EP1003715A1 - Inhibiteurs du transport des polyamines - Google Patents

Inhibiteurs du transport des polyamines

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Publication number
EP1003715A1
EP1003715A1 EP98918385A EP98918385A EP1003715A1 EP 1003715 A1 EP1003715 A1 EP 1003715A1 EP 98918385 A EP98918385 A EP 98918385A EP 98918385 A EP98918385 A EP 98918385A EP 1003715 A1 EP1003715 A1 EP 1003715A1
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EP
European Patent Office
Prior art keywords
polyamine
synthetic derivative
spermine
methyl
desc
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.)
Withdrawn
Application number
EP98918385A
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German (de)
English (en)
Inventor
Richard Poulin
Marie Audette
René CHAREST-GAUDREALT
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universite Laval
Original Assignee
Universite Laval
Ilex Oncology Inc
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Publication date
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Publication of EP1003715A1 publication Critical patent/EP1003715A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C211/00—Compounds containing amino groups bound to a carbon skeleton
    • C07C211/01—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms
    • C07C211/02—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C07C211/14—Amines containing amino groups bound to at least two aminoalkyl groups, e.g. diethylenetriamines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C211/00—Compounds containing amino groups bound to a carbon skeleton
    • C07C211/01—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms
    • C07C211/26—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms of an unsaturated carbon skeleton containing at least one six-membered aromatic ring
    • C07C211/27—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms of an unsaturated carbon skeleton containing at least one six-membered aromatic ring having amino groups linked to the six-membered aromatic ring by saturated carbon chains
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/10—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by nitrogen atoms not being part of nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C323/39—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton at least one of the nitrogen atoms being part of any of the groups, X being a hetero atom, Y being any atom
    • C07C323/40—Y being a hydrogen or a carbon atom
    • C07C323/41—Y being a hydrogen or an acyclic carbon atom

Definitions

  • the present invention relates to a novel class of competitive inhibitors of natural polyamine transport in mammalian cells.
  • the present invention is more particularly directed to low molecular weight, high-affinity, specific, impermeant, pure antagonists of polyamine transport of a structure different to that of endogenous polyamines.
  • the novel inhibitors of the present invention exhibit an effect on cultured tumor cells essentially cytostatic, with minor non-specific effects.
  • the present invention is also directed to the use of such novel inhibitors of polyamine transport to evaluate the antitumor efficacy of polyamine depletion strategies with minimal systemic cytotoxic effects or to control and treat disorders involving unrestrained cell proliferation and/or cell differentiation wherein polyamine transport is required.
  • Natural polyamines such as putrescine (1,4-butane-diamine), spermidine (N-3[- aminopropyl]-l,4-diaminobutane) and spermine (NN'-bis-fS-aminopropylJ-l ⁇ -butane- diamine) play essential roles in the control of macromolecular synthesis and growth processes in eukaryotic cells.
  • Cells maintain appropriate polyamine concentrations principally by de novo synthesis from amino acids wherein ornithine decarboxylase catalyzes conversion of ornithine to putrescine, which is then converted to spermidine and spermine.
  • Most tissues also possess a specific plasma membrane transport system allowing for utilization of plasma sources of polyamines.
  • Inhibitors of polyamine biosynthesis such as -difluoromethylornithine (DFMO), which inhibits ornithine decarboxylase, cause an extensive depletion of polyamines followed by growth arrest in virtually all known mammalian cell types in vitro. Since tissues such as tumor cells and other transformed or rapidly proliferating cells exhibit a tissues such as tumor cells and other transformed or rapidly proliferating cells exhibit a high demand for polyamines, these properties have encouraged an extensive assessment of such inhibitors for the treatment of proliferative diseases, including several types of tumors, in experimental models and in clinical trials. Unfortunately, the antitumor efficacy of such inhibitors in vivo has been disappointing.
  • DFMO -difluoromethylornithine
  • spermidine as low as 300 nM, i.e., such as those found in human plasma (Moulinoux, J.-P., Quemener, V., and Khan, N.A. 1991. Cell. Mol. Biol. 37: 773-783;
  • polymers of Aziz et al. as well as their charge would cause their adsorption to the cellular surface, which bears negative charges due to the presence of glycoproteins, e.g. sialic acid.
  • Poly-L-lysine a commercially used compound analogous to high molecular weight polymers of polyamines by its positive charges, is known to promote a strong electrostatic interaction between the cell and its substrate, as in the induction of positive charges of gamma irradiation of synthetic polymers used to produce dishes for tissue culture.
  • the polyamine transport inhibitors of Aziz et al. present the additional drawback of being highly cytotoxic. It is noteworthy that their spermine polymer is effective in decreasing contents of polyamines in cells even when not used in combination with DFMO and at concentrations much higher than those required to block polyamine uptake, which indicates inherent high toxicity of the compound toward the cell by a mechanism independent of polyamine transport per se. The cytotoxicity of the spermidine polymer of Aziz et al. is most probably explained by a non-specific effect on cellular physiology such as the cellular membrane.
  • This spermine polymer for specifically blocking polyamine accumulation is therefore uncertain in view of its marked cytotoxicity.
  • Cysteamine and aliphatic monoamines of similar chain length such a n-butylamine and n-pentylamine have a low but significant ability to antagonize putrescine uptake (Gordonsmith, R.H., Brooke-Taylor, S., Smith, L.L. and Cohen, G.M. 1983. Biochem. Pharmacol. 32, 431-437), although the mode of inhibition of these compounds has not been reported.
  • polyamine-like structure known to interact non-competitively with the polyamine transport system is pentamidine, an aromatic diamidine (Jones, H.E., Blundell, G.K., Wyatt, I., John, R.A., Farr, SJ. and Richards, R.J. 1992. Biochem. Pharmacol. 43, 431-437), but the structural basis of its inhibitory activity is not yet clear. It follows that there still exists a need for effective polyamine transport inhibitors which, while inhibiting the transport of polyamines, will not be internalized by the transport system and will not be toxic to the cell. The availability of low molecular weight inhibitors of polyamine transport would provide for the possibility of better renal elimination, as well as lower risks of being immunogenic.
  • Plasma polyamines are partly derived from various plasma sources (7, 12, 18, 58-60, 62, 70) and from the activity of the gastrointestinal microflora, which produces and excretes very high amounts of putrescine and cadaverine (1, 17, 45, 50, 62, 70), which can enter the general circulation through the enterohepatic pathway (6, 45). Other systemic contributions can also be attributed to polyamine excretion by peripheral tissues, including dying tumor cells (32, 35, 41, 42, 63, 64, 67, 79, 80). The enhanced uptake of polyamines by tumor cells results both from the increased polyamine transport activity that accompanies the malignant phenotype ( 11 , 43 ,
  • polyamine transport inhibitors having a low molecular weight, less susceptible to immunogenicity and to nonspecific interactions with the cellular membrane. These inhibitors have high affinity, are specific, impermeant, pure antagonists of polyamine transport in mammalian cells while exhibiting minimal cytotoxic effects.
  • the original polyamine is modified to comprise an amido group immediately linked to a carbon atom of said original polyamine, said synthetic derivatives inhibiting the cellular uptake of natural polyamines by specifically binding cellular transporters for said natural polyamines.
  • the immediate vicinity of the amido group to the backbone of the original polyamine preserves the specificity of the derivative towards the transporter while conferring thereto an impermeant character, providing a true antagonist.
  • the amido group is located between two internal nitrogen atoms of the original polyamine.
  • the synthetic derivative comprises a dimer wherein monomers of said dimer are linked together by a spacer side chain anchored to the amido group of each monomer.
  • R, and R,' independently represent a hydrogen atom or an alkyl group having 1 to 2 carbon atoms
  • R 2 , R 2 ', or R 3 and R 3 ' independently represent a hydrogen atom or a methyl group
  • w and z independently represent an integer of 3 or 4
  • x represents an integer from 0 to n
  • y represents an integer from 0 to n
  • n represents an integer from 3 to 6
  • L represents a hydrogen atom or a molecule which cannot be captured by said natural polyamine transporter.
  • the side chain or linker L may be labeled and be used as a marker for a polyamine transporter.
  • the side chain L can be varied to increase the affinity of the derivative for the transporter.
  • the side chain L may also become a spacer molecule useful in the formation of a dimer.
  • This spacer side chain comprises a linear hydrocarbon-containing backbone of 3 to 8 atoms.
  • the backbone may comprise sulfur, oxygen, or nitrogen atoms.
  • the original polyamine is spermine.
  • N-(2-mercaptoethyl)spermine-5-carboxamide (MESC)
  • DSC 2,2'-dithiobis(N-ethyl-spermine-5-carboxamide)
  • DEASC N-[2,2'-dithio(ethyl, 1 -aminoethyl)]spermine-5-carboxamide
  • the method may be described as comprising the step of contacting said transporter with an inhibitory effective amount of said synthetic derivative. This inhibition results in the control of the treatment of disorders involving unrestrained cell proliferation and/or differentiation where control of polyamine transport is required, when used in combination with an inhibitor of polyamine synthesis such as DFMO.
  • the above sequence of steps results in the diagnosis of a disorder involving unrestrained cell proliferation and/or differentiation where control of polyamine transport is required.
  • compositions for treating disorders wherein control of polyamine transport is required comprising anyone of the above derivatives in adjunction with an acceptable pharmaceutical carrier.
  • this composition also comprises an inhibitor of polyamine synthesis, such as DFMO.
  • the applicants have unexpectedly discovered that the presence of a lateral amido group immediately linked to a carbon atom of the polyamine backbone of a synthetic derivative of an original polyamine confers impermeant properties to the so derived synthetic polyamine against the mammalian cell. It follows that the synthetic polyamine derivatives of the present invention, by exhibiting high affinity for diamine and polyamine transport systems, block the transport of natural polyamines by competing therewith, while in the same time acting as poor substrate for intracellular uptake. The affinity of the polyamine derivative for the transporter system is further enhanced by increasing the length of a side chain anchored to the amido group of the derivative.
  • the best affinity is achieved by dimerizing the polyamine derivative with the aid of a spacer molecule anchored at both ends to the amido group of each monomer.
  • the flexibility of the chemical structure of the inhibitors of the present invention permits better optimization of the activity and affinity than a simple polymeric structure such as (TS) n .
  • modifications to the polyamine backbone as taught by the present invention such as methylation of CI and C12, lowers the possibility of oxidation of the primary amides by the serum amine oxidase, which is present in mammalian sera.
  • Additional modifications including adjunction to the lateral chain of alkylating groups that irreversibly modify residues that are essential to the activity of the polyamine transporter, such as carboxylic moieties of the carrier protein, are also contemplated in the present invention (Torossian, K., Audette, M., and Poulin R., 1996. Biochem. J. 319: 21-28).
  • the inhibitory action of the derivatives of the present invention is thus enhanced.
  • additional modifications to the side chain that can be of potential therapeutic interest include the incorporation of reactive groups to the side chain that would allow the covalent modification of residues in the polyamine transporter by the principle of affinity labeling, and its subsequent irreversible inactivation.
  • Specific transport inhibition by polyamines dimerized via a side group on an integral atom of the polyamine chain is not limited to spermine or spermine-like dimers, but can also be obtained with dimers of triamines such as spermidine [N - (3-aminopropyl)-
  • one spermidine or yw-norspermidine chain is cross-linked to a second spermidine or sym-norspermidine chain with an ⁇ -alkyl spacer side group anchored to the secondary amino group of said spermidineor .sym-norspermidine chain.
  • said spacer side group is an aliphatic carbon chain or an aromatic carbon chain.
  • FIG 3 graphically illustrates the inhibition of [ 3 H] spermidine uptake by spermine and DESC in ZR-75-1 cells.
  • the rate of spermidine uptake was measured in ZR-75-1 cells grown as monolayers in 24-well culture plates in the presence of the indicated concentrations of spermine (O) and DESC (•) using 3 ⁇ M [ 3 H]putrescine (A) or 1 ⁇ M [ 3 H]spermidine (B) as substrate.
  • Data are the mean ⁇ SD of triplicate determinations from a representative experiment;
  • FIG 4 illustrates graphically the Lineweaver-Burke analysis of putrescine transport inhibition by DESC and DEASC in ZR-75-1 cells.
  • the rate of [ ⁇ Jputrescine uptake was determined in ZR-75-1 cell cultures with increasing concentrations of substrate (A) in the presence of 0 ⁇ M DESC (O), 3 ⁇ M DESC (•), 30 ⁇ M DESC (D) or 100 ⁇ M DESC ( ⁇ ) or (B) in the presence of 0 ⁇ M DEASC (O), 20 ⁇ M DEASC (•), 50 ⁇ M DEASC (D) or
  • FIG 5 illustrates graphically the structure of MESC thioether derivatives and their K j values with respect to spermidine uptake in CHO-K1 cells.
  • the various conjugates were prepared from MESC as described supra, and structure and name of the substituents are given in the first two columns from the left, wherein R corresponds to the group attached to sulfur in MESC (structure VII, Fig. 1).
  • the rate of spermidine uptake was determined in CHO-K1 cells in the presence of increasing concentrations of the various MESC derivatives, using 1 ⁇ M [ 3 H] spermidine as substrate.
  • FIG 6 graphically represents the effect of DESC and MESC on the intracellular accumulation of [ 3 H]spermidine in ZR-75-1 cells, wherein at time 0 (A), 5 ⁇ M
  • spermidine was added to ZR-75-1 cell cultures grown in 24-well plates (1 ml/well) in the presence of 200 ⁇ M MESC (•), 50 ⁇ M DESC (D) or 200 ⁇ M DESC ( ⁇ ), and accumulation of radio-labeled spermidine determined after the indicated interval.
  • Control cells (O) received vehicle only.
  • B same as in A, except that 200 ⁇ M CHX was added at time 0 in the presence of 0 (•), 50 (D) or 200 ⁇ M DESC ( ⁇ ).
  • Data are the mean ⁇ SD of triplicate determinations;
  • FIG 7 illustrates the effect of spermine, MESC, DESC and DEASC on ZR-75-1 cell proliferation.
  • Cells were incubated for 11 days in MEZR medium with the indicated concentration of spermine, DESC, MESC, or DEASC in the presence (shaded bars) or absence (plain bars) of 1 mM of aminoguanidine, and DNA content per culture was then determined.
  • Data represent the mean ⁇ SD of triplicate determinations;
  • FIG 8 represents the effect of DESC on the reversal of DFMO-induced growth inhibition by exogenous spermidine in ZR-75-1 cells.
  • Cells were incubated for 11 days in SD medium with the indicated concentrations of spermidine in the presence of 50 ⁇ M DESC (•), ImM DFMO (D), or the combination thereof ( ⁇ ), or in the absence of drugs
  • FIG 9 represents the chromatographic profile of DESC and its degradation products in IMEM or PBS.
  • DESC 50 ⁇ M was added to 1ml of IMEM containing 10% fetal bovine serum in the absence (A) or presence (B) of ImM aminoguanidine, or 1 ml PBS (C) in 24- well culture plates in the absence of cells. Media were analyzed after 20 minutes (solid lines) or 48 hours (dotted lines) of incubation at 37°C in 95% air: 5% CO 2 , water-saturated atmosphere for amine composition by ion-pair reversed-phase HPLC as described supra. Peaks 1 and 2 are degradation products of DESC, whereas peak 3 is a minor amount of DEASC initially present in the DESC preparation. Note the disappearance of peak 3 (DEASC) and the appearance of a shoulder (indicated by the arrow) at 42 minutes on the
  • FIG 10 represents the time course of degradation of DESC in growth medium.
  • 50 ⁇ M DESC was added to 1ml of IMEM in 24-well culture plates and the content in DESC (O), compound 1 (Comp 1, •) and compound 2 (Comp 2, D) determined by HPLC after the indicated incubation period at 37°C in a 5% CO 2 atmosphere.
  • Data represent the mean of triplicate determinations from a representative experiment.
  • FIG 11 Structures of putrescine, of the natural polyamines spermidine and spermine, and of three cell-impermeant inhibitors of polyamine transport (DESC, DEASC and MESC).
  • FIG. 12 Structure and scheme for the synthesis of unmethylated spermine analogs as polyamine transport inhibitors with a linker attached via amide bonds to the polyamine chains (BS-3, BS-4, BS-5 and BS-6 compounds). The method of synthesis is described in greater detail in Example 1.
  • FIG 13 Initial route of synthesis of terminal C-methylated, dimeric spermine analogs as transport inhibitors with a linker attached via an alkyl bond to the polyamine chains (BMS-3, BMS-4, BMS-5 and BMS-6). The steps presented in this figure describe the complete route of synthesis leading to the precursor N', N 4 , N 8 , N /2 -tetra (Boc)-l, 12- dimethylspermine-5-carbinol (XV).
  • FIG 16. The final step of the synthesis of BMS compounds (XX); the Boc- protected, cross-linked 1, 12-dimethylspermine dimer is deprotected to generate the BMS compounds.
  • BMS-3, BMS-4, BMS-5 and BMS-6 correspond to N", N"-bis ([1, 12- dimethyl-spermine]-5-methyl)-diaminoalkanes where the diaminoalkane linker is 1,3- diaminopropane, 1 ,4-diaminobutane, 1,5-diaminopentane, and 1 ,6-diaminohexane, respectively.
  • FIG 17 A, 17B and 17C presents three classes of dimeric polyamine transport inhibitors according to the site of attachment of the linker (L) to the polyamine chain.
  • FIG 17A - C-linked dimeric analogs is H, methyl, ethyl or propyl; R 2 is H or methyl; R 3 is an alkyl, amide, keto, ether, thioether, phosphono or sulfonyl group; x is greater than 2 and less than 5 (2 ⁇ x ⁇ 5), and the sum of y+z is greater than or equal to 2 and less than or equal to 6 (2 ⁇ y + z ⁇ 6).
  • the linker L is any chemical structure covalently linked to the R 3 groups and which prevents the uptake of the analog.
  • FIG 17B N-linked dimeric analogs.
  • R is H, methyl, ethyl or propyl;
  • R 2 is H or methyl;
  • x is greater than 2 and less than 8 (2 ⁇ x ⁇ 8), and
  • w is greater than 2 and less than 7 (2 ⁇ w ⁇ 7).
  • the linker L is any chemical structure covalently linked to one internal amino group of each polyamine chain and which prevents the uptake of the analog.
  • FIG 17C C-linked/N-linked mixed dimeric analogs.
  • R is H, methyl, ethyl or propyl; R 2 is H or methyl; x is greater than 2 and less than 5 (2 ⁇ x ⁇ 5), the sum of y+z is greater than or equal to 2 and less than or equal to 6 (2 ⁇ y + z ⁇ 6), and w is greater than 2 and less than 8 (2 ⁇ w ⁇ 8).
  • the linker L is any chemical structure covalently linked to one internal amino group of one polyamine chain and to the R 3 of the other polyamine chain, and which prevents the uptake of the analog.
  • FIG 18. Initial route of synthesis of unmethylated, ⁇ -alkylated dimeric spermine analogs (FIG 17B). Steps leading to the synthesis of the intermediate N 1 -benzyl, N 3 , N 12 - di(CBZ)-spermine.
  • FIG 19. Final steps for the synthesis of unmethylated, N-alkylated dimeric spermine analogs (FIG 17B, represented by type compound XXIX).
  • FOG 17B For the aliphatic linker
  • FIG 20 Initial route of synthesis of terminal C-methylated, N-alkylated dimeric spermine analogs (FIG 17B). Steps leading to the synthesis of the intermediate N", N"-bis (N-[N-Boc-3-amino, 3-methylpropyl], N-[4-aminobutyl])- ⁇ -diminoalkane. For the aliphatic linker-(CH 2 ) n -, 2 ⁇ n ⁇ 51.
  • FIG 21 Final steps for the synthesis of terminal C-methylated, N-alkylated dimeric spermine analogs (FIG 17B, represented by type compound XXXVIII).
  • FOG 17B Final steps for the synthesis of terminal C-methylated, N-alkylated dimeric spermine analogs (FIG 17B, represented by type compound XXXVIII).
  • aliphatic linker-(CH 2 ) n -, 2 ⁇ n ⁇ 51 For the aliphatic linker-(CH 2 ) n -, 2 ⁇ n ⁇ 51.
  • FIG 22 Initial route of synthesis of 1,12-dimethylspermine dimers cross-linked through jV-alky 1/5 -alkyl attachments of the linker (FIG 17C). Steps leading to the synthesis of the intermediate N /N-Boc-3-amino, 3-methylpropyl], N-[N-FMOC-4- aminobutyl]), N°-[5-(N', N 4 , N 8 , N 12 -tetra (Boc)-spermine)-methyl]- , ⁇ -diaminoalkane.
  • FIG 23 Intermediate route of synthesis of 1,12-dimethylspermine dimers cross- linked through N-alkyl/5 -alkyl attachments of the linker (FIG 17C). Steps leading to the synthesis of the intermediate N N-Boc-3-amino, 3-methylpropyl], N-[8-amino-5-aza-
  • FIG 24 Final route of synthesis of 1,12-dimethylspermine dimers cross linked through N 4 -alkyl/5-alkyl attachments of the linker (FIG 17C represented by type compound XLV).
  • linker (CH 2 ) n -, 2 ⁇ n ⁇ 51.
  • FIG 25 illustrates the structure of representative dimeric transport inhibitors with a triamine backbone that are included in the present invention.
  • BABAC is NN'-bis(3- aminopropyl), N, N'-bis(4-aminobutyl)cystamine, a dimeric spermidine derivative with a diethyl disulfide linker;
  • B ⁇ Spd-(n+2) (standing for 6/ ym-norspermidine)molecules with a carbon chain length of n+2 atoms; 0 ⁇ n ⁇ 7) represents the general structure of dimeric sym-norspermidine-derived transport inhibitors with an aliphatic linker;
  • BSpd-(n+2) standing for bw(spermidine) molecules with a carbon chain length of n+2 atoms;
  • 0 ⁇ n ⁇ 7) represents the general structure of dimeric spermidine-derived transport inhibitors with an aliphatic linker;
  • TADAX is N,N,N'
  • FIG 26 illustrates the structure of other representaive dimeric transport inhibitors with a triamine backbone that are included in the present invention.
  • BABA-trans and BABA-cis stands for the trans and cis isomers of N,N'-NN'-bis(3-aminopropyl),NN'- bis(4-aminobutyl) derivatives of ⁇ , ⁇ '-diaminoalkenes, which are dimeric derivatives of either .sy/w-homospermidine, .sym-norspermidine or spermidine.
  • BABA-yne stands for
  • N,N'-N, N '-bis(3 -aminopropy 1),N N '-bis(4-arninobutyI) derivatives of ⁇ , ⁇ ' -diaminoalkynes which are dimeric derivatives of either syw-homospermidine, 5ym-norspermidine or spermidine.
  • FIG 28 illustrates synthetic Scheme 2 used to obtain dimeric .sym-homospermidine, sym-norspermidine-, or spermidine-derived polyamine transport inhibitors (XIV) from the parent triamine (XI) cross-linked with a linker L via symmetrical N-alkylation of the secondary amino group of said triamine, and typical examples of said linker.
  • FIG 29 illustrates synthetic Scheme 3 used to obtain dimeric .sym-homospermidine-
  • XIV 5y -norspermidine-, or spermidine-derived polyamine transport inhibitors (XIV) from the trityl-protected parent triamine (XII) cross-linked with a linker L via symmetrical amidation of the secondary amino group of said triamine followed by reduction of the amide bonds, and typical examples of said linker.
  • THF tetrahydrofuran.
  • FIG 30 illustrates synthetic Scheme 4 used to obtain dimeric sjr ⁇ -homospermidine-, .sym-norspermidine-, or spermidine-derived polyamine transport inhibitors (XVIII) from the parent triamine (XI) cross-linked via a diethyl disulfide linker L by symmetrical alkylation of the secondary amino group of said triamine.
  • TFA trifluoroacetyl.
  • FIG 31 illustrates a comparison between the structures of NN'-bis(3- aminopropyl),NN'-bis(4-aminobutyl)cystamine (BAB AC), a dimeric spermidine derivative with a diethyldisulfide linker, of its monomeric thiol form N-(3-aminopropyl)JV-(4- aminobutyl)cysteamine (AAC) and of 2,2'-dithiobis[N-ethyl-spermine 5-carboxamide (DESC).
  • FIG 32 [A-32C] represents the inhibition of [ 3 H]putrescine (32A), [ 3 H] spermidine
  • FIG 33 represents the inhibition of [ 14 C]spermine uptake by spermine (SPM), DESC and TADAX in ZR-75-1 human breast cancer cells. Data are the mean ⁇ SD of triplicate determinations from a representative experiment.
  • FIG 34 represents the relative cytotoxicity of .sym-norspermidine ( ⁇ Spd), BABAC and TADAX in ZR-75-1 human breast cancer cells. Data are the mean ⁇ SD of triplicate determinations from a representative experiment.
  • Sym-norspermidine, ornithine dihydrochloride and other reagents for organic syntheses were purchased from Aldrich (Milwaukee, WI) and Sigma (St. Louis, MO).
  • Reversed phase silica gel liquid chromatography was performed with a LichroprepTM RP- 18 C lg silica gel column (40-63 ⁇ M ; BDH, St. Laurent, Qc, Canada) using a gradient of CH 3 CN:MeOH:H 2 O (25:35:40 to 50:30:20) as eluent. Homogeneity of synthetic products was assessed by thin-layer chromatography performed on 0.20 mm F 254 silica gel 60 plates or 0.25 mm F 254 S RP-18 reversed phase silica gel plates (E. Merck, Darmstadt, Germany).
  • FIR spectra were obtained on a Perkin-Elmer 1600 spectrophotometer (FTIR series) and were expressed in cm "1 .
  • FTIR series Perkin-Elmer 1600 spectrophotometer
  • 13 C were recorded at 75.47 MHz.
  • Chemical shifts ( ⁇ in ppm) were referenced to CDC1 3 (7.26 ppm for ⁇ and 77.00 ppm for 13 C).
  • Mass spectra were recorded at the Mass Spectrometry Regional Center (University of Montreal, Montreal, Qc, Canada) by fast atomic bombardment mass spectrometry (FABMS) or liquid secondary ion mass spectrometry (LSMIS), using a VG AutoSpecQTM and a Kratos MS50 TCTA, respectively.
  • FABMS fast atomic bombardment mass spectrometry
  • LSMIS liquid secondary ion mass spectrometry
  • [2,3- 3 H(N]putrescine dihydrochloride (4.1 x 10 4 Cl/mol) and [l,8- 3 H(N)]spermidine trihydrochloride (1.5 x 10 4 Cl/mol) were obtained from Dupont- ⁇ ew England Nuclear (Lachine, Qc, Canada).
  • [5,8- 14 C]spermine tetrahydrochloride (108 Cl/mol)) was purchased from Amersham (Arlington Heights, IL).
  • DFMO was provided by the Marion Merrell Dow Research Institute (Cincinnati, OH).
  • Fetal bovine serum (FBS) and CosmicTM calf serum were from Hyclone (Logan, UT).
  • the heterobifunctional reagent l(-p-azidosalicylamido)- 4-iodoacetamido)butane (ASIB) as obtained from Pierce (Rockford, IL). Lucifer Yellow (OY) iodoacetamide was purchased from Molecular Probes (Eugene, OR). Putrescine dihydrochloride, spermidine trihydrochloride, spermine tetrahydrochloride, iodoacetamide, 5,5'-dithio(2-nitrobenzoic acid) and 3,4-diaminobenzoic acid as well as tissue culture reagents were purchased from Sigma.
  • Ort/zo-phthaldialdehyde was purchased from Fluka (Ronkonkoma, NY) and other reagents for high-performance liquid chromatography (HPLC) were from Fisher Scientific (Montreal, Qc, Canada) or Aldrich (Milwaukee, WI).
  • DTT-free solution of MESC (20 mM in H 2 ) were added 50 ⁇ l of 50 mM Tris-HCl (pH 7.0) and 105 ⁇ l of a 40 mM solution of either iodoacetamide, LY iodoacetamide or ASIB in a light-protected microcentrifuge tube, and the mixture was incubated for 2 hours at 37 °C.
  • the extent of thiol modification was assessed by measuring the amount of thiol remaining at the end of the incubation with 5,5'- dithio- ⁇ s-(2-nitrobenzoic acid) as described above, and was determined to be essentially complete.
  • ZR-75-1 cells were maintained in phenol red-free RPMI 1640 medium supplemented with 10% fetal bovine serum, 2mM L-glutamine, 1 mM sodium pyruvate, 15 mM Hepes, 10 nM 17 ⁇ - estradiol, and antibiotics [MEZR medium] (Huber, M. and Poulin, R. 1995. Cancer Res., 55, 934-943).
  • CHO-K1 cells were routinely grown in -Minimal Essential Medium supplemented with 10% CosmicTM calf serum in a 5% CO 2 humid atmosphere at 37°C.
  • ZR-75-1 cells were cultured in MEZR medium or in phenol red- free RPMI 1640 supplemented with 2mM L-glutamine, 1 mM sodium pyruvate, 15 mM
  • the effect of the transport inhibitors on cell growth was measured by incubating ZR-75-1 cells for 11 days in medium supplemented with antagonist, polyamines and/or 1 mM DFMO as indicated, followed by colorimetric determination of DNA content with 3,4-diaminobenzoic acid (Simard, J., Dauvois, S., Haagensen, D.E., Levesque C, Merand, Y. and Labrie, F. 1990.
  • ZR-75-1 cells were plated in 100 mm culture dishes at 5 x 10 5 cells/dish in MEZR medium and grown for 5 days with medium changes every other day. Fresh MEZR medium containing the indicated concentration of transport antagonist was then added, plus or minus 200 ⁇ M cycloheximide (CHX), and cells were incubated for 1 or 6 hours.
  • CHX cycloheximide
  • Trichloroacetic acid was then added to DTT-containing samples to a final concentration of 10% (wt/v). Samples were dispersed for 2 minutes in a sonicating water bath, and pelleted in a microcentrifuge for 5 minutes. The trichloroacetic acid-insoluble pellet was solubilized in 300-500 ⁇ l of 1 N NaOH and used to determine protein content using bovine serum albumin (fraction V) as standard.
  • DESC stability was tested by incubating the compound dissolved (at 50 ⁇ M) in PBS or in IMEM medium containing 10% (v/v) fetal bovine serum plus or minus 1 mM aminoguanidine in a humid 5% CO 2 atmosphere at 37°C and in the absence of cells. At indicated times, trichloroacetic acid was added to aliquots of this solution to a final concentration 10% (w/v) and the samples directly analyzed by HPLC as above.
  • the rate of putrescine and spermidine transport was determined in ZR-75-1 cells incubated in serum-free RPMI 1640 medium as described (Lessard, M., Zhao, C, Singh, S.M. and Poulin, R. 1995. J. Biol. Chem. 270: 1685-1694), using [ 3 H ]putrescine (30 Ci/mol) and [ 3 H]spermidine (20 Ci/mol), respectively as substrates for a 20 minute-assay period.
  • Spermine uptake was similarly determined, using 1 ⁇ M [ 14 C]spermine (32 Ci/mol) as substrate.
  • Uptake activity was expressed per amount of DNA as fiuorometrically determined using 3,4-diaminobenzoic acid (Simard, J., Dauvois, S., Haagensen, D.E., Levesque, C, Merand, Y. and Labrie, F. 1990. Endocrinology, 126: 3223-3231).
  • spermidine uptake activity in CHO-K1 cells 80% confluent cell monolayers were rinsed twice with PBS and incubated for 20 minutes at 37°C in 400 ⁇ l of buffer A (20 mM Tris-HCl, pH 7.4; 0.42 mM CaCl 2 ; 0.41 mM MgSO 4 ; 103 mM NaCl;
  • K ?? K; and V max values were then estimated by Lineweaver-Burke analysis.
  • K f values were also estimated by measuring uptake activity in the presence of logarithmically increasing concentrations of antagonist, and using the Cheng-Prusoff equation (Cheng, Y.-C. and Prusoff, W.H. 1973. Biochem. Pharmacol. 22: 3099-3108) by iterative curve fitting for a sigmoidal curve.
  • Cheng-Prusoff equation Cheng-Prusoff equation
  • Prusoff W.H. 1973. Biochem. Pharmacol. 22: 3099-3108
  • v, s, and are the transport velocity, substrate concentration and inhibitor concentration respectively, was used to calculate the inhibition constants for inhibitor/carrier complex formation (K f ) and carrier/inbibitor/substrate complex formation (K j 1 ) (Dixon, M. and Webb, E.C. 1976. Enzymes, 3rd Ed., Academic Press, San Diego, CA).
  • K f inhibitor/carrier complex formation
  • K j 1 carrier/inbibitor/substrate complex formation
  • the value of K j for a mixed competitor/non-competitor was estimated from the intersect of equations V 1 vs / at two different substrate concentrations (Dixon, M. and Webb, E.C. 1976. Enzymes, 3rd Ed., Academic Press, San Diego, CA).
  • the time course of intracellular accumulation of spermidine in the presence of transport antagonists was determined by incubating ZR-75-1 cells in 24-well plates with DESC (50 or 200 ⁇ M) or MESC (200 ⁇ M) in dissolved in MEZR medium containing 5 ⁇ M [ 3 H]spermidine in the presence or absence of cycloheximide (CHX, 200 ⁇ M), and harvesting at the indicated times for the determination of intracellular radioactive contents, as described above for polyamine uptake assays.
  • DESC was the most potent antagonist of [ 14 C]spermine transport in ZR- 75-1 cells, with a K, value about 5-fold and 16-fold lower than that of DEASC and MESC, respectively.
  • the ability of spermine to compete against [ 3 H]putrescine and [ 3 H]spermidine uptake was in fact only about 7-fold higher than that of DESC (Fig. 3).
  • DESC (Fig. 4A) and MESC (data not shown) were pure competitive inhibitors of [ 3 H]putrescine uptake at concentrations up to 100 and 200 ⁇ M, respectively.
  • K values determined for DESC, MESC and DEASC toward putrescine, spermidine and/or spermine uptake, in relation with the mutual transport interactions between the latter substrates.
  • K values of the three spermine conjugates with respect to putrescine uptake were 3-fold to 5-fold higher than for spermine uptake, unlike spermidine and spermine which both inhibited the uptake of either substrate with similar potency, and with a K, roughly equal to their K,,, as substrate.
  • MESC was thus derivatized with substituting groups of different sizes and charges through thioether linkage with three different iodoacetamides, namely LY iodoacetamide, ASIB and iodoacetamide itself, and the ability of the resulting complexes (MESC-LY, MESC-ASIB, and MESC-acetamide, respectively) to inhibit spermidine uptake was then evaluated.
  • LY iodoacetamide namely LY iodoacetamide, ASIB and iodoacetamide itself
  • MESC-acetamide MESC-acetamide
  • ZR-75-1 cells The ability of ZR-75-1 cells to accumulate DESC and MESC was determined. Since DESC was eluted as a late, broad peak in the HPLC system used, DTT was added to cell extracts to reduce DESC to MESC and decrease the detection threshold. Results are shown in Table II. ZR-75-1 cells were incubated for 1 or 8 hours in MEZR medium in the presence of 50 or 200 ⁇ M DESC or MESC prior to determination of polyamine contents. CHX was added at 200 ⁇ M where indicated. Other details are provided under "Materials and Methods.” Values are the mean ⁇ SD of triplicate determinations from 2 independent experiments.
  • cycloheximide which is known to upregulate polyamine uptake by preventing the synthesis of a polyamine-induced feedback repressor of transport (Lessard, M., Zhao, C, Singh, S.M. and Poulin, R. 1995. J. Biol. Chem. 270: 1685-1694; Mitchell, J.L.A., Diveley, R.R., Jr. and Bareyal-Leyser, A. 1992. Biochem. Biophys. Res. Commun. 186: 81-88), did not enhance DESC internalization, in marked contrast with its effect on spermidine accumulation under similar conditions (Fig.
  • spermidine accumulation in the presence of either inhibitor followed a pattern similar to that of control cells, i.e. a rapid phase during the first 60 minutes, followed by a much slower rate of accumulation thereafter, which was nearly independent of antagonist concentration.
  • This pattern suggests that even cellular levels of newly internalized spermidine as low as 20% of those found under control conditions, e.g., in cells treated with 200 ⁇ M DESC, may induce a near maximal degree of feedback repression of polyamine transport. Nevertheless, even a 40-fold excess of the most potent antagonist (i.e. 200 ⁇ M DESC) only decreased net spermidine accumulation by only 50% after 6 hours.
  • the most potent antagonist i.e. 200 ⁇ M DESC
  • DESC was only mildly growth inhibitory at 50 ⁇ M, there was an abrupt, aminoguanidine-resistant increase in toxicity at 200 ⁇ M.
  • spermine was acutely cytotoxic at 50 ⁇ M, an effect that was only partly prevented by aminoguanidine.
  • MESC was considerably less toxic than its dimer, with a 35% decrease in cell growth at 200 ⁇ M which was not blocked by aminoguanidine.
  • DESC and to a much lesser degree, its thiol monomer MESC, are cytotoxic toward breast cancer cells at high concentrations through a mechanism that does not involve BSAO.
  • DESC is indeed a potent antagonist of polyamine accumulation
  • the slow residual uptake that occurred even at a 40-fold molar excess of inhibitor might be sufficient to counteract polyamine depletion by inhibitors of polyamine biosynthesis.
  • DESC solutions (20 ⁇ M) made in PBS or in sterile IMEM medium enriched with 10% (v/v) FBS were incubated for 20 minutes or 48 hours under cell-free conditions at 37 °C in a humid 5% CO 2 atmosphere, and the polyamine analog was then analyzed by ion-pair reversed-phase HPLC. After 48 hours, degradation of DESC to two new amine-containing derivatives occurred in IMEM (Fig. 9 A, B) but not in PBS (Fig.
  • DESC a novel type of spermine derivative, is shown to be endowed with high affinity for the polyamine transport system while being highly resistant to cellular uptake. The combination of these two attributes confers unique characteristics to DESC as a pure competitive antagonist of polyamine uptake.
  • MESC thioethers as diverse in size as MESC-LY, MESC-ASIB, or MESC- acetamide had K; values virtually identical to that of MESC, indicating that the thiol group of MESC does not specifically determine its lower affinity as a polyamine transport inhibitor as compared with DESC.
  • MESC- cysteamine mixed disulfide DEASC
  • DEASC MESC- cysteamine mixed disulfide
  • DESC The biochemical properties of DESC clearly illustrate that the binding affinity of a compound can be dissociated from its ability to serve as a substrate for the polyamine transporter.
  • the large size of DESC cannot be the main factor preventing its internalization through the channel-like portion of the transporter since MESC was also virtually impermeant.
  • MESC was also virtually impermeant.
  • the mere attachment of an amido side chain on the spermine backbone would appear to be responsible per se for the impaired internalization of MESC and its derivatives.
  • TV-alkylated spermidine derivatives are far better competitors of spermidine uptake than their N-acyl counte ⁇ arts in mouse leukemia cells, in support of the notion that charged secondary amino groups are important in the interaction with the
  • chlorambucil-spermidine which bears a N-propyl chlorambucil carboxamide side chain on the central nitrogen of spermidine, is a good substrate of the polyamine transport system, with a K,,, averaging that of spermidine (Holley, J.L., Mather, A., Wheelhouse, R.T, Cullis, P.M., Hartley, J.A., Bingham, J.P., and Cohen, G.M. 1992. Cancer Res. 52: 4190-4195).
  • a spermidine conjugate with a chlorambucil carboxamide side chain directly attached at the C5 position of the spermidine head is a very poor substrate of the polyamine uptake system (Stark, P.A., Thrall, B.D., Meadows, G.G., and Abdel-Monam, M.M. 1992. J. Me Chem. 35: 4264-
  • MESC-ASIB might serve as a photoaffinity label to detect polyamine-binding proteins, including the polyamine carrier. Experiments are currently conducted with 125 I-labeled MESC-ASIB to assess its usefulness as a probe to identify the mammalian polyamine transporter.
  • the basic features of this molecule should be useful for the design of potent transport inhibitors with minor non-specific effects on cell viability.
  • the inherent structural features of DESC that confer its high affinity and resistance to uptake should thus provide a useful framework for the design of potent irreversible inhibitors of polyamine transport, which could inco ⁇ orate an alkylating group such as that used in the design of specific suicide substrates of mammalian glucose transporters (Clark, A.E., and Holman, G.D. 1990. Biochem. J. 269: 615-622; Lehmann, J., and Scheuring, M. 1995. Carbohydrate Res. 276: 57-74)].
  • Eflornithine Eflornithine. These molecules are based on the overall design of a prototype, 2, 2'- dithiobis(N-ethyl-spermine-5-carboxamide) (DESC). DESC has recently been reported to act as a competitive and potent antagonist of polyamine uptake in leukemia and breast cancer cells. DESC is proposed here to potentiate the chemotherapeutic efficacy of DFMO. While not intending to be limited to any particular theory, it is proposed that such effect is provided by preventing the replenishment of DFMO-treated tumor cells with polyamines from exogenous sources. Structural modifications to the molecule will improve it to a pharmacologically useful compound.
  • DESC 2, 2'- dithiobis(N-ethyl-spermine-5-carboxamide)
  • DESC analogs Two types of DESC analogs will be synthesized, and characterized for their ability to inhibit polyamine transport and to enhance the therapeutic action of DFMO in various
  • the first type of analogs will be simply obtained by substituting the original cystamine side chain of DESC with ⁇ , ⁇ -diamine cross-linkers of varying length. The synthesis of these analogs will help in the short term to optimize the length of the cross-linker chain, and to rapidly evaluate their relative ability to potentiate DFMO action in vitro.
  • the second type of analogs will be made according to a new route of synthesis to introduce methyl groups at the extremities of the spermine- like backbone, and will also inco ⁇ orate alkylation instead of acylation of the aliphatic, ⁇ , ⁇ -diamine cross-linker in order to improve their affinity for the polyamine transport system, their potency as antagonists of uptake and as enhancers of DFMO therapeutic action.
  • the pharmacological evaluation of the second-type analogs will be conducted in a standard mouse model bearing L1210 leukemia tumor cells treated with DFMO.
  • DESC was designed for biochemical use. It was found to degraded in physiological media due to thiol-disulfide reaction with compounds such as L-cystine. DESC cannot efficiently counteract the ability of exogenous spermidine to reverse DFMO-induced cytostasis in breast cancer cells as a result of this instability (21). DESC is also subject to attack by serum amine oxidase (SAO), an ubiquitous plasma enzyme which oxidatively
  • DESC analogs are prepared with unmodified spermine backbones but different side chain lengths as lead compounds to guide us in the design of methylated analogs described herein. This series of compounds will be synthesized in order to: (i) Perform a structure-function study in the short-term to determine the optimal length of the cross-linker for inhibition of polyamine uptake.
  • BS-3, BS-4, BS-5 and BS-6; Fig. 12 The kinetic properties of these DESC analogs (abbreviated as BS-3, BS-4, BS-5 and BS-6; Fig. 12), as inhibitors of polyamine transport will be determined by uptake assays of radiolabeled putrescine, spermidine and spermine, according to procedures in Huber et al. (1996), J. Biol. Chem., 271: 27556-27563, which is specifically inco ⁇ orated herein by reference. These structures are shown below.
  • These compounds are expected to be stable under cell culture conditions in the presence of aminoguanidine, a SAO inhibitor (13, 28, 40, 46, 49, 66, 67).
  • SAO inhibitor 13, 28, 40, 46, 49, 66, 67.
  • These polyamine transport inhibitors will be evaluated using ZR-75-1 human breast cancer cells and L1210 mouse leukemia cells. Briefly, the rate of cell proliferation will be determined in ZR-75- 1 and L1210 cells grown in the presence or absence of DFMO (1 and 5 mM, respectively), and of the transport inhibitor candidate to be analyzed, in the presence of increasing concentrations of putrescine or spermidine.
  • the ability of the transport antagonist to prevent the reversal of DFMO-induced growth inhibition by exogenous putrescine or spermidine will provide a valid measurement of the pharmacological potential of these compounds as enhancers of DFMO action in vivo.
  • These studies will also include (a) dose- response experiments to evaluate the cytotoxicity of these analogs and the optimal concentration for their use as inhibitors of polyamine uptake, and (b) measurement of the uptake of the transport inhibitors during incubation with tumor cells by HPLC, along with their effect on polyamine pools.
  • This improved scheme also includes the use of mono-FMOC-protected diamines as building blocks for cross-linking the dimethylspermine-5-methyl precursors, as described above for the unmethylated DESC analogs.
  • the resulting compounds are abbreviated as BMS-3, BMS-4, BMS-5 and BMS-6 (Fig. 16; compounds XXa to Xxd).
  • n 3, 4, 5 or 6.
  • Protocol I - Toxicity will first be determined by single i.v. and i.p. injections of logarithmically increasing drug concentrations to mice and estimating the LD 50 . Blood samples will be taken at intervals to measure the plasma drug concentration by ion pairing reverse-phase HPLC (22, 23). Body weight and liquid consumption will also be monitored for 10 days, at the end of which period animals will be sacrificed to evaluate the incidence of liver and kidney damage. A similar experiment will be conducted by dissolving the drug in the drinking water with free access to the animals.
  • mice will be injected with LI 210 cells, with concomitant treatment with DFMO or vehicle, plus or minus 2 different sublethal doses of the transport antagonist on a daily schedule. Oral, i.v. and i.p. routes will be compared for the transport antagonist. Survival will be evaluated for up to 120 days, with regular body weight measurements and blood sampling to determine the steady-state plasma concentrations of inhibitor.
  • L1210 cells are strongly immunogenic tumors and cured animals develop extended immunity against this leukemia (1). Thus, to evaluate the curative potential of the drug combination, survivors will be rechallenged with L1210 cells in the absence of treatment and survival monitored.
  • the present example demonstrates the utility of the present invention with the use of compounds that are analogs of spermine that include two chains connected to one another through a linker.
  • the linker molecule that attaches the two spermine chains may be any spacer chain that is capable of bridging the polyamine chains.
  • the two chains may attach to the linker at an internal C atom or an N group within the chain. It is also possible for one chain to be connected to the linker through one of its carbon molecules, while the second chain attaches to the linker molecule through an N group within its chain.
  • the general structure of compounds claimed include the following characteristics:
  • the central carbon chain of the spermine backbone can have between 3 and 7 methylene groups or carbon atoms. This is the range of central chain length that can be accommodated with good affinity by the mammalian polyamine transporter (81).
  • Each methylene group of the polyamine chains can be modified by methyl groups without compromising the ability of the inhibitor to interact with the polyamine transporter.
  • the linkage between the polyamine chains and the spacer may comprise any type of linkage compatible with a K ⁇ ⁇ 20 ⁇ M (relative to spermine) for the resulting inhibitor, such as direct alkyl substitution or ether group on the central methylene groups (Structure 1), or alkylation on the secondary amino (Structure 2) groups of the polyamine chain.
  • a K ⁇ ⁇ 20 ⁇ M relative to spermine
  • R is H, methyl, ethyl or propyl
  • R 2 is H or methyl
  • x is greater than two and less than five (2 ⁇ x ⁇ 5)
  • the sum of y+z is greater than or equal to 2 and less than or equal to 6 (2 ⁇ y+z ⁇ 6).
  • L a chemical structure (the linker) connecting covalently the two polyamine chains via alkyl, amide, ether or thioether bonds with a substituent group (R 3 ) attached on a carbon atom located between the two most internal amino groups of the polyamine chain.
  • R is H, methyl, ethyl or propyl
  • R 2 is H or methyl
  • x is greater than two and less than five (2 ⁇ x ⁇ 5)
  • w is greater than 2 and less than 8 (2 ⁇ x ⁇ 8)
  • the sum of y+z is greater than or equal to 2 and less than or equal to 6 (2 ⁇ y+z ⁇ 6).
  • R is H, methyl, ethyl or propyl
  • R 2 is H or methyl
  • x is greater than two and less than five (2 ⁇ x ⁇ 5)
  • w is greater than 2 and less than 8 (2 ⁇ x ⁇ 8)
  • the sum of y+z is greater than or equal to 2 and less than or equal to 6 (2 ⁇ y+z ⁇ 6).
  • Alkylation can be preferred over amidation because the former allows a greater flexibility to the polyamine chain to adopt the optimal conformation to interact with the polyamine transporter (81).
  • the Linker (L) can be of any nature or chain length, as long as the total mass of the final structure does not exceed 3,000. These molecules may in other embodiments be described as having a total mass of between about 50 to about 2,500, or about between 500 to about 1500 or about 1,000 as a total mass.
  • linkers may comprise alkyl, ether, a thioether, amide, phosphono, keto, amine, or sulfonyl groups or a combination thereof.
  • the linker may comprise a carbon chain by a length of 2 to 50 carbons.
  • the carbon chain will have a length of between 5 to about
  • the synthetic original polyamine is either sym-norspermidine, yym-homospermidine, or spermidine, and is cross linked to a second original polyamineby a side group on the central amino group of said polyamines.
  • spermidine dimers cross-linked by an ⁇ , ⁇ '-dialkyl disulfide side chain via N-alkyl bonds with the central amino group of each triamine chain as depicted for type compound I, NN'-bis(3-aminopropyl),NN'-bis(4- aminobutyl)cystamine or BABAC (structure I, Fig. 25),
  • B .sym-norspermidine dimers
  • the two polyamine backbones are either spermidine [N- (3-aminopropyl)- 1,4-diaminobutane], yym-homospermidine [N- (4-aminobutyl)-l,4-diaminobutane] or sym- norspermidine [N- (3-aminopropyl)-l,3-diaminopropane].
  • Each methylene group of the polyamine chain can be modified by methyl groups without compromising the ability of the inhibitor to interact with the polyamine transporter.
  • Each primary amino group of the polyamine chains can be modified by methyl, ethyl or propyl groups without compromising the ability of the inhibitor to interact with the polyamine transporter.
  • the central secondary amino groups of the two polyamine chains are connected via N-alkyl bonds by a linker (L) so that the resulting inhibitor has a K,- ⁇ 20 ⁇ M
  • the linker (L) can be of any nature or chain length, as long as the total final structure does not exceed 3,000. These molecules may in other embodiments be described as having a total mass of about 50 to about 2,500, or about between 500 to about 1 ,500, or about 1,000 as a total mass. By way of example, such linkers may comprise alkyl, aryl,
  • these synthetic derivatives comprise a structure of a first polyamine chain and a second polyamine chain according to the structure.
  • R is H, methyl, ethyl or propyl
  • R 2 is H or methyl
  • x is greater than two and less than five (2 ⁇ x ⁇ 5)
  • y is greater than two and less than five (2 ⁇ y ⁇ 5)
  • L is a chemical structure (the linker) covalently connecting said second polyamine chain to said second chain through an alkyl bond, such as a oc, ⁇ , - diamine cross-linker.
  • ZR-75-1 cells were grown for four days in twenty-four well plates in RPMI 1640 medium supplemented with 10% fetal bovine serum, InM estradiol, 2mM L-glutamine, 1 mM sodium pyruvate, 15 mM Hepes and antibiotics, and specific uptake of [ 3 H] putrescine, [ 3 H] spermidine and [ 14 C]spermine was measured as described (Lessard, et al.), using 20 ⁇ M and 3 ⁇ M substrate, respectively.
  • ZR-75-1 cells are a convenient system to assess the potential of polyamine transport inhibitors because they exhibit elevated polyamine uptake activity (Lessard, et al.).
  • BABAC was a potent inhibitor of putrescine, spermidine and spermine uptake in ZR-75-1 cells, with calculated apparent K ⁇ values of 0.15 ⁇ M, 0.68 and 2.1 ⁇ M, respectively.
  • spermidine relative to spermine can still generate potent polyamine transport inhibitors, and that the nature of the linker L as well as its site of covalent attachment on the polyamine backbone can strongly influence the potency of the dimer to inhibit polyamine uptake.
  • a comparison between the structures of DESC and BABAC suggests that a N-alkyl type of attachment leads to superior properties of polyamine transport inhibition than an amide linkage on a methylene group of the polyamine backbone, and that this effect dominates over the effect of elongating the polyamine chains.
  • TADAX N,NN'N'-tetrakis(3-aminopropyl)- ⁇ -xylylenediamine
  • TADAX (FIG 25) was synthesized according to the Scheme 2 described above (FIG 26). Briefly, into a solution of norspermidine (2.55 g) and diethylamine (8.0 mL) in chloroform (100 mL) was added trityl chloride (10.87 g) portionwise. After addition, stirring was continued for 24 h. The reaction mixture was washed with water and dried over anhydrous potassium carbonate. The solution was concentrated under reduced pressure and the residue was re-crystallized from dichloromethane-methanol to obtain N'.N'-bis trity norspermidine.
  • N N, N ', N '-tetrakis(N-trity 1-3 -aminopropy 1) /7-xylylene diamine (374 mg) was suspended in a solution of HCI 6 M (20 mL) and the mixture was refluxed for 24 hours. The solid was removed by filtration and the aqueous phase concentrated to 2-3 mL by rotary evaporation. Addition of ethanol into the concentrated solution afforded the hexahydrochloride salt of N,N,N',N'-tetrakis(3-aminopropyl) P-xylylenediamine, or TADAX (FIG 25).
  • TADAX triamine dimers
  • IC 50 >500 ⁇ M cytotoxicity of TADAX
  • BABAC disulfide BABAC
  • cytotoxicity of ⁇ ym-norspermidine has been well documented (Bergeron and Seligshon, Komori and Ohsugi, Porter and Bergeron), a less pronounced, but yet significant cytotoxicity of DESC has been found using ZR-75- 1 breast cancer cells. That cytotoxicity may well be related to the reactivity of the disulfide groups of DESC and BABAC with biological thiols and disulfides found in growth media and at the cell surface. Thus, despite the presence of four 3-aminopropyl groups on the TADAX backbone, and its two .sym-norspermidine-derived moieties, it is remarkably inert toward biological functions.
  • BS-3, BS-4, BS-5 and BS-6 correspond to the forms where the diaminoalkane linker is 1,3-diaminopropane, 1, 4-diaminobutane, 1, 5- diaminopentane and 1, 6-diaminohexane, respectively.
  • the spacer is going to be alkylated to the polyamine chain, the carboxyl group used as an acceptor in an amidation reaction is first reduced to an alcohol with LiAlH 4 . After protecting the amine groups with carbobenzoxy (CBZ) groups, the alcohol is then converted to a bromide with PBr 3 .
  • the resulting CBX-protected spermine bromide is then reacted with a diamine spacer with a 2: 1 stoichiometry to generate the CBZ-protected spermine dimer.
  • This dimer is finally deprotected by catalytic hydrogenation with Pd/C (82) to generate the unmethylated spermine dimer (the transport inhibitor).
  • Pd/C the transport inhibitor
  • the alcohol obtained as above is then converted to an alkoxide with sodium metal, and then reacted with an alkyl dihalide (e.g.
  • Methylated spermine analogs (FIG 17A): For example, ornithine methylester (X, FIG 13) is synthesized as described (89) and is diamidated with two equivalents of 3- azidobutyric acid (XII, FIG 13) using DCC/OHB, (90) to generate N ; , N -bis (3- azidobutyryl)-ornithine methylester (XIII, FIG 13). The latter is then reduced using BH 3 /THF (90) to obtain 1, 12-dimethylspermine-5 carbinol (XIV, FIG13).
  • the carbinol group is activated with PBr 3 to generate 1, 12-dimethyl-N', N 4 , N 8 , N /2 -tetra (Boc)-5-bromomethyl spermine (XVI, FIG 14), and reacted with FMOC- ⁇ H-(CH 2 ) n ⁇ H 2 (where 3 ⁇ n ⁇ 6) to
  • Unmethylated, N-alkylated spermine analogs (FIG 17B): A symmetrical dimer that can be made where the linker (L) bridges two polyamine derivative chains through one of the innermost, secondary nitrogens of each polyamine chain.
  • N-benzyl-l,3-diaminopropane (XXI, FIG 18) is first obtained by catalytic hydrogenation of 3-(benzylamino)propiononitrile with Raney nickel as described (84).
  • N-benzyl-l,3-diaminopropane is then N-alkylated with 3- bromobutyronitrile to generate N 1 -benzyl, N 3 -(3-cyanopropyl)-l,3-diaminopropane (XXII, FIG 18) (85).
  • N 1 -benzyl, N 3 -(3-cyanopropyl)-l,3,-diaminopropane is protected with a Boc group (86) to generate N 1 -benzyl, N 3 -Boc, N 3 -(3-cyanopropyl)-l,3,- diaminopropane (XXIII, FIG 18)
  • N 1 -benzyl, N 3 -Boc, ⁇ 3 -(3-cyanopropyl)- 1,3, -diaminopropane is reduced to N 1 -benzyl, N -Boc-spermidine ( XXIV, FIG 18) by catalytic hydrogenation with Raney nickel (84).
  • N 1 -benzyl, N 4 -Boc-spermidine is then cyanoethylated with acrylonitrile to generate N 1 -benzyl, N'-Boc, ⁇ -cyanoethyl- -spermidine, and reduced to N 1 -benzyl, N 4 -Boc-spermine by catalytic hydrogenation with Raney nickel (84) (XXV, FIG 18).
  • N 1 -benzyl, N-Boc, N 8 , N 12 -di(CBZ)-spermine is then deprotected to N 1 -benzyl, N 8 , N 12 -di(CBZ)-spermine with trifiuoroacetic acid as described (87) (XXVII, FIG 18).
  • N-benzyl, N 8 , N 12 -di(CBZ)-spermine can then be cross-linked with an ⁇ , ⁇ -dibromoalkane of the desired chain length to generate the corresponding bis(N'-benzyl, N 8 , N ,2 -di(CBZ)-spermine) dimer (XXVIII, FIG 19), which is then deprotected by catalytic hydrogenation with Pd/C (87) to generate the unmethylated, N-alkylated spermine dimer (the transport inhibitor) (XXIX, FIG 19).
  • XXIX the transport inhibitor
  • Dimeric polyamine transport inhibitors of a different type can be generated by cross-linking one polyamine chain to a linker through a N-alkyl bond as in Examples 3 and
  • N'-Boc-N ⁇ FMOC-l-methylspermidine (XXXII, FIG 20), obtained as described above (Example 4, steps a to b), is N ⁇ -alkylated using an ⁇ - bromoalkylphthalimide of the desired length as described (92), to generate the corresponding N-Boc, N-alkylphthalimide, N ⁇ FMOC-l -methylspermidine (XXXIX, FIG 22).
  • This scheme is used to prepare symmetrical dimers of sym-norspermidine by Michael addition via total cyanoethylation of hydrochloride salts of aliphatic or aromatic ⁇ , ⁇ -diamines.
  • This route is the simplest one to generate polyamine dimers since no amine protection is necessary.
  • the diamine hydrochloride of the desired nature and chain length (VIII, FIG 27) to be used as a crosslinker is stirred with a 4-fold excess of acrylonitrile in the presence of triethylamine (Et 3 N) in aqueous solution, yielding a N,N,N',N'- tetrakis(cyanoethyl)- , ⁇ -diamine (IX, FIG 27).
  • Cyanoethyl groups are then reduced with NaBH 4 in the presence of methanolic CoCl 2 as a reducing agent, resulting in the formation of a .sym-norspermidine dimer (X, FIG 27) with the desired linker L.
  • the compound is
  • linkers can be used according to that scheme, including aliphatic ⁇ , ⁇ - diaminoalkanes (preferably with a chain length greater than two and less than ten) which may inco ⁇ orate one double bond in a cis or trans configuration, or one triple bond, and aromatic diamines where the amino groups are present as aminoalkyl substituents at two different positions of the aromatic cycle, such as the para configuration shown in FIG 27.
  • spermidine, .sym-homospermidine and sym-norspermidine dimers can be prepared by protecting the primary amino groups of the precursor triamine with trityl groups, and directly alkylating the secondary amino group of the triamine with a dibromoalkene, a dibromoalkyne or a dibromoarene with the desired crosslinking carbon chain (Zang and
  • the diamide is then reduced to its N-alkyl form with LiAlH 4 in dry THF under nitrogen to obtain the trityl- protected form of the triamine dimer (XIII, FIG 28 and FIG 29).
  • the primary amine groups are then freed with 6 ⁇ HCI as in Scheme 2 to generate the desired dimeric polyamine transport inhibitor XIV (FIG 28 and FIG 29).
  • Triamines can be dimerized via N-alkyl bonds with a linker L containing a disulfide bond, generating polyamine transport inhibitors with even higher potency than DESC, which is a dimeric spermine disulfide cross-linked through amide bonds with position C5 of each spermine skeleton.
  • the following scheme allows the synthesis of dimeric triamines cross-linked via N-alkyl bonds with a diethyl disulfide chain.
  • the primary amino groups of the desired triamine XI (FIG 28 and FIG 30) are first protected with ethyl trifluoroacetate in aqueous acetonitrile to obtain the bis- trifluoroacetyl)-protected triamine XVI (FIG 30).
  • Gastrointestinal Tract edited by R. H. Dowling, U. R. Folsch and C. Loser. Dordrecht: Kluwer Academic Publ., 1992, p. 435-445.
  • Pegg, A. E., R. Poulin, and J. K. Coward Use of aminopropy ltransferase inhibitors and of non-metabolizable analogs to study polyamine regulation and function. Int. J. Biochem. Cell. Biol. 27: 425-442, 1995. 49. Pegg, A. E., R. Wechter, R. Poulin, P. M. Woster, and J. K. Coward. Effect of S- adenosyl-l,12-diamino-3-thio-9-azadodecane, a multisubstrate adduct inhibitor of spermine synthase, on polyamine metabolism in mammalian cells.

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Abstract

Cette invention se rapporte à la conception, à la synthèse et à l'utilisation thérapeutique d'une variété de nouveaux inhibiteurs de transport des polyamines. La caractéristique principale de cette classe d'inhibiteurs de transport consiste à incorporer un segment de liaison ou une chaîne latérale qui empêche l'assimilation des polyamines et contribue à conjuguer des analogues de polyamines pour former des dimères ayant un pouvoir inhibiteur élevé contre l'assimilation des polyamines. Ces nouveaux composés incorporent des caractéristiques qui sont conçues pour maximiser leur stabilité chimique et métabolique et leur capacité à se fixer au transporteur de polyamines, ainsi qu'à minimiser leur toxicité en empêchant leur absorption par les cellules. L'objectif de ces inhibiteurs est d'empêcher l'assimilation ou la récupération des polyamines en circulation par des cellules proliférant rapidement, telles que les cellules tumorales, afin de renforcer l'effet des inhibiteurs thérapeutiques de la biosynthèse des polyamines, telle que l'alpha-difluorométhylornithène.
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US6646149B1 (en) * 1997-07-15 2003-11-11 Nicolaas M. J. Vermeulin Polyamine analogues as therapeutic and diagnostic agents
ATE335719T1 (de) 1999-04-30 2006-09-15 Cellgate Inc Polyamine und ihre therapeutische verwendung
USRE43327E1 (en) 2001-01-08 2012-04-24 Aminex Therapeutics, Inc. Hydrophobic polyamine analogs and methods for their use
EP1453787A1 (fr) 2001-12-07 2004-09-08 SLIL Biomedical Corporation Polyamines substituees par un cycloalkyle pour le traitement du cancer, et procedes de synthese de celles-ci
CN102659605B (zh) * 2012-05-08 2013-11-13 天津市普莱克医药科技有限公司 一种亚精胺的合成方法
EP3432914B1 (fr) 2016-03-25 2020-09-23 Aminex Therapeutics, Inc. Polyamides biodisponibles
WO2020236562A1 (fr) 2019-05-17 2020-11-26 Cancer Prevention Pharmaceuticals, Inc. Méthodes de traitement de la polypose adénomateuse familiale
WO2022072586A1 (fr) 2020-09-30 2022-04-07 Aminex Therapeutics, Inc. Combinaison de substances médicamenteuses à base d'inhibiteur de transport de polyamine et de dfmo

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US5242947A (en) * 1988-02-10 1993-09-07 New York University Use of polyamines as ionic-channel regulating agents
US5456908A (en) * 1994-03-01 1995-10-10 The University Of Kentucky Research Foundation Polyamine-polyamine and polyamine-protein transport inhibitor conjugates and their use as pharmaceuticals and in research relating to polyamine transport
US6083496A (en) * 1996-10-22 2000-07-04 Universite Laval Polyamine transport inhibitors

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