USE OF A 6-ARYL PYRIMIDINE COMPOUND FOR TREATING MULTIPLE SCLEROSIS

METHODE DE TRAITEMENT DE LA SCLEROSE EN PLAQUES

English Abstract

A method for treating multiple sclerosis by systemic administration of a 6-
aryl pyrimidine compound or a pharmaceutically acceptable salt thereof in
association with a pharmaceutical carrier to a human having symptoms of
multiple sclerosis.

French Abstract

L'invention concerne une méthode de traitement de la sclérose en plaques par administration systémique à un humain présentant des symptômes de sclérose en plaques, d'un composé de 6-aryl pyrimidine ou d'un sel de ce dernier acceptable du point de vue pharmaceutique, associé à un excipient pharmaceutique.

Claims

CLAIMS
1. A method for treating multiple sclerosis comprising the systemic
administration of an amount effective for treating multiple sclerosis of a
compound
of the formula:
<IMG>
wherein X3 is equal to X, X4 or X5 wherein X4 is fluoro, chloro, bromo or
iodo, and X5
is mono-, di or trihalomethyl, mono, di or trifluoroethyl, perfluoropropyl,
and
wherein X is alkyl of from 1 to 3 carbon atoms, inclusive, including isomeric
forms, 2
propynyl and 2-propenyl, and X1 is a member selected from the group consisting
of
(a) phenyl,
(b) a monosubstituted phenyl of the formula
<IMG>
wherein one of the groups R, R1, R2, R3, R4, is not hydrogen and wherein R or
R4 is
alkyl of from 1 to 8 carbon atoms, inclusive, including isomeric forms, alkoxy
of from
1 to 8 carbon atoms, inclusive, including isomeric forms, fluoro, chloro,
bromo, iodo
or vitro; R1 or R3 is fluoro, chloro, bromo, iodo, nitro; trifluoromethyl or
alkoxy of
from 1 to 8 carbon atoms, inclusive, including isomeric forms, alkoxyethyloxy
wherein alkoxy is from 1 to 5 carbon atoms, inclusive, including isomeric
forms, or
<IMG>
wherein R5 and R6 are the same or different and are alkyl of from 1 to 8
carbon
-15-

atoms, inclusive, including isomeric forms, benzyl, or taken together with -N>
are a
saturated cycloalkylamino group
<IMG>
wherein n' is 3, 4, 5 or 6 or dialkyl substituted cycloalkylamino wherein each
alkyl is
from 1 to 3 carbon atoms, inclusive; including isomeric forms, and R2 is
chloro,
fluoro, bromo, iodo, or alkyl of from 1 to 3 carbon atoms, inclusive;
(c) a disubstituted phenyl of the formula:
<IMG>
wherein any two of R, R1, R2, R3 and R4 are not hydrogen and are the same or
different and are fluoro, chloro, bromo, iodo, alkyl of from 1 to 8 carbon
atoms,
inclusive, including isomeric forms, alkoxy of from 1 to 8 carbon atoms,
inclusive,
including isomeric forms, vitro and trifluoromethyl;
(d) a trihalo substituted phenyl wherein halo is chloro, bromo, iodo, or
fluoro;
(e) .alpha.-naphthyl of the formula:
<IMG>
wherein R7 is substituted in either ring and is hydrogen, alkyl of from 1 to 8
carbon
atoms, inclusive, including isomeric forms, alkoxy of from 1 to 8 carbon
atoms,
inclusive, including isomeric forms, fluoro, chloro, iodo, bromo or nitro-
provided that
when X3 is X, X1 is not .alpha.-napthyl;
(f) 2-furyl,
-16-

(g) 3-pyridyl,
(h) 2-pyridyl, and
(i) 2-pyrazyl, provided that when X3 is X, 2 propynyl or 2-propenyl, X1 is
not 2-furyl, 3-pyridyl, 2-pyridyl or 2-pyrazyl, or a salt thereof, in
association with a
pharmaceutical carrier to a human having symptoms of multiple sclerosis.
2. The method of Claim 1 wherein the amount of compound administered, in
divided doses, is from about 50 to about 1,000 mg/kg/week. of body weight of
the
human.
3. The method of Claim 1 wherein the compound administered is selected from
the group consisting of
2-amino-5-iodo-6-(3-bromophenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(3-fluorophenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(3-ethoxyethylphenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(2-methoxyphenyl)-4-pyrimidinol;
2-amino-5-chloro-6-(2-methoxyphenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(2-methoxyphenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(3-chlorophenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3-chlorophenyl)-4-pyrimidinol;
2-amino-5-chloro-6-(3-chlorophenyl)-4-pyrimidinol;
2-amino-5-chloro-6-(2-fluorophenyl)-4-pyrimidinol;
2-amino-5-chloro-6-(3-fluorophenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(2-fluorophenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3-fluorophenyl)-4-pyrimidinol;
2-amino-5-iodo-6-phenyl-4-pyrimidinol;
2-amino-5-chloro-6-phenyl-4-pyrimidinol;
2-amino-5-bromo-6-phenyl-4-pyrimidinol;
2-amino-5-chloro-6-(3-methoxyphenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(3-methoxyphenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3-methoxyphenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(2-pyridyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3,4-dichlorophenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(.alpha.-naphthyl)-4-pyrimidinol;
2-amino-5-chloro-6-(3-nitrophenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3-nitrophenyl)-4-pyrimidinol;
-17-

2-amino-5-iodo-6-(3-trifluoromethylphenyl-4-pyrimidinol;
2-amino-5-ethyl-6-phenyl-4-pyrimidinol;
2-amino-5-bromo-6-(3,5-dimethoxyphenyl)-4-pyrimidinol and
2-amino-5-chloro-6-(3-propyloxyphenyl)-4-pyrimidinol.
4. The method of Claim 2 wherein the compound is 2-amino-5-bromo-6-phenyl-
4(1H)-pyrimidinone.
5. The method of Claim 4 wherein the compound is administered orally.
6. The method of Claim 4 wherein the compound is nonhygroscopic.
7. The method of Claim 6 wherein the compound is administered orally.
8. Use of a compound of the formula:
<IMG>
wherein X3 is equal to X, X4 or X5 wherein X4 is fluoro, chloro, bromo or
iodo, and X6
is mono-, di or trihalomethyl, mono, di or trifluoroethyl, perfluoropropyl,
and
wherein X is alkyl of from 1 to 3 carbon atoms, inclusive, including isomeric
forms, 2
propynyl and 2-propenyl, and X1 is a member selected from the group consisting
of
(a) phenyl,
(b) a monosubstituted phenyl of the formula
<IMG>
wherein one of the groups R, R1, R2, R3, R4 is not hydrogen and wherein R or
R4 is
alkyl of from 1 to 8 carbon atoms, inclusive, including isomeric forms, alkoxy
of from
-18-

1 to 8 carbon atoms, inclusive, including isomeric forms, fluoro, chloro,
bromo, iodo
or vitro; R1 or R3 is fluoro, chloro, bromo, iodo, vitro; trifluoromethyl or
alkoxy of
from 1 to 8 carbon atoms, inclusive, including isomeric forms, alkoxyethyloxy
wherein alkoxy is from 1 to 5 carbon atoms, inclusive, including isomeric
forms, or
<IMG>
wherein R5 and R6 are the same or different and are alkyl of from 1 to 8
carbon
atoms, inclusive, including isomeric forms, benzyl, or taken together with -N>
are a
saturated cycloalkylamino group
<IMG>
wherein n' is 3, 4, 5 or 6 or dialkyl substituted cycloalkylamino wherein each
alkyl is
from 1 to 3 carbon atoms, inclusive; including isomeric forms, and R2 is
chloro,
fluoro, bromo, iodo, or alkyl of from 1 to 3 carbon atoms, inclusive;
(c) a disubstituted phenyl of the formula:
<IMG>
wherein any two of R, R1, R2, R3 and R4 are not hydrogen and are the same or
different and are fluoro, chloro, bromo, iodo, alkyl of from 1 to 8 carbon
atoms,
inclusive, including isomeric forms, alkoxy of from 1 to 8 carbon atoms,
inclusive,
including isomeric forms, nitro and trifluoromethyl;
(d) a trihalo substituted phenyl wherein halo is chloro, bromo, iodo, or
fluoro;
(e) .alpha.-naphthyl of the formula:
-19-

<IMG>
wherein R7, is substituted in either ring and is hydrogen, alkyl of from 1 to
8 carbon
atoms, inclusive, including isomeric forms, alkoxy of from 1 to 8 carbon
atoms,
inclusive, including isomeric forms, fluoro, chloro, iodo, bromo or nitro-
provided that
when X3 is X, X1 is not .alpha.-napthyl;
(f) 2-furyl,
(g) 3-pyridyl,
(h) 2-pyridyl, and
(i) 2-pyrazyl, provided that when X3 is X, 2 propynyl or 2-propenyl, X1 is
not 2-furyl, 3-pyridyl, 2-pyridyl or 2-pyrazyl, or a salt thereof, to prepare
a
medicament to treat multiple sclerosis.
9. The use of Claim 8 wherein the amount of compound administered, in divided
doses, is from about 50 to about 1,000 mg/kg/week. of body weight of the
human.
10. The use of Claim 9 wherein the compound administered is selected from the
group consisting of:
2-amino-5-iodo-6-(3-bromophenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(3-fluorophenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(3-ethoxyethylphenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(2-methoxyphenyl)-4-pyrimidinol;
2-amino-5-chloro-6-(2-methoxyphenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(2-methoxyphenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(3-chlorophenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3-chlorophenyl)-4-pyrimidinol;
2-amino-5-chloro-6-(3-chlorophenyl)-4-pyrimidinol;
2-amino-5-chloro-6-(2-fluorophenyl)-4-pyrimidinol;
2-amino-5-chloro-6-(3-fluorophenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(2-fluorophenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3-fluorophenyl)-4-pyrimidinol;
2-amino-5-iodo-6-phenyl-4-pyrimidinol;
2-amino-5-chloro-6-phenyl-4-pyrimidinol;
-20-

2-amino-5-bromo-6-phenyl-4-pyrimidinol;
2-amino-5-chloro-6-(3-methoxyphenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(3-methoxyphenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3-methoxyphenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(2-pyridyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3,4-dichlorophenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(.alpha.-naphthyl)-4-pyrimidinol;
2-amino-5-chloro-6-(3-nitrophenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3-nitrophenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3-trifluoromethylphenyl-4-pyrimidinol;
2-amino-5-ethyl-6-phenyl-4-pyrimidinol;
2-amino-5-bromo-6-(3,5-dimethoxyphenyl)-4-pyrimidinol and
2-amino-5-chloro-6-(3-propyloxyphenyl)-4-pyrimidinol.
11. The use of Claim 9 wherein the compound is 2-amino-5-bromo-6-phenyl-
4(1H)-pyrimidinone.
12. The use of Claim 11 wherein the compound is administered orally.
13. The use of Claim 11 wherein the compound is nonhygroscopic.
14. The use of Claim 13 wherein the compound is administered orally.
-21-

Description

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WO 98/25596 PCT/US97/21402
METHOD FOR TREATING MULTIPLE SCLEROSIS
BACKGROUND OF THE INVENTION
The present invention provides a new use of known compounds. More
particularly the present invention provides a method of treating multiple
sclerosis
using a series of known pyrimidiols including bropirimine.
The preparation and use of 2-amino-5-halo-6-alkyl-4-pyrimidiols as antiviral
agents is known US Patent 3,956,302 and Nicols, Weed and Underwood,
Antimicrobial Agents, Chemo. Ther. 9 433, 1976.
The preparation and use of 2-amino-6-aryl-4-pyrimidinol compounds for
alteration of the immunoregulatory system of the host animal is disclosed in
US
Patent 4,543,248. US Patent 4,543,248 claims a process for treating acquired
or
congenital hypogammaglobulinemia, acquired or congenital agammaglobulinemia
with 2-amino-6-aryl-4-pyrimidinol compounds.
US Patent 4,50?,302 discloses that the immunoregulatory system is altered
or arthritis treated by systemic administration of 2-amino-6-aryl-4-
pyrimidinol
compounds.
US Patent 4,619,933 claims a process of treating aplastic anemia with 2-
amino-6-aryl-4-pyrimidinol compounds and discloses that alteration of the
immunoregulatory system of the host animal and that administration of the
active
compounds increases antibody formation and be used to treat acquired or
congenital
hypogammaglobulinemia; activates macorphages and can be used to treat or
prevent
intracellular or extracellular parasitic infections, including bacterial and
protozoal;
increases hematopoietic stem cells in bone marrow and spleen and can be used
to
treat or prevent aplastic anemia; and decreases generation of allospecific
killer cells
and can be used to prevent rejection of organ and skin grafts.
US Patent 5,002,951 claims a method for treating bacterial and protozoal
infections with 2-amino-6-aryl-4-pyrimidinol compounds.
US Patent 5,434,157 claims various 6-aryl pyrimidinol compounds and
compositions thereof as well a method for treating viral infections and
inducing
interferon production. In addition, this patent also discloses that the
compounds
have immunoregulatory activity; for example the compounds increased antibody
formation and decreased delayed hypersensitivity and are useful in the
treatment of
parasitic diseases, organ transplants and skin graft rejections, and immune
deficiencies including those caused as a side effect of therapy with cytotoxic
agents
and radiation.
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WO 98I25596 PCT/US97/21402
US Patent Application Serial No. 08/419,963 claims the process of treating
susceptible forms of cancer with 2-amino-6-aryl-4-pyrimidinol compounds and
discloses the alteration of the immunoregulatory system of the host animal and
that
administration of the active compounds increases antibody formation and be
used to
treat acquired or congenital hypogammaglobulinemia; increases natural killer
cells
and can be used to treat various forms of cancer; activates macorphages and
can be
used to treat or prevent intracellular and can be used to treat or prevent
intracellular or extracellular parasitic infections, including bacterial and
protozoal;
increases hematopoietic stem cells in bone marrow and spleen and can be used
to
treat or prevent aplastic anemia; and decreases generation of allospecific
killer cells
and can be used to prevent rejection of organ and skin grafts.
Bropirimine (2-amino-5-bromo-6-phenyl-4(1H)-pyrimidinone; ABPP) which
was selected from this group of 6-arylpyrimidinones for further development,
is an
orally active inducer of endogenous interferon, with established antiviral,
immunostimulatory and antitumor actii~ity. Regulatory approval from the United
States Food and Drug Administration is being sought to market bropirimine
tablets
for the treatment of superficial bladder cancer (CIS).
A variety of compounds have been proposed for use in the treatment of
multiple sclerosis (MS) including matrix metalloproteinase inhibitors (MMPIs)
[Gijbels, K.,Galardy, R.E., Steinman, L., Reversal of Experimental Autoimmune
Encephalomyelitis with a Hydroxamate Inhibitor of Matrix Metalloproteinases.
[J.
Clin. Invest. 94: 217?-2182 (l994)1, lymphokines, such as interferon-beta
[Swanborg, R.H., Experimental Autoimmune Encephalomyelitis in Rodents as a
Model for Human Demyelinating Disease. Clin. Immunol. Immunopath. ??: 4-13
(1995) and Yu, M., Nishiyama, A., Trapp, B.D. and Tuohy) V.K., Interferon-beta
Inhibits Progression of Relapsing-Remitting Experimental Autoimmune
Encephalomyelitis. J. Neuroimmunol. 64: 91-100 (1996)], and antibodies against
surface markers such as VLA-4 [Baron, J.L., Madri, J.A., Ruddle, N.H., Hashim,
G.,
Janeway, Jr., C.A., Surface Expression of Alpha-4 Integrin by CD4 T cells is
Required for their Entry into Brain Parenchyma. J. Esp. Med. 17?: 5?-68
(1993)].
Recently, linomide has entered clinical trials after demonstrated efficacy in
the
animal model of MS, experimental autoimmune encephalomyelitis (EAE) [Karussis,
D.M., Lehmann, D., Slavin, S., Vourka-Karussis, U., Mizrachi-Koll, R., Ovadia,
H.,
Kalland, T., and Abramsky, O., Treatment of Chronic-Relapsing Experimental
Autoimmune Encephalomyelitis with the Synthetic Immunomodulator Linomide
(quinoline-3-carboximide). Proc. Natl. Acad. Sci. 90: 6400-6404 (1993) and
-2-

CA 02269681 1999-04-22
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Karussis, D.M., Lehmann, D., Slavin, S., Vourka-Karussis, U., Mizrachi-Koll,
R.,
Ovadia, H., Ben-Nun, A. Kalland, T., and Abramsky, O., Inhibition of acute,
Experimental Autoimmune Encephalomyelitis by the Synthetic Immunomodulator
Linomide. Ann. Neurol. 34: 654-660 (199S)].
The most recently approved biologicals for the treatment of MS are from the
cytokine/lymphokine family, i.e. 13-interferons. However, dosing of the
interferons is
restricted to parenteral injections, which must be repeated regularly, and
there are
indications that the administration of the interferons is unpleasant enough to
result
in a relatively high dropout rate for patients who initially attempt to use
this
therapy. Furthermore, the efficacy reported is weak enough to warrant
continuing
the search for better therapy to treat the disease. One concern associated
with the
direct use of interferons is the failure of the natural regulation systems to
be able to
control the concentrations of the cytokine. Since lymphokine/cytokine systems
are
usually tightly regulated, such a lack of internal control systems may result
in side
effects and less than optimal efficacy. Toxicological problems associated with
interferons include neurological problems, such as changes in
electroencephalograms, sleep patterns and cognition) and a flu-like syndrome
[Spiegel, R.J., The alpha interferons: Clinical overview. Seminars in Oncology
14:
1-12 (1987) and Rohatiner, A.Z.S., Prior) P.F., and Burton, A.C., Central
nervous
system toxicity of interferon. Br. J. Cancer 47: 418-422 (1983)1. Furthermore,
the
full benefit of the cytokine may not be realized because the exogenous
interferon
concentration is determined strictly by timing of the doses and the dose
administered. The role of naturally occurring interferon in MS is not
understood.
Experimental autoimmune encephalomyelitis (EAE) has been an accepted
animal model of MS for many years. A review of the literature containing
extensive
citations was recently published comparing EAE and MS [Swanborg, R.H.,
Experimental autoimmune encephalomyelitis in rodents as a model for human
demyelinating disease. Clin. Immunol. Immunopath. 77: 4-13 (1995)]. The most
common models of EAE are those developed using rodents. Although EAE and MS
are not identical, there are a number of important features in common between
EAE
and MS. For example, using mice, a chronic-remitting form of EAE, closely
resembling the clinical appearance of MS in humans, has been developed. EAE in
mice is often accompanied by demyelination in addition to the perivascular
mononuclear cell infiltrates in the CNS. In contrast, in rats perivascular
mononuclear cell infiltrates are readily observed in EAE) but the lesions are
not
usually accompanied by demyelination. Since the number and type of samples
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CA 02269681 1999-04-22
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obtained from human MS patients is necessarily limited, the use of the animal
models has significantly contributed to our understanding of some of the
cellular
events critical for initiating the disease. For example, a transient form of
EAE can
be transmitted between animals by transferring appropriately selected CD4+ T
lymphocytes. This indicates a key role for the CD4+ T cells. Furthermore,
results
from the animal models have shown that particular amino acid sequences from
MBP
and proteolipid protein (PLP), which are the major protein constituents of
myelin,
may be key antigenic determinants in initiation and/or progression of EAE. The
importance of the immunoreactivity to these proteins has subsequently been
confirmed in patients with MS. Furthermore, the genetics associated with
susceptibility to disease and the biochemical consequences of gene expression
may be
studied in considerably greater detail using animal models than may be carried
out
in human populations, especially by making use of a number of inbred strains
of
mice. Another parallel between EAE and MS was recently demonstrated by showing
that administration of interferon-beta to mice with EAE resulted in a decrease
in
symptoms and a decreased relapse rate (Yu, M., Nishiyama, A., Trapp, B.D. and
Tuohy, V.K., Interferon-betas Inhibits Progression of Relapsing-Remitting
Experimental Autoimmune Encephalomyelitis. J. Neuroimmunol. 64: 91~100
(1996)], similar to the results observed in MS patients.
The cells comprising a lesion have been studied in both EAE and in MS.
Conclusions about the sequence of cell types that infiltrate lesions in the
brain or
spinal cord vary somewhat from author to author, but the early infiltration of
CD4+
T-lymphocytes is well established (Fabry, Z., Raine, C.S.) and Hart, M.N. ,
Nervous
Tissue as an hnmune Compartment: the Dialect of the Immuiae Response i~r tlxe
CNS.
Immunol. Today 15: 218-224 (1994)l. At a later stage of disease, CD8+ T cells
infiltrate lesions. In most demyelinating lesions, macrophage-like cells are
present
in large numbers. The origin of the macrophage-like cells appears to be a
combination of cells from the periphery and differentiated microglial cells
[Huitinga,
L, Ruuls, S.R., Jung, S., Van Rooijen, N., Hartung, H.-P., and Dijkstra, C.D.,
Macrophages in T Cell Line-Mediated, Demyelinating, and Chronic Relapsing
Experimental Autoirnmune Encephalomyelitis in Leivis Rats. Clin. Exp. Immunol.
100: 344-3b1 (1996)] and Bauer, J., Huitinga, L, Zhao, W., Lassmann, H.,
Hickey,
W., and Dijkstra, C.D., The Role of Macrophages, Perivascular Cells, and
Microglial
Cells in the Pathogenesis of Experimental Autoimmune Encephalomyelitis. Glia
15:
437-446 (199b)]. Suppressor cells also appear to play an important role in
progression and control of EAE [Sun, D., Qin, Y., Chluba, J.) Epplen, J.T.,
and
-4-

CA 02269681 1999-04-22
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Wekerle, H., Suppression of Experimentally Induced Autoimmune
Encephalomyelitis
by Cytolytic ?'-T Cell Interactions. Nature 332: 843-846 (18S8)] ) although
the
particular type of cell involved in the suppression activity is only beginning
to
emerge [Fabry, Z., Raine, C.S., and Hart, M.N., Nervous tissue as an immune
compartment: the dialect of the immune response in the CNS. Immunol. Today 15:
21&224 (1994)].
Following intraperitoneal (i.p.) or oral (p.o.) administration of bropirimine
to
mice, high interferon-alpha concentrations were observed in the serum and may
have been produced predominantly in the spleen and thymus [Stringfellow, D.A.
and
Weed, S.D., 5-halo-6-phenyl pyrimidinones: A New Series of Interferon-Inducing
Agents. In: Khan, A. Hill, N.O., Dorn, G.L., editors. Interferons: Properties
and
clinical uses. Proceedings Internatioaal Symposium, Cleland Fikes
Foundation Press, Dallas, TX 31S-326 (1980) (Weed, S.D., Kramer, G.D., and
Stringfellow, D.A. ( 1980) Antiviral Properties of 6-arylpyrimidinones. In:
Nelson,
J.D., and Grassi, C., editors. American Society Microbiology, 1980, 1408-1409;
and Stringfellow, D.A., 6-Arylpyrimidinols: Interferon inducers-immuno-
modulators-
antiviral and antineoplastic agents. Prog. Cancer Res. Ther. 16: 215-228
(1981)].
2',5'-oligoadenylate synthetase activity, which is induced by interferon, was
also
increased following bropirimine treatment of mice, consistent with interferon
induction. Bropirimine induced production of IFN-alpha in vitro in cultured
murine
thymocytes and in bone marrow, and spleen cells [Stringfellow, D.A. and Weed,
S.D.,
5-halo-6-phenyl pyrimidinones: A New Series of Interferon-Inducing Agents. In:
Khan, A. Hill, N.O., Dorn, G.L., editors. Interferons: Properties and clinical
uses. Proceedings International Symposium, Cleland Fikes Foundation
Press, Dallas, TX 315-326 (1980) and Hamilton, R.D.) Buthala, D.A., Eidson,
E.E.,
Tomilo, A., and Andrews, J.C., Interferon induction in vitro with 6-methyl and
6-
aryl-pyrimidines. In: Nelson, J.D. and Grassi, C., editors. 11th Current
Chemotherapy Infectious Disease, Proceedings International Congress
Chemotherapy, American Society Microbiology, Washington (DC). 1980,2:
1409-1411). IFN-alpha was induced by bropirimine in human tonsillar tissue
[Stringfellow, D.A., Vanderberg, H.C., Weed, S.D., Interferon Induction by 5-
halo-6-
phenyl Pyrimidinones. J. Interferon Res. 1:I-14 (1980)] and in peripheral
mononuclear cells [Kita, M. and Imanishi, J., Induction of Interferon by
Bropirimine
in Human Peripheral Blood Mononuclear Cell Culture. Pharmaeol. Ther. (Japan)
20: 37-42 (1992)]. Furthermore, IFN-alpha was detected in the sera of
bropirimine-
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treated patients. Induction of natural killer cell activity [Lotzova, E.,
Savary, C.A.,
and Stringfellow) D.A., 5-halo-6-phenyl-pyri.midinones; New molecules witty
cancer
therapeutic potential and interferon-inducing capacity are strong inducers of
murine
natural killer cells. J. ImmunoL 13D: 965-969 (1983); Lotzova, E., Savary,
C.A.,
Khan, A., and Stringfellow, D.A., Stimulation of Natural Killer Cells in Two
Random-bred Strains of Atlaymic Rats by Interferon-Inducing Pyrinxidinones. J.
Immunol. 132: 2566-2570 (1984); and Lotzova, E., Savary, C.A., Lowlachi, IVL)
and
Murasko, D.M., Cytatoxic grad Morphologic Profile of Endogenous and
Pyrimidinone-
Activated Murine NK Cells. J. Immunol. 136: 732-740 (1986)] and macrophage
tumoricidal activity [Li, L., Wallace, T.L., Richard, K.A., and Tracey, D.E.,
Mechanisms of Antitum.or Action of Pyrimidinones in the Treatment of B16
Melanoma and P388 Leukenaia. Caneer Res. 45: 532-538 (1985)] have also been
associated with bropirimine administration. Anti-inflammatory effects, typical
of
interferon-alpha inducers) have been demonstrated for bropirimine in chronic T
cell
mediated granuloma formation [Dunn. C.J., Galinet, L.A., Gibbons, A.J., and
Shields, S.K., Murine Delayed-Type H~~persensitiuity Granuloma: An improved
Model
for the Identification and Evaluation of Different Classes of Anti-
inflammatory
Drugs. Int. J. Immunopharm. 12: 899-904 (1990)].
BRIEF DESCRIPTION OF THE INVENTION
This invention relates to the use of 6-aryl pyrimidine compounds for the
treatment of multiple sclerosis (MS).
DETAILED DESCRIPTION OF THE INVENTION
The present invention a method of treating multiple sclerosis (MS)
comprising the systemic administration of an effective amount of a compound
represented by the formula
HO
X3
N~
N X
H2N
wherein X3 is equal to X, X4 or X5 wherein X4 is fluoro, chloro, bromo or
iodo, and
X6 is mono-, di or trihalomethyl, mono, di or trifluoroethyl, perfluoropropyl,
and
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CA 02269681 1999-04-22
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wherein X is alkyl of from 1 to 3 carbon atoms, inclusive, including isomeric
forms, 2
propynyl and 2-propenyl, and Xl is a member selected from the group consisting
of
(a) phenyl,
(b) a morosubstituted phenyl of the formula
R R~
\
R4 R3
wherein one of the groups R, R1) R2, R3, R4 is not hydrogen and wherein R or
R4 is
alkyl of from 1 to 8 carbon atoms, inclusive, including isomeric forms, alkoxy
of from
1 to 8 carbon atoms, inclusive) including isomeric forms, fluoro, chloro,
bromo, iodo
or vitro; Rl or R3 is fluoro, chloro, bromo, iodo, vitro; trifluoromethyl or
alkoxy of
from 1 to 8 carbon atoms, inclusive, including isomeric forms, alkoxyethyloxy
wherein alkoxy is from 1 to 5 carbon atoms, inclusive, including isomeric
forms, or
~s
Rs
wherein R5 and Rs are the same or different and are alkyl of from 1 to 8
carbon
atoms, inclusive, including isomeric forms, benzyl, or taken together with -N>
are a
saturated cycloalkylamino group
-N C~1
wherein n' is 3, 4, 5 or 6 or dialkyl substituted cycloalkyIamino wherein each
alkyl is
from 1 to 3 carbon atoms, inclusive; including isomeric forms, and R2 is
chloro,
fluoro, bromo, iodo, or alkyl of from 1 to 3 carbon atoms) inclusive;
(c) a disubstituted phenyl of the formula:
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R Rl
R2
Ra Rs
wherein any two of R, R1, R2, R3 and R4 are not hydrogen and are the same or
different and are fluoro, chloro, bromo, iodo, alkyl of from 1 to 8 carbon
atoms,
inclusive,including isomeric forms, alkoxy of from 1 to 8 carbon atoms,
inclusive,
including isomeric forms, nitro and trifluoromethyl;
(d) a trihalo substituted phenyl wherein halo is chloro, bromo, iodo) or
fluoro;
(e) a-naphthyl of the formula:
/ \
\ /
R~
wherein R~ is substituted in either ring and is hydrogen, alkyl of from 1 to 8
carbon
atoms, inclusive, including isomeric forms, alkoxy of from 1 to 8 carbon
atoms,
inclusive, including isomeric forms, fluoro, chloro, iodo, bromo or nitro-
provided that
when X3 is X, Xl is not a-napthyl;
(f) 2-furyl,
(g) 3-pyridyl,
(h) 2-pyridyl, and
(i) 2-pyrazyl, provided that when X3 is X, 2 propynyl or 2-propenyl, Xl is
not 2-furyl, 3-pyridyl, 2-pyridyl or 2-pyrazyl, or a salt thereof, in
association with a
pharmaceutical carrier.
Preferred compounds are:
2-amino-5-iodo-6-(3-bromophenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(3-fluorophenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(3-ethoxyethylphenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(2-methoxyphenyl)-4-pyrimidinol;
2-amino-5-chloro-6-(2-methoxyphenyl)-4-pyrimidinol;
_g_

CA 02269681 1999-04-22
gyp gg/~35g6 PCT/US97/Z1402
2-amino-5-iodo-6-(2-methoxyphenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(3-chlorophenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3-chlorophenyi)-4-gyrimidinol;
2-amino-5-chloro-6-(3-chlorophenyl)-4-pyrimidinol;
2-amino-5-chloro-6-(2-fluorophenyl)-4-pyrimidinol;
2-amino-5-chloro-6-(3-fluorophenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(2-fluorophenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3-fluorophenyl)-4-pyrimidinol;
2-amino-5-iodo-6-phenyl-4-pyrimidinol;
2-amino-5-chloro-6-phenyl-4-pyrimidinol;
2-amino-5-bromo-6-phenyl-4-pyrimidinol;
2-amino-5-chloro-6-(3-rnethoxyphenyl )-4-pyrimidinol;
2-amino-5-bromo-6-(3-methoxyphenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3-methoxyphenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(2-pyridyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3,4-dichlorophenyl)-4-pyrimidinol;
2-amino-5-bromo-6-(a-naphthyl)-4-pyrimidinol;
2-amino-5-chloro-6-(3-nitrophenyl)-4-pyrimidinol;
2-amino-5-iodo-6-(3-nitrophenyl )-4-pyrimidinol;
2-amino-5-iodo-6-(3-trifluoromethylphenyl-4-pyrimidinol;
2-amino-5-ethyl-6-phenyl-4-pyrimidinol;
2-amino-5-bromo-6-(3,5-dimethoxyphenyl)-4-pyrimidinol and
2-amino-5-chloro-6-(3-propyloxyphenyl)-4-pyrimidinol.
Bropirimine (2-amino-5-bromo-6-phenyl-4( 1H)-pyrimidinone; ABPP) is
particularly preferred and exists as an odorless white crystalline powder
which
melts with decomposition near 270 C. Bropirimine exists in several crystal
forms
comprising two general classes. One form, the hydroscopic, can undergo
transition
to at least two other polymorphic crystal forms; the other is a nonhydroscopic
form
stable to polymorphic transition. At 100% relative humidity, the hydroscopic
form of
bropirimine gains about 6.3% water, which is equivalent to the amount of water
required to form a monohydrate. The nonhydroscopic drug does not show similar
behavior and is preferred.
Applicant hereby incorporates by reference, in their entirety, US Patents
5,434,157 and 4,619,933. The 6-aryl pyrimidine compounds used in the present
invention can be prepared by the general procedures) disclosed therein.
The hydroscopic crystal form of bropirimine can be synthesized by treating 2-
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amino-6-phenyl-4(3H)-pyrimidine with bromine in acetic acid. The material is
filtered from the mixture and dissolved in aqueous base. The material is
reprecipitated by neutralization with acetic acid, filtered, washed with
water, and
dried.
The non-hydroscopic crystal form of bropirimine can be obtained by heating
an aqueous suspension of the hydroscopic crystal form above ?0~ C. The
material is
filtered, washed with water, and dried.
Procedure 1: Preparation of Bropirimine
Part A: A mixture of guanidine carbonate, (39.4 g) n-butyl alcohol, and ethyl
benzoylacetate (60 g) is reacted at or above 90~C. The reaction is monitored
by
HPLC until the reaction is judged to be complete. The mixture is cooled and
then
acetic acid is added to pH 5.8-8.0 to complete the crystallization. The slurry
is
cooled further (0-5~C) and then the slurry is filtered. The cake is washed
with n-
butyl alcohol, and then with water. The cake of 2-amino-6-phenyl-4(3H)-
pyrimidine
may or may not be dried.
Part B: Liquid bromine (about 41.6 g) is added slowly to a slurry of 2-amino-
6-phenyl-4(3H)-pyrimidine (about 45.0 g on a dry basis) in acetic acid (273.7
ml) and
water (273.0 ml) until the reaction is judged to be complete by HPLC. The
reaction
is quenched with the addition of 50% sodium hydroxide (36.2 g). The reaction
mixture is heated to ?5-90~C for 1 or more hours. The slurry is cooled and
filtered.
The cake is washed with water and dried to yield the nonhydroscopic crystal
form of
bropirimine.
The mode of administration of the compounds of Formula I can be by the
means disclosed in US Patent 5,434,157, preferably orally.
The dosage administered will be dependent upon the condition of the patient,
its age, health, weight, kind of concurrent treatment, if any, frequency of
treatment,
therapeutic ratio, and tolerance. In addition, the timing of drug
administration can
be important in maintenance of activity in the treatment of multiple
sclerosis.
Illustratively, dosage levels of the administered compounds of Formula I (i.e.
"active ingredients") can be intravenous, 0.1 to about 50 mglkg.;
subcutaneous, 0.1
to about 150 mg./kg.; intramuscular, 0.1 to about 150 mg/kg.; and about 0.1 to
about
400 mg/kg orally, preferably 15 to about 150 mg/kg of animal (body) weight.
The active ingredients to be employed in the treatment of MS can be easily
prepared in unit dosage form with the employment of pharmaceutical materials
as
disclosed in US Patent 5,434,157.
Illustratively, the active ingredients (compounds of Formula I) are
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CA 02269681 1999-04-22
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administered, in divided doses) of about 50 to about 1,000 mg/kg/week of human
body weight.
A preferred oral dosage regimen of the present invention, is the
administration of three grams per day (divided over a six hour period) for
three
consecutive days per week, e.g. one gram of the active ingredient (preferably
presented in unit dosage form) every two hours for three doses on days 1, 2
and 3 of
each week for about three to about twelve months, or longer. An alternative
preferred oral dosage regimen is the administration of three grams of the
active
ingredient (preferably presented in unit dosage form) per day (divided over a
six
hour period) every other day for up to about one year, or longer.
Bropirimine, which is representative of the compounds of Formula I, has been
evaluated in an EAE model (Procedure A) and representative results shown in
Figures 1-3.
Figure 1: Effect of ABPP on EAE in mice. Mice were dosed with 250
milligrams per kilogram ( mpk) on the days indicated by arrows. Two priming
doses
were administered on consecutive days, and only these doses were given
intraperitoneally (i.p.) into mice. Subsequent doses) all were administered
orally via
a gavage tube followed at 3 or 4 day intervals to result in a twice-a-week
dosing
regimen. The mean scores for 40 mice in each group were statistically
significantly
less in ABPP treated mice beginning at day 11 and continued through day 29,
that
is 7 days after dosing was stopped during the first dosing interval. No
significant
decline in disease scores was detected during the second dosing interval.
Figure 2: Effect of two different doses of ABPP on EAE in mice. A11 doses of
ABPP were administered orally using a gavage tube. Two priming doses on days 7
and 8 were followed with doses given twice a week for a total of three weeks.
The
mean disease in mice treated with 400 milligrams per kilogram (mpk) ABPP was
statistically significantly less than in mice treated with vehicle alone or
with 100
mpk. The mean disease was statistically highly significantly lower for the
group
treated with 400 mpk beginning on approximately day 15 and continuing through
day 37, i.e. approximately 1 week after dosing was stopped. The mean disease
for
mice treated with 100 mpk ABPP was statistically significantly less than in
the
vehicle-treated group only on days 16 and 17. Each group began with 65 mice
per
group, and 5 mice were removed at three times during the course of the
experiment
for histological evaluation of the lower spinal cords. Additionally, 5 mice
from each
group were processed for histology on days 43 and 56.
Figure 3: Dose response for ABPP. Two priming doses of ABPP were given
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CA 02269681 1999-04-22
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via gavage intubation on days 6 and 7 after injection of MBP to initiate EAE.
Dosing was continued at 3 day intervals through day 25. The days that doses
were
administered to the mice are indicated by arrows just above the abscissa on
the
graph. Approximately equivalent and statistically significant suppression of
disease
was observed beginning on day 6 for a11 3 doses of ABPP used. After day 16,
the
degree of disease suppression was greatest in mice treated with 400 mpk ABPP)
and
the mice treated with 400 mpk had significantly less disease than the mice
treated
with either 100 mpk or 200 mpk. However, it was difficult to distinguish
between
the degree of effect of the lower two doses. Disease was suppressed to a
statistically
significant amount relative to vehicle-treated mice for a period extending for
approximately 6 days after the last dose for the 100 mpk and 200 mpk doses and
for
a period of up to 9 days in mice treated with 400 mpk ABPP.
In the four independent assays, treatment with bropirimine (ABPP) orally,
once daily every 3-4 days over 2-3 week periods resulted in a highly
statistically
significant moderation of EAE. Treatment with bropirimine was begun either 5,
6,
or 7 days after induction of disease with myelin basic protein (MBP?.
Histopathology of formalin-fixed cross sections of the lower spinal cord of
mice from
the tenth thoracic vertebra to the fourth lumbar vertebra, was also assessed;
first, to
establish and quantify the correlation between the pattern of the clinical
scores and
the infiltration of leukocytes and, second, to assess how bropirimine affected
the
leukocyte infiltration. The results show that there is a small but
statistically
significant increase in the amount of leukocyte infiltration in the spinal
cords of
mice with EAE when they are treated with high doses of bropirimine compared to
the amount of infiltration expected for mice with equivalent levels of
clinical disease
but not receiving bropirimine.
Procedure A:
12-20 week old female B10.PIJJ mice (Jackson Laboratories, Bar Harbor,
Maine) are injected intramuscularly in a rear flank on day 0 with 150 lzg of
purified
guinea pig myelin basic protein (MBP) in complete Freund's adjuvant
supplemented
with 2.5 mg/ml M. tuberculosis. One day later, 0.1 ml of adsorbed B. pertussis
vaccine (Michigan Dept. of Health) is injected subcutaneously at the base of
the tail.
A second dose of B. pertussis is injected two days later, on day 3. The first
signs of
disease are usually observed in a few mice beginning at day 6-7 and the mean
day of
disease onset is approximately day 10. By day 15-20 clinical signs of EAE has
been
observed in a11 of the mice.
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After induction of EAE with MBP and injection of B. pertussis) the mice are
scored approximately 6 times per week for changes in motor activity according
to the
following scale:
Score
0 = Normal/asymptomatic
1 = limpness or weakness in tail
2 = weakness in one hind limb (hemiparesis/paraparesis)
3 = weakness in both hind limbs (paraparesis)
4 = paralysis in both hind limbs (paraparalysis)
5 = paralysis in both hind limbs, weakness in one or both forelimbs
(paraparalysis/paraparesis)
6 = Death
Dosing for the test compound is via a gavage tube inserted into the stomach
of a mouse, unless indicated otherwise. ' The volume administered is 0.5 ml.
The
vehicle used with bropirimine (ABPP) contained 5.26 mg carboxymethylcellulose,
4.0
mg poiysorbate 80 NF, 9.0 mg sodium chloride, and 9.18 mg benzylalcohol NF.
Histolo~v end Histolo~ical Evaluation
The mice are perfused with formalin to fix tissues in situ. Eight vertebrae,
thoracic 10-13 and lumbar 1-4) are removed from the carcass, further fixed in
formalin, decalcified using a 2 x 4 hr incubation in formic acid, carefully
separated
into individual vertebra and placed in cassettes for paraffin embedding,
sectioning,
and staining with hematoxyiin and eosin (H&E). Five, 5 um thick spinal cord
cross
sections are cut, each I00 microns apart, from each of the 8 vertebrae for a
total of
40 cross sections per mouse. The cross sections are scored for the presence
and
degree of infiltration. For an individual mouse, the results are presented as
the
proportion of cross sections with leukocyte infiltration, regardless of the
degree of
infiltration, or as the sum of the histological scores for all 40 cross
sections.
Histology samples were scored according to the following scale:
Score
0 = normal histology
1 = 0-10 infiltrating leukocytes in any 1 area
2 = 10-20 infiltrating leukocytes in any 1 area
3 = 20-50 infiltrating leukocytes in any 1 area
4 = 50 or more infiltrating leukocytes in any 1 area
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Interferon Assav
Murine interferon determinations are performed using a modification of the
method published previously [Stringfellow, D.A., Vanderberg, H.C., Weed, S.D.,
Interferon induction by 5-halo-6-phenyl pyrimidinones. J. Interferon ftes. 1:1-
14
(1980)]. L929 (ATCC) cells are used at 90% confluence in 24 well plates.
Samples
for interferon determination are diluted into DMEM (Gibco) tissue culture
medium
containing 10% fetal bovine sera and antibiotics. Dilutions of samples are
then
added to wells of L929 cells and the cells became 100% confluent overnight.
The
growth medium is removed and the cells are infected with approximately 50
plaque
forming units per well of vesicular stomatitis virus (VSV). Virus is allowed
to
adsorb to cells for one hour, and the wells are overlaid with 0.9% agarose
prepared
in tissue culture medium containing 2% sera. Cell cultures are incubated for
40-44
hrs, and VSV plaques are counted after 3 hrs of staining with neutral red.
Included
in each interferon assay are samples of interferon standards obtained from the
NIAID Repository, NIH. The standards included murine alpha interferon
(Ga0290I511 ) and a mixture of murine alpha/beta interferon (Gu02901511 ). In
order to quantify the concentration of interferon in samples, inhibition
curves
generated from the sample dilution series are compared to inhibition curves
generated using the interferon standards. Data is reported as unitslml of
marine
interferon.
Figure 4. Interferon concentrations in peripheral blood in vehicle- and ABPP
treated mice. Blood samples were collected on the days indicated and assessed
for
interferon concentration. Results shown correspond to the experiment shown in
Figure 2.
Figure 5. Interferon concentrations in peripheral blood in vehicle- and ABPP
treated mice. Blood samples were collected on the days indicated and assessed
for
interferon concentration. Results shown correspond to the experiment shown in
Figure 3.
-14-