Reversal of multi-drug resistance by triphenyl-azacycloalkane derivatives

Certain triphenylpiperidines reverse drug resistance in multi-drug resistant tumors. These compounds apparently function by inhibiting a p-glycoprotein pump which becomes activated in late stage tumor development and which is inherently present in tumors from certain origins.

Effective tumor treatment is frequently thwarted by the lack of sensitivity 
of certain tumors to standard chemotherapeutic agents (intrinsic 
resistance) or by the ability of certain tumors to develop a lack of 
chemotherapeutic sensitivity during the course of treatment (acquired or 
extrinsic resistance). The cause of these phenomena has been linked to the 
existence of an energy-dependent efflux pump which acts to remove the 
chemotherapeutic agent from the target cell. The pump consists of the 
P-glycoprotein found as a constituent of cell membrane, and it has been 
suggested that the normal function of the P-glycoprotein is to remove 
toxins from within the cell. This theory is supported by the observation 
that P-glycoprotein is found as a cell membrane constituent in cells such 
as liver, kidney, colon, and jejunum. It has been suggested that 
P-glycoprotein in the cell membrane of such normal tissues could act to 
remove toxins or to assist in the transport of nutrients and solutes and 
in secreting a variety of protein and steroid substances. Natural presence 
of P-glycoprotein in tumor cells derived from these tissues as well as its 
presence in tumor cells derived from other tissue types could explain, at 
least in part, resistance of various tumors to therapy with standard 
chemotherapeutic agents. 
Disubstituted piperidine and pyrrolidine derivatives have already been 
disclosed in the art. In this respect, reference may be made to the 
following documents: 
WO 91/10651 which relates to certain disubstituted piperidine and 
pyrrolidine derivatives useful as selective muscarinic receptor 
antagonists; 
EP-A2-0235463 which relates to certain N-substituted arylalkyl and 
arylalkylenepyrrolidines, peperidines and homopiperidines useful in 
methods of treating cardiovascular dysfunctions, countering the effects of 
histamine in allergies and countering gastric secretion excesses; 
FR-A1-188403 which relates to certain disubstituted piperidine derivatives; 
Acta Pharmaceutica Sinica, vol. 19, 671-675 (1984) which relates to the 
synthesis of 2',2'-diphenylethylazacycloalkane derivatives and their uses 
as anticholinergics. 
There are also many multidrug resistance reversing agents and 
anti-neoplastic enhancing agents known in the art. Hence, reference may be 
made to the following documents: 
WO-A-90/15599 which relates to the use of certain aryl-substituted amine 
derivatives as adjuvant chemotherapy for neoplasias resistant to multiple 
drugs; 
EP-A3-0467435 which relates to the use of certain benzylhydryl derivatives 
useful for the treatment of patients suffering from calmodulin-influenced 
diseases; 
Annu. Rev, Biochem, 58, 137-171 (1989) which relates the biochemistry of 
P-glycoprotein-mediated multidrug resistance; 
Pharmacol. Rev., 42, 155-199 (1990) which relates to the pharmacology of 
drugs that alter multidrug resistance in cancer. 
The use of additional therapeutic agents which inactivate the 
P-glycoprotein pump would be invaluable in the treatment of multidrug 
resistant tumors. 
SUMMARY OF THE INVENTION 
Compounds of formula 
##STR1## 
wherein Ph.sub.1, Ph.sub.2, and Ph.sub.3 are each a phenyl group 
optionally substituted with up to a combination of three members of the 
group consisting of F, Br, Cl, OH, CF.sub.3, NH.sub.2, N(CH.sub.3)2, 
(C.sub.1-4)alkyl, (C.sub.1-4)alkoxy, and(C.sub.1-4)alkylthio; 
n is an integer of from 1 to 5; 
m is 0 or an integer of from 1 to 2; and 
q is an integer of from 1 to 2; 
or an acid addition salt thereof reverse multidrug resistance in multidrug 
resistant tumor cells and are thus useful adjuvants in the treatment of 
tumors. 
DETAILED DESCRIPTION OF THE INVENTION 
This invention relates to the compounds of Formula 1 as well as their use 
as agents effective in reversing drug resistance in multi-drug resistant 
tumors. The compounds of formula 1 can be administered together with 
standard chemotherapeutic agents, can be used in the treatment of tumors 
which are intrinsically or extrinsically multi-drug resistant, and can be 
used to reverse resistance in experimental multi-drug resistant tumor cell 
lines. Multi-drug resistance is defined to be that condition of a tumor 
cell in which the cell is resistant to a wide variety of unrelated 
anti-cancer drugs such as vinca alkaloids, epipodophyllotoxins, 
dactinomycin, and anthracycline classes as well as colchicine. (Goodman 
and Gilman, 7th Ed., p. 1278). This broad based, cross-resistance can 
develop after administration of a single agent of either the vinca 
alkaloid, epipodophyllotoxins, dactinomycin, and anthracycline classes as 
well as colchicine and is characterized by resistance to the other members 
of these drug classes. Examples of anti-tumor drugs of the vinca alkaloid 
class include the naturally occurring vincristine and vinblastine as well 
as the synthetic derivative vindesine. Examples of anti-tumor drugs of the 
epipodophyllotoxins class include etoposide and teniposide. Example of 
anti-tumor drugs of the anthracycline class is daunorubicin. An example of 
an anti-tumor drugs of the dactinomycin class include actinomycin A and 
actinomycin D. 
As used herein, the term "(C.sub.1-4)alkoxy" means a straight or branched 
chain alkoxy group having from one to four carbon atoms such as methoxy, 
ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, 
tert-butoxy. 
The compounds of Formula 1 may contain one or more asymmetric centers and 
may therefore exist as enantiomers and diastereomers. In particular if the 
carbon atom of the piperidine ring to which the 
diphenyl(C.sub.1-2)alkylene group (i.e. the 
(Ph.sub.2)(Ph.sub.3)CH(CH.sub.2).sub.q-- group) is attached is at the 2 or 
3-position in relation to the nitrogen then this carbon atom is an 
asymetric center. Moreover, when Ph.sub.2 and Ph.sub.3 are not identical, 
the carbon atom to which these phenyl groups are attached is an asymmetric 
center. References to the compounds of formula 1, or any intermediate 
thereof, may therefore be construed as covering a specific optical isomer, 
a racemic mixture or a diastereomeric mixture. The specific optical 
isomers can be synthesized or can be separated and recovered by techniques 
known in the art such as chromatography on chiral stationary phases, 
resolution via chiral salt formation and subsequent separation by 
selective crystallization, as is known in the art. Alternatively, a 
chirally pure starting material may be utilized. 
The expression "a pharmaceutically acceptable acid addition salt" is 
intended to apply to any non-toxic organic or inorganic acid addition salt 
of the base compounds. Illustrative inorganic acids which form suitable 
salts include hydrochloric, hydrobromic, sulfuric, and phosphoric acids 
and acid metal salts such as sodium monohydrogen orthophosphate and 
potassium hydrogen sulfate. Illustrative organic acids which form suitable 
salts include the mono, di, and tricarboxylic acids. Illustrative of such 
acids are, for example, acetic, glycolic, lactic, pyruvic, malonic, 
succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, 
hydroxymalic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, and 
2-phenoxybenzoic acids. Other organic acids which form suitable salts are 
the sulfonic acids such as methane sulfonic acid and 2-hydroxyethane 
sulfonic acid. These salts and the base compounds can exist in either a 
hydrated or a substantially anhydrous form. The acid salts are prepared by 
standard techniques such as by dissolving the free base in aqueous or 
aqueous-alcohol solution or other suitable solvent containing the 
appropriate acid and isolating by evaporating the solution, or by reacting 
the free base in an organic solvent in which case the salt separates 
directly or can be obtained by concentration of the solution or in a 
solvent such as water which is then removed in vacuo or by freeze-drying, 
or by exchanging the cations of an existing salt for another cation on a 
suitable ion exchange resin. In general the acid addition salts of the 
compounds of this invention are crystalline materials which are soluble in 
water and various hydrophilic organic solvents and which in comparison to 
their free base forms, demonstrate higher melting points and an increased 
solubility. 
As is true for most classes of therapeutically effective compounds, certain 
subclasses and certain species are especially effective and are preferred 
over others. In this instance, those compounds of Formula 1 wherein 
Ph.sub.1 is an unsubstituted phenyl or is a phenyl substituted with one or 
two alkoxy groups are preferred. More preferred are those formula 1 
compounds wherein n is the integer 2, as well as those compounds of 
formula 1 wherein Ph.sub.1 is a phenyl group substituted with one or two 
methoxy groups. Most preferred is the compound of formula 1 wherein n is 
the integer 2, m is the integer 1, q is the integer 1, Ph.sub.1 is a 
3,4-dimethoxyphenyl group and wherein Ph.sub.2 and Ph.sub.3 are each an 
unsubstituted phenyl group. 
Applicants have prepared compounds of formula 1 as illustrated in scheme 1 
by reacting a suitably substituted 2-,3-, or 4- diphenylalkyl substituted 
cyclic amine of formula 2 with a suitably substituted 
phenylalkylcarbonylchloride of formula 3. The resulting amide of formula 4 
is then reduced by, for example, treatment with diborane to yield the 
desired product of formula 1. 
Alternatively, the compounds of formula 1 can be made as illustrated in 
scheme 2 by direct alkylation of a suitably substituted 2-,3-, or 4- 
diphenylalkyl substituted cyclic amine of formula 2 with a suitably 
substituted aralkyl halide of formula 5. Further illustrations of the 
preparation of the compounds of this invention are provided in the 
examples. 
##STR2## 
The ability of the triphenylpiperidines derivatives of this invention to 
reverse drug resistance in multi-drug resistant tumors can be demonstrated 
by the ability of test compounds to reduce cell growth in a vinblastine 
(VBL) resistant tumor cell line. 
The ability of compounds of this invention to reverse multi drug resistance 
can be determined using standard test procedures. The ability of the test 
compounds to inhibit the growth of multidrug resistant human epidermoid 
carcinoma (KBV1) cells in the presence of both Vinblastine and the 
compound was determined. These cells are at least 1,000 times resistant to 
vinblastine compared to wild type KB cells. Vinblastine (0.25 .mu.g/ml) 
alone showed 0-10% growth inhibition. Compound alone at different 
concentrations did not significantly inhibit any growth of KBV1 cells. The 
results are tabulated in Table 1. 
TABLE 1 
______________________________________ 
MODULATION OF DRUG RESISTANCE IN HUMAN 
EPIDERMOID CARCINOMA (KBV1) CELLS 
BY1-(3,4-DIMETHOXYPHENETHYL-2-(2,2- 
DIPHENYLETHYL)PIPERIDINE 
% Inhibition of Growth 
Concentration Compound Compound 
(.mu.M) Alone + VLB* 
______________________________________ 
0.23 12.2 94.2 
0.18 8.1 91.6 
0.12 0 60.0 
0.06 1.0 30.4 
______________________________________ 
*VLB at 0.25 .mu.g/ml showed 0-10% growth inhibition. 
The term "patient" used herein is taken to mean mammals such as primates, 
including humans, sheep, horses, cattle, pigs, dogs, cats, rats and mice. 
The amount of the triphenylpiperidine derivative of formula 1 to be 
administered can vary widely according to the particular dosage unit 
employed, the period of treatment, the age and sex of the patient treated, 
the nature and extent of the multidrug resistance in the tumor to be 
treated, and the particular triphenylpiperidine derivative selected. The 
triphenylpiperidine derivative is used in conjunction with other 
chemotherapeutic agents known to be useful in the treatment of tumors. The 
amount of a triphenylpiperidine derivative of formula 1 effective to 
reverse multidrug resistance will generally range from about 15 mg/kg to 
500 mg/kg. A unit dosage may contain from 25 to 500 mg of the 
triphenylpiperidine derivative, and can be taken one or more times per 
day. The triphenylpiperidine derivative can be administered with a 
pharmaceutical carrier using conventional dosage unit forms either orally 
or parenterally. 
Treatment of tumors by the method of this invention requires that an 
anti-tumor effective amount of a chemotherapeutic agent be administered 
together with a compound of formula 1. Tumors which can be treated by the 
method of this invention include both benign and malignant tumors or 
neoplasms, and include melanomas, lymphomas, leukemias, and sarcomas. 
Illustrative examples of tumors are cutaneous tumors, such as malignant 
melanomas and mycosis fungoides; hematologic tumors such as leukemias, for 
example, acute lymphoblastic, acute myelocytic or chronic myelocytic 
leukemia; lymphomas, such as Hodgkin's disease or malignant lymphoma; 
gynecologic tumors, such as ovarian and uterine tumors; urologic tumors, 
such as those of the prostate, bladder or testis; soft tissue sarcomas, 
osseus or non-osseus sarcomas, breast tumors; tumors of the pituitary, 
thyroid and adrenal cortex; gastrointestinal tumors, such as those of the 
esophagus, stomach, intestine and colon; pancreatic and hepatic tumors; 
laryngeae papillomestasas and lung tumors. Of course those tumors which 
typically are or become multi-drug resistant are most beneficially treated 
with the method of this invention. Such tumors include colon tumors, 
breast tumors, lung tumors, stomach tumors, and liver tumors. 
The chemotherapeutic agents used together with the triphenylpiperidines of 
formula I are those cytotoxic agents commonly used in the treatment of 
tumors. Illustrative examples of chemotherapeutic agents are: 
cyclophosphamide, methotrexate, prednisone, 6-mercaptopurine, 
procarbazine, daunorubicin, vincristine, vinblastine, chlorambucil, 
cytosine arabinoside, 6-thioguanine, thio TEPA, 5-fluorouracil, 
5-fluoro-2-deoxyudirinde, 5-azacytidine, nitrogen mustard, 
1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 
(1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea) (CCNU), busulfan, 
adriamycin, bleomycin, vindesine, cycloleucine or methylglyoxal 
bix(guanylhydrazone) (i.e., MGBG). The effective amount of 
chemotherapeutic agent used in the method of this invention varies widely 
and depends on factors such as the patient, the tumor tissue type and its 
size, and the particular chemotherapeutic agent selected. The amount is 
any effective amount and can be readily determined by those skilled in the 
art. In general, less chemotherapeutic agent will be required when 
administered with the triphenylpiperidine of formula 1, primarily because 
the problem of drug resistance need not addressed by the addition of 
larger quantities of chemotherapeutic agent. Of course mixtures of 
chemotherapeutic agents may be employed and surgical excision and 
radiation therapy may be useful adjuvants as in any tumor therapy. While 
the compound of formula 1 and the chemotherapeutic agent are said to be 
administered together, this does not necessarily mean that the compounds 
are formulated into the same dosage form or are administered concurrently. 
Rather, the expression "together" means that a compound of formula 1 and 
the chemotherapeutic agent(s) are administered in a combined dosage form 
or separately during the course of therapy. 
The preferred route of administration is oral administration. For oral 
administration the triphenylpiperidinederivative can be formulated into 
solid or liquid preparations such as capsules, pills, tablets, troches, 
lozenges, melts, powders, solutions, suspensions, or emulsions. The solid 
unit dosage forms can be a capsule which can be of the ordinary hard- or 
soft-shelled gelatin type containing, for example, surfactants, 
lubricants, and inert fillers such as lactose, sucrose, calcium phosphate, 
and cornstarch. In another embodiment the compounds of this invention can 
be tableted with conventional tablet bases such as lactose, sucrose, and 
cornstarch in combination with binders such as acacia, cornstarch, or 
gelatin, disintegrating agents intended to assist the break-up and 
dissolution of the tablet following administration such as potato starch, 
alginic acid, corn starch, and guar gum, lubricants intended to improve 
the flow of tablet granulations and to prevent the adhesion of tablet 
material to the surfaces of the tablet dies and punches, for example, 
talc, stearic acid, or magnesium, calcium, or zinc stearate, dyes, 
coloring agents, and flavoring agents intended to enhance the aesthetic 
qualities of the tablets and make them more acceptable to the patient. 
Suitable excipients for use in oral liquid dosage forms include diluents 
such as water and alcohols, for example, ethanol, benzyl alcohol, and the 
polyethylene alcohols, either with or without the addition of a 
pharmaceutically acceptably surfactant, suspending agent, or emulsifying 
agent. 
The triphenylpiperidine derivatives of this invention may also be 
administered parenterally, that is, subcutaneously, intravenously, 
intramuscularly, or intraperitoneally, as injectable dosages of the 
compound in a physiologically acceptable diluent with a pharmaceutical 
carrier which can be a sterile liquid or mixture of liquids such as water, 
saline, aqueous dextrose and related sugar solutions, an alcohol such as 
ethanol, isopropanol, or hexadecyl alcohol, glycols such as propylene 
glycol or polyethylene glycol, glycerol ketals such as 
2,2-dimethyl-1,3-dioxolane-4-methanol, ethers such as poly(ethyleneglycol) 
400, an oil, a fatty acid, a fatty acid ester or glyceride, or an 
acetylated fatty acid glyceride with or without the addition of a 
pharmaceutically acceptable surfactant such as a soap or a detergent, 
suspending agent such as pectin, carbomers, methylcellulose, 
hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying 
agent and other pharmaceutically adjuvants. Illustrative of oils which can 
be used in the parenteral formulations of this invention are those of 
petroleum, animal, vegetable, or synthetic origin, for example, peanut 
oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, 
petrolatum, and mineral oil. Suitable fatty acids include oleic acid, 
stearic acid, and isostearic acid. Suitable fatty acid esters are, for 
example, ethyl oleate and isopropyl myristate. Suitable soaps include 
fatty alkali metal, ammonium, and triethanolamine salts and suitable 
detergents include cationic detergents, for example, dimethyl dialkyl 
ammonium halides, alkyl pyridinium halides, and alkylamines acetates; 
anionic detergents, for example, alkyl, aryl, and olefin sulfonates, 
alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates; 
nonionic detergents, for example, fatty amine oxides, fatty acid 
alkanolamides, and polyoxyethylenepolypropylene copolymers; and amphoteric 
detergents, for example, alkylbeta-aminopropionates, and 
2-alkylimidazoline quarternary ammonium salts, as well as mixtures. The 
parenteral compositions of this invention will typically contain from 
about 0.5 to about 25% by weight of the oxazolone derivative of formula 1 
in solution. Preservatives and buffers may also be used advantageously. In 
order to minimize or eliminate irritation at the site of injection, such 
compositions may contain a non-ionic surfactant having a 
hydrophile-lipophile balance (HLB) of from about 12 to about 17. The 
quantity of surfactant in such formulations ranges from about 5 to about 
15% by weight. The surfactant can be a single component having the above 
HLB or can be a mixture of two or more components having the desired HLB. 
Illustrative of surfactants used in parenteral formulations are the class 
of polyethylene sorbitan fatty acid esters, for example, sorbitan 
monooleate and the high molecular weight adducts of ethylene oxide with a 
hydrophobic base, formed by the condensation of propylene oxide with 
propylene glycol. 
The compounds of this invention can also be administered topically. This 
can be accomplished by simply preparing a solution of the compound to be 
administered, preferably using a solvent known to promote transdermal 
absorption such as ethanol or dimethyl sulfoxide (DMSO) with or without 
other excipients. Preferably topical administration will be accomplished 
using a patch either of the reservoir and porous membrane type or of a 
solid matrix variety. 
Some suitable transdermal devices are described in U.S. Pat. Nos. 
3,742,951, 3,797,494, 3,996,934, and 4,031,894. These devices generally 
contain a backing member which defines one of its face surfaces, an active 
agent permeable adhesive layer defining the other face surface and at 
least one reservoir containing the active agent interposed between the 
face surfaces. Alternatively, the active agent may be contained in a 
plurality of microcapsules distributed throughout the permeable adhesive 
layer. In either case, the active agent is delivered continuously from the 
reservoir or microcapsules through a membrane into the active agent 
permeable adhesive, which is in contact with the skin or mucosa of the 
recipient. If the active agent is absorbed through the skin, a controlled 
and predetermined flow of the active agent is administered to the 
recipient. In the case of microcapsules, the encapsulating agent may also 
function as the membrane. 
In another device for transdermally administering the compounds in 
accordance with the present invention, the pharmaceutically active 
compound is contained in a matrix from which it is delivered in the 
desired gradual, constant and controlled rate. The matrix is permeable to 
the release of the compound through diffusion or microporous flow. The 
release is rate controlling. Such a system, which requires no membrane is 
described in U.S. Pat. No. 3,921,636. An least two types of release are 
possible in these systems. Release by diffusion occurs when the matrix is 
non-porous. The pharmaceutically effective compound dissolves in and 
diffuses through the matrix itself. Release by microporous flow occurs 
when the pharmaceutically effective compound is transported through a 
liquid phase in the pores of the matrix.