Note: Descriptions are shown in the official language in which they were submitted.
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ANALOGS EXHIBITING INHIBITION OF CELL PROLIFERATION,
METHODS OF MAKING, AND USES THEREOF
The present application is a continuation-in-part and claims priority to and
benefit of
U.S. Patent Application Serial No. 10/992,175, which claims the priority
benefit of
provisional U.S. Patent Application Serial No. 601523,079, filed November 18,
2003, both of
which are hereby incorporated by reference in their entirety.
The present application claims priority to and benefit of provisional U.S.
Patent
Application Serial No. 60/543,724, filed February 11, 2004, and provisional
U.S. Patent
Application Serial No. 60/555,803, filed March 24, 2004, both of which are
hereby
incorporated by reference in their entirety. .
This application was made, at least in part, with funding received from the
U.S.
Department of Defense under grant DAMD 17-O1-1-0830. The U.S. government may
retain
certain rights in this invention.
BACKGROUND
Prostate cancer accounts for 33% of all newly diagnosed malignancies among men
in
the United States (American Cancer Society: Cancer Facts and Figures (2003)).
According to
the American Cancer Society, an estimated 230,110 men will be diagnosed with
prostate
cancer in 2004, and 29,900 men will die of it (American Cancer Society: Cancer
Facts and
Figures (2004)). The incidence of prostate cancer varies worldwide, with the
highest rates
found in the United States, Canada, and Scandinavia, and the lowest rates
found in China and
other parts of Asia (Quinn and Babb, "Patterns and Trends in Prostate Cancer
Incidence,
Survival, Prevalence and Mortality. Part: International Comparisons," BJU Int.
90:162-173
(2002); Gronberg, "Prostate Cancer Epidemiology," Lancet 361:859-864 (2003)).
These
differences are caused by genetic susceptibility, exposure to unknown external
risk factors,
differences in health care and cancer registration, or a combination of these
factors.
Cancer of the prostate is multifocal and it is conunonly observed that the
cancerous
gland contains multiple independent lesions, suggesting the heterogeneity of
the disease
(Foster et al., "Cellular and Molecular Pathology of Prostate Cancer
Precursors," Scand. J.
Uf~ol. Nephrol. 205:19-43 (2000)). Determinants responsible for the pathologic
growth of the
prostate remain poorly understood, although steroidal androgens and peptide
growth factors
have been implicated (Agus et al., "Prostate Cancer Cell Cycle Regulators:
Response to
Androgen Withdrawal and Development of Androgen Independence," J. Natl.
Cancer. Inst.
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WO 2005/086638 PCT/US2005/004759
91:1869-1876 (1999); Djakiew, "Dysregulated Expression of Growth . Factors and
Their
Receptors in the Development of Prostate Cancer," Prostate 42:150-160 (2000)).
As long as
the cancer is confined to the prostate, it can be successfully controlled by
surgery or
radiation, but in metastatic disease, few options are available beyond
androgen ablation
(Frydenberg et al., "Prostate Cancer Diagnosis and Management," Lancet
349:1681-1687
(1997)), the mainstay of treatment in the case of lymph node involvement or
disseminated
loci. Once tumor cells have become hormone refractory, the standard cytotoxic
agents are
marginally effective in slowing disease progression, although they do provide
some degree of
palliative relief. Current chemotherapeutic regimens, typically two or more
agents, afford
response rates in the range of only 20-30% (Beedassy et al., "Chemotherapy in
Advanced
Prostate Cancer," Sern. Oncol. 26:428-438 (1999); Raghavan et al., "Evolving
Strategies of
Cytotoxic Chemotherapy for Advanced Prostate Cancer," Eur. J. Cancer 33:566-
574 (1997)).
One promising drug development strategy for prostate cancer involves
identifying and
testing agents that interfere with growth factors and other molecules involved
in the cancer
cell's signaling pathways. G-protein coupled receptors ("GPCRs") are a family
of membrane-
bound proteins that are involved in the proliferation and survival of prostate
cancer cells
initiated by binding of lysophospholipids ("LPLs") (Raj et at., "Guanosine
Phosphate
Binding Protein Coupled Receptors in Prostate Cancer: A Review," J. U~ol.
167:1458-1463
(2002); Kue et al., "Essential Role for G Proteins in Prostate Cancer Cell
Growth and
Signaling," J. Urol. 164:2162-2167 (2000); Guo et al., "Mitogenic Signaling in
Androgen
Sensitive and Insensitive Prostate Cancer Cell Lines," J. Urol. 163:1027-1032
(2000); Barki-
Harrington et al., "Bradykinin Induced Mitogenesis of Androgen Independent
Prostate
Cancer Cells," J. Urol 165:2121-2125 (2001)). The importance of G protein-
dependent
pathways in the regulation of growth and metastasis in vivo is corroborated by
the
observation that the growth of androgen-independent prostate cancer cells in
mice is
attenuated by treatment with pertussis toxin, an inhibitor of Gi/o proteins
(Hex et al.,
"Influence of Pertussis Toxin on Local Progression and Metastasis After
Orthotopic
Implantation of the Human Prostate Cancer Cell Line PC3 in Nude Mice,"
Prostate Cancer
Prostatic Dis. 2:36-40 (1999)). Lysophosphatidic acid ("LPA") and sphingosine
1-phosphate
("SIP") are lipid mediators generated via the regulated breakdown of membrane
phospholipids that are . known to stimulate GPCR-signaling.
LPL binds to GPCRs encoded by the Edg gene family, collectively referred to as
LPL
receptors, to exert diverse biological effects. LPA stimulates phospholipase D
activity and
PC-3 prostate cell proliferation (Qi et al., "Lysophosphatidic Acid Stimulates
Phospholipase
2
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D Activity and Cell Proliferation in PC-3 Human Prostate Cancer Cells," J.
CeII. Physiol.
174:261-272 (1998)). Further, prior studies have shown that LPA is mitogenic
in prostate
cancer cells and that PC-3 and DU-145 express LPAl, LPA2, and LPA3 receptors
(Daaka,
"Mitogenic Action of LPA in Prostate," Biochim. Biophys. Acts 1582:265-269
(2002)).
Advanced prostate cancers express LPL receptors and depend on
phosphatidylinositol 3-
kinase ("PI3K") signaling for growth and progression to androgen independence
(Kue and
Daaka, "Essential Role for G Proteins in Prostate Cancer Cell Growth and
Signaling," J. Ural.
164:2162-2167 (2000)). Thus, these pathways are widely viewed as one of the
most
promising new approaches to cancer therapy (Vivanco et al., "The
Phosphatidylinositol 3-
Kinase AKT Pathway in Human Cancer," Nat. Rev. Cancer 2:489-501 (2002)) and
provide
an especially novel approach to the treatment of advanced, androgen-refractory
prostate
cancer. Despite the promise of this approach, there are no clinically
available therapies that
selectively exploit or inhibit LPA or PI3K signaling.
The present invention is directed to overcoming these and other deficiencies
in the
prior art.
SUMMARY
A first aspect of the present invention relates to compounds according to
formula (I)
and formula (II)
C2 C2
R2\ ~1 J(1
(I) R1 (II)
wherein
Xl and X2 are each optional, and each can be oxygen;
X3 and X4 are each optional, and each can be oxygen or sulfur;
l is an integer from 1 to 12;
Rl is selected from the group of saturated or unsaturated cyclic hydrocarbons,
saturated or unsaturated N-heterocyeles, saturated or unsaturated O-
heterocycles, saturated or
unsaturated S-heterocycles, saturated or unsaturated mixed heterocycles,
aliphatic or non-
aliphatic straight- or branched-chain C1 to C30 hydrocarbons, or
3
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S aH
or-(CH2)m Yl where era is an integer from 0 to 10 and Yl is a
saturated or unsaturated cyclic hydrocarbon, saturated or unsaturated N-
heterocycle, saturated
or unsaturated O-heterocycle, saturated or unsaturated S-heterocycle, or
saturated or
unsaturated mixed heterocycle;
RZ is hydrogen, an aliphatic or non-aliphatic straight- or branched-chain C1
to C30
hydrocarbon, Rl°-N(Z)-hydrocarbon- or Rl°-hydrocarbon- where the
hydrocarbon
group is an aliphatic or non-aliphatic straight- or branched-chain Cl to C30
hydrocarbon, a
saturated or unsaturated cyclic hydrocarbon, a saturated or unsaturated N-
heterocycle, a
saturated or unsaturated 0-hetexocycle, a saturated or unsaturated S-
heterocycle, a saturated
R13
or unsaturated mixed heterocycle, or or -(CH2)ri Y2 where
n is an integer from 0 to 10 and YZ is a saturated or unsaturated cyclic
hydrocarbon, saturated
or unsaturated N-heterocycle, saturated or unsaturated O-heterocycle,
saturated or
unsaturated S-heterocycle, or saturated or unsaturated mixed heterocycle;
R3 is hydrogen or an aliphatic or non-aliphatic straight- or branched-chain Cl
to C10
hydrocarbon;
R4 is optional, or can be hydrogen, an aliphatic or non-aliphatic straight- or
branched-
chain Cl to C10 hydrocarbon, aryl, acetyl, or mesyl;
R5, R6, R~, R8, R9, Rl l, Ria, Ri3, Rlø, and R15 are independently selected
from the
group of hydrogen, hydroxyl, an aliphatic or non-aliphatic straight-or
branched-chain C1 to
C10 hydrocarbon, alkoxy, aryloxy, nitro, cyano, chloro, fluoro, bromo, iodo,
haloallcyl,
dihaloalkyl, trihaloalkyl, amino, alkylamino, diallcylamino, acylamino,
arylamido, amido,
allcylamido, diallcylamido, arylamido, aryl, CS to C7 cycloalkyl, arylalkyl;
Rl° is H(Z)N-, H(Z)N-hydrocarbon-, H(Z)N-hydrocarbon-N(Z)
-hydrocarbon-, H(Z)N-hydrocarbon-, O hydrocarbon-,
hydrocarbon-O-hydrocarbon-, hydrocarbon-N(Z) hydrocarbon-,
H(Z)N-hydrocarbon-carbonyl-hydrocarbon-, hydrocarbon-carbonyl-hydrocarbon,
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H(Z)N-phenyl-, H(Z)N-phenylalkyi-, H(Z)N-phenylalkyl-N(Z)-hydrocarbon-,
H(Z)N-phenylalkyl-O-hydrocarbon-, phenylalleyl-O-hydrocarbon-,
phenylalkyl-N(Z) -hydrocarbon-, H(Z)N-phenylalkyl-carbonyl-hydrocarbon-, or
phenylalltyl-carbonyl-hydrocarbon-, wherein each hydrocarbon is independently
an aliphatic
or non-aliphatic straight- or branched-chain C 1 to C 10 group, and wherein
each alkyl is a C 1
to C 10 alkyl; and
Z is independently hydrogen or t-butoxycarbonyl.
A second aspect of the present invention relates to compounds according to
formula
(V) and formula (VI)
(V) '
wherein
Xl and X2 are each optional, and each can be oxygen;
XS is optional, and can be oxygen;
Rl is selected from the group of saturated or unsaturated cyclic hydrocarbons,
saturated or unsaturated N-heterocycles, saturated or unsaturated O-
heterocycles, saturated or
unsaturated S-heterocycles, saturated or unsaturated mixed heterocycles,
aliphatic or non-
aliphatic straight- or branched-chain C1 to C30 hydrocarbons, or
~cH2)
or-(CHZ)m Yl where rn is an integer from 0 to 10 and Yl is a
saturated or unsaturated cyclic hydrocarbon, saturated or unsaturated N-
heterocycle, saturated
or unsaturated O-heterocycle, saturated or unsaturated S-heterocycle, or
saturated or
unsaturated mixed heterocycle;
RZ is hydrogen, an aliphatic or non-aliphatic straight- or branched-chain C1
to C30
hydrocarbon, Rl°-N(Z)-hydrocarbon- or Rl°-hydrocarbon- where the
hydrocarbon
group is an aliphatic or non-aliphatic straight- or branched-chain Cl to C30
hydrocarbon, a
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saturated or unsaturated cyclic hydrocarbon, a saturated or unsaturated N-
heterocycle, a
saturated or unsaturated 0-heterocycle, a saturated or unsaturated S-
heterocycle, a saturated
R~~~ R12
(CH2)n R~s
or unsaturated mixed heterocycle, or R1s R~4 or -(CH2)ri Y2 where
n is an integer from 0 to 10 and YZ is a saturated or unsaturated cyclic
hydrocarbon, saturated
or unsaturated N-heterocycle, saturated or unsaturated O-heterocycle,
saturated or
unsaturated S-heterocycle, or saturated or unsaturated mixed heterocycle;
R3 is nothing, hydrogen or an aliphatic or non-aliphatic straight- or branched-
chain Cl
to C10 hydrocarbon;
R4 is optional, or can be hydrogen, an aliphatic or non-aliphatic straight- or
branched-
chain C1 to C10 hydrocarbon, aryl, acetyl, or mesyl;
R5, R6, R', R8, R9, Rl l, Rlz, R13, R14, and R15 are independently selected
from the
group of hydrogen, hydroxyl, an aliphatic or non-aliphatic straight-or
branched-chain C 1 to
CIO hydrocarbon, alkoxy, aryloxy, nitro, cyano, chloro, fluoro, bromo, iodo,
haloa.lkyl,
dihaloalkyl, trihaloalkyl, amino, alkylamino, dialkylamino, acylamino,
arylamido, amido,
alkylamido, dialkylamido, arylamido, aryl, CS to C7 cycloalkyl, arylalkyl;
Rl° is H(Z)N-, H(Z)N-hydrocarbon-, H(Z)N-hydrocarbon-N(Z)
-hydrocarbon-, H(Z)N-hydrocarbon-, O hydrocarbon-,
hydrocarbon-O-hydrocarbon-, hydrocarbon-N(Z) hydrocarbon-,
H(Z)N-hydrocarbon-carbonyl-hydrocarbon-, hydrocarbon-carbonyl-hydrocarbon,
H(Z)N-phenyl-, H(Z)N-phenylalkyi-, H(Z)N-phenylallcyl-N(Z)-hydrocarbon-,
H(Z)N-phenylallcyl-O-hydrocarbon-, phenylallcyl-O-hydrocarbon-,
phenylalkyl-N(Z) -hydrocarbon-, H(Z)N-phenylallcyl-carbonyl-hydrocarbon-, or
phenylallcyl-carbonyl-hydrocarbon-, wherein each hydrocarbon is independently
an aliphatic
or non-aliphatic straight- or branched-chain C1 to C10 group, and wherein each
alkyl is a C1
to C 10 alkyl; and
Z is independently hydrogen or t-butoxycarbonyl.
A third aspect of the present invention relates to compounds according to
formula
(VII)
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R2W
R~ (VII)
wherein
X3 is optional and can be oxygen;
X6 is oxygen or nitrogen;
Rl is selected from the group of saturated or unsaturated cyclic hydrocarbons,
saturated or unsaturated N-heterocyeles, saturated or unsaturated O-
heterocycles, saturated or
unsaturated S-heterocycles, saturated or unsaturated mixed heterocycles,
aliphatic or non-
aliphatic straight- or branched-chain C1 to C30 hydrocarbons, or
(CH2)m R~
or-(CHZ)m Yl where m is an integer from 0 to 10 and Yl is a
saturated or unsaturated cyclic hydrocarbon, saturated or unsaturated N-
heterocycle, saturated
or unsaturated O-heterocycle, saturated or unsaturated S-heterocycle, or
saturated or
unsaturated mixed heterocycle;
R2 is hydrogen, an aliphatic or non-aliphatic straight- or branched-chain C1
to C30
hydrocarbon, Rl°-N(Z)-hydrocarbon- or Rl°-hydrocarbon- where the
hydrocarbon
group is an aliphatic or non-aliphatic straight- or brmched-chain Cl to C30
hydrocarbon, a
saturated or unsaturated cyclic hydrocarbon, a saturated or unsaturated N-
heterocycle, a
saturated or unsaturated 0-heterocycle, a saturated or unsaturated S-
heterocycle, a saturated
R~~~ R~2
(CH2)n R~s
or unsaturated mixed heterocycle, or R~5 R~4 or -(CH2)n- YZ where
n is an integer from 0 to 10 and Y2 is a saturated or unsaturated cyclic
hydrocarbon, saturated
or unsaturated N-heterocycle, saturated or unsaturated O-heterocycle,
saturated or
unsaturated S-heterocycle, or saturated or unsaturated mixed heterocycle;
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R3 is nothing, hydrogen or an aliphatic or non-aliphatic straight- or branched-
chain Cl
to CIO hydrocarbon;
R4 is optional, or can be hydrogen, an aliphatic or non-aliphatic straight- or
branched-
chain C 1 to C 10 hydrocarbon, aryl, acetyl, or mesyl;
R5, R6, R', R8, R9, R11, Riz' R13, Ri4, and R15 are independently selected
from the
group of hydrogen, hydroxyl, an aliphatic or non-aliphatic straight-or
branched-chain C1 to
C10 hydrocarbon, alkoxy, aryloxy, nitro, cyano, chloro, fluoro, bromo, iodo,
haloalkyl,
dihaloalkyl, trihaloalkyl, amino, alkylamino, dialkylamino, acylamino,
arylamido, amido,
alkylamido, dialkylarnido, arylamido, aryl, CS to C7 cycloall~yl, arylalkyl;
IO Ri° is H(Z)N-, H(Z)N-hydrocarbon-, H(Z)N-hydrocarbon-N(Z)
-hydrocarbon-, H(Z)N-hydrocarbon-, O hydrocarbon-,
hydrocarbon-O-hydrocarbon-, hydrocarbon-N(Z) hydrocarbon-,
H(Z)N-hydrocarbon-carbonyl-hydrocarbon-, hydrocarbon-carbonyl-hydrocarbon,
H(Z)N-phenyl-, H(Z)N-phenylalkyi-, H(Z)N-phenylalkyl-N(Z)-hydrocarbon-,
H(Z)N-phenylalkyl-O-hydrocarbon-, phenylalkyl-O-hydrocarbon-,
phenylalkyl-N(Z) -hydrocarbon-, H(Z)N-phenylalkyl-carbonyl-hydrocarbon-, or
phenylalkyl-carbonyl-hydrocarbon-, wherein each hydrocarbon is independently
an aliphatic
or non-aliphatic straight- or branched-chain C 1 to C 10 group, and wherein
each allcyl is a C 1
to C 10 alkyl; and
Z is independently hydrogen or t-butoxycarbonyl.
A fourth aspect of the present invention relates to compounds of Formula
(VIII)
O
R~
~N NH-(CH2)n-N
R~ ~Xa
(VIII)
wherein
X$ is O or S;
n is between 1 and 30;
Rl is selected from the group of saturated or unsaturated cyclic hydrocarbons,
saturated or unsaturated N-heterocyeles, saturated or unsaturated O-
heterocycles, saturated or
unsaturated S-heterocycles, saturated or unsaturated mixed heterocycles,
aliphatic or non-
aliphatic straight- or branched-chain C1 to C30 hydrocarbons, or
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(CH2)m R7
R9~ R$ or-(CH2)m Yl where m is an integer from 0 to 10 and Yl is a
saturated or unsaturated cyclic hydrocarbon, saturated or unsaturated N-
heterocycle, saturated
or unsaturated O-heterocycle, saturated or unsaturated S-heterocycle, or
saturated or
unsaturated mixed heterocycle;
R4 is optional, or can be hydrogen, an aliphatic or non-aliphatic straight- or
branched-
chain C1 to C10 hydrocarbon, aryl, acetyl, or mesyl; and
R5, R6, R', R8, and R9 are independently selected from the group of hydrogen,
hydroxyl, an aliphatic or non-aliphatic straight-or branched-chain C1 to C10
hydrocarbon,
allcoxy, aryloxy, nitro, cyano, chloro, fluoro, bromo, iodo, haloalkyl,
dihaloallcyl, trihaloalkyl,
amino, alkylamino, dialkylamino, acylamino, arylamido, amido, allcylamido,
diall~ylamido,
arylamido, aryl, CS to C7 cycloalkyl, arylallcyl.
A fifth aspect of the present invention relates to compounds having Formula
X9
'R16 /R16
HO NH NH/'
NH
R17~ \R18 (IX) ..
(X)
NH R16 NH R1s
HO
O (X~ O
(XII)
wherein
X' is P03H or O-benzyl;
X9 is O or nothing;
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R16 is a C1 to C30 aliphatic or non-aliphatic, straight-, cyclic- or branched-
chain,
substituted or unsubstituted, C1 to C30 hydrocarbon;
Rl' and Rl8 are independently nothing, hydrogen, -S02R19, CORl9, and R19; and
Rl9 is an aliphatic or non-aliphatic, straight-, cyclic- or branched-chain,
substituted or
unsubstituted, C1 to C30 hydrocarbon or a substituted or unsubstituted aryl.
A sixth aspect of the present invention relates to a compound of Formula (XIV)
amd
(XV)
O O C~2H2s~0 O
~ ~ Oi~" -
HO NH(CH2)~~HN~OH O ~P=ONH+a
NHBoc NHBoc (X~) ~ONH+4 (XV) .
A seventh aspect of the present invention relates to a pharmaceutical
composition
including a pharmaceutically acceptable carrier and a compound according to
the first,
second, third, fourth, fifth, and sixth aspects of the present invention.
A eighth aspect of the present invention relates to a method of destroying a
cancer cell
that includes the steps of providing a compound according to the first,
second, third, fourth,
fifth, and sixth aspects of the present invention and contacting a cancer cell
with the
compound under conditions effective to destroy the contacted cancer cell.
A ninth aspect of the present invention relates to a method of treating or
preventing a
cancerous condition that includes the steps of providing a compound according
to the first,
second, third, fourth, fifth, and sixth aspects of the present invention and
administering an
amount of the compound to a patient in a manner effective to treat or prevent
a cancerous
condition.
A tenth aspect of the present invention relates to a method of making a
compound
according to formula (I) that includes the steps of: reacting an intermediate
according to
formula (III),
X3
X4
S N
l OH
(III)
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where l, Rl, X3, and X4 are defined as above, with either (i) a suitable
primary or secondary
amine according to the formula (HNR2R3) where R2 and R3 are defined as above,
or (ii)
ammonia in the presence of an RZ-H containing compound, under conditions
effective to
form the compound according to formula (I).
A eleventh aspect of the present invention relates to a method of making a
compound
according to formula (II) that includes the steps of: reacting an intermediate
according to
formula (IV),
X3
CHs
S NH
where R1 and X3 are defined as above, with a primary or secondary amine
according to the
formula (HNRaR3) where RZ and R3 are defined as above, under conditions
effective to form
the compound according to formula (II).
A twelfth aspect of the present invention relates to intermediate compounds
according
to formula (III) and formula (IV).
The present invention affords a significant improvement over previously
identified
cancer therapeutics that are known to be useful for the inhibition of prostate
cancer cell
growth. In a previous report, it was shown that cytotoxic compounds were
obtained by
replacing the glycerol backbone in LPA with serine amide in five prostate
cancer cell lines
(Gududuru et al., "Synthesis and Biological Evaluation of Novel Cytotoxic
Phospholipids for
Prostate Cancer," Btaorg. Med. Chem. Lett. 14:4919-4923 (2004), which is
hereby
incorporated by reference in its entirety). The most potent compounds reported
in Gududuru
et al. (cited above) were non-selective and potently lcilled both prostate
cancer and control
cell lines. The present invention affords compounds that possess similar or
even improved
potency, but more importantly, improved selectivity, particularly with respect
to prostate
cancer cell lines. Compounds of the present invention are shown to be
effective against
prostate cancer cells and ovarian cancer cells.
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BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
The following detailed description of embodiments of the present invention can
be
best understood when read in conjunction with the following drawings, where
like structure is
indicated with like reference numerals and in which:
Figure 1 illustrates one approach (scheme 1) for the synthesis of thiazolidine
carboxylic acid amides. The thiazolidine carboxylic acid intermediate (2a-v)
is formed upon
reacting L-cysteine with various aldehydes under reported conditions (Seki et
al., "A Novel
Synthesis of(+)-Biotin from L-Cysteine,"J. Org. Chem. 67:5527-5536 (2002),
which is
hereby incorporated by reference in its entirety). The intermediate carboxylic
acid is reacted
with an amine to form the corresponding amide (3-27);
Figure 2 illustrates one approach (scheme 2) for the synthesis of N-30 aryl
and N-
sulfonyl derivatives of the thiazolidine carboxylic acid amides. The N-acyl
and N-sulfonyl
derivatives (compounds 28 and 29) were synthesized from compound 5 by standard
procedures;
Figure 3 illustrates one approach (scheme 3) for the synthesis of thiazole
carboxylic
acid amides. The thiazolidine carboxylic acid methyl ester was converted to
the thiazole
carboxylic acid methyl ester following a reported procedure (Badr et al"
"Synthesis of
Oxazolidines, Thiazolidines, and 5,6,7,8-Tetrahydro-1H, 3H pyrrolo[1,2-cJ
Oxazole (or
Thiazole)-1,3-diones from (3-Hydroxy- or ~3-Mercapto-a-amino Acid Esters,"
Bull. Clzefya.
Soc. JprZ. 54:1844-1847 (1981), which is hereby incorporated by reference in
its entirety), and
then converted to the alkylamide;
Figures 4A-B illustrate agarose gel electrophoresis of total DNA extracted
from 2 x
106 LNCaP cells following treatment with thiazolidine compounds 4 (Figure 4A)
and 5
(Figure 4B) for 24 to 108 hours. The results show the effects of treatment on
DNA
fragmentation, indicating progression of cell death. In Figure 4A, the dose
and exposure time
are indicated for compound 4 as follows: lane 1, 100 by DNA marlcer; lane 2, 5
~.M for 36 h;
lane 3, 3 ~M for 24 h; lane 4, 3 ~M for 24 h; lane 5, 3 ~.M for 48 h; lane 6,
3 ,uM for 72 h;
lane 7, 3 ~.M for 108 h; and lane 8, SO ~.M for 36 h. In Figure 4B, the dose
and exposure time
are indicated for compound 5 as follows: lane 1, 100 by DNA marker; lane 2,
S~,M for 24 h;
lane 3, 5 ACM for 48 h; lane 4, 5 ~,M for 72 h; lane 5, 5 ~M for 96 h; lane 6,
'3 pM for 96 h;
lane 7, 8 ~.M for 48 h; and lane 8, 8 ~.M for 72 h;
Figures 5A-B demonstrate AKT inhibitory effects of thiazolidine compounds, as
measured by inhibition of AKT phosphorylation. Figure 5A shows the immunoblot
results
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using anti-phospho AKT (5473) or anti-AKT antibodies. The immunoblots were
visualized
by enhanced chemiluminescence, and changes of relative levels of phospho-AKT
compared
to total AKT by analog treatment were quantified by densitometric analysis.
Figure SB
graphically illustrates the immunological detection of AI~T using anti-AKT and
anti-phospo-
AKT, shown in Figure SA;
Figure 6 illustrates one approach (scheme 4) for the synthesis of 4-
thiazolidinone
carboxylic acids, and their conversion to corresponding amides by reaction
with primary or
secondary amines (HNR2R3). As shown in this reaction scheme, different
starting materials
(where 1 differs) can be used to prepare various compounds of the invention;
Figure 7 illustrates a second approach (scheme 5) for the synthesis of 4-
thiazolidinone
carboxylic acids, and their conversion to corresponding amides by reaction
with RZ-CNO;
Figure 8 illustrates three approaches for modifying the core structure of the
thiazolidinone compounds of the present invention (scheme 6) to afford ring-
bound sulfone
or sulfoxide groups (steps a and b, respectively), as well as the complete
reduction of
carbonyl groups (step c);
Figure 9 illustrates a process for the synthesis of polyamine conjugates of
thiazolidinone amides;
Figure 10 illustrates a scheme for the synthesis of thiazolidinone ethers and
esters;
Figure 11 illustrates a scheme for the synthesis of oxazoline amides;
Figure 12 illustrates a scheme for the synthesis of thiazolidinone dimers;
Figure 13 illustrates a scheme for the synthesis of serine amide alcohols and
phosphates. The reagents and conditions can be: (i) CH3(CH2)"NH2, EDC, HOBt,
CHZCIz, rt,
5 h (ii) TFA, CH2C12, rt, 0.5 h (iii) tetrazole, dibenzyl
diisopropylphosphoramidite, CHZC12,
rt, 0.5 h, HZOz, rt, 0.5 h (iv) H2, 10% Pd/C, EtOH, rt, 3 h;
Figure 14 illustrates a scheme for the synthesis of unsaturated serine amide
alcohols
and phosphates. The reagents and conditions can be: (i) C8H1~ (CH:
CH)C$H16NH2, EDC,
HOBt, CH2C12, rt, 5 h (ii) 2M HCI/EtzO, rt, overnight (iii) tetrazole, di test
butyl
diisopropylphosphoramidite, CH2CI2, rt, 0.5 h, H20z, rt, 0.5 h (iv) TFA,
CHzCl2, rt, 0.5 h;
Figure 15 illustrates a scheme for the synthesis of serine diamide phosphates
and
other amide analogs. The reagents and can be conditions: (i) RZNHz, EDC, HOBt,
CHZC12,
rt, 5 h (ii) TFA, CHZCl2, rt, 0.5 h (iii) TEA, R3SOzCl or R3NC0 or R3COCl (iv)
Ha, 10%
Pd/C, EtOH, rt, 3 h (v) tetrazole, dibenzyl diisopropylphosphoramidite,
CHaCl2, rt, 0.5 h,
H202, rt, 0.5 h (vi) H2, 10% Pd/C, EtOH, rt, 3 h; and
13
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Figure 16 illustrates a scheme for the preparation of amino alcohol analogs.
The
Reagents and conditions: (i) TFA, CHZCIa, rt, 0.5 h (ii) a. LAH, Et20, reflux,
7 h, b. HCl
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
The present invention will now be described with occasional reference to the
specific
embodiments of the invention. This invention may, however, be embodied in
different forms
and should not be construed as limited to the embodiments set forth herein.
Rather, these
embodiments are provided so that this disclosure will be thorough and
complete, and will
fully convey the scope of the invention to those skilled in the art.
Unless otherwise defined, all technical and scientific terms used herein have
the same
meaning as commonly understood by one of oxdinary skill in the art to which
this invention
belongs. The terminology used in the description of the invention herein is
for describing
particular embodiments only and is not intended to be limiting of the
invention. As used in
the description of the invention and the appended claims, the singular forms
"a," "an," and
"the" are intended to include the plural forms as well, unless the context
clearly indicates
otherwise. All publications, patent applications, patents, and other
references mentioned
herein are incorporated by reference in their entirety.
Unless otherwise indicated, all numbers expressing quantities of ingredients,
properties such as molecular weight, reaction conditions, and so forth as used
in the
specification and claims are to be understood as being modified in all
instances by the term
"about." Accordingly, unless otherwise indicated, the numerical properties set
forth in the
following specification and claims are approximations that may vary depending
on the
desired properties sought to be obtained in embodiments of the present
invention.
Notwithstanding that the numerical ranges and parameters setting forth the
broad scope of the
invention are approximations, the numerical values set forth in the specif c
examples are
reported as precisely as possible. Any numerical values, however, inherently
contain ceutain
errors necessarily resulting from error found in their respective
measurements.
One aspect of the invention relates to compounds according to formulae (I) and
(II)
below
14
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RW
R2
\N
R3 R1 (~ R1 (TI)
wherein
Xl and XZ are each optional, and each can be oxygen;
X3 and X4 are each optional, and each can be oxygen or sulfur;
l is an integer from 1 to 12;
Rl is selected from the group of saturated or unsaturated cyclic hydrocarbons,
saturated or unsaturated N-heterocyeles, saturated or unsaturated O-
heterocycles, saturated or
unsaturated S-heterocycles, saturated or unsaturated mixed heterocycles,
aliphatic or non-
aliphatic straight- or branched-chain C1 to C30 hydrocarbons, or
(CH2 R7
or-(CHz)m Yi where m is an integer from 0 to 10 and Yl is a
saturated or unsaturated cyclic hydrocarbon, saturated or unsaturated N-
heterocycle, saturated
or unsaturated O-heterocycle, saturated or unsaturated S-heterocycle, or
saturated or
unsaturated mixed heterocycle;
R2 is hydrogen, an aliphatic or non-aliphatic straight- or branched-chain C1
to C30
hydrocarbon, Rl°-N(Z)-hydrocarbon- or Rl°-hydrocarbon- where the
hydrocarbon
group is an aliphatic or non-aliphatic straight- or branched-chain Cl to C30
hydrocarbon, a
saturated or unsaturated cyclic hydrocarbon, a saturated or unsaturated N-
heterocycle, a
saturated or unsaturated 0-heterocycle, a saturated or unsaturated S-
heterocycle, a saturated
o~~
(CH 13
or unsaturated mixed heterocycle, or ~ - or -(CH2)"- Ya where
n is an integer from 0 to 10 and Y2 is a saturated or unsaturated cyclic
hydrocarbon, saturated
IS
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or unsaturated N-heterocycle, saturated or unsaturated O-heterocycle,
saturated or
unsaturated S-heterocycle, or saturated or unsaturated mixed heterocycle;
R3 is hydrogen or an aliphatic or non-aliphatic straight- or branched-chain Cl
to C10
hydrocarbon;
R4 is optional, or can be hydrogen, an aliphatic or non-aliphatic straight- or
branched-
chain C1 to C10 hydrocarbon, aryl, acetyl, or mesyl;
R5, R6, R', R8, R9, Rll, Ria, Ri3, Ri4, and Rls are independently selected
from the
group of hydrogen, hydroxyl, an aliphatic or non-aliphatic straight-or
branched-chain C1 to
C10 hydrocarbon, alkoxy, aryloxy, nitro, cyano, chloro, fluoro, bromo, iodo,
haloalkyl,
dihaloalkyl, trihaloalkyl, amino, alkylamino, dialkylamino, acylamino,
arylamido, amido,
alkylamido, dialkylamido, arylamido, aryl, CS to C7 cycloalleyl, arylallcyl;
Rl° is H(Z)N-, H(Z)N-hydrocarbon-, H(Z)N-hydrocarbon-N(Z)
-hydrocarbon-, H(Z)N-hydrocarbon-, O hydrocarbon-,
hydrocarbon-O-hydrocarbon-, hydrocarbon-N(Z) hydrocarbon-,
H(Z)N-hydrocarbon-carbonyl-hydrocarbon-, hydrocarbon-carbonyl-hydrocarbon,
H(Z)N-phenyl-, H(Z)N-phenylalkyi-, H(Z)N-phenylallcyl-N(Z)-hydrocarbon-,
H(Z)N-phenylalkyl-O-hydrocarbon-, phenylalkyl-O-hydrocarbon-,
phenylalkyl-N(Z) -hydrocarbon-, H(Z)N-phenylalkyl-carbonyl-hydrocarbon-, or
phenylalkyl-carbonyl-hydrocarbon-, wherein each hydrocarbon is independently
an aliphatic
or non-aliphatic straight- or branched-chain C1 to C10 group, and wherein each
alkyl is a C1
to C 10 alkyl; and
Z is independently hydrogen or t-butoxycarbonyl.
As used herein, "aliphatic or non-aliphatic straight- or branched-chain
hydrocarbon"
refers to both allcylene groups that contain a single carbon and up to a
defined upper limit, as
well as allcenyl groups and alkynyl groups that contain two carbons up to the
upper limit,
whether the carbons are present in a single chain or a branched chain. Unless
specifically
identified, a hydrocarbon can include up to about 30 carbons, or up to about
20 hydrocarbons,
or up to about 10 hydrocarbons.
As used herein, the term "alkyl" can be any straight- or branched-chain alkyl
group
containing up to about 30 carbons unless otherwise specified. The alkyl group
can be a sole
constituent or it can be a component of a larger constituent, such as in an
alkoxy, arylallcyl,
alkylamino, etc.
As used herein, "saturated or unsaturated cyclic hydrocarbons" can be any such
cyclic
hydrocarbon, including but not limited to phenyl, biphenyl, triphenyl,
naphthyl, cycloalkyl,
16
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cycloall~enyl, cyclodienyl, etc.; "saturated or unsaturated N-heterocycles"
can be any such N-
containing heterocycle, including but not limited to aza- and diaza-
cycloalkyls such as
aziridinyl, azetidinyl, diazatidinyl, pyrrolidinyl, piperidinyl, piperazinyl,
and azocanyl,
pyrrolyl, pyrazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl,
pyridazinyl, triazinyl,
tetrazinyl, pyrrolizinyl, indolyl, unsaturated O-heterocycles" can be any such
O-containing heterocycle including but not limited to oxiranyl, oxetanyl,
tetrahydrofuranyl,
tetrahydropyranyl, dioxanyl, furanyl, pyrylium, benzofuranyl, etc.; "saturated
or unsaturated
S-heterocycles" can be any such S-containing heterocycle, including but not
limited to
thiranyl, thietanyl, tetrahydrothiophenyl, dithiolanyl, tetrahydrothiopyranyl,
thiophenyl,
thiepinyl, thianaphthenyl, etc.; "saturated or unsaturated mixed heterocycles"
can be any .
heterocycle containing two or more S-, N-, or O-heteroatoms, including but not
limited to
oxathiolanyl, morpholinyl, thioxanyl, thiazolyl, isothiazolyl, thiadiazolyl,
oxazolyl,
isoxazolyl, oxadiaziolyl, etc.
Another aspect of the present invention relates to compounds according to
formula
(V) and formula (VI)
R2\ R2\
O
R
(V) ~ - (VI)
wherein
Xl and X2 are each optional, and each can be oxygen;
XS is optional, and can be oxygen;
RI is selected from the group of saturated or unsaturated cyclic hydrocarbons,
saturated or unsaturated N-heterocyeles, saturated or unsaturated O-
heterocycles, saturated or
unsaturated S-heterocycles, saturated or unsaturated mixed heterocycles,
aliphatic or non-
aliphatic straight- or branched-chain C1 to C30 hydrocarbons, or
Rs
(CH2)m R7
or-(CH2)m Yl where m is an integer from 0 to 10 and YI is a
saturated or unsaturated cyclic hydrocarbon, saturated or unsaturated N-
heterocycle, saturated
17
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or unsaturated O-heterocycle, saturated or unsaturated S-heterocycle, or
saturated or
unsaturated mixed heterocycle;
RZ is hydrogen, an aliphatic or non-aliphatic straight- or branched-chain C1
to C30
hydrocarbon, Rl°-N(Z)-hydrocarbon- or Rl°-hydrocarbon- where the
hydrocarbon
group is an aliphatic or non-aliphatic straight- or branched-chain Cl to C30
hydrocarbon, a
saturated or unsaturated cyclic hydrocarbon, a saturated or unsaturated N-
heterocycle, a
saturated or unsaturated 0-heterocycle, a saturated or unsaturated S-
heterocycle, a saturated
R~lv R12
(CH2)n R~3
or unsaturated mixed heterocycle, or R15~ R~4 or -(CH2)p YZ where
n is an integer from 0 to 10 and YZ is a saturated or unsaturated cyclic
hydrocarbon, saturated
or unsaturated N-heterocycle, saturated or unsaturated O-heterocycle,
saturated or
unsaturated S-heterocycle, or saturated or unsaturated mixed heterocycle;
R3 is nothing, hydrogen or an aliphatic or non-aliphatic straight- or branched-
chain Cl
to C10 hydrocarbon;
R4 is optional, or can be hydrogen, an aliphatic or non-aliphatic straight- or
branched-
chain C 1 to C 10 hydrocarbon, aryl, acetyl, or mesyl;
R5, R6, R~, R8, R9, Rll, R12, Ri3, Ri4, and RIS are independently selected
from the
group of hydrogen, hydroxyl, an aliphatic or non-aliphatic straight-or
branched-chain C 1 to
C10 hydrocarbon, allcoxy, aryloxy, nitro, cyano, chloro, fluoro, bromo, iodo,
haloalkyl,
dihaloalkyl, trihaloallcyl, amino, allcylamino, dialkylamino, acylamino,
arylamido, amido,
allcylamido, diallcylarnido, arylamido, aryl, CS to C7 cycloalkyl, arylalkyl;
Rl° is H(Z)N-, H(Z)N-hydrocarbon-, H(Z)N-hydrocarbon-N(Z)
-hydrocarbon-, H(Z)N-hydrocarbon-, O hydrocarbon-,
hydrocarbon-O-hydrocarbon-, hydrocarbon-N(Z) hydrocarbon-,
H(Z)N-hydrocarbon-carbonyl-hydrocarbon-, hydrocarbon-carbonyl-hydrocarbon,
H(Z)N-phenyl-, H(Z)N-phenylallcyi-, H(Z)N-phenylallcyl-N(Z)-hydrocarbon-,
H(Z)N-phenylallcyl-O-hydrocarbon-, phenylallcyl-O-hydrocarbon-,
phenylall~yl-N(Z) -hydrocarbon-, H(Z)N-phenylallcyl-carbonyl-hydrocarbon-, or
phenylall~yl-carbonyl-hydrocarbon-, wherein each hydrocarbon is independently
an aliphatic
or non-aliphatic straight- or branched-chain C 1 to C 10 group, and wherein
each alkyl is a C 1
3 0 to C 10 allcyl; and
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Z is independently hydrogen or t-butoxycarbonyl.
Another aspect of the present invention relates to compounds according to
formula
(VII)
R
(VII)
wherein
X3 is optional and can be oxygen;
X6 is oxygen or nitrogen;
l is an integer from 1 to 12;
Rl is selected from the group of saturated or unsaturated cyclic hydrocarbons,
saturated or unsaturated N-heterocyeles, saturated or unsaturated O-
heterocycles, saturated or
uazsaturated S-heterocycles, saturated or u~.zsaturated mixed heterocycles,
aliphatic or non-
aliphatic straight- or branched-chain C1 to C30 hydrocarbons, or
S aH2
or-(CHa)m Yl where m is an integer from 0 to 10 and YI is a
saturated or unsaturated cyclic hydrocarbon, saturated or unsaturated N-
heterocycle, saturated
or unsaturated O-heterocycle, saturated or unsaturated S-heterocycle, or
saturated or
unsaturated mixed heterocycle;
R2 is hydrogen, an aliphatic or non-aliphatic straight- or branched-chain C1
to C30
hydrocarbon, Rl°-N(Z)-hydrocarbon- or Rl°-hydrocarbon- where the
hydrocarbon
group is an aliphatic or non-aliphatic straight- or branched-chain Cl to C30
hydrocarbon, a
saturated or unsaturated cyclic hydrocarbon, a saturated or unsaturated N-
heterocycle, a
saturated or unsaturated 0-heterocycle, a saturated or unsaturated S-
heterocycle, a saturated
19
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R~~~ R~2
(CH~)n~~ ~~R~s
or unsaturated mixed heterocycle, or R15 R'4 or -(CH2)ri Y2 where
n is an integer from 0 to 10 and YZ is a saturated or unsaturated cyclic
hydrocarbon, saturated
or unsaturated N-heterocycle, saturated or unsaturated O-heterocycle,
saturated or
unsaturated S-heterocycle, or saturated or unsaturated mixed heterocycle;
R3 is notlung, hydrogen or an aliphatic or non-aliphatic straight- or branched-
chain Cl
to C 10 hydrocarbon;
R4 is optional, or can be hydrogen, an aliphatic or non-aliphatic straight- or
branched-
chain C1 to C10 hydrocarbon, aryl, acetyl, or mesyl;
R5, R6, R', R8, R9, Rl l, Rl~, R13, R14, and Rls are independently selected
from the
group of hydrogen, hydroxyl, an aliphatic or non-aliphatic straight-or
branched-chain C 1 to
C10 hydrocarbon, alkoxy, aryloxy, nitro, cyano, chloro, fluoro, bromo, iodo,
haloalkyl,
dihaloalkyl, trihaloalkyl, amino, alkylamino, dialkylamino, acylamino,
arylarnido, amido,
alkylamido, dialkylamido, arylamido, aryl, CS to C7 cycloalkyl, arylalkyl;
Rl° is H(Z)N-, H(Z)N-hydrocarbon-, H(Z)N-hydrocarbon-N(Z)
-hydrocarbon-, H(Z)N-hydrocarbon-, O hydrocarbon-,
hydrocarbon-O-hydrocarbon-, hydrocarbon-N(Z) hydrocarbon-,
H(Z)N-hydrocarbon-carbonyl-hydrocarbon-, hydrocarbon-carbonyl-hydrocarbon,
H(Z)N-phenyl-, H(Z)N-phenylalkyi-, H(Z)N-phenylalkyl-N(Z)-hydrocarbon-,
H(Z)N-phenylalkyl-O-hydrocarbon-, phenylalkyl-O-hydrocarbon-,
phenylalkyl-N(Z) -hydrocarbon-, H(Z)N-phenylalkyl-carbonyl-hydrocarbon-, or
phenylallcyl-carbonyl-hydrocarbon-, wherein each hydrocarbon is independently
an aliphatic
or non-aliphatic straight- or branched-chain C 1 to C 10 group, and wherein
each alkyl is a C I
to C I 0 alkyl; and
Z is independently hydrogen or t-butoxycarbonyl.
Yet another aspect of the present invention relates to compounds of Formula
(VIII)
CA 02559333 2006-09-11
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H-(CH2)n-N
F
wherein
XBisOorS;
n is between 1 and 30;
1 (VIII)
Rl is selected from the group of saturated or unsaturated cyclic hydrocarbons,
saturated or unsaturated N-heterocyeles, saturated or unsaturated O-
heterocycles, saturated or
unsaturated S-heterocycles, saturated or unsaturated mixed heterocycles,
aliphatic or non-
aliphatic straight- or branched-chain C1 to C30 hydrocarbons, or
(CH2
or-(CH2)m-Yl where m is an integer from 0 to 10 and Yl is a
saturated or unsaturated cyclic hydrocarbon, saturated or unsaturated N-
heterocycle, saturated
or unsaturated O-heterocycle, saturated or mzsaturated S-heterocycle, or
saturated or
unsaturated mixed heterocycle;
R4 is optional, or can be hydrogen, an aliphatic or non-aliphatic straight- or
branched-
chain C1 to C10 hydrocarbon, aryl, acetyl, or mesyl; and
R5, R6, R', R8, and R9 are independently selected from the group of hydrogen,
hydroxyl, an aliphatic or non-aliphatic straight-or branched-chain C1 to C10
hydrocarbon,
all~oxy, aryloxy, nitro, cyano, chloro, fluoro, bromo, iodo, haloall~yl,
dihaloallcyl, trihaloalkyl,
amino, allcylamino, diallcylamino, acylamino, arylamido, amido, allcylamido,
diall~ylamido,
arylamido, aryl, CS to C7 cycloallcyl, arylalkyl.
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Another aspect of the present invention relates to compounds having Formula
X9
H/R16 H/R1s
H
(~) (~17~ \R18
(X)
NH R16 NH R1s
HO X7
O (XI) O
(XII)
wherein
X' is POSH or O-benzyl;
X9 is O or nothing;
R16 is a Cl to C30 aliphatic or non-aliphatic, straight-, cyclic- or branched-
chain,
substituted or unsubstituted, C1 to C30 hydrocarbon;
Rl' and Rl8 are independently nothing, hydrogen, -SO2R19, COR19, and R19; and
R19 is an aliphatic or non-aliphatic, straight-, cyclic- or branched-chain,
substituted or
unsubstituted, C1 to C30 hydrocarbon or a substituted or unsubstituted aryl.
In one example,
the compound of Formula (X) is limited such that R16 is not C14Hz9 when X' is
P03H and X8
is O.
A further aspect of the present invention relates to a compound of Formula
(XIV) and
(XV)
O O C12H25~~
~ A 0~~~~ -O-
HO NH(CH2)12HN- Y \pH O ~P -ONH+a
NHBoc NHBoc (X~7) -ONH+4 (XV) .
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It will be understood that the dotted lines in the structures of Formulae II
and VII
indicate the presence or absence of a bond.
Preferred Rl groups include benzyl, furanyl, indolyl, pyridinyl, phenyl, or
substituted
phenyl (with Rs-R9 as defined above).
Preferred Ra groups include aliphatic or non-aliphatic straight- or branched-
chain C 1
to C30 hydrocarbons, phenyl, phenylalkyls, substituted phenyls and substituted
phenylalkyls
with Rll-Ris groups as defined above. Preferred aliphatic or non-aliphatic
straight- or
branched-chain hydrocarbons are CS to C24 hydrocarbons, including C10 to C20
alkyls,
more preferably C 14 to C 18 allcyls.
Preferred R3 groups include hydrogen and C 1 to C 10 alkyls.
Preferred R4 groups include hydrogen, acyl, acetyl, and mesyl.
Preferred Rl° groups are polyarnines.
The integer l is preferably from 1 to 10, more preferably 1 to 8, 1 to 6, or 1
to 4. The
integer In is preferably from 0 to 8, 0 to 6, 0 to 4, or 0 to 2. The integer h
is preferably from 0
to8,Oto6,Oto4,orOto2.
Exemplary compounds according to formula (I) include, without limitation: 2-(4-
oxo-
2-phenylthiazolidin-3-yl)acetamide (compound 65), N-decyl-2-(4-oxo-2-
phenylthiazolidin-3-
yl)acetamide (compound 66), N-tetradecyl-2-(4-oxo-2-phenylthiazolidin-3-
yl)acetamide
(compound 67), N-octadecyl-2-(4-oxo-2-phenylthiazolidin-3-yl)acetamide
(compound 68),
N-octadecyl-2-(4-oxo-2-biphenylthiazolidin-3-yl)acetamide (compound 69), 2-(2-
(1-
(dimethylamino)naphthalen-4-yl)-4-oxothiazol idin-3-yl)-N-octadecylacetamide
(compound
70), 2-(2-(4-methoxyphenyl)-4-oxothiazolidin-3-yl)-N-octadecylacetamide
(compound 71),
2-(2-(2,6-dichlorophenyl)-4-oxothiazolidin-3 yl)-N-octadecylacetamide
(compound 72), N-
octadecyl-2-(4-oxo-2-phenyl-1-sulfoxide-thiazolidin-3-yl)acetamide (compound
80), N-
octadecyl-2-(4-oxo-2-phenyl-1-sulfonyl-thiazolidin-3-yl)acetamide (compound
81), N-(3,5-
difluorophenyl)-2-(4-oxo-2-phenylthiazolidin-3-yl)acetamide (compound 73), N-
(3,5-
difluorophenyl)-2-(4-oxo-2-phenylthiazolidin-3-yl)ethanethioamide, N-(3,5-
bis(trifluoromethyl)phenyl)-2-(4-oxo-2-phenylthiazolidin-3-yl)acetamide
(compound 74), N-
(3,5-dichlorophenyl)-2-(4-oxo-2-phenylthiazolidin-3-yl)acetamide (compound
75), N-(2,4-
dimethoxyphenyl)-2-(4-oxo-2-phenylthiazolidin-3-yl)acetarnide (compound 76), N-
(naphthalen-1-yl).2-(4-oxo-2-phenylthiazolidin-3-yl)acetamide (compound 77), 3-
(2-
(octadecylamino)ethyl)-2-phenylthiazolidin-4-one (compound 79), N-(2-(2-
phenylthiazolidin-3-yl)ethyl)octadecan-1 -amine, and salts thereof.
Preferred compounds according to formula (I) include compounds 68, 71, 80, and
81.
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Exemplary compounds according to formula (II) include, without limitation:
(4R)-2-
(4-methoxyphenyl)-N-octadecylthiazolidine-4-carboxamide (compound 15); (4R)-2-
(4-
ethoxyphenyl)-N-octadecylthiazolidine-4-carboxamide; N-octadecyl-2-
phenyithiazole-4-
carboxamide (compound 34); (4R)-2-(3,5-difluorophenyl)-N-octadecylthiazolidine-
4-
carboxamide (compound 23); (4R)-2-(4-cyanophenyl)-N-octadecylthiazolidine-4-
carboxamide (compound 22); (4R)-N-octadecyl-N-mesyl-2-phenylthiazolidine-4-
carboxamide (compound 29); (4R)-N-octadecyl-N-acetyl-2-phenylthiazolidine-4-
carboxamide (compound 28); (4R)-N-heptyl-2-phenylthiazolidine-4-carboxamide
(compound
3); (4R)-N-octadecyl-2-phenylithiazolidine-4-carboxamide (compound 5, R-
isomer); (4S)-N-
octadecyl-2-phenylthiazolidine-4-carboxamide (compound 5, S-isomer); (4R)-N-
tetradecyl-
2-phenylithiazolidine-4-carboxamide hydrochloride (compound 4); (4R)-N-
octadecyl-2-
biphenylthiazolidine-4-carboxamide (compound 27); (4R)-2-dodecyi-N-
octadecylthiazolidine-4-carboxamide (compound 7); (4R)-N-octadecyl-2-(pyridin-
3-
yl)thiazolidine-4-carboxamide (compound 11); 2-(furan-3-yl)-N-
octadecylthiazolidine-4-
carboxamide (compound 12); (4R)-N-nonadecyl-2-phenylthiazolidine-4-carboxamide
(compound 6); (4R)-2-(4-hydroxyphenyl)-N-octadecylthiazolidine-4-carboxamide;
2-(3-
hydroxyphenyl)-N-octadecylthiazolidine-4-carboxamide (compound 14); (4R)-2-
(2,4,6-
dimethoxyphenyl) N-octadecylthiazolidine-4-carboxamide; 2-(3,4-
dimethoxyphenyl)-N-
octadecylthiazolidine-4-carboxamide (compound 18); (4R)-2-(4-fluorophenyl)-N-
octadecylthiazotidine-4-carboxamide (compound 19); (4R)-2-(2,6-dichlorophenyl)-
N-
octadecylthiazolidine-4-carboxamide (compound 24); (4R)-2-(4-bromophenyl)-N-
octadecylthiazolidine-4-carboxamide (compound 20); (4R)-N-octadecyl-2-p-
tolylthiazolidine-4-carboxamide (compound 26); (4R)-2-cyclohexyl-N-
octadecylthiazolidine-
4-carboxamide (compound 8); 2-(4-nitrophenyl)-N-octadecylthiazolidine-4-
carboxamide
(compound 21); (4R)-2-(4-(dimethylamino)phenyl)-N-octadecylthiazolidine-4-
carboxamide
(compound 13); (4R)-2-(1H-indol-3-yl)-N-octadecylthiazolidine-4-carboxamide
(compound
10); (4R)-2-benzyl-N-octadecylthiazolidine-4-carboxamide (compound 9); (4R)-2-
(3-bromo-
4-fluorophenyl)-N-octadecylthiazolidine-4-carboxamide (compound 2S); (4R)-2-
(3,4,5-
trimethoxyphenyl)-N,N-dioctylthiazolidine-4-carboxamide; and salts thereof.
Preferred compounds according to formula (II) include compounds 5 (R-isomer),
13,
14, 16, 17, 18, 19, 25, and 26.
Compounds of Formula V include, but are not limited to:
24
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O
NH ~O C~gl"i37 NH ~O C~$Hs7
S \ S
OH O
HN ,~'ILOH
HN ''''~OH
C~2H2 ~~
Me0
> >
O
,.' O O O
HN ILOH
F ~ ~ HN ~''I~OH HN ,~'ILON CI HN '''[LOH
~ ~ ~ ~ w
F ANC ~ / ~F I / ; ~ / CI
O
HN ~''I~OH
iJ
and N
Compounds of Formula (VII) include, but are not limited to:
O
HCI. NH- CH nCH
NH ( 2) s
O
wherein n= 6, 13, and 17.
Compounds of Formulae (IX), (X), (XI), and (XII) include, but are not limited
to
those found in Table 9 of the Examples section.
The compounds of the present invention and their intermediates can be
synthesized
using commercially available or readily synthesized reactants.
By way of example, the compounds according to formula (I) can be synthesized
according to scheme 4 illustrated in Figure 6. According to one approach, an
intermediate
acid according to formula (III)
CA 02559333 2006-09-11
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H
(III)
(where l, Rl, X3, and X4 are as defined above) is reacted with appropriate
amines in the
presence of EDC/HOBt under standard conditions. The intermediate acids can be
prepared
initially via condensing mercaptoacetic acid, glycine methyl ester, and
aromatic aldehydes in
a one-pot reaction, followed by basic hydrolysis of the ester (Holrnes et al.,
"Strategies for
Combinatorial Organic Synthesis: Solution and Polymer-Supported Synthesis of 4-
Thiazolidinones and 4-Metathiazanones Derived from Amino Acids," J. O~g. Chew.
60:7328-7333 (1995), which is hereby incorporated by reference in its
entirety). By
substituting glycine methyl ester with analogs containing longer carbon
backbones, it
becomes possible to prepare compounds according to formula (III) and,
ultimately, formula
(I), with l being greater than 1 (i.e., containing an alkylene group that is
longer than
methylene). According to a second approach, the thiazolidinone amides of
formula (I) can
also be prepared by a simple and direct method (Schuemacher et al.,
"Condensation Between
Isocyanates and Carboxylic Acids in the Presence of 4-Dimethylaminopyridine .
(DMAP), a
Mild and Efficient Synthesis of Amides," Synthesis 22:243-246 (2001), which is
hereby
incorporated by reference in its entirety), which involves reaction of the
intermediate acid
with desired isocyanates in the presence of a catalytic amount of DMAP (Figure
7) (scheme
5),
Further modification of the thiazolidinone compounds can be achieved by, e.g.,
exhaustive reduction of using BH3 THF under reflux conditions to eliminate
carbonyl or
sulfoxide groups X3 and X4 (Figure 8) (scheme 6c), as well as oxidation of a
compound using
H202 and I~.Mn04 to afford sulfoxides or sulfones, respectively, as shown in
scheme 6a and
6b.
Also by way of example, compounds according to foi~nula (II) can be prepared
by
reacting an intermediate acid according to formula (IV),
X3
CH3
S NH
26
CA 02559333 2006-09-11
WO 2005/086638 PCT/US2005/004759
where compound (IV) can be either the R- or S-stereoisomer and Rl and X3 are
defined as
above, with appropriate amines in the presence of EDC/HOBt under standard
conditions. The
intermediate acids can be prepared via reaction of L-cysteine with desired
aldehydes under
reported conditions (Sell et al., "A Novel Synthesis of (+)-Biotin from L-
Cysteine," J. O~g.
Chem. 67:5527-5536 (2002), which is hereby incorporated by reference in its
entirety).
The compounds of the present invention can also be modified to contain a
polymeric
conjugate. Suitable polymeric conjugates include, without limitation,
poly(alkyl)amines,
poly(alkoxy)amine, polyamines, etc. It is also well known that polyamine
containing
compounds exhibit a number of biological activities and have been utilized as
chemotherapeutic agents. Exemplary conjugates include those containing the
naturally
occurring polyamines like putrescine, spermidine, and spermine, as well as
synthetic
polyamines.
According to one approach, a compound of the present invention can be
conjugated to
a polyamine by reacting the intermediate acid or a nitrophenyl derivative
thereof with a
polyamine NH2-R2 where RZ is Rl°-N(Z) - hydrocarbon-. or. Rl°-
hydrocarbon- , with Rlo
and Z being as defined above. An exemplary synthesis scheme is illustrated in
Figure 9.
By way of example, compounds of Formulae (V) and (VI) can be formed in
accordance with the exemplary synthesis scheme illustrated in Fig. 10. The
compound can be
made in any other suitable manner.
By way of example, oxazoline analog compounds of Formula (VII) can be formed
in
accordance with the scheme illustrated in Fig. 11. Additionally, the compounds
of Formula
(VII) can be formed using the methods outlined above with respect to the
compounds of
Formula (II). The compounds may also be made in any other suitable manner.
By way of example, compounds of Formula (VIII) can be formed in accordance
with
the scheme illustrated in Fig. 12. Additionally, the compounds can be made in
any other
suitable manner.
By way of example, compounds of Formulae (IX) and (X) can be made in
accordance with
the general synthesis of serine amide phosphates (SAPs), serine amide alcohols
(SAAs), and serine
diamide phosphates (SDAPs) shown in the schemes of Figs. 13-16. Commercially
available N-
Boc-serine (R or .S form) is allowed to react with an appropriate amine in
presence of EDC/HOBt
to form an amide. The amide is treated with TFA to give an SAA analog.
Phosphorylation of the
amide and concurrent removal of protecting groups under hydrogenolysis
conditions using Pd/C in
ethanol gave an SAP. Unsaturated analogues of SAA and SAP can synthesized by
similar
procedures as shown in the scheme of Fig. 14. Serine diamide phosphates
(SDAPs) and other
27
CA 02559333 2006-09-11
WO 2005/086638 PCT/US2005/004759
amine derivatives can be synthesized starting from O-benzyl N-Boc-serine as
shown in the scheme
of Fig. 15. LAH mediated reduction of an amine compound gives long chain N-
alkyl amino
alcohols as shown in the scheme of Fig. 16. Compounds of Formula (XI) and
(XII) which have an
ethanolamine amide backbone rather than the serine amide backbone can be
synthesized according
to the reported procedure Lynch, K. R. H., D. W.Carlisle, S. J.Catalano, T.
G.Zhang,
M.MacDonald, T. L. Mol. Pha~macol. 1997, 52, 75-81, which is incorporated by
reference in its
entirety.
The compounds can also be in the form of a salt, preferably a pharmaceutically
acceptable salt. The term "pharmaceutically acceptable salt" refers to those
salts that retain
I O the biological effectiveness and properties of the free bases or free
acids, which are not
biologically or otherwise undesirable. The salts are formed with inorganic
acids such as
hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric
acid and the like,
and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic
acid, oxylie acid,
malefic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric
acid, benzoic acid,
cirmamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-
toluenesulfonic
acid, salicylic acid, N-acetylcysteine and the like. Other salts are known to
those of skill in
the ant and can readily be adapted for use in accordance with the present
invention.
The compounds of the present invention can be present in the form of a racemic
mixture, containing substantially equivalent amounts of stereoisomers. In
another
embodiment, the compounds of the present invention can be prepared or
otherwise isolated,
using known procedures, to obtain a stereoisomer substantially free of its
corresponding
stcreoisomer (i.e., substantially pure). By substantially pure, it is intended
that a stereoisomer
is at least about 95% pure, more preferably at least about 98% pure, most
preferably at least
about 99% pure.
Another aspect of the present invention relates to pharmaceutical compositions
that
contain one or more of the above-identified compounds of the present
invention. Generally,
the pharmaceutical composition of the present invention will include a
compound of the
present invention or its pharmaceutically acceptable salt, as well as a
pharmaceutically
acceptable carrier. The term "pharmaceutically acceptable carrier" refers to
any suitable
adjuvants, carriers, excipients, or stabilizers, and can be in solid or liquid
form such as,
tablets, capsules, powders, solutions, suspensions, or emulsions.
In one example, the composition will contain from about 0.01 to about 99
percent or
from about 20 to about 75 percent of active compound(s), together with the
adjuvants,
tamers and/or excipients. For example, application to mucous membranes can be
achieved
28
CA 02559333 2006-09-11
WO 2005/086638 PCT/US2005/004759
with an aerosol spray containing small particles of a compound of . this
invention in a spray
or dry powder form.
The solid unit dosage forms can be of any suitable type. The solid form can be
a
capsule and the lilce, such as an ordinary gelatin type containing the
compounds of the present
invention and a carrier, for example, lubricants and inert fillers such as,
lactose, sucrose, or
cornstarch. In another embodiment, these compounds are tableted with
conventional tablet
bases such as lactose, sucrose, or cornstarch in combination with binders
lilce acacia,
cornstarch, or gelatin, disintegrating agents, such as cornstarch, potato
starch, or alginic acid,
and a lubricant, life stearic acid or magnesium stearate.
IO The tablets, capsules, and the life can also contain a binder such as gum
tragacanth,
acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent
such as corn starch, potato starch, alginic acid; a lubricant such as
magnesium stearate; and a
sweetening agent such as sucrose, lactose, or saccharin. When the dosage unit
form is a
capsule, it can contain, in addition to materials of the above type, a liquid
carrier such as a
fatty oil.
Various other materials may be present as coatings or to modify the physical
form of
the dosage unit. For instance, tablets can be coated with shellac, sugar, or
both. A syrup can
contain, in addition to active ingredient, sucrose as a sweetening agent,
methyl and
propylparabens as preservatives, a dye, and flavoring such as cherry or orange
flavor,
For oral therapeutic administration, these active compounds can be
incorporated with
excipients and used in the form of tablets, capsules, elixirs, suspensions,
syrups, and the like.
Such compositions and preparations can contain at least 0.1 % of active
compound. The
percentage of the compound in these compositions can, of course, be varied and
can
conveniently be between about 2% to about 60% of the weight of the unit. The
amount of
active compound in such therapeutically useful compositions is such that a
suitable dosage
will be obtained. In one example, compositions according to the present
invention are
prepared so that an oral dosage unit contains between about I mg and 800 mg of
active
compound. .
The active compounds of the present invention may be orally administered, for
example, with an inert diluent, or with an assimilable edible Garner, or they
can be enclosed
in hard or soft shell capsules, or they can be compressed into tablets, or
they can be
incorporated directly with the food of the diet.
The pharmaceutical forms suitable for injectable use include sterile aqueous
solutions
or dispersions and sterile powders for the extemporaneous preparation of
sterile injectable
29
CA 02559333 2006-09-11
WO 2005/086638 PCT/US2005/004759
solutions or dispersions. In all cases, the form should be sterile and should
be fluid to the
extent that easy syringability exists. It should be stable under the
conditions of manufacture
and storage and should be preserved against the contaminating action of
microorgaiv.sms,
such as bacteria and fungi. The carrier can be a solvent or dispersion medium
containing, for
example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid
polyethylene
glycol), suitable mixtures thereof, and vegetable oils.
The compounds or pharmaceutical compositions of the present invention may also
be
administered in injectable dosages by solution or suspension of these
materials in a
physiologically acceptable diluent with a pharmaceutical adjuvant, carrier or
excipient. Such
adjuvants, carriers and/or excipients include, but are not limited to, sterile
liquids, such as
water and oils, with or without the addition of a surfactant and other
pharmaceutically and
physiologically acceptable components. Illustrative oils are those of
petroleum, animal,
vegetable, or synthetic origin, for example, peanut oil, soybean ail, or
mineral oil. W general,
water, saline, aqueous dextrose and related sugar solution, and glycols, such
as propylene
glycol or polyethylene glycol, are preferred liquid carriers, particularly for
injectable
solutions.
These active compounds may also be administered parenterally. Solutions or
suspensions of these active compounds can be prepared in water suitably mixed
with a
surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in
glycerol,
liquid polyethylene glycols, and mixtures thereof in oils. Illustrative oils
are those of
petroleum, animal, vegetable, or synthetic origin, for example, peanut oil,
soybean oil, or
mineral oil. In general, water, saline, aqueous dextrose and related sugar
solution, and glycols
such as, propylene glycol or polyethylene glycol, are preferred liquid
carriers, particularly for
injectable solutions. Under ordinary conditions of storage and use, these
preparations contain
a preservative to prevent the growth of microorganisms.
For use as aerosols, the compounds of the present invention in solution or
suspension
may be packaged in a pressurized aerosol container together with suitable
propellants, for
example, hydrocarbon propellants like propane, butane, or isobutane with
conventional
adjuvants. The materials of the present invention also . may be administered
in a non-
pressurized form such as in a nebulizer or atomizer.
The compounds of the present invention are particularly useful in the
treatment or
prevention of various forms of cancer, particularly prostate cancer, breast
cancer, and ovarian
cancer. It is believed that other forms of cancer will lilcewise be treatable
or preventable upon
administration of the compounds or compositions of the present invention to a
patient.
CA 02559333 2006-09-11
WO 2005/086638 PCT/US2005/004759
Preferred compounds of the present invention are selectively disruptive to
cancer cells,
causing ablation of cancer cells but not normal cells. Significantly, harm to
normal cells is
minimized because the cancer cells are susceptible to disruption at much lower
concentrations of the compounds of the present invention.
Thus, a further aspect of the present invention relates to a method of
destroying a
cancerous cell that includes: providing a compound of the present invention
and then
contacting a cancerous cell with the compound under conditions effective to
destroy the
contacted cancerous cell. According to various embodiments of destroying the
cancerous
cells, the cells to be destroyed can be located either in vivo or ex vivo
(i.e., in culture).
I O A still further aspect of the present invention relates to a method of
treating or
preventing a cancerous condition that includes: providing a compound of the
present
invention and then administering an effective amount of the compound to a
patient in a
manner effective to treat or prevent a cancerous condition. An effective
amount will be
understood as referring to an amount of the compound that is effective at
reducing,
15 preventing, ameliorating, or improving at least one symptom of the
condition for which the
compound is administered. It will be further understood that the term
"prevent" shall be
understood as referring to the prevention of the development of at least one
symptom related
to the condition for which the compound is administered.
According to one embodiment, the patient to be treated is characterized by the
20 presence of a precancerous condition, and the administering of the compound
is effective to
prevent development of the precancerous condition into the cancerous
condition. This can
occur by destroying the precancerous cell prior to or concurrent with its
further development
into a cancerous state.
According to another embodiment, the patient to be treated is characterized by
the
25 presence of a cancerous condition, and the administering of the compound is
effective either
to cause regression of the cancerous condition or to inhibit growth of the
cancerous condition.
This preferably occurs by destroying cancer cells, regardless of their
location in the patient
body. That is, whether the cancer cells are located at a primary tumor site or
whether the
cancer cells have metastasized and created secondary tumors within the patient
body.
30 As used herein, patient refers to any mammalian patient, including without
limitation,
humans and other primates, dogs, cats, horses, cows, sheep, pigs, rats, mice,
and other
rodents.
When administering the compounds of the present invention, they can be
administered systemically or, alternatively, they can be administered directly
to a specific site
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WO 2005/086638 PCT/US2005/004759
where cancer cells or precancerous cells are present. Thus, administering can
be
accomplished in any manner effective for delivering the compounds or the
pharmaceutical
compositions to the cancer cells or precancerous cells. Exemplary modes of
administration
include, without limitation, administering the compounds or compositions
orally, topically,
transdermally, parenterally, subcutaneously, intravenously, intramuscularly,
intraperitoneally,
by intranasal instillation, by intracavitary or intravesical instillation,
intraocularly,
intraarterially, intralesionally, or by application to mucous membranes, such
as, that of the
nose, throat, and bronchial tubes.
When the compounds or pharmaceutical compositions of the present invention are
administered to treat or prevent a cancerous condition, the pharmaceutical
composition can
also contain, or can be administered in conjunction with, other therapeutic
agents or treatment
regimen presently known or hereafter developed for the treatment of various
types of cancer.
Examples of other therapeutic agents or treatment regimen include, without
limitation,
radiation therapy, chemotherapy, surgical intervention, and combinations
thereof.
Compositions within the scope of this invention include all compositions
wherein the
compound of the present invention is contained in an amount effective to
achieve its intended
purpose. While individual needs may vary, determination of optimal ranges of
effective
' amounts of each component is within the skill of the art. Typical dosages
comprise about
0.01 to about 100 mg/kg body wt. The most preferred dosages comprise about 0.1
to about
100 mg/kg body wt. Treatment regimen for the administration of the compounds
of the
present invention can also be determined readily by those with ordinary skill
in art. That is,
the frequency of administration and size of the dose can be established by
routine
optimization, preferably while minimizing any side effects.
EXAMPLES
The Examples set forth below are for illustrative purposes only and are not
intended
to limit, in any way, the scope of the present invention.
Example 1 - Synthesis of Thiazolidine Carboxylic Acid Amides
All reagents and solvents used were reagent grade or were purified by standard
methods before use. Moisture-sensitive reactions were carried under an argon
atmosphere.
Progress of the reactions was followed by thin-layer chromatography (TLC)
analysis. Flash
column chromatography was carried out using silica gel (200-425 mesh) supplied
by Fisher.
Melting points were measured in open capillary tubes on a Thomas-Hoover
melting point
apparatus and are uncorrected. All compounds were characterized by NMR and MS
(ESI).
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WO 2005/086638 PCT/US2005/004759
1H NMR spectra were recorded on a Varian 300 instrument. Chemical shifts are
reported as
S values relative to MeaSi as internal standard. Mass spectra were obtained in
the
electrospray (ES) mode using Esquire-LC (Broker) spectrometer. Elemental
analyses were
performed by Atlantic Microlab Inc. (Norcross, GA).
All the compounds described in this study were prepared following
straightforward
chemistry. Reaction of L-cysteine with various aldehydes under reported
conditions (Seki et
al., "A Novel Synthesis of (+)-Biotin from L-Cysteine," J. Org. Chem. 67:5527-
5536 (2002),
which is hereby incorporated by reference in its entirety) afforded
corresponding acids
(Figure 1, 2a-v), which were isolated as diastereomeric mixtures. These
mixtures were used
directly for the formation of corresponding amides by reacting with
appropriate alkyl amines
using EDC/HOBt as shown in Scheme 1. All compounds thus prepared were
characterized as
diastereomeric mixtures (Table 1).
A mixture of appropriate carboxylic acid (Figure 1, 2a-2v, 0.3-0.5 g), EDC
(1.25
equiv) and HOBt (1 equiv) in CH~C12 (25-50 mL) was stirred for 10 min. To this
solution,
appropriate allcyl amine (1 equiv) was added and stirnng continued at room
temperature for
6-8 h. Reaction mixture was diluted with CHZC12 (100-150 mL) and sequentially
washed with
water, satd. NaHC03, brine and dried over Na2S04. The solvent was removed
under reduced
pressure to yield a crude solid, which was purified by column chromatography.
The purified
compounds (3-6, 12, 15-18 & 27) were converted to corresponding hydrochlorides
using 2M
HC1/Et20.
(2RS, 4R)-2-Phenylthiazolidine-4-carboxylic acid heptylamide Hydrochloride
(compound 3~HCl): 1H NMR (DMSO-d6) 8 8.72 (s, IH), 7.65 (m, 2H), 7,43 (m, 3H),
5.89 (s,
0.6H), 5.84 (s, 0.4H), 4.66 (t, T= 6.3 Hz, 0.6H), 4.46 (t, J= 6.9 Hz, 0.4H),
3.55-3.71 (m, IH),
3.24-3.34 (m, IH), 3.13 (d, J-- 5.7 Hz, 2H), 1.44 (m, 2H), 1.25 (s, 8H), 0.83
(t, J= 6.9 Hz,
3H); MS (ESI) m/z calcd for C1~HZ~N20S 307.47 (M+1), obsd 307.10.
(2RS, 4R)-2-Phenylthiazolidine-4-carboxylic acid tetradecylamide Hydrochloride
(compound 4~HCI): 1H NMR (DMSO-d6) 8 8.69 (m, 1H), 7.64-7.71 (m, 2H), 7.45 (m,
3H),
5.89 (s, 0.6H), 5.84 (s, 0.4H), 4,67 (t, J= 6.6 Hz, 0.6H), 4.47 (t, J= 7.2 Hz,
0.4H), 3.55-3.71
(m, 1H), 3.25-3.35 (m, 1H), 3.1,0-3.16 (m, 2H), 1.44 (m, 2H), 1.23 (s, 22H),
0.85 (t, J-- 63
Hz, 3H); MS (ESI) mlz calcd for C24H4oNaOS 404.65 (M+), obsd 427.30 (M+Na).
(2RS, 4R)-2-Phenylthiazolidine-4-carboxylic acid octadecylamide Hydrochloride
(compound 5~HCI): 1H NMR (DMSO-d6) 8 8.59 (d, J-- 5.1 Hz, 1H), 7.63 (d, J= 3.9
Hz, 2H),
7.42-7.47 (in, 3H), 5.86 (s, 0.6H), 5.81(s, 0.4H), 4.60 (t, J= 6.3 Hz, 0.6H),
4.39 (t, J= 6.9
33
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WO 2005/086638 PCT/US2005/004759
Hz, 0.4H), 3.52-3.66 (m, 1H), 3.24-3.30 (m, 1H), 3.10-3.16 (m, 2H), 1.42 (m,
2H), 1.23 (s,
30H), 0.85 (t, J 6.3 Hz, 3H); MS (ESl~ nzlz calcd for Cz$H49N20S 461.76 (M+I),
obsd
461.50.
(2RS, 4R)-2-Phenylthiazolidine-4-carboxylic acid nonadecylamide Hydrochloride
(compound 6~HC1): 'H NMR (DMSO-d6) 8 8.51 (s, IH), 7.62 (m, 2H), 7.41-7.46
(in, 3H),
5.83 (s, 0.6H), 5.78 (s, 0.4H), 4.53 (m, 0.6H), 4.32 (m, 0.4H), 3.48-3.61 (m,
1H), 3.24-3.29
(m, 1H), 3.11-3.15 (m, 2H), 1.43 (m, 2H), 1.23 (s, 32H), 0.85 (t, .I--- 6.3
Hz, 3H); MS (EST)
m/z calcd for Cz9HsoNzOS 474.79 (M+), obsd 497.40 (M+Na).
(2RS, 4R)-2-Dodecylthiazolidine-4-carboxylic acid octadecylamide (compound 7):
IH NMR (CDC13) 8 7.18 (m, 1H), 4.20-4.27 (m, 1H),3.79 (m, 0.3H), 3.54-3.59 (m,
0.7H),
3.08-3.34 (m, 4H), 1.65-1.78 (m, 2H), 1.43-1.51 (m, 4H), 1.27 (brs, 48H), 0.89
(t, J-- 6 Hz,
6H); MS (ESI) m/z calcd for C34H69NzOS 553.98 (M+1), obsd 553.60.
(2RS, 4R)-2-Cyclohexylthiazolidine-4-carboxylic acid octadecylamide (compound
8):
1H NMR (CDC13) & 7.I7 (m, IH), 4.10-4.20 (m, 1H), 3.76 (m, 0.3H), 3.54 (dd, J
11.1, 3.6
Hz, 0.7H), 2.97-3.34 (m, 4H), 2.02 (m, 1H), 1.68-1.78 (m, 4H), ~ 1.48-1.54 (m,
2H), 1.27
(brs, 36H), 0.87 (t, .I--- 6.9 Hz, 3H); MS (ESI) m/z calcd for CzBHssNzOS
467.81 (M+1), obsd
467.60.
(2RS, 4R)-2-Benzylthiazolidine-4-carboxylic acid octadecylamide (compound 9):
1H
NMR (CDC13) 8 7.28-7.33 (m, SH), 7,03 (s, 0,7H), 6.48 (s, 0.3H), 4.55 (brs,
O.SH), 4.18 (brs,
O.SH), 3.82 (brs, 0.3H), 3.54 (dd, J I I.1, 3.6 Hz, 0.7H), 2.99-3.31 On, 6H),
1.46-1.51 (In,
2H),1.27 (brs, 30H), 0.89 (t, J-- 6.3 Hz, 3H); MS (ESI) nz/z calcd for
Cz9HsoNzOS 475.79
(M+1), obsd 475.50.
(2RS, 4R)-2-(1H-Indol-3yI)-thiazolidine-4-carboxylic acid octadecylamide
(compound 10): 1H NMR (CDCI3) 8 7.86 (m, 0.6H), 7.77 (m, 0.4H), 7.41-7.48 (m,
4H), 7.29-
7.34 (m,I H), 6.0 (s, 0.3H), 5.69 (s, 0.7H), 4.37-4.41 (m, O.SH), 3.76 (dd, J--
11.1, 4.2 Hz,
O.SH), 3.23-3.52 (m, 3H), 2.79-3.04 (in, IH), 1.43 (m, 2H),I.27 (s, 30H), 0.89
(t, J= 6.6 Hz,
3H); MS (ESI) m/z calcd for C3oHsoNsOS 500.80 (M+1), obsd 500.60.
(2RS, 4R)-2-Pyridin-3-yl-thiazolidine-4-carboxylic acid octadecylamide
(compound
11): 1H NMR (CDC13) b 8.74 (d, J-- 2.1 Hz, 1H), 8.60 (d, J-- 4.8 Hz, IH), 7.84
(d, J= 7.8 Hz,
1H), 7.31-7.36 (m, 1H), 7.08 (m, 1H), 5.44 (s, O.SH), 5.40 (s, O.SH), 4.28-
4.35 (m, 1H), 3.72
(dd, J=11.1, 4.2 Hz, 1H), 3.27-3.45 (m, 3H), 2.57 (m, 1H), 1.53-1.57 (m, 2H),
1.26 (s, 30H),
0.89 (t, J-- 6.6 Hz, 3H); MS (ESL) rnlz calcd for Cz~H49N30S 462.75 (M+1),
obsd 462.40.
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WO 2005/086638 PCT/US2005/004759
(2RS, 4R)-2-Furan-3-yl-thiazolidine-4-carboxylic acid Hydrochloride (compound
12~HC1): 1H NMR (DMSO-4) 8 8.59 (d, J--15.6 Hz, 1H), 7.89 (d, J-- 8.1 Hz, 1H),
7.72 (s,
1H), 5.86 (s, 0.71-1), 5.78 (s, 0.3H), 4.37-4.56 (m, 1H), 3.50-3.63 (nay, IH),
3.11-3.23 (m,
3H), 1.43 (m, 2H), 1.23 (s, 30H), 0.85 (t,J--- 6.6 Hz, 3H); MS (ESI) mlz calcd
for
C26H48N20zS 4S 1.72 (M+1), obsd 4S 1.60.
(2RS, 4R)-2-(4-Dimethylamino-phenyl)-thiazolidine-4-carboxylic acid
octadecylamide (compound 13): 1H NMR (CDCl3) 8 7.34-7.41 (m, 2H), 6.70-6.74
(m, 2H),
S.S7 (s, 0.3H), 5.28 (s, 0.7H), 4.34 (m, 0.7H), 3.90 (m, 0.3H), 3.69 (dd, J--
11,1, 4.2 Hz, 1H),
3.41-3.47 (m, 1H), 3.20-3.33 (m, 2H), 2.97 (d, J= 3.6 Hz, 6H), 1.48-1.SS (m,
2H), 1.27 (s,
30H), 0.89 (t, J-- 6.3 Hz, 3H); MS (ESl~ mlz calcd for C3oH54N30S 504.83
(M+1), obsd
5 04.60.
(2RS, 4R)-2-(3-Hydroxy-phenyl)-thiazolidine-4-carboxylic add octadecylamide
(compound 14): 1H NMR (DMSO-d6) ~ 8.59 (s, 1H), 7.22 (t, J-- 6.6 ~ Hz, 1H),
7.02 (d, J=
6.3 Hz, 2H), 6.82 (d, J = 7.S Hz, 1H), 5.77 (s, 0.7H), 5.71 (s, 0.3H), 4.S4S
(m, 0.7H), 4.37 (m,
1S 0.3H), 3.49-3.59 (m, 1H), 3.13-327 (m, 3H), 1:43 (brs, 2H), 1.23 (s, 30H),
0,85 (t, J-- 6.3 Hz,
3H); MS (ESI) nalz calcd for Ca8H49NzO2S 477.76 (M+1), obsd 477.60.
(2RS, 4R)-2-(4-Methoxy-phenyl)-thiazolidine-4-carboxylic acid octadecylamide
Hydrochloride (compound 1S HCl): 1H NMR (DMSO-d6) 8 8.61 On, 1H), 7,57 (d, J--
8.4 Hz,
2H), 6.98 (d, J 9 Hz, 211), 5.83 (s, 0.711), 5.78 (s, 0.311), 4.61 (t, J 6.3
Hz, 0.7H), 4,40
(m, 0.3H), 3.77 (s, 311), 3.51-3.70 (m, 1H), 3.22-3.31 (m, 1H), 3.1.1 (m, 2H),
1.43 (m, 2H),
1.23 (s, 30H), 0.84 (t, J= 6.6 Hz, 3H); MS (ESI) m/z calcd for C29HsiNaOaS
491.79 (M+1),
obsd 491.60.
(2RS, 4R)-2-(3,4-Dimethoxy-phenyl)-thiazolidine-4-carboxylic add
octadecylamide
Hydrochloride (compound 16 HC1): 1H NMR (DMSO-d6) 8 8.58 (m, 1H), 7.33 (d, J--
4.2 Hz,
2S 111), 7.14 (t, J-- 7.S Hz, 1H), 6.97 (d, J-- 8.4 Hz, 1H), 5.81 (s, 0.8H),
5.77 (s, 0.2H), 4.62 (m,
0.711), 4.40 (m, 0.3H), 3.78 (d, J= 7.8 Hz, 6H), 3.52-3.68 (m, 1H), 3.23-3.29
On, 1H), 3.22-
3.13 (m, 2H), 1.43 (m, 2H), 1.23 (s, 30H), 0.85 (t, J 6.6 Hz, 3H); MS (ESI)
m/z calcd for
C3oHssNaOsS 521.81 (Mil), obsd 521.60.
(2RS, 4R)-2-(3,4,5-Trimethoxy-phenyl)-thiazolidine-4-carboxylic acid
octadecylamide Hydrochloride (compound 17 HC1): 1H NMR (DMSO-d6) ~ 8.59 (m,
1H),
7.01 (d, J= S.7 Hz, 2H), 5.80 (s, 0.8H), 5.76 (s, 0.2H), 4.63 (m, 0.7H), 4.37
(m, 0.3H), 3.80
(d, J-- S.7 Hz, 611), 3.66 (s, 3H), 3.23-3.28 (m, 1H), 3.12-3.I3 (m, 2H), 1.43
(m, 2H), 1.23 (s,
3S
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30H), 0.85 (t, J-- 6 Hz, 3H); MS (ESA m/z calcd for C3iHssNzO4S 551.84 (M+1),
obsd
551.60.
(2RS, 4R)-2-(4-Acetylamino-phenyl)-thiazolidine-4-carboxylic acid
octadecylamide
Hydrochloride (compound 18~HC1): 1H NMR (DMSO-d6) 8 10.18 (s, 1H), 8.61 (m,
1H),
7.54-7.64 (m, 4H), 5.82 (s, 0.7H), 5.77 (s, 0.3H), 4.60 (m, 0.8H), 4.42 (m,
0.2H), 3.56-3.64
(m, IH), 3.12-3.26 (m, 3H), 2.05 (s, 3H), 1.43 (m, 2H), 1.23 (s, 30H), 0.84
(t, J-- 6 Hz, 3H);
MS (ESA m/z calcd for C3oHszN3O2S 518.81 (M+1), obsd 518.70.
(2RS, 4R)-2-(4-Fluoro-phenyl)-thiazolidine-4-carboxylic acid octadecylamide
(compound 19): 1H NMR (CDC13) 8 7.46-7.54 (m, 2H), 7.13-7.20 (m, 1H), 7.01-
7.08 (m,
2H), 5.60 (s, 0.3H), 5.34 (s, 0.7H), 4.76 (m, 0.3H), 4.34 (m, 0.7H), 3.69 (dd,
J=11.1, 6.9 Hz,
1H), 3.21-3.52 (m, 3H), 1.49 (in, 2H), 1.26 (s, 30H), 0.89 (t, J-- 6.3 Hz,
3H); MS (EST) m/z
calcd for CzsH48FNzOS 479.75 (M+1), obsd 479.60.
(2RS, 4R)-2-(4-Bromo-phenyl)-thiazolidine-4-carboxylic acid octadecylamide
(compound 20): 1H NMR (CDC13) 8 7.48-7.62 (m, 2H), 7.36-7.42 (m, 2H), 7.14 (m,
0.7H),
6.40 (m, 0.3), 5.57 (d, J--10.2 Hz, 0.3H), 5.33 (d, J--11.1 Hz, 0.7H), 4.32
(m, 0.7H), 3.94 (in,
0.3H), 3.70 (dd, J-- 11.1, 4.2 Hz, 1H), 3.20-3.44 (m, 3H), 1.49 (m, 2H), 1.27
(s, 30H), 0.89 (t,
J-- 6.3 Hz, 3H); MS (ESL) m/z calcd for Cz3H4~BrNzOS 539.66 (M~), obsd 539.70.
(2RS, 4R)-2-(4-Nitro-phenyl)-thiazolidine-4-carboxylic acid octadecylamide
(compound 21): 1H NMR (CDC13) 8 8.24 (d, J-- 8.7 Hz, 2H), 7.67 (d, J-- 8.7 Hz,
2H), 6.92
(m, 1H), 5.54 (s, O.SH), 5.50(s, O.SH), 4.24-4.31 (in, 1H), 3.67 (dd, J--10.8,
4.8 Hz, 1H), 3,27-
3.44 (m, 3H), 1.55 On, 2.H), 1.26 (s, 30H), 0.89 (t, J-- 6.3 Hz, 3H); MS (ESI)
m/z calcd for
CzsH4~N303S 506.76 (M+I), obsd 506.60.
(2RS, 4R)-2-(4-Cyano-phenyl)-thiazolidine-4-carboxylic acid octadccylarnide
(compound 22): 1H NMR (CDCl3) 8 7.60-7.70 (m, 4H), 6.94 (m, 0.6H), 6.37 (m,
0.4), 5.64
(s, 0.4H), 5.46 (s, 0.6H), 4.27 (m, 0.6H), 3.96 (m, 0.4H), 3.65-3.70 (m, 1H),
3.20-3.45 (m,
3H), 1.54 On, 2H), 1.26 (s, 30H), 0.89 (t, ,l= 6.3 Hz, 3H); MS (ES 1) m/z
calcd for
Cz~Hø~N3OS 485.77 (M.), obsd 508.50 (M+Na).
(2RS, 4R)-2-(3,5-Difluoro-phenyl)-thiazolidine-4-carboxylic acid
octadecylamide
(compound 23): 1H NMR (CDCI3) 8 7.04-7.08 (in, 2H), 6.97 (m, 1H), 6.79 (m,
1H), 5.40 (s,
0.5H), 5.36 (s, 0.5H), 4.23-4.30 (rn, 1H), 3.66 (dd, .I--11.I, 4.5 Hz, 1H),
3.26-3.42 On, 3H),
1.33 (m, 2H), 1.26 (s, 30H), 0.89 (t, J-- 6.3 Hz, 3H); MS (ESI) m/z calcd for
Cz8H4~FZNz02S
497.74 (M+1), obsd 497.50.
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(2RS, 4R)-2-(2,6-Dichloro-phenyl)-thiazolidine-4-carboxylic acid
octadecylamide
(compound 24): 1H NMR (CDCl3) ~ 7.34-7.38 (m, 2H), 7.15-7.28 (m, 2H), 6.29 (s,
0.5H),
6.25 (s, 0.511), 4.25 (t, J-- 5,7 Hz, 1H), 3.94 (dd, J--10.5, 1.8 Hz, 1H),
3.26-3.52 (m, 3H),
1.52 (m, 2H), 1.26 (s, 30H), 0.89 (t, J 6 Hz, 3H); MS (ESI) m/z calcd for
C2gH46C12N20zS
529.65 (M'), obsd 529.70.
(2RS, 4R)-2-(3-Bromo-4-fiuoro-phenyl)-thiazolidine-4-carboxylic acid
octadecylamide (compound 25): 1H NMR (CDC13) S 7.71 On, 1H), 7.42 (m, 1H),
7.06-7.16
(m, 2I1), 5.56 (d, J-- 9.3 Hz, 0.2H), 5.34 (d, J--10.2 Hz, 0.8H), 4.29 (d, J=
4.5 Hz, 0.811),
3.94 (m, 0.2H), 3.69 (dd, J--11,1, 4.2 Hz, 1H), 3.21-141 (m, 3H), 1.52 (m,
2H), 1.26 (s, 30H),
0.89 (t, .J--- 6.3 Hz, 3H); MS (ESI) mlz calcd for CZ8H4~BrFNzOS 558.65 (M+1),
obsd 558,70.
(2RS, 4R)-2 p-Tolyl-thiazolidine-4-carboxylic acid octadecylamide (compound
26):
1H NMR (CDC13) ~ 7.34-7.43 (m, 2H), 7.14-7.21 (m, 3H), 5.59 (s, 0.2H), 5.32
(s, 0.8H), 4.76
(in, 0.2H), 4.35 (m, 0.8H), 3.70 (dd, J--11.1, 3.9 Hz, 1H), 3.21-3.43 (m,
311), 2.36 (d, J-- 2.7
Hz, 3H), 1.51 (m, 2H), 1.27 (s, 30H), 0.89 (t, J-- 6.3 Hz, 3H); MS (ESI) nalz
calcd for
C29HsiNaOS 475.79 (M+1), obsd 475.60.
(2RS, 4R)-2-Biphenyl-4-yl-thiazolidine-4-carboxylic acid octadecylamide
Hydrochloride (compound 27~HC1): 1H NMR~(DMSO-d6) b 8.59 (m, 1H), 7,66-7.73
(m,
5H), 7.37-7.51 (m, 4H), 5.92 (s, 0.7H), 5.87 (s, 0.3H), 4.62 (m, 0.7H), 4.41
(m, 0.3H), 3.53-
3.64 (m, 1H), 3.26-3.32 (m, 1H), 3.13-3.17 (m, 2H), 1.44 (m, 2H), 1.22 (s,
30H), 0.84 (t, J=
6.3 Hz, 3H); MS (ESI) m/z calcd for C34Hs3NaOS 537.86 (M+1), obsd. 537.70.
Example 2 - Synthesis of N-Acyt and N-sulfonyl Derivatives Thiazolldine
Carboxylic Acid
Amides
N-Acyl and N-sulfonyl derivatives (compounds 28 and 29) were synthesized from
compound 5 by standard procedures (scheme 2). Briefly, (2RS, 4R)-2-
phenylthiazolidine-4-
carboxylic acid octadecylamide (compound 5) was reacted with either acetic
anhydride or
methyl sulfonyl chloride, in pyridine, to afford the desired derivatives.
(2RS, 4R)-3-Acetyl-2-phenylthiazolidine-4-carboxylic acid octadecylamide
(compound 28): 1H NMR (CDC13) 8 7.31-7.41 (m, 5H), 6.01 (s, 1H), 5.12 (s, 1H),
3.73 (m,
1H), 3.40 (m, 1H), 3.31 (m, I H), 3.11-3.17 (m, 1H), 2.00 (s, 3H), 1.27-1.33
(m, 32H), 0.89 (t,
J= 6.3 Hz, 3H); MS (ESI) nalz calcd for C3oHsoNaOzS 502.80 (M+), obsd 502.60.
(2RS,4R)-3-Methanesulfonyl-2-phenylthiazolidine-4-carboxylic acid
octadecylamide
(compound 29): 1H NMR (CDCI3) b 7.65-7.68 (m, 2H), 7.32-7.36 (m, 3H), 6.20 (s,
1H), 4.63
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WO 2005/086638 PCT/US2005/004759
(dd, J= 9, 6 Hz, 1H), 3.67 d, J=12, 6 Hz, 1H), 3.47 (dd, J= 12.3, 8.1 Hz, 1H),
3.04-3.13 (m,
2H), 3.02 (s, 3H), 1.27 (m, 32H), 0.89 (t, J= 6.3 Hz, 3H); MS (ESI] m/z calcd
for
C29HSON2o3s2 538.85 (M+), obsd 538.70.
Based on the foregoing synthesis, it is expected that other acyl anhydrides
(e.g.,
S containing larger all~yl groups) can also be prepared according to this same
synthesis
procedure (Badr et al., "Synthesis of Oxazolidines, Thiazolidines, and 5,6,7,8-
Tetrahydro-lII,
3H pyrrolo[1,2-c] oxazole (or thiazole)-1,3-diones from (3-Hydroxy- or,~-
Mercapto-a-amino
Acid Esters," Bull. Chem. Soc. Jpn. 54:1844-1847 (1981), which is hereby
incorporated by
reference in its entirety).
Example 3 - Synthesis of Thiazole Carboxylic Acid Amides
The synthesis of thiazole derivative (compound 34) was accomplished starting
from
cysteine as shown in scheme 3.
To a solution of DL-cysteine (3g, 24.76 mmol) in MeOH (SO mL) at 0°C,
SOC12 (2.76
1S mL, 37.14 mmol) was slowly added and warmed to room temperature then
refluxed for 3 h.
The reaction mixture was concentrated in vacuo to yield a residue. This
residue was taken in
to aqueous EtOH (1:1, 30 mL), NaHC03 (2.28 g, 27.23 mmol) was added, after 10
min
benzaldehyde (2.S mL, 24.76 mmol) was added and stirnng continued for 3 h.
CHCl3 (200
mL) was added to the reaction mixture and washed with water, brine, dried
(Na2SO4) and
solvent was removed in vacuo. The crude product was purified by column
chromatography
to afford 2-phenylthiazolidine-4-carboxylic acid methyl ester (compound 31):
yield 4.7 g,
85%; 1H NMR (CDC13) 8 7.51-7.62 (m, 2H), 7.32-7.42 (m, 3H), 5.84 (s, 0.4H),
S.SB (x,
0.4H), 4.24 (t, J= 6.3 Hz, 0.4H), 4.01 (t, J= 7.S Hz, 0.6H), 3.83 (s, 3H),
3.39-3.SS (m, 1H),
3.10-3.26 (m, 1H); MS (ES17 rnlz 224 (M+1).
2S Beginning with compound 31, 2-phenylthiazole-4-carboxylic acid methyl ester
(compound 32) was synthesized following a reported procedure (Kue et al.,
"Essential Role
for G Proteins in Prostate Cancer Cell Growth and Signaling" J. Urol. 164:2162-
2167 (2000),
which is hereby incorporated by reference in its entirety). Yield 033 g, 68%;
1H NMR
(CDC13) 8 8.20 (s,IIH), 8.0-8.04 (m, 2H), 7.45-7.50 (m, 3H), 4.0 (s, 3H); MS
(ES1~ m/z 220
(M+1).
To a solution of compound 32 (0.5 g, 2.28 mmol) in MeOH (10 mL) at
0°C,1N NaOH
(5 mL) was added and stirred for 2 h. To the reaction mixture EtOAc (30 mL),
was added and
acidified with 1N HCl. Extracted with EtOAc (3XS0 mL), combined extracts were
washed
38
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with water, brine, dried (NaZS04) and solvent was - removed under vacua to
give crude acid
(compound 33), which was converted to 2-phcnylthiazole-4-carboxylic acid
octadecylamide
(compound 34) following the general procedure described in Example I above.
Yield 0.30 g,
68%; 1H NMR (CDCl3) 6 8.10 (s, 1H), 7.96.7.93 (m, 2H), 7.46-7.50 (m, 3H), 3.49
(dd, J=
13.5, 6.9 Hz, 2H), 1.69 (m, 2H), 1.27 (m, 301-1), 0.89 (t, J 6.3 Hz, 3H); MS
(BSI) m/z calcd
for CZ8H4sNzOS 457.73 (M+1), obsd 457.60.
Table 1:
compoundR' Rz R4 mp yield formula
(C) (%)
3-HCI Phenyl C~HIS H ND 80 CI~Hz~C1N20S
4-HCI Phenyl C14Hz8 H 95 83 Cz4HaiCINzOS
5-HCI Phenyl Cl$H3~ H 93 70 Cz$H49C1NzOS
6-HCI Phenyl Ci9Hs9 H 85 78 Cz9HsICTNzOS
7 n-dodecyl Cl$H3~ H 86 69 C34H68NzOS
8 Cyclohyxyl C18H3~ H 60 75 Cz$Hs4NzOS
9 Benzyl Cl$H3~ H 80 81 Cz9HsoNzOS
3-indolyl Cl$H3~ H 125 65 C3oHd9N30S
11 3-pyridinyl Cl$H3~ H 94 63 Cz~H4~N30S
12-HCI 3-furanyl CI$H3~ H 99 60 Cz6Ha7C1NzOzS
13 4-dimethylaminophenylCI$H3~ H 75 75 C3oHssNsOS
14 3-hydroxyphenyl Cl$H3~ H 50 69 Cz$H4gN20zS
15-HCI 4-methoxyphenyl CI$H3~ H 95 70 Cz9Hs,CINzO2S
16-HCI 3,4-dimethoxyphenylC18H3~ H 103 83 C3oHssClNzOsS
17-HCI 3,4,5-trimethoxyphenylCI8H3~ H 115 70 C3lHssClNzOaS
18-HCI 4-acetamidophenylCl$H3~ H 170 63 C3oHszCIN30zS
19 4-fluorophenyl C~SH3~ H 65 73 Cz$H4~FNZOS
4-bromophenyl Cl$H3~ H 81 77 Cz$Hd~BrNZOS
21 4-nicrophenyl Cl$H3~ H 115 60 Cz$H~~N303S
22 4-cyanophenyl Cl$H3~ H 90 70 Cz9H4~N30S
23 3,5-difluorophenylC18H3~ H 113 70 Cz$H46FZNZOS
24 2,6-difluorophenylCI$H3~ H 49 80 Cz8H46C~zN20S
ZS 3-bromo-4-fluorophenylCI$H3~ H 100 78 Cz8H46BrFN20S
26 4-methylphenyl C18H3~ H 120 73 Cz9HsoNzOS
27-HCI Biphenyl Cl$H3~ H 130 70 C3Hs3CINzOS
28 Phenyl Cl$H3~ COCH3 90 95 C3oHsoNzOzS
29 Phenyl C,8H3~ SOZMe 55 90 Cz9HsoNzOsSz
Examine 4 - Analysis of Selected Prostate Cancer Cell Lines by RT-PCR for LPA
Receptor
Ex ression
DU-145, PC-3, and LNCaP human prostate cancer cells, and RH7777 rat hepatoma
cells were obtained from American Type Culture Collection (Manassas, VA). Dr.
Mitchell
39
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WO 2005/086638 PCT/US2005/004759
Steiner at University of Tennessee Health Science Center, kindly provided PPG-
1 and TSU-
PrI cells. Prostate cancer cells and RH7777 cells were maintained in RPMI 1640
medium and
DMEM (Mediatech, Inc., Herndon, VA), respectively, supplemented with 10% fetal
bovine
serum (Gibco, Grand Island, NY) in 5% CO2 / 95% air humidified atmosphere at
37°C.
Total RNA was extracted using Trizol~ reagent (Invitrogen Corp., Carlsbad, CA)
according to the manufacturer's instruction. 0.5 ~,g (LPAI) or 1 (LPA2 and
LPA3) of total
RNA was used to perform RT-PCR using SuperScriptT"" One-Step RT-PCR with
Platinum~
Tag (Invitrogen Corp., Carlsbad, CA) with 0.2 ~M of primers. The following
primer pairs
were used:
LPAI forward 5'-GCTCCACACACGGATGAGCAACC-3' (SEQ ID NO: I), and
LPA, reverse 5'-GTGGTCATTGCTGTGAACTCCAGC-3' (SEQ ~ NO: 2);
LPA2 forward 5'-CTGCTCAGCCGCTCCTATTTG-3' (SEQ ID NO: 3), and
LPAZ reverse 5'-AGGAGCACCCACAAGTCATCAG-3' (SEQ ID NO: 4);
LPA3 forward 5'-CCATAGCAACCTGACCAAAAAGAG-3' (SEQ ID NO: 5), and
LPA3 reverse 5'-TCCTTGTAGGAGTAGATGATGGGG-3' (SEQ ID NO: 6);
(3-actin forward 5'-GCTCGTCGTCGACAACGGCTC-3' (SEQ ID NO: 7), and
(3-actin reverse 5'-CAAACATGATCTGGGTCATCTTCTC-3' (SEQ ID NO: 8).
PCR conditions were as follows: After 2 min denaturation step at 94 °C,
samples were
subjected to 34 to 40 cycles at 94 °C for 30 sec, 60 °C (LPAI)
or SS °C (LPAZ and LPA3) for
30 sec, and 72 °C for I min, followed by an additional elongation step
at 72 °C for 7 min.
Primers were selected to span at least one intron of the genomic sequence to
detect genomic
DNA contamination. The PCR products were separated on 1.5% agaxose gels,
stained with
ethidium bromide, and the band intensity was quantified using Quantity One
Software (Bio-
Rad Laboratories, Inc., Hercules, CA). Expression levels of each receptor
subtype in different
cell lines were expressed as ratios compared to i-actin mRNA level.
LPL receptor expression in these cell lines was determined to validate their
use as i~.
vitro models (see Table 2 below). 1 ~,g of total RNA was subjected to RT-PCR,
the PCR
products were separated on agarose gels, and relative expression level of each
receptor
subtype compared to (3-actin was quantified by Quantity One Software (Bio-
Rad). LPAI was
the predominant LPL receptor expressed in these cell lines. However, LNCaP
cells did not
express this receptor subtype. LPA3 receptor was uniquely expressed in
prostate cancer cell
lines. RH7777 cells do not express any of the known LPL receptors.
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Table 2: LPL Receptor mRNA Expression
LPL Old Expression
level
relative
to ~3-actin
ReceptorName RH777 DU145 PC-3 LNCaP PPC-1 TSU-P1S
LPAI EDG-2 UDa 2.16 2.53 UD 2.29 2.13
LPAz EDG-4 UD 0.33 0.43 0.32 0.41 0.19
LPA3 EDG-7 UD 0.07 0.27 0.28 0.15 UD
Sum LPAI_3 0 2.56 3.23 0.60 2.85 2.32
aUD=under detection limit
Example 5 - Cytotoxicity Assay in Prostate Cancer Cells
For in vitro cytotoxicity screening, 1000 to 5000 cells were plated into each
well of
96-well plates depending on growth rate, and exposed to different
concentrations of a test
compound for 96 h in three to five replicates. All the compounds were
dissolved in dimethyl
sulfoxide at 5 to 20 mM, and diluted to desired concentrations in complete
culture medium.
Cell numbers at the end of the drug treatment were measured by the SRB assay
(Gududuru et
al., "Synthesis and Biological Evaluation of Novel Cytotoxic Phospholipids for
Prostate
Cancer," Bi~o~g. Med. Chem. Lett. 14:4919-4923 (2004); Rubinstein et at.,
"Comparison of
in vitf°o Anticancer-Drug-Screening Data Generated with a Tetrazolium
Assay Versus a
Protein Assay Against a Diverse Panel of Human Tumor Cell Lines," J. Naatl,
Cancer Tnst.
82:1113-1118 (1990), each of which is hereby incorporated by reference in its
entirety).
1 S Briefly, the cells were fixed with 10% of trichioroacetic acid, stained
with 0.4% SRB, and the
absorbances at 540 nm was measured using a plate reader (DYNEX Technologies,
Chantilly,
VA). Percentages of cell survival versus drug concentrations were plotted and
the ICSo
(concentration that inhibited cell growth by 50% of untreated control) values
were obtained
by nonlinear regression analysis using WinNonlin (Pharsight Corporation,
Mountain View,
CA). 5-fluorouracil was used as a positive control to compare potencies of the
new
compounds.
A sandwich ELISA (Roche, Mannheim, Germany) utilizing monoclonal antibodies
specific for DNA and histones was used to quantify degree of apoptosis induced
by the
analogs after 72 h exposure. This assay measures DNA-histone complexes (mono-
and
oligonucleosomes) released into cytoplasm from the nucleus during apoptosis.
RH7777 cells
were employed because of nonspecific cytotoxicity of compound 4 in receptor-
negative cells
as well as receptor-positive prostate cancer cells.
The ability of 2-aryl-thiazolidine derivatives (ATCAAs) to inhibit the growth
of five
human prostate cancer cell lines (DU-145, PC-3, LNCaP, PPG-1, and TSU-Prl) was
assessed
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using the sulforhodamine B (SRB) assay (described above). A control cell line
(RH7777) that
does not express LPL receptors (Svetlov et al., "EDG Receptors and Hepatic
Pathophysiology of LPA and EDG-ology of Liver Injury," Biochimica et
Biophysica ACT
1582:251-256 (2002), which is hereby incorporated by reference in its
entirety) was also
utilized to understand whether the antiproliferative activity of these
derivatives is mediated
through inhibition of LPL receptors.
The diastereomeric mixtures of the target compounds 3-29 were used as such to
evaluate their irc vit~~o inhibitory activity against prostate cancer cell
lines, and the results are
summarized in Tables 3 and 4 below. 5-Fluorouracil was used as the reference
drug. To
deduce sound structure-activity relationships, ICSOS should on principle be
determined on
pure isomers. One drawback of testing mixtures of stereoisomers, unavoidable
in this case,
was that the effect of each stereoisomer on the biological activity could not
be assessed. On
the other hand, the TCSO values calculated can be used as a screening method
to select
promising selective cytotoxic agents and to identify the diastereomeric
mixture with the best
availability to inhibit the growth of prostate cancer cells. Many of these
thiazolidine analogs
were very effective in killing prostate cancer cell lines with IC50 values in
the low/sub
micromolar range (Table 3). Examination of the cytotoxic effects of compounds
3-5 shows
that as the chain length increases from C7 to C,B, the potency also increases.
However, a
further increase in the allcyl chain length by one carbon unit (i.e., Cl8 to
C19) caused a
significant loss in cytotoxicity. Interestingly, C14 derivative (compound 4)
demonstrated
higher potency than compound 5, but was 8-fold less selective against RH7777
cell line.
Thus, an alkyl chain with a C18 unit is optimal for maintaining the potency
and selectivity
observed in this series of compounds. N-Acyl and N-sulfonyl derivatives
(compounds 28 and
29) were less cytotoxic than parent compound 5. Replacement of the phenyl ring
with an
allcyl or cyclohexyl group reduced the potency (compounds 7 and 8) relative to
the
thiazolidine (compound 5) derivative. Introduction of a methylene spacer
separating the
phenyl ring and the thiazolidine ring furnished a compound 9, which was less
active than the
parent compound 5.
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Table 3: Antiproliferative effects of compounds 3-17 on prostate cancer cell
lines
ICSO - _
Compd (~cM)
~7777a DU-145 PC-3 LNCaP PPC-1 TSU-Prl
3-HCI 52.2 44.9 38.5 12.4 34.7 28.0
4-HCI 3.4 2.4 3.0 1.4 1.3 2.0
5-HCI 25.6 5.4 7.8 2.1 2.0 5.0
6-HCI NA >20 NA I3.6 16.8 >20
7 ~20 8.9 15.0 1I.9 13.0 10.7
8 >20 >20 >20 12.8 9.3 >20
9 >20 15.3 16.4 4.4 4.0 11.2
>20 8.9 11.5 2.1 1.3 4.4
11 10.5 7.5 9.2 3.6 2.9 7.8
12-HCI 10.4 6.6 8.1 1.7 1.1 4.2
13 >20 5.3 6.0 1.6 1.1 3.0
14 31.0 5.7 6.7 1.7 1.2 4.0
15-HCI >20 8.7 ~20 2.1 1.5 ND
16-HCI 10.3 4.5 5.2 0.85 0/58 2.4
17-HCI 11.4 3.9 4.0 0.82 0148 2.4
5-FU ND 11.9 12.0 4.9 6.4 3.6
aControl cell line. °Prostate cancer cell lines. ND=not detectable.
NA=no activity.
Table 4: Antiproliferative effects of compounds 18-29 and 34 on prostate
cancer cell line
ICso (wM)
Compd a
~7777 DU-145 PC-3 LNCaP PPC-1 TSU-Prl
18-HCI 21.1 3.1 5.6 1.3 0.55 0.94
19 17.4 5.7 6/8 1.9 2.1 5.4
>20 13.8 17.3 5.1 3.7 18.3
21 ~20 15.3 ~20 8.4 15.3 15.9
22 >20 >20 >20 5.9 5.0 >20
23 >20 >20 >20 11.2 10.6 >20
24 >20 >20 >20 13.1 17.1 ~20
~20 11.3 13.5 3.0 4.7 14.0
26 >20 10.5 12.8 1.9 1.9 8.0
27-HCI >20 >20 >20 >20 >20 >20
28 >20 ~20 ~20 16.1 12.6 >20
29 >20 >20 >20 >20 >20 >20
34 >20 >20 >20 >20 >20 >20
5-FU ND 11.9 12.0 4.9 6.4 3.6
5 aControl cell line. nProstate cancer cell lines.
To understand the effect of unsaturation on potency and selectivity, and to
overcome
the problems associated with stereoisomers, the central thiazolidine core in
compound 5 was
replaced with a thiazole ring. However, thiazole derivative (compound 34) did
not show any
10 activity below 20 ~,M in both prostate and RH7777 cells, which indicates
that thiazolidine
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ring with two chiral centers plays an important role in providing potency and
selectivity.
Replacements of the phenyl ring with a heterocycle, such as an indole,
pyridine or fixran ring
was investigated by synthesizing analogs (compounds 10-12). The furanyl
derivative
(compound 12) showed equivalent cytotoxicity as compound 5, but was 3-fold
less selective
against RH7777 cells.
The cytotoxicity data of compounds 13-27 provides a summary of a broad survey
of
phenyl ring substituted analogs, Examination of the ICSO values of these
analogs demonstrates
a greater tolerance for diverse substituents in the phenyl ring. In general,
the most potent
analogues possessed electron-donating substituents, as exemplified by
comparison of
compound 13, and compounds 16-1 ~, relative to compound 5. One of the most
active
compounds (compound 18) with an ICSO of 0.55 ~,M was 38-fold more selective in
PPC-1
cells compared to RH7777 cells. On the other hand, thiazolidine analogs
(compounds 19-25),
with electron-withdrawing substituents demonstrated less cytotoxicity.
Comparison of the
potencies of compound 26 and compound 27, suggest that substitution of the
phenyl ring with
a bulky group reduces the activity.
From the LPL receptor mRNA expression studies (Table 2), it was evident that
these
cell lines serve as an excellent model system to explore the effects of LPL
receptor. Given the
structural similarity of SAPS to ceramide (and the lrnown ability of ceramide
to induce
apoptosis), it was then determined whether the antiproliferative effects of
thiazolidine analogs
were mediated via apoptotla events. The ability of the analogs to induce
apoptosis in LNCaP,
PC-3, and RH7777 cells was examined using a quantitative sandwich ELISA that
measures
DNA-histone complex released during apoptosis. The enrichment factor
calculated (as ratio
of OD405 in treated and un-treated cells) provides a quantitative assessment
of the degree of
apoptosis induced. Initially, only two compounds (4 & 5) were used for this
study. Apoptotic
activity of analog (compound 4) was selective in prostate cancer cells despite
nonselective
cytotoxicity in RH7777 negative control cells (see Table 5 below). Analog
compound 5
induced apoptosis in PC-3 and LNCaP cells, but to a lesser extent in PC-3
cells perhaps due
to lower potency in this cell line. This data suggests that thiazolidine
analogs may act as
potent inducers of apoptosis and selectively kill a variety of prostate cancer
cell lines.
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Table 5: Thiazolidine Amides-Induced Apoptosis
Compound for 72 h PC-3 LNCaP RH7777
2 ACM 1.8 14.1 2.6
4 5 ~,M 18.7 75.4 3.2
~,M 54.0 80.7 2.5
2 ~M 1.4 4.5
5 ~M 2.3 45.2
10 ~,M 3.4 37.1
~,M 12.7 26.1
These results are consistent with the assay testing LNCaP cells for DNA
5 fragmentation by agarose gel electrophoresis. LNCaP cells were treated With
a thiazolidine
derivative (compound 4 or 5) for 24 to 108 hours, and then total DNA was
extracted from 2 x
106 cells by simple centrifugation method, treated with RNase and Proteinase
K. After
precipication in ethanol, DNA was reconstituted in Tris-EDTA buffer, separated
on agarose
gels, and visualized by ethidium bromide staining ~ (Hemnann et al., "A Rapid
and Simple
10 Method for the Isolation of Apoptotic DNA Fragments," Nucl. Acids Res.
22:5506-5507
(1994), which is hereby incorporated by reference in its entirety). The
results, shown in
Figures 4A-B, demonstrate that both of these compounds induce cell apoptosis
in the LNCaP
prostate cancer cell line.
As another assessment of cytotoxicity, AKT inhibition was measured. 30 pg of
total
15 cellular protein from untreated control cells and compound-treated cells
were separated by
SDS-PAGE, transferred to nitrocellulose membrane, and total AKT and phospho-
AKT were
probed with anti-AKT and anti-phospho AKT antibody specific for AKT
phosphorylated at
Ser 473, respectively (Cell Signaling Technology, Beverly, MA). The
immunoblots were
visualized by enhanced, chemiluminescence, and changes of relative levels of
phospho-AKT
20 compared to total AKT by analog treatment were quantified by densitometric
analysis. Figure
SB graphically illustrates the immunological detection of AKT using anti-AKT
and anti-
phospo-AKT, shown in Figure SA.
From the foregoing, it should be appreciated that the introduction of ring
activating
groups on the phenyl ring resulted in increasing potencies for prostate cancer
cell lines. The
above results demonstrate several new anticancer agents (represented by
compounds I6, 17,
and 18) with low/sub micromolar cytoxicity and high selectivity. From this
study, compound
18 emerged as one of the most potent and selective cytotoxic agents with an
ICSO of 0.55 ~,M
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and 38-fold selectivity in PPC-1 cells. Further, the ability of these analogs
to induce apoptosis
in LNCaP, PC-3 and RH7777 cells provides an important clue to understand their
mechanism
of action.
S Exam lp_e 6 - Synthesis of Thiazolidinone Amides
The synthesis of thiazolidinone derivatives (comnounds 6S -72) utilized
straightforward chemistry as shown in scheme 4 (Figure 6), where l is 1.
Various 4-
thiazolidinones were synthesized following a reported procedure of condensing
mercaptoacetic acid, glycine methyl ester, and aromatic aldehydes in a one-pot
reaction,
followed by basic hydrolysis of the ester (Holmes et al., "Strategies for
Combinatorial
Organic Synthesis: Solution and Polymer-supported Synthesis of 4-
thiazolidinones and 4-
metathiazanones Derived from Amino Acids," J. Org. Chem. 60:7328-7333 (1995),
which is
hereby incorporated by reference in its entirety). Thiazolidinone amides were
obtained by the
treatment with appropriate amines in the presence of EDC/HOBt under standard
conditions.
1 S Compound 6S that has no side chain was synthesized from the corresponding
acid as shown
in Figure 6 (scheme 4). Thiazolidinone amides (compounds 73 -77) were
synthesized by a
simple and direct method (Schuemacher et al., "Condensation Between
Isocyanates and
Carboxylic Acids in the Presence of 4-dimethylaminopyridine (DMAP), a Mild and
Efficient
Synthesis of Amides," Synthesis 22:243-246 (2001), which is hereby
incorporated by
reference in its entirety), which involves reaction of the acid compound 64a
with different
isocyanates in the presence of a catalytic amount of DMAP (Figure 7)(scheme
S). Exhaustive
reduction of compound 68 using BH3 THF under reflex conditions gave compound
79
(Figure 8) (scheme 6). Oxidation of 68 using H2O2 and with I~Mn04 afforded
sulfoxide
(compound 80) and sulfone (compound 81), respectively, as shown in scheme 6.
All
2S compounds were characterized by 1H and 13C NMR, mass spectroscopy and, in
certain cases,
elemental analysis.
Compounds were obtained as mixtures of diastereomers and were used as such for
the
biological studies. Characteristic data for exemplary compounds 68, 71, 72,
and 81 are
provided below.
N-octadecyl-2-(4-oxo-2-phenylthiazolidin-3-yl)acetamide (compound 68): 1H NMR
(300 MHz, CDC13): 8 0.89 (t, J-- 6.0 Hz, 3H), 1.26 (br s, 30H), 1.46 (m, 2H),
3.16-3.29 (m,
3H), 3,82 (d, .I--- 1.S Hz, 2H), 4.20 (s, O.SH), 4,25 (s, O.SH), 5.83-5.85 (m,
2H), 7.27-7.41 (m,
SH); 13C NMR (300 MHz, CDCl3): ~ 13.SS, 22.13, 26.30, 28.69, 28.80, 28.88,
28.99, 29.03,
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29.10, 29.14, 31.37, 32.13, 39.08, 45.88, 63.67, 127.05, 128.58, 128.96,
137.61, 166.30,
171.61; MS (ESI) rnlz 5I 1 [M+Na). Anal. Calcd for C29H48N~OZS; C, 71.26; H,
9.90; N,
5.73. Found: C, 71.18; H, 10.03; N, 5.79.
2-(2-4methoxyphenyl)-4-oxothiazolidin-3-yl)-N-ocatadecylacetamide (compound
71): 1H NMR (300 MHz, CDCl3): 8 0.89 (t, J= 6.0 Hz, 3H), 1.26 (br s, 30H),
1.33 (s, 2H),
3.16-3.19 (m, 1H), 3.2-3.29 (m, 2H), 3,80 (d, J= 0.9 Hz, 2H), 3.83 (s, 3H),
4.16 (s, O.SH),
4.21 (s, 0.47H), 5.82 (s, 1H), 6.9 (dd, J-- I.8 Hz, 2H), 7.29 (dd, .I--- I.5
Hz, 2H); 13C NMR
(300 MHz, CDC13): c~ 13.53, 22.12, 26.31, 28.70, 28.74, 28.79, 28.89, 28.99,
29.03, 29.09,
29.13, 31.36, 32.23, 39.06, 45.74, 54.79, 63.44, 128.64, 129.11, 159.97,
166.41, 171.47; MS
(ESI) m/z 541 [M+Na]. Anal. Calcd for C3o Hso N2 03 S: C, 69.45; H, 9.71; N,
5.40. Found:
C, 69.30; H, 9.86; N, 5.43.
2-(2-(2,6-dichlorophenyl)-4-oxothiazolidin-3-yl)-N-octadecylacetamide
(compound
72): 1H NMR (300 MHz, CDC13): ~ 3.54 (d, J--15.3 Hz, IH), 3.87 (s, 2H), 4.25
(d, J--15.3
Hz, 1H), 5.88 (s, 1H), 7.10 (t, J-- I.8 Hz, 1H), 7.36-7.43 (m, 7H), 8.29 (s,
1H); 13C NMR (300
MHz, CDC13): ~ 32.35, 46.73, 64.40, 117,37, 123.85, 127.29, 128.74, 129.32,
134.59,
136.87, 138.61, 165.14, 172.60; MS (ESI) nZ/z 403 [M+Na]. Anal. Calcd for Cl~
Hi4 Cia N2
02 S: C, 53.55; H, 3.70; N, 7.35. Found: C, 53.39; H, 3.47; N, 7.36.
N-octadecyl-2-(4-oxo-2-phenyl-1-sulfonyl-thiazolidin-3-yl)acetamide (compound
81):
1H NMR (300 MHz, CDC13): ~ 0.89 (t, J= 6.0 Hz, 3H), 1.26 (br s, 32H), 3.19-
3.34 (rn, 3H),
3.88-4.03 (dd, J-- 16.S Hz, 2H), 4.66 (s, 0.51), 4.72 (s, O.SH), 5.67 (br s,
1H), 5.95 (s, 1H),
7.38 (m, 2H), 7.50-7.53 (m, 3H); 13C NMR (300 MHz, CDC13): S 13.54, 22.12,
26.26, 28.66,
28.79, 28.96, 29.02, 29.09, 29,14, 31.36, 39.30, 44.35, 49.85, 81.32, 125.77,
128.43, 128.91,
130.55, 163.23, 165.30; MS (ESI) mlz 519 [M-H]. Anal. Calcd for Ca9 Ha8 Na 04
S: C, 66.88;
H, 9.29; N, 5.38. Found: C, 66.68; H, 9.27; N, 5.41.
Example 7 - Cytotoxicity Assay
The antiproliferative activity of all the synthesized compounds was evaluated
against
five human prostate cancer cell lines and in RH7777 cells (negative control)
using the
sulforhodamine B (SRB) assay (see description in Example 5 above). S-
Fluorouracil (5-FLT)
was used as reference drug. As shown in Table 6, 4-thiazolidinone carboxylic
acids
(compounds 64a and 64b) were unable to inhibit the growth of any of the five
prostate cancer
cells below 50 ~,M. However, the corresponding amides (compounds 66 -68)
showed higher
activities. It was observed that an increase in the all~yl chain length
[compounds 66 (10), 67
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(C14), and 68 (C18)~ enhances the antiproliferative activity of these analogs
in prostate
cancer cells. Interestingly, the simple amide 65 without any long alkyl chain
is not cytotoxic
below 100 ~,M, which indicates that the absence of an alkyl side chain causes
a considerable
decrease in antiproliferative effect. On the other hand, replacement of the
alkyl chain with
various aryl side chains (compounds 73 -78) reduced the biological activity.
Among this
series, compound 73 is moderately cytotoxic, where as analogs (compounds 76 -
78)
displayed poor cytotoxicity in several prostate cancer cell lines. However, it
is noteworChy to
mention that thiazolidinone amides (compounds 74 and 75), with electron-
withdrawing
substituents on the aryl ring showed cytotoxicity in the range of 13-29 ACM
against all five
prostate cancer cell lines.
Table 6; Antiproliferative effects of compounds 64a --64b and 65 -78
Compd Rl Y lCSO
a (~M) ~
RH7777 DU-145 PC-3 LNCaP P C-1'TSIT-P
64a phenyl OH ND >50 >50 >50 >50 >50
64b biphenyl OH >100 >100 >100 >100 >I00 >100
65 phenyl NHZ >100 >100 >100 >100 >100 >100
66 phenyl NH-CloHzi20.0 22.4 20.3 14.1 15.8 19.7
67 phenyl NH-Ct~H2916.4 I9.6 13.5 14.1 10.1 13.4
68 phenyl NH-CI$H3~39.6 12.6 11.1 9.3 7.1 8.5
69 biphenyl NH-Ci$H3~>50 >50 >50 >50 >50 >50
70 dimethylaminoNH-Cl$H3~>50 >50 >50 >50 >50 >50
naphtalen-4-yl
71 4-methoxy NH-CI$H3~31.1 14.8 12.6 11.8 10.7 17.5
phenyl
72 2,6-dichloroNH-C,$H3~>50 >50 >50 >50 >50 >50
phynyl
73 phenyl NH-3,5- 70.9 69.0 74.1 24.1 46.2 53.2
difluoro
phenyl
74 phenyl NH-3,5-di(tri25.4 16.2 18.1 14.5 13.1 16.1
fluoromethyl)
phenyl
75 phenyl NH-3,5-di34.9 24.0 28.6 I3.2 20.5 17.2
chlorophenyl
76 phenyl NH-2,4- >100 >100 >100 82.5 >100 60/8
dimethoxy
phenyl
77 phenyl NH-naphthyl>100 >100 >100 31.4 >100 69.9
78 phenyl 2,4dimethoxy>100 >100 >100 >100 >100 >100
phenylethyl
5-FU ND I1.9 12.0 4.9 6.4 3.6
aControl cell line. °Prostate cancer cell lines.
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Table 7: Antiproliferative effects of compounds 79-81
ICso (N~M)
Compd a
~7777 DU-145 PC-3 LNCaP PPC-1 TSU
79 >20 15.8 >20 >20 12.0 6.1
80 11.5 11.2 6.5 7.9 5.4 6.4
81 22.1 15.5 8.5 10.9 5.5 9.3
5-FU ND 11.9 12.0 4.9 6.4 3.6
aControl cell line. °Prostate cancer cell lines. ND=not detectable.
NA=no activity.
Thiazolidinone derivatives (compounds 69 and 70) with bullcy biphenyl or
naphthalene groups demonstrated low cytotoxicity compared to compound 68
(Table 6).
Compounds 71 and 72 were synthesized to understand the effects of aromatic
ring
substitution in compound 68. Tt was observed that electron-donating
substituents maintained
good activity while the ortho electron-withdrawing substituents substantially
decrease the
antiproliferative activity of these derivatives (Table 6). Compound 79, which
has no amide
groups, showed significantly good potency in all five prostate cancer cell
lines. Notably,
compounds 80 and 81 bearing sulfoxide or sulfone moiety displayed higher
cytotoxic potency
comparable to that of the reference drug 5-FU against both PC-3 and PPC-I cell
lines (Table
7).
Tn summary, a series of novel and cytotoxic 4-thiazolidinone amides were
prepared
and identified. Among this series, detailed structure activity relationship
studies of type I
compounds (Figure 6) were performed to evaluate their antiproliferative
activity against five
prostate cancer cell lines and RH7777 cells (negative controls). The
cytotoxicity study shows
that the antiproliferative activity is sensitive to 2-aryl ring substitutions,
the length of the
alkyl side chain, and the removal or replacements of the lipophilic allcyl
side chain. Sulfur
oxidation is well tolerated as compounds 80 and 81 showed significant
cytotoxicity compared
to 5-FU. This study resulted in the discovery of potent cytotoxic 4-
thiazolidinones
(compounds 68, 80, and 81), which inhibit the growth of all five human
prostate cancer Bell
lines (DU-145, PC-3, LNCaP, PPC-l, and TSU) with 2-5-fold lower selectivity
compared to
RH7777 cell line. These 4-thiazolidinone derivatives are a significant
improvement on the
SAP moiety in that they are less cytotoxic but demonstrated improved
selectivity in non-
tumor cells.
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Example 8 - Cytotoxicity Assax in Breast and Ovarian Cancer Cells
The most potent compounds from each structural formula were selected and
tested for
their growth inhibitory activity in a human breast cancer cell line (MCF-7)
and three human
ovarian cancer cell lines (CHO-I, CaOv-3, SKOv-3, and OVCAR-3). Ih
vitf°o cytotoxicity
assay was performed by the same sulforhodamine B (SRB) assay (described
above). The
compounds shown in Table 8 below where tested for activity against the breast
cancer and
ovarian cancer cell lines.
Table 8: Antiproliferative effects of compounds on breast and ovarian cancer
cell lines
ICso (!~M)
Compd a
MCF-7 CHO-1 CaOv-3 OVCAR-3 SKOv-3
3-HCI 50.3 NT 19.2 34.0 47.8
4-HCI 2.4 NT 13.9 1.6 2.1
5-HCI (R) 4.2 NT 2.5 4.5 8.5
5-HCI (S) 7.4 NT 18.0 5.2 18.0
6-HCI >20 NT NT NT NT
7 10.4 NT NT NT NT
8 ~20 NT NT NT NT
9 18.7 NT NT NT NT
10 1-/7 NT NT NT NT
11 9.3 NT NT NT NT
12 NT NT 7.7 2.3 5.4
13 13.5 NT NT NT NT
14-HCI NT NT 18.3 8.1 11.0
IS-HCI 16.3 NT NT NT NT
16-HCI NT NT 5.5 1.2 3.6
17-HCI NT NT 4.4 1.4 2.7
18-HCI NT NT 4.9 2.0 2.6
19 8.8 NT 5.5 2.3 4.2
16.6 NT NT NT NT
21 16.3 NT NT NT NT
24 17.7 NT NT NT NT
15.3 NT NT NT NT
26 10.3 NT NT NT NT
27-HCI >20 NT NT NT NT
28 16.3 NT NT NT NT
29 >20 NT NT NT NT
34 >20 NT NT NT NT
66 13.5 21.0 NT NT NT
67 8.9 11.4 NT NT NT
68 15.4 23.5 NT NT NT
69 >20 >20 NT NT NT
70 >20 >20 NT NT NT
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71 13.0 15.2 NT NT NT
72 ~3 0 >3 0 NT NT NT
80 14.3 11.6 NT NT NT
81 8.9 9.8 NT NT NT
aBreast cancer cell line. °Ovarian cancer cell lines. NT=not tested.
Stereoselectivity of compound S was observed (compare the (R) and (S) isomers)
in
CaOV-3 and SKOv-3 cells. Substitutions on 2-phenyl ring generally increased
cytotoxicity of
the compounds.
Exam lp a 9 - Synthesis and Testing-of Spermine-conjugated Thiazolidine Amide
As illustrated in Figure 9, a mixture of 4-thiazolidinone acid (where Rl is
phenyl and
1 is 1) (1.5 g, 6.32 m mol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride
(1.51 g, 7.9 m mol) and 1-hydroxybenzotriazole (0.85 g, 6.32 rn mol) in CH2C12
was cooled
in an ice bath was stirred for 10 min. To this solution 4-nitrophenol (0.78 g,
5.61 m mot) was
added and stirred for 2h. The reaction mixture was diluted with CH2Clz washed
sequentially
with cold 5% HCt, saturated NaHCO3, water, brine, dried (anhydrous Na2S04) and
solvent
was removed ira vacuo. The nitrophenyl ester product (compound 100) was
purified by flash
chromatography (silica gel) using EtOAc/Hexanes to afford 1.76 g (78%). 1H NMR
(CDCI3)
8 3.70 (d, J=18 Hz, 1H), 3.85 (d, J--1.2 Hz, 2H), 4.64 (d, J--17.7 Hz, IH),
5.88 (s, 1H), 7.24
(d, J=2.1 Hz, 1H), 7.26 (d, .I--2.4 Hz, 1H), 7.40-7.46 (m, 5H), 8.26 (d, J--
1.8 Hz, 1H), 8.28 (d,
J--2.1 Hz, 1H).
To a solution of the nitrophenyl ester (compound 100) (0.5 g, 1.39 m mol) in
CH3OH
(35 mL) at room temperature, a solution of spennine (0.33 g, 1.63 m mol, in
CH30H) was
added slowly and stirred for lh. The reaction mixture was concentrated in
vacuo, and to the
concentrated reaction mixture 1:1 (CHC 13: CH30H) was added and filtered
through celite.
Solvent was removed in vacuo and the residue was purified by flash column
chromatography
(silica gel) using CHC13:CH30H/i-PrNH2 to give 0.2 g (50%) of spermine
conjugate
(compound 101), which was converted to the corresponding hydrochloride salt
using 2M
HCI/Et20. jH NMR (DMSO-d6) S I.71-I.76 (m, 6H), L95-2.0 (m, 2H), 2.89-3.0 (m,
lOH),
3.0-3.15 (m, 4H), 3,74 (d, J--15.6 Hz, 1H), 3.87 (d, J--15.3 Hz, IH), 4,10(d,
J--16.5 Hz, IH),
7.35-7.44 (m, 5H), 8.0-8.18 (m, 4H), 8.89 (Ins, 2H), 9.15 (brs, 2H). ESI1VIS
m/z 422.4
(M++1).
Compound 101 demonstrated more potent activity against prostate cancer cells
compared to ovarian and MCF-7 breast cancer cells, with ICSO (~.M) values as
follows:
51
CA 02559333 2006-09-11
WO 2005/086638 PCT/US2005/004759
RH7777 (>100), DU145 (12.4), PC-3 (11.1), LNCaP (26.2), PPC-1 (11.7), TSU-Prl
(5.0),
MCF-7 (>100), CaOv-3 (39.3), OVCAR-3 (39.7), and SKOv-3 (>100).
Example 10
The antiproliferative effects of 3, 4, SR, and SS were compared to that
observed with
four active serine amide phosphates (SAPs) derivatives and 5-fluorouracil (5-
FU, positive
control) in human prostate cancer cell lines (PC-3, DU 145, LNCaP, PPG-l, TSU-
Prl). A
control cell line (RH7777) that does not express LPL receptors4 and MCF-7 (a
human breast
cancer cell line) was included to gauge their selectivity. The chosen cell
lines represent
different basal levels of active AKT and LPL receptor expression (discussed
later). Cells
were exposed to a wide range of concentrations (0 to 100 ~M) of the indicated
compound for
96 h. Cell numbers at the end of treatment were measured using the
sulforhodamine B (SRB)
assays. ICso (i.e., concentration that inhibited cell growth by 50% of
untreated control) values
were obtained by nonlinear regression analysis (WinNonlin, Pharsight Corp.).
As previously observed in our laboratory, the SAP derivatives (compounds S1-S4
in
the Table below were potent inhibitors of tumor cell proliferation with ICso
values ranging
from 1.1 to ~ 20 ~M (ND= not determined).
Differences in SAP and thiazolidine antiproliferative activity were observed.
The
thiazolidine derivatives (3, 4, 5R, and SS) also potently inhibited prostate
and breast cancer
cell growth, but were 2- to 12-fold less potent in LPL receptor negative
RH7777 cells,
suggesting that thiazolidine analogs demonstrate more potent and selective
antiproliferative
activity. Two important structure-activity relationships were suggested in
this small series of
compounds. First, analogs containing long allcyl chains (i.e., C18; 5R, and
SS) were more
potent and selective than derivatives with shorter allcyl chain lengths (i.e.,
C7 and C14; 3 and
4). Secondly, the ICSO for the R-isomer (SR) were less than the ICSO for the S-
isomer (5S) in
all of the tumor cell lines, except for RH7777. This suggests a stereospecific
interaction with
a molecular target that is absent or less critical in RH7777 cells.
hnportantly, analogs 4, SR,
and SS were as potent inhibitors of tumor cell proliferation as 5-FU, and were
measurably
better in many cell lines.
52
CA 02559333 2006-09-11
WO 2005/086638 PCT/US2005/004759
m u~ S~triicture
RH7777 DU PC-3 NCaF = P~?C=1TSI7 ' MCF-7'"
y 145 ~~ . Fxl
~
k. .' " ~
' *.,Y
M . ~ . 7
v. ~' ' ~ ~~ Y~ .,
. _ '
y ~ 5 15 5.8 1 5 5
7 3 8 0 8
HO-P-O~H-C~H3e . . . . .
OH NH
S2 II
ND 10.8 ~20 2.6 1.6 11.1 ~20
HO-~P-O~N-C
H
Z
4~
+H
OH NH
3
S3 H~~~~ H" 2.5 3.2 2.4 3.3 1.6 1.1 2.9
S4 Ho~'hN~-czH",2.9 4.1 2.6 5.1 1.9 2.2 4.6
HN ~'ILN-
CIN
H
.
3 C~ 52.2 44.9 38.5 12.4 34.7 28.0 50.3
iS
7 H
~S
I~
CIH
HN ~~ILN-
H
.
4 C~q 3.4 2.4 3.0 1.4 1.3 2.0 4.2
29
7 H
~S
I~
~L
.,
5R - ie 3~ 25.6 5.4 7.8 2.1 2.0 5.0 4.2
7 ~ c H
~
I s
5S ~IH.HN~H-~~H3~19.1 7.1 > 6.3 4.0 ~10 7.4
J 10
s
5-FU ND 11.9 12.0 4.9 6.4 3.6 ND
Example 11
The cytotoxicity of thiazolidine and SAP derivatives in five human prostate
cancer
cell lines (DU-145, PC-3, LNCaP, PPC-1, TSU-Prl) and in a negative control
cell line
(RH7777) that laclcs LPL receptor was examined using the sulforhodamine B
(SRB) assay.
Cells were exposed to a wide range of concentrations (0 to 100 ~,M) of the
particular
compound for 96 h in 96 well plates. Cells were fixed with 10% trichloroacetic
acid, washed
five times with water. The plates were air dried overnight and fixed cells
were stained with
SRB solution. The cellular protein-bound SRB was measured at 540 iun using a
plate reader.
Cell numbers at the end of the treatment were measured. IC50 (i.e.
concentration that
inhibited cell growth by 50% of untreated control) values were obtained by
nonlinear
regression analysis using WinNonlin. For comparative purposes and to
understand the
53
CA 02559333 2006-09-11
WO 2005/086638 PCT/US2005/004759
degree of cytotoxicity 5-fluorouracil (5-F~ was tested against all five
prostate cancer cell
lines. Compounds showing more potent antiproliferative activity will display
low ICso
values comparable to that of 5-fluorouracil.The results are summarized below.
ICso
(wM)
Structure CHO- DU PPC- TSU- MCF-
RH7777 PC-3 LNCaP
KI 145 1 Prl 7
O
Not
OH > 20 ~ 20 16.1 12.6 ~ 16.3
20
C~~H25 g tested20
C~2H25--~O Not > No No > >
On,.
O
- _
O P 'ONH+4 ~ 100 >
100
'ONH+ tested100 toxicitytoxicity100 100
4
O
~'~I~OH
HN Not > > >
> 100 > 100 71.0 ~
100
testedI00 100 100
O
'~I~'
OH Not ~ > >
HN ~ > 100 > 100 ~ 50 ~ 100
~S
tested100 100 100
Me0
O
~'~ILOH
HN Not > > >
w >100 >100 >100 >100
tested100 100 100
F
O
'~~L
OH Not > > >
HN ' >loo >IOO >IOO >IOo
~
s
tested100 100 100
NC
54
CA 02559333 2006-09-11
WO 2005/086638 PCT/US2005/004759
O
NN ''~I~OH Not > > >
> 100 > 100 > 100 > 100
~S
tested 100 100 100
F
O
CI HN '°ILOH Not > > >
> 100 > 100 > 100 > 100
~S
tested 100 100 100
CI
O
HN ''~I~OH Not > >
> 100 > 100 > 100 > 100
~~ ~S
tested 100 100 100
N
/C18H37
HO ~ ~NH/ Not > > >
>100 >100 >100 >100
NHMe tested 100 100 100
HCI
/C1sH37
HO ~ ~NH
2.5 I.7 3.2 2.4 3.3 1.6 1.1 2.9
NHZ
HCI
O O Not >
HO~NH(CH2)12HN~pH 8.5 > 20 12.5 > 20 3.7 10.6
NHBoc NHBoc tested 20
Example I2
Prostate cell line LNCaP cells were treated with 30 ~.M of the compound of
Formula:
/C~aH37
HO NH
CA 02559333 2006-09-11
WO 2005/086638 PCT/US2005/004759
for the indicated period and time. The active form of AI~T (Pi-AKT) and the (3-
Actin were
quantified by Western blot analysis. The compound inhibited AT~T
phosphorylation to 50%
by 12 hours of treatment. The compound of Formula VIII had an ICso equal to
10.3 ~,M. The
results of the experiment are given below.
Treatment with Relative abundance
30 p,M (Pi-AI~T/,~-Actin)
(-) control 1.00
(+) control 2.01
1 h 1.29
2 h 1.01
6 h 0.71
12 h 0.50
24 h 0.32
48 h <0.10
72 h <0.10
Example 13
Prostate cell line LNCaP cells were treated with 10 ~.M of the compound of
Formula:
\ /C18H37
HO 'NH
NHMe
HCI
for the indicated period and time. The active form of AI~T (Pi-AKT) and AKT
were
quantified by Western blot analysis. The compound of Formula IX almost
completely
inhibited AI~T phosphoiylation within 6 hours of treatment. The compound an
ICSO equal to
3.3 ~M. The results of the experiment are given below.
Treatment with Relative abundance
10 ~,M Formula (Pi-AKT/AI~T)
IX
(-) control 1.00
6 h 0.07
12 h 0.31
24 h 1.02
3 6h 0.63
48 h 0.94
56
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WO 2005/086638 PCT/US2005/004759
Exam lp a 14
Prostate cell line LNCaP cells were treated with 10 ~.M of the compound of
Formula
\ /C~sHs7
HO 'NH
NHMe
HCI
for the indicated period and time. The active form of AKT (Pi-AKT), AKT and (3-
Actin were
quantified by Western blot analysis. The compound almost completely inhibited
AKT
phosphorylation by 1 hour of treatment. The compound had an ICSO equal to 3.3
~M. The
results of the experiment are given below.
Treatment with Relative abundanceRelative abundance
~M Formula (Pi-AKT/AKT (Pi-AK
IX ) T/,~-Actin)
(-) control _ _
1.00 1.00
lh 0.07 0.08
2h 0.06 0.06
3h 0.04 0.04
4h 0.12 0.11
6h 0.12 0.10
8h 0.14 0.11
10 h 0.14 0.43
EXAMPLE 15
10 The cytotoxicity of synthesized compounds in five human prostate cancer
cell lines (DU-145,
PC-3, LNCaP, PPC-1, and TSU) and in two negative control cell lines (CHO and
RH7777) was
examined using the sulforhodamine B (SRB) assay (Rubinstein, L. V. S., R.
H.Paull, K. D.Sirnon,
R. M.Tosini, S.Sl~ehan, P.Scudiero, D. A.Monlcs, A.Boyd, M. R. J. Natl.
Ca~r.cer. Inst. 1990, 82,
1113-1118, which is incorporated by reference herein). Cells were exposed to a
wide range of
concentrations {0 to 100 ~.M) of the particular compound for 96 h in 96-well
plates. Cells were
fixed with 10% trichloroacetic acid, washed eve times with water. The plates
were air dried
overnight and fixed cells were stained with SRB solution. The cellulax protein-
bound SRB was
measured at 540 nm using a plate reader. Cell numbers at the end of the
treatment were calculated
as a percentage of untreated control. ICso (i.e. concentration that inhibited
cell growth by 50% of
untreated control) values were obtained by nonlinear regression analysis using
WinNonlin. For
57
CA 02559333 2006-09-11
WO 2005/086638 PCT/US2005/004759
comparative purposes and to understand the degree of cytotoxicity 5-
fluorouracil was tested
against all five prostate cancer cell lines. The results are summarized in
Table 1.
From the cytotoxicity data it is clear that most of the compounds tested
showed good
anticancer activity against aII five prostate cancer cell lines. SAAs (306b,
306e, 306 without a
phosphate group are as effective as SAPs. A direct relationship was observed
between length of the
alkyl chain and cytotoxicity of the tested compounds. Accordingly, all of
these compounds showed
an allcyl chain length dependent cytotoxicity. Compounds with shorter alkyl
chains (302a, 306b,
315d, 316d) are less cytotoxic than analogues with longer alkyl chains (see
Table 1). Compound
302f emerged as one of the most potent SAPS tested so far with an ICso of 1.8
~,M against PPC-1
cell line. However, SAAB are more potent than corresponding SAPS when the
alkyl chain length is
below 18C, but no significant difference in the cytotoxicity is observed
between SAAs and SAPS
with alkyl chain more than I BC. ICSO values for enantiomers of SAAs (306c,
306d) and SAPS
(302b, 302c) are approximately equivalent which suggests that chirality is not
important for the
antiproliferative activity of these compounds in prostate cancer. Introduction
of a double bond in
the alkyl chain lowered the potency of both SAA 309 and SAP 311. To understand
the importance
of the amine functionality we derivatized the amine group to the corresponding
Set B amide,
sulfonamide and urea derivatives. Serine diamide phosphate 316d with a shorter
allcyl chains failed
to demonstrate cytotoxicity below 100 ~,M in four prostate cancer cell lines
except TSU prostate
cell line. The inhibitory activity of sulfonamide derivatives 315b and 316b
and urea derivative
315c in all five prostate cancer cell lines showed a general decreasing trend
suggesting that
derivatization of C2 amine group is not tolerable for their ability to kill
prostate cancer cells.
To further investigate the extent of structural tolerance permitted in the
serine amide
backbone region we replaced the serine amide group with simple ethanolamine
amide by
synthesizing compounds 319 and 320. However, these ethanolamine amide analogs
were less
potent and particularly compound 319 did not show any activity against DU-145,
PC-3, and
LNCaP prostate cancer cell lines. When the amide group in SAAB was reduced to
produce long
chain N-allcyl amino alcohols 317 and 318, these analogues retained
cytotoxicity and were very
effective in killing prostate cancer cell lines with low micromolar
cytotoxicity. To determine the
selectivity, several of sy~zthesized compounds were also examined for their
cytotoxicity in CHO
and RH7777 cells as negative controls. Many of the potent compounds showed
similar cytotoxicity
and were non selective in their action against prostate cancer cell lines and
non-tumor negative
control cells.
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Table 9: ICso (p.M) of various compounds
0 0
H
RIO NHRz R~O~NHRZ R~O~NHRZ R~O~N~Rz
Set A3 NHR3 NHZ R3+ O
Set B Set C Set D
ICso
(
M)
Compd
Set Rl Rz R3 CHO ~ DU- PC-3 LNCa PPC_1 TSU
7777 145 P
302a
(2R) P03H CloHz1- ND ND 50.2 36.0 44.7 22.1 31.5
302b(2R)POSH CI4Hz9- ND ND 20.6 >50 10.1 >10 >10
302c(2S)P03H C14Hz9- ND ND 32.0 >50 19.7 >10 >10
302d(2R)P03H C18H3~- ND ND 11.7 19.1 7.2 5.6 4.8
302e(2R)POSH C19Hs9- 3.7 ND 5.7 15.3 5.8 1.8 5.0
302f(2S)POSH CzoH1 - 7.8 ND 10.8 >20 3.6 1.8 11.1
306a(2S)H C8H,7 - >100 ND >100 >100 >100 >100 >100
306b(2R)H CloHzi- ND ND 52.2 35.0 31.0 15.9 26.0
306c(2R)H C14Hz9- ND ND 8.2 10.2 8.1 6.3 7.5
306d(2S)H C14Hz9- ND ND 6.9 10.3 10.0 6.2 9.2
306e(2R)H C18H3~- 2.5 2.6 5.4 5.2 3.8 2.2 4.4
306f(2R)H C,9H39- 2.4 3.2 5.1 5.3 5.3 1.8 3.9
306g(2S)H CzoHdl- 4.1 ND 7.0 6.6 3.9 2.6 6.6
CsHm_
309(2S) H CH:CH-- 5.2 6.8 6.9 5.9 6.6 5.1 5.5
CsHis
CBH,~-
311(2S) P03H cH:CH-- 11.9 28.6 16.0 39.2 12.2 21.1 12.4
CeH~s
314a(2S)OBn C,$H3~H 3.0 9.9 11.2 6.2 10.9 2.9 6.8
314b(2S)OBn Cl$H3~SOZMe >50 >50 >50 47.3 A .~e 16.7 >50
CO
314c(2S)OBn C18H3~NH 18.5 >20 20 >20 >20 >20 15.9
Ph
(3,5-
difluoro
314d(2S)OBn C$Hl~ COC,H,S9.2 12.9 22.9 31.3 35.0 4.0 10.0
315b(2S)H C18H3~SOZMe 12.9 9.2 23.1 13.6 16.0 10.2 20.5
CO
315c(2S)H C18H3~NH 20 >20 20 >20 >20 >20 15.3
Ph
(3,5-
difluoro
315d(2S)Ii C$Hl~ COC,H,s>100 ND >100 81.5 >100 81.2 93.8
316b(2S)POSH C18H3~SOzMe >50 50 43.2 >50 15.1 17.8 35.7
316d(2S)P03H C8H1~ CoC,H,s>100 >100 >100 >100 A .~e >100 79.0
317(2R) H CI8H3~H 2.2 2.9 4.1 2.6 5.1 1.9 2.2
318(2R) H Cl$H3~Me 1.7 2.5 3.2 2.4 3.3 1.6 1.1
59
CA 02559333 2006-09-11
WO 2005/086638 PCT/US2005/004759
c$H"- Not Not Not Not
319 H CH:CH-- >20 >20 >20
ActiveActiveActive Active
C
H
ia -
~
CsHir
320 P03H CH:CH-- >50 >50 >50 >50 50 >50
C~Hm Active
5-FU - - - - - 11.9 12.0 4.9 6.4 3.6
Although various embodiments have been depicted and described in detail
herein, it
will be apparent to those skilled in the relevant art that various
modifications, additions,
substitutions, and the like can be made without departing from the spirit of
the invention and
these are therefore considered to be within the scope of the invention as
defined in the claims
which follow.
