Polyamine analogs that activate antizyme frameshifting Number:7,144,920 from the United States Patent and Trademark Office (PTO) owispatent Novel polyamines, their synthesis and use in pharmacological, cosmetic or agricultural applications are provided. The polyamines induce antizyme production which in turn down regulates both the production of polyamines by omithine decarboxylase (ODC) and the transport of polyamines by its corresponding polyamine transporter. These compounds will preferably enter the cell independent of the polyamine transporter. As drugs, these compounds are used to treat any disease associated with cellular proliferation including but not limited to cancer.<H frameshifting, Polyamine, Polyamine, analo, 7144920.html, Patent, pto, 7144920

Title: Polyamine analogs that activate antizyme frameshifting

Abstract: Novel polyamines, their synthesis and use in pharmacological, cosmetic or agricultural applications are provided. The polyamines induce antizyme production which in turn down regulates both the production of polyamines by omithine decarboxylase (ODC) and the transport of polyamines by its corresponding polyamine transporter. These compounds will preferably enter the cell independent of the polyamine transporter. As drugs, these compounds are used to treat any disease associated with cellular proliferation including but not limited to cancer.

Patent Number: 7,144,920 Issued on 12/05/2006 to Burns, et al.

Inventors: Burns; Mark R. (Shoreline, WA), Graminski; Gerard F. (Shoreline, WA)
Assignee: MediQuest Therapeutics, Inc. (Bothell, WA)

Appl. No.: 10/810,649

Filed: March 29, 2004

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
10251819	Sep., 2002	6914079

Current U.S. Class: 514/649 ; 564/336

Current International Class: A61K 31/135 (20060101); C07D 211/18 (20060101)

Field of Search: 564/336 514/649

References Cited [Referenced By]

U.S. Patent Documents


3755455	August 1973	Houlihan
4605765	August 1986	Miyamoto et al.
4720789	January 1988	Shander
5648394	July 1997	Boxall et al.
6001824	December 1999	Nakanishi et al.
2004/0058954	March 2004	Burns et al.

Foreign Patent Documents


0 645 370	Mar., 1995	EP
WO-96/22982	Aug., 1996	WO
WO-00/46187	Aug., 2000	WO
WO-00/46187	Aug., 2000	WO
WO-01/92218	Dec., 2001	WO

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Primary Examiner: Desai; Rita J.
Attorney, Agent or Firm: Connolly, Bove, Lodge & Hutz, LLP

Parent Case Text

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent application Ser. No. 10/251,819, filed Sep. 23, 2002 now U.S. Pat. No. 6,914,079.

Claims

What is claimed is:

1. A polyamine having the structure ##STR00013## wherein, n can be 0 to 8 and the aminomethyl functionality can be ortho, meta or para substituted, R is hydrogen, 2-aminoethyl, 3-aminopropyl, 4-aminobutyl, 5-aminopentyl, 6-aminohexyl, 7-aminoheptyl, or 8-aminooctyl and R.sub.1 is hydrogen and wherein said polyanilne is non-symmetrical.

2. A polyamine selected from the group consisting of one of the following compounds: ##STR00014##

3. A pharmaceutical composition comprising a polyamine according to any one of claims 1 or 2 and a pharmaceutically acceptable excipient, diluent or vehicle.

4. The composition of claim 3 wherein said excipient, diluent or vehicle is pharmaceutically acceptable.

5. The composition of claim 3 wherein said excipient, diluent or vehicle is for topical or intra-aural administration.

6. The composition of claim 3 formulated for intravenous, subcutaneous, intramuscular, intracranial, intraperitoneal, topical, transdermal, intravaginal, intranasal, intrabronchial, intracranial, intraocular, intraaural, rectal, or parenteral administration.

7. The polyamine of claim 1 wherein said structure is ##STR00015##

8. The polyamine of claim 1 wherein is ##STR00016##

9. The polyarnine of claim 1 wherein said structure is ##STR00017##

Description

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable

REFERENCE TO A COMPACT DISK APPENDIX

Not applicable

TECHNICAL FIELD

The present invention relates to novel polyamines, their synthesis and use in pharmacological, cosmetic or agricultural applications. The instant invention provides polyamines that induce antizyme production which in turn down regulates both the production of polyamines by ornithine decarboxylase (ODC) and the transport of polyamines by its corresponding polyamine transporter. These compounds will preferably enter the cell independent of the polyamine transporter. As drugs, these compounds are used to treat any disease associated with cellular proliferation including but not limited to cancer. As such, they will be useful as drugs to treat diseases where components of the immune system undergo undesired proliferation. These compounds will also be effective for the treatment of unwanted proliferation of hair on skin. The present invention also identifies key structural elements expected to comprise the antizyme inducing motif(s) of small molecules related to polyamines.

BACKGROUND OF THE INVENTION

The endogenous polyamines, putrescine, spermidine, and spermine contribute to many essential cellular functions through their interactions with DNA, RNA, proteins, and lipids (Pegg, A. E. Cancer Res. 48:759 774 (1988); Heby, O. et. al., Trends Biochem. Sci. 15:153 158 (1990); Janne, J. et. al., Ann. Med. 23:241 259 (1991); Brooks, W. H. Med Hypotheses 44:331 338 (1995); Igarashi, K. et. al., Biochem. Biophys. Res. Commun. 271:559 564 (2000); Casero, R. A. et. al., J. Med. Chem. 44:1 26 (2001)). Polyamines are essential for cell proliferation through their involvement in DNA replication, cell cycle regulation, and protein synthesis. Depletion of intracellular polyamine levels inhibits cell growth. Antizyme regulates polyamine levels both by inhibiting polyamine biosynthesis and uptake/import. The importance of their function is highlighted by the fact that specific biosynthesis, degradation, uptake and excretion pathways tightly control cellular polyamine levels (Heby, O. Differentiation 19:1 20 (1981); Seiler, N. et. al., Int. J. Biochem. 22:211 218 (1990); Seiler, N. et. al., J. P. Int. J. Biochem. Cell Biol. 28:843 861 (1996)). Excessive cell growth has been correlated with high levels of intracellular polyamines (Pegg, A. E. Cancer Res. 48:759 774 (1988)). Numerous tumor cell types have been analyzed and shown to have higher polyamine levels than normal, non-tumorigenic cells. Within a single tumor type, the more highly malignant tumors often have higher polyamine levels (Kurihara, H. et. al., Neurosurgery 32:372 375 (1993)). For these reasons, depletion of intracellular polyamine levels is an attractive approach for the inhibition of uncontrolled or undesirable cell growth.

Omithine decarboxylase (ODC) is the rate-limiting enzyme of cellular polyamine synthesis, converting omithine to putrescine. Putrescine is then converted to both spermidine and spermine by the sequential transfer of an aminopropyl group from decarboxylated --S-adenosylmethionine. Increasing concentrations of intracellular polyamine levels induce the production of antizyme which negatively regulates ODC by binding to it and targeting it for destruction. Antizyme has also been shown to inhibit polyamine uptake (Mitchell, J. L. et. al., Biochem. J. 299:19 22 (1994); Suzuki, T. et. al., Proc. Natl. Acad. Sci. USA 91: 8930 8934 (1994); Sakata, K. et. al., Biochem. Biophys. Res. Commun 238:415 419 (1997)) and recent evidence suggests that antizyme may increase polyamine excretion (Sakata, K. et. al., Biochem J. 347:297 303 (2000)). Therefore, antizyme can very effectively limit the accumulation of cellular polyamines.

Antizyme has been found in vertebrates, fungi, nematodes, insects and eukaryotes (Ivanov, I. et. al., Nucleic Acids Res. 28:3185 3196 (2000)). Three antizyme isoforms, AZ1, AZ2 and AZ3, have now been identified among vertebrates. Both AZ1 and AZ2 have wide tissue distribution but AZ2 mRNA is less abundantly expressed. AZ3 is expressed only in the testis germ cells (Ivanov, I. et. al., Proc. Natl. Acad. Sci. USA 97: 4808 4813 (2000); Tosaka, Y. et. al., Genes to Cells 5:265 276 (2000)) where expression begins early in spermiogenesis and finishes in the late spermatid phase. Antizyme production is controlled by a unique regulatory mechanism known as translational frameshifting (Matsufuji, S. et. al., Cell 80: 51 60 (1995)). The antizyme gene consists of two overlapping open reading frames (ORFs). The bulk of the coding sequence is encompassed in the second (ORF2) but it does not contain an initiation codon. ORF1 is short but contains two AUG initiation codons. Either one of the initiation codons can be used to initiate translation but normally little full length mRNA is made unless a +1 frameshift occurs just before the ORF1 UGA stop codon enabling translation to continue. Only minute quantities of antizyme are generally present in mammalian tissues. Polyamines and agniatine have been found to greatly enhance the efficiency of frameshifting (Hayashi, S. et. al., Trends Biochem. Sci. 21:27 30 (1996); Satriano, J. et. al., J. Biol. Chem. 273:15313 15316 (1998)). Vertebrates possess three elements that control frameshifting, the UGA stop codon in ORF1, a stem-loop structure 3' to the ORF1 UGA that can base pair with a portion of the loop and conserved sequence motifs within the 3' region of ORF1 (Matsufuji, S. et. al., Cell 80: 51 60 (1995)). It is unclear how or if polyamines interact directly with these structural elements to induce frameshifting. It is possible that there are unknown mediators that may involve the ribosome.

ODC is enzymatically active only as a homodimer since the active site contains structural contributions from both monomers. The interaction between the monomers is weak; whereas, antizyme has a high affinity for the ODC monomer. Antizyme binding disrupts the homodimer interface leading to the formation of two antizyme-ODC heterodimers that are now enzymatically inactive (Kameji, T. et. al., Biochim. Biophys. Acta 717:111 117 (1982); Kern, A. D. et. al., Struct. Fold. Des. 7:567 581 (1999)). Antizyme directs the ODC monomer to the proteosome where it is degraded without ubiquitination (Murakami, Y. et. al., Nature 360:597 599 (1992); Tokunaga, F. et. al., J. Biol. Chem. 269:17382 17385 (1994)). Antizyme is then released and free to interact with and destroy additional ODC monomers in a catalytic fashion. The AZ2 isoform has not been shown to catalytically induce the degradation of ODC, although AZ2 has been shown to inhibit both ODC and polyamine uptake equipotently (Zhu, C. et. al., J. Biol. Chem. 274:26425 26430 (1999). AZ3 is the most recently discovered antizyme and has also been shown to inhibit ODC (Ivanov, I. et. al., Proc. Natl. Acad. Sci. USA 97:4808 4813 (2000); Tosaka, Y. et. al., Genes to Cells 5:265 276 (2000)).

Antizyme is regulated by antizyme inhibitor, which has a higher affinity towards antizyme than ODC (Fujita, K. et. al., J. Biol. Chem. 274:26424 26430 (1982); Kitani, T. et. al., Biochim. Biophys. Acta 991:44 49 (1989); Murakami, Y. et. al., Biochem. J. 259:839 845 (1989)). Thus it may rescue ODC from degradation by displacing it from antizyme. Antizyme inhibitor, like ODC, forms a homodimer and has a high degree of sequence homology with ODC. However, it does not form heterodimers with ODC (Murakami, Y. et. al. J. Biol. Chem. 271:3340 3342 (1996)) and lacks ODC activity. Antizyme inhibitor has been shown to be rapidly induced in growth-stimulated fibroblasts and release ODC from antizyme suppression (Nilsson, J. et. al., Biochem. J. 346:699 704 (2000)).

Frameshifting can be detected using a dual luciferase reporter system that measures the efficiency of antizyme translational frameshifting (Grentzmann, G. et. al., RNA 4:479 486 (1998); Howard, M. et. al., Genes to Cells 6:931 941 (2001)). Frameshifting efficiency is determined by comparing the ratio of firefly luciferase to renilla luciferase activity in cells transfected in parallel using a control vector containing a constitutive +1 frameshift (AZ-IF) that measures the in-frame translation efficiency and a vector containing the inducible 0 to +1 frameshift (AZ1) construct. In these constructs, the renilla luciferase gene is upstream of the firefly luciferase gene which are separated by a short cloning sequence containing the portions of antizyme 1 and 2 known to contain the mRNA signals for polyamine stimulated frameshifting. Using a 96-well format, this assay system gives a quantitative measure of the efficiency of the polyamines, polyamine analogs and other compounds to induce frameshifting in a cell-based bioassay. Cells must be pretreated with .alpha.-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, prior to screening to decrease the basal antizyme frameshifting levels and increase the sensitivity to polyamine or compound-mediated stimulation of antizyme frameshifting.

In one of the first systematic assessments of antizyme induction by polyamine analogs, oligoamines such as octamines, decamines and dodecamines were found to induce antizyme to varying degrees (Mitchell, J. L. A. et. al., Biochem. J. Vol. 366, p. 663 671, 2002). These levels correlated with the cellular levels of antizyme as measured by Western blotting. The differences in the levels of antizyme appeared to be a result of dissimilar rates of protein synthesis since the half-life of antizyme (T1/2.about.75 min.) did not appear to be controlled by the polyamine analog. Therefore, it is presumable that the analogs have varying abilities to stimulate the +1 translational frameshift. A number of compounds such as bisethylnorspermine, bisethylhomospermine and 1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) were found to induce antizyme as well as spermine. However, certain conformational restrictions within the polyamine analogs such as three, four and five-membered rings or triple bonds between the central nitrogens negatively affected antizyme induction. Many of the oligoamines greatly exceeded spermine in their ability to induce antizyme (super-induction) when tested at the same concentration (10 .mu.M). The amount of antizyme frameshifting was found to correlate with the degree of growth inhibition. The oligoamines induced immediate cessation of cell growth, which was speculated to result from the super-induction of frameshifting. However, the authors also noted that these compounds might have other mechanisms of action leading to their observed cytotoxicity.

It is plausible that some antizyme inducers will also directly inhibit the enzymatic activity of ODC. A number of putrescine analogs have been found to be potent reversible inhibitors of ODC. For example, 1,4-diamino-trans-2-butene inhibits ODC with a K.sub.i of 2 .mu.M and 1,4-phenylenediamine somewhat less potently inhibits ODC with a K.sub.i of 46 .mu.M (Relyea, N. et. al., Biochem. Biophys. Res. Comm. 67:392 402 (1975); Solano, F. et. al., Int. J. Biochem. 20:463 470 (1988). Compounds of this nature may enhance polyamine depletion because ODC is inhibited in both a direct and indirect manner through induction of antizyme.

Polyamines may arrest prostate cell growth in the G1 phase by inducing antizyme. The prostate is the only vertebrate organ that synthesizes polyamines for export. As such, this tissue is exposed to higher concentrations of the polyamines. Spermine has been found to be a naturally occurring inhibitor of prostatic carcinoma cell growth in vitro and in vivo (Smith, R. C. et. al., Nature Med. 1:1040 1045 (1995)). Subsequently, it was found that spermine could induce G1 arrest in poorly metastatic prostatic carcinomas but not in highly malignant cells (Koike, C et. al., Cancer Res. 59:6109 6112, (1999)). Furthermore, antizyme could be induced only in the poorly metastatic prostatic carcinomas. Antizyme was later found to affect the cell-cycle of prostatic carcinoma cells with the discovery that it could interact with G1 cyclin D1 and its associated cyclin-dependent kinase, cdk4 (Coffino, P. Nat. Rev. Mol. Cell. Biol. 2:188 194 (2001)). The degradation of cdk4 and cyclin D1 were dependent on antizyme and independent of ubiquitin using in vitro purified proteasomes. The steady-state levels of the cyclin and kinase decreased when the polyamine levels were experimentally raised in the cultured cells. It has been proposed that prostatic cells that lose the ability to activate antizyme may eventually become malignant (Koike, C et. al., Cancer Res. 59:6109 6112, (1999)).

A number of studies have looked at both transient and inducible overexpression of antizyme in cell lines and animal models. Anti-tumor activity was shown in a study by Iwata and colleagues (Iwata, S. et. al., Oncogene 18:165 172 (1999)) using ectopically expressed inducible antizyme. In this study, nude mice were inoculated with H-ras transformed NIH3T3 cells expressing an inducible antizyme vector. Induction of antizyme blocked tumor formation in these mice and induced cell death in vitro. Intracellular polyamine levels were also measured. Both putrescine and spermine were completely depleted within 12 hours of induction. Spermine was also significantly reduced but over a slower time frame. Some of these observations were verified in another report that used a glucocorticoid (dexamethasone)--inducible promoter to force expression of antizyme in HZ7 cells (Murakami, Y. et. al., Biochem. J. 304:183 187 (1994)). Dexamethasone inhibited growth of this cell line, depleted putrescine levels, severely decreased spermidine levels but did not affect spermine levels. Addition of exogenous putrescine restored the intracellular putrescine levels and partially restored spermidine levels. In a third study, Tsuji and colleagues (Tsuji, T. et. al., Oncogene 20:24 33 (2001)) developed a hamster malignant oral keratinocyte (HCPC-1) cell line that stably expressed antizyme. Ectopic expression of antizyme suppressed tumor mass in nude mice by about 50%. In vitro, ectopic expression significantly increased the doubling time of antizyme transfectants and the antizyme transfectants demonstrated significantly less growth in soft agar. There was also a substantial increase in G1 phase cells with a corresponding decrease in S phase cells. These cells also showed morphological alterations suggesting terminal differentiation. This was accompanied by an increase in demethylation of DNA CCGG sites of 5-methyl cytosines. It was proposed that antizyme mediates a novel mechanism in tumor suppression by reactivating key cellular genes silenced by DNA hypermethylation during cancer development. In yet another example, transgenic mice that overexpress ODC in keratinocytes have been shown to develop a high rate of spontaneous and induced skin cancer (Megosh, L. et. al., Cancer Res. 55:4205 4209 (1995)). A reduction in the frequency of induced skin-tumors was observed in the skin of these transgenic mice expressing antizyme (Feith, D. et. al. Cancer Res. 61:6073 6081 (2001)).

Polyamines have been found to play a central role in hair follicle cell growth, a highly proliferative tissue, with a cell turnover time of between 18 23 hours. ODC plays a functional role in hair follicle growth, which is characterized by cyclic transformations from active growth and hair fiber production (anagen) through regression (catagen) into a resting phase (telogen). In mice, ODC is expressed in ectodermal cells at sites where hair follicles develop during embryonic development (Nancarrow, M. J., et. al., Mech. Dev. 84: 161 164 (1999); Schweizer, J. In: Molecular Biology of the Skin: The Keratinocyte, Darmon M, et. al., Eds., Academic Press, New York, 1993, pp 33 78). In proliferating bulb cells of anagen follicles, ODC is abundantly expressed except for a pocket of cells at its base. ODC protein expression is down regulated when the hair follicle enters catagen and is not detected in telogen. ODC protein expression does not resume until new follicle formation commences. A more complex expression of ODC is found in vibrissae (beard hair). ODC is expressed in the keratinocytes of the vibrissal hair shaft as well as in the bulb and outer root sheath cells near the follicle bulge. In comparison, ODC expression is very low in interfollicular epidermis.

Numerous studies have shown that inhibition of ODC with DMFO, an irreversible inhibitor of ODC, reduces hair growth in mammals. Mice were found to have reduced hair growth when DFMO was systemically delivered via the drinking water (Takigawa, M. et. al., Cancer Res. 43:3732 3738 (1983)). Intravenous administration of DFMO decreased wool growth in sheep (Hynd, P. I. et. al., J. Invest. Dermatol. 106:249 253 (1996)) and oral administration of DFMO in cats and dogs produced alopecia and dermatitis (Crowell, J. A. et. al., Fundam. Appl. Toxicol. 22:341 354 (1994)). Additional evidence that ODC plays a role in hair follicle regulation resulted from a study in humans that were being treated for acute Trypanosoma brucei infections (African sleeping sickness) (Pepin, J. et. al. Lancet 2:1431 1433 (1987)) using DFMO. Patients using this treatment showed signs of hair loss mainly on the scalp but it was reversible after discontinuing treatment.

The development of a number of transgenic mice either overexpressing spermidine/spermine N1-acetytransferase (SSAT) or ODC have contributed additional evidence that distorted tissue polyamine pools leads to hair loss (Pietila, M. et. al., J. Biol. Chem. 272:18746 18751 (1997); Suppola, S. et. al., Biochemistry 7338:311 316 (1999); Megosh, L. et. al., Cancer Res. 55:4205 4209 (1995)). SSAT is a key enzyme in the catabolism of polyamines that is rate-limiting for the conversion of spermine to spermidine and spermidine to putrescine. Both transgenic animal models showed permanent hair loss in which the normal hair follicles were transformed into dermal cysts that progressively increased in size as the animals aged (Pietila, M. et. al., J. Biol. Chem. 272:18746 18751 (1997); Suppola, S. et. al., Biochemistry 7338:311 316 (1999); Soler, A. P. et. al., J. Invest. Dermatol. 106, 1108 1113 (1996); Megosh, L. et. al., Cancer Res. 55:4205 4209 (1995)). This was manifest as a thickening and excessive skin folding of the epidermis. The common phenotypic feature that each of these animal models shared was a massive over accumulation of putrescine in the skin (Pietila, M. et. al., J. Invest. Dermatol. 116:801 805 (2001)). It was proposed that elevated levels of polyamines and especially putrescine favor continuous proliferation of epithelial cells leading to the formation of follicular cysts and hair loss. Low levels of putrescine favor differentiation of the outer root sheath keratinocytes and are not permissive for proliferation.

Polyamine biosynthesis has also been shown to be essential during the activation of immunocompetent cells (Fillingame, R. H. et. al., Proc. Natl. Acad. Sci. USA 72:4042 4045, (1975); Korpela, H. et. al., Biochem. J. 196:733 738 (1981)). Studies with DFMO confirm that polyamine depletion therapy can inhibit the immune response and may be a successful therapy against a number of autoimmune diseases. Both humoral and cell-mediated immune responses were affected by the anti-proliferative effect of polyamine depletion. DFMO treatment of mice challenged with tumor allografts resulted in modified cytotoxic T-lymphocyte and antibody responses (Ehrke, J. M. et. al., Cancer Res. 46:2798 2803 (1986)). Reports by Singh et al. indicate that DFMO treatment may also ameliorate acute lethal graft versus host (ALGVH) disease in mice (Singh, A. B. et. al., Clin. Immunol. Immunopathol. 65:242 246 (1992)). Murine ALGVH represents a model of human GVH that contributes to the morbidity and mortality of bone marrow transplantation in humans and is characterized by anemia and the loss of T cell function and numbers. In this study, treatment of ALGVH mice with DFMO decreased mortality and anemia while preserving the cytotoxic T cell and natural killer cell population of the host. Polyamine depletion therapy using DFMO has also been shown to benefit lupus-prone female NZB/W mice (Thomas, T. J. et. al., J. Rheumatol. 18:215 222 (1991)). Anti-DNA antibody production, immunoglobulin G and A synthesis, proteinuria and blood urea nitrogen were significantly reduced in treated mice.

Chemotherapeutics and radiation therapies target rapidly dividing cancer cells but they inadvertently affect the rapidly dividing epithelial cells of the mouth and intestine, hair follicles and hematopoietic cells in bone marrow. If the epithelial cells of the mouth or intestine become damaged and depleted, thinning and ulceration can result (mucositis) leading to pain and potential infection. Oral mucositis is also the result of damaged stem cells. Oral tissues are particularly painful if damaged.

Under normal conditions, the lining of the intestine is continuously being renewed through the proliferation of epithelial stem cells and their progeny in the crypts of villi (Booth, D, et. al., J Natl Cancer Inst Monogr 29:16 20 (2001)). When damage occurs (e.g., radiation or cytotoxic insult), a burst of proliferation/regeneration occurs in undamaged stem cells. A number of proposals to limit the damage to stem cells and enhance regeneration have been made. One strategy has been to arrest the cell cycle progression and accumulate cells in G.sub.0 or G.sub.1 during radiation or chemotherapy treatment to make them more resistant to damage. Other strategies include increasing the number of stem cells prior to potential damage or enhancing proliferation after damage (Farrell, C. L. et. al., Cancer Res. 58: 933 939 (1998)). Polyamines are taken up from the gut by normal and neoplastic epithelial cells of the gut mucosa, especially during periods of cell proliferation (Milovic V. et. al., Eur J Gastroenterol Hepatol. 13:1021 5 (2001)). The involvement of polyamines in proliferation of intestinal epithelial cells has been demonstrated using the nontransformed small intestinal cell line from rats, IEC-6, where polyamines increased DNA synthesis (Olaya, J. et. al. In Vitro Cell Dev Biol. Anim. 35:43 8, (1999)). The chemotherapeutic agent camptothecin, a DNA topoisomerase I inhibitor, can induce apoptosis in IEC-6 cells. However, reducing polyamines can have a protective effect. When IEC-6 cellular polyamines were reduced with DFMO, apoptosis due to camptothecin was delayed (Ray, R. M. et. al., Am J Physiol Cell Physiol 278:C480 489 (2000)). This may be due to G.sub.1 cell cycle arrest, which has been demonstrated to occur in IEC-6 cells incubated with DFMO (Ray R. M. et. al. Am. J. Physiol. 276:C684 91 (1999)). A more efficient depletion of polyamines with synthesis and uptake inhibition through induction of antizyme could provide significant protection against mucositis after radiation or chemotherapy.

BRIEF SUMMARY OF THE INVENTION

Ideal polyamine analogs should not substitute for the normal physiological functions of polyamines such as having the ability to rescue cells from DFMO-induced growth inhibition in vitro. It is also desirable that these compounds not be readily metabolized to regenerate polyamines. Identifying compounds that induce frameshifting and ultimately increase full length antizyme protein levels will be useful for depleting intracellular polyamine levels. These compounds should be an effective therapy for any disease associated with cellular proliferation including but not limited to cancer. As such, they are useful as drugs in a number of diseases where components of the immune system undergo undesired proliferation. Non-limiting examples include asthma, inflammation, autoimmune diseases, psoriasis, restenosis, rheumatoid arthritis, scleroderma, systemic and cutaneous lupus erythematosus, Type I insulin dependent diabetes, tissue transplantation, osteoporosis, hyperparathyroidism, treatment of peptic ulcer, glaucoma, Alzheimer's disease, Crohn's disease and other inflammatory bowel diseases. Other disease states associated with the proliferation of fungal, bacterial, viral and parasitic agents such as African sleeping sickness are also included. These compounds will also be effective for the treatment of unwanted proliferation of hair on skin. Antizyme inducers will be useful in the treatment of diseases involving the cell cycle by pausing the cell cycle progression during radiation or chemotherapy treatment. The appropriate cells will accumulate in G.sub.0 or G.sub.1, protecting them from radiation or chemotherapy induced hair loss (alopecia) and mucositis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a tabular representation of a large number of polyamine analogs A-S (25 .mu.M) that were tested for their ability to induce antizyme frameshifting using the dual luciferase reporter assay.

FIG. 2 shows the frameshifting induced by 25 .mu.M of various compounds in HEK-293 cells.

FIG. 3 shows the dose-dependent induction of frameshifting in HEK-293 cells with various compounds.

FIG. 4 shows the growth inhibition of HEK-293 cells with compound A.

FIG. 5 gives a comparison of the ability of antizyme frameshifters (25 .mu.M) to rescue cells from 2.5 mM DFMO-induced growth inhibition compared to 25 .mu.M spermidine (SPD) in a 6-day assay in HEK-293 cells.

FIG. 6 is a graph showing the effect of a 6-day incubation of compound A on HEK-293 cellular polyamine levels and cell growth.

FIG. 7 illustrates the effect of extracellular compound A on the intracellular concentration of compound A in HEK-293 cells as determined by HPLC.

FIG. 8 shows the reaction scheme for the synthesis of compound A. Conditions and reagents: (a) CH.sub.2.dbd.CHCN 1.2 equiv., CH.sub.3OH (b) LiAlH.sub.4 in THF.

FIG. 9 shows the reaction scheme for the synthesis of compound B.

FIG. 10 shows the reaction scheme for synthesis of intermediate R groups for FIG. 11.

FIG. 11 shows the reaction scheme for the synthesis of compounds C--R.

BEST AND VARIOUS MODES FOR CARRYING OUT INVENTION

The analogs and derivatives that can be used according to the present disclosure include those encompassed by the following formula I:

##STR00001## wherein, n can be 0 to 8 and the aminomethyl functionality can be ortho, meta or para substituted, R is hydrogen, --CH.sub.3, --CH.sub.2CH.sub.3, 2-aminoethyl, 3-aminopropyl, 4-aminobutyl, 5-aminopentyl, 6-aminohexyl, 7-aminoheptyl, 8-aminooctyl, N-methyl-2-aminoethyl, N-methyl-3-aminopropyl, N-methyl-4-aminobutyl, N-methyl-5-aminopentanyl, N-methyl-6-aminohexyl, N-methyl-7-aminoheptyl, N-methyl-8-aminooctyl, N-ethyl-2-aminoethyl, N-ethyl-3-aminopropyl, N-ethyl-4-aminobutyl, N-ethyl-5-aminopentyl, N-ethyl-6-aminohexyl, N-ethyl-7-aminoheptyl or N-ethyl-8-aminooctyl and R.sub.1 is a moiety selected from the group consisting of a hydrogen or a straight or branched C1 20 saturated or unsaturated aliphatic; aliphatic amine but not propylamine when R.dbd.H, n=1 and the aminomethyl functionality is para substituted; an alicyclic; single or multi-ring aromatic; single or multi-ring aryl substituted aliphatic; aliphatic-substituted single or multi-ring aromatic; a single or multi-ring heterocyclic, a single or multi-ring heterocyclic-substituted aliphatic; an aliphatic-substituted aromatic; and halogenated forms thereof.

The compounds induce expression of full-length antizyme without replacing the functionality of the native polyamines.

In preferred embodiments of the invention, the analogs and derivatives that can be used according to this disclosure can be further modified as described in formula II:

##STR00002##

wherein n can be 0 to 8, R and R.sub.1 are described as above, R.sub.2 can be independently selected from hydrogen, --CH.sub.3 or --CH.sub.2CH.sub.3 and R.sub.3 and R.sub.4 may be the same or different and are independently selected from hydrogen, or flourine.

An additional preferred embodiment of compounds that can be used according to this disclosure are described in formula III:

##STR00003##

wherein, m and n can be 0 to 7 independently, but m cannot equal n when R.sub.1 equals R.sub.2 and R.sub.3 equals R.sub.4, R can be 2 to 4, R can be independently selected from H, --CH.sub.3 or --CH.sub.2CH.sub.3, R.sub.1 and R.sub.2 can be independently selected from hydrogen, --CH.sub.3 or --CH.sub.2CH.sub.3 and R.sub.3 and R.sub.4 may be the same or different and are independently selected from hydrogen or fluorine.

Another aspect of the present invention are compounds of formula IV:

##STR00004##

wherein, R is hydrogen, --CH.sub.3, or --CH.sub.2CH.sub.3, m and n can be 0 to 7 independently and o can be 2 to 4, R.sub.2 can be independently selected from hydrogen, --CH.sub.3 or --CH.sub.2CH.sub.3 and R.sub.3 and R.sub.4 may be the same or different and are independently selected from hydrogen or fluorine.

In a further aspect of the invention, compounds of the present invention are represented by formula V

##STR00005##

wherein, R is hydrogen, --CH.sub.3, or --CH.sub.2CH.sub.3, m can be 0 to 7, n can be 0 to 8 and o can be 2 to 4, R.sub.2 can be independently selected from hydrogen, --CH.sub.3 or --CH.sub.2CH.sub.3 and R.sub.3 and R.sub.4 may be the same or different and are independently selected from hydrogen or fluorine.

The present disclosure also relates to novel compounds of formulae I, II, III, IV and V above with the further proviso that the novel compounds are non-symmetrical substituted xylene derivatives.

In other words, in formula I,

##STR00006## differs from

##STR00007## In formula II,

##STR00008## differs from

##STR00009## and in formula III

##STR00010## differs from

##STR00011##

Preferred novel compounds according to the present disclosure are those compounds wherein only one side of the xylene ring contains a group other than with the most preferred compounds being B, T and U as shown in FIG. 1.

As drugs, the polyamine analogs decrease cellular polyamine levels and can be used to treat disorders of undesired cell proliferation, including cancer, viral infections and bacterial infections. The invention additionally encompasses the stabilization of polyamine analogs by modifying them to resist enzymatic degradation. Such modifications include substitution of primary amine groups with alkyl groups, the addition of alkyl groups to the terminal amino groups and the addition of fluorine atoms .mu. to the terminal amino groups.

Additionally, it is desirable that the polyamine analogs of the invention enter cells by pathways other than those of active polyamine transport regulated by antizyme. Thus, an additional embodiment of the invention are analogs that are not imported into cells primarily by the polyamine transporters. Frameshifting activity is only one of the requirements for a good antizyme inducer. According to the present invention, it is preferable that the compounds enter the cell independent of the polyamine transporter since antizyme expression is known to inhibit polyamine transport. It has been determined by the present inventors that ideal candidates should not substitute for the normal physiological functions of polyamines such as having the ability to rescue cells from DFMO induced growth inhibition in vitro. Moreover, according to the present invention, it is also desirable that these compounds not be readily metabolized to regenerate polyamines. It is believed, pursuant to this invention, that any compound with frameshifting activity that could substitute for or degrade to a polyamine would be expected to defeat the goal of decreasing polyamine levels. Compounds, as determined by the present inventors, should be selective for antizyme frameshifting activity, exhibiting little affinity for the biosynthetic or catabolic enzymes associated with polyamine regulation such as ODC or SSAT. Compounds that fit into the above categories should deplete intracellular polyamine levels at concentrations known to induce frameshifting.

The present invention also relates to pharmaceutical compositions comprising an effective amount of at least one of the above disclosed compounds.

A further aspect of the present invention relates to treating a condition associated with cellular proliferation by administering at least one of the compounds described above.

The present disclosure also relates to treating one or more conditions associated with cellular proliferation comprising administration of at least one of B, T or U shown in FIG. 1. These conditions include, but are not limited to condition is selected from the group consisting of cancer, mucositis, asthma, inflammation, autoimmune disease, psoriasis, restentosis, rheumatoid arthritis, scleroderma, systemic and cutaneous lupus erythematosus, Type I insulin dependent diabetes, tissue transplantation, osteoporosis, hyperparathyroidism, treatment of peptic ulcer, glaucoma, Alzheimer's disease, and inflammatory bowel diseases.

The administration can be systemic, for example, and can be oral. In addition, the administration can be via a time-release vehicle, if desired. Also, if desired, the compounds R and S can be formulated as a cosmetic.

Another aspect of the present invention relates to inhibiting hair growth comprising topical administration of at least one of B, T or U shown in FIG. 1 to a subject in need of hair growth inhibition.

The present invention also relates to of inhibiting hair loss (alopecia) comprising topical administration of at least one of B, T or U shown in Figure to a subject undergoing radiation or chemotherapy.

Compounds B, T and U can also be used to treat conditions from fungal, bacterial, viral and parasitic agents.

Listed below are definitions of various terms used to describe this invention. These definitions apply to the terms as they are used throughout this specification, unless otherwise limited in specific instances, either individually or as part of a larger group.

The term "aryl" refers to monocyclic or bicyclic aromatic hydrocarbon groups having 6 to 12 carbon atoms in the ring portion, such as phenyl, naphthyl, biphenyl and diphenyl and diphenyl groups, each of which may be substituted.

The term "alkyl" refers to straight or branched chain unsubstituted hydrocarbon groups of 1 to 20 carbon atoms, preferably 1 to 8 carbon atoms. The expression "lower alkyl" refers to unsubstituted alkyl groups of 1 to 4 carbon atoms.

Examples of suitable alkyl groups include methyl, ethyl and propyl. Examples of branched alkyl groups include isopropyl and t-butyl.

The term "halogen" or "halo" refers to fluorine, chlorine, bromine and iodine.

The alkoxy groups typically contains about 1 8 carbon atoms and more typically about 1 4 carbon atoms. Examples of suitable alkoxy groups are methoxy, ethoxy and propoxy.

Examples of some suitable alkaryl groups include phenyl C.sub.1-3 alkyl such as benzyl.

Examples of some substitution groups are NO.sub.2, alkyl, CF.sub.3, alkoxy and halo.

Examples of suitable cycloalkyl groups typically contain 3 8 carbon atoms and include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

Examples of fused bicyclic unsatured ring groups are 2-quinolinyl, 3-quinolinyl, 5-quinolinyl, 6-quinolinyl, 7-quinolinyl, 1-isoquinolinyl, 3-isoquinolinyl, 6-isoquinolinyl, 7-isoquinolinyl, 3-cinnolyl, 6-cinnolyl, 7-cinnolyl, 2-quinazolinyl, 4-quinazolinyl, 6-quinazolinyl, 7-quinazolinyl, 2-quinoxalinyl, 5-quinoxalinyl, 6-quinoxalinyl, 1-phthalaonyl, 6-phthalazinyl, 1 5-naphthyridin-2-yl, 1,5-naphthyridin-3-yl, 1,6-naphthyridin-3-yl, 1,6-naphthyridin-7-yl, 1,7-naphthyridin-3-yl, 1,7-naphth7yridin-6-yl, 1,8-naphthyrdiin-3-yl, 2,6-naphthyridin-6-yl, 2,7-naphthyridin-3-yl, indolyl, 1H-indazolyl, purinyl and pteridinyl. Substitutions for each of the fused ring groups include the above noted group of substituents described herein.

Examples of mono- and multi-ring groups include aryl and bicyclic fused aryl-cycloalkyl groups. The aryl groups include an aromatic substituent which can be a single ring of multiple rings (up to three rings) which are fused together or linked covalently. The rings may each contain from zero to four heteroatoms selected from N, O and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quartemized. Non-limiting examples of aryl groups include phenyl, 1-naphthyl, 2-naphthyl, biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-uinoxalinyl, 3-quinolyl and 6-quinolyl. Substitutions for each of the above noted aryl systems include the above noted group of substitutents described herein.

The "bicyclic fused aryl-cycloalkyl" groups are those groups in which an aryl ring (or rings) is fused to a cycloalkyl group (including cycloheteroalkyl groups. The group can be attached to the remainder of the molecule through either an available valence on the aryl portion of the group, or an available valence on the cycloalkyl portion of the group. Examples of such benzotetrahydropyranyl and 1,2,3,4-tetrahydronaphthyl. Substitutents for each of the above noted groups include the group of substituents described herein.

The compounds of the present invention can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic agents or in a combination of therapeutic agents. They can be administered alone, but generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

The present invention includes the free base or acid forms, as well as salts thereof, of the polyamines and derivatives described by the above formulas. The invention also includes the optical isomers of the above described analogs and derivatives. In a further embodiment of the invention, mixtures of enantiomers and/or diastereoisomers, resulting from a single preparative step, combination, or interconversion are encompassed.

Prodrug forms of the compounds bearing various nitrogen functions (amino, hydroxyamino, hydrazino, guanidino, amidino, amide, etc.) may include the following types of derivatives where each R group individually may be hydrogen, substituted or unsubstituted alkyl, aryl, alkenyl, alkynyl, heterocycle, alkylaryl, aralkyl, aralkenyl, aralkynl, cycloalkyl or cycloalkenyl groups as defined beginning on Page 7.

(a) Carboxamides, --NHC(O)R

(b) Carbamates, --NHC(O)OR

(c) (Acyloxy)alkyl Carbamates, NHC(O)OROC(O)R

(d) Enamines, --NHCR(.dbd.CHCRO.sub.2R) or --NHCR(.dbd.CHCRONR.sub.2)

(e) Schiff Bases, --N.dbd.CR.sub.2

(f) Mannich Bases (from carboximide compounds), RCONHCH.sub.2NR.sub.2

Preparations of such prodrug derivatives are discussed in various literature sources (examples are: Alexander et al., J. Med. Chem. 1988, 31, 318; Aligas-Martin et al., PCT WO pp/41531, p.30). The nitrogen function converted in preparing these derivatives is one (or more) of the nitrogen atoms of a compound of the invention.

Prodrug forms of carboxyl-bearing compounds of the invention include esters (--CO.sub.2R) where the R group corresponds to any alcohol whose release in the body through enzymatic or hydrolytic processes would be at pharmaceutically acceptab