U.S. patent application number 14/803650 was filed with the patent office on 2015-12-24 for method for treating macrophage migration inhibitory factor (mif)-implicated diseases and conditions with iodo pyrimidine derivatives.
This patent application is currently assigned to ADVANCED CANCER THERAPEUTICS, LLC. The applicant listed for this patent is ADVANCED CANCER THERAPEUTICS, LLC, UNIVERSITY OF LOUISVILLE RESEARCH FOUNDATION, INC.. Invention is credited to Pooran Chand, Robert A. Mitchell, Gilles Hugues Tapolsky, John O. Trent.
Application Number | 20150368207 14/803650 |
Document ID | / |
Family ID | 43796505 |
Filed Date | 2015-12-24 |
United States Patent
Application |
20150368207 |
Kind Code |
A1 |
Mitchell; Robert A. ; et
al. |
December 24, 2015 |
METHOD FOR TREATING MACROPHAGE MIGRATION INHIBITORY FACTOR
(MIF)-IMPLICATED DISEASES AND CONDITIONS WITH IODO PYRIMIDINE
DERIVATIVES
Abstract
Compounds useful for the inhibition of macrophage migration
inhibitory factor (MIF) are provided herein, having the Formula I:
##STR00001## 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, NH.sub.2, 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.
Inventors: |
Mitchell; Robert A.;
(Louisville, KY) ; Trent; John O.; (Louisville,
KY) ; Chand; Pooran; (Birmingham, AL) ;
Tapolsky; Gilles Hugues; (Lousiville, KY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF LOUISVILLE RESEARCH FOUNDATION, INC.
ADVANCED CANCER THERAPEUTICS, LLC |
Louisville
Louisville |
KY
KY |
US
US |
|
|
Assignee: |
ADVANCED CANCER THERAPEUTICS,
LLC
Louisville
KY
UNIVERSITY OF LOUISVILLE RESEARCH FOUNDATION, INC.
Louisville
KY
|
Family ID: |
43796505 |
Appl. No.: |
14/803650 |
Filed: |
July 20, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13498036 |
Dec 12, 2012 |
9162987 |
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PCT/US2010/050206 |
Sep 24, 2010 |
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14803650 |
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61245481 |
Sep 24, 2009 |
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Current U.S.
Class: |
514/274 ;
514/256; 514/275; 544/315; 544/330; 544/333; 544/334 |
Current CPC
Class: |
C07D 239/42 20130101;
C07D 401/04 20130101; C07D 409/04 20130101; C07D 239/38 20130101;
C07D 239/30 20130101; C07D 405/04 20130101 |
International
Class: |
C07D 239/30 20060101
C07D239/30; C07D 239/42 20060101 C07D239/42; C07D 409/04 20060101
C07D409/04; C07D 239/38 20060101 C07D239/38; C07D 405/04 20060101
C07D405/04; C07D 401/04 20060101 C07D401/04 |
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: ##STR00064## wherein: (i) A is selected from
the group consisting of: (a) 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; (b) substituted or unsubstituted bicyclic
ring; (c) substituted or unsubstituted polycyclic rings; and (d)
substituted or unsubstituted alkenes and alkynes having 2 to 10
carbon atoms with 1 to 3 double or triple bonds; and B is H, OH,
OR, SR, NH.sub.2, NHR, or alkyl or substituted alkyl, wherein R is
H, alkyl, or substituted alkyl of 2 to 20 carbon atoms; or (ii) A
is H or halo; and B is selected from the group consisting of: (a)
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; (b)
substituted or unsubstituted bicyclic ring; (c) substituted or
unsubstituted polycyclic rings; and (d) substituted or
unsubstituted alkenes and alkynes having 2 to 10 carbon atoms with
1 to 3 double or triple bonds; and 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 the MIF inhibitory compound is
selected from the group consisting of:
4-Iodo-6-(2,3-difluoro-4-methoxyphenyl)pyrimidine;
4-Iodo-6-(2-fluoro-4-methoxyphenyl)pyrimidine;
4-Iodo-6-(2-fluorophenyl)pyrimidine;
4-Iodo-6-(4-fluorophenyl)pyrimidine;
4-Iodo-6-(furan-3-yl)pyrimidine; 4-Iodo-6-(pyridin-3-yl)pyrimidine;
4-Iodo-6-(3-fluorophenyl)pyrimidine;
4-Iodo-6-(4-tert-butyloxymethylphenyl)pyrimidine;
4-Iodo-6-(2-fluoropyridin-3-yl)pyrimidine;
4-Iodo-6-(furan-2-yl)pyrimidine;
4-Iodo-6-(4-fluoropyrimidin-3-yl)pyrimidine;
4-Iodo-6-(3-fluoro-4-methoxyphenyl)pyrimidine;
4-Iodo-6-(2-chloropyridin-5-yl)pyrimidine;
4-Iodo-6-(2-hydroxyphenyl)pyrimidine;
4-Iodo-6-(2,4-difluorophenyl)pyrimidine;
4-Iodo-6-(2-fluoro-6-methoxyphenyl)pyrimidine;
4-Iodo-6-(2-chlorophenyl)pyrimidine;
4-Iodo-6-(3-acetylaminophenyl)pyrimidine;
4-Iodo-6-(thiophen-3-yl)pyrimidine;
4-Iodo-6-(3-hydroxymethylphenyl)pyrimidine;
4-Iodo-6-(isoquinolin-4-yl)pyrimidine;
4-Iodo-6-(2,4,5-trifluorophenyl)pyrimidine;
4-Iodo-6-(2,4-difluoropyridin-3-yl)pyrimidine;
4-Iodo-6-(4-methoxypyridin-3-yl)pyrimidine;
4-Iodo-6-(thiophen-2-yl)pyrimidine;
4-Iodo-6-(3,4-difluorophenyl)pyrimidine;
4-Iodo-6-(4-ethoxyphenyl)pyrimidine;
4-Iodo-6-(4-aminocarbonylphenyl)pyrimidine;
4-Iodo-6-(3-aminocarbonylphenyl)pyrimidine;
4-Iodo-6-(quinolin-4-yl)pyrimidine;
4-Iodo-6-(quinolin-8yl)pyrimidine;
4-Iodo-6-(quinolin-3-yl)pyrimidine;
4-Iodo-6-(isoquinolin-5-yl)pyrimidine;
2-Methylthio-4-iodo-6-phenylpyrimidine;
2-Ethylthio-4-iodo-6-phenylpyrimidine;
2-Isopropylthio-4-iodo-6-phenylpyrimidine;
2-n-Butylthio-4-iodo-6-phenylpyrimidine;
2-Methylamino-4-iodo-6-phenylpyrimidine;
2-Ethylamino-4-iodo-6-phenylpyrimidine;
2-Propylamino-4-iodo-6-phenylpyrimidine;
2-Isopropylamino-4-iodo-6-phenylpyrimidine;
2-n-Butylamino-4-iodo-6-phenylpyrimidine;
4-Iodo-6-(benzothiophen-2-yl)pyrimidine;
4-Iodo-6-(benzofuran-2-yl)pyrimidine;
4-Iodo-6-(4-hydroxybenzothiophen-2-yl)pyrimidine;
4-Iodo-6-(4-acetylaminobenzothiophen-2-yl)pyrimidine;
4-Iodo-6-(4-aminocarbonylbenzothiophen-2-yl)pyrimidine;
4-Iodo-6-(5-acetylaminopyridin-3-yl)pyrimidine;
4-Iodo-6-(5-aminocarbonylpyridin-3-yl)pyrimidine;
4-Iodo-6-(4-fluoropyridin-3-yl)pyrimidine;
4-Iodo-6-(4-acetylaminothiophen-2-yl)pyrimidine;
4-Iodo-6-(4-aminocarbonylthiophen-2-yl)pyrimidine; and
4-Iodo-6-(4-methoxythiophen-2-yl)pyrimidine.
7. The method of claim 1, wherein: A is a substituted or
unsubstituted bicyclic ring selected from the group consisting of a
quinoline, an isoquinoline, a benzofuran, and a benzothiophene; 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.
8. The method of claim 7, wherein the MIF inhibitory compound is
selected from the group consisting of:
4-Iodo-6-(isoquinolin-4-yl)pyrimidine;
4-Iodo-6-(quinolin-4-yl)pyrimidine;
4-Iodo-6-(quinolin-8-yl)pyrimidine;
4-Iodo-6-(quinolin-3-yl)pyrimidine;
4-Iodo-6-(isoquinolin-5-yl)pyrimidine;
4-Iodo-6-(benzothiophen-2-yl)pyrimidine; and
4-Iodo-6-(benzofuran-2-yl)pyrimidine.
9. The method of claim 1, wherein the administering is via oral
administration, intravenous administration, intraperitoneal
administration, or a combination thereof.
10. 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: ##STR00065## wherein:
(i) A is selected from the group consisting of: (a) 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; (b) substituted or
unsubstituted bicyclic ring; (c) substituted or unsubstituted
polycyclic rings; and (d) substituted or unsubstituted alkenes and
alkynes having 2 to 10 carbon atoms with 1 to 3 double or triple
bonds; and B is H, OH, OR, SR, NH.sub.2, NHR, or alkyl or
substituted alkyl, wherein R is H, alkyl, or substituted alkyl of 2
to 20 carbon atoms; or (ii) A is H or halo; and B is selected from
the group consisting of: (a) 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; (b) substituted or unsubstituted bicyclic
ring; (c) substituted or unsubstituted polycyclic rings; and (d)
substituted or unsubstituted alkenes and alkynes having 2 to 10
carbon atoms with 1 to 3 double or triple bonds; and 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.
11. The method of claim 10, 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-fluoro-4-methoxyphenyl)pyrimidine;
4-Iodo-6-(2-fluorophenyl)pyrimidine;
4-Iodo-6-(4-fluorophenyl)pyrimidine;
4-Iodo-6-(furan-3-yl)pyrimidine; 4-Iodo-6-(pyridin-3-yl)pyrimidine;
4-Iodo-6-(3-fluorophenyl)pyrimidine;
4-Iodo-6-(4-tert-butyloxymethylphenyl)pyrimidine;
4-Iodo-6-(2-fluoropyridin-3-yl)pyrimidine;
4-Iodo-6-(furan-2-yl)pyrimidine;
4-Iodo-6-(4-fluoropyrimidin-3-yl)pyrimidine;
4-Iodo-6-(3-fluoro-4-methoxyphenyl)pyrimidine;
4-Iodo-6-(2-chloropyridin-5-yl)pyrimidine;
4-Iodo-6-(2-hydroxyphenyl)pyrimidine;
4-Iodo-6-(2,4-difluorophenyl)pyrimidine;
4-Iodo-6-(2-fluoro-6-methoxyphenyl)pyrimidine;
4-Iodo-6-(2-chlorophenyl)pyrimidine;
4-Iodo-6-(3-acetylaminophenyl)pyrimidine;
4-Iodo-6-(thiophen-3-yl)pyrimidine;
4-Iodo-6-(3-hydroxymethylphenyl)pyrimidine;
4-Iodo-6-(isoquinolin-4-yl)pyrimidine;
4-Iodo-6-(2,4,5-trifluorophenyl)pyrimidine;
4-Iodo-6-(2,4-difluoropyridin-3-yl)pyrimidine;
4-Iodo-6-(4-methoxypyridin-3-yl)pyrimidine;
4-Iodo-6-(thiophen-2-yl)pyrimidine;
4-Iodo-6-(3,4-difluorophenyl)pyrimidine;
4-Iodo-6-(4-ethoxyphenyl)pyrimidine;
4-Iodo-6-(4-aminocarbonylphenyl)pyrimidine;
4-Iodo-6-(3-aminocarbonylphenyl)pyrimidine;
4-Iodo-6-(quinolin-4-yl)pyrimidine;
4-Iodo-6-(quinolin-8yl)pyrimidine;
4-Iodo-6-(quinolin-3-yl)pyrimidine;
4-Iodo-6-(isoquinolin-5-yl)pyrimidine;
2-Methylthio-4-iodo-6-phenylpyrimidine;
2-Ethylthio-4-iodo-6-phenylpyrimidine;
2-Isopropylthio-4-iodo-6-phenylpyrimidine;
2-n-Butylthio-4-iodo-6-phenylpyrimidine;
2-Methylamino-4-iodo-6-phenylpyrimidine;
2-Ethylamino-4-iodo-6-phenylpyrimidine;
2-Propylamino-4-iodo-6-phenylpyrimidine;
2-Isopropylamino-4-iodo-6-phenylpyrimidine;
2-n-Butylamino-4-iodo-6-phenylpyrimidine;
4-Iodo-6-(benzothiophen-2-yl)pyrimidine;
4-Iodo-6-(benzofuran-2-yl)pyrimidine;
4-Iodo-6-(4-hydroxybenzothiophen-2-yl)pyrimidine;
4-Iodo-6-(4-acetylaminobenzothiophen-2-yl)pyrimidine;
4-Iodo-6-(4-aminocarbonylbenzothiophen-2-yl)pyrimidine;
4-Iodo-6-(5-acetylaminopyridin-3-yl)pyrimidine;
4-Iodo-6-(5-aminocarbonylpyridin-3-yl)pyrimidine;
4-Iodo-6-(4-fluoropyridin-3-yl)pyrimidine;
4-Iodo-6-(4-acetylaminothiophen-2-yl)pyrimidine;
4-Iodo-6-(4-aminocarbonylthiophen-2-yl)pyrimidine; and
4-Iodo-6-(4-methoxythiophen-2-yl)pyrimidine.
12. The method of claim 10, wherein: A is a substituted or
unsubstituted bicyclic ring selected from the group consisting of a
quinoline, an isoquinoline, a benzofuran, and a benzothiophene; 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.
13. The method of claim 12, wherein the MIF inhibitory compound is
selected from the group consisting of:
4-Iodo-6-(isoquinolin-4-yl)pyrimidine;
4-Iodo-6-(quinolin-4-yl)pyrimidine;
4-Iodo-6-(quinolin-8-yl)pyrimidine;
4-Iodo-6-(quinolin-3-yl)pyrimidine;
4-Iodo-6-(isoquinolin-5-yl)pyrimidine;
4-Iodo-6-(benzothiophen-2-yl)pyrimidine; and
4-Iodo-6-(benzofuran-2-yl)pyrimidine.
14. The method of claim 10, 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.
15. The method of claim 14, 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.
16. The method of claim 15, wherein the pharmaceutical composition
is pharmaceutically acceptable for use in a human.
17. A method for inhibiting autoimmune-associated activation of a T
cell, the method comprising contacting a T cell subject to
autoimmune-associated activation with an effective amount of a MIF
inhibitory compound or its enantiomeric or diastereomeric form or a
pharmaceutically acceptable salt, prodrug, or metabolite thereof of
claim 1, whereby autoimmune-associated activation of the T cell is
inhibited.
18. The method of claim 18, wherein the T cell subject to
autoimmune-associated activation is present within a mammal.
19. A compound selected from the group consisting of:
4-Iodo-6-(2,3-difluoro-4-methoxyphenyl)pyrimidine;
4-Iodo-6-(2-fluoro-4-methoxyphenyl)pyrimidine;
4-Iodo-6-(4-fluorophenyl)pyrimidine;
4-Iodo-6-(furan-3-yl)pyrimidine; 4-Iodo-6-(pyridin-3-yl)pyrimidine;
4-Iodo-6-(3-fluorophenyl)pyrimidine;
4-Iodo-6-(4-tert-butyloxymethylphenyl)pyrimidine;
4-Iodo-6-(2-fluoropyridin-3-yl)pyrimidine;
4-Iodo-6-(furan-2-yl)pyrimidine;
4-Iodo-6-(4-fluoropyrimidin-3-yl)pyrimidine;
4-Iodo-6-(3-fluoro-4-methoxyphenyl)pyrimidine;
4-Iodo-6-(2-chloropyridin-5-yl)pyrimidine;
4-Iodo-6-(2-hydroxyphenyl)pyrimidine;
4-Iodo-6-(2,4-difluorophenyl)pyrimidine;
4-Iodo-6-(2-fluoro-6-methoxyphenyl)pyrimidine;
4-Iodo-6-(2-chlorophenyl)pyrimidine;
4-Iodo-6-(3-acetylaminophenyl)pyrimidine;
4-Iodo-6-(thiophen-3-yl)pyrimidine;
4-Iodo-6-(3-hydroxymethylphenyl)pyrimidine;
4-Iodo-6-(2,4,5-trifluorophenyl)pyrimidine;
4-Iodo-6-(2,4-difluoropyridin-3-yl)pyrimidine;
4-Iodo-6-(4-methoxypyridin-3-yl)pyrimidine;
4-Iodo-6-(thiophen-2-yl)pyrimidine;
4-Iodo-6-(3,4-difluorophenyl)pyrimidine;
4-Iodo-6-(4-ethoxyphenyl)pyrimidine;
4-Iodo-6-(4-aminocarbonylphenyl)pyrimidine;
4-Iodo-6-(3-aminocarbonylphenyl)pyrimidine;
2-Methylthio-4-iodo-6-phenylpyrimidine
2-Ethylthio-4-iodo-6-phenylpyrimidine;
2-Isopropylthio-4-iodo-6-phenylpyrimidine;
2-n-Butylthio-4-iodo-6-phenylpyrimidine;
2-Methylamino-4-iodo-6-phenylpyrimidine;
2-Ethylamino-4-iodo-6-phenylpyrimidine;
2-Propylamino-4-iodo-6-phenylpyrimidine;
2-Isopropylamino-4-iodo-6-phenylpyrimidine;
2-n-Butylamino-4-iodo-6-phenylpyrimidine;
4-Iodo-6-(4-hydroxybenzothiophen-2-yl)pyrimidine;
4-Iodo-6-(4-acetylaminobenzothiophen-2-yl)pyrimidine;
4-Iodo-6-(4-aminocarbonylbenzothiophen-2-yl)pyrimidine;
4-Iodo-6-(5-acetylaminopyridin-3-yl)pyrimidine;
4-Iodo-6-(5-aminocarbonylpyridin-3-yl)pyrimidine;
4-Iodo-6-(4-fluoropyridin-3-yl)pyrimidine;
4-Iodo-6-(4-acetylaminothiophen-2-yl)pyrimidine;
4-Iodo-6-(4-aminocarbonylthiophen-2-yl)pyrimidine; and
4-Iodo-6-(4-methoxythiophen-2-yl)pyrimidine.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/498,036, filed Mar. 23, 2012, now pending; 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.
[0002] 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.
[0003] 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.
[0004] 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.
[0005] 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.
[0006] 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:
##STR00002##
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, NH.sub.2, 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.
[0007] 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.
[0008] 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.
[0009] 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
[0010] FIG. 1 depicts MIF liver enzyme inhibition as a percent of
control, comparing ACT-MIF-003, ACT-MIF-002, and 4-IPP.
[0011] FIG. 2 depicts MIF tumor enzyme inhibition as a percent of
control, comparing ACT-MIF-003, ACT-MIF-002, and 4-IPP.
[0012] 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.
[0013] FIG. 4 depicts a comparison of IC50 values across the tumor
cell lines DU145, MDA-MB-231, Miapaca, CAOV03, and HUVEC.
[0014] FIG. 5 depicts p53 regulation of compounds compared to
control (DMSO), 4-IPP, and ISO-1 at 10 .mu.M 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.
[0015] 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.
[0016] 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
.mu.M).
[0017] 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.
[0018] 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.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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.times.10.sup.6 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 .mu.M 4-IPP
(FIG. 13C), or 25 .mu.M 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.
[0023] 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.
[0024] 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.times.10.sup.6 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 .mu.M 4-IPP
(FIG. 15C), or 25 .mu.M 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.
[0025] 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.
[0026] 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.times.10.sup.6 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 .mu.M 4-IPP
(FIG. 17C), or 25 .mu.M 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.
[0027] 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.
[0028] 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.times.10.sup.6 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 .mu.M 4-IPP
(FIG. 19C), or 25 .mu.M 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.
[0029] 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.times.10.sup.6 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 .mu.M 4-IPP
(FIG. 20C), or 25 .mu.M 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.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] "Halo" or "halogen" refers to fluoro, chloro, bromo, or
iodo.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] Compounds according to the present invention have the
following generic structure:
##STR00003##
wherein: [0051] A is selected from the group consisting of: [0052]
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; [0053] ii)
substituted or unsubstituted bicyclic ring; [0054] iii) substituted
or unsubstituted polycyclic rings; and [0055] iv) substituted or
unsubstituted alkenes and alkynes having 2 to 10 carbon atoms with
1 to 3 double or triple bonds; [0056] B is H, OH, OR, SR, NH.sub.2,
NHR, alkyl or substituted alkyl or A, but when B is A, A is H or
halo; [0057] R is H, alkyl or substituted alkyl of 2 to 20 carbon
atoms; and [0058] X and Y are independently N or CH, but one of X
and Y must be N.
[0059] 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.
[0060] 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, NH.sub.2, NHR, alkyl, or substituted
alkyl; X and Y are both N.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] 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, NH.sub.2, NHR, alkyl or substituted
alkyl; X is N and Y is CH.
[0065] 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, NH.sub.2, NHR, alkyl, or substituted
alkyl; X is CH; and Y is N.
[0066] In another embodiment, the compound is selected from the
group set forth in Table 1.
TABLE-US-00001 TABLE 1 EXAM- ACT-MIF PLE NO. CHEMICAL NAME 2
ACT-MIF- 4-Iodo-6-(2,3-difluoro-4-methoxyphenyl) 001 pyrimidine 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) 010 pyrimidine 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
[0067] In another embodiment, X and Y are both N. In another
embodiment, when X and Y are both N, Bis H.
[0068] 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.
[0069] 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.
[0070] 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.
[0071] In a specific embodiment, A is selected from the group
consisting of indole, quinoline, and naphthalene.
[0072] In a very specific embodiment, the compound is
4-Iodo-6-(2-fluorophenyl)pyrimidine or
4-Iodo-6-(3-aminocarbonylphenyl)pyrimidine.
[0073] In another embodiment, a pharmaceutical composition is
provided, comprising: [0074] 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:
[0074] ##STR00004## [0075] wherein: [0076] A is selected from the
group consisting of: [0077] 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; [0078] ii) substituted or unsubstituted
bicyclic ring; [0079] iii) substituted or unsubstituted polycyclic
rings; and [0080] iv) substituted or unsubstituted alkenes and
alkynes having 2 to 10 carbon atoms with 1 to 3 double or triple
bonds; [0081] B is H, OH, OR, SR, NH.sub.2, NHR, alkyl or
substituted alkyl or A, but when B is A, A is H or halo; [0082] R
is H, alkyl or substituted alkyl of 2 to 20 carbon atoms; and
[0083] X and Y are independently N or CH, but one of X and Y must
be N; and [0084] b) one or more pharmaceutically acceptable
excipients.
[0085] In one embodiment, the compound is selected from the group
set forth in Table 1.
[0086] In another embodiment, X and Y are both N. In another
embodiment, when X and Y are both N, Bis H.
[0087] 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.
[0088] 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.
[0089] 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.
[0090] In a specific embodiment, A is selected from the group
consisting of indole, quinoline, and naphthalene.
[0091] In a very specific embodiment, the compound is
4-Iodo-6-(2-fluorophenyl)pyrimidine or
4-Iodo-6-(3-aminocarbonylphenyl)pyrimidine.
[0092] 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:
##STR00005##
wherein: [0093] A is selected from the group consisting of: [0094]
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; [0095] ii)
substituted or unsubstituted bicyclic ring; [0096] iii) substituted
or unsubstituted polycyclic rings; and [0097] iv) substituted or
unsubstituted alkenes and alkynes having 2 to 10 carbon atoms with
1 to 3 double or triple bonds; [0098] B is H, OH, OR, SR, NH.sub.2,
NHR, alkyl or substituted alkyl or A, but when B is A, A is H or
halo; [0099] R is H, alkyl or substituted alkyl of 2 to 20 carbon
atoms; and [0100] X and Y are independently N or CH, but one of X
and Y must be N.
[0101] In one embodiment, the compound is selected from the group
set forth in Table 1.
[0102] In another embodiment, X and Y are both N. In another
embodiment, when X and Y are both N, B is H.
[0103] 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.
[0104] 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.
[0105] 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.
[0106] In a specific embodiment, A is selected from the group
consisting of indole, quinoline, and naphthalene.
[0107] In a very specific embodiment, the compound is
4-Iodo-6-(2-fluorophenyl)pyrimidine or
4-Iodo-6-(3-aminocarbonylphenyl)pyrimidine.
[0108] In one embodiment, the MIF-implicated disease is selected
from the group consisting of inflammatory disease and cancer.
[0109] 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.
[0110] 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
[0111] 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
[0112] Methods for the Preparation of 4-Iodo-6-arylpyrimidine
Derivatives, where Aryl is Substituted Phenyl, Heterocyclic, or
Bicyclic Ring
##STR00006##
[0113] General Procedure:
[0114] 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.degree. 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.
[0115] 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)
##STR00007##
[0117] 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.
[0118] .sup.1H NMR (DMSO-d.sub.6): .delta. 8.98 (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)
##STR00008##
[0120] 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.
[0121] .sup.1H NMR (DMSO-d.sub.6): .delta. 8.91 (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)
##STR00009##
[0123] The compound was prepared according to EXAMPLE 1.
Specifically, the following method was employed:
##STR00010##
1. Preparation of 4-chloro-6-(2-fluoro-phenyl)-pyrimidine (3)
(TRM/AP/005/127)
[0124] 4,6-dichloropyrimidine (20.3 g, 136.3 mmol), 2-fluorophenyl
boronic acid (20.0 g, 142.9 mmol, 1.05 equiv), Na.sub.2CO.sub.3
(23.4 g, 106.0 mmol, 1.8 equiv) and Pd(PPh.sub.3).sub.2Cl.sub.2
(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%).
[0125] .sup.1H NMR (CDCl.sub.3): 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)
[0126] 4,6-dichloropyrimidine (5.1 g, 34.1 mmol), 2-fluorophenyl
boronic acid (5.0 g, 35.7 mmol, 1.05 equiv), Na.sub.2CO.sub.3 (6.9
g, 65.0 mmol, 1.8 equiv) and Pd(PPh.sub.3).sub.2Cl.sub.2 (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)
[0127] 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 .about.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%).
[0128] .sup.1H NMR (DMSO-d.sub.6): 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)
[0129] HPLC=98.55%
Example 5
4-Iodo-6-(4-fluorophenyl)pyrimidine (ACT-MIF-004)
##STR00011##
[0131] 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.
[0132] .sup.1H NMR (DMSO-d.sub.6): .delta. 8.90 (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)
##STR00012##
[0134] 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.
[0135] .sup.1H NMR (DMSO-d.sub.6): .delta. 8.79 (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)
##STR00013##
[0137] 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.
[0138] .sup.1H NMR (DMSO-d.sub.6): .delta. 9.40 (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)
##STR00014##
[0140] 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.
[0141] .sup.1H NMR (DMSO-d.sub.6): .delta. 8.95 (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)
##STR00015##
[0143] 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.
[0144] .sup.1H NMR (CDCl.sub.3): .delta. 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)
##STR00016##
[0146] 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.
[0147] .sup.1H NMR (DMSO-d.sub.6): .delta. 8.94 (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)
##STR00017##
[0149] 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.
[0150] .sup.1H NMR (DMSO-d.sub.6): .delta. 8.80 (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)
##STR00018##
[0152] 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.
[0153] .sup.1H NMR (CDCl.sub.3): .delta. 8.71 (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)
##STR00019##
[0155] 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.
[0156] .sup.1H NMR (CDCl.sub.3): .delta. 8.89 (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)
##STR00020##
[0158] 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.
[0159] .sup.1H NMR (DMSO-d.sub.6): .delta. 9.20 (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)
##STR00021##
[0161] 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.
[0162] .sup.1H NMR (DMSO-d.sub.6): .delta. 12.70 (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)
##STR00022##
[0164] 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.
[0165] .sup.1H NMR (DMSO-d.sub.6): .delta. 9.01 (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)
##STR00023##
[0167] 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.
[0168] .sup.1H NMR (DMSO-d.sub.6): .delta. 8.89 (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)
##STR00024##
[0170] 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-d.sub.6):
.delta. 9.0 (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)
##STR00025##
[0172] 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.
[0173] .sup.1H NMR (DMSO-d.sub.6): .delta. 10.15 (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)
##STR00026##
[0175] 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.
[0176] .sup.1H NMR (DMSO-d.sub.6): .delta. 8.88 (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)
##STR00027##
[0178] 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.
[0179] .sup.1H NMR (DMSO-d.sub.6): .delta. 8.92 (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)
##STR00028##
[0181] 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.
[0182] .sup.1H NMR (DMSO-d.sub.6): .delta. 9.69 (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)
##STR00029##
[0184] 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.
[0185] .sup.1H NMR (DMSO-d.sub.6): .delta. 9.0 (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)
##STR00030##
[0187] 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.
[0188] .sup.1H NMR (DMSO-d.sub.6): .delta. 9.05 (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)
##STR00031##
[0190] 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.
[0191] .sup.1H NMR (DMSO-d.sub.6): .delta. 9.10 (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)
##STR00032##
[0193] The compound was prepared according to EXAMPLE 1.
Specifically, the following method was employed:
##STR00033##
1. Preparation of 4-chloro-6-thiophen-2-yl-pyrimidine (3)
[0194] 4,6-dichloropyrimidine (22.2 g, 149.1 mmol),
thiophene-2-boronic acid (20.0 g, 156.3 mmol, 1.05 equiv),
Na.sub.2CO.sub.3 (28.8 g, 271.4 mmol, 1.8 equiv) and
Pd(PPh.sub.3).sub.2Cl.sub.2 (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%).
[0195] .sup.1H NMR (CDCl.sub.3): 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)
[0196] 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 .about.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%).
[0197] HPLC=93.1%
[0198] 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%).
[0199] .sup.1H NMR (CDCl.sub.3): 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).
[0200] HPLC=99.12%
Example 27
4-Iodo-6-(3,4-difluorophenyl)pyrimidine (ACT-MIF-032)
##STR00034##
[0202] 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.
[0203] .sup.1H NMR (DMSO-d.sub.6): .delta. 8.94 (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)
##STR00035##
[0205] 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.
[0206] .sup.1H NMR (DMSO-d.sub.6): .delta. 8.89 (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)
##STR00036##
[0208] 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.
[0209] .sup.1H NMR (DMSO-d.sub.6): .delta. 8.99 (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)
##STR00037##
[0211] 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.
[0212] .sup.1H NMR (DMSO-d.sub.6): .delta. 8.95 (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)
##STR00038##
[0214] 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.
[0215] .sup.1H NMR (DMSO-d.sub.6): .delta. 9.11 (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
##STR00039##
[0217] 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.
[0218] .sup.1H NMR (DMSO-d.sub.6): .delta. 8.80 (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
##STR00040##
[0220] 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.
[0221] .sup.1H NMR (DMSO-d.sub.6): .delta. 9.65 (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
##STR00041##
[0223] 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.
[0224] .sup.1H NMR (DMSO-d.sub.6): .delta. 9.41 (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
##STR00042##
[0226] The compounds of Examples 36-39 are prepared using the
method of EXAMPLE 35.
Example 36
2-Methylthio-4-iodo-6-phenylpyrimidine
##STR00043##
[0228] The compound was prepared according to EXAMPLE 35 using
methyl iodide as one of the reactants.
[0229] .sup.1H NMR (CDCl.sub.3): .delta. 8.03-8.06 (m, 2H), 7.82
(s, 1H), 7.49-7.54 (m, 3H), 2.62 (s, 3H).
Example 37
2-Ethylthio-4-iodo-6-phenylpyrimidine
##STR00044##
[0231] The compound was prepared according to EXAMPLE 35 using
ethyl iodide as one of the reactants.
[0232] .sup.1H NMR (CDCl.sub.3): .delta. 7.95-7.96 (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
##STR00045##
[0234] The compound was prepared according to EXAMPLE 35 using
isopropyl iodide as one of the reactants.
[0235] .sup.1H NMR (DMSO-d.sub.6): .delta. 8.25 (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
##STR00046##
[0237] The compound was prepared according to Scheme-2 using
n-butyl iodide as one of the reactant.
[0238] .sup.1H NMR (DMSO-d.sub.6): .delta. 8.26 (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
##STR00047##
[0240] The compounds of Examples 41-45 were prepared according to
Scheme 3 of EXAMPLE 40.
Example 41
2-Methylamino-4-iodo-6-phenylpyrimidine
##STR00048##
[0242] The compound was prepared according to EXAMPLE 40 using
methylamine as RNH.sub.2.
[0243] .sup.1H NMR (CDCl.sub.3): .delta. 7.98-8.00 (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
##STR00049##
[0245] The compound was prepared according to EXAMPLE 40 using
ethylamine as RNH.sub.2.
[0246] .sup.1H NMR (CDCl.sub.3): .delta. 7.97-7.99 (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, 1.2 Hz, 2H), 1.28 (t, J=7.2 Hz, 3H).
Example 43
2-Propylamino-4-iodo-6-phenylpyrimidine
##STR00050##
[0248] The compound was prepared according to EXAMPLE 40 using
propylamine as RNH.sub.2.
[0249] .sup.1H NMR (CDCl.sub.3): .delta. 7.92 (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, 7.3 Hz, 2H), 0.93 (t, J=7.3 Hz,
3H).
Example 44
2-Isopropylamino-4-iodo-6-phenylpyrimidine
##STR00051##
[0251] The compound was prepared according to EXAMPLE 40 using
isopropylamine as RNH.sub.2.
[0252] .sup.1H NMR (CDCl.sub.3): .delta. 8.04-8.07 (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
##STR00052##
[0254] The compound was prepared according to EXAMPLE 40 using
n-butylamine as RNH.sub.2.
[0255] .sup.1H NMR (CDCl.sub.3): .delta. 7.97 (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).
[0256] The compounds of Examples 46-56 are also prepared according
the Scheme 1.
Example 46
4-Iodo-6-(benzothiophen-2-yl)pyrimidine
##STR00053##
[0257] Example 47
4-Iodo-6-(benzofuran-2-yl)pyrimidine
##STR00054##
[0258] Example 48
4-Iodo-6-(4-hydroxybenzothiophen-2-yl)pyrimidine
##STR00055##
[0259] Example 49
4-Iodo-6-(4-acetylaminobenzothiophen-2-yl)pyrimidine
##STR00056##
[0260] Example 50
4-Iodo-6-(4-aminocarbonylbenzothiophen-2-yl)pyrimidine
##STR00057##
[0261] Example 51
4-Iodo-6-(5-acetylaminopyridin-3-yl)pyrimidine
##STR00058##
[0262] Example 52
4-Iodo-6-(5-aminocarbonylpyridin-3-yl)pyrimidine
##STR00059##
[0263] Example 53
4-Iodo-6-(4-fluoropyridin-3-yl)pyrimidine
##STR00060##
[0264] Example 54
4-Iodo-6-(4-acetylaminothiophen-2-yl)pyrimidine
##STR00061##
[0265] Example 55
4-Iodo-6-(4-aminocarbonylthiophen-2-yl)pyrimidine
##STR00062##
[0266] Example 56
4-Iodo-6-(4-methoxythiophen-2-yl)pyrimidine
##STR00063##
[0267] Example 57
Solubility and Stability
[0268] 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-US-00002 TABLE 2 Solubilities of the Compounds of the
Invention at 20-22.degree. 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
[0269] 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.RTM. 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-US-00003 TABLE 3 Cell Permeability and Transport R(Caco-2)
Caco-2 Permeability Papp (10.sup.4 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 Cell
Permeability and Transport Results (MDR1-MDCK) MDR1-MDCK
Permeability Papp (10.sup.4 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
[0270] Stability in human liver microsomes was tested over 24 hours
at 37.degree. 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.degree. 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-US-00004 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
[0271] 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-US-00005 TABLE 5 Human Plasma Protein Binding 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
[0272] 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 .mu.l
of corn oil every day for 3 days. Mice were sacrificed 6 hours
after the last injection and livers were harvested. .about.1 gram
pieces of liver were lysed in PBS containing 1 mM NaVO.sub.4, 2 mM
NaF and a protease inhibitor cocktail (Roche Biochemical,
Indianapolis, Ind.) using dounce-homogenization on ice. 500 .mu.g
of liver lysates were added to a final volume of 700 .mu.l 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 .mu.L of L-dopachrome
methyl ester was then immediately added to the cuvettes; the
OD.sub.475 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
[0273] 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
[0274] 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.times.10.sup.5 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-US-00006 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
[0275] The up regulation of p53 was determined using a commercially
available p53 luciferase assay kit. 1.times.10.sup.5 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 .mu.g/well
of p53-responsive luciferase promoter plasmid (Promega, Madison,
Wis.) together with 0.0125 .mu.g/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 in 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
[0276] 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-US-00007 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
[0277] 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 .mu.M). 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-Angio Genic Properties in the Chick
Chorioallantoic Membrane (CAM) Assay
[0278] 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.degree. C. for 3 days. On
Day 3, eggs were cracked under sterile conditions and embryos were
placed into 20.times.100 mm plastic plates and cultivated at
37.degree. 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 .mu.L 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.times.. 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-US-00008 TABLE 8 Blood Vessel Densities 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 .+-. 3.4 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
[0279] 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-US-00009 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
[0280] 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
[0281] 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.times.10.sup.6 cells/100 .mu.l. 4 groups of 10 mice each were
used in the experiment. DU145 cells suspended in 100 .mu.l 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
mm.sup.3, 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.times.W.times.H.times..pi./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
[0282] 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
[0283] 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.
[0284] 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
[0285] 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).
[0286] 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 .about.12% inhibition in liver extracts to
.about.76.2% inhibition in the lungs.
[0287] 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
[0288] 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.times.10.sup.6 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 .mu.M 4-IPP or 25 .mu.M 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.
[0289] 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.
[0290] 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.
[0291] 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.
[0292] 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.
[0293] 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.
[0294] 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.
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