WO1999065875A1 - Telomerase inhibitors - Google Patents

Abstract

Methods and compositions for treating cancer are provided. In one aspect, the present invention includes methods and compositions for treating cancer that include telomerase-inhibiting compounds from among the generic class of compounds shown in formula (1).

Description

TELOMERASE INHIBITORS 1 Notice of U.S. Government Rights

A portion of the work described herein was funded in part by SBIR Grant No. 1 R43 CA 5 01. The U.S. Government may therefore have certain rights relating to this invention.

3 Background of the Invention 3.1 Field of the Invention

The present invention relates to human telomerase, a ribonucleoprotein enzyme involved in human telomere DNA syntheses, and to compounds that inhibit telomerase activity. The invention provides methods and compositions relating to the fields of molecular biology, chemistry, pharmacology, oncology, and medicinal screening and diagnostic technology.

3.2 The Related Art

The search for novel and effective treatments for cancer has continued 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 relatively constant over the past forty years. If the current epidemiological trends continue, cancer will likely overtake cardiovascular disease as the leading cause of death in the United States.

Much progress has been made towards understanding cancer and the mechanisms underlying the onset, progression, and treatment. As a consequence, 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 among 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 surgical ablation, radiation, immunotherapy, and a variety of cytoreductive- and hormone-based drugs protocols that are used alone or in combination. Some oncolytic drugs are also available, but the efficacy of these drugs varies widely among cancer types. Most chemotherapeutic agents proposed for the treatment of cancer can only modify the course of the disease or alleviate some of the symptoms and generally exhibit significant dose-limiting toxicity. Commonly, the toxicity of the treatment 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. Chromosomes in normal mortal cells lose about 50 to 200 nucleotides of DNA per cell division at their end (telomeric) portions. The loss of these end-portion nucleotides, or "telomeres", appears to function as a mitotic clock whereby the number of cell divisions is recorded, and ultimately signals the onset of replicative senescence in normal cells. Therefore, the maintenance of telomeres appears to be necessary for cells to escape replicative senescence and proliferate indefinitely (Harley 1991). In most cancers, cell immortality results from the maintenance of short, but stable, telomeres through the action of the enzyme telomerase.

Telomerase is a novel molecular target consisting of 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 (Blackburn 1992). 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 (West, Shay et al. 1993; West, Shay et al. 1993; West, Shay et al. 1993; Kim, Piatyszek et al. 1994; West, Shay et al. 1994; West, Shay et al. 1994; Villeponteau, Feng et al. 1996; West, Shay et al. 1996).

Clearly, the inhibition of telomerase is an important and novel therapeutic modality for the treatment of human disease and malignancies. Reinitiating telomere shortening by telomerase inhibition is expected to result in telomeres rapidly reaching a critically short length leading to the end of the cells' proliferative lifespan and, ultimately, cell death. Thus, the identification of 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.

4 Summary of the Invention

The present invention provides methods and compositions that are highly unique, specific and effective for treating malignant conditions by targeting cells having telomerase activity. The methods 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 one aspect, the present invention includes methods and compositions for treating cancer that include telomerase-inhibiting compounds from among the generic class of compounds shown below

and the pharmaceutically acceptable salts of such compounds. Ri is selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, heterocycle, aralkyl, heteroaralkyl, arylcarbonylalkyl, alkylcarbonylalkyl, alkylcarbonyl, arylcarbonyl, arylamidoalkryl, alkylamidoalkyl, arylcarbonylaminoalkyl, and alkylcarbonylaminoaklkyl. Z_\ is selected from the group consisting of -C(R₂R₃)-, -C(=C(R₄R₅))-, and -C(=Xι)-, where Xi is selected from the group consisting of NR₆, O, and S, and where R₂-R₅ are selected independently from the group consisting of hydrogen, alkyl, aryl, aralkyl, alkoxyl, aryloxyl, heteroaryl, heteroaralkyl, alkylcarbonyl, arylcarbonyl, arylamino, alkylamino, dialkylamino, diarylamino, alkylarylamino, carboxyl, alkylcarbonyloxy, arylcarbonyloxy, alkylthio, arylthio, thio, alkylaminosulfonyl, and arylaminosulfonyl; R₆ is selected from the group consisting of hydrogen, hydroxyl, aryl, alkyl, aralkyl, heteroaryl, heteroaralkyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy, and heteroaralkylcarbonyloxy; and wherein Rt and R₅ can be points of attachment for the group -Z6-(Z₇)_n-Z₈- where n is an integer between 1 and 3 to form thereby a 4-, 5-, or 6-membered ring respectively. Finally, Z₂- Z₈ are selected independently from the group consisting of methine, quaternary carbon, and nitrogen.

In one embodiment, Z- is -C(O)- defining thereby compounds that are derivatives of isatin. In one embodiment, these isatin derivatives include compounds in which Ri is selected from the group consisting of hydrogen, aralkyl, and heteroaralkyl. Particularly useful isatin derivatives used in the present invention include those compounds having the general structure:

where R₇-Rn are selected independently from the group consisting of hydrogen, halogen, hydroxyl, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, nitro, cyano, alkoxyl, aryloxyl, heteroaryloxyl, alkylthio, arylthio, alkoxycarbonyl, aryloxycarbonyl, sulfonylamido, amidosulfonyl, aralkoxycarbonylamino, a ido, amino, alkylamino, arylamino, dialkylamino, diarylamino, arylalkylamino, heterocycleamino, heterocyclealkylamino, heterocycle, carboxyl. In addition, adjacent substituents together can form a 5- or 6-membered cyclic alkyl ether or carbocyclic fused ring system. Particular isatin derivatives having good anti-telomerase properties include the following compounds.

In another embodiment, the compounds used in the methods and compositions of the invention include those for which Z* of the generic structure above is -C=NR₆-, defining thereby isatin oxime derivatives. Embodiments of these oximes having good anti-telomerase properties include acyloxime derivatives, and more particularly, those acyloximes shown below.

Still other useful oxime derivatives include the following compounds:

These and other aspects and advantages will become apparent when the Description below is read in conjunction with the accompanying Examples.

5 Description of Some Embodiments of the Invention

5.1 Definitions

5.1.1 Alkyl

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, buten-1-yl, 2-cyclopenten-l-yl, 1,3-cyclohexadien-l-yl, 3-cyclohexen-l-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 (-CF₃)); nitro (- N0₂); 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); carboxaldehyde (-C(O)H); aryl- or alkylcarbonyl (RC(O)-); iminyl, or aryl- or alkyliminyl (-C(=NR)R'); sulfonate (-S0₂OR); alkyl- or arylsulfonyl (- S0₂R); hydroximinyl, or aryl- or alkoxyiminyl (-C(=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.

5.1.2 Methylene

The term "methylene" refers to the group -CH₂-.

5.1.3 Methine

The term "methine" refers to a methylene group for which one hydrogen atom has been replaced by a substituent such as described above in Section 5.1.1. The term "methine" can also refer to a methylene group for which one hydrogen atom is replaced by a bond to form an sp²- hybridized carbon center (i.e., -CH=). 5.1.4 Quaternary Carbon

The term "quaternary carbon" refers to a methylene group in which both hydrogen atoms are replaced by two independent substituents such as described above in Section 5.1.1. The term "quaternary carbon" can also refer to a methylene group for which one hydrogen atom is replaced by a bond to form an sp²- hybridized carbon center and the other hydrogen atom is replaced by a substituent as described above (i.e., -CR=), or a methylene group in which both hydrogen atoms are replaced by bonds to form an sp-hybridized carbon center (i.e., -C≡).

5.1.5 Halogen

The term "halogen" as used herein refers to the substituents fluoro, bromo, chloro, and iodo.

5.1.6 Carbonyl

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 (- S0₂-), phosphonyl (-P0₂-), and methylene (-C(CH₂)-). Other carbonyl equivalents will be familiar to those having skill in the medicinal and organic chemical arts.

5.1.7 Aryl

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, alkylamino, arylamino, dialkyl-, diaryl-, or arylalkylamino; aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl or arylalkyla inocarbonyl; carboxyl, or alkyl- or aryloxycarbonyl; carboxaldehyde, aryl- or alkylcarbonyl; iminyl, or aryl or akyliminyl; sulfo, alkyl- or arylsulfonyl; hydroximinyl, or aryl- or alkoxyiminyl. 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. 5.1.8 Aralkyl

The term "aralkyl" as used herein refers to an aryl group that is joined to a parent structure by alkyl group as described above, e.g., benzyl, α-methylbenzyl, phenethyl, and the like.

5.1.9 Heterocycle 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. Aromatic heterocycles will also be referred to herein as "heteroaryl". For example, heterocyclic 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, naphthyl, benzofuranyl, benzothienyl, indolyl, indolinyl, indolizinyl, indazolyl, quinolizinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, pteridinyl, quinuclidinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, purinyl, benzimidazolyl, benzoxazolyl, 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-, dialkyl-, diaryl-, or arylalkylamino; aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl or arylalkylaminocarbonyl; carboxyl, or alkyl- or aryloxycarbonyl; carboxaldehyde; 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 system. Substituents include heterocyclic groups (e.g., heterocycleoxy, heteroaryloxy, and heteroaralkylthio) are defined by analogy to the above-described terms.

5.1.10 Heterocyclealkyl

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. 5.2 Telomerase-lnhibiting Isatin Derivatives

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), retinal and blood tumor cell lines, to remain immortal has been demonstrated by analysis of telomerase activity (Kim, et al, cited above). This analysis, supplemented by data that indicates that the shortening of telomere length can provide the signal for replicative senescence in normal cells, 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. In addition, compositions capable of inhibiting telomerase activity in tumor cells offer benefits with respect to a wide variety of conditions other than cancer 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 (e.g., fungal infections). Telomerase inhibitors can also be used to inhibit telomerase activity in germ line cells, which may be useful for various non-disease conditions, e.g., for contraceptive purposes.

Thus, in one aspect, the present invention provides treatment methods and compositions that can serve as important weapons against many types of malignancies. In particular, the treatment methods and compositions 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 treatment methods and compositions of the present invention can be effective in discriminating 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. 5.2.1 Monoisatin Derivatives

The present invention includes telomerase-inhibiting compounds from among the isatin derivatives shown as Compound I,

Compound I

and their pharmaceutically acceptable salts, as well as compositions and methods for treating cancer that include compounds selected from those compounds. R- is selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, heterocycle, aralkyl, heteroaralkyl, arylcarbonylalkyl, alkylcarbonylalkyl, alkylcarbonyl, arylcarbonyl, arylamidoalkryl, alkylamidoalkyl, arylcarbonylaminoalkyl, and alkylcarbonylaminoaklkyl. Z* is selected from the group consisting of -C(R₂R₃)-, -C(=C(R R₅))-, and -C(=Xj)-, where X, is selected from the group consisting of NR₆, O, and S, and where R₂-R₅ are selected independently from the group consisting of hydrogen, alkyl, aryl, aralkyl, alkoxyl, aryloxyl, heteroaryl, heteroaralkyl, alkylcarbonyl, arylcarbonyl, arylamino, alkylamino, dialkylamino, diarylamino, alkylarylamino, carboxyl, alkylcarbonyloxy, arylcarbonyloxy, alkylthio, arylthio, thio, alkylaminosulfonyl, and arylaminosulfonyl; R₆ is selected from the group consisting of hydrogen, hydroxyl, aryl, alkyl, aralkyl, heteroaryl, heteroaralkyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy, and heteroaralkylcarbonyloxy; and wherein R₄ and R₅ can be points of attachment for the group -Z₆-(Z₇)_n-Z₈- where n is an integer between 1 and 3 to form thereby a 4-, 5-, or 6-membered ring respectively. Finally, Z₂- Z₈ are selected independently from the group consisting of methine, quaternary carbon, and nitrogen.

Particular embodiments of the present invention include compounds, compositions, and methods for which Z-. is

More particular embodiments of the present invention include derivative of Compound I for which Z_\ is -C(=X - and Xi is oxygen or sulfur, and, still more particularly, those derivative for which X- is oxygen. Still more particular embodiment are those for which Zi is -C(O)- and Ri is selected from the group consisting of hydrogen, aralkyl, and heteroaralkyl. In some embodiments, Zi is -C(O)- and Ri is aralkyl or heteroaralkyl. Of these embodiments, useful telomerase-inhibiting properties have been found among those compounds for which Ri is aralkyl, and, more particularly, among those compounds having the structure shown below (Compound II).

Compound II

R₇-R_n are selected independently from the group consisting of hydrogen, halogen, hydroxyl, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, nitro, cyano, alkoxyl, aryloxyl, heteroaryloxyl, alkylthio, arylthio, alkoxycarbonyl, aryloxycarbonyl, sulfonylamido, amidosulfonyl, aralkoxycarbonylamino, amido, amino, alkylamino, arylamino, dialkylamino, diarylamino, arylalkylamino, heterocycleamino, heterocyclealkyl amino, heterocycle, carboxyl. In addition, adjacent substituents together can form a 5- or 6-membered cyclic alkyl ether or carbocyclic fused ring system.

Compounds having the generic structure of Compound I above that have been found to embody anti-telomerase activity include those derivatives of Compound II for which each of Z₂-Z₅ independently is quaternary carbon. More particular derivatives of these embodiments of the compounds of the invention include those for which Z-*— Z₅ are selected independently from the group consisting of -CH- and -CRι₂-, where R₁₂ is alkyl or halogen. Still more particular embodiments are those derivatives of Compound II for which Z₂-Z₅ are selected independently from the group consisting of -CH- and -CR12-, R12 is alkyl or halogen, and R₇-Rπ are selected independently from the group consisting of hydrogen, halogen, and alkyl. Useful telomerase- inhibiting properties have been found for those derivatives of Compound H. in which at least one of Z₂-Z₅ is -CR12- and R_]2 is a moiety considered by those of skill in the art to be an electron withdrawing group. Examples of such groups include, but are not limited to, halogen atoms, nitro groups, carbonyl groups, carboxyl groups, sulfonyl groups, carbamido groups, sulfonamido groups, and cyano groups. Three particularly useful derivatives having the substituent pattern just described include Compound HI, Compound IV, and Compound V.

Compound V

In other embodiments, the present invention includes compounds having the general structural formula of Compound I above for which Z- is -C(=NR₆)-. In general, it will be appreciated that such embodiments can comprise mixtures of the Z- and E-isomeric oximes, or, following appropriate synthetic and/or purification protocols for the preparation of the pure structural isomers, substantially pure Z- or E-isomeric oximes. Therefore, the discussion herein of those embodiments of the present invention for which Zi is -C(=NR₆)- will be assumed to include both the case of mixtures of the Z- and E-isomeric oximes (and their derivatives) in addition to the case of pure Z- or pure E-isomers unless otherwise indicated.

Of these derivatives, good anti-telomerase properties have been found among those compounds for which R₆ is selected from the group consisting of hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy, and heteroaralkylcarbonyloxy, and, more particularly, among those derivatives for which R₆ is alkylcarbonyloxy, arylcarbonyloxy, or aralkylcarbonyloxy. Of the latter compounds, those for which Z₂-Z₅ are selected independently from the group consisting of -CH- and -CR]₃-, where R_!3 is alkyl, have good telomerase-inhibiting properties. More specific derivatives include those in which Z- is -C(=NR₆)-, where R₆ is alkylcarbonyloxy, arylcarbonyloxy, or aralkylcarbonyloxy, and, more particularly, where R₆ is alkylcarbonyloxy, arylcarbonyloxy, or aralkylcarbonyloxy. Particularly useful anti-telomerase properties are found among those compounds for which R*is 2,6-dichlorobenzyl (Compound VI).

Compound VI

In Compound VI Rι is hydrogen, alkyl or aryl. Still more specific oxime derivatives having anti-telomerase properties include those acyloximes as shown in Compound VI for which Rι₄ is selected from the group consisting of 4-chlorophenyl, 4-(trifluoromethyl)phenyl, 2-methylprop- 1-yl, 1-butyl, and methyl. Of these compounds, those for which Rι is 1-butyl (Compound VII), 2-methylprop-l-yl (Compound VHT), and 4-chlorophenyl (Compound LX) have especially good anti-telomerase properties.

Compound VIII

Compound IX

Embodiments of the compound having the structure shown as Compound I above for which

Zi is oxime (i.e., -C(=NOH)-) have also been found to have useful telomerase-inhibiting properties. More specifically, useful oxime derivatives are those derivatives for which Z₂-Z₅ are selected independently from the group consisting of -CH- and -CR₁₅-, where R₁₅ is heterocycle or alkylthio. Still more specific useful oxime derivatives are those for which Z₄ is -CR15-, where R_J5 is piperazin-1-yl or alkylpiperazin-1-yl. Particularly useful isatin oximes include those for which Z₂, Z₃, and Z₅ are -CH- and 7* is 4-methylpiperazin-l-yl (Compound X), and those for which Z₂, Z₃, and Z₅ are -CH- and Z₄ is piperazinyl (Compound XI)

Compound X

Compound XI

Still more useful oxime derivatives (i.e., those derivatives of Compound I above for which Zi is -C(=NOH)-) include those for which Z₄ is -CR₁₅-, where R_]5 is alkylthio. More specific useful alkylthioisatin oxime derivatives are those derivatives of Compound I above for which Zi is (-C(=NOH)-), Z₂, Z₃, and Z₅ are -CH-, and Z4 is methylthio (CH₃S-). Especially useful are those alkylthioisatin oximes just described for which Ri is 2,6-dichlorobenyl (Compound XII):

Compound XII

Those having skill in the medicinal chemical and pharmaceutical arts will appreciate that the observation that both the acyloxime and unsubstituted oxime derivatives of Compound I shown above demonstrate good anti-telomerase properties is consistent with a model of activity in which the unsubstituted oxime is (i.e.,

is -C(=NOH)-) the active species. Although not wishing to be bound by any particular theory of pharmaceutical action, the present invention further contemplates the use of groups having lability to hydrolysis similar to acyl moieties (e.g., phosphonates) for R₆.

In another aspect, the present invention includes novel isatin derivatives of Compound I having the structure shown generically as Compound XIII

Compound XIII

and their pharmaceutically acceptable salts. X₂ is selected from the group consisting of oxygen, sulfur and NR₂₅, where R₂₅ is hydrogen, hydroxyl, aryl, alkyl, aralkyl, heteroaryl, heteroaralkyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy, or heteroaralkylcarbonyloxy. R₂₀ is hydrogen or aralkyl. R21 and R₂2 are selected independently from the group consisting of hydrogen, alkyl, aryl, aralkyl, alkoxyl, aryloxyl, heteroaryl, heteroaralkyl, alkylcarbonyl, arylcarbonyl, arylamino, alkylamino, dialkylamino, diarylamino, alkylarylamino, carboxyl, alkylcarbonyloxy, arylcarbonyloxy, alkylaminosulfonyl, and arylaminosulfonyl.

In one embodiment, X₂ is oxygen. In another embodiment, X₂ is oxygen and R₂₀ is benzyl or benzyl substituted optionally with at least one substituent selected from the group consisting of halogen or trifluoromethyl. In more particular embodiments, X₂ is oxygen and R₂₀ is 3, 4- dichlorobenzyl, 2, 6-dicholorobenzyl, or 3-(trifluoromethyl)phenyl. Still more particular embodiments are those in which X₂ is oxygen, R₂₀ is 3, 4-dichlorobenzyl, 2, 6-dicholorobenzyl, or 3-(trifluoromethyl)phenyl, and R_2] is hydrogen or halogen. Of the latter embodiments, those for which R21 is hydrogen or chloro have particularly useful telomerase-inhibiting properties and more particularly those for which X₂ is oxygen, R2₀ is 3, 4-dichlorobenzyl, 2, 6- dicholorobenzyl, or 3-(trifluoromethyl)phenyl, R₂ι is hydrogen or chloro, and R₂₂ is thio or alkylthio. Specific embodiments of the telomerase-inhibiting compounds having the generic structural formula of Compound XIII above include those for which X₂ is oxygen, R₂₀ is 2, 6- dichlorobenzyl, R₂ι is hydrogen and R₂₂ is tert-butylthio, or rc-butylthio (Compounds XIV, and XV respectively); and those for which X₂ is oxygen, R20 is 3, 4-dichlorobenzyl, R₂₎ is chloro, and R₂₂ is thio or methylthio (Compounds XVI, and XVII respectively).

Other telomerase-inhibiting isatin derivative provided by the present invention include those derivatives of Compound XEI for which X2 is oxygen, R₂₀ is 3, 4-dichlorobenzyl, 2, 6- dicholorobenzyl, or 3-(trifluoromethyl)phenyl, R₂ι is hydrogen or chloro, and R₂₂ is heterocycle. More particular telomerase-inhibiting isatin derivatives of the invention include compounds having the substituent pattern just described for which R₂₂ is selected from the group consisting of piperazinyl, alkylpiperazinyl, and arylpiperazinyl, and more particularly, those for which R₂₀ is 2, 6-dichlorobenzyl and R22 is piperazin-1-yl or 4-phenylpiperazin-l-yl. One particular derivative is that for which X2 is oxygen, R20 is 2, 6-dichlorobenzyl, R21 is chloro, and R₂₂ is 4-phenylpiperazin-l-yl (Compound XVHT).

Still other telomerase-inhibiting isatin derivative provided by the present invention include those derivatives of Compound XIII for which X₂ is oxygen, R₂₀ is 3, 4-dichlorobenzyl, 2, 6- dicholorobenzyl, or 3-(trifluoromethyl)phenyl, R₂ι is hydrogen or chloro, and R₂₂ is alkylsulfonyl or arylsulfonyl, and, more particularly, those for which R₂₂ is methylsulfonyl, tert-butylsulfonyl, or phenylsulfonyl. Yet other telomerase-inhibiting isatin derivatives of the invention includes those for which X2 is oxygen, R20 is 3, 4-dichlorobenzyl, 2, 6- dicholorobenzyl, or 3-(trifluoromethyl)phenyl, R₂ι is hydrogen and R₂₂ is arylcarbonyl, and more^' particularly, R₂₂ is phenylcarbonyl or 4-chlorophenylcarbonyl. Still other telomerase- inhibitors of the present invention are those for which X₂ is oxygen, R₂₀ is 3, 4-dichlorobenzyl, 2, 6-dicholorobenzyl, or 3-(trifluoromethyl)phenyl, R₂₁ is arylaminosulfonyl and R₂₂ is hydrogen, and more particularly, those having this substituent pattern for which R₂ι is 3, 4- dichlorophenylaminosulfonyl (Compounds XLX, XX, and XXI respectively).

5.2.2 Diisatin Derivatives

Another embodiment of the present invention includes compounds having the structural formula shown below (Compound XXII).

Compound XXII

including their pharmaceutically acceptable salts. X₃ is selected from the group consisting of oxygen, sulfur and NR₃₆, where R₃₆ is hydrogen, hydroxyl, aryl, alkyl, aralkyl, heteroaryl, heteroaralkyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy, or heteroaralkylcarbonyloxy. R₃₀ and R₃₃ are is selected independently from the group consisting of hydrogen, alkyl, aryl, alkylcarbonyl, aralkylcarbonyl, arylcarbonyl, and aralkyl. R₃1, R₃2_> and R₃₄-R₃₅ are selected independently from the group consisting of hydrogen, hydroxyl, nitro, cyano, thio, alkylthio, arylthio, alkyl, aryl, aralkyl, alkoxyl, aryloxyl, heteroaryl, heteroaralkyl, alkylcarbonyl, arylcarbonyl, arylamino, alkylamino, carboxyl, carbonyl, dialkylamino, diarylamino, alkylarylamino, carboxyl, alkylcarbonyloxy, arylcarbonyloxy, and arylaminosulfonyl; and further wherein R₃₅ and R₃₆ together are selected from the group consisting of a bond, oxygen, sulfur, -NR₃₈-, and -(Ze),,-, where R₃₈ is selected from the group consisting of hydrogen, hydroxyl, aryl, alkyl, aralkyl, heteroaryl, heteroaralkyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy, or heteroaralkylcarbonyloxy, Z_$ is methylene, methine, or quaternary carbon, and n is an integer between 1 and 3. In one embodiment, X₃ is oxygen. In another embodiment, X₃ is oxygen and each of R₃*, R₃₂,^'and R₃ -R₃₇ is hydrogen. Still more embodiments include those for which X₃ is oxygen, each of R₃ι, R₃₂, and R₃ -R₃₇ is hydrogen, and R₃₀ and R₃₃ independently are benzyl or benzyl substituted optionally with at least one substituent selected from the group consisting of halogen or trifluoromethyl. Specific exemplary embodiments having strong telomerase- inhibiting character include those derivatives of Compound XXII for which X₃ is oxygen, each of R₃ι, R₃2, and R₃ -R₃₇ is hydrogen, and each of R₃₀ and R₃₃ is 3, 4-dichlorobenzyl (Compound XXIH), each of R₃₀ and R₃₃ is 2, 6-dichlorobenzyl (Compound XX1N), and each of R₃o and R₃₃ is 3-trifluoromethylbenzyl (Compound XXV). Another specific embodiment of Compound XXII is that for which X₃ is oxygen and each of R₃o-R₃₇ is hydrogen (Compound XXVI).

5.3 Synthesis of Isatin and Diisatin Derivatives

The compounds of the present invention can be synthesized using techniques and material known to those of skill in the art, such as described, for example, in March, ADVANCED ORGANIC CHEMISTRY 4^th Ed., (Wiley 1992); Carey and Sundberg, ADVANCED ORGANIC

CHEMISTRY 3^rd Ed., Vols. A and B (Plenum 1992), and Green and Wuts, PROTECTIVE GROUPS LN ORGANIC SYNTHESIS 2^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, WI), Sigma Chemical Co. (St. Louis, MO), Lancaster Synthesis (Windham, NH), Apin Chemicals Ltd. (New Brunswick, NJ), Ryan Scientific (Columbia, SC), Maybridge (Cornwall, England), Arcos (Pittsburgh, PA) 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), the resolution of enantiomers (e.g., using diastereomeric salt formation, "chiral HPLC", gas chromatography (GC), enzymatic resolution, and enantioselective synthetic procedures), 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 (^!H and ¹³C NMR), optionally with the use of chiral shift reagents, infrared and ultraviolet spectroscopy (IR and UV, X-ray crystallography, elemental analysis (EA), GC, HPLC, optical rotation 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. Furthermore, it will be familiar to those having skill in the medicinal chemical arts that the synthetic procedures described herein can include the formation of various derivatives to control the solubility of the subject compounds. Techniques for controlling solubility include, but are not limited to, the formation of acid/base addition salts, quaternary ammonium salts, and inclusion complexes such as by incorporation of the subject compound within a cyclodextrin or other carrier molecule or complex.

Compounds of the class represent by Compound I can be synthesized using the general procedures described in Section 6.1 hereinbelow. An example of the general procedures for the synthesis of both isatin derivatives and N-benzyl isatin acyloximes is provided in Scheme 1 below. However, it will be appreciated that the general procedures discussed below can be extended to make the other compounds described herein. The use of the substituent identifiers R, R', and R" is merely to indicate the presence of one or more substituents at the position or moiety indicated and can be determined by reference to the specific moieties described above in connection with the structure of Compound I.

Starting from the appropriately substituted isatin 1, prepared from standard methods and procedures or purchased commercially (e.g., Maybridge), reaction with an alkylating agent, e.g., a benzyl halide, in the presence of a base such as sodium hydride, provides the corresponding N-alkylated isatin 2 in high yield. See, Webber et al, 1996, J. Med. Chem. 39:5072-5082, incorporated herein by reference. Preparation of isatin oxime derivatives 3 can be performed by reaction of the appropriately N-alkylated isatin 2 with hydroxylamine-HCl while heating. Acylation of the oxime to provide compound 4 can be performed by reaction of oxime 3 with the appropriate acid chloride in the presence of base.

Diisatin compounds can be made using the general methodology illustrated below in Scheme

2.

Scheme 2

According to one embodiment of the method of the invention, reaction of an appropriately substituted ort io-halonitrobenzene 5 (X = Cl, F) with diethylmalonate (e.g., combination of 5 with CH₂(C02Et)2 and NaH) provides 2-nitrophenylmalonate diester 6. Closure of the ring to provide indolinone 7 can be performed by treatment of 6 with Sn/HCl. Reaction of 7 with pyridinium bromide perbromide or similar perbromide reagent provides geminal dibromide 8 which is transformed to the desired isatin 9 by hydrolysis (e.g., by reaction with H₂0/MeOH). Other methods of performing the above-described transformations will be familiar to those of skill in the art of organic and medicinal chemistry.

5.4 Anti-Tumor Activity of the Telomerase Inhibitors of the Invention

The compounds of the present invention demonstrate inhibitory activity against telomerase in vivo, as has been and can be demonstrated as described below. The in vitro activities of the compounds of the invention can also be demonstrated using the methods described herein. 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 IC₅₀ (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, IC₅₀ values for several of the compounds of the present invention were determined. The values reported in Table 1 below are only approximate values; more exact IC₅0 value can be obtained by repetitive testing.

Table 1

Compound IC_M (μM)

m 51 rv 24 v 18

vπ 14

vm 11

IX 19

x 26

XI 16 xπ 32

X1N 29 xv 42

XVI 21

xvm 50 Compound I , (μM)

XLX 5

XX 5

XXI 7

xxm 9

XXIV 9

XXV 7

XXVI 47

All of the compounds of Table lhave anti-telomerase activity (i.e., an IC₅₀< 100 μM). Two of the compounds of the invention (Compound VII and Compound VUI) are good telomerase inhibitors, each having an approximate IC₅0 value of less than about 15 μM.

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 B J cells of fibroblast origin, the observed reduction in telomere length is expected to be substantially no different from cells treated with a control substance, e.g., dimethylsulfoxide (DMSO). The compounds of the invention also are expected to demonstrate no significant cytotoxic effects at concentrations below about 5 μM in the tumor cells. In addition, the specificity of the compounds of the present invention for telomerase can be determined by comparing their activity (IC₅₀) 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 π, T3 RNA Polymerase, MMLV Reverse Transcriptase, Topoisomerase I, Topoisomerase π, Terminal Transferase and Single-Stranded DNA Binding Protein (SSB). Compounds having lower IC₅₀ values for telomerase as compared the IC₅₀ values 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 Section 6.2.2 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 regimes involving administration of a compound of the invention. For such purposes, it may be helpful to perform a terminal repeat fragment (TRF) analysis in which DNA from tumor cells is analyzed by digestion with restriction enzymes specific for sequences other than the telomeric (T₂AG₃)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.

5.5 Telomerase-inhibiting Compositions and Methods for Treating Diseases with the Same

The pharmaceutical compositions for treating cancer and other conditions in which inhibition of telomerase is an effective therapy include a therapeutically effective amount of a telomerase inhibiting compound having the structure of Compound I above in a pharmaceutically acceptable carrier or salt.

In addition, it will be appreciated that therapeutic benefits from 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. Patent Application Serial Nos. 08/549,597, filed October 27, 1995; 08/548,005, filed January 11, 1996; 08/539,93 filed October 6, 1995; 08/425,043, filed April 18, 1995; and U.S. Patent Applications Serial Nos. 08/554,788, filed November 7, 1995; 08/535,988, filed September 29, 1995; and 08/424,813, filed April 18, 1995, all of which are 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 treatment). One regimen for reducing tumor size includes administration of a topoisomerase-II inhibitor, including those topoisomerase-IJ inhibitors described in the above-cited co-pending U.S. Patent Applications. In addition, in some cases it may be advisable to combine telomerase inhibiting agent of the invention with one or more agents that treat the side effects of disease, e.g., an analgesic, or agents effective to stimulate the patient's own immune response (colony stimulating factor).

In another such method, a pharmaceutical formulation comprises a telomerase inhibitor of the invention with an anti-angiogenesis agent, such as fumagillin, fumagillin derivatives, or AGM1470. 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, mechlorethamine 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, erythromycin, 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, α-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 mustard, MNNG and NMS; and intercalating agents such as acridine dyes, actinomycines, 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 β-1-D-arabinoside, amethopterin, aminopterin, 2-aminopurine, aphidicolin, 8- azaguanine, azaserine, 6-azauracil, 2'-azido-2'-deoxynucleosides, 5-bromodeoxycytidine, cytosine β-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, α-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. Still more additional agents include tubulin inhibitors such as taxol and epothilone and their derivatives. 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 addition to the application of the telomerase inhibitors of the present invention to the treatment of mammalian diseases characterized by telomerase activity, telomerase inhibitors such as those disclosed herein, or in the above-cited co-pending U.S. Patent Applications can be applied to agricultural phytopathogenic organisms that are characterized by telomerase activity. These organisms include nematodes such as Ceanorhabditis elegans, in which telomerase activity has been found, and in fungi which are expected to have telomerase activity based on the determination that the DNA of the fungus Ustilago maydis exhibits telomeres having the tandem TTAGGG repeats that are maintained by telomerase. The telomerase-inhibiting compounds of the invention can be administered to plants and soil infected with phytopathogenic organisms having telomerase activity alone, or in combination with other telomerase-inhibiting agents (such as those in the above-cited U.S. Patent Applications or other telomerase inhibiting agents) and/or other agents used to control plant diseases. The determination of the compositions used to control such phytopathogenic organisms and the appropriate modes of delivering such compositions will be known to those having skill in the agricultural arts.

The determination that nematodes and possibly fungi have telomerase activity also indicates that the telomerase inhibitors provided by the present invention and the above-cited co-pending U.S. Patent Applications can be used to treat nematode infections in humans animals of veterinary interest such as dogs and cats. Nematode infection in humans and animals often in the form of hookworm or roundworm infection and leads to a host of deadly secondary illnesses such as meningitis, myocarditis, and various neurological diseases. Thus, it will be appreciated that administration of the telomerase-inhibiting compounds such as those of the invention or those described in the above-cited U.S. Patent Applications alone, or in combination with other telomerase-inhibiting agents and/or other therapeutic agents, can be used to control nematode and fungal infections in humans and animals.

In general, a suitable effective does 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/kg of body weight per day, more preferably between about 0.1 and 100 mg/kg of body weight per day and still more preferably in the range of between 0.1 to 10 mg/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 present once per day at a dosing at least equal to TID.

The compositions 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 solution or suspensions, liposomes, and injectable and infusible solutions. The preferred form depends on the intended mode of administration and therapeutic application. The compositions also prefer; include conventional pharmaceutically acceptable carriers and adjuvants, as is well known to those of skill in the art. See, e.g., REMINGTON'S PHARMACEUTICAL SCIENCE, Mack Publishing Co.; Easton, PA, 17^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 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, 8^th 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. Oral administration is a preferred mode of delivery. The pharmaceutical compositions of the invention 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 case directly onto the backing material or can be applied to the skin to form an adherent coating. See, e.g., U.S. Patent Nos. 4,310,509; 4,560,555; and 4,542,012.

In other embodiments, the compounds 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. Patent Nos. 4,597,961; 4,485,097; 4,608,249; 4,5005,891; 3,843,480; 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, and enhancers, vehicles, stabilizers, and the like are combined with the foam precursors. The foam is allowed to cure to produce a tack, elastomeric matrix which can be directly affixed to the backing material. See, e.g., U.S. Patent Nos. 4,542,013; 4,460,562; 4,466,953; 4,482,543; 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 or cream formulations. Ointment and creams may, for example, be formulated with an aqueous oil 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 more of the following: stabilizing agents, emulsifying agents, dispersing agents, suspending or 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 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 also be preferred for treating diseases such as skin cancer and fungal infections of the skin.

The topical pharmaceutical compositions according to this invention may also include one or more preservatives or bacteriostatic agents, e.g., methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocreosol, 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. Patent 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. Patent 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 solution or suspensions, solid forms suitable for solutions 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. Patent 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 dosages 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 pharmaceutically acceptable nontoxic composition is formed by incorporating any of the often employed excipients, such as those carriers previously listed, and generally 0.1-95% of the acting ingredient, e.g., 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 agents for longer periods or at higher concentration 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 compound 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.

6 Examples

The following Examples are provided to illustrate certain aspects of the present invention and to aid those of skill in the art in the art in practicing the invention. These Examples are in no way to be considered to limit the scope of the invention in any manner.

6.1 Chemical Synthesis

6.1.1 General Procedure A: N-Alkylation of Isatins

Into a 100 mL oven dried round-bottom flask equipped with a magnetic stir bar and under a positive pressure of nitrogen were placed the desired isatin (5.56 mmol) and 15 mL of anhydrous dimethylformamide (DMF). The mixture was stirred under nitrogen until the isatin completely dissolved. Into another oven dried round-bottom flask equipped with a magnetic stir bar and under a positive pressure of nitrogen was placed sodium hydride (6.67 mmol). The sodium hydride was washed with anhydrous pentane (3 x 5 mL). The dissolved isatin was added slowly with cooling (ice bath) to the sodium hydride. After one hour of stirring, the desired substituted benzylchloride (7.23 mmol) was added to the reaction mixture. The mixture was then stirred at room temperature overnight. The reaction was quenched with the addition of 20 mL of ice cold water (5 °C). A precipitate formed and was isolated by suction filtration. The solid was washed with water and then with hexane and air-dried. The desired compound was isolated and analyzed by 1H NMR (DMSO-d₆) and TLC (50:50 ethylacetate:hexane on SiO_∑/silica). 6.1.2 General Procedure B: Preparation of Oximes from Isatins

Into a 100 mL oven dried round-bottom flask equipped with a magnetic stir bar and under a positive pressure of nitrogen were placed the N-alkylated isatin (6.54 mmol) and two equivalents of hydroxylamine-HCl (13.09 mmole). To the mixture was added 15 mL of anhydrous pyridine. The reaction mixture was stirred overnight at 60 °C. To the reaction was added another equivalent of hydroxylamine-HCl (6.54 mmol). The reaction was stirred with heating at 60 °C overnight. Again, another equivalent of hydroxylamine-HCl (6.54 mmol) was added. Alternatively, the hydroxylamine-HCl portions can be added simultaneously. The reaction was monitored by TLC (50:50 ethyl acetate:hexane on Si0₂/silica) until complete. Upon completion the reaction was cooled to room temperature and quenched with the addition of 15 mL of ice cold water. A precipitate formed and was isolated by suction filtration. The isolated solid was washed sequentially with water, ether, and sodium bisulfate and then again with water and ether. The product was air-dried. The desired oxime was isolated and analyzed by Η NMR (DMSO-de) and TLC (50:50 ethyl acetate:hexane on Si0₂/silica).

6.1.3 General Procedure C: Acylation of Isatin Oximes

Into an oven-dried round-bottom flask, equipped with a magnetic stir bar and under a positive pressure of nitrogen, were placed the isatin oxime (341 μmol) and 1.7 ml of anhydrous pyridine. The appropriate acyl chloride (409 μmol) was added and the mixture was heated to 60 °C for 12-14 hours. One additional equivalent (341 μmol) of acyl chloride was necessary for some compounds and was added after this time. In these cases, the reaction was continued for another 24 hours. The reaction mixture was diluted with 2 mL ice-cold water and the precipitated product was collected by suction filtration washed with sodium bisulfate solution, water and hexane. The product was air-dried.

6.1.4 Specific Synthesis Examples 6.1.4.1 2-(4-Benzoyl-2-nitrophenyl)malonic acid diethyl ester.

Sodium hydride, 60% dispersion in mineral oil (1.10 g, 27.4 mmol) was placed in a 100 mL three-necked flask, the flask was flushed with nitrogen and the material was washed three times with pentane. Anhydrous DMSO (25 mL) was added, followed by 4.2 mL of diethyl malonate in 6 mL of dry DMSO. The mixture was heated to 100° C, then 4-chloro-3-nitrobenzophenone (3.26 g, 12.5 mmol) in 12 mL of dry DMSO was added and the dark red mixture was stirred for 14 h. The mixture was then poured into 320 mL of H₂0 and extracted with Et₂0 (3 x 122 mL). The extracts were washed with H₂O (4 x 122 mL), dried over Na2Sθ₄, filtered and concentrated to afford 3.52 g of an orange oil, contaminated with diethyl malonate (60%): ^]H NMR (400 MHz, OMSO-d₆) δ 1.18 (td, 6H, 7 =6.8, 1.9 Hz), 4.18 (qd, 4H, 7=7.2, 5.2 Hz), 5.57 (s, 1H), 7.60 (uneven t, 2H), 7.73 (m, 2H), 7.81 (d, 2H, 7 =8.0 Hz), 8.11 (dt, 1H, 7=8.0, 1.9 Hz), 8.36 (s, 1H).

6.1.4.2 6-Benzoyl-1 ,3-dihydroindol-2-one (6-Benzoyl-2-oxindole).

Benzophenone malonate adduct (2.88 g, 7.48 mmol)) was dissolved in 19 mL of EtOH and 8.1 mL of 12 M HC1 (97.2 mmol) in a 100 mL one-necked flask. Tin powder (2.84 g, 23.9 mmol) was added and the mixture was stirred at reflux for 3 h. The hot reaction mixture was then decanted into an Erlenmeyer flask and crystals formed upon cooling. More yellow crystals formed upon cooling the mixture in an ice bath. The product was collected by filtration and washed with cold EtOH, then with H₂O until the washings were neutral, then with hexane to afford 1.70 g of yellow needles (96%): -H NMR (400 MHz, DMSO-J₆) δ 3.60 (s, 2H), 7.13 (s, 1H), 7.31 (d, 1H7 =7.6 Hz), 7.37 (d, 1H, 7 =7.6 Hz), 7.55 (uneven t, 2H), 7.68 (m, 3H), 10.54 (br s, 1H); C{^lH} NMR (100 MHz, DMSO-^) δ 35.9, 109.4, 123.6, 124.3, 128.5, 129.4,

131.3, 132.4, 136.3, 137.3, 144.1, 176.1, 195.5; EI-MS m/z 237 (M+, 100), 160 (36), 105 (38), 77 (32); Anal. Calcd. for Ci5H_nN0₂: C, 75.93; H, 4.68; N, 5.90. Found: C, 75.93; H, 4.71; N, 5.71; TLC EtOAc-hexane (50:50 v/v) fl/0.21.

6.1.4.3 6-Benzoyl-3,3-dibromo-1 ,3-dihydroindol-2-one (6-Benzoyl-3,3-dibromo-2-oxindole)

Benzoyl oxindole (1.00 g, 4.21 mmol) was suspended in 42 mL of tert-BuOH in a 100 mL one-necked flask. H2O (180 μL, 10.1 mmol) and pyridinium bromide perbromide (5.39 g, 16.9 mmol) were added and the reaction mixture was stirred for 14 h. The reaction was quenched with 42 mL of H2O and the resulting solid was collected by filtration, washed with water until the washings were neutral, then with hexane to afford 1.67 g of a yellow powder (100%): ^JH NMR (400 MHz, DMSO-Λ₆) δ 7.18 (d, 1H, 7 =1.2 Hz), 7.47 (dt, 1H, 7 =8.0, 1.6 Hz), 7.57 (m, 2H), 7.69 (m, 1H), 7.76 (m, 3H), 11.43 (br s, 1H); TLC EtOAc-hexane (50:50 v/v) R 0.53. 6.1.4.4 6-Benzoyl-1 H-indol-2,3-dione (6-Benzoylisatin)

Benzoyl dibromooxindole (1.67 g, 4.21 mmol) was dissolved in 12 mL of 4:1 MeOH/H₂θ in a 50 mL one-necked flask and stirred at reflux for 2 days. The mixture was cooled to ambient temperature, then in an ice bath to afford a dark orange precipitate. The product was collected by filtration, washed with H₂O until the washings were neutral, then with hexane to give 798 mg of an orange solid, (75%): ^!H NMR (400 MHz, OMSO-d₆) δ 7.11 (t, 1H, 7 =0.8 Hz), 7.32 (dd, 1H, 7=7.6, 1.2 Hz), 7.59 (t, 2H, 7 =8.0 Hz), 7.65 (d, lH, 7 =7.6 Hz), 7.74 (m, 3H), 11.17 (br s, 1H); ¹3C{ -*H} NMR (100 MHz, DMSO- ) δ 112.2, 120.3, 123.9, 124.6, 128.8, 129.7, 133.4, 136.1, 144.9, 150.3, 159.1, 184.1, 194.9; EI-MS m/z 251 (M+, 96), 223 (100), 105 (43), 77 (30); Anal. Calcd. for C-_.5H9NO₃: C, 71.70; H, 3.62; N, 5.58. Found: C, 71.59; H, 3.51; N, 5.35; TLC EtOAc-hexane (50:50 v/v) fyθ.35.

6.1.4.5 Benzophenone dimalonate adduct.

4,4'-Dichloro-3,3'-dinitrobenzophenone (5.00 g, 14.7 mmol) was treated in a manner analogous to 4-chloro-3-nitrobenzophenone above, except 4.4 eq each of diethyl malonate and sodium hydride were employed. The product was isolated as 5.76 g of an off-white powder (67%).

6.1.4.6 Oxo-linked dioxindole.

The benzophenone dimalonate adduct (5.76 g, 9.80 mmol) was treated in a manner analogous to the benzoyl malonate adduct, except 6.4 eq of tin and 26 eq of HC1 were employed. The product was isolated as 1.78 g of a white solid (62%).

6.1.4.7 Oxo-linked tetrabromodioxindole

The dioxindole (1.77 g, 6.07 mmol) was treated in a manner analogous to the benzoyl oxindole, except 8.0 eq of pyridinium bromide perbromide were utilized. No precipitate formed upon quenching and the mixture was extracted with EtOAc (3 x 27 mL). The organic extracts were washed with H₂O (3 x 100 mL) and saturated NaCl solution (1 x 100 mL) and dried over Na2S0₄, filtered and concentrated to afford 3.67 g of a yellow solid (99%). 6.1.4.8 Oxo-linked diisatin

The tetrabromodioxindole (3.66 g, 6.00 mmol) was subjected to hydrolysis condition analogous to those of the benzoyl dibromooxindole. The crude product was recrystallized from acetone to afford 1.56 g of an orange solid (81%).

6.1.4.9 N-(2',6'-dichlorobenzyl)-6-morpholinoisatin.

N-(2',6'-Dichlorobenzyl)-6-chloroisatin (201 mg, 590 μmol), K₂C0₃ (122 mg, 885 μmol) and morpholine (51 μL, 590 μmol) were placed in a one-necked round-bottom flask, the flask was capped and flushed with nitrogen and 3 mL of dry DMF were added. The contents were heated at 105° C for 15 h, after which time the reaction was judged to be complete as evidenced by TLC. The reaction mixture was cooled to ambient temperature and quenched with 3 mL of ice-cold H₂O. The mixture was then extracted with CHCI₃ (3 x 3 mL) and the extracts were washed with H₂O (3 x 3 mL), dried over Na₂S0₄, filtered and concentrated to afford 154 mg of an orange solid (67%).

6.1.4.10 N-(2',6'-dichlorobenzyl)-6-(methylsulfido)isatin.

N-(2',6'-Dichlorobenzyl)-6-chloroisatin (400 mg, 1.17 mmol) and sodium methanethiolate

(82 mg, 1.17 mmol) were placed in a one-necked round-bottom flask, the flask was capped and flushed with nitrogen and 6 mL of dry DMF were added. The contents were stirred for 2 days and heated at 105° C. The reaction mixture was cooled to ambient temperature and quenched with 6 mL of ice-cold H2O. The green precipitate was collected by filtration and purified by silica gel chromatography (EtOAc/hexane 20:80 v/v to 40:60 v/v). The product was obtained as 116 mg of an orange solid (28%).

6.2 Anti-tumor Activity

6.2.1 Ex Vivo Studies

6.2.1.1 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 10⁶ 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 telomerase-inhibiting compound at a predetermined concentration (e.g., between about 5 μM and about 20 μM) 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 periodically (dependent on the compounds half-life) 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 T₂AG₃ 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 telomerase- inhibiting compounds described above have no effect 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 telomerase-inhibiting compounds. Tumor cells exposed to the control 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 μM and about 20 μM using the protocol just described. Cells are expected to enter crisis (i.e., 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.

6.2.1.2 Specificity

Telomerase-inhibiting compounds are screened for activity (ICjo) against telomerase and several enzymes have nucleic acid binding or modifying activities related to telomerase using standard techniques. The enzymes being screened include Telomerase, DNA Polymerase I, HeLa RNA Polymerase π, T3 RNA Polymerase, MMLV Reverse Transcriptase, Topoisomerase I, Topoisomerase II, Terminal Transferase, and Single-Stranded DNA Binding Protein (SSB). The specificity of a compound for telomerase is determined by comparing the IC₅₀ of the compound with respect to telomerase with the IC₅₀s of the compound for each of the enzymes being screened. The compound is determined to have high specificity for telomerase if the IC50 of the compound for telomerase is lower than the IC₅₀ value for each of the enzymes screened.

6.2.1.3 Cytotoxicity The XTT 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 μM to 1,000 μM. During this period, the optical density (OD) of the samples is determined for light at 540 nanometer (nm). No significant cytotoxic effects are expected to be observed at concentrations less than about 5 μM. In addition, other known cytotoxicity assays, such as the MTT assay, can be used to determine the cytotoxicity of the compounds of the invention.

Some compounds may induce G2 arrest at concentrations above about 5 μM (i.e., at 10 μM- 20 μM 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. 6.2.2 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 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.

7 Bibliography

The following references are incorporated herein by reference in their entirety and for all purposes.

Blackburn, E. 1992. "Telomerases." Annu. Rev. Biochem. 61: 113-129.

Harley, C. 1991. "Telomere Loss: Mitotic Clock or Genetic Time Bomb?" Mutation

Research 256: 271-282.

Kim, N., Piatyszek, M., et al 1994. "Specific Association of Human Telomerase With Immortal Cells and Cancer." Science 266: 2011-2014.

Villeponteau, B., Feng, J., et al. December 10, 1996. U.S. Patent No. 5,583,016

West, M., Shay, J., et al. . U.S. Patent Application Serial No. 08/255,774 Filed June 7, 1994.

West, M., Shay, J., et al. . U.S. Patent Application Serial No. 08/315,216 Filed September 28, 1994.

West, M., Shay, J., et al. February 6, 1996. U.S. Patent No. 5,489,508

West, M., Shay, J, et al November 25, 1993. PCT Application Patent No. WO 93/23572

West, M., Shay, J., et al. U.S. Patent Application Serial No. 08/151,477 Filed November 12,

1993.

West, M., Shay, J., et al. U.S. Patent Application Serial No. 08/060,952 Filed May 13, 1993.

Claims

WHAT IS CLAIMED:

1. A method for treating cancer in a mammal, comprising the step of administering to such mammal a therapeutically effective amount of a compound having the structure shown below, or a pharmaceutically acceptable salt thereof, to inhibit telomerase activity in cancer cells such that telomeres is said cancer cells are reduced over successive cell divisions:

wherein:

Ri is selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, heterocycle, aralkyl, heteroaralkyl, arylcarbonylalkyl, alkylcarbonylalkyl, alkylcarbonyl, arylcarbonyl, arylamidoalkryl, alkylamidoalkyl, arylcarbonylaminoalkyl, and alkylcarbonylaminoaklkyl;

Zi is selected from the group consisting of -C(R₂R )-, -C(=C(R_,R₅))-, and -C(=Xj)-, where Xj is selected from the group consisting of NR₆, O, and S, and where R₂-R₅ are selected independently from the group consisting of hydrogen, alkyl, aryl, aralkyl, alkoxyl, aryloxyl, heteroaryl, heteroaralkyl, alkylcarbonyl, arylcarbonyl, arylamino, alkylamino, dialkylamino, diarylamino, alkylarylamino, carboxyl, alkylcarbonyloxy, arylcarbonyloxy, alkylthio, arylthio, thio, alkylaminosulfonyl, and arylaminosulfonyl; R₆ is selected from the group consisting of hydrogen, hydroxyl, aryl, alkyl, aralkyl, heteroaryl, heteroaralkyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy, and heteroaralkylcarbonyloxy; and wherein > and R₅ can be points of attachment for the group -Z6-(Z )„-Z₈- where n is an integer between 1 and 3 to form thereby a 4-, 5-, or 6-membered ring respectively; and

Z₂-Z₈ are selected independently from the group consisting of methine, quaternary carbon, and nitrogen.

2. The method of claim 1, wherein Z_\ is -C(=Xι .

3. The method of claim 1, wherein X] is oxygen or sulfur.

4. The method of claim 3, wherein Xi is oxygen.

5. The method of claim 4, wherein R] is selected from the group consisting of hydrogen, aralkyl, and heteroaralkyl.

6. The method of claim 5, wherein Ri is aralkyl or heteroaralkyl.

7. The method of claim 6, wherein R_\ is aralkyl.

8. The method of claim 7, wherein said compound has the structure:

wherein R -Rπ are selected independently from the group consisting of hydrogen, halogen, hydroxyl, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, nitro, cyano, alkoxyl, aryloxyl, heteroaryloxyl, alkylthio, arylthio, alkoxycarbonyl, aryloxycarbonyl, sulfonylamido, amidosulfonyl, aralkoxycarbonylamino, amido, amino, alkylamino, arylamino, dialkylamino, diarylamino, arylalkylamino, heterocycleamino, heterocyclealkylamino, heterocycle, carboxyl, and wherein adjacent substituents together can form a 5- or 6-membered cyclic alkyl ether or carbocyclic fused ring system.

9. The method of claim 8, wherein each of Z2-Z₅ independently is quaternary carbon.

10. The method of claim 9, wherein Z₂-Z₅ are selected independently from the group consisting of -CH- and -CR -, where R12 is alkyl or halogen.

1. The method of claim 10, wherein R -Rπ are selected independently from the group consisting of hydrogen, halogen, and alkyl.

12. The method of claim 11, wherein said compound has the structure:

13. The method of claim 11, wherein said compound has the structure:

14. The method of claim 11, wherein said compound has the structure:

15. The method of claim 2, wherein X is NRβ.

16. The method of claim 15, wherein R₆ is selected from the group consisting of hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy, and heteroaralkylcarbonyloxy.

17. The method of claim 16, wherein R₆ is alkylcarbonyloxy, arylcarbonyloxy, or aralkylcarbonyloxy.

18. The method of claim 17, wherein Z₂-Z₅ are selected independently from the group consisting of -CH- and -CRι₃-, where R-₃ is alkyl.

19. The method of claim 18, wherein each of Z₂-Z₅ is -CH-.

20. The method of claim 19 wherein said compound has the structure:

wherein R₁₄ is alkyl or aryl.

21. The method of claim 20, wherein R-₄ is selected from the group consisting of 4- chlorophenyl, 4-(trifluoromethyl)phenyl, 2-methylprop-l-yl, 1-butyl, and methyl.

22. The method of claim 16, wherein R$ is hydroxyl.

23. The method of claim 22, wherein Z₂-Z₅ are selected independently from the group consisting of -CH- and -CR₁₅-, where R1₅ is heterocycle or alkylthio.

24. The method of claim 23, wherein Z- is -CR₁₅-, where R₁₅ is piperazin-1-yl or alkylpiperazin- 1 -yl.

25. The method of claim 24, wherein said compound has the structure:

26. The method of claim 24, wherein said compound has the structure:

27. The method of claim 22, wherein Z₄ is -CR₁₅-, where R_]5 is alkylthio.

28. The method of claim 27, wherein said compound has the structure:

29. A composition for treating cancer in a mammal comprising a therapeutically effective amount of the compound of claim 1 or a pharmaceutically acceptable salt thereof in a pharmaceutically acceptable carrier.

30. A compound of claim 1 having the structure:

and its pharmaceutically acceptable salts, wherein

X₂ is selected from the group consisting of oxygen, sulfur and NR₂₅, where R₂₅ is hydrogen, hydroxyl, aryl, alkyl, aralkyl, heteroaryl, heteroaralkyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy, or heteroaralkylcarbonyloxy;

R₂₀ is hydrogen or aralkyl; and

R₂₎ and R₂₂ are selected independently from the group consisting of hydrogen, alkyl, aryl, aralkyl, alkoxyl, aryloxyl, heteroaryl, heteroaralkyl, alkylcarbonyl, arylcarbonyl, arylamino, alkylamino, dialkylamino, diarylamino, alkylarylamino, carboxyl, alkylcarbonyloxy, arylcarbonyloxy, alkylaminosulfonyl, and arylaminosulfonyl.

31. The compound of claim 30, wherein X₂ is oxygen.

32. The compound of claim 31, wherein R₂₀ is benzyl or benzyl substituted optionally with at least one substituent selected from the group consisting of halogen or trifluoromethyl.

33. The compound of claim 32, wherein R₂₀ is 3, 4-dichlorobenzyl, 2, 6-dicholorobenzyl, or 3-(trifluoromethyl)phenyl.

34. The compound of claim 33, wherein R21 is hydrogen or halogen.

35. The compound of claim 34, wherein R21 is hydrogen or chloro.

36. The compound of claim 35, wherein R22 is thio or alkylthio.

37. The compound of claim 36, wherein R₂₀ is 3, 4-dichlorobenzyl, R₂₁ is chloro, and R₂₂ is thio or methylthio.

38. The compound of claim 36, wherein R₂₀ is 2, 6-dichlorobenzyl, R₂ι is hydrogen and R₂ is tert-butylthio, or π-butylthio.

39. The compound of claim 35, wherein R₂₂ is heterocycle.

40. The compound of claim 39, wherein R₂₂ is selected from the group consisting of piperazinyl, alkylpiperazinyl, and arylpiperazinyl.

41. The compound of claim 40, wherein R20 is 2, 6-dichlorobenzyl and R₂₂ is piperazin-1-yl or 4-phenylpiperazin-l-yl.

42. The compound of claim 35, wherein R₂₂ is alkylsulfonyl or arylsulfonyl.

43. The compound of claim 42, wherein R₂₂ is methylsulfonyl, tert-butylsulfonyl, or phenylsulfonyl.

44. The compound of claim 43, wherein R₂ι is hydrogen and R₂₂ is arylcarbonyl.

45. The compound of claim 44, wherein R₂₂ is phenylcarbonyl or 4-chlorophenylcarbonyl.

46. The compound of claim 33, wherein R₂ι is arylaminosulfonyl and R₂₂ is hydrogen.

47. The compound of claim 46, wherein R₂- is 3, 4-dichlorophenylaminosulfonyl.

48. A compound having the structure:

and its pharmaceutically acceptable salts, wherein X₃ is selected from the group consisting of oxygen, sulfur and NR₃₆₎ where R₃₆ is hydrogen, hydroxyl, aryl, alkyl, aralkyl, heteroaryl, heteroaralkyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy, or heteroaralkylcarbonyloxy;

R₃o and R₃₃ are is selected independently from the group consisting of hydrogen, alkyl, aryl, alkylcarbonyl, aralkylcarbonyl, arylcarbonyl, and aralkyl; and

R31. R₃2. and R₃₄-R₃₅ are selected independently from the group consisting of hydrogen, hydroxyl, nitro, cyano, thio, alkylthio, arylthio, alkyl, aryl, aralkyl, alkoxyl, aryloxyl, heteroaryl, heteroaralkyl, alkylcarbonyl, arylcarbonyl, arylamino, alkylamino, carboxyl, carbonyl, dialkylamino, diarylamino, alkylarylamino, carboxyl, alkylcarbonyloxy, arylcarbonyloxy, and arylaminosulfonyl; and further wherein R₃₅ and R₃₆ together are selected from the group consisting of a bond, oxygen, sulfur, - NR₃₈-, and -(Ze)_n-, where R₃₈ is selected from the group consisting of hydrogen, hydroxyl, aryl, alkyl, aralkyl, heteroaryl, heteroaralkyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy, or heteroaralkylcarbonyloxy, Z_δ is methylene, methine, or quaternary carbon, and n is an integer between 1 and 3.

49. The compound of claim 48, wherein X₃ is oxygen.

50. The compound of claim 49, wherein each of R₃ι, R₃∑, and R₃₄-R₃₇ is hydrogen.

51. The compound of claim 51, wherein R₃₀ and R₃₃ independently are benzyl or benzyl substituted optionally with at least one substituent selected from the group consisting of halogen or trifluoromethyl.

52. The compound of claim 50, wherein each of R30 and R₃₃ is 3, 4-dichlorobenzyl.

53. The compound of claim 50, wherein each of R₃₀ and R₃₃ is 2, 6-dichlorobenzyl.

54. The compound of claim 50, wherein each of R₃₀ and R₃₃ is 3-trifluoromethylbenzyl.

55. The compound of claim 51, wherein each of R₃₀ and R₃₃ is hydrogen.

6. A method for synthesizing isatin or an isatin derivative, comprising the steps of (a) reacting an ortΛo-halonitrobenzene with a dialkyl malonate under conditions effective to cause addtion of said malonate to said nitrobenzene to form a 2-nitrophenylmalonate diester; (b) reacting said 2-nitrophenylmalonate diester under conditions effective to form an indolin-2-one; (c) brominating said indolin-2-one with a perbromide reagent under conditions effective to form a 3, 3-dibromomindolin-2-one; and (d) hydrolyzing said 3, 3- dibromomindolin-2-one under conditions effective to form isatin or an isatin derivative.