U.S. patent application number 11/264981 was filed with the patent office on 2006-03-30 for triazine compounds.
This patent application is currently assigned to Synta Pharmaceuticals Corp.. Invention is credited to Wenli Ding, David A. James, M. Ono, Teresa Przewloka, Lijun Sun, Yumiko Wada, Shijie Zhang.
Application Number | 20060069090 11/264981 |
Document ID | / |
Family ID | 31996388 |
Filed Date | 2006-03-30 |
United States Patent
Application |
20060069090 |
Kind Code |
A1 |
Ono; M. ; et al. |
March 30, 2006 |
Triazine compounds
Abstract
This invention relates to triazine compounds of formula (I):
##STR1## R.sub.1 is ##STR2## aryl, or heteroaryl; each of R.sub.2,
R.sub.4, and R.sub.5, independently, is R.sup.c, halogen, nitro,
nitroso, cyano, azide, isothionitro, SR.sup.c, or OR.sup.c; R.sub.3
is R.sup.c, alkenyl, alkynyl, aryl, heteroaryl, cyclyl,
heterocyclyl, OR.sup.c, OC(O)R.sup.c, SO.sub.2R.sup.c, S(O)R.sup.c,
S(O.sub.2)NR.sup.cR.sup.d, SR.sup.c, NR.sup.cR.sup.d,
NR.sup.cCOR.sup.d, NR.sup.cC(O)OR.sup.d,
NR.sup.cC(O)NR.sup.cR.sup.d, NR.sup.cSO.sub.2, COR.sup.c,
C(O)OR.sup.c, or C(O)NR.sup.cR.sup.d; n is 0, 1, 2, 3, 4, 5, 6, or
7; X is O, S, S(O), S(O.sub.2), or NR.sup.c; Y is a covalent bond,
CH.sub.2, C(O), C.dbd.N--R.sup.c, C.dbd.N--OR.sup.c,
C.dbd.N--SR.sup.c, O, S, S(O), or S(O.sub.2); Z is N; and W is O,
S, S(O), S(O.sub.2), NR.sup.c, or NC(O)R.sup.c; in which each of
R.sup.a and R.sup.b, independently, is H, alkyl, aryl, heteroaryl;
and each of R.sup.c and R.sup.d, independently, is H, alkyl, or
alkylcarbonyl.
Inventors: |
Ono; M.; (Lexington, MA)
; Sun; Lijun; (Harvard, MA) ; Zhang; Shijie;
(Nashua, NH) ; Przewloka; Teresa; (Tewksbury,
MA) ; James; David A.; (Cambridge, MA) ; Ding;
Wenli; (Worcester, MA) ; Wada; Yumiko;
(Billerica, MA) |
Correspondence
Address: |
EDWARDS & ANGELL, LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Assignee: |
Synta Pharmaceuticals Corp.
Lexington
MA
|
Family ID: |
31996388 |
Appl. No.: |
11/264981 |
Filed: |
November 1, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10656360 |
Sep 5, 2003 |
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11264981 |
Nov 1, 2005 |
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10006624 |
Nov 30, 2001 |
6680315 |
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10656360 |
Sep 5, 2003 |
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09594362 |
Jun 15, 2000 |
6384032 |
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10006624 |
Nov 30, 2001 |
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Current U.S.
Class: |
514/227.8 ;
514/235.5; 514/241 |
Current CPC
Class: |
A61K 31/541 20130101;
C07D 403/12 20130101; C07D 417/12 20130101; A61P 17/06 20180101;
A61P 29/00 20180101; A61K 31/53 20130101; C07D 231/12 20130101;
C07D 401/14 20130101; C07D 409/14 20130101; C07D 409/12 20130101;
C07D 403/14 20130101; A61P 25/28 20180101; A61K 31/5377 20130101;
C07D 233/56 20130101; C07D 405/12 20130101; A61P 19/02 20180101;
A61P 3/10 20180101; C07D 405/14 20130101; C07D 249/08 20130101 |
Class at
Publication: |
514/227.8 ;
514/235.5; 514/241 |
International
Class: |
A61K 31/541 20060101
A61K031/541; A61K 31/5377 20060101 A61K031/5377; A61K 31/53
20060101 A61K031/53 |
Claims
1. A method for treating an interleukin-12 overproduction-related
disorder, comprising administering to a subject in need thereof an
effective amount of the compound of formula (I): ##STR12## wherein
R.sub.1 is ##STR13## aryl, orheteroaryl; each of R.sub.2, R.sub.4,
and R.sub.5, independently, is R.sup.c, halogen, nitro, nitroso,
cyano, azide, isothionitro, SR.sup.c, or OR.sup.c; R.sub.3 is
R.sup.c, alkenyl, alkynyl, aryl, heteroaryl, cyclyl, heterocyclyl,
OR.sup.c, OC(O)R.sup.c, SO.sub.2R.sup.c, S(O.sub.2)R.sup.c,
S(O.sub.2)NR.sup.cR.sup.d, SR.sup.c, NR.sup.cR.sup.d,
NR.sup.cCOR.sup.d, NR.sup.cC(O)OR.sup.c,
NR.sup.cC(O)NR.sup.cR.sup.d, NR.sup.cSO.sub.2R.sup.d, COR.sup.c,
C(O)OR.sup.c, or C(O)NR.sup.cR.sup.d; n is 0, 1, 2, 3, 4, 5, 6, or
7; X is O, S, S(O), S(O.sub.2), or NR.sup.c; Y is a covalent bond,
CH.sub.2, C(O), C.dbd.N--R.sup.c, C.dbd.N--OR.sup.c,
C.dbd.N--SR.sup.c, O, S, S(O), S(O.sub.2), or NR.sup.c; Z is N or
CH; and W is O, S, S(O), S(O.sub.2), NR.sup.c, or NC(O)R.sup.c; in
which each of R.sup.a and R.sup.b, independently, is H, alkyl,
aryl, heteroaryl; and each of R.sup.c and R.sup.d, independently,
is H, alkyl, or alkylcarbonyl; or a phamaceutically acceptable salt
thereof.
2. The method of claim 1, wherein R.sub.1 is ##STR14##
3. The method of claim 2, wherein W is O.
4. The method of claim 3, wherein R.sub.5 is H or alkyl.
5. The method of claim 2, wherein X is NR.sup.c.
6. The method of claim 2, wherein R.sup.c is H, methyl, ethyl, or
acetyl.
7. The method of claim 2, wherein Y is O or CH.sub.2, and n is 0,
1, 2, 3, or 4.
8. The method of claim 7, wherein R.sub.3 is aryl or
heteroaryl.
9. The method of claim 8, wherein R.sub.3 is pyridinyl.
10. The method of claim 7, wherein R.sub.3 is OR.sup.c, SR.sup.c,
C(O)OR.sup.c, or C(O)NR.sup.cR.sup.d.
11. The method of claim 7, wherein R.sub.3 is ##STR15## in which
each of A and A', independently, is O, S, or NH; each of R.sup.e
and R.sup.f, independently is H, alkyl, aryl, or heteroaryl; and m
is 1 or 2.
12. The method of claim 2, wherein one of R.sup.a and R.sup.b is
##STR16## in which B is NR.sup.i, O, or S; B' is N or CR.sup.i;
R.sup.g is H, alkyl, or alkoxyl; R.sup.h is halogen, CN, hydroxyl,
alkyl, aryl, heteroaryl, alkoxyl, aryloxyl, or heteroaryloxyl;
R.sup.i is H, alkyl, or alkylcarbonyl; p is 0, 1, or 2; and q is 0,
1, 2, 3, or 4.
13. The method of claim 12, wherein one of R.sup.a and R.sup.b is
##STR17## the other of R.sup.a and R.sup.b is alkyl.
14. The method of claim 13, wherein R.sup.g is H, methyl, ethyl,
methoxy, or ethoxy; Rh is F, Cl, CN, methoxy, methyl, or ethoxy;
R.sup.i is H, methyl, ethyl, or acetyl, and q is 0, 1, or 2.
15. The method of claim 14, wherein R.sup.g is methyl or methoxy;
R.sup.i is H; and q is 0.
16. The method of claim 14, wherein W is O; and R.sub.5 is H or
alkyl.
17. The method of claim 16, wherein X is NR.sup.c; and R.sup.c is
H, methyl, ethyl, or acetyl.
18. The method of claim 14, wherein Y is O or CH.sub.2, and n is 0,
1, 2, 3, or 4.
19. The method of claim 18, wherein R.sub.3 is aryl or
heteroaryl.
20. The method of claim 19, wherein R.sub.3 is pyridinyl.
21. The method of claim 20, wherein R.sub.1 is ##STR18## in which D
is O, S, or NR.sup.m; D' is N or CR.sup.m; R.sup.j is halogen, CN,
hydroxyl, alkyl, aryl, heteroaryl, alkoxyl, aryloxyl, or
heteroaryloxyl; R.sup.k is aryl or heteroaryl; R.sup.l is H, alkyl,
or alkylcarbonyl; R.sup.m is H, alkyl, or alkylcarbonyl; r is 0, 1,
or 2; s is 0 or 1; t is 0, 1, 2, 3, or 4; and u is 0, 1, 2, 3, 4,
or 5.
22. The method of claim 14, wherein R.sub.3 is OR.sup.c, SR.sup.c,
C(O)OR.sup.c or C(O)NR.sup.cR.sup.d.
23. The method of claim 14, wherein R.sub.3 is ##STR19## in which
each of A and A', independently, is O, S, or NH; each of R.sup.e
and R.sup.f, independently is H, alkyl, aryl, or heteroaryl; and m
is 1 or 2.
24. The method of claim 1, wherein Z is CH.
25. The method of claim 24, wherein R.sub.1 is ##STR20##
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/656,360, filed on Sep. 5, 2003, pending, which is a
continuation of U.S. application Ser. No. 10/006,624, filed on Nov.
30, 2001, now U.S. Pat. No. 6,680,315, which is a
continuation-in-part of U.S. application Ser. No. 09/594,362, filed
on Jun. 15, 2000, now U.S. Pat. No. 6,384,032, all of which are
incorporated herein by reference in their entirety.
BACKGROUND
[0002] Interleukin-12 (IL-112) is a heterodimeric cytokine (p70)
composed of two subunits (p35 and p40), and plays key roles in
immune responses by bridging innate resistance and antigen-specific
adaptive immunity. Trinchieri (1993) Immunol Today 14: 335. For
example, it promotes type 1 T helper cell (Th1) responses and,
hence, cell-mediated immunity. Chan et al. (1991) J Exp Med 173:
869; Seder et al. (1993) Proc Natl Acad Sci USA 90: 10188; Manetti
et al. (1993) J Exp Med 177: 1199; and Hsieh et al. (1993) Science
260: 547. Overproduction of IL-12 causes excessive Th1 responses,
and may result in inflammatory disorders, insulin-dependent
diabetes mellitus, multiple sclerosis, rheumatoid arthritis,
psoriasis, Crohn's disease, or sepsis. See, for example, Gately et
al. (1998) Annu Rev Immunol. 16: 495; and Abbas et al. (1996)
Nature 383: 787. Thus, inhibiting IL-12 overproduction is an
approach to treat the just-mentioned diseases. Trembleau et al.
(1995) Immunol. Today 16: 383; and Adorini et al. (1997) Chem.
Immunol. 68: 175. For example, overproduction of IL-12 and the
resultant excessive Th1 type responses can be suppressed by
modulating IL-12 production. A compound that down-regulates IL-12
production can be used for treating inflammatory diseases. Ma et
al. (1998) Eur Cytokine Netw 9: 54.
SUMMARY
[0003] This invention is based on the identification of new
compounds from a library of 80,000 compounds, which were screened
for their abilities to inhibit IL-12 overproduction. In one aspect,
this invention features triazine compounds of formula (I) ##STR3##
wherein R.sub.1 is ##STR4## [referred to hereinafter as
NC(R.sup.aR.sup.b)], aryl, or heteroaryl; each of R.sub.2, R.sub.4,
and R.sub.5, independently, is R.sup.c, halogen, nitro, nitroso,
cyano, azide, isothionitro, SR.sup.c, or OR.sup.c; R.sub.3 is
R.sup.c, alkenyl, alkynyl, aryl, heteroaryl, cyclyl, heterocyclyl,
OR.sup.c, OC(O)R.sup.c, SO.sub.2R.sup.c, S(O)R.sup.c,
S(O.sub.2)NR.sup.cR.sup.d, SR.sup.c, NR.sup.cR.sup.d,
NR.sup.cCOR.sup.d, NR.sup.cC(O)OR.sup.c, NR.sup.cC(O)NR.sup.cR,
NR.sup.cSO.sub.2R.sup.d, COR.sup.c, C(O)OR.sup.c, or
C(O)NR.sup.cR.sup.d; n is 0, 1, 2, 3, 4, 5, 6, or 7; X is O, S,
S(O), S(O.sub.2), or NR.sup.c; Y is a covalent bond, CH.sub.2,
C(O), C.dbd.N--R.sup.c, C.dbd.N--OR.sup.c, C.dbd.N--SR.sup.c, O, S,
S(O), or S(O.sub.2); Z is N; and W is O, S, S(O), S(O.sub.2),
NR.sup.c, or NC(O)R.sup.c; in which each of R.sup.a and R.sup.b,
independently, is H, alkyl, aryl, heteroaryl; and each of R.sup.c
and R.sup.d, independently, is H, alkyl, or alkylcarbonyl. Note
that the left atom shown in any substituted group described above
is closest to the tirazine ring. Also note that when n is 2 or
greater, the just-described triazine compound may have two or more
different C(R.sup.2R.sup.4) moieties. The same rule applies to
other similar situations.
[0004] Referring to formula (I), a subset of the triazine compounds
of this invention is featured by that R.sup.1 is
NC(R.sup.aR.sup.b). In these compounds, W can be 0; R.sub.5 can be
H or alkyl; X can be NR.sup.c; R.sup.c can be H, methyl, ethyl, or
acetyl; Y can be O or CH.sub.2, and n can be 0, 1, 2, 3, or 4. In
some embodiments, R.sub.3 is aryl, heteroaryl (e.g., pyridinyl),
OR.sup.c, SR.sup.c, C(O)OR.sup.c, or C(O)NR.sup.cRd. In other
embodiments, R.sub.3 is ##STR5## in which each of A and A',
independently, is O, S, or NH; each of R.sup.e and R.sup.f,
independently, is H, alkyl, aryl, or heteroaryl; and m is 1 or
2.
[0005] In this subset of triazine compounds, R.sup.a or R.sup.b,
preferably, is ##STR6## in which B is NR.sup.i, O, or S; B' is N or
CR.sup.i; R.sup.g is H, alkyl, or alkoxyl; R.sup.h is halogen, CN,
hydroxyl, alkyl, aryl, heteroaryl, alkoxyl, aryloxyl, or
heteroaryloxyl; R.sup.i is H, alkyl, or alkylcarbonyl; p is 0, 1,
or 2; and q is 0, 1, 2, 3, or 4. Preferably, B is NR.sup.i; B' is
CH; R.sup.g is H, methyl, ethyl, methoxy, or ethoxy; R.sup.h is F,
Cl, CN, methoxy, methyl, or ethoxy; R.sup.i is H, methyl, ethyl, or
acetyl; and q is 0, 1, or 2.
[0006] Another subset of the triazine compounds of this invention
is featured by that R.sup.1 is aryl or heteroaryl. In these
compounds, W can be 0; R.sub.5 can be H or alkyl; X can be
NR.sup.c; R.sup.c can be H, methyl, ethyl, or acetyl; Y can be O or
CH.sub.2, and n can be 0, 1, 2, 3, or 4. In some embodiments,
R.sub.3 is aryl, heteroaryl (e.g., pyridinyl), OR.sup.c, SR.sup.c,
C(O)OR.sup.c, or C(O)NR.sup.cR.sup.d. In other embodiments, R.sub.3
is ##STR7## in which each of A and A', independently, is O, S, or
NH; each of R.sup.e and R.sup.f, independently, is H, alkyl, aryl,
or heteroaryl; and m is 1 or 2.
[0007] In this second subset of triazine compounds, R.sub.1,
preferably, is ##STR8## in which D is O, S, or NR.sup.m; D' is N or
CR.sup.m; R.sup.j is halogen, CN, hydroxyl, alkyl, aryl,
heteroaryl, alkoxyl, aryloxyl, or heteroaryloxyl; R.sup.k is aryl
or hetereoaryl; R.sup.l is H, alkyl, or alkylcarbonyl; R.sup.m is
H, alkyl, or alkylcarbonyl; r is 0, 1, or 2; s is 0 or 1; t is 0,
1, 2, 3, or 4; and u is 0, 1, 2, 3, 4, or 5. Preferably, R.sub.1 is
##STR9## and R.sup.j is methyl, ethyl, propyl, or benzyl; and r can
be 1 or 2.
[0008] In another aspect, this invention also features triazine
compounds of formula (I), wherein R.sub.1 is NC(R.sup.aR.sup.b),
aryl, or heteroaryl; each of R.sub.2, R.sub.4, and R.sub.5,
independently, is R.sup.c, halogen, nitro, nitroso, cyano, azide,
isothionitro, SR.sup.c, or OR.sup.c; R.sub.3 is R.sup.c, alkenyl,
alkynyl, aryl, heteroaryl, cyclyl, heterocyclyl, OR.sup.c,
OC(O)R.sup.c, SO.sub.2R.sup.c, S(O)R.sup.c,
S(O.sub.2)NR.sup.cR.sup.d, SR.sup.c, NR.sup.cR.sup.d,
NR.sup.cCOR.sup.d, NR.sup.cC(O)OR.sup.d,
NR.sup.cC(O)NR.sup.cR.sup.d, NRCSO.sub.2R.sup.d, COR.sup.c,
C(O)OR.sup.c, or C(O)NR.sup.cR.sup.d; n is 0, 1, 2, 3, 4, 5, 6, or
7; X is O, S, S(O), S(O.sub.2), or NR.sup.c; Y is a covalent bond,
CH.sub.2, C(O), C.dbd.N--R.sup.c, C.dbd.N--OR.sup.c,
C.dbd.N--SR.sup.c, O, S, S(O), S(O.sub.2), or NR.sup.c; Z is CH;
and W is O, S, S(O), S(O.sub.2), NR.sup.c, or NC(O)R.sup.c; in
which each of R.sup.a and R.sup.b, independently, is H, alkyl,
aryl, heteroaryl; and each of R.sup.c and R.sup.d, independently,
is H, alkyl, or alkylcarbonyl. A subset of the triazine compounds
is featured by that R.sup.1 is NC(R.sup.aR.sup.b); and another
subset is featured by that R.sup.1 is aryl or heteroaryl.
[0009] Alkyl, alkenyl, alkynyl, aryl, heteroaryl (e.g., pyridinyl),
cyclyl, heterocyclyl mentioned herein include both substituted and
unsubstituted moieties. The term "substituted" refers to one or
more substituents (which may be the same or different), each
replacing a hydrogen atom. Examples of substituents include, but
are not limited to, halogen, hydroxyl, amino, alkylamino,
arylamino, dialkylamino, diarylamino, cyano, nitro, mercapto,
carbonyl, carbamido, carbamyl, carboxyl, thioureido, thiocyanato,
sulfoamido, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkenyl,
C.sub.1-C.sub.6 alkoxy, aryl, heteroaryl, cyclyl, heterocyclyl,
wherein alkyl, alkenyl, alkoxy, aryl, heteroaryl cyclyl, and
heterocyclyl are optionally substituted with C.sub.1-C.sub.6 alkyl,
aryl, heteroaryl, halogen, hydroxyl, amino, mercapto, cyano, or
nitro. The term "aryl" refers to a hydrocarbon ring system having
at least one aromatic ring. Examples of aryl moieties include, but
are not limited to, phenyl, naphthyl, and pyrenyl. The term
"heteroaryl" refers to a hydrocarbon ring system having at least
one aromatic ring which contains at least one heteroatom such as O,
N, or S. Examples of heteroaryl moieties include, but are not
limited to, furyl, fluorenyl, pyrrolyl, thienyl, oxazolyl,
imidazolyl, thiazolyl, pyridinyl, pyrimidinyl, quinazolinyl, and
indolyl.
[0010] Set forth below are exemplary compounds (Compounds 1-12) of
this invention: ##STR10## ##STR11##
[0011] In still another aspect, this invention features a
pharmaceutical composition that contains a pharmaceutically
acceptable carrier and an effective amount of at least one of the
above-described triazine compounds.
[0012] In further another aspect, the present invention features a
method for treating an IL-12 overproduction-related disorder (e.g.,
rheumatoid arthritis, sepsis, Crohn's disease, multiple Sclerosis,
psoriasis, or insulin-dependent diabetes). The method includes
administering to a subject in need thereof an effective amount of a
triazine compound of formula (I), wherein R.sub.1 is
NC(R.sup.aR.sup.b), aryl, or heteroaryl; each of R.sub.2, R.sub.4,
and R.sub.5, independently, is R.sup.c, halogen, nitro, nitroso,
cyano, azide, isothionitro, SR.sup.c, or OR.sup.c; R.sub.3 is
R.sup.c, alkenyl, alkynyl, aryl, heteroaryl, cyclyl, heterocyclyl,
OR.sup.c, OC(O)R.sup.c, SO.sub.2R.sup.c, S(O)R.sup.c,
S(O.sub.2)NR.sup.cR.sup.d, SR.sup.c, NR.sup.cR.sup.d,
NR.sup.cCOR.sup.d, NR.sup.cC(O)OR.sup.d,
NR.sup.cC(O)NR.sup.cR.sup.d, NR.sup.cSO.sub.2R.sup.d, COR.sup.c,
C(O)OR.sup.c, or C(O)NR.sup.cR.sup.d; n is 0, 1, 2, 3, 4, 5, 6, or
7; X is O, S, S(O), S(O.sub.2), or NR.sup.c; Y is a covalent bond,
CH.sub.2, C(O), C.dbd.N--R.sup.c, C.dbd.N--OR.sup.c,
C.dbd.N--SR.sup.c, O, S, S(O), S(O.sub.2), or NR.sup.c; Z is N or
CH; and W is O, S, S(O), S(O.sub.2), NR.sup.c, or NC(O)R.sup.c; in
which each of R.sup.a and R.sup.b, independently, is H, alkyl,
aryl, heteroaryl; and each of R.sup.c and R.sup.d, independently,
is H, alkyl, or alkylcarbonyl.
[0013] The triazine compounds described above include the compounds
themselves, as well as their salts and their prodrugs, if
applicable. The salts, for example, can be formed between a
positively charged substituent (e.g., amino) on a compound and an
anion. Suitable anions include, but are not limited to, chloride,
bromide, iodide, sulfate, nitrate, phosphate, citrate,
methanesulfonate, trifluoroacetate, and acetate. Likewise, a
negatively charged substituent (e.g., carboxylate) on a compound
can form a salt with a cation. Suitable cations include, but are
not limited to, sodium ion, potassium ion, magnesium ion, calcium
ion, and an ammonium cation such as teteramethylammonium ion.
Examples of prodrugs include esters and other pharmaceutically
acceptable derivatives, which, upon administration to a subject,
are capable of providing the triazine compounds described
above.
[0014] In addition, some of the just-described triazine compounds
have one or more double bonds, or one or more asymmetric centers.
Such compounds can occur as racemates, racemic mixtures, single
enantiomers, individual diastereomers, diastereomeric mixtures, and
cis- or trans- or E- or Z-double bond isomeric forms.
[0015] Also within the scope of this invention are a composition
containing one or more of the compounds described above for use in
treating an IL-12 overproduction-related disorder, and the use of
such a composition for the manufacture of a medicament for the
just-described use. Other features or advantages of the present
invention will be apparent from the following detailed description
of several embodiments, and also from the appending claims.
DETAILED DESCRIPTION
[0016] The compounds described above can be prepared by methods
well known in the art, as well as by the synthetic routes disclosed
herein. For example, a triazine compound of this invention (e.g.,
Compound 1) can be prepared in a stepwise manner by using cyanuric
chloride as a starting material and replacing its three chloro
groups with various substitutes by the methods described above. Due
to the symmetry of cyanuric chloride, the order of displacement is
not of particular importance. For example, a chloro group of
cyanuric chloride can be substituted with a nucleophile
X--R.sub.1--H, wherein X is O or S, thus forming an ether linkage.
In another example, a compound of formula (I), wherein Y is
CH.sub.2 (e.g., Compound 7), can be prepared by reacting the
cyanuric chloride with a Grignard reagent, an organotin reagent, an
organoboric acid, an organocopper reagent or an organozinc reagent
in the presence of an organopalladium compound as a catalyst. If
preferred, other types of linkages can be prepared by similar
nucleophilic reactions. Sensitive moieties on the triazinyl
intermediates and on the nucleophiles can be protected prior to
coupling. For suitable protecting groups, see, e.g., Greene (1981)
Protective Groups in Organic Synthesis, John Wiley & Sons,
Inc., New York. A triazine compound thus synthesized can be further
purified by flash column chromatography, high performance liquid
chromatography, or crystallization.
[0017] Also within the scope of this invention is a pharmaceutical
composition that contains an effective amount of at least one
triazine compound of this invention and a pharmaceutically
acceptable carrier. Further, the present invention covers a method
of administering an effective amount of one or more triazine
compounds described in the "Summary" section to a subject in need
of treatment of IL-12 overproduction related diseases (e.g.,
rheumatoid arthritis, sepsis, or Crohn's disease, multiple
sclerosis, psoriasis, or insulin-dependent diabetes mellitus). "An
effective amount" refers to the amount of the compound which is
required to confer a therapeutic effect on the treated subject. The
interrelationship of dosages for animals and humans (based on
milligrams per meter squared of body surface) is described in
Freireich et al., (1966) Cancer Chemother Rep 50: 219. Body surface
area may be approximately determined from height and weight of the
patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals,
Ardley, N.Y., 1970, 537. An effective amount of the compound of
this invention can range from about 0.001 mg/Kg to about 1000
mg/Kg. Effective doses will also vary, as recognized by those
skilled in the art, depending on the diseases treated, route of
administration, excipient usage, and the possibility of co-usage
with other therapeutic treatments such as use of other agents.
[0018] To practice the method of the present invention, a triazine
compound, as a component of a pharmaceutical composition, can be
administered orally, parenterally, by inhalation spray, topically,
rectally, nasally, buccally, vaginally or via an implanted
reservoir. The term "parenteral" as used herein includes
subcutaneous, intracutaneous, intravenous, intramuscular,
intraarticular, intraarterial, intrasynovial, intrastemal,
intrathecal, intralesional and intracranial injection or infusion
techniques.
[0019] A sterile injectable composition, for example, a sterile
injectable aqueous or oleaginous suspension, can be formulated
according to techniques known in the art using suitable dispersing
or wetting agents (such as, for example, Tween 80) and suspending
agents. The sterile injectable preparation can also be a sterile
injectable solution or suspension in a non-toxic parenterally
acceptable diluent or solvent, for example, as a solution in
1,3-butanediol. Among the acceptable vehicles and solvents that can
be employed are mannitol, water, Ringer's solution and isotonic
sodium chloride solution. In addition, sterile, fixed oils are
conventionally employed as a solvent or suspending medium (e.g.,
synthetic mono- or diglycerides). Fatty acids, such as oleic acid
and its glyceride derivatives are useful in the preparation of
injectables, as are natural pharmaceutically-acceptable oils, such
as olive oil or castor oil, especially in their polyoxyethylated
versions. These oil solutions or suspensions can also contain a
long-chain alcohol diluent or dispersant, or carboxymethyl
cellulose or similar dispersing agents. Other commonly used
surfactants such as Tweens or Spans or other similar emulsifying
agents or bioavailability enhancers which are commonly used in the
manufacture of pharmaceutically acceptable solid, liquid, or other
dosage forms can also be used for the purposes of formulation. A
composition for oral administration can be any orally acceptable
dosage form including, but not limited to, capsules, tablets,
emulsions and aqueous suspensions, dispersions and solutions. In
the case of tablets for oral use, carriers which are commonly used
include lactose and corn starch. Lubricating agents, such as
magnesium stearate, are also typically added. For oral
administration in a capsule form, useful diluents include lactose
and dried corn starch. When aqueous suspensions or emulsions are
administered orally, the active ingredient can be suspended or
dissolved in an oily phase combined with emulsifying or suspending
agents. If desired, certain sweetening, flavoring, or coloring
agents can be added. A nasal aerosol or inhalation composition can
be prepared according to techniques well-known in the art of
pharmaceutical formulation and can be prepared as solutions in
saline, employing benzyl alcohol or other suitable preservatives,
absorption promoters to enhance bioavailability, fluorocarbons,
and/or other solubilizing or dispersing agents known in the art. A
triazine compound can also be administered in the form of
suppositories for rectal administration.
[0020] The carrier in the pharmaceutical composition must be
"acceptable" in the sense of being compatible with the active
ingredient of the formulation (and preferably, capable of
stabilizing it) and not deleterious to the subject to be treated.
For example, solubilizing agents such as cyclodextrins, which form
specific, more soluble complexes with the compounds of this
invention, or one or more solubilizing agents, can be utilized as
pharmaceutical excipients for delivery of a triazine compound.
Examples of other carriers include colloidal silicon dioxide,
magnesium stearate, cellulose, sodium lauryl sulfate, and D&C
Yellow # 10.
[0021] The biological activities of a triazine compound can be
evaluated by a number of cell-based assays. One of such assays can
be conducted using cells from human peripheral blood cells (PBMC)
or human monocytic cell line (THP-1). The cells are stimulated with
a combination of human interferon-.gamma. and lipopolysaccharide or
a combination of IFN.gamma. and Staphylococcus aureus Cowan I in
the presence of a test compound. The level of inhibition of IL-12
production can be measured by determining the amount of p70 by
using a sandwich ELISA assay with anti-human IL-12 antibodies.
IC.sub.50 of the test compound can then be determined.
Specifically, PBMC or THP-1 cells are incubated with the test
compound. Cell viability was assessed using the bioreduction of MTS
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl-
)-2H-tetrazolium] (Promega, Madison, Wis.).
[0022] A triazine compound can also be evaluated by animal studies.
For example, one of such studies involves the ability of a test
compound to treat adjuvant arthritis (i.e., a IL-12 overproduction
related disorder) in rats.
[0023] Without further elaboration, it is believed that the above
description has adequately enabled the present invention. The
following specific embodiments are, therefore, to be construed as
merely illustrative, and not limitative of the remainder of the
disclosure in any way whatsoever. All of the publications cited
herein are hereby incorporated by reference in their entirety.
EXAMPLE 1
Preparation of Compound 1:
N-(1H-indol-3-ylmethylene)-N'-[4-morpholin-4-yl-6-(2-pyridin-2-yl-ethoxy)-
-[1,3,5]triazin-2-yl]-hydrazine
[0024] Cyanuric chloride (13.66 g, 74 mmol) was dissolved in
methylene chloride (100 mL) at -78.degree. C., followed by the
addition of diisopropylethylamine (12.9 mL, 74 mmol). The reaction
mixture was stirred for 5 minutes. Morpholine (6.46 mL, 74 mmol)
was added dropwise into the reaction mixture in 10 min. The
resulting white precipitate was filtered, washed with water, and
dried to afford the desired intermediate in quantitative yield (17
g, 100%).
[0025] 2-(2-Hydroxyethyl)pyridine (2 g, 16.2 mmol) was dissolved in
THF (20 mL) at 0.degree. C. 6.5 mL of 2.5 M n-butyl lithium (16.2
mmol) was added into the pyridine solution dropwise in 5 min. The
resulting solution was then added dropwise via cannula to a
triazine dichloride solution (3.8 g, 16.2 mmol, in THF) at
-78.degree. C. The reaction was allowed to warm to room temperature
for overnight to yield the triazine monochloride intermediate (2.8
g, 54%) as a white powder. Hydrazine (0.5 mL, 15.5 mmol) was
dissolved in 10 mL ethanol at room temperature. The triazine
monochloride intermediate (1 g, 3.11 mmol) was added to a solution
of ethanol (20 mL) and heated to 60.degree. C. before adding into
the hydrazine solution. After stirring for 30 min, white crystals
precipitated, which were then filtered, washed with water and air
dried to yield the triazine hydrazine intermediate (781 mg, 78%) as
a white powder.
[0026] Indole-3-aldehyde (1.05 g, 7.25 mmol) and the triazine
hydrazine intermediate (2.3 g, 7.25 mmol) were added to 30 mL of
methanol at room temperature. 5 mL of acetic acid was added to the
reaction mixture and was refluxed for 5 min. Upon cooling, a white
precipitate was formed, which was filtered and washed with water to
yield Compound 1 as a white powder (1.7 g, 52%).
[0027] .sup.1H NMR (CDCl.sub.3), .delta. (ppm): 3.28 (t, J=6.9,
2H); 3.7 (broad s, 4H); 3.86 (broad s, 4H); 4.73 (broad t, 2H);
7.14-7.24 (m, 2H); 7.27-7.30 (m, 3H); 7.37 (d, J=8.1, 1H); 7.45 (d,
J=2.4, 1H); 7.59 (t, J=7.5, 1H); 8.14 (s, 1H); 8.42 (d, J=7.8, 1H);
8.49 (s, 1H); and 8.56 (d, J=8.5, 1H).
[0028] MS (ESI): m/z 445.2 (M+H).
EXAMPLE 2
Preparation of Compound 2:
2,3-dimethyl-1H-indol-5-yl)-[4-morpholin-4-yl-6-(2-pyridin-2-yl-ethoxy)-[-
1,3,5]triazin-2-yl]-amine
[0029] To a solution of cyanuric chloride (0.922 g, 5.00 mmol, 1.00
equiv.) in 15 mL CH.sub.2Cl.sub.2 at 0.degree. C. was added slowly
DIPEA (1.422 g, 11.00 mmol, 2.20 equiv.) during a period of 10
minutes. Ice bath was removed, and 2-(2-hydroxyethyl)pyridine
(0.677 g, 5.50 mmol, 1.10 equiv.) was added, and the reaction
mixture was stirred at room temperature for 15 minutes.
5-Amino-2,3-dimethylindole (0.641 g, 4.00 mmol, 0.80 equiv.) was
then added, and stirred for 4 hours at room temperature. A light
brown solid precipitated out after 10 mL of water was added to the
reaction mixture and stirred for about 10 minutes. The light brown
solid was collected by filtration, washed with 2.times.10 mL water,
5 mL EtOAc and dried (1.50 g, 3.80 mmol, 95%). This solid was then
added to a solution of morpholine (0.827 g, 9.5 mmol, 2.50 equiv.)
in 30 mL THF, and stirred at 60.degree. C. for 4 hours. Usual
workup and flash chromatography purification gave Compound 2 as an
off-white solid (1.30 g, 2.92 mmol, 77%).
[0030] .sup.1H NMR (300 MHz, DMSO-d.sub.6), .delta. ppm: 10.50 (s,
1H); 9.29 (br s, 1H); 8.51 (d, J=4.8 Hz, 1H); 7.70-7.79 (m, 2H);
7.22-7.34 (m, 2H); 7.10 (s, 2H); 4.63 (t, J=6.9 Hz, 2H); 3.71 (br
s, 4H); 3.63 (br s, 4H); 3.16 (t, J=6.9 Hz, 2H); 2.78 (s, 3H), 2.07
(br s, 3H); MS (ESI): m/z 446.2 (M+H).sup.+.
EXAMPLE 3
Preparation of Compound 3:
N-(1H-indol-3-ylmethylene)-N'-[4-morpholin-4-yl-6-(2-pyridin-3-yl-ethoxy)-
-[1,3,5]triazin-2-yl]-hydrazine
[0031] Compound 3 was prepared in a similar manner as described in
Example 1.
[0032] .sup.1H NMR (300 MHz, CDCl.sub.3), .delta. ppm: 9.10 (br s,
1H); 8.55 (d, J=1.8 Hz, 1H); 8.47-8.49 (m, 2H); 8.34-8.41 (m, 1H);
8.07 (s, 1H); 7.60 (dt, J=1.8 Hz, 7.5 Hz, 1H); 7.34-7.39 (m, 2H);
7.14-7.25 (m, 3H); 4.58 (br s, 2H); 3.86 (br s, 4H); 3.75 (br s,
4H); 3.09 (t, J=7.2 Hz, 1H); MS (ESI): m/z 445.1 (M+H).sup.+.
EXAMPLE 4
Preparation of Compound 4:
N-(3-Methoxy-benzylidene)-N'-[4-morpholin-4-yl-6-(2-pyridin-2-yl-ethoxy)--
[1,3,5]triazin-2-yl]-hydrazine
[0033] Compound 4 was prepared in a similar manner as described in
Example 1.
[0034] .sup.1H NMR (300 MHz, DMSO-d.sub.6), .delta. ppm: 11.19 (s,
1H); 8.52 (dd, J=3.9 Hz, 0.9 Hz, 1H); 8.07 (s, 1H); 7.73 (m, 1H);
7.19-7.36 (m, 4H); 6.95 (dd, J=7.8 Hz, 2.4 Hz, 1H); 4.64 (t, J=6.3
Hz, 2H); 3.64-3.78 (m, 11H); 3.17 (t, J=6.3 Hz, 2H); MS (ESI): m/z
436.2 (M+H).sup.+.
EXAMPLE 5
Preparation of Compound 5:
N-(3-methyl-benzylidene)-N'-[4-morpholin-4-yl-6-(2-pyridin-2-yl-ethoxy)-[-
1,3,5]triazin-2-yl]-hydrazine
[0035] Compound 5 was prepared in a similar manner as described in
Example 1.
[0036] .sup.1H NMR (300 MHz, DMSO-d.sub.6), .delta. ppm: 11.14 (s,
1H); 8.52 (dd, J=3.9 Hz, 0.9 Hz, 1H); 8.07 (s, 1H); 7.73 (m, 1H);
7.17-7.45 (m, 6H); 4.64 (t, J=6.3 Hz, 2H); 3.63-3.73 (m, 8H); 3.17
(t, J=6.3 Hz, 2H); 2.33 (s, 3H); MS (ESI): m/z 420.2
(M+H).sup.+.
EXAMPLE 6
Preparation of Compound 6:
4-{4-[N'-(1H-indol-3-ylmethylene)-hydrazino]-6-morpholin-4-yl-[1,3,5]tria-
zin-2-yl}-butan-1-ol
[0037] Compound 6 was prepared in a similar manner as described in
Example 7.
[0038] .sup.1H NMR (300 MHz, CDCl.sub.3+DMSO-d.sub.6, 8:1), .delta.
ppm: 10.16 (br s, 1H); 9.17 (br s, 1H); 8.37-8.47 (m, 1H); 8.21 (s,
1H); 7.36-7.47 (m, 3H); 7.17-7.26 (m, 2H); 3.93 (br s, 4H); 3.77
(br s, 4H); 3.65 (t, J=6.3 Hz, 2H); 2.62 (br s, 2H); 1.84-1.92 (m,
2H); 1.62-1.71 (m, 2H); MS (ESI): m/z 396.2 (M+H).sup.+.
EXAMPLE 7
Preparation of Compound 7:
N-{4-[3-(3,4-dimethoxy-phenyl)-propyl]-6-morpholin-4-yl-1,3,5-triazin-2-y-
l}-N'-[1-(1H-indol-3-yl)-meth-(E)-ylidene]-hydrazine
[0039] To a solution of 3-(3,4-dimethoxyphenyl)-propyl iodide
(1.224 g, 4.00 mmol, 1.00 equiv.) in 20 mL dry THF was added highly
active zinc (suspension in THF, Rieke metal from Aldrich, 5.2 mL
0.05 g/mL, 4.00 mmol, 1.00 equiv.). The mixture was stirred at room
temperature overnight. 2,4-dichloro-6-morpholin-4-yl-1,3,5-triazine
(0.936 g, 4.0 mmol, 1.00 equiv.) and
trans-benzyl-(chloro)-bis-(triphenylphosphine)palladium(II) (0.03
g, 0.04 mmol, 0.01 equiv.) were added, and the reaction mixture was
stirred at room temperature for 8 hours. Usual workup and flash
chromatography purification gave
4-chloro-2-[3-(3,4-dimethoxyphenyl)propyl]-6-morpholin-4-yl-1,3,5-triazin-
e as a light yellow solid which was treated with hydrazine
following the typical procedure to yield
{4-[3-(3,4-Dimethoxy-phenyl)-propyl]-6-morpholin-4-yl-1,3,5-triazin-2-yl}-
-hydrazine as a white solid (0.85 g, 2.27 mmol, 57%). MS (ESI): m/z
375.2 (M+H).sup.+.
[0040] A mixture of
{4-[3-(3,4-dimethoxy-phenyl)-propyl]-6-morpholin-4-yl-1,3,5-triazin-2-yl}-
-hydrazine (0.75 g, 2.00 mmol, 1.00 equiv.),
indole-3-carboxaldehyde (0.29 g, 2.00 mmol, 1.00 equiv.), and AcOH
(80 mg, cat.) in 15 mL MeOH was stirred at 75.degree. C. for 4
hours. Solvent was removed and the residue was subjected to flash
chromatography purification and crystallization in MeOH to yield
Compound 7 as an off-white solid (0.72 g, 1.44 mmol, 72%).
[0041] .sup.1H NMR (300 MHz, CDCl.sub.3), .delta. ppm: 8.57 (br s,
1H); 8.45 (br s, 1H); 8.29-8.32 (m, 1H); 8.00 (s, 1H); 7.39-7.43
(m, 2H); 7.23-7.34 (m, 2H); 6.74-6.80 (m, 3H); 6.30 (s, 1H); 3.86
(s, 3H); 3.85 (s, 3H); 3.78-3.84 (m, 4H); 3.67-3.70 (m, 4H);
2.63-2.71 (m, 4H), 2.03-2.13 (m, 2H); MS (ESI): m/z 502.2
(M+H).sup.+.
EXAMPLE 8
Preparation of Compound 8:
N-{4-[2-(2,2-Dimethyl-[1,3]dioxolan-4-yl)-ethoxy]-6-morpholin-4-yl-[1,3,5-
]triazin-2-yl}-N'-(1H-indol-3-ylmethylene)-hydrazine
[0042] Compound 8 was prepared in a similar manner as described in
Example 1.
[0043] .sup.1H NMR (300 MHz, CD.sub.3Cl) .delta. (ppm): 8.50 (s,
1H), 8.42 (d, J=8.4 Hz, 1H), 8.24 (s, 1H), 8.09 (s, 1H), 7.44 (d,
J=3.0 Hz, 1H), 7.38 (d, 1H, J=7.2 Hz), 7.20-7.26 (m, 2H), 4.55
(br., 2H), 4.28 (d, J=7.4 Hz, 1H) 3.84 (m, 4H), 3.71 (m, 4H), 3.60
(t, J=7.4 Hz, 2H), 2.03 (m, 2H), 1.42 (s, 3H), 1.35 (s, 3H). MS
(ESI): m/z 468.3 (M+H).sup.+.
EXAMPLE 9
Preparation of Compound 9:
N-[4-(4,5-dihydro-oxazol-2-ylmethoxy)-6-morpholin-4-yl-[1,3,5]triazin-2-y-
l]-N'-(1H-indol-3-ylmethylene)-hydrazine
[0044] Compound 9 was prepared in a similar manner as described in
Example 1.
[0045] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. (ppm): 11.40 (s,
1H), 10.91 (s, 1H), 8.32-8.28 (m, 2H), 7.68 (bs, 1H), 7.40-7.37 (m,
1H), 7.21-7.05 (m, 2H), 4.80-4.66 (m, 4H), 3.75-3.55 (m, 8H), 3.15
(s, 2H); MS (ESI): m/z 423.1.
EXAMPLE 10
Preparation of Compound 10:
{4-[N'-(1H-indol-3-ylmethylene)-hydrazino]-6-morpholin-4-yl-[1,3,5]triazi-
n-2-yloxy}-acetic acid ethyl ester
[0046] Compound 10 was prepared in a similar manner as described in
Example 1.
[0047] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. (ppm): 8.62-8.60
(m, 1H), 8.42 (d, 1H, J=9.0 Hz), 8.09 (s, 1H), 7.45 (bs, 1H),
7.39-7.36 (m, 1H), 7.28-7.20 (m, 3H), 4.84 (s, 2H), 4.27-4.19 (m,
2H), 3.80-3.65 (m, 8H), 1.25 (t, 3H, J=7.2 Hz); MS (ESI): m/z
426.1.
EXAMPLE 11
Preparation of Compound 11:
N-(2-hydroxy-ethyl)-2-{4-[N'-(1H-indol-3-ylmethylene)-hydrazino]-6-morpho-
lin-4-yl-[1,3,5]triazin-2-yloxy}-acetamide
[0048] Compound 11 was prepared in a similar manner as described in
Example 1.
[0049] .sup.1H NMR (DMSO-d.sub.6) 6 (Ppm): 11.40 (s, 1H), 10.92 (s,
1H), 8.32-8.28 (m, 2H), 8.00-7.93 (m, 1H), 7.69 (bs, 1H), 7.40-7.37
(m, 1H), 7.21-7.05 (m, 2H), 4.75-4.60 (m, 4H), 3.75-3.55 (m, 8H),
3.20-3.10 (m, 2H); MS (ESI): m/z 441.1.
EXAMPLE 12
Preparation of Compound 12:
4-[4-(2,3-Dimethyl-1H-indol-5-ylamino)-6-morpholin-4-yl-[1,3,5]triazin-2--
yloxy]-benzonitrile
[0050] Compound 11 was prepared in a similar manner as described in
Example 2.
[0051] .sup.1H-NMR (300 MHz, DMSO-d.sub.6), .delta. (ppm): 1.93 (s,
1H), 2.08 (s, 2H), 2.27 (s, 3H), 3.74-3.27 (m, 8H), 6.99 (s, 1H),
7.09 (s, 1H), 7.46 (d, J=8.7 Hz), 7.79 (s, 1H), 7.91 (d, J=8.7 Hz),
9.46 (s, 1H), 10.51 (s, 1H). MS (ESI): m/z 441.2 (M+H).sup.+.
EXAMPLE 13
In Vitro Assays
[0052] Reagents. Staphylococcus aureus Cowan I (SAC) was obtained
from Calbiochem (La Jolla, Calif.), and lipopolysaccharide (LPS,
Serratia marscencens) was obtained from Sigma (St. Louis, Mo.).
Human and mouse recombinant IFN.gamma. were purchased from
Boehringer Mannheim (Mannheim, Germany) and Pharmingen (San Diego,
Calif.), respectively.
[0053] Human In Vitro Assay. Human PBMC were isolated by
centrifugation using Ficoll-Paque (Pharmacia Biotech, Uppsala,
Sweden) and prepared in RPMI medium supplemented with 10% fetal
calf serum (FCS), 100 U/mL penicillin, and 100 .mu.g/mL
streptomycin. PBMC were plated in wells of a 96-well plate at a
concentration of 5.times.10.sup.5 cells/well, and primed by adding
IFN.gamma. (30 U/mL) for 22 h and stimulated by adding LPS (1
.mu.g/mL), or by adding IFN.gamma. (100 U/mL) and then stimulated
by adding SAC (0.01%). A test compound was dissolved in DMSO, and
added to wells of the 96 well plate. The final DMSO concentration
was adjusted to 0.25% in all cultures, including the compound-free
control. Human THP-1 cells were plated in wells, primed by adding
IFN.gamma. (100 U/mL) for 22 h and stimulated by adding SAC
(0.025%) in the presence of different concentrations of the test
compound. Cell-free supernatants were taken 18 h later for
measurement of cytokines. Cell viability was assessed using the
bioreduction of MTS. Cell survival was estimated by determining the
ratio of the absorbance in compound-treated groups versus
compound-free control.
[0054] The supernatant was assayed for the amount of IL-112p40,
IL-12p70, or IL-10 by using a sandwich ELISA with anti-human
antibodies, i.e., a Human IL-12 p40 ELISA kit from R&D Systems
(Berkeley, Calif.), and a Human IL-12 p70 or IL-10 ELISA kit from
Endogen (Cambridge, Mass.). Assays were based on the manufacturer's
instructions.
[0055] Murine In Vitro Assay. Balb/c mice (Taconic, Germantown,
N.Y.) were immunized with Mycobacterium tuberculosis H37Ra (Difco,
Detroit, Mich.). The splenocytes were harvested 5 days and prepared
in RPMI medium supplemented with 10% FCS and antibiotics in a flat
bottom 96-well plate with 1.times.10.sup.6 cells/well. The
splenocytes were then stimulated with a combination of IFN.gamma.
(60 ng/mL) and SAC (0.025%) [or LPS (20 .mu.g/mL)] in the presence
of a test compound. Cell-free supernatants were taken 24 h later
for the measurement of cytokines. The preparation of compound and
the assessment of cell viability were carried out as described
above. Mouse IL-12 p70, IL-10, IL-1.beta., and TNF.alpha. were
measured using ELISA kits from Endogen, according to the
manufacturer's instructions.
[0056] The biological activities of triazine compounds were tested
on human PBMC or THP-1 cells. At least 240 compounds have IC.sub.50
values of at least 5 .mu.M. Unexpectedly, some of the test
compounds have IC.sub.50 values as low as <1 nM.
EXAMPLE 14
In Vivo Assays
[0057] Treatment of adjuvant arthritis in rats: Adjuvant arthritis
(AA) was induced in female Lewis rats by the intracutaneous
injection (base of the tail) of 0.1 mL of a 10 mg/mL bacterial
suspension made from ground, heat-killed Mycobacterium tuberculosis
H37Ra suspended in incomplete Freund's adjuvant. Rats were given a
test compound orally once a day for 12 days, starting the day
following the induction. The development of polyarthritis was
monitored daily by macroscopic inspection and assignment of an
arthritis index to each animal, during the critical period (days 10
to 25 post-immunization).
[0058] The intensity of polyarthritis was scored according to the
following scheme: (a) Grade each paw from 0 to 3 based on erythema,
swelling, and deformity of the joints: 0 for no erythema or
swelling; 0.5 if swelling is detectable in at least one joint; 1
for mild swelling and erythema; 2 for swelling and erythema of both
tarsus and carpus; and 3 for ankylosis and bony deformity. Maximum
score for all 4 paws was thus 12. (b) Grade for other parts of the
body: for each ear, 0.5 for redness and another 0.5 if knots are
present; 1 for connective tissue swelling (saddle nose); and 1 for
the presence of knots or kinks in the tail. The highest possible
arthritic index was 16.
[0059] Experiments with the AA model were repeated four times. Oral
administration of triazine compounds described above (e.g.,
Compounds 1 and 2) reproducibly reduced the arthritic score and
delayed the development of polyarthritis in a dose-dependent
manner. The arthritis score used in this model was a reflection of
the inflammatory state of the structures monitored and the results
therefore show the ability of the test compound to provide relief
for this aspect of the pathology.
[0060] Treatment of Crohn's disease in dinitrobenzene sulfonic
acid-induced inflammatory bowel syndrome model rats: Wistar derived
male or female rats weighing 200.+-.20 g and fasted for 24 hours
were used. Distal colitis was induced by intra-colonic instillation
of 2,4-dinitrobenzene sulfonic acid (DNBS, 25 mg in 0.5 mL ethanol
30%) after which air (2 mL) was gently injected through the cannula
to ensure that the solution remained in the colon. A test compound
and/or vehicle was administered orally 24 and 2 hours before DNBS
instillation and then daily for 5 days. One control group was
similarly treated with vehicle alone while the other is treated
with vehicle plus DNBS. The animals were sacrificed 24 hours after
the final dose of test compound administration and each colon was
removed and weighed. Colon-to-body weight ratio was then calculated
for each animal according to the formula: Colon (g)/BW.times.100.
The "Net" increase in ratio of Vehicle-control+DNBS group relative
to Vehicle-control group was used as a base for comparison with
test substance treated groups and expressed as "% Deduction."
Triazine compounds described above (e.g., Compounds 1 and 2)
reproducibly had about 30% deduction. A 30% or more reduction in
colon-to-body weight ratio, relative to the vehicle treated control
group, was considered significant.
[0061] Rats treated with test substance orally showed a marked
reduction in the inflammatory response. These experiments were
repeated three times and the effects were reproducible.
[0062] Treatment of Crohn's disease in CD4.sup.+ CD45Rb.sup.high T
cell-reconstituted SCID colitis model mice: Spleen cells were
prepared from normal female BALB/c mice. For cell purification, the
following anti-mouse antibodies were used to label non-CD4.sup.+ T
cells: B220 (RA3-6B2), CD11b (M1/70), and CD8 cc (53-6.72). All
antibodies were obtained from BioSource (Camarillo, Calif.). M450
anti-rat IgG-coated magnetic beads (Dynal, Oslo, Norway) were used
to bind the antibodies and negative selection was accomplished
using an MPC-1 magnetic concentrator. The enriched CD4.sup.+ cells
were then labeled for cell sorting with FITC-conjugated CD45RB
(16A, Pharmingen, San Diego, Calif.) and PE-conjugated CD4 (CT-CD4,
Caltag, Burlingame, Calif.). CD4.sup.+ CD45RBhigh cells were
operationally defined as the upper 40% of CD45Rb-staining CD4.sup.+
cells and sorted under sterile conditions by flow cytometry.
Harvested cells were resuspended at 4.times.10.sup.6/mL in PBS and
injected 100 .mu.L intraperitoneally into female C.B-17 SCID mice.
Triazine compounds described above (e.g., Compounds 1 and 2) and/or
vehicle was orally administered once a day for 5 days per week,
starting the day following the transfer. The transplanted SCID mice
were weighed weekly and their clinical condition was monitored.
[0063] Colon tissue samples were fixed in 10% buffered formalin and
embedded in paraffin.
[0064] Sections (4 .mu.m) collected from ascending, transverse, and
descending colon were cut and stained with hematoxylin and eosin.
The severity of colitis was determined based on histological
examination of the distal colon sections, whereby the extent of
colonic inflammation was graded on a scale of 0-3 in each of four
criteria: crypt elongation, cell infiltration, depletion of goblet
cells, and the number of crypt abscesses.
[0065] LP lymphocytes were isolated from freshly obtained colonic
specimens. After removal of payer's patches, the colon was washed
in Ca/Mg-free HBSS, cut into 0.5 cm pieces and incubated twice in
HBSS containing EDTA (0.75 mM), DTT (1 mM), and antibiotics
(amphotericin 2.5 .mu.g/mL, gentamicin 50 .mu.g/mL from Sigma) at
37.degree. C. for 15 min. Next, the tissue was digested further in
RPMI containing 0.5 mg/mL collagenase D, 0.01 mg/mL DNase I
(Boehringer Manheim), and antibiotics at 37.degree. C. LP cells
were then layered on a 40-100% Percoll gradient (Pharmacia,
Uppsala, Sweden), and lymphocyte-enriched populations were isolated
from the cells at the 40-100% interface.
[0066] To measure cytokine production, 48-well plates were coated
with 10 .mu.g/mL murine anti-CD3.epsilon. antibody (145-2C11) in
carbonate buffer (PH 9.6) overnight at 4.degree. C.
5.times.10.sup.5 LP cells were then cultured in 0.5 ml of complete
medium in precoated wells in the presence of 1 .mu.g/mL soluble
anti-CD28 antibody (37.51). Purified antibodies were obtained from
Pharmingen. Culture supernatants were removed after 48 h and
assayed for cytokine production. Murine IFN.gamma. was measured
using an ELISA kit from Endogen (Cambridge, Mass.), according to
the manufacturer's instructions.
[0067] Histological analysis showed that oral administration of
triazine compounds described above (e.g., Compounds 1 and 2)
reduced colonic inflammation as compared to vehicle control. The
suppressive effect was dose-dependent with a substantial reduction
at a dose of 10 mg/kg. The calculated colon-to-body weight ratio
was consistent with the histological score, showing attenuation by
treatment with the test compound. Furthermore, analysis of
cytokines from LP cells in response to anti-CD3 antibody and
anti-CD28 antibody demonstrated that LP cells from vehicle control
produced an augmented level of IFN.gamma. and treatment with test
substance greatly diminished the production. These results clearly
demonstrated the potential of the test substance in treatment of
inflammatory bowel disease represented by Crohn's disease.
Other Embodiments
[0068] All of the features disclosed in this specification may be
combined in any combination. Each feature disclosed in this
specification may be replaced by an alternative feature serving the
same, equivalent, or similar purpose. Thus, unless expressly stated
otherwise, each feature disclosed is only an example of a generic
series of equivalent or similar features.
[0069] From the above description, one skilled in the art can
easily ascertain the essential characteristics of the present
invention, and without departing from the spirit and scope thereof,
can make various changes and modifications of the invention to
adapt it to various usages and conditions. For example, compounds
structurally analogous a triazine compound described in the
specification also can be made, screened for their inhibiting IL-12
activities, and used to practice this invention. Thus, other
embodiments are also within the claims.
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