CA1332742C - Cell growth inhibitors and methods of treating cancer and cell proliferative diseases - Google Patents
Cell growth inhibitors and methods of treating cancer and cell proliferative diseasesInfo
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- CA1332742C CA1332742C CA000573139A CA573139A CA1332742C CA 1332742 C CA1332742 C CA 1332742C CA 000573139 A CA000573139 A CA 000573139A CA 573139 A CA573139 A CA 573139A CA 1332742 C CA1332742 C CA 1332742C
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- compound
- hydroxyphenyl
- methyl
- phenyl group
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/075—Ethers or acetals
- A61K31/085—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/12—Ketones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C59/40—Unsaturated compounds
- C07C59/42—Unsaturated compounds containing hydroxy or O-metal groups
- C07C59/48—Unsaturated compounds containing hydroxy or O-metal groups containing six-membered aromatic rings
Abstract
CELL GROWTH INHIBITORS AND METHODS OF TREATING
CANCER AND CELL PROLIFERATIVE DISEASES
ABSTRACT
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 agents, as inhibitors of proliferative cell growth and as prophylactic agents to inhibit and prevent cancer and non-malignant cell growth.
CANCER AND CELL PROLIFERATIVE DISEASES
ABSTRACT
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 agents, as inhibitors of proliferative cell growth and as prophylactic agents to inhibit and prevent cancer and non-malignant cell growth.
Description
:
. 5 ., .
'.j 1 0 J,l ~1 .
CEI,L GROWTH INHIBITORS AND METHODS OF TREATING
' CANCER ~ND CELL PP~OLIFERATIVE DISEASES
,: 15 ' E~eld of the Invention i,, This invention relates to the use of new and useul compounds which include methyl p-hydroxyphenyllactate (MeHPLA), its analogues~ chemical derivatives and chemically related compounds as antitumor agents and as inhibitors of proliferative cell growth.
I
¦ Backqround of tha Invention ~ 26 There are two types o~ nuclear estrogen binding ;1 sites in normal and malignant tissues. Type I sites represent the classical estrogen receptor and nuclear Type ,- II sites appear to mediate a speciflc nuclear response to I estr~ge~ic hormones. ~Etex estrogen administration, Type ¦ 30 I r~cepto~ ~ites bind astradiol and this receptor-estrogen complex interacts with nuclear acceptor sites before the , inltiation of the transcriptional events khat are ;~ associated with estrogen stimulation of tissue growth. In contrast, Type II sites bind estrogen with a hi~her capacity and a lower affinity than the classical estrogen . .
;
. 5 ., .
'.j 1 0 J,l ~1 .
CEI,L GROWTH INHIBITORS AND METHODS OF TREATING
' CANCER ~ND CELL PP~OLIFERATIVE DISEASES
,: 15 ' E~eld of the Invention i,, This invention relates to the use of new and useul compounds which include methyl p-hydroxyphenyllactate (MeHPLA), its analogues~ chemical derivatives and chemically related compounds as antitumor agents and as inhibitors of proliferative cell growth.
I
¦ Backqround of tha Invention ~ 26 There are two types o~ nuclear estrogen binding ;1 sites in normal and malignant tissues. Type I sites represent the classical estrogen receptor and nuclear Type ,- II sites appear to mediate a speciflc nuclear response to I estr~ge~ic hormones. ~Etex estrogen administration, Type ¦ 30 I r~cepto~ ~ites bind astradiol and this receptor-estrogen complex interacts with nuclear acceptor sites before the , inltiation of the transcriptional events khat are ;~ associated with estrogen stimulation of tissue growth. In contrast, Type II sites bind estrogen with a hi~her capacity and a lower affinity than the classical estrogen . .
;
-2- ~ 3~7il ~
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 5 after hormone administration. Nuclear Type II sites appear to mediate a specific nuclear response to estrogenic hormones 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 proliEerative tissue has an increased number of nuclear Type II sites. Because the stimulation of nuclear Type II sites is closely correlated with the 15 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
.1 . ., Synthesls.
~ uclear 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 p-hydroxyphenyllactate (MeHPLA or methyl 3-~4-hydroxyphenyl)-2-hydroxypropionate). When MeHPLA
binds to Type II sites, ccll growth and proliferation o~
non-malignant tissues are slowed down or stopped.
Conversely, malignant cells metabolize Me~PLA and thus there is insu~icient binding to the nuclear Type II sites ¦ 30 and the regulation o~ cell growth and proll~eration is lost. Consequently all tumor cell populations examined have very high levels of unbound nuclear Type II sites.
1, These si'es should represent targets for the analogues of MeHPLA.
.1 _3_ ~3~
,.
This invention discloses compounds which are not metabolized by malignant cells but which bind to nuclear Type II sites with high affinity. These compounds are very effective inhibitors of tumor cell proliferation and 5 DNA synthesis. Since nuclear Type II sites have been observed in a variety of tumor cells, it is likely that analogues of MeHPLA and chemically related compounds will be effective inhibitors of a broad spectrum of tumors.
Consequently, any tumor which contains nuclear Type II
o sites should respond to treatment with MeHPLA, its analogues, derivatives and chemically related compounds, including cancers of the pancreas, cervix, liver, brain, pituitary, prostate and other organ or tissue sites, as well as other cancers, such as leukemias, lymphomas, 15 stromal myomas and leiomyomas, among others. Since MeHPLA
will also block estrogen stimulation of normal cell growth such as that in the rat uterus tTahle I), analogues of this compound may also be useful for the treatment of i uterine hyperplasia, cervical hyperplasia, endometriosis and benign prostatic hypertrophy. Because non-proliferating non-malignant cells normally have their i 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, MeHPL~, its analogues, derivatives and chemically related compounds are also useful as prophylactic agents in the inhibition and preventlon o~ cancer and non-malignant cell growth.
The precise physiological role of Type II sites i8 unknown, but inhibition of the nuclear Type II sites is associated with antagonism of uterotropic responses to estrogen. l'his is true for steroid antagonists such as dexamethasone, progesterone and triphenylethylene derivatives such as nafo~idine and clomiphene. While there is at least one endogenous inhibitor of estradiol binding to nuclear Type II sites, the specific inhibitor -4- ~3 32'7' for the nuclear Type II sites has not been identified previous to this invention. Furthermore, the inhibitor of the present invention is specific to nuclear Typ~ II sites and does not interfere with estradiol binding to 5 cytoplasmic or nuclear Type I estrogen receptors.
The inhibitor is identified as methyl 3-(4-hydrogyphenyl)-2-hydroxypropionate and is an important regulator of cell growth and proliferation in normal and malignant tissues. The inhibitor is also known as methyl p-hydroxyphenyllactate or MeHPLA. These terms may be used interchangeably. Cell growth inhlbition by this compound resides in its ability to interact with the high-affinity nuclear binding sites in normal and malignant cells which may be involved in the regulation of cellular proliferation and DNA synthesis. When MeHPLA is bound to nuclear Type II sites, cell growth and proliferation are inhibited. The endogenous 3-(4-hydroxyphenyl~-2-hydroxypropionic acid inhibits the cell growth much less effectively. This activity 20 correlates with its low binding affinity for nuclear Type II sites.
An additional ob]ect of the present invention is i a means for the prevention of cancer. Since MeHPLA is a normal constituent of mammalian cells, but metaboliæed by malignant cell~, MeHPLA, structural analogues and ch~mically related compounds as describad herein, which are not metaholized b~ tumors should be useful in the prevention of malignancy. These compounds should possess little if any side efPects, and i~ taken in a low level maintenance dose should inhibit the proliEeration o~
malignant cells.
8ecause MePHLA is such a potent inhibitor of cell growth, this compound/ as well as its analogues and chemically related compounds were used as potential ~, ~ , . . .. , . , ., . ~ . . . . .
-5~
antitumor agents. The present invention discloses the potent antitumor activity of these compounds.
Summary of the Invention An object of the present invention is a treatment for cancer.
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.
o A further object of the present invention is a method for inhibiting the growth of estrogen responsive tissues.
An additional object of the present invention 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.
Thus, in accomplishing the foregoing objects, t~lere is provided in accordance with one aspect of the present i~vention a method of treating cancer comprising the step of administerillg a therapeutic dose of MeHPLA, its analogues, chemical derivatives or chemically related compounds. More specifically the compound is selected from the ~roup consistin~ of the formulae:
~a ~CO.O~ ~,CO ~) Q~
35 1~, R~--i , 1 .
i ~,' '''I
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 5 after hormone administration. Nuclear Type II sites appear to mediate a specific nuclear response to estrogenic hormones 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 proliEerative tissue has an increased number of nuclear Type II sites. Because the stimulation of nuclear Type II sites is closely correlated with the 15 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
.1 . ., Synthesls.
~ uclear 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 p-hydroxyphenyllactate (MeHPLA or methyl 3-~4-hydroxyphenyl)-2-hydroxypropionate). When MeHPLA
binds to Type II sites, ccll growth and proliferation o~
non-malignant tissues are slowed down or stopped.
Conversely, malignant cells metabolize Me~PLA and thus there is insu~icient binding to the nuclear Type II sites ¦ 30 and the regulation o~ cell growth and proll~eration is lost. Consequently all tumor cell populations examined have very high levels of unbound nuclear Type II sites.
1, These si'es should represent targets for the analogues of MeHPLA.
.1 _3_ ~3~
,.
This invention discloses compounds which are not metabolized by malignant cells but which bind to nuclear Type II sites with high affinity. These compounds are very effective inhibitors of tumor cell proliferation and 5 DNA synthesis. Since nuclear Type II sites have been observed in a variety of tumor cells, it is likely that analogues of MeHPLA and chemically related compounds will be effective inhibitors of a broad spectrum of tumors.
Consequently, any tumor which contains nuclear Type II
o sites should respond to treatment with MeHPLA, its analogues, derivatives and chemically related compounds, including cancers of the pancreas, cervix, liver, brain, pituitary, prostate and other organ or tissue sites, as well as other cancers, such as leukemias, lymphomas, 15 stromal myomas and leiomyomas, among others. Since MeHPLA
will also block estrogen stimulation of normal cell growth such as that in the rat uterus tTahle I), analogues of this compound may also be useful for the treatment of i uterine hyperplasia, cervical hyperplasia, endometriosis and benign prostatic hypertrophy. Because non-proliferating non-malignant cells normally have their i 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, MeHPL~, its analogues, derivatives and chemically related compounds are also useful as prophylactic agents in the inhibition and preventlon o~ cancer and non-malignant cell growth.
The precise physiological role of Type II sites i8 unknown, but inhibition of the nuclear Type II sites is associated with antagonism of uterotropic responses to estrogen. l'his is true for steroid antagonists such as dexamethasone, progesterone and triphenylethylene derivatives such as nafo~idine and clomiphene. While there is at least one endogenous inhibitor of estradiol binding to nuclear Type II sites, the specific inhibitor -4- ~3 32'7' for the nuclear Type II sites has not been identified previous to this invention. Furthermore, the inhibitor of the present invention is specific to nuclear Typ~ II sites and does not interfere with estradiol binding to 5 cytoplasmic or nuclear Type I estrogen receptors.
The inhibitor is identified as methyl 3-(4-hydrogyphenyl)-2-hydroxypropionate and is an important regulator of cell growth and proliferation in normal and malignant tissues. The inhibitor is also known as methyl p-hydroxyphenyllactate or MeHPLA. These terms may be used interchangeably. Cell growth inhlbition by this compound resides in its ability to interact with the high-affinity nuclear binding sites in normal and malignant cells which may be involved in the regulation of cellular proliferation and DNA synthesis. When MeHPLA is bound to nuclear Type II sites, cell growth and proliferation are inhibited. The endogenous 3-(4-hydroxyphenyl~-2-hydroxypropionic acid inhibits the cell growth much less effectively. This activity 20 correlates with its low binding affinity for nuclear Type II sites.
An additional ob]ect of the present invention is i a means for the prevention of cancer. Since MeHPLA is a normal constituent of mammalian cells, but metaboliæed by malignant cell~, MeHPLA, structural analogues and ch~mically related compounds as describad herein, which are not metaholized b~ tumors should be useful in the prevention of malignancy. These compounds should possess little if any side efPects, and i~ taken in a low level maintenance dose should inhibit the proliEeration o~
malignant cells.
8ecause MePHLA is such a potent inhibitor of cell growth, this compound/ as well as its analogues and chemically related compounds were used as potential ~, ~ , . . .. , . , ., . ~ . . . . .
-5~
antitumor agents. The present invention discloses the potent antitumor activity of these compounds.
Summary of the Invention An object of the present invention is a treatment for cancer.
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.
o A further object of the present invention is a method for inhibiting the growth of estrogen responsive tissues.
An additional object of the present invention 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.
Thus, in accomplishing the foregoing objects, t~lere is provided in accordance with one aspect of the present i~vention a method of treating cancer comprising the step of administerillg a therapeutic dose of MeHPLA, its analogues, chemical derivatives or chemically related compounds. More specifically the compound is selected from the ~roup consistin~ of the formulae:
~a ~CO.O~ ~,CO ~) Q~
35 1~, R~--i , 1 .
i ~,' '''I
R ~J~ R,X~--~ , R,~
R~X~O ,CO.OQ, R~O~,,.cHaaa~
wherein, Rl is selected fr~m the group consisting of H, alkyl groups containing 1 to 6 carbons, and aryl groups; ~2 and R3 are selected from the group ~0 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 used to practice the present invention may be selected from the group consisting of methyl 3-~4-hydro~yphenyl)-2-hydro~ypropionate, n-propyl 3-(4-hydroxyphenyl)-2-hydroxypropionate, ; n-butyl 3-(4-hydroxyphenyl)-2-hydro~ypropionate, , 3-(4-hydroxyphenyl)-2-propenoic acid, , 4-(4-hydroxyphenyl)-2-butanone, 1-(4-hydro~yphenyl)-3-pentanone, methyl (4-hydroxyphenoxy)acetate, and methyl 3 (3,4-dihydro~yphenyl)-2-propenoate.
Another aspect of the invention involves the inhibition of the growth of proliferating cells which 2s include a Type II nuclear estrogen binding site by tha step o~ administsring a biolo~ical inhibiting dose of MeHPLA, its analogues, chemical derivatives or chemically related compounds to the proliferating cells.
An additional aspect o~ the present invention is the lnhibition of the proli~erative growth o estrogen responsivQ tissues such as uterus, mammary gland, uterine tumors and mammary tumors. In one specific aspect, the above-mentioned compounds have been used for the treatment of human breast cancer cells. The compounds inhibit the growth of human breast cancer cells.
.1 ..
'~ .
.1 133~7~2 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 analogues, chemical derivatives or chemically 5 related compounds.
Another aspect of the present invention is the provision of an antitumor agent which comprises MeHPLA, its analogues, chemical derivatives or chemically related compounds.
Another aspect of the present invention is prophylactic agents to inhibit and prevent cancer and non-malignant cell growth. These prophylactic agents include the above-mentioned MeHPLA, its analogues, chemical derivatives or chemically related compounds.
Other and further objects, features and advantages will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.
., :
Brief DescriptiQ~Qf~ Figures Figure 1 demonstrates the effect of dilution on [3H]estradiol binding in uterine nuclear fractions from estradiol-17~ implanted adult ovariectomized rats.
Figure 2 demonstrates that tumor cytosol preparations contain lower levels of inhibitor activity for nuclear Type II slte, than cytosol preparations from uterus or mammary gland.
Figure 3 shows the chromatographic profile of rat uterine cytosol inhibitor preparations on Sephade~ G-25 (panels A, C, and D) or LH-20 ~panel B) columns.
-' Figure 4 demonstrates the chromatographic profile of bovine serum egtract on a preparative LH-20 column.
Figure 5 demonstrates the chromatographic profile , of a and ~ Type II ligand by HPLC.
*trademark : `
.1 i -8- ~3~
Figure 6 demonstrates the mass spectra of the trimethylsilyl derivatives of the peak (A) purified from fetal bovine serum and authentic p-hydroxyphenyllactic acid (B;HPLA).
Figure 7 represents the mass spectra of the trimethylsilyl derivatives of the ~ peak (A) purified from fetal bovine serum and authentic methyl p-hydro~yphenyllactate (B;MeHPLA).
Figure 8 demonstrates the competition of type II
1O ligand a and ~ peaks and authentic HPhA and MeHPLA for Type II sites.
Figure 9 represents the competition of authentic HPLA and MeHPLA for [3H]estradiol binding to nuclear Type II sites.
~5 Figure 10 represents the chromatographic profile of mouse mammary gland (A) and mouse mammary tumor (B) cytosols on LH-20 columns.
Figure 11 represents the chromatography of human breast cancer cytosol on an LH-20 column.
Figure 12 demonstrates the effects of authentic HPLA and MeHPLA on MCF-7 human breast cancer cell growth.
Figura 13 represents the efects of LH-20 ~
pe~k fractions on the growth of MCF-7 human breast cancer cells in culture. Panel A shows the dose response curve ~or the ~ peak from rat liver. Panel B shows the time course o~ inhibition following the addition of 33 ~l/ml o~ the LH-20 ~ peak ~ractions.
Figure 1~ demonstrates t~le effects of MeHPLA ~A) and HPLA ~B) on uterine growth.
Fi~ure 15 demonstrates the e~fects o~
p-hydroxyphenylbutanone (1-(4-hydroxyphenyl)-3-butanone) on uterine growth.
"~ .
,'~ ' !
`'1 -9- ~33~7~2 1 DetailQd Description of the SPecific Embodiment Definitions ~ Methyl 3-(4-hydro~yphenyl)-2-hydroxypropionate"
is also known as methyl p-hydroxyphenyllactate or MeHPLA.
5 The term "MeHPLA~' is meant to also include its analogues, chemical derivatives, and chemically related compounds which bind to the nuclear Type II receptors and by so doing inhibit cell proliferation.
The term "chemically related cornpounds" refers to 1~ the derivatives and analogues of p-coumaric acid, p-hydroxyphenylbutanone, (4-hydroxyphenoxy)acetate and the arylpropenaldehydes, alkyl arylethenyl ketones, aryl arylethenyl ketones, aryl butenaldehydes, alkyl arylpropenyl ketones and arylpropenyl ketones which are ~5 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 analogues and derivatives include compounds where R
' represents the methyl, ethyl, n-propyl, n-butyl, isopropyl, tert-butyl or aryl group and R2 and R3 represent H, OH or OCH3 groups and R4 is H or an alkyl group of 1 to 6 carbons. Speciic analogues o~ each class 1 ~5 o~ these structurally relatad compounds to MeHPL~ have been demonstrated to possess biological activity ~Table I) as defined ~lerein and therefore mimic MaHPL~ as an efEecti~e inhibitor o~ cell proli~eration and tumor cell ~rowth, The term "~ndividual" is meant to include animals and humans.
The term ~biologically inhibiting" or "inhibition" of tha growth of proliferating cells is meant to include partial or total growth inhibition and also is meant to include decreases in the rate of proliferation or .
3~
1 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 culture (see Figure 12), uterine 5 growth in the animal (see Figures 14 and 15) or tumor growth in the animal as previously described by Markaverich et al., Cancer Research 43:3208-3~11 (1983~, or any other method known to those of ordinary skill in the art.
0 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 15 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 cor.current treatment, if any, and nature of the malignancy. The effective compound useful in the method ;, oE the present invention may be employed in such orms as capsules, tablets, liquid solutions, suspensions, or elixirs, for oral administration, or sterile liquid forms l such as solutions, suspensions or emulsions. ~ny inert ;l carrier is preferably used, such as saline, or phespha~e-buEered saline, or any such carrier in which the compounds used in tha method of the present invention have suitable solubility properties.
i The compounds o~ the present invention may be ~i administered in a biologically effective carrier. The b;ologically effective carriers may include any solvent with which the compounds of the present invention are ~
l compatible and which are non-to~ic to the individuals treated at the amounts administered.
The term "antitumor agent" is meant to include ; agents which decrease cell growth, or inhibit the 5 proliferation of tumor cells when administered to said tumor cells in an effective dose.
One specific embodiment of this invention is an antitumor agent including MeHPL~i, its analogues, chemical derivatives or chemically related compounds. Specific 10 examples of MeHPL~ analoques are derivatives of the general formula R
. 15 One specific example of this type of compound is methyl 3-(4-hydro~yphenyl~-2-hydroxypropionate wherein Rl is CH3, R2 is H, R3 is OH and R4 is H. This is a 20 naturally occurring endogenous compound which was isolated and characterized and identified as the present invention. Other examples of analogues include the compounds in which the Rl group has been replaced by an ethyl, n-propyl, n-butyl, isopropyl, tert-butyl or aryl 2s group; R2 and/or R3 have been replaced with an ~I, OH
or OC~i3 group and R4 is H or an alkyl group of 1 to 6 carbons. Each o~ these esters can exist in the D and L
I orm.
Anather group of derivative compounds includes those with ~he ~ormula:
' , ~ R2~f _Ç
~Q3 .
~' ~:
i.
-12- ~3~
1 E~amples of these compounds are p-coumaric acid, 3-(4-hydro~yphenyl)-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.
5 Additional esters include compounds wherein Rl is methyl, ethyl, n-propyl, n-butyl, isopropyl, tert-butyl or aryl and R~ and/or R3 is a H, OH or OCH3 `group.
; Additional analogues include caffeic acid, 3-~3,4-dihydro~yphenyl~-2-propenoic acid, wherein Rl is H and R2 and R3 are both OH. Additionally the methoxy compound is very effective as an antitumor agent.
Other compounds with antitumor activity ars the derivatives of 1-(4-hydro~yphenyl)-3-butanone, such as compou~ds with the formulae:
R ~ R~ `U
~ 20 ,, O - .
Q~R R2~3 Q~ Q ~ RI
1 25 RJ , R3 an~ R~
~i ICetone derivatives include compounds with a methyl, ethyl, n-propyl, n-butyl, i~opropyl, tert-butyl or aryl group at 1 30 the Rl po~ition, H, OH or OCH3 group at the R2 and f R3 positions; and most preferably H at thelR~ position ¦ and O~ at the R3 position.
I Additionally, as can be seen by ~he formulae~ the number of CH2 groups between the aromatic entity and the 35 keto group can be ~aried. Specific examples of compounds .j .
-13- ~33~7~2 1 are 1-~4-hydro~yphenyl)-3-pentanone and 1-(4-hydroxyphenyl)-3-butanone. These compounds have been shown to bind to Type II sites and to have antitumor proliferative activity in the uterotropic assay.
Another group of compounds which show antitumor proliferative activity in the rat uterus is described by the formula:
R2 ~ O~COOR, R,~
These compounds are ether-linked analogues o (4~hydrosyphenosy)acetic acid, for example, methyl (4-hydro~yphenoxy)acetate. All of these compounds bind to the Type II binding sites. These ether-linked compounds include analogues wherein Rl is H, a Cl to C6 alkyl carbon chain or an aryl group, R2 and R3 are H, OH or OCH3. An additional variation on the pheno~y compounds include the ether compound~, for e~ample, 2-(4-hydro~ypheno~y)ethyl methyl ether, wherein Rl can ~e H or any Cl to C6 alkyl carbon chain or an aryl group, R2 and R3 ars H, OH or OCH3 in the formula:
Q2~ CH~OR, R~ ~
Additionally useful is the compound o~ the ~ormula:
R~ ~ CO.~
wherein Rl is a Cl to C6 alkyl chain or an aryl group, R2 and R3 are H, OH or OCH3.
`
Another group of compounds which show tumor anti-proliferative action is described by the general ; formulas:
s R ~ R~
0 wherein Rl is from the group consisting of H, alkyl groups containing l to 6 carbons, and substituted or unsubstituted aryl groups; and R2 and R3 are selected from the group consisting of H, OH and OCH3. Preferred compounds of this group which may be used to practice the 15 present invention are:
, CU
~ ~3 H~--~3CH=CH~ ~~1 2s ~O ~ ~I=C~I _ ~ and H0 ~ _~H s-C~ - C ~3 o~
:l 30 ` Most preferred compounds of this group for practicing the ! present invention are 3-~4-Hydroxyphenyl)-l-phenyl-`~ 2-propen-l-one and 4-(4-Hydroxyphenyl)-3-buten-2-one, ~ 35 analogs, chemical derivatives and chemically related i,l .
.j .
'I .
-15- ~3~7~
1 compounds and pharmaceutically acceptable salts thereof.
Another embodiment o~ the present invention includes a rnethod for treating cancer comprising the ~tep of administering a therapeutic dose of MeHPLA, its analogues, 5 chemical derivatives or chemically related compounds.
This compound, can be any of the above-described antitumor compounds.
In addition to being used as a tr~atment for cancer, these antitumor agents are also useful as 0 inhibitors of cell growth and proliferation in those cells which include a Type II nuclear estrogen binding site.
These compounds bind to Type II nuclear estrogen b;nding sites and r2gulate cell growth. Specific proliferating cells which are sensitive to the binding of these compounds include estrogen responsive tissues such as uterus, mammary gland, uterine tumors and mammary tumors.
The above-described compounds inhibit the proliferative ; capacity of human breast cancer cells and thus provide an effective therapy for this disease.
~` 20 Benign prosta~ic hyperplasia is another example o~ a prolifera~ive tissue disease in which the above-described compoundæ can successfully be used in the f treatment.
;~ .
-~ 25 ~in~i~gLAag~
I Tis~ue was dissected ~rom host animals, weighed, I rinsed in saline, and homo~enized in 10 mM Tris-1.5 mM
EDTA, by appro~imat~ly four 30-sec bursts in a Sorva~
Omnimi~er with i~termittent cooling for about 5 min. This crude homogena~ was transferred to a Xontes ground glass homogenizer and further homogenized with interrnittent cooling until no ti~sue aggregates were visible. It was imperative that the homogenate be kept cold, about 4C
through all homogenization s~eps. The final homogenate was centrifuged at 800~g for appro~imately 20 min to *Trademarks ~ i 1 obtain the crude nuclear pellet, which was then washed by gentle resuspension in a Dounc~ homogenizer and then centrifuged at- 800~g for appro~imately 7 min. The final washed nuclear pellet was resuspended in 10 mM Tris-1.5 mM
5 EDTA in a concentration ranging from 5 to 50 mg of fresh tissue equivalents/ml. By the term Uresh tissue equivalent" is meant the amount of material which represents the initial concentration in the ~resh uterine tissue.
To assess total and nonspecific binding, aliquots of the nuclear suspension were incubated at 37C for 30 min with various concentrations ranging from ~ to 40 nM of t3H]estradiol with and without a large molar escess of diethylstilbestrol. The diethylstilbestrol concentration ranged from 0.6 to 12.0 ~M. Following incubation, the nuclear pellets were resuspended in about 1 ml of lOmM
Tris-1~5mM EDTA buffer and centrifuged at 800xg for about 7 min. The pellet was extracted with 1 ml of ethanol at about 30C ~or about 30 min, and counted in 4 ml of 20 ~iquifluor. Speci~lc binding was d~termined by subtraction of nonspecific binding; i.e., not competable I by diethylstilbestrol from the total quantity of : ~3~]estradiol bound. Results were e~pressed as sites/cell, assuming a p9 of DNA/cell nucleus and that each binding site binds one molecule of ~H]estradiol.
.
~nhi~itio~ A8S~Y
~ A variety of rat tissues pos es~ an endogenous ; ligand which blocks L3H~estradiol binding to nuclear Type II estrogen binding sites; however, this compound does not interfere with ~3H]estradiol binding to the i estro~en recep~or. In order to assess the levels of this inhibitor activity in various tissue cy~osols and LH-20 column chromatography fractions from these cytosols~ the following inhibition assay was used, Uterine nuclei were *Trademarks . ~,,' .~
r.~
-17- ~ ~327 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 th,e endogenous inhibitor were minimal, and 5 nuclear Type II sites bound maximum quantities of [3H]estradiol. Aliquots of these nuclei and the inhibitor sample were incubated at about 4C for approximately 60 min in the presence of 4',0 nM of [3H]estradiol with and without 12 ~M
0 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 ~m~ 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.
, The numbers of nuclear Type II sites measured in ' 20 the binding assay increased significantly, four-to f eight-fold, with nuclear dilution. Thus, more nuclear ~-1 Type II sites are measured in dilute than in concentrated nuclei. Figure 1 shows the effects of the dilution on ~3H]estradiol binding in uterine nuclear ~ractions from est~adiol~l7~ implanted adult ovariectomized rats. The uterine nuclei were preparad as previousl~ described in M~rkaverich, 3. M. et al., J. Biol. Chem. 258:11663-11671 ~1983), ~riefly, uterine nuclei after preparation were diluted in 10 m~ Tris, 1.5 mM EDTA buffer in final volumes equivalent to 10 ~ 20 or 40 mg fresh uterine equivalents per ml, and assayed for estrogen binding sites by [3H]estradiol exchange at 37C for 30 minutes. Specific binding was determined by subtraction of non-specific binding, i.e., non-competable with 300-fold excess ;:
diethylstil~estrol, from the total quantity of [3H]estradiol bound. The results shown in Figure 1, panel D demonstrate the specific bindin~ measured at 10, 20 or 40 mg nuclear equivalents per ml corrected or the dilution effect (pmoles~uterus). These results suggest that rat uterine nuclear preparations contain a factor which inhibits [3H]estradiol binding to nuclear Type II
sites and that the interaction of this factor with Type II
sites is decreased by dilution. Using an ~inhibition 10 assay~ it was found that rat uterine cytosol contained a factor which inhibits [3H]estradiol bindiDg to nuclear Type II sites. Briefly, acid-precipitated, boiled cytosol from rat uterus, mouse mammary tumor and normal mouse ; mammary gland was diluted as shown on Figure 2 and 15 evaluated for inhi~itor activity. ~igure 2 shows that this factor is very effective and completely inhibits the Type II sit~ at very dilute cytosol concentrations. ~he results represented by Figure 2 represent ~he mean ~
standard error of the mean for triplicate determinations ; 20 in four replicate e~periments for each preparation. This inhibitor activity was stable to acid and was not destroyed by trypsin, proteinase K, RN~se or DNAse.
l The inhibitor responsible for this activity was J~. characterized as methyl 3-(~-hydro~yphenyl~-2-;s 25 hydro~ypropionate. The identification and ~haracterization procedure involved chromatography o~
cyto~ol preparations on Saphade~*~-25 and Sephade~*LH-20 column~ ~Figure 3). Column ractions were assayed for inhibitory a~t~vity by incubating with dilute nu~lear ~raction~ ~rom estra~iol lmplanted rats. Under these con~itions ma~imum C H]estradiol binding to nuclear Type II sites was observed in the absence of inhibitor activlty. Sephade~*G-25 chromatography resulted in two ma~or peak~ (a and ~) of inhibitor activity eluting in the inclusion volume o~ the column. Chromatography of the :
A~ Trademarks :
.:
-19- 3 h 7 1 h same cytosol preparation on Sephadez LH-~0 also revealed two peaks showing inhibitor activity. When the LH-20 peaks were individually rechromatographed on the Sephadex G-25, the LH-20 ~ and ~ peaks eluted in positions 5 identical to the components previously seen on the Sephade~ G-25. Further characterization of the compounds ~ with the inhibition assay demonstrated that the ;;1, compound was much more inhibitory than was the ~
~l component and thus is a preferred compound for use in cell : 1O growth regulation.
The inhibitor molecules for nuclear Type II sites were isolated from bovine serum. Appro~imately 500 ml of - bovine serum were diluted with an equal volume of HPLC
grade water and the pH was adjusted to 1.0 with high purity HCl. The acidified serum was e~tracted three times with equal volumes of ethyl acetate and the pooled ethyl acetate extracts were dried under vacuum at 60C.
Following evaporation, the extract was redissolved in 100 ml of ethyl acetate and washed with an equal volume of acidified HPLC grade water (pH 1.0). This ethyl acetate fraction was then dried under vacuum, redissolved in the buffer, and chromatographed on a preparative LH-20 column to separate the a and ~ components (Figure 4). The column fractions were collected and and ~ peaks were separately pooled, adjusted to pH 1.0, extracted with ' ethyl acetate, dried, and further purified by HPI~C on an Ultrasphere-octyl*(C8) reversed phase column (Figure 5).
Structural identi~ication was determined by gas chromatography-mass spectrometry o~ the trimethylsilyl derivatives o~ th~e u and ~ peak components (Figures 6 and 7). The peak component mass spectrum was identical to that of HPLA (Figure 6). The identity was confirmed with known authentic samples of p-hydro~yphenyllactic acid. The ~ peak component mass ' *trademark ' J ~ .
, .
spectrum was identified as methyl p-hydroxyphenyllactate (Figure 7).
Once the peaks were identified, the relative binding with nuclear Type II sites was more precisely determined by competition analysis with known amounts of the two authentic compounds. From Figures 8 and 9 it can be seen that MeHPLA is approximately 20-fold more effective in inhibiting ~3H]estradiol binding to nuclear Type II sites than the free acid (HPLA).
Binding assays have shown that the mouse mammary tumor and human breast cancer preparations had high levels of ~ree nuclear Type II sites relative to non-malignant tissues. Figures 2 and 10 demonstrate that the normal rat mammary glands contain very high levels of total inhibitor activity relative to mouse mammary tumors. Figure 11 shows that human breast cancer 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 deficiency in inhibitor activity explains the high levels of free nuclear Type II sites observed in these tumor tissue populations as well as their rapid rate of proliferation, cell growth and DNA synthesis. The high inhibitor activity found in the normal tissues was characterized as previously described on tha LH-20 column and revealed that the rat uterus and normal mouse mammary ~land contained both a and ~ inhibitor peak components. Comparison o~
the ~ and ~ peaks seen in the mouse mammary tumors with those observed ln normal tissues showed a high correlation betwean a de~iclency in the ~ peak component and increased unbound nuclear Type II sites in mouse mammary tumor tissue. In the rat and mouse mammary tumor tissues which have been analyzed, the ~ inhibitor component is consistently very low and, in some cases, undetectable. The low to undetectable ~ inhibitor level i ,; , ~ " .
-2l- ~ 3 ~ ~ 7 ~ ~
is also obs~rved in human breast cancer tissue.
E~amination of a variety o normal tissues, such as rat sQrum, spleen,- diaphragm, liver and uterus, however, shows the presence of both the a and ~ inhibitor peak 5 components. These results demonstrate that the active ;nhibitor component, with respect to the regulation of cell proliferation, is the ~ inhibitor peak component.
MCF-7 Cancer Cell AssaY
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 r e~cellent model system to assess the effects of hormones and drugs on human cancer cell growth and proliferation.
The MCF-7 c~lls have both Type I and nuclear Type II
estrogen receptor sites and respond in a proliferative fashion ~o estrogenic hormones. Furthermore, they are inhibited by well-known ant$-estrogens such as Tamo~ifen.
The MCF-7 cells were plated at 5 ~ 105 cells~dish in 30 20 mm petri dishes and grown in Dulbecco's Modified Eagles Medium containing about 10% charcoal stripped fetal bov-ine serum for approximately 48 hours. During this interval, ~he cells attached to the plastic dishes and then underwent e~ponential growth with a cell-doubling time of appro3imately 24 hours. The plated cells were allowed to attach ~or appro~imately 4a hours snd the medium was replaced (Uday zeroN). The cell~ were allowed to grow exponentially or about 6 days. At day zero the cells were treatsd with do~e~ ranging ~rom l-10 ~g~ml o~ the compound o~ interest, ~or e~ample, methyl p-hydroxyphenyllactat~, in lO ~l of ethanol. ~he 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 harvested, counted on a hemocytometer and DNA content per dish was determined by :, ~! *Trademarks -22- ~ ~3 1 the Burton assay (Burton, K., Biochem. J. 62:315-323, 1956). Results are expressed as cells/dish or D~A content ~g/dish~ at 6 days following treatment. The results are shown in Figure 12.
' 5 Cultures of rat uterine or mammary tumor cells with cytosol preparations from rat uterus or liver containing a and ~ components showed cell growth inhibition within about 24 hours. Conversely, when the cells were cultured with cytosol preparations from mouse 0 mammary tumors which contained the ~ but not the ~
component, there was no substantial inhibition of cell growth across the wide range of concentrations tested.
These data, consistent with the resu:Lts of the competition experiments shown on Figures 8 and 9, demonstrated that the cytosol preparations from a normal mammary gland contain 15- to 20-fold more total inhibitor activity than those from tumors (Figure 2). One major diEference between cytosol preparations from normal and malignant tissues was that the malignant tissues were deficient in t.he ~ peaX inhibitor component (Figure 10). Hence, the peak component was the most important compound with respact to potential regulation of cell proliferation. As described in the present invention the ~ peak component was identified as methyl p-hydro~yphenyllactate.
Further, characterization and demonstration o~
the antitumor activity of the ~ inhibitor component was seen in examples using rat liver isolates. After the i~olation, the ~ inhibltor material was suspended in 2 ml of tissue culture medlum and add*d to cultures o MCF-7 hu~an breast cancer cell linesl The MCF 7 cells were plated in 3 ml o~ DMEM contalning 10% charcoal stripped ~etal bovine serum and allowed to attach to the surface of petri dishes. Following the establishment of monolayar cultures, approximately 24 to 36 hours, the floating cells were removed by aspiration, and 3 ml of , :, . . ~ ,. .. . . , "
, -23- ~ 33~ 7~
1 fresh DMEM were added to each culture. At this time various amounts, ranging from 7 to 32 ~l~ml, of the pooled ~ peak fractions or control medium were added to the cultures. The cells were cultured for an additional 5 24 hours. At the end of this culture period, the floating cells were remo~ed by aspiration and the attached cells resuspended in DMEM following mild trypsinization. The cells were then counted on a hemocytometer. with the increasing concentrations of the ~ peak inhibitor 1O material, the cell number was reduced in a dose dependent fashion within about 24 hours (Figure 13A). The control samples did not exhibit a similar decrease. The time course of the inhibition activity was also examined.
Approximately 32 ~1 of control medium or ~ peak inhibitor material was added to the cells and the floating cells were removed. Within approximately four hours of inhibitor addition, the inhibition of cell growth had begun. Ma~imum inhibition was observed in approximately 12 hours (Figure 13B).
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 rsgrow to a full monolayer.
It is postulated that tumor cell proliferation is very rapid because the tumor cell me~abolizes or inactivates the ~ peak inhibitor. This is supported by the observation that methyl p-hydroxyphenyllactate is Eound bound to Type II sites in normal tissues but is not ll Eound in malignant tissue. Cell proli~eration is - 30 regulated by ligand bindin~ ~o nuclear Type II sites. The number of unbound sites determines the rate of j proli~eration since tumor cells have an increased number of unbound nuclear Type II sites and thus are not inhibited, so tumor cell proliferation is dramatically accelerated. These cells are brought back into regulation ,, .
, -24- ~3~27~,~
and cell proliferation is decreased by administering a therapeutic dose of the inhibitors described in the present invention.
In addition to testing purified biological 5 preparations, the authentic compounds, methyl p-hydroxyphenyllactate and hydroxyphenyllactic acid, were added to cell cultures. As can be seen in Figure 12, methyl p-hydro~yphenyllactate inhibited MCF-7 human hreast cancer cells, but hydroxyphenyllactic acid did not.
o Furthermore, it can be seen that the methyl p-hydroxyphenyllactate inhibited MCF~7 breast cancer cells in a similar dose-dependent fashion. Results obtained by using the authentic compound were in agreement with the data from biological preparations.
UterotroPic Assay The rat uterus is e~quisitively sensitive to estrogen, and this hormone stimulates 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 ; rats with saline-sthanol vehicle, estradiol-17~ and the test compound of interest. Control rats were injec-ted only with saline-ethanol vehicle and estradiol-17~. 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 oE changes in cell proliferation and DNA synthesis.
~ he results of these experiments with various compound~ are shown in Table I.
Additional i~ vivo measurements using the uterotrDpic assay show the utility o these compounds for inhibiting cell proliferation. Low doses o methyl ,1 :
~3~7~
p-hydroxyphenyllactate, but not hydroxyphenyllactic acid, block estradiol stimulation of uterine growth in the immature rat (Figure 14 and Table I). However, higher doses of hydroxyphenyllactic acid showed some partial 5 antagonism. This is not surprising since it is known that hydroxyphenyllactic acid binds to nuclear Type II sites with a 20-fold lower affinity than methyl p-hydroxyphenyllactate.
These results demonstrate that the bound/unbound 10 ratio of nuclear Type II sites is important in the regulation of cell growth and that the primary bound inhibitor in normal cells is methyl p-hydroxyphenyllactate. Since the data demonstrated that tumor cells have the ability to inactivate methyl p-hydroxyphenyllactate, the analogues described in this invention were synthesized to avoid this inactivation.
Thus, as can be seen in Table I, administration of compounds with various side-chains and various substituents on the aromatic ring resulted in inhibition ~ uterine growth. One compound 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 ViVQ. Furthermore, e~periments with 1-(4-hydro~yphenyl)-3-butanone demonstrated that it binds to the nuclear Type II sites with a high affinity and blocks estradiol stimulation of uterine growth when injected into lmmature rats ~Figure 15). Thus 1-~4-hydroxyphenyl)-3-butanone is an e~ective inhibitor of tumor growth and regulator of cell proliferation.
. .
Table I. MeHPLA Analogue and Related Compound Effects on Uterine Growth and Nuclear Type II Site Binding rnhibition GROWTHa ~YPE IIb C~MPOUND INHIBITION (%) INHIBITION
5 methyl 3-(4-hydroxyphenyl) 2-hydroxypropionate 90 0.8 3-(4-hydro~yphenyl)-2-hydroxypropionic acid 0 80.0 l-(4-hydrogyphenyl)-3-butanone 96 2.0 methyl 3-(3,4-dihydroxyphenyl)-2-propenoate 70 l.0 methyl 3-~4-hydroxy-3-metho~yphenyl)-2~propenoate 56 6.0 methyl (4-hydroxyphenoxy)acetate 70 0.8 a Determined by the ability of the compound (l0~g) to block estradiol stimulation in the rat uterotropic assay.
b The concentration (nM) times 10-2 of the compound to inhibit nuclear Type II binding sites by 50~.
One skilled 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 prese~tly representative o~ the pre~erred emhodiments, are intended to be e~emplary, ~nd are not intended as limitations 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 o~ the invention and are defined by the scope of the appended claims.
What is claimed is:
.'.i
R~X~O ,CO.OQ, R~O~,,.cHaaa~
wherein, Rl is selected fr~m the group consisting of H, alkyl groups containing 1 to 6 carbons, and aryl groups; ~2 and R3 are selected from the group ~0 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 used to practice the present invention may be selected from the group consisting of methyl 3-~4-hydro~yphenyl)-2-hydro~ypropionate, n-propyl 3-(4-hydroxyphenyl)-2-hydroxypropionate, ; n-butyl 3-(4-hydroxyphenyl)-2-hydro~ypropionate, , 3-(4-hydroxyphenyl)-2-propenoic acid, , 4-(4-hydroxyphenyl)-2-butanone, 1-(4-hydro~yphenyl)-3-pentanone, methyl (4-hydroxyphenoxy)acetate, and methyl 3 (3,4-dihydro~yphenyl)-2-propenoate.
Another aspect of the invention involves the inhibition of the growth of proliferating cells which 2s include a Type II nuclear estrogen binding site by tha step o~ administsring a biolo~ical inhibiting dose of MeHPLA, its analogues, chemical derivatives or chemically related compounds to the proliferating cells.
An additional aspect o~ the present invention is the lnhibition of the proli~erative growth o estrogen responsivQ tissues such as uterus, mammary gland, uterine tumors and mammary tumors. In one specific aspect, the above-mentioned compounds have been used for the treatment of human breast cancer cells. The compounds inhibit the growth of human breast cancer cells.
.1 ..
'~ .
.1 133~7~2 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 analogues, chemical derivatives or chemically 5 related compounds.
Another aspect of the present invention is the provision of an antitumor agent which comprises MeHPLA, its analogues, chemical derivatives or chemically related compounds.
Another aspect of the present invention is prophylactic agents to inhibit and prevent cancer and non-malignant cell growth. These prophylactic agents include the above-mentioned MeHPLA, its analogues, chemical derivatives or chemically related compounds.
Other and further objects, features and advantages will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.
., :
Brief DescriptiQ~Qf~ Figures Figure 1 demonstrates the effect of dilution on [3H]estradiol binding in uterine nuclear fractions from estradiol-17~ implanted adult ovariectomized rats.
Figure 2 demonstrates that tumor cytosol preparations contain lower levels of inhibitor activity for nuclear Type II slte, than cytosol preparations from uterus or mammary gland.
Figure 3 shows the chromatographic profile of rat uterine cytosol inhibitor preparations on Sephade~ G-25 (panels A, C, and D) or LH-20 ~panel B) columns.
-' Figure 4 demonstrates the chromatographic profile of bovine serum egtract on a preparative LH-20 column.
Figure 5 demonstrates the chromatographic profile , of a and ~ Type II ligand by HPLC.
*trademark : `
.1 i -8- ~3~
Figure 6 demonstrates the mass spectra of the trimethylsilyl derivatives of the peak (A) purified from fetal bovine serum and authentic p-hydroxyphenyllactic acid (B;HPLA).
Figure 7 represents the mass spectra of the trimethylsilyl derivatives of the ~ peak (A) purified from fetal bovine serum and authentic methyl p-hydro~yphenyllactate (B;MeHPLA).
Figure 8 demonstrates the competition of type II
1O ligand a and ~ peaks and authentic HPhA and MeHPLA for Type II sites.
Figure 9 represents the competition of authentic HPLA and MeHPLA for [3H]estradiol binding to nuclear Type II sites.
~5 Figure 10 represents the chromatographic profile of mouse mammary gland (A) and mouse mammary tumor (B) cytosols on LH-20 columns.
Figure 11 represents the chromatography of human breast cancer cytosol on an LH-20 column.
Figure 12 demonstrates the effects of authentic HPLA and MeHPLA on MCF-7 human breast cancer cell growth.
Figura 13 represents the efects of LH-20 ~
pe~k fractions on the growth of MCF-7 human breast cancer cells in culture. Panel A shows the dose response curve ~or the ~ peak from rat liver. Panel B shows the time course o~ inhibition following the addition of 33 ~l/ml o~ the LH-20 ~ peak ~ractions.
Figure 1~ demonstrates t~le effects of MeHPLA ~A) and HPLA ~B) on uterine growth.
Fi~ure 15 demonstrates the e~fects o~
p-hydroxyphenylbutanone (1-(4-hydroxyphenyl)-3-butanone) on uterine growth.
"~ .
,'~ ' !
`'1 -9- ~33~7~2 1 DetailQd Description of the SPecific Embodiment Definitions ~ Methyl 3-(4-hydro~yphenyl)-2-hydroxypropionate"
is also known as methyl p-hydroxyphenyllactate or MeHPLA.
5 The term "MeHPLA~' is meant to also include its analogues, chemical derivatives, and chemically related compounds which bind to the nuclear Type II receptors and by so doing inhibit cell proliferation.
The term "chemically related cornpounds" refers to 1~ the derivatives and analogues of p-coumaric acid, p-hydroxyphenylbutanone, (4-hydroxyphenoxy)acetate and the arylpropenaldehydes, alkyl arylethenyl ketones, aryl arylethenyl ketones, aryl butenaldehydes, alkyl arylpropenyl ketones and arylpropenyl ketones which are ~5 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 analogues and derivatives include compounds where R
' represents the methyl, ethyl, n-propyl, n-butyl, isopropyl, tert-butyl or aryl group and R2 and R3 represent H, OH or OCH3 groups and R4 is H or an alkyl group of 1 to 6 carbons. Speciic analogues o~ each class 1 ~5 o~ these structurally relatad compounds to MeHPL~ have been demonstrated to possess biological activity ~Table I) as defined ~lerein and therefore mimic MaHPL~ as an efEecti~e inhibitor o~ cell proli~eration and tumor cell ~rowth, The term "~ndividual" is meant to include animals and humans.
The term ~biologically inhibiting" or "inhibition" of tha growth of proliferating cells is meant to include partial or total growth inhibition and also is meant to include decreases in the rate of proliferation or .
3~
1 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 culture (see Figure 12), uterine 5 growth in the animal (see Figures 14 and 15) or tumor growth in the animal as previously described by Markaverich et al., Cancer Research 43:3208-3~11 (1983~, or any other method known to those of ordinary skill in the art.
0 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 15 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 cor.current treatment, if any, and nature of the malignancy. The effective compound useful in the method ;, oE the present invention may be employed in such orms as capsules, tablets, liquid solutions, suspensions, or elixirs, for oral administration, or sterile liquid forms l such as solutions, suspensions or emulsions. ~ny inert ;l carrier is preferably used, such as saline, or phespha~e-buEered saline, or any such carrier in which the compounds used in tha method of the present invention have suitable solubility properties.
i The compounds o~ the present invention may be ~i administered in a biologically effective carrier. The b;ologically effective carriers may include any solvent with which the compounds of the present invention are ~
l compatible and which are non-to~ic to the individuals treated at the amounts administered.
The term "antitumor agent" is meant to include ; agents which decrease cell growth, or inhibit the 5 proliferation of tumor cells when administered to said tumor cells in an effective dose.
One specific embodiment of this invention is an antitumor agent including MeHPL~i, its analogues, chemical derivatives or chemically related compounds. Specific 10 examples of MeHPL~ analoques are derivatives of the general formula R
. 15 One specific example of this type of compound is methyl 3-(4-hydro~yphenyl~-2-hydroxypropionate wherein Rl is CH3, R2 is H, R3 is OH and R4 is H. This is a 20 naturally occurring endogenous compound which was isolated and characterized and identified as the present invention. Other examples of analogues include the compounds in which the Rl group has been replaced by an ethyl, n-propyl, n-butyl, isopropyl, tert-butyl or aryl 2s group; R2 and/or R3 have been replaced with an ~I, OH
or OC~i3 group and R4 is H or an alkyl group of 1 to 6 carbons. Each o~ these esters can exist in the D and L
I orm.
Anather group of derivative compounds includes those with ~he ~ormula:
' , ~ R2~f _Ç
~Q3 .
~' ~:
i.
-12- ~3~
1 E~amples of these compounds are p-coumaric acid, 3-(4-hydro~yphenyl)-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.
5 Additional esters include compounds wherein Rl is methyl, ethyl, n-propyl, n-butyl, isopropyl, tert-butyl or aryl and R~ and/or R3 is a H, OH or OCH3 `group.
; Additional analogues include caffeic acid, 3-~3,4-dihydro~yphenyl~-2-propenoic acid, wherein Rl is H and R2 and R3 are both OH. Additionally the methoxy compound is very effective as an antitumor agent.
Other compounds with antitumor activity ars the derivatives of 1-(4-hydro~yphenyl)-3-butanone, such as compou~ds with the formulae:
R ~ R~ `U
~ 20 ,, O - .
Q~R R2~3 Q~ Q ~ RI
1 25 RJ , R3 an~ R~
~i ICetone derivatives include compounds with a methyl, ethyl, n-propyl, n-butyl, i~opropyl, tert-butyl or aryl group at 1 30 the Rl po~ition, H, OH or OCH3 group at the R2 and f R3 positions; and most preferably H at thelR~ position ¦ and O~ at the R3 position.
I Additionally, as can be seen by ~he formulae~ the number of CH2 groups between the aromatic entity and the 35 keto group can be ~aried. Specific examples of compounds .j .
-13- ~33~7~2 1 are 1-~4-hydro~yphenyl)-3-pentanone and 1-(4-hydroxyphenyl)-3-butanone. These compounds have been shown to bind to Type II sites and to have antitumor proliferative activity in the uterotropic assay.
Another group of compounds which show antitumor proliferative activity in the rat uterus is described by the formula:
R2 ~ O~COOR, R,~
These compounds are ether-linked analogues o (4~hydrosyphenosy)acetic acid, for example, methyl (4-hydro~yphenoxy)acetate. All of these compounds bind to the Type II binding sites. These ether-linked compounds include analogues wherein Rl is H, a Cl to C6 alkyl carbon chain or an aryl group, R2 and R3 are H, OH or OCH3. An additional variation on the pheno~y compounds include the ether compound~, for e~ample, 2-(4-hydro~ypheno~y)ethyl methyl ether, wherein Rl can ~e H or any Cl to C6 alkyl carbon chain or an aryl group, R2 and R3 ars H, OH or OCH3 in the formula:
Q2~ CH~OR, R~ ~
Additionally useful is the compound o~ the ~ormula:
R~ ~ CO.~
wherein Rl is a Cl to C6 alkyl chain or an aryl group, R2 and R3 are H, OH or OCH3.
`
Another group of compounds which show tumor anti-proliferative action is described by the general ; formulas:
s R ~ R~
0 wherein Rl is from the group consisting of H, alkyl groups containing l to 6 carbons, and substituted or unsubstituted aryl groups; and R2 and R3 are selected from the group consisting of H, OH and OCH3. Preferred compounds of this group which may be used to practice the 15 present invention are:
, CU
~ ~3 H~--~3CH=CH~ ~~1 2s ~O ~ ~I=C~I _ ~ and H0 ~ _~H s-C~ - C ~3 o~
:l 30 ` Most preferred compounds of this group for practicing the ! present invention are 3-~4-Hydroxyphenyl)-l-phenyl-`~ 2-propen-l-one and 4-(4-Hydroxyphenyl)-3-buten-2-one, ~ 35 analogs, chemical derivatives and chemically related i,l .
.j .
'I .
-15- ~3~7~
1 compounds and pharmaceutically acceptable salts thereof.
Another embodiment o~ the present invention includes a rnethod for treating cancer comprising the ~tep of administering a therapeutic dose of MeHPLA, its analogues, 5 chemical derivatives or chemically related compounds.
This compound, can be any of the above-described antitumor compounds.
In addition to being used as a tr~atment for cancer, these antitumor agents are also useful as 0 inhibitors of cell growth and proliferation in those cells which include a Type II nuclear estrogen binding site.
These compounds bind to Type II nuclear estrogen b;nding sites and r2gulate cell growth. Specific proliferating cells which are sensitive to the binding of these compounds include estrogen responsive tissues such as uterus, mammary gland, uterine tumors and mammary tumors.
The above-described compounds inhibit the proliferative ; capacity of human breast cancer cells and thus provide an effective therapy for this disease.
~` 20 Benign prosta~ic hyperplasia is another example o~ a prolifera~ive tissue disease in which the above-described compoundæ can successfully be used in the f treatment.
;~ .
-~ 25 ~in~i~gLAag~
I Tis~ue was dissected ~rom host animals, weighed, I rinsed in saline, and homo~enized in 10 mM Tris-1.5 mM
EDTA, by appro~imat~ly four 30-sec bursts in a Sorva~
Omnimi~er with i~termittent cooling for about 5 min. This crude homogena~ was transferred to a Xontes ground glass homogenizer and further homogenized with interrnittent cooling until no ti~sue aggregates were visible. It was imperative that the homogenate be kept cold, about 4C
through all homogenization s~eps. The final homogenate was centrifuged at 800~g for appro~imately 20 min to *Trademarks ~ i 1 obtain the crude nuclear pellet, which was then washed by gentle resuspension in a Dounc~ homogenizer and then centrifuged at- 800~g for appro~imately 7 min. The final washed nuclear pellet was resuspended in 10 mM Tris-1.5 mM
5 EDTA in a concentration ranging from 5 to 50 mg of fresh tissue equivalents/ml. By the term Uresh tissue equivalent" is meant the amount of material which represents the initial concentration in the ~resh uterine tissue.
To assess total and nonspecific binding, aliquots of the nuclear suspension were incubated at 37C for 30 min with various concentrations ranging from ~ to 40 nM of t3H]estradiol with and without a large molar escess of diethylstilbestrol. The diethylstilbestrol concentration ranged from 0.6 to 12.0 ~M. Following incubation, the nuclear pellets were resuspended in about 1 ml of lOmM
Tris-1~5mM EDTA buffer and centrifuged at 800xg for about 7 min. The pellet was extracted with 1 ml of ethanol at about 30C ~or about 30 min, and counted in 4 ml of 20 ~iquifluor. Speci~lc binding was d~termined by subtraction of nonspecific binding; i.e., not competable I by diethylstilbestrol from the total quantity of : ~3~]estradiol bound. Results were e~pressed as sites/cell, assuming a p9 of DNA/cell nucleus and that each binding site binds one molecule of ~H]estradiol.
.
~nhi~itio~ A8S~Y
~ A variety of rat tissues pos es~ an endogenous ; ligand which blocks L3H~estradiol binding to nuclear Type II estrogen binding sites; however, this compound does not interfere with ~3H]estradiol binding to the i estro~en recep~or. In order to assess the levels of this inhibitor activity in various tissue cy~osols and LH-20 column chromatography fractions from these cytosols~ the following inhibition assay was used, Uterine nuclei were *Trademarks . ~,,' .~
r.~
-17- ~ ~327 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 th,e endogenous inhibitor were minimal, and 5 nuclear Type II sites bound maximum quantities of [3H]estradiol. Aliquots of these nuclei and the inhibitor sample were incubated at about 4C for approximately 60 min in the presence of 4',0 nM of [3H]estradiol with and without 12 ~M
0 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 ~m~ 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.
, The numbers of nuclear Type II sites measured in ' 20 the binding assay increased significantly, four-to f eight-fold, with nuclear dilution. Thus, more nuclear ~-1 Type II sites are measured in dilute than in concentrated nuclei. Figure 1 shows the effects of the dilution on ~3H]estradiol binding in uterine nuclear ~ractions from est~adiol~l7~ implanted adult ovariectomized rats. The uterine nuclei were preparad as previousl~ described in M~rkaverich, 3. M. et al., J. Biol. Chem. 258:11663-11671 ~1983), ~riefly, uterine nuclei after preparation were diluted in 10 m~ Tris, 1.5 mM EDTA buffer in final volumes equivalent to 10 ~ 20 or 40 mg fresh uterine equivalents per ml, and assayed for estrogen binding sites by [3H]estradiol exchange at 37C for 30 minutes. Specific binding was determined by subtraction of non-specific binding, i.e., non-competable with 300-fold excess ;:
diethylstil~estrol, from the total quantity of [3H]estradiol bound. The results shown in Figure 1, panel D demonstrate the specific bindin~ measured at 10, 20 or 40 mg nuclear equivalents per ml corrected or the dilution effect (pmoles~uterus). These results suggest that rat uterine nuclear preparations contain a factor which inhibits [3H]estradiol binding to nuclear Type II
sites and that the interaction of this factor with Type II
sites is decreased by dilution. Using an ~inhibition 10 assay~ it was found that rat uterine cytosol contained a factor which inhibits [3H]estradiol bindiDg to nuclear Type II sites. Briefly, acid-precipitated, boiled cytosol from rat uterus, mouse mammary tumor and normal mouse ; mammary gland was diluted as shown on Figure 2 and 15 evaluated for inhi~itor activity. ~igure 2 shows that this factor is very effective and completely inhibits the Type II sit~ at very dilute cytosol concentrations. ~he results represented by Figure 2 represent ~he mean ~
standard error of the mean for triplicate determinations ; 20 in four replicate e~periments for each preparation. This inhibitor activity was stable to acid and was not destroyed by trypsin, proteinase K, RN~se or DNAse.
l The inhibitor responsible for this activity was J~. characterized as methyl 3-(~-hydro~yphenyl~-2-;s 25 hydro~ypropionate. The identification and ~haracterization procedure involved chromatography o~
cyto~ol preparations on Saphade~*~-25 and Sephade~*LH-20 column~ ~Figure 3). Column ractions were assayed for inhibitory a~t~vity by incubating with dilute nu~lear ~raction~ ~rom estra~iol lmplanted rats. Under these con~itions ma~imum C H]estradiol binding to nuclear Type II sites was observed in the absence of inhibitor activlty. Sephade~*G-25 chromatography resulted in two ma~or peak~ (a and ~) of inhibitor activity eluting in the inclusion volume o~ the column. Chromatography of the :
A~ Trademarks :
.:
-19- 3 h 7 1 h same cytosol preparation on Sephadez LH-~0 also revealed two peaks showing inhibitor activity. When the LH-20 peaks were individually rechromatographed on the Sephadex G-25, the LH-20 ~ and ~ peaks eluted in positions 5 identical to the components previously seen on the Sephade~ G-25. Further characterization of the compounds ~ with the inhibition assay demonstrated that the ;;1, compound was much more inhibitory than was the ~
~l component and thus is a preferred compound for use in cell : 1O growth regulation.
The inhibitor molecules for nuclear Type II sites were isolated from bovine serum. Appro~imately 500 ml of - bovine serum were diluted with an equal volume of HPLC
grade water and the pH was adjusted to 1.0 with high purity HCl. The acidified serum was e~tracted three times with equal volumes of ethyl acetate and the pooled ethyl acetate extracts were dried under vacuum at 60C.
Following evaporation, the extract was redissolved in 100 ml of ethyl acetate and washed with an equal volume of acidified HPLC grade water (pH 1.0). This ethyl acetate fraction was then dried under vacuum, redissolved in the buffer, and chromatographed on a preparative LH-20 column to separate the a and ~ components (Figure 4). The column fractions were collected and and ~ peaks were separately pooled, adjusted to pH 1.0, extracted with ' ethyl acetate, dried, and further purified by HPI~C on an Ultrasphere-octyl*(C8) reversed phase column (Figure 5).
Structural identi~ication was determined by gas chromatography-mass spectrometry o~ the trimethylsilyl derivatives o~ th~e u and ~ peak components (Figures 6 and 7). The peak component mass spectrum was identical to that of HPLA (Figure 6). The identity was confirmed with known authentic samples of p-hydro~yphenyllactic acid. The ~ peak component mass ' *trademark ' J ~ .
, .
spectrum was identified as methyl p-hydroxyphenyllactate (Figure 7).
Once the peaks were identified, the relative binding with nuclear Type II sites was more precisely determined by competition analysis with known amounts of the two authentic compounds. From Figures 8 and 9 it can be seen that MeHPLA is approximately 20-fold more effective in inhibiting ~3H]estradiol binding to nuclear Type II sites than the free acid (HPLA).
Binding assays have shown that the mouse mammary tumor and human breast cancer preparations had high levels of ~ree nuclear Type II sites relative to non-malignant tissues. Figures 2 and 10 demonstrate that the normal rat mammary glands contain very high levels of total inhibitor activity relative to mouse mammary tumors. Figure 11 shows that human breast cancer 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 deficiency in inhibitor activity explains the high levels of free nuclear Type II sites observed in these tumor tissue populations as well as their rapid rate of proliferation, cell growth and DNA synthesis. The high inhibitor activity found in the normal tissues was characterized as previously described on tha LH-20 column and revealed that the rat uterus and normal mouse mammary ~land contained both a and ~ inhibitor peak components. Comparison o~
the ~ and ~ peaks seen in the mouse mammary tumors with those observed ln normal tissues showed a high correlation betwean a de~iclency in the ~ peak component and increased unbound nuclear Type II sites in mouse mammary tumor tissue. In the rat and mouse mammary tumor tissues which have been analyzed, the ~ inhibitor component is consistently very low and, in some cases, undetectable. The low to undetectable ~ inhibitor level i ,; , ~ " .
-2l- ~ 3 ~ ~ 7 ~ ~
is also obs~rved in human breast cancer tissue.
E~amination of a variety o normal tissues, such as rat sQrum, spleen,- diaphragm, liver and uterus, however, shows the presence of both the a and ~ inhibitor peak 5 components. These results demonstrate that the active ;nhibitor component, with respect to the regulation of cell proliferation, is the ~ inhibitor peak component.
MCF-7 Cancer Cell AssaY
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 r e~cellent model system to assess the effects of hormones and drugs on human cancer cell growth and proliferation.
The MCF-7 c~lls have both Type I and nuclear Type II
estrogen receptor sites and respond in a proliferative fashion ~o estrogenic hormones. Furthermore, they are inhibited by well-known ant$-estrogens such as Tamo~ifen.
The MCF-7 cells were plated at 5 ~ 105 cells~dish in 30 20 mm petri dishes and grown in Dulbecco's Modified Eagles Medium containing about 10% charcoal stripped fetal bov-ine serum for approximately 48 hours. During this interval, ~he cells attached to the plastic dishes and then underwent e~ponential growth with a cell-doubling time of appro3imately 24 hours. The plated cells were allowed to attach ~or appro~imately 4a hours snd the medium was replaced (Uday zeroN). The cell~ were allowed to grow exponentially or about 6 days. At day zero the cells were treatsd with do~e~ ranging ~rom l-10 ~g~ml o~ the compound o~ interest, ~or e~ample, methyl p-hydroxyphenyllactat~, in lO ~l of ethanol. ~he 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 harvested, counted on a hemocytometer and DNA content per dish was determined by :, ~! *Trademarks -22- ~ ~3 1 the Burton assay (Burton, K., Biochem. J. 62:315-323, 1956). Results are expressed as cells/dish or D~A content ~g/dish~ at 6 days following treatment. The results are shown in Figure 12.
' 5 Cultures of rat uterine or mammary tumor cells with cytosol preparations from rat uterus or liver containing a and ~ components showed cell growth inhibition within about 24 hours. Conversely, when the cells were cultured with cytosol preparations from mouse 0 mammary tumors which contained the ~ but not the ~
component, there was no substantial inhibition of cell growth across the wide range of concentrations tested.
These data, consistent with the resu:Lts of the competition experiments shown on Figures 8 and 9, demonstrated that the cytosol preparations from a normal mammary gland contain 15- to 20-fold more total inhibitor activity than those from tumors (Figure 2). One major diEference between cytosol preparations from normal and malignant tissues was that the malignant tissues were deficient in t.he ~ peaX inhibitor component (Figure 10). Hence, the peak component was the most important compound with respact to potential regulation of cell proliferation. As described in the present invention the ~ peak component was identified as methyl p-hydro~yphenyllactate.
Further, characterization and demonstration o~
the antitumor activity of the ~ inhibitor component was seen in examples using rat liver isolates. After the i~olation, the ~ inhibltor material was suspended in 2 ml of tissue culture medlum and add*d to cultures o MCF-7 hu~an breast cancer cell linesl The MCF 7 cells were plated in 3 ml o~ DMEM contalning 10% charcoal stripped ~etal bovine serum and allowed to attach to the surface of petri dishes. Following the establishment of monolayar cultures, approximately 24 to 36 hours, the floating cells were removed by aspiration, and 3 ml of , :, . . ~ ,. .. . . , "
, -23- ~ 33~ 7~
1 fresh DMEM were added to each culture. At this time various amounts, ranging from 7 to 32 ~l~ml, of the pooled ~ peak fractions or control medium were added to the cultures. The cells were cultured for an additional 5 24 hours. At the end of this culture period, the floating cells were remo~ed by aspiration and the attached cells resuspended in DMEM following mild trypsinization. The cells were then counted on a hemocytometer. with the increasing concentrations of the ~ peak inhibitor 1O material, the cell number was reduced in a dose dependent fashion within about 24 hours (Figure 13A). The control samples did not exhibit a similar decrease. The time course of the inhibition activity was also examined.
Approximately 32 ~1 of control medium or ~ peak inhibitor material was added to the cells and the floating cells were removed. Within approximately four hours of inhibitor addition, the inhibition of cell growth had begun. Ma~imum inhibition was observed in approximately 12 hours (Figure 13B).
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 rsgrow to a full monolayer.
It is postulated that tumor cell proliferation is very rapid because the tumor cell me~abolizes or inactivates the ~ peak inhibitor. This is supported by the observation that methyl p-hydroxyphenyllactate is Eound bound to Type II sites in normal tissues but is not ll Eound in malignant tissue. Cell proli~eration is - 30 regulated by ligand bindin~ ~o nuclear Type II sites. The number of unbound sites determines the rate of j proli~eration since tumor cells have an increased number of unbound nuclear Type II sites and thus are not inhibited, so tumor cell proliferation is dramatically accelerated. These cells are brought back into regulation ,, .
, -24- ~3~27~,~
and cell proliferation is decreased by administering a therapeutic dose of the inhibitors described in the present invention.
In addition to testing purified biological 5 preparations, the authentic compounds, methyl p-hydroxyphenyllactate and hydroxyphenyllactic acid, were added to cell cultures. As can be seen in Figure 12, methyl p-hydro~yphenyllactate inhibited MCF-7 human hreast cancer cells, but hydroxyphenyllactic acid did not.
o Furthermore, it can be seen that the methyl p-hydroxyphenyllactate inhibited MCF~7 breast cancer cells in a similar dose-dependent fashion. Results obtained by using the authentic compound were in agreement with the data from biological preparations.
UterotroPic Assay The rat uterus is e~quisitively sensitive to estrogen, and this hormone stimulates 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 ; rats with saline-sthanol vehicle, estradiol-17~ and the test compound of interest. Control rats were injec-ted only with saline-ethanol vehicle and estradiol-17~. 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 oE changes in cell proliferation and DNA synthesis.
~ he results of these experiments with various compound~ are shown in Table I.
Additional i~ vivo measurements using the uterotrDpic assay show the utility o these compounds for inhibiting cell proliferation. Low doses o methyl ,1 :
~3~7~
p-hydroxyphenyllactate, but not hydroxyphenyllactic acid, block estradiol stimulation of uterine growth in the immature rat (Figure 14 and Table I). However, higher doses of hydroxyphenyllactic acid showed some partial 5 antagonism. This is not surprising since it is known that hydroxyphenyllactic acid binds to nuclear Type II sites with a 20-fold lower affinity than methyl p-hydroxyphenyllactate.
These results demonstrate that the bound/unbound 10 ratio of nuclear Type II sites is important in the regulation of cell growth and that the primary bound inhibitor in normal cells is methyl p-hydroxyphenyllactate. Since the data demonstrated that tumor cells have the ability to inactivate methyl p-hydroxyphenyllactate, the analogues described in this invention were synthesized to avoid this inactivation.
Thus, as can be seen in Table I, administration of compounds with various side-chains and various substituents on the aromatic ring resulted in inhibition ~ uterine growth. One compound 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 ViVQ. Furthermore, e~periments with 1-(4-hydro~yphenyl)-3-butanone demonstrated that it binds to the nuclear Type II sites with a high affinity and blocks estradiol stimulation of uterine growth when injected into lmmature rats ~Figure 15). Thus 1-~4-hydroxyphenyl)-3-butanone is an e~ective inhibitor of tumor growth and regulator of cell proliferation.
. .
Table I. MeHPLA Analogue and Related Compound Effects on Uterine Growth and Nuclear Type II Site Binding rnhibition GROWTHa ~YPE IIb C~MPOUND INHIBITION (%) INHIBITION
5 methyl 3-(4-hydroxyphenyl) 2-hydroxypropionate 90 0.8 3-(4-hydro~yphenyl)-2-hydroxypropionic acid 0 80.0 l-(4-hydrogyphenyl)-3-butanone 96 2.0 methyl 3-(3,4-dihydroxyphenyl)-2-propenoate 70 l.0 methyl 3-~4-hydroxy-3-metho~yphenyl)-2~propenoate 56 6.0 methyl (4-hydroxyphenoxy)acetate 70 0.8 a Determined by the ability of the compound (l0~g) to block estradiol stimulation in the rat uterotropic assay.
b The concentration (nM) times 10-2 of the compound to inhibit nuclear Type II binding sites by 50~.
One skilled 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 prese~tly representative o~ the pre~erred emhodiments, are intended to be e~emplary, ~nd are not intended as limitations 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 o~ the invention and are defined by the scope of the appended claims.
What is claimed is:
.'.i
Claims (24)
1. The use of methyl p-hydroxyphenyllactate and pharmaceutically acceptable salts thereof for the preparation of a medicament for the treatment of cancer.
2. The use of a compound for the preparation of a medicament for the treatment of cancer wherein said compound is selected from the group consisting of the formulae:
, , , , , , and wherein, R1 is selected from the group consisting of H, alkyl groups containing 1 to 6 carbons, a phenyl group, a hydroxy-substituted phenyl group, a methoxy-substituted phenyl group, or a combination thereof;
R2 and R3 are selected from the group consisting of H, OH and OCH3; and R4 is selected from the group consisting of H and an alkyl group of 1 to 6 carbons.
, , , , , , and wherein, R1 is selected from the group consisting of H, alkyl groups containing 1 to 6 carbons, a phenyl group, a hydroxy-substituted phenyl group, a methoxy-substituted phenyl group, or a combination thereof;
R2 and R3 are selected from the group consisting of H, OH and OCH3; and R4 is selected from the group consisting of H and an alkyl group of 1 to 6 carbons.
3. The use of a compound for the preparation of a medicament for the treatment of cancer wherein said compound is 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, 1-(4-hydroxyphenyl)-3-butanone, 1-(4-hydroxyphenyl)-3-pentanone, methyl-(4-hydroxyphenoxy)acetate, and methyl 3-(3,4-dihydroxyphenyl)-2-propenoate .
4. The use of a compound for the preparation of a medicament for the inhibition of the growth of proliferating cells which include a Type II nuclear estrogen binding site wherein said compound is 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.
5. The use of a compound for preparation of a medicament for the inhibition of the growth of proliferating cells which include a Type II nuclear estrogen binding site wherein said compound is a biologically inhibiting dose of a compound selected from the formulae:
, , , , , , and wherein, R1 is selected from the group consisting of H, alkyl groups containing 1 to 6 carbons, a phenyl group, a hydroxy-substituted phenyl group, a methoxy-substituted phenyl group, or a combination thereof;
R2 and R3 are selected from the group consisting of H, OH and OCH3; and R4 selected from the group consisting of H
and an alkyl group of 1 to 6 carbons.
, , , , , , and wherein, R1 is selected from the group consisting of H, alkyl groups containing 1 to 6 carbons, a phenyl group, a hydroxy-substituted phenyl group, a methoxy-substituted phenyl group, or a combination thereof;
R2 and R3 are selected from the group consisting of H, OH and OCH3; and R4 selected from the group consisting of H
and an alkyl group of 1 to 6 carbons.
6. The use of a compound for preparation of a medicament for the inhibition of the growth of proliferating cells which include a Type II nuclear estrogen binding site wherein said compound is a biologically inhibiting dose of a compound 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, 1-(4-hydroxyphenyl)-3-butanone, 1-(4-hydroxyphenyl)-3-pentanone, methyl-(4-hydroxyphenoxy)acetate, and methyl 3-(3,4-dihydroxyphenyl)-2-propenoate.
7. The use according to claim 5, wherein said proliferating cells are estrogen responsive tissue selected from the group consisting of uterus, mammary gland, uterine tumors and mammary tumors.
8. The use according to Claim 7, wherein said estrogen responsive tissue is human breast cancer cells.
9. The use of a compound for preparation of a medicament for the treatment of benign prostatic hyperplasia wherein said compound is selected from the group consisting of methyl p-hydroxyphenyllactate analogues of methyl p-hydroxyphenyllactate, chemical derivatives of methyl p-hydroxyphenyllactate, and chemically related compounds.
10. The use according to Claim 9, wherein said compound is selected from the group consisting of the formulae:
, , , , , , and wherein, R1 is selected from the group consisting of H, alkyl groups containing 1 to 6 carbons, a phenyl group, a hydroxy-substituted phenyl group, a methoxy-substituted phenyl group, or a combination thereof;
R2 and R3 are selected from the group consisting of H, OH and OCH3; and R4 is selected from the group consisting of H and alkyl groups of 1 to 6 carbons.
, , , , , , and wherein, R1 is selected from the group consisting of H, alkyl groups containing 1 to 6 carbons, a phenyl group, a hydroxy-substituted phenyl group, a methoxy-substituted phenyl group, or a combination thereof;
R2 and R3 are selected from the group consisting of H, OH and OCH3; and R4 is selected from the group consisting of H and alkyl groups of 1 to 6 carbons.
11. The use according to Claim 10, wherein said compound is 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, 1-(4-hydroxyphenyl)-3-butanone, 1-(4-hydroxyphenyl)-3-pentanone, methyl-(4-hydroxyphenoxy)acetate, and methyl 3-(3,4-dihydroxyphenyl)-2-propenoate acid.
12. The use of a compound for the preparation of a medicament for use as an antitumor agent wherein said compound is selected from the group consisting of methyl p-hydroxyphenyllactate, analogues of methyl p-hydroxyphenyllactate, chemical derivatives of methyl p-hydroxyphenyllactate, and chemically related compounds.
13. The use according to Claim 12, wherein said compound is selected from the group consisting of the formulae:
, , , , , , and wherein, R1 selected from the group consisting of H, alkyl groups containing 1 to 6 carbons, a phenyl group, a hydroxy-substituted phenyl group, a methoxy-substituted phenyl group, or a combination thereof;
R2 and R3 are selected from the group consisting of H, OH and OCH3; and R4 is selected from the group consisting of H and alkyl groups of 1 to 6 carbons.
, , , , , , and wherein, R1 selected from the group consisting of H, alkyl groups containing 1 to 6 carbons, a phenyl group, a hydroxy-substituted phenyl group, a methoxy-substituted phenyl group, or a combination thereof;
R2 and R3 are selected from the group consisting of H, OH and OCH3; and R4 is selected from the group consisting of H and alkyl groups of 1 to 6 carbons.
14. The use according to Claim 13, wherein said compound is 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, 1-(4-hydroxyphenyl)-3-butanone, 1-(4-hydroxyphenyl)-3-pentanone, methyl-(4-hydroxyphenoxy)acetate, and methyl 3-(3,4-dihydroxyphenyl)-2-propenoate.
15. A compound of the formula wherein, R1 is CH3;
R2 is H;
R3 is OH; and R4 is CH3.
R2 is H;
R3 is OH; and R4 is CH3.
16. The use of a compound for the manufacture of a medicament for the treatment of proliferative diseases and cancer wherein said compound is selected from the group consisting of:
, and , , and pharmaceutically acceptable salts thereof, wherein, R1 is selected from the group consisting of H, alkyl groups containing 1 to 6 carbons, a phenyl group, a hydroxy-substituted phenyl group, a methoxy-substituted phenyl group, or a combination thereof; and R2 and R3 are not both H and are selected from the group consisting of H, OH and OCH3.
, and , , and pharmaceutically acceptable salts thereof, wherein, R1 is selected from the group consisting of H, alkyl groups containing 1 to 6 carbons, a phenyl group, a hydroxy-substituted phenyl group, a methoxy-substituted phenyl group, or a combination thereof; and R2 and R3 are not both H and are selected from the group consisting of H, OH and OCH3.
17. The use according to claim 16 wherein said compound is selected from the group consisting of , , , and .
18. The use according to claim 16 wherein said compound is selected from the group consisting of 3-(4-Hydroxyphenyl)-1-penyl-2-propen-1-one and 4-(4-Hydroxyphenyl)-3-buten-2-one, analogs, chemical derivatives and chemically related compounds and pharmaceutically acceptable salts thereof to an individual in need of said treatment.
19. The use of a compound for the manufacture of a medicament for inhibiting the growth of proliferating cells wherein said compound is used in a biologically inhibiting dose and is selected from the group consisting of:
, , and and pharmaceutically acceptable salts thereof, wherein, R1 is selected from the group consisting of H, alkyl groups containing 1 to 6 carbons, a phenyl group, a hydroxy substituted phenyl group, a methoxy-substituted phenyl group, or a combination thereof; and R2 and R3 are not both H and are selected from the group consisting of H, OH and OCH3.
, , and and pharmaceutically acceptable salts thereof, wherein, R1 is selected from the group consisting of H, alkyl groups containing 1 to 6 carbons, a phenyl group, a hydroxy substituted phenyl group, a methoxy-substituted phenyl group, or a combination thereof; and R2 and R3 are not both H and are selected from the group consisting of H, OH and OCH3.
20. The use according to claim 19 wherein said compound is selected from the group consisting of , , and .
21. The use according to claim 19 wherein said compound is selected from the group consisting of 3-(4-Hydroxyphenyl)-1-phenyl-2-propen-1-one and 4-(4-Hydroxyphenyl)-3-buten-2-one, analogs, chemical derivatives and chemically related compounds to the proliferating cells.
22. The use of a compound for the manufacture of a medicament for treating benign prostatic hyperplasia wherein said compound is selected from the group consisting of:
, and , , and pharmaceutically acceptable salts thereof, wherein, R1 is selected from the group consisting of H, alkyl groups containing 1 to 6 carbons, a phenyl group, a hydroxy-substituted phenyl group, a methoxy-substituted phenyl group, or a combination thereof; and R2 and R3 are not both H and are selected from the group consisting of H, OH and OCH3.
, and , , and pharmaceutically acceptable salts thereof, wherein, R1 is selected from the group consisting of H, alkyl groups containing 1 to 6 carbons, a phenyl group, a hydroxy-substituted phenyl group, a methoxy-substituted phenyl group, or a combination thereof; and R2 and R3 are not both H and are selected from the group consisting of H, OH and OCH3.
23. The use according to Claim 22, wherein said compound is selected from the group consisting of , , and .
24. The use according to Claim 22, wherein said compound is selected from the group consisting of 3-(4-Hydroxyphenyl)-1-propen-1-one and 4-(4-Hydroxyphenyl)-3-buten-2-one, and their analogs, chemical derivatives and chemically related compounds and pharmaceutically related salts thereof.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US7919987A | 1987-07-28 | 1987-07-28 | |
| US079,199 | 1987-07-28 | ||
| US219,680 | 1988-07-15 | ||
| US07/219,680 US5216024A (en) | 1987-07-28 | 1988-07-15 | Cell growth inhibitors and methods of treating cancer and cell proliferative diseases |
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| CA1332742C true CA1332742C (en) | 1994-10-25 |
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| Country | Link |
|---|---|
| US (1) | US5216024A (en) |
| EP (1) | EP0374184B1 (en) |
| JP (1) | JPH03501249A (en) |
| AT (1) | ATE99931T1 (en) |
| AU (1) | AU625313B2 (en) |
| CA (1) | CA1332742C (en) |
| DE (1) | DE3887151T2 (en) |
| WO (1) | WO1989000849A1 (en) |
Families Citing this family (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4698225A (en) * | 1985-04-15 | 1987-10-06 | Morrison David G | Granular binding agent for pellets |
| US6515009B1 (en) | 1991-09-27 | 2003-02-04 | Neorx Corporation | Therapeutic inhibitor of vascular smooth muscle cells |
| US5811447A (en) | 1993-01-28 | 1998-09-22 | Neorx Corporation | Therapeutic inhibitor of vascular smooth muscle cells |
| US6251920B1 (en) | 1993-05-13 | 2001-06-26 | Neorx Corporation | Prevention and treatment of cardiovascular pathologies |
| US6306421B1 (en) | 1992-09-25 | 2001-10-23 | Neorx Corporation | Therapeutic inhibitor of vascular smooth muscle cells |
| US6663881B2 (en) | 1993-01-28 | 2003-12-16 | Neorx Corporation | Therapeutic inhibitor of vascular smooth muscle cells |
| US5981568A (en) | 1993-01-28 | 1999-11-09 | Neorx Corporation | Therapeutic inhibitor of vascular smooth muscle cells |
| US6491938B2 (en) | 1993-05-13 | 2002-12-10 | Neorx Corporation | Therapeutic inhibitor of vascular smooth muscle cells |
| CA2162586C (en) * | 1993-05-13 | 2006-01-03 | David J. Grainger | Prevention and treatment of pathologies associated with abnormally proliferative smooth muscle cells |
| IL112205A0 (en) * | 1994-01-06 | 1995-03-15 | Res Dev Foundation | Curcumin, analogues of curcumin and novel uses thereof |
| CA2198752A1 (en) | 1994-08-31 | 1996-03-07 | Jason Scott Sawyer | Methods for identifying and treating resistant tumors |
| US5710179A (en) * | 1995-04-05 | 1998-01-20 | Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo | Antitumor agent |
| US7611533B2 (en) | 1995-06-07 | 2009-11-03 | Cook Incorporated | Coated implantable medical device |
| WO1997008934A2 (en) * | 1995-06-19 | 1997-03-13 | Ontogen Corporation | Aryl acrylic acid derivatives useful as protein tyrosine phosphatase inhibitors |
| GB0505509D0 (en) * | 2005-03-17 | 2005-04-27 | Ic Innovations Ltd | Compounds |
| WO2007026875A1 (en) * | 2005-09-02 | 2007-03-08 | Ube Industries, Ltd. | PROCESS FOR PRODUCING OPTICALLY ACTIVE (S OR R)-α-HYDROXY ACID AND OPTICALLY ACTIVE (R OR S)-α-HYDROXY ACID ESTER |
| ES2376277T3 (en) * | 2006-09-01 | 2012-03-12 | Piramal Life Sciences Limited | USE OF THE CA? CAFEIC ACID AND DERIVATIVES AS ANTI-TARGET AGENTS. |
| CN102697757B (en) * | 2012-05-18 | 2014-06-04 | 广州军区广州总医院 | Application of p-hydroxy benzylidene acetone in preparation of drugs for preventing and/or treating encephalopathy |
| CN113480418B (en) * | 2021-07-13 | 2022-09-06 | 上海健康医学院 | Compound and application thereof in preparing medicine for treating cancer |
Family Cites Families (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2451149A (en) * | 1942-11-16 | 1948-10-12 | Nipa Lab Ltd | Manufacture of materials resistant to or active against microorganisms |
| GB1438652A (en) * | 1972-07-19 | 1976-06-09 | Shell Int Research | Hydroxyl phenyl-substituted ketones and their preparation |
| US4124724A (en) * | 1975-12-08 | 1978-11-07 | Agoro John W | Crystalline caffeic acid derivatives and compositions and method for treating snakebite |
| NL7804290A (en) * | 1977-05-12 | 1978-11-14 | Henkel Kgaa | IGNITION RETARDANT FOR COSMETIC PREPARATIONS. |
| GB1591063A (en) * | 1977-07-11 | 1981-06-10 | Ici Ltd | Monoalkylation of dihydroxybenzenes |
| JPS54160740A (en) * | 1978-06-09 | 1979-12-19 | Takeda Chem Ind Ltd | Anti-fibrotic agent |
| US4219569A (en) * | 1978-10-10 | 1980-08-26 | The Upjohn Company | Process for treating inflammation |
| FR2481270A1 (en) * | 1980-04-24 | 1981-10-30 | Roussel Uclaf | NOVEL SUBSTITUTED DERIVATIVES OF 4-PHENYL 4-OXO 2-HYDROXY BUTANOIC ACID, PROCESS FOR THEIR PREPARATION AND APPLICATIONS AS MEDICAMENTS |
| IT1171604B (en) * | 1981-10-22 | 1987-06-10 | Roussel Maestretti Spa | 4-FENYL-4-OSSOBUTEN-2-OICO ACID DERIVATIVES EQUIPPED WITH PHARMACOLOGICAL PROPERTIES AND THEIR PREPARATION PROCEDURE |
| DE3150233A1 (en) * | 1981-12-18 | 1983-06-30 | Basf Ag, 6700 Ludwigshafen | METHOD FOR PRODUCING 2- (HYDROXIPHENOXI) CARBONIC ACID ESTERS |
| JPS59104340A (en) * | 1982-12-07 | 1984-06-16 | Ube Ind Ltd | Preparation of benzylacetone |
| GB2141626B (en) * | 1983-04-25 | 1987-01-28 | Kao Corp | Cinnamic acid derivatives for lightening melanin pigmentation of skin |
| JPS60222437A (en) * | 1984-04-17 | 1985-11-07 | Ube Ind Ltd | Production of 1-phenyl-2-alkanone |
| US4733002A (en) * | 1984-05-23 | 1988-03-22 | Green Cross Corporation | Lipoxygenase inhibitor |
| JPS60248645A (en) * | 1984-05-23 | 1985-12-09 | Green Cross Corp:The | Caffeic acid derivative |
| JPH0623130B2 (en) * | 1984-07-10 | 1994-03-30 | 三井東圧化学株式会社 | Method for producing α-keto acid |
| IT1190340B (en) * | 1986-06-06 | 1988-02-16 | Roussel Maestretti Spa | DERIVATIVES OF 4-FENYL 4-BONE 2-BUTENOIC ACID, THEIR PREPARATION PROCEDURE AND THEIR USE AS MEDICINAL PRODUCTS |
| WO1987007835A1 (en) * | 1986-06-21 | 1987-12-30 | Dainippon Ink And Chemicals, Inc. | Drug for treatment and prophylaxis of kidney and liver diseases |
-
1988
- 1988-07-15 US US07/219,680 patent/US5216024A/en not_active Expired - Fee Related
- 1988-07-20 JP JP63506993A patent/JPH03501249A/en active Pending
- 1988-07-20 EP EP88908058A patent/EP0374184B1/en not_active Expired - Lifetime
- 1988-07-20 DE DE3887151T patent/DE3887151T2/en not_active Expired - Fee Related
- 1988-07-20 WO PCT/US1988/002476 patent/WO1989000849A1/en active IP Right Grant
- 1988-07-20 AU AU23152/88A patent/AU625313B2/en not_active Ceased
- 1988-07-20 AT AT88908058T patent/ATE99931T1/en not_active IP Right Cessation
- 1988-07-27 CA CA000573139A patent/CA1332742C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| WO1989000849A1 (en) | 1989-02-09 |
| AU2315288A (en) | 1989-03-01 |
| US5216024A (en) | 1993-06-01 |
| DE3887151T2 (en) | 1994-06-30 |
| EP0374184A1 (en) | 1990-06-27 |
| JPH03501249A (en) | 1991-03-22 |
| DE3887151D1 (en) | 1994-02-24 |
| EP0374184B1 (en) | 1994-01-12 |
| ATE99931T1 (en) | 1994-01-15 |
| AU625313B2 (en) | 1992-07-09 |
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