chosen to facilitate administration of the compound.
• Examples of carriers include calcium carbonate, calcium phosphate, various sugars such as lactose, glucose, or sucrose, or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents.
• Examples of physiologically compatible solvents include sterile solutions of water for injection (WFI), saline solution, and dextrose.
• the compounds can be administered by different routes including intravenous, intraperitoneab subcutaneous, intramuscular, oral, transmucosal, rectal, or transdermal. Oral administration is preferred.
• the compounds can be formulated into conventional oral dosage forms such as capsules, tablets, and liquid preparations such as syrups, elixirs, and concentrated drops.
• compositions for oral use can be obtained, for example, by combining the active compounds with solid excipients, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
• suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose (CMC), and/or polyvinylpyrrolidone (PVP: povidone).
• disintegrating agents maybe added, such as the cross — linked polyvinylpyrrolidone, agar, or alginic acid, or a salt thereof such as sodium alginate.
• Dragee cores are provided with suitable coatings.
• suitable coatings For this pu ⁇ ose, concentrated sugar solutions may be used, which may optionally contain, for example, gum arabic, talc, poly-vinylpyrrolidone, carbopol gel, polyethylene glycol (PEG), and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
• Dye-stuffs or pigments maybe added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
• compositions that can be used orally include push-fit capsules made of gelatin (“gelcaps”), as well as soft, sealed capsules made of gelatin, and a plasticizer, such as glycerol or sorbitol.
• the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
• the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols (PEGs).
• PEGs liquid polyethylene glycols
• stabilizers may be added.
• injection parenteral administration
• the compounds of the invention are formulated in sterile liquid solutions, preferably in physiologically compatible buffers or solutions, such as saline solution, Hank's solution, or Ringer's solution.
• physiologically compatible buffers or solutions such as saline solution, Hank's solution, or Ringer's solution.
• the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms can also be produced.
• Administration can also be by transmucosal or transdermal means.
• penetrants appropriate to the barrier to be permeated are used in the formulation.
• penetrants are generally known in the art, and include, for example, for transmucosal administration, bile salts and fusidic acid derivatives.
• detergents may be used to facilitate permeation.
• Transmucosal administration for example, may be through nasal sprays or suppositories (rectal or vaginal).
• the amounts of various compound to be administered can be determined by standard procedures taking into account factors such as the compound IC 50 , the biological half-life of the compound, the age, size, and weight of the patient, and the disorder associated with the patient. The importance of these and other factors are well known to those of ordinary skill in the art. Generally, a dose will be between about 0.01 and 50 mg/kg, preferably 0.1 and 20 mg/kg of the patient being treated. Multiple doses may be used.
• Compounds with the chemical stracture of Formula I can be prepared in a number of different synthetic routes, including, for example, the synthetic schemes described herein for groups of compounds within Formula I. Additional synthetic routes can be utilized by one skilled in chemical synthesis.
• Step 1- Preparation of compound of formula IV: [0307]
• Compound IV was prepared by reacting commercially available aldehyde III with an activated phosphonate ester in an inert solvent (e.g. tetrahydrofuran) under reflux conditions, typically for 16-24 h, as described by Garati et al in Arch. Pharm, 1988, 321, 377-83).
• Compound III in turn, can be prepared by reacting compound V under Vilsmeier (POCl 3 and DMF) conditions as described by in March 's Advanced Organic Chenistry, 5 l Edition, p.715.
• Key intermediate II was prepared by the reduction of IV in an inert solvent (i.e. tetrahydrofuran) by catalytic hydrogenation (typically 10% palladium on activated carbon and atmospheric hydrogen) as described by Garati et al in Arch. Pharm, 1988, 321, 377- 83).
• an inert solvent i.e. tetrahydrofuran
• catalytic hydrogenation typically 10% palladium on activated carbon and atmospheric hydrogen
• Compound VI was prepared conventionally by a reacting commercially available compound of formula V with an N,N-dialkyl amine hydrochloride in a polar solvent (e.g. i-Propanol), in the presence of formaldehyde and heated, typically near 90 °C, typically for 24h, as described by Snyder et al, JACS, 73, 970.
• a polar solvent e.g. i-Propanol
• Compound of formula VII was prepared by heating compound VI with diethyl malonate and a catalytic amount of sodium metal, typically at 120° C as described by Robinson et. al, JACS, 78, 1247, followed by flash chromatography purification.
• Compound of formula la was prepared by hydrolyzing compound VII using aqueous base (e.g. NaOH) followed by the decarboxylation under reflux conditions (JACS, 78, 1247).
• aqueous base e.g. NaOH
• Intermediate II was prepared by Fisher esterification of compound la with alcohol (e. g. Methanol) and catalytic amount of an acid (e.g. HCl) under reflux, typically for 16-24 h.
• alcohol e. g. Methanol
• an acid e.g. HCl
• Compound VIII can be prepared by a reacting commercially available of a compound of formula V with bromine in an inert solvent (e.g. DMF) (Bocchi and Palla; Synthesis, 1982, pi 096).
• an inert solvent e.g. DMF
• Step 2 - Preparation of formula IV [0316]
• Compound IV can be prepared by a reacting compound of formula VIII with methacrylate under Heck coupling conditions as described by Sznaidman et. at " , in Bioorg. Med. Chem. Lett, 13, 2003, 1517.
• Key intermediate II was prepared by the reduction of IV in an inert solvent (i.e. tetrahydrofuran) by catalytic hydrogenation (typically 10%> palladium on activated carbon and atmospheric.hydrogen) as described by Aarati et. al in Arch. Pharm, 321, 1988, 377- 83.
• an inert solvent i.e. tetrahydrofuran
• catalytic hydrogenation typically 10%> palladium on activated carbon and atmospheric.hydrogen
• Compound of formula la was prepared by the hydrolysis of key intermediate of formula II with aqueous base (e. g. aq. NaOH), typically for 6-15 h and isolating the product by conventional methods (e.g. aqueous work up and purification by chromatography) Jerry March in March 's Advanced Organic Chenistry, 5 l Edition, p.715.
• aqueous base e. g. aq. NaOH
• Compound of formula IXa was prepared by treating intermediate of formula II with a base (e. g. sodium hydride) in an inert solvent N,N-Dimethylformamide, followed by the addition of R 2 W, where "W” is a leaving group (e.g. chloro, bromo), and stirring at RT, typically for 16 to 24 h (Jerry March in March 's Advanced Organic Chenistry, 5 l ' Edition, p576). The product was obtained by column chromatography (e. g. silica gel) after workup using conventional methods.
• a base e. g. sodium hydride
• N,N-Dimethylformamide N,N-Dimethylformamide
• Compound of formula lb was prepared by the hydrolysis of compound of formula V with aqueous base (e. g. aq. NaOH), typically for 6-15 h and isolating the product by conventional methods (e.g. aqueous work up and purification by chromatography) .
• aqueous base e. g. aq. NaOH
• Compound of formula IXb was prepared by treating intermediate of formula II with a base (e. g. sodium hydride) in an inert solvent (DMF) followed by the addition of R 16 NCZ, where "Z" is oxygen or sulfur, and stirring at RT, typically for 16 to 24 h (Jerry March in Marc 's Advanced Organic Chenistry, 5 th Edition, pi 191).
• a base e. g. sodium hydride
• R 16 NCZ where "Z” is oxygen or sulfur
• Compound of formula IXb can also be prepared by treating intermediate of formula II with R 16 NCZ, where "Z” is oxygen or sulfur, in an inert solvent (THF) followed by the addition of catalytic amount of DMAP (N,N,-dimethylaminopyridine) and stirring at RT, typically for 16 to 24 h.
• DMAP N,N,-dimethylaminopyridine
• the product can be obtained by column chromatography (e. g. silica gel) after workup using conventional methods.
• Compound of formula lc was prepared by the hydrolysis of compound of formula IXb with aqueous base (e. g. aq. NaOH), typically for 6-15 h and isolating the product by conventional methods (e.g. aqueous work up and purification by chromatography).
• aqueous base e. g. aq. NaOH
• Compound of formula IXd was prepared from compound of formula IXc by reacting it with aryl boronic acids under Suzuki reaction conditions (March 's Advanced Organic Chemistry, 5 th Edition, p8) and heating the reaction mixture, typically 90° C, for 24 and isolating the product by conventional methods (e.g. aqueous work up and purification by chromatography).
• Compound of formula Id was prepared by the hydrolysis of compound of formula IXd with aqueous base (e. g. aq. NaOH), typically for 6-15 h and isolating the product by conventional methods (e.g. aqueous work up and purification by chromatography) .
• aqueous base e. g. aq. NaOH
• Compound of formula V was prepared from commercially available compound of formula Va by reacting it with aryl boronic acids under Kumada reaction conditions as described by Hayashi et. al, JACS, 106(1984), 158 -163, and heating the reaction mixture, typically 90° C, for 24 and isolating the product by conventional methods (e.g. aqueous work up and purification by chromatography).
• Compound VI was prepared conventionally by a reacting commercially available compound of formula V with an N,N-dialkyl amine hydrochloride in a polar solvent (e.g. i-Propanol), in the presence of formaldehyde and heated, typically near 90 °C, typically for 24h, as described previously for compound VI.
• a polar solvent e.g. i-Propanol
• Compound of formula la was prepared by hydrolyzing compound VII using aqueous base (e.g. NaOH) followed by the decarboxylation under reflux conditions as described previously.
• aqueous base e.g. NaOH
• Intermediate II was prepared by Fisher esterification of compound la with alcohol (e. g. Methanol) and catalytic amount of an acid (e.g. HCl) under reflux, typically for 16-24h.
• alcohol e. g. Methanol
• an acid e.g. HCl
• Compound of formula lb was prepared by the hydrolysis of compound of formula IXa with aqueous base (e. g. aq. NaOH), typically for 6-15 h and isolating the product by conventional methods (e.g. aqueous work up and purification by chromatography).
• aqueous base e. g. aq. NaOH
• Compound XI was prepared from the compound V reacting with ⁇ -butyrolatone in an inert solvent with potassium hydroxide under reflux conditions, usually 4 to 24 hours, as described by Fritz et al, (J. Org. Chem., 1963, 28, 1384-1385).
• Compound XII was prepared by the carboxylic acid XI reacting in either a catalytic amount of sulfuric acid in methanol under reflux conditions, or activated methylene moiety such as diazomethane.
• Compound XIII was prepared by treating intermediate of formula XII with a base (e. g. sodium hydride) in an inert solvent (DMF) followed by the addition of R 2 W, where "W” is a leaving group (e.g. chloro, bromo), and stirring at RT, typically for 16 to 24 h (Jerry March in March 's Advanced Organic Chenistry, 5 th Edition, p576).
• a base e. g. sodium hydride
• R 2 W inert solvent
• R 2 W a leaving group
• the product was obtained by column chromatography (e. g. silica gel) after workup using conventional methods.
• Step 4 - Preparation of intermediate X Compound of formula X was prepared by the hydrolysis of compound of formula XIII with aqueous base (e. g. aq. NaOH), typically for 6-15 h and isolating the product by conventional methods (e.g. aqueous work up and purification by chromatography).
• aqueous base e. g. aq. NaOH
• Compound XV can be prepared from the corresponding aldehyde III reacting with a reducting agent such as sodium borohydride in an inert solvent (e.g. tetrahydrofuran).
• a reducting agent such as sodium borohydride in an inert solvent (e.g. tetrahydrofuran).
• Compound XVI can be prepared by reacting the methanol XV with silyl ketene acetal in presence of a catalyst such as magnesium triflimide or perchlorate at ambient temperature for 1-2 hours as described by Grieco et al in Tetrahdron Letts (1997, 35, 2645-2648).
• a catalyst such as magnesium triflimide or perchlorate
• Step 3 Preparation of Intermediate XVII
• a base e. g. sodium hydride
• R 2 W inert solvent
• R 2 W a leaving group
• the product was obtained by column chromatography (e. g. silica gel) after workup using conventional methods.
• Compound of formula XIV was prepared by the hydrolysis of compound of fonnula XVII with aqueous base (e. g. aq. NaOH), typically for 6-15 h and isolating the product by conventional methods (e.g. aqueous work up and purification by chromatography) .
• aqueous base e. g. aq. NaOH
• Indazole-3-propionic acid 5-isopropoxy-3-(l-Benzene-sulfonyl-indol-3yl)-propionic acid,
• the homogenous Alpha screen assay was used in the agonist mode to detennine the ligand dependent interaction of the PPARs ( ⁇ , ⁇ , ⁇ ) with the coactivator peptides (SRC or DRIP205). Briefly 15ul of the reaction mix (50mM Tris pH 7.5,50mM Kcb 0.05% Tween 20,lmM DTT,0.1% BSA and 10nM-200nMPPAR and 10nM-200nM coactivator peptide) was added to the test compound (lul compound in DMSO) and preincubated for l-6hr.Next, 5ul of the Alpha screen beads were added. The reactions were incubated for 2 hrs before taking the reading in the Fusion alpha instrument. In the antagonist mode compounds were assayed for inhibition of the co-activator binding signal caused by the control agonists for each receptor.
• the controls agonists used were WY- 14643 (PP AR( ⁇ ), farglitazar (PPAR ( ⁇ ) and bezafibrate (PPAR ( ⁇ ).
• compound 6 is selective for PPAR ⁇ , with activity on PPAR ⁇ of approximately 8 micromolar and activity on PPAR ⁇ and ⁇ of at least 200 micromolar.
• 293T cells were transfected for 4-5hr in serum free DMEM media using cell fectin reagent. Each well was transfected with lug each of the reporter plasmid ( pFR-Luc from stratagene)and PPAR constructs (Gal4-PPAR-LBD). After 24hrs of recovery in serum medium the cells were treated with compounds for 48 hrs then assayed for luciferase activity using luciferase reporter gene assay kit (Roche).
• This assay serves to confirm the observed biochemical activity on the modulation of intended target molecule(s) at the cellular level.
• Step 3 Preparation of 3-[5-Methoxy-l-(4-methoxy-benzenesulfonyl)-lH-indol-3-ylJ- propionic acid methyl ester 5:
• Step 4 Preparation of 3-[5-Methoxy-l-(4-methoxy-benzenesulfonyl)-lH-indol-3-yl]- propionic acid 1 :
• fr ⁇ dole-3 -propionic acid 6 was synthesized from the commercially available 5- bromo-indole 7 in eight steps as shown in Scheme 8.
• Step 1 Preparation of5-Bromo-l-triisopropylsilanyl-lH-indole 8.
• Step 2 Preparation of5-Ethyl-l-triisopropylsilanyl-lH-indole 9.
• Step 4 Preparation of(5-Ethyl-lH-indol-3-ylmethyl)-dimethyl-amine 11.
• Step 5 Preparation of2-(5-Ethyl-lH-indol-3-yhnethyl)-malonic acid diethyl ester 12.
• the 5-Ethylgramine (1.25 g, 6.18 mmol) was combined with diethyl malonate (2.85 mL, 18.54 mmol) and heated to 120° C until a homogeneous solution was formed.
• sodium metal 100 mg, 4.36 mmol
• TLC indicated the completion of the reaction.
• the reaction was allowed to cool to room temperature and a solution of 5%> HCl (aqueous) was slowly added to the mixture and the resulting product was extracted with EtOAc.
• Step 6 Preparation of2-(5-Ethyl-lH-indol-3-ylmethyl)-malonic acid 13.
• the crade diethyl malonylindole 12 (1.67 g, 5.26 mmol) was dissolved in THF (20 mL) and a solution of NaOH (1.0 g, 25.5 mmol) in H 2 O (20mL) was added. MeOH (5 mL) was also added to the reaction to make the solution homogeneous. The mixture was warmed to 50° C and stirred overnight. The mixture was allowed to cool to room temperature, the organic layer was evaporated and the residue was acidified with 2N H PO 4 , and the product was extracted with a mixture of 3:1 / CHCl 3 :MeOH.
• Step 7 Preparation of3-(5-Ethyl-lH-indol-3-yl)-propionic acid 14.
• Step 8 Preparation of3-(l-Benzenesulfonyl-5-ethyl-lH-indol-3-yl)-propionic acid 6.
• the indole propionic acid 14 (100 mg, 0.46 mmol) was dissolved in THF (5.0 mL) and cooled to -78° C.
• n-butyllithium n-BuLi; 0.4 mL, 1.0 mmol, 2.4 M in hexanes
• Indazole-3 -propionic acid 16 was prepared from commercially available indazole-3-carboxylic acid 17 in 5 steps as described in Scheme 9.
• indazole-3 -carboxylic acid 17 (3.95 g, 24.4 mmol) in tetrahydrofuran (THF, 300 ml) under nitrogen, lithium aluminum hydride (LAH; 1.9 g, 50.5 mmol) was added in one portion.
• LAH lithium aluminum hydride
• the resulting alcohol 17 was isolated through quenching the reactive LAH with water, until no hydrogen evolution was observed and the solution was then filtered, washed with THF, and concentrated to to give alcohol 18 as a light brown solid (2.63 g, 72%).
• Indazole-3 -propionic acid methyl ester was prepared from compound 20 as described in Step 2, Example 3.
• Propionic acid 22 was preparedfrom commercially available 5-hydroxy-indole 23 in 5 steps as shown in Scheme 10.
• Step 3 Synthesis of2-(5-Isopropox)>-lH-Indol-3-ylmethyl)-malonic acid diethyl ester 26
• Step 4 Synthesis of 5 -Is opropoxy-indole-3 -propionic acid 27
• h ⁇ dole-3 -propionic acid 28 was prepared through the commercially available indole-3-carboxyaldehyde as described in Example 3. (M-l, 188.2)
• Compound XVIII can be prepared through coupling of compound III with benzene sulfonyl chloride in a bi-phasic solvent condition e.g. toluene and water, in presence of a base, e.g. an aqueous potassium hydroxide solution with a phase transfer catalyst, e.g. tetrabutylammonium hydrogen sulfate, similar to conditions as described Gribble et al, in J. Org. Chem., 2002, 63, pg 1001-1003.
• a base e.g. an aqueous potassium hydroxide solution with a phase transfer catalyst, e.g. tetrabutylammonium hydrogen sulfate, similar to conditions as described Gribble et al, in J. Org. Chem., 2002, 63, pg 1001-1003.
• Compound XIX was prepared through conventionally Knoevenagel reaction reacting compound XVIII with malonic acid piperidine in pyridine at 80°C for 3-4 hours, as described in Vangvera et al in J. Med. Chem., 1998, 41, pg 4995-5001.
• Compound la was prepared from compound XIX through reduction via catalytic hydrogenation (typically with 10% palladium on activated carbon in an inert solvent (see preparation of intermediate II, vide supra).
• Example 80 Alternate synthesis of 3-[5-methoxy-l-(4-methoxy-benzenesulfonyl)-lH- indol-3-yl]- propionic acid 1
• Step 1 Preparation of l-(4-Methoxy benzenesulfonyl)-5-methoxy-lH- indole-3- carboxy aldehyde) (117)
• Step 3 Preparation of 3-[l-(4-Methoxy benzenesulfonyl)-5-methoxy-lH-indol-3-yl]- propionic acid (1)
• Step 3 Preparation of 3-[5-Methoxy-l-(4-methoxy-benzenesulfonyl)-lH-indol-3-yl] ⁇ propionic acid methyl ester 120:
• Step 4 Preparation of 3-[5-Methoxy-l-(4-methoxy-benzenesulfonyl)-lH-indol-3-yl]-2,2- dimethyl-propionic acid 119:
• Example 82 Synthesis of 3-[l-(3,4-Dimethoxy-benzenesulfonyl)-5-methoxy-lH-indol- 3 -yl] -propionic acid 101
• Example 84 Synthesis of 3-[l-(3-chloro-4-methyl-benzenesulfonyl)-5-methoxy-lH- indol-3-yl]-propionic acid 103
• Example 98 Synthesis of 3-[l-(3,4-Dichloro-benzenesulfonyl)-5-methoxy-lH-indol-3- yl]-2,2-dimethyl-propionic acid methyl ester 122.
• 3-Chloro-4-methoxy-benzenesulfonyl chloride was in turn prepared by reacting 2-chloroanisole with chlorosulfonic acid (neat at 0 °C, 4h) following the literature procedure (Cremlyn,RJ.W.; Hornby,R; JChem.Soc.C; 1969; 1341-1345)
• Example 104 Synthesis of Synthesis of 3-[l-(4-Methoxy-benzenesulfonyl)-7-methyl- 1 H-indol-3 -yl] -propionic acid 127
• Compound 128 is synthesized from commercially available 6-methyl-indole-3- carboxaldehyde following synthetic steps shown in Scheme 12.
• Compound 132 is synthesized from 6-methoxy-indole-3-carboxaldehyde, which in turn is synthesized from commercially available 6-methoxy-indole using Nilsmeier- Haack reaction (Advanced organic chemistry, Jerry March, 2 nd Ed. P715), following synthetic steps shown in Scheme 12.
• Example 110 Synthesis of 3-[l-(4-Methoxy-benzenesulfonyl)-5,6-dimethoxy-lH-indol- 3 -yl] -propionic acid 133
• Compound 133 is synthesized from 5,6-dimethoxy-indole-3-carboxaldehyde, which in turn is synthesized from commercially available 5,6-dimethoxy-indole using Nilsmeier-Haack reaction (Advanced organic chemistry, Jeny March, 2 n Ed. P715), following synthetic steps shown in Scheme 12.
• Example 111 Synthesis of 3-[l-(4-Methoxy-benzenesulfonyl)-6-bromo-lH-indol-3-yl]- propionic acid 134
• Example 112 Synthesis of 3-[l-(4-Methoxy-benzenesulfonyl)-5-methoxy-lH-indazol-3- yl]-propionic acid 135
• Compound 140 is prepared by heating the bromide 139 with sodium methoxide in dimethyl formamide in presence of cuprous bromide as described by Mazeas, Daniel; GuiUaumet, Gerald; Marie- Claude Niaud, Heterocycles, 1999, v50 (2), 1065-1080, from which the aldehyde 138 is prepared by Nilsmeier-Haack reaction.
• Analogs of compound 1 can be synthesized, e.g., by using the commercially available compounds shown in Table 3 as described in Example 3 or Example 109.
• the carboxylic acid functional group of the propionic acid moiety at position 3 can advantageously be replaced with any of a number of carboxylic acid bioisosteres in compounds of Formula I.
• the following moieties can be used, which are shown with respect to Formula 1-1, but which can also be incorporated in other bicyclic rings systems within Formula I.
• Compound XX can be prepared through a Knoevenagel coupling of thiazohdione or related compounds in presence of an inert solvent, e.g. ethanol, with catalytic amount of piperidine with starting compound III.
• an inert solvent e.g. ethanol
• Step 2 [0499] Compound XXI can be prepared from compound XX through a reduction process using palladium on activated carbon, or a metal reduction reaction (e.g. magnesium). (B.C. Cantello, J. Med. Chem., 1994, 37, 3977-85.)
• Compound XXII can be prepared through either amide bond formation reaction with la or lb or nucleophilic displacement of the ester II or IXa with n-hydroxyamine. (Hurd et al, J. Am. Chem. Soc, 1954, 76, 2791 and Dmh, T.Q., Tet. Lett. 1996, 37, 1161- 4).
• Tetrazole isostere of the carboxylic acid can be prepared through 3 steps from the corresponding acetic or propanoic acid (depending on linker size).
• the propionamide XXIII can be converted to the nitrile XXIV by treating the amide with methyl magnesium iodide (Wilson et al, J. Chem Soc, 1923, 123, 2615) or with formic acid in acetonitrile (Heck, M.-P., J. Org. Chem., 1996, 61, 6486-7).
• the hydroxyiso-oxazole compound XXVIII can be derived in 5 steps. Using starting material indole-3 -acetic acid XXVI, compound XXVII can be prepared through reactions in Example 4. Activation of the acid group with bis-imidazole-carbonyl leads to compound XXVIII (Eils et al, Synthesis, 1999, 275-81). The reaction with ethyl malonic acid affords XXIX. Cyclization with hydroxylamine provides the hydroxy protected iso- oxazole XXX. The deprotection of the hydroxy functionality arrives at the desired compound XXXI. (Frolund et al, J. Med. Chem., 2002, 45, 2454-2468)
• the hydroxyiso-oxazole compound XXXI can be derived in 4 steps.
• the first step involves direct coupling of the 3-unsubstituted indole V with a protected hydroxy iso- oxazole methyl halogen (chloride or bromide) with a base (e.g. sodium hydroxide) in an alcohol solvent (e.g. methanol) system.
• a base e.g. sodium hydroxide
• alcohol solvent e.g. methanol
• Compound XXXVIII can be prepared through a two step process starting from either la or lb.
• the carboxylic acid group in la or lb can be converted to acyl halide XXXVII through the use of reagents (e.g. thionyl chloride, phosphorous pentachloride, or phosphorous trichloride) in an inert solvent (e.g. dichloromethane).
• reagents e.g. thionyl chloride, phosphorous pentachloride, or phosphorous trichloride
• an inert solvent e.g. dichloromethane
• acyl cyanamide functionality can be introduced via coupling of the cyanamide with compound XXXVII to yield the desired product XXXVIII.
• the sulfonamide bio-isostere for carboxylic acid can be prepared in 6 steps from from indolyl-3-acetic acid or propionic acid (if the linker is to be extended)
• Compound XXVII can be transformed to the corresponding alcohol XXXIX tlirough treatment with reducing reagent such as lithium aluminum hydride in an inert solvent such as THF.
• reducing reagent such as lithium aluminum hydride in an inert solvent such as THF.
• the corresponding alcohol can be converted to mesylate or halogen with the proper reagents such as methane sulfonyl chloride or Phosphorous tribromide respectively.
• Intermediate XL can be prepared by treating XXXIX with sodium hydrogen sulfide, hexabutyldistannathian, or 1 -(2 -hydroxyethyl)-4,6-diphenylpyridine-2 -thione to get to the ethanethiol or propanethiol.
• XXXIX sodium hydrogen sulfide, hexabutyldistannathian, or 1 -(2 -hydroxyethyl)-4,6-diphenylpyridine-2 -thione
• the thiol XL can be oxidized to the corresponding sulfonic acid with oxidative reagents such as hydrogen peroxide to afford intermediate XLI.
• Step 5 [0515] Compound XL can be treated to reagents (e.g. thionyl chloride or phosphorous pentachloride) to convert the sulfonic acid to the corresponding sulfonyl chloride to arrive at intermediate XLII.
• reagents e.g. thionyl chloride or phosphorous pentachloride
• Sulfonamide isosteres of the carboxylic acid is then generated through coupling of the sulfonyl chloride XLIIIa with amine reagents (e.g. sodium amide or methylamine).
• amine reagents e.g. sodium amide or methylamine
• Acetyl-sulfonamides XLIV can be prepared through the sulfonyl chloride XLII in two steps.
• Benzenesulfonic acid XLV can be reacted with aryl halides, e.g., iodobenzene benzyl bromide etc., under Buckwald reaction conditions and SN reaction conditions respectively, or with alcohols, e.g. benzyl alcohols under Mitsunobu reaction conditions, or other coupling reactions to afford XLVII.
• aryl halides e.g., iodobenzene benzyl bromide etc.
• alcohols e.g. benzyl alcohols under Mitsunobu reaction conditions, or other coupling reactions to afford XLVII.
• Compound of formula XL TI can be converted to the corresponding sulfonly chloride with reagents such as PC1 3 , PC1 5 , POCl 3 , or SOCl 2 .
• Compound of formula L can be prepared by reacting the sulfonyl chloride XLNIII with 5-methoxy-indole-3-propionic ester in presence of a base, e.g. aq. Potassium hydroxide, in THF.
• a base e.g. aq. Potassium hydroxide
• Compound 143 can be prepared through methods described in Scheme 23, using 4-hydroxybenzenesulfonic acid and 3-hydroxypyridine to prepare the corresponding sulfonyl chloride.
• Example 117 Synthesis of 3- ⁇ 5-Methoxy-l-[4-(pyridin-4-yloxy)-benzenesulfonyl]-lH- indol-3-yl ⁇ -propionic acid 144.
• Compound 144 can be prepared through methods described in Scheme 23, using 4-hydroxybenzenesulfonic acid and 4-hydroxypyridine to prepare the conesponding sulfonyl chloride. The various coupling of the sulfonyl chloride to 5-methoxy-indole-3- p;ropionic ester or the conesponding acid as described in Scheme 7, 10, or 12.
• Example 118 Synthesis of 3- ⁇ 5-Methoxy-l-[4-(pyridin-4-ylmethoxy)-benzenesulfonyl]- 1 H-indol-3 -yl ⁇ -propionic acid 145. 145
• Compound 145 can be prepared through methods described in Scheme 23, using 4-hydroxybenzenesulfonic acid and 4-pyridylcarbinol to prepare the conesponding sulfonyl chloride. The various coupling of the sulfonyl chloride to 5-methoxy-indole-3- p;ropionic ester or the corresponding acid as described in Scheme 7, 10, or 12.
• Compound 146 can be prepared by reacting 5-methoxy-indole-3-p;ropionic ester or the conesponding acid through methods with 3,5-dichlorobenzenesulfonyl chloride as described in Scheme 7, 10, or 12.
• Example 120 Synthesis of 3-[l-(3,5-Dimethoxy-benzenesulfonyl)-5-methoxy-lH-indol- 3 -yl] -propionic acid 147
• Compound 147 can be prepared by reacting 5-methoxy-indole-3-p;ropionic ester or the conesponding acid through methods with 3,5-dimethoxybenzenesulfonyl chloride as described in Scheme 7, 10, or 12.
• Compound LII can be prepared through coupling of indole (2 or 4) with sulfonyl chloride LI from methodologies described in Scheme 7 or 12.
• Compound LIV or LV can be prepared through nucleophilic displacement of the bromomethyl group under basic conditions, in an inert solvent such as DMF.
• Example 121 Synthesis of 3- ⁇ 5-Methoxy-l-[4-(quinolin-7-ylaminomethyl)- benzenesulfonyl]-! H-indol-3 -yl ⁇ -propionic acid 148
• Compound 148 can be prepared via coupling of compound LII with the conesponding Quinol-7-ylamine with the bromomethyl moiety in Scheme 24.
• Example 122 Synthesis of 3- ⁇ l-[4-(Isoquinolin-3-ylaminomethyl)-benzenesulfonyl]-5- methoxy-lH-indol-3-yl ⁇ -propionic acid 149
• Compound 149 can be prepared via coupling of compound LII with the conesponding isoquinolin-3-yl-amine with the bromomethyl moiety in Scheme 24.
• Compound 149 can be prepared via coupling of compound LII with the conesponding quinolin-6-yl amine with the bromomethyl moiety in Scheme 24.
• Example 124 Synthesis of 3-[5-Methoxy-l-(4-py ⁇ olo[2,3-b]pyridin-l-ylmethyl- benzenesulfonyl)-lH-indol-3-yl]-propionic acid 151
• Compound 151 can be prepared via coupling of compound LII with the conesponding 7-azaindole with the bromomethyl moiety in Scheme 24.
• Compound 152 can be prepared via coupling of compound LII with the conesponding phenol with the bromomethyl moiety in Scheme 24.
• the intermediate LVII can be prepared through either similar methods as described in step 1 of preparation of XLVII, or through nucleophilic displacement of a fluoro group.
• the sulfonic acid can be converted to the conesponding sulfonyl chloride with PC1 3 , POC ⁇ 3) PCi5, or SOCb2.
• nitrile moiety can be further converted to either amide through hydrolysis or amine through reduction.
• Example 126 Synthesis of 3- ⁇ 5-Methoxy-l-[4-(pyridin-3-ylmethoxy)-benzenesulfonyl]- lH-indol-3-yl ⁇ -propionic acid 153
• Compound 153 can be prepared through methods described in Scheme 23, using 4-hydroxybenzenesulfonic acid and 3-pyridinemethanol to prepare the conesponding sulfonyl chloride.
• the various coupling of the sulfonyl chloride to the indole-moiety are described in Scheme 7, 10, or 12.
• Example 127 Synthesis of 3- ⁇ l-[4-(4-Aminomethyl-benzyloxy)-benzenesulfonyl]-5- methoxy-lH-indol-3-yl ⁇ -propionic acid 154
• Compound 154 can be prepared through reduction of the nitrile group, as described in Scheme 25.
• the nitrile functionality can be prepared through coupling of the sulfonyl chloride with the 5-methoxyindole-3-propionic acid methyl ester.
• the sulfonyl chloride can be prepared through coupling of the 4-hydroxy benzenesulfonic acid with 4- cyanobenzyl bromide.
• Example 128 Synthesis of 3- ⁇ l-[4-(4-Carbamoyl-benzyloxy)-benzenesulfonyl]-5- methoxy-lH-indol-3-yl ⁇ -propionic acid 155

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