Telomerase inhibitors

BACKGROUND OF THE INVENTION
1. The Field of the Invention
The present invention relates to human telomerase, a ribonucleoprotein enzyme involved in human telomere DNA synthesis, and to compounds that inhibit telomerase activity. The invention provides methods, compounds and compositions relating to the fields of molecular biology, chemistry, pharmacology, oncology and medicinal and diagnostic technology.
2. Description of Related Disclosures
The war on cancer has raged for over two decades. Yet, despite the expenditure of over a billion dollars for research and development of new technologies to diagnose and treat malignancies, the age-adjusted cancer mortality rate in the U.S. has remained largely unchanged for the past forty years. Indeed, if current epidemiological trends continue, it appears likely that cancer will overtake cardiovascular disease as the leading cause of death in the United States.
To be sure, some battles have been won and much has been learned about the enemy. A few cancers (e.g., Hodgkin's disease) are now considered curable, and treatment regimes for many other cancers have improved over the last decade. In addition, there has been an explosion of information describing the regulatory mechanisms involved with the onset of malignancy, including the roles of growth factors, receptors, signal transduction pathways, oncogenes, and tumor suppressor genes in the control of cell growth and differentiation. However, these successes are overshadowed by the fact that cancer is a highly heterogeneous disease in which profound differences exist in the mechanisms by which different cell types become malignant. Thus, although we know more about the mechanisms by which cells become malignant than ever before, each type of cancer presents a unique set of problems in terms of treatment.
Because the cellular mechanisms leading to cancer are so heterogeneous, research on such mechanisms is unlikely to yield a general approach to cancer treatment that is effective and well tolerated by cancer patients. Presently, a variety of non-specific treatment modalities are available, including surgery, radiation, and a variety of cytoreductive and hormone-based drugs, used alone or in combination. Some oncolytic drugs are also available, but the efficacy of these drugs varies among cancer types. Thus, patients suffering from cancer often are forced to undergo treatments that are highly non-specific and highly toxic. Commonly, the toxicity of the treatments produces severe side effects, including nausea and vomiting, hair loss, diarrhea, fatigue, ulcerations and the like, which severely impact the patient's quality of life. In some cases, the impact on the patient's quality of life can be so great that the patient is unable to continue the full course of therapy or opts out of treatment entirely.
Recently, however, an understanding of the mechanisms by which normal cells reach the state of senescence, i.e., the loss of proliferative capacity that cells normally undergo in the cellular aging process, has begun to emerge. The DNA at the ends, or telomeres, of the chromosomes of eukaryotes usually consists of tandemly repeated simple sequences. Scientists have long known that telomeres have an important biological role in maintaining chromosome structure and function. More recently, scientists have speculated that the cumulative loss of telomeric DNA over repeated cell divisions may act as a trigger of cellular senescence and aging, and that the regulation of telomerase, an enzyme involved in the maintenance of telomere length, may have important biological implications. See Harley, 1991, Mutation Research, 256:271-282, incorporated herein by reference.
Telomerase is a ribonucleoprotein enzyme that synthesizes one strand of the telomeric DNA using as a template a sequence contained within the RNA component of the enzyme. See Blackburn, 1992, Annu. Rev. Biochem, . 61:113-129, incorporated herein by reference. Methods for detecting telomerase activity, as well as for identifying compounds that regulate or affect telomerase activity, together with methods for therapy and diagnosis of cellular senescence and immortalization by controlling telomere length and telomerase activity, have also been described. See, Feng, et al., 1995, Science, 269:1236-1241; Kim, et al., 1994, Science, 266:2011-2014; PCT patent publication No. 93/23572, published Nov. 25, 1993; U.S. patent application Ser. Nos.08/288, 501, filed Aug. 10, 1994; 08/330, 123, filed Oct. 27, 1994; 08/272,102, filed Jul. 7, 1994; 08/255,774, filed Jun. 7, 1994; 08/315,214 and 08/315,216, both of which were filed Sept. 28, 1994; 08/151,477 and 08/153,051, both of which were filed Nov. 12, 1993; 08/060, 952, filed May 13, 1993; and 08/038, 766, filed Mar. 24, 1993. Each of the foregoing patent applications and reference is incorporated herein by reference.
The identification of compounds that inhibit telomerase activity provides important benefits to efforts at treating human disease. Compounds that inhibit telomerase activity can be used to treat cancer, as cancer cells express telomerase activity and normal human somatic cells do not express telomerase activity at biologically relevant levels (i.e., at levels sufficient to maintain telomere length over many cell divisions). Unfortunately, few such compounds have been identified and characterized. Hence, there remains a need for compounds that act as telomerase inhibitors and for compositions and methods for treating cancer and other diseases in which telomerase activity is present abnormally. The present invention meets these and other needs.
SUMMARY OF THE INVENTION
The present invention provides methods, compounds and compositions that are highly unique, specific and effective for treating malignant conditions by targeting cells having telomerase activity. The methods, compounds and compositions of the invention can be applied to a wide variety of malignant cell types and avoid the problems inherent in current cancer treatment modalities, which are non-specific and excessively toxic.
In a first aspect, the present invention provides methods and compositions for treating cancer in which a therapeutically effective amount of a compound having the formula shown below is administered to a mammal. ##STR2## In Compound I, X.sub.1 is selected from the group consisting of oxygen, sulfur, sulfone, sulfinyl and --NR--, where R selected from the group consisting of hydrogen, alkyl, aryl, and aralkyl. R.sub.1 is --Y.sub.n R.sub.6 where n is an integer between 0 and 10 and each Y.sub.n for n greater than 0 independently is methylene, methine, or quarternary carbon. R.sub.6, for any value of n (i.e., n zero or non-zero), is alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, alkylcarbonyl, arylcarbonyl, heteroalkylcarbonyl, heteroaralkylcarbonyl, aralkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl, arylalkylaminocarbonyl, carboxyl, carboxaldehyde, alkoxycarbonyl, aryloxycarbonyl, sulfo, alkylsulfonyl, arylsulfonyl, alkylsulfinyl, and arylsulfinyl. In addition, R.sub.6 can be a linker L selected from the group consisting of alkyl, aryl, and heterocycle, to which linker between 1 and 3 compounds having the structure shown above are attached. R.sub.2 is hydrogen, alkyl, aryl, hydroxyl, alkoxyl, aryloxyl, halogen, cyano, amino, alkylamino, arylamino, dialkylamino, diarylamino, arylalkylamino, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl, arylalkylaminocarbonyl, alkylcarbonyl, arylcarbonyl, alkylcarbonyloxy, arylcarbonyloxy, carboxyl, alkoxycarbonyl, aryloxycarbonyl, sulfo, alkylsulfonyl, or arylsulfonyl. R.sub.3 and R.sub.4 are selected independently from the group consisting of hydrogen, amino, alkylamino, arylamino, heterocycleamino, aralkylamino, dialkylamino, diarylamino, heterocylcealkylamino, alkylcarbonyl, arylcarbonyl, nitro, halogen, hydroxyl, aryloxyl, alkoxyl, lower alkyl, aryl, heteroaryl, aralkyl, cyano, carboxyl, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, and heteroaralkyl. R.sub.5 is selected from the group consisting of iminyl, hydroximinyl, alkyliminyl, aryliminyl, aralkyliminyl, alkoximinyl, aryloximinyl, heterocycleimninyl, cyclic iminyl; --HC.dbd.NNHR.sub.7 where R.sub.7 is selected from the group consisting of hydrogen, alkyl, aryl, aralkyl, heterocycle, heterocyclealkyl, and --C(.dbd.X.sub.2)(X.sub.3).sub.p R.sub.8 where p is 0 or 1, X.sub.2 is oxygen or sulfur, X.sub.3 is selected from the group consisting of oxygen, sulfur, and --NR.sub.9 --, where R.sub.8 and R.sub.9 are selected independently from the group consisting of hydrogen, alkyl, aryl, aralkyl, heterocycle, and heterocyclealkyl. Finally, R.sub.4 and R.sub.5 can be points of attachment for the group --CR'.dbd.N--Q.sub.k --, where k is 1 or 2, R' is selected from the group consisting hydrogen, alkyl, aryl, aralkyl, heterocycle, and heterocyclealkyl, and each Q.sub.k is selected independently from the group consisting of methylene, methine, and quarternary carbon, to form thereby a isopyrolo�4, 3-b!pyridine or pyrido�3, 4-b!pyridine derivative, respectively.
In another aspect, the present invention provides novel compounds relating to a class of telomerase inhibiting agents that are derivatives or analogues of pyridine imines, hydrazides, semicarbazides, and thiosemicarbazides. These telomerase inhibiting compounds have the general structure shown in Compound I above, with the exception that R.sub.5 is not 4-methylphenyliminyl when X.sub.1 is sulfur, n is 1, Y is methylene, R.sub.3 is cyano, R.sub.3 and R.sub.4 are hydrogen, and R6 is 4-chlorophenyl.
In one embodiment of Compound 1, X.sub.1 is sulfur, n is 1, Y is methylene, R.sub.2 is cyano, R.sub.3 and R.sub.4 are hydrogen, R.sub.5 is phenyliminyl or substituted phenyliminyl, and R.sub.6 is phenyl or substituted phenyl. These compounds have the structure below (Compound II). ##STR3## Here, R.sub.10 -R.sub.14 are selected independently from the group consisting of hydrogen, halogen, hydroxyl, heterocycle, alkyl, aryl, aralkyl, heterocyclealkyl, alkoxyl, aryloxyl, heterocycleoxyl, nitro, cyano, alkylthio, arylthio, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyl, aryloxycarbonyl, carboxyl, and heterocyclethio. R.sub.15 -R.sub.19 are selected independently from the group consisting of hydrogen, halogen, hydroxyl, aryl, lower alkyl, alkoxyl, aryloxyl, arylthio, nitro, cyano, alkylamino, dialkylamino. R.sub.16 and R.sub.17 together also may be points of attachment for the groups --C(O)--NR.sub.20 C--(O)-- and --O--(CH.sub.2).sub.m --O--, where m is 1 or 2 and R.sub.20 is hydrogen, aryl, aralkyl or alkyl, to form thereby iminyl phthalamides and 3, 4-(methylenedioxy)phenyl imines or 3, 4-(ethylenedioxy)phenyl imines, respectively.
Useful embodiments of the present invention include compounds having the general structure of Compound II in which R.sub.10 -R.sub.1 l are selected from the group consisting of hydrogen and halogen. In more particular embodiments of Compound II, R.sub.10, R.sub.11, R.sub.14, R.sub.18, and R.sub.19 are hydrogen; R.sub.12 is chloro; R.sub.13 is hydrogen or chloro; R.sub.15 is hydrogen, phenoxy, or 4-methylphenoxy; R.sub.16 is hydrogen, chloro, fluoro, nitro, or trifluoromethyl; R.sub.17 is selected from the group consisting of methoxy, cyano, bromo, chloro, fluoro, trifluoromethyl, and dimethylamino. In addition, R.sub.16 and R.sub.17 together can be points of attachment for the groups --C(O)--NR.sub.20 C--(O)-- and --O--(CH.sub.2).sub.2 --O--, where R.sub.20 is hydrogen, ethyl or benzyl. Still more particular embodiments are those in which R.sub.13 is chloro and R.sub.15 is hydrogen.
Other useful embodiments are those in which X.sub.1 is sulfur, n is 1, Y is methylene, R.sub.2 is cyano, R.sub.3 and R.sub.4 are hydrogen, R.sub.5 is pyridyliminyl or substituted pyridyliminyl, and R.sub.6 is phenyl or substituted phenyl. One particularly useful embodiment having this general formula, is shown below: ##STR4## In this structure, R.sub.21 -R.sub.25 are selected independently from the group consisting of hydrogen, halogen, hydroxyl, heterocycle, alkyl, aryl, aralkyl, heterocyclealkyl, alkoxyl, aryloxyl, heterocycleoxyl, nitro, cyano, alkylthio, arylthio, alkylcarbonyloxy, alkoxycarbonyl, aryloxycarbonyl, arylcarbonyloxy, and heterocyclethio. In addition, adjacent substituents on the benzylthioether ring can be points of attachment for the group --Z.sub.1 --Z.sub.2 --Z.sub.3 --Z.sub.4 --, where Z.sub.1 -Z.sub.4 are selected independently from the group consisting of methylene, methine, quarternary carbon, and oxygen. R.sub.26 -R.sub.29 are selected independently from the group consisting of hydrogen, halogen, hydroxyl, aryl, lower alkyl, alkoxyl, aryloxyl, arylthio, nitro, cyano, alkylamino, and dialkylamino.
In one embodiment of the above-described pyridyliminyl pyridinethioethers, R.sub.21 -R.sub.25 are selected independently from the group consisting of hydrogen and halogen. More specific embodiments include those for which R.sub.21, R.sub.22, and R.sub.25 are hydrogen; R.sub.23 and R.sub.24 are chloro; and R.sub.26 -R.sub.29 independently are hydrogen, halogen, or lower alkyl.
Yet another embodiment of Compound I is the dimeric derivative shown below, which is obtained when n is 1, Y is methylene, and R.sub.6 is a linker L to which linker a second compound having the formula of Compound I is bound: ##STR5## In one useful embodiment, X.sub.1 is sulfur, R.sub.2 is cyano, R.sub.3 and R.sub.4 are hydrogen, R.sub.5 is heteroaryliminyl, and L is alkyl or aryl. Particularly useful embodiments are those for which R.sub.5 is 3-methyl-2-pyridyliminyl and R.sub.6 selected from the group consisting of ethylene, ethenylene, and ethynylene.
The above-illustrated derivatives have many valuable uses as inhibitors of deleterious telomerase activity, most importantly, the use to treat cancer in humans. The pharmaceutical compositions of this invention can be employed in treatment regimens in which cancer cells are killed, in vivo, as demonstrated by the use of telomerase inhibitors of the invention to kill cancer cells ex vivo. Thus, this invention provides therapeutic compositions for treating cancer, and methods for treating cancer in humans and other mammals (e.g., cows, horses, sheep, steer, pigs and animals of veterinary interest such as cats and dogs).
These and other features of the invention will be described in detail below with reference to the associated structures and tables.
DESCRIPTION OF SPECIFIC EMBODIMENTS
I. Definitions
The term "alkyl" as used herein refers to a straight, branched, or cyclic hydrocarbon chain fragment or radical containing between about one and about twenty carbon atoms, more preferably between about one and about ten carbon atoms (e.g., methyl, ethyl, n-propyl, iso-propyl, cyclopropyl, n-butyl, iso-butyl, tert-butyl, cyclobutyl, adamantyl, noradamantyl and the like). Straight, branched, or cyclic hydrocarbon chains having eight or fewer carbon atoms will also be referred to herein as "lower alkyl". The hydrocarbon chains may further include one or more degrees of unsaturation, i.e., one or more double or triple bonds (e.g., vinyl, propargyl, allyl, 2-buten-1-yl, 2-cyclopenten-1-yl, 1,3-cyclohexadien-1-yl, 3-cyclohexen-1-yl and the like). Alkyl groups containing double bonds such as just described will also be referred to herein as "alkenes". Similarly, alkyl groups having triple bonds will also be referred to herein as "alkynes". However, as used in context with respect to cyclic alkyl groups, the combinations of double and/or triple bonds do not include those bonding arrangements that render the cyclic hydrocarbon chain aromatic.
In addition, the term "alkyl" as used herein further includes one or more substitutions at one or more carbon atoms of the hydrocarbon fragment or radical. Such substitutions include, but are not limited to: aryl; heterocycle; halogen (to form, e.g., trifluoromethyl, --CF.sub.3); nitro (--NO.sub.2); cyano (--CN); hydroxyl (also referred to herein as "hydroxy"), alkoxyl (also referred herein as alkoxy) or aryloxyl (also referred to herein as "aryloxy", --OR); thio or mercapto, alkyl, or arylthio (--SR); amino, alkylamino, arylamino, dialkyl-- or diarylamino, or arylalkylamino (--NRR'); aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl or arylalkylaminocarbonyl (--C(O)NRR'); carboxyl, or alkyl-- or aryloxycarbonyl (--C(O)OR); aldehyde; aryl-- or alkylcarbonyl (RC(O)--); imninyl, or aryl-- or alkyliminyl (--C(.dbd.NR)R'); sulfo (--SO.sub.2 OR); alkyl-- or arylsulfonyl (--SO.sub.2 R); hydroximinyl, or aryl-- or alkoximinyl (--C(.dbd.NOR)R'); where R and R' independently are hydrogen, aryl or alkyl as defined herein. Substituents including heterocyclic groups (i.e., heterocycle, heteroaryl, and heteroaralkyl) are defined by analogy to the above-described terms. For example, the term "heterocycleoxy" refers to the group --OR, where R is heterocycle as defined below.
The term "methylene" refers to the group --CH.sub.2 --.
The term "methine" refers to a methylene group for which one hydrogen atom has been replaced by a substituent as described above.
The term "quarternary carbon" refers to a methylene group in which both hydrogen atoms are replaced by two independent substituents as described above.
The term "cyclic iminyl" refers to a cyclic group having the general structure shown below ##STR6## where n is 3 or 4 and "C" represents a methylene, methine, or quarternary carbon ring constituent.
The term "halogen" as used herein refers to the substituents fluoro, bromo, chloro, and iodo.
The term "carbonyl" as used herein refers to the functional group --C(O)--. However, it will be appreciated that this group may be replaced with well-known groups that have similar electronic and/or steric character, such as thiocarbonyl (--C(S)--); sulfinyl (--S(O)--); sulfonyl (SO.sub.2 --), phosphonyl (--PO.sub.2 --), and methylene (--C(CH.sub.2)--). Other carbonyl equivalents will be familiar to those having skill in the medicinal and organic chemical arts.
The term "aryl" as used herein refers to cyclic aromatic carbon chains having twenty or fewer carbon atoms, e.g., phenyl, naphthyl, biphenyl and anthracenyl. One or more carbon atoms of the aryl group may also be substituted with, e.g.: alkyl; aryl; heterocycle; halogen; nitro; cyano; hydroxyl, alkoxyl or aryloxyl; thio or mercapto, alkyl-, or arylthio; amino, alkylamnino, arylamino, dialkyl-, diaryl-, or arylalkylamino; aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl or arylalkylaminocarbonyl; carboxyl, or alkyl- or aryloxycarbonyl; aldehyde; aryl- or alkylcarbonyl; iminyl, or aryl- or alkyliminyl; sulfo; alkyl- or arylsulfonyl; hydroxirninyl, or aryl- or alkoximinyl. In addition, two or more alkyl or heteroalkyl substituents of an aryl group may be combined to form fused aryl-alkyl or aryl-heteroalkyl ring systems (e.g., tetrahydronaphthyl). Substituents including heterocyclic groups (e.g., heterocycleoxy, heteroaryloxy, and heteroaralkylthio) are defined by analogy to the above-described terms.
The term "aralkyl" as used herein refers to an aryl group that is joined to a parent structure by an alkyl group as described above, e.g., benzyl, .alpha.-methylbenzyl, phenethyl, and the like.
The term "heterocycle" as used herein refers to a cyclic alkyl group or aryl group as defined above in which one or more carbon atoms have been replaced by a non-carbon atom, especially nitrogen, oxygen, or sulfur. Non-aromatic heterocycles will also be referred to herein as "cyclic heteroalkyl". Aromatic heterocycles are also referred to herein as "heteroaryl". For example, such groups include furyl, tetrahydrofuryl, pyrrolyl, pyrrolidinyl, thienyl, tetrahydrothienyl, oxazolyl, isoxazolyl, triazolyl, thiazolyl, isothiazolyl, pyrazolyl, pyrazolidinyl, oxadiazolyl, thiadiazolyl, imidazolyl, imidazolinyl, pyridyl, pyridazinyl, triazinyl, piperidinyl, morpholinyl, thiomorpholinyl, pyrazinyl, piperazinyl, pyrimidinyl, naphthyridinyl, benzofuranyl, benzothienyl, indolyl, indolinyl, indolizinyl, indazolyl, quinolizinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, pteridinyl, quinuclidinyl, carbazolyl, acridiniyl, phenazinyl, phenothiazinyl, phenoxazinyl, purinyl, benzimidazolyl and benzthiazolyl.
The above heterocyclic groups may further include one or more substituents at one or more carbon and/or non-carbon atoms of the heteroaryl group, e.g.: alkyl; aryl; heterocycle; halogen; nitro; cyano; hydroxyl, alkoxyl or aryloxyl; thio or mercapto, alkyl- or arylthio; amino, alkyl-, aryl-, dialkyl- diaryl-, or arylalkylamino; aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl or arylalkylaminocarbonyl; carboxyl, or alkyl- or aryloxycarbonyl; aldehyde; aryl- or alkylcarbonyl; iminyl, or aryl- or alkyliminyl; sulfo; alkyl- or arylsulfonyl; hydroximinyl, or aryl- or alkoximinyl. In addition, two or more alkyl substituents may be combined to form fused heterocycle-alkyl ring systems. Substituents including heterocyclic groups (e.g., heterocycleoxy, heteroaryloxy, and heteroaralkylthio) are defined by analogy to the above-described terms.
The term "heterocyclealkyl" refers to a heterocycle group that is joined to a parent structure by one or more alkyl groups as described above, e.g., 2-piperidylmethyl, and the like. The term "heteroaralkyl" as used herein refers to a heteroaryl group that is joined to a parent structure by one or more alkyl groups as described above, e.g., 2-thienylmethyl, and the like.
II. Telomerase Inhibitors
As noted above, the immortalization of cells involves inter alia the activation of telomerase.
More specifically, the connection between telomerase activity and the ability of many tumor cell lines, including skin, connective tissue, adipose, breast, lung, stomach, pancreas, ovary, cervix, uterus, kidney, bladder, colon, prostate, central nervous system (CNS), retina and blood tumor cell lines, to remain immortal has been demonstrated by analysis of telomerase activity (Kim, et al, incorporated herein by reference above). This analysis, supplemented by data that indicates that the shortening of telomere length can provide the signal for replicative senescence in normal cells, see PCT Application No. 93/23572 (incorporated herein by reference above), demonstrates that inhibition of telomerase activity can be an effective anti-cancer therapy. Thus, telomerase activity can prevent the onset of otherwise normal replicative senescence by preventing the normal reduction of telomere length and the concurrent cessation of cell replication that occurs in normal somatic cells after many cell divisions. In cancer cells, where the malignant phenotype is due to loss of cell cycle or growth controls or other genetic damage, an absence of telomerase activity permits the loss of telomeric DNA during cell division, resulting in chromosomal rearrangements and aberrations that lead ultimately to cell death. However, in cancer cells having telomerase activity, telomeric DNA is not lost during cell division, thereby allowing the cancer cells to become immortal, leading to a terminal prognosis for the patient. Agents capable of inhibiting telomerase activity in tumor cells offer therapeutic benefits with respect to a wide variety of cancers and other conditions (e.g., fungal infections) in which immortalized cells having telomerase activity are a factor in disease progression or in which inhibition of telomerase activity is desired for treatment purposes. The telomerase inhibitors of the invention can also be used to inhibit telomerase activity in germ line cells, which may be useful for contraceptive purposes.
Thus, in one aspect, the present invention provides compounds that can serve as an important weapons against many types of malignancies in the war against cancer. In particular, the compounds of the present invention can provide a highly general method of treating many--if not most--malignancies, as demonstrated by the highly varied human tumor cell lines and tumors having telomerase activity. More importantly, the compounds of the present invention can be effective in providing treatments that discriminate between malignant and normal cells to a high degree, avoiding many of the deleterious side-effects present with most current chemotherapeutic regimes which rely on agents that kill dividing cells indiscriminately.
In one aspect, the present invention provides pharmaceutical compositions and methods relating to compounds having the general structure shown as Compound I below: ##STR7## and its pharmaceutically acceptable salts. In Compound I, X.sub.1 is selected from the group consisting of oxygen, sulfur, sulfone, sulfinyl and --NR--, where R selected from the group consisting of hydrogen, alkyl, aryl, and aralkyl. R.sub.1 is --Y.sub.n R.sub.6, where n is an integer between 0 and 10 and each Y.sub.n for n greater than 0 independently is methylene, methine, or quarternary carbon. R.sub.6 for any value of n (i.e., n zero or non-zero), is alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, alkylcarbonyl, arylcarbonyl, heteroalkylcarbonyl, heteroaralkylcarbonyl, aralkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl, arylalkylaminocarbonyl, carboxyl, carboxaldehyde, alkoxycarbonyl, aryloxycarbonyl, sulfo, alkylsulfonyl, arylsulfonyl, alkylsulfinyl, and arylsulfinyl. In addition, R.sub.6 can also be a linker L selected from the group consisting of alkyl, aryl, and heterocycle, to which linker between 1 and 3 compounds having the structure shown above can be attached. R.sub.2 is hydrogen, alkyl, aryl, hydroxyl, alkoxyl, aryloxyl, halogen, cyano, amino, alkylamino, arylamino, dialkylamino, diarylamino, arylalkylamino, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl, arylalkylaniinocarbonyl, alkylcarbonyl, arylcarbonyl, alkylcarbonyloxy, arylcarbonyloxy, carboxyl, alkoxycarbonyl, aryloxycarbonyl, sulfo, alkylsulfonyl, or arylsulfonyl. R.sub.3 and R.sub.4 are selected independently from the group consisting of hydrogen, amino, alkylarnino, arylamino, heterocycleamino, aralkylamino, dialkylamino, diarylamino, heterocylcealkylamino, alkylcarbonyl, arylcarbonyl, nitro, halogen, hydroxyl, aryloxyl, alkoxyl, lower alkyl, aryl, heteroaryl, aralkyl, cyano, carboxyl, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, and heteroaralkyl. R.sub.5 is selected from the group consisting of iminyl, hydroximinyl, alkyliminyl, aryliminyl, aralkyliminyl, alkoximinyl, aryloximinyl, heterocycleiminyl, cyclic irninyl, carboxyl, alkyl, alkylcarbonyl, arylcarbonyl, arylalkylarninomethyl, arylaminomethyl, alkylaminomethyl, alkoxycarbonyl, and aryloxycarbonyl; --HC.dbd.NNHR.sub.7 where R.sub.7 is selected from the group consisting of hydrogen, alkyl, aryl, aralkyl, heterocycle, heterocyclealkyl, and --C(.dbd.X.sub.2)(X.sub.3).sub.p R.sub.8 where p is 0 or 1, X.sub.2 is oxygen or sulfur, X.sub.3 is selected from the group consisting of oxygen, sulfur, and --NR.sub.9 --, where R.sub.8 and R.sub.9 are selected independently from the group consisting of hydrogen, alkyl, aryl, aralkyl, heterocycle, and heterocyclealkyl. Finally, R.sub.4 and R.sub.5 can be points of attachment for the group --CR'.dbd.N--Q.sub.k --, where k is 1 or 2, R' is selected from the group consisting hydrogen, alkyl, aryl, aralkyl, heterocycle, and heterocyclealkyl, and each Q.sub.k is selected independently from the group consisting of methylene, methine, and quarternary carbon, to form thereby an isopyrolo�4, 3-b!pyridine or pyrido�3, 4-b!pyridine derivative, respectively.
In another aspect, the present invention also provides compounds of the general formula of Compound I. However, R.sub.5 is not 4-methylphenyliminyl when X.sub.1 is sulfur, n is 1, Y is methylene, R.sub.2 is cyano, R.sub.3 and R.sub.4 are hydrogen, and R.sub.6 is 4-chlorophenyl.
In one embodiment of Compound I, n is 1 or 2 and R.sub.6 is selected from the group consisting of alkyl, aryl, aralkyl, arylcarbonyl, and heterocycle. A more specific embodiment is that in which n is 1 or 2, R.sub.6 is selected from the group consisting of alkyl, aryl, aralkyl, arylcarbonyl, and heterocycle, and X.sub.1 is oxygen or sulfur. Another more specific embodiment is that having the substituent pattern just recited and in which R.sub.2 is cyano. Still more specific embodiments include those for which n is 1 or 2 and R.sub.6 is selected from the group consisting of alkyl, aryl, aralkyl, arylcarbonyl, and heterocycle, R.sub.2 is cyano, X.sub.1 is oxygen or sulfur, and R.sub.5 is iminyl, hydroximinyl, alkyliminyl, aryliminyl, aralkyliminyl, alkoximinyl, aryloximinyl, or heteroaryliminyl, and especially those embodiments for which R.sub.5 is alkyliminyl, aryliminyl, aralkyliminyl, or heteroaryliminyl.
In some of the embodiments of Compound I in which R.sub.6 is selected from the group consisting of alkyl, aryl, aralkyl, arylcarbonyl, and heterocycle, R.sub.2 is cyano, and R.sub.5 is alkyliminyl, aryliminyl, aralkyliminyl, or heteroaryliminyl, R.sub.3 and R.sub.4 are hydrogen and X.sub.1 is sulfur. Of these embodiments, several derivatives for which n is 1, Y is methylene, and R.sub.6 is aryl or heteroaryl have been found to have useful telomerase-inhibiting properties. Particularly useful embodiments of Compound I having this latter substituent pattern are those in which R.sub.5 is phenyliminyl, substituted phenyliminyl, naphthyliminyl or substituted naphthyliminyl. Compounds in which R.sub.5 and R.sub.6 are independently phenyl or substituted phenyl (Compound II below) have especially useful properties. ##STR8## In Compound II, R.sub.10 -R.sub.14 are selected independently from the group consisting of hydrogen, halogen, hydroxyl, heterocycle, alkyl, aryl, aralkyl, heterocyclealkyl, alkoxyl, aryloxyl, heterocycleoxyl, nitro, cyano, alkylthio, arylthio, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyl, aryloxycarbonyl, carboxyl, and heterocyclethio. R.sub.15 -R.sub.19 are selected independently from the group consisting of hydrogen, halogen, hydroxyl, aryl, lower alkyl, alkoxyl, aryloxyl, arylthio, nitro, cyano, alkylarnino, and dialkylamino. Furthermore, R.sub.16 and R.sub.17 together may be points of attachment for the groups --C(O)--NR.sub.20 C--(O)-- and --O--(CH.sub.2).sub.m --O--, where m is 1 or 2 and R.sub.20 is hydrogen, aryl, aralkyl or alkyl, to form thereby iminyl phthalamides and 3,4-(methylenedioxy)phenyl imines or 3,4-(ethylenedioxy)phenyl imines, respectively.
In one embodiment, the present invention provides compounds of the general formula shown in Compound II above in which R.sub.10 -R.sub.11, are selected from the group consisting of hydrogen and halogen. In more particular embodiments of Compound II, R.sub.10, R.sub.11, R.sub.14, R.sub.18, and R.sub.19 are hydrogen; R.sub.12 is chloro; R.sub.13 is hydrogen or chloro; R.sub.15 is hydrogen, phenoxy, 4-methylphenoxy; R.sub.16 is hydrogen, chloro, fluoro, nitro, or trifluoromethyl; R.sub.17 is selected from the group consisting of methoxy, cyano, bromo, chloro, fluoro, trifluoromethyl, and dimethylamino. In addition, R.sub.16 and R.sub.17 together can be points of attachment for the groups --C(O)--NR.sub.20 C--(O)-- and --O--(CH.sub.2).sub.2 --O--, where R.sub.20 is ethyl or benzyl. Still more particular embodiments are those in which R.sub.13 is chloro and R.sub.15 is hydrogen. Several specific embodiments are shown Table 1 below.
TABLE 1______________________________________Compound R.sub.16 R.sub.17______________________________________III H ClIV H OCH.sub.3V Cl CNVI F FVII CF.sub.3 BrVIII ##STR9##IX ##STR10## ##STR11## In another embodiment, the present invention provides compounds of thegeneral formula shown in Compound I in which R.sub.2 is cyano, R.sub.3and R.sub.4 are hydrogen, R.sub.5 is heteroaryliminyl, R.sub.6 is aryl orheteroaryl, n is 1, Y is methylene, and X.sub.1 is sulfur; and, moreparticularly, in which R.sub.5 is pyridyliminyl, substitutedpyridyliminyl, isoquinolylininyl, or substituted isoquinolyliminyl.Particularly useful compounds are those in which R.sub.5 is2-pyridyliminyl or substituted 2-pyridyliminyl, and R.sub.6 is phenyl orsubstituted phenyl (Compound XI): ##STR12## Compound XI In Compound XI, R.sub.21 -R.sub.25 are selected independently from the group consisting of hydrogen, halogen, hydroxyl, heterocycle, alkyl, aryl, aralkyl, heterocyclealkyl, alkoxyl, aryloxyl, heterocycleoxyl, nitro, cyano, alkylthio, arylthio, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyl, aryloxycarbonyl, and heterocyclethio. In addition, adjacent substituents on the benzylthioether ring can be points of attachment for the group --Z.sub.1 --Z.sub.2 --Z.sub.3 --Z.sub.4 --, where Z.sub.1 -Z.sub.4 are selected independently from the group consisting of methylene, methine, quarternary carbon, and oxygen. R.sub.26 -R.sub.29 are selected independently from the group consisting of hydrogen, halogen, hydroxyl, aryl, lower alkyl, alkoxyl, aryloxyl, arylthio, nitro, cyano, alkylamino, and dialkylamino.
In one embodiment of Compound XI, R.sub.21 -R.sub.25 are selected independently from the group consisting of hydrogen and halogen. More specific embodiments include those for which R.sub.21, R.sub.22, and R.sub.25 are hydrogen; R.sub.23 and R.sub.24 are chloro; and R.sub.26 -R.sub.29 independently are hydrogen, halogen, or lower alkyl. Still more specific embodiments are provided in Table 2 below.
TABLE 2______________________________________Compound R.sub.26 R.sub.27 R.sub.28 R.sub.29______________________________________XII --H --H --Br --CH.sub.3XIII --H --H --CH.sub.3 --HXIV --CH.sub.3 --H --H --HXV --H --CH.sub.3 --H --H______________________________________
Additional embodiments of Compound XI in which R.sub.26 is methyl and R.sub.27 -R.sub.29 are hydrogen include those for which R.sub.21 -R.sub.25, together with the phenyl ring to which they are attached, define the following moieties: 2,6-dichlorophenyl (Compound XVI), 3,5-bis(trifluoromethyl)phenyl (Compound XVII), 2-biphenyl (Compound XVIII), 2-trifluoromethylphenyl (Compound XIX), 4-(2-phenylethenyl)phenyl (Compound XX), 4-isopropylphenyl (Compound XXI), 4-biphenyl (Compound XXII), 2-bromophenyl (Compound XXIII), 2-tolulyl (Compound XXIV), 6-chlorobenzo�3,4-e!-1,3-dioxin-8-yl (Compound XXV), 2,5-dimethylphenyl (Compound XXVI), 2,4-bis(trifluoromethyl)phenyl (Compound XXVII), 2-fluoro-3-chlorophenyl (Compound XXVII), 3,5-dimethylphenyl (Compound XXIX), 4-tert-butylphenyl (Compound XXX), 2,4-dimethylphenyl (Compound XXXI), 3,5-dichlorophenyl (Compound XXXII), 2,3,4,5,6-pentamethylphenyl (Compound XXXIII), 3,4-dimethylphenyl (Compound XXXIV), 2-bromo-5-fluorophenyl (Compound XXXV), 3-bromophenyl (Compound XXXVI), 3,5-dichlorophenyl (Compound XXXVII), 3,4-bis(benzyloxy)phenyl (Compound XXXVI), 4-(trifluoromethyl)phenyl (Compound XXXIX), 2-chlorophenyl (Compound XL), 4-(trifluoromethoxy)phenyl (Compound XLI), 3-chlorophenyl (Compound XLII), 3,4-(1,1,4,4-tetramethylbutylene)phenyl (Compound XLIII), 2-ethyl-4,5-(1,1,4,4-tetramethylbutylene)phenyl (Compound XLIV), and 3,4-(methylenedioxy)phenyl (Compound XLV).
Also included are derivatives of Compound I in which n is 1, Y is methylene, X.sub.1 is sulfur, R.sub.2 is cyano, R.sub.3 and R.sub.4 are hydrogen, R.sub.5 is 3-methyl-2-pyridyliminyl, 5-bromo-2-pyridiminyl, 5-methoxy-2-pyridiminyl, and R.sub.6 is aryl or heteroaryl. In one embodiment having this general substitution pattern, R.sub.5 is 3-methyl-2-pyridyliminyl and R.sub.6 is 2-methylnaphthyl (Compound XLVI). In another embodiment of this general substitution pattern, R.sub.6 is heteroaryl. These compounds include the derivatives for which R.sub.5 is 3-methyl-2-pyridyliminyl and R.sub.6 is 5-chlorobenzo�4,3-b!thiophen-3-yl (Compound XLVII), 2-(3-chloro-4-fluorophenyl)thiazol4-yl (Compound XLVI), 2-(4-chlorophenyl)thiazol-4-yl (Compound XLIX), 5-(3-(trifluoromethyl)phenyl)-1,2,4-oxadizol-3-yl (Compound L), and 2-chlorothien-5-yl (Compound LI). Also included are those derivatives for which R.sub.5 is 5-bromo-2-pyridiminyl, and R.sub.6 is 2-chlorothien-5-yl (Compound LII); and those in which R.sub.5 is 5-methoxy-2-pyridiminyl, and R.sub.6 is 2-chlorothien-5-yl (Compound LIII). Another embodiment of Compound I is that in which X.sub.1 is sulfur, n is 2 and each Y is methylene, R.sub.2 is cyano, R.sub.3 and R.sub.4 are hydrogen, R.sub.5 is 5-methoxy-2-pyridiminyl, and R.sub.6 is 4-fluorophenyl.
In still another embodiment of Compound I, R.sub.2 is cyano, R.sub.3 and R.sub.4 are hydrogen, X.sub.1 is sulfur, n is 1, Y is methylene, R.sub.5 is alkylirinyl, and R.sub.6 is phenyl or substituted phenyl. In one embodiment, R.sub.5 is alkyliminyl and R.sub.6 is 3,4-dichlorophenyl. More particular embodiments include those for which R.sub.5 is adamantyliminyl, chloroadamantyliminyl, noradamantyliminyl, norbornyliminyl, spiro�4.5!decanyliminyl, 2-ethynylcyclohexyliminyl, bornyliminyl, or - (cis)-myrtanyliminyl.
In yet another embodiment of Compound I, R.sub.2 is cyano, R.sub.3 and R.sub.4 are hydrogen, X.sub.1 is sulfur, n is 1, Y is methylene, R.sub.5 is aralkyliminyl. More specific embodiments are those in which R.sub.6 is 4-chlorophenyl, 2, 6-dichlorophenyl, or 3, 4-dichlorophenyl, and R.sub.5 is benzyliminyl, substituted benzyliminyl, 2-phenylethyliminyl, or phenyl-substituted 2-phenylethylininyl.
Other embodiments of the present invention include those in which n is 1 or 2, R.sub.6 is alkyl, aryl, arallyl, arylcarbonyl, or heterocycle, X.sub.1 is oxygen or sulfur, R2 is cyano, and R.sub.5 is --CH.dbd.NNHR.sub.7 where R.sub.7 is aryl. One useful embodiment of this general formula is that in which n is 1, Y is methylene, X.sub.1 is sulfur, R.sub.3 and R.sub.4 are hydrogen, R.sub.6 is 3,4-dichlorophenyl and R.sub.7 is 4-methylphenyl (Compound LIV) or 4-chlorophenyl (Compound LV). Also provided by the present invention are derivatives of Compound I in which n is 1 or 2, R.sub.6 is alkyl, aryl, aralkyl, arylcarbonyl, or heterocycle, X.sub.1 is oxygen or sulfur, R.sub.2 is cyano, and R.sub.5 is --CH.dbd.NNHR.sub.7 where R.sub.7 is --C(S)NHR.sub.8 and R.sub.8 is aryl. Specific embodiments having this general formula are those for which n is 1, Y is methylene, X.sub.1 is sulfur, R.sub.3 and R.sub.4 are hydrogen, R.sub.6 is 3,4-dichlorophenyl and R.sub.7 is 4-methylphenyl or 4-fluorophenyl (Compounds LVI and LVII).
Still other embodiments of Compound I provided by the present invention include those for which n is 1, Y is methylene, X.sub.1 is sulfur, R.sub.2 is cyano, R.sub.3 and R.sub.4 are hydrogen, R.sub.6 is 3,4-dichlorophenyl and R.sub.5 is quinuclidyliminyl. Also provided in the present invention are embodiments of Compound I for which n is 1, Y is methylene and R.sub.6 is a linker L as shown below (Compound LVIII): ##STR13## In one embodiment of Compound LVII, X.sub.1 is sulfur, R.sub.2 is cyano, R.sub.3 and R.sub.4 are hydrogen, R.sub.5 is heteroaryliminyl, and L is alkyl or aryl. Particularly useful embodiments of Compound LV having this substituent pattern are those for which R.sub.5 is 3-methyl-2-pyridyliminyl, and R.sub.6 selected from the group consisting of ethylene, ethenylene, and ethynylene (Compounds LIX, LX and LXI).
III. Synthesis of Telomerase Inhibitors
The compounds of the present invention can be synthesized using techniques and materials known to those of skill in the art, such as described, for example, in March, ADVANCED ORGANIC CHEMISTRY 4.sup.th Ed., (Wiley 1992); Carey and Sundberg, ADVANCED ORGANIC CHEMISTY 3.sup.rd Ed., Vols. A and B (Plenum 1992), and Green and Wuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS 2.sup.nd Ed. (Wiley 1991), each of which is incorporated herein by reference. Starting materials for the compounds of the invention may be obtained using standard techniques and commercially available precursor materials, such as those available from Aldrich Chemical Co. (Milwaukee, Wis.), Sigma Chemical Co. (St. Louis, Mo.), Lancaster Synthesis (Windham, N.H.), Apin Chemicals, Ltd. (New Brunswick, N.J.), Ryan Scientific (Columbia, S.C.), Maybridge (Cornwall, England) and Trans World Chemicals (Rockville, Md.).
The procedures described herein for synthesizing the compounds of the invention may include one or more steps of protection and deprotection (e.g., the formation and removal of acetal groups). In addition, the synthetic procedures disclosed below can include various purifications, such as column chromatography, flash chromatography, thin-layer chromatography (TLC), recrystallization, distillation, high-pressure liquid chromatography (HPLC) and the like. Also, various techniques well known in the chemical arts for the identification and quantification of chemical reaction products, such as proton and carbon-13 nuclear magnetic resonance (.sup.1 H and .sup.13 C NMR), infrared and ultraviolet spectroscopy (IR and UV), X-ray crystallography, elemental analysis (EA), HPLC and mass spectroscopy (MS) can be used as well. Methods of protection and deprotection, purification and identification and quantification are well known in the chemical arts.
Compounds of the class represented by Compound I can be synthesized using the following general procedure (Scheme 1). The use of the substituent identifiers R, R', R" and R"' is merely to indicate the presence of one or more substituents at the position or moiety indicated. The values of R, R', R" and R"' shown in Schemes 1 and 2 below can be determined by reference to the specific moieties described above in connection with the structure of Compound I. ##STR14##
With the appropriately substituted derivative of commercially available 2-thio-3-cyano-5- (dimethoxymethyl)pyridine 1, reaction with an appropriate alkylating agent or other nucleophile under basic conditions (e.g., potassium carbonate (K.sub.2 CO.sub.3) in dimethylformamnide (DMF) followed by addition of R'X, where X is a halide or other suitable leaving group such as tosyl) provides the desired thioether 2. Removal of the acetal group using standard conditions (e.g., p-toluenesulfonic acid in benzene), provides aldehyde 3 (pathway A).
Formation of imine 4 can be accomplished by conversion of the acetal 2 or aldehyde 3 by reaction with an appropriately substituted amine under standard conditions, e.g., by heating the acetal or aldehyde with the amine in benzene with 3.ANG. molecular sieves in the presence of an acid (pathways B and D). Similarly, reaction of the acetal 2 with an appropriately substituted hydrazide or semicarbazide and acid (e.g., HCl), provides the desired hydrazide 5 (pathway C). Additional synthetic routes to compounds 3,4,and 5 will be apparent to those having skill in the art based on the disclosure herein.
A more specific example of the general synthetic procedure just described above is presented below (Scheme 2). Again, R, R' and R" are as defined above. ##STR15##
Starting from commercially available 2-thio-3-cyano-6-(dimethoxymethyl)pyridine 1 (Ryan Scientific), removal of the thiol proton using base (B:, e.g., K.sub.2 CO.sub.3 in DMF) and introduction of the appropriately substituted benzylchloride derivative 6 under substantially anhydrous conditions provides the benzylthioether 7. Removal of the dimethylacetal group under acidic conditions, followed by imine formation with aniline derivative 8 produces the desired arylimine 9. The benzyl chloride and aniline derivatives can be purchased commercially (e.g., Aldrich) or made using standard methods. The reactions to form the thioether 7 and the imine 8 are performed using standard methods.
Formation of the above-described di-, tri-, or tetrakisiminylpyridylthioethers (e.g., compound LVIII) is accomplished by analogy to the reactions illustrated in Scheme 1 above in which R' is a nucleophile corresponding to the desired combination of n, Y.sub.n, and linker L. One equivalent of starting dimethoxymethylpyridine thiol 1 is combined with the nucleophile R' for each leaving group available on the nucleophile. Following the coupling of the nucleophile to the starting dimethoxymethylpyridine thiol 1, the dimethoxymethyl group of each of the linked pyridylthioethers is converted to the corresponding imine 4. For example, reaction of two equivalents of 1 (R=H) with one equivalent of .alpha., .alpha.'-dichloroxylene as described above for Scheme 1 provides the corresponding dialdehyde which is then converted into the desired bis-imine using standard procedures.
Protocols from which the individual Compounds described above can be synthesized are provided in the Examples below.
IV. Anti-Tumor Activity of the Telomerase Inhibitors of the Invention
The compounds of the present invention demonstrate inhibitory activity against telomerase in vitro and in vivo, as has been and can be demonstrated as described below. As used herein, the term "in vitro" refers to tests performed using living cells in tissue culture. Such procedures are also known as "ex vivo".
One method used to identify compounds of the invention that inhibit telomerase activity involves placing cells, tissues, or preferably a cellular extract or other preparation containing telomerase in contact with several known concentrations of a test compound in a buffer compatible with telomerase activity. The level of telomerase activity for each concentration of test compound is measured and the IC.sub.50 (the concentration of the test compound at which the observed activity for a sample preparation was observed to fall one-half of its original or a control value) for the compound is determined using standard techniques. Other methods for determining the inhibitory concentration of a compound of the invention against telomerase can be employed as will be apparent to those of skill in the art based on the disclosure herein.
With the above-described methods, IC.sub.50 values for several of the compounds of the present invention were determined. The values reported in Table 3 below are only approximate values; more exact IC.sub.50 values can be obtained by repetitive testing.
TABLE 3______________________________________Compound IC.sub.50 (.mu.M) Compound IC.sub.50 (.mu.M)______________________________________III 6.5 XXIV 3.0IV 4.9 XXV 4.0V 10.0 XXVI 0.9VI 10.0 XXVII 5.0VII 12.0 XXVIII 2.0VIII 10.0 XXIX 2.0IX 8.0 XXX 2.0X 9.0 XXXI <3.2XII 7.0 XXXII <3.2XIII 7.0 XXXVII <3.2XIV <3.2 XLIII <3.2XV 8.0 XLVI 3.0XVI 1.0 XLVII <3.2XVII 2.0 LII <3.2XVIII 2.0 LIII 5.0XIX 3.0 LIV 17.0XX 8.0 LV 19.2XXI 2.0 LVI 18.0XXII 2.0 LVII 18.0XXIII 2.0 LXI <3.2______________________________________
As shown in Table I, all of the compounds have strong anti-telomerase activity (i.e., an IC.sub.50 <20 .mu.M). Several of the compounds of the invention (IV, XIV, XVI-XIX, XXI-XXXII, XXXVII, XLIII, XLVI, XLVII, LII, LIII, and, LXI) are potent telomerase inhibitors, having approximate IC50 values of less than about 5 .mu.M.
With respect to the treatment of malignant diseases using the compounds described herein, compounds of the present invention are expected to induce crisis in telomerase-positive cell lines. Treatment of telomerase-positive cell lines, such as HEK-293 and HeLa cells, with a compound of the invention is also expected to induce a reduction of telomere length in the treated cells.
Compounds of the invention are also expected to induce telomere reduction during cell division in human tumor cell lines, such as the ovarian tumor cell lines OVCAR-5 and SK-OV-3. Importantly, however, in normal human cells used as a control, such as BJ cells of fibroblast origin, the observed reduction in telomere length is expected to be no different from cells treated with a control substance, e.g., dimethyl sulfoxide (DMSO). The compounds of the invention also are expected to demonstrate no significant cytotoxic effects at concentrations below about 5 .mu.M in the tumor cells.
In addition, the specificity of the compounds of the present invention for telomerase can be determined by comparing their activity (IC.sub.50) with respect to telomerase to other enzymes having similar nucleic acid binding or modifying activity similar to telomerase in vitro. Such enzymes include DNA Polymerase I, HeLa RNA Polymerase II, T3 RNA Polymerase, MMLV Reverse Transcriptase, Topoisomerase I, Terminal Transferase and Single-Stranded DNA Binding Protein (SSB). Compounds having lower IC.sub.50 s for telomerase as compared to IC.sub.50 s toward the other enzymes being screened are said to possess specificity for telomerase.
In vivo testing can also be performed using a mouse xenograft model in which OVCAR-5 tumor cells are grafted onto nude mice. As discussed in Example B. 2 below, mice treated with a compound of the invention are expected to have tumor masses that, on average, may increase for a period following the initial dosing, but will begin to shrink in mass with continuing treatment. In contrast, mice treated with a control (e.g., DMSO) are expected to have tumor masses that continue to increase.
From the foregoing those skilled in the art will appreciate that the present invention also provides methods for selecting treatment regimens involving administration of a compound of the invention. For such purposes, it may be helpful to perform a TRF analysis in which DNA from tumor cells is analyzed by digestion with restriction enzymes specific for sequences other than the telomeric (T.sub.2 AG.sub.3).sub.N sequence. Following digestion of the DNA, gel electrophoresis is performed to separate the restriction fragments according to size. The separated fragments are then probed with nucleic acid probes specific for telomeric sequences to determine the lengths of the telomeres of the cells in the sample. By measuring the length of telomeric DNA, one can estimate how long a telomerase inhibitor should be administered and whether other methods of therapy (e.g., surgery, chemotherapy and/or radiation) should also be employed. In addition, during treatment, one can test cells to determine whether a decrease in telomere length over progressive cell divisions is occurring to demonstrate treatment efficacy.
V. Telomerase Inhibiting Compositions and Methods for Treating Diseases With the Same
The present invention also provides pharmaceutical compositions for treating cancer and other conditions in which inhibition of telomerase is an effective therapy. These compositions include a therapeutically effective amount of a telomerase inhibiting compound of the invention in a pharmaceutically acceptable carrier or salt.
In one preferred embodiment, the present invention provides a composition effective for treating cancer in a mammal. The composition includes a therapeutically effective amount of a compound having the structure of Compound I shown above in a pharmaceutically acceptable carrier.
In addition, it will be appreciated that therapeutic benefits for treatment of cancer can be realized by combining a telomerase inhibitor of the invention with other anti-cancer agents, including other inhibitors of telomerase such as described in co-pending U.S. Pat. Application Ser. No. 08/535,988, entitled Telomerase Inhibitors, by inventors Federico C. A. Gaeta, and Elaine C. Stracker, filed Sep. 29, 1995, which is incorporated herein by reference for every purpose. The choice of such combinations will depend on various factors including, but not limited to, the type of disease, the age and general health of the patient, the aggressiveness of disease progression, the TRF length and telomerase activity of the diseased cells to be treated and the ability of the patient to tolerate the agents that comprise the combination. For example, in cases where tumor progression has reached an advanced state, it may be advisable to combine a telomerase inhibiting compound of the invention with other agents and therapeutic regimens that are effective at reducing tumor size (e.g. radiation, surgery, chemotherapy and/or hormonal treatments). One regimen for reducing tumor size includes administration of a topoisomerase II-inhibitor, including those topoisomerase-II inhibitors described in the above-cited co-pending U.S. Pat. Application Ser. No. 08/535,988. In addition, in some cases it may be advisable to combine a telomerase inhibiting agent of the invention with one or more agents that treat the side effects of a disease, e.g., an analgesic, or agents effective to stimulate the patient's own immune response (e.g., colony stimulating factor).
In one such method, a pharmaceutical formulation comprises a telomerase inhibitor of the -invention with an anti-angiogenesis agent, such as fumagillin, fumagillin derivatives, or AGM-1470. The latter compound is available from Takeda Chemical Industries, Ltd., while the former compounds are described in Ingber, et al., 6 Dec. 1990, "Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumor growth", Nature 348:555-557, incorporated herein by reference for all purposes. Other combinations may include, but are not limited to, a telomerase inhibitor of the invention in addition to one or more antineoplastic agents or adjuncts (e.g., folinic acid or mesna).
Antineoplastic agents suitable for combination with the compounds of the present invention include, but are not limited to, alkylating agents including alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines, such as a benzodizepa, carboquone, meturedepa and uredepa; ethylenimines and methylmelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cyclophosphamide, estramustine, iphosphamide, mechlorethamine, mechlorethamnine oxide hydrochloride, melphalan, novembichine, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitroso ureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine. Additional agents include dacarbazine, mannomustine, mitobronitol, mitolactol and pipobroman. Still other classes of relevant agents include antibiotics, hormonal antineoplastics and antimetabolites. Yet other combinations will be apparent to those of skill in the art.
Additional agents suitable for combination with the compounds of the present invention include protein synthesis inhibitors such as abrin, aurintricarboxylic acid, chloramphenicol, colicin E3, cycloheximide, diphtheria toxin, edeine A, emetine, erytliromycin, ethionine, fluoride, 5-fluorotryptophan, fusidic acid, guanylyl methylene diphosphonate and guanylyl imidodiphosphate, kanamycin, kasugamycin, kirromycin, and O-methyl threonine. Additional protein synthesis inhibitors include modeccin, neomycin, norvaline, pactamycin, paromomycine, puromycin, ricin, .alpha.-sarcin, shiga toxin, showdomycin, sparsomycin, spectinomycin, streptomycin, tetracycline, thiostrepton and trimethoprim. Inhibitors of DNA synthesis, including alkylating agents such as dimethyl sulfate, mitomycin C, nitrogen and sulfur mustards, MNNG and NMS; intercalating agents such as acridine dyes, actinomycins, adriamycin, anthracenes, benzopyrene, ethidium bromide, propidium diiodide intertwining agents such as distamycin and netropsin, can also be combined with compounds of the present invention in pharmaceutical compositions. DNA base analogs such as acyclovir, adenine .beta.-1-D-arabinoside, amethopterin, aminopterin, 2-aminopurine, aphidicolin, 8-azaguanine, azaserine, 6-azauracil, 2'-azido-2'-deoxynucleosides, 5-bromodeoxycytidine, cytosine .beta.-1-D-arabinoside, diazooxynorleucine, dideoxynucleosides, 5-fluorodeoxycytidine, 5-fluorodeoxyuridine, 5-fluorouracil, hydroxyurea and 6-mercaptopurine also can be used in combination therapies with the compounds of the invention. Topoisomerase inhibitors, such as coumermycin, nalidixic acid, novobiocin and oxolinic acid, inhibitors of cell division, including colcemide, colchicine, vinblastine and vincristine, and RNA synthesis inhibitors including actinomycin D, .alpha.-amanitine and other fungal amatoxins, cordycepin (3'-deoxyadenosine), dichlororibofuranosyl benzimidazole, rifampicine and streptovaricin and streptolydigin also can be combined with the compounds of the invention to provide pharmaceutical compositions.
In another embodiment, the present invention includes compounds and compositions in which a telomerase inhibitor is either combined with or covalently bound to a cytotoxic agent bound to a targeting agent, such as a monoclonal antibody (e.g., a murine or humanized monoclonal antibody). It will be appreciated that the latter combination may allow the introduction of cytotoxic agents into cancer cells with greater specificity. Thus, the active form of the cytotoxic agent (i.e., the free form) will be present only in cells targeted by the antibody. Of course, the telomerase inhibitors of the invention may also be combined with monoclonal antibodies that have therapeutic activity against cancer.
In general, a suitable effective dose of a compound of the invention will be in the range of 0.001 to 1000 milligram (mg) per kilogram (kg) of body weight of the recipient per day, preferably in the range of 0.001 to 100 mg per kg of body weight per day, more preferably between about 0.1 and 100 mg per kg of body weight per day and still more preferably in the range of between 0.1 to 10 mg per kg of body weight per day. The desired dosage is preferably presented in one, two, three, four, or more subdoses administered at appropriate intervals throughout the day. These subdoses can be administered as unit dosage form, for example, containing 5 to 10,000 mg, preferably 10 to 1000 mg of active ingredient per unit dosage form. Preferably, the dosage is presented once per day at a dosing at least equal to TID.
The composition used in these therapies can be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, liposomes, and injectable and infusible solutions. The preferred form depends on the intended mode of administration and therapeutic application. The compositions also preferably include conventional pharmaceutically acceptable carriers and adjuvants, as is well known to those of skill in the art. See, e.g., REMINGTON's PHARMACEUTICAL SCIENCES, Mack Publishing Co.: Easton, Pa., 17.sup.th Ed. (1985). Preferably, administration will be by oral or parenteral (including subcutaneous, intramuscular, intravenous, and intradermal) routes. More preferably, the route of administration will be oral. The therapeutic methods and agents of this invention can of course be used concomitantly or in combination with other methods and agents for treating a particular disease or disease condition.
While it is possible to administer the active ingredient of this invention alone, it is preferable to present a therapeutic agent as part of a pharmaceutical formulation or composition. The formulations of the present invention comprise at least one telomerase activity-inhibiting compound of this invention in a therapeutically or pharmaceutically effective dose together with one or more pharmaceutically or therapeutically acceptable carriers and optionally other therapeutic ingredients. Various considerations for preparing such formulations are described, e.g., in Gilman et al. (eds.) GOODMAN AND GILMAN's: THE PHARMACOLOGICAL BASES OF THERAPEUTICS, 8th Ed., Pergamon Press (1990); and REMINGTON's supra, each of which is incorporated herein by reference for all purposes. Methods for administration are discussed therein, e.g., for oral, intravenous, intraperitoneal, intramuscular, and other forms of administration. Generally, oral administration is preferred.
Typically, methods for administering pharmaceutical compositions will be either topical, parenteral, or oral administration methods for prophylactic and/or therapeutic treatment. Oral administration is a preferred. The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. As noted above, unit dosage forms suitable for oral administration include powders, tablets, pills, and capsules.
One can use topical administration to deliver a compound of the invention by percutaneous passage of the drug into the systemic circulation of the patient. The skin sites include anatomic regions for transdermally administering the drug, such as the forearm, abdomen, chest, back, buttock, and mastoidal area. The compound is administered to the skin by placing on the skin either a topical formulation comprising the compound or a transdermal drug delivery device that administers the compound. In either embodiment, the delivery vehicle is designed, shaped, sized, and adapted for easy placement and comfortable retention on the skin.
A variety of transdermal drug delivery devices can be employed with the compounds of this invention. For example, a simple adhesive patch comprising a backing material and an acrylate adhesive can be prepared. The drug and any penetration enhancer can be formulated into the adhesive casting solution. The adhesive casting solution can be cast directly onto the backing material or can be applied to the skin to form an adherent coating. See, e.g., U.S. Pat. Nos. 4,310,509; 4,560,555; and 4,542,012.
In other embodiments, the compound of the invention will be delivered using a liquid reservoir system drug delivery device. These systems typically comprise a backing material, a membrane, an acrylate based adhesive, and a release liner. The membrane is sealed to the backing to form a reservoir. The drug or compound and any vehicles, enhancers, stabilizers, gelling agents, and the like are then incorporated into the reservoir. See, e.g., U.S. Pat. Nos. 4,597,961; 4,485,097; 4,608,249; 4,505,891; 3,843,480; 3,948,254; 3,948,262; 3,053,255; and 3,993,073.
Matrix patches comprising a backing, a drug/penetration enhancer matrix, a membrane, and an adhesive can also be employed to deliver a compound of the invention transdermally. The matrix material typically will comprise a polyurethane foam. The drug, any enhancers, vehicles, stabilizers, and the like are combined with the foam precursors. The foam is allowed to cure to produce a tacky, elastomeric matrix which can be directly affixed to the backing material. See, e.g., U.S. Pat. Nos. 4,542,013; 4,460,562; 4,466,953; 4,482,534; and 4,533,540.
Also included within the invention are preparations for topical application to the skin comprising a compound of the invention, typically in concentrations in the range from about 0.001% to 10%, together with a non-toxic, pharmaceutically acceptable topical carrier. These topical preparations can be prepared by combining an active ingredient according to this invention with conventional pharmaceutical diluents and carriers commonly used in topical dry, liquid, and cream formulations. Ointment and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Such bases may include water and/or an oil, such as liquid paraffin or a vegetable oil, such as peanut oil or castor oil. Thickening agents that may be used according to the nature of the base include soft paraffin, aluminum stearate, cetostearyl alcohol, propylene glycol, polyethylene glycols, woolfat, hydrogenated lanolin, beeswax, and the like.
Lotions may be formulated with an aqueous or oily base and will, in general, also include one or more of the following: stabilizing agents, emulsifying agents, dispersing agents, suspending agents, thickening agents, coloring agents, perfumes, and the like. Powders may be formed with the aid of any suitable powder base, e.g., talc, lactose, starch, and the like. Drops may be formulated with an aqueous base or non-aqueous base also comprising one or more dispersing agents, suspending agents, solubilizing agents, and the like. Topical administration of compounds of the invention may also be preferred for treating diseases such as skin cancer and fungal infections of the skin (pathogenic fungi typically express telomerase activity).
The topical pharmaceutical compositions according to this invention may also include one or more preservatives or bacteriostatic agents, e.g., methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkonium chlorides, and the like. The topical pharmaceutical compositions also can contain other active ingredients such as antimicrobial agents, particularly antibiotics, anesthetics, analgesics, and antipruritic agents.
The compounds of the present invention can also be delivered through mucosal membranes. Transmucosal (i.e., sublingual, buccal, and vaginal) drug delivery provides for an efficient entry of active substances to systemic circulation and reduces immediate metabolism by the liver and intestinal wall flora. Transmucosal drug dosage forms (e.g., tablet, suppository, ointment, pessary, membrane, and powder) are typically held in contact with the mucosal membrane and disintegrate and/or dissolve rapidly to allow immediate systemic absorption. Note that certain such routes may be used even where the patient is unable to ingest a treatment composition orally. Note also that where delivery of a telomerase inhibitor of the invention would be enhanced, one can select a composition for delivery to a mucosal membrane, e.g., in cases of colon cancer one can use a suppository to deliver the telomerase inhibitor.
For delivery to the buccal or sublingual membranes, typically an oral formulation, such as a lozenge, tablet, or capsule, will be used. The method of manufacture of these formulations is known in the art, including, but not limited to, the addition of the pharmacological agent to a pre-manufactured tablet; cold compression of an inert filler, a binder, and either a pharmacological agent or a substance containing the agent (as described in U.S. Pat. No. 4,806,356); and encapsulation. Another oral formulation is one that can be applied with an adhesive, such as the cellulose derivative hydroxypropyl cellulose, to the oral mucosa, for example as described in U.S. Pat. No. 4,940,587. This buccal adhesive formulation, when applied to the buccal mucosa, allows for controlled release of the pharmacological agent into the mouth and through the buccal mucosa.
Parenteral administration is generally characterized by injection, either subcutaneously, intramuscularly, or intravenously. Thus, this invention provides compositions for intravenous administration that comprise a solution of a compound of the invention dissolved or suspended in an acceptable carrier. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, buffered water, saline, dextrose, glycerol, ethanol, or the like. These compositions will be sterilized by conventional, well known sterilization techniques, such as sterile filtration. The resulting solutions can be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, etc. Such formulations will be useful in treating ovarian cancers.
Another method of parenteral administration employs the implantation of a slow-release or sustained-release system, such that a constant level of dosage is maintained. See, e.g., U.S. Pat. No. 3,710,795, incorporated herein by reference.
Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc., an active compound as defined above and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, olive oil, and other lipophilic solvents, and the like, to form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc. Actual methods of preparing such dosage forms are known and will be apparent to those skilled in this art; for example, see REMINGTON's PHARMACEUTICAL SCIENCES, supra. The composition or formulation to be administered will contain an effective amount of an active compound of the invention.
For solid compositions, conventional nontoxic solid carriers can be used and include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 0.1-95% of active ingredient, preferably about 20%.
The compositions containing the compounds of the invention can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, compositions are administered to a patient already suffering from a disease, as described above, in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. An amount adequate to accomplish this is defined as a "therapeutically effective amount or dose." Amounts effective for this use will depend on the severity of the disease and the weight and general state of the patient.
In addition to internal (in vivo) administration, the compounds and compositions of the invention may be applied ex vivo to achieve therapeutic effects, as for example, in the case of a patient suffering from leukemia. In such an application, cells to be treated, e.g., blood or bone marrow cells, are removed from a patient and treated with a pharmaceutically effective amount of a compound of the invention. The cells are returned to the patient following treatment. Such a procedure can allow for exposure of cells to concentrations of therapeutic agent for longer periods or at higher concentrations than otherwise available.
Once improvement of the patient's conditions has occurred, as, for example, by the occurrence of remission in the case of a cancer patient, a maintenance dose is administered, if necessary. Subsequently, the dosage or the frequency of administration, or both, can be reduced, as a function of the systems, to a level at which the improved condition is retained. When the symptoms have been alleviated to the desired level, treatment can cease. Patients can, however, require additional treatment upon any recurrence of the disease symptoms.
In prophylactic applications (e.g. chemoprevention), compositions containing the compounds of the invention are administered to a patient susceptible to or otherwise at risk of a particular disease. Such an amount is defined to be a "prophylactically effective amount or dose." In this use, the precise amounts again depend on the patient's state of health and weight.
As will be apparent to those of skill in the art upon reading of this disclosure, the present invention provides valuable reagents relating to human telomerase. The above description of necessity provides a limited and merely illustrative sampling of specific compounds, and should not be construed as limiting the scope of the invention. Other features and advantages of the invention will be apparent from the following examples and claims.

EXAMPLES
The following examples describe specific aspects of the invention to illustrate the invention and also provide a description of the methods used to identify and test compounds that inhibit the activity of telomerase to aid those of skill in the art in understanding and practicing the invention. The examples should not be construed as limiting the invention, in any manner.
A. Chemical Syntheses
The following procedures illustrate the syntheses of compounds in the class defined by the structure of Compound I.
General Procedure A: Synthesis of 6-Dimethoxymethyl-3-Cyano-2-Thioetherpyridines
The following demonstrates the synthesis of Compound 2 of Scheme 1. Into a round-bottom flask equipped with a magnetic stir bar and under a positive pressure of nitrogen were placed 3-cyano-2-mercaptopyridine-6-carboxaldehyde dimethyl acetal (1.0513 g, 5.0 mmol), DMF (5 ml), K.sub.2 CO.sub.3 (0.7602 g, 5.5 mmol), and the corresponding alkylating agent (5.2 mmol). The heterogeneous mixture was stirred at room temperature for 3 hr. The reaction mixture was poured into ice/H.sub.2 O (50 ml) and extracted twice with ethyl acetate (EtOAc.) The combined organic layers were washed with saturated sodium chloride (NaCl, brine), dried over magnesium sulfate (MgSO.sub.4), filtered, and the solvent removed by rotary evaporation to yield the coupled product. Isolated yields range from 87-96% The product was analyzed by .sup.1 H NMR and TLC.
General Procedure B: Acetal Deprotection of 6-Dimethoxymethyl-3-Cyano-2-Thioetherpyridines
The following demonstrates the conversion of Compound 2 of Scheme 1 into Compound 3 of the same Scheme. Into a round-bottom, three-necked flask equipped with a magnetic stir bar, thermometer, reflux condenser, and under a positive pressure of nitrogen, were placed the corresponding dimethyl acetal (5.0 mmol), benzene (60 ml), and p-toluenesulfonic acid (1.9975 g, 10.5 mmol). The mixture was refluxed at 73-78 .degree. C. for 48 hours (h). The reaction was quenched by pouring the reaction mixture into saturated aqueous (sat. aq.) sodium bicarbonate (NaHCO.sub.3) and then extracted twice with methylene chloride. The combined organic layers were washed with sat. NaCl (brine), dried over MgSO.sub.4, filtered, and the solvent removed by rotary evaporation to yield the desired aldehyde. The product was analyzed by H.sup.1 NMR and TLC.
General Procedure C: Synthesis of 6-Imino-3-Cyano-2-Thioetherpyridines
This procedure demonstrates the conversion of Compound 2 or Compound 3 of Scheme 1 into Compound 4 of that Scheme. Into a round bottom, three-necked flask equipped with a magnetic stir bar, thermometer, reflux condenser, and under a positive pressure of nitrogen, were placed the corresponding dimethyl acetal or aldehyde (1.0 mmol), 3.ANG. molecular sieves (5.0 g), benzene (12 ml), and p-toluenesulfonic acid (0.3995g, 2.1 mmol), and the amine (1.0 mmol). The mixture was refluxed at 73.degree.-78.degree. C. for 24-48 h. Upon cooling, the molecular sieves were filtered off and washed with methylene chloride. The filtrate was poured into sat. aq. NaHCO.sub.3 and extracted twice with methylene chloride. The combined organic layers were washed with sat. NaCl (brine), dried over MgSO.sub.4 (or sodium sulfate (Na.sub.2 SO.sub.4)), filtered, and the solvent removed by rotary evaporation to yield the crude product. The crude product was recrystallized from acetonitrile. The final product was analyzed by .sup.1 H NMR and TLC.
General Procedure D: Deprotection of Dimethoxyacetal Pyridine Thioethers
This procedure also illustrates the conversion of compound 2 of Scheme 1 into compound 3 of that Scheme. Into a round bottom, three-necked flask equipped with a magnetic stir bar, thermometer and under a positive nitrogen pressure, were introduced the corresponding acetal (7.0 mmol) and chloroform (CHCl.sub.3, 63 mL). To this mixture a second mixture of aqueous trifluoroacetic acid (TFA/H.sub.2 O, 31 mL/31 mL) was added at 0 .degree. C. The reaction mixture was stirred vigorously at 35 .degree. C. until completion, as determined by TLC analysis (3-78 h.) The reaction was quenched with sat. aq. NaHCO.sub.3 and the solution extracted twice with EtOAc. The combined organic layers from each extraction were washed with sat. aq. NaCl (brine), then dried over sodium sulfate (Na.sub.2 SO.sub.4) and filtered. The solvent was removed by rotary evaporation, and the product analyzed by .sup.1 H NMR and TLC.
General Procedure E: Synthesis of Hydrazide and Semicarbazide Derivatives
This procedure illustrates the formation of compound 5 of Scheme 1. Into a reaction vessel equipped with a magnetic stir bar, were placed the corresponding dimethyl acetal (0.25 mmol), semicarbazide or hydrazide (0.25 mmol), methanol (2.5 mL), and 2 drops of concentrated hydrochloric acid (HCl.) The vessel was sealed with a rubber septa and provided with a pressure release needle. The vessel was placed in a sonicator and heated to 50.degree. C. for two days. Upon cooling, the solids were isolated by filtration and washed with methanol. The product was dried overnight in a high vacuum oven.
B. Anti-tumor Activity
1. Ex vivo Studies
a. Reduction of Telomere Length in Tumor Cells
Colonies of the tumor cell lines, such as the ovarian tumor cell lines OVCAR-5 and SK-OV-3, and normal human cells used as a control (e.g., normal human BJ cells) are prepared using standard methods and materials. In one test, the colonies are prepared by seeding 15-centimeter dishes with about 10.sup.6 cells in each dish. The dishes are incubated to allow the cell colonies to grow to about 80% confluence, at which time each of the colonies are divided into two groups. One group is exposed to a subacute dose of a compound of the invention at a predetermined concentration (e.g., between about 5 .mu.M and about 20 .mu.M) for a period of about 4-8 hours after plating following the split; the other group is exposed to a control (e.g., DMSO).
Each group is then allowed to continue to divide, and the groups are split evenly again (near confluence). The same number of cells are seeded for continued growth. The compound or control is added every fourth day to the samples at the same concentration delivered initially. Remaining cells are analyzed for telomere length. As the untested cell cultures near confluence, the samples are split again as just described. This sequence of cell doubling and splitting is continued for about 20 to 25 doublings. Thus, a determination of telomere length as a function of cell doublings is obtained.
Telomere length is determined by digesting the DNA of the cells using restriction enzymes specific for sequences other than the repetitive T.sub.2 AG.sub.3 sequence of human telomeres. The digested DNA is separated by size using standard techniques of gel electrophoresis to determine the lengths of the telomeric repeats, which appear, after probing, on the gel as a smear of high-molecular weight DNA (approximately 2 Kb-15 Kb).
The results of the telomere length analysis are expected to indicate that the compounds of the invention have no affect on the rate of decrease in telomere length for control cells as a function of progressive cell doublings. With respect to the tumor cell lines, however, measurable decreases in telomere length are expected to be determined for tumor cells exposed to the compounds of the invention. Tumor cells exposed to the control are expected to maintain steady telomere lengths. Thus, the compounds of the invention are expected to cause resumption of the normal loss of telomere length as a function of cell division in tumor cells.
In another experiment, HEK-293 cells are incubated with a compound of the invention and a control at concentrations between about 1 .mu.M and about 20 .mu.M using the protocol just described. Cells are expected to enter crisis (ie., the cessation of cell function) within several weeks following administration of the test compound of the invention. In addition, TRF analyses of the cells using standard methodology is expected to show that the test compounds of the invention are effective to cause reductions in telomere length. In addition to the HEK-293 cells described above, this assay can be performed with any telomerase-positive cell line, such as HeLa cells.
In one uncontrolled experiment, Compound XWV was administered to telomerase-positive cells with a dosing period of about 48 hours. No significant shorting of telomere length was observed. However, additional data suggest that the half-life for Compound XIV is on the order of about 12 hours. Thus, it is believed that cells treated with Compound XIV would exhibit telomere shortening if the testing regimen includes a dosing frequency of about once every 12 hours, or more frequently, or if the cells are exposed to a constant concentration of Compound XIV of between about 5 .mu.M and about 10 .mu.M.
b. Specificity
Compounds of the invention are screened for activity (IC.sub.50) against telomerase and several enzymes having nucleic acid binding or modifying activities related to telomerase using standard techniques. The enzymes being screened include Telomerase, DNA Polymerase I, HeLa RNA Polymerase II, T3 RNA Polymerase, MMLV Reverse Transcriptase, Topoisomerase I, Terminal Transferase and Single-Stranded DNA Binding Protein (SSB). The specificity of a compound of the invention for telomerase is determined by comparing the IC.sub.50 of the compound with respect to telomerase with the IC.sub.50 s of the compound for each of the enzymes being screened. The compound is determined to have high specificity for telomerase if the IC.sub.50 for telomerase of the compound is lower than the IC.sub.50 s for each of the enzymes being screened.
c. Cytotoxicity
The MTT assay for cytotoxicity is performed using tumor cells lines such as the ovarian tumor cell lines OVCAR-5 and SK-OV-3. Cells from the normal human cell lines (e.g., normal human BJ cells) are used as a control. The cell lines used in the assay are exposed to a compound of the invention for 72 hours at concentrations ranging from 3 .mu.M to 1,000 .mu.M. During this period, the optical density (OD) of the samples is determined for light at 540 nanometers (nm). No significant cytotoxic effects are expected to be observed at concentrations less than about 5 .mu.M.
Some compounds may induce G2 arrest at concentrations above about 5 JIM (i.e., at 10 .mu.M-20 .mu.M concentrations or higher). Preferably, to observe any telomerase inhibiting effects the compounds should be administered at a concentration below the level of cytotoxicity. Nevertheless, since the effectiveness of many cancer chemotherapeutics derives from their cytotoxic effects, it is within the scope of the present invention that the compounds of the present invention be administered at any dose for which chemotherapeutic effects are observed.
2. In vivo Studies
A human tumor xenograft model in which OVCAR-5 tumor cells are grafted into nude mice can be constructed using standard techniques and materials. The mice are divided into two groups. One group is treated intraperitoneally with a compound of the invention. The other group is treated with a control comprising a mixture of either DMSO or ethanol and emulphor (oil) and phosphate buffer solution (PBS). The average tumor mass for mice in each group is determined periodically following the xenograft using standard methods and materials.
In the group treated with a compound of the invention, the average tumor mass is expected to increase following the initial treatment for a period of time, after which time the tumor mass is expected to stabilize and then begin to decline. Tumor masses in the control group are expected to increase throughout the study. Thus, the compounds of the invention are expected to lessen dramatically the rate of tumor growth and ultimately induce reduction in tumor size and elimination of the tumor.
Thus, the present invention provides novel compounds, compositions and methods for inhibiting telomerase activity and treating disease states in which telomerase activity has deleterious effects, especially cancer. The compounds of the invention provide a highly selective and effective treatment for malignant cells that require telomerase activity to remain immortal; yet, without affecting non-malignant cells.
Although certain embodiments and examples have been used to describe the present invention, it will be apparent to those of skill in the art that changes may be made to those described embodiments and examples without departing from the scope or spirit of the invention or the following claims.

Number	Name	Date
4269846	Huang et al.	May 1981
4611059	Sih	Sep 1986
4863923	Ho et al.	Sep 1989

Number	Date	Country
0 050957 A1	May 1982	EPX
0 069521 A2	Jan 1983	EPX
0 158380 B1	Oct 1985	EPX
0 483647 A1	May 1992	EPX
0 512349 A1	Nov 1992	EPX
0 568289 A2	Nov 1993	EPX
0 572712 A2	Dec 1993	EPX
0 619297 A1	Oct 1994	EPX
1936721	May 1969	DEX
1927393	Dec 1969	DEX
9203427	Mar 1992	JPX
9302037	Feb 1993	WOX
9323572	Nov 1993	WOX

Telomerase inhibitors

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Entry
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