WO2010140902A1 - Inhibiteurs du facteur d'inhibition de la migration des macrophages - Google Patents

Inhibiteurs du facteur d'inhibition de la migration des macrophages Download PDF

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Publication number
WO2010140902A1
WO2010140902A1 PCT/NZ2010/000102 NZ2010000102W WO2010140902A1 WO 2010140902 A1 WO2010140902 A1 WO 2010140902A1 NZ 2010000102 W NZ2010000102 W NZ 2010000102W WO 2010140902 A1 WO2010140902 A1 WO 2010140902A1
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substituted
mif
disease
compound
alkyl
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PCT/NZ2010/000102
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English (en)
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Mark Hampton
Robin Andrew James Smith
Kristen Brown
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Mark Hampton
Robin Andrew James Smith
Kristen Brown
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Publication of WO2010140902A1 publication Critical patent/WO2010140902A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/26—Cyanate or isocyanate esters; Thiocyanate or isothiocyanate esters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C331/00—Derivatives of thiocyanic acid or of isothiocyanic acid
    • C07C331/16—Isothiocyanates
    • C07C331/18—Isothiocyanates having isothiocyanate groups bound to acyclic carbon atoms
    • C07C331/22—Isothiocyanates having isothiocyanate groups bound to acyclic carbon atoms of an unsaturated carbon skeleton
    • C07C331/24—Isothiocyanates having isothiocyanate groups bound to acyclic carbon atoms of an unsaturated carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates generally to uses of isothiocyanate and isothioselenate compounds that are inhibitors of macrophage migration inhibitory factor (MIF).
  • MIF macrophage migration inhibitory factor
  • this invention relates to used of isothiocyanate and isothioselenate compounds to treat disorders in which it is desirable to decrease MIF activity.
  • MIF The protein MIF is a cytokine released by many cell types including T-lymphocytes and macrophages. MIF proteins have been identified in several species including mammals and are generally 12-13 kDa in size. MIF levels increase during physiological stress or systemic inflammatory conditions. MIF plays an important role in septic shock and delayed-type hypersensitivity reaction, possibly due to its ability to act as an endogenous regulator of glucocorticoid action within the immune system. Deletion of the MIF gene or immunoneutralisation of MIF protects against septic shock.
  • MIF MIF-induced levels
  • cardiovascular disease cardiovascular disease
  • diabetes diabetes, sepsis and many cancer types.
  • genetic ablation of MIF has been shown to attenuate various disease states in murine models. While details surrounding the mechanisms of MIF action are still in question, the clinical significance of MIF expression is such that targeted approaches to modulate the biological activities of MIF are currently in development.
  • MIF is also known to have a proinflammatory role in arthritis and glomerulonephritis. Inhibition of proinflammatory cytokine activity using monoclonal antibodies has been shown to improve disease outcomes in mouse models of rheumatoid arthritis, sepsis, cardiovascular disease, and inflammatory bowel disease. Additional biological activities for MIF include tumour invasion, metastasis and angiogenesis, insulin release, cell growth and apoptosis, regulation of T-cell and macrophage activation and IgE synthesis. MIF also acts as a tautomerase. While the biological significance of this activity is still under debate, the tautomerase site appears to be important for regulating protein-protein interactions that mediate MIF activity.
  • MIF MIF is known to be involved in numerous pathological events
  • inhibition of MIF may have several therapeutic effects.
  • Candidate MIF inhibitors have been obtained from a variety of sources and include antibodies and small molecules.
  • MIF inhibitors can be classified as non-covalent modifiers, such as ISOl and its analogs, (13, 29, 30) phenyl pyruvic derivatives, ketones, (10) and coumarin derivatives, (12) and as covalent modifiers, including NAPQI, (7) 2-OBP, (31) 4-IPP, (32, 33) PMSF, (34) ITCs, (19, 35,
  • MIF's tautomerase activity in vitro In some cases, they have also resulted in the inhibition of glucocorticoids overriding activity7 and have improved survival in an animal model of sepsis (13, 14) in vivo.
  • Isothiocyanates are a class of phytochemicals with many well-recognised biological properties.
  • isothiocyanates are proposed to have chemotherapeutic potential (Conaway, C. C;
  • Isoselenocyanates are structurally and electronically similar to isothiocyanates and are also known to have anti-tumor activity (Sharma, A.K. et al. J. Med. Chem., 2008, 51, pp 7820-7826).
  • Isoselenocyanates like isothiocyanates, are known to react predominantly with thiols.
  • the active form of these compounds in animals is generally attributed to circulating N-acetyl-L- cysteine conjugates formed by reversible reaction of glutathione with the isothiocyanate or isoselenocyanate.
  • isothiocyanate and isoselenocyanate compounds have therapeutic applications in conditions in which MIF has a mediating function.
  • isothiocyanates and isothioselenates can bind to MIF and therefore decrease MIF activity.
  • isothiocyanates and isothioselenates represent classes of compounds that can be useful in treating disorders in which it is desirable to decrease MIF activity.
  • disorders include, for example, cancer and inflammatory diseases.
  • the invention provides a use of an isothiocyanate or isothioselenate compound or pharmaceutically acceptable salt or prodrug thereof for reducing MIF activity.
  • the invention provides an isothiocyanate of isothioselenate compound or a pharmaceutically acceptable salt or prodrug thereof for treating a disease or condition mediated- by MIF in a subject in need thereof.
  • the invention provides an isothiocyanate or isothioselenate compound or a pharmaceutically acceptable salt or prodrug thereof for reducing MIF activity in a subject in need thereof.
  • the invention provides a method for treating a disease or condition mediated by MIF in a subject in need thereof comprising administering to a subject a therapeutically effective amount of an isothiocyanate or isothioselenate compound or a pharmaceutically acceptable salt or prodrug thereof.
  • the invention provides a method for reducing MIF activity in a subject in need thereof comprising administering to a subject a therapeutically effective amount of an isothiocyanate or isothioselenate compound or a pharmaceutically acceptable salt or prodrug thereof.
  • the invention provides a use of an isothiocyanate or isothioselenate compound or a pharmaceutically acceptable salt or prodrug thereof in the manufacture of a medicament for treating a disease or condition mediated by MIF. In one aspect the invention provides a use of an isothiocyanate or isothioselenate compound or a pharmaceutically acceptable salt or prodrug thereof in the manufacture of a medicament for reducing MIF activity.
  • the invention provides the use of a compound of formula (I)
  • X is S or Se
  • R is selected from the group comprising alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, arylalkyl, substituted arylalkyl, allyl, substituted allyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, arylalkenyl, substituted arylalkenyl, acyl, substituted acyl, acyloxy, substituted acyloxy, alkyloxycarbonyloxy, substituted alkyloxycarbonyloxy, aryloxycarbonyloxy, substituted aryloxycarbonyloxy, alkoxycarboylacyl, substituted alkoxycarbonylacyl, alkylcarbonylalkyl, substituted alkylcarbonylalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
  • R is selected from the group comprising alkyl, alkylaryl, aryl, alkoxy alkylaryl, allyl and alkylsulfinylalkyl, alkylamino and substituted versions thereof.
  • the substituent is selected from the group comprising hydrogen, alkyl, aryl, alkylaryl, heteroaryl, acyloxy, alkoxy, and cycloalkyl.
  • R is arylalkyl, or substituted arylalkyl, preferably phenylalkyl, or substituted phenylalkyl, more preferably phenyl-(Ci to C 6 ) alkyl wherein the phenyl ring is unsubstituted or substituted with 0-3 methoxy groups.
  • the compound of formula (I) is selected from the group comprising:
  • R 1 , R 2 and R 3 are independently selected from the group comprising hydrogen, halogen, hydroxy, cyano, nitro, amino, alkylamino, dialkylamino, alkyl, alkoxy, alkylthio, haloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl,
  • R is a substituted group
  • the substituent is selected from the unsubstituted groups in the definition of "substituent" herein.
  • R 1 , R 2 and R 3 are independently selected from the group comprising hydrogen, alkyl, alkoxy and alkylamino.
  • R 1 is (Q-C ⁇ alkyl, (C 1 -C 6 )alkoxy or (C 1 - C 6 )alkylphenyl.
  • n 0 to 6.
  • X S.
  • the compound of formula (I) is selected from the group comprising phenyl isothiocyanate (PITC), 2-(3-(2-aminoethyl)phenyl)ethyl isothiocyanate (amino-PEITC) benzyl isothiocyanate (BITC), phenethylisothiocyanate (PEITC), phenylhexylisothiocyanate, 3,4,5- trimethyloxybenzyl isothiocyanate, allyl isothiocyanate, erucin, erysolin and sulforaphane.
  • PITC phenyl isothiocyanate
  • Amino-PEITC 2-(3-(2-aminoethyl)phenyl)ethyl isothiocyanate
  • benzyl isothiocyanate benzyl isothiocyanate
  • PEITC phenethylisothiocyanate
  • the compound of formula (I) is 4-hydroxy phenethyl ITC.
  • the invention provides a compound of formula (I) as defined above, or a pharmaceutically acceptable salt or prodrug thereof for reducing MIF activity.
  • the invention provides a compound of formula (I) as defined above, or a pharmaceutically acceptable salt or prodrug thereof for treating a disease or condition mediated by MIF.
  • the invention provides a method for reducing MIF activity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of formula (I) as defined above or a pharmaceutically acceptable salt or prodrug thereof.
  • the invention provides a method for treating a disease or condition mediated by MIF in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of formula (I) as defined above or a pharmaceutically acceptable salt or prodrug thereof.
  • the invention provides the use of a compound of formula (I) as defined above or a pharmaceutically acceptable salt or prodrug thereof in the manufacture of a medicament for reducing MIF activity in a subject in need thereof.
  • the invention provides the use of a compound of formula (I) as defined above or a pharmaceutically acceptable salt or prodrug thereof in the manufacture of a medicament for treating a disease or condition mediated by MIF.
  • reduction of MIF activity is via binding of the N-terminal proline residue of MIF.
  • the disease or condition mediated by MIF is an inflammatory disease. In another embodiment, it is an autoimmune disease. In yet another embodiment it is an infectious disease.
  • the disease or condition mediated by MIF is selected from diseases or conditions where one or more of the following has been demonstrated;
  • the disease or condition mediated by MIF is selected from the group comprising rheumatoid arthritis, osteoarthritis, juvenile idiopathic arthritis, inflammatory bowel disease such as Crohn's disease and ulcerative colitis; cardiovascular disease, conjestive heart failure, atherosclerosis, autoimmune myocarditis, autoimmune hepatitis, Alzheimer's disease, Parkinson's disease, ir-iscular dystrophy, granuloma, alopecia, acute pancreatitis, bacterial infection, endotoxemia, glomerulonephritis, inflammatory disease, inflammation, malaria, sepsis, tissue rejection vitreoretinopathy, autoimmune graft versus host disease, multiple sclerosis, endotoxic shock, metastasis, asthma, Cushing's disease, atopic dermatitis, atopy, otitis media, acute respiratory distress syndrome, delayed-type hypersensitivity, contact hypersensitivity type I and II diabetes, endometriosis,
  • the disease or condition mediated by MIF is selected from the group comprising atherosclerosis, rheumatoid arthritis, juvenile idiopathic arthritis, Crohn's disease, ulcerative colitis, sepsis, endotoxic shock, obesity, asthma, acute respiratory distress syndrome, atopy, delayed type hypersensitivity and contact hypersensitivity reactions, systemic lupus erythematusus, psoriasis and sarcoidosis.
  • the invention provides a method for treating an inflammatory disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of formula (I) or pharmaceutically acceptable salt or prodrug thereof.
  • the inflammatory disease is selected from the group comprising rheumatoid arthritis, osteoarthritis, juvenile idiopathic arthritis, glomerulonephritis and inflammatory bowel disease including Crohn's disease and ulcerative colitis.
  • the invention provides a method for treating an autoimmune disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or prodrug thereof.
  • the autoimmune disease is selected from the group comprising diabetes, multiple sclerosis, Crohn's disease, ulcerative colitis, autoimmune graft versus host disease, systemic lupus erythematosus, rheumatoid arthritis, autoimmune myocarditis and autoimmune hepatitis.
  • the invention provides a method of treating an infectious disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or prodrug thereof.
  • the infectious disease is selected from the group comprising bacterial infection and endotoxemia.
  • the invention provides a method for preventing metastasis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt of prodrug thereof.
  • the invention provides a compound of formula (II)
  • the isothiocyanate or isothioselenate MIF inhibitor is substantially purified.
  • isothiocyanates and isothioselenates of this invention can be ingested in food or as food additives.
  • FIG. 1 shows the loss of Jurkat cell viability following a 24 hour exposure to PEITC (•) and amino-PEITC (o). Cell viability was assessed by flow cytometric determination of the uptake of propidium iodide, (mean ⁇ SE from three experiments).
  • Figure 2 shows (A) SDS-PAGE analysis of protein from Jurkat cell lysates eluted from blocked Affi-Gel or Affi-PEITC resins. The highlighted protein band was excised from the gel and
  • Figure 3 shows the mass spectroscopy results for: (A) rhMIF before (1) and after (2) incubation with a 10-fold excess of PEITC for 10 minutes. (A) Wild type rhMIF before (i) and after (ii)
  • FIG. 4 shows (A) Time course of the inhibition of rhMIF tautomerase activity by 10 ⁇ M (•), 20 ⁇ M (o) and 30 ⁇ M (A) PEITC (results are representative of three experiments). (B) in silico modeling of MIF in the presence of PEITC. (C) Immunoreactivity of rhMIF following a 10 min exposure to 20 ⁇ M PEITC as measured using a commercial ELISA. Values are mean ⁇ SE of three experiments.
  • Figure 5 shows (A) Time course of the inhibition of cellular MIF tautomerase activity by 1 ⁇ M (•), 2 ⁇ M (o) and 4 ⁇ M (A) PEITC (mean ⁇ SE of three experiments).
  • B Immunobinding of MIF monoclonal (mAb) and polyclonal (pAb) antibodies to extracts prepared from Jurkat cells treated with increasing concentrations of PEITC for 50 minutes. Images are representative of results from three experiments.
  • C Cells were treated with 15 ⁇ M PEITC for 1 h before detectable levels of extracellular MIF (black bars) and intracellular MIF (grey bars) were measured using a commercial ELISA (mean ⁇ SE from three experiments).
  • Figure 6 shows in silico modeling (I) of PEITC at the tautomerase active site. MIF without PEITC bound is shown in dark grey. The unreacted N-terminal proline is not shown. PEITC is bound to the tautomerase active site of MIF via covalent modification of the N-terminal proline residue. MIF with PEITC bound is shown in light grey).
  • Figure 7 depicts the concentration-dependence of inhibition of cellular MIF tautomerase activity following a 30 min exposure to PEITC (O), sulforaphane (T) or benzyl isothiocyanate (V). Values are mean ⁇ SE of at least three experiments.
  • Figure 8 depicts a gel showing progressinve inhibition of MIF binding to PEITC.
  • Jurkat cells were treated with increasing concentrations of PEITC for 30 min.
  • Cell lysates were prepared and the ability of MIF to bind Affi-PEITC was measured.
  • a representative gel is shown.
  • Figure 9 depicts results of a study in human volunteers. Three volunteers consumed 50 g of watercress. Blood was drawn just prior to eating (0), one hour after eating (1) and two hours (2) after eating. Plasma fractions were prepared immediately after venipuncture. Plasma levels of isothiocyanates and their corresponding dithiocarbamates were measured at all time points in two of the volunteers using a cyclocondensation reaction with 1,2-benzenedithione. Values are mean ⁇ range.
  • FIG 10 depicts plasma MIF in the volunteer subjects of Figure 9.
  • MIF levels (o) were measured by a commercial ELISA just prior to eating and 2 hours after ingestion of watercress.
  • Figure 11 depicts the chemical reaction between Affi-Gel and Amino PEITC, a derivative of PEITC, to generate the Affi-PEITC probe of this invention.
  • Figure 12 depicts the tautomerase reaction used to evaluate MIF activity.
  • MIF catalyses the tautomerisation of the chromogenic substrate dopachrome methyl ester to a colourless indole derivative.
  • Figure 13 depicts In silico modeling (II) of PEITC (yellow) bound to the tautomerase active site of MIF via covalent modification of the N-terminal proline residue.
  • Figure 13 A depicts the MIF homotrimer shown with PEITC docked in one of the tautomerase active sites. MIF without PEITC bound is shown in magenta (pdb3B9S).
  • Figure 13B depicts a close-up image of a portion of Figure 13A, and shows conformational shifts of the catalytic proline by 2 A (arrow 1) and lysine 32 by 1.6 A (arrow 2).
  • Figure 14 depicts mass spectra of rhMIF-PEITC adduct formation. rhMIF was reacted with 10"- fold excess of PEITC for 10 mintues before analysis of PEITC-adduct formation.
  • Figure 14A is wild type rhMIF before (i) and after (ii) incubation with PEITC. A peak with a mass of 12,508 Da, corresponding to MIF (12,345 Da) with the addition of 1 PEITC (163 Da) resulted. In addition, a peak with a mass of 12.248 Da was also apparent.
  • Figure 14B shows rhMIF with a C60S mutation (i), a C57A mutation (ii) or a C81S mutation (iii) (12,329 Da) after incubation with PEITC.
  • a peak with a mass of 12,232 Da was also formed.
  • Figure 14C shows a P2A rhMIF before (i) and after (ii) incubation with PEITC. Only the mass of the parent protein (12,319 Da) was observed.
  • Figure 15 depicts mass spectromerty analysis of the modification of MIF by PEITC in the abasence of acid. MIF was reacted with a 10-fold excess of PEITC for 10 min before analysis of the PEITC-adduct formation by mass spectrometry in a formic acid-free buffer.
  • Figure 15A depicts rhMIF before and Figure 15B depicts rhMIF after incubation with PEITC.
  • Figure 15A shows rhMIF in its un-conjugated form with a mass of 12,345 Da.
  • Figure 15B shows a peak with m/z 12,508, corresponding to MIF (m/z 12,346) with addition of one PEITC resulted. Sodiated species (+23 Da) were also formed due to the composition of the buffer.
  • MIF activity means an activity or effect mediated at least in part by MIF and includes, but is not limited to, inhibition of macrophage migration, tautomerase activity, 5 endotoxin induced shock, inflammation, glucocorticoid counter regulation, enhanced proliferation, induction of ERK phosphorylation and MAP kinase activity.
  • MIF inhibitor means a molecule (either natural or synthetic) that can alter the conformation of MIF and/or compete with a monoclonal antibody to MIF and can decrease at least one activity of MIF or its export from a cell when compared to the activity or export in the D absence of the inhibitor.
  • An inhibitor alters the conformation, activity or export of MIF 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.
  • MIF inhibitors include the compounds 1- 73 listed herein, as well as others having MIF inhibiting activity.
  • mediated by MIF means a condition or disease in which elevated MIF activity is believed to play a role.
  • Conditions or diseases mediated by MIF are targets for the MIF inhibitors of formula (I) because inhibition of MIF is likely to have a positive effect on the condition or disease. Examples of conditions and diseases mediated by MIF include inflammation, diabetes, sepsis, cardiovascular disease,
  • MIF D rheumatoid arthritis and the like.
  • Diseases or conditions mediated by MIF also include those where one or more of the following has been demonstrated;
  • D increased associated with polymorphisms in the MIF gene and/or its promoter region.
  • e increased MIF associated with mutations or duplications in the MIF gene and/or its promoter region.
  • alkyl means, unless otherwise stated, a straight or branched chain, noncyclic or cyclic hydrocarbon radical, which may be fully saturated, mono- or 5 polyunsaturated, including di- and multivalent radicals, and may have the number of carbon atoms designated (i.e. Ci-Ci 0 means one to ten carbons).
  • saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n- hexyl and the like.
  • saturated branched chain alkyls include isopropyl, isobutyl, sec- butyl, test-butyl, isopentyl and the like.
  • Representative saturated cyclic alkyls include
  • alkyl group is one having one or more double or triple bonds.
  • unsaturated straight chain alkyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4- pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.
  • alkyl unless otherwise stated, is also meant to include those derivatives of alkyl defined
  • alkenyl alkynyl
  • cycloalkyl alkylene
  • alkylene as used herein means, unless otherwise stated, a divalent radical derived from an alkane, as exemplified by -CH 2 CH 2 CH 2 CH 2 -. Typically, an alkylene group will have from 1 to 24 carbon atoms.
  • a “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
  • cycloalkyl as used herein means, unless otherwise stated, a cyclic version of “alkyl”, and includes di-and poly-homocyclic rings such as decalin and adamentane.
  • examples of cycloalkyls include cyclopentyl, cyclohexyl, 1 -cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like.
  • alkenyl means, unless otherwise stated, a hydrocarbon radical having '.5 at least one double bond including, but not limited to, ethenyl, propenyl, 1-butenyl, 2-butenyl and the like.
  • alkynyl means, unless otherwise stated, a hydrocarbon radical having at least one triple bond including, but not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl and the like.
  • aryl alone or in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) as used herein means, unless otherwise stated, an aromatic carbocyclic moiety which can be a single ring or multiple rings (for example, three rings) which are fused together or linked covalently for example phenyl or napthyl.
  • the rings may each contain from zero to four heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. Therefore, the term "aryl” unless otherwise stated, is also meant to include those aryl groups containing heteroatoms defined in more detail below as “heteroaryl”. The heteroaryl groups can be attached to the remainder of the molecule through a heteroatom.
  • Non-limiting examples of aryl groups include phenyl, 1- naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4- imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4- isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3- thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl 4-pyrimidyl, 2-benzothiazolyl, 5- benzothiazolyl, 2-benzoxazolyl, 5-benzoxazolyl, purinyl, 2-benz
  • heteroaryl means, unless otherwise stated, an aromatic heterocycle ring of 5- to 10 members and having at least one heteroatom selected from nitrogen, oxygen and sulfur, and containing at least 1 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, benzothioazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl.
  • aralkyl or "arylalkyl” as used herein means, unless otherwise stated, an alkyl group having at least one hydrogen atom replaced with an aryl moiety such as a phenyl or naphthyl including mono-, di-, and poly-homocyclic aromatic ring systems, for example, (C 6 - J4 aryl).
  • Preferred arylalkyls comprise a lower alkyl group attached to the aryl group.
  • suitable arylalkyl groups include benzyl, phenylhexyl, 2-phenylethyl and naphthalenylmethyl.
  • the bond to the parent moiety is through the alkyl.
  • heteroarylalkyl means, unless otherwise stated, an alkyl group having at least one alkyl hydrogen atom replaced with a heteroaryl moiety, for example, -CH 2 pyridinyl, -CH 2 pyrimidinyl, and the like.
  • heterocycle and “heterocycle ring,” as used herein, mean, unless otherwise stated, a 3- to 7-membered monocyclic, or 6- to 14-membered polycyclic, heterocycle ring which is either saturated, unsaturated or aromatic, and which contains from 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulphur, and wherein the nitrogen and sulphur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quarternized, 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 may be attached via any heteroatom or carbon atom.
  • 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.
  • heterocyclealkyl means, unless otherwise stated, an alkyl having at least one alkyl hydrogen atom replaced with a heterocycle, for example -CH 2 morpholinyl, and the like.
  • halo or halogen
  • fluorine chlorine, bromine, or iodine atom.
  • fluoroalkyl are meant to include monofluoroalkyl and poly fluoroalkyl.
  • haloalkyl as used herein means, unless otherwise stated, an alkyl group having at least one hydrogen atom replaced with a halogen, for example trifluoromethyl, and the like.
  • alkoxy means, unless otherwise stated, an O-alkyl group wherein “alkyl” is defined herein, for example, methoxy, ethoxy, and the like.
  • thioalkyl means, unless otherwise stated, an alkyl moiety attached through a sulfur bridge (ie., -S-alkyl) such as methylthio, ethylthio, and the like.
  • alkylsulfonyl means, unless otherwise stated, an alkyl moiety attached through a sulfonyl bridge (i.e, -SO2-alkyl) such as methylsulfonyl, ethylsulfonyl, and the like.
  • alkylsulfinyl as used herein means, unless otherwise stated, an alkyl moiety attached through a sulfinyl bridge (-S(O)-alkyl) where methylsulfinyl, ethylsulfinyl, propylsulfinyl, butylsulfinyl, and the like.
  • alkylamino and dialkyl amino as used herein means, unless otherwise stated, 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 means, unless otherwise stated, an alkyl substituted with at least one hydroxyl group.
  • alkyloxyalkyl means, unless otherwise stated, an alkyl substituted with an -O-alkyl group.
  • alkylthioalkyl means, unless otherwise stated, an alkyl substituted with a -S-alkyl group.
  • di(alkyl)aminoalkyl means, unless otherwise stated, an alkyl substituted with a mono- or di(alkyl)amino.
  • substituted includes halogen, hydroxy, cyano, nitro, amino, alkylamino, dialkylamino, alkyl, alkoxy, alkylthio, haloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, heterocycle.
  • Ra and Rb are the same or different and independently hydrogen, alkyl, haloalkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, 5 substituted heteroaryl, heteroarylalkyl, substituted heteroaryl
  • terapéuticaally-effective amount means that amount of an active compound, or a material, composition or dosage form comprising an active compound, which is effective for producing some desired therapeutic or prophylactic effect, commensurate with a O reasonable benefit/risk ratio.
  • treatment as used herein in the context of treating a condition or disease, relates generally to treatment and therapy, whether of human or animal, in which some desired therapeutic effect is achieved, for example, the inhibition of progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the 5 condition, and cure of the condition.
  • Treatment as a prophylactic measure i.e., prophylaxis is also included.
  • Treatment also includes combination treatments and therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously.
  • a therapeutically effective amount of a compound of formula (I) could be combined with or used O in conjunction with radiation therapy or chemotherapy in the treatment of cancer.
  • prodrug means, unless otherwise stated a compound that undergoes chemical conversion in the body to become an active pharmacological agent with the defined chemical properties.
  • a prodrug of a compound of formula (I) is metabolised or otherwise converted to a compound of formula (I) as defined above.
  • prodrugs 5 within the scope of the invention include ester and amide derivatives that are hydrolysed to form isothiocyanate and isothioselenate compounds of formula (I) in the body.
  • N- acetylcysteine conjugates of the isothiocyanate and isothioselenate compounds are hydrolysed in the body to release N-acetylcysteine and the isothiocyanate or isothioselenate compound, including compounds of formula (I).
  • pharmaceutically acceptable refers to compounds, ingredients, materials, compositions, dosage forms and the like, which are within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g. human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Each carrier, diluent, exipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
  • subject refers to a human or non-human mammal.
  • non-human mammals include livestock animals such as sheep, horses, cows, pigs, goats, rabbits, deer, ostriches and emus; and companion animals such as cats, dogs, rodents, and horses.
  • livestock animals such as sheep, horses, cows, pigs, goats, rabbits, deer, ostriches and emus
  • companion animals such as cats, dogs, rodents, and horses.
  • the subject is a human.
  • a certain compound may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; (+) and (-) forms; keto-, enol-, and enolate- forms; ⁇ - and ⁇ -forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair- forms; and combinations thereof, hereinafter collectively referred to as "isomers” (or "isomeric forms").
  • Some compounds of formula (I) have at least one asymmetrical carbon atom and therefore all isomers, including enantiomers, stereoisomers, rotamers, tautomers and racemates of the compounds are contemplated as being part of this invention.
  • the invention includes d and 1 isomers in both pure form and in admixture, including racemic mixtures.
  • Isomers can be prepared using conventional techniques, either by reacting optically pure or optically enriched starting materials or by separating isomers of a compound of the invention. Isomers may also include geometric isomers, e.g., when a double bond is present.
  • isomers are structural (or constitutional) isomers (i.e., isomers which differ in the connections between atoms rather than merely by the position of atoms in space).
  • a reference to a methoxy group, -OCH 3 is not to be construed as a reference to its structural isomer, a hydroxymethyl group, -CH 2 OH.
  • a reference to ortho — chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl.
  • Ci -7 alkyl includes w-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and/? ⁇ r ⁇ -methoxyphenyl).
  • keto-, enol-, and enolate- forms as in, for example, the following tautomeric pairs: keto/enol, imine/enamine, amide/imino alcohol, amidine/amidine nitroso/oxime, thioketone/enethiol, N- nitroso/hyroxyazo, and nitro/aci-nitro.
  • H may be in any isotopic form, including H, H (D), and H (T); C may be in any isotopic form, including 12 C, 13 C, and 14 C; O may be in any isotopic form, including 16 O and 18 O; and the like.
  • a reference to a particular compound includes all such isomeric forms, including racemic and other mixtures thereof.
  • Methods for the preparation (e.g., L5 asymmetric synthesis) and separation (e.g., fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein in a known manner.
  • a reference to a particular compound also includes ionic, salt, hydrate, and protected forms of thereof, for example, as discussed below.
  • a salt may be formed with a suitable anion.
  • suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.
  • suitable organic anions include, but are not limited to, anions from the following
  • organic acids acetic, propionic, succinic, gycolic, stearic, lactic, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetyoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and valeric.
  • solvate is used herein in the conventional sense to refer to a
  • solute e.g., active compound, salt of active compound
  • solvent water
  • the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.
  • chemically protected form pertains to a compound in which one or more reactive functional groups are protected from undesirable chemical reactions, that is, are in the form of a protected or protecting group (also known as masked or masking group).
  • a protected or protecting group also known as masked or masking group.
  • Isothiocyanates are a class of phytochemicals with widely reported anti-cancer and anti- inflammatory activity. However, knowledge of their activity at a molecular level is limited.
  • This invention relates to isothiocyantes and their uses to treat disease.
  • this invention uses phenethyl isothiocyanate (PEITC) to determine which biological targets are affected by isothiocyanates.
  • PEITC phenethyl isothiocyanate
  • An analogue of PEITC, amino-PEITC was synthesised to enable conjugation to a solid-phase resin.
  • MIF pleiotropic cytokine macrophage migration inhibitory factor
  • isothiocyanates Although the mechanisms of action of isothiocyanates are not completely known, they represent a class of phytochemicals with recognised anti-cancer activity. They can act in a chemopreventive capacity via inhibition of carcinogen-activating phase I enzymes (1) and induction of phase II detoxification enzymes (2). Isothiocyanates are also active in the post- initiation phase of tumorigenesis and are therefore proposed to have chemotherapeutic potential (3,4). Isothiocyanate-mediated disruption of cancer progression is achieved by a variety of mechanisms including modulation of cell growth (5), inhibition of angiogenesis (6), suppression 5 of metastasis (7) and induction of apoptosis (8,9). Isothiocyanates can also modulate inflammatory pathways via inhibition of the transcription factor nuclear factor- ⁇ B (NF- ⁇ B) (10).
  • NF- ⁇ B transcription factor nuclear factor- ⁇ B
  • Isothiocyanates can be advantageously used to treat a variety of disorders associated with MIF activity.
  • this invention includes use of known isothiocyanates and novel
  • Table 2 below depicts other compounds and their sources useful as inhibitors or precursors of inhibitors of MIF.
  • Table 3 below below depicts other compounds and their sources useful as inhibitors or precursors of inhibitors of MIF.
  • Table 4 below depicts other compounds and their sources useful as inhibitors or precursors of inhibitors of MIF.
  • Isothiocyanate and isothioselenate inhibitors of MIF may be prepared using any convenient synthetic process.
  • isothiocyanate compounds can be prepared by reacting a primary amine with thiophosgene.
  • Example 1 Another example of the synthesis of an isothiocyanate compound of formula (I) is provided in Example 1 herein below.
  • Amino-PEITC [2-(3-(2-aminoethyl)phenyl)ethyl isothiocyanate] (Informal name: Compound 67) was prepared from the diamine precursor [l,3-bis(2-aminoethyl)benzene] (Ruggli, P. & Prijs, B. Helvetica Chimica Acta, 1945, 28, pp 674-90) by selective protection as a mono t-Boc derivative (Callahan, J.F. et al.
  • l,3-Bis(2-aminoethyl)benzene was obtained by reduction of l,3-bis(cyanomethyl)benzene (Dewey, T.M., Du Bois, J. & Raymond, K.N. "Ligands for oxovanadium(IV): bis(catecholamide) coordination and intermolecular hydrogen bonding to the oxo atom", Inorganic Chemistry, 1993, 32, pp 1729-1738) with a nickel-modified borohydride reagent (Caddick, S., Haynes, A.K.D.K., Judd, D.B. & Williams, M.R.V. "Convenient synthesis of protected primary amines from nitriles", Tetrahedron Letters, 2000, 41, pp 3513-3516). Such a synthetic scheme can be used to prepare novel inhibitors of MIF.
  • Isothioselenate compounds can be prepared from their corresponding formamides using methods known in the art. See for example, Barton, D. H. R.; Parekh, S. L; Tajbakhsh, M.; Thodorakis, E.A.; Tse, C. -L. Tetrahedron 1994, 50, pp 639-654; Fernandez-Bolanos, J.G.; Lopez, O.; Ulgar, V.; Maya, L; Fuentes, J., Tetrahedron Lett. 2004, 45, pp 4081-4084.
  • prodrugs of isothiocyanate and isothioselenate inhibitors of MIF include prodrugs of isothiocyanate and isothioselenate inhibitors of MIF.
  • N-acetylcysteine conjugates can be prepared by treatment of the corresponding isothicyanate or isothioselenate with N-acetylcysteine in THF in the presence of NaOH.
  • isothiocyanates are generated by administering a glucosinolate prodrug and the enzyme thioglucosidase (also known as myrosinase).
  • Scheme 2 describes the general structure of glucosinolates, their intermediate and final degradation products.
  • the stars denote positions in the glucose moiety known to be acylated in certain glucosinolates.
  • the conditions favouring the formation of isothiocyantes include a pH of 7.0.
  • administration into the circulation favors the reaction at pH of about 7, whereas administration into the stomach, which 5 has a resting pH of about 1-2, the reaction favored produces nitriles.
  • the pH of the stomach can by near 7, which could, under desired conditions, produce the desired isothiocyanates.
  • co-administration of an antiacid could favor reactions producing isothiocyanates.
  • Electrophilic isothiocyanates and isoselenocyanates are generally thought to react with biological nucleophiles such as cysteine resides in proteins and the tripeptide glutathione. Reaction with amines to form stable thiourea derivatives is known, but is considered to be a less favourable reaction at physiological pH.
  • isothiocyanate compound phenethyl isothiocyanate (PEITC) L5 binds to the N-terminal proline of MIF, as described in Examples 4 and 5.
  • an amine linker was used to immobilise PEITC to a solid phase resin to form Aff ⁇ -PEITC.
  • MIF was identified as a major biological target of the Affi-PEITC. Mass spectroscopy and site-directed mutagenesis were used to identify the target residue of MIF and confirm that binding at this site alters the enzymology and conformational integrity of MIF.
  • Example 9 the inhibitory effects of several different isothiocyanate compounds of formula (I) were investigated. All were found to inhibit MIF. Benzylthiocyanate provided a control and showed no inhibition. The results obtained demonstrate that compounds of formula (I) are potent inhibitors of MIF. Therefore, compounds of formula (I) have potential in the treatment of conditions and diseases mediated by MIF.
  • MIF is involved in many physiological processes, the person skilled in the art will be able to ascertain whether a disease or condition is one that is "mediated by MIF", as discussed in the definition of this term.
  • MIF isothiocyanate and isoselenocyanate inhibitors of MIF have a variety of uses.
  • MIF is involved in the shock response of mammals. Accordingly, inhibition of MIF may provide protection against lethal shock in animals exposed to high concentrations of endotoxin.
  • the MIF inhibitors may be able to be used at late stages where treatments such as anti-TNF therapy are ineffective.
  • Isothiocyanates and isoselenocyanates may also be used to prevent the MIF-dependent migration, anchorage-dependent growth and invasion of tumours (metastasis).
  • MIF is known to play a role in numersous disorders, including the infectious diseases caused by Pseudomonas aeruginosa, Influenza H5N1, Schistosoma mansoni, Granulomas, malaria.
  • MIF is also important in auto-immune diseases, inflammatory bowel disease, Crohn's disease, multiple sclerosis, autoimmune uveitis, Guillain-Barre syndrome, experimental allergic neuritis (EAN), autoimmune glomerulonephritis (experimental model), systemic lupus erythematosus (SLE), experimental allergic neuritis, autoimmune diabetes mellitus (experimental model), systemic sclerosis (SSc), ANCA-associated vasculitides, sarcoidosis, adult-onset Still's Disease, Cushing's Disease, graft versus host Disease, alopecia, kidney disease, psoriasis, atopic dermatitis, endometriosis, otitis media, rheumatoid arthritis, glomerulonephritis, and vitreoretinopathy.
  • EAN allergic neuritis
  • SLE systemic lupus erythematosus
  • autoimmune diabetes mellitus
  • isothiocyanates and isothioselenates can be used to treat any of the above disorders, or others, in which MIF is involved in the pathogenesis of disease.
  • isothiocyanates and isoselenocyanate compounds can be used to treat diseases and conditions mediated by MIF.
  • Such methods include administering an isothiocyanate or isothioselenate compound, such as a compound of formula (I) to a subject in a therapeutically effective amount.
  • Such methods include systemic administration of an inhibitor of MIF, 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 may also include flavorants, preservatives, suspending, thickening, and emulsifying agents, and other pharmaceutically acceptable additives.
  • flavorants for parental administration, the compounds of preferred embodiments can be prepared in aqueous injection solutions that may contain, in addition to the inhibitor of MIF activity, and/or export, buffers, antioxidants, bacteriostats, and other additives commonly employed in such solutions.
  • the inhibitors of MIF can also be administered in the form of natural plant extracts that contain the inhibitory compounds, provided that the extract contains sufficient concentration of the inhibitor to achieve a therapeutic benefit.
  • the invention also provides uses of isothiocyanate and isothioselenate compounds to reduce MIF activity.
  • the activity may be in vitro or in vivo.
  • the MIF inhibitor may be used as a research tool in an assay to investigate further aspects of MIF functioning.
  • the inhibitor may be used to investigate the biological role of MIF in intracellular and extracellular signalling pathways, and the role of MIF in disease models.
  • compositions comprising Isothiocyanate and Isothioselenate MIF Inhibitors
  • the isothiocyanate and isothioselenate MIF inhibitors may be administered simultaneously or sequentially with one or more additional pharmaceutically active compounds.
  • compositions containing the MIF inhibitors can be manufactured according to conventional methods such as by mixing, granulating, coating and dissolving the active agent.
  • the MIF inhibitor or its salt or prodrug is present in the composition in an amount that is effective to treat a particular disease or condition. For example, an amount that is sufficient to achieve MIF inhibition, reduce MIF activity, and/or to decrease or eliminate symptoms of the disease or condition in a subject, preferably with acceptable toxicity to the subject.
  • the 5 composition comprises a prodrug of a MIF inhibitor
  • the prodrug is present in an amount that will result in release of a therapeutically effective amount of the MIF inhibitor compound.
  • compositions of the invention comprising the isothiocyanate or isoselenocyanate MIF inhibitors of formula (I) or salts or prodrugs thereof will contain about
  • LO present, the intended rate and frequency of administration, and the subject's disease or condition.
  • compositions of the invention will contain MIF inhibitors of formula (I) or salts or prodrugs thereof in an amount of at least 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 15, 20, 30, 40, 50, 60, 70, 80, 90 ,100, 120, 140 ,160, 180, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, .5 700, 750, 800, 850, 900, 950 to 1000 mg MIF inhibitor. In certain embodiments lower or higher dosages may be appropriate. Appropriate concentrations and dosages can readily be determined by those skilled in the art.
  • Suitable pharmaceutically acceptable carriers and/or diluents are familiar to those skilled in the art.
  • acceptable carriers and/or diluents include
  • compositions can also be formulated as pills, capsules, granules, tablets (coated or uncoated), (injectable) solutions, solid solutions, suspensions, dispersions, solid dispersions (e.g., in the form of ampoules, vials, creams, gels, pastes, inhaler powder, foams, tinctures, lipsticks, drops, sprays, or suppositories).
  • the formulation can contain (in addition to
  • One skilled in this art may further formulate the inhibitor of MIF in an appropriate
  • MIF inhibiting compounds including compounds of formula (I) may 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 mediated by MIF.
  • 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 mediated by MIF for example, 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 mediated by MIF.
  • IBD inflammatory bowel disease
  • Such pharmaceutically active agents include, but are not limited to, for example, steroids, glucocorticoids, nonsteroidal anti-inflammatory drugs, anti-infective drugs, beta stimulants, antihistamines, anti-cancer drugs, asthma drugs, anti-sepsis drugs, anti-arthritis drugs, and immunosuppressive drugs, inhibitors of other inflammatory cytokines (e.g, anti-TNF ⁇ antibodies, anti-IL-1 antibodies, anti-IFN- ⁇ antibodies), and other cytokines such as IL-IRA or IL-10, and other MIF inhibitors.
  • steroids glucocorticoids
  • nonsteroidal anti-inflammatory drugs e.g, anti-inflammatory drugs, anti-infective drugs, beta stimulants, antihistamines, anti-cancer drugs, asthma drugs, anti-sepsis drugs, anti-arthritis drugs, and immunosuppressive drugs
  • inhibitors of other inflammatory cytokines e.g, anti-TNF ⁇ antibodies, anti-IL-1 antibodies, anti-IFN- ⁇
  • Combination therapies can include fixed combinations, in which two or more pharmaceutically active agents are in the same formulation; kits, in which two or more pharmaceutically active agents in separate formulations are sold in the same package, e.g, with instructions for coadministration; and free combinations in which the pharmaceutically active agents are packaged separately, but instruction for simultaneous or sequential administration are provided.
  • kit components can include diagnostics, assays, multiple dosage forms for sequential or simultaneous administration, instructions and materials for reconstituting a lyophilized or concentrated form of a pharmaceutical composition, apparatus for administering the pharmaceutically active agents, and the like.
  • 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 include, but are not limited to, celecoxib; rofecosib; NSAIDs, for example, aspirin, choline magnesium trisalicylate, diclofenac potassium, 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, methylprednisolone, prednisone, pred
  • one or more MIF inhibiting compounds are present in combination with one or more beta stimulants, inhalation corticosteroids, antihistamines,
  • 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, azatidine,
  • one or more MIF inhibiting compounds are present in combination with pharmaceutical compounds for treating IBD, such as azathioprine or .5 corticosteriods, in a pharmaceutical compositon.
  • 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
  • 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
  • SLE Systemic Lupus Erythematosus
  • Such drugs may include protease inhibitors, for example, indinavir,
  • nucleoside reverse transcriptase inhibitors for example, zidovudine, abacavir, lamivudine, idanosine, zalcitabine, and stavudine
  • nucleotide reverse transcriptase inhibitors for example, tenofovir disproxil fumarate
  • non nucleoside reverse transcriptase inhibitors for example delavirdine, efavirenz, and nevirapine
  • biological response modifiers for example, etanercept, infliximab, and other compounds that
  • one or more MIF inhibiting compounds are present in combination with pharmaceutical compounds for treating sepsis, such as steroids or anti- infective agents.
  • steroids examples include corticosteriods, for example, cortisone, hydrocortisone, methylprednisone, prednisone, prednisolone, betamethesone, beclomethasone dipropionate, budesonide, dexamethasone sodium phosphate, flunisolide, fluticasone, fluticasone propionate, triamcinolone acetonide, betamethasone, fluocinolone, fluocinonide, betamethasone dipropionate, betamethasone valerate, desonide, desoximetasone, fluocinolone, triamcinalone, triamcinolone acetonide, clobetasol propionate, and dexamethasone.
  • corticosteriods for example, cortisone, hydrocortisone, methylprednisone, prednisone, prednisolone, betamethesone, beclomethasone dipropionate
  • anti-infective agents include anthelmintics (mebendazole), antibiotics including aninocylcosides (gentamicin, neomycin, tobramycin, antifungal antiobiotics (amphotericin b, fluconazole, griseofulvin, itraconazole, ketoconazole, nystatin, micatin, tolnafitate), 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, min
  • 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, xylocaine, and phenazopyridine.
  • an anesthetic for example, ethanol, bupivacaine, chloroprocaine, levobupivacaine, lidocaine, mepivacaine, procaine, ropivacaine, tetracaine, des
  • the compounds of preferred embodiments can generally be employed as the free acid or the free base. Alternatively, the compounds of the preferred embodiments can preferably be in the form of acid or base addition salts.
  • pharmaceutically acceptable salt of structures (I), (II), and (III) is intended to encompass any and all acceptable salt forms. While salt forms of the preferred embodiments are preferably pharmaceutically acceptable salts, pharmaceutically unacceptable salts can be employed (e.g., for preparation, isolation, and/or purification purposes).
  • MIF was originally identified as a lymphocyte-derived factor with cytokine-like activity (28,29). MIF has since been shown to be constitutively expressed in numerous tissues and cell types (30) and have a variety of biological activities, including pivotal roles in the regulation of immune and inflammatory responses and promotion of tumorigenesis (31). Elevated levels of MIF have been observed in a number of disease states including cardiovascular disease (32), arthritis (33), diabetes (34), sepsis (35) and many cancer types (36,37). Furthermore, genetic ablation of MIF has been shown to attenuate various disease states in murine models (38-40). While details surrounding the mechanisms of MIF action are still in question, the clinical significance of MIF expression is such that targeted approaches to inhibit the biological activities of MIF are currently in development (41,42).
  • MIF thiol protein oxidoreductase
  • TPOR thiol protein oxidoreductase
  • MIF has three cysteine residues, two of which (C57 and C60) are critical to the TPOR activity of MIF, while a third cysteine residue (C81) is believed to play a role in maintenance of protein conformation (50).
  • cysteines were considered prime candidates for direct modification based on the known reactivity of isothiocyanates with thiols.
  • mutant rhMIF in which the cysteine residues were isosterically mutated to serine we have shown that PEITC is not dependent on any of these thiols. Instead, the
  • N-terminal proline was identified as the critical binding site of PEITC.
  • the sensitivity of the proline to attack by isothiocyanates can partly be explained by its unusually low pK a of 5.6, a value which is almost four pH units less than the pK a of free proline (48).
  • the increased nucleophilicity of the N-terminal proline allows it to function as a general base catalyst in the tautomerase reaction (51) and favours reactivity with electrophiles such as isothiocyanates.
  • Affi-Gel ® 10 and D c protein assay kit were purchased from BioRad Laboratories (Hercules, CA, USA). PEITC and Z-3,4-dihydroxyphenylalanine methyl ester were sourced from Sigma Chemical Co. (St Louis, MO, USA). Cell culture materials were from Invitrogen New Zealand Ltd. (Auckland, New Zealand). CompleteTM protease inhibitors and CHAPS were from Roche Diagnostics (Mannheim, Germany). SYPRO ® Ruby protein gel stain was obtained from Invitrogen Molecular Probes (Eugene, OR, USA).
  • a goat polyclonal antibody to human MIF, a mouse monoclonal antibody to human MIF and a human MIF ELISA kit were obtained from R & D systems (Minneapolis, MN, USA).
  • Hybond-PVDF membrane and enhanced chemiluminescence (ECLTM) western blotting system were from Amersham Biosciences (Buckinghamshire, England). All other chemicals and reagents were from Sigma Chemical Co. (St Louis, MO, USA) and BDH Laboratory supplies (Poole, England).
  • MIF and MIF mutant proteins were prepare ⁇ from pETl lb vector as previously described (Bernhagen, J.; Mitchell, R. A.; Calandra, T.; Voelter, W.; Cerami, A.; Bucala, R. Purification, Bioactivity, and Secondary Structure-Analysis of Mouse and Human Macrophage-Migration Inhibitory Factor (Mif). Biochemistry 33:14144-14155; 1994.) (Kleemann, R.; Kapurniotu, A.; Frank, R. W.; Gessner, A.; Mischke, R.; Flieger, O.; Juttner, S.; Brunner, H.; Bernhagen, J.
  • MIF macrophage migration inhibitory factor
  • the Jurkat T-lymphocyte cell line was obtained from American Type Culture Collection (Rockville, MD) and was maintained in RPMI- 1640 containing 10% fetal bovine serum, 100 units/mL penicillin and 100 ⁇ g/mL streptomycin. Cells were grown at 37°C in a humidified atomosphere with 5% CO 2 . Fresh antibiotic-free medium was added to cells 1 h before treatment. Working solutions of isothiocyanates were prepared in DMSO and added to cells so that the final concentration of DMSO in the media was kept constant at 0.1%. Cells were treated at a density of 1 x 10 6 cells/mL.
  • cytotoxicity of amino-PEITC was tested.
  • Amino PEITC retained cytotoxicity but with 0 slightly reduced potency compared to the parent PEITC. Cytotoxicity was indicated by plasma membrane integrity, as monitored using propidium iodide (PI) staining. After a 24 h treatment with each isothiocyanate, 5 ⁇ g PI was added to cells and samples were allowed to incubate in the dark for 10 min. Cell fluorescence was measured using a FC500 MPL Flow Cytometry system (Beckman Coulter Inc., Fullerton, CA). The results are shown in Figure 1.
  • Affi-Gel ® 10 activated immunoaff ⁇ nity support was thoroughly washed with 0.1 M NaHCO 3 and resuspended in 500 ⁇ L of the same buffer.
  • the Affi-Gel suspension was reacted with 7 ⁇ L of amino-PEITC (100 mg/mL in DMSO) for 1 h at room temperature with constant rotation. Remaining reactive ester groups were blocked by addition of 50 ⁇ L of 1 M i0 ethanolamine (pH 8.0). The suspension was then incubated for a further 1 h at room temperature with constant rotation.
  • An unreactive resin (blocked Affi-Gel) was prepared by incubating 100 ⁇ L AffiGel-10 in 500 ⁇ L 0.1 M NaHCO 3 containing 50 ⁇ L of 1 M ethanolamine (pH 8.0) for 2 h with constant rotation.
  • binding/wash buffer 0.1 M KCl, 20 mM HEPES, pH 7.6, 0.1 M EDTA, 0.1% NP-40, 0.25 rnM PMSF
  • Jurkat cells were collected and lysed in a buffer consisting of 25 mM HEPES, pH 7.5, 150 mM NaCl, 1% NP-40, 10 mM MgCl 2 , 1 mM EDTA, 10% glycerol and CompleteTM protease inhibitors. Protein concentration was determined using a D c protein assay and if necessary adjusted to 2 mg/mL with additional buffer.
  • Affi-PEITC and blocked Affi-Gel prepared in Example 3 were incubated with 1 mL of cell lysate for 1 h at room temperature with constant rotation. Following incubation the resins were thoroughly washed with binding/wash buffer.
  • Bound protein was eluted by boiling the resin in the presence of 100 ⁇ L of reducing sample buffer (62.5 mM Tris-HCl, pH 6.8, 10 % glycerol, 2% SDS, 0.025% bromophenol blue and 700 mM ⁇ -mercaptoethanol). The resin was pelleted and the resulting supernatant was resolved by SDS-PAGE. Total protein was stained with SYPRO ® Ruby and visualised using a Molecular Imager ® FX (BioRad Laboratories, Hercules, CA, USA).
  • MALDI-TOF mass spectrometry analysis of the excised band identified the protein as the 12.5 kDa cytokine MIF ( Figure 2B).
  • eluates were resolved by SDS-PAGE and immunoblotted with a polyclonal anti-MIF antibody.
  • Samples were transferred to PVDF membrane which were then blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween 2 o (TBST 20 ). Blots were probed with goat anti-human MIF antibody (0.2 ⁇ g/mL) or mouse anti-human MIF antibody (2 ⁇ g/mL) in TBST 20 containing 2% skim milk.
  • Immunoblotted proteins were visualised using horseradish-peroxidase conjugated secondary antibodies and the ECL system. Images were obtained using a ChemiDoc XRS system (BioRad Laboratories, Hercules, CA, USA). While antibody binding was absent in the elution fraction from blocked Affi-Gel resin, MIF was clearly shown to associate with the Affi-PEITC resin ( Figure 2C). Pre-treatment of lysates with PEITC, prior to incubation with Affi-PEITC, prevented the capture of MIF indicating that isothiocyanate modification was sufficient to inhibit subsequent binding of protein to the resin.
  • rhMIF human MIF
  • mutant rhMIF proteins were reacted with a 10-fold excess of PEITC for 20 min and adduct formation was monitored by mass spectroscopy.
  • Samples were passed through spin columns pre- equilibrated with water and then diluted 1 :1 with acetonitrile containing 0.1% formic acid.
  • Mass spectrometry was performed using an LC QTM DECA ⁇ p plus ion trap instrument (ThermoFinnigan, San Jose, CA). Samples were directly infused using a Hamilton syringe at a flow rate of 5 ⁇ L/min. A full scan for the mass range 100-2000 m/z was monitored. Data was collected for 1 min before deconvolution using BioworksBrowser 3.1 SRl (ThermoFinnigan).
  • the N-terminal proline plays a critical role as a catalytic residue in the tautomerase active site of MIF (Bendrat, K.; AlAbed, Y.; Callaway, D. J. E.; Peng, T.; Calandra, T.; Metz, C. N.; Bucala, R. Biochemical and nutational investigations of the enzymatic activity of macrophage migration inhibitory factor. Biochemistry 36:15356-15362; 1997).
  • the tautomerase activity of rhMIF was investigated using a dopachrome tautomerization assay.
  • a I-dopachrome methyl ester solution was prepared just before use by combining 72 ⁇ L of a sodium periodate stock (20 mM) with 108 ⁇ L of I-3,4-dihydroxyphenylalanine methyl ester solution (4 mM) in 1620 ⁇ L of sodium phosphate buffer (10 mM sodium phosphate, 1 mM
  • rhMIF was diluted to 1 ⁇ M with sodium phosphate buffer and 20 ⁇ L aliquots were transferred to a 96 well plate. Appropriate dilutions of PEITC (0.5 ⁇ L) were added to the rhMIF and incubated at room temperature for 5, 10 or 30 min at which point 180 ⁇ L of dopachrome solution was added to all wells and the change in absorbance at 475 nm due to dopachrome tautomerization was monitored for 2 min. A blank containing sodium phosphate buffer and 0.5 ⁇ L of DMSO was included in all experiments.
  • Figure 4 depicts results of these studies. Co-incubation of rhMIF with PEITC resulted in a dose- and time-dependent loss of MIF tautomerase activity (Figure 4A). While the biological significance of inhibiting MIF tautomerase activity is controversial, a previous study recognized that covalent modification of the N-terminal proline residue can disrupt the integrity of epitope(s) critical to the biological activity of MIF, as measured by the ability of an monoclonal anti-MIF antibody to bind the protein (Senter, P. D.; Al-Abed, Y.; Metz, C. N.; Benigni, F.; Mitchell, R. A.; Chesney, J.; Han, J. L.; Gartner, C.
  • MIF macrophage migration inhibitory factor
  • Inhibition of MIF tautomerase activity was further examined in Jurkat cells. Following treatment, 1.5 ⁇ l ⁇ 6 Jurkat cells were collected and resuspended in 60 ⁇ L of lysis buffer (40 mM HEPES, pH 7.4, containing 50 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1.6 mg/niL CompleteTM protease inhibitors and 1% CHAPS). Insoluble material was removed by centrifugation at 15 000 x g for 4 min. Protein extracts (25 ⁇ L) were transferred to a 96 well plate in duplicate. 200 ⁇ L of dopachrome solution was added to each well and the change in absorbance at 475 nm was monitored for 2 min.
  • lysis buffer 40 mM HEPES, pH 7.4, containing 50 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1.6 mg/niL CompleteTM protease inhibitors and 1% CHAPS.
  • Insoluble material was
  • PEITC was shown to inhibit cellular MIF tautomerase activity in a time- and concentration-dependent manner with an IC 50 of 1.9 ⁇ M ( ⁇ 0.1 ⁇ M) following a 30 min exposure to PEITC ( Figure 5A).
  • PEITC was shown to reduce the immunoreactivity of rhMIF as measured by an ELISA using an anti-MIF monoclonal antibody ( Figure 4C). rhMIF was reacted with PEITC for 10 min before detectable MIF levels were determined by a commercial ELISA according to manufacturer's instructions. 10 6 Jurkat cells (10 6 cells/mL) were resuspended in fresh media and incubated for 4 h at 37 °C before addition of 15 ⁇ M PEITC for 1 h. A control sample was prepared by resuspending Jurkat cells in media and incubating for 5 h at 37 0 C. Following treatment cells were pelleted by centrifugation at 10 000 x g for 1 min.
  • Isothiocyanates were built within sybyl8.0.3 using sketcher and minimised using MMFF forcefield with 1000 iterations of conjugate gradient method. Modified isothiocyanate compounds were docked into an active site of the homotrimer of MIF covalently bound to inhibitor 4-iodo-6-phenylpyrimidine (4-IPP) (pdb3B9S) using GOLD4.0.1 using the covalent bond constraint.
  • Modeling of PEITC at the tautomerase active site revealed a conformational change with the catalytic proline shifting 2 A compared to that seen in the unmodified structure.
  • lysine 32 which sits above the active site shifted by 1.6 A ( Figure 4B).
  • OHPE 2 can be prepared from the amine using conventional chemical methods, which need not be further disclosed herein.
  • Amine precursor OHPE 9 is a novel compound and can be prepared from a corresponding amine. The synthesis of this compound is complex, but can be achieved using conventional methods.
  • OHPE 10 is a novel compound that can be prepared from an amine which is available from Aldrich Chemical Company.
  • OHBN 1 is available commercially from Santa Cruz Biotechnology Inc.
  • O OHBN 2 can be made from commercially available hydroxyl precursor such as 4-(l- hydroxypropyl) phenol which is available from UkrOrgSynthesis.
  • OHBN 3 can be prepared from commercially available amine precursor such as 4- [amino(phenyl)methyl] phenol which is available from UkrOrgSynethesis.
  • Ami ⁇ o-PEITC [2-(3-(2-aminoethyl)phenyl)ethyl isothiocyanate] was prepared as in Example 1 from the diamine precursor [l,3-bis(2-aminoethyl)benzene] (17) by selective protection as a mono t-Boc derivative (18) followed by reaction with thiophosgene (19) and deprotection.
  • 1,3- Bis(2-aminoethyl)benzene was obtained by reduction of l,3-bis(cyanomethyl)benzene (20) with a nickel-modified borohydride reagent (21).
  • Nuclear magnetic resonance (NMR) spectra were obtained on a Varian Unity 500 spectrometer.
  • Example 17 Culture of Jurkat T-Lymphocytes.
  • the Jurkat T-lymphocyte cell line was obtained from American Type Culture Collection (Rockville, MD) and was maintained in RPMI 1640 containing 10% fetal bovine serum, 100 units/mL penicillin and 100 ⁇ g/mL streptomycin (Invitrogen New Zealand Ltd., Auckland, New Zealand). Cells were grown at 37°C in a humidified atmosphere with 5% CO 2 .
  • An unreactive resin (blocked Aff ⁇ -Gel) was prepared by incubating 100 ⁇ L AffiGel-10 in 500 ⁇ L 0.1 M NaHCO 3 containing 50 ⁇ L of 1 M ethanolamine (pH 8.0) for 2 hr with constant rotation.
  • the resulting Affi-PEITC and blocked Affi-Gel preparations were thoroughly washed with binding buffer (0.1 M KCl, 20 mM HEPES, pH 7.6, 0.1 M EDTA, 0.1% NP-40, 0.25 mM PMSF) before use.
  • binding buffer 0.1 M KCl, 20 mM HEPES, pH 7.6, 0.1 M EDTA, 0.1% NP-40, 0.25 mM PMSF
  • Recombinant human MIF and MIF mutant proteins were expressed, purified, and renatured from pETl lb vector as previously described (22). 10 ⁇ g recombinant human MIF and mutant MIF proteins were reacted with a 10-fold molar excess of PEITC (Sigma Chemical Co., St Louis, MO, USA) for 20 min. Samples were passed through spin columns pre-equilibrated with water and then diluted 1:1 with acetonitrile containing 0.1% formic acid. Mass spectrometry was performed using an LCQTM DECA ⁇ p pIus ion trap instrument (ThermoFinnigan, San Jose, CA, USA).
  • Jurkat cells were treated with PEITC for 1 hr before cells were lysed in a buffer consisting of 40 mM HEPES, pH 7.4, containing 50 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1.6 mg/mL CompleteTM protease inhibitors and 1% CHAPS (Roche Diagnostics, Mannheim, Germany). Insoluble material was removed by centrifugation at 15 000 x g for 4 min. Samples were diluted in reducing sample buffer and resolved by SDS-PAGE before immunoblot analysis as described above.
  • Plasma MIF levels were determined by a commercial ELISA according to manufacturer's instructions (see Figures 9 & 10).
  • Plasma isothiocyanate and dithiocarbamate derivatives were determined by a cyclocondensation reaction as previously described (23). Fractions were analysed using a Waters 2690 HPLC system (Waters, Milford, MA 5 USA) with a reverse phase HPLC column (Luna 5 ⁇ Cl 8 column, 250 x 4.6 mm, Phenomenex, San Jose, CA, USA) and eluted with 80% methanol/20% water at a rate of 0.6 mL/min. The l,3-benzodithiole-2-thione was eluted at approximately 12 min, and the peak was detected and integrated by a Photodiode array detector (Waters Model 996) at 365 run. The instrument was calibrated with pure l,3-benzodithiole-2-thione and plasma samples spiked with known concentrations of isothiocyanates were included to ensure completion of the 5 cyclocondensation reaction (see Figure 9).
  • MIF possesses a catalytic tautomerase activity and can convert the methyl ester of Z-dopachrome to an indole derivative ( Figure 12).
  • the N-terminal proline lies within the tautomerase active site and plays a critical role as a catalytic residue in the tautomerase reaction (24).
  • Co-incubation of rhMIF with PEITC resulted in a dose- and time-dependent loss of MIF tautomerase activity (Figure 4A).
  • PEITC also inhibited MIF tautomerase activity in Jurkat cells in a time- and concentration-dependent manner with a half-maximal inhibitory concentration (IC 5 o) of 2 ⁇ M following a 30 min treatment ( Figure 5A).
  • Jurkat cells (1 x 10 6 /ml) were treated with varying doses of selected isothiocyanates for 30 min. The cells were then harvested, and 1.5 x 10 6 resuspended in 60 ⁇ L of lysis buffer consisting of 40 mM HEPES, pH 7.4, containing 50 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1.6 mg/mL CompleteTM protease inhibitors and 1% CHAPS. Insoluble material was removed by centrifugation at 15,000 x g for 4 min. Cell extracts (25 ⁇ L) were transferred to a 96 well plate in duplicate.
  • lysis buffer consisting of 40 mM HEPES, pH 7.4, containing 50 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1.6 mg/mL CompleteTM protease inhibitors and 1% CHAPS. Insoluble material was removed by centrifugation at 15,000 x g for 4 min. Cell extracts (25 ⁇
  • Example 30 In sttico Fitness Scores of Isothiocyanates Docking to MIF
  • Isothiocyanates were built within SYBYL8.0 using sketcher and minimized MMFF forcefield with 1000 iterations of conjugate gradient method. Modified isothiocyanate compounds were docked into an active site of the homotrimer of MIF covalently bound to inhibitor 4-iodo-6- phenylpyrimidine (4-IPP) (pdb3B9S) using GOLD4.0.1 using the covalent bond constraint.
  • Figure 6 depicts an example of results of such modeling.
  • Figure 6A depicts the MIF molecule with an isothiocyanate compound bound thereto.
  • Figure 6B depicts an enlarged image of a portion of the image shown in Figure 6 A.
  • the Goldscore fitness functions generated from the docking shows a definite trend positively correlating to the biological assay IC 50 values in Examples 9, 28 and 29. There is a slight inconsistency where the Goldscore of Benzyl ITC is lower than the score of Phenethyl ITC, however some variability between in silico and in vitro experiments can be expected and the procedure appears valid in principle.
  • Table 8 below depicts Goldscores for selected isothiocyanates or related compounds.

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Abstract

L'invention concerne l'utilisation de composés d'isothiocyanate et d'isosélénocyanate dans le traitement de maladies et d'affections induites par le facteur d'inhibition de la migration de macrophages.
PCT/NZ2010/000102 2009-06-02 2010-06-02 Inhibiteurs du facteur d'inhibition de la migration des macrophages WO2010140902A1 (fr)

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US9931314B2 (en) 2012-07-26 2018-04-03 The William M. Yarbrough Foundation Method for treating skin cancer
WO2014049044A1 (fr) * 2012-09-26 2014-04-03 INSERM (Institut National de la Santé et de la Recherche Médicale) Méthodes et compositions pharmaceutiques pour le traitement de la colite ulcéreuse
CN108601754A (zh) * 2015-12-01 2018-09-28 宝洁公司 抑制肉毒碱向三甲胺（tma）转化的方法
US10786479B2 (en) 2015-12-01 2020-09-29 The Procter & Gamble Company Methods for inhibiting conversion of carnitine to trimethylamine (TMA)
US10780072B2 (en) 2015-12-01 2020-09-22 The Procter & Gamble Company Methods for inhibiting conversion of choline to trimethylamine (TMA)
CN108601753A (zh) * 2015-12-01 2018-09-28 宝洁公司 抑制胆碱向三甲胺(tma)的转化的方法
US11246844B2 (en) 2016-06-29 2022-02-15 The Procter & Gamble Company Methods for inhibiting conversion of choline to trimethylamine (TMA)
CN113181160A (zh) * 2020-01-14 2021-07-30 无锡杰西医药股份有限公司 异硫氰酸酯类化合物的用途

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