WO2004103270A2 - Compounds and methods for treatment of thrombosis - Google Patents

Compounds and methods for treatment of thrombosis Download PDF

Info

Publication number
WO2004103270A2
WO2004103270A2 PCT/US2004/010349 US2004010349W WO2004103270A2 WO 2004103270 A2 WO2004103270 A2 WO 2004103270A2 US 2004010349 W US2004010349 W US 2004010349W WO 2004103270 A2 WO2004103270 A2 WO 2004103270A2
Authority
WO
WIPO (PCT)
Prior art keywords
group
alkyl
carbon atoms
compound
optionally substituted
Prior art date
Application number
PCT/US2004/010349
Other languages
French (fr)
Other versions
WO2004103270A3 (en
Inventor
Sherin S. Abdel-Meguid
Robert E. Babine
Hongfeng Deng
Lei Jin
Jian Lin
Scott R. Magee
Harold V. Meyers
Pramod Pandey
Michael J. Rynkiewicz
David T. Weaver
Original Assignee
Suntory Pharmaceutical Research Laboratories Llc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Suntory Pharmaceutical Research Laboratories Llc filed Critical Suntory Pharmaceutical Research Laboratories Llc
Publication of WO2004103270A2 publication Critical patent/WO2004103270A2/en
Publication of WO2004103270A3 publication Critical patent/WO2004103270A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Blood coagulation is the first line of defense against blood loss following injury.
  • the blood coagulation "cascade” involves a number of circulating serine protease zymogens, regulatory cofactors and inhibitors, as shown in Figure 1.
  • Each enzyme once generated from its zymogen, specifically cleaves the next zymogen in the cascade to produce an active protease. This process is repeated until finally thrombin cleaves the fibrinopeptides from fibrinogen to produce fibrin that polymerizes to form a blood clot.
  • efficient clotting limits the loss of blood at a site of trauma, it also poses the risk of systemic coagulation resulting in massive thrombosis.
  • hemostasis maintains a balance between clot formation (coagulation) and clot dissolution (fibrinolysis).
  • coagulation coagulation
  • clot dissolution clot dissolution
  • Cardiovascular diseases e.g., acute myocardial infarction, stroke, and pulmonary embolism
  • cardiovascular diseases disable or kill more people in the developed world than any other disease.
  • Over two million patients are hospitalized each year in the U.S. for acute arterial thrombosis and stroke.
  • the worldwide population for acute arterial antithrombotic therapy is five to six million, while over 25 million patients have chronic arterial thrombosis.
  • Over 10 million individuals are candidates for venous thrombosis therapy.
  • thrombin inhibitors The first generation of thrombin inhibitors to reach the clinic were polypeptides derived from natural sources, such as the potent anticoagulant, hirudin, which is a leech peptide. Potent, orally available, small molecule thrombin inhibitors have been discovered over the past few decades. Some of these are now in the clinic or are ready to be marketed. Efforts to develop potent Factor Xa inhibitors are not far behind. Targeting enzymes involved in propagation (e.g., hirudin) does not appear to be ideal since inhibitors of this phase of the coagulation cascade are associated with severe bleeding. This is further supported by findings that Factor V and Factor X deficiencies are associated with severe bleeding episodes.
  • the present invention features compounds (e.g., peptidomimetics and non-peptides) that inhibit Factor Xla and methods for preventing or treating undesired thrombosis by administering one or more of these compounds alone or in combination with other molecules to a mammal.
  • the invention also provides three-dimensional structures of Factor Xla and methods for designing or selecting additional Factor Xla inhibitors using these structures. Desirably, these compounds have certain structural, physical, and spatial characteristics that enable the compounds to interact with specific residues of the active site of Factor Xla.
  • the invention features a compound of formula I, or a pharmaceutically acceptable salt or prodrug thereof:
  • R 1 is selected from the group consisting of:
  • R 4 is H or C 1-6 alkyl
  • R D is H, C ]-6 alkyl, OH, NH 2 , NO 2 , CO 2 R 3a
  • R 5a is C 1-6 alkyl, or R 5 taken together with R 4 forms a 5- or 6-membered ring
  • R 6 is H, OH, C ]-6 alkyl, or when taken together with R 4 , forms a 5- or 6- membered ring
  • R 7 is H, OH, SH, NH 2 , NO 2 , optionally substituted C 1-6 alkyl, halogen, or CF 3
  • Y is O, S, or NR 4
  • a is an integer from 0-5
  • b is an integer from 0-2, and Z is NR 5 or (H,H);
  • R 2 is H or an optionally substituted C 1-6 alkyl, C 2-6 alkenyl, C 3 . 8 cycloalkyl, C 7 . 16 aralkyl, C 8-16 aralkenyl, C 6 or C 10 aryl, C 2- heterocyclyl, C 2 . 15 heteroaralkyl, or C 3-15 heteroaralkenyl, or when taken together with X and R 3 forms an optionally substituted C 6 aryl group or an optionally substituted C 3 -C 5 heteroaryl group;
  • X is C or N;
  • R 3 is H or optionally substituted C ]-6 alkyl, or together with X and A forms an optionally substituted C 3-5 heterocyclic ring, or together with X and R 2 forms an optionally substituted C 6 aryl group or an optionally substituted C 2 -C 5 heteroaryl group;
  • A is R 8 -AA 2 , where AA 2 is a covalent bond or a peptide chain of one to five natural or unnatural alpha-amino acid residues of D- or L- configuration, preferably one or two residues, where R 8 is H, C 2- acyl, C 7-11 aroyl, C 3-10 heteroaroyl, C 2- alkoxycarbonyl, C 4-9 cycloalkoxycarbonyl, C 8-17 aralkoxycarbonyl, C 7 or C ⁇ aryloxycarbonyl, aminocarbonyl, C 2-7 alkylaminocarbonyl, C 3-13 dialkylaminocarbonyl, C -9 cycloalkylaminocarbonyl, Cg- ⁇ aralkyl aminocarbonyl, C 7 or C arylaminocarbonyl, C 3-10 heterocyclylaminocarbonyl, C 3- ⁇ heteroaralkylaminocarbonyl, C 2-7 alkylthiocarbonyl, C 7- ⁇ aryl
  • R° is an optionally substituted C 1-6 alkyl, C 2-6 alkenyl, C 3-8 cycloalkyl, C 7-]6 aralkyl, C 8-16 aralkenyl, C 6 or C 10 aryl, C 1-9 heterocyclyl, C 2-15 hetero aralkyl, C 3- ⁇ 5 heteroaralkenyl, or C(O)R 9 , wherein R 9 is an optionally substituted C ⁇ _ 6 alkyl, C 2 ⁇ alkenyl, C ⁇ -8 cycloalkyl, C 7 . 16 aralkyl, C 8 .
  • desirable R° heterocyclyl substituents include 2-thiazole, 2-(4-methylthiazole), 2-(5- methylthiazole), 2-(4,5-dimethylthiazole), 2-(5-(2-hydroxyethyl)thiazole), or 2- N-methylimidazole.
  • R° is H, CHO, CN, or B(OR 9 ) 2 , wherein R 9 is H, C 1-6 alkyl, or taken together forms a C 2-4 cyclic boronate ester, or R° is an optionally substituted C ]-6 alkyl, C 2-6 alkenyl, C 3-8 cycloalkyl, C 7-16 aralkyl, C 8-16 aralkenyl, C 6 or Cio aryl, C ]-9 heterocyclyl, C 2- i 5 heteroaralkyl, or C 3-15 heteroaralkenyl, with the proviso that the amino acid of AA 2 that is covalently linked to X is not D-cyclohexylglycine when R 8 is C ]-6 alkylsulfonyl or C 7-16 aralkylsulfonyl.
  • R is H or a substituted C 1-6 alkyl, C 2-6 alkenyl, C 3-8 cycloalkyl, C 7- ⁇ 6 aralkyl, C 8- ⁇ 6 aralkenyl, C 6 or C 10 aryl, C 1-9 heterocyclyl, C 2-15 heteroaralkyl, or C 3- ⁇ 5 heteroaralkenyl;
  • X is N; a is an integer from 1-4; b is an integer from 0-2; AA 2 is a covalent bond and R 8 is B-Y 1 -(CH 2 ) n -K-C(O)-, wherein n is an integer from 0-6, K together with -C(O)-, X, and R forms an optionally substituted C 2-5 heterocyclyl group or K is a single bond, NR 9 , O, S, C(O), CH(OR 10 ), or -CH(NHR n ), wherein R 9 is H, OH, or C 1-6 al
  • dialkylaminocarbonyl C 4 . 9 cycloalkylaminocarbonyl, C 8-17 arallcylammocarbonyl, C 7 or C ⁇ arylaminocarbonyl, C 3 . 10 heterocyclylaminocarbonyl, C 3- ⁇ 6 heteroaralkylaminocarbonyl, C 2-7 alkylthiocarbonyl, C 7- ⁇ arylthiocarbonyl, C 3 .
  • R is H or C ]-6 alkyl
  • B is an optionally substituted C] -6 alkyl, C 2-6 alkenyl, C 3-8 cycloalkyl, C 6 or o aryl, C 1-9 heterocyclyl, C 7-16 aralkyl, C 8- ⁇ 6 aralkenyl, C 2- i 5 heteroaralkyl, C 3-15 heteroaralkenyl, C 1-6 alkylsulfonyl amino, and when Y 1 is O, S, or NR , and B is an optionally substituted C ]-6 alkyl, C 2-6 alkenyl, C 3-8 cycloalkyl, C 7- ⁇ 6 aralkyl, C 8-]6 aralkenyl, C 6 or C 10 aryl, C 1-9 heterocyclyl, C 2-15 heteroaralkyl, C 3-15 heteroaralkenyl,
  • R 3 is H or optionally substituted C 1-6 alkyl, or together with X and A forms an optionally substituted C 2-5 heterocyclic ring; and R° is -E-J, where when m is 0, E is a single bond to J, where J is H or an optionally substituted C ]-6 alkyl, and when m is 1 , E is selected from the group consisting of a single bond to J, -C(O)O-, -C(O)S-, and -C(O)NR 10 -, wherein R 10 is H or C ] -6 alkyl and J is an optionally substituted C ]-6 alkyl, C 2-6 alkenyl, C 3-8 cycloalkyl. C 7 .] 6 aral
  • R 8 is B-Y'-(CH 2 ) n -K-C(O)-, K is oxo or CHOH, n is 0, where Y 1 is a covalent bond and B is phenyl, 4- chlorophenyl, 3-chlorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl, 3- fluorophenyl, 4-fluorophenyl, 3,4-difluorophenyl, and 3,5-difluorophenyl.
  • AA 2 is a covalent bond and R is B-Y -(CH 2 ) ⁇ -K- C(O)-, where n is 0 and K, -C(O)-, X, and R 3 together forms a heterocyclyl group having the formula X:
  • R is hydrogen or an optionally substituted C )-6 alkyl, C 2-6 alkenyl, C 3-8 cycloalkyl, C 7-16 aralkyl, C 8-16 aralkenyl, C 6 or C ⁇ 0 aryl, C 1-9 heterocyclyl, C 2 - 15 heteroaralkyl, Q- ⁇ alkylthio, C 3-8 cycloalkylthio, C 6 or do arylthio, C 7 .] 6 aralkylthio, C 2-9 heterocyclylthio, C 2-15 heteroaralkylthio, C 1-6 alkylamino, C 2- ] 2 dialkylamino C 3-8 cycloalkyl amino, C 7-16 aralkylamino, C 6 or C 10 arylamino, or C 2-9 heterocyclylamino.
  • Y 1 is O, S, NR 18 , or a covalent bond, where R 18 is H or C 1-6 alkyl.
  • B is an optionally substituted C 1-6 alkyl, C 2-6 alkenyl, C 3-8 cycloalkyl, C 6 or C 10 aryl, C 1-9 heterocyclyl, C 7-16 aralkyl, C 8 . ]6 aralkenyl, C 2 . 15 heteroaralkyl, C 3- ⁇ 5 heteroaralkenyl, C 1-6 alkylsulfonyl amino, C 3 . 8 cycloalkylsulfonylamino, C 7 .
  • B is an optionally substituted C ] -6 alkyl, C 2-6 alkenyl, C 3-8 cycloalkyl, C 7-] 6 aralkyl, C 8-]6 aralkenyl, C 6 or C] 0 aryl, C 1-9 heterocyclyl, C 2- ⁇ 5 heteroaralkyl, C 3-15 heteroaralkenyl, C 2-7 acyl, C 7- ⁇ aroyl, C 3-]0 heteroaroyl, C 2-7 alkoxycarbonyl, C 4-9 cycloalkoxycarbonyl, C 8-17 aralkoxycarbonyl, C 7 or C ⁇ aryloxycarbonyl, heterocyclyloxycarbonyl, aminocarbonyl, C 2-7 alkylammocarbonyl, C 3-13 dialkylaminocarbonyl, C 4-9 cycloalkylaminocarbonyl, C 8-17 aralkylaminocarbonyl, C 7 or
  • aralkylaminothiocarbonyl C 7 or C ⁇ arylaminothiocarbonyl, C 3- ⁇ o heterocyclylaminothiocarbonyl, C 3- ⁇ 6 heteroaralkylaminothiocarbonyl, C 1-6 alkylsulfonyl, C 3-8 cycloalkylsulfonyl, C 7- 16 aralkylsulfonyl, C 6 or C ]0 arylsulfonyl, C 2-9 heterocyclylsulfonyl, or C 2-15 heteroaralkylsulfonyl.
  • Desirable compounds include those where m is 1 , R° is an optionally substituted 2-thiazole or 2-benzthiazole ring, R 2 is H or optionally substituted C 1-6 alkyl, R 17 is C 6 or C 10 aryl or C 1-9 heteroaryl, Y 1 is a bond, and B is an optionally substituted C ]-6 alkyl, C 7-16 aralkyl, C 6 or C ⁇ 0 aryl, C ]-9 heterocyclyl, or C 2- ⁇ 5 heterocyclylalkyl; those where m is 1, R° is an optionally substituted 2- thiazole or 2-benzthiazole ring, R is H or optionally substituted C 1-6 alkyl, R is C or C 10 aryl or .Q heteroaryl, Y 1 is NH, and B is C ]-9 heterocyclyl or C 2- ⁇ 5 heterocyclylalkyl; those where m is 1, R° is 2-thiazole, R 1 is 3-
  • R is H, R is C 6 or C 10 aryl, or C 1-9 heteroaryl, Y is NH, and B is C 1- heterocyclyl or C 2 . ⁇ 5 heterocyclylalkyl; those where Y 1 is O or S;
  • R is optionally substituted C 6 or C ⁇ 0 aryl, C].
  • Y 1 is NH
  • B is C 1- heterocyclyl or C 2-15 heterocyclylalkyl.
  • m is 1, X is N, R 3 is H, and R 1 is 3-guanidinopropyl, with the carbon bearing R 1 having an (R)- or (S)- configuration, and R 2 is selected from the group consisting of:
  • the carbon bearing any of these substituents in either the (R)- or (S)- configuration is of the (S)-configuration.
  • A is R 8 -AA 2 , with R 8 is selected from the group consisting of:
  • each of R 11 , R 12 , R 13 , and R 14 is, independently, C 2- alkanoyl, C 1-6 alkyl, C ]-6 alkenyl, C 1-6 alkoxy, C 2 .
  • arylalkyl wherein the alkylene group is of one to six carbon atoms, -(CH 2 ) q SO 2 R , wherein q is zero to four and R D is selected from the group consisting of (a) hydrogen, (b) C 1-6 alkyl, (c) C 6 or Cio aryl and (d) C 7-16 arylalkyl, wherein the alkylene group is of one to six carbon atoms, -(CH 2 ) q SO 2 NR E R F , wherein q is zero to four and R E and R F are independently selected from the group consisting of (a) hydrogen, (b) C ⁇ .
  • R G and R H are independently selected from the group consisting of (a) hydrogen, (b) an N-protecting group, (c) alkyl of one to six carbon atoms, (d) alkenyl of two to six carbon atoms, (e) alkynyl of two to six carbon atoms, (f) C 6 or do aryl, (g) C 7-16 arylalkyl, wherein the alkylene group is of one to six carbon atoms, (h) cycloalkyl of three to eight carbon atoms, and (i) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms, and the alkylene group is of one to ten
  • AA 2 is selected from the group consisting of:
  • AA 2 is L-3- -pyridylalanine, R ! is
  • AA 2 is
  • R 8 is selected from the group consisting of: ⁇ -toluenesulfonyl, (methyl-3- benzoate)methanesulfonyl, 2-nitro- ⁇ -toluenesulfonyl, 1-propanesulfonyl, 3- carboxyl- ⁇ -toluenesulfonyl, and 3-chloropropanesulfonyl.
  • A is selected from the group consisting of:
  • m is 1, R° is an optionally substituted 2-thiazole or 2-benzthiazole ring; R is H or optionally substituted C 1-6 alkyl, G is an optionally substituted C ⁇ profession 6 alkyl, aralkyl, or B-Y-, wherein Y is NH and B is C ⁇ -9 heterocyclyl or C 2-15 heteroaralkyl; and R 17 is C 6 or C 10 aryl, or d. 9 heteroaryl.
  • R 1 is selected from the group consisting of: , wherein a is an integer from 1-4, b is an integer from 0-2,
  • R 4 and R 5 are H or C ⁇ 6 alkyl
  • R 6 is H, OH, C 1-6 alkyl, or together with R 4 forms a 5- or 6-membered ring
  • R 7 is H, OH, SH, NH 2 , NO 2 , optionally substituted C ]-6 alkyl, halogen, or CF 3 ;
  • R is selected from the group consisting of:
  • X is N; R 3 is H; and A is
  • R 3a is H, C ⁇ -6 alkyl, or together with M forms a 5- or 6-membered ring
  • D is C(O), C(S), or S(O) 2
  • q is an integer from 0-4
  • Q is an optionally substituted C ]-6 alkyl, C 3-8 cycloalkyl, C 1-9 heterocyclyl, C 6 or C 10 aryl, C 1-6 alkyloxy, C 3-8 cycloalkyloxy, Q or C 10 aryloxy, C 2-9 heterocyclyloxy, C 1-6 alkylthio, C 3-8 cycloalkylthio, C 6 or C 10 arylthio, d.
  • n is 1, R° is an optionally
  • R is 3-guanidinopropyl
  • R is 2- methylpropyl
  • R 3a is H
  • D is S(O) 2
  • M is a single bond
  • q is 0,
  • Q is phenyl, cyclohexyl, cyclopentyl, 4-pyridyl, 3-pyridyl, or 2-pyridyl
  • U is selected from the group consisting of: benzyl, 3-methylcarboxybenzyl, 3- carboxybenzyl, 2-nitrobenzyl, 1 -propyl, and 3-chloropropyl.
  • m is 1, R° is an optionally substituted 2-thiazole or 2- benzthiazole ring, R 1 is 3-guanidinopropyl, R 3a is H, D is C(O), M is NR 3b , where R is H, q is 0, Q is selected from the group consisting of:
  • U is selected from the group consisting of: 2-chlorobenzyl, 3-chlorobenzyl, 4- chlorobenzyl, 2,3-dichlorobenzyl, 2,4-dichlorobenzyl, 2,5-dichlorobenzyl, 2,6- dichlorobenzyl, 3,4-dichlorobenzyl, 3,5-dichlorobenzyl, 2-fluorobenzyl, 3- fluorobenzyl, 4-fluorobenzyl, 2,3-difluorobenzyl, 2,4-difluorobenzyl, 2,5- difluorobenzyl, 2,6-difluorobenzyl, 3,4-difluorobenzyl, 3,5-difluorobenzyl, (R)- ⁇ -methyl-4-bromobenzyl, (S)- ⁇ -methyl-4-bromobenzyl, (R)- ⁇ -methyl-4-chlorobenzyl,
  • Preferred compounds include those where R 2 is isopropyl, Q is selected from the group consisting of: cyclohexyl, 2-pyridylmethyl, 3-ureidopropyl, 4-hydroxybenzyl, and acetamide, and U is (R)- ⁇ -methyl-4-bromobenzyl.
  • U is CHR 3c R 3d , wherein R 3c is an optionally substituted C 6 or C 10 aryl or C 1-9 heterocyclyl, and R 3d is H, C 1-6 alkyl, C2-6 alkenyl, or together with R 3c forms a fused bicyclic ring.
  • m is 0 and R° is H.
  • Preferred compounds include those where R is H or an optionally substituted C 1-6 alkyl, C 2-6 alkenyl, C 3-8 cycloalkyl, C 7- ⁇ 6 aralkyl, C 8-16 aralkenyl, C 6 or C 10 aryl, C 2-9 heterocyclyl, C 2- i 5 heteroaralkyl, or C 3 . ]5 heteroaralkenyl; R 3 is H or C].
  • X is N; and
  • A is R 8 -AA 2 , wherein AA 2 is a single natural or unnatural alpha-amino acid residue and R 8 is selected from the group consisting of:
  • each of R n , R 12 , R 13 , and R 14 is, independently, C 2-7 alkanoyl
  • C ⁇ _ 6 alkyl C 1-6 alkenyl, C 1-6 alkoxy, C . ⁇ 2 alkoxyalkyl, C ⁇ -6 alkylsulfinyl, C 2-12 alkylsulfinylalkyl, d -6 alkylsulfonyl, C 6 or C 10 aryl, C 7-16 arylalkyl, amino, C 1-6 aminoalkyl, C 7 or C ⁇ aryloyl, azido, C ]-6 azidoalkyl, carboxaldehyde, carboxamide, carboxyl, C 2-7 (carboxaldehyde)alkyl, C 3-8 cycloalkyl, C -14 cycloalkylalkyl, halo, C ]-6 haloalkyl, C ]-9 heterocyclyl, d.
  • (CH 2 ) q CONR B R c wherein q is zero to four and R B and R c are independently selected from the group consisting of (a) hydrogen, (b) C ]-6 alkyl, (c) C 6 or do aryl and (d) C 7-16 arylalkyl, wherein the alkylene group is of one to six carbon atoms, -(CH 2 ) q SO 2 R D , wherein q is zero to four and R D is selected from the group consisting of (a) hydrogen, (b) C ]-6 alkyl, (c) C 6 or C 10 aryl and (d) C -16 arylalkyl, wherein the alkylene group is of one to six carbon atoms, - (CH 2 ) q SO 2 NR E R F , wherein q is zero to four and R E and R F are independently selected from the group consisting of (a) hydrogen, (b) C 1-6 alkyl, (c) C 6 or C 10 aryl
  • each of R 15 and R 16 is, independently, H, d_ 6 alkyl; C 1-6 hydroxyalkyl; C 1- perfluoroalkyl, cyano, halo, -(CH 2 ) q CO 2 R A , -(CH 2 ) q CONR B R c , -(CH 2 ) q SO 2 R D , -(CH 2 ) q SO 2 NR E R F , - (CH 2 ) q NR G R H , wherein q is zero to two and R A , R B , R c , R D , R E , R F , R G , and R H are as defined above.
  • R 1 is selected from the group consisting of:
  • AA 2 is selected from the group consisting of:
  • R 2 is selected from the group consisting of:
  • R 8 is benzylsulfonyl, 3- methylcarboxybenzylsulfonyl, 3-carboxybenzylsulfonyl, 2-nitrobenzylsulfonyl, propylsulfonyl, and 3-chloropropylsulfonyl and AA 2 is as previously defined.
  • the invention features a compound of formula II:
  • R 19 is H, C 1-6 alkyl, OH, NH 2 , NO 2 , CO 2 R 19a , wherein R 19a is C ]-6 alkyl;
  • R 20 is H, OH, SH, NH 2 , NO 2 , optionally substituted C 1-6 alkyl, halogen, C 1-3 perfluoroalkyl, or when together with Z' forms a fused 5- or 6-membered ring;
  • e is N, NO, or CR 21a , wherein R 21a is H, halogen, or C 1-3 perfluoroalkyl;
  • g is N, NO, or CR 21 , wherein R 1b is H, halogen, C 1-6 alkyl, C 1-3 perfluoroalkyl, C 1-3 alkenyl, C 7 .
  • T is a bond, O, S, or NR 23 , where R 23 is H, OH, or optionally substituted
  • C 1-6 alkyl C 1-6 alkenyl, C 7 . n aralkyl, C 8-16 aralkenyl, C 2- ⁇ 5 heteroaralkyl, or C 3-15 heteroaralkenyl.
  • B is a natural or unnatural alpha-amino acid residue of D- or L-configuration, or an optionally substituted C ⁇ -6 alkyl, C 3-8 cycloalkyl, C ]-6 alkenyl, C 7-16 aralkyl, C 8-]7 aralkylcarbonyl, C 8-]6 aralkenyl, C 6 or C]o aryl, C 2- ⁇ 5 heteroaralkyl, C 3-16 heteroaralkylcarbonyl, C 3- ⁇ 5 heteroaralkenyl, C 2-7 acyl, C 4-9 cycloalkylcarbonyl, C 7- ⁇ aroyl, C 2-]0 heterocyclyl oyl, C -9 cycloalkoxycarbonyl, C 7 or C n aryloxycarbonyl, C 3-10 heterocyclyloxycarbonyl, C 3-16 heteroaralkyloxycarbonyl, aminocarbonyl, C 2-7 al
  • B is OH, SH, NH 2 , NO 2 , SO 3 H, CO 2 H or an optionally substituted C 1-6 alkyl, C 1-6 alkenyl, C 7- ⁇ 6 aralkyl, C 8- ⁇ 6 aralkenyl, C 2 . 15 heteroaralkyl, C 3 - 15 heteroaralkenyl, C 2 . 7 acyl, C 7- ⁇ aroyl, C 3-10 heteroaroyl, C 2-7 alkoxycarbonyl, C - cycloalkoxycarbonyl, Cg.
  • V is H or an optionally substituted C 1-6 alkyl, C 7- ⁇ 6 aralkyl, C 2 _ ⁇ 5 heteroaralkyl, C 2-7 acyl, C 7-n aroyl, C 3-]0 heteroaroyl, C 2-7 alkoxycarbonyl, C 4-9 cycloalkoxycarbonyl, C 8- ⁇ aralkoxycarbonyl, C 7 or C ⁇ aryloxycarbonyl, C 3 .] 6 heteroaralkyloxycarbonyl, C 2-1 o heterocyclyloxycarbonyl, aminocarbonyl, C 2- alkylaminocarbonyl, C 3 .] 3 dialkylaminocarbonyl, C 4-9 cycloalkylaminocarbonyl, C 8-17 aralkylaminocarbonyl, C 7 or C ⁇ arylaminocarbonyl, C 3- ⁇ 0 heterocyclylaminocarbonyl, C 3-] 6 heteroa
  • V is H, OH, SH, NH 2 , SO 3 H, CO 2 H, or an optionally substituted C 1-6 alkyl, hydroxy-C ]-6 alkyl, C 3-8 cycloalkyl, C 1-6 alkenyl, C -16 aralkyl, C 8 . ]6 aralkenyl, C 2-15 heteroaralkyl, C 3-15 heteroaralkyl, C 6 or C ]0 aryl, C]- heterocyclyl, C 2- alkylaminocarbonyl, C 3 .
  • aralkylaminosulfonyl C 6 or C ]0 arylaminosulfonyl, C ⁇ heterocyclylaminosulfonyl, d-isheteraralkylaminosulfonyl, C ]-6 alkylsulfinyl, C 3-8 cycloalkylsulfinyl, d. ⁇ aralkylsulfinyl, C 6 or C 10 arylsulfinyl, C 2 _ 9 heterocyclylsulfinyl, or C 2-15 heteroaralkylsulfinyl.
  • T is NH
  • B is an optionally substituted optionally substituted C 2-7 acyl, C -9 cycloalkylcarbonyl, C 7 or C n aroyl, C 2-10 heterocyclylcarbonyl, C 3 .] 0 heteroaroyl, C 2-7 alkoxycarbonyl, C 4 .
  • T is NH
  • B is an optionally substituted C 6 or Cio aroyl or an optionally substituted C ⁇ -9 heteroaroyl. Examples include those compounds in which B is
  • each of R 25 , R 26 ,and R 27 is, independently H, C 2-7 alkanoyl, C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkoxy, C 2-12 alkoxyalkyl, C ]-6 alkylsulfinyl, C 2-12 alkylsulfinylalkyl, C ]-6 alkylsulfonyl, C 6 or C ]0 aryl, C 7-] 6 arylalkyl, amino, d -6 aminoalkyl, C 7 or C n aryloyl, azido, d -6 azidoalkyl, carboxaldehyde, carboxamide, carboxyl, C 2- (carboxaldehyde)alkyl, d -8 cycloalkyl, C 4-]4 cycloalkylalkyl, halo, C 1-6 haloalkyl, C 1-9 heterocyclyl, C 1-9 (heterocyclyl)
  • Preferred compounds in which B is a 2,3-disubstituted or 2,3,4- trisubstituted aryloyl include those in which f is a bond and V is an optionally substituted C 1-6 alkyl, hydroxy-C 1-6 alkyl, C 3-8 cycloalkyl, C ] -6 alkenyl, C 7-] 6 aralkyl, C 8-16 aralkenyl, C 2- ⁇ 5 heteroaralkyl, C 3 .
  • alkylthiocarbonyl C 4-9 cycloalkylthiocarbonyl, C 7- ⁇ arylthiocarbonyl, C 8-17 aralkylthiocarbonyl, C 2-1 o heterocyclylthiocarbonyl, C 3 . ⁇ 6 heteroaralkylthiocarbonyl, C 2-7 acyl, C 7- ⁇ aroyl, C 3 . ⁇ 0 heteroaroyl, C 1 -6 alkylsulfonyl, C 3- cycloalkylsulfonyl, C 7-1 aralkylsulfonyl, C 6 or C 10 arylsulfonyl, C 2-9 heterocyclylsulfonyl, or C 2-15 heteroaralkylsulfonyl, d -6 alkylaminosulfonyl, C 3-8 cycloalkylaminosulfonyl, C 2-12 dialkylaminosulfonyl, C 7-17 aralky
  • aralkylsulfinyl C 6 or C 10 arylsulfinyl, C 2-9 heterocyclylsulfinyl, or C 2- ⁇ s heteroaralkylsulfinyl.
  • B is a 2,3-disubstituted or 2,3,4-trisubstituted aryloyl include those where f is a bond and V is optionally substituted C 6 or C ]0 arylaminocarbonyl or C 7- ⁇ 7 aralkylaminocarbonyl.
  • f is a bond and V is H.
  • examples include those compounds in which B is
  • each of R 25 , R 26 ,and R 27 is, independently H, C 2-7 alkanoyl, d -6 alkyl, C 1-6 alkenyl, C ! -6 alkoxy, d. ⁇ alkoxyalkyl, C 1-6 alkylsulfinyl, C 2-12 alkylsulfinylalkyl, C 1-6 alkylsulfonyl, C 6 or C ]0 aryl, C 7-]6 arylalkyl, amino, C ]-6 aminoalkyl, C or C aryloyl, azido, C 1-6 azidoalkyl, carboxaldehyde, carboxamide, carboxyl, C 2-7 (carboxaldehyde)alkyl, C 3 .g cycloalkyl, C 4-]4 cycloalkylalkyl, halo, C 1-6 haloalkyl, C 1-9 heterocyclyl, C 1-9 (heterocyclyl)
  • a compound of the invention can further be attached to a linker (e.g. to any of the groups R°, R 2 , or A of compounds of formula I or to any of the groups B or V of formula II), where the linker is reactive to a blood component, such as, for example, an erythrocyte, a lymphocyte, a blood platelet, an immunoglobulin, serum albumin, ferritin, corticosteroid-binding globulin, sex hormone-binding globulin, transferrin, thyroxin-binding protein, and alpha-2-macroglobulin.
  • a blood component such as, for example, an erythrocyte, a lymphocyte, a blood platelet, an immunoglobulin, serum albumin, ferritin, corticosteroid-binding globulin, sex hormone-binding globulin, transferrin, thyroxin-binding protein, and alpha-2-macroglobulin.
  • linkers attached to a group reactive to a blood component are maleimide- (CH 2 ) bb C(O)NHCH 2 CH 2 (OCH 2 CH 2 ) aa OCH 2 C(O)-, maleimide- (CH 2 ) bb C(O)NHCH 2 CH 2 (OCH 2 CH 2 ) aa NHCH 2 C(O)-, maleimide- (CH 2 ) b C(O)NHCH 2 CH 2 (OCH 2 CH 2 ) aa NHC(S)-, maleimide-(CH 2 ) bb NHC(S)-, maleimide-(CH 2 ) bb C(O)-, and maleimide (CH 2 ) b -where aa is 1-10 and bb is 1- 4.
  • a compound of the invention can further be attached to a polyethylene glycol moiety (e.g. to
  • polyethylene glycol moieties include H(OCH 2 CH 2 ) cc O-, H 3 C(OCH 2 CH 2 ) cc OC(O), H(OCH 2 CH 2 ) cc OC(O), H 3 C(OCH 2 CH 2 ) cc NHC(O), H(OCH 2 CH 2 ) Cc NHC(O), H 3 C(OCH 2 CH 2 ) cc NHC(S), H(OCH 2 CH 2 ) cc NHC(S), H 3 C(OCH 2 CH 2 ) Cc C(O), H(OCH 2 CH 2 ) cc C(O), H 3 C(OCH 2 CH 2 ) cc NHCH 2 C(O), H(OCH 2 CH 2 ) cc NHCH 2 C(O), H(OCH 2 CH 2 ) cc NHCH 2 C(O), H(OCH 2 CH 2 ) cc NHCH 2 C(O), H(OCH 2 CH 2 )
  • the invention features a pharmaceutical composition that includes a pharmaceutically acceptable carrier and a therapeutically effective amount of any compound of the invention, or a pharmaceutically acceptable salt or prodrug thereof.
  • the invention also features a method of treating a patient in need of thromboembolic disorder treatment that includes administering to the patient a therapeutically effective amount of any compound of the invention, or a pharmaceutically acceptable salt or prodrug thereof.
  • the thromboembolic disorder can be arterial cardiovascular thromboembolic disorders, venous cardiovascular thromboembolic disorders, and thromboembolic disorders in the chambers of the heart; including unstable angina, an acute coronary syndrome, first myocardial infarction, recurrent myocardial infarction, ischemic sudden death, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary arterial thrombosis, cerebral arterial thrombosis, cerebral embolism, kidney embolism, pulmonary embolism, and thrombosis resulting from (a) prosthetic valves or other implants, (b) ind
  • Factor Xla inhibitors are useful in the treatment and prevention of other diseases in which the generation of thrombin has been implicated as playing a physiologic role.
  • thrombin has been implicated in contributing to the morbidity and mortality of chronic and degenerative diseases such as cancer, arthritis, atherosclerosis, and Alzheimer's disease by its ability to regulate many different cell types through specific cleavage and activation of a cell surface thrombin receptor, mitogenic effects, diverse cellular functions such as cell proliferation, for example, abnormal proliferation of vascular cells resulting in restenosis or angiogenesis, release of PDGF, and DNA synthesis.
  • FXIcat residues are assigned numbers corresponding to those of trypsin, these numbers are shown in regular type and apply only to residues in the FXIcat.
  • FXIcat is assigned numbers corresponding to those of the native zymogen found in the bloodstream.
  • the initiator methionine is residue -18, and the protein begins with Glul (SEQ ID NO: 2). These numbers are shown in bold in the text.
  • the 18 amino acid signal sequence which is included in the translated sequence in Genbank, is removed co-translationally to yield the N-terminus of the mature enzyme.
  • Figure 2 shows the conversion between these two numbering systems.
  • the invention features a computer system that includes (a) a memory having stored therein data indicative of atomic coordinates in any one of the files listed in Figure 16, or surrogates thereof, of the non-hydrogen atoms of residues serine 195, histidine 57, and aspartic acid 102 of FXIcat or atomic coordinates that have a root mean square deviation from the backbone atoms of the residues of less than 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 A, and (b) a processor in electrical communication with the memory.
  • This processor includes a program for generating and displaying a three-dimensional model representative of FXIcat.
  • the stored data is x-ray diffraction data
  • the invention features a computer system that includes a memory having stored therein data indicative of a pharmacophore model of a Factor Xla ligand.
  • the pharmacophore model includes a three-dimensional model that includes 3 or more of the following: (i) an electrophilic carbon atom that binds to the side chain oxygen atom (OG) of Ser 195 such that the electrophilic carbon atom is 1.4-4.0 A (e.g., 1.8 to 3.5 A or 2 to 3 A) from the oxygen atom; (ii) at least one hydrogen bond donor group to interact with Asp 189 such that the non-hydrogen atoms of the hydrogen bond donor groups are within 2.0 to 4.2 A (e.g., 2.5 to 3.5 A or 2.8 to 3.2 A ) away from the side chain oxygens of Aspl89; (iii) a hydrophobic group that makes van der Waals contacts with residues of the active site and is less than 5.0
  • the invention features a computer readable memory having stored therein data indicative of a pharmacophore model of the invention or atomic coordintes of the invention.
  • Information from the three-dimensional structure of FXIcat can be used in a variety of methods for identifying, manufacturing, or evaluating Factor Xla ligands.
  • the invention includes a method of selecting or designing a candidate ligand for Factor Xla. This method involves generating in a computer a three-dimensional structure of FXIcat based on the atomic coordinates in one or more of the files listed in Figure 16, or surrogates thereof, of the non-hydrogen atoms of residues serine 195, histidine 57, and aspartic acid 102 or atomic coordinates that have a root mean square deviation from the backbone atoms of the residues of less than 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 A.
  • a candidate ligand that has sufficient surface complementary to the structure to bind Factor Xla (e.g., binding in an aqueous solution such as an aqueous solution at pH 7.0) is designed, selected or identified.
  • Surface complementarities can be assessed with a number of commercially available software packages, including, but not limited to, Insight II and Quanta (Accelrys Inc: P.O. Box 5350, Princeton, NJ 08543-5350, USA), DOCK (Gschwend, D.A. and LD. Kuntz, J. Comput-Aided Mol. Design, 1996) and
  • Designed ligands are then manufactured. If desired, the ability of any candidate ligand designed by the methods described herein to bind Factor Xla in vitro or in vivo can be optionally measured to confirm this binding and/or to compare the affinity of the ligand to other Factor Xla ligands. Compounds so prepared can also be used to determine their ability to inhibit Factor Xla, using inhibitition assays as described herein.
  • the invention provides a method for manufacturing a Factor Xla ligand.
  • This method includes crystallizing FXIcat or a mutant of FXIcat with a ligand and obtaining the atomic coordinates, or surrogates thereof, of at least a portion of FXIcat with at least a portion of the ligand.
  • One or more moieties in the ligand are selected for modification such that the modified ligand maintains the ability to bind Factor Xla.
  • the ligand is modified based on this determination. Such modifications include moieties known by those skilled in the art to improve aqueous solubility, improve pharmacodynamic properties, such as increased circulation half-life or cell membrane permeability. Modifications can also be made to increase ligant affinity to FXIcat, as described in more detail elsewhere herein.
  • the invention provides another method for manufacturing a Factor Xla ligand.
  • This method involves manufacturing a ligand that binds FXIcat and that is designed or selected based on information obtained using a model of the atomic coordinates, or surrogates thereof, of at least a portion of the three-dimensional structure of FXIcat.
  • the ligand is designed or selected using a computer system of the invention.
  • the method involves (i) using information from a model generated from the atomic coordinates, or surrogates thereof, of at least a portion of the three-dimensional structure of FXIcat and (ii) designing, selecting, and/or manufacturing a ligand for Factor Xla based on the information.
  • the invention features a method of evaluating the ability of a candidate ligand to bind Factor Xla.
  • This method involves (a) generating in a computer a three-dimensional structure of FXIcat based on the atomic coordinates in any one of the files listed in Figure 16, or surrogates thereof, of the non-hydrogen atoms of residues serine 195, histidine 57, and aspartic acid 102 or atomic coordinates that have a root mean square deviation from the backbone atoms of the residues of less than 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 A, and (b) employing a computational means to measure the interaction between the candidate ligand and FXIcat.
  • the invention provides a method of identifying a candidate ligand for Factor Xla.
  • This method includes generating a three- dimensional model of a pharmacophore model of a FXIcat ligand using a computer system of the invention, and selecting or designing a candidate ligand satisfying the criteria of the pharmacophore model.
  • the invention also provides methods for excluding a candidate compound from further drug development.
  • candidate compounds for indications other than excess or undesired thrombosis can be evaluated for their ability to inhibit Factor Xla.
  • candidate compounds that inhibit Factor Xla and that are being developed for other indications are desirably modified to reduce their affinity for Factor Xla or eliminated from further drug development.
  • the invention features a method of evaluating the ability of a candidate compound to bind Factor Xla.
  • This method involves (a) generating in a computer a three-dimensional structure of FXIcat based on the atomic coordinates in one or more of the files listed in Figure 16, or surrogates thereof, of the non-hydrogen atoms of residues serine 195, histidine 57, and aspartic acid 102 or atomic coordinates that have a root mean square deviation from the backbone atoms of the residues of less than 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 A, and (b) employing a computational means to measure the interaction between the candidate compound and FXIcat or to measure the surface complementarity between the candidate compound and FXIcat.
  • the candidate compound inhibits the activity of another protease (e.g., tryptase and elastase) and is either subsequently modified to reduce its affinity for Factor Xla or eliminated from further drug development.
  • another protease e.g., tryptase and elastase
  • the three-dimensional structural information of the present invention can be used to model the three-dimensional structure of other proteins, such as FXIcat from other mammals of laboratory, domestic or veterinary interest (e.g., rat, rabbit, mouse, cat, dog, cow, goat, pig, or sheep). Additionally, the structure of FXIcat can be used to model the structure of related proteins, such as other proteases with at least 30, 40, 50, 60, 70, 80, 90, 95, or 100% sequence identity to the catalytic domain of human Factor XI or to full-length human Factor XL Accordingly, in one such aspect, the invention features a method of generating a three-dimensional model of a FXIcat of interest.
  • other proteins such as FXIcat from other mammals of laboratory, domestic or veterinary interest (e.g., rat, rabbit, mouse, cat, dog, cow, goat, pig, or sheep).
  • the structure of FXIcat can be used to model the structure of related proteins, such as other proteases with at least 30, 40, 50
  • This method includes homology modeling using at least a portion of the atomic coordinates in any one of the files listed in Figure 16 (e.g., atomic coordinates of a structure of the invention), or surrogates thereof, and at least a portion of the amino acid sequence of the Factor XI of interest, thereby generating a model of the FXIcat of interest.
  • the invention features a method for experimentally determining the three-dimensional structure of the FXIcat of interest.
  • This method involves crystallizing a FXIcat of interest, generating an X-ray diffraction pattern from the crystallized FXIcat of interest, and (i) applying at least a portion of the atomic coordinates of the files listed in Figure 16 (e.g., atomic coordinates of a structure of the invention), or surrogates thereof, to the diffraction pattern to generate a three-dimensional electron density map of at least a portion of the FXIcat of interest, or (ii) utilizing de novo crystallographic methods such as isomo ⁇ hous replacement or MAD phasing to generate such an electron density map.
  • the three- dimensional structure also includes the hydrogen atoms of residues serine 195, histidine 57, and/or aspartic acid 102.
  • the three- dimensional structure further includes the non-hydrogen atoms, or both the non-hydrogen and hydrogen atoms, of one or more of the following residues Tyr5901, Ala97, Glu98, Aspl02, Leul46, Hisl74, Aspl 89, Alal90, Cysl91, Lysl92, Glyl93, Aspl94, Ser214, T ⁇ 215, Gly216, Glu217, and Gly218.
  • the three-dimensional structure further includes the non- hydrogen atoms, or both the non-hydrogen and hydrogen atoms, of one or more of the following residues: Arg3704, His38, Leu39, Cys40, Cys58, Tyr94, Gly 142, Tyr 143, Tyrl72, Cys219, Ala220, and Arg224.
  • the three-dimensional structure further includes the non- hydrogen atoms, or both the non-hydrogen and hydrogen atoms, of one or more of the following residues: Phe59, Ilel 5 l, Alal 83, Gly 196, Gly 197, Thr213, Pro225, Gly226, Val227, and Tyr228.
  • the structure further includes coordinates for the non-hydrogen or both the non-hydrogen and hydrogen atoms of one or more of the following residues: Arg3704, His38, Leu39, Cys40, His57, Cys58, Phe59, Tyr5901, Tyr94, Ala97, Glu98, Aspl02, Glyl42, Tyrl43, Leul46, Uel51, Tyrl72, Hisl 74, Alal83, Aspl 89, Alal90, Cysl91, Lysl92, Glyl93, Aspl94, Serl95, Glyl96, Glyl97, Thr213, Ser214, T ⁇ 215, Gly216, Glu217, Gly218, Cys219, Ala220, Arg224, Pro225, Gly226, Val227, and Tyr228, In desirable embodiments, the structure includes the coordinates for all of the residues in the FXIcat.
  • the atomic coordinates have a root mean square deviation from the atomic coordinates of the backbone atoms of the corresponding residues in any one of the files listed in Figure 16 of less than 1.2, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, or 0.3 A.
  • the resolution of the structure is at least 5, 4, 3, 2.5, 2.0, 1.8, or 1.5 A (in order of increasing resolution and increasing preference).
  • Exemplary surrogates for atomic coordinates of the invention include any modification (e.g., mathematical modification, addition of a constant or scaling) of the coordinates that preserves the relative relationships among the coordinates.
  • Desirable portions of atomic coordinates of Factor Xlcat include at least 50, 60, 70, 80, 90, 95, or 100% of the atomic coordinates for Factor Xlcat.
  • the portion of the atomic coordinates of Factor Xlcat includes at least 50, 60, 70, 80, 90, 95, or 100% of the atomic coordinates for the non-hydrogen atoms of Factor Xlcat or for all the atoms of Factor Xlcat.
  • the compound possesses at least one hydrogen bond donor group that interacts (e.g., forms a hydrogen bond) with Aspl 89 of FXIcat.
  • the compound possesses .an electrophilic carbon and at least one hydrogen bond donor group that interacts with Aspl 89 of FXIcat.
  • the electrophilic carbon of the compound is desirably separated by approximately 5.9 A (e.g., 5.5 to 6.3 A) and 8.3 A (e.g., 8.0 to 8.6 A) from the hydrogen bond donor group of the compound and Aspl 89, respectively.
  • the electrophilic carbon atom is desirably 1.4 to 4.0 A (e.g., 2 to 3.5 A or 2.5 to 3 A) from the side chain oxygen atom (OG) of Ser 195.
  • the non-hydrogen atoms of the hydrogen bond donor groups are desirably within 2.0 to 4.2 A away from the side chain oxygens of Aspl 89. These groups can either interact directly with Aspl 89 or interact indirectly through water molecules.
  • the compound has one or more of the following:
  • an electrophilic carbon atom that binds to the side chain oxygen atom (OG) of Ser 195 such that the electrophilic carbon atom is 1.4-4.0 A from the oxygen atom;
  • a hydrophobic group that makes van der Waals contacts with residues of the active site and is less than 5.0 A (e.g., less than 4.5, 4, 3, 2, or 1 A) of the CB atom of Ala97;
  • a hydrogen bond donor group which makes a hydrogen bond with the main chain carbonyl of Ser214 such that the distance between the hydrogen-bonded atoms is less than 4.0 A (e.g., less than 3.5, 3, 2, or 1 A);
  • a hydrogen bond donor group that makes a hydrogen bond with the main chain carbonyl of Gly216 such that
  • the compound has at least one hydrogen bond donor group that interacts with Aspl 89.
  • the non-hydrogen atoms of the hydrogen bond donor groups are desirably within 2.0 to 4.2 A away from the side chain oxygens of Aspl 89.
  • the method also includes determining the ability of the candidate ligand to bind Factor Xla in vitro or in vivo. In some embodiments, the method further includes determining the ability of the candidate ligand to inhibit the enzymatic activity of Factor Xla in vitro or in vivo.
  • the affinity of the candidate ligand for Factor Xla is at least 2, 5, 10, 20, or 30-fold higher than its affinity for another protease such as thrombin, trypsin, Factor IXa, Factor Vila and/or Factor Xa.
  • Affinity can be reported as, e.g., K m , K ⁇ j, Kj or IC 50 values in which lower values indicate higher affinity.
  • the affinity of the candidate ligand for Factor Xla is at least 10 ⁇ M, l ⁇ M, 100 nM, 10 nM, or 1 nM.
  • the amino acid sequence of FXIcat present in the structure is at least 70, 80, 92, 94, 96, 98, or 100% identical to the amino acid sequence of the corresponding region of human Factor XI (SEQ ID NO: 2).
  • the invention also features crystals of FXIcat that are useful for determining the three-dimensional structure of FXIcat or a protein with substantial sequence identity (e.g., at least 30, 40, 50, 60, 70, 80, 90, 95, or 100% sequence identity) to FXIcat.
  • substantial sequence identity e.g., at least 30, 40, 50, 60, 70, 80, 90, 95, or 100% sequence identity
  • the invention features a crystal (e.g., a cubic crystal) of FXIcat that is at least 10, 12, 15, 18, 20, or 25 ⁇ in the shortest dimension and diffracts to better than 3.5, 3.2, 3.0, 2.8, 2.5, or 2.0 A resolution, or a crystal (e.g, a needle-like crystal) that has a size of at least 10, 12, 15, 18, 20, or 25 ⁇ in the smallest dimension and diffracts to 3.5, 3.2, 3.0, 2.8, 2.5, or 2.0 A resolution or better.
  • a crystal e.g., a cubic crystal
  • FXIcat e.g., a cubic crystal
  • a crystal e.g., a needle-like crystal
  • the invention also features crystals of a quadruple mutant FXIcat protein in which each of residues Ser434, Thr475 and Lys437 has been mutated to an alanine, and Cys482 has been mutated to serine (FXIcat-
  • the invention features a crystal (e.g., a cubic crystal) that is at least 10, 12, 15, 18, 20, or 25 ⁇ in the shortest dimension and diffracts to better than 3.5, 3.2, 3.0, 2.8, 2.5, or 2.0 A resolution, or a crystal (e.g.. a needle-like crystal) that is at least 10, 12, 15, 18, 20, or 25 ⁇ in the smallest dimension and diffracts to 3.5, 3.2, 3.0, 2.8, 2.5, or 2.0 A resolution or better.
  • a crystal e.g., a cubic crystal
  • a crystal e.g. a needle-like crystal
  • the invention further features crystals of a mutant FXIcat protein in which one or more of the following residues Ser434, Thr475, Lys422, Lys437, Lys486, Lys505, Lys509, Arg479 and/or Asp476 are mutated (e.g., mutated to alanine), and/or Cys482 has been mutated (e.g., mutated to serine) and that are useful for determining the three-dimensional structure.
  • the invention features a crystal (e.g., a cubic crystal) of FXIcat-
  • S434A,T475A,C482S,K505A that is at least 10, 12, 15, 18, 20, or 25 ⁇ in the shortest dimension and diffracts to better than 3.5, 3.2, 3.0, 2.8, 2.5, or 2.0 A resolution
  • a crystal e.g., a plate-like crystal
  • FXIcat- S434A,T475A,C482S,D476A that is at least 10, 12, 15, 18, 20, or 25 ⁇ in the smallest dimension and diffracts to 3.5, 3.2, 3.0, 2.8, 2.5, or 2.0 A resolution or better.
  • the invention provides a purified, less than full-length protein fragment of Factor XI, such as a protein consisting essentially of the catalytic domain of Factor XI.
  • the catalytic domain is identified as the residues carboxy] to the activating protease cleavage site designated as KPR JVG (where the arrow denotes the proteolytic cleavage).
  • the residues that make up this domain are 370-607 for the human, 372-606 for the mouse and 369-606 for the rabbit.
  • the sequence for entire Factor XI rat gene is not yet known, but the catalytic domain comprises residues 1-234.
  • the human protein fragment consists of residues 370-607 of SEQ ID NO: 1 (GenBank Accession No. P03951).
  • the protein fragment consists of residues 369-606 of SEQ ID NO: 2 (GenBank Accession No. AF395821).
  • the protein fragment consists of residues 372- 606 of SEQ ID NO: 3 (GenBank Accession No. AF356627).
  • the protein fragment consists of residues 1-234 of SEQ ID NO: 4.
  • the invention features a purified Factor XI protein or fragment thereof including (a) a mutation of a residue that is otherwise post- translationally modified either naturally or in an organism used for recombinant expression, (b) a mutation that alters the charge of Factor XI, (c) a mutation that eliminates a free, reactive sulfhydryl group of a cysteine, (d) a combination of mutations that together alter the distribution of charge density Without altering the overall charge of Factor XI, (e) a mutation of the NH 2 - or COOH- terminal residue of Factor XI, and/or (f) a mutation that alters the folding and/or crystal packing of Factor XL
  • the protein is enzymatically de-glycosylated.
  • one or more residues that would otherwise lead to glycosylation are mutated to another residue that results in no glycosylation.
  • the protein has at least 2, 3, 4, 5, 6, 7, 8, or 9 residues that are mutated compared to a naturally occurring Factor XI protein or the catalytic domain of a naturally-occurring Factor XI protein.
  • the mutation(s) allow crystallization or increase the resolution of the corresponding three-dimensional structure of the mutant protein compared to the structure of Factor XI without the mutation(s). For example, the mutations may increase the size or order of the resulting crystals, resulting in a higher resolution structure.
  • Desirable mutant Factor XI proteins or fragments are at least 80, 85, 90, 92, 94, 96, 98, or 99% identical to any one of SEQ ID NOs: 1 -4.
  • the invention provides a purified protein including a region that is at least 92, 94, 96, 98, or 100% identical to the amino acid sequence of SEQ ID NO: 1 (human Factor XI) or SEQ ID NO: 3 (rat Factor XI).
  • the protein has an amino acid sequence at least 92, 94, 96, 98, or 100% identical to the amino acid sequence of SEQ ID NO: 1, over the entire length of SEQ ID NO: 1 or 3.
  • the invention features a purified nucleic acid encoding one or more of the proteins or protein fragments of the invention.
  • the nucleic acid is contained within a vector and operably linked to a promoter that regulates transcription of the nucleic acid.
  • acyl or "alkanoyl,” as used interchangeably herein, represent an alkyl group, as defined herein, or hydrogen attached to the parent molecular group through a carbonyl group, as defined herein, and is exemplified by formyl, acetyl, propionyl, butanoyl and the like.
  • exemplary unsubstituted acyl groups are of from 2 to 7 carbons.
  • acylamino represents an acyl group attached to the parent molecular group through a nitrogen atom.
  • exemplary unsubstituted acylamino groups are of from 2 to 7 carbons.
  • acyloxy represents an acyl group attached to the parent molecular group through an oxygen atom.
  • exemplary unsubstituted acyloxy groups are of from 2 to 7 carbons.
  • alkenyl represents monovalent straight or branched chain groups of, unless otherwise specified, from 2 to 6 carbons containing one or more carbon-carbon double bonds and is exemplified by ethenyl, 1-propenyl, 2-propenyl, 2-methyl- 1-propenyl, 1-butenyl, 2-butenyl and the like and may be optionally substituted with one, two, three or four substituents independently selected from the group consisting of: (1) alkoxy of one to six carbon atoms; (2) alkylsulfinyl of one to six carbon atoms; (3) alkylsulfonyl of one to six carbon atoms; (4) amino; (5) aryl; (6) arylalkoxy, wherein the alkylene group is of one to six carbon atoms; (7) aryloyl; (8) azido; (9) carboxaldehyde; (10) cycloalkyl of three to eight carbon atoms; (11)
  • alkoxy or "alkyloxy,” as used interchangeably herein, represent an alkyl group attached to the parent molecular group through an oxygen atom. Exemplary unsubstituted alkoxy groups are of from 1 to 6 carbons.
  • alkoxyalkyl or “alkyloxyalkyl,” as used interchangeably herein, represent an alkyl group to which is attached an alkoxy group. Exemplary unsubstituted alkoxyalkyl groups are of from 2 to 12 carbons.
  • alkoxycarbonyl or "alkyloxycarbonyl,” as used interchangeably herein, represent an ester group; i.e. an alkoxy group, attached to the parent molecular group through a carbonyl group and is exemplified by methoxycarbonyl, ethoxycarbonyl and the like.
  • exemplary unsubstituted alkoxycarbonyl groups are of from 2 to 7 carbons.
  • alkyl represents a monovalent group derived from a straight or branched chain saturated hydrocarbon of, unless otherwise specified, from 1 to 6 carbons and is exemplified by methyl, ethyl, n- and isopropyl, n-, sec-, iso- and tert-butyl, neopentyl and the like and may be optionally substituted with one, two, three or, in the case of alkyl groups of two carbons or more, four substituents independently selected from the group consisting of: (1) alkoxy of one to six carbon atoms; (2) alkylsulfinyl of one to six carbon atoms; (3) alkylsulfonyl of one to six carbon atoms; (4) amino; (5) aryl; (6) arylalkoxy; (7) aryloyl; (8) azido; (9) carboxaldehyde; (10) cycloalkyl of three to eight carbon atoms
  • alkylamino represents an alkyl group attached to the parent molecular group through a nitrogen atom.
  • exemplary unsubstituted alkylamino groups are of from 1 to 6 carbons.
  • alkylaminocarbonyl represents an alkylamino group attached to the parent molecular group through a carbonyl group.
  • exemplary unsubstituted alkylaminocarbonyl groups are of from 2 to 7 carbons.
  • alkylaminosulfonyl represents an alkylamino group attached to the parent molecular group through an -SO 2 - group.
  • exemplary unsubstituted alkylaminosulfonyl groups are of from 1 to 6 carbons.
  • alkylene represents a saturated divalent hydrocarbon group derived from a straight or branched chain saturated hydrocarbon by the removal of two hydrogen atoms, and is exemplified by methylene, ethylene, isopropylene and the like.
  • alkylsulfinyl represents an alkyl group attached to the parent molecular group through an -S(O)- group. Exemplary unsubstituted alkylsulfinyl groups are of from 1 to 6 carbons.
  • alkylsulfinylalkyl represents an alkyl group, as defined herein, substituted by an alkylsulfinyl group. Exemplary unsubstituted alkylsulfinylalkyl groups are of from 2 to 12 carbons.
  • alkylsulfonyl represents an alkyl group attached to the parent molecular group through an -SO 2 - group.
  • exemplary unsubstituted alkylsulfonyl groups are of from 1 to 6 carbons.
  • alkylsulfonylalkyl represents an alkyl group, as defined herein, substituted by a alkylsulfonyl group.
  • exemplary unsubstituted alkylsulfonylalkyl groups are of from 2 to 12 carbons.
  • alkylthio represents an alkyl group attached to the parent molecular group through a sulfur atom. Exemplary unsubstituted alkylthio groups are of from 1 to 6 carbons.
  • alkynyl represents monovalent straight or branched chain groups of from two to six carbon atoms containing a carbon- carbon triple bond and is exemplified by ethynyl, 1-propynyl, and the like and may be optionally substituted with one, two, three or four substituents independently selected from the group consisting of: (1) alkoxy of one to six carbon atoms; (2) alkylsulfinyl of one to six carbon atoms; (3) alkylsulfonyl of one to six carbon atoms; (4) amino; (5) aryl; (6) arylalkoxy, wherein the alkylene group is of one to six carbon atoms; (7) aryloyl; (8) azido; (9) carboxaldehyde; (10) cycloalkyl of three to eight carbon atoms; (11) halo; (12) heterocycle; (13) (heterocycle)oxy; (14) (heterocycle)oy
  • R A is selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, as defined
  • R C herein; and R and R are independently selected from the group consisting of: (a) hydrogen, (b) optionally substituted alkyl, (c) optionally substituted cycloalkyl, (d) optionally substituted aryl, (e) optionally substituted arylalkyl, (f) optionally substituted heterocyclyl, and (g) optionally substituted heterocyclylalkyl, each of which is as defined herein.
  • R and R are independently selected from the group consisting of: (a) hydrogen, (b) optionally substituted alkyl, (c) optionally substituted cycloalkyl, (d) optionally substituted aryl, (e) optionally substituted arylalkyl, (f) optionally substituted heterocyclyl, and (g) optionally substituted heterocyclylalkyl, each of which is as defined herein.
  • R is H and R corresponds to those side chains of natural amino acids found in nature, or their antipodal configurations.
  • exemplary natural amino acids include alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, aspartamine, ornithine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine, each of which, except glycine, as their D- or L-form.
  • the present invention also contemplates non-naturally occuring (i.e., unnatural) amino acid residues in their D- or L-form such as, for example, homophenylalanine, phenylglycine, cyclohexylglycine, cyclohexylalanine, cyclopentyl alanine, cyclobutylalanine, cyclopropylalanine, cyclohexylglycine, norvaline, norleucine, thiazoylalanine (2-, 4- and 5- substituted), pyridylalanine (2-, 3- and 4-isomers), naphthalalanine (1- and 2-isomers) and the like.
  • non-naturally occuring amino acid residues in their D- or L-form such as, for example, homophenylalanine, phenylglycine, cyclohexylglycine, cyclohexylalanine, cyclopentyl alanine, cycl
  • Stereochemistry is as designated by convention, where a bold bond indicates that the substituent is oriented toward the viewer (away from the page) and a dashed bond indicates that the substituent is oriented away from the viewer (into the page). If no stereochemical designation is made, it is to be assumed that the structure definition includes both stereochemical possibilities.
  • amino represents an -NH 2 group.
  • aminoalkyl represents an alkyl group, as defined herein, substituted by an amino group.
  • aryl represents a mono- or bicyclic carbocyclic ring system having one or two aromatic rings and is exemplified by
  • arylalkenyl or “aralkenyl,” as used interchangeably herein, represent an aryl group attached to the parent molecular group through an alkenyl group.
  • exemplary unsubstituted arylalkenyl groups are of from 8 to 16 carbons.
  • arylalkoxy or “aralkoxy,” as used interchangeably herein, represent an arylalkyl group attached to the parent molecular group through an oxygen atom.
  • exemplary unsubstituted arylalkoxy groups are of from 7 to 16 carbons.
  • arylalkoxycarbonyl or “aralkoxycarbonyl,” as used interchangeably herein, represent an arylalkoxy group attached to the parent molecular group through a carbonyl group.
  • Exemplary unsubstituted arylalkoxycarbonyl groups are of from 8 to 17 carbons.
  • arylalkyl or “aralkyl,” as used interchangeably herein, represent an aryl group attached to the parent molecular group through an alkyl group.
  • exemplary unsubstituted arylalkyl groups are of from 7 to 16 carbons.
  • arylalkylamino or “aralkylamino,” as used interchangeably herein, represent an arylalkyl group attached to the parent molecular group through a nitrogen atom.
  • exemplary unsubstituted arylalkylamino groups are of from 7 to 16 carbons.
  • arylalkylaminocarbonyl or “aralkylaminocarbonyl,” as used interchangeably herein, represents an arylalkylamino group attached to the parent molecular group through a carbonyl group.
  • exemplary unsubstituted arylalkylaminocarbonyl groups are of from 8 to 17 carbons.
  • aryl alkylsulfinyl or “aralkylsulfmyl,” as used interchangeably herein, represent an arylalkyl group attached to the parent molecular group through an -SO- group.
  • exemplary unsubstituted arylalkylsulfmyl groups are of from 7 to 16 carbons.
  • arylalkylsulfonyl or “aralkylsulfonyl,” as used interchangeably herein, represent an aralkyl group attached to the parent molecular group through an -SO 2 - group.
  • exemplary unsubstituted arylalkylsulfonyl groups are of from 7 to 16 carbons.
  • arylalkylthio or " aralkylthio,” as used interchangeably herein, represents an arylalkyl group attached to the parent molecular group through a sulfur atom.
  • exemplary unsubstituted arylalkylthio groups are of from 7 to 16 carbons.
  • arylamino represents an aryl group which is attached to the parent molecular group through a nitrogen atom.
  • exemplary unsubstituted arylamino groups are of 6 or 10 carbons.
  • arylaminocarbonyl represents an arylamino group attached to the parent molecular group through a carbonyl group.
  • exemplary unsubstituted arylaminocarbonyl groups are of from from 7 or 11 carbons.
  • arylaminosulfonyl represents an arylamino group attached to the parent molecular group through an -SO 2 - group.
  • exemplary unsubstituted arylaminosulfonyl groups are of 6 or 10 carbons.
  • aryloxy represents an aryl group which is attached to the parent molecular group through an oxygen atom.
  • exemplary unsubstituted aryloxy groups are of 6 or 10 carbons.
  • aryloxycarbonyl represents an aryloxy group which is attached to the parent molecular group through a carbonyl group.
  • Exemplary unsubstituted aryloxycarbonyl groups are of 7 or 11 carbons.
  • aryloyl or “aroyl,” as used interchangeably herein, represent an aryl group which is attached to the parent molecular group through a carbonyl group.
  • exemplary unsubstituted aryloxycarbonyl groups are of 7 or 11 carbons.
  • aryloylamino or "aroylamino,” as used interchangeably herein, represent an aroyl group which is attached to the parent molecular group through a nitrogen atom.
  • exemplary unsubstituted aryloylamino groups are of 7 or 11 carbons.
  • arylsulfinyl represens an aryl group attached to the parent molecular group through an -SO- group.
  • exemplary unsubstituted arylsulfinyl groups are of 6 or 10 carbons.
  • arylsulfonyl represens an aryl group attached to the parent molecular group through an -SO 2 - group.
  • Exemplary unsubstituted arylsulfonyl groups are of 6 or 10 carbons.
  • arylthio represents an aryl group which is attached to the parent molecular group through a sulfur atom.
  • exemplary unsubstituted arylthio groups are of 6 or 10 carbons.
  • azidoalkyl represents an alkyl group, as defined herein, substituted by an azido group.
  • blood component is meant a biological entity normally ound in blood, such as, for example cells, such as erythrocytes, leukocytes, and platelets, or proteins such as immunoglobulins, serum albumin, ferritin, steroid binding proteins, such as corticosteroid-binding globulin and sex hormone- binding globulin, transferrin, thyroxin binding protein, and alpha-2- macroglobulin.
  • Blood components also include glycans, including glycosylamino glycans.
  • Preferred blood components are those that have reactive organic functionality, such as thiols or amines.
  • carboxylate or “carbamyl,” as used interchangeably herein, represent a R A OC(O)NR B - group, or a -OC(O)NR B - linkage, depending on the chemical context in which this term is used, wherein R is selected from the group consisting of (a) alkyl, (b) aryl, and (c) arylalkyl; and R B is selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, as defined herein.
  • carbonate represents a -R OC(O)O- group, or a - OC(O)O- linkage, depending on the chemical context in which this term is used, wherein R A is selected from the group consisting of (a) alkyl, (b) aryl, and (c) arylalkyl, as defined herein.
  • carboxydehyde represents a -CHO group.
  • (carboxaldehyde)alkyl represents an alkyl group, as defined herein, substituted by a carboxaldehyde group.
  • Carboxy represents a -CO 2 H group.
  • Carboxyalkyl represents an alkyl group, as defined herein, substituted by a carboxy group.
  • cycloalkenyl represents a monovalent cyclic hydrocarbon of from three to eight carbons, unless otherwise specified, having at least one carbon-carbon double bond.
  • the cycloalkenyl groups of this invention can be optionally substituted with (1) alkanoyl of one to six carbon atoms; (2) alkyl of one to six carbon atoms; (3) alkoxy of one to six carbon atoms; (4) alkoxyalkyl, wherein die alkyl and alkylene groups are independently of one to six carbon atoms; (5) alkylsulfinyl of one to six carbon atoms; (6) alkylsulfinylalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (7) alkylsulfonyl of one to six carbon atoms; (8) alkylsulfonylalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (9)
  • cycloalkyl represents a monovalent saturated or unsaturated non-aromatic cyclic hydrocarbon group of from three to eight carbons, unless otherwise specified, and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1.]heptyl and the like.
  • the cycloalkyl groups of this invention can be optionally substituted with (1) alkanoyl of one to six carbon atoms; (2) alkyl of one to six carbon atoms; (3) alkoxy of one to six carbon atoms; (4) alkoxyalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (5) alkylsulfinyl of one to six carbon atoms; (6) alkylsulfinylalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (7) alkylsulfonyl of one to six carbon atoms; (8) alkylsulfonylalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (9) aryl; (10) arylalkyl, wherein the alkyl group is of one to six carbon atoms; (11) amino; (12) aminoalkyl of one to six carbon atoms
  • cycloalkylamino represents a cycloalkyl group attached to the parent molecular group through a nitrogen atom.
  • exemplary unsubstituted cycloalkylamino groups are of from 3 to 8 carbons.
  • cycloalkylaminocarbonyl represents a cycloalkylamino group attached to the parent molecular group through a carbonyl group.
  • exemplary unsubstituted cycloalkylaminocarbonyl groups are of from 4 to 9 carbons.
  • cycloalkyloxy or "cycloalkoxy,” as used interchangeably herein, represent a cycloalkyl group, as defined herein, attached to the parent molecular group through an oxygen atom.
  • exemplary unsubstituted cycloalkyloxy groups are of from 3 to 8 carbons.
  • cycloalkyloxycarbonyl or “cycloalkoxycarbonyl,” as used interchangeably herein, represent a cycloalkyloxy group, as defined herein, attached to the parent molecular group through a carbonyl group.
  • exemplary unsubstituted cycloalkyloxycarbonyl groups are of from 4 to 9 carbons.
  • cycloalkylalkoxy represents an alkoxy group, as defined herein, to which is attached a cycloalkyl group.
  • exemplary unsubstituted cycloalkylalkoxy groups are of from 4 to 14 carbons.
  • cycloalkylalkyl represents a cycloalkyl group, as defined herein, attached to the parent molecular group through an alkyl group.
  • exemplary unsubstituted cycloalkylalkyl groups are of from 4 to 14 carbons.
  • cycloalkylsulfinyl represents a cycloalkyl group attached to the parent molecular group through an -SO- group.
  • exemplary unsubstituted cycloalkylsulfinyl groups are of from 3 to 8 carbons.
  • cycloalkylsulfonyl represents a cycloalkyl group attached to the parent molecular group through an -SO 2 - group.
  • Exemplary unsubstituted cycloalkylsulfonyl groups are of from 3 to 8 carbons.
  • dialkylamino represents an N,N- dialkylsubstituted amine attached to the parent molecular group through the nitrogen atom.
  • the two alkyl substituents of a dialkylamino group can be the same or different, or can be joined together to form a ring.
  • Exemplary dialkylamino groups are of from 2 to 12 carbons and include dimethylamino, diethylamino, pyrrolidino, and piperidino.
  • haloalkyl represents an alkyl group, as defined herein, substituted by one, two, or three halogen atoms and is exemplified by chloromethyl, bromoethyl, trifluoromethyl and the like.
  • halogen represents F, CI, Br and I.
  • heteroaryl represents that subset of heterocycles, as defined herein, which are aromatic: i.e., they contain 4n+2 pi electrons within the mono- or multicyclic ring system.
  • exemplary unsubstituted heteroaryl groups are of from 1 to 9 carbons.
  • heteroarylalkenyl or “heteroaralkenyl,” or as used interchangeably herein, represent a heteroaryl group, as defined herein, attached to the parent molecular group through an alkenyl group.
  • Exemplary unsubstituted heteroarylalkenyl groups are of from 3 to 15 carbons.
  • heteroarylalkyl or “heteroaralkyl,” as used interchangeably herein, represent a heteroaryl group, as defined herein, attached to the parent molecular group through an alkyl group.
  • exemplary unsubstituted heteroarylalkyl groups are of from 2 to 15 carbons.
  • heteroarylalkylamino or “heteroaralkylamino,” as used interchangeably herein, represent a heteroarylalkyl group, as defined herein, attached to the parent molecular group through a nitrogen atom.
  • exemplary unsubstituted heteroarylalkylamino groups are of from 2 to 15 carbons.
  • heteroarylalkylaminocarbonyl or
  • heteroarylalkylaminocarbonyl or as used interchangeably herein, represent a heteroarylalkylamino group, as defined herein, attached to the parent molecular group through a carbonyl group.
  • exemplary unsubstituted heteroarylalkylaminocarbonyl groups are of from 3 to 16 carbons.
  • heteroaryloyl or “heteroaroyl,” or as used interchangeably herein, represent a heteroaryl group, as defined herein, attached to the parent molecular group through a carbonyl group.
  • exemplary unsubstituted heteroaryloyl groups are of from 2 to 10 carbons.
  • heteroarylalkyloxy or “heteroaralkoxy,” or as used interchangeably herein, represent a heteroarylalkyl group, as defined herein, attached to the parent molecular group through an oxygen atom.
  • exemplary unsubstituted heteroarylalkyloxy groups are of from 2 to 15 carbons.
  • heteroarylalkyloxycarbonyl or “heteroaralkoxycarbonyl,” as used interchangeably herein, represent a heteroaralkoxy group, as defined herein, attached to the parent molecular group through a carbonyl group.
  • exemplary unsubstituted heteroarylalkyloxycarbonyl groups are of from 3 to 16 carbons.
  • heteroarylalkylsulfonyl or “heteroaralkylsulfonyl,” as used interchangeably herein, represent a heteroarylalkyl group attached to the parent molecular group through an -SO 2 - group.
  • exemplary unsubstituted heteroarylalkylsulfonyl groups are of from 2 to 15 carbons.
  • heteroarylamino represents a heteroaryl group attached to the parent molecular group through a nitrogen atom.
  • exemplary unsubstituted heteroarylamino groups are of from 1 to 9 carbons.
  • heteroarylaminocarbonyl represents a heteroarylamino group attached to the parent molecular group through a carbonyl group.
  • exemplary unsubstituted heteroarylaminocarbonyl groups are of from 2 to 10 carbons.
  • heteroarylaminosulfonyl represents a heteroarylamino group attached to the parent molecular group through an -SO 2 - group.
  • exemplary unsubstituted heteroarylaminosulfonyl groups are of from 1 to 9 carbons.
  • heteroaryloxy represents a heteroaryl group attached to the parent molecular group through an oxygen atom.
  • Exemplary unsubstituted heteroaryloxy groups are of from 1 to 9 carbons.
  • heteroaryloxycarbonyl represents a heteroaryloxy group attached to the parent molecular group through a carbonyl group.
  • exemplary unsubstituted heteroaryloxycarbonyl groups are of from 1 to 9 carbons.
  • heteroarylsulfonyl represents a heteroaryl group attached to the parent molecular group through an -SO 2 - group.
  • exemplary unsubstituted heteroarylsulfonyl groups are of from 1 _to 9 carbons.
  • heteroarylthio represents a heteroaryl group attached to the parent molecular group through a sulfiir atom.
  • Exemplary unsubstituted heteroaryloxy groups are of from 1 to 9 carbons.
  • heterocycle or “heterocyclyl,” as used interchangeably herein represent a 5-, 6- or 7-membered ring, unless otherwise specified, containing one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur.
  • the 5-membered ring has zero to two double bonds and the 6- and 7-membered rings have zero to three double bonds.
  • heterocycle also includes bicyclic, tricyclic and tetracyclic groups in which any of the above heterocyclic rings is fused to one or two rings independently selected from the group consisting of an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring and another monocyclic heterocyclic ring such as indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl, benzothienyl and the like.
  • Heterocyclics include pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, i idazolidinyl, pyridyl, piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidiniyl, mo ⁇ holinyl, thiomo ⁇ holinyl, thiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, furyl, thieny
  • F is selected from the group consisting of -CH 2 -, -CH 2 O- and -O-
  • G is selected from the group consisting of -C(O)- and -(C(R')(R")) V -
  • R' and R" are independently selected from the group consisting of hydrogen or alkyl of one to four carbon atoms
  • v is one to three and includes groups such as 1,3-benzodioxolyl, 1,4-benzodioxanyl and the like.
  • any of the heterocycle groups mentioned herein may be optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of: (1) alkanoyl of one to six carbon atoms; (2) alkyl of one to six carbon atoms; (3) alkoxy of one to six carbon atoms; (4) alkoxyalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (5) alkylsulfinyl of one to six carbon atoms; (6) alkylsulfinylalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (7) alkylsulfonyl of one to six carbon atoms; (8) alkylsulfonylalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (9) aryl; (10) arylalkyl, wherein the alkyl group is of one to six carbon atoms
  • heterocyclylalkyl represents a heterocyclyl group attached to the parent molecular group through an alkyl group.
  • exemplary unsubstituted heterocyclylalkyl groups are of from 2 to 15 carbons.
  • heterocyclylamino or “(heterocycle)amino,” as used interchangeably herein, represents a heterocycle group, as defined herein, attached to the parent molecular group through nitrogen.
  • exemplary unsubstituted heterocyclylamino groups are of from 1 to 9 carbons.
  • heterocyclyloxy or “(heterocycle)oxy,” as used interchangeably herein, represents a heterocycle group, as defined herein, attached to the parent molecular group through an oxygen atom.
  • exemplary unsubstituted heterocyclyloxy groups are of from 1 to 9 carbons.
  • heterocyclyloxycarbonyl or “(heterocycle)oxycarbonyl,” as used interchangeably herein, represents a heterocycloxy group, as defined herein, attached to the parent molecular group through a carbonyl group.
  • Exemplary unsubstituted heterocyclyloxycarbonyl groups are of from 2 to 10 carbons.
  • heterocyclyloyl or “(heterocycle)oyl,” as used interchangeably herein, represents a heterocycle group, as defined herein, attached to the parent molecular group through a carbonyl group.
  • exemplary unsubstituted heterocyclyloyl groups are of from 2 to 10 carbons.
  • heterocyclylsulfonyl represents a heterocyclyl group attached to the parent molecular group through an -SO 2 - group.
  • exemplary unsubstituted heterocyclylsulfonyl groups are of from 1 to 9 carbons.
  • heterocyclylthio represents a heterocyclyl group attached to the parent molecular group through a sulfur atom.
  • Exemplary unsubstituted heteroaryloxy groups are of from 1 to 9 carbons.
  • hydroxy represents an -OH group.
  • hydroxyalkyl represents an alkyl group, as defined herein, substituted by one to three hydroxy groups, with the proviso that no more than one hydroxy group may be attached to a single carbon atom of the alkyl group and is exemplified by hydroxymethyl, dihydroxypropyl and the like.
  • N-protected amino refers to an amino group, as defined herein, to which is attached an N-protecting or nitrogen-protecting group, as defined herein.
  • N-protected aminoalkyl refers to an alkyl group, as defined herein, which is substituted by an N-protecting or nitrogen- protecting group, as defined herein.
  • N-protecting group or “nitrogen protecting group” as used herein, represent those groups intended to protect an amino group against undersirable reactions during synthetic procedures. Commonly used N- protecting groups are disclosed in Greene, “Protective Groups In Organic Synthesis, 3 rd Edition” (John Wiley & Sons, New York, 1999), which is inco ⁇ orated herein by reference.
  • N-protecting groups comprise acyl, aroyl, or carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2- chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o- nitrophenoxyacetyl, ⁇ -chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4- bromobenzoyl, 4-nitrobenzoyl and chiral auxiliaries such as protected or unprotected D, L or D, L-amino acids such as alanine, leucine, phenylalanine and the like; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl and the like; carbamate forming groups such as benzyloxycarbonyl, p- chlor
  • N-protecting groups are formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t- butyloxycarbonyl (Boc) and benzyloxycarbonyl (Cbz).
  • nitro represents an -NO 2 group.
  • nitroalkyl represents an alkyl group substituted by an -NO group.
  • perfluoroalkyl represents an alkyl group, as defined herein, wherein each hydrogen radical bound to the alkyl group has been replaced by a fluoride radical.
  • Perfluoroalkyl groups are exemplified by trifluoromethyl, pentafluoroethyl, and the like.
  • perfluoroalkoxy refers to a perfluoroalkyl group, as defined herein, attached to the parent molecular group through an oxygen atom.
  • pharmaceutically acceptable salt represents those salts which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, S. M Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences 66:1-19, 1977.
  • the salts can be prepared in situ during the final isolation and purification of the compounds of the invention or separately by reacting the free base group with a suitable organic acid.
  • Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphersulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- na
  • alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine and the like.
  • pharmaceutically acceptable ester represents esters which hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof.
  • Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl group preferably has not more than 6 carbon atoms.
  • Examples of particular esters includes formates, acetates, propionates, butyates, acrylates and ethylsuccinates.
  • prodrugs as used herein, represents those prodrugs of the compounds of the present invention which are, within the scope of sound medical judgement, suitable for use in contact with with the tissues of humans and animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention.
  • prodrug represents compounds which are rapidly transformed in vivo to the parent compound of the above formula, for example, by hydrolysis in blood.
  • ring system substituent is meant a substituent attached to an aromatic or non-aromatic ring system.
  • ring system substituent When a ring system is saturated or partially saturated the "ring system substituent” further includes methylene (double bonded carbon), oxo (double bonded oxygen) or thioxo ⁇ double bonded sulfur).
  • spiroalkyl represents an alkylene diradical, both ends of which are bonded to the same carbon atom of the parent group to form a spirocyclic group.
  • sulfonyl represents an -SO 2 - group.
  • thioalkoxy represents represents an alkyl group attached to the parent molecular group through a sulfur atom. Exemplary unsubstituted thioalkoxy groups are of from 1 to 6 carbons.
  • thioalkoxyalkyl represents an alkyl group substituted by a thioalkoxy group.
  • exemplary unsubstituted thioalkoxyalkyl groups are of from 2 to 12 carbons.
  • thiocarbonyl is meant a -C(S)- group.
  • thiol is meant an -SH group.
  • Asymmetric or chiral centers may exist in the compounds of the present invention.
  • the present invention contemplates the various stercoisomers and mixtures thereof.
  • Individual stereoisomers of compounds or the present invention are prepared synthetically from commercially available starting materials which contain asymmetric or chiral centers or by preparation of mixtures of enantiometic compounds followed by resolution well-known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a racemic mixture of enantiomers, designated (+/-), to a chiral auxiliary, separation of the resulting diastereomers by recrystailization or chromatography and liberation of the optically pure product from the auxiliary or (2) direct separation of the mixture of optical enantiomers on chiral chromatographic columns.
  • Enantiomers are designated herein by the symbols "R” or "S,” depending on the configuration of substituents around the chiral carbon atom.
  • Geometric isomers may also exist in the compounds of the present invention.
  • the present invention contemplates the various geometric isomers and mixtures thereof resulting from the arrangement of substituents around a carbon-carbon double bond and designates such isomers as of the Z or E configuration, wherein the term "Z” represents substituents on the same side of the carbon-carbon double bond and the term “E” represents substituents on opposite sides of the carbon-carbon double bond. It is also recognized that for structures in which tautomeric forms are possible, the description of one tautomeric form is equivalent to the description of both, unless otherwise specified.
  • Figure 1 is a simplified diagrammatic representation of the coagulation cascade showing the role of Factor XI in this pathway. All three thick arrows represent the amplification phase. The initiation and propagation phases are also labled. Flla is also known as thrombin. The arrow from Flla to FXIa indicates that FXI is activated by Flla and FXIIa.
  • Figure 2 is a table that lists the conversion between the amino acid numbering of FXIcat in the native zymogen found in the bloodstream and trypsin. Residue numbers greater than 999 under "trypsin” denote amino acid insersions in FXIcat relative to trypsin, while missing numbers under "trypsin” denote amino acid deletions in FXIcat relative to trypsin.
  • Figure 3 is a table describing the Protein Data Bank (PDB) format used to list the atomic coordinates of the various structures of this invention shown in the files listed in Figure 16).
  • PDB Protein Data Bank
  • Figure 4A is a table that lists the residues of human Factor Xla that are altered to other amino acids or removed by mutagenesis/engineering strategies.
  • the AVC-terminal truncation mutation is the removal of the C-terminal Ala606 and Val607 residues, such that the last residue of the mutant is Gln605.
  • N.D is meant that the enzymatic activity of the mutant has not been determined or that crystalization of the mutant has not been undertaken or has not produced crystals of sufficient quality to determine the three-dimensional structure.
  • Figure 4B is a table that lists relevant crystallographic data derived from crystals of the invention. Crystallization conditions are also listed.
  • Figure 5 A is a ribbon diagram of human FXIcat, as derived from its crystal structure with ecotin. The amino- and carboxy-termini are indicated by N and C. The drawing was produced using the program Insight II.
  • Figure 5B is a ribbon diagram of human FXIcat, as derived from its crystal structure with benzamidine. The amino- and carboxy-termini are indicated by N and C. The drawing was produced using the program Insight II.
  • Figure 5C is a stereoview of the active site of human FXIcat, as derived from its crystal structure with ecotin. For clarity, no hydrogen atoms or water molecules are shown.
  • Figure 5D is a stereoview of the active site of human FXIcat, as derived from its crystal structure with benzamidine. For clarity, no hydrogen atoms or water molecules are shown.
  • Figure 6 shows the position of the oligonucleotides chosen for the PCR approach to isolate rat FXIcat and the rat FXIcat nucleotide sequence that was derived.
  • Figure 7 is an alignment of amino acid sequences of FXIcat proteins from human, mouse, rabbit and rat.
  • Figure 8 is a schematic diagram of an expression plasmid for FXIcat in Pichia pastoris .
  • a fusion between the ⁇ -factor secretion signal sequence and FXIcat was constructed in order to create a fusion protein with an N-terminal ⁇ -factor, signal peptide, and FXIcat. This gene is expressed from the methanol-regulated AOX promoter.
  • Figure 9 lists the signal peptide cleavage site of pPICZ ⁇ FXIcat.
  • Figure 10 shows the effect of continuous intravenous administration of compound 22 in a rat model of venous thrombosis.
  • venous thrombosis was induced by inco ⁇ orating stenosis of the inferior vena cava with rapid infusion of hypotonic saline followed by partial stenosis.
  • Compound 22 was administered as a continuous intravenous infusion via the femoral vein commenced 15 minutes prior to the thrombotic challenge. Thrombus mass was assessed at 60 minutes following challenge. Results are expressed as the percentage of the vehicle mean.
  • Figure 11 is a schematic illustration of an exemplary computer.
  • Figure 12 is a table containing data on the supe ⁇ osition of the three- dimensional structure of FXIcat complexed with compound 144 and that of complexes with other ligands.
  • the first row labeled “rms/loop,” is the root mean squared (rms) deviation of just the 48 backbone atoms constituting the loop when backbone atoms (number of atoms are listed on second row) of the two structures are superimposed.
  • the second row labeled “rms/fit,” is the rms deviation and number of backbone atoms used for supe ⁇ osition.
  • the third row labeled “rms/loop,” is the rms deviation of just the 48 backbone atoms constituting the loop using a best fit of all backbone atoms that do not deviate by more than 3.8 A between the two structures.
  • the fourth row labeled “rms/fit,” is the rms deviation of all backbone atom pairs that do not deviate by more than 3.8 A.
  • Figure 13 is a table of the rms deviation (values to the right of the diagonal) of the supe ⁇ osition of FXIcat structures solved in the presence of the indicated compounds. Values to the left are the number of backbone atom pairs used in the supe ⁇ osition. Rms values are calculated only for pairs of atoms that do not deviate from each others by more than 3.8 A.
  • Figure 14 is the same as Figure 13 except that all backbone atom pairs are used in the supe ⁇ osition.
  • Figure 15 is a table of the rms deviation between Serl95, His 57, and Asp 102 in FXIcat structures solved in the presence of the indicated compounds using a best fit of backbone atoms (values to the right) and all backbone atoms (values to the left).
  • Figure 16 is a list of an electronic file contained on 2 identical compact discs (Copy 1 and Copy 2) as "Table of Figure 16 listings", which is hereby inco ⁇ orated by reference.
  • “Listing 4" contains the file atomic coordinates of the complex between two molecules of human FXIcat and two molecules of ecotin M84R.
  • “Listing 5" contains the coordinates of the complex between two molecules of human FXIcat and two molecules of wild type ecotin.
  • “Listing 6” contains the coordinates of the complex between two molecules of human FXIcat and two molecules of ecotin IX-D.
  • “Listing 7” contains the atomic coordinates of the complex between human FXIcat and benzamidine.
  • “Listing 8” contains the atomic coordinates of the complex between human FXIcat and compound 77.
  • “Listing 9” contains the atomic coordinates of the complex between human FXIcat and compound 85.
  • “Listing 10” contains the atomic coordinates of the complex between human FXIcat and compound 25.
  • “Listing 11” contains the atomic coordinates of the complex between human FXIcat and compound 79.
  • “Listing 12” contains the atomic coordinates of the complex between human FXIcat and compound 86.
  • “Listing 13” contains the atomic coordinates of the complex between human FXIcat and compound 144.
  • “Listing 14” contains the atomic coordinates of the complex between human FXIcat and compound 141.
  • “Listing 15” contains the atomic coordinates of the complex between human FXIcat and compound 145.
  • the numbering system is based upon structural homology to trypsin (see Figure 2 for conversion between Factor XI numbering system and trypsin). The format is as described in Figure 3. DETAILED DESCRIPTION
  • Human coagulation Factor Xla is a serine protease that results from activation of Factor XI by thrombin, Factor Xlla, or autoactivation.
  • Factor XI is an upstream serine protease in the amplification phase of the intrinsic anticoagulant cascade that circulates in blood plasma as a homo-dimeric zymogen ( Figure 1). Although the protease domain exhibits homology with other coagulation serine proteases, the remaining domains are not shared.
  • Factor XI is not gamma carboxylated (i.e., not vitamin K-dependent) and does not have EGF domains, but possesses an N-terminal series of 'apple' domains and a C-terminal catalytic domain.
  • Inhibiting enzymes of the amplification phase of the clotting cascade represents an attractive strategy for novel antithrombotic agents. On the one hand this approach may allow for sufficient anticoagulant activity to prevent unwanted thrombotic episodes, but also allow enough clotting to occur so as to prevent or minimize bleeding.
  • complete deficiency of factors involved in the amplification phase of the coagulation cascade can result in bleeding (e.g., hemophilia A and B are caused by deficiencies in Factors VIII and IX, respectively)
  • low levels of activity generally in the range of 5% of normal, are sufficient to minimize any bleeding diathesis and confer a low probability of suffering acute thrombotic disorders such as AMI or stroke.
  • Factor XI deficiency results in a mild-to-moderate bleeding disorder, especially in tissues with high levels of local fibrinolytic activity, such as the urinary tract, nose, oral cavity, and tonsils.
  • the important role of Factor XI in coagulation is also indicated by the recent observation that high levels of Factor XI are associated with increased venous thrombosis (Meijers et al, N. Engl J. Med. 342:696, 2000).
  • This structural information can be used to identify functional groups within a ligand that can be modified to increase the affinity and selectivity of the ligand for Factor Xla or to identify functional groups within the ligand that can be modified to increase the bioavailability of the ligand without adversely affecting its affinity for Factor Xla.
  • Compounds that may be tested for their ability to decrease Factor X ⁇ a activity include, but are not limited to, synthetic organic molecules, naturally occurring organic molecules and their semi-synthetic derivatives, nucleic acid molecules, biosynthetic proteins or peptides, naturally occurring peptides or proteins, or antibodies.
  • the organic compound has a molecular weight contained in one of the following ranges: 100-3,000 daltons; 100-2,000 daltons; 100-1,000 daltons; 100-750 daltons; 250-3,000 daltons; 250-2,000 daltons; 250-1 ,000 daltons; 250-750 daltons; 400-3,000 daltons; 400-2,000 daltons; 400-1 ,000 daltons; or 400-750 daltons, inclusive.
  • the present invention includes a class of peptidomimetics and non-peptides that inhibit the activity of Factor Xla (e.g., the compounds of formula I or formula II, as described herein).
  • any of the compounds of the invention are useful for treating or preventing diseases for which inhibition of Factor Xla is desirable, such as, but not limited to, arterial (e.g., unstable angina, acute myocardial infarction, percutaneous coronary interventions, or peripheral arterial occlusions), venous (e.g., deep venous thrombosis) and cardiac (e.g., atrial fibrillation) conditions and resulting ischemic conditions (e.g., acute myocardial infarction, pulmonary embolism, or stroke), and heparin induced complications (e.g., heparin induced thromobocytopenia).
  • arterial e.g., unstable angina, acute myocardial infarction, percutaneous coronary interventions, or peripheral arterial occlusions
  • venous e.g., deep venous thrombosis
  • cardiac e.g., atrial fibrillation
  • resulting ischemic conditions e.g., acute myocardial in
  • the invention provides a purified, less than full-length protein fragment of Factor XI, such as a protein consisting essentially of the catalytic domain of Factor XI.
  • the catalytic domain is identified as the residues carboxyl to the activating protease cleavage site designated as
  • the human protein fragment consists of residues 370-607 of SEQ ID NO: 1 (GenBank Accession No. P03951).
  • the protein fragment consists of residues 369-606 of SEQ ID NO: 2 (GenBank Accession No. AF395821).
  • the protein fragment consists of residues 372- 606 of SEQ ID NO: 3 (GenBank Accession No. AF356627).
  • the protein fragment consists of residues 1-234 of SEQ ID NO: 4.
  • the invention features a purified Factor XI protein or fragment thereof including (a) a mutation of a residue that is otherwise post- translationally modified either naturally or in an organism used for recombinant expression, (b) a mutation that alters the charge of Factor XI, (c) a mutation that eliminates a free, reactive sulfhydryl group of a cysteine, (d) a combination of mutations that together alter the distribution of charge density without altering the overall charge of Factor XI, (e) a mutation of the NH 2 - or COOH- terminal residue of Factor XT, and/or (f) a mutation that alters the folding and/or crystal packing of Factor XL
  • the protein is enzymatically de-glycosylated.
  • one or more residues that would otherwise lead to glycosylation are mutated to another residue that results in no glycosylation.
  • the protein has at least 2, 3, 4, 5, 6, 7, 8, or 9 residues that are mutated compared to a naturally occurring Factor XI protein or the catalytic domain of a naturally-occurring Factor XI protein.
  • the mutation(s) allow crystallization or increase the resolution of the corresponding three-dimensional structure of the mutant protein compared to the structure of Factor XI without the mutation(s). For example, the mutations may increase the size or order of the resulting crystals, resulting in a higher resolution structure.
  • Desirable mutant Factor XI proteins or fragments are at least 80, 85, 90, 92, 94, 96, 98, or 99% identical to any one of SEQ ID NOs: 1-4.
  • the invention provides a purified protein including a region that is at least 92, 94, 96, 98, or 100% identical to the a nino acid sequence of SEQ ID NO: 1 (human Factor XI) or SEQ ID NO: 3 (rat Factor XI).
  • the protein has an amino acid sequence at least 92, 94, 96, 98, or 100% identical to the amino acid sequence of SEQ ID NO: 1, over the entire length of SEQ ID NO: 1 or 3.
  • the invention features a purified nucleic acid encoding one or more of the proteins or protein fragments of the invention.
  • the nucleic acid is contained within a vector and operably linked to a promoter that regulates transcription of the nucleic acid.
  • FXIcat was expressed in a heterologous organism and purified for three- dimensional structural determination. The ability of individual residues or combinations of residues to influence the folding and packing of FXIcat into protein crystals was also evaluated.
  • the light chain segment of Factor Xla was expressed as a recombinant protein in the absence of Factor Xla heavy chain.
  • a human FXI cDNA fragment encompassing amino acids 370-607 of SEQ ID NO. 1 was derived by PCR amplification from a cDNA encoding the full length protein, and subcloned into pPICZ ⁇ A (Invitrogen, Cat. No. VI 95-20). The resulting plasmid was designated pPICZ ⁇ FXIcat ( Figure 8). During PCR cloning, the coding sequence of FXIcat was arranged so that a fusion protein with the S. cerevisiae ⁇ -factor was generated.
  • a single KEX-2 signal peptide cleavage site was present, allowing post- translational proteolytic cleavage at this site during the process of secretion.
  • the cleavage site was placed so that cleavage generates the same light chain segment as is found for human Factor Xla protein.
  • the pPICZ ⁇ FXIcat plasmid was transformed into the methanolotrophic yeast Pichia pastoris Mut strain, X33 (Invitrogen, Cat. No. CI 80-00) by standard methods, and transformants selected on Zeocin-containing selection plates.
  • Methanol induction of recombinant protein expression was screened by expanding 10-20 colonies each in 30 mL cultures of buffered methanol complex (BMMY) medium by standard growth conditions (Invitrogen). Methanol was added to 0.5% at 12 hour intervals for 72 hours.
  • BMMY buffered methanol complex
  • Cell supernatants were analyzed for FXIcat expression by several methods.
  • FXIcat Large-scale preparation of FXIcat was conducted in a 10 L fermentor using 1% glycerol to achieve high cell density, followed by continuous infusion of methanol (0.5-0.7%) for 100 hours. Conditioned medium was collected at intervals throughout the methanol induction phase. Alternatively, cells harboring the FXIcat vector were grown in flasks in a shaking incubator, and FXIcat expression was induced by the addition of methanol to a final concentration of 0.5% every 12 hours. Conditioned medium was collected by centrifugation after 72 hours of induction.
  • the bacterial ecotin gene and mutants thereof were expressed as recombinant proteins in the plasmid, pTacTac (see Yang, et al, J. Mol. Biol. 279:945-957, 1998).
  • the periplasmic fraction was isolated by centrifugation. Ecotin was purified by heating the lysate to 100°C for ten minutes and centrifuging to remove precipitated denatured protein. The supernatant was fractionated by reversed-phase HPLC. The protein peak corresponding to ecotin was analyzed by SDS-PAGE, and fractions containing pure ecotin were concentrated by centrifugal filtration with a Centriprep-10.
  • Recombinant FXIcat for use in crystallography was purified from Pichia pastoris conditioned medium by one of two methods.
  • the recombinant enzyme was collected from conditioned medium by ammonium sulfate precipitation.
  • the medium was first brought to 50% of saturation by the addition of crystalline (NH 4 ) 2 SO .
  • the resulting slurry was mixed at room temperature for 30 minutes, and the precipitated proteins removed by centrifugation at 20,000 x g from 20 minutes.
  • the supernatant solution was isolated and brought to 80% of saturation by the addition of (NH 4 ) 2 SO 4 .
  • the slurry was again mixed at room temperature for 30 minutes, and the precipitated protein was collected by centrifugation as described above.
  • the supernatant solution was discarded, and the precipitate was redissolved in a minimal amount of 20 mM Tris-HCl, pH 7.4.
  • the redissolved protein was dialyzed against two changes (at least 50 volumes each) of the same buffer at 4° C.
  • the dialyzed protein was applied to a 2.6 x 8 cm Chelating Sepharose Fast Flow column (Amersham Bioscience, P/N 17-0575-01) charged with Zn +2 .
  • the column was run at room temperature at a flow rate of ⁇ 8 mL/minutes. Following loading, the column was washed with additional 20 mM Tris-HCl, pH 7.4 until the A 280 of the effluent returned to baseline.
  • the column was eluted with a 250 mL gradient from 0 to 50 mM imidazole in 20 mM Tris-HCl, pH 7.4.
  • the pH of the fraction containing eluted, recombinant FXIcat was adjusted to pH ⁇ 6 by the addition of 1/200 volume of 20% CH 3 COOH.
  • the protein was then enzymatically deglycosylated by the addition of Endoglycosidase H (New England Biolabs, Cat. No. P0703S) to a final concentration of 500-1000 mU/mL. Digestion was carried out at room temperature for 1.5-2 hours.
  • the pH of the solution was brought up to pH ⁇ 7.4 by the addition of 1/200 volume of 2 M Tris, and the fraction was applied to a 1.6 x 8 cm SP-Sepharose Fast Flow column (Amersham Bioscience, P/N 17- 0729-01) equilibrated in 20 mM Tris-HCl, pH 7.4 at a flow rate of 8 mL/minutes. After the column was loaded, it was washed with additional 20 mM Tris-HCl, pH 7.4 buffer. The column was then washed with 0.1 M NaCl in the same buffer to remove minor contaminants. Recombinant FXIcat was eluted from the column with a step to 0.2 M NaCl in 20 mM Tris-HCl, pH 7.4.
  • various mutant forms of FXIcat were purified from concentrated, diafiltered P. pastoris conditioned medium. Cells and cell debris were removed by centrifugation followed by filtration through 0.8 ⁇ m mixed ester cellulose nitrate filters. The filtered medium (7-15 L) was concentrated to ⁇ 1 L using a Centramate PE apparatus equipped with a 1 OK NMWCO membrane cassette (Pall-Gelman, P/N OS010C10). The concentrate was diluted with 4 L of 20 mM Tris-HCl, pH 7.4 and reconcentrated to ⁇ 1 L. The dilution and concentration were repeated once more to yield the final concentrated, diafiltered medium. Recombinant FXIcat was purified by a combination of cation exchange and heparin-affinity chromatography. The concentrated, diafiltered medium was applied directly to a 1.6 x 8 cm SP-
  • Sepharose Fast Flow column equilibrated in 20 mM Tris-HCl, pH 7.4 at a flow rate of 8-10 mL/minutes. After the column was loaded, it was washed with additional 20 mM Tris-HCl, pH 7.4 buffer. The column was then washed with 0.1 M NaCl in the same buffer to remove minor contaminants. Recombinant FXIcat was eluted from the column with a step to 0.2 M NaCl in 20 M Tris- HCl, pH 7.4.
  • Recombinant FXIcat eluted from the SP-column was diluted 1 :1 with 20 mM Tris-HCl, pH 7.4 and applied to a 5 mL HiTrapTM Heparin column (Amersham Bioscience, P/N 17-0407-01) equilibrated in 0.1 M NaCl; 20 mM Tris-HCl, pH 7.4 at a flow rate of 3-4 mLl/minutes.
  • the protein was eluted from this column with a 50 mL gradient from 0.1 to 1 M NaCl in the same buffer.
  • the purity and concentration of the enzyme was determined by RP- HPLC and SDS-PAGE. Typically, the concentration was ⁇ 1 mg/mL, and the purity was at least 95%.
  • a third purifcation step was included.
  • the heparin column eluate was applied to a 2.6 x 90 cm Superdex 75 column equilibrated in 20 mM Tris-HCl, pH 7.4; 0.2 M NaCl at a flow rate of 4-5 mL/minutes.
  • the enzyme eluted between 70 and 90 minutes, consistent with an apparent molecular weight of 30,000.
  • rat Factor XI cDN A was derived.
  • the rat cDNA sequence was determined to be located in the vicinity of the residues corresponding to the boundaries of the light chain protein, conserved between human, rat, mouse, and rabbit proteins. Expression studies were continued as described above. DNA sequence analysis revealed that there was a significant percentage of identity between species for rat and human (79%), mouse and human (79%), and rabbit and human (91%) FXIcat.
  • modification of the protein structure of human FXIcat can be achieved by mutational change of the DNA plasmid prior to the introduction into the organism for gene expression.
  • the pPICZ ⁇ FXIcat plasmid was altered at a single codon using PCR-based mutagenesis with synthesized oligonucleotides bearing the changes.
  • one or more additional residues were altered by the same methodology in a sequential fashion.
  • pPICZ ⁇ FXIcat mutant plasmids were then transformed into the methanolotrophic yeast Pichia pastoris Mut+ strain, X33 (Invitrogen) by standard methods.
  • the mutant proteins were purified as described above and characterized in terms of their purity, activity, and crystallographic properties.
  • the substitution of an Ala for Ser434 was accomplished by the above PCR methodologies. Following the transformation and expression of the S434A mutant protein in Pichia pastoris, the migration of the protein was analyzed using a standard gel assay. The mobility of the S434A protein differed from that of the fully glycosylated (wild-type, WT) protein and that of the protein resulting from endoglycosidase H treatment of the expressed recombinant protein.
  • S434A and T475A mutations were prepared by serial mutagenesis of the Pichia pastoris expression plasmid, pPICZ ⁇ FXIcat.
  • Individual mutagenesis of either S434A or T475A reduced glycosylation of the mutant protein as evidenced by a lower apparent molecular weight by SDS-PAGE analysis.
  • the ability of the double mutations to dramatically reduce the extent of glycosylation was demonstrated by SDS-PAGE and Western blot analysis. The increased mobility of the protein in SDS-PAGE was indicative of a lack of glycosylation.
  • the single mutants had mobility intermediate to wild-type and the double mutant which migrated with an apparent molecular weight slightly lower than that of the Endoglycosidase H treated, wild-type FXIcat.
  • wild-type FXIcat yielded four distinct peaks on RP-HPLC analysis. These peaks collapsed into two major peaks following Endoglycosidase H treatment. These peaks had significantly longer retention times than the wild-type peaks.
  • the double glycosylation mutant showed a single major peak with a longer retention time than any of the enzymatically deglycosylated forms. This increased hydrophobicity is consistent with the loss of surface-exposed, hydrophilic carbohydrate moieties. Taken together with the observed absence of carbohydrate moieties in the solved structures for FXIcat, these observation suggest strongly that the mutations resulted in the complete elimination of glycosylation on this form of the protein.
  • one or more mutants were generated to eliminate free sulfhydryls within FXIcat.
  • Unpaired cysteine residues may impact folding and crystal formation.
  • the free sulfhydryl group may change the nature of the FXIcat.
  • the presence of free sulfhydryl groups on FXIcat monomers may also influence solubility, aggregation, and crystallization properties.
  • FXIcat has a single cysteine residue (Cys482) that is not involved in a disulfide bond. Cys482 forms a disulfide bond with Cys362 in intact Factor XI and thus is unpaired in the FXIcat.
  • Cys482 forms a disulfide bond with Cys362 in intact Factor XI and thus is unpaired in the FXIcat.
  • FXIcat-C482S was generated to replace the free sulfhydryl group with an isosteric serine residue.
  • K437A substitution was generated in FXIcat.
  • the neutral alanine substitution replaces the positively charged lysine residue leading to a net loss of charge in that location. Additional modifications leading to a change in charge on the surface of FXIcat can be used to further characterize the influence of Lys437 on the structure and packing of FXIcat.
  • a D476A substitution was also created using the methodology described above. The alanine substitution replaces the negatively charged aspartic acid, leading to a local change of charge; however, the charge change is opposite to that of K437A.
  • a series of substitution mutations that do not alter the net surface charge of the FXIcat monomer, but change the charge distribution were generated.
  • a localized region of FXIa monomers is important to the packing of crystals, changes in the charge distribution is expected to impact crystal integrity.
  • the double mutation N571D, E572Q may change the charge distribution in one local region of the protein monomer. This alteration of the charge distribution may impact packing of crystals.
  • a related strategy involved the generation of a series of substitution mutants that affect the packing of FXIcat monomers in crystals by influencing van der Waals interactions between hydrophobic residues and the protein backbone of another monomer in the crystal.
  • the residue Tyr416 is oriented such that the active site is at least partially occluded, and such that this residue interacts with the peptide backbone of another FXIcat monomer in the crystal packing. Since this position is a serine residue in the mouse and rat FXI proteins, substitution of Y416S was expected to retain the activity of the protein. The Y416S substitution disrupts the interaction of the tyrosine residue with the peptide backbone of another monomer.
  • substitution mutations that combine the different features of the strategies described above are examined to yield a different outcome than any of the substitution mutations by itself. For example, mutations that alter the charge distribution of the protein surface can be combined with mutations that block glycosylation of the protein and the availability of free sulfhydryl groups on the surface. Therefore, the FXIcat-
  • S434A,T475A,C482S triple mutant was made.
  • mutations with the same functional effects on protein folding and/or packing were examined in different combinations.
  • a serial substitution at K437A and K505A is a means to combine the effects of eliminating positively charged lysine residues at two different locations on the surface of FXIcat.
  • the quadruple mutant S434A, T475A, K437A, C482S has proven quite robust for obtaining useful crystals of various inhibitors as exemplified in the examples that follow.
  • FXIcat-ecotin complex was formed by incubating a stoichiometric quantity of each component at 4 °C overnight and subsequently isolated by chromatography on a Superose 12 column (Amersham Bioscience) in 20 mM Tris-HCl, pH7.8 and 0.1 M NaCl on BioCAD 700E.
  • the FXIcat-ecotin M84R complex was crystallized at room temperature by the hanging drop vapor diffusion method (McPherson, Methods Biochem. Anal. 23:249, 1976) from 0.2 M NaCl, 20% (w/v) PEG-1000 and 0.1 M Na/K phosphate, pH 6.2.
  • Example 8 Structure Determination, Model Building, and Refinement
  • the structure of FXIcat-ecotin M84R was solved by molecular replacement using A ore.
  • the search model was created from the coordinates of the tetrameric rat granzyme B [N66Q] - ecotin [81-84 ⁇ EPD] with PDB code 1FI8 (Waugh et al, Nat. Struct. Biol. 7:762, 2000).
  • the conserved core structure of serine proteases was identified first by structure alignment of several serine proteases. All the side chain atoms beyond C ⁇ , all surface loops in granzyme B, and all water molecules were deleted from the model.
  • Desirable inhibitors have excellent steric and electrostatic complementarity to Factor Xla, a fair amount of buried hydrophobic surface, and sufficient conformational rigidity to minimize entropy loss upon binding (see, for example, Babine and Bender, Chem. Rev., 97:1359-1472, 1997).
  • a target site in FXIcat is defined.
  • This region can be any place important or essential for the protease activity such as, but not limited to, the active site cavity of Factor Xla. Because the FXIcat crystal structure was first determined in the presence of ecotin, a protein inhibitor, the spatial and chemical properties of the active site cavity of Factor Xla were clearly delineated and aped.
  • the target site can be a region other than the active site, such as a region that when bound by an inhibitor prevents a substrate from binding Factor Xla or prevents Factor Xla from adopting a three-dimensional structure that enables full catalytic activity.
  • a small molecule or other candidate molecule is docked onto the target site of Factor Xla.
  • Many methods can be used for this pu ⁇ ose such as, but not limited to, fast shape matching (Dock [Kuntz et al, J. Mol. Biol, 161 :269-288, 1982]; Eudock [Perola et al, J. Med. Chem., 43:401- 408, 2000]); incremental construction (FlexX [Rarey et al, J. Mol. Biol, 261 :470-89, 1996]; Hammerhead [Welch et al, Chem.
  • the peptidolytic activity of recombinant FXIcat was measured using the chromogenic substrate pyroGlu-Pro-Arg-p-nitroanaline (DiaPharma, Cat. No. S-2366).
  • the substrate was diluted to 1 mM in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.02% Tween20 (assay buffer) and mixed with an equal volume of diluted enzyme in a microtiter plate (Wallac Isoplate, P/N 1450-514). The final volume in the wells was 100 ul.
  • the plate was placed immediately into a ThermoMax plate reader (Molecular Devices), and the time course of hydrolysis was monitored for 10 minutes at 405 nm. The progress curves were visually inspected and the linear portion of the curves was used to determine the activity of the enzyme.
  • Factor Xla Potential inhibitors of Factor Xla were evaluated using a modification of the above assay. Briefly, the inhibitors were tested against native Factor Xla (Haematological Associates, P/N FXIA01) using a custom made analog of S- 2366. This substrate had the/?-nitroanaline group replaced with 7- methylaminocoumarin (AMC). The final concentration of the substrate in the assay was 100 ⁇ M, and the final concentration of the enzyme was 0.25 nM. Inhibitors were tested by serial dilution over an appropriate range to yield a dose response curve for determination of the inhibitors' IC 50 values. The assay mixture was incubated for 30 minutes at 30° C, and the fluorescent yield was measured using a PE- Wallac Victor 2 Plate Reader.
  • the compounds have an IC 50 value better than 1.0 micromolar, 0.1 micromolar, 0.01 micromolar or 0.001 micromolar (in order of increasing preference).
  • the FXIcat-wild type ecotin complex was crystallized at room temperature by the hanging drop vapor diffusion method (McPherson, Methods Biochem. Anal. 23:249-345, 1976) from 0.1 M (NH 4 ) 2 SO 4 , 22% ⁇ w/v) PEGMME-2000 and 0.1 M Na cacodylate, pH 6.2.
  • the coordinates of FXIcat-ecotin M84R were used for the calculation of the difference electron density map by the program CNX (Accelrys Inc., San
  • the FXIcat-ecotin IX-D complexe was crystallized at 10°C by the hanging drop vapor diffusion method (McPherson, Methods Biochem. Anal. 23:249-345, 1976) from 0.1 M (NH 4 ) 2 SO 4 , 22% (w/v) PEGMME-2000 and 0.1 M Na cacodylate, pH 6.2.
  • FXIcat-ecotin M84R The coordinates of FXIcat-ecotin M84R were used for the calculation of the difference electron density map by the program CNX (Accelrys Inc., San Diego, California) for FXIcat-ecotin IX- D. Subsequent model building was carried out in the program Quanta2000 (Accelrys Inc., San Diego, California) and refinement was done by CNX.
  • Example 20 Crystallization of FXIcat with SPRL-122624 A ten-fold molar excess of 10 mM SPRL-122624 in DMSO was added to the FXIcat-S434A,T475A,C482S,K437A, incubated 30 minutes at 20 °C and then concentrated to 20 mg/mL. Cubic crystals were obtained using the hanging drop method (McPherson, Methods Biochem. Anal. 23:249-345, 1976) with 20% (w/v) PEG monomethyl ether 5000, 0.1 M ammonium sulfate, and 0.1 M Tris-HCl, pH 7.6 at 10 °C.
  • Example 21 Crystallization of FXIcat with SPRL-123586 A ten-fold molar excess of 10 mM SPRL-123586 in DMSO was added to the FXIcat-S434A,T475A,C482S,K437A, incubated 30 minutes at 4°C and then concentrated to 20 mg/mL. Needle-like crystals were obtained using the hanging drop method (McPherson, Methods Biochem. Anal. 23:249-345, 1976) with 1.4 M tri-sodium citrate, 0.1 M HEPES, pH 7.5 at 10 °C.
  • Diffraction data were measured to 2.7 A and processed by HKL2000.
  • the orientations of the two monomers were determined by AmoRe using the structure described in Example 13 as model.
  • the solutions were refined in CNX (Accelrys Inc., San Diego, California).
  • the difference electron density map was also calculated in CNX.
  • Example 22 Soaking SPRL-130421 into preformed FXIcat-benzamidine crystal
  • the preformed FXIcat-benzamidine crystals were obtained in a condition similar to Example 13. Benzamidine was added to the FXIcat- S434A,T475A,C482S,K437A protein to a concentration of 1 mM and the complex was concentrated to 20 mg/mL. Cubic crystals were obtained by the hanging-drop method in 2.0 M ammonium sulfate and 0.1 M Tris-HCl, pH 8.5 at 10 °C. A preformed FXIcat-benzamidine crystal was transferred into a drop containing its mother liquor with 5 mM of SPRL-130421 and incubated at 10 °C overnight.
  • Diffraction data were measured to 1.9 A and processed by HKL2000.
  • Phases were calculated using the structure of FXIcat-SPRL-123545 described in Example 19 as the model, and CNX (Accelrys Inc., San Diego, California) was used for calculating the difference electron density map. Model building was performed using Quanta (Accelrys Inc., San Diego, California).
  • the compounds of formula I may be synthesized by either solid or liquid phase methods described and referenced in standard textbooks, or by a combination of both methods. These methods are well known to those skilled in the art, (see, for example, Bodanszky, In “The Principles of Peptide Synthesis”, Hafner, Rees, Trost, Lehn, Schleyer, Zahradnik, Eds., Springer- Verlag, Berlin, 1984; Stewart and Young, Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, 111. (1984); and U.S. Pat. Nos. 4,105,603; 3,972,859; 3,842,067; and 3,862,925).
  • compounds of formula I can be prepared by standard amide bond forming reactions, including fragment condensation when compounds of formula la are coupled to compounds of formula lb, wherein A, R°, R 1 , R 2 , and R 3 are as previously defined herein.
  • Peptide alpha-keto acids, esters, thio esters, and amides can be prepared as described by Medhi et al., Biochem. Biophys. Res. Commun. 166:595-600, 1990; Angelastro et al, J. Med. Chem. 33:11-13, 1990; Wipf et al, J. Org. Chem. 58:5592-5594, 1993; or Webb et al, PCT Publication WO 9408941.
  • Ketothiazoles can be prepared by solid-phase synthesis as described by Parlow et al, J. Med. Chem. 46:4043-9, 2003.
  • the pyrimidinones of formula I can be prepared as described by Veale et al, J. Med.Chem. 38:98-108, 1995; Tamura et al, PCT Publication WO 9618644, South et al, PCT Publication WO 0069832; Ewin, et al, PCT Publication WO 0032590; and Akahoshi et al, J. Med Chem. 44:1286-96, 2000.
  • the functional groups of the amino acid derivatives used in these methods are protected by blocking groups to prevent cross reaction during the amide bond- forming procedure.
  • blocking groups examples of suitable blocking groups and their use are described in "The Peptides: Analysis, Synthesis, Biology," Academic Press, Gross & Meienhofer, Eds., Vol. 3 (1981) and Vol. 9 (1987).
  • the reaction products are isolated and purified by conventional methods, typically by solvent extraction into a compatible solvent, or by using a washing protocol for resin bound intermediates.
  • the products may be further purified by column chromatography or other appropriate methods, including medium pressure or high pressure liquid chromatography.
  • compounds of the invention can be conveniently prepared using solid phase synthesis methodology (Merrifield J. Am. Chem.
  • Solid phase synthesis begins at the carboxy terminus of the putative compound by forming a bond (e.g., an amide or ester bond) between a protected amino acid, or other carboxylic acid-containing compound, and an inert solid support.
  • the inert solid support can be any macromolecule capable of serving as an anchor for the C-terminus of the initial amino acid.
  • the macromolecular support is a cross-linked polymeric resin (e.g. a polyamide or polystyrene resin).
  • the C-terminal amino acid is coupled to a polystyrene resin to form a benzyl ester.
  • Particularly useful benzyl-type resins such as trityl resin, chlorotrityl resin, and Wang resin, are those in which the linkage of the carboxy group (or carboxamide) to the resing is acid-lable.
  • a macromolecular support is selected such that the peptide anchor link is stable under the conditions used to deprotect the ⁇ -amino group of the blocked amino acids in peptide synthesis. If a base-labile ⁇ -protecting group is used, then it is desirable to use an acid-labile link between the peptide and the solid support.
  • an acid-labile ether resin is effective for base-labile Fmoc-amino acid peptide synthesis as described by Stewart and Young, vide supra.
  • a peptide anchor link and ⁇ -protecting group that are differentially labile to acidolysis can be used.
  • an aminomethyl resin such as the phenylacetamidomethyl (Pam) resin works well in conjunction with Boc-amino acid peptide synthesis.
  • the ⁇ - amino protecting group of the initial amino acid is removed with, for example, trifluoroacetic acid (TFA) in methylene chloride and neutralizing in, for example, triethylamine (TEA).
  • TFA trifluoroacetic acid
  • TAA triethylamine
  • the next . ⁇ -amino and side chain protected amino acid in the synthesis is added.
  • the remaining ⁇ -amino protected and, if necessary, side chain protected amino acids are then coupled sequentially in the desired order by condensation to obtain an intermediate compound connected to the solid support.
  • some amino acids may be coupled to one another in solution to form a fragment of the desired peptide followed by addition of the peptide fragment to the growing solid phase peptide chain.
  • the condensation reaction between two amino acids, or an amino acid and a peptide, or a peptide and a peptide can be carried out according to the usual condensation methods such as the azide method, acyl halide method (preferably acyl fluoride), mixed acid anhydride method, DCC (N,N- dicyclohexylcarbodiimide) or DIC (N,N'-diisopropylcarbodiimide) methods, active ester method, p-nitrophenyl ester method, BOP (benzotriazole-1-yl-oxy- tris[dimethylamino] phosphonium hexafluorophosphate)method, N- hydroxysuccinicacid imido ester method, O-benzotriazolyl-N,N,N',N'- tetra
  • Suitable protecting groups for ⁇ -amino and side chain amino groups are exemplified by benzyloxycarbonyl (abbreviated Z), isonicotinyloxycarbonyl (iNOC), o-chlorobenzyloxycarbonyl [Z(2C1)], p-nitrobezyloxycarbonyl
  • Protective groups for the carboxy functional group are exemplified by benzyl ester, (OBz), cyclohexyl ester (Chx), 4-nitrobenzyl ester (ONb), t-butyl ester (OtBu), 4-pyridylmethyl ester (OPic), and the like. It is often desirable that amino acids possessing a functional group other than amino and carboxy groups, such as, for example, arginine, cysteine, histidine, asparagine, glutamine, threonine, tyrosine, tryptophan and serine, be protected by a suitable protecting group.
  • the guanidino group may be protected with nitro, p-toluenesulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl, p- methoxybenzenesulfonyl, 4-methoxy-2, 6-dimethylbenzenesulfonyl (Nds), 1,3,5-trimethylphenysulfonyl (Mts), and 2,3,6-trimethyl-4- methoxyphenylsulfonyl (Mtr), and the like.
  • the thiol group can be protected with p-methoxybenzyl, trityl, and the like.
  • Other suitable protecting groups for hydroxy, phenoxy, the indole amine, carboxamide, the imidazole nitrogen can be found in Greene, vide supra.
  • the compounds of the invention are synthesized with the help of blocking groups that protect the side chain amide bond- forming substituents of the N-terminal and C-terminal flanking residues.
  • the protecting group or groups used for the side chain amide bond-forming substituents of the N-terminal and C-terminal flanking residues can be the same or different than the protecting group or groups used to block the side chain functional groups of other residues in the peptide.
  • the protecting group or groups used to block the side chain amide bond- forming substituents is (are) differentially removable with respect to the protecting groups used for other side chain functional groups, i.e.
  • the side chain amide bond-forming substituents can be deprotected without deprotecting the other side chain functional groups, in addition to being differentially removable with respect to the ⁇ -amino protecting group used in peptide synthesis.
  • the side chain amide bond-forming substituents of the flanking residues are orthogonally protected with respect to each other such that the side chain amide bond-forming substituent of one flanking residue can be deprotected without deprotecting the side chain amide bond-forming substituent of the other flanking residue.
  • Suitable protecting groups for use in orthogonally protecting the side chain amide bond-forming substituents of the flanking residues with respect to other functional groups and/or with respect to each other include pairs of differentially removable carboxy protective groups, such as a reduction-labile carboxy protective group, e.g. allyl or benzyl esters, paired with a base-labile carboxy protective group, e.g. fluorenylmethylesters, methyl or other primary alkyl esters.
  • a reduction-labile carboxy protective group e.g. allyl or benzyl esters
  • a base-labile carboxy protective group e.g. fluorenylmethylesters, methyl or other primary alkyl esters.
  • Fluorenylmethyl, methyl or other primary alkyl groups or other base-labile carboxy protective groups can be removed from their corresponding esters to yield a free carboxy group (without deprotecting allyl or benzyl esters or other reduction-labile esters) by saponification of the esters with a suitable base such as piperidine and sodium hydroxide in a suitable solvent such as dimethylacetamide, or methanol and water, for a period of 10 to 120 minutes, and preferably 20 minutes, 0°C to 50°C.
  • the allyl or benzyl or other reduction- labile esters can be removed when desired by reduction in the presence of a suitable transition metal catalyst, such as Pd(PPh 3 ) 4 , Pd(PPh 3 ) 2 Cl 2 , Pd(OAc) 2 or Pd on carbon with a source of hydrogen, e.g. H 2 gas, in a suitable solvent such as dimethylacetamide, dimethylformamide, N-methylpyrrolidinoneor methanol for a period of 10 to 500 minutes, and preferably 100 minutes, at 0°C to 50°C.
  • a suitable transition metal catalyst such as Pd(PPh 3 ) 4 , Pd(PPh 3 ) 2 Cl 2 , Pd(OAc) 2 or Pd on carbon with a source of hydrogen, e.g. H 2 gas
  • a suitable solvent such as dimethylacetamide, dimethylformamide, N-methylpyrrolidinoneor methanol for a period of 10 to 500 minutes, and preferably 100 minutes,
  • the Pd catalyst can be restored by reaction with a suitable nucleophile, such as piperidine or tributyltin hydride.
  • a suitable nucleophile such as piperidine or tributyltin hydride.
  • the carboxy substituents of the flanking residues can be orthogonally protected with respect the other functional groups and/or with respect to each other by using an acid-labile protecting group, such as a tertiary alkyl ester, e.g. t-butyl ester, in combination with a reduction-labile protecting group, such as the allyl or benzyl esters described above.
  • an acid-labile protecting group such as a tertiary alkyl ester, e.g. t-butyl ester
  • a reduction-labile protecting group such as the allyl or benzyl esters described above.
  • the tertiary alkyl or other acid-labile ester group can be removed by acidolysis, e.g. with trifluoroacetic acid in methylene chloride, and the ally] or benzyl or other reduction-labile esters can be removed by reduction in the presence of a transition metal catalyst as described above.
  • the carboxy substituents of the flanking residues can be orthogonally protected with respect to other functional groups and/or with respect to each other by using a fluoride ion-labile protecting group, such as 2-(trimethylsilyl)ethyl and silyl esters, in combination with a reduction- labile protecting group, such as the ally] or benzyl esters described above, or in combination with a base-labile protecting group, such as the fluorenylmethyl, methyl or other primary alkyl esters described above, without deprotecing the reduction-labile or base-labile esters.
  • a fluoride ion-labile protecting group such as 2-(trimethylsilyl)ethyl and silyl esters
  • a reduction- labile protecting group such as the ally] or benzyl esters described above
  • a base-labile protecting group such as the fluorenylmethyl, methyl or other primary alkyl esters described above
  • the 2-(trimethylsilyl)ethyl, silyl or other fluoride-labile ester group can be removed by reaction with a suitable fluoride ion source, such as tetrabutyl ammonium fluoride in the presence of a suitable solvent, such as dimethylacetamide (DMA), dimethylformamide (DMF), tetrahydrofuran (THF), or acetonitrile.
  • a suitable fluoride ion source such as tetrabutyl ammonium fluoride
  • a suitable solvent such as dimethylacetamide (DMA), dimethylformamide (DMF), tetrahydrofuran (THF), or acetonitrile.
  • Suitable protecting groups for use in orthogonally protecting the side chain amide bond-forming substituents of the flanking residues with respect to other functional groups and/or with respect to each other also include pairs of differentially removable amino protective groups, such as an allyloxycarbonyl or other reduction-labile amino protective group paired with a t-butoxycarbonyl (Boc) or other acid-labile amino protective group, and a reduction-labile amino protective group paired with a fluorenylmethoxycarbonyl (Fmoc) or other base- labile amino protective group.
  • An allyloxycarbonyl (or other reduction-labile blocking group) protected amino group can be deprotected by reduction using a transition metal catalyst as in the procedure for removing reduction-labile carboxy protective groups described above, without deprotecting a Boc or
  • an acid-labile amino protective group and a base-labile amino protective group can be removed by acidolysis and base saponification, respectively, without removing a reduction-labile amino protective group.
  • all amino protective groups that are removable by Pd-catalyzed methods which result in the reduction of the protected amino substitutent are included in the term "reduction-labile protective groups" as used herein, even though such Pd- catalyzed deprotection methods may not result in the reduction of the protective group in question.
  • the amino substituents of the flanking residues can be orthogonally protected with respect to other functional groups and/or with respect to each other by using a fluoride-labile protecting group, such as 2-trimethylsilylethylcarbamate (Teoc), in combination with a reduction-labile protecting group, such as allyloxylcarbonyl, or in combination with a base- labile protecting group, such as fluorenylmethoxycarbonyl, as described above.
  • a fluoride-labile protecting group such as 2-trimethylsilylethylcarbamate (Teoc)
  • Teoc 2-trimethylsilylethylcarbamate
  • a reduction-labile protecting group such as allyloxylcarbonyl
  • a base- labile protecting group such as fluorenylmethoxycarbonyl
  • the Teoc or other fluoride-labile group can be removed by reaction a with a suitable fluoride ion source, such as tetrabutyl ammonium fluoride, as in the procedures for removal of fluoride-labile carboxy protective groups described above, without deprotecting an allyloxycarbonyl or fluorenylmethoxycarbonyl protected amino group.
  • a reduction-labile amino protective group and a base-labile amino protective group can be removed by reduction and base saponification, respectively, without removing a fluoride-labile amino protective group.
  • the carboxy substituent and the amino substituent can be orthogonally protected with respect to each other by using a reduction-labile protecting group to block one substituent, e.g. allyl ester or allyloxycarbonyl, and a fluoride-labile, acid-labile or base-labile protecting group to block other substituent, e.g. silyl ester, t-butyl ester, fluorenylmethyl ester, Teoc, Boc, or Fmoc.
  • a reduction-labile protecting group to block one substituent
  • a fluoride-labile, acid-labile or base-labile protecting group to block other substituent, e.g. silyl ester, t-butyl ester, fluorenylmethyl ester, Teoc, Boc, or Fmoc.
  • a reduction-labile protecting group is used to block the side chain amide bond-forming substituent of one flanking residue and the protecting group for the side chain amide bond-forming substituent of the other flanking residue is selected such that it provides orthogonal protection with respect to both the reduction-labile protecting group and the ⁇ -amino protecting group used in the synthesis.
  • orthogonal protection of the side chain amide bond-forming substituents would be provided by a reduction-labile protecting group and an acid-labile protecting group.
  • orthogonal protection of the side chain amide bond-forming substituents would be provided by a reduction-labile protecting group and a base-labile protecting group.
  • the side chain amide bond- forming substituents of the flanking residues are orthogonally protected with respect to each other, with respect to ⁇ -amino protecting group used in the synthesis, and with respect to the protecting groups used to block other side chain functional groups in the peptide chain.
  • the side chain amide bond-forming substituents of the flanking residues are orthogonally protected with respect to each other, and with respect to ⁇ -amino protecting group, but only one of the side chain amide bond-forming substituents is orthogonally protected with respect to the protecting groups used to block other side chain functional groups.
  • the amide bond-forming reaction will be specific to the desired side chain amide bond-forming substituent, and will reduce the production of unwanted difunctional linker derivatives.
  • cyclization will require the deprotection of the side chain amide bond-forming substituent of the other flanking residue, and may cause concomitant deprotection of other side chain functional groups, unwanted derivatives are less likely to form given that the peptide chains are anchored to a solid support and that the linker length will regioselectively favor a amide bond-forming reaction between the unbound functional group of the linker and the side chain amide bond-forming substituent of the other flanking residue.
  • any side chain functional groups on other amino acid residues left unprotected by the cyclization reactions can be reprotected before chain synthesis is resumed.
  • Many of the blocked amino acids described above can be obtained from commercial sources such as Novabiochem (San Diego, Calif), Bachem Calif. (Torrence, Calif.) or Peninsula Labs (Belmont, Calif).
  • functionalized or protected amino acids, including unnatural amino acids can be prepared by methods known in the art.
  • the compounds of the invention can be prepared by, or in conjunction with, solution phase peptide synthesis, for example, the solution phase peptide synthesis methods described in Principles of Peptide Synthesis, 2d ed, M.
  • R° is an optionally substituted alkyl, alkenyl, cycloalkyl, aralkyl, aralkenyl, aryl, heterocyclyl, heteroaralkyl, or heteroaralkenyl, or C(O)R 9 , where R 9 is as previously defined herein)
  • R 9 is as previously defined herein
  • appropriately protected amino acid derivatives having a free carboxyl group e.g., an appropriately alpha-amine protected analog of formula lb, where m is 1 and R° is OH
  • an alkoxyamine under amide bond-forming conditions, as described herein, to form Weinreb amides of formula III, where P is a nitrogen protecting group.
  • the Weinreb amide can be reacted with alkyl, alkenyl, cycloalkyl, aralkyl, aralkenyl, aryl, heterocyclyl, heteroaralkyl, heteroaralkenyl, or heteroaryl carbanions such as lithium salts or Grignard reagents to form ketones of formula IV, which can be subsequently deprotected at the alpha-amine position to form compounds of formula lb, where m is 1 and R° is alkyl, alkenyl, cycloalkyl, aralkyl, aralkenyl, aryl, heterocyclyl, heteroaralkyl, heteroaralkenyl, or heteroaryl.
  • These ketone compounds can then be coupled to the carboxyl- containing compounds of formula la to produce the corresponding compounds of formula I.
  • ketone derivatives of the formula I can be prepared from Weinreb amide III by reduction of this amide to the corresponding aldehyde with a hydride reagent, such as DIBAL-H or LAH, to produce compounds of formula V, to which an R group can be introduced by the addition of a nucleophile, such as the carbanions, organolithio reagents, or Grignard reagents described before in the nucleophilic addition to Weinreb amides.
  • a nucleophile such as the carbanions, organolithio reagents, or Grignard reagents described before in the nucleophilic addition to Weinreb amides.
  • the resulting compound can then be reacted with compounds of formula la, as previously described, followed by oxidation of the secondary alcohol, such as with Dess- Martin reagent, to yield a compound of formula IV.
  • compounds of formula I where m is 1 and R° is 2-thiazole, are prepared by reacting aldehydes of formula V with 2-trimethylsilyl (TMS) thiazole.
  • TMS 2-trimethylsilyl
  • the resulting TMS ether is deprotected with fluoride and subsequent oxidation of the resulting secondary alchohol, followed by removal of the amine protecting group and reaction with compounds of formula la, provides thiazole ketones of formula I (i.e., m is 1 and R° is 2-thiazole).
  • TMS 2-trimethylsilyl
  • R 9 is, for example, an optionally substituted alkyloxy, aryloxy, heteroaryloxy, aralkyloxy, alkylthio, cycloalkylthio, arylthio, aralkylthio, alkylamino, alkylamino, dialkylamino, aralkylamino, arylamino, heterocyclylamino, or heterocyclylakylamino.
  • the acetoxy and amine protecting groups of the compounds of formula VI can be removed and the amine coupled to compounds of formula la to form the corresponding compounds of formula VII, which can be further transformed by oxidation, with oxidants such as Dess-Martin reagent, to provide ketones of formula I, wherein m is 1 and R° is, for example, an alkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, or aralkoxycarbonyl.
  • the acetoxy protecting group can be retained and carried through the coupling step and removed later.
  • thiouracil is methylated (methyl iodide) then halogenated (e.g., with 1 2 or Br 2 ), and the resulting halopyrimidinone XI is alkylated with electrophile XII, where Y 1 is Br, I, or OTf and P 1 is a carboxylic acid protecting group, to produce compounds of the formula XIII.
  • amine groups including primary and secondary amines of the formula B-N(R 18 )H, where R is H or alkyl and B is an optionally substituted alkyl, alkenyl, cycloalkyl, aralkyl, aryl, heterocyclyl, heterocyclylalkyl, or a suitable nitrogen protecting group, can be inco ⁇ orated into the pyrimidinone scaffold utilizing, for example, the palladium-mediated amide bond-forming method of Buchwald, et al, J. Org. Chem. 65:1144-1157, 2000, to produce compounds of the formula XlYa.
  • B is a nitrogen protecting group, it can be removed and the free amine thus generated further functionalized such that B can be an acyl, aroyl, heteroaroyl, alkoxycarbonyl, cycloalkoxycarbonyl, aralkoxycarbonyl, aryloxycarbonyl, heterocyclyloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, alkylthiocarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl, alkoxythiocarbonyl, cycloalkoxythiocarbonyl, aralkoxythiocarbonyl, aryloxythiocarbonyl, aminothiocarbonyl, alkylaminothiocarbonyl, dialkylaminothiocarbonyl, cycloalkyla
  • Substituents R 17 which includes, but is not limited to, substituted aromatic rings, can be introduced at the 2-position of the pyrimidinone scaffold of compound XlVa by utilizing, for example, C-C palladium-mediated bond forming conditions (see, for example, Lindley, Tetrahedron 40:1433, 1984) to produce compounds of the formula XlVb after removal of the carboxyl protecting group, P 1 .
  • Other intermediates of formula XlVb can be prepared by alkylating thiouracil with another appropriately activated compound other than
  • R is alkylthio, cycloalkylthio, arylthio, aralkylthio, heterocyclylthio, or heteroaralkylthio.
  • the thiomethyl group, or the methyl sulfoxide or methyl sulfone derived therefrom, of compounds of formula XlVa can be displaced with other nucleophilic groups, such as, for example, the NH 2 group of various amines, to produce compounds for formula XlVb where R17 is alkylamino, cycloalkylamino, aralkylamino, arylamino, or heterocyclylamino.
  • naphthyl amidines or amines i.e., each of e and g is C-H
  • naphthyl amidines or amines can be prepared, at least in part, as described by Geye et al, U.S. Patent No. 6,258,822 and U.S. Patent No. 6,284,796 Bl ; Klinghofer et al, Biochemistry 40:9125-9131, 2001; and Atwell et al, EP Patent 0206802 and U.S. Patent No. 4,904,659.
  • Quinazoline amidines or amines i.e., each of e and g is N
  • T is NH
  • B is an alkyl, substituted carbonyl or substituted sulfonyl group
  • f is a bond
  • V is H
  • R 19 is H
  • Z 1 is NH
  • 8-substituted naphthalene-2- carboxamidines can be prepared starting from 8-amino-naphthalen-2-ol, which is first N-protected, such as with the carbonylbenzyloxy group (Cbz), and then activated at the 2-position, such as by conversion of the hydroxyl group to a trifiate.
  • the triflate can be cyanated under Pd-mediated C-C bond forming conditions (see (a) Takagi et al, Chem. Lett.
  • the naphthylamine can be derivatized with acid halides, sulfonyl chlorides, chloroformates, isocyanates, isothiocyanates, alkyl halides, or coupled to amino acid derivatives to provide compounds of the formula XVI where T is NH and B is a natural or unnatural alpha-amino acid residue of D- or L-configuration, or an optionally substituted alkyl, cycloalkyl, alkenyl, aralkyl, aralkylcarbonyl, aralkenyl, aryl, heteroaralkyl, heteroaralkylcarbonyl, heteroaralkenyl, acyl, cycloalkylcarbonyl, aroyl, heterocyclyloyl, cycloalkoxycarbonyl, aryloxycarbonyl, heterocyclyloxycarbonyl, heteroaralkyloxycarbonyl, aminocarbonyl, alkylamino
  • the nitrile group can then be transformed to an amidine group under a variety of conditions, including acidic conditions in alcohol (e.g., Pinner conditions followed by aminolysis as described by (a) Pinner, Die Imino ⁇ ther und emp Derivate, Verlag, R. Oppenheim: Berlin, 1892; (b) Roger et al, Chem. Rev. 61 :179, 1961; (c) Schaefer et al, J. Org. Chem. 26:412, 1961), or treatment with hydroxylamine followed by acetylation of the N-OH amidine group and subsequent reduction under hydrogenation conditions (see Judkins et al, Synth. Commun.
  • acidic conditions in alcohol e.g., Pinner conditions followed by aminolysis as described by (a) Pinner, Die Imino ⁇ ther und Hä Derivate, Verlag, R. Oppenheim: Berlin, 1892; (b) Roger et al, Chem. Rev. 61 :179, 1961; (c) Schaefer
  • Compounds of formula II, where T is a bond, B is H, each of e and g is C-H, f is O, and Z is NH can be prepared from 2-cyano-6-hydroxynaphthalene, which is first O-alkylated with an alkyl halide, aralkyl halide, or heteroaralkyl halide, where the halide group is chloro, bromo, or iodo, to give compounds of the formula XVIII.
  • O-alkylation can be achieved with alkyl alcohols, aralkyl alcohols, or heteroaralkyl alcohols under Mitsunobu conditions using diispropylazodicarboxylate and triphenylphosphine.
  • amidine, hydroxyamidine, or aminoamidine groups can be obtained from the nitrile, as previously described herein, to afford compounds of the formula XIX, where R 19 is H, and R 22 is H, alkyl, OH, or NH 2 , and V is an alkyl, aralkyl, or heteroaralkyl group.
  • R is H, alkyl, OH, or NH 2
  • V is an alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyi, heterocyclylaminocarbonyl, alkoxycarbonyl, cycloalkoxycarbonyl, aralkoxycarbonyl, aryloxycarbonyl, heterocyclyloxycarbonyl, or heteroaralkyloxycarbonyl.
  • the primary amine of compounds of formula XXIII can be monoalkylated via reaction with an alkyl halide or via reductive amination procedures, followed by treatment with an activated carboxylic acid, to produce compounds of formula XXIV, where T is NR 23 , where R 23 is alkyl.
  • Amidine, hydroxyamidine, or aminoamidine groups can be obtained from the nitrile as described previously to afford compounds of the formula XXV, where R 19 is H and R 22 is H, alkyl, OH, or NH , and V is an alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, or heterocyclylaminocarbonyl group.
  • the compounds of formula XXV in which T is a bond, B is NO 2 , V is methoxycarbonyl, R 22 is H, and R 19 is H, alkyl, OH, CO 2 -alkyl or NH 2 can be obtained directly from 6-cyano-4-nitro-naphthalene-2-carboxylic acid methyl ester from the nitrile as described herein or the methoxycarbonyl group can be transformed to compounds of formula XXV in which T is a bond, B is NO 2 , V is an alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, or heterocyclylaminocarbonyl group, followed by transformation of the cyano moiety to an amidine, hydroxyamidine, or aminoamidine group.
  • the nitro group of 6-cyano-4-nitro-naphthalene-2- carboxylic acid methyl ester can be reduced and optionally acylated as described herein to produce compounds of formula XXV, wherein V is methoxycarbonyl.
  • Those compounds of formula XXII in which V is an alkoxycarbonyl can be further saponified and reduced with borane or LiBH 4 to provide, after the cyano is converted to an amidine, compounds of the formula XXV is which V is hydroxylmethyl, or derivative thereof.
  • Quinoline compounds of formula II, where e is N and g is C-H can be prepared via the condensation product XXIX derived from 4-iodoaniline, pyruvic acid, and O-benzyl protected salicylaldehyde (Doebner-Miller protocol, see M. Matsugi et al, Tet. Lett. 41 :8523-8525, 2000).
  • the resulting quinoline carboxylic acid is reacted with CuCN to displace the iodo group with a cyano group after the carboxyl group is converted by a Curtius rearrangement to a carbamoylated amine.
  • the cyano- aminoquinoline intermediate, XXX can be acylated (or otherwise derivatized), followed by subsequent conversion of the nitrile group to an amidine group as previously described to provide quinoline compounds of the formula II, where T is NR 23 ;
  • B is acyl, cycloalkylcarbonyl, aroyl, heterocyclyloyl, cycloalkoxycarbonyl, aryloxycarbonyl, heterocyclyloxycarbonyl, heteroaralkyloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, heteroaralkylaminocarbonyl, alkylthiocarbonyl, cycloalkylthiocarbonyl, arylthiocarbonyl, aralkylthiocarbony
  • Compounds of formula II in which e is N, f is a bond, and V is an optionally substituted alkyl, cycloalkyl, alkenyl, aralkyl, aralkenyl, heteroaralkyl, heteroaralkenyl, aryl, or heterocyclyl can be prepared by using the appropriate aldehyde.
  • a particularly useful aldehyde is 2-furaldehyde, as the furyl group can be subsequently oxidized with RuCl 3 /NaIO 4 and the resulting carboxyl group transformed to amides, esters, or thioesters, as described herein.
  • 4-aminobenzonitrile can be converted to 6-cyano-4- hydroxyquinoline-2-carboxylic acid, methyl ester, by Michael addition to dimethylacetylene dicarboxylate, followed by thermal cycloaddition (see Jaen et al., J. Med. Chem. 38:4439-45, 1995).
  • the resulting hydroxyquinoline is converted to 4-amino-6-cyanoquinoline-2-carboxylic acid, methyl ester by bro ination with tetrabutyl ammonium bromide/phosphorus pentoxide (see Kato et al, Tetrahedron Lett.
  • the amine can be acylated ⁇ or otherwise derivatized, such as, for example, to an amide), followed by subsequent conversion of the nitrile group to an amidine group as previously described for compounds of formula XXX to provide quinoline compounds of formula II, where V-f- is, for example, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, or heteroaralkylaminocarbonyl.
  • V-f- is, for example, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, or heteroaralkylaminocarbonyl.
  • Quinazoline compounds of the formula II, where each of e and g is N can be prepared via iodination of 2-aminobenzonitrile with N-iodosuccinimide, followed by acylation with 2-acetoxybenzoyl chloride to form compound XXXI.
  • Cyclization of XXXI can be mediated with a variety of primary amines, followed by conversion of the quinazolyl iodide to the quinazolyl cyanide, under cross coupling conditions with a cyanide source.
  • aminoquinazoline XXXII can be obtained by reacting compound XXXI with 4-methoxybenzylamine and subsequent conversion to the quinazolyl cyanide.
  • benzylic cleavage can be affected under oxidative or acidic conditions and the resulting aminoquinazolines can be derivatized, for example, with electrophilic reagents to prepare amides, ureas, carbamates, or sulfonamides after phenol deprotection.
  • Example 24 Synthesis of compounds of formula lb, where m is 1, R° is 2-thiazole, and R 1 is 3-N g -Mtr-guanidinopropyl.
  • compounds of formula lb, where m is 1, R 1 is 3-N g -Mtr- guanidinopropyl, and R° is can also be prepared by reaction of compound XXXIII with the appropriate organometallic reagent.
  • the furyl group can be further synthetically manipulated by an oxidation procedure to produce an alpha-keto moiety.
  • the resin was subjected to DMF (2 mL) and piperidine (500 ul) for 2 hours at room temperature, filtered, and washed four times with DMF to remove the N-terminal Fmoc-protecting group.
  • the resin was treated with methylene chloride (2.25 mL) and 3,4-dichlorobenzylisocyanate (105 ul, 0.72 mmol, 6.0 equiv), and the reaction mixture was shaken overnight at room temperature to produce resin-supported 3,4- dichlorobenzylaminocarbonyl-Leu-Ue.
  • N-terminal ureas can be synthesized by reaction of the amine terminus of the resin with p-nitrophenylchloro formate (5-10 equiv) in the presence of diisopropylethylamine, followed by the addition of excess (5 equiv) of a primary or secondary amine.
  • the resin supported Leu-He dipeptide was so treated with 4-Br- ⁇ -methyl benzylamine to provide resin-supported compound XXXVI.
  • the resin was filtered and washed sequentially with CH 2 C1 2 , DMF, CH 3 OH, CH 2 C1 2 , DMF, and CH 2 C1 2 , and taken to dryness under a high vacuum.
  • To the dry resin was added 50% trifluoroacetic acid/50% methylene chloride (3 mL total volume), and the reaction mixture was shaken at room temperature for 1.5 hours.
  • the resin was filtered, washed with methylene chloride once, and the filtrate was concentrated in vacuo.
  • Representative compounds of formula la where X is N, R 3 is H, A is an ⁇ -amino acid AA 2 , and R is an aminocarbonyl substituent on the ⁇ -nitrogen of AA 2 , are provided in Table 1 below.
  • Additional compounds of formula la can be prepared in which X is N, R is H, A is an ⁇ -amino acid AA 2 , and R is an aminocarbonyl substituent acyl, aroyl, heteroaroyl, or alkoxycarbonyl moiety on the ⁇ -nitrogen of AA .
  • These compounds can be prepared by reaction of the N-terminal amine of the resin-supported amino acid as described previously when R 8 is an aminocarbonyl substituent, or with the appropriate carbonyl chloride compounds, chloroformates, or by reacting the N-terminal amine with the appropriate carboxylic acid in a coupling step.
  • Compounds prepared in this manner include, but are not limited to, compounds of formula la wherein X is N, R 3 is H, AA 2 is Leu, Phe, Val, Ala, cyclohexylglycine (Chg), Arg, Asn, 3- pyridylalanine (3-Pal), 4-pyridylalanine (4-Pal), T ⁇ , He, Lys, Glu, His, or Gin, and R 8 is a substituent on the ⁇ -nitrogen of AA 2 and can be
  • Compounds of formula la can be prepared in which A is an aminocarbonyl substituent (i.e., the step in which AA 2 is coupled to the resin-supported amino acid is omitted and an N-terminal urea is formed directly to the alpha amine of the amino acid bearing the R side-chain by methods previously described herein).
  • Compounds prepared in this manner include compounds of formula la in which X is N, R 2 is isopropyl, R 3 is H, and A is
  • Additional compounds of the invention formed from the reaction of compounds of formula la with those of formula lb, followed by deprotection, include those of formula I where m is 1, R° is 2-thiazole, R 1 is 3- guanidinopropyl, X is N, R 2 is isopropyl, R 3 is H, and A is selected from the following group:
  • the compounds of formula I where m is 0 can also be prepared by either using solid-phase approaches or solution phase approaches.
  • resin supported amine XXXVIII is prepared by treating ⁇ -bromo-j ⁇ -tolunitrile with NaN 3 and Bu 4 NCl to form the corresponding ⁇ -azido-jP-tolunitrile, followed by conversion of the nitrile group to the amidine group employing typical Pinner and aminolysis conditions, as described herein.
  • the p- azidomethyl benzamidine thus produced is attached to an activated p- nitrophenylcarbonate Wang resin, followed by reduction of the azide group to an amine by SnCl 2 to provide compound XXXVIII.
  • compounds of formula I where m is 0 and R 1 is H can be prepared in solution by condensing an amine, such as 4-cyanobenzylamine, to compounds of the formula la, followed by conversion of the cyano group to an amidine moiety, as described herein.
  • Representative compounds of formula I, where m is 0, R 1 is H, X is N, R 3 is H, A is ⁇ -amino acid AA 2 , and R 8 is a substituent on the ⁇ -nitrogen of AA are given in Table 5.
  • 2-Thiouracil (33.3 g, 0.26 mol) was dissolved in an aqueous solution of sodium hydroxide (20.8 g in 183 mL of water; ⁇ 0.52 mol). The mixture was placed in an ice bath until the internal reaction temperature was 0°C. Methyl iodide (18.5 mL, 0.29 mol) was slowly added. The resulting reaction mixture was allowed to return to room temperature and then stirred for 16 hours. The pale yellow solution was cooled to 0°C and acidified with glacial acetic acid. The white precipitate that formed was collected by vacuum filtration, washed with cold water (3 x 150 mL), and dried to afford 2-methylsulfanyl-3H- pyrimidin-4-one as a white powder. (37.4 g, 98 %).
  • the reaction mixture was then heated at reflux for 2 hours, cooled to room temperature, and slowly poured in small portions into ice water (600 mL). The mixture was vacuum-filtered, and the filtrate extracted with methylene chloride (200 mL). The aqueous layer was separated and extracted with methylene chloride (2 x 150 mL). The combined methylene chloride layers were then extracted with H 2 O (3 x 200 mL), dried over MgSO , and concentrated to give an off white solid (9 g). The solid was triturated in 10% CH 2 C1 2 / hexanes (500 mL) and filtered. The filtrate was collected and saved.
  • This amine can be acylated, carbamylated, or sulfonylated as previously described here to provide compounds of formula XL where Y 1 is NH and B is alkoxycarbonyl, cycloalkoxycarbonyl, aralkoxycarbonyl, aryloxycarbonyl, heterocyclyloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, heteroaralkylaminocarbonyl, alkylthiocarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl, alkoxythiocarbonyl, cycloalkoxythiocarbonyl, aralkoxythiocarbonyl, aryloxythiocarbonyl, aminothiocarbonyl, alkylaminothiocarbonyl, dial
  • l-(3- Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 1.9 equiv.) was added in one portion, and the reaction was stirred at room temperature overnight.
  • the reaction was diluted with ethyl acetate (2 mL), washed with 5% aqueous acetic acid (l x l mL), saturated aqueous NaHCO 3 (2 x 1 mL), water (l x l mL), and brine (l x l mL).
  • the organic layer was dried over Na 2 SO 4 , filtered, and concentrated in vacuo.
  • Example 31 Exemplary synthesis of compounds of formula II.
  • the amidine (1.5-2 equiv) was dissolved in a 1:1 CH 2 C1 2 /DMF (1 mL total volume) solution, which was then added to a vessel containing p- nitrophenyl carbonate Wang resin (1 equiv).
  • DIEA was added (50 ul) and the mixture shaken overnight.
  • the resin was filtered and washed sequentially with CH 2 C1 2 , DMF, MeOH, CH 2 C1 2 , DMF, and again with CH 2 C1 2 .
  • Added to the resin was 1 :1 CH 2 Cl 2 /MeOH (1 mL total volume) with 5% DIEA, and the mixture was shaken for an additional 1 hour. The washing protocol was repeated.
  • Compounds in which f is O, and V is an optionally substituted acyl, aroyl, heterocyclylcarbonyl, alkoxycarbonyl, cycloalkoxycarbonyl, aralkoxycarbonyl, aryloxycarbonyl, heteroaralkyloxycarbonyl, heteroaryloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, or heteroaralkylaminocarbonyl can be prepared by from 6-hydroxynaphthalene-2- carbonitrile, with subsequent unmasking of the amidine groups as described herein.
  • amidine can be unmasked prior to C6-O functionalization, followed by protection with a suitable protecting group such as Boc; subsequent O-acylation, carbonylation, or carbamoylation provides, after amidine deprotection, compounds of the formula II.
  • a suitable protecting group such as Boc
  • T is a bond
  • each of g and e is C-H
  • each of B, R 19 , and R 20 is H
  • Z is NH
  • f is a covalent bond
  • V is an optionally substituted aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, or heteroaralkylaminocarbonyl.
  • T is NH
  • B is an optionally substituted natural or unnatural alpha-amino acid residue of D- or L-configuration, or an optionally substituted acyl, aroyl, araalkanoyl, heteroaraalkanoyl, or heteroaroyl
  • each of g and e is C-H
  • each of R 19 and R 20 is H
  • Z is NH
  • f is a covalent bond
  • V is an optionally substituted aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, or heteroaralkylaminocarbonyl.
  • Nitration of 6-cyano-naphthalene-2-carboxylic acid methyl ester is carried out with KNO 3 in sulfuric acid at 0°C for 30 min.
  • the reaction mixture is poured into ice and filtered, and the compound is washed with water and dried.
  • the product, 6- cyano-4-nitro-naphthalene-2-carboxylic acid methyl ester is obtained and used without further purification.
  • the methyl ester is treated with LiOH (4 equiv.) in THF/H 2 O for 30 min, acidified with oxalic acid, and extracted with EtOAc. After removal of the solvent, the product is obtained as a solid, which is added into SOCl 2 and stirred at 70°C for 1 hour. Removal of excess thionyl chloride gives the desired acid chloride, 6-cyano-4-nitro-naphthalene-2-carbonyl chloride.
  • a mixture of the acid chloride, THF, K 2 CO 3 (3 equiv.) and a primary or secondary amine (3 equiv.) is stirred at room temperature for 2 hours.
  • the reaction mixture is loaded onto a preparative thick layer chromatography plate and developed with hexane/ethyl acetate (2: 1) to give a compound of formula XL VI, where f is a covalent bond and V is an optionally substituted aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, or heteroaralkylaminocarbonyl.
  • a compound of formula XL VI where f is a bond and V is benzylaminocarbonyl (50,mg, 0.151 mmol), is dissolved in methanol (3 mL) and hydrogenated in the presence of 10% Pd/C (5 mg) for 6 hours.
  • the resulting amine is obtained after removal of the catalyst and solvent (MS: m/z 302, M+ 1; calculated mass: 301.3).
  • Amides of formula XLVII are subjected to similar reaction conditions previously described herein for the conversion of a cyano group to an amidine group.
  • the compound of formula XLVII where f is a bond, V is benzylaminocarbonyl, T is NH and B is 2-methyl-3-acetyloxyphenylcarbonyl (20 mg) is treated with an HCl/methanol solution (7 N, 2 mL) at room temperature for 2 hours.
  • a solid product is obtained after removal of the solvent in vacuo, which is treated with a solution of ammonia in methanol (3.5 N, 3 mL) at 50 °C for 1.5 hours.
  • T is NH
  • B is an optionally substituted natural or unnatural alpha- amino acid residue of D- or L-configuration, or an optionally substituted acyl, aroyl, aralkanoyl, heteroaralkanoyl, or heteroaroyl
  • g is C-H
  • e is N
  • each of R 19 and R 20 is H
  • Z is NH
  • f is a covalent bond
  • V is an optionally substituted alkoxycarbonyl, cycloalkoxycarbonyl, aralkoxycarbonyl, aryloxycarbonyl, heterocyclyloxycarbonyl, heteroaralkyloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, or heteroaralkylaminocarbonyl.
  • T is NH
  • B is an optionally substituted natural or unnatural alpha-amino acid residue of D- or L-configuration, or an optionally substituted acyl, aroyl, aralkanoyl, heteroaralkanoyl, or heteroaroyl
  • g is C-H
  • e is N
  • each of R 19 and R 20 is H
  • Z is NH
  • f is a covalent bond
  • V is methoxycarbonyl
  • the methyl ester of the 4-bromo or 4-azido intermediate can be saponified to the carboxylic, then coupled with various alcohols under ester bond-forming conditions known to those skilled in the art, followed by conversion of the 4- bromo or -azido functional group to the 4-amino group with subsequent functionalization as previously described, to afford compounds of formula II, where T is NH; B is an optionally substituted natural or unnatural alpha-amino acid residue of D- or L-configuration, or an optionally substituted
  • the 2-carboxylic acid methyl ester substituent can be directly converted to an amide by treatment with an amine in the presence of Me 3 Al (see Akakura and Yamamoto, Syn. Lett. 3:277- 278, 1997).
  • T is NH
  • B is an optionally substituted natural or unnatural alpha-amino acid residue of D- or L-configuration, or an optionally substituted acyl, aroyl, aralkanoyl, heteroaralkanoyl, or heteroaroyl
  • g is C-H
  • e is N
  • each of R 19 and R 20 is H
  • Z is NH
  • f is a covalent bond
  • V is an optionally substituted aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, or heteroaralkylaminocarbonyl (e.g., V is benzylaminocarbonyl, B is 3-hydroxy- 2-methylphenylcarbonyl; MS: m
  • T is NH
  • B is an optionally substituted natural or unnatural alpha-amino acid residue of D- or L-configuration, or an optionally substituted alkyl, alkenyl, aralkyl, aralkenyl, heteroaralkyl, heteroaralkenyl, acyl, aroyl, aralkanoyl, heteroaralkanoyl, or heteroaroyl; each of e and g is N; each of R 19 and R 20 is H; Z is NH, f is a covalent bond, and V is an optionally substituted C 1-6 alkyl, cycloalkyl, alkenyl, aralkyl, aralkenyl, heteroaralkyl, heteroaralkenyl, aryl, or heterocyclyl.
  • 2-Aminobenzonitrile (2.0 g, 16.9 mmol, 1 equiv.) was dissolved in dry N,N-dimethylformamide (30 mL), and the solution was cooled to 0 °C.
  • a solution of N-iodosuccinimide, NIS (3.81g, 16.9 mmol, 1 equiv.) in 30 mL of DMF was added dropwise with an addition funnel. After the NIS/DMF solution was added, the ice bath was removed, and the reaction was stirred at room temperature overnight. The reaction was quenched by pouring into 3N NaOH (300 mL) and diluted with an additional 200 mL of water.
  • 2-Amino-5- iodobenzonitrile formed as a precipitate, which was collected by filtration.
  • 2-Amino-5-iodobenzonitrile 500 mg, 2.05 mmol, 1 equiv.
  • DMAP 50 mg, 0.41mmol, 0.2 equiv.
  • triethylamine 285 ul, 2.05 mmol, 1 equiv.
  • O-Acetylsalicyloyl chloride 2.0 g, 10.07 mmol, 4.9 equiv
  • reaction mixture was diluted with CH 2 C1 2 , washed with water, dried with MgSO , filtered, and concentrated.
  • the resulting residue was purified by flash column chromatography (20% EtO Ac/80% hexane) to provide compound XLLX (260 mg, 31% yld.), where f is a covalent bond and V is 2-acetoxyphenyl.
  • Compounds of formula L where T is NH and B is a substituted carbonyl or sulfonyl derivative can be prepared by conversion of the iodo-quinazoline to the cyano-quinazoline, removal of the 4-methoxybenzyl group, and subsequent acylation or sulfonylation of the resulting free amine; conversion of the nitrile to the amidine is then effected as previously described herein. Alternatively, it can be advantageous in some cases to unmask and protect the amidine prior to effecting acylation or sulfonylation.
  • the organic layer was washed with brine, dried over MgSO , filtered, and then concentrated to afford the compound in which the iodo group was displaced with a cyano group to form the corresponding cyanoquinazoline.
  • This compound was treated with 4N HCl/dioxane in MeOH to provide the intermediate methyl imidate ester, followed by aminolysis with ammonia in MeOH to provide, after concentration in vacuo, the quinazoline amidine.
  • the cyanoquinazoline can be allowed to react with hydroxylamine, followed by acylation of the N-hydroxyamidine with, for example, acetic anhydride.
  • acids which can be used to prepare the acid addition salts desirably include those which produce, when combined with the free base, pharmaceutically acceptable salts, that is, salts whose anions are non-toxic to the patient in pharmaceutical doses of the salts so that the beneficial effects inherent in the free base are not modulated by side effects ascribable to the anions.
  • Exemplary pharmaceutically acceptable salts are those derived from the following acids: acetic acid, benzenesulfonic acid, citric acid, cyclohexylsulfamic acid, ethanesulfonic acid, fumaric acid, gentisic acid, hydrochloric acid, hydrobromic acid, lactic acid, malonic acid, methanesulfonic acid, quinic acid, phosphoric acid, sulfamic acid, sulfuric acid, p- toluenesulfonic acid, and trifluoroacetic acid.
  • Acid addition salts of the compounds of the invention are prepared by reaction of the free base with the appropriate acid by the application or adaption of methods known to those skilled in the art.
  • base addition salts may be formed.
  • the bases which can be used to prepare the base addition salts desirably include those which produce, when combined with the free acid, pharmaceutically acceptable salts, that is, salts whose effects inherent in the free acid are not modulated by side effects ascribable to the cations.
  • Pharmaceutically acceptable salts are those derived from the following bases: aluminum hydroxide, ammonia, arginine, N-benzylphenethylamine, calcium hydroxide, chloroprocaine, choline, N, N'-dibenzylethylenediamine, diethanloamine, diethylamine, tris(hydroxymethyl)aminomethane, lithium hydroxide, lysine, magnesium hydroxide, N-methylglucamine, ornithine, piperazine, potassium hydroxide, procaine, sodium hydride, sodium hydroxide, tetramethylammonium hydroxide, triethylammonia, trimethylammonia, zinc hydroxide, and the like.
  • bases aluminum hydroxide, ammonia, arginine, N-benzylphenethylamine, calcium hydroxide, chloroprocaine, choline, N, N'-dibenzylethylenediamine, diethanloamine, diethylamine, tris(
  • Metal salts of compounds of the invention may be obtained by contacting a hydride or hydroxide of the chosen metal in an aqueous or organic solvent with the free acid form of the compound.
  • Amine salts of compounds of the invention may be obtained by contacting an amine in an aqueous or organic solvent with the free acid form of the compounds.
  • a number of methods are useful for the preparation of the salts described above and are known to those skilled in the art. For example, reaction of the free acid or free base form of a compound of the invention with one or more molar equivalents of the desired acid or base in a solvent or solvent mixture in which the salt is insoluble, or in a solvent like water after which the solvent is removed by evaporation, distillation, or freeze drying.
  • the free acid or base form of the product may be passed over an ion exchange resin to form the desired salt, or one salt form of the product may be converted to another using the same general process.
  • Isomers Compounds of this invention may exhibit stereoisomerism by virtue of the presence of one or more asymmetric or chiral centers in the compounds.
  • the present invention includes the various stereoisomers and mixtures thereof. Desired isomers may be obtained by chiral synthesis or resolution of enantiomeric mixtures or chromatographic separation of enantiomers on chiral chromatography columns. Diastereomers can be resolved using, e.g., standard chromatography columns or crystallization. Compounds of this invention may also exhibit geometrical isomerism. Geometrical isomers include the cis and trans form of compounds of the invention having, for example, alkenyl moieties. Compounds of this invention may also exhibit rotameric or tautomeric isomerism.
  • the present invention comprises the individual stereoisomers, geometrical isomers, rotameric and tautomeric isomers, and mixtures thereof.
  • a pharmaceutical composition comprising a therapeutically effective amount of a compound of the formula I and II, or a pharmaceutically acceptable salt, pharmaceutically acceptable solvate, pharmaceutically active metabolite, or prodrug thereof, can be chemically modified to include linkers that can covalently bind blood components including, but not limited to, cells (e.g., red blood cells and platelets) and proteins (e.g., immunoglobulins including IgG and IgM, serum albumin, ferritin, steroid binding proteins, such as corticosteroid-binding globulin and sex hormone-binding globulin, transferrin, thyroxin binding protein, and alpha-2-macroglobulin: see, for example, rantz et al, U.S.
  • linkers that can covalently bind blood components including, but not limited to, cells (e.g., red blood cells and platelets) and proteins (e.g., immunoglobulins including IgG and IgM, serum albumin,
  • the linker is ordinarily a hydrophilic synthetic polymer, i.e., a polymer not otherwise found in nature.
  • hydrophilic polyvinyl polymers fall within the scope of this invention, e.g. polyvinylalcohol and polyvinylpyrrolidone.
  • polyalkylene ethers such as polyethylene glycol (PEG); polyelkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene (Pluronics); polymethacrylates; carbomers; branched or unbranched polysaccharides which comprise the saccharide monomers D-mannose, D- and L-galactose, fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic acid (e.g.
  • polymannuronic acid or alginic acid
  • D- glucosamine, D-galactosamine, D-glucose and neuraminic acid including homopolysaccharides and heteropolysaccharides such as lactose, amylopectin, starch, hydroxyethyl starch, amylose, dextrane sulfate, dextran, dextrins, glycogen, or the polysaccharide subunit of acid mucopolysaccharides, e.g. hyaluronic acid; polymers of sugar alcohols such as polysorbitol and polymannitol; heparin or heparan.
  • the polymer prior to cross-linking need not be, but preferably is, water soluble, but the final conjugate must be water soluble.
  • the polymer should not be highly immunogenic in the conjugate form, nor should it possess viscosity that is incompatible with intravenous infusion or injection if it is intended to be administered by such routes.
  • the linker group can be attached to compounds of the formula I and II through the appropriate handle (e.g., by the acylation of amino groups) and inco ⁇ orate at the end of its tether a functionality, desirably a maleimide, reactive towards biomolecules. Once reacted, the conjugated compound of the invention should form a stable covalent linkage to the biomolecule via the linker moiety.
  • the maleimide of the linker reacts with a cysteine residue (C34) of human serum albumin to form a stable, conjugated species in vivo.
  • the linker is typically a chain of varying length, e.g., maleimide- (CH 2 ) bb C(O)NHCH 2 CH 2 (OCH 2 CH 2 ) aa OCH 2 C(O)-, maleimide- (CH 2 ) b C(O)NHCH 2 CH 2 (OCH 2 CH 2 ) aa NHCH 2 C(O)-, maleimide- (CH 2 ) bb C(O)NHCH 2 CH 2 (OCH 2 CH 2 ) aa NHC(S)-, maleimide-(CH 2 ) bb NHC(S)-, maleimide-(CH 2 ) b C(O)-, or maleimide (CH 2 ) bb -where aa is 1-10 and bb is 1-4, and desirably is
  • the length of the linker is between 6 and 100 atoms, inclusive.
  • the reactive moiety e.g., maleimide
  • a compound of the invention, or a pharmaceutically acceptable salt, pharmaceutically acceptable solvate, pharmaceutically active metabolite, or prodrug thereof can be further modified to include only a polyethylglycol moiety.
  • Such compounds are also known to increase blood serum half-life (see Yoo et al., Chem. Pharm. Bull. 48:1921- 1924, 2000).
  • the polyethylene glycol moiety can be selected from the following group: H(OCH 2 CH 2 ) cc O-, H 3 C(OCH 2 CH 2 ) cc OC(O), H(OCH 2 CH 2 ) cc OC(O), H 3 C(OCH 2 CH 2 ) cc NHC(O), H(OCH 2 CH 2 ) cc NHC(O), H 3 C(OCH 2 CH 2 ) cc NHC(S), H(OCH 2 CH 2 ) cc NHC(S), H 3 C(OCH 2 CH 2 ) cc C(O), H(OCH 2 CH 2 ) cc C(O), H 3 C(OCH 2 CH 2 ) cc NHCH 2 C(O), H(OCH 2 CH 2 ) cc NHCH 2 C(O), H 3 C(OCH 2 CH 2 ) cc OC(O)C(CH 3 ) 2 -, and
  • cc is a range of numbers that results in an average molecular weight of said polyethylene glycol moiety of between 1 ,000-40,000, preferably 20,000 or 40,000.
  • compounds modified with a linker e.g., a linker with a group for conjugation to a molecule of interest
  • a linker e.g., a linker with a group for conjugation to a molecule of interest
  • X is N, m is 1 ;
  • A- is L-(CH 2 ) n -K-C(O)-;
  • R 3 is H, C ⁇ -6 alkyl, or together with K forms a 5- or 6-membered ring;
  • R° is -E-J;
  • R 1 is b ' ;
  • R is selected from the group consisting of optionally substituted alkyl, alkenyl, cycloalkyl, aralkyl, aralkenyl, aryl, heterocyclyl, heteroaralkyl, heteroaralkenyl, and heteroaryl;
  • R 4 , R 5 , R 6 , R 7 , "a", "b", "n", E, and J are as previously defined herein; and at least one of J, R 2 , or L further includes a linker attached to a moiety reactive to a free amine or sulfhydryl contained on a blood component.
  • Exemplary linkers can be chosen from
  • R 4 and R 5 are H or C 1-6 alkyl
  • R 6 is H, OH, C ]-6 alkyl, or together with R 4 forms a 5- or 6-membered ring
  • R 7 is H, OH, SH, NH 2 , NO 2 , optionally substituted d_ 6 alkyl, halogen, or CF 3
  • R 2 is and optionally substituted 2-propyl, 2-butyl, CH 2 Ph, - CH 2 (C 6 H 4 )-4-OH, cyclohexyl, or Ph
  • X is N
  • R 3 is H or an optionally substituted C 1-6 alkyl
  • A is an alpha-amino acid of the formula: where
  • R 13 is H, d- 6 alkyl, or together with M forms a 5- or 6-membered ring, D is C(O) or S(O) 2 , q is an integer from 0-4; Q is an optionally substituted C 1-6 alkyl, C 3-8 cycloalkyl, C 1-9 heterocyclyl, C 6 or C ]0 aryl, C 1-6 alkyloxy, C 3-8 cycloalkyloxy, C 6 or C] 0 aryloxy, C 2-9 heterocyclyloxy, C 1-6 alkylthio, C 3-8 cycloalkylthio, C 6 or Cio arylthio, C 1-6 alkylamino, C 2-12 dialkylamino, C 7-16 aralkylamino, C 6 or Cio arylamino, C 2 .
  • M is single bond, NR 14 , O, or S, wherein R 14 is H, C 1-6 alkyl, or when taken together with R forms a 5- or 6-membered ring;
  • U is an optionally substituted C 1-6 alkyl, C 2-6 alkenyl, C 3-8 cycloalkyl, C 1- heterocyclyl, C 7-16 aralkyl, C 8- ⁇ 6 aralkenyl, C 6 or C 10 aryl, C 2-15 heteroaralkyl, or C 3-15 heteroaralkenyl, wherein when m is 1, R° is a thiazole, and when m is 0, R° is H or Cj- 6 alkyl; and least one of U, R 2 , or M when M is NR 14 further includes a linker attached to a moiety reactive
  • linkers can be chosen from the polyethylene glycol moieties described herein, which can, for example, be linked to a compound of the invention by attachment to an amino group contained in U, R 2 , or M when M is NR 14 .
  • compounds modified with a linker have formula
  • linker attached to a moiety reactive to a free amine or sulfhydryl contained on a blood component.
  • linkers can be chosen from the polyethylene glycol moieties described herein, which can, for example, be linked to a compound of the invention by attachment to an amino group contained in B or V.
  • exemplary compounds of the invention further include a polyethylene glycol moiety.
  • the polyethylene glycol increases the half-life of the compound in vivo and/or increases the bioavailability of the compound.
  • the linear or branched polyethylene glycol is bonded to a compound of the invention via a spacer and/or has a molecular weight between 1,000 and 40,000 daltons, inclusive (e.g., approximately 1,000 daltons, 1,500 daltons, 2,000 daltons, 3,500 daltons, 5,000 daltons, 8,000 daltons, 10,000 daltons, 20,000 daltons, or 40,000 daltons).
  • polyethylene glycol moiety can be inco ⁇ orated into the compounds of the invention by attachment to the A, R , or R substituents of the compounds of formula I or to the B or V substituents of the compounds of formula II in the same manner as attachment of the linkers as described herein.
  • the present invention also includes within its scope the pharmaceutical formulations which comprise at least one of the compounds of formula I or II in association with a pharmaceutically acceptable carrier or coating (e.g., pegylation).
  • a pharmaceutically acceptable carrier or coating e.g., pegylation
  • a compound described in any of the embodiments of the above aspects of the invention may be used in any of the following clinical applications.
  • the compounds of the invention are useful for the treatment, stabilization, or prevention of a variety of medical disorders where anticoagulant therapy is indicated in the treatment or prevention of thrombotic conditions such as coronary artery and cerebro- and peripheral vascular disease.
  • Indications include, but are not limited to, myocardial infarction, venous or arterial thrombosis, the formation of atherosclerotic plaques, coagulation syndromes, endarterectomy, including carotid endarterectomy, envascular injury including reocclusion and restenosis following angioplasty and coronary artery bypass surgery, thrombus formation after the application of blood vessel operative techniques, the introduction of artificial heart valves or on the recirculation of blood, cerebral infarction, cerebral thrombosis, transient ischemic attacks, stroke, cerebral embolism, pulmonary embolism, ischaemia, and angina, including unstable angina.
  • DVT deep vein thrombosis
  • DIC Disseminated intravascular coagulopathy
  • Factor Xla inhibitors are useful in the treatment and prevention of other diseases in which the generation of thrombin has been implicated as playing a physiologic role.
  • thrombin has been implicated in contributing to the morbidity and mortality of chronic and degenerative diseases such as cancer, arthritis, atherosclerosis, and Alzheimer's disease by its ability to regulate many different cell types through specific cleavage and activation of a cell surface thrombin receptor, mitogenic effects, diverse cellular functions such as cell proliferation, for example, abnormal proliferation of vascular cells resulting in restenosis or angiogenesis, release of PDGF, and DNA synthesis.
  • one or more compounds of the invention can be used in the manufacture of medicaments for use in the production of an antithrombotic or anticoagulant.
  • the invention features a method of treating, stabilizing, or preventing a disease, disorder, or condition associated with undesirable or excess thrombosis in a mammal (e.g., a human). This method involves administering a compound of the invention to the mammal in an amount sufficient to treat, stabilize, or prevent the disease, disorder, or condition. The compound may be administered to the mammal before, during, or after the occurrence of the condition.
  • a compound that binds to Factor XI or Factor Xla decreases the activity of Factor Xla, the binding of a Factor Xla to another molecule (e.g., a substrate for Factor Xla), or the half-life of a Factor XI protein, as measured using standard methods (see, for example, Coligan, et al. Current Protocols in Protein Chemistry, Chapters 19 and 20, John Wiley & Sons, New York, 2000; Ausubel et al, Current Protocols in Molecular Biology, Chapter 9, John Wiley & Sons, New York, 2000).
  • the compound may competitively, noncompetitively, or uncompetitively inhibit the ability of Factor XI or Factor Xla to bind one or more of its endogenous substrates.
  • the level of protein may be determined using standard Western, blot immunoassay, or immunohistochemical analysis (see, for example, Coligan, et al, supra; Ausubel et al, supra).
  • the compound decreases Factor Xla activity by at least 20, 40, 60, 80, 90 or 95%.
  • the level of Factor Xla activity is at least 2, 3, 5, 10, 20, or 50-fold lower in the presence of the compound.
  • the compound is administered in a dose that is sufficient to reduce thrombosis but does not eliminate normal clotting resulting from external injuries or does not induce bleeding complications.
  • the half-life of an injected compound is less than 7, 6, 5, 4, 3, 2, 1, or 0.5 hours. In some embodiments, the half-life is contained within one of the following ranges: 4-6 hours, 2-4 hours, 30-120 minutes, or 30-60 minutes, inclusive. In desirable embodiments, the half-life of an oral compound is less than 24, 20, 16, 12, 8, or 4 hours. In some embodiments, the half- life is contained within one of the following ranges: 20-28 hours, 14-20 hours, 10-14 hours, 6-10 hours, 2-6 hours or 30-120 minutes, inclusive. In desirable embodiments, the compound has better inhibitory activity than benzamidine for Factor Xla, such as an IC 5 o value of less than 100, 10, 1, 0.1, 0.01, or 0.001 ⁇ M.
  • the administration of compounds to a mammal be limited to a particular mode of administration, dosage, or frequency of dosing; the present invention contemplates all modes of administration, including oral, intraperitoneal, intramuscular, intravenous, intraarticular, intralesional, subcutaneous, or any other route sufficient to provide a dose adequate to prevent or treat excess or undesired Factor Xla activity (e.g., excess or undesired clotting).
  • One or more compounds may be administered to the mammal in a single dose or multiple doses. When multiple doses are administered, the doses may be separated from one another by, for example, several hours, one day, one week, one month, or one year.
  • compositions containing at least one compound of the invention that is suitable for use in human or veterinary medicine may be presented in forms permitting administration by a suitable route.
  • These compositions may be prepared according to the customary methods, using one or more pharmaceutically acceptable adjuvants or excipients.
  • the adjuvants comprise, inter alia, diluents, sterile aqueous media, and various non-toxic organic solvents.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical field, and are described, for example, in Remington: The Science and Practice of Pharmacy (20th ed.), ed. A.R. Gennaro, Lippincott Williams & Wilkins, 2000, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York.
  • compositions may be presented in the form of tablets, pills, granules, powders, aqueous solutions or suspensions, injectable solutions, elixirs, or syrups, and the compositions may optionally contain one or more agents chosen from the group comprising sweeteners, flavorings, colorings, and stabilizers in order to obtain pharmaceutically acceptable preparations.
  • excipients such as lactose, sodium citrate, calcium carbonate, and dicalcium phosphate and disintegrating agents such as starch, alginic acids, and certain complex silicates combined with lubricants (e.g., magnesium stearate, sodium lauryl sulfate, and talc) may be used for preparing tablets.
  • lubricants e.g., magnesium stearate, sodium lauryl sulfate, and talc
  • lactose and high molecular weight polyethylene glycols When aqueous suspensions are used, they may contain emulsifying agents which facilitate suspension.
  • Diluents such as sucrose, ethanol, polyethylene glycol, propylene glycol, glycerol, chloroform, or mixtures thereof may also be used.
  • emulsions, suspensions, or solutions of the compositions of the invention in vegetable oil e.g., sesame oil, groundnut oil, or olive oil
  • aqueous-organic solutions e.g., water and propylene glycol
  • injectable organic esters e.g., ethyl oleate
  • sterile aqueous solutions of the pharmaceutically acceptable salts are used.
  • the solutions of the salts of the compositions of the invention are especially useful for administration by intramuscular or subcutaneous injection.
  • Aqueous solutions that include solutions of the salts in pure distilled water may be used for intravenous administration with the proviso that (i) their pH is adjusted suitably, (ii) they are appropriately buffered and rendered isotonic with a sufficient quantity of glucose or sodium chloride, and (iii) they are sterilized by heating, irradiation, or microfiltration.
  • Suitable compositions containing the compounds of the invention may be dissolved or suspended in a suitable carrier for use in a nebulizer or a suspension or solution aerosol, or may be absorbed or adsorbed onto a suitable solid carrier for use in a dry powder inhaler.
  • Solid compositions for rectal administration include suppositories formulated in accordance with known methods and containing at least one compound of formula I or II.
  • Dosage formulations of the compounds of this invention to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile membranes (e.g., 0.2 micron membranes) or by other conventional methods. Formulations typically are stored in lyophilized form or as an aqueous solution.
  • the pH of the compositions of this invention is typically between 3 and 11, more desirably between 5 and 9, and most desirably between 7 and 8, inclusive. While a desirable route of administration is by injection such as intravenously (bolus and/or infusion), other methods of administration may be used.
  • compositions may be administered subcutaneously, intramuscularly, colonically, rectally, nasally, or intrapertoneally in a variety of dosage forms such as suppositories, implanted pellets or small cylinders, aerosols, oral dosage formulations, and topical formulations such as ointments, drops, and dermal patches.
  • dosage forms such as suppositories, implanted pellets or small cylinders, aerosols, oral dosage formulations, and topical formulations such as ointments, drops, and dermal patches.
  • the compounds of the invention are desirably inco ⁇ orated into shaped articles such as implants, including but not limited to valves, stents, tubing, and prostheses, which may employ inert materials such as synthetic polymers or silicones, (e.g., Silastic, silicone rubber, or other commercially available polymers).
  • Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxy-propyl- methacrylamide-phenol, polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues.
  • the Factor Xla inhibitors of the invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example polylactic acid, polyglycolic acid, copolymers of polylaclic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and cross linked or amphipathic block copolymers of hydrogels.
  • the compounds of the invention may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles.
  • Liposomes can be formed from a variety of lipids, such as cholesterol, stearylamine, or phosphatidylcholines.
  • the compounds of the invention may also be delivered using antibodies, antibody fragments, growth factors, hormones, or other targeting moieties to which the compound molecules are coupled (e.g., see Remington: The Science and Practice of Pharmacy, vide supra), including in vivo conjugation to blood components of a suitably modified compound of the formula I or II which possesses a metastable or reactive functional group as described above.
  • Dosage levels of active ingredients in the pharmaceutical compositions of the invention may be varied to obtain an amount of the active compound(s) that achieves the desired therapeutic response for a particular patient, composition, and mode of administration.
  • the selected dosage level depends upon the activity of the particular compound, the route of administration, the severity of the condition being treated, and the condition and prior medical history of the patient being treated.
  • the doses are generally from about 0.01 to about 100 mg/kg, desirably about 0.1 to about 1 mg/kg body weight per day by inhalation, from about 0.01 to about 100 mg/kg, desirably 0.1 to 70 mg/kg, more desirably 0.5 to 10 mg/kg body weight per day by oral administration, and from about 0.01 to about 50 mg/kg, desirably 0.1 to 1 mg/kg body weight per day by intravenous administration.
  • Doses are determined for each particular case using standard methods in accordance with factors unique to the patient, including age, weight, general state of health, and other factors which can influence the efficacy of the compound(s) of the invention.
  • compositions of the invention may be as frequent as necessary to obtain the desired therapeutic effect. Some patients may respond rapidly to a higher or lower dose and may find much weaker maintenance doses adequate. Other patients, however, receive long-term treatments at the rate of 1 to 4 doses per day, in accordance with the physiological requirements of each patient.
  • the active product may be administered, e.g., orally 1 to 4 times daily.
  • Factor Xla and related serine proteases such as Factor Xa, thrombin, trypsin, Factor Vila, plasma kallikrein, tissue-plasminogen activator (t-PA), urokinase-plasminogen activator (u-PA), plasmin, and activated protein C (APC) were evaluated by determining the concentration of inhibitor which resulted in a 50% reduction in enzyme activity (IC 50 ) using purified enzyme. Potential inhibitors of Factor Xla were evaluated using the following assay.
  • the inhibitors were tested against native Factor Xla (Haematological Associates, P/N FXIA01) using a custom made analog of S-2366, pyroGlu-Pro-Arg-7- methylamidocoumarin (AMC) available from American Peptide, Inc.
  • AMC pyroGlu-Pro-Arg-7- methylamidocoumarin
  • This analog is based on the well described substrate pyro-Glu-Pro-Arg-pNA, a.k.a. S-2366, available from Diapharma Group, Inc. (Columbus, OH). This substrate had the 9-nitroanaline group replaced with 7-methylaminocoumarin (AMC).
  • the final concentration of the substrate in the assay was 100 ⁇ M, and the final concentration of the enzyme was 0.25 nM.
  • Inhibitors were tested by serial dilution over an appropriate range to yield a dose response curve for determination of the inhibitors' IC 5 o value.
  • the assay mixture was incubated for 30 minutes at 30° C, and the fluorescent yield measured using a PE-Wallac Victor 2 Plate Reader. Dose response curves were fit to equation 1 below in which A is the maximum inhibition, B is the minimum inhibition, C is the IC 5 o, and D is the Hill coefficient.
  • Desirable compounds have an IC 50 value for inhibiting Factor Xla of less than 1 micromolar, 100 nanomolar, 10 nanomalar, or 1 nanomolar (in order of increasing preference).
  • Table 12 shows the IC 50 values for some of the compounds of formula I, where X is N, R 1 is 3-guanidinopropyl, m is 1, X is N, R 3 is H, and R° is 2-thiazole.
  • Table 13 shows the IC 50 values for compounds of formula I, where X is N, W is N, R 2 is H, R 1 is 3-guanidinopropyl, m is 1, R° is 2-thiazole, R 3 together with A forms a ring having the formula:
  • Table 14 shows the IC 50 values for compounds of formula II, where Z' is NH, each of e and g is C-H, each of R 19 and R 20 is H, f is a covalent bond, and T is NH.
  • An activated partial thomboplastin time ⁇ aPTT assay was used to measure the ability of compounds to inhibit the contact coagulation pathway.
  • This pathway involves Factor XII, kallikrein, and Factor XI, which activates Factor IX and Factor VIII, leading to activation of Factor X and Factor V, and then activation of Factor II to form a blood clot ( Figure 1).
  • CaCl 2 (30 mM) was placed in a large central reagent position of a Thromboscreen 400C instrument, allowing it to equilibrate to 37°C. Plasma (50 ul) and compounds of the invention were added at different concentrations to cuvettes.
  • aPTT reagent (ALEXIN, Sigma) was added (50 ul) and incubated an additional three minutes. The cuvettes were transferred to a measuring position; prewarmed CaCl 2 reagent (50 ul) was added, and readings were then taken over a maximum of 300 seconds. A dose response curve was generated, and the concentration at which the clotting time was doubled (2 x aPTT) was determined.
  • Compounds which inhibit Factor Xla in the desired range desirably have an effect at less than 50 uM, more desirably at less than 10 uM.
  • a prothrombin time (PT) assay was also used to measure inhibition of coagulation.
  • the assay measures inhibition of Factor Vila, Factor Xa, and thrombin, but not FXI.
  • This assay measures the ability of Factor Vila to activate Factor X, which activates Factor II to form a blood clot.
  • the thromboplastin reagent ThromboMax with Calcium, Sigma
  • Plasma 50 ul of plasma prewarmed for three minutes
  • compounds of the invention different concentrations were added to cuvettes.
  • the cuvettes were transferred to a measuring position.
  • the prewamied Thromboplastin reagent 100 ul was then added, and readings were then taken over 300 seconds.
  • a dose response curve was generated, and the concentration at which the clotting time was doubled (2 x PT) was determined.
  • Both iliolumbar veins were ligated with suture (3-0 silk).
  • a loose ligature (3-0 silk) was placed around the vena cava distal to the renal veins.
  • a 25G butterfly bent at a 90-degree angle and attached to a 5 cc syringe filled with 2.5 mL of hypotonic saline (0.225%) was inserted into the vena cava at the level of the iliolumbar veins and glued into place using VetbondTM.
  • a 26G needle bent to a 90-degree angle was then tied tightly to the vena cava, using the loose ligature.
  • vena cava proximal to the iliac bifurcation, and the hypotonic saline manually infused at a rate of 10 mL/minutes.
  • the atraumatic clamp was removed, along with the 26G needle, creating a 26G stenosis.
  • Organs were carefully replaced, and animals were kept normofhermic until sacrifice. Animals were sacrificed by anesthetic overdose at 10 or 60 minutes post thrombus induction. Following sacrifice, the vena cava was isolated using hemostats, and the clot(s) was dissected out. Clots were removed, blotted on gauze, and then immediately weighed using an analytical balance.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Plural Heterocyclic Compounds (AREA)

Abstract

The present invention provides compounds that inhibit Factor XIa and methods of preventing or treating undesired thrombosis by administering a compound of the invention to a mammal. The invention also provides three-dimensional structures of Factor XIa and methods for designing or selecting additional Factor XIa inhibitors using these structures.

Description

COMPOUNDS AND METHODS FOR TREATMENT OF
THROMBOSIS
BACKGROUND OF THE INVENTION
Blood coagulation is the first line of defense against blood loss following injury. The blood coagulation "cascade" involves a number of circulating serine protease zymogens, regulatory cofactors and inhibitors, as shown in Figure 1. Each enzyme, once generated from its zymogen, specifically cleaves the next zymogen in the cascade to produce an active protease. This process is repeated until finally thrombin cleaves the fibrinopeptides from fibrinogen to produce fibrin that polymerizes to form a blood clot. Although efficient clotting limits the loss of blood at a site of trauma, it also poses the risk of systemic coagulation resulting in massive thrombosis. Under normal circumstances, hemostasis maintains a balance between clot formation (coagulation) and clot dissolution (fibrinolysis). However, in certain disease states such as acute myocardial infarction and unstable angina, the rupture of an established atherosclerotic plaque results in abnormal thrombus formation in the coronary arterial vasculature.
Despite the availability of a number of approved anticoagulant therapies, myocardial infarction, unstable angina, atrial fibrillation, stroke, pulmonary embolism, and deep vein thrombosis represent areas of major medical need. Cardiovascular diseases (e.g., acute myocardial infarction, stroke, and pulmonary embolism) disable or kill more people in the developed world than any other disease. Over two million patients are hospitalized each year in the U.S. for acute arterial thrombosis and stroke. The worldwide population for acute arterial antithrombotic therapy is five to six million, while over 25 million patients have chronic arterial thrombosis. Over 10 million individuals are candidates for venous thrombosis therapy.
A large medical need exists for novel anticoagulation drugs that lack some or all of the side effects of currently available drugs, such as the risk of bleeding episodes and patient-to-patient variability that results in the need for close monitoring and titration of therapeutic doses. Current anticoagulant therapies that dominate the market include injectable unfractionated and low _ molecular weight (LMW) heparin, and orally administered warfarin (coumadin). Three phases of the coagulation cascade can be described, namely initiation, amplification, and propagation (see Figure 1). Inhibiting enzymes of 'the propagation phase, i.e., Factor Xa and Factor Ila (thrombin), has been an area of active interest in the pharmaceutical industry for some time. The first generation of thrombin inhibitors to reach the clinic were polypeptides derived from natural sources, such as the potent anticoagulant, hirudin, which is a leech peptide. Potent, orally available, small molecule thrombin inhibitors have been discovered over the past few decades. Some of these are now in the clinic or are ready to be marketed. Efforts to develop potent Factor Xa inhibitors are not far behind. Targeting enzymes involved in propagation (e.g., hirudin) does not appear to be ideal since inhibitors of this phase of the coagulation cascade are associated with severe bleeding. This is further supported by findings that Factor V and Factor X deficiencies are associated with severe bleeding episodes.
Several new treatments under development are aimed at the initiation phase that involves Factor VII and tissue factor (TF). These include an active site-blocked Factor Vila, a high affinity neutralizing antibody against TF, and a nematode protein (NAPcc) that inhibits Factor VIIa/TF. Because these approaches target the very start of the coagulation cascade, they may lead to bleeding episodes. Due to the limited efficacy and adverse side-effects of some current therapeutics for the inhibition of undesirable thrombosis (e.g., deep vein thrombosis and stroke), improved compounds and methods are needed for preventing or treating undesirable thrombosis.
SUMMARY OF THE INVENTION
The present invention features compounds (e.g., peptidomimetics and non-peptides) that inhibit Factor Xla and methods for preventing or treating undesired thrombosis by administering one or more of these compounds alone or in combination with other molecules to a mammal. The invention also provides three-dimensional structures of Factor Xla and methods for designing or selecting additional Factor Xla inhibitors using these structures. Desirably, these compounds have certain structural, physical, and spatial characteristics that enable the compounds to interact with specific residues of the active site of Factor Xla.
Compounds of the Invention
Accordingly, in a first aspect, the invention features a compound of formula I, or a pharmaceutically acceptable salt or prodrug thereof:
Figure imgf000004_0001
where R1 is selected from the group consisting of:
Figure imgf000005_0001
where R4 is H or C1-6 alkyl, RD is H, C]-6 alkyl, OH, NH2, NO2, CO2R3a, where R5a is C1-6 alkyl, or R5 taken together with R4 forms a 5- or 6-membered ring, R6 is H, OH, C]-6 alkyl, or when taken together with R4, forms a 5- or 6- membered ring, R7 is H, OH, SH, NH2, NO2, optionally substituted C1-6 alkyl, halogen, or CF3, Y is O, S, or NR4, a is an integer from 0-5; b is an integer from 0-2, and Z is NR5 or (H,H);
R2 is H or an optionally substituted C1-6 alkyl, C2-6 alkenyl, C3.8 cycloalkyl, C7.16 aralkyl, C8-16 aralkenyl, C6 or C10 aryl, C2- heterocyclyl, C2.15 heteroaralkyl, or C3-15 heteroaralkenyl, or when taken together with X and R3 forms an optionally substituted C6 aryl group or an optionally substituted C3-C5 heteroaryl group;
X is C or N; R3 is H or optionally substituted C]-6 alkyl, or together with X and A forms an optionally substituted C3-5 heterocyclic ring, or together with X and R2 forms an optionally substituted C6 aryl group or an optionally substituted C2-C5 heteroaryl group;
A is R8-AA2, where AA2 is a covalent bond or a peptide chain of one to five natural or unnatural alpha-amino acid residues of D- or L- configuration, preferably one or two residues, where R8 is H, C2- acyl, C7-11 aroyl, C3-10 heteroaroyl, C2- alkoxycarbonyl, C4-9 cycloalkoxycarbonyl, C8-17 aralkoxycarbonyl, C7 or Cπ aryloxycarbonyl, aminocarbonyl, C2-7 alkylaminocarbonyl, C3-13 dialkylaminocarbonyl, C -9 cycloalkylaminocarbonyl, Cg-π aralkyl aminocarbonyl, C7 or C arylaminocarbonyl, C3-10 heterocyclylaminocarbonyl, C3-ι heteroaralkylaminocarbonyl, C2-7 alkylthiocarbonyl, C7-π arylthiocarbonyl, C3-ι0 heteroarylthiocarbonyl, C2-7 alkoxythiocarbonyl, C -9 cycloalkoxythiocarbonyl, C8-π aralkoxythiocarbonyl, C7 or Cπ aryloxythiocarbonyl, aminothiocarbonyl, C2-7 alkylaminothiocarbonyl, C3-1 dialkylaminothiocarbonyl, C4-9 cycloalkylaminothiocarbonyl, C8-]7 aralkylaminothiocarbonyl, C7 or Cπ arylaminothiocarbonyl, C3-ιo heterocyclylaminothiocarbonyl, C3-ι heteroaralkylaminothiocarbonyl, C1-6 alkylsulfonyl, C3-8 cycloalkylsulfonyl, C - ]6 aralkylsulfonyl, C6 or C10 arylsulfonyl, C2-9 heterocyclylsulfonyl, orC2-]5 heteroaralkylsulfonyl, or A together with X and R forms an optionally substituted C2.5 heterocyclic ring; and m is an integer from 0-1 , where when m is 1 , R° is an optionally substituted C1-6 alkyl, C2-6 alkenyl, C3-8 cycloalkyl, C7-]6 aralkyl, C8-16 aralkenyl, C6 or C10 aryl, C1-9 heterocyclyl, C2-15 hetero aralkyl, C3-ι5 heteroaralkenyl, or C(O)R9, wherein R9 is an optionally substituted Cι_6 alkyl, C2^ alkenyl, Cι-8 cycloalkyl, C7.16 aralkyl, C8.16 aralkenyl, C6 or C10 aryl, .9 heterocyclyl, C2-15 heteroaralkyl, C3-15 heteroaralkenyl, C]-6 alkyloxy, C6 or C10 aryloxy, Cι-9 heteroaryloxy, C7-ι6 aralkyloxy, C1-6 alkylthio, C3-8 cycloalkylthio, C6 or C]0 arylthio, C7-16 aralkylthio, Cι-6 alkylamino, C2-i2 dialkylamino, C3.8 cycloalkyl amino, C7.ι6 aralkylamino, C6 or CJO arylamino, C2-9 heterocyclylamino, or C2-15 heteroaralkylamino, with the proviso that the amino acid of AA2 that is covalently linked to X is not D-phenylalanine when R8 is C]-6 alkylsufonyl or C7-]6 aralkylsulfonyl. When m is 1 , desirable R° heterocyclyl substituents include 2-thiazole, 2-(4-methylthiazole), 2-(5- methylthiazole), 2-(4,5-dimethylthiazole), 2-(5-(2-hydroxyethyl)thiazole), or 2- N-methylimidazole.
When m is 0, R° is H, CHO, CN, or B(OR9)2, wherein R9 is H, C1-6 alkyl, or taken together forms a C2-4 cyclic boronate ester, or R° is an optionally substituted C]-6 alkyl, C2-6 alkenyl, C3-8 cycloalkyl, C7-16 aralkyl, C8-16 aralkenyl, C6 or Cio aryl, C]-9 heterocyclyl, C2-i5 heteroaralkyl, or C3-15 heteroaralkenyl, with the proviso that the amino acid of AA2 that is covalently linked to X is not D-cyclohexylglycine when R8 is C]-6 alkylsulfonyl or C7-16 aralkylsulfonyl.
In one embodiment, R is H or a substituted C1-6 alkyl, C2-6 alkenyl, C3-8 cycloalkyl, C7-ι6 aralkyl, C8-ι6 aralkenyl, C6 or C10 aryl, C1-9 heterocyclyl, C2-15 heteroaralkyl, or C3-ι5 heteroaralkenyl; X is N; a is an integer from 1-4; b is an integer from 0-2; AA2 is a covalent bond and R8 is B-Y1-(CH2)n-K-C(O)-, wherein n is an integer from 0-6, K together with -C(O)-, X, and R forms an optionally substituted C2-5 heterocyclyl group or K is a single bond, NR9, O, S, C(O), CH(OR10), or -CH(NHRn), wherein R9 is H, OH, or C1-6 alkyl, each of R10 Rn is, independently, H, C]-6 alkyl, C2-6 alkenyl, C3-8 cycloalkyl, C7-]6 aralkyl, C8-ι6 aralkenyl, C6 or C10 aryl, C1-9 heterocyclyl, C2-i5 heteroaralkyl, C3- ]5 heteroaralkenyl, C2- acyl, C7.π aroyl, C3-1o heteroaroyl, C2-7 alkoxycarbonyl, C -9 cycloalkoxycarbonyl, C8-1 aralkoxycarbonyl, C7 or Cπ aryloxycarbonyl, aminocarbonyl, C2-7 alkylaminocarbonyl, C3.13 dialkylaminocarbonyl, C4.9 cycloalkylaminocarbonyl, C8-17 arallcylammocarbonyl, C7 or Cπ arylaminocarbonyl, C3.10 heterocyclylaminocarbonyl, C3-ι6 heteroaralkylaminocarbonyl, C2-7 alkylthiocarbonyl, C7-π arylthiocarbonyl, C3. 10 heteroarylthiocarbonyl, C2-7 alkoxythiocarbonyl, C4-9 cycloalkoxythiocarbonyl, C8-!7 aralkoxythiocarbonyl, C7 or Cπ aryloxythiocarbonyl, aminothiocarbonyl, C2.7 alkylaminothiocarbonyl, C3.13 dialkylaminothiocarbonyl, C4-9cycloalkylaminothiocarbonyl, C8-17 aralkylaminothiocarbonyl, C7 or Cπ arylaminothiocarbonyl, C3.10 heterocyclylaminothiocarbonyl, C3-16 heteroaralkylaminothiocarbonyl, C1-6 alkylsulfonyl, C3-8 cycloalkylsulfonyl, C7-16 aralkylsulfonyl, C6 or C]0 arylsulfonyl, C2-9 heterocyclylsulfonyl, or C2-ι5 heteroaralkylsulfonyl, Y1 is O,
S, NR , or a covalent bond, wherein R is H or C]-6 alkyl, wherein when Y is a covalent bond, B is an optionally substituted C]-6 alkyl, C2-6 alkenyl, C3-8 cycloalkyl, C6 or o aryl, C1-9 heterocyclyl, C7-16 aralkyl, C8-ι6 aralkenyl, C2-i5 heteroaralkyl, C3-15 heteroaralkenyl, C1-6 alkylsulfonyl amino, and when Y1 is O, S, or NR , and B is an optionally substituted C]-6 alkyl, C2-6 alkenyl, C3-8 cycloalkyl, C7-ι6 aralkyl, C8-]6 aralkenyl, C6 or C10 aryl, C1-9 heterocyclyl, C2-15 heteroaralkyl, C3-15 heteroaralkenyl, C2-7 acyl, C7-π aroyl, C3-ιo heteroaroyl, C2- alkoxycarbonyl, C4-9 cycloalkoxycarbonyl, C8-17 aralkoxycarbonyl, C7 or Cπ aryloxycarbonyl, C2-ι0 heterocyclyloxycarbonyl, aminocarbonyl, C2-7 alkylammocarbonyl, C3.13 dialkylaminocarbonyl, C4-9 cycloalkylaminocarbonyl, C8.1 aralkylaminocarbonyl, C7 or Cπ arylaminocarbonyl, C3-ιo heterocyclylaminocarbonyl, C3-16 heteroaralkylaminocarbonyl, C2-7 alkylthiocarbonyl, C7-π arylthiocarbonyl, C3- 0 heteroarylthiocarbonyl, C2-7 alkoxythiocarbonyl, C4-9 cycloalkoxythiocarbonyl, C8-π aralkoxythiocarbonyl, C7 or Cπ aryloxythiocarbonyl, aminothiocarbonyl, C2-7 alkylaminothiocarbonyl, C3-ι 3 dialkylaminothiocarbonyl, C -9 cycloalkylaminothiocarbonyl, C8.17 aralkylaminothiocarbonyl, C7 or Cπ arylaminothiocarbonyl, C3-]0 heterocyclylaminothiocarbonyl, C3-16 heteroaralkylaminothiocarbonyl; R3 is H or optionally substituted C1-6 alkyl, or together with X and A forms an optionally substituted C2-5 heterocyclic ring; and R° is -E-J, where when m is 0, E is a single bond to J, where J is H or an optionally substituted C]-6 alkyl, and when m is 1 , E is selected from the group consisting of a single bond to J, -C(O)O-, -C(O)S-, and -C(O)NR10-, wherein R10 is H or C] -6 alkyl and J is an optionally substituted C]-6 alkyl, C2-6 alkenyl, C3-8 cycloalkyl. C7.]6 aralkyl, C8-16 aralkenyl, C6 or C10 aryl, C1-9 heterocyclyl, C2-15 heteroaralkyl, or C3-ι5 heteroaralkenyl.
In one example of this embodiment, R8 is B-Y'-(CH2)n-K-C(O)-, K is oxo or CHOH, n is 0, where Y1 is a covalent bond and B is phenyl, 4- chlorophenyl, 3-chlorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl, 3- fluorophenyl, 4-fluorophenyl, 3,4-difluorophenyl, and 3,5-difluorophenyl.
In another example, AA2 is a covalent bond and R is B-Y -(CH2)π-K- C(O)-, where n is 0 and K, -C(O)-, X, and R3 together forms a heterocyclyl group having the formula X:
Figure imgf000009_0001
11 where W is CH or N. R is hydrogen or an optionally substituted C)-6 alkyl, C2-6 alkenyl, C3-8 cycloalkyl, C7-16 aralkyl, C8-16 aralkenyl, C6 or Cι0 aryl, C1-9 heterocyclyl, C2-15 heteroaralkyl, Q-ό alkylthio, C3-8 cycloalkylthio, C6 or do arylthio, C7.]6 aralkylthio, C2-9 heterocyclylthio, C2-15heteroaralkylthio, C1-6 alkylamino, C2-]2 dialkylamino C3-8 cycloalkyl amino, C7-16 aralkylamino, C6 or C10 arylamino, or C2-9 heterocyclylamino.
Y1 is O, S, NR18, or a covalent bond, where R18 is H or C1-6 alkyl. When Y1 is a covalent bond, B is an optionally substituted C1-6 alkyl, C2-6 alkenyl, C3-8 cycloalkyl, C6 or C10 aryl, C1-9 heterocyclyl, C7-16 aralkyl, C8.]6 aralkenyl, C2.15 heteroaralkyl, C3-ι5 heteroaralkenyl, C1-6 alkylsulfonyl amino, C3.8 cycloalkylsulfonylamino, C7.16 aralkylsulfonylamino, C6 or C10 arylsulfonylamino, C2-9 heterocyclylsulfonylamino, or C2.15 heteroaralkylsulfonylamino. When Y1 is O, S, or NR18, B is an optionally substituted C] -6 alkyl, C2-6 alkenyl, C3-8 cycloalkyl, C7-] 6 aralkyl, C8-]6 aralkenyl, C6 or C]0 aryl, C1-9 heterocyclyl, C2-ι5 heteroaralkyl, C3-15 heteroaralkenyl, C2-7 acyl, C7-π aroyl, C3-]0 heteroaroyl, C2-7 alkoxycarbonyl, C4-9 cycloalkoxycarbonyl, C8-17 aralkoxycarbonyl, C7 or Cπ aryloxycarbonyl,
Figure imgf000010_0001
heterocyclyloxycarbonyl, aminocarbonyl, C2-7 alkylammocarbonyl, C3-13 dialkylaminocarbonyl, C4-9 cycloalkylaminocarbonyl, C8-17 aralkylaminocarbonyl, C7 or Cπ arylaminocarbonyl, C3-10 heterocyclylaminocarbonyl, C3-] 6 heteroaralkylaminocarbonyl, C2- alkylthiocarbonyl, C7-π arylthiocarbonyl, C3-10 heteroarylthiocarbonyl, C2- alkoxythiocarbonyl, C4-9 cycloalkoxythiocarbonyl, C8-17 aralkoxythiocarbonyl, C or C] i aryloxythiocarbonyl, aminothiocarbonyl, C2- alkylaminothiocarbonyl, C3-13 dialkylaminothiocarbonyl, C4-9 cycloalkylaminothiocarbonyl, Cs-π aralkylaminothiocarbonyl, C7 or Cπ arylaminothiocarbonyl, C3-ιo heterocyclylaminothiocarbonyl, C3-16 heteroaralkylaminothiocarbonyl, or B is a natural or unnatural amino acid of L- or D-configuration substituted on the alpha-amine with H, C2- acyl, C7-11 aroyl, C3-ιo heteroaroyl, C2- alkoxycarbonyl, C4-9 cycloalkoxycarbonyl, C8-]7 aralkoxycarbonyl, C7 or Cπ aryloxycarbonyl, C2-ιo heterocyclyloxycarbonyl, C3-]5 heteroaralkoxycarbonyl, aminocarbonyl, C2-7 alkylammocarbonyl, C3-13 dialkylaminocarbonyl, C - cycloalkylaminocarbonyl, C8-17 aralkylaminocarbonyl, C7 or Cπ arylaminocarbonyl, C3-10 heterocyclylaminocarbonyl, C3. 6 heteroaralkylaminocarbonyl, C - alkylthiocarbonyl, C7-π arylthiocarbonyl, C3-]0 heteroarylthiocarbonyl, C2- alkoxythiocarbonyl, C4-9 cycloalkoxythiocarbonyl, C8-ι7 aralkoxythiocarbonyl, C7 or Cπ aryloxythiocarbonyl, aminothiocarbonyl, C2-7 alkylaminothiocarbonyl, C3-13 dialkylaminothiocarbonyl, C4-9 cycloalkylaminothiocarbonyl, C8.17 aralkylaminothiocarbonyl, C7 or Cπ arylaminothiocarbonyl, C3-ιo heterocyclylaminothiocarbonyl, C3-ι6 heteroaralkylaminothiocarbonyl, C1-6 alkylsulfonyl, C3-8 cycloalkylsulfonyl, C7- 16 aralkylsulfonyl, C6 or C]0 arylsulfonyl, C2-9 heterocyclylsulfonyl, or C2-15 heteroaralkylsulfonyl. Desirable compounds include those where m is 1 , R° is an optionally substituted 2-thiazole or 2-benzthiazole ring, R2 is H or optionally substituted C1-6 alkyl, R17 is C6 or C10 aryl or C1-9 heteroaryl, Y1 is a bond, and B is an optionally substituted C]-6 alkyl, C7-16 aralkyl, C6 or Cι0 aryl, C]-9 heterocyclyl, or C2-ι5 heterocyclylalkyl; those where m is 1, R° is an optionally substituted 2- thiazole or 2-benzthiazole ring, R is H or optionally substituted C1-6 alkyl, R is C or C10 aryl or .Q heteroaryl, Y1 is NH, and B is C]-9 heterocyclyl or C2-ι5 heterocyclylalkyl; those where m is 1, R° is 2-thiazole, R1 is 3-
9 17 1 guanidinopropyl, R is H, R is C6 or C10 aryl, or C1-9 heteroaryl, Y is NH, and B is C1- heterocyclyl or C2.ι5 heterocyclylalkyl; those where Y1 is O or S;
17 or those where R is optionally substituted C6 or Cι0 aryl, C].9 heterocyclyl, . 6 alkylthio, C3-8 cycloalkylthio, C6 or C]0 arylthio, C7-ι6 aralkylthio, C2-9 heterocyclylthio, or C2-ι5heteroaralkylthio, Y1 is NH, and B is C1- heterocyclyl or C2-15 heterocyclylalkyl.
In another embodiment of the first aspect, m is 1, X is N, R3 is H, and R1 is 3-guanidinopropyl, with the carbon bearing R1 having an (R)- or (S)- configuration, and R2 is selected from the group consisting of:
Figure imgf000011_0001
where the carbon bearing any of these substituents in either the (R)- or (S)- configuration. Preferably, the carbons bearing R1 is of the (S)-configuration.
Preferably, A is R8-AA2, with R8 is selected from the group consisting of:
Figure imgf000012_0001
where c is 0 or 1 ; each of R11, R12, R13, and R14 is, independently, C2- alkanoyl, C1-6 alkyl, C]-6 alkenyl, C1-6 alkoxy, C2.]2 alkoxy alkyl, C]-6 alkylsulfmyl, C2-ι2 alkylsulfinylalkyl, C1-6 alkylsulfonyl, C6 or C]0 aryl, C7-16 arylalkyl, amino, C1-6 aminoalkyl, C7 or Cπ aryloyl, azido, C1-6 azidoalkyl, carboxaldehyde, carboxamide, carboxyl, C2-7 (carboxaldehyde)alkyl, C3-8 cycloalkyl, C4-14 cycloalkylalkyl, halo, C]-6haloalkyl, C1-9 heterocyclyl, Cι- (heterocyclyl)oxy, C2-10 (heterocyclyl)oyl, hydroxy, C]-6 hydroxyalkyl, nitro, C]-6nitroalkyl, N- protected amino, N-protected aminoalkyl, C1-6thioalkoxy, C -12 thioalkoxyalkyl, C]- perfluoroalkyl, C1-4 perfiuoroalkoxy, C6 or C10 aryloxy, C3-8 cycloalkoxy, C -] cycloalkyl alkoxy, or C7-ι 6 arylalkoxy, -(CH2)qCO2RA, wherein q is zero to four and R is selected from the group consisting of (a) . 6 alkyl, (b) C6 or o aryl and (c) C7-ι6 arylalkyl, wherein the alkylene group is of one to six carbon atoms, -(CH2)qCONRBRc, wherein q is zero to four and RB and Rc are independently selected from the group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) C6 or C]0 aryl and (d) C7.]6 arylalkyl, wherein the alkylene group is of one to six carbon atoms, -(CH2)qSO2R , wherein q is zero to four and RD is selected from the group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) C6 or Cio aryl and (d) C7-16 arylalkyl, wherein the alkylene group is of one to six carbon atoms, -(CH2)qSO2NRERF, wherein q is zero to four and RE and RF are independently selected from the group consisting of (a) hydrogen, (b) C\.6 alkyl, (c) C6 or C10 aryl and (d) C7-16 arylalkyl, wherein the alkylene group is of one to six carbon atoms, or -(CH2)qNRGRH, wherein q is zero to four and RG and RH are independently selected from the group consisting of (a) hydrogen, (b) an N-protecting group, (c) alkyl of one to six carbon atoms, (d) alkenyl of two to six carbon atoms, (e) alkynyl of two to six carbon atoms, (f) C6 or do aryl, (g) C7-16 arylalkyl, wherein the alkylene group is of one to six carbon atoms, (h) cycloalkyl of three to eight carbon atoms, and (i) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms, and the alkylene group is of one to ten carbon atoms, with the proviso that no two groups are bound to the nitrogen atom through a carbonyl group or a sulfonyl group; and each of R15 and R16 is, independently, H, C1-6 alkyl; C1-6hydroxyalkyl; C]-4 perfluoroalky], cyano, halo, -(CH2)qCO2RA, -(CH2)qCONRBRc, -(CH2)qSO2RD, -(CH2)qSO2NRERF, -(CH2)qNRGRH, wherein q is 0-2 and RA, RB, Rc, RD, RE, RF, RG, and RH are as defined above.
Most preferably, AA2 is selected from the group consisting of:
Figure imgf000013_0001
Figure imgf000014_0001
In one example, AA2 is L-3- -pyridylalanine, R ! is
Figure imgf000014_0002
In other examples, AA2 is
Figure imgf000014_0003
and R8 is selected from the group consisting of: α-toluenesulfonyl, (methyl-3- benzoate)methanesulfonyl, 2-nitro-α-toluenesulfonyl, 1-propanesulfonyl, 3- carboxyl-α-toluenesulfonyl, and 3-chloropropanesulfonyl.
In other preferred compounds, A is selected from the group consisting of:
Figure imgf000015_0001
Figure imgf000016_0001
Preferably, m is 1, R° is an optionally substituted 2-thiazole or 2-benzthiazole ring; R is H or optionally substituted C1-6 alkyl, G is an optionally substituted Cι„6 alkyl, aralkyl, or B-Y-, wherein Y is NH and B is Cι-9 heterocyclyl or C2-15 heteroaralkyl; and R17 is C6 or C10 aryl, or d.9 heteroaryl.
In another embodiment of the first aspect, R1 is selected from the group consisting of:
Figure imgf000017_0001
, wherein a is an integer from 1-4, b is an integer from 0-2,
R4 and R5 are H or Cμ6 alkyl, R6 is H, OH, C1-6 alkyl, or together with R4 forms a 5- or 6-membered ring, and R7 is H, OH, SH, NH2, NO2, optionally substituted C]-6 alkyl, halogen, or CF3;
R is selected from the group consisting of:
Figure imgf000017_0002
X is N; R3 is H; and A is
Figure imgf000017_0003
where R3a is H, Cι-6 alkyl, or together with M forms a 5- or 6-membered ring, D is C(O), C(S), or S(O)2, q is an integer from 0-4, Q is an optionally substituted C]-6 alkyl, C3-8 cycloalkyl, C1-9 heterocyclyl, C6 or C10 aryl, C1-6 alkyloxy, C3-8 cycloalkyloxy, Q or C10 aryloxy, C2-9 heterocyclyloxy, C1-6 alkylthio, C3-8 cycloalkylthio, C6 or C10 arylthio, d.6alkylamino, C2-j2 dialkylamino, C7-ι6 aralkylamino, C6 or C]0 arylamino, C2-9 heterocyclylamino, aminocarbonyl, C2-7 alkylaminocarbonyl, C3-13 dialkylaminocarbonyl, amino, amidino, guanidino, ureido, hydroxy, or carboxy, with the proviso that when q is 1, Q is not phenyl, M is single bond, NR3b, O, or S, wherein R3b is H, C1-6 alkyl, or when taken together with M, D, N, and R3a forms a 5- or 6-membered ring, and U is an optionally substituted C]-6 alkyl, C2-6 alkenyl, C3-8 cycloalkyl, C1-9 heterocyclyl, C7-ι6 aralkyl, C8-ι6 aralkenyl, C6 or C]0 aryl, C2-15 heterocyclylalkyl, or C3-15 heteroaralkenyl, where when m is 1, R° is an optionally substituted C6 or C10 aryl or C1-9 heterocyclyl, or when m is 0, R° is H or C1-6 alkyl.
In some examples of preferred compounds, m is 1, R° is an optionally
1 7 substituted 2-thiazole or 2-benzthiazole ring, R is 3-guanidinopropyl, R is 2- methylpropyl, R3a is H, D is S(O)2, M is a single bond, q is 0, Q is phenyl, cyclohexyl, cyclopentyl, 4-pyridyl, 3-pyridyl, or 2-pyridyl, and U is selected from the group consisting of: benzyl, 3-methylcarboxybenzyl, 3- carboxybenzyl, 2-nitrobenzyl, 1 -propyl, and 3-chloropropyl.
In other examples, m is 1, R° is an optionally substituted 2-thiazole or 2- benzthiazole ring, R1 is 3-guanidinopropyl, R3a is H, D is C(O), M is NR3b, where R is H, q is 0, Q is selected from the group consisting of:
Figure imgf000018_0001
Figure imgf000019_0001
U is selected from the group consisting of: 2-chlorobenzyl, 3-chlorobenzyl, 4- chlorobenzyl, 2,3-dichlorobenzyl, 2,4-dichlorobenzyl, 2,5-dichlorobenzyl, 2,6- dichlorobenzyl, 3,4-dichlorobenzyl, 3,5-dichlorobenzyl, 2-fluorobenzyl, 3- fluorobenzyl, 4-fluorobenzyl, 2,3-difluorobenzyl, 2,4-difluorobenzyl, 2,5- difluorobenzyl, 2,6-difluorobenzyl, 3,4-difluorobenzyl, 3,5-difluorobenzyl, (R)- α-methyl-4-bromobenzyl, (S)-α-methyl-4-bromobenzyl, (R)-α-methyl-4- chlorobenzyl, (S)-α-methyl-4-chlorobenzyl, (R)-α-methyl-4-fluorobenzyl, (S)- α-methyl-4-fluorobenzyl, 2-trifluoromethylbenzyl, 2-methylbenzyl, 3- methylbenzyl, 4-methylbenzyl, 2-phenyl acetic acid, 2-pyridylmethyl, 3- pyridylmethyl, 4-pyridylmethyl, phenyl, and indanyl. Preferred compounds include those where R2 is isopropyl, Q is selected from the group consisting of: cyclohexyl, 2-pyridylmethyl, 3-ureidopropyl, 4-hydroxybenzyl, and acetamide, and U is (R)-α-methyl-4-bromobenzyl.
In still some other examples, U is CHR3cR3d, wherein R3c is an optionally substituted C6 or C10 aryl or C1-9 heterocyclyl, and R3d is H, C1-6 alkyl, C2-6 alkenyl, or together with R3c forms a fused bicyclic ring.
In yet another embodiment of the first aspect, m is 0 and R° is H. Preferred compounds include those where R is H or an optionally substituted C1-6 alkyl, C2-6 alkenyl, C3-8 cycloalkyl, C7-ι6 aralkyl, C8-16 aralkenyl, C6 or C10 aryl, C2-9 heterocyclyl, C2-i5 heteroaralkyl, or C3.]5 heteroaralkenyl; R3 is H or C].6 alkyl; X is N; and A is R8-AA2, wherein AA2 is a single natural or unnatural alpha-amino acid residue and R8 is selected from the group consisting of:
Figure imgf000020_0001
wh c is 0 or 1 ; each of Rn, R12, R13, and R14 is, independently, C2-7 alkanoyl,
Cι_6 alkyl, C1-6 alkenyl, C1-6 alkoxy, C .ι2 alkoxyalkyl, Cι-6 alkylsulfinyl, C2-12 alkylsulfinylalkyl, d-6 alkylsulfonyl, C6 or C10 aryl, C7-16 arylalkyl, amino, C1-6 aminoalkyl, C7 or Cπ aryloyl, azido, C]-6azidoalkyl, carboxaldehyde, carboxamide, carboxyl, C2-7 (carboxaldehyde)alkyl, C3-8 cycloalkyl, C -14 cycloalkylalkyl, halo, C]-6 haloalkyl, C]-9 heterocyclyl, d.9 (heterocyclyl)oxy, C2.ιo(heterocyclyl)oyl, hydroxy, C1-6 hydroxyalkyl, nitro, C1-6nitroalkyl, N- protected amino, N-protected aminoalkyl, C1-6thioalkoxy, C2.ι2 thioalkoxyalkyl, -(CH2)qCO2RA, wherein q is zero to four and RA is selected from the group consisting of (a) C]-6 alkyl, (b) C or C10 aryl and (c) C -16 arylalkyl, wherein the alkylene group is of one to six carbon atoms, -
(CH2)qCONRBRc, wherein q is zero to four and RB and Rc are independently selected from the group consisting of (a) hydrogen, (b) C]-6 alkyl, (c) C6 or do aryl and (d) C7-16 arylalkyl, wherein the alkylene group is of one to six carbon atoms, -(CH2)qSO2RD, wherein q is zero to four and RD is selected from the group consisting of (a) hydrogen, (b) C]-6 alkyl, (c) C6 or C10 aryl and (d) C -16 arylalkyl, wherein the alkylene group is of one to six carbon atoms, - (CH2)qSO2NRERF, wherein q is zero to four and RE and RF are independently selected from the group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) C6 or C10 aryl and (d) C -16 arylalkyl, wherein the alkylene group is of one to six carbon atoms, -(CH2)qNRGRH, wherein q is zero to four and RG and RH are independently selected from the group consisting of (a) hydrogen, (b) an N- protecting group, (c) alkyl of one to six carbon atoms, (d) alkenyl of two to six carbon atoms, (e) alkynyl of two to six car(bon atoms, (f) C6 or Cio aryl, (g) C7- 16 arylalkyl, wherein the alkylene group is of one to six carbon atoms, (h) cycloalkyl of three to eight carbon atoms, and (i) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms, and the alkylene group is of one to ten carbon atoms, with the proviso that no two groups are bound to the nitrogen atom through a carbonyl group or a sulfonyl group, C1-4 perfluoroalkyl, C1-4 perfluoroalkoxy; C6 or C10 aryloxy, C3-8 cycloalkoxy, C .14 cycloalkylalkoxy, or C7-]6 arylalkoxy; and each of R15 and R16 is, independently, H, d_6 alkyl; C1-6 hydroxyalkyl; C1- perfluoroalkyl, cyano, halo, -(CH2)qCO2RA, -(CH2)qCONRBRc, -(CH2)qSO2RD, -(CH2)qSO2NRERF, - (CH2)qNRGRH, wherein q is zero to two and RA, RB, Rc, RD, RE, RF, RG, and RH are as defined above.
Other preferred compounds include those where R1 is selected from the group consisting of:
Figure imgf000021_0001
Figure imgf000022_0001
AA2 is selected from the group consisting of:
Figure imgf000022_0002
R2 is selected from the group consisting of:
Figure imgf000023_0001
Most preferred compounds are those in which R is
Figure imgf000023_0002
where b is 0 or 1 and R7 is H, and A is R8-AA2, where R8 is benzylsulfonyl, 3- methylcarboxybenzylsulfonyl, 3-carboxybenzylsulfonyl, 2-nitrobenzylsulfonyl, propylsulfonyl, and 3-chloropropylsulfonyl and AA2 is as previously defined.
In a second aspect, the invention features a compound of formula II:
Figure imgf000023_0003
or a pharmaceutically acceptable salt or prodrug thereof, where R19 is H, C1-6 alkyl, OH, NH2, NO2, CO2R19a, wherein R19a is C]-6 alkyl; R20 is H, OH, SH, NH2, NO2, optionally substituted C1-6 alkyl, halogen, C1-3 perfluoroalkyl, or when together with Z' forms a fused 5- or 6-membered ring; e is N, NO, or CR21a, wherein R21a is H, halogen, or C1-3 perfluoroalkyl; g is N, NO, or CR21 , wherein R 1b is H, halogen, C1-6 alkyl, C1-3 perfluoroalkyl, C1-3 alkenyl, C7.]6 aralkyl, C2-15 heteroaralkyl, C1-6 alkoxy, C7-16 aralkoxy, C2-15heteroaralkoxy, C2- η alkoxycarbonyl, C8-ι7 aralkoxycarbonyl, Q.^heteroaralkoxycarbonyl, C2-7 alkylaminocarbonyl, C8-π aralkylaminocarbonyl, C3-ι heteroaralkylaminocarbonyl, C]-6 alkylthio, C7-i6 ralkylthio, C2.15 heteroaralkylthio, C2-7 alkylthiocarbonyl, C8-]7 aralkylthiocarbonyl, C3-16 heteroaralkylthiocarbonyl; T is a bond, O, S, or NR23, where R23 is H, OH, or optionally substituted
C1-6 alkyl, C1-6 alkenyl, C7.n aralkyl, C8-16 aralkenyl, C2-ι5 heteroaralkyl, or C3-15 heteroaralkenyl.
When T is O, S, or NR23, B is a natural or unnatural alpha-amino acid residue of D- or L-configuration, or an optionally substituted Cι-6 alkyl, C3-8 cycloalkyl, C]-6 alkenyl, C7-16 aralkyl, C8-]7 aralkylcarbonyl, C8-]6 aralkenyl, C6 or C]o aryl, C2-ι5 heteroaralkyl, C3-16 heteroaralkylcarbonyl, C3-ι5 heteroaralkenyl, C2-7 acyl, C4-9 cycloalkylcarbonyl, C7-π aroyl, C2-]0 heterocyclyl oyl, C -9 cycloalkoxycarbonyl, C7 or Cn aryloxycarbonyl, C3-10 heterocyclyloxycarbonyl, C3-16 heteroaralkyloxycarbonyl, aminocarbonyl, C2-7 alkylaminocarbonyl, C3- 3 dialkylaminocarbonyl, C -9 cycloalkylaminocarbonyl, C8-π aralkylaminocarbonyl, C7 or Cπ arylaminocarbonyl, C3-ι0 heterocyclylaminocarbonyl, C3-16 heteroaralkylaminocarbonyl, C2-7 alkylthiocarbonyl, C4-9 cycloalkylthiocarbonyl, C7-π arylthiocarbonyl, C8-ι7 aralkylthiocarbonyl, C -]0 heterocyclylthiocarbonyl, C3-]6 heteroaralkylthiocarbonyl, aminothiocarbonyl, C2-7 alkylaminothiocarbonyl, C3- j 3 dialkylaminothiocarbonyl, C4-9 cycloalkylaminothiocarbonyl, C8-]7 aralkylaminothiocarbonyl, C7 or Cπ arylaminothiocarbonyl, C3-10 heterocyclylaminothiocarbonyl, C3-ι6 heteroaralkylaminothiocarbonyl, C1-6 alkylsulfonyl, C3-8 cycloalkylsulfonyl, C7-16 aralkylsulfonyl, C6 or C10 arylsulfonyl, C2-9 heterocyclylsulfonyl, or C2-] 5 heteroaralkylsulfonyl.
When T is a bond, B is OH, SH, NH2, NO2, SO3H, CO2H or an optionally substituted C1-6 alkyl, C1-6 alkenyl, C7-ι6 aralkyl, C8-ι6 aralkenyl, C2.15 heteroaralkyl, C3-15 heteroaralkenyl, C2.7 acyl, C7-π aroyl, C3-10 heteroaroyl, C2-7 alkoxycarbonyl, C - cycloalkoxycarbonyl, Cg.17 aralkoxycarbonyl, C7 or Cn aryloxycarbonyl, C3-ι6 heteroaralkyloxycarbonyl, C2-ιo heterocyclyloxycarbonyl, aminocarbonyl, C2-7 alkylaminocarbonyl, C3.13 dialkylaminocarbonyl, C -9 cycloalkylaminocarbonyl, C?.17 aralkylaminocarbonyl, C or Cπ arylaminocarbonyl, C3.10 heterocyclylaminocarbonyl, C3-] 6 heteroaralkylaminocarbonyl, C2-7 alkylthiocarbonyl, C4-9 cycloalkylthiocarbonyl, C7-π arylthiocarbonyl, C8-π aralkylthiocarbonyl, C2-10 heterocyclylthiocarbonyl, C3-16 heteroaralkylthiocarbonyl, C]-6 alkylsulfonyl, C3-8 cycloalkylsulfonyl, C7-1 aralkylsulfonyl, C6 or C10 arylsulfonyl, C2-9 heterocyclylsulfonyl, or C2-] 5 heteroaralkylsulfonyl. f is a bond, O, S, or NR24, wherein R24 is H, OH, or C1-6 alkyl.
When f is O, S, or NR24, V is H or an optionally substituted C1-6 alkyl, C7-ι6 aralkyl, C2_ι5 heteroaralkyl, C2-7 acyl, C7-n aroyl, C3-]0 heteroaroyl, C2-7 alkoxycarbonyl, C4-9 cycloalkoxycarbonyl, C8-π aralkoxycarbonyl, C7 or Cπ aryloxycarbonyl, C3.]6 heteroaralkyloxycarbonyl, C2-1o heterocyclyloxycarbonyl, aminocarbonyl, C2- alkylaminocarbonyl, C3.]3 dialkylaminocarbonyl, C4-9 cycloalkylaminocarbonyl, C8-17 aralkylaminocarbonyl, C7 or Cπ arylaminocarbonyl, C3-ι0 heterocyclylaminocarbonyl, C3-] 6 heteroaralkylaminocarbonyl, C -7 alkylthiocarbonyl, C -9 cycloalkylthiocarbonyl, C7-11 arylthiocarbonyl, C8-17 aralkylthiocarbonyl, C2-10 heterocyclylthiocarbonyl, C3-ι6 heteroaralkylthiocarbonyl, C1-6 alkylsulfonyl, C3-8 cycloalkylsulfonyl, C7-ι6 aralkylsulfonyl, C6 or C10 arylsulfonyl, C2-9 heterocyclylsulfonyl, or C -15 heteroaralkylsulfonyl. When f is a bond, V is H, OH, SH, NH2, SO3H, CO2H, or an optionally substituted C1-6 alkyl, hydroxy-C]-6 alkyl, C3-8 cycloalkyl, C1-6 alkenyl, C -16 aralkyl, C8.]6 aralkenyl, C2-15 heteroaralkyl, C3-15 heteroaralkyl, C6 or C]0 aryl, C]- heterocyclyl, C2- alkylaminocarbonyl, C3.13 dialkylaminocarbonyl, C -9 cycloalkylaminocarbonyl, C8-1 aralkylaminocarbonyl, C7 or Cπ arylaminocarbonyl, C -1o heterocyclylaminocarbonyl, C2- alkoxycarbonyl, C4-9 cycloalkoxycarbonyl, C8-17 aralkoxycarbonyl, C7 or Cυ aryloxycarbonyl, C3-10 heterocyclyloxycarbonyl, C3.ι6 heteroaralkyloxycarbonyl, C2-7 alkylthiocarbonyl, C4-9 cycloalkylthiocarbonyl, C7-11 arylthiocarbonyl, C8-ι7 aralkylthiocarbonyl, C2-ιo heterocyclylthiocarbonyl, C3.]6 heteroaralkylthiocarbonyl, C2-7 acyl, C7-π aroyl, C3.10 heteroaroyl, C1-6 alkylsulfonyl, C3-8 cycloalkylsulfonyl, C7-i6 aralkylsulfonyl, C6 or C10 arylsulfonyl, C2-9 heterocyclylsulfonyl, C -]5 heteroaralkylsulfonyl, C]-6 alkylaminosulfonyl, C3-8 cycloalkylaminosulfonyl, C2-12 dialkylaminosulfonyl, Cη. η aralkylaminosulfonyl, C6 or C]0 arylaminosulfonyl, Cμ heterocyclylaminosulfonyl, d-isheteraralkylaminosulfonyl, C]-6 alkylsulfinyl, C3-8 cycloalkylsulfinyl, d.^ aralkylsulfinyl, C6 or C10 arylsulfinyl, C2_9 heterocyclylsulfinyl, or C2-15 heteroaralkylsulfinyl.
In one embodiment, T is NH, B is an optionally substituted optionally substituted C2-7 acyl, C -9 cycloalkylcarbonyl, C7 or Cn aroyl, C2-10 heterocyclylcarbonyl, C3.]0 heteroaroyl, C2-7 alkoxycarbonyl, C4.9 cycloalkoxycarbonyl, C8-17 aralkoxycarbonyl, C7 or Cπ aryloxycarbonyl, C3-ι0 heterocyclyloxycarbonyl, C3-16 heteroaralkyloxycarbonyl, C2-7 alkylthiocarbonyl, C4-9 cycloalkylthiocarbonyl, C7-17 aralkylthiocarbonyl, C7 or Cπ arylthiocarbonyl, C3-]6 heteroaralkylthiocarbonyl, C3-10 heterocyclylthiocarbonyl, aminothiocarbonyl, C2- alkylaminocarbonyl, C3-13 dialkylaminocarbonyl, C -9 cycloalkylaminocarbonyl, C8-17 aralkylaminocarbonyl, C or Cπ arylaminocarbonyl, C3.10 heterocyclylaminocarbonyl, C3-16 heteroaralkylaminocarbonyl, Cι-6 alkylsulfonyl, C3-8 cycloalkylsulfonyl, C7.]6 aralkylsulfonyl, C6 or C]0 arylsulfonyl, C2- heterocyclylsulfonyl, or C2-]5 heteroaralkylsulfonyl; and f is O, S, or NR24. In another embodiment, T is NH, and B is an optionally substituted C6 or Cio aroyl or an optionally substituted Cι-9 heteroaroyl. Examples include those compounds in which B is
Figure imgf000027_0001
where each of R25, R26,and R27 is, independently H, C2-7 alkanoyl, C1-6 alkyl, C1-6 alkenyl, C1-6 alkoxy, C2-12 alkoxyalkyl, C]-6 alkylsulfinyl, C2-12 alkylsulfinylalkyl, C]-6 alkylsulfonyl, C6 or C]0 aryl, C7-] 6 arylalkyl, amino, d-6 aminoalkyl, C7 or Cn aryloyl, azido, d-6azidoalkyl, carboxaldehyde, carboxamide, carboxyl, C2- (carboxaldehyde)alkyl, d-8 cycloalkyl, C4-]4 cycloalkylalkyl, halo, C1-6 haloalkyl, C1-9 heterocyclyl, C1-9 (heterocyclyl)oxy, C2-ιo (heterocycly])oyl, hydroxy, C1-6 hydroxyalkyl, nitro, Cι-6nitroalkyl, N- protected amino, N-protected aminoalkyl, C1-6thioalkoxy, C2-] thioalkoxyalkyl, thiol, -(CH2)qCO2RA, wherein q is zero to four and RA is selected from the group consisting of (a) C]-6 alkyl, (b) C6 or Cj0 aryl and (c) C7-ι6 arylalkyl, wherein the alkylene group is of one to six carbon atoms, - (CH )qCONR R , wherein q is zero to four and R and R are independently selected from the group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) C6 or C]0 aryl and (d) C7-] 6 arylalkyl, wherein the alkylene group is of one to six carbon atoms, -(CH2)qSO2RD, wherein q is zero to four and RD is selected from the group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) C6 or C10 aryl and (d) C7-16 arylalkyl, wherein the alkylene group is of one to six carbon atoms, - (CH2)qSO2NRERF, wherein q is zero to four and RE and RF are independently selected from the group consisting of (a) hydrogen, (b) C]-6 alkyl, (c) C6 or C10 aryl and (d) C7-16 arylalkyl, wherein the alkylene group is of one to six carbon atoms, -(CH2)qNRGRH, wherein q is zero to four and RG and RH are independently selected from the group consisting of (a) hydrogen, (b) an N- protecting group, (c) alkyl of one to six carbon atoms, (d) alkenyl of two to six carbon atoms, (e) alkynyl of two to six carbon atoms, (f) C6 or C10 aryl, (g) C7- 16 arylalkyl, wherein the alkylene group is of one to six carbon atoms, (h) cycloalkyl of three to eight carbon atoms, and (i) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms, and the alkylene group is of one to ten carbon atoms, with the proviso that no two groups are bound to the nitrogen atom through a carbonyl group or a sulfonyl group, C -4 perfluoroalkyl, C]-4 perfluoroalkoxy; C6 or C10 aryloxy, C3-8 cycloalkoxy, C -ι4 cycloalkylalkoxy, or C7-]6 arylalkoxy.
Preferred compounds in which B is a 2,3-disubstituted or 2,3,4- trisubstituted aryloyl include those in which f is a bond and V is an optionally substituted C1-6 alkyl, hydroxy-C1-6 alkyl, C3-8 cycloalkyl, C] -6 alkenyl, C7-] 6 aralkyl, C8-16 aralkenyl, C2-ι5 heteroaralkyl, C3.15 heteroaralkyl, C6 or do aryl, C1- heterocyclyl, C -7 alkylaminocarbonyl, C3-13 dialkylaminocarbonyl, C -9 cycloalkylaminocarbonyl, C8-π aralkylaminocarbonyl, C or Cπ arylaminocarbonyl, C2-ι0 heterocyclylaminocarbonyl, C2-7 alkoxycarbonyl, C4-9 cycloalkoxycarbonyl, C8-]7 aralkoxycarbonyl, C7 or Cπ aryloxycarbonyl, C3-ι0 heterocyclyloxycarbonyl, C3-16 heteroaralkyloxycarbonyl, C2. alkylthiocarbonyl, C4-9 cycloalkylthiocarbonyl, C7-π arylthiocarbonyl, C8-17 aralkylthiocarbonyl, C2-1o heterocyclylthiocarbonyl, C3.ι6 heteroaralkylthiocarbonyl, C2-7 acyl, C7-π aroyl, C3.ι0 heteroaroyl, C1 -6 alkylsulfonyl, C3- cycloalkylsulfonyl, C7-1 aralkylsulfonyl, C6 or C10 arylsulfonyl, C2-9 heterocyclylsulfonyl, or C2-15 heteroaralkylsulfonyl, d-6 alkylaminosulfonyl, C3-8 cycloalkylaminosulfonyl, C2-12 dialkylaminosulfonyl, C7-17 aralkylaminosulfonyl, C6 or C10 arylaminosulfonyl, C1-9 heterocyclylaminosulfonyl, C2-ι5 heteraralkylaminosulfonyl, C]-6 alkylsulfinyl, C3-8 cycloalkylsulfinyl, C7.16 aralkylsulfinyl, C6 or C10 arylsulfinyl, C2-9 heterocyclylsulfinyl, or C2-ιs heteroaralkylsulfinyl. Most preferred compounds in which B is a 2,3-disubstituted or 2,3,4-trisubstituted aryloyl include those where f is a bond and V is optionally substituted C6 or C]0 arylaminocarbonyl or C7-ι7 aralkylaminocarbonyl.
In another embodiment of the second aspect, f is a bond and V is H. Examples include those compounds in which B is
Figure imgf000029_0001
where each of R25, R26,and R27 is, independently H, C2-7 alkanoyl, d-6 alkyl, C1-6 alkenyl, C! -6 alkoxy, d.^ alkoxyalkyl, C1-6 alkylsulfinyl, C2-12 alkylsulfinylalkyl, C1-6 alkylsulfonyl, C6 or C]0 aryl, C7-]6 arylalkyl, amino, C]-6 aminoalkyl, C or C aryloyl, azido, C1-6 azidoalkyl, carboxaldehyde, carboxamide, carboxyl, C2-7 (carboxaldehyde)alkyl, C3.g cycloalkyl, C4-]4 cycloalkylalkyl, halo, C1-6 haloalkyl, C1-9 heterocyclyl, C1-9 (heterocyclyl)oxy, C2.ιo (heterocyclyl)oyl, hydroxy, C1-6 hydroxyalkyl, nitro, cyano, C1-6nitroalkyl, N-protected amino, N-protected aminoalkyl, Cι-6thioalkoxy, C2-ι2 thioalkoxyalkyl, thiol, -(CH2)qCO2RA, wherein q is zero to four and RA is selected from the group consisting of (a) Cj-6 alkyl, (b) C6 or C10 aryl and (c) C7-ι6 arylalkyl, wherein the alkylene group is of one to six carbon atoms, - (CH2)qCONR R , wherein q is zero to four and R and R are independently selected from the group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) C6 or C10 aryl and (d) C7.16 arylalkyl, wherein the alkylene group is of one to six carbon atoms, -(CH2)qSO2RD, wherein q is zero to four and RD is selected from the group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) C6 or C]0 aryl and (d) C7-]6 arylalkyl, wherein the alkylene group is of one to six carbon atoms, - (CH2)qSO NRERF, wherein q is zero to four and RE and RF are independently selected from the group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) C6 or C10 aryl and (d) C7-16 arylalkyl, wherein the alkylene group is of one to six carbon atoms, -(CH2)qNRGRH, wherein q is zero to four and R° and RH are independently selected from the group consisting of (a) hydrogen, (b) an N- protecting group, (c) alkyl of one to six carbon atoms, (d) alkenyl of two to six carbon atoms, (e) alkynyl of two to six carbon atoms, (f) C6 or C]0 aryl, (g) C7- 16 arylalkyl, wherein the alkylene group is of one to six carbon atoms, (h) cycloalkyl of three to eight carbon atoms, and (i) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms, and the alkylene group is of one to ten carbon atoms, with the proviso that no two groups are bound to the nitrogen atom through a carbonyl group or a sulfonyl group, C1-4 perfluoroalkyl, C perfluoroalkoxy; C6 or C]0 aryloxy, C3„8 cycloalkoxy, C4-]4 cycloalkylalkoxy, or C7-16 arylalkoxy. A preferred B substituent for any of the compounds of formula II is a
2,3,-disubstituted aroyl group in which the 2-substituent is halo or C1-6 alkyl and the 3-substituent is hydroxyl.
In an embodiment of either the first aspect or second aspect of the invention, a compound of the invention can further be attached to a linker (e.g. to any of the groups R°, R2, or A of compounds of formula I or to any of the groups B or V of formula II), where the linker is reactive to a blood component, such as, for example, an erythrocyte, a lymphocyte, a blood platelet, an immunoglobulin, serum albumin, ferritin, corticosteroid-binding globulin, sex hormone-binding globulin, transferrin, thyroxin-binding protein, and alpha-2-macroglobulin. Some examples of linkers attached to a group reactive to a blood component are maleimide- (CH2)bbC(O)NHCH2CH2(OCH2CH2)aaOCH2C(O)-, maleimide- (CH2)bbC(O)NHCH2CH2(OCH2CH2)aaNHCH2C(O)-, maleimide- (CH2)b C(O)NHCH2CH2(OCH2CH2)aaNHC(S)-, maleimide-(CH2)bbNHC(S)-, maleimide-(CH2)bbC(O)-, and maleimide (CH2)b -where aa is 1-10 and bb is 1- 4.
In another embodiment of either the first or second aspect, a compound of the invention can further be attached to a polyethylene glycol moiety (e.g. to
Λ 7 any of the groups R , R , or A of compounds of formula I or to any of the groups B or V of formula II). Examples of polyethylene glycol moieties include H(OCH2CH2)ccO-, H3C(OCH2CH2)ccOC(O), H(OCH2CH2)ccOC(O), H3C(OCH2CH2)ccNHC(O), H(OCH2CH2)CcNHC(O), H3C(OCH2CH2)ccNHC(S), H(OCH2CH2)ccNHC(S), H3C(OCH2CH2)CcC(O), H(OCH2CH2)ccC(O), H3C(OCH2CH2)ccNHCH2C(O), H(OCH2CH2)ccNHCH2C(O),
H3C(OCH2CH2)ccOC(O)C(CH3)2-, and H(OCH2CH2)ccOC(O)C(CH3)2-, wherein cc is a range of numbers that results in an average molecular weight of said polyethylene glycol moiety of between 1 ,000-40,000, preferabley 20,-000 or 40,000. In another aspect, the invention features a pharmaceutical composition that includes a pharmaceutically acceptable carrier and a therapeutically effective amount of any compound of the invention, or a pharmaceutically acceptable salt or prodrug thereof.
The invention also features a method of treating a patient in need of thromboembolic disorder treatment that includes administering to the patient a therapeutically effective amount of any compound of the invention, or a pharmaceutically acceptable salt or prodrug thereof. The thromboembolic disorder can be arterial cardiovascular thromboembolic disorders, venous cardiovascular thromboembolic disorders, and thromboembolic disorders in the chambers of the heart; including unstable angina, an acute coronary syndrome, first myocardial infarction, recurrent myocardial infarction, ischemic sudden death, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary arterial thrombosis, cerebral arterial thrombosis, cerebral embolism, kidney embolism, pulmonary embolism, and thrombosis resulting from (a) prosthetic valves or other implants, (b) indwelling catheters, (c) stents, (d) cardiopulmonary bypass, (e) hemodialysis, or (f) other procedures in which blood is exposed to an artificial surface that promotes thrombosis. In addition to their use in anticoagulant therapy, Factor Xla inhibitors are useful in the treatment and prevention of other diseases in which the generation of thrombin has been implicated as playing a physiologic role. For example, thrombin has been implicated in contributing to the morbidity and mortality of chronic and degenerative diseases such as cancer, arthritis, atherosclerosis, and Alzheimer's disease by its ability to regulate many different cell types through specific cleavage and activation of a cell surface thrombin receptor, mitogenic effects, diverse cellular functions such as cell proliferation, for example, abnormal proliferation of vascular cells resulting in restenosis or angiogenesis, release of PDGF, and DNA synthesis. Inhibition of Factor Xla effectively blocks thrombin generation and therefore neutralizes any physiologic effects of thrombin on various cell types. The representative indications discussed above include some, but not all, of the potential clinical situations amenable to treatment with a Factor Xla inhibitor.
Three-dimensional structures of Factor Xla and methods for designing or selecting additional Factor Xla inhibitors using these structures
For the purpose of comparison to other serine proteases in the trypsin superfamily, two distinct residue numbering systems are used herein. For three-dimensional structure related descriptions, FXIcat residues are assigned numbers corresponding to those of trypsin, these numbers are shown in regular type and apply only to residues in the FXIcat. For primary structure (sequence) related descriptions, e.g., mutations or catalytic domain termini, FXIcat is assigned numbers corresponding to those of the native zymogen found in the bloodstream. Thus, the initiator methionine is residue -18, and the protein begins with Glul (SEQ ID NO: 2). These numbers are shown in bold in the text. The 18 amino acid signal sequence, which is included in the translated sequence in Genbank, is removed co-translationally to yield the N-terminus of the mature enzyme. Figure 2 shows the conversion between these two numbering systems.
Computer Systems In one aspect, the invention features a computer system that includes (a) a memory having stored therein data indicative of atomic coordinates in any one of the files listed in Figure 16, or surrogates thereof, of the non-hydrogen atoms of residues serine 195, histidine 57, and aspartic acid 102 of FXIcat or atomic coordinates that have a root mean square deviation from the backbone atoms of the residues of less than 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 A, and (b) a processor in electrical communication with the memory. This processor includes a program for generating and displaying a three-dimensional model representative of FXIcat. In one example, the stored data is x-ray diffraction data In another aspect, the invention features a computer system that includes a memory having stored therein data indicative of a pharmacophore model of a Factor Xla ligand. The pharmacophore model includes a three-dimensional model that includes 3 or more of the following: (i) an electrophilic carbon atom that binds to the side chain oxygen atom (OG) of Ser 195 such that the electrophilic carbon atom is 1.4-4.0 A (e.g., 1.8 to 3.5 A or 2 to 3 A) from the oxygen atom; (ii) at least one hydrogen bond donor group to interact with Asp 189 such that the non-hydrogen atoms of the hydrogen bond donor groups are within 2.0 to 4.2 A (e.g., 2.5 to 3.5 A or 2.8 to 3.2 A ) away from the side chain oxygens of Aspl89; (iii) a hydrophobic group that makes van der Waals contacts with residues of the active site and is less than 5.0 A (e.g., less than 4.5, 4, 3, 2, or 1 A) away from the CB atom of Ala97; (iv) a hydrogen bond donor group which makes a hydrogen bond with the main chain carbonyl of Ser214 such that the distance between the hydrogen-bonded atoms is less than 4.0 A (e.g., less than 3.5, 3, 2, or 1 A); (v) a hydrogen bond donor group that makes a hydrogen bond with the main chain carbonyl of Gly216 such that the distance between the hydrogen-bonded atoms is less than 4.0 A (e.g., less than 3.5, 3, 2, or 1 A); (vi) a hydrogen bond acceptor group that makes a hydrogen bond with the main chain nitrogen of Gly216 such that the distance between the hydrogen-bonded atoms is less than 4.0 A (e.g., less than 3.5, 3, 2, or 1 A); (vii) a hydrogen bond acceptor group that makes a hydrogen bond with a side chain nitrogen of His57 such that the distance between the hydrogen-bonded atoms is less than 4.0 A (e.g., less than 3.5, 3, 2, or 1 A); (viii) a group that makes van der Waals contacts with the side chains of residues of Lys 192 and/or Leul46; (ix) a group that makes van der Waals contacts with the side chains of residues of Cysl91 and/or Cys219; and (x) a group or groups that make one or more polar interactions with the side chains of residues of Hisl74, Glu98 and/or Glu217. The computer also has a processor that is in electrical communication with the memory and that has a program for generating the three-dimensional model.
In another aspect, the invention features a computer readable memory having stored therein data indicative of a pharmacophore model of the invention or atomic coordintes of the invention.
Methods for Identifying, Designing, Manufacturing, or Evaluating Factor Xla Ligands
Information from the three-dimensional structure of FXIcat can be used in a variety of methods for identifying, manufacturing, or evaluating Factor Xla ligands. In one such aspect, the invention includes a method of selecting or designing a candidate ligand for Factor Xla. This method involves generating in a computer a three-dimensional structure of FXIcat based on the atomic coordinates in one or more of the files listed in Figure 16, or surrogates thereof, of the non-hydrogen atoms of residues serine 195, histidine 57, and aspartic acid 102 or atomic coordinates that have a root mean square deviation from the backbone atoms of the residues of less than 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 A. A candidate ligand that has sufficient surface complementary to the structure to bind Factor Xla (e.g., binding in an aqueous solution such as an aqueous solution at pH 7.0) is designed, selected or identified. Surface complementarities can be assessed with a number of commercially available software packages, including, but not limited to, Insight II and Quanta (Accelrys Inc: P.O. Box 5350, Princeton, NJ 08543-5350, USA), DOCK (Gschwend, D.A. and LD. Kuntz, J. Comput-Aided Mol. Design, 1996) and
Sybyl (Tripos, Inc.: 1699 South Hanley Rd., St. Louis, MO, 63144-2913, USA; Han et al., Bioorg. Med. Chem. Lett, 10:39-43, 2000). Designed ligands are then manufactured. If desired, the ability of any candidate ligand designed by the methods described herein to bind Factor Xla in vitro or in vivo can be optionally measured to confirm this binding and/or to compare the affinity of the ligand to other Factor Xla ligands. Compounds so prepared can also be used to determine their ability to inhibit Factor Xla, using inhibitition assays as described herein.
In another aspect, the invention provides a method for manufacturing a Factor Xla ligand. This method includes crystallizing FXIcat or a mutant of FXIcat with a ligand and obtaining the atomic coordinates, or surrogates thereof, of at least a portion of FXIcat with at least a portion of the ligand. One or more moieties in the ligand are selected for modification such that the modified ligand maintains the ability to bind Factor Xla. The ligand is modified based on this determination. Such modifications include moieties known by those skilled in the art to improve aqueous solubility, improve pharmacodynamic properties, such as increased circulation half-life or cell membrane permeability. Modifications can also be made to increase ligant affinity to FXIcat, as described in more detail elsewhere herein.
In a related aspect, the invention provides another method for manufacturing a Factor Xla ligand. This method involves manufacturing a ligand that binds FXIcat and that is designed or selected based on information obtained using a model of the atomic coordinates, or surrogates thereof, of at least a portion of the three-dimensional structure of FXIcat. In some embodiments, the ligand is designed or selected using a computer system of the invention. In desirable embodiments, the method involves (i) using information from a model generated from the atomic coordinates, or surrogates thereof, of at least a portion of the three-dimensional structure of FXIcat and (ii) designing, selecting, and/or manufacturing a ligand for Factor Xla based on the information.
In yet another aspect, the invention features a method of evaluating the ability of a candidate ligand to bind Factor Xla. This method involves (a) generating in a computer a three-dimensional structure of FXIcat based on the atomic coordinates in any one of the files listed in Figure 16, or surrogates thereof, of the non-hydrogen atoms of residues serine 195, histidine 57, and aspartic acid 102 or atomic coordinates that have a root mean square deviation from the backbone atoms of the residues of less than 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 A, and (b) employing a computational means to measure the interaction between the candidate ligand and FXIcat.
In another aspect, the invention provides a method of identifying a candidate ligand for Factor Xla. This method includes generating a three- dimensional model of a pharmacophore model of a FXIcat ligand using a computer system of the invention, and selecting or designing a candidate ligand satisfying the criteria of the pharmacophore model.
The invention also provides methods for excluding a candidate compound from further drug development. For example, candidate compounds for indications other than excess or undesired thrombosis can be evaluated for their ability to inhibit Factor Xla. Candidate compounds that inhibit Factor Xla and that are being developed for other indications are desirably modified to reduce their affinity for Factor Xla or eliminated from further drug development. In one such aspect, the invention features a method of evaluating the ability of a candidate compound to bind Factor Xla. This method involves (a) generating in a computer a three-dimensional structure of FXIcat based on the atomic coordinates in one or more of the files listed in Figure 16, or surrogates thereof, of the non-hydrogen atoms of residues serine 195, histidine 57, and aspartic acid 102 or atomic coordinates that have a root mean square deviation from the backbone atoms of the residues of less than 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 A, and (b) employing a computational means to measure the interaction between the candidate compound and FXIcat or to measure the surface complementarity between the candidate compound and FXIcat. In some embodiments, the candidate compound inhibits the activity of another protease (e.g., tryptase and elastase) and is either subsequently modified to reduce its affinity for Factor Xla or eliminated from further drug development. Methods for Determining the Structure of FXIcat
The three-dimensional structural information of the present invention can be used to model the three-dimensional structure of other proteins, such as FXIcat from other mammals of laboratory, domestic or veterinary interest (e.g., rat, rabbit, mouse, cat, dog, cow, goat, pig, or sheep). Additionally, the structure of FXIcat can be used to model the structure of related proteins, such as other proteases with at least 30, 40, 50, 60, 70, 80, 90, 95, or 100% sequence identity to the catalytic domain of human Factor XI or to full-length human Factor XL Accordingly, in one such aspect, the invention features a method of generating a three-dimensional model of a FXIcat of interest. This method includes homology modeling using at least a portion of the atomic coordinates in any one of the files listed in Figure 16 (e.g., atomic coordinates of a structure of the invention), or surrogates thereof, and at least a portion of the amino acid sequence of the Factor XI of interest, thereby generating a model of the FXIcat of interest.
In a related aspect, the invention features a method for experimentally determining the three-dimensional structure of the FXIcat of interest. This method involves crystallizing a FXIcat of interest, generating an X-ray diffraction pattern from the crystallized FXIcat of interest, and (i) applying at least a portion of the atomic coordinates of the files listed in Figure 16 (e.g., atomic coordinates of a structure of the invention), or surrogates thereof, to the diffraction pattern to generate a three-dimensional electron density map of at least a portion of the FXIcat of interest, or (ii) utilizing de novo crystallographic methods such as isomoφhous replacement or MAD phasing to generate such an electron density map. Desirable Embodiments Using the FXIa Three-Dimensional Structure
In some embodiments of any of the aspects of the invention in which information from the three-dimensional structure of FXIcat is used for identifying, manufacturing, or evaluating a Factor Xla ligand, the three- dimensional structure also includes the hydrogen atoms of residues serine 195, histidine 57, and/or aspartic acid 102. In certain embodiments, the three- dimensional structure further includes the non-hydrogen atoms, or both the non-hydrogen and hydrogen atoms, of one or more of the following residues Tyr5901, Ala97, Glu98, Aspl02, Leul46, Hisl74, Aspl 89, Alal90, Cysl91, Lysl92, Glyl93, Aspl94, Ser214, Tφ215, Gly216, Glu217, and Gly218. In some embodiments, the three-dimensional structure further includes the non- hydrogen atoms, or both the non-hydrogen and hydrogen atoms, of one or more of the following residues: Arg3704, His38, Leu39, Cys40, Cys58, Tyr94, Gly 142, Tyr 143, Tyrl72, Cys219, Ala220, and Arg224. In some embodiments, the three-dimensional structure further includes the non- hydrogen atoms, or both the non-hydrogen and hydrogen atoms, of one or more of the following residues: Phe59, Ilel 5 l, Alal 83, Gly 196, Gly 197, Thr213, Pro225, Gly226, Val227, and Tyr228. In various embodiments, the structure further includes coordinates for the non-hydrogen or both the non-hydrogen and hydrogen atoms of one or more of the following residues: Arg3704, His38, Leu39, Cys40, His57, Cys58, Phe59, Tyr5901, Tyr94, Ala97, Glu98, Aspl02, Glyl42, Tyrl43, Leul46, Uel51, Tyrl72, Hisl 74, Alal83, Aspl 89, Alal90, Cysl91, Lysl92, Glyl93, Aspl94, Serl95, Glyl96, Glyl97, Thr213, Ser214, Tφ215, Gly216, Glu217, Gly218, Cys219, Ala220, Arg224, Pro225, Gly226, Val227, and Tyr228, In desirable embodiments, the structure includes the coordinates for all of the residues in the FXIcat.
Desirably, the atomic coordinates have a root mean square deviation from the atomic coordinates of the backbone atoms of the corresponding residues in any one of the files listed in Figure 16 of less than 1.2, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, or 0.3 A. In various embodiments, the resolution of the structure is at least 5, 4, 3, 2.5, 2.0, 1.8, or 1.5 A (in order of increasing resolution and increasing preference). Exemplary surrogates for atomic coordinates of the invention include any modification (e.g., mathematical modification, addition of a constant or scaling) of the coordinates that preserves the relative relationships among the coordinates. Desirable portions of atomic coordinates of Factor Xlcat include at least 50, 60, 70, 80, 90, 95, or 100% of the atomic coordinates for Factor Xlcat. In some embodiments, the portion of the atomic coordinates of Factor Xlcat includes at least 50, 60, 70, 80, 90, 95, or 100% of the atomic coordinates for the non-hydrogen atoms of Factor Xlcat or for all the atoms of Factor Xlcat.
In some embodiments, the compound possesses at least one hydrogen bond donor group that interacts (e.g., forms a hydrogen bond) with Aspl 89 of FXIcat. In some embodiments, the compound possesses .an electrophilic carbon and at least one hydrogen bond donor group that interacts with Aspl 89 of FXIcat. The electrophilic carbon of the compound is desirably separated by approximately 5.9 A (e.g., 5.5 to 6.3 A) and 8.3 A (e.g., 8.0 to 8.6 A) from the hydrogen bond donor group of the compound and Aspl 89, respectively. The electrophilic carbon atom is desirably 1.4 to 4.0 A (e.g., 2 to 3.5 A or 2.5 to 3 A) from the side chain oxygen atom (OG) of Ser 195. The non-hydrogen atoms of the hydrogen bond donor groups are desirably within 2.0 to 4.2 A away from the side chain oxygens of Aspl 89. These groups can either interact directly with Aspl 89 or interact indirectly through water molecules.
In various embodiments, the compound has one or more of the following:
(i) an electrophilic carbon atom that binds to the side chain oxygen atom (OG) of Ser 195 such that the electrophilic carbon atom is 1.4-4.0 A from the oxygen atom; (ii) at least one hydrogen bond donor group to interact with Aspl 89 such that the non-hydrogen atoms of the hydrogen bond donor groups are within 2.0 to 4.2 A away from the side chain oxygens of Aspl 89; (iii) a hydrophobic group that makes van der Waals contacts with residues of the active site and is less than 5.0 A (e.g., less than 4.5, 4, 3, 2, or 1 A) of the CB atom of Ala97; (iv) a hydrogen bond donor group which makes a hydrogen bond with the main chain carbonyl of Ser214 such that the distance between the hydrogen-bonded atoms is less than 4.0 A (e.g., less than 3.5, 3, 2, or 1 A); (v) a hydrogen bond donor group that makes a hydrogen bond with the main chain carbonyl of Gly216 such that the distance between the hydrogen-bonded atoms is less than 4.0 A (e.g., less than 3.5, 3, 2, or 1 A); (vi) a hydrogen bond acceptor group that makes a hydrogen bond with the main chain nitrogen of Gly216 such that the distance between the hydrogen-bonded atoms is less than 4.0 A (e.g., less than 3.5, 3, 2, or 1 A); (vii) a hydrogen bond acceptor group that makes a hydrogen bond with a side chain nitrogen of His57 such that the distance between the hydrogen-bonded atoms is less than 4.0 A (e.g., less than 3.5, 3, 2, or 1 A); (viii) a group that makes van der Waals contacts with the side chains of residues of Lysl92 and/or Leul46; (ix) a group that makes van der Waals contacts with the side chains of residues of Cysl91 and/or Cys219; and (x) a group or groups that make one or more polar interactions with the side chains of residues of Hisl74, Glu98 and/or Glu217. Desirably, the inhibitors of Factor Xla used in the present invention comprise 3, 4, 5, or 6 or more of the above.
In other embodiments, the compound has at least one hydrogen bond donor group that interacts with Aspl 89. In this compound, the non-hydrogen atoms of the hydrogen bond donor groups are desirably within 2.0 to 4.2 A away from the side chain oxygens of Aspl 89.
In a desirable embodiment of any of the aspects of the invention in which information from the three-dimensional structure of FXIcat is used for identifying, manufacturing, or evaluating a Factor Xla ligand, the method also includes determining the ability of the candidate ligand to bind Factor Xla in vitro or in vivo. In some embodiments, the method further includes determining the ability of the candidate ligand to inhibit the enzymatic activity of Factor Xla in vitro or in vivo. Desirably, the affinity of the candidate ligand for Factor Xla is at least 2, 5, 10, 20, or 30-fold higher than its affinity for another protease such as thrombin, trypsin, Factor IXa, Factor Vila and/or Factor Xa. Affinity can be reported as, e.g., Km, K<j, Kj or IC50 values in which lower values indicate higher affinity. Desirably, the affinity of the candidate ligand for Factor Xla is at least 10 μM, lμM, 100 nM, 10 nM, or 1 nM. In various embodiments, the amino acid sequence of FXIcat present in the structure is at least 70, 80, 92, 94, 96, 98, or 100% identical to the amino acid sequence of the corresponding region of human Factor XI (SEQ ID NO: 2).
FXIcat Crystals
The invention also features crystals of FXIcat that are useful for determining the three-dimensional structure of FXIcat or a protein with substantial sequence identity (e.g., at least 30, 40, 50, 60, 70, 80, 90, 95, or 100% sequence identity) to FXIcat. In one such aspect, the invention features a crystal (e.g., a cubic crystal) of FXIcat that is at least 10, 12, 15, 18, 20, or 25 μ in the shortest dimension and diffracts to better than 3.5, 3.2, 3.0, 2.8, 2.5, or 2.0 A resolution, or a crystal (e.g, a needle-like crystal) that has a size of at least 10, 12, 15, 18, 20, or 25 μ in the smallest dimension and diffracts to 3.5, 3.2, 3.0, 2.8, 2.5, or 2.0 A resolution or better.
The invention also features crystals of a quadruple mutant FXIcat protein in which each of residues Ser434, Thr475 and Lys437 has been mutated to an alanine, and Cys482 has been mutated to serine (FXIcat-
S434A,T475A,K437A,C482S) that are useful for determining the three- dimensional structure. In one such aspect, the invention features a crystal (e.g., a cubic crystal) that is at least 10, 12, 15, 18, 20, or 25 μ in the shortest dimension and diffracts to better than 3.5, 3.2, 3.0, 2.8, 2.5, or 2.0 A resolution, or a crystal (e.g.. a needle-like crystal) that is at least 10, 12, 15, 18, 20, or 25 μ in the smallest dimension and diffracts to 3.5, 3.2, 3.0, 2.8, 2.5, or 2.0 A resolution or better. The invention further features crystals of a mutant FXIcat protein in which one or more of the following residues Ser434, Thr475, Lys422, Lys437, Lys486, Lys505, Lys509, Arg479 and/or Asp476 are mutated (e.g., mutated to alanine), and/or Cys482 has been mutated (e.g., mutated to serine) and that are useful for determining the three-dimensional structure. In one such aspect, the invention features a crystal (e.g., a cubic crystal) of FXIcat-
S434A,T475A,C482S,K505A that is at least 10, 12, 15, 18, 20, or 25 μ in the shortest dimension and diffracts to better than 3.5, 3.2, 3.0, 2.8, 2.5, or 2.0 A resolution, or a crystal (e.g., a plate-like crystal) of FXIcat- S434A,T475A,C482S,D476A that is at least 10, 12, 15, 18, 20, or 25 μ in the smallest dimension and diffracts to 3.5, 3.2, 3.0, 2.8, 2.5, or 2.0 A resolution or better.
Factor Xla Proteins and Nucleic acids
In one aspect, the invention provides a purified, less than full-length protein fragment of Factor XI, such as a protein consisting essentially of the catalytic domain of Factor XI. The catalytic domain is identified as the residues carboxy] to the activating protease cleavage site designated as KPR JVG (where the arrow denotes the proteolytic cleavage). The residues that make up this domain are 370-607 for the human, 372-606 for the mouse and 369-606 for the rabbit. The sequence for entire Factor XI rat gene is not yet known, but the catalytic domain comprises residues 1-234. In some embodiments, the human protein fragment consists of residues 370-607 of SEQ ID NO: 1 (GenBank Accession No. P03951). For the rabbit, the protein fragment consists of residues 369-606 of SEQ ID NO: 2 (GenBank Accession No. AF395821). For the mouse, the protein fragment consists of residues 372- 606 of SEQ ID NO: 3 (GenBank Accession No. AF356627). For the rat, the protein fragment consists of residues 1-234 of SEQ ID NO: 4. In another aspect, the invention features a purified Factor XI protein or fragment thereof including (a) a mutation of a residue that is otherwise post- translationally modified either naturally or in an organism used for recombinant expression, (b) a mutation that alters the charge of Factor XI, (c) a mutation that eliminates a free, reactive sulfhydryl group of a cysteine, (d) a combination of mutations that together alter the distribution of charge density Without altering the overall charge of Factor XI, (e) a mutation of the NH2- or COOH- terminal residue of Factor XI, and/or (f) a mutation that alters the folding and/or crystal packing of Factor XL In certain embodiments, the protein is enzymatically de-glycosylated. In other embodiments, one or more residues that would otherwise lead to glycosylation are mutated to another residue that results in no glycosylation. In some embodiments, the protein has at least 2, 3, 4, 5, 6, 7, 8, or 9 residues that are mutated compared to a naturally occurring Factor XI protein or the catalytic domain of a naturally-occurring Factor XI protein. In desirable embodiments, the mutation(s) allow crystallization or increase the resolution of the corresponding three-dimensional structure of the mutant protein compared to the structure of Factor XI without the mutation(s). For example, the mutations may increase the size or order of the resulting crystals, resulting in a higher resolution structure. Desirable mutant Factor XI proteins or fragments are at least 80, 85, 90, 92, 94, 96, 98, or 99% identical to any one of SEQ ID NOs: 1 -4.
In another aspect, the invention provides a purified protein including a region that is at least 92, 94, 96, 98, or 100% identical to the amino acid sequence of SEQ ID NO: 1 (human Factor XI) or SEQ ID NO: 3 (rat Factor XI). In some embodiments, the protein has an amino acid sequence at least 92, 94, 96, 98, or 100% identical to the amino acid sequence of SEQ ID NO: 1, over the entire length of SEQ ID NO: 1 or 3.
In another aspect, the invention features a purified nucleic acid encoding one or more of the proteins or protein fragments of the invention. Desirably, the nucleic acid is contained within a vector and operably linked to a promoter that regulates transcription of the nucleic acid.
Definitions
As used throughout this specification and the appended claims, the following terms have the meanings specified:
The terms "acyl" or "alkanoyl," as used interchangeably herein, represent an alkyl group, as defined herein, or hydrogen attached to the parent molecular group through a carbonyl group, as defined herein, and is exemplified by formyl, acetyl, propionyl, butanoyl and the like. Exemplary unsubstituted acyl groups are of from 2 to 7 carbons.
The term "acylamino," as used herein, represents an acyl group attached to the parent molecular group through a nitrogen atom. Exemplary unsubstituted acylamino groups are of from 2 to 7 carbons.
The term "acyloxy," as used herein, represents an acyl group attached to the parent molecular group through an oxygen atom. Exemplary unsubstituted acyloxy groups are of from 2 to 7 carbons.
The term "alkenyl," as used herein, represents monovalent straight or branched chain groups of, unless otherwise specified, from 2 to 6 carbons containing one or more carbon-carbon double bonds and is exemplified by ethenyl, 1-propenyl, 2-propenyl, 2-methyl- 1-propenyl, 1-butenyl, 2-butenyl and the like and may be optionally substituted with one, two, three or four substituents independently selected from the group consisting of: (1) alkoxy of one to six carbon atoms; (2) alkylsulfinyl of one to six carbon atoms; (3) alkylsulfonyl of one to six carbon atoms; (4) amino; (5) aryl; (6) arylalkoxy, wherein the alkylene group is of one to six carbon atoms; (7) aryloyl; (8) azido; (9) carboxaldehyde; (10) cycloalkyl of three to eight carbon atoms; (11) halo; (12) heterocycle; (13) (heterocycle)oxy; (14) (heterocycle)oyl; (15) hydroxy; (16) N-protected amino; (17) nitro; (18) oxo; (19) spiroalkyl of three to eight carbon atoms; (20) thioalkoxy of one to six carbon atoms; (21) thiol; (22) - CO2RA, wherein RA is selected from the group consisting of (a) alkyl, (b) aryl and (c) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (23) -C(O)NRBRc, wherein RB and Rc are independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (24) -SO RD, wherein RD is selected from the group consisting of (a) alkyl, (b) aryl and (c) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (25) -SO NRERF, wherein RE and RF are independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, wherein the alkylene group is of one to six carbon atoms; and (26) -NRGRH, wherein RG and RH are independently selected from the group consisting of (a) hydrogen; (b) an N- protecting group; (c) alkyl of one to six carbon atoms; (d) alkenyl of two to six carbon atoms; (e) alkynyl of two to six carbon atoms; (f) aryl; (g) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (h) cycloalkyl of three to eight carbon atoms and (i) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms, and the alkylene group is of one to ten carbon atoms, with the proviso that no two groups are bound to the nitrogen atom through a carbonyl group or a sulfonyl group.
The terms "alkoxy" or "alkyloxy," as used interchangeably herein, represent an alkyl group attached to the parent molecular group through an oxygen atom. Exemplary unsubstituted alkoxy groups are of from 1 to 6 carbons. The terms "alkoxyalkyl" or "alkyloxyalkyl," as used interchangeably herein, represent an alkyl group to which is attached an alkoxy group. Exemplary unsubstituted alkoxyalkyl groups are of from 2 to 12 carbons.
The terms "alkoxycarbonyl" or "alkyloxycarbonyl," as used interchangeably herein, represent an ester group; i.e. an alkoxy group, attached to the parent molecular group through a carbonyl group and is exemplified by methoxycarbonyl, ethoxycarbonyl and the like. Exemplary unsubstituted alkoxycarbonyl groups are of from 2 to 7 carbons.
The term "alkyl," as used herein, represents a monovalent group derived from a straight or branched chain saturated hydrocarbon of, unless otherwise specified, from 1 to 6 carbons and is exemplified by methyl, ethyl, n- and isopropyl, n-, sec-, iso- and tert-butyl, neopentyl and the like and may be optionally substituted with one, two, three or, in the case of alkyl groups of two carbons or more, four substituents independently selected from the group consisting of: (1) alkoxy of one to six carbon atoms; (2) alkylsulfinyl of one to six carbon atoms; (3) alkylsulfonyl of one to six carbon atoms; (4) amino; (5) aryl; (6) arylalkoxy; (7) aryloyl; (8) azido; (9) carboxaldehyde; (10) cycloalkyl of three to eight carbon atoms; (1 1) halo; (12) heterocyclyl; (13) (heterocycle)oxy; (14) (heterocycle)oyl; (15) hydroxyl; (16) N-protected amino; (17) nitro; (18) oxo; (19) spiroalkyl of three to eight carbon atoms; (20) thioalkoxy of one to six carbon atoms; (21) thiol; (22) -CO2RA, wherein RA is selected from the group consisting of (a) alkyl, (b) aryl and (c) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (23) -C(O)NRBRc, wherein R and R are independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (24) -SO2RD, wherein RD is selected from the group consisting of (a) alkyl, (b) aryl and (c) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (25) -SO2NRERF, wherein RE and RF are independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, wherein the alkylene group is of one to six carbon atoms; and (26) -NRGRH, wherein RG and RH are independently selected from the group consisting of (a) hydrogen; (b) an N-protecting group; (c) alkyl of one to six carbon atoms; (d) alkenyl of two to six carbon atoms; (e) alkynyl of two to six carbon atoms; (f) aryl; (g) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (h) cycloalkyl of three to eight carbon atoms and (i) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms, and the alkylene group is of one to ten carbon atoms, with the proviso that no two groups are bound to the nitrogen atom through a carbonyl group or a sulfonyl group.
The term "alkylamino," as used herein, represents an alkyl group attached to the parent molecular group through a nitrogen atom. Exemplary unsubstituted alkylamino groups are of from 1 to 6 carbons.
The term "alkylaminocarbonyl," as used herein, represents an alkylamino group attached to the parent molecular group through a carbonyl group. Exemplary unsubstituted alkylaminocarbonyl groups are of from 2 to 7 carbons.
The term "alkylaminosulfonyl," as used herein, represents an alkylamino group attached to the parent molecular group through an -SO2- group. Exemplary unsubstituted alkylaminosulfonyl groups are of from 1 to 6 carbons.
The term "alkylene," as used herein, represents a saturated divalent hydrocarbon group derived from a straight or branched chain saturated hydrocarbon by the removal of two hydrogen atoms, and is exemplified by methylene, ethylene, isopropylene and the like. The term "alkylsulfinyl," as used herein, represents an alkyl group attached to the parent molecular group through an -S(O)- group. Exemplary unsubstituted alkylsulfinyl groups are of from 1 to 6 carbons. The term "alkylsulfinylalkyl," as used herein, represents an alkyl group, as defined herein, substituted by an alkylsulfinyl group. Exemplary unsubstituted alkylsulfinylalkyl groups are of from 2 to 12 carbons.
The term "alkylsulfonyl," as used herein, represents an alkyl group attached to the parent molecular group through an -SO2- group. Exemplary unsubstituted alkylsulfonyl groups are of from 1 to 6 carbons.
The term "alkylsulfonylalkyl," as used herein, represents an alkyl group, as defined herein, substituted by a alkylsulfonyl group. Exemplary unsubstituted alkylsulfonylalkyl groups are of from 2 to 12 carbons. The term "alkylthio," as used herein, represents an alkyl group attached to the parent molecular group through a sulfur atom. Exemplary unsubstituted alkylthio groups are of from 1 to 6 carbons.
The term "alkynyl," as used herein, represents monovalent straight or branched chain groups of from two to six carbon atoms containing a carbon- carbon triple bond and is exemplified by ethynyl, 1-propynyl, and the like and may be optionally substituted with one, two, three or four substituents independently selected from the group consisting of: (1) alkoxy of one to six carbon atoms; (2) alkylsulfinyl of one to six carbon atoms; (3) alkylsulfonyl of one to six carbon atoms; (4) amino; (5) aryl; (6) arylalkoxy, wherein the alkylene group is of one to six carbon atoms; (7) aryloyl; (8) azido; (9) carboxaldehyde; (10) cycloalkyl of three to eight carbon atoms; (11) halo; (12) heterocycle; (13) (heterocycle)oxy; (14) (heterocycle)oyl; (15) hydroxy; (16) N-protected amino; (17) nitro; (18) oxo; (19) spiroalkyl of three to eight carbon atoms; (20) thioalkoxy of one to six carbon atoms; (21) thiol; (22) -CO2RA, wherein RA is selected from the group consisting of (a) alkyl, (b) aryl and (c) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (23) - C(O)NRBRc, wherein RB and Rc are independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (24) -SO2R , wherein R is selected from the group consisting of (a) alkyl, (b) aryl and (c) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (25) -SO2NR R , wherein RE and RF are independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, wherein the alkylene group is of one to six carbon atoms; and (26) -NRGRH, wherein RG and RH are independently selected from the group consisting of (a) hydrogen; (b) an N- protecting group; (c) alkyl of one to six carbon atoms; (d) alkenyl of two to six carbon atoms; (e) alkynyl of two to six carbon atoms; (f) aryl; (g) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (h) cycloalkyl of three to eight carbon atoms and (i) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms, and the alkylene group is of one to ten carbon atoms, with the proviso that no two groups are bound to the nitrogen atom through a carbonyl group or a sulfonyl group. The term "alpha-amino acid residue," as used herein, represents a -
N(RA)C(RB)(Rc)C(O)- linkage, wherein RA is selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, as defined
R C herein; and R and R are independently selected from the group consisting of: (a) hydrogen, (b) optionally substituted alkyl, (c) optionally substituted cycloalkyl, (d) optionally substituted aryl, (e) optionally substituted arylalkyl, (f) optionally substituted heterocyclyl, and (g) optionally substituted heterocyclylalkyl, each of which is as defined herein. For natural amino acids,
R C
R is H and R corresponds to those side chains of natural amino acids found in nature, or their antipodal configurations. Exemplary natural amino acids include alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, aspartamine, ornithine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine, each of which, except glycine, as their D- or L-form. As used herein, for the most part, the names of naturally-occuring amino acids and aminoacyl residues used herein follow the naming conventions suggested by the TUPAC Commission on the Nomenclature of Organic Chemistry and the TUP AC-TUB Commission on Biochemical Nomenclature as set out in Nomenclature of α- Amino Acids (Recommendations, 1974), Biochemistry 14 (2), 1975. The present invention also contemplates non-naturally occuring (i.e., unnatural) amino acid residues in their D- or L-form such as, for example, homophenylalanine, phenylglycine, cyclohexylglycine, cyclohexylalanine, cyclopentyl alanine, cyclobutylalanine, cyclopropylalanine, cyclohexylglycine, norvaline, norleucine, thiazoylalanine (2-, 4- and 5- substituted), pyridylalanine (2-, 3- and 4-isomers), naphthalalanine (1- and 2-isomers) and the like.
Stereochemistry is as designated by convention, where a bold bond indicates that the substituent is oriented toward the viewer (away from the page) and a dashed bond indicates that the substituent is oriented away from the viewer (into the page). If no stereochemical designation is made, it is to be assumed that the structure definition includes both stereochemical possibilities.
The term "amino," as used herein, represents an -NH2 group.
The term "aminoalkyl," as used herein, represents an alkyl group, as defined herein, substituted by an amino group.
The term "aryl," as used herein, represents a mono- or bicyclic carbocyclic ring system having one or two aromatic rings and is exemplified by
I phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl and the like and may be optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of: (1) alkanoyl of one to six carbon atoms; (2) alkyl of one to six carbon atoms; (3) alkoxy of one to six carbon atoms; (4) alkoxyalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (5) alkylsulfinyl of one to six carbon atoms; (6) alkylsulfinylalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (7) alkylsulfonyl of one to six carbon atoms; (8) alkylsulfonylalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (9) aryl; (10) arylalkyl, wherein the alkyl group is of one to six carbon atoms; (11) amino; (12) aminoalkyl of one to six carbon atoms; (13) aryl; (14) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (15) aryloyl; (16) azido; (17) azidoalkyl of one to six carbon atoms; (18) carboxaldehyde; (19) (carboxaldehyde)alkyl, wherein the alkylene group is of one to six carbon atoms; (20) cycloalkyl of three to eight carbon atoms; (21) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms and the alkylene group is of one to ten carbon atoms; (22) halo; (23) haloalkyl of one to six carbon atoms; (24) heterocyclyl; (25) (heterocyclyl)oxy; (26) "(heterocyclyl)oyl; (27) hydroxy; (28) hydroxyalkyl of one to six carbon atoms; (29) nitro; (30) nitroalkyl of one to six carbon atoms; (31) N-protected amino; (32) N-protected aminoalkyl, wherein the alkylene group is of one to six carbon atoms; (33) oxo; (34) thioalkoxy of one to six carbon atoms; (35) thioalkoxyalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (36) - (CH2)qCO2RA, wherein q is zero to four and RA is selected from the group consisting of (a) alkyl, (b) aryl and (c) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (37) -(CH2)qCONRBRc, wherein RB and Rc are independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (38) -(CH2)qSO2RD, wherein RD is selected from the group consisting of (a) alkyl, (b) aryl and (c) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (39) -(CH2)qSO2NRERF, , wherein RE and RF are independently selected from the group consisting of (a) hydrogen, (b) alkyl, -(c) aryl and (d) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (40) - (CH2)qNRGRH, wherein RG and RH are independently selected from the group consisting of (a) hydrogen; (b) an N-protecting group; (c) alkyl of one to six carbon atoms; (d) alkenyl of two to six carbon atoms; (e) alkynyl of two to six carbon atoms; (f) aryl; (g) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (h) cycloalkyl of three to eight carbon atoms and (i) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms, and the alkylene group is of one to ten carbon atoms, with the proviso that no two groups are bound to the nitrogen atom through a carbonyl group or a sulfonyl group; (41) oxo; (42) thiol; (43) perfluoroalkyl; (44) perfluoroalkoxy; (45) aryloxy; (46) cycloalkoxy; (47) cycloalkylalkoxy; and (48) arylalkoxy.
The terms "arylalkenyl" or "aralkenyl," as used interchangeably herein, represent an aryl group attached to the parent molecular group through an alkenyl group. Exemplary unsubstituted arylalkenyl groups are of from 8 to 16 carbons.
The terms "arylalkoxy" or "aralkoxy," as used interchangeably herein, represent an arylalkyl group attached to the parent molecular group through an oxygen atom. Exemplary unsubstituted arylalkoxy groups are of from 7 to 16 carbons. The terms "arylalkoxycarbonyl" or "aralkoxycarbonyl," as used interchangeably herein, represent an arylalkoxy group attached to the parent molecular group through a carbonyl group. Exemplary unsubstituted arylalkoxycarbonyl groups are of from 8 to 17 carbons.
The terms "arylalkyl" or "aralkyl," as used interchangeably herein, represent an aryl group attached to the parent molecular group through an alkyl group. Exemplary unsubstituted arylalkyl groups are of from 7 to 16 carbons.
The terms "arylalkylamino" or "aralkylamino," as used interchangeably herein, represent an arylalkyl group attached to the parent molecular group through a nitrogen atom. Exemplary unsubstituted arylalkylamino groups are of from 7 to 16 carbons.
The terms "arylalkylaminocarbonyl" or "aralkylaminocarbonyl," as used interchangeably herein, represents an arylalkylamino group attached to the parent molecular group through a carbonyl group. Exemplary unsubstituted arylalkylaminocarbonyl groups are of from 8 to 17 carbons.
The terms "aryl alkylsulfinyl" or "aralkylsulfmyl," as used interchangeably herein, represent an arylalkyl group attached to the parent molecular group through an -SO- group. Exemplary unsubstituted arylalkylsulfmyl groups are of from 7 to 16 carbons.
The terms "arylalkylsulfonyl" or "aralkylsulfonyl," as used interchangeably herein, represent an aralkyl group attached to the parent molecular group through an -SO2- group. Exemplary unsubstituted arylalkylsulfonyl groups are of from 7 to 16 carbons.
The term "arylalkylthio" or " aralkylthio," as used interchangeably herein, represents an arylalkyl group attached to the parent molecular group through a sulfur atom. Exemplary unsubstituted arylalkylthio groups are of from 7 to 16 carbons. The term "arylamino," as used herein, represents an aryl group which is attached to the parent molecular group through a nitrogen atom. Exemplary unsubstituted arylamino groups are of 6 or 10 carbons.
The term "arylaminocarbonyl," as used herein, represents an arylamino group attached to the parent molecular group through a carbonyl group. Exemplary unsubstituted arylaminocarbonyl groups are of from from 7 or 11 carbons.
The term "arylaminosulfonyl," as used herein, represents an arylamino group attached to the parent molecular group through an -SO2- group. Exemplary unsubstituted arylaminosulfonyl groups are of 6 or 10 carbons. The term "aryloxy," as used herein, represents an aryl group which is attached to the parent molecular group through an oxygen atom. Exemplary unsubstituted aryloxy groups are of 6 or 10 carbons. The term "aryloxycarbonyl," as used herein, represents an aryloxy group which is attached to the parent molecular group through a carbonyl group. Exemplary unsubstituted aryloxycarbonyl groups are of 7 or 11 carbons.
The terms "aryloyl" or "aroyl," as used interchangeably herein, represent an aryl group which is attached to the parent molecular group through a carbonyl group. Exemplary unsubstituted aryloxycarbonyl groups are of 7 or 11 carbons.
The terms "aryloylamino" or "aroylamino," as used interchangeably herein, represent an aroyl group which is attached to the parent molecular group through a nitrogen atom. Exemplary unsubstituted aryloylamino groups are of 7 or 11 carbons.
The term "arylsulfinyl," as used herein, represens an aryl group attached to the parent molecular group through an -SO- group. Exemplary unsubstituted arylsulfinyl groups are of 6 or 10 carbons. The term "arylsulfonyl," as used herein, represens an aryl group attached to the parent molecular group through an -SO2- group. Exemplary unsubstituted arylsulfonyl groups are of 6 or 10 carbons.
The term "arylthio," as used herein, represents an aryl group which is attached to the parent molecular group through a sulfur atom. Exemplary unsubstituted arylthio groups are of 6 or 10 carbons.
The term "azido," as used herein, represents an -N3 group.
The term "azidoalkyl," as used herein, represents an alkyl group, as defined herein, substituted by an azido group.
By "blood component" is meant a biological entity normally ound in blood, such as, for example cells, such as erythrocytes, leukocytes, and platelets, or proteins such as immunoglobulins, serum albumin, ferritin, steroid binding proteins, such as corticosteroid-binding globulin and sex hormone- binding globulin, transferrin, thyroxin binding protein, and alpha-2- macroglobulin. Blood components also include glycans, including glycosylamino glycans. Preferred blood components are those that have reactive organic functionality, such as thiols or amines. The terms "carbamate" or "carbamyl," as used interchangeably herein, represent a RAOC(O)NRB- group, or a -OC(O)NRB- linkage, depending on the chemical context in which this term is used, wherein R is selected from the group consisting of (a) alkyl, (b) aryl, and (c) arylalkyl; and RB is selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, as defined herein.
The term "carbonate," as used herein represents a -R OC(O)O- group, or a - OC(O)O- linkage, depending on the chemical context in which this term is used, wherein RA is selected from the group consisting of (a) alkyl, (b) aryl, and (c) arylalkyl, as defined herein. The term "carbonyl," as used herein, represents a C=O group.
The term "carboxaldehyde," as used herein, represents a -CHO group.
The term "(carboxaldehyde)alkyl," as used herein, represents an alkyl group, as defined herein, substituted by a carboxaldehyde group.
The term "carboxy," as used herein, represents a -CO2H group. The term "carboxyalkyl," as used herein, represents an alkyl group, as defined herein, substituted by a carboxy group.
The term "cycloalkenyl," as used herein represents a monovalent cyclic hydrocarbon of from three to eight carbons, unless otherwise specified, having at least one carbon-carbon double bond. The cycloalkenyl groups of this invention can be optionally substituted with (1) alkanoyl of one to six carbon atoms; (2) alkyl of one to six carbon atoms; (3) alkoxy of one to six carbon atoms; (4) alkoxyalkyl, wherein die alkyl and alkylene groups are independently of one to six carbon atoms; (5) alkylsulfinyl of one to six carbon atoms; (6) alkylsulfinylalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (7) alkylsulfonyl of one to six carbon atoms; (8) alkylsulfonylalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (9) aryl; (10) arylalkyl, wherein the alkyl group is of one to six carbon atoms; (11) amino; (12) aminoalkyl of one to six carbon atoms; (13) aryl; (14) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (15) aryloyl; (16) azido; (17) azidoalkyl of one to six carbon atoms; (18) carboxaldehyde; (19) (carboxaldehyde)alkyl, wherein the alkylene group is of one to six carbon atoms; (20) cycloalkyl of three to eight carbon atoms; (21) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms and the alkylene group is of one to ten carbon atoms; (22) halo; (23) haloalkyl of one to six carbon atoms; (24) heterocyclyl; (25) (heterocyclyl)oxy; (26) (heterocyclyl)oyl; (27) hydroxy; (28) hydroxyalkyl of one to six carbon atoms; (29) nitro; (30) nitroalkyl of one to six carbon atoms; (31) N-protected amino; (32) N-protected aminoalkyl, wherein the alkylene group is of one to six carbon atoms; (33) oxo; (34) thioalkoxy of one to six carbon atoms; (35) thioalkoxyalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (36) -(CH2)qCO2RA, wherein q is zero to four and RA is selected from the group consisting of (a) alkyl, (b) aryl and (c) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (37) -(CH2)qCONRBRc, wherein RB and Rc are independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (38) -(CH2)qSO2RD, wherein RD is selected from the group consisting of (a) alkyl, (b) aryl and (c) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (39) - (CH2)qSO2NRERF, , wherein RE and RF are independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (40) -(CH2)qNRGRH, wherein RG and RH are independently selected from the group consisting of (a) hydrogen; (b) an N-protecting group; (c) alkyl of one to six carbon atoms; (d) alkenyl of two to six carbon atoms; (e) alkynyl of two to six carbon atoms; (f) aryl; (g) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (h) cycloalkyl of three to eight carbon atoms and (i) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms, and the alkylene group is of one to ten carbon atoms, with the proviso that no two groups are bound to the nitrogen atom through a carbonyl group or a sulfonyl group; (41) oxo; (42) thiol; (43) perfluoroalkyl; (44) perfluoroalkoxy; {45) aryloxy; (46) cycloalkoxy; (47) cycloalkylalkoxy; and (48) arylalkoxy.
The term "cycloalkyl," as used herein represents a monovalent saturated or unsaturated non-aromatic cyclic hydrocarbon group of from three to eight carbons, unless otherwise specified, and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1.]heptyl and the like. The cycloalkyl groups of this invention can be optionally substituted with (1) alkanoyl of one to six carbon atoms; (2) alkyl of one to six carbon atoms; (3) alkoxy of one to six carbon atoms; (4) alkoxyalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (5) alkylsulfinyl of one to six carbon atoms; (6) alkylsulfinylalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (7) alkylsulfonyl of one to six carbon atoms; (8) alkylsulfonylalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (9) aryl; (10) arylalkyl, wherein the alkyl group is of one to six carbon atoms; (11) amino; (12) aminoalkyl of one to six carbon atoms; (13) aryl; (14) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (15) aryloyl; (16) azido; (17) azidoalkyl of one to six carbon atoms; (18) carboxaldehyde; (19) (carboxaldehyde)alkyl, wherein the alkylene group is of one to six carbon atoms; (20) cycloalkyl of three to eight carbon atoms; (21) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms and the alkylene group is of one to ten carbon atoms; (22) halo; (23) haloalkyl of one to six carbon atoms; (24) heterocyclyl; (25) (heterocyclyl)oxy; (26) (heterocyclyl)oyl; (27) hydroxy; (28) hydroxyalkyl of one to six carbon atoms; (29) nitro; (30) nitroalkyl of one to six carbon atoms; (31) N-protected amino; (32) N-protected aminoalkyl, wherein the alkylene group is of one to six carbon atoms; (33) oxo; (34) thioalkoxy of one to six carbon atoms; (35) thioalkoxyalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (36) - (CH )qCO2RA, wherein q is zero to four and RA is selected from the group consisting of (a) alkyl, (b) aryl and (c) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (37) -(CH2)qCONRBRc, wherein RB and Rc are independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (38) -(CH2)qSO2RD, wherein RD is selected from the group consisting of (a) alkyl, (b) aryl and (c) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (39) -(CH2)qSO2NRERF, , wherein RE and RF are independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (40) - (CH2)qNRGRH, wherein RG and RH are independently selected from the group consisting of (a) hydrogen; (b) an N-protecting group; (c) alkyl of one to six carbon atoms; (d) alkenyl of two to six carbon atoms; (e) alkynyl of two to six carbon atoms; (f) aryl; (g) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (h) cycloalkyl of three to eight carbon atoms and (i) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms, and the alkylene group is of one to ten carbon atoms, with the proviso that no two groups are bound to the nitrogen atom through a carbonyl group or a sulfonyl group; (41) oxo; (42) thiol; (43) perfluoroalkyl; (44) perfluoroalkoxy; (45) aryloxy; (46) cycloalkoxy; (47) cycloalkylalkoxy; and (48) arylalkoxy. The term "cycloalkylamino," as used herein, represents a cycloalkyl group attached to the parent molecular group through a nitrogen atom. Exemplary unsubstituted cycloalkylamino groups are of from 3 to 8 carbons.
The term "cycloalkylaminocarbonyl," as used herein, represents a cycloalkylamino group attached to the parent molecular group through a carbonyl group. Exemplary unsubstituted cycloalkylaminocarbonyl groups are of from 4 to 9 carbons.
The terms "cycloalkyloxy" or "cycloalkoxy," as used interchangeably herein, represent a cycloalkyl group, as defined herein, attached to the parent molecular group through an oxygen atom. Exemplary unsubstituted cycloalkyloxy groups are of from 3 to 8 carbons.
The terms "cycloalkyloxycarbonyl" or "cycloalkoxycarbonyl," as used interchangeably herein, represent a cycloalkyloxy group, as defined herein, attached to the parent molecular group through a carbonyl group. Exemplary unsubstituted cycloalkyloxycarbonyl groups are of from 4 to 9 carbons.
The term "cycloalkylalkoxy," as used herein, represents an alkoxy group, as defined herein, to which is attached a cycloalkyl group. Exemplary unsubstituted cycloalkylalkoxy groups are of from 4 to 14 carbons.
The term "cycloalkylalkyl," as used herein, represents a cycloalkyl group, as defined herein, attached to the parent molecular group through an alkyl group. Exemplary unsubstituted cycloalkylalkyl groups are of from 4 to 14 carbons.
The term "cycloalkylsulfinyl," as used herein, represents a cycloalkyl group attached to the parent molecular group through an -SO- group. Exemplary unsubstituted cycloalkylsulfinyl groups are of from 3 to 8 carbons.
The term "cycloalkylsulfonyl," as used herein, represents a cycloalkyl group attached to the parent molecular group through an -SO2- group.
Exemplary unsubstituted cycloalkylsulfonyl groups are of from 3 to 8 carbons. The term "dialkylamino," as used herein, represents an N,N- dialkylsubstituted amine attached to the parent molecular group through the nitrogen atom. The two alkyl substituents of a dialkylamino group can be the same or different, or can be joined together to form a ring. Exemplary dialkylamino groups are of from 2 to 12 carbons and include dimethylamino, diethylamino, pyrrolidino, and piperidino.
The term "haloalkyl," as used herein, represents an alkyl group, as defined herein, substituted by one, two, or three halogen atoms and is exemplified by chloromethyl, bromoethyl, trifluoromethyl and the like. The term "halogen," as used herein, represents F, CI, Br and I.
The term "heteroaryl," as used herein, represents that subset of heterocycles, as defined herein, which are aromatic: i.e., they contain 4n+2 pi electrons within the mono- or multicyclic ring system. Exemplary unsubstituted heteroaryl groups are of from 1 to 9 carbons. The terms "heteroarylalkenyl" or "heteroaralkenyl," or as used interchangeably herein, represent a heteroaryl group, as defined herein, attached to the parent molecular group through an alkenyl group. Exemplary unsubstituted heteroarylalkenyl groups are of from 3 to 15 carbons.
The terms "heteroarylalkyl" or "heteroaralkyl," as used interchangeably herein, represent a heteroaryl group, as defined herein, attached to the parent molecular group through an alkyl group. Exemplary unsubstituted heteroarylalkyl groups are of from 2 to 15 carbons.
The terms "heteroarylalkylamino" or "heteroaralkylamino," as used interchangeably herein, represent a heteroarylalkyl group, as defined herein, attached to the parent molecular group through a nitrogen atom. Exemplary unsubstituted heteroarylalkylamino groups are of from 2 to 15 carbons.
The terms "heteroarylalkylaminocarbonyl" or
"heteroaralkylaminocarbonyl," or as used interchangeably herein, represent a heteroarylalkylamino group, as defined herein, attached to the parent molecular group through a carbonyl group. Exemplary unsubstituted heteroarylalkylaminocarbonyl groups are of from 3 to 16 carbons.
The terms "heteroaryloyl" or "heteroaroyl," or as used interchangeably herein, represent a heteroaryl group, as defined herein, attached to the parent molecular group through a carbonyl group. Exemplary unsubstituted heteroaryloyl groups are of from 2 to 10 carbons.
The terms "heteroarylalkyloxy" or "heteroaralkoxy," or as used interchangeably herein, represent a heteroarylalkyl group, as defined herein, attached to the parent molecular group through an oxygen atom. Exemplary unsubstituted heteroarylalkyloxy groups are of from 2 to 15 carbons.
The terms "heteroarylalkyloxycarbonyl" or "heteroaralkoxycarbonyl," as used interchangeably herein, represent a heteroaralkoxy group, as defined herein, attached to the parent molecular group through a carbonyl group. Exemplary unsubstituted heteroarylalkyloxycarbonyl groups are of from 3 to 16 carbons.
The terms "heteroarylalkylsulfonyl" or "heteroaralkylsulfonyl," as used interchangeably herein, represent a heteroarylalkyl group attached to the parent molecular group through an -SO2- group. Exemplary unsubstituted heteroarylalkylsulfonyl groups are of from 2 to 15 carbons.
The term "heteroarylamino," as used herein, represents a heteroaryl group attached to the parent molecular group through a nitrogen atom. Exemplary unsubstituted heteroarylamino groups are of from 1 to 9 carbons.
The term "heteroarylaminocarbonyl," as used herein, represents a heteroarylamino group attached to the parent molecular group through a carbonyl group. Exemplary unsubstituted heteroarylaminocarbonyl groups are of from 2 to 10 carbons. The term "heteroarylaminosulfonyl," as used herein, represents a heteroarylamino group attached to the parent molecular group through an -SO2- group. Exemplary unsubstituted heteroarylaminosulfonyl groups are of from 1 to 9 carbons. The term "heteroaryloxy," as used herein, represents a heteroaryl group attached to the parent molecular group through an oxygen atom. Exemplary unsubstituted heteroaryloxy groups are of from 1 to 9 carbons.
The term "heteroaryloxycarbonyl," as used herein, represents a heteroaryloxy group attached to the parent molecular group through a carbonyl group. Exemplary unsubstituted heteroaryloxycarbonyl groups are of from 1 to 9 carbons.
The term "heteroarylsulfonyl," as used herein, represents a heteroaryl group attached to the parent molecular group through an -SO2- group. Exemplary unsubstituted heteroarylsulfonyl groups are of from 1 _to 9 carbons. The term "heteroarylthio," as used herein, represents a heteroaryl group attached to the parent molecular group through a sulfiir atom. Exemplary unsubstituted heteroaryloxy groups are of from 1 to 9 carbons.
The terms "heterocycle" or "heterocyclyl," as used interchangeably herein represent a 5-, 6- or 7-membered ring, unless otherwise specified, containing one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur. The 5-membered ring has zero to two double bonds and the 6- and 7-membered rings have zero to three double bonds. The term "heterocycle" also includes bicyclic, tricyclic and tetracyclic groups in which any of the above heterocyclic rings is fused to one or two rings independently selected from the group consisting of an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring and another monocyclic heterocyclic ring such as indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl, benzothienyl and the like. Heterocyclics include pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, i idazolidinyl, pyridyl, piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidiniyl, moφholinyl, thiomoφholinyl, thiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl, isoindazoyl, triazolyl, tetrazolyl, oxadiazolyl, uricyl, thiadiazolyl, pyrimidyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroinidolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl, benzothienyl and the like. Heterocyclic groups also include compounds of the formula
Figure imgf000064_0001
F is selected from the group consisting of -CH2-, -CH2O- and -O-, and G is selected from the group consisting of -C(O)- and -(C(R')(R"))V-, wherein R' and R" are independently selected from the group consisting of hydrogen or alkyl of one to four carbon atoms, and v is one to three and includes groups such as 1,3-benzodioxolyl, 1,4-benzodioxanyl and the like. Any of the heterocycle groups mentioned herein may be optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of: (1) alkanoyl of one to six carbon atoms; (2) alkyl of one to six carbon atoms; (3) alkoxy of one to six carbon atoms; (4) alkoxyalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (5) alkylsulfinyl of one to six carbon atoms; (6) alkylsulfinylalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (7) alkylsulfonyl of one to six carbon atoms; (8) alkylsulfonylalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (9) aryl; (10) arylalkyl, wherein the alkyl group is of one to six carbon atoms; (11) amino; (12) aminoalkyl of one to six carbon atoms; (13) aryl; (14) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (15) aryloyl; (16) azido; (17) azidoalkyl of one to six carbon atoms; (18) carboxaldehyde; (19) (carboxaldehyde)alkyl, wherein the alkylene group is of one to six carbon atoms; (20) cycloalkyl of three to eight carbon atoms; (21) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms and the alkylene group is of one to ten carbon atoms; (22) halo; (23) haloalkyl of one to six carbon atoms; (24) heterocycle; (25) (heterocycle)oxy; (26) (heterocycle)oyl; (27) hydroxy; (28) hydroxyalkyl of one to six carbon atoms; (29) nitro; (30) nitroalkyl of one to six carbon atoms; (31) N-protected amino; (32) N-protected aminoalkyl, wherein the alkylene group is of one to six carbon atoms; (33) oxo; (34) thioalkoxy of one to six carbon atoms; (35) thioalkoxyalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (36) - (CH2)qCO2RA, wherein q is zero to four and RA is selected from the group consisting of (a) alkyl, (b) aryl and (c) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (37) -(CH2)qCONHBRc, wherein RB and Rc are independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (38) -(CH2)qSO2RD, wherein RD is selected from the group consisting of (a) alkyl, (b) aryl and (c) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (39) -(CH2)qSO2NRERF, , wherein RE and RF are independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (40) - (CH2)qNRGRH, wherein RG and RH are independently selected from the group consisting of (a) hydrogen; (b) an N-protecting group; (c) alkyl of one to six carbon atoms; (d) alkenyl of two to six carbon atoms; (e) alkynyl of two to six carbon atoms; (f) aryl; (g) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (h) cycloalkyl of three to eight carbon atoms and (i) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms, and the alkylene group is of one to ten carbon atoms, with the proviso that no two groups are bound to the nitrogen atom through a carbonyl group or a sulfonyl group; (41) oxo; (42) thiol; (43) perfluoroalkyl; (44) perfluoroalkoxy; (45) aryloxy; (46) cycloalkoxy; (47) cycloalkylalkoxy; and (48) arylalkoxy.
The term "heterocyclylalkyl" represents a heterocyclyl group attached to the parent molecular group through an alkyl group. Exemplary unsubstituted heterocyclylalkyl groups are of from 2 to 15 carbons.
The terms "heterocyclylamino" or "(heterocycle)amino," as used interchangeably herein, represents a heterocycle group, as defined herein, attached to the parent molecular group through nitrogen. Exemplary unsubstituted heterocyclylamino groups are of from 1 to 9 carbons.
The terms "heterocyclyloxy" or "(heterocycle)oxy," as used interchangeably herein, represents a heterocycle group, as defined herein, attached to the parent molecular group through an oxygen atom. Exemplary unsubstituted heterocyclyloxy groups are of from 1 to 9 carbons. The terms "heterocyclyloxycarbonyl" or "(heterocycle)oxycarbonyl," as used interchangeably herein, represents a heterocycloxy group, as defined herein, attached to the parent molecular group through a carbonyl group. Exemplary unsubstituted heterocyclyloxycarbonyl groups are of from 2 to 10 carbons. The term "heterocyclyloyl" or "(heterocycle)oyl," as used interchangeably herein, represents a heterocycle group, as defined herein, attached to the parent molecular group through a carbonyl group. Exemplary unsubstituted heterocyclyloyl groups are of from 2 to 10 carbons.
The term "heterocyclylsulfonyl," as used herein, represents a heterocyclyl group attached to the parent molecular group through an -SO2- group. Exemplary unsubstituted heterocyclylsulfonyl groups are of from 1 to 9 carbons. The term "heterocyclylthio," as used herein, represents a heterocyclyl group attached to the parent molecular group through a sulfur atom. Exemplary unsubstituted heteroaryloxy groups are of from 1 to 9 carbons.
The term "hydroxy" as used herein, represents an -OH group. The term "hydroxyalkyl," as used herein, represents an alkyl group, as defined herein, substituted by one to three hydroxy groups, with the proviso that no more than one hydroxy group may be attached to a single carbon atom of the alkyl group and is exemplified by hydroxymethyl, dihydroxypropyl and the like. The term "methine" as used herein, represents a =C(H)- group.
The term "N-protected amino," as used herein, refers to an amino group, as defined herein, to which is attached an N-protecting or nitrogen-protecting group, as defined herein.
The term "N-protected aminoalkyl," as used herein, refers to an alkyl group, as defined herein, which is substituted by an N-protecting or nitrogen- protecting group, as defined herein.
The terms "N-protecting group" or "nitrogen protecting group" as used herein, represent those groups intended to protect an amino group against undersirable reactions during synthetic procedures. Commonly used N- protecting groups are disclosed in Greene, "Protective Groups In Organic Synthesis, 3rd Edition" (John Wiley & Sons, New York, 1999), which is incoφorated herein by reference. N-protecting groups comprise acyl, aroyl, or carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2- chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o- nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4- bromobenzoyl, 4-nitrobenzoyl and chiral auxiliaries such as protected or unprotected D, L or D, L-amino acids such as alanine, leucine, phenylalanine and the like; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl and the like; carbamate forming groups such as benzyloxycarbonyl, p- chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p- nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p- bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5- dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4- methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5- trimethoxybenzyloxycarbonyl, 1 -(p-biphenylyl)- 1 -methylethoxycarbonyl, α,α- dimethyl-3 ,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t- butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2,- trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxy carbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and the like, arylalkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl and the like and silyl groups such as trimethylsilyl and the like. Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t- butyloxycarbonyl (Boc) and benzyloxycarbonyl (Cbz).
The term "nitro," as used herein, represents an -NO2 group.
The term "nitroalkyl," as used herein, represents an alkyl group substituted by an -NO group.
The term "oxo," as used herein, represents =O.
The term "perfluoroalkyl," as used herein, represents an alkyl group, as defined herein, wherein each hydrogen radical bound to the alkyl group has been replaced by a fluoride radical. Perfluoroalkyl groups are exemplified by trifluoromethyl, pentafluoroethyl, and the like.
The term "perfluoroalkoxy," as used herein, refers to a perfluoroalkyl group, as defined herein, attached to the parent molecular group through an oxygen atom. The term "pharmaceutically acceptable salt," as use herein, represents those salts which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences 66:1-19, 1977. The salts can be prepared in situ during the final isolation and purification of the compounds of the invention or separately by reacting the free base group with a suitable organic acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphersulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine and the like. The term "pharmaceutically acceptable ester," as used herein, represents esters which hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof. Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl group preferably has not more than 6 carbon atoms. Examples of particular esters includes formates, acetates, propionates, butyates, acrylates and ethylsuccinates. The term "pharmaceutically acceptable prodrugs" as used herein, represents those prodrugs of the compounds of the present invention which are, within the scope of sound medical judgement, suitable for use in contact with with the tissues of humans and animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention.
The term "prodrug," as used herein, represents compounds which are rapidly transformed in vivo to the parent compound of the above formula, for example, by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, and Judkins et al, Synthetic Communications 26(23):4351-4367, 1996), each of which is incoφorated herein by reference. By "ring system substituent" is meant a substituent attached to an aromatic or non-aromatic ring system. When a ring system is saturated or partially saturated the "ring system substituent" further includes methylene (double bonded carbon), oxo (double bonded oxygen) or thioxo {double bonded sulfur). The term "spiroalkyl," as used herein, represents an alkylene diradical, both ends of which are bonded to the same carbon atom of the parent group to form a spirocyclic group.
The term "sulfonyl," as used herein, represents an -SO2- group. The term "thioalkoxy," as used herein, represents represents an alkyl group attached to the parent molecular group through a sulfur atom. Exemplary unsubstituted thioalkoxy groups are of from 1 to 6 carbons.
The term "thioalkoxyalkyl," as used herein, represents an alkyl group substituted by a thioalkoxy group. Exemplary unsubstituted thioalkoxyalkyl groups are of from 2 to 12 carbons.
By "thiocarbonyl" is meant a -C(S)- group.
By "thiol" is meant an -SH group.
Asymmetric or chiral centers may exist in the compounds of the present invention. The present invention contemplates the various stercoisomers and mixtures thereof. Individual stereoisomers of compounds or the present invention are prepared synthetically from commercially available starting materials which contain asymmetric or chiral centers or by preparation of mixtures of enantiometic compounds followed by resolution well-known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a racemic mixture of enantiomers, designated (+/-), to a chiral auxiliary, separation of the resulting diastereomers by recrystailization or chromatography and liberation of the optically pure product from the auxiliary or (2) direct separation of the mixture of optical enantiomers on chiral chromatographic columns. Enantiomers are designated herein by the symbols "R" or "S," depending on the configuration of substituents around the chiral carbon atom.
Geometric isomers may also exist in the compounds of the present invention. The present invention contemplates the various geometric isomers and mixtures thereof resulting from the arrangement of substituents around a carbon-carbon double bond and designates such isomers as of the Z or E configuration, wherein the term "Z" represents substituents on the same side of the carbon-carbon double bond and the term "E" represents substituents on opposite sides of the carbon-carbon double bond. It is also recognized that for structures in which tautomeric forms are possible, the description of one tautomeric form is equivalent to the description of both, unless otherwise specified. For example, amidine structures of the formula -C(=NRQ)NHRT and -C(NHRQ)=NRT, where Rτ and RQ are different, are equivalent tautomeric structures and the description of one inherently includes the other.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a simplified diagrammatic representation of the coagulation cascade showing the role of Factor XI in this pathway. All three thick arrows represent the amplification phase. The initiation and propagation phases are also labled. Flla is also known as thrombin. The arrow from Flla to FXIa indicates that FXI is activated by Flla and FXIIa.
Figure 2 is a table that lists the conversion between the amino acid numbering of FXIcat in the native zymogen found in the bloodstream and trypsin. Residue numbers greater than 999 under "trypsin" denote amino acid insersions in FXIcat relative to trypsin, while missing numbers under "trypsin" denote amino acid deletions in FXIcat relative to trypsin.
Figure 3 is a table describing the Protein Data Bank (PDB) format used to list the atomic coordinates of the various structures of this invention shown in the files listed in Figure 16).
Figure 4A is a table that lists the residues of human Factor Xla that are altered to other amino acids or removed by mutagenesis/engineering strategies.
Relative enzymatic activity and crystallization results for the different recombinant FXIcat proteins are listed. The numbering system is that of Factor
XI native zymogen found in the bloodstream. The AVC-terminal truncation mutation is the removal of the C-terminal Ala606 and Val607 residues, such that the last residue of the mutant is Gln605. By "N.D." is meant that the enzymatic activity of the mutant has not been determined or that crystalization of the mutant has not been undertaken or has not produced crystals of sufficient quality to determine the three-dimensional structure.
Figure 4B is a table that lists relevant crystallographic data derived from crystals of the invention. Crystallization conditions are also listed. Figure 5 A is a ribbon diagram of human FXIcat, as derived from its crystal structure with ecotin. The amino- and carboxy-termini are indicated by N and C. The drawing was produced using the program Insight II.
Figure 5B is a ribbon diagram of human FXIcat, as derived from its crystal structure with benzamidine. The amino- and carboxy-termini are indicated by N and C. The drawing was produced using the program Insight II.
Figure 5C is a stereoview of the active site of human FXIcat, as derived from its crystal structure with ecotin. For clarity, no hydrogen atoms or water molecules are shown.
Figure 5D is a stereoview of the active site of human FXIcat, as derived from its crystal structure with benzamidine. For clarity, no hydrogen atoms or water molecules are shown.
Figure 6 shows the position of the oligonucleotides chosen for the PCR approach to isolate rat FXIcat and the rat FXIcat nucleotide sequence that was derived. Figure 7 is an alignment of amino acid sequences of FXIcat proteins from human, mouse, rabbit and rat.
Figure 8 is a schematic diagram of an expression plasmid for FXIcat in Pichia pastoris . A fusion between the α-factor secretion signal sequence and FXIcat was constructed in order to create a fusion protein with an N-terminal α-factor, signal peptide, and FXIcat. This gene is expressed from the methanol-regulated AOX promoter.
Figure 9 lists the signal peptide cleavage site of pPICZα FXIcat. Figure 10 shows the effect of continuous intravenous administration of compound 22 in a rat model of venous thrombosis. In anesthetized male Sprague-Dawley rats venous thrombosis was induced by incoφorating stenosis of the inferior vena cava with rapid infusion of hypotonic saline followed by partial stenosis. Compound 22 was administered as a continuous intravenous infusion via the femoral vein commenced 15 minutes prior to the thrombotic challenge. Thrombus mass was assessed at 60 minutes following challenge. Results are expressed as the percentage of the vehicle mean.
Figure 11 is a schematic illustration of an exemplary computer. Figure 12 is a table containing data on the supeφosition of the three- dimensional structure of FXIcat complexed with compound 144 and that of complexes with other ligands. The first row, labeled "rms/loop," is the root mean squared (rms) deviation of just the 48 backbone atoms constituting the loop when backbone atoms (number of atoms are listed on second row) of the two structures are superimposed. The second row, labeled "rms/fit," is the rms deviation and number of backbone atoms used for supeφosition. The third row, labeled "rms/loop," is the rms deviation of just the 48 backbone atoms constituting the loop using a best fit of all backbone atoms that do not deviate by more than 3.8 A between the two structures. The fourth row, labeled "rms/fit," is the rms deviation of all backbone atom pairs that do not deviate by more than 3.8 A.
Figure 13 is a table of the rms deviation (values to the right of the diagonal) of the supeφosition of FXIcat structures solved in the presence of the indicated compounds. Values to the left are the number of backbone atom pairs used in the supeφosition. Rms values are calculated only for pairs of atoms that do not deviate from each others by more than 3.8 A.
Figure 14 is the same as Figure 13 except that all backbone atom pairs are used in the supeφosition. Figure 15 is a table of the rms deviation between Serl95, His 57, and Asp 102 in FXIcat structures solved in the presence of the indicated compounds using a best fit of backbone atoms (values to the right) and all backbone atoms (values to the left). Figure 16 is a list of an electronic file contained on 2 identical compact discs (Copy 1 and Copy 2) as "Table of Figure 16 listings", which is hereby incoφorated by reference. "Listing 4" contains the file atomic coordinates of the complex between two molecules of human FXIcat and two molecules of ecotin M84R. "Listing 5" contains the coordinates of the complex between two molecules of human FXIcat and two molecules of wild type ecotin.
"Listing 6" contains the coordinates of the complex between two molecules of human FXIcat and two molecules of ecotin IX-D. "Listing 7" contains the atomic coordinates of the complex between human FXIcat and benzamidine. "Listing 8" contains the atomic coordinates of the complex between human FXIcat and compound 77. "Listing 9" contains the atomic coordinates of the complex between human FXIcat and compound 85. "Listing 10" contains the atomic coordinates of the complex between human FXIcat and compound 25. "Listing 11" contains the atomic coordinates of the complex between human FXIcat and compound 79. "Listing 12" contains the atomic coordinates of the complex between human FXIcat and compound 86. "Listing 13" contains the atomic coordinates of the complex between human FXIcat and compound 144. "Listing 14" contains the atomic coordinates of the complex between human FXIcat and compound 141. "Listing 15" contains the atomic coordinates of the complex between human FXIcat and compound 145. The numbering system is based upon structural homology to trypsin (see Figure 2 for conversion between Factor XI numbering system and trypsin). The format is as described in Figure 3. DETAILED DESCRIPTION
Human coagulation Factor Xla is a serine protease that results from activation of Factor XI by thrombin, Factor Xlla, or autoactivation. Factor XI is an upstream serine protease in the amplification phase of the intrinsic anticoagulant cascade that circulates in blood plasma as a homo-dimeric zymogen (Figure 1). Although the protease domain exhibits homology with other coagulation serine proteases, the remaining domains are not shared. Factor XI is not gamma carboxylated (i.e., not vitamin K-dependent) and does not have EGF domains, but possesses an N-terminal series of 'apple' domains and a C-terminal catalytic domain.
Inhibiting enzymes of the amplification phase of the clotting cascade represents an attractive strategy for novel antithrombotic agents. On the one hand this approach may allow for sufficient anticoagulant activity to prevent unwanted thrombotic episodes, but also allow enough clotting to occur so as to prevent or minimize bleeding. Although complete deficiency of factors involved in the amplification phase of the coagulation cascade can result in bleeding (e.g., hemophilia A and B are caused by deficiencies in Factors VIII and IX, respectively), low levels of activity, generally in the range of 5% of normal, are sufficient to minimize any bleeding diathesis and confer a low probability of suffering acute thrombotic disorders such as AMI or stroke. Furthermore, Factor XI deficiency results in a mild-to-moderate bleeding disorder, especially in tissues with high levels of local fibrinolytic activity, such as the urinary tract, nose, oral cavity, and tonsils. The important role of Factor XI in coagulation is also indicated by the recent observation that high levels of Factor XI are associated with increased venous thrombosis (Meijers et al, N. Engl J. Med. 342:696, 2000).
To facilitate the identification and/or design of high affinity inhibitors for Factor Xla, several three-dimensional structures of the human Factor XI catalytic domain (FXIcat) bound to a ligand have been determined {Figures 3, 4A, 4B, 5 A, 5B, 5C, 5D, and 12-16). These structures can be used to homology model the structure of other candidate inhibitors with FXIcat. In addition, the methods described herein for the crystallization and structural determination of complexes of FXIcat with a ligand can be used to experimentally determine the structure of other ligands bound to FXIcat. This structural information can be used to identify functional groups within a ligand that can be modified to increase the affinity and selectivity of the ligand for Factor Xla or to identify functional groups within the ligand that can be modified to increase the bioavailability of the ligand without adversely affecting its affinity for Factor Xla.
Compounds that may be tested for their ability to decrease Factor XΪa activity include, but are not limited to, synthetic organic molecules, naturally occurring organic molecules and their semi-synthetic derivatives, nucleic acid molecules, biosynthetic proteins or peptides, naturally occurring peptides or proteins, or antibodies. In various embodiments of any of the aspects of the invention, the organic compound has a molecular weight contained in one of the following ranges: 100-3,000 daltons; 100-2,000 daltons; 100-1,000 daltons; 100-750 daltons; 250-3,000 daltons; 250-2,000 daltons; 250-1 ,000 daltons; 250-750 daltons; 400-3,000 daltons; 400-2,000 daltons; 400-1 ,000 daltons; or 400-750 daltons, inclusive.
Accordingly, in addition to providing compounds designed based on the structure of FXIcat, the present invention includes a class of peptidomimetics and non-peptides that inhibit the activity of Factor Xla (e.g., the compounds of formula I or formula II, as described herein). Any of the compounds of the invention are useful for treating or preventing diseases for which inhibition of Factor Xla is desirable, such as, but not limited to, arterial (e.g., unstable angina, acute myocardial infarction, percutaneous coronary interventions, or peripheral arterial occlusions), venous (e.g., deep venous thrombosis) and cardiac (e.g., atrial fibrillation) conditions and resulting ischemic conditions (e.g., acute myocardial infarction, pulmonary embolism, or stroke), and heparin induced complications (e.g., heparin induced thromobocytopenia).
The following non-limiting examples are provided to further describe various aspects and embodiments of the present invention.
Factor Xla Proteins and Nucleic acids
In one aspect, the invention provides a purified, less than full-length protein fragment of Factor XI, such as a protein consisting essentially of the catalytic domain of Factor XI. The catalytic domain is identified as the residues carboxyl to the activating protease cleavage site designated as
KPR TVG (where the arrow denotes the proteolytic cleavage). The residues that make up this domain are 370-607 for the human, 372-606 for the mouse and 369-606 for the rabbit. The sequence for entire Factor XI rat gene is not yet known, but the catalytic domain comprises residues 1-234. In some embodiments, the human protein fragment consists of residues 370-607 of SEQ ID NO: 1 (GenBank Accession No. P03951). For the rabbit, the protein fragment consists of residues 369-606 of SEQ ID NO: 2 (GenBank Accession No. AF395821). For the mouse, the protein fragment consists of residues 372- 606 of SEQ ID NO: 3 (GenBank Accession No. AF356627). For the rat, the protein fragment consists of residues 1-234 of SEQ ID NO: 4.
In another aspect, the invention features a purified Factor XI protein or fragment thereof including (a) a mutation of a residue that is otherwise post- translationally modified either naturally or in an organism used for recombinant expression, (b) a mutation that alters the charge of Factor XI, (c) a mutation that eliminates a free, reactive sulfhydryl group of a cysteine, (d) a combination of mutations that together alter the distribution of charge density without altering the overall charge of Factor XI, (e) a mutation of the NH2- or COOH- terminal residue of Factor XT, and/or (f) a mutation that alters the folding and/or crystal packing of Factor XL In certain embodiments, the protein is enzymatically de-glycosylated. In other embodiments, one or more residues that would otherwise lead to glycosylation are mutated to another residue that results in no glycosylation. In some embodiments, the protein has at least 2, 3, 4, 5, 6, 7, 8, or 9 residues that are mutated compared to a naturally occurring Factor XI protein or the catalytic domain of a naturally-occurring Factor XI protein. In desirable embodiments, the mutation(s) allow crystallization or increase the resolution of the corresponding three-dimensional structure of the mutant protein compared to the structure of Factor XI without the mutation(s). For example, the mutations may increase the size or order of the resulting crystals, resulting in a higher resolution structure. Desirable mutant Factor XI proteins or fragments are at least 80, 85, 90, 92, 94, 96, 98, or 99% identical to any one of SEQ ID NOs: 1-4.
In another aspect, the invention provides a purified protein including a region that is at least 92, 94, 96, 98, or 100% identical to the a nino acid sequence of SEQ ID NO: 1 (human Factor XI) or SEQ ID NO: 3 (rat Factor XI). In some embodiments, the protein has an amino acid sequence at least 92, 94, 96, 98, or 100% identical to the amino acid sequence of SEQ ID NO: 1, over the entire length of SEQ ID NO: 1 or 3. In another aspect, the invention features a purified nucleic acid encoding one or more of the proteins or protein fragments of the invention. Desirably, the nucleic acid is contained within a vector and operably linked to a promoter that regulates transcription of the nucleic acid. GTGTTCGGAGGA
Example 1. Expression of Human Coagulation FXIcat
FXIcat was expressed in a heterologous organism and purified for three- dimensional structural determination. The ability of individual residues or combinations of residues to influence the folding and packing of FXIcat into protein crystals was also evaluated.
The light chain segment of Factor Xla was expressed as a recombinant protein in the absence of Factor Xla heavy chain. A human FXI cDNA fragment encompassing amino acids 370-607 of SEQ ID NO. 1 was derived by PCR amplification from a cDNA encoding the full length protein, and subcloned into pPICZαA (Invitrogen, Cat. No. VI 95-20). The resulting plasmid was designated pPICZα FXIcat (Figure 8). During PCR cloning, the coding sequence of FXIcat was arranged so that a fusion protein with the S. cerevisiae α-factor was generated. After translation of this fusion protein, a single KEX-2 signal peptide cleavage site was present, allowing post- translational proteolytic cleavage at this site during the process of secretion. The cleavage site was placed so that cleavage generates the same light chain segment as is found for human Factor Xla protein. The pPICZα FXIcat plasmid was transformed into the methanolotrophic yeast Pichia pastoris Mut strain, X33 (Invitrogen, Cat. No. CI 80-00) by standard methods, and transformants selected on Zeocin-containing selection plates. Methanol induction of recombinant protein expression was screened by expanding 10-20 colonies each in 30 mL cultures of buffered methanol complex (BMMY) medium by standard growth conditions (Invitrogen). Methanol was added to 0.5% at 12 hour intervals for 72 hours.
Cell supernatants were analyzed for FXIcat expression by several methods. First, enzymatic activity was measured at defined culture intervals in a Factor Xla peptidolysis assay as described below. Second, Western blot analysis with anti-human Factor Xla-specific antisera identified transformants with the highest expression of FXIcat. N-terminal sequence analysis of the purified protein yielded the sequence TVGGTA(S)VxGExP, consistent with cleavage at the expected signal peptide processing site resulting in a protein with the authentic amino terminus of Factor Xla light chain. Large-scale preparation of FXIcat was conducted in a 10 L fermentor using 1% glycerol to achieve high cell density, followed by continuous infusion of methanol (0.5-0.7%) for 100 hours. Conditioned medium was collected at intervals throughout the methanol induction phase. Alternatively, cells harboring the FXIcat vector were grown in flasks in a shaking incubator, and FXIcat expression was induced by the addition of methanol to a final concentration of 0.5% every 12 hours. Conditioned medium was collected by centrifugation after 72 hours of induction.
Example 2. Expression and Purification of Ecotin
For expression and purification of ecotin for structural studies with FXIcat, the bacterial ecotin gene and mutants thereof were expressed as recombinant proteins in the plasmid, pTacTac (see Yang, et al, J. Mol. Biol. 279:945-957, 1998). Two liters of E. coli, transformed with the appropriate plasmid, were grown at 37°C to OD60o = 0.7 and then induced with 0.25 mM IPTG for 12 hours. Cells were harvested by centrifugation, and cell pellets were suspended in 100 mL of 25% sucrose, 10 mM Tris, pH 8, 6.8 mM EDTA, and 20 μg/mL lysozyme. The periplasmic fraction was isolated by centrifugation. Ecotin was purified by heating the lysate to 100°C for ten minutes and centrifuging to remove precipitated denatured protein. The supernatant was fractionated by reversed-phase HPLC. The protein peak corresponding to ecotin was analyzed by SDS-PAGE, and fractions containing pure ecotin were concentrated by centrifugal filtration with a Centriprep-10.
Ecotin was further purified using a POROS HQ column (Applied Biosystems) on a BioCAD 700E in buffer containing 5 mM MgCl2 in 10 mM Tris-HCl, pH 7.8. Ecotin did not bind to the matrix under these conditions, while impurities were retained on the column. Example 3. Purification of Human Coagulation FXIcat
Recombinant FXIcat for use in crystallography was purified from Pichia pastoris conditioned medium by one of two methods. In one method, the recombinant enzyme was collected from conditioned medium by ammonium sulfate precipitation. The medium was first brought to 50% of saturation by the addition of crystalline (NH4)2SO . The resulting slurry was mixed at room temperature for 30 minutes, and the precipitated proteins removed by centrifugation at 20,000 x g from 20 minutes. The supernatant solution was isolated and brought to 80% of saturation by the addition of (NH4)2SO4. The slurry was again mixed at room temperature for 30 minutes, and the precipitated protein was collected by centrifugation as described above. The supernatant solution was discarded, and the precipitate was redissolved in a minimal amount of 20 mM Tris-HCl, pH 7.4. The redissolved protein was dialyzed against two changes (at least 50 volumes each) of the same buffer at 4° C. The dialyzed protein was applied to a 2.6 x 8 cm Chelating Sepharose Fast Flow column (Amersham Bioscience, P/N 17-0575-01) charged with Zn+2. The column was run at room temperature at a flow rate of ~8 mL/minutes. Following loading, the column was washed with additional 20 mM Tris-HCl, pH 7.4 until the A280 of the effluent returned to baseline. The column was eluted with a 250 mL gradient from 0 to 50 mM imidazole in 20 mM Tris-HCl, pH 7.4. The pH of the fraction containing eluted, recombinant FXIcat was adjusted to pH~6 by the addition of 1/200 volume of 20% CH3COOH. The protein was then enzymatically deglycosylated by the addition of Endoglycosidase H (New England Biolabs, Cat. No. P0703S) to a final concentration of 500-1000 mU/mL. Digestion was carried out at room temperature for 1.5-2 hours. The pH of the solution was brought up to pH~7.4 by the addition of 1/200 volume of 2 M Tris, and the fraction was applied to a 1.6 x 8 cm SP-Sepharose Fast Flow column (Amersham Bioscience, P/N 17- 0729-01) equilibrated in 20 mM Tris-HCl, pH 7.4 at a flow rate of 8 mL/minutes. After the column was loaded, it was washed with additional 20 mM Tris-HCl, pH 7.4 buffer. The column was then washed with 0.1 M NaCl in the same buffer to remove minor contaminants. Recombinant FXIcat was eluted from the column with a step to 0.2 M NaCl in 20 mM Tris-HCl, pH 7.4. Alternatively, various mutant forms of FXIcat were purified from concentrated, diafiltered P. pastoris conditioned medium. Cells and cell debris were removed by centrifugation followed by filtration through 0.8 μm mixed ester cellulose nitrate filters. The filtered medium (7-15 L) was concentrated to ~1 L using a Centramate PE apparatus equipped with a 1 OK NMWCO membrane cassette (Pall-Gelman, P/N OS010C10). The concentrate was diluted with 4 L of 20 mM Tris-HCl, pH 7.4 and reconcentrated to ~1 L. The dilution and concentration were repeated once more to yield the final concentrated, diafiltered medium. Recombinant FXIcat was purified by a combination of cation exchange and heparin-affinity chromatography. The concentrated, diafiltered medium was applied directly to a 1.6 x 8 cm SP-
Sepharose Fast Flow column equilibrated in 20 mM Tris-HCl, pH 7.4 at a flow rate of 8-10 mL/minutes. After the column was loaded, it was washed with additional 20 mM Tris-HCl, pH 7.4 buffer. The column was then washed with 0.1 M NaCl in the same buffer to remove minor contaminants. Recombinant FXIcat was eluted from the column with a step to 0.2 M NaCl in 20 M Tris- HCl, pH 7.4. Recombinant FXIcat eluted from the SP-column was diluted 1 :1 with 20 mM Tris-HCl, pH 7.4 and applied to a 5 mL HiTrap™ Heparin column (Amersham Bioscience, P/N 17-0407-01) equilibrated in 0.1 M NaCl; 20 mM Tris-HCl, pH 7.4 at a flow rate of 3-4 mLl/minutes. The protein was eluted from this column with a 50 mL gradient from 0.1 to 1 M NaCl in the same buffer. The purity and concentration of the enzyme was determined by RP- HPLC and SDS-PAGE. Typically, the concentration was ~1 mg/mL, and the purity was at least 95%. In some instances, a third purifcation step was included. The heparin column eluate was applied to a 2.6 x 90 cm Superdex 75 column equilibrated in 20 mM Tris-HCl, pH 7.4; 0.2 M NaCl at a flow rate of 4-5 mL/minutes. The enzyme eluted between 70 and 90 minutes, consistent with an apparent molecular weight of 30,000.
Example 4. Expression of FXIcat of Other Mammalian Species in Non- glycosylated Forms
In another strategy for producing alternative forms of FXIcat monomers, a comparison was made between the Factor Xla light chain domains of other organisms, either from databases publicly available or by discovery. Utilizing the cDNA sequence of human Factor XI in the regions A, B, C, and D shown in Figure 7, oligonucleotides composing these sequences were prepared. Using standard PCR approaches, genes encoding the putative Factor XI catalytic domains from rat, mouse, and rabbit were derived from liver cDNA libraries, and subsequently cloned into the yeast Pichia pastoris expression vector, pPICZα, according to the procedure described above {Figure 9). The regions of homology corresponding to human FXIcat were cloned and identified (Figure 7). For example, for the Rat FXIcat, a 5' oligonucleotide consisting of the sequence GTGTTCGGAGGAGCTGCGTCTGTTCACGGCGAG (SEQ ID NO. 5) and a 3' oligonucleotide consisting of the sequence GTGGACTGGATTTTGGAGAAAACTCAGTCGGAATGA (SEQ ID NO. 6) were chosen (Figure 6). Alternatively, by choosing a position of the oligonucleotides that was spaced further towards the N-terminus of the gene for the 5' oligonucleotide ATGGATAATGTGTGCACAACTAAAATC (SEQ ID NO. 7), and spaced into the 3' LTR for the 3' oligonucleotide TCCAGGGCCACAAAGTGATACCAGTTGAAC (SEQ ID NO. 8), a larger portion of the rat Factor XI cDN A was derived. Using DNA sequencing approaches to determine the ends of this cloned DNA, the rat cDNA sequence was determined to be located in the vicinity of the residues corresponding to the boundaries of the light chain protein, conserved between human, rat, mouse, and rabbit proteins. Expression studies were continued as described above. DNA sequence analysis revealed that there was a significant percentage of identity between species for rat and human (79%), mouse and human (79%), and rabbit and human (91%) FXIcat. Importantly, this information also demonstrated variation between FXIcat structures that was concentrated to surface regions of the proteins, based on homology models. Therefore, examination of the crystallographic properties of these additional FXIcat proteins is a means to investigate further the role of surface variations in controlling crystal integrity. Mutational analysis of rat, mouse, and rabbit Factor XI was utilized to alter residues potentially governing N-linked glycosylation of these proteins when they are expressed in Pichia pastoris . In rat FXIcat, there are two potential N-linked glycosylation sites at residues Asn60 and AsnlOl. PCR based mutational substitution of Ala for residues in the glycosylation consensus sequence (i.e., Ser62 and Thr 103) in tandem created a non-glycosylated version of the rat FXIcat protein.
Example 5. Mutations to FXIcat to Improve Crystallization Quality
If desired, modification of the protein structure of human FXIcat can be achieved by mutational change of the DNA plasmid prior to the introduction into the organism for gene expression. In one such method, the pPICZα FXIcat plasmid was altered at a single codon using PCR-based mutagenesis with synthesized oligonucleotides bearing the changes. In another approach, one or more additional residues were altered by the same methodology in a sequential fashion. Following mutagenesis and DNA sequencing to determine the sequence integrity of the plasmid, pPICZα FXIcat mutant plasmids were then transformed into the methanolotrophic yeast Pichia pastoris Mut+ strain, X33 (Invitrogen) by standard methods. The mutant proteins were purified as described above and characterized in terms of their purity, activity, and crystallographic properties.
A series of amino acid substitution mutants that alter the ability of recombinant human FXIcat to be glycosylated in the host used for expression of the recombinant protein were generated. Expression of recombinant FXIcat in Pichia pastoris leads to production of a protein that is glycosylated by an N- linked glycosylation pathway. By altering residues in the consensus epitope for N-linked glycosylation (Asn-Xaa-(Ser/Thr) where Xaa is any amino acid except Pro), the protein would not be glycosylated. As expressed in Pichia pastoris, FXIcat has two potential N-linked glycosylation sites based on the N- X-S/T motif. In one example, the substitution of an Ala for Ser434 was accomplished by the above PCR methodologies. Following the transformation and expression of the S434A mutant protein in Pichia pastoris, the migration of the protein was analyzed using a standard gel assay. The mobility of the S434A protein differed from that of the fully glycosylated (wild-type, WT) protein and that of the protein resulting from endoglycosidase H treatment of the expressed recombinant protein.
Additionally, multiple mutations that more profoundly reduced the ability of FXIcat to be glycosylated were generated. In this method, S434A and T475A mutations were prepared by serial mutagenesis of the Pichia pastoris expression plasmid, pPICZα FXIcat. Individual mutagenesis of either S434A or T475A reduced glycosylation of the mutant protein as evidenced by a lower apparent molecular weight by SDS-PAGE analysis. The ability of the double mutations to dramatically reduce the extent of glycosylation was demonstrated by SDS-PAGE and Western blot analysis. The increased mobility of the protein in SDS-PAGE was indicative of a lack of glycosylation. The single mutants had mobility intermediate to wild-type and the double mutant which migrated with an apparent molecular weight slightly lower than that of the Endoglycosidase H treated, wild-type FXIcat. In addition, wild-type FXIcat yielded four distinct peaks on RP-HPLC analysis. These peaks collapsed into two major peaks following Endoglycosidase H treatment. These peaks had significantly longer retention times than the wild-type peaks. The double glycosylation mutant showed a single major peak with a longer retention time than any of the enzymatically deglycosylated forms. This increased hydrophobicity is consistent with the loss of surface-exposed, hydrophilic carbohydrate moieties. Taken together with the observed absence of carbohydrate moieties in the solved structures for FXIcat, these observation suggest strongly that the mutations resulted in the complete elimination of glycosylation on this form of the protein.
Additionally, one or more mutants were generated to eliminate free sulfhydryls within FXIcat. Unpaired cysteine residues may impact folding and crystal formation. Alternatively, by covalent association with reactive proteins or small molecules, the free sulfhydryl group may change the nature of the FXIcat. The presence of free sulfhydryl groups on FXIcat monomers may also influence solubility, aggregation, and crystallization properties. For example, FXIcat has a single cysteine residue (Cys482) that is not involved in a disulfide bond. Cys482 forms a disulfide bond with Cys362 in intact Factor XI and thus is unpaired in the FXIcat. Thus, FXIcat-C482S was generated to replace the free sulfhydryl group with an isosteric serine residue.
In an additional strategy to enhance crystallization, a series of mutants that alter individual charged residues on the surface of FXIcat were generated by alanine substitution mutagenesis. The methodologies are technically parallel to those described above. Surface charged residues were identified by the inspection of the FXIcat/ecotin structure. In one particular example, a
K437A substitution was generated in FXIcat. The neutral alanine substitution replaces the positively charged lysine residue leading to a net loss of charge in that location. Additional modifications leading to a change in charge on the surface of FXIcat can be used to further characterize the influence of Lys437 on the structure and packing of FXIcat. A D476A substitution was also created using the methodology described above. The alanine substitution replaces the negatively charged aspartic acid, leading to a local change of charge; however, the charge change is opposite to that of K437A. In another variation of this method, a series of substitution mutations that do not alter the net surface charge of the FXIcat monomer, but change the charge distribution were generated. If a localized region of FXIa monomers is important to the packing of crystals, changes in the charge distribution is expected to impact crystal integrity. For example, the double mutation N571D, E572Q may change the charge distribution in one local region of the protein monomer. This alteration of the charge distribution may impact packing of crystals.
A related strategy involved the generation of a series of substitution mutants that affect the packing of FXIcat monomers in crystals by influencing van der Waals interactions between hydrophobic residues and the protein backbone of another monomer in the crystal. For example, the residue Tyr416 is oriented such that the active site is at least partially occluded, and such that this residue interacts with the peptide backbone of another FXIcat monomer in the crystal packing. Since this position is a serine residue in the mouse and rat FXI proteins, substitution of Y416S was expected to retain the activity of the protein. The Y416S substitution disrupts the interaction of the tyrosine residue with the peptide backbone of another monomer.
In an additional strategy, a series of substitution mutations that combine the different features of the strategies described above are examined to yield a different outcome than any of the substitution mutations by itself. For example, mutations that alter the charge distribution of the protein surface can be combined with mutations that block glycosylation of the protein and the availability of free sulfhydryl groups on the surface. Therefore, the FXIcat-
S434A,T475A,C482S triple mutant was made. In another method, mutations with the same functional effects on protein folding and/or packing were examined in different combinations. For example, a serial substitution at K437A and K505A is a means to combine the effects of eliminating positively charged lysine residues at two different locations on the surface of FXIcat. The quadruple mutant S434A, T475A, K437A, C482S has proven quite robust for obtaining useful crystals of various inhibitors as exemplified in the examples that follow.
Example 6. Formation of FXIcat-ecotin Complexes FXIcat-ecotin complex was formed by incubating a stoichiometric quantity of each component at 4 °C overnight and subsequently isolated by chromatography on a Superose 12 column (Amersham Bioscience) in 20 mM Tris-HCl, pH7.8 and 0.1 M NaCl on BioCAD 700E.
Example 7. Crystallization of FXIcat-Ecotin M84R Complex
The FXIcat-ecotin M84R complex was crystallized at room temperature by the hanging drop vapor diffusion method (McPherson, Methods Biochem. Anal. 23:249, 1976) from 0.2 M NaCl, 20% (w/v) PEG-1000 and 0.1 M Na/K phosphate, pH 6.2. The crystals belong to the space group P21212] with unit cell parameters of a=44.6 A, b=92.7 A, and c=l 86.9 A. There are two FXIcat and two ecotin molecules forming a tetramer in the asymmetric unit, and the diffraction data extends to 2.2 A.
Example 8. Structure Determination, Model Building, and Refinement The structure of FXIcat-ecotin M84R was solved by molecular replacement using A ore. The search model was created from the coordinates of the tetrameric rat granzyme B [N66Q] - ecotin [81-84 ϊEPD] with PDB code 1FI8 (Waugh et al, Nat. Struct. Biol. 7:762, 2000). The conserved core structure of serine proteases was identified first by structure alignment of several serine proteases. All the side chain atoms beyond Cβ, all surface loops in granzyme B, and all water molecules were deleted from the model. A clear solution was obtained using data of 20-5 A with correlation coefficient of 52.4 and R factor of 48.9%. This solution also persisted in calculations performed using data in different resolution ranges. Structure refinement was carried out using CNX (Accelrys Inc., San Diego, California) and model building was performed in Quanta (Accelrys Inc., San Diego, California).
Example 9. Design of Factor Xla Inhibitors
The three-dimensional atomic structures reported herein can be readily used as a template for selecting potent inhibitors. Various computer programs and databases can be used for this puφose. Desirable inhibitors have excellent steric and electrostatic complementarity to Factor Xla, a fair amount of buried hydrophobic surface, and sufficient conformational rigidity to minimize entropy loss upon binding (see, for example, Babine and Bender, Chem. Rev., 97:1359-1472, 1997).
The approach usually comprises the following steps. First, a target site in FXIcat is defined. This region can be any place important or essential for the protease activity such as, but not limited to, the active site cavity of Factor Xla. Because the FXIcat crystal structure was first determined in the presence of ecotin, a protein inhibitor, the spatial and chemical properties of the active site cavity of Factor Xla were clearly delineated and aped. Alternatively, the target site can be a region other than the active site, such as a region that when bound by an inhibitor prevents a substrate from binding Factor Xla or prevents Factor Xla from adopting a three-dimensional structure that enables full catalytic activity. Second, a small molecule or other candidate molecule is docked onto the target site of Factor Xla. Many methods can be used for this puφose such as, but not limited to, fast shape matching (Dock [Kuntz et al, J. Mol. Biol, 161 :269-288, 1982]; Eudock [Perola et al, J. Med. Chem., 43:401- 408, 2000]); incremental construction (FlexX [Rarey et al, J. Mol. Biol, 261 :470-89, 1996]; Hammerhead [Welch et al, Chem. Biol, 3:449-462, 1996]), Tabu search (ProJLeads [Baxter et al, Proteins, 33:367-382, 1998]; SFDock [Hou et al, Protein Eng., 12:639-647, 1999]), genetic algorithms (GOLD [Gold et al, J. Mol. Biol, 267:727-748, 1997]; AutoDock 3.0 [Morris et al, J. Comput Chem., 19:1639-1662, 1998]; Gambler [Charifson et al, J. Med. Chem. , 42 :5100-5109, 1999]), evolutionary programming {Gehlhaar et al, Chem. Biol, 2:317-324, 1995], simulated annealing (AutoDock 2.4 [Goodsell et al, Proteins, 8:195-202, 1990]), Monte Carlo simulations (MCDock [Liu et al, J. Comput-Aided Mol. Des., 13:435-451, 1999]; QXP [McMartin et al, J. Comput.-Aided Mol. Des., 11:333-344, 1997]), and distance geometry (Dockit [Metaphorics LLC, Piemont, CA, USA]). The profiles of hydrogen bond donor-acceptor and lipophilic points of the candidate compounds can also be used to complement those of the target site in FXIcat. Those skilled in the art can readily identify many small molecules or fragments as hits. If desired, one can link the different functional groups or small molecules identified by the above procedure into a single, larger molecule. The resulting molecule is likely to be more potent and have higher specificity. The affinity and/or specificity of a hit can also be improved by adding more atoms or fragments that will interact with the target protein. The originally defined target site can be readily expanded to allow further necessary extension. The differences in three-dimensional structure between FXIcat and related proteins with known structure can be used to optimize selectivity of an inhibitor for Factor Xla. In addition to the structural differences described herein, other differences between FXIcat and other proteins can also be identified by a skilled artisan. Promising compounds can be selected through the process. They can then be synthesized and assayed for their inhibitory properties. The success rate can sometimes be as high as 20% and may be even higher due to the rapid progress in standard computing methods.
Example 10. Inhibition of Factor Xla
The peptidolytic activity of recombinant FXIcat was measured using the chromogenic substrate pyroGlu-Pro-Arg-p-nitroanaline (DiaPharma, Cat. No. S-2366). The substrate was diluted to 1 mM in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.02% Tween20 (assay buffer) and mixed with an equal volume of diluted enzyme in a microtiter plate (Wallac Isoplate, P/N 1450-514). The final volume in the wells was 100 ul. The plate was placed immediately into a ThermoMax plate reader (Molecular Devices), and the time course of hydrolysis was monitored for 10 minutes at 405 nm. The progress curves were visually inspected and the linear portion of the curves was used to determine the activity of the enzyme.
Potential inhibitors of Factor Xla were evaluated using a modification of the above assay. Briefly, the inhibitors were tested against native Factor Xla (Haematological Associates, P/N FXIA01) using a custom made analog of S- 2366. This substrate had the/?-nitroanaline group replaced with 7- methylaminocoumarin (AMC). The final concentration of the substrate in the assay was 100 μM, and the final concentration of the enzyme was 0.25 nM. Inhibitors were tested by serial dilution over an appropriate range to yield a dose response curve for determination of the inhibitors' IC50 values. The assay mixture was incubated for 30 minutes at 30° C, and the fluorescent yield was measured using a PE- Wallac Victor 2 Plate Reader. Dose response curves were fit to Equation 1 in which A is the maximum inhibition, B is the minimum inhibition, C is the IC50, and D is the Hill coefficient. [{A ~%+ (i D)] + B (E(3uation l)
Desirably, the compounds have an IC50 value better than 1.0 micromolar, 0.1 micromolar, 0.01 micromolar or 0.001 micromolar (in order of increasing preference).
Example 1 1. Crystallization of FXIcat- Wild Type Ecotin Complex
The FXIcat-wild type ecotin complex was crystallized at room temperature by the hanging drop vapor diffusion method (McPherson, Methods Biochem. Anal. 23:249-345, 1976) from 0.1 M (NH4)2SO4, 22% {w/v) PEGMME-2000 and 0.1 M Na cacodylate, pH 6.2. The crystals belong to the space group P212121 with unit cell parameters of a=44.3 A, b=91.9 A, and c=186.2 A. There are two FXIcat and two ecotin molecules forming a tetramer in the asymmetric unit, and the diffraction data extends to 2.6 A. The coordinates of FXIcat-ecotin M84R were used for the calculation of the difference electron density map by the program CNX (Accelrys Inc., San
Diego, California) for FXIcat-wild type ecotin. Subsequent model building was carried out in the program Quanta2000 (Accelrys Inc., San Diego, California) and refinement was done by CNX.
Example 12. Crystallization of FXIcat-Ecotin IX-D Complex
The FXIcat-ecotin IX-D complexe was crystallized at 10°C by the hanging drop vapor diffusion method (McPherson, Methods Biochem. Anal. 23:249-345, 1976) from 0.1 M (NH4)2SO4, 22% (w/v) PEGMME-2000 and 0.1 M Na cacodylate, pH 6.2. The crystals belong to the space group P2]2121 with unit cell parameters of a=45.4 A, b=91.4 A, and c=l 88.6 A. There are two FXIcat and two ecotin molecules forming a tetramer in the asymmetric unit, and the diffraction data extends to 3.0 A. The coordinates of FXIcat-ecotin M84R were used for the calculation of the difference electron density map by the program CNX (Accelrys Inc., San Diego, California) for FXIcat-ecotin IX- D. Subsequent model building was carried out in the program Quanta2000 (Accelrys Inc., San Diego, California) and refinement was done by CNX.
Example 13. Crystallization of FXIcat-S434A,T475A,K437A with Benzamidine
The FXIcat triple mutant S434A, T475A, K437A was cloned and purified as described above. Benzamidine was added to the protein to a concentration of 1 mM and the complex was concentrated to about 18-20 mg/mL. Cubic crystals were obtained in 2.0 M ammonium sulfate and 0.1 M Tris-HCl, pH 7.5-9 at 20 °C. The crystals belong to the cubic space group 123 with unit cell parameters of a=b=c= 120.1 A. There is one FXIcat molecule in the asymmetric unit, and the X-ray diffraction data extends to 1.96 A. The initial solution of this structure was found by molecular replacement using an automated package for molecular replacement (AmoRe,: Navaza J, Acta Cryst. A50: 157-163, 1994). The search model was one FXIcat molecule from the tetramer structure of FXIcat-ecotin M84R complex. To avoid model bias, all the surface loops, the side chain atoms beyond Cp, and water molecules were removed from the model. A clear solution was obtained using data of 20-5 A with correlation coefficient of 50.6 and R factor of 34.8%. Structure refinement was performed using CNX (Accelrys Inc., San Diego, California), and model building was performed using Quanta (Accelrys Inc., San Diego, California)). Example 14. Crystallization of FXIcat with SPRL-122599
A ten-fold molar excess of 10 mM SPRL-122599 in DMSO was added to the FXIcat quadruple mutant S434A, T475A, C482S, K437A, incubated at 4°C and then concentrated to 15 mg/mL. Cubic crystals were obtained using the hanging drop method (McPherson, Methods Biochem. Anal. 23:249-345, 1976) with 23% (w/v) PEG 4000, 0.2 M Li2SO4, 0.1 M Tris-HCl, pH 8.5 at 10 °C. The crystals belong to the space group 123 with unit cell parameters of a=b=c=121.5 A. Diffraction data were measured to 2.8 A resolution and processed by HKL2000. Phases were calculated using the structure described in Example 13 as the model, and CNX (Accelrys Inc., San Diego, California) was used for calculating the difference electron density map.
Example 15. Crystallization of FXIcat with SPRL-121995
A ten-fold molar excess of 10 mM SPRL-121995 in DMSO was added to diluted FXIcat-S434A,T475A,C482S,K437A, incubated at 4 °C, and then concentrated to 15 mg/mL. Cubic crystals were obtained using the hanging drop method (McPherson, Methods Biochem. Anal. 23:249-345, 1976) with 24% w/v polyethylene glycol 4000, 0.16 M lithium sulfate, and 0.08 M Tris- HCl, pH 8.5 at 10 °C. The crystals belong to space group 123 with unit cell parameters of a=b=c=121.7 A. Diffraction data were measured to 2.1 A resolution and processed by HKL2000. Phases were calculated using the structure described in Example 13 as the model, and CNX (Accelrys Inc., San Diego, California) was used for calculating the difference electron density map.
Example 16. Crystallization of FXIcat with SPRL-124336
A ten-fold molar excess of 10 mM SPRL-121995 in DMSO was added to diluted FXIcat-S434A,T475A,C482S,K437A, incubated at 4 °C, and then concentrated to 20 mg/mL. Cubic crystals were obtained using the hanging drop method (McPherson, Methods Biochem. Anal. 23:249-345, 1976) with 30% PEGMME 5000, 0.1 M Tris pH 7.9, 0.2 M (NH4)2SO4, pH 8.5 at 10 °C. The crystals belong to space group P32 with unit cell parameters of a=b=41.9, c=103.8 A. Diffraction data were measured to 3.0 A resolution and processed by HKL2000. Phases were calculated using the structure described in Example 13 as the model, and CNX (Accelrys Inc., San Diego, California) was used for calculating the difference electron density map.
Example 17. Crystallization of FXIcat with SPRL-123682
A ten-fold molar excess of 10 mM SPRL-123682 in DMSO was added to diluted FXIcat-S434A,T475A,C482S,K437A, incubated at 4 °C, and then concentrated to 15 mg/mL. Cubic crystals were obtained using the hanging drop method (McPherson, Methods Biochem. Anal. 23:249-345, 1976) with 18% (w/v) PEG 4000, 0.2 M Li2SO4, 0.1 M Tris-HCl, pH 8.5 at 10 °C. The crystals belong to space group 123 with unit cell parameters of a=b=c=121.3 A. The diffraction data were measured to 2.0 A resolution and processed by HKL2000. Phases were calculated using the structure described in Example 13 as the model, and CNX (Accelrys Inc., San Diego, California) was used for calculating the difference electron density map.
Example 18. Crystallization of FXIcat with SPRL-123672
A ten-fold molar excess of 10 mM SPRL-123672 in DMSO was added to diluted FXIcat-S434A,T475A,C482S,K437A, incubated at 4 °C, and then concentrated to 15 mg/mL. Cubic crystals were obtained using the hanging drop method (McPherson, Methods Biochem. Anal 23:249, 1976) with 2.0 M ammonium sulfate, 0.1 M Tris-HCl, pH 8.5 at 10 °C. The crystals belong to space group 123 with unit cell parameters of a=b=c=121.4 A. Diffraction data were measured to 2.2 A resolution and processed by HKL2000. Phases were calculated using the structure described in Example 13 as the model, and CNX (Accelrys Inc., San Diego, California) was used for calculating the difference electron density map.
Example 19. Crystallization of FXIcat with SPRL-123545
A ten-fold molar excess of 10 mM SPRL-123545 in DMSO was added to diluted FXIcat-S434A,T475A,C482S,K437A, incubated at 4 °C, and then concentrated to 15 mg/mL. Cubic crystals were obtained using the hanging drop method (McPherson, Methods Biochem. Anal. 23:249, 1976) with 30% (w/v) PEG 4000, 0.2 M Li2SO4, 0.1 M Tris-HCl, pH 8.5. at 10 °C. The crystals belong to space group 123 with unit cell parameters of a=b=c=120.7 A. Diffraction data were measured to 2.05 A and processed by HKL2000. Phases were calculated using the structure described in Example 13 as the model, and CNX (Accelrys Inc., San Diego, California) was used for calculating the difference electron density map.
Example 20. Crystallization of FXIcat with SPRL-122624 A ten-fold molar excess of 10 mM SPRL-122624 in DMSO was added to the FXIcat-S434A,T475A,C482S,K437A, incubated 30 minutes at 20 °C and then concentrated to 20 mg/mL. Cubic crystals were obtained using the hanging drop method (McPherson, Methods Biochem. Anal. 23:249-345, 1976) with 20% (w/v) PEG monomethyl ether 5000, 0.1 M ammonium sulfate, and 0.1 M Tris-HCl, pH 7.6 at 10 °C. The crystals belong to the space group 123 with unit cell parameters of a=b=c=122.4 A. Diffraction data were measured to 2.05 A and processed by HKL2000. Phases were calculated using the structure described in Example 13 as the model, and CNX (Accelrys Inc., San Diego, California) was used for calculating the difference electron density map.
Example 21. Crystallization of FXIcat with SPRL-123586 A ten-fold molar excess of 10 mM SPRL-123586 in DMSO was added to the FXIcat-S434A,T475A,C482S,K437A, incubated 30 minutes at 4°C and then concentrated to 20 mg/mL. Needle-like crystals were obtained using the hanging drop method (McPherson, Methods Biochem. Anal. 23:249-345, 1976) with 1.4 M tri-sodium citrate, 0.1 M HEPES, pH 7.5 at 10 °C. The crystals belong to the space group P21 with unit cell parameters of a=55.8 A, b=70.3 A, c=62.1 A, α= γ= 90° and β=l 02.2°. Diffraction data were measured to 2.7 A and processed by HKL2000. There are two FXIcat molecules in the asymmetric unit. The orientations of the two monomers were determined by AmoRe using the structure described in Example 13 as model. The solutions were refined in CNX (Accelrys Inc., San Diego, California). The difference electron density map was also calculated in CNX.
Example 22. Soaking SPRL-130421 into preformed FXIcat-benzamidine crystal The preformed FXIcat-benzamidine crystals were obtained in a condition similar to Example 13. Benzamidine was added to the FXIcat- S434A,T475A,C482S,K437A protein to a concentration of 1 mM and the complex was concentrated to 20 mg/mL. Cubic crystals were obtained by the hanging-drop method in 2.0 M ammonium sulfate and 0.1 M Tris-HCl, pH 8.5 at 10 °C. A preformed FXIcat-benzamidine crystal was transferred into a drop containing its mother liquor with 5 mM of SPRL-130421 and incubated at 10 °C overnight. The crystal is in the space group 123 with unit cell parameters of a=b=c= 121.0 A. Diffraction data were measured to 1.9 A and processed by HKL2000. Phases were calculated using the structure of FXIcat-SPRL-123545 described in Example 19 as the model, and CNX (Accelrys Inc., San Diego, California) was used for calculating the difference electron density map. Model building was performed using Quanta (Accelrys Inc., San Diego, California).
Example 23. General Synthetic Methods for the Compounds of the Present Invention
Preparation of Compounds of Formula I
Figure imgf000099_0001
The compounds of formula I may be synthesized by either solid or liquid phase methods described and referenced in standard textbooks, or by a combination of both methods. These methods are well known to those skilled in the art, (see, for example, Bodanszky, In "The Principles of Peptide Synthesis", Hafner, Rees, Trost, Lehn, Schleyer, Zahradnik, Eds., Springer- Verlag, Berlin, 1984; Stewart and Young, Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, 111. (1984); and U.S. Pat. Nos. 4,105,603; 3,972,859; 3,842,067; and 3,862,925).
As described herein, compounds of formula I can be prepared by standard amide bond forming reactions, including fragment condensation when compounds of formula la are coupled to compounds of formula lb, wherein A, R°, R1, R2, and R3 are as previously defined herein.
Figure imgf000099_0002
Peptide alpha-keto acids, esters, thio esters, and amides can be prepared as described by Medhi et al., Biochem. Biophys. Res. Commun. 166:595-600, 1990; Angelastro et al, J. Med. Chem. 33:11-13, 1990; Wipf et al, J. Org. Chem. 58:5592-5594, 1993; or Webb et al, PCT Publication WO 9408941. Ketothiazoles can be prepared by solid-phase synthesis as described by Parlow et al, J. Med. Chem. 46:4043-9, 2003. The pyrimidinones of formula I can be prepared as described by Veale et al, J. Med.Chem. 38:98-108, 1995; Tamura et al, PCT Publication WO 9618644, South et al, PCT Publication WO 0069832; Ewin, et al, PCT Publication WO 0032590; and Akahoshi et al, J. Med Chem. 44:1286-96, 2000.
During the synthesis of the compounds of the present invention, the functional groups of the amino acid derivatives used in these methods are protected by blocking groups to prevent cross reaction during the amide bond- forming procedure. Examples of suitable blocking groups and their use are described in "The Peptides: Analysis, Synthesis, Biology," Academic Press, Gross & Meienhofer, Eds., Vol. 3 (1981) and Vol. 9 (1987). The reaction products are isolated and purified by conventional methods, typically by solvent extraction into a compatible solvent, or by using a washing protocol for resin bound intermediates. The products may be further purified by column chromatography or other appropriate methods, including medium pressure or high pressure liquid chromatography. In one example, compounds of the invention can be conveniently prepared using solid phase synthesis methodology (Merrifield J. Am. Chem. Soc, 85:2149, 1964; Houghten, Proc. Natl. Acad. Sci. USA, 82:5132, 1985). Solid phase synthesis begins at the carboxy terminus of the putative compound by forming a bond (e.g., an amide or ester bond) between a protected amino acid, or other carboxylic acid-containing compound, and an inert solid support. The inert solid support can be any macromolecule capable of serving as an anchor for the C-terminus of the initial amino acid. Typically, the macromolecular support is a cross-linked polymeric resin (e.g. a polyamide or polystyrene resin). In one embodiment, the C-terminal amino acid is coupled to a polystyrene resin to form a benzyl ester. Particularly useful benzyl-type resins, such as trityl resin, chlorotrityl resin, and Wang resin, are those in which the linkage of the carboxy group (or carboxamide) to the resing is acid-lable. A macromolecular support is selected such that the peptide anchor link is stable under the conditions used to deprotect the α-amino group of the blocked amino acids in peptide synthesis. If a base-labile α-protecting group is used, then it is desirable to use an acid-labile link between the peptide and the solid support. For example, an acid-labile ether resin is effective for base-labile Fmoc-amino acid peptide synthesis as described by Stewart and Young, vide supra. Alternatively, a peptide anchor link and α-protecting group that are differentially labile to acidolysis can be used. For example, an aminomethyl resin such as the phenylacetamidomethyl (Pam) resin works well in conjunction with Boc-amino acid peptide synthesis.
After the initial amino acid is coupled to an inert solid support, the α- amino protecting group of the initial amino acid is removed with, for example, trifluoroacetic acid (TFA) in methylene chloride and neutralizing in, for example, triethylamine (TEA). Following deprotection of the initial amino acid's α-amino group, the next . α-amino and side chain protected amino acid in the synthesis is added. The remaining α-amino protected and, if necessary, side chain protected amino acids are then coupled sequentially in the desired order by condensation to obtain an intermediate compound connected to the solid support. Alternatively, some amino acids may be coupled to one another in solution to form a fragment of the desired peptide followed by addition of the peptide fragment to the growing solid phase peptide chain. The condensation reaction between two amino acids, or an amino acid and a peptide, or a peptide and a peptide can be carried out according to the usual condensation methods such as the azide method, acyl halide method (preferably acyl fluoride), mixed acid anhydride method, DCC (N,N- dicyclohexylcarbodiimide) or DIC (N,N'-diisopropylcarbodiimide) methods, active ester method, p-nitrophenyl ester method, BOP (benzotriazole-1-yl-oxy- tris[dimethylamino] phosphonium hexafluorophosphate)method, N- hydroxysuccinicacid imido ester method, O-benzotriazolyl-N,N,N',N'- tetramethyluronium hexafluorophosphate (HBTU) or O-(7-azabenzotriazol-l- yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and Woodward reagent K method.
Alternatively, other functional groups, can be introduced on the liberated N-terminus of the resin. For example, reaction with commercially available carboxylic acids or acid chlorides, sulfonyl chlorides, or isocyanates under standard conditions known in the art produce compounds of the invention containing an amide, sulfonamide, or urea bond, respectively. An alternative to using isocyanates in preparing urea-containing compounds is to activate the deprotected amine terminus with, for example, phosgene, triphosgene, carbonyl di-imidazole, or p-NO2 phenylchloro formate followed by reaction with primary or secondary amines employed in excess.
It is common in both solid-phase and solution-phase synthesis to protect any reactive side-chain groups of the amino acid with suitable protecting groups. Ultimately, these protecting groups are removed after the desired compounds have been sequentially assembled. Also common is the protection of the α-amino group on an amino acid or a fragment while that entity reacts at the carboxy group followed by the selective removal of the α-amino protecting group to allow subsequent reaction to take place at that location. Accordingly, it is common that an intermediate compound is produced which contains each of the amino acid residues located in the desired sequence in the peptide chain with various of these residues having side chain protecting groups attached. These protecting groups are then commonly removed at substantially the same time so as to produce the desired product following cleavage from the resin. Protecting groups and procedure for their use in peptide synthesis are reviewed in Protective Groups in Organic Synthesis, 3d ed., Greene, T. W. and Wuts, P. G. M., Wiley & Sons (New York: 1999).
Suitable protecting groups for α-amino and side chain amino groups are exemplified by benzyloxycarbonyl (abbreviated Z), isonicotinyloxycarbonyl (iNOC), o-chlorobenzyloxycarbonyl [Z(2C1)], p-nitrobezyloxycarbonyl
[Z(NO2)], p-methoxybenzyloxycarbonyl [Z(OMe)], t-butoxycarbonyl (Boc), t- amyloxycarbonyl(Aoc), isobornyloxycarbonyl, adamantyloxycarbonyl, 2-(4- biphenyl)-2-propyloxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl(Fmoc), methylsulfonyethoxycarbonyl (Msc), trifluoroacetyl, phthalyl, formyl, 2- nitrophenylsulfenyl (NPS), diphenylphosphinothioyl (Ppt), and dimethylphosphinothioyl (Mpt) groups, and the like.
Protective groups for the carboxy functional group are exemplified by benzyl ester, (OBz), cyclohexyl ester (Chx), 4-nitrobenzyl ester (ONb), t-butyl ester (OtBu), 4-pyridylmethyl ester (OPic), and the like. It is often desirable that amino acids possessing a functional group other than amino and carboxy groups, such as, for example, arginine, cysteine, histidine, asparagine, glutamine, threonine, tyrosine, tryptophan and serine, be protected by a suitable protecting group. For example, the guanidino group may be protected with nitro, p-toluenesulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl, p- methoxybenzenesulfonyl, 4-methoxy-2, 6-dimethylbenzenesulfonyl (Nds), 1,3,5-trimethylphenysulfonyl (Mts), and 2,3,6-trimethyl-4- methoxyphenylsulfonyl (Mtr), and the like. The thiol group can be protected with p-methoxybenzyl, trityl, and the like. Other suitable protecting groups for hydroxy, phenoxy, the indole amine, carboxamide, the imidazole nitrogen can be found in Greene, vide supra.
In one embodiment, the compounds of the invention are synthesized with the help of blocking groups that protect the side chain amide bond- forming substituents of the N-terminal and C-terminal flanking residues. The protecting group or groups used for the side chain amide bond-forming substituents of the N-terminal and C-terminal flanking residues can be the same or different than the protecting group or groups used to block the side chain functional groups of other residues in the peptide. In a preferred embodiment, the protecting group or groups used to block the side chain amide bond- forming substituents is (are) differentially removable with respect to the protecting groups used for other side chain functional groups, i.e. the side chain amide bond-forming substituents can be deprotected without deprotecting the other side chain functional groups, in addition to being differentially removable with respect to the α-amino protecting group used in peptide synthesis. In another preferred embodiment, the side chain amide bond-forming substituents of the flanking residues are orthogonally protected with respect to each other such that the side chain amide bond-forming substituent of one flanking residue can be deprotected without deprotecting the side chain amide bond-forming substituent of the other flanking residue. Suitable protecting groups for use in orthogonally protecting the side chain amide bond-forming substituents of the flanking residues with respect to other functional groups and/or with respect to each other include pairs of differentially removable carboxy protective groups, such as a reduction-labile carboxy protective group, e.g. allyl or benzyl esters, paired with a base-labile carboxy protective group, e.g. fluorenylmethylesters, methyl or other primary alkyl esters. Fluorenylmethyl, methyl or other primary alkyl groups or other base-labile carboxy protective groups can be removed from their corresponding esters to yield a free carboxy group (without deprotecting allyl or benzyl esters or other reduction-labile esters) by saponification of the esters with a suitable base such as piperidine and sodium hydroxide in a suitable solvent such as dimethylacetamide, or methanol and water, for a period of 10 to 120 minutes, and preferably 20 minutes, 0°C to 50°C. The allyl or benzyl or other reduction- labile esters can be removed when desired by reduction in the presence of a suitable transition metal catalyst, such as Pd(PPh3)4, Pd(PPh3)2Cl2, Pd(OAc)2 or Pd on carbon with a source of hydrogen, e.g. H2 gas, in a suitable solvent such as dimethylacetamide, dimethylformamide, N-methylpyrrolidinoneor methanol for a period of 10 to 500 minutes, and preferably 100 minutes, at 0°C to 50°C. For the sake of simplicity and convenience, all carboxy protective groups that are removable by Pd-catalyzed methods which result in the reduction of the protected carboxy substitutent are included n the term "reduction-labile protective groups" as used herein, even though such Pd-catalyzed deprotection methods may not result in the reduction of the protective group in question.
In embodiments wherein Pd catalysis involves the formation of intermediates of Pd derivatized with reduction-labile protecting groups, e.g. Pd- allyl derivatives, the Pd catalyst can be restored by reaction with a suitable nucleophile, such as piperidine or tributyltin hydride. When such reduction- labile groups are used to provide orthogonal protection in combination with base-labile protecting groups, it is preferable to either (1) utilize a synthetic scheme that calls for the removal of the base-labile protecting groups before the removal of the reduction-labile protecting groups or (2) restore the Pd catalyst with a nucleophile that does not deprotect the base-labile protecting groups.
Alternatively, the carboxy substituents of the flanking residues can be orthogonally protected with respect the other functional groups and/or with respect to each other by using an acid-labile protecting group, such as a tertiary alkyl ester, e.g. t-butyl ester, in combination with a reduction-labile protecting group, such as the allyl or benzyl esters described above. The tertiary alkyl or other acid-labile ester group can be removed by acidolysis, e.g. with trifluoroacetic acid in methylene chloride, and the ally] or benzyl or other reduction-labile esters can be removed by reduction in the presence of a transition metal catalyst as described above.
In another embodiment, the carboxy substituents of the flanking residues can be orthogonally protected with respect to other functional groups and/or with respect to each other by using a fluoride ion-labile protecting group, such as 2-(trimethylsilyl)ethyl and silyl esters, in combination with a reduction- labile protecting group, such as the ally] or benzyl esters described above, or in combination with a base-labile protecting group, such as the fluorenylmethyl, methyl or other primary alkyl esters described above, without deprotecing the reduction-labile or base-labile esters. The 2-(trimethylsilyl)ethyl, silyl or other fluoride-labile ester group can be removed by reaction with a suitable fluoride ion source, such as tetrabutyl ammonium fluoride in the presence of a suitable solvent, such as dimethylacetamide (DMA), dimethylformamide (DMF), tetrahydrofuran (THF), or acetonitrile. Suitable protecting groups for use in orthogonally protecting the side chain amide bond-forming substituents of the flanking residues with respect to other functional groups and/or with respect to each other also include pairs of differentially removable amino protective groups, such as an allyloxycarbonyl or other reduction-labile amino protective group paired with a t-butoxycarbonyl (Boc) or other acid-labile amino protective group, and a reduction-labile amino protective group paired with a fluorenylmethoxycarbonyl (Fmoc) or other base- labile amino protective group. An allyloxycarbonyl (or other reduction-labile blocking group) protected amino group can be deprotected by reduction using a transition metal catalyst as in the procedure for removing reduction-labile carboxy protective groups described above, without deprotecting a Boc or
Fmoc protected amino group. Likewise, an acid-labile amino protective group and a base-labile amino protective group can be removed by acidolysis and base saponification, respectively, without removing a reduction-labile amino protective group. For the sake of simplicity and convenience, all amino protective groups that are removable by Pd-catalyzed methods which result in the reduction of the protected amino substitutent are included in the term "reduction-labile protective groups" as used herein, even though such Pd- catalyzed deprotection methods may not result in the reduction of the protective group in question. In another embodiment, the amino substituents of the flanking residues can be orthogonally protected with respect to other functional groups and/or with respect to each other by using a fluoride-labile protecting group, such as 2-trimethylsilylethylcarbamate (Teoc), in combination with a reduction-labile protecting group, such as allyloxylcarbonyl, or in combination with a base- labile protecting group, such as fluorenylmethoxycarbonyl, as described above. The Teoc or other fluoride-labile group can be removed by reaction a with a suitable fluoride ion source, such as tetrabutyl ammonium fluoride, as in the procedures for removal of fluoride-labile carboxy protective groups described above, without deprotecting an allyloxycarbonyl or fluorenylmethoxycarbonyl protected amino group. Likewise, a reduction-labile amino protective group and a base-labile amino protective group can be removed by reduction and base saponification, respectively, without removing a fluoride-labile amino protective group. In embodiments that use a carboxy substituent as the side chain amide bond-forming substituent of one flanking residue and that use an amino substituent as the side chain amide bond-forming substituent of the other flanking residue, the carboxy substituent and the amino substituent can be orthogonally protected with respect to each other by using a reduction-labile protecting group to block one substituent, e.g. allyl ester or allyloxycarbonyl, and a fluoride-labile, acid-labile or base-labile protecting group to block other substituent, e.g. silyl ester, t-butyl ester, fluorenylmethyl ester, Teoc, Boc, or Fmoc.
In a preferred embodiment, a reduction-labile protecting group is used to block the side chain amide bond-forming substituent of one flanking residue and the protecting group for the side chain amide bond-forming substituent of the other flanking residue is selected such that it provides orthogonal protection with respect to both the reduction-labile protecting group and the α-amino protecting group used in the synthesis. For example, in an embodiment using Fmoc chemistry for peptide synthesis, orthogonal protection of the side chain amide bond-forming substituents would be provided by a reduction-labile protecting group and an acid-labile protecting group. Likewise, in an embodiment using Boc chemistry for peptide synthesis, orthogonal protection of the side chain amide bond-forming substituents would be provided by a reduction-labile protecting group and a base-labile protecting group.
In yet another preferred embodiment, the side chain amide bond- forming substituents of the flanking residues are orthogonally protected with respect to each other, with respect to α-amino protecting group used in the synthesis, and with respect to the protecting groups used to block other side chain functional groups in the peptide chain.
In still another preferred embodiment, the side chain amide bond- forming substituents of the flanking residues are orthogonally protected with respect to each other, and with respect to α-amino protecting group, but only one of the side chain amide bond-forming substituents is orthogonally protected with respect to the protecting groups used to block other side chain functional groups. In this embodiment, it is preferable to use the side chain amide bond-forming substituent with fully orthogonal protection as the target for initial attachment of the compound to the difunctional linker. Since the side chain amide bond-forming substituent with fully orthogonal protection can be deprotected without deprotecting other functional groups, the amide bond- forming reaction will be specific to the desired side chain amide bond-forming substituent, and will reduce the production of unwanted difunctional linker derivatives. Although cyclization will require the deprotection of the side chain amide bond-forming substituent of the other flanking residue, and may cause concomitant deprotection of other side chain functional groups, unwanted derivatives are less likely to form given that the peptide chains are anchored to a solid support and that the linker length will regioselectively favor a amide bond-forming reaction between the unbound functional group of the linker and the side chain amide bond-forming substituent of the other flanking residue. If further peptide chain synthesis is desired after cyclization, any side chain functional groups on other amino acid residues left unprotected by the cyclization reactions can be reprotected before chain synthesis is resumed. Many of the blocked amino acids described above can be obtained from commercial sources such as Novabiochem (San Diego, Calif), Bachem Calif. (Torrence, Calif.) or Peninsula Labs (Belmont, Calif). Alternatively, functionalized or protected amino acids, including unnatural amino acids, can be prepared by methods known in the art. In addition, the compounds of the invention can be prepared by, or in conjunction with, solution phase peptide synthesis, for example, the solution phase peptide synthesis methods described in Principles of Peptide Synthesis, 2d ed, M. Bodanszky, Springer-Verlag (1993) or in The Practice of Peptide Synthesis, 2d ed, M. Bodanszky and A. Bodanszky, Springer-Verlag (1994). It will be appreciated that solution phase peptide synthesis methods can be easily modified to incoφorate the desired flanking residues, with or without orthogonally-protected side chain amide bond-forming substituents, into the compound of interest, using procedures similar to those used in the solid phase synthesis methods described herein. It will be further appreciated that all references to amide bond-forming reactions herein encompass both solid phase and solution (or liquid) phase peptide synthesis methods, unless otherwise indicated.
The preparation of ketone derivatives of the formula I (for example, m is
1, R° is an optionally substituted alkyl, alkenyl, cycloalkyl, aralkyl, aralkenyl, aryl, heterocyclyl, heteroaralkyl, or heteroaralkenyl, or C(O)R9, where R9 is as previously defined herein) can be prepared by reacting appropriately protected amino acid derivatives having a free carboxyl group (e.g., an appropriately alpha-amine protected analog of formula lb, where m is 1 and R° is OH) with an alkoxyamine under amide bond-forming conditions, as described herein, to form Weinreb amides of formula III, where P is a nitrogen protecting group. The Weinreb amide can be reacted with alkyl, alkenyl, cycloalkyl, aralkyl, aralkenyl, aryl, heterocyclyl, heteroaralkyl, heteroaralkenyl, or heteroaryl carbanions such as lithium salts or Grignard reagents to form ketones of formula IV, which can be subsequently deprotected at the alpha-amine position to form compounds of formula lb, where m is 1 and R° is alkyl, alkenyl, cycloalkyl, aralkyl, aralkenyl, aryl, heterocyclyl, heteroaralkyl, heteroaralkenyl, or heteroaryl. These ketone compounds can then be coupled to the carboxyl- containing compounds of formula la to produce the corresponding compounds of formula I.
Figure imgf000110_0001
Alternatively, ketone derivatives of the formula I can be prepared from Weinreb amide III by reduction of this amide to the corresponding aldehyde with a hydride reagent, such as DIBAL-H or LAH, to produce compounds of formula V, to which an R group can be introduced by the addition of a nucleophile, such as the carbanions, organolithio reagents, or Grignard reagents described before in the nucleophilic addition to Weinreb amides. The resulting compound can then be reacted with compounds of formula la, as previously described, followed by oxidation of the secondary alcohol, such as with Dess- Martin reagent, to yield a compound of formula IV. In one example, compounds of formula I, where m is 1 and R° is 2-thiazole, are prepared by reacting aldehydes of formula V with 2-trimethylsilyl (TMS) thiazole. The resulting TMS ether is deprotected with fluoride and subsequent oxidation of the resulting secondary alchohol, followed by removal of the amine protecting group and reaction with compounds of formula la, provides thiazole ketones of formula I (i.e., m is 1 and R° is 2-thiazole). When an appropriately protected Weinreb amide of formula III is reacted with 2-lithiofuran, compounds of formula IV are produced, wherein m is 1 and R° is a 2-furyl moiety. Subsequent reduction of this ketone with sodium borohydride provides a mixture of antipodal alcohols. Acetylation of the alcohol, followed by oxidative degradation of the furan with RuCl3 and NaIO , produces a compound of formula VI, where R9 is OH. Condensation of this carboxylic acid with amines, alcohols, or thiols, provides the alpha-acetoxy amides, esters, or thioesters, respectively, of formula VI, where R9 is, for example, an optionally substituted alkyloxy, aryloxy, heteroaryloxy, aralkyloxy, alkylthio, cycloalkylthio, arylthio, aralkylthio, alkylamino, alkylamino, dialkylamino, aralkylamino, arylamino, heterocyclylamino, or heterocyclylakylamino.
Figure imgf000111_0001
The acetoxy and amine protecting groups of the compounds of formula VI can be removed and the amine coupled to compounds of formula la to form the corresponding compounds of formula VII, which can be further transformed by oxidation, with oxidants such as Dess-Martin reagent, to provide ketones of formula I, wherein m is 1 and R° is, for example, an alkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, or aralkoxycarbonyl. Alternatively, the acetoxy protecting group can be retained and carried through the coupling step and removed later.
Compounds of the formula I, wherein m is 0 and R° is H, can be prepared through condensation of compounds of the formula la with amine derivatives of formula H2NCH2-R'. In one example, appropriately protected compounds of the formula la are reacted with appropriately protected amines of the formula VIII, using amide-bond-forming conditions as described herein. Subsequent removal of any protecting groups provides compounds of formula IX, wherein Z, A, R2, R3, R4, and R7 are as previously defined herein.
Figure imgf000112_0001
Compounds of formula I, where R3 together with A forms a ring having formula (X) are prepared as exemplified by the synthesis of pyrimidinones, where W is N.
Figure imgf000112_0002
Accordingly, thiouracil is methylated (methyl iodide) then halogenated (e.g., with 12 or Br2), and the resulting halopyrimidinone XI is alkylated with electrophile XII, where Y1 is Br, I, or OTf and P1 is a carboxylic acid protecting group, to produce compounds of the formula XIII. Various amine groups, including primary and secondary amines of the formula B-N(R18)H, where R is H or alkyl and B is an optionally substituted alkyl, alkenyl, cycloalkyl, aralkyl, aryl, heterocyclyl, heterocyclylalkyl, or a suitable nitrogen protecting group, can be incoφorated into the pyrimidinone scaffold utilizing, for example, the palladium-mediated amide bond-forming method of Buchwald, et al, J. Org. Chem. 65:1144-1157, 2000, to produce compounds of the formula XlYa. If B is a nitrogen protecting group, it can be removed and the free amine thus generated further functionalized such that B can be an acyl, aroyl, heteroaroyl, alkoxycarbonyl, cycloalkoxycarbonyl, aralkoxycarbonyl, aryloxycarbonyl, heterocyclyloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, alkylthiocarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl, alkoxythiocarbonyl, cycloalkoxythiocarbonyl, aralkoxythiocarbonyl, aryloxythiocarbonyl, aminothiocarbonyl, alkylaminothiocarbonyl, dialkylaminothiocarbonyl, cycloalkylaminothiocarbonyl, aralkylaminothiocarbonyl, arylaminothiocarbonyl, heterocyclylaminothiocarbonyl, heteroaralkylaminothiocarbonyl, alkylsulfonyl, cycloalkylsulfonyl, aralkylsulfonyl, arylsulfonyl, heterocyclylsulfonyl, or a heterocyclylalkylsulfonyl group. The free amine can also be condensed with the carboxyl group of amino acid derivatives.
Figure imgf000113_0001
Figure imgf000113_0002
Substituents R17, which includes, but is not limited to, substituted aromatic rings, can be introduced at the 2-position of the pyrimidinone scaffold of compound XlVa by utilizing, for example, C-C palladium-mediated bond forming conditions (see, for example, Lindley, Tetrahedron 40:1433, 1984) to produce compounds of the formula XlVb after removal of the carboxyl protecting group, P1. Other intermediates of formula XlVb can be prepared by alkylating thiouracil with another appropriately activated compound other than
17 methyl iodide to produce compounds in which R is alkylthio, cycloalkylthio, arylthio, aralkylthio, heterocyclylthio, or heteroaralkylthio. Alternatively, the thiomethyl group, or the methyl sulfoxide or methyl sulfone derived therefrom, of compounds of formula XlVa can be displaced with other nucleophilic groups, such as, for example, the NH2 group of various amines, to produce compounds for formula XlVb where R17 is alkylamino, cycloalkylamino, aralkylamino, arylamino, or heterocyclylamino. Subsequent amide bond formation by the condensation of compounds of formula XlVb with the alpha amine of amino acid derivatives, including Weinreb amides, with subsequent functional group manipulation, as described herein, and removal of any protecting groups used, provides pyrimidinone compounds of formula 1 (e.g., compounds of formula XV). Alternatively, subsequent amide bond formation by the condensation of compounds of the formula XlVb with amine derivatives of formula H2NCH2-R1, provides pyrimidinone compounds of the formula I (m is 0, R is H).
Figure imgf000114_0001
Preparation of Compounds of Formula II
Figure imgf000114_0002
For compounds of the formula II, naphthyl amidines or amines (i.e., each of e and g is C-H) can be prepared, at least in part, as described by Geye et al, U.S. Patent No. 6,258,822 and U.S. Patent No. 6,284,796 Bl ; Klinghofer et al, Biochemistry 40:9125-9131, 2001; and Atwell et al, EP Patent 0206802 and U.S. Patent No. 4,904,659. Quinazoline amidines or amines (i.e., each of e and g is N) can be prepared according to procedures detailed by Mederski et al, WO 0224667 and Ahn et al, U.S. Patent No. 5,658,902. In one example, compounds of formula II, where each of e and g is C-H;
T is NH, B is an alkyl, substituted carbonyl or substituted sulfonyl group, f is a bond, V is H, R19 is H, and Z1 is NH (i.e., 8-substituted naphthalene-2- carboxamidines), can be prepared starting from 8-amino-naphthalen-2-ol, which is first N-protected, such as with the carbonylbenzyloxy group (Cbz), and then activated at the 2-position, such as by conversion of the hydroxyl group to a trifiate. The triflate can be cyanated under Pd-mediated C-C bond forming conditions (see (a) Takagi et al, Chem. Lett. 1973:471 ; (b) Sekiya et al., Chem. Lett. 1975:277; (c) Tschaen et al, Synth. Commun. 24:887, 1994; and (d) Sundermeier et al, Tetrahedron Lett. 42:6707, 2001). After removal of the N-protecting group, the naphthylamine can be derivatized with acid halides, sulfonyl chlorides, chloroformates, isocyanates, isothiocyanates, alkyl halides, or coupled to amino acid derivatives to provide compounds of the formula XVI where T is NH and B is a natural or unnatural alpha-amino acid residue of D- or L-configuration, or an optionally substituted alkyl, cycloalkyl, alkenyl, aralkyl, aralkylcarbonyl, aralkenyl, aryl, heteroaralkyl, heteroaralkylcarbonyl, heteroaralkenyl, acyl, cycloalkylcarbonyl, aroyl, heterocyclyloyl, cycloalkoxycarbonyl, aryloxycarbonyl, heterocyclyloxycarbonyl, heteroaralkyloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, heteroaralkylaminocarbonyl, alkylthiocarbonyl, cycloalkylthiocarbonyl, arylthiocarbonyl, aralkylthiocarbonyl, heterocyclylthiocarbonyl, heteroaralkylthiocarbonyl , aminothiocarbonyl, alkylaminothiocarbonyl, dialkylaminothiocarbonyl, cycloalkylaminothiocarbonyl, aralkylaminothiocarbonyl, arylaminothiocarbonyl, heterocyclylaminothiocarbonyl, heteroaralkylaminothiocarbonyl, alkylsulfonyl, cycloalkylsulfonyl, aralkylsulfonyl, arylsulfonyl, heterocyclylsulfonyl, or heteroaralkylsulfonyl. In those cases where B is aryl, the use of organometallic chemistry coupling techniques involving a palladium catalyst in the reaction of an amine (e.g., an amine on a naphthyl ring of formula II) with an aryl triflate is recommended.
Figure imgf000116_0001
The nitrile group can then be transformed to an amidine group under a variety of conditions, including acidic conditions in alcohol (e.g., Pinner conditions followed by aminolysis as described by (a) Pinner, Die Iminoάther und ihre Derivate, Verlag, R. Oppenheim: Berlin, 1892; (b) Roger et al, Chem. Rev. 61 :179, 1961; (c) Schaefer et al, J. Org. Chem. 26:412, 1961), or treatment with hydroxylamine followed by acetylation of the N-OH amidine group and subsequent reduction under hydrogenation conditions (see Judkins et al, Synth. Commun. 26:4351, 1996), to provide compounds of the formula XVII, where Z is NH and R19 is H. For those compounds of formula II where Z is NR22, where R22 is alkyl, OH, or NH2, the nitrile is treated with an alkyl amine, hydroxylamine or hydrazine, respectively. Carbamoylation of the amidine group with a suitable chloroformate reagent under basic conditions
77 provides compounds of the formula II where R is CO2-R. Other syntheses of amidines from nitriles are described by Garigipati et al, Tetrahedron Lett.
31 :1969, 1990; Moss et al, Tetrahedron Lett. 36:8761, 1995; Rousselet et al, Tetrahedron Lett. 34: 6395, 1993; and Fosberg et al, J. Org. Chem. 52:1017, 1987.
Compounds of formula II, where T is a bond, B is H, each of e and g is C-H, f is O, and Z is NH can be prepared from 2-cyano-6-hydroxynaphthalene, which is first O-alkylated with an alkyl halide, aralkyl halide, or heteroaralkyl halide, where the halide group is chloro, bromo, or iodo, to give compounds of the formula XVIII. Alternatively, O-alkylation can be achieved with alkyl alcohols, aralkyl alcohols, or heteroaralkyl alcohols under Mitsunobu conditions using diispropylazodicarboxylate and triphenylphosphine. The amidine, hydroxyamidine, or aminoamidine groups can be obtained from the nitrile, as previously described herein, to afford compounds of the formula XIX, where R19 is H, and R22 is H, alkyl, OH, or NH2, and V is an alkyl, aralkyl, or heteroaralkyl group.
Figure imgf000117_0001
Compounds of formula II, where T is a bond, B is H, each of e and g is C-H, f is a bond, V is a substituted carbonyl group, and Z is NH can be prepared as follows. Hydrolysis of naphthalene-2,6 dicarboxylic acid dimethyl ester with one equivalent of a base such as KOH, followed by acidification of the product with an acid such as HCl, affords the naphthalene mono carboxylic acid. After conversion of the carboxylic acid to an acid chloride with a reagent such as thionyl chloride and subsequent reaction with ammonia in methylene chloride, the resulting 6-carbamoyl-naphthalene-2-carboxylic acid methyl ester is reacted with triphosgene in trimethylphosphate to provide 6-cyano- naphthalene-2-carboxylic acid methyl ester. Hydrolysis of the ester with LiOH in aqueous THF solution, followed by reaction with thionyl chloride, affords 6- cyano-naphthalene-2-carbonyl chloride, which can then be coupled with alcohols or amines to provide compounds of the formula XX. The amidine, hydroxyamidine, or aminoamidine groups can be obtained from the nitrile as described previously to afford compounds the formula XXI, where R19 is H,
77 and R is H, alkyl, OH, or NH2, and V is an alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyi, heterocyclylaminocarbonyl, alkoxycarbonyl, cycloalkoxycarbonyl, aralkoxycarbonyl, aryloxycarbonyl, heterocyclyloxycarbonyl, or heteroaralkyloxycarbonyl.
Figure imgf000117_0002
Compounds of formula II, where each of e and g is C-H; T is NH, B is an alkyl, substituted carbonyl or substituted sulfonyl group, f is a bond, V is a substituted carbonyl group, and Z is NH can be prepared as follows. Nitration of 6-cyano-naphthalene-2-carboxylic acid methyl ester with NO3/H2SO4 gives of 6-cyano-4-nitro-naphthalene-2-carboxylic acid methyl ester. Hydrolysis of the ester with LiOH in aqueous THF solution, followed by treatment with thionyl chloride, gives 6-cyano-4-nitro-naphthalene-2-carbonyl chloride, which is coupled with a primary or secondary amine to give amides of the formula XXII, where V is an alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, or heterocyclylaminocarbonyl group. Hydrogenation of compounds of the formula XXII in the presence of Pd/C (10%) in methanol affords amines of the formula XXIII. Compounds of the formula XIII in the presence of a base, such as, for example, diisopropylethylamine or K2CO3 in THF, are treated with acid chlorides, chloroformates, isocyanates, or sulfonyl chlorides, or coupled with carboxylic acids as described herein, to afford compounds of the formula XXIV, where T is NR23, where R23 is H, OH, or optionally substituted alkyl, alkenyl, aralkyl, aralkenyl, heteroaralkyl, or heteroaralkenyl, and B is acyl, aroyl, heterocyclylcarbonyl, alkoxycarbonyl, cycloalkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, alkylaminosulfonyl, cycloalkylaminosulfonyl, aralkylaminosulfonyl, or B is a protected natural or unnatural alpha-amino acid residue of D- or L-configuration.
Alternatively, the primary amine of compounds of formula XXIII can be monoalkylated via reaction with an alkyl halide or via reductive amination procedures, followed by treatment with an activated carboxylic acid, to produce compounds of formula XXIV, where T is NR23, where R23 is alkyl. Amidine, hydroxyamidine, or aminoamidine groups can be obtained from the nitrile as described previously to afford compounds of the formula XXV, where R19 is H and R22 is H, alkyl, OH, or NH , and V is an alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, or heterocyclylaminocarbonyl group.
Figure imgf000119_0001
Similarly, the compounds of formula XXV in which T is a bond, B is NO2, V is methoxycarbonyl, R22 is H, and R19 is H, alkyl, OH, CO2-alkyl or NH2 can be obtained directly from 6-cyano-4-nitro-naphthalene-2-carboxylic acid methyl ester from the nitrile as described herein or the methoxycarbonyl group can be transformed to compounds of formula XXV in which T is a bond, B is NO2, V is an alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, or heterocyclylaminocarbonyl group, followed by transformation of the cyano moiety to an amidine, hydroxyamidine, or aminoamidine group. Alternatively, the nitro group of 6-cyano-4-nitro-naphthalene-2- carboxylic acid methyl ester can be reduced and optionally acylated as described herein to produce compounds of formula XXV, wherein V is methoxycarbonyl. Those compounds of formula XXII in which V is an alkoxycarbonyl can be further saponified and reduced with borane or LiBH4 to provide, after the cyano is converted to an amidine, compounds of the formula XXV is which V is hydroxylmethyl, or derivative thereof.
Compounds of formula II in which T is O can be prepared in a modification of the procedure of Fuganti et al., J. Chem. Res. Miniprint 10:2769-2782, 1998. Emmons-Horner reaction of4-cyanobenzaldehyde with compound XXVI to produce compound XXVII, and subsequent cyclization by treatment with acetic acid in acetic anhydride yields compound XXVIII. Compound XXVIII carried forward to compounds of formula XXV, where T is O and B is alkyl, alkenyl, cycloalkyl, aralkyl, aralkenyl, heteroaralkyl, heteroaralkenyl, acyl, aroyl, heteroaroyl, alkoxycarbonyl, cycloalkoxycarbonyl, aralkoxycarbonyl, aryloxycarbonyl, heterocyclyloxycarbonyl, heteroaralkyloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, heteroaralkylaminocarbonyl, alkylsulfonyl, cycloalkylsulfonyl, aralkylsulfonyl, arylsulfonyl, heterocyclylsulfonyl, or heteroaralkylsulfonyl by saponification of the acetyl group, refunctionalization of the resulting hydroxyl group by methods known in the art or those described herein, and tranformation of the nitrile moiety to an amidine, hydroxyamidine, or aminoamidine group as described herein.
Figure imgf000120_0002
(XXVI)
Figure imgf000120_0001
(XXVII)
Figure imgf000120_0003
Quinoline compounds of formula II, where e is N and g is C-H can be prepared via the condensation product XXIX derived from 4-iodoaniline, pyruvic acid, and O-benzyl protected salicylaldehyde (Doebner-Miller protocol, see M. Matsugi et al, Tet. Lett. 41 :8523-8525, 2000). The resulting quinoline carboxylic acid is reacted with CuCN to displace the iodo group with a cyano group after the carboxyl group is converted by a Curtius rearrangement to a carbamoylated amine. After deprotecting this amine, the cyano- aminoquinoline intermediate, XXX can be acylated (or otherwise derivatized), followed by subsequent conversion of the nitrile group to an amidine group as previously described to provide quinoline compounds of the formula II, where T is NR23; B is acyl, cycloalkylcarbonyl, aroyl, heterocyclyloyl, cycloalkoxycarbonyl, aryloxycarbonyl, heterocyclyloxycarbonyl, heteroaralkyloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, heteroaralkylaminocarbonyl, alkylthiocarbonyl, cycloalkylthiocarbonyl, arylthiocarbonyl, aralkylthiocarbonyl, heterocyclylthiocarbonyl, heteroaralkylthiocarbonyl, aminothiocarbonyl, alkylaminothiocarbonyl, dialkylaminothiocarbonyl, cycloalkylaminothiocarbonyl, aralkylaminothiocarbonyl, arylaminothiocarbonyl, heterocyclylaminothiocarbonyl, heteroaralkylaminothiocarbonyl, alkylsulfonyl, cycloalkylsulfonyl, aralkylsulfonyl, arylsulfonyl, heterocyclylsulfonyl, or heteroaralkylsulfonyl; f is a bond; and V is aryl.
Compounds of formula II in which e is N, f is a bond, and V is an optionally substituted alkyl, cycloalkyl, alkenyl, aralkyl, aralkenyl, heteroaralkyl, heteroaralkenyl, aryl, or heterocyclyl can be prepared by using the appropriate aldehyde. A particularly useful aldehyde is 2-furaldehyde, as the furyl group can be subsequently oxidized with RuCl3/NaIO4 and the resulting carboxyl group transformed to amides, esters, or thioesters, as described herein.
Figure imgf000121_0001
Alternatively, 4-aminobenzonitrile can be converted to 6-cyano-4- hydroxyquinoline-2-carboxylic acid, methyl ester, by Michael addition to dimethylacetylene dicarboxylate, followed by thermal cycloaddition (see Jaen et al., J. Med. Chem. 38:4439-45, 1995). The resulting hydroxyquinoline is converted to 4-amino-6-cyanoquinoline-2-carboxylic acid, methyl ester by bro ination with tetrabutyl ammonium bromide/phosphorus pentoxide (see Kato et al, Tetrahedron Lett. 42:4849-52, 2001), azide displacement of the bromo group (see Sashida et al, Chem. Pharm. Bull 38:2919-2925, 1990), and sodium borohydride reduction of the azide to the amine (see Outt et al, J. Org. Chem. 63:5762-68, 1998). The amine can be acylated {or otherwise derivatized, such as, for example, to an amide), followed by subsequent conversion of the nitrile group to an amidine group as previously described for compounds of formula XXX to provide quinoline compounds of formula II, where V-f- is, for example, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, or heteroaralkylaminocarbonyl.
Quinazoline compounds of the formula II, where each of e and g is N, can be prepared via iodination of 2-aminobenzonitrile with N-iodosuccinimide, followed by acylation with 2-acetoxybenzoyl chloride to form compound XXXI. Cyclization of XXXI can be mediated with a variety of primary amines, followed by conversion of the quinazolyl iodide to the quinazolyl cyanide, under cross coupling conditions with a cyanide source. In one example, aminoquinazoline XXXII can be obtained by reacting compound XXXI with 4-methoxybenzylamine and subsequent conversion to the quinazolyl cyanide. After orthogonal protection of the phenol, benzylic cleavage can be affected under oxidative or acidic conditions and the resulting aminoquinazolines can be derivatized, for example, with electrophilic reagents to prepare amides, ureas, carbamates, or sulfonamides after phenol deprotection. Subsequent conversion of the nitrile group to an amidine group as previously described provides compounds of the formula II, where each of e and g is N, and T is NR ; and B is acyl, cycloalkylcarbonyl, aroyl, heterocyclyloyl, cycloalkoxycarbonyl, aryloxycarbonyl, heterocyclyloxycarbonyl, heteroaralkyloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, heteroaralkylaminocarbonyl, alkylthiocarbonyl, cycloalkylthiocarbonyl, arylthiocarbonyl, aralkylthiocarbonyl, heterocyclylthiocarbonyl, heteroaralkylthiocarbonyl, aminothiocarbonyl, alkylaminothiocarbonyl, dialkylaminothiocarbonyl, cycloalkylaminothiocarbonyl, aralkylaminothiocarbonyl, arylaminothiocarbonyl, heterocyclylaminothiocarbonyl, heteroaralkylaminothiocarbonyl, alkylsulfonyl, cycloalkylsulfonyl, aralkylsulfonyl, arylsulfonyl, heterocyclylsulfonyl, or heteroaralkylsulfonyl; t is a bond; and V is 2-hydroxyphenyl. Compounds in which V is as described above can be prepared by the replacement in the synthesis of 2-acetoxybenzoyl chloride with other carbonyl chloride moieties.
Figure imgf000123_0001
For the quinazolines just described, it may be necessary to covert the nitrile to an appropriately protected amidine earlier in the synthesis, as Pinner conditions used for its conversion can result in partial hydrolysis of a B-T- substituent amide bond.
Representative Examples
Example 24. Synthesis of compounds of formula lb,
Figure imgf000124_0001
where m is 1, R° is 2-thiazole, and R1 is 3-Ng-Mtr-guanidinopropyl.
(L)-Boc-NH-Arg(Mtr)OH (1.14g, 2.34 mmol, 1.0 equiv), N,O- dimethylhydroxyl amine hydrochloride (460 mg, 4.72 mmol, 2.0 equiv), and 1- hydroxybenzotriazole (HOBt, 350 mg, 2.59 mmol, 1.1 equiv) were dissolved in 25 mL THF at room temperature. N,N-Diisopropylethylamine (DIEA, 1.22 mL, 7.00 mmol, 3 equiv) was added via syringe, and the solution was stirred until it was homogeneous. l-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 470 mg, 2.45 mmol, 1.05 equiv) was added in one portion, and the reaction was allowed to stir at room temperature overnight.
The reaction was diluted with ethyl acetate (150 mL), washed with 5% aqueous acetic acid (1 x 75 mL), sat. aq. NaHCO3 (2 x 75 mL), water (1 x 75 mL), and brine (1 x 75 mL). The organic layer was dried with Na2SO , filtered, and concentrated. The yield was 1.14g (92%) for the desired Weinreb amide (XXXIII) as a white powder (MS: m/z 530.1, M+l, calculated mass: 529.3).
Figure imgf000124_0002
(XXXIII)
To thiazole (5.85 mL, 82.5 mmol) and TMEDA (11.4 mL, 75.6 mmol) in THF (180 mL) at -78 °C was added nBuLi in hexane (30 mL, 75 mmol, 2.5 M) at a rate that raised the internal temperature to -50 °C. The reaction flask was placed in a dry ice/acetonitrile bath to give an internal temperature of -41 °C. The reaction was stirred for 25 minutes and then cooled to -78 °C. Compound XXXffl (9.54 g, 18 mmol) in THF (100 mL) was added to the reaction mixture and stirred for 45 minutes. The reaction was poured over a saturated aqueous ammonium chloride solution (600 mL), shaken vigorously, and then extracted with ethyl acetate (2 x 500 mL). The combined organic phases were washed with water (200 mL) and brine (200 mL), dried with sodium sulfate, filtered, and concentrated in vacuo. The residue was purified on a silica gel column, eluting with 75% ethyl acetate in hexane to 100% ethyl acetate to afford ketothiazole XXXIV (7.5 g, 75%) as a white foam (MS: m/z 554.1, M+l, calculated mass: 553.7).
The Boc-protected ketothiazole (4.0 g) was stirred in CH2C12 (30 mL) at 0°C, and trifluoroacetic acid (TFA, 10 mL) was added slowly. The ice bath was immediately removed after the addition of TFA. The resulting solution was stirred at room temperature for 1.5 hours, then a small amount of isopropyl alcohol (2 mL) was added. Concentration of the solution under reduced pressure gave the trifluoroacetic acid salt as a brown gum. The brown gum was triturated with diethyl ether. The ether supernatant was decanted, and the remaining solid was dried under vacuum to yield compound XXXV as a light yellow solid (4.01 g; 98% recovered yield, MS: m z 454, M+l, calculated mass: 453.2).
Figure imgf000125_0001
(XXXV)
Alternatively, compounds of formula lb, where m is 1, R1 is 3-Ng-Mtr- guanidinopropyl, and R° is
Figure imgf000126_0001
can also be prepared by reaction of compound XXXIII with the appropriate organometallic reagent. As described elsewhere herein, the furyl group can be further synthetically manipulated by an oxidation procedure to produce an alpha-keto moiety.
Example 25. Solid-phase synthesis of compounds of formula la
The following representative procedure is for compounds of formula la in which R2 is 2-butyl (i.e., the side chain of isoleucine), X is N, R3 is H and A is 3,4-dichlorobenzylleucine. This procedure can be readily adapted for the synthesis of related compounds of the invention. Commercially available Fmoc-(L)-Ue Wang resin (100-200 mesh, 200 mg, 0.6 mmol/g, 0.12 mmol) was placed in a peptide synthesis vessel. N,N-Dimethylformamide (DMF, 2.0 mL) and then piperidine (500 ul) were added. The reaction mixture was shaken at room temperature for 2 hours. The resin was then filtered and washed four times with dry DMF.
A solution of Fmoc-Leu-OH (250 mg, 0.72 mmol, 6.0 equiv), 1- hydroxybenzotriazole (HOBt, 100 mg, 0.74 mmol, 6 equiv), and diisopropylcarbodiimide (DIC, 115 ul, 0.72 mmol, 6 equiv) in THF (2 mL) or DMF or DMF/CH2C12 (2/1, v/v) was added to the resin. The reaction mixture was shaken at room temperature overnight, filtered, and washed sequentially with CH2C12, DMF, CH3OH, CH2C12, DMF, and CH2C12, and pumped to dryness under a high vacuum.
The resin was subjected to DMF (2 mL) and piperidine (500 ul) for 2 hours at room temperature, filtered, and washed four times with DMF to remove the N-terminal Fmoc-protecting group. In one example, the resin was treated with methylene chloride (2.25 mL) and 3,4-dichlorobenzylisocyanate (105 ul, 0.72 mmol, 6.0 equiv), and the reaction mixture was shaken overnight at room temperature to produce resin-supported 3,4- dichlorobenzylaminocarbonyl-Leu-Ue. As an alternative to the isocyanate approach, N-terminal ureas can be synthesized by reaction of the amine terminus of the resin with p-nitrophenylchloro formate (5-10 equiv) in the presence of diisopropylethylamine, followed by the addition of excess (5 equiv) of a primary or secondary amine. In one example, the resin supported Leu-He dipeptide was so treated with 4-Br-α-methyl benzylamine to provide resin-supported compound XXXVI.
Figure imgf000127_0001
The resin was filtered and washed sequentially with CH2C12, DMF, CH3OH, CH2C12, DMF, and CH2C12, and taken to dryness under a high vacuum. To the dry resin was added 50% trifluoroacetic acid/50% methylene chloride (3 mL total volume), and the reaction mixture was shaken at room temperature for 1.5 hours. The resin was filtered, washed with methylene chloride once, and the filtrate was concentrated in vacuo. Representative compounds of formula la, where X is N, R3 is H, A is an α-amino acid AA2, and R is an aminocarbonyl substituent on the α-nitrogen of AA2, are provided in Table 1 below.
Figure imgf000127_0002
Table 1. Compounds of formula la (X is N, R3 is H, A is an α-amino acid AA2, and R8 is an aminocarbonyl substituent on the α-nitrogen of AA2)
Figure imgf000128_0001
Figure imgf000129_0002
Figure imgf000129_0001
(b) The combined purity percentages of the two α-methyl diastereomeric isomers which were not separated for subsequent amide bond-formation .
Additional compounds of formula la can be prepared in which X is N, R is H, A is an α-amino acid AA2, and R is an aminocarbonyl substituent acyl, aroyl, heteroaroyl, or alkoxycarbonyl moiety on the α-nitrogen of AA . These compounds can be prepared by reaction of the N-terminal amine of the resin-supported amino acid as described previously when R8 is an aminocarbonyl substituent, or with the appropriate carbonyl chloride compounds, chloroformates, or by reacting the N-terminal amine with the appropriate carboxylic acid in a coupling step. Compounds prepared in this manner include, but are not limited to, compounds of formula la wherein X is N, R3 is H, AA2 is Leu, Phe, Val, Ala, cyclohexylglycine (Chg), Arg, Asn, 3- pyridylalanine (3-Pal), 4-pyridylalanine (4-Pal), Tφ, He, Lys, Glu, His, or Gin, and R8 is a substituent on the α-nitrogen of AA2 and can be
Figure imgf000130_0001
Compounds of formula la can be prepared in which A is an aminocarbonyl substituent (i.e., the step in which AA2 is coupled to the resin- supported amino acid is omitted and an N-terminal urea is formed directly to the alpha amine of the amino acid bearing the R side-chain by methods previously described herein). Compounds prepared in this manner include compounds of formula la in which X is N, R2 is isopropyl, R3 is H, and A is
Figure imgf000131_0001
Compounds of formula la can also be prepared in which A is an acyl, such as the substituents selected from the following group:
Figure imgf000131_0002
Compounds of formula la, where X is N, R is H, A is α-amino acid AA2, and R is a sulfonyl substituent on the α-nitrogen of AA2 are prepared in a similar fashion, except the penultimate step before removal of the peptide from the resin with TFA involves treatment of the N-terminal amine with a sulfonyl chloride in the presence of a base. In one example, resin-supported D- cyclohexylglycine-leucine (0.20 mmol) was treated with (methyl-3- benzoate)methanesulfonyl chloride (304 mg, 1.2 mmol) in 3 mL of 2,6- lutidine/CH2Cl2 (1 :9). The resulting slurry was agitated for 3 h at room temperature. The resin was filtered and washed with DMF/MeOH (4x), CH2C12 (2x), and then dried under vacuum overnight. After swelling in 3 mL of CH C12, the solvent was drained and the resin treated with 50 % TFA /DCM (5 mL) at room temperature for 2 hrs. The resin was filtered and washed with CH2C1 (2x). The collected cleavage product, was combined with washes and mixed with 0.3 mL of isopropanol. After concentration of solvents in vacuo,
0.89 g of a yellow residue was collected (LCMS: m/z = 483.1, M+l).
Representative compounds of formula la, where X is N, R3 is H, A is α-amino acid AA2, and R8 is a sulfonyl substituent on the α-nitrogen of AA , are provided in Table 2.
Table 2. Compounds of formula la (X is N, R3 is H, A is α-amino acid AA2, and R8 is a sulfonyl substituent on the α-nitrogen of AA2)
Figure imgf000132_0001
a) The purity was determined by HPLC analysis without purification Example 26. Exemplary coupling reactions of compounds of formula la to compounds of formula lb
The following procedure is reprensentative of the condensation of carboxylic acids of formula la with amine compounds of formula lb.
3,4-dichlorobenzylcarbamyl-Leu-Phe-OH (17 mg, 0.036mmol, 1.0 equiv) and HOBt (10.5 mg, 0.078 mmol, 1.1 equiv) were dissolved in 1.0 mL THF at room temperature. DIEA (37 ul, 0.213 mmol, 3 equiv) was added via syringe, and the solution was stirred until it was homogeneous. 1 -(3- Dimethylaminopropyl)-3-ethy]carbodiimide hydrochloride (EDCI, 14.5 mg, 0.075 mmol, 1.05 equiv) was then added in one portion, and the reaction was allowed to stir at room temperature. The Mtr-protected Arg monomer amine prepared in Example 24 (compound XXXV, 1.0-2.0 equiv.) was added and the reaction stirred for 3 hours. The reaction mixture was diluted with ethyl acetate (2 mL), washed with 5% aqueous acetic acid (1 mL), saturated aqueous
NaHCO3 (2 x 1 mL), water (1 mL), and brine (1 mL). The organic layer was dried over Na2SO4, filtered, and concentrated and the resulting residue (compound XXXVII) used in the following deprotection reaction (Example 27) without further purification.
Figure imgf000133_0001
(XXXVII) Example 27. Synthesis of exemplary compounds of the formula I (m = 1)
The following procedure is representative of the Mtr-deprotection of compounds produced by the procedure of Example 26. Compound XXXVII (10 mg, 0.01 lmmol) was dissolved in thioanisole (25 ul) and trifluoroacetic acid (475 ul). The reaction was shaken at room temperature in a sealed vial for 4 hours. The liquid was removed in vacuo overnight, and the residue was triturated with Et2O (2 X), then dissolved in 50% acetonitrile/50% water and the product purified by reverse phase preparative HPLC. Representative compounds of formula I, where X is N, R is H, R is 3-guanidmopropyl, R is
2-thiazole, and A is α-amino acid AA2, wherein R is a substituent on the α- nitrogen of AA2, are provided in Table 3a.
Table 3a. Compounds of formula I (X is N, R is H, R is 3-guanidinopropyl R R° iiss 22--tthhiiaazzoollee,, aanndd AA is α-amino acid AA2, wherein R is a substituent on the α-nitrogen of AA2)
Figure imgf000134_0001
Figure imgf000135_0001
Figure imgf000136_0001
Figure imgf000137_0001
Figure imgf000138_0001
Figure imgf000139_0001
(a) The purity percentage was determined by HPLC analysis using a CIS (3x30 mm, 3 μ) column with AcCN:H2O (0.1%TFA) as mobile phase.
(b) The purity of a single α-methyl isomer of unknown configuration.
(c) Prior to the Mtr deprotection step, the benzoate ester was saponified (NaOH, THF/water).
Additional compounds of the invention formed from the reaction of compounds of formula la with those of formula lb, followed by deprotection, include those of formula I where m is 1, R° is 2-thiazole, R1 is 3- guanidinopropyl, X is N, R2 is isopropyl, R3 is H, and A is selected from the following group:
Figure imgf000140_0001
Other compounds of the invention formed from the reaction of compounds of formula la with those of formula lb, followed by further functional group synthetic manipulation and/or deprotection include those of formula I, where X is N, R3 is H, m is 1, R1 is 3-guanidinopropyl, and A is α- amino acid AA2, wherein R8 is a substituent on the α-nitrogen of AA2, where R8 AA2, R2, and R° are as shown in Table 3b. Table 3b. Compounds of formula I (X is N, R3 is H, is 1, R1 is 3- guanidinopropyl, and A is α-amino acid AA2, R is a substituent on the α- nitrogen of AA2)
Figure imgf000141_0001
Figure imgf000142_0001
Example 28. Synthesis of exemplary compounds of the formula I (m = 0)
The compounds of formula I where m is 0 can also be prepared by either using solid-phase approaches or solution phase approaches. In one example, resin supported amine XXXVIII, is prepared by treating α-bromo-jσ-tolunitrile with NaN3 and Bu4NCl to form the corresponding α -azido-jP-tolunitrile, followed by conversion of the nitrile group to the amidine group employing typical Pinner and aminolysis conditions, as described herein. The p- azidomethyl benzamidine thus produced is attached to an activated p- nitrophenylcarbonate Wang resin, followed by reduction of the azide group to an amine by SnCl2 to provide compound XXXVIII.
Figure imgf000143_0001
(XXXVIII)
Reaction of this amine with a compound of formula la under amide bond forming conditions, as described herein, produces compounds of formula I, where m is 0, R1 is H,
Figure imgf000143_0002
Alternatively, compounds of formula I where m is 0 and R1 is H can be prepared in solution by condensing an amine, such as 4-cyanobenzylamine, to compounds of the formula la, followed by conversion of the cyano group to an amidine moiety, as described herein. Representative compounds of formula I, where m is 0, R1 is H, X is N, R3 is H, A is α-amino acid AA2, and R8 is a substituent on the α-nitrogen of AA , are given in Table 5.
Table 5. Compounds of the formula I (m is 0, R1 is H, X is N, R3 is H, A is α- amino acid AA2, and R8 is a substituent on the α-nitrogen of AA2)
Figure imgf000143_0003
Figure imgf000144_0002
Figure imgf000144_0001
Example 29. Synthesis of exemplary compounds of formula I (m is 1, A is acyl)
The following procedure is representative of the preparation of compounds of formula I where m is 1, X is N, R3 is H, and A is acyl. Valine methyl ester (HCl salt) (176 mg, 1.05 mmol) was dissolved in 6 mL of methylene chloride at room temperature. (R)-rn-chloro mandelic acid (187 mg, 1.00 mmol), 261 ul of diisopropylethylamine (1.50 mmol), 162 mg of hydroxybenzotriazole hydrate (1.20 mmol), and 231 mg of the amide bond- forming reagent, EDC (1.20 mmol) were added sequentially. The mixture was maintained at room temperature for 6 hours and diluted with water (10 mL) and ethyl acetate (10 mL). The water layer was extracted with 2x10 mL portions of ethyl acetate. The combined organic layers were washed with 0.1N HCl and brine. Concentration of the organic layer in vacuo and flash chromatographic purification of the residue on silica gel using a gradient (1 :2 ethyl acetate/hexane, up to 100% ethyl acetate) gave compound XXXIX (255 mg, 85% yld., MS: m/z 300, M+l).
Figure imgf000145_0001
A portion of this ester (121 mg, 0.42 mmol) was dissolved in 3 mL of a 1 :1 :1 mixture of tetrahydrofuran methanol/water. Lithium hydroxide (101 mg, 4.2 mmol) was added. After 2 hours at room temperature, 10 mL of 0.5N HCl was added dropwise, followed by 10 mL of ethyl acetate. The aqueous layer was separated and washed with 2x10 mL portions of ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated in vacuo to give the pure crude carboxylic acid which was used without further purification (98 mg, 95% yld., MS: m/z 286, M+l).
This carboxylic acid (a compound of formula la) was converted to an arginine-ketothiazole (a compound of formula I) by amide bond-forming to compound XXXV and subsequent Mtr-deprotection as previously described herein (product MS: m/z 509, M+l). Representative compounds of formula I, where m is 1, X is N, R3 is H, R2 is isopropyl, R1 is 3-guanidinopropyl, R° is 2- thiazole, A is R8 (R8-AA2, where AA2 is a covalent bond and R8 is acyl) are given in Table 6.
Table 6. Compounds of formula I (X is N, R3 is H, R2 is isopropyl, R1 is 3- guanidinopropyl, R° is 2-thiazole, and A is R8)
Figure imgf000145_0002
Figure imgf000146_0001
(a) The purity percentage was determined by HPLC analysis using a C18 (3x30 mm, 3 μ) column with AcCN:H2O (0.1%TFA) as mobile phase.
(b) Assigned as hydrate of desired structure.
Example 30. Synthesis of exemplary compounds of the formula I where R3 together with A forms a ring having the formula XL
Figure imgf000147_0001
The following procedure is representative of the preparation of compounds of formula I that conform to formula XL, wherein R1 is 3- guandinopropyl, R° is 2-thiazole, m is 1, X is N, W is N, R17 is aryl, R2 is H, Y1 is NH and B is an optionally substituted alkyl, alkenyl, cycloalkylaralkyl, aralkenyl, aryl, C1- heterocyclyl, heteroaralkyl, acyl, aroyl, or heteroaroyl.
2-Thiouracil (33.3 g, 0.26 mol) was dissolved in an aqueous solution of sodium hydroxide (20.8 g in 183 mL of water; ~ 0.52 mol). The mixture was placed in an ice bath until the internal reaction temperature was 0°C. Methyl iodide (18.5 mL, 0.29 mol) was slowly added. The resulting reaction mixture was allowed to return to room temperature and then stirred for 16 hours. The pale yellow solution was cooled to 0°C and acidified with glacial acetic acid. The white precipitate that formed was collected by vacuum filtration, washed with cold water (3 x 150 mL), and dried to afford 2-methylsulfanyl-3H- pyrimidin-4-one as a white powder. (37.4 g, 98 %).
A solution of 2-methylsulfanyl-3H-pyrimidin-4-one (36 g, 0.25 mol) in glacial acetic acid (500 mL) was cooled to 0°C in an ice bath and treated with Br2 (15.6 mL, 0.3 mol). The reaction mixture changed in color from yellow to brown within 0.5 hours, and was allowed to warm to room temperature where it stirred overnight. When all the starting material had been consumed (as indicated by TLC and HPLC UV detection at λ=254 nm), the reaction mixture was slowly poured into a mixture of ice/H2O (2 liters). The resulting white precipitate was collected by vacuum filtration and washed with H2O (200 mL) and Et2O (2 x 250 mL). The off-white solid was then suspended in anhydrous toluene (250 mL) and concentrated to dryness under reduced pressure in a -60°C H2O bath. 5-Bromo-2-methylsulfanyl-3H-pyrimidin-4-one was obtained as a white solid (30.4 g, 55% yield).
A suspension of calcium hydride (2.3 g, 0.045 mol) in anhydrous THF (200 mL) was cooled to an internal temperature of 0°C under an argon atmosphere, and 5-bromo-2-methylsulfanyl-3H-pyrimidin-4-one (10 g, .045 mol) was slowly added. The internal reaction temperature was maintained at 0°C. After 30 minutes t-butyl bromoacetate (8 mL, 0.054 mol) was added via syringe and stirred at 0°C for 1.5 hours, then the reaction mixture was allowed to return to room temperature and stir for an additional 2 hours. The reaction mixture was then heated at reflux for 2 hours, cooled to room temperature, and slowly poured in small portions into ice water (600 mL). The mixture was vacuum-filtered, and the filtrate extracted with methylene chloride (200 mL). The aqueous layer was separated and extracted with methylene chloride (2 x 150 mL). The combined methylene chloride layers were then extracted with H2O (3 x 200 mL), dried over MgSO , and concentrated to give an off white solid (9 g). The solid was triturated in 10% CH2C12/ hexanes (500 mL) and filtered. The filtrate was collected and saved. The trituration was repeated with the collected solid, and the combined filtrate after concentration gave compound XLI, where B-Y1 is Br and R17 is SCH3, as a white solid (6.7 g, 45%, MS: m/z 336.2, M+l, calculated is 335.2).
Figure imgf000148_0001
(5-Bromo-2-methylsulfanyl-6-oxo-6H-pyrimidin- 1 -yl)acetic acid, tert- butyl ester (250 mg, 0.75 mmol), cesium carbonate (269 mg, 0.83 mmol), and 2-(di-t-butylρhosphino)biphenyl (23 mg, 0.08 mmol) or 2-
(dicylcohexylρhosphino)biρhenyl (28 mg, 0.08 mmol), tris(dibenzylideneacetone)dipalladium (34 mg, 0.04 mmol) or dihydrogen dichlorobis(di-t-butylphosphinito)palladate (40 g, 0.08 mmol) were combined in a high pressure flask under an argon atmosphere, and 10% DMF/toluene (5 mL) or dioxane (5 mL) was added. 4-Methoxybenzylamine (1.5 mmol, 2 eq.) was added, and the reaction stirred magnetically in a preheated 100°C oil bath for 4-16 hours. The heterogenous mixture was then cooled, filtered, washed with EtOAc and the filtrate concentrated. The crude material was purified by silica gel column chromatography using a 10-20% EtOAc/hexanes to -60-70% EtOAc/hexanes gradient as eluent. In several cases, 100% EtOAc was required to elute the product. Representative compounds of formula XLI, where W is N and R17 is -SMe, are provided in Table 7.
Table 7. Compounds of formula XLI (W is N, R 1'7 ' is -SMe)
Figure imgf000149_0001
(a) Purity estimated by HPLC analysis, 5 minutes gradient elution with 10% acetonitrile to 90% acetonitrile/water, 0.1% TFA, 1 mL/minute, Phenomenex Luna CI 8 column (30 x 3 mm, 4 micron).
The compound of formula XLI where B-Y1 is benzyl (1 equiv.) was placed in a vial with a Teflon screw cap (8 mL), and 3-methylphenyl boronic acid (1.2 equiv.), Cu(I)-2-thiophenecarboxylate (2 equiv.), Pd(PPh3)4 (2.7 mole %), and THF were added under a head of argon. The reaction was heated at 70 0 C with orbital shaking overnight, cooled to room temperature, and concentrated, followed by suspension in methylene chloride, filtration through Celite, and purification by silica gel flash chromatography. Compounds of formula XL where Y1 is NH and B is a substituted carbonyl or sulfonyl derivative can be prepared by methods analogous to those of Veale, vide supra, or South, vide supra, via Curtius rearrangement of a carboxylic acid to generate pyrimidinones with an amine at this position after hydrolysis of the amide. This amine can be acylated, carbamylated, or sulfonylated as previously described here to provide compounds of formula XL where Y1 is NH and B is alkoxycarbonyl, cycloalkoxycarbonyl, aralkoxycarbonyl, aryloxycarbonyl, heterocyclyloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, heteroaralkylaminocarbonyl, alkylthiocarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl, alkoxythiocarbonyl, cycloalkoxythiocarbonyl, aralkoxythiocarbonyl, aryloxythiocarbonyl, aminothiocarbonyl, alkylaminothiocarbonyl, dialkylaminothiocarbonyl, cycloalkylaminothiocarbonyl, aralkylaminothiocarbonyl, arylaminothiocarbonyl, heterocyclylaminothiocarbonyl, or hetero aralkyl aminothiocarbonyl .
(5-Benzylamino-2-(3-methylphenyl)-6-oxo-6H-pyrimidin-l-yl)acetic acid, tert-butyl ester was suspended in 1 :1 TFA DCM for 1 hour to remove the t-butyl ester. The reaction was then concentrated, and the resulting carboxylic acid (1.0 equiv), compound XXXV (2.0 equiv.), and HOBt (1.7 equiv.) were dissolved in THF (1 mL) at room temperature. DIEA (3 equiv.) was added via syringe, and the solution was stirred until homogeneity was obtained. l-(3- Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 1.9 equiv.) was added in one portion, and the reaction was stirred at room temperature overnight. The reaction was diluted with ethyl acetate (2 mL), washed with 5% aqueous acetic acid (l x l mL), saturated aqueous NaHCO3 (2 x 1 mL), water (l x l mL), and brine (l x l mL). The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. Removal of the Mtr protecting group was affected with TFA (w/5% thioanisole), as previously described herein, to provide compounds of the formula XL. Representative compounds of formula XL, where R is H, R is 3-guanidinopropyl, and R is 2-thiazole are provided in Table 8.
Table 8. Compounds of formula XL (R2 is H, R1 is 3-guanidinopropyl, and R°
Figure imgf000151_0002
(a) Purity estimated by HPLC analysis, 5 minute gradient elution with 10% acetonitrile to 90% acetonitrile/water, 0.1% TFA, 1 mL/minute, Phenomenex Luna CI 8 column (30 x 3 mm, 4 micron).
Example 31. Exemplary synthesis of compounds of formula II.
Figure imgf000151_0001
I) 8-Substituted 2-naphthamidines The following representative procedure is for a compound of formula II, where f is a bond; each of g and e is C-H; each of V, R19, and R20 is H; Z is NH, T is NH, and B is an optionally substituted natural or unnatural alpha- amino acid residue of D- or L-configuration, or an optionally substituted acyl, aroyl, aralkanoyl, heteroaralkanoyl, or heteroaroyl. 8-Amino-2-naphthol (5.0 g, 31.4 mmol) was dissolved in 1 ,4-dioxane
(80 mL) and triethylamine (6.0 mL). A solution of N- phenyltrifluoromethanesulfonimide (11.2 g, 31.4 mmol) in dioxane (60 mL) was added slowly via an addition funnel, and the brown solution was stirred for 4 hours. The reaction was then concentrated in vacuo and triturated two times with hexanes (2 x 75 mL). The resulting crude naphthyl triflate {9.0 g, 30.9 mmol) was dissolved in pyridine (100 mL) and, while stirring, dimethylaminopyridine (DMAP, 250 mg, 2.0 mmol) was added in one portion. The solution was cooled to 0°C, and benzyl chloroformate (18 mL, 123 mmol) was added slowly via syringe. The reaction mixture was then allowed to warm to room temperature, stirred overnight, then quenched with H2O (100 mL) and extracted with ethyl acetate (3 x 250 mL). The combined organic fractions were washed sequentially with saturated NaHCO3 (200 mL), water (100 mL), and brine (100 mL), and then dried with MgSO4. After filtration and concentration, the crude residue was purified by flash column chromatography with 15% ethyl acetate/85% hexane as eluent, which yielded compound XLH as a brown solid.
Figure imgf000152_0001
A Schlenk tube was flame dried under high vacuum, backfilled with Argon, and charged with Cul (110 mg), NaCN (575 mg), and Pd(PPh3)4 (335mg). Compound XLII (2.5g) was dissolved in dry acetonitrile (15 mL) and added to the Schlenk flask via syringe. The solution was stirred vigorously at 80°C for 1.5 hours. The reaction mixture was then cooled to room temperature, diluted with ethyl acetate, filtered through Celite, and the resulting filtrate was washed with water and brine. After drying with MgSO , the organic layer was concentrated to provide 1.42 g of 8-benzyloxycarbonylamino napththalene-2-carbonitrile which was subsequently utilized without further purification. The Cbz group was removed by placing the Cbz-protected compound in a flask under argon. A 30% (w/w) solution of HBr/AcOH (5 mL) was added via syringe, and the solution was stirred at room temperature for 2 hours. Ether
(40 mL) was added, and the solution was stirred for 10 minutes. Filtration and collection of the precipitate provided 8-aminonaρththalene-2-carbonitrile
(compound XLIII) as an orange powder.
Figure imgf000153_0001
Compound XLIII was dissolved in CH2CI2 and triethylamine (3 equiv) and DMAP (0.05 equiv) was added. The respective acid chloride (1.5 equiv), was added in one portion and stirred overnight. The reaction mixture was quenched with water, extracted with CH2C1 , and dried with MgSO4. Filtration and concentration in vacuo provides the 8-amidonapththalene-2-carbonitrile intermediate.
A solution of 6N HCl/MeOH was generated by cooling a solution of anhydrous MeOH (9 mL) to 0°C. Acetyl chloride (6 mL) was added via syringe. The ice bath was removed, and the reaction was stirred for 30 minutes. To a cyanonaphthamide derivative (20-30 mg) in a 4 mL vial was placed 2 mL of the methanolic HCl solution. The vial was sealed and shaken for 3 hours. Concentration in vacuo provided a methyl imidate ester intermediate to which 2 mL of a 7N NH3 MeOH solution was added. The vial was sealed and heated to 50°C with shaking overnight. Concentration in vacuo provided a naphthamidine, which can be purified by standard chromatographic techniques.
The amidine (1.5-2 equiv) was dissolved in a 1:1 CH2C12/DMF (1 mL total volume) solution, which was then added to a vessel containing p- nitrophenyl carbonate Wang resin (1 equiv). DIEA was added (50 ul) and the mixture shaken overnight. The resin was filtered and washed sequentially with CH2C12, DMF, MeOH, CH2C12, DMF, and again with CH2C12. Added to the resin was 1 :1 CH2Cl2/MeOH (1 mL total volume) with 5% DIEA, and the mixture was shaken for an additional 1 hour. The washing protocol was repeated. The amidine was then cleaved from the resin with 1 :1 TFA/CH2C12 (1.5 mL total volume) for 1 hour, and then the reaction mixture was filtered and concentrated in vacuo. Compounds of formula II, where f is a bond; each of g and e is C-H; each of V, R19, and R20 is H; Z is NH, and T is NH, are provided in Table 9.
Table 9. Compounds of formula II (f is a bond; g and e are C-H; V, R19, and R20 is H; Z is NH, and T is NH)
Figure imgf000154_0001
(a) Purity estimated by HPLC analysis, 5 minutes gradient elution with 10% acetonitrile to 90% acetonitrile/water, 0.1% TFA, 1 mL/minute, Phenomenex Luna CI 8 column (30 x 3 mm, 4 micron).
II) 6-Oxy-substituted naρhthalene-2-carboxamidines
The following representative procedure is for a compound of formula II, where T is a bond; each of g and e is C-H; each of B, R19, and R20 is H; Z is NH, f is O, and V is an optionally substituted alkyl, aralkyl, or heteroaralkyl
To 6-hydroxynaphthalene-2-carbonitrile (80 mg, 0.473 mmol) in acetone (2.0 mL) in the presence of potassium carbonate (196 mg) is added 2- bromo-1-phenyl-ethanone (95 mg, 1.2 equiv.), and the reaction mixture is stirred at room temperature overnight. The solvent is removed in vacuo and the resulting mixture is purified by preparative thick layer chromatography (EtOAc Hexane, 1 :4) to give compound XLIV. MS: m/z 288, M+l, calculated mass: 287.3.
Figure imgf000155_0001
The product (30 mg, 0.104 mmol) is dissolved in an HCl/methanol solution (6 N) at room temperature and stirred for 1 hour. The solvent is removed in vacuo and the resulting crude mixture is immediately submitted to an ammonia/methanol solution (3.5 N) and stirred for 2 hours. The crude reaction mixture is purified with reverse-phase HPLC to give compound XLV (MS: m/z 305, M+l, calculated mass: 304.3, 85% purityb). The identity (m/z) of a representative sample of similarly prepared compounds is provided in Table 10 (compounds 146-148). Compounds in which f is O, and V is an optionally substituted acyl, aroyl, heterocyclylcarbonyl, alkoxycarbonyl, cycloalkoxycarbonyl, aralkoxycarbonyl, aryloxycarbonyl, heteroaralkyloxycarbonyl, heteroaryloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, or heteroaralkylaminocarbonyl can be prepared by from 6-hydroxynaphthalene-2- carbonitrile, with subsequent unmasking of the amidine groups as described herein. Alternatively, the amidine can be unmasked prior to C6-O functionalization, followed by protection with a suitable protecting group such as Boc; subsequent O-acylation, carbonylation, or carbamoylation provides, after amidine deprotection, compounds of the formula II.
Ill) 6-Amido-substituted naphthalene-2-carboxamidines The following representative procedure is for a compound of formula II, where T is a bond; each of g and e is C-H; each of B, R19, and R20 is H; Z is NH, f is a covalent bond, and V is an optionally substituted aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, or heteroaralkylaminocarbonyl.
A solution of 6-cyano-naphthalene-2-carbonyl chloride (1 mmol), prepared as described in U.S. Patent No. 6,284,796, in THF (2 mL) in the presence of K CO3 (3 equiv.) and benzyl amine (3 equiv.) was stirred overnight. The crude mixture is purified by preparative thick layer chromatography (Hexane/EtOAc 1 : 1 ) to give 6-cyano-2- benzylamidonaphthalene (m/z 287, M+l, calculated mass: 286.3).
Conversion of the cyano group to an amidine group is achieved as previously described herein to give 6-amidino-2-benzylamidonaphthalene. The crude reaction mixture is purified with reverse-phase HPLCa to give the amidine product (MS: m/z 304.3, M+l, calculated mass: 303.4).
Representative compounds of formula II, where T is a bond; each of g and e is C-H; each of B, R19, and R20 is H;and Z is NH, can be prepared in a similar manner and are provided in Table 10.
Table 10. Compounds of formula II (T is a bond; g and e are C-H; B, R .19 , and R20 is H; and Z is NH)
Figure imgf000157_0001
(a) Acetonitrile/water, 0.1% TFA, 12 mL/minute, 45 minute gradient elution (various methods), VyDac C18 column (250 x 15 mm, 5 micron). (b) Purity estimated by HPLC analysis, 5 minute gradient elution with 0%10% acetonitrile to 90-100% acetonitrile/water, 0.1% TFA, 1 mL/minute, Phenomenex Luna C18 column (30 x 3 mm, 4 micron).
IV) 4,6-Disubstituted-2-carboxamidines The following representative procedure is for a compound of formula II, where T is NH; B is an optionally substituted natural or unnatural alpha-amino acid residue of D- or L-configuration, or an optionally substituted acyl, aroyl, araalkanoyl, heteroaraalkanoyl, or heteroaroyl; each of g and e is C-H; each of R19 and R20 is H; Z is NH, f is a covalent bond, and V is an optionally substituted aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, or heteroaralkylaminocarbonyl.
Nitration of 6-cyano-naphthalene-2-carboxylic acid methyl ester, prepared as described in U.S. Patent No. 6,284,796, is carried out with KNO3 in sulfuric acid at 0°C for 30 min. The reaction mixture is poured into ice and filtered, and the compound is washed with water and dried. The product, 6- cyano-4-nitro-naphthalene-2-carboxylic acid methyl ester is obtained and used without further purification.
The methyl ester is treated with LiOH (4 equiv.) in THF/H2O for 30 min, acidified with oxalic acid, and extracted with EtOAc. After removal of the solvent, the product is obtained as a solid, which is added into SOCl2 and stirred at 70°C for 1 hour. Removal of excess thionyl chloride gives the desired acid chloride, 6-cyano-4-nitro-naphthalene-2-carbonyl chloride.
A mixture of the acid chloride, THF, K2CO3 (3 equiv.) and a primary or secondary amine (3 equiv.) is stirred at room temperature for 2 hours. The reaction mixture is loaded onto a preparative thick layer chromatography plate and developed with hexane/ethyl acetate (2: 1) to give a compound of formula XL VI, where f is a covalent bond and V is an optionally substituted aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, or heteroaralkylaminocarbonyl.
Figure imgf000158_0001
In a representative example, a compound of formula XL VI, where f is a bond and V is benzylaminocarbonyl (50,mg, 0.151 mmol), is dissolved in methanol (3 mL) and hydrogenated in the presence of 10% Pd/C (5 mg) for 6 hours. The resulting amine is obtained after removal of the catalyst and solvent (MS: m/z 302, M+ 1; calculated mass: 301.3).
To a solution of the amine (15 mg, 0.031 mmol) in THF (2 mL) in the presence of K2CO3 (3 equiv.) is added 2-methyl-3-acetyloxybenzoic chloride (2 equiv.) and the reaction is stirred overnight. Preparative thick layer chromatography of the reaction mixture with hexane/ethyl acetate (1 :1) gives a compound of formula XL VII, where f is a bond, V is benzylaminocarbonyl, T is NH and B is 2-methyl-3-acetyloxyphenylcarbonyl {MS: m/z 478, M+l; calculated mass: 477.5).
Figure imgf000159_0001
(XLVII)
Amides of formula XLVII are subjected to similar reaction conditions previously described herein for the conversion of a cyano group to an amidine group. For example, the compound of formula XLVII, where f is a bond, V is benzylaminocarbonyl, T is NH and B is 2-methyl-3-acetyloxyphenylcarbonyl (20 mg) is treated with an HCl/methanol solution (7 N, 2 mL) at room temperature for 2 hours. A solid product is obtained after removal of the solvent in vacuo, which is treated with a solution of ammonia in methanol (3.5 N, 3 mL) at 50 °C for 1.5 hours. Preparative reverse phase HPLCa of the crude reaction mixture affords the desired amidine product of formula XL VIII, where f is a covalent bond and V is benzylaminocarbonyl, as a trifluoroacetic acid salt (87% purityb; MS: m/z 453.2, M+l; calculated mass: 452.5). Representative compounds of formula XL VIII are shown in Table 11.
Figure imgf000160_0001
(XLVIII)
Table 11. Identity and purity of compounds of the formula XLVIII
Figure imgf000160_0002
Figure imgf000161_0001
Figure imgf000162_0001
Figure imgf000163_0001
(a) Acetonitrile/water, 0.1% TFA, 12 mL/minute, 45 minute gradient elution (various methods), VyDac C18 column (250 x 15 mm, 5 micron).
(b) Purity estimated by HPLC analysis, 5 minute gradient elution with 0%-10% acetonitrile to 90-100% acetonitrile/water, 0.1% TFA, 1 mL/minute, Phenomenex Luna C18 column (30 x 3 mm, 4 micron)..
V) Quinolines
The following representative procedures are for compounds of formula II, where T is NH; B is an optionally substituted natural or unnatural alpha- amino acid residue of D- or L-configuration, or an optionally substituted acyl, aroyl, aralkanoyl, heteroaralkanoyl, or heteroaroyl; g is C-H; e is N; each of R 19 and R20 is H; Z is NH, f is a covalent bond, and V is an optionally substituted alkoxycarbonyl, cycloalkoxycarbonyl, aralkoxycarbonyl, aryloxycarbonyl, heterocyclyloxycarbonyl, heteroaralkyloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, or heteroaralkylaminocarbonyl.
4-Amino-benzonitrile was converted to 6-cyano-4-hydroxy-quinoline-2 carboxylic acid methyl ester by Michael addition to dimethylacetylene dicarboxylate followed by thermal cyclization (see Jaen et al, J. Med. Chem.
38:4439-4445, 1995). The resulting 4-hydroxy quinoline was converted to 4- amino-6-cyano-quinoline-2-carboxylic acid methyl ester by bromination with n-butylammonium bromide and phosphorus pentoxide (see Kato et al,
Tetrahedron Lett. 42:4849-4852, 2001). The 4-bromo compound is treated with with sodium azide in DMSO (see Sashida et al., Chem. Pharm. Bull. 38:2919-2925, 1990), which is subsequently reduced with sodium borohydride (see Outt et al, J. Org. Chem. 63:5762-5768, 1998). Acylation of the amine and subsequent conversion of the cyano group to an amidine group, as previously described herein, affords compounds of formula II, where T is NH; B is an optionally substituted natural or unnatural alpha-amino acid residue of D- or L-configuration, or an optionally substituted acyl, aroyl, aralkanoyl, heteroaralkanoyl, or heteroaroyl; g is C-H; e is N; each of R19 and R20 is H; Z is NH, f is a covalent bond, and V is methoxycarbonyl; alternatively, the methyl ester of the 4-bromo or 4-azido intermediate can be saponified to the carboxylic, then coupled with various alcohols under ester bond-forming conditions known to those skilled in the art, followed by conversion of the 4- bromo or -azido functional group to the 4-amino group with subsequent functionalization as previously described, to afford compounds of formula II, where T is NH; B is an optionally substituted natural or unnatural alpha-amino acid residue of D- or L-configuration, or an optionally substituted acyl, aroyl, aralkanoyl, heteroaralkanoyl, or heteroaroyl; g is C-H; e is N; each of R19 and R20 is H; Z is NH, f is a covalent bond, and V is an optionally substituted alkoxycarbonyl, cycloalkoxycarbonyl, aralkoxycarbonyl, aryloxycarbonyl, or heterocyclyloxycarbonyl, or heteroaralkyloxycarbonyl (e.g., V is methoxycarbonyl, B is 3-hydroxy-2-methylphenylcarbonyl; MS: m/z 378.2, M+l, calculated mass: 377.1; HPLC purity = 92%). Alternatively, prior to the conversion of the cyano group to an amidine, the 2-carboxylic acid methyl ester substituent can be directly converted to an amide by treatment with an amine in the presence of Me3Al (see Akakura and Yamamoto, Syn. Lett. 3:277- 278, 1997). Subsequent conversion of the cyano group to an amidine using the Pinner protocol also provides compounds of the formula II, where T is NH; B is an optionally substituted natural or unnatural alpha-amino acid residue of D- or L-configuration, or an optionally substituted acyl, aroyl, aralkanoyl, heteroaralkanoyl, or heteroaroyl; g is C-H; e is N; each of R19 and R20 is H; Z is NH, f is a covalent bond, and V is an optionally substituted aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cycloalkylaminocarbonyl, aralkylaminocarbonyl, arylaminocarbonyl, heterocyclylaminocarbonyl, or heteroaralkylaminocarbonyl (e.g., V is benzylaminocarbonyl, B is 3-hydroxy- 2-methylphenylcarbonyl; MS: m/z 454.2, M+l, calculated mass: 453.2; HPLC purity = 74%).
VI) Quinazolines
The following representative procedure is for a compound of formula II, where T is NH; B is an optionally substituted natural or unnatural alpha-amino acid residue of D- or L-configuration, or an optionally substituted alkyl, alkenyl, aralkyl, aralkenyl, heteroaralkyl, heteroaralkenyl, acyl, aroyl, aralkanoyl, heteroaralkanoyl, or heteroaroyl; each of e and g is N; each of R19 and R20 is H; Z is NH, f is a covalent bond, and V is an optionally substituted C1-6 alkyl, cycloalkyl, alkenyl, aralkyl, aralkenyl, heteroaralkyl, heteroaralkenyl, aryl, or heterocyclyl.
2-Aminobenzonitrile (2.0 g, 16.9 mmol, 1 equiv.) was dissolved in dry N,N-dimethylformamide (30 mL), and the solution was cooled to 0 °C. A solution of N-iodosuccinimide, NIS (3.81g, 16.9 mmol, 1 equiv.) in 30 mL of DMF was added dropwise with an addition funnel. After the NIS/DMF solution was added, the ice bath was removed, and the reaction was stirred at room temperature overnight. The reaction was quenched by pouring into 3N NaOH (300 mL) and diluted with an additional 200 mL of water. 2-Amino-5- iodobenzonitrile formed as a precipitate, which was collected by filtration. 2-Amino-5-iodobenzonitrile (500 mg, 2.05 mmol, 1 equiv.), DMAP (50 mg, 0.41mmol, 0.2 equiv.), and triethylamine (285 ul, 2.05 mmol, 1 equiv.) were dissolved in methylene chloride (10 mL). O-Acetylsalicyloyl chloride (2.0 g, 10.07 mmol, 4.9 equiv) was added in one portion, and the reaction was stirred at room temperature for 1 hour. The reaction mixture was diluted with CH2C12, washed with water, dried with MgSO , filtered, and concentrated. The resulting residue was purified by flash column chromatography (20% EtO Ac/80% hexane) to provide compound XLLX (260 mg, 31% yld.), where f is a covalent bond and V is 2-acetoxyphenyl.
Compound XLIX (20 mg, 0.050 mmol, 1 equiv.) was dissolved in THF (500 ul). 4-Methoxybenzylamine (14 ul, 0.108 mmol, 2.2 equiv.) was added, and the reaction vial sealed and heated to 65° C overnight with shaking. Concentration in vacuo provided the compound of formula L, where f is a covalent bond, V is 2-hydroxyphenyl, T is NH and B is 4-methoxybenzyl (24 mg)-
Compounds of formula L where T is NH and B is a substituted carbonyl or sulfonyl derivative can be prepared by conversion of the iodo-quinazoline to the cyano-quinazoline, removal of the 4-methoxybenzyl group, and subsequent acylation or sulfonylation of the resulting free amine; conversion of the nitrile to the amidine is then effected as previously described herein. Alternatively, it can be advantageous in some cases to unmask and protect the amidine prior to effecting acylation or sulfonylation.
Figure imgf000166_0001
The compound for formula L, where where f is a covalent bond, V is 2- hydroxyphenyl, T is NH and B is 4-methyoxybenzyl (24 mg, 0.050 mmol, 1 equiv.) was dissolved in NMP (1 mL) and CuCN (9 mg, 0.10 mmol, 2 equiv.) was added. The vial was sealed and heated to 130 °C, while shaking overnight. The reaction mixture was then poured into water (10 mL) and extracted with ethyl acetate. The organic layer was washed with brine, dried over MgSO , filtered, and then concentrated to afford the compound in which the iodo group was displaced with a cyano group to form the corresponding cyanoquinazoline. This compound was treated with 4N HCl/dioxane in MeOH to provide the intermediate methyl imidate ester, followed by aminolysis with ammonia in MeOH to provide, after concentration in vacuo, the quinazoline amidine. Alternatively, the cyanoquinazoline can be allowed to react with hydroxylamine, followed by acylation of the N-hydroxyamidine with, for example, acetic anhydride. Reduction under hydrogenolysis conditions (5% Pd/C at 50 psi under a H2 atmosphere for 2 hours) resulted in the compound of formula II, where T is NH; B is 4-methoxybenzyl; each of e and g is N; each of R19 and R20 is H; Z is NH; f is a covalent bond; and V is 2-hydroxyphenyl (MS: m/z 400.2, M+l, calculated mass: 399.2).
Example 32. Pharmaceutical Compositions Salts
Compounds of the invention may be prepared as salts. If a compound of this invention is substituted with a basic moiety, acid addition salts may be formed. The acids which can be used to prepare the acid addition salts desirably include those which produce, when combined with the free base, pharmaceutically acceptable salts, that is, salts whose anions are non-toxic to the patient in pharmaceutical doses of the salts so that the beneficial effects inherent in the free base are not modulated by side effects ascribable to the anions. Exemplary pharmaceutically acceptable salts are those derived from the following acids: acetic acid, benzenesulfonic acid, citric acid, cyclohexylsulfamic acid, ethanesulfonic acid, fumaric acid, gentisic acid, hydrochloric acid, hydrobromic acid, lactic acid, malonic acid, methanesulfonic acid, quinic acid, phosphoric acid, sulfamic acid, sulfuric acid, p- toluenesulfonic acid, and trifluoroacetic acid. Acid addition salts of the compounds of the invention are prepared by reaction of the free base with the appropriate acid by the application or adaption of methods known to those skilled in the art.
If a compound of the invention is substituted by an acidic moiety, base addition salts may be formed. The bases which can be used to prepare the base addition salts desirably include those which produce, when combined with the free acid, pharmaceutically acceptable salts, that is, salts whose effects inherent in the free acid are not modulated by side effects ascribable to the cations. Pharmaceutically acceptable salts (e.g., alkali and alkaline earth metal salts) are those derived from the following bases: aluminum hydroxide, ammonia, arginine, N-benzylphenethylamine, calcium hydroxide, chloroprocaine, choline, N, N'-dibenzylethylenediamine, diethanloamine, diethylamine, tris(hydroxymethyl)aminomethane, lithium hydroxide, lysine, magnesium hydroxide, N-methylglucamine, ornithine, piperazine, potassium hydroxide, procaine, sodium hydride, sodium hydroxide, tetramethylammonium hydroxide, triethylammonia, trimethylammonia, zinc hydroxide, and the like.
Metal salts of compounds of the invention may be obtained by contacting a hydride or hydroxide of the chosen metal in an aqueous or organic solvent with the free acid form of the compound. Amine salts of compounds of the invention may be obtained by contacting an amine in an aqueous or organic solvent with the free acid form of the compounds.
A number of methods are useful for the preparation of the salts described above and are known to those skilled in the art. For example, reaction of the free acid or free base form of a compound of the invention with one or more molar equivalents of the desired acid or base in a solvent or solvent mixture in which the salt is insoluble, or in a solvent like water after which the solvent is removed by evaporation, distillation, or freeze drying. Alternatively, the free acid or base form of the product may be passed over an ion exchange resin to form the desired salt, or one salt form of the product may be converted to another using the same general process.
Isomers Compounds of this invention may exhibit stereoisomerism by virtue of the presence of one or more asymmetric or chiral centers in the compounds. The present invention includes the various stereoisomers and mixtures thereof. Desired isomers may be obtained by chiral synthesis or resolution of enantiomeric mixtures or chromatographic separation of enantiomers on chiral chromatography columns. Diastereomers can be resolved using, e.g., standard chromatography columns or crystallization. Compounds of this invention may also exhibit geometrical isomerism. Geometrical isomers include the cis and trans form of compounds of the invention having, for example, alkenyl moieties. Compounds of this invention may also exhibit rotameric or tautomeric isomerism. The present invention comprises the individual stereoisomers, geometrical isomers, rotameric and tautomeric isomers, and mixtures thereof.
Modifications to increase half-life Compounds of the invention may also be rendered reactive for in vivo conjugation to biological targets to substantially increase their duration of action. A pharmaceutical composition comprising a therapeutically effective amount of a compound of the formula I and II, or a pharmaceutically acceptable salt, pharmaceutically acceptable solvate, pharmaceutically active metabolite, or prodrug thereof, can be chemically modified to include linkers that can covalently bind blood components including, but not limited to, cells (e.g., red blood cells and platelets) and proteins (e.g., immunoglobulins including IgG and IgM, serum albumin, ferritin, steroid binding proteins, such as corticosteroid-binding globulin and sex hormone-binding globulin, transferrin, thyroxin binding protein, and alpha-2-macroglobulin: see, for example, rantz et al, U.S. Patent No. 5,942,620, 1999; Krantz et al, J. Drug Target 7 113, 1999; and Holmes et al, Bioconjug. Chem. 11, 439, 2000). Compounds of formulas LI and LII exemplify the types of modifications that can be made to compounds of the present invention to render them susceptible to selective, in vivo conjugation with the aforementioned cells and proteins. In exemplary embodiments, the linker is attached to R°, R2, or A, of compounds of the formula I; or to B or V of compounds of the formula II.
Figure imgf000170_0001
The linker is ordinarily a hydrophilic synthetic polymer, i.e., a polymer not otherwise found in nature. However, polymers which exist in nature and are produced by recombinant or in vitro methods are useful, as are polymers which are isolated from native sources. Hydrophilic polyvinyl polymers fall within the scope of this invention, e.g. polyvinylalcohol and polyvinylpyrrolidone.
Particularly useful are polyalkylene ethers such as polyethylene glycol (PEG); polyelkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene (Pluronics); polymethacrylates; carbomers; branched or unbranched polysaccharides which comprise the saccharide monomers D-mannose, D- and L-galactose, fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic acid (e.g. polymannuronic acid, or alginic acid), D- glucosamine, D-galactosamine, D-glucose and neuraminic acid including homopolysaccharides and heteropolysaccharides such as lactose, amylopectin, starch, hydroxyethyl starch, amylose, dextrane sulfate, dextran, dextrins, glycogen, or the polysaccharide subunit of acid mucopolysaccharides, e.g. hyaluronic acid; polymers of sugar alcohols such as polysorbitol and polymannitol; heparin or heparan. The polymer prior to cross-linking need not be, but preferably is, water soluble, but the final conjugate must be water soluble. In addition, the polymer should not be highly immunogenic in the conjugate form, nor should it possess viscosity that is incompatible with intravenous infusion or injection if it is intended to be administered by such routes.' The linker group can be attached to compounds of the formula I and II through the appropriate handle (e.g., by the acylation of amino groups) and incoφorate at the end of its tether a functionality, desirably a maleimide, reactive towards biomolecules. Once reacted, the conjugated compound of the invention should form a stable covalent linkage to the biomolecule via the linker moiety. In one example, the maleimide of the linker reacts with a cysteine residue (C34) of human serum albumin to form a stable, conjugated species in vivo. The linker is typically a chain of varying length, e.g., maleimide- (CH2)bbC(O)NHCH2CH2(OCH2CH2)aaOCH2C(O)-, maleimide- (CH2)b C(O)NHCH2CH2(OCH2CH2)aaNHCH2C(O)-, maleimide- (CH2)bbC(O)NHCH2CH2(OCH2CH2)aaNHC(S)-, maleimide-(CH2)bbNHC(S)-, maleimide-(CH2)b C(O)-, or maleimide (CH2)bb-where aa is 1-10 and bb is 1-4, and desirably is attached to atoms or groups of atoms on the inhibitor which have minimal impact on the binding of the inhibitor to its protein target (e.g., atoms or groups of atoms known to have no direct or indirect binding interactions with the protein target, or solvent exposed functional groups). Desirably, the length of the linker is between 6 and 100 atoms, inclusive. In some embodiments, the reactive moiety (e.g., maleimide) covalently binds a sulfur atom in a protein, such as human serum albumin. In other embodiments, a compound of the invention, or a pharmaceutically acceptable salt, pharmaceutically acceptable solvate, pharmaceutically active metabolite, or prodrug thereof, can be further modified to include only a polyethylglycol moiety. Such compounds are also known to increase blood serum half-life (see Yoo et al., Chem. Pharm. Bull. 48:1921- 1924, 2000). The polyethylene glycol moiety can be selected from the following group: H(OCH2CH2)ccO-, H3C(OCH2CH2)ccOC(O), H(OCH2CH2)ccOC(O), H3C(OCH2CH2)ccNHC(O), H(OCH2CH2)ccNHC(O), H3C(OCH2CH2)ccNHC(S), H(OCH2CH2)ccNHC(S), H3C(OCH2CH2)ccC(O), H(OCH2CH2)ccC(O), H3C(OCH2CH2)ccNHCH2C(O), H(OCH2CH2)ccNHCH2C(O), H3C(OCH2CH2)ccOC(O)C(CH3)2-, and
H(OCH2CH2)ccOC(O)C(CH3)2-, wherein cc is a range of numbers that results in an average molecular weight of said polyethylene glycol moiety of between 1 ,000-40,000, preferably 20,000 or 40,000.
In one example, compounds modified with a linker (e.g., a linker with a group for conjugation to a molecule of interest) have formula I:
Figure imgf000172_0001
where
X is N, m is 1 ; A- is L-(CH2)n-K-C(O)-; R3 is H, Cι-6 alkyl, or together with K forms a 5- or 6-membered ring; R° is -E-J; R1 is
Figure imgf000173_0001
b ' ; R is selected from the group consisting of optionally substituted alkyl, alkenyl, cycloalkyl, aralkyl, aralkenyl, aryl, heterocyclyl, heteroaralkyl, heteroaralkenyl, and heteroaryl; R4, R5, R6, R7 , "a", "b", "n", E, and J are as previously defined herein; and at least one of J, R2, or L further includes a linker attached to a moiety reactive to a free amine or sulfhydryl contained on a blood component. Exemplary linkers can be chosen from the polyethylene glycol moieties described herein, which can, for example, be linked to a compound of the invention by attachment to an amino group contained in J, R2 or L.
In another example, compounds modified with a linker have formula I where R is
Figure imgf000173_0002
where a is an integer from 1-4, b is an integer from 0-2, R4 and R5 are H or C1-6 alkyl, R6 is H, OH, C]-6 alkyl, or together with R4 forms a 5- or 6-membered ring, and R7 is H, OH, SH, NH2, NO2, optionally substituted d_6 alkyl, halogen, or CF3; R2 is and optionally substituted 2-propyl, 2-butyl, CH2Ph, - CH2(C6H4)-4-OH, cyclohexyl, or Ph; X is N; R3 is H or an optionally substituted C1-6 alkyl; A is an alpha-amino acid of the formula:
Figure imgf000174_0001
where
R13 is H, d-6 alkyl, or together with M forms a 5- or 6-membered ring, D is C(O) or S(O)2, q is an integer from 0-4; Q is an optionally substituted C1-6 alkyl, C3-8 cycloalkyl, C1-9 heterocyclyl, C6 or C]0 aryl, C1-6 alkyloxy, C3-8 cycloalkyloxy, C6 or C]0 aryloxy, C2-9 heterocyclyloxy, C1-6 alkylthio, C3-8 cycloalkylthio, C6 or Cio arylthio, C1-6 alkylamino, C2-12 dialkylamino, C7-16 aralkylamino, C6 or Cio arylamino, C2.9 heterocyclylamino, C2-7 alkylaminocarbonyl, C3-13 dialkylaminocarbonyl, amino, amidino, guanidino, hydroxy, or carboxy; M is single bond, NR14, O, or S, wherein R14 is H, C1-6 alkyl, or when taken together with R forms a 5- or 6-membered ring; U is an optionally substituted C1-6 alkyl, C2-6 alkenyl, C3-8 cycloalkyl, C1- heterocyclyl, C7-16 aralkyl, C8-ι6 aralkenyl, C6 or C10 aryl, C2-15 heteroaralkyl, or C3-15 heteroaralkenyl, wherein when m is 1, R° is a thiazole, and when m is 0, R° is H or Cj-6 alkyl; and least one of U, R2, or M when M is NR14 further includes a linker attached to a moiety reactive to a free amine or sulfhydryl contained on a blood component. Exemplary linkers can be chosen from the polyethylene glycol moieties described herein, which can, for example, be linked to a compound of the invention by attachment to an amino group contained in U, R2, or M when M is NR14. In yet another example, compounds modified with a linker have formula
II:
Figure imgf000174_0002
as previously defined herein and and least one of B, or V further includes a linker attached to a moiety reactive to a free amine or sulfhydryl contained on a blood component. Exemplary linkers can be chosen from the polyethylene glycol moieties described herein, which can, for example, be linked to a compound of the invention by attachment to an amino group contained in B or V.
Other exemplary compounds of the invention further include a polyethylene glycol moiety. Desirably, the polyethylene glycol increases the half-life of the compound in vivo and/or increases the bioavailability of the compound. In various embodiments, the linear or branched polyethylene glycol is bonded to a compound of the invention via a spacer and/or has a molecular weight between 1,000 and 40,000 daltons, inclusive (e.g., approximately 1,000 daltons, 1,500 daltons, 2,000 daltons, 3,500 daltons, 5,000 daltons, 8,000 daltons, 10,000 daltons, 20,000 daltons, or 40,000 daltons). The polyethylene glycol moiety can be incoφorated into the compounds of the invention by attachment to the A, R , or R substituents of the compounds of formula I or to the B or V substituents of the compounds of formula II in the same manner as attachment of the linkers as described herein.
The present invention also includes within its scope the pharmaceutical formulations which comprise at least one of the compounds of formula I or II in association with a pharmaceutically acceptable carrier or coating (e.g., pegylation). Administration of Compounds of the Invention for the Treatment or Prevention of Excess or Undesired Thrombosis
A compound described in any of the embodiments of the above aspects of the invention may be used in any of the following clinical applications. For example, the compounds of the invention are useful for the treatment, stabilization, or prevention of a variety of medical disorders where anticoagulant therapy is indicated in the treatment or prevention of thrombotic conditions such as coronary artery and cerebro- and peripheral vascular disease. Indications include, but are not limited to, myocardial infarction, venous or arterial thrombosis, the formation of atherosclerotic plaques, coagulation syndromes, endarterectomy, including carotid endarterectomy, envascular injury including reocclusion and restenosis following angioplasty and coronary artery bypass surgery, thrombus formation after the application of blood vessel operative techniques, the introduction of artificial heart valves or on the recirculation of blood, cerebral infarction, cerebral thrombosis, transient ischemic attacks, stroke, cerebral embolism, pulmonary embolism, ischaemia, and angina, including unstable angina. In addition, pathologic thrombus formation often occurs in the venous vasculature of the lower extremities following knee, hip, and abdominal surgery (e.g., deep vein thrombosis, DVT). DVT further predisposes the patient to a higher risk of pulmonary thromboembolism. Disseminated intravascular coagulopathy (DIC), a systemic condition that commonly occurs in vascular systems during septic shock, certain viral infections, and cancer, is also indicated for treatment by compounds of the present invention. This condition is characterized by a rapid consumption of coagulation factors and their plasma inhibitors which can result in the formation of life-threatening clots throughout the microvasculature of several organ systems. Another application of Factor Xla inhibitors is the enhancement of fibrinolysis by tissue plasminogen activator. In addition to their use in anticoagulant therapy, Factor Xla inhibitors are useful in the treatment and prevention of other diseases in which the generation of thrombin has been implicated as playing a physiologic role. For example, thrombin has been implicated in contributing to the morbidity and mortality of chronic and degenerative diseases such as cancer, arthritis, atherosclerosis, and Alzheimer's disease by its ability to regulate many different cell types through specific cleavage and activation of a cell surface thrombin receptor, mitogenic effects, diverse cellular functions such as cell proliferation, for example, abnormal proliferation of vascular cells resulting in restenosis or angiogenesis, release of PDGF, and DNA synthesis. Inhibition of Factor Xla effectively blocks thrombin generation and therefore neutralizes any physiologic effects of thrombin on various cell types. The representative indications discussed above include some, but not all, of the potential clinical situations amenable to treatment with a Factor Xla inhibitor. Thus, one or more compounds of the invention can be used in the manufacture of medicaments for use in the production of an antithrombotic or anticoagulant. In one aspect, the invention features a method of treating, stabilizing, or preventing a disease, disorder, or condition associated with undesirable or excess thrombosis in a mammal (e.g., a human). This method involves administering a compound of the invention to the mammal in an amount sufficient to treat, stabilize, or prevent the disease, disorder, or condition. The compound may be administered to the mammal before, during, or after the occurrence of the condition.
In various embodiments, a compound that binds to Factor XI or Factor Xla decreases the activity of Factor Xla, the binding of a Factor Xla to another molecule (e.g., a substrate for Factor Xla), or the half-life of a Factor XI protein, as measured using standard methods (see, for example, Coligan, et al. Current Protocols in Protein Chemistry, Chapters 19 and 20, John Wiley & Sons, New York, 2000; Ausubel et al, Current Protocols in Molecular Biology, Chapter 9, John Wiley & Sons, New York, 2000). For example, the compound may competitively, noncompetitively, or uncompetitively inhibit the ability of Factor XI or Factor Xla to bind one or more of its endogenous substrates. The level of protein may be determined using standard Western, blot immunoassay, or immunohistochemical analysis (see, for example, Coligan, et al, supra; Ausubel et al, supra). Desirably, the compound decreases Factor Xla activity by at least 20, 40, 60, 80, 90 or 95%. In another desirable embodiment, the level of Factor Xla activity is at least 2, 3, 5, 10, 20, or 50-fold lower in the presence of the compound. In some embodiments, the compound is administered in a dose that is sufficient to reduce thrombosis but does not eliminate normal clotting resulting from external injuries or does not induce bleeding complications. In desirable embodiments, the half-life of an injected compound is less than 7, 6, 5, 4, 3, 2, 1, or 0.5 hours. In some embodiments, the half-life is contained within one of the following ranges: 4-6 hours, 2-4 hours, 30-120 minutes, or 30-60 minutes, inclusive. In desirable embodiments, the half-life of an oral compound is less than 24, 20, 16, 12, 8, or 4 hours. In some embodiments, the half- life is contained within one of the following ranges: 20-28 hours, 14-20 hours, 10-14 hours, 6-10 hours, 2-6 hours or 30-120 minutes, inclusive. In desirable embodiments, the compound has better inhibitory activity than benzamidine for Factor Xla, such as an IC5o value of less than 100, 10, 1, 0.1, 0.01, or 0.001 μM.
With respect to the therapeutic methods of the invention, it is not intended that the administration of compounds to a mammal be limited to a particular mode of administration, dosage, or frequency of dosing; the present invention contemplates all modes of administration, including oral, intraperitoneal, intramuscular, intravenous, intraarticular, intralesional, subcutaneous, or any other route sufficient to provide a dose adequate to prevent or treat excess or undesired Factor Xla activity (e.g., excess or undesired clotting). One or more compounds may be administered to the mammal in a single dose or multiple doses. When multiple doses are administered, the doses may be separated from one another by, for example, several hours, one day, one week, one month, or one year. It is to be understood that, for any particular subject, specific dosage regimes should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. If desired, conventional treatments such as heparin may be used in combination with the compounds of the present invention. Exemplary mammals that can be treated using the methods of the invention include humans, primates such as monkeys, animals of veterinary interest (e.g., cows, sheep, goats, buffalos, and horses), and domestic pets (e.g., dogs and cats).
For clinical applications, compounds of the present invention may generally be administered, e.g., parenterally, intravenously, subcutaneously, intramuscularly, colonically, nasally, intraperitoneally, rectally, buccally, or orally. Compositions containing at least one compound of the invention that is suitable for use in human or veterinary medicine may be presented in forms permitting administration by a suitable route. These compositions may be prepared according to the customary methods, using one or more pharmaceutically acceptable adjuvants or excipients. The adjuvants comprise, inter alia, diluents, sterile aqueous media, and various non-toxic organic solvents. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical field, and are described, for example, in Remington: The Science and Practice of Pharmacy (20th ed.), ed. A.R. Gennaro, Lippincott Williams & Wilkins, 2000, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York. The compositions may be presented in the form of tablets, pills, granules, powders, aqueous solutions or suspensions, injectable solutions, elixirs, or syrups, and the compositions may optionally contain one or more agents chosen from the group comprising sweeteners, flavorings, colorings, and stabilizers in order to obtain pharmaceutically acceptable preparations.
The choice of vehicle and the content of active substance in the vehicle are generally determined in accordance with the solubility and chemical properties of the product, the particular mode of administration, and the provisions to be observed in pharmaceutical practice. For example, excipients such as lactose, sodium citrate, calcium carbonate, and dicalcium phosphate and disintegrating agents such as starch, alginic acids, and certain complex silicates combined with lubricants (e.g., magnesium stearate, sodium lauryl sulfate, and talc) may be used for preparing tablets. To prepare a capsule, it is advantageous to use lactose and high molecular weight polyethylene glycols. When aqueous suspensions are used, they may contain emulsifying agents which facilitate suspension. Diluents such as sucrose, ethanol, polyethylene glycol, propylene glycol, glycerol, chloroform, or mixtures thereof may also be used.
For parenteral administration, emulsions, suspensions, or solutions of the compositions of the invention in vegetable oil (e.g., sesame oil, groundnut oil, or olive oil), aqueous-organic solutions (e.g., water and propylene glycol), injectable organic esters (e.g., ethyl oleate), or sterile aqueous solutions of the pharmaceutically acceptable salts are used. The solutions of the salts of the compositions of the invention are especially useful for administration by intramuscular or subcutaneous injection. Aqueous solutions that include solutions of the salts in pure distilled water may be used for intravenous administration with the proviso that (i) their pH is adjusted suitably, (ii) they are appropriately buffered and rendered isotonic with a sufficient quantity of glucose or sodium chloride, and (iii) they are sterilized by heating, irradiation, or microfiltration. Suitable compositions containing the compounds of the invention may be dissolved or suspended in a suitable carrier for use in a nebulizer or a suspension or solution aerosol, or may be absorbed or adsorbed onto a suitable solid carrier for use in a dry powder inhaler. Solid compositions for rectal administration include suppositories formulated in accordance with known methods and containing at least one compound of formula I or II.
Dosage formulations of the compounds of this invention to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile membranes (e.g., 0.2 micron membranes) or by other conventional methods. Formulations typically are stored in lyophilized form or as an aqueous solution. The pH of the compositions of this invention is typically between 3 and 11, more desirably between 5 and 9, and most desirably between 7 and 8, inclusive. While a desirable route of administration is by injection such as intravenously (bolus and/or infusion), other methods of administration may be used. For example, compositions may be administered subcutaneously, intramuscularly, colonically, rectally, nasally, or intrapertoneally in a variety of dosage forms such as suppositories, implanted pellets or small cylinders, aerosols, oral dosage formulations, and topical formulations such as ointments, drops, and dermal patches. The compounds of the invention are desirably incoφorated into shaped articles such as implants, including but not limited to valves, stents, tubing, and prostheses, which may employ inert materials such as synthetic polymers or silicones, (e.g., Silastic, silicone rubber, or other commercially available polymers). Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxy-propyl- methacrylamide-phenol, polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the Factor Xla inhibitors of the invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example polylactic acid, polyglycolic acid, copolymers of polylaclic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and cross linked or amphipathic block copolymers of hydrogels. The compounds of the invention may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of lipids, such as cholesterol, stearylamine, or phosphatidylcholines. The compounds of the invention may also be delivered using antibodies, antibody fragments, growth factors, hormones, or other targeting moieties to which the compound molecules are coupled (e.g., see Remington: The Science and Practice of Pharmacy, vide supra), including in vivo conjugation to blood components of a suitably modified compound of the formula I or II which possesses a metastable or reactive functional group as described above.
Dosage levels of active ingredients in the pharmaceutical compositions of the invention may be varied to obtain an amount of the active compound(s) that achieves the desired therapeutic response for a particular patient, composition, and mode of administration. The selected dosage level depends upon the activity of the particular compound, the route of administration, the severity of the condition being treated, and the condition and prior medical history of the patient being treated. For adults, the doses are generally from about 0.01 to about 100 mg/kg, desirably about 0.1 to about 1 mg/kg body weight per day by inhalation, from about 0.01 to about 100 mg/kg, desirably 0.1 to 70 mg/kg, more desirably 0.5 to 10 mg/kg body weight per day by oral administration, and from about 0.01 to about 50 mg/kg, desirably 0.1 to 1 mg/kg body weight per day by intravenous administration. Doses are determined for each particular case using standard methods in accordance with factors unique to the patient, including age, weight, general state of health, and other factors which can influence the efficacy of the compound(s) of the invention.
Administration of compositions of the invention may be as frequent as necessary to obtain the desired therapeutic effect. Some patients may respond rapidly to a higher or lower dose and may find much weaker maintenance doses adequate. Other patients, however, receive long-term treatments at the rate of 1 to 4 doses per day, in accordance with the physiological requirements of each patient. The active product may be administered, e.g., orally 1 to 4 times daily. Example 33. Enzyme Assays
The ability of compounds of the present invention to inhibit Factor Xla and related serine proteases such as Factor Xa, thrombin, trypsin, Factor Vila, plasma kallikrein, tissue-plasminogen activator (t-PA), urokinase-plasminogen activator (u-PA), plasmin, and activated protein C (APC) were evaluated by determining the concentration of inhibitor which resulted in a 50% reduction in enzyme activity (IC50) using purified enzyme. Potential inhibitors of Factor Xla were evaluated using the following assay. Briefly, the inhibitors were tested against native Factor Xla (Haematological Associates, P/N FXIA01) using a custom made analog of S-2366, pyroGlu-Pro-Arg-7- methylamidocoumarin (AMC) available from American Peptide, Inc. This analog is based on the well described substrate pyro-Glu-Pro-Arg-pNA, a.k.a. S-2366, available from Diapharma Group, Inc. (Columbus, OH). This substrate had the 9-nitroanaline group replaced with 7-methylaminocoumarin (AMC).
The final concentration of the substrate in the assay was 100 μM, and the final concentration of the enzyme was 0.25 nM. Inhibitors were tested by serial dilution over an appropriate range to yield a dose response curve for determination of the inhibitors' IC5o value. The assay mixture was incubated for 30 minutes at 30° C, and the fluorescent yield measured using a PE-Wallac Victor 2 Plate Reader. Dose response curves were fit to equation 1 below in which A is the maximum inhibition, B is the minimum inhibition, C is the IC5o, and D is the Hill coefficient.
r (A -BY ] + B EQN. 1
'(! + (£)*> Desirable compounds have an IC50 value for inhibiting Factor Xla of less than 1 micromolar, 100 nanomolar, 10 nanomalar, or 1 nanomolar (in order of increasing preference). Table 12 shows the IC50 values for some of the compounds of formula I, where X is N, R1 is 3-guanidinopropyl, m is 1, X is N, R3 is H, and R° is 2-thiazole.
Table 12. Selective inhibition of factor Xla by compounds of formula I (X is N, R1 is 3-guanidinopropyl, m is 1, X is N, R3 is H, and R° is 2-thiazole)
Figure imgf000184_0001
Table 13 shows the IC50 values for compounds of formula I, where X is N, W is N, R2 is H, R1 is 3-guanidinopropyl, m is 1, R° is 2-thiazole, R3 together with A forms a ring having the formula:
Figure imgf000185_0001
Table 13. Selective inhibition of factor Xla by compounds of formula I
Figure imgf000185_0002
Table 14 shows the IC50 values for compounds of formula II, where Z' is NH, each of e and g is C-H, each of R19 and R20 is H, f is a covalent bond, and T is NH.
Table 14. Selective inhibition of factor Xla by compounds of formula II (Z' is NH, each of e and g is C-H, each of R19 and R20 is H, f is a covalent bond, T is NH)
Figure imgf000185_0003
Figure imgf000186_0001
Example 34. Clotting Assays
Human Plasma Based Clotting Assay
An activated partial thomboplastin time {aPTT) assay was used to measure the ability of compounds to inhibit the contact coagulation pathway. This pathway involves Factor XII, kallikrein, and Factor XI, which activates Factor IX and Factor VIII, leading to activation of Factor X and Factor V, and then activation of Factor II to form a blood clot (Figure 1). For this assay, CaCl2 (30 mM) was placed in a large central reagent position of a Thromboscreen 400C instrument, allowing it to equilibrate to 37°C. Plasma (50 ul) and compounds of the invention were added at different concentrations to cuvettes. After incubation for two minutes, aPTT reagent (ALEXIN, Sigma) was added (50 ul) and incubated an additional three minutes. The cuvettes were transferred to a measuring position; prewarmed CaCl2 reagent (50 ul) was added, and readings were then taken over a maximum of 300 seconds. A dose response curve was generated, and the concentration at which the clotting time was doubled (2 x aPTT) was determined. Compounds which inhibit Factor Xla in the desired range desirably have an effect at less than 50 uM, more desirably at less than 10 uM. A prothrombin time (PT) assay was also used to measure inhibition of coagulation. In this assay, the Factor XI dependent steps are bypassed. Hence, the assay measures inhibition of Factor Vila, Factor Xa, and thrombin, but not FXI. This assay measures the ability of Factor Vila to activate Factor X, which activates Factor II to form a blood clot. For this assay, the thromboplastin reagent (ThromboMax with Calcium, Sigma) was placed in a central reagent position in a Thromboscreen 400C instrument, and allowed to equilibrate to 37 °C. Plasma (50 ul of plasma prewarmed for three minutes) and compounds of the invention (different concentrations) were added to cuvettes. The cuvettes were transferred to a measuring position. The prewamied Thromboplastin reagent (100 ul) was then added, and readings were then taken over 300 seconds. A dose response curve was generated, and the concentration at which the clotting time was doubled (2 x PT) was determined.
Example 36. Experimental In Vivo Rat Venous Thrombosis Model
The model of venous thrombosis used incoφorated stenosis of the inferior vena cava with rapid infusion of hypotonic saline followed by partial stasis of blood flow (Millet, et al., (1987). Thromb. Res., 45, 123-133; Schumacher et al., (1996). J. Cardiovasc. Pharmacol, 28, 19-25). Briefly, male Sprague-Dawley rats (300 - 400 g) were anesthetized using ketamine/xylazine I.M. A midline laparotomy was performed, and the vena cava was visualized and gently separated from the descending aorta using cotton swabs. Both iliolumbar veins were ligated with suture (3-0 silk). A loose ligature (3-0 silk) was placed around the vena cava distal to the renal veins. A 25G butterfly bent at a 90-degree angle and attached to a 5 cc syringe filled with 2.5 mL of hypotonic saline (0.225%) was inserted into the vena cava at the level of the iliolumbar veins and glued into place using Vetbond™. A 26G needle bent to a 90-degree angle was then tied tightly to the vena cava, using the loose ligature. An atraumatic clamp was placed on the vena cava proximal to the iliac bifurcation, and the hypotonic saline manually infused at a rate of 10 mL/minutes. The atraumatic clamp was removed, along with the 26G needle, creating a 26G stenosis. Organs were carefully replaced, and animals were kept normofhermic until sacrifice. Animals were sacrificed by anesthetic overdose at 10 or 60 minutes post thrombus induction. Following sacrifice, the vena cava was isolated using hemostats, and the clot(s) was dissected out. Clots were removed, blotted on gauze, and then immediately weighed using an analytical balance. Compounds under investigation along with vehicles and positive controls (heparin, enoxaparin) were administered intravenously via a catheter (PE-50 tubing) implanted in the jugular or femoral vein. In some studies, rats were administered a bolus of the test article (4 mL/kg) via the venous catheter immediately prior to initiation of intravenous infusion (4 mL/kg/h). The rats were subjected to the challenge 15 minutes post bolus/infusion initiation. All test article infusions continued to the end of the study period.
Figure imgf000188_0001
Continuous intravenous administration of compound 22 suppressed the thrombus formation induced by stenosis of the inferior vena cava with rapid infusion of hypotonic saline followed by partial stenosis in the rat (Figure 10).
Administration of compound XLXI commenced 15 minutes prior to thrombotic challenge, with determination of thrombus mass at 60 minutes post challenge.
Efficacy was observed at a total dose exposure of 1.4 mg/kg with no further decrease in thrombus size at 4.0 mg/kg. Despite the fact that the specificity of compound XLXI for rat Factor Xla is 11-fold less than that observed against native human Factor Xla, statistically significant efficacy was observed in a rat model of venous thrombosis.
Other Embodiments
From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adapt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
All publications mentioned in this specification are herein incoφorated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incoφorated by reference.
What is claimed is:

Claims

1. A compound of formula I
Figure imgf000190_0001
(I), or a pharmaceutically acceptable salt or prodrug thereof, wherein
R1 is selected from the group consisting of:
Figure imgf000190_0002
R4 is H or C1-6 alkyl, R5 is H, C1-6 alkyl, OH, NH2, NO2, CO2R5a, wherein R5a is C1-6 alkyl, or R5 taken together with R4 forms a 5- or 6- membered ring, R6 is H, OH, C1-6 alkyl, or when taken together with R4, forms a 5- or 6-membered ring, R7 is H, OH, SH, NH2, NO2, optionally substituted . 6 alkyl, halogen, or CF3, Y is O, S, or NR4, and Z is NR5 or (H,H);
R2 is H or an optionally substituted C1-6 alkyl, C2-6 alkenyl, C3-8 cycloalkyl, C7-ι6 aralkyl, C8-ι6 aralkenyl, C6 or Cio aryl, C2-9 heterocyclyl, C2-ι5 heteroaralkyl, or C3-15 heteroaralkenyl, or when taken together with X and R3 forms an optionally substituted C6 aryl group or an optionally substituted C3-C5 heteroaryl group; a is an integer from 0-5; b is an integer from 0-2;
X is C or ;
R3 is H or optionally substituted C1-6 alkyl, or together with X and A forms an optionally substituted C2-5 heterocylic ring, or together with X and R forms an optionally substituted C6 aryl group or an optionally substituted C3-C5 heteroaryl group;
A is R8-AA2, where AA2 is a covalent bond or a peptide chain of one to five natural or unnatural alpha-amino acid residues of D- or L- configuration, wherein R8 is H, C2-7 acyl, C7-π aroyl, C3-1o heteroaroyl, C2- alkoxycarbonyl, C4- cycloalkoxycarbonyl, C8-]7 aralkoxycarbonyl, C or Cπ aryloxycarbonyl, aminocarbonyl, C2-7 alkylaminocarbonyl, C3-ι3 dialkylaminocarbonyl, C4-9 cycloalkylaminocarbonyl, C8-17 aralkylaminocarbonyl, C7 or Cπ arylaminocarbonyl, C3-10 heterocyclylaminocarbonyl, C3-16 heteroaralkylaminocarbonyl, C2-7 alkylthiocarbonyl, C7-11 arylthiocarbonyl, C3, ιo heteroarylthiocarbonyl, C2-7 alkoxythiocarbonyl, C4-9 cycloalkoxythiocarbonyl, C8-π aralkoxythiocarbonyl, C7 or Cπ aryloxythiocarbonyl, aminothiocarbonyl, C2.7 alkylaminothiocarbonyl, C3-13 dialkylaminothiocarbonyl, C4-9 cycloalkylaminothiocarbonyl, C8-1 aralkylaminothiocarbonyl, C or Cπ arylaminothiocarbonyl, C3-10 heterocyclylaminothiocarbonyl, C3-ι6 heteroaralkylaminothiocarbonyl, C1-6 alkylsulfonyl, C3-8 cycloalkylsulfonyl, C7.ι6 aralkylsulfonyl, C6 or C10 arylsulfonyl, C2-9 heterocyclylsulfonyl, or C2.ι5 heteroaralkylsulfonyl, or A together with X and R3 forms an optionally substituted C2-5 heterocylic ring; and m is 0 or 1, wherein when m is 1, R° is an optionally substituted Cι-6 alkyl, C2-6 alkenyl, C3-8 cycloalkyl, C7-ι6 aralkyl, C8-ι6 aralkenyl, C6 or Cio aryl, C]-9 heterocyclyl, C2-15 heteroaralkyl, C3-15 heteroaralkenyl, or C(O)R9, wherein R9 is an optionally substituted C]-6 alkyl, C2-6 alkenyl, C]-8 cycloalkyl, C7-ι6 aralkyl, C8-ι6 aralkenyl, C6 or Cio aryl, C]-9 heterocyclyl, C2-]5 heteroaralkyl, C3-ι5 heteroaralkenyl, Cι-6 alkyloxy, C6 or C10 aryloxy, Cι-9 heteroaryloxy, C -ι6 aralkyloxy, Cι-6 alkylthio, C3-8 cycloalkylthio, C6 or C]0 arylthio, C7-]6 aralkylthio, Cι-6 alkylamino, C2-]2 dialkylamino, C3-8 cycloalkylamino, C7-]6 aralkylamino, C6 or CJQ arylamino, C2-9 heterocyclylamino, or C2-ι5 heteroaralkylamino, with the proviso that the amino acid of AA2 that is covalently linked to X is not D-phenylalanine when R8 is C1-6 alkylsufonyl or C7-16 aralkylsulfonyl; when m is 0, R° is H, CHO, CN, or B(OR9)2, wherein R9 is H, C,-6 alkyl, or taken together forms a C2-4 cyclic boronate ester, or R° is an optionally substituted C1-6 alkyl, C2-6 alkenyl, C3-8 cycloalkyl, C7-ι6 aralkyl, C8.]6 aralkenyl, C6 or Cio aryl, d-9 heterocyclyl, C2-i5 heteroaralkyl, or C3.15 heteroaralkenyl, with the proviso that the amino acid of AA2 that is covalently linked to X is not D-cyclohexylglycine when R8 is C]-6 alkylsulfonyl or C7-ι6 aralkylsulfonyl.
2. The compound of claim 1, wherein R° is 2-thiazole, 2-(4- methylthiazole), 2-(5-methylthiazole), 2-(4,5-dimethylthiazole), 2-(5-(2- hydroxyethyl)thiazole), or 2-N-methylimidazole.
3. The compound of claim 1, wherein
R2 is H or a substituted Cμ6 alkyl, C2-6 alkenyl, C3-8 cycloalkyl, C7-ι6 aralkyl, C8-16 aralkenyl, C6 or Cio aryl, C1-9 heterocyclyl, C2-i5 heteroaralkyl, or C3-15 heteroaralkenyl; X is ; a is an integer from 1-4; b is an integer from 0-2;
AA2 is a covalent bond and R8 is B-Y1-(CH2)n-K-C(O)-, wherein n is an integer from 0-6, K together with -C(O)-, X, and R3 forms an optionally substituted C2-5 heterocyclyl group or K is a single bond, NR , O, S, C(O), CH(OR10), or -CH(NHRn), wherein R9 is H, OH, or Cι-6 alkyl, each of R10 R11 is, independently, H, Cι-6 alkyl, C2-6 alkenyl, C3-8 cycloalkyl, C7.]6 aralkyl, C8-]6 aralkenyl, C6 or Cio aryl, d- heterocyclyl, C2-15 heteroaralkyl, C3.15 heteroaralkenyl, C2-7 acyl, C7-π aroyl, C3-10 heteroaroyl, C2-7 alkoxycarbonyl, C4.9 cycloalkoxycarbonyl, C8-π aralkoxycarbonyl, C7 or Cπ aryloxycarbonyl, aminocarbonyl, C2-7 alkylaminocarbonyl, C3-ι 3 dialkylaminocarbonyl, C4-9 cycloalkylaminocarbonyl, C8-ι aralkylaminocarbonyl, C7 or Cπ arylaminocarbonyl, C3-ιo heterocyclylaminocarbonyl, C3_ι6 heteroaralkylaminocarbonyl, C2-7 alkylthiocarbonyl, C7-π arylthiocarbonyl, C3- 10 heteroarylthiocarbonyl, C2-7 alkoxythiocarbonyl, C4-9 cycloalkoxythiocarbonyl, C8-17 aralkoxythiocarbonyl, C7 or Cπ aryloxythiocarbonyl, aminothiocarbonyl, C2-7 alkylaminothiocarbonyl, C3-ι3 dialkylaminothiocarbonyl, C -9 cycloalkylaminothiocarbonyl, C8-π aralkylaminothiocarbonyl, C7 or Cπ arylaminothiocarbonyl, C3-10 heterocyclylaminothiocarbonyl, C3.16 heteroaralkylaminothiocarbonyl, Cι-6 alkylsulfonyl, C3-8 cycloalkylsulfonyl, C7-16 aralkylsulfonyl, C6 or Cio arylsulfonyl, C2-9 heterocyclylsulfonyl, or C2-ι5 heteroaralkylsulfonyl, Y1 is O, S, NR18, or a covalent bond, wherein R18 is H or Cι-6 alkyl, wherein when Y1 is a covalent bond, B is an optionally substituted Cι-6 alkyl, C2-6 alkenyl, d-s cycloalkyl, C6 or Cio aryl, C1-9 heterocyclyl, C7.]6 aralkyl, C8-16 aralkenyl, C2-ι5 heteroaralkyl, C3-15 heteroaralkenyl, Cι-6 alkylsulfonyl amino, and when Y1 is O, S, or NR18, and B is an optionally substituted C]-6 alkyl, C2-6 alkenyl, C3-8 cycloalkyl, C -ι6 aralkyl, C8.16 aralkenyl, C6 or Cι0 aryl, C1-9 heterocyclyl, C2-15 heteroaralkyl, C3-]5 heteroaralkenyl, C2-7 acyl, C7-π aroyl, C3.10 heteroaroyl, C2-7 alkoxycarbonyl, C4-9 cycloalkoxycarbonyl, C8-π aralkoxycarbonyl, C7 or Cπ aryloxycarbonyl, C2.10 heterocyclyloxycarbonyl, aminocarbonyl, C2-7 alkylaminocarbonyl, C3.]3 dialkylaminocarbonyl, C4-9 cycloalkylaminocarbonyl, Cg.17 aralkylaminocarbonyl, C7 or Cπ arylaminocarbonyl, C3-ι0 heterocyclylaminocarbonyl, C3.16 heteroaralkylaminocarbonyl, C2-7 alkylthiocarbonyl, C7-π arylthiocarbonyl, C3-10 heteroarylthiocarbonyl, C2-7 alkoxythiocarbonyl, C4-9 cycloalkoxythiocarbonyl, C8.17 aralkoxythiocarbonyl, C7 or Cπ aryloxythiocarbonyl, aminothiocarbonyl, C2-7 alkylaminothiocarbonyl, C3-]3 dialkylaminothiocarbonyl, C4-9 cycloalkylaminothiocarbonyl, C8-17 aralkylaminothiocarbonyl, C7 or Cπ arylaminothiocarbonyl, C3.10 heterocyclylaminothiocarbonyl, C3-ι6 heteroaralkylaminothiocarbonyl;
R3 is H or optionally substituted C]-6 alkyl, or together with X and A forms an optionally substituted C -5 heterocylic ring;
R is -E-J, wherein when m is 0,
E is a single bond to J, wherein J is H or an optionally substituted C]-6 alkyl; and when m is 1,
E is selected from the group consisting of a single bond to J, -C(O)O-, - C(O)S-, and -C(O)NR10-, wherein R10 is H or C1-6 alkyl and J is an optionally substituted C1-6 alkyl, C2-6 alkenyl, C3-8 cycloalkyl, C7-ι6 aralkyl, C8-]6 aralkenyl, C6 or Cio aryl, d-9 heterocyclyl, C2-15 heteroaralkyl, or C3-15 heteroaralkenyl.
4. The compound of claim 3, wherein R8 is B-Y1-(CH )n-K-C(O)-, wherein K is oxo or CHOH, n is 0, and B-Y1 is phenyl, 4-chlorophenyl, 3-chlorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl, 3-fluorophenyl, 4-fluorophenyl, 3,4- difluorophenyl, and 3,5-difluorophenyl.
5. The compound of claim 1 , wherein m is 1 ; R° is an optionally substituted 2-thiazole or 2-benzthiazole ring;
Figure imgf000195_0001
R2 is selected from the group consisting of:
Figure imgf000195_0002
X is N; and R3 is H.
6. The compound of claim 5, wherein
A is R8-AA2, wherein AA2 is a single natural or unnatural alpha-amino acid residue and R8 is selected from the group consisting of:
Figure imgf000195_0003
wherein c is 0 or 1 ; each of R11, R12, R13, and R14 is, independently, C2.7 alkanoyl, C1-6 alkyl,
C1-6 alkenyl, C1-6 alkoxy, C2.12 alkoxyalkyl, C1-6 alkylsulfinyl, C2-ι2 alkylsulfinylalkyl, C1-6 alkylsulfonyl, C6 or C]0 aryl, C7-]6 arylalkyl, amino, d_6 aminoalkyl, C7 or Cn aryloyl, azido, C].6azidoalkyl, carboxaldehyde, carboxamide, carboxyl, C2-7 (carboxaldehyde)alkyl, C3-8 cycloalkyl, C -ι4 cycloalkylalkyl, halo, Cι-6 haloalkyl, C]-9 heterocyclyl, d-9 (heterocyclyl)oxy, C2.ιo (heterocyclyloyl, hydroxy, C1-6 hydroxyalkyl, nitro, Cι-6 nitroalkyl, N- protected amino, N-protected aminoalkyl, Cι-6 thioalkoxy, C2-ι2 thioalkoxyalkyl, -(CH2)qCO2RA, wherein q is zero to four and RA is selected from the group consisting of (a) Cι-6 alkyl, (b) C6 or Cj0 aryl and (c) C -ι6 arylalkyl, wherein the alkylene group is of one to six carbon atoms, - (CH2)qCONRBRc, wherein q is zero to four and RB and Rc are independently selected from the group consisting of (a) hydrogen, (b) Cι-6 alkyl, (c) C6 or C]0 aryl and (d) C7-ι6 arylalkyl, wherein the alkylene group is of one to six carbon atoms, -(CH2)qSO2RD, wherein q is zero to four and RD is selected from the group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) C6 or do aryl and (d) C7-ι6 arylalkyl, wherein the alkylene group is of one to six carbon atoms, - (CH2)qSO2NRERF, wherein q is zero to four and RE and RF are independently selected from the group consisting of (a) hydrogen, (b) Cι-6 alkyl, (c) C6 or Cio aryl and (d) C7-]6 arylalkyl, wherein the alkylene group is of one to six carbon
I atoms, -(CH2)qNRGRH, wherein q is zero to four and RG and RH are independently selected from the group consisting of (a) hydrogen, (b) an N- protecting group, (c) alkyl of one to six carbon atoms, (d) alkenyl of two to six carbon atoms, (e) alkynyl of two to six carbon atoms, (f) C6 or Cio aryl, (g) C - ]6 arylalkyl, wherein the alkylene group is of one to six carbon atoms, (h) cycloalkyl of three to eight carbon atoms, and (i) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms, and the alkylene group is of one to ten carbon atoms, with the proviso that no two groups are bound to the nitrogen atom through a carbonyl group or a sulfonyl group, CM perfluoroalkyl, C^ perfluoroalkoxy; C6 or do aryloxy, Q-s cycloalkoxy, C4-14 cycloalkylalkoxy, or C7.16 arylalkoxy; and each of R15 and R16 is, independently, H, C]-6 alkyl; C1-6 hydroxyalkyl; CM perfluoroalky], cyano, halo, -(CH2)qCO2RA, -(CH2)qCONRBRc, - (CH2)qSO2RD, -(CH2)qSO2NRERF, -(CH2)qNRGRH, wherein q is zero to two and RA, RB, Rc, RD, RE, RF, RG, and RH are as defined above.
7. The compound of claim 6, wherein AA2 is selected from the group consisting of:
Figure imgf000197_0001
The compound of claim 7, wherein
Figure imgf000197_0002
Figure imgf000198_0001
9. The compound of claim 5, wherein AA2 is
Figure imgf000198_0002
Figure imgf000198_0003
and R8 is selected from the group consisting of: α-toluenesulfonyl, (methyl-3-benzoate)methanesulfonyl, 2-nitro-α-toluenesulfonyl, 1- propanesulfonyl, 3-carboxyl-α-toluenesulfonyl, and 3-chloropropanesulfonyl.
10. The compound of claim 5, wherein A is selected from the group consisting of:
Figure imgf000198_0004
Figure imgf000199_0001
Figure imgf000200_0001
11. The compound of claim 1 , wherein
R1 is selected from the group consisting of:
Figure imgf000200_0002
, wherein a is an integer from 1-4, b is an integer from 0-2,
R4 is H or C1-6 alkyl, R5 is H, C1-6 alkyl, OH, NH2, NO2, CO2R5a, wherein R5a is Ci-6 alkyl, or R5 taken together with R4 forms a 5- or 6-membered ring, and R7 is H, OH, SH, NH2, NO2, optionally substituted d-6 alkyl, halogen, or CF3; R2 is selected from the group consisting of:
Figure imgf000201_0001
X is ;
R3 is H;
A is
Figure imgf000201_0002
wherein
R3a is H, Cι-6 alkyl, or together with M forms a 5- or 6-membered ring,
D is C(O), C(S), or S(O)2, q is an integer from 0-4,
Q is an optionally substituted d-6 alkyl, C3-8 cycloalkyl, C]-9 heterocyclyl, C6 or C]0 aryl, Cι-6 alkyloxy, C3-8 cycloalkyloxy, C6 or Cι0 aryloxy, C2- heterocyclyloxy, C]-6 alkylthio, C3-8 cycloalkylthio, C6 or Cio arylthio, Cι-6 alkylamino, C2-ι 2 dialkylamino, C -16 aralkylamino, C6 or C10 arylamino, C - heterocyclylamino, aminocarbonyl, C2-7 alkylaminocarbonyl, d-i 3 dialkylaminocarbonyl, amino, amidino, guanidino, ureido, hydroxy, or carboxy, with the proviso that when q is 1 , Q is not phenyl,
M is single bond, NR3 , O, or S, wherein R3 is H, C1-6 alkyl, or when taken together with M, D, N, and R3a forms a 5- or 6-membered ring, and
U is an optionally substituted d-6 alkyl, C2-6 alkenyl, C3.8 cycloalkyl, d_ 9 heterocyclyl, C7-ι6 aralkyl, C8-ι6 aralkenyl, C6 or Cio aryl, C2-ι5 heterocyclyalkyl, or C3-15 heteroaralkenyl, wherein when m is 1 , R° is an optionally substituted C6 or Cio aryl Cι-9 heterocyclyl, or when m is 0, R° is H or d-6 alkyl.
12. The compound of claim 11 , wherein m is 1 ; R° is an optionally substituted 2-thiazole or 2-benzthiazole ring;
Figure imgf000202_0001
R2 is 2-methylpropyl;
R3a is H;
D is S(O)2;
M is a single bond; q is 0;
Figure imgf000202_0002
U is selected from the group consisting of: benzyl, 3- methylcarboxybenzyl, 3-carboxybenzyl, 2-nitrobenzyl, 1 -propyl, and 3- chloropropyl.
13. The compound of claim 11 , wherein m is 1 ;
R° is an optionally substituted 2-thiazole or 2-benzthiazole ring;
Figure imgf000202_0003
R3a is H;
D is C(O);
M is NR3b, wherein R3b is H; q is 0;
Q is selected from the group consisting of:
Figure imgf000203_0001
U is selected from the group consisting of: 2-chlorobenzyl, 3- chlorobenzyl, 4-chlorobenzyl, 2,3-dichlorobenzyl, 2,4-dichlorobenzyl, 2,5- dichlorobenzyl, 2,6-dichlorobenzyl, 3,4-dichlorobenzyl, 3,5-dichlorobenzyl, 2- fluorobenzyl, 3-fluorobenzyl, 4-fluorobenzyl, 2,3-difluorobenzyl, 2,4- difluorobenzyl, 2,5-difluorobenzyl, 2,6-difluorobenzyl, 3,4-difluorobenzyl, 3,5- difluorobenzyl, (R)-α-methyl-4-bromobenzyl, (S)-α-methyl-4-bromobenzyl, (R)-α-methyl-4-chlorobenzyl, (S)-α-methyl-4-chlorobenzyl, (R)-α-methyl-4- fluorobenzyl, (S)-α-methyl-4-fluorobenzyl, 2-trif uoromethylbenzyl, 2- methylbenzyl, 3-methylbenzyl, 4-methylbenzyl, 2-ρhenylacetic acid, 2- pyridylmethyl, 3-pyridylmethyl, 4-pyridylmethyl, phenyl, and indanyl.
14. The compound of claim 13, wherein
R2 is ^\ ;
Q is selected from the group consisting of:
Figure imgf000204_0001
U is (R)-α-methyl-4-bromobenzyl.
15. The compound of claim 11 , wherein
U is CHR3cR3d, wherein R3c is an optionally substituted C6 or Cio aryl or Ci-9 heterocyclyl, and R3d is H, d-6 alkyl, C2-6 alkenyl, or together with R3c forms a fused bicyclic ring.
16. The compound of claim 3, wherein AA2 is a covalent bond and R8 is B- Y1-(CH2)n-K-C(O)-, wherein K-C(O)-, X, and R3 together forms a heterocyclyl group having the formula X:
Figure imgf000204_0002
(X), wherein
W is CH or N;
R17 is hydrogen or an optionally substituted Cι-6 alkyl, C2-6 alkenyl, C3-8 cycloalkyl, C7.16 aralkyl, C8-ι6 aralkenyl, C6 or Cio aryl, Cι-9 heterocyclyl, C2-ι5 heteroaralkyl, Cι-6 alkylthio, C3-8 cycloalkylthio, C6 or Cio arylthio, C7-ι6 aralkylthio, C2-9 heterocyclylthio, C2.ι5 heteroaralkylthio, Cι-6 alkylamino, C2-ι2 dialkylamino C3-8 cycloalkylamino, C7-ι6 aralkylamino, C6 or C10 arylamino, or C2-9 heterocyclylamino;
Y1 is O, S, NR18, or a covalent bond, wherein R18 is H or C1-6 alkyl, wherein when Y1 is a covalent bond, B is an optionally substituted Cι-6 alkyl, C2.6 alkenyl, C3.8 cycloalkyl, C6 or do aryl, C1-9 heterocyclyl, C7.16 aralkyl, C8-]6 aralkenyl, C2.15 heteroaralkyl, C3.15 heteroaralkenyl, C1-6 alkylsulfonyl amino, C3-8 cycloalkylsulfonylamino, C7-]6aralkylsulfonylamino, C6 or C10 arylsulfonylamino, C2-9 heterocyclylsulfonylamino, or C2-ι5 heteroaralkylsulfonylamino, or when Y1 is O, S, or NR18, B is an optionally substituted Cι-6 alkyl, C2-6 alkenyl, C3-8 cycloalkyl, C7.16 aralkyl, C8-ι6 aralkenyl, C6 or Cio aryl, Cι-9 heterocyclyl, C2-15 heteroaralkyl, C3-15 heteroaralkenyl, C2-7 acyl, C7-π aroyl, C3.]0 heteroaroyl, C2-7 alkoxycarbonyl, C4-9 cycloalkoxycarbonyl, C8-ι7 aralkoxycarbonyl, C7 or Cπ aryloxycarbonyl, C2-ιo heterocyclyloxycarbonyl, aminocarbonyl, C2-7 alkylaminocarbonyl, C3-ι 3 dialkylaminocarbonyl, C -9 cycloalkylaminocarbonyl, C8-]7 aralkylaminocarbonyl, C7 or Cπ arylaminocarbonyl, C3-ι0 heterocyclylaminocarbonyl, C3-ι6 heteroaralkylaminocarbonyl, C2.7 alkylthiocarbonyl, C7-π arylthiocarbonyl, C3- 10 heteroarylthiocarbonyl, C2-7 alkoxythiocarbonyl, C4-9 cycloalkoxythiocarbonyl, C8-π aralkoxythiocarbonyl, C7 or Cπ aryloxythiocarbonyl, aminothiocarbonyl, C2-7 alkylaminothiocarbonyl, C3-13 dialkylaminothiocarbonyl, C4-9 cycloalkylaminothiocarbonyl, C8-ι7 aralkylaminothiocarbonyl, C7 or On arylaminothiocarbonyl, C3-ιo heterocyclylaminothiocarbonyl, C3-ι6 heteroaralkylaminothiocarbonyl, or B is a natural or unnatural amino acid of L- or D-configuration substituted on the alpha-amine with H, C2-7 acyl, C -π aroyl, C3.10 heteroaroyl, C2-7 alkoxycarbonyl, C4-9 cycloalkoxycarbonyl, C8-17 aralkoxycarbonyl, C7 or Cπ aryloxycarbonyl, C2-1o heterocyclyloxycarbonyl, C3.15 heteroaralkoxycarbonyl, aminocarbonyl, C2.7 alkylaminocarbonyl, 03.] 3 dialkylaminocarbonyl, C .9 cycloalkylaminocarbonyl, C8.17 aralkylaminocarbonyl, C7 or Cπ arylaminocarbonyl, C3.10 heterocyclylaminocarbonyl, C3-ι6 heteroaralkylaminocarbonyl, C2-7 alkylthiocarbonyl, C _π arylthiocarbonyl, C3- 10 heteroarylthiocarbonyl, C2-7 alkoxythiocarbonyl, C4-9 cycloalkoxythiocarbonyl, C8-17 aralkoxythiocarbonyl, C7 or Cπ aryloxythiocarbonyl, aminothiocarbonyl, C2.7 alkylaminothiocarbonyl, C3.13 dialkylaminothiocarbonyl, C -9 cycloalkylaminothiocarbonyl, C8-ι7 aralkylaminothiocarbonyl, C7 or Cn arylaminothiocarbonyl, C3-ι0 heterocyclylaminothiocarbonyl, C3.]6 heteroaralkylaminothiocarbonyl, Cι-6 alkylsulfonyl, C3-8 cycloalkylsulfonyl, C7-16 aralkylsulfonyl, C6 or Cio arylsulfonyl, C2-9 heterocyclylsulfonyl, or C2-ι5 heteroaralkylsulfonyl.
17. The compound of claim 16, wherein m is 1 , R° is an optionally substituted 2-thiazole or 2-benzthiazole ring; R2 is H or optionally substituted Cι-6 alkyl, R17 is C6 or C]0 aryl, or C]-9 heteroaryl; and Y1 is a bond and B is an optionally substituted C1-6 alkyl, C .]6 aralkyl, C6 or Cio aryl, C1-9 heterocyclyl, or d-15 heterocyclylalkyl; or Y1 is NH and B is C]-9 heterocyclyl or C2-i5 heterocyclylalkyl.
18. The compound of claim 17, wherein m is 1, R is 2-thiazole; R is H; R is C6 or Cio aryl, or d-9 heteroaryl; Y1 is NH and B is Cι-9 heterocyclyl or C2-15 heterocyclylalkyl, and R1 is 3-guanidinopropyl.
19. The compound of claim 16, wherein Y1 is O or S.
20. The compound of claim 16, wherein
17
R is optionally substituted C6 or Cio aryl, Cι_9 heterocyclyl, Ci-6 alkylthio, C3-8 cycloalkylthio, C6 or C10 arylthio, C7-ι6 aralkylthio, C2-9 heterocyclylthio, or C2-i5 heteroaralkylthio;
Y1 is NH and B is C]- heterocyclyl or C2-ι5 heterocyclylalkyl.
21. The compound of claim 16, wherein m is 1; W is N;Y] is NH; and R17 is an optionally substituted C3-8 cycloalkyl, C6 or Cio aryl, or C1-9 heterocyclyl.
22. The compound of claim 1, wherein m is 0 and R° is H.
23. The compound of claim 22, wherein
R2 is H or an optionally substituted Cι-6 alkyl, C2,6 alkenyl, C3-8 cycloalkyl, C7_,6 aralkyl, C8.]6 aralkenyl, C6 or Cio aryl, C2.9 heterocyclyl, C2-ι5 heteroaralkyl, or C3-ι5 heteroaralkenyl;
R3 is H or Cι-6 alkyl;
X is N; and
A is R8-AA2, wherein AA2 is a single natural or unnatural alpha-amino acid residue and R8 is selected from the group consisting of:
Figure imgf000207_0001
wherein c is 0 or 1 ; each of R11, R12, R13, and R14 is, independently, C2-7 alkanoyl, Cι-6 alkyl, Ci-6 alkenyl, Cι-6 alkoxy, C2-ι2 alkoxyalkyl, C\.6 alkylsulfinyl, C2.i2 alkylsulfinylalkyl, C]-6 alkylsulfonyl, C6 or Cio aryl, C7-ι6 arylalkyl, amino, Cι_6 aminoalkyl, C7 or Cπ aryloyl, azido, Cι-6azidoalkyl, carboxaldehyde, carboxamide, carboxyl, C2-7 (carboxaldehyde)alkyl, C3-8 cycloalkyl, C4.]4 cycloalkylalkyl, halo, Cι-6 haloalkyl, C]-9 heterocyclyl, Cι-9 (heterocyclyl)oxy, C2.1o(heterocyclyl)oyl, hydroxy, Cj.6 hydroxyalkyl, nitro, C1-6 nitroalkyl, N- protected amino, N-protected aminoalkyl, d-6 thioalkoxy, C2-i2 thioalkoxyalkyl, C]-4 perfluoroalkyl, C perfluoroalkoxy, C6 or Cio aryloxy, C3-8 cycloalkoxy, C4.ι cycloalkylalkoxy, or C7-16 arylalkoxy, -(CH2)qCO2RA, wherein q is zero to four and RA is selected from the group consisting of (a) C
6 alkyl, (b) C6 or Cio aryl and (c) C7_ι6 arylalkyl, wherein the alkylene group is of one to six carbon atoms, -(CH2)qCONRBRc, wherein q is zero to four and RB and Rc are independently selected from the group consisting of (a) hydrogen, (b) C1-6 alkyl, (c) C6 or C10 aryl and (d) C7.16 arylalkyl, wherein the alkylene group is of one to six carbon atoms, -(CH2)qSO2RD, wherein q is zero to four and RD is selected from the group consisting of (a) hydrogen, (b) d-6 alkyl, (c) C6 or Cio aryl and (d) C7-]6 arylalkyl, wherein the alkylene group is of one to six carbon atoms, -(CH2)qSO2NRERF, wherein q is zero to four and RE and RF are independently selected from the group consisting of (a) hydrogen, (b) Cι-6 alkyl, (c) C6 or Cι0 aryl and (d) C7-]6 arylalkyl, wherein the alkylene group is of one to six carbon atoms, or -(CH2)qNRGRH, wherein q is zero to four and RG and RH are independently selected from the group consisting of (a) hydrogen, (b) an N-protecting group, (c) alkyl of one to six carbon atoms, (d) alkenyl of two to six carbon atoms, (e) alkynyl of two to six carbon atoms, (f) C6 or d0 aryl, (g) C7-ι6 arylalkyl, wherein the alkylene group is of one to six carbon atoms, (h) cycloalkyl of three to eight carbon atoms, and (i) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms, and the alkylene group is of one to ten carbon atoms, with the proviso that no two groups are bound to the nitrogen atom through a carbonyl group or a sulfonyl group; and each of R15 and R16 is, independently, H, Cι_6 alkyl; Cμ6 hydroxyalkyl; CM perfluoroalkyl, cyano, halo, -(CH2)qCO2RA, -(CH2)qCONRBRc, - (CH2)qSO2RD, -(CH2)qSO2NRERF, -(CH2)qNRGRH, wherein q is 0-2 and RA, RB, Rc, RD, RE, RF, RG, and RH are as defined above.
24. The compound of claim 23, wherein
R1 is selected from the group consisting of:
Figure imgf000208_0001
Figure imgf000209_0001
AA2 is selected from the group consisting of:
Figure imgf000209_0002
Figure imgf000210_0001
R2 is selected from the group consisting of:
Figure imgf000210_0002
25. The compound of claim 24, wherein
RAs:
Figure imgf000210_0003
wherein b is 0 or 1 and R is H; and
A is R8-AA2, wherein R8 is benzylsulfonyl, 3-methylcarboxybenzylsulfonyl, 3- carboxybenzylsulfonyl, 2-nitrobenzylsulfonyl, 1-proρylsulfonyl, and 3- chloropropylsulfonyl.
26. The compound of claim 1 , wherein said compound further comprises a linker attached to any of the groups R°, R2, or A, wherein said linker is attached to a group reactive to a blood component.
27. The compound of claim 26, wherein said blood component is an erythrocyte, a lymphocyte, a blood platelet, an immunoglobulin, serum albumin, ferritin, corticosteroid-binding globulin, sex hormone-binding globulin, transferrin, thyroxin-binding protein, or alpha-2-macroglobulin.
28. The compound of claim 26, wherein said linker attached to a group reactive to a blood component is maleimide- (CH2)b C(O)NHCH2CH2(OCH2CH2)aaOCH2C(O)-, maleimide- (CH2)bbC(O)NHCH2CH2(OCH2CH2)aaNΗCH2C(O)-, maleimide- (CH2)bbC(O)NHCH2CH2(OCH2CH2)aaNHC(S)-, maleimide-(CH2)bbNΗC(S)-, maleimide-(CH2)bbC(O)-, or maleimide (CH2)bb-wherein aa is 1-10 and bb is 1- 4.
29. The compound of claim 1, wherein said compound further comprises a polyethylene glycol moiety attached to any of the groups R , R , or A.
30. The compound of claim 29, wherein said said polyethylene glycol moiety is selected from the following group: H(OCH2CH2)ccO-, H3C(OCH2CH2)ccOC(O), H(OCH2CH2)ccOC(O), H3C(OCH2CH2)ccNHC(O), H(OCH2CH2)ccNHC(O), H3C(OCH2CH2)ccNHC(S), H(OCH2CH2)ccNHO(S), H3C(OCH2CH2)ccC(O), H(OCH2CH2)ccC(O), H3C(OCH2CH2)CCNHOH2C{O), H(OCH2CH2)ccNHCH2C(O), H3C(OCH2CH2)ccOCj(O)C(CH3)2-, and H(OCH2CH2)ccOC(O)C(CH3)2-, wherein cc is a range of numbers that results in an average molecular weight of said polyethylene glycol moiety of between 1,000-40,000.
31. The compound of claim 30, wherein cc is a range of numbers that results in an average molecular weight of said polyethylene glycol moiety of 20,000.
32. The compound of claim 30, wherein cc is a range of numbers that results in an average molecular weight of said polyethylene glycol moiety of 40,000.
33. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of claim 1, or a pharmaceutically acceptable salt or prodrug thereof.
34. A method of treating a patient in need of thromboembolic disorder treatment comprising administering to said patient a therapeutically effective amount of a compound of claim 1, or a pharmaceutically acceptable salt or prodrug thereof.
35. The method of claim 34, wherein the thromboembolic disorder is selected from the group consisting of arterial cardiovascular thromboembolic disorders, venous cardiovascular thromboembolic disorders, and thromboembolic disorders in the chambers of the heart.
36. The method of claim 34, wherein the thromboembolic disorder is selected from unstable angina, an acute coronary syndrome, first myocardial infarction, recurrent myocardial infarction, ischemic sudden death, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary arterial thrombosis, cerebral arterial thrombosis, cerebral embolism, kidney embolism, pulmonary embolism, and thrombosis resulting from (a) prosthetic valves or other implants, (b) indwelling catheters, (c) stents, {d) cardiopulmonary bypass, (e) hemodialysis, or (f) other procedures in which blood is exposed to an artificial surface that promotes thrombosis.
37. A compound of formul a II :
Figure imgf000213_0001
(II), or a pharmaceutically acceptable salt or prodrug thereof, wherein
R19 is H, CM alkyl, or when taken together with R20 forms a fused 5- or 6-membered ring;
R20 is H, OH, SH, NH2, NO2, optionally substituted C] -6 alkyl, halogen, Cι-3 perfluoroalkyl, or when together with Z' forms a fused 5- or 6-membered ring; e is N, NO, or CR21a, wherein R21a is H, halogen, or C]-3 perfluoroalkyl; g is N, NO, or CR21b, wherein R21b is H, halogen, C,-6 alkyl, C]-3 perfluoroalkyl, d-3 alkenyl, C7-]6 aralkyl, C -15 heteroaralkyl, C1-6 alkoxy, C7-16 aralkoxy, C2-ι5 heteroaralkoxy, C2.7 alkoxycarbonyl,
C8.]7 aralkoxycarbonyl, C3-] 6 heteroaralkoxycarbonyl, C2-7 alkylaminocarbonyl, C8-π aralkylaminocarbonyl, C3.16 heteroaralkylaminocarbonyl, C]-6 alkylthio, C -ι6 aralkylthio, C2_ι5 heteroaralkylthio, C2-7 alkylthiocarbonyl, C8-17 aralkylthiocarbonyl, 03.16 heteroaralkylthiocarbonyl;
Z' is NR22, (H, H), wherein R22 is H, Cι.6 alkyl, OH, NH2, NO2, CO2R22a, wherein R22a is Cι.6 alkyl;
T is a bond, O, S, or NR23, where R23 is H, OH, or optionally substituted d-6 alkyl, Cι-6 alkenyl, C7-π aralkyl, C8-ι6 aralkenyl, C2-ι5 heteroaralkyl, or 03.15 heteroaralkenyl, wherein when T is O, S, or NR , B is a natural or unnatural alpha-amino acid residue of D- or L-configuration, or an optionally substituted C1-6 alkyl, C3-8 cycloalkyl, ^ alkenyl, C7.16 aralkyl, C8.ι7 aralkylcarbonyl, C8.16 aralkenyl, C6 or Cio aryl, C2-ι5 heteroaralkyl, C3.16 heteroaralkylcarbonyl, C3.]5 heteroaralkenyl, C2-7 acyl, C4-9 cycloalkylcarbonyl, C7.π aroyl, C2-]0 heterocyclyloyl, C4-9 cycloalkoxycarbonyl, C7 or Cπ aryloxycarbonyl, C3.10 heterocyclyloxycarbonyl, C3-ι6 heteroaralkyloxycarbonyl, aminocarbonyl, C2.7 alkylaminocarbonyl, 03.13 dialkylaminocarbonyl, C -9 cycloalkylaminocarbonyl, Cs-17 aralkylaminocarbonyl, C or Cπ arylaminocarbonyl, 03.10 heterocyclylaminocarbonyl, 03.16 heteroaralkylaminocarbonyl, C2-7 alkylthiocarbonyl, C -9 cycloalkylthiocarbonyl, C7-π arylthiocarbonyl, C8-ι7 aralkylthiocarbonyl, C2-ι0 heterocyclylthiocarbonyl, C3.16 heteroaralkylthiocarbonyl, aminothiocarbonyl, C2-7 alkylaminothiocarbonyl, C3- ]3 dialkylaminothiocarbonyl, C4-9 cycloalkylaminothiocarbonyl, C8-π aralkylaminothiocarbonyl, C7 or On arylaminothiocarbonyl, C3.10 heterocyclylaminothiocarbonyl, C3.16 heteroaralkylaminothiocarbonyl, Cι-6 alkylsulfonyl, C3-8 cycloalkylsulfonyl, C7.]6 aralkylsulfonyl, C6 or Cj0 arylsulfonyl, C -9 heterocyclylsulfonyl, or C2-ι5 heteroaralkylsulfonyl, or when T is a bond, B is OH, SH, NH2, NO2, SO3H, CO2H or an optionally substituted d.6 alkyl, C e alkenyl, C7-ι6 aralkyl, C8-ι6 aralkenyl, C2- 5 heteroaralkyl, C3-ι5 heteroaralkenyl, C2-7 acyl, C7.π aroyl, C3.ιo heteroaroyl, C2.7 alkoxycarbonyl, C4-9 cycloalkoxycarbonyl, C8-ι7 aralkoxycarbonyl, C7 or On aryloxycarbonyl, C3-]6 heteroaralkyloxycarbonyl, C2-ιo heterocyclyloxycarbonyl, aminocarbonyl, C -7 alkylaminocarbonyl, C3-ι3 dialkylaminocarbonyl, C4.9 cycloalkylaminocarbonyl, C8.ι7 aralkylaminocarbonyl, C or Cπ arylaminocarbonyl, C3.10 heterocyclylaminocarbonyl, C3.]6 heteroaralkylaminocarbonyl, C2.7 alkylthiocarbonyl, C4.9 cycloalkylthiocarbonyl, C .π arylthiocarbonyl, C8.π aralkylthiocarbonyl, C2.ι0 heterocyclylthiocarbonyl, C3.16 heteroaralkylthiocarbonyl, d_6 alkylsulfonyl, C3-8 cycloalkylsulfonyl, C7-ι6 aralkylsulfonyl, C6 or C10 arylsulfonyl, C2.9 heterocyclylsulfonyl, or C2_ι5 heteroaralkylsulfonyl; f is a bond, O, S, or NR24, wherein R24 is H, OH, or C]-6 alkyl, wherein when f is O, S, or NR24, V is H or an optionally substituted C]-6 alkyl, C7-i6 aralkyl, C2-ι5 heteroaralkyl, C2-7 acyl, C7.π aroyl, C3-ι0 heteroaroyl, C2-7 alkoxycarbonyl, C - cycloalkoxycarbonyl, C8.17 aralkoxycarbonyl, C7 or Cπ aryloxycarbonyl, C3.ι6 heteroaralkyloxycarbonyl, C2.]0 heterocyclyloxycarbonyl, aminocarbonyl, C2.7 alkylaminocarbonyl, C3-i3 dialkylaminocarbonyl, C4- cycloalkylaminocarbonyl, C8-ι7 aralkylaminocarbonyl, C7 or Cπ arylaminocarbonyl, d-io heterocyclylaminocarbonyl, C3.16 heteroaralkylaminocarbonyl, C2- alkylthiocarbonyl, C4-9 cycloalkylthiocarbonyl, C7.π arylthiocarbonyl, C8-ι7 aralkylthiocarbonyl, C2.ιo heterocyclylthiocarbonyl, C3.ι6 heteroaralkylthiocarbonyl, d.6 alkylsulfonyl, C3.8 cycloalkylsulfonyl, C7.16 aralkylsulfonyl, C6 or C]0 arylsulfonyl, C2-9 heterocyclylsulfonyl, or C2.]5 heteroaralkylsulfonyl, or when f is a bond, V is H, OH, SH, NH2, SO3H, CO2H, or an optionally substituted CM alkyl, hydroxy-C].6 alkyl, C3.8 cycloalkyl, C alkenyl, C7.ι6 aralkyl, C8_ι6 aralkenyl, C2.15 heteroaralkyl, C3-ι5 heteroaralkyl, C6 or Cio aryl, C]-9 heterocyclyl, C2- alkylaminocarbonyl, C3.ι3 dialkylaminocarbonyl, C4-9 cycloalkylaminocarbonyl, C8-ι7 aralkylaminocarbonyl, C7 or Cπ arylaminocarbonyl, C2-ιo heterocyclylaminocarbonyl, C2.7 alkoxycarbonyl, C .9 cycloalkoxycarbonyl, C8. ]7 aralkoxycarbonyl, C7 or Cπ aryloxycarbonyl, C3.10 heterocyclyloxycarbonyl, C3.]6 heteroaralkyloxycarbonyl, C2.7 alkylthiocarbonyl, C4.9 cycloalkylthiocarbonyl, C7.π arylthiocarbonyl, C8.]7 aralkylthiocarbonyl, C2_ιo heterocyclylthiocarbonyl, C3.]6 heteroaralkylthiocarbonyl, C2.7 acyl, C7.π aroyl, C3.10 heteroaroyl, C alkylsulfonyl, C3.8 cycloalkylsulfonyl, C7.ι6 aralkylsulfonyl, C6 or C10 arylsulfonyl, C .9 heterocyclylsulfonyl, C2.ι5 heteroaralkylsulfonyl, C].6 alkylaminosulfonyl, C3.8 cycloalkylaminosulfonyl, C2-12 dialkylaminosulfonyl, C7.17 aralkylaminosulfonyl, C6 or do arylaminosulfonyl, C].9 heterocyclylaminosulfonyl, C2-15 heteraralkylaminosulfonyl, Cι.6 alkylsulfinyl, C3.8 cycloalkylsulfinyl, C7.] 6 aralkylsulfinyl, C6 or C]0 arylsulfinyl, C2.9 heterocyclylsulfmyl, or C2.ι5 heteroaralkylsulfinyl.
38. The compound of claim 37, wherein T is NR23, wherein R23 is H;
B is an optionally substituted optionally substituted C2.7 acyl, C4.9 cycloalkylcarbonyl, C7-π aroyl, C2-ι0heterocyclylcarbonyl, C3.10 heteroaroyl, C2-7 alkoxycarbonyl, C4- cycloalkoxycarbonyl, C8-π aralkoxycarbonyl, C7 or Cπ aryloxycarbonyl, C3-ι0 heterocyclyloxycarbonyl, C3-ι6 heteroaralkyloxycarbonyl, C2-7 alkylthiocarbonyl, C4-9 cycloalkylthiocarbonyl, C7-π aralkylthiocarbonyl, C7 or Cπ arylthiocarbonyl, C3-ι6 heteroaralkylthiocarbonyl, C3-ιo heterocyclylthiocarbonyl, aminothiocarbonyl, C2-7 alkylaminocarbonyl, C3-ι3 dialkylaminocarbonyl, C4-9 cycloalkylaminocarbonyl, C8-ι7 aralkylaminocarbonyl, C7 or Cn arylaminocarbonyl, C3-ι0 heterocyclylaminocarbonyl, C3-16 heteroaralkylaminocarbonyl, Cμ6 alkylsulfonyl, C3-8 cycloalkylsulfonyl, C7-ι6 aralkylsulfonyl, C6 or Cio arylsulfonyl, C2-9 heterocyclylsulfonyl, or C2-15 heteroaralkylsulfonyl; and f is O, S, or NR24.
39. The compound of claim 37, wherein T is NR23, wherein R23 is H, and B is an optionally substituted C6 or Cj0 aroyl or an optionally substituted C1-9 heteroaroyl.
40. The compound of claim 39, wherein B is
Figure imgf000217_0001
, wherein each of R25, R 6,and R27 is, independently H, C2-7 alkanoyl, C]-6 alkyl, C,. 6 alkenyl, C]-6 alkoxy, C2-ι2 alkoxyalkyl, C1-6 alkylsulfinyl, C2-i2 alkylsulfinylalkyl, Cj.6 alkylsulfonyl, C6 or C]0 aryl, C7-]6 arylalkyl, amino, C]-6 aminoalkyl, C7 or Cn aryloyl, azido, C]-6 azidoalkyl, carboxaldehyde, carboxamide, carboxyl, C2-7 (carboxaldehyde)alkyl, C3-8 cycloalkyl, C4.]4 cycloalkylalkyl, halo, Cι-6 haloalkyl, C1-9 heterocyclyl, Cι_9 (heterocyclyl)oxy, C2-ιo (heterocyclyl)oyl, hydroxy, C1-6 hydroxyalkyl, nitro, cyano, Cι-6 nitroalkyl, N-protected amino, N-protected aminoalkyl, C thioalkoxy, C2.12 thioalkoxyalkyl, thiol, -(CH2)qCO2RA, wherein q is zero to four and RA is selected from the group consisting of (a) C alkyl, (b) C6 or Cι0 aryl and (c) C7.16 arylalkyl, wherein the alkylene group is of one to six carbon atoms, - (CH2)qCONRBRc, wherein q is zero to four and RB and Rc are independently selected from the group consisting of (a) hydrogen, (b) d.6 alkyl, (c) C6 or do aryl and (d) C7.j6 arylalkyl, wherein the alkylene group is of one to six carbon atoms, -(CH2)qSO RD, wherein q is zero to four and RD is selected from the group consisting of (a) hydrogen, (b) C]-6 alkyl, (c) C6 or Cio aryl and (d) C7-ι6 arylalkyl, wherein .the alkylene group is of one to six carbon atoms, - (CH2)qSO2NRERF, wherein q is zero to four and RE and RF are independently selected from the group consisting of (a) hydrogen, (b) Cι-6 alkyl, (c) C or do aryl and (d) C7-]6 arylalkyl, wherein the alkylene group is of one to six carbon atoms, -(CH2)qNRGRH, wherein q is zero to four and RG and RH are independently selected from the group consisting of (a) hydrogen, (b) an N- protecting group, (c) alkyl of one to six carbon atoms, (d) alkenyl of two to six carbon atoms, (e) alkynyl of two to six carbon atoms, (f) C6 or C]0 aryl, (g) C7- 16 arylalkyl, wherein the alkylene group is of one to six carbon atoms, (h) cycloalkyl of three to eight carbon atoms, and (i) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms, and the alkylene group is of one to ten carbon atoms, with the proviso that no two groups are bound to the nitrogen atom through a carbonyl group or a sulfonyl group, C]- perfluoroalkyl, C]-4 perfluoroalkoxy; C6 or C]0 aryloxy, C3-8 cycloalkoxy, C4-ι4 cycloalkylalkoxy, or C7-ι6 arylalkoxy.
41. The compound of claim 40, wherein f is a bond and V is an optionally substituted C]-6 alkyl, hydroxy-C]-6 alkyl, C3-8 cycloalkyl, C1-6 alkenyl, C7.]6 aralkyl, C8-i6 aralkenyl, C2.]5 heteroaralkyl, C3.15 heteroaralkyl, C6 or do aryl, C1.9 heterocyclyl, C2.7 alkylaminocarbonyl, C3-ι3 dialkylaminocarbonyl, C -9 cycloalkylaminocarbonyl, C8.]7 aralkylaminocarbonyl, C7 or Cπ arylaminocarbonyl, C2-ιo heterocyclylaminocarbonyl, C2-7 alkoxycarbonyl, C4-9 cycloalkoxycarbonyl, C8-ι7 aralkoxycarbonyl, C7 or Cπ aryloxycarbonyl, C3-ι0 heterocyclyloxycarbonyl, C3-16 heteroaralkyloxycarbonyl, C2.7 alkylthiocarbonyl, C4. cycloalkylthiocarbonyl, C7.π arylthiocarbonyl, C8.ι7 aralkylthiocarbonyl, d-io heterocyclylthiocarbonyl, C3.16 heteroaralkylthiocarbonyl, C2.7 acyl, C7.π aroyl, C3.10 heteroaroyl, C alkylsulfonyl, C3.8 cycloalkylsulfonyl, C7.ι6 aralkylsulfonyl, C6 or Cι0 arylsulfonyl, C - heterocyclylsulfonyl, or C2-]5 heteroaralkylsulfonyl, Cι-6 alkylaminosulfonyl, d-s cycloalkylaminosulfonyl, C2-12 dialkylaminosulfonyl, C7-17 aralkylaminosulfonyl, C6 or C10 arylaminosulfonyl, Cι_9 heterocyclylaminosulfonyl, C2.i5 heteraralkylaminosulfonyl, Cι.6 alkylsulfinyl, d.8 cycloalkylsulfinyl, C7.ι6 aralkylsulfinyl, C6 or C10 arylsulfinyl, C2. heterocyclylsulfinyl, or C2-15 heteroaralkylsulfinyl.
42. The compound of claim 41, wherein f is a bond and V is optionally substituted C6 or C10 arylaminocarbonyl or C7_ι7 aralkylaminocarbonyl.
43. The compound of claim 41, wherein R25 is H, halo, or C]-6 alkyl; R26 is OH; and R27 is H.
44. The compound of claim 37, wherein f is a bond and V is H.
45. The compound of claim 44, wherein
B is
Figure imgf000219_0001
, wherein each of R25, R26,and R27 is, independently H, C2.7 alkanoyl, CM alkyl, Ci. 6 alkenyl, d_6 alkoxy, C2.ι2 alkoxyalkyl, Cι_6 alkylsulfinyl, C2-12 alkylsulfinylalkyl, C].6 alkylsulfonyl, C6 or C]0 aryl, C7-ι6 arylalkyl, amino, CM aminoalkyl, C7 or Cπ aryloyl, azido, C].6 azidoalkyl, carboxaldehyde, carboxamide, carboxyl, C2-7 (carboxaldehyde)alkyl, C3.8 cycloalkyl, C .]4
I cycloalkylalkyl, halo, C]-6 haloalkyl, Cμ9 heterocyclyl, Cι-9 (heterocyclyl)oxy, C2.ιo(heterocyclyl)oyl, hydroxy, d_6 hydroxyalkyl, nitro, C].6 nitroalkyl, N- protected amino, N-protected aminoalkyl, C1-6 thioalkoxy, C2.12 thioalkoxyalkyl, thiol, -(CH2)qCO2RA, wherein q is zero to four and RA is selected from the group consisting of (a) C]-6 alkyl, (b) C6 or Cio aryl and (c) -iό arylalkyl, wherein the alkylene group is of one to six carbon atoms, - (CH2)qCONRBRc, wherein q is zero to four and RB and Rc are independently selected from the group consisting of (a) hydrogen, (b) Cι-6 alkyl, (c) C6 or Cio aryl and (d) C7-] 6 arylalkyl, wherein the alkylene group is of one to six carbon atoms, -(CH2)qSO2RD, wherein q is zero to four and RD is selected from the group consisting of (a) hydrogen, (b) d_6 alkyl, (c) C6 or C10 aryl and (d) C7_]6 arylalkyl, wherein the alkylene group is of one to six carbon atoms, - (CH2)qSO2NRERF, wherein q is zero to four and RE and RF are independently selected from the group consisting of (a) hydrogen, (b) CM alkyl, (c) C6 or Cio aryl and (d) C7.16 arylalkyl, wherein the alkylene group is of one to six carbon atoms, -(CH2)qNRGRH, wherein q is zero to four and RG and RH are independently selected from the group consisting of (a) hydrogen, (b) an N- protecting group, (c) alkyl of one to six carbon atoms, (d) alkenyl of two to six carbon atoms, (e) alkynyl of two to six carbon atoms, (f) C6 or Cio aryl, (g) C7. ι6 arylalkyl, wherein the alkylene group is of one to six carbon atoms, (h) cycloalkyl of three to eight carbon atoms, and (i) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms, and the alkylene group is of one to ten carbon atoms, with the proviso that no two groups are bound to the nitrogen atom through a carbonyl group or a sulfonyl group, C perfluoroalkyl, CM perfluoroalkoxy; C6 or Cio aryloxy, C3.8 cycloalkoxy, C4.]4 cycloalkylalkoxy, or C7.]6 arylalkoxy.
46. The compound of claim 45, wherein R25 is H, halo, or d.6 alkyl; R26 is OH; and R27 is H.
47. The compound of claim 37, wherein said compound further comprises a linker attached to any of the groups B or V, wherein said linker is attached to a group reactive to a blood component.
48. The compound of claim 47, wherein said blood component is an erythrocyte, a lymphocyte, a blood platelet, an immunoglobulin, serum albumin, ferritin, corticosteroid-binding globulin, sex hormone-binding globulin, transferrin, thyroxin-binding protein, or alpha-2-macroglobulin.
49. The compound of claim 47, wherein said linker attached to a group reactive to a blood component is maleimide- (CH2)bbC(O)NHCH2CH2(OCH2CH2)aaOCH2C(O)-, maleimide- (CH2)bbC(O)NHCH2CH2(OCH2CH2)aaNHCH2C(O)-, maleimide- (CH2)bbC(O)NHCH2CH2(OCH2CH2)aaNHC(S)-, maleimide-(CH2)bbNHC(S), maleimide-(CH )bbC(O)-, or maleimide-(CH2)b -, wherein aa is 1-10 and bb is 1-4.
50. The compound of claim 37, wherein said compound further comprises a polyethylene glycol moiety attached to any of the groups B or V.
51. The compound of claim 50, wherein said said polyethylene glycol moiety is selected from the following group: H(OCH2CH2)ccO-, H3C(OCH2CH2)ccOC(O), H(OCH2CH2)ccOC(O), H3C(OCH2CH2)ccNHC(O), H(OCH2CH2)ccNHC(O), H3C(OCH2CH2)ccNHC(S), H(OCH2CH2)ccNHC(S), H3C(OCH2CH2)CcC(O), H(OCH2CH2)ccC(O), H3C(OCH2CH2)ccNHCH2C(O), H(OCH2CH2)ccNHCH2C(O), H3C(OCH2CH2)ccOC(O)C(CH3)2-, and H(OCH CH2)ccOC(O)C(CH3)2-, wherein cc is a range of numbers that results in an average molecular weight of said polyethylene glycol moiety of between 1,000-40,000.
52. The compound of claim 50, wherein cc is a range of numbers that results in an average molecular weight of said polyethylene glycol moiety of 20,000.
53. The compound of claim 51, wherein cc is a range of numbers that results in an average molecular weight of said polyethylene glycol moiety of 40,000.
54. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of claim 37, or a pharmaceutically acceptable salt or prodrug thereof.
55. A method of treating a patient in need of thromboembolic disorder treatment comprising administering to said patient a therapeutically effective amount of a compound of claim 37, or a pharmaceutically acceptable salt or prodrug thereof.
56. The method of claim 55, wherein the thromboembolic disorder is selected from the group consisting of arterial cardiovascular thromboembolic disorders, venous cardiovascular thromboembolic disorders, and thromboembolic disorders in the chambers of the heart.
57. The method of claim 55, wherein the thromboembolic disorder is selected from unstable angina, an acute coronary syndrome, first myocardial infarction, recurrent myocardial infarction, ischemic sudden death, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary arterial thrombosis, cerebral arterial thrombosis, cerebral embolism, kidney embolism, pulmonary embolism, and thrombosis resulting from (a) prosthetic valves or other implants, (b) indwelling catheters, (c) stents, (d) cardiopulmonary bypass, (e) hemodialysis, or (f) other procedures in which blood is exposed to an artificial surface that promotes thrombosis.
58. A computer comprising a processor in electrical communication with a memory; said memory having stored therein
(i) atomic coordinates, or surrogates thereof, in one or more of the files listed in Figure 16 for all of the non-hydrogen atoms of residues serine 195, histidine 57, and aspartic acid 102 of Factor XI catalytic domain or atomic coordinates that have a root mean square deviation from the backbone atoms of said residues of less than 0.4 A; and
(ii) a program for displaying a three-dimensional model of said Factor XI catalytic domain.
59. A computer comprising a processor in electrical communication with a memory; said memory having stored therein
(i) x-ray diffraction data for all of the non-hydrogen atoms of residues serine 195, histidine 57, and aspartic acid 102 of Factor XI catalytic domain or x-ray diffraction data for amino acids that have a root mean square deviation from the backbone atoms of said residues of less than 0.4 A; and
(ii) a program for displaying a three-dimensional model of said Factor XI catalytic domain.
60. A computer comprising a processor in electrical communication with a memory; said memory having stored therein a pharmacophore model of Factor XI ligands and a program for displaying said model, said model comprising at least three of the following:
(i) an electrophilic carbon atom that binds to the side chain oxygen atom (OG) of Ser 195 such that the electrophilic carbon atom is 1.4 to 4.0 A from said oxygen atom;
(ii) at least one hydrogen bond donor group to interact with Aspl 89 such that the non-hydrogen atoms of the hydrogen bond donor groups are 2.0 to 4.2 A away from the side chain oxygens of Aspl 89;
(iii) a hydrophobic group that makes one or more van der Waals contacts with residues of the active site and is less than 5.0 A away from the CB atom of Ala97;
(iv) a hydrogen bond donor group which makes a hydrogen bond with the main chain carbonyl of Ser214 such that the distance between the hydrogen-bonded atoms is less than 4.0 A;
(v) a hydrogen bond donor group that makes a hydrogen bond with the main chain carbonyl of Gly216 such that the distance between the hydrogen- bonded atoms is less than 4.0 A; (vi) a hydrogen bond acceptor group that makes a hydrogen bond with the main chain nitrogen of Gly216 such that the distance between the hydrogen-bonded atoms is less than 4.0 A;
(vii) a hydrogen bond acceptor group that makes a hydrogen bond with a side chain nitrogen of His57 such that the distance between the hydrogen- bonded atoms is less than 4.0 A;
(viii) a group that makes one or more van der Waals contacts with the side chains of residues ofLysl92 and/or Leul46;
(ix) a group that makes one or more van der Waals contacts with the side chains of residues of Cysl91 and/or Cys219; and
(x) a group or groups that make participate in one or more polar interactions with the side chain of residue of Hisl74, Glu98, and or Glu217.
61. A method of selecting or designing a candidate ligand for Factor Xla, said method comprising the steps of:
(a) generating in a computer a three-dimensional structure of Factor XI catalytic domain based on the atomic coordinates in one or more of the files listed in Figure 16 for all of the non-hydrogen atoms of residues serine 195, histidine 57, and aspartic acid 102 or atomic coordinates that have a root mean square deviation from the backbone atoms of said residues of less than 0.4 A; and
(b) selecting or designing a candidate ligand having sufficient surface complementary to said structure to bind Factor Xla in an aqueous solution.
62. The method of claim 61, further comprising determining the ability of said candidate ligand to bind said Factor Xla in vitro or in vivo.
63. The method of claim 61, further comprising determining the ability of said candidate ligand to inhibit the enzymatic activity of said Factor Xla in vitro or in vivo.
64. The method of claim 61, wherein said three-dimensional structure further comprises the hydrogen atoms of residues serine 195, histidine 57, and aspartic acid 102.
65. The method of claim 64, wherein said three-dimensional structure further comprises the non-hydrogen atoms of residues Tyr5901, Ala97, Glu98, ASP 102, Ilel46, Hisl74, Aspl89, Alal90, Cysl91, Lysl92, Glyl93, Aspl94, Ser214, Tφ215, Gly216, Glu217, and Gly218.
66. The method of claim 65, wherein said three-dimensional structure further comprises the hydrogen atoms of residues Tyr5901, Ala97, Glu98, ASP 102, Ilel46, Hisl74, Aspl89, Alal90, Cysl91, Lysl92, Glyl93, Aspl94, Ser214, Tφ215, Gly216, Glu217, and Gly218.
67. A method for manufacturing a Factor Xla ligand, said method comprising the steps of:
(a) obtaining the atomic coordinates of at least a portion of Factor XI catalytic domain with a ligand;
(b) selecting one or more moieties in said ligand to be modified; wherein said modified ligand maintains the ability to bind said Factor XI; and
(c) modifying said ligand based on said determination.
68. The method of claim 67, further comprising determining the ability of said candidate ligand to bind said Factor Xla in vitro or in vivo.
69. The method of claim 67, further comprising determining the ability of said candidate ligand to inhibit the enzymatic activity of said Factor Xla in vitro or in vivo.
70. The method of claim 67, wherein said atomic coordinates further comprises the hydrogen atoms of residues serine 195, histidine 57, and aspartic acid 102.
71. The method of claim 70, wherein said atomic coordinates further comprises the non-hydrogen atoms of residues Tyr5901, Ala97, Glu98, ASP 102, Ilel46, Hisl74, Aspl89, Alal90, Cysl91, Lysl92, Glyl93, Aspl94, Ser214, Tφ215, Gly216, Glu217, and Gly218.
72. The method of claim 71, wherein said atomic coordinates further comprises the hydrogen atoms of residues Tyr5901, Ala97, Glu98, ASP 102, Hel46, Hisl74, Aspl89, Alal90, Cysl91, Lysl92, Glyl93,< Aspl94, Ser214, Tφ215, Gly216, Glu217, and Gly218.
73. The method of claim 67, further comprising crystallizing Factor XI catalytic domain (FXIcat) with a ligand.
74. The method of claim 67, wherein said ligand specifically binds said FXIcat.
75. The method of claim 67, wherein said modification increases the affinity of said ligand for said FXIcat.
76. The method of claim 67, wherein said modification increases the aqueous solubility of said ligand.
77. The method of claim 67, wherein said modification increases the blood circulation half-life of said ligand in vivo.
78. A method for manufacturing a Factor XI catalytic domain ligand, said method comprising manufacturing a ligand that binds Factor Xla; wherein said ligand is designed or selected based on information obtained using a model of the atomic coordinates of at least a portion of said Factor XI catalytic domain.
79. A method of evaluating the ability of a candidate ligand to bind Factor Xla, the method comprising the steps of:
(a) generating in a computer a three-dimensional structure of Factor XI catalytic domain based on the atomic coordinates in one or more of the files listed in Figure 16 of all of the non-hydrogen atoms of residues serine 195, histidine 57, and aspartic acid 102 or atomic coordinates that have a root mean square deviation from the backbone atoms of said residues of less than 0.4 A; and
(b) employing computational means to measure the interaction between said candidate ligand and said Factor XI catalytic domain.
80. The method of claim 79, further comprising determining the ability of said candidate ligand to bind said Factor Xla in vitro or in vivo.
81. The method of claim 79, further comprising determining the ability of said candidate ligand to inhibit the enzymatic activity of said Factor Xla in vitro or in vivo.
82. The method of claim 79, wherein said three-dimensional structure further comprises the hydrogen atoms of residues serine 195, histidine 57, and aspartic acid 102.
83. The method of claim 82, wherein said three-dimensional structure further comprises the non-hydrogen atoms of residues Tyr5901, Ala97, Glu98, ASP 102, Ilel46, Hisl74, Aspl89, Alal90, Cysl91, Lysl92, Glyl93, Aspl94, Ser214, Tφ215, Gly216, Glu217, and Gly218.
84. The method of claim 83, wherein said three-dimensional structure further comprises the hydrogen atoms of residues Tyr5901, Ala97, Glu98, ASP 102, Ilel46, Hisl74, Aspl89, Alal90, Cysl91, Lysl92, Glyl93, Aspl94, Ser214, Tφ215, Gly216, Glu217, and Gly218.
85. A method of identifying a candidate ligand for Factor Xla, the method comprising the steps of:
(a) generating a three-dimensional pharmacophore model of Factor XI catalytic domain ligands using a computer of claim 60; and
(b) selecting a candidate ligand satisfying the criteria of said pharmacophore model.
86. The method of claim 85, further comprising determining the ability of said candidate ligand to bind said Factor Xla in vitro or in vivo.
87. The method of claim 85, further comprising determining the ability of said candidate ligand to inhibit the enzymatic activity of said Factor Xla in vitro or in vivo.
88. The method of claim 85, wherein said three-dimensional pharmacophore model further comprises the hydrogen atoms of residues serine 195, histidine 57, and aspartic acid 102.
89. The method of claim 88, wherein said three-dimensional pharmacophore model further comprises the non-hydrogen atoms of residues Tyr5901, Ala97, Glu98, ASP 102, Ilel46, Hisl74, Aspl89, Alal90, Cysl91, Lysl92, Glyl93, Aspl94, Ser214, Tφ215, Gly216, Glu217, and Gly218.
90. The method of claim 89, wherein said three-dimensional pharmacophore model further comprises the hydrogen atoms of residues Tyr5901, Ala97, Glu98, ASP 102, He 146, His 174, Aspl 89, Ala 190, Cysl91, Lys 192, Gly 193, Aspl94, Ser214, Tφ215, Gly216, Glu217, and Gly218.
91. A crystal of Factor XI that has a size of at least 10 μ in the smallest dimension. /
92. A method of obtaining a structural model of a Factor XI catalytic domain of interest, said method comprising homology modeling using at least a portion of the atomic coordinates in one or more of the files listed in Figure 16 and at least a portion of the amino acid sequence of said Factor XI catalytic domain of interest, thereby generating a model of said Factor XI catalytic domain of interest.
93. A method of determining the three-dimensional structure of a Factor XI of interest, said method comprising the steps of:
(a) crystallizing said Factor XI catalytic domain of interest;
(b) generating an X-ray diffraction pattern from said crystallized Factor XI catalytic domain of interest; and (c) applying at least a portion of the atomic coordinates in one or more of the files listed in Figure 16 to said diffraction pattern to generate a three- dimensional electron density map of at least a portion of said Factor XI catalytic domain of interest.
94. A purified, less than full-length fragment of Factor XI comprising residues 370-607 of SEQ ID NO: 1 (human Factor Xla).
95. A nucleic acid encoding a protein of claim 94.
96. A purified, less than full-length fragment of Factor XI comprising residues 369-606 of SEQ ID NO: 2 (rabbit Factor Xla).
97. A nucleic acid encoding a protein of claim 96.
98. A purified, less than full-length fragment of Factor XI comprising residues 372-606 of SEQ ID NO: 3 (mouse Factor Xla) or residues 1-234 of SEQ ID NO: 4 (rat Factor Xla).
99. A nucleic acid encoding a protein of claim 98.
100. A purified Factor XI protein or fragment thereof comprising a mutation, wherein said mutation is
(a) a mutation that enhances the ability of Factor XI catalytic domain to crystallize,
(b) a mutation of a residue that is otherwise post-translationally modified in an organism used for recombinant expression,
(c) a mutation that alters the charge of Factor XI, (d) a mutation that eliminates a free, reactive sulfhydryl group of a cysteine,
(e) a combination of mutations that together alter the distribution of charge density without altering the overall charge of Factor XI,
(f) a mutation of the NH2- or COOH-terminal residue of Factor XI, or
(g) a mutation that alters the folding of Factor XI.
101. A nucleic acid encoding a protein of claim 100.
102. The protein or fragment of claim 100 comprising one of the following mutations:
(i) S434A;
(ii) T475A;
(iii) S434A, T475A;
(iv) S434A, T475A, K422A
(v) S434A, T475A, K437A;
(vi) S434A, T475A, K486A;
(vii) S434A, T475A, K505A;
(viii) S434A, T475A, K509A;
(ix) S434A, T475A, C482S;
(x) S434A, T475A, C482S, K437A;
(xi) S434A, T475A, C482S, R479A;
(xii) S434A, T475A, C482S, K505A;
(xiii) S434A, T475A, C482S, D476A;
(xiv) S434A, T475A, AVC-terminal trucation; or
(xv) S434A, T475A, C482S, Y416S.
103. A nucleic acid encoding a protein of claim 102.
PCT/US2004/010349 2003-04-02 2004-04-02 Compounds and methods for treatment of thrombosis WO2004103270A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US45991003P 2003-04-02 2003-04-02
US60/459,910 2003-04-02

Publications (2)

Publication Number Publication Date
WO2004103270A2 true WO2004103270A2 (en) 2004-12-02
WO2004103270A3 WO2004103270A3 (en) 2005-05-12

Family

ID=33159711

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/US2004/010300 WO2004089297A2 (en) 2003-04-02 2004-04-02 Compounds and methods for treatment of thrombosis
PCT/US2004/010349 WO2004103270A2 (en) 2003-04-02 2004-04-02 Compounds and methods for treatment of thrombosis

Family Applications Before (1)

Application Number Title Priority Date Filing Date
PCT/US2004/010300 WO2004089297A2 (en) 2003-04-02 2004-04-02 Compounds and methods for treatment of thrombosis

Country Status (2)

Country Link
US (1) US7217794B2 (en)
WO (2) WO2004089297A2 (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1757290A1 (en) 2005-08-16 2007-02-28 Zentaris GmbH Novel triazole derivatives as ghrelin analogue ligands of growth hormone secretagogue receptors
EP2431035A1 (en) 2010-09-16 2012-03-21 Æterna Zentaris GmbH Novel Triazole Derivatives with Improved Receptor Activity and Bioavailability Properties as Ghrelin Antagonists of Growth Hormone Secretagogue Receptors
US10465011B2 (en) 2015-06-26 2019-11-05 Novartis Ag Factor XI antibodies and methods of use
US10647780B2 (en) 2016-05-25 2020-05-12 Novartis Ag Reversal binding agents for anti-factor XI/XIa antibodies and uses thereof
US11168147B2 (en) 2016-12-23 2021-11-09 Novartis Ag Factor XI antibodies and methods of use

Families Citing this family (48)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7217794B2 (en) * 2003-04-02 2007-05-15 Daiamed, Inc. Compounds and methods for treatment of thrombosis
UA87854C2 (en) * 2004-06-07 2009-08-25 Мерк Энд Ко., Инк. N-(2-benzyl)-2-phenylbutanamides as androgen receptor modulators
US9512125B2 (en) 2004-11-19 2016-12-06 The Regents Of The University Of California Substituted pyrazolo[3.4-D] pyrimidines as anti-inflammatory agents
DK2004654T3 (en) 2006-04-04 2013-07-22 Univ California Pyrazolopyrimidine derivatives for use as kinase antagonists
MX2009012847A (en) * 2007-06-13 2009-12-08 Bristol Myers Squibb Co Dipeptide analogs as coagulation factor inhibitors.
WO2009046448A1 (en) 2007-10-04 2009-04-09 Intellikine, Inc. Chemical entities and therapeutic uses thereof
US8703777B2 (en) 2008-01-04 2014-04-22 Intellikine Llc Certain chemical entities, compositions and methods
US8193182B2 (en) 2008-01-04 2012-06-05 Intellikine, Inc. Substituted isoquinolin-1(2H)-ones, and methods of use thereof
JP5547099B2 (en) * 2008-03-14 2014-07-09 インテリカイン， エルエルシー Kinase inhibitors and methods of use
WO2009114874A2 (en) 2008-03-14 2009-09-17 Intellikine, Inc. Benzothiazole kinase inhibitors and methods of use
AU2009268611B2 (en) 2008-07-08 2015-04-09 Intellikine, Llc Kinase inhibitors and methods of use
US20110224223A1 (en) 2008-07-08 2011-09-15 The Regents Of The University Of California, A California Corporation MTOR Modulators and Uses Thereof
JP5731978B2 (en) 2008-09-26 2015-06-10 インテリカイン， エルエルシー Heterocyclic kinase inhibitor
ES2570429T3 (en) 2008-10-16 2016-05-18 Univ California Condensed Ring Heteroaryl Kinase Inhibitors
US8476282B2 (en) 2008-11-03 2013-07-02 Intellikine Llc Benzoxazole kinase inhibitors and methods of use
JP5789252B2 (en) 2009-05-07 2015-10-07 インテリカイン， エルエルシー Heterocyclic compounds and uses thereof
EP2305289A1 (en) 2009-09-16 2011-04-06 Bio-Products & Bio-Engineering Aktiengesellschaft Medicinal products for the treatment of blood coagulation disorders
US8980899B2 (en) 2009-10-16 2015-03-17 The Regents Of The University Of California Methods of inhibiting Ire1
AU2011255218B2 (en) 2010-05-21 2015-03-12 Infinity Pharmaceuticals, Inc. Chemical compounds, compositions and methods for kinase modulation
EP2637669A4 (en) 2010-11-10 2014-04-02 Infinity Pharmaceuticals Inc Heterocyclic compounds and uses thereof
JP2014501790A (en) 2011-01-10 2014-01-23 インフィニティー ファーマシューティカルズ， インコーポレイテッド Process for the preparation of isoquinolinone and solid form of isoquinolinone
US9295673B2 (en) 2011-02-23 2016-03-29 Intellikine Llc Combination of mTOR inhibitors and P13-kinase inhibitors, and uses thereof
EP2734520B1 (en) 2011-07-19 2016-09-14 Infinity Pharmaceuticals, Inc. Heterocyclic compounds and uses thereof
AR088218A1 (en) 2011-07-19 2014-05-21 Infinity Pharmaceuticals Inc USEFUL HETEROCICLICAL COMPOUNDS AS PI3K INHIBITORS
RU2014111823A (en) 2011-08-29 2015-10-10 Инфинити Фармасьютикалз, Инк. HETEROCYCLIC COMPOUNDS AND THEIR APPLICATIONS
JP6342805B2 (en) 2011-09-02 2018-06-13 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア Substituted pyrazolo [3,4-D] pyrimidine and uses thereof
US8940742B2 (en) 2012-04-10 2015-01-27 Infinity Pharmaceuticals, Inc. Heterocyclic compounds and uses thereof
US8828998B2 (en) 2012-06-25 2014-09-09 Infinity Pharmaceuticals, Inc. Treatment of lupus, fibrotic conditions, and inflammatory myopathies and other disorders using PI3 kinase inhibitors
RU2015115631A (en) 2012-09-26 2016-11-20 Дзе Риджентс Оф Дзе Юниверсити Оф Калифорния MODULATION IRE1
US9481667B2 (en) 2013-03-15 2016-11-01 Infinity Pharmaceuticals, Inc. Salts and solid forms of isoquinolinones and composition comprising and methods of using the same
US9751888B2 (en) 2013-10-04 2017-09-05 Infinity Pharmaceuticals, Inc. Heterocyclic compounds and uses thereof
PL3052485T3 (en) 2013-10-04 2022-02-28 Infinity Pharmaceuticals, Inc. Heterocyclic compounds and uses thereof
SG11201607705XA (en) 2014-03-19 2016-10-28 Infinity Pharmaceuticals Inc Heterocyclic compounds for use in the treatment of pi3k-gamma mediated disorders
US20150320755A1 (en) 2014-04-16 2015-11-12 Infinity Pharmaceuticals, Inc. Combination therapies
WO2016020880A2 (en) 2014-08-07 2016-02-11 Novartis Ag Angiopoietin-like 4 antibodies and methods of use
TW201613977A (en) 2014-08-07 2016-04-16 Novartis Ag Angiopoetin-like 4 (ANGPTL4) antibodies and methods of use
US9708348B2 (en) 2014-10-03 2017-07-18 Infinity Pharmaceuticals, Inc. Trisubstituted bicyclic heterocyclic compounds with kinase activities and uses thereof
US11066481B2 (en) 2015-07-23 2021-07-20 The Regents Of The University Of California Antibodies to coagulation factor XIa and uses thereof
EP3324992A4 (en) * 2015-07-23 2018-12-26 The Regents of The University of California REVERSAL AGENTS FOR FXIa INHIBITORS
US10160761B2 (en) 2015-09-14 2018-12-25 Infinity Pharmaceuticals, Inc. Solid forms of isoquinolinones, and process of making, composition comprising, and methods of using the same
WO2017161116A1 (en) 2016-03-17 2017-09-21 Infinity Pharmaceuticals, Inc. Isotopologues of isoquinolinone and quinazolinone compounds and uses thereof as pi3k kinase inhibitors
WO2017211818A1 (en) * 2016-06-06 2017-12-14 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Proteasome inhibitors
JP2019525896A (en) * 2016-06-06 2019-09-12 マックス−プランク−ゲゼルシャフト・ツア・フェルデルング・デア・ヴィッセンシャフテン・エー・ファオ Proteasome inhibitor
US10919914B2 (en) 2016-06-08 2021-02-16 Infinity Pharmaceuticals, Inc. Heterocyclic compounds and uses thereof
JP7164521B2 (en) 2016-06-21 2022-11-01 オリオン・オフサルモロジー・エルエルシー carbocyclic prolinamide derivatives
CN114685453A (en) 2016-06-21 2022-07-01 奥瑞恩眼科有限责任公司 Heterocyclic prolinamide derivatives
SG10201912456RA (en) 2016-06-24 2020-02-27 Infinity Pharmaceuticals Inc Combination therapies
CN113527224B (en) * 2020-04-21 2023-07-04 宁夏苏融达化工有限公司 Thiazole derivative and application thereof in agriculture

Family Cites Families (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2589289A (en) * 1948-03-31 1952-03-18 Du Pont High-pressure reaction of carbon monoxide with hydrazine
DE942510C (en) * 1954-07-15 1956-05-03 Glanzstoff Ag Process for the preparation of aminothiocarboxylic acids containing imino groups
GB796894A (en) * 1956-01-10 1958-06-18 American Cyanamid Co Preparation of alkyl substituted thioctic acids
US4461724A (en) * 1981-10-28 1984-07-24 Nippon Zoki Pharmaceutical Co., Ltd. Peptide compounds, a process for manufacturing them, pharmaceutical compositions containing them, and methods for treating ulcer and thrombus with them
NZ212525A (en) 1985-06-24 1989-02-24 Dfc New Zealand Ltd Formerly D Nitrogen-containing heterocyclic compounds, and pharmaceutical compositions containing such
US5033179A (en) * 1988-04-29 1991-07-23 Shim-A-Line, Inc. Tool and method for adjusting camber and caster
US4886165A (en) * 1989-02-07 1989-12-12 Medical Concepts Development, Inc. Hinged container for surgical articles
EP0387738A1 (en) * 1989-03-13 1990-09-19 Matsushita Electric Industrial Co., Ltd. Flat panel display device
US5286637A (en) * 1989-08-07 1994-02-15 Debiopharm, S.A. Biologically active drug polymer derivatives and method for preparing same
US5078287A (en) * 1990-12-17 1992-01-07 Holmes Iii Wendell R Variable size nursing bottle
US5121838A (en) * 1991-10-23 1992-06-16 Intepac Technologies Inc. Package having product clamping for card-like products
US5311990A (en) * 1992-02-19 1994-05-17 Ethicon, Inc. Thermoformed blister package having quick disconnect locking cover mechanism
US5407070A (en) * 1993-08-18 1995-04-18 Bascos; Christine M. One-time resealable package for needled medical devices
BR9509994A (en) 1994-12-13 1997-12-30 Corvas Int Inc Aromatic heterocyclic derivatives as enzyme inhibitors
US5658902A (en) 1994-12-22 1997-08-19 Warner-Lambert Company Quinazolines as inhibitors of endothelin converting enzyme
USD450391S1 (en) * 1995-07-07 2001-11-13 Arrow International, Inc. Medical instrument tray
SE9503380D0 (en) * 1995-09-29 1995-09-29 Pharmacia Ab Protein derivatives
JP3359220B2 (en) * 1996-03-05 2002-12-24 キヤノン株式会社 Lithium secondary battery
US5840733A (en) 1996-07-01 1998-11-24 Redcell, Canada, Inc. Methods and compositions for producing novel conjugates of thrombin inhibitors and endogenous carriers resulting in anti-thrombins with extended lifetimes
US6258822B1 (en) 1997-08-06 2001-07-10 Abbott Laboratories Urokinase inhibitors
US5971152A (en) * 1998-07-01 1999-10-26 Ray Products, Inc. Container with reinforced tab and method
US6284796B1 (en) 1998-08-06 2001-09-04 Abbott Laboratories Ukokinase inhibitors
JP2003529531A (en) 1998-11-25 2003-10-07 アヴェンティス ファーマシューティカルズ インコーポレイテッド Substituted oxoazaheterocyclyl factor Xa inhibitors
FR2793247B1 (en) 1999-05-04 2001-06-22 Synthelabo DERIVATIVES OF 6 - [[(ARYL AND HETEROARYL) OXY] METHYL] NAPHTALENE-2- CARBOXIMIDAMIDE, THEIR PREPARATION AND THEIR THERAPEUTIC APPLICATION
JP2002544262A (en) 1999-05-19 2002-12-24 ファルマシア・コーポレイション Substituted polycyclic aryl and heteroaryl pyrimidinones as useful anticoagulants
US6608026B1 (en) * 2000-08-23 2003-08-19 Board Of Regents, The University Of Texas System Apoptotic compounds
CN1474815A (en) 2000-09-20 2004-02-11 Ĭ��ר���ɷ����޹�˾ 4-amino-quinazolines
US7217794B2 (en) * 2003-04-02 2007-05-15 Daiamed, Inc. Compounds and methods for treatment of thrombosis

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AKTIMUR A ET AL: 'The Factor IX ... and Factor XIa' J BIOL CHEM vol. 278, no. 10, March 2003, pages 7981 - 7987 *
BAIRD T R ET AL: 'The Interaction of Factor XIa with ... Blood Coagulation' J. BIOL. CHEM. vol. 277, no. 41, October 2002, pages 38462 - 38467 *
MINNNEMA M C ET AL: 'Activation of clotting factor XI without detectable contact activation in experimental human endotoxemia' BLOOD vol. 92, no. 9, November 1998, pages 3294 - 3301 *
SALOMON O ET AL: 'Prevalence, causes, and characterization of Factor IX inhibitors in patients with inherited factor XI deficiency' BLOOD vol. 101, no. 12, June 2003, pages 4783 - 4788 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7829724B2 (en) 2005-08-15 2010-11-09 Zentaris Gmbh Triazole derivatives as ghrelin analogue ligands of growth hormone secretagogue receptors
US8710089B2 (en) 2005-08-15 2014-04-29 Zentaris Gmbh Triazole derivatives as ghrelin analogue ligands of growth hormone secretagogue receptors
EP1757290A1 (en) 2005-08-16 2007-02-28 Zentaris GmbH Novel triazole derivatives as ghrelin analogue ligands of growth hormone secretagogue receptors
EP2431035A1 (en) 2010-09-16 2012-03-21 Æterna Zentaris GmbH Novel Triazole Derivatives with Improved Receptor Activity and Bioavailability Properties as Ghrelin Antagonists of Growth Hormone Secretagogue Receptors
WO2012035124A1 (en) 2010-09-16 2012-03-22 Æterna Zentaris Gmbh Novel triazole derivatives with improved receptor activity and bioavailability properties as ghrelin antagonists of growth hormone secretagogue receptors
US10465011B2 (en) 2015-06-26 2019-11-05 Novartis Ag Factor XI antibodies and methods of use
US10647780B2 (en) 2016-05-25 2020-05-12 Novartis Ag Reversal binding agents for anti-factor XI/XIa antibodies and uses thereof
US11168147B2 (en) 2016-12-23 2021-11-09 Novartis Ag Factor XI antibodies and methods of use
US12012464B2 (en) 2016-12-23 2024-06-18 Novartis Ag Factor XI antibodies and methods of use

Also Published As

Publication number Publication date
WO2004089297A3 (en) 2006-03-30
US7217794B2 (en) 2007-05-15
WO2004089297A2 (en) 2004-10-21
WO2004103270A3 (en) 2005-05-12
US20050143317A1 (en) 2005-06-30

Similar Documents

Publication Publication Date Title
US7217794B2 (en) Compounds and methods for treatment of thrombosis
KR100337271B1 (en) Antithrombotic agents
EP0772590B1 (en) Thrombin inhibitors
US20020102235A1 (en) Novel imidazolidinones as NS3-serine protease inhibitors of hepatitis C virus
JP4152900B2 (en) β-sheet mimetics and their use as protease inhibitors
JP2002521386A (en) Urokinase and angiogenesis inhibitors
CA2418204A1 (en) Novel peptides as ns3-serine protease inhibitors of hepatitis c virus
JP2000505437A (en) Serine protease inhibitor
EP0994892A1 (en) SELECTIVE FACTOR Xa INHIBITORS
JP2846963B2 (en) Antithrombotic amidinotetrahydropyridylalanine derivative
JP2002542164A (en) Small molecule inhibitors of complement protease
CA2268621A1 (en) 2-pipirazinone-1-acetic acid derivative, production and use thereof
JPH07258287A (en) Antithrombotic agent
JP2000506837A (en) Serine protease inhibitor
EP0858262B1 (en) Thrombin inhibitors
JP2004506648A (en) Non-covalent inhibitors of urokinase and angiogenesis
US5798377A (en) Thrombin inhibitors
US20040087511A1 (en) Peptide derivatives
JPH11507366A (en) Thrombin inhibitors
US6069232A (en) Polyfluoroalkyl tryptophan tripeptide thrombin inhibitors
EP1212300B1 (en) Azacycloalkanone serine protease inhibitors
JPH09509936A (en) Antithrombotic agent
KR100367389B1 (en) Acylated enol derivatives as prodrugs of elastase inhibitors
WO2001079262A1 (en) Pyrazinone and pyridinone derivatives as thrombin inhibitors
JPH09509943A (en) Antithrombotic agent

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 69(1) EPC. (EPO COMMUNICATION FORM 1205A HAS BEEN SENT ON17-01-2006)

122 Ep: pct application non-entry in european phase