Note: Descriptions are shown in the official language in which they were submitted.
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C-PROTE~ASE INHIBITORS
FO~ T~ TREATMENT OF DISORDER~ LATED TO T~
OVERPRODUCTION OF COLLAGEN
.
This application is related to and is a contiml~tion-in-part application of
the U.S. application Serial Number 08/601,203, entitled "C-Proteinase
Innibitors ~or The Tre~tm~nt Of Disorders 3?elated To The Overproduction Of
Collagen," filed Fe~ruary 14, 1996, which is a contin-l~tion-in-part application
of tne provisional U.S. application Serial Number 60/002,038, filed August 8,
10 199~.
1. ~IELD OF T~ INVENTION
Collagen is integral to, among other things, the proper formation of
connective tissue. Thus, the over- or under-production of collagen or the
production of abnormal collagen (including incorrectly processed collagen) has
been linked witn numerous connt~ctive tissue diseases and disorders.
Mounting evidence suggests that C-~ult:h~ase is an ecsenti~l key enzyme for
the proper maturation of collagen, and Ll~el~fo~e appears to be an ideal target
for the inhibition, control and/or modulation of collagen formation.
The present invention relates to organic molecules capable of inhibiting
C-y~hlase activity in order to regulate, modlll~te and/or inhibit abnormal
collagen formation. More specifically, the invention relates to the use of
compounds and pharm~relltir~l compositions thereof for the tr~tm~ont of
various rlice~cec relating to the indl~-uL~liate or unregulated production of
2:~ collagen.
2. BACKGROUND O~ THE INVENTlON
Collagen Structure. At present nineteen types of collagens have been
ic~entifi~ ~l. These collagens, including fibrillar collagen types I, II, III are
30 syntht~ci7ed as procollagen precursor molecules which contain amino- and
3~
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carboxy-terminal peptide extensiorls. These peptide extensions, referred to as
"pro-regions," are designated as N- and C-propeptides, respectively.
The pro-regions are typically cleaved upon secretion of the procollagen
triple helical precursor molecule from the cell to yield a mature triple helicalcollagen molecule. ~Jpon cleavage, the "mature" collagen molecule is capable
of association, for example, into highly structured collagen fibers. See e.g.,
Fessler and Fessler, 1978, Annu. ~ev. Biochem. 47:129-162; Bornstein and
Traub, 1979, in: The Proteins (eds. Neurath, H. and Hill, ~.H.), Ac~c'.emic
Press, New York, pp. 412-632; Kivirikko et al., 1984, in: ~xtracellular
10 Matrix ~iochemistry (eds. Piez, K.A. and ~eddi, A.H.), Elsevier Science
Publishing Co., Inc., New York, pp. 83-118; Prockop and Kiviri~co, 1984,
N. ~:ngl. J. Med. 311:376-383; Kuhn, 1987, in: Structure and ~unction of
Collagen Types (eds. Mayne, R. and Burgeson, ~.E.), Academic Press, Inc.,
Orlando, Florida, pp. 142.
1~ Diseases Associa~ed Wit~ The Abnormal Productzon of Collagen. An
array of critical diseases has been associated witn the hla~ o~7liate or
unregulated production of collagen, including pathological fibrosis or scarring,including endocardial sclerosis, idiopathic interstitial fibrosis, i~ iLial
pulmonary fibrosis, perim--cc~ r fibrosis, Symmers' fibrosis, pericentral
20 fi'orosis, hepatitis, de~ tofibroma, billary cirrhosis, alcoholic cirrhosis, acute
pulmonary fibrosis, idiopathic pu~nonary fibrosis, acute l~ h~Loly distress
syndrome, kidney fibrosis/glomerulonephritis, kidney fibrosis/diabetic
nephropathy, scleroderma/systemic, scleroderma/local, keloids, hy~.clllu~hic
scars, severe joint adhesions/artnritis, myelofibrosis, comeal scarring, cystic
2~; fibrosis, mllcclll~ dystrophy (~Illcht~nn~'s), cardiac fibrosis, mllcclll,.rfibrosis/retinal separation, esophageal stricture, payronles disease. Further
fibrotic disorders may be inrl~lred or initi,.terl by surgery, including scar
revision/plastic surgeries, glaucoma, cataract fibrosis, corneal scarring, jointadhesions, graft vs. host disease, tendon surgery, nerve ~ Ll~ ent,
30 du~uyLl~ll's contracture, OB/GYN adhesions/fibrosis, pelvic adhesions,
peridural fibrosis, restenosis. One strategy for the treatrnent of these rlice,.ces
3~
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is to in'nibit the pathological overproduction of collagen. Thus, identificationand isolation of molecules which control, inhibit and/or modulate the
production of collagen are of major mPrlir~l interest.
~ ek~onship Between Coll~gen Forrr.a~zon and C-Prot~ .n~e. ~ecent
evidence suggests that C-proteinase is the essential key enzyme that catalyzes
the cleavage of the C-propeptide of, for example, fibrillar collagens, includingtype I, type II, and type III collagen. See, U.S. Application Ser. No.
60/002,038, filed August 8, 1995 as a provisional application, and references
disclosed therein.
C-proteinase was first observed in the culture media of human and
mouse fibroblasts (Goldberg et al., 1975, Cell 4:45-50; Kessler and
Goldberg, 1978, A~zal. Biochem. 86:463-469), and chick tendon fibroblasts
(Duskin et al., 1978, Arch. Biochem. Biophys. 185:326 332; Leung et al.,
1979, J. Biol. Chem. 254:224-232). An acidic proteinase which removes the
C-terminal propeptides from type I procollagen has also been identified.
Davidson et al., 1979, ~:ur. J. Biochem. 100:5~1.
A partially purified protein having C-~ tei~-ase activity was obtained
from chick calvaria in 1982. Njieha et al., 1982, Biochemistry 23:757-764.
In 198~, chicken C-proteinase was isolaled, purified and characterized from
conditioned media of chick embryo tendons. EIojima et al., 1985, J. Biol.
Chem. 260:15996-16003. Murine C-~lo~il~se has been subseqnently purified
from media of cultured mouse fibroblasts. Kessler et al., 1986, Collagen
~'elat. kes. 6:249-266; Kessler and Adar, 1989, ~ur. J. Biochem. 186:115
121. Finally, the cDNA enrotlina human C-proteinase has been iriPntifi~l, as
set forth in the above-referenced related applications and references disclosed
therein.
Experiments conrll~rted with these purified forms of chick and mouse
C-proteinase have indicated that tne enzyme is instr.~-mPnf~l in the formation
~ of functional collagen fibers. Fertala et ~1., 1994, J. Biol. Chem. 269:11584.
~ C-Protei~ e Inhi~itors. As a consequence of the enzyme's apparent
importance to collagen production, scientists have identified a number of C-
3~
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proteinase inhibitors. See e.g., Hojima et al., szlpra. For example, several
metal chelators have demonstrated activity as a C-proteinase inhibitor.
Likewise, chymostatin and pepstatin A were found to be relatively strong
inhibitors of C-proteinase. Additionally, ~x;!-Macroglobulin, ovostatin, and
~i fetal bovine serum appear to at least partially inhibit C-proteinase activity.
Dithiothreitol, SDS, concanavalin A, Zn'+, C~ , and Cd~+ are
similarily reported to be inhibitory at low concentrations. Likewise, some
reducing agents, several amino acids, phosphate, and ammonium sulfate were
inhibitory at concentratio~s of 1-10mM. Further, the enzyme was shown to
be inhibited by the basic amino acids lysine and arginine. Leung et al.,
s~pra; Ryhanen et al., 1982, Arch. ~iochem. Biophys. ~ 230-236. ~inally,
high concentrations of NaCl or Tris-HCl buffer were found to inhibit C-
proteinase's activity. ~or example, it is reported that, with 0.2, 0.3, and
O.~M NaCl, the activity of C-~loL~h~ase was reduced 66, 38, and 25%,
1:~ respectively, of that observed with the standard assay concentration of 0.1~M.
Tris-HCl buffer in a cvnc~ dLion of 0.2-O.~M markedly inhibited activity.
~Iojima et al., s~pra. In contrast, microbial inhibitors such as leupeptin,
phosphoramidon, ~ntip~in ~-ost~tin, el~ctin~l, and ~m~ct~tin, are considered to
have weak or no effect on the activity of C-proteinase.
C-proteinase activity and its inhibition have been deterrnined using a
wide array of assays. See e.g., Kessler and Goldberg, 1978, Anal. Biochem.
86:463; Njieha et al., 1982, Biochemistry 21:7~7-764. Despite the
availability of such assays, large scale review and testin~ of potential C-
~lolt~ ase inhibitors has not ~een performed to date due to the limited
2~ availability of human C-~loteinase. As artir~ 3t~1 in numerous publications,
the enzyme is difficult to isolate by conventional biochemical means and the
identity of the cDNA sequence encoding such enzyme was not known until
reported in the above-referenced and related patent applications.
Development Of Compol~nds ~o Inhi~it C-Proteinase Activity. In
30 view of its ~ssenti~l role in the formation and maturation of collagen, C-
proteinase appears to be an ideal target for the treatment of disorders
3~
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associated with the ~ ,iate or unregulated production and maturation of
collagen. ~Iowever, none of the inhibitors so far irl~ntifi~rl has proven an
effective therapeutic for the tre~fm~nt of collagen-related diseases or even an
inhibitor to C-~loL~ ase activity.
The identification of effective compounds which specifically inhibit the
activity of C-~loLe~.lase to regulate and modulate abnormal or il~L~loL~iate
collagen production is therefore desirable and the o~ject of this invention.
3. SUMMARY OF T~E ~VENTION
The present invention relates to organic molecules capable of
mofl~ ting, regulating and/or inhibiting production and/or maturation of
collagen by affecting C-proteinase activity. Specifically, the compounds of
the present invention have the formulae:
a. Inhi~itor A
y~<N~x~E~4
Rl R2
or:
o
2~ Y)~C~ >
N--SO~ ~4
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wherein:
Rl is selected from the group con~i~ting of H, lower alkyl, mono- or
poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, halo sub~ d
aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl,
acyloxyalkyl, mercaptoalkyl, (amino, mono- or dia1kylamino)alkyl,
acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
heterocycloalkylalkyl, alkyl-(thio, sulfinyl or sulfonyl)-alkyl;
~2 is selected from the group consisting of H, lower alkyl;
~3iS selected from the group consisting of H, lower alkyl, mono- or
poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, halo ~ul,~ lrd
aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl,
acyloxyalkyl, mercaptoalkyl, (amino, mono- or diaLkylamino)alkyl,
acy~minr~lkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
heterocycloaLkylalkyl, alkyl-(thio, sulfinyl or sulfonyl)-alkyl;
~ is selected from the group eol~ Lillg of aryl, heteroaryl, alkyl,
aralkyl, h~teludldlkyl, alkylamino, arylalkylamino;
X is selected from the group Co~ li,.g of SO2, C--O;
Y is selected from the group consisting of OH,
HOHN(hydroxylamine), H2N, alkylamino;
Z is a direct bond; methylene, oxygen, sulfor, amino;
nis 0 or 1;
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or:
b. Inhibitor B
R2
HOHN ~ N 7~R~
~1 ~ R3 R4
whe~
Rl is selected from the group concicting of H, lower alkyl, mono- or
1_, poly-haloalkyl, carboxyalkyl, aryl, heleroaryl, aralkyl, heteroaralkyl, biaryl,
biarylalkyl, hydroxyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl,
mclca~,Loalkyl, (amino, mono- or dialkylamino)alkyl, acyl"mino~lkyl,
cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-
(thio, sulfinyl or sulfonyl3-aLkyl;
R2 is selected from the group consisting of H, lower alkyl, mono- or
poly-haloalkyl, carboxyalkyl, aryl, h~ .ualyl, aralkyl, heteroaralkyl, biaryl,
biarylalkyl, hydroxyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl,
mercaptoalkyl, (amino, mono- or dialkylamino3alkyl, acyl"mino~lkyl,
cycloaLkyl, heLt:lo~;ycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-
2~; (thio, sulfmyl or sulfonyl)-alkyl;
R3iS selectecl from the group conci.cting of H, lower alkyl, mono- or
poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl,
biarylalkyl, hydroxyaLkyl, alkyoxyaLkyl, acyloxyalkyl, mercaptoalkyl, (amino,
- mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl,
cycloalkylalkyl, heterocycloaLkylalkyl, alkyl-(thio, sulfinyl or sulfonyl)-alkyl;
R4 is selected ~rom the group consisting of H, lower alkyl;
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R5 is selected from the group consisting of H, lower alkyl,
carboxyaLkyl, (mono- or dialkylamino)aLkyl, alkyl-(thio, sufmyl or
sulfonyl)alkyl, alkoyalkylacylalkyl;
or:
c. ~nh~ or C
R2 ~
HS~ ~ ~ Rs
R~ O R~
wherein:
3~1 is select~cl from the group concictin~ of H, lower alkyl, mono- or
1~ poly-haloallyl, carboxyalkyl, aryl, hel~lodlyl, aralkyl, h~Lt;loa,dlkyl, biaryl,
biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino,
mono- or dialkylamino)alkyl, acyl~mino~lkyl, cycloalkyl, heterocycloalkyl,
cycloaLkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfinyl or sulfonyl~-alkyl;
~. is selected from the group co".~ i"g of ~, lower alkyl, mono- or
20 poly-haloaL~cyl, carboxyaL~cyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl,
biarylalkyl, hydroxyalkyl, alkyoxyallyl, acyloxyalkyl, mercaptoalkyl, (amino,
mono- or dialkylamino)alkyl, acyl~mino~lkyl, cycloalkyl, heterocycloalkyl,
cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfinyl or sulfonyl)-aLkyl;
R3 is selected from the group co~ g of ~I, lower aLkyl, mono- or
2~ poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, he~eroaralkyl, biaryl,
biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino,
mono- or dialkylarnino)alkyl, acyl~mino~lkyl, cycloalkyl, heterocycloalkyl,
cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfinyl or sulfonyl)-alkyl;
R4 is selected from the group consisting of H, lower alkyl;
3~
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Rs is selected from the group consisting of H, lower alkyl,
carboxyalkyl, (mono- or dialkylamino)aLkyl, alkyl-(tnio, sufinyl or
sulfonyl)aLkyl, alkoyalkylacylalkyl;
or:
d. Inhibitor D
0 R,~ / ~R5
O ~, R.,
1~. wherein:
Rl is selected from the group con~icting of ~I, lower alkyl, mono- or
poly-haloalkyl, carboxyalkyl, aryl, helclualy-l, aralkyl, hclelodldlkyl, biaryl,biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mcl~a~lodlkyl, (amino,
mono- or dialkylamino)alkyl, acyl~mino~lkyl, cycloalkyl, heterocycloalkyl,
20 cycloalkylalkyl, heterocycloaLkylalkyl, alkyl-(thio, sulfinyl or sulfonyl~-alkyl;
R~ is selected from the group COl~si~ g of H, lower allcyl, mono- or
poly-haloalkyl, cal'Lo~ydlkyl, aryl, heteroaryl, aralkyl, ht:lcl-~alalkyl, biaryl,
biarylalkyl, hydlu~yalkyl, alkyoxyalkyl, acyloxyalkyl, lllcl~d~loalkyl, (amino,
mono- or dialkylamino)alkyl, acyl~mino~lkyl, cycloalkyl, heterocycloalkyl,
2~ cycloalkylalkyl, hetcrocycloalkylalkyl, alkyl-(thio, sulfinyl or sulfonyl~-alkyl;
R3 is selected from the group consisting of ~, lower alkyl, mono- or
poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, hctelua~dlkyl, biaryl,
biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino,
~ mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl,
30 cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfinyl or sulfonyl~-alkyl;
R~ is selected from the group consisting of H, lower alkyl;
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3~ is selected from the group concicting of ~I, lower alkyl,
carboxyalkyl, (mono- or dialkylamino)aLcyl, aLkyl-(thio, sufinyl or
sulfonyl)alkyl, alkoyaLkylacylalkyl;
or:
e. ~nhibitor E
O R2 ~
0 HO X/~ N
O R~ R.
wherein:
Rl is selected from the group concicting of OH, alkoxyl, lower aLkyl,
15 alkylamino, peptide;
X is selected from the group col~i~Lillg of N, C;
R2 is select~(l from the group consisting of H, lower alkyl, mono- or
poly-haloalkyl, carboxyaL~cyl, aryl, heteroaryl, aralkyl, h~teludldlkyl, biaryl,~iarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino,
20 mono- or dialkylamino)alkyl, acyl~mino~lkyl~ cycloalkyl, heterocycloalkyl,
cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfinyl or sulfonyl)-alkyl;
R3 is selPct~?rl from the group consisting of H, lower alkyl, mono- or
poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl,
~iarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino,
2~ mono- or dialkylamino)alkyl, acyl~mino~lkyl, cycloalkyl, heterocycloalkyl,
cycloalkylalkyl, heterocycloalkylalkyl, allcyl-(thio, snlfinyl or sulfonyl)-alkyl;
R~ is selected from the group consisting of ~I, lower alkyl;
Rs is selected from the group concicting of H, lower alkyl,
carboxyalkyl, (mono- or dialkylamino)alkyl, aLkyl-(thio, sufinyl or
30 sulfonyl)alkyl, alkoyalkylacylalkyl.
3~
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The present invention is further directed to pharm~relltir~l
compositions comprising a pharm~re--tir~lly effective arnount of the above-
described compounds and a ph~ rellrir~lly acceptable carrier or excipient.
Such a composition should modulate the production and/or ~ lulaLion of
collagen by inhibi~ g C-yloL~ ase activity.
The present invention is also directed to the use of the disclosed
compounds and compositions for the tre~tm~ont of disorders associated with t_e
i.la~lu~lial~ or unregulated production of collagen by mo~ ting, inhibiting
and/or regulating C-proteinase activity.
More particularly, the compositions of the present invention may be
included in methods for treating diseases associated with ~a~ opliaLe or
unregulated production of collagen, including, but not limited to, rhr~-m~tQid
alll"iLis, scleroderma, pathological fibrosis or scarring.
15 4. D~:FINll IONS
"C-~u~ ase" shall be construed to mean an enzyme capable of
procrssing collagen molecules, derivatives or fragments, or their precursors
by cleaving through -Ala ~ Asp-Asp- and/or -Gly ~ Asp-Glu-. The term shall
include human C-~ hldse and derivatives, analogs, fragments and variams
20 thereof having C-proteinase-like activity.
"ph~ re~ltir~lly acceptable salts" refers to those salts which retain
the biûlogical effectiveness and properties of the free acids and which are
obtained by reaction with inorganic or organic bases such as sodium
hydroxide, m~g~ ,., hydroxide, ammorlia, trialkylamine, dialkylamine,
2~ monoalkylamine, dibasic amino acids, sodium acetate, potassium bell20aLt:,
triethanol amine and the like.
"Alkyl" refers to a saturated aliphatic hydrocarbon, including straight-
chain, branched-chain, and cyclic allcyl groups. Prefera~ly, the alkyl group
has 1 to 12 carbons. More preferably, it is a lower alkyl of from 1 to 7
3() carbons, more ~ Ç~ldbly 1 to 4 carbons. Typical alkyl groups includes
methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl
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and the like. The alkyl group may be substituted or unsubstituted. When
substituted the substituted group(s) is preferably, carboxyl, hydroxyl,
mercapto, cycloaLkyl, heterocycloaLkyl, halo, aLkoxyl, alkylamino.
"Aryl" refers to an aromatic group which has at least one ring having a
conjugated pi electron system and includes carbocylic aryl, heterocyclic aryl
and biaryl groups, all of which may be optionally substituted. Preferably, the
aryl is a substituted or unsubstituted phenyl or pyridyl. Preferred aryl
substituent(s) preferably phenyl or pydridyl are halogen, trihalomethyl,
hydroxyl, S~I, NO" amine, thioether, cyano, alkoxy, and groups.
~0
. DETAILl~D DESCRIPTION OF THE ~V~NTION
The present invention relates to compounds capable of regulating
and/or mor~ tin~ collagen formation by inhibiting C-proteinase activity.
More particularly, the present invention is directed to compounds
which inhibit C-plote~lase activity as a therapeutic approach to cure or
manage various connective tissue disorders, including fibrotic disorders,
arthritic disorders, or disorders inrll-red or initi~tto~ by surgical or dramatic
insults.
~.1. The Compounds
The invention is generally directed to compounds and/or
compositions co~ g compounds having the formulae:
a. Irz*i~itor A
2~ G R3
y~><N~x~3
Rl R2
or:
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~ >
Y I--N R~
~2 SO2
wherein:
Rl is selected from the group consisting of ~, lower alkyl, mono- or
poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, halo sllbstititllted
10 aralkyl, ht:LeloaLdlkyl, biaryl, biarylalkyl, hydroxyaLkyl, allcyoxyalkyl,
acyloxyalkyl, mercaptoaLIcyl, (amino, mono- or dialkylamino)alkyl,
acyl~mino~lkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
heterocycloalkylalkyl, alkyl-(thio, sulfinyl or sulfonyl3-aLkyl;
~2is selecterl from the group consisting of H, lower alkyl;
1~; ~3iS selected from the group consisling of ~I, lower alkyl, mono- or
poly-haloalkyl, carboxyalkyl, aryl, h~ oalyl, aralkyl, halo substitinlte~l
aralkyl, h~lel.~a,alkyl, biaryl, biarylalkyl, hydroxyalkyl, alkyoxyalkyl,
acyloxyalkyl, mcl.;ayLoalkyl, (amino, mono- or dialkylamino)alkyl,
acyl~mino~lkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
20 heterocycloalkylalkyl, alkyl-(thio, sulfinyl or sulfonyl3-alkyl;
~ 4 is selectPcl from the group CQ.~ lg of aryl, hclel-~dlyl, alkyl,
aralkyl, heteroaralkyl, alkylamino, arylalkylamino;
X is selected from the group col~ ling of SO2, C=O;
Y is selectP~l from the group consisting of O~I,
2~ HOHN(hydroxylamine), H2N, alkylamino;
Z is a direct bond; methylene, oxygen, sulfor, amino;
n is 0 or 1;
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or:
. Inhi~i~or B
HOHN/ ~' ~N
Rl O R3 ~4
wherein:
Rl is selected from the group consisting of H, lower alkyl, mono- or
poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl,
biarylalkyl, hydroxyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl,
mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acyl~rnino"lkyl,
cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-
1~; (thio, sulfinyl or sulfonyl)-alkyl;
R. is selected from the group cnncicting of H, lower alkyl, mono- or
poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl,
biarylalkyl, hydroxyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl,
mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acy1~mino~lkyl,
20 cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-
(thio, sulfinyl or sulfonyl3-alkyl;
3~3iS selected from the group consisting of H, lower alkyl, mono- or
poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl,
biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino,
2~ mono- or dialkylamino)alkyl, acyl~minn~lkyl, cycloalkyl, heterocycloalkyl,
cycloalkylalkyl, heterocycloalkylalkyl, allyl-(thio, sulfinyl or sulfonyl)-alkyl;
is selected from the grol~p consisting of H, lower alkyl;
~ 5 iS selected from the group CulLsi~,Lillg of H, lower alkyl,
carboxyalkyl, (mono- or dialkylamino)alkyl, alkyl-(thio, sufinyl or
30 sulfonyl)alkyl, alkoyalkylacylalkyl;
14
3~i
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or:
c. Inh~i~or C
HS ~ ~ N ~ ~ R!s
P~, O R
wherein:
R~ is selected from the group col~sisL~Ilg of H, lower alkyl, mono- or
poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, h~ dldlkyl, biaryl,
biarylalkyl, hydlv~yalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoaIkyl, (amino,
mono- or diaL~ylamino3aIkyl, acyl~mino~lkyl, cycloalkyl, heterocycloalkyl,
15 cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfinyl or sulfonyl3-alkyl;
R~ is selçctec~ from the group consisting of H, lower alkyl, mono- or
poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl,
biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, melca~Lualkyl, (amino,
mono- or dialkylamino)alkyl, acyl~minn~lkyl, cycloalkyl, heterocycloalkyl,
20 cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfinyl or sulfonyl)-alkyl;
R3 is selected from the group consisting of H, lower alkyl, mono- or
poly-haloaLkyl, carboxyaL~cyl, aryl, heteroaryl, aralkyl, hetelodldlkyl, biaryl,biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino,
mono- or dialkylamino3alkyl, acyl~mino~llcyl, cycloalkyl, heterocycloalkyl,
2~; cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfinyl or sulfonyl3-alkyl;
R~ is selected from the group consisting of H, lower alkyl;
R5 is selected from the group consisting of H, lower alkyl,
carboxyalkyl, (mono- or dialkylamino)alkyl, alkyl-(thio, sufinyl or
~ sulfonyl)alkyl, alkoyalkylacylalkyl;
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or:
d. Inhi~i~or D
R, ~ ~ ,Rs
O R3 R~
wherein:
~ 1 is selected from the group consisting of H, lower alkyl, mono- or
poly-haloaL~yl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl,
biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (arnino,
mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl,
cycloaLkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfinyl or sulfonyl)-alkyl;
R, is select~l from the group concicting of H, lower alkyl, mono- or
poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, helelualdlkyl, biaryl,
biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino,
20 mono- or dialkylamino)alkyl, acyl~nino~lkyl, cycloalkyl, heterocycloalkyl,
cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfinyl or sulfonyl~-aLkyl;
~ 3iS selecte~l from the group co,~ g of H, lower alkyl, mono- or
poly-haloalkyl, carboxyalkyl, aryl, h~lûdlyl, aralkyl, h~L~lualdlkyl, biaryl,
biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino,
2~; mono- or dialkylamino)alkyl, acyl~mino~lkyl, cycloalkyl, heterocycloalkyl,
cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfinyl or sulfonyl)-alkyl;
R~ is selected from the group consisting of H, lower alkyl;
~ 5iS selected from the group consisting of H, lower alkyl,
carboxyalkyl, (mono- or dialkylamino~alkyl, alkyl-(thio, sufinyl or
30 sulfonyl)alkyl, alkoyalkylacylalkyl;
16
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or:
e. Inhi~itor E
o R,2 ~''
~; I I HN
R~ \ R5
HO X T N
o R3 li~
wherein:
Rl is selected from the group consisting of O~, alkoxyl, lower alkyl,
alkylamino, peptide;
X is selected from the group COllSi~,LLlg of N, C;
R2 is selected from the group consisting of H, lower alkyl, mono- or
15 poly-haloalkyl, carboxyalkyl, aryl, hLLelodlyl7 aralkyl, heteroaralkyl, 'oiaryl,
biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, m~ l.;ayl~,alkyl, (amino,mono- or dialkylamino)alkyl, acy1~mino"l'Kyl, cycloaL~cyl, heterocycloalkyl,
cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(taio, sulfinyl or sulfonyl3-alkyl;
R3iS selected from the group consisting of H, lower alkyl, mono- or
20 poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl,
biarylalkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, melcaylualkyl, (amino,
mono- or dialkylamino~alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl,
cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfmyl or sulfonyl3-alkyl;
~ is selected from the group consisting of H, lower alkyl;
2~ Rs is selected from the group consisting of ~I, lower alkyl,
carboxyalkyl, (mono- or dialkylamino)alkyl, alkyl-(thio, sufinyl or
sulfonyl)alkyl, alkoyalkylacylalkyl.
In specific em~odiments of the invention, the compounds of the present
invention may have tibe following formulae:
CA 02229098 1998-02-09
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~0~Cl
ItO S~ OMe
and ph~ elltit~.~lly accepta~le salts thereof;
or:
Cl
HOHN J~-- S~ OM~
and ph~ relltir~lly accepta~le salts thereof;
2~
18
3~
,
CA 02229098 1998-02-09
PCTAUS96/12876
W O 97/05865
or:
HOHN J~ 'S~ OMe
10and ph~nn~reutir,~lly accep~le salts thereof;
or:
OMe
'S--I~
- O
HOHN O
20and pharm~reutir~lly acceptable salts thereof;
or:
CO2H
2~ O
~o J~,N~S--~ OMe
30and ph~nn~relltic.~lly acceptable salts thereof;
19
3~
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or:
CO2H
HO2C~ ~ H ~
H ~f N~ NHMe
O \, CO2H
and ph~ relltir~lly acceptable salts thereof;
or.
SH O -/~CO2H
~ O
o
and phanT~re~-tir~lly acceptable salts thereof;
3~
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WO 97/0586~
or:
~CO2tBu
HOtlN J~-- S ~OMe
and ph~relltir~lly acceptable salts thereof;
or:
1~
NOltNJ~/N~ ~OM-
and phaIm~rel~tir~lly acceptable salts thereof;
2~ or:
CA 02229098 1998-02-09
WO 97/05865 PCTAJS96/12876
f ~CF3
HOHNJ~ S~OMe
~0
and pharrn~relltir~lly accepta~le salts thereof;
or:
~ ~ ~
HOHNJ~ ~OMe
2~; and ph~ relltir~lly accepta~le salts thereof;
CA 02229098 1998-02-09
WO 97/05865 PCT~US96/12876
or:
CI
tlOHN ,1 , ~
and phs~ relltir~lly ~crept~hle salts thereof;~~
or:
~
HOHN N S~OMe
c2~
and ph~ rt--ltir~lly acceptable salts thereof.
The ch~mir~l formulae referred herein may exhibit the phenomenon of
tautomerism. As the formulae drawing within this specification can only
30 ~ esent one of the possible tautomeric forms, it should be understood that
the invention encompasses any tautomeric form which possesses the ability to
3~
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regulate and/or modulate the production and/or maturation of collagen by
inhibiting C-~loteiu,ase activity. Further, the invention is understood to
include all possible stereoisomers of each of the compounds described.
In addition to the above compounds and their pharm~rentir~.lly
acceptable salts, the invention is further directed, where applicable, to
solvated as well as unsolvated forms of the compounds (e.g. hydrated forms)
having the ability to inhibit, regulate and/or modulate the production and/or
maturation of collagen by inhibiting C-~luLeillase activity.
The compounds described above may be prepared by any process
10 known to be applicable to the ~re~aldlion of chemically-related compounds.
Suitable processes are illustrated by the fo,llowing representative examples.
Necessary starting materials may be obtained by standard procedures of
organic cl ,~ l, y.
An individual compound's relevant activity and efficiency as an agent
to affect C-~LoLeinase activity may be determined using available techniques.
Preferentially, a colll~oulld is subjected to a series of screens to d~L~llllille the
compound's ability to modulate, regulate and/or inhibit the production and
lllaLuldlion of collagen. These screens include biochemir~l assays, cell culture
assays and animal models.
~i.2. ~n-lir~ti~nc
Disorders associated with the hld~lu~lidLe or unregulated
production and/or maturation of collagen, inrilltling arthritic disorders, fibrotic
disorders, and other connective tissue disorders, can be treated with the
25 compounds and compositions of the present invention.
These diseases or disorders include pathological fibrosis or scarring,
such as endocardial sclerosis, idiopathic hlL~l~.LiLial fibrosis, interstitial
pulmonary fiblusis, periml-scl-lar fibrosis, Symmers' fibrosis, pericentral
fibrosis, hepatitis, derlnatofibroma, billary cirrhosis, alcoholic cirrhosis, acute
30 pulmonary fibrosis, idiopatnic pulmonary fibrosis, acute respiratory distress syndrome, kidney fibrosis/glomerulonephritis, kidney fibrosis/diabetic
24
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nephlo~dllly, scleroderma/systemic, scleroderma/local, keloids, hypertrophic
scars, severe joint adhesions/arthritis, myelofibrosis, corneal scarring, cysticfibrosis, mn~ r dystrophy (duchenne's), cardiac fibrosis, mncc~ r
fibrosis/retinal separation, esophageal stricture, payronles disease. Further,
~; fibrotic disorders may be inrlncer1 or initi~tr~f1 by surgery such as scar
revision/plastic surgeries, glaucoma, cataract fibrosis, corneal scarring, jointadhesions, graft vs. host disease, tendon surgery, nerve e~ d~ ent,
duyuyLlcll's contracture, OB/GYN adhesions/fibrosis, pelvic adhesions,
peridural fibrosis, restenosis. Still further fibrotic disorders may be inrl~lced
10 by chemotherapy, including, for example lung fibrosis and the like.
.3. Pharm~~el-ti~ ~l Form~ t;crlc And Routes Of ~ i . dlion
The identified compounds can be ~r1mi"i~leled to a patient in
need, by itself, or in ph~rm~re~ tir~l compositions where it is mixed with
l~i suitable carriers or excipient(s) at doses to treat or ameliorate a variety of
disorders. A therape~ltir~lly effective dose further refers to that amount of the
compound sufficient to result in amelioration of symptoms. Techniques for
fonmll~tion and ~rlminictration of the compounds of the instant application
may be found in "Remington's Pharm~celltir~l Sciences," Mack ~ublishing
20 Co., Easton, PA, latest edition.
.3.1. Routes Of Atlrninictration.
Suitable routes of ~r~ ,dlion may, for example,
include oral, rectal, tr~ncmllros~l, or intr~stin~ dtion; ~drC~ dl
2~; delivery, including intr~mllcclll~r, subcutaneous, intr~m~rlllll~ry injections, as
well as illl~dll~ecal, direct intraventricular, intravenous, intraperitoneal,
intranasal, or intraocular injections.
Alternately, one may ~lrnini~t~r the compound in a local rather than
systemic manner, for example, via injection of the compound directly into a
30 arthritic joints or in fibrotic tissue, often in a depot or sn~t~int~(l release
formulation. In order to prevent the scarring process frequently occurring as
3~
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complication of glaucoma surgery, the compounds may be atlminist~red
topically, for exarnple, as eye drops.
Furtherrnore, one may ~1minicter the drug in a targeted drug delivery
system, for example, in a liposome coated with a specific antibody, L~lgeLillg,
for exatnple, arthritic or fibrotic tissue. The liposomes will be targeted to and
taken up selectively by the ,.fflir~ecl tissue.
.3.2. Composition/Form~ tion.
The ph~ relltir,.l compositions of the present
10 invention may be m~nnf~rn-red in a l~lal~ that is itself known, e.g., by
means of conventional mixing, dissolving, gr~m~l~Jing, dragee-making,
levigating, emulsifying, encaps~ ting, ellL~dLJpillg or Iyophilizing processes.
Pharm~relltir:,.l compositions for use in accul-lallce with the present
invention thus may be fo~nlll~trA in conventional manner using one or more
l~i physiologically acceptable carriers COlll~li.illg excipients and auxiliaries which
facilitate processin~ of the active compounds into ~ alations which can be
used ph~rm~relltir~lly. Proper forrnulation is dependent upon the route of
aArninistration chosen.
~or injection, the agents of the invention may be form~ t.oA in aqueous
20 solutions, preferably in physiologically compatible buffers such as ~anks's
solution, ~inger's solution, or physiological saline buffer. ~or ~n~mllrosal
."i"i~l,ation, p~ . a~o~liate to the barrier to be perrneated are used
in the forrmll~tion. Such penetrants are generally known in the art.
For oral ar'mini~tration, the compounds can be forrmll,.tt~rl readily by
2_ combining the active compounds with pharm~reutir~lly acceptable carriers
well ~nown in tne art. Such carriers enable the compounds of the invention to
be for,n~ tt-cl as tablets, pills, dragees, capsules, liquids, gels, syrups,
slurries, suspensions and the like, for oral ingestion by a patient to be treated.
Pharm~ce~ltir~l preparations for oral use can be obtained solid excipient,
3~ optionally grinding a r~slll~ing mixture, and processing the mixture of
granules, after adding suitable auxiliaries, if desired, to obtain tablets or
26
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dragee cores. Suitable excipients are, in particular, fillers such as sugars,
including lactose, sucrose, m~nnitol, or sorbitol; cellulose ~l~paldLiorls such
as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin,
gum tr~gAr~nth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium
~i carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,
disintegrating agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium ~lgin~tr-.
Dragee cores are provided with suitable coatings. For this purpose,
concelllldl~d sugar solutions may be used, which may optionally contain gum
10 arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or
th~nillm dioxide, lacquer solutions, and suitable organic solvents or solvent
mixtures. Dyestuffs or pigm~-nt~ may be added to the tablets or dragee
coatings for idellLiflcdLion or to characterize dirr~l~l-L combinations of active
compound doses.
ph~rm~relltir~ dldLions which can be used orally include push-fit
r~pslllr~5 made of gelatin, as well as soft, sealed capsules made of gelatin anda plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain
the active ingredients in admixture with filler such as lactose, binders such asstarches, and/or lubricants such as talc or m~gnr-sillm stearate and, optionally,
2~ stabilizers. In soft capsules, the active compounds may be dissolved or
suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid
polyethylene glycols. In addition, stabilizers may be added. All forml-l~rions
for oral ~ .aLion should be in dosages suitable for such ~rl~"i~ dlion
For buccal a~lminicfration,the compositions may take the form of
2~ tablets or lozenges formulated in conventional manner.
For ~ ni~tration by inhalation, the compounds for use according to
the present invention are conveniently delivered in the form of an aerosol
spray presentation from pressurized packs or a nebuliser, with the use of a
~ suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
30 dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of
a ~ie~uli~ed aerosol the dosage unit may be determined by providing a valve
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to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use
in an inhaler or incllffl~t~r may be forn~ tt~d cont~inin~ a powder mix of the
compound and a suitable powder base such as lactose or starch.
The compounds may be form~ teA for parenteral a~lmini~tration by
5 injection, e.g., by bolus injection or continuous infusion. Formulations for
injection may be presented in unit dosage form, e.g., in ampoules or in multi-
dose containers, with an added preservative. The compositions may take such
forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and
may contain formlll~tory agents such as suspending, stabilizing and/or
10 dispersing agents.
ph~rm~celltic~l formulations for parenteral ~rlminictration include
aqueous solutions of the active compounds in water-soluble form.
Additionally, suspensions of the active compounds may be prepared as
a~luyliat~ oily injection suspensions. Suitable lipophilic solvents or vehicles
include fatty oils such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions
may contain substances which increase the viscosity of the suspension, such as
sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the
suspension may also contain suitable stabilizers or agents which increase the
20 solubility of the compounds to allow for the preparation of highly concel~Lldtt:d
solutions.
Alternatively, tne active ingredient may be in powder form for
col~.LiLuLion with a suitable vehicle, e.g., sterile pyrogen-free water, before
use.
2_, The compounds may also be form~ t~-~d in rectal compositions such as
suppositories or retention enemas, e.g., cont~inin~ conventional suppository
bases such as cocoa butter or other glycerides.
In addition to tne forrnulations described previously, the compounds
may also be formnl~t~d as a depot preparation. Such long acting formulations
30 may ~e ~lmini~tered by implantation (for example subcutaneously or
intr~mlls~ rly) or by intr~mllcc~ r injection. Thus, for example, the
28
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compounds may be formulated with suita~le polymeric or hydrophobic
materials (for example as an emulsion in an acceptable oil) or ion exchange
resins, or as sparingly soluble derivatives, for example, as a 5p~ringly solublesalt.
A ph~ relltir~l carrier for the hydrophobic compounds of the
invention is a cosolvent system comprising benzyl alcohol, a nonpolar
surfactant, a water-miscible organic polymer, and an aqueous phase. The
cosolvent system may be the VPD co-solvent system. VPD is a solution of
3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80,
10 and 6~ ~ w/v polyethylene glycol 300, made up to volume in absolute ethanol.
The VPD co-solvent system (VPD:~W) consists of VPD diluted 1:1 with a
~% dextrose in water solution. This co-solvent system dissolves hydrophobic
compounds well, and itself produces low toxicity upon systemic
dLion~ Naturally, the proportions of a co-solvent system may be
varied considerably without destroying its solubility and toxicity
characteristics. Furthermore, the identity of the co-solvent components may
be varied: for example, other low-toxicity nonpolar surfactants may be used
instead of polysorbate 80; the fraction size of polyethylene glycol may be
varied; other bioco~ aLible polymers may replace polyethylene glycol, e.g.
20 polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
Alternatively, other delivery systems for hydrophobic ph~rm~relltir~l
compounds may be employed. Liposomes and emulsions are well known
examples of delivery vehicles or carriers for hydrophobic drugs. Certain
2~; organic solvents such as dimethylsulfoxide also may be employed, although
usually at the cost of greater toxicity. Additionally, the compounds may be
delivered using a 5llct~inrcl-relea5e system, such as semiper neable matrices ofsolid hydrophobic polymers cont~ining the therapeutic agent. Various of
- sllst~int~d-release materials have been established and are well known by those
30 skilled in the art. Sustained-release capsules may, depending on their
chrmir~l nature, release the compounds for a few weeks up to over 100 days.
29
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Depending on the cht?mir~l nature and the biological stability of the
therapeutic reagent, additional strategies for protein stabilization may be
employed.
The pharm~reutir~l compositions also may comprise suitable solid or
5 gel phase carriers or excipients. Examples of such carriers or excipients
include but are not limited to calcil~m carbonate, calcium phosphate, various
sugars, starches, cellulose derivatives, gelatin, and polymers such as
polyethylene glycols.
Many of the C-l~,oleillase inhibiting compounds of the invention may
10 be provided as salts with ph~rm~reutir~lly compatible counteriorLs. Such
ph~ remir~lly acceptable base addition salts are ~hose salts which retain the
biological effectiveness and ~u~elLies of the free acids and which are obtained
by reaction with inorganic or ûrganic bases such as sodium hydroxide,
m~nrsillm hydroxide, ammonia, trialkylamine, dialkylamine,
1~; monoa~ylamine, dibasic amino acids, sodium acetate, potassium benzoate,
triethanol amine and the like.
.3.3. Effective Dosage.
ph~rm~relltir~l compositions suitable for use in the
20 present invention include compositions wherein the active ingredients are
contained in an effective amount to achieve its intended purpose. More
specifically, a theraFeutir~lly effective amount means an arnount effective to
prevent development of or to alleviate the existing symptoms of the subject
being treated. Determination of the effective amounts is well within the
25 capability of those skilled in the art, especially in light of the det~ d
disclosure provided herein.
For any compound used in the method of the invention, the
therapeutir~lly effective dose can be ~ frd initially from cell culture
assays. For example, a dose can be form-ll~red in ani~nal models to achieve a
30 circ~ ting concen~ration range that includes the IC50 as determined in cell
culture (i.e., the concentration of the test compound which achieves a half-
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maximal innibition of the C-ploteillase activity). Such information can be
used to more accurately ~leter7lnin~ useful doses in h-lm~nc.
A therape-lr;r~lly effective dose refers to that amount of the compound
that results in amelioration of symptoms or a prolongation of survival in a
patient. Toxicity and therapeutic efficacy of such compounds can be
determined by standard ph~rn~rellti~l procedures in cell cultures or
experimental ~nim~l~, e.g., for deL~ g the LD50 (the dose lethal to 50%
of the population) and the 3~D50 (the dose the,d~e~,lir~lly effective in 50% of
the population). The dose ratio between toxic and therapeutic effects is t'ne
therapeutic index and it can be expressed as the ratio between LD50 and FD50.
Compounds which exhibit high therapeutic indices are L~.er~ d. The data
obtained from these cell culture assays and anirnal studies can be used in
formnl~ting a range of dosage for use in human. The dosage of such
compounds lies preferably within a range of circlll~ting concentrations t'nat
include the ~D50 with little or no toxicity. The dosage may vary witnin this
range depending upon the dosage form employed and tne route of
~rlminictration lltili7-o~l The exact forrnulation, route of ~rimini~tration anddosage can be chosen by the individual physician in view of the patient's
condition. See, e.g., Fingl et al., 197~, in "The ph~rm~rological Basis of
Therapeutics", Ch. 1 p.l.
Dosage amount and interval may be adjusted individually to provide
plasma levels of the active moiety which are sufficient to m~int~in the C-
uLeil~ase h.llibi~illg effects, or minim~l effective concentration (MEC). Ihe
MEC will vary for each compound but can be e~ t~rl from in vitro data;
for example, tne collcelllldLion n~c.oc~ry to achieve ~0-90% inhi~ition of the
C-~ hlase using the assays described herein. Dosages n~cesS~ry to achieve
the M3~C will depend on individual characteristics and route of ~rlminict~ation
However, HPLC assays or bioassays can be used to determine plasma
concentrations .
Dosage intervals can also be determined using M~C value.
Compounds should be a~ ir~ied using a regimen which m~int~inc plasma
31
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levels above the MEC for 10-90% of the time, preferably between 30-90%
and most preferably between ~0-90%.
In cases of local ~rlministration or selective uptake, the effective local
concentration of the drug may not be related to plasma concentration.
The ~mollnt of composition ~rlmini.stt-red will, of course, be dependent
on the subject being treated, on the subject's weight, the severity of the
affliction, the manner of ~rlminictration and the j~ m~nt of the prescribing
physician.
0 5,3.4, P~ck~n~
The compositions may, if desired, be presented in a pack
or dispenser device which may contain one or more unit dosage forms
cont~inin~ the active ingredient. The pack may, for example, comprise metal
or plastic foil, such as a blister pack. The pack or dispenser device may be
1~; accomr~ni~l by instructions for ~rlmini~tration. Compositions comprising a
compound of the invention forrn~ ttod in a compatible ph~nn~r~llric~l carrier
may also be ~ al~d, placed in an a~lopliate container, and labelled for
treatment of an inrlir~l condition. Suitable conditions intlir~t~l on the label
may include treatment of arthritis or any other fibrotic disorder.
6. EXAMPLES
The compounds of the present invention may be syntht-si7torl according
to known terhnirlul-s. The following represent ~ r~ d methods for
synth~si7in~ and testing the compounds of the claimed invention:
6.1. F.Y~ Compound Synthesis
6.1.1. Synthesis Of N-l~ydl-~xy-2-[~ 4-
m~thclxyD~ ..lfonyl~-N'-(4-
chlorobenzyl)]amino~ et~mide
The preferred method for synth~si7.in~ N-hydroxy-2-
[[N'-(4-methoxy'~enzenesulfonyl)-N'(4-chloro~enzyl)]amino]-~cet~micle (~)
3~
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(specific compounds in this specification may ~e referred to, for nomenclature
purposes as "C~", wherein "#" is an arabic number), also rle~ign~tecl as
~G047, (~xample for Inhi~itor A) is as follows:
¦¦ ~ N~EB ~ a
~N~ ~ a 7.r~
2 EtO NH 3
uo~ ~ OMc ~C~
a ~ON~,HCl ,~ a
~ (} G (14 7) 4
Synt*esis Of Et*yl-2~(4c*10robenz;ylJarnino~-acetate (3). To a cold
solution of glycine ethyl ester hydrochloride (1) (1.00 g, 7.16mmol) and 4-
chloro~en7~ hyde (2) (1.00 g, 7.16 mmol) in anlly(ll~us MeOH (10 ml) was
2- added anhydrous ZnCl, solid (75 mg, 0.~5 mmol) followed ~y NaBCN~I3
solid (0.45 g, 7.16 mmol). After stirring at room ~ e~ re for 18 h, the
reaction mixture was quenched with 1 N HCI (20 ml) aqueous solution, and
stirred for another 30 min. The mixture was concentrated on a rotary
evaporator to remove most MeOH solvent and then extraeted with ether (20
3~ ml). The aqueous layer was carefully basified in an ice bath with 45%(w/w)
of KO~I aqueous solution to pH 10, and extracted with 3~tOAc (2 x 50 ml).
3~
-
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The combirled ~tOAc organic layers was washed with brine, dried over
Na2SO4, and concentrated. The crude product was purified by silica gel c
chromatography (4/1 hexanes/EtOAc) to get ethyl-2[(4-chlorobenzyl)amino]-
acetate (3) as an oil.
Synthesis Of Ethyl-2-1lZV-(4methoxybenzene-sulfonylJ-N-(4
chloroben~ arninol~etnte (4). Triethylamine (294 mg, 2.90 mmol) was
added dropwise to a solution of ethyl-2[(4-chlorobenzyl)amino]-acetate (3)
(600 mg, 2.64 mmol) and p-metho~yl,~ enesulfonyl chloride (545 mg, 2.64
mmol) in allllydl~us CH~Cl2 (7 rnl). The llli~lule was stirred at room
temperature for 1~ h and treated with 1 N HCI (20 ml) solution. The
res-~ltin" two phases were separated, and the aqueous layer was extracted with
CH2Cl,. The combined organic layer was washed with brine, dried over
MgSO4, and collcellLldll:d. The crude product was purified by silica gel
1~ cl~ ,atography (3/1 to 2/1 h~x~n~s/EtOAc) to get ethyl-2-~[N-(4-
methoxyberl7ene-sulfonyl)-N-(4-chloro~enzyl)3-amino3acetate (4) as an oil.
Synt*esis Of N-hydroxy-21llV'-(4metho~y-benz.ones~lfonyl)-N'(4
chloroben~ ]arnino~ etn~ e (5). Se~dldte solutions of hydroxylamine
hydrochloride (171 mg, 2.46 mmol) in MeOH (1.3 ml) and of KOH (207 mg,
3.69 mmol) in MeOH (1.3 ml) were prepared at the ~oiling point, cooled to
40~C, and then the latter solution was added to the former. After cooling the
reaction lni~lul~ in an ice bath for 30 min, pot~ n chloride solid was
filtered off. To the filtrate was added e~hyl ester (4) (487 mg, 1.23 mmol).
2~i After stirring at room temperature for 6 h, the reaction ~ Lul~ was treatedwith 1 N HCl (2û ml) solution, and extracted with CH2C12. The organic layer
was washed with ~rine, dried over MgS04, and concentrated to get the crude
product. The residue was L~ ldlt:d in ether and the white solid was collected
to get the hydroxarnate, i.e., N-hydroxy-2[~N'-(4-methoxy-benzenesulfonyl)-
N'(4-chlorobenzyl)~-amino]-~cet~mirle (~), also decign~t~d as ~G-047.
3~
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mp: 124 - 125~C; MS (ES) (M+H): 38~; lH NM3~ (360 MHz,
DMSO-d6)~ 10.47 (s, 1 H, O~I), 8.81 (s, 1 H, NH). 7.81 - 7.08 (m, 8 H,
Ph), 4.34 (s, 2 H, CH7CO), 3.8~ (s, 3 H, OMe), 3.63 (s, 2 H, CH2Ph).
6.1.2. Synthesis Of N-lly~llo~y-2-[1N'-(4-
mPthoxyben~.on~snlfonyl3-N'-(4-fluorobenzyl~amino3 -
...ide
The ~ler~:lled method for 5ynth~osi7ing N-hydroxy-2-
~[N'-(4-metho~y~e.~ slllfonyl)-N'-(4-fluoro'oenzyl)]amino]-~eet~mirl~o (6),
also ~le~i,7n~te~1 as FG0~3, (~xample for Inhi'oitor A) is essentially as
described for the synthesis of N-hydroxy-2-[~N'-(4-methoxyben7~n~slllfonyl)-
N'(4-chlorobenzyl)3amino3-?lcet~mi~, using as starting material 4-
fluorobenzaldehyde instead of 4-chlorob~n7~k'~hyde. See, Section 6.1.1.
~F
HOHN)~/ o _~OUo
6 (~ 0S3)
MS (~S) (M-1)-:367; lH NM3:~ (360 MHz, DMSO-d6) ~ 10.4~ (s, 1 H,
OH), 8.83 (s, 1 H, NH).7.81-7.08 (m, 8 H, Ph), 4.33 (s, 2 ~, CEI CO), 3.85
(s, 3 H, OMe), 3.62 (s, 2 H, C~I7Ph).
6.1.3. Synthesis Of Hy(ll <~Y~ le
The ~lef~llcd me~od for synthesizing hydroxamate (11)
(Example for Inhi'oitor B) is as follows:
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O O
~~ ~H2C02~ ~ ~ 1. ~0~ ~ } COOH
H+
7 8 9
~0 * ~ 0 COOEh 1.IPA
GI~OEh~NHMe ~hO2C ~N - ~ A~O
0 D~r~nRt10 ~ 3. N~OH
~ =COOBn
tlOHN ~ NH~N~1U-
1~ o ~~ H O
AL~ylation of malonic ester (n with tert-butyl bromoacetate provides
tetraester (8). Upon sap~llirlcaLion and decarboxylation, the resnlting aid (9)
is coupled with Glu (OBn) NHMe using peptide coupling reaction to give
(10). After subsequent hydrolysis, anhydride formation, and addition of
2- N~,OH, (10) can be converted to the desired hydroxamate (11).
6.1.4. Synthesis Of N-Carbo~y~ lhyl Dipeptide
The preferred method for synth~ci7incg N-carboxymethyl
dipeptide (16), also designated as FG0~7, (Example for Inhibitor D) is as
30 follows:
36
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COOBn
~COOBn ~ 1. TFA
H'N--b--~H + ~I~Nl~fNHMe 2. DCC/HOBt
~ ~; Boc O BOC O
12 13
~COOBn 1. TE~A BnO2C ~COOBn
~I~N~ CONHMe 2. BrCH2CO2Bn NJ\n'NH CON~IMe
- NMM ~ -
Boc O ~COOBn O ~COOBn
14 15
~I2/Pd
HO2C ~COOH
~,NH CONHMe
O ~OE
16 (FG 057)
The building blocks (12) and (13) which are readily available by
2~ cl~Lulc procedures, are coupled by the cl~Csic~l DCC/HOBt method leading
to the dipeptide (14) in 78% yield. De~ Lc-;Lion of (14) using TFA in C~I2Cl,
and subsequent alkylation with benzyl brom~rer~te in the presence of NMM
resulted in the formation of the N-carboxymethyl dipeptide (1~), which was
converted to the free acid (163 by catalytic hydrogenation.
Boc-Glu (OBn)O~I and Boc-Asp(OBn)O~I have been synthesized using
methods readily known in the art.
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Syn~*eszs Of Boc-Glu(OBn)NHMe (13): Triethylamine (2.0 g, 20
mmol) was added to a solution of Boc-Glu (OBn)OH (6.7 g, 20 rnmol) in dry
THF (100 ml). The solution was cooled to -78~C under argon atmosphere,
then ethyl chloroformale (2.2 g, 20mmol) was added dropwise. The reaction
mixture was allowed to warm to -30~C over 2h, then a 40% aquoeus solution
of methyl amine (22 mmol) was add, and the reaction n,i~Lule was allowed to
warm to room temperature. After the reaction had stirred for a further hour,
diethyl ether (~0 ml) and water (70 ml) were added. The organic layer was
se~dl~d, washed with 1 M NaHCO3, 10% citric acid, and saturated NaCI
solution, and dried over MgSO~. The solvent was evaporated in vacuo to give
a white solid (~.0 g, 72% yield).
IH-NMR (200 MHz, CDC13): ~ 1.38 (s, 9H, CH3); 1.8~ 9 (m,
4H, C~); 2.7~ (d, 3H, NCH3); 4.17 (m, 1~I, C~I); 5.08 (~, 2H, OCH2); 5.33
I, NH); 6.30 (~s, lH, NH); 7.31 (s, ~I, Ph-H).
1~
Synt*esis Of Boc-Asp(OBn)-Glu(OBn~NHMe (14): To a solution of
tert-butoxycarbonyl arnino ester (13) (700 mg, 2 mmol) in 10 ml CH~Cl. was
added 1.~ ml TFA and the reaction ~ Lule was stirred for 1 h at room
eldlule under argon atmosphere. The excess acid was evaporated under
~~ vacuurn, the residue was treated several times with diethyl ether and
concentrated under reduced pressure, to give a colourless oil, which was
without further p~-rifir~tion. The TFA-salt, Boc-Asp(OBn)OH (446 mg, 2
mmol), HOBt (170 mg, 2 mmol) and NMM (202 mg, 2 mmol) in C~I2CI2 (10
ml) was added and the reaction was stirred overnight at room temperature
2~ under argon atmosphere. The precipitate of dicyclohexylurea was removed by
filtration and the filtrate was collc~llLldled under reduced pressure. The
residue was diluted with ~tOAc (30 ml), filtrated again and washed with 1 M
NaHCO7, 10% citric acid, and saturated NaCI solution. The organic layer
was dried over MgSO.~, and concentrated in vacuo, to give a white solid in
78% yield (867 mg).
38
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~H-NMR (200 MHz, CDC13): ~ 1.41 (s, 9H, CH3); 1.85 - 3.05 (m,
9H, 3CH2, NCH3); 4.40 (m, 2H, CH); 5.01 (m, 4H, OCH2); 5.~3 (d, lH,
NH); 6.48 (bs, lH, NH); 7.15 - 7.38 (m, 10H, Ph-H).
Synthesis Of N-Carboxymethyl-D~peptide fl~): To a solution of Boc-
Asp(OBn~-Glu(OBn)NHMe (14) (~55 mg, 1 mmol) in 10 ml CH2Cl2 was
added 1 ml TFA and the reaction mixture was stirred for 1 h at room
L~ eldlul~ under argon atmosphere. The excess acid was evaporated under
vacuum, the residue was treated several tirnes with diethyl ether and
conce~ dted under reduced pressure, to give a colourless oil, which was
dissolved in dry THF (20 ml). To this solution was added NMM (101 mg, 1
mmol) and ~enzyl bromo~ret~t~- (230 mg, 1 mmol), and the reaction was
stirred overnight at room temperature under argon atmosphere. The reaction
llPLX.Lul~ was collcelll.dl~d under reduced ~le.,~ule. The residue was diluted
1~; with ~tOAc (20 ml), and washed with 1 M NaHCO3, 10% citric acid, and
saturared NaC1 solution. The organic layer was dried over MgSO2, and then
cullcellLldLt:d to give a colourless oil, which was purified by flash
cl~,olllatography on silica gel (ethyl acetate/MeOH 10:1) to afford (1~) (410
mg) as a white solid in 88% yield.
'H-NMR (200 MHz, CDCI3): ~ 1.83 - 2.95 (m, 9H, 3CH2, NCH3);
3.30 - 3.60 (m, 4H, CH & N CH2); 4.39 (m, 2H, CH); 5.05 (m, 6H, OCH2);
6.64 (bs, lH, N~); 7.29 - (m, 15~I, Ph-H). 8.02 (bs. -lH, NH); l3C-NMR
(60 MHz, CDCl3); ô 26.17, 27.24, 30.~9, 36.~7 (3CH2, NCH3); 4g.59
(NCH2); ~2.54, 59.07 (2CH); 66.46, 66.71, 66.86 (30CH~); 128.17, 128.26,
2~ 128.34, 128.55, 128.80, 135.27, 135.44, 135.79 (Ph-C); 171.2~, 171.34,
172.01, 172.61, 173.0~ (~C=O).
Synthesis Of N-Car~oxymethyl-Asp-GI~l-NMe (16J: To a solution of
benzyl-~lote~;L~d N-carboxymethyl dipeptide (1:~) (68 mg, 0.113 mmol) in
mçth~n~ rnl) was added portionwise Pd/C powder (~0 mg). The mixture
was s~irred under H2 atmosphere (balloon pressure) at room temperature for
39
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20 h. Catalyst was filtered of through a pad of celite and nnsed with
methanol. The filtrate was concentrated and the crude solid was recr
from EtOAc/MeOH at -20~C to afford the product N-Carboxymethyl-Asp-
Glu-NMe (16) (23 mg, 0.06~ mmol) as a white solid in 58 % yield.
mp: 147-149 ~C; NMR (360 M~Iz, DMSO-d6) ~ 8.19 (d, J = 8.5 Hz,
1 H), 7.72 (m, 1 H), 4.20 (m, 1 H), 3.45-3.20 (m, 4 H), 2.63-2.44 (m, 5 H),
2.20 (m, 2 H), 1.93 (m, 1 H), 1.72 (m, 1 H).
6.1.~. Symthesis O f M e.ca~Lo Co m pound
The pl~r~ d method for synth.osi7in~ mercapto
compound (22), also ~~eC;crn~t~ as FG-074 (Example for Inhi~itor C) is as
follows:
01 0 BrCH;~OC~ IOI O
P ~ C~'t N~H ~~kP
17 18 ~ ~
K~CO3 CH2O
~COS~ 1. LIOEI~o~Et
~~ ~ ~ ~ RCOSH
19
1. ~u(Oan).~EMe
2. DKX~HOBt
2~
RCO,S O - COOB~ SH Q -~-~CO,H
~N~e 1 . TF~ ~ ~ NE~e
~~~1~~ 3ti~I O
O O
21 22
(FG~74)
3~
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ALI~ylation of the diethyl phl~sph~te (17) with tert-butyl brom~et~te in
the presence of NaH provided the phosphonate (18) in 90 % yield. Horner-
3~r~nons reaction of (18) with formaldehyde using K.CO3, as a base resulted
in the formation of the unsaturated ester (19). Saponification of (19) with
LIOH and sul~sequent Michael addition of thiolacetic acid provided the acid
(20). Coupling of (20) with Glu(OBn)NHMe using the DCC/HO~t method
afforded the dipeptide (21) as a mixture of diastereomers by flash
chromatography. De~loleL;lion of (21) with TFA in CH2Cl~ provided the
mono acid (22).
Syntkesis l-Ethyl-2-diethylphosphono-srr~cini~ acid 4tert-butyl ester
(18): To a suspension of NaH (0.48 g, 20 rnmol) in dry T H F (80 ml) was
added dropwise a solution of triethyl phosphono~ret~t~ (4.48 g, 20 mrnol) in
THF (20 rnl) and after 3 h at room L~ t;ldl-lre tert.-butyl bromo~rPt~t~o in
1~; TEF (20 ml). The reslllting s ~cpencion was stirred for 3 h at room
temperature, water (~0 rnl) was added and the reaction mixture was acidified
with 1 M HCl to pH 3. After addition of diethyl ether (70 rnl), the organic
was separated, washed with sdluldted NaCI solution, dried over MgSO, and
the solvent was evaporated in vacuo, to give a colourless oil in 90 % yield.
Synthesis Of l-Ethyl-2-methylene-sr~ acid 4-tert.-hutyl ester (19):
A mixture of compound (18) (~.4 g, 16 rnrnol)l K.C03 (6.9 g, ~0 mmol), and
a 30 % aqueous solution of form~ hyde (3.2 g, 100 mmol) was refluxed for
3 h. After cooling, the l~ Ul~ was extracted with hexane, the organic layer
2~ was washed with water and brine and dried over MgSO.~. After filtration and
evaporation in vacuo, an oily residue was o~tained in 80% yield (2.7 g).
'H-NM~ (200 MHz, CDCl3): ~ 1.29 (t, j = 7 Hz, 3H, CH2); 1.43 (s,
9H, CH3); 3.24 (s, 2H, CH2; 4.20 (q, J = Hz, 2H, OCH7); ~.63, 6.28 (s,
2 H, = C H2)
41
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Synthesis Of 2-Acetylsulfanylmet*yl-s1r~inic acid 4tert.-~u~yl ester
(20): The unsaLulcl~d ester (19) (2.3 g, 11 rnmol) was dissolved in THF (50
ml) and a 0.2 M aqueous solution of LIOH (264 mg, 11 mmol) was added at
0~C. The mixture was allowed to come to room temperature and was stirred
additional 3 h. The organic solvent was evaporated and the aqueous layer was
extracted with diethyl ether (30 rnl). The aqueous layer was acidified with 1
M HCI to pH 3 and extracted with EtOAc. The organic layer was washed
with water and brine and dried over MgSO.,. After filtration and evaporation
of the solvent, the acid was obtained as a white solid in 60% yield.
lH-NMR (200 MHz, CDC13); ~ 1.44 (s, 9~, CH3); 3.26 (s, 2H, CH~);
5.78, 6.42 (s, 2H, =CH~); 10.01 (~s. lH, COOH).
The acid (1.0 g, ~.4 mmol) was dissolved in C~Cl3 and thioacetic acid
(1.4~ g, 19 rnmol) was added. The ~ was stirred at 60~C for49 h.
The solvent was eva~dted in vacuo, and the compound (20) was obtained
l~i with a y~ t~ti~e yield as a colourless oil.
~ H-NMR (200 MHz, CDC13); ~ 1.44 (s, 9~, CH3); 7.34 (s, 3H, CH3);
2.45 - 2.75 (m, 2H, CH2; 2.96 - 3.33 (m, 3H, CH" CH); 9.74 (bs, lH,
COOH).
Synthesis Of 3-acetylthiomethyl-4axo-5-~-6-(R)-methylcar~amoyl-8-
benzyloxycarbonyl-octanoic Acid, tert-butyl Ester t21): To a solution of tert.-
butoxycarbonyl amino ester (13) (700 mg, 2 rmnol) in 10 ml CH~Cl2 was
added 1 ml TFA and the reaction ~ LuL~ was stirred for 1 h at room
temperature under argon ~trnosph~re. The excess acid was evaporated under
2~ vacuum, the residue was treated several times with diethyl ether (1~ ml) and
concentrated under reduced pressure, to give a colourless oil, which was used
without further purification. The TFA-salt, acid (20) (~2~ mg, 2 mmol)
HOBt (170 mg, 2 mmol) and NMM (202 mg, 2 mmol) were dissolved in
CH2CI2 (40 ml). A solution of DCC (412 mg, 2 rnmol) in CH2CH, (10 ml)
30 was added and the reaction was stirred overnight at room temperature under
argon atmosphere. The precipitate of dicyclohexylurea was removed by
42
3~
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W O 97/05865 PCTAJS96/12876
filtration and tne f1ltrate was concentrated under reduced pressure. The
residue was diluted with EtOAc (30 ml), filtrated again and washed with 1 M
NaHCO3, 10% citric acid, and saturated NaCl solution. The organic layer
was dried over MgSO4 and concentrated in vacuo, to give (21) as a colourless
oil, which was purified ~y silica gel chromatography using FtO.Ac as eluant.
The afforded yield was 72% (710 mg), as a mixture of diastereomers. lH-
NMR (200 MHz, CDCl3); ~ 1 36, 1.39 (2s, 9H, CH3); 1.87 - 3.2 (m, 14H,
4C~I3, CH3, NCH3); 4.40 (m, 2H, CH); 5.08, 5.11 (2s, 2H, OCH2); 6.43,
6.76, 7.07 (bs, -2H, NH); 6.76 (2d, lH, NH); 7.31 (m, 15H, Ph-H).
Synt*esis of 3-Thiomet*yl-40xo-5-aza-6-(R)-met*ylcar~amoyl-8-
carboxy-ocfanoic Acid (22) (FG 074): To a solution of tert-butoxycarbonyl
amino ester 21 (495 mg, 1 mmol) in 15 mL CH,CI2 was added TFA (1.5 mL)
and the reaction mixture was stirred for lh at room temperature under argon
15 ~trnosph~re. The solvent and excess acid were evaporated under vacuum, tne
residue was treated several tirnes with diethyl ether (10 mL) and conc~ dlt:d
under reduced ~r~ .ule, to give a colourless oil, which was purified ~y flash
cluulllatography on silica gel (ethyl acetate/MeOH 10:1, c~ A;..illo 1% acetic
acid) to yield the interme~ te as a white solid 84~ (370mg) yield.
To a solution of the intern~ te (30 mg, 0.07 mmol) in 1 mL of
(1.5/1) l.~rll.A..nl/H2O at room temperature was added LiOH.H O (12 mg,
0.27 mmol). After stirring for 3 hours at room temperature, the reaction
lll~Lul~ was quenched with 0.5mT of 1 N HCI aqueous solution and extracted
with ~tOAc (2 x 10 mL). The combined orgar~ic layers were washed with
2~ brine, dried over MgSO." and conc~ d~d to get the thio compound 22 as a
gummy solid (19 mg, 0.06 mmol) in 86% yield.
MS (~S) (M + H)+: 307
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CO2H
HS~ll~ N J~NHMe
O \,CO2~
22 (FG-074)
6.1.6. Synthesis Of 2-[1N-(4-methoxybenzenesulfonyl3-N-(4-
chlorobenzyl)~amino3acetic Acid
The LJl.,r~.lcd method for syn~hP~i7ing 2-~[N-(4-
methoxyl,e~ esulfonyl~-N-(4-chlolob~l.,yl)3-amino]aceIic acid (23), also
designated as FG046, (Example for Inhibitor A) is as follows:
1~
o ~ LiO~zO o ~
E O ~ ~ \ OM ~ N\ ~ OMe
4 23 ~& OU~
2~ Synthesis Of 2-fi[N-( 1 . et~to~enz,onesl~lfonyl~-N-(4
chloro~enzyl)laminolace~ic Acid (23): To a suspension ~ cLulc of ethyl ester
(4) (300 mg, 0.7~ mmol) in 1.~:1 MeO~ O (4ml) was added LiOH/H,O.
After stirring at room temperature for ~ h, the mixture was quenched with 1
N ~ICl solution (20 ml) and extracted with C~I2Cl, (2x20 ml). The combined
30 organic layer was washed with brine, dried over MgSO,~ and concentrated.
44
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The crude solid was recryst~lli7to~1 from hot ether to afford (23) as a white
solid.
mp: 139.5-140~C; lH NMR (360 MHz, DMSO-d6) ~7.79 - 7.08
(m,4H,Ph), 4.37 (s,2 H,CH2), 3.85 (s, 3H, OCH3), 3.83 (s,2H, CH2).
6.1.7. Sy-nthesis Of N-hy~lrvxy-2[rN'-(~
m~tho~yb...~ r~ fonyl)-
N~(carboxyl~lelllyl)lamino]-~et~mide
The ~ler~lled method for synthesizing N-hydroxy-2~[N'-
O (4-methoxybenzenesulfonyl)-N'(carboxymethyl)]amino~aeet~mitle (30), also
~ecigT-~tt-rl as FG0~, (Example for Inhibitor A) is as follows:
~ NH2 l Cl~O~I NEt3 ~ ~ 5,~
24 2S 02
1~ ~
N~H, ~ Br CO2tBu
O O
ll HONEI2/HCI. ll
~--O~ Kn~ U ~0~
HOHNJ~ 7 ~OM~
T~A,CH2~ ~
JLOH
2~ HOHN~ ~2
29 ~FG O~i5)
Synthesis Of Ethyl 2-~V-(4methoxyhenzen-sulfonyl)laminolacetate
(26): To a llPLxlule of glycine ethyl ester hydrochloride (24) (3.0 g, 21.
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rnmol) and 4-methoxyberLzenesulfonyl chloride (2~) (4.4 g, 21.3 mmol) in
anhydrous CH~Cl2 (60 rnl) was added triethylamine (4.79 g, 47.3 rnmol).
After stirring at room temperature for 15 h, the reaction ~ Lule was
q~ nrh~-l with 1 N HCl (120 ml) and extracted with CH C12 (2x100 ml). The
5 combined organic layer was washed with brine, dried over MgSO4, arld
concentrated. The crude solid was ~ lli7~oA from EtOAc/h~-x~n~os to give
sulfonamide (263 as a white solid.
Synthesis Of Et*yl 2-fi~ (4methoxybenzene-sulfonylJ-N-te~t-
10 bu~yloxycarbonylmethyl)Jamino]~cetnte (27): To a slurry of sodium hydride
(60% dispersed in mineral oil) (162 mg, 4.03 mmol) in anhydrous THF (10
rnl) in an ice bath was added sulfonamide (26) (1.0 g, 3.66 mmol), followed
by bromo tert-butylacetate (785 mg, 4.03 mmol). The mixture was stirred
vigorously at 0~C for 30 min and then at rt for 15 h. After cooling the
1~; reaction mixture in an ice bath, it was quenched with water (25 ml) and
extracted with ether (2x50 ml). The combined organic layer was washed with
brine, dried over MgS04, and co,lcelllldLed. The residue was purified by silica
gel chromatography (2:1 hexanes:EtOAc) to give (273 as a colorless syrup.
Synthesis OfN-hydroxy-2[l2V'4-met)~orybenzenesulfonyl3-N'-(tert-
hutyloxycarbonyl-methyl)lamino~ tc~ e (28): Separate solutions of
hydroxylarnine hydrochloride (377 mg, 5.43 mmol) in MeOH (2.7 ml) and of
KOH (4~6 mg, 8.13 mmol) in MeOH (2.7 ml) were prepared at the boiling
point, cooled to 40~C, and then the latter solution was added to the former.
2~ After cooling in an ice bath for 30 min, potassium chloride solid was filtered
off. To the filtrate was added ethyl ester (27) (1.05 g, 2.71 mmol). After
stirring at room temperature for 6 h, the reaction mixture was neutralized with
1 N HCl solution to pH 4, and partitioned between C~I,CI.(60 ml) and water
(20 ml). The r~s--lting two phases were separated and the aqueous layer was
30 extracted with CH,Cl, (60 ml). The combined organic layer was washed with
brine, dried over MgS0." and concentrated. The residue was puri~led by silica
46
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gel chromatography (11:1 CH2Cl7:MeOH) to give ester hydroxamate (28) also
~lesif~n~t~ as FG-058, as a white solid.
Synthesis Of N-hydroxy-2[~ (4metho~ybenzenesulfonyl)-
N'fcar~o~ymethyl3~-aminol~etn~ le (29): A solution of tert-butyl ester
hydroxamate (28) (520 mg, mmol) in 35% CF3COOH/CH7Cl2 (9 ml) was
stirred at 0~C for 10 min and then at room ~ ,Ld~llre for 1.5 h. The
mixture was concell~ldted and dried in vacuo. The residue was ~ uldLed in
LtOAc and the solid was collected which was recryst~lli7~1 from
10 EtOAc/MeOHthexanes to give acid hydroxamate (29) as a white solid.
mp: 160-161 ~C; MS (ES) (M + H)+: 319; 'H NM~ (360 MHz,
DMSO-d6) ~ 12.08 (brs, 1 H, CO2H), 10.69 (s, 1 H, OH), 8.96 (s, 1 H,
NH), 7.76 (d, J = 8.7 Hz, 2 H, Ph), 7.09 (d, J = 8.7 Hz, 2 H, Ph), 4.01 (s,
2 H, CH2), 3.84 (s, 3 H, OMe), 3.83 (s, 2 H, CH2).
1~
6.1.8. Synthesis Of N-(~Methoxybenzene-sulfonyl3-~proline
Hy~ e
The preferred method for synthesizing N-(4-
Methoxybenzenesulfonyl)-L-proline hydroxamate, also designated as ~G0~4,
zo (Example for Inhibitor A) is as follows:
2:~
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OIH ~CI02S~\ OM~ c~CI2 ~ - N
MoO~O ~O~O
3 1 32
~{ONH2~C5 ~N ~/
~ s
HOHN ~O
33 (~Y; 054)
Synthesis Of N-~4-Met*o~b~,~ z~nesr~fonyl3-L-Proline Met*yl Ester
1~ To a solutio~:L-proline methyl ester hydrochloride (30) (1.00 g, 6.03
mrnol) and 4-methoxyb~n7~n~sl-1fonyl chloride (31) (1.19 g, 5.75 mrnol) in
anhydrous CH~Cl, (17 ml) was added triethylamine (1.22 g, 12.06 mmol).
After stirring at room tL~ eldLIlre for 15 h, the reaction mixture was
quenched with 1 N HCI (30 rnl) in aqueous solution and extracted with EtOAc
(2 x 100 ml). The combined ~tOAc organic layers was washed with brine,
dried over Mg,SO" and conce~ Led to afford (32). This product was
directly used for the next reaction without further purification.
Synt*esis Of N-(4Met*oxyhenz~nes~lfonyl)-L-Proline Hydro~n~nnte
2~ (33): Separate solutions of hydroxylamine hydrochloride (465 mg, 6.68
mmol) in MeOH (3.4 ml) and of KOH (~61 mg, 10.0 rnmol) in MeO~I (3.4
ml) were prepared at the hoiling point, cooled to 40~C, and then tne latter
solution was added to the former. After cooling in an ice ~ath for 30 min,
potassium chloride solid was filtered off. To the filtrate was added ethyl ester
(32) (1.0 g, 3.34 mmol). After stirring at room temperature for 15 h, the
reaction mixture was treated with 1 N ~ICI (40 ml) solution, and extracted
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with CH2Clz/MeO~I (10:1) The organic layer was washed with brine, dried
over MgSO4, and concentrated to get the crude product. The crude solid was
recrystalized from hot MeOH/EtOAc to afford the hydroxamate (33) as a
solid.
6.1.9. Synthesis Of N-llyllr~y-2-[~N'-(4-
m~th~xyl~ esulfonyl)-N'-(4-
trifluo~ ylbenzyl)3aIl~ino3-~et~
The preferred method for sy~th~osi7ing N-hydroxy-2-
10 t[N'-(4-methoxybenzenesulfonyl~-N'-(4-trifluoromethylbenzyl)]amino]-
acet~mi~le (34), also ~lesign~ted as FG-066 (~xample for Inhibitor A) is
~sse~ti~lly as described for the synthesis of 5ynth~5i7ing N-hydroxy-2-r[N'-(4-
methoxybenzenesulfonyl)-N'-(4-chlorobenzyl)3amino3-~e~mirl~, using as
starting material 4-trifluoromethylben~lrlehyde instead of 4-
15 chlorobenzaldehyde. See, Section 6.1.1., supra.
lH NMR (360 MHz, DMSO-d6) ~ 10.50 (s, 1 H, OH), 8.83 (s, 1 H,
NH). 7.81 - 7.08 (m, 8 H, Ph), 4.45 (s, 2 H, C~2CO), 3.86 (s, 3 H, OMe),
3.67 (s, 2 H, CH2Ph).
2~ HOHN J~ S ~OM~3
34(~G~66)
-
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6.1.10. Synthesis Of N-hydroxy-2-[[N'-(4-
m~th~ryben~n~cl-lfonyl3-N'-(4-
mf~th- ~rybenzyl)3amino] ~~et~mi-le
The preferred method for synthesizing N-
hydroxy-2-[[N'-(4-methoxyben_enesulfonyl)-N'-(4-methoxybenzyl)~arnino]-
aeet~mi-le (3:~), also ~lesi~n~tecl as 3~G-067 (Example for Inhibitor A) is
essentially as descrihed for the synthesis of synth~ci7in~ N-hydroxy-2-rrN'-(4-
methoxybenzenesulfonyl3-N-(4-chlorobenzyl)]amino]-~cet~mi~le, using as
starting material 4-methoxybenzaldehyde instead of 4-chloro~enzaldehyde.
O See, Section 6.1.1., sllpra.
lH NMR (360 MHz, DMSO-d6) ~ 10.42 (s, 1 II, OH), 8.79 (s, 1 H,
NH). 7.81 - 6.86 (m, 8 H, Ph), 4.29 (s, 2 H, CH2CO), 3.85 (s, 3 H, OMe),
3. 73 (s, 3 H, OMe), 3.28 (st 2 H, C~7Ph).
~ '
tlOHNJ~ o~OM~
35(FG~67)
6.1.11. ~,ynthesis Of N-hydro~y-2-[rN'-(4-
2~ b~n7~n~c--1fonyl3-N'-(4-chlorobenzyl)3arnino3-
~cet~mide
The pl~re~ d method for synrhPci7ing N-
hydroxy-2-[[N '-(4-~enzenesulfonyl)-N'-(4-chlorobenzyl)~amino]-~ret~mide
(36), also designated as ~G-080 (~xample for Inhibitor A) is essentially as
30 described for the synthesis of synthPsi7in~ N-hydroxy-2-[[N'-(4-
methoxy~enzenesulfonyl)-N'-(4-chlorobenzyl)]amino]-~ret~mi~le, using as
~0
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starting material benzenesulfonyl chloride instead 4-methoxybenzenesulfonyl
chloride. See, Section 6.1.1., supra.
MS (ES) (M + ~I)+: 3~.
HOHN /~/ ~2 --6
36(FG~80)
1~
6.1.12. Sy~ ~is Of N-hy~ xy-2-~ (4-
methoxybe..,...~..lfonyl)-N'-(benzyl)]all~ino3-
Z~''etZ~m ~P
The ~,er~lled method for syntht-si7ing N-
20 hydroxy-2-[[N~-(4-methoxyhel~e,le~ulfonyl)-N~-(benzyl)]amino3-~ret~m~
(37), also ~leci~n~te~ as FG-061 (Example for Inhibitor A) is esse~ti~lly as
described for t_e synthesis of synthr~i7in~ N-hydroxy-2-~[N'-(4-
methoxyben_enesulfonyl)-N'-(carboxymethyl)]arnino3-~ret~mi-lf, using as
starting benzyl bromide instead of bromo tert-butylacetate. See, section
2~ 6.1.7., supra.
MS (ES) (M - H)-:349
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HOHN J~, 02 ~OMe
37 (FG-061)
6.1.13. Synthesis OI N-(4-Methoxybenzenesulfonyl3-
,B-benzyl-(L3-Aspartic Acid
The ~lcf~ d method for synth~ci7ing
N-(4-Methoxybenzenesulfonyl3-b-benzyl-(L3-Aspartic Acid (42), also
1:~ designated as FG084, (Exarnple for Inhibitor A) is as follows:
Synthesis of F~; 084 (Inhibitor A)
HOJ~ NH2 + O CIS 3 OMe ~I t~ol~ ~2~--OMe
CO2E3n CO2Bn
~8 39 40
DIC,~OBT ~ _~ ~ OMe ~ ,H, HOHN ~ N-~ ~ Me
NEM CO2Bn CO2H
2~ 41 42 Inhibi orA
N-(4MefhoxybenzenesulfonyZ)-,8-benzyl- fl,~-aspartic Acid (40). To a
suspension l~ lul~ of b-benzyl-(L~-aspartic acid HCl salt (38) (2.00 g, 8.96
mmol) and p-methoxybenzenesulfonyl chloride (39) (1.76 g, 8.~3 mmol) in
anhydrous CH;~Cl;! was added triethylamine (1.81 g, 17.91 rnmol) at room
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temperature. After stirring for 1~ hrs the reaction lni~lul~ was quenched with
1 N HCI (60 rnl), and extracted with C~I~CI2 (3 x ~0 rnl). The combined
organic layers was washed with brine, dried over MgSO4, and concentrated to
give N-(4- methoxybenzenesulfonyl)-b-benzyl-(L)-aspartic acid (3.14 g, 7.99
mmol, 94% yield) as a gurnmy product.
N-Benz;yloxy-N'-~4Methoxy~en~nes~lfonyl)-,~-ben~yl-(L)-aspar~ic
amide (41). To a mixture of N-(4-methoxyben7en~sl-lfonyl)-~-benzyl-
(L)-aspartic acid (300 mg, 0.76 mmol) and O- benzylhydroxylamine/~ICl in an
anhydride solution of (7/3) T~IF/DM~ (10 ml) was added
N-hydroxybenzotriazole (HOBT) (103 mg, 0.76 rnmol), N-ethylmorpholine
(204 mg, 1.68 mmol) and then diisopropylcarbotliimide (106 mg, 0.84 mmol)
at room te nperature. After stirring over the weekend (2.5 days), the reaction
mixture was diluted with (1/1) hexanes/EtOAc (40 rnl), washed successively
1~ with 1 N ~ICl (2 x ~0 ml), sdLuldl~d Na~ICO3 aqueous solution (2 x 20 mT.)
and brine. The organic layer was dried over MgSO.,, and concentrated. The
residue was purified by silica gel flash chromatography ((1/1) EtOAc/h~ nt?s)
to get N-~enzyloxy-NO-(4-methoxyb~..,elle~ulfonyl3~ enzyl-(L)-aspartic
amide (114 m~, 0.22 mmol, 30% yield) as a white solid.
mp: 128 -129 .C; MS (~S) (M + ~I)+: 499
N-~lydroxy-N'-(4Met*oxybenz~nes~lfonyl)-(L)-aspartic amide (42).
A mixture of N-benzyloxy-NO-(4-methoxyben7~n~sl-lfonyl)-
b-benzyl-(L~-aspartic arnide (102 mg, 0.20 rnmol) and 10% Pd/C (43 mg) in
25 m~th~nol (7 ml) was stirred vigorously in a hydrogen atmosphere ~balloon
pressure) for 20 hrs. The catalyst was filtered off through a pad of celite and
the filtrate was ~;ollcelll, dted. The residue was Iyophilized from water to getN-hydroxy-NO-(4-methoxybenzenesulfonyl)-(L)-aspartic amide (50 mg, 0.16
rnmol, 77% yield) as a hygroscopic fluffy powder.
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lH NM~ (360 MHz, DMSO-d6) ô 10.60 (s, 1 ~I), 8.78 (s, 1 H), 7.87
(d, J = 8.2 Hz, 1 H), 3.93 (m, 1 H), 3.82 (s, 3 H), 2.50 (m, 1 ~I), 2.21 (dd,
J = 16.0, 6.~ IIz, 1 H).
6.2. Example 2: C~ tein~ce Assays
6.2.1. In Vitro ~ssay For The Det~ Of C-
Proteinase Activity And The ICso Of Inhibitors
The following assay may be used to determine the level
of activity and effect of the dirrelcn~ compounds of the present invention on
10 C-~ leillase activity.
About 125 ~Lg radiolabeled ('~C) procollagen is added to 10 units/ml of
chicken C-L)loteillase in a solution of 0.1 M Tris-HCl, 0.1 M NaCl, 0.02%
Brij-3~, and ~ rnM CaCl. in a total volume of 10 ~Ll. The reaction is allowed
to proceed for 1~ mimlt~ at 35~ C and is stopped with one-half volume of 3x
1~; stop/loading buffer (30 mM EDTA, 30% glycerol, 6% SDS, 0.006%
Bromophenol-blue). Subsequently, the samples are heated to 100~C for 4
s, and resolved by SDS-PAGE (Novex) using 6% polyacry1e~mirlt- gels.
The protein bands are detect~d by autoradiography. The amount of enzyme
activity is based on the disappearance of the band corresponding to uncleaved
20 procollagen.
The IC50 of inhibitors can be deterrnined by plotting the % activity
versus inhibitor concentration and estim~ting the inhibitor concentration which
results in 50% activity.
The IC50 value of the h~ ols which have been tested is shown in
2:~ TABLE I.
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TABLE I:
IC~jo Of Various Trl~ntified C-Pr~t~in~ce Inhibitors.
Inhil3itor Generic Group ICso
FG-047 A 50~M
FG-061 A 100,uM
FG-053 A 100,~M
FG-052l B 125~M
FG-066 A 150~LM
FG-067 A 150~M
~G-086 (cbz-Pro-Leu-Gly-hydroxamate') A 200~M
FG-087 (Ac-PYYG-hydroxamate) A 335~M
3~G-088 (A~ u~ 350,uM
~G-054 A 400~M
FG-057 D 1500~M
FG-058' A 2100~LM
FG-055 A 2600~M
FG-0515 C > > 100~LM
FG-046 A > > 1000~M
Cu~ ILially available from Peptides International (I~IN-3850-PI).
' collull~l~ially availa~le from Sigma (C-8~37).
3 ccll.lllL.~.ially available from Sigma (A-6671).
4 intermediate compound (28), see, section 6.1.7
5 coll~ L~ially available from Peptides Tnt~ tional (ISN-3835-PI)
6.2.2. In Vi~ro ELISA Assay For The Determin~ion Of C-
Pr~in~ce Actiqity And The ICso Of Inhibitors
The IC50 value of the inhibitors can also be determined
by a filtration ~LISA assay. In this assay about 25 ng of unlabeled human
procollagen I were ;~ with the C-Proteinase as, see, section 6.2.1 but
for one hour. The reaction was stopped with the addition of 40 ~l
~l~cil,iLaLion buffer (0.5 X ~eaction buffer, 0.1 mg/ml chicken collagen II, 10
,ug/ml BSA, 7.5 rnM ~DTA). 25 ,u1 of 75% ethanol was added and the
reactions were mixed and inc~lb~te~l on ice for one hour to precipitate the
procollagen. The soluble c-propeptide was separated from the precipitated
O collagen by filtering through a Millipore multiscreen-HV 0.45 ~Lm hydrophilic
plate using a Millipore multiscreen vacuum manifold. ~0 ~Ll of the filtrate was
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removed and the amount of cleaved c~ e~lide was determined by using the
procollagen type I C-peptide (PIP) EIA kit from Talcara ~3iomedicals.
For inhibition of hBMP-1 about '70 n(7 radiolabeled (l~5I) human
procollagen I was added to 1 to 2 ~Ll of 5 times concentrated recombinant
hBMP-l cell media (Kessler et al. (1996) Science 271:360) in reaction buffer
in a total volume of 10 Ill. The reaction was let to proceed for one hour at 3
~C and then stopped with one-half volume of 3x stop/loading buffer and
analyzed on SDS-PAGE as above.
The IC50 of the inhibitors was determined by plotting the % activity
versus inhibitor concentration and estim~ting the inhibitor concentration which
gives ~0 % activity. IC50 values are shown in TABLE II.
TABLE II:
IC50 Of Various Identified C-Proteinase Inhibitors
As Determined by ELISA
lS
Inhibitor Generic Group IC5~ 3~LISA
FG-061 A 12~LM
FG-047 A 13 ~lM
FG-053 A 22~LM
FG-067 A 37!1M
FG-066 A 48~LM
6.2.3. Tissue Culture Assay For The Determination Of C-
Protein~e Activity And The IC50 Of Inhibitors
C-proteinase activity and the IC50 of inhibitors in vivo
2~ may be ~lete~rrnin.-A in tissue culture assays by measurement of the production
of procollagen and mature collagen in conditioned medium before and after
treatment with a compound. The ratio of collagen and procollagen ~ill
directly correlate to the cellular con~ersion of the precursor to the mature
collagen product, and as such indicate the C-proteinase activity.
Alternatively, the media content of C-propeptide/cell may be
det~mine-l, and compared for untreated cells and inhibitor-treated cells.
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6.3. Animal Models ~or The Determination Of C-Prol~i~ase
Activity And The Efficacy Of Inhibitors
Several animal models which mimic clinical disorders related to
unregulated or ina~p..,~liate collagen production are known in the art and may
, be employed to deterrnine the in vivo efficacy of the compounds of the
invention. These animal models include a wound cham'oer model in rats
(Schilling et al., 1959, Surgery 46:70~-710), an estradiol 5tim~ te~1 uterus
expansion model (Mandell et al., 1982, The Journal of Biological Chemistry
257:5~68-5273), and an in~ ced angiogenesis model (Matrigel) (Pas~ i et
al., 1992, Laboratory Investigation 67:519-528). Further animal models
include clinical disorder models lil~e liver fibrosis models (Tsukamoto et al.,
1990, SeminarinLiverDisease 10:56-65; Kock-Weser, l957, Laboratory
Investigation 1:324-33l; Marrione, l949, American Journal of Pathology
25:273-285; Tams, 1957, American Journal of Pathology 33:13-27; Wahl et
1~ al., 1986, Journal of ~xperimental Medicine 163:884-902), a pulmonary
fibrosis model (Kelly et al., 1980, Journal of Laboratory Clinical A~Iedicine
96-954-964), arterial restenosis models (Jackson, 1994, Trends of
Cardiovascular Medicine 4:122-130; Clowes et al., 1983, Laboratory
Investigation 49:327-333), a ~idney fibrosis model (Yarnamoto et al., 1987,
~idney International 3~:514-525), a tendon reyai~ g model (~ranklin et al.,
1986, The Journal of Laboratory and Clinical Medicine 108:103-108), a turnor
growth model (Kiohs, et al., 1985, JNCL 75:353-359), a trabeculectomy model
(Lahery et al., 1989, Journal of Ocular Pharmacology 5:1~5-179), and an
abdominal adhesions model (Williams et al., 1992, Journal of Surgical
2~ Research 52:65-70)
6.4. Example 4: Measurement Of Cytotoxicity
Potential inhibitors are studied in cytotoxicity assays in order to
deterrnine whether there is an effect on cell survi~-al or proliferation. These
30 assays may involve the use of rapidly proliferating or quiescent cells. A
~nown nurnber of cells is seeded and exposed for increasing periods of time to
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a concenkation range of potential inhibitors. Cell numbers are determined by
cell counting or staining (e.g Crystal Violet).
Cytotoxicit,v is evaluated as a function of cellular survival and cell
proliferation. Cellular survival involves the use of quiescent cells and is
5 determined by cell number (counting or st~ining). A decrease in cell number
indicates cell loss, and thus an effect on cell survival. Cell proliferation
involves the use of rapidly proliferating cells and is, as well, cletPrmin~ by
cell number. ~ere a decrease in cell number relative to the untreated controls
indicates an effect on cell proliferation.
The present invention is not to be limited in scope by the exemplified
emborlim~nts which are intPn-lP~ as illustrations of single aspects of the
invention, and any compounds and methods for the use thereof which are
functionally equivalent are within the scope of the invention. Indeed, various
1~; modifications of the invention in addition to those described herein will
become a~a.ellL to those skilled in the art from the foregoing description and
accompanying drawings. Such modifications are int.onrl~d to fall within the
scope of the appended claims.
All references cited herein are hereby incorporated by reference in their
entirety.
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