# Patent

## Title
Dolastatin 15 derivatives

## Abstract

Compounds of the present invention include cell growth inhibitors which are peptides of Formula I,

A-B-D-E-F-(G)r-(K)s-L,  (I)

and acid salts thereof, wherein A, B, D, E, F, G and K are α-amino acid residues, and s and r are each, independently, 0 or 1. L is a monovalent radical, such as, for example, an amino group, an N-substituted amino group, a β-hydroxylamino group, a hydrazido group, an alkoxy group, a thioalkoxy group, an aminoxy group, or an oximato group. The present invention also includes a method for treating cancer in a mammal, such as a human, comprising administering to the mammal an effective amount of a compound of Formula I in a pharmaceutically acceptable composition.

## Background
RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 09/618,694, filed Jul. 18, 2000, now issued as U.S. Patent No. 6,458,765, which is a continuation of U.S. application Ser. No. 08/896,394, filed Jul. 18, 1997, now issued as U.S. Pat. No. 6,143,721. The contents of the aforementioned applications are hereby incorporated herein by reference.


BACKGROUND OF THE INVENTION
A number of short peptides with significant activity as inhibitors of cell growth have been isolated from the Indian Ocean sea hare Dolabella auricularia (Bai et al., Biochem. Pharmacology,40: 1859–1864 (1990); Beckwith et al., J. Natl. Cancer Inst., 85: 483–488 (1993) and references cited therein). These include Dolastatins 1–10 (U.S. Pat. No. 4,816,444, issued to Pettit et al.) and Dolastatin-15 (European Patent Application No. 398558). Dolastatin 15, for example, markedly inhibits the growth of the National Cancer Institute's P388 lymphocytic leukemia (PS system) cell line, a strong predictor of efficacy against various types of human malignancies.
The exceedingly small amounts of the various Dolastatin peptides present in Dolabella auricularia (about 1 mg each per 100 kg sea hare) and the consequent difficulties in purifying amounts sufficient for evaluation and use, have motivated efforts toward the synthesis of these compounds (Roux et al., Tetrahedron 50: 5345–5360 (1994); Shioiri et al., Tetrahedron 49: 1913–24 (1993); Patino et al., Tetrahedron 48: 4115–4122 (1992) and references cited therein). Synthetic Dolastatin 15, however, suffers from drawbacks which include poor solubility in aqueous systems and the need for expensive starting materials for its synthesis. These, in turn, have led to the synthesis and evaluation of structurally modified Dolastatin 15 derivatives [cf.: Biorg. Med. Chem. Lett. 4: 1947–50 (1994); WO 93 03054; JP-A-06234790; WO 93 23424].
However, there is a need for synthetic compounds with the biological activity of Dolastatin 15 which have useful aqueous solubility and can be produced efficiently and economically.
SUMMARY OF THE INVENTION
Compounds of the present invention include cell growth inhibitors which are peptides of Formula I,

A-B-D-E-F-(G)r-(K)s-L  (I),

and acid salts thereof, wherein A, B, D, E, F, G and K are α-amino acid residues, and s and r are each, independently, 0 or 1. L is a monovalent radical, such as, for example, an amino group, an N-substituted amino group, a β-hydroxylamino group, a hydrazido group, an alkoxy group, a thioalkoxy group, an aminoxy group, or an oximato group.

Another aspect of the present invention includes pharmaceutical compositions comprising a compound of Formula I and a pharmaceutically acceptable carrier.
An additional embodiment of the present invention is a method for treating cancer in a mammal, such as a human, comprising administering to the mammal an effective amount of a compound of Formula I in a pharmaceutically acceptable composition.


DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to peptides having antineoplastic activity. It also includes pharmaceutical compositions comprising these compounds and methods for treating cancer in a mammal, including a human, by administration of these compositions to the mammal.
Dolastatin 15, a peptide isolated from the sea hare Dolabella auricularia, is a potent inhibitor of cell growth. This compound, however, is present in trace quantities in the sea hare, and is thus difficult to isolate. Dolastatin 15 is also expensive to synthesize and suffers from poor aqueous solubility. As shown herein, however, Dolastatin 15 can serve as a starting point for the development of compounds which overcome these disadvantages while retaining antineoplastic activity or exhibiting greater antineoplastic activity than the natural product. Applicants have discovered that certain structural modifications of Dolastatin 15 provide compounds with a surprisingly improved therapeutic potential for the treatment of neoplastic diseases as compared to Dolastatin 10 and Dolastatin 15. Furthermore, the compounds of the present invention can be conveniently synthesized, as described below in detail.
For the purposes of the present invention, the term “monovalent radical” is intended to mean an electrically neutral molecular fragment capable of forming one covalent bond with a second neutral molecular fragment. Monovalent radicals include the hydrogen atom, alkyl groups, such as methyl, ethyl and propyl groups, halogen atoms, such as fluorine, chlorine and bromine atoms, aryl groups, such as phenyl and naphthyl groups, and alkoxy groups, such as methoxy and ethoxy groups. Two monovalent radicals on adjacent sigma-bonded atoms can also together form a pi bond between the adjacent atoms. Two monovalent radicals may also be linked together, for example, by a polymethylene unit, to form a cyclic structure. For example, the unit —N(R)R′, wherein R and R′ are each a monovalent radical, can, together with the nitrogen atom, form a heterocyclic ring. In addition, two monovalent radicals bonded to the same atom can together form a divalent radical, such as an oxygen atom or an alkylidene group, for example, a propylidene group.
For the purposes of the present invention, the term “normal alkyl” refers to an unbranched, or straight chain, alkyl group, for example, normal propyl (n-propyl, —CH2CH2CH3).
The compounds of the present invention can be represented by Formula I,

A-B-D-E-F-(G)r-(K)s-L  (I),

wherein A, B, D, E, F, G, and K are α-amino acid residues; s and r are each, independently, 0 or 1; and L is a monovalent radical such as an amino group, an N-substituted amino group, a β-hydroxylamino group, a hydrazido group, an alkoxy group, a thioalkoxy group, an aminoxy group, or an oximato group.

The peptides of Formula I are generally composed of L-amino acids but they can contain one or more D-amino acids. In the following discussion, reference to a particular amino acid includes both enantiomers unless a specific enantiomer is indicated. The present compounds can also be present as salts with physiologically-compatible acids, including hydrochloric acid, citric acid, tartaric acid, lactic acid, phosphoric acid, methanesulfonic acid, acetic acid, formic acid, maleic acid, fumaric acid, malic acid, succinic acid, malonic acid, sulfuric acid, L-glutamic acid, L-aspartic acid, pyruvic acid, mucic acid, benzoic acid, glucuronic acid, oxalic acid, ascorbic acid and acetylglycine.
The following is a description of the present invention, including a detailed description of individual components and of methods of using the claimed compounds.
Compounds of the Present Invention
Identity of A
In one embodiment, A is a proline derivative of Formula IIa,




where na is an integer, preferably 0, 1, 2, or 3. Ra is a monovalent radical, such as a hydrogen atom or an unsubstituted or fluorine-substituted alkyl group, for example a normal, branched or cyclic C1–C3-alkyl group which is, optionally, substituted by from 1 to about 3 fluorine atoms; suitable examples include methyl, ethyl, isopropyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, 1-methyl-2-fluoroethyl, 1-fluoromethyl-2-fluoroethyl or cyclopropyl; methyl, ethyl or isopropyl are preferred;

In this embodiment, R1a is a monovalent radical, such as a hydrogen atom, an alkyl group, such as a methyl, ethyl or propyl group, or a phenyl group. The phenyl group can be substituted; suitable substituents include one or more halogen atoms, with fluorine, chlorine and bromine atoms preferred, C1–C4-alkyl groups, methoxy, ethoxy, trifluoromethyl or nitro groups. Ra and R1a together can also form a propylene bridge.
R2a, R3a, R4a and R5a are each, independently, a monovalent radical, such as a hydrogen atom or an alkyl, preferably, methyl, group.
In another embodiment, A is a substituted glycine derivative of Formula IIIa,




where Ra has the meaning stated for Ra in Formula IIa and, R1a is a monovalent radical, for example, a hydrogen atom or a C1–C6-alkyl group, preferably a methyl, ethyl or propyl group.

In this embodiment, R6a is a monovalent radical, such as an alkyl, substituted alkyl, alkenyl, phenyl or substituted phenyl group. Suitable examples include methoxymethyl, 1-methoxyethyl, 1,1-dimethyl-hydroxymethyl, 1-trifluoromethylethyl, 1-trifluoromethyl-2,2,2-trifluoroethyl, vinyl, and 1-methylvinyl. Phenyl substituents can include one or more halogen atoms, preferably fluorine, chlorine or bromine atoms, and alkyl, methoxy, ethoxy, trifluoromethyl, and nitro groups.
When R1a is an alkyl group, R6a can also be a C1–C6-alkyl, cycloalkyl, unsubstituted benzyl or substituted benzyl group. Suitable benzyl substituents include one or more halogen atoms, such as fluorine, chlorine or bromine atoms, C1–C4-alkyl groups, and methoxy, ethoxy, trifluoromethyl and nitro groups.
R7a is a monovalent radical, preferably a methyl, ethyl or isopropyl group.
In another embodiment, A is an α-amino acid derivative of Formula IVa,




where ma is an integer, preferably 1 or 2, and Ra and R7a have the meanings stated for these substituents in Formula IIIa.

In another embodiment, A is an α-amino acid derivative of Formula Va,




where Ra and R7a have the meanings stated for Ra and R7a in Formula IIIa.

In a further embodiment, A is a substituted proline derivative of Formula VIa,




where Ra and R1a have the meanings stated for Ra and R1a in Formula IIa, and Xa is a monovalent radical, preferably a hydroxyl, alkoxy, for example, methoxy or ethoxy, group or a fluorine atom.

In another embodiment, A is a thiaprolyl derivative of Formula VIIa,




where Ra, R1a, R2a, R3a, R4a and R5a have the meanings stated for the respective substituents in Formula IIa.

In another embodiment, A is a 1,3-dihydroisoindole derivative of Formula VIIIa 




where Ra has the meaning stated for Ra for Formula IIa.

In another embodiment, A is a 2-azabicyclo[2.2.1]heptane-3-carboxylic acid derivative of Formula IXa,




where Za is a single or double bond and Ra has the meaning stated for Formula IIa. The 3-carbonyl substituent can have either the exo or endo orientation.

In another embodiment, A is an α-amino acid derivative of Formula Xa,




where na has the meaning as stated for na for Formula IIa, and R7a and Ra have the meanings as stated for R7a and Ra for Formula IIIa.

Identity of B

B is a valyl, isoleucyl, allo-isoleucyl, norvalyl, 2-tert-butylglycyl or 2-ethylglycyl residue. B can also be an α-amino acid residue of Formula IIb,




in which R1b and R2b are each a monovalent radical. R1b is, preferably, a hydrogen atom and R2b is, for example, an alkyl, alkoxyalkyl or alkenyl group. In preferred embodiments, R2b is a cyclopropyl group, a normal or branched butyl, preferably tertiary-butyl, group, a methoxymethyl group, a 1-methoxyethyl group or a 1-methylvinyl group. Additionally, R1b and R2b together can be an isopropylidene group.

Identity of D

D is an N-alkylvalyl, N-alkyl-2-ethylglycyl, N-alkyl-2-tert-butylglycyl, N-alkyl-norleucyl, N-alkyl-isoleucyl, N-alkyl-allo-isoleucyl or N-alkyl-norvalyl residue, where the N-alkyl group is preferably a methyl group or an ethyl group.
In another embodiment, D is an α-amino acid residue of Formula IId,




where Rd has the meaning stated for Ra in Formula IIIa, R1d is a monovalent radical, preferably a hydrogen atom, and R2d is a monovalent radical, for example, an alkyl, alkoxyalkyl or alkenyl group. In preferred embodiments, R2d is a cyclopropyl group, a normal or branched butyl, preferably tertiary-butyl, group, a methoxymethyl group, a 1-methoxyethyl group or a 1-methylvinyl group, such as a cyclopropyl group, a methoxymethyl group, a 1-methoxyethyl group or a 1-methylvinyl group. Additionally, R1d and R2d together can form an isopropylidene group.

Alternatively, D can be a proline derivative of Formula IIId,




where nd is an integer, for example, 1 or 2, and R3d has the meaning stated for R1a in Formula IIIa. Xd is a monovalent radical, preferably a hydrogen atom, and, in the case where nd equals 1, can also be a hydroxy or alkoxy, for example, methoxy or ethoxy, group or a fluorine atom.

Identity of E

E is a prolyl, thiazolidinyl-4-carbonyl, homoprolyl or hydroxyprolyl residue, or a cyclic α-amino carboxylic acid residue of Formula IIe,




where ne is an integer, preferably 0, 1 or 2. R1e has the meaning stated for R1a in Formula IIIa. R2e and R3e are each a monovalent radical, and can be, independently, a hydrogen atom or an alkyl, preferably methyl, group. R4e, is a monovalent radical, preferably a hydrogen atom, a hydroxy, alkoxy, for example, methoxy or ethoxy, group or a fluorine atom. R5e is a monovalent radical, preferably a hydrogen atom or a fluorine atom. In the case where ne is 1, R3e and R4e can together form a double bond, or R4e and R5e can together be a double-bonded oxygen radical. In the case where ne has the value 1 or 2, R1e and R2e can together form a double bond.

In another embodiment, E is a 2- or 3-amino-cyclopentanecarboxylic acid residue of Formula IIIe,




where Re is an alkyl group, such as methyl or ethyl, and R1e has the meaning stated for R1a in Formula IIIa.

Identity of F

F is a prolyl, thiazolidinyl-4-carbonyl, homoprolyl or hydroxyprolyl residue. F can also be a cyclic α-amino acid residue of Formula IIf,




where nf is an integer, preferably 0, 1 or 2. R1f has the meaning stated for R1a in Formula IIIa. R2f and R3f are each a monovalent radical, and can be, independently, a hydrogen atom or an alkyl, preferably methyl, group. R4f is a monovalent radical, preferably a hydrogen atom, a hydroxy, alkoxy, for example, methoxy or ethoxy, group or a fluorine atom. R5f is a monovalent radical, preferably a hydrogen atom or a fluorine atom. In the case where nf has the value 1, R3f and R4f together can form a double bond or R4f and R5f can together be a double-bonded oxygen radical. In the case where nf has the value 1 or 2, R1f and R2f can together form a double bond.

In another embodiment, F is a 2- or 3-amino-cyclopentanecarboxylic acid residue of Formula IIIf 




where Rf is a monovalent radical, such as a methyl or ethyl group, and R1f has the meaning stated for R1a in Formula IIIa.

In another embodiment, F is an N-alkylglycyl or N-alkylalanyl residue, and the alkyl group is, preferably, a methyl group or an ethyl group.
Identity of G
G is an α-amino acid residue of Formula IIg,




wherein R1g is a hydrogen atom, or an alkyl group, for example, methyl, ethyl or n-propyl. R2g can be, for example, a hydrogen atom, or an alkyl, arylalkyl, heteroarylalkyl or aryl group. Preferably, R2g is an ethyl, isopropyl, tert-butyl, isobutyl, 2-methylpropyl, cyclohexylmethyl, benzyl, thiazolyl-2-methyl, pyridyl-2-methyl, n-butyl, 2,2-dimethylpropyl, naphthylmethyl, or n-propyl group, or a substituted or unsubstituted phenyl group. Suitable phenyl substituents include one or more halogen, preferably fluorine, chlorine or bromine, atoms, C1–C4-alkyl groups, methoxy, ethoxy, nitro or trifluoromethyl groups or a dioxomethylene group. Alternately, R1g and R2g can, together with the α-carbon atom, form a cyclopentane or cyclohexane ring or a benzo-fused cyclopentane ring, such as, for example, the indanyl group.

Identity of K

K is an α-amino acid residue of Formula IIk,




wherein R1k has the identity stated for R1g in Formula IIg, and R2k has the identity stated for R2g in Formula IIg.

Identity of L

In one embodiment, L is an amino or substituted amino group of Formula IIl,




where R1l is a monovalent radical, such as a hydrogen atom, a normal or branched, saturated or unsaturated C1–C18-alkoxy group, a substituted or unsubstituted aryloxy group, a substituted or unsubstituted aryl-C1–C6-alkoxy group, or a substituted or unsubstituted aryloxy-C1–C6- alkoxy or heteroaryl-C1–C6-alkoxy group. The aryl group is preferably a phenyl or naphthyl group. The heteroaryl group is a 5- or 6-membered, preferably nitrogen-, oxygen- or sulfur-containing, ring system, such as, for example, a heteroaryl group derived from imidazole, isoxazole, isothiazole, thiazole, oxazole, pyrazole, thiophene, furan, pyrrole, 1,2,4- or 1,2,3-triazole, pyrazine, indole, benzofuran, benzothiophene, indole, isoindole, indazole, quinoline, pyridazine, pyrimidine, benzimidazole, benzopyran, benzothiazole, oxadiazole, thiadiazole or pyridine. Suitable aryl substituents include one or more halogen, preferably fluorine, bromine or chlorine, atoms, C1–C4-alkyl groups, methoxy, ethoxy or trifluoromethyl groups, a dioxymethylene group or nitro groups.

R2l is a monovalent radical, such as a hydrogen atom, a normal or branched, saturated or unsaturated C1–C18-alkyl group, a C3–C10-cycloalkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group. The aryl group is preferably a phenyl or naphthyl group. The heteroaryl group is a 5- or 6-membered, preferably nitrogen-, oxygen- or sulfur- containing, ring system, such as, for example, a heteroaryl group derived from imidazole, isoxazole, isothiazole, thiazole, oxazole, pyrazole, thiophene, furan, pyrrole, 1,2,4- or 1,2,3-triazole, pyrazine, indole, benzofuran, benzothiophene, indole, isoindole, indazole, quinoline, pyridazine, pyrimidine, benzimidazole, benzopyran, benzothiazole, oxadiazole, thiadiazole or pyridine. Suitable aryl substituents include one or more halogen, preferably fluorine, bromine or chlorine, atoms, C1–C4-alkyl groups, methoxy, ethoxy or trifluoromethyl groups, a dioxymethylene group or nitro groups.
R2l can, alternately, be of Formula IIr,




where al is an integer, such as 0, 1, 2, 3, 4 or 5. R3l is a monovalent radical, preferably a lower alkyl group, such as a methyl, ethyl, propyl or isopropyl group. R4l is a monovalent radical, which can be a saturated or partially unsaturated carbocyclic system comprising from about 3 to about 10 carbon atoms, a substituted or unsubstituted aryl or heteroaryl group, with aryl and heteroaryl and preferred substituents having the meaning stated for R2l in Formula IIl.

R2l can also be a substituent of Formula IIIr,

—(CH2)2—Wl—R5l  (IIIr),

wherein Wl is an oxygen or sulfur atom or an N—R6l group. R5l is a monovalent radical, such as a hydrogen atom, a C1–C4-alkyl or C3–C7-cycloalkyl group or a substituted or unsubstituted aryl or arylmethyl group, with aryl and its preferred substituents having the meaning stated for R2l from Formula IIl. R6l is a monovalent radical, preferably a hydrogen atom, a C1–C4-alkyl group or a C3–C7-cycloalkyl group, a C1–C18-alkanoyl group, a benzoyl group or a substituted or unsubstituted aryl or arylmethyl group, with aryl and its preferred substituents having the meaning stated for R2l in Formula IIl.

R2l can, alternately, be a substituent of Formula IVr,

—(CH2)bl—Zl  (IVr),

where bl is an integer, preferably 2, 3 or 4. Zl can be a monovalent radical such as a formyl, aminocarbonyl or hydrazinocarbonyl group, or a cyclic or acyclic acetal or thioacetal group.

R2l can also be a substituent of Formula Vr,




in which bl has the above-mentioned meaning. R7l can be a monovalent radical, such as a polyglycol group of the formula —O—(CH2—CH2—O)dl—CH3, where dl is an integer, preferably in the range from about 2 to about 4 or from about 40 to about 90.

R2l can further be a carbohydrate of Formula VIr,




where R8l is a monovalent radical, such as a hydrogen atom, a C1–C4-alkanoyl or alkyl group, a benzoyl group or a benzyl group.

L can also be a β-hydroxylamino group of Formula IIIl,




where R9l is a monovalent radical such as a hydrogen atom, a C1–C6-alkyl group or a substituted or unsubstituted aryl group, with aryl and its preferred substituents having the meaning stated for R2l. R10l is a monovalent radical, preferably a hydrogen atom, alkyl, for example, methyl, or a phenyl group.

When r and/or s is 1, L can also be an amino group of Formula IVl,




where R2l and R4l are each a monovalent radical. R2l and R4l can also be linked by a carbon-carbon bond.

Another subclass of compounds of this invention includes peptides of Formula I wherein L is a hydrazido group of Formula Vl,




and R11l is a monovalent radical, preferably a hydrogen atom. R12l can be a monovalent radical such as a hydrogen atom, a normal or branched C1–C8-alkyl group, a C3–C8-cycloalkyl group, a C3–C8-cycloalkyl-C1–C4-alkyl group or a substituted or unsubstituted aryl, heteroaryl, aryl-C1–C4-alkyl or heteroaryl-C1–C4-alkyl group, where aryl, heteroaryl and their preferred substituents can be selected from among the options listed for R2l.

When r and/or s is 1, R11l can also be selected from among the options listed above for R12l, and the two radicals together can additionally form a propylene or butylene bridge.
Another subclass of compounds of this invention includes peptides of Formula I wherein L is a monovalent radical of the formula —O—R13l or the formula —S—R13l, where R13l is a monovalent radical, such as a C3–C10-cycloalkyl group, a normal or branched C2–C16-alkenylmethyl group or a C1–C16-alkyl group which can be substituted by from 1 to about 5 halogen, preferably fluorine, atoms.
R13l can also be the radical —(CH2)e—R14l, where e is an integer, preferably 1, 2 or 3. R14l is a monovalent radical, preferably a saturated or partially unsaturated C3–C10-carbocycle.
R13l can further be the monovalent radical —[CH2—CH═C(CH3)—CH2]f—H, where f is an integer, preferably 1, 2, 3 or 4.
R13l can also be the radical —[CH2—CH2—O]g—CH3, where g is an integer, preferably in the range from 1 to about 5.
R13l can also be the radical —(CH2)h-aryl or —(CH2)h-heteroaryl, where aryl and heteroaryl can also be substituted and, along with their preferred substituents, can be selected from the group listed for R2l. h is an integer, preferably 0, 1, 2 or 3.
R13l can further be the radical —(CH2)b—Wl—R5l. b, Wl and R5l can each be selected from among the options described for Formula IVl.
Another subclass of compounds of this invention includes peptides of Formula I in which L is an aminoxy group of the formula —O—N(R15l)(R16l), where R15l and R16l are each a monovalent radical, which can independently be a hydrogen atom, a normal or branched C1–C8-alkyl group, which can be substituted by halogen, preferably fluorine, atoms, a C3–C8-cycloalkyl group, a C3–C8-cycloalkyl-C1–C4-alkyl group, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted aryl-C1–C4-alkyl group. Aryl and heteroaryl groups and the preferred substituents thereof can be selected from the options listed for R2l. R16l can be selected from among the options listed for R15l. Additionally, R15l and R16l can together form a 5-, 6- or 7-membered heterocycle. The compounds of the present invention further comprise the salts of the compounds described above with physiologically tolerated acids.
Another subclass of compounds of this invention includes peptides of Formula I wherein L is an oximato group of the formula —O—N═C(R15l)(R16l), R15l and R16l can be selected from among the options listed above and, additionally, can together form a cyclic system comprising, preferably, from about 3 to about 7 ring atoms. This cyclic system can additionally be fused to one or more aromatic rings. Particularly preferred cyclic systems are shown below.







In one embodiment, the invention provides compounds of Formula I wherein A is an amino acid derivative selected from among N-alkyl-D-prolyl, N-alkyl-L-prolyl, N-alkyl-D-piperidine-2-carbonyl, N-alkyl-L-piperidine-2-carbonyl, N,N-dialkyl-D-2-ethyl-2-phenylglycyl and N,N-dialkyl-L-2-ethyl-2-phenylglycyl, wherein alkyl is methyl, ethyl or isopropyl; and B is a valyl, isoleucyl or 2-t-butyl-L-glycyl residue.
Preferred compounds of the invention include compounds of Formula I wherein r and s are each 0. A is an amino acid derivative selected from among D-N-methyl-piperidine-2-carbonyl, L-N-methyl-piperidine-2-carbonyl, N,N-dimethylamino-iso-butyryl, N-methyl-L-prolyl, N-methyl-L-thiazolidine-4-carbonyl, N,N-dimethyl-glycyl, L-prolyl, L-piperidine-2-carbonyl, N-propyl-D-piperidine-2-carbonyl, D-piperidine-2-carbonyl, N-ethyl-D-piperidine-2-carbonyl, N-methyl-[2,2,5,5-tetramethyl]-L-thiazolidine-2-carbonyl, N-isopropyl-D-piperidine-2-carbonyl, N,N-dimethyl-2-cyclopropylglycyl, N,N-dimethyl-L-2-ethyl-2-phenylglycyl, N,N-dimethyl-D-2-ethyl-2-phenylglycyl, D-prolyl, N-methyl-D-prolyl, N,N-dimethyl-2-(2-fluorophenyl)glycyl, 1-aza-[3,3,0]bicyclooctyl-5-carbonyl, N,N-dimethyl-2-[4-fluoro]phenyl-glycyl, N-methyl-[2,2,5,5-tetramethyl]-thiazolidine-2-carbonyl, 2-(R,S)-ethyl-2-phenylglycyl, D,L-1-aminoindane-1-carbonyl, N,N-dimethyl-2-(R,S)-methyl-2-phenylglycyl, 2-[N,N-dimethylamino]indane-2-carbonyl, 5-[N,N-dimethylamino]-5,6,7,8-tetrahydro-naphthalene-5-carbonyl, N-isopropyl-2-(R,S)-ethyl-2-phenylglycyl, 1-[N,N-dimethyl-amino]indane-2-carbonyl, N,N-dimethyl-2-propyl-2-phenylglycyl, N,N-dimethyl-2-[4-methoxy]phenyl-glycyl, N-methyl-3-hydroxy-D,L-valyl, N,N-dimethyl-D,L-2-isopropyl-2-phenylglycyl, N-methylpiperidine-2-carbonyl, N-methyl-L-prolyl, N-methyl-1,2,3,4-tetrahydroisoquinoline-1-carbonyl, N-methylazetidine-2-carbonyl, N-isopropylazetidine-2-carbonyl, N,N-dimethyl-[O-methyl]seryl, N,N-dimethyl-[O-methyl]threonyl, N-methyl-1,2,3,4-tetrahydroisoquinoline-3-carbonyl, 1-[N,N-dimethylamino]cyclohexyl-1-carbonyl, 1-[N,N-dimethylamino]cyclopentyl-1-carbonyl and 1,2,3,4-tetrahydroisoquinoline-3-carbonyl. B is valyl, isoleucyl or 2-tert-butylglycyl. D is N-methylvalyl, N-methyl-2-t-butylglycyl or N-methylisoleucyl. E and F are each, independently, prolyl, thiaprolyl, homoprolyl, hydroxyprolyl, 3,4-didehydroprolyl, 4-fluoroprolyl, and 3-methylprolyl. L is an alkoxy group or an amino group of the formula R1l—N—R2l, wherein R1l and R2l are independently selected from the group consisting of hydrogen, alkoxy, hydroxy, alkyl and alkylaryl.
In a particularly preferred subset of the compounds of the invention, r and s are each 0. A is an amino acid derivative selected from among D-N-methyl-piperidine-2-carbonyl, N-ethyl-D-piperidine-2-carbonyl, N-isopropyl-D-piperidine-2-carbonyl, N,N-dimethyl-2-cyclopropyl-glycyl, N-methyl-D-prolyl, 1-aza-[3,3,0]bicyclooctyl-5-carbonyl, N-methyl-[2,2,5,5-tetramethyl]-thiazolidine-2-carbonyl, 2-(R,S)-ethyl-2-phenylglycyl, D,L-1-aminoindane-1-carbonyl, N,N-dimethyl-2-(R,S)-methyl-2-phenylglycyl, 5-[N,N-dimethylamino]-5,6,7,8-tetrahydro-naphthalene-5-carbonyl, 1-[N,N-dimethylamino]indane-2-carbonyl, N,N-dimethyl-2-propyl-2-phenylglycyl, N,N-dimethyl-L-2-ethyl-2-phenylglycyl, N,N-dimethyl-D-2-ethyl-2-phenylglycyl, N-methyl-3-hydroxy-D,L-valyl, N,N-dimethyl-D,L-2-isopropyl-2-phenylglycyl, N-methyl-piperidine-2-carbonyl, N-methyl-D,L-prolyl, N-methyl-1,2,3,4-tetra-hydroisoquinoline-1-carbonyl, N-methylazetidine-2-carbonyl, N-isopropylazetidine-2-carbonyl, N,N-dimethyl-[O-methyl]seryl, 1-[N,N-dimethylamino]cyclohexyl-1-carbonyl and 1-[N,N-dimethylamino]cyclopentyl-1-carbonyl. B is valyl; D is N-methylvalyl; and E and F are each prolyl. L is a C1–C6-alkoxy group or an amino group of the formula R1l—N—R2l, wherein R1l and R2l are each independently selected from the group consisting of hydrogen, C1–C6-alkoxy, hydroxy, normal, cyclic or branched C1–C12-alkyl, and phenylalkyl.
Synthetic Methods
The compounds of the present invention can be prepared by known methods of peptide synthesis. Thus, the peptides can be assembled sequentially from individual amino acids or by linking suitable small peptide fragments. In sequential assembly, the peptide chain is extended stepwise, starting at the C-terminus, by one amino acid per step. In fragment coupling, fragments of different lengths can be linked together, and the fragments in turn can be obtained by sequential assembly from amino acids or by fragment coupling of still shorter peptides.
In both sequential assembly and fragment coupling it is necessary to link the units by forming an amide linkage, which can be accomplished via a variety of enzymatic and chemical methods. Chemical methods for forming the amide linkage are described in detail in standard references on peptide chemistry, including Müller, Methoden der organischen Chemie Vol. XV/2, 1–364, Thieme Verlag, Stuttgart, (1974); Stewart and Young, Solid Phase Peptide Synthesis, 31–34 and 71–82, Pierce Chemical Company, Rockford, Ill.(1984); Bodanszky et al., Peptide Synthesis, 85–128, John Wiley & Sons, New York, (1976). Preferred methods include the azide method, the symmetric and mixed anhydride method, the use of in situ generated or preformed active esters, the use of urethane protected N-carboxy anhydrides of amino acids and the formation of the amide linkage using coupling reagents, such as carboxylic acid activators, especially dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethoxycarbonyl-2- ethoxy-1,2-dihydroquinoline (EEDQ), pivaloyl chloride, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), n-propanephosphonic anhydride (PPA), N,N-bis(2-oxo-oxazolidinyl)amidophosphoryl chloride (BOP-Cl), bromo-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBrop), diphenyl-phosphoryl azide (DPPA), Castro's reagent (BOP, PyBop), O-benzotriazolyl-N,N,N′,N′-tetramethyluronium salts (HBTU), O-azabenzotriazolyl-N,N,N′,N′-tetramethyluronium salts (HATU), diethylphosphoryl cyanide (DEPCN), 2,5-diphenyl-2,3- dihydro-3-oxo-4-hydroxythiophene dioxide (Steglich's reagent; HOTDO), and 1,1′-carbonyldi-imidazole (CDI). The coupling reagents can be employed alone or in combination with additives such as N,N-dimethyl-4-aminopyridine (DMAP), N-hydroxy-benzotriazole (HOBt), N-hydroxyazabenzotriazole (HOAt), N-hydroxybenzotriazine (HOOBt), N-hydroxysuccinimide (HOSu) or 2-hydroxypyridine.
Although the use of protecting groups is generally not necessary in enzymatic peptide synthesis, reversible protection of reactive groups not involved in formation of the amide linkage is necessary for both reactants in chemical synthesis. Three conventional protective group techniques are preferred for chemical peptide synthesis: the benzyloxycarbonyl (Z), the t-butoxycarbonyl (Boc) and the 9-fluorenylmethoxy-carbonyl (Fmoc) techniques. Identified in each case is the protective group on the α-amino group of the chain-extending unit. A detailed review of amino-acid protective groups is given by Müller, Methoden der organischen Chemie Vol. XV/1, pp 20–906, Thieme Verlag, Stuttgart (1974). The units employed for assembling the peptide chain can be reacted in solution, in suspension or by a method similar to that described by Merrifield, J. Am. Chem. Soc. 85: (1963) 2149.
Solvents suitable for peptide synthesis include any solvent which is inert under the reaction conditions, especially water, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile, dichloromethane (DCM), 1,4-dioxane, tetrahydrofuran (THF), N-methyl-2-pyrrolidone (NMP) and mixtures of these solvents.
Peptide synthesis on the polymeric support can be carried out in a suitable inert organic solvent in which the amino acid derivatives starting materials are soluble. However, preferred solvents additionally have resin- swelling properties, such as DMF, DCM, NMP, acetonitrile and DMS0, and mixtures of these solvents. Following synthesis, the peptide is removed from the polymeric support. The conditions under which this cleavage is accomplished for various resin types are disclosed in the literature. The cleavage reactions most commonly used are acid- or palladium-catalyzed, the former being conducted in, for example, liquid anhydrous hydrogen fluoride, anhydrous trifluoromethanesulfonic acid, dilute or concentrated trifluoroacetic acid, and acetic acid/dichloromethane/trifluoroethanol mixtures. The latter can be carried out in THF or THF-DCM-mixtures in the presence of a weak base, such as morpholine. Certain protecting groups are also cleaved off under these conditions.
Partial deprotection of the peptide may also be necessary prior to certain derivatization reactions. For example, peptides dialkylated at the N-terminus can be prepared by coupling the appropriate N,N-di-alkylamino acid to the peptide in solution or on the polymeric support, by reductive alkylation of the resin-bound peptide in DMF/1% acetic acid with NaCNBH3 and the appropriate aldehyde or by hydrogenation of the peptide in solution in the presence of the appropriate aldehyde or ketone and Pd/carbon.
The various non-naturally occurring amino acids as well as the various non-amino acid moieties disclosed herein can be obtained from commercial sources or synthesized from commercially available staring materials using methods known in the art. For example, amino acid building blocks with R1 and R2 groups can be prepared according to the method described by Wuensch and Weyl, Methoden der Organischen Chemie, vol. XV, Springer Verlag: Stuttgart, p. 306 (1974) and references cited therein.
Methods of Use of the Claimed Compounds
In another embodiment, the present invention comprises a method for partially or totally inhibiting formation of, or otherwise treating (e.g., reversing or inhibiting the further development of) solid tumors (e.g., tumors of the lung, breast, colon, prostate, bladder, rectum, or endometrial tumors) or hematological malignancies (e.g., leukemias, lymphomas) in a mammal, for example, a human, by administering to the mammal a therapeutically effective amount of a compound or a combination of compounds of Formula I. The compound(s) may be administered alone or in a pharmaceutical composition comprising the compound(s) and an acceptable carrier or diluent. Administration can be by any of the means which are conventional for pharmaceutical, preferably oncological, agents, including oral and parenteral means, such as subcutaneously, intravenously, intramuscularly and intraperitoneally, nasally or rectally. The compounds may be administered alone or in the form of pharmaceutical compositions containing a compound or compounds of Formula I together with a pharmaceutically accepted carrier appropriate for the desired route of administration. Such pharmaceutical compositions may be combination products, i.e., they may also contain other therapeutically active ingredients.
The dosage to be administered to the mammal, such as a human, will contain a therapeutically effective amount of a compound described herein. As used herein, “therapeutically effective amount” is an amount sufficient to inhibit (partially or totally) formation of a tumor or a hematological malignancy or to reverse development of a solid tumor or other malignancy or prevent or reduce its further progression. For a particular condition or method of treatment, the dosage is determined empirically, using known methods, and will depend upon factors such as the biological activity of the particular compound employed; the means of administration; the age, health and body weight of the recipient; the nature and extent of the symptoms; the frequency of treatment; the administration of other therapies; and the effect desired. A typical daily dose will be from about 0.05 to about 50 milligrams per kilogram of body weight by oral administration and from about 0.01 to about 20 milligrams per kilogram of body weight by parenteral administration.
The compounds of the present invention can be administered in conventional solid or liquid pharmaceutical administration forms, for example, uncoated or (film-)coated tablets, capsules, powders, granules, suppositories or solutions. These are produced in a conventional manner. The active substances can for this purpose be processed with conventional pharmaceutical aids such as tablet binders, fillers, preservatives, tablet disintegrants, flow regulators, plasticizers, wetting agents, dispersants, emulsifiers, solvents, sustained release compositions, antioxidants and/or propellant gases (cf. H. Sücker et al.: Pharmazeutische Technologie, Thieme-Verlag, Stuttgart, 1978). The administration forms obtained in this way typically contain from about 1 to about 90% by weight of the active substance.
The present invention will now be illustrated by the following examples, which are not limiting.
EXAMPLES
The proteinogenous amino acids are abbreviated in the examples using the known three-letter code. Other abbreviations employed are: TFA=trifluoroacetic acid, Ac=acetic acid, DCM=dichloromethane, DMSO=dimethylsulfoxide, Bu=butyl, Et=ethyl, Me=methyl, Bzl=benzyl. In the compounds listed, all proteinogenous amino acids are L-amino acids unless otherwise noted. Other abbreviations used: Me2Val=N,N-dimethylvaline, MeVal=N-methylvaline, Bn=benzyl, Me2Aib=[2-N,N-dimethylamino]-isobutyric acid.
General Procedures
The peptides of the invention are synthesized either by classical solution synthesis using standard Z- and Boc-methodology as described above or by standard methods of solid-phase synthesis using Boc and Fmoc protective group techniques.
In the case of solid phase synthesis, the N,N-dialkyl-penta- or hexapeptide acids are liberated from the solid support and further coupled with the corresponding C-terminal amines in solution. BOP-Cl and PyBrop were used as reagents for coupling of the amino acid following the N-methylamino acids. The reaction times were correspondingly increased. For reductive alkylation of the N-terminus, the peptide-resin was deprotected at the N terminus and then reacted with a 3-fold molar excess of aldehyde or ketone in DMF/1% acetic acid with addition of 3 equivalents of NaCNBH3. After the reaction was complete (negative Kaiser test) the resin was washed several times with water, isopropanol, DMF and dichloromethane.
In solution synthesis, the use of either Boc-protected amino acid NCAs (N-tert-butyloxycarbonyl-amino acid-N-carboxy-anhydrides), Z-protected amino acid NCAs (N-benzyloxycarbonyl-amino acid-N-carboxy-anhydrides), or the use of pivaloyl chloride as condensing agent respectively is most advantageous for coupling of the amino acid following the N-methylamino acids. Reductive alkylation of the N terminus can e.g. be achieved by reaction of the N-terminally deprotected peptides or amino acids with the corresponding aldehydes or ketones using NaCNBH3 or hydrogen-Pd/C.
Valyl-N-methylvalyl-prolyl-prolylbenzylamide hydrochloride for example was prepared according to methods disclosed in German Patent Application No. DE 19527575 A1.
Purification and Characterization of the Peptides
Peptide purification was carried out by gel chromatography (SEPHADEX G-10, G-15/10% HOAc, SEPHADEX LH20/MeOH), medium pressure chromatography (stationary phase: HD-SIL C-18, 20–45 micron, 100 Angstrom; mobile phase: gradient with A=0.1% TFA/MeOH, B=0.1% TFA/water), preparative HPLC (stationary phase: Waters Delta-Pak C-18, 15 micron, 100 Angstrom; mobile phase: gradient with A=0.1% TFA/MeOH, B=0.1% TFA/water), or by crystallization.
The purity of the resulting products was determined by analytical HPLC (stationary phase: 100 2.1 mm VYDAC C-18, 5 micron, 300 A; mobile phase: acetonitrile-water gradient, buffered with 0.1% TFA, 40° C.; or 3.9 mm VYDAC C-18, 30° C.). Characterization was by fast atom bombardment mass spectroscopy and NMR-spectroscopy.
Example 1
Synthesis of [N-Methyl-L-piperidine-2-carbonyl]-Val-MeVal-Pro-Pro-NHBn (Compound 1) and [N-Methyl-D-piperidine-2-carbonyl]-Val-MeVal-Pro-Pro-NHBn (Compound 2)
Preparation of N-methyl-piperidine-2-carboxylic acid
N-Methyl-piperidine-2-carboxylic acid ethyl ester (5.1 g) was dissolved in a mixture of 100 ml methanol and 10 ml water. NaOH (8 g) was added and the reaction mixture was stirred at room temperature overnight. The solution was then neutralized with hydrochloric acid, evaporated to dryness, and evaporated four times with toluene. The resulting powdery residue was used directly in the next step.
Preparation of [N-Methyl-piperidine-2-carbonyl]-Val-MeVal-Pro-Pro-NHBn
The residue prepared as described above (5.05 g) and H-Val-MeVal-Pro-Pro-NHBn×HCl (4.88 g) were dissolved in 50 ml dry DMF. After cooling the solution in an ice bath, 1.52 g DEPCN and 2.66 ml triethylamine were added. The reaction mixture was stirred at 0° C. for 2 h and then at room temperature overnight. The DMF was removed by evaporation under reduced pressure. The residue was diluted with dichloromethane and the organic phase was washed with aqueous hydrochloric acid (pH 2) and water, dried over sodium sulfate and evaporated to dryness. The diastereomeric mixture was then separated by flash chromatography with a gradient using heptane/ethyl acetate and dichloromethane/methanol. Under the HPLC conditions described in the previous section (C-18 reverse phase) isomer 1 has a retention time of 14.9 minutes, and isomer 2 has a retention time of 15.8 minutes. Both isomers were characterized by fast atom bombardment mass spectrometry ([M+H]+=639).
Example 2
Preparation of Me2Aib-Val-MeVal-Pro-Pro-NHBn (Compound 3)
Preparation of 2-[N,N-dimethylamino]-isobutyric acid
2-Amino-isobutyric acid (10.3 g) was dissolved in 200 ml methanol. After addition of 25 ml aqueous formaldehyde and 1 g 10% Pd/C, the reaction mixture was hydrogenated overnight at room temperature. The catalyst was filtered, and the filtrate was evaporated to dryness. The residue was crystallized from isopropanol to give 4.8 g of the desired product.
Preparation of Me2Aib-Val-MeVal-Pro-Pro-NHBn×HCl
2-[N,N-Dimethylamino]-isobutyric acid (1.3 g, 10 mmol) and 5.5 g (10 mmol) H-Val-MeVal-Pro-Pro-NHBn×HCl were dissolved in 50 ml dry DMF. After cooling to 0° C., 1.6 g DEPCN (10 mmol) and 2.9 ml triethylamine were added to the reaction mixture. The resulting mixture was stirred at 0° C. for 2 h and at room temperature overnight. Ice water (50 mL) was then added, and the resulting mixture was extracted twice with diethyl ether. The ether extracts were washed with 1 N NaOH (1×) and aqueous NaCl (3×), then dried over sodium sulfate and evaporated to dryness under reduced pressure. The product was crystallized from 100 ml diethyl ether with HCl/ether, and recrystallized from acetone to give 1.2 g of the desired product, which was characterized by fast atom bombardment mass spectrometry ([M+H]+=627).
Example 3
Preparation of [N,N-dimethyl-2-ethyl-2-phenylglycyl]-Val-MeVal-Pro-Pro-NHBn×HCl (Compound 4)
Preparation of [N,N-dimethyl-2-ethyl-2-phenylglycyl]-Val-MeVal-Pro-Pro-NHBn×HCl
2.07 g (10 mmol) N,N-Dimethyl-2-ethyl-2-phenylglycine and 5.5 g (10 mmol) H-Val-MeVal-Pro-Pro-NHBn×HCl were dissolved in 100 ml dry DMF. After cooling to 0° C., 1.6 g DEPCN (10 mmol) and 2.9 ml triethylamine were added. The reaction mixture was stirred at 0° C. for 2 h and at room temperature overnight, then worked up as described above. The crude product was crystallized from diethyl ether with HCl/ether to give 4 g of the desired product, which was characterized by fast atom bombardment mass spectrometry ([M+H]+=703).
Example 4
Preparation of [N-Methyl-D-Pro]-Val-MeVal-Pro-Pro-NHBn (Compound 5)
Preparation of Z-D-Pro-Val-MeVal-Pro-Pro-NHBn
3.74 g Z-D-Pro-OH (15 mmol, BACHEM) and 8.25 g H-Val-MeVal-Pro-Pro-NHBn×HCl (15 mmol) were dissolved in 80 ml dry DMF. After cooling to 0° C., 2.4 g DEPCN (2.25 ml, 15 mmol) and 4.2 ml triethylamine (30 mmol) were added. The reaction mixture was stirred at 0° C. for several hours and room temperature overnight, then the DMF was evaporated under reduced pressure. The residue was diluted with ethyl acetate and thoroughly washed with dilute aqueous HCl (pH 2), water, dilute aqueous NaOH (pH 9–10), and water. The organic phase was dried over sodium sulfate and evaporated to dryness to yield 9.2 g of the desired protected pentapeptide.
Preparation of D-Pro-Val-MeVal-Pro-Pro-NHBn×HCl
8.2 g (11 mmol) Z-D-Pro-Val-MeVal-Pro-Pro-NHBn was dissolved in 70 ml methanol. After addition of 0.7 ml concentrated hydrochloric acid and 0.3 g 10% Palladium/charcoal to the solution, the resulting mixture was hydrogenated. Filtration and evaporation of the solvent gave a residue which was dissolved in water, adjusted to pH 2 and extracted twice with ethyl acetate. The aqueous phase was adjusted to pH 9–10 and extracted twice with dichloromethane. The organic extracts were evaporated and the residue was redissolved in diethylether and crystallized by addition of HCl/ether as the hydrochloride salt to give 6.5 g of the desired product.
Preparation of [N-methyl-D-Pro]-Val-MeVal-Pro-Pro-NHBn×HCl
1.94 g (3 mmol) of D-Pro-Val-MeVal-Pro-Pro-NHBn×HCl was dissolved in 30 ml methanol. To this solution was then added 0.3 g 10% Pd/charcoal and 1.5 ml aqueous formaldehyde solution and the reaction mixture was hydrogenated. Following filtration and evaporation of the solvents, the resulting residue was dissolved in water, adjusted to pH 2 and extracted twice with diethyl ether and several additional times with dichloromethane. The aqueous phase was adjusted to pH 9–10 and extracted twice with dichloromethane. The organic extracts were dried over sodium sulfate and evaporated to dryness. The residue was crystallized as the hydrochloride salt to give 0.5 g of the desired product which was characterized by fast atom bombardment mass spectrometry ([M+H]+=625).
The compounds in Table 1 were prepared according to the methods described in Examples 1–4. Where compounds are referred to as “isomer 1” or “isomer 2”, isomer 1 is the diastereomer with the shorter retention time on the reversed phase analytical HPLC system. Fast atom bombardment-mass spectrometry results for selected compounds are provided in Table 2.








TABLE 1





Compound No.
Compound







 6
Xah Val Xaa Pro Xab


 7
Xai Val Xaa Pro Xab


 8
Xae Val Xaa Pro Xab


 9
Xad Val Xaa Pro Xbr


 10
Xam Val Xaa Pro Xab


 11
Xaw Ile Xaa Pro Xbx


 12
Xao Val Xaa Pro Xab


 13
Xad Val Xaa Pro Xap


 14
Xaq Val Xaa Pro Xab


 15
Xar Val Xaa Pro Xab


 16
Xas Val Xaa Pro Xab


 17
Xat Val Xaa Pro Xab isomer 1


 18
Xat Val Xaa Pro Xab isomer 2


 19
Xaf Val Xaa Pro Xab


 20
Xav Val Xaa Pro Xab


 21
Xag Val Xaa Pro Xab


 22
Xax Val Xaa Pro Xab isomer 1


 23
Xax Val Xaa Pro Xab isomer 2


 24
Xay Val Xaa Pro Xab


 25
Xaz Val Xaa Pro Xab isomer 1


 26
Xaz Val Xaa Pro Xab isomer 2


 27
Xba Val Xaa Pro Xab


 28
Xbb Val Xaa Pro Xab


 29
Xbc Val Xaa Pro Xab


 30
Xbd Val Xaa Pro Xab isomer 1


 31
Xbd Val Xaa Pro Xab isomer 2


 32
Xbe Val Xaa Pro Xab isomer 1


 33
Xbe Val Xaa Pro Xab isomer 2


 34
Xbf Val Xaa Pro Xab isomer 1


 35
Xbg Val Xaa Pro Xab


 36
Xbh Val Xaa Pro Xab isomer 1


 37
Xbh Val Xaa Pro Xab isomer 2


 38
Xbi Val Xaa Pro Xab isomer 1


 39
Xbi Val Xaa Pro Xab isomer 2


 40
Xbk Val Xaa Pro Xab isomer 1


 41
Xbk Val Xaa Pro Xab isomer 2


 42
Xbl Val Xaa Pro Xab


 43
Xbf Val Xaa Pro Xab isomer 2


 44
Xbm Val Xaa Pro Xab


 45
Xaw Val Xaa Pro Xbn


 46
Xbo Val Xaa Pro Xbn isomer 1


 47
Xbo Val Xaa Pro Xbn isomer 2


 48
Xaw Val Xaa Pro Xbp


 49
Xbo Val Xaa Pro Xbp isomer 1


 50
Xbo Val Xaa Pro Xbp isomer 2


 51
Xaw Val Xaa Pro Xbq


 52
Xaw Val Xaa Pro Xbr


 53
Xbs Val Xaa Pro Xbt isomer 1


 54
Xbl Val Xaa Pro Xab isomer 1


 55
Xbl Val Xaa Pro Xab isomer 2


 56
Xbu Val Xaa Pro Xab isomer 1


 57
Xbv Val Xaa Pro Xab


 58
Xbw Val Xaa Pro Xab isomer 1


 59
Xbw Val Xaa Pro Xab isomer 2


 60
Xbs Val Xaa Pro Xbt isomer 2


 61
Xbu Val Xaa Pro Xab isomer 2


 62
Xbo Val Xaa Pro Xbr isomer 1


 63
Xbo Val Xaa Pro Xbr isomer 2


 64
Xbo Val Xaa Pro Xbq isomer 1


 65
Xbo Val Xaa Pro Xbq isomer 2


 66
Xaw Val Xaa Pro Xbx


 67
Xby Val Xaa Pro Xab


 68
Xbz Val Xaa Pro Xab


 69
Xca Val Xaa Pro Xab isomer 1


 70
Xca Val Xaa Pro Xab isomer 2


 71
Xbo Val Xaa Pro Xbx isomer 1


 72
Xbo Val Xaa Pro Xbx isomer 2


 73
Xau Val Xaa Pro Xbp


 74
Xau Val Xaa Pro Xbx


 75
Xbi Val Xaa Pro Xbx isomer 2


 76
Xau Val Xaa Pro Xab isomer 1


 77
Xau Val Xaa Pro Xab isomer 2


 78
Xau Val Xaa Pro Xcb


 79
Xbi Val Xaa Pro Xcb isomer 1


 80
Xbi Val Xaa Pro Xcb isomer 2


 81
Xbi Val Xaa Pro Xcc isomer 1


 82
Xbi Val Xaa Pro Xcc isomer 2


 83
Xbi Val Xaa Pro Xcd


 84
Xbk Val Xaa Pro Xcc isomer 1


 85
Xbk Val Xaa Pro Xcc isomer 2


 86
Xax Val Xaa Pro Xbp isomer 1


 87
Xax Val Xaa Pro Xbp isomer 2


 88
Xbk Val Xaa Pro Xcb isomer 1


 89
Xbk Val Xaa Pro Xcb isomer 2


 90
Xau Val Xaa Pro Xcc


 91
Xau Val Xaa Pro Xcd


 92
Xba Val Xaa Pro Xcb isomer 1


 93
Xba Val Xaa Pro Xcb isomer 2


 94
Xbo Val Xaa Pro Xbp isomer 1


 95
Xbo Val Xaa Pro Xbp isomer 2


 96
Xau Val Xaa Pro Xbp isomer 1


 97
Xau Val Xaa Pro Xbp isomer 2


 98
Xbi Val Xaa Pro Xcd isomer 2


 99
Xbk Val Xaa Pro Xcd


100
Xba Val Xaa Pro Xbp isomer 1


101
Xba Val Xaa Pro Xbp isomer 2


102
Xba Val Xaa Pro Xcc isomer 1


103
Xba Val Xaa Pro Xcc isomer 2


104
Xba Val Xaa Pro Xcd


105
Xce Val Xaa Pro Xab


106
Xcf Val Xaa Pro Xab


107
Xcg Val Xaa Pro Xab isomer 1


108
Xcg Val Xaa Pro Xab isomer 2


109
Xaw Val Xaa Pro Xch


110
Xaw Val Xaa Pro Xci


111
Xaw Val Xaa Pro Xck


112
Xaw Val Xaa Pro Xcl


113
Xaw Val Xaa Pro Xcm


114
Xaw Val Xaa Pro Xcn


115
Xaw Val Xaa Pro Xco


116
Xaw Val Xaa Pro Xcp


117
Xaw Val Xaa Pro Xcq


118
Xaw Val Xaa Pro Xcr


119
Xad Val Xaa Pro Xch


120
Xad Val Xaa Pro Xci


121
Xad Val Xaa Pro Xck


122
Xad Val Xaa Pro Xcl


123
Xad Val Xaa Pro Xcm


124
Xad Val Xaa Pro Xcn


125
Xad Val Xaa Pro Xco


126
Xad Val Xaa Pro Xcp


127
Xad Val Xaa Pro Xcq


128
Xad Val Xaa Pro Xcr


129
Xad Val Xaa Pro Xbx


130
Xau Val Xaa Pro Xch


131
Xau Val Xaa Pro Xci


132
Xau Val Xaa Pro Xck


133
Xau Val Xaa Pro Xcl


134
Xau Val Xaa Pro Xcm


135
Xau Val Xaa Pro Xcn


136
Xau Val Xaa Pro Xco


137
Xau Val Xaa Pro Xcp


138
Xau Val Xaa Pro Xcq


139
Xau Val Xaa Pro Xcr


140
Xau Val Xaa Pro Xbr


141
Xad Val Xaa Xal Xbx


142
Xau Val Xaa Xal Xbx


143
Xaw Val Xaa Xal Xbx


144
Xad Val Xaa Xal Xch


145
Xau Val Xaa Xal Xch


146
Xaw Val Xaa Xal Xch


147
Xad Val Xaa Xal Xcr


148
Xau Val Xaa Xal Xcr


149
Xaw Val Xaa Xal Xcr


150
Xad Val Xaa Xan Xbx


151
Xau Val Xaa Xan Xbx


152
Xaw Val Xaa Xan Xbx


153
Xad Val Xaa Xan Xch


154
Xau Val Xaa Xan Xch


155
Xaw Val Xaa Xan Xch


156
Xad Val Xaa Xan Xcr


157
Xau Val Xaa Xan Xcr


158
Xaw Val Xaa Xan Xcr


159
Xau Ile Xaa Pro Xbx


160
Xad Ile Xaa Pro Xbx


161
Xaw Ile Xaa Pro Xch


162
Xad Ile Xaa Pro Xch


163
Xau Ile Xaa Pro Xch


164
Xaw Xcs Xaa Pro Xch


165
Xad Xcs Xaa Pro Xch


166
Xau Xcs Xaa Pro Xch


167
Xaw Xcs Xaa Pro Xbx


168
Xad Xcs Xaa Pro Xbx


169
Xau Xcs Xaa Pro Xbx


170
Xaw Val Xct Pro Xch


171
Xad Val Xct Pro Xch


172
Xau Val Xct Pro Xch


173
Xaw Val Xct Pro Xbx


174
Xad Val Xct Pro Xbx


175
Xau Val Xct Pro Xbx









The symbols Xaa in Table 1 represent the following amino acids or residues thereof:













Xaa:
N-methyl-valine


Xab:
Prolyl N-benzylamide


Xac:
L-N-methyl-piperidine-2-carboxylic acid


Xad:
D-N-methyl-piperidine-2-carboxylic acid


Xae:
N-methyl-L-proline


Xaf:
N-methyl-L-thiazolidine-4-carboxylic acid


Xag:
N,N-dimethylglycine


Xah:
L-proline


Xai:
L-piperidine-2-carboxylic acid


Xak:
2-[N,N-dimethylamino]-isobutyric acid


Xal:
L-thiazolidine-4-carboxylic acid


Xam:
N-propyl-D-piperidine-2-carboxylic acid


Xan:
L-3,4-didehydroproline


Xao:
D-piperidine-2-carboxylic acid


Xap:
proline tert.butylester


Xaq:
N-ethyl-D-piperidine-2-carboxylic acid


Xar:
N-methyl-[2,2,5,5-tetramethyl]-L-thiazolidine-2-carboxylic acid


Xas:
N-isopropyl-D-piperidine-2-carboxylic acid


Xat:
N,N-dimethyl-2-cyclopropyl-glycine


Xau:
N,N-dimethyl-2-ethyl-2-phenyl-glycine


Xav:
D-proline


Xaw:
N-methyl-D-proline


Xax:
N,N-dimethyl-2-[2-fluoro]phenyl-glycine


Xay:
1-aza-[3,3,0]bicyclooctyl-5-carboxylic acid


Xaz:
N,N-dimethyl-2-[4-fluoro]phenyl-glycine


Xba:
N-methyl-[2,2,5,5-tetramethyl]-thiazolidine-2-carboxylic acid


Xbb:
2-(R,S)-ethyl-2-phenyl-glycine


Xbc:
D,L-1-aminoindane-1-carboxylic acid


Xbd:
N,N-dimethyl-2-(R,S)-methyl-2-phenyl-glycine


Xbe:
2-[N,N-dimethylamino]indane-2-carboxylic acid


Xbf:
5-[N,N-dimethylamino]-5,6,7,8-tetrahydro-naphthalene-5-



carboxylic acid


Xbg:
N-isopropyl-2-(R,S)-ethyl-2-phenyl-glycine


Xbh:
1-[N,N-dimethylamino]indane-2-carboxylic acid


Xbi:
N,N-dimethyl-2-propyl-2-phenyl-glycine


Xbk:
N,N-dimethyl-2-[4-methoxy]phenyl-glycine


Xbl:
N-methyl-3-hydroxy-D,L-valine


Xbm:
N,N-dimethyl-D,L-2-isopropyl-2-phenyl-glycine


Xbn:
proline-N-methoxy-N-methyl-amide


Xbo:
N-methyl-piperidine-2-carboxylic acid


Xbp:
proline-isopropylamide


Xbq:
proline-isoxazolidinyl


Xbr:
proline-N-methoxy-N-benzylamide


Xbs:
N-methyl-D,L-proline


Xbt:
proline-[5-phenyl]isoxazolidinyl


Xbu:
N-methyl-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid


Xbv:
N-methyl-azetidine-2-carboxylic acid


Xbw:
N-isopropyl-azetidine-2-carboxylic acid


Xbx:
proline-tert-butylamide


Xby:
N,N-dimethyl-[O-methyl]serine


Xbz:
N,N-dimethyl-[O-methyl]threonine


Xca:
N-methyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid


Xcb:
proline-pentyl(3)amide


Xcc:
proline-(R)-phenethylamide


Xcd:
proline-(S)-phenethylamide


Xce:
1-[N,N-dimethylamino]cyclohexyl-1-carboxylic acid


Xcf:
1-[N,N-dimethylamino]cyclopentyl-1-carboxylic acid


Xcg:
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid


 


Xch:






 


Xci:






 


Xck:






 


Xcl:






 


Xcm:






 


Xcn:






 


Xco:






 


Xcp:






 


Xcq:






 


Xcr:






 


Xcs:
L-2-tert-butyl-glycine


Xct:
N-methyl-L-Isoleucine
















TABLE 2







Results of FAB-MS analysis of selected compounds










Compound No.
Mol. weight measured







 1
639



 2
639



 3
627



 4
703



 5
625



 6
611



 7
625



 8
625



10
667



12
625



13
606



14
653



15
699



16
667



17
639



18
639



19
643



20
611



21
599



22
693



23
693



24
651



25
693



26
693



27
699



28
675



29
673



30
689



31
689



32
701



33
701



34
715



35
717



36
701



37
701



38
717



39
717



40
705



41
705



42
643



43
715



44
703



45
579



46
593



47
593



48
577



49
591



50
591



51
591



52
655



53
667



54
657



55
657



56
687



57
611



58
639



59
639



60
667



61
687



62
669



63
669



64
605



65
605



66
591



67
643



68
657



69
687



70
687



71
605



72
605



73
655



74
669



75
683



76
703



77
703



78
683



79
697



80
697



81
731



82
731



83
731



84
719



85
719



86
645



87
645



88
685



89
685



90
717



91
717



92
679



93
679



94
591



95
591



96
655



97
655



98
731



99
719



100 
651



101 
651



102 
713



103 
713



104 
713



105 
666



106 
653



107 
687



108 
687










Example 5
Evaluation of Biological Activity
In vitro Methodology
Cytotoxicity was measured using a standard methodology for adherent cell lines, such as the microculture tetrazolium assay (MTT). Details of this assay have been published (Alley, M. C. et al., Cancer Research 48: 589–601, (1988)). Exponentially growing cultures of HT-29 colon carcinoma cells were used to make microtiter plate cultures. Cells were seeded at 5000–20,000 cells per well in 96-well plates (in 150 mL of media), and grown overnight at 37° C. Test compounds were added, in 10-fold dilutions varying from 10−4 M to 10−10 M. Cells were then incubated for 48 hours. To determine the number of viable cells in each well, the MTT dye was added (50 mL of a 3 mg/mL solution of 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide in saline). This mixture was incubated at 37° C. for 5 hours, and then 50 mL of 25% SDS, pH 2, was added to each well. After an overnight incubation, the absorbance of each well at 550 nm was read using an ELISA reader. The values for the mean +/− SD of data from replicated wells were calculated, using the formula % T/C (% viable cells treated/control). The concentration of test compound which gives a T/C of 50% growth inhibition was designated as the IC50.
Table 3 presents the IC50 values determined in the HT-29 assay for a series of compounds of the invention.








TABLE 3





Compound No.
HT-29 [IC50]







 1
4.7 × 10−8 


 2
6.8 × 10−10


 3
3.5 × 10−8 


 4
1.2 × 10−9 


 5
5.0 × 10−9 


 8
5.1 × 10−7 


10
1.3 × 10−7 


12
3.7 × 10−7 


13
1.0 × 10−9 


14
1.5 × 10−9 


15
1.7 × 10−7 


16
7.3 × 10−10


17
6.3 × 10−8 


18
8.8 × 10−9 


22
6.4 × 10−7 


24
2.8 × 10−8 


27
3.7 × 10−8 


28
4.9 × 10−8 


29
3.6 × 10−8 


30
6.1 × 10−9 


31
2.0 × 10−7 


32
8.5 × 10−7 


33
1.2 × 10−6 


34
5.0 × 10−9 


35
1.4 × 10−7 


36
6.2 × 10−9 


37
1.9 × 10−7 


38
7.3 × 10−7 


39
2.5 × 10−8 


40
5.6 × 10−7 


41
7.3 × 10−6 


42
3.4 × 10−7 


43
5.9 × 10−8 


44
4.8 × 10−8 


45
5.6 × 10−8 


46
7.2 × 10−7 


47
2.3 × 10−8 


48
2.5 × 10−8 


49
8.8 × 10−8 


50
8.9 × 10−8 


51
4.6 × 10−8 


52
3.4 × 10−7 


53
5.0 × 10−9 


54
4.2 × 10−9 


55
5.6 × 10−8 


57
2.5 × 10−8 


58
6.3 × 10−8 


59
1.9 × 10−7 


60
1.8 × 10−9 


62
9.9 × 10−8 


63
5.6 × 10−8 


64
1.7 × 10−6 


65
9.7 × 10−8 


66
3.4 × 10−7 


67
3.4 × 10−7 


68
4.2 × 10−7 


70
7.1 × 10−6 


72
1.2 × 10−7 


73
1.4 × 10−9 


74
5.1 × 10−8 


75
8.5 × 10−7 


76
2.3 × 10−10


77
7.2 × 10−9 


78
4.3 × 10−9 


79
1.7 × 10−6 


80
6.7 × 10−8 


81
1.3 × 10−7 


82
1.1 × 10−8 


83
1.3 × 10−7 


84
1.2 × 10−6 


85
9.5 × 10−6 


90
9.3 × 10−10


91
8.3 × 10−10


92
1.5 × 10−6 


93
1.8 × 10−6 


94
3.0 × 10−6 


95
1.1 × 10−8 


96
1.7 × 10−9 


97
3.2 × 10−8 


98
6.0 × 10−9 


99
3.8 × 10−6 


100 
2.3 × 10−6 


101 
2.1 × 10−6 


102 
1.2 × 10−7 


103 
1.1 × 10−7 


104 
3.5 × 10−6 


105 
1.8 × 10−8 


106 
9.7 × 10−8 


108 
7.1 × 10−6 









In vivo Methodology

Compounds of this invention may be further tested in any of the various preclinical assays for in vivo activity which are indicative of clinical utility. Such assays are conducted with nude mice into which tumor tissue, preferably of human origin, has been transplanted (“xenografted”), as is well known in this field. Test compounds are evaluated for their anti-tumor efficacy following administration to the xenograft-bearing mice.
More specifically, human tumors grown in athymic nude mice can be transplanted into new recipient animals, using tumor fragments which are about 50 mg in size. The day of transplantation is designated as day 0. Six to ten days later, the mice are treated with the test compounds given as an intravenous or intraperitoneal injection, in groups of 5–10 mice at each dose. Compounds are given daily for 5 days, 10 days or 15 days, at doses from 10–100 mg/kg body weight. Tumor diameters and body weights are measured twice weekly. Tumor masses are calculated using the diameters measured with Vernier calipers, and the formula:

(length×width2)/2=mg of tumor weight

Mean tumor weights are then calculated for each treatment group, and T/C values are determined for each group relative to the untreated control tumors.

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed in the scope of the following claims.

## Claims

1. A compound of the formula


A-B-D-E-F-(G)r-(K)s-L,

wherein
r and s are each independently, 0 or 1;
A is a proline derivative of Formula IIa,




 wherein na is 0 to 3; Ra is hydrogen, or unsubstituted or fluorine-substituted normal, branched or cyclic C1–C3-alkyl; R1a is hydrogen, C1–C3-alkyl, phenyl, or substituted phenyl; or Ra and R1a together form a propylene bridge; and R2a, R3a, R4a and R5a are each, independently, hydrogen or alkyl; or
an α-amino acid derivative of Formula IIIa,




 wherein Ra is hydrogen or unsubstituted or fluorine-substituted C1–C3-alkyl; R1a a is C1–C4-alkyl; R6a is alkyl, substituted alkyl, alkenyl, phenyl or substituted phenyl; or R1a is an alkyl group and R6a is C1–C6-alkyl, cycloalkylmethyl, benzyl or substituted benzyl; and R7a is hydrogen or alkyl; or
an α-amino acid derivative of Formula IVa,




 wherein ma is 1 or 2; R7a is hydrogen or alkyl; Ra is hydrogen, or unsubstituted or fluorine-substituted alkyl; or
an α-amino acid derivative of Formula Va,




 wherein R7a is hydrogen or alkyl and Ra is hydrogen, or unsubstituted or fluorine-substituted alkyl; or
an α-amino acid of Formula Via,




 wherein Ra is hydrogen, or unsubstituted or fluorine-substituted alkyl; R1a is hydrogen, alkyl, phenyl, or substituted phenyl; or Ra and R1a together form a propylene bridge; and Xa is hydroxy, alkoxy or fluorine; or
an α-amino acid of Formula VIIa,




 wherein Ra is hydrogen, or unsubstituted or fluorine-substituted alkyl; R1a is hydrogen, alkyl, phenyl, or substituted phenyl; or Ra and R1a together form a propylene bridge; and R2a, R3a, R4a and R5a are each, independently, hydrogen or alkyl; or
an α-amino acid residue of Formula VIIIa,




 wherein Ra is hydrogen, or unsubstituted or fluorine-substituted alkyl; or
a 2-azabicyclo[2.2.1]heptane-3-carboxylic acid derivative of Formula IXa,




 wherein the 3-carbonyl moiety is in the endo or exo position, Za is a single bond or a double bond, and Ra is hydrogen or unsubstituted or fluorine-substituted alkyl; or
an α-amino acid residue of Formula Xa,




 wherein na is 1, 2 or 3, and R7a is hydrogen or alkyl and Ra is hydrogen, unsubstituted alkyl or fluorine-substituted alkyl;
B is a valyl, isoleucyl, allo-isoleucyl, norvalyl, 2-tert-butylglycyl or 2-ethylglycyl residue; or
an α-amino acid residue of Formula IIb,




 wherein R1b is hydrogen, and R2b is alkyl or alkenyl; or R1b and R2b together form an isopropylidene group;
D is an N-alkylvalyl, N-alkyl-2-ethylglycyl, N-alkyl-2-tert-butylglycyl, N-alkylnorleucyl, N-alkylisoleucyl, N-alkyl-allo-isoleucyl or N-alkylnorvalyl residue; or
an α-amino acid residue of Formula IId,




 wherein Rd is hydrogen, or unsubstituted or fluorine-substituted alkyl; R1d is hydrogen; and R2d is alkyl, substituted alkyl or alkenyl; or R1d and R2d together form an isopropylidene group; or
an α-amino acid residue of Formula IIId,




 wherein nd is 1 or 2; R3d is hydrogen, alkyl or fluorine-substituted alkyl; and Xd is hydrogen; or nd is 1 and Xd is fluorine, hydroxy, methoxy, or ethoxy;
E is a prolyl, thiazolidinyl-4-carbonyl, homoprolyl, or hydroxyprolyl residue; or
an α-amino acid residue of Formula IIe,




 wherein ne is 0, 1 or 2, R1e is hydrogen, or unsubstituted or fluorine-substituted alkyl; R2e and R3e are each, independently, hydrogen or alkyl; R4e is hydrogen, hydroxy or alkoxy; and R5e is hydrogen or fluorine; or ne is 1 and R3e and R4e together form a double bond; or ne is 1 and R4e and R5e together form a double-bonded oxygen diradical; or ne is 1 or 2 and R1e and R2e together form a double bond; or
an aminocyclopentanecarboxylic acid residue of Formula IIIe,




 wherein Re is alkyl and R1e is hydrogen, or unsubstituted or fluorine-substituted alkyl;
F is a prolyl, thiazolidinyl-4-carbonyl, homoprolyl or hydroxyprolyl residue; or
an α-amino acid residue of Formula IIf,




 wherein nf is 0, 1 or 2, R1f is hydrogen, or unsubstituted or fluorine-substituted alkyl; R2f and R3f are each, independently, hydrogen or methyl; R4f is hydrogen, hydroxy, alkoxy, or fluorine; R5f is hydrogen or fluorine; or nf is 1 and R3f and R4f together form a double bond; or nf is 1 and R4f and R5f together form a double-bonded oxygen diradical; or nf is 1 or 2 and R1f and R2f together form a double bond; or
a 2- or 3-aminocyclopentanecarboxylic acid residue of Formula IIIf,




 wherein Rf is alkyl and R1f is hydrogen, or unsubstituted or fluorine-substituted alkyl; or an N-alkylglycyl or N-alkylalanyl residue;
G is an α-amino acid residue of Formula IIg,




 wherein R1g is hydrogen or alkyl and R2g is hydrogen, alkyl, arylalkyl, heteroarylalkyl, phenyl or substituted phenyl; or R1g and R2g, together with the α-carbon atom, form a C5–C6 ring or a benzo-fused C5 ring;
K is an α-amino acid of Formula IIk,




 wherein R1k is hydrogen or alkyl; and R2k is hydrogen, alky, arylakyl, heteroarylalkyl, phenyl or substituted phenyl; or R1g and R2g, together with the α-carbon atom, form a cyclopentane ring or a benzo-fused cyclopentane ring; and
L is a substituted or unsubstituted amino, hydrazido, aminoxy or oximato group provided that when s and r are each 0, and A is formula IIa where na is 0 or 1 R1a, R2a, R3a, R4a, and R5a are each hydrogen, the L is a substituted or unsubstituted amino or hydrazido group.