Note: Descriptions are shown in the official language in which they were submitted.
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Description
PYRIMIDINE CARBOXAMIDES AND RELATED COMPOUNDS AND METHODS
FOR TREATING INFL.AMMATORY CONDITIONS
s
Technical Field
The present invention relates generally to compounds that block
intr~ce~ r signal tr~ned~ction and activation of ~ s.;~ lion factors, and to m~h- -l,e
for prc ~elllil.g or ll~,alillg immllnoinfl~n~ln~lQly and ~utQimmllne ~lieç:le~,e,
Background of the Invention
Signals n~cçse~ry for cell growth, di~el elllialion, re~l,onse to
bioregulatory molecules, infectious agents and physiological stress involve f.11~nges in the
rates of gene c~.css;on. The ability to respond applopliately to such eign~ling events
15 challenge the survival of the cell and llhim~tçly the or~ ~ism. Perturbations in the
normal re~ll~ti~ n of these specific genetic responses can result in pathogenic events
which lead to acute and chronic disease.
In certain auk~ e ~iee~ees or chronic infl~mm~tQry states,
contim-ous activation of T-cells eventually leads to a self-perpet~fing destruction of
20 normal tissues or organs. This is caused by the induction of adhesion molecules,
cllcl..ola~is of leukocytes, activation of leukocytes and the production of merii~tQrs of
il~lli.. ~;on. All of these events are re~ teA at the level of ll~ls~ ion for the
production of new proteins, in~ 1ing cytokines. The production of cytokines, as well
as a number of other cellular regulators, is controlled by a family of proteins known as
25 transcription factors (TFs). These transcription factors, when activated, bind to specific
regions on the DNA and act as molecular switches or m~osSçng~rs to induce or
upregulate gene c~les~ion. The activation of these TFs is caused by a variety ofexternal signals incl~ltlinp physiological stress, infectious agents and other bioregulatory
molecules. Once the plasma .-.el--l~-~ne .~ceplc.~ are activated, a c~eciqde of protein
30 kinases and second m~seengers are indl~ced which, in turn, result in the production of
RNA transcripts. The end result is the production of proinfl~mm~tory proteins via
translation and processing of the RNA transcripts.
This activation system can, at times, be very robust. For example, a
specific set of external signals could result in a single Ll~ls~ ,lion factor to induce many
35 proteins responsible for a given disease. Therefore, re~ll~ting this process by disrupting
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the production of activated TF(s) has the l)otel~Lial to ~tten~te the pro~luction of the
associated pathological ploleil-s, thereby halting or reversing the course ofthe disease.
Two l~ansc,ipLion factors, NFlcB and AP-l, have been shown to regulate
the prod~-ction of many p-v;~ln;1~ ory cytokines and related proteins that are elevated
5 in immm~o;i.n,....,..~loly ~ie~A~es These TFs regulate interleukin-l (~-1), interleukin-2
(IL-2), tumor necrosis factor-a (TNFa), interleukin-6 (IL-6) and interleukin-8 (IL-8)
levels in a variety of cell types. For example, NFlcB and other related co ~! 9 are
involved in the rapid inrl~ction of genes whose products function in prolecLi~e and
proliferative responses upon exposure of cells to external stimuli. Similarly, AP-l has a
10 significant role in the re~-l~ti-)n of interleukin-2 (IL-2) and tumor necrosis factor~
(TNF-a) L-~lsc-il.lion during T-cell activation. In addition, TNF-a and IL-l are strong
activators of coll~g~n~eç7 g~l~tin~ee and stromelysin gene cA~ulG~ion~ which require a
single AP-l binding site in the promoter region of these genes. ThGlel;JlG, an inhibitor of
NFIcB and/or AP-l activation would coordinately repress the activities of a series of
15 protçin~eeS In addition, cell adhesion mol~c~ e are also controlled by these TFs. All of
these p-~,lehls have been shown to play a role in ~liee~ee~, inc~ in51~ osteoarthritis,
transplant reje-ction iec.l~ reperfusion injury, trauma, certain cancers and viral
disorders, and autoimm-lne tlicç~ees such as rhe-lm~toid arthritis, multiple sclerosis,
psoriasis, ;~nS~ ory bowel disease, glomerulonephritis, lupus and juvenile diabetes.
20 In summary, the role of these TFs is to act as a tr~nedl~c~r for certain stimuli that lead to
imm~n~ n~ Ory~ and acute phase .es~onses.
Since many ~liee~ees are caused by the hlapplop.iate production of
proteins, conventional therapeutic approaches have focused on inhibiting function or
activity of individual effector proteins. These tre~tm~onte have not always proved to be
25 effective and, at times, are associated with many undesirable side effects. Therefore,
there is a need for new therapies for the prevention and/or tr.o~tment of
imm-.. ~o;.. n~.. ~lory and ~-toimm--ne ~liee~es More specifically, there is a need for
compounds that prevent, preferably by inhibiting transcription at an early stage, the
production of p-oteins ~esori~ted with imm--noil.llA.. ~o~y and auto;.. ~ -e .I;eç~ees
Furthermore, these compounds should inhibit the kinase(s) that regulate the activation of
TFs such as NFlcB and AP-l. The present invention fulfills these needs and provides
further related adv~nt~q~es
Swul~a~y ofthe Invention
In brief, this invention is directed to compounds that block the activation
of transcription factors (TFs), particularly NFlcB and AP-l, and are believed to function
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through inhibition of a family of specific kinases. This results in a decrease in a number
of p~o;~ oly proteins, inC~ ing IL 1, IL-2, IL-8 and/or TNFa, which are
~ poll~;ble for tissue and organ ~l~m~gç associated with tli~e~ces such as ,l.e.. ~l~id
arthritis, osteo_llllilis~ related autoimm--ne disorders and tissue rejection Acco..li..~,ly,
5 compounds of the present invention are useful in, for example, the prevention of organ
and tissue rejection ~c,co~ ted with tr~nCpls~ t~tion Furthermore, the compounds of this
invention also have utility in the prevention and/or lIeAI~ I of imm~.l)oi..n~.,,,,.,.lQly
and ~toimm~ne ~ ec, as well as having general activity as anti-infl~mm~tory agents.
In one embodiment of this invention, compounds are disclosed having the
10 following general structure (I):
R4
N ~ B
R2
(I)
wheleill A is C-R6 when B is N, and A is N when B is C-RI, and wherein R" R2, R4, R5
and R6 are as defined in the following detailed description.
In another embodiment, a pharmaceutical composition is disclosed
co..lAil)iog one or more compounds of this invention in co.,.binalion with a
pharm~e~tic~lly or prophylactically acceptable carrier or diluent.
In a further embodiment, methods are disclosed for preventing and/or
L-ealillg infl~mm~tory conditions by ~ illg to a warm-blooded animal in need
thereof an effective amount of a compound of this invention. Such ;.~nh.......... ~lory
conditions include both imm~ in~.. ~tory conditions and autoimmlme tli~ç~es Inthe practice of the disclosed methods, the compounds are preferably ~r1mini~tçred to the
warm-blooded animal in the form of a pharm~e~lflcal composition.
These and other aspects of this invention will become evident upon
25 reference to the attached figures and the following detailed description.
Brief Description of the Drawin~s
Figure I illustrates a reaction scheme for the synthesis of repleselllali~e
pyrimidine-co..l~;..;.-g compounds ofthis invention.
Figure 2 illustrates a reaction scheme for the synthesis of lel).esel.lali~e
pyrazine-co..l;.;..;..g compounds ofthis invention.
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Figure 3 illustrates the ability of a re~.esellLa~ e compound of this
invention to inhibit the activation of NFKB and AP~
Figure 4 illustrates the ability of a It;pres~ ali~e compound of this
invention to inhibit IL-2 and IL-8.
S Figure S illustrates the ability of a l~,pr~se~ e compound of this
invention to cause a dose-depen-lçnt supp~e~;on of ~lloanti~en_in-lllced PLN
proliferation.
Detailçd Des~iliplion ofthe Invention
As mentioned above, the compounds ofthis invention block activation of
scliplion factors (TFs), and thus have utility as anti-i.,ll,...~ lory agents in general,
and in the prevention and/or tre~tmlont of a variety of conditions, inclu~ing (but not
limited to) immllno;--ll~------~lQry and autoimmllne ~liCç~ces The compounds are believed
to function by h hibilillg, at an early stage, llanscliplion of deleterious ploteills
15 associated with such conditions or ~l;ce~c~s It is believed that this is achieved by
inhibiling the kinase(s) that regulate the activation of TFs, such as NFKB and/or AP-l.
By disrupting the production of these activated TFs, synthesis of pathological proLei-ls,
in~ in~ ploi~ oly cytokines, associated with a series of imml~oi~n~ Qry
and autoimmlme ~iice~ces are effectively blocked at a l,ans~ lional level. Accoldillgly,
20 the compounds of this invention have activity in both the prevention and lle~ of
immlmoinfl~.. ~c.ly ~lice~ces such as rhellm~toid arthritis, osteoarthritis and lla~.c~l~
rejection (tissue and organ), as well as autoimm~lne ~1ice~cçs such as multiple sclerosis.
The compounds of this invention are generally represented by the
following general structure (I):
R4~
N~B
R2
(I)
wherein A is C-R6 when B is N, and A is N when B is C-RI, and wherein Rl, R2, R4, R5
and R6 are as defined below. Thus, when A is C-R6 and B is N, structure (I) is apyrimidine-co.~ g compound having structure (II), and when A is N and B is C-R"
30 structure (1) is a pyrazine-co~ g compound having structure (III):
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'
R~a ~ R6 R4 ~ N
N ~ N N ~ Rl
R2a R2b
~ (~I)
In structures (I), (II) and (III) above, Rs is selected from the following
che,.~G~I moieties (i) through (iv):
o~N~ S~ 'R7 ~ O ~ S
(i) (ii) (iii) (iv)
wherein
R~ is sçlected from hydrogen, -CH3 and -CH2C6H5; and
R8 is selected from hydrogen and an unsubstituted or substituted
Cl~alkyl, C6 l2aryl, C, l2aralkyl, C3 l2heterocycle and a C4 l6heterocyclealkyl.The compounds of this invention further include pharm~cel~ti~ lly and
prophyl~.tic~lly acceptable salts of compounds of structure (I). Compounds of structure
(I) may contain proton donating groups (e.g, a carboxylic acid group) and/or proton
accepting groups (e.g., a group with a nitrogen atom having a free lone pair of electrons,
such as an amine group), and the salts of compounds of structure (I) may be formed and
utilized in the practice of the invention. Thus, compounds of the invention may be in the
form of a base addition salt (i.e., a salt of a proton donating group) or in the form of an
acid addition salt (i.e., a salt of a proton accepting group), as well as the free acid or free
base forms thereof.
Acid addition salts of a free base amino compound of the invention may
be prepared by methods well known in the art, and may be formed from organic andhlo.~,al ic acids. Suitable organic acids include acetic, ascorbic, bçn7~nes~l1fonic,
benzoic, fumaric, maleic, meth~nes~llfonic, and succinic acids. Suitable inorganic acids
include hydrochloric, hydrobromic, sulfuric, phosphoric and nitric acids. Base addition
salts of a free acid carboxylic acid compound of the invention may also be p.~;pa.ed by
methods well known in the art, and may be formed from organic and inorganic bases.
Thus, the compounds of this invention also include those salts derived from inorganic
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bases such as the hydroxide or other salt of so~ m pot~ m~ lithium, ~m m ~Im
c~lr;~1m ...~.~;.1m iron, zinc, copper, ~ f~e,S~ m;mIm, and the like, and organic
bases such as sul,sliluled allllllOILulll salts.
As used herein, the above terms have the following ~
S A "Cl 8alkyl" is a straight chain or branched, cyclic or non-cyclic,
saturated or unsaturated carbon chain Co.-~Ai~ from I to 8 carbon atoms. In one
embo~im~nt, the Cl.8alkyl is a fully saturated, straight chain alky1 s~1ected from methyl,
ethyl, n-propyl, n-butyl, n-pentyl and n-hexyl. In another embo-lim~nt the C"~alkyl is a
fully salu,~led cyc1ic alkyl s~lected from (but not 1imited to) ~,yclop~ Jyl, cyclobutyl,
10 cyclopentyl, cyclohexyl, methylenecyclopropyl and methylenecyclohexyl. In still a
further embodiment, the Cl salkyl is a fully saturated, branched alkyl selected from (but
not limited to) isoplul yl, sec-butyl, isobutyl, tert-butyl, isopentyl and isohexyl. In yet a
further embodiment, the Cl 8alkyl is an unsaturated straight chain alkyl s~lecte(~ from
(but not limited to) ethylenyl, propylenyl, I-butenyl, I-pentenyl and l-hexenyl.A "C6 l2aryl" is an aromatic moiety co.~lAi~ ~om 6 to 12 carbon
atoms. In one embo~lim~nt~ the C6 l2aryl is sr1ected from (but not limited to) phenyl,
tetralinyl, and napthalenyl. In a p,~lled embodiment, the C6 l2aryl is phenyl.
A "C7 l2aralkyl" is an arene CQl~ from 7 to 12 carbon atoms, and
has both aliphatic and aromatic units. In one embodiment, the C, l2aralkyl is se1ected
20 from (but not limited to) benzyl, ethylbenzyl, prowlbenzyl and isobutylbenzyl.
A "C3 l2heterocycle" is a compound that co~lah~s a ring made up of more
than one kind of atom, and which conLai,ls 3 to 12 carbon atoms. In one embodiment,
the C3 l2heterocycle is selected from (but not limited to) pyrrolyl, furanyl, thienyl,
imid~7c)1yl, oxazolyl, thiazolyl, pyrazolyl, pyrrolidinyl, pyridinyl, pyrimidinyl and purinyl.
25 In a further embo-lim~nt the C3 l2heterocycle inr1udes the following structures:
- N ~ ¦ - N ~
A "C4 l6heterocyclealkyl" is a compound that contains a C3 l2heterocycle
linked to a Cl 8alkyl. In one embodim.ont the C4 l6heterocyclealkyl is a methylene furan
having the following structure:
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A "substit~tecl~ Cl 8alkyl, C6 l2ary!, C~ uaralkyl, C3 l2heterocycle or
C~ l6heterocyclealkyl is a Cl salkyl~ C6 l2aryl, C7 l2aralkyl, C3 l2heterocycle or
C, l6h~terocyclealkyl having one or more hydrogens replaced with a s~lbstituçnt s~lected
from halogen (inr,l~lrlin~ -F, -Cl, -Br and -I), -OH, -R, -OR, -COOH, -COOR, -COR,
S -CONH2, -NH2,-NHR, -NRR, -SH, -SR, -SOOR, -SO3R and -SOR, where each
occurrence of R is intlepenrlently s~lected from an Im~llbstit~lted or s~lbstituted Cl 8alkyl,
C6 l2aryl, C, l2aralkyl, C3 l2heterocycle or C~ l6h~terocyclealkyl as defined above. In one
embo-1im~nt the substituted Cl salkyl is a Cl sh~lO~lkyl in~ linp (but not limited to)
-CF3 and -C2F5.
In structure (II) above, R2~ is sçlected from h~log~n an Im~lbstit~lted or
substituted Cl 8alkyl, C6 l2aryl, C, l2aralk,vl, C3 l2heterocycle or C~l6heterocyclealkyl,
-CN, -OR, -NHR, -NRR and -NRNCOR, wherein each occurrence of R is indep~nrl~nt1ys~lected from an Im~lbstitllted or sl~bstituted Cl 8alk,vl, C6 l2aryl, C~ l2aralkyl,
C3 l2heterocycle or C~l6heterocyclealkyl as defined above. In one embo~limpnt R~, is
15 s~lected from -Cl, -F, -CN and -CF3.
In structure (III) above, R2b is halogen, such as -Cl or -F.
In structure (II) above, R4, is selected from hydrogen, halogen, an
unsubstituted or sllhstit~lted Cl 8alkyl, C6 l2aryl, C~ ~2aralkyl, C3 ~2heterocycle or
C1 l6h~,t~rocyclealkyl, -CN, -OR, -NHR, -NRR and -NRNCOR, wherein each
20 occurrence of R is indep~ond~ntly selected from an Imc~lhstit~lted or s -hsfit~lted Cl 8alkyl,
C6 ,2aryl, C~ l2aralkyl, C3 ~2heterocycle or C, l6het~rocyclealkyl as defined above. In one
embodiment, R4, is s~lected from hydrogen, -CH3, -CF3, -C2Fs, -C2H5, -C6H5 and
-CH2C6H5
In structure (III) above, R4b is selected from hydrogen, halogen, -CN, and
25 an lm~lbstitllted or s ~hsti1nted C~ 8alkyl, C6 12aryl, C"2aralkyl, C3 ~2heterocycle or
C~ lcheterocyclealkyl
In structures (I) and (II) above, R6 is selected from hydrogen, halogen,
and an unsubstituted or substituted Cl 8alkyl, in~ltl-lin~ (but not limited to) a
Cl 8h~10~1kyl (such as -CF3 and -C2F5). In one emborlim~ont, R6 is selectecl from
30 hydrogen, -Cl, -F, -CH3 and -CF3.
In structures (I) and (m) above, Rl is selected from hydrogen, -CH3,
-CF3 and -C2H5.
In one embodiment, the compounds of this invention have structure (II)
above, wherein Rl is the chlomic~l moiety (i). In this embodiment, the compounds35 disclosed herein have the following structure (IV):
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Oq~N~R
N ~ N
R2a
(IV)
where R2~, R4" R6, R, and R8 are as defined above. In a plerelled embodiment,
leplese,lla~ e compounds of structure (IV) contain R2., R4,,, R6, R7 and Rg moieties as
S idPntifi~cl in Table 1 below.
Table 1
Compounds of Structure (IV)
R2, R4~ R6 R, R8
-Cl -CF3 -H -H
-OCH3 -Cl -CF3 -CH3 ~x
z
-H -F -CH3
-N(CH3~2 -CH3 -Cl
-CF3 -H
-CN -C2F5
-NHNH2 ~X
z
-NHPh
wherein X Y and Z are the same or dirre~ e"L, and independently selected from
hydrogen, -OH, -R, -OR, -COOH, -COOR, -COR, -CON H2, -NH2, -NHR,
-NRR,-SH, -SR, -SOOR, -SO3R and -SOR, where each occurrence of R is
independently selected from an unsubstituted or sllbstitllted Cl galkyl, C6 l2aryl,
C~ l2aralkyl, C3 l2heterocycle or C~6heterocyclealkyl.
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- In a p~rGllcd embodiment of the compounds disclosed in Table 1 above,
X Y and Z are the same or ~ rclelll~ and indepPn~lently selected from -H, -Cl, -F, -CF3,
-OH, -CH3 and -OCH3 In a further p,erG-,cd embodiment, R8 is a 3,5-
S bis(trifluo-u-,-~.Ll-rl)phenyl moiety or a 3-trifluor~ Gll~yl-s-halo-phenyl moiety
As m~ntiorle(l above, in one embodiment of this invention the compounds
have structure (II) Within one aspect of this embodiment, R4~ is -CF3 and R2, is -Cl
Such compounds include (but are not limited to): 2-chloro-4-trifluo,u...cll.yl-s-N-(3~s~-
bistrifluo,-,-,.~,Lllyl~he.-yl)pyrimidine carbo ~e; 2-chloro-4-trifluo-u...~,ll.yl-5-N-(3',5'-
10 dichlorophenyl)pyrimidine carboxamide; 2-chloro-4-trifluol c,lllcLllyl-5-N-(4'-
trifluol u~ hylphenyl)pyrimidine carboxamide; 2-chloro-4-trifluoromethyl-5-N-
(phenyl)pyrimidine carboxamide; 2-chloro-4-trifluolc,.ncll.yl-5-N-(cyclohexyl)pyrimidine
carboxamide; 2-chloro-4-trifluo.u-nGll-yl-5-N-(3',4',5'-trichlorophenyl)pyrimidine
c~l,ox~ itle; 2-chloro4-trifluoro---cll-yl-5-N-(benzyl)pyrimidine ca.l,o~ ..;de; 2-chloro-
15 4-trifluo.o,l,~,lllyl-5-N-(4'-(2',1',3'-bG-~o~ 7Ole))pyrimidine carbox~mide; 2-chloro-
4-trifluoromethyl-5-N-(3',5'-dichloro-6'-hydloxy~,henyl)pyrimidine carboxamide; 2-
chloro-4-trifluolo".Gll-yl-5-N-(5'-(3'-methylisox~zole))pyrimidine carbox~mi~; 2-
chloro-4-trifluoro.,.~,ll,yl-5-N-(3'-N-acyl-4'-fluoro~niline)pyrimidine carboxamide; 2-
chloro-4-trifluoro..,t;Ll-yl-5-N-(3 -trifluol c,mell-yl-5'-ethoxycarbonylphenyl)pyrimidine
20 carboxamide; 2-chloro4-trifluoromethyl-5-N-(3'-trifluo.v.neLl.yl-5-
(carboY~mide)phenyl) pyrimidine ca.l,ux~ ide; 2-chloro-4-trifluoromethyl-5-N-(3',5'-
dichlorophenyl)-N-(methyl)pyrimidine carbox~mide; and 2-chloro-4-trifluoromethyl-5-
N-(3 ', 5'-dichlorophenyl)-N (benzyl)pyrimidine carboxamide
Within another aspect of this embodiment, R4. is -CF3 and R2, is a moiety
25 other than -Cl Such compounds include (but are not limited to): 2-fluoro-4-
trifluolu.l.cll.yl-5-N-(3',5'-bistrifluolunlt;lhyl)pyrimidine carboxamide, 5-(3',5'-
bis(trifluoromethyl)phenacyl)-2-methoxy-4-trifluoromethylpyrimi~line; 4-trifluoromethyl-
5-N-(3',5'-dichlorophenyl)pyrimidine carbox~mide; 2-dimethylamino-4-trifluoromethyl-
5-N-(3',5'-dichlorophenyl)pyrimidine carboxamide; 2-triethylammonium chloride-4-
30 trifluo.u.--ell,yl-5-N-(3',5'-dichlo-ol)he--yl)pyrimidine carboxamide; 2-cyano-4-
trifluo-u-..~;ll.yl-5-N-[3',5'-(bistrifluoromethyl)phenyl]pyrimidine carboxamide; 2-
hydrazino-4-trifluoromethyl-5-[N-(3',5'-dichlorophenyl)pyrimidine-5-carboxamide; 2-[N-
(1-Aminocitr~con~mide)]-4-trifluololllell-yl-5-tN-(3',5'-dichlorophenyl) pyrimidine-5-
carboxamide; and 2-aminophenyl-4-trifluolo---elhyl-N-(3',5'-dichlorophenyl)pyrimidine-
35 5-carboY~mide.
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Within yet a further aspect of this embodiment, R2, is -Cl and R4, is a
moiety other than -CF3. Such compounds include (but are not limited to): 5-N-(3',5'-
bis(trifluol u~ Lllyl)phenyl)-2~4-dichloro-6-methyl-pyrimidine carboxamide; 2-chloro~-
methyl-5-N-(3',5'-(bistrifluol~ tllyl)phenyl)pyrimidine c~l,o~l~ide; 2,4-dichloro-5-N-
(3~,5~-bis(trifluolonlelllyl)benzyl)pyrimidine-5-carboA ~~e; and 2-chloro~-phenyl-5-N-
(3',5'-(bistrifluorc,ll.~,lllyl)phenyl)pyrimidine carb,. '1e
In another embo~lim~nt the compounds of this invention have structure
(III) above. Within one aspect of this embodiment, R~ is s~lected from hydrogen, -CH3
and -CF3 . Such compounds include (but are not limited to) ~yl ~h~e-co~ g
compounds which correspond to the pyrimidine-co.~ p compounds disclosed above.
In one embodiment of structure (III), R2b is -Cl, R4b is -CF3 and R5 is a moiety of
structure (i) above.
A small number of compounds which fall within structure (I) above have
been previously disclosed and/or are commercially available. However, such colll~oullds
have not been ~ccoçiste~1 with the utilities of the present invention, or possess no
recognized utility. Accoldi-l~,ly, compounds that fall within the scope of structure (I),
and which have recognized utility, are spec.ific~lly excluded from the novel compounds
of structure (I). However, to the extent such compounds have not been disclosed for the
utilities of the present invention, they are incl~detl in the various methods of this
invention.
To this end, the novel compounds of this invention do not include
compounds of structure (IV) above where R, and Rg are both hydrogen, and where R2,
is selected from an unsubstitlltef~ straight chain or branched, non-cyclic, saturated Cl 3
alkyl (i.e.,-CH3, -CH2CH3,-(CH2)2CH3 and -CH(CH3)2), -N(CH3)2, -N(CH2CH3)2 and
-OR, where R is as defined above. Similarly, the novel compounds of structure (IV) are
subject to the following provisos: (a) when R2, is -Cl and R6 is -H, R4, is not -CF3, -Cl,
-CH3 or -C(CH3)3, (b) when R2l is -Cl and both R4, and R6 are -H, R8 is not
-CH(CN)C6H5 or -(CH2)5CH3, and (c) when R2, is -Cl and R4,, is -Cl, R6 is not -Cl or
-CH2Cl.
The novel compounds of this invention also do not include compounds of
structure (II) when R5 is moiety (iii) and (a) R2~ is -CH3, -OCH3 or -N(CH3)2, or (b) Rg
is -H or -CH3.
Fulll.t;ll..ole, the novel compounds of structure (III) when R5 is moiety
(i) are subject to the following proviso: when R2b is -Cl, R4b and R, are not both
35 hydrogen.
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The compounds of this invention may be made by one skilled in organic
,Ll~;s by known te~ h.~ ee as well as by the synthetic routes ~ c~c!sed herein. For
ose of conv~"-,el-ce, the compounds have been separated into pyrimidine-co..~ g
- (structure (II)) and l~Y~ ~ine-co.~lA~ -g (structure (III)) compounds as set forth below.
5 The pyrimidine-co..li.;..;.~g compounds ofthis invention may be p-t;pared as illustrated by
the reaction scheme of Figure 1. In general, cc.. -.-.,cially available ~-keto esters 1 are
heated at elevated le-npel~lu--,s (75-110~C) with a mixture of urea and
triethylorthoru--ndle (or a s~lbstit~lted orthorc~ lale) to provide ureido derivatives 2.
Trç~A~ of these intermP~ tes with sodium alkoxides, such as sodium ethoxide in an
10 alcoholic solvent at 35-100~C, gives 2-hydroxypyrimidine esters 3 which, uponwith a chlorinating agent such as phosphorous oxychloride at elevated
telllpe.~ res (75-120~C), yields 2-chlolupy.i.ll;dine esters 4. The 2-hydr~,~y~,yrimidine
esters 3 may also be treated with a mild base, such as lithium hydroxide, sodiumhydroxide or potassium carbonate to provide the corresponding acid 3A, which may15 then be converted with a chlo- illaLing agent, such as phosphorous oxychloride or thionyl
chloride in an inert solvent or neat at 25-75~C, to the acid chloride 5. Compounds of
structure 6 may be p.ep~t;d using standard conditions known in the art by reacting the
acid chloride 5 with an amine in the presence of a base, such as potassium ca-l,onale or
dimethyla...hlo~,yridine (DMAP), in a non-protic solvent, such as methylene chloride or
20 EtOAc at 25~0~C, followed by standard workup.
Alternatively, pyrimidine-co..l;.;.~;.-g compounds of this invention may
also be made by the following combinatorial procedure. Commercially available and/or
readily synth~ ed amines, anilines and related compounds may be reacted with the acid
chloride 5 in EtOAc in the presence of basic Amberlyst 21 resin. The reactions are
25 qllçn-~hed with 50 ~lL of water and the final products are obtained in the organic layer
and concentrated. This procedure may be done in a 96 well (1 mL deep well) plate and
the final products i~ol~ted as dry powders. TLC analysis is performed on each
co---pûu--d and indicates the purity, and GC/MS and HPLC analysis den.onsl.ales that
the desired products are synthçci7ed (mass spectral analysis, molecular weight) and are
30 greater than 80% pure. By this method, eighty distinct pyrimidine-co~ g
compounds may be routinely syntheci7ed at the same time in one 96 well plate.
In addition, compound 4 may be reacted with various nucleophiles in an
aprotic solvent and at ambient tt;l,lpelalllre to provide derivatives 7. These compounds
can be hydrolyzed with base to yield co--lpounds having structure 8. Compounds of
35 structure 8 can be converted to the acid chloride as described above, and reacted with
various amines to give compounds having structure 9 using known conditions, in(~ ding
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the co~ at~lial approach described above. Alternatively, compounds of structure 7
can a1so be plt;p~Gd by reacting the ,I~-keto ester 1 in a seq~l~nti~l fashion with
triethylorthorolllldLe and acetic anhydride or N,N-dimethyl~l..-~-..;de dimethyl acetal in
DMF to give h~ltl---P,~ e 10. Re~cting intermPAi~te 10 with a variety of r -linps in
s s~lr,oh~1ic solvents provides hlLt;l..lP,.~ e 11 which, upon ~d~itiQn of base, provides
col.lpc,unds of structure 7
P~l~hlc co..~ g colllpou~-ds of structure (III) may be plepaled as
illustrated by the reaction scheme of Figure 2. The ~yllLl~~;s of these compounds may
begin with readily &va,lable pyruvic acid derivatives 12. These compounds are
10 conr1P.n~ed with conllllelcially available 2-cyano-1,2-diamino-2-substit~ted ethenes 13 in
an alcoholic solvent (such as MeOH) in the plest;llce of an acid (such as HCI) at ambient
tc~ ,lal~res (25-60~C) to provide the cyano pyrazines of structure 14. The ~yl~il~es
may then be converted to the collc;~,onding carboxylic acids 15 using a strong base such
as sodium hydroxide in water, or a strong acid such as HCI, at elevated temperatures
15 (70-110~C). These carboxylic acids may then be converted to 5-chloro-2-carbonyl acid
chloride derivatives 16 using a chlorinating agent such as POC13 or SOC12. Tre~tmPnt Of
16 with various amines or anilines at ambient temperatures in an inert solvent such as
EtOAc or CH2C12 provides coll-poùllds of structure 17.
The carboxylic acids of structure 15 can also be converted to the hydroxy
20 ester 18 by lIe~ III with SOCI2 and MeOH at a temperature of 25-60~C . TI~AI~
of 18 with a chlolinalillg agent such as SOCI2 or POCI3 in the presence of DMF gives
the chloro ester 19. Compound 19 can also be converted to the acid chloride 16 using a
mild base such as potassium carbonate in an a protic solvent such as MeOH, followed by
tre~tmPn~ with a chlorinating agent such as oxalyl chloride in an inert solvent such as
25 methylene chloride at ambient It---pe-~L~Ires.
The pyra_ine-co--~ -g compounds of this invention may also be
synthP,ci7ed by appl op. iate co...binaLo. ial techniques as described. In short,
co..l...ercially available and/or readily synthesi7Pd amines, anilines and related
coull)oul~ds may be reacted with the acid chloride 16 in EtOAc in the presence of basic
30 Amberlyst 21 resin. The reactions are quen~hp~d with 50 ~L of water and the final
products are obtained in the organic layer and concellLI~led. This procedure may be
done in a 96 well (1 mL deep well) plate and the final products isolated as dry powders.
TLC analysis is pe-rolmed on each compound and in-lic~fes the purity, and GC and~'LC analysis ~le~..ol.~ Les that the desired products are synthPei7~d (mass spectral
35 analysis, molecular weight) and are greater than 80% pure. By this method, eighty
pyrazine-co..~ g compounds may be routinely synthP~i7ed in one 96 well plate.
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Once synthe,ci7pt1 the compounds of this invention may be form~ te~l for
~ r. ~ .alion to a warm-biooded animal by a variety of techn:qlles known to those
skilled in the art. In one emborlim~nt~ the compound is in the form of a phal...Ace~ltic~l
composition for prophylactic or therapeutic use, and which co~l;.;..c at least one
S conl~oulld of this invention nn co,ll~inalion with a pharrn~ ltic~lly acceptable carrier or
diluent. The compound is present in the composition in an ~molmt which, upon
s~ .cl.~lion to the animal, is effective in preventing or treating the con-1ition of
interest. Plc;rel~bly, the composition inc~ ec a compound ofthis invention in an amount
l~y,h~g from 0.01 mg to 250 mg per dosage, depending upon the route of
10 ~ .cl.~lion~ and more preferably from 1 mg to 60 mg. Applopli~le concentrations,
dosages and modes of ~-lminictration may be readily determined by one skilled in the art.
Suitable carriers or ~illl~ntc are familiar to those skilled in the rolmul~lion
field. For compositions forrn~ fed as liquid solutions, acceptable carrier or diluents
include saline and sterile water, and may optionally include antioxidants, buffers,
15 bacteriostats and other common additives. The compositions of this invention may also
be form~ ted as pills, capsules, granules or tablets which cont~in7 in addition to the
compound of this invention, ~ t~entc~ dispersing and surface active agents, binders and
lubricants. One skilled in the art may further formulate the compounds of this invention
in any appro~l;ate manner, and in accordance with accepted practices, such as those
20 disclosed in Remington 's Pharmn< et~ n~ Sciences, Gennaro, Ed., Mack Publishing Co.,
Easton, PA, 1990 (incorporated herein by reference).
In another embodiment, the present invention provides methods for
preventing or treating a variety of conditions. Such methods include ~lminictering a
compound of this invention to a warm-blooded animal in need thereof in an amount25 sl!ffif i~nt to prevent or treat the condition. Such methods include systemicion of a compound of this invention, preferably in the form of a composition
as disclosed above. As used herein, systemic ~A.,.i.~ lion in~.hlcles oral and parental
methods of ~rl.,~ on For oral ~tlmini.~tration, suitable pharm~c~lti~
compositions include powders, granules, pills, tablets and capsules, as well as liquids,
30 syrups, s lcp~ncions and emulsions. These compositions may also include flavorants,
- preservatives, suspending, thickening and emulsifying agents, and other pharm~ce~ltiç~ily
acceptable additives. For parental ~flministration, the compounds of the presentinvention may be plt;~ ed in aqueous injectable solutions which may contain, in addition
to the compound of this invention, buffers, antioxidants, bacteriostats and other additives
35 commonly employed in such solutions.
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As m~ntioned above, col-lpoullds of the present invention can be used to
prevent or treat a wide variety of disorders, .I;ce~es and/or illn~sses In particular, the
compounds may be ~ le~ed to a warm-blooded animal for pl~c,nLion or L,e,.~
of rhellm~toi~1 arthritis, osteoalLlllilis, tissue and/or organ transplant rejection, sepsis,
S ARDS, asthma, trauma, oxidative stress, cell death, irradiation ~l~m~ r.l~.":sreperfusion, cancer, viral infection, and ~ltQ;~ ne tli~e~es such as l)Soli~;S,
h~m~ ly bowel disease, glomerulonephritis, lupus, uveitis and chronic he~ ;l;c
Co~ >ounds of this invention may be scr~ned by known and accepled
teçhni(ll~es for their ability to function as prophylactically and/or thc.~ .l;r.~lly active
10 agents. For example, the compounds may be evaluated in in vitro and/or in vivo assays
indicative of the compound's ~I;.. 11:1.. ~tQry and immllnos~.pple~ re properties. To
this end, such compounds may first be evaluated in a number of cell-based assays which
determine the ability of a compound to prevent activation of NFlcB and AP-l(see
Example 56). Next, the compound's ability to ~ttenll~te cytokine levels (such as IL-2
15 and IL-8), which are known to be elevated in certain disease states, may be deterrnined
(see Example 57). The compounds may then be evaluated in an applopliate animal
model, inrl~lrliny rodent models of ;..ll;....".~l;on and immlmclsuppression (see Example
58).
It should be recognized that, for example, in the case of
20 immllnos~pplt;~ e drugs and other agents which have utility for the Ll~ ,lll of
rhellm~toid al~hlilis(RA)~ llul~elo~ls studies have been pe.ro----ed directed to the activity
of such drugs. To this end, cyclosporin A has been used in clinical trials since the late
1970's as a second-line drug and is recomm~n-led to be used only in patients with active
RA. Thus, E~ lhl.~ 58 was pelrc,lllled ~ltili7ing cyclosporin A as a positive control.
25 A recent review of such immllnos~ pressive drugs, inrl~lrling relevant assays for the
same, is presented by R.P. Carlson in Exp. Opi~L Invest. Drugs 4(9):853-859, 1995
(incorporated herein by It;relt;l-ce in its entirety, inrl~l<lin~ cited references).
The following examples are presented for purpose of illustration, not
limitation.
EXAMPLES
To sn~ dGe the examples that follow, Examples 1-54 fii~çlose the
~ylllhe:jis of repr~cent~tive compounds of this invention, as well as intermerli~tes thereof;
Example 55 discloses the synthesis of iepl~senlaLi~e compounds by colllbill~Lional
35 rl~ y techniques; Examples 56-57 disclose the ability of representative compounds
of this invention to inhibit NFlcB, AP-I and cytokines; and Example 58 discloses the
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activity of a ~épreeselllaLi~e compound of this invention in both graft versus host disease
~ and contact sensitivity mo~
Example 1
52-CHLORO-4~U~ m-5-N-
[3',5'-BIS(TR~LUOROMETHYL)PHENYL]PYRIMIDINE CARBOXAMIDE
To a mixture of 3,5-bistrifluoromethylaniline (0.20 g, 0.92 mmol),
Amberlyst A-21 ion ~ ge resin (0.02 g) in EtOAc (5 mL) was added a sol~tion of 2-
chloro-4-trifluor~,,,,eLl-ylpyrimidine-S-carbonyl chloride (0.27 g, 1.13 mmol) in EtOAc
10 (5 mL). The mixture was stirred for 0.5 h, then qllerlched with water (0.20 rnL). The
organic layer was separated, dried over MgSO4, filtered and the solvent removed under
reduced pressure. The rçs~llfing oil was ,ec,y~l~lli7~cl from EtOH/H20 to provide the
title compound (0.21 g, 53% yield) as a white solid; m.p. 162-163~C.
15Example 2
2-~HLoRO-4-TRIFLUOROMETHYL-S -N-
(4'-TRIF LUoROMETHYLPHENYL)PYR~DlNE CARl3OXAMIDE
The title compound was p, epa, èd as described in Example 1, but
emp1Oying4-trifluol~"--e~hy-laniline (0.1 g, 0.41 mmol) in place of 3,5-
20 bistrifluoromethylaniline and the acid ch1Oride (0.10 g, 0.41 mmol), resulting in a 24%
yield; m.p. 172- 173~C.
Example 3
2-CHLORO-4-TRIFLUOROMETHYL-
25 S-N-(PHENYL)PYRIMIDINE CARBOXAMIDE
The title compound was prepared as described in Example 1, but
employing aniline (0.04 g, 0.39 mmol) and the acid chloride (0.22 g, 0.90 mmol), re~lllfing in a 62% yield; m.p. 108-181~C.
Example 4
2-CHLoRo-4-TRn~LuoRo~THYL-
S-N-(CYCLOHEXYL)PYR~DrNE CARBOXAMIDE
The title compound was prepared as described in Example 1, but
employingcyclohexylamine (0.02 g, 0.18 mmol) and the acid chloride (0.05 g,
35 0.22 mmol), reslllting in a 33% yield; m.p. 150-151~C.
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16
Example 5
2-CHLORO 4-TRIFLUOROMETHYL-
5-N-1BENZYL)PYRIMIDINE CARBOXAMIDE
The title compound was p-t;pal~d as described above in Example 1, but
5 employing benzylamine (0.09 g, 0.92 mmol) and the acid chloride (0.25 g, 1.0 mmol),
reslllting in a 78% yield; m.p. 152-153~C
Example 6
2-CHLORO-4-TRIFLUOROMETHYL-5-N-(3',4',5'-
TRICHLOROPHENYL)PYRIMIDINE CARBOXAMIDE
The title compound was p-t;p~ed as described in Example 1, but
employing 3,4,5-trichloroaniline (0.15 g, 0.61 mmol) and the acid chloride (0.15 g,
0.61 mmol), reslllting in a 55% yield; m.p. 200-201~C.
Example 7
2-CHLoRo-4-TRlFLuoRoMETHyL-5-N-(4-(2~ 1 ',3'
-BENZOTHIADIAZOLE))PYRIMIDINE CARBOXAMIDE
The title con-pound was prepared as described above in Example 1, bu
employing 4-amino-2~1~3-benzoth~ 7ole (0.01 g, 0.07 mmol) and the acid chloride
20 (0.025 g, 0.10 mmol), r~s llting in a 60% yield; m.p. 179-180~C.
Example 8
2-CHLORO-4-TRIFLUOROMETHYL-5-N-(3',5'-DICHLORO-
6'-HYDROXYPHENYL)PYRIMIDINE CARBOXAMIDE
The title compound was prepared as described in Example 1, but
employing 3,5-dichloro-6-h~dlo~yalliline (0.02g, 0.11 mmol) and the acid chloride
(0.04g, 0.16mmol), and purified by cl~.,---alography (sio2, 1:1 h~Y~n~/EtOAc) toprovide the compound in a 10% yield; m.p. 211-213~C.
Example 9
2-cHLoRo-4-TRIFLuoRoMETHyL-5-N-[5~-(3~-METHyL
ISOXAZOLE)]PYR~DINE CARBOXAMIDE
The title compound was p- epa- c d as described in Example 1, but
employing 5-amino-3-methylisoxazole (0.02 g, 0.17 mmol) and the acid chloride (0.03 g,
35 0.10 mmol), res~-ltin~ in a 75% yield; m.p. 170-171~C.
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Example 10
- 2-CHLoRo-4-TRIFLuoRoMETHYL-5-N-(3'-N-AcYL-
4'-FLUORO-ANILINE)PYRIMIl~INE CARBOXAMIDE
A sol~lfion of 2-fluoro-5-nitroaniline (1.97g, 12.60mmol) and a 1:1
5 mixture of Ac20/pyridine (20 mL) was stirred for 18 h. The r~ lting precipitate was
filtered and washed with MeOH to provide N-acyl-2-fluoro-5-nitro~niline.
The N-acyl-2-fluoro-5-nitroaniline (0.99 g, 5.00 mmol) was dissolved in
EtOH (25 mL), and then 10% Pd/C (0.12 g) was added and the sol~-tion stirred under H2
for 5 h. The ~uspcnsion was filtered through celite and the filtrate evaporated to
10 dryness. The rP~slllting oil was chro~alographed (SiOz, 1:3 hPY~nPC/EtOAc) to provide
3-N-acyl-4-fluoro-aniline as a yellow oil. The aniline derivative was then coupled to 2-
chloro-4-trifluo,o",eLl,yl~yrimidine-5-carbonyl chloride as described in Example 1 to
provide the title compound in a 47% yield; m.p. 126-127~C.
Example 11
2-CHLORO-4-TRIFLUOROMETHYL-5 -N-(3 '-TRIFLUOROMETHYL-
5'-CARBOXAMIDEPHENYL) PYRIMIDINE CARBOXAM~E
To a solution of 3-nitro-5-trifluo,ull,elllylbenzoic acid (1.00 g 4.25
mmol) in CH2C12 (50 mL) was added oxalyl chloride (1.45 g 13.8 mmol) followed by20 D~ (3 drops). An imme~ te evolution of gas occurred and the reaction was stirred
for 18 h. The solvent was removed under reduced pressure, the reslllting oil wasdissolved in THF (80 mL) and cooled to 0~C. To the cold solution NH4OH (22 mL) in
THF (15 mL) was added dropwise and the mixture was stirred 18 h at room
temperature. The mixture was concentrated to remove the THF and the reslllting
25 preci~ Le was filtered and dried. The solid was dissolved in EtOH (25 mL) and 10%
Pd/C (0.12 g) was added, and the suspension was stirred 15 h under a blanket of H2.
The reaction was filtered through celite and the filtrate evaporated to dryness to provide
3-carboxamide-5-trifluoromethylaniline as a yellow oil. This compound was then
coupled to 2-chloro-4-trifluoromethylpyrimidine-5-carbonyl chloride as desc, il,ed in
30Example 1 to provide the title compound in a 55% yield; m.p. 218-219~C.
Example 12
~ 2-CHLORO4-~FLUOROMETHYL-5 -N-(3 '-TRIF LUOROMETHYL-
5~-ETHOXYcARBONYLPHENYL) PYRIMIDINE CARl30XAMIDE
35To a solution of 3-nitro-5-trifluolulllt;lllylbenzoic acid (0.36 g, 1.53
mmol) in CH2CI2 (20 mL) was added oxalyl chloride (0.58 g 4.60 mmol) followed by
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DMF (3 drops). An imm~i~te evolution of gas occurred and the reaction was stirred
for 18 h. The solvent was removed under reduced pressure, the re~ , oil was
dissolved in THF (80 mL) and cooled to 0~C. To the cooled solution was added EtOH
(5 mL) in THF (15 mL) and the ~ LuJc was stirred for 18 h at room tcmpcl~lule. The
S n~ ult; was co~ ed to remove the THF and the res -ltin~ ple_ipi~nle was filtered
and dried. The solid was dissolved in EtOH (25 mL) and 10% Pd/C (0.12 g) was added
and the s~-~p~n~ion was stirred for 15 h under a blanket of H2. The reaction was filtered
through celite and the filtrate evaporated to dryness to provide 3-clho~yc&lbonyl-5-
trifluo~ clLylaniline as a yellow oil. This compound was then coup1ed to 2-chloro-4-
trifluoromethyl pyrimidine-S-carbonyl chloride as described above to provide the title
compound in a 12% yield; m.p. 67-71~C.
Example 13
2-CHLORO-4-TRIFLUOROMETHYL-S-N-(3',5'-DICHLOROPE~ENYL)-
5-N-(METHYL)PYR~D]NE CARBOXAMIDE
To a solution of 2-chloro-4-trifluol u-l~clhyl-5-N-(3,5-dichlorophenyl)-
pyrimidine carboxamide (0.086 g, 0.23 mmol) in DMF (20 mL) was added NaH (0.02 g,
0.53 mmol). The mixture was stirred for 0.3 h at room temperature and then MeI (0.100
mL, 1.61 mmol) was added and stirring continued for 2 h. The solution was acidified
with 2N HCI and then extracted with EtOAc (3X). The combined organic layers weredried over MgSO4, filtered and the solvent removed under reduced pressure. The
reslllting oil was chl-o-n~lographed (SiO2, 7: 1 hexanes/EtOAc) to provide the title
compound ~6% yield) as a white solid; m.p. 124-125~C.
Example 14
2-CE~OR~4 -TRIFI UOROMETHYL-S -N-(3 ', S '-DICHLOROP~NYT )-
5-N-(BENZYL)PYR~DINE CARBOXAMIDE
A mixture of b~n7~1~çhyde (1.04 g, 9.40 mmol), 3,5-dichloro~nilin~ (1.71
g, 10.60 mmol), and HOAc (0.20 mL) in MeOH (35 mL) was cooled to 0~C. Then a
solution of NaBH3CN (28.0 mL, 28.0 mmol, 1.0 M solution in THF) was added
dropwise via a syringe pump over 0.25 h. The solution was allowed to stir an additional
0.3 h at 0~C, and then room temperature for 18 h. The excess NaBH3CN was q~lçnr.hed
with HCI and the solvent was removed under reduced pressure. The resulting oil was
dissolved in EtOAc/H20, basified with NaOH, and extracted with EtOAc. The
combined organic layers were washed with brine, dried over MgSO4, filtered and the
solvent removed under reduced pressure. The r.osl-lting oil was purified by
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19
cl~ o~aphy (SiO2,15:1 hexanes/EtOAc) to provide N-benzyl-3,5-dichloro~nilin~ as
- a white solid. This compound was coupled to 2-chloro-4-trifluo~mell~rlpyrimidine-5
carbonyl chloride as desc,il,ed and purified by ~hlo,~,alography (SiO2, 9:1
~ h. .~n~sfEtoAc) to provide the title compound (15% yield) as a white foam; m.p. 102-
5 104~C.
Example 15
5-N-[3',5'-BIS(TRIFI,UOROMETHYL)PH~NYL]-
2,4-DICH~ORo-6-METHYLP~DrNE CARBOXAMIDE
5-Carbethoxy-6-methyluracil was p,~;~aled as reported in the literature
(Lamon, J. Het. Chem., 261, 1969); m.p. 180-182~C. The ethyl ester was then
hydrolyzed as described for 2-hydroxy-4-methylpyrimidine-5-carboxylic acid to provide
2,4-dihydroxy-6-methylpyrimidine-5-carboxylic acid in a 95% yield; m.p. >230~C.
The 2,4-dihydroxy-6-methyl pyrimidine-5-carboxylic acid was heated at
15 reflux with POCI3. The reaction mixture was concentrated and 2,4-dichloro-6-
methylpyrimidine-5-carbonyl chloride was obtained by ~ictill~tion (b.p. 70-80~C, 1.5
mm/Hg). The 2,4-dichloro-6-methylpyrimidine-5-carbonyl chloride (0.15 g, 0.67 mmol)
was immerli~t~ly reacted with 3,5-bis(trifluoromethyl)aniline (0.15 g, 0.67 mmol) in a
similar manner to that described in Example 1 to provide the title compound (0.06 g,
20 24% - based upon starting 2,4-dihydroxy-6-methylpyrimidine-5-carboxylic acid); m.p.
174-176~C.
Example 16
2,4-DICHLOROPYR~DINE-S-CARBONYL CHLORIDE
The title comlpound was prepared as described in the literature (Smith and
Chrictenc~n J. Org C*em. 20:829, 1955) starting from 2,4-dihydroxypyrimidine-5-
carboxylic acid. The compound was obtained by ~lictill~tjon; b.p. 90-100~C (1.5
mm/Hg) in a yield of 46%; 'HN~ (CDC13) ~ 9.29.
Example 17
- ETHYL UREIDOMETHYLENE ACETOACETATE
A mixture of ethyl ~cetoacet~te (200 g, 1.54 mol), urea (105 g, 1.54
mole) and triethyl orthor~"maLe (228 g, 1.54 mol) was heated at 140~C under N2 for 22
h. The reaction mixture was cooled and filtered to provide the title compound in a 51%
35 yield (156 g); m.p. 173-174~C.
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Example 18
ETHYL UREIDOMETHYL~E BENZOYLACETATE
The title con,l)oulld was p, t;l~ c;d as described in Example 17, but
employing ethyl benzoyl~cet~te (30 g, 156 mmol), resllltin~ in a yield of 21% (12 g);
m.p.l24-126~C.
E~ lç 19
ETHYL 2-HlrDRoXY~-METHYT PYRLMIDrNE-s-cARsoxyLATE
A solution of ethyl ureidomethylene ~qceto~c-et~te (50 g, 250 mmol)
10 NaOEt (22.1 g, 325 mmol) in EtOH (500 mL) was stirred at room te---pe~ re under N2
for 3 days. The resl-lting solid was filtered and dried to yield the title compound as a
sodium salt in a yield of 88% (45 g); m.p. >220~C (dec.).
Example 20
ETHYL 2-HYDRoxY~-PHENYLpyRMIDr~E-s-cA~soxyLATE
The title compound was prepared as described in Example 19, but
employing ethyl ureidomethylene benzoyl acetate (12 g, 45 mmol), resl-ltin~ in a yield of
15% (6 g); m.p. >260~C, (dec.).
Example 21
ETHYL 2-cHLoRo-4-METHYLPYRIMIDrNE-s-cARsoxyLATE
A solution of ethyl 2-hydroxy-4-methylpyrimidine-5-carboxylate (5 g,
27.5 mmol) and POCI3 (84 g, 550 mmol) was heated at reflux under N2 for 1 h. Thereaction was cooled and concentrated. The residue was partitioned between CHCI3 and
25 H20 and the organic layer was dried (Na2SO4), filtered, and concentrated to yield the
title colllpound in a yield of 27% (1.5 g); IHNMR (CDCI3) ~ 9.04 (s, lH), 4.42 (q, 2H),
2.85 (s, 3H), 1.43 (t, 3H).
Example 22
E~YL 2-c~oRo-4-PHENYLPYR~Dn~E-s-cARsoxyLAlE
The title compound was prepared as described in Example 21, but
employing 2-hyd,-oxy-4-phenylpyrimidine-5-carboxylate (6 g, 25 mmol) to give the title
compound (5.5 g, 18%); m.p. 45-47~C.
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Example 23
2-CLORO~-METHYLPYR~DINE-5-CARBOXYLIC ACID
A solution of ethyl 2-chloro-4-methylpyrimidine-5-carboxylate (1.0 g, 5
mmol), NaOH (0.24 g, 6 mmol) in H20 (30 mL) was stirred at room temperature for 3
5 h. The soh~tion was ~ci~lified with 6N HCl and the reSllltin~ solid was filtered and dried
to give the title compound ~0 67 g 78%), IHNMR (DMSO-d6) o 9.01 (s, lH), 2.75 (s,
3H).
Example 24
2-CHLoRo-4-PHENyLpyRIMIDlNE-5-cARsox~rLIc ACID
The title compound was p- c;pal ~d as described in Example 23, but
emp1Oying 2-chloro-4-phenylpyrimidine-5-carboxylate (4.5 g, 17 mmol), rçslllting in a
yield of 87% (3.9 g); m.p. 105-110~C.
Example 25
2-CHLORO-4-METHYLPYRIMIDINE-5-CARBONYL CHLORIDE
A solution of 2-chloro-4-methylpyrimidine-5-carboxylic acid (0.81 g,
4.70 mmol), oxalyl chloride (0.89 g, 7.05 mmol), DMF (2 drops) in CH2CI2 (23 mL) was
stirred at room te,-lpt;-~ re under N2 for 4 h. The solution was concenllaled and
20 distilled to give the title compound (0.55 g,61%); b.p. 90-100~C, 1.3 mm/Hg; 'HNMR
(CDCI3) o d 9.02 (s, lH), 2.74 (s, 3H).
Example 26
2-CHLoRo-4-PHEN~rLP~MIDINE-5-cARsoNYL CHLORIDE
The compound was prepared as described above in Example 25, but
employing 2-chloro-4-phenylpyrimidine-5-carboxylic acid (3.8 g, 14 mmol), res~-lting in
a yield of 53 %; m.p. 42~C.
Example 27
2-CHLOROPYRIMIDD~E-5-CARBONYLC~ORIDE
The compound was plt;pared as described in the literature (see, Arukwe,
J. Undheim, K. Ac~a Chemica Scand. B40:764, 1986).
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Example 28
E~L ETHoXYMETHYLENE-4,4,4-TRIFLUOROACETOACETATE
A solution of 4,4,4-trifluoro~cetoacet~te (46 g, 0.25 mol) triethyl
orthoro""ale (74 g, 0.50 mol) and Ac2O (77 g, 0.75 mol) was heated at 120-140~C for 7
5 h. The mixture was concenL,~led and distilled to give the title compound in a 98% yield
(58.6 g); b.p. 80-90~C, 1.5 mm/Hg.
Example 29
2,4-BIs(TRIFLuoRoMETHYL)PYRIMIDrNE-S-cARsoNYL CHLORIDE
A solution of ethyl ethoxymethylene-4,4,4-trifluoro~ceto~c.et~te (15 g,
62.5 mmol) and trifluoro~cet~midine (12.6 g, 112.5 mmol) in EtOH (50 mL) was heated
at reflux for 24 h under N2. The reaction mixture was cooled and concentrated.
Chromatography (SiO2, 20% EtOAc/hexane) afforded ethyl-2,4-bis
(trifluo,u",ell,.yl)pyrimidine-5-carboxylate as an oil (7.0 g, 39 %), ~HNMR (CDCI3) o
9.37 (s, lH), 3.70 (q, 2H), 1.27 (t, 3H).
A solution of ethyl-2,4-bis(trifluoromethyl)pyrimidine-5-carboxylate (5.0
g, 17 mmol) and NaOH (0.72 g, 18 mmol) in EtOH (20 mL) and H20 (50 mL) was
stirred at room te,l,l)e,~ re for 1 h. The solution was acidified ~HCI) and the res-llting
solid was filtered and dried to give 2,4-bis (trifluoromethyl)-pyrimidine-5-carboxylic acid
(1.5 g, 25%), m.p. 59~C, IHNMR (DMSO-d6) ~ 9.62 (s, lH).
The desired acid chloride was obtained from 2,4-bis(trifluoromethyl)-
pyrimidine-S-carboxylic acid in a manner similar to that described in Example 25 in a
yield of 44%; b.p. 105~C (1.5 mm/Hg); ~HNMR (CDCI3) o 9.12 (s, lH).
Example 30
2-CHLoRo-4-TRIFLuoRo~THYLPYRIMIDrNE-5-cARsoxYLIc ACID
A solution of 2-chloro-4-trifluoromethylpyrimidine-5-carbonyl chloride
(2.1 g, 8.6 mmol) in H20 (10 mL) was stirred at 0~C under N2 for 0.5 h. The r~cllltin~
solid was filtered and dried to give the title compound (1.91 g, 98% yield); m.p. 232-
234~C (dec.).
Example 31
2-CYANO-4-TRIFLUOROMETHYLPYRIMIDrNE-S -cARsoNYLcHLoRIDE
To a solution of 2-chloro-4-trifluoromethylpyrimidine-5-carboxylic acid
(2.80 g, 12.4 mmol) in THF (50 mL) at 0~C. was added Me3N (bubbled for 5 mimltes).
The reaction was kept at 0~C for 0.25 h and the reslllting solid was filtered to provide 2-
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L~ ly~ o~ m chloride-4-trifluoru.--~l1.ylpyrimidine-5-carboxylic acid (3.40 g, 97%
yield); m.p. 120-121~C (dec.).
A solution of 2-tlilll~tllyl~mmoni.lm chloride-4-
~ trifluolo.,l~,ll"tlpyrimidine-S-carboxylic acid (3.62 g, 12.7 mrnol) and KCN (0.99 g, 15.2
5 mmol) in DM~ (36.5 mL) and H20 (18.3 mL) was stirred at room te.llpG,~lur~ under N2
for 0.25 h. The reaction l~ lw~ was concentrated and dissolved in EtOAc (400 mL).
The EtOAc layer was washed with H20 (4 X 100 mL), brine (100 mL) and dried
(Na2SO4). The EtOAc layer was filtered and concenll~led to yield 2-cyano-4-
trifluoromethylpyrimidine-S-carboxylic acid (2.03 g, 74% yield); m.p. 148-149~C (dec.).
A solution of 2-cyano-4-trifluolunlGLl.ylpyrimidine-S-carboxylic acid (2.0
g, 9.2 mmol), oxalyl chloride (1.4 g, 11 mmol) and DMF (4 drops) in CH2Ck (46 mL)
was stirred at room telllpe-~ re under N2 for 0.75 h. The reaction was ccncwlLl~led
and ~ tilled (b.p. 100~C, 1.5 mm/Hg) to give the title compound (1.8 g, 82% yield);
IHNMR (CDCl3) o 9.49 (s, IH).
Example 32
2-PHENYLPYRIMIDrNE-5-cARsoNYL CHLORIDE
A solution of ethyl 3-N,N-dimethylamino-2-formylacrylate (4.0 g, 23
mmol) (Arnold, Coll. Czech. Chem. Commu~.~ 26:3051, 1961), bç~-7~...;-iin.o
hydrochloride (4.0 g, 26 mmol) and sodium (0.65 g, 28 mmot) in EtOH (40 mL) was
heated at reflux for I h. The solution was filtered and concentrated and the residue
partitioned between EtOAc and dilute HCI (10%). The organic layer was dried
(Na2SO4), and concentrated to give ethyl 2-phenylpyrimidine-5-carboxylate (4.0 g, 75%
yield); m.p. >220~C (dec.).
The corresponding 2-phenylpyrimidine-5-carboxylic acid was plt;p~t;d in
a yield of 80% (0.35 g) starting from ethyl 2-phenylpyrimidine-5-carboxylate in a similar
manner to that described in Example 23; m.p. > 220~C (dec.).
The title compound was prepared in a c~ ntit~tive yield from 2-
phenylpyrimidine-S-carboxylic acid in a similar manner to that described in Example 25;
m.p. 135~C.
Example 33
E~YL 2-TRIF LuoRoMETHYL-4-HYDRoxYpyRIMIDrNE-s -CARBOXYL ATE
A solution of diethyl ethoxymethylenemalonate (35.0 g, 162 mmol),
trifluoro~cet~mitiine (18 g, 162 mmol) and NaOEt (11.0 g, 162 mmol) in EtOH (200mL) was heated at reflux for 6 h. The reaction mixture was concentrated and H20 (48
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24
mL) was added. The ~ g solid was filtered, washed with Et20 (300 mL) and H20
(200 mL), and dried to give the title compound (21 g, 50% yield); m.p. >220~C (dec.);
IHNMR (I)MSO-d6) o 8.38, 4.16 (q, 2H), 1.25 (q, 3H) .
Example 34
2-TR1FLUOROMErHYL-4-CHLOROPYRIMIDn~E-S-CARBONYL C~ORIDE
A solution of ethyl 2-trifluor~,l,GLl,yl-4-hydroxypyrimidine-5-carboxylate
(5.00 g, 19.4 mmol) and NaOH (0.93 g, 23.3 mmol) in H20 (20 mL) was stirred at 60~C
for 15 h. The reaction was ac;~lifie~ (conc. HCI) and conce~ Led until a solid began to
forrn. The solid was filtered and dried to give 2-trifluoromethyl-4-hyd,c,~y~,yrimidine-5-
carboxylic acid (2.1 g, 53% yield); IHNMR (DMSO-d6) o 8.83 (s, lH).
A solution of 2-trifluo[c".,GI1l~1-4-hydroxypyrimidine-5-carboxylic acid
(2.0 g, 10.4 mmol), POCI3 (32 g, 212 mmol) and SOCk (25 g, 212 mmol) was heated at
reflux for 4 days. The reaction was conc~ntrated and distilled (b.p. 90-95~C, 1.5
mm/Hg) to provide the title co"~pou,ld (2.1 g, 81% yield), IHNMR (CDCI3) o 9.45 (s,
1H).
Example 35
2-CHLORO-4-PENTAFLUOROETHYLPYRU~DnNE-S-cARBoNyLcElLoRn~E
A solution of ethyl 2-hydroxy-4-p~nt~fl-~oroethylpyrimidine-5-
c~l,oxylale (4.0 g, 13 mmol) and NaOH (1.60 g, 39 mmol) in EtOH (20 mL) and H20
(45 mL) was heated at reflux for 1 h. The solution was cooled and acidified (conc.
HCI). The res~lting solid was filtered and dried to provide 2-hydroxy-4-
p~.nt~fll-oroethylpyrimidine-5-carboxylic acid (3.3 g, 98% yield); 'H NMR (DMSO-d6) o
9.90 (bs, lH), 8.43 (s, lH).
A solution of 2-hydroxy-4-p~nt~fl~-oroethylpyrimidine-5-carboxylic acid
(3.33 g, 12.9 mmol) in SOCI2 (27.7 g, 233 mmol) was heated at reflux for 0.5 h. Then
POCI3 (35.6 g, 233 mmol) was added to the reaction mixture and heating continued for
36 h. The reaction mixture was conc~ ed and distilled (b.p. 80-85~C, I mrn/Hg) to
give the title compound (1.2 g, 35% yield). ~H NMR (DMSO-d6) o 9.18 (s, lH).
Example 36
4-TRIFLUOROMETHYL-S -N-
(3 ',5 ~-DlC~OROP~NYL)PYRIMIDrNE CARBOXAMIDE
A solution of 2-chloro-4-trifluoromethyl-5-N-(3,5-
dichlorophenyl)pyrimidine carboxamide (0.10 g, 0.27 mmol), Mg2O (0.024 g, 0.59
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mmol) and 5% Pd/C (0.01 g,l in EtOH (1.8 mL) and water (0.9 mL) was stirred at room
temperature under a blanket of H2 for 2.5 h. The reaction mixture was concentrated and
chro-,-alographed (SiO2, 9% EtOAc/hexane) to yield the title compound (0.05 g, 53%
- yield); m.p. 189-190~C.
F~xample 37
2-DIMETHYL~NO-4-TRIFLUOROMETHYL-
5-N-(3',5'-DICHLOROPHENYL) PYRIMI~lNE CARBOXAMIDE
A solution of 2-chloro-4-trifluol~,..~ll.yl-5-N-(3~5-
10 dichlolophe--yl)pyrimidine carboxamide (0.13 g, 0.36 mmol) and dimethyl amine (0.10 g,
2.20 mmol) in MeOH was stirred at room te~ re for 3 h. The reaction mixture
was concentrated and chlc,-l~alographed (SiO2, 5% EtOAc/hexane) to afford the title
compound (0.022 g, 16% yield); m.p. 163-164~C.
Example 38
2-TRIETHYLAMMONIUM CHLORIDE-4-TRIF LUOROMETHYL-
5-N-(3',5'-DICHLOROPHENYL) PYRIMIDINE CARBOXAMIDE
A solution of 2-chloro-4-trifluorolllelllyl-5-N-(3',5'-
dichlorophenyl)pyrimidine carboxamide (0.10 g, 0.27 mmol) and triethylamine (0.027 g,
0.27 mmol) in dry THF was stirred for 24 h. The solid was filtered, washed with Et20,
and dried to afford the title compound (0.031 g, 24% yield); m.p. 158-159~C.
Example 39
2-C~ORO-4-METHYL-5 -N-[3 ',5'-BIS(TRIFLUORO-
METHYL)PHENYL]PYR~DlNE cARsoxAMIDE
A solution of 2-chloro-4-methylpyrimidine-5-carbonyl chloride (0.10 g,
0.53 mmol), 3,5-bis(trifluoromethyl)aniline (0.12 g, 0.53 mmol) and Amberlyst A-21
resin (0.lOg) in EtOAc (5.3 mL) was stirred at room telllpt;la~,lre for 1 h. The solution
was filtered, concentrated and chromatographed (SiO2, 10% EtOAc/hexane) to afford
the title compound (0.17 g, 84% yield); m.p. 156-157~C.
Example 40
2,4-DICHLoRO-5-N-[3',5'-BIS(TRIFLUORO-
METHYL)sENzYL~PyR~D~E-5 -cARsoxAMIDE
The title compound was prepared as described in Example 1, but
employing 2,4-dichloropyrimidine-5-carbonylchloride (0.10 g, 0.40 mmol) and 3,5-
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26
bistrifluor~."~,lh~rlbenzylamine (0.10 g, 0.45 mmol) to give the compound in a 61% yield
(0.12 g); m.p. 144-145~C.
Example 41
2,4-DICHLORO-S-N-[3',5'-BIS(TR~LUORO-
METHYL)PHENYL]PYR~DINE-S~ARBOXAMIDE
The title compound was p- Gpal ~;d as described in Example 1, but
employing 2,4-dichloropyrimidine-5-carbonyl chloride to give the compound in a 97%
yield (0.28 g); m.p. 104-105~C.
Example 42
2-cyANo-4-TRIFLuoRoMETHyL-s-N-[3 ', S'-BIS(TRIFLUORO-
METHyL)pHENyL]pyRIMlDr~E CARBOXAMlDE
The title compound was pl ep~ ~d as described in Example 1, but
employing 2-cyano-4-trifluoromethylpyrimidine-5-carbonyl chloride (0.11 g, 0.46 mmol)
to give the compound in a 96% yield (0.19 g); m.p. 146-147~C.
Example 43
2-CHLORO-4-PHENYL-S-N-[3',5'-BIS(TRIFLUORO-
METHYL)PHENYL]PYRIMIDrNE CARBOXAMIDE
A solution of 2-chloro-4-phenylpyrimidine-5-carbonyl chloride (0.10 g,
0.40 mmol), 3,5-bis(trifluolon,t;lh~l) aniline (0.08 g, 0.40 mmol) and Et3N (0.04 g, 0.40
mmol) in EtOAc was stirred at room temperature for 2 h. The solution was
concenLl ~led and chl omalographed (siO2, 5% EtOH/CHCI3) to afford the title
compound (0.08 g, 45% yield); m.p. 154~C.
Example 44
2-HYDRAZINO-4-TRIFLUOROMETHYL-S -N-
(3~s'-DlcE~LoRo~HFNyL)pyRlMlDrNE-s-cARBoxAMlDE
A solution of 2-chloro-4-trifluoromethyl-5-N-(3',5'-
dichlorophenyl)pyrimidine carboxamide (0.10 g, 0.27 mmol) and hydrazine (0.009 g,
0.54 mmol) in THF was stirred under N2 at room temperature for 14 h. The solution was
filtered, conce"l-~ted and chromatographed (siO2, 20% EtOAc/hexane) to afford the
title compound (0.08 g, 79% yield), ~HNMR (acetone-d6) ~ 10.08 (bs, lH), 9.64 (bs,
lH), 8.89 (s, lH), 7.80 (s, 2H), 7.24 (s, lH), 2.79 (bs, 2H).
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Example 45;
2-[N-( I -AMlNOClTRACONAMIDE)]-4-TR~LUOROMETHYL-
S -tN-(3 ', S'-DIC~OROPHENYL)] -PYR~DINE-S -CARE3OXAMIDE
A solution of 2-hydrazino-4-trifluor~.l,lclll~l-S-[N-(3',5'-
S dichlc,~vph~;"yl)pyrimidine carboxamide (0.08 g, 0.21 mmol) and citraconic anhydride
(0.024 g, 0.21 mmol) in CHCl3 (2.1 mL) was heated at reflux under N2 for 24 h. The
solution was conce-~ led and ch~ alographed (SiO2, 33% EtOAc/hexane) to afford
the title compound (0.06 g, 62% yield); m.p. 182-183~C.
E~all",le 46
2-PHENYLAMINo-4-TRIFLuoRoMETHYL-
S-N-(3',5'-DICHLOROPHENYL)-PYRIMIDINE-S-CARBOXAMIDE
A solution of 2-chloro-4-trifluo~ ,lhyl-S-N-(3',5'-
dichlorophenyl)pyrimidine carboxamide (0.10 g, 0.27 mmol) and aniline (0.06 g, 0.59
mmol) in dry THF (2.7 mL) was stirred at room telllpel~ re under Nz for 18 h. The
reaction mixture was filtered, conce"L, ~ted and cl-l u~alographed (SiO2, 50%
CHClJhexane) to afford the title compound (0.10 g, 91% yield); m.p. 228-229~C.
Example 47
METHYL S-cHLoRo-6-~THYL-2-PYRAzr~E CARBOXYLATE.
To a solution of methyl 4,5-dihydro-6-methyl-5-oxo-2-pyrazine
carboxylate (M. Mano, T. Seo, K. Imai, Chem. Pharm. Bull 10:3057-3063, 1980) in
DMF (20 mL) was added POCl3 (20 rnL). The reaction was refluxed for 0.5 h and then
poured into ice. The aqueous layer was extracted with CHCl3 dried (MgS04) and
concentrated. The residue was chl-"lldlographed (SiO2, CHCl3) to provide the title
compound (2.34 g, 52% yield); m.p. 49-50~C.
Example 48
S-CHLoRo-6-METHYL-2-PYRAzrNEcARsoxYLIc ACID
A mixture of methyl S-chloro-6-methyl-2-pyrazine carboxylate (0.16 g,
0.86 mmol), K2C03 (0.31 g, 2.18 mmol) and H2O was stirred for 2 h at room
Lelllpel~L~Ire. The reaction was filtered and acidified (20% HCI), and the rçsulting solid
collected to provide the title compound (0.057 g, 39% yield); m.p. 116-117~C.
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28
Example 49
2-CHLORO-5-N-(BISTRIFLUOROMETHYL ANILINE)
PYRAzn~E CARBOXAMIDE
The title compound was p,epdr~d in a yield of 51% (0.08 g) using the
S same procedure as o~tlined in Example 1, except substitllting 2-chloro-5-pyrazine
carbonyl chloride (0.1 g, 0.57 mmol.) in place of the pyrimidine carbonyl chloride; m.p.
101-102~C.
Example 50
2-TRIMETHYLAMMONIUM CHLORIDE-4-TRIFLUOROMETHYL-
5-PYR~DINE CARBOXYLIC ACID
A solution of 2-chloro-4-trifluoromethylpyrimidine-5-carboxylic acid (6.0
g, 27 mmol) and excess l~ yl amine in THF (60 mL) was stirred for S min. The
solid was filtered and dried to yield 97% (7.1 g) of the title compound; lH NM~
(DMSO-d6) o 9.19 (s, lH), 2.72 (s, 9H).
Example 51
2-FLuoRo-4-l~FLuoRoMETHYL-
5-PYRIMIDINE CAR130XYLIC AcID
A mixture of 2-l. i~ llylammonium chloride-4-trifluoromethyl-5-
pyrimidine carboxylic acid (4.3 g, 15 mmol), KF (1.8 g, 30 mmol), DM~ (40 mL) and
H20 (20 mL) was stirred for 0.5h. The mixture was conce~ led~ acidified and
extracted with Et20. The Et20 layer was concentrated to yield 47% (1.6 g) of the title
compound; IH NMR (DMS0-d6) o 9.41 (s, lH).
Example 52
2-FLuoRo-4-TRIFLuoRoMETHYL-
5-PYR~DINE CARBONYL CHLORIDE
The title compound was pl ~;p~ t;d as described in Example 25, but
employing a solution of 2-fluoro-4-trifluo~on~ell-ylpyrimidine-5-carboxylic acid (1.5 g,
7.1 mmol) and oxalyl chloride (1.0 g, 8 mmol), DMF (2 drops) in CH2CI2 (30 mL)
resulted in a 75% yield (1.2 g); lH NMR (CDCI3) ~ 9.42 (s, 1H).
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29
Example 53
- 2-FLUORO 4-TR~LUOROMETHYL-5-N-[3~5'-BIS(TR~LUOROMETHYL)P~NYL]
PYR~INE CARsoxA~nDE
- The title compound was l~ep~èd as described in FY~ 1, but
employing a solution of 2-fluoro-4-trifluoro~ ,Lllylpyrimidine-5-carbonyl chloride (0.05
g, 0.22 mmol) and 3~5-bis(trifluo~u,~u-lll.yl)aniline (45 mg, 0.2 mmol) in EtOAc (2 mL)
resulted in a 22% yield (0.02 g); m.p. 133-135~C.
Example 54
2-CHLoRo-4-TRIFLuoRoMETHYL-
5-PYR~ E CARBONYL CHLORIDE
The title compound was p,ep~ed as described in Example 25, but
employing a solution of 2-chloro-4-trifluoromc;ll.ylpyrimidine-5-carboxylic acid (1.5 g,
7.1 mmol) and oxalyl chloride (1.0 g, 8 mmol) in CH2CI2 (30 mL) resulted in a 70%
yield ( l . lg); IH NMR (CDCI3) o 9.31 (s, lH).
Fxample 55
S~rHEsls OF REPRESENTATIVE COMPOUNDS
BY COMBINATORIAL CHEMISTRY TECHNIQUES
This example illustrates the synthesis of a lepleselllaLi~e class of
compounds of this invention by co",bi,lalorialrh~mi~try. It should be understood that,
while a specific class of compounds are illustrated in this example, the following
procedure may be employed to syntheci7e other compounds of this invention.
Into wells 2-11 of a 96 well 1 mL plate (rows 1 and 12 left open as
controls) was added 5 mg of Amberlyst 21 resin, 0.2 mL of EtOAc and 22.4 ~mol of 80
difrelélll amine derivatives. Then to each well was added 25.0 ~mol of the app,.,~,iaLe
5-carbonyl chloride (for example 2-chloro~-trifluol c)lllelLylpyrimidine-5-carbon
chloride). The 96 well plate was sonicated for 0.3 h and 50 ~LL of H20 was added to
each well. The plate was sonicated for an additional 0.25 h, and the EtOAc layer from
each well was removed and concentrated to provide 80 individual compounds. Thin-layer cl,. ~"na~ography, HPLC and GC/MS analysis indicated that the desired compounds
had been produced at >90% purity. This approach can be used to generate large
~ numbers of derivatives for each substituted pyrimidine prepared, and can be used to
routinely prepare >160 derivatives for each ofthe dirrelelll 5-carbonyl pyrim~ nes
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Example 56
~BITION OF THE ACTIVATION OF NFKB AND AP- 1
A. NFKB ASSAY
S Stable human Jurkat T-cells co.. ~ an NFKB binding site (from the
MHC promoter) fused to a minim~l Sv-40 promoter driving luciferase CA~C~;OI1 were
used in this c~Jclilllclll. Cells were split to 3 x 105 cells/mL every 2-3 days (cell
col-c~ lion should not exceed 1 x 106 cells/mL to keep the cells prolirc,~lhl~, in log
phase). These cells were co~-nte~, resllsp~n-led in fresh medium co,~ g 10% Serum-
10 Plus at a density of 1 x 106 cells/mL and plated in 96 well round bottom plates (200 ~LL
per well) 18 hours prior to starting the ~ GI illlGlll.
Compounds of this invention, dissolved in dimethyl sulfoxide (3.3, 0.33
and 0.03 ~:Lg/mL), were then added to the 96 well plates co..l~ the cells and the
plates are incub~ted for 0.5 h at 37~C. Then 50 ng/mL of phorbol 12-myristate-13-
15 acetate (PMA) and 1 llg/mL of phytoh~m~ ltinin (PHA) was added to each well andthe cells were inc~lb~ted for an additional 5 h at 37~C. The plates were centrifuged at
2200 R~M for 3 mimlt~ at room temperature and then the merlillm was removed. To
each well was added 60 ~L of cell lysis buffer and the plates were left at room
temperature for 0.25 h. Then 40 ,uL of each cell extract was ~ nsre"Gd to a black 96
20 well plate and 50 ~L of luciferase substrate buffer was added. I.llminescPnr:e was
immerli~tely measured using a Packard TopCount.
B. AP-1 ASSAY
For AP-1, the assay was run as described above for NFKB except stable
25 Jurkat T-cells were used that co.~ ;ed a coll~g~n~ce promoter driving luciferase
CA~.JI es:jion. In addition, the concentration of PMA used was S ng/mL.
C. RESULTS
The results of the above assays for a representative compound of this
invention, 2-chloro-4-trifluoromethyl-5-N-(3',5'-bistrifluoromethylphenyl)pyrimidine
carbox~mide7 as percent inhibition versus control are presented in Figure 3. This figure
also in~lic~tes activity of ,B-actin which was employed in these assays as a control cell line
in~1ic~tin~ effects on l-~s~ ion. The lack of ,13-actin activity evidences selectivity of
the test compounds for the L, ~1sc~ ion factors AP- l and NFlcB .
Expressed as ICso's, the results of these assays on ~qd~1ition~1 test
compounds are s-.. ~ .ed in Table 2 below.
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- Table 2
Test Con"~vund NFKB/AP-1
(Example #) IGn
0.03
2 0.75
6 0.8
8 6.0
1.0
11 50
12
13 5.0
0.8
39 0.075
41 0.6
42 >10
43 0.5
2.0
Based on the results of this tA~elilllent, ,eplt;se~ e compounds of this
5 invention were found to be effective at inhibiting the activation of L~ns.,iplion factors
(i.e., NF~B and AP-1) involved in gene transcription and therefore have utility as, for
example, immllnos~lpple~si~e agents.
Example 57
INHmBI~ON OF CYTOKnNES
To determine the effects of compounds on PMA/PHA-in~ ced cytokine
production supelllal~llls from either the NFlcB (for IL-8) an~d AP-1 (for IL-2) reporter
gene assays of Example 56 were collected and saved. Cytokine levels in the
s~pe~ lanl~ (25-50 ~IL aliquots) were determined by ELISA. The results of this
15 experiment for a .ep,ese-llative compound of this invention 2-chloro-4-trifluo.~."elllyl-
5-N-(3' 5'-bistrifluo,~""ell"~lphenyl)pyrimidine carboxamide is presented in Figure 4
(eA~l~essed as percent inhibition versus control).
Example 58
I~ Vl~o AC~V~Y OFREPRESENTA~VE COMPOU~D
The murine popliteal Iymph node (PLN) assay is a graft vs. host model
that predicts activity of compounds in blocking human transplant rejection. The delayed-
type hypel ~eulsili~ity response to oxazolone is a standard contact sensitivity model. Both
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of these models are used routinely to evaluate compounds that are used clinically. For
~ -;t...ple, cyclosporin and cyclophosphamide are active in these models and are used
clinically (Morris et al., Tr~rnsplantation Proceedings 22(Suppl. 1):110-112, 1990).
5 A. POPLr~EAL LYMPH NODE MODEL
Spleens were removed from donor BALB/c mice and splenocytes were
icQ!sted then irradiated (3,000 rads) to prevent donor cell proliferation. After washing
and a~ cting cell density, 2.5x106 cells were injected subc~-t~neously into the left hind
footpad of C3H mice. On day 4, the mice were sacrificed and left ,~,oplileal Iymph nodes
~PLNS) were weighed.
The compound of Example 1, 2-chloro-4-trifluor~,lllelllyl-5-N-(3',5'-
bistrifluoromethylphenyl)pyrimidine carbox~midç, was 2sdminictered once daily byl~eliIolleal injection be~ one day before footpad injection (day 0) through day
4. The compound was suspended, immerliatçly prior to use, at a concentration of 5
15 mg/mL in 0.25% methyl cellulose (Sigrna) using a glass-teflon homogenizer. For doses
of 10, 20 and 30 mg/kg, appropliale ~ ltionc of the stock solution were made so that
0.1 mL/10 g body weight was a~minictçred by hl~l~peliLoneal injection.
The results of this experiment, presented in Figure 5, demonstrate that a
representative compound of this invention caused a dose-dependent suppression of~IIO~nt;~n-;n~I1Ced PLN proliferation. The lowest dose of this compound, 10 mg/kg,
caused a 52% inhibition of proliferation whereas cyclosporin A, at 12 mg/kg, caused a
35% inhibition.
B. DELAYED TYPE HYPERSENSmVITY STI1DY
On day 0, ox~olone (100 ~LL of a 3% solution) was applied to the
shaved abdomen of mice. On day 7, a challenge application of ox~olone was applied
(10 ,uL) around the right ear. The compound of Example 1, 2-chloro-4-trifluorolllt;lllyl-
5-N-(3,5-bistrifluoromethylphenyl)pyrimidine carboxamide, was at1minictered from days
-2 to 7 by ;l~ pG,iloneal injection. It was prepared immefli~tçly prior to use by
sllcp~n~ling it in 0.25% methyl cellulose (Sigma) using a glass-teflon homogenizer. For
each dose, 0.1 mL/l 0 g body weight of the suspension was a~lminictçred. The
compound was pl~dl~d at the highest concentration for that study and apl)lopliate
dilutions of the stock solution were made so that 0.1 mL/10 g body weight was
d. Twenty four hours later, the difference in right vs. Ieft ear thickness was
35 measured. The results ofthis ~ Iilllent are presented in Table 3 below.
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Table 3
Effect on the DTH Re;.~,onse to Oxazolone
Compound Dose Right-Left Ear P Value
(mg/kg) (mean ' SEM) (vs. vehicle)
Vehicle only --- 0.30 ' 0.02 ---
Test Cpd. 10 (i.p.) 0.27 ~ 0.01 0.163
Test Cpd. 30 (i.p.) 0.13 + 0.02 <0.001*
Cyclophosphamide50 (i.p.)0.08 ' 0.01 <0.001
*One animal died during study
S The test compound (30 mg/kg i.p.) and cyclophosphamide (50 mg/kg
i.p.) ~ignific~ntly ~tten~ted the delayed-type response to oxazo1One by 56% and 73%,
~ ~,pe~ ely.
It will be app~eci~led that, although specific emborliment~ of this
invention have been described herein for purpose of illustration, various modifications
may be made ~,vithout departing from the spirit and scope of the invention.
