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WO1999047529A1 - Heterocyclic signal transduction inhibitors, compositions containing them - Google Patents

Heterocyclic signal transduction inhibitors, compositions containing them Download PDF

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Publication number
WO1999047529A1
WO1999047529A1 PCT/US1999/005970 US9905970W WO9947529A1 WO 1999047529 A1 WO1999047529 A1 WO 1999047529A1 US 9905970 W US9905970 W US 9905970W WO 9947529 A1 WO9947529 A1 WO 9947529A1
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Prior art keywords
compound
substituted
unsubstituted
aliphatic
nrr
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French (fr)
Inventor
John L. Buchanan
Regine Bohacek
Chi B. Vu
George P. Luke
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Ariad Pharmaceuticals Inc
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Ariad Pharmaceuticals Inc
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Priority to JP2000536724A priority Critical patent/JP2002506873A/en
Priority to CA002319493A priority patent/CA2319493A1/en
Priority to EP99912685A priority patent/EP1064289A1/en
Publication of WO1999047529A1 publication Critical patent/WO1999047529A1/en
Anticipated expiration legal-status Critical
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    • C07—ORGANIC CHEMISTRY
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    • C07D271/00—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms
    • C07D271/02—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms not condensed with other rings
    • C07D271/06—1,2,4-Oxadiazoles; Hydrogenated 1,2,4-oxadiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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    • A—HUMAN NECESSITIES
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    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/22—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
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    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/22—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C07D277/30—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
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    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
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    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
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    • C07F9/6527—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having nitrogen and oxygen atoms as the only ring hetero atoms
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    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6527—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07F9/653—Five-membered rings
    • C07F9/65306—Five-membered rings containing two nitrogen atoms
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    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6536—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having nitrogen and sulfur atoms with or without oxygen atoms, as the only ring hetero atoms
    • C07F9/6539—Five-membered rings
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    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
    • C07F9/65515—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a five-membered ring
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    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having sulfur atoms, with or without selenium or tellurium atoms, as the only ring hetero atoms
    • C07F9/655345—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having sulfur atoms, with or without selenium or tellurium atoms, as the only ring hetero atoms the sulfur atom being part of a five-membered ring
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    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65583—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system each of the hetero rings containing nitrogen as ring hetero atom
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    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom

Definitions

  • This invention concerns a new class of compounds which have a broad range of useful biological and pharmacological activities.
  • these compounds are useful for inhibiting intracellular signal transduction, especially intracellular signal transduction mediated by one or more molecular interactions involving a phosphotyrosine-contaimng protein
  • This invention also relates to pharmaceutical compositions containing the compounds and prophylactic and therapeutic methods involving pharmaceutical and veterinary administration of the compounds.
  • Cellular signal transduction i.e., the se ⁇ es of events leading from extracellular events to intracellular sequelae, is an aspect of cellular function in both normal and disease states.
  • Numerous proteins that function as signal transducing molecules have been identified, including receptor and non-receptor tyrosine kinases, phosphatases and other molecules with enzymatic or regulatory activities. These molecules generally demonstrate the capacity to associate specifically with other proteins to form a signaling complex that can alter cell activity.
  • Signaling proteins often contain domam(s) of conserved sequence which constitute catalytic domains such as kinase or phosphatase domains, or serve as non-catalytic modules that direct protein 'protein or other inter- or intramolecular interactions du ⁇ ng signal transduction.
  • Such domains include among others, Src homology 2 ("SH2") and phosphotyrosine interaction (“PI”) domains.
  • SH2 and PI domains recognize, i.e., bind to, proteins containing characteristic peptide sequences which include one or more phosphorylated tyrosine (“pTyr”) residues.
  • Src tyrosine kinases reveals that each family member contains an N-terminal membrane anchor, a poorly conserved "unique" region of 40-70 ammo acids, a Src homology 3 (SH3) domain of about sixty ammo acids capable of protein-protein interactions with prolme- ⁇ ch sequences and a Src homology 2 (SH2) domain composing about 100 ammo acid residues which mediates binding of the Src family member of phosphotyrosme-(pTyr) containing peptides and proteins (reviewed in Superti-Furga, FEBS Lett. 369 62-66 (1995).
  • SH3 Src homology 3
  • SH2 Src homology 2
  • SH2 domains including, e.g., ZAP-70, Syk, She, Tsk, Btk, VAV, Grb2, Crk, STATs
  • PI domam-contammg proteins see WO 97/39326 and references cited therein
  • SH2 domain binding specificity is thought to be influenced significantly by the specific ammo acids located carboxy-terminal to the pTyr residue.
  • the pp60c-src, Fyn, Lck and Fgr SH2 domains prefer the sequence pTyr-Glu-Glu-Ile.
  • Many signaling pathways which play cntical roles in disease processes are mediated by the binding of a phosphotyrosine-contaming protein or protein domain with an SH2 or other protein receptor for a tyrosme-phosphorylated domain.
  • Pharmaceutical agents which interfere with signaling mediated by such molecules e.g., which interfere with the formation or stability of such signaling complexes, may be used for precise intervention in these complex biological processes in order to treat or prevent the diseases or pathological effects mediated by such signaling.
  • Such interference may be achieved through a vanety of mechanisms, including competitive inhibition of a phosphotyrosine-contaming ligand with its receptor (e.g., with an SH2-conta ⁇ n ⁇ ng protein), inhibition of phosphorylation of the tyrosine residue of such a ligand, inhibition of activation of a kinase which catalyzes the phosphorylation of a ligand in a signaling pathway, etc.
  • a vanety of mechanisms including competitive inhibition of a phosphotyrosine-contaming ligand with its receptor (e.g., with an SH2-conta ⁇ n ⁇ ng protein), inhibition of phosphorylation of the tyrosine residue of such a ligand, inhibition of activation of a kinase which catalyzes the phosphorylation of a ligand in a signaling pathway, etc.
  • This invention concerns compounds of Formula I, or pharmaceutically acceptable derivatives thereof:
  • G is -O-, -S- or -NR-
  • each occurrence of M is an independently selected, substituted or unsubstituted, methylene moiety, and any M-M' moiety may be electronically saturated or unsaturated and/or may be part of a 3-8-membered ring.
  • M moieties include -CH 2 -, -CHF-, — CF 2 -,
  • n is independently 0, 1, 2, 3, 4 or 5 (in many embodiments n is 0, 1 or 2);
  • each m is independently 0, 1 or 2;
  • J and K are independently selected from the group consisting of -APO.RR , -OPO RR , -ASO 3 R, -OSO 3 R, -ACO 2 R, -A-tetrazole, -ASO 2 NRR', -(M) NRR' and -(M) n OR;
  • Z is a halogen (i.e., F, Cl, Br or I);
  • R 7 and R 8 are independently R, -CN, -NO 2 , Z, J, -A(M) aliphatic, -G(M) aliphatic ,
  • Each occurrence of R represents hydrogen or an aliphatic, heteroahphatic, aryl, heteroaryl, (aryl)al ⁇ phat ⁇ c-, or (heteroaryl)a phat ⁇ c- moiety, each of which (other than hydrogen) may be substituted or unsubstituted, e.g , with any of the va ⁇ ous substituents listed, illustrated or otherwise disclosed herein. While each occurrence of "R” withm a given compound is thus independently selected, where multiple R groups are depicted m the same figure or moiety, the va ⁇ ous R groups are generally marked R, R', R" and so on, as a reminder that they may be the same or different.
  • n M groups m a "M_" moiety may be the same or different from one another.
  • q is an integer from 1 to 8, and in many embodiments is 1, 2 or 3,
  • R is hydrogen, al ⁇ phat ⁇ c.-(M) -cycloaliphatic, -(M) -aryl, or -(M) -heterocyclic, each of which, other than H, may be substituted or unsubstituted (including, e g. moieties such as -(M) n CO 2 R, -(M) n C(O)NRR', -(M) n Z, -(M) n CN, -(M) n tetrazole, etc.),
  • R is hydrogen or substituted or unsubstituted aliphatic, which is optionally covalently linked with R to form a ⁇ ng, or R or R are covalently linked either to B or a substituent of B to form a 4 - 10- membered, saturated or unsaturated, ring, or to the N depicted in Formula I above to form a 5, 6 or 7-membered, saturated or unsaturated, ring;
  • X is:
  • R 3 is hydrogen, R(CO)NR'-, RR'N(CO)NR"-, R'SO 2 NR- R'CSNR- RR''NCSNR"-, RR''NS0 2 NR"-, R'OCONR- RR'N- or
  • R is hydrogen, aliphatic (which may be branched, unbranched or cyclic), cycloaliphatic-(M) n - aryl-(M) n - heterocyclic-(M) n -, RSO 2 (M n )- , (CO 2 R)(M) n - or (RR'N-CO)(M) n , where the aliphatic, cycloaliphatic, aryl and heterocyclic groups are substituted or unsubstituted;
  • HET is a heterocyclic moiety
  • R 9 , R 10 and R 11 are independently -(M) Z, -(M) R, -(M) n GR, -(M) n WR,
  • SR -CHO, -COR, -COOH (or amide, ester, carbamate, urea, oxime or carbonate thereof), -NH 2 (or substituted amine, amide, urea, carbamate or guanidino derivative therof), halo, trihaloalkyl, cyano, -SO 2 -CF 3 , -OSO 2 F, -OS(O) 2 R, -SO 2 -NHR, -NHSO 2 R, sulfate, sulfonate, aryl and heteroaryl moieties.
  • R 12 is independently selected from -(M) Z, -(M) R, -(M) GR, -(M) n WR, -(M) n WGR, and -(M) n W-COR;
  • R 14 is R;
  • U and W are independently -CO-, -CS-, -M-, -SO-, or -SO 2 -.
  • One subgenus includes compounds in which at least one R 4 moiety is H and at least one R moiety is either H or NH 2 .
  • Compounds of the latter sort include those in which X is
  • R comprises a substituted or unsubstituted, lower (i.e., containing 1 - 8 carbon atoms) aliphatic or alkoxyl group, or is a substituted or unsubstituted -(M) ⁇ -aryl or -(M) n -heterocyclic (including e.g., substituted and unsubstituted phenyl or benzyl group, or a homolog and heterocyclic analog thereof, including e.g., 2-naphthyl, 3-indolyl, and 1- imidazolyl);
  • R comprises -(M) n CH 3 , -(M) ⁇ aryl, -(M) ⁇ heterocyclic, -(M) n CN, -(M) n COOR, where n is 0, 1, 2, 3, 4, or 5.
  • n is 0, 1, 2, 3, 4, or 5.
  • R is a substituted or unsubstituted methyl, ethyl, n-propyl, l-propyl, n-butyl, sec- butyl, t- butyl, n-pentyl, sec- pentyl, l-pentyl, cyclo pentyl, etc. or benzyl moiety.
  • R comp ⁇ ses -(CH,.) CH., -(CH 2 )(CH 2 ) n aryl, -(CH 2 )(CH 2 ) n heterocycl ⁇ c, -(CH 2 )(CH 2 ) n CN or -(CH 7 )(CH 2 ) n COOR, where n again is 0, 1, 2, 3, 4, or 5.
  • R 5 comp ⁇ ses -CH 2 CN, -(CH 2 )CO 2 R, -(CH 2 ) 2 CO ? R,
  • R is H, lower alkyl or benzyl and those in which R 5 comp ⁇ ses -O-(subst ⁇ tuted or unsubstituted lower alkyl or benzyl).
  • Another subgenus of interest includes amides of the formula:
  • R1 R where R 4 is hydrogen, substituted or unsubstituted aliphatic (which may be branched, unbranched or cyclic), substituted or unsubstituted aryl-(M) n -, substituted or unsubstituted heterocychc-(M) n -, or (CO 2 R)(M) n -.
  • R 4 is hydrogen, substituted or unsubstituted aliphatic (which may be branched, unbranched or cyclic), substituted or unsubstituted aryl-(M) n -, substituted or unsubstituted heterocychc-(M) n -, or (CO 2 R)(M) n -.
  • R 4 is -(M) (CO)OR, -(M) n SO 2 R, -(M) (C0)NRR', or -(M) (tetrazole), including, for example, compounds in which R 4 is -CH 2 COOR, -CH 2 SO 2 R, -CH 2 (CO)NRR', or
  • Simple members of this subgenus are those in which the R group(s) of R is (are independently) H, lower alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tbutyl, etc.) or benzyl.
  • R group(s) of R is (are independently) H, lower alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tbutyl, etc.) or benzyl.
  • Another subgenus includes ureas of the formula: r R 14
  • R 1 , R 2 , R 4 , R 14 , Y and m are defined as above.
  • R 4 may be simply H or may be a more complex R moiety such as are noted above.
  • Another subgenus includes amides of the formula: R 14
  • R moieties are H. Also, in many compounds of interest, R is H.
  • One subgenus of interest includes compounds of Formula I, including the examples described or illustrated above, in which m is 1, R 1 is H, and R 2 comprises H, -(M) H,
  • R 1 is H
  • R 2 is methyl, ethyl, i-propyl, n-propyl, n-butyl, isobutyl, n-amyl, sec-amyl, isoamyl, substituted benzyl, -CH 2 -(3-indolyl), -CH 2 -(4-imidazolyl), -CH 2 CH 2 COOR,
  • R ⁇ and R ⁇ are independently selected, substituted or unsubstituted lower aliphatic groups, usually of 1 - 8 contiguous carbon atoms, or R and R are covalently linked to each other to form a ring, which may be a substituted or unsubstituted, aliphatic or heterocyclic ring or ring system (e.g. a bicyclic moiety), generally containing 3 - 10 annular atoms.
  • q is an integer from 1 to 8:
  • Another subgenus includes compounds of Formula I, including the examples described or illustrated above, in which m is 2, and each of R 1 , R 1 ', R 2 , and R 2 ' is independently selected from H, -(M) n H, -(M) n -(substituted or unsubstituted lower alkyl), -(M) n -(substituted or unsubstituted aryl), -(M) n -(substituted or unsubstituted heterocyclic), -(M) -COOR and -(M) (CO)NRR .
  • each of R ⁇ R ⁇ R 2 , and R 2 ' is H.
  • R and R is methyl, ethyl, i-propyl, n-propyl, n-butyl, isobutyl, n-amyl, sec-amyl, isoamyl, substituted benzyl, -CH 2 -(3-indolyl), -CH 2 -(4-imidazolyl), -CH 2 CH 2 COOR, -CH 2 CH 2 CONH 2 ,
  • R 1 , R 1 ' , R 2 , and R 2' is methyl, ethyl, i-propyl, n-propyl, n-butyl, isobutyl, n-amyl, sec-amyl, isoamyl, substituted benzyl, -CH 2 -(3-indolyl),
  • R 1 , R 1' , R 2 , and R 2' are covalently linked to form a ring, which as in other cases, may be a substituted or unsubstituted, aliphatic or heterocyclic ring or ring system (e.g. a bicyclic moiety), generally containing 4 - 10 annular atoms.
  • a ring which as in other cases, may be a substituted or unsubstituted, aliphatic or heterocyclic ring or ring system (e.g. a bicyclic moiety), generally containing 4 - 10 annular atoms.
  • Compounds containing 3-, 5- and 6-membered rings are illustrated by the following formulas:
  • One subgenus of compounds of this invention i.e., of compounds of Formula I. including among others the members of the various illustrative classes of compounds noted above, includes those compounds of Formula I in which m is 0:
  • R comprises -OR, -APO 3 RR , -OPO RR , -ASO R, -OSO3R, -ACO 2 R, -A-tetrazole, -ASO 2 NRR', -ACOCF3, -C(R)(J)(K) or -C(Z)(J)(K); and, R 7 and R 8 are independently H, -CN, -NO , halogen, J,
  • R comprises -OR, -APO 3 RR', -OPO 3 RR', -ACO 2 R, -ACOCF 3 , or -C(R)(J)(K);
  • A comprises -M m - (e.g., -CH 2 - -CF 2 - -CHF- , -CHOH-, -CH 2 CF 2 - etc), -GM- (e.g.
  • each R and R' is H, or substituted or unsubstituted lower alkyl or substituted or unsubstituted benzyl; and, R 7 and R 8 are independently H, J, -A-(M) n substituted or unsubstituted aliphatic, -(M) n COCF 3 , -(M) n OH, -(M) n COOR,
  • R 6 comprises -OH, -PO RR ' , -OPO RR ' ,
  • -CF 2 CO 2 R -CH 2 SO 3 R, -CF 2 SO 3 R, -CH 2 COCF 3 , -CF 2 COCF 3> -CH(PO 3 RR') 2 , -CH(OH)(PO 3 RR'), -CH(NH 2 )(PO 3 RR'), -CH(CO 2 R) 2 , -CF(CO 2 R) 2 ,
  • R 6 is -NRM m CO R' are illustrated by compounds in which R is H, -M m CO 2 R', -M m SO 0 R" or another substituted or unsubstituted lower aliphatic moiety. In some such compounds, one or more
  • -CH(PO 3 RR')(SO 3 R"), -CH(PO 3 RR')(SO 2 NH 2 ), -CH(SO 2 NH 2 ) 2 , or -CH(SO 3 RR') 2 moiety is H.
  • one or more of those R groups is -(M) m -CH 2 Z, -(M) m -CHZ 2 , -(M) m -CZ 3 , -R 15 , -M-O-CO-OR 15 or -M-O-CO-R 15 , where Z is halogen and R 15 is substituted or unsubstituted lower aliphatic, aryl or heterocyclic.
  • R is methyl, ethyl, n-propyl, i-propyl, n-butyl, isobutyl, t-butyl, n-amyl, sec-amyl, benzyl or substituted benzyl
  • M is CH 2 , CHR (e.g. CHCH 3 etc.) and the like. Further illustrations include -CH 2 -O-CO-OEt, -CH(Me)-O-CO-OEt, -CH 2 - O-CO-t-butyl, etc.
  • R and R are both H.
  • R is J, -A-(M) (substituted or unsubstituted aliphatic, aryl or heterocyclic),
  • R 8 is H.
  • R is lower alkyl, lower alkenyl, -OH,
  • -CH 2 PO 3 RR', -CF 2 PO 3 RR', -OCH 2 CO 2 R, -NHCH 2 CO 2 R, -CH 2 CO 2 R, -CF 2 CO 2 R, -SO 3 R, -CH 2 SO 3 R, or -CF 2 SO 3 R is H.
  • one or more of those R groups is -(M) m -CH 2.Z, -(M) m -CHZ 2, -(M) m -CZ, 3, -R 15 , -M-O-CO-OR 15 or -M-O-CO-R 15 ,
  • Z is halogen and R is substituted or unsubstituted lower aliphatic, aryl or
  • R is methyl, ethyl, n-propyl, i-propyl, n-butyl, isobutyl, t-butyl, n-amyl, sec-amyl, benzyl or substituted benzyl
  • M is CH 2 , CHR (e.g. CHCH 3 etc.) and the like.
  • R 6 comp ⁇ ses -APO RR ' (e.g., -OPO H ) and R 7 is
  • R and R are independently selected from J and K.
  • R is -C(R)(J)(K).
  • Illustrative compounds of this subgenus include those in which R 6 is -CH(J)(K) and those m which R 6 is -C(R)(PO R'R'')(K). The latter compounds are illustrated by embodiments in which none, one, two or three of the R groups of the -C(R)(PO R'R'')(K) moiety are H.
  • compounds of this invention which contain a moiety J, e.g., compounds of Formula I m which R is -C(R)(J)(K), include among others embodiments in which one or both of R and R' (e.g., of a -PO RR ' moiety) are R 15 ,
  • Z is halogen and R is substituted or unsubstituted lower aliphatic, aryl or heterocyclic (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, isobutyl, t-butyl, n-amyl, sec-amyl, benzyl or substituted benzyl), and M is CH 2 , CHR (e.g. CHCH 3 etc.) and the like.
  • R is substituted or unsubstituted lower aliphatic, aryl or heterocyclic (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, isobutyl, t-butyl, n-amyl, sec-amyl, benzyl or substituted benzyl)
  • M is CH 2 , CHR (e.g. CHCH 3 etc.) and the like.
  • each of R 9 , R 10 and R 11 is independently Z, R, -GR, -COR, -CO 2 R, or
  • -(M) n W-NRR' and R 12 is independently selected from -(M) Z, -(M) R, -(M) GR, -(M) n WR , and -(M) n WGR.
  • one or more of the R, R' and R" groups of R >9 , D R10 and R »11 comprise a halo, hydroxy, aliphatic, amino, amido or sulfonamido moiety.
  • one or more of R , R , R , and R is a substituted aliphatic moiety containing at least one substituent selected from substituted or unsubstituted cycloaliphatic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -COR, -CO 2 R, -CO-NRR', and -OR.
  • substituents selected from substituted or unsubstituted cycloaliphatic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -COR, -CO 2 R, -CO-NRR', and -OR.
  • R , R , R , and R comprises -(M) (cycloaliphatic), n
  • R 12 is -(M) OR, n is 1 or greater.)
  • R 11 , and R 12 comprise methyl, -(CH ) R 13 where q is 1-8 13
  • R comprises methyl; i-propyl; i-butyl; t-butyl; cycloaliphatic; phenyl; substituted phenyl; naphthyl; substituted naphthyl; a 5, 6 or 7-membered heterocyclic ring or a bicyclic heterocylic moiety.
  • R 12 comprises a formyl group on a ring nitrogen.
  • Possible substituents on the R, R' and R" groups include, among others, halo, hydroxy, alkyl, amino, amido and sulfonamido moieties. Other potential substituents are as disclosed elsewhere herein, including in the numerous specific examples.
  • Compounds of particular interest include those in which B is selected from the following:
  • compounds of particular interest include those compounds of Formula I, including those of the various subgenera and examples herein, which have the following structures:
  • R and R each comp ⁇ se an independently selected, substituted or unsubstituted lloowweerr aalliipphhaattiicc mmooiieettyy,, aanndd RR aanndd RR aarree eeaacchh iinnddependently selected from H. and a substituted or unsubstituted lower aliphatic moiety.
  • R and R are independently selected from H; linear, branched or cyclic lower aliphatic; -(CH 2 ) n COOR; -(CH 2 ) n CONRR ⁇ -(CH 2 ) n cycloahphat ⁇ c (substituted or unsubstituted); -(CH 2 ) n aryl (substituted or unsubstituted) or -(CH 2 ) n heterocycl ⁇ c (substituted or unsubstituted).
  • R comp ⁇ ses a linear, branched or cyclic lower aliphatic, -(CH ) 2 COOR, -(CH 2 ) 2 aryl (substituted or unsubstituted) or -(CH 2 ) 2 heterocychc
  • R comp ⁇ ses a linear, branched or cyclic lower aliphatic; and R and R are independently selected from H; linear, branched or cyclic lower aliphatic; -(CH 2 ) 2 COOR, -(CH 2 ) 2 CONH 2 ; -(CH 2 )cycloal ⁇ phat ⁇ c (substituted or unsubstituted); -(CH 2 )aryl (substituted or unsubstituted) or -(CH 2 )heterocycl ⁇ c
  • Thiazoles of Formula II may be useful as inhibitors of cellular signaling mediated by Src or Src family kinases, and thus may be useful in treating diseases such as osteoporosis and other bone resorptive disorders in patients in need thereof.
  • Such compounds may be used for example as immunosuppressants in patients who are going to receive, or have received, an organ or tissue transplant or in patients suffering an autoimmune disorder.
  • R 1 and R 5 each comprise an independently selected, substituted or unsubstituted lower aliphatic moiety, and R comprises H or a substituted or unsubstituted lower aliphatic moiety.
  • R comprises a linear, branched or cyclic lower aliphatic, -(M) n aliphatic (which may be substituted or unsubstituted), -(M) n COOR, -(M) n OR, -(M) n aryl (which may be substituted or unsubstituted) or -(CH 2 ) n heterocyclic (which may be substituted or unsubstituted);
  • R comprises a linear, branched or cyclic lower aliphatic; and R comprises H; linear, branched or cyclic lower aliphatic; -(M) COOR;
  • M is CH 2 .
  • R comprises a linear, branched or cyclic lower aliphatic, -(CH-) 2 COOR, -(CH 2 )aryl (substituted or unsubstituted) or -(CH 2 )heterocyclic (substituted or unsubstituted);
  • R comprises a linear, branched or cyclic lower aliphatic (substituted or unsubstituted); and R comprises H; linear, branched or cyclic lower aliphatic; -(CH 2 ) 2 COOR; -(CH 2 ) 2 CONH 2 ; -(CH 2 )cycloaliphatic (substituted or unsubstituted);
  • Oxadiazoles of Formula HI may be useful as inhibitors of cellular signaling mediated by Src or Src family kinases, and thus may be useful in treating diseases such as osteoporosis and other bone resorptive disorders in patients in need thereof.
  • Oxadiazoles of Formula III are further illustrated by the following types of compounds:
  • M is CH 2 .
  • R moieties are illustrated by the following:
  • one set of illustrative compounds of particular interest include those of the following sorts of structures:
  • Such oxadiazoles may be useful as inhibitors of cellular signaling mediated by ZAP-70 or ZAP- 70 family kinases, and thus may be useful in treating or preventing an inflammatory response in patients in need thereof.
  • Such compounds may be used for example as immunosuppressants in patients who are going to receive, or have received, an organ or tissue transplant or in patients suffering an autoimmune disorder.
  • Another set of illustrative oxadiazoles of particular interest include the following types of compounds:
  • R 6 comprises -PO 3 RR', -OPO 3 RR', -OSO 2 NRR', -(CH 2 )PO 3 RR ⁇ ⁇ -(CF 0 )PO RR' or -CRJK; and R comprises R (including among others, H, alkyl, alkenyl, etc.) -CN, amido, acylamino, J (e.g. -CO 2 R), or -CHO.
  • R comprises -PO 3 RR', -OPO 3 RR', -OSO 2 NRR', -(CH 2 )PO 3 RR ⁇ ⁇ -(CF 0 )PO RR' or -CRJK
  • R comprises R (including among others, H, alkyl, alkenyl, etc.) -CN, amido, acylamino, J (e.g. -CO 2 R), or -CHO.
  • J e.g. -CO 2 R
  • R comprises -OPO RR' or -(CF )PO RR' and R is H.
  • R groups including R', R", etc
  • R comprises -(M) -CH Z
  • R is substituted or unsubstituted lower aliphatic, aryl or heterocyclic.
  • R is methyl, ethyl, n-propyl, i-propyl, n-butyl, isobutyl, t-butyl, n-amyl, sec-amyl, benzyl or substituted benzyl
  • M is CH 2 , CHR (e.g. CHCH 3 etc.) and the like.
  • SH2 domains of current interest include those of a Src, Fyn, Lck. Yes, Blk. Lyn, Fgr, Hck, Yrk, ZAP-70, Syk, STAT or Abl protein.
  • compositions comprising a compound of this invention, or a pharmaceutically acceptable derivative thereof, and one or more pharmaceutically acceptable excipients.
  • Compounds of this invention can be administered to cells or to animals, preferably a mammal in need thereof, as a method for inhibiting SH2-mediated signal transduction therein.
  • the SH2-mediated signal transduction is mediated by a PDGF receptor protein, EGF receptor protein,
  • HER2/Neu receptor protein 20 HER2/Neu receptor protein, fibroblast growth factor receptor protein, focal adhesion kinase protein, pl30 protein, or p68 protein.
  • Cases in which a mammal may be m need of inhibition of SH2-med ⁇ ated signaling include cases in which the mammal has a prohferative disease, cancer, restenosis, osteoporosis, inflammation, allergies, or cardiovascular disease.
  • admimste ⁇ ng a therapeutically effective amount of the composition to the mammal, preferably to a human patient will constitute treating or preventing the prohferative disease, cancer, restenosis, osteoporosis, inflammation, allergic reaction, or cardiovascular disease in the recipient or a method for causing lmmunosuppression in the recipient.
  • prefened compounds of this invention include any of the foregoing compounds which yield an observable IC 50 value, when tested against an SH2 domain of interest and a pTyr-containmg peptide ligand (or mimic thereof) for that SH2 domain, of 50 ⁇ M or better, preferably 5 ⁇ M or better, more preferably 1 ⁇ M or better, and even more preferably, 500 nM or better, as determined by any scientifically valid measure, especially when the SH2 domain is from a Src, Fyn, Lck, Yes, Blk, Lyn, Fgr, Hck, Yrk, ZAP, Syk, STAT or Abl protein.
  • a pharmaceutical composition may be prepared containing a compound of this invention (including a pharmaceutically acceptable de ⁇ vative thereof) together with one or more pharmaceutically acceptable excipients.
  • a compound of this invention may be administered to a mammal in need thereof, preferably a human patient, as a method for inhibiting SH2-med ⁇ ated signal transduction m the recipient mammal.
  • the compound may be selected based on its ability to specifically bind to an SH2 domain, e.g of Src, ZAP-70, Syk, or STAT 6, etc., or on its ability to inhibit a signal transduction pathway mediated by an SH2 domam-contaming protein.
  • Such use of an approp ⁇ ately selected compound of this invention thus provides a method for inhibiting SH2-med ⁇ ated signal transduction which is mediated by a PDGF receptor protein, EGF receptor protein, HER2/Neu receptor protein, fibroblast growth factor receptor protein, focal adhesion kinase protein, pi 30 protein, or p68 protein.
  • Use of a compound of this invention may be particularly advantageous m cases in which the mammal has a prohferative disease, cancer, restenosis, osteoporosis, inflammation, allergies, or cardiovascular disease.
  • a therapeutically effective amount of a compound of this invention preferably in the form of a pharmaceutical composition, provides a method for
  • this invention provides a novel class of compounds useful as inhibitors of signal transduction pathways mediated by the interaction of protein receptors for phosphotyrosine-contaming proteins, such as proteins containing one or more SH2 domains, with their phosphotyrosine-contaming hgands.
  • Compounds of this invention comp ⁇ se those of Formula I, set forth above, and are illustrated in part by the va ⁇ ous classes, subgenera and subsets of compounds noted above, and by the va ⁇ ous subgenera and species disclosed elsewhere herein.
  • the compound may be m the form of an individual enantiomer, diastereomer or geomet ⁇ c isomer, or may be in the form of a mixture of stereoisomers.
  • pharmaceutically acceptable de ⁇ vatives of the foregoing compounds, where the phrase "pharmaceutically acceptable de ⁇ vative" denotes any pharmaceutically acceptable salt, ester, or salt of such ester, of such compound, or any other adduct or de ⁇ vative which, upon administration to a patient, is capable of providing (directly or indirectly) a compound as otherwise desc ⁇ bed herein, or a metabolite or residue thereof, preferably one which is a signal transduction inhibitor.
  • Pharmaceutically acceptable de ⁇ vatives thus include among others pro-drugs.
  • a pro-drug is a de ⁇ vative of a compound, usually with significantly reduced pharmacological activity, which contains an additional moiety which is susceptible to removal w vivo yielding the parent molecule as the pharmacologically active species
  • An example of a pro-drug is an ester which is cleaved in vivo to yield a compound of interest.
  • Pro-drugs of a va ⁇ ety of compounds, and mate ⁇ als and methods for denvatizmg the parent compounds to create the pro-drugs are known and may be adapted to the present invention.
  • aliphatic as used herein includes both saturated and unsaturated, straight chain (i.e., unbranched), branched, cyc c, or polycychc aliphatic hydrocarbons, which are optionally substituted with one or more functional groups.
  • alkyl, other aliphatic, alkoxy and acyl groups preferably contain 1-8, and m many cases 1- 6, contiguous aliphatic carbon atoms.
  • Illustrative aliphatic groups thus include, for example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, -CH 2 -cyclopropyl, allyl, n- butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl, -CH 2 -cyclobutyl, n-pentyl, sec-pentyl,
  • Aliphatic, heteroaliphatic, aryl and heterocyclic substituents may themselves be substituted or unsubstituted (e.g. mono-, di- and tri-alkoxyphenyl; methylenedioxyphenyl or ethylenedioxyphenyl; halophenyl; or -phenyl-C(Me) 2 -CH 2 -O-CO-[C3-C6] alkyl or alkylamino). Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples which follow.
  • alkyl includes both straight, branched and cyclic alkyl groups.
  • alkenyl alkynyl
  • alkynyl alkynyl
  • the language “alkyl”, “alkenyl”, “alkynyl” and the like encompasses both substituted and unsubstituted groups.
  • alkyl refers to groups usually having one to eight, preferably one to six carbon atoms.
  • alkyl may refer to methyl, ethyl, n-propyl, isopropyl, cyclopropyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, pentyl, isopentyl tert-pentyl, cyclopentyl, hexyl, isohexyl, cyclohexyl, and the like.
  • Suitable substituted alkyls include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 3- fluoropropyl, hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, benzyl, substituted benzyl and the like.
  • alkenyl refers to groups usually having two to eight, preferably two to six carbon atoms.
  • alkenyl may refer to prop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl, hex-5-enyl, 2,3-dimethylbut-2-enyl, and the like.
  • alkynyl which also refers to groups having two to eight, preferably two to six carbons, includes, but is not limited to, prop-2-ynyl, but-2-ynyl, but-3-ynyl, pent-2-ynyl, 3-methylpent-4-ynyl, hex-2-ynyl, hex-5-ynyl, and the like.
  • cycloalkyl refers specifically to groups having three to seven, preferably three to ten carbon atoms. Suitable cycloalkyls include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in
  • heteroaliphatic refers to aliphatic moieties which contain one or more oxygen, sulfur, nitrogen, phosphorous or silicon atoms, e.g., m place of carbon atoms. Heteroaliphatic moieties may be branched, unbranched or cyclic and include heterocycles such as morpho no, pyrro dinyl, etc.
  • heterocycle refers to cyclic heteroaliphatic and heteroaryl groups and preferably three to ten ⁇ ng atoms total, mcludes, but is not limited to heteroaliphatic moieties such as oxetane, tetrahydrofuranyl, tetrahydropyranyl, azi ⁇ dme, azetidme, pyrrolidme, pipe ⁇ dme, morpho ne, piperazme and the like, and heteroaryl moieties as desc ⁇ bed below.
  • aryl and “heteroaryl” as used herein refer to stable mono- or polycychc, heterocyclic, polycychc, and polyheterocyc c unsaturated moieties havmg 3 - 14 carbon atom which may be substituted or unsubstituted.
  • Substituents include any of the previously mentioned substituents
  • Non-limiting examples of useful aryl ⁇ ng groups include phenyl, halophenyl, alkoxyphenyl, dialkoxyphenyl, t ⁇ alkoxyphenyl, alkylenedioxyphenyl, naphthyl, phenanthryl, anthryl, phenanthro and the like.
  • heteroaryl ⁇ ngs examples include 5-membered monocychc ⁇ ng groups such as thienyl, pyrrolyl, lmidazolyl, pyrazolyl, furyl, isothiazolyl, furazanyl, oxazolyl, isoxazolyl, thiazolyl, oxadiazolyl and the like; 6-membered monocychc groups such as py ⁇ dyl, pyrazmyl, py ⁇ midmyl, py ⁇ dazinyl, t ⁇ azmyl and the like; and polycychc heterocyclic ⁇ ng groups such as benzo[b]th ⁇ enyl, naphtho[2,3-b]th ⁇ enyl, thianthrenyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathienyl, mdo zinyl, isoindolyl, indolyl, ind
  • the aryl or heteroaryl moieties may be substituted with one to five members selected from the group consisting of hydroxy, C1-C8 alkoxy, C1-C8 branched or straight-chain alkyl, acyloxy, carbamoyl, ammo, N-acylamino, mtro, halo, t ⁇ halomethyl, cyano, and carboxyl
  • Aryl moieties thus include, e.g phenyl; substituted phenyl bea ⁇ ng one or more substituents selected from groups including: halo such as chloro or fluoro, hydroxy, C1-C6 alkyl, acyl, acyloxy, C1-C6 alkoxy (such as methoxy or ethoxy, including among others dialkoxyphenyl moieties such as 2,3-, 2,4-, 2,5-, 3,4- or 3,5-d ⁇ methoxy or diethoxy phenyl or such
  • a "halo" substituent may be fluoro, chloro, bromo or iodo.
  • Compounds of this invention may be evaluated in a variety of assays to determine their relative ability to bind to a receptor for a pTyr-containing ligand, such as a protein containing one or more SH2 or PI domains, or to otherwise inhibit an intermolecular interaction mediated by such a domain. See e.g. US 5667980 (Pawson; competitive binding assays), PCT/US97/02635 (Rickles et al; cell-based assays) and PCT/US97/06746 (Lynch et al, FP assays). Compounds may also be evaluated for their selectivity of binding to one such receptor (or family of receptors) relative to another such receptor (or family of receptors).
  • a pTyr-containing ligand such as a protein containing one or more SH2 or PI domains
  • the compounds of this invention can be further evaluated by conventional methods for possible therapeutic applications, including evaluations of toxicological and pharmacological activity.
  • the compounds may further be evaluated for activity in inhibiting cellular or other biological events mediated by a pathway involving the molecular interaction of interest using a suitable cell-based assay or an animal model.
  • Cell-based assays and animal models suitable for evaluating inhibitory activity of a test compound with respect to a wide variety of cellular and other biological events are known
  • compounds which bind to an SH2 domain involved in the transduction of a signal leading to asthma or allergic episodes may be evaluated in a mast cell or basophil degranulation assay.
  • the inhibitory activity of a test compound identified as an SH2 inhibitor by the method of this invention with respect to cellular release of specific mediators such as histamine, leukotrienes, hormonal mediators and/or cytokines, as well as its biological activity with respect to the levels of phosphatidylinositol hydrolysis or tyrosine phosphorylation can be characterized with conventional in vitro assays as an indication of biological activity.
  • mediators such as histamine, leukotrienes, hormonal mediators and/or cytokines
  • histamine release can be measured by a radioimmunoassay using a kit available from AMAC Inc. (Westbrook, ME).
  • AMAC Inc. Westbrook, ME
  • Inhibitors of this invention may also be tested in an ex vivo assay, e.g., for their ability to block antigen-stimulated contraction of sensitized guinea pig tracheal strip tissue. Activity in this assay has been shown to be useful in predicting the efficacy of potential anti-asthma drugs.
  • compounds which bind to an SH2 or other domain of interest involved in the transduction of a signal involved in the initiation, maintenance or spread of cancerous growth may be evaluated in relevant conventional in vitro and in vivo assays. See e.g., Ishii et al., I. Antibiot.. XLII: 1877- 1878 (1989); and US Patent 5,206,249 (issued 27 April 1993).
  • Compounds which bind to a Src SH2 domain or which otherwise inhibit Src- mediated signaUng may be evaluated for activity in a variety of assays considered predictive of activity in treating or preventing osteoporosis.
  • assays include the various pit assays and calvaria assays, among others. Illustrative assays are described below.
  • MURINE CALVARIA ASSAY In osteoporosis, excessive bone resorption results in decreased bone density. In vivo and in vitro models of bone resorption are used to study the processes leading to osteoporosis. In vitro, fetal rat long bone and murine calvaria cultures are routinely used. Both models display similar responses to parathyroid hormone (PTH), a physiological modulator of bone reso ⁇ tion (Stern, P.H. and N.S. Krieger. Comparison of fetal rat limb bones and neonatal mouse calvaria: effects of parathyroid hormone and 1,25- dihydroxyvitamin D 3 . Calcif. Tissue Int. 35: 172-176, 1983).
  • PTH parathyroid hormone
  • Stern, P.H. and N.S. Krieger Comparison of fetal rat limb bones and neonatal mouse calvaria: effects of parathyroid hormone and 1,25- dihydroxyvitamin D 3 . Calcif. Tissue Int. 35: 172-176
  • the calvaria model of bone reso ⁇ tion can be successfully used to screen osteotropic compounds as has been previously shown (Green, J.R., K. Muller and K. Jaeggi. Preclinical pharmacology of CGP 42'446, a new, potent, heterocyclic bisphosphonate compound. J. Bone Miner. Res. 9: 745-751, 1994.).
  • calvaria culture model tests the ability of anti-reso ⁇ tive compounds to prevent reso ⁇ tion (prophylactic model).
  • a second model tests the ability of these compounds to terminate ongoing reso ⁇ tion (therapeutic model). Cytotoxicity may be assessed in both models using a lactate dehydrogenase (LDH) assay.
  • LDH lactate dehydrogenase
  • DMEM Dulbecco's Modified Eagle's Medium
  • D-012 Dulbecco's Modified Eagle's Medium
  • a lx solution is prepared using ultrafiltered water.
  • a suitable media contains 15% heat inactivated horse serum (Sigma, H 1270).
  • 27 concentration is adjusted to 1.65 to 1.83 mM using 0.2 M CaCl 2 .
  • Penicillin (100 U/ml) and streptomycin (0.1 mg/ml) are added to the final media preparation.
  • Indomethacin is prepared to 0.5 mg/ml (1.397 x 10 M) in ethanol, and is added to an aliquot of DMEM to produce a final concentration of 0.5 ⁇ M.
  • Bovine parathyroid hormone (1-34) may be obtained from Bachem (PCAL 100).
  • PTH is solubilized in 0.1 % BSA and is then diluted in DMEM to produce a final concentration of 10 M PTH.
  • Ten-fold serial dilutions are performed down to 10 M.
  • CD-I mice may be obtained from Charles River and are subjected to parturition. Neonatal mice (4-6 days) are cleansed with betadine and then euthanized by decapitation. Adherent skin is cleared away from the skull, exposing the calvaria. The calvaria are dissected away from the skull using a 12B scalpel. Calvaria are immediately placed into a glass petri dish containing room temperature Tyrode's Salt Solution (Sigma, T-2397). The calvaria are trimmed free of cartilage and bisected with a scalpel along the sagital suture. After dissection of all calvaria, calvaria are transferred into 24 well plates containing 0.5 ⁇ M indomethacin (Sigma, 1-7378).
  • calvaria are thoroughly washed in indomethacin-free DMEM. Calvaria are then transferred to new wells containing various PTH concentrations, and are cultured for an additional 72 hours. Media samples (30 ⁇ l) are obtained every 24 hours and assayed for calcium and LDH activity.
  • the calvaria are washed free of indomethacin using DMEM. Calvaria are then transferred to new wells containing DMEM or various concentrations of PTH. After 24 hours calvaria are transferred into new wells
  • a commercially available diagnostic calcium assay (Sigma, No. 588-3), modified for use in a microtiter format, may be used to determine circulating serum calcium concentrations.
  • This colorimetric assay is dependent on the specific, high affinity complexation of calcium with arsenazo HI dye under acidic conditions, which occurs with 1:1 stoichiometry and absorbs at 600 nm (Bauer, P.J. Affinity and stoichiometry of calcium binding by Arsenazo HI. Anal Biochem, 110:61, 1981; Michaylova, V and P Ilkova. Photometric determination of micro amounts of calcium with Arsenazo HI. Anal Chim Acta, 53: 194, 1971). Magnesium has very low affinity for arsenazo HI.
  • Phosphate buffer is prepared in distilled water (0.26 M K 2 HPO 4 '3H 2 O, 0.26 M
  • Ten ⁇ l of media samples obtained from incubated calvaria are added to 96 well plates. Wells containing 10 ⁇ l of DMEM serve as blanks. To each well, 90 ⁇ l distilled water and 150 ⁇ l phosphate mix is added. 50 ⁇ l NADH is added using an eight channel pipette immediately before the plate is read on a microtiter plate reader at 340 nm. A kinetic assay is performed for 10 minutes, with a read interval of 20 seconds.
  • Parathyroid hormone (PTH) replacement in thyroparathyroidectomized (TPTX) rats is routinely used as an in vivo model of controlled bone reso ⁇ tion. Rats are the species of choice since the mechanisms of bone modeling in the rat resemble those in humans. In addition, hormones and pharmacologic agents have similar effects on both rat and human bone (Frost, H.M. and W.S.S. Jee. On the rat model of human osteopenias and osteoporoses. Bone and Mineral, 18: 227-236, 1992). Removal of the thyroid and parathyroid glands results in a rapid loss of parathyroid hormone (PTH) from the circulation.
  • PTH parathyroid hormone
  • mice Female Wistar rats (226-250 gm, Charles River) are fasted overnight and anesthetized with 0.15 ml of 1.2% tribromoethanol (TBE).
  • TBE tribromoethanol
  • the ventral neck area is shaved and swabbed with betadine and isopropanol.
  • a midline incision is made in the neck through the skin and superficial muscle layer, as well as in the stemohyoid muscle. Blunt dissection is performed to expose the thyroid gland.
  • the thyroid gland is carefully isolated from the trachea, thyrohyoid muscle, as well as adjacent nerves and blood vessels, using blunt dissection.
  • the thyroid gland is excised one lobe at a time. Cautery is performed for hemostasis.
  • Rats are fed at least 5 grams, but not more than 10 grams, of food Rats consuming less than 3.0 grams of food receive the nutritional supplement Nut ⁇ -Cal p.o. (Evsco; ⁇ 0.0033% calcium).
  • rats which are found to be hypocalcemic, based on day 2 serum calcium levels, are implanted with PTH-contammg Alzet mini-osmotic pumps (ALZA, model 2001D) which pumps at a rate of 1 ⁇ l/h.
  • the rats are anesthetized with ketarmne (50 mg/kg, l.p.) and acepromazine (1.67 mg/kg, ⁇ .p.).
  • the scapula region is shaved and prepared for surgery with betadine and isopropanol. A lateral incision of approximately 2 cm in length is made between the scapulae.
  • Bovme parathyroid hormone 1-34 (Bachem California, PCAL100) is prepared in vehicle (10 "3 N HCl, 0.15 M NaCl, 20 mg/ml cysteme ⁇ Cl) at the following concentrations: 0 156, 0.47, 1.56, 4.7, 15.6, and 156 ⁇ M.
  • Alzet mini-osmotic pumps are filled with the PTH solution and maintained in 37 » C saline for 4 hours p ⁇ or to implantation.
  • Rats are anesthetized by CO 2 from dry ice and daily blood samples are obtained via cardiac puncture using a 27 gauge needle. Baseline samples are taken just p ⁇ or to TPTX Daily samples are obtained m the morning. Samples are allowed to clot on their side for several hours and subsequently spun at lOOOxg for 15 minutes to obtain serum Serum is ahquoted and stored in the ref ⁇ gerator until assayed for serum calcium Serum calcium is measured (see above) daily for at least 7 days following TPTX.
  • Compounds of this invention which bind to an SH2 domain of interest may be used as biological reagents in assays as desc ⁇ bed herein for functional classification of a pTyr- bindmg domain (e g. SH2 or PI domain) of a particular protein, particularly a newly discovered protein. Families or classes of such proteins which bind to pTyr-contaimng hgands may now be defined functionally, with respect to ligand specificity Moreover, compounds of this invention can be used to inhibit the occurrence of biological events resulting from molecular interactions mediated by the protein of interest Inhibiting such
  • Such compounds would be useful, for example, in the diagnosis, prevention or treatment of conditions or diseases resulting from a cellular processes mediated by the binding of a pTyr-containing ligand with a receptor therefor.
  • a patient can be treated to prevent the occurrence or progression of osteoporosis or to reverse its course by administering to the patient in need thereof an SH2inhibitor which selectively binds Src SH2 or otherwise interferes with Src-mediated signaling.
  • Still other relevant applications include the prevention of interferon-, growth factor-, or cytokine-mediated diseases (e.g. inflammatory diseases) by targeting the interaction of STAT proteins with their pTyr-containing ligands or otherwise inhibiting their signal transduction pathways.
  • cytokine-mediated diseases e.g. inflammatory diseases
  • Agents that block the SH2 domains of ZAP-70 or otherwise inhibit ZAP-70-mediated signaling would be candidates for the treatment of immune-related disorders such as rejection of transplanted bone marrow, skin or other organs; rheumatoid arthritis; inflammatory bowel disease; and systemic lupus erythmatosis, and a variety of autoimmune diseases.
  • compounds of this invention which inhibit cellular signal transduction may be used in pharmaceutical compositions and methods for treatment or prevention in a subject in need thereof.
  • Such inhibitors can be used to treat or reduce the risk of the diseases or their pathological effects mediated by such interactions.
  • drugs that completely block one of the two ZAP SH2 domains should effectively prevent ZAP from associating with the activated TCR and thus block T cell activation.
  • a ZAP antagonist or inhibitor would specifically inhibit T cells and avoid the toxicity of the currently used immunosuppressive drugs, FK506 and cyclosporin, which target the more ubiquitously expressed protein, calcineurin. Since calcineurin is required for cellular activities in several tissues in addition to T cells, cyclosporin and FK506 cause side effects in the kidney and central nervous system which limit their application largely to patients with organ transplant rejection.
  • compositions of this invention can exist in free form or, where approp ⁇ ate, m salt form.
  • Pharmaceutically acceptable salts of many types of compounds and their preparation are well-known to those of skill in the art.
  • the pharmaceutically acceptable salts of compounds of this invention include the conventional non-toxic salts or the quaternary ammonium salts of such compounds which are formed, for example, from inorganic or organic acids of bases.
  • the compounds of the invention may form hydrates or solvates. It is known to those of skill in the art that charged compounds form hydrated species when lyophilized with water, or form solvated species when concentrated in a solution with an approp ⁇ ate organic solvent.
  • This invention also relates to pharmaceutical compositions comp ⁇ sing a therapeutically (or prophylactically) effective amount of the compound, and a pharmaceutically acceptable earner or excipient.
  • Car ⁇ ers include e g. saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof, and are discussed in greater detail below.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffe ⁇ ng agents.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • the composition can be formulated as a suppository, with traditional binders and car ⁇ ers such as t ⁇ glyce ⁇ des.
  • Oral formulation can include standard car ⁇ ers such as pharmaceutical grades of manmtol, lactose, starch, magnesium stearate, sodium saccha ⁇ ne, cellulose, magnesium carbonate, etc. Formulation may involve mixing, granulating and compressing or dissolving the ingredients as approp ⁇ ate to the desired preparation.
  • the pharmaceutical earner employed may be, for example, either a solid or liquid
  • Illustrative solid earner include lactose, te ⁇ a alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stea ⁇ c acid and the like.
  • a solid earner can include one or more substances which may also act as flavo ⁇ ng agents, lub ⁇ cants, solubilizers, suspending agents, fillers, ghdants, compression aids, binders or tablet-disintegrating agents; it can also be an encapsulating mate ⁇ al.
  • the earner is a finely divided solid which is in admixture with the finely divided active ingredient.
  • the active ingredient is mixed with a earner havmg the necessary compression properties in suitable proportions ,and compacted m the shape and size desired
  • the powders and tablets preferably contain up to 99% of the active ingredient.
  • Suitable solid earners include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dext ⁇ n, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrro dme, low melting waxes and ion exchange resins.
  • Illustrative liquid carriers include syrup, peanut oil, olive oil, water, etc. Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions.
  • the active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats.
  • the liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators.
  • liquid carriers for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil).
  • the carrier can also be an oily ester such as ethyl oleate and isopropyl myristate.
  • Sterile liquid carders are useful in sterile liquid form compositions for parenteral administration.
  • the liquid carrier for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellant.
  • Liquid pharmaceutical compositions which are sterile solutions or suspensions can be utilized by, for example, intramuscular, intraperitoneal or subcutaneous injection. Sterile solutions can also be administered intravenously.
  • the compound can also be administered orally either in liquid or solid composition form.
  • the carrier or excipient may include time delay material well known to the art, such as glyceryl monostearate or glyceryl distearate along or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate and the like.
  • time delay material such as glyceryl monostearate or glyceryl distearate along or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate and the like.
  • Tween 80 in PHOSAL PG-50 phospholipid concentrate with 1,2-propylene glycol, A. Nattermann & Cie. GmbH
  • PHOSAL PG-50 phospholipid concentrate with 1,2-propylene glycol, A. Nattermann & Cie. GmbH
  • a wide variety of pharmaceutical forms can be employed. If a solid carrier is used, the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form or in the form of a troche or lozenge. The amount of solid carrier will vary widely but preferably will be from about 25 mg to about 1 g. If a liquid carrier is used, the preparation will be in the form of a syrup, emulsion, soft gelatin capsule, sterile injectable solution or suspension in an ampule or vial or nonaqueous liquid suspension.
  • a pharmaceutically acceptable salt of the compound may be dissolved in an aqueous solution of an organic or inorganic acid, such as a 0.3M solution of succinic acid or citric acid.
  • an organic or inorganic acid such as a 0.3M solution of succinic acid or citric acid.
  • acidic derivatives can be dissolved in suitable basic solutions. If a soluble salt form is not available, the
  • suitable cosolvents include, but are not limited to, alcohol, propylene glycol, polyethylene glycol 300, polysorbate 80, glycerin, polyoxyethylated fatty acids, fatty alcohols or glycerin hydroxy fatty acids esters and the like in concentrations ranging from 0-60% of the total volume.
  • Various delivery systems are known and can be used to administer the compound, or the various formulations thereof, including tablets, capsules, injectable solutions, encapsulation in liposomes, microparticles, microcapsules, etc.
  • Methods of introduction include but are not limited to dermal, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, pulmonary, epidural, ocular and (as is usually preferred) oral routes.
  • the compound may be administered by any convenient or otherwise appropriate route, for example by infusion or bolus injection, by abso ⁇ tion through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • prefened routes of administration are oral, nasal or via a bronchial aerosol or nebulizer.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic to ease pain at the side of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • Administration to an individual of an effective amount of the compound can also be accomplished topically by administering the compound(s) directly to the affected area of the
  • the compound is administered or applied in a composition including a pharmacologically acceptable topical carrier, such as a gel, an ointment, a lotion, or a cream, which includes, without limitation, such carriers as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oils.
  • a pharmacologically acceptable topical carrier such as a gel, an ointment, a lotion, or a cream, which includes, without limitation, such carriers as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oils.
  • Topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5%) in water, or sodium lauryl sulfate (5%) in water.
  • Other materials such as anti-oxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary.
  • Percutaneous penetration enhancers such as Azone may also be included.
  • the compound may be disposed within devices placed upon, in, or under the skin.
  • Such devices include patches, implants, and injections which release the compound into the skin, by either passive or active release mechanisms.
  • Materials and methods for producing the various formulations are well known in the art and may be adapted for practicing the subject invention. See e.g. US Patent Nos. 5,182,293 and 4,837,311 (tablets, capsules and other oral formulations as well as intravenous formulations) and European Patent Application Publication Nos. 0 649 659 (published April 26, 1995; illustrative formulation for IV administration) and 0 648 494 (published April 19, 1995; illustrative formulation for oral administration).
  • the effective dose of the compound will typically be in the range of about 0.01 to about 50 mg/kgs, preferably about 0.1 to about 10 mg/kg of mammalian body weight, administered in single or multiple doses.
  • the compound may be administered to patients in need of such treatment in a daily dose range of about 1 to about 2000 mg per patient.
  • the amount of compound which will be effective in the treatment or prevention of a particular disorder or condition will depend in part on the nature and severity of the disorder or condition, which can be determined by standard clinical techniques.
  • in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the precise dosage level should be determined by the attending physician or other health care provider and will depend upon well known factors, including route of administration, and the age, body weight, sex and general health of the individual; the nature, severity and clinical stage of the disease; the use (or not) of concomitant therapies.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of
  • the invention Optionally associated with such contamer(s) can be a notice m the form presc ⁇ bed by a governmental agency regulating the manufacture, use or sale of pharmaceutical products, which notice reflects approval by the agency of manufacture, use or sale for human administration
  • Compounds of this invention can be prepared by convergent synthesis as illustrated in the following examples.
  • a carboxylic acid e.g., Ac-Tyr(PO3Bn2)- OH or Boc-Tyr(PO3Bn2)-OH, for example, is coupled with a heterocyclic amine using standard materials and methods for peptide coupling, including any necessary or desired protection and subsequent deprotection.
  • Compounds such as Compound ZZ1 can be prepared according to General Methods A-G, as exemplified by the following examples.
  • General Methods C and D exemplify typical coupling strategies, which can be modified with obvious standard manipulations and as shown here in General Methods A-G as well as in the following General Methods H-ZT.
  • thiazoles and oxazoles can be prepared via standard Hantzsch thiazole synthesis with alpha halo ketones and thioamides or amides.
  • Compounds such as Compound A can be prepared using thiazole formation Conditions A followed by Coupling Conditions C. Expe ⁇ mental details for the synthesis of Compound A are as follows
  • Bromomethyl ketone 2 can be prepared according to the procedure found in Hajos, Z G , Wachter, M P , Werblood, H. M.; Adams, R. E. J. Org. Chem 1984, 49, 2600.
  • the intermediate amide can be prepared according to the procedure found in Nozaki, S.; Muramatsu, I. Bull. Chem. Soc. Jpn. 1988, 61, 2647.
  • Thiazole 4 can be prepared according to Aguilar, E.; Meyers, A. I. Tetrahedron Lett. 1994, 35, 2473 and Bredenkamp, M. W.; Holzapfel, C. W.; van Zyl, W. J. Synth. Commun. 1990, 20, 2235.
  • Compounds such as Compound B can be prepared using thiazole formation Conditions A followed by Coupling Conditions D. Experimental details for the synthesis of compound B are as follows:
  • Bromoketone 9 was prepared as bromoketone 2 except that TMSCI was used rather than the centrifugate from 1:1 Et3N-TMSCl. Flash chromatography (elution with 6:1 hexanes- Et2 ⁇ ) gave 1.8 g (50.2%) of a very volatile pale oil: Rf 0.38 (6:1 hexanes-Et2 ⁇ ).
  • Thiazole 10 can be prepared as shown for the preparation of thiazole 4. Flash chromatography (elution with 3:2 hexanes-Et2 ⁇ ) gave 0.95 g (80.5%) of a pale oil: Rf 0.50 (1:1 hexanes-Et2 ⁇ ).
  • Dibenzyl phosphate 12 can be prepared according to the procedure found in Silverberg, L. J.; Dillon, J. L.; Vemishetti, P. Tetrahedron Lett. 1996, 37, 111.
  • Compounds such as Compound C can be prepared using thiazole formation Conditions B followed by Coupling Conditions D. Experimental details for the synthesis of Compound C are as follows:
  • Thioamide 15 can be prepared following the procedure described for the preparation of thioamide 3 using the commercially available N-Cbz-glycinamide as the starting material. Flash chromatography (elution with 2:1 hexanes-EtOAc) provided 4.05 g (75.1%) of a white solid: Rf 0.50 (1: 1 hexanes-EtOAc). Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 449 (M+H).
  • Coupling product 18 can be prepared as shown for the preparation of coupling product 6. Flash chromatography (elution with 10:1 CH2Cl2-MeOH) gave 260.7 mg (51.0%) of a pale oil: Rf 0.59 (9:1 CH2CI2 -MeOH).
  • Compounds such as Compound D can be prepared using thiazole formation Conditions A followed by Coupling Conditions C. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 496 (M+H).
  • Compounds such as Compound E can be prepared using thiazole formation Conditions B followed by Coupling Conditions D. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 854 (2M-H).
  • Compounds such as Compound F can be prepared using thiazole/oxazole formation Conditions B, substituting the appropriate keto amide for the bromo ketone and substituting toluene as solvent, followed by Coupling Conditions D. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 412 (M+H).
  • Compounds such as Compound G can be prepared using thiazole formation Conditions B followed by Coupling Conditions D. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 476 (M+H).
  • Compounds such as Compound H can be prepared using thiazole formation Conditions B followed by Coupling Conditions D. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 472 (M+H).
  • Compounds such as Compound I can be prepared using thiazole formation Conditions B followed by Coupling Conditions D. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 550 (M-H).
  • Compounds such as Compound J can be prepared using thiazole formation Conditions B followed by Coupling Conditions D with the appropriate side chain manipulations as shown in the following scheme. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 574 (M+CH3CN).
  • Compounds such as Compound K can be prepared using thiazole formation Conditions A followed by Coupling Conditions D. Compound K was isolated as a 60/40 mixture of diastereomers, presumably at the NHAc stereocenter. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 542 (M+H).
  • thiazoles, oxazoles and imidazoles can be prepared via the conesponding keto amides according to the cyclization conditions described in Gordon, T. D. et al. (Tetrahedron Lett. 1993. 34, 1901) as shown generically below.
  • Compounds such as Compound L can be prepared according to General Method E, followed by Coupling Conditions D. Experimental details for the synthesis of compound L are as follows:
  • Compound L can then be prepared using amine 27 according to Coupling Conditions D Electrospray Mass Spectrum (50/50 acetomt ⁇ le/water + 0.1% ammonium hydroxide) m/z 595 (M+H)
  • Compounds such as Compound M can be prepared using General Method E using the commercially available amino ketone followed by Coupling Conditions D. Electrospray Mass Spectrum (50/50 acetonit ⁇ le/water + 0.1% ammonium hydroxide) m/z 476 (M+H).
  • Compounds such as Compound N can be prepared using General Method E followed by Coupling Conditions D. Compound N was isolated as a 60/40 mixture of diastereomers, presumably at the NHAc stereocenter. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 582 (M+H).
  • Compounds such as Compound O can be prepared using General Method E followed by Coupling Conditions D with the appropriate side chain manipulations as shown in the following scheme. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 500 (M+H).
  • Compounds such as Compound P can be prepared using General Method E using the commercially available amino ketone followed by Coupling Conditions D. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 546 (M-H).
  • Compounds such as Compound Q can be prepared using General Method E followed by Coupling Conditions D. Reverse phase HPLC resulted in the separation of two diastereomers, presumably at the NHAc stereocenter. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 566 (M+H).
  • Compounds such as Compound R can be prepared using General Method E followed by Coupling Conditions D with the appropriate side chain manipulations as shown in the following scheme. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 575 (M+H).
  • Compounds such as Compound S can be prepared using General Method E followed by Coupling Conditions D with the appropriate side chain manipulations included according to the following scheme using the aldehyde (30) described for the preparation of compound R. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 603 (M+H).
  • Compounds such as Compound T can be prepared using General Method E followed by Coupling Conditions D similar to the preparation of Compound O with the approp ⁇ ate side chain manipulations as shown in the following scheme. Electrospray Mass Spectrum (50/50 acetonit ⁇ le/water + 0.1% ammonium hydroxide) m/z 485 (M+H).
  • Compounds such as Compound W can be prepared using General Method E followed by Coupling Conditions D using the approp ⁇ ate am o alcohol as desc ⁇ bed m the preparation of Compounds U and R. Electrospray Mass Spectrum (50/50 acetonit ⁇ le/water + 0.1% ammonium hydroxide) m/z 542 (M+H).
  • Compounds such as Compound X can be prepared using General Method E followed by Coupling Conditions D with the approp ⁇ ate side chain manipulations as shown in the following scheme using the intermediate alcohol 29 found in the preparation of Compound R.
  • Electrospray Mass Spectrum (50/50 acetonit ⁇ le/water + 0.1% ammonium hydroxide) m/z 589 (M+H)
  • Compounds such as Compound Y can be prepared using amino alcohol 28, prepared using General Method E as found in the preparation of Compound U, followed by Coupling Conditions D with the appropriate side chain manipulations as shown in the following scheme. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 576 (M+H).
  • Compounds such as Compound Z can be prepared using General Method E followed by Coupling Conditions D along with the appropriate side chain manipulations as shown in the scheme shown for the preparation of Compound S. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 583 (M+H).
  • Compounds such as Compound AA can be prepared using General Method E followed by Coupling Conditions D with the approp ⁇ ate side chain manipulations analogous to those shown for the preparation of Compound L. Electrospray Mass Spectrum (50/50 acetomt ⁇ le/water + 0.1% ammonium hydroxide) m/z 637 (M-H).
  • Compounds such as Compound AB can be prepared using General Method E followed by Coupling Conditions D. Electrospray Mass Spectrum (50/50 acetonit ⁇ le/water + 0 1% ammonium hydroxide) m/z 512 (M+H).
  • Compounds such as Compound AC can be prepared using General Method E followed by Coupling Conditions D using the appropriate ammo alcohol as in the preparation of Compounds U and W. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 542 (M+H).
  • Compounds such as Compound AD can be prepared using General Method E followed by Coupling Conditions D similar to the preparation of Compound O with the approp ⁇ ate side chain manipulations as shown in the following scheme Electrospray Mass Spectrum (50/50 acetonit ⁇ le/water + 0.1% ammonium hydroxide) m/z 466 (M-H)
  • Compounds such as Compound AE can be prepared using General Method E followed by Coupling Conditions D as in the preparation of Compounds U and W, using the appropriate amino alcohol which can be prepared as shown in the scheme below. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 582 (M-H).
  • BocHN ' 1. 3 N HCI ⁇ -BuLi, DME 2. Boc-Ala-OSu -78 °C
  • Compounds such as Compound AF can be prepared using General Method E followed by Coupling Conditions D with side chain manipulations as shown for the preparation of
  • oxadiazoles can be prepared via cyclization of the appropriate O-acylamidoxime according to the method described in Borg et al. (J. Org. Chem. 1995, 60, 3112).
  • the required amidoxime can be prepared via the conesponding nitrile as shown below.
  • the resulting amine can then be further elaborated according to Coupling Conditions D.
  • Boc-Glu(OBn)-OSu 500 mg, 1.15 mmol was dissolved in 7 mL of ethylene glycol dimethyl ether along with 1.15 mmol of amidoxime 31. The resulting reaction mixture was stined at rt for 15 h. It was then concentrated under reduced pressure.
  • Compounds such as Compounds BA-BH can be prepared according to General Method F as exemplified above for the preparation of Compound BC.
  • Compounds such as Compounds CA-FBG and GA-GC can be prepared according to General Method F followed by Coupling Conditions D as exemplified below by the preparation of Compound CD.
  • Amidoxime 36 was prepared using the same procedure that was outlined for compound BC substituting isovaleronitrile. Amidoxime 36 (150 mg, 1.15 mmol) was then dissolved in 2 mL of DMF and 6 mL of CH 2 C1 along with Boc-Gln-OH (237 mg, 0.958 mmol). HOBT (176 mg, 1.15 mmol) and EDC-HCI (220 mg, 1.15 mmol) were added, followed by DIEA (0.25 mL, 1.44 mmol). The resulting reaction mixture was stined at rt for 8 h. It
  • Compounds such as Compounds CA-FBG can be prepared according to General Method G as exemplified above for the preparation of Compound CD.

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Abstract

This invention concerns compounds for inhibiting intracellular signal transduction, especially intracellular signal transduction mediated by one or more molecular interactions involving a phosphotyrosine-containing protein. This invention also relates to pharmaceutical compositions containing the compounds and prophylactic and therapeutic methods involving pharmaceutical and veterinary administration of the compounds. The compounds are of formula (I) as defined herein.

Description

Heterocyclic Signal Transduction Inhibitors, Compositions Containing Them
Field of the Invention
This invention concerns a new class of compounds which have a broad range of useful biological and pharmacological activities. In particular, these compounds are useful for inhibiting intracellular signal transduction, especially intracellular signal transduction mediated by one or more molecular interactions involving a phosphotyrosine-contaimng protein This invention also relates to pharmaceutical compositions containing the compounds and prophylactic and therapeutic methods involving pharmaceutical and veterinary administration of the compounds.
Background of the Invention
Cellular signal transduction, i.e., the seπes of events leading from extracellular events to intracellular sequelae, is an aspect of cellular function in both normal and disease states. Numerous proteins that function as signal transducing molecules have been identified, including receptor and non-receptor tyrosine kinases, phosphatases and other molecules with enzymatic or regulatory activities. These molecules generally demonstrate the capacity to associate specifically with other proteins to form a signaling complex that can alter cell activity.
Signaling proteins often contain domam(s) of conserved sequence which constitute catalytic domains such as kinase or phosphatase domains, or serve as non-catalytic modules that direct protein 'protein or other inter- or intramolecular interactions duπng signal transduction. Such domains include among others, Src homology 2 ("SH2") and phosphotyrosine interaction ("PI") domains. SH2 and PI domains recognize, i.e., bind to, proteins containing characteristic peptide sequences which include one or more phosphorylated tyrosine ("pTyr") residues. Significant information related to such domains, proteins containing them, the production of proteins containing such domains (including protein fragments and fusion proteins), the charactenstic peptide sequences which they recognize and the biological and/or clinical role played by the interactions of such proteins has been descnbed in the scientific literature. See e.g. US 5667980, PCT/US97/02635 ("Cell-Based Assay") and WO 97/39326 ("In Vitro Fluorescence
Polanzation Assay") and references cited therein for additional background information on SH2 and PI domains, inhibition of intermolecular interactions mediated by such domains, assays and related topics. The protein domains of the tyrosine kinase, Src, gave πse to the "Src homology" ("SH") nomenclature and illustrate this class of proteins. At least nine members of the Src family of tyrosine kinases have been identified to date m vertebrates including Src (alternatively known as c-src and pp60c-src), Fyn, Yes, Lyn, Hck, Fgr, Blk and Yrk. Sequence analysis of the Src tyrosine kinases reveals that each family member contains an N-terminal membrane anchor, a poorly conserved "unique" region of 40-70 ammo acids, a Src homology 3 (SH3) domain of about sixty ammo acids capable of protein-protein interactions with prolme-πch sequences and a Src homology 2 (SH2) domain composing about 100 ammo acid residues which mediates binding of the Src family member of phosphotyrosme-(pTyr) containing peptides and proteins (reviewed in Superti-Furga, FEBS Lett. 369 62-66 (1995). Several cognate phosphoprotems known to bind the Src SH2 domain include middle T antigen, PDGF receptor, EGF receptor, and focal adhesion kinase (FAK). See Courtneidge et al, J. Virol. 65:3301-3308 (1991), Moi et al. EMBO J 12:2257-2264 (1993); Luttrell et al. Proc. Natl. Acad. Sci. USA 91.83-87 (1994), and Xing et al, Mol. Biol. Cell 5.413-421 (1994). For additional information on other SH2 domains (including, e.g., ZAP-70, Syk, She, Tsk, Btk, VAV, Grb2, Crk, STATs) and PI domam-contammg proteins, see WO 97/39326 and references cited therein
The molecular structure of several SH2 domains has been solved and, m particular, the molecular structure of certain SH2 domains in complex with a phosphotyrosme- containing peptide or peptide analog has been elucidated. See Waksman et al, Cell 72:779- 790 (1993); Xu et al. Biochemistry 34:2107-2121 (1995); Hatada et al, Nature 377(6544), 32-38 (1995). Whereas the general consensus sequence of Src family SH2-bιndιng peptides, for example, compπses a pTyr-X-X-(Leu/Ile) motif, SH2 domain binding specificity is thought to be influenced significantly by the specific ammo acids located carboxy-terminal to the pTyr residue. For example, the pp60c-src, Fyn, Lck and Fgr SH2 domains prefer the sequence pTyr-Glu-Glu-Ile. See Songyang et al, Cell 72.767-778 (1993) Crystallographic data concerning pp60c-src SH2 in complex with synthetic peptides has revealed, in particular, two important binding determinants for binding to phosphotyrosine-contaming proteins or peptides: the phosphotyrosme binding site which is electropositive in nature such that phosphotyrosme binding is stabilized and the lipophilic binding site which stabilizes binding of pTyr+3 residues within the phosphotyrosine- contaming peptides via hydrophobic contacts. Reviewed by Brown and Cooper. Biochim. Biophys. Acta 1287 (2-3): 121-149 (1996).
Structural studies of phosphotyrosine-contaming peptides complexed with isolated SHE domains has provided information regarding the molecular interactions of peptide gands with the SH2 domain peptidyl binding site. Recent attempts have been made to extrapolate these data to design novel peptide hgands and peptidomimetic agonists of SH2- mediated signaling. For example, Plummer et al reported that incorporation of C-terminal D-amino acid residues to tnpeptide SH2 domain hgands increases affinity relative to their L-amino acid-containing counterparts. See Plummer et al, Drug Design Discovery 13:75-81 (1996). Burke et al reported that hexapeptides containing difluoro- (phosphonomethyl)phenylalanme bound SH2 domains with high relative affinity compared to analogous pTyr peptides and were resistant to naturally-occurring cellular phosphatases. Studies of the pTyr residue of peptide agonists of the Src SH2 domain have shown that that phosphate ester is important for molecular recognition, and that significant loss in binding occurs when it is replaced with sulfate, carboxylate, nitro, hydroxy or amino groups. See Gilmer et al, J Biol Chem 269:31711-31719 (1994).
Many signaling pathways which play cntical roles in disease processes are mediated by the binding of a phosphotyrosine-contaming protein or protein domain with an SH2 or other protein receptor for a tyrosme-phosphorylated domain. Pharmaceutical agents which interfere with signaling mediated by such molecules, e.g., which interfere with the formation or stability of such signaling complexes, may be used for precise intervention in these complex biological processes in order to treat or prevent the diseases or pathological effects mediated by such signaling. Such interference may be achieved through a vanety of mechanisms, including competitive inhibition of a phosphotyrosine-contaming ligand with its receptor (e.g., with an SH2-contaιnιng protein), inhibition of phosphorylation of the tyrosine residue of such a ligand, inhibition of activation of a kinase which catalyzes the phosphorylation of a ligand in a signaling pathway, etc.
Compounds that can enter cells and block a signal transduction pathway of interest, such as an SH2-medιated pathway, would be of great interest as reagents for biological research and for pharmaceutical and veterinary uses.
Summary of the Invention
This invention concerns compounds of Formula I, or pharmaceutically acceptable derivatives thereof:
R 14
B
U'Vπ m
I R1 R2 in which
Y is
R8
R - =1-= R? or
R (kJ-wnf H (M r
(a) (b)
Figure imgf000006_0001
G is -O-, -S- or -NR-
R° comprises -OR, -APO3RR , -OPO3RR , -ASO3R, -OSO3R, -ACO2R, -A- tetrazole, -ASO2NRR', -ACOCF3, -(C=O)J, -C(R)(J)(K) or -C(Z)(J)(K);
where each occurrence of A is independently a covalent bond, -G-M- or -(M) m
each occurrence of M is an independently selected, substituted or unsubstituted, methylene moiety, and any M-M' moiety may be electronically saturated or unsaturated and/or may be part of a 3-8-membered ring. Illustrative "M" moieties include -CH2-, -CHF-, — CF2-,
-CHOH-, -CH(Me)-, etc.
Each n is independently 0, 1, 2, 3, 4 or 5 (in many embodiments n is 0, 1 or 2);
each m is independently 0, 1 or 2;
J and K are independently selected from the group consisting of -APO.RR , -OPO RR , -ASO3R, -OSO3R, -ACO2R, -A-tetrazole, -ASO2NRR', -(M) NRR' and -(M)nOR; Z is a halogen (i.e., F, Cl, Br or I); R7 and R8 are independently R, -CN, -NO2, Z, J, -A(M) aliphatic, -G(M) aliphatic ,
-(M)nCOCF3, -(M) OH, -(M)πCOOR, -A-(M) NRR', -G-(M) NRR', -(M) CHO,
-A(M)nN(R)(CO)R', -A(M)πN(R)(CO)GR', -G(M)qN(R)(CO)R\
-G-(M) N(R)(CO)G'R', -A-(M) -CO-NRR', or -G-(M) -CO-NRR', where the
aliphatic groups may be substituted or unsubstituted; or R is a covalent bond to an R substituent of X forming an aliphatic, aryl or heterocyclic πng of 4 to 8 atoms (including, for example a 5-membered nitrogen-containing πng of an indole moiety); additionally, R and R may be of the structure -(C=O)R so long as R is not alkenyl or alkynyl or B is not a thiazole.
Each occurrence of R (unnumbered) represents hydrogen or an aliphatic, heteroahphatic, aryl, heteroaryl, (aryl)alιphatιc-, or (heteroaryl)a phatιc- moiety, each of which (other than hydrogen) may be substituted or unsubstituted, e.g , with any of the vaπous substituents listed, illustrated or otherwise disclosed herein. While each occurrence of "R" withm a given compound is thus independently selected, where multiple R groups are depicted m the same figure or moiety, the vaπous R groups are generally marked R, R', R" and so on, as a reminder that they may be the same or different. (The same is true m the case of numbered "R" groups and other vaπables such as "m", "n", "M", etc where apostrophes are used for the same purpose Note also that the n M groups m a "M_" moiety may be the same or different from one another.)
q is an integer from 1 to 8, and in many embodiments is 1, 2 or 3,
R is hydrogen, alιphatιc.-(M) -cycloaliphatic, -(M) -aryl, or -(M) -heterocyclic, each of which, other than H, may be substituted or unsubstituted (including, e g. moieties such as -(M)nCO2R, -(M)nC(O)NRR', -(M)nZ, -(M)nCN, -(M)ntetrazole, etc.),
*2
R is hydrogen or substituted or unsubstituted aliphatic, which is optionally covalently linked with R to form a πng, or R or R are covalently linked either to B or a substituent of B to form a 4 - 10- membered, saturated or unsaturated, ring, or to the N depicted in Formula I above to form a 5, 6 or 7-membered, saturated or unsaturated, ring;
X is:
R3 R4
Figure imgf000008_0001
or R4-Nϊ •*•
R3 is hydrogen, R(CO)NR'-, RR'N(CO)NR"-, R'SO2NR- R'CSNR- RR''NCSNR"-, RR''NS02NR"-, R'OCONR- RR'N- or
NCONR—
R is hydrogen, aliphatic (which may be branched, unbranched or cyclic), cycloaliphatic-(M)n- aryl-(M)n- heterocyclic-(M)n-, RSO2(Mn)- , (CO2R)(M)n- or (RR'N-CO)(M)n, where the aliphatic, cycloaliphatic, aryl and heterocyclic groups are substituted or unsubstituted;
B is -{HET]R9R10R R12, where
HET is a heterocyclic moiety;
R9, R10 and R11 are independently -(M) Z, -(M) R, -(M)nGR, -(M)nWR,
-(M)nWGR, or -(M)nW-COR, including, among others, moieties such as R, -OR,-
SR, -CHO, -COR, -COOH (or amide, ester, carbamate, urea, oxime or carbonate thereof), -NH2 (or substituted amine, amide, urea, carbamate or guanidino derivative therof), halo, trihaloalkyl, cyano, -SO2-CF3, -OSO2F, -OS(O)2R, -SO2-NHR, -NHSO2R, sulfate, sulfonate, aryl and heteroaryl moieties.
R12 is independently selected from -(M) Z, -(M) R, -(M) GR, -(M)nWR, -(M)nWGR, and -(M)nW-COR; R14 is R; and,
U and W are independently -CO-, -CS-, -M-, -SO-, or -SO2-.
Compounds of Formula I thus include compounds having the following structures:
R14 R14 ' fit4' R14 R14
B
Figure imgf000009_0001
Figure imgf000009_0002
Figure imgf000009_0003
or Y"
Figure imgf000009_0004
0 R1R2 O O R1 R2
and comprise a number of subgenera of particular interest. Representative subgenera are illustrated in the examples which follow.
One subgenus includes compounds in which at least one R4 moiety is H and at least one R moiety is either H or NH2. Compounds of the latter sort include those in which X is
¥ NH2.
Also of interest are the subgenera of compounds in which the nitrogen atom of the moiety X is further elaborated, as depicted below:
Y R1 Y p14 Y R14
R 0Y^R\5PV F R 0Y^R5 O R ^ C R50 y R R2
O
where R comprises a substituted or unsubstituted, lower (i.e., containing 1 - 8 carbon atoms) aliphatic or alkoxyl group, or is a substituted or unsubstituted -(M)π-aryl or -(M)n-heterocyclic (including e.g., substituted and unsubstituted phenyl or benzyl group, or a homolog and heterocyclic analog thereof, including e.g., 2-naphthyl, 3-indolyl, and 1- imidazolyl);
Such compounds are further illustrated by the subset thereof in which R comprises -(M)nCH3, -(M)πaryl, -(M)πheterocyclic, -(M)nCN, -(M)nCOOR, where n is 0, 1, 2, 3, 4, or 5. For instance, m some such compounds R is a substituted or unsubstituted methyl, ethyl, n-propyl, l-propyl, n-butyl, sec- butyl, t- butyl, n-pentyl, sec- pentyl, l-pentyl, cyclo pentyl, etc. or benzyl moiety. In other such compounds R compπses -(CH,.) CH., -(CH2)(CH2)naryl, -(CH2)(CH2)nheterocyclιc, -(CH2)(CH2)nCN or -(CH7)(CH2)nCOOR, where n again is 0, 1, 2, 3, 4, or 5. Examples of such compounds include those in which R5 compπses -CH2CN, -(CH2)CO2R, -(CH2)2CO?R,
-(CH2)3CO2R, -(CH2)4CO2R, where R is H, lower alkyl or benzyl and those in which R5 compπses -O-(substιtuted or unsubstituted lower alkyl or benzyl).
Another subgenus of interest includes amides of the formula:
Y R14 ^B
R
Figure imgf000010_0001
O _ 2
R1 R where R4 is hydrogen, substituted or unsubstituted aliphatic (which may be branched, unbranched or cyclic), substituted or unsubstituted aryl-(M)n-, substituted or unsubstituted heterocychc-(M)n-, or (CO2R)(M)n-. Such compounds are illustrated by those in which
R4 is -(M) (CO)OR, -(M)nSO2R, -(M) (C0)NRR', or -(M) (tetrazole), including, for example, compounds in which R4 is -CH2COOR, -CH2SO2R, -CH2(CO)NRR', or
-tetrazole. Simple members of this subgenus are those in which the R group(s) of R is (are independently) H, lower alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tbutyl, etc.) or benzyl.
Another subgenus includes ureas of the formula: r R14
O RI\R2 where R1, R2, R4, R14, Y and m are defined as above. Thus, R4 may be simply H or may be a more complex R moiety such as are noted above.
Another subgenus includes amides of the formula: R14
Figure imgf000011_0001
R1R
In many examples of the foregoing compounds, one or more R moieties (R' , R" etc) are H. Also, in many compounds of interest, R is H.
One subgenus of interest includes compounds of Formula I, including the examples described or illustrated above, in which m is 1, R 1 is H, and R 2 comprises H, -(M) H,
-(M)n-(substituted or unsubstituted lower alkyl), -(M)n-(substituted or unsubstituted aryl), -(M)n-(substituted or unsubstituted heterocyclic), -(M)n-COOR, or
-(M) (CO)NRR ' . That subclass is illustrated by compounds in which R 1 is H, and R 2 is methyl, ethyl, i-propyl, n-propyl, n-butyl, isobutyl, n-amyl, sec-amyl, isoamyl, substituted benzyl, -CH2-(3-indolyl), -CH2-(4-imidazolyl), -CH2CH2COOR,
-CH2CH2CONH2, -CH2COOR or -CH2CONH2.
Another subgenus includes compounds of Formula I, including compounds of the sort described or illustrated above, in which R ι and R ι are independently selected, substituted or unsubstituted lower aliphatic groups, usually of 1 - 8 contiguous carbon atoms, or R and R are covalently linked to each other to form a ring, which may be a substituted or unsubstituted, aliphatic or heterocyclic ring or ring system (e.g. a bicyclic moiety), generally containing 3 - 10 annular atoms. Compounds containing an unsubstituted 3-10- membered ring are illustrated by the following formula, where q is an integer from 1 to 8:
Figure imgf000011_0002
Another subgenus includes compounds of Formula I, including the examples described or illustrated above, in which m is 2, and each of R1, R1', R2, and R2' is independently selected from H, -(M)nH, -(M)n-(substituted or unsubstituted lower alkyl), -(M)n-(substituted or unsubstituted aryl), -(M)n-(substituted or unsubstituted heterocyclic), -(M) -COOR and -(M) (CO)NRR . In some such compounds, each of R^ R^ R2, and R2' is H.
Another subgenus of compounds of interest are compounds of Formula I, including the examples described or illustrated above, in which at least one of R and R is methyl, ethyl, i-propyl, n-propyl, n-butyl, isobutyl, n-amyl, sec-amyl, isoamyl, substituted benzyl, -CH2-(3-indolyl), -CH2-(4-imidazolyl), -CH2CH2COOR, -CH2CH2CONH2,
-CH.COOR or -CH2CONRR', or R1 and R2 are covalently linked to form a πng. In some cases, at least one of R 1 , R 1 ' , R 2 , and R 2' is methyl, ethyl, i-propyl, n-propyl, n-butyl, isobutyl, n-amyl, sec-amyl, isoamyl, substituted benzyl, -CH2-(3-indolyl),
-CH2-(4-imidazolyl), -CH2CH2COOR, -CH2CH2CONH2, -CH2COOR or
-CH CONRR', or two of R 1 , R 1' , R 2 , and R 2' are covalently linked to form a ring, which as in other cases, may be a substituted or unsubstituted, aliphatic or heterocyclic ring or ring system (e.g. a bicyclic moiety), generally containing 4 - 10 annular atoms. Compounds containing 3-, 5- and 6-membered rings are illustrated by the following formulas:
,^ \JX
Figure imgf000012_0001
fl' R2
Figure imgf000012_0003
Figure imgf000012_0002
One subgenus of compounds of this invention, i.e., of compounds of Formula I. including among others the members of the various illustrative classes of compounds noted above, includes those compounds of Formula I in which m is 0:
Y" ^ιT Έ
Compounds of Formula I, including, among others, compounds of the various subgenera described above, include those in which Y comprises
10 R'
R er '^L n Rkfc J mr
(M)-τj-
Figure imgf000013_0002
R7 ψ R7 ^ or - R7
Figure imgf000013_0001
Such compounds in which R comprises -OR, -APO3RR , -OPO RR , -ASO R, -OSO3R, -ACO2R, -A-tetrazole, -ASO2NRR', -ACOCF3, -C(R)(J)(K) or -C(Z)(J)(K); and, R7 and R8 are independently H, -CN, -NO , halogen, J,
-A-(M)nsubstituted or unsubstituted aliphatic, -(M)nCOCF3, -(M)nOH, -(M)πCOOR,
-A-(M) NRR\ -(M) CHO, -A-(M)nN(R')(CO)R" or -A-(M) -CO-NRR' are of particular interest. This set of compounds is illustrated by those in which R comprises -OR, -APO3RR', -OPO3RR', -ACO2R, -ACOCF3, or -C(R)(J)(K); A comprises -Mm- (e.g., -CH2- -CF2- -CHF- , -CHOH-, -CH2CF2- etc), -GM- (e.g. -OCH-,-) or a covalent bond; each R and R' is H, or substituted or unsubstituted lower alkyl or substituted or unsubstituted benzyl; and, R 7 and R 8 are independently H, J, -A-(M)nsubstituted or unsubstituted aliphatic, -(M)nCOCF3, -(M)nOH, -(M)nCOOR,
-A-(M) NRR', -(M) CHO, -A-(M)nN(R)(CO)R' or -A-(M) -CO-NRR'. For
example, in some such cases, R 6 comprises -OH, -PO RR ' , -OPO RR ' ,
-CH.P0,RR', l i -CF 1P0α5RR', -OCH 1 O^1R, -NRM COlR', -CH 1 01R,
-CF2CO2R, -CH2SO3R, -CF2SO3R, -CH2COCF3, -CF2COCF3> -CH(PO3RR')2, -CH(OH)(PO3RR'), -CH(NH2)(PO3RR'), -CH(CO2R)2, -CF(CO2R)2,
-CH(PO3RR')(CO2R"), -CH(PO3RR')(SO3R"), -CH(PO3RR')(SO2NH2), -CH(SO2NH2)2, or -CH(SO3RR')2. Compounds in which R6 is -NRMmCO R' are illustrated by compounds in which R is H, -MmCO2R', -MmSO0R" or another substituted or unsubstituted lower aliphatic moiety. In some such compounds, one or more
11 of R, R' and R" in the -PO3RR , -OPO3RR , -CH2PO3RR , -CF2PO3RR , -OCH2CO2R, -NHCH2CO2R, -CH2CO2R, -CF2CO2R, -CH2SO3R, -CF2SO3R,
-CH2COCF3, -CF2COCF3, -CH(PO3RR')2, -CH(OH)(PO3RR'),
-CH(NH2)(PO3RR'), -CH(CO2R)2, -CF(CO2R)2, -CH(PO3RR')(CO2R"),
-CH(PO3RR')(SO3R"), -CH(PO3RR')(SO2NH2), -CH(SO2NH2)2, or -CH(SO3RR')2 moiety is H. In others, one or more of those R groups is -(M)m-CH2Z, -(M)m-CHZ2, -(M)m-CZ3, -R15, -M-O-CO-OR15 or -M-O-CO-R15, where Z is halogen and R15 is substituted or unsubstituted lower aliphatic, aryl or heterocyclic. For example, in various embodiments, R is methyl, ethyl, n-propyl, i-propyl, n-butyl, isobutyl, t-butyl, n-amyl, sec-amyl, benzyl or substituted benzyl, and M is CH2, CHR (e.g. CHCH3 etc.) and the like. Further illustrations include -CH2-O-CO-OEt, -CH(Me)-O-CO-OEt, -CH2- O-CO-t-butyl, etc.
In one subgenus of the foregoing compounds, R and R are both H. In another subgenus, R is J, -A-(M) (substituted or unsubstituted aliphatic, aryl or heterocyclic),
-(M)nCOCF3, -(M)πOH, -(M)nCOOR, -A-(M) NRR'', -<M)nCHO,
-A-(M)nN(R)(CO)R', -A-(M) -NRR' or -A-(M)π-CO-NRR'; and R8 is H. The latter subgenus is illustrated by compounds in which R is lower alkyl, lower alkenyl, -OH,
-NH2, -NO2, -CN, -NHR, -NHCOR, -CHO, -CH2CHO, -PO..RR', -OPO3RR', -CH2PO3RR', -CF2PO3RR', -OCH2CO2R, -NHCH2CO2R, -CH2CO2R, -CF2CO2R, -SO3R, -CH2SO3R, -CF2SO3R, -COCF3, -COCH2F, -COCF2H, -CF2COCF3 OR
-SO NH2. In some such compounds, one or both of R and R' in -PO3RR', -OPO3RR',
-CH2PO3RR', -CF2PO3RR', -OCH2CO2R, -NHCH2CO2R, -CH2CO2R, -CF2CO2R, -SO3R, -CH2SO3R, or -CF2SO3R is H. In others, one or more of those R groups is -(M) m -CH 2.Z, -(M) m -CHZ 2, -(M) m -CZ, 3, -R15, -M-O-CO-OR15 or -M-O-CO-R15,
where Z is halogen and R is substituted or unsubstituted lower aliphatic, aryl or
12 heterocyclic. For example, in individual cases, R is methyl, ethyl, n-propyl, i-propyl, n-butyl, isobutyl, t-butyl, n-amyl, sec-amyl, benzyl or substituted benzyl, and M is CH2, CHR (e.g. CHCH3 etc.) and the like.
In an illustrative subgenus, R 6 compπses -APO RR ' (e.g., -OPO H ) and R 7 is
-A-(M)nsubstιtuted or unsubstituted aliphatic.
f 7
In another subgenus, R and R are independently selected from J and K.
In another subgenus, R is -C(R)(J)(K). Illustrative compounds of this subgenus include those in which R6 is -CH(J)(K) and those m which R6 is -C(R)(PO R'R'')(K). The latter compounds are illustrated by embodiments in which none, one, two or three of the R groups of the -C(R)(PO R'R'')(K) moiety are H.
As in previously mentioned cases, compounds of this invention which contain a moiety J, e.g., compounds of Formula I m which R is -C(R)(J)(K), include among others embodiments in which one or both of R and R' (e.g., of a -PO RR ' moiety) are R 15 ,
-(M) m -CH 2.Z, -(M) ym -CHZ 2, -(M) m -CZ- 3,, -M-O-CO-OR15 or -M-O-CO-R15, where
Z is halogen and R is substituted or unsubstituted lower aliphatic, aryl or heterocyclic (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, isobutyl, t-butyl, n-amyl, sec-amyl, benzyl or substituted benzyl), and M is CH2, CHR (e.g. CHCH3 etc.) and the like.
The compounds of Formula I, including the vaπous subgenera and illustrative examples descπbed above, all contain a heterocyclic moiety, B, as that term is defined below and as is illustrated by the following group:
13 o
.S--/R9 .O^R9
1 . ^
N^R 10 l\ ^R 10 W^ -R ^y * V^R"
■,10
-10
■ N R *1°1 t "N,NJ1 I f -&
S^ OΨ^RH° .0 3I2 v° ,10 n10
e 1 )11 P- 11 l n 4t ]
.-x \^
R^R- -5- - X ,ιo .9 F 10 "
including, among others, the following specific cases
&— ,R
AA I .R, -K -Λ- &. t^ r" ^R,
R9 R9 R9 R9
,10 A^R
=> ,11 □11 R RU l 12 R R11
» J5V" JW" JW" ? " *fT
R o o
*/r\R., ? ,, ? R„ ^- , j --f--.
R" R R9 R ° ,^— * Rιo R9 R
wherein each of R9, R10 and R11 is independently Z, R, -GR, -COR, -CO2R, or
-(M)nW-NRR' and R12 is independently selected from -(M) Z, -(M) R, -(M) GR, -(M)nWR , and -(M)nWGR. In some embodiments, one or more of the R, R' and R" groups of R >9 , D R10 and R »11 comprise a halo, hydroxy, aliphatic, amino, amido or sulfonamido moiety. In some embodiments, one or more of R , R , R , and R is a substituted aliphatic moiety containing at least one substituent selected from substituted or unsubstituted cycloaliphatic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -COR, -CO2R, -CO-NRR', and -OR. In some embodiments of particular
9 10 11 12 interest, one or more of R , R , R , and R comprises -(M) (cycloaliphatic), n
14 -(M)n(substituted or unsubstituted aryl), -(M)n(substituted or unsubstituted heteroaryl), -(M)nCHO, -(M)nCONH2, -(M)nCSNH2, -(M)nSONH2, -(M)nSO2NRR', -(M)nOR,
-(M)n(lower aliphatic), -(M)n-C(OR)R'R", or -(M)n-C=CRR'. (In examples in which
R 12 is -(M) OR, n is 1 or greater.) For example, in some cases, one or more of R 9 , R 10 ,
R 11 , and R 12 comprise methyl, -(CH ) R 13 where q is 1-8 13
1 q and R comprises methyl; i-propyl; i-butyl; t-butyl; cycloaliphatic; phenyl; substituted phenyl; naphthyl; substituted naphthyl; a 5, 6 or 7-membered heterocyclic ring or a bicyclic heterocylic moiety. In some cases, R 12 comprises a formyl group on a ring nitrogen. Possible substituents on the R, R' and R" groups include, among others, halo, hydroxy, alkyl, amino, amido and sulfonamido moieties. Other potential substituents are as disclosed elsewhere herein, including in the numerous specific examples.
Compounds of particular interest include those in which B is selected from the following:
fS~-/R r i O. -v- 9 - P _ nιι
^ ^NT^-R 10 N^R 10 I** \>
10
R12 10 R 10 \ 10
,Ra ' hr Γ^DH s* 1 N^R10 ^RDi1ι
J12
From the perspective of Y moieties, compounds of particular interest include those compounds of Formula I, including those of the various subgenera and examples herein, which have the following structures:
R14
- and •
Figure imgf000017_0001
m R1R2
R 1Ήb2
Figure imgf000017_0002
One subgenus of compounds of Formula I which is of particular interest includes thiazoles of Formula II:
H
< O FT
II
15 where R and R each compπse an independently selected, substituted or unsubstituted lloowweerr aalliipphhaattiicc mmooiieettyy,, aanndd RR aanndd RR aarree eeaacchh iinnddependently selected from H. and a substituted or unsubstituted lower aliphatic moiety.
This subgenus is illustrated by compounds of Formula II in which R compπses a linear, branched or cyclic lower aliphatic moiety, -(CH2)nCOOH, -(CH2)naryl (substituted or unsubstituted) or -(CH ) heterocyclic (substituted or unsubstituted); R compπses a
linear, branched or cyclic lower aliphatic moiety; and R and R are independently selected from H; linear, branched or cyclic lower aliphatic; -(CH2)nCOOR; -(CH2)nCONRR\ -(CH2)ncycloahphatιc (substituted or unsubstituted); -(CH2)naryl (substituted or unsubstituted) or -(CH2)nheterocyclιc (substituted or unsubstituted). For example, in some cases, R compπses a linear, branched or cyclic lower aliphatic, -(CH )2COOR, -(CH2)2aryl (substituted or unsubstituted) or -(CH2)2heterocychc
(substituted or unsubstituted); R compπses a linear, branched or cyclic lower aliphatic; and R and R are independently selected from H; linear, branched or cyclic lower aliphatic; -(CH2)2COOR, -(CH2)2CONH2; -(CH2)cycloalιphatιc (substituted or unsubstituted); -(CH2)aryl (substituted or unsubstituted) or -(CH2)heterocyclιc
(substituted or unsubstituted). Thiazoles of Formula II, e.g. compounds such as those shown immediately below, may be useful as inhibitors of cellular signaling mediated by Src or Src family kinases, and thus may be useful in treating diseases such as osteoporosis and other bone resorptive disorders in patients in need thereof.
Figure imgf000018_0001
C02H
Y H $
H N N
- o ^
Figure imgf000018_0002
Figure imgf000018_0003
The following thiazoles further illustrate the compounds of Formula II:
16
Figure imgf000019_0001
Figure imgf000019_0002
and may be useful as inhibitors of cellular signaling mediated by ZAP-70 or ZAP-70 family kinases, and thus may be useful in treating or preventing an inflammatory response in patients in need thereof. Such compounds may be used for example as immunosuppressants in patients who are going to receive, or have received, an organ or tissue transplant or in patients suffering an autoimmune disorder.
Another subgenus of compounds of Formula I which is of particular interest includes oxadiazoles of Formula HI:
"
Figure imgf000019_0003
Figure imgf000019_0004
O
R5
III where R1 and R5 each comprise an independently selected, substituted or unsubstituted lower aliphatic moiety, and R comprises H or a substituted or unsubstituted lower aliphatic moiety. In some cases, R comprises a linear, branched or cyclic lower aliphatic, -(M)naliphatic (which may be substituted or unsubstituted), -(M)nCOOR, -(M)nOR, -(M)naryl (which may be substituted or unsubstituted) or -(CH2)nheterocyclic (which may be substituted or unsubstituted); R comprises a linear, branched or cyclic lower aliphatic; and R comprises H; linear, branched or cyclic lower aliphatic; -(M) COOR;
-(M)nCONRR'; -(M)ncycloaliphatic (which may be substituted or unsubstituted); -(M)naryl (which may be substituted or unsubstituted) or -(M)nheterocyclic (which may be substituted or unsubstituted). In its simplest form, M is CH2. For example, in some embodiments, R comprises a linear, branched or cyclic lower aliphatic, -(CH-)2COOR, -(CH2)aryl (substituted or unsubstituted) or -(CH2)heterocyclic (substituted or unsubstituted); R comprises a linear, branched or cyclic lower aliphatic (substituted or unsubstituted); and R comprises H; linear, branched or cyclic lower aliphatic; -(CH2)2COOR; -(CH2)2CONH2; -(CH2)cycloaliphatic (substituted or unsubstituted);
17 -(CH,)aryl (substituted or unsubstituted) or -(CH2)2heterocyclιc (substituted or unsubstituted). Oxadiazoles of Formula HI may be useful as inhibitors of cellular signaling mediated by Src or Src family kinases, and thus may be useful in treating diseases such as osteoporosis and other bone resorptive disorders in patients in need thereof. Oxadiazoles of Formula III are further illustrated by the following types of compounds:
7 H o-., l-k
Figure imgf000020_0001
CO H γvT
Figure imgf000020_0002
co2H
Naπous compounds of Foπnula I (including each of the foregoing subgenera thereof)
9 containing a substituent R which compπses -(M) -aryl (which may be substituted or unsubstituted) or -(M)n-heterocychc (which may be substituted or unsubstituted). Again, in its simplest form, M is CH2.
Such R moieties are illustrated by the following:
CF3 R
Figure imgf000020_0003
Returning to the subject of oxadiazoles, one set of illustrative compounds of particular interest include those of the following sorts of structures:
18
Figure imgf000021_0001
Figure imgf000021_0002
Such oxadiazoles may be useful as inhibitors of cellular signaling mediated by ZAP-70 or ZAP- 70 family kinases, and thus may be useful in treating or preventing an inflammatory response in patients in need thereof. Such compounds may be used for example as immunosuppressants in patients who are going to receive, or have received, an organ or tissue transplant or in patients suffering an autoimmune disorder.
Another set of illustrative oxadiazoles of particular interest include the following types of compounds:
Figure imgf000021_0003
Figure imgf000021_0004
Compounds of this invention of particular interest, including the various thiazoles and oxadiazoles discussed above, include, among others, embodiments of the type in which Y comprises:
19 or
Figure imgf000022_0001
Figure imgf000022_0002
where R6 comprises -PO3RR', -OPO3RR', -OSO2NRR', -(CH2)PO3RR\ η -(CF0)PO RR' or -CRJK; and R comprises R (including among others, H, alkyl, alkenyl, etc.) -CN, amido, acylamino, J (e.g. -CO2R), or -CHO. For example, in some
(\ 7 cases, R comprises -OPO RR' or -(CF )PO RR' and R is H. In some embodiments
one or more R groups (including R', R", etc) of R comprises -(M) -CH Z,
-(M) m -CHZ 2, -(M) m -CZ, 3, -R15, -M-O-CO-OR15 or -M-O-CO-R15, where Z is H or
halogen and R is substituted or unsubstituted lower aliphatic, aryl or heterocyclic. For example, in individual cases, R is methyl, ethyl, n-propyl, i-propyl, n-butyl, isobutyl, t-butyl, n-amyl, sec-amyl, benzyl or substituted benzyl, and M is CH2, CHR (e.g. CHCH3 etc.) and the like.
Compounds of this invention which are of special interest include those which bind to a given SH2 domain (or protein containing such SH2 domain) with a IC50 value of less than
50 μM, preferably less than 20 μM, as determined by any scientifically valid method, in vitro or in vivo. SH2 domains of current interest include those of a Src, Fyn, Lck. Yes, Blk. Lyn, Fgr, Hck, Yrk, ZAP-70, Syk, STAT or Abl protein.
Also of interest are pharmaceutical compositions comprising a compound of this invention, or a pharmaceutically acceptable derivative thereof, and one or more pharmaceutically acceptable excipients.
Compounds of this invention (or a composition containing such a compound) can be administered to cells or to animals, preferably a mammal in need thereof, as a method for inhibiting SH2-mediated signal transduction therein. In particular cases, it will be advantageous to carry out that method using a pharmaceutical composition containing a compound which specifically binds to an SH2 domain of Src, ZAP-70, Syk, or STAT 6. In other cases it will be advantageous to carry out that method where the SH2-mediated signal transduction is mediated by a PDGF receptor protein, EGF receptor protein,
20 HER2/Neu receptor protein, fibroblast growth factor receptor protein, focal adhesion kinase protein, pl30 protein, or p68 protein.
Cases in which a mammal may be m need of inhibition of SH2-medιated signaling include cases in which the mammal has a prohferative disease, cancer, restenosis, osteoporosis, inflammation, allergies, or cardiovascular disease. In such cases, admimsteπng a therapeutically effective amount of the composition to the mammal, preferably to a human patient, will constitute treating or preventing the prohferative disease, cancer, restenosis, osteoporosis, inflammation, allergic reaction, or cardiovascular disease in the recipient or a method for causing lmmunosuppression in the recipient.
Generally prefened compounds of this invention include any of the foregoing compounds which yield an observable IC50 value, when tested against an SH2 domain of interest and a pTyr-containmg peptide ligand (or mimic thereof) for that SH2 domain, of 50 μM or better, preferably 5 μM or better, more preferably 1 μM or better, and even more preferably, 500 nM or better, as determined by any scientifically valid measure, especially when the SH2 domain is from a Src, Fyn, Lck, Yes, Blk, Lyn, Fgr, Hck, Yrk, ZAP, Syk, STAT or Abl protein.
A pharmaceutical composition may be prepared containing a compound of this invention (including a pharmaceutically acceptable deπvative thereof) together with one or more pharmaceutically acceptable excipients.
A compound of this invention, preferably in the form of a pharmaceutical composition, may be administered to a mammal in need thereof, preferably a human patient, as a method for inhibiting SH2-medιated signal transduction m the recipient mammal. In some cases, the compound may be selected based on its ability to specifically bind to an SH2 domain, e.g of Src, ZAP-70, Syk, or STAT 6, etc., or on its ability to inhibit a signal transduction pathway mediated by an SH2 domam-contaming protein. Such use of an appropπately selected compound of this invention thus provides a method for inhibiting SH2-medιated signal transduction which is mediated by a PDGF receptor protein, EGF receptor protein, HER2/Neu receptor protein, fibroblast growth factor receptor protein, focal adhesion kinase protein, pi 30 protein, or p68 protein. Use of a compound of this invention may be particularly advantageous m cases in which the mammal has a prohferative disease, cancer, restenosis, osteoporosis, inflammation, allergies, or cardiovascular disease. In such cases, administeπng to the patient a therapeutically effective amount of a compound of this invention, preferably in the form of a pharmaceutical composition, provides a method for
21 treating or preventing a prohferative disease, cancer, restenosis, osteoporosis, inflammation, allergies, or cardiovascular disease in the patient
Detailed Description of the Invention
Compounds and Definitions
As mentioned above, this invention provides a novel class of compounds useful as inhibitors of signal transduction pathways mediated by the interaction of protein receptors for phosphotyrosine-contaming proteins, such as proteins containing one or more SH2 domains, with their phosphotyrosine-contaming hgands. Compounds of this invention compπse those of Formula I, set forth above, and are illustrated in part by the vaπous classes, subgenera and subsets of compounds noted above, and by the vaπous subgenera and species disclosed elsewhere herein. The compound may be m the form of an individual enantiomer, diastereomer or geometπc isomer, or may be in the form of a mixture of stereoisomers.
Also included are pharmaceutically acceptable deπvatives of the foregoing compounds, where the phrase "pharmaceutically acceptable deπvative" denotes any pharmaceutically acceptable salt, ester, or salt of such ester, of such compound, or any other adduct or deπvative which, upon administration to a patient, is capable of providing (directly or indirectly) a compound as otherwise descπbed herein, or a metabolite or residue thereof, preferably one which is a signal transduction inhibitor. Pharmaceutically acceptable deπvatives thus include among others pro-drugs. A pro-drug is a deπvative of a compound, usually with significantly reduced pharmacological activity, which contains an additional moiety which is susceptible to removal w vivo yielding the parent molecule as the pharmacologically active species An example of a pro-drug is an ester which is cleaved in vivo to yield a compound of interest. Pro-drugs of a vaπety of compounds, and mateπals and methods for denvatizmg the parent compounds to create the pro-drugs, are known and may be adapted to the present invention.
The term "aliphatic" as used herein includes both saturated and unsaturated, straight chain (i.e., unbranched), branched, cyc c, or polycychc aliphatic hydrocarbons, which are optionally substituted with one or more functional groups. Unless otherwise specified, alkyl, other aliphatic, alkoxy and acyl groups preferably contain 1-8, and m many cases 1- 6, contiguous aliphatic carbon atoms. Illustrative aliphatic groups thus include, for example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, -CH2-cyclopropyl, allyl, n- butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl, -CH2-cyclobutyl, n-pentyl, sec-pentyl,
22 isopentyl, tert-pentyl, cyclopentyl, -CH2-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl,- CH9-cyclohexyl moieties and the like, which again, may bear one or more substituents.
Some examples of substituents of aliphatic (and other) moieties of compounds of this invention include: R, -OH, -OR, -SH, -SR,-CHO, =O, -COR, -COOH (or amide, ester, carbamate, urea, oxime or carbonate thereof), -NH2 or substituted amine, amide, urea, carbamate or guanidino derivative therof), halo, trihaloalkyl, cyano, -SO->-CF3,- OSO2F, -OS(O)2R, -SO2-NHR, -NHSO2R, sulfate, sulfonate, aryl and heteroaryl moieties. Aliphatic, heteroaliphatic, aryl and heterocyclic substituents may themselves be substituted or unsubstituted (e.g. mono-, di- and tri-alkoxyphenyl; methylenedioxyphenyl or ethylenedioxyphenyl; halophenyl; or -phenyl-C(Me)2-CH2-O-CO-[C3-C6] alkyl or alkylamino). Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples which follow.
The term "aliphatic" is thus intended to include alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. As used herein, the term "alkyl" includes both straight, branched and cyclic alkyl groups. An analogous convention applies to other generic terms such as "alkenyl", "alkynyl" and the like. Furthermore, as used herein, the language "alkyl", "alkenyl", "alkynyl" and the like encompasses both substituted and unsubstituted groups.
The term "alkyl" refers to groups usually having one to eight, preferably one to six carbon atoms. For example, "alkyl" may refer to methyl, ethyl, n-propyl, isopropyl, cyclopropyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, pentyl, isopentyl tert-pentyl, cyclopentyl, hexyl, isohexyl, cyclohexyl, and the like. Suitable substituted alkyls include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 3- fluoropropyl, hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, benzyl, substituted benzyl and the like.
The term "alkenyl" refers to groups usually having two to eight, preferably two to six carbon atoms. For example, "alkenyl" may refer to prop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl, hex-5-enyl, 2,3-dimethylbut-2-enyl, and the like. The language "alkynyl," which also refers to groups having two to eight, preferably two to six carbons, includes, but is not limited to, prop-2-ynyl, but-2-ynyl, but-3-ynyl, pent-2-ynyl, 3-methylpent-4-ynyl, hex-2-ynyl, hex-5-ynyl, and the like.
The term "cycloalkyl" as used herein refers specifically to groups having three to seven, preferably three to ten carbon atoms. Suitable cycloalkyls include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in
23 the case of other aliphatic or heteroaliphatic or heterocyclic moieties, may optionally be substituted
The term "heteroaliphatic" as used herein refers to aliphatic moieties which contain one or more oxygen, sulfur, nitrogen, phosphorous or silicon atoms, e.g., m place of carbon atoms. Heteroaliphatic moieties may be branched, unbranched or cyclic and include heterocycles such as morpho no, pyrro dinyl, etc.
The term "heterocycle" as used herein refers to cyclic heteroaliphatic and heteroaryl groups and preferably three to ten πng atoms total, mcludes, but is not limited to heteroaliphatic moieties such as oxetane, tetrahydrofuranyl, tetrahydropyranyl, aziπdme, azetidme, pyrrolidme, pipeπdme, morpho ne, piperazme and the like, and heteroaryl moieties as descπbed below.
The terms "aryl" and "heteroaryl" as used herein refer to stable mono- or polycychc, heterocyclic, polycychc, and polyheterocyc c unsaturated moieties havmg 3 - 14 carbon atom which may be substituted or unsubstituted. Substituents include any of the previously mentioned substituents Non-limiting examples of useful aryl πng groups include phenyl, halophenyl, alkoxyphenyl, dialkoxyphenyl, tπalkoxyphenyl, alkylenedioxyphenyl, naphthyl, phenanthryl, anthryl, phenanthro and the like. Examples of typical heteroaryl πngs include 5-membered monocychc πng groups such as thienyl, pyrrolyl, lmidazolyl, pyrazolyl, furyl, isothiazolyl, furazanyl, oxazolyl, isoxazolyl, thiazolyl, oxadiazolyl and the like; 6-membered monocychc groups such as pyπdyl, pyrazmyl, pyπmidmyl, pyπdazinyl, tπazmyl and the like; and polycychc heterocyclic πng groups such as benzo[b]thιenyl, naphtho[2,3-b]thιenyl, thianthrenyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathienyl, mdo zinyl, isoindolyl, indolyl, indazolyl, puπnyl, isoquinolyl, qumolyl, phthalazinyl, naphthyπdinyl, quinoxa nyl, quinazo nyl, benzothiazole, benzimidazole, tetrahydroqumo ne cmno nyl, pteπdmyl, carbazolyl, beta-carbo nyl, phenanthπdinyl, acπdmyl, peπmidmyl, phenanthrolinyl, phenazmyl, isothiazolyl, phenothiazmyl, phenoxazmyl, and the hke(see e.g. Katπtzky, Handbook of Heterocyclic Chemistry). The aryl or heteroaryl moieties may be substituted with one to five members selected from the group consisting of hydroxy, C1-C8 alkoxy, C1-C8 branched or straight-chain alkyl, acyloxy, carbamoyl, ammo, N-acylamino, mtro, halo, tπhalomethyl, cyano, and carboxyl Aryl moieties thus include, e.g phenyl; substituted phenyl beaπng one or more substituents selected from groups including: halo such as chloro or fluoro, hydroxy, C1-C6 alkyl, acyl, acyloxy, C1-C6 alkoxy (such as methoxy or ethoxy, including among others dialkoxyphenyl moieties such as 2,3-, 2,4-, 2,5-, 3,4- or 3,5-dιmethoxy or diethoxy phenyl or such as methylenedioxyphenyl, or 3-methoxy-5-ethoxyphenyl; or tπsubstituted phenyl, such as tπalkoxy (e.g., 3,4,5-tπmethoxy or ethoxyphenyl), 3,5-dιmethoxy-4- chloro-phenyl, etc.), ammo, -SO2NH2, -SO2NH(ahphatιc), -SO2N(alιphatιc)2, -O-
24 aliphatic-COOH, and -O-aliphatic-NH2 (which may contain one or two N-aliphatic or N- acyl substituents).
A "halo" substituent may be fluoro, chloro, bromo or iodo.
With respect to nomenclature, note that asymmetric moieties such as "-G-M-" are written in the direction or order in which they are intended to be read into a given structure. Thus, "-G-M-" is distinct from "-M-G-". For example, in "Ar-A-COOR", where A is -G-M-, the structure Ar-G-M-COOR, not Ar-M-G-COOR, is intended.
Synthesis Those of ordinary skill in this art will appreciate that compounds of this invention may be produced using any of a variety of synthetic strategies. We typically use a convergent synthetic scheme in which an intermediate comprising the desired "YXU" moiety, protected as appropriate, is condensed with a second intermediate comprising the desired amino moiety HR N(CR R ) B, again, protected as appropriate, to yield (following any necessary deprotection steps) the desired compound of Formula I. A variety of methods and materials for effecting the relevant chemical transformations, product recovery, purification and formulation are known in the art which may be adapted to use in the practice of this invention. The detailed examples which follow illustrate such syntheses and should provide helpful guidance to the practitioner.
Assays for Comparative Functional Evaluation of Compounds
Compounds of this invention may be evaluated in a variety of assays to determine their relative ability to bind to a receptor for a pTyr-containing ligand, such as a protein containing one or more SH2 or PI domains, or to otherwise inhibit an intermolecular interaction mediated by such a domain. See e.g. US 5667980 (Pawson; competitive binding assays), PCT/US97/02635 (Rickles et al; cell-based assays) and PCT/US97/06746 (Lynch et al, FP assays). Compounds may also be evaluated for their selectivity of binding to one such receptor (or family of receptors) relative to another such receptor (or family of receptors). The compounds of this invention can be further evaluated by conventional methods for possible therapeutic applications, including evaluations of toxicological and pharmacological activity. For example, the compounds may further be evaluated for activity in inhibiting cellular or other biological events mediated by a pathway involving the molecular interaction of interest using a suitable cell-based assay or an animal model. Cell-based assays and animal models suitable for evaluating inhibitory activity of a test compound with respect to a wide variety of cellular and other biological events are known
25 in the art. New assays and models are regularly developed and reported in the scientific literature.
By way of non-limiting example, compounds which bind to an SH2 domain involved in the transduction of a signal leading to asthma or allergic episodes may be evaluated in a mast cell or basophil degranulation assay. The inhibitory activity of a test compound identified as an SH2 inhibitor by the method of this invention with respect to cellular release of specific mediators such as histamine, leukotrienes, hormonal mediators and/or cytokines, as well as its biological activity with respect to the levels of phosphatidylinositol hydrolysis or tyrosine phosphorylation can be characterized with conventional in vitro assays as an indication of biological activity. [See, e.g., Edward L. Barsumian et al, Eur. J. Immunol..11:317-323 (1981); M. J. Forrest, Biochem. Pharmacol.. 42:1221-1228 (1991) (measuring N-acetyl-betaglucosamin-adase from activated neutrophils); and Stephan et al., J. Biol. Chem.. 267:5434-5441 (1992)].
For example, histamine release can be measured by a radioimmunoassay using a kit available from AMAC Inc. (Westbrook, ME). One can thus evaluate the biological activity of compounds of this invention and compare them to one another and to known active compounds or clinically relevant compounds which can be used as positive controls.
Generally speaking, in such assays IC50 scores of 20 μM or less are considered of special interest, scores below 1 μM are considered of particular interest and scores below about 500 nM are of high interest. Inhibitors of this invention may also be tested in an ex vivo assay, e.g., for their ability to block antigen-stimulated contraction of sensitized guinea pig tracheal strip tissue. Activity in this assay has been shown to be useful in predicting the efficacy of potential anti-asthma drugs.
Numerous animal models of asthma have been developed and can be used [for reviews, see Larson, "Experimental Models of Reversible Airway Obstruction", in THE LUNG, Scientific Foundations, Crystal, West et al. (eds.), Raven Press, New York, pp. 953-965 (1991); Warner et al., Am. Rev. Respir. Pis.. 141:253-257 (1990)]. Species used in animal models of asthma include mice, rats, guinea pigs, rabbits, dogs, sheep and primates. Other in vivo models available are described in Cross et al., Lab Invest., 63:162-170 (1990); and Koh, et al., Science. 256:1210-1213 (1992).
By way of further example, compounds which bind to an SH2 or other domain of interest involved in the transduction of a signal involved in the initiation, maintenance or spread of cancerous growth may be evaluated in relevant conventional in vitro and in vivo assays. See e.g., Ishii et al., I. Antibiot.. XLII: 1877- 1878 (1989); and US Patent 5,206,249 (issued 27 April 1993).
26 Compounds which bind to a ZAP SH2 domain or which otherwise inhibit ZAP-70- mediated signaling may be evaluated for immunosuppressive activity, e.g., in any of the well-known in vitro or in vivo immunosuppression assays.
Compounds which bind to a Src SH2 domain or which otherwise inhibit Src- mediated signaUng may be evaluated for activity in a variety of assays considered predictive of activity in treating or preventing osteoporosis. Such assays include the various pit assays and calvaria assays, among others. Illustrative assays are described below.
MURINE CALVARIA ASSAY In osteoporosis, excessive bone resorption results in decreased bone density. In vivo and in vitro models of bone resorption are used to study the processes leading to osteoporosis. In vitro, fetal rat long bone and murine calvaria cultures are routinely used. Both models display similar responses to parathyroid hormone (PTH), a physiological modulator of bone resoφtion (Stern, P.H. and N.S. Krieger. Comparison of fetal rat limb bones and neonatal mouse calvaria: effects of parathyroid hormone and 1,25- dihydroxyvitamin D3. Calcif. Tissue Int. 35: 172-176, 1983). The calvaria model of bone resoφtion can be successfully used to screen osteotropic compounds as has been previously shown (Green, J.R., K. Muller and K. Jaeggi. Preclinical pharmacology of CGP 42'446, a new, potent, heterocyclic bisphosphonate compound. J. Bone Miner. Res. 9: 745-751, 1994.).
In one modification of the conventional calvaria model, calvaria are not labeled with
Ca++. Instead, calvarial calcium release into the media is assessed using a microtiter colorimetric calcium assay. This modification can yield more consistent responses than the radioactive methodology and provides results which are comparable to literature values for Ca++ assays.
One calvaria culture model tests the ability of anti-resoφtive compounds to prevent resoφtion (prophylactic model). A second model tests the ability of these compounds to terminate ongoing resoφtion (therapeutic model). Cytotoxicity may be assessed in both models using a lactate dehydrogenase (LDH) assay. These in vitro models of bone resoφtion may be used for routine screening and evaluation of compounds for their ability to alter osteoclast-mediated bone resoφtion.
Media preparation
Calcium free Dulbecco's Modified Eagle's Medium (DMEM) may be obtained in a 5x solution (Specialty Media, D-012). A lx solution is prepared using ultrafiltered water. A suitable media contains 15% heat inactivated horse serum (Sigma, H 1270). Calcium
27 concentration is adjusted to 1.65 to 1.83 mM using 0.2 M CaCl2. Penicillin (100 U/ml) and streptomycin (0.1 mg/ml) are added to the final media preparation. Indomethacin is prepared to 0.5 mg/ml (1.397 x 10 M) in ethanol, and is added to an aliquot of DMEM to produce a final concentration of 0.5 μM. Bovine parathyroid hormone (1-34) may be obtained from Bachem (PCAL 100). PTH is solubilized in 0.1 % BSA and is then diluted in DMEM to produce a final concentration of 10 M PTH. Ten-fold serial dilutions are performed down to 10 M.
Calvaria dissection Pregnant CD-I mice may be obtained from Charles River and are subjected to parturition. Neonatal mice (4-6 days) are cleansed with betadine and then euthanized by decapitation. Adherent skin is cleared away from the skull, exposing the calvaria. The calvaria are dissected away from the skull using a 12B scalpel. Calvaria are immediately placed into a glass petri dish containing room temperature Tyrode's Salt Solution (Sigma, T-2397). The calvaria are trimmed free of cartilage and bisected with a scalpel along the sagital suture. After dissection of all calvaria, calvaria are transferred into 24 well plates containing 0.5 μM indomethacin (Sigma, 1-7378).
Culture conditions Calvaria are incubated in 1.5 ml DMEM in 24 well tissue culture plates at 37*C, 5%
CO /air. Plates are rocked in the incubator using a Bellco rocker platform. Calvaria are pre-incubated in 0.5 μM indomethacin for 24 hours. For each experiment, 6 to 8 random calvaria halves are used for each group. Both halves from a single mouse are never in the same group. Experiments are repeated at least three times.
Prophylactic calvaria experiment
After the 24h pre-incubation period, calvaria are thoroughly washed in indomethacin-free DMEM. Calvaria are then transferred to new wells containing various PTH concentrations, and are cultured for an additional 72 hours. Media samples (30 μl) are obtained every 24 hours and assayed for calcium and LDH activity.
Therapeutic calvaria experiment
At the end of the 24h pre-incubation period, the calvaria are washed free of indomethacin using DMEM. Calvaria are then transferred to new wells containing DMEM or various concentrations of PTH. After 24 hours calvaria are transferred into new wells
28 with fresh media (PTH or DMEM) and cultured an additional 48 hours before addition of control vehicle. This may be accomplished by adding 3 μl of DMSO to new wells, and transferring each calvaria along with its media into wells. Culture continues for a further 24 hours. Media samples are obtained after 72 hours and 96 hours in culture with PTH and assayed for calcium. Additional samples are obtained after 48, 72, and 96 hours in culture with PTH and assayed for LDH.
Calcium Assay
A commercially available diagnostic calcium assay (Sigma, No. 588-3), modified for use in a microtiter format, may be used to determine circulating serum calcium concentrations. This colorimetric assay is dependent on the specific, high affinity complexation of calcium with arsenazo HI dye under acidic conditions, which occurs with 1:1 stoichiometry and absorbs at 600 nm (Bauer, P.J. Affinity and stoichiometry of calcium binding by Arsenazo HI. Anal Biochem, 110:61, 1981; Michaylova, V and P Ilkova. Photometric determination of micro amounts of calcium with Arsenazo HI. Anal Chim Acta, 53: 194, 1971). Magnesium has very low affinity for arsenazo HI.
Briefly, 15 μl of media or rat sera (see below) is diluted 18-fold with ultrafiltered water (nearly calcium-free). Fifty μl of this solution are pipetted into microtiter wells (Nunc, Maxisoφ, flat-bottom, 0.4 ml/well). Standards of 0, 0.5, 1, 2.5, 3.75, 5, 6.25, and 7.5 mg/dl (mg%) calcium, diluted 8-fold with ultrafiltered water from control standards (Sigma, 360-11), are used to construct standard curves. Once all standards and samples are pipetted onto the plate, 150 μl of diagnostic reagent is added to initiate complexation. Optical density measurements are obtained on a microtiter plate reader (Molecular Devices, ThermoMax) at 600 nm.
Lactate dehydrogenase assay
Phosphate buffer is prepared in distilled water (0.26 M K2HPO4'3H2O, 0.26 M
KH9PO4; pH 7.4). A mix consisting of: 22 ml of phosphate buffer, 6 ml distilled water and 2.0 ml of 0.01 M pyruvate is prepared. NADH is prepared to 0.4 mg/ml in phosphate buffer.
Ten μl of media samples obtained from incubated calvaria are added to 96 well plates. Wells containing 10 μl of DMEM serve as blanks. To each well, 90 μl distilled water and 150 μl phosphate mix is added. 50 μl NADH is added using an eight channel pipette immediately before the plate is read on a microtiter plate reader at 340 nm. A kinetic assay is performed for 10 minutes, with a read interval of 20 seconds.
29 THYROID/PARATHYROIDECTOMIZED RAT MODEL of BONE RESORPTION
Parathyroid hormone (PTH) replacement in thyroparathyroidectomized (TPTX) rats is routinely used as an in vivo model of controlled bone resoφtion. Rats are the species of choice since the mechanisms of bone modeling in the rat resemble those in humans. In addition, hormones and pharmacologic agents have similar effects on both rat and human bone (Frost, H.M. and W.S.S. Jee. On the rat model of human osteopenias and osteoporoses. Bone and Mineral, 18: 227-236, 1992). Removal of the thyroid and parathyroid glands results in a rapid loss of parathyroid hormone (PTH) from the circulation. Since PTH induces osteoclast-mediated bone resoφtion, this process is inhibited in TPTX animals. In addition, PTH mediates calcium reabsoφtion from the kidneys and absoφtion from the small intestines. The lack of these activities work in concert to decrease serum calcium levels. In the absence of PTH, rats remain in a hypocalcemic state. Restriction of dietary calcium limits intestinal calcium absoφtion and renal calcium filtration such that serum calcium levels are primarily influenced by bone resoφtion. Controlled PTH replacement therapy results in a controlled return of serum calcium to baseline levels. When replacement occurs, concomitantly with a low calcium diet, serum calcium increase is due to PTH-induced osteoclast-mediated bone resoφtion. In this model, drugs which inhibit bone resoφtion prevent the PTH-mediated return of serum calcium to baseline levels.
Female Wistar rats (226-250 gm, Charles River) are fasted overnight and anesthetized with 0.15 ml of 1.2% tribromoethanol (TBE). The ventral neck area is shaved and swabbed with betadine and isopropanol. A midline incision is made in the neck through the skin and superficial muscle layer, as well as in the stemohyoid muscle. Blunt dissection is performed to expose the thyroid gland. The thyroid gland is carefully isolated from the trachea, thyrohyoid muscle, as well as adjacent nerves and blood vessels, using blunt dissection. The thyroid gland is excised one lobe at a time. Cautery is performed for hemostasis. Care is taken to avoid damaging the recurrent laryngeal nerve since damage to it is shown to affect serum calcium concentrations (Hirsch, P.F., G.F. Gauthier and P.L. Munson. Thyroid hypocalcemic principle and recurrent laryngeal nerve injury as factors affecting the response to parathyroidectomy in rats. Endocrinology, 73: 244-252, 1963. et al., 1963). The incisions are closed using 3-0 vicryl. The wound is coated with triple antibiotic ointment (Fougera; 400 units/g bacitracin zinc, 5 mg/g neomycin sulfate, 5000 units/g polymyxin B sulfate). Following TPTX, rats are pair fed a low calcium diet
(Harlan Teklad TD 95065; <0.003% Ca++, <0.04% PO ) such that each rat receives the
30 same quantity of food. Rats are fed at least 5 grams, but not more than 10 grams, of food Rats consuming less than 3.0 grams of food receive the nutritional supplement Nutπ-Cal p.o. (Evsco; <0.0033% calcium).
PTH Dose Response/Pump implantation
Three days post TPTX, rats which are found to be hypocalcemic, based on day 2 serum calcium levels, are implanted with PTH-contammg Alzet mini-osmotic pumps (ALZA, model 2001D) which pumps at a rate of 1 μl/h. The rats are anesthetized with ketarmne (50 mg/kg, l.p.) and acepromazine (1.67 mg/kg, ι.p.). The scapula region is shaved and prepared for surgery with betadine and isopropanol. A lateral incision of approximately 2 cm in length is made between the scapulae. Using hemostats, a subcutaneous pocket is created into which the Alzet pump is inserted The wound is closed either with nylon suture or with staples. Tπple antibiotic ointment is applied as descπbed previously. Bovme parathyroid hormone 1-34 (PTH) (Bachem California, PCAL100) is prepared in vehicle (10"3 N HCl, 0.15 M NaCl, 20 mg/ml cystemeΗCl) at the following concentrations: 0 156, 0.47, 1.56, 4.7, 15.6, and 156 μM. Alzet mini-osmotic pumps are filled with the PTH solution and maintained in 37»C saline for 4 hours pπor to implantation.
Serum Samples
Rats are anesthetized by CO2 from dry ice and daily blood samples are obtained via cardiac puncture using a 27 gauge needle. Baseline samples are taken just pπor to TPTX Daily samples are obtained m the morning. Samples are allowed to clot on their side for several hours and subsequently spun at lOOOxg for 15 minutes to obtain serum Serum is ahquoted and stored in the refπgerator until assayed for serum calcium Serum calcium is measured (see above) daily for at least 7 days following TPTX.
Uses of Compounds of This Invention Compounds of this invention which bind to an SH2 domain of interest may be used as biological reagents in assays as descπbed herein for functional classification of a pTyr- bindmg domain (e g. SH2 or PI domain) of a particular protein, particularly a newly discovered protein. Families or classes of such proteins which bind to pTyr-contaimng hgands may now be defined functionally, with respect to ligand specificity Moreover, compounds of this invention can be used to inhibit the occurrence of biological events resulting from molecular interactions mediated by the protein of interest Inhibiting such
31 interactions can be useful in research aimed at better understanding the biology of events mediated by the binding of pTyr-containing Hgands to their receptors.
Such compounds would be useful, for example, in the diagnosis, prevention or treatment of conditions or diseases resulting from a cellular processes mediated by the binding of a pTyr-containing ligand with a receptor therefor. For example, a patient can be treated to prevent the occurrence or progression of osteoporosis or to reverse its course by administering to the patient in need thereof an SH2inhibitor which selectively binds Src SH2 or otherwise interferes with Src-mediated signaling.
There are many other conditions for which such signal transduction inhibitors may be useful therapeutically, including, e.g., breast cancer where the SH2 domain-containing proteins Src, PLCgamma and Grb7 have been implicated. Other relevant conditions include prostate cancer, in which case targeting Grb2, PLCgamma, and PI3K, all of which contain SH2 domains, may be useful in treatment or prevention of the disease. Inhibition of the interaction of Grb2 or Abl SH2 domains with BCR-abl may be useful to treat chronic myelogenous leukemia (CML) or acute myelogenous leukemia (AML).
Still other relevant applications include the prevention of interferon-, growth factor-, or cytokine-mediated diseases (e.g. inflammatory diseases) by targeting the interaction of STAT proteins with their pTyr-containing ligands or otherwise inhibiting their signal transduction pathways. Agents that block the SH2 domains of ZAP-70 or otherwise inhibit ZAP-70-mediated signaling would be candidates for the treatment of immune-related disorders such as rejection of transplanted bone marrow, skin or other organs; rheumatoid arthritis; inflammatory bowel disease; and systemic lupus erythmatosis, and a variety of autoimmune diseases.
By virtue of the capacity to inhibit protein-protein interactions or a relevant kinase or phosphatase activity required for cellular events of pharmacologic importance, compounds of this invention which inhibit cellular signal transduction may be used in pharmaceutical compositions and methods for treatment or prevention in a subject in need thereof. Such inhibitors can be used to treat or reduce the risk of the diseases or their pathological effects mediated by such interactions. For example, drugs that completely block one of the two ZAP SH2 domains should effectively prevent ZAP from associating with the activated TCR and thus block T cell activation. A ZAP antagonist or inhibitor would specifically inhibit T cells and avoid the toxicity of the currently used immunosuppressive drugs, FK506 and cyclosporin, which target the more ubiquitously expressed protein, calcineurin. Since calcineurin is required for cellular activities in several tissues in addition to T cells, cyclosporin and FK506 cause side effects in the kidney and central nervous system which limit their application largely to patients with organ transplant rejection.
32 Therapeutic/Prophylactic Administration & Pharmaceutical Compositions
Compounds of this invention can exist in free form or, where appropπate, m salt form. Pharmaceutically acceptable salts of many types of compounds and their preparation are well-known to those of skill in the art. The pharmaceutically acceptable salts of compounds of this invention include the conventional non-toxic salts or the quaternary ammonium salts of such compounds which are formed, for example, from inorganic or organic acids of bases.
The compounds of the invention may form hydrates or solvates. It is known to those of skill in the art that charged compounds form hydrated species when lyophilized with water, or form solvated species when concentrated in a solution with an appropπate organic solvent.
This invention also relates to pharmaceutical compositions compπsing a therapeutically (or prophylactically) effective amount of the compound, and a pharmaceutically acceptable earner or excipient. Carπers include e g. saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof, and are discussed in greater detail below. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffeπng agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carπers such as tπglyceπdes. Oral formulation can include standard carπers such as pharmaceutical grades of manmtol, lactose, starch, magnesium stearate, sodium sacchaπne, cellulose, magnesium carbonate, etc. Formulation may involve mixing, granulating and compressing or dissolving the ingredients as appropπate to the desired preparation. The pharmaceutical earner employed may be, for example, either a solid or liquid
Illustrative solid earner include lactose, teπa alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, steaπc acid and the like. A solid earner can include one or more substances which may also act as flavoπng agents, lubπcants, solubilizers, suspending agents, fillers, ghdants, compression aids, binders or tablet-disintegrating agents; it can also be an encapsulating mateπal. In powders, the earner is a finely divided solid which is in admixture with the finely divided active ingredient. In tablets, the active ingredient is mixed with a earner havmg the necessary compression properties in suitable proportions ,and compacted m the shape and size desired The powders and tablets preferably contain up to 99% of the active ingredient. Suitable solid earners include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextπn, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrro dme, low melting waxes and ion exchange resins.
33 Illustrative liquid carriers include syrup, peanut oil, olive oil, water, etc. Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid carriers for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the carrier can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid carders are useful in sterile liquid form compositions for parenteral administration. The liquid carrier for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellant. Liquid pharmaceutical compositions which are sterile solutions or suspensions can be utilized by, for example, intramuscular, intraperitoneal or subcutaneous injection. Sterile solutions can also be administered intravenously. The compound can also be administered orally either in liquid or solid composition form.
The carrier or excipient may include time delay material well known to the art, such as glyceryl monostearate or glyceryl distearate along or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate and the like. When formulated for oral administration, 0.01% Tween 80 in PHOSAL PG-50 (phospholipid concentrate with 1,2-propylene glycol, A. Nattermann & Cie. GmbH) has been recognized as providing an acceptable oral formulation for other compounds, and may be adapted to formulations for various compounds of this invention.
A wide variety of pharmaceutical forms can be employed. If a solid carrier is used, the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form or in the form of a troche or lozenge. The amount of solid carrier will vary widely but preferably will be from about 25 mg to about 1 g. If a liquid carrier is used, the preparation will be in the form of a syrup, emulsion, soft gelatin capsule, sterile injectable solution or suspension in an ampule or vial or nonaqueous liquid suspension.
To obtain a stable water soluble dosage form, a pharmaceutically acceptable salt of the compound may be dissolved in an aqueous solution of an organic or inorganic acid, such as a 0.3M solution of succinic acid or citric acid. Alternatively, acidic derivatives can be dissolved in suitable basic solutions. If a soluble salt form is not available, the
34 compound is dissolved in a suitable cosolvent or combinations thereof. Examples of such suitable cosolvents include, but are not limited to, alcohol, propylene glycol, polyethylene glycol 300, polysorbate 80, glycerin, polyoxyethylated fatty acids, fatty alcohols or glycerin hydroxy fatty acids esters and the like in concentrations ranging from 0-60% of the total volume.
Various delivery systems are known and can be used to administer the compound, or the various formulations thereof, including tablets, capsules, injectable solutions, encapsulation in liposomes, microparticles, microcapsules, etc. Methods of introduction include but are not limited to dermal, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, pulmonary, epidural, ocular and (as is usually preferred) oral routes. The compound may be administered by any convenient or otherwise appropriate route, for example by infusion or bolus injection, by absoφtion through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. For treatment or prophylaxis of nasal, bronchial or pulmonary conditions, prefened routes of administration are oral, nasal or via a bronchial aerosol or nebulizer.
In certain embodiments, it may be desirable to administer the compound locally to an area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, by injection, by means of a catheter, by means of a suppository, or by means of a skin patch or implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
In a specific embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the side of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
Administration to an individual of an effective amount of the compound can also be accomplished topically by administering the compound(s) directly to the affected area of the
35 skin of the individual. For this puφose, the compound is administered or applied in a composition including a pharmacologically acceptable topical carrier, such as a gel, an ointment, a lotion, or a cream, which includes, without limitation, such carriers as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oils.
Other topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5%) in water, or sodium lauryl sulfate (5%) in water. Other materials such as anti-oxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary. Percutaneous penetration enhancers such as Azone may also be included.
In addition, in certain instances, it is expected that the compound may be disposed within devices placed upon, in, or under the skin. Such devices include patches, implants, and injections which release the compound into the skin, by either passive or active release mechanisms. Materials and methods for producing the various formulations are well known in the art and may be adapted for practicing the subject invention. See e.g. US Patent Nos. 5,182,293 and 4,837,311 (tablets, capsules and other oral formulations as well as intravenous formulations) and European Patent Application Publication Nos. 0 649 659 (published April 26, 1995; illustrative formulation for IV administration) and 0 648 494 (published April 19, 1995; illustrative formulation for oral administration).
The effective dose of the compound will typically be in the range of about 0.01 to about 50 mg/kgs, preferably about 0.1 to about 10 mg/kg of mammalian body weight, administered in single or multiple doses. Generally, the compound may be administered to patients in need of such treatment in a daily dose range of about 1 to about 2000 mg per patient.
The amount of compound which will be effective in the treatment or prevention of a particular disorder or condition will depend in part on the nature and severity of the disorder or condition, which can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. The precise dosage level should be determined by the attending physician or other health care provider and will depend upon well known factors, including route of administration, and the age, body weight, sex and general health of the individual; the nature, severity and clinical stage of the disease; the use (or not) of concomitant therapies.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of
36 the invention Optionally associated with such contamer(s) can be a notice m the form prescπbed by a governmental agency regulating the manufacture, use or sale of pharmaceutical products, which notice reflects approval by the agency of manufacture, use or sale for human administration
The representative examples which follow are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, vaπous modifications of the invention and many further embodiments thereof, in addition to those shown and descπbed herein, will become apparent to those stalled in the art from the full contents of this document, including the examples which follow and the references to the scientific and patent literature cited herein It should also be appreciated that those cited references are incoφorated herein by reference to help illustrate the state of the art
In addition, the full contents of US Patent Applications USSN 09/190,424 (Weigele et al, "Novel Signal Transduction Inhibitors, Compositions Containing Them & Uses Thereof, filed November 12, 1998), and USSN 60/108,106 (Shakespeare et al, "Bicyclic Signal Transduction Inhibitors, Compositions Containing Them & Uses Thereof, filed November 12, 1998) are incoφorated by reference herein. Those documents provide additional synthetic and other guidance which may be of interest to the practitioner of the subject invention
The following examples contain important additional information, exemplification and guidance which can be adapted to the practice of this invention m its vaπous embodiments and the equivalents thereof
37 Examples
General: Compounds of this invention can be prepared by convergent synthesis as illustrated in the following examples. Typically, a carboxylic acid, e.g., Ac-Tyr(PO3Bn2)- OH or Boc-Tyr(PO3Bn2)-OH, for example, is coupled with a heterocyclic amine using standard materials and methods for peptide coupling, including any necessary or desired protection and subsequent deprotection.
Abbreviations. The following abbreviations are used in this document.
Abu alpha-aminobutyric acid
Ac acetyl aq aqueous
Bn benzyl
Boc tertiary butyloxycarbonyl
BOC-ON (CH3)3COCO2N=C(C6H5)CN
Cbz benzyloxycarbonyl
CDI 1 , 1 '-carbonyldiimidazole
Chx cyclohexyl
DCM dichloromethane dba dibenzylidene acetone
DBU l,8-diazabicyclo[5.4.0]undec-7-ene
Dess-Martin periodinane 1,1,1 -triacetoxy- 1 , 1 -dihydro- 1 ,2-benziodoxol-3 ( 1 H)-one
DIBAL-H diisobutylaluminum hydride
DIEA N,N-diisopropylethylamine
DMAP 4-dimethylaminopyridine
DME ethylene glycol dimethyl ether
DMF N,N-dimethylformamide
DMSO dimethylsulfoxide or methyl sulfoxide dppp l,3-bis(diphenylphosphino)propane
EDC l-(3-dimethylaminopropyl)-3-ethylcarbodiimide
Gin glutamine
Glu glutamic acid
Gly glycine
HMDS 1 , 1 , 1 ,3 ,3 ,3-hexamethyldisilazane
HOBT 1 -hydroxybenzotriazole
HPLC high performance liquid chromatography imid imidazole
Lawesson's Reagent [2,4-bis(4-methoxyphenyl)-l,3-dithia-2,4-diphosphetane-2,4- disulfide]
LiHMDS lithium hexamethyldisilazide or lithium bis(trimethylsilyl) amide
MeCN acetonitrile
MS mass spectrometry
Ms methanesulfonyl (mesyl)
NBS N-bromosuccinimide
NMR nuclear magnetic resonance
Pd/C palladium on carbon
Rochelle salt potassium sodium tartrate satd saturated
Su succinimide
Figure imgf000040_0001
pyr pyridine
38 RT room temperature
It room temperature
TBAF tetrabutylammonium fluoride
TBS tertiarybutyldimethylsilyl
Tf trifluoromethanesulfonyl or trifluoromethanesulfonate
TFA trifluoroacetic acid
TFAA trifluoroacetic anhydride
THF tetrahydrofiiran
Thr threonine
TLC thin layer chromatography
TMS trimethylsilyl
TMSE trimethylsilylethanol
Figure imgf000041_0002
Tyr tyrosine
« « « «
General Methods A-G: Preparation of N-acetyl phosphate derivatives
H2O3P
ZZ1
Figure imgf000041_0001
R1 R2
Compounds such as Compound ZZ1 can be prepared according to General Methods A-G, as exemplified by the following examples. General Methods C and D exemplify typical coupling strategies, which can be modified with obvious standard manipulations and as shown here in General Methods A-G as well as in the following General Methods H-ZT.
General Methods A and B: Preparation of thiazoles and oxazoles via the Hantzsch synthesis:
Many thiazoles and oxazoles can be prepared via standard Hantzsch thiazole synthesis with alpha halo ketones and thioamides or amides.
39 Standard thiazole formation - Conditions A and B:
NI-UHCO3, DME Lawesson's
OSu *- NH2
PHN PHN PHN or standard methods Reagent
O trom ammo acid
P = Boc or Cbz
0_R9 standard
PHN ^R9 deprotection H N* conditions A orB // conditions -A ,10 1
A* KHCO3, DME free amine then or salt
TFAA, pyr
B. EtOH
RT to reflux
General Method C - Coupling Conditions C:
O R1 amine or salt 1 EDC-HCI, HOBT
OH
DIEA, CH2CI2
Figure imgf000042_0001
Figure imgf000042_0002
H -ζ
P = Boc
(commercially available) 1. standard deprotection O R1 conditions
2. standard acetylation etc. H203P
Figure imgf000042_0003
S ιo conditions
General Method D - Coupling Conditions D:
1 (BnO)2P(0)H amine or salt 1 CCI4, DIEA EDC'HCI, HOBT
OCH3 OH
Figure imgf000042_0004
DMAP, CH3CN DIEA, CH2CI2
HO 2. LιOH«H20 Bn2θ3P
Figure imgf000042_0005
THF-H20 13 commercially available
O R standard
N-_R9 deprotection //
Bn203P
Figure imgf000042_0006
conditions
Figure imgf000042_0007
10
40 1. Compound A
Figure imgf000043_0001
A
Compounds such as Compound A can be prepared using thiazole formation Conditions A followed by Coupling Conditions C. Expeπmental details for the synthesis of Compound A are as follows
Preparation of Bromomethyl Ketone 2:
1 LiHMDS, THF TMSCI-Et3N (1 1 )
Br
2 NBS NaHC03, THF
Bromomethyl ketone 2 can be prepared according to the procedure found in Hajos, Z G , Wachter, M P , Werblood, H. M.; Adams, R. E. J. Org. Chem 1984, 49, 2600.
To a solution of 8.56 mL (8.56 mmol) of LiHMDS (1 0 M solution in THF) in 25 mL of THF at -78 °C (in a flame dπed 250 mL round bottom flask) was added 1 0 g (7 13 mmol) of cyclohexylacetone m 50 mL of THF via cannula over 10 mm followed by a 5 mL πnse The resulting bπght yellow solution was kept at -78 °C for 40 min whereupon 5 9 mL (21 4 mmol) of the supernatant of a 1:1 mixture of TMSCl-Et3N (after centπfugation) was added via syπnge. The resulting mixture was stirred at -78 °C for 1 5 h, quenched with 450 mg NaHCO3, followed by 45 mL saturated aqueous NaHCO3, allowed to warm to RT, and then diluted with Et2θ, water and bπne. The aqueous layer was extracted with Et2θ The combined organic layers were washed with brine, dried over Na2SO4 and concentrated. The volatile crude silyl enol ether was used directly in the next step
To a solution of the crude silyl enol ether m 40 mL THF at RT was added 719 mg (8.56 mmol) of NaHCO3, and the resulting slurry was cooled to -78 °C. At this time, 1.27 g (7 13 mmol) of NBS was added in one portion, and the resulting mixture stined at -78 °C for 30 min, warmed to RT, quenched with 450 mg NaHCO3, followed by 45 mL saturated aqueous NaHCO3, and then diluted with Et2θ, water and bπne. The aqueous layer was extracted with Et2θ. The combined organic layers were washed with bπne, dπed over
41 Na2SO4 and concentrated. Flash chromatography (elution with 7:1 hexanes-Et2θ) gave 1.23 g (78.8%) of a volatile pale oil: Rf 0.39 (7:1 hexanes-Et2θ).
Preparation of S-4*-(benzyloxycarbonyl)-2-(tert-butoxycarbonylamino)thiobutyramide (3): pθ2Bn
1. NH4HCθ3 ΛSu
BocHN f 2 |_awesson's BocHN
Reagent
Figure imgf000044_0001
The intermediate amide can be prepared according to the procedure found in Nozaki, S.; Muramatsu, I. Bull. Chem. Soc. Jpn. 1988, 61, 2647.
To a mixture of 4.0 g (9.21 mmol) of Boc-Glu(OBn)-OSu in 45 mL of CH2CI2 and 5 mL of dioxane at RT was added 2.2 g (27.6 mmol) of ammonium hydrogen carbonate. The resulting mixture was stined at RT for 19 h, diluted with CH2CI2 and water and stined until clear. The aqueous layer was extracted twice with CH2CI2. The combined organic layers were dried over Na2SO4 and concentrated. The crude amide was used directly in the next step.
To a mixture of the crude amide in 50 mL of DME and 20 mL of THF at RT was added 2.05 g (5.07 mmol) of Lawesson's reagent. The resulting mixture was stined at RT for 14 h and concentrated. Flash chromatography (elution with 2:1 hexanes-EtOAc) gave 3.14 g (96.6%) of a white solid: Rf 0.56 (3:1 hexanes-EtOAc). Electrospray Mass Spectrum (50/50 acetonitrile/water) m z 351 (M-H).
Preparation of Thiazole 4:
?02Bn CC 02Bn
BrCH2C(0)CH2Chx (2) J
KHC03, DME ,NH2
BocHN' then BocNH
Thiazole 4 can be prepared according to Aguilar, E.; Meyers, A. I. Tetrahedron Lett. 1994, 35, 2473 and Bredenkamp, M. W.; Holzapfel, C. W.; van Zyl, W. J. Synth. Commun. 1990, 20, 2235.
To a solution of 348 mg (0.987 mmol) of thioamide 3 in 5 mL of DME at -15 °C was added 791 mg (7.90 mmol) of KHCO3. The resulting mixture was stined at -15 °C for 5 min whereupon 516 mg (2.35 mmol) of bromide 2 in 2 mL of DME was added via syringe
42 followed by two 0.5 mL DME rinses. The resulting mixture was stined at -15 °C for 1 h, at RT for 2.5 h, and then recooled to -15 °C. At this point, a cooled solution of 0.558 mL (3.95 mmol) of TFAA in 0.639 mL (7.90 mmol) of pyridine was added via syringe. The resulting mixture was stined at -15 °C for 50 min, warmed to RT, then diluted with CH2CI2 and H2O. The aqueous layer was extracted twice with CH2CI2. The combined organic layers were dried with Na2SO4 and concentrated. Flash chromatography (elution with 2: 1 hexanes-Et2θ then 1 : 1 hexanes-Et2θ) gave 423 mg (90.6%) of a pale oil: Rf 0.29 (1:1 hexanes-Et2θ). Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 946 (M- H).
Preparation of amine 5 TFA salt: θ2Bn
TFA
BocNH -r" *•
VI T I CH2CI2
Figure imgf000045_0001
To a solution of 423 mg (0.895 mmol) of Boc-protected amine 4 in 6 mL of CH2CI2 at RT was added 0.5 mL of TFA. The resulting mixture was stined at RT for 30 min whereupon an additional 0.5 mL of TFA was added. The resulting mixture was stined at RT for 1.75 h and concentrated in vacuo to give 497 mg of a white foam. The crude amine salt was used directly in the next step.
Preparation of coupling product 6:
amine salt 5
EDC'HCI, HOBT -
„ ,-.
Figure imgf000045_0002
DIEA, CH2CI2
Figure imgf000045_0003
To a solution of 497 mg of crude amine salt 5 (assume 0.895 mmol), 0.648 mL ( 3.72 mmol) of DIEA and 403 mg (0.745 mmol) of Boc-Tyr(PO3Bn2)-OH in 7.5 mL of CH2CI2 at RT was added 171 mg (1.12 mmol) of HOBT followed by 214 mg (1.12 mmol) of EDC'HCl. The resulting mixture was stined at RT for 4 h, then diluted with CH2CI2 and water. The aqueous layer was extracted twice with CH2CI2. The combined organic layers were dried over Na2SO4 and concentrated. Flash chromatography (elution with 1: 1 hexanes-EtOAc) gave 404 mg (60.6%) of a white foam: Rf 0.30 (1: 1 hexanes- EtOAc). Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 897 (M+H).
43 Preparation of amme 7 TFA salt:
95 % TFA PhOMe
RT, 2 h
Figure imgf000046_0001
Figure imgf000046_0002
To 337 mg (0.376 mmol) of Boc-protected amine 6 and 0.5 mL of anisole was added 7.5 mL of 95% TFA (aq.). The resulting solution was stined at RT for 2 h and concentrated to a pale gold oil. White granular crystals were formed by adding Et2θ and scraping and crushing the solid in the flask. The crystals were πnsed three times with Et2θ (supernatant removed via pipet) to give 190 mg (69% assuming TFA salt) of a granular white solid. The combined supernatants were combined with those from another batch, concentrated and crystallized as above to yield a second crop. Electrospray Mass Spectrum (50/50 acetonitπle/water + 0.1% ammonium hydroxide) m/z 616 (M-H)
Preparation of acetamide 8:
Ac20 DIEA
DMF, HCl H203P
Figure imgf000046_0004
Figure imgf000046_0003
To 189 mg (0.259 mmol) of amine salt 7 in 2.5 mL of DMF at 0 °C was added via synnge 0.226 mL (1.30 mmol) of DIEA followed by 0.049 mL (0.52 mmol) of Ac2θ. The resulting solution was stined at 0 °C for 1.25 h and then at RT for 2 h whereupon it was quenched with 1.5 mL 2 N HCl. The resulting mixture was stined at RT for 15 mm and then diluted with EtOAc. The organic layer was washed with water. The combined aqueous layers were extracted three times with EtOAc. The combined organic layers were dπed over Na2SO4 and concentrated. HPLC and MS indicated desired product (658 (M+H)) as well as over acetylation product (700.35 (M+H)). The residue was dissolved in 3 mL DMF, stined with 0.5 mL 2 N HCl for 30 mm, and worked up as above. White granular crystals were formed by adding Et2θ and scraping and crushing the solid in the flask. The crystals were πnsed three times with Et2θ (supernatant removed via pipet) to give 114 mg (67%) of a granular white solid. The combined supernatants were concentrated to yield 31 mg (18%) of a second crop. Electrospray Mass Spectrum (50/50 acetonitπle/water + 0.1% ammonium hydroxide) m/z 658 (M-H).
44 Preparation of Compound A:
10 % Pd-C
HC02NH4
Figure imgf000047_0001
MeOH
Figure imgf000047_0002
To a sluny of 114 mg (0.173 mmol) of benzyl ester 8 in 2.5 mL of MeOH at RT was added 55 mg (0.867 mmol) of NH4CO2H followed by 100 mg of 10% Pd-C. The resulting mixture was heated at 67 °C for 35 min, cooled to RT, filtered through Celite and the filtrate concentrated. The resulting concentrate was dissolved in H2O and lyophilized (repeated several times) until the mass remained constant, yielding 105 mg of a fluffy white solid. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 568 (M+H).
2. Compound B
?02H
' SJ
Figure imgf000047_0003
B
Compounds such as Compound B can be prepared using thiazole formation Conditions A followed by Coupling Conditions D. Experimental details for the synthesis of compound B are as follows:
Preparation of Bromomethyl Ketone 9:
1. LiHMDS, THF TMSCI, -78 °C Bt*
2. NBS, NaHC03 THF, -78 °C
Bromoketone 9 was prepared as bromoketone 2 except that TMSCI was used rather than the centrifugate from 1:1 Et3N-TMSCl. Flash chromatography (elution with 6:1 hexanes- Et2θ) gave 1.8 g (50.2%) of a very volatile pale oil: Rf 0.38 (6:1 hexanes-Et2θ).
45 Preparation of Thiazole 10:
C02Bn
Br0H2C(O)CH2CH(CH 3)2
3 KHC03, DME NH2 10
BocH " then BocNH TFAA, pyr
Figure imgf000048_0001
Thiazole 10 can be prepared as shown for the preparation of thiazole 4. Flash chromatography (elution with 3:2 hexanes-Et2θ) gave 0.95 g (80.5%) of a pale oil: Rf 0.50 (1:1 hexanes-Et2θ).
Preparation of amine 11:
TFA, CH2CI2,
11
Figure imgf000048_0002
NaHC03, EtOAc
Figure imgf000048_0003
To a solution of 0.95 g (2.20 mmol) of Boc-protected amine 10 m 15 mL of CH2CI2 at RT was added 1.5 mL of TFA. The resulting mixture was stined at RT for 1.3 h whereupon an additional 1.5 mL of TFA was added. The resulting mixture was stined at RT for 1.7 h and then concentrated. The resulting oil was dissolved in EtOAc and 5% aqueous NaHCO3. The organic layer was dπed over Na2SO4 and concentrated. Flash chromatography (elution with 12.1 CH2C -MeOH) gave 634 mg (86.8%) of a white foam: Rf 0.46 (9 1 CH2CI2- MeOH). Electrospray Mass Spectrum (50/50 acetomtπle/water) m/z 333 (M+H)
Preparation of Dibenzyl phosphate 12: o (BnO)2P(0)H O CCI4, DIEA 12
HQX NHAc DMAP, CH3CN
Figure imgf000048_0004
Dibenzyl phosphate 12 can be prepared according to the procedure found in Silverberg, L. J.; Dillon, J. L.; Vemishetti, P. Tetrahedron Lett. 1996, 37, 111.
To a mixture of 5 g (21.1 mmol) of N-acetyl-L-tyrosme methyl ester in 300 mL of CH3CN at -10 °C was added 10.2 mL (105.5 mmol) of CCI4. The resulting mixture was stined at -10 °C for 5 mm whereupon 8.1 mL (46.5 mmol) of DIEA, 70 mg (0.57 mmol) of DMAP and 8.29 g (31.6 mmol) of dibenzyl phosphite were added. The resulting mixture was stined at -10 °C for 1 h and then at 0 °C for 30 mm. The mixture was then quenched with
46 25 mL 0.5 M KH2PO4 and allowed to warm to RT. The quenched reaction mixture was diluted with H2O, concentrated, then extracted three times with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated. Flash chromatography (elution with 5% MeOH in CH2CI2) gave 9.2 g (87.6%) of a pale oil: Rf 0.66 (5% MeOH in CH2CI2).
Preparation of Acid 13:
LiOH-H20
O I CH, 13
NHAc THF, H2O
Bn203PC Bn2θ3 < σ^ NHAc
To a solution of 9.2 g (18.5 mmol) of methyl ester 12 in 150 mL of THF at 0 °C was added via addition funnel a solution of 0.855 g (20.4 mmol) of LiOH*Η2θ dissolved in 100 mL of H2O over 20 min. The resulting mixture was stined at 0 °C for 1.5 h and concentrated. The aqueous reaction mixture was washed two times with EtOAc and acidified to pH 2-3. The acidified aqueous layer was then extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4 and concentrated, giving 8.2 g (91.7%) of a thick oil: Rf 0.24 (5% MeOH in CH2CI2). Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 482 (M-H).
Preparation of coupling product 14:
amine 11 EDC-HCI, HOBT r, Λ
Figure imgf000049_0001
DIEA, CH2Ci2
Bn203PO^ ^--^
Figure imgf000049_0002
To a solution of 418 mg (0.865 mmol) of Ac-Tyr(PO3Bn2)-OH (13) and 345 mg (1.04 mmol) of amine 11 in 6 mL of CH2CI2 at RT was added 0.452 mL (2.59 mmol) of DIEA followed by 159 mg (1.04 mmol) of HOBT and 199 mg (1.04 mmol) of EDC-HCI. The resulting mixture was stined at RT for 5.75 h (TLC indicated the presence of both starting materials) whereupon an additional 20 mg each of HOBT and EDC»HC1 were added. The resulting mixture was stined at RT for lh and concentrated (no change from previous TLC). The residue was then dissolved in EtOAc and 5% NaHCO3. The organic layer was washed with H2O and 10% citric acid, dried over Na2SO4 and concentrated. Flash chromatography (elution with 12:1 CH2CI2 -MeOH) gave 368 mg (53.3%) of a pale oil: Rf 0.54 (9:1 CH2Cb-MeOH).
47 Preparation of B:
10 % Pd-C
HC02NH4
Figure imgf000050_0001
MeOH
Figure imgf000050_0002
To a mixture of 100 mg (0.125 mmol) of benzyl ester 14 in 2.5 mL MeOH at RT was added 39.5 mg (0.627 mmol) of NH4CO2H followed by 50 mg of 10% Pd-C. The resulting mixture was heated at 67 °C for 50 min, cooled to RT, filtered through a pad of Celite with MeOH and the resulting filtrate was concentrated. The slightly blackened oil was filtered through a pipet with a pad of Celite and a cotton plug, then through another pad of Celite, both with MeOH and a little CH2CI2. The filtrate was concentrated and the residue was dissolved in H2O and lyophilized to a glassy solid. The solid was dissolved again in H2O and lyophilized until the mass remained constant (one more time), yielding 72.6 mg of a fluffy pale yellow solid. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 528 (M+H).
3. Compound C
Figure imgf000050_0003
Compounds such as Compound C can be prepared using thiazole formation Conditions B followed by Coupling Conditions D. Experimental details for the synthesis of Compound C are as follows:
Preparation of thioamide 15:
Lawβsson's
NH2
CbzH NH,
Reagent CbzHN'^i**-" 15 S
Thioamide 15 can be prepared following the procedure described for the preparation of thioamide 3 using the commercially available N-Cbz-glycinamide as the starting material. Flash chromatography (elution with 2:1 hexanes-EtOAc) provided 4.05 g (75.1%) of a white solid: Rf 0.50 (1: 1 hexanes-EtOAc). Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 449 (M+H).
48 Preparation of thiazole 16:
PhC(0)CHBrMe
NH2
CbzHN'"^*-^ CbzHN
EtOH "" 16 S RT to reflux
Figure imgf000051_0001
To a solution of 1.0 g (4.46 mmol) of thioamide 15 in 20 mL of EtOH at RT was added 0.882 mL (5.80 mmol) of 2-bromopropιophenone (90% tech.). The resulting mixture was stined at RT for 2.75 h, at 78 °C for 7 h, and then cooled to RT and concentrated. Flash chromatography (elution with 2:1 hexanes-EtOAc followed by 1:1 hexanes-EtOAc, then EtOAc, then 2:1 CH2Cl2-MeOH) provided 0.80 g (53.0%) of a white foam [Rf 0.50 (1:1 hexanes-EtOAc)] and 0.86 g of slightly impure mateπal which was retained for additional punfication.
Preparation of amine 17:
30% HBr in HOAc
17
NaHC03, EtOAc
Figure imgf000051_0002
's Me
To 800 mg (2.36 mmol) of neat Cbz-protected amine 16 at RT was added 12 mL of 30% HBr in HOAc. The resulting mixture was stmed at RT for 1 h and then concentrated (CH2CI2 added for transfer and dissolution and hexanes added for hexanes-HOAc azeotrope). The resulting red-orange solid was slunied in EtOAc and 10% aqueous NaHCO3 was added until the solution became clear. The organic layer was washed with water. The combined aqueous layers were extracted three times with EtOAc, saturated with solid (NH4)2SO4 and extracted once more with EtOAc. The combined organic layers were dπed over Na2SO4 and concentrated. Flash chromatography (elution with 1 IT CH2CI2- MeOH followed by 6:1 CH2Cl2-MeOH then 3:1 CH2Cl2-MeOH) gave 187 mg (38.7%) of a pale foam: Rf 0.40 (3:1 CH2Cl2-MeOH). Electrospray Mass Spectrum (50/50 acetonitπle/water) m/z 339 (M+H).
Preparation of coupling product 18:
amine 17 EDC-HCI, HOBT
OH 18
DIEA, CH2CI2
Figure imgf000051_0003
Figure imgf000051_0004
49 Coupling product 18 can be prepared as shown for the preparation of coupling product 6. Flash chromatography (elution with 10:1 CH2Cl2-MeOH) gave 260.7 mg (51.0%) of a pale oil: Rf 0.59 (9:1 CH2CI2 -MeOH).
Preparation of C:
0 y^
A**y ^r χ 95 % aq TFA
PhOMe
Figure imgf000052_0001
-
Figure imgf000052_0003
BnjOgPC ^^ A -0
Me^ H2og>cr c "Λ Me
To 254.7 mg (0.380 mmol) of benzyl protected phosphate 18 and 0.4 mL of anisole was added 5 mL of 95% TFA (aq). The resulting solution was stined at RT for 2.75 h and then concentrated to a pale gold oil. White granular crystals were formed by adding Et2θ and scraping and crushing the solid in the flask. The crystals were rinsed three times with Et2θ (supernatant removed via pipet) to give 197.3 mg of a granular white solid. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 490 (M+H).
4. Compound D
Figure imgf000052_0002
D
Compounds such as Compound D can be prepared using thiazole formation Conditions A followed by Coupling Conditions C. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 496 (M+H).
5. Compound E
^ oά^j-
Compounds such as Compound E can be prepared using thiazole formation Conditions B followed by Coupling Conditions D. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 854 (2M-H).
50 6. Compound F
^ rir -
Compounds such as Compound F can be prepared using thiazole/oxazole formation Conditions B, substituting the appropriate keto amide for the bromo ketone and substituting toluene as solvent, followed by Coupling Conditions D. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 412 (M+H).
7. Compound G
o
Figure imgf000053_0001
H^P ^ NHA° S
Compounds such as Compound G can be prepared using thiazole formation Conditions B followed by Coupling Conditions D. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 476 (M+H).
8. Compound H
C02Et
H2o3pr-
Figure imgf000053_0002
H
Compounds such as Compound H can be prepared using thiazole formation Conditions B followed by Coupling Conditions D. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 472 (M+H).
9. Compound I
Figure imgf000053_0003
51 Compounds such as Compound I can be prepared using thiazole formation Conditions B followed by Coupling Conditions D. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 550 (M-H).
10. Compound J
o
H2θ3PcA^ NHAC S
Figure imgf000054_0001
Compounds such as Compound J can be prepared using thiazole formation Conditions B followed by Coupling Conditions D with the appropriate side chain manipulations as shown in the following scheme. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 574 (M+CH3CN).
>JO CbzHN^γ NH,
Br2 S
\-^^r Et,0
CO2H
Figure imgf000054_0002
EtOH RT to reflux
CbzHN I . UOH+I2O CbzHN 2. BH3-THF
Figure imgf000054_0003
Figure imgf000054_0004
11. Compound K
Figure imgf000054_0005
K
Compounds such as Compound K can be prepared using thiazole formation Conditions A followed by Coupling Conditions D. Compound K was isolated as a 60/40 mixture of diastereomers, presumably at the NHAc stereocenter. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 542 (M+H).
52 General Method E: Preparation of thiazoles, oxazoles and imidazoles via keto amide intermediates:
Many thiazoles, oxazoles and imidazoles can be prepared via the conesponding keto amides according to the cyclization conditions described in Gordon, T. D. et al. (Tetrahedron Lett. 1993. 34, 1901) as shown generically below.
PHN R*** HN(Me)OMe-HCI rnnyri R10MgX PHN^ ,R9
O Y^OH DIEA O^NMe
CDI, THF THF, 0 °C .
OMe
P = Boc or Cbz any protected standard H2Nv ammo acid I 1 ' H ! Q
N^ ^R9
PHN deprotection
0^R1° standard conditions coupling conditions O ϊ' free amine or salt
cyclization 1. side chain conditions manipulation
PHN H2
Figure imgf000055_0001
2. standard
Figure imgf000055_0002
La wesson's deprotection
Reagent (S) conditions
Dehydration reagent (O) X = S, O, NR12 X = S, O, NR12
NH2R12 etc. (NR12)
12. Compound L
H2θ*3P
Figure imgf000055_0003
Figure imgf000055_0004
Compounds such as Compound L can be prepared according to General Method E, followed by Coupling Conditions D. Experimental details for the synthesis of compound L are as follows:
Preparation of Amide 19:
BocHN-^^. HN(Me)θMe.HCI B°°HN
DIEA rjJMe CDI, THF XXA - OMe
53 To a mixture of 4.0 g (14.7 mmol) of Boc-beta-cyclohexyl-L-alanme in 75 mL of CH2CI2 at 0 °C was added 2.63 g (16.2 mmol) of CDI. The resulting mixture was warmed to RT (bath removed) over 10 mm whereupon 3.85 mL (22.1 mmol) of DIEA was added via syπnge followed by 1.58 g (16.2 mmol) of HN(Me)OMeeHCl. The resulting mixture was stined at RT for 22.5 h, concentrated to a viscous oil and then diluted with EtOAc and water. The orgamc layer was washed once with bπne. The combined aqueous layers were extracted once with EtOAc. The combined organic layers were dπed with Na2SO4 and concentrated. Flash chromatography (elution with EtOAc) gave 3.82 g (82.4%) of a colorless oil: Rf 0.58 (9: 1 CH2d2-MeOH). Electrospray Mass Spectrum (50/50 acetonitπle/water) m/z 315 (M+H) .
Preparation of Ketone 20:
^^Nv /V -A^^~^M o O^-^NljJMS- —^ ^ T THHFF, n 0 °°πC 20
OMe
Figure imgf000056_0001
To a solution of 3.82 g (12.1 mmol) of amide 19 m 60 mL of THF at 0 °C was added 72.9 mL (36.5 mmol) of 3-butenylmagnesιum bromide (0.5 M in THF). The resulting mixture was stined at 0 °C for 10 mm, and at RT for 25.5 h. The reaction was then diluted with Et2θ, quenched with saturated aqueous NH4CI, then diluted with more Et2θ and water. Note: At this point, the reaction mixture was combined with another reaction performed in parallel (5.66 mmol scale). The combined organic layers were washed once with bπne. The combined aqueous layers were extracted twice with Et2θ. The combined organic layers were dπed with Na2SO4 and concentrated. Flash chromatography (elution with 1: 1 hexanes-EtOAc) gave 3.84 g (69.7%, combined reactions) of a pale oil. Rf 0.49 (1.1 hexanes-EtOAc)
Preparation of Amine Salt 21:
BocH ""N"^■Y^^-y^^ 33NN HHCCll HH2j-NN--.^*^^.
O^- . ^-N -^ EtOAc, dioxane -*-**^\ \/ 21
^
^ -HCI ^
To a mixture of 3.84 g (12.4 mmol) of protected amine 20 in 20 mL of EtOAc and 20 mL of dioxane at RT was added 60 mL of 3 N HCl. The resulting mixture was stined at RT for 4.5 days and then concentrated. The off white solid was tπturated with Et2θ (3 times with decanting) and dπed m vacuo giving 2.68 g (87.9%) of a fluffy white solid. Electrospray Mass Spectrum (50/50 acetonitπle/water) m/z 210 (M+H).
54 Preparation of Ketoamide 22:
Boc-Abu-OH
BocH
EDC-HCI, HOBT DIEA, CH2CI2
•HCl ^ ***-***
Figure imgf000057_0001
To 1.34 g (5.45 mmol) of amine salt 21 in 30 mL of CH2CI2 at RT was added 3.2 mL (18.2 mmol) of DIEA followed by 0.922 g (4.54 mmol) of Boc-Abu-OH. The resulting mixture was stined at RT for 5 min whereupon 1.043 g (6.81 mmol) of HOBT and 1.305 g (6.81 mmol) of EDC'HCl were added in one portion. The resulting mixture was stined at RT for 23 h and then concentrated to a viscous oil. The resulting oil was then diluted with EtOAc and 5% aqueous NaHCO3. The organic layer was then washed with 10% citric acid and brine. The combined aqueous layers were extracted once with EtOAc. The combined organic layers were dried with Na2SO4 and concentrated. Flash chromatography (elution with 3:1 hexanes-EtOAc) gave 1.59 g (88.8%) of a white solid: Rf 0.42 (1:1 hexanes- EtOAc). Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 395 (M+H).
Preparation of Thiazole 23:
Mθ Me
Lawesson's Reagent
BocHN BocHN
THF, reflux
Figure imgf000057_0002
23
Figure imgf000057_0003
To a solution of 1.59 g (4.03 mmol) of ketoamide 22 in 20 mL of THF at RT was added 3.34 g (8.26 mmol) of Lawesson's reagent. The resulting mixture was stined at 67 °C for 2.5 h, cooled to RT and then concentrated. Flash chromatography (elution with 3:1 hexanes-EtOAc) gave 1.28 g (81.0%) of a yellowish oil: Rf 0.60 (1:1 hexanes-EtOAc). Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 393 (M+H).
Preparation of Aldehyde 24:
Me
Os0 , pyr
BocHN NalQ BocHN
THF-H20
24
Figure imgf000057_0004
"•CHO
55 To a solution of 1.28 g (3.26 mmol) of alkene 23 in 40 mL of 1:1 THF-H2O at RT was added 0.025 mL (0.309 mmol) of pyridine followed by 2.05 mL (0.163 mmol) of Osθ4 (2.5 wt. % in 2-methyl-2-propanol). The resulting brown mixture was stined at RT for 10 min when 1.57 g (7.33 mmol) of NaIO4 was added in 3 portions over the next 30 min. The resulting creamy tan/white slurry was stined at RT for an additional 30 min, then diluted with Et2θ and H2O. The organic layer was washed once each with H2O and brine. The combined organic layers were dried with Na2SO4 and concentrated. Flash chromatography (elution with 2:1 hexanes-EtOAc) gave 1.01 g (78.3%) of a pale oil which turned brown upon standing: Rf 0.67 (1:1 hexanes-EtOAc). Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 395 (M+H).
Preparation of Acid 25:
Me ^ e
NaCI02
BocHN •N\--*- - 50 % H2O2 „ , ,„,
BocHN CH3CN, HaO
25 HO >;,H
To a solution of 1.01 g (2.56 mmol) of aldehyde 24 in 7.5 mL of CH3CN at 0 °C was added over 1 min, a solution of 0.463 g (5.12 mmol) of NaClθ2 in 4.5 mL H2O followed by 1.2 mL of 50% H2O2. The resulting mixture was stined at 0 °C for 10 min and at RT for 1.3 h, whereupon an additional 58 mg (0.64 mmol) of NaClθ2 was added. The resulting mixture was stined at RT for an additional 40 min, quenched with solid Na2SO3, stined an additional 5 min and then concentrated. The residue was diluted with EtOAc and H2O. The organic layer was washed once with brine, dried with Na2S O4 and concentrated. The crude acid was used in the next step without purification: Rf 0.33 (9: 1 CH2C -MeOH). Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 411 (M+H).
Preparation of Amide 26:
„Me Me
Bot^O, pyr J
BOCHN- W V , NH4HC°3
S— -l dioxane S
Figure imgf000058_0001
Figure imgf000058_0002
26
To a solution of crude acid 25 in 20 mL of dioxane and 2.0 mL of pyridine at RT was added 2.79 g (12.8 mmol) of Boc2θ followed by 1.02 g (12.8 mmol) of NH4HCO3. The resulting mixture was stined at RT for 13.5 h, then diluted with EtOAc and H2O. The organic layer was washed once with brine. The combined aqueous layer and washing were
56 extracted three times with EtOAc. The combined organic layers were dπed with Na2SO4 and concentrated. Flash chromatography (elution with EtOAc) gave 0.693 g (66.1% from aldehyde 24) of a yellowish oil: Rf 0.47 (EtOAc). Electrospray Mass Spectrum (50/50 acetomtπle/water) m/z 410 (M+H).
Preparation of Amine 27:
Me
TFA, CH2CI2,
BocHf* H2
Figure imgf000059_0002
then NaHC03 EtOAc
Figure imgf000059_0001
27
Figure imgf000059_0003
To a solution of 693 mg (1.69 mmol) of protected amine 26 in 15 mL of CH2CI2 at RT was added 3 mL of TFA. The resulting mixture was stmed at RT for 2 h and then concentrated. To the resulting viscous residue was added EtOAc and H2O followed by 10% aqueous NaHCO3 until pH paper indicated the aqueous phase was slightly basic. The organic layer was washed once with bnne. The combined aqueous layer and washing were extracted twice with EtOAc. The combined organic layers were dπed with Na2SO4 and concentrated giving 483 mg (92.2%) of a gold solid: Rf 0.14 (9:1 CH2Cl2-MeOH). Electrospray Mass Spectrum (50/50 acetonitπle/water) m/z 310 (M+H).
Compound L can then be prepared using amine 27 according to Coupling Conditions D Electrospray Mass Spectrum (50/50 acetomtπle/water + 0.1% ammonium hydroxide) m/z 595 (M+H)
13. Compound M
Figure imgf000059_0004
M
Compounds such as Compound M can be prepared using General Method E using the commercially available amino ketone followed by Coupling Conditions D. Electrospray Mass Spectrum (50/50 acetonitπle/water + 0.1% ammonium hydroxide) m/z 476 (M+H).
57 14. Compound N
Figure imgf000060_0001
N
Compounds such as Compound N can be prepared using General Method E followed by Coupling Conditions D. Compound N was isolated as a 60/40 mixture of diastereomers, presumably at the NHAc stereocenter. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 582 (M+H).
15. Compound O
Figure imgf000060_0002
O Compounds such as Compound O can be prepared using General Method E followed by Coupling Conditions D with the appropriate side chain manipulations as shown in the following scheme. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 500 (M+H).
1. H-Thr-OMe-HCI 1. Lawesson's Me Et3N, DME
Su Reagent
CbzHN
Figure imgf000060_0003
2. Dess-Martin CbzHN
2. DIBAL-H CbzHN peπodinane
Figure imgf000060_0004
Me
1. Dess-Martin JVte peπodinane, CH2CI2 1. DIBAL-H, THF
CbzHN H2N
2. Ph3P=CHC02Me
2. HC02NH4 CH2CI2 - _ C02Me e 10 % Pd-C
Figure imgf000060_0005
16. Compound P
Figure imgf000060_0006
Figure imgf000060_0007
58 Compounds such as Compound P can be prepared using General Method E using the commercially available amino ketone followed by Coupling Conditions D. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 546 (M-H).
17. Compounds Q
C02H
Figure imgf000061_0001
H" O
Figure imgf000061_0002
Compounds such as Compound Q can be prepared using General Method E followed by Coupling Conditions D. Reverse phase HPLC resulted in the separation of two diastereomers, presumably at the NHAc stereocenter. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 566 (M+H).
18. Compound R
Figure imgf000061_0003
R
Compounds such as Compound R can be prepared using General Method E followed by Coupling Conditions D with the appropriate side chain manipulations as shown in the following scheme. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 575 (M+H).
59 Me
BocHN BCI, BOC-ON sNN- " >Bn
Figure imgf000062_0001
H
Et,N -
Figure imgf000062_0004
Figure imgf000062_0008
^Ph 28 Ph 29
,Me
NaCIO
Mnθ2 50 % H2θ2
BocHN' BocH CH2CI2
S CH3CN, H 0
30
Figure imgf000062_0002
Ph
Figure imgf000062_0003
Ph
B0C2O, pyr Me NH4HCO3
BocHN dioxane
Figure imgf000062_0005
Ph
19. Compound S
Figure imgf000062_0006
Compounds such as Compound S can be prepared using General Method E followed by Coupling Conditions D with the appropriate side chain manipulations included according to the following scheme using the aldehyde (30) described for the preparation of compound R. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 603 (M+H).
4
BocHN* Ph3P=CHC02Bn HC02NH
" V-CHO
Figure imgf000062_0007
CH2CI2 c >"'''V-C02Bn 10%Pd-C
MeOH-EtOAc
30 ~Ph Ph reflux
Me Me
Boc2Ot pyr NH4HC03
BocH N BocHN'
CO2H dioxane >^ -.CONH2
Ph
60 20. Compound T
Figure imgf000063_0001
Compounds such as Compound T can be prepared using General Method E followed by Coupling Conditions D similar to the preparation of Compound O with the appropπate side chain manipulations as shown in the following scheme. Electrospray Mass Spectrum (50/50 acetonitπle/water + 0.1% ammonium hydroxide) m/z 485 (M+H).
Me
1 LιOH-H20
CbzHN' ^cθ2Me -
Figure imgf000063_0002
CONH2
Sχ-ζ 2 Boc20, pyr
Me NH4HC03 Me dioxane
21. Compound U
Figure imgf000063_0003
U Compounds such as Compound U can be prepared using General Method E followed by Coupling Conditions D using the ammo alcohol 28 formed in the preparation of Compound R and shown below. Electrospray Mass Spectrum (50/50 acetonitπle/water + 0.1% ammonium hydroxide) m/z 562 (M+H).
Me
H2N
S^-^OH
28 'Ph
22. Compound V
Figure imgf000063_0004
V
61 Compounds such as Compound N can be prepared using General Method E followed by Coupling Conditions D. Electrospray Mass Spectrum (50/50 acetonitπle/water + 0.1% ammonium hydroxide) m/z 614 (M+H).
23. Compound W
Figure imgf000064_0001
W
Compounds such as Compound W can be prepared using General Method E followed by Coupling Conditions D using the appropπate am o alcohol as descπbed m the preparation of Compounds U and R. Electrospray Mass Spectrum (50/50 acetonitπle/water + 0.1% ammonium hydroxide) m/z 542 (M+H).
24. Compound X
Figure imgf000064_0002
X
Compounds such as Compound X can be prepared using General Method E followed by Coupling Conditions D with the appropπate side chain manipulations as shown in the following scheme using the intermediate alcohol 29 found in the preparation of Compound R. Electrospray Mass Spectrum (50/50 acetonitπle/water + 0.1% ammonium hydroxide) m/z 589 (M+H)
Me Me
50 % H2θ2
1 MsCI, Et3N
BocH cat K2C03
Figure imgf000064_0003
BocHN BocHN OH 2 NaCN sl ^CN DMSO
29 DMSO
Figure imgf000064_0004
Ph Λ^Ph Ph
62 25. Compound Y
Figure imgf000065_0001
Compounds such as Compound Y can be prepared using amino alcohol 28, prepared using General Method E as found in the preparation of Compound U, followed by Coupling Conditions D with the appropriate side chain manipulations as shown in the following scheme. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 576 (M+H).
1. Mn02 ,CH2CI2
2. NaCI02 , 50 % H202 H 0 3. 95 % TFA
Figure imgf000065_0002
Figure imgf000065_0003
26. Compound Z
Figure imgf000065_0004
Compounds such as Compound Z can be prepared using General Method E followed by Coupling Conditions D along with the appropriate side chain manipulations as shown in the scheme shown for the preparation of Compound S. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 583 (M+H).
27. Compound AA
Figure imgf000065_0005
"CONH2
AA
63 Compounds such as Compound AA can be prepared using General Method E followed by Coupling Conditions D with the appropπate side chain manipulations analogous to those shown for the preparation of Compound L. Electrospray Mass Spectrum (50/50 acetomtπle/water + 0.1% ammonium hydroxide) m/z 637 (M-H).
28. Compound AB
Figure imgf000066_0001
AB
Compounds such as Compound AB can be prepared using General Method E followed by Coupling Conditions D. Electrospray Mass Spectrum (50/50 acetonitπle/water + 0 1% ammonium hydroxide) m/z 512 (M+H).
29. Compound AC
Q Ma Me
NHAc
H2O3PC 5
AC
Compounds such as Compound AC can be prepared using General Method E followed by Coupling Conditions D using the appropriate ammo alcohol as in the preparation of Compounds U and W. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 542 (M+H).
29B. Compound AD
M Mee
N-> CONH2
»,* rx'" *K Me
AD
Compounds such as Compound AD can be prepared using General Method E followed by Coupling Conditions D similar to the preparation of Compound O with the appropπate side chain manipulations as shown in the following scheme Electrospray Mass Spectrum (50/50 acetonitπle/water + 0.1% ammonium hydroxide) m/z 466 (M-H)
64 ,Me 1. "CH3AICINH2"
H NH4OAc PhCH3, 50 °C
CbzHN' OMe CbzHN
HOAc >— C02Me 2. HC02NH4
Figure imgf000067_0001
ONH2
° o^ reflux HN---/ 10 % Pd-C
90 Me MeOH-EtOAc Me
29C. Compound AE
AE
Figure imgf000067_0002
Compounds such as Compound AE can be prepared using General Method E followed by Coupling Conditions D as in the preparation of Compounds U and W, using the appropriate amino alcohol which can be prepared as shown in the scheme below. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 582 (M-H).
Bu-jSnCHjOBn
BocHN' 1. 3 N HCI π-BuLi, DME 2. Boc-Ala-OSu -78 °C
Figure imgf000067_0003
Figure imgf000067_0004
1. Lawesson's , , . , /
BocH Reagent
2. BCI3, CH2CI2
444
Figure imgf000067_0005
Figure imgf000067_0006
29D. Compound AF
AF
Figure imgf000067_0007
Compounds such as Compound AF can be prepared using General Method E followed by Coupling Conditions D with side chain manipulations as shown for the preparation of
65 Compounds R and S, using the appropriate amino alcohol (444), which can be prepared as previously described in the preparation of Compound AE. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m z 625 (M+H).
General Method F: Preparation of oxadiazoles - hydrogenation procedure:
Many oxadiazoles can be prepared via cyclization of the appropriate O-acylamidoxime according to the method described in Borg et al. (J. Org. Chem. 1995, 60, 3112). The required amidoxime can be prepared via the conesponding nitrile as shown below. The resulting amine can then be further elaborated according to Coupling Conditions D.
The general scheme shown below can be used for the preparation of compounds such as Compounds BA-BH and is exemplified by the preparation of Compound BC.
NH2OH*ΗCI MU-
N≡- -R9 aq. NaOH H2N"^R9 EtOH 43
BocW Is: o-N-rR
43 pyridine BocHN^^Λ*- /-***■ R9
BocHN^J**-. NH2 \
1 DME
CC*2Bn s heat
CO∑Bn
C02Bn
O-N TFA H2N--^AN -R9
\ 44
Figure imgf000068_0002
CO∑Bn
30. Compound BC
Figure imgf000068_0001
- Cι02H (BC)
Preparation of Amidoxime 31:
H C ,
NH2OH-HCI
N=- H2N' aq. NaOH
31 EtOH
66 In a typical procedure, benzyl cyanide (0.03755 mol) was dissolved in 80 mL of EtOH. A solution of hydroxylamine hydrochloride (0.04506 mol) and NaOH (0.04506 mol) in 20 mL of water was added and the resulting reaction mixture was heated at reflux for two days. The reaction mixture was then cooled to room temperature and concentrated under reduced pressure to remove the volatile solvent. The resulting residue was diluted with 50 mL of H2O and extracted with EtOAc. The combined organic layers were washed with brine, dried (Na2SO4), and concentrated under reduced pressure to give 5.30 g of product 31.
Preparation of Amine 34:
31
H f^ ϊι
OSu T
H2N^^ BocHN^^s**^ BocHN^ o NHz pyridine s DME heat
^| 32
C02Bn C 02Bn
N A O-N
O-N BocHN. J^ y~ TFA y^ A
Figure imgf000069_0001
s 34
33 COzBn
C02Bn
In a typical procedure, Boc-Glu(OBn)-OSu (500 mg, 1.15 mmol) was dissolved in 7 mL of ethylene glycol dimethyl ether along with 1.15 mmol of amidoxime 31. The resulting reaction mixture was stined at rt for 15 h. It was then concentrated under reduced pressure.
The resulting residue was taken up in EtOAc and washed with dilute NaHCO3, brine, dried
(Na2SO4) and concentrated under reduced pressure to give the intermediate product 32.
This material was dissolved in 4 mL of pyridine and heated at reflux for 30 min. The reaction mixture was cooled to rt and diluted with EtOAc. The organic layer was washed with 10% citric acid to remove all the pyridine. It was then washed with brine, dried
(Na SO4), and concentrated under reduced pressure to give protected oxadiazole 33. The resulting residue was dissolved in 6 mL of 25% TFA in CH2CI2 and allowed to stand at rt for 3 h. It was then concentrated under reduced pressure. The light yellow, gummy solid was taken up in EtOAc and carefully neutralized with dilute NaHCO3. The organic layer was then washed with brine, dried (Na2SO4), and concentrated under reduced pressure to afford 350 mg of product 34.
67 Preparation of BC:
Bn203P
Acid 13
34
EDOHCI HOBT, DIEA
Figure imgf000070_0001
C 02Bn
H2O3P
H2 Pd(OH)2 ! or
1. LiOH
2. TFA
Figure imgf000070_0002
C 02H
In a typical procedure (analogous to Coupling Conditions D), 252 mg of acid 13 (0.523 mmol) was dissolved in 5 mL of CH2CI2 and 1 mL of DMF along with 220 mg of amine 34 (0.627 mmol). HOBT (96 mg, 0.627 mmol) and EDC'HCl (120 mg, 0.627 mmol) were added at rt. After 0.136 mL of DIEA (0.784 mmol) was added, the resulting reaction mixture was stined at rt for 4 h. It was then concentrated under reduced pressure. The resulting residue was taken up in EtOAc and washed successively with dilute NaHCO3, brine, 10% citric acid and brine. The organic layer was dried (Na2SO4) and concentrated under reduced pressure to give 410 mg of product 35. A portion of product 35 (100 mg, 0.122 mmol) was dissolved in 3 mL of MeOH and 3 mL of EtOAc. A catalytic amount of 20% Pd(OH)2 over activated carbon (30 mg) was added and the resulting reaction mixture was hydrogenated at rt under atmospheric pressure using a hydrogen balloon for 40 min. The reaction mixture was filtered through Celite. The filtrate was concentrated under reduced pressure. The resulting crude product was purified by preparative HPLC using aqueous CH3CN containing 0.1% TFA to afford 12 mg of oxadiazole product B C (retention time = 18 min; solvent gradient = 0 min, 95:5 H2O/CH3CN; 5 min, 95:5 H2O/CH3CN; 25 min, 50:50 H2O/CH3CN, 50 min, 0:100 H2O/CH3CN). Electrospray mass spectrum (50:50 H2O/CH3CN + 0.1% aq NH4OH) m z 545 (M-H). lH NMR (DMSO) d 1.8 (s, 3 H), 2-2.5 (m, 6 H), 4.15 (s, 2 H), 4.4-4.5 (m, 1 H), 5.1-5.2 (m, 1 H), 7-7.6 (m, 9 H), 8.0-8.1 (m, 1 H), 8.7-8.8 (m, 1 H).
68 31. Compounds BA-BH
Compounds such as Compounds BA-BH can be prepared according to General Method F as exemplified above for the preparation of Compound BC.
Figure imgf000071_0001
CO2H
Compound R9 MS
BA -CH2Chx 552 (M-H)
BB -(CH2)2CH(CH3)2 525 (M-H)
BC -CH2Ph 545 (M-H)
BD -CH2CH(CH3)2 511 (M-H)
BE -CH2-cyclopropyl 509 (M-H)
BF -(CH2)6CH3 554 (M-H)
BG -(CH2)4CH3 525 (M-H)
BH -(CH2)5CH3 562 (M+MeCN)
Figure imgf000071_0003
31A. Compounds BN-BW
Similarly, compounds such as Compounds BN-BW can be prepared according to General Method F as exemplified above for the preparation of Compound BC.
• 2 N-- R9
Figure imgf000071_0002
"C02H
Compound R9 MS
BN -CH2(C6H4)-4-Cl 565 (M-H)
BO -CH2-(2-naphthyl) 581 (M-H)
BP -CH2(C6H4)-4-CF3 599 (M-H)
BQ -CH2(C6H4)-4-OCH3 561 (M-H)
BR -CH2(C6H4)-4-F
Figure imgf000071_0004
549 (M-H)
69 BS -CH2(C6H4)-3,4-Cl2 600 (M-H)
BT -CH2(C6H4)-4-CH3 545 (M-H)
BU -CH2(C6H4)-4-Br 610 (M-H)
BN -CH2(C6H4)-3-Cl 566 (M-H)
BW -CH2(C6H4)-3-CF3 599 (M-H)
Figure imgf000072_0003
General Method G: Preparation of oxadiazoles - TFA procedure:
Compounds such as Compounds CA-FBG and GA-GC can be prepared according to General Method F followed by Coupling Conditions D as exemplified below by the preparation of Compound CD.
32. Compound CD
Figure imgf000072_0001
Preparation of amine 39:
BocH s COΝH2
BocHI
Figure imgf000072_0002
Amidoxime 36 was prepared using the same procedure that was outlined for compound BC substituting isovaleronitrile. Amidoxime 36 (150 mg, 1.15 mmol) was then dissolved in 2 mL of DMF and 6 mL of CH2C1 along with Boc-Gln-OH (237 mg, 0.958 mmol). HOBT (176 mg, 1.15 mmol) and EDC-HCI (220 mg, 1.15 mmol) were added, followed by DIEA (0.25 mL, 1.44 mmol). The resulting reaction mixture was stined at rt for 8 h. It
70 was then concentrated under reduced pressure and then redissolved in 50 mL of EtOAc The organic layer was washed with dilute aqueous NaHCO3, bπne, dπed (Na2SO4), and concentrated under reduced pressure to obtain 245 mg of product 37. Compound 37 was then subjected to the same reaction sequence outlined in the preparation of compound BC in order to obtain 95 mg of compound 39
Preparation of compound CD:
Acid 13
39
EDC-HCI HOBT, DIEA
H203Pi
TFA
Figure imgf000073_0001
CONH2
In a typical procedure, 150 mg of acid 13 (0.313 mmol) was dissolved in 5 mL of CH2CI2 and 1 mL of DMF along with 90 mg of amine 39 (0.375 mmol). HOBT (57 mg, 0.375 mmol) and EDC-HCI (72 mg, 0.375 mmol) were added at rt. After 0 082 mL of DIEA (0.470 mmol) was added, the resulting reaction mixture was stined at rt for 4 h. It was then concentrated under reduced pressure. The resulting residue was taken up in EtOAc and washed successively with dilute NaHCO3, bπne, 10% citπc acid and bπne. The organic layer was then dπed (Na2SO4) and concentrated under reduced pressure to give 190 mg of product 40. This mateπal was dissolved m 4 mL of a solution containing 95% TFA, 2.5% H2O, 2.5% anisole. The resulting reaction mixture was allowed to stand at rt for 2h. It was then concentrated and the crude product CD was precipitated out as a solid using Et2θ. A portion of this crude product was puπfied by preparative HPLC using aqueous CH3CN containing 0.1% TFA to afford 9 mg of oxadiazole CD (retention time = 20 mm; solvent gradient = 0 mm, 95:5 H2O/CH3CN; 5 mm, 95:5 H2O/CH3CN; 25 min, 60:40 H2O/CH3CN, 50 min, 0:100 H2O/CH3CN). Electrospray mass spectrum (50:50 H2O/CH3CN + 0.1% aq NH4OH) m/z 525 (M-H). *H NMR (DMSO) d 0.8 (s, 3H), 0.85 (s, 3 H), 1 3-2.5 (m, 11 H), 1.7 (s, 3 H), 4.3-4.4 (m, 1 H), 5.0-5.1 (m, 1 H), 6.8 (br s, 1 H), 7 0 (d, 2 H), 7.15 (d, 2 H), 7.25 (br s, 1 H), 8.0-8.1 (m, 1 H). 8.8-8.9 (m, 1 H)
71 33. Compounds CA-FBG
Compounds such as Compounds CA-FBG can be prepared according to General Method G as exemplified above for the preparation of Compound CD.
Figure imgf000074_0001
Compound Rl R9 MS
CA -(CH2)2CO2CH2Ph -CH2CH(CH3)2 601 (M-H)
CB -(CH2)2CONH2 -CH2Ph 545 (M-H)
CC -(CH2)2CONH2 -CH2Chx 550 (M-H)
CD -(CH2)2CONH2 -(CH2)2CH(CH3)2 525 (M-H)
CE -CH2(C6H4)-4-OH -CH2Ph 579 (M-H)
CF -CH2(C6H4)-4-OH -CH2Chx 586 (M-H)
CG -CH2Ph -CH2Ph 563 (M-H)
CH -CH2Ph -CH2Chx 569 (M-H)
Cl -CH2(C6H4)-4-OH -(CH2)2CH(CH3)2 560 (M-H)
CJ -CH2Ph -(CH2)2CH(CH3)2 543 (M-H)
CK -(CH2)2CONH2 Op 586 (M-H)
CL H -CH2Chx 479 (M-H)
CM -(CH2)2CONH2 -(CH2)3CH3 511 (M-H)
CN -(CH2)2CONH2 -Ph 530 (M-H)
CO -(CH2)2CONH2 i^O 548 (M-H)
CP -(CH2)2CONH2 -(CH2)5CH3 538 (M-H)
CQ -Ph -CH2Ph 549 (M-H)
CR -(CH2)3CH3 -CH2Ph 529 (M-H) +
CS -CH2Ph 639 (M-H)
CT -(CH2)2SO2CH3 -(CH2)2CH(CH3)2 561 (M-H)
CU -(CH2)2CONH2 -CH2(C6H4)-4-CF3 607 (M-H)
|cv -CH2CH3 -(CH2)2CH(CH3)2
Figure imgf000074_0002
481 (M-H)
72 CW -(CH2)2COΝH2 586 (M-H) J^
CX -(CH2)2CONH2 601 (M-H)
CY -(CH2)2CONH2 -CH2-(1 -naphthyl) 595 (M-H)
CZ -(CH2)2CONH2 O" 613 (M-H)
DA -(CH2)3CH3 -(CH2)2CH(CH3)2 509 (M-H)
DB -CH2-(3-indole) -(CH2)2CH(CH3)2 582 (M-H)
DC -Ph -CH2Chx 555 (M-H)
DD -CH2CH(CH3)2 -CH2Chx 536 (M-H)
DE -(CH2)3CH3 -CH2Chx 535 (M-H)
DF -CH2-(3-indole) -CH2Chx 609 (M-H)
DG -CH2CH3 -CH2Ph 502 (M-H)
DH -(CH2)2SCH3 -(CH2)2CH(CH3)2 527 (M-H) +
DI -(CH2)2CH(CH3)2 618 (M-H)
DJ -CH2CH(CH3)2 -CH2Ph 529 (M-H)
DK -CH2-(3-indole) -CH2Ph 603 (M-H)
DL -CH2CH(CH3)2 -(CH2)2CH(CH3)2 509 (M-H)
DM -(CH2)2CONH2 -CH2(C6H4)-4-OCH3 574 (M-H)
DN -(CH2)2CONH2 -(CH2)2Ph 558 (M-H)
DO -(CH2)2SCH3 -CH2Chx 553 (M-H) i +
DP -CH2Chx 645 (M-H)
DQ -Ph -(CH2)2CH(CH3)2 529 (M-H)
DR -(CH2)2CONH2 U 534 (M-H)
DS -(CH2)2CONH2 •(CH2) CCH 522 (M+H)
DT -(CH2)2CONH2 -(CH2)3CH=CH2 524 (M+H)
DU -CH2CH(CH3)2 k 544 (M-H)
DN -CH2CH(CH3)2 k r 544 (M-H)
DW -CH2-(3-indole) k 543 (M-H)
Figure imgf000075_0001
73 PX •CH2CH(CH3)2 544 (M-H)
Figure imgf000076_0001
PY ^CH2CH(CH3)2 572 (M-H)
Figure imgf000076_0002
PZ (CH2)2CONH2 CH2)6CH3 1552 (M-H)
EA •CH2-(3-indole) K17 (M-H)
EB H2-(3-indole) ■CH2-(l-naphthyl) K53 (M-H)
EC CH2-(3-indole) c 0' fe71 (M-H)
ED hCH2-(3-indole) 45 (M-H)
Figure imgf000076_0003
EE ^CH2-(3-indole) (CH2)4CH3 1583 (M-H)
EF CH2-(3-indole) (CH2)5CH3 597 (M-H)
EG (CH2)2CONH2 547 (M+H)
Figure imgf000076_0004
EH (CH2)2CONH2 575 (M+H)
Figure imgf000076_0005
Pi CH2-(3-indole) ^09 (M-H)
Figure imgf000076_0006
EJ CH2-(3-indole) CH2-(2-naphthyl) 653 (M-H)
EK CH2-(3-indole) k517 (M-H)
Figure imgf000076_0007
ET . CH2-(3-indole) (CH2)6CH3 |611 (M-H)
EM CH2-(3-indole) 613 (M-H)
EN CH2-(3-indole) fell (M-H)
EO CH2-(3-indole) KCH2)3CH(CH3)2 [599 (M-H)
EP CH2-(3-indole) 597 (M-H)
§Q CH2CH3 (CH2)4CH3 W82 (M-H)
ER kCH2CH3 kCH2)5CH3 W96 (M-H)
ES CH2CH3 kCH2)6CH3 509 (M-H)
ET -CH. CH2Chx |493 (M-H)
EU CH (CH2)5CH3 |481 (M-H)
EV -CH2(C6H4)-4-OH (CH2)5CH3 574 (M-H)
JEW CH2(C6H4)-4-OPO3H2 (CH2)5CH3 653 (M-H)
74 EX -CH3 -(CH2)6CH3 495 (M-H)
EY -CH2(C6H4)-4-OH -(CH2)6CH3 587 (M-H)
EZ -CH3 -CH2Ph 488 (M-H)
FA -CH2(C6H4)-4-OPO3H2 -(CH2)6CH3 667 (M-H)
FB -CH3 -(CH2)4CH3 467 (M-H)
FC -CH3 K^γ^ 482 (M-H)
FD -CH3 530 (M-H)
FE -CH3 -CH2-(2-naphthyl) 538 (M-H)
FF -CH2CONH2 -CH2-(2-naphthyl) 580 (M-H)
FG -CH2CONH2 -CH2(C6H4)-4-CF3 599 (M-H)
FH -CH3 -CH2(C6H4)-4-CF3 556 (M-H)
FI -CH2CONH2 -CH2(C6H4)-4-OCH3 560 (M-H)
FJ -CH2CONH2 588 (M-H)
I\^*N
FK H -CH2(C6H4)-4-OCH3 503 (M-H)
FL H -CH2(C6H4)-4-CF3 541 (M-H)
FM H -CH2(C6H4)-4-Cl 508 (M-H)
FN -CH2CONH2 -CH2(C6H4)-4-Cl 565 (M-H)
FO -CH2CONH2 co 574 (M-H)
FP H 531 (M-H)
X^
FQ H -CH2-(2-naphthyl) 524 (M-H)
FR -(CH2)2CONH2 uos 588 (M-H)
FS -CH2CN -CH2(C6H4)-4-CF3 580 (M-H)
FT -CH3 -CH2(C6H4)-4-CH3 501 (M-H)
FU -CH3 -CH2(C6H4)-4-Cl 522 (M-H)
FV -(CH2)2CONH2 -CH2(C6H4)-4-Cl 579 (M-H)
FW -CH3 -CH2(C6H4)-3,4-Cl2 555 (M-H)
FX -CH3 -(CH2)5CH3 467 (M+H)
FY -CH2OH -CH2(C6H4)-4-CF3 571 (M-H)
FZ |-CH2CONH2 -CH2(C6H4)-4-CH3 546 (M+H)
Figure imgf000077_0001
75 FAA -(CH2)2CONH2 -CH2(C6H4)-4-CH3 558 (M-H)
FAB -CH3 -CH2(C6H4)-3,4-(OMe)2 547 (M-H)
FAC -CH3 -CH2(C6H4)-3-Cl 521 (M-H)
FAD -CH3 -CH2(C6H4)-3-CF3 555 (M-H)
FAE -CH2CONH2 -CH2(C6H4)-3,4-Cl2 598 (M-H)
FAF -CH2CONH2 -CH2(C6H4)-3-Cl 566 (M+H)
FAG -(CH2)2CONH2 -CH2(C6H4)-3,4-Cl2 614 (M+H)
FAH -(CH2)2CONH2 -CH2(C6H4)-3-Cl 578 (M-H)
FAI -CH2OH -CH2(C6H4)-3,4-Cl2 571 (M-H)
FAJ -CH2CONH2 -CH2(C6H4)-3,4-(OMe)2 592 (M+H)
FAK -geminal-Me2 -CH2(C6H4)-4-CF3 571 (M+H)
FAL -CH3 -CH2(C6H4)-4-F 505 (M-H)
FAM -CH2CONH2 -CH2(C6H4)-3-CF3 600 (M+H)
FAN -(CH2)2CONH2 -CH2(C6H4)-3-CF3 614 (M+H)
FAO -CH3 -CH2(C6H4)-4-I 613 (M-H)
FAP -CH3 * f 540 (M-H)
FAQ -CH3 -CH2(C6H4)-3,4-F2 523 (M-H)
FAR -CH3 -CH2(C6H4)-3,5-Cl2 556 (M-H)
FAS -(CH2)2CONH2 -CH2(C6H4)-4-Br 622 (M-H)
FAT -CH2OH -CH2(C6H4)-4-F 523 (M+H)
FAU -CH2CH3 -CH2(C6H4)-3,4-Cl2 571 (M+H)
FAV -CH2OH -CH2(C6H4)-4-Cl 539 (M+H)
FAW -CH2OH -CH2-(2-naphthyl) 555 (M+H)
FAX -CH(OH)CH3 -CH2(C6H4)-4-Br 597 (M+H)
FAY -CH3 -CH2(C6H4)-4-Br 566 (M-H)
FAZ -(CH2)2Ph -CH2(C6H4)-4-CF3 645 (M-H)
FBA -(CH2)2Ph -CH2(C6H4)-4-(O-n-Pr) 635 (M-H)
FBB -(CH2)2Ph -CH2(C6H4)-3,4-Cl2 645 (M-H)
FBC -CH2OH -CH2(C6H4)-3-Cl, 4-1 662 (M-H)
FBD -CH2OH -CH2(C6H4)-4-Br 565 (M-H)
FBE -CH3 -CH2(C6H4)-3-Cl, 4-1
Figure imgf000078_0001
647 (M-H)
76 FBF -(CH2)2CONH2 -CH2-(2-naphthyl) 594 (M-H)
FBG -(CH2)2Ph -CH2-(2-naphthyl) 627 (M-H)
Figure imgf000079_0003
34. Compounds GA-GC
Compounds such as Compounds GA-GC can be prepared using General Methods F and G using the appropriate nitrile and with the substitution of beta alanine for a typical alpha amino acid.
Figure imgf000079_0001
T !L-ζ N
Compound R9 MS
GA -CH2(C6H4)-4-CF3 555 (M-H)
GB -CH2-(2-naphthyl) 537 (M-H)
GC 544 (M-H)
Figure imgf000079_0004
General Method H: Preparation of benzamides:
Figure imgf000079_0002
o
ZZ2 R1 R2
Compounds such as Compound ZZ2 can be prepared according to the scheme below using the appropriate acid coupling partner along with the appropriate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example.
77 HO.,
^ ^Mβ
H2 tT R
Figure imgf000080_0001
1. EDC-HCI 1. EDC-HCI O HOBT
Figure imgf000080_0002
HOBT
O R1 2. LiOH 2. pyr, heat
- 1. phosphorylate
Figure imgf000080_0003
*N R9 2. deprotect
Figure imgf000080_0004
O R1
35. Compounds HA-HG
^L
Figure imgf000080_0005
Rη
Compound Rl R9 MS
HA -(CH2)2CO2H -(CH2)2CH(CH3)2 440 (M-H)
HB -CH2CH3 -(CH2)2CH(CH3)2 396 (M-H)
HC -CH2CH3 -(CH2)6CH3 424 (M-H)
HD -(CH ) CO2H -(CH2)6CH3 468 (M-H)
HE -CH2CH3 -(CH2)2Ph 430 (M-H)
HF -(CH2)2CO2H -(CH2)2Ph 474 (M-H)
Figure imgf000080_0007
|HG -(CH2)2CO2Me -(CH2)2Ph 488 (M-H)
General Method I: Preparation of branched benzamides:
Compounds such as Compounds IA-IC can be prepared according to General Methods F, G and H with the appropriate side chain modifications as shown below.
1. Ru-CHO, Eψ O -*^^r--T N*-*$^ NH2 mol. sieves _
•HCl 2. NaBH(OAc)3
3. Ac20 or
Boc-Gly-OH etc.
36 Compounds IA-IC
O-N
Figure imgf000080_0006
°γRP
78 Compound Rl RP RQ MS
IA -CH2CH3 -CH3 -(CH2)5CH3 495 (M-H)
IB -(CH2)2CO2H -CH3 -(CH2)5CH3 539 (M-H)
Figure imgf000081_0005
k -CH2CH3 -CH2NH -(CH2)5CH3 510 (M-H)
General Method J: Preparation of benzamides with aldehydes:
B
OH
Figure imgf000081_0001
m o ZZ3 R1 R2
Compounds such as Compound ZZ3 (JA-JC below) can be prepared according to General Methods H and I with the appropriate substitution of the starting material as shown below.
Figure imgf000081_0002
37. Compounds JA-JC
Figure imgf000081_0003
01
Compound Rl R9 MS
JA -CH2CH3 -(CH2)2Ph 458 (M-H)
JB -(CH2)2CO2H -(CH2)6CH3 496 (M-H)
JC -CH2CH3 523 (M-H)
Figure imgf000081_0006
f^^ (CHj-)6CH3
General Method JA: Preparation of difluorophosphonate derivatives:
H2θ3
Figure imgf000081_0004
79 Compounds such as Compound ZZ4 (KA-KG below) can be prepared using Coupling Conditions C and D using the appropriate acid coupling partner along with the appropriate amine coupling partner, which can be prepared according to General Methods A. B. E, F and G. for example. The difluorophosphonate acid coupling partner can be prepared according to literature procedures (see for example Burke et al. J. Org. Chem. 1993, 58, 1336 and Smythe and Burke Tetrahedron Leu. 1994, 35, 551) and as described in WO 97/12903.
38. Compounds KA-KG
H2θ3
Figure imgf000082_0001
Compound Rl R9 MS |
KA -CH2CH3 -(CH2)5CH3 529 (M-H)
KB -CH2-(3-indole) -(CH2)5CH3 631 (M-H)
KC -CH3 -CH2(C6H4)-4-CF3 589 (M-H)
KD -(CH2)2CONH2 -CH2(C6H4)-4-CF3 646 (M-H)
KE -CH OH -CH2(C6H4)-4-CF3 605 (M-H)
KF -CHΪOH -CH (C6H4)-3.4-Cl2 605 (M-H)
Figure imgf000082_0003
KG -CH3 -CH2(C6H4)-3,4-Cl2 589 (M-H)
General Method JB: Preparation of difluorophosphonate derivatives:
H2O3
Figure imgf000082_0002
Compounds such as Compound ZZ5 (KM-KS below) with an NH substituent other than acetyl can be prepared according to Conditions C and D as shown in the scheme below using the appropriate acid coupling partner along with the appropriate amine coupling
80 partner, which can be prepared according to General Methods A, B, E, F and G, for example, substituting the appropriate reagent (for example: acid, acid chloride, acid anhydride, sulfonyl chloride) and conditions. The difluorophosphonate acid coupling partner can be prepared according to literature procedures (see for example Burke et al. J. Org. Chem. 1993, 55, 1336 and Smythe and Burke Tetrahedron Lett. 1994, 35, 551) and as described in WO 97/12903. The free amine shown below is analogous to that which is acetylated in Coupling Conditions C.
acylation etc. reagent
H203 and DIEA or EDC-HCI HOBT, DIEA
2. standard
Figure imgf000083_0001
deprotection methods
Figure imgf000083_0002
38A. Compounds KM-KS
Figure imgf000083_0003
Cmpd R9 Rl R9 MS
KM -C(O)CH2Ph -CH2OH -CH2(C6H4)-4-CF3 681 (M-H)
KN -CO2CH2Ph -CH3 -CH2(C6H4)-4-CF3 665 (M-H)
KO -CO2CH2Ph -CH2OH -CH2(C6H4)-3,4-Cl2 681 (M-H)
KP -CO2CH2Ph -CH3 -CH2(C6H4)-3,4-Cl? 665 (M-H)
KO -CO2CH2Ph -CH2OH -CH2-(2-naphthyl) 665 (M+H)
KR -CO2CH2Ph -CH2OH -CH2(C6H4)-4-Cl 647 (M-H)
KS -C(O)CH2-(2-thienyl) -CH2OH -CH2-(2-naphthyl) 671 (M+H)
Figure imgf000083_0005
General Method K: Preparation of aldehyde deriviatives related to pTyr:
Figure imgf000083_0004
81 Compounds such as Compounds ZZ6 and ZZ12 can be prepared according to the general schemes shown below (for R" = Ac), using the appropriate acid coupling partner along with the appropriate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example, followed by standard deprotection methods.
1. CI2CHOMe 1. (BnO)2P(0)H TiCU CCI4, DIEA. DMAP
2. LiOH 2' BCl3 3. HSCH2CH2SH
BFg-OEt,
Figure imgf000084_0001
Figure imgf000084_0002
1. amine (1 , 44, or 39 etc.) standard H20***P coupling conditions
<
2. standard deprotection methods
Figure imgf000084_0003
Figure imgf000084_0004
39. Compound LA
H O- N
Figure imgf000084_0005
" e
LA
Compounds such as Compound LA can be prepared using General Methods F. G and K using the appropriate amine coupling partner. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 523 (M-H).
40. Compound LB
Figure imgf000084_0006
Compounds such as Compound LB can be prepared using General Methods F. G and K using the appropriate amine coupling partner and the appropriate manipulations as shown in the scheme below. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 636 (M-H).
82 amine (1 , 39 or 44 etc.) standard coupling conditions
Figure imgf000085_0002
1. CIS02NCO
2. heat
3. water
4. standard deprotection
Figure imgf000085_0001
methods
Figure imgf000085_0003
General Method L: Preparation of aryl phosphonates
ZZ7
Figure imgf000085_0004
Compounds such as Compound ZZ7 can be prepared according to Coupling Conditions C and D substituting the appropriate carboxylic acid coupling partner along with the appropriate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. The carboxylic acid coupling partner can be prepared according to literature methods with the appropriate modifications. See for example: WO 97/12903; Stankovic et al. Bioorg. Med. Chem. Lett. 1997, 7, 1909; Petrakis et al. J. Am. Chem. Soc. 1987, 109, 2831.
41. Compound MA
CF3
Figure imgf000085_0005
Compounds such as Compound MA can be prepared according to General Method L. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 539 (M-H).
83 41A. Compound MB
Figure imgf000086_0001
CONH2
Compounds such as Compound MB can be prepared according to General Method L. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 596 (M-H).
General Method LA: Preparation of aryl phosphonates:
Figure imgf000086_0002
ZZ8 R R1 R*
Compounds such as Compound ZZ8 with an NH substituent (R99) other than acetyl can be prepared according to General Methods JB and L according to the general scheme shown below, substituting the appropriate reagent (for example: acid, acid chloride, acid anhydride, sulfonyl chloride) and conditions. The free amine shown below is analogous to that which is acetylated in Coupling Conditions C.
1. acylation etc. reagent and DIEA or EDC-HCI
HOBT, DIEA
2. standard
Figure imgf000086_0003
deprotection
O R1 methods
Figure imgf000086_0004
41B. Compound MC
UO O
Figure imgf000086_0005
MC
Compounds such as Compound MC can be prepared according to General Method LA. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m z 547 (M-H).
84 General Method M: Preparation of ureido analogs
H203P
Figure imgf000087_0001
ZZ9
Compounds such as Compound ZZ9 (NA-NC below) with a urea linkage similar to that shown above can be prepared according to the scheme below using the appropπate amine coupling partner, which can be prepared according to General Methods A. B, E. F and G. for example and according to literature methods. See for example: WO 97/12903: Plummer et al. J. Med. Chem. 1997, 40, 3719. tπphosgene DIEA then
Figure imgf000087_0002
Figure imgf000087_0003
NH,
R □ 11 R2 H Ύ o
R1 R2
1. (BnO)2P(0)H CCU, DMAP, DIEA
2. standard
Figure imgf000087_0004
deprotection „N- Hi V; conditions
42. Compounds NA-NC
H-03P
Figure imgf000087_0005
H A A^ N F
O R1
Compound Rl R9 MS |
NA -CH2-(3-indole) -(CH2)5CH3 540 (M-H)
NB -CHT -CH2(C6H4)-4-CF3 499 (M-H)
NC -CH2CH3 -(CH2)5CH3
Figure imgf000087_0006
439 (M-H)
85 General Method MA: Preparation of naphthyl ureido analogs
Figure imgf000088_0001
ZZ10
Compounds such as Compound ZZIO (NM-NQ below) with a urea linkage similar to that shown above can be prepared according to General Method M according to the scheme below and according to literature methods (see for example: WO 97/12903; Plummer et al. J. Med. Chem. 1997, 40, 3719), using the appropriate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example, and according to literature methods. tπphosgene DIEA then
Figure imgf000088_0003
Figure imgf000088_0002
NH2 H*tt . B R1 R2 H-~Λ R* R'*
1. (BnO)2P(0)H CCU. DMAP, DIEA
2. standard
Figure imgf000088_0004
deprotection conditions "Y Rft1 R'2
42. Compounds NM-NQ
Figure imgf000088_0005
H' O ^ R1 R9
Compound Rl R9 MS
NM -CΆ-S -(CH2)5CH3 501 (M-H)
NO -CH3 -CH2(C6H4)-3,4-Cl2 535 (M-H)
NP -CHoOH -CH2(C6H4)-3,4-Cl2 551 (M-H)
NO -CH-7OH -CH2(C6H4)-4-CF3 551 (M-H)
Figure imgf000088_0006
86 General Method N: Preparation of N-acyl or N-sulfonyl related analogs:
H203PC
„99 O / \
ZZ11 R ' R<*
Compounds such as Compound ZZl l with an NH substituent other than acetyl can be prepared according to Coupling Conditions C and D as shown in the scheme below, substituting the appropriate reagent (for example: acid, acid chloride, acid anhydride, sulfonyl chloπde) and conditions The free amme shown below is analogous to that which is acetylated in Coupling Conditions C
1 acylation etc reagent H203P and DIEA or EDC-HCI
HOBT, DIEA
2 standard deprotection
Figure imgf000089_0001
methods
Figure imgf000089_0002
43. Compounds OA-OZM
Figure imgf000089_0003
Cmpd R99 Rl R9 MS
OA -C(O)CH2Ph -CH3 -CH2(C6H4)-4-CF3 632 (M-H)
OB -CO2CH2Ph -CH3 -CH2(C6H4)-4-CF3 648 (M-H)
OC -C(O)CH2Ph -CH3 -CH2(C6H4)-4-Cl 598 (M-H)
OD -C(O)CH2CN -CH3 -CH2(C6H4)-4-CF3 580 (M-H)
OE -C(O)(CH2)3CO2H -CH3 -CH2(C6H4)-4-CF3 627 (M-H)
OF -C(O)(CH2)2CO2H -CH3 -CH2(C6H4)-4-CF3 613 (M-H)
OG -C(O)(CH2)2CO2Et CH3 -CH2(C6H4)-4-CF3 642 (M-H)
OH -SO2CH2Ph -CH3 -CH2(C6H4)-4-CF3 667 (M-H)
OI -SO2CH2Ph -CH3 -CH2(C6H4)-4-Cl 634 (M-H)
OJ -SO2CH3 -CH3 -CH2(C6H4)-4-Cl 558 (M-H)
OK -C(O)CH2Ph -CH2OH -CH2(C6H4)-4-CF3
Figure imgf000089_0004
647 (M-H)
87 OL -C(O)CH2Ph -CH2OH -CH2(C6H4)-3,4-Cl2 647 (M-H)
OM -C(O)CH2Ph -CH2CO2H -CH2(C6H4)-4-Cl 642 (M-H)
ON -C(O)CH2Ph -CH3 -CH2(C6H4)-3,4-Cl2 632 (M-H)
00 -C(O)CH2Ph -gem-Me2 -CH2(C6H4)-4-CF3 647 (M+H)
OP -C(O)CH2Ph -CH2CO2H -CH2(C6H4)-4-CF3 677 (M+H)
00 -C(O)CH2Ph -CH2OH -CH2-(2-naphthyl) 631 (M+H)
OR -C(O)CH2Ph -CH2OH -CH2(C6H4)-4-Cl 613 (M-H)
OS -C(O)CH2-(2-thienyl) -CH2OH -CH2(C6H4)-4-CF3 637 (M-H)
OT -C(O)CH2(C6H4)-3,4-F2 -CH3 -CH2(C6H4)-4-CF3 667 (M-H)
OU -C(O)CH2(C6H4)-4-OMe -CH3 -CH2(C6H4)-4-CF3 661 (M-H)
ON -C(O)CH2-(3-indole) -CH3 -CH2(C6H4)-4-CF3 670 (M-H)
OW -C(O)CH2-( 1 -imidazole) -CH3 -CH2(C6H4)-4-CF3 621 (M-H)
OX -C(O)CH2-(2-naphthyl) -CH3 -CH2(C6H4)-4-CF3 681 (M-H)
OY -C(O)CH2Ph -CH?OH -CH2(C6H4)-4-F 599 (M+H) oz -C(O)CH2Ph -CH2OH -CH2(C6H4)-3-CF3 649 (M+H)
OZA -C(O)CH2Ph -CH2OH -CH2(C6H4)-4-I 705 (M-H)
OZB -C(O)CH2-(2-thienyl) -CH2OH -CH2(C6H4)-4-I 711 (M-H)
OZC -C(O)CH2Ph -CH2OH -CH2(C6H4)-4-Br 657 (M-H)
OZD -C(O)CH2Ph -CH(OH)CH3 -CH2(C6H4)-4-CF3 663 (M+H)
OZE -C(O)CH2Ph -CH(OH)CH3 -CH2(C6H4)-3,4-Cl2 661 (M-H)
OZF -C(O)CH2-(2-thienyl) -CH OH -CH2-(2-naphthyl) 636 (M+H)
OZG -C(O)CH2Ph -CH(OH)CH3 -CH2-(2-naphthyl) 645 (M+H)
OZH -C(O)CH2Ph -CH(OH)CH3 -CH2(C6H4)-4-Br 673 (M+H)
OZI -C(O)CH2Ph -CH3 -CH2(C6H4)-4-(O-/2-Pr) 622 (M-H)
OZJ -C(O)CH2Ph -i-Bu -CH2(C6H4)-3,4-Cl2 673 (M-H)
OZK -C(O)CH2Ph -i-Bu -CH2(C6H4)-4-CF3 673 (M-H)
OZL -C(O)CH2Ph -CH3 -CH2(C6H4)-4-Br 641 (M-H)
OZM -C(O)CH2Ph -CH3 -(CH2)3Ph 591 (M-H)
OZΝ -C(O)CH2Ph -CH2OH -CH2(C6H4)-3-Cl, 4-1 739 (M-H)
Figure imgf000090_0001
88 General Method NA: Preparation of ortho alkyl substituted phosphates:
Figure imgf000091_0001
Compounds such as Compound OZZY can be prepared according to the scheme shown below using the appropriate acid coupling partner along with the appropπate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. This method is analogous to Coupling Conditions C with obvious differences in protecting group strategies and is similar to General Method N, for example.
1 MeOH, H+
2 acylation etc reagent 170 °C
OH and DIEA Me 1 2-dιchloro
Figure imgf000091_0004
benzene
Figure imgf000091_0002
or EDC-HCI R99 O HOBT, DIEA
Figure imgf000091_0006
1 standard
1 (BnO 2P(0)H coupling CCI4, DIEA, DMAP conditions
2 LiOH 2 standard
OH deprotection conditions
Figure imgf000091_0003
Figure imgf000091_0005
Figure imgf000091_0007
Preparation of Acid 58
58
Figure imgf000091_0008
Commercially available L-tyrosme (5.0 g) was suspended in 100 mL of MeOH and HCl gas was bubbled through the reaction mixture for 10 min. The reaction mixture was stirred at rt for 45 m and then concentrated under reduced pressure. The resulting solid was dissolved in 20 mL of DMF and cooled to 0 °C. Phenylacetyl chloπde (4 7 mL, 1.05 eq) was added, followed by DIEA (17.5 mL, 3 eq) at 0 °C. The reaction mixture was stined at 0 °C for 45 mm and then diluted with EtOAc and washed with 3 x 20 mL of H2O, 2 x 20 mL of 10% aqueous citπc acid, bπne, dπed (Na2SO4) and concentrated under reduced pressure to afford 5.2 g of the N-phenyl acetyl tyrosine methyl ester.
89 N-phenylacetyl tyrosine methyl ester was dissolved in 50 mL of anhydrous CH3CN. Anhydrous potassium carbonate (4.6 g, 2 eq) was added, followed by allyl bromide (1.7 mL, 1.2 eq). The resulting reaction mixture was stirred at rt for 15 h. It was then quenched with H2O and concentrated under reduced pressure. The aqueous phase was extracted with EtOAc. The combined organic layers were washed with brine, dried (Na2SO- , and concentrated under reduced pressure. A portion of this material (2.0 g) was dissolved in 6 mL of 1 ,2-dichlorobenzene in a sealed tube. The reaction tube was purged with N2 and sealed. It was then stirred at 170 °C for 2 days. Upon cooling to rt, the crude material was purified by chromatography (2:1 hexanes/EtOAc) to afford 526 mg of the desired ortho- allyl phenol derivative. This ortho-allyl phenol derivative (526 mg, 1.49 mmol) was dissolved in 15 mL of anhydrous CH3CN and cooled to 0 °C. CCI4 (0.720 mL, 5 eq) was added, followed by DIEA (0.545 mL, 2.1 eq) and a catalytic amount of DMAP (5 mg). After dibenzyl phosphite (0.480 mL, 1.5 eq) was added, the reaction mixture was stined at 0 °C. After 45 min, the resulting reaction mixture was quenched with an aqueous solution of KH2PO4 (0.5 M). The volatile solvent was removed by concentration under reduced pressure, and the aqueous phase was extracted with EtOAc. The organic layer was washed with brine, dried (Na2SU4), and concentrated under reduced pressure. Purification by chromatography (1: 1 hexanes/EtOAc) afforded 450 mg of the dibenzyl phosphate derivative. This material was dissolved in a 1:1 mixture of THF/H2O and treated with 1 eq of LiOH at 0 °C. When all the starting material was consumed by TLC, the reaction mixture was quenched with dilute citric acid and extracted with EtOAc. The organic layer was washed with brine, dried (Na SU4) and concentrated under reduced pressure to afford the corresponding acid.
The above procedure could also be repeated using commercial N-acetyl tyrosine methyl ester instead of N-phenylacetyl tyrosine methyl ester in order to prepare the corresponding N-acetyl derivatives.
90 43A. Compounds OZZA-OZZD
Figure imgf000093_0001
Cmpd R99 Rl R9 MS
OZZA -C(O)CH2Ph -CH2OH -CH2(C6H4)-4-CF3 669 (M-H)
OZZB -C(O)CH3 -CH3 -CH2(C6H4)-3,4-Cl2 595 (M-H)
OZZC -C(O)CH3 -CH2OH -CH2(C6H4)-4-CF3 611 (M-H)
Figure imgf000093_0009
OZZD -C(O)CH3 -CH2OH -CH2-(2-naphthyl) 593 (M-H)
General Method NB: Preparation of ortho alkyl substituted phosphates:
Figure imgf000093_0002
R
Compounds such as Compound OZZZ can be prepared according to General Method NA according to the scheme below using the appropriate acid coupling partner (59) along with the appropπate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. This method is analogous to Coupling Conditions C with obvious differences in protecting group strategies and is similar to General Method N, for example.
1 MeOH, H + 1 Bu3SnCH=CH2 2 DIEA, Boc2θ PdCI2(PPh3)2 3 TBSCI 2 TBAF imid
Figure imgf000093_0004
Figure imgf000093_0003
Boc O
1 standard 1 standard H203P phosphorylation coupling conditions conditions
O e 2. standard 2. standard
Figure imgf000093_0006
hydrolysis deprotection o conditions
Figure imgf000093_0007
acylation etc/
Boc deprotection
Figure imgf000093_0008
conditions
91 Preparation of Acid 59
59
Figure imgf000094_0001
Commercially available 3 -lodo-L- tyrosine (5.0 g) was suspended in 100 mL of MeOH and HCl gas was bubbled through the reaction mixture for 10 mm. The reaction mixture was stined at rt for 45 mm and then concentrated under reduced pressure. The resulting solid was dissolved in 20 mL of DMF and cooled to 0 °C. Boc2O (1.05 eq) was added, followed by DIEA (3 eq) at 0 °C. The reaction mixture was stirred at 0 °C for 45 min and then diluted with EtOAc and washed with 3 x 20 mL of H2O, 2 x 20 mL of 10% aqueous citπc acid and bπne. The organic extract was dπed (Na SU4) and concentrated under reduced pressure to affored the N-Boc 3-ιodo-L-tyrosιne methyl ester. A portion of this mateπal (2.7 mmol) was dissolved in 15 mL of anhydrous DMF along with TBSC1 (1.05 eq) and lmidazole (1.05 eq). The reaction mixture was stirred at rt for 15 h. It was then diluted with EtOAc and washed with 3 x 10 mL of H2O, bπne, dπed (Na2SO4) and concentrated under reduced pressure. Puπfication by chromatography (2:1 hexanes/EtOAc) afforded the fully protected 3-ιodo-L tyrosine deπvative. This mateπal (1.55 mmmol) was dissolved in 8 mL of anhydrous DMF along with tπbutylvmyltm (2.33 mmol, 1.5 eq). Lithium chloπde (3 eq) was added, followed by 2 mg of 2,6-dι-tert-butyl-4-methylphenol and 54 mg of PdCl2(PPh3)2 (0.05eq). The resulting reaction mixture was stirred at 80-85 °C for 1 h. It was then cooled to rt and diluted with EtOAc and washed with H2O, brine, dried (Na2Sθ4), and concentrated under reduced pressure. Puπfication by chromatography (2:1 hexanes/EtOAc) afforded 430 mg of the ortho-vinyl deπvative. The TBS protecting group of this ortho-vinyl deπvative was removed by dissolving 1 mmol of this mateπal m 6 mL of anhydrous THF. TBAF was added as a 1 M solution m THF (1.2 eq) at rt and the reaction mixture was stirred at rt until all starting mateπal was consumed, as determined by TLC. The reaction mixture was concentrated and purified by chromatography (1: 1 hexanes/EtOAc) to afford the desired N-Boc-3-vιnyl-L-tyrosme methyl ester. This mateπal was subjected to the same reaction phosphorylation conditions as before to obtain the dibenzylphosphate deπvative. Standard hydrolysis of the methyl ester was performed as descπbed previously to afford the acid. This acid was subjected to the same reaction conditions (Coupling Conditions C and D) that were outlined previously in order to obtain the desired phosphates.
92 43B. Compound OZZG
Figure imgf000095_0001
Cmpd R99 Rl R9 MS
OZZG -C(O)CH2Ph -CH2OH -CH2-(2-naphthyl) 655 (M-H)
Figure imgf000095_0008
General Method NC: Preparation of ortho amino substituted phosphates:
H„ ^R98 N'
Figure imgf000095_0002
^ R1 R2
Compounds such as Compound OZZX can be prepared as exemplified in the scheme shown below (with R98 = Ac) using the appropriate acid coupling partner along with the appropriate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. This method is analogous to Coupling Conditions C with obvious differences in protecting group strategies and is similar to General Method N, for example.
1 MeOH, H+ 1. H2, Pd-C H
2. standard 2. Ac2θ acyladon etc. conditions
Figure imgf000095_0003
Figure imgf000095_0004
Figure imgf000095_0005
1. standard 1. standard phosphorylation Bn 0 A coupling conditions " conditions
2. standard 2. standard hydrolysis deprotection conditions conditions
Figure imgf000095_0006
Figure imgf000095_0007
93 Preparation of Acid 60
60
Figure imgf000096_0001
Commercially available 3-nitro-L-tyrosine (5.0 g, 0.02 mol) was suspended in 100 mL of MeOH, and HCl gas was bubbled through the reaction mixture for 10 min. The reaction mixture was stirred at rt for 45 min and then concentrated under reduced pressure. The resulting solid was dissolved in 20 mL of DMF and cooled to 0 °C. Phenylacetyl chloride (4.2 mL, 1.05 eq) was added, followed by DIEA (16 mL, 3 eq) at 0 °C. The reaction mixture was stirred at 0 °C for 45 min and then diluted with EtOAc and washed with 3 x 20 mL of H2O, 2 x 20 mL of 10% aqueous citric acid, brine, dried (Na2SU4) and concentrated under reduced pressure to afford 6.5 g of the N-phenylacetyl 3-nitro-L-tyrosine methyl ester.
N-phenylacetyl 3-nitro-L-tyrosine methyl ester (1.0 g) was dissolved in 50 mL of MeOH. 10% Pd-C (100 mg) was then added, followed by 2 mL of concentrated HCl. The reaction mixture was hydrogenated at rt under 50 psi of H2 for 15 h. It was then filtered through Celite, and the filtrate was concentrated under reduced pressure. The residue was dissolved in 50 mL of DMF along with Ac2O (1.05 eq) and cooled to 0 °C. DIEA (3 eq) was then added and the resulting reaction mixture was stirred at 0 °C for 45 min. It was then diluted with EtOAc and washed with dilute citric acid, 3 x 20 mL of H2O, brine, dried (Na2SO4) and concentrated under reduced pressure. Purification by chromatography (1 : 1 EtOAc/hexanes) afforded 330 mg of the ortho-N-acetyl tyrosine methyl ester. Standard hydrolysis of the methyl ester was performed as described previously to afford the acid (60). This acid was subjected to the same reaction conditions (Coupling Conditions C and D) that were outlined previously in order to obtain the desired phosphates.
94 43C. Compounds OZZJ
Figure imgf000097_0001
O- N
H-_ X
R99
Cmpd R99 Rl R9 MS |
Figure imgf000097_0002
|θZZJ -C(O)CH2Ph -CH2OH -CH2-(2-naphthyl) 686 (M-H)
General Method ND: Preparation of additional heterocycle amines - General Methods O-U
Ή* Y^uA r m
/
R1 R2 R1
Figure imgf000097_0003
ZZ12 ZZ13
Compounds such as Compound ZZ13 prepared according to General Methods A-NC and N-ZT with representative heterocyclic amine tails such as ZZ12 as described within General Methods A-ΝC could also include many other heterocyclic amine tails, for example those described below in General Methods O-U with specific examples shown within General Methods S-TC, for example.
General Method O: Preparation of pyrones - hetero Diels Alder:
R1
--"- ORc H!N OR
Many substituted pyrones can be prepared via the hetero Diels Alder reaction according to the method described in Midland et al. (J. Am. Chem. Soc. 1989, 111, 4368) according to the scheme shown below. The resulting amine can then be further elaborated according to Coupling Conditions C and D with the appropriate carboxylic acid coupling partner.
95 DMe OMe
R9-. 1 (RO)3CH, H+ a ^4- TMS NPP'
Lewis acid
2. LDA, TMSCI RO ^-
R1 P'PN- °γ0 1. aromatize
2. standard ^ζ deDrotection
OR OR
R1 standard deprotection H2N^ ° C
OR
General Method P: Preparation of pyrones - cyclization:
Alternately, the pyrone can be prepared via a beta, gamma diketo ester with subsequent cyclization according to the method described in Hiyama et al. Tetrahedron Lett. 1988, 29, 6467 and Lygo Tetrahedron 1995, 51, 12859 as shown in the scheme below. The resulting amine can then be further elaborated according to Coupling Conditions C and D with the appropriate carboxylic acid coupling partner.
o °u Na o o o
OR cyclization
OR
NPP'
NPP'
1. alkylation
2. deprotection
Figure imgf000098_0001
OH A
NPP'
PhNTfc P'PN 1. Pd(0) coupling
EtøN 2. deprotection
Figure imgf000098_0002
-)Tf
General Method Q: Preparation of pyrazoles and isoxazoles:
R1 R
H2N^J ^ R9 H2I -
N-N N /rRa / -N
^R 2 J12
H2h ^ .R9 "12' II N-O O-N
96 Many substituted pyrazoles and isoxazoles can be prepared according to the following schemes. The resulting amine can then be further elaborated according to Coupling Conditions C and D with the appropπate carboxylic acid coupling partner.
Preparation of diketone 41:
R1 R1 f^N V9
PHN
Q H CDI
PHN iHh- oN^ PHN' form enolate Q Q and
P r- = Boc or C-b.z add to ac .y 'l 1 imidazoe
Diketone 41 can then be used for pyrazole formation.
R1 R1 R
I R9 R12NHNH2 I I
PH -^Y^ " - PHN^^ R9 and HN- ^ *-R9
0 O -2H20 N-N N-N
R12 /
41
1. side chain manipulation
H 2 N^ C R9 and H 2 N- ^ F
2. standard -N^B12 N_N deprotection R p'12 conditions
Diketone 41 can also be used for isoxazole formation.
R1 NH2OH I1
Figure imgf000099_0002
R9 ► pH
PHN PH -^-^/^^---.R"9 a __n.d. D PUHMNΛ' . R9
-2H20
Figure imgf000099_0001
N-O O-N
41
1. side chain manipulation R1 R1
2. standard H2N' W^ and H^"^*^-"9 deprotection N-0 N-O conditions
97 General Method R: Preparation of isoxazoles:
Alternately, the isoxazoles can be prepared according to the scheme shown below. The resulting amine can then be further elaborated according to Coupling Conditions C and D with the appropriate carboxylic acid coupling partner.
R1 R1
0H nitrilβ oxide | Rlf> — R9 PHN ■V T formation PHN ^
P = Boc or Cbz
R1 R
PHN^NT "9 rnn W / m 1 a sniidpeul cahtiaoinn H2ι>T ", V ' R9
N-O N-O
. 2 standard ,n and deprotection and
R1 R9 conditions R1 R9
PHN- V"10 H 2 N- VR10
N-O N-O
General Method S: Preparation of pyridines:
Many substituted pyridines can be prepared as exemplified by the scheme shown below for the preparation of Compound PA. The resulting amine can then be further elaborated according to Coupling Conditions C and D with the appropriate carboxylic acid coupling partner. The starting diol allows for obvious side chain manipulations that can be used for the preparation of additional analogs.
O H MnQ2 y^A> » Cs2C03 S^°~
NaCI02
OH
Br ^0
EDC-HCI HOBT ^- ,o^ N BS eT CO!H DIEA
CONH2 v*"4
NH,OH
Figure imgf000100_0001
B
DMSO \\ \ 1 ) MeMgCI
Figure imgf000100_0003
CH2CI2, EtsN OHC" "N' "CONH2 2) Mnθ2
Me,N0
Figure imgf000100_0002
NHjOH'HCI
— pyπdin ~e~
Figure imgf000100_0004
98 50. Compound PA
Figure imgf000101_0001
PA
Compounds such as Compound PA can be prepared from amine 42, prepared according to General Method S, using Coupling Conditions D. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 675 (M-H).
51. Compound PB
H2O3P
Figure imgf000101_0002
' f Nt CONH2
O ° PB
Compounds such as Compound PB can be prepared from the appropriate amine, prepared according to General Method S (omitting the addition of the Grignard reagent), using Coupling Conditions D. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 661 (M-H).
51A. Compound PC
Figure imgf000101_0003
Compounds such as Compound PC can be prepared from amine 42, prepared according to General Method S, using General Method K. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 590 (M-H).
51B. Compound PD
Figure imgf000101_0004
N- " ^ ^CONH., PD
Compounds such as Compound PD can be prepared from the appropriate amine, prepared according to General Method S (omitting the addition of the Grignard reagent), using
99 Coupling Conditions D and the carboxylic acid used in General Method H. Electrospray Mass Spectrum (50/50 acetonitπle/water + 0.1% ammonium hydroxide) m/z 462 (M-H)
General Method T: Preparation of furans:
Many substituted furans can be prepared as shown below in the following scheme. The resulting amine can then be further elaborated according to Coupling Conditions C and D with the appropπate carboxylic acid coupling partner. Substitution of the 3 and/or 4 positions of the starting furan aldehyde allows for obvious side chain manipulations that can be used for the preparation of additional analogs.
// « RMgX NaBH„ // DMF
HO OHC
TFA Λ
OH POCI,
R 'MgX DBU N3
Figure imgf000102_0001
Figure imgf000102_0002
Figure imgf000102_0003
(PhO)2PON3 Pd/C
52. Compound QA
H203P
Figure imgf000102_0004
Compounds such as Compound QA can be prepared according to General Method T, substituting the appropriate Grignard reagents, followed by Coupling Conditions D Electrospray Mass Spectrum (50/50 acetonitπle/water + 0.1% ammonium hydroxide) m/z 493 (M-H)
General Method TA: Preparation of thiophenes:
Many substituted thiophenes can be prepared as shown in the following scheme. The resulting amme can then be further elaborated according to Coupling Conditions C and D with the appropπate carboxylic acid coupling partner. Substitution of the 3 and/or 4 positions of the starting thiophene allows for obvious side chain manipulations that can be used for the preparation of additional analogs.
100
Figure imgf000103_0001
53. Compound QB
CF3
Figure imgf000103_0002
QB
Compounds such as Compound QB can be prepared according to General Method TA followed by Coupling Conditions D. Compound QB was isolated as a 1:1 mixture of diastereomers. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 569 (M-H).
53B. Compound QD
CF3
Figure imgf000103_0003
QD
Compounds such as Compound QD can be prepared according to General Methods TA and N. Compound QD was isolated as a 1: 1 mixture of diastereomers. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 645 (M-H).
General Method TB: Preparation of thiophenes:
Alternately, many substituted thiophenes can be prepared as shown below in the following scheme starting with ketone 80, prepared in Method TA. The resulting amine can then be further elaborated according to Coupling Conditions C and D with the appropriate
101 carboxylic acid coupling partner. Substitution of the 3 and/or 4 positions of the starting thiophene allows for obvious side chain manipulations that can be used for the preparation of additional analogs.
Figure imgf000104_0002
Figure imgf000104_0001
1. NaBH4
2. DBU
Figure imgf000104_0003
(PhO)2PON3
Figure imgf000104_0004
OTBS 3. LiAIH4 OTBS
54. Compound QC
Figure imgf000104_0005
QC
Compounds such as Compound QC can be prepared according to General Method TB followed by Coupling Conditions D. Compound QC was isolated as a 1: 1 mixture of diastereomers. The two individual diastereomers could be separated by reverse-phase HPLC using a mixture of H2O/CH3CN with 0.1% TFA as the buffer. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 585 (M-H).
General Method TC: Preparation of aminothiazoles
Many substituted aminothiazoles can be prepared according to General Methods A and B, and as exemplified by the scheme shown below, substituting thiourea for the conesponding thioamide, as described in Patt, W. C. et al. Synth. Commun. 1990, 20, 3097 and Leanna, M. R. et al. Tetrahedron Lett. 1993, 34, 4485. The resulting amine or amine salt can then be further elaborated according to Coupling Conditions C and D with the appropriate carboxylic acid coupling partner.
R9
X... ϊ acetone
H2N NH2 O^R10 RT to reflux H -ι2,ι A R1°
•HBr
102 Preparation of amine salt 101
Me
Br^ Ji/le acetone
A.
Figure imgf000105_0001
Ph
H2N-^NH2 O^Ph RT to reflux
•HBr 101
To a solution of 1.43 mL (9.39 mmol) of 2-bromopropιophenone (90% tech ) in 50 mL of acetone at RT was added 0.79 g (10.3 mmol) of thiourea. The resulting mixture was stined at RT for 22 h. The white solid was isolated by filtration, πnsed several times with acetone, and dπed in vacuo. MS and -Η NMR indicated incomplete reaction. A portion (500 mg) of this solid was suspended in 10 mL of acetone and heated to mild reflux overnight. After cooling, the white solid was isolated by filtration, πnsed several times with acetone, and dπed in vacuo. MS and -Η NMR indicated that the reaction had gone to completion. Electrospray Mass Spectrum (50/50 acetomtπle/water) m/z 191 (M+H).
General Method U: Preparation of pyrroles:
Many substituted pynoles can be prepared as shown in the following scheme. The resulting amme can then be further elaborated according to Coupling Conditions C and D with the appropπate carboxylic acid coupling partner. Substitution of the 3 and/or 4 positions of the starting pynole allows for obvious side chain manipulations that can be used for the preparation of additional analogs.
Ph— B Mff→^-CF3
Figure imgf000105_0002
NaOH
2) Nal, TMSCI
Figure imgf000105_0003
Figure imgf000105_0004
BrMg EtMgBr
Figure imgf000105_0005
Figure imgf000105_0007
N)'
H2N
Ra-Ni
Figure imgf000105_0006
Figure imgf000105_0008
103 General Method V: Preparation of acid aldehydes:
Figure imgf000106_0001
Compounds such as Compounds ZA-ZI can be prepared according to the preparation of Compound ZA described below using the appropriate acid coupling partner along with the appropriate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. This method is analogous to Coupling Conditions C with obvious differences in protecting group strategies.
55. Compound ZA: (2-Formyl-4-((S)-2-acetylamino-2-r(S -l-carbamoyl-(5-hexyl- 1.2.4-oxadiazol-3-yl)-2-(3-indolyl)ethyllethyl }benzoic acid)
1. BF «OEt2 HS(CH2)2SH
2. Tf2NPh Et3N, CH2CI2
3. CO, Pd(OAc)2
Ph2P(CH2)3PPh2
DMSO, MeOH
4. LiOH«H20
Figure imgf000106_0002
THF-H20
46
1. EDOHCI, HOBT DMF, CH2CI2
2. Hg(CI04)2«xH20
CH3CN, H2O
Figure imgf000106_0003
3. LiOH«H20
THF-H20
0 °C
Figure imgf000106_0004
(a) 3-(l,3-Dithiolan-2-yl)-N-acetyl-L-tyrosine methyl ester
To L-3-formyl-N-acetyltyrosine methyl ester (US patent 4,022,910) (0.13 g, 0.48 mmol) in methylene chloride (5 mL) at 0 °C was added boron trifluoride etherate (0.12 mL, 0.96 mmol) followed by ethanedithiol (0.044 mL, 0.53 mmol). The mixture was allowed to warm to room temperature and stined for 1 hour. The solution was poured into water and the layers separated. The aqueous layer was extracted with methylene chloride, and the combined extracts were washed with water, dried over magnesium sulfate and concentrated
104 to a solid. The solid was recrystal zed from ethyl acetate/hexane (0 14 g, 82%). mp 97-99 °C
(b) 3-( 1.3-Dιthιolan-2-yl)-4-(tπfluoromethansulfonyloxy)-N-acetyl-L-phenylalanιne methyl ester
To 3-(l,3-dιthιolan-2-yl)-N-acetyltyrosιne methyl ester (0.50 g, 1.46 mmol) and tπethylamine (0.22 mL, 1.61 mmol) in methylene chloπde (10 mL) at 0 °C was added N- phenyl-bιs(tπfluoromethanesulfonιmιde) (0.58 g, 1.61 mmol). The mixture was allowed to stir for five days then washed sequentially with 1 N NaOH, 1 N HCl, and bπne. The organic layer was dπed over magnesium sulfate and concentrated to a solid. The solid was recrystalhzed from ethyl acetate/hexane (0.61 g, 87%). mp 116-118 °C.
(c) 3-(1.3-Dιthιolan-2-yl)-4-(carboxymethyl)-N-acetyl-L-phenylalanιne methyl ester
To 3-(l,3-dιthιolan-2-yl)-4-(tπfluoromethansulfonyloxy)-N-acetyl-L-phenylalanιne methyl ester (0.51 g, 1.08 mmol) in DMSO/MeOH (3:2, 5 mL) was added tπethylam e (0.33 mL, 2.36 mmol) followed by palladium acetate (0.0073 g, 0 033 mmol) and 1,3- bιs(dιphenylphosphmo)propane (0.013 g, 0.034 mmol). Carbon monoxide was bubbled through for 3 mm, and the mixture was heated to 80 °C for 24 hours. After cooling, the solution was diluted with water and extracted with ethyl acetate. The combined extracts were washed with water, dπed over magnesium sulfate and concentrated to a solid. The solid was recrystalhzed from ethyl acetate/hexane (0.30 g, 72%). mp 113-119 °C
(d) 3-(l,3-Dιthιolan-2-yl)-4-(carboxymethlyVN-acetyl-L-phenylalanme (46)
To the 3-(l,3-dιthιolan-2-yl)-4-(carboxymethyl)-N-acetyl-L-phenylalanιne methyl ester (0.22 g, 0.57 mmol) m THF (10 mL) at 0 °C was added lithium hydroxide (0.025 g, 0.60 mmol, 1 mL water). The mixture was stined for 45 mm., diluted with water, made acidic with 1 N HCl, and extracted with ethyl acetate. The combined extracts were washed with water, dried over magnesium sulfate and concentrated to a glassy solid which was homogeneous by RP HPLC (0.18 g, 86%). MS [M - H] 368.
(e) 2-Formyl-4-((S)-2-acetylamιno-2-r(S)-l-carbamoyl-(5-hexyl-1.2.4-oxadιazol-3-yl)-2- (3-ιndolyl)ethyllethyl}benzoιc acid (ZA)
3-(l,3-Dιthιolan-2-yl)-4-(carboxymethyl)-N-acetyl-L-phenylalanιne (46) (0.5 mmol) was dissolved in 2 mL of DMF and 5 mL of CH2CI2 along with (S)-l-ammo-l-(5-hexyl-l,2,4-
105 oxadιazol-3-yl)-2-(3-ιndolyl) ethane (0.6 mmol) (see general Method G). HOBT (0.6 mmol) and EDC (0.6 mmol) were added, followed by DIEA (0.75 mmol). The resulting reaction mixture was stmed at rt for 4 h. It was then concentrated, and the residue was taken up m EtOAc. The organic layer was washed with satd aq NaHCO3, dilute aq citπc acid. bπne. dπed (Na2SO4), and concentrated under reduced pressure. The resulung product was dissolved in 5 mL of CH3CN. Hg(ClO4)2 was added dropwise at rt as a solution in 3 mL of water. The resulting reaction mixture was stmed at rt for 10 mm and then filtered through a pad of Celite. The filtrate was concentrated. The residue was dissolved m 2 mL of THF and 0.5 mmol of LiOH was added as a solution in 2 mL of water at 0 °C. The reaction mixture was stmed at 0 °C for 1 h. It was then acidified with 10% aq citπc acid and extracted with EtOAc. The combined organic layers were washed with bπne, dπed (Na2SO4), and concentrated under reduced pressure. A portion of the crude product was puπfied by reverse phase HPLC (H2O/CH3CN) to afford 6 mg of the desired product (ZA). Electrospray Mass Spectrum (50/50 MeCN/water) m z 572 (M-H).
56. Compounds ZB-ZG
CHO
HO.
H O*
Figure imgf000108_0001
N. ~ »°
A*
Compound II*1 9 MS
ZB -(CΑ-> CO)H -CH2Chx 527 (M-H)
ZC -(CH2)2CO H -(CH2)6CH3 531 (M+H)
ZD -ClfrCHi -(CH2)5CH3 473 (M+H)
ZE -CH^CH^ -(CH2)6CH3 487 (M+H
ZF -CH3 -(CH2)5CH3 457 (M-H)
ZG -CH2CH3 k^ϊ^ 471 (M-H)
Figure imgf000108_0002
106 57. Compound ZH: (2-Formyl-4-f (S)-2-acetylamino-2-r(S)-l-carbamoyl-(5-hexyl-1.3- thiazol-2-ylV2-(3-indolyl)ethyllethyl } benzoic acid)
CHO
Figure imgf000109_0001
ZH
Compounds such as Compound ZH can be prepared according to General Method V, exemplified by the preparation of Compound ZA, substituting the appropriate amine coupling partner, which can be prepared according to General Method E. Flash chromatography (elution with 7: 1 CH2Cl2-MeOH followed by 3:1 then 2:1 CH2CI2- MeOH) gave 68.8 mg of a white solid: Rf 0.33 (3:1 CH Cl2-MeOH). Electrospray Mass Spectrum (50/50 MeCN/water + 0.1% formic acid) m/z 557 (M-H).
58. Compound ZI:
(2-Formyl-4-((S)-2-acetylamino-2-l(S)-l-carbamoyl-(4-(3-(propionamide))(5-hexyl)-1.3- thiazol-2-yl)-2-(3-indolyl)ethynethyl Ibenzoic acid)
CHO
H S-
Figure imgf000109_0002
Figure imgf000109_0003
ZI
Compounds such as Compound ZI can be prepared according to General Method V, exemplified by the preparation of Compound ZA, substituting the appropriate amine coupling partner which can be prepared according to General Method E. Compound ZI was purified by reverse phase preparative HPLC. Electrospray Mass Spectrum (50/50 MeCN/water) m/z 589 (M+H).
107 General Method W: Preparation of acetic acid aldehydes
QHO H02C^/0„ 1
ZM-ZU
Figure imgf000110_0002
A0J R1 R2
Compounds such as Compounds ZM-ZU can be prepared according to the preparation of Compound ZM descπbed below using the appropπate acid coupling partner along with the appropπate amme coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. This method is analogous to Coupling Conditions C with obvious differences in protecting group strategies and is similar to General Method N
59. Compound ZM: (2-Formyl-4-l (S -2-acetylamιno-2-r(S)-l-carbamoyl-(5-hexyl- 1.2.4-oxadιazol-3-yl)-2-(3-ιndolyl)ethyllethyπphenoxyacetιc acid
CHO
Hθ2α^o
Figure imgf000110_0001
In a typical procedure, 3-(l,3-dιthιolan-2-yl)-Ν-acetyl-L-tyrosme methyl ester (General Method V) was hydrolyzed to the conesponding acid using the standard procedure involving 2 equivalents of LiOH. The resulting acid (0.62 mmol) was dissolved in 2 mL of DMF and 5 mL of CH2CI2 along with 0.62 mmol of (S)-l -ammo- l-(5-hexyl- 1,2,4- oxadιazol-3-yl)-2-(3-ιndolyl)ethane (prepared according to General Method G) HOBT (0.74 mmol) and EDC (0 74 mmol) were added, followed by DIEA (0 93 mmol). The resulting reaction mixture was st ed at rt for 4 h. It was then concentrated, and the residue was taken up m EtOAc. The organic layer was washed with sat NaHCO3, dilute aq citπc acid, bπne, dπed (Na2SO4) and concentrated under reduced pressure. The resulting crude product was dissolved m 5 mL of CH3CN along with 1.86 mmol of t-butyl bromoacetate and 2 0 mmol of K2CO3. The resulting reaction mixture was stmed at rt for 2 days. It was then diluted with EtOAc and washed with bπne, dπed (Na2SO4) and concentrated The resulting product was dissolved m 4 mL 25% TFA in CH2CI2 and allowed to stand at rt for 1 h. It was then concentrated and dissolved in 4 mL of CH3CN Hg(ClO4)2 (1 10 mmol) was then added dropwise as a solution in 2 mL of water. The resulting reaction mixture was stined at rt for 10 min and then filtered through a pad of Celite. The filtrate was concentrated & a portion was then puπfied by RP HPLC (H2O/CH3CN). MS (M-H) 602.
108 60. Compounds ZN-ZS
CHO
Hθ2C^,0
H O-N
Figure imgf000111_0001
V 9
Compound R9 MS
ZN -(CH )2CO H -CH2Chx 559 (M+H)
ZO -CH2-(3-indole) 602 (M-H)
ZP -CH^CHT -(CH2)4CH3 503 (M+H)
ZQ -CHoCH3 -(CH2)5CH3 558 (M+Na+ O)
ZR -(CH2)2CO H -(CH2)6CH3 561 (M+H)
Figure imgf000111_0003
ZS -(CH2)2CO2H -(CH2)4CH3 533 (M+H)
61. Compound ZT: (S)-4-f3-fonnyl-4-carboxymethoxy-phenyl-carbonylarninol- 4-(5- heptyl- 1.2,4-oxadiazol-3-ylVbutyric acid
Figure imgf000111_0002
(a) 3-(1.3-Dithiolan-2-vP-4-hvdroxybenzoic acid
This compound was prepared analogously to 3-(l,3-dithiolan-2-yl)-N-acetyl-L-tyrosine methyl ester (General Method V) using 3-formyl-4-hydroxybenzoic acid (Pfaltz & Bauer) to provide a solid. MS (M-H) 241.
(h) 3-( 1 ■3-Dithiolan-2-yl)-4-hvdroxyphenyl-carhonyl-L-Glu(OtBu)-OMe
This compound was prepared using the standard EDC/HOBT coupling conditions (as in Coupling Conditions C and D) with 3-(l,3-dithiolan-2-yl)-4-hydroxybenzoic acid and L- Glu(OtBu)-OMe to provide a white solid. MS (M-H) 440.
109 (c) 3-(1.3-Dithiolan-2-yl)-4-hvdroxyphenyl-carbonyl-L-Glu(OtBu)-OH
This compound was prepared using the standard LiOH hydrolysis conditions to provide a solid. MS (M-H) 426.
(d) t-Butyl-(SV4-r3-(1.3-dithiolan-2-yl)-4-hydroxy phenyl-carbonylaminol- 4-(5-heptyl- 1.2.4-oxadiazol-3-yl)-butyrate
To 3-(l,3-dithiolan-2-yl)-4-hydroxypheny-carbonyl-L-Glu(OtBu)-OH (0.31 g, 0.72 mmol), HOBT (0.17 g, 0.87 mmol), EDC (0.13 g, 0.87 mmol), and n-heptamidoxime
(0.11 g, 0.72 mmol) (prepared according to General Method F) was added 6.0 mL
CH2C12/DMF (3:1) at 0 °C followed by DIEA (0.19 mL, 1.09 mmol). The resulting solution was stined overnight at ambient temperature. The reaction mixture was diluted with EtOAc and washed successively with saturated NaHCO3 and brine, dried over magnesium sulfate and concentrated. The crude material was dissolved in 2.5 mL of pyridine and heated at reflux for 30 min, diluted with EtOAc and washed successively with
10% aqueous citric acid and brine, dried over magnesium sulfate, concentrated and chromatographed over silica gel (1% MeOH/CHCl3) to provide a white solid (0.13 g). MS
(M-H) 548.
(e) t-Butyl-4-r(S)-3-(1.3-Dithiolan-2-yl)-4-carboxymethoxy-phenyl-carbonylaminol- 4-(5- heptyl-1.2.4-oxadiazol-3-yl)-butyrate
This compound was prepared using t-butyl-4-[(S)-3-(l,3-dithiolan-2-yl)-4-hydroxy -phenyl-carbonylamino]- 4-(5-heptyl-l,2,4-oxadiazol-3-yl)-butyrate and t- butylbromoacetate under standard alkylating (see General Method W) conditions to provide a solid. MS (M+H) 664.
(f) 4-r(S)-3-formyl-4-carboxymethoxy-phenyl-carbonylaminol- 4-(5-heptyl-1.2.4- oxadiazol-3-ylVbutyric acid
This compound was prepared using standard mercury perchlorate and TFA deprotection conditions (see General Methods V and W). Purification by reverse phase HPLC (CH3CN/H2O) provided a white solid. MS (M-H) 474.
110 61A. Compound ZU: 2-Formyl-4-((S)-l-r5-(N-acetyl-N-heptyl-aminomethyl)-1.2.4- oxadiazol-3-yll-propylcarbamoyπ-phenoxyacetic acid
HO2C
Figure imgf000113_0001
Compounds such as compound ZU can be prepared analogously to Compound ZT and according to General Method H using the appropriate oxime (N-acetyl-N-heptyloxime - prepared according to General Method I) and amino acid (L-Abu-OMe). Purification by reverse phase HPLC (CH3CN/H2O) provided a white solid. MS (M-H) 501.
General Method WI: Preparation of acetic acid acetic aldehydes
ZU1
Figure imgf000113_0002
Compounds such as Compound ZUl can be prepared according to General Methods W and NA, substituting obvious manipulations (ozonolysis of the allyl side chain and O-alkylation of the phenol) to install the appropriate acetic acid/aldehyde functional groups.
61B. Compound ZU2
H02C ^O
O-N w
ZU2
Figure imgf000113_0003
Compounds such as Compound ZU2 can be prepared according to General Method WI. Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 503 (M+H).
I l l General Method WA: Preparation of naphthyl hydroxy furanones
0 i ^1
Ni#X.
T 1
HO 1 >X
Figure imgf000114_0002
N rnB zv ° R V,2
Compounds such as Compound ZV can be prepared according to the preparation of Compound ZA described above using the appropriate acid coupling partner (55) along with the appropriate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. This method is analogous to Coupling Conditions C with obvious differences in protecting group strategies and is similar to General Methods V and K.
Preparation of Acid 55
Figure imgf000114_0001
(a) 5-Formyl-6-hydroxy-2-naphthoic acid
To a mixture of 941 mg (5.0 mmol) of 6-hydroxynaphthoic acid in 20 mL of (CH2C1)2 at 0 °C under N2 was added 1.0 mL (11.0 mmol) of CHCbOMe followed by 1.15 mL (10.5 mmol) of TiCl-j (added slowly). The mixture was stined at rt overnight, then cooled to 0 °C and diluted with 25 mL of 10% aq HCl. The mixture was extracted with 3:1 EtOAc -THF and the extract washed with 25 mL of 1 M aq HCl. The first aqueous layer, which had suspended solids, was filtered and the pink solid washed with 1 M aq HCl. The solid was dried under high vacuum over P2O5 affording 197 mg. The filtrate was reextracted once with 3:1 EtOAc -THF and washed with the second aqueous layer. The combined extracts were concentrated in vacuo affording an additional 752 mg of about 90% purity. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) m/z 215 (M-H).
112 (b) Benzyl 5-formyl-6-hydroxy-2-naphthoate
To a suspension of 5-formyl-6-hydroxy-2-naphthoιc acid (1.12 g, 5.18 mmol) in 7.3 mL DMF at rt under N2 was added 569 mg (5.69 mmol) of K2CO3 and 1.11 mL (9 32 mmol) of benzyl bromide. The mixture was stmed for 3 days, at which point it was diluted with H2O and extracted with EtOAc. The extract was washed with saturated aqueous NaHCO , dπed over MgSO4 and concentrated in vacuo. The crude mateπal was puπfied by flash chromatography on silica gel, using 85:15 hexanes-EtOAc as the eluent, to give 410 mg (25%)
(c) Benzyl 5-fonnyl-6-[[(tπfluoromethyl)sulfonyl1oxyl-2-naphthoate
To a mixture of benzyl 5-formyl-6-hydroxy-2-naphthoate (410 mg, 1 33 mmol) in 6.25 mL of CH2C12 at it under N2 was added 0.28 mL (2.00 mmol) of Et3N followed by 717 mg (2.00 mmol) of N-phenyl-bιs(tπfluoromethanesulfonιmιde). The mixture was stined for 18 h at it. diluted with saturated aqueous NaHCO3 and extracted with CH2C12 The extract was dπed over MgSO4 and concentrated in vacuo. The crude mateπal was puπfied by flash chromatography on silica gel. Elution with 4:1 hexanes-Et2O afforded 560 mg (96%).
(d) 7-Benzyloxycarbonyl-l-methoxynaphthoπ.2-c1furan-3(lH)-one
To a solution of benzyl 5-formyl-6-[[(tπfluoromethyl)sulfonyl]oxy]-2-naphthoate 560 mg (1.27 mmol) in 3 6 mL dry of DMSO and 2.54 mL of dry MeOΗ at rt was added 0 39 mL (2.8 mmol) of Et3N, 8.5 mg (0.038 mmol) of Pd(OAc)2, and 15.6 mg (0.038 mmol) of dppp. The mixture stmed under an atmosphere of CO (balloon) at rt for 1 5 h and then at 60 °C for 2 h. The mixture was then cooled to rt, diluted with Η2O and extracted with EtOAc followed by CHC13. The combined extracts were dπed over MgSO4 and concentrated in vacuo. The crude mateπal was puπfied by flash chromatography on silica gel. Elution with 85:15 hexanes-EtOAc afforded a mixture of products. The mixture was diluted with Et2O, washed with 10 mL of 1.0 M aqueous NaOH, H2O and saturated aqueous NH4C1 The orgamc extract was dπed over MgSO and concentrated in vacuo affording 33 mg (10%).
(e) 7-Hydroxycarbonyl-l-methoxynaphtho 1.2-clfuran-3(lH)-one (55)
A mixture of 7-benzyloxycarbonyl-l-methoxynaphtho[l,2-e]furan-3(lH)-one (33 mg, 0.095 mmol) and a catalytic amount of 10% Pd/C in 2 mL of EtOΗ was stined at rt under an atmosphere of Η2 (balloon) for 2 h. The mixture was then diluted with THF and filtered
113 through a pad of Celite. The filtrate was then concentrated in vacuo. The crude material was dissolved in saturated aqueous NaHCO3 and washed with EtOAc. The aqueous layer was then acidified with 1 M aqueous HCl and extracted with EtOAc. The extract was dried over MgSO4 and concentrated in vacuo affording 19 mg (79%).
General Method WB: Preparation of naphthyl hydroxy furanones
Figure imgf000116_0001
Compounds such as Compound ZW can be prepared according to the preparation of Compound ZA described above using the appropriate acid coupling partner (56) along with the appropriate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. This method is analogous to Coupling Conditions C with obvious differences in protecting group strategies and is similar to General Methods V and K.
Preparation of Acid 56
Figure imgf000116_0002
(a) Methyl 3.5-Dihydroxy-2-naphthoate
To a solution of 3,5-dihydroxy-2-naphthoic acid (5.05 g) in MeOH (100 mL) was added TsOH (93 mg). The reaction mixture was stined at reflux for 18 hours and then CH(OMe)3 (1 mL) was added. The reaction mixture was stined at reflux for an additional 5 hours, at which point H2SO4 (10 drops) was added. After an additional 18 h, the reaction mixture was cooled to rt, poured into saturated aqueous NaHCO3 and extracted with EtOAc. The extract was dried over MgSO and concentrated in vacuo to give the title compound as a yellow-white solid (5.19 g, 96%).
114 (b) Methyl 3.5-Bis[f(trifluoromethyl sulfonynoxyl-2-naphthoate
To a solution of methyl 3,5-dihydroxy-2-naphthoate (2.0 g, 9.17 mmol) in CH2C12 (43 mL) at rt was added Et3N (4.0 g, 5.6 mL, 40.3 mmol) followed by PhNTf2 (7.2 g, 20.2 mmol). The solution was then stined at rt for 18 hours. The reaction mixture was diluted with Et2O and washed with 1.0 M aqueous HCl. The extract was dried over MgSO4 and concentrated in vacuo. The crude material was chromatographed on silica gel (20:1 hexanes:EtOAc) to give a 1 : 1 mixture of the title compound and PhNHTf.
(c) Methyl 3.5-Diethenyl-2-naphthoate
To a solution of a 1:1 mixture of methyl 3,5-bis[[(trifluoromethyl)sulfonyl]oxy]-2- naphthoate and PhNHTf (3.24g, 4.5 mmol) in DMF (11.25 mL) was added (PPh3)2PdCl2 (158 mg, 0.23 mmol) and LiCl (1.14 g, 27.0 mmol). The mixture was stined at rt for 20 min at which point Bu3SnCH=CH2 (2.6 mL, 9.45 mmol) was added. The reaction mixture was heated for 90 min whereupon saturated aqueous KF was added. The resulting precipitate was removed by filtration and the filtrate extracted with EtOAc. The organic layer was washed with 1.0 M aqueous HCl, dried over MgSO4 and concentrated in vacuo. The crude material was chromatographed on silica gel (2% to 5% EtOAc/hexanes) to give the title compound (slightly impure as judged by Η-NMR) as a clear oil. This material was used in the next reaction without further purification.
(d) Methyl 3.5-diformyl-2-naphthoate
O3 was bubbled through a solution of slightly impure methyl 3,5-diethenyl-2-naphthoate (from the previous reaction) in CH2C12 (30 mL) and pyridine (2 mL) at -78 °C until a blue color persisted. Next, Me2S (2 mL) was added, producing a yellow color. The mixture warmed to rt over 18 h. All volatiles were then removed in vacuo. The crude material was chromatographed on silica gel (20:1 to 85:15 hexanes :EtOAc) to give the title compound (slightly impure as judged by 1H-NMR). The material was used in the next step without further purification.
(e 5-Formyl-3-hydroxynaphthor2.3-clfuran-l(3H)-one
To a solution of the impure methyl 3,5-diformyl-2-naphthoate in TΗF (10 mL) at rt was added 1.0 M aqueous LiOΗ (5 mL, 5 mmol). After 1.5 hours, the reaction mixture was diluted with Η2O and washed with Et2O. The aqueous layer was acidified with 1.0 M aqueous HCl and extracted with EtOAc. The organic layer was dried over MgSO4 and
115 concentrated in vacuo to give the title compound (200 mg, 19% from methyl 3,5-diethenyl- 2-naphthoate) as a white solid.
(f) 3-Acetoxy-5-formylnaphthor2.3-clfuran-l(3H)-one
To a solution of 5-formyl-3-hydroxynaphtho[2,3-c]furan-l(3H)-one (190 mg, 0.83 mmol) in pyridine (1.66 mL) at rt was added Ac2O (126 mg, 0.117 mL, 1.25 mmol). The solution was stined at rt for 18 hours and then poured into brine and extracted with EtOAc. The extract was dried over MgSO4 and concentrated. The crude material was chromatographed on silica gel (3:1 hexanes:EtOAc) to give the title compound (100 mg, 44%).
(g) 3-Acetoxy-5-hydroxycarbonylnaphthor2.3-c1furan-l(3H)-one (56)
To a solution of 3-acetoxy-5-formylnaphtho[2,3-c]furan-l(3H)-one (100 mg, 0.377 mmol) in MeCN (0.373 mL) and Η2O (0.150 mL) at rt was added NaH2PO4 (15.3 mg, 0.097 mmol) followed by 50% H2O2 (0.026 mL). NaClO2 (1.0 M in H2O, 0.518 mL) was added over the course of 2 h via syringe pump. The reaction mixture was then diluted with saturated aqueous NH4C1 and extracted with EtOAc. The organic layer was dried over MgSO and concentrated to give the title compound 56 (87 mg, 80%).
General Method WC: Preparation of naphthyl hydroxy furanones
zx
Figure imgf000118_0001
R1 R2
Compounds such as Compound ZX can be prepared according to the preparation of Compound ZA described above using the appropriate acid coupling partner (57) along with the appropriate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. This method is analogous to Coupling Conditions C with obvious differences in protecting group strategies and is similar to General Methods N and K.
Preparation of Acid 57
Figure imgf000118_0002
116 (a) 5-Formyl-6-hydroxy-l-naphthoic acid
To a solution of 6-hydroxy-l-naphthoic acid (5.0 g, 37.6 mmol) in CH2C12 (60 mL) at 0 °C was added 1.0 M TiCl4 in CH2C12 (80 mL, 80 mmol) dropwise. After 5 min, Cl2CHOCH3 (10.16 g, 88.4 mmol, 8 mL) was added. The reaction mixture was then warmed to room temperature and stined overnight. The reaction mixture was cooled to 0 °C and quenched with 100 mL H2O, followed by 100 mL 1.0 M aqueous HCl. The resulting mixture was filtered, and the remaining solid was dissolved in 100 mL 1.0 M aqueous NaOH and filtered. The basic filtrate was acidified with 1.0 M aqueous HCl, and the resulting red solid was filtered and washed with 1.0 M aqueous HCl and Et2O to give the title compound.
(b) Methyl 5-formyl-6-hydroxy-l-naphthoate
To a solution of 5-formyl-6-hydroxy-l-naphthoic acid (1.89 g, 8.75 mmol) in THF (200 mL) and EtOH (200 mL) was slowly added CH2N2 in Et2O until no SM remained by HPLC. The excess CH2N2 was quenched with a small amount of AcOH, and the volatiles were then removed in vacuo. The resulting crude material was chromatographed on silica gel (85:15 hexanes :EtO Ac) to give the title compound.
(c) Methyl 5-formyl-6-r(trifluoromethyl)sulfonynoxy-l-naphthoate
To a solution of methyl 5-formyl-6-hydroxy-l-naphthoate (500 mg, 2.17 mmol) in CH2C12
(10.2 mL) at rt was added Et3N (483 mg, 4.78 mmol, 0.664 mL) followed by PhNTf2 (928 mg, 2.6 mmol). The mixture was stined for 18 h and then diluted with Et2O and washed with 1.0 M HCl. The extract was dried with MgSO4 and concentrated in vacuo.
The crude material was chromatographed on silica gel (20:1 hexanes/EtOAc), and the material thus obtained was recrystalhzed from hexanes to give 1.0 g of a 3:2 mixture of the title compound and PhNHTf (-90% yield).
(d) l-Methoxy-6-methoxycarbonylnaphthori.2-clfuran-3(lH)-one
To a solution of a 3:2 mixture of methyl 5-formyl-6-[(trifluoromethyl)sulfonyl]oxy-l- naphthoate (1.5 mmol ) and PhNHTf in DMSO (4.5 mL) at rt was added Pd2(dba)3-CHC13 (155 mg, 0.217 mmol), and MeOH (4.34 mL). CO gas was bubbled through the mixture for 3 min and then Et3N (0.522 mL, 3.75 mmol) was added. The mixture was then heated to 60 °C. CO gas was bubbled through the mixture for another 10 min, and the mixture was stined for an additional 30 min. The mixture was then poured into 1.0 M aqueous HCl and
117 extracted with EtOAc. The organic layer was dπed over MgSO4 and concentrated in vacuo The crude mateπal was chromatographed on silica gel (20:1 hexanes:EtOAc) to give the title compound.
(e) l-Hydroxy-6-hvdroxycarbonylnaphtho 1.2-clfuran-3(lH)-one
To a solution of l-methoxy-6-methoxycarbonylnaphtho[l,2-c]furan-3(lH)-one (160 mg, 0.588 mmol) in TΗF (10 mL) at rt was added 1.0 M aqueous LiOΗ (2 mL, 2 mmol). The mixture was st ed for 2 h, acidified with 1.0 M ΗC1 and extracted with EtOAc. The extract was dπed over MgSO4 and concentrated in vacuo. The resulting mateπal was earned into next reaction.
(f) 6-Ηydroxycarbonyl-l-methoxycarbonylnaphthoπ.2-clfuran-3(lH)-one (57)
To a solution of l-hydroxy-6-hydroxycarbonylnaphtho[l,2-c]furan-3(lH)-one in dry MeOΗ (15 mL) was added TsOΗ (150 mg) and CΗ(OMe)3 (0.5 mL). The mixture was stined at reflux for 2 h. The mixture was then cooled to rt, diluted with saturated aqueous NaHCO3, acidified with AcOH and extracted with EtOAc. The extract was dπed over MgSO4 and concentrated in vacuo. The crude mateπal was chromatographed on silica gel (85:15:1 to 66:33:1 hexanes:EtOAc:AcOH) to give the title compound (57) (120 mg, 79% from (d)).
General Method WD: Preparation of naphthyl aldehydes
Figure imgf000120_0001
Figure imgf000120_0002
Compounds such as Compounds ZY and ZZ can be prepared according to the scheme below using the appropπate acid coupling partner along with the appropπate amme coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. This method is analogous to Coupling Conditions C and D with obvious differences m protecting group strategies and is similar to General Methods V and K.
118 HS(CH2)2SH •
H BF3.OEt2 EDC, HOBT
Figure imgf000121_0001
Figure imgf000121_0003
1 phosphorylate
(Coupling Conditions D) or alkylate
Figure imgf000121_0002
(General Method W)
2 standard deprotection conditions
Figure imgf000121_0004
General Method WDl: Preparation of naphthyl and indole phosphates
H2θ3p ^
Figure imgf000121_0005
. m o ZZ2 H O
ZZ1 R1 R2 R1 R2
Compounds such as ZZl and ZZ2 can be prepared according to General Method WD substituting the appropπate hydroxy acid starting mateπal.
62. Compound ZZ3
Figure imgf000121_0006
Compounds such as Compound ZZ3 can be prepared according to General Method ZA. Electrospray Mass Spectrum (50/50 acetonitπle/water) m/z 508 (M-H).
General Method WE: Preparation of succinic acid derivatives
Figure imgf000121_0007
ZZA
R1 R2
119 Compounds such as Compounds ZZA can be prepared according to the scheme below using the appropriate acid coupling partner along with the appropriate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. This method is analogous to Coupling Conditions C and D with obvious differences in protecting group strategies and is similar to General Method WD.
I- *-
Figure imgf000122_0002
A /-butyl bromoacetate
2. BnOϋ
3. H,
115
Figure imgf000122_0003
61
1. standard H Q p phosphorylation 2 3 conditions
Figure imgf000122_0001
standard H „ standard coupling l /Sr^ ) m deP«ϊ**on c coonnddrtitmionnss I ϊ ,VV,m conditions
!-Bu02C
Figure imgf000122_0004
Preparation of Acid 61
Figure imgf000122_0005
In a typical procedure, methyl 3-(4-hydroxyphenyl)propionate (0.05 mol) was dissolved in 100 mL of methyl ethyl ketone along with BnBr (1.05 eq) and DIEA (2 eq). The reaction mixture was stined at reflux until all starting material was consumed, as observed by TLC (2 days). It was then quenched with H2O and concentrated under reduced pressure. The aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried (Na2SO- and concentrated under reduced pressure to afford 13.50 g of the benzylated product. This material was dissolved 100 mL of THF and LiOH (1.1 eq) was added as a solution in 100 mL of H2O at 0 °C. The reaction mixture was warmed to rt and stined for 2 h. It was then concentrated to remove all the volative solvent and acidified to pH 2 with 3 N HCl. The aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried (Na2SO4), and concentrated under reduced pressure to afford the acid. This material (12.70 g, 49.6 mmol) was suspended in 150 mL of CH2CI2, and oxalyl chloride was added (2 eq). After a catalytic amount of DMF (0.050 mL) was added, the reaction mixture was stined at rt for 3 h. It was then concentrated under reduced pressure and dried under high vacuum to afford to acid chloride. In a
120 separate, oven-dried flask, 8.8 g of (S)-(-)-(4)-benzyl-2-oxazolidinone (Aldrich) was dissolved in 300 mL of anhydrous THF and cooled to -78 °C. n-BuLi (1 eq, 2.5 M solution in hexanes) was added slowly at -78 °C over a period of about 10 min. The reaction mixture was stined at -78 °C for 20 min, and the freshly prepared 3-(4-benzyloxyphenyl)propionyl chloride was added as a solution in 200 mL of anhydrous THF. The reaction mixture was warmed to rt and stined for 1 h. It was then quenched with brine, and concentrated to remove most of the THF. The aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried (Na2SO- , and concentrated under reduced pressure to afford 18.3 g of the chiral auxiliary reagent. A portion of this reagent (8.0 g, 19.3 mmol) was dissolved in 300 mL of anhydrous THF and cooled to -78 °C. Next, LDA solution (1.2 eq) was added at -78 °C as a solution in 200 mL of THF. t-Butyl bromoacetate (3.4 mL, 1.2 eq) was added at -78 °C, and the reaction mixture was warmed slowly to -45 °C and stined at that temperature for 3 h. It was then cooled to -78 °C and quenched with 2 eq of HOAc (2.2 mL). The reaction mixture was warmed to rt, diluted with H2O and then concentrated to remove most of the THF. The aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried (Na2SO- , and concentrated under reduced pressure. Purification by chromatography (3:1 hexanes/EtOAc) afforded 8.70 g of the succinyl derivative. This material (8.7 g, 16.4 mmol) was dissolved in 100 mL of anhydrous THF and cooled to 0 °C. In a separate flask, benzyl alcohol (2.2 mL, 1.3 eq) was dissolved in 100 mL of anhydrous THF and cooled to 0 °C. π-BuLi (1.1 eq) was then added at 0 °C to this benzyl alcohol solution, and the resulting solution was stined at 0 °C for 30 min. This freshly prepared alkoxide solution was then added at 0 °C to the succinyl derivative above. After lh, the reaction was quenched with brine and concentrated to remove most of the THF. The aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried (Na2SO4), and concentrated under reduced pressure. Purification by chromatography (6:1 hexanes/EtOAc) afforded 6.10 g of the dibenzyl derivative.
A portion of this dibenzyl derivative (1.80 g) was dissolved in 50 mL of EtOAc. 10% Pd-C (50 mg) was added and the resulting reaction mixture was stined under 1 atm of H2 for 24 h. The reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure to afford 1.2 g of the desired acid (61). This acid could be subjected to the standard amide coupling, phosphorylation, and deprotection conditions (as in Coupling Conditions C and D) to afford the desired phosphate derivative.
62A. Compounds ZZB-ZZE
121
Figure imgf000124_0001
Compound Rl R9 MS
ZZB -CHT -CH2(C6H4)-4-CF3 556 (M-H)
ZZC -CH OH -CH2(C6H4)-4-CF3 574 (M+H)
ZZD -CHoOH -CH2(C6H4)-3,4-Cl2 574 (M+H)
Figure imgf000124_0005
ZZE -CHT -CH2(C6H4)-3.4-Cl2 556 (M-H)
General Method WEA: Preparation of succimamide derivatives
H203P
Figure imgf000124_0002
ZZM
R2N R * R2
Compounds such as Compounds ZZM (R = H or Me, for example) can be prepared according to the scheme below using the appropπate acid coupling partner along with the appropπate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G. for example. This method is analogous to Coupling Conditions C and D with obvious differences in protecting group strategies and is similar to General Method WE.
1 LDA
(-butyl bromoacetate
2. TFA
S.oxalyl chloπde: then RjNH
A BnOϋ
Figure imgf000124_0003
5 H2 " r Y0H
R-rAo °
1. standard phosphorylation typical amine conditions standard 2. standard coupling deprotection X \ R conditions r V '7m ZZM conditions \
Figure imgf000124_0004
Figure imgf000124_0006
R,r*r X^*O 0 R /' R2
122 General Method WF: Preparation of sulfone tyrosine derivatives
H-O3P
Figure imgf000125_0001
ZZE
A, sv, o
0 ° R1 R2
Compounds such as Compounds ZZE can be prepared according to the scheme below using the appropπate acid coupling partner along with the appropπate amme coupling partner, which can be prepared according to General Methods A, B, E, F and G. This method is analogous to Coupling Conditions C and D with obvious differences in protecting group strategies and is similar to General Method WE.
OMe
Figure imgf000125_0002
1 standard A s. o
Ht2O3P phosphorylation typical amine conditions standard 2 standard coupling deprotection conditions conditions
Figure imgf000125_0003
Figure imgf000125_0004
Preparation of Acid 62
62
Figure imgf000125_0005
In a typical procedure, methyl acetoacetate (2.3 mL, 21.5 mmol) was dissolved in 20 mL of MeOH and 1 eq of NaOMe (0.5 M in MeOH, Aldπch) was added. The reaction mixture was stined at rt for 15 mm and 4-benzyloxy benzyl chloπde was added The resulting reaction mixture was stmed at reflux for 4 h. It was cooled to rt, quenched with H2O and concentrated to remove most of the volatiles. The aqueous layer was extracted with EtOAc The combined organic layers were washed with bπne, dπed (Na2SO- and concentrated
123 Purification by chromatography (6: 1 hexanes/EtOAc) afforded 2.80 g of the alkylated product. This material was then dissolved in 30 mL of anhydrous THF and added at -78 °C to a freshly prepared solution of LDA in 30 mL of anhydrous THF. The reaction mixture was warmed to rt, and 1 g of paraformaldehyde was added. After stirring at rt for lh, the reaction mixture was heated to a reflux for 4 h. It was then cooled to rt and filtered. The filtrate was concentrated, and the residue was stined in 20 mL of saturated aqueous NaHCU3 for lh. The aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried (Na2SO- , and concentrated under reduced pressure. Purification by chromatography (neutral alumina, 3:1 hexanes/EtOAc) afforded 1.4 g of the alpha-beta unsaturated ester. This material was immediately dissolved in 50 mL of absolute ethanol and tert-butyl thiol (0.54 mL, 1 eq) was added. After 6 mg of NaH (60% by wt in mineral oil) was added, the reaction mixture was stined at rt for 15 h. The reaction mixture was diluted with 40 mL of H O and OXONE (8.8 g, 3 eq, Aldrich) was added at 0 °C. The reaction mixture was warmed to rt and stined for 15 h. It was then diluted with EtOAc and H2O and stined vigorously for 15 min. The mixture was filtered and the filtrate was concentrated. The resulting aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried (Na2SO4), and concentrated under reduced pressure. Purification by chromatography (Siθ2, 1:1 hexanes/EtOAc) afforded 900 mg of the sulfone derivative. This sulfone was dissolved in 20 mL of EtOAc and 20 mL of MeOH, and 50 mg of 10% Pd/C was added. The reaction mixture was hydrogenated under 1 atm of H2 at it for 4 h. It was then filtered through Celite. The filtrate was concentrated under reduced pressure, and the resulting residue was dissolved in 6 mL of 6N HCl and 2 mL of glacial acetic acid. The reaction mixture was heated at reflux for 15 h. It was then cooled to rt, diluted with H2O and extracted with EtOAc. The combined organic layers were washed with brine, dried (Na2SO- and concentrated under reduced pressure to afford 500 mg of the acid (62). This acid could be subjected to the standard amide coupling, phosphorylation, and deprotection to afford the desired phosphate derivative. The individual diastereomers could be separated by reverse-phase HPLC using a mixture of H2O/CH3CN containing 0.1% TFA.
62B. Compounds ZZG-ZZI
H203P
ZZE "^"AR9
Figure imgf000126_0001
124 Compound Rl R9 MS
ZZG -CH^ -(CH2)5CH3 560 (M+H)
ZZH -CH? -CH2(C6H4)-4-CF3 634 (M+H)
Figure imgf000127_0006
771 -(CH2)2CONH2 -CH2(C6H4)-4-CF3 689 (M-H)
General Method X: Preparation of phosphonate derivatives
P03H2
H2O-3
Figure imgf000127_0001
Y °
Compounds such as Compounds ZAA-ZAI can be prepared according to the preparation of Compound ZAA described below using the appropriate acid coupling partner along with the appropriate amine coupling partner, which can be prepared according to General Methods A, B. E, F and G, for example. This method is analogous to Coupling Conditions C with obvious differences in protecting group strategies. Use of the appropriate acylation reagent analogous to Method N provides R".
63. Compound ZAA
P03H2
H 2-O*--3.
ZAA
Figure imgf000127_0002
Preparation of acid 47
P03Et
Et-O.
1. Et2NH Et 1. Cs2C03, Mel Et,0. *-»
2. BoCjO 2. LiHMDS (EtO)2POCI 3. LiOH
Figure imgf000127_0003
Figure imgf000127_0004
Figure imgf000127_0005
125 (a) pfCHoPO^EtoVL-Phe-OH
Fmoc-p(CH PO3Et )-L-Phe-OH (5.0 g, 9.3 mmol) was dissolved in 170 mL of THF and 50 mL of diethyl amine, and the mixture was stined vigorously at rt for 3 h. Solvents were removed under reduced pressure, and the solid was resuspended in anhydrous ether, filtered, and dried on high vacuum to afford 2.8 g (94%) of p(CH2PO Et )-L-Phe-OH as white solid, which was used without purification in the next step.
(b) N-Boc-p(CH2PO3Et2)-L-Phe-OH (51)
To a solution of p(CH PO3Et )-L-Phe-OH (5.0 g, 16.7 mmol) in a 1: 1 mixture of DME/water (140 mL) at 0 °C was added NaHCO3 (3.1 g, 36.8 mmol) followed by Boc O (4.0 g, 18.4 mmol). The mixture was stined at 0 °C for 30 min, then warmed to rt and stined for 1 h. About 50 mL of DME was removed by evaporation, and then the remaining aqueous solution was washed with EtOAc (2 x 50 mL). The aqueous layer was brought to pH 4 with 1 N HCl and extracted with EtOAc (3 x 100 mL). The combined extracts were washed with water, dried over MgSO4 and concentrated to a colorless oil (51) (6.2 g, 90%). MS [M - H] 414.
(c) N-Boc-prCH PO2Et L-Phe-OMe
To a solution of N-Boc-p(CH2PO3Et )-L-Phe-OH (5.1 g, 12.2 mmol) in DMF (60 mL) was added Cs CO3 (4.8 g, 14.7 mmol) followed by Mel (0.76 mL, 12.2 mmol). The mixture was stined for 1 h, diluted with water (600 mL) and extracted with EtOAc (3 x 100 mL). The combined extracts were washed with water, 10% NaHSO , dried over MgSO4 and concentrated to a solid which was recrystalhzed from EtOAc/hexane to give a white solid (4.6 g, 88%). MS [M - H] 428. mp 104-105 °C.
(d) N-Boc-prCHrPO^Et ^-L-Phe-OMe
To a suspension of N-Boc-p(CH2PO Et )-L-Phe-OMe (7.0 g, 16.3 mmol) in 185 mL of anhydrous DME, purged with N2 and cooled to -42 °C (CH3CN/dry ice), was added dropwise lithium bis(trimethylsilyl)amide (1 M THF, 48.9 mL, 48.9 mmol). The reaction mixture was stined at -42 °C for 15 min. Diethyl chlorophosphate (4.7 mL. 32.6 mmol) was added, and the orange solution was stined at -42 °C for an additional 20 min before being quenched with 1 N HCl (20 mL). The mixture was further diluted with water and extracted with EtOAc (3 x 100 mL). The combined extracts were washed with water, dried
126 over MgSO4, concentrated, and chromatographed over silica gel (3% MeOH/CH2Cl2) to give a colorless oil (6.0 g, 65%). MS [M - H] 564.
(e) N-Boc-prCH(PO3Et2)2l-L-Phe-OH
To a solution of N-Boc-p[CH(PO3Et2)2]-L-Phe-OMe (0.490 g, 0.966 mmol) in 5 mL of THF cooled to 0 °C was added dropwise a solution of lithium hydroxide monohydrate (49.0 mg, 1.17 mmol) in 1.0 mL of water. The reaction mixture was stmed at 0 °C for 1 h. THF was removed under reduced pressure to give a yellow oil which was diluted with 10 mL of 1 N HCl. The aqueous phase was extracted with CH2C12 (8 x 15 mL), and the extracts were combined, dπed over Na SO4, and concentrated to afford 0.453 g (95%) of N-Boc-p[CH(PO3Et2) ]-L-Phe-OH (47) as a crystalline white solid. MS [M - H] 550. mp 84-87 °C
Preparation of Compound 48
CONH2
Et Et∑Os
S-* EDC-HCI, HOBT
Figure imgf000129_0001
27
Figure imgf000129_0002
(f) To 249 mg (0.451 mmol) of acid 47 in 10 mL of CH2C12 and 2.5 mL of DMF at RT was added a combined portion of 95 mg (0.497 mmol) of EDC-HCI and 76 mg (0.497 mmol) of HOBT. The resulting mixture was stined at RT for 10 mm, at which point 154 mg (0.497 mmol) of amine 27 (General Method E) was added in 1 portion.The resulting mixture was cooled to 0 °C. After stirπng at 0 °C for 2 h, an additional 0.3 eq each of EDC'HCl and HOBT were added, and the reaction mixture was stmed for 1.1 h at RT (additional reagent had no effect by TLC analysis). At this point, the reaction mixture was partially concentrated and then diluted with EtOAc and 5% NaHCO3. The organic layer was then washed with 10% citπc acid and bπne. The organic layer was then dried over Na2SO4 and concentrated. Flash chromatography (elution with 9:1 CH2Cb-MeOH) gave 342 mg (90.1%) of a pale foam: Rf 0.52 (9: 1 CH2Cl2-MeOH). Electrospray Mass Spectrum (50/50 MeCN/water) m/z 843 (M+H).
127 Preparation of Compound 49
P03Et2 P03Et2
CONH2 CONH?
Et20-. Et203
/ 1 TFA, CH2CI2 ) - 2 Acj-O, DIEA
Figure imgf000130_0002
r \ CH2CI2
Figure imgf000130_0001
48 A 49
(g) To a solution of 342 mg (0.406 mmol) of Compound 48 in 2.5 mL of CH2CI2 at RT was added 0.5 mL of TFA. The resulting mixture was stmed at RT for 2.3 h (additional TFA added at 1.25 h (0.2 mL) and 2 h (0.05 mL) as TLC analysis revealed the presence of starting mateπal) and then concentrated. The resulting residue was taken five times through a sequence of dissolving in a small amount of CH2CI2, concentration via a N2 stream and concentration in vacuo. The resulting final residue was pumped on overnight. Rf 0.10 (9: 1 CH2Cl2-MeOH). Electrospray Mass Spectrum (50/50 MeCN/water) m/z 743 (M+H).
(h) To the crude amme/TFA salt above m 4 mL of CH2CI2 at 0 °C was added 0.057 mL (0.609 mmol) of AC2O followed by 0.212 mL (1.22 mmol) of DIEA. The resulting mixture was stmed at 0 °C for 2.5 h, then diluted with CH2CI2 and washed with 10% NaHCO3 followed by IN HCl. Each of the washings was back extracted once with CH2CI2. The combined organic layers were then washed once with bπne and dπed over Na2SO4, filtered through glass wool and concentrated. An additional amount of product was isolated separately from saturating the combined aqueous layers with (NH4)2SO4 and extracting with CH2CI2. Flash chromatography (separately again, elution with 9:1 CH2Ch-MeOH) gave 249 mg (78.3% combined) of a pale foam: Rf 0.24 (9: 1 CH2Cl2-MeOH). Electrospray Mass Spectrum (50/50 MeCN/water) m/z 785 (M+H).
Preparation of Compound ZAA
EfeOs HzOs
TMSI
CH3CN
Figure imgf000130_0004
Figure imgf000130_0003
ZAA
(1) To 249 mg (0.317 mmol) of Compound 49 in 2.5 mL of CH3CN at -20 °C was added 0.9 mL (6.34 mmol) of TMSI. The resulting mixture was stined at -20 °C for 1.1 h whereupon an additional 0.2 mL of TMSI was added. The mixture was stmed an additional
128 15 min and quenched by the addition of 3 mL of 5% NaHCO3 followed by 3 mL of DMF and several crystals of solid Na2S2θ5 to turn the dark orange color to pale yellow. Reverse phase HPLC followed by lyophilization gave 110 mg (51.6%) of a fluffy white solid. Electrospray Mass Spectrum (50/50 MeCN/water) m z 673 (M+H).
64. Compound ZAB
Figure imgf000131_0001
ZAB
Compounds such as Compound ZAB can be prepared according to General Method X, exemplified by the procedure described for the preparation of compound ZAA with the appropriate amine coupling partner, which can be prepared according to General Method E. Reverse phase HPLC followed by lyophilization gave a fluffy white solid. Electrospray Mass Spectrum (50/50 MeCN/water) m/z 590 (M+H).
65. Compound ZAC
Figure imgf000131_0002
ZAC
Compounds such as Compound ZAC can be prepared directly from compounds such as Compound 48 according to the procedure for the preparation of Compound ZAA in General Method X. Reverse phase HPLC followed by lyophilization gave a fluffy white solid. Electrospray Mass Spectrum (50/50 MeCN/water) m/z 631 (M+H).
129 66. Compound ZAD
ZAD
Figure imgf000132_0001
Compounds such as Compound ZAD can be prepared according to General Method X, exemplified by the procedure described for the preparation of Compound ZAA, substituting the appropriate carboxylic acid coupling partner (50 - see below) with the appropπate amme coupling partner (27), which can be prepared according to General Method E. Reverse phase HPLC followed by lyophilization gave a fluffy white solid. Electrospray Mass Spectrum (50/50 MeCN/water) m/z 659 (M+H).
Preparation of acid coupling partner 50
1 socι2
MeOH 1 Tf2NPh, Et3N ». ».
2. Boc20 2. (EtO)2P(0)H NaHC03 4-Me-morpholιne
Pd(PPh3)4
3. LiOH
Figure imgf000132_0002
Figure imgf000132_0003
Figure imgf000132_0004
(a) r3-(3,4-(Dihydroxyphenyl)-L-alaninel methyl ester
To 3-(3,4-(Dihydroxyphenyl)-L-alanine (5 g, 25.4 mmol MeOH (40 mL) at 0 °C was added thionyl chloπde (3.8 mL, 50.7 mmol). The mixture was allowed to stir for 16 h at rt, and then the solvent was removed under reduced pressure. The resulting mixture was then diluted with water, and extracted with EtOAc. The organic layer was dried over magnesium sulfate and concentrated to a solid. The solid was recrystalhzed from ethyl acetate/hexane (5 g, 92%). MS [M + H] 212.
(b) r3-(3.4-(DihydroxyphenylVN-Boc-L-alanme1 methyl ester (118)
To [3-(3,4-(Dihydroxyphenyl)-L-alanine] methyl ester (5.4 g, 25.4 mmol) and di-tert-butyl dicarbonate (5.5 g, 25.4 mmol) in a mixture of THF (20 mL) and water (20 mL) at rt was added sodium bicarbonate (3.2 g, 38.1 mmol). The mixture was allowed to stir for 16 h and then washed with water and extracted with EtOAc. The organic layer was dried over magnesium sulfate and concentrated to a solid. The solid was recrystalhzed from ethyl acetate/hexane (7 g, 88%). MS [M + H] 312.
130 (c) \ 3-(3 ^-(DitrifluoromethanesulfonyloxyphenyD-N-Boc-L-alaninel methyl ester
To [3-(3,4-(Dihydroxyphenyl)-N-Boc-L-phenylalanine] methyl ester (118) (12 g, 38.6 mmol) and triethylamine (13 mL, 88.7 mmol) in methylene chloride (100 mL) at 0 °C was added N-phenyl-bis(trifluoromethanesulfonimide) (31.6 g, 88.7 mmol). The mixture was allowed to stir for two days and then washed sequentially with 1 N NaOH, 1 N HCl, and brine. The organic layer was dried over magnesium sulfate and concentrated to a solid. The solid was recrystalhzed frome dichloromethane/hexane. MS [M + Na] 598.
(d) 3.4-(Diethylphosphonyl)-N-Boc-L-phenylalanine methyl ester
To [3-(3,4-(Ditrifluoromethanesulfonyloxyphenyl)-N-Boc-L-alanine] methyl ester (2 g, 3.47 mmol), diethyl phosphite (1 mL, 7.65 mmol) and 4-methyl morpholine (0.93 mL, 8.3 mmol) in MeCN (10 mL) was added Pd(Ph3)4 (167 mg, 0.15 mmol). The mixture was allowed to stir for two days at 95 °C. It was then diluted with saturated NH4CI and extracted with EtOAc. The organic layer was dried over magnesium sulfate. concentrated, and chromatographed over silica gel (5% MeOH EtOAc) to an oil (0.2 g, 37% yield). MS [M + H] 552 and [M + Na] 574.
(e) 3.4-(Diethylphosphonyl)-N-Boc-L-phenylalanine
Acid 50 can be prepared from 3,4-(Diethylphosphonyl)-N-Boc-L-phenylalanine methyl ester with LiOH according to the method described above for the preparation of acid 47 as in General Method X-(e).
67. Compound ZAE
Figure imgf000133_0001
Compounds such as compound ZAE can be prepared according to General Method X, exemplified by the procedure described for the preparation of compounds ZAA and ZAD, substituting pivaloyl anhydride for acetic anhydride in the step analogous to that for the preparation of Compound 49 as in General Method X-(h).
131 68. Compounds ZAF and ZAE
Figure imgf000134_0001
Figure imgf000134_0002
ZAF
Alternatively, compounds such as Compound ZAE and Compound ZAF can be prepared according to General Method X, exemplified by the procedure described for the preparation of Compounds ZAA and ZAE with the appropriate modifications according to the scheme shown below. Reverse phase HPLC of either Compound ZAE or Compound ZAF followed by lyophilization gave a fluffy white solid. Compound ZAE: Electrospray Mass Spectrum (50/50 MeCN/water) m/z 701 (M+H). Compound ZAF: Electrospray Mass Spectrum (50/50 MeCN/water) m/z 683 (M+H).
Et,0-
E O-
Figure imgf000134_0003
K2C03 50 % H202
DMSO
Figure imgf000134_0004
Figure imgf000134_0005
69. Compound ZAG
Figure imgf000134_0006
ZAG
Compounds such as Compound ZAG can be prepared according to General Method X, exemplified by the procedure described for the preparation of Compound ZAA,
132 substituting the appropriate carboxylic acid coupling partner (50) with the appropπate amme coupling partner, which can be prepared according to General Method G Reverse phase HPLC followed by lyophilization gave a fluffy white solid Electrospray Mass Spectrum (50/50 MeCN/water) m/z 559 (M-H).
70. Compound ZAH
Figure imgf000135_0001
Compounds such as Compound ZAH can be prepared according to General Method X, exemplified by the procedure described for the preparation of Compound ZAA, substituting the appropπate carboxylic acid coupling partner (99) with the appropπate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example.
Preparation of acid coupling partner 99
Figure imgf000135_0002
(a) 2-Acetylamιno-3-(3-bromo-4-hydroxy-phenyl)-propιonιc acid methyl ester
To N-acetyl-L-tyrosine methyl ester (6 g, 25.3 mmol) in THF (60 mL) at it was added N- bromosuccinimide (5.4 g, 30.3 mmol) followed by five drops of sulfuπc acid. The mixture was stined for 16 h at rt. The solvent was removed under reduced pressure and then water was added. The aqueous layer was extracted twice with EtOAc, and the combined extracts were dπed over magnesium sulfate and concentrated to a solid. The solid was recrystalhzed from ethyl acetate/hexane (6 g, 75%). MS [M + H] 316
133 (b) 2-Acetylamιno-3-r3-("dιethoxy-phosphoryl)-4-hydroxy-phenyll-propιonιc acid methyl ester
To 2-acetylamιno-3-(3-bromo-4-hydroxy-phenyl)-propιonιc acid methyl ester (3 g, 9 mmol), diethyl phosphite (1.6 mL, 11 mmol) and 4-methylmorphohne (1 5 mL, 13 5 mmol) in toluene (10 mL) and MeCN (10 mL) was added Pd(Ph3)4 (0.5 g, 0.45 mmol) The mixture was allowed to stir for two days at 100 °C. It was then diluted with saturated NH C1 and extracted with EtOAc. The organic layer was dπed over magnesium sulfate. concentrated, and chromatographed over silica gel (5% MeOH/CHCl3) affording an oil. MS [M + H] 374 and [M - H] 372.
(c 2-Acetylamιno-3-[3-(dιethoxy-phosphoryl)-4-(dιethoxy-phosphoryloxy -phenyll- propionic acid methyl ester
To 2-Acetylarmno-3-[3-(dιethoxy-phosphoryl)-4-hydroxy-phenyl]-propιonιc acid methyl ester (0.15 g, 0.25 mmol) in MeCN (10 mL) was added diethyl chlorophosphate (0.05 mL, 0.3 mmol) followed by K2CO3 (0.07 g, 0.5 mmol) at rt. The reaction was stined for 4 h before H2O (10 mL) and EtOAc (20 mL) were added. The organic layer was dπed over magnesium sulfate, concentrated, and chromatographed over silica gel (20% MeOH/CHCl3) affording an oil. MS [M + H] 510.
(d) 2-Acetylamιno-3-r3-(dιethoxy-phosphoryl)-4-(dιethoxy-phosphoryloxy -phenvn- propionic acid (99)
Acid 99 can be prepared from 2-acetylamιno-3-[3-(dιethoxy-phosphoryl)-4-(dιethoxy- phosphoryloxy)-phenyl]-propιonιc acid methyl ester with LiOH according to the method descπbed above for the preparation of acid 47.
71. Compound ZAI
H203
Figure imgf000136_0001
Compounds such as Compound ZAI can be prepared according to General Method X, exemplified by the procedure described for the preparation of Compound ZAA , substituting the appropπate carboxylic acid coupling partner, 51 from General Method X-
134 (b), with the appropriate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. Electrospray Mass Spectrum (50/50 MeCN/water) m/z 647 (M+H).
General Method XA: Preparation of bisphosphate derivatives
H2o3pα
Y^ii
H203PCr ΛJ H
■ JJ- A q™
Figure imgf000137_0002
ZIT R" o 1
R1
Compounds such Compound ZIT can be prepared according to General Method X using the appropriate acid coupling partner (119) along with the appropriate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. This method is analogous to Coupling Conditions C with obvious differences in protecting group strategies. Use of the appropriate acylation reagent analogous to Method N provides R".
Preparation of Acid 119
1. CIP03Et2
Et203PC ;^\ K2C03 2. LiOH EtaOaPO-^^S
Figure imgf000137_0001
BocHN AyA>H
118 119
(a) 3-(S)-r3.4-Bis-(diethoxy-phosphoryloxy)-phenyll-2-tert-butoxycarbonylamino- propionic acid methyl ester
To 2-tert-Butoxycarbonylamino-(S)-3-(3,4-dihydroxy-phenyl)-propionic acid methyl ester (118) (2 g, 6.4 mmol) in MeCN (30 mL) was added diethyl chlorophosphate (1.1 mL, 7.7 mmol) followed by K2CO3 (3.5 g, 25.6 mmol) at rt. The reaction was stined for 8 h before H2O (50 mL) and EtOAc (50 mL) were added. The organic layer was dried over magnesium sulfate, concentrated, and chromatographed over silica gel (5% MeOH/CHCl3) affording an oil (2.3 g, 62%). MS [M + H] 584.
135 (c) 3-(Syr3.4-Bis-Cdiethoxy-phosphoryloxy -phenyπ-2-tert-butoxycarbonylamino- propionic acid (119)
The title compound (119) can be prepared from 3-(S)-[3,4-Bis-(diethoxy-phosphoryloxy)- phenyl]-2-tert-butoxycarbonylamino-propionic acid methyl ester with LiOH according to the method described previously for the preparation of acid 47 in General Method X-(h). The product was obtained as a colorless powder. Electrospray Mass Spectrum (50/50 acetonitrile/water + 0.1% ammonium hydroxide) MS [M + H] 673.
General Method XB: Preparation of other phosphate derivatives
H203P> H203P--^0
H
"^
Figure imgf000138_0001
H2O3PO 0
R ' R*** R R2 R1 R2
ZAT ZBT ZCT
Compounds such as Compounds ZAT-ZCT can be prepared according to the following scheme using the appropriate acid coupling partner along with the appropπate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. This method is analogous to Coupling Conditions C and D with obvious differences in protecting group strategies.
typical amme standard standard phosphorylation B
Figure imgf000138_0002
coupling
Figure imgf000138_0003
conditions m O conditions O H2θ3PO o
R1 R2 R2
1. typical amine, standard coupling
1. standard conditions H'jOaPO^^ protecting group 2. standard conditions phosphorylation or
OMe OH
Figure imgf000138_0004
2. ArCH2OH
Figure imgf000138_0005
alkylation i\
O Mitsunobu conditions A (Et203PCH2l)
O O
3. standard deprotection conditions w/0 o
R1 R2 conditions 3. standard
4. LiOH deprotection conditions H^Xy
136 General Method Y: Preparation of acetic acid phosphonate derivatives
P0 '33Hπ2
ZAJ
Figure imgf000139_0001
R R2
Compounds such as Compounds ZAJ can be prepared according to the scheme below using the appropπate acid coupling partner (63) along with the appropπate amme coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. This method is analogous to Coupling Conditions C and D with obvious differences in protecting group strategies and is similar to General Method WD.
I P03Et2 P03Et2
HC ^ CIPQ3E12 π-BuLl ■J chloramine-T HCL BnBr
A ? κ ,22co*-* :3 Nal
Figure imgf000139_0002
Figure imgf000139_0003
jf^ , CsCOs
Zn-I D --M^ _-O ■** BocHN on e o" 1. H2, Pd/C
Figure imgf000139_0004
(PhCN)2PdCI2, (o-tol)3P 2. LiOH
OMe
Figure imgf000139_0005
P03Et2
1 standard deprotection/ H02C typical amine acylation etc. conditions ^^^ standard 2. standard alkylation coupling conditions conditions 3. standard deprotection
Figure imgf000139_0006
conditions ZAJ
Figure imgf000139_0007
Figure imgf000139_0009
R1 R2 R,
Preparation of acid coupling partner 63
P03Et2
63
Figure imgf000139_0008
137 (a Phosphoric acid diethyl ester 2-iodo-phenyl ester
CIP03Et*-*
K2C03
Figure imgf000140_0001
Figure imgf000140_0002
To a mixture of 2-iodophenol (14.1 g, 64.1 mmol) and potassium carbonate (17.6 g, 128 mmol) in MeCN (100 mL) was added diethyl chlorophosphate (11.1 mL, 76.7 mmol). The mixture was allowed to stir at RT for 5 h. The solvent was removed under reduced pressure, and the residue diluted with water and extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was chromatographed over silica gel (elution with a stepwise gradient 25-40% EtOAc-hexanes) to give 21.7 g (95%) of a pale oil. Rf 0.47 (1:1 EtOAc-hexanes). Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 357 (M+H).
(b) (2-hydroxy-phenyl)-phosphonic acid diethyl ester
P03Et2 "-BuLi
Figure imgf000140_0003
Figure imgf000140_0004
(2-Hydroxy-phenyl)-phosphonic acid diethyl ester was prepared according to Casteel, D. A.; Peri, S. P. Synthesis 1991, 691.
To a cooled (-78 °C) solution of phosphoric acid diethyl ester 2-iodo-phenyl ester (21.7 g, 61.0 mmol) in dry THF (500 mL) under N2 was added a 2.5 M solution of rc-BuLi (40 mL, 100 mmol). After 20 min, the reaction was treated with satd aq NH4CI (50 mL) and allowed to warm to RT. The mixture was diluted with H2O (50 mL) and extracted twice with EtOAc. The organic extracts were pooled, washed with brine, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was chromatographed over silica gel (20% EtOAc-hexanes) to give 13.1 g (93%) of a pale oil. Rf 0.60 (1: 1 EtOAc- hexanes). Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 231 (M+H).
(c) (2-hvdroxy-5-iodo-phenyl)-phosphonic acid diethyl ester
°3Et2 chloramine ■-T HOv
Nal --L s-.
Figure imgf000140_0005
138 (2-Hydroxy-5-iodo-phenyl)-phosphonic acid diethyl ester was prepared according to Kometani, T.; Watt, D. S.; Ji, T. Tetrahedron Lett. 1985, 26, 691.
To a cooled (0 °C) mixture of (2-hydroxy-phenyl)-phosphonic acid diethyl ester (13.1 g, 56.9 mmol) and Nal (10.2 g, 68.3 mmol) in DMF (200 mL) was added chloramine-T trihydrate (19.2 g, 68.3 mmol) over 5 min. The ice bath was removed after 10 min, and the mixture was allowed to stir at RT for 2 h. The mixture was diluted with H2O (50 mL), acidified using 0.5 N HCl and extracted twice with EtOAc. The organic extracts were pooled and washed sequentially with satd. Na2S2θ3/brine (1/1) and then brine. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was chromatographed over silica gel (elution with a stepwise gradient 5-15% EtOAc-hexanes) to give 14.9 g (73%) of a colorless solid after recrystallization (EtOAc- hexanes). Rf 0.30 (20% EtOAc-hexanes). Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 357 (M+H).
(d) (2-benzyloxy-5-iodo-phenylVphosphonic acid diethyl ester
BnBr
Figure imgf000141_0001
CsCO
To a mixture of (2-hydroxy-5-iodo-phenyl)-phosphonic acid diethyl ester (14.7 g, 41.2 mmol) and CS2CO3 (17.4 g, 53.5 mmol) in DMF (150 mL) was added benzyl bromide (6.40 mL, 54 mmol). The reaction was allowed to stir for 2.5 days at RT at which point the reaction was concentrated under reduced pressure. The residue was diluted with 0.5 N HCl (30 mL) and extracted twice with EtOAc. The pooled organic extracts were washed with brine, dried over anhydrous Na2SU4 and concentrated under reduced pressure to give 17.4 g (91%) of a colorless solid after recrystallization (EtOAc-hexanes). Rf 0.36 (1:1 EtOAc- hexanes). Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 447 (M+H).
(e) 3-r4-benzyloxy-3-(diethoxy-phosphoryl)-phenyH-(SV2-tert-butoxycarbonylamino- propionic acid methyl ester
Zn-I
P03Et2 BocHN^V3Mθ s„ i o _ ^ (PhCN)2PdCI2, (o-tol)3P
OMe
Figure imgf000141_0003
139 To a mixture of Zn dust (403 mg, 6.17 mmol) in dry DMA (1 mL) and dry THF (1 mL) was added dibromoethane (0.055 mL, 0.64 mmol) and chlorotrimethylsilane (0.080 mL, 0.63 mmol). The mixture was sonicated for 15 min under N2. To this mixture was added a solution of Boc-iodoalanine-methyl ester (1.45 g, 4.41 mmol) in dry DMA (1 mL) and dry THF (1 mL). The mixture was sonicated for 30 min and then heated (65 °C) for 45 min under N2. To the heated mixture was added a solution (2-benzyloxy-5-iodo-phenyl)- phosphonic acid diethyl ester (1.37 g, 2.94 mmol), fos(benzonitrile)dichloropalladium (II) (67 mg, 0.17 mmol) and tri-σrt/io-tolylphosphine (148 mg, 0.486 mmol) in dry DMA (1 mL) and THF (1 mL) over 5 min. The mixture was allowed to stir for 3 h at 65 °C under N2 at which point it was allowed to cool to RT, diluted with 0.5 N HCl and extracted twice with Et2θ. The organic extracts were pooled, washed with brine, filtered through Celite, dried over anhydrous MgSO4 and concentrated under reduced pressure. An initial chromatographic purification over silica gel (elution with a stepwise gradient 15-40% acetone-hexanes) was followed by a second chromatographic purification over silica gel (70% EtOAc-hexanes) to give 958 mg (63%) of a pale oil. Rf 0.33 (80% EtOAc-hexanes). Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 520 (M-H).
(f) (S)-2-tert-butoxycarbonylamino-3-r3-(diethoxy-phosphorylV4-hydroxy-phenvπ- propionic acid methyl ester
P03Et2
^OMe
Figure imgf000142_0001
Figure imgf000142_0002
To a solution of 3-[4-benzyloxy-3-(diethoxy-phosphoryl)-phenyl]-(5)-2-tert- butoxycarbonylamino-propionic acid methyl ester (956 mg, 1.84 mmol) in MeOH (50 mL) was added 10% Pd/C (50 mg). The mixture was degassed under reduced pressure and allowed to stir at RT under H2 for 18 h. The catalyst was removed by filtration through Celite, and the filtrate concentrated under reduced pressure. The residue was chromatographed over silica gel (elution with 35% EtOAc-hexanes) to give 782 mg (99%) of a colorless oil. Rf 0.41 (1:1 EtOAc-hexanes). Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 432 (M+H).
140 (g) C5 -2-tert-butoxycarbonylamino-3-r3-(diethoxy-phosphoryl)-4-hvdroxy-phenyll- propionic acid (63)
P03Et2 03Et2
LiOH
Figure imgf000143_0001
Figure imgf000143_0002
To a cooled (0 °C) solution (S)-2-tert-butoxycarbonylamino-3-[3-(diethoxy-phosphoryl)-4- hydroxy-phenyl]-propionic acid methyl ester (423 mg, 0.98 mmol) in MeOH (4 mL) was added LiOH monohydrate (84 mg, 2.0 mmol) in H2O (4 mL). After 1 h, the ice bath was removed and the reaction was allowed to stir at RT for 1.5 h. The solution was carefully acidified to pH 2 using 0.5 N HCl and extracted twice with EtOAc. The organic extracts were pooled, washed with brine, dried over anhydrous Na2SO4 and concentrated to give 379 mg (91%) of a yellow foam. The crude material was canied on without further purification. Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 416 (M-H).
General Method Z: Preparation of O-suIfur and O-alkyI derivatives
H02C^O
ZAK
Figure imgf000143_0004
Figure imgf000143_0003
R,1 R2
Compounds with substitution other than phosphate such as Compounds ZAK and ZAL can be prepared according to General Method Y, substituting the appropriate carboxylic acid coupling partner (N-acetyl tyrosine, for example) with the appropriate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. O-derivatization can be performed via standard O-alkylation (as in General Method W) and O-sulfonamidation (as in General Method K), for example.
72. Compounds ZAM-ZAO
HQ2C-vO
Figure imgf000143_0005
141 Compound Rl R9 MS
ZAM -CH2-(3-indole) -(CH2)5CH3 574 (M-H)
ZAN -(CH2)2CO H -CH2Chx 531 (M+H)
Figure imgf000144_0006
|ZA0 -(CH2)2CO H -(CH2)5CH3 519 (M-H+H2O)
73. Compound ZAP
Figure imgf000144_0001
Compounds such as Compound ZAP can be prepared according to General Method Z. Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 555 (M-H).
General Method ZA: Preparation of hydrocinnamic acid phosphate and phosphonate analogs
H203P
Figure imgf000144_0002
Figure imgf000144_0003
R1 R2 R1 R2
Figure imgf000144_0004
Compounds such as Compounds ZAQ-ZAT can be prepared according to General Methods such as General Methods C, D, X and Y, for example, with obvious substitution of the appropriate 3-(4-hydroxyphenyl)propionic acid derivative as starting material.
74. Compound ZAQ1
Figure imgf000144_0005
Compounds such as Compound ZAQ1 can be prepared according to General Method ZA. Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 514 (M-H).
142 75. Compound ZAQ2
Figure imgf000145_0001
Compounds such as Compound ZAQ2 can be prepared according to General Method ZA. Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 499 (M-H).
General Method ZB: Preparation of hydrocinnamic acid phosphonate analogs
P03H2
H203R^O
ZAU
Figure imgf000145_0002
R1 R2
Compounds such as Compound ZAU can be prepared according to General Method ZA, substituting the appropriate acid (64) which can be prepared according to the scheme shown below.
ClPθ(θEt), 1 LDA
Figure imgf000145_0003
2 NHjCl
Figure imgf000145_0004
O Pθ3Et2
BrCH-,Pθ3(*-Pr)2 ,pr.0 ^, o. OMe CS2CO3
Figure imgf000145_0005
Figure imgf000145_0006
POjEtj
LιθH-H2θ '•Pr-OsP-.
OH
64
Figure imgf000145_0007
Preparation of acid coupling partner 64
Pθ3Et2
/-Pr2θ3P\
Figure imgf000145_0008
143 (a) S-^-Diethoxy-phosphoryl-oxyphenyl) propionic acid methyl ester
To methyl 3-(4-hydroxyphenyl) propionate (11 g, 61 mmol) in dry ether (250 mL) at 0 °C, was added NaH (1.8g, 75 mmol) portionwise. The reaction mixture was stined at room temperature for 1 h and then diethyl chlorophosphate (10.6 mL, 73 mmol) was added. Stirring was continued for another 2 h. The reaction was quenched by slow addition of water. The layers were separated and the aqueous layer was extracted with ether twice. The combined organic layers were washed with 4 N NaOH, brine and dried over MgSO4. Removal of the solvent resulted in the crude product as a colorless oil (19.0 g, 98%).
(b) 3-(3-Diethoxy-phosphoryl-4-hydroxyphenyl) propionic acid methyl ester (66)
To a solution of diisopropylamine (7.2 mL, 52.1 mmol) in dry THF (200 mL) at -78 °C was added n-BuLi (32.8 mL, 1.6 M in hexanes, 52.5 mmol) via syringe. The reaction was stined at -78 °C for 30 min. 3-(4-Diethoxy-phosphoryloxyphenyl) propionic acid methyl ester (4.66 g, 14.7 mmol) in dry THF (250 mL) was added via cannula. Stirring was continued at -78 °C for 1 h. The reaction was allowed to warm to room temperature and then quenched with saturated NH4C1 (50 mL). The mixture was extracted with ether 3 times, and the combined organic layers were dried over MgSO4 and concentrated. The residue was purified by flash column chromatography over silica gel (50% EtOAc/hexane, Rf = 0.43) to give product (66) as a clear oil (2.3 g, 50%).
(c) 3-f3-Diethoxyphosphoryl-4-diisopropoxyphosphorylmethoxyphenyl) propionic acid methyl ester
3-(3-Diethoxyphosphoryl-4-hydroxphenyl) propionic acid methyl ester (66) (316 mg, 1 mmol) was dissolved in 4 mL of DMF, and Cs2CO3 (429 mg, 1.32 mmol) was added, followed by diisopropyl bromomethylphosphonate (337 mg, 1.32 mmol). The reaction mixture was stined at 75 °C under N2 overnight. The reaction mixture was then partitioned between EtOAc and H2O and the organic layer was dried over Na2SO4 and concentrated. The residue was further purified by flash column chromatography over silica gel (10% MeOH/EtOAc, Rf = 0.45) to obtain the pure product as a clear oil (474 mg, 96%).
(d) 3-(3-Diethoxyphosphoryl-4-diisopropoxyphosphoryl-methoxyphenyl) propionic acid (64)
To a solution of 3-(3-Diethoxyphosphoryl-4-diisopropoxyphosphoryl-methoxyphenyl) propionic acid methyl ester (474 mg, 0.96 mmol) in 5 mL THF at 0 °C was added
144 LiOH»H2O (60 mg, 1.43 mmol) in 1 mL H2O. The reaction mixture was stined at 0 °C for 1 h. THF was removed in vacuo and 5 mL IN HCl was added. The aqueous phase was extracted with DCM (8 X 10 mL). The combined organics were dried over Na2SO4 and concentrated to yield crude 64 (400 mg, 87%) as a clear oil. Electrospray mass spectrum: m/z 479.50 (M-H).
General Method ZC: Preparation of hydrocinnamic acid phosphonate analogs
Figure imgf000147_0001
Compounds such as Compound ZAN can be prepared according to General Method ZA, substituting the appropriate acid (65) which can be prepared according to the scheme shown below.
P03Et, ^^SnBUa Pθ,E
HO- 3 ' PhΝ(Tf\ TfO. A -_ r
^T il ^
** ^ /OMe A^^,<Me Pd(PPh3)2Cl2 X ^-^ OMe π
66 o 67 I 0
Pθ3Efe POsEfe NaI04 OHc l NaBH3CN HO^γ*^j, CBr4* PPh3
RuCl3 ' ^-^O e ^^υMe 106 « O
POaEfe Pθ3Et, Pθ3E
1 3 2 LiθH-tt,θ
Et-03P- k ^-^OMe /^^O θ π Y II 0H
Figure imgf000147_0002
0 T 65 0
0
145 Preparation of acid coupling partner 65
Figure imgf000148_0001
(a) 3-f3-diethoxyphosphoiyl-4-tnfluoromethanesulfonyloxy-phenyl)-propiomc acid methyl ester (67)
3-(3-Dιethoxyphosphoryl-4-hydroxyphenyl)-propιomc acid methyl ester (66) (3.16 g, 10 mmol) and PhNTf2 (3.93 g, 11 mmol) were dissolved in 30 mL dry DCM. The mixture was cooled to 0 °C, and NEt3 (1.67 mL, 12 mmol) was added dropwise. The reaction mixture was stined at 0 °C for 1 h, and then slowly warmed to rt. The reaction mixture was diluted with 85 mL ether, and then washed with H2O, 1 N NaOH, H2O and bπne. The organic layer was dπed over MgSO and concentrated. The crude product was puπfied by flash column chromatograghy over silica gel (EtOAc/hexane 3.1. Rf = 0.38) to obtain an oil (3.41 g 78%).
(b) 3-(3-Dιethoxyphosphoryl-4-vmyl-pheny propionic acid methyl ester
3-(3-dιethoxyphosphoryl-4-tπfluoromethanesulfonyloxy-phenyl)-propιonιc acid methyl ester (67) (1 g, 2.23 mmol) was dissolved in dry dioxane (25 mL) together with vinyl tπbutyltin (0.67 mL, 2.29 mmol), LiCl (283 mg, 6.7 mmol), Pd(PPh3)2Cl2 (47 mg, 0.067 mmol) and a crystal of 2,6-dι-tert-butyl-4-methylphenol. The reaction mixture was degassed with argon, heated to 98 °C and allowed to stir for 2 h. The reaction mixture was then cooled to rt and diluted with excess ether and 10 mL saturated aqeous KF solution. The resulting mixture was stmed at rt overnight. The mixture was then filtered through Celite, and the layers were separated. The orgamc layer was washed with 1 N HCl and bπne, dπed over Na2SO and concentrated. The brownish oil was puπfied by flash column chromatography (EtOAc/hexane 90:10, Rf = 0.50) to give an oil (470 mg, 65%)
(c) 3-(3-Dιethoxyphosphoryl-4-formyl-phenyl') propionic acid methyl ester
3-(3-Dιethoxyphosphoryl-4-vιnyl-phenyl) propionic acid methyl ester (200 mg, 0.61 mmol) was dissolved m 3.9 mL CC14 and 3.9 mL of MeCN. A solution of NaIO4 (388 mg,
1.81 mmol) m 6 mL H2O was added. The mixture was stmed vigorously and RuCl3 (10 mg, 0.05 mmol) was then added. The mixture was stmed for 1 h at rt The reaction mixture
146 was diluted with DCM, and the organic layer was separated, dried over Na2SO and concentrated. The residue was purified by flash column chromatography over silica gel (4/1 EtOAc/hexane, Rf = 0.37) to give pure product as an oil (153 mg, 76%).
(d) 3-(3-Diethoxyphosphoryl-4-hydroxymethyl-phenyl) propionic acid methyl ester
3-(3-Diethoxyphosphoryl-4-formyl-phenyl) propionic acid methyl ester ( 328.3 mg, 1 mmol) and NaBH3CN (67.0 mg, 1.1 mmol) were dissolved in 3 mL MeOH. A trace of methyl orange was added, and 2 N HCl/MeOH was then added dropwise with stirring to maintain the red color. After ca. 15 min. the color changed very slowly. The stirring was continued for an additional 45 min when the solvent was evaporated. The residue was taken up in 3 mL of H2O, saturated with NaCl, and extracted with 3 mL (4 times) of ether. The combined organic layers were dried over MgSO4 and concentrated to give crude product (330 mg, 100%).
Ce") 3-(3-Diethoxyphosphoryl-4-bromomethyl-phenyl) propionic acid methyl ester
3-(3-Diethoxyphosphoryl-4-hydroxymethyl-phenyl) propionic acid methyl ester (330 mg, 1 mmol), PPh3 (289 mg, 1.1 mmol) and CBr4 (365 mg, 1.1 mmol) were dissolved in 5 mL dry THF. The reaction mixture was stined at 25 °C for 1 h. The mixture turned cloudy. The mixture was filtered, and the filtrate concentrated. The residue was purified by flash column chromatography over silica gel (EtOAC/hexane 3/1, Rf = 0.40). Pure product was obtained as an oil (150 mg, 38%).
(T) 3-G-Diethoxyphosphoryl-4-diethoxyphosphorylmethyl-phenyl) propionic acid methyl ester
3-(3-Diethoxyphosphoryl-4-bromomethyl-phenyl) propionic acid methyl ester (150 mg, 0.38 mmol) was dissolved in P(OEt)3 (2 mL, 11.4 mmol) and the reaction mixture was heated at 130 °C for 1 h. The volatile component was evaporated by N2 flow. The product was obtained as a clear oil (172 mg, 100%).
(g) 3-(3-Diethoxyphosphoryl-4-diethoxyphosphorylmethyl-phenyl) propionic acid (65)
This compound can be prepared according to the standard LiOH procedure described in General Method ZB (d).
147 General Method ZD: Preparation of hydrocinnamic acid phosphonate analogs l 03H2
Figure imgf000150_0001
Figure imgf000150_0002
R1 R2 R
Compounds such as Compounds ZAW-ZAY can be prepared according to General Method ZA with appropriate modifications as shown in the scheme below.
1. LiOH-HjO 1 LiOH-HjO
2. typical amine. 2 typical amine.
ZAX ZAW standard coupling "^ - ^ΥOMθ standard coupling conditions II
66 o conditions
3. Tf2NPh 3. standard
4. standard deprotecuon deprotection 1. L1OHΗ2O mediods methods 2. typical amine, standard coupling conditions
3. BrCH^CN
4 NaN3 "
5. standard deprotecUon
Figure imgf000150_0004
■ methods
ZAY
General Method ZE: Preparation of ureido phosphonates
P03H2 H 03P^ ^
^ H
Figure imgf000150_0003
k
ZBB O
R1 R2
Compounds such as Compounds ZB A and ZBB can be prepared according to General Methods M and MA, substituting the appropriate amines (69, 70), which can be prepared according to the scheme below.
148 -p03Et2 Et2θ3P^,Pθ3Et2 Et203P^P03Et2
1 ) LiN(SιMe3)2 JL NBS
2) ClPO(OEt)2 ^T (PhCθθ)2
E-aO-j ^ /POgE^
NaN3 .A*. Hl
Figure imgf000151_0002
Br
H° ^ NBS Et-OjPO- 1 n-BuLi
**Ϊ^ \
Figure imgf000151_0001
Figure imgf000151_0003
2 ClP(0)(0Et)2
Pθ3Et2 NBs EIA O Λ NaN3
Figure imgf000151_0005
Figure imgf000151_0004
(PhCOO)2 i^ ^Br 2. H1? Pd C
70
Preparation of amine 69 -P03Et2
Figure imgf000151_0006
(a) [(4-Methylphenyl)-(diethoxy-phosphorylVmethyl1-phosphonic acid diethyl ester
Diethyl 4-methylbenzylphosphonate (4.55 g, 18.8 mmol) was dissolved in 150 mL dry DME under N2 and cooled to -42 °C. LiHDMS (1.0 M in THF, 56.5 mL, 56.5 mmol) was added via syringe, and the mixture was stirred at -42 °C for 15 min. Diethyl chlorophosphate (5.5 mL, 37.7 mmol) was then added. The reaction was slowly warmed to 0 °C and stined for 20 min. The reaction was quenched by 4 N HCl and extracted with DCM (3 times 500 mL). The combined organic layers were dried over Na2SO and concentrated. The crude product was distilled using a Kugelrohr apparatus to give pure product as a clear oil (7.0 g, 98%).
(b) r(4-Bromomethylphenyl)-(diethoxy-phosphoryl)-methyll-phosphonic acid diethyl ester
[(4-Methylphenyl)-(diethoxy-phosphoryl)-methyl]-phosphonic acid diethyl ester (1.92 g, 5.1 mmol) was dissolved in 20 mL CC14 with NBS (1.0 g, 5.6 mmol) and benzoyl peroxide (48 mg, 0.2 mmol). The reaction mixture was refluxed for 2 h. The solid was
149 filtered off and the filtrate was concentrated. The residue was further purified by flash column chromatography over silica gel (EtOAc/MeOH 10:0.5, Rf = 0.41) to give 1.8 g (77%) of an oil.
(c) [f4-Azidomethylphenyl)-(diethoxy-phosphoryl)-methyl]-phosphonic acid diethyl ester
To 3.4 mL of a 0.5 M NaN3/DMSO solution (1.71 mmol) at rt was added [(4- bromomethylphenyl)-(diethoxy-phosphoryl)-methyl]-phosphonic acid diethyl ester (0.71 g, 1.55 mmol). The reaction mixture was stined at rt for 2 h and then quenched with 8 mL of H2O. The reaction was extracted with ether and the organic layer was dried over MgSO4. Evaporation of the solvent gave crude product (0.47 g, 72%).
(d) r("4-Aminomethylphenyl)-(diethoxy-phosphorylVmethyl1-phosphonic acid diethyl ester
[(4-Azidomethylphenyl)-(diethoxy-phosphoryl)-methyl]-phosphonic acid diethyl ester (1.02 g, 2.43 mmol) was dissolved in 15 mL EtOAc. Next, 120 mg Pd/C (10%) was added. The reaction mixture was hydrogenated under 50 psi H2 for 2 h. After removal of the solid, the solvent was removed to give 0.95 g (100%) of the title product (69).
Preparation of amine 70
P03Et2
Figure imgf000152_0001
NH
70
(e) 2-Bromo-4-methyl phenol
p-Cresol (5.4 g, 50 mmol) was dissolved in 120 mL of MeCN, and NBS (7 g, 39 mmol) was added. The reaction was stined at rt for 2 h. The precipitate was removed by filtration and the filtrate concentrated. The residue was further purified by flash column chromatography (DCM/hexane 1:3, Rf = 0.26) to give the title product as a clear oil (5.3 g, 72%).
150 (T) Phosphoric acid 2-bromo-4-methyl-phenyl ester diethyl ester
This compound can be prepared according to the method described in General Method ZB (a).
(g) 2-(Diethoxy-phosphoryloxy)-5-methyl-phenyn-phosphonic acid diethyl ester
Phosphoric acid 2-bromo-4-methyl-phenyl ester diethyl ester (646 mg, 2 mmol) was dissolved in dry ether (10 mL) at 0 °C. n-BuLi (1.6 M in hexane, 1.25 mL, 2 mmol) was added dropwise via syringe. The mixture was stined at 0 °C for 1 h, when diethyl chlorophosphate (0.35 mL, 2.4 mmol) was added. The reaction mixture was stined at rt overnight and then partitioned between EtOAc and H2O. The organic layer was separated, washed with 4 N NaOH and brine, and dried over Na2SO4. Removal of the solvent resulted in the crude product as a clear oil (0.71 g, 93%).
(h) |"2-(Diethoxy-phosphoryloxy)-5-bromomethyl-phenyll-phosphonic acid diethyl ester
This compound can be prepared according to the method described in General Method ZE (b).
(T) r2-(Diethoxy-phosphoryloxy)-5-azidomethyl-phenyl]-phosphonic acid diethyl ester
This compound can be prepared according to the method described in General Method ZE (c).
(j) f2-(Oiethoxy-phosphoiyloxyV5-aminomethyl-phenyl1-phosphonic acid diethyl ester
This compound can be prepared according to the method described in General Method ZE ( ).
151 General Method ZF: Preparation of acid phosphonate derivatives
C02H
H∑QjP^ ^
^K H .
■» 7m
ZBC R" 0 1
Figure imgf000154_0005
R1 \ R2 2
Compounds such as Compounds ZBC can be prepared according to the scheme below using the appropπate acid coupling partner (71) along with the appropπate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. This method is analogous to Coupling Conditions C and D with obvious differences m protecting group strategies.
Zn-I
Cθ2**Bu BocHN
°ABu I . NaH, DMF ζ .OBn
Et203l Et203F
2 Cul (PhCN)2PdCI2, (o-tol)3P 1 ,4 -Dnodobenzene 72
typical amine standard coupling conditions
Figure imgf000154_0002
deprotection/ etc. conditions H,0, deprotection conditions
R
Figure imgf000154_0003
Preparation of acid coupling partner 71
C02.-Bu
Et,0:
Figure imgf000154_0004
152 (a) Dιethoxy-phosphoryl-(4-ιodophenyl')acetιc acid t-butyl ester (72)
C02**Bu
To diethoxyphosphoryl acetic acid t-butyl ester (5.04 g, 20 mmol) DMF (25 mL) was added sodium hydπde (0.84 g, 60 mmol) in small portions under nitrogen. After 30 mm, a solution of 1,4-duodobenzene (3.3 g, 10 mmol) and copper(I) iodide (3.8 g, 10 mmol) in DMF (10 mL) was added, and nitrogen was bubbled through this solution for 10 mm. The tube was sealed and heated to 100 °C for 6 h. After cooling, the reaction mixture was poured into 10% hydrochloπc acid (200 mL). The solution was filtered through Celite and the Celite was repeatedly washed with ethyl acetate (3 x 50 mL). The ethyl acetate layer was separated and the aqueous solution was extracted with ethyl acetate (2 x 25 mL). The combined ethyl acetate extracts were washed with water (10 mL), dπed (Na2SO4) and concentrated to give a yellow gum, which was purified by column chromatography on silica gel. Elution with hexane/acetone (7/3) to gave a pale yellow gum which, solidified duπng storage in the freezer, mp 68 °C (3.28 g, 72%).
(b) 4-((t-butoxycarbonyl-dιethoxyphophonyl)-methyl-N-(t-butoxycarbonyl-S-phenyl alanine benzyl ester
Figure imgf000155_0001
Zinc (0 1438 g, 2.2 mmol) was covered with THF/DMA (2 mL) and heated to 60 °C. 1,2- dibromoethane (0.022 mL) was added and the flask was removed from the oil bath. TMS chloπde (0.030 mL) was added, and the mixture was sonicated at rt for 15 min after which it was again heated to 60 °C m an oil bath. N-t-butoxycarbonyl-2-ιodo-L-alamne benzyl ester (0.8105 g, 2 mmol) in THF/DMA (4 mL, 1/1) was added to the activated zinc at 60 °C and the reaction mixture was again sonicated for 30 mm after which it was heated to 60 °C After lh, a mixture of dιethoxy-phosphoryl)-(4-ιodophenyl)acetιc acid t-butyl ester (0.4542 g, 1 mmol) bιs(benzonιtπle) palladium (II) chloπde (21.86 mg, 0.057 mmol) and tπ-o- tolylphosphme (33.17 mg, 0.109 mmol) in THF/DMA (8 mL, 1/1) was added and the reaction mixture was heated to 60 °C for 1 h, then diluted with excess ethyl acetate (-200 mL) and IN HCl (20 mL) and the resulting mixture was filtered through Celite. The
153 organic layer was separated, washed (water, 10 mL) , dried (Na2SO ) and concentrated in vacuo. The resulting gum was purified by flash chromatography over silica gel using hexane/ethyl acetate (85/15) to give recovered starting material (120 mg, 22%), and the product (424 mg, 70%). mp 89 °C.
(c) 4-{ (t-butoxycarbonyl-diethoxyphophonyl)-methyl-N-(t-butoxycarbonyl)-S-phenyl alanine (71)
Figure imgf000156_0001
To 4- { (t-butoxycarbonyl-diethoxyphophonyl)-methyl-N-(t-butoxycarbonyl-S-pheny l alanine benzyl ester (420 mg, 0.695 mmol) was added ethyl acetate (45 mL) followed by 10% Pd/C (75 mg) carefully in an inert atmosphere. The flask was fitted with a balloon containing hydrogen and stined at rt for 5 h. The reaction mixture was filtered through a pad of Celite, which was washed with ethyl acetate (3 x 10 mL). The combined filtrate was concentrated to give 348.2 mg (97%). mp 138 °C.
General Method ZG: Preparation of hydrocinnamic acid derivatives
C02H
HaOgP^ r*^ ^ ^1 H - <([
ZBD
Figure imgf000156_0002
0 R1
Compounds such as Compounds ZBD can be prepared according to the scheme below using the appropriate acid coupling partner (73) along with the appropriate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. This method is analogous to Coupling Conditions C and D with obvious differences in protecting group strategies.
154 C02t*Bu » Et 2,0u3* Et203
Figure imgf000157_0001
Pd(PPh3)2CI2
72
Figure imgf000157_0002
Figure imgf000157_0004
C02**Bu standard typical amine H203 *- deprotection standard conditions coupling conditions
Figure imgf000157_0003
Figure imgf000157_0005
Preparation of acid coupling partner 73
C02/-Bu
Et O
Figure imgf000157_0006
(a) 3-(4-tert-butoxycarbonyl-(dιethoxyphosphonyl)-methyl-phenyl-)-acryhc acid benzyl ester
C02/-Bu
E α
Figure imgf000157_0007
To dιethoxy-phosphoryl-(4-ιodophenyl)acetιc acid t-butyl ester (72) (500 mg, 1 76 mmol) was added benzyl acrylate (589 mg, 3 52 mmol), Pd(PPh3)2Cl2 (81.5 mg, 0.106 mmol) followed by tπethylamme (0.740 mL, 5.28 mmol). This mixture was heated to 70 °C under nitrogen for 4 h. The reaction mixture was diluted with excess ethyl acetate and water and filtered through a pad of Celite. The ethyl acetate layer was separated, washed (water, 5 mL), dried (Na2SO4) and concentrated. The resulting gum was purified by column chromatography over silica gel using hexane/acetone (25%) to give a yellow liquid (449 mg, 65%) which is a mixture of E and Z isomers, which was used as such in the next step. Electrospray MS: 489 (M+H).
155 (b) 3-(4-tert-butoxycarbonyl-(dιethoxyphosphonyl)-methyl-phenyl-)propιonιc acid (73)
Figure imgf000158_0001
Title compound was hydrogenolyzed using the same conditions used m General Method ZF (c) above. Glassy gum. Electrospray MS: 401 (M+H)
General Method ZH: Preparation of hydroxy phosphonate derivatives
H-jO-P^ rl
H203P'' | H
ZUA Ύ R
Figure imgf000158_0002
R*-**-*S °Λ rfm Rι V
Compounds such as Compounds ZUA can be prepared according to the scheme below using the appropπate acid coupling partner (102) along with the appropπate amme coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. This method is analogous to Coupling Conditions C and D with obvious differences m protecting group strategies.
156 Zn-I .OMe
1 CIPO(OEt)2 BocHN ^ HC /^ K2C03, MeCN H BnBr
^ 2. π-Bul-i, THF
Figure imgf000159_0001
I Cs2C03 xx (PhCN)2PdCI2, (o-tol)3P
BnC /^.
^SnBu3
1 H2, Pd-C Et203f^^,
Me 2. Tf2NPh Me Pd(PPh3)2CI2
BocH 2. Nal04 RuCI3
Figure imgf000159_0005
Figure imgf000159_0002
1 HPO(OEt)2
DIEA typical amine Et
2. LiOH standard
OH coupling conditions
Figure imgf000159_0003
Figure imgf000159_0006
1 standard deprotection/ acylation etc. conditions
2 standard deprotection H conditions
Figure imgf000159_0004
General Method ZI: Preparation of aldehyde phosphonate derivatives
Figure imgf000159_0007
R1 R2
Compounds such as Compounds ZUB can be prepared according to the scheme below using the appropriate acid coupling partner (104) along with the appropriate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. This method is analogous to Coupling Conditions C and D with obvious differences in protecting group strategies.
157 typical amine standard coupling conditions
Figure imgf000160_0001
Figure imgf000160_0002
1 standard deprotection/ acylation etc conditions -.
2 standard deprotection conditions
Figure imgf000160_0004
,
Figure imgf000160_0003
R2
General Method ZJ: Preparation of hydrocinnamic acid derivatives
Figure imgf000160_0005
Compounds such as Compounds ZUC can be prepared according to the scheme below using the appropriate acid coupling partner (105) along with the appropπate amme coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. This method is analogous to Coupling Conditions C and D with obvious differences m protecting group strategies.
1 Mel, NaHC03 BnO
Figure imgf000160_0006
I 2 BnBr, Cs2C03 Me02C ^
111 t-Bl
1 Tf2NPH typical amine
2 HPO(OEt)2 standard
Pd(PPh3)4 coupling
3 TFA conditions
Figure imgf000160_0007
Figure imgf000160_0008
R R2 standard deprotection conditions
Figure imgf000160_0009
158 General Method ZK: Preparation of hydrocinnamic acid derivatives
Figure imgf000161_0001
Compounds such as Compounds ZUD can be prepared according to the scheme below using the appropπate acid coupling partner (107) along with the appropriate amme coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. This method is analogous to Coupling Conditions C and D with obvious differences in protecting group strategies.
1 HS(CH2)2SH BF-3'OEte typical amine
Et203 ^^ 2 LiOH standard ^OMe coupling conditions
106 o
Figure imgf000161_0003
1 standard deprotection/ acylation etc conditions
2 standard deprotection conditions
Figure imgf000161_0002
Figure imgf000161_0004
General Method ZL: Preparation of acid phosphonate derivatives
H02C
■ ^
H203P"' H N^ n?
ZUE
Figure imgf000161_0005
R" o A 1
R1 V
Compounds such as Compounds ZUE can be prepared according to the scheme below using the appropriate acid coupling partner (109) along with the appropπate amine coupling partner, which can be prepared according to General Methods A, B, E. F and G, for example. This method is analogous to Coupling Conditions C and D with obvious differences in protecting group strategies.
159 TfO 1. CO, dppp
•^ Pd(OAc)2 typical amine
Et3N, TMSE Et203
Me 2. LiOH standard
BocHN' coupling conditions
108
Figure imgf000162_0002
1. standard deprotection acylation etc. conditions
2. standard deprotection conditions
Figure imgf000162_0001
Figure imgf000162_0003
General Method ZM: Preparation of hydrocinnamic acid derivatives
Figure imgf000162_0004
Compounds such as Compounds ZUF can be prepared according to the scheme below using the appropriate acid coupling partner (120) along with the appropriate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. This method is analogous to Coupling Conditions C and D with obvious differences in protecting group strategies.
1. CO, dppp
Pd(OAc)2 typical amine
Et3N, TMSE 2. LiOH standard coupling conditions
Figure imgf000162_0005
Figure imgf000162_0006
1. standard deprotection/ acylation etc. conditions
2. standard deprotection conditions
Figure imgf000162_0007
Figure imgf000162_0008
160 General Method ZN: Preparation of acid phosphonate derivatives
H203 . ^il
H02C" 1 H
ZUG Y □
Figure imgf000163_0006
R 99Y 0 Rι V
Compounds such as Compounds ZUG can be prepared according to the scheme below using the appropriate acid coupling partner (110) along with the appropπate amme coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. This method is analogous to Coupling Conditions C and D with obvious differences in protecting group strategies.
,Zn-l
BocHN A jOMe
Bn HO- ^i) 1 Tf2NP
Figure imgf000163_0001
(P CN)2PdCI2, (σ-tol)3P Me02' 2 H2, Pd-C λ 2 HPO(OEt)2
Me W(PPf*3>
111 BocHN^ Y01 3 LiOH
Figure imgf000163_0002
1 standard deprotection/ typical amine acylation etc conditions » standard 2 standard deprotection coupling conditions conditions
Figure imgf000163_0003
Figure imgf000163_0004
R1 R2 R R2
General Method ZO: Preparation of aldehyde phosphonate derivatives
Figure imgf000163_0005
Compounds such as Compounds ZUH can be prepared according to the scheme below using the appropriate acid coupling partner (112) along with the appropriate amme coupling partner, which can be prepared according to General Methods A, B, E, F and G. for example. This method is analogous to Coupling Conditions C and D with obvious differences in protecting group strategies.
161 1 BF3-OEt2
HS(CH2)2SH
2 Tf2NPh
3 HPO(OEt)2
OMe Pd(PPh3)4
Figure imgf000164_0001
4 LlOH-H20
Λ°
Figure imgf000164_0002
see General Method V
standard typical amine deprotection standard conditions coupling B
Figure imgf000164_0004
m conditions
ZUH
Figure imgf000164_0003
A) ° Rl R2
General Method ZP: Preparation of hydrocinnamic acid derivatives
Figure imgf000164_0005
Compounds such as Compounds ZUl can be prepared according to the scheme below using the appropriate acid coupling partner (113) along with the appropπate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G, for example. This method is analogous to Coupling Conditions C and D with obvious differences in proecting group strategies.
1 Mel, K2C03 2 BF3-OEt2
CHCI, HS(CH2)2SH
NaOH, H20 3 Tf2NPh
4 HPO(OEt)2
Pd(PPh3)4
Figure imgf000164_0006
Figure imgf000164_0007
o o 5 LiOH
Figure imgf000164_0008
Jung, M E et al J Org Chem 1997, 62, 1553
typical amine standard standard deprotection coupling B conditions conditions
Figure imgf000164_0009
Figure imgf000164_0010
R1 R2
162 General Method ZQ: Preparation of cyano phosphonate derivatives
Figure imgf000165_0001
Compounds such as Compounds ZUJ can be prepared according to the scheme below using the appropπate acid coupling partner (114) along with the appropπate amme couphng partner, which can be prepared according to General Methods A, B, E, F and G, for example. This method is analogous to Coupling Conditions C and D with obvious differences m protecting group strategies.
1
2 Mel, 3 BnBr, OMe
Figure imgf000165_0002
Figure imgf000165_0004
1 Tf2NPh Et203
2 HPO(OEt)2 H .
Pd(PPh3)4
3 LiOH γ
114
R1 R2
1 standard acylation
2. standard
Figure imgf000165_0003
R1 R2
General Method ZR: Preparation of nitro or dinitro tyrosine derivatives
B
Figure imgf000165_0005
m
ZBE R99 O / \
R1 R2
Figure imgf000165_0006
Figure imgf000165_0008
R1
Figure imgf000165_0007
163 Compounds such as Compounds ZBE, ZBF, ZBG and ZBH (R95 = -PO3H2, -CH2CO2H -CH2PO3H2, for example) can be prepared according to Coupling Conditions C and D as shown in the scheme below (for the N-Ac dinitro example), using the appropriate acid coupling partner along with the appropriate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G. This method is similar to General Methods N and WD.
1. typical amine 1 amine protecting group deprotection
2. typical amine, 2. standard standard acylation etc. coupling conditions
Figure imgf000166_0001
conditions
Figure imgf000166_0002
1. standard alkylation or phosphorylation conditions
2. standard deprotection
Figure imgf000166_0003
methods
Figure imgf000166_0004
76. Compound ZBT
ZBT
Figure imgf000166_0005
Compounds such as Compound ZBT can be prepared according to General Method ZR. Electrospray Mass Spectrum (50/50 acetonitrile/water) m z 565 (M-H).
77. Compound ZBU
HO
Figure imgf000166_0006
ZBU
Compounds such as Compound ZBU can be prepared according to General Method ZR. Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 625 (M+H).
164 General Method ZS: Preparation of other phosphonate derivatives
P03H2
H20.
Figure imgf000167_0001
ZBI ZBJ N
Figure imgf000167_0002
R1 R-2 f03H2 f?03H2
H203F T*^ X= NH, O, !
H203 H
X H H203
ML AB
Figure imgf000167_0003
ZBL Λ^ R1 R'2 ZBM ft ZBN
R1 R2
Compounds such as Compounds ZBI-ZBN can be prepared according to General Method X using the appropriate acid coupling partner (74 - 79) along with the appropriate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G.
Preparation of Acid 74
1 - (EtO)3P 2. (Me3Si)2NLi F?03Et2 (EtO)2POCI
Et∑O-3
3. CO, Pd(0)
TMS(CH2)2OH
4. TBAF
Figure imgf000167_0004
Preparation of Acid 75
1. CS2C03, BnBr 2. CBr4, PPh3 1. H2 Pd(OH)2
HO Et, 20>->3.
3. (EtO)3P
Figure imgf000167_0005
2. BrCH2C0 H
OH
Figure imgf000167_0006
4. (Me3Si)2NLi OBn (EtO)2POCI 75 H
165 Preparation of Acid 76
1 Cs2C03 4-nιtrobenzyl
P03Et2 P03Et2 bromide 2 CBr4, PPh3 1 H*VPd(OH)2
HO' » Et 2O*-*.3 Et, 20ι-* 3r
Figure imgf000168_0001
2 Hg(OAc)2
SH 3 (EtO)3P 4 (Me3Sι)2NLι 3 BrCH2C0 H (EtO)2POCI Ar 76
O
Preparation of Acid 77
1 Cs2C03, BnBr
2 (Mθ3Sι)2N ι PQ3Et2 (EtO)2POCI
Figure imgf000168_0002
«.
3 (Mθ-jS aNLi
Figure imgf000168_0003
.OH (EtO)2POCI
4 H2/Pd(OH)2 77 O O
Preparation of Acid 78
(?03Et2 P03Et2
OBn X = NH, O, S
Et2o3p^γ:: j| Cl' Et,0, l^XH
Figure imgf000168_0004
CS2C03 2 H2 78 _^°H O O
Preparation of Acid 79
1 Tf2NPh, Et3N Et203 >.
2 (EtO)2P(0)H
Figure imgf000168_0005
4-Me-morpholιne
Figure imgf000168_0006
Pd(PPh3)4 3 LiOH 79
166 General Method ZT: Preparation of Misc. Acid analogs
Figure imgf000169_0001
Figure imgf000169_0002
R1 R2 R1 R2
Figure imgf000169_0003
Compounds such as Compounds ZBN, ZBO, ZBP and ZBQ can be prepared according to Coupling Conditions C and D using the appropπate acid couphng partner, which can be prepared according to literature methods (Burke, T. R., Jr. et al. Tetrahedron 1998, 54, 9981 and references cited therein) along with the appropπate amine coupling partner, which can be prepared according to General Methods A, B, E, F and G.
Inhibition of Binding to Src SH2:
The binding affinities of compounds of the present invention to Src SH2 can be determined according to the method of Lynch et al. PCT/US 97/06746. The following representative compounds of the present invention were determined to have an IC50 less than 50 micromolar. Compound A, Compound L, Compound V, Compound Z, Compound AB, Compound ZAA. Compound ZAB, Compound BA, Compound CP, Compound EF, Compound EU, Compound BF and Compound EW.
Inhibition of Binding to Zap-70 SH2:
The binding affinities of compounds of the present invention to Zap-70 SH2 can be determined according to the method of Lynch et al. PCT/US97/06746. The following representative compounds of the present invention were determined to have an IC50 less than 50 micromolar: Compound FH, Compound FW, Compound FY, Compound FE, Compound FAI, Compound FAW, Compound FAZ, Compound FBB, Compound OA, Compound OL, Compound OK, Compound ON, Compound OQ and Compound AE.
C « « «
167

Claims

Claims
1. A compound of the formula:
R 14
U m
R1 R' wherein Y is
R8 6 r
R 7-
RkJ-^f R& G. or R Ra
Figure imgf000170_0001
(a) (b) ( fcr (C)
G is -O-, -S- or -NR-
R° comprises -OR, -APO3RR , -OPO3RR , -ASO3R, -OSO3R, -ACO2R, -A- tetrazole, -ASO2NRR', -ACOCF3, -C(O)J, -C(R)(J)(K) or -C(Z)(J)(K);
where each occurrence of A is independently a covalent bond, -G-M- or -(M)m-;
each occuπence of M is an independently selected, substituted or unsubstituted, methylene moiety, and any M-M' moiety may be electronically saturated or unsaturated;
each n is independently 0, 1, 2, 3, 4 or 5;
each m is independently 0, 1 or 2;
J and K are independently -APO3RR , -OPO3RR , -ASO3R, -OSO3R, -ACO2R,
-A-tetrazole, -ASO2NRR', -(M) -NRR' or -(M) -OR;
Z is a halogen;
R7 and R8 are independently R, -CN, -NO2, Z, J, -A(M)naliphatic, -G(M)naliphatic ,
-(M) n COCF^ 3, -(M) n OH, -(M) n COOR, -A-(M) n NRR', -G(M) A NRR', -(M) n CHO,
-A(M)nN(R)(CO)R', -A(M)nN(R)(CO)GR', -G(M) N(R)(CO)R',
168 -G-(M) N(R)(CO)G'R', -A-(M)π*-CO-NRR', or -G(M)n-CO-NRR', where the aliphatic groups may be substituted or unsubstituted; or R 7 is a covalent bond to an R 4 substituent of X to form an aliphatic, aryl or heterocyclic πng of 4 to 8 atoms, additionally, R and R may be of the structure -(C=O)R so long as R is not alkenyl or alkynyl or B is not a thiazole
each occurrence of R (unnumbered) represents hydrogen or an aliphatic, heteroaliphatic, aryl, heteroaryl, (aryl)ahphatιc-, or (heteroaryl)alιphatιc- moiety, each of which (other than hydrogen) may be substituted or unsubstituted;
q is an integer from 1 to 8;
R is hydrogen, alιphatιc.-(M) -cycloaliphatic, -(M) -aryl, or -(M) -heterocyclic, each
2 of which, other than H, may be substituted or unsubstituted, and R is hydrogen or substituted or unsubstituted aliphatic;
or R and R are covalently linked together to form a πng;
or R or R are covalently linked either to B or a substituent of B to form a 4 - 10- membered, saturated or unsaturated, πng, or to the N depicted in Formula I above to form a 5, 6 or 7-membered, saturated or unsaturated, πng;
X is -(CR3R4) or -NR"
R3 is hydrogen, R(CO)NR'-, RR'N(CO)NR"-, R'SO NR-, R'CSNR- RR''NCSNR"-, RR''NSO2NR"-, R'OCONR-, RR'N-, or
< J NCONR—
169 R is hydrogen, aliphatic, cycloaliphatic-(M) -, aryl-(M) -, heterocyclic-(M) -, R-SO2Mn- , (RO-CO)(M)n- or (RR'N-CO)(M)n-, where the aliphatic, cycloaliphatic, aryl or heterocyclic moiety is substituted or unsubstituted;
B is -{HET]R9R10RnR12, where HET is a heterocyclic moiety;
R9, R10 and R11 are independently -(M)nZ, -(M)nR, -(M)nGR, -(M)nWR, or
-(M)nWGR;
R12 is independently selected from -(M) Z, -(M) R, -(M) GR, -(M) WR, and
-(M)nWGR;
R14 is R; and,
U and W are independently -CO-, -CS-, -M-, -SO-, or -SO2-;
or a pharmaceutically acceptable derivative thereof.
2. A compound of claim 1 of the formula:
514 R14 B a14 'Y or
OV
Figure imgf000172_0001
R'^2 O O R1 2
R1 XQ2
Figure imgf000172_0002
3. A compound of claim 2 containing a R moiety which is H.
4. A compound of claim 2 in which R and R are H.
5. A compound of claim 1 or 2 wherein n is 0, 1 or 2.
6. A compound of claim 1 wherein X is -CH(NH2)-.
170 A compound of claim 1 of the formula
Y RM Y R14 i n yyn ι n vym 0r i
Figure imgf000173_0001
0^S.R5O R1X R 2 0*f-R5θ R
O where R5 compπses a substituted or unsubstituted lower aliphatic or alkoxyl or is a substituted or unsubstituted -(M)n-aryl or -(M)n-heterocychc group.
8. A compound of claim 7 wherein R compπses -(M) CH , -(M) aryl, -(M)nheterocychc, -(M)nCN or -(M)nCOOR, where n is 0, 1, 2, 3, 4, or 5.
9 A compound of claim 7 wherein R is a methyl, ethyl, propyl, butyl, pentyl, hexyl, benzyl, aryl, heterocyclic, -(CH2)aryl or -(CH2)heterocychc moiety, which may be substituted or unsubstituted.
10. A compound of claim 7 wherein R compπses -(CH ) 11 CH J , -(CH )(CH A, ) II aryl,
-(CH2)(CH2)nheterocyclιc, -(CH2)(CH2)nCN, or -(CH2)(CH2)nCOOR, where n is 0, 1, 2, 3, 4. or 5.
11 A compound of claim 10 wherein R compπses -CH CN, -(CH COOR. -(CH2)2COOR, -(CH2)3COOR, -(CH2)4COOR, where R is H, lower alkyl or benzyl
12. A compound of claim 10 wherein R compπses -O-(substιtuted or unsubstituted lower alkyl or benzyl).
13. A compound of claim 1 of the formula
Figure imgf000173_0002
171 where R is hydrogen, substituted or unsubstituted aliphatic (which may be branched, unbranched or cyclic), substituted or unsubstituted aryl-(M)n-, substituted or unsubstituted heterocyclic-(M)n-, or (CO2R)(M)n-.
14. A compound of claim 13 wherein R4 is -(M)n(CO)OR, -(M) SO2R, -(M) (CO)NRR', or -(M) (tetrazole).
15. A compound of claim 14 wherein R4 is -CH2COOR, -CH SO2R, -CH2(CO)NRR', or -tetrazole.
16. A compound of claim 14 wherein each R and R' is independently H, lower alkyl or benzyl.
17. A compound of claim 1 of the formula
r R14
R^Ύ^8
where R is hydrogen, substituted or unsubstituted aliphatic (which may be branched, unbranched or cyclic), substituted or unsubstituted aryl-(M)n-, substituted or unsubstituted heterocyclic-(M)n- or (CO2R)(M)π-.
18. A compound of claim 17 wherein R is hydrogen.
19. A compound of claim 1 of the formula
R 14
N B
0 R
20. A compound of any of claims 7 - 12 wherein R and R' are H.
172
21. A compound of any of claims 1 - 20 wherein R14 is H.
22. A compound of any of claims 1 - 21, having the formula:
R 4
I A J /B
R1 R2
wherein R 1 is H, and R 2 comprises H, -(M) H, -(M) -(substituted or unsubstituted lower alkyl), -(M)n-(substituted or unsubstituted aryl), -(M)n-(substituted or unsubstituted heterocyclic), -(M) -COOR, or -(M) (CO)NRR'.
23. A compound of claim 22, wherein R is methyl, ethyl, i-propyl, n-propyl, n-butyl, isobutyl, n-amyl, sec-amyl, isoamyl, substituted benzyl, -CH2-(3-indolyl),
-CH2CH2COOR, -CH2CH2CONH2, -CH2COOR or -CH2CONH2.
24. A compound of claim 1-21, wherein R 1 and R 1 are independently selected, substituted or unsubstituted lower aliphatic groups, or R and R are covalently linked to each other to form a ring.
25. A compound of claim 1 - 21, having the formula
R1« R1'R2'
R1 R2 wherein each of R1, R1', R2, and R2' is independently selected from H, -(M)nH, -(M)n-(substituted or unsubstituted lower alkyl), -(M)n-(substituted or unsubstituted aryl), -(M)π-(substituted or unsubstituted heterocyclic), -(M)n-COOR and
-(M) (CO)NRR'.
26. A compound of claim 25, wherein each of R , R , R , and R is H.
173 1 7
27. A compound of claim 1 - 24, wherein at least one of R and R is methyl, ethyl, i-propyl, n-propyl, n-butyl, isobutyl, t-butyl, n-amyl, sec-amyl, isoamyl, substituted benzyl, -CH2-(3-indolyl), -CH2CH2COOR, -CH2CH2CONH2, -CH2COOR or
-CH-CONRR', or R 1 and R 2 are covalently linked to form a ring.
28. A compound of claim 25, wherein at least one of R1, R , R , and R2' is methyl, ethyl, i-propyl, n-propyl, n-butyl, isobutyl, t-butyl, n-amyl, sec-amyl, isoamyl, substituted benzyl, -CH2-(3-indolyl), -CH2CH2COOR, -CH2CH2CONH2, -CH2COOR or -CH2CONRR', or two of R1, R1', R2, and R2' are covalently linked to form a ring.
29. A compound of any of claims 1 - 28, wherein Y comprises
« A£ ι «& . ι "β3 "m
R° AA'A ."rrΛ ^ Ύ Y
Figure imgf000176_0001
30. A compound of claim 29 wherein
R6 comprises -OR, -APO3RR', -OPO3RR', -ASOJR, -OSO3R, -ACO^R, -A- tetrazole, -ASO2NRR\ -ACOCF3, -C(R)(J)(K) or -C(Z)(J)(K); and
R 7 and R 8 are independently H, -CN, -NO , halogen, J, -A-(M) aliphatic,
-G-(M)naliphatic, -(M)nCOCF3> -(M) OH, -(M)fiCOOR, -A-(M) NRR',
-G-(M)nNRR', -(M) CHO, -A-(M)nN(R)(CO)R', -G-(M)nN(R)(CO)R',
-A-(M) -CO-NRR', or -G-(M) -CO-NRR', where the aliphatic groups may be
substituted or unsubstituted; or R is a covalent bond to an R substituent of X to form an aliphatic, aryl or heterocyclic ring of 4 to 8 atoms.
31. A compound of claim 29 wherein
R6 comprises -OR, -AP03RR', -0P03RR', -ACO2R, -ACOCF3 or -C(R)(J)(K);
174 A comprises -CH2-, -OCH2-, -CF2-, -CHF- , -CHOH- or a covalent bond:
each R and R' is H, or substituted or unsubstituted lower alkyl or substituted or unsubstituted benzyl; and,
R 7 and R 8 are independently H, J, -A-(M) substituted or unsubstituted aliphatic,
-G-(M)nsubstituted or unsubstituted aliphatic, -(M)πCOCF3, -(M)nOH, -(M)nCOOR,
-A-(M)πNRR', -(M)nCHO, -A-(M)nN(R)(CO)R' or -A-(M)n-CO-NRR'.
32. A compound of claim 29 wherein R6 comprises -OH, -PO3RR', -OPO3RR', -CH2PO3RR', -CF2PO3RR', -OCH2CO2R, -NRCH2CO2R', -N(CH2CO R)2,
-CH2CO2R, -CF2CO2R, -CH2SO3R, -CF2SO3R, -CH2COCF3, -CF2COCF3, -CH(PO3RR')2, -CH(OH)(PO3RR'), -CH(NH2)(PO3RR'), -CH(CO2R)2,
-CF(CO2R)2, -CH(PO3RR')(CO2R"), -CH(PO3RR')(SO3R"), -CH(PO3RR')(SO2NH2), -CH(SO2NH2)2, or -CH(S03RR')2.
33. A compound of claim 32 in which one or more of R, R' and R" in the -PO RR ,
-OPO3RR', -CH2PO3RR', -CF2PO3RR', -OCH2CO2R, -NRCH2CO2R', -N(CH2CO2R)2, -CH2CO2R, -CF2CO2R, -CH2SO3R, -CF2SO3R, -CH2COCF3, -CF2COCF3, -CH(PO3RR')2, -CH(OH)(PO3RR'), -CH(NH2)(PO3RR'),
-CH(CO2R)2, -CF(CO2R)2, -CH(PO3RR')(CO2R"), -CH(PO3RR')(SO3R"), -CH(PO3RR')(SO2NH2), -CH(SO2NH2)2, or -CH(SO3RR')2 moiety is H.
34. A compound of claim 32 in which one or more of R, R' and R" in the -PO RR ,
-OPO3RR', -CH2PO3RR', -CF2PO3RR', -OCH2CO2R, -NRCH2CO2R', -N(CH2CO2R)2, -CH2CO2R, -CF2CO2R, -CH2SO3R, -CF2SO3R, -CH2COCF3,
-CF2COCF3, -CH(PO3RR')2, -CH(OH)(PO3RR'), -CH(NH2)(PO3RR'),
175 -CH(CO2R)2, -CH(PO3RR )(CO2R"), -CH(PO3RR )(SO3R"), -CH(PO3RR')(SO2NH2), -CH(SO2NH2)2, or -CH(SO3RR')2 moiety is -(M)mCH2Z,
-(M)mCHZ2, -(M) CZ , -R15, -M-O-CO-R15 or -M-O-CO-OR15, where Z is halogen and R is substituted or unsubstituted lower aliphatic, aryl or heterocyclic.
35. A compound of claim 34 in which R is methyl, ethyl, n-propyl, i-propyl, n-butyl, isobutyl, t-butyl, n-amyl, sec-amyl, benzyl or substituted benzyl.
36. A compound of claim 32 - 35 wherein R 7 and R 8 are H.
η
37. A compound of claim 32 - 35 wherein R is J, -A-(M) (substituted or unsubstituted aliphatic, aryl or heterocyclic), -G-(M)π(substituted or unsubstituted aliphatic, aryl or heterocyclic), -(M)nCOCF3> -(M) OH, -(M)nCOOR, -A-(M) NRR'',
-(M)nCHO, -A(M) N(R)(CO)R\ -A(M)πNRR' or -A-(M) -CO-NRR'; and R8 is H.
38. A compound of claim 32 - 35 wherein R is lower alkyl, lower alkenyl, -OH,
-NH2, -NO , -CN, -NHR, -NHCOR, -CHO, -CH2CHO, -PO3RR', -OPO3RR',
-CH2PO3RR', -CF2PO3RR', -OCH2CO2R, -NHCH2CO2R, -CH2CO2R, -CF2CO2R, -SO3R, -CH2SO3R, -CF2SO3R, -COCF3, -COCH2F, -COCF2H, -CF2COCF3 OR
-SO2NH2.
39. A compound of claim 38 in which one or both of R and R' in -PO RR ,
-OPO3RR', -CH2PO3RR', -CF2PO3RR', -OCH2CO2R, -NHCH2CO2R,
-CH2CO2R, -CF2CO2R, -SO3R, -CH2SO3R, or -CF2SO3R is H.
40. A compound of claim 38 in which one or both of R and R' in -PO.RR ,
-OPO RR', -CH PO RR', -CF PO RR', -OCH CO R, -NHCH CO R,
3 2 3 2 3 2 2 2 2
176 -CH*,CO2R, -CF2CO2R, -SO3R, -CH2SO3R, or -CF2SO3R is -(M)m-CH2Z,
-(M) m -CHZ„ 2, -(M) m -CZ„ 3 -R15, -M-O-CO-R15 or -M-O-CO-OR15, where Z is
halogen and R is substituted or unsubstituted lower aliphatic, aryl or heterocyclic.
41. A compound of claim 40 in which R is methyl, ethyl, n-propyl, i-propyl, n-butyl, isobutyl, t-butyl, n-amyl, sec-amyl, benzyl or substituted benzyl.
42. A compound of claim 29 wherein R 6 comprises -APO RR ' or -OPO RR ' and R 7 is
-A-(M)n- aliphatic or -G-(M)n- aliphatic, where the aliphatic moiety is substituted or unsubstituted.
43. A compound of claim 36 or 42 wherein R comprises -OPO H
44. A compound of claim 29 wherein R and R are independently selected from J and K.
45. A compound of claim 29 wherein R6 is -C(R)(J)(K).
46. A compound of claim 45 wherein R is H.
47. A compound of claim 45 or 46 wherein J is -PO RR
48. A compound of claim 47 in which one or both of R and R' are H.
49. A compound of claim 47 in which one or both of R and R' are R , -(M) -CH Z, -(M) -CHZ , -(M)m-CZ3, -M-O-CO-R15 or -M-O-CO-OR15, where Z is halogen
and R is substituted or unsubstituted lower aliphatic, aryl or heterocyclic.
50. A compound of claim 49 in which R is methyl, ethyl, n-propyl, i-propyl, n-butyl, isobutyl, t-butyl, n-amyl, sec-amyl, benzyl or substituted benzyl.
177
51. A compound of claim 1-50, comprising a moiety B selected from the group consisting of:
s-^R" ,0-/ s
Tt NT^R 10 ■i N^R f£ ^R10 ^ V^R"
Rs
.10
10 10
4 / r R A N- pH 0' R" J 2 S^T T -,v 1100 R1s0 «
,* * ■>ι ι .11
JLR11
10 -i
■^2 H ^ -_ 10 »
wherein each of R9, R10 and R11 is independently -(M)nZ, -(M)nR. -(M)nGR, -(M)nCN, -(M)nWR, or -(M)nWGR (including, e.g., halo, R, -OR, -SR, -NRR',
-COR,-CO2R, or -(M)nW-NRR'), and,
R12 is independently selected from -(M) Z, -(M) R, -(M) GR, -(M) WR, and -(M)nWGR.
52. A compound of claim 51 wherein one or more of the R, R' and R" groups contains at least one substituent selected from halo, hydroxy, aliphatic, amino, amido and sulfonamido moieties.
53 . A compound of claim 51 wherein one or more of R , R and R is a substituted aliphatic moiety containing at least one substituent selected from substituted or unsubstituted cycloaliphatic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -CO7R, -CO-NRR', -COR, and -OR.
54. A compound of claim 51, wherein one or more of R , R and R comprises
-(M)n(cycloaliphatic), -(M)n(substituted or unsubstituted aryl), -(M)n(substituted or unsubstituted heteroaryl), -(M)πCHO, -(M)nCONH2, -(M)πCSNH2, -(M)nSONH2,-(M)nSO2NRR\ -(M)nOR, -(M)n(lower aliphatic), -(M)n-C(OR)R'R", or -(M)n-C=CRR'.
178
55. A compound of claim 51, wherein one or more of R , R , R , and R comprises methyl, -(CH 3 13
1 ) q R 1 where q is 1-8 and R comprises methyl; i-propyl; i-butyl; t-butyl; cycloaliphatic; phenyl; substituted phenyl; naphthyl; substituted naphthyl; a 5, 6 or 7-membered heterocyclic ring or a bicyclic heterocylic moiety.
56. A compound of claim 1 of the formula:
Figure imgf000181_0001
c ° n
57. A compound of claim 56 in which R is H; a linear, branched or cyclic lower aliphatic; -(M)nOR, -(M)nCOOR, or -(M)πCONRR'.
58. A compound of claim 57 in which one or more R groups of R are H;
59. A compound of claim 56 - 58 in which n in R is 0, 1 or 2, and/or each M in R is CH-,.
60. A compound of any of claims 56 - 59 in which R1 is H, lower aliphatic (which may be branched, unbranched or cyclic), -(M)nOR, -(M)nCOOR, -(M)nCONRR\ -(M)naryl or -(M)nheterocyclic.
61. A compound of claim 60 in which each M of R is CH ; n is 1, 2 or 3; and/or one
or more R groups of R is H.
62. A compound of any of claims 56 - 61 in which R5 is a linear, branched or cyclic lower aliphatic moiety.
63. A compound of any of claims 56 - 62 in which R is H, -M -branched, unbranched or cyclic aliphatic, -(M)nOR, -(M)nCOOR, or -(M)nCONRR'.
179
64. A compound of claim 63 in which each M moiety in R 10 i.s CH,
65. A compound of claim 63 or 64 in which n in R 10 is 0, 1, 2 or 3.
66. A compound of claim 63 - 65 in which one or more R groups in R 10 is H
67. A compound of claim 56, selected from the set consisting of:
Figure imgf000182_0001
68. A compound of any of claims 56 - 62 in which R ,10 i.s -M -aryl or
-M-j-heterocyclic, where the aryl or heterocyclic moiety may be substituted or unsubstituted.
69. A compound of claim 68 in which each M of R is CH and/or n is 1, 2 or 3.
70. A compound of claim 68 selected from the set consisting of:
Figure imgf000182_0002
Figure imgf000182_0003
71. A compound of claim 1 of the formula:
180
Figure imgf000183_0002
I
Figure imgf000183_0001
5 o
72. A compound of claim 71 wherein R comprises a substituted or unsubstituted lower aliphatic moiety, which may be branched, unbranched or cyclic.
73. A compound of claim 71 or 72 wherein R is H, lower aliphatic (which may be branched, unbranched or cyclic), -(M)nOR, -(M)nCOOR, -(M)nCONRR' , -(M)naryl or -(M)nheterocyclic, wherein the aryl or heterocyclic moiety may be substituted or unsubstituted.
74. A compound of claim 73 in which each M of R is CH ; n is 1, 2 or 3: and/or one or more R groups of R is H.
75. A compound of claim 71 - 74 in which R comprises H or a substituted or unsubstituted lower aliphatic moiety.
76. A compound of claim 71 - 74, wherein R comprises a linear, branched or cyclic lower aliphatic; -(M)nCOOR; -<M)πCONRR'; or -(M)ncycloaliphatic, in which the aliphatic or cycloaliphatic moiety is substituted or unsubstituted.
a
77. A compound of claim 76 in which each M of R is CH ; and/or n is 0, 1, 2 or 3.
78. A compound of claim 71 , selected from the group consisting of:
181
Figure imgf000184_0001
79. A compound of claim 71 - 74, wherein R9 comprises -(M)naryl or -(M)nheterocyclic, in which the aryl and heterocyclic moiety may be substituted or unsubstituted.
9
80. A compound of claim 79 in which each M of R is CH ; and/or n is 0, 1, 2 or 3.
81. A compound of claim 80, wherein R^ is selected from the group consisting of:
CF*** .cι
Figure imgf000184_0002
Figure imgf000184_0003
rO "Br
Cl Br
Figure imgf000184_0005
Cl
Figure imgf000184_0007
CF3 Br
Figure imgf000184_0004
Cl
Figure imgf000184_0008
R Cl Br r^ 0R Ύ
Figure imgf000184_0006
Figure imgf000184_0009
Figure imgf000184_0010
Figure imgf000184_0013
Figure imgf000184_0012
m RZ = HC, ZorCf RR'N"" F3
Figure imgf000184_0014
Figure imgf000184_0011
82. A compound of claim 71, selected from the group consisting of:
182
Figure imgf000185_0001
83. A compound of claim 71, selected from the group consisting of:
Figure imgf000185_0002
*
84. A compound of any of claims 51 to 83, containing a Y moiety comprising:
or
Figure imgf000185_0003
Figure imgf000185_0004
where R6 comprises -PO3RR\ -OPO3RR',-MPO3RR', -SO2NRR', -OSO2NRR', -ACOoR, -A-tetrazole, or -CRJK.
183
85. A compound of claim 84, wherein R comprises -PO RR' .
86. A compound of claim 84, wherein R comprises -OPO RR'.
87. A compound of claim 84, wherein R comprises -CH PO RR'.
88. A compound of claim 84, wherein R comprises -CF PO RR' .
89. A compound of claim 84, wherein R comprises -OCH PO RR'.
90. A compound of claim 84, wherein R comprises -COOR or -tetrazole.
91. A compound of claim 84, wherein R comprises -OCH COOR or -OCH,tetrazole.
92. A compound of claim 84, wherein R comprises -CHJK or -CZJK.
93. A compound of claim 92, wherein J and K are independently selected from the group consisting of -PO3RR', -COOR, and -SO2NRR'.
7
94. A compound of any of claims 84 - 93 wherein R comprises R,-CN, -NO , halogen, J, -A-(M)naliphatic, -G-(M)naliphatic , -(M)nCOCF3, -(M)nOH,
-(M)nCOOR, -A-(M)πNRR', -G-(M)nNRR', -(M)nCHO, -A-(M)πN(R)(CO)R',
-G-(M)nN(R)(CO)R', -A-(M)n-CO-NRR', or -G-(M)π-CO-NRR', where the
aliphatic groups may be substituted or unsubstituted; or R 7 is a covalent bond to an R 4 substituent of X to form an aliphatic, aryl or heterocyclic ring of 4 to 8 atoms.
95. A compound of claim 94 wherein R comprises H, lower alkyl, lower alkenyl or -CHO.
184
96. A compound of claim 94 wherein R comprises J.
97. A compound of any of claims 84 to 94 wherein one or more R groups (including R', R", etc) of R6 or R7 comprise -(M) -CHjZ, -<M) -CHZ , -(M) -CZ , -R15,
-M-O-CO-R15 or -M-O-CO-OR15, where Z is halogen and R15 is a substituted or unsubstituted lower aliphatic, aryl or heterocyclic group.
98. A compound of any of claims 84 to 94 wherein one or more R groups (including R', R", etc) of R6 or R7 comprise H.
99. A compound of any of claims 1-98 which binds to a given SH2 domain with a IC50 value of less than about 50 μM.
100. A compound of claim 99, which binds to a given SH2 domain with a IC50 value of less than about 20 μM.
101. A compound of claim 99 wherein the SH2 domain is from a Src, Fyn, Lck, Yes, Blk, Lyn, Fgr, Hck, Yrk, ZAP-70, Syk, STAT or Abl protein.
102. A composition comprising a compound of any of claims 1-101 , or a pharmaceutically acceptable derivative thereof; and a pharmaceutically acceptable excipient.
103. A method for inhibiting SH2-mediated signal transduction in a mammal in need thereof which comprises administering to the mammal a composition of claim 102.
104. The method of claim 103, wherein the composition contains a compound which specifically binds to an SH2 domain of Src, ZAP-70, Syk, or STAT 6.
105. The method of claim 103, wherein said SH2-mediated signal transduction is mediated by a PDGF receptor protein, EGF receptor protein, HER2/Neu receptor protein, fibroblast growth factor receptor protein, focal adhesion kinase protein, pi 30 protein, or p68 protein.
185
106. The method of claim 103, wherein the mammal has a prohferative disease, cancer, restenosis, osteoporosis, inflammation, allergies, or cardiovascular disease.
107. A method of treating or preventing a prohferative disease, cancer, restenosis, osteoporosis, inflammation, allergic reaction, or cardiovascular disease in a patient, the method comprising administering to the patient a therapeutically effective amount of a composition of claim 102.
108. A method for inducing immunosuppression in a patient, the method comprising administering to the patient an amount of a composition of claim 102 sufficient to cause immunosuppression.
109. A compound of having a structure selected from the group consisting of: p9 R10
H2f ζ Rl0 • yCJ -, H2f^ζ R9
R1 R and R1 wherein each of R9 and R10 is independently -(M)--Z, ~(M)nR, -(M)nGR, -(M)nWR, or -(M)nWGR (including, e.g., halo, R, -OR, -SR, -NRR', -COR,-CO2R, or
-(M)nW-NRR').
186
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