METHOD FOR TREATING MACROPHAGE MIGRATION INHIBITORY FACTOR (MIF)-IMPLICATED DISEASES AND CONDITIONS WITH IODO PYRIMIDINE DERIVATIVES

Compounds useful for the inhibition of macrophage migration inhibitory factor (MIF) are provided herein, having the Formula I: wherein A is selected from the group consisting of aromatic or non-aromatic rings, bicyclic rings, polycyclic rings, alkenes or alkynes; B is H, OH, OR, SR, NH2, NHR, or alkyl; R is H or alkyl, and X and Y are independently N or CH, but one of X and Y must be N. Also provided are pharmaceutical compositions that contain a Formula I compound and methods for the treatment of MIF-implicated diseases or conditions that include administering a safe and effective amount of a Formula I compound.

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Description
CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 14/803,650, filed Jul. 20, 2015, now pending; which itself is a divisional of U.S. patent application Ser. No. 13/498,036, filed Mar. 23, 2012, now U.S. Pat. No. 9,162,987; which itself is a United States National Stage application of PCT International Patent Application Serial No. PCT/US2010/050206, filed Sep. 24, 2010; which itself claims the benefit of U.S. Provisional Patent Application Ser. No. 61/245,481, filed Sep. 24, 2009. The disclosure of each of these applications is incorporated herein by reference in its entirety.

The acquisition of migratory and invasive properties by tumor cells is a central and often fatal step in neoplastic disease progression. While normal, non-transformed cells have strict growth factor and adhesive requirements for motility, malignant cells have overcome these requirements through multiple mechanisms including gain of function oncogene mutations, growth factor receptor overexpression and/or constitutive deregulation of extracellular matrix degrading enzymes. Not coincidentally, many solid cancers also possess very low oxygen tensions.

Hypoxia can induce macrophage migration inhibitory factor (MIF) expression. It has been demonstrated that MIF expression is increased in pre-malignant, malignant, and metastatic tumors. Breast, prostate, colon, brain, skin, and lung-derived tumors have all been shown to contain significantly higher levels of MIF message and protein than their non-cancerous cell counterparts. MIF expression closely correlates with tumor aggressiveness and metastatic potential, possibly suggesting an important contribution to disease severity by MIF. MIF has been indirectly implicated in tumor growth and progression by stimulating tumor-dependent stromal processes such as neovascularization. Further, MIF has been implicated in macrophage and lymphocyte activation and survival and may play a role in inflammatory disorder progression.

Thus, certain aggressive tumors appear to possess an important functional requirement for MIF in maintaining optimal growth and progression. MIF therefore provides a valuable target for development of therapeutics for the treatment of cancer. Further, MIF may be important in the progression of inflammatory disorders. The need exists to develop therapeutic molecules that target MIF and modulate one or more biological activities of MIF for the treatment of cancers and other inflammatory disorders.

Moreover, MIF is produced by several different pathogens including parasitic helminths, spirochetes, and plasmodium. As such, irreversible inhibitors of MIF such as 4-iodo-6-phenylpyrimidine (4-IPP) and analogs may be excellent antagonists of parasite-derived MIF. The need exists to develop therapeutic molecules that target MIF and ameliorate the disease-causing pathologies associated with these and other MIF-producing pathogens.

In one embodiment of the invention, a compound or its enantiomeric or diastereomeric form or a pharmaceutically acceptable salt, prodrug, or metabolite thereof is provided, said compound having the formula:

wherein: A is selected from the group consisting of: i) substituted or unsubstituted 5, 6 or 7-membered aromatic or nonaromatic rings having 0 or 1 to 4 heteroatoms selected from the group consisting of N, O, S, and combinations thereof; ii) substituted or unsubstituted bicyclic ring; iii) substituted or unsubstituted polycyclic rings; and iv) substituted or unsubstituted alkenes and alkynes having 2 to 10 carbon atoms with 1 to 3 double or triple bonds; B is H, OH, OR, SR, NH2, NHR, alkyl or substituted alkyl or A, but when B is A, A is H or halo; R is H, alkyl or substituted alkyl of 2 to 20 carbon atoms; and X and Y are independently N or CH, but one of X and Y must be N.

In another embodiment, a pharmaceutical composition is provided, comprising (a) an effective amount of a Formula I compound or its enantiomeric or diastereomeric form or a pharmaceutically acceptable salt, prodrug, or metabolite thereof, and (b) one or more pharmaceutically acceptable excipients.

In another embodiment, a method for treating a macrophage migration inhibitory factor (MIF)-implicated disease or condition is provided, the method comprising administering to a patient in need thereof an effective amount of a Formula I compound, or its enantiomeric or diastereomeric form or a pharmaceutically acceptable salt, prodrug, or metabolite thereof.

These and other objects, features, embodiments, and advantages will become apparent to those of ordinary skill in the art from a reading of the following detailed description and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts MIF liver enzyme inhibition as a percent of control, comparing ACT-MIF-003, ACT-MIF-002, and 4-IPP.

FIG. 2 depicts MIF tumor enzyme inhibition as a percent of control, comparing ACT-MIF-003, ACT-MIF-002, and 4-IPP.

FIG. 3 depicts a comparison of IC50 values across the tumor cell lines H23, MCF7, MDA-MB-231, H-460, SKOV-3, PC3, DU145, Miapaca, LnCap, Capan 1, Capan 2, and CAOV3.

FIG. 4 depicts a comparison of IC50 values across the tumor cell lines DU145, MDA-MB-231, Miapaca, CAOV03, and HUVEC.

FIG. 5 depicts p53 regulation of compounds compared to control (DMSO), 4-IPP, and ISO-1 at 10 μM concentration. Compounds tested included ACT-MIF-030, ACT-MIF-035, ACT-MIF-038, ACT-MIF-029, ACT-MIF-033, ACT-MIF-034, ACT-MIF-003, and ACT-MIF-028. Results indicate the compounds are implicated in p53 regulation.

FIG. 6 depicts inhibition of cell proliferation IC50 values for ACT-MIF-006, ACT-MIF-035, and ACT-MIF-038 in the LOX-IMV1 tumor cell line.

FIGS. 7A-7D depict inhibition of cell migration in the LOX-IMV1 tumor cell line at 72 hrs, for ACT-MIF-006 (A), ACT-0035 (B and D), and ACT-MIF-038 (C). Results indicate a significant inhibition of migration, even at low concentrations (0.03 μM).

FIG. 8 depicts tumor growth inhibition of DU145 human prostate xenografts in athymic nude mice treated with ACT-MIF-001, ACT-MIF-002, and ACT-MIF-003. Results show ACT-MIF-003 significantly inhibited tumor growth.

FIGS. 9A-9D depict tumor slices from animals treated with control (A), ACT-MIF-002 (B), ACT-MIF-001 (C), and ACT-MIF-003 (D). Blood vessel density of the tumor tissues was measured by immunohistochemistry. Results indicated a decrease in microvessel density with respect to the tumors of the control group with a statistically meaningful difference for the ACT-MIF-003 treated group.

FIGS. 10A and 10B depict tumor growth inhibition (A) and survival data (B) in a pancreatic tumor model treated with control, ACT-MIF-002, and ACT-MIF-003. Results indicate the tested compounds had significant impact on survival and limited metastatic tumor burden.

FIGS. 11A and 11B depict histopathological slides comparing bone marrow from pancreatic tumor model animals treated with control (A) and ACT-MIF-002 (B). Bone marrow of the vehicle treated mice is consistent with bone metastases (1) with evidence of surrounding skeletal muscle metastases from invading marrow tumor cells (2 and 3). No evidence of bone metastases was observed with spinal column sections from ACT-MIF-002 treated mice.

FIG. 12 depicts a comparison of MIF enzyme inhibition in the liver, brain, and lung of healthy animals administered ACT-MIF-002 either intraperitoneally (IP) or per oral (PO). Results indicate the compound is orally bioavailable, crosses the brain blood barrier, and inhibits MIF enzymatic activity in both the brain and the lungs.

FIGS. 13A-13D depict 4-IPP-based MIF antagonists effects on primary T lymphocyte activation/proliferation. Fresh, primary human T lymphocytes was collected by aphaeresis and separated by Ficoll gradients. 1×106 lymphocytes were added to immobilized anti-CD3 tissue culture plates in the presence of nothing (control; FIG. 13A), vehicle control (0.1% DMSO; FIG. 13B), 25 μM 4-IPP (FIG. 13C), or 25 μM ACT-003 (FIG. 13D). 48 hours later cells were collected, washed, and stained with anti-CD4 and ant-CD8 labeled antibodies followed by flow cytometric analyses. Shown are the relative percentages of CD4/CD8 lymphocytes.

FIGS. 14A and 14B depict the data from FIG. 15 as an overlay of relative fluorescence intensity of expression of CD4 (FIG. 14A) or CD8 (FIG. 14B) in PBMCs activated with plate bound anti-CD3 for 48 hours.

FIGS. 15A-15D depict 4-IPP-based MIF antagonists effects on primary T lymphocyte activation/proliferation. Fresh, primary human T lymphocytes was collected by aphaeresis and separated by Ficoll gradients. 1×106 lymphocytes were added to immobilized anti-CD3 tissue culture plates in the presence of nothing (control; FIG. 15A), vehicle control (0.1% DMSO; FIG. 15B), 25 μM 4-IPP (FIG. 15C), or 25 μM ACT-003 (FIG. 15D). 48 hours later cells were collected, washed, and stained with an anti-CD25-labeled antibody followed by flow cytometric analyses. CD25 (high affinity IL-2 receptor) is a commonly used marker for T lymphocyte activation. Shown are the relative percentages of CD25+(i.e., activated) treated vs. untreated lymphocytes.

FIG. 16 depicts the data from FIG. 17 as an overlay of fluorescence intensity of expression of CD25 in PBMCs activated with plate bound anti-CD3 for 48 hours.

FIGS. 17A-17D depict 4-IPP-based MIF antagonists effects on primary T lymphocyte activation/proliferation. Fresh, primary human T lymphocytes was collected by aphaeresis and separated by Ficoll gradients. 1×106 lymphocytes were added to immobilized anti-CD3 tissue culture plates in the presence of nothing (control; FIG. 17A), vehicle control (0.1% DMSO; FIG. 17B), 25 μM 4-IPP (FIG. 17C), or 25 μM ACT-003 (FIG. 17D). 16 hours later cells were collected, washed, and stained with an anti-CD69-labeled antibody followed by flow cytometry analysis. CD69 is an early marker of lymphocyte activation and the lack of a large effect on early lymphocyte activation suggests that treatment of established T cell-dependent autoimmune diseases with 4-IPP-based anti-MIF antagonists is feasible. Shown are the relative percentages of CD69 on treated vs. untreated lymphocytes.

FIG. 18 depicts the data from FIG. 19 as an overlay of fluorescence intensity of expression of CD69 in PBMCs activated with plate bound anti-CD3 for 48 hours.

FIGS. 19A-19D depict 4-IPP-based MIF antagonists' effects on primary T lymphocyte activation/proliferation. Fresh, primary human T lymphocytes was collected by aphaeresis and separated by Ficoll gradients. 1×106 lymphocytes were added to immobilized anti-CD3 tissue culture plates in the presence of nothing (control; FIG. 19A), vehicle control (0.1% DMSO; FIG. 19B), 25 μM 4-IPP (FIG. 19C), or 25 μM ACT-003 (FIG. 19D). 48 hours later labeled-BrdU was added to the cells briefly, then washed, stained with labeled-anti-CD8 antibodies and analyzed for BrdU incorporation into DNA (readout for proliferation) by flow cytometry.

FIGS. 20A-20D depict 4-IPP-based MIF antagonists' effects on primary T lymphocyte activation/proliferation. Fresh, primary human T lymphocytes was collected by aphaeresis and separated by Ficoll gradients. 1×106 lymphocytes were added to immobilized anti-CD3 tissue culture plates in the presence of nothing (control; FIG. 20A), vehicle control (0.1% DMSO; FIG. 20B), 25 μM 4-IPP (FIG. 20C), or 25 μM ACT-003 (FIG. 20D). 48 hours later labeled-BrdU was added to the cells briefly, then washed, stained with labeled-anti-CD4 antibodies and analyzed for BrdU incorporation into DNA (readout for proliferation) by flow cytometry.

The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document.

While the following terms are believed to be well understood by one of ordinary skill in the art, definitions are set forth to facilitate explanation of the presently-disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently-disclosed subject matter belongs.

Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.

As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.

The terms “enantiomer” and “diastereomer” have the standard art recognized meanings (see e.g., Hawley's Condensed Chemical Dictionary, 14th ed.). The illustration of specific protected forms and other derivatives of the compounds of the instant invention is not intended to be limiting. The application of other useful protecting groups, salt forms, etc. is within the ability of the skilled artisan.

The term “prodrug” refers to any covalently bonded carriers which release the active parent drug according to the Formula I described above in vivo when such prodrug is administered to a subject. Prodrugs of the compounds are prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compounds.

The term “substituted” is defined herein as “encompassing moieties or units which can replace one or more hydrogen atoms of a hydrocarbyl moiety. The term “hydrocarbyl” is defined herein as any organic unit or moiety which is comprised of carbon atoms and hydrogen atoms.

“Halo” or “halogen” refers to fluoro, chloro, bromo, or iodo.

The term “aromatic ring” refers to an aromatic hydrocarbon ring system. Suitable aromatic rings of embodiments of the present invention contain 5, 6, or 7 carbon atoms in the ring. Aromatic rings can also contain 0 or 1-4 heteroatoms selected from the group consisting of N, O, S, and combinations thereof. Non-limiting examples of suitable aromatic rings include phenyl, pyridinyl, pyrimidinyl, pyridazinyl, furanyl, thiophenyl, pyrrolyl, imidazolyl, pyrazolyl, and thiadiazolyl. Aromatic rings of the present invention can be unsubstituted or substituted with from 1 to 3 substituents. Non-limiting examples of suitable substituents include halo, hydroxyl, alkoxy, amino, substituted amino, carboxylic acid, ester, amide, substituted amide, nitro, alkyl, substituted alkyl, combinations thereof, or functional equivalents thereof.

The term “non-aromatic ring” refers to a non-aromatic saturated or unsaturated hydrocarbon ring system. Suitable non-aromatic rings of embodiments of the present invention contain 5, 6, or 7 carbon atoms in the ring. Non-aromatic rings can also contain 0 or 1-4 heteroatoms selected from the group consisting of N, O, S, and combinations thereof. Non-aromatic rings of the present invention can be unsubstituted or substituted with from 1 to 3 substituents. Non-limiting examples of suitable substituents include halo, hydroxyl, alkoxy, amino, substituted amino, carboxylic acid, ester, amide, substituted amide, nitro, alkyl, substituted alkyl, combinations thereof, or functional equivalents thereof.

The term “bicyclic ring” refers to two fused hydrocarbon rings that may optionally include one or more heteroatoms as ring members. A bicyclic ring can be substituted or unsubstituted, including single or multiple substitutions. The rings can independently show a different degree of saturation and may be saturated, unsaturated, or aromatic. Fusion of the rings can occur in three ways: across a bond between two atoms; across a sequence of atoms (bridgehead); or at a single atom (spirocyclic). Bicyclic rings of the present invention include, but are not limited to, 6-5, 6-6, 6-7, 5-5, 5-6, 5-7, 7-5, and 7-6 ring systems, wherein the integers refer to the number of carbon atoms or heteroatoms in each ring in the structure. Bicylic rings of the present invention can be unsubstituted or substituted with from 1 to 4 substituents. Non-limiting examples of suitable substituents include halo, hydroxyl, alkoxy, amino, substituted amino, carboxylic acid, ester, amide, substituted amide, nitro, alkyl, substituted alkyl, combinations thereof, or functional equivalents thereof. Non-limiting examples of suitable bicyclic rings of the present invention include indole, quinoline, and naphthalene.

The term “polycyclic ring” refers to three or more fused hydrocarbon rings that may optionally include one or more heteratoms as ring members. A polycyclic ring can be substituted or unsubstituted, including single or multiple substitutions. The rings can independently show a different degree of saturation and may be saturated, unsaturated, or aromatic. Fusion of the rings can occur in three ways: across a bond between two atoms; across a sequence of atoms (bridgehead); or at a single atom (spirocyclic). Polycyclic rings of the present invention can be unsubstituted or substituted with from 1 to 4 substituents. Non-limiting examples of suitable substituents include halo, hydroxyl, alkoxy, amino, substituted amino, carboxylic acid, ester, amide, substituted amide, nitro, alkyl, substituted alkyl, combinations thereof, or functional equivalents thereof.

The term “alkene” refers herein to a hydrocarbon chain having from 1 to 3 carbon-carbon double bonds and having 2 to 10 carbon atoms. Alkenes of the present invention can be unsubstituted or substituted with from 1 to 3 substituents. Non-limiting examples of suitable substituents include halo, hydroxyl, alkoxy, amino, substituted amino, carboxylic acid, ester, amide, substituted amide, nitro, alkyl, substituted alkyl, combinations thereof, or functional equivalents thereof.

The term “alkyne” refers herein to a hydrocarbon chain having from 1 to 3 carbon-carbon triple bonds and having 2 to 10 carbon atoms. Alkynes of the present invention can be unsubstituted or substituted with from 1 to 3 substituents. Non-limiting examples of suitable substituents include halo, hydroxyl, alkoxy, amino, substituted amino, carboxylic acid, ester, amide, substituted amide, nitro, alkyl, substituted alkyl, combinations thereof, or functional equivalents thereof.

The term “alkyl” refers to a saturated hydrocarbon chain having 2 to 20 carbon atoms. Alkyls of the present invention can be substituted or unsubstituted. Non-limiting examples of suitable substituents include hydroxyl, amino, thiol, morpholino, pyrrolidino, piperidino, glycol, and polyethyleneglycol (PEG) having molecular weight of 200 to 20,000.

The term “pharmaceutically-acceptable excipient,” as used herein, means any physiologically inert, pharmacologically inactive material known to one skilled in the art, which is compatible with the physical and chemical characteristics of the particular CEL inhibitor selected for use. Pharmaceutically-acceptable excipients include, but are not limited to, polymers, resins, plasticizers, fillers, lubricants, diluents, binders, disintegrants, solvents, co-solvents, buffer systems, surfactants, preservatives, sweetening agents, flavoring agents, pharmaceutical grade dyes or pigments, and viscosity agents.

The term “MIF-implicated disease or condition” refers to a disease or condition for which MIF is a factor in the onset and/or progression of the disease or condition.

The term “safe and effective amount” of a Formula (I) compound is an amount that is effective to inhibit the MIF enzyme in an animal, specifically a mammal, more specifically a human subject, without undue adverse side effects (such as toxicity, irritation, or allergic response), commensurate with a reasonable benefit/risk ratio when used in the manner of this invention. The specific “safe and effective amount” will, obviously, vary with such factors as the particular condition being treated, the physical condition of the patient, the duration of treatment, the nature of concurrent therapy (if any), the specific dosage form to be used, the excipient employed, the solubility of the Formula (I) compound therein, and the dosage regimen desired for the composition.

The term “inflammatory disease” refers to a disease characterized by inflammation, or the complex vascular and immune response to harmful stimuli. Inflammatory diseases include those diseases in which inflammation and immune cells are involved in the pathology of the disease. In a specific embodiment, the inflammatory disease is selected from the group consisting of dermatitis, arthritis, rheumatoid arthritis, insulin-dependent diabetes, proliferative vascular disease, acute respiratory distress syndrome, sepsis, septic shock, psoriasis, asthma, cytokine related toxicity, lupus, multiple sclerosis, transplant-host response, and autoimmune disorders.

Compounds according to the present invention have the following generic structure:

wherein:

A is selected from the group consisting of:

    • i) substituted or unsubstituted 5, 6 or 7-membered aromatic or nonaromatic rings having 0 or 1 to 4 heteroatoms selected from the group consisting of N, O, S, and combinations thereof;
    • ii) substituted or unsubstituted bicyclic ring;
    • iii) substituted or unsubstituted polycyclic rings; and
    • iv) substituted or unsubstituted alkenes and alkynes having 2 to 10 carbon atoms with 1 to 3 double or triple bonds;

B is H, OH, OR, SR, NH2, NHR, alkyl or substituted alkyl or A, but when B is A, A is H or halo;

R is H, alkyl or substituted alkyl of 2 to 20 carbon atoms; and

X and Y are independently N or CH, but one of X and Y must be N.

In one embodiment, A is selected from the group consisting of: substituted or unsubstituted 5, 6 or 7-membered aromatic or nonaromatic rings having none or 1 to 4 heteroatoms which could be a single atom or the combination of N, O and S; substituted or unsubstituted bicyclic ring, for example indole, quinoline and naphthalene; substituted or unsubstituted polycyclic rings; and substituted or unsubstituted alkenes and alkynes having 2 to 10 carbon atoms with 1 to 3 double or triple bonds; wherein substitutions for any of the above are selected from the group consisting of halo, hydroxyl, alkoxy, amino, substituted amino, carboxylic acid, ester, amide, substituted amide, nitro, alkyl, substituted alkyl, combinations thereof, or functional equivalents thereof; B is H; and X and Y are both N.

In another embodiment, A is selected from the group consisting of: substituted or unsubstituted 5, 6 or 7-membered aromatic or nonaromatic rings having none or 1 to 4 heteroatoms which could be a single atom or the combination of N, O and S; substituted or unsubstituted bicyclic ring, for example indole, quinoline and naphthalene; substituted or unsubstituted polycyclic rings; and substituted or unsubstituted alkenes and alkynes having 2 to 10 carbon atoms with 1 to 3 double or triple bonds; wherein substitutions for any of the above are selected from the group consisting of halo, hydroxyl, alkoxy, amino, substituted amino, carboxylic acid, ester, amide, substituted amide, nitro, alkyl, substituted alkyl, combinations thereof, or functional equivalents thereof; B is H, OH, OR, SR, NH2, NHR, alkyl, or substituted alkyl; X and Y are both N.

In another embodiment, A is halo, B is selected from the group consisting of: substituted or unsubstituted 5, 6 or 7-membered aromatic or nonaromatic rings having none or 1 to 4 heteroatoms which could be a single atom or the combination of N, O and S; substituted or unsubstituted bicyclic ring, for example indole, quinoline and naphthalene; substituted or unsubstituted polycyclic rings; and substituted or unsubstituted alkenes and alkynes having 2 to 10 carbon atoms with 1 to 3 double or triple bonds; wherein substitutions for any of the above are selected from the group consisting of halo, hydroxyl, alkoxy, amino, substituted amino, carboxylic acid, ester, amide, substituted amide, nitro, alkyl, substituted alkyl, combinations thereof, or functional equivalents thereof, and X and Y are both N.

In another embodiment, A is selected from the group consisting of: substituted or unsubstituted 5, 6 or 7-membered aromatic or nonaromatic rings having none or 1 to 4 heteroatoms which could be a single atom or the combination of N, O and S; substituted or unsubstituted bicyclic ring, for example indole, quinoline and naphthalene; substituted or unsubstituted polycyclic rings; and substituted or unsubstituted alkenes and alkynes having 2 to 10 carbon atoms with 1 to 3 double or triple bonds; wherein substitutions for any of the above are selected from the group consisting of halo, hydroxyl, alkoxy, amino, substituted amino, carboxylic acid, ester, amide, substituted amide, nitro, alkyl, substituted alkyl, combinations thereof, or functional equivalents thereof; B is H; X is N, and Y is CH.

In still another embodiment, A is selected from the group consisting of: substituted or unsubstituted 5, 6 or 7-membered aromatic or nonaromatic rings having none or 1 to 4 heteroatoms which could be a single atom or the combination of N, O and S; substituted or unsubstituted bicyclic ring, for example indole, quinoline and naphthalene; substituted or unsubstituted polycyclic rings; and substituted or unsubstituted alkenes and alkynes having 2 to 10 carbon atoms with 1 to 3 double or triple bonds; wherein substitutions for any of the above are selected from the group consisting of halo, hydroxyl, alkoxy, amino, substituted amino, carboxylic acid, ester, amide, substituted amide, nitro, alkyl, substituted alkyl, combinations thereof, or functional equivalents thereof; B is H; X is CH; and Y is N.

In another embodiment, A is selected from the group consisting of: substituted or unsubstituted 5, 6 or 7-membered aromatic or nonaromatic rings having none or 1 to 4 heteroatoms which could be a single atom or the combination of N, O and S; substituted or unsubstituted bicyclic ring, for example indole, quinoline and naphthalene; substituted or unsubstituted polycyclic rings; and substituted or unsubstituted alkenes and alkynes having 2 to 10 carbon atoms with 1 to 3 double or triple bonds; wherein substitutions for any of the above are selected from the group consisting of halo, hydroxyl, alkoxy, amino, substituted amino, carboxylic acid, ester, amide, substituted amide, nitro, alkyl, substituted alkyl, combinations thereof, or functional equivalents thereof; B is H, OH, OR, SR, NH2, NHR, alkyl or substituted alkyl; X is N and Y is CH.

In still another embodiment, A is selected from the group consisting of: substituted or unsubstituted 5, 6 or 7-membered aromatic or nonaromatic rings having none or 1 to 4 heteroatoms which could be a single atom or the combination of N, O and S; substituted or unsubstituted bicyclic ring, for example indole, quinoline and naphthalene; substituted or unsubstituted polycyclic rings; and substituted or unsubstituted alkenes and alkynes having 2 to 10 carbon atoms with 1 to 3 double or triple bonds; wherein substitutions for any of the above are selected from the group consisting of halo, hydroxyl, alkoxy, amino, substituted amino, carboxylic acid, ester, amide, substituted amide, nitro, alkyl, substituted alkyl, combinations thereof, or functional equivalents thereof; B is H, OH, OR, SR, NH2, NHR, alkyl, or substituted alkyl; X is CH; and Y is N.

In another embodiment, the compound is selected from the group set forth in Table 1.

TABLE 1 ACT-MIF EXAMPLE NO. CHEMICAL NAME 2 ACT-MIF- 4-Iodo-6-(2,3-difluoro-4-methoxyphenyl)pyrimidine 001 3 ACT-MIF- 4-Iodo-6-(2-fluoro-4-methoxyphenyl)pyrimidine 002 4 ACT-MIF- 4-Iodo-6-(2-fluorophenyl)pyrimidine 003 5 ACT-MIF- 4-Iodo-6-(4-fluorophenyl)pyrimidine 004 6 ACT-MIF- 4-Iodo-6-(furan-3-yl)pyrimidine 005 7 ACT-MIF- 4-Iodo-6-(pyridin-3-yl)pyrimidine 006 8 ACT-MIF- 4-Iodo-6-(3-fluorophenyl)pyrimidine 008 9 ACT-MIF- 4-Iodo-6-(4-tert-butyloxymethylphenyl)pyrimidine 010 10 ACT-MIF- 4-Iodo-6-(2-fluoropyridin-3-yl)pyrimidine 011 11 ACT-MIF- 4-Iodo-6-(furan-2-yl)pyrimidine 012 12 ACT-MIF- 4-Iodo-6-(4-fluoropyrimidin-3-yl)pyrimidine 013 13 ACT-MIF- 4-Iodo-6-(3-fluoro-4-methoxyphenyl)pyrimidine 014 14 ACT-MIF- 4-Iodo-6-(2-chloropyridin-5-yl)pyrimidine 015 15 ACT-MIF- 4-Iodo-6-(2-hydroxyphenyl)pyrimidine 016 16 ACT-MIF- 4-Iodo-6-(2,4-difluorophenyl)pyrimidine 017 17 ACT-MIF- 4-Iodo-6-(2-fluoro-6-methoxyphenyl)pyrimidine 018 18 ACT-MIF- 4-Iodo-6-(2-chlorophenyl)pyrimidine 019 19 ACT-MIF- 4-Iodo-6-(3-acetylaminophenyl)pyrimidine 021 20 ACT-MIF- 4-Iodo-6-(thiophen-3-yl)pyrimidine 022 21 ACT-MIF- 4-Iodo-6-(3-hydroxymethylphenyl)pyrimidine 023 22 ACT-MIF- 4-Iodo-6-(isoquinolin-4-yl)pyrimidine 025 23 ACT-MIF- 4-Iodo-6-(2,4,5-trifluorophenyl)pyrimidine 027 24 ACT-MIF- 4-Iodo-6-(2,4-difluoropyridin-3-yl)pyrimidine 028 25 ACT-MIF- 4-Iodo-6-(4-methoxypyridin-3-yl)pyrimidine 029 26 ACT-MIF- 4-Iodo-6-(thiophen-2-yl)pyrimidine 030 27 ACT-MIF- 4-Iodo-6-(3,4-difluorophenyl)pyrimidine 032 28 ACT-MIF- 4-Iodo-6-(4-ethoxyphenyl)pyrimidine 033 29 ACT-MIF- 4-Iodo-6-(4-aminocarbonylphenyl)pyrimidine 034 30 ACT-MIF- 4-Iodo-6-(3-aminocarbonylphenyl)pyrimidine 035 31 ACT-MIF- 4-Iodo-6-(quinolin-4-yl)pyrimidine 036 32 4-Iodo-6-(quinolin-8-yl)pyrimidine 33 4-Iodo-6-(quinolin-3-yl)pyrimidine 34 4-Iodo-6-(isoquinolin-5-yl)pyrimidine 36 2-Methylthio-4-iodo-6-phenylpyrimidine 37 2-Ethylthio-4-iodo-6-phenylpyrimidine 38 2-Isopropylthio-4-iodo-6-phenylpyrimidine 39 2-n-Butylthio-4-iodo-6-phenylpyrimidine 41 2-Methylamino-4-iodo-6-phenylpyrimidine 42 2-Ethylamino-4-iodo-6-phenylpyrimidine 43 2-Propylamino-4-iodo-6-phenylpyrimidine 44 2-Isopropylamino-4-iodo-6-phenylpyrimidine 45 2-n-Butylamino-4-iodo-6-phenylpyrimidine 46 4-Iodo-6-(benzothiophen-2-yl)pyrimidine 47 4-Iodo-6-(benzofuran-2-yl)pyrimidine 48 4-Iodo-6-(4-hydroxybenzothiophen-2-yl)pyrimidine 49 4-Iodo-6-(4-acetylaminobenzothiophen-2- yl)pyrimidine 50 4-Iodo-6-(4-aminocarbonylbenzothiophen-2- yl)pyrimidine 51 4-Iodo-6-(5-acetylaminopyridin-3-yl)pyrimidine 52 4-Iodo-6-(5-aminocarbonylpyridin-3-yl)pyrimidine 53 4-Iodo-6-(4-fluoropyridin-3-yl)pyrimidine 54 4-Iodo-6-(4-acetylaminothiophen-2-yl)pyrimidine 55 4-Iodo-6-(4-aminocarbonylthiophen-2-yl)pyrimidine 56 4-Iodo-6-(4-methoxythiophen-2-yl)pyrimidine

In another embodiment, X and Y are both N. In another embodiment, when X and Y are both N, B is H.

In still another embodiment, A is halo, B is A, and X and Y are both N. In a specific embodiment, A is I, B is A, and X and Y are both N.

In another embodiment, X is N and Y is CH. In still another embodiment, when X is N and Y is CH, B is H.

In another embodiment, X is CH and Y is N. In a further embodiment, when X is CH and Y is N, B is H.

In a specific embodiment, A is selected from the group consisting of indole, quinoline, and naphthalene.

In a very specific embodiment, the compound is 4-Iodo-6-(2-fluorophenyl) pyrimidine or 4-Iodo-6-(3-aminocarbonylphenyl)pyrimidine.

In another embodiment, a pharmaceutical composition is provided, comprising:

a) a safe and effective amount of a compound or its enantiomeric or diastereomeric form or a pharmaceutically acceptable salt, prodrug, or metabolite thereof, said compound having the formula:

    • wherein:
    • A is selected from the group consisting of:
      • i) substituted or unsubstituted 5, 6 or 7-membered aromatic or nonaromatic rings having 0 or 1 to 4 heteroatoms selected from the group consisting of N, O, S, and combinations thereof;
      • ii) substituted or unsubstituted bicyclic ring;
      • iii) substituted or unsubstituted polycyclic rings; and
      • iv) substituted or unsubstituted alkenes and alkynes having 2 to 10 carbon atoms with 1 to 3 double or triple bonds;
    • B is H, OH, OR, SR, NH2, NHR, alkyl or substituted alkyl or A, but when B is A, A is H or halo;
    • R is H, alkyl or substituted alkyl of 2 to 20 carbon atoms; and
    • X and Y are independently N or CH, but one of X and Y must be N; and

b) one or more pharmaceutically acceptable excipients.

In one embodiment, the compound is selected from the group set forth in Table 1.

In another embodiment, X and Y are both N. In another embodiment, when X and Y are both N, B is H.

In still another embodiment, A is halo, B is A, and X and Y are both N. In a specific embodiment, A is I, B is A, and X and Y are both N.

In another embodiment, X is N and Y is CH. In still another embodiment, when X is N and Y is CH, B is H.

In another embodiment, X is CH and Y is N. In a further embodiment, when X is CH and Y is N, B is H.

In a specific embodiment, A is selected from the group consisting of indole, quinoline, and naphthalene.

In a very specific embodiment, the compound is 4-Iodo-6-(2-fluorophenyl) pyrimidine or 4-Iodo-6-(3-aminocarbonylphenyl)pyrimidine.

In a further embodiment, a method for treating a macrophage migration inhibitory factor (MIF)-implicated disease or condition is provided, the method comprising administering to a patient in need thereof a safe and effective amount of a compound or its enantiomeric or diastereomeric form or a pharmaceutically acceptable salt, prodrug, or metabolite thereof, said compound having the formula:

wherein:

A is selected from the group consisting of:

    • i) substituted or unsubstituted 5, 6 or 7-membered aromatic or nonaromatic rings having 0 or 1 to 4 heteroatoms selected from the group consisting of N, O, S, and combinations thereof;
    • ii) substituted or unsubstituted bicyclic ring;
    • iii) substituted or unsubstituted polycyclic rings; and
    • iv) substituted or unsubstituted alkenes and alkynes having 2 to 10 carbon atoms with 1 to 3 double or triple bonds;

B is H, OH, OR, SR, NH2, NHR, alkyl or substituted alkyl or A, but when B is A, A is H or halo;

R is H, alkyl or substituted alkyl of 2 to 20 carbon atoms; and

X and Y are independently N or CH, but one of X and Y must be N.

In one embodiment, the compound is selected from the group set forth in Table 1.

In another embodiment, X and Y are both N. In another embodiment, when X and Y are both N, B is H.

In still another embodiment, A is halo, B is A, and X and Y are both N. In a specific embodiment, A is I, B is A, and X and Y are both N.

In another embodiment, X is N and Y is CH. In still another embodiment, when X is N and Y is CH, B is H.

In another embodiment, X is CH and Y is N. In a further embodiment, when X is CH and Y is N, B is H.

In a specific embodiment, A is selected from the group consisting of indole, quinoline, and naphthalene.

In a very specific embodiment, the compound is 4-Iodo-6-(2-fluorophenyl) pyrimidine or 4-Iodo-6-(3-aminocarbonylphenyl)pyrimidine.

In one embodiment, the MIF-implicated disease is selected from the group consisting of inflammatory disease and cancer.

In a specific embodiment, the inflammatory disease is selected from the group consisting of dermatitis, arthritis, rheumatoid arthritis, insulin-dependent diabetes, proliferative vascular disease, acute respiratory distress syndrome, sepsis, septic shock, psoriasis, asthma, cytokine related toxicity, lupus, multiple sclerosis, transplant-host response, and autoimmune disorders.

MIF is produced by several different pathogens, including parasitic helminths, spirochetes, and plasmodium. Thus, irreversible inhibitors of MIF, such as the MIF inhibitors of Formula I, are useful as antagonists of parasite-derived MIF. Accordingly, in a further embodiment, the MIF-implicated condition is caused by a MIF-producing pathogen. In a specific embodiment, the MIF-producing pathogen is selected from the group consisting of parasitic helminths, spirochetes, and plasmodium.

EXAMPLES

These following exemplary embodiments and synthetic schemes are provided by way of illustration only and are in no way intended to limit the scope of the present invention.

Example 1 Methods for the Preparation of 4-Iodo-6-arylpyrimidine Derivatives, Where Aryl is Substituted Phenyl, Heterocyclic, or Bicyclic Ring

General Procedure: 4,6-Dichloropyrimidine (1) is reacted with corresponding aryl boronic acid (2) in dioxane- and aqueous sodium carbonate in the presence of a catalyst used for Suzuki coupling at 50 to 100° C. temperature. The resultant 4-chloro-6-arylpyrimidine (3) is isolated by crystallization or column chromatography on silica gel and is converted to corresponding 4-iodo-6-arylpyrimidine (4) using hydroiodic acid. Further treatment of HI may be needed when the reaction is not complete.

The compounds of Examples 2-34 are prepared using Scheme 1.

Example 2 4-Iodo-6-(2,3-difluoro-4-methoxyphenyl)pyrimidine (ACT-MIF-001)

The compound was prepared according to EXAMPLE 1 using 2,3-difluoro-4-methoxyphenylboronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ8.98 (s, 1H), 8.30 (s, 1H), 7.92 (m, 1H), 7.21 (m, 1H), 3.98 (s, 3H).

Example 3 4-Iodo-6-(2-fluoro-4-methoxyphenyl)pyrimidine (ACT-MIF-002)

The compound was prepared according to EXAMPLE 1 using 2-fluoro-4-methoxyphenylboronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ8.91 (s, 1H), 8.23 (s, 1H), 8.05 (m, 1H), 7.01 (m, 2H), 3.88 (s, 3H).

Example 4 4-Iodo-6-(2-fluorophenyl)pyrimidine (ACT-MIF-003)

The compound was prepared according to EXAMPLE 1. Specifically, the following method was employed:

1. Preparation of 4-chloro-6-(2-fluoro-phenyl)-pyrimidine (3) (TRM/AP/005/127)

4,6-dichloropyrimidine (20.3 g, 136.3 mmol), 2-fluorophenyl boronic acid (20.0 g, 142.9 mmol, 1.05 equiv), Na2CO3 (23.4 g, 106.0 mmol, 1.8 equiv) and Pd(PPh3)2Cl2 (1.0 g, 1.4 mmol, 0.01 equiv) were refluxed in dimethoxyethane-water (817:272 mL) mixed solvent system for 6.5 h. Reaction was monitored by TLC (using ethyl acetate:n- hexane, 1:9). Reaction mixture was cooled and the subject compound was extracted using dichloromethane. Subject compound was purified by flash chromatography (2.5% ethyl acetate:n-hexane) to yield 4.5 g (Yield=15.8%).

1H NMR (CDC13): 9.07 (s, 1H), 8.19 (t, J=7.8 Hz, 1H), 7.91 (s, 1H), 7.48-7.55 (m, 1H), 7.18-7.35 (m, 2H)
4,6-dichloropyrimidine (5.1 g, 34.1 mmol), 2-fluorophenyl boronic acid (5.0 g, 35.7 mmol, 1.05 equiv), Na2CO3 (6.9 g, 65.0 mmol, 1.8 equiv) and Pd(PPh3)2Cl2 (0.3 g, 0.4 mmol, 0.01 equiv) were refluxed in dimethoxyethane-water (204:69 mL) mixed solvent system for 4 h. Reaction was monitored by TLC (using ethyl acetate-hexane, 1:9). Reaction mixture was cooled and the subject compound was extracted using dichloromethane. Subject compound was purified by flash chromatography (2.5% ethyl acetate in n-hexane) to yield 3.7 g (Yield=52.0%).

2. Preparation of 4-(2-fluoro-phenyl)-6-iodo-pyrimidine (4) (TRM/AP/006/064)

A solution of 4-chloro-6-(2-fluoro-phenyl)-pyrimidine (7.0 g, 33.6 mmol) in 350 mL acetone was charged with sodium iodide (25.9 g, 172.8 mmol, 5.1 equiv) and aqueous solution of HI (241.9 g, 1.9 mol, 56.4 equiv) and stirred continually for 15 h. Reaction mixture was then made slightly alkaline (pH˜10) by using 5% NaOH solution. Subject compound was precipitated out, filtered, washed well with distilled water and dried under vacuum to yield 10.0 g of 4 (Yield=99.3%).

1H NMR (DMSO-d6): 9.02 (s, 1H), 8.35 (s, 1H), 8.01-8.07 (m, 1H), 7.60-7.65 (m, 1H), 7.38-7.44 (m, 2H)

HPLC=98.55% Example 5 4-Iodo-6-(4-fluorophenyl)pyrimidine (ACT-MIF-004)

The compound was prepared according to EXAMPLE 1 using 4-fluorophenylboronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ8.90 (s, 1H), 8.61 (s, 1H), 8.30 (m, 2H), 7.38 (m, 2H).

Example 6 4-Iodo-6-(furan-3-yl)pyrimidine (ACT-MIF-005)

The compound was prepared according to EXAMPLE 1 using furan-3-boronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ8.79 (s, 1H), 8.60 (s, 1H), 8.39 (s, 1H), 7.85 (s, 1H), 7.15 (s, 1H).

Example 7 4-Iodo-6-(pyridin-3-yl)pyrimidine (ACT-MIF-006)

The compound was prepared according to EXAMPLE 1 using pyridine-3-boronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ9.40 (s, 1H), 8.96 (s, 1H), 8.72 (m, 2H), 8.53 (m, 1H), 7.52 (m, 1H).

Example 8 4-Iodo-6-(3-fluorophenyl)pyrimidine (ACT-MIF-008)

The compound was prepared according to EXAMPLE 1 using 3-fluorophenylboronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ8.95 (s, 1H), 8.70 (s, 1H), 8.10 (m, 2H), 7.65 (m, 1H), 7.45 (m, 1H).

Example 9 4-Iodo-6-(4-tert-butyloxymethylphenyl)pyrimidine (ACT-MIF-010)

The compound was prepared according to EXAMPLE 1 using 4-tert-butyloxymethylphenylboronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (CDCl3): 6 8.8 (s, 1H), 8.10 (s, 1H), 7.98 (m, 2H), 7.42 (m, 2H), 4.71 (s, 2H), 1.50 (s, 9H).

Example 10 4-Iodo-6-(2-fluoropyridin-3-yl)pyrimidine (ACT-MIF-011)

The compound was prepared according to EXAMPLE 1 using 2-fluoropyridine-3-boronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ8.94 (s, 1H), 8.70 (m, 1H), 8.40 (s, 1H), 7.69 (s, 1H), 7.42 (m, 1H).

Example 11 4-Iodo-6-(furan-2-yl)pyrimidine (ACT-MIF-012)

The compound was prepared according to EXAMPLE 1 using furan-2-boronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ8.80 (s, 1H), 8.22 (s, 1H), 8.01 (s, 1H), 7.50 (s, 1H), 6.79 (s, 1H).

Example 12 4-Iodo-6-(4-fluoropyrimidin-3-yl)pyrimidine (ACT-MIF-013)

The compound was prepared according to EXAMPLE 1 using 2-fluoropyridine-5-boronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (CDCl3): δ8.71 (s, 1H), 8.39 (s, 1H), 8.25 (s, 1H), 8.15 (m, 1H), 6.50 (m, 1H).

Example 13 4-Iodo-6-(3-fluoro-4-methoxyphenyl)pyrimidine (ACT-MIF-014)

The compound was prepared according to EXAMPLE 1 using 3-fluoro-4-methoxyphenylboronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (CDCl3): δ8.89 (s, 1H), 8.60 (s, 1H), 8.12 (m, 2H), 7.31 (m, 1H), 3.92 (s, 3H).

Example 14 4-Iodo-6-(2-chloropyridin-5-yl)pyrimidine (ACT-MIF-015)

The compound was prepared according to EXAMPLE 1 using 2-chloropyridine-5-boronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ9.20 (m, 1H), 9.0 (s, 1H), 8.70 (s, 1H), 8.60 (m, 1H), 7.72 (m, 1H).

Example 15 4-Iodo-6-(2-hydroxyphenyl)pyrimidine (ACT-MIF-016)

The compound was prepared according to EXAMPLE 1 using 2-trifluoromethoxyphenylboronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ12.70 (s, 1H), 8.31 (s, 1H), 7.80 (m, 1H), 7.55 (m, 3H), 6.61 (s, 1H).

Example 16 4-Iodo-6-(2,4-difluorophenyl)pyrimidine (ACT-MIF-017)

The compound was prepared according to EXAMPLE 1 using 2,4-difluorophenylboronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ9.01 (s, 1H), 8.39 (s, 1H), 7.80 (m, 1H), 7.41 (m, 2H).

Example 17 4-Iodo-6-(2-fluoro-6-methoxyphenyl)pyrimidine (ACT-MIF-018)

The compound was prepared according to EXAMPLE 1 using 2-fluoro-6-methoxyphenylboronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ8.89 (s, 1H), 8.15 (s, 1H), 7.49 (m, 1H), 7.0 (m, 2H).

Example 18 4-Iodo-6-(2-chlorophenyl)pyrimidine (ACT-MIF-019)

The compound was prepared according to EXAMPLE 1 using 2-chlorophenylboronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ9.0 (s, 1H), 8.31 (s, 1H), 7.67 (m, 2H), 7.57 (m, 2H). Example 19 4-Iodo-6-(3-acetylaminophenyl)pyrimidine (ACT-MIF-021)

The compound was prepared according to EXAMPLE 1 using 3-acetylaminophenylboronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ10.15 (s, 1H), 9.10 (s, 1H), 8.50 (s, 1H), 8.25 (s, 1H), 7.90 (m, 2H), 7.55 (m, 1H), 2.10 (s, 3H).

Example 20 4-Iodo-6-(thiophen-3-yl)pyrimidine (ACT-MIF-022)

The compound was prepared according to EXAMPLE 1 using thiophene-3-boronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ8.88 (s, 1H), 8.58 (s, 1H), 8.50 (s, 1H), 7.88 (m, 1H), 7.71 (m, 1H).

Example 21 4-Iodo-6-(3-hydroxymethylphenyl)pyrimidine (ACT-MIF-023)

The compound was prepared according to EXAMPLE 1 using 3-tert-butyloxymethylphenylboronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ8.92 (s, 1H), 8.58 (s, 1H), 8.22 (m, 1H), 8.19 (m, 1H), 7.50 (m, 2H), 4.60 (s, 2H).

Example 22 4-Iodo-6-(isoquinolin-4-yl)pyrimidine (ACT-MIF-025)

The compound was prepared according to EXAMPLE 1 using isoquinoline-4-boronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ9.69 (s, 1H), 9.30 (m, 1H), 9.05 (s, 1H), 8.90 (s, 1H), 8.15 (m, 2H), 7.90 (m, 1H), 7.70 (m, 1H).

Example 23 4-Iodo-6-(2,4,5-trifluorophenyl)pyrimidine (ACT-MIF-027)

The compound was prepared according to EXAMPLE 1 using 2,4,5-trifluorophenylboronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ9.0 (s, 1H), 8.32 (s, 1H), 8.12 (m, 1H), 7.81 (m, 1H).

Example 24 4-Iodo-6-(2,4-difluoropyridin-3-yl)pyrimidine (ACT-MIF-028)

The compound was prepared according to EXAMPLE 1 using 2,6-difluoropyridine-3-boronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ9.05 (s, 1H), 8.75 (m, 1H), 8.36 (s, 1H), 7.40 (m, 1H).

Example 25 4-Iodo-6-(4-methoxypyridin-3-yl)pyrimidine (ACT-MIF-029)

The compound was prepared according to EXAMPLE 1 using 2-methoxypyridine-5-boronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ9.10 (d, 1H), 8.90 (s, 1H), 8.61 (s, 1H), 8.45 (m, 1H), 7.0 (m, 1H), 3.92(s, 3H).

Example 26 4-Iodo-6-(thiophen-2-yl)pyrimidine (ACT-MIF-030)

The compound was prepared according to EXAMPLE 1. Specifically, the following method was employed:

1. Preparation of 4-chloro-6-thiophen-2-yl-pyrimidine (3)

4,6-dichloropyrimidine (22.2 g, 149.1 mmol), thiophene-2-boronic acid (20.0 g, 156.3 mmol, 1.05 equiv), Na2CO3 (28.8 g, 271.4 mmol, 1.8 equiv) and Pd(PPh3)2Cl2 (2.9 g, 4.2 mmol, 0.03 equiv) were refluxed in dimethoxyethane-water (727:238 mL) mixed solvent system for 16 h. Reaction was monitored by TLC (using ethyl acetate:n- hexane, 1:9). Reaction mixture was cooled and the subject compound was extracted using dichloromethane. Subject compound was purified by flash chromatography (5% ethyl acetate:n-hexane) to yield 18.4 g of 3 (Yield=62.8%).

1H NMR (CDCl3): 8.90 (d, J=0.9 Hz 1H), 7.79-7.80 (dd, J=3.9, 1.2 Hz, 1H), 7.58-7.60 (m, 2H), 7.18-7.20 (m, 1H).

2. Preparation of 4-iodo-6-thiophen-2-yl-pyrimidine (4)

Aqueous solution of HI (63.5 g, 496.5 mol, 13.9 equiv) was charged to 4-chloro-6-thiophen-2-yl-pyrimidine (3, 7.0 g, 35.6 mmol) and stirring was continued for 20 h. Reaction mixture was then made slightly alkaline (pH˜10) by using 5% NaOH solution. Subject compound was precipitated out, filtered, washed well with distilled water and dried under vacuum to yield 9.6 g of 4 (Yield=94.1%).

HPLC=93.1%

To convert the unreacted chloro-, the product was again treated with HI (6.1 g, 47.7 mmol, 13.9 equiv) by following the same procedure as mentioned above to get 10.0 g of 4 (Yield=98.0%).

1H NMR (CDCl3): 8.76 (s, 1H), 8.02 (s, 1H), 7.76 (d, J=3.9 Hz, 1H), 7.58 (d, J=4.8 Hz, 1H), 7.16-7.19 (m, 2H).

HPLC=99.12% Example 27 4-Iodo-6-(3,4-difluorophenyl)pyrimidine (ACT-MIF-032)

The compound was prepared according to EXAMPLE 1 using 3,4-difluorophenylboronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ8.94 (s, 1H), 8.69 (s, 1H), 8.31 (m, 1H), 8.13 (m, 1H), 7.68 (m, 1H).

Example 28 4-Iodo-6-(4-ethoxyphenyl)pyrimidine (ACT-MIF-033)

The compound was prepared according to EXAMPLE 1 using 3-fluoro-4-ethoxyphenylboronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ8.89 (s, 1H), 8.60 (s, 1H), 8.05 (m, 1H), 7.21 (m, 2H), 4.20 (m, 2H), 1.32 (m, 3H).

Example 29 4-Iodo-6-(4-aminocarbonylphenyl)pyrimidine (ACT-MIF-034)

The compound was prepared according to EXAMPLE 1 using 4-aminocarbamoylphenylboronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ8.99 (s, 1H), 8.7 (s, 1H), 8.30 (m, 2H), 8.12 (s, 1H), 8.0 (m, 2H), 7.51 (s, 1H).

Example 30 4-Iodo-6-(3-aminocarbonylphenyl)pyrimidine (ACT-MIF-035)

The compound was prepared according to EXAMPLE 1 using 3-aminocarbamoylphenylboronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ8.95 (s, 1H), 8.65 (m, 2H), 8.40 (m, 1H), 8.19 (s, 1H), 8.08 (m, 1H), 7.62 (m, 2H).

Example 31 4-Iodo-6-(quinolin-4-yl)pyrimidine (ACT-MIF-036)

The compound was prepared according to EXAMPLE 1 using quinoline-4-boronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ9.11 (s, 1H), 9.0 (s, 1H), 8.47 (s, 1H), 8.12 (m, 2H), 7.81 (m, 1H), 7.7 (s, 1H), 7.61 (m, 1H).

Example 32 4-Iodo-6-(quinolin-8yl)pyrimidine

The compound was prepared according to EXAMPLE 1 using quinolin-8-boronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ8.80 (s, 1H), 8.49 (m, 1H), 8.30 (s, 1H), 7.98 (m, 1H), 7.88 (s, 1H), 7.62 (m, 2H), 7.52 (m, 1H).

Example 33 4-Iodo-6-(quinolin-3-yl)pyrimidine

The compound was prepared according to EXAMPLE 1 using quinolin-3-boronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ9.65 (s, 1H), 9.23 (s, 1H), 9.01 (s, 1H), 8.87 (s, 1H), 8.01 (m, 2H),7.90 (m, 1H), 7.66 (m, 1H).

Example 34 4-Iodo-6-(isoquinolin-5-yl)pyrimidine

The compound was prepared according to EXAMPLE 1 using isoquinolin-5-boronic acid and 4,6-dichloropyrimidine. The resultant chloro compound was converted to iodo with hydroiodic acid as described in the general procedure.

1H NMR (DMSO-d6): δ9.41 (s, 1H), 9.09 (s, 1H), 8.51 (m, 1H), 8.42 (s, 1H), 8.30 (m, 1H), 8.10 (m, 2H), 7.80 (m, 1H).

Example 35 Methods for the Preparation of 2-alkylthio Derivatives

The compounds of Examples 36-39 are prepared using the method of EXAMPLE 35.

Example 36 2-Methylthio-4-iodo-6-phenylpyrimidine

The compound was prepared according to EXAMPLE 35 using methyl iodide as one of the reactants.

1H NMR (CDCl3): δ8.03-8.06 (m, 2H), 7.82 (s, 1H), 7.49-7.54 (m, 3H), 2.62 (s, 3H).

Example 37 2-Ethylthio-4-iodo-6-phenylpyrimidine

The compound was prepared according to EXAMPLE 35 using ethyl iodide as one of the reactants.

1H NMR (CDCl3): δ7.95-7.96 (m, 2H), 7.74 (s, 1H), 7.39-7.48 (m, 3H), 3.14 (q, J=7.2 Hz, 2H), 1.38 (t, J=7.2 Hz, 3H).

Example 38 2-Isopropylthio-4-iodo-6-phenylpyrimidine

The compound was prepared according to EXAMPLE 35 using isopropyl iodide as one of the reactants.

1H NMR (DMSO-d6): δ8.25 (s, 1H), 8.17-8.20 (m, 2H), 7.51-7.59(m, 3H), 3.89-3.99 (h, J=6.9 Hz, 1H), 1.42 (d, J=6.9 Hz, 6H).

Example 39 2-n-Butylthio-4-iodo-6-phenylpyrimidine

The compound was prepared according to Scheme-2 using n-butyl iodide as one of the reactant.

1H NMR (DMSO-d6): δ8.26 (s, 1H), 8.18-8.20 (m, 2H), 7.54-7.59(m, 3H), 3.18 (t, J=7.2 Hz, 2H), 1.65-1.73 (m, J=7.2 Hz, 2H), 1.41-1.49 (m, J=7.2 Hz, 2H), 0.93 (t, J=7.2 Hz, 3H).

Example 40 Methods for the Preparation of 2-alkylamino Derivatives

The compounds of Examples 41-45 were prepared according to Scheme 3 of EXAMPLE 40.

Example 41 2-Methylamino-4-iodo-6-phenylpyrimidine

The compound was prepared according to EXAMPLE 40 using methylamine as RNH2.

1H NMR (CDCl3): δ7.98-8.00 (br s, 2H), 7.43-7.49 (m, 3H), 7.40 (s, 1H), 5.24 (br s, 1H), 3.06 (d, J=3.0 Hz).

Example 42 2-Ethylamino-4-iodo-6-phenylpyrimidine

The compound was prepared according to EXAMPLE 40 using ethylamine as RNH2.

1H NMR (CDCl3): δ7.97-7.99 (m, 2H), 7.44-7.48 (m, 3H), 7.39 (s, 1H), 5.20 (br s, 1H), 3.48-3.57 (m, J=7.2 Hz, 1.2Hz, 2H), 1.28(t, J=7.2Hz, 3H).

Example 43 2-Propylamino-4-iodo-6-phenylpyrimidine

The compound was prepared according to EXAMPLE 40 using propylamine as RNH2.

1H NMR (CDCl3): δ7.92 (br s, 2H), 7.35-7.44 (m, J=6.6 Hz, 3H), 7.31 (s, 1H), 5.21 (br s, 1H), 3.38 (q, J=6.9 Hz, 2H), 1.53-1.65(m, J=6.9, 7.3Hz, 2H), 0.93 (t, J=7.3 Hz, 3H).

Example 44 2-Isopropylamino-4-iodo-6-phenylpyrimidine

The compound was prepared according to EXAMPLE 40 using isopropylamine as RNH2.

1H NMR (CDCl3): δ8.04-8.07 (m, 2H), 7.54-7.56 (m, 3H), 7.41 (s, 1H), 6.98 (br s, 1H), 4.29-4.36 (m, J=6.9, 3.3 Hz, 1H), 1.34 (d, J=6.9, 6H).

Example 45 2-n-Butylamino-4-iodo-6-phenylpyrimidine

The compound was prepared according to EXAMPLE 40 using n-butylamine as RNH2.

1H NMR (CDCl3): δ7.97 (br s, 2H), 7.45-7.48 (m, 3H), 7.38 (s, 1H), 5.30 (br s, 1H), 1.57-1.64 (m, J=6.0 Hz, 2H), 1.40-1.47(h, J=6.0, 2H), 0.96 (t, J=6.0 Hz, 3H).

The compounds of Examples 46-56 are also prepared according the Scheme 1.

Example 46 4-Iodo-6-(benzothiophen-2-yl)pyrimidine

Example 47 4-Iodo-6-(benzofuran-2-yl)pyrimidine

Example 48 4-Iodo-6-(4-hydroxybenzothiophen-2-yl)pyrimidine

Example 49 4-Iodo-6-(4-acetylaminobenzothiophen-2-yl)pyrimidine

Example 50 4-Iodo-6-(4-aminocarbonylbenzothiophen-2-yl)pyrimidine

Example 51 4-Iodo-6-(5-acetylaminopyridin-3-yl)pyrimidine

Example 52 4-Iodo-6-(5-aminocarbonylpyridin-3-yl)pyrimidine

Example 53 4-Iodo-6-(4-fluoropyridin-3-yl)pyrimidine

Example 54 4-Iodo-6-(4-acetylaminothiophen-2-yl)pyrimidine

Example 55 4-Iodo-6-(4-aminocarbonylthiophen-2-yl)pyrimidine

Example 56 4-Iodo-6-(4-methoxythiophen-2-yl)pyrimidine

Example 57 Solubility and Stability

Solubility of exemplary compounds in varying solvents is shown in Table 2. The stability of the compounds in solution was examined by HPLC concomitantly. Results indicated no degradation after 2 months stored at room temperature.

TABLE 2 Solubilities of the Compounds of the Invention at 20-22° C. (mg/ml) MIF MIF MIF MIF MIF MIF 001 002 003 006 035 038 Ethanol 85.0 1.4 4.7 DMSO 216 — 150 Propane-diol 25 2.7 PEG-300 66 13.3 19.7 Corn oil 15 <6 <6 Ethanol/Tween 20 10 10 80 Cremophor 15 14.5

Example 58 Cell Permeability and Transport

Cell permeability and transport mechanisms in Caco-2 and MDR1-MDCK monolayers experiments were performed in triplicate in the apical-to-basolateral and basolateral-to-apical direction using TRANSWELL® wells containing either Caco-2 or MDR1-MDCK monolayers. A modified Hanks buffer pH 7.4 was used in both reservoir and receiver wells with the addition of 1% BSA in the receiver side. Confluent monolayers were used and their integrity was verified using reference compounds (Atenolol as a low permeability reference compound and Propanolol as a high permeability reference compound). A sample in the basolateral and apical sides was taken after 2 hours and the concentration measured by LC/MS-MS. Results are summarized in Table 3. The results also suggest that the compounds are not P-gp substrates and may cross the blood brain barrier.

TABLE 3A Cell Permeability and Transport R(Caco-2) Caco-2 Permeability Papp (104 cm/s) Permeability A-B B-A Efflux Class ACT-MIF-001 <0.1 <0.1 — Low ACT-MIF-002 0.2 04 1.7 Low ACT-MIF-003 2.1 2.6 1.2 High ACT-MIF-006 5.4 7.8 1.4 High ACT-MIF-011 12.6 7.8 0.6 High ACT-MIF-025 0.4 0.6 1.5 Low ACT-MIF-029 3.1 3.1 1.0 High ACT-MIF-033 2.7 3.0 1.1 High ACT-MIF-035 3.1 3.3 1.1 High ACT-MIF-038 0.9 06 0.7 High

TABLE 3B Cell Permeability and Transport Results (MDR1-MDCK) MDR1-MDCK Permeability Papp (104 cm/s) P-gp A-B B-A Substrate Efflux Brain (1) ACT-MIF-001 2.3 1.9 No Low Low ACT-MIF-001 + 3.3 3.1 CSA ACT-MIF-002 1.0 0.7 No High Low ACT-MIF-002 + 1.9 2.0 CSA ACT-MIF-003 3.3 3.3 No High High ACT-MIF-003 + 5.2 4.7 CSA (1) Brain penetration classification (2)

Example 59 Microsomal Stability

Stability in human liver microsomes was tested over 24 hours at 37° C. using pooled mixed gender human liver microsomes. Liver microsomes were prepared at 1.0 mg/ml of microsomal protein in a 100 mM potassium phosphate pH 7.4 buffer with 1 mM NADPH. The media was incubated at 37° C. with the compound in solution in DMSO. The concentration of the compound was followed by LC/MS-MS as a function of time. Samples were assayed at t=0, 30, 60 and 120 minutes. Testosterone was used as a positive control. The same experiment was performed with mouse liver microsomes instead of human liver microsomes. Results are summarized in Table 4.

TABLE 4 Metabolic Stability Determined from Stability in Human Microsomes Metabolic Stability in Human Microsomes % Remaining 0 min 15 min 60 min ACT-MIF-001 100 <2 ACT-MIF-002 100 <2 ACT-MIF-003 100 57 ACT-MIF-006 100 82 ACT-MIF-017 100 0 ACT-MIF-021 100 73 ACT-MIF-029 100 6.4 ACT-MIF-033 100 82 ACT-MIF-035 100 92 ACT-MIF-038 100 47 Testosterone 100 56

Example 60 Plasma Protein Binding

Plasma protein binding was ascertained using dialysis equilibrium methods known in the art. Results are summarized in Table 5. Warfarin was used a high protein binding control.

TABLE 5 Human Plasma Protein Binding % Bound Compound Warfarin ACT-MIF-001 98.3 99.0 ACT-MIF-002 97.9 98.9 ACT-MIF-003 96.2 98.9

Example 61 MIF Liver Lysates Enzymatic Activity

This experiment, using an ex-vivo approach and the tautomeric reaction of L-dopachrom, was designed to ascertain the level of inhibition of MIF following administration of the compounds of this invention via oral, IV, IP or any other route of administration. Mice were used in the example illustrated below, but other animals could be used as well. Groups of 3 mice were administered IP 1 mg of 4-IPP and ACT-002 resuspended in 100 μl of corn oil every day for 3 days. Mice were sacrificed 6 hours after the last injection and livers were harvested. ˜1 gram pieces of liver were lysed in PBS containing 1 mM NaVO4, 2 mM NaF and a protease inhibitor cocktail (Roche Biochemical, Indianapolis, Ind.) using dounce-homogenization on ice. 500 μg of liver lysates were added to a final volume of 700 μl PBS in plastic cuvettes. 4 mM L-3, 4-dihydroxyphenylalanine methyl ester and 8 mM sodium periodate (Sigma-Aldrich) were combined in a 3:2 ratio to form L-dopachrome methyl ester. 300 μL of L-dopachrome methyl ester was then immediately added to the cuvettes; the OD475 nm was measured 2 min and 4 min after addition of the L-dopachrome. As shown in FIG. 1 (DMSO was used as a negative control—no inhibition of MIF), there is a significant in vivo inhibition of MIF indicating that the compound interacts with the MIF binding pocket.

Example 62 MIF Tumor Lysate Enzymatic Activity

The ex vivo MIF enzymatic activity of tumor extracts/lysates following in vivo dosing can be estimated in a manner similar to the method of EXAMPLE 61. Tumor bearing mice were administered 1 mg/kg daily for 3 days. 6 hours following the last dose, animals were sacrificed and tumors were resected and processed as described in EXAMPLE 61. Inhibition was also ascertained as in EXAMPLE 61. Results, shown in FIG. 2, demonstrate significant inhibition of MIF in tumor lysates.

Example 63 Inhibition of Tumor Cells Proliferation

Inhibition of the proliferation of tumor cells was investigated in vitro in several tumor cell lines. Cells of the desired tumor cell line were plated at 2×105 cells/ml in 96 well plates. Twice the indicated concentrations of the compounds of the invention were added to cells the following day in an equal volume of media. 72 hours later, cells were lysed and subjected to ATP determination using the CellTiter Glo-Luminescent Cell Viability Assay kit (Promega, Madison, Wis.). Experiments were done in triplicate. Results for the inhibition of cells proliferation are reported as IC50 (the concentration leading to a 50% inhibition of proliferation of the cell population) and are listed in Table 6. FIGS. 3 and 4 show bar graphs comparing the IC50s of specific embodiments of compounds of the invention across multiple tumor cell lines.

TABLE 6 IC50s for Compounds in Selected Tumor Cell Lines IC50 (microM) Du 145 ACT-MIF-001 <10 ACT-MIF-002 24.9 ACT-MIF-003 16.5 ACT-MIF-006 36.7 ACT-MIF-017 <10 ACT-MIF-022 <40 ACT-MIF-029 <20 ACT-MIF-033 <5 ACT-MIF-034 <100 ACT-MIF-035 21.7 ACT-MIF-038 9.2

Example 64 p53 Up Regulation

The up regulation of p53 was determined using a commercially available p53 luciferase assay kit. 1×105 cells/ml were plated in a 24 well plate and allowed to adhere overnight. MIF antagonists were added to the cells at the indicated concentrations for 16 hours and transiently co-transfected with 0.125 μg/well of p53-responsive luciferase promoter plasmid (Promega, Madison, Wis.) together with 0.0125 μg/well Renilla pRL-null plasmid (Promega) using Lipofectamine (Invitrogen) transfection reagent. After 24 hrs, Firefly and Renilla luciferase activities were measured by the Dual Luciferase Reporter Assay System (Promega, Madison, Wis.) on a TD-20/20 luminometer (Turner Designs). Results represented in FIG. 5 indicate the compounds of the invention are implicated in p53 regulation.

Example 65 MIF Cell Lysate Enzymatic Inhibition

Normal or transformed cell lysates can be used to determine the concentration inhibiting the enzymatic activity of MIF present in cell lysates. Cells are cultured in the appropriate media to the required number of cells, collected, and lysed. Compounds to be characterized are solubilized in DMSO and serial dilutions are performed in order to obtain a range of concentrations including complete and no quantifiable inhibition. Results, reported as IC50 (concentration leading to an inhibition of 50% of the MIF enzymatic activity), are summarized in Table 7.

TABLE 7 IC50 Values for MIF Cell Lysate Enzymatic Activity Inhibition IC50 (nM) 4-IPP >2000 ACT-MIF-001 37 ACT-MIF-002 70 ACT-MIF-003 200 ACT-MIF-006 250 ACT-MIF-017 190 ACT-MIF-021 570 ACT-MIF-029 140 ACT-MIF-033 115 ACT-MIF-034 270 ACT-MIF-035 185 ACT-MIF-036 230 ACT-MIF-037 >1000 ACT-MIF-039 195

Example 66 Inhibition of Cell Migration and Invasion

The LOX-IMV1 tumor cell line was used to determine the inhibition of cell migration using the Oris Cell Migration Assay kit (Promega, Mich.). Briefly, adherent cells were seeded into each well of the kit according to kit instructions. Concentrations of cells in the migration zone were determined to calculate IC50 values. Prior to the migration assay, cell proliferation IC50s were determined to differentiate between inhibition of proliferation and migration. Results are shown in FIGS. 6 and 7. Results show a significant inhibition of migration even at very low concentration (0.03 μM). A slightly modified method was also used to determine the inhibition of invasion. As shown in FIG. 7, invasion was also inhibited.

Example 67 Determination of the Anti-angiogenic Properties in the Chick Chorioallantoic Membrane (CAM) assay

8 groups with 10 embryos in each group were used in the experiment described below. Fresh fertile eggs were incubated for 3 days in a standard egg incubator at 37° C. for 3 days. On Day 3, eggs were cracked under sterile conditions and embryos were placed into 20×100 mm plastic plates and cultivated at 37° C. in an embryo incubator with a water reservoir on the bottom shelf. Air was continuously bubbled into the water reservoir using a small pump so that the humidity in the incubator is kept constant. On Day 6, a sterile silicon “o” ring was placed on each CAM and test compound dissolved in 0.5% methylcellulose was placed into each “o” ring in a sterile hood. Paclitaxel was used as a positive control. Embryos were returned to the incubator after addition of test material. Control embryos received 10 μL of vehicle alone. On Day 8, embryos were removed from the incubator and kept at room temperature while blood vessel density were determined under each “o” ring using an image capturing system at a magnification of 160×. The blood vessel density was measured using an angiogenesis scoring system in that arithmetic numbers 0 to 5 (or exponential numbers 1 to 32) are used to indicate number of blood vessels present at the treatment sites on the CAM. Number 5 represents the highest density and 0 represents no angiogenesis. The percent of inhibition at each dosing site was calculated using the score recorded for that site divided by the mean score obtained from the appropriate control samples for each individual experiment. The percent of inhibition for each dose of a given compound was calculated by pooling all results obtained for that dose from 8-10 embryos. Results are summarized in Table 8 below and demonstrate that among others, compounds ACT-MIF-001, ACT-MIF-002, and ACT-MIF-003 have high anti-angiogenic properties.

TABLE 8 Blood Vessel Density Conc per CAM — 6 nM 0.3 nM 3 nM 30 nM Control 14.0 ± 3.2 Paclitaxel 2.8 ± 0.7 ACT-MIF-001 10.5 ± 3.4  4.1 ± 0.8 1.8 ± 0.3 ACT-MIF-002 9.4 ± 2.4 8.6 ± 2.6 4.4 ± 1.3 ACT-MIF-003 11.6 ± 1.2  4.2 ± 1.2 4.1 ± 0.7

Another experiment was performed using a protocol similar to the one described above but using matrigel plugs instead of o ring to deliver the test material to the CAM. Results are summarized in Table 9 below and show a statistically significant inhibition of angiogenesis at the high concentrations of test material.

TABLE 9 Blood Vessel Densities Blood Vessel Counts Conc per CAM — 2 nM 0.3 nM 3 nM 30 nM Control 39.3 ± 1.3 Paclitaxel 15.5 ± 2.1 ACT-MIF-006 38.8 ± 3.5 35.8 ± 5.4 33.5 ± 1.7 ACT-MIF-030 35.6 ± 1.0 32.7 ± 3.2 28.1 ± 2.0 ACT-MIF-035 28.3 ± 1.7 27.3 ± 2.4 26.7 ± 1.9 ACT-MIF-038 31.8 ± 5.8 33.6 ± 1.7 31.0 ± 4.1

Example 68 Pharmacokinetics Parameters

The pharmacokinetic parameters of several compounds were investigated in rodents. Both oral and iv administration were investigated in rats. Blood samples were collected over time; plasma was analyzed using an LC/MS-MS method. Pharmacokinetic parameters were calculated using Win-NonLin. Terminal plasma half-lives were 7.10 hr for ACT-MIF-001, 1.66 hr for ACT-MIF-002, and 1.50 hr for ACT-MIF-003. After i.v. administration, the clearance values were 45753 mL/hr/kg for ACT-MIF-001, 7911 mL/hr/kg for ACT-MIF-002, and 11827 mL/hr/kg for ACT-MIF-003. The volume of distribution values were 72666 mL/kg for ACT-MIF-001, 2118 mL/kg for ACT-MIF-002, and 1926 mL/kg for ACT-MIF-003.

Example 69 In Vivo Efficacy in Xenograft Tumor Models

Athymic nude mice at 7-8 weeks of age were used for the study. Mice were housed in microisolator housing, with food and water provided as libitum, and quarantined for 4 days prior to the initiation of the study. DU145 cells were maintained in McCoy's 5A medium supplemented with 10% fetal bovine serum and 2 mM glutamine. Cells at 80% confluence were harvested using 0.25% trypsin/EDTA solution, washed once with PBS and resuspended in a mixture of serum-free medium/Matrigel (1:1 by volume) at a density of 3×106 cells/100 μl. 4 groups of 10 mice each were used in the experiment. DU145 cells suspended in 100 μl of a mixture of medium/Matrigel (1:1) were subcutaneously implanted in the right flank region. Animals were monitored for tumor growth daily after cell implantation. When tumor volumes reached 80-100 mm3, mice were randomized into 4 groups of 10 mice each using only mice having tumor volumes closest to the mean value. Tumor volumes were measured using the formula V=L×W×H×π/6, where L and W represent the longer and shorter diameters of the tumor and H represents the height of the tumor. Treatment began the day after randomization. Act-MIF-001, ACT-MIF-002, and ACT-MIF-003 were administered daily by IP injection at a dose of 40 mg/kg for 4 weeks. Throughout the entire study, tumor volumes were measured twice weekly and body weights once weekly. Animals were observed for possible toxic effect from the drug treatment. Results illustrated below in FIG. 8 demonstrated that ACT-MIF-003 significantly inhibited tumor growth.

Example 70 Determination of the Microvessel Density in Xenograft Tumors

At the end of the experiment described in EXAMPLE 69 above, tumors in each group were removed and sliced. Blood vessel density of the tumor tissues was measured by immunohistochemistry. Results indicated a decrease in microvessel density with respect to the tumors of the control group with a statistically meaningful difference for the ACT-MIF-003 treated group. These in vivo results confirmed that the compounds described in this application inhibit angiogenesis. Representative pictures of the stained tissues are showed in FIG. 9.

Example 71 Efficacy Study in a Pancreatic Tumor Model

The activity of the compounds of the invention was investigated in a pancreatic tumor model using an experiment similar to the one described in EXAMPLE 69. Compounds ACT-MIF-002 and ACT-MIF-003 were dosed daily at 40 mg/kg via IP administration. Results shown in FIG. 10 indicated that the compounds of the invention tested in this experiment had a significant impact on survival and that limited the metastatic tumor burden as shown in the survival graph and representative histopathologic slides (FIG. 11) of the lumbar region of control and treated animals. In addition, animal weights were monitored throughout the study; there was no body weight loss and no clinical signs of toxicity indicating that these compounds are very well tolerated.

Lumbar regions of the control and treated groups were excised and sent for histopathological evaluation. As shown in FIG. 11, there were significant differences between control and treated groups as there was no evidence of bone metastases in the ACT-MIF-002 treated group. In the example shown in FIG. 11, bone marrow of the vehicle treated mice is consistent with bone metastases (1) with evidence of surrounding skeletal muscle metastases from invading marrow tumor cells (2 and 3). No evidence of bone metastases was observed with spinal column sections from ACT-MIF-002 treated mice.

Example 72 Oral Bioavailability

The compounds were administered orally (PO) and intraperitoneally (IP) to healthy animals. The inhibition of the MIF liver enzymatic activity determined ex vivo following IP and PO dosing is similar, indicating high oral bioavailability. Furthermore, brain and lung tissues were collected and processed to determine MIF enzymatic activity in these organs. Results also shown in FIG. 12 are indicative of an excellent tissue distribution and demonstrate significant MIF inhibition in both the brain and lungs. As shown in FIG. 12, MIF-002, is orally bioavailable. Inhibition of MIF enzyme was determined in vitro following dosing of MIF-002 at 40 mg/kg once a day for three days, both IP and PO (normal C57BL6 mice, n=3). Tissues were collected at sacrifice and processed. Liver, lung, and brain tissues were collected, processed, and used for the determination of MIF enzyme activity. Values are expressed as a percentage calculated using DMSO as control (no inhibition).

Two additional compounds were tested, MIF-035 and MIF-041. Results (data not shown) indicated that these compounds were also orally bioavailable, crossed the blood brain barrier, and inhibited MIF enzymatic activity very efficiently in all three organs with results varying ˜12% inhibition in liver extracts to ˜76.2% inhibition in the lungs.

Results indicate compounds of the invention are orally bioavailable, cross the brain blood barrier, and inhibit MIF enzymatic activity in both the brain and the lungs.

Example 73 4-IPP and ACT-003 Inhibit T lymphocyte Activation

In order to assess the ability of MIF antagonists to disrupt autoimmune-associated T cell activation, primary human T lymphocytes were prepared using standard Ficoll-gradient preparations. 1×106 lymphocytes/ml were resuspended in RPMI/10% FCS and plated onto anti-CD3 antibodies previously immobilized onto tissue culture plates. Control, vehicle control (0.1% DMSO), 25 μM 4-IPP or 25 μM ACT-003 were added to cells and allowed to incubate for 48 hours. Cells were lifted, washed and stained with anti-CD4 or anti-CD8 antibodies and then analyzed by flow cytometry. As shown in FIGS. 13 and 14, cells treated with MIF antagonists 4-IPP and ACT-003 during anti-CD3 lymphocyte activation had significantly fewer CD4 and CD8 T lymphocytes suggesting defective anti-CD3 induced activation/proliferation in MIF inhibitor treated lymphocytes.

To validate the effects of MIF antagonists on T lymphocyte activation, experiments were set up exactly as described above and, 48 hours later, treated and untreated lymphocytes were stained with an anti-CD25 antibody. CD25 is also known as the high affinity IL-2 receptor—a very well characterized and frequently marker of T lymphocyte activation. As shown in FIGS. 15 and 16, 4-IPP and ACT-003 almost completely blocked the anti-CD3-induced CD25 expression suggesting a nearly complete block of T lymphocyte activation.

In order to investigate the relative kinetics of when 4-IPP and ACT-003 are acting in blocking T lymphocyte activation, we repeated the experiment described above but harvested lymphocytes only 16 hours after anti-CD3 plating. At this early time point during T lymphocyte activation, CD69 is found to be expressed and is usually considered to be an “early marker” of lymphocyte activation. As shown in FIGS. 17 and 18, treatment with MIF antagonists had only a marginal effect on CD69 expression suggesting that MIF inhibitors are acting at a relatively late stage in the activation process. This is important because it suggests that therapeutic use of 4-IPP-based MIF inhibitors in autoimmune diseases can be used at later stages and aren't likely to be required to be delivered in the early stages of disease onset.

Finally, to confirm that proliferation of CD4+ and CD8+ T lymphocytes is blocked by 4-IPP-based MIF antagonists, we repeated the experiment as described above, added labeled-BrdU to cells, stained with either labeled anti-CD4 or anti-CD8 antibodies and then assessed relative CD4/CD8 and BrdU labeling in each treatment group. As shown in FIGS. 19 and 20, 4-IPP and ACT-003 almost completely blocked BrdU labeling in both CD8+ and CD4+ T lymphocytes.

Combined, these results suggest that targeting MIF using these 4-IPP-based small molecules may have profound inhibitory effects on T lymphocyte-dependent autoimmune disorders.

All documents cited are incorporated herein by reference; the citation of any document is not to be construed as an admission that it is prior art with respect to the present invention.

While particular embodiments of the present invention have been illustrated and described, it would be obvious to one skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims

1. A method for treating a macrophage migration inhibitory factor (MIF)-implicated disease or condition, comprising administering to a patient in need thereof a safe and effective amount of a migration inhibitory factor (MIF) inhibitory compound or its enantiomeric or diastereomeric form or a pharmaceutically acceptable salt, prodrug, or metabolite thereof, said compound having the formula:

wherein: A is a substituted or unsubstituted 5, 6, or 7-membered aromatic or nonaromatic ring having 0 or 1 to 4 heteroatoms selected from the group consisting of N, O, S, and combinations thereof; B is H, OH, OR, SR, NH2, NHR, or alkyl or substituted alkyl, wherein R is H, alkyl, or substituted alkyl of 2 to 20 carbon atoms; X and Y are independently N or CH, wherein at least one of X and Y is N.

2. The method of claim 1, wherein the MIF-implicated disease is selected from the group consisting of inflammatory disease and cancer.

3. The method of claim 2, wherein the inflammatory disease is selected from the group consisting of dermatitis, arthritis, rheumatoid arthritis, insulin-dependent diabetes, proliferative vascular disease, acute respiratory distress syndrome, sepsis, septic shock, psoriasis, asthma, cytokine related toxicity, lupus, multiple sclerosis, transplant-host response, and autoimmune disorders.

4. The method of claim 1, wherein the MIF-implicated condition is caused by a MIF-producing pathogen.

5. The method of claim 4, wherein the MIF-producing pathogen is selected from the group consisting of parasitic helminths, spirochetes, and plasmodium.

6. The method of claim 1, wherein:

A is a substituted or unsubstituted 5- or 6-membered aromatic ring having 0 or 1 heteroatoms selected from the group consisting of N and S;
B is H;
X and Y are both N,
and further wherein the MIF inhibitory compound interacts with a MIF polypeptide present in the patient in need thereof to inhibit an enzymatic activity of the MIF polypeptide.

7. The method of claim 1, wherein the MIF inhibitory compound is selected from the group consisting of:

4-Iodo-6-(2,3-difluoro-4-methoxyphenyl)pyrimidine;
4-Iodo-6-(2-fluorophenyl)pyrimidine;
4-Iodo-6-(pyridin-3-yl)pyrimidine;
4-Iodo-6-(3-fluorophenyl)pyrimidine;
4-Iodo-6-(2-fluoropyridin-3-yl)pyrimidine;
4-Iodo-6-(3-fluoro-4-methoxyphenyl)pyrimidine;
4-Iodo-6-(2,4-difluorophenyl)pyrimidine;
4-Iodo-6-(2-chlorophenyl)pyrimidine;
4-Iodo-6-(2,4,5-trifluorophenyl)pyrimidine;
4-Iodo-6-(2,4-difluoropyridin-3-yl)pyrimidine;
4-Iodo-6-(thiophen-2-yl)pyrimidine; and
4-Iodo-6-(3,4-difluorophenyl)pyrimidine.

8. The method of claim 1, wherein the administering is via oral administration, intravenous administration, intraperitoneal administration, or a combination thereof

9. A method for inhibiting proliferation, cell migration, metastasis, and/or invasion of a tumor cell, and/or angiogenesis associated with the presence of the tumor cell, the method comprising contacting the tumor cell with an effective amount of a MIF inhibitory compound or its enantiomeric or diastereomeric form or a pharmaceutically acceptable salt, prodrug, or metabolite thereof, said compound having the formula:

wherein: A is a substituted or unsubstituted 5, 6, or 7-membered aromatic or nonaromatic ring having 0 or 1 to 4 heteroatoms selected from the group consisting of N, O, S, and combinations thereof; B is H, OH, OR, SR, NH2, NHR, or alkyl or substituted alkyl, wherein R is H, alkyl, or substituted alkyl of 2 to 20 carbon atoms; X and Y are independently N or CH, wherein at least one of X and Y is N,
whereby proliferation, cell migration, metastasis, and/or invasion of the tumor cell and/or angiogenesis associated with the presence of the tumor cell is inhibited.

10. The method of claim 9, wherein:

A is a substituted or unsubstituted 5- or 6-membered aromatic ring having 0 or 1 heteroatoms selected from the group consisting of N and S;
B is H;
X and Y are both N,
and further wherein the MIF inhibitory compound interacts with a MIF polypeptide present in the patient in need thereof to inhibit an enzymatic activity of the MIF polypeptide.

11. The method of claim 9, wherein the MIF inhibitory compound is selected from the group consisting of:

4-Iodo-6-(2,3-difluoro-4-methoxyphenyl)pyrimidine;
4-Iodo-6-(2-fluorophenyl)pyrimidine;
4-Iodo-6-(pyridin-3-yl)pyrimidine;
4-Iodo-6-(3-fluorophenyl)pyrimidine;
4-Iodo-6-(2-fluoropyridin-3-yl)pyrimidine;
4-Iodo-6-(3-fluoro-4-methoxyphenyl)pyrimidine;
4-Iodo-6-(2,4-difluorophenyl)pyrimidine;
4-Iodo-6-(2-chlorophenyl)pyrimidine;
4-Iodo-6-(2,4,5-trifluorophenyl)pyrimidine;
4-Iodo-6-(2,4-difluoropyridin-3-yl)pyrimidine;
4-Iodo-6-(thiophen-2-yl)pyrimidine; and
4-Iodo-6-(3,4-difluorophenyl)pyrimidine.

12. The method of claim 9, wherein the tumor cell is present within a subject and the contacting results from administering the MIF inhibitory compound or its enantiomeric or diastereomeric form, or the pharmaceutically acceptable salt, prodrug, or metabolite thereof to the subject orally, intravenously, intraperitoneally, or a combination thereof.

13. The method of claim 12, wherein the MIF inhibitory compound or its enantiomeric or diastereomeric form, or the pharmaceutically acceptable salt, prodrug, or metabolite thereof is administered as part of a pharmaceutical composition comprising a safe and effective amount of the MIF inhibitory compound and one or more pharmaceutically acceptable excipients.

14. The method of claim 13, wherein the pharmaceutical composition is pharmaceutically acceptable for use in a human.

15. A migration inhibitory factor (MIF) inhibitory compound, or its enantiomeric or diastereomeric form or a pharmaceutically acceptable salt, prodrug, or metabolite thereof, said MIF inhibitory compound having the formula:

wherein: A is a substituted or unsubstituted 5, 6, or 7-membered aromatic or nonaromatic ring having 0 or 1 to 4 heteroatoms selected from the group consisting of N, O, S, and combinations thereof; B is H, OH, OR, SR, NH2, NHR, or alkyl or substituted alkyl, wherein R is H, alkyl, or substituted alkyl of 2 to 20 carbon atoms; X and Y are independently N or CH, wherein at least one of X and Y is N,
and further wherein the MIF inhibitory compound interacts with a MIF polypeptide to inhibit an enzymatic activity of the MIF polypeptide.

16. The MIF inhibitory compound of claim 15, wherein:

A is a substituted or unsubstituted 5- or 6-membered aromatic ring having 0 or 1 heteroatoms selected from the group consisting of N and S;
B is H;
X and Y are both N,
and further wherein the MIF inhibitory compound interacts with a MIF polypeptide present in the patient in need thereof to inhibit an enzymatic activity of the MIF polypeptide.

17. The MIF inhibitory compound of claim 15, wherein the MIF inhibitory compound is selected from the group consisting of:

4-Iodo-6-(2,3 -difluoro-4 -methoxyphenyl)pyrimidine;
4-Iodo-6-(2-fluorophenyl)pyrimidine;
4-Iodo-6-(pyridin-3-yl)pyrimidine;
4-Iodo-6-(3-fluorophenyl)pyrimidine;
4-Iodo-6-(2-fluoropyridin-3-yl)pyrimidine;
4-Iodo-6-(3-fluoro-4-methoxyphenyl)pyrimidine;
4-Iodo-6-(2,4-difluorophenyl)pyrimidine;
4-Iodo-6-(2-chlorophenyl)pyrimidine;
4-Iodo-6-(2,4,5-trifluorophenyl)pyrimidine;
4-Iodo-6-(2,4-difluoropyridin-3-yl)pyrimidine;
4-Iodo-6-(thiophen-2-yl)pyrimidine; and
4-Iodo-6-(3,4-difluorophenyl)pyrimidine.

18. A pharmaceutical composition comprising the MIF inhibitory compound of claim 15, or an enantiomeric or diastereomeric form thereof, or a pharmaceutically acceptable salt, prodrug, or metabolite thereof, and one or more pharmaceutically acceptable excipients.

Patent History
Publication number: 20180009764
Type: Application
Filed: Sep 19, 2017
Publication Date: Jan 11, 2018
Inventors: Robert A. Mitchell (Louisville, KY), John O. Trent (Louisville, KY), Pooran Chand (Birmingham, AL), Gilles Hugues Tapolsky (Lousiville, KY)
Application Number: 15/708,827
Classifications
International Classification: C07D 239/30 (20060101); C07D 401/04 (20060101); C07D 239/38 (20060101); C07D 239/42 (20060101); C07D 409/04 (20060101); C07D 405/04 (20060101);