CA2244112A1 - Methods of inhibiting colon tumors - Google Patents

Abstract

A method of inhibiting colon cancer comprising administering to a mammal in need thereof an effective amount of a compound having formula (I), wherein R1 and R3 are independently hydrogen, -CH3, (a), or (b), wherein Ar is optionally substituted phenyl; R2 is selected from the group consisting of pyrrolidine, hexamethyleneimino, and piperidino; or a pharmaceutically acceptable salt or solvate thereof.

Description

METHODS OF INHIBITING COLON TUMORS

r Backaround of the Invention Colon cancer, which includes rectal and colorectal adenocarcinoma, primary or metastatic adenocarcinoma, and the like, is a major health concern today in terms of disease incidence. It has been estimated that one out of twenty-five Americans will develop some form of colon cancer during the course of his lifetime.
Sugarbaker, P.H. et al., in Cancer, DeVita, V.T., et al.
(Eds.), Lippincott Publ. Philadelphia, pp. 795-884 (1985).
In terms of treatment, surgery has been widely used with good, mixed or less than favorable results.
Particularly among colon cancer patients with locally advanced or metastatic disease, the prognosis is extremely unfavorable with a high rate of mor~idity and mortality.
Drug therapies have been tried with less than favorable results. The anti-neoplastic compound, 5-fluorouracil (5-FU), has been the major drug of choice in treating colon cancer, and its use has proven to be only marginally effective. 5-FU may reduce colon tumor size temporarily but there has been little evidence to show that the survival times o~ patients have been substantially prolonged or that "cures'~ are obtained (based on five year periods of remission). Chemotherapy with 5-FU has been used in patients with metastases to the liver, but temporary improvement is observed in only 25 percent or less of such cases, and the overall, survival is not significantly affected. LaMont, J.T. and Isselbacher, K., in Harrison's Principles of Internal Medicine (lOth ed.) McGraw-Hill, New York, p. 1764, (1983). Despite the marginal effectiveness of 5-FU, no other drugs or combination therapy has been convincingly shown to be more effective. Sugarbaker P.H. et al., supra. Woolley, P.V., et al., New Eng. ~. Med., 312:1465 (1985).

W 097/26877 PCTrUS97/02286 Drug therapies have also been evaluated with respect to treating human cancer, e.g., human colon cancer xenograft lines, in which human tumors are serially heterotransplanted into immunode~icient, so call ~nude~
mice, and the mice then tested ~or their responsiveness to a speci~ic drug. Giovanella, B.C., et al., Cancer 5(7):1146 (1~83). The data obtained in these studies strongly support the validity o~ heterotransplanted human tumors, including colon tumors, into ;mmllnode~icient m~mm~ ~ S, such as ~'nude" mice, as a predictive model for testing the e~fectiveness o~ anticancer agents.
The sodium salt of a naturally occurring alkaloid, camptothecin, was used in a brief clinical trial to evaluate toxic e~ects on patients with advanced incurable cancers. Gottlieb, G.A , et al., Cancer Chemotherapy Rep. 54:461 (1970). Few conclusions could be drawn from this study, although median survival ~or those patients responding to the treatment increased ~rom about two months to about 3.5+ months.
Camptothecin derivatives or analogs have been synthesized and employed as antileukemic agents in mice (see e.g., Wani, M.C., et al., ~. Med. Chem. 23:544, 1980;
Wani M.C., et al,., ~. Med. Chem. 30:1774 (1987); and Wani, M.C. et al., J. Med. Chem. 30:2317 (1987).
In U S. Pat. Nos. 4,473,692 and 4,545,880, Miyasaka et al. disclose 10-substituted camptothecin derivatives and process ~or their preparation. The 10-substituted camptothecin derivatives are said to possess anti-tumor activity with reduced or slight toxicity in comparison to the parent camptothecin compound. Miyasaka et al. do not disclose speci~ic tumor targets nor do they indicate what level o~ reduced or slight toxicity is achieved by using their 10-substituted camptothecin compounds.
Recently, the enzyme, human topoisomerase I, has been examined in various human cancers, e.g., leukemia, CA 02244ll2 l998-07-24 W 097126877 PCT~USg7/02286 lymphoma. Potmesil M. et al., Cancer Res. 48:3537 (1988).
Human topoisomerase I is known to be a monomeric protein with an apparent molecular weight of lOO,OQ0 daltons. The swivel-like function of the enzyme has been implicated in various DNA transactions (replication, transcription and recombination ). Purified m~mm~l ian topoisomerase I
relaxes positively-supercoiled as well as negatively-supercoiled DNA in a mechanism which involves the transient breakage of one of the two DNA strands and the formation of a covalent topoisomerase I-DNA complex. In this complex, the enzyme is covalently linked to the 3'-phosphoryl end of the broken DNA backbone.
Recently, it was disclosed that topoisomerase I
enzyme levels were on average higher in cancerous tissue, e. g., surgical specimens o~ colorectal carcinoma, in comparison to the enzyme level in normal mucosa (Hsiang, Y.-H., et al., Proc Ann Meet of~ the Amer. Assoc. Cancer Res. 29:172 (1988) Abstract. Although it was stated that topoisomerase I could be considered as an alternative target in chemotherapy of this (colorectal carcinoma) disease, no disclosure or suggestion at all was made as to any specific topoisomerase I interacting drugs.
In view of very poor 5 -year survival rates (approximately 50 percent or less) ~or patients undergoing conventional treatment for colonic cancer, e. g., surgical resection, or chemotherapy with 5-FU, it would be extremely useful to discover a new way to effectively treat human malignant colon tumors using drugs or compounds, ~ollowing surgery, for example, which helps to establish the diagnosis and removes the bulk of cancer. The drug treatment could al~o be help~ul for patients with advanced disease which has metastasized or spread to various organs, so that surgery is not feasible to remove all the cancerous ~ tissues.

W O 97/26877 PCTrUS97/02286 SU~n~ARY OF THE I ~ ENTION

The invention provides methods for inhibiting colon cancer in m~mm~l 5 by administering to the mammal in need thereof of an effective amount of a compound of formula I.

~\~ OCH2CH2--R2 Rlo~ ~ '~ } oR3 (I) wherein R1 and R3 are independently hydrogen, O O
-CH3 -C-(Cl-C6 alkyl), or -C-Ar , wherein Ar is optionally substituted phenyl;
R2 is selected from the group consisting of pyrrolidino, hexamethyleneimino, and piperidino; and pharmaceutically acceptable salts and solvates thereo~.

~F.TATT.F.n DESCRIPTION OF THE T ~ ENTION
The current invention concerns the discovery that a select group of 2-phenyl-3-aroylbenzothiophenes (benzothiophenes), those of formula I, are useful for inhibiting colon cancer.
The methods of use provided ~y this invention are practiced by administering to a human in need thereof a dose of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof, that is effective to inhibit colon cancer. The term ~inhibit~ includes its -W 097/26877 PCTrUS97102286 generally accepted me~ni ng which includes prohibiting, preventing, restraining, and slowing, stopping, or reversing progression, severity, or a resultant symptom or effect.
Raloxi~ene, a compound of this invention wherein it is the hydrochloride salt of a compound of formula 1, Rl and R3 are hydrogen and R2 is l-piperidinyl, is a nuclear regulatory molecule. Raloxifene has been shown to bind to the estrogen receptor and was originally thought to be a molecule whose function and pharmacology was that of an anti-estrogen in that it blocked the ability of estrogen to activate uterine tissue and estrogen dependent breast cancers. Indeed, raloxifene does block the action of estrogen in some cells; however in other cell types, raloxifene activates the same genes as estrogen does and displays the same pharmacology, e.gr, osteoporosis, hyperlipidemia. As a result, raloxifene has been referred to as an anti-estrogen with mixed agonist-antagonist properties. The unique profile which raloxifene displays and differs from that of estrogen is now thought to be due to the uni~ue activation and/or suppression of various gene functions by the raloxifene-estrogen receptor complex as opposed to the activation and/or suppression of genes by the estrogen-estrogen receptor complex. Therefore, although raloxifene and estrogen utilize and compete for the same receptor, the pharmacological outcome from gene regulation o~ the two is not easily predicted and is uni~ue to each.
Generally, the compound is formulated with common excipients, diluents or carriers, and compressed into tablets, or formulated as elixirs or solutions for convenient oral ~m; ni stration, or administered by the intramuscular or intravenous routes. The compounds can be administered transdermally, and may be formulated as sustained release dosage forms and the like.

WO 97/~6877 PCTnUS97/02286 The compounds used in the methods of the current invention can be made according to established procedures, such as those detailed in U.S. Patent Nos. 4,133,814, 4,418,068, and 4,380,635 all o~ which are incorporated by re~erence herein. In general, the process starts with a benzo[b]thiophene having a 6-hydroxyl group and a 2-(4-hydroxyphenyl) group. The starting compound is protected, acylated, and deprotected to form the formula I compounds.
Examples of the preparation o~ such compounds are provided in the U.S. patents discussed above. Optionally substituted phenyl includes phenyl and phenyl substituted once or twice with Cl-C6 alkyl, Cl-C4 alkoxy, hydroxy, nitro, chloro, fluoro, or tri~chloro or ~luoro)methyl.
The compounds used in the methods of this invention form pharmaceutically acceptable acid and base addition salts with a wide variety of organic and inorganic acids and bases and include the physiologically acceptable salts which are o~ten used in pharmaceutical chemistry.
Such salts are also part of this invention. Typical inorganic acids used to ~orm such salts include hydrochloric, hydrobromic, hydroiodic, nitric, sul~uric, phosphoric, hypophosphoric and the like. Salts derived from organic acids, such as aliphatic mono and dicarboxylic acids, phenyl substituted alkanoic acids, hydroxyalkanoic and hydroxyalkandioic acids, aromatic acids, aliphatic and aromatic sul~onic acids, may also be used. Such pharmaceutically acceptable salts thus include acetate, phenylacetate, tri~luoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate, B-hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate, caprate, caprylate, chloride, c;nn~m~te~
citrate, ~ormate, ~umarate, glycollate, heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, isonicotinate, -WO 97/26877 PCTrUS97/02286 nitrate, oxalate, phthalate, terephthalate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate, propionate, phenylpropionate, salicylate, sebacate, succinate, suberate, sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate, benzene-sulfonate, p-bromophenylsulfonate, chlorobenzenesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, methanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, p-toluenesulfonate, xylenesulfonate, tartarate, and the like. A preferred salt is the hydrochloride salt.
The pharmaceutically acceptable acid addition salts are typically formed by reacting a compound of formula I with an equimolar or excess amount of acid. The reactants are generally combined in a mutual solvent such as diethyl ether or benzene. The salt normally precipitates out of solution within about one hour to 10 days and can be isolated by filtration or the solvent can be stripped off by conventional means.
Bases commonly used for formation of salts include ammonium hydroxide and alkali and alkaline earth metal hydroxides, carbonates, as well as aliphatic and primary, secondary and tertiary amines, aliphatic diamines.
sases especially useful in the preparation of addition salts include ammonium hydroxide, potassium carbonate, methylamine, diethylamine, ethylene diamine and cyclohexylamine.
The pharmaceutically acceptable salts generally have enhanced solubility characteristics compared to the compound from which they are derived, and thus are often more ~m~n~hle to formulation as liquids or emulsions.
Pharmaceutical formulations can be prepared by procedures known in the art. For example, the compounds can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, suspensions, powders, and the like. Examples of excipients, diluents, and carriers that are suitable for such formulations include the following: fillers and extenders such as starch, sugars, mannitol, and silicic derivatives; binding agents such as carboxymethyl cellulose and other cellulose derivatives, alginates, gelatin, and polyvinyl pyrrolidone;
moisturizing agents such as glycerol; disinteyrating agents such as calcium carbonate and sodium bicarbonate; agents for retarding dissolution such as paraffin; resorption accelerators such as ~uaternary ammonium compounds; surface active agents such as cetyl alcohol, glycerol monostearate;
adsorptive carriers such as kaolin and bentonite; and lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols.
The compounds can also be formulated as elixirs or solutions for convenient oral ~mi ni stration or as solutions appropriate for parenteral ~mi n; stration, for instance by intramuscular, subcutaneous or intravenous routes. Additionally, the compounds are well suited to formulation as sustained release dosage forms and the like.
The formulations can be so constituted that they release the active ingredient only or preferably in a particular part of the intestinal tract, possibly over a period of time. The coatings, envelopes, and protective matrices may be made, for example, ~rom polymeric substances or waxes.
The particular dosage of a compound of formula I
required to inhibit colon cancer, or any other use disclosed herein, and according to this invention will depend upon the severity of the condition, the route of administration, and related factors that will be decided by the attending physician. Generally, accepted and effective daily doses will be from about 0.1 to about 1000 mg/day, and more typically from about 50 to about 200 mg/day. Such dosages will be ~min; stered to a subject in need thereof from once to about three times each day, or more often as needed to effectively inhibit colon cancer.

CA 02244ll2 l998-07-24 W O 97/26877 PCTnUS97tO2286 It is usually preferred to administer a compound o~ ~ormula I in the form of an acid addition salt, as is t customary in the administration of pharmaceuticals bearing a basic group, such as the piperidino ring. It is also advantageous to administer such a compound by the oral route. For such purposes the following oral dosage forms are available.

Formulations In the formulations which follow, ~Active ingredient~ means a compound of formula I.

Formulation 1: Gelatin Capsules Hard gelatin capsules are prepared using the following:

IngredientQuantity (mg/capsule) Active ingredient0.1 - 1000 Starch, NF O - 650 Starch ~lowable powderO - 650 Silicone fluid 350 centistokes O - 15 The ingredients are blended, passed through a No. 45 mesh U.S. sieve, and filled into hard gelatin capsules.
Examples of specific capsule formulations of 2~ raloxifene that have been made include those shown below:

Formul~tion 2: Raloxifene capsule IngredientQuantity (mg/capsule) Raloxi~ene Starch, NF 112 Starch ~lowable powder225.3 Silicone ~luid 350 centistokes 1.7 CA 02244ll2 l998-07-24 W 097l26877 PCTAUS97/02286 Formulation 3: Raloxi~ene capsule IngredientQuantity (ma/capsule) Raloxifene 5 Starch, NF 108 Starch flowable powder225.3 Silicone fluid 350 centistokes 1.7 Formulation 4: Raloxi~ene capsule IngredientQuantity (mg/capsule) Raloxifene 10 Starch, NF 103 Starch flowable powder225.3 Silicone fluid 350 centistoke~ 1.7 Formlllatlon 5: Raloxi~ene capsule IngredientQuantity (mg/capsule) Raloxifene 50 Starch, NF 150 Starch flowable powder397 Silicone fluid 350 centistokes 3.0 The specific formulations above ma~ be changed in compliance with the reasonable variations provided.
A tablet formulation is prepared using the ingredients below:

CA 02244ll2 l998-07-24 W O 97t26877 PCT~US97/02286 Formulation 6: Tablets Ingredient Quantity (mq/tablet) Active ingredient 0.1 - 1000 ~ Cellulose, microcrystalline 0 - 650 Silicon dioxide, fumed0 - 6S0 Stearate acid 0 - 15 The components are blended and compressed to form tablets.
Alternatively, tablets each containing 0.1 -1000 mg o~ active ingredient are made up as follows:

Formulation 7: Tablets Ingredient Quantity (mg/tablet) Active ingredient 0.1 - 1000 Starch ~5 Cellulose, microcrystalline 35 Polyvinylpyrrolidone 4 (as 10~ solution in water) Sodium car~oxymethyl cellulose 4.5 Magnesium stearate o 5 Talc The active ingredient, starch, and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with the resultant powders which are then passed through a No. 14 mesh U.S. sieve. The granules so produced are dried at 50~-60~ C and passed through a No. 18 mesh U.S. sieve.
The sodium carboxymethyl starch, magnesium stearate, and talc, previously passed through a No. 60 U.S. sieve, are then added to the granules which, a~ter mixing, are compressed on a tablet machine to yield tablets.
Suspensions each containing 0.1 - 1000 mg of medicament per 5 mL dose are made as follows:

CA 02244ll2 l998-07-24 WO 97/26877 PCT~US97/02286 Formulation 8: Suspensions InqredientQuantity (mg/5 ml) Active ingredient0.1 - 1000 mg Sodium c~rboxymethyl cellulo~e 50 mg Syrup 1.25 mg Benzoic acid solution0.10 mL
Flavor ~.v.
Co~or q.v.
Purified water to 5 mL

The medicament is passed through a No. 45 mesh U.S. sieve and mixed with the sodium carboxymethyl cellulose and syrup to form a smooth paste. The benzoic acid solution, flavor, and color are diluted with some of the water and added, with stirring. Sufficient water is then added to produce the required volume.
Desmoid tumors are rare non metastatic tumors of fibrous origin. Clinical correlates suggest that steroid hormones may have a role in the natural history of these tumors: it is predominantly seen in female patients of child-bearing age and regression of these tumors have been associated with menopause or with antiestrogen therapy.
The aim of this work was to identify estrogen receptors in desmoid tumor primary cells, and to evaluate the effect o~ a compound of formula I on desmoid cells in primary culture.
Because sometimes desmoid tumors develop in patients with Familial Adenomatous Polyposis (FAP), that can degenerate in colon or rectal cancer, we have tested the inhibitory effect of Compound Ia on cellular growth of an adenocarcinoma cell line, (HCT8), and fibroblasts from colon cancer bioptic specimens.
Compound Ia is a compound of formula I wherein R2 is pyrrolidino, and R1 and R3 are hydrogen.

W O 97/26877 PCT~US97/02286 sinding studies are performed using intact cells. Desmoid cells are plated on 6-well plates in growth medium (Coon's modified Ham's F12 supplemented with 10%
FCS). A~ter 24 hours, the growth medium is substituted with steady state medium without phenol red, and cells are maintained in starvation for 24 hours. Then cells are incubated for one hour with 1 ml of medium without phenol red cont~;n;ng 25 mM HEPES and 0.5% EtOH (binding buffer) and increasing concentrations (0.05- 10 nM) of [3H]17BE2 with or without a 500-fold excess of unlabeled 17BE2 and Compound Ia. After incubation, cells are washed two times with 800 ~l of binding buffer and lysed with lN NaOH at 70~C for 30 minutes. Four N HC1 is then added to each well for neutralization. Radioactivity is measured by liquid scintillation spectroscopy. ER binding affinity and binding capacity are evaluated by Scatchard analysis.
All subse~uent steps are performed at 0-4~C.
The pulverized tissue is homogenized with two 10-sec burst in a polytron homogenizer separated by a 30-sec cooling period in the following buffer: 10 mM Tris-HCl, 5 mM EDTA, 10 mM sodium molybdate, 10 mM dithiothreitol, 10% glycerol (v/v), pH 7.4. The homogenate is centrifuged at 7000 g for 20 min and the pellet is discarded, the supernatant was recentrifuged at 105000 g for 60 min to obtain cytosol for estrogen receptor analysis. Cytosol is diluted to 1-2 mg protein/ml. Cytosol protein is determined according to the method of sradford. For estrogen receptor assessment, cytosol is incubated for 16 hr at 4~C over a concentration range of 0.05-5 nM of [3H]17BE2 with or without a 500-fold excess of unlabeled 17BE2 and Compound Ia. ER b; n~; ng affinity and binding capacity are evaluated by Scatchard analysis.
Cells are plated on 6 well plates at a density of 8x104 cell for well in growth medium. After 24 hours, the cells are stimulated in growth medium without phenol CA 02244ll2 l998-07-24 W O 97/26877 PCTrUS97/02286 red supplemented with 0.1% DMF, 0.1% EtOH, and different concentrations of Compound Ia (2x10-5M, 10-5M, 5x10-6M, 10-6M) .
Cells are incubated six days, detached with trypsin/ethylenediamine tetracetic acid solution and then the growth is evaluated by counting to the microscope. The same method is used ~or colon cancer primary ~ibroblasts cell line and ~or HCT8 cell line: this line is cultured in RPMI and incubated 4 days after stimulation.
Collagen type I in culture media and cell layers are measured using an enzyme-linked immunoassay (ELISA).
srie~ly, cells are incubated ~or 24 hrs in supplement-~ree Coon's modified Ham F12 medium containing 50~g/ml ascorbic acid and 100~1g/ml R~minQpropionitryl fumarate.
Culture media are harvested and appropriately diluted in 0 1 M carbonate/bicarbonate bui~er (pH 9. 6) and then used for coating the ELISA plates, overnight at 4~C
ELISA plates are incubated 1.5 hrs at 37~C in PBS
containing 5% o~ milk powder (PBS Blotto) to saturate non specific binding sites, 2 hrs at 37~C with PBS slotto containing the specific polyclonal antibody, and 1.5 hrs at 37~C in PBS Blotto cont~;n,n~ goat antirabbit IgG-alkaline phosphatase conjugated complex (Sigma Chemical Co., st.
Louis, MO).
Samples are then exposed to 10~ diethanolamine (pH 9.8) with 5~g/ml Mg++ and 1 mg/ml paranitrophenyl-phosphate as a substrate of alkaline phosphatase at room temperature. optical density is read at 405 nM, and concentrations calculated on the basis of the st~n~rd curve. Cell monolayers are harvested in 0.5 N NaOH and sonicated to determine cellular Collagen type I. Cell extracts are then diluted in 0.1 M carbonate/bicarbonate bu~er (pH 9.6) and used for the coating o~ the ELISA
plates. Standards and samples are assayed in triplicate.
Results are expressed as ~g protein/~g cellular DNA. DNA
content is spectro~luorimetrically measured.

W 097/26877 PCT~US97/02286 sinding experiments are performed using [3H]17~E2 as ligand in primary desmoid tumors cells and in ~rozen specimens of desmoid tumor.
In both experiments, [3H]17gE2 binding was slightly (approximately 10%) displaced by 500-fold excess of both unlabeled estrogen and Compound Ia. Scatchard analysis o~ [3H]17BE2 binding data using the computer b; n~ i n~ program LIGAND (Munson P.J., Rodbard D. Anal.
Bioc~em. 1980; 107:220-39.) shows the presence o~ ER in three di~erent cultures and in two di~erent cytosol preparation from bioptic specimens of desmoid tumors.
In the growth assay, desmoid tumors primary cells are stimulated when exposed to various concentrations o~ Compound Ia. The result is a cellular growth inhibition with increasing Compound Ia concentrations (Table 1).
Similar results are obtained with the HCT8 cell line ~Table 2) and with a colon cancer ~ibroblastic cell line (Table 3).
Desmoid cells are inhibited in a dose-dependent fashion by Compound Ia at concentrations o~ 10-5M, 5x10-6M, 10-6M, with maximal inhibitory e~fect at lQ-~;M
concentration (Table 4).
Compound Ia is able to displace 17~E2 binding to desmoid tissue only at very high concentrations (500-~old excess).
Compound Ia is able to signi~icantly inhibit desmoid cell proli~eration at micromolar concentrations.
In addition, at similar concentrations the compound inhibits the proli~eration o~ epithelial and ~ibroblastic cells derived ~rom human colon cancer.
Type I collagen production is also signi~icantly reduced in desmoid cell in primary culture by the Compound Ia.
At all the conditions ~electroporation, Ca/P
precipitation, lyposomes) tested ~or trans~ection o~
desmoid cells with the estrogen responsive elements, the CA 02244112 l99X-07-24 W 097/26877 PCT~US97/02286 cells are damaged, resulting not suitable ~or "in vitro~
analysis.
Table 1 Com~ound Ia (Mol/L)Cell x 10-4 Control 12.3 2.10-5 0.1 10-5 2.8 5,10-6 7.0 10-6 10 . O

~able 2 Com~ound Ia (Mol/L) Cell x 10-4 Control 150 2.10-5 3 5,10-6 115 ~able 3 Com~ound Ia (Mol/L) Cell x 10-4 Control 7.6 2.10-5 0.1 10-5 5,4 5,10-6 6.3 lO-6 7.6 Table 4 DNA DNA CollagenCollagen Pvalue~
(O:D.) (~q) Type i Type I
(pg/well) (~g/~g DNA) Con~rol 4.85iO.321.36iO.06 47.82+4.15 35.00~1.41 Compound Ia 1 ~M 8 20iO.231 97iO.05 43 78i5 23 22 00+1.46 Pc0 005 ~nd Ia 5 pM 6.90iO.501 85iO.24 38.01+6 24 20.50iO.61 P~0 005 Compound Ia 10 ~M 7.90+1.461.96iO 29 35.16i2.44 18.00il.41 P<0 005

Claims (4)

We claim:

1. A method of inhibiting colon cancer comprising administering to a mammal in need thereof an effective amount of a compound having the formula wherein R1 and R3 are independently hydrogen, -CH3, , or , wherein Ar is optionally substituted phenyl;
R2 is selected from the group consisting of pyrrolidine, hexamethyleneimino, and piperidino; or a pharmaceutically acceptable salt of solvate thereof.

2. The method of Claim 1 wherein said mammal is a human.

3. The method of Claim 2 wherein said compound is the hydrochloride salt thereof.

4. The method of Claim 3 wherein said compound is or its hydrochloride salt.