WO2005032594A2

WO2005032594A2 - Alkylators linked to polyamides as dna binding agents

Info

Publication number: WO2005032594A2
Application number: PCT/US2004/032617
Authority: WO
Inventors: Zoltan Szekely; Humcha K. Hariprakasha; Christopher J. Michejda
Original assignee: The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority date: 2003-10-03
Filing date: 2004-10-01
Publication date: 2005-04-14
Also published as: WO2005032594A3

Abstract

The invention provides an alkylator that is an analog of a compound based on 5-oxo-1a,2,3,5-tetrahydro-1H-3-aza-cyclopropa[c]indene-7-carboxylic acid, 9a-chloromethyl-4-oxo-2,4,9,9a-tetrahydro-1H-2-aza-cyclopropa[1,5]cyclopenta[1,2-a]naphthalene-7-carboxylic acid, or 4-oxo-1,2,4,5,8,8a-hexahydrocyclopropa[c]pyrrolo[3,2-e]indole-6-carboxylic acid and conjugates thereof.

Description

BUILDING BLOCKS FOR DNA BINDING AGENTS

FIELD OF THE INVENTION [0001] The present invention relates to alkylators and conjugates comprising one or more alkylators(s) linked to one or more polyamide(s) that binds to a polynucleotide sequence. The alkylators and conjugates of the present invention and compositions thereof are useful in the inhibition of gene expression and in the treatment of cancer in a mammal.

BACKGROUND OF THE INVENTION [0002] There are many potent antitumor compounds with a wide spectrum of activities. However, these compounds are very toxic and affect healthy cells and tissue as well as cancerous cells. Many researchers have proposed increasing selectivity, while maintaining a high degree of toxicity. In fact, many effective antitumor drugs function by inhibiting nucleic acid (DNA or RNA) or protein synthesis in a sequence-specific manner. [0003] Polyamides that contain pyπole and/or imidazole carboxamide subunits are known to bind specifically to the minor groove of DNA (see, for example, Dervan, Bioorganic and Medicinal Chemistry, 9: 2215-2235 (2001); Soto et al., Nucleic Acids Research, 29(17): 3638-3645 (2001); and Reddy et al., Current Medicinal Chemistry, 8: 475-508 (2001)). Such polyamides can be designed so that they bind to DNA or RNA in a sequence-specific manner. These polyamide minor groove binders can inhibit, suppress, or alter gene functions. The polyamide minor groove binders bis-lexitropsins (Reddy et al., Current Medicinal Chemistry, 8: 475-508 (2001)), for example, have shown enhanced cytotoxic activity against KB human nasopharyngeal carcinoma. It also has been shown that double-stranded hairpin polyamides can permeate cellular and nuclear membranes of eukaryotes and, when targeted to promoter regions, can inhibit specific gene expression (Gottesfeld et al., Nature, 387: 202-205 (1997); and Dickinson et al, Proc. Natl. Acad. Sci. U.S.A., 95: 12890-12895 (1998)). In addition to the double-stranded hairpin polyamides, the single-stranded analogues have been proven to cany out inhibition of gene expression in Drosophila (Maeshima et al., The EMBO Journal, 20: 3218-3228 (2001)). [0004] Researchers have linked toxic compounds to polyamide sequences, such as netropsin, distamycin and lexitropsin (see, for example, Chang et al., J Am. Chem. Soc, 122: 4856-4864 (2000); Gupta et al., Anti-Cancer Drug Design, 11(8): 581-596 (1996); Jia et al., Heterocyclic Commun., 4(6): 557-560 (1998); Jia et al., Chem. Commun., (2): 119- 120 (1999); Jia et al., Sv«/ett, (5): 603-606 (2000); and Wang et al., Gene, 149(1): 63-67 (1994)). While these conjugates show some antitumor activity, they have geometric and/or electronic parameters that hinder binding to the minor groove of DNA. Poor binding can result in a lower efficacy or selectivity as well as result in a higher occuπence of side effects, due to nonspecific binding to untargeted genomic elements, DNA sequences, or RNA sequences.

[0005] Thus, there remains a need for therapeutic conjugates that have improved antitumor selectivity and nucleic acid sequence-specific binding properties. Ideally, these conjugates would elicit fewer side effects and less harm to healthy cells and tissue. Effective therapeutic conjugates can be designed rationally because an understanding of the geometry of the conjugates enables better binding of the conjugate to the shape of the minor groove pocket, thereby increasing the conjugate's specificity and the activity of the alkylator subunit. The present invention provides such conjugates. The conjugates of the present invention bind to the minor groove of DNA in a sequence-specific manner to deliver effectively an alkylator. This and other objects and advantages of the present invention, as well as additional inventive features will become apparent from the detailed description provided herein.

BRIEF SUMMARY OF THE INVENTION [0006] The present invention provides an alkylator of the formula:

wherein Ri is hydroxyl, a halogen, an alkylester, a sulfonate, a silyloxy, or an alkoxy; R₂ is hydrogen, an alkylhalogen, an alkylester, a methylsulfonate, or an alkoxy; R₃ is hydrogen, an arylcarbonyl, a vinylogous carbonyl, or a linker, L'; > is hydroxyl, an amino, a carbonate, a carbamate, or an alkoxy; R₅ is hydrogen; _* is a carbonyl, an alkylcarbonyl, a carbonylalkenyl, a vinylogous carbonyl, a carbonylalkyl, an alkylcarbonylalkyl, an alkylcarbonylalkenyl, or an alkyl, wherein R^ is optionally substituted with a linker, L; or R₅ and R_ό, taken together, form an aryl group, a heterocycle, or a saturated ring. [0007] In addition, the present invention provides for an alkylator of the formula:

wherein R₂ is hydrogen, a halogen, an alkylhalogen, an alkylester, or an alkoxy; R₃ is hydrogen, an arylcarbonyl, a vinylogous carbonyl, or a linker, L'; I ; is an oxygen or an imino group; R₅ is hydrogen; R$ is a carbonyl, an alkylcarbonyl, a carbonylalkenyl, a vinylogous carbonyl, a carbonylalkyl, an alkylcarbonylalkyl, an alkylcarbonylalkenyl, or an alkyl; or R₅ and R^, taken together, form an aryl group, a heterocycle, or a saturated ring, with the proviso that, when R₅ and R_ό form an unsubstituted aryl group, R is a halogen, an alkylester or an alkoxy. [0008] Moreover, the present invention provides for a conjugate of the formula:

A-P (I)

in which A is an alkylator and P is a polyamide, with the proviso that A comprises a linker by which A is conjugated to P.

[0009] The present invention further provides a conjugate of the formula: A -P-A" (II)

1 in which A and A , which can be the same or different, are alkylators, with the proviso that each of A¹ and A²comprises a linker, by which A¹ and A² are independently conjugated to P. [0010] In addition, the present invention provides a conjugate of the formula:

P'-A-T (III)

in which A is an alkylator and P¹ and P² are polyamides, which can be the same or different, with the proviso that A comprises two linkers, which can be the same or different and by each of which A is conjugated to P and P .

[0011] Further provided by the present invention is a composition comprising a conjugate of any of formulae (I)-(III) and a carrier.

[0012] Still further provided by the present invention is a method of inhibiting cancer in a mammal. The method comprises administering to the mammal a cancer-inhibiting effective amount of a conjugate of any of formulae (I)-(III), and/or a composition thereof. [0013] Also provided by the present invention is a method of inhibiting gene expression in a mammal. The method comprises administering to the mammal a gene expression- inhibiting effective amount of a conjugate of any of formulae (I)-(III), and/or a composition thereof.

BRIEF DESCRIPTION OF THE DRAWINGS [0014] Figure 1 depicts the synthetic scheme for protected and activated 5-oxo-la,2,3,5- tetrahydro-lH-3-aza-cyclopropa[c]indene-7-carboxylic acid ("CI") analogs. [0015] Figure 2 depicts the synthetic scheme for the protected and activated la- chloromethyl-5-oxo-la,2,3,5-tetrahydro-lH-3-aza-cyclopropa[c]indene-7-carboxylic acid ("C₂I") analogs.

[0016] Figure 3 depicts the synthetic scheme for the protected and activated 9a- chloromethyl-4-oxo-2,4,9,9a-tetrahydro-lH-2-aza-cyclopropa[l,5]cyclopenta[l,2- a]naphthalene-7-carboxylic acid ("C₂BI") analogs. [0017] Figure 4 depicts the synthetic scheme of alkylator-minor groove binder conjugate 40.

[0018] Figure 5 depicts the synthetic scheme of alkylator-minor groove binder conjugate 48.

[0019] Figure 6 depicts the cytotoxicity of the conjugates Z-120, Z-121, and Z-122 against human colon tumor cells HCT166 at various concentrations (MTT-120h).

[0020] Figure 7 depicts the cytotoxicity of the conjugate Z-120 against several cancerous cell lines at various concentrations (MTT-120h).

[0021] Figure 8 depicts the cytotoxicity of the conjugate Z-120 against human colon tumor cells HCT166 at various concentrations (MTT-120h).

DETAILED DESCRIPTION OF THE INVENTION [0022] The present invention provides alkylators and conjugates that comprise one or more polyamide(s) linked to one or more alkylator(s). The conjugates bind to a given polynucleotide sequence with specificity, thereby inhibiting the polynucleotide's functions. Therefore, the alkylators and conjugates are useful as inhibitors of gene expression, including inhibitors of cancer. [0023] In one aspect, the invention provides an alkylator of the formula:

wherein Ri is hydroxyl, a halogen, an alkylester, a sulfonate, a silyloxy, or an alkoxy; R₂ is hydrogen, an alkylhalogen, an alkylester, a methyl sulfonate, or an alkoxy; R₃ is hydrogen, an arylcarbonyl, a vinylogous carbonyl, or a linker, L'; R_t is hydroxyl, an amino, a carbonate, a carbamate, or an alkoxy; R₅ is hydrogen; Rό is a carbonyl, an alkylcarbonyl, a carbonylalkenyl, a vinylogous carbonyl, a carbonylalkyl, an alkylcarbonylalkyl, an alkylcarbonylalkenyl, or an alkyl, wherein Rβ is optionally substituted with a linker, L; or R₅ and R_ό, taken together, form an aryl group, a heterocycle, or a saturated ring, any one of which is optionally substituted with a carbonyl, an alkylcarbonyl, a carbonylalkyl, an alkylcarbonylalkyl, a carbonylalkenyl, an alkylcarbonylalkenyl, an alkyl, or a linker, L; wherein L and L' can be the same or different. R₆ can be Α., ^., ^^^L, or ^ . In another embodiment, if R is hydrogen, and if R₅ and R taken together form an aryl group, then the aryl group is not substituted with L. [0024] In another aspect, the present invention provides for an alkylator of the formula:

wherein R₂ is hydrogen, a halogen, an alkylhalogen, an alkylester, or an alkoxy; R₃ is hydrogen, an arylcarbonyl, a vinylogous carbonyl, or a linker, L'; R__t oxygen or an imino group; R₅ is hydrogen; R₆ is a carbonyl, an alkylcarbonyl, a carbonylalkenyl, a vinylogous carbonyl, a carbonylalkyl, an alkylcarbonylalkyl, an alkylcarbonylalkenyl, or an alkyl, wherein R₆ is optionally substituted with a linker, L; or R₅ and R , taken together, form an aryl group, a heterocycle or a saturated ring, any of which is optionally substituted with a carbonyl, an alkylcarbonyl, a carbonylalkyl, an alkylcarbonylalkyl, a carbonylalkenyl, an alkylcarbonylalkenyl, an alkyl, or a linker, L; wherein L and L' can be the same or different; with the proviso that, when R₅ and R_ό, taken together, form an unsubstituted aryl group, R₂ is a halogen, an alkylester or an alkoxy. R₆ can be Α., --N., ^"^L, or / . In another embodiment, if R is hydrogen, and if R₅ and Re taken together form an aryl group, then the aryl group is not substituted with L. [0025] In a further aspect, the invention provides for an alkylator of the formula:

wherein R_\ is hydroxyl, a halogen, an alkylester, a sulfonate, a silyloxy or an alkoxy; R₂ is hydrogen, an alkylhalogen, an alkylester, or an alkoxy; R₃ is hydrogen, an arylcarbonyl, a vinylogous carbonyl, or a linker, L'; j is hydroxyl, an amino, a carbonate, a carbamate, or an alkoxy; R is a carbonyl, an alkylcarbonyl, a carbonylalkenyl, a carbonylalkyl, an alkylcarbonylalkyl, an alkylcarbonylalkenyl, or an alkyl, wherein R₆ is optionally substituted with a linker, L; wherein L and L' can be the same or different. Re can be ^ ,

/ , ^/^L, or / . In another embodiment, R₃ is hydrogen and Re is not substituted with L. [0026] In another aspect, the invention provides for an alkylator of the formula:

wherein R₂ is hydrogen, a halogen, an alkylester, or an alkoxy; R is hydrogen, an arylcarbonyl, a vinylogous carbonyl, or a linker, L'; R; is oxygen or an imino group; Re is a carbonyl, an alkylcarbonyl, a carbonylalkenyl, a carbonylalkyl, an alkylcarbonylalkyl, an alkylcarbonylalkenyl, or an alkyl, wherein R₆ is optionally substituted with a linker, L; wherein L and L' can be the same or different. R_ό can be Α, ^ , ^^^L, or / . In another embodiment, R₃ is hydrogen and R is not substituted with L.

[0027] In another aspect, the invention provides for an alkylator of the formula:

wherein Ri is hydroxyl, a halogen, an alkylester, a sulfonate, a silyloxy, or an alkoxy; R₂ is hydroxyl, an amino, a carbonate, a carbamate, or an alkoxy; R₃ is hydrogen, an arylcarbonyl, a vinylogous carbonyl, or a linker, L'; R₄ is a carbonyl, an alkylcarbonyl, a carbonylalkenyl, a carbonylalkyl, an alkylcarbonylalkyl, an alkylcarbonylalkenyl, or an alkyl, wherein R_ό is optionally substituted with a linker, L; wherein L and L' can be the same or different. R can be - ., ^_, ^^L, or / . In another embodiment, R is hydrogen and R__t is not substituted with L.

[0028] In another aspect, the invention provides for an alkylator of the formula:

wherein R is hydrogen, an arylcarbonyl, a vinylogous carbonyl, or a linker, L'; ; is a carbonyl, an alkylcarbonyl, a carbonylalkenyl, a carbonylalkyl, an alkylcarbonylalkyl, an alkylcarbonylalkenyl, or an alkyl, wherein RΔ, is optionally substituted with a linker, L; wherein L and L' can be the same or different. _t can be Α, / ₅ ^^^L, or /^L. In another embodiment, R₃ is hydrogen and R is not substituted with L.

[0029] Referring to terminology used generically herein, the term "alkyl" means a straight-chain or branched alkyl substituent containing from, for example, about 1 to about 12 carbon atoms, preferably from about 1 to about 8 carbon atoms, more preferably from about 1 to about 6 carbon atoms. Examples of such substituents include methyl, ethyl, propyl, isopropyl, ..-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isoamyl, hexyl, octyl, dodecanyl, and the like.

[0030] The term "alkenyl" means a straight-chain or branched alkenyl substituent containing from, for example, about 1 to about 12 carbon atoms, preferably from about 1 to about 8 carbon atoms, more preferably from about 1 to about 6 carbon atoms. Examples of such substituents include methyl, ethyl, propyl, isopropyl, w-butyl, sec-butyl, isobutyl, tert- butyl, pentyl, isoamyl, hexyl, octyl, dodecanyl, and the like.

[0031] The term "halo" or "halogen," as used herein, means a substituent selected from Group VIIA, such as, for example, fluorine, bromine, chlorine, and iodine. Preferably, the halo is chlorine or bromine.

[0032] The term "aryl" refers to an unsubstituted or substituted aromatic carbocyclic substituent, as commonly understood in the art, and includes monocyclic and polycyclic aromatics such as, for example, phenyl, biphenyl, toluenyl, anisolyl, naphthyl, anthracenyl and the like. An aryl substituent generally contains from, for example, about 3 to about 30 carbon atoms, preferably from about 6 to about 18 carbon atoms, more preferably from about 6 to about 14 carbon atoms and most preferably from about 6 to about 10 carbon atoms. It is understood that the term aryl applies to cyclic substituents that are planar and comprise 4n+2 π electrons, according to Huckel's Rule. A saturated ring is a fully substituted cycloalkyl, for example, cyclohexyl, or a heterocycloalkyl, for example, morpholinyl, and the like.

[0033] The term "heterocycle" means a substituent defined by an aromatic heterocyclic ring, as is commonly understood in the art, including monocyclic and polycyclic heteroaryls containing from, for example, about 3 to about 30 carbon atoms, preferably from about 5 to about 10 carbon atoms, more preferably from about 5 to about 6 carbon atoms. Monocyclic heteroaryls include, for example, imidazolyl, thiazolyl, pyrazolyl, pynolyl, furanyl, pyrazolinyl, thiophenyl, oxazolyl, isoxazolyl, pyridinyl, pyridonyl, pyrimidinyl, pyrazinyl, and triazinyl substituents. Polycyclic heteroaryls include, for example, quinolinyl, isoquinolinyl, benzofiiranyl, indolyl, purinyl, benzimidazolyl, benzopyπolyl, and benzothiazolyl.

[0034] The term "alkoxy" embraces linear or branched alkyl groups that are attached to divalent oxygen. The alkyl group is the same as described herein. Examples of such substituents include methoxy, ethoxy, t-butoxy, and the like.

[0035] The term "carbonyl" refers to the group -C(O)H. The term "carbonylalkyl" refers to the group -RC(O)H that is connected to the compound through the alkyl R group. The term "alkylcarbonyl" refers to the group -C(O)R that is connected to the compound through the carbonyl carbon. The term "alkenylcarbonyl" refers to the group -C(O)R₂, in which R₂ is an alkenyl group as described herein, that is connected to the compound through the carbonyl carbon. The term "arylcarbonyl" refers to the group -C(O)R₃, in which R₃ is an aryl group as described herein, that is connected to the compound through the carbonyl carbon. The term "carbonylalkenyl" refers to the group - R₂C(O)H that is connected to the compound through the R₂ alkenyl carbon. The term "alkylcarbonylalkyl" refers to the group -RC(O)R', in which R and R' are alkyl groups as described herein, that is connected to the compound through the alkyl R group. The term "alkylcarbonylalkenyl" refers to the group -R₂C(O)R, in which R is an alkyl group and R₂ is an alkenyl group as described herein, that is connected to the compound through the alkenyl R₂ group. [0036] Moreover, the present invention provides for a conjugate of the formula:

A-P (I)

[0037] The present invention further provides a conjugate of the formula:

A'-P-A² (II) 1 in which A and A , which can be the same or different, are alkylators, and P is a polyamide, with the proviso that each of A¹ and A² comprises a linker, by which A¹ and A² are independently conjugated to P. [0038] In addition, the present invention provides a conjugate of the formula:

P'-A-P² (III)

in which A is an alkylator, and P¹ and P² are polyamides, with the proviso that A comprises two linkers, which can be the same or different, and by each of which A is conjugated to P¹ and P

[0039] In addition, the present invention provides a conjugate of the formula:

P^,-(Aⁿ-P^m)_k-A-P² where k, n, and m ≥l (IV)

in which A and Aⁿ, which can be the same or different, are alkylators, and P¹, P², and P^m are polyamides, with the proviso that A and A" also comprise two linkers, which can be the same or different, and by each of which A or A" is conjugated to P¹, P², or P^m. Preferably k, n, and m are integers from 1 to about 10 (e.g., 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 to 2). [0040] In another embodiment, the conjugate is further conjugated by way of a linker between P¹ and/or P² to another polyamide P³ such that from one to five P'-A-P² conjugates are conjugated to P³.

[0041] The alkylator (A, A¹, A², and/or Aⁿ) is any suitable polynucleotide sequence- specific alkylating agent as described herein. Preferably, the alkylators are analogs of compounds based on 5-oxo-la,2,3,5-tetrahydro-lH-3-aza-cyclopropa[c]indene-7-carboxylic acid ("CI"), 9a-chloromethyl-4-oxo-2,4,9,9a-tetrahydro- lH-2-aza- cyclopropa[l,5]cyclopenta[l,2-a]naphthalene-7-carboxylic acid ("C₂BI"), or 4-oxo- 1,2,4,5, 8, 8a-hexahydrocyclopropa[c]pyπolo[3,2-e]indole-6-carboxylic acid ("DA") that are known to be cytotoxic (e.g., IC₅₀ in the nano- to picomolar range). Preferably, the alkylator is a rigid, flexible, vinyl, or an alkyl alkylator. One of ordinary skill in the art will understand that the terms rigid, flexible, vinyl, and alkyl refer to the nature of the bond formed between the alkylator and the polyamide. [0042] The present invention also provides conjugates comprising the above-described alkylators. The conjugates that comprise the CI, C₂I, C₂BI, and DA alkylators have a higher binding affinity to the minor groove of DNA than the conjugates of the prior art. The higher binding affinity to the minor groove of DNA results from the use of alkylators, which are structurally similar to the polyamide of the conjugate. This creates a conjugate with a uniform polyamide-type structure throughout and results in a higher binding affinity to the minor groove of DNA. In addition, the conjugates of the present invention are synthesized to have an optimal geometry for binding to the minor groove of DNA that is not present in the conjugates of the prior art.

[0043] In the case of CI analogs, the linking moiety or polyamide groove binder is preferably connected at the C3 and/or C5 positions, as shown in the following structure:

[0044] With respect to C₂BI and DA analogs, preferably the linking moiety or polyamide groove binder is connected at the C3 and/or C7 positions, as shown in the following structure:

[0045] Conjugates in which the connectivity of the C₂BI and DA analog is through the C3 and C8 positions are generally not prefeπed because the geometry of the conjugate is not ideal for binding to the minor groove of DNA. [0046] The alkylator is preferably 5-oxo-la,2,3,5-tetrahydro-lH-3-aza- cyclopropa[c]indene-7-carboxylic acid ("CI"), or 3-chloromethyl-6-hydroxy-2,3-dihydro- lH-indole-4-carboxylic acid ("seco-CI"), or analogs thereof.

[0047] Examples of CI analogs are a rigid analog ("CIr"), flexible analog ("Clf), a vinyl analog ("Civ"), and an alkyl analog ("Cla"). One of ordinary skill in the art will understand that the terms rigid, flexible, vinyl, and alkyl refer to the nature of the bond formed between the CI ring and the carboxyl group of the polyamide' s N-methyl-pyπole. [0048] The following chemical structures are depictions of these bonds:

CIr bond with Polyamide Clf bond with Polyamide Cla bond with Polyamide

[0049] In one aspect of the invention, CIr can be optionally substituted, protected, and/or in its seco (i.e.'Open") or closed form:

protected, activated building block seco form "closed" form

[0050] In another aspect of the invention, Clf can be optionally substituted, protected, and/or in its seco (i.e.'Open") or closed form:

protected, activated building block seco form "closed" form

[0051] In another aspect of the invention, Civ can be optionally substituted, protected, and/or in its seco (i.e.'Open") or closed form:

protected, activated building block seco form "closed" form

[0052] In another aspect of the invention, Cla can be optionally substituted, protected, and/or in its seco (i.e.'Open") or closed form:

"closed" form [0053] In another embodiment, the alkylator is preferably la-chloromethyl-5-oxo- la,2,3,5-tetrahydro-lH-3-aza-cyclopropa[c]indene-7-carboxylic acid ("C₂I"), or 3,3-bis- chloromethyl-6-hydroxy-2,3-dihydro-lH-indole-4-carboxylic acid("seco-C2l"), or analogs thereof.

[0054] Examples of C₂I analogs are a rigid analog ("C₂Ir"), flexible analog ("C₂If '), a vinyl analog ("C₂IV"), and an alkyl analog ("C₂Ia"). One of ordinary skill in the art would understand that the terms rigid, flexible, vinyl, and alkyl refer to the nature of the bond formed between the C₂I ring and the carboxyl group of the polyamide' s N-methyl-pyπole. [0055] In one aspect of the invention, C₂lr can be optionally substituted, protected, and or in its seco (i.e.'Open") or closed form:

protected, activated building block seco form "closed" form

[0056] In another aspect of the invention, C₂lf can be optionally substituted, protected, and/or in its seco (i.e.'Open") or closed form:

protected, activated building block seco form "closed" form [0057] In another aspect of the invention, C₂IV can be optionally substituted, protected, and/or in its seco (i.e.'Open") or closed form:

protected, activated building block seco form "closed" form

[0058] In another aspect of the invention, C₂la can be optionally substituted, protected, and/or in its seco (i.e.'Open") or closed form:

protected, activated building block seco form "closed" form

[0059] In another embodiment, the alkylator is preferably 9a-chloromethyl-4-oxo- 2,4,9,9a-tetrahydro- 1 H-2-aza-cyclopropa[ 1 ,5]cyclopenta[ 1 ,2-a]naphthalene-7-carboxylic acid ("C₂BI"), or l,l-Bis-chloromethyl-5-hydroxy-2,3-dihydro-lH-benzo[e]indole-8- carboxylic acid ("seco-C₂BI"), or analogs thereof.

[0060] Examples of C₂BI analogs are a rigid analog ("C₂BIr"), flexible analog ("C₂BIf ' ), a vinyl analog ("C₂BIv"), and an alkyl analog ("C₂BIa"). One of ordinary skill in the art will understand that the terms rigid, flexible, vinyl, and alkyl refer to the nature of the bond formed between the C₂BI ring and the carboxyl group of the polyamide's N- methyl-pyπole. [0061] In one aspect of the invention, C₂BIr can be optionally substituted, protected, and/or in its seco (i.e.'Open") or closed form:

protected, activated building block seco form "closed" form

[0062] In another aspect of the invention, C₂BIf can be optionally substituted, protected, and/or in its seco (i.e.'Open") or closed form:

protected, activated building block seco form "closed" form

[0063] In another aspect of the invention, C₂BIv can be optionally substituted, protected, and/or in its seco (i.e.'Open") or closed form:

protected, activated building block seco form "closed" form

[0064] In another aspect of the invention, C₂BIa can be optionally substituted, protected, and/or in its seco (i.e.'Open") or closed form:

protected, activated building block seco form "closed" form

[0065] In another embodiment, the alkylator is preferably 4-oxo- 1,2,4,5, 8,8a- hexahydrocyclopropa[c]pyπolo[3,2-e]indole-6-carboxylic acid ("DA"), or 8-chloromethyl- 4-hydroxy-3,6,7,8-tetrahydro-3,6-diaza-as-indacene-2-carboxylic acid ("seco-DA"), or analogs thereof.

[0066] Examples of DA analogs are a rigid analog ("DAr"), flexible analog ("DAf '), a vinyl analog ("DAv"), and an alkyl analog ("DAa"). One of ordinary skill in the art will understand that the terms rigid, flexible, vinyl, and alkyl refer to the nature of the bond formed between the C₂BI ring and the carboxyl group of the polyamide' s N-methyl-pyπole. [0067] In one aspect of the invention, DAr can be optionally substituted, protected, and/or in its seco (i.e.'Open") or closed form:

protected, activated building block

"closed" form

[0068] In another aspect of the invention, DAf can be optionally substituted, protected, and/or in its seco (i.e.'Open") or closed form:

protected, activated building block

"closed" form

[0069] In another aspect of the invention, DAv can be optionally substituted, protected, and/or in its seco (i.e.'Open") or closed form:

protected, activated building block

closed" form

[0070] In another aspect of the invention, DAa can be optionally substituted, protected, and/or in its seco (i.e.'Open") or closed form:

prote

cted, activated building block "closed" form

[0071] The CI, C2l, C₂BI, and DA analogs described above comprise an amino acid or amino alkyl moiety, thereby allowing them to be part of the polyamide minor groove binder sequence. Therefore, conjugates that comprise such amino acid or amino alkyl alkylators do not necessarily require an additional linker to link the alkylator to the polyamide. [0072] The polyamide group binds to a polynucleotide sequence, for example a DNA or RNA sequence, in a sequence-specific manner. The polynucleotide's sequence-specific binding characteristic creates a conjugate with specificity for a particular polynucleotide sequence. Therefore, the conjugate can bring its linked alkylator within close proximity to the targeted polynucleotide sequence, which can result in a therapeutic response. [0073] The polyamide group that binds to the minor groove of DNA (P, P¹, P² and/or P^m) is any suitable polyamide sequence that recognizes specific DNA sequences through minor groove binding. Preferably, the polyamide sequence comprises one or more substituted or unsubstituted polypyπole carboxamide, one or more substituted or unsubstituted polyimidazole carboxamide, or combinations thereof. More preferably, the polyamide minor groove binder comprises subunits of 4-amino-l-methylpyπole-2- carboxylic acid, 3-(4-amino-l-methyl-lH-pyπol-2-yl)-acrylic acid, 4-amino-l- methylimidazole-2-carboxylic acid, 3 -(4-amino-l -methyl- lH-imidazol-2-yl)-acrylic acid, 4- amino-l-methyl-3-hydroxypyπole-2-carboxylic acid, γ-amino-butyric acid, α,γ-diamino- butyric acid, glutamic acid, 8-amino-3,6-dioxaoctanoic acid, β-alanine, 4-amino-benzoic acid, 3-amino-benzoic acid, 2-aminothiazole-5-carboxylic acid, 4-aminothiophene-2- carboxylic acid, 5-aminobenzthiophene-2-carboxylic acid, 5-aminobenzoxazole-2- carboxylic acid, 5-aminobenzimidazole-2-carboxylic acid or combinations thereof. It is understood that the longer the polyamide sequence, the more DNA-selective it is considered to be. The polyamide minor groove binder preferably contains about 20 or fewer amide subunits, more preferably about 10 or fewer amide subunits, more preferably about 8 or fewer amide subunits, more preferably about 6 or fewer amide subunits, and most preferably about 5 or fewer amide subunits. A prefeπed conjugate will contain one or more polyamide minor groove binder(s) made up of about 4 or 5 amide subunits. [0074] When one alkylator subunit is present in the conjugate, it is considered the n^l unit of the conjugate. The units from (n-k) to (n-1) and (n+1) to (n+k), wherein k represents the total number of subunits (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) can comprise apyπolyl, an imidazolyl, and or residues containing acyclic, mono-, bi- and tri-cyclic heteroatom- containing subunits. The (n+1) subunit, i.e., the first subunit immediately to the right of the alkylator subunit, preferably comprises a heteroatom-containing bicyclic group, such as benzofuranyl and indolyl. The (n+1) subunit most preferably comprises benzofuranyl. When more than one alkylator subunit is present in the conjugate, the foregoing embodiments apply.

[0075] The linker comprises any moiety that can form a chemical bond between the polyamide (i.e. P, P¹, and/or P²) and the alkylator (i.e. A, A¹, and/or A²). The linker can optionally bind to the polynucleotide, but can have little to no ability for sequence-specific interaction. The linker can be of any suitable charge, length and/or rigidity, but preferably the linker is bifunctional and/or comprises one or more amino groups, benzofuranyl and/or indolyl. At physiological pH, the amino group is protonated and can also bind to DNA. The amino group can be primary, secondary or tertiary. Preferably, the amino group comprises a moiety selected from the group consisting of amino,

alkylamino, -C12 dialkylamino, cycloalkylamino, piperazinyl, piperidinyl, pyrazinyl, purinyl, pyridazinyl, pyπolidinyl, oxazolyl, isooxazolyl, quinolinyl, isoquinolinyl, byrimidinyl, morpholinyl, thiazolyl, isothiazolyl, quinoxalinyl, quinazolinyl, pyπolyl, imidazolyl and an amino acid residue. Specific examples of suitable linkers include, but are not limited to, benzofuranyl, indolyl, N,N-bis(aminopropyl)piperazine, N,N-bis(aminopropyl)methylamine, 8-amino- 3,6-dioxaoctanoic acid, spermidine and β-alanine.

[0076] With respect to the above, a C_\-Cn alkyl can be straight chain or branched chain. In addition, the -C₁2 alkyl can be optionally substituted with substituents such as, for example, hydroxy, Cι-ι₂ alkoxy, acyloxy, halo or benzyl, acetyl, carboxyl, carboxy-Cι_ι₂ alkyl, such as carboxymethyl, carboxyethyl, carboxy-Cj-π alkylamido, carboxy-Cι.12 dialkylamido, carboxamido, amino, C_1-12 alkylamino, C_1-12 dialkylamino, C1-1₂ alkylcarbonyl, C₆.₃₀ arylamino, C_{6- 0} diarylamino, cyano, tolyl, xylyl, mesityl, anisyl, pyπolidinyl, formyl, dioxane, thio, C_M2 alkylthio, C_6-3o aryl, C_5-30 heteroaryl, such as pyranyl, pyπolyl, furanyl, thiophenyl, thiazolyl, pyrazolyl, pyridinyl, or pyrimidinyl, phenoxy, benzyloxy, phenylcarbonyl, benzylcarbonyl, nitrophenyl C_1-12 trialkylsilyl, nitro, sulfonyl, nitrobenzyl, C_1-12 trialkylammonium, C_M2 alkyl, C_3-8 cycloalkyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl and morpholinyl. Typical examples of a -C₁₂ alkyl are methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, neo-pentyl, hexyl, octyl, decyl, etc. [0077] Preferably a conjugate of the invention is one of the following formula:

[0078] In a prefeπed embodiment, the conjugate of any of formulae (I)-(III) binds to DNA, more preferably in the minor groove. The conjugate is sequence-selective and binds to about 5 or more base pairs in DNA, preferably to about 7 or more base pairs in DNA, more preferably to about 9 or more base pairs in DNA, and most preferably to about 10 or more base pairs in DNA. Depending on the specific polyamide minor groove binder and alkylator selected for the conjugate, the conjugate binds to a G-C rich DNA sequence or to an A-T rich DNA sequence. Those skilled in the art will understand how to make such selections for each element of the conjugate in order to bind to a G-C or A-T rich DNA sequence. For example, DNA alkylators such as CI, C₂I, C₂BI, and/or DA analogs, as descobed herein, bind covalently to adenine residues through the minor groove. Therefore, conjugates comprising CI, C₂I, C₂BI, and/or DA analogs can be used to bind selectively to A-T rich DNA sequences. Alternatively, conjugates that compose a phthalimide or anthracycline residue tend to favor G-C rich DNA sequences. The selection of subunits of the polyamide allows for the preparation of sequences that can preferentially bind to G-C or A-T rich DNA sequences. For example, N-methylimidazole preferably will bind to guanosine, whereas N-methylpyπole preferably will bind to cytosine, adenine, and thymidine.

[0079] The conjugates of formulae (I)-(III) can be prepared by any suitable method. The examples included herein are merely exemplary methods, and the synthetic routes for the conjugates are in no way limited to these methods. Typically, the alkylator-minor groove binder conjugates are prepared via a solid support method (see, for example, WO 03/072058) or a solution phase assembly (see, for example, Junhua Xiao, Gu Yuan and Weiqiang Huang, J Org. Chem. 2000, 65, 5506-5513). In Xiao et al., a "haloform reaction" is used to synthesize pyπole/imidazole oligomers targeting certain DNA sequences (binding to the minor groove of those DNA sequences).

[0080] Although not required, the conjugates are preferably prepared by heterogeneous methods using solid supports. In general, the minor groove binder oligomers are synthesized using solid-phase synthesis (see, for example, Dervan et al., Org.Lett., 3: 1201- 1203 (2001)). Depending on the post-solid-phase steps (e.g., coupling the minor groove binders to doxorubicin), different resins are used, such as, for example, Wang-resin, hydroxymethyl-benzoic acid resin, trityl-resin, etc.

[0081] Preferably, all of the monomers (e.g., pyπole-, imidazole-amino acids and alkylator subunits) are introduced as protected residues (e.g., Fmoc-protected, or 4,4'- dimethoxytrityl(DMT)-protected). Piperidine or piperidine/DBU typically are used to deprotect the Fmoc-protected monomers and dichloroacetic acid to deprotect DMT- protected monomers. For coupling, the monomers are preferably preactivated or activated in situ. In the case of aromatic carboxylic acids, stable benztirazyl active esters are isolated for the coupling reaction, whereas, in the case of aliphatic carboxylic acids, various activating agents (e.g., HBTU, HATU, HOBT/DCC) are used for activation. The alkylator residue CIr, Clf, Civ, Cla, C₂Ir, C₂If, C₂Iv, C₂Ia, C2BIr, C₂BIf, C₂BIv, C₂BIa, or DA is preferably modified to form the pro-drug equivalent (seco-form) in three steps: (i) removal of the TBDMS group; (ii) converting the alcohol into a chloro group; and (iii) deprotection of the t-Butyl ether to release the phenol. The oligomer is generally cleaved from the resin using either acidolytic (e.g., Wang- or trityl resins) or nucleophilic (e.g., HMBA resin) conditions. [0082] The present invention also provides compositions, including pharmaceutical compositions, comprising the conjugate of formulae (I)-(III) or combinations thereof, and a carrier, alone or in further combination with other active agents, such as adjuvants and anti- cancer agents. Preferably, the pharmaceutical compositions further comprise a pharmaceutically acceptable carrier.

[0083] Also provided is a method of inhibiting or altering gene expression in a mammal by administering to a mammal a gene expression-inhibiting (or a gene expression-altering) effective amount of the conjugate of formulae (I)-(III) or combinations thereof, and a carrier, alone or in further combination with other active agents. Preferably, the pharmaceutical compositions further comprise a pharmaceutically acceptable carrier. A method of altering gene expression by administering to a mammal a gene expression- altering effective amount of the conjugate of formulae (I)-(III) or combinations thereof, and a carrier, alone or in further combination with other active agents includes, for example, inhibition or up-regulation of a gene such that a beneficial result (e.g., tumor growth is suppressed) is realized.

[0084] One ordinarily skilled in the art will appreciate that suitable methods of administering a conjugate or composition thereof to a mammal, such as a human, are known, and, although more than one route can be used to administer a particular composition, a particular route can provide a more immediate and more effective reaction than another route. If the cancer is in the form of a tumor, preferably the conjugate or composition thereof is administered intratumorally or peritumorally. Pharmaceutically acceptable carriers are also well-known in the art. The choice of carrier will be determined, in part, by the particular conjugate or composition and by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of the pharmaceutical compositions of the present invention.

[0085] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the conjugate of any of formulae (I)-(III) dissolved in diluents, such as water or saline, (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules, (c) suspensions in an appropriate liquid, and (d) suitable emulsions. [0086] Tablet forms can include one or more of lactose, mannitol, cornstarch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoong agents, and pharmacologically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, such carriers as are known in the art. [0087] The conjugates of the present invention, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, hydro fluorocarbon (such as HFC 134a and/or 227), propane, nitrogen and the like.

[0088] Formulations suitable for parenteral administration include aqueous and non- aqueous solutions, iso tonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0089] The dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a prophylactic or therapeutic response in the mammal over a reasonable time frame. The dose will be determined by the strength of the particular composition employed (taking into consideration, at least, the bioactivity of any decomposition products derived from the conjugates) and the condition of the mammal (e.g., human), as well as the body weight of the mammal (e.g., human) to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse side effects that might accompany the administration of a particular composition. A suitable dosage for internal administration is 0.01 to 100 mg/kg of body weight per day, such as 0.01 to 35 mg/kg of body weight per day or 0.05 to 5 mg/kg of body weight per day. Although in some instances a dose such as 0.001 to 100 μg/kg of body weight per day would be suitable, depending on the strength of the composition. A suitable concentration of the conjugate in pharmaceutical compositions for topical administration is 0.05 to 15% (by weight), preferably 0.02 to 5%, and more preferably 0.1 to 3%.

[0090] The conjugates of any of formulae (I)-(III) or compositions thereof are useful for treating a mammal, such as a human, for cancer. The method comprises administering to the mammal, e.g., human, a cancer-inhibiting effective amount of a conjugate of any of formulae (I)-(III) or a composition thereof, whereupon the mammal is treated for cancer. The treatment can be prophylactic or therapeutic. By "prophylactic" is meant any degree in inhibition of the onset of cancer, including complete inhibition. By "therapeutic" is meant any degree in inhibition of the growth or metastasis of the cancer in the mammal (e.g., human).

[0091 ] The method can be used in combination with other known treatment methods, such as radiation, surgery, or the administration of other active agents, such as adjuvants or other anti-cancer agents and their prodrugs. Examples of cyotoxic agents and their prodrugs include genistein, okadaic acid, 1-β-D-arabinofuranosyl-cytosine, arabinofuranosyl-5-aza- cytosine, cisplatin, carboplatin, actinomycin D, asparaginase, bis-chloro-ethyl-nitroso-urea, bleomycin, chlorambucil, cyclohexyl-chloro-ethyl-nitroso-urea, cytosine arabinoside, daunomycin, etoposide, hydroxyurea, melphalan, mercaptopurine, mitomycin C, nitrogen mustard, procarbazine, teniposide, thioguanine, thiotepa, vincristine, 5-fluorouracil, 5- fluorocytosine, adriamycin, cyclophosphamide, methotrexate, vinblastine, doxorubicin, leucovorin, taxol, anti-estrogen agents such as tamoxifen, intracellular antibodies against oncogenes, the flavonol quercetin, Guan-mu-tong extract, retinoids such as fenretinide, nontoxid retinoid analogues such as N-(4-hydroxyphenyl)-retinamide (HPR), and monoterpenes such as limonene, perillyl alcohol and sobrerol.

[0092] The method of treating cancer with a conjugate of any of formulae (I)-(III) or composition thereof can be combined with still other methods of prophylactic and therapeutic treatment. Such methods include those that target destruction of cancer cells, e.g., by targeting of cell-surface markers, receptor ligands, e.g., ligands to gastrin-releasing peptide-like receptors, tumor-associated antigens, e.g., the 57 kD cytokeratin or the antigen recognized by the monoclonal antibody GB24, the extracellular matrix glycoprotein tamascin, antisense oncogenes such as c-fos, homeobox genes that are expressed in cancer cells but not normal cells, tumor-infiltrating lymphocytes that express cytokines, RGD- containing peptides and proteins, which are administered following surgery, lipophilic drug- containing liposomes to which are covalently conjugated monoclonal antibodies for targeting to cancer cells, low fat diet, moderate physical exercise and hormonal modulation. For prostate cancer, anti-testosterone agents can be used as well as an inhibitor of cellular proliferation produced by prostatic stromal cells and C-CAM, an epithelial cell adhesion molecule.

[0093] The conjugates of any of formulae (I)-(III) preferably are useful for targeting coding regions or control regions of genes, such as promoters or enhancers, and inhibiting transcription. Cancers that are suitable to treatment with conjugates of the present invention include those in which specific genes are known to be over-expressed and necessary for the survival of the cancer cell. Preferably, the cancer comprises an oncogene that is involved in one or more of the following molecular pathogenesis: (i) signal transducers (e.g. K-Ras); (ii) growth factor receptors (e.g. Her-2-Neu); (iii) nuclear oncoproteins (e.g. c-Myc); (iv) antagonists of apoptosis (e.g. BCL2); and (v) antagonists of tumor suppressors (e.g., MDM2). For instance, typical cancers that are to be treated in conjunction with the conjugates and compositions of the present invention include breast cancer, metastatic melanoma, colorectal cancer, pancreatic cancer, myeloid leukemia, follicular thyroid carcinoma, prostate cancer, hepatoblastoma, and hepatocellular carcinoma.

EXAMPLES

[0094] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

[0095] The following abbreviations are used herein:

BOC t-Butyloxycarbonyl,

DBU 1 ,8-Diazabicyclo[5.4.0.]undec-7-ene,

DCC 1,3-Dicyclohexylcarbodiimide, DCM Dichloromethane,

D1PEA Diisopropylethylamine,

DMT 4,4'-Dimethoxytrityl,

DPPA Diphenylphosphoryl azide,

EDC l-Ethyl-3(3'-dimehtylaminopropyl)carbodiimide - HC1

Fmoc 9-Fluorenylmethoxycarbonyl,

FmocCl 9-Fluorenylmethoxycarbonylchloride,

Fmoc-Im-OBt ( 1 H-Benzotriazole- 1 -yl) 4-(9-fluorenylmethoxycarbonyl)amino- 1 methylimidazole-2-carboxylate,

Fmoc-Py-OBt (1 H-Benzotriazole-1 -yl) 4-(9-fluorenylmethoxycaτbonyl)amino-l methyJpyπole-2-earboxylate,

HATU 2-( 1 H-7- Azabenzotriazole- 1 -yl)- 1,1,3,3 tetra ethyluromum hexafluorophosphate,

HBTU 2-(l H-Benzotriazole-1 -yl)-l,l,3,3-tetramethyluronium hexafluorophosphate,

HFIP Hexafiuoroisopropanol,

HMBA Hydroxymethylbenzoicacid,

HOBt N-Hydroxybenzotoazole,

Im Imidazole,

NIS N-Iodosuccinimide,

NMP N-Methylpyπolidone.

Py Pyπcle,

ΪBDMS t-Butyldimethylsilyl,

TBDMSC1 t-Butyldimethylsilylchloride,

TBTH Tributylt inhydride, t-BuOH t-Butanol,

TEMPO 2,2,6,6-tetramethyl-l-piperidinyloxy,

TFA Trifluoroacetic acid, and

TsOH p-Toluenesulfonic acid.

Example 1 [0096] This examples describes the synthesis of 5-oxo-la,2,3,5-tetrahydro-lH-3-aza- cyclopropa[c]indene-7-carboxylic acid ("CI") analogs (Fig. 1).

[0097] Commercially available 1 was converted to the bromo derivative 3 through the protection of the amino group as its Boc-derivative followed by NBS-mediated bromination. Allylation of 3 was performed using allyl-bromide in the presence of NaH. Transmetallation of the bromide 4 using t-BuLi, followed by zirconation with zirconocenemethylchloride and iodine, resulted in the desired dihydroindole derivative 5. Selective substitution of the primary iodide, followed by protection of the alcohol, gave the dibenzyl derivative 7. Halogen-metal exchange and electrophilic quenching led to the benzyl ester 8. Changing the amino protection from Boc to Fmoc was achieved in two steps. Complete hydrogenolysis of the benzyl groups, protection of the hydroxyl groups as their TBS ether, (using TBS-Cl in the presence of DIPEA), and deprotection of the partially formed TBS-ester resulted in the carboxylic acid, which was converted to its benztriazole- ester 11. Compound 11 is a stable intermediate and is ready-to-use for further solid-phase synthesis.

Example 2

[0098] This example describes the solution phase synthesis of conjugate 40. The following synthesis is easy to scale up to industrial-sized applications.

[0099] Commercially available ethyl 5-nitrobenzofuran-2-carboxylate 30 was hydrogenated on Pd-C and acylated using commercially available 4-nitro-N-methyl-2- trichloroacetyl-pyπole 32 to form 33. Hydrogenation of 33 followed by acylation with 32 afforded the nitro compound 35, which upon hydrogenation yielded 36 as the amino compound. Acetylation of the amino group of 36 followed by base catalyzed hydrolysis resulted in the acid 37. Coupling of 37 with the hydrochloride salt 39 (obtained by deprotection of the BOC derivative 38) using EDC afforded the ester 40. See Figure 4.

Example 3

[0100] This example describes the solution phase synthesis of conjugate 48.

[0101] The methyl ester conjugate 40, prepared in Example 2, was hydrolyzed with base to form the carboxylic acid 41. NN-dimethylpropanediamine 42 was coupled with 32 using haloform conditions to give 43, which upon hydrogenation followed by further coupling with 32 afforded 45. Hydrogenation of 45 and amide coupling with the intermediate 41 resulted in the conjugate 47. Further hydrogenation of 47 on Pd-C and ammonium formate as the hydrogen source afforded 48, the "seco" form of the final conjugate, ready to be used for biological studies. See Figure 5.

Example 4

[0102] This example demonstrates the cytotoxic effect of the present inventive conjugates against cancerous cells.

[0103] In order to determine the cytotoxicity of the compounds, "Cell Titer 96" non- radioactive cell proliferation assay (Mossman, J Immunol. Meth. 65:55 (1983)) was used. Cellular growth in presence (T) or absence (C) of experimental agents was determined using the Microculture Tefrazolium-based (MTT), CellTiter96 Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI) according to instructions provided by the supplier of the kit with small modifications (Monks et al., J. Nat/. Cancer Inst. 1991, 83, 757-766). Briefly, cells in exponential growth phase were harvested, counted and inoculated at the appropriate concentrations (100 μl volume) into 96-well flat bottom microtiter plates using a multichannel pipet. After 24 h (T₀), drugs were applied (100 μl volume) to sixplicate culture wells, and cultures were incubated for 96 or 120 h at 37 °C. After that, 15 μl of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was dispensed into to all wells and the plates were incubated 4 hours at 37 °C. At the end of the incubation, 75 μl of the Solubilization/Stop Solution was added to solubilize the MTT-formazan product. The next day, the absorbance of the wells was recorded by FLUOstar/POLARstar Galaxy (BMG Labtechnologies GmbH) MicroplateReader at 544 nm. During the time at which the drugs were added, assays were performed on extra reference plates to determine the cell population density at time 0 (T₀). Cellular responses were calculated from the data as described (1): 100 x [(T-T₀)/(C-T₀)] for T>T₀ and 100 x [(T-T₀)/T₀] for T<T₀. [0104] Three exemplary conjugates of the present invention, Z-120, Z- 121, and Z-122 were tested for cytotoxicity against the human colon cancer cell line HCT116. See Figure 6. The negative % growth means the number of tumor cell decreased at a particular drug concentration. TGI (total growth inhibition) is the drug concentration at which all cells stopped growing (aπested), LCioo is the drug concentration at which all cells are dead, LC ₀ is the drug concentration at which 50% of the cells are dead and 50% of the cells stopped growing, and IC₅₀ is the drug the concentration at which 50% of the cells stopped growing. All three conjugates proved to be cytotoxic with Z-121 as the most potent. Compound Z-121 had a TGI value of about 1 nM and an IC₅₀ value of about 0.25 nM. [0105] In addition, compound Z-120 was tested against human colon cancer (HCT116) and several human liver cancer cell lines: HepG2, Hep3B, Maklavu, and PLC. See Figures 7 and 8. Compound Z-120 had a TGI value of 10 nM, an IC₅₀ value of about 2-3 nM, and an LC₅o value of 30 nM against HCT116 cells.

[0106] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0107] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0108] Prefeπed embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations of those prefeπed embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

WHAT IS CLAIMED IS:

1. An alkylator of the formula:

wherein Ri is hydroxyl, a halogen, an alkylester, a sulfonate, a silyloxy, or an alkoxy; R₂ is hydrogen, an alkylhalogen, an alkylester, a methylsulfonate, or an alkoxy; R₃ is hydrogen, an arylcarbonyl, a vinylogous carbonyl, or a linker, L'; R₄ is hydroxyl, an amino, a carbonate, a carbamate, or an alkoxy; R₅ is hydrogen; R₆ is a carbonyl, an alkylcarbonyl, a carbonylalkenyl, a vinylogous carbonyl, a carbonylalkyl, an alkylcarbonylalkyl, an alkylcarbonylalkenyl, or an alkyl, wherein Re is optionally substituted with a linker, L; or R₅ and Re, taken together, form an aryl group, a heterocycle, or a saturated ring which is optionally substituted with a carbonyl, an alkylcarbonyl, a carbonylalkyl, an alkylcarbonylalkyl, a carbonylalkenyl, an alkylcarbonylalkenyl, an alkyl, or a linker, L; wherein L and L' can be the same or different.

2. The alkylator of claim 1, wherein R₆ is ^, --N., ^^^L, or ^^L.

3. The alkylator of claim 1 or 2, wherein R₃ is hydrogen; Re is not substituted with L; and R₅ and R₆ are taken together to form an aryl group which is not substituted with L.

4. An alkylator of the formula:

wherein R₂ is hydrogen, a halogen, an alkylhalogen, an alkylester, or an alkoxy; R₃ is hydrogen, an arylcarbonyl, a vinylogous carbonyl, or a linker, L'; R is an oxygen or an imino group; R₅ is hydrogen; Re is a carbonyl, an alkylcarbonyl, a carbonylalkenyl, a vinylogous carbonyl, a carbonylalkyl, an alkylcarbonylalkyl, an alkylcarbonylalkenyl, or an alkyl, wherein Re is optionally substituted with a linker, L; or R₅ and R₆, taken together, form an aryl group, a heterocycle, or a saturated ring which is optionally substituted with a carbonyl, an alkylcarbonyl, a carbonylalkyl, an alkylcarbonylalkyl, a carbonylalkenyl, an alkylcarbonylalkenyl, an alkyl, or a linker, L; wherein L and L' can be the same or different; with the proviso that, when R₅ and Re, taken together, form an unsubstituted aryl group, R₂ is a halogen, an alkylester or an alkoxy.

5. The alkylator of claim 3, wherein Re is -^ , -^ , ^^ or / .

6. The alkylator of claim 4 or 5, wherein R₃ is hydrogen; Re is not substituted with L; and R₅ and R₆ are taken together to form an aryl group which is not substituted with L.

7. An alkylator of the formula:

wherein Ri is hydroxyl, a halogen, an alkylester, a sulfonate, a silyloxy, or an alkoxy; R₂ is hydrogen, an alkylhalogen, an alkylester, or an alkoxy; R₃ is hydrogen, an arylcarbonyl, a vinylogous carbonyl, or a linker, L'; R₄ is hydroxyl, an amino, a carbonate, a carbamate, or an alkoxy; R₆ is a carbonyl, an alkylcarbonyl, a carbonylalkenyl, a carbonylalkyl, an alkylcarbonylalkyl, an alkylcarbonylalkenyl, or an alkyl, wherein R₆ is optionally substituted with a linker, L; wherein L and L' can be the same or different.

The alkylator of claim 7, wherein R₆ is A X ^ or

9. The alkylator of claim 7 or 8, wherein R₃ is hydrogen and Re is not substituted with L.

10. An alkylator of the formula:

wherein R₂ is hydrogen, a halogen, an alkylester, or an alkoxy; R₃ is hydrogen, an arylcarbonyl, a vinylogous carbonyl, or a linker, L¹; > is an oxygen or an imino group; Re is a carbonyl, an alkylcarbonyl, a carbonylalkenyl, a carbonylalkyl, an alkylcarbonylalkyl, an alkylcarbonylalkenyl, or an alkyl, wherein R₆ is optionally substituted with a linker, L; wherein L and L¹ can be the same or different.

11. The alkylator of claim 7, wherein R is ^, - ., ^^^L, or / .

12. The alkylator of claim 10 or 11 , wherein R₃ is hydrogen and Re is not substituted with L.

13. An alkylator of the formula:

wherein Ri is hydroxyl, a halogen, an alkylester, a sulfonate, a silyloxy, or an alkoxy; R₂ is hydroxyl, an amino, a carbonate, a carbamate, or an alkoxy; R₃ is hydrogen, an arylcarbonyl, a vinylogous carbonyl, or a linker, L'; j is a carbonyl, an alkylcarbonyl, a carbonylalkenyl, a carbonylalkyl, an alkylcarbonylalkyl, an alkylcarbonylalkenyl, or an alkyl, wherein Re is optionally substituted with a linker, L; wherein L and L' can be the same or different.

14. The alkylator of claim 13, wherein R₄ is ^, -- , ^^L, or

15. The alkylator of claim 13 or 14, wherein R₃ is hydrogen and R₄ is not substituted with L.

16. An alkylator of the formula:

wherein R₃ is hydrogen, an arylcarbonyl, a vinylogous carbonyl, or a linker, L'; R is a carbonyl, an alkylcarbonyl, a carbonylalkenyl, a carbonylalkyl, an alkylcarbonylalkyl, an alkylcarbonylalkenyl, or an alkyl, wherein R_, is optionally substituted with a linker, L; wherein L and L' can be the same or different.

17. The alkylator of claim 16, wherein R_t is ■* ., ^., ^^^L, or / .

18. The alkylator of claim 16 or 17, wherein R₃ is hydrogen and _t is not substituted with L.

19. A conjugate of the formula:

A-P (I)

in which A is an alkylator of any of claims 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, or 17, and P is a polyamide, with the proviso that A comprises a linker by which A is conjugated to P.

20. The conjugate of claim 19, wherein P binds to a polynucleotide in a sequence-specific manner.

21. The conjugate of claim 19, wherein the polyamide comprises a polypyπole carboxamide, polyimidazole carboxamide, or a combination thereof, wherein any of the foregoing are optionally substituted.

22. The conjugate of claim 19, wherein the polyamide comprises subunits selected from the group consisting of 4-amino-l-methylpyπole-2-carboxylic acid, 3-(4- amino- 1 -methyl- 1 H-pyπol-2-yl)-acrylic acid, 4-amino- 1 -methylimidazole-2-carboxylic acid, 3-(4-amino-l-methyl-lH-imidazol-2-yl)-acrylic acid, 4-amino- l-methyl-3- hydroxypyπole-2-carboxylic acid, γ-amino-butyric acid, α,γ-diamino-butyric acid, glutamic acid, 8-amino-3,6-dioxaoctanoicacid, β-alanine, 4-amino-benzoic acid, 3-amino-benzoic acid, 2-aminothiazole-5-carboxylic acid, 4-aminothiophene-2-carboxylic acid, 5- aminobenzthiophene-2-carboxylic acid, 5-aminobenzoxazole-2-carboxylic acid, 5- aminobenzimidazole-2-carboxylic acid, and combinations thereof.

23. The conjugate of claim 19, wherein any one of the linker(s) comprises benzofuranyl, indolyl, N,N-bis(aminopropyl)piperazine, N,N- bis(aminopropyl)methylamine, 8-amino-3,6-dioxaoctanoic acid, spermidine, or β-alanine.

24. The conjugate of claim 21, wherein any one of the linker(s) comprises benzofuranyl, indolyl, N,N-bis(aminopropyl)piperazine, N,N- bis(aminopropyl)methylamine, 8-amino-3,6-dioxaoctanoic acid, spermidine, or β-alanine.

25. The conjugate of claim 22, wherein any one of the linker(s) comprises benzofuranyl, indolyl, N,N-bis(aminopropyl)piperazine, N,N- bis(aminopropyl)methylamine, 8-amino-3,6-dioxaoctanoic acid, spermidine, or β-alanine.

26. A conj ugate of the formula:

A'-P-A (II)

in which A¹ and A², which can be the same or different, are alkylators of any of claims 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, or 17, and P is a polyamide, with the proviso that each of A¹ and A² comprises a linker, by which A¹ and A² are independently conjugated to P.

27. The conjugate of claim 26, wherein P binds to a polynucleotide in a sequence-specific manner.

28. The conjugate of claim 26 or 27, wherein the polyamide comprises a polypyπole carboxamide, polyimidazole carboxamide, or a combination thereof, wherein any of the foregoing are optionally substituted.

29. The conjugate of claim 26, wherein the polyamide comprises subunits selected from the group consisting of 4-amino- l-methylpyπole-2-carboxylic acid, 3-(4- amino- 1 -methyl- 1 H-pyπol-2-yl)-acrylic acid, 4-amino- 1 -methylimidazole-2-carboxylic acid, 3 -(4-amino- 1 -methyl- lH-imidazol-2-yl)-acrylic acid, 4-amino- l-methyl-3- hydroxypyπole-2-carboxylic acid, γ-amino-butyric acid, α,γ-diamino-butyric acid, glutamic acid, 8-amino-3,6-dioxaoctanoicacid, β-alanine, 4-amino-benzoic acid, 3-amino-benzoic acid, 2-aminothiazole-5-carboxylic acid, 4-aminothiophene-2-carboxylic acid, 5- aminobenzthiophene-2-carboxylic acid, 5-aminobenzoxazole-2-carboxylic acid, 5- aminobenzimidazole-2-carboxylic acid, and combinations thereof.

30. The conjugate of claim 26, wherein any one of the linker(s) comprises benzofuranyl, indolyl, N,N-bis(aminopropyl)piperazine, NN- bis(aminopropyl)methylamine, 8-amino-3,6-dioxaoctanoic acid, spermidine, or β-alanine.

31. The conjugate of claim 28, wherein any one of the linker(s) comprises benzofuranyl, indolyl, N,N-bis(aminopropyl)piperazine, NN- bis(aminopropyl)methylamine, 8-amino-3,6-dioxaoctanoic acid, spermidine, or β-alanine.

32. The conjugate of claim 29, wherein any one of the linker(s) comprises benzofuranyl, indolyl, N,N-bis(aminopropyl)piperazine, NN- bis(aminopropyl)methylamine, 8-amino-3,6-dioxaoctanoic acid, spermidine, or β-alanine.

33. A conjugate of the formula:

P'-A-P² (III)

in which A is an alkylator of any of claims 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, or 17, P¹ and P² are polyamides, which can be the same or different, with the proviso that A comprises two linkers, which can be the same or different and by each of which A is conjugated to P¹ and P².

• • 1 ^*) 34. The conjugate of claim 33, wherein P and P binds to a polynucleotide in a sequence-specific manner.

35. The conjugate of claim 33, wherein the polyamide comprises a polypyπole carboxamide, polyimidazole carboxamide, or a combination thereof, wherein any of the foregoing are optionally substituted.

36. The conjugate of claim 33, wherein the polyamide comprises subunits selected from the group consisting of 4-amino- l-methylpyπole-2-carboxylic acid, 3-(4- amino- 1 -methyl- 1 H-pyπol-2-yl)-acrylic acid, 4-amino- 1 -methylimidazole-2-carboxylic acid, 3-(4-amino-l -methyl- lH-imidazol-2-yl)-acrylic acid, 4-amino- l-methyl-3- hydroxypyπole-2 -carboxylic acid, γ-amino-butyric acid, α,γ-diamino-butyric acid, glutamic acid, 8-amino-3,6-dioxaoctanoicacid, β-alanine, 4-amino-benzoic acid, 3-amino-benzoic acid, 2-aminothiazole-5 -carboxylic acid, 4-aminothiophene-2-carboxylic acid, 5- aminobenzthiophene-2-carboxylic acid, 5-aminobenzoxazole-2-carboxylic acid, 5- aminobenzimidazole-2-carboxylic acid, and combinations thereof.

37. The conjugate of claim 33, wherein any one of the linker(s) comprises benzofuranyl, indolyl, N,N-bis(aminopropyl)piperazine, N,N- bis(aminopropyl)methylamine, 8-amino-3,6-dioxaoctanoic acid, spermidine, or β-alanine.

38. The conjugate of claim 35, wherein any one of the linker(s) comprises benzofuranyl, indolyl, N,/V-bis(aminopropyl)piperazine, NN- bis(aminopropyl)methylamine, 8-amino-3,6-dioxaoctanoic acid, spermidine, or β-alanine.

39. The conjugate of claim 36, wherein any one of the linker(s) comprises benzofuranyl, indolyl, N,N-bis(aminopropyl)piperazine, N,N- bis(aminopropyl)methylamine, 8-amino-3,6-dioxaoctanoic acid, spermidine, or β-alanine.

40. The conjugate of claim 33, which is further conjugated by way of a linker between P¹ and or P² to another polyamide P³ such that from one to five P'-A-P² conjugates are conjugated to P³.

41. The conjugate of claim 40, wherein P¹, P² and/or P³ binds to a polynucleotide in a sequence-specific manner.

42. The conjugate of claim 40, wherein the polyamide comprises a polypyπole carboxamide, polyimidazole carboxamide, or a combination thereof, wherein any of the foregoing are optionally substituted.

43. The conjugate of claim 40, wherein the polyamide comprises subunits selected from the group consisting of 4-amino- l-methylpyπole-2-carboxylic acid, 3-(4- amino- 1 -methyl- 1 H-pyπol-2-yl)-acrylic acid, 4-amino- 1 -methylimidazole-2-carboxylic acid, 3-(4-amino-l -methyl- lH-imidazol-2-yl)-acrylic acid, 4-amino- l-methyl-3- hydroxypyπole-2-carboxylic acid, γ-amino-butyric acid, α,γ-diamino-butyric acid, glutamic acid, 8-amino-3,6-dioxaoctanoicacid, β-alanine, 4-amino-benzoic acid, 3-amino-benzoic acid, 2-aminothiazole-5 -carboxylic acid, 4-aminothiophene-2-carboxylic acid, 5- aminobenzthiophene-2-carboxylic acid, 5-aminobenzoxazole-2-carboxylic acid, 5- aminobenzimidazole-2-carboxylic acid, and combinations thereof.

44. The conjugate of claim 40, wherein any one of the linker(s) comprises benzofuranyl, indolyl, N,N-bis(aminopropyl)piperazine, N,N- bis(aminopropyl)methylamine, 8-amino-3,6-dioxaoctanoic acid, spermidine, or β-alanine.

45. The conjugate of claim 42, wherein any one of the linker(s) comprises benzofuranyl, indolyl, N,N-bis(aminopropyl)piperazine, NN- bis(aminopropyl)methylamine, 8-amino-3,6-dioxaoctanoic acid, spermidine, or β-alanine.

46. The conjugate of claim 43, wherein any one of the linker(s) comprises benzofuranyl, indolyl, N,N-bis(aminopropyl)piperazine, N,N- bis(aminopropyl)methylamine, 8-amino-3,6-dioxaoctanoic acid, spermidine, or β-alanine.

47. A composition comprising the conjugate of claim 19 and a carrier.

48. A composition comprising the conjugate of claim 26 and a carrier.

49. A composition comprising the conjugate of claim 33 and a carrier.

50. A composition comprising the conjugate of claim 40 and a carrier.

51. A method of inhibiting cancer in a mammal having cancer, which method comprises administering to the mammal a cancer-inhibiting effective amount of the conjugate of claim 19 or a composition comprising same, whereupon cancer in the mammal is inhibited.

52. The method of claim 51 , wherein the cancer comprises an oncogene involved in the pathogenesis of one or more molecules selected from the group consisting of: signal transducers; growth factor receptors; nuclear oncoproteins; antagonists of apoptosis; and antagonists of tumor suppressors.

53. The method of claim 51 , wherein the cancer is colorectal cancer, pancreatic cancer, myeloid leukemia, follicular thyroid carcinoma, breast cancer, metastatic melanoma, or prostate cancer.

54. A method of inhibiting cancer in a mammal having cancer, which method comprises administering to the mammal a cancer-inhibiting effective amount of the conjugate of claim 26 or a composition comprising same, whereupon cancer in the mammal is inhibited.

55. The method of claim 54, wherein the cancer comprises an oncogene involved in the pathogenesis of one or more molecules selected from the group consisting of: signal transducers; growth factor receptors; nuclear oncoproteins; antagonists of apoptosis; and antagonists of tumor suppressors.

56. The method of claim 54, wherein the cancer is colorectal cancer, pancreatic cancer, myeloid leukemia, follicular thyroid carcinoma, breast cancer, metastatic melanoma, or prostate cancer.

57. A method of inhibiting cancer in a mammal having cancer, which method comprises administering to the mammal a cancer-inhibiting effective amount of the conjugate of claim 33 or a composition comprising same, whereupon cancer in the mammal is inhibited.

58. The method of claim 57, wherein the cancer comprises an oncogene involved in the pathogenesis of one or more molecules selected from the group consisting of: signal transducers; growth factor receptors; nuclear oncoproteins; antagonists of apoptosis; and antagonists of tumor suppressors.

59. The method of claim 57, wherein the cancer is colorectal cancer, pancreatic cancer, myeloid leukemia, follicular thyroid carcinoma, breast cancer, metastatic melanoma, or prostate cancer.

60. A method of inhibiting cancer in a mammal having cancer, which method comprises administering to the mammal a cancer-inhibiting effective amount of the conjugate of claim 40 or a composition composing same, whereupon cancer in the mammal is inhibited.

61. The method of claim 60, wherein the cancer comprises an oncogene involved in the pathogenesis of one or more molecules selected from the group consisting of: signal transducers; growth factor receptors; nuclear oncoproteins; antagonists of apoptosis; and antagonists of tumor suppressors.

62. The method of claim 60, wherein the cancer is colorectal cancer, pancreatic cancer, myeloid leukemia, follicular thyroid carcinoma, breast cancer, metastatic melanoma, or prostate cancer.

63. A method of inhibiting cancer in a mammal having cancer, which method comprises administering to the mammal a cancer-inhibiting effective amount of the alkylator of claim 9 or a composition comprising same, whereupon cancer in the mammal is inhibited.

64. The method of claim 63, wherein the cancer comprises an oncogene involved in the pathogenesis of one or more molecules selected from the group consisting of: signal transducers; growth factor receptors; nuclear oncoproteins; antagonists of apoptosis; and antagonists of tumor suppressors.

65. The method of claim 63, wherein the cancer is colorectal cancer, pancreatic cancer, myeloid leukemia, follicular thyroid carcinoma, breast cancer, metastatic melanoma, or prostate cancer.

66. A method of inhibiting cancer in a mammal having cancer, which method comprises administering to the mammal a cancer-inhibiting effective amount of the alkylator of claim 12 or a composition comprising same, whereupon cancer in the mammal is inhibited.

67. The method of claim 66, wherein the cancer comprises an oncogene involved in the pathogenesis of one or more molecules selected from the group consisting of: signal transducers; growth factor receptors; nuclear oncoproteins; antagonists of apoptosis; and antagonists of tumor suppressors.

68. The method of claim 66, wherein the cancer is colorectal cancer, pancreatic cancer, myeloid leukemia, follicular thyroid carcinoma, breast cancer, metastatic melanoma, or prostate cancer.

69. A method of inhibiting cancer in a mammal having cancer, which method comprises administering to the mammal a cancer-inhibiting effective amount of the alkylator of claim 15 or a composition comprising same, whereupon cancer in the mammal is inhibited.

70. The method of claim 69, wherein the cancer comprises an oncogene involved in the pathogenesis of one or more molecules selected from the group consisting of: signal transducers; growth factor receptors; nuclear oncoproteins; antagonists of apoptosis; and antagonists of tumor suppressors.

71. The method of claim 69, wherein the cancer is colorectal cancer, pancreatic cancer, myeloid leukemia, follicular thyroid carcinoma, breast cancer, metastatic melanoma, or prostate cancer.

72. A method of inhibiting cancer in a mammal having cancer, which method comprises administering to the mammal a cancer-inhibiting effective amount of the alkylator of claim 18 or a composition comprising same, whereupon cancer in the mammal is inhibited.

73. The method of claim 72, wherein the cancer comprises an oncogene involved in the pathogenesis of one or more molecules selected from the group consisting of: signal transducers; growth factor receptors; nuclear oncoproteins; antagonists of apoptosis; and antagonists of tumor suppressors.

74. The method of claim 72, wherein the cancer is colorectal cancer, pancreatic cancer, myeloid leukemia, follicular thyroid carcinoma, breast cancer, metastatic melanoma, or prostate cancer.

75. A method of inhibiting gene expression in a mammal in need thereof, which method comprises administering to the mammal a gene expression-inhibiting effective amount of the conjugate of claim 19, whereupon gene expression in the mammal is inhibited.

76. A method of inhibiting gene expression in a mammal in need thereof, which method comprises administering to the mammal a gene expression-inhibiting effective amount of the conjugate of claim 26, whereupon gene expression in the mammal is inhibited.

77. A method of inhibiting gene expression in a mammal in need thereof, which method comprises administering to the mammal a gene expression-inhibiting effective amount of the conjugate of claim 33, whereupon gene expression in the mammal is inhibited.

78. A method of inhibiting gene expression in a mammal in need thereof, which method comprises administering to the mammal a gene expression-inhibiting effective amount of the conjugate of claim 40, whereupon gene expression in the mammal is inhibited.

79. A conjugate of the formula:

P -(Aⁿ-P^m) -A-P where k, n, m >l (IV)

in which A and A", which can be the same or different, are alkylators of any of claims 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, or 17, and P¹, P², and P^m are polyamides, with the proviso that A and A" also comprise two linkers, which can be the same or different, and by each of which A or A" is conjugated to P¹, P², or P^m.

80. The conjugate of claim 79, wherein P binds to a polynucleotide in a sequence-specific manner.

81. The conjugate of claim 79 or 80, wherein the polyamide comprises a polypyπole carboxamide, polyimidazole carboxamide, or a combination thereof, wherein any of the foregoing are optionally substituted.

82. The conjugate of claim 79, wherein the polyamide comprises subunits selected from the group consisting of 4-amino- l-methylpyπole-2-carboxylic acid, 3-(4- amino- 1 -methyl- 1 H-pyπol-2-yl)-acrylic acid, 4-amino- 1 -methylimidazole-2-carboxylic acid, 3-(4-amino-l-methyl-lH-imidazol-2-yl)-acrylic acid, 4-amino- l-methyl-3- hydroxypyπole-2-carboxylic acid, γ-amino-butyric acid, α,γ-diamino-butyric acid, glutamic acid, 8-amino-3,6-dioxaoctanoicacid, β-alanine, 4-amino-benzoic acid, 3-amino-benzoic acid, 2-aminothiazole-5-carboxylic acid, 4-aminothiophene-2-carboxylic acid, 5- aminobenzthiophene-2-carboxylic acid, 5-aminobenzoxazole-2-carboxylic acid, 5- aminobenzimidazole-2-carboxylic acid, and combinations thereof.

83. The conjugate of claim 79, wherein any one of the linker(s) comprises benzofuranyl, indolyl, N,N-bis(aminopropyl)piperazine, NJV- bis(aminopropyl)methylamine, 8-amino-3,6-dioxaoctanoic acid, spermidine, or β-alanine.

84. The conjugate of claim 81, wherein any one of the linker(s) comprises benzofuranyl, indolyl, N,N-bis(aminopropyl)piperazine, NN- bis(aminopropyl)methylamine, 8-amino-3,6-dioxaoctanoic acid, spermidine, or β-alanine.

85. The conjugate of claim 82, wherein any one of the linker(s) comprises benzofuranyl, indolyl, N,N-bis(aminopropyl)piperazine, NN- bis(aminopropyl)methylamine, 8-amino-3,6-dioxaoctanoic acid, spermidine, or β-alanine.

86. A composition comprising the conjugate of claim 79 and a carrier.

87. A method of inhibiting cancer in a mammal having cancer, which method composes administering to the mammal a cancer-inhibiting effective amount of the conjugate of claim 79 or a composition comprising same, whereupon cancer in the mammal is inhibited.

88. The method of claim 87, wherein the cancer comprises an oncogene involved in the pathogenesis of one or more molecules selected from the group consisting of: signal transducers; growth factor receptors; nuclear oncoproteins; antagonists of apoptosis; and antagonists of tumor suppressors.

89. The method of claim 87, wherein the cancer is colorectal cancer, pancreatic cancer, myeloid leukemia, follicular thyroid carcinoma, breast cancer, metastatic melanoma, or prostate cancer.

90. A method of inhibiting gene expression in a mammal in need thereof, which method comprises administering to the mammal a gene expression-inhibiting effective amount of the conjugate of claim 79, whereupon gene expression in the mammal is inhibited.

91. A conjugate of the formula

92. A conjugate of the formula

93. A conjugate of the formula

94. A composition comprising the conjugate of any of claims 91-93 and a carrier.

95. A method of inhibiting cancer in a mammal having cancer, which method comprises administering to the mammal a cancer-inhibiting effective amount of the composition of claim 94, whereupon cancer in the mammal is inhibited.

96. The method of claim 95, wherein the cancer comprises an oncogene involved in the pathogenesis of one or more molecules selected from the group consisting of: signal transducers; growth factor receptors; nuclear oncoproteins; antagonists of apoptosis; and antagonists of tumor suppressors.

97. The method of claim 95, wherein the cancer is colorectal cancer, pancreatic cancer, myeloid leukemia, follicular thyroid carcinoma, breast cancer, metastatic melanoma, or prostate cancer.