EP1305299A1 - Composes de polyamine cyclique pour la cancerotherapie - Google Patents

Composes de polyamine cyclique pour la cancerotherapie

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Publication number
EP1305299A1
EP1305299A1 EP01956110A EP01956110A EP1305299A1 EP 1305299 A1 EP1305299 A1 EP 1305299A1 EP 01956110 A EP01956110 A EP 01956110A EP 01956110 A EP01956110 A EP 01956110A EP 1305299 A1 EP1305299 A1 EP 1305299A1
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European Patent Office
Prior art keywords
alkyl
compound
group
independently selected
formula
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP01956110A
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German (de)
English (en)
Inventor
Benjamin Frydman
Manfred Hesse
Armin Guggisberg
Kasmin Popaj
Konstantin Drandarov
Hirak Basu
Subhra Bhattacharya
Yu Wang
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SLIL Biomedical Corp
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SLIL Biomedical Corp
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Publication of EP1305299A1 publication Critical patent/EP1305299A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D255/00—Heterocyclic compounds containing rings having three nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D249/00 - C07D253/00
    • C07D255/02—Heterocyclic compounds containing rings having three nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D249/00 - C07D253/00 not condensed with other rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
    • C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings

Definitions

  • This invention is directed to compounds and methods useful for treating cancer and other diseases caused by uncontrolled cell proliferation. More specifically, this invention is directed to cyclic polyamine compounds which display anti-tumor activity in vitro, as well as methods of making and using those compounds.
  • Cancer is one of the leading causes of death in the developed world. Approximately one-quarter of the deaths in the United States in 1997 were due to cancer, making it the second most common cause of death after heart disease. Accordingly, development of new and effective treatments for cancer is a high priority for health care researchers.
  • Cancer is often treated by using chemotherapy to selectively kill or hinder the growth of cancer cells, while having a less deleterious effect on normal cells.
  • Chemotherapeutic agents often kill rapidly dividing cells, such as cancer cells; cells which are dividing less rapidly are affected to a lesser degree.
  • Other agents, such as antibodies attached to toxic agents, have been evaluated for use against cancers. These agents target the cancer cells by making use of a characteristic specific to the cancer, for example, higher-than-normal rates of cell division, or unique antigens expressed on the cancer cell surface.
  • One peculiar distinguishing characteristic of malignant cells is their high rate of glycolysis, even in the presence of oxygen (so-called aerobic glycolysis, or the Warburg effect).
  • glucose transporter GLUT1 The expression of the glucose transporter GLUT1 is also increased in cancer cells. See Grover-McKay, M. et al, "Role for glucose transporter 1 protein in human breast cancer," Pathol. Oncol. Res. (1998)
  • Glucose utilization through the glycolytic pathway in cancer cells leads to pyruvate formation as is the case in normal cells, but in the absence of oxygen, pyruvate is not metabolized through the tricarboxylic cycle. This deprives the cancer cells of the efficient production of ATP by oxidative phosphorylation. In cancer cells pyruvate is reduced (by NADPH) to lactate, leading to the acidic environment of tumors. The cytosolic pH of tumor cells, however, is maintained as it is in normal cells. See Dang, CN. and Semenza, G.L., "Oncogenic alterations of metabolism," Trends Biochem. Sci. (1999) 24(2):68-72.
  • hypoxia is a strong selective force, and it regulates glycolysis by modulating oncogenes and tumor suppressing genes.
  • Tumor angiogenesis is stimulated by hypoxia and hypoglycemia, which induce expression of angiogenic factors that recruit microvessels to allow delivery of nutrients and oxygen, to support expansion of the tumor mass.
  • Angiostatin binds ATP synthase on the surface of human endothelial cells
  • Proc. ⁇ atl. Acad. Sci. USA (1999) 96(6):2811-6 the new microvessels are limited and disorganized, and the oxygen consumption rate exceeds the supply rate.
  • Glucose deprivation is a potent inducer of necrosis in transformed cells, and physiological and oncogenic transcription factors that stimulate glycolysis by increasing glucose transport as well as the activity of key glycolytic enzymes (e.g., hexokinase II, lactate dehydrogenase A) play a crucial role in promoting the survival of cancer cells in adverse tumor microenvironments.
  • key glycolytic enzymes e.g., hexokinase II, lactate dehydrogenase A
  • Cancer cells thus depend mainly on the glycolytic pathway to generate the necessary ATP to grow, even in the presence of oxygen. It is known that the energy provided by one mole of ATP is needed to produce 10 g of cells. While the aerobic oxidation of one mole of glucose to carbon dioxide results in a net gain of ca. 38 moles of ATP, the anaerobic (glycolytic) transformation of 1 mole of glucose into pyruvate and lactate only results in the gain of 2 moles of ATP, 19 times less than in aerobic oxidation. It is clear that ATP is very much at a premium in cancer cells due to the incomplete oxidation of glucose in the cells, which in turn requires an elevated rate of glycolysis in tumor cells.
  • Cyclic polyamines have been observed to have surprising effects on ATP hydrolysis. Cyclic polyamines, when protonated, bind ATP, ADP, and AMP stably and enhance the rate of hydrolysis of ATP by several orders of magnitude over a wide pH range. Linear polyamines, which do not bind ATP, do not increase the rate of hydrolysis. Hydrolysis catalyzed by a cyclic compound yields orthophosphate and ADP as products; the ADP is then hydrolyzed slowly to AMP. In the cleavage of ATP, the formation of an intermediate phosphoramidate was detected and the possible form of an initial "perched" complex and a mechanism of hydrolysis were postulated. See Merthes, M.P.
  • ATPases The hydrolysis of ATP involved an exchange of oxygen at the beta-phosphate of ATP and occurred in the presence of calcium. Under these conditions, subsequent hydrolysis of ADP was decreased and the phosphorylated cyclic compound accumulated. When this reaction mixture was adjusted to pH
  • pyrophosphate was formed.
  • the cyclic phosphoramidate was shown to be capable of phosphorylating ADP to give ATP.
  • phosphatase activity of the cyclic polyamines was also studied using other biological phosphate esters. It was shown that a cyclic polyamine catalyst cleaved acetyl phosphate to orthophosphate; the reaction then proceeded to the synthesis of pyrophosphate. It has been observed that a cyclic polyamine could activate formate in an ATP-dependent reaction in the presence of Ca++ or
  • cyclic polyamine alkaloids also called macrocyclic aminolactams
  • spermine-derived alkaloids are homaline and the mixture of alkaloids called pithecolobines. The latter were isolated from extracts of Pithecolobium saman
  • the cyclic polyamines of the present invention represent a new approach to cancery therapy. Additionally, no syntheses of the budmunchiamine compounds have been reported, and work with budmunchiamines has typically been performed with plant extracts containing a mixture of compounds.
  • the present invention provides methods that allow the synthesis of individual compounds similar to the structures proposed for the pithecolobines and the budmunchiamines, in order to design new compounds with ATP-ase-like activity in vivo and permit study of the isolated compounds. Such new compounds were created with the methods of the present invention for use in treating cancer and other pathological conditions.
  • the invention provides compounds and compositions for treating diseases caused by uncontrolled proliferation of cells, such as cancer, especially prostate cancer, and for inducing intracellular ATP hydrolysis for treatment of other disorders. [0016] In one embodiment, the invention provides compounds of the
  • each A 2 (if present), and A 3 are independently selected from Ci- C 8 alkyl; where each Y is independently selected from H or C 1 -C 4 alkyl; where M is selected from C ⁇ -C 4 alkyl; where k is 0, 2, or 3; and where R is selected from C1-C32 alkyl; and all stereoisomers and salts thereof.
  • the Y group is -H or -CH 3 .
  • a l5 each A 2 (if present), and A 3 are independently selected from C 2 -C 4 alkyl.
  • M is -CH 2 -.
  • the invention also includes compositions of one or more of the compounds above in combination with a pharmaceutically-acceptable carrier. [0017] The invention also provides compounds of the formula:
  • a t and A 3 are independently selected from -Cs alkyl; where A 2 is independently selected from -C 3 alkyl or C 5 -C 8 alkyl; where each Y is independently selected from H or C1-C 4 alkyl; where M is selected from C1-C 4 alkyl; and where R is selected from C ⁇ -C 32 alkyl; and all stereoisomers and salts thereof.
  • the Y group is -H or -CH 3 .
  • a t and A 3 are independently selected from C2-C 4 alkyl, and A 2 is selected from the group consisting of C 2 -C 3 alkyl and C 5 alkyl.
  • M is -CH 2 -.
  • the invention also includes compositions of one or more of the compounds above in combination with a pharmaceutically-acceptable carrier. [0018] The invention also provides compounds of the formula:
  • Ai and A 3 are independently selected from -Cs alkyl; where A 2 is independently selected from -Cs alkyl; where each Y is independently selected from H or C 2 - alkyl; where M is selected from C ⁇ -C 4 alkyl; and where R is selected from C 1 -C 32 alkyl; and all stereoisomers and salts thereof.
  • each Y group is -H.
  • a t and A 3 are independently selected from C 2 - alkyl, and A 2 is selected from the group consisting of C 2 -C 5 alkyl.
  • M is -CH2-.
  • the invention also includes compositions of one or more of the compounds above in combination with a pharmaceutically-acceptable carrier.
  • the invention also provides a method of synthesizing a compound of the formula
  • Ai each A 2 (if present), and A 3 are independently selected from - C 8 alkyl; where each Y is independently selected from H or C 1 -C 4 alkyl; where M is selected from C C 4 alkyl; where k is 0, 1, 2, or 3; and where R is selected from C ⁇ -C 32 alkyl; where the method comprises the steps of reacting an ⁇ -halo alkyl alkanoate with an aldehyde or ketone-containing compound to give an alkene- containing alkanoate compound; reacting the alkene-containing alkanoate compound with a compound containing two primary amino groups and optionally containing secondary amino groups to effect addition of one of the amino groups across the double bond; cyclizing the other amino group with the alkanoate group to form an amide bond; and optionally alkylating the secondary amino groups if present.
  • the ⁇ -halo alkyl alkanoate is ethyl bromoacetate.
  • the aldehyde or ketone-containing compound is an aldehyde-containing compound.
  • the step of reacting an ⁇ -halo alkyl alkanoate with an aldehyde or ketone-containing compound to give an alkene-containing alkanoate compound is performed by reacting the ⁇ - halo alkyl alkanoate with triphenylphosphine.
  • the compound containing two primary amino groups is selected from the group consisting of H 2 N-A 1 -(NH-A 2 ) k -NH-A 3 -NH 2 where A ls each A 2 (if present), and A 3 are independently selected from -Cs alkyl and k is 0, 1, 2, or 3.
  • the compound containing two primary amino groups can be selected from the group consisting of spermine, spermidine, and putrescine in still another embodiment.
  • the step of cyclizing the other amino group with the alkyl alkanoate group to form an amide bond can be performed by reacting the compound with antimony (III) ethoxide in yet another embodiment.
  • the step of optionally alkylating any secondary amino groups can be performed by reacting the compound first with an aliphatic aldehyde to result in a Schiff base, then reducing the Schiff base, resulting in alkylation of the secondary amino groups; the step of reducing the Schiff base can be performed by using the reagent NaCNBH 3 .
  • the invention also provides a method of synthesizing a compound of the formula:
  • At is C 3 alkyl, and each A 2 (if present) and A 3 are independently selected from -Cs alkyl; where each Y is independently selected from H or d- C 4 alkyl; where M is selected from C 1 -C 4 alkyl; where k is 0, 1, 2, or 3; and where R is selected from C 1 -C 32 alkyl; where the method comprises the steps of condensing a compound comprising a primary amino group and a hexahydropyrimidine moiety with an ⁇ , ⁇ -unsaturated ester compound, such that the primary amino group adds at the ⁇ -position of the unsaturated ester compound, whereby the primary amino group is converted to a secondary amino group; cleaving the methylene bridge of the hexahydropyrimidine moiety to generate a secondary amino group and a newly-generated primary amino group; and condensing the newly-generated primary amino group with the ester group to form an amide group.
  • the compound comprising a primary amino group and a hexahydropyrimidine moiety is of the formula
  • each A 2 (if present) and A 3 are independently selected from -Cs alkyl; where each Y is independently selected from H or C 1 -C 4 alkyl; and where j is 0, 1, 2, or 3. In a preferred embodiment, j is 0. In another preferred embodiment, A 3 is C 4 alkyl.
  • the step of cleaving the methylene bridge of the hexahydropyrimidine moiety can be performed with anhydrous HC1 in an alcoholic solvent.
  • the step of condensing the newly-generated primary amino group with the ester group to form an amide group can be performed with the reagent B(N(CH 3 ) 2 ) 3 .
  • t is a nonentity.
  • X is -Z, and -Z is -H.
  • Y is -CH 3 .
  • M is -CH 2 -.
  • k is 1.
  • A] and A 3 are -CH 2 CH 2 CH 2 -.
  • R is -C 0 H 27 .
  • one or more of the specific limitations on A 4 , X, Z, Y, M, k, A l5 A 3 , and R are combined.
  • a 4 is -Cs alkyl
  • X is -NHZ
  • Z is selected from one of the 20 genetically encoded amino acids (alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine), a peptide of the formula acetyl-SKLQL-, a peptide of the formula acetyl-SKLQ- ⁇ -alanine-, or a peptide of the formula acetyl-SKLQ-.
  • amino acids alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, methionine, asparagine, proline, glut
  • the invention also provides methods of synthesizing compounds of the formula
  • each A 2 (if present), and A 3 are independently selected from Ci- C 8 alkyl; where each Y is independently selected from H or C J. -C 4 alkyl; where M is selected from C 1 -C 4 alkyl; where k is 0, 1, 2, or 3; and where R is selected from -C 32 alkyl; and all stereoisomers and salts thereof.
  • Lithium aluminum hydride may be used as the reducing agent.
  • Diborane may also be used as the reducing agent.
  • the invention also provides a method of synthesizing a compound of the formula
  • each A 2 (if present), and A 3 are independently selected from Ci- C 8 alkyl; where each Y is independently selected from C1-C4 alkyl; where M is selected from C 1 -C 4 alkyl; where k is 0, 1, 2, or 3; and where R is selected from C 1 -C 32 alkyl, comprising reacting a compound of the formula
  • the invention also provides a method of synthesizing compounds of the formula
  • each A 2 (if present), and A3 are independently selected from Cj;- C 8 alkyl; where each Y is independently selected from C 1 -C4 alkyl; where M is selected from C 1 -C 4 alkyl; where k is 0, 1, 2, or 3; and where R is selected from Cj-C 32 alkyl, by reducing the nitrile group of a compound of the formula
  • a preferred reducing reagent is gaseous hydrogen (H 2 ) over Raney nickel.
  • the invention also provides methods of treating diseases characterized by uncontrolled cell proliferation, such as cancer, especially prostate cancer, by administration of one or more of the compounds described above.
  • the invention also provides methods of depleting ATP, particularly in a cancerous cell, by administration of one or more of the compounds described above.
  • the invention also includes compositions of one or more of the compounds described above in combination with a pharmaceutically-acceptable carrier, or with another therapeutic agent.
  • Fig. 1 is a graph showing the in vitro effect of increasing concentration of SL-11174 on the growth of cultured human prostate cancer cell DuPro.
  • Fig.2 is a graph showing the in vitro effect of increasing concentration of SL- 11197 on the growth of cultured human prostate cancer cell DuPro.
  • Fig.3 is a graph showing the in vitro effect of increasing concentration of SL-11199 on the growth of cultured human prostrate cancer cell
  • Fig.4 is a graph showing the in vitro effect of increasing concentration of SL- 11200 on the growth of cultured human prostrate cancer cell
  • Fig. 5 is a graph showing the in vitro effect of increasing concentration of SL-11208 on the growth of cultured human prostrate cancer cell
  • Fig.6 is a graph showing the effect of SL-11174 on the survival of cultured human prostate cancer cell DuPro.
  • Fig. 7 is a graph showing the effect of SL-11197 on the survival, of cultured human prostate cancer cell DuPro.
  • Fig. 8 is a graph showing the effect of SL-11199 on the survival of cultured human prostate cancer cell DuPro.
  • Fig.9 is a graph showing the effect of SL-11200 on the survival of cultured human prostate cancer cell DuPro.
  • Fig. 10 is a graph showing the effect of SL-11208 on the survival of cultured human prostate cancer cell DuPro.
  • Fig. 11 depicts the effect of SL-11238 on DuPro cell growth.
  • Fig. 12 depicts the effect of SL-11239 on DuPro cell growth.
  • Fig. 13 depicts the effect of SL-11238 on survival of DuPro cells.
  • Fig. 14 depicts the effect of SL-11239 on survival of DuPro cells.
  • Fig. 15 depicts the in vitro effect of spermine (control)
  • Fig. 16 depicts the in vitro effect of spermine (control)
  • Fig. 17 depicts the in vitro effect of spermine (control)
  • Fig. 18 depicts the in vitro effect of spermine (control)
  • Fig. 19 depicts the in vitro effect of spermine (control)
  • Fig.20 depicts the in vitro effect of SL- 11238 on ATP hydrolysis.
  • Fig.21 depicts the in vitro effect of SL- 11239 on ATP hydrolysis.
  • Fig.22 depicts the mean relative changes in luciferin/luciferase activities and standard deviations in the presence of extracts of 50,000 cultured human prostate tumor cells (DuPro) treated with varying concentrations of
  • Fig. 23 depicts the effect of SL- 11238 on cellular ATP measured by the luciferin/luciferase reaction.
  • Fig.24 depicts the effect of SL-11239 on cellular ATP measured by the luciferin/luciferase reaction.
  • Pharmaceutically acceptable salts are those salts which retain the biological activity of the free bases and which are not biologically or otherwise undesirable.
  • the desired salt may be prepared by methods known to those of skill in the art by treating the polyamine with an acid.
  • inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid.
  • organic acids include, but are not limited to, formic acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, sulfonic acids, and salicylic acid.
  • Salts of the polyamines with amino acids, such as aspartate salts and glutamate salts, can also be prepared.
  • the invention also includes all stereoisomers of the compounds, including diastereomers and enantiomers, as well as mixtures of stereoisomers, including, but not limited to, racemic mixtures. Unless stereochemistry is explicitly indicated in a structure, the structure is intended to embrace all possible stereoisomers of the compound depicted.
  • alkyl refers to saturated aliphatic groups including straight-chain, branched-chain, cyclic groups, and combinations thereof, having the number of carbon atoms specified, or if no number is specified, having up to
  • alkyl groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n- pentyl, neopentyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and adamantyl.
  • Cyclic groups can consist of one ring, including, but not limited to, groups such as cycloheptyl, or multiple fused rings, including, but not limited to, groups such as adamantyl or norbornyl.
  • Alkyl groups may be unsubstituted, or may be substituted with one or more substituents including, but not limited to, groups such as halogen (fluoro, chloro, bromo, and iodo), alkoxy, acyloxy, amino, hydroxyl, mercapto, carboxy, benzyloxy, phenyl, benzyl, cyano, nitro, thioalkoxy, carboxaldehyde, carboalkoxy and carboxamide, or a functionality that can be suitably blocked, if necessary for purposes of the invention, with a protecting group.
  • substituents including, but not limited to, groups such as halogen (fluoro, chloro, bromo, and iodo), alkoxy, acyloxy, amino, hydroxyl, mercapto, carboxy, benzyloxy, phenyl, benzyl, cyano, nitro, thioalkoxy, carboxaldehyde, carbo
  • alkenyl refers to unsaturated aliphatic groups including straight-chain, branched-chain, cyclic groups, and combinations thereof, having the number of carbon atoms specified, or if no number is specified, having up to
  • alkynyl refers to unsaturated aliphatic groups including straight-chain, branched-chain, cyclic groups, and combinations thereof, having the number of carbon atoms specified, or if no number is specified, having up to 12 carbon atoms, which contain at least one triple bond ( -C ⁇ C-).
  • Hydrocarbon chain or “hydrocarbyl” refers to any combination of straight-chain, branched-chain, or cyclic alkyl, alkenyl, or alkynyl groups, and any combination thereof.
  • Substituted alkenyl refers to the respective group substituted with one or more substituents, including, but not limited to, groups such as halogen, alkoxy, acyloxy, amino, hydroxyl, mercapto, carboxy, benzyloxy, phenyl, benzyl, cyano, nitro, thioalkoxy, carboxaldehyde, carboalkoxy and carboxamide, or a functionality that can be suitably blocked, if necessary for purposes of the invention, with a protecting group.
  • substituents including, but not limited to, groups such as halogen, alkoxy, acyloxy, amino, hydroxyl, mercapto, carboxy, benzyloxy, phenyl, benzyl, cyano, nitro, thioalkoxy, carboxaldehyde, carboalkoxy and carboxamide, or a functionality that can be suitably blocked, if necessary for purposes of the invention, with a protecting group.
  • Aryl or “Ar” refers to an aromatic carbocyclic group having a single ring (including, but not limited to, groups such as phenyl) or multiple condensed rings (including, but not limited to, groups such as naphthyl or anthryl), and includes both unsubstituted and substituted aryl groups.
  • Substituted aryls can be substituted with one or more substituents, including, but not limited to, groups such as alkyl, alkenyl, alkynyl, hydrocarbon chains, halogen, alkoxy, acyloxy, amino, hydroxyl, mercapto, carboxy, benzyloxy, phenyl, benzyl, cyano, nitro, thioalkoxy, carboxaldehyde, carboalkoxy and carboxamide, or a functionality that can be suitably blocked, if necessary for purposes of the invention, with a protecting group.
  • substituents including, but not limited to, groups such as alkyl, alkenyl, alkynyl, hydrocarbon chains, halogen, alkoxy, acyloxy, amino, hydroxyl, mercapto, carboxy, benzyloxy, phenyl, benzyl, cyano, nitro, thioalkoxy, carboxaldehyde, carboalk
  • Heteroalkyl refers to alkyl, alkenyl, and alkynyl groups, respectively, that contain the number of carbon atoms specified (or if no number is specified, having up to 12 carbon atoms) which contain one or more heteroatoms as part of the main, branched, or cyclic chains in the group. Heteroatoms include, but are not limited to, N, S, O, and P; N and O are preferred. Heteroalkyl, heteroalkenyl, and heteroalkynyl groups may be attached to the remainder of the molecule either at a heteroatom (if a valence is available) or at a carbon atom.
  • heteroalkyl groups include, but are not limited to, groups such as -O-CH 3 , -CH 2 -O-CH 3 , -CH 2 -CH 2 -O-CH 3 , -S-CH 2 -CH 2 -CH 3 , -CH 2 -CH(CH 3 )-S-CH 3 , -CH2-CH2-NH-CH2-CH2-,l-ethyl-6- propylpiperidino, 2-ethylthiophenyl, and morpholino.
  • heteroalkenyl groups include, but are not limited to, groups such as
  • Heteroaryl or “HetAr” refers to an aromatic carbocyclic group having a single ring (including, but not limited to, examples such as pyridyl, thiophene, or furyl) or multiple condensed rings (including, but not limited to, examples such as imidazolyl, indolizinyl or benzothienyl) and having at least one hetero atom, including, but not limited to, heteroatoms such as N, O, P, or S, within the ring.
  • Heteroalkyl, heteroalkenyl, heteroalkynyl and heteroaryl groups can be unsubstituted or substituted with one or more substituents, including, but not limited to, groups such as alkyl, alkenyl, alkynyl, benzyl, hydrocarbon chains, halogen, alkoxy, acyloxy, amino, hydroxyl, mercapto, carboxy, benzyloxy, phenyl, benzyl, cyano, nitro, thioalkoxy, carboxaldehyde, carboalkoxy and carboxamide, or a functionality that can be suitably blocked, if necessary for purposes of the invention, with a protecting group.
  • substituents including, but not limited to, groups such as alkyl, alkenyl, alkynyl, benzyl, hydrocarbon chains, halogen, alkoxy, acyloxy, amino, hydroxyl, mercapto, carboxy, benzyloxy, phenyl
  • the heteroatom(s) as well as the carbon atoms of the group can be substituted.
  • the heteroatom(s) can also be in oxidized form.
  • heteroalkyl, heteroalkenyl, heteroalkynyl, and heteroaryl groups have between one and five heteroatoms and between one and twenty carbon atoms.
  • alkylaryl refers to an alkyl group having the number of carbon atoms designated, appended to one, two, or three aryl groups.
  • alkoxy refers to an alkyl, alkenyl, alkynyl, or hydrocarbon chain linked to an oxygen atom and having the number of carbon atoms specified, or if no number is specified, having up to 12 carbon atoms. Examples of alkoxy groups include, but are not limited to, groups such as methoxy, ethoxy, and t-butoxy.
  • ⁇ -haloalkyl alkanoate refers to an alkyl alkanoate bearing a halogen atom on the alkanoate carbon atom furthest from the carboxyl group; thus, ethyl ⁇ -bromo propionate refers to ethyl 3-bromopropionate, methyl ⁇ -chloro n-butanoate refers to methyl 4-chloro n-butanoate, etc.
  • halo and halogen as used herein refer to Cl, Br, F or
  • Protecting group refers to a chemical group that exhibits the following characteristics: 1) reacts selectively with the desired functionality in good yield to give a protected substrate that is stable to the projected reactions for which protection is desired; 2) is selectively removable from the protected substrate to yield the desired functionality; and 3) is removable in good yield by reagents compatible with the other functional group(s) present or generated in such projected reactions. Examples of suitable protecting groups can be found in Greene et al. (1991) Protective Groups in Organic Synthesis, 2nd Ed. (John Wiley & Sons, Inc., New York).
  • Preferred amino protecting groups include, but are not limited to, benzyloxycarbonyl (CBz), t-butyloxycarbonyl (Boc), t- butyldimethylsilyl (TBDIMS), 9-fluorenylmethyloxycarbonyl (Fmoc), tosyl, benzenesulfonyl, 2-pyridyl sulfonyl, or suitable photolabile protecting groups such as 6-nitroveratryloxy carbonyl (Nvoc), nitropiperonyl, pyrenylmethoxycarbonyl, nitrobenzyl, dimethyl dimethoxybenzil, 5-bromo-7- nitroindolinyl, and the like.
  • CBz benzyloxycarbonyl
  • Boc t-butyloxycarbonyl
  • TBDIMS t- butyldimethylsilyl
  • Fmoc 9-fluorenylmethyloxycarbonyl
  • tosyl benz
  • Preferred hydroxyl protecting groups include Fmoc, TBDIMS, photolabile protecting groups (such as nitroveratryl oxymethyl ether (Nvom)), Mom (methoxy methyl ether), and Mem (methoxy ethoxy methyl ether).
  • Particularly preferred protecting groups include NPEOC (4- nitrophenethyloxycarbonyl) and NPEOM (4-nitrophenethyloxymethyloxycarbonyl).
  • protein and the like are used interchangeably herein to refer to any polymer of amino acid residues of any length, i.e., polymers of two or more amino acids.
  • the polymer can be linear or non-linear (e.g., branched), it can comprise modified amino acids or amino acid analogs, and it can be interrupted by chemical moieties other than amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.
  • Amino acids include the twenty encoded amino acids (including proline, an imino acid), other alpha-amino acids, and other natural and artificial amino acids such as p-iodotyrosine and beta-alanine.
  • Cyclic polyamine derivatives that can affect the hydrolysis of ATP in vivo are constructed by condensing spermine (spm), its isomers and its higher and lower homologues, as well as spermidine (spd) and its isomers and higher and lower homologues, with an ⁇ , ⁇ -unsaturated fatty acid chain (Scheme 1).
  • Scheme 1 is very likely the pathway for biogenesis of cyclic polyamines (e.g., pithecolobines and budmunchiamines), the practical synthetic approach follows a different route. The latter is depicted in Scheme 2 for several analogs of these series.
  • cyclic polyamines e.g., pithecolobines and budmunchiamines
  • Wittig salt 2 was obtained.
  • the a, ⁇ -unsaturated esters 4a-4c were obtained in ca. 90% yield.
  • reaction of 4a-4c with spermine (or a spermine analog) one equivalent of the base adds to the double bond by its primary amino group and the amino esters 5a-5c are obtained in ca. 40% yield. Lactamization of 5a-5c to 6a-6c was achieved using antimony (III) ethoxide in 76% yield.
  • N-methylation of the secondary amino residues of 6a-6c is desired, it can be achieved by a reductive alkylation reaction using formaldehyde and sodium cyanoborohydride to give 7a-7c. Yields for this reaction are ca. 80%. N-alkylation with homologues of formaldehyde will give the higher homologues of 7a-7c.
  • synthesis of compounds of the invention proceeds by reacting a haloalkyl alkanoate, preferably an ⁇ -haloalkyl alkanoate, with triphenylphosphine to give a phosphonium salt.
  • the phosphonium salt is condensed with an aldehyde or ketone-containing compound, preferably an aldehyde-containing compound, to give an ⁇ , ⁇ -unsaturated alkenyl alkanoate following the general reaction protocol of the Wittig reaction.
  • Addition of a polyamine containing at least two primary amino groups across the double bond yields a ⁇ -aminoalkyl alkanoate, where one of the primary amino groups has added to the double bond and the other amino group remains free. Condensation of the free amino group with the ester function gives the cyclic compound. Derivatization of secondary amino groups, if present in the cycle, can then be carried out if desired.
  • Scheme 3 [0070] As can be readily appreciated, the synthesis following Scheme 3 utilizes a compound (8) comprising a primary amino group and a hexahydropyrimidine moiety.
  • the hexahydropyrimidine moiety can be considered a protected form of 1,3-diaminopropane; the methylene bridge between the two nitrogens in the hexahydropyrimidine ring is readily cleaved to yield the free amino groups.
  • the portion of the molecule containing the free primary amino group is attached to one of the hexahydropyrimidine nitrogens; the primary amine can be linked to the hexahydropyrimidine nitrogen by any linker arm.
  • the linker arm contains at least one carbon atom.
  • the linker arm can be of the form -A 3 -(NY-A )j-, where each A 2 (if present) and A3 are independently selected from -Cs alkyl, where each Y is independently selected from H or C 1 -C alkyl, and where j is 0, 1, 2, or 3; this compound is represented by the structural formula
  • the linker arm is -CH 2 CH2CH2- as in compound 8 of Scheme 3.
  • hydride reagents such as lithium aluminum hydride and other reducing agents known in the art
  • the amide group will be reduced to a secondary amino group, while the other nitrogens will be present as tertiary amino groups, and this difference can be exploited to perform further chemistry at the secondary amino group.
  • the reduction is illustrated in Scheme 4, where Y a i k indicates an alkyl group (e.g., excluding hydrogen).
  • the resulting secondary amine can then be reacted with a compound such as acrylonitrile to derivatize the secondary amine, as outlined in Scheme 5.
  • the resulting secondary amine can then be reacted with a compound such as an ⁇ -haloalkyl nitrile, for example, but not limited to, where the alkyl group is a Ci-C 8 alkyl group and the halogen is iodo or bromo.
  • the acyl group can be reduced with lithium aluminum hydride or other organometallic agents to form an alkyl group.
  • the secondary amine can be alkylated by alkyl halides in the presence of bases.
  • Polypeptides are conveniently made by chemical synthesis, such as the Fmoc or Boc synthesis methods. See, for example, Atherton and Sheppard, Solid Phase Peptide Synthesis: A Practical Approach, New York: IRL Press, 1989; Stewart and Young: Solid-Phase Peptide Synthesis 2nd Ed., Rockford, Illinois: Pierce Chemical Co., 1984; and Jones, The Chemical Synthesis of Peptides, Oxford: Clarendon Press, 1994.
  • the polypeptides can be produced by an automated polypeptide synthesizer employing the solid phase method, such as those sold by Perkin Elmer- Applied Biosystems, Foster City, California, or can be made in solution by methods known in the art.
  • Peptides can also be attached to the cyclic polyamines by coupling of small protected peptide fragments, using the widely- known techniques for fragment condensation methods in peptide synthesis.
  • Individual amino acids such as leucine, can also be coupled to the cyclic polyamimes simply by stopping the peptide synthesis procedure after attachment of the first amino acid.
  • Longer peptides such as acetyl-Ser-Lys-Leu-Gln-Leu-, can be attached to the cyclic polyamines by either stepwise synthesis or fragment coupling methods.
  • SL-11243 was synthesized.
  • Peptides of interest for use in the peptide-derivatized compounds described above include peptides which are substrates of prostate specific antigen (PSA) or cathepsin B. Peptides of length 25 amino acids or less, or 10 amino acids or less, can be used.
  • sequences cleaved by PSA are HSSKLQ, SKLQ- ⁇ -alanine,SKLQL, or SKLQ, with or without N- terminal protecting or capping groups such as Boc, Fmoc, acetyl, or other acyl capping groups, and with or without side-chain protecting groups (such as carbobenzyloxy carbonyl, Boc or Fmoc on the ⁇ -amino group of lysine).
  • N- terminal protecting or capping groups such as Boc, Fmoc, acetyl, or other acyl capping groups
  • side-chain protecting groups such as carbobenzyloxy carbonyl, Boc or Fmoc on the ⁇ -amino group of lysine.
  • Examples of polypeptides recognized and cleaved by cathepsin B include the peptide sequence Z 1 -P2-P1-, where Z ⁇ is hydrogen, an amino- protecting group, or an amino-capping group attached to the N-terminus of P 2 ; where P 2 is the N-terminal amino acid and Pj. is the C-terminal amino acid; and where P 2 is a hydrophobic amino acid and Pi is a basic or polar amino acid.
  • the peptide sequence is Z 1 -P 2 -P 1 -Y-, where Zi is hydrogen, an amino-protecting group, or an amino-capping group attached to the N-terminus of P 2 ;
  • P 2 is a hydrophobic amino acid;
  • Pt is a basic or polar amino acid;
  • Y is leucine, ⁇ -alanine, or a nonentity.
  • Z ⁇ is a 4- morpholinocarbonyl group.
  • P 2 is selected from the group consisting of leucine, isoleucine, valine, methionine, and phenylalanine; and P] is selected from the group consisting of lysine, arginine, glutamine, asparagine, histidine and citrulline.
  • the subject polyamines induce cell growth inhibitions in several cultured human prostate tumor cell lines such as LnCap, DuPro, and PC-3 as determined by an accepted MTT assay (Table 2). All three cell lines are sensitive to the cyclic polyamines, with IDs 0 values ranging from 500 nM to 2,600 nM. The results with the DuPro cell line are given; these results are representative of the results with human prostate cell lines.
  • the cyclic polyamines of the present invention have been shown to inhibit cell growth and to cause cell death in accepted in vitro test cultures of human prostate cancer cell lines as shown in Figs. 1-14. The figures are described in detail in the Example section below.
  • the uptake of the cyclic polyamines by the DuPro cells and the resultant changes in the cellular polyamine levels are shown in Tables 3a and 3b.
  • the hydrolysis of ATP is believed to be one of the probable causes of cell kill. In a standardized method for measuring acid hydrolysis of ATP, a marked increase was observed in ATP hydrolysis in the presence of the cyclic polyamines (see Figs. 15-21) as compared to the lack of ATP hydrolysis in the presence of the naturally occurring linear polyamine spermine.
  • the cyclic polyamines were also found to hydrolyze ATP in vivo in the cancer cells (Fig. 22- 24).
  • the concentration of cyclic polyamines required to hydrolyze intracellular ATP in the cancer cells parallels the concentrations at which they produce cell kill (Figs. 6-14), lending support to the hypothesis that cell kill is due to intracellular ATP depletion.
  • the invention is not to be construed as limited by any particular theory of biological or therapeutic activity.
  • Polyamine analogs of the present invention are likely to be useful for treatment of a variety of diseases caused by uncontrolled proliferation of cells, including cancer, particularly prostate cancer and other cancer cell lines.
  • the analogs are used to treat mammals, preferably humans.
  • "Treating" a disease using a cyclic polyamine of the invention is defined as administering one or more cyclic polyamines of the invention, with or without additional therapeutic agents, in order to prevent, reduce, or eliminate either the disease or the symptoms of the disease, or to retard the progression of the disease or of symptoms of the disease.
  • “Therapeutic use” of the cyclic polyamines of the invention is defined as using one or more cyclic polyamines of the invention to treat a disease, as defined above.
  • the compounds can be first tested against appropriately chosen test cells in vitro.
  • polyamine analogs can be tested against tumor cells, for example, prostate tumor cells.
  • Exemplary experiments can utilize cell lines capable of growing in culture as well as in vivo in athymic nude mice, such as LNCaP. Horoszewicz et al. (1983) Cancer Res. 43:1809-1818.
  • Analysis begins with IC 50 determinations based on dose-response curves ranging from 0.1 to 1000 ⁇ M performed at 72 hr. From these studies, conditions can be defined which produce about 50% growth inhibition and used to: (a) follow time-dependence of growth inhibition for up to 6 days, with particular attention to decreases in cell number, which may indicate drug-induced cell death; (b) characterize analog effects on cell cycle progression and cell death using flow cytometry (analysis to be performed on attached and detached cells); (c) examine analog effects on cellular metabolic parameters. Analog effects can be normalized to intracellular concentrations (by HPLC analysis), which also provide an indication of their relative ability to penetrate cells.
  • Analogs found to have potent anti-proliferative activity in vitro towards cultured carcinoma cells can be evaluated in in vivo model systems.
  • the first goal is to determine the relative toxicity of the analogs in non-tumor-bearing animals, such as DBA/2 mice. Groups of three animals each can be injected intraperitoneally with increasing concentrations of an analog, beginning at, for example, 10 mg/kg. Toxicity as indicated by morbidity is closely monitored over the first 24 hr.
  • a well-characterized polyamine analog, such as BE-333 can be used as an internal standard in these studies, since a data base has already been established regarding acute toxicity via a single dose treatment relative to chronic toxicity via a daily x 5 d schedule. Thus, in the case of new analogs, single dose toxicity relative to BE-333 is used to project the range of doses to be used on a daily x 5 d schedule.
  • tumors can be subcutaneously implanted into nude athymic mice by trocar and allowed to reach
  • analogs can be given in a range between 10 and 200 mg/kg. Analogs can be evaluated at three treatment dosages with 10-15 animals per group (a minimum of three from each can be used for pharmacodynamic studies, described below).
  • mice can be monitored and weighed twice weekly to determine tumor size and toxicity. Tumor size is determined by multi-directional measurement from which volume in mm 3 is calculated. Tumors can be followed until median tumor volume of each group reaches 1500 mm 3 (i.e., 20% of body weight), at which time the animals can be sacrificed.
  • the initial anti-tumor studies focuses on a daily x 5 d schedule, constant infusion can be performed via Alzet pump delivery for 5 days since this schedule dramatically improves the anti-tumor activity of
  • polyamine analogs of the present invention can be administered to a mammalian, preferably human, subject via any route known in the art, including, but not limited to, those disclosed herein.
  • administration of the novel polyamine analogs is intravenous.
  • Other methods of administration include but are not limited to, oral, intrarterial, intratumoral, intramuscular, topical, inhalation, subcutaneous, intraperitoneal, gastrointestinal, and directly to a specific or affected organ.
  • novel polyamine analogs described herein are administratable in the form of tablets, pills, powder mixtures, capsules, granules, injectables, creams, solutions, suppositories, emulsions, dispersions, food premixes, and in other suitable forms.
  • the compounds can also be administered in liposome formulations.
  • the compounds can also be administered as prodrugs, where the prodrug undergoes transformation in the treated subject to a form which is therapeutically effective. Additional methods of administration are known in the art.
  • the pharmaceutical dosage form which contains the compounds described herein is conveniently admixed with a non-toxic pharmaceutical organic carrier or a non-toxic pharmaceutical inorganic carrier.
  • Typical pharmaceutically- acceptable carriers include, for example, mannitol, urea, dextrans, lactose, potato and maize starches, magnesium stearate, talc, vegetable oils, polyalkylene glycols, ethyl cellulose, poly(vinylpyrrolidone), calcium carbonate, ethyl oleate, isopropyl myristate, benzyl benzoate, sodium carbonate, gelatin, potassium carbonate, silicic acid, and other conventionally employed acceptable carriers.
  • the pharmaceutical dosage form can also contain non-toxic auxiliary substances such as emulsifying, preserving, or wetting agents, and the like.
  • a suitable carrier is one which does not cause an intolerable side effect, but which allows the novel cyclic polyamine analog(s) to retain its pharmacological activity in the body.
  • Formulations for parenteral and nonparenteral drug delivery are known in the art and are set forth in Remington 's Pharmaceutical Sciences, 18th Edition, Mack
  • Solid forms such as tablets, capsules and powders, can be fabricated using conventional tableting and capsule-filling machinery, which is well known in the art.
  • Solid dosage forms including tablets and capsules for oral administration in unit dose presentation form, can contain any number of additional non-active ingredients known to the art, including such conventional additives as excipients; dessicants; colorants; binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrollidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulphate.
  • the tablets can be coated according to methods well known in standard pharmaceutical practice.
  • Liquid forms for ingestion can be formulated using known liquid carriers, including aqueous and non-aqueous carriers, suspensions, oil-in-water and/or water-in-oil emulsions, and the like.
  • Liquid formulations can also contain any number of additional non-active ingredients, including colorants, fragrance, flavorings, viscosity modifiers, preservatives, stabilizers, and the like.
  • novel cyclic polyamine analogs can be administered as injectable dosages of a solution or suspension of the compound in a physiologically acceptable diluent or sterile liquid carrier such as water or oil, with or without additional surfactants or adjuvants.
  • a physiologically acceptable diluent or sterile liquid carrier such as water or oil, with or without additional surfactants or adjuvants.
  • carrier oils would include animal and vegetable oils (peanut oil, soy bean oil), petroleum-derived oils (mineral oil), and synthetic oils.
  • liquid carriers for injectable unit doses, water, saline, aqueous dextrose and related sugar solutions, and ethanol and glycol solutions such as propylene glycol or polyethylene glycol are preferred liquid carriers.
  • the pharmaceutical unit dosage chosen is preferably fabricated and administered to provide a final concentration of drug at the point of contact with the cancer cell of from 1 ⁇ M to
  • Cyclic polyamine analogs can be administered as the sole active ingredient, or can be administered in combination with another active ingredient, including, but not limited to, cytotoxic agents, antibiotics, antimetabolites, nitrosourea, vinca alkaloids, polypeptides, antibodies, cytokines, etc.
  • IR measurements are presented in units of [cm "1 ] and were recorded on a Perkin-Elmer 781 instrument.
  • NMR spectra were recorded on Bruker-300 or Bruker AMX-600 instruments with ⁇ in ppm and using the appropriate solvent as internal standard.
  • MS spectra were generated on Finnigan MAT SSO 700 or Finnigan MAT 90 instruments using chemical ionization (CI) with NH 3 and electron impact (El; 70 eV), and on a Finnigan TSQ 700 instrument using electrospray ionisation (ESI).
  • Numerals included in the structure drawings denote atom numbers for specfroscopic data which are not otherwise identified; e.g., the numbers 1 and 2 for the compound 2 in Example 1 identify carbons 1 and 2 for the carbon nuclear magnetic resonance assignments.
  • ⁇ I-MS 254 (5, [ + • ]), 209 (9, [M- O ⁇ f]+), 157 (18), 127 (46), 113 (27), 99 (47), 81 (37), 67 (24), 55 (58), 43 (100).
  • EI-MS 452 (40, [M+ • ]), 437 (28, [M- CH 3 ]+), 380 (16), 366 (31), 295 (25), 273 (19), 243 (31), 238 (20), 226 (20), 212 (19), 200 (28), 186 (16), 169 (15), 149 (33), 127 (18), 112 (21), 100 (29), 98 (35), 86 (76), 84 (100), 70 (39), 58 (57), 49 (95), 43 (69).
  • EI-MS 424 (51, [M+ • ]), 409 (28, [M- CH 3 ]+), 352 (19), 338 (51), 297 (41), 281 (15), 224 (14), 212 (25), 210 (38), 198 (23), 184 (36), 169 (27), 155 (17), 112 (25), 100 (32), 98 (59), 86 (62), 84 (100), 72 (36), 70 (43), 58 (75), 57 (29), 43 (29).
  • CI-MS 283 ( ⁇ 5, [M+ 1]+), 282 (16, [ + NH 4 - H 2 O]+), 237 (37, [M- O ⁇ t]+), 194 (16), 127 (61), 114 (21), 101 (59), 99 (51), 88 (43), 81 (33), 69 (35), 57 (49), 43 (89), 41 (100).
  • EI-MS 481 (18 [ + 1]+), 480 (68 [M+ • ]), 465 (37 [ - CH 3 ]+), 408 (21), 394 (56), 339 (16), 297 (58), 266 (42), 254 (38), 240 (50), 238 (30), 226 (36), 169 (32), 155 (22), 112 (27), 100 (35), 98 (69), 86 (69), 84 (100), 72 (32), 70 (41), 58 (62), 43 (25).
  • N-(4-aminobutyl)hexahydropyrimidine (8, 1.57 g, 10 mmol; see McManis, J.S., Ganem, B., J. Org. Chem. (1980), 45: 2042 and U.S. Pat. No. 5,869,734) in 400 ml of abs. EtOH was stirred for 4 days at 40°. After evaporation the residue was purified by column chromatography (SiO 2 , CH 2 Cl 2 /MeOH/25% aq. NH 4 OH 100:10:1) to give 1.21 g (27%) of 9 as a colorless oil.
  • Example 18 Cyclic Polyamines as Anti-neoplastic Agents [0114] To assess the utility of the subject compounds in the treatment of neoplastic cell growth, the ability of the compounds to inhibit the in vitro growth characteristics of several commonly used cancer models were studied.
  • the subject polyamines induce cell growth inhibitions in several cultured human prostate tumor cell lines such as LnCap, DuPro, and PC-3 as determined by the accepted MTT assay (Table 2) (Hansen, M.B. et al., "Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill," J. Immunol. Methods (1989)119(2):203-10). All three cell lines are sensitive to the cyclic polyamines with ID 50 values ranging between 500 nM to 2,600 nM. The results with the DuPro cell line are representative of the results with human prostate cell lines.
  • Figs. 1-14 the cyclic polyamines of the present invention have been shown to inhibit cell growth and even to cause cell death in accepted in vitro test cultures of human prostate cell cancer.
  • the Figures are described in more detail below.
  • the uptake of the cyclic polyamines by the DuPro cells and the resultant changes in the cellular polyamine levels are shown in Tables 3a and 3b.
  • Assays to measure ATP hydrolysis were carried out. In a standardized method for measuring hydrolysis of ATP, a marked increase in ATP hydrolysis was observed in the presence of the cyclic polyamines (Figs. 15-21) as compared to the naturally occurring linear polyamine spermine.
  • Figs. 1-5 and Figs. 11-22 depict the X-axes depict the number of days after seeding DuPro cells and the Y-axes depict the number of cells harvested under control (no drug) conditions (Figs. 1-5) and in the presence of 10 ⁇ M of the drug SL-11174 (Fig. 1), 5 ⁇ M SL-11197 (Fig. 2), 5 ⁇ M SL-11199 (Fig. 3), 10 ⁇ M SL-11200 (Fig. 4) and 5 ⁇ M SL-11208 (Fig. 5), 2 ⁇ m of SL-11238 (Fig. 11) and 5 ⁇ m of SL-11239 (Fig. 12).
  • Figs. 6-10 and Figs. 13-14 depict the concentrations of the cyclic polyamines and the Y-axes depict the fraction of surviving cells after 5 days treatment with the drug SL-11174 (Fig. 6), SL-11197 (Fig. 7), SL-11199 (Fig. 8), SL-11200 (Fig. 9), SL-11208 (Fig. 10), SL-11238 (Fig. 13), and SL- 11239 (Fig. 14) as determined by the colony forming efficiency (CFE) assay (Wilson A.P., "Cytotoxicity and viablity assays.” See Freshney, R.I. (ed) Animal Cell Culture: A Practical Approach.
  • CFE colony forming efficiency
  • Figs. 15-21 depict the concentrations of the polyamines and the Y-axes depict the relative increase in inorganic phosphate (PPi) released from 100 ⁇ M ATP in 24 hour in the presence of spermine and SL-11174 (Fig. 15), SL-11197 (Fig. 16), SL-11199 (Fig. 17), SL-11200 (Fig. 18),
  • PPi inorganic phosphate
  • SL-11208 (Fig. 19), SL-11238 (Fig. 20), and SL-11239 (Fig. 21) as compared to the inorganic phosphate released from the same amount of ATP under identical conditions in the absence of any polyamines.
  • Figs. 1-14 The following standardized protocol was used to evaluate the test cultures and to generate data shown in Figs. 1-14.
  • Figs. 1-5 and Figs. 11-12 cells were seeded into 75 cm culture flasks with 15 ml of Eagle's minimal essential medium supplemented with 10% fetal calf serum and nonessential amino acids. The flasks were then incubated in a humidified 95% air/5% CO 2 atmosphere. The cells were grown for at least 24 h to ensure that they were in the log phase of growth, then treated with the polyamine analogs. Cells were harvested by treatment for 5 min with STV (saline A, 0.05% trypsin, 0.02%
  • the Petri dishes were prepared not more than 24 hr in advance with 4 ml of supplemented Eagle's minimum essential medium containing 5-10% fetal bovine serum (standardized for each cell line). Cells were incubated for the previously standardized number of days in a 95% air/5% CO 2 atmosphere. The plates were stained with 0.125% crystal violet in methanol and counted.
  • intracellular polyamine levels were determined using a standard protocol. About 0.5-1 x 10 6 cells were taken from harvested samples and centrifuged at 1000 rpm at 4°C for 5 min. The cells were washed twice with chilled Dulbecco's isotonic phosphate buffer (pH 7.4) by centrifugation at 1000 rpm at 4°C and resuspended in the same buffer. After the final centrifugation, the supernatant was decanted, and 250 ⁇ l of 8% sulfosalycilic acid was added to the cell pellet. The cells were then sonicated, and the mixture was kept at 4° C for at least 1 h.
  • Enliten ATP Assay System containing luciferase/luciferin mixture in assay buffer was injected and each well was read for 5 seconds after a one-second delay time.
  • the relative changes in cellular ATP content were measured as relative light units (RLU) generated by the luciferase/luciferin reaction.

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Abstract

L'invention concerne de nouveaux composés de polyamine cyclique de formule (I) dans laquelle A1, chaque A2 (si existant) et A3 sont sélectionnés indépendamment à partir d'alkyle C1-C8, chaque Y est sélectionné indépendamment entre H ou alkyle C1-C4, M est sélectionné à partir d'alkyle C1-C4, k est 0, 2 ou 3, R est sélectionné à partir d'alkyle C1-C32, ainsi que tous leurs stéréoisomères et leurs sels. L'invention concerne également des composés additionnels, où k est 1 et A2 est sélectionné indépendamment entre alkyle C1-C3 ou alkyle C5-C8, des polyamines cycliques, où le groupe amide est réduit à un groupe amino secondaire, différents dérivés de ces composés, ainsi que des procédés de synthèse desdits composés. Ces composés sont utiles dans le traitement de maladies causées par la prolifération incontrôlée de cellules, telles que le cancer, notamment le cancer de la prostate, ainsi que dans l'induction de l'hydrolyse ATP intracellulaire pour traiter d'autres troubles.
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CA2417064A1 (fr) 2002-02-07
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AU2001278143B2 (en) 2007-08-09
WO2002010142A1 (fr) 2002-02-07
AU7814301A (en) 2002-02-13

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