CA2555850A1 - Methods of treating cancer and the pain associated therewith using endothelin antagonists - Google Patents

Abstract

The instant invention is directed to methods for the inhibition of bone metastases, methods for the prevention of growth of new metastases, methods for the inhibition of bone turnover, and methods for the prevention of bone loss in patients, including cancer patients, using an endothelin ET-A receptor antagonist.

Description

W/O 02/11713 PCT/UHOl/2471~
Methods of Treating Cancer And The Pain Associated Therewith Using Endothelin Antagonists Field of the Invention The instant invention is directed to the inhibition of bone metastases, the prevention of growth of new metastases, the inhibition of bone turnover, and the inhibition of net to bone loss in humans with cancer, using an endothelin ET-A
receptor antagonist.
Background of the Invention is Endothelin (ET), a 21 amino acid peptide, is produced by enzymatic cleavage of a precursor peptide by an endothelin converting enzyme. First discovered in vascular endothelial cells, ET and ET/ET receptor binding are now known to modulate smooth muscle tone, blood flow, cell proliferation and 2o differentation, protein synthesis, and metabolic function in a variety of tissues and cell types such as ovary, prostate, skin, and brain.

VVO 02/11713 ' PCT/US01/2~716 ET/ET receptor binding has been shown to constrict arteries and veins; increase mean arterial blood pressure;
decrease incardiac output; increase cardiac contractility in vitro; stimulate mitogenesis in vascular smooth muscle cells s in vitro; contract non-vascular smooth muscle such as guinea pig trachea, human urinary bladder strips and rat uterus in vitro; increase airway resistance in vivo; induce formation of gastric ulcers; stimulate release of atrial natriuretic factor in vitro and in vivo; increase plasma levels of vasopressin, to aldosterone, and catecholamines; inhibit release of renin in vitro; and stimulate release of gonadotropins in vitro.
ET/ET receptor binding also causes vasoconstriction on vascular smooth muscle (Nature 332 411 (1988), FEB~ Letters 231 440 (1988) and Biochem. Biophys. Res. Commun. 154 8~8 i5 (1988)). In fact, an anti-ET antibody has been shown to ameliorate adverse effects of renal ischemia on renal vascular resistance and glomerular filtration rate (J. Clin. Invest. 83 17b2 (1989)). In addition, an anti-ET antibody attenuat-ed both the nephrotoxic effects of intravenously administered 2o cyclosporin (Kidney Int. 37 1487 (1990)) and the infarct size in a coronary artery ligation-induced myocardial infarction model (Nature 344 114 (1990) ) .

3 PCTlUS01/~.1716 A nonpeptide ET antagonist prevents post-ischaemic renal vasoconstriction in rats, prevents the decrease in cerebral blood flow due to subarachnoid hemorrhage i.n rats; and decreases MAP in sodium-depleted squirrel monkeys. when dosed orally (Nature 365: 759-761 (1993)). A similar effect of an ET antagonist on arterial calibera has also been recently reported (Biechem. Biophys. Res. Comm., 195: 969-75 (1993).
An ET receptor antagonist reduced injury in a rat model of colitis (EUR. J. Pharmacol. 1996, 309, 261-269) and io prevented ischemia-reperfusion injury in kidney transplantation (Transplant Int 1996, 9, 201-207). The use of ET antagonists in the treatment of angina, pulmonary hypertension, Raynaud's disease, and migraine has also been suggested (Drugs 1996, 51,12-27). In malignant growth i5 disorders, ET and its growth-promoting effects have been best characterized,in prostate cancer, (Nature N~edicine 1995, 1, 944-949) wherein ET acts as a modulator in osteoblastic bone lesion (UROLOGY 53:1063-1069, 1999).
Given the results from these and other reports which 2o illuminate the role of ET/ET receptor binding in disease states, and the knowledge that blocking ET/ET receptor binding results in improvement or reversal of endothelin-induced WO 02/11713 . PCT/USO1/2~716 disease states, agents which antagonize ET/ET receptor binding activity, designated as ET receptor antagonists, can provide substantial benefit in many therapeutic area's.
Summary of the Invention ~ , In one embodiment of the instant invention, therefore, is disclosed a method for inhibiting bone metastases in a patient which comprises administering to the patient in need thereof a therapeutically effective amount~of an endothelin ET-A
to receptor antagonist.
In another embodiment of the invention is disclosed a method for preventing new bone metastases in a~patient which comprises administering to the patient in need thereof a therapeutically effective amount of an endothelin ET-A
receptor antagonist.
In another embodiment of the instant invention, therefore, is disclosed a method for inhibiting metastatic growth in a patient which comprises administering to the patient in need thereof a therapeutically effective amount of 2o an endothelin ET-A receptor antagonist.
In another embodiment of the invention is disclosed a method for inhibiting bone loss in a patient which comprises WO 0?/11713 PCT/USOI/~,.1716 administering to the patient in need thereof a therapeutically effective amount of an endothelia ET-A receptor antagonist.
In another embodiment of the instant invention, is disclosed a method for inhibiting bone turnover in a patient s which comprises administering to the patient in need thereof a therapeutically effective amount of an endothelia ET-A
receptor antagonist.
In another embodiment of the invention is disclosed a method for the reduction of cancer 'related pain in a patient io in need thereof which comprises administering to the patient a therapeutically effective amount of an endothelia ET-A
receptor antagonist.
In another embodiment of the instant invention is disclosed therapeutically acceptable formulations of an endothelia ET-A receptor antagonist, optionally in the presence of a co-therapeutic agent, for use in these methods.
Brief Description of the Drawings Figure 1 illustrates levels of interleul~in-6 (IL-6) in a zo subject population treated with a placebo or 2.5 mg or la mg ABT-627.

V1'O 02/11713 PCT/USO1/2~71G

Figure 2 illustrates levels of prostate specific antigen (PSA) in a subject population treated with a placebo or 2.5 mg or 10 mg of ABT-627.
s Figure 3 illustrates VAS score levels relating to pain assessment in a subject population treated with a placebo or 2.5 mg or 1~0 mg of ABT-627.
Figure 4 illustrates crosslinked N-telopeptides lo (degradation) in a subject population treated with a placebo or 10 mg ABT-627.
Figure 5 illustrates bone alkaline phosphatase (BAP)(formation) in a subject population treated with a is placebo or 10 mg ABT-627.
Figure 6 illustrates skeletal involvement in a subject population treated with.a placebo or 10 mg ABT-627.
2o Figure 7 illustrates acid phosphatase levels in a subject population treated with a placebo or 1D mg ABT-627.

- 1~V0 02/11713 PCT/USOI/~~71G

Detailed Description of the Invention Endothelin receptor antagonists are employed in the practice of the instant invention. Endothelins are a family of peptides mainly synthesized and released by endothelial s cells. The term "endothelin" refers to a family of homologous 21-amino acid peptides found in 3 distinct isoforms: ET-1, ET-2, and ET-3.
The term "endothelin ET-A receptor antagonist" includes both compounds which antagonize the ET-A receptor in a io selective manner, as well as compounds which antagonize the ET-A receptor in a non-selective manner. An example of the latter type of compound would be a compound that antagonizes the ET-A receptor and also antagonizes the ET-B receptor.
The term "primary cancer" means cancer in a specific is tissue, which is first in time or in order of development.
Primary cancers include, but are not limited to, breast, prostate, lung, kidney, thyroid, brain, heart, intestine, ovary, myeloma, lymphoma, sarcoma, and osteosarcoma.
The term "cancer-related pain" includes pain which arises 2o from direct invasion or expansion of a tumor into tissue, such as bone or nerve; pain which arises from the consequences of tumor invasion or expansion, such as bone collapse due to CA 02555850 2001-08-06 _ V1'O 02/11713 . PCT/USO1/2~716 _ . -' cancer erosion or secretion of noxious agents which modulate or produce pain; and pain mediated by ischemia, i.e. reduced blood flow.
Specifically, a compound of formula I may be employed, in s the practice of the instant invention R2 Z ~ ~ Ra N
(CH~)n R
R~
' I
wherein Zo R is -(CH2)m-W;
Z is selected from -C(RIg)(R19)- and -C(O)-;
R1 and R2 are independently selected from hydrogen, loweralkyl,, alkenyl, alkynyl, alkoxyalkyl, alkoxycarbonylalkyl, hydroxyalkyl, haloalkyl, haloalkoxyalkyl, Zs alkoxyalkoxyalkyl, thioalkoxyalkoxyalkyl, cycloalkyl, cycloalkylal.kyl, aminocarbonylalkyl, alkylaminocarbonylalkyl, dialkylaminocarbonylalkyl, aminocarbonylalkenyl, alkylaminocarbonylalkenyl, dialkylaminocarbonylalkenyl, hydroxyalkenyl, aryl, arylalkyl, aryloxyalkyl, - WO 02/11713 PCT/USO1/2.1716 arylalkoxyalkyl, (N-alkanoyl-N-alkyl)aminoalkyl;
alkylsulfonylamidoalkyl, heterocyclic, (heterocyclic)alkyl, and (Raa)(Rbb)N-Rcc-, with the proviso that one or both of R1 and R2 is other than hydrogen;
R3 is selected from R4-C(O)-R5-, R~-R5a-, R4-C(O)-R5-N(R6) -, R6-S (O) 2-R~- R26-S (O) -R27-. R22-O-C (O) -R23-.
loweralkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, aryloxyalkyl, heterocyclic, to (heterocyclic)alkyl, alkoxyalkyl, alkoxyalkoxyalkyl, and R13-C(O) -CH(R~4) -:
R4 and R6 are independently selected from (Rll)(Rl2)N-.
loweralkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclic, (heterocyclic)alkyl, i~ alkoxyalkyl, hydroxyalkyl, haloalkyl, haloalkenyl, haloalkoxyalkyl, haloalkoxy, alkoxyhaloalkyl, alkylaminoalkyl, dialkylaminoa13~y1, aikoxy, and ~ H
' (CHy)'Z \N N~R7a' O
R5 is selected from a covalent bond, alkylene, V1'O 02/11713 PCT/USO1/2~716 alkenylene, -N(R20)-Rg-, -R8a-N(R2~)-R8-, -O-Rg-, and -Rga-O-R9-R6 is selected from loweralkyl, haloalkyl, alkoxyalkyl, haloalkoxyalkyl, aryl or arylalkyl;
s R~ is a covalent bond, alkylene, alkenylene -N(R~1)-Rl.p-, and -RlOa-N(R21)-R10-~
Rg is selected from alkylene and alkenylene;
R9 is alkylene;
Rlp is selected from alkylene and alkenylene;
is R11 and Rl2,are independently selected from hydrogen, loweralkyl, haloalkyl, alkoxyalkyl, haloalkoxy'alkylalkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, heterocyclic, arylalkyl, (heterocyclic)alkyl, hydroxyalkyl, alkoxy, aminoalkyl,trialkylaminoalkyl, alkylarninoalkyl, z5 dialkylaminoalkyl, and carboxyalkyl;
R13 is selected from amino, alkylamino and dialkylamino;
R14 is selected from aryl and R15-C(O)-;
R15 is selected from amino, alkylamino and dialkylamino;
R16 is selected from loweralkyl, haloalkyl, aryl and 2o dialkylamino;
R1~ is loweralkyl;

V1'O 02111713 PCT/US01/2.1716 Rlg and Rlg are independently selected from hydrogen and loweralkyl;
R2p is selected from hydrogen, loweralkyl, alkenyl, haloalkyl, alkoxyalkyl, haloalkoxyalkyl, cylcoalkyl and s cycloalkylalkyl;
R21 is selected from hydrogen, loweralkyl, alkenyl, haloalkyl, alkoxyalkyl, haloalkoxyalkyl, aryl and arylalkyl;
R22 is selected from a carboxy protecting group and heterocyclic;
to R23 is selected from covalent bond, alkylene, alkenylene.
and -N(R24)-R25-%
R2g is selected from hydrogen anal loweralkyl;
R~5 is alkylene;
R26 is selected from loweralkyl, haloalkyl, alkenyl, is alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclic, (heterocyclic)alkyl, alkoxyalkyl and alkoxy-substituted haloalkyl;
R2~ is selected from alkylene and alkenylene;
R5a is selected from alkylerre and alkenylene;
2o Rya is alkylene;
R8a is selected from alkylene and alkenylene;

WO 02/11713 ~ PCT/US01/2.t71G , R9a is alkylene;
Rl~a is selected from alkylene and alkenylene;
Raa is selected from'aryl and arylalkyl;
Rbb is selected from hydrogen and .aTkanoyl;
s Rcc is alkylene; ~ ' m is 0-6;
n is ~ or 7.;
z is 0-5;
E is selected from hydrogen, loweralkyl and arylalkyl;
to G is selected from hydrogen and a carboxy protecting group; and W is selected from -C (O) 2-G; -P03H2, -P (O) {OH) (E) , -CN, -C(O)NHR1~, alkylaminocarbonyl, dialkylaminocarbonyl, tetrazolyl, hydroxy, alkoxy, sulfonamido, -C(O)NHS(O)2R16, -15 S (O) 2NHC (O) R16, HO O O
O OH _~~ Nw - ~ ,~ \ N _~ NH O
NH N
--~~~w H
O HO O ~ O O~ fl O
, , . , NCO ~IJ
DSO~ ~ >--Cf3 NHS02CF3 H ~~ H
, , and , WO 02/11713 PCT/US01/?716 or a pharmaceutically acceptable salt thereof.
A preferred embodiment of the a compound of formula I is a compound of formula II
R2 Z ~N i R3 R\'~,~- (CHZ)n II
wherein the substituents -R2, -R and -R1 exist in a trans,trans relationship and Z, n, R, R1, R2, and R3 are as deffined above.
to Compounds of formulas I and II are endothelia antagonists, specifically ETA-selective endothelia antagonists.
Another preferred~embodiment of the invention is a compound of formula I or II wherein n is 0 and Z is _CH2_.
is Another preferred embodiment of the invention is a compound of formula I or II wherein n is 1 and Z is _CH2_.
Another preferred embodiment of the invention is a compound of formula I or II wherein n is 0, Z is -CH2-, and R3 WO 02/11713 ' PCT/LSOI/2~716 is R4-C(O)-R5- , R6-S(O)2-R7- or R26-S(0)-R27- wherein R4, R5, R6, R7, R2~ and R27 are as defined above.
Another preferred embodiment of the invention is a compound of formula I or II wherein n is '0, Z is -CH2-, and~R3 s is alkoxyal.kyl or alkoxyalkoxyalkyl.
A more preferred embodiment of the invention is a compound of formula I or II wherein n is 0, Z is -CH2-, and R3 is R4-C(O)-R5- wherein R4 is (R11)1R12)N- as defined ak~ove and R5 is alkylene or R3 is R6-S(O)2-R7- or R26-S(O)-R27- wherein to R~ is alkylene, R27 is alkylene and Rs and R26 are defined as above.
Another more preferred embodiment of the invention is a compound of formula I or II wherein n is 0, Z is -CH2- and R~ is R4-C10)-N(R20)-Rg- or 15 R~-S(0)2-N(R21)-R10- wherein Rg and R10 are alkylene and R4, R6, R20 and R21 are defined as above.
An even more preferred embodiment of the invention is a compound of formula I or II wherein n is 0, R is tetrazolyl or -Clfl)2-G wherein G is hydrogen ar a carboxy protecting group 20 or R is tetrazolyl or R is -C(0)-NHS(0)2R1~ wherein R16 is loweralkyl, haloalkyl or aryl, WO 02/11713 PCT/USO1/2.1716 Z is -CH2-; R1 and R2 are independently selected from (i) loweralkyl, (ii) cycloalkyl, (iii) substituted aryl wherein aryl is phenyl substituted with one, two or three substituents independently selected from loweralkyl, alkoxy, halo, 5 alkoxyalkoxy and carboxyalkoxy, (iv) substituted or unsubstituted heterocyclic, (v) alkenyl, (vi) heterocyclic (alkyl), (vii) arylalkyl, (viii) aryloxyalkyl, (ix) (N-alkanoyl-N-alkyl)aminoalkyl and (x) alkylsulfonylamidoalkyl, and R3 is R4-Cy0)-R5- wherein R4 is (R11)(R12)N- wherein R11 to and R12 are independently selected from loweralkyl, haloalkyl, alkoxyalkyl, haloalkoxyalkyl, ai~-yl, arylalkyl, heterocyclic, hydroxyalkyl, alkoxy, aminoalkyl, and trialkylaminoalkyl, and R5 is alkylene; or R3 is R4-C(O)-N(R2Q)-Rg- or R~-S(O)2-N(R21)-R1p- wherein R4 is loweralkyl, aryl, alkoxy, 15 alkylamino, aryloxy or arylalkoxy and Rg is loweralkyl, haloalkyl, alkoxyalkyl, haloalkoxyalkyl, aryl or arylalkyl, Rg and R1p are alkylene and R2p and R21 are loweralkyl; or R3 is R.g-S(O)2-R~- or R26-S(O)-R2~- wherein R6 is loweralkyl or haloalkyl, R~ is alkylene, R2g is loweralkyl and R2~ is 2o alkylene.
A yet more preferred embodiment of the invention is a WO 02/1171 ~ PCT/USOl/2~716 , compound of formula I or II wherein n is 0, R is -C{O)2-G
wherein G is hydrogen or a carboxy protecting group, tetrazolyl or -C(O)-NHS(O)2R16 wherein R16 is loweralkyl, haloalkyl or aryl, Z is -CH2-, R1 is (i) loweralkyl, (ii) s alkenyl, (iii) alkoxyalkyl, (iv) cycloalkyl, {v) phenyl,, (vi) pyridyl, (vii) furanyl,' (viii) substituted or unsubstituted 4-methoxyphenyl, 4-fluorophenyl, 3-fluorophenyl, 4-ethoxyphenyl, 4-ethylphenyl, 4-methylphenyl, 4-trifluoromethylphenyl, 4-pentafluoroethylphenyl, 3-fluoro-4-methoxyphenyl, 3-fluoro-4-to ethoxyphenyl, 2-fluorophenyl, 4-methoxymethoxyphenyl, 4-hydroxyphenyl, 4-t-butylphenyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl or dihydrobenzofuranyl wherein the substituent is selected from alkoxy, alkoxyalkoxy and carboxyalkoxy, (ix) heterocyclic (alkyl), (x) arylalkyl, (xi) aryloxyalkyl, (xii) Zs (N-alkanoyl-N-alkyl)aminoalkyl, or (xiii) alkylsulfonylamidoalkyl, R2 is substituted or unsubstituted 1,3-benzodioxolyl, 7-methoxy-1,3-benzodioxolyl, 1,4-benzodioxanyl, 8-methoxy-1,4-benzodioxanyl, dihydrobenzofuranyl, benzofurnayl, 4-methoxyphenyl, 2o dimethoxyphenyl, fluorophenyl or difluorophenyl and R3 is R4-C{O) -N(R20) -Rg- or N'O 02/11713 PCT/L~S01h~71G

R6-S(0)2-N(R21)-R10- wherein Rg and R10 are alkylene, R20 and R21 are loweralkyl, R4 is loweralkyl, aryl, alkoxy, alkylamino, aryloxy or arylalkoxy and R6 is loweralkyl, haloalkyl, alkoxyalkyl, aryl or arylalkyl.
s Another yet more preferred embodiment of the invention is a compound of formula I or II wherein n is 0, R is -C(O)2-G
wherein G is hydrogen or a carboxy protecting group, tetrazolyl or -C(O)-NHS(O)2R16 wherein Rlb is loweralkyl, haloalkyl ar aryl, Z is -CH2-, R1 is (i) loweralkyl, (ii) to alkenyl, (i.ii) alkoxyalkyl, (iv) cycloalkyl, (v) phenyl, (vi) pyridyl, (vii) furanyl, (viii) substituted or unsubstituted 4-methoxyphenyl, 4-fluorophenyl, 3-fluorophenyl, 4-ethoxyphenyl, 4-ethylphenyl, 4-methylphenyl, 4-trifluoromethylphenyl, 4-pentafluoroethylphenyl, 3-fluoro-4-methoxyphenyl, 3-fluoro-4-Zs ethoxyphenyl, 2-fluorophenyl, 4-methoxymethoxyphenyl, 4-hydroxyphenyl, 4-t-butylphenyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl or dihydrobenzofuranyl wherein the substituent is selected from alkoxy, alkoxyalkoxy and carboxyalkoxy, (ix) heterocyclic (alkyl), ~(x) arylalkyl, (xi) aryloxyalkyl, (xii) 20 (N-alkanoyl-N-alkyl)aminoalkyl, or (xiii) alkylsulfonylamidoalkyl, R2 is substituted or unsubstituted WO 02/11713 ' PCT/US01J2~716 , 1,3-benzodioxolyl, 7-methoxy-1,3-benzodioxolyl, 1,4-benzodioxanyl, 8-methoxy-1,4-benzodioxanyl, dihydrobenzofuranyl, benzofurnayl, 4-methoxyphenyl, dimethoxyphenyl, fluorophenyl or difluorophenyl and R3 is R4-C(0)-R5- wherein R5 is alkylene and R4 is (R11)(R12)N- wherein R11 and R12 are independently selected from loweralkyl, haloalkyl, alkoxyalkyl, haloalkoxyalkyl, aryl, arylalkyl, heterocyclic, hydroxyalkyl, alkoxy, aminoalkyl, and trialkylaminoalkyl.
io Another yet more preferred embodiment of the invention is a compound of formula I or II wherein n is 0, R is -C(O)2-~G
wherein G is hydrogen or a carboxy protecting group, tetrazolyl or -C(O)-NHS(O)2R16 wherein R16 is loweralkyl, haloalkyl or aryl, Z is -C~I2-, R1 is (i) loweralkyl, (ii) alkenyl, (iii) heterocyclic(alkyl), (iv) aryloxyalkyl, (v) arylalkyl, (vi) aryl, (vii) (N-alkanoyl-N-alkyl)aminoalkyl, or (viii) alkylsulfonylamidoalkyl, R2 is substituted or unsubstituted 1,3-benzodioxolyl, 7-methoxy-1,3-benzodioxolyl, 1,4-benzodioxanyl, 8-methoxy-1,4-benzodioxanyl, 2o dihydrobenzofuranyl, benzofurnayl, 4-methoxyphenyl,-dimethoxyphenyl, fluorophenyl or dif luorophenyl wherein the WO 02/11713 PCT/US01/2~716 substituent is selected from loweralkyl, alkoxy and halogen and R3 is R4-C(0)-R5- wherein R~ is alkylene and R4 is (R11)(R12)N- wherein R11 is loweralkyl and R12 is aryl, arylalkyl, hydroxyalkyl, alkoxy, aminoalkyl, s trialkylaminoalkyl, or heterocyclic.
Another yet more preferred embodiment of the invention is a compound of formula I or II wherein n is 0, R is -C(O)2-G
wherein G is hydrogen or a carboxy protecting group, tetrazolyl or -C(O)-NHS(0)2R16 wherein R1~ is loweralkyl, to haloalkyl or aryl, Z is -CH2-, R1 is (i) loweralkyl, (ii) alkenyl, (iii) heterocyclic (alkyl), (iv) aryloxyalkyl, (v) arylalkyl, (vi) (N-alkanoyl-N-alkyl)aminoalkyl, or (vii) alkylsulfonylamidoalkyl,(vii) phenyl, or (ix) substituted or unsubstituted 4-methoxyphenyl, 3-fluoro-4-methoxyphenyl, 3-is fluorophenyl, 3-fluoro-4-ethoxyphenyl, 2-fluorophenyl, 4-methoxymethoxyphenyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl or dihydrobenzofuranyl wherein the substituent is selected from loweralkyl, haloalkyl, alkoxy, alkoxyalkoxy and carboxyalkoxy, R2 is substituted or unsubstituted 1,3-benzodioxolyl, 7-2o methoxy-1,3-benzodioxolyl, 1,4-benz.odioxanyh, 8-methoxy-1,4-benzodioxanyl, dihydrobenzofuranyl, 4-methoxyphenyl, VI'O (1/11713 ' PCT/USOl/~.1716 2fl dimethoxyphenyl, fluorophenyl or difluorophenyl wherein the substituent is selected from loweralkyl, alkoxy and halogen and R3 is Rg-S(O)2-N(R21)-R10- wherein R10 is alkylene, R6 is loweralkyl, haloalkyl, alkoxyalkyl, haloalkoh-yalkyl, aryl~or s arylalkyl and R21 is loweralkyl, haloalkyl, alkoxyalkyl, haloalkoxyalkyl, aryl or arylalkyl.
Another yet more preferred embodiment of the invention is a compound of formula I or II wherein n is 0, R is -C(O)2-G
wherein G is hydrogen or a carboxy protecting group, io tetrazolyl or -C(O)-NHS(O)2R16 wherein R16 is loweralkyl, haloalkyl or aryl, Z is -CH2-, R1 is (i) substituted or unsubstituted 4-methoxyphenyl, 3-fluoro-4-methoxyphenyl, 3-fluorophenyl, 3-fluoro-4-ethoxyphenyl, 4-methoh-ymethoh-yphenyl, 1,3-benzodioxolyl or 1,4-benzodioxanyl wherein the substituent 15 is selected from loweralkyl, haloalkyl, alkoxy and alkoxyalkoxy, (ii) loweralkyl, (iii) alkenyl, (iv) heterocyclic (alkyl), (v) aryloxyalkyl, (vi) arylalkyl, (vii) (N-alkanoyl-N-alkyl)aminoalkyl, (viii) alkylsulfonylamidoalkyl,or (ix) phenyl, R2 is, substituted or 2o unsubstituted 1,3-benzodioxolyl, 7-methoxy-1,3-benzodioxolyl, 1,4-benzodioxanyl, 8-methoxy-1,4-benzodioxanyl, V1'O 02/11713 PCT/US01/2:1716 dihydrobenzofuranyl, 4-methoxyphenyl, dimethoxyphenyl;
fluorophenyl or difluorophenyl wherein the substituent is selected from loweralkyl, alkoxy and halogen and R3 is alkoxycarbonyl or R6-S(O)2-N(R21)-R10- wherein R10 is s alkylene, R6 is loweralkyl, haloalkyl, alkoxyalkyl or haloalkoxyalkyl and R21 is loweralkyl, haloalkyl, alkoxyalkyl or haloalkoxyalkyl.
Another yet more preferred embodiment of the invention is a compound of formula I or II wherein n is 0, R is -C(O)2-G
to wherein G is hydrogen or a carboxy protecting group, tetrazolyl or -C(O)-NHS(O)2R1~ wherein R1& is loweralkyl or haloalkyl, ~ is -CH2-, R1 is loweralkyl,alkenyl, heterocyclic (alkyl), aryloxyalkyl, aryalkyl, aryl, (N-alkanoyl-N-alkyl)aminoalkyl " or alkylsulfonylamidoalkyl, and R3 is R4-is C(O)-R5- wherein R5 is alkylene and R4 is (R11) (R12)N- wherein R11 and R12 are independently selected from alkyl, aryl, hydroxyalkyl, alkoxy, aminoalkyl, trialkylaminoalkyl, and heterocyclic.
A still more preferred embodiment of the invention is a 2o compound of formula I or II wherein n is 0, R is -C(0)2-G whereir_ G is hydrogen or a carboxy protecting group, WO 02/11713 . PCT/USU1/2.1716 tetrazolyl or -C(0)-NHS(0)2R16 wherein R16 is loweralkyl or haloalkyl, Z is -CH2-, R1 is substituted or unsubstituted 4-methoxyphenyl, 4-fluorophenyl, 2-fluorophenyl, 4-methylphenyl, 4-trifluoromethylphenyl, 4-pentafluoroethylphenyl, 4-s methoxymethoxyphenyl, 4-hydroxyphenyl, 4-ethylphenyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl or dihydrobenzofuranyl wherein the substituent is selected from alkoxy, alkoxyalkoxy and carboxyalkoxy, (ii) loweralkyl, (iii) alkenyl, (iv) heterocyclic (alkyl), (v) aryloxyalkyl, (vi) arylalkyl, (vii) io (N-alkanoyl-N-alkyl)aminoalkyl, (viii) alkylsulfonylamidoalkyl,or (ix) phenyl, R2 is 1,3-benzodioxolyl, 1,4-benzodioxanyl, dihydrobenzofuranyl, benzofuranyl, 4-methoxyphenyl, dimethoxyphenyl, fluorophenyl or difluorophenyl and R3 is R4-C~(O)-R5- wherein R5 is alkylene ?s and R4 is (R11)(R12)N- wherein R11 and R12 are independently selected from loweralkyl, aryl, arylalkyl, hydroxyalkyl, alkoxy, aminoalkyl, trialkylaminoalkyl, or heterocyclic.
Another still more preferred embodiment of the invention is a compound of formula I or II wherein n is 0, R is -C(O)2-G
2o wherein G is hydrogen or a carboxy protecting group, tetrazolyl or -C(0)-NHS(O)2R1~ wherein R1~ is loweralkyl or V1'O 02/11713 PCT/US01/2.t716 haloalkyl, Z is -CH2-, R1 is loweralkyl, alkenyl, heterocyclic (alkyl), aryloxyalkyl, arylalkyl, (N-alkanoyl-N-alkyl)aminoalkyl, alkylsulfonylamidoalkyl, phenyl,. or alkoxyalkyl, R2 is 1,3-benzodioxolyl, 1,4-benzodioxanyl, s dihydrobenzofuranyl, benzofuranyl, 4-methoxyphenyl, dimetho:cyphenyl, fluorophenyl or difluorophenyl and R3 is R4-Cd0)-R5- wherein R5 is alkylene and R4 is (R11)(R12)N- where in R11 and R12 are independently selected from loweralkyl, aryl, arylalkyl, hydroxyalkyl, alkoxy, aminoalkyl, to trialkylaminoalkyl, or heterocyclic.
A most highly preferred embodiment of the invention is a compound of formula I or II wherein n is 0, R is -C(O)2-G
wherein G is hydrogen or a carboxy protecting group, Z is -CH2-, R1 is substituted or unsubstituted 4-methoxyphenyl, 4-2s fluorophenyl, 2-fluorophenyl, 4-methylphenyl, 4-trifluoromethylphenyl, 4-pentafluoroethylphenyl, 4-methoxymethoxyphenyl, 4-hydroxyphenyl, 4-ethylphenyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl or dihydrobenzofuranyl wherein the substituent is selected from alkoxy, alkoxyalkoxy 2o and carboxyalkoxy, R2 is 1,3-benzodioxolyl, 1,4-benzodioxanyl, dihydrobenzofuranyl, benzofuranyl, 4-methoxyphenyl, WO 02/11713 ~ PCT/USOI/2-1716 dimethoxyphenyl, fluorophenyl or difluorophenyl and R3 is R4-C(a)-R5- wherein R5 is alkylene and R4 is (R11)(R12)N- wherein R11 and R12 are independently selected from loweralkyl.
Another most highly preferred embodiment of the invention s is a compound of formula I or II wherein n is fl, R is -C(O)2-~
wherein G is hydrogen or a carboxy protecting group, Z is -CH2-, R1 is substituted or unsubstituted 4-methoxyphenyl, 4-fluorophenyl, 2-fluorophenyl, 4-methylphenyl, 4-trifluoromethylphenyl, 4-pentafluoroethylphenyl, 4-io methoxymethoxyphenyl, 4-hydroxyphenyl, 4-ethylphenyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl or dihydrobenzofuranyl wherein the substituent is selected from alkoh-y, alkoxyalkoxy and carboxyalkoxy, R2 is 1,3-benzodioxolyl, 1,4-benzodioxanyl, dihydrobenzofuranyl, benzofuranyl, 4-methoxyphenyl, 1s dimethoxyphenyl, fluorophenyl or difluorophenyl and R3 is R4-C(4)-R5- wherein R5 is alkylene and R4 is (R11)(R12)N- wherein R11 is loweralkyl and R12 is aryl.
Another most highly preferred embodiment of the invention is a compound of formula I or II wherein n is~0, R is -C(0)2-G
2o wherein G is hydrogen or a carboxy protecting group, Z is -CH2-, R1 is substituted or unsubstituted 4-methoxyphenyl, 3-VVO 02/11713 ' PCT/US01/2.i716 fluoro-4-methoxyphenyl, 3-fluorophenyl, 2-fluorophenyl, 3-fluoro-4-ethoxyphenyl, 4-methoxymethoxyphenyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl or dihydrobenzofuranyl , wherein the substituent is selected from loweralkyl, haloalkyl, alkoxy, alkoxyalkoxy and carboxyalkoxy, R2 is substituted or unsubstituted 1,3-benzodioxolyl, 7-methoxy-1,3-benzodioxolyl, 1,4-benzodioxanyl, 8-methoay-1,4-benzodioxanyl, dihydrobenzofuranyl, 4-methoxyphenyl, dimethoxyphenyl, fluorophenyl or difluorophenyl wherein the substituent is selected from loweralkyl, alkoxy and halogen and R3 is R6-S(O)2-N(R~1)-R1fl- wherein Rlp is alkylene, R6 is loweralkyl, haloalkyl, alkoxyalkyl or haloalkoxyalkyl and R21 is loweralkyl, haloalkyl or alkoxyalkyl.
Another most highly preferred embodiment of the invention is is a compound of formula I or II wherein n is 0, R is -~C(O)~-~
wherein .G is hydrogen or a carboxy protecting group, Z is -CH2-, R1 is substituted or unsubstituted 4-methoxyphenyl, 3-fluoro-4-methoxyphenyl, 3-fluorophenyl, 2-fluorophenyl, 3-fluoro-4-ethoxyphenyl, 4-methoxymethoxyphenyl,,l,3-' 2o benzodioxolyl, 1,4-benzodioxanyl or dihydrobenzofuranyl wherein the substituent is selected from loweralkyl, WO 02/11713 PCT/US01/2~716 , haloalkyl, alkoxy,, alkoxyalkoxy and carboxyalkoxy, R2 is substituted or unsubstituted 1,3-benzodioxolyl, 7-methoxy-1,3-benzodioxolyl, 1,4-benzodioxanyl, 8-methoxy-1,4-benzodioxanyl, dihydrobenzofuranyl, 4-methoxyphenyl, dimethoxyphenyl, s fluorophenyl or difluorophenyl wherein the substituent is selected from loweralkyl, alkoxy and halogen and R3 is R4-C(O)-R5- wherein R5 is alkylene and R4 is (R11)(R12)N- wherein R11 is alkyl and R12 is selected from aryl, aminoalkyl, trialkylaminoalkyl, and heterocyclic.
to Another most highly preferred embodiment of the invention is a compound of formula I or II wherein n is fl, R is -C(O)2-G
wherein G is hydrogen or a carboxy protecting group, Z is -CH2-, R1 is loweralkyl,alkenyl, heterocyclic (alkyl), aryloxyalkyl, aryalkyl, aryl, (N-alkanoyl-N-alkyl)aminoalkyl, i5 or alkylsulfonylamidoalkyl, and R3 is R4-C(O)-R5- wherein R~
is alkylene and.R4 is (R11)(R12)N- wherein R11 and R12 are independently selected from alkyl, aryl, hydroxyalkyl, alkoxy, aminoalkyl, trialkylaminoalkyl, and heterocyclic, with the proviso that one or R11 and R12 is alkyl.
20 ,Another most highly preferred embodiment of the invention is a compound of formula I or II wherein n is 0, Z is -CFi2-, WO 02/11713 ' PCT/USU1/2~71G

and R3 is R4-C{0)-R5- 'wherein R4 is (R11) (R12)N- as defined therein and R5 is alkylene.
Another most highly preferred embodiment of the invention is a compound of formula I or II wherein n is 0, Z is -C~I2-, R1 is loweralkyl, and R3 is R4-C(O)-R5- wherein R4 is (R11)(R12)N- as defined therein and R5 is alkylene.
Another most highly preferred embodiment of the invention is a compound of formula I or II wherein n is 0, Z is -CH2-, ~R1 is alkenyl, and R3 is R4-C(O)-R5- wherein R4 is (R11)(R12)N- as defined therein and R5 is alkylene.
Another most highly preferred embodiment o,f the invention is a compound of formula I or II wherein n is 0, Z is -CH2-, R1 is heterocyclic (alkyl), and R3 is R~-C(O)-RS- wherein R4 is (R11){R12)N- as defined therein and i5 R5 is alkylene.
Another most highly preferred embodiment of the invention is a compound of formula I or II wherein n is 0, Z is --CH2-, R1 is aryloxyalkyl, and R3 is R4-C(O)-R5- wherein R4 is (R11)(R12)N° as defined therein and R5 is alkylene.
Another most highly preferred embodiment~of the invention is a compound of formula I or II wherein n is 0. Z i-s -Ci~2-, WO 02/11713 PCT/USO1l2.1716 R1 is arylalkyl, and R3 is R4-C(O)-RS- wherein R4 is (R11)(R12)N- as defined therein and R5 is alkylene.
Another most highly preferred embodiment of, the invention is a compound of formula I or II wherein n is 0, Z is -CH2-, s R1 is aryl, and R3 is R4-C(O)-R5- wherein R4 is (R11)(R12)N-as defined therein and R5 is alkylene.
Another most highly preferred embodiment of the invention is a compound of formula I or II wherein n is 0, Z is -CH2-, R1 is {N-al.kanoyl-N-alkyl)aminoalkyl, and R3 is R4-C(O).-R5-1o wherein R4 is (R11)(R12)N- as defined therein and R5 is alkylene..
Another most highly preferred embodiment of the invention is a compound of formula I or II wherein n is 0, Z is -CH2-, R1 is alkylsulfonylamidoalkyl, and R3 is R~-Ct0)-R5- wherein 15 R4 is (R11)(R12)N- as defined therein and R5 is alkylene.
A particularly preferred compound of formula i is a compound of formula III, also known as ,ACT-627:

i~1/O 02/11713 P~CT/USO1/24716 2~
~"1~
III
Compounds of formula I, 1I, and III may be synt3~.esized by methods provided in commonly owned U.S. Patewt I~o. 5,767,144, filed May 30, 1995.
Other suitable endothelin ~T-A receptor antac~o~ists may WO 02/11713 PCT/USOl/2.t716 be used, such as those disclosed in U.S. Patent Nos.

6,048,893, 6,017,951, and 5,998,468.
The term "inhibit" is defined to include its. generally accepted meaning which includes preventing, prohibiting, s restraining, and slowing, stopping or reversing progression, or severity, and holding in check and/or treating existing characteristics. The present method includes both medical therapeutic and/or prophylactic treatment, as appropriate.
The methods of the present invention are useful in men as io well as in women. Preferably, however, the methods of the present invention are useful in men, more preferably men with prostate cancer.
The ability of the compounds of formula I, II, and III to treat cancers can be demonstrated according to the method 15 described in J. Clin. Invest. 87 1867 (1991). Types of cancer includes primary cancer such as breast, prostate, lung, kidney, thyroid, myeloma, lymphoma, sarcoma, osteosarcoma, anal ovarian.
The ability of the compounds of the invention to treat 2o nociception can be demonstrated according to the method described in J. Pharmacol. Exp. Therap. 271 156 (1994).
The compounds of the present invention can be used in the WO 02/11713 PCT/USO1/?-1716 form of salts derived from inorganic or organic acids. These salts include but are not limited to the following: acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, heptancate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxy-ethanesulfonate, lactate, maleate, methanesulfonate, ~.o nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, p-toluenesulfonate and undecanoate. Also, the basic nitrogen-containing groups can be quaternized with such agents as loweralkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides, and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorir~es, bromides and iodides, aralkyl halides like benzyl and 2o phenethyl bromides, and others. Water or oil-soluble or dispersible products are thereby obtained.
Examples of acids which may be employed to form WO 02/11713 PCT/1~501~2.1716 pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid, sulphuric acid and phosphoric acid and such organic acids as oxalic acid, malefic acid, succinic acid and citric acid.
Basic addition salts can be prepared in situ during the final isolation and purification of the compounds of formula I, or separately by reacting the carboxylic acid function with a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation or io with ammonia, or an organic primary, secondary or tertiary amine. Such pharmaceutically acceptable salts include, but are not limited to, cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, aluminum salts and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.
Other representative organic amines useful for the formation of base addition salts include diethylamine, ethyl.enediamine, ethanolamine, diethanolamine, piperazine and the like.
The compounds of formulas I, II and III are useful for WO 02/11713 ' PCT/US01/2~716 antagonizing endothelin in humans or other mammals. Total daily dose administered to a host in single or divided doses may be in amounts, for example, from 0.001 to 1000 mg/kg body weight daily and more usually 0.1 to 100 mg/kg for oral s administeration or 0.01 to 10 mg/kg for parenteral administeration. Dosage~unit compositions may contain such amounts of submultiples thereof to make up the daily dose.
Pharmaceutical formulations may be prepared by procedures known in the art. The amount of active ingredient that may be io combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administeration.
It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of is factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administeration, route of administeration, rate of excretion, drug combination, and the severity of the particular disease undergoing therapy.
2o The compounds of the present invention may be administered.orally, buccally, parenterally, sublingually, by inhalation spray, rectally, or topically in dosage unit CA 02555850 2001-08-06 _ WO 0?/11713 ~ PCT/USOl/2~716 formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired.
Topical administeration may also involve the use of transdermal administeration such as transdermal patches or s iontophoresis devices. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection, transcutaneous, intradermal, or infusion techniques.
Injectable preparations, for example, sterile injectable io aqueous or oleagenous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for 15 example, as a solution in 1,3-propanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer s solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose 2o any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

WO 02/11713 ' PCT/USO1/2~716 Suppositories for rectal administeration of the drug can be prepared by mixing the drug with a suitable nonirritating excipient such as cocoa butter and polyethylene glycols which are solid at ordinary temperatures but liquid at the rectal, s temperature and will therefore melt in the rectum and release the drug.
Solid dosage forms for oral administeration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound may be admixed with at to least one inert diluent such as sucrose lactose or starch.
Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also is comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
Liquid dosage forms for oral administeration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in 2o the art, such as water. Such compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.

WO U~/11713 PCT/USOI/2~716 The compounds of the present invention can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or s multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a compound of the present invention, Zo stabilizers, preservatives, excipients, and the like. The preferred lipids are the phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic.
Methods to form liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, i5 Academic Press, New York, N.Y. (1976), p. 33 et seq.
A representative solid dosage form, for example, a tablet or a capsule, comprises:
Compound of the invention: 35% w/w Starch, Pregelatin~ized, NF 50% w/w so Microcrystalline Cellulose, NF 10% w/w Talc, Powder, USP 5% w/w While the compounds of the invention can be administered WO 02/11713 PCT/U'SO1/2.1716 as the sole active therapeutic agent, they can also be used in combination with one or more co-therapeutic agents, such as anticancer drugs or methods including, but not limited to, hormonal agents, such as leuprolide (Lupron ); gonado.relin s antagonists, such as goserelin (Zoladex°°) and abarelix;
bicalutamide; nilutamide; flutamide; vitamin D; vitamin D
analogues; estrogen and estrogen analogues, such as diethylstibestrol; prednisone; hydrocortisone; ketoconazole;
cyproterone acetate; progesterone; 5-alpha reductase 1.o inhibitors, such as finasteride; bone-seeking radionuclides, such as samarium (Quadramet~), strontium (Metastro~ ), and iesrhenium; external beam radiation, including three dimensional conformal radiation; brachytherapy, which is the implantation of radioactive seeds directly into the prostate;
15 monoclonal antibodies such as trastu~umab (Hercepti~ ); anti-angiogenic agents such as thrombospondin peptide or kringle 5;
matrix metalloproteinase inhibitors: farnesyl transferase inhibitors; lycopenes; urokinase; plasminogen activator inhibitors; plasminogen activator receptor blockers; ap~t.os~.s 2o inducers; selective and non-selective alpha Mockers; platinur.~
agents, such as cis-platinum and carbo-platinum; iaxane class agents, such as docitaxil and paclitaxil; estramustine;

V1'O 02/11713 PCT/USOll3~71G

gemcytabine; adriamycin; doxorubicin; daunorubicin;
mitoxantrone; vinblastine; vincristi:~.e; capecitabine;
irinotecan; topotecan;
5-fluorouracil; interferons; cytoxan; methotrexate;
cytokines, such as IL-2; PPAR agonists, such as thiazolidine diones; retinoid-type agents, 5-lipooxygenase inhibitors, such as zyfo (Ziluetori), COX-2 inhibitors; gene-therapy based therapeutics, including sense and anti-sense genes;
cholesterol lowering drugs, such as lovastatin, pravastatin, to and simvistatin; bisphosphonates; osteoprotegrin; and antibodies, both monoclonal and polyclonal; antibody-coupled radionucleotides; antibody-coupled cytotoxi-c agents; antibody-coupled radionucleotides; viral-vector delivered agents;
vaccines directed at protein, carbohydrate, or nucleic acid i~ targets; aminoglutethimide; and suramin.
These combinations can be administered as separate compositions or as a single dosage form containing both or all agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions, which are 20~ given at the same time or different times, or the therapeutic agents can be given as a single composition.
In addition, the compounds invention can be used in V1'O 02/11713 PCT/US01/~~716 combination with one or more co-therapeutic agents which impede net bone loss, such as estrogens, bisphosphonates, and estrogen receptor modulators, such as raloxifene, and calcitonin.
s The compounds of the invention can additionally be administered in combination with surgery, such as radical prostatectomy, cryotherapy, transurethral resection of the prostate as an adjuvant, and the life, or priflr to surgery as a neoadjuvant agent.
to The current major diseases or conditions of bone which are of public concern include, but are not limited to, post-menopausal osteoporosis, ovariectomy patients, senile osteoporosis, patients undergoing long-term treatment of corticosteroids, side effects from glucocorticoid or steroid is treatment, patients suffering from Cushings's syndrome, gonadal dysgenesis; periarticular erosions in rheumatoid arthritis, osteoarthritis, Paget's disease, osteohalist-eresis, osteomalacia, hypercalcemia of malignancy, osteopenia due to bone metastases, periodontal disease, hyperparathyroidism, 20 osteroperosis from Lupron therapy, anal starvation. All of these conditions are characterized by bone loss, resulting from an imbalance between the degradation of bone (bone WO 02/1 3 713 ' PCT/US01/2~71G
resorption) and the formation of new healthy bone. This turnover of bone continues normally throughout life and is the mechanism by which bone regenerates. However, the conditions stated above will tip the balance towards. bone loss such that s the amount of bone resorbed is inadequately replaced with new .
bone, resulting in net bone loss.
Examples Studies were performed on male subjects with asymptomatic to hormone refractory prostate cancer with rising PSA levels and on male subjects with symptomatic hormone refractory prostate cancer with rising PSA levels and pain. Subjects in the phase II studies had castrate levels of testosterone, either due to pharmacologic intervention, via leuprolide (Luprori ) or is goserelin (Zoladex""), or via surgical castration. Subjects received ABT-627 or placebo. The following tests were conducted:
ABT-627 was formulated in 2.5 and 10 mg doses. An oral liquid formulation of ABT-627 was also prepared as follows: 1 2o mg/ml ABT-627, 50% glycerin, 14% alcohol, and water. Matching placebos were also provided.
A number of recognized or putative biochemical markers of disease progression have been used to monitor treatment of individuals with prostate cancer. Among these markers are serum Prostate Specif is Antigen (PSA), serum acid Phosphatase, Interleukin-6, and Chromagranin-A. As currently accepted, s favorable treatment is marked by a decrease or slower rate of increase for PSA, acid phosphatase, and Interleukin-6, while a favorable response is marked by an increase in Chromagrar_in-A.
Serum samples were obtained from subjects during treatment with the ET antagonist ABT-627 in order to determine to PSA, acid phosphatase, IL-6, and Chromagranin-A values.
Prostate Specific Antigen Level Assay The effect of ABT-627 administeration on prostate specific antigen (PSA) levels in human subject serum samples is was determined using the procedure described in the Chiron Diagnostics ACS: Centaur PSA2 Assay. This assay i~s a two-site sandwich immunoassay which uses direct chemiluminescense arad constant amounts of two antibodies. The (first antibody, the Lite Reagent, is an affinity purified polyclonal sheep anti-2o PSA antibody labeled with acridinium ester. The Lite Reagent is purchased as a 5.0 mL reagent pack comprising the polyclonal sheep anti-PSA antibody (3.1 fig) in buffered salir~e WO 0211171 ~ PCT/USO1/2.t716 with sodium azide (0:1%). The second antibody, the Solid Phase, is a monoclonal mouse anti-PSA antibody covalently coupled to paramagnetic particles. The Solid Phase is purchased as a 25.0 mL reagent pack comprising the covalently s coupled monoclonal mouse anti-PSA antibody (316 ~.g) in buffered saline with sodium azide (0.1%). The assay was performed at Quintiles Laboratories (Smyrna, ~A) using Chiron Diagnostics ACS: Centaur Automated Chemiluminescence Systems.
Briefly, a subject population was treated with a placebo to or 2.5 mg or 10 mg of ABT-627. Blood samples were collected, allowed to adequately clot, centrifuged at 1000 x g for 15-20 minutes, and stored at -20 °C if not assayed within 48 hours.
A cuvette was charged sequentially with serum, Lite Reagent (50 ~L) , and Solid Phase (250 ~,L) . The resulting mixture was is incubated for 7.5 minutes at 37 °C, separated, and treated with the solution of Acid Reagent and Base Reagent to initiate the chemiluminescent reaction. A direct relationship exists between the amount of PSA present in the patient sample and the RLU's (relative light units) detected. As show~z by the 2o area under the curve (AUC) in Figure 2, the rate of increase of PSA in the serum samples decreases after the adminstex~ation of ABT-627, demonstrating the effectivness of ABT-627 as an WO 0?/11713 PCT/USO1/?716 agent for treating prostate cancer.
Acid Phosphatase Levels The effect of ABT-627 administeration on Acid Phosphatase s levels in human subject serum samples was determined at Quintiles Laboratories using the chemical test described in S ~a Diagnostics Acid Phosphatase (ACP) Procedure No. 435.
The enzyme Acid Phosphatase (ACP) catalyzes the hydrolysis of alpha-naphthyl phosphate to alpha-naphthol and inorganic Zo phosphate. The alpha-naphthol immediately reacts with fast red TR salt. to produce a yellow chromophore with an absorbance maximum at 405 nm. The rate of increase in absorbance at 405 nm is directly proportional to ACP activity in the sample.
ACP activity was determined in the presence and absence of L-15 tartrate, the difference being attributed to prostatic acid phosphatase activity.
Briefly, a subject population was treated with a placebo or 2.5 mg or 10 mg of ABT-627. Blood samples were collected, allowed to adequately clot, centrifuged at 1000 x g for 15-20 2o minutes, and stored at -20 °C if not assayed within 48 hours.
Assays were performed on a Hitachi Spectrophotomer. A cuvette was charged sequentially with AcP reagent (1 mL), prepared as WO 0/11713 ~ PCT/USOl/2~716 . -described in the assay protocol, and serum (0.1 mL). The mixture was agitated and incubated for 5 minutes, and an absorbance (A) at 405 nm (versus water as a reference) was read to provide an initial absorbance. The mixture was s incubated for another 5 minutes, and a second absorbanc.e was read to provide a final~absorbance. A change A/5 minute value was obtained by subtracting the initial absorbance from the final absorbance and was used to calculate total ACP activity.
To provide the tartrate-resistant acid phosphatase to activity, the above procedure was repeated with the addition of ACP tartrate reagent (0.01 mL) to the cuvette containing the ACP reagent and mixing before adding the serum. Prostatic acid phosphatase activity was calculated by subtracting the the tartrate-resistant acid phosphatase activity from the ACP
i5 activity. As shown shown by the (AUC) in Figure 7, the rate of increase and the average change from baseline for acid phosphatase was decreased in those subjects treated with ABT-627, again demonstrating the effectivness of ABT-627 as an agent for treating prostate cancer.
Chromagrania-A Levels V1'O ~?/11713 ° ~~. PCTlUSOII?4716 The effect of ABT-627 admi~atrati~n on C~rorr~agranin-A w levels in human serum samples was taet~ermin~d by pr-oprietary assay conducted at the Nichols Institute. The procedure is a two site chemiluminescence assay (ICMA) using one monoclonal antibody conjugated with biotin, another monoclonal antibody labeled with an acridinium ester, and an avidin-coated solid phase. The antibody/Chromagranin-A/antibody complex is 3~ound to the solid phase by the avidin-biotin interaction and unbound materials are removed by washing. The 3~ound, acridinium--labeled material produces light that is detected in a luminomet:er after addition of triggering agents. The Limit of Detectian (LOD) for the assay was 0.07 ng/mL. The average change from baseline for Chromagranin-A was higher for subjects treated with 2.5 mg/day of ABT-627, again ~5 demonstrating the effectivness of ABT-627 as an agent for treating prostate cancer.
Interleukin-b Levels The effect of ABT-627 ad.minstr.ation ~on Interleukin-6 levels in human serum samples was determined at Quintiles Laboratcries using a sandwich immu~oassay. ~-:uman sDrum samples and standards were incubated in microtiter plate wells WO 0?/11713 ~ PCT/US01/2~716 . . -coated with a monoclonal anti-IL-6 antibody, in the presence of a second monoclonal anti IL-6 antibody, linked to acetylcholinesterase. After incubation, the wells were washed, and the bound enzymatic activity was measured using a s chromogenic substrate. The intensity of the color was proportional to the concentration of IL-6 in the sample or standard. As shown by the AUC Figure 1, the average change in baseline for Interleukin-6 was lower in those subjects treated with ABT-627, demonstrating the effectivness of ABT-627 as an io agent for .reducing inflammation and ameliorating pain.
Bone Scan Methodology Bone scans were performed with an NDA approved, Tc-99m phosphonate type radiopharmaceutical. This technique uses is whole body format (skull to feet) so that anterior and posterior :images are presented when using a 510 K-approved gamma camera. Alternatively, spot views covering both anterior and posterior projections of the total body can be obtained. Interpretation was performed according to standard 2o nuclear medicine criteria, on a bone by bone basis, by recording the number of lesions at each site. Each site was evaluated against a confidence scare of 1 to 5, where 1 is WO 02/11713 PCTNSU1/~.171G

negative, 2 is probably negative, 3 is equivocal, 4 is probably positive, and 5 is definitely positive. The MSI~CC
(Clin. Can. Res. 1998; 4:1765-1772) was used to record these findings. For the purposes of scoring the extent of disease s or the response to treatment, lesions with a confidence score of 4 and 5 were considered positive, and all other lesions were considered negative. In addition, in a blinded study, a reference nuclear medicine physician interpreted the bone scans quantitatively as follows: the percent of involved bone to was estimated for each individual bone, and the individual bone involvement was summed to calculate a global percent bone scan index (BSI). More specifically, the bone scan was separated into three indices. The first was the appindicular scan which involved arms and legs (i.e. the humorous and all 15 bones distal to the humerous and the femur and everything distal to the femur). The second was the axial (everything but the arms and the legs). The results of these scans were combined to provide the total BSI.
Bone scans were conducted on each subject on day one of 2o the study, and on the final day of the study, and the changes from baseline in bone scan index scores were analysed by mean change and mean percent change, adjusting for baseline WO 02/11713 ' PCT/1iS01/?..1716 .

characteristics as co-variates using SAS version XXX software.
As shown in Figure &, bone scans indicated a decrease in the proportion of total skeketal involvement in those subjects receiving ABT-627 versus placebo, demonstrating the s effectivness of ABT-627 as an agent for reducing the fraction of total skeletal involvement by tumor.
VAS Methodology/Administeration/Analysis The Visual Analog Scale (VAS) is a common instrument of to pain assessment performed by having a subject draw a vertical line on a IO em scale at the point that best describes his or her pain on average in the last 24 hours. A diagram of the scale is shown below:
is No pain .r------------------------I Pain as bad as it could possibly be (not to scale) During the course of the study, pain ase-ssments were done daily, at bedtime, by the subject. If the subject was unable 2o to maintain the log, a caregiver could complete the log on his or her behalf. The log also contained a table on which was recorded all daily pain medication consumed by the patient.

WO 02/11713 PCT/ITS01/2~716 The logs of daily VAS scores and analgesic consumption were collected at biweekly visits of the subject to the clinic when a new log was distributed. Clinical personnel who received the logs measured the score by measuring the distance tin mm) s from the "no pain" end mark t~o the point where the subject's line crossed the VAS line. The number was written into the case report. form next to the date the subj-ect completed that page of the logbook.
Subjects with pain were initially stabilized in their to pain so that their pain was treated to a tolerable and constant level. For this study, "tolerable and constant"
refers to a pain score less than or equal to 5 cm on the 'VAS
for an average of seven successive days while using four or less rescue doses of pain medication per day. A rescue is medication dose refers to a dose equal to one single dose a patient used for common timed pain relief.
The weekly VAS scores were calculated excluding the lowest and highest score for each week and averaging the remaining five scores. If there were two days with the same 2o VAS score, the day with the highest analgesic use was discarded.
The weekly mean VAS score was used to define subjects as WO 02/11713 PCT/US01/2.t716 responders or non-responders. A subject was~considered a responder based on the reduction in the pain intensity: a weekly VAS score reduction of greater than or equal to 25%
during at least two consecutive weeks without an increase of s analgesic use during the same period tcompared to baseline).
Alternatively, a subject was considered a responder if his pain analgesic consumption was reduced by at least 25% during at least two consecutive weeks without a concomitant increase in VAS score.
to The percentage of responders in each treatment group was compared to evaluate drug efficacy. The comparisoaz was subjected to an adjustment for baseline characteristics an3 prognostic factors as co-variates, and the analysis was performed using the Cochran-Mantel-Haenszel test or a 1~ generalized linear model.
Weekly VAS scores are examined using a longitudinal analysis method to explore trends over time. The duration of the response, defined as the time from baseline to the last weekly assessment for which the responder definition was 2o satisfied, was analyzed using the I~aplan-Meier methodology and logrank test. Cox proportional hazard models were used as needed (see U.S. Department of Health and Human Services.

WO 02/11713 PCT/US01/2.~71G

Management of Cancer Pain Clinical Practice Guidelines. AHCPR
Publication #94-0592, Rockville, MD (1994). As shown by the AUC in Figure 3, VAS scores showed a decrease in pain, independent of the effects of morphine, after treatment with with ABT-627, demonstrating the effectivness of ABT-627 as an agent for ameliorating pain.
Osteoblastic Activity and Bone Markers Markers of osteoblastic activity were assessed using io urine samples. Bone markers include bone alkaline phosphatase (BAP), deoxypridinoline, and N-telopeptide of Type I collagen.
Blood samples were collected prior to dosing on Day 1, Day 42, Day 84, Day 168, and every 28 days after Day 168, with a final collection on the last day of the study.
is Bone Alkaline Phosphatase Bone Alkaline Phosphatase levels were determined using the bone-specific Alkphase-B° assay published by Metra Biosystems (Mountain View, CA). As shown by the AUC in Figure 20 5, BAP levels decreased in subjects treated with ABT-627, demonstrating the effectivness of ABT-627 as an agent for inhibiting abnormal bone remodeling.

WO 0?/11713 PCT/USO1/?.1716 Crosslinked N-Telopeptide Levels:
Cross--linked N-telopeptide levels were determined using the DiaSorin (Stillwater, MN) assay for the quantitative s determination of carboxyterminal cross-linked telopeptide of type I col7_agen (ICTP) in human serum by equilibrium radioimmunaassay (RIA). Briefly, samples were incubated with the lasl ICTP tracer and ICTP primary antibody for 2 hours at 37 °C. Following the 2 hour incubation, a pre-precipitated to second antibody complex was added to separate the bound from free tracer'. The assay was then centrifuged and decanted after a 30 minute incubation at room temperature. The bound tracer in the pellet was counted with a gamma counter. Counts were inversely proportional to the amount of ICTP present in ~s each sample. As shown by the AUC in Figure 4, Crosslinked N-telopeptide levels decreased in subjects treated with ABT-~G27, demonstrating the effectivness of ABT-X27 as an agent for inhibiting the bone remodeling associated with bone diseases.

Claims

-53-

1. The use of a therapeutically effective amount of a compound of formula I to inhibit bone metastases in a human:
wherein R is -(CH2)m-W;
Z is selected from -C(R18)(R19)- and -C(O)-;
R1 and R2 are independently selected from hydrogen, loweralkyl, alkenyl, alkynyl, alkoxyalkyl, alkoxycarbonylalkyl, hydroxyalkyl, alkoxyalkoxyalkyl, thioalkoxyalkoxyalkyl, cycloalkyl, aminocarbonylalkyl, alkylaminocarbonylalkyl, dialkylaminocarbonylalkyl, aminocarbonylalkenyl, alkylaminocarbonylalkenyl, dialkylaminocarbonylalkenyl, hydroxyalkenyl, aryl, arylalkoxyalkyl, heterocyclic, and (R aa)(R bb)N-R cc-, with the proviso that one or both of R1 and R2 is other than hydrogen;
R3 is selected from R4-C(O)-R5-, R6-S(O)2-R7-, R26-S(O)-R27-, R22-O-C(O)-R23-, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, aryloxyalkyl, heterocyclic, (heterocyclic)alkyl, alkoxyalkyl, alkoxyalkoxyalkyl, and R13-C(O)-CH(R14)-;
R4 and R6 are independently selected from (R11)(R12)N-, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclic, (heterocyclic)alkyl, and alkoxyalkyl;
R5 is selected from a covalent bond, alkylene, alkenylene, -N(R20)-R8-, -R8a-N(R20)-R8-, -O-R9-, and -R9a-O-R9-;
R7 is a covalent bond, alkylene, alkenylene, or -N(R21)-R10-;

R8 is alkylene;
R9 is alkylene;
R10 is alkylene;
R11 and R12 are independently selected from hydrogen, alkyl, alkoxyalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, heterocyclic, arylalkyl, and (heterocyclic)alkyl;
R13 is selected from amino, alkylamino and dialkylamino;
R14 is selected from aryl and R15-C(O)-;
R15 is selected from amino, alkylamino and dialkylamino;
R16 is selected from alkyl, haloalkyl, phenyl and dialkylamino;
R17 is alkyl;
R18 and R19 are independently selected from hydrogen and alkyl;
R20 is selected from hydrogen, alkyl, alkenyl, cylcoalkyl and cycloalkylalkyl;
R21 is selected from hydrogen and alkyl;
R22 is selected from alkyl and heterocyclic;
R23 is selected from covalent bond, alkylene, alkenylene and -N(R24)-R25-;
R24 is selected from hydrogen and alkyl;
R25 is alkylene;
R26 is selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclic, (heterocyclic)alkyl, alkoxyalkyl and alkoxy-substituted haloalkyl;
R27 is selected from alkylene and alkenylene;
R8a is alkylene;
R9a is alkylene;
R aa is selected from aryl and arylalkyl;
R bb is selected from hydrogen and alkanoyl;
R cc is alkylene;
m is 0;
n is 0;
E is selected from hydrogen, alkyl and arylalkyl;
G is hydrogen; and W is selected from -C(O)2-G; -PO3H2, -P(O)(OH)(E), -CN, -C(O)NHR17, dialkylaminocarbonyl, tetrazolyl, hydroxy, alkoxy, sulfonamido, -C(O)NHS(O)2R16, -S(O)2NHC(O)R16, or a pharmaceutically acceptable salt thereof.