GROWTH INHIBITORS AND METHODS OF TREATING CANCER AND CELL PROLIFERATIVE DISEASES

INHIBITEURS DE CROISSANCE ET METHODE DE TRAITEMENT DE CANCER ET DES MALADIES A PROLIFERATION CELLULAIRE

English Abstract

2084208 9117749 PCTABS00008
The present invention discloses new and useful compounds
including methyl p-hydroxyphenyllactate, its analogues, chemical
derivatives and chemically related compounds and their use as antitumor
and immune suppressive agents, as inhibitors of proliferative cell
growth and as prophylactic agents to inhibit and prevent cancer
and non-malignant cell growth.

Claims

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Claims:
1. A method of treating cancer, comprising the step of administering a
therapeutic dose of a compound selected from the group consisting of:
<IMG> <IMG> ,
,
<IMG> ,
<IMG> ,
and <IMG>
Wherein, R1 and R4 are selected from the group consisting of H and OH;
R2, R3 and R6 are selected from the group consisting of H, OH, OCH3, amino,
alkylamino and alkyl groups containing 1 to 6 carbons, acyloxy, and halogens; R5is selected from the group consisting of H, OH, OCH3, acyloxy and halogens; R'1
and R'2 are selected from the group consisting of H, OH, OCH3, amino, cyano,
alkyl or alkylamino groups of 1 to 6 carbon atoms, acyloxy, halogens, .ang.-azido and
aziridine derivatives, 3,4-dihydroxyphenylmethylene, p-hydroxyphenylmethylene
and other substituted phenyl groups preferably with acyloxy or halogen
substinuents; R'3, R'4, R'5, R"1 and R"2 are selected from the group consisting of
H, OH, OCH3, amino, cyano, alkyl or alkylamino groups of 1 to 6 carbon atoms,
acyloxy, halogens, .ang.-azido, aziridine, acyloxy and halogen substituted derivatives.

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2. The method of Claim 1, wherein said compound is selected from the
group consisting of the formulae:
<IMG> <IMG>
and
Wherein R1, and R4 are H; OH, acyloxy or halogens, R2 and R3 are OH,
OCH3, amino, alkyl or alkylamino groups of 1 to 6 carbon atoms or halogens, R'1,and R'2 are H, CH3, or phenylmethylene having p-hydroxy, 3,4-dihydroxy,
acyloxy and halogen ring subtituents and R'3 is H, OH, CH3 ,OCH3 and alkyl
groups from 1-6 carbon atoms.
3. The method of Claim 1, wherein said compound is:
<IMG>
Wherein, R1 and R4 are selected from the group consisting of H and OH;
R2, and R3 are selected from the group consisting of H, OH, OCH3, amino,
alkylamino and alkyl groups containing 1 to 6 carbons; and R'1 and R'2 are
selected from the group consisting of H, CH3, OH, OCH3, 3,4
dihydroxyphenylmethylene, p-hydroxyphenylmethylene and other substituted
phenyl groups.

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4. The method of Claim 1, wherein said compound is
<IMG>
Wherein, R1 and R4 are selected from the group consisting of H and OH;
R2, R3 and R'3 are selected from the group consisting of H, OH, OCH3, amino,
alkylamino and alkyl groups containing 1 to 6 carbons; and R'1 and R'2 are
selected from the group consisting of H, CH3, OH, OCH3, 3,4
dihydroxyphenylmethylene, p-hydroxyphenylmethylene and other substituted
phenyl groups.
5. The method of Claim 1, wherein said compound is:
<IMG>
Wherein R1, R2 and R3 are H, OH, OCH3, amino, alkyl or alkylamino
groups of 1 to 6 carbon atoms, acyloxy and halogens, and R6 is H, or alkyl groupof 1 to 6 carbon atoms.
6. The method of Claim 1, wherein said compound is:
<IMG>
Wherein R1-R4 and R'1-R'5 are H, OH, OCH3, amino, alkyl or alkylamino
groups of 1 to 6 carbon atoms.

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7. The method of Claim 1, wherein said compound is:
<IMG>
Wherein R1-R5, R'1-R's, R"1 and R"2 are H, OH, OCH3, amino, cyano,
alkyl or alkylamino groups of 1 to 6 carbon atoms, acyloxy, halogens, .ang.-azido,
aziridine, acyloxy and halogen substituted derivatives.
8. A method for inhibiting the growth of proliferating cells which include
a Type II nuclear estrogen bindiag site comprising the step of administering a
biologically inhibiting dose of a compound selected from the group consisting of:
<IMG> , <IMG> ,
<IMG> , <IMG> ,
and <IMG>
Wherein, R1 and R4 are selected from the group consisting of H and OH;
R2, R3 and R6 are selected from the group consisting of H, OH, OCH3, amino,

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alkylamino and alkyl groups containing 1 to 6 carbons, acyloxy, and halogens; R5is selected from the group consisting of H, OH, OCH3, acyloxy and halogens; R'1
and R'2 are selected from the group consisting of H, CH3, OH, OCH3, 3,4-
dihydroxyphenylmethylene, p-hydroxyphenylmethylene and other substituted
phenyl groups preferably with acyloxy or halogen substituents; R'3, R'4, R's, R"1
and R"2 are selected from the group consisting of H, OH, OCH3, amino, cyano,
alkyl or alkylamino groups of 1 to 6 carbon atoms, acyloxy, halogens, .ang.-azido,
aziridine, acyloxy and halogen substituted derivatives.
9. The method of Claim 8, wherein said compound is selected from the
group consisting of the formulae:
<IMG> and <IMG>
10. The method of Claim 9, wherein said compound is selected from the
group consisting of the formulae:
<IMG> <IMG>
and
Wherein, R1and R4 are selected from the group consisting of H and OH;
R2, R3 and R'3 are selected from the group consisting of H, OH, OCH3, amino,
alkylamino and alkyl groups containing 1 to 6 carbons; and R'1 and R'2 are
selected from the group consisting of H, CH3, OH, OCH3, 3,4
dihydroxyphenylmethylene, p-hydroxyphenylmethylene and other substituted
phenyl groups.

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11. The method of Claim 9, wherein said compound is:
<IMG>
Wherein, R1 and R4 are selected from the group consisting of H and OH;
R2, and R3 are selected from the group consisting of H, OH, OCH3, amino,
alkylamino and alkyl groups containing 1 to 6 carbons; and R'1 and R'2 are
selected from the group consisting of H, CH3, OH, OCH3, 3,4
dihydroxyphenylmethylene, p-hydroxyphenylmethylene and other substituted
phenyl groups.
12. The method of Claim 8, wherein said compound is
<IMG>
Wherein R1, and R4 are H; OH, acyloxy or halogens, R2 and R3 are OH,
OCH3, amino, alkyl or alkylamino groups of 1 to 6 carbon atoms or halogens, R'1
and R'2 are H, CH3, or phenylmethylene having p-hydroxy, 3,4-dihydroxy,
acyloxy and halogen ring subtituents and R'3 is H, OH, CH3,OCH3 and alkyl
groups from 1-6 carbon atoms.
13. The method of Claim 8, wherein said compound is:
<IMG> ,

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Wherein R1, R2 and R3 are H, OH, OCH3, amino, alkyl or alkylamino
groups of 1 to 6 carbon atoms, acyloxy and halogens, and R6 is H, or alkyl groupof 1 to 6 carbon atoms.
14. The method of Claim 8, wherein said compound is:
<IMG>
Wherein R1-R4 and R'1-R's are H, OH, OCH3, amino, alkyl or alkylamino
groups of 1 to 6 carbon atoms.
15. The method of Claim 8, wherein snid compound is:
<IMG>
Wherein R1-R5 and R'1-R'5, R"1 and R"2 are H, OH, OCH3, amino, cyano,
alkyl or alkylamino groups of 1 to 6 carbon atoms, acyloxy, halogens, .ang.-azido,
aziridine, acyloxy and halogen substituted derivatives.
16. The method of Claim 8, wherein said proliferating cells are estrogen
responsive tissue selected from the group consisting of uterus, mammary gland,
utesine tumoss and mammary tumors.
17. The method of Claim 16, wherein said estrogen responsive tissue is
human breast cancer cells.

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18. A method for treating benign prostatic hyperplasia, comprising the
step of administering a therapeutic dose of a compound selected from the group
consisting of:
<IMG> , <IMG> ,
<IMG> , <IMG> ,
and <IMG>
Wherein, R1 and R4 are selected from the group consisting of H and OH;
R2, R3 and R6 are selected from the group consisting of H, OH, OCH3, amino,
alkylamino and alkyl groups containing 1 to 6 carbons, acyloxy, and halogens; R5is selected from the group consisting of H, OH, OCH3, acyloxy and halogens; R'1
and R'2 are selected from the group consisting of H, CH3, OH, OCH3, 3,4-
dihydroxyphenylmethylene, p-hydroxyphenylmethylene and other substituted
phenyl groups preferably with acyloxy or halogen substituents; R'3, R'4, R'5, R"1
and R"2 are selected from the group consisting of H, OH, OCH3, amino, cyano,
alkyl or alkylamino groups of 1 to 6 carbon atoms, acyloxy, halogens, .ang.-azido,
aziridine, acyloxy and halogen substituted derivatives.

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19. The method of Claim 18, wherein said compound is selected from the
group consisting of the formulae:
<IMG> <IMG>
and
Wherein R1, and R4 are H; OH, acyloxy or halogens, R2 and R3 are OH,
OCH3, arnino, alkyl or aLkylamino groups of 1 to 6 carbon atoms or halogens, R'1,
and R'2 are H, CH3, or phenylmethylene having p-hydroxy, 3,4-dihydroxy,
acyloxy and halogen ring subtituents and R'3 is H, OH, CH3 ,OCH3 and alkyl
groups from 1-6 carbon atoms.
20. The method of Claim 18, wherein said compound is:
<IMG>
Wherein, R1 and R4 are selected from the group consisting of H and OH;
R2, and R3 are selected from the group consisting of H, OH, OCH3, amino,
alkylamino and alkyl groups containing 1 to 6 carbons; and R'1 and R'2 are
selected from the group consisting of H, CH3, OH, OCH3, 3,4
dihydroxyphenylmethylene, p-hydroxyphenylmethylene and other substituted
phenyl groups.

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21. The method of Claim 18, wherein said compound is
<IMG>
Wherein, R1 and R4 are selected from the group consisting of H and OH;
R2, R3 and R'3 are selected from the group consisting of H, OH, OCH3, amino,
alkylamino and alkyl groups containing 1 to 6 carbons; and R'1 and R'2 are
selected from the group consisting of H, CH3, OH, OCH3, 3,4
dihydroxyphenylmethylene, p-hydroxyphenylmethylene and other substituted
phenyl groups.
22. The method of Claim 18, wherein said compound is:
<IMG>
Wherein R1, R2 and R3 are H, OH, OCH3, amino. alkyl or alkylamino
groups of 1 to 6 carbon atoms, acyloxy and halogens, and R6 is H, or alkyl groupof 1 to 6 carbon atoms.
23. The method of Claim 18, wherein said compound is:
<IMG>
Wherein R1-R4 and R'1-R'5 are H, OH, OCH3, amino, alkyl or alkylamino
groups of 1 to 6 carbon atoms.

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24. The method of Claim 18, wherein said compound is:
<IMG>
Wherein R1-R5, R'1-R'5, R"1 and R"2 are H, OH, OCH3, amino, cyano,
aLkyl or alkylamino groups of 1 to 6 carbon atoms, acyloxy, halogens, .ang.-azido,
aziridine, acyloxy and halogen substituted derivatives.
25. A method of administration of a therapeutic dose of a compound
selected from the group consisting of:
<IMG> , <IMG> ,
<IMG> ,
<IMG> ,
and <IMG>

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Wherein, R1 and R4 are selected from the group consisting of H and OH;
R2, R3 and R6 are selected from the group consisting of H, OH, OCH3, amino,
alkylamino and alkyl groups containing 1 to 6 carbons, acyloxy, and halogens; R5is selected from the group consisting of H, OH, OCH3, acyloxy and halogens; R'1
and R'2 are selected from the group consisting of H, CH3, OH, OCH3, 3,4-
dihydroxyphenylmethylene, p-hydroxyphenylmethylene and other substituted
phenyi groups preferably with acyloxy or halogen substituents; R'3, R'4, R'5, R"1
and R"2 are selected from the group consisting of H, OH, OCH3, amino, cyano,
alkyl or alkylamino groups of 1 to 6 carbon atoms, acyloxy, halogens, .ang.-azido,
aziridine, acyloxy and halogen substituted derivatives.
26. A method for inhibiting the growth of proliferating cells comprising
administration of a biologically inhibiting dose of a compound selected from thegroup consisting of:
<IMG>
, <IMG> ,
<IMG> ,
<IMG> ,
and <IMG>

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Wherein, R1 and R4 are selected from the group consisting of H and OH;
R2, R3 and R6 are selected from the group consisting of H, OH, OCH3, amino,
alkylamino and alkyl groups containing 1 to 6 carbons, acyloxy, and halogens; R5is selected from the group consisting of H, OH, OCH3, acyloxy and halogens; R'1
and R'2 are selected from the group consisting of H, CH3, OH, OCH3,3,4-
dihydroxyphenylmethylene, p-hydroxyphenylmethylene and other substituted
phenyl groups preferably with acyloxy or halogen substituents; R'3, R'4, R'5, R"1
and R"2 are selected from the group consisting of H, OH, OCH3, amino, cyano,
alkyl or alkylamino groups of 1 to 6 carbon atoms, acyloxy, halogens, .ang.-azido,
aziridine, acyloxy and halogen substituted derivatives.
27. A method of treating autoimmune disease, comprising the
administradon of a therapeutic dose of a compound selected from the group
consisting of:
<IMG> , <IMG> ,
<IMG> , <IMG> ,
and <IMG>
Wherein, R1 and R4 are selected from the group consisting of H and OH;
R2, R3 and R6 are selected from the group consisting of H, OH, OCH3, amino,
alkylamino and alkyl groups containing 1 to 6 carbons, acyloxy, and halogens; R5
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is selected from the group consisting of H, OH, OCH3, acyloxy and halogens; R'1
and R'2 are selected from the group consisting of H, CH3, OH, OCH3, 3,4-
dihydroxyphenylmethylene, p-hydroxyphenylmethylene and other substituted
phenyl groups preferably with acyloxy or halogen substituents; R'3, R'4, R'5, R"1
and R"2 are selected from the group consisting of H, OH, OCH3, amino, cyano,
alkyl or alkylamino groups of 1 to 6 carbon atoms, acyloxy, halogens, .ang.-azido,
aziridine, acyloxy and halogen substituted derivatives.
28. The method of Claim 27, wherein said compound is selected from the
group consisting of the formulae:
<IMG> <IMG>
and
Wherein R1, and R4 are H; OH, acyloxy or halogens, R2 and R3 are OH,
OCH3, amino, alkyl or alkylamino groups of 1 to 6 carbon atoms or halogens, R'1
and R'2 are H, CH3, or phenylmethylene having p-hydroxy, 3,4-dihydroxy,
acyloxy and halogen ring subtituents and R'3 is H, OH, CH3,OCH3 and alkyl
groups from 1-6 carbon atoms.
29. The method of Claim 27, wherein said compound is:
<IMG>
Wherein R1, and R4 are H; OH, acyloxy or halogens, R2 and R3 are OH,
OCH3, amino, alkyl or alkylamino groups of 1 to 6 carbon atoms or halogens, R'1,and R'2 are H, CH3, or phenylmethylene having p-hydroxy, 3,4-dihydroxy,
acyloxy and halogen ring subtituents and R'3 is H, OH, CH3,OCH3 and alkyl
groups from 1-6 carbon atoms.

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30. The method of Claim 27, wherein said compound is
<IMG>
Wherein R1, and R4 are H; OH, acyloxy or halogens, R2 and R3 are OH,
OCH3, amino, alkyl or alkylamino groups of 1 to 6 carbon atoms or halogens, R'1,and R'2 are H, CH3, or phenylmethylene having p-hydroxy, 3,4-dihydroxy,
acyloxy and halogen ring subtituents and R'3 is H, OH, CH3 ,OCH3 and alkyl
groups from 1-6 carbon atoms.
31. The method of Claim 27, wherein said compound is:
<IMG>
Wherein R1, R2 and R3 are H, OH, OCH3. amino, alkyl or alkylamino
groups of 1 to 6 carbon atoms, acyloxy and halogens, and R6 is H, or alkyl groupof 1 to 6 carbon atoms.
32. The method of Claim 27, wherein said compound is:
<IMG>

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Wherein R1-R4 and R'1-R's are H, OH, OCH3, amino, alkyl or alkylamino
groups of 1 to 6 carbon atoms.
33. The method of Claim 27, wherein said compound is selected from the
group consisting of:
<IMG>
Wherein R1-Rs and R'1-R's, R"1 and R"2 are H, OH, OCH3, amino, cyano,
alkyl or alkylamino groups of 1 to 6 carbon atoms, acyloxy, halogens, .ang.-azido,
aziridine, acyloxy and halogen substituted derivatives.
34. The method of any of claims 27-33 wherein said disease is selected
from the group consisting of rheumatoid arthritis, multiple sclerosis, myasinia
gravis, diabetes mellitus, thyroiditis, uveoretinitis, systemic lupus erythemytosus,
Sjorgins Syndrone, autoimmune skin diseases and graft versus host disease and
organ and tissue transplant rejections.
35. A method of treating cancer, comprising the step of administering a
therapeutic dose of a compound selected from the group consisting of: 2-
(hydroxybenzylidene)-5-methyl-cyclopentanone (MV-1), 2,6-bis(4-
hydroxybenzylidene)-4-methyl-cyclohexanone (MV-17), 2,6-bis(3,4-
dihydroxybenzylidene)-4-methyl cyclohexanone (MV-18), 4-hydroxy-?-methyl-?-
nitrostyrene (MV-N1), 3,4-dihydroxy-?-methyl-?-nitrostyrene (MV-N3), aurone
(MV-19), 4'-hydroxyaurone (MV-20), 3',4-dihydroxyaurone (MV-21), 2'-
hydroxychalcone (MV-72), 2',4',4-trihydroxychalcone (RV-40), 4-
hydroxychalcone (RV-73), 4,4'-hydroxychalcone (MV-88), .alpha.-azido-2'-
hydroxychalcone (MV-35), 3,4-dihydroxy-4'-cyanochalcone (MV-46) and 4'-
cyanochalcone (MV47).

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36. A method for inhibiting the growth of proliferating cells which
include a Type II nuclear estrogen binding site comprising the step of
administering a biologically inhibiting dose of a compound selected from the
group consisting of: 2-(hydroxybenzylidene)-5-methyl-cyclopentanone (MV-1),
2,6-bis(4-hydroxybenzylidene)-4-methyl-cyclohexanone (MV-17), 2,6-bis(3,4-
dihydroxybenzylidene)-4-methyl cyclohexanone (MV-18), 4-hydroxy-?-methyl-?-
nitrostyrene (MV-N1), 3,4-dihydroxy-?-methyl-?-nitrostyrene (MV-N3), aurone
(MV-19), 4'-hydroxyaurone (MV-20), 3',4-dihydroxyaurone (MV-21),2'-
hydroxychalcone (MV-72), 2',4',4-trihydroxychalcone (RV-40),4-
hydroxychalcone (RV-73), 4,4'-hydroxychalcone (MV-88), .ang.-azido-2'-
hydroxychalcone (MV-35), 3,4-dihydroxy-4'-cyanochalcone (MV-46) and 4'-
cyanochalcone (MV-47).
37. A method for treating benign prostatic hyperplasia, comprising the
step of administering a therapeutic dose of a compound selected from the group
consisting of: 2-(hydroxybenzylidene)-5-methyl-cyclopentanone (MV-1),2,6-
bis(4-hydroxybenzylidene)-4-methyl-cyclohexanone (MV-17), 2,6-bis(3,4-
dihydroxybenzylidene)-4-methyl cyclohexanone (MV-18), 4-hydroxy-?-methyl-?-
nitrostyrene (MV-N1),3,4-dihydroxy-?-methyl-?-nitrostyrene (MV-N3), aurone
(MV-19), 4'-hydroxyaurone (MV-20), 3',4-dihydroxyaurone (MV-21),2'-
hydroxychalcone (MV-72), 2',4',4-trihydroxychalcone (RV-40), 4-
hydroxychalcone (RV-73), 4,4'-hydroxychalcone (MV-88), .ang.-azido-2'-
hydroxychalcone (MV-35), 3,4-dihydroxy-4'-cyanochalcone (MV-46) and 4'-
cyanochalcone (MV-47).
38. A method for inhibiting the growth of proliferating cells comprising
administration of a biologically inhibiting dose of a compound selected from thegroup consisting of: 2-(hydroxybenzylidene)-5-methyl-cyclopentanone (MV-1),
2,6-bis(4-hydroxybenzylidene)-4-methyl-cyclohexanone (MV-17), 2,6-bis(3,4-
dihydroxybenzylidene)-4-methyl cyclohexanone (MV-18), 4-hydroxy-?-methyl-?-
nitrostyrene (MV-N1),3,4-dihydroxy-?-methyl-?-nitrostyrene (MV-N3), aurone
(MV-19), 4'-hydroxyaurone (MV-20), 3',4-dihydroxyaurone (MV-21),2'-
hydroxychalcone (MV-72), 2',4',4-trihydroxychalcone (RV-40),4-
hydroxychalcone (RV-73), 4,4'-hydroxychalcone (MV-88), .ang.-azido-2'-

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hydroxychalcone (MV-35), 3,4-dihydroxy-4'-cyanochalcone (MV-46) and 4'-
cyanochalcone (MV-47).
39. A method of treating autoimmune disease, comprising the
adtninistration of a therapeutic dose of a compound selected from the group
consisting of: 2-(hydroxybenzylidene)-5-methyl-cyclopentanone (MV-1), 2,6-
bis(4-hydroxybenzylidene)-4-methyl-cyclohexanone (MV-17), 2,6-bis(3,4-
dihydroxybenzylidene)-4-methyl cyclohexanone (MV-18), 4-hydroxy-?-methyl-?-
nitrostyrene (MV-N1), 3,4-dihydroxy-?-methyl-?-nitrostyrene (MV-N3), aurone
(MV-19), 4'-hydroxyaurone (MV-20), 3',4-dihydroxyaurone (MV-21),2'-
hydroxychalcone (MV-72), 2',-1',4-trihydroxychalcone (RV-40), 4-
hydroxychalcone (RV-73), 4,4'-hydroxychalcone (MV-88), a-azido-2'-
hydroxychalcone (MV-35), 3,4-dihydroxy-4'-cyanochalcone (MV-46) and 4'-
cyanochalcone (MV-47).
40. A method for treating autoimmune disease comprising the step of
administering a biologically inhibiting dose of a compound selected from the
group consisting of methyl p-hydroxyphenyllactate, analogues of methyl p-
hydroxyphenyllactate, chemical derivatives of methyl p-hydroxyphenyllactate,
and chemically related compounds to the proliferating cells.

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41. The method of claim 40, wherein said compound is selected from
the group consisting of the formulae:
<IMG> , <IMG> , <IMG> ,
<IMG> , <IMG> , <IMG> ,
<IMG> , <IMG> , <IMG> ,
<IMG> and <IMG>
wherein, R1 is selected from the group consisting of H, alkyl groups
containing 1 to 6 carbons and aryl groups; R2 and R3 are selected from the groupconsisting of H, OH, OCH3; and R4 selected from the group consisting of H and a
alkyl group of 1 to 6 carbons.

Description

0 91/17749 PCI-/VS91/03130
20s4208
GROWTH INHIBITORS AND METHODS OF TREATING CANCER AND
CELL PROLIFERATIVE DISEASES
Field of the Invention
This invention relates to the use of new and useful bioflavonoid
compounds and related compounds which include methyl p-hydroxyphenyllactate
(MeHPLA), its analogs, chemical derivatives and chemically related compounds,
phenylmethylene ketones, nitroallcenes, aurones, and chalcones as antitumor
1 0 agents, inhibitors of proliferative cell growth and immunosuppressive agents.
Background of the Invention
There are two types of nuclear estrogen binding sites in normal and
1 5 malignant tissues. Type I sites represent the classical estrogen receptor and
nuclear Type II sites appear to mediate a specific nuclear response to estrogenic
hormones. After estrogen adrninistration, Type I receptor sites bind estradiol and
this receptor-estrogen complex interacts with nuclear acceptor sites before the
initiation of ~he transcriptional events that are associated with estrogen stimulation
2 0 of tissue growth. In contrast, Type II sites bind estrogen with a higher capacity
and a lower affinity than the classical estrogen receptor and do not appear to be
translocated from the cytoplasm to the nucleus. Thus, although the levels of
nuclear Type II sites are increased by estrogen administration, Type II sites remain
in the cytoplasm after hormone adrninistration. Nuclear Type II sites appear to
2 5 mediate a specific nuclear response to estrogenic horrnones and are highly
correlated with uterine cellular hypertrophy and hyperplasia. Additionally,
nuclear Type II sites are highly stimulated in malignant tissues such as mouse
mammary tumors and human breast cancer. This observation is consistent with
the findings that highly proliferative tissue has an increased number of nuclearO Type II sites. Because the stimulation of nuclear Type II sites is closely co~elated
with the stimulation of uterine growth, it has been postulated that the Type II sites
are the location for the mechanisms by which estrogens cause uterotropic
stimulation. Furthermore, the presence of Type II sites on the nuclear matrix
suggests a potential role in the regulation of DNA synthesis.
~5

WO91/17749 84'~8 -2- PCr/US91/03~1)
Nuclear Type II sites are constituents of many, if not all, non-malignant
cells. Normally, Type II sites in non-malignant cells are occupied by methyl
hydroxyphenyllactate (MeHPLA or methyl 3-(4-hydroxyphenyl)-2-
hydroxypropionate). Methyl p-hydroxyphenyllactate (MeHPLA) is an
5 endogenous ligand for nuclear type II binding sites in normal and malignant cells,
as well as in Iymphocyte cells of the immune system, and this compound regula~escell growth and prolife ation through this binding interaction. MeHPLA may be
derived endogenously from bioflavonoid ~Griffiths and Smith (1972) Biochem, J.,
128:901) and/or tyrosine metabolism ~Karoum (1985) Biogenic Amines 2:269).
1 0 Additionally, MeHPLA is metabolized by malignant cells, and the resulting
deficiency of this compound in tumors is directly correlated with the loss of cell
growth regulation. When MeHPLA binds to Type II sites, cell growth and
proliferation of non-malignant tissues are slowed down or stopped. Conversely,
malignant cells metabolize MeHPLA and, thus, there is insufficient binding to the
1 5 nuclear Type II sites and the regulation of cell growth and proliferation is lost.
Consequently, all tumor cell populations exarnined have very high levels of
unbound nuclear Type II sites. This same metabolic activity is herein proposed as
the probable mechanism wherein the compounds of the present invention regulate
the cell proliferative activity in the immune system. These sites should represent
2 0 targets for the analogs of MeHPLA as anti-proliferative agents.
This invention discloses compounds which are not metabolized by
malignant or other rapidly proliferating cells such as those of the immune system
but which bind to nuclear Type U sites with high affinisy. These compounds are
25 very effective inhibitors of tumor cell proliferation, DNA synthesis and
lymphocyte activa~ion. Therefore, the compounds of the present invention are
also useful as immunosuppressive agents. Nuclear Type II sites have been
observed in a variety of tumor and other proliferating cells such as those of she
immune system. In addition, analogs and chemically related compounds such as
3 0 phenylmethylene ketones, nitroalkenes, aurones, and chalcones are effective
inhibitors of a broad spectrum of tumors and other rapidly proliferating cells such
as activated Iymphocytes. These compounds will be therapeutically effective in
treating a wide vaTiety of autoimmune diseases, as well as other pathological
conditions of the irnrnune system wherein inhibition of cell proliferation is
5 desirable or necessary to treat the pathological condition. Consequently, any

~0 91/17749 PCI/US91/03130
3 2 ~ 8 4 2 0 8
tumor which contains nuclear Type II sites should respond to treatment with
MeHPLA, its analogs, such as phenylmethylene ketones, nitroalkenes, aurones,
and chalcones, derivatives and chemically related compounds, including cancers
of the pancreas, cervix, liver, brain, pituitary, prostate and other organ or tissue
5 sites, as well as other cancers, such as leukernias, lymphomas, stromal myomasand leiomyomas, among others. Since MeHPLA also blocks estrogen stimulation
of normal cell growth such as that in the rat uterus (Table I), analogs and
chemically related compounds of MeHPLA are also useful for the treatment of
uterine hyperplasia, cervical hyperplasia, endometriosis and benign prostatic
l 0 hypert~ophy. Because non-proliferating non-malignant cells normally have their
Type II sites bound with MeHPLA, the effects of the proposed compounds on
non-malignant cell populations will be minimal to non-existent. For this reason,MeHPLA, its analogs and chemically related compounds, such as
phenylmeehylene ketones, nitroalkenes, aurones, and chalcones, derivatives and
1 5 chemically related compounds and physiologically acceptable salts thereof are
also useful as prophylactic agents in the inhibition and prevention of cancer,
autoimmune disease, graft vs. host disease and abnormal proliferation of nor~
malignant cells.
The precise physiological role of Type II sites is unknown, but inhibition
of the nuclear Type II sites is associated with antagonism of uterotropic responses
to estrogen. This is true for steroid antagonists such as dexamethasone,
progesterone and triphenylethylene derivatives such as nafoxidine and
clomiphene. While there is at least one endogenous inhibitor of estt~diol binding
to nuclear Type II sites, no specific inhibitors for the nuclear Type II sites had
been identified previous to those identified by some of the inventors of the present
invention. Furtherrnore, the inhibitors of the present invention are specific tonuclear Type II sites and do not interfere with estradiol binding to cytoplasmic or
nuclear Type I estrogen receptors.
The inhibitors are identified as methyl 3-(~ hydroxyphenyl)-2-
hydroxyp,ropionate, its analogs, derivatives and chemically related compounds,
such as phenylmethylene ketones, nitroalkenes, aurones, and chalcones, and
physiologically acceptable salts thereof and are potent regulators of cell growth
and proliferation in normal and malignant tissues, as well as in the regulation of

2 ~ 8 4 ~ ~ PCI /US91/03130
immune function. One inhibitor is also known as methyl p-hydroxyphenyllactate
or MeHPLA. These terms may be used interchangeably. Cell growth inhibition
by these compounds resides in their ability to interact with the high-affinity
nuclear binding sites in normal and malignant cells which may be involved in the5 regulation of cellular proliferation and DNA synthesis. When MeHPLA is bound
to nuclear Type II sius, cell growth and proliferation are inhibited. The
endogenous 3-(4-hydroxyphenyl)-2-hydroxypropionis acid inhibits the cell growth
much less effectively. This activity correlates with its low binding affinity for
nuclear Type II sites.
An additional object of the present invention is a means for the prevention
of cancer. Since MeHPBA is a normal constituent of mammalian cells, but
metaboli~ed by malignant cells, MeHPLA, structural analogs and chemically
related compounds as described herein, including, but not limited to
15 phenylmethylene ketones, nitroalkenes, aurones, and chalcones, derivatives and
chemically related compounds and physiologically acceptable salts thereof, whichare not metabolized by tumors or other rapidly proliferating cells such as those of
the immune system should be useful in the prevention of malignancy and in the
treatment of many pathological conditions of the immune system, including, but
2 0 not limited to autoimmune diseases. These compounds possess little if any side
effects, and if taken in a low level maintenance dose should inhibit the
proliferation of malignant cells, as well as Iymphocytes of the immune system.
Because MeHPLA is such a potent inhibitor of cell growth, this
2 5 compound, as well as its analogs and chemically related compounds were used as
potential antitumor agents. The present invention discloses the potent antitumorand immunosuppressive activity of these compounds.
An object of the present invention is a treatment for cancer.
' ~0
An additional object of the present invention is a procedure to inhibit the
growth of proliferating cells which include a Type II nuclear estrogen binding
site. A further object of the present invention is a method for inhibiting the
growth of estrogen responshe tissues.
~35

,wo 91/1774g PCI/US9t/03130
2~42~8
An additional object of the present inventior~ is the treatment of human
breast cancer, and other malignancies which contain unbound nuclear Type II
sites.
.:
Another object of the present invention is the treatment of benign prostatic
hyperplasia, cervical hyperplasia, uterine hyperplasia and endometriosis.
An additional object of the present invention is to provide an
immunosuppressive agent.
Summary of the Invention
Many anti-cancer drugs possess immunosuppressive- activity (Seldin and
Steinberg (1988) In: "Inflammation Basic Principles and Clinical Correlates",
l 5 (Galin, Goldstein, Snyderman, eds.) Raven Press, Ltd., New York). Many of the
commonly used immunosuppressive agents were originally designed as anti-
cancer drugs. Immunosuppressive drugs have proven to be therapeutically
effective in treating a variety of autoimmune diseases.
An additional object of this invention is the treatment of autoimmune
diseases, including, but not limited to, rheumatoid arthIitis, multiple sclerosis,
myasthenia gravis, diabetes mellitis, thyroiditis, uveoretinitis, systemic lupuserythematosus, Sjorgen's syndrome, autoimmune skin diseases, and others.
2 5 An additional object of the present invention is to provide a treatment for
graft vs. host disease and to prevent transplant rejec~ions.
Thus, in accomplishing the foregoing objects, there is provided iD
accordance with one aspect of the present invention a method of treating cancer
and autoimmune disease comprising the step of adrninistering a therapeutic dose
of MeHPLA, its analogs, chernical derivatives or chemically related compounds.
More specifically the compound is selected from the group consisting of the
formula:

WO 91/17749 PCr/US91/031~0
2084~8 -6-
R3~oRC400R1 R3 OOR1 R2~COOR
R2~R, R3 R~ R2~R
R3'~ R3 R
R2~ and ~ Rl
Wherein, Rl is selected from the group consisting of H, alkyl groups
S containing 1 to 6 carbons, and aryl groups; R2 and R3 are selected from the group
consisting of H, OH and OCH3 and R4 is selected from the group consisting of H,
or aL~cyl group containing 1 to 6 carbons. Preferred compounds which may be
used to practice the present invention may be selected from the group consistingof methyl 3-(4-hydroxyphenyl)-2-hydroxypropionate, n-propyl 3-(4-
10 hydroxyphenyl)-2-hydroxypropionate, n-butyl 3-(4-hydroxyphenyl)-2-
hydroxypropionate, 3-(4-hydroxyphenyl)-2-propenoic acid, 4-(4-hydroxyphenyl~
2-butanone, 1-(4-hydroxyphenyl)-3-pentanone, methyl (4
hydroxyphenoxy)acetate, and methyl 3-(3,4-dihydroxyphenyl)-2-propenoate.
1 5 Another aspect of the invention provides a method of treating cancer and
pathological conditions of the immune system, including, but not limited to,
autoimmune diseases and graft vs. host disease, comprising administeTing a
therapeutic dose of a compound selected from the group consisting of the
formulas:

WO 91/17749 PCl/US91/03130
-7- 2~3~$
~R~ ~R,
Rz~CH--C--R6 ~=CH~R'~
and ~R,
Wherein, Rl and R4 are selected from the group consisting of H and OH;
R2 and R3 are selected from the group consisting of H, OH, OCH3, amino,
5 aLt~ylamino and aLtcyl groups containing 1 to 6 carbons, acyloxy, and halogens; Rs
is selected from the group consisting of H, OH, OCH3, acyloxy and halogens; R'
and R'2 are selected from the group consisting of H, OH, OCH3, amino, cyano,
alkylamino groups of I to 6 carbon atoms, acyloxy, halogens and a-azido and
aziridine derivatives, 3,4-dihydroxyphenylmethylene, p-hydroxyphenylmethylene
1 0 and other substituted phenyl groups preferably with acyloxy or halogen
substituents; R'3, R'4, R's, R"1 and R"2 are selected from the group consisting of H,
OH, OCH3, amino, cyano, aL~cylamino groups of 1 to 6 carbon atoms, acyloxy,
halogens and a-azido and aziridine derivatives and halogen substituted derivatives.
When the chalcone R"l and R"2 is substituted to form an aziridine ring system, a1 5 three membered ring structure comprising the R"l and R"2 carbons and a nitrogen
atom is formed. Prefe~ed compounds which may be used to practice the present
invention may be selected from phenylmethylene ketones, nitroalkenes, aurones
and chalcones.

WO 91/17749 PCltUS91/031~Q
2~8~2~ . ,
Phenylmethylene ketones most preferred for practicing the present
invention include
R' ~2 R-2
R4 R~3 R4
s
Wherein Rl, and R4 are H; OH, acyloxy or halogens, R2 and R3 are OH,
OCH3, amino, alkyl or alkylamino groups of 1 to 6 carbon atoms or halogens, R~
and R'2 are H, CH3, or phenylmethylene having p-hydroxy, 3,4-dihydroxy,
acyloxy and halogen ring subtituents and R'3 is H, OH, CH3 ,OCH3 and aL'cyl
10 groups from 1-6 carbon atoms.
Most preferably the phenylmethylene ketones which may be used to
practice the present invention are selected from the group consisting of
MV-1
CH3 ~ ~ --~`
OHHO CH, OH
MV-1
MV-17
HO~OH
CH3
l 5 MV-18

~vo 91/17749 PSr/VS91/03130
~9~ 2084208
Nitroalkenes most preferred for practicing the present invention include
Rl
R2 ~CH =C--R6
R3 NO2
S Wherein Rl, R2 and R3 are H, OH, OCH3, amino, allcyl or aL~cylamino
groups of 1 to 6 carbon atoms, acyloxy and halogens, and R6is H, or alkyl gTOUp
of 1 to 6 carbon atoms.
Among the nitroalkenes most preferably used to practice the present
l 0 invention are: -
HO
Ho~3CH= f_ CH3 H~CH=f_ CH3
NO2 NO2
MV-N1 MV-N3
Among the aurones most preferable for practicing the present invention
are:
Rl R1 ~ 2
RR~,~=CH~R',
R4
Wherein Rl-R4 and R'l-R's are H, OH, OCH3, amino, aL~cyl or alkylamino
groups of 1 to 6 carbon atoms.
The most preferable aurones useful in practicing the present invention are
MV-19, MV-20 and MV-21.

WO91/17749 2~84Z08 PCI/VS91/03130
-10-
O~H~ ~=CH~ OH
MV-20
MV-19
OH
and~=CH~OH
MV-21
Among the chalcones most preferable for practicing the present invention
are:
s
~ ~R3
Wherein R1-Rs, R'l-R's, R"l and R"2 are H, OH, OCH3, amino,cyano,
alkyl or allcylamino groups of 1 to 6 carbon atoms, acyloxy and halogens and a-
10 azido and aziridine derivatives.

710 91/17749 , . PCI/US91/03130
The most preferable chalcones useful in practicing the present invention
are:
~3 HO ~ OH
O
2'-hydroxychalcone 2',4',4-trihydroxychalcone
~3OH HO ~--OH
o
4-hydroxychalcone
4-4'-dihydroxychalcone
N C ~--410H
o ~ o
MV-35 MV-46
and
MV-47
Another aspect of the invention involves the inhibition of the growth of
proliferating eells which include a Type II nuclear estrogen binding site by thestep of administeIing a biological inhibiting dose of MeHPLA, its analogs,
chemical derivatives or chemically related compounds such as phenylmethylene
1 0 ketones, nitroalkenes, analogs and chalcones to the proliferating cells.
An additional aspect of the present invention is the inhibition of the
proliferative growth of estrogen responsive tissues such as uterus, mammary
gland, uterine tumors and mammary tumors. In one specific aspect, the abov~

WO 91/17749 pcr/us91/o3l3o
2~)84208 -12-
mentioned compounds have been used for the treatment of human breast cancer
cells. The compounds inhibit the growth of human breast cancer cells.
An additional aspect of the present invention is the provision of an
5 immunosuppressive agent. In one aspect these compounds are selected from the
group consisting of MeHPLA, its analogs, derivatives and chemically related
compounds including, but not limited to, phenylmethylene ketones, nitroalkenes,
aurones, and chalcones, and physiologically acceptable salts thereof.
1 0 Another specific aspect of the present invention is a method for treating
benign prostatic hyperplasia comprising the step of administering a therapeutic
dose of MeHPLA, its analogs, derivatives and chemically related compounds
including, but not limited to, phenylmethylene ketones. nitroalkenes, aurones, and
chalcones, and physiologically acceptable salts thereof.
Another specific aspect of the present invention is a method for treating
autoimmune diseases, including, but not limited to, rheumatoid arthritis, multiple
sclerosis, myasthenia gravis, diabetes mellitis, thyroiditis, uveoretinitis, systemic
lupus erythematosus, Sjogren's syndrome, autoimmune skin diseases, and others
2 0 comprising the step of administering a therapeutic dose of MeHPLA, its analogs,
derivatives and chemically related compounds including, but not limited to,
phenylmethylene ketones, nitroalkenes, aurones, and chalcones, and
physiologically acceptable salts thereof.
2 5 Another specific aspect of the present invention is a method for treating
autoimmune diseases, including, but not lirnited to, rheumatoid arthritis, multiple
sclerosis, myasthenia gravis, diabetes mellitis, thyroiditis, uveoretinitis, systemic
lupus erythematosus, Sjogren's syndrome, autoimmune skin diseases, and others
comprising the step of administering a therapeutic dose of a compound selected
:;0 from the group consisting of

~0 91/17749 PCr/US91/03130
-13- 208~208
o~OOR1 R2X~CR R2~COOR,
R2~R, R3 R~ R2XD~R,
R3 O~,COOR1 2~0~CH20R
R3 and R2 Rl
Wherein, Rl is selected from the group consisting of H, alkyl groups
5 containing 1 to 6 carbons, and aryl groups; R2 and R3 are selected from the group
consisting of H, OH and OCH3 and R4 is selecud from the group consisting of H,
or alkyl group containing 1 to 6 carbons. Preferred compounds which may be
used to practice the present invention may be selected from the group consistingof methyl 3-(4-hydroxyphenyl)-2-hydroxypropionate, n-propyl 3-(4-
l 0 hydroxyphenyl)-2-hydroxypropionate, n-butyl 3-(4-hydroxyphenyl)-2-
hydroxypropionate, 3-(4-hydroxyphenyl)-2-propenoic acid, 4-(4-hydroxyphenyl~
2-butanone, 1-(4-hydroxyphenyl)-3-pentanone, methyl (4
hydroxyphenoxy)acetate, and methyl 3-(3,4-dihydroxyphenyl)-2-propenoate.
and
,~ .

WO 91/17749 PCI/US9t/03~0
208 ~2~8 -14-
R2~ R' ~RZ
R4 ' R~3 R4
R2 ~CH : C--R6 ~=CH~R~3
R4 R~ R, R2
R ~Rs
Wherein, Rl and R4 are selected from the group consisting of H and OH;
R2, R3 and R6 are selected from the group consisting of H, OH, OCH3, amino,
5 alkylamino and alkyl groups containing 1 to 6 carbons, acyloxy, and halogens; Rs
is selected from the group consisting of H, OH, OCH3, acyloxy and halogens; R'
and R'2 are selected from the group consisting of H, CH3 OH, OCH3, 3,4-
dihydroxyphenylmethylene, p-hydroxyphenylmethylene and other substituted
phenyl groups preferably with acyloxy or halogen substituents; R'3, R'4, R's, R" I
1 0 and R"2 are selected from the group consisting of H, OH, CH3 and OCH3, acyloxy
and halogen substituted derivatives. PrefeIred compounds which may be used to
pMctice the present invention may be selected from phenylmethylene ketones,
nitroalkenes, aurones and chalcones.
1 5 Another aspect of the present invention is the provision of an ansitumor
agent which comprises analogs of MeHPLA, including, but not limited to,
phenylmethylene ketones, nitroalkenes, aurones, and chalcones, derivatives and
chemically related compounds and physiologically acceptable salts thereof.

U~O 91/17749 PCT/US91/03130
-15- ~ 2~8 ~
Another aspect of the present invention is the provision of an
immunosuppressive agent which comprises analogs of MeHPLA, including, but
not limited to, phenylmethylene ketones, nitroalkenes, aurones, and chalcones,
derivatives and chetnically related compounds and physiologically acceptable salts
5 thereof.
Another aspect of the present invention is prophylactic agents to inhibit
and prevent cancer, autoimmune disease, graft versus host disease and non~
malignant cell growth. These prophylactic agents include the above-mentioned
1 0 MeHPLA, its analogs, including but not limited to, phenylmethylene ketones,
nitroalkenes, aurones, and chalcones, chemical derivatives or chemically relatedcompounds and pharmaceutically acceptable salts thereof.
Other and further objects, features and advantages will be apparent from
l 5 the following description of the presently preferred embodiments of the invention
given for the purpose of disclosure.
Brief Description of the Figures
Figure I represents the competition of MV-3, MV-12 and MV-88 for
[3H]estradiol binding to nuclear Type II sites.
Figure 2 represents the analysis of nuclear type II binding sites in popliteal
Lymph node nuclei.
Figure 3 represents the effects of MV-3, MV-12, and MV-88 on MCF-7
human breast cancer cell proliferation.
Figure 4 represents the effects of the compounds of the present invention
3 0 on Mouse Mammary tumor growth in vivo.
Figure 5 represents the effects of MV-19, MV-20 and MY-21 on mouse
mammary tumor growth in vivo.

~ - WO 9t~17749 PCI/US91/03L30
( ,,
2,~842~8 -16-
Figure 6 demonstrates the effects of cyclodextrin encapsulated MV-88 on
mouse mammary tumor growth in vivo.
Figure 7 demonstrates the inhibition of the development of autoimmune
5 uveoretinitis.
Detailed Description of the Specific Embodiment
De~lnitions.
"Methyl 3-(4-hydroxyphenyl)-2-hydroxypropionate" is also known as
methyl p-hydroxyphenyllactate or MeHPLA. The term "MeHPLA" is meant to
also include its analogs, chemical derivatives, and chemically related compoundswhich bind to the nuclear Type II receptors and by so doing inhibit cell
l 5 proliferation.
The terrn "chemically related compounds" refers to the derivatives and
analogs of p-coumaric acid, p-hydroxyphenylbutanone, (~
hydroxyphenoxy)acetate and the arylpropenaldehydes, alkyl arylethenyl ketones,
2 0 aryl arylethenyl ketones, aryl butenaldehydes, alkyl arylpropenyl ketones and
arylpropenyl ketones which are structurally related to MeHPLA and disclosed
herein. These chemically related compounds include the cis and trans isomers of
said compounds and their esters, ethers, ketones and derivatives containing S or N
in place of O atoms. More specifically these structurally related analogs and
2 5 derivatives include compounds where Rl represents the methyl, ethyl, n-propyl, n-
butyl, isopropyl, tert-butyl or aryl group and R2 and R3 represent H, OH or OCH3groups and R4 is H or an alkyl group of 1 to 6 carbons. Specific analogs of eachclass of these structurally related compounds to MeHPLA have been
demonstrated to possess biological activity (Tables I and II) as defined herein and
:~ O therefore mimic MeHPLA as an effective inhibitor of cell proliferation, tumor cell
growth and as irnmunosuppressive agents. Preferably, the analogs and chemically
related compounds effective in practicing the present invention include, but arenot limited to, phenylmethylene ketones, nitroalkenes, aurones, and chalcones.
Most preferably, these analogs are selected from the group consisting of the
:~5 general fo~nulas:

WO 91/17749 PCI'/US91/03130
-17- ' 2~)g~20~
R3~ORC~OOR' R2~,COOR ~COOR
R2~R~ R3X3~R, R3~
RRa2~ O_COOR1 R~ O~CH2OR~ R2~R,
R2~ and R2 R,
Wherein, Rl is selected from the group consisting of H, alkyl groups
containing 1 to 6 carbons, and aryl groups; R2 and R3 are selected from the group
consisting of H, OH and OCH3 and R4 is selected from the group consisting of H,
or aL~cyl group containing 1 to 6 carbons. Preferred compounds which may be
used to practice the present invention may be selected from the group consistingof methyl 3-t4-hydroxyphenyl)-2-hydroxypropionate, n-propyl 3-(4-
hydroxyphenyl)-2-hydroxypropionate, n-butyl 3-(4-hydroxyphenyl)-2-
hydroxypropionate, 3-(4-hydroxyphenyl)-2-propenoic acid, 4-(4 hydroxyphenyl~
2-butanone, 1-(4-hydroxyphenyl)-3-pentanone, methyl (4
hydroxyphenoxy)acetate, and methyl 3-(3,4-dihydroxyphenyl)-2-propenoate.
Another aspect of the invention provides a method of treating cancer and
pathological conditions of the immune system, including, but not limited to,
autoimmune diseases and graft vs. host disease, comprising administering a
therapeutic dose of a compound selected from the group consisting of the
2 0 formulas:

- wo 9l/!7749 ~ 42~ PCI~/US91/03130
-18-
~ , ~[R,
N2 , ~=CH~ R~3
and ~RJ
Wherein, Rl and R4 are selected from the group consisting of H and OH;
5 R2 and R3 are selected from the group consisting of H, OH, OCH3, amino,
alkylamino and alkyl groups containing 1 to 6 carbons, acyloxy, and halogens; Rsis selected from the group consisting of H, OH, OCH3, acyloxy and halogens; R'
and R'2 are selected from the group consisting of H, CH3 OH, OCH3, 3,4-
dihydroxyphenylmethylene, p-hydroxyphenylmethylene and other substituted
l 0 phenyl groups preferably with acyloxy or halogen substituents; R3, R'4, R's, R"
and R"2 are selected from the group consisting of H, OH, OCH3, amino, cyano,
alkylamino groups of 1 to 6 carbon atoms, acyloxy, halogens and a-azido and
a~idine derivatives, acyloxy and halogen substituted derivatives. Preferred
compounds which may be used to practice the present invention may be selected
1 5 from phenylmethylene ketones, nitroaLIcenes, aurones and chalcones.

WO 91/17749 PCI`/US91/03130
, ~ -19- ` 20842~8
Phenylmethylene ketones most preferred for practicing the present
invention include
R' ~ R' ~$F~
R~3
Wherein Rl, and R4 are H; OH, acyloxy or halogens, R2 and R3 are OH,
OCH3, amino, allcyl or alkylamino groups of 1 to 6 carbon atoms or halogens, R'l.
and R'2 are H, CH3, or phenylmethylene having p-hydroxy, 3,4-dihydroxy,
1 0 acyloxy and halogen ring subtituents and R'3 is H, OH, CH3 ,OCH3 and aLkyl
groups from 1-6 carbon atoms.
Most preferably the phenylmethylene ketones which may be used to
practice the present invention are selected from the group consisting of
MoV-1
CH, ~OH HO~LOH
CH3
MV-1
MV-17
O
H~OH
CH3
MV-18

wo 91/17749 PCI`/US91/03130
2~& 4~~ -20-
Among the nitroalkenes most preferably used to practice the present
invention are:
HO
HO~CH=C--CH3 H~ ~CH=C--CH3
NO2 NO2
MV-N1 MV-N3
s
Among the aurones most preferable for practicing the present invention
are:
0~H ~ ~CH~ OH
MV-20
MV-19
OH
and~cCH~OH
l O MV-21

WO 91/17749 PCI'/US91/û3130
'` -21- 2084208
Among the chalcones most preferable for practicing the present invention
are:
~3 HO~--OH
2'-hydroxychalcone 2',4',4-trihydroxychalcone
~OH HO~W~OH
4-hydroxychalcone
4-4'-dlhydroxychalcone
NC ~OH
MV-35 MV-46
and
o
MV-47
The terrn "individual" is meant to include animals and humans.
The term "biologically inhibiting" or "inhibition" of the growt'n of
. 1 0 proliferating cells is meant to include partial or total growth inhibition and also is
meant to include decreases in the rate of proliferation or growth of the cells. The
biologically inhibitory dose of the compounds of the present invention may be
determined by assessing the effects of the test compound on malignant cell growth
in tissue cultore (see Figure 3), uterine growth in the animal (see Figures 14 and

WO 91/17749 2 0 8 4 2 0 8 PCr/US91/03130
-22-
15) or tumor growth in the animal as previously described by Markaverich et al.,Cancer Research 43:3208-3211 (1983), or any other method known to thos~ of
ordinary skill in the art. These methods have also been fully described in U.S.
Patent Application No. 219,680 which is incorporated herein by reference as if it
5 appeared in full.
The term 'timrnunosuppresive" or "irnmunosuppressing" or "suppression of
the immune system" is meant to include partial or total immune suppression and is
also meant to include changes in immune function such that "abnorrnal" immune
l 0 functions become more normalized. The imrnunosuppressive dose of the
compounds of the present invention may be deteImined by assessing the effects ofthe test compound in the established rat model of experimental autoimmune
uveoretinitis as described by Gery et al., (1986) Invest. Ophthalmol. Vis. Sci.
~7: 1296, or any other method known to those of ordinary skill in the art.
The term "prophylactic agent" is meant to include agents which may be
used for partial or total inhibition or prevention of disease and the spread of
disease and also is meant to include agents which may be used as a precaution
against disease and for preventive treatment of disease.
Administration of the compounds useful in the method of the present
invention may be by topical, parenteral, oral, intranasal, intravenous,
intramuscular, subcutaneous, or any other suitable means. The dosage
administered is dependent upon the age, weight, kind of concurrent treatment, if2 5 any, and nature of the malignancy or the pathological immune condition. The
effective compound useful in the method of the present invention may be
employed in such forms as capsules, tablets, liquid solutions, suspensions, or
elixirs, for oral administration, or sterile liquid forrns such as solutions,
suspensions or emulsions. Any inert carrier is preferably used, such as saline, or
0 phosphate-buffered saline, or any such ca~ier in which the compounds used in the
method of the present invention have suitable solubility properties.
The compounds of the present invention may be administered in a
biologically effective ca~ier. The biologically effective ca~iers may include any

WO 91~17749 PCr/US91/03130
-23- 2~2~
solvent with which the compounds of the present invention are compatible and
which are non-toxic to the individuals treated at the amounts administered.
Most preferably, the compounds of the present invention are adrninistered
5 as an encapsulated composition. Due to the low aqueous solubility of many of the
compounds effective in carrying out the present invention, another aspect of thepresent invention comprises the drug delivery system of the compounds of the
present invention encapsulated in cyclodextrin, liposomes or as silastic implants.
However, the compounds of the present invention may be encapsulated by other
1 0 methods and with other compounds by methods known to those skilled in the art.
The term "antitumor agent" is meant to include agents which decrease cell
growth, or inhibit the proliferation of tumor cells when administered to said tumor
cells in an effective dose.
One specific embodiment of tnis invention is an antitumor agent including
MeHPLA, its analogs, chemical derivatives or chemically related compounds.
Specific examples of MeHPLA analogs are derivatives of the general formula
Ra ~ ORC4OOR~ R2~ ~ ,COOR, R2 ~ COOR,
R2~R~ R2~R~ R2~ R
R3 O _COOR~ 2~ O~_CH2OR R3~R,
R3 and R3 R~

W O 91/17749 P ~ /VS91/03130
2~84208 -24- t~
Wherein, Rl is selected from the group consisting of H, alkyl groups
containing 1 to 6 carbons, and aryl groups; R2 and R3 are selected from the group
consisting of H, OH and OCH3 and R4 is selected from the group consisting of H,
or alkyl group containing 1 to 6 carbons. Preferred compounds which may be
5 used to practice the present invention may be selected from the group consisting
of methyl 3-(4-hydroxyphenyl)-2-hydroxypropionate, n-propyl 3-(4-
hydroxyphenyl)-2-hydroxypropionate, n-butyl 3-(4-hydroxyphenyl)-2-
hydroxypropionate, 3-(4-hydroxyphenyl)-2-propenoic acid, 4-(4-hydroxyphenyl~
2-butanone, 1-(4-hydroxyphenyl)-3-pentanone, methyl (4
l 0 hydroxyphenoxy)acetate, and methyl 3-(3,4-dihydroxyphenyl)-2-propenoate.
One specific example of this type of compound is wherein Rl is CH3, R2
is H, R3 is OH and R4 is H. This is a naturally occurring endogenous compound
which was isolated and characterized and identified as the present invention.
l 5 Other examples of analogs include the compounds in which the R group has been
replaced by an ethyl, n-propyl, n-butyl, isopropyl, tert-butyl or aryl group; R2and/or R3 have been replaced with an H, OH or OCH3 group and R4 is H or an
alkyl group of 1 to 6 carbons. Each of these esters can exist in the D and L form.
2 0 Another group of derivative compounds includes those with the formula:
R2~, COOR1
R
Exarnples of these compounds are p-coumaric acid, 3-(4-hydroxyphenyl~
25 2-propenoic acid, and its esters. These substances exist as cis and trans isomers.
In coumaric acid Rl and R2 are hydrogen and R3 is OH. Addi~ional esters include
compounds wherein Rl is methyl, ethyl, n-propyl, n-butyl, isopropyl, tert-butyl or
aryl and R2 and/or R3 is a H, OH or OCH3 group. Additional analogs include
caffeic acid, 3-(3,4-dihydroxyphenyl)-2-propenoic acid, wherein R1 is H and R2
0 and R3 are both OH.

~0 91/17749 PCI/US91/03130
-25- 2084208
Other compounds with antitumor activity are the derivatives of 1-(4
hydroxyphenyl)-3-butanone, such as compounds with the formula:
R~, R~ Rl R~ ¢R,
~,~R~ R3 and ~ R~
Wherein, Rl is selected from the group consisting of H, alkyl groups
containing I to 6 carbons, and aryl groups; R2 and R3 are selected from the group
consisting of H, OH and OCH3 and R4 is selected from the group consisting of
1 0 H, or aL~yl group containing I to 6 carbons. Preferred compounds which may be
used to practice the present invention may be selected from the group consistingof methyl 3-(4-hydroxyphenyl)-2-hydroxypropionate, n-propyl 3-(4-
hydroxyphenyl)-2-hydroxypropionate**, n-butyl 3-(4-hydroxyphenyl)-2-
hydroxypropionate, 3-(4-hydroxyphenyl)-2-propenoic acid, 4-(4-hydroxyphenyl~
2-butanone, 1-(4-hydroxyphenyl)-3-pentanone, methyl (4-
hydroxyphenoxy)acetate, and methyl 3-(3,4-dihydroxyphenyl)-2-propenoate.
Ketone derivatives include compounds with a methyl, ethyl, n-propyl, n-
butyl, isopropyl, tert-butyl or aryl group at the Rl position, H, OH or OCH3 group
2 0 at the R2 and R3 positions; and most preferably H at the R2 posidon and OH at the
R3 position.
Additionally, as can be seen by the formulae, the number of CH2 groups
between the aromatic entity and the keto group can be varied. Specific examples
of compounds are 1-(4-hydroxyphenyl)-3-pentanone and 1-(4-hydroxyphenyl)-~
butanone. These compounds have been shown to bind to Type II sites and to have
andtumor and and-proliferative acdvity in the uterotropic assay.

WO 91/t7749 PCT/US91/03130
2~8 4io8 -26-
Another group of compounds which show anti-proliferative activity in the
rat uterus is described by the formula:
R2~ O~,COORl
These compounds are ether-linked analogs of (4-hydroxyphenoxy)acetic
acid, for example, methyl (4-hydroxyphenoxy)acetate. All of these compounds
bind to the Type II binding sites. These ether-linked compounds include analogs
wherein Rl is H, a Cl to C6 alkyl carbon chain or an aryl group, R2 and R3 are H,
10 OH or OCH3. An additional variation on the phenoxy compounds include the
çther compounds, for example, 2-(4-hydroxyphenoxy) ethyl methyl ether, wherein
Rl can be H or any Cl to C6 alkyl carbon chain or an aryl group, R2 and R3 are H,
OH or OCH3 in the formula:
R2~,~ O~,CH20R
R3'W
Additionally useful is the compound of the formula:
R:~ COOR
Wherein Rl is a Cl to C6 alkyl chain or an aryl group, R2 and R3 are H,
OH or OCH3.

WO 91/17749 PCr/VS91/03130
-27- 2~8420~
Another group of compounds which show tumor anti-proliferative action is
described by the general formulas:
F~2 122~ R2~ R~
Wherein R~ is from the group consisting of H, aLlcyl groups containing 1 to
6 carbons, and substituted or unsubsdtuted aryl groups; and R2 and R3 are selected
from the group consisting of H, OH and OCH~ Preferred compounds of this
group which may be used to pracdce the present invention are:
~ Ho~3--OH
Z-hydroxychalcone 2',4',4-trihydroxychalcone
~3OH HO~OH
o
4-hydroxychalcone 4-4~-dihydroxychalcone
OH
¢~ NC ~OH
O ~ O
MV-35 MV-46
NC
MV-47

WO 91/17749 PCI/I 'S91/03130
208420~ -28-
Most preferred compounds of this group for practicing the present
invention are 3-(4-hydroxyphenyl)-l-phenyl-2-propen-1-one and 4-~4
hydroxyphenyl)-3-buten-2-one, analogs, chemical derivatives and chemically
related compounds and pharmaceutically acceptable salts thereof.
Another embodiment of the present invention includes a method for
treating cancer comprising the step of administering a therapeutic dose of
MeHPLA, its analogs, chernical derivatives or chemically related compounds.
This compound, can be any of the above-described antitumor compounds.
In addition to being used as a treatrnent for cancer, these antitumor agents
are also useful as inhibitors of cell growth and proliferation in those cells which
include a Type II nuclear estrogen binding site.
1 5 These compounds bind to Type II nuclear estrogen binding sites and
regulate cell growth. Specific proliferating cells which are sensitive to the binding
of these compounds include estrogen responsive tissues such as uterus, marnmary
gland, uterine tumors and mammary tumors. The above-described compounds
inhibit the proliferative capacity of human breast cancer cells and thus provide an
2 0 effective therapy for this disease. Benign prostatic hyperplasia is another example
of a proliferative tissue disease in which the above-described compounds can
successfully be used in the treatment.

WO 91/17749 PCl/US91/03~30
29 . I
Another group of compounds which show tumor anti-proliferative and
immunosuppressive action is described by the general formulas:
R3~COOR1 F~COORl R3~CooR,
R2~R, F~ R~ R3~
R3,~3,0~C00~, R~3~o~cH2oR~ 2~,
R2~ and R2~ R1
Wherein Rl is from the group consisting of H, alkyl groups containing 1 to
6 carbons, and substituted or unsubstituted aryl groups; and R2and R3 are selected
from the group consisting of H, OH and OCH~ Preferred compounds of this
10 group which may be used to practice the present invention are:
",

WO 91/17749 PCI/US91/03130
208420~ -30-
~ HO ~ OH
2'-hydroxychalcone 2',4',4-trihydroxychalcone
~3OH H~30H
O O
4-hydroxychalcone
4-4'-dlhydroxychalcone
NC ~HOH
O ~ O
MV-3~ MV-46
and
O
MV-47
:`
Most preferred compounds of this group for practicing the presen~ invention are
2'-hydroxychalcone, 2',4',4,-trihydroxychalcone, 4-hydroxychalcone, 4-4'
dihydroxychalcone, MV-35, MV-46, and MV-47, analogs, chernical derivatives
and chemically related compounds and pharmaceutically acceptable salts thereof.
Another embodiment of the present invention includes a method for
1 0 treating autoimmune diseases comprising the step of administering a therapeutic

Vl O 91/1774g PCI /US91/03130
-31- 2~8`4208
dose of MeHPLA, its analogs, including, but not limited to, phenylmethylene
ketones, nitroalkenes, aurones, and chalcones, chemical derivatives or chemically
related compounds. This compound, can be any of the above-described antitumor
or imrnunosuppressive compounds.
Example 1
[3H]Estradiol Binding Assay
l 0 A variety of rat tissues possess an endogenous ligand which blocks
[3H]estradiol binding to Nuclear Type II estrogen binding sites; however, this
compound does not interfere with [3H]estradiol binding to the estrogen receptor.Uterine tissue from estradiol-implanted rats was dissected from host anirnals. The
uterine nuclei were prepared as previously described in Markaverich, B. M. et al.,
l 5 J. Biol. Chem. 258: 11663-11671 (1983), the disclosure of which is herein
incorporated by reference. Uterine nuclei were prepared from estrogen-implanted,adult-ovariectomized rats. The washed nuclear pellet was diluted to 10 mg of fresh
uterine equivalents/ml. At this concentration the effects of the endogenous
inhibitor were rninimal, and Nuclear Type II sites bound maximum quantities of
2 0 [3H]estradiol. Aliquots of these nuclei and various concentrations of the
compounds of the present invention were incubated at about 4C for
approximately 60 min in the presence of 40 mM of [3H]estradiol with and without
12 uM diethylstilbestrol. Under these conditions, nuclear Type II sites were
quantitatively measured without interference from Type I sites. The nuclear
pellets were resuspended in 1 ml of 10 mM Tris-1.5 mM EDTA and centrifuged,
ethanol extracted, and counted. The results were expressed as the percentage of
~3H~estradiol bound as compared to the buffer control, or as the percentage of
inhibition where 100% bound was 0% inhibition and was equivalent to
approximately 45,000 cpm. Figure 1 demonstrates representative data showing
0 that concentrations of these compounds above lOnM competed for the
[3H]estradiol Nuclear Type II sites. The results represented by Figure 1 r present
the mean + standard error of the mean for triplicate detemlinations in four
replicate experiments for each preparation.
.

WO 91/17749 PCI/US91/03130
2~8~0~ -32- !
Binding assays have shown that the mouse mamrnary tumor and human
breast cancer preparations had high levels of free nuclear Type II sites relative to
non-malignant dssues. Normal rat mammary glands contain very high levels of
total inhibitor activity relative to mouse mammary tumors. Human breast cancer
5 contains low levels of inhibitor. Thus, the evidence shows that malignant tissues
have high levels of free nuclear Type II sites and are deficient in the inhibitor
activity. This deficiencv in inhibitor activity explains the high levels of freenuclear Type II sites observed in these tumor tissue populations as well as their
rapid rate of proliferation, cell growth and DNA synthesis.
It has been previously demonstrated (U.S. Patent Application No. 079,199,
incorporated herein by reference~ that two endogenous inhibitors (a and J) are
prescnt in no~nal dssues such as rat uterus an~ normal mouse mammary gland. A
high correlation between a deficiency in the J-peak component and increased
1 5 unbound Nuclear Type II sites was also demonstrated in rat, mouse and human
mamrnary tumors.
The data presented in Figure 1 demonstrate that compounds such as MV-3,
MV-12, and MV-88 bind to nuclear type II binding sites with a very high binding
20 affinity (Kd-1-10 nM), as does MeHPLA. Similar binding inhibition curves wereobtained for all of the compounds presented in Table I, below. The Ki's for the
binding interaction was determined. The Ki is the concentration of drug which
inhibits [3H]estradiol binding to nuclear type II binding sites by 50%. The datafor all compounds presented in Table I show a good correlation between binding
25 affinity and cell growth inhibitory activity.

yvo 91/17749 PCl/US91/03130
~33^ 2~08420~
Table I Effects of MV-Compounds on Type II Binding Sites, MCF-7
Human Breast Cancer Cell Proliferation in ~itro, and Mouse Mammary
Tumor Growth in vivo.
S Compound Type II Sitesa Cell Proliferationb Tumor Gro~vthC
Phenylmethylene Ketones
MV-1 34.00 Inactive Not Tested
l O MV-3 0.06 1.00 1 l I
MV-17 0.500 1.60 Not Tested
MV-18 0.800 1.00 Not Tested
.
NitroaLIcenes
MV-NI ?I0.0 1.40 Not Tested
MV-N3 0.220 0.80 Not Tested
.
Aurones
MV-I9 I0.0 8 00 Negative
MV-20 2.800 1.90 +
MVo2I 0.08 0.78 ++~
Chalcone-Cyclodextrin Complex
MV-88CD NotTested 3.00 l l l
aKi is the concentration (mM) of the compound required to inhibit [3H~estradiol binding to
Nuclear Type II sites by 50% (sec Figure 5) bKi is the concentration (mglml) ~equired to inhibit
3 0 MCF-7 human breast cancer cell proliferation by 50% (see Figure 5). CThis compound inhibited
the growth of well differentiated mouse mamm~y adenocarcinomas in vivo (see Figures 7-9).
In order to test whether Nuclear Type II binding sites were also present in
tissues of the imrnune system, popliteal Iymph node nuclei were prepared and
:~ 5 analysis of Nuclear Type II binding sites were performed by [3Hlestradiol
exchange. Popliteal lysnph nodes were removed from mature female mice,
dissected from ext~aneous tissue, weighed and homogenized in TE buffer (10 rnM
Tris; 1.5 mM EDTA, pH 7.4). The homogenate was centrifuged at 800 x g for 20
.

WO 91/17749 PCI/US91/03130
2~ 4208 t'-
-34-
minutes to obtain the nuclear pel}et. The nuclear pellet was washed three times by
resuspension and centrifugation in TE buffer (800 x g x 7 rninutes), and the final
washed nuclear preparation was diluted to a final volume of 30 mg tissue wet
weight equivalents/ml (8.6 ml) and assayed for Type II binding sites by [3H]
estradiol exchange (Markaverich et al, Endocrinology 109: 62-69 (1981)).
Briefly, this consisted of incubating aliquots (250 ml) of the nuclear suspension
with [3H] eslradiol (4-40 nM) in the absence (total binding) or presence (non-
specific binding; NS) at 4C for 60 minutes. Following incubation, the nuclei
were washed 4 times by resuspension and centrifugation in TE buffer, and the
I 0 final washed nuclear pellets were extracted with 1 ml of ethanol (100%). The
ethanol extract was decanted to mini-vials and radioactivity deterrnined by liquid
scintillation counting. Specific binding was determined by subtraction of non-
specific binding (NS) for the total binding in the system. Results were expressed
as picomoles of ~3Hl estradiol bound per gram wet weight of Iymph node tissue
I 5 and 1 pmole represented 70265 cpm. Figure 2 demonstrates the presence of
nuclear type II estrogen binding sites in the popliteal lymph nodes. These type II
binding sites in popliteal lymph nodes possessed an equivalent binding affinity
(Kd20nM) to those sites obseIved in other tissues such as the rat uterus, and were
present in equivalent numbers as compared to those measured in other non-
2 0 estrogenized, non-malignant tissues. Popliteal Iymph nodes contain almost
exclusively Iymphocyte cells and these cells should therefore be inhibited by
MeHPLA related analoges and deriva~ives through binding interactions with
nuclear type II sites.
Example 2
In Vitro Inhibition of Cell Proliferation
A. MCF-7 Breast Cancer Cell Proliferation Assay
' ~0
To assay for tumor growth sensitivity, the MCF-7 human breast cancer cell
line in tissue culture was used. One skilled in the art will recognize that this is an
excellent model system to assess the effects of hormones and drugs on human
cancer cell growth and proliferation. The MCF-7 cells have both Type I and
:~ 5 nuclear Type II estrogen receptor sites and respond in a proliferative fashion to

~vo 91/17749 PC~rtUS91/03130
35 2~8~208
estrogenic hormones. Furthermore, they are inhibited by well-known anti-
estrogens such as Tamoxifen. The MCF-7 cells were plated at 5 x 10~ cells/dish
in 30 mm petri dishes and grown in Dulbecco's Modified Eagles Medium
containing about 10% charcoal stripped fetal bovine serum for approximately 48
hours. During this interval, the cells attached to the plastic dishes and then
underwent exponential growth with a cell-doubling time of approximately 24
hours. The plated cells were allowed to atta.,h for approximalely 48 hours and the
medium was replaced ("day zerol'). The cells were allowed to grow exponentially
for about 6 days. At day zero the cells were treated with doses ranging from 0.1-
l 0 10 ug/ml of the compound of interest, for example, methyl p-
hydroxyphenyllactate, in 10 ul of ethanol. The medium was changed at about 2
and 4 days. The control and test solutions were also re-added when the medium
was changed. On day 6 the cells were haTvested, counted on a hemocytometer
and DNA content per dish was determined by the Burton assay (BuIton, K.,
l 5 Biochem. J. 62:315-323,1956). Results are expressed as cells/dish orDNA
content (ug/dish) at 6 days following treatment. The results are shown in Figure 3.
The effects of compounds MV-3, MV-12, and MV-88 on MCF-7 cell
proliferation were assessed. MCF-7 cells were plated at 2 x 104cells/well in
Dulbecco's Modified Eagles Medium (DMEM) containing 10% fetal calf serum.
After 24 hours, the cells were treated with concentrations of MV-3, MV-12 or
MV-88 at concentrations ranging from 102 to 105 ng/ml dissolved in 10 ul
ethanol. Controls were treated with an equivalent volume of ethanol. The cell
number was determined 48 hrs following treatment. As demonstrated in Figure 3,
MV-3, MV-12, and MV-88 also inhibited the proliferation of MCF-7 human
breast cancer cells. The proliferation of Y-79 retinoblastoma cells, ME- 180
human cervical cancer cells and human melanoma cells was also inhibited (data
not shown). Utilizing similar data derived from inhibition curves for all of thecompounds shown in Table II, the Ki's for cell inhibition was determined. The KiO for inhibition is defined as the concentration of drug (~lg/ml) required to inhibit
cFII proliferation by 50%-

WO 91/t7749 PCl/l,'S91/031~0
208420~ -36-
Table II. INHIBlTION OF HUMAN MELANOMA (HSO294 CELLS) AND
BREAST CANCER (MCF-7 CELLS) CELL PROLIFERATION BY
CHALCONES AND THED~ DERIYATIVES.
5Compound Melanoma Cells a MCF-7Cells a
MV-3g 0 9
MV-46 5.0 5.0
1 0MV-47 0.9 6.0
MV-72 3.0 30
MV-88 1.5 l.0
aValues represent the Ki for inhibition where Ki is the concentration
1 5 (~l~/ml) required to inhibit cell proliferation by 50% (see Figure 5) relative to
control (untreated cells).
The effects of inhibition were reversible. It took approximately 24 hours
for the cells to recover and about 7 to 24 days after the removal of the ¦-peak
inhibitor to regrow to a full monolayer.
B. Melanoma Cells
Analogous experiments were conducted with human melanoma cells, and
MV-39, MV46, MV-47, MV-72 and MV-88 inhibited the proliferation of these
cells at relatively low concentrations (Table II). Therefore, chalcones ~MV-72 and
MV-88), and their cyano (MV-46, MV-47) and azido (MV-35) derivatives also
inhibit the proliferation of malignant cells.
It is postulated that tumor cell proliferation is very rapid because the tumor
cell metabolizes or inactivates the 1- peak innibitor. This is supported by the
O observation that methyl p-hydroxyphenyllactate is found bound to Type II sites in
normal tissues but is not found in malignant tissue. Cell proliferation is regulated
by ligand binding to nuclear Type II sites. The number of unbound sites
detem~ines the rate of proliferation. Since tumor cells have an increased numberof unbound nuclear Type II sites and thus, their growth is not inhibited by the
amount of endogenous inhibitor normally present in the cells. Therefore, tumor
cell proliferation is dramatically accelerated as compared to the rate of normal cell

~vo 91/17749 P ~ /US91/03130
37 ` : :
proliferadon (i.e., cells with fewer or no unbound nuclear Type II sites). Theserapidly proliferadng cells are brought back into regulation and cell proliferation is
decreased by administering a therapeutic dose of the inhibitors described in thepresent invention. The experimental protocol utilized for assessing the effects on
5 human melanoma cells (HS0294 cells) was idendcal to that described above for
MCF-7 cells.
Example 3
1 0 In Vivo Inhibition of Mammary Tumor Gro~th
To assess drug effects on tumor growth in vivo, a transplantable mouse
mammary tumor model system was udlized. This tumor model and its use as an
assay for anti-tumor drugs has been previously described by Markaverich et al.
l 5 Cancer Res. 43:3208(1983) and is incorporated herein by reference. Briefly,syngeneic female mice bearing transplanted mammary tumors were divided into
experimental groups as indicated. When the tumors were approximately 0.5 x 0.5
crn in size (length x width) (day 0), the animals were treated with blank silastic
capsules (controls), or silastic capsules containing 25 mg of the test compound.Tumor size (length x width) was monitored at the indicated times following
treatment. These implants condnuously released 450 ng of the compound daily.
Figure 4 demonstrates that M V-3, M V-12 and M V-88 inhibited the growth of
these tumors as compared to controls. Similarly, an excellent correlation was
observed between binding affinity (Table I) and the anti-tumor activity of MV-19,
M V-20 and M V-21 ~Figure 19).
Example 4
Utertropic As~ay
3J
The rat uterus is exquisitely sensitive to esFogen, and this hormone
stirnulates uterine cellular hypertrophy, hyperplasia and DNA synthesis within 24
hours following a single injection. Estradiol stimulation of nuclear Type II sites is
a prerequisite for these responses. This assay includes injecting immature female
3 5 rats with saline-ethanol vehicle, estradiol-17l and the test compound of interest.

WO 91/17749 PCI/US91/03130
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Control rats were injected only with saline-ethanol vehicle and estradiol-171. The
rats were sacrificed 24 hours later and the uterine wet and dry weights were
determined. The wet and dry weight measurements are well defined biochemical
end points of estrogen action and are a direct index of changes in cell proliferation
5 and DNA synthesis. The results of these experiments with various compounds are shown in Table m.
Table III. MeHPLA Analogue and Related Compound Effects on Uterine
Growth and Nuclear Type II Site Binding Inhibition
GROWTH TYPE II
COMPOUND INHIBITION a INHIBlTION b
methyl 3-(4-hydroxyphenyl)
2-hydroxypropionate 90.0 0.8
3-(4-hydroxyphenyl)-
2-hydroxypropionic acid 0.0 80.0
2 0 1 -(4-hydroxyphenyl)-3-butanone 96.0 2.0
methyl 3-(3,4-dihydroxyphenyl)-
2-propenoate 70.0 1.0
methyl 3-(4-hydroxy-3-methoxyphenyl)-
2-propenoate 56.0 6.0
methyl (4-hydroxyphenoxy)acetate 70.0 0.8
,
3 o a Detennined by the ability of the compound (10~1g) to block estradiol slimulation in the rat
uterotropic assay.
b The concent~ation (nM x lo-2) of the compound to inhibil nuclear Type II binding sites by 50%.
Additional in vivo measurements using the uterotropic assay show the
5 utility of these compounds for inhibiting cell proliferation. Low doses of methyl
p-hydroxyphenyllactate, but not hydroxyphenyllactic acid, block estradiol
stimulation of uterine growth in the immature rat (Figure 14 and Table m).
However, higher doses of hydroxyphenyllactic acid showed some partial
antagonism. This is not surprising since it is known that hydroxyphenyllactic acid

WO 91/17749 PCI/US91/03130
~39~ 2~84208
binds to nuclear Type II sites with a 20-fold lower affinity than methyl p-
hydroxyphenyllactate.
Previous results demonstrated that the bound/unbound ratio of nuclear
5 Type II sites is important in the regulation of cell growth and that the primary
bound inhibitor in normal cells is methyl p-hydroxyphenyllactate (Markaverich etal. 1988).
Since the data demonstrated that tumor cells have the ability to inactivate
l 0 methyl p-hydroxyphenyllactate, the analogues and chemically related compounds
described in this invention were synthesized to avoid this inactivation. Thus,
administration of compounds with various side-chains and various substituents onthe arornatic ring resulted in inhibition of uterine growth.
l 5 One compound 1-(4-hydroxyphenyl)-3-butanone (p-
hydroxyphenylbutanone) which includes a C-terminal methyl group is not subject
to the esterase cleavage since the methyl group is attached by a C-C bond. This
compound is more stable and thus a better inhibitor in culture and in vivo.
Furthermore, experiments with 1-(4-hydroxyphenyl)-3-butanone demonstrated
2 0 that it binds to the nuclear Type II sites with a high affinity and blocks estradiol
stimulation of uterine growth when injected into immature rats. Thus, 1-(4
hydroxyphenyl)-3-butanone is an effective inhibitor of tumor growth and regulator
of cell proliferation.
Many of the compounds of the present invention, particularly
phenylmethylene ketones, nitroalkenes, aurones, and chalcones, have very low
solubility in aqueous solutions such as those commonly used as injection vehicles.
This insolubility causes major problems with drug delivery and potential use
clinically. To improve drug solubility and delivesy, the compounds may be
3 0 encapsulated to improve their delivery. Among the types of drug delivery systems
useful in the present invention are incorporation into liposomes and encapsulation
in cyclodextrins.

WO 91/1774g PCr/US91/03130
-40-
2a8~'~08
Example 5
Enhancement of Delivery of Compounds of Present Invention
Cyclodextrins solubilize hydrophobic compounds and improve
gastrointestinal absorption (Szejtli, (1982) In: "Cyclodextrins and Their Inclusion
Complexes", Akadmiai Kiado, Budapest, Hungary). Bioflavonoids such as
quercetin (closely related to MV-88) have recently been administered by this
method (Yan, et al (1988) Zhongcaoyoa, 19:492). The lack of cytotoxicity of 2-
1 0 hydroxypropyl J-cyclodextrin and poly J-cyclodextrin (a soluble forrn of '~-cyclodextrin) has been demonstrated in animals (Pitha and Pitha (1985) J. Pharm.Sci. 19:492) and to a limited extent in humans (Pitha, J. (1984) Third Internat.Sympos. on Clathrate Compounds, Tokyo, p69). These compounds increased the
efficacy and sustained delivery of the MV-88 r~lated enones and chalcones.
1 5 Cyclodextrin was condensed with propylene oxide in aqueous alkali to give 2-hydroxycyclodextrin as describcd in Pitha and Pitha (1985). The drug was added
in excess (2 fold excess, i.e., 200 mg drug to 100 mg 2-hydroxycyclodextrin) to a
solution of 2-hydroxycyclodextrin dissolved in water or saline. The drug was
added to the aqueous cyclodextrin solution in methanol. The suspension was then
2 0 stirred at room temperature, and the excess non-solubilized drug was removed by
centrifugation and ultrafiltration as described in (Pitha and Pitha (1985) J. Pharm.
Sci. 19:492). Quantitatio& of drug encapsulated in the cyclodextrin preparationswas by high performance liquid chromatography (HPLC). Briefly, this consisted
of weighing out the dried cyclodextrin-drug mixture and dissolving known
25 amounts (1 mg/ml) of the preparation in methanol. This procedure extracted the
drug from the cyclodextrin such that MV-88 could be quantitated by HPLC. For
HPLC analysis, known concentrations of MV-88 standards (1-20ug), or aliquots
(5-50ul) of the cyclodextrin-MV-88 preparation were injected onto a Wa~ers
uBondapak C18 column and eluted with water: methanol (30:70) at a flow rate of
0 1 ml per rninute. The area of the sample peaks detected at 268 nM versus the
known MV-88 standards allowed quantitation of the concentration of the MV-88
encapsulated in the cyclodexttin. Peak areas of the MV-88 standards and sample
preparadon injecdons were determined by measuring the peak height (in cm) and
multiplying by the peak width (in cm) at 1/2 height (area-cm2). In this manner a~5 standard curve was constructed (MV-B8 concentradon versus OD 268 reading)

~vo 91117749 PCI/US9t/03130
-41- 2~84208
and the concentration of MV-88 in the sample (OD 268 reading) was determined
from the slope of the standard curve. Typical values were approximately 1 mg
MV-88 per 12 mg cyclodextrin MV-88 complex.
To test the effectiveness of the compounds of the present invention to
inhibit tumor growth when encapsulated in cyclodextrin, MV-88 encapsulated in
cyclodextrin was dissolved in saline vehicle and administered by subcutaneous
injection to mamrnary tumor bearing mice. MV-88 is insoluble under aqueous
conditions. However, MV-88 cyclodextrin (MV-88 CD; Table II) inhibited mouse
1 0 mammary tumor growth in a dose-dependent fashion (Figure 6). When
administered by injection in other vehicles such as dimethylsulfoxide saline, MV-
88 was not as effective at inhibiting the growth of this tumor as when administered
after cyclodex¢in encapsuladon. Howeverj MV- 88, when adrninistered in a
continuous fashion by silastic implant (Figure 4), significantly inhibited tumor1 5 growth.
Mice bearing transplantable marnmary tumors were implanted with silastic
capsules containing 25 mg of the test compound. Controls were implanted with
blank capsules and tumor size (length x width) was determined at the 0, 3, 6 and2 0 14 days following implantation. The capsules releases approximately 450 ng of
test compound per day (about 10-15 mg/Kg of body weight). No significant
effects on the body weights of the treated animals relative to controls were
observed throughout the experimental period. Figure 4 demonstrates that
treatment of mouse mammaTy tumors with MV-3, MV-12, and MV-88 in silastic
2 5 implants caused a complete inhibition of the growth of the marnmary tumor in
vivo. However, treatment of mice bearing transplantable mammary tumor with
silastdc capsules containing MV-l9 and MV-20 caused no significant growth
inhibition reladve to the controls because MV- 19 and MV-20 are not released
efficiently from the silastdc capsules. Therefo~e, these compounds were
3 0 administered by cyclodextrin encapsulation procedures which enhanced their
solubility in aqueous injectdon vehicles. These structure actdvity studies
demonstrated that there was a precise structure/activity relationship between
binding affinity and tumor growth inhibitdon observed with MV-l9, MV-20 and
MV-21. MV-l9 and MV-20 did not bind to nuclear type II sites with high
affinity, and did not inhibit rnammary tumor growth. However, MV-21 was found
,; .

WO 91/17749 P(~/US91/03130
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to bind with a relatively high affinity, and treatrnent with MV-21 caused a
significant inhibition of the tumor growth. (Figure 5)]. It is likely that alteration
of the treatrnent regime and dtug dosage will cause complete inhibition of tumorgrowth.
Example 6
Immunosuppressive Activity
1 0 Many anti-cancer drugs possess immunosuppressive activity (Seldin and
Steinberg (1988) In: "Inflammation Basic Principles and Clinical Conelates",
~Galin, J.I., Goldstein, M., Snyderrnan, R., eds.) Raven Press, Ltd., New York).Irrlrnunosuppressive drugs have proven to be therapeutically effective in treating a
variety of autoitnrnune diseases. In the United States, 10-15% of all blindness is
l 5 caused directly or indirectly by inflatnmation of the uveal tract or uveitis. Unless
therapeutic intervention in uveoretinitis is initiated, irreversible darnage can occur
resulting in reduced retinal function andlor blindness. The administration of
corticosteroids is currently the most effective treatment for uveoretinitis.
However, use of corticosteroids is often associated with side effects such as
2 0 elevated intraocular pressure and cataract formation. Imrnunosuppressive drugs
used heretofore including cyclosporin-A (csA) which blocks the proliferation of
T-lymphocytes by interference with the Interleukin-2 receptor expression produceadverse side effects such as hepatotoxicity and nephrotoxicity.
The compounds of the present invention such as MV-3 were tested for
immunosuppressive activity using the established rat model of experimental
autoimmune uveoretinitis (Gery et al. (1986) Invest. Opthalmol. Vis. Sci. 103:
1559). Female Lewis rats weighing 100-200 grarns were immunized with
Interphotoreceptor Retinoid Binding Protein (IRBP) peptide corresponding to
~30 amino acid positions 523-538 of the bovine and human molecules. This sequence
designated #896 has the amino acid sequence LTSHRTATAAEEFAFL. the rats
were immunized by a single foot pad injection of 50 micrograms of ~BP #896
emulsified in complete Freund's adjuvant containing 2.0 mglml M. tuberculosis
H37Ra (Difco). The rats simultaneously received 10x109 heat-killed B. pertussis
;~5 organisms by intraperitoneal injection. 100% of the control, untreated animals

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developed severe, bilateral anterior uveitis and posterior uveoretinitis by day 9 or
day 10 following immunization. Experimental animals were given intraperitoneal
injections of l0 mg of MV-3 on Days 0, 3 and 7.
The MV-3 potent immunosuppressive activity is demonstrated by
experiments illustrated in Figure 7. Figures 7a and 7b demonstrate that 9 days
after immunization of Lewis rats wi~h interphotoreceptor retinoid binding protein
(IRBP) peptide #896 there is a 100% induction of severe, bilateral, panuveitis. In
contrast, the disease in MV-3 treated rats (Figure 7c) was totally blocked. There
1 0 was only a mild uveitic response in some of the MV-3 treated rats at Day lS(Figure 7 c). These results indicate that there is suppression of the autoimrnune
response at its usual point of onset with a lag period in which only mild disease
develops approximatdy 6 days later, apparently occurnng after active levels of the
MV-3 have diminished. In addition, ~he typical inflammatory response at the foot1 5 pad imtnunization site in MV-3 treated rats was also inhibited. These results
suggest that MV-3 and related compounds also have anti-inflammatory properties.
MV-3 as well as other compounds of the present invention provide a useful
method for treating and/or preventing other autoimmune diseases, including, but
not limited to, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, diabetes
mellitis, thyroiditis, systemic lupus erythematosus, Sjorgen's syndrome,
autoimmune skin diseases, and others. In addition, MV-3 and related compounds
provide a useful treatment for graft vs host disease and prevention of transplant
rejections.
2 5 One slcilled in the art will readily appreciate that the present invention is
well adapted to carry out the objects and obtain the ends and advantages
mentioned, as well as those inherent therein. The compounds, methods,
procedures and techniques described herein are presently representative of the
preferred embodiments, are intended to be exemplary, and are not intended as
3 0 lirnitations on the scope of the present invention. Changes therein and other uses
will occur to those skilled in the art which are encompassed within the spirit of the
invention and are defined by the scope of the appended claims.
One skilled in the art will readily appreciate that the present invention is
3 5 well adapted to carry out the objects and obtain the ends and advantages

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mentioned, as well as those inherent therein. The compounds, methods,
procedures and techniques described herein are presently representative of the
preferred embodiments, are intended to be exemplary, and are not intended as
limitations on the scope of the present invention. Changes therein and other uses
5 will occur to those skilled in the art which are encompassed within the spirit of the
invention and are defined by the scope of the appended claims.