US20050124604A1 - Substituted naphthyridine derivatives as inhibitors of macrophage migration inhibitory factor and their use in the treatment of human diseases - Google Patents

Substituted naphthyridine derivatives as inhibitors of macrophage migration inhibitory factor and their use in the treatment of human diseases Download PDF

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US20050124604A1
US20050124604A1 US10/920,031 US92003104A US2005124604A1 US 20050124604 A1 US20050124604 A1 US 20050124604A1 US 92003104 A US92003104 A US 92003104A US 2005124604 A1 US2005124604 A1 US 2005124604A1
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substituted
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Jagadish Sircar
Sunil K.C.
Wenbin Ying
Timothy Davis
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Avanir Pharmaceuticals Inc
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Avanir Pharmaceuticals Inc
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Definitions

  • MIF macrophage migration inhibitory factor
  • MIF macrophage migration inhibitory factor
  • MIF was first characterized as being able to block macrophage migration, MIF also appears to effect macrophage adherence; induce macrophage to express interleukin-1-beta, interleukin-6, and tumor necrosis factor alpha; up-regulate HLA-DR; increase nitric oxide synthase and nitric oxide concentrations; and activate macrophage to kill Leishmania donovani tumor cells and inhibit Mycoplasma avium growth, by a mechanism different from that effected by interferon-gamma.
  • MIF can act as an immunoadjuvant when given with bovine serum albumin or HIV gp120 in incomplete Freunds or liposomes, eliciting antigen induced proliferation comparable to that of complete Freunds.
  • MIF has been described as a glucocorticoid counter regulator and angiogenic factor.
  • glucocorticoids As one of the few proteins that is induced and not inhibited by glucocorticoids, it serves to attenuate the immunosuppressive effects of glucocorticoids. As such, it is viewed as a powerful element that regulates the immunosuppressive effects of glucocorticoids.
  • MIF is also thought to act on cells through a specific receptor that in turn activates an intracellular cascade that includes erk phosphorylation and MAP kinase and upregulation of matrix metalloproteases, c-jun, c-fos, and IL-1 mRNA (see Onodera et al., J. Biol. Chem. 275:444-450, 2000), it also possesses endogenous enzyme activity as exemplified by its ability to tautomerize the appropriate substrates (e.g., dopachrome). Further, it remains unclear whether this enzymatic activity mediates the biological response to MIF and the activities of this protein in vitro and in vivo.
  • MIF is known for its cytokine activity concentrating macrophages at sites of infection, and cell-mediated immunity. Moreover, MIF is known as a mediator of macrophage adherence, phagocytosis, and tumoricidal activity. See Weiser et al., J. Immunol. 147:2006-2011, 1991. Hence, the inhibition of MIF results in the indirect inhibition of cytokines, growth factors, chemokines, and lymphokines that the macrophage can otherwise bring to a site of inflammation.
  • MIF cDNA has been isolated from a T-cell line, and encodes a protein having a molecular mass of about 12.4 kDa with 115 amino acid residues that form a homotrimer as the active form (Weiser et al., Proc. Natl. Acad. Sci. USA 86:7522-7526, 1989). While MIF was originally observed in activated T-cells, it has now been reported in a variety of tissues including the liver, lung, eye lens, ovary, brain, heart, spleen, kidney, muscle, and others. See Takahashi et al., Microbiol. Immunol. 43(1):61-67, 1999. Another characteristic of MIF is its lack of a traditional leader sequence (i.e., a leaderless protein) to direct classical secretion through the ER/Golgi pathway.
  • a leader sequence i.e., a leaderless protein
  • a MIF inhibitor (and a method to identify MIF inhibitors) that act by neutralizing the cytokine activity of MIF presents significant advantages over other types of inhibitors.
  • the link between tautomerase activity alone and the inflammatory response is controversial.
  • inhibitors that act intracellularly are often toxic by virtue of their action on related targets or the activities of the target inside cells.
  • Small molecule inhibitors of the ligand receptor complex are difficult to identify let alone optimize and develop.
  • the ideal inhibitor of a cytokine like MIF is one that alters MIF itself so that when released from the cell it is effectively neutralized.
  • a small molecule with this activity is superior to antibodies because of the fundamental difference between proteins and chemicals as drugs. See, Metz and Bucala (supra); Swope and Lolis, Rev.
  • MIF has been identified in a variety of tissues and has been associated with numerous pathological events
  • pharmaceutical compositions containing such inhibitors as well as methods relating to the use thereof to treat, for example, immune related disorders or other MIF induced pathological events, such as tumor associated angiogenesis.
  • the preferred embodiments can fulfill these needs, and provide other advantages as well.
  • inhibitors of MIF are provided that have the following general structures (Ia), (Ib), (Ic), and (Id): including stereoisomers, prodrugs, and pharmaceutically acceptable salts thereof, wherein n, R, R 1 , R 2 , X, Y and Z are as defined below.
  • the MIF inhibitors of preferred embodiments have utility over a wide range of therapeutic applications, and can be employed to treat a variety of disorders, illnesses, or pathological conditions including, but not limited to, a variety of immune related responses, tumor growth (e.g., cancer, such as prostate cancer, breast cancer, lung cancer, liver cancer, skin cancer, brain cancer, bone cancer, colon cancer, testicular cancer, etc.), glomerulonephritis, inflammation, malarial anemia, septic shock, sepsis, tumor associated angiogenesis, vitreoretinopathy, psoriasis, graft versus host disease (tissue rejection), atopic dermatitis, rheumatoid arthritis, inflammatory bowel disease, inflammatory lung disease, otitis media, Crohn's disease, acute respiratory distress syndrome, delayed-type hypersensitivity, transplant rejection, immune-mediated and inflammatory elements of CNS disease (e.g., Alzheimer's, Parkinson's, multiple sclerosis, etc.), muscular dystrophy, diseases of hemostas
  • Such methods include administering an effective amount of one or more inhibitors of MIF as provided by the preferred embodiments, preferably in the form of a pharmaceutical composition, to an animal in need thereof.
  • Pharmaceutical compositions are provided containing one or more inhibitors of MIF of preferred embodiments in combination with a pharmaceutically acceptable carrier and/or diluent.
  • a compound for inhibiting macrophage migration inhibitory factor having a structure selected from the group consisting of: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof, wherein R is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl, substituted acylalkyl, heterocycle, substituted heterocycle, —(CH 2 ) m C( ⁇ O)Ar, and —(CH 2 ) m NR 4 R 5 ; R 1 is selected from the group consisting of —CN, —NO, —NO 2 , —C( ⁇ O)R 3 , —C( ⁇ O)OH, —NHC( ⁇ O)R 3 , —C
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof wherein R is selected from the group consisting of hydrogen, C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 3 -C 12 cycloalkyl, C 6 -C 12 aryl, C 7 -C 12 arylalkyl, C 7 -C 12 alkylaryl, C 2 -C 12 acylalkyl, C 3 -C 12 heterocyclealkyl, C 3 -C 12 alkylheterocycle, and C 2 -C 12 heterocycle, wherein R is substituted with one or more substituents selected from the group consisting of hydrogen, —F, —Cl, —CN, —NO, —NO 2 , —C( ⁇ O)R 3 , —C( ⁇ O)OR 3 , —OC( ⁇ O)R 3 , —C( ⁇ O)NR
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof wherein R is selected from the group consisting of hydrogen, C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 3 -C 12 cycloalkyl, C 6 -C 12 aryl, C 7 -C 12 arylalkyl, C 7 -C 12 alkylaryl, C 2 -C 12 acylalkyl, C 3 -C 12 heterocyclealkyl, C 3 -C 12 alkylheterocycle, and C 2 -C 12 heterocycle, wherein R is substituted with one or more substituents selected from the group consisting of hydrogen, —F, —Cl, —CN, —NO, —NO 2 , —NHSO 2 R 3 , —C( ⁇ O)R 3 , —C( ⁇ O)OR 3 , —OC( ⁇ O)R 3
  • R 1 comprises —(CH 2 ) m C( ⁇ O)Ar.
  • R 1 comprises —C( ⁇ O)OCH 2 CH 3 .
  • R 1 comprises —NH—C( ⁇ O)CH 3 .
  • R 1 comprises —CN.
  • R 1 comprises —NO 2 .
  • R 1 comprises —NH 2 .
  • R 2 comprises
  • R 2 comprises
  • R comprises —(CH 2 ) m C( ⁇ O)Ar.
  • X is selected from the group consisting of hydrogen, fluorine, and chlorine; wherein Y is selected from the group consisting of hydrogen, fluorine, and chlorine; and wherein Z is selected from the group consisting of hydrogen, fluorine, and chlorine.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof in an aspect of the first embodiment, a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure having a structure:
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • a compound having a structure: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof is provided.
  • the compound of the first embodiment in combination with a pharmaceutically acceptable carrier or diluent is provided.
  • a method for reducing MIF activity in a patient in need thereof comprising administering to the patient an effective amount of a compound, the compound having a structure selected from the group consisting of: or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof, wherein R is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl, substituted acylalkyl, heterocycle, substituted heterocycle, —(CH 2 ) m C( ⁇ O)Ar, and CH 2 ) m NR 4 R 5 ; R 1 is selected from the group consisting of —CN, —NO, —NO 2 , —C( ⁇ O)R 3 , —C( ⁇ O)OH, —
  • a method for treating inflammation in a warm-blooded animal comprising administering to the animal an effective amount of the compound of the first embodiment.
  • a method for treating septic shock in a warm-blooded animal comprising administering to the animal an effective amount of the compound of the first embodiment.
  • a method for treating arthritis in a warm-blooded animal comprising administering to the animal an effective amount of the compound of the first embodiment.
  • a method for treating cancer in a warm-blooded animal comprising administering to the animal an effective amount of the compound of the first embodiment.
  • a method for treating acute respiratory distress syndrome in a warm-blooded anima comprising administering to the animal an effective amount of the compound of the first embodiment.
  • a method for treating an inflammatory disease in a warm-blooded animal comprising administering to the animal an effective amount of the compound of the first embodiment.
  • the inflammatory disease can be selected from the group consisting of rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, and asthma.
  • a method for treating a cardiac disease in a warm-blooded animal comprising administering to the animal an effective amount of the compound of the first embodiment.
  • the cardiac disease can be selected from the group consisting of cardiac dysfunction, myocardial infarction, congestive heart failure, restenosis, and atherosclerosis.
  • a method for treating an autoimmune disorder in a warm-blooded animal comprising administering to the animal an effective amount of the compound of the first embodiment.
  • the autoimmune disorder can be selected from the group consisting of diabetes, asthma, and multiple sclerosis.
  • a method for suppressing an immune response in a warm-blooded animal comprising administering to the animal an effective amount of the compound of the first embodiment.
  • a method for decreasing angiogenesis in a warm-blooded animal comprising administering to the animal an effective amount of the compound of the first embodiment.
  • a method for treating a disease associated with excess glucocorticoid levels in a warm-blooded animal comprising administering to the animal an effective amount of the compound of the first embodiment.
  • the disease can be Cushing's disease.
  • a process for preparing a compound comprising the steps of: reacting POCl 3 with a compound of Formula (3): wherein R is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl, subtituted acylalkyl, heterocycle, substituted heterocycle, —(CH 2 ) m C( ⁇ O)Ar, and CH 2 ) m NR 4 R 5 ; R 1 is selected from the group consisting of —CN, —NO, —NO 2 , —C( ⁇ O)R 3 , —C( ⁇ O)OH, —NHC( ⁇ O)R 3 , —C( ⁇ O)OR 3 , —C( ⁇ O)NR 4 R 5 ,
  • R 1 comprises —(CH 2 ) m C( ⁇ O)Ar.
  • R 1 comprises —C( ⁇ O)OCH 2 CH 3 .
  • R 1 comprises —NH—C( ⁇ O)CH 3 .
  • R 1 comprises —CN.
  • R 1 comprises —NO 2 .
  • R 1 comprises —NH 2 .
  • R 2 comprises
  • R 2 comprises
  • R comprises CH 2 ) m C( ⁇ O)Ar.
  • X is selected from the group consisting of hydrogen, fluorine, and chlorine; wherein Y is selected from the group consisting of hydrogen, fluorine, and chlorine; and wherein Z is selected from the group consisting of hydrogen, fluorine, and chlorine.
  • a process for preparing a compound comprising the steps of reacting a compound of Formula (13): wherein R is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl, subtituted acylalkyl, heterocycle, substituted heterocycle, —(CH 2 ) m C( ⁇ O)Ar, and CH 2 ) m NR 4 R 5 ; R 4 and R 5 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl, acylalkyl, sub
  • R 2 comprises
  • R 2 comprises
  • R comprises —(CH 2 ) m C( ⁇ O)Ar.
  • X is selected from the group consisting of hydrogen, fluorine, and chlorine; wherein Y is selected from the group consisting of hydrogen, fluorine, and chlorine; and wherein Z is selected from the group consisting of hydrogen, fluorine, and chlorine.
  • a process for preparing a compound comprising the steps of reacting a compound of Formula (23): wherein R 1 is selected from the group consisting of —CN, —NO, —NO 2 , —C( ⁇ O)R 3 , —C( ⁇ O)OH, —NHC( ⁇ O)R 3 , —C( ⁇ O)OR 3 , —C( ⁇ O)NR 4 R 5 , —NR 3 C( ⁇ O)R 3 , —SO 2 NR 4 R 5 , —NR 3 SO 2 R 3 , —NHSO 2 R 3 , —S(O) m R 3 , —(CH 2 ) m NR 4 R 5 , and —(CH 2 ) m C( ⁇ O)Ar; R 3 is independently selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, wherein R 1 is selected from the group consist
  • R 1 comprises —(CH 2 ) m C( ⁇ O)Ar.
  • R 1 comprises —C( ⁇ O)OCH 2 CH 3 .
  • R 1 comprises —NH—C( ⁇ O)CH 3 .
  • R 1 comprises —CN.
  • R 1 comprises —NO 2 .
  • R 1 comprises —NH 2 .
  • R 2 comprises
  • R 2 comprises
  • R comprises CH 2 ) m C( ⁇ O)Ar.
  • X is selected from the group consisting of hydrogen, fluorine, and chlorine; wherein Y is selected from the group consisting of hydrogen, fluorine, and chlorine; and wherein Z is selected from the group consisting of hydrogen, fluorine, and chlorine.
  • R comprises benzyl
  • a process for preparing a compound comprising the steps of reacting a compound of Formula (3a): with POCl 3 , thereby yielding a compound of Formula (4a): reacting the compound of Formula (4a) with piperazine, thereby yielding a compound of Formula (5a): reacting the compound of Formula (5a) with a compound having the formula R 2 —C( ⁇ O)Cl, wherein R 2 is selected from the group consisting —CH 2 R 3 , —NR 4 R 5 , —OR 3 , and —R 3 , wherein R 3 is selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl, subtituted acylalkyl, heterocycle, substituted heterocycle, and
  • R 2 comprises
  • a process for preparing a compound comprising the steps of reacting a compound of Formula (13a): with cyclohexylamine, thereby yielding a compound of Formula (14): reacting the compound of Formula (14a) with POCl 3 , thereby yielding a compound of Formula (15a): reacting the compound of Formula (15a) with piperazine, thereby yielding a compound of Formula (16a): reacting the compound of Formula (16a) with a compound having the formula R 2 —C( ⁇ O)Cl, wherein R 2 is selected from the group consisting —CH 2 R 3 , —NR 4 R 5 , —OR 3 , and —R 3 , wherein R 3 is selected from the group consisting of R 3 alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted
  • R 2 comprises
  • a process for preparing a compound comprising the steps of reacting a compound of Formula (23a): with POCl 3 and trifluoroacetic acid, thereby yielding a compound of Formula (24a): reacting the compound of Formula (24a) with a compound of formula wherein R 2 is selected from the group consisting —CH 2 R 3 , —NR 4 R 5 , —OR 3 , and —R 3 , wherein R 3 is selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl, subtituted acylalkyl, heterocycle, substituted heterocycle; R 4 and R 5 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted substituted alkenyl, substitute
  • R 2 comprises
  • R 2 comprises
  • R comprises CH 2 ) m C(O)Ar.
  • R comprises benzyl
  • MIF activity is a broad term and is used in its ordinary sense, including, without limitation, to refer to an activity or effect mediated at least in part by macrophage migration inhibitory factor. Accordingly, MIF activity includes, but is not limited to, inhibition of macrophage migration, tautomerase activity (e.g., using phenylpyruvate or dopachrome), endotoxin induced shock, inflammation, glucocorticoid counter regulation, induction of thymidine incorporation into 3T3 fibroblasts, induction of erk phosphorylation and MAP kinase activity.
  • tautomerase activity e.g., using phenylpyruvate or dopachrome
  • endotoxin induced shock e.g., using phenylpyruvate or dopachrome
  • endotoxin induced shock e.g., using phenylpyruvate or dopachrome
  • endotoxin induced shock e.g
  • export is a broad term and is used in its ordinary sense, including, without limitation, to refer to a metabolically active process, which may or may not be energy-dependent, of transporting a translated cellular product to the cell membrane or the extracellular space by a mechanism other than standard leader sequence directed secretion via a canonical leader sequence.
  • export unlike secretion that is leader sequence-dependent, is resistant to brefeldin A (i.e., the exported protein is not transported via the ER/Golgi; brefeldin A is expected to have no direct effect on trafficking of an exported protein) and other similar compounds.
  • export can also be referred to as “non-classical secretion.”
  • leaderless protein is a broad term and is used in its ordinary sense, including, without limitation, to refer to a protein or polypeptide that lacks a canonical leader sequence, and is exported from inside a cell to the extracellular environment.
  • Leaderless proteins in the extracellular environment refer to proteins located in the extracellular space, or associated with the outer surface of the cell membrane.
  • leaderless proteins include naturally occurring proteins, such as macrophage migration inhibitory factor and fragments thereof as well as proteins that are engineered to lack a leader sequence and are exported, or proteins that are engineered to include a fusion of a leaderless protein, or fraction thereof, with another protein.
  • inhibitor is a broad term and is used in its ordinary sense, including, without limitation, to refer to a molecule (e.g., natural or synthetic compound) that can alter the conformation of MIF and/or compete with a monoclonal antibody to MIF and decrease at least one activity of MIF or its export from a cell as compared to activity or export in the absence of the inhibitor.
  • an “inhibitor” alters conformation and/or activity and/or export if there is a statistically significant change in the amount of MIF measured, MIF activity or in MIF protein detected extracellularly and/or intracellularly in an assay performed with an inhibitor, compared to the assay performed without the inhibitor.
  • binding agent as used herein is a broad term and is used in its ordinary sense, including, without limitation, to refer to any molecule that binds MIF, including inhibitors.
  • MIF inhibitors inhibit the physiological function of MIF, and thus are useful in the treatment of diseases where MIF is pathogenic.
  • inhibitors of MIF are provided that have the following structures (Ia), (Ib), (Ic), and (Id): or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof, wherein R is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl, subtituted acylalkyl, heterocycle, substituted heterocycle, —(CH 2 ) m C( ⁇ O)Ar, and CH 2 ) m NR 4 R 5 ; R 1 is selected from the group consisting of —CN, —NO, —NO 2 , —C( ⁇ O)R 3 , —C( ⁇ O)OH, —NHC( ⁇ O)R 3 , —C
  • methods for reducing MIF activity in a patient in need thereof by administering to the patient an effective amount of a compound having the following structure (Ia), (Ib), (Ic), or (Id): or a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof, wherein R is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl, subtituted acylalkyl, heterocycle, substituted heterocycle, —(CH 2 ) m C( ⁇ O)Ar, and CH 2 ) m NR 4 R 5 ; R 1 is selected from the group consisting of N, —NO, —NO 2 , —C( ⁇ O)R 3 , —C( ⁇ O)
  • alkyl As used herein, the above terms have the following meanings.
  • alkyl is a broad term and is used in its ordinary sense, including, without limitation, to refer to a straight chain or branched, acyclic or cyclic, unsaturated or saturated aliphatic hydrocarbon containing from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more carbon atoms, while the term “lower alkyl” has the same meaning as alkyl but contains from 1 to 2, 3, 4, 5, 6 carbon atoms.
  • saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.
  • Unsaturated alkyls contain at least one double or triple bond between adjacent carbon atoms (referred to as an “alkenyl” or “alkynyl,” respectively).
  • Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like; while representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.
  • cycloalkyl is a broad term and is used in its ordinary sense, including, without limitation, to refer to alkyls that include mono-, di-, or poly-homocyclic rings. Cycloalkyls are also referred to as “cyclic alkyls” or “homocyclic rings.” Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, —CH 2 cyclopropyl, —CH 2 cyclobutyl, —CH 2 cyclopentyl, —CH 2 cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like. Cyclic alkyls include decalin, adamantane, and the like.
  • aryl is a broad term and is used in its ordinary sense, including, without limitation, to refer to an aromatic carbocyclic moiety such as phenyl or naphthyl.
  • the aryl group contains from 6 to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more carbon atoms.
  • arylalkyl is a broad term and is used in its ordinary sense, including, without limitation, to refer to an alkyl having at least one alkyl hydrogen atom replaced with an aryl moiety, such as benzyl, —CH 2 (1 or 2-naphthyl), —(CH 2 ) 2 phenyl, —(CH 2 ) 3 phenyl, —CH(phenyl) 2 , and the like.
  • heteroaryl as used herein is a broad term and is used in its ordinary sense, including, without limitation, to refer to an aromatic heterocycle ring of 5 or 6 to 10 members and having at least one heteroatom (or 2, 3, or 4 or more heteroatoms) selected from nitrogen, oxygen and sulfur, and containing at least one carbon atom, including both mono and bicyclic ring systems.
  • heteroaryls include (but are not limited to) furyl, benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl, isoindolyl, azaindolyl, pyridyl, quinolinyl, isoquinolinyl, oxazolyl, isooxazolyl, benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl; cinnolinyl, phthalazinyl, and quinazolinyl.
  • heteroarylalkyl as used herein is a broad term and is used in its ordinary sense, including, without limitation, to refer to an alkyl having at least one alkyl hydrogen atom replaced with a heteroaryl moiety, such as —CH 2 pyridinyl, —CH 2 pyrimidinyl, and the like.
  • heterocycle and “heterocycle ring,” as used herein, are broad terms and are used in their ordinary sense, including, without limitation, to refer to a 5, 6, or 7 membered monocyclic heterocyclic ring, or a 7, 8, 9, 10, 11, 12, 13, to 14 or more membered polycyclic heterocyclic ring.
  • the ring can be saturated, unsaturated, aromatic, or nonaromatic, and contains 1, 2, 3, or 4 or more heteroatoms independently selected from nitrogen, oxygen, and sulfur.
  • the nitrogen and sulfur heteroatoms can be optionally oxidized, and the nitrogen heteroatom can be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring as well as tricyclic (and higher) heterocyclic rings.
  • the heterocycle can be attached via any heteroatom or carbon atom of the ring or rings.
  • Heterocycles include heteroaryls as defined above.
  • heterocycles also include (but are not limited to) morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like. Also included are heterocycles of the following structures:
  • heterocyclealkyl is a broad term and is used in its ordinary sense, including, without limitation, to refer to an alkyl having at least one alkyl hydrogen atom replaced with a heterocycle, such as —CH 2 morpholinyl, and the like.
  • substituted is a broad term and is used in its ordinary sense, including, without limitation, to refer to any of the above groups (e.g., alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocycle or heterocyclealkyl) wherein at least one hydrogen atom is replaced with a substituent.
  • a keto substituent for example —C( ⁇ O)—, two hydrogen atoms are replaced.
  • substituted within the context of preferred embodiment, include halogen, hydroxy, cyano, nitro, amino, alkylamino, dialkylamino, alkyl, alkoxy, alkylthio, haloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl, —NR a R b , —NR a C( ⁇ O)R b , —NR a C( ⁇ O)NR b R c , —NR a C( ⁇ O)OR b , —NR a SO 2 R b , —OR a , —C( ⁇ O)R a , —C( ⁇ O)OR a , —C( ⁇ O)NR a R
  • halogen is a broad term and is used in its ordinary sense, including, without limitation, to refer to fluoro, chloro, bromo, and iodo.
  • haloalkyl is a broad term and is used in its ordinary sense, including, without limitation, to refer to an alkyl having at least one hydrogen atom replaced with halogen, such as trifluoromethyl and the like.
  • alkoxy is a broad term and is used in its ordinary sense, including, without limitation, to refer to an alkyl moiety attached through an oxygen bridge (i.e., (alkyl) such as methoxy, ethoxy, and the like.
  • thioalkyl as used herein is a broad term and is used in its ordinary sense, including, without limitation, to refer to an alkyl moiety attached through a sulfur bridge (i.e., —S-alkyl) such as methylthio, ethylthio, and the like.
  • alkylsulfonyl as used herein is a broad term and is used in its ordinary sense, including, without limitation, to refer to an alkyl moiety attached through a sulfonyl bridge (i.e., —SO 2 -alkyl) such as methylsulfonyl, ethylsulfonyl, and the like.
  • alkylamino and dialkyl amino are broad terms and are used in their ordinary sense, including, without limitation, to refer to one alkyl moiety or two alkyl moieties, respectively, attached through a nitrogen bridge (i.e., —N-alkyl) such as methylamino, ethylamino, dimethylamino, diethylamino, and the like.
  • hydroxyalkyl as used herein is a broad term and is used in its ordinary sense, including, without limitation, to refer to an alkyl substituted with at least one hydroxyl group.
  • cycloalkyl is a broad term and is used in its ordinary sense, including, without limitation, to refer to a methyl group substituted with one or two cycloalkyl groups, such as cyclopropylmethyl, dicyclopropylmethyl, and the like.
  • alkylcarbonylalkyl or “acylalkyl” as used herein are broad terms and are used in their ordinary sense, including, without limitation, to refer to an alkyl substituted with a —C( ⁇ O)alkyl group.
  • alkylcarbonyloxyalkyl as used herein is a broad term and is used in its ordinary sense, including, without limitation, to refer to an alkyl substituted with a —C( ⁇ O)O-alkyl group or a —OC( ⁇ O)alkyl group.
  • alkyloxyalkyl as used herein is a broad term and is used in its ordinary sense, including, without limitation, to refer to an alkyl substituted with an —O-alkyl group.
  • arylcarbonylaryl is a broad term and is used in its ordinary sense, including, without limitation, to refer to an aryl substituted with a —C( ⁇ O)aryl group.
  • arylcarbonyloxyaryl as used herein is a broad term and is used in its ordinary sense, including, without limitation, to refer to an aryl substituted with a —C( ⁇ O)O-aryl group or a —OC( ⁇ O)aryl group.
  • aryloxyaryl as used herein is a broad term and is used in its ordinary sense, including, without limitation, to refer to an alkyl substituted with an aryl group.
  • alkylcarbonylaryl is a broad term and is used in its ordinary sense, including, without limitation, to refer to an alkyl substituted with a —C( ⁇ O)aryl group.
  • alkylcarbonyloxyaryl as used herein is a broad term and is used in its ordinary sense, including, without limitation, to refer to an alkyl substituted with a —C( ⁇ O)O-aryl group or a —OC( ⁇ O)aryl group.
  • alkyloxyaryl as used herein is a broad term and is used in its ordinary sense, including, without limitation, to refer to an alkyl substituted with an —O-aryl group.
  • arylcarbonylalkyl is a broad term and is used in its ordinary sense, including, without limitation, to refer to an aryl substituted with a —C( ⁇ O)alkyl group.
  • arylcarbonyloxyalkyl as used herein is a broad term and is used in its ordinary sense, including, without limitation, to refer to an aryl substituted with a —C( ⁇ O)O-alkyl group or a —OC( ⁇ O)alkyl group.
  • aryloxyalkyl as used herein is a broad term and is used in its ordinary sense, including, without limitation, to refer to an aryl substituted with an —O-alkyl group.
  • alkylthioalkyl as used herein is a broad term and is used in its ordinary sense, including, without limitation, to refer to an alkyl substituted with a —S-alkyl group.
  • di(alkyl)aminoalkyl as used herein is a broad term and is used in its ordinary sense, including, without limitation, to refer to an alkyl substituted with a mono- or di(alkyl)amino.
  • cyclic systems referred to herein include fused ring, bridged ring, and spiro ring moieties, in addition to isolated monocyclic moieties.
  • the nitrogen atom of the naphthyridine ring can occupy the 5, 6, 7, or 8 ring position.
  • Chemical structures for representative compounds of the preferred embodiments are provided below. In these structures, the following symbol is employed to represent a pyridine ring wherein the nitrogen atom can occupy either the 5, 6, 7, or 8 ring position:
  • the pyridine ring so depicted includes as a substituent a methyl group or a chlorine atom.
  • a substituent a substituent that occupies either the 6, 7, or 8 ring position.
  • the nitrogen atom of the pyridine ring occupies the 6 ring position then the substituent occupies either the 5, 7, or 8 ring position.
  • the nitrogen atom of the pyridine ring occupies the 7 ring position then the substituent occupies either the 5, 6, or 8 ring position.
  • the nitrogen atom of the pyridine ring occupies the 8 ring position then the substituent occupies either the 5, 6, or 7 ring position.
  • the nitrogen atom of the pyridine ring occupies the 8 ring position, and a substituent, if present, occupies the 6 ring position.
  • the compounds of preferred embodiments can generally be employed as the free acid or free base.
  • the compounds of preferred embodiments can preferably be in the form of acid or base addition salts.
  • Acid addition salts of the free base amino compounds of preferred embodiments can be prepared by methods well known in the art, and can be formed from organic and inorganic acids. Suitable organic acids include maleic, fumaric, benzoic, ascorbic, succinic, methanesulfonic, acetic, oxalic, propionic, tartaric, salicylic, citric, gluconic, lactic, mandelic, cinnamic, aspartic, stearic, palmitic, glycolic, glutamic, and benzenesulfonic acids.
  • Suitable inorganic acids include hydrochloric, hydrobromic, sulfuric, phosphoric, and nitric acids.
  • Base addition salts of the free acid can similarly be prepared by methods well known in the art, and can be formed from suitable bases, such as cations chosen from the alkali and alkaline earth metals (e.g., lithium, sodium, potassium, magnesium, barium or calcium) as well as the ammonium cation.
  • suitable bases such as cations chosen from the alkali and alkaline earth metals (e.g., lithium, sodium, potassium, magnesium, barium or calcium) as well as the ammonium cation.
  • pharmaceutically acceptable salt of structure (Ia), (Ib), (Ic), or (Id) is intended to encompass any and all acceptable salt forms.
  • the compounds of structure (Ia), (Ib), (Ic), and (Id) can be made according to the organic synthesis techniques known to those skilled in this field, as well as by the representative methods set forth in the Example.
  • compounds of structure (Ia) can be made according to the following Reaction Schemes. It is noted that in these synthetic routes the nitrogen atom of the naphthyridine ring occupies the 8 ring position. However, the synthetic routes are also effective in preparation of compounds of the preferred embodiments where in the nitrogen atom of the naphthyridine ring occupies the 5, 6, or 7 ring position, as discussed below.
  • a preferred intermediate in the preparation of compounds of formula (Ia) is 1-benzyl-4-hydroxy-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester, depicted by formula (3) below.
  • the starting material used for this synthesis was 2-chloro-3-pyridinecarboxylic acid.
  • benzylamine is employed as a starting material. However, it is noted that other secondary amines can also be employed as starting materials, including substituted benzylamines such as 4-methoxybenzylamine or 4-fluorobenzylamine.
  • 2-Benzylamino nicotinic acid (1) can then be employed to prepare 1-benzyl-1H-pyrido[2,3-d][1,3]oxazine-2,4-dione, depicted by formula (2) as shown in Scheme 2.
  • diesters can be employed in this reaction if a different carboxylate substituent is preferred for the substituent R 1 of the resulting compound of formula (Ia) (or formula (Ib), (Ic), or (Id)), for example, dimethyl malonate, dipropyl malonate, and the like. Otherwise, the carboxylate group can be substituted by another moiety, for example cyano, as discussed below.
  • One compound of a preferred embodiment can be prepared using the intermediate 1-benzyl-4-hydroxy-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester as a starting material. This compound was prepared by applying sequence of reactions as shown in Scheme 4.
  • 2-thiophene carbonyl chloride is reacted with 1-benzyl-2-oxo-4-piperazin-1-yl-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester to yield the target compound, 1-benzyl-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester, depicted by formula (6) in Scheme 4. If it is preferred that the substituent R is other than 2-thiophene, another carbonyl chloride compound can be substituted in the reaction.
  • a compound of formula (Ia) including a cyano group as the substituent R 1 can be prepared starting from intermediate 1-benzyl-4-hydroxy-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester.
  • the intermediate is functionalized with cyano by the reactions as shown in Scheme 5:
  • the final product is then prepared by reaction with piperazine, followed by reaction with 2-thiophene carbonyl chloride, as discussed above in the preparation of the compounds of formulae (5) and (6) in Scheme 4.
  • the substituent R is other than 2-thiophene
  • another carbonyl chloride compound can be substituted in the reaction.
  • the reaction sequences used for the preparation of 1-benzyl-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-3-cyano[1,8]-naphthyridine is shown in Scheme 6.
  • Preferred compounds of formula (Ia) include 1-R-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-[1,8]-naphthyridines-3-carboxylic acid ethyl ester wherein the substituent R on the 1 position of the naphthyridinyl ring is hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl, subtituted acylalkyl, heterocycle, substituted heterocycle, —(CH 2 ) m C( ⁇ O)Ar, or —(CH 2 ) m NR 4 R 5, , wherein m is 0, 1, 2, 3, or 4.
  • the starting materials for this synthesis are 2-chloro-3-pyridinecarboxylic acid and 4-methoxy
  • piperazine moiety is other than 2-thiophene corresponding N-acyl piperazine, prepared from acylation of t-butyl-1-piperazinecarboxylate with corresponding acid chloride followed by deprotection, can be used instead of piperazine-1-yl-thiophene-2-yl-methanone.
  • Preferred compounds of formula (Ia) include 1-R-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-[1,8]naphthyridine-3-carbonitriles wherein the substituent R on the 1 position of the naphthyridinyl ring is hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl, subtituted acylalkyl, heterocycle, substituted heterocycle, —(CH 2 ) m C( ⁇ O)Ar, or —(CH 2 ) m NR 4 R 5, , wherein m is 0, 1, 2, 3, or 4.
  • piperazine moiety is other than 2-thiophene corresponding N-acyl piperazine, prepared from acylation of t-butyl-1-piperazinecarboxylate with corresponding acid chloride followed by deprotection, can be used instead of piperazine-1-yl-thiophene-2-yl-methanone.
  • 2,6 Dichloro-5-fluoro-nicotinic acid was selected as a starting material to make the compounds of structure (Ia) with fluoro substitution at 6-position of naphthyridine moiety.
  • 2,6 Dichloro-5-fluoro-nicotinic acid was esterified by treating with thionyl chloride followed by refluxing with dry ethanol to yield 2,6-dichloro-5-fluoro-nicotinic acid ethyl ester, depicted by formula 21 in Scheme 10.
  • 6-Ethylsulfanyl-5-fluoro-2-(4-methoxy-benzylamino)-nicotinic acid ethyl ester depicted by formula 23 in Scheme 10 was prepared by animation of 2-chloro-6-ethylsulfanyl-5-fluoro-nicotinic acid ethyl ester, which was converted into 5-fluoro-2-(4-methoxy-benzylamino)-nicotinic acid ethyl ester, depicted by formula 24 in Scheme 10, by refluxing with ethanol and raney nickel.
  • 2-Hydroxy nicotinic acid was selected as a starting material for the synthesis of compounds with chloro substitution at 6-position of naphthyridine moiety. Chlorination of 2-hydroxynicotinic acid by sodium hypochlorite gave 5-chloro-2-hydroxy-nicotinic acid, depicted by formula 26 in Scheme 11. This intermediate was treated with thionyl chloride followed by refluxing with methanol to yield 2,5-dichloro-nicotinic acid methyl ester, depicted by formula 27 in Scheme 11.
  • piperazine moiety is other than 2-thiophene corresponding N-acyl piperazine, prepared from acylation of t-butyl-1-piperazinecarboxylate with corresponding acid chloride followed by deprotection, can be used instead of piperazine-1-yl-thiophene-2-yl-methanone in Scheme 13 and 14.
  • Preferred compounds of formula (Ic) include 1-R-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-[1,6]-naphthyridines-3-carboxylic acid ethyl ester wherein the substituent R on the 1 position of the naphthyridinyl ring is hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl, subtituted acylalkyl, heterocycle, substituted heterocycle, —(CH 2 ) m C( ⁇ O)Ar, or —(CH 2 ) m NR 4 R 5, , wherein m is 0, 1, 2, 3, or 4.
  • the starting materials for this synthesis was 4-aminopyridine which was protected by boc group and converted to 4-tert-butoxycarbonylamino-nicotinic acid, depicted as formula 35 in Scheme 15, by ortholithiation followed by quenching with dry ice.
  • This intermediate was reacted with trichloromethyl chloroformate to yield 1H-pyrido[4,3-d][1,3]oxazine-2,4-dione, depicted by formula 36 in Scheme 15, which was then converted to 4-chloro-2-oxo-1,2-dihydro-[1,6]-naphthyridine-3-carboxylic acid ethyl ester, depicted by formula 37 in Scheme 15.
  • piperazine moiety is other than 2-thiophene corresponding N-acyl piperazine, prepared from acylation of t-butyl-1-piperazinecarboxylate with corresponding acid chloride followed by deprotection, can be used instead of piperazine-1-yl-thiophene-2-yl-methanone.
  • the intermediate 4-chloro-2-oxo-1,2-dihydro-[1,6]-naphthyridine-3-carboxylic acid ethyl ester depicted by formula 37 in Scheme 15 can be prepared from 4-chloro pyridine or nicotinic acid as shown in Scheme 16.
  • Ortholithiation of 4-chloropyridine by LDA followed by quenching with dry ice or lithiation of nicotinic acid followed by quenching with hexachloroethane can give chloronicotinic acid intermediate, depicted by formula 39 in Scheme 16.
  • Preferred compounds of formula (Ic) include 1-R-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-[1,6]-naphthyridines-3-carbonitrile wherein the substituent R on the 1 position of the naphthyridinyl ring with nitrile group at 3-position is hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl, subtituted acylalkyl, heterocycle, substituted heterocycle, —(CH 2 ) m C( ⁇ O)Ar, or CH 2 ) m NR 4 R 5, , wherein m is 0, 1, 2, 3, or 4.
  • piperazine moiety is other than 2-thiophene corresponding N-acyl piperazine, prepared from acylation of t-butyl-1-piperazinecarboxylate with corresponding acid chloride followed by deprotection, can be used instead of piperazine-1-yl-thiophene-2-yl-methanone.
  • Preferred compounds of formula (Ib) include 1-R-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-[1,7]-naphthyridines-3-carboxylic acid ethyl ester wherein the substituent R on the 1 position of the naphthyridinyl ring is hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl, subtituted acylalkyl, heterocycle, substituted heterocycle, —(CH 2 ) m C( ⁇ O)Ar, or —(CH 2 ) m NR 4 R 5, , wherein m is 0, 1, 2, 3, or 4.
  • pyridine 3,4-dicarboxylic acid can react with acetic anhydride to give furo[3,4-c]pyridine-1,3-dione, depicted by formula 43 in Scheme 18, which can be converted to pyrrolo[3,4-c]pyridine-1,3-dione, depicted by formula 44 in Scheme 18, by reacting with acetamide.
  • 3-Amino isonicotinic acid can be prepared from Hoffmann degradation of this intermediate. Reductive amination of 3-amino isonicotinic acid can give 3-(4-methoxy-benzylamino) isonicotinic acid, depicted by formula 46 in Scheme 18.
  • This intermediate can also be prepared from alkylation of 3-amino isonicotinic acid by using lithium hexamethyl disilazide and p-methoxybenzylchloride as shown in Scheme 18.
  • Preferred compounds of formula (Ib) include 1-R-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-[1,7]-naphthyridines-3-carbonitrile wherein the substituent R on the 1 position of the naphthyridinyl ring with nitrile group at 3-position is hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl, subtituted acylalkyl, heterocycle, substituted heterocycle, —(CH 2 ) m C( ⁇ O)Ar, or CH 2 ) m NR 4 R 5, , wherein m is 0, 1, 2, 3, or 4.
  • piperazine moiety is other than 2-thiophene corresponding N-acyl piperazine, prepared from acylation of t-butyl-1-piperazinecarboxylate with corresponding acid chloride followed by deprotection, can be used instead of piperazine-1-yl-thiophene-2-yl-methanone.
  • Preferred compounds of formula (Id) include 1-R-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-[1,5]-naphthyridines-3-carboxylic acid ethyl ester wherein the substituent R on the 1 position of the naphthyridinyl ring is hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl, subtituted acylalkyl, heterocycle, substituted heterocycle, —(CH 2 ) m C( ⁇ O)Ar, or —(CH 2 ) m NR 4 R 5, , wherein m is 0, 1, 2, 3, or 4.
  • pyridine 2,3-dicarboxylic acid can react with acetic anhydride to give furo[3,4-b]pyridine-5,7-dione, depicted by formula 48 in Scheme 21, which can be converted to pyrrolo[3,4-b]pyridine-5,7-dione, depicted by formula 49 in Scheme 21, by reacting with acetamide.
  • 3-Amino pyridine-2-carboxylic acid can be prepared from Hoffmann degradation of this intermediate.
  • Reductive amination of 3-Amino pyridine-2-carboxylic acid can give 3-(4-methoxy-benzylamino)-pyridine-2carboxylic acid, depicted by formula 51 in Scheme 21.
  • This intermediate can also be prepared from alkylation of 3-amino isonicotinic acid by using lithium hexamethyl disilazide and p-methoxybenzylchloride as shown in Scheme 21.
  • Preferred compounds of formula (Id) include 1-R-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-[1,5]-naphthyridines-3-carbonitrile wherein the substituent R on the 1 position of the naphthyridinyl ring with nitrile group at 3-position is hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl, subtituted acylalkyl, heterocycle, substituted heterocycle, —(CH 2 ) m C( ⁇ O)Ar, or CH 2 ) m NR 4 R 5, , wherein m is 0, 1, 2, 3, or 4.
  • piperazine moiety is other than 2-thiophene corresponding N-acyl piperazine, prepared from acylation of t-butyl-1-piperazinecarboxylate with corresponding acid chloride followed by deprotection, can be used instead of piperazine-1-yl-thiophene-2-yl-methanone.
  • Macrophage migration inhibitory factor can be well suited for analysis as a drug target as its activity has been implicated in a variety of pathophysiological conditions. For instance, MIF has been shown to be a significant mediator in both inflammatory responses and cellular proliferation. In this regard, MIF has been shown to play roles as a cytokine, a pituitary hormone, as glucocorticoid-induced immunomodulator, and just recently as a neuroimmunomodulator and in neuronal function. Takahashi et al., Mol. Med. 4:707-714, 1998; Bucala, Ann. N.Y. Acad. Sci. 840:74-82, 1998; Bacher et al., Mol. Med.
  • anti-MIF antibodies have a variety of uses, notably decreased tumor growth, along with an observed reduction in angiogenesis. Ogawa et al., Cytokine 12(4):309-314, 2000; Metz and Bucala (supra). Accordingly, small molecules that can inhibit MIF have significant value in the treatment of inflammatory responses, reduction of angiogenesis, viral infection, bacterial infection, treatment of cancer (specifically tumorigenesis and apoptosis), treatment of graft versus host disease and associated tissue rejection.
  • a MIF inhibitor can be particularly useful in a variety of immune related responses, tumor growth, glomerulonephritis, inflammation, malarial anemia, septic shock, tumor associated angiogenesis, vitreoretinopathy, psoriasis, graft versus host disease (tissue rejection), atopic dermatitis, rheumatoid arthritis, inflammatory bowel disease, inflammatory lung disorders, otitis media, Crohn's disease, acute respiratory distress syndrome, delayed-type hypersensitivity.
  • a MIF inhibitor can also be useful in the treatment of stress and glucocorticoid function disorders, e.g., counter regulation of glucocorticoid action; or overriding of glucocorticoid mediated suppression of arachidonate release (Cys-60 based catalytic MIF oxidoreductase activity or JABI/CSNS-MIF-interaction based mechanism).
  • MIF may likely be produced by activated T-cells and macrophages during the proinflammatory stage of endotoxin-induced shock, e.g., as part of the localized response to infection.
  • a pro-inflammatory stimulus e.g., low concentrations of LPS, or by TNF- ⁇ and IFN- ⁇
  • macrophage-derived MIF may be the probable source of MIF produced during the acute phase of endotoxic shock.
  • Inhibitors of preferred embodiments inhibit lethality in mice following LPS challenge and likely attenuate IL-1 ⁇ and TNF- ⁇ levels. Accordingly, a variety of inflammatory conditions can be amenable to treatment with a MIF inhibitor.
  • the inhibition of MIF activity and/or release can be employed to treat inflammatory response and shock. Beneficial effects can be achieved by intervention at both early and late stages of the shock response.
  • anti-MIF studies have demonstrated that introduction of anti-MIF antibodies is associated with an appreciable (up to 35-40%) reduction in circulating serum TNF- ⁇ levels.
  • MIF inhibition therapy can be beneficial at the early stages of the inflammatory response.
  • MIF also plays a role during the post-acute stage of the shock response, and therefore, offers an opportunity to intervene at late stages where other treatments, such as anti-TNF- ⁇ therapy, are ineffective.
  • Inhibition of MIF can protect against lethal shock in animals challenged with high concentrations of endotoxin (i.e., concentrations that induce release of pituitary MIF into the circulation), and in animals challenged with TNF- ⁇ . Accordingly, the ability to inhibit MIF and protect animals challenged with TNF indicates that neutralization of MIF during the later, post-acute phase of septic shock can be efficacious.
  • TNF- ⁇ and IL-1 ⁇ levels are correlated at least in some instances to MIF levels.
  • an anti-MIF small molecule can be useful in a variety of TNF- ⁇ and/or IL-1 ⁇ associated disease states including transplant rejection, immune-mediated and inflammatory elements of CNS disease (e.g., Alzheimer's, Parkinson's, multiple sclerosis, etc.), muscular dystrophy, diseases of hemostasis (e.g., coagulopathy, veno occlusive diseases, etc.), allergic neuritis, granuloma, diabetes, graft versus host disease, chronic renal damage, alopecia (hair loss), acute pancreatitis, joint disease, congestive heart failure, cardiovascular disease (restenosis, atherosclerosis), joint disease, and osteoarthritis.
  • CNS disease e.g., Alzheimer's, Parkinson's, multiple sclerosis, etc.
  • diseases of hemostasis e.g., coagulopathy, veno occlusive diseases, etc.
  • MIF inhibitors in combination with steroidal therapy for the treatment of cytokine mediated pathophysiological conditions, such as inflammation, shock, and other cytokine-mediated pathological states, particularly in chronic inflammatory states such as rheumatoid arthritis.
  • cytokine mediated pathophysiological conditions such as inflammation, shock, and other cytokine-mediated pathological states, particularly in chronic inflammatory states such as rheumatoid arthritis.
  • Such combination therapy can be beneficial even subsequent to the onset of pathogenic or other inflammatory responses.
  • the administration of steroids subsequent to the onset of septic shock symptoms has proven of little benefit. See Bone et al., N. Engl. J. Med. 317: 653-658, 1987; Spring et al., N. Engl. J. Med. 311: 1137-1141, 1984.
  • Combination steroids/MIF inhibition therapy can be employed to overcome this obstacle.
  • such therapies can be tailored to inhibit MIF release and/or activity locally and/or systemically.
  • Suitable inhibitors of preferred embodiments are capable of decreasing one or more activities associated with MIF and/or MIF export.
  • a compound of structure (Ia), (Ib), (Ic), (Id) or any other structure can be assessed for activity as an inhibitor of MIF by one or more generally accepted assays for this purpose, including (but not limited to) the assays described below.
  • the assays can generally be divided into three categories, including assays which monitor export, those that monitor effector or small molecule binding, and those that monitor MIF activity. However, combinations of these assays are within the scope of the preferred embodiments.
  • epitope mapping of MIF acts as surrogate for biological activity.
  • the presence of a candidate inhibitor blocks the detection of export of MIF from cells (e.g., THP-1 cells) measured using a monoclonal antibody such as that commercially available from R&D systems (Minneapolis, Minn.) whereas a polyclonal antibody demonstrates that MIF is present.
  • cellular based or in vitro assays can be employed to demonstrate that these potential inhibitors inhibit MIF activity.
  • these two assays i.e., binding and activity assays
  • binding and activity assays can be combined into a singular assay which detects binding of a test compound (e.g., the ability to displace monoclonal antibodies or inhibit their binding) while also affecting MIF activity.
  • assays include combining an ELISA type assay (or similar binding assay) with a MIF tautomerism assay or similar functional assay.
  • the exact assay employed is irrelevant, provided it is able to detect the ability of the compound of interest to bind MIF.
  • the assay preferably detects the ability of the compound to inhibit a MIF activity because it selects for compounds that interact with biologically active MIF and not inactive MIF.
  • MIF inhibitory activity can also be recognized as a consequence of interfering with the formation of a polypeptide complex that includes MIF; disturbing such a complex can result in a conformational change inhibiting detection.
  • assays that monitor conformational changes in MIF are advantageous when employed either in addition to assays measuring competition between compounds, such as small molecules with mAb, or as a replacement of such an assay.
  • assays include, calorimetry, circular-dichroism, fluorescence energy transfer, light-scattering, nuclear magnetic resonance (NMR), surface plasmon resonance, scintillation proximity assays (see U.S. Pat. No. 5,246,869), and the like. See also WO02/07720-A1 and WO97/29635-A1.
  • any assay that indicates binding and preferably conformational change or placement near the active site of MIF can be utilized.
  • Descriptions of several of the more complicated proximity assays and conformational assays are set forth below, this discussion is merely exemplary and in no way should be construed as limiting to the type of techniques that can be utilized in preferred embodiments.
  • Circular dichroism can be utilized to determine candidate inhibitor binding.
  • Circular dichroism is based in part on the fact that most biological protein macromolecules are made up of asymmetric monomer units, L-amino acids, so that they all possess the attribute of optical activity. Additionally, rigid structures like DNA or an alpha helical polypeptide have optical properties that can be measured using the appropriate spectroscopic system. In fact, large changes in an easily measured spectroscopic parameter can provide selective means to identify conformational states and changes in conformational states under various circumstances, and sometimes to observe the perturbation of single groups in or attached to the macromolecule. Further, CD analysis has been frequently employed to probe the interactions of various macromolecules with small molecules and ligands. See Durand et al., Eur.
  • Plane polarized light is a combination of left circularly polarized light and right circularly polarized light traveling in phase.
  • the interaction of this light with an asymmetric molecule results in a preferential interaction of one circularly polarized component which, in an absorption region, is seen as a differential absorption (i.e., a dichroism).
  • a differential absorption i.e., a dichroism
  • Circular dichroism is an absorptive phenomenon that results when a chromophore interacts with plane polarized light at a specific wavelength.
  • the absorption band can be either negative or positive depending on the differential absorption of the right and left circularly polarized components for that chromophore.
  • ORD optical rotatory dispersion
  • CD offers the advantage of measuring optical events at the wavelength at which the event takes place.
  • Circular dichroism is specific to the electronic transition of the chromophore. See Berova and Woody, Circular Dichroism: Principles and Applications, John Wiley & Sons, N.Y., (2000).
  • alpha helical fibrous proteins show absorption curves closely resembling those of alpha helical polypeptides, but in globular proteins of known structure, like lysozyme and ribonuclease, the helical structures are in rather poor agreement with X-ray crystallography work.
  • a further source of difficulty in globular proteins is the prevalence of aromatic chromophores on the molecules around 280 nm.
  • An interesting example of helical changes has been demonstrated using myoglobin and apomyoglobin. After removing the prosthetic group heme, the apoprotein remaining has a residual circular dichroic ellipticity reduced by 25%. This loss of helix is attributable to an uncoiling of 10-15 residues in the molecule.
  • Other non-peptide, optically active chromophores include tyrosine, tryptophan, phenylalanine, and cysteine when located in the primary amino acid sequence of a macromolecule. Examples of non-peptide ellipticities include the disulfide transition in ribonuclease and the cysteine transitions of insulin.
  • circular dichroism can be employed to screen candidate inhibitors for the ability to affect the conformation of MIF.
  • MIF-binding agent or inhibitor complex formation can be determined by detecting the presence of a complex including MIF and a detectably labeled binding agent.
  • fluorescence energy signal detection for example by fluorescence polarization, provides determination of signal levels that represent formation of a MIF-binding agent molecular complex.
  • fluorescence energy signal-based comparison of MIF-binding agent complex formation in the absence and in the presence of a candidate inhibitor provides a method for identifying whether the agent alters the interaction between MIF and the binding agent.
  • the binding agent can be a MIF substrate, an anti-MIF antibody, or a known inhibitor, while the candidate inhibitor can be the compound to be tested or vice versa.
  • fluorescence energy signal-based determination of MIF-binding agent complex formation can be employed. Fluorescence energy signal detection can be, for example, by fluorescence polarization or by fluorescence resonance energy transfer, or by other fluorescence methods known in the art.
  • the MIF polypeptide can be labeled as well as the candidate inhibitor and can comprise an energy transfer molecule donor-acceptor pair, and the level of fluorescence resonance energy transfer from energy donor to energy acceptor is determined.
  • the candidate inhibitor and/or binding agent can be detectably labeled, and in particularly preferred embodiments the candidate inhibitor and/or binding agent is capable of generating a fluorescence energy signal.
  • the candidate inhibitor and/or binding agent can be detectably labeled by covalently or non-covalently attaching a suitable reporter molecule or moiety, for example any of various fluorescent materials (e.g., a fluorophore) selected according to the particular fluorescence energy technique to be employed, as known in the art and based upon the methods described herein.
  • Fluorescent reporter moieties and methods for as provided herein can be found, for example in Haugland (1996 Handbook of Fluorescent Probes and Research Chemicals—Sixth Ed., Molecular Probes, Eugene, Oreg.; 1999 Handbook of Fluorescent Probes and Research Chemicals—Seventh Ed ., Molecular Probes, Eugene, Oreg., http://www.probes.com/lit/) and in references cited therein.
  • Particularly preferred for use as such a fluorophore in preferred embodiments are fluorescein, rhodamine, Texas Red, AlexaFluor-594, AlexaFluor-488, Oregon Green, BODIPY-FL, and Cy-5.
  • any suitable fluorophore can be employed, and in certain embodiments fluorophores other than those listed can be preferred.
  • a fluorescence energy signal includes any fluorescence emission, excitation, energy transfer, quenching, dequenching event, or the like.
  • a fluorescence energy signal can be mediated by a fluorescent detectably labeled candidate inhibitor and/or binding agent in response to light of an appropriate wavelength.
  • generation of a fluorescence energy signal generally involves excitation of a fluorophore by an appropriate energy source (e.g., light of a suitable wavelength for the selected fluorescent reporter moiety, or fluorophore) that transiently raises the energy state of the fluorophore from a ground state to an excited state.
  • the excited fluorophore in turn emits energy in the form of detectable light typically having a different (e.g., usually longer) wavelength from that preferred for excitation, and in so doing returns to its energetic ground state.
  • the methods of preferred embodiments contemplate the use of any fluorescence energy signal, depending on the particular fluorophore, substrate labeling method and detection instrumentation, which can be selected readily and without undue experimentation according to criteria with which those having ordinary skill in the art are familiar.
  • the fluorescence energy signal is a fluorescence polarization (FP) signal.
  • the fluorescence energy signal can be a fluorescence resonance energy transfer (FRET) signal.
  • the fluorescence energy signal can be a fluorescence quenching (FQ) signal or a fluorescence resonance spectroscopy (FRS) signal.
  • FRET fluorescence resonance energy transfer
  • FQ fluorescence quenching
  • FRS fluorescence resonance spectroscopy
  • FP a measurement of the average angular displacement (due to molecular rotational diffusion) of a fluorophore that occurs between its absorption of a photon from an energy source and its subsequent emission of a photon, depends on the extent and rate of rotational diffusion during the excited state of the fluorophore, on molecular size and shape, on solution viscosity and on solution temperature (Perrin, 1926 J. Phys. Rad. 1:390). When viscosity and temperature are held constant, FP is directly related to the apparent molecular volume or size of the fluorophore.
  • the polarization value is a ratio of fluorescence intensities measured in distinct planes (e.g., vertical and horizontal) and is therefore a dimensionless quantity that is unaffected by the intensity of the fluorophore.
  • Low molecular weight fluorophores such as the detectably labeled candidate inhibitor and/or binding agent provided herein, are capable of rapid molecular rotation in solution (i.e., low anisotropy) and thus give rise to low fluorescence polarization readings.
  • the fluorophore moiety of the substrate associates with a complex that exhibits relatively slow molecular rotation in solution (i.e., high anisotropy), resulting in higher fluorescence polarization readings.
  • This difference in the polarization value of free detectably labeled candidate inhibitor and/or binding agent compared to the polarization value of MIF:candidate inhibitor and/or binding agent complex can be employed to determine the ratio of complexed (e.g., bound) to free.
  • This difference can also be employed to detect the influence of a candidate agent (i.e., candidate inhibitor) on the formation of such complexes and/or on the stability of a pre-formed complex, for example by comparing FP detected in the absence of an agent to FP detected in the presence of the agent.
  • FP measurements can be performed without separation of reaction components.
  • one aspect of a preferred embodiment utilizes the binding or displacement of a monoclonal antibody, known inhibitor, or other binding agent and/or complex formation of the candidate inhibitor with MIF to provide a method of identifying an inhibitor that alters the activity of MIF.
  • a class of compounds demonstrated the ability to inhibit/decrease monoclonal antibody binding to a biologically active MIF that is naturally produced from cells while not affecting the antibody's ability to recognize inactive (recombinant) MIF (as is available from commercial sources) and also demonstrated pronounced modulation of MIF activity in vivo.
  • antibody binding can be preferred as a surrogate for enzyme activity, thus eliminating the need to run expensive and complex enzymatic assays on each candidate compound.
  • the ability to avoid enzymatic assays leads to an assay that can be extremely well suited for high throughput use.
  • an assay can be employed outside of the MIF context and wherever enzyme or biological activity can be replaced by a binding assay.
  • any enzyme or other polypeptide whose biologically active form is recognized by a monoclonal antibody that does not recognize the inactive form e.g., small molecule inhibited form
  • the monoclonal antibody can bind the active site, but be displaced by a small molecule.
  • any small molecule that displaces the antibody can be a strong lead as a potential enzyme inhibitor.
  • the antibody can recognize an epitope that changes conformation depending on the active state of the enzyme, and that binding of a small molecule such that it precludes antibody binding to this epitope can also act as a surrogate for enzymatic activity even though the epitope may not be at the active site.
  • the type of assay utilized herein can be expanded to be employed with essentially any polypeptide wherein antibody displacement is predictive of activity loss.
  • any polypeptide e.g., enzyme and its associated neutralizing antibody can be employed to screen for small molecules that displace this antibody, thereby identifying likely inhibitors.
  • a MIF-binding agent/candidate inhibitor complex can be identified by any of a variety of techniques known in the art for demonstrating an intermolecular interaction between MIF and another molecule as described above, for example, co-purification, co-precipitation, co-immunoprecipitation, radiometric or fluorimetric assays, western immunoblot analyses, affinity capture including affinity techniques such as solid-phase ligand-counterligand sorbent techniques, affinity chromatography and surface affinity plasmon resonance, NMR, and the like (see, e.g., U.S. Pat. No. 5,352,660). Determination of the presence of such a complex can employ antibodies, including monoclonal, polyclonal, chimeric and single-chain antibodies, and the like, that specifically bind to MIF or the binding agent.
  • Labeled MIF and/or labeled binding agents/candidate inhibitors can also be employed to detect the presence of a complex.
  • the molecule of interest can be labeled by covalently or non-covalently attaching a suitable reporter molecule or moiety, for example any of various enzymes, fluorescent materials, luminescent materials, and radioactive materials.
  • suitable enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, and acetylcholinesterase.
  • fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, Texas Red, AlexaFluor-594, AlexaFluor-488, Oregon Green, BODIPY-FL and Cy-5.
  • luminescent materials include, but are not limited to, luminol and suitable radioactive materials include radioactive phosphorus [ 32 P], iodine [ 125 I or 131 I] or tritium [ 3 H].
  • MIF and the binding agent and/or the candidate inhibitor are combined under conditions and for a time sufficient to permit formation of an intermolecular complex between the components.
  • Suitable conditions for formation of such complexes are known in the art and can be readily determined based on teachings provided herein, including solution conditions and methods for detecting the presence of a complex and/or for detecting free substrate in solution.
  • Association of a detectably labeled binding agent(s) and/or candidate inhibitor(s) in a complex with MIF, and/or binding agent or candidate inhibitor that is not part of such a complex can be identified according to a preferred embodiment by detection of a fluorescence energy signal generated by the substrate.
  • an energy source for detecting a fluorescence energy signal is selected according to criteria with which those having ordinary skill in the art are familiar, depending on the fluorescent reporter moiety with which the substrate is labeled.
  • the test solution containing (a) MIF and (b) the detectably labeled binding agent and/or candidate inhibitor, is exposed to the energy source to generate a fluorescence energy signal, which is detected by any of a variety of well known instruments and identified according to the particular fluorescence energy signal.
  • the fluorescence energy signal is a fluorescence polarization signal that can be detected using a spectrofluorimeter equipped with polarizing filters.
  • the fluorescence polarization assay is performed simultaneously in each of a plurality of reaction chambers that can be read using an LJL CRITERIONTM Analyst (LJL Biosystems, Sunnyvale, Calif.) plate reader, for example, to provide a high throughput screen (HTS) having varied reaction components or conditions among the various reaction chambers.
  • LJL CRITERIONTM Analyst LJL Biosystems, Sunnyvale, Calif.
  • Examples of other suitable instruments for obtaining fluorescence polarization readings include the POLARSTARTM (BMG Lab Technologies, Offenburg, Germany), BEACONTM (Panvera, Inc., Madison, Wis.) and the POLARIONTM (Tecan, Inc., Research Triangle Park, N.C.) devices.
  • Determination of the presence of a complex that has formed between MIF and a binding agent and/or a candidate inhibitor can be performed by a variety of methods, as noted above, including fluorescence energy signal methodology as provided herein and as known in the art. Such methodologies can include, by way of illustration and not limitation FP, FRET, FQ, other fluorimetric assays, co-purification, co-precipitation, co-immunoprecipitation, radiometric, western immunoblot analyses, affinity capture including affinity techniques such as solid-phase ligand-counterligand sorbent techniques, affinity chromatography and surface affinity plasmon resonance, circular dichroism, and the like. For these and other useful affinity techniques, see, for example, Scopes, R.
  • MIF can interact with a binding agent and/or candidate inhibitor via specific binding if MIF binds the binding agent and/or candidate inhibitor with a K a of greater than or equal to about 10 4 M ⁇ 1 , preferably of greater than or equal to about 10 5 M ⁇ 1 , more preferably of greater than or equal to about 10 6 M ⁇ 1 and still more preferably of greater than or equal to about 10 7 M ⁇ 1 to 10 11 M ⁇ 1 .
  • Affinities of binding partners can be readily calculated from data generated according to the fluorescence energy signal methodologies described above and using conventional data handling techniques, for example, those described by Scatchard et al., Ann. N.Y. Acad. Sci. 51:660 (1949).
  • fluorescence energy signal is a fluorescence polarization signal
  • fluorescence anisotropy (in polarized light) of the free detectably labeled candidate inhibitor and/or binding agent can be determined in the absence of MIF
  • fluorescence anisotropy (in polarized light) of the fully bound substrate can be determined in the presence of a titrated amount of MIF.
  • Fluorescence anisotropy in polarized light varies as a function of the amount of rotational motion that the labeled candidate inhibitor and/or binding agent undergoes during the lifetime of the excited state of the fluorophore, such that the anisotropies of free and fully bound candidate inhibitor and/or binding agent can be usefully employed to determine the fraction of candidate inhibitor and/or binding agent bound to MIF in a given set of experimental conditions, for instance, those wherein a candidate agent is present (see, e.g., Lundblad et al., 1996 Molec. Endocrinol. 10:607; Dandliker et al., 1971 Immunochem. 7:799; Collett, E., Polarized Light: Fundamentals and Applications, 1993 Marcel Dekker, New York).
  • Certain of the preferred embodiments pertain in part to the use of intermolecular energy transfer to monitor MIF-binding agent complex formation and stability and/or MIF-candidate inhibitor complex formation.
  • Energy transfer is generated from a resonance interaction between two molecules: an energy-contributing “donor” molecule and an energy-receiving “acceptor” molecule.
  • Energy transfer can occur when (1) the emission spectrum of the donor overlaps the absorption spectrum of the acceptor and (2) the donor and the acceptor are within a certain distance (for example, less than about 10 nm) of one another.
  • the efficiency of energy transfer is dictated largely by the proximity of the donor and acceptor, and decreases as a power of 6 with distance. Measurements of ET thus strongly reflect the proximity of the acceptor and donor compounds, and changes in ET sensitively reflect changes in the proximity of the compounds such as, for example, association or dissociation of the donor and acceptor.
  • the method can employ any suitable ET donor molecule and ET acceptor molecule that can function as a donor-acceptor pair.
  • a detectable signal that is generated by energy transfer between ET donor and acceptor molecules results from fluorescence resonance energy transfer (FRET), as discussed above.
  • FRET occurs within a molecule, or between two different types of molecules, when energy from an excited donor fluorophore is transferred directly to an acceptor fluorophore (for a review, see Wu et al., Analytical Biochem. 218:1-13, 1994).
  • the ability of a candidate inhibitor to effect MIF export is tested.
  • test cells expressing MIF are employed (e.g., THP-1 cells). Either the test cells can naturally produce the protein or produce it from a transfected expression vector. Test cells preferably normally express the protein, such that transfection merely increases expressed levels. Thus, for expression of MIF and IL-1, THP1 cells are preferred.
  • virally-derived proteins such as HIV tat
  • the test cells do not “naturally” produce the protein, they can readily be transfected using an appropriate vector, so that the test cells express the desired protein, as those of skill in the art readily appreciate.
  • MIF is detected by any one of a variety of well-known methods and procedures. Such methods include staining with antibodies in conjunction with flow cytometry, confocal microscopy, image analysis, immunoprecipitation of cell cytosol or medium, Western blot of cell medium, ELISA, 1- or 2-D gel analysis, HPLC, bioassay, or the like. A convenient assay for initial screening is ELISA. MIF export can be confirmed by one of the other assays, preferably by immunoprecipitation of cell medium following metabolic labeling.
  • cells expressing MIF protein are pulse labeled for 15 minutes with 35 S-methionine and/or 35 S-cysteine in methionine and/or cysteine free medium and chased in medium supplemented with excess methionine and/or cysteine.
  • Media fractions are collected and clarified by centrifugation, such as in a microfuge.
  • Lysis buffer containing 1% NP-40, 0.5% deoxycholate (DOC), 20 mM Tris, pH 7.5, 5 mM EDTA, 2 mM EGTA, 10 nM PMSF, 10 ng/ml aprotinin, 10 ng/ml leupeptin, and 10 ng/ml pepstatin is added to the clarified medium.
  • Immune complexes are pelleted and washed with ice-cold lysis buffer. Complexes are further washed with ice-cold IP buffer (0.15 M NaCl, 10 mM Na-phosphate, pH 7.2, 1% DOC, 1% NP-40, 0.1% SDS).
  • Immune complexes are eluted directly into SDS-gel sample buffer and electrophoresed in SDS-PAGE.
  • the gel is processed for fluorography, dried and exposed to X-ray film.
  • cells can be engineered to produce a MIF that is tagged with a reporter so that the presence of an active MIF can be through the surrogate activity of the reporter.
  • the present inhibitors function by interacting directly with the naturally produced MIF complex in such a fashion as to alter the protein's conformation enough to block its biological activity. This interaction can be mapped by X-ray crystallography of MIF-compound co-crystals to determine the exact site of interaction. One site localizes to the pocket that is responsible for the tautomerase activity of MIF.
  • Screening assays for inhibitors of MIF export vary according to the type of inhibitor and the nature of the activity that is being affected. Assays can be performed in vitro or in vivo. In general, in vitro assays are designed to evaluate MIF activity, or multimerization, and in vivo assays are designed to evaluate MIF activity, extracellular localization, and intracellular localization in a model cell or animal system. In any of the assays, a statistically significant increase or decrease compared to a proper control is indicative of enhancement or inhibition.
  • One in vitro assay can be performed by examining the effect of a candidate compound on the ability of MIF to inhibit macrophage migration. Briefly, human peripheral blood monocytes are preferred as indicator cells in an agarose-droplet assay system essentially as described by Weiser et al., Cell. Immunol. 90:167-178, 1985 and Harrington et al., J. Immunol. 110:752-759, 1973. Other assay systems of analyzing macrophage migration can also be employed. Such an assay is described by Hermanowski-Vosatka et al., Biochem. 38:12841-12849, 1999.
  • In vivo assays can be performed in cells transfected either transiently or stably with an expression vector containing a MIF nucleic acid molecule, such as those described herein. These cells are preferred to measure MIF activity (e.g., modulation of apoptosis, proliferation, etc.) or extracellular and intracellular localization in the presence or absence of a candidate compound.
  • MIF activity e.g., modulation of apoptosis, proliferation, etc.
  • extracellular and intracellular localization in the presence or absence of a candidate compound.
  • assaying for apoptosis a variety of cell analyses can be employed, including, for example, dye staining and microscopy to examine nucleic acid fragmentation and porosity of the cells.
  • assays can be performed in model cell or animal systems by providing to the system a recombinant or naturally occurring form of MIF or inducing endogenous MIF expression in the presence or absence of test compound, thereby determining a statistically significant increase or decrease in the pathology of that system.
  • LPS can be employed to induce a toxic shock response.
  • a test cell can express the MIF naturally (e.g., THP-1 cells) or following introduction of a recombinant DNA molecule encoding the protein.
  • Transfection and transformation protocols are well known in the art and include Ca 2 PO 4 -mediated transfection, electroporation, infection with a viral vector, DEAE-dextran mediated transfection, and the like.
  • chimeric MIF proteins i.e., proteins prepared by fusion of MIF protein with another protein or protein fragment
  • protein sequences engineered to lack a leader sequence can be employed.
  • a fusion can be constructed to direct secretion, export, or cytosolic retention.
  • Any and all of these sequences can be employed in a fusion construct with MIF to assist in assaying inhibitors.
  • the host cell can also express MIF as a result of being diseased, infected with a virus, and the like.
  • Secreted proteins that are exported by virtue of a leader sequence are well known and include, human chorionic gonadatropin (hCG ⁇ ), growth hormone, hepatocyte growth factor, transferrin, nerve growth factor, vascular endothelial growth factor, ovalbumin, and insulin-like growth factor.
  • cytosolic proteins are well known and include, neomycin phosphotransferase, ⁇ -galactosidase, actin and other cytoskeletal proteins, and enzymes, such as protein kinase A or C.
  • the most useful cytosolic or secreted proteins are those that are readily measured in a convenient assay, such as ELISA.
  • the three proteins leaderless, secreted, and cytosolic
  • cells can be stably transformed or express the protein transiently.
  • Immunoprecipitation is one such assay that can be employed to determine inhibition. Briefly, cells expressing MIF naturally or from an introduced vector construct are labeled with 35 S-methionine and/or 35 S-cysteine for a brief period of time, typically 15 minutes or longer, in methionine- and/or cysteine-free cell culture medium. Following pulse labeling, cells are washed with medium supplemented with excess unlabeled methionine and cysteine plus heparin if the leaderless protein is heparin binding. Cells are then cultured in the same chase medium for various periods of time. Candidate inhibitors or enhancers are added to cultures at various concentrations. Culture supernatant is collected and clarified.
  • Supernatants are incubated with anti-MIF immune serum or a monoclonal antibody, or with anti-tag antibody if a peptide tag is present, followed by a developing reagent such as Staphylococcus aureus Cowan strain I, protein A-Sepharose®, or Gamma-bindTM G-Sepharose®.
  • Immune complexes are pelleted by centrifugation, washed in a buffer containing 1% NP-40 and 0.5% deoxycholate, EGTA, PMSF, aprotinin, leupeptin, and pepstatin. Precipitates are then washed in a buffer containing sodium phosphate pH 7.2, deoxycholate, NP-40, and SDS.
  • Immune complexes are eluted into a SDS gel sample buffer and separated by SDS-PAGE. The gel is processed for fluorography, dried, and exposed to x-ray film.
  • ELISA can be preferred to detect and quantify the amount of MIF in cell supernatants.
  • ELISA is preferred for detection in high throughput screening. Briefly, 96-well plates are coated with an anti-MIF antibody or anti-tag antibody, washed, and blocked with 2% BSA. Cell supernatant is then added to the wells. Following incubation and washing, a second antibody (e.g., an antibody to MIF) is added. The second antibody can be coupled to a label or detecting reagent, such as an enzyme, or to biotin. Following further incubation, a developing reagent is added and the amount of MIF determined using an ELISA plate reader.
  • a second antibody e.g., an antibody to MIF
  • the developing reagent is a substrate for the enzyme coupled to the second antibody (typically an anti-isotype antibody) or for the enzyme coupled to streptavidin.
  • Suitable enzymes are well known in the art and include horseradish peroxidase, which acts upon a substrate (e.g., ABTS) resulting in a colorimetric reaction.
  • the anti-MIF antibody can be directly coupled to the horseradish peroxidase, or other equivalent detection reagent.
  • cell supernatants can be concentrated to raise the detection level.
  • detection methods such as ELISA and the like can be employed to monitor intracellular as well as extracellular levels of MIF. When intracellular levels are desired, a cell lysate is preferred. When extracellular levels are desired, media can be screened.
  • ELISA can also be readily adapted for screening multiple candidate inhibitors or enhancers with high throughput.
  • an assay is conveniently cell-based and performed in 96-well plates.
  • Test cells that naturally or stably express MIF are plated at a level sufficient for expressed product detection, such as, about 20,000 cells/well. However, if the cells do not naturally express the protein, transient expression is achieved, such as by electroporation or Ca 2 PO 4 -mediated transfection.
  • 100 ⁇ l of a mixture of cells (e.g., 150,000 cells/ml) and vector DNA (5 ⁇ g/ml) is dispensed into individual wells of a 96-well plate.
  • the cells are electroporated using an apparatus with a 96-well electrode (e.g., ECM 600 Electroporation System, BTX, Genetronics, Inc.). Optimal conditions for electroporation are experimentally determined for the particular host cell type. Voltage, resistance, and pulse length are the typical parameters varied. Guidelines for optimizing electroporation can be obtained from manufacturers or found in protocol manuals, such as Current Protocols in Molecular Biology (Ausubel et al. (ed.), Wiley Interscience, 1987). Cells are diluted with an equal volume of medium and incubated for 48 hours. Electroporation can be performed on various cell types, including mammalian cells, yeast cells, bacteria, and the like.
  • ELISA ELISA is employed to detect the protein. An initial concentration of 50 ⁇ M is tested. If this amount gives a statistically significant reduction of export or reduction of monoclonal Ab detection, the candidate inhibitor is further tested in a dose response.
  • concentrated supernatant can be electrophoresed on a SDS-PAGE gel and transferred to a solid support, such as nylon or nitrocellulose.
  • MIF is then detected by an immunoblot (see Harlow, Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory, 1988), using an antibody to MIF containing an isotopic or non-isotopic reporter group.
  • reporter groups include, but are not limited to enzymes, cofactors, dyes, radioisotopes, luminescent molecules, fluorescent molecules, and biotin.
  • the reporter group is 125 I or horseradish peroxidase, which can be detected by incubation with 2,2′-azino-di-3-ethylbenzthiazoline sulfonic acid.
  • detection assays described above are readily adapted for use if MIF contains a peptide tag.
  • the antibody binds to the peptide tag.
  • Other assays include size or charge-based chromatography, including HPLC and affinity chromatography.
  • a bioassay can be employed to quantify the amount of active MIF present in the cell medium.
  • the bioactivity of the MIF can be measured by a macrophage migration assay. Briefly, cells transfected with an expression vector containing MIF are cultured for approximately 30 hours, during which time a candidate inhibitor or enhancer is added. Following incubation, cells are transferred to a low serum medium for a further 16 hours of incubation. The medium is removed and clarified by centrifugation. A lysis buffer containing protease inhibitors is added to the medium or, in the alternative, cells are lysed and internal levels are determined as follows. Bioactivity of MIF is then measured by macrophage migration assay, isomerase activity, or a proliferation assay.
  • a proliferation assay is performed by adding various amounts of the eluate to cultured quiescent 3T3 cells. Tritiated thymidine is added to the medium and TCA precipitable counts are measured approximately 24 hours later. Reduction of the vital dye MTT is an alternative way to measure proliferation.
  • purified recombinant human FGF-2 can be employed. Other functions can be assayed in other appropriate bioassays available in the art, such as CPS induced toxic shock, TSST-1 induced toxic shock, collagen induced arthritis, etc.
  • in vitro angiogenic assays include bioassays that measure proliferation of endothelial cells within collagen gel (Goto et al., Lab Invest. 69:508, 1993), co-culture of brain capillary endothelial cells on collagen gels separated by a chamber from cells exporting the MIF protein (Okamure et al., B.B.R.C. 186:1471, 1992; Abe et al., J. Clin. Invest. 92:54, 1993), or a cell migration assay (see Warren et al., J. Clin. Invest. 95:1789, 1995).
  • antibody is a broad term and is used in its ordinary sense, including, without limitation, to refer to polyclonal, monospecific, and monoclonal antibodies, as well as antigen binding fragments of such antibodies.
  • antigen as used herein is a broad term and is used in its ordinary sense, including, without limitation, to refer to a macrophage migration inhibitory factor polypeptide or a target polypeptide, variant, or functional fragment thereof.
  • An anti-MIF/target antibody, or antigen binding fragment of such an antibody can be characterized as having specific binding activity for the target polypeptide or epitope thereof of at least about 1 ⁇ 10 5 M ⁇ 1 , generally at least about 1 ⁇ 10 6 M ⁇ 1 , and preferably at least about 1 ⁇ 10 8 M ⁇ 1 .
  • Fab, F(ab′) 2 , Fd and Fv fragments of an anti-MIF/target antibody, which retain specific binding activity for a MIF/target polypeptide-specific epitope are encompassed within preferred embodiments.
  • the active site of an enzyme can be the epitope for a particular antibody and upon binding of a small molecule at or near the active site, immunoreactivity of the antibody is lost, thereby allowing the use of loss of immunoreactivity with an antibody as a surrogate marker for enzyme activity.
  • antibody as used herein is a broad term and is used in its ordinary sense, including, without limitation, to refer to naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric, bifunctional and humanized antibodies, as well as antigen-binding fragments thereof.
  • non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly, or can be obtained, for example, by screening combinatorial libraries including variable heavy chains and variable light chains (Huse et al., Science 246:1275-1281 (1989)).
  • an anti-MIF/target antibody can be raised using as an immunogen, for example, an isolated peptide including the active site region of MIF or the target polypeptide, which can be prepared from natural sources or produced recombinantly, as described above, or an immunogenic fragment of a MIF/target polypeptide (e.g., immunogenic sequences including 8-30 or more contiguous amino acid sequences), including synthetic peptides, as described above.
  • an immunogen for example, an isolated peptide including the active site region of MIF or the target polypeptide, which can be prepared from natural sources or produced recombinantly, as described above, or an immunogenic fragment of a MIF/target polypeptide (e.g., immunogenic sequences including 8-30 or more contiguous amino acid sequences), including synthetic peptides, as described above.
  • a non-immunogenic peptide portion of a MIF/target polypeptide can be made immunogenic by coupling the hapten to a carrier molecule such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), or by expressing the peptide portion as a fusion protein.
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • Various other carrier molecules and methods for coupling a hapten to a carrier molecule are well known in the art (Harlow and Lane, supra, 1992).
  • polyclonal antibodies for example, in a rabbit, goat, mouse, or other mammal
  • monoclonal antibodies can be obtained using methods that are well known and routine in the art (Harlow and Lane, supra, 1992).
  • spleen cells from a target polypeptide-immunized mammal can be fused to an appropriate myeloma cell line such as SP/02 myeloma cells to produce hybridoma cells.
  • Cloned hybridoma cell lines can be screened using a labeled target polypeptide or functional fragment thereof to identify clones that secrete target polypeptide monoclonal antibodies having the desired specificity.
  • Hybridomas expressing target polypeptide monoclonal antibodies having a desirable specificity and affinity can be isolated and utilized as a continuous source of the antibodies, which are useful, for example, for preparing standardized kits.
  • a recombinant phage that expresses, for example, a single chain anti-target polypeptide also provides a monoclonal antibody that can be employed for preparing standardized kits.
  • Candidate inhibitors of MIF have a variety of applicable uses, as noted above.
  • Candidate inhibitors of MIF can be isolated or procured from a variety of sources, such as bacteria, fungi, plants, parasites, libraries of chemicals (small molecules), peptides or peptide derivatives, and the like. Further, one of skill in the art recognizes that inhibition has occurred when a statistically significant variation from control levels is observed.
  • MIF is a mediator of endotoxemia
  • anti-MIF antibodies fully protected mice from LPS-induced lethality. See Bernhagen et al., Nature 365:756-759, 1993; Calandra et al., J. Exp. Med. 179:1895-1902, 1994; Bernhagen et al. Trends Microbiol. 2:198-201, 1994.
  • anti-MIF antibodies have markedly increased survival in mice challenged with gram-positive bacteria that induces septic shock. Bernhagen et al., J. Mol. Med.
  • MIF is a counterregulator of glucocorticoid action
  • pathological conditions including autoimmunity, inflammation, endotoxemia, and adult respiratory distress syndrome, inflammatory bowel disease, otitis media, inflammatory joint disease, and Crohn's disease.
  • compositions of preferred embodiments can be formulated for the manner of administration indicated, including for example, for oral, nasal, transmucosal, transcutaneous, venous, intracranial, intraperitoneal, subcutaneous, or intramuscular administration.
  • the compositions described herein can be administered as part of a sustained release implant.
  • compositions of preferred embodiments can be formulized as a lyophilizate, utilizing appropriate excipients that provide stability as a lyophilizate, and subsequent to rehydration.
  • compositions containing one or more inhibitors of MIF are provided.
  • the compounds of preferred embodiments can be formulated as pharmaceutical compositions.
  • Pharmaceutical compositions of preferred embodiments comprise one or more MIF inhibitors of preferred embodiments (i.e., a compound of structure (Ia) or (Ib)) and a pharmaceutically acceptable carrier and/or diluent.
  • the inhibitor of MIF is present in the composition in an amount which is effective to treat a particular disorder, that is, in an amount sufficient to achieve decreased MIF levels or activity, symptoms, and/or preferably with acceptable toxicity to the patient.
  • the pharmaceutical compositions of preferred embodiments can include an inhibitor of MIF in an amount from less than about 0.01 mg to more than about 1000 mg per dosage depending upon the route of administration, preferably about 0.025, 0.05, 0.075, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 mg to about 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 375, 400, 425, 450, 500, 600, 700, 800, or 900 mg, and more preferably from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg to about 30, 35, 40, 45, 50, 55, or 60 mg.
  • concentrations and dosages can be readily determined by one skilled in the art.
  • compositions formulated as liquid solutions include saline and sterile water, and can optionally include antioxidants, buffers, bacteriostats, and other common additives.
  • the compositions can also be formulated as pills, capsules, granules, or tablets that contain, in addition to an inhibitor or inhibitors of MIF, diluents, dispersing and surface-active agents, binders, and lubricants.
  • One skilled in this art can further formulate the inhibitor of MIF in an appropriate manner, and in accordance with accepted practices, such as those described in Remington's Pharmaceutical Sciences, Gennaro, Ed., Mack Publishing Co., Easton, Pa. 1990.
  • prodrugs are also included within the context of preferred embodiments.
  • Prodrugs are any covalently bonded carriers that release a compound of structure (Ia) or (Ib) in vivo when such prodrug is administered to a patient.
  • Prodrugs are generally prepared by modifying functional groups in a way such that the modification is cleaved, either by routine manipulation or in vivo, yielding the parent compound.
  • the compounds of structures (Ia), (Ib), (Ic), and (Id) can have chiral centers and can occur as racemates, racemic mixtures and as individual enantiomers or diastereomers. All such isomeric forms are included within preferred embodiments, including mixtures thereof. Furthermore, some of the crystalline forms of the compounds of structures (Ia), (Ib), (Ic), and (Id) can exist as polymorphs, which are included in preferred embodiments. In addition, some of the compounds of structures (Ia), (Ib), (Ic), and (Id) can also form solvates with water or other organic solvents. Such solvates are similarly included within the scope of the preferred embodiments.
  • a method for treating a variety of disorders or illnesses, including inflammatory diseases, arthritis, immune-related disorders, and the like. Such methods include administering of a compound of preferred embodiments to a warm-blooded animal in an amount sufficient to treat the disorder or illness.
  • Such methods include systemic administration of an inhibitor of MIF of preferred embodiments, preferably in the form of a pharmaceutical composition.
  • systemic administration includes oral and parenteral methods of administration.
  • suitable pharmaceutical compositions of an inhibitor of MIF include powders, granules, pills, tablets, and capsules as well as liquids, syrups, suspensions, and emulsions.
  • compositions can also include flavorants, preservatives, suspending, thickening and emulsifying agents, and other pharmaceutically acceptable additives.
  • the compounds of preferred embodiments can be prepared in aqueous injection solutions that can contain, in addition to the inhibitor of MIF activity and/or export, buffers, antioxidants, bacteriostats, and other additives commonly employed in such solutions.
  • administration of a compound of preferred embodiments can be employed to treat a wide variety of disorders or illnesses.
  • the compounds of preferred embodiments can be administered to a warm-blooded animal for the treatment of inflammation, cancer, immune disorders, and the like.
  • MIF inhibiting compounds can be used in combination therapies with other pharmaceutical compounds.
  • the MIF inhibiting compound is present in combination with conventional drugs used to treat diseases or conditions wherein MIF is pathogenic or wherein MIF plays a pivotal or other role in the disease process.
  • pharmaceutical compositions are provided comprising one or more MIF inhibiting compounds, including, but not limited to compounds of structures (Ia), (Ib), (Ic), and (Id), in combination with one or more additional pharmaceutical compounds, including, but not limited to drugs for the treatment of various cancers, asthma or other respiratory diseases, sepsis, arthritis, inflammatory bowel disease (IBD), or other inflammatory diseases, immune disorders, or other diseases or disorders wherein MIF is pathogenic.
  • one or more MIF inhibiting compounds are present in combination with one or more nonsteroidal anti-inflammatory drugs (NSAIDs) or other pharmaceutical compounds for treating arthritis or other inflammatory diseases.
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • Preferred compounds include, but are not limited to, celecoxib; rofecoxib; NSAIDS, for example, aspirin, celecoxib, choline magnesium trisalicylate, diclofenac potasium, diclofenac sodium, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, melenamic acid, nabumetone, naproxen, naproxen sodium, oxaprozin, piroxicam, rofecoxib, salsalate, sulindac, and tolmetin; and corticosteroids, for example, cortisone, hydrocortisone,
  • one or more MIF inhibiting compounds are present in combination with one or more beta stimulants, inhalation corticosteroids, antihistamines, hormones, or other pharmaceutical compounds for treating asthma, acute respiratory distress, or other respiratory diseases.
  • Preferred compounds include, but are not limited to, beta stimulants, for example, commonly prescribed bronchodilators; inhalation corticosteroids, for example, beclomethasone, fluticasone, triamcinolone, mometasone, and forms of prednisone such as prednisone, prednisolone, and methylprednisolone; antihistamines, for example, azatadine, carbinoxamine/pseudoephedrine, cetirizine, cyproheptadine, dexchlorpheniramine, fexofenadine, loratadine, promethazine, tripelennamine, brompheniramine, cholopheniramine
  • one or more MIF inhibiting compounds are present in combination with pharmaceutical compounds for treating cancer, such as paclitaxel, in a pharmaceutical composition.
  • one or more MIF inhibiting compounds are present in combination with immunosuppresive compounds in a pharmaceutical composition.
  • one or more MIF inhibiting compounds are present in combination with one or more drugs for treating an autoimmune disorder, for example, Lyme disease, Lupus (e.g., Systemic Lupus Erythematosus (SLE)), or Acquired Immune Deficiency Syndrome (AIDS).
  • SLE Systemic Lupus Erythematosus
  • AIDS Acquired Immune Deficiency Syndrome
  • Such drugs can include protease inhibitors, for example, indinavir, amprenavir, saquinavir, lopinavir, ritonavir, and nelfinavir; nucleoside reverse transcriptase inhibitors, for example, zidovudine, abacavir, lamivudine, idanosine, zalcitabine, and stavudine; nucleotide reverse transcriptase inhibitors, for example, tenofovir disoproxil fumarate; non nucleoside reverse transcriptase inhibitors, for example, delavirdine, efavirenz, and nevirapine; biological response modifiers, for example, etanercept, infliximab, and other compounds that inhibit or interfere with tumor necrosing factor; antivirals, for example, amivudine and zidovudine.
  • protease inhibitors for example, indinavir, amprenavir, saquin
  • one or more MIF inhibiting compounds are present in combination with pharmaceutical compounds for treating sepsis, such as steroids or anti-infective agents.
  • steroids include corticosteroids, for example, cortisone, hydrocortisone, methylprednisolone, prednisone, prednisolone, betamethesone, beclomethasone dipropionate, budesonide, dexamethasone sodium phosphate, flunisolide, fluticasone propionate, triamcinolone acetonide, betamethasone, fluocinolone, fluocinonide, betamethasone dipropionate, betamethasone valerate, desonide, desoximetasone, fluocinolone, triamcinolone, triamcinolone acetonide, clobetasol propionate, and dexamethasone.
  • corticosteroids for example, cortisone, hydrocortisone, methylprednisolone, pre
  • anti-infective agents include anthelmintics (mebendazole), antibiotics including aminoclycosides (gentamicin, neomycin, tobramycin), antifungal antibiotics (amphotericin b, fluconazole, griseofulvin, itraconazole, ketoconazole, nystatin, micatin, tolnaftate), cephalosporins (cefaclor, cefazolin, cefotaxime, ceftazidime, ceftriaxone, cefuroxime, cephalexin), beta-lactam antibiotics (cefotetan, meropenem), chloramphenicol, macrolides (azithromycin, clarithromycin, erythromycin), penicillins (penicillin G sodium salt, amoxicillin, ampicillin, dicloxacillin, nafcillin, piperacillin, ticarcillin), tetracyclines (doxycycline, minocycl
  • a MIF inhibitor in combination with an anesthetic, for example, ethanol, bupivacaine, chloroprocaine, levobupivacaine, lidocaine, mepivacaine, procaine, ropivacaine, tetracaine, desflurane, isoflurane, ketamine, propofol, sevoflurane, codeine, fentanyl, hydromorphone, marcaine, meperidine, methadone, morphine, oxycodone, remifentanil, sufentanil, butorphanol, nalbuphine, tramadol, benzocaine, dibucaine, ethyl chloride, xylocalne, and phenazopyridine.
  • an anesthetic for example, ethanol, bupivacaine, chloroprocaine, levobupivacaine, lidocaine, mepivacaine, procaine, ropivacaine, tetracaine,
  • the inhibitors of MIF of preferred embodiments were prepared by the methods described in Example 1.
  • Benzylamine 14 mL, 126.8 mmol was added to a solution of chloronicotinic acid (10 g, 63.4 mmol) in pyridine and refluxed overnight.
  • the pyridine was distilled and the residue was dissolved in 1N NaOH.
  • the solution was diluted with water to adjust the pH to 10 to 11 and washed by dichloromethane.
  • the aqueous phase was neutralized with cold aqueous 10% HCl solution to adjust the pH to 6 to 7.
  • the solids formed were filtered, washed with cold water, and dried in a vacuum oven to yield 12.2 g (84%) of 2-benzylamino nicotinic acid (1) as white solids.
  • Diethyl malonate (0.6 mL, 4 mmol) was added slowly to a suspension of NaH (60% in mineral oil, 164 mg, 4.1 mmol) in dimethylacetamide (20 mL) and stirred at room temperature for 0.5 h under inert atmosphere.
  • 1-Benzyl-1H-pyrido[2,3-d][1,3]oxazine-2,4-dione (2) (1 g, 4 mmol) was added to the solution and heated at 110° C. for 4 h (TLC control). The solution was cooled and poured into ice water. The pH of the solution was adjusted to 3 by cold 10% HCl.
  • 2-Thiophene carbonyl chloride (0.16 mL, 1.5 mmol) was added to a stirred solution of 1-benzyl-2-oxo-4-piperazin-1-yl-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester (5) (392 mg, 1 mmol) in pyridine (5 mL) at 0° C. under inert atmosphere. The solution was allowed to come at room temperature and further stirred for 18 h.
  • Cyclohexylamine (1.18 mL, 10.35 mmol) was added to a stirred solution of 1-benzyl-4-hydroxy-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester (3) (1.68 g, 5.17 mmol) in xylene and heated at 140° C. for 3 h. The solution was cooled and the solvent was evaporated under vacuum. The residue was suspended in water and extracted by dichloromethane.
  • 2-Thiophene carbonyl chloride (0.16 mL, 1.5 mmol) was added to a stirred solution of 1-benzyl-2-oxo-4-piperazin-1-yl-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (9) (345 mg, 1 mmol) in pyridine at 0° C. The solution was allowed to come at room temperature and further stirred overnight at room temperature. The solution was poured into ice water and the solids formed were filtered, washed by water, and dried.
  • p-Methoxybenzylamine (8.24 mL, 63.5 mmol) was added to a solution of 2-chloronicotinic acid (5 g, 31.7 mmol) in pyridine and refluxed overnight.
  • the pyridine was distilled and the residue was dissolved in 1N NaOH.
  • the solution was diluted with water to adjust the pH to 10 to 11 and washed by dichloromethane.
  • the aqueous phase was neutralized with cold aqueous 10% HCl solution to adjust the pH to 4 to 5.
  • the solids formed were filtered, washed with cold water, and dried in a vacuum oven to yield 6.32 g (77%) of 2-(4-methoxy-benzylamino)-nicotinic acid (11) as white solids.
  • Diethyl malonate (2.37 mL, 15.61 mmol) was added slowly to a suspension of NaH (60% in mineral oil, 0.62 g, 15.61 mmol) in dimethylacetamide (40 mL) and stirred at room temperature for 0.5 h under inert atmoshphere.
  • 1-(4-Methoxy-benzyl)-1H-pyrido[2,3-d][1,3]oxazine-2,4-dione (12) (4.47 g, 15.61 mmol) was added to the solution and heated at 110° C. for 3 h (TLC control). The solution was cooled and poured into ice water. The pH of the solution was adjusted to 3 by cold 10% HCl.
  • Cyclohexylamine (1.18 mL, 10.35 mmol) was added to a stirred solution of 4-hydroxy-1-(4-methoxy-benzyl)-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester (13) (1.80 g, 5.17 mmol) in xylene and heated at 140° C. for 3 h. The solution was cooled and the solvent was evaporated under vacuum. The residue was suspended in water and extracted by dichloromethane.
  • 1,4-Diazabicyclo[2.2.2]-octane (8.60 g, 77 mmol) was added to a solution of 4-chloro-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (31) (7.9 g, 38 mmol) and piperazin-1-yl-thiophene-2-yl-methanone (11.30 g, 57 mmol) in dimethylacetamide at room temperature. The solution was heated at 110° C. overnight. The solution was cooled and poured into ice cold 10% NH 4 Cl solution.
  • the compounds referred to as compound 34 through 48 were prepared from 2-oxo-4-[4-(thiophene-2-carbonyl)-piperazine-1-yl]-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (33) (400 mg, 1.1 mmol) and corresponding alkyl halides by applying either General Procedure A or General Procedure B as described above.
  • the compounds referred to as 49 through 55 were prepared from 1-benzyl-2-oxo-4-piperazin-1-yl-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester (5) by applying either General Procedure C or General Procedure D.
  • the compounds referred to as 56 through 64 were prepared from 1-benzyl-2-oxo-4-piperazin-1-yl-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (9) by applying either General Procedure C or General Procedure D described above.
  • Ethanethiol (15.08 mL, 204 mmol) was added slowly to a stirred suspension of NaH (60% in Mineral oil, 8.15 g, 204 mmol) in THF and stirred for 30 min at room temperature to yield a white thick suspension.
  • This suspension was diluted by cold THF, cooled to 0° C. and transferred to an already cooled solution of 2,6-dichloro-5-fluoro-nicotinic acid ethyl ester (65) (48.5 g, 204 mmol) in THF at ⁇ 20° C. under argon by maintaining the temperature below ⁇ 10° C. The solution was stirred at ⁇ 20° C. for 15 min and allowed to come at room temperature slowly.
  • Freshly activated raney nickel 50 g was added to a solution of 6-ethylsulfanyl-5-fluoro-2-(4-methoxy-benzylamino)-nicotinic acid ethyl ester (67) (31 g, 85 mmol) in anhydrous ethanol and refluxed for 48 h. The solution was cooled and filtered through celite. The filtrate was evaporated under reduced pressure to yield 24 g (93%) of 5-fluoro-2-(4-methoxy-benzylamino)-nicotinic acid ethyl ester (68) as a viscous oil which solidified to a white solids after keeping several days under vacuum at room temperature. Mp 90° C.
  • Diethyl malonate (10.79 mL, 71.38 mmol) was added slowly to a suspension of NaH (60% in mineral oil, 3.13 g, 78.24 mmol) in dimethylacetamide (200 mL) and stirred at room temperature for 0.5 h under inert atmoshphere.
  • 6-Fluoro-1-(4-methoxy-benzyl)-1H-pyrido-[2,3-d]-[1,3]-oxazine-2,4-dione (69) (21.5 g, 71.31 mmol) was added to the solution and heated at 110° C. for 4 h (TLC control). The solution was cooled and poured into ice water.
  • Diethyl malonate (6.02 mL, 71.38 mmol) was added slowly to a suspension of NaH (60% in mineral oil, 3.13 g, 78.24 mmol) in dimethylacetamide (200 mL) and stirred at room temperature for 0.5 h under argon atmoshphere.
  • 6-Chloro-1-(4-methoxy-benzyl)-1H-pyrido-[2,3-d]-[1,3]-oxazine-2,4-dione (73) (21.5 g, 71.31 mmol) was added to the solution and heated at 110° C. for 4 h (TLC control). The solution was cooled and poured into ice water.
  • the compounds referred to as 80 through 82 were prepared from either 4-chloro-6-fluoro-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester (78) or 4,6-dichloro-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester (79) by reacting with corresponding piperazine derivative according to general procedure E.
  • This compound was prepared from 4-chloro-6-fluoro-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester (78) and piperazine-1-yl-thiophene-2-yl-methanone according to general procedure E.
  • This compound was prepared from 4-chloro-6-fluoro-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester (78) and 2-furoyl piperazine according to general procedure E.
  • This compound was prepared from 4,6-dichloro-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester (79) and piperazine-1-yl-thiophene-2-yl-methanone according to general procedure E.
  • the compounds referred to as compound 83 through 91 were prepared from either 6-fluoro-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester (80), or 6-fluoro-4-[4-(furan-2-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester (81) or 6-chloro-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester (82) and corresponding alkyl halides by applying General Procedure B.
  • This compound was prepared from 6-fluoro-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester (80) and benzyl bromide according to General Procedure B.
  • This compound was prepared from 6-fluoro-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester (80) and 3-fluorobenzyl bromide according to General Procedure B.
  • This compound was prepared from 6-fluoro-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester (80) and 2-bromoacetophenone according to General Procedure B.
  • This compound was prepared from 6-fluoro-4-[4-(furan-2-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester (81) and benzyl bromide according to General Procedure B.
  • This compound was prepared from 6-fluoro-4-[4-(furan-2-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester (81) and 3-fluorobenzyl bromide according to General Procedure B.
  • This compound was prepared from 6-fluoro-4-[4-(furan-2-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester (81) and 2-bromoacetophenone according to General Procedure B.
  • This compound was prepared from 6-chloro-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester (82) and benzyl bromide according to General Procedure B.
  • This compound was prepared from 6-fluoro-6-chloro-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester (82) and 3-fluorobenzyl bromide according to General Procedure B.
  • This compound was prepared from 6-chloro-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester (82) and 2-bromoacetophenone according to General Procedure B.
  • Cyclohexylamine (6.14 mL, 53.65 mmol) was added to a stirred solution of 6-fluoro-4-hydroxy-1-(4-methoxy-benzyl)-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester (74) (10 g, 26.82 mmol) in xylene and heated at 140° C. for 3 h. The solution was cooled and the solvent was evaporated under vacuum. The residue was suspended in water and extracted by dichloromethane.
  • Cyclohexylamine (6.15 mL, 53.70 mmol) was added to a stirred solution of 6-chloro-4-hydroxy-1-(4-methoxy-benzyl)-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester (75) (10.44 g, 26.85 mmol) in xylene and heated at 140° C. for 3 h. The solution was cooled and the solvent was evaporated under vacuum. The residue was suspended in water and extracted by dichloromethane.
  • the compounds referred to as 98 through 101 were prepared from either 4-chloro-6-fluoro-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (96) or 4,6-dichloro-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (97) by reacting with corresponding piperazine derivative according to general procedure E described above.
  • This compound was prepared from 4-chloro-6-fluoro-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (96) and piperazin-1-yl-thiophene-2-yl-methanone according to general procedure E to yield 4.85 g (79%) of 6-fluoro-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazine-1-yl]-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (98) as yellow solids.
  • This compound was prepared from 4-chloro-6-fluoro-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (96) and 2-furoyl piperazine according to general procedure E to yield 2.87 g (71%) of 6-fluoro-2-oxo-4-[4-(furan-2-carbonyl)-piperazine-1-yl]-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (99) as white solids.
  • This compound was prepared from 4,6-dichloro-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (97) and piperazin-1-yl-thiophene-2-yl-methanone according to general procedure E to yield 6.8 g (74%) of 6-chloro-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazine-1-yl]-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (100) as yellow solids.
  • This compound was prepared from 4,6-dichloro-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (97) and 2-furoyl piperazine according to general procedure E to yield 2.27 g (71%) of 6-chloro-2-oxo-4-[4-(furan-2-carbonyl)-piperazine-1-yl]-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (101) as yellow solids.
  • the compounds referred to as compound 102 through 113 were prepared from either 6-fluoro-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazine-1-yl]-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (98) or 6-fluoro-2-oxo-4-[4-(furan-2-carbonyl)-piperazine-1-yl]-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (99) or 6-chloro-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazine-1-yl]-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (100) or 6-chloro-2-oxo-4-[4-(furan-2-carbonyl)-piperazine-1-yl]-1,2-dihydro-[1,8]-nap
  • This compound was prepared from 6-fluoro-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazine-1-yl]-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (98) and benzyl bromide according to General Procedure B.
  • This compound was prepared from 6-fluoro-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazine-1-yl]-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (98) and 3-fluorobenzyl bromide according to General Procedure B.
  • This compound was prepared from 6-fluoro-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazine-1-yl]-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (98) and 2-bromoacetophenone according to General Procedure B.
  • This compound was prepared from 6-fluoro-2-oxo-4-[4-(furan-2-carbonyl)-piperazine-1-yl]-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (99) and benzyl bromide according to General Procedure B.
  • This compound was prepared from 6-fluoro-2-oxo-4-[4-(furan-2-carbonyl)-piperazine-1-yl]-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (99) and 3-fluorobenzyl bromide according to General Procedure B.
  • This compound was prepared from 6-fluoro-2-oxo-4-[4-(furan-2-carbonyl)-piperazine-1-yl]-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (99) and 2-bromoacetophenone according to General Procedure B.
  • This compound was prepared from 6-chloro-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazine-1-yl]-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (100) and benzyl bromide according to General Procedure B.
  • This compound was prepared from 6-chloro-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazine-1-yl]-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (100) and 3-fluorobenzyl bromide according to General Procedure B.
  • This compound was prepared from 6-chloro-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazine-1-yl]-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (100) and 2-bromoacetophenone according to General Procedure B.
  • This compound was prepared from 6-chloro-2-oxo-4-[4-(furan-2-carbonyl)-piperazine-1-yl]-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (101) and benzyl bromide according to General Procedure B.
  • This compound was prepared from 6-chloro-2-oxo-4-[4-(furan-2-carbonyl)-piperazine-1-yl]-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (101) and 3-fluorobenzyl bromide according to General Procedure B.
  • This compound was prepared from 6-chloro-2-oxo-4-[4-(furan-2-carbonyl)-piperazine-1-yl]-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (101) and 2-bromoacetophenone according to General Procedure B.
  • Ethyl methanesulfonyl acetate (1.3 mL, 9.83 mmol) was added slowly to a suspension of NaH (60% in mineral oil, 433 mg, 10.81 mmol) in dimethylacetamide (20 mL) and stirred at room temperature for 0.5 h under argon.
  • 1-Benzyl-1H-pyrido[2,3-d][1,3]oxazine-2,4-dione (2) (2.5 g, 9.83 mmol) was added to the solution and heated at 110° C. for 4 h (TLC control). The solution was cooled and poured into ice water. The pH of the solution was adjusted to 4 by cold 10% HCl.
  • Triethylamine (1.2 mL, 8.6 mmol) was added to a suspension of of 1-benzyl-4-hydroxy-3-(methylsulfonyl)-[1,8]-naphthyridin-2(1H)-one (114) (0.94 g, 2.9 mmol) in neat POCl 3 and heated at 90° C. for 3 h. The solution was cooled and the excess POCl 3 was distilled under vacuum. The residue was suspended in water, neutralized by solid NaHCO 3 , and extracted by dichloromethane.
  • DABCO (0.57 g, 5.0 mmol) was added to a solution of 1-benzyl-4-chloro-3-(methylsulfonyl)-[1,8]-naphthyridin-2(1H)-one (115) (0.88 g, 2.5 mmol) and piperazine-1-yl-thiophene-2-yl-methanone (0.60 g, 3.08 mmol) in N-methylpyrrilidone at room temperature. The solution was heated at 110° C. for 15 min. The solution was cooled and poured into ice cold 10% ammonium chloride solution in water.
  • n-Buli (1.6 M soln, 155 mL, 249 mmol) was added to a stirred solution of TMEDA (37.36 mL, 249 mmol) in THF at ⁇ 40° C. The solution was allowed to come at room temperature over 10 min and stirred for another 10 min. The solution was cooled to ⁇ 78° C. A solution of pyridine-4-yl-carbamic acid-tert-butyl ester (117) (22 g, 113.26 mmol) in THF was added slowly. The solution was allowed to come at room temperature within 3 h. After stirring at room temperature for 15 min the solution was again cooled to ⁇ 78° C. and a freshly crushed dry ice was added.
  • Diethyl malonate (13.77 mL, 91 mmol) was added slowly to a suspension of NaH (60% in mineral oil, 3.63 mg, 91 mmol) in dimethylacetamide and stirred at room temperature for 0.5 h under inert atmosphere.
  • 1H-pyrido[4,3-d][1,3]oxazine-2,4-dione (119) (15 g, 91 mmol) was added to the solution and heated overnight at 110° C. The solution was cooled and poured into ice water. Basified by saturated NaHCO 3 solution and extracted by dichloromethane. The pH of the aqueous phase was adjusted to 3 by cold 10% HCl and extracted by n-BuOH.
  • DABCO (0.7 g, 6.3 mmol) was added to a solution of 4-chloro-2-oxo-1,2-dihydro-[1,6]-naphthyridine-3-carboxylic acid ethyl ester (120) (0.8 g, 3.16 mmol) and piperazine-1-yl-thiophene-2-yl-methanone (0.93 g, 4.74 mmol) DMA at room temperature. The solution was heated at 110° C. for 2 h. The solution was cooled and poured into ice cold 10% ammonium chloride solution in water.
  • the compounds referred to as compound 122 through 124 were prepared from 2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-[1,6]-naphthyridin-3-carboxylic acid ethyl ester (121) and corresponding alkyl halides by applying General Procedure B as described above.
  • This compound was prepared from 2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-[1,6]-naphthyridin-3-carboxylic acid ethyl ester (121) and 4-fluorobenzyl bromide according to General Procedure B.
  • This compound was prepared from 2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-[1,6]-naphthyridin-3-carboxylic acid ethyl ester (121) and 3-fluorobenzyl bromide according to General Procedure B.
  • This compound was prepared from 2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-[1,6]-naphthyridin-3-carboxylic acid ethyl ester (121) and 2-bromoacetophenone according to General Procedure B.
  • Triethylamine (12.2 mL, 88 mmol) was added to a suspension of of 1-benzyl-4-hydroxy-3-nitro-1,8-naphthyridin-2(1H)-one (125) (8.7 g, 29 mmol) in neat POCl 3 and heated at 90° C. for 3 h. The solution was cooled and the excess POCl 3 was distilled under vacuum. The residue was suspended in water, neutralized by solid NaHCO 3 , and extracted by dichloromethane. The organic layer was subsequently washed by saturated NaHCO 3 solution, water and brine, dried over Na 2 SO 4 , and evaporated to yield yellow solid. Yield 9.2 g, 98%, Mp ca 215° C. (not sharp).
  • This compound was prepared from 2-furyl chloride and 1-benzyl-2-oxo-4-piperazin-1-yl-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (9) according to General Procedure C.
  • the reaction yielded 1-benzyl-4-[4-(furan-2-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-[1,8]-naphthyridine-3-carbonitrile (129) as white solids.
  • Macrophage migration is measured by using the agarose droplet assay and capillary method as described by Harrington and Stastny et al., J. Immunol. 110(3):752-759, 1973. Briefly, macrophage-containing samples are added to hematocrit tubes, 75 mm long with a 1.2 mm inner diameter. The tubes are heat-sealed and centrifuged at 100 ⁇ G for 3 minutes, cut at the cell-fluid interface and imbedded in a drop of silicone grease in Sykes-Moore culture chambers. The culture chambers contain either a control protein (BSA) or samples. Migration areas are determined after 24 and 48 hours of incubation at 37° C. by tracing a projected image of the macrophage fans and measuring the areas of the migration by planimetry.
  • BSA control protein
  • each well of a 96-well plate is pre-coated with one microliter of liquid 0.8% (w/v) Sea Plaque Agarose in water dispensed onto the middle of each well.
  • the plate is then warmed gently on a light box until the agarose drops are just dry.
  • Two microliters of macrophage containing cell suspensions of up to 25% (v/v) in media (with or without MIF or other controls), containing 0.2% agarose (w/v) and heated to 37° C. is added to the precoated plate wells and cooled to 4° C. for 5 min.
  • Each well is then filled with media and incubated at 37° C. under 5% CO 2 -95% air for 48 hr.
  • Migration from the agarose droplets is measured at 24 and 48 hr by determining the distance from the edge of the droplet to the periphery of migration.
  • Monocyte migration inhibitory activities of recombinant murine and human wild-type and murine mutant MIF are analyzed by use of human peripheral blood mononuclear cells or T-cell depleted mononuclear cells in a modified Boyden chamber format.
  • Calcein AM-labeled monocytes are suspended at 2.5 to 5 ⁇ 10 6 /mL in RPMI 1640 medium, with L-glutamine (without phenol red) and 0.1 mg/mL human serum albumin or bovine serum albumin.
  • An aliquot (200 ⁇ L) of cell suspension is added to wells of a U-bottom 96-well culture plate (Costar, Cambridge, Mass.) prewarmed to 37° C.
  • MIF in RPMI 1640 is added to the cell suspension to yield final concentrations of 1, 10, 100, and 1000 ng/mL.
  • the culture plate is placed into the chamber of a temperature-controlled plate reader, mixed for 30 s, and incubated at 37° C. for 10-20 min.
  • 28 ⁇ L of prewarmed human monocyte chemotactic protein 1 (MCP-1; Pepro Tech., Inc., Rocky Hill, N.J.) at 10 or 25 ng/mL or RPMI 1640 with 0.1 mg/mL HSA is added to the bottom well of a ChemoTX plate (Neuro Probe Inc., Gaithersburg, Md.; 3 mm well diameter, 5 ⁇ M filter pore size).
  • the filter plate is carefully added to the base plate. Treated cell suspensions are removed from the incubator and 30 ⁇ L is added to each well of the filter plate. The assembled plate is incubated for 90 min. at 37° C. in a humidified chamber with 5% CO 2 . Following incubation, the cell suspension is aspirated from the surface of the filter and the filter is subsequently removed from the base plate and washed three times by adding 50 ⁇ L of 1 ⁇ HBSS ⁇ to each filter segment. Between washes, a squeegee (NeuroProbe) is employed to remove residual HBSS ⁇ . The filter is air-dried and then read directly in the fluorescent plate reader, with excitation at 485 nm and emission at 535 nm.
  • Chemotactic or random migration indices are defined as average filter-bound fluorescence for a given set of wells divided by average fluorescence of filters in wells containing neither MCP-1 nor MIF. Titration of fluorescently-labeled cells revealed that levels of fluorescence detected in this assay have a linear relationship to cell number (not shown).
  • the tautomerization reaction is carried out essentially as described by Rosengren et al., Mol. Med. 2(1):143-149, 1996. D-dopachrome conversion to 5,6-dihydroxyindole-2-carboxylic acid is assessed. 1 ml sample cuvettes containing 0.42 mM substrate and 1.4 ⁇ g of MIF in a sample solution containing 0.1 mM EDTA and 10 mM sodium phosphate buffer, pH 6.0 are prepared and the rate of decrease in iminochrome absorbance is followed at 475 nm. L-dopachrome is employed as a control.
  • reaction products can be followed using an HPLC, utilizing a mobile phase including 20 mM KH 2 PO 4 buffer (pH 4.0) and 15% methanol with a flow rate of 1.2 ml/min. Fluorimetric detection is followed at 295/345 mm.
  • the tautomerization reaction utilizing phenylpyruvate or (p-hydroxyphenyl)pyruvate is carried out essentially as described by Johnson et al., Biochem. 38:16024-16033, 1999.
  • the assay mixture contains 50 mM Na 2 HPO 4 buffer (1 mL, pH 6.5) and an aliquot of a solution of MIF sufficiently dilute (0.5-1.0 ⁇ L of a 2.3 mg/mL solution, final concentration of 93-186 nM) to yield an initial liner rate.
  • the assay is initiated by the addition of a small quantity (1-3.3 ⁇ L) of either phenylpyruvate or (p-hydroxyphenyl)pyruvate from stock solutions made up in ethanol.
  • the crystalline forms of phenylpyruvate and (p-hydroxyphenyl)pyruvate exist exclusively as the enol isomers (Larsen et al., Acta Chem. Scand. B 28:92-96, 1974).
  • the concentration of substrate can range from 10 to 150 M, with no significant inhibition of MIF activity by ethanol observed at less than 0.5% v/v.
  • lysis buffer 1% NP40, 0.5% deoxycholate, 20 mM Tris pH 7.5, 5 mM EDTA, 2 mM EGTA, 0.01 mM phenylmethylsufonyl fluoride, 10 ng/ml aprotinin, 10 ng/ml leupeptin, 10 ng/ml peptstatin
  • lysis buffer 1% NP40, 0.5% deoxycholate, 20 mM Tris pH 7.5, 5 mM EDTA, 2 mM EGTA, 0.01 mM phenylmethylsufonyl fluoride, 10 ng/ml aprotinin, 10 ng/ml leupeptin, 10 ng/ml peptstatin
  • Immune complexes are sedimented by microfuge centrifugation, washed three times with lysis buffer, and four times with ice cold immunoprecipitation wash buffer (0.15M NaCl, 0.01 M Na-phosphate pH 7.2, 1% deoxycholate, 1% NP-40, 0.1% sodium dodecyl sulfate).
  • Immune complexes are dissociated directly in SDS gel sample buffer 125 mM Tris, pH 6.8, 4% SDS, 10% glycerol, 0.004% bromphenol blue, 2 mM EGTA, and separated by 12% SDS-PAGE.
  • the gel is processed for fluorography, dried, and exposed to X-ray film at ⁇ 70° C.
  • a rabbit anti-NPT antibody (5Prime-3Prime, Boulder, Colo.) is employed.
  • proteins are transferred from the 12% SDS-PAGE gel to a nitrocellulose membrane (pore size 0.45 ⁇ m in cold buffer containing 25 mM 3-[dimethyl(hydroxymethyl)methylamino]-2-hydroxypropane-sulfonic acid, pH 9.5, 20% methanol for 90 minutes at 0.4 amps.
  • a nitrocellulose membrane pore size 0.45 ⁇ m in cold buffer containing 25 mM 3-[dimethyl(hydroxymethyl)methylamino]-2-hydroxypropane-sulfonic acid, pH 9.5, 20% methanol for 90 minutes at 0.4 amps.
  • the media is centrifuged (10 minutes at 800 g) and the supernatants concentrated 10-fold by membrane filtration (10 kDa cut-off, Centricon-10 Amicon).
  • Membranes are blocked in 10 mM Tris, pH 7.5, 150 mM NaCl, 5 mM NaN 3 , 0.35% polyoxyethylene-sorbitan monolaurate, and 5% nonfat dry milk (Carnation Co., Los Angeles, Calif.) for 1 hr at room temperature. Membranes are incubated with a monoclonal antibody (Catalog Number MAB289, purchased from R&D Systems, Minneapolis, Minn.) or polyclonal (goat polyclonal serum, R&D Systems cat#AF-289-PB).
  • a monoclonal antibody Catalog Number MAB289, purchased from R&D Systems, Minneapolis, Minn.
  • polyclonal goat polyclonal serum, R&D Systems cat#AF-289-PB.
  • membranes are washed at room temperature with 10 changes of buffer containing 150 mM NaCl, 500 mM sodium phosphate pH 7.4, 5 mM NaN 3 , and 0.05% polyoxyethylene-sorbitan monolaurate.
  • buffer containing 150 mM NaCl, 500 mM sodium phosphate pH 7.4, 5 mM NaN 3 , and 0.05% polyoxyethylene-sorbitan monolaurate When using monoclonal antibodies, membranes are then incubated in blocking buffer containing 1 ⁇ g/ml rabbit anti-mouse IgG (H+L, affinipure, Jackson Immuno Research Laboratories, West Grove, Pa.) for 30 minutes at room temperature. For polyclonal probing, incubation employed rabbit anti-goat (Sigma, Catalog Number G5518).
  • Membranes are subsequently washed in 1 L of buffer described above, and incubated for 1 hr in 100 ml of blocking buffer containing 15 ⁇ Ci 125 ]-protein A (ICN Biochemicals, Costa Mesa, Calif.), and washed with 1 L of buffer. The radiosignal is visualized by autoradiography.
  • mouse macrophage RAW 264.7 cells (American Type Culture Collection, Manassas, Va.) are selected.
  • Raw 264.7 macrophage (3 ⁇ 10 6 cells per well) are plated in 12-well tissue culture plates (Costar) and are cultured in RPMI/1% heat-inactivated fetal bovine serum (FBS) (Hyclone Laboratories, Logan, Utah). After three hours of incubation at 37° C. in a humidified atmosphere with 5% CO 2 , nonadherent cells are removed and wells are washed twice with RPMI/1% FBS.
  • FBS heat-inactivated fetal bovine serum
  • LPS (011:B4) or TSST-1 (Toxin Technology, Sarasota, Fla.), that are approximately 95% pure and are resuspended in pyrogen-free water, at a concentration ranging from 1 pg/ml to 1000 ng/ml (for the dose response experiment).
  • conditioned media of parallel cultures are removed at 0.5, 1, 2, 4, 8 and 24 hours intervals after stimulation with 1 ng/ml TSST-1 or LPS.
  • RAW 264.7 cells (3 ⁇ 10 6 cells per well) are incubated for 24 hours with 1 ng/ml of LPS (0111:B4) or 1 ng/ml of TSST-1 in the presence of 0.01 ⁇ M to 10 ⁇ M candidate compound or buffer (as control).
  • the MIF in cell-conditioned media is concentrated on filters and the MIF remaining in the samples is analyzed by Western blotting and MIF band densities are also measured by Stratagene Eagle EyeTM.
  • RAW cells are induced to express MIF by addition of either 1 ng/ml TSST-1 or LPS and are cultured for 24 hours.
  • MIF in conditioned media is measured as described above.
  • MIF inhibiting compounds reduce immunodetectable MIF levels in conditioned media in a concentration dependent manner, as compared to cells incubated with buffer only.
  • Target cells obtained from the American Type Culture Collection (ATCC No. CRL 1650) are cultured overnight in a 48-well plate in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 nM nonessential amino acids, and 50 ⁇ g/ml gentamycin.
  • the target cells are then transfected with 2 ⁇ g/ml of CsCl-purified plasmid DNA in transfection buffer (140 mM NaCl, 3 mM KCl, 1 mM CaCl 2 , 0.5 mM MgCl 2 , 0.9 mM Na 2 HPO 4 , 25 mM Tris, pH 7.4.
  • plasmid of interest is co-transfected with pMAMneo (Clontech, Palo Alto, Calif.), which contains the selectable marker neomycin phosphotransferase.
  • pMAMneo Clontech, Palo Alto, Calif.
  • the target cells are metabolically pulse-labeled for 15 minutes with 100 ⁇ Ci of 35 S-methionine and 35 S-cysteine (Trans 35 S-label, ICN Biomedicals, Irvine, Calif.) in 1 ml of methionine and cysteine free DMEM. Following labeling, the cell monolayers are washed once with DMEM supplemented with excess (10 mM) unlabeled methionine and cysteine for 1-2 minutes. Cells are then cultured in 2 ml of this medium for the indicated lengths of time and the cell supernatants are immunoprecipitated for the presence of leaderless protein. For the indicated cultures, chase medium is supplemented with modulator at the indicated concentrations.
  • the target cells are washed once with 250 ⁇ l of 0.1 M sodium carbonate, pH 11.4, for 1 to 2 minutes and immediately aspirated.
  • a high salt solution can alternatively be preferred.
  • the cells are washed with media containing 0.5% FBS plus 25 ⁇ g/ml heparin and then the cells are incubated in this same medium for the indicated lengths of time.
  • chase medium is supplemented with a modulator.
  • the carbonate wash and heparin containing medium can be omitted.
  • the high throughput screening assay for MIF inhibitors is performed in a 96-well format using MIF produced by THP-1 cells and is performed as follows. MIF assays are performed by ELISA as indicated above. THP-1 cells are resuspended to approx. 5 ⁇ 10 6 cells/ml in RPMI medium containing 20 ⁇ g/ml of bacterial LPS and the cells incubated for 18-20 hours. Subsequently cell supernatant is collected and incubated with putative inhibitors. Briefly, a 96-well plate (Costar Number 3590) ELISA plate is coated with a MIF monoclonal antibody (R&D Systems Catalog Number MAB289) at a concentration of 4 ⁇ g/ml for two hours at 37° C.
  • MIF monoclonal antibody R&D Systems Catalog Number MAB289
  • Undiluted culture supernate is added to the ELISA plate for a two-hour incubation at room temperature.
  • the wells are then washed, a biotinylated MIF polyclonal antibody (R&D Systems #AF-289-PB) is added followed by Streptavidin-HRP and a chromogenic substrate.
  • the amount of MIF is calculated by interpolation from an MIF standard curve.
  • test compounds such as MIF inhibitors
  • RP-HPLC is performed with a Hewlett-Packard Model HP-1100 unit using Symmetry Shield RP-8 (4.6 ⁇ 75 mm id, Waters, Milford, Mass.).
  • the mobile phase is an isocratic solution of 35% acetonitrile/water containing 0.1% trifluroacetic acid. Absorbance is monitored at 235 nm.
  • the sample serum proteins are first separated using 50% Acetonitrile (4° C. overnight) followed by centrifugation at 14000 rpm for 30 minutes. The supernatant is then analyzed by the RP-HPLC and the compound concentration is calculated based on a calibration curve of known standard.
  • reverse phase HPLC is employed to detect candidate compounds in a linear range of 1.5-800 ng using spiked test samples.
  • the above analytical technique is applied to blood serum from animals receiving candidate compounds (0.4 mg/20 gram mouse), circulating concentrations of candidate compounds are quantitatively measured.
  • the purpose for in vivo experiments is to confirm initial in vitro assay results using candidate compounds to inhibit MIF.
  • LPS-induced toxicity appears to be related to an overproduction of MIF as well as TNF- ⁇ and IL-1, since animals can be protected from endotoxin shock by neutralizing or inhibiting these inflammation mediators.
  • the present model is chosen because it provides reproducible and rapid lethal models of sepsis and septic shock.
  • LPS lipopolysaccharide
  • mice Ten 10-week-old (20 ⁇ 2 gram) female BALB/c mice (Charles River Laboratories, Kingston, N.Y.) are housed in a group of 5 per cage with free access to food and water and are acclimatized for at least one week prior to experimentation. On the day of experiment, mice are weighed and randomly distributed into groups of 10 animals of equal mean body weight. Mice are injected i.p. with 200 ⁇ L of formulated candidate compound or buffer alone immediately before the i.p. injection of LPS ( Escherichia coli 0111:B4, 10 mg/kg or 5 mg/kg body weight) and ⁇ -D-galactosamine (50 mg/kg body weight).
  • LPS Escherichia coli 0111:B4, 10 mg/kg or 5 mg/kg body weight
  • ⁇ -D-galactosamine 50 mg/kg body weight
  • Each dose of LPS (0.2 ml for 20 gram mouse) is administered intraperitoneally and is mixed with a final concentration of ⁇ -D-galactosamine of 50 mg per ml. Following collection of blood specimens taken from cardiac puncture, the animal is sacrificed. Typical collections are performed at 4 hours post LPS treatment. The serum is separated in a serum separator (Microtainer® Becton Dickinson, Minneapolis, N.J.) according to the manufacturer's protocol. Mouse serum Il-1 ⁇ and TNF- ⁇ are measured by ELISA using a “mouse IL 1 ⁇ immunoassay” or “mouse TNF- ⁇ immunoassay” kit (R&D System Minneapolis, Minn.) following manufacturer's direction. Serum MIF concentrations in mouse serum are quantified by a sandwich ELISA (ChemiKine MIF Kit, Chemicon, San Diego, Calif.). Samples are analyzed in duplicate, and results are averaged.
  • mice Ten 8 to 10 week-old (20 ⁇ 2 gram) female BALB/c mice are housed and acclimatized as described above. On the day of the experiments, the mice are weighed and randomly distributed into groups of 5 animals of equal mean body weight. Mice are injected with 200 ⁇ l of formulated candidate compound or its Buffer (average 20 mg/kg compound) following i.p. injection of LPS ( E. Coli 055B5, Sigma) (40, 10, 5, 2 or 0.5 mg/kg body weight) and 50 mg/kg of ⁇ -D-galactosamine. Mice are observed every two hours during the first 18 hours and twice a day for seven days. For these studies Kaplan-Meier estimation methods are employed to assess animal survival.
  • LPS E. Coli 055B5, Sigma
  • An initial control experiment is conducted to determine the base line levels of endogenous MIF in the murine model system (female Balb/c mice), and further to determine the rate and extent of increase in endogenous MIF following treatment with LPS (10 mg/kg).
  • Female Balb/c mice are treated with LPS (Sigma 0111:B1) admixed with 50 mg/kg ⁇ -D-galactosamine.
  • the level of MIF in serum is measured by HPLC as described above at 0, 2, 5 and 6 hours following LPS/galactosamine treatment.
  • the baseline level of endogenous MIF is approximately 45 ng/ml.
  • mice When mice are treated with candidate compound (formulated in 50% aqueous solution) and 10 mg/kg of LPS there is a significant decrease in the level of circulating MIF that can be detected.
  • BALB/c mice are injected i.p. with 20 mg/kg body weight of candidate compound at time of LPS administration. Blood samples are collected 5.5 hours later. The results demonstrate that animals treated with the candidate compound have a decreased ability to respond to LPS and lowered MIF levels are detected.
  • serum MIF is determined four hours following treatment. This data reveals decrease in MIF.
  • both MIF and IL-1 ⁇ are measured in mouse serum via ELISA.
  • the experiments show a direct and highly significant correlation between MIF and IL-1 ⁇ . This correlation is also observed between MIF and TNF- ⁇ . In a similar experiment, reductions in serum IL-1 ⁇ level and serum TNF- ⁇ level are observed following administration of 20 mg/kg of candidate compound.
  • Exogenous recombinant human MIF when administered with candidate compounds can reverse the beneficial effects of the compounds, supporting the hypothesis that candidate compounds act to increase animal resistance to LPS by modulating MIF levels in mice serum.
  • Mice are treated with the standard LPS protocol except that in addition to 1 mg/kg LPS and 20 mg/kg of the candidate compound, some animals also receive 300 ⁇ g/kg human recombinant MIF. At 12 hours, significantly more mice survive the LPS with candidate compound, but this survival is neutralized by the administration of MIF.
  • mice Twenty DBA/1LacJ mice, age 10-12 weeks, are immunized on day 0 at base of the tail with bovine collagen type II (CII 100 ⁇ g) emulsified in Freunds complete adjuvant (FCA; GibcoBRL). On day 7, a second dose of collagen is administrated via the same route (emulsified in Freunds incomplete adjuvant). On Day 14 mice are injected subcutaneously with 100 mg of LPS (055:B5). On day 70 mice are injected 40 ⁇ g LPS (0111:B4) intraperitoneally. Groups are divided according paw thickness, which is measured by a caliper, after randomization, to create a balanced starting group.
  • FCA bovine collagen type II
  • FCA Freunds complete adjuvant
  • Candidate compound in buffer is given to mice on days 71, 72, 73, and 74 (total of eight doses at 0.4 mg/dose, approximately 20 mg/kg of body weight). Mice are then examined on day 74 by two observers for paw thickness. In this experiment, subsided mice (decline of full-blown arthritis) are treated with a final i.p. injection of LPS on day 70 to stimulate cytokine production as well as acute inflammation. Candidate compound treated mice develop mildly reduced edema of the paw compared with vehicle only treated controls. In the late time point, the animals in the treated group do not reach a full-blown expression of collagen induced arthritis as compared to controls.
  • mice In another experiment, fifteen DBA/1J mice, age 10-12 weeks are immunized on day 0 at the base of the tail with bovine collagen type II (CII 100 ⁇ g), emulsified in Freunds complete adjuvant (FCA; GibcoBRL). On day 21, a second dose of collagen is administered via the same route, emulsified in Freunds incomplete adjuvant.
  • FCA emulsified in Freunds incomplete adjuvant
  • the mice On day 28 the mice are injected subcutaneously with 100 ⁇ g of LPS (055:B5). On day 71 the mice are injected i.p. with 40 ⁇ g LPS (0111:B4). Groups and treatment protocol are the same as described as above. On day 74 blood samples are collected and cytokines are measured. The candidate compound reduces serum MIF levels as compared to untreated CIA samples. An even more significant inhibition of serum TNF- ⁇ levels is detected.

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