Note: Descriptions are shown in the official language in which they were submitted.
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POLSIAMINE ANALOGS THAT ACTIVATE ANTIZY1VIE FRAMESHIFTING
DESCRIPTION
TECHNICAL FIELD
The present invention relates to novel polyamines, their synthesis and use in
pharmacological, cosmetic or agricultural applications. The instayit 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 motifs) of small molecules
related to
polyamines.
BACKGROUND OF THE INVE°NTION
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., Trertds
Biochenr. 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., Irat. J. Biochern. 22:211-218 (1990); Seiler, N. et. al.,
J. P. Int. J: Bioclaem. 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
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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., Neuf°osurgefy 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.
Ornithine decarboxylase (ODC) is the rate-limiting enzyme of cellular
polyamine
synthesis, converting ornithine 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., Pf°oc. Natl. Acad.
Sci. ZISA 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 AZl 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. LISA 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
colon. ORF1 is short but contains two AUG initiation colons. Either one of the
initiation
colons 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 colon enabling translation
to continue.
Only minute quantities of antizyme are generally present in mammalian tissues.
Polyamines and
agmatine 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. Claem.
273:15313-15316
(1998)). Vertebrates possess three elements that control frameshifting, the
UGA stop colon in
ORFl, a stem-loop structure 3' to the ORF1 UGA that can base pair with a
portion of the loop
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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); Fern,
A.D. et. al., StruGt. 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., .I. Biol. Chena. 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., ,I.
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. Claem. 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., Bioclaem. 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-1F) 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
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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
frarneshifting.
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 a-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., Bioehena. .I. 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 (Tl/2 ~ 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 spennine in their ability to induce antizyme (super-
induction) when tested at
the same concentration (10 ~.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; of 2 ~M
and 1,4-
phenylenediamine somewhat less potently inhibits ODC with a K; of 46 ~,M
(Relyea, N. et. al.,
Biochem. Biophys. Res. Conarn. 67:392-402 (1975); Solano, F. et. al., Int. J.
Biochena. 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.
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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 iu 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 D 1 and
its associated cyclin-dependent kinase, cdk4 (Coffino, P. Nat. Rev. Mol. Cell.
Biol. 2:188-194
(2001 )). The degradation of cdk4 and cyclin D 1 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., Cahce~° 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., Oyacogene 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 H~7 cells (Murakami, Y. et. al., BiocheTn. J.
304:183=187
(1994)). Dexamethasone inhibited growth ofthis 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., Oszcogene 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
vitf-o, 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 Gl phase cells with a corresponding decrease in S phase cells.
These cells also
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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., Cancey~ Res.
55:4205-4209 (1995)).
A reduction in the frequency of induced skin-tumors was observed in the skin
of these transgenic
mice expressing antizylne (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: M~lecular Biology of the Skin: The Keratin~cyte, 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 DEMO decreased
wool
growth in sheep (Hynd, P. I. et. al., J. Iravest. De~matol. 106:249-253
(1996)) and oral
administration of DFMO in cats and dogs produced alopecia and dermatitis
(Crowell, J. A, et.
al., Fundafn. 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
Tiyparaosoma 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.
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The development of a number of transgenic mice either overexpressing
spermidine/spermine Nl-acetytransferase (SSAT) or ODC have contributed
additional evidence
that distorted tissue polyamine pools leads to hair loss (Pietila, M. et. al.,
J. Biol. Claem.
272:18746-18751 (1997); Suppola, S. et. al., Biocher~zistry 7338:311-316
(1999); Megosh, L. et.
al., Caracef~ 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. Claem. 272:18746-18751 (1997); Suppola, S. et,
al., Biochefnistry
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.
Dernzat~l. 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., PY~c.Natl.Acad.Sci. USA
72:4042-4045,
(1975); T~orpela, 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.Inamunol. Inznzunopathol. 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.Rlzeunzatol. 18:215-222 (1991)). Anti-DNA
antibody
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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 (Eooth, .
D, et. al., JNatl Cancer Inst Moraog~ 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 Go or Gl 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 278:0480-489 (2000)).
This may be due
to G~ cell cycle arrest, which has been demonstrated to occur in IEC-6 cells
incubated with
DFMO (Ray R. M. et. al. An2. J. Playsiol. 276:0684-91 (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.
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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 DFM~-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
Go or Gl,
protecting them from radiation or chemotherapy induced hair loss (alopecia)
and mucositis.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a tabular representation of a large number of polyamine analogs A-
S (25
p,M) that were tested for their ability to induce antizyme frameshifting using
the dual luciferase
reporter assay.
Figure 2 shows the frameshifting induced by 25 p,M of various compounds in HEK-
293 cells.
Figure 3 shows the dose-dependent induction of frameshifting in HEK-293 cells
with
various compounds.
Figure 4 shows the growth inhibition of HEK-293 cells with compound A.
Figure 5 gives a comparison of the ability of antizyme frameshifters (25 ~,M)
to rescue
cells from 2.5 mM DFMO-induced growth inhibition compared to 25 ~,M spermidine
(SPD) in a
6-day assay in HEK-293 cells. ,
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Figure 6 is a graph showing the effect of a 6-day incubation of compound A on
HEK-293
cellular polyamine levels and cell growth.
Figure 7 illustrates the effect of extracellular compound A on the
intracellular
concentration of compound A in HEK-293 cells as determined by HPLC.
Figure 8 shows the reaction scheme for the synthesis of compound A. Conditions
and
reagents: (a) CHZ=CHCN 1.2 equiv., CH30H (b) LiAlH4 in THF.
Figure 9 shows the reaction scheme for the synthesis of compound B.
Figure 10 shows the reaction scheme for synthesis of intermediate R groups for
Figure
11.
Figure 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:
RHN H I ~H
N. R~
wherein, n can be 0 to 8 and the aminomethyl functionality can be ortho, meta
or para
substituted, R is hydrogen, -CH3, -CHZCH3, 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-aininopentanyl, 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 Rl is a moiety selected from the group consisting of
a hydrogen or a
straight or branched Cl-20 saturated or unsaturated aliphatic; aliphatic amine
but not
propylamine when R =H, n=1 and the aminomethyl functionality is para
substituted; an alicyclic;
single or mufti-ring aromatic; single or mufti-ring aryl substituted
aliphatic; aliphatic-substituted
single or mufti-ring aromatic; a single or mufti-ring heterocyclic, a single
or mufti-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.
CA 02555862 2006-08-11
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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:
R2
RHN N~~~~ H
R3 R4 nH I ~~N'R1 II
wherein n can be 0 to 8, R and Rl are described as above, RZ can be
independently
selected from hydrogen, -CH3 or -CHZCH3 and R3 and R4 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:
R3 R4
RHN ~ Rs Ra H
R~ m H I / H N R
n R
2 o III
wherein, m and n can be 0 to 7 independently, but m cannot equal n when Rl
equals R2
and R3 equals R4, o can be 2 to 4, R can be independently selected from H, -
CH3 or -CHaCH3, Rl
and R2 can be independently selected from hydrogen, -CH3 or -CHZCH3 and R3 and
R4 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:
R3 R4
RHN
R2 H ~ /~N N R
m \ /n o IV
wherein, R is hydrogen, -CH3, or -CHZCH3, m and n can be 0 to 7 independently
and o
can be 2 to 4, R2 can be independently selected from hydrogen, -CH3 or -CHZCH3
and R3 and R4
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
R3 R4
RHN ~ H
R2 \/mH n H\ I ~No V
wherein, R is hydrogen, -CH3, or -CHaCH3, m can be 0 to 7, n can be 0 to 8 and
o
can be 2 to 4, Ra can be independently selected from hydrogen, -CH3 or -CHZCH3
and R3 and R4
may be the same or different and are independently selected from hydrogen or
fluorine.
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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, ,N. differs from RHN'~ ~ . In formula II3
/ 1
differs from ~ R2 'R~ and in formula III \ Ra R4 N R R
RHN N~ H n R
R ~~~H \/ 2 .a
3 R4
R3 R4
differs from RHN
R~ / m -
Preferred novel compounds according to the present disclosure are those
compounds
wherein only one side of the xylene ring contains a group other than -CHzNH2
with the most
preferred compounds being B, T and U as shown in Figure 1.
As drugs, the polyamine analogs decrease cellular polyamine levels and can be
used to
15 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 ~ to the terminal amino groups.
20 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
25 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 uit~~o. Moreover,
according to the present
invention, it is also desirable that these compounds not be readily
metabolized to regenerate
30 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 SEAT.
Compounds that
12
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WO 2005/105729 PCT/US2004/009582
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 Figure 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 Figure 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
Biphenyl and
Biphenyl groups, each of which may be substituted.
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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 C1_3 alkyl such as
benzyl.
Examples of some substitution groups are NOZ, alkyl, CF3, 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 atoms) are optionally
quarternized. 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-thienyl, 2-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
14
CA 02555862 2006-08-11
WO 2005/105729 PCT/US2004/009582
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.
S 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 earner 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 andlor 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(=CHCR02R) or-NHCR(=CHCRONRz)
(e) Schiff Bases, -N=CRz
(f) Mannich Bases (from carboximide compounds), RCONHCHzNRz
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
(-COzR) where the R group corresponds to any alcohol whose release in the body
through
enzymatic or hydrolytic processes would be at pharmaceutically acceptable
levels. Another prodrug
derived from a carboxylic acid form of the invention may be a quaternary salt
type
15.
CA 02555862 2006-08-11
WO 2005/105729 PCT/US2004/009582
RC(=~)~C O~ ~O
R
of structure described by Bodor et al., J. Med. Chem. 190, 23, 469.
It is of course understood that the compounds of the present invention relate
to all optical
isomers and stereo-isomers at the various possible atoms of the molecule.
S The compounds used in the methods of this invention form pharmaceutically
acceptable
acid and base addition salts with a wide variety of organic and inorganic
acids and bases and
includes the physiologically acceptable salts which are often used in
pharmaceutical chemistry.
Such salts are also part of this invention. Typical inorganic acids used to
form such salts include
hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric,
hypophosphoric and the lilce.
Salts derived from organic acids, such as aliphatic mono and dicarboxylic
acids, phenyl substituted
alkonic acids, hydroxyalkanoic and hydroxyalkandioic acids, aromatic acids,
aliphatic and aromatic
sulfonic acids, may also be used. Such pharmaceutically acceptable salts thus
include acetate,
phenylacetate, trifluoroacetate, acrylate, ascorbate, benzoate,
chlorobenzoate, dinitrobenzoate,
hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate,
naphthalene-2-benzoate,
bromide, isobutyrate, phenylbutyrate, ~3-hydroxybutyrate, butyne-1,4-dioate,
hexyne-1,4-dioate,
cabrate, caprylate, chloride, cinnamate, citrate, fonnate, fumarate,
glycollate, heptanoate, hippurate,
lactate, malate, maleate, hydroxymaleate, rnalonate, mandelate,'mesylate,
nicotinate, isonicotinate,
nitrate, oxalate, phthalate, teraphthalate, phosphate, monohydrogenphosphate,
dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate, propionate,
phenylpropionate,
salicylate, sebacate, succinate, suberate, sulfate, bisulfate, pyrosulfate,
sulfite, bisulfate, sulfonate,
benzene-sulfonate, p-bromobenzenesulfonate, chlorobenzenesulfonate,
ethanesulfonate, 2-
hydroxyethanesulfonate, methanesulfonate, naphthalene-1-sulfonate, naphthalene-
2-sulfonate, p-
toleunesulfonate, xylenesulfonate, tartarate, and the like.
Bases commonly used for formation of salts include ammonium hydroxide and
alkali and
allcaline earth metal hydroxides, carbonates, as well as aliphatic and
primary, secondary and tertiary
amines, aliphatic diamines. Bases especially useful in the preparation of
addition salts include
sodium hydroxide potassium hydroxide, ammonium hydroxide, potassium carbonate,
methylamine,
diethylamine, and ethylene diamine.
The invention also provides prodrug forms of the above described compounds and
derivatives, wherein the prodrug is metabolized in vivo to produce a compound
or derivative as
set forth above. Indeed, some of the above described compounds or derivatives
may be a
prodrug for another compound or derivative. This scenario is plausible if the
prodrug is a
16
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WO 2005/105729 PCT/US2004/009582
substrate for either spermidine synthase or spermine synthase which are
enzymes that transfer an
aminopropyl group to putrescine and spernidine, respectively.
The compounds may be utilized alone or in combination with other agents,
particularly
other inhibitors of polyamine synthesis or transport, but including other
inhibitors of cell
proliferation. Without being bound by theory, the polyamines compound of the
invention may
decrease polyamine levels by inducing antizyrne (A~), which in turn down
regulates both the
production of polyamines by ornithine decarboxylase (ODC) and the transport of
polyamines by
its corresponding transporter. Polyamine levels may also decrease because
antizyme may induce
increases in polyamine excretion. The invention further defines structural
elements/motifs
within these compounds that appear key to their induction of antizyme as
determined by assays.
Because polyamines are absolutely essential for DNA replication and are
essential to cellular
homeostasis, there is an interest in preventing cell proliferation by lowering
intracellular
polyamine levels. Sufficiently low polyamine levels can lead to cell death.
Thus any agent able
to lower polyamine levels, particularly by inhibiting both polyamine
biosynthesis and
uptake/import, offers the opportunity to target a variety of disease or
undesirable conditions
related to cell proliferation, including cancer.
The compounds of the invention are not necessarily metabolized like naturally
occurring polyamines. As such, the compounds of the invention may have the
advantage of not
being readily metabolized to regenerate polyamines. Administration of
polyamine compounds,
which are subject to conversion to putrescine. and other polyamines, would be
expected to defeat
the goal of decreasing polyamine levels. Thus one aspect of the invention is
the production and
use of polyamines compound that are not metabolized to putrescine or any other
naturally
occurring polyamine metabolite. In addition, ideal candidates should not
substitute for the
normal physiological functions of polyamines such as having the ability to
rescue cells from
DFM~ induced growth inhibition ifz vitro.
In another aspect of the invention, compositions containing the above
described
compounds and derivatives are provided. Preferably, the compositions are
formulated to be
suitable for pharmaceutical or agricultural use by the inclusion of
appropriate carriers or
excipients.
Figure 1 is a tabular representation of a large number of polyamines A-S (25
p,M)
that were tested for their ability to induce antizyme frameshifting using the
dual luciferase
reporter assay. The percent relative frameshifting value (% RF) gives a
comparison of the ability
of a compound to induce frameshifting compared to 25 p,M spermidine. The % RF
was
calculated as follows. The background percent frameshifting activity
determined from the 2.5
17
CA 02555862 2006-08-11
WO 2005/105729 PCT/US2004/009582
mM DFMO negative control was subtracted from the percent frameshifting
activity for all
compounds including the spermidine control. The background corrected
frameshifting activity
of each compound was then divided by the background corrected frameshifting
activity induced
by 25 ~M spermidine and multiplied by 100. The compounds were also evaluated
for their
ability to rescue cells from DFMO-induced growth inhibition by determining
their rescue
coefficient. The rescue coefficient represents the ratio of the cell growth,
as measured by O.D.,
in the presence of the test compound with 2.5 mM DFMO to the growth in the
presence of 2.5
mM DFMO alone. The frameshift-rescue factor (FRF) is a useful factor to
compare the
effectiveness of the various antizyme frameshifters by taking into account
their potency for
inducing frameshifting and ability to rescue cells from DFMO-induced growth
inhibition. The
FRF was calculated by multipling the %RF value by the inverse of the rescue
coefficient.
Figure 2 shows the frameshifting induced by 25 ~,M of various compounds in HEK-
293
cells. The percent relative frameshifting (%RF) activity was determined as
described above.
Results are expressed as the mean ~ standard deviation from these independent
determinations.
Figure 3 shows the dose-dependent induction of frameshifting in HEK-293 cells
with
various compounds. Values represent percent frameshifting following transient
transfection of
an AZ1-IF control and the inducible 0 to +1 AZ1 plasmid construct into HEK-293
cells grown in
the presence of 2.5 mM DFMO. The compounds were added to the cells after
transfection and
incubated overnight before assaying the next day. Each value represents
triplicate
measurements.
Figure 4 shows the growth inhibition of HEK-293 cells with compound A. HEK-293
cells were incubated with 1 mM aminoguanidine and various concentrations of
compound A
with or without 1 ~M spermidine during a 6-day growth assay. Cell number was
determined by
MTSlPMS assay from triplicate wells.
Figure 5 gives a comparison of the ability of antizyme frameshifters (25 ~M)
to rescue
cells from 2.5 mM DFMO-induced growth inhibition compared to 25 ~,M spermidine
(SPD) in a
6-day assay in HEK-293 cells. The rescue coefficient represents the ratio of
cell growth, as
measured by O.D., of the test compound with 2.5 mM DFMO versus 2.5 mM DFMO
alone.
Figure 6 is a graph showing the effect of a 6-day incubation of compound A on
HEK-293
cellular polyamine levels and cell growth. All flasks received 1 mM
aminoguanidine. Cells
were washed, counted, lysed in perchloric acid, dansylated and polyamine
levels determined by
HPLC. Each value represents the average of triplicate values with the error
bars representing
standard deviation.
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Figure 7 illustrates the effect of extracellular compound A on the
intracellular
concentration of compound A in HEK-293 cells as determined by HPLC. Cells were
prepared as
in Figure 6. Peak area was normalized by dividing the peak area of the
compound divided by the
peak area of the internal standard, diaminoheptane. Values represent at least
triplicate
measurements with the error bars representing standard deviation.
Figure 8 shows the reaction scheme for the synthesis of compound A. Conditions
and
reagents: (a) CHZ=CHCN 1.2 equiv., CH3OH (b) LiAlH4 in THF.
Figure 9 shows the reaction scheme for the synthesis of compound B. Conditions
and
reagents: (a)p-xylylenediamine (10 equiv), CHaCIa (b) 2-
nitrobenzenesulfonylchloride, Et3N,
CHZC12 (c) HOCHaCH2CHzCHaNPth, Ph3P, DIAD, THF (d) NHZNH2, EtOH (e) PhSH,
KZCO3,
DMF (fJ TFA/CHZCHZ/iFr3SiH, 20:78:2 (g) HOCHZCHzCH2NPth, Ph3P, DIA.D, THF.
Figure 10 shows the reaction scheme for synthesis of intermediate R groups for
Figure
11. Conditions and reagents: (a) Phthalic anhydride, EtOH, reflux (b)
'BuOCOCI, NaBH4.
Figure 11 shows the reaction scheme for the synthesis of compounds C-R.
Conditions
and reagents: (a) 3-aminopropanol (10 equiv), CHzCl2 (b) 1,4-bis-(2-
nitrobenzenesulfonamide)-
xylylene, Ph3P, DIAD, THF (c) Intermediate 3.2, Ph3Ph, DIAD, THF (d) K2C03,
PhSH, DMF
(e) TFA/CHZCHZ/iPr3SiH, 20:78:2.
The following non-limiting examples are presented to further illustrate the
present
invention. The general procedures suitable for preparing compounds of the
present invention are
illustrated in the following non-limiting examples.
E~~AMPLE 1
Synthesis of Compound A
N (2-cyanoethyl)-xylylenediamine (1.1) - To the slightly cloudy solution of
6.81 g (50
mmoL) ofp-xylylenediamine [539-48-0] in 250 mL of dry CH30H is added 3.95 mL
(3.18 g, 60
mrnol, 1.2 equiv.) of acrylonitrile dropwise at 25°C under an
atmosphere of argon. The reaction
flask is shielded from light and allowed to stir for 5 h. Thin layer
chromatography
(CHC13:'PrOH:concd. NH40H 80/18/2) shows a mixture of di-(2-cyanoethyl)
product (Rf= 0.63)
and mono-(2-cyanoethyl) product (Rf= 0.26) materials is formed. Only a slight
trace ofp-
xylylenediamine (Rf= 0.02) remains. The solvents are evaporated and the
resulting oil is
purified by silica gel chromatography using the following ratios of
CHC13:'PrOH:concd.
NH40H: 85/13/2 (1L), 80/1812 (1L) then 75:23:2 (1L) to give 3.90 g (32%) ofthe
di-(2-
cyanoethyl) product as a white solid. The desired mono-(2-cyanoethyl) product
eluted in later
column fractions and weighs 3.06 g (32%) as a colorless oil.
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WO 2005/105729 PCT/US2004/009582
N (3-aminopropyl)-xylylenediamin (Compound A) - To the clear homogeneous
solution of 1.97 g (10.4 mmole) of N (2-cyanoethyl)-xylylenediamine in 50 mL
of dry THF is
added 30 mL (30 mmole) of a 1M solution of lithium aluminum hydride (LiAlH4)
in THF
dropwise at 25°C under an atmosphere of argon. Bubbles form and a white
precipitate forms
immediately. A pink color is noted. After stirring for 1 h the heterogeneous
reaction mixture is
placed in an oil bath and heated to reflux for 18 h. The reaction is allowed
to cool and 10 mL of
H20 is carefully added. This is followed by the addition of 10 mL of 4N NaOH.
The resulting
mixture is heated to reflux for 4h when it is filtered over a pad of Celite
after only slight cooling.
The white precipitate on the Celite pad is washed twice each with CH30H, THF
then CH2Cl2.
The combined filtrates are evaporated to give 4.20 g oily crude product. This
is purified by SiO2
column chromatography using the following concentrations of concd. NH40H in
CH3CN: 5%
(1L) and 20% (1L) to give 1.52 g (76%) ofproduct as a colorless clear oil.
This is dissolved in
50 mL of EtOH and treated with 10 mL of 6N HCl followed by evaporation. The
resulting white
solid is suspended in 50 mL of hot EtOH and treated with just enough H20 to
form a complete
solution. This solution is stored at -20°C to produce the first crop of
crystals that are filtered and
dried to give 0.52 g (16%) pure product. The mother liquor is evaporated and
treated as above
with less solvent to give 0.34 g (11%) as the second crop crystals (Fig. 8).
EXAMPLE 2
Synthesis of Compound B
Resin 2.2 - To a 100 mL solid-phase peptide synthesis vessel containing 10 g
(Rapp
Polymere, 14 mmole, 1.4 mmole/g) of polystyrene-based trityl chloride resin in
30 mL of
CHZCl2 is added a solution of 19.07 g (140 mmole, 10 equiv) ofp-
xylylenediamine in 30 mL of
CHZCl2 dropwise at 25°C over 30 min. During this addition the resin is
agitated via the
introduction of a slow stream of argon through the bottom frit of the vessel.
Copious amounts of
white precipitate form over the course of the 8 h reaction. Following this
time the resin is
filtered and washed with CHzCl2, 'PrOH, DMF, THF then CHZCl2 (3 x 75 mL each).
The dried
resin is treated with 10 mL of diethylamine in 40 mL of CH~C12 for 2 h to
completely cap the
resin. The same washing procedure as above followed by a thorough overnight
vacuum drying
process takes place to give the product resin. This resin gives a positive
Kaiser amine test
reaction.
Resin 2.3 - All of the resin from the step are suspended in 50 mL of CHzCl2 in
a 100 mL
solid-phase peptide synthesis vessel and treated with 9.31 g (42 mmole, 3
equiv) of 2-
nitrobenzenesulfonyl chloride and 5.85 mL (4.25 g, 42 mmole, 3 equiv) of
triethylamine at 25°C.
CA 02555862 2006-08-11
WO 2005/105729 PCT/US2004/009582
The resin is agitated via the introduction of a slow stream of argon through
the bottom frit of the
vessel for 4 h when the resin is filtered and washed with CHZC12, 'PrOH, DMF,
THF then
CH2C12 (3 x 75 mL each). The resin is dried under vacuum overnight to give a
product that is
negative by the Kaiser amine test reaction.
Resin 2.4 - To all of the resin obtained above suspended in 30 mL of dry THF
is added as
solids 11.02 g (42 mmole, 3 equiv) of triphenylphosphine and 9.21 g (42 mmole,
3 equiv) of 4-
phthalimide-1-butanol. The resulting suspension is agitated with a slow stream
of argon through
the bottom of the vessel while 8.49 g (42 mmole, 3 equiv) of
diisopropylazodicarboxyate is
introduced in a dropwise fashion. The resulting suspension is agitated for 18
h at 25°C when the
solvents are filtered and washed with CH2C12,'PrOH, DMF, THF then CHZCIz (3 x
75 mL each).
The resin is dried under vacuum overnight to give a product that is negative
by the Kaiser amine
test reaction.
Resin 2.5 - To all of the resin produced above is added 50 mL of absolute EtOH
and 50
mL of hydrazine hydrate in a solid-phase peptide synthesis vessel. The
resulting suspension is
heated to 60°C and rotated in a Robins Scientific ChemFlex Model 404
oven. After 18 h at this
temperature the vessel is allowed to cool and the resin is filtered and washed
with H20, EtOH,
CH2Clz,'PrOH, DMF, THF then CHZCIZ (3 x 75 mL each). The resin is dried under
vacuum
overnight to give a product that is positive by the Kaiser amine test
reaction.
Resin 2.6 - Resin Z.5 is treated in a similar manner as that described for
resin 2.3 above
to give resin 2.6 as product. The resin is dried under vacuum overnight to
give a product that is
negative by the Kaiser amine test reaction.
Resin 2.7 - To all of the resin 2.6 obtained above suspended in 30 mL of dry
THF is
added as solids 11.02 g (42 mrnole, 3 equiv) of triphenylphosphine and 8.62 g
(42 mmole, 3
equiv) of N (3-hydroxypropyl)phthalimide. The resulting suspension is agitated
with a slow
stream of argon through the bottom of the vessel while 8.49 g (42 mmole, 3
equiv) of
diisopropylazodicarboxyate is introduced in a dropwise fashion. The resulting
suspension is
agitated for 18 h at 25°C when the solvents are filtered and washed
with CHaCl2, 'PrOH, DMF,
THF then CH2C12 (3 x 75 mL each). The resin is dried under vacuum overnight to
give a product
that is negative by the Kaiser amine test reaction.
N (4-amino-1-butane-N (3-aminopropyl-)-xylylenediamine (Compound B) - One
gram of Resin 2.7 obtained above is treated with 10 mL each of EtOH and
hydrazine hydrate at
60°C for 18 as above for Resin 2.5. After 18 h at this temperature the
vessel is allowed to cool
and the resin is filtered and washed with H20, EtOH, CHaCIa,'PrOH, DMF, THF
then CH2Cla (3
x 75 mL .each). The resin is dried under vacuum overnight to give a product
that ,is;positive by. ..
21
CA 02555862 2006-08-11
WO 2005/105729 PCT/US2004/009582
the Kaiser amine test reaction. The resulting resin is treated with 1.74 g
(12.6 mmole, 9 equiv)
of KZC03 and 1.29 mL (12.6 mmole, 9 equiv) of thiophenol in 10 mL of dry DMF
at 25°C for
3 h. The resin is filtered and washed with CHZCl2,'PrOH, DMF, THF then CH2Cl2
(3 x 15 mL
each) to give a product that gave a positive Kaiser amine test reaction. This
resin is suspended in
10 mL of TFA/CHZCIzfPr3SiH 20:78:2 and agitated for 30 min. on a platform
shaker. The resin
is filtered and washed 3 times with 20 mL of CHZCIz then 3 times with CH30H.
The combined
filtrates are evaporated to give compound B as its crude tetra-
trifluoroacetate salt. This could be
purified over silica gel using a 5 to 40% gradient of concd NH40H in CH3CN
using a fraction
collector. The resulting fractions are analyzed by TLC (Rf= 0.34 in 70:30
CH3CN/concd
NH4OH) and those containing pure product are combined and evaporated to give
pure
Compound B as a clear oil. This is totally converted to its tetrahydrochloride
salt by dissolving
in 20 mL of CH30H and treating with an excess of 6N HCI. Evaporation of the
solvents gives
149 mg (26%) white solid (Fig. 9).
Resin precursor to Compounds C through R (Resin 4.2) - To a 100 mL solid-phase
peptide synthesis vessel containing 10 g (Rapp Polymer 14 mmole, 1.4 mmole/g)
of polystyrene-
based trityl chloride resin in 30 mL CHZCl2 is added a solution of 10.51 g
(140 mmole, 10 equiv)
of 3-aminopropanol in 30 mL of CH2C12 dropwise at 25°C over 30 min.
During this addition the
resin is agitated via the introduction of a slow stream of argon through the
bottom frit of the
vessel. The reaction is allowed to proceed for 5 h. Following this time the
resin is filtered and
washed with CH2C12, 'PrOH, DMF, THF then CHZCl2 (3 x 75 mL each). The dried
resin is
treated with 10 mL of diethylamine in 40 mL of CHaCl2 for 2 h to completely
cap the resin. The
same washing procedure as above followed by a thorough overnight vacuum drying
process take
place to give the product resin. This resin gives a negative Kaiser amine test
reaction.
1,4-bis-(2-nitrobenzenesulfonamide)-xylylene - To 13.62 g (100 mmole) of 1,4-
xylylenediamine in 120 mL of dry pyridine in an ice bath under a stream of
argon is added 48.76
g (220 mmole) of 2-nitrobenzenesulfonylchloride portionwise as a solid. The
reaction is allowed
to warm to room temperature and stir for 18 hr. A large portion of the
pyridine is removed by
rotoevaporation and the oily residue is dissolved in 250 mL of CHZC12 and
washed with satd
CuS04 then H20 and brine. Drying and evaporation gives a crude product as a
yellowish solid.
Purification by silica gel column chromatography using CHCl3/CH30H 98:2 gives
39.5 g (78%)
of product as a white solid.
Resin 4.2 - To 2 g of Resin 4.1 produced above is added 4.2 g (3 equiv) of 1,4-
bis-(2-
nitrobenzenesulfonamide)-xylylene and 2.20 g (3 equiv) of triphenylphosphine
in 20 mL of dry
THF. To the resulting suspension is added 1.65 g of diisopropyl-
azodicarboxylate dropwise at
22
CA 02555862 2006-08-11
WO 2005/105729 PCT/US2004/009582
room temperature over 45 min. The resin is agitated over the next 18 h by the
introduction of a
slow stream of argon through the bottom frit of the vessel. After this time
the resin is filtered
and washed with CH2C12,'PrOH, DMF, THF then CH2C12 (3 -x 25 mL each). The
resin gives a
negative Kaiser amine test.
EXAMPLE 3
Synthesis of Compound C
Resin 4.3 - To 0.5 g of Resin 4.2 and 0.55 g (3 equiv) of triphenylphosphine
suspended in 10
mL of dry THF is added 0.43 g of 2-methyl-2-phthalimido-1-ethanol produced via
the route
shown in Figure 10 (other compounds in figure 1 are produced by use of the
appropriate
precursor molecule). To the resulting suspension is added 0.42 g of
diisopropyl-
azodicarboxylate dropwise at room temperature over 45 min. The resin is
agitated over the next
18 h by the introduction of a slow stream of argon through the bottom frit of
the vessel. After
this time the resin is filtered and washed with CHaCl2, 'PrOH, DMF, THF then
CH2C12 (3 x 25
mL each). The resin gives a negative Kaiser amine test.
N (3-aminopropyl) N'-(2-methyl-2-aminoethyl)-1,4-xylylenediamine (Compound C)
- To a 0.25 gram portion of Resin 4.2 obtained above is added 10 mL each of
EtOH and
hydrazine hydrate. The resin is heated at 60°C for 18 as for Resin 2.5
above. After 18 h at this
temperature the vessel is allowed to cool and the resin is filtered and washed
with HZO, EtOH,
CHZC12, 'PrOH, DMF, THF then CHZC12 (3 x 75 mL each). The resin is dried under
vacuum
overnight to give a product that is positive by the Kaiser amine test
reaction. The resulting resin
is treated with 0.435 g (3.15 mmole, 9 equiv) of KZC03 and 0.32 mL (3.15
mmole, 9 equiv) of
thiophenol in 10 mL of dry DMF at 25°C for 3 h. The resin is filtered
and washed with CHZCl2,
'PrOH, DMF, THF then CH2C12 (3 x 15 mL each) to gives a product that gives a
positive Kaiser
amine test reaction. This resin is then suspended in 10 mL of
TFA/CHZCIzfPr3SiH 20:78:2 and
agitated for 30 min. on a platform shaker. The resin is filtered and washed 3
times with 20 mL
of CH2ClZ then 3 times with CH30H. The combined filtrates are evaporated to
give compound
C as its crude tetra-trifluoroacetate salt. This could be purified over silica
gel using a 5 to 40%
gradient of concd NH40H in CH3CN using a fraction collector. The resulting
fractions are
analyzed by TLC (Rf= 0.18 in 70:30 CH3CN/concd NH4OH) and those containing
pure product
are combined and evaporated to give pure compound C as a clear oil in its free
base form. This
is totally converted to its tetrahydrochloride salt by dissolving in 20 mL of
CH30H and treating
with an excess of 6N HCl. Evaporation of the solvents gives 30 mg (22%) white
solid (Fig. 11).
23
CA 02555862 2006-08-11
WO 2005/105729 PCT/US2004/009582
EXAMPLE 4
Cell Culture and Rea ents
All cell lines are obtained from ATCC (Manassas, VA) and cultured in the
recommended
media, serum, and COa concentration. Medias are obtained from Mediatech, Inc.
(Herndon,
S WA) and serums from Gibco BRL (Gaithersburg, MD). 50 U/mL penicillin, 50
mglmL
streptomycin and 2 mM L-glutamine (all from BioWhittaker, Walkersville, MD)
are included in
all cultures. When cells are cultured with polyamines or polyamine analogs, 1
mM
aminoguanidine (AG; Sigma) is included to inhibit serum amine oxidase
activity. DFMO is
obtained from Advanced ChemTech (Louisville, Kentucky).
EXAMPLE 5
Polyamine Analogs Induce Antizyme Frameshiftin~
A series of compounds are screened for their ability to induce frameshifting
using the
dual luciferase reporter assay. HEK-293 cells are plated in white sided, clear-
bottomed 96-well
assay plates at 15,000 cells per well in 100 ~L of medium (DMEM supplemented
with 10% fetal
bovine serum (Gibco), 1 % penicillin, streptomycin and L-glutamine) containing
2.5 mM DFMO.
The cells are incubated for 2-days at 37°C in an atmosphere of 5% COZ.
The medium is
removed and the cells are then transfected with lipofectamine reagent
(LifeTechnologies) using
serum free DMEM. All cells are transfected overnight with 100 ng of the
appropriate plasmid
DNA and 0.3 p.L lipofectamine in 50 ~.L of serum free DMEM containing 2.5 mM
DFMO. The
next day, 40 ~.L of medium is added per well containing 3.1 mM DFMO, 2.5 mM
aminoguanidine and 25% FBS. Compounds are diluted in either water or medium to
0.25 mM
and 10 p,L is added per well such that the final concentration is 25 p.M. The
positive control
contains 25 ~M spermidine and for the negative control no compound is added.
The cells are
incubated overnight at 37°C in an atmosphere of 5% COa, washed once
with 1X PBS, lysed with
50 ~,L of passive lysis buffer (Promega) and assayed for renilla and firefly
luciferase activity
using the Dual-Luciferase Reporter Assay System (Promega). The percent
frameshifting activity
is determined by dividing the ratio of the firefly luciferase to renilla
luciferase activity in cells
transfected with the inducible 0 to +1 AZ1 construct by the ratio of the
firefly luciferase to
renilla luciferase activity in cells transfected with the control vector AZ-IF
and multiplied by
100.
The percent relative frameshifting value (% RF) gives a comparison of the
ability of a
compound to induce frameshifting compared to 25 ~,M spermidine. The % RF is
calculated as
follows. The background percent frameshifting activity determined from the 2.5
ri'i DFMO
24
CA 02555862 2006-08-11
WO 2005/105729 PCT/US2004/009582
negative control is subtracted from the percent frameshifting activity for all
compounds
including the spermidine control. The background corrected frameshifting
activity of each
compound is then divided by the background corrected frameshifting activity
induced by 25 ~,M
spermidine and multiplied by 100.
From the above screenings, a number of compounds are found to induce
frameshifting
(Fig. 1 and Fig. 2). Some of these compounds induce frameshifting
substantially better than
spermidine at the same concentration (25 ~M). To determine the potency for
inducing antizyme
frameshifting, the polyamines spermine, spermidine, putrescine and compound A
are also titrated
using the dual luciferase reporter assay (Fig. 3). ECso represents the
concentration of the
compound that resulted in 50% of the maximum percent frameshifting as measured
by the
plateau values in Figure 3. The titration indicates that EC50 values for
induction of frameshifting
of all three polyamines are close to 1 ~,M with a rank order potency of
spermidine (0.56 ~M) >
spermine (0.68 p,M) > putrescine (0.95 ~M). Compound A has a less potent ECso
for
frameshifting of about 120 ~M. The maximum levels of frameshifting also vary
between
putrescine, spermidine, spermine and compound A.
It has generally been reported that the rank order potency for frameshift
induction by the
polyamines beginning with the most potent is spermine, followed by spermidine
then putrescine.
However in this study (Fig. 3), the polyamines all have a similar potency with
ECSO values
between 0.5-1.0 ~M. Spermine has been reported to have a similar value
(Mitchell, J. L. A. et.
czl., Bioclaem. ,I. Vol. 366, p.663-671, 2002). In the cell-based assay, both
putrescine and
spermidine can be converted intracellularly into spermidine and spermine via
the transfer of an
aminopropyl group by spermidine synthase or spermine synthase, respectively.
It is conceivable
the frameshifting activities observed for putrescine and spermidine may
actually reflect a
combination of activities for the polyamines synthesized during the course of
the assay.
25
CA 02555862 2006-08-11
WO 2005/105729 PCT/US2004/009582
EXAMPLE 6
Growth Inhibition Assay
Cells are plated in 96-well plates such that they would be in log growth for
the duration
of the assay. The day after plating, the polyamine analogs are added to the
cells, and growth, if
any, permitted to continue for six days in the presence of 1 mM AG and 1 ~.M
SPD when
appropriate. At the end of six days, cell growth is measured by MTS/PMS dye
assay (Cell Titer
96 Aqueous Non-Radioactive Cell Proliferation Assay; Promega, Madison, WI).
ICSO refers to
the concentration of the polyamine analog that results in 50% of maximum cell
growth
inhibition.
The cell growth of HEK-293 cells is inhibited by compound A (Fig. 4). An ICSO
of
approximately 60 ~,M is found for growth inhibition with this polyamine
analog. Addition of 1
p,M spermidine did rescue the cells from this growth inhibition between 3 -100
p.M of
compound A (Fig 4). These results suggest that spermidine competes with
compound A for
entry into the cell and that compound A enters the cell at least in part
through the polyamine
transporter.
EXAMPLE 7
Polyamine Analogs and Rescue from DFMO induced growth inhibition
Compounds found to induce antizyme frameshifting are evaluated for the
undesired
ability to rescue cells from DFMO-induced growth inhibition (Fig. 1 and Fig.
5). HEK-293 cells
are plated in 96-well assay plates at 1,000 cells per well in 100 pL of medium
(DMEM
supplemented with 10% fetal bovine serum (Gibco), 1 % penicillin, streptomycin
and L-
glutamine). The cells are incubated overnight at 37°C in an atmosphere
of 5% COa.
Compounds are added the next day to a final concentration of 25 pM along with
1 rnM
aminoguanidine (inhibits serum amine oxidase) and 2.5 mM DFMO in a final
volume of 200 ~,L
medium. The positive control contained 25 ~M spermidine. The cells are allowed
to incubate
for 6 days before cell growth is measured by MTS/PMS dye assay (Cell Titer 96
Aqueous Non-
Radioactive Cell Proliferation Assay, Promega). The rescue coefficients in
Fig. 1 and Fig. 5 are
expressed as the ratio of the O.D. ((compound + 2.5 mM DFMO)/O.D. (2.5 mM DFMO
alone)).
Therefore, DFMO alone has a value of one. If the drug has the desired effect
of no rescue then it
has a value close to DFMO alone (i.e. about 1). If the value is less than one,
then the compound
is growth inhibitory. If the value is 0, the compound is cytotoxic. Compounds
that rescued cells
from DFMO induced growth inhibition give ratios higher than one and are less
desirable as
therapeutics.
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CA 02555862 2006-08-11
WO 2005/105729 PCT/US2004/009582
As shown in Fig 5, neither compound A nor B significantly rescue cells from
DFMO-
induced growth inhibition. Compound A is the least effective in comparison to
the spermidine
control. Agmatine is somewhat better than either A or B in rescuing cells from
DFMO induced
growth inhibition.
A useful factor to compare the effectiveness of the various antizyme
frameshifters takes
into account their potency for inducing frameshifting and inability to rescue
cells from DFMO-
induced growth inhibition. This measure is referred to as the frameshift-
rescue factor (FRF).
The FRF is calculated by multipling the %RF value by the inverse of the rescue
coefficient. This
method greatly increases the simplicity of analyzing multiple data parameters.
If either the %RF
value is low or the rescue coefficient high, the compound does not stand out
as a potential
candidate. Using this analysis with compounds A, B and agmatine, FRF values of
120, 83 and
17 are obtained, respectively. Based on this method of analysis, compound A
potentially has the
greatest ability to deplete polyamine levels and inhibit cell growth.
EXAMPLE 8
Depletion of Intracellular Polyamine Levels with Compound A
Compound A is further evaluated to determine if it depletes intracellular
polyamine levels
in HEK-293 cells in a dose-dependent fashion (Fig. 6). HEK-293 cells are
plated in 75 cm2
flasks at 300,000 cells/mL to insure they would be in log growth for the
duration. of the
experiment. After overnight incubation at 37°C in an atmosphere of 5%
CO2, compound A is
added along with 1 mM aminoguanidine. The flasks are then incubated for 6 days
at 37°C in an
atmosphere of 5% C02. The cells are harvested by washing twice with ice-cold
1X PBS,
trypsinized, counted, and lysed in 0.4 N perchloric acid. Diaminoheptane is
used in all
dansylation reactions as an internal standard. Peak area is normalized by
dividing the peak area
of the compound divided by the peak area of the internal standard. The HPLC
method for the
fluorometric detection of polyamines from the cell extracts is based on the
procedure by Kabra
(Kabra, P. M. et. al. J. Chromatogr. 380:19-32 (1986)).
It is expected that if compound A induces frameshifting and therefore elevates
the level
of antizyme, intracellular levels of polyamines would decrease. This is
observed in HEK-293
cells, which show significant reductions of intracellular levels of polyamines
(Fig. 6). Putrescine
is most sensitive to the treatment and is non- detectable at the lowest
concentration tested.
Spemidine levels are down by about 90% at 10 ~M and are non-detectable when
incubated with
100 ~M of compound A. Cell growth is also affected, decreasing by 32% at 10
~,M and 38.5%
at 30 ~,M. Spermine levels initially remained steady as has been generally
found:fox'man~,. ,
27~
CA 02555862 2006-08-11
WO 2005/105729 PCT/US2004/009582
polyamine depletion therapies involving ~DC inhibition. However, spermine
levels are reduced
by 50% at 100 ~M. The intracellular levels of compound A also increase dose-
dependently (Fig.
7).
Cell growth inhibition positively correlates with the decreasing polyamine
levels (Fig. 6).
The huge drop in the intracellular levels of putrescine and spermidine may
lead to the initial
inhibition of cell growth. At compound A levels above 30 ~,M, spermine levels
also begin to
diminish to approximately 50% of normal. It is possible that if spermine
levels fall below 50%
of normal then cell death will occur. This is supported by the fact that
although the intracellular
spermine concentration levels off at about 50% of normal, cell growth
continues to diminish.
Perhaps as the extracellular levels of Compound A increase, fewer cells are
able to maintain
intracellular spermine levels at the critical level of 50% of normal. The
evidence for the 50%
threshold level arises from the fact that only those cells that are living
(still attached to the plate)
are harvested and their intracellular polyamine levels determined. Presumably,
the dead cells
expire because their spermine levels fell below 50%.
EXAMPLE 9
Potency of Polyamine Analogs versus A atine
Agmatine is an additional compound reported to induce antizyme frameshifting
(Satriano, J. et. al., J. Biol. Chem. 273:15313-15316 (1998)). "It has a
reported maximum
frameshifting efficiency at 4 mM. It is also found to inhibit mouse kidney
proximal tubule
(MCT) cell proliferation with maximum growth inhibition observed by 1 mM.
Studies at
MediQuest Therapeutics have found that agmatine inhibits growth with an ICSO
of approximately
2 mM in MDA-MB-231 cells, 5 mM in the prostate PC-3 cells line and 0.21 mM in
HEK-293
cells (data not shown). Both compound A and B are found to be more potent than
agmatine in
inducing antizyme frameshifting (Fig 2). Compound A is also a more potent
inhibitor of HEK-
293 cell growth with an ICSo of 60 ~,M and is cytotoxic at 1 mM. It has been
previously shown
that forced expression of antizyme can result in cell death. The toxicity of
compound A
observed at 1 mM suggests that a potent antizyme inducer can be cytotoxic when
the antizyme
levels reach a sufficiently high threshold value.
EXAMPLE 10
Polyamine Analogs that Inhibit Polyamine Transport and Induce Antizyme
Frameshiftin~
A number of compounds have been developed in recent years that inhibit
polyamine
transport ( Huber, M. et. al., J. Biol. Chem. 271:27556-27563 (1996);
Covassin, L. et. al.,
28
CA 02555862 2006-08-11
WO 2005/105729 PCT/US2004/009582
Bioofg. Med. Chena. Lett. 9:1709-1714 (1999); Zharig, M, et. al., J. Mol. Med.
5:595-605 (1999);
Aziz, S. M. et. al J. Pharmacol. Exp. Tl2er~. 274:181-186 (1995); Tomasi, S.
et. al., Bioorg. Med.
Claena. Lett. 8:635-640 (1998); Cullis, P. M. et. al., Chem. Biol. 6:717-729
(1999); Chao, J. et.
al., Mol. Pharmacol. 51:861-871 (1997); Weeks, R. S. et. al., Exp. Cell. Res.
261:293-302
(2000); Burns, M. R. et. al., J. Med. Chem. 44:36232-3644 (2001); Graminski,
G. F. et. al.,
BiooYg. Med. Chem. Lett. 12:35-40 (2002)). These compounds axe generally
thought to bind to
the transporter but are not in themselves substrates for the transporter. It
is conceivable that a
number of these reported transport inhibitors may inhibit the transporter
indirectly by activating
antizyme. For example, Poulin has described potent transport inhibitors that
crosslink syrra-
norspermidine via its secondary amino groups with compounds such as a planarp-
xylyl
crosslinker (Covassin, L. et. al., BiooYg. Med. Claem. Lett. 9:1709-1714
(1999)). The 1,4-
bis[bis(3-aminopropyl)xylene diamine analog (Compound S) has been found to
induce antizyme
frameshifting using the dual luciferase reporter assay. At a concentration of
25 ~.M, this
compound is found to induce frameshifting with a %RF of 100. When tested for
its ability to
rescue from 2.5 mM DFMO induced growth inhibition, it shows no ability to
rescue at 25 ~.M
(Fig. 1). These results suggest that 1,4-bis[bis(3-aminopropyl) xylene
diaxnine can enter the cell
and inhibit transport at least in part through induction of full length
antizyme.
The pharmaceutically acceptable carriers described herein, for example,
vehicles,
adjuvants, excipients, or diluents, are well-known to those who are skilled in
the art. Typically,
the pharmaceutically acceptable earner is chemically inert to the active
compounds and has no
detrimental side effects or toxicity under the conditions of use. The
pharmaceutically acceptable
earners can include polymers and polymer matrices.
The compounds of this invention can be administered by any conventional method
available for use in conjunction with pharmaceuticals, either as individual
therapeutic agents or
in a combination of therapeutic agents.
The dosage administered well, of course, vary depending upon known factors,
such as the
pharmacodynamic characteristics of the particular agent and its mode and route
of
administration; the age, health and weight of the recipient; the nature and
extent of the
symptoms; the kind of concurrent treatment; the frequency of treatment; and
the effect desired.
A daily dosage of active ingredient can be expected to be about 0.001 to 1000
milligrams (mg)
per kilogram (kg) of body weight, with the preferred dose being 0.1 to about
30 mg/kg.
Dosage forms (compositions suitable for administration) contain from about 1
mg to
about 500 mg of active ingredient per unit. In these pharmaceutical
compositions, the active
29
CA 02555862 2006-08-11
WO 2005/105729 PCT/US2004/009582
ingredient will ordinarily be present in an amount of about 0.5-95% weight
based on the total
weight of the composition.
The active ingredient can be administered orally in solid dosage forms, such
as capsules,
tablets, and powders, or in liquid dosage forms, such as elixirs, syrups and
suspensions. It can
also be administered parenterally, in sterile liquid dosage forms. The active
ingredient can also
be administered intranasally (nose drops) or by inhalation of a drug powder
mist. Other dosage
forms are potentially possible such as administration transdermally, via patch
mechanism or
ointment.
Formulations suitable for oral administration can consist of (a) liquid
solutions, such as
an effective amount of the compound dissolved in diluents, such as water,
saline, or orange juice;
(b) capsules, sachets, tablets, lozenges, and troches, each containing a
predetermined amount of
the active ingredient, as solids or granules; (c) powders; (d) suspensions in
an appropriate liquid;
and (e) suitable emulsions. Liquid formulations may include diluents, such as
water and
alcohols, for example, ethanol, benzyl alcohol, propylene glycol, glycerin,
and the polyethylene
alcohols, either with or without the addition of a pharmaceutically acceptable
surfactant,
suspending agent, or emulsifying agent. Capsule forms can be of the ordinary
hard- or soft-
shelled gelatin type containing, for example, surfactants, lubricants, and
inert fillers, such as
lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can include
one or more of
the following: lactose, sucrose, mannitol, corn starch, potato starch, alginic
acid, microcrystalline
cellulose, acacia, gelatin, guar gurn, colloidal silicon dioxide,
croscarmellose sodium, talc,
magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other
excipients, colorants,
diluents, buffering agents, disintegrating agents, moistening agents,
preservatives, flavoring
agents, and pharmacologically compatible carriers. Lozenge forms can comprise
the active
ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as
pastilles comprising
the active ingredient in an inert base, such as gelatin and glycerin, or
sucrose and acadia,
emulsions, and gels containing, in addition to the active ingredient, such
carriers as are known in
the art.
The compounds of the present invention, alone or in combination with other
suitable
components, can be made into aerosol formulations to be administered via
inhalation. These
aerosol formulations can be placed into pressurized acceptable propellants,
such as
dichlorodifluoromethane, propane, and nitrogen. They also may be formulated as
pharmaceuticals for non-pressured preparations, such as in a nebulizer or an
atomizer.
Formulations suitable for parenteral administration include aqueous and non-
aqueous,
isotonic sterile injection solutions, which can contain anti-oxidants,
buffers, bacteriostats,.and
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solutes that render the formulation isotonic with the blood of the intended
recipient, and aqueous
and non-aqueous sterile suspensions that can include suspending agents,
solubilizers, thickening
agents, stabilizers, and preservatives. The compound can be administered in a
physiologically
acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or
mixture of liquids,
including water, saline, aqueous dextrose and related sugar solutions, an
alcohol, such as ethanol,
isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or
polyethylene glycol such
as poly(ethyleneglycol) 400, glycerol ketals, such as 2,2-dimethyl-1,3-
dioxolane-4-methanol,
ethers, an oil, a fatty acid, a~fatty acid ester or glyceride, or an
acetylated fatty acid glyceride
with or without the addition of a pharmaceutically acceptable surfactant, such
as a soap or a
detergent, suspending agent, such as pectin, carbomers, methylcellulose,
hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents
and other
pharmaceutical adjuvants.
Oils, which can be used in parenteral formulations include petroleum, animal,
vegetable,
or synthetic oils. Specific examples of oils include peanut, soybean, sesame,
cottonseed, corn,
olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral
formulations include
oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl
myristate are examples of
suitable fatty acid esters. Suitable soaps for use in parenteral formulations
include fatty alkali
metal, ammonium, and triethanolamine salts, and suitable detergents include
(a) cationic
detergents such as, for example, dimethyldialkylaxnmonium halides, and
alkylpyridinium
halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin
sulfonates, alkyl,
olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic
detergents such as,
for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene
polypropylene
copolymers, (d) amphoteric detergents such as, for example, alkyl 13-
aminopropionates, and 2-
alkylimidazoline quaternary ammonium salts, and (e) mixtures thereof.
The parenteral formulations typically contain from about 0.5% to about 25% by
weight of
the active ingredient in solution. Suitable preservatives and buffers can be
used in such
formulations. In order to minimize or eliminate irritation at the site of
injection, such
compositions may contain one or more nonionic surfactants having a hydrophile-
lipophile
balance (HLB) of from about 12 to about 17. The quantity of surfactant in such
formulations
ranges from about 5% to about 15% by weight. Suitable surfactants include
polyethylene
sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular
weight adducts of
ethylene oxide with a hydrophobic base, formed by the condensation of
propylene oxide with
propylene glycol.
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Pharmaceutically acceptable excipients are also well-known to those who are
skilled in
the art. The choice of excipient will be determined in part by the particular
compound, as well as
by the particular method used to administer the composition. Accordingly,
there is a wide
variety of suitable formulations of the pharmaceutical composition of the
present invention. The
following methods and excipients are merely exemplary and are in no way
limiting. The
pharmaceutically acceptable excipients preferably do not interfere with the
action of the active
ingredients and do not cause adverse side-effects. Suitable carriers and
excipients include
solvents such as water, alcohol, and propylene glycol, solid absorbants and
diluents, surface
active agents, suspending agent, tableting binders, lubricants, flavors, and
coloring agents.
The formulations can be presented in unit-dose or multi-dose sealed
containers, such as
ampules and vials, and can be stored in a freeze-dried (lyophilized) condition
requiring only the
addition of the sterile liquid excipient, for example, water, for inj ections,
immediately prior to
use. Extemporaneous injection solutions and suspensions can be prepared from
sterile powders,
granules, and tablets. The requirements for effective pharmaceutical carriers
for injectable
compositions are well known to those of ordinary skill in the art. See
Plaa~maceutics and
Pharmacy Practice, J.B. Lippincott Co., Philadelphia, PA, Banker and Chalmers,
Eds., 238-250
(1982) and ASHP Handbook on Trajectable Drugs, Toissel, 4th ed., 622-630
(1986).
Formulations suitable for topical administration include lozenges comprising
the active
ingredient in a flavor, usually sucrose and acacia or tragacanth; pastilles
comprising the active
ingredient in an inert base, such as gelatin and glycerin, or sucrose and
acacia; and mouthishes
comprising the active ingredient in a suitable liquid carrier; as well as
creams, emulsions, and
gels containing, in addition to the active ingredient, such carriers as are
known in the art.
Additionally, formulations suitable for rectal administration may be presented
as
suppositories by mixing with a variety of bases such as emulsifying bases or
water-soluble bases.
Formulations suitable for vaginal administration may be presented as
pessaries, tampons, creams,
gels, pastes, foams, or spray formulas containing, in addition to the active
ingredient, such
carriers as are known in the art to be appropriate.
Suitable pharmaceutical carriers are described in Remington's Pharmaceutical
Sciences,
Mack Publishing Company, a standard reference text in this field.
The dose administered to an animal, particularly a human, in the context of
the present
invention should be sufficient to effect a therapeutic response in the animal
over a reasonable
time frame. One skilled in the art will recognize that dosage will depend upon
a variety of
factors including a condition of the animal, the body weight of the animal, as
well as the
condition being treated.
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A suitable dose is that which will result in a concentration of the active
agent in a patient
which is known to effect the desired response.
The size of the dose also will be determined by the route, timing and
frequency of
administration as well as the existence, nature, and extend of any adverse
side effects that might
accompany the administration of the compound and the desired physiological
effect.
Useful pharmaceutical dosage forms for administration of the compounds
according to
the present invention can be illustrated as follows:
Hard Shell Capsules
A large number of unit capsules are prepared by filling standard two-piece
hard gelatine
capsules each with 100 mg of powdered active ingredient, 150 mg of lactose, 50
mg of cellulose
and 6 mg of magnesium stearate.
Soft Gelatin Capsules
A mixture of active ingredient in a digestible oil such as soybean oil,
cottonseed oil or
olive oil is prepared and injected by means of a positive displacement pump
into molten gelatin
to form soft gelatin capsules containing 100 mg of the active ingredient. The
capsules are fished
and dried. The active ingredient can be dissolved in a mixture of polyethylene
glycol, glycerin
and sorbitol to prepare a water miscible medicine mix.
Tablets
A large number of tablets are prepared by conventional procedures so that the
dosage unit
is 100 mg of active ingredient, 0.2 mg. of colloidal silicon dioxide, 5 mg of
magnesium stearate,
275 mg of microcrystalline cellulose, 11 mg. of starch, and 98.8 mg of
lactose. Appropriate
aqueous and non-aqueous coatings may be applied to increase palatability,
improve elegance and
stability or delay absorption.
Immediate Release Tablets/Capsules
These are solid oral dosage forms made by conventional and novel processes.
These
units are taken orally without water for immediate dissolution and delivery of
the medication.
The active ingredient is mixed in a liquid containing ingredient such as
sugar, gelatin, pectin and
sweeteners. These liquids are solidified into solid tablets or caplets by
freeze drying and solid
state extraction techniques. The drug compounds may be compressed with
viscoelastic and
thermoelastic sugars and polymers or effervescent components to produce porous
matrices
intended for immediate release, without the need of water.
Moreover, the compounds of the present invention can be administered in the
form of
nose drops, or metered dose and a nasal or buccal inhaler. The drug is
delivered from a nasal
solution as a fine mist or from a powder as an aerosol.
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The foregoing description of the invention illustrates and describes the
present invention.
Additionally, the disclosure shows and describes only the preferred
embodiments of the
invention but, as mentioned above, it is to be understood that the invention
is capable of use in
various other combinations, modifications, and environments and is capable of
changes or
modifications within the scope of the inventive concept as expressed herein,
commensurate with
the above teachings and/or the skill or knowledge of the relevant art. The
embodiments
described hereinabove are further intended to explain best modes known of
practicing the
invention and to enable others skilled in the art to utilize the invention in
such, or other,
embodiments and with the various modifications required by the particular
applications or uses
of the invention. Accordingly, the description is not intended to limit the
invention to the form
disclosed herein. Also, it is intended that the appended claims be construed
to include alternative
embodiments.
34
