U.S. patent application number 09/748622 was filed with the patent office on 2002-08-29 for telomerase inhibitors and methods of their use.
Invention is credited to Akama, Tsutomu, Holcomb, Ryan, Tolman, Richard L..
Application Number | 20020120144 09/748622 |
Document ID | / |
Family ID | 25010219 |
Filed Date | 2002-08-29 |
United States Patent
Application |
20020120144 |
Kind Code |
A1 |
Akama, Tsutomu ; et
al. |
August 29, 2002 |
Telomerase inhibitors and methods of their use
Abstract
Thiazolidinedione compounds, compositions, and methods of
inhibiting telomerase activity in vitro and treatment of telomerase
mediated conditions or diseases ex vivo and in vivo are provided.
The methods, compounds and compositions of the invention may be
employed alone, or in combination with other pharmacologically
active agents in the treatment of conditions or diseases mediated
by telomerase activity, such as in the treatment of cancer. Also
disclosed are methods for assaying or screening for inhibitors of
telomerase activity.
Inventors: |
Akama, Tsutomu; (Redwood
City, CA) ; Holcomb, Ryan; (San Carlos, CA) ;
Tolman, Richard L.; (Los Altos, CA) |
Correspondence
Address: |
GERON CORPORATION
230 CONSTITUTION DRIVE
MENLO PARK
CA
94025
|
Family ID: |
25010219 |
Appl. No.: |
09/748622 |
Filed: |
December 22, 2000 |
Current U.S.
Class: |
546/114 ;
544/350; 546/115; 546/117 |
Current CPC
Class: |
C07D 417/14 20130101;
C07D 417/10 20130101 |
Class at
Publication: |
546/114 ;
546/115; 546/117; 544/350 |
International
Class: |
C07D 498/02; C07D
491/02; C07D 471/02 |
Claims
We claim:
1. A compound comprising the formula: 69wherein X is O or S;
L.sub.1 is a direct bond, --CHR.sub.1--, or .dbd.CR.sub.1--,
wherein R.sub.1 is H or alkyl; A is aryl or heteroaryl; L.sub.2 is
a direct single bond or a linking group having from 1 to 3 atoms
independently selected from unsubstituted or substituted carbon, N,
O or S; W is selected from the group consisting of 0, NR.sub.5, and
S, wherein R.sub.5 is selected from the group consisting of H,
alkyl, aryl, and aralkyl; Y and Z are independently selected to be
C or N; and R.sub.2, and R.sub.3 are independently selected from
the group consisting of hydrogen, hydroxyl, halogen, alkyl, aryl,
alkoxyl, cyano, nitro, alkylthio, arylthio, aralkyl, and
heteroaryl; or a pharmaceutically acceptable salt thereof.
2. A compound of claim 1, wherein X is O.
3. A compound of claim 2, wherein L.sub.1 is a direct bond.
4. A compound of claim 2, wherein R.sub.1 is H.
5. A compound of claim 2, wherein A is selected from the group
consisting of aryl, substituted or unsubstituted pyridine,
substituted or unsubstituted thiophene, substituted or
unsubstituted furan, substituted or unsubstituted naphthalene,
substituted or unsubstituted quinoline, and substituted or
unsubstituted indole.
6. A compound of claim 5, wherein A is selected from the group
consisting of phenyl, naphthalene, pyridine, indole, quinoline,
furan, or thiophene.
7. A compound of claim 2, wherein W is NR.sub.5.
8. A compound of claim 7, wherein R.sub.2 and R.sub.3 are
halogen.
9. A compound of claim 8, wherein at least one of R.sub.2, R.sub.3,
or R.sub.5 is: 70wherein n is 0 or 1.
10. A compound comprising the formula: 71wherein L.sub.1 is a
direct bond, --CHR.sub.1--, or .dbd.CR.sub.1--, wherein R.sub.1 is
H or alkyl; A is aryl or heteroaryl; L.sub.2 is a direct single
bond or a linking group having from 1 to 3 atoms independently
selected from unsubstituted or substituted carbon, N, O or S; W is
selected from the group selected from O, N R.sub.5, and S, wherein
R.sub.5 is selected from the group consisting of H, alkyl, aryl,
and aralkyl; Y and Z are independently selected to be C or N; and
R.sub.2 and R.sub.3 are independently selected from the group
consisting of hydrogen, hydroxyl, halogen, alkyl aryl, alkoxyl,
cyano, nitro, alkylthio, arylthio, aralkyl, and heteroaryl; or a
pharmaceutically acceptable salt thereof.
11. A compound of claim 10, wherein W is NR.sub.5.
12. A compound of claim 11, wherein R.sub.5 is selected from the
group consisting of methyl, ethyl, propyl, butyl, allyl,
ethoxycarbonylmethyl, and 2-(dimethylamino)ethyl.
13. A compound of claim 11, wherein R.sub.5 is selected from the
group consisting of benzyl, 3,4-dichlorobenyzl, 4-formylbenzyl,
4-methoxycarbonylbenzyl, 2-naphtylmethyl, and
5-chlorothiophen-2-ylmethyl- .
14. A compound of claim 10, wherein at least one of R.sub.2,
R.sub.3, or R.sub.5 is: 72wherein n is 0 or 1.
15. A compound of claim 10, wherein L.sub.1 is .dbd.CH--, L.sub.2
is a direct bond, W is S, Y is CH, and Z is CH.
16. A compound comprising the formula: 73wherein L.sub.1 is a
direct bond, --CHR.sub.1--, or .dbd.CR.sub.1--, wherein R.sub.1 is
H or alkyl; A is aryl or heteroaryl; L.sub.2 is a direct single
bond or a linking group having from 1 to 3 atoms independently
selected from unsubstituted or substituted carbon, N, O or S; Y and
Z are independently selected to be C or N; and R.sub.2, R.sub.3 and
R.sub.4 are independently selected from the group consisting of
hydrogen, hydroxyl, halogen, alkyl, aryl, alkoxyl, cyano, nitro,
alkylthio, arylthio, aralkyl, and heteroaryl; or a pharmaceutically
acceptable salt thereof.
17. A compound of claim 16, wherein R4 is H, methyl, phenyl, or
SCH.sub.3.
18. A compound of claim 16, wherein at least one of R.sub.2,
R.sub.3, or R.sub.4 is: 74wherein n is 0 or 1.
19. A compound of claim 2, wherein A is aryl, L.sub.1 and L.sub.2
are direct bonds, W is NR.sub.5 and R.sub.2 and R.sub.3 are
halogen.
20. A compound of claim 19, wherein A is phenyl, pyridine, indole,
quinoline, furan, or thiophene.
21. A compound of claim 20, wherein R.sub.5 is hydrogen, and
R.sub.2 and R.sub.3 are chloride.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to thiazolidinone compounds
that inhibit telomerase activity, to pharmaceutical compositions
containing the compounds and to the use of the compounds and
compositions, alone or in combination with other pharmaceutically
active agents, in the treatment of telomerase-mediated conditions
or diseases, such as cancer.
BACKGROUND OF THE INVENTION
[0002] Telomerase catalyzes the synthesis of telomeres. Telomeres
are characteristic tandem repeats (TTAGGG in mammals) found at the
ends of most eukaryotic chromosomes, that may be 15-25 kilobases
long in human germline cells. With each cell division, about 60-100
bases are lost from the ends of the chromosomes, and as the
telomeres shorten, cells eventually reach crisis and apotosis is
triggered. See Harley et al., (1991) Mutation Res. 256: 271-282.
Telomerase acts to maintain the telomere length just above the
crisis level, and are thus responsible for chromosome stability and
are involved in the regulation of the cell cycle.
[0003] Telomerase is a ribonucleoprotein reverse transcriptase that
contains its own RNA template for the synthesis of telomeric DNA.
See Blackburn, 1992, Annu. Rev. Biochem., 61:113-129. Telomerase is
present in stem and germline cells of normal tissues, and at much
higher levels in over 85% of tumors (Kim et al., 1994, Science,
266:2011-2014). Thus, drugs targeted towards telomerase potentially
will have a high selectivity for tumor over healthy tissues.
Consequently, telomerase inhibition has been proposed as a new
approach to cancer therapy.
[0004] The inhibition of telomerase activity by antisense
strategies directed towards the telomerase RNA component, for
example peptide nucleic acids (Norton et al., (1996) Nature
Biotech. 14: 615-619) and phosphorothioate oligonucleotides has
been reported. Since telomerase is a reverse transcriptase, the use
of inhibitors of reverse transcriptases, such as AZT, and other
nucleosides has also been reported. Telomerase inhibition by
cisplatin, possibly due to crosslinking of the telomeric repeat
sequences, is also known (Burger et al., (1997) Eur. J. Cancer 33:
638-644).
[0005] We are interested in inhibitors of telomerase that are small
molecules, such as thiazolidinediones (see U.S. Ser. No.
09/608,636). Thiazolidinediones comprise a group of structurally
related antidiabetic compounds that increases the insulin
sensitivity of target tissues (skeletal muscle, liver, adipose) in
insulin resistant animals. In addition to these effects on
hyperglycemia, thiazolidinediones also reduce lipid and insulin
levels in animal models of NIDDM. Recently, the thiazolidinedione
troglitazone was shown to have these same beneficial effects in
human patients suffering from impaired glucose tolerance, a
metabolic condition that precedes the development of NIDDM, as in
patients suffering from NIDDM (Nolan et al., (1994) N. Eng. J. Med.
331, 1188-1193). While their mechanism of action remains unclear,
it is known that the thiazolidinediones do not cause increases in
insulin secretion or in the number or affinity of insulin receptor
binding sites, suggesting that thiazolidinediones amplify
post-receptor events in the insulin signaling (Colca, J. R., and
Morton, D. R. (1990) in New Antidiabetic Drugs (C. J. Bailey and P.
R. Flatt, eds.). Smith-Gordon, New York, 255-261; Chang et al.
(1983) Diabetes 32, 839-845).
[0006] Thiazolidinediones have been found to be efficacious
inducers of differentiation in cultured pre-adipocyte cell lines
(Hiragun et al. (1988) J. Cell Physiol. 134, 124-130; Sparks et al.
(1991) J. Cell. Physiol. 146, 101-109; Kleitzien et al. (1992) Mol.
Pharmacol. 41, 393-398). Additionally, thiazolidinediones have been
implicated in appetite regulation disorders (see WO 94/25026 A1),
and in increase of bone marrow fat content. In addition,
thiazolidinedione compounds have been suggested for use in the
treatment of psoriasis (U.S. Pat. No. 5,824,694) and climacteric
symptoms and mesenchymal tumors (U.S. Pat. No. 5,814,647).
[0007] The identification of compounds that inhibit telomerase
activity provides important benefits to efforts at treating human
disease. Compounds that inhibit telomerase activity can be used to
treat telomerase-mediated disorders, such as cancer, since cancer
cells express telomerase activity and normal human somatic cells do
not possess telomerase activity at biologically relevant levels
(i.e., at levels sufficient to maintain telomere length over many
cell divisions). Unfortunately, few such compounds, especially
compounds with high potency or activity and compounds that are
orally bioavailable, have been identified and characterized. Hence,
there remains a need for compounds that act as telomerase
inhibitors that have relatively high potency or activity and that
are orally bioavailable, and for compositions and methods for
treating cancer and other diseases in which telomerase activity is
present abnormally. The present invention meets these and other
needs.
SUMMARY OF THE INVENTION
[0008] The present invention provides methods, compounds and
compositions that are specific and effective for treating
telomerase-mediated disorders, such as malignant conditions by
targeting cells having telomerase activity. The methods, compounds,
and compositions of the invention can be applied to a wide variety
of malignant cell types and avoid the problems inherent in current
cancer treatment modalities which are non-specific and excessively
toxic.
[0009] In a first aspect, the present invention is based on the
finding that thiazolidinone compounds are effective in the
inhibition of telomerase enzyme activity, in vitro, ex vivo and in
vivo. Thus, in certain aspects, the present invention provides
methods of inhibiting telomerase by contacting telomerase with the
compounds described herein. In particular embodiments, the
telomerase to be inhibited is a mammalian telomerase, such as a
human telomerase. A related aspect of the present invention is the
discovery that thiazolidinone compounds inhibit the proliferation
of cells that have telomerase activitiy, such as many cancer cells.
Thus, this aspect of the present invention provides methods of
inhibiting telomerase activity in a patient, preferably a mammal,
suffering from a telomerase-mediated condition or disease,
comprising administering to the patient a therapeutically effective
amount of a telomerase inhibiting thiazolidinone compound, or a
pharmaceutically acceptable salt thereof.
[0010] In another aspect, the present invention provides compounds
having the formula: 1
[0011] or their pharmaceutically acceptable salts, wherein X is O
or S; L.sub.1 is a direct bond, --CHR.sub.1--, or .dbd.CR.sub.1--,
wherein R.sub.1 is H or alkyl; L.sub.2 is a direct bond or a
linking group having from 1 to 3 atoms independently selected from
unsubstituted or substituted carbon, N, O or S; A is aryl or
heteroaryl; W is selected from the group consisting of O, NR.sub.5,
and S, wherein R.sub.5 is selected from the group consisting of H,
alkyl, aryl, and aralkyl; Y and Z are independently selected to be
C or N; and R.sub.2 and R.sub.3 are independently selected from the
group consisting of hydrogen, hydroxyl, halogen, alkyl, aryl,
alkoxyl, cyano, nitro, alkylthio, arylthio, aralkyl, and
heteroaryl. The compounds find use in methods and compositions for
inhibiting a telomerase enzyme, where the telomerase enzyme is
contacted with a compound or a composition containging the compound
of the invention.
[0012] In another aspect, the present invention provides compounds,
or their pharmaceutically acceptable salts, having the formula:
2
[0013] wherein L.sub.1 is a direct bond, --CHR.sub.1--, or
.dbd.CR.sub.1--,wherein R.sub.1 is H or alkyl; A is aryl or
heteroaryl; L.sub.2 is a direct bond or a linking group having from
1 to 3 atoms independently selected from unsubstituted or
substituted carbon, N, O or S; W is selected from the group
consisting of O, NR.sub.5, and S, wherein R.sub.5 is selected from
the group consisting of H, alkyl, aryl, and aralkyl; Y and Z are
independently selected to be C or N; and R.sub.2 and R.sub.3 are
independently selected from the group consisting of hydrogen,
hydroxyl, halogen, alkyl, aryl, alkoxyl, cyano, nitro, alkylthio,
arylthio, aralkyl, and heteroaryl
[0014] In another aspect, the present invention provides a compound
of formula: 3
[0015] or their pharmaceutically acceptable salts, wherein L.sub.1
is a direct bond, --CHR.sub.1--, or .dbd.CR.sub.1--, wherein
R.sub.1 is H or alkyl; A is aryl or heteroaryl; L.sub.2 is a direct
bond or a linking group having from 1 to 3 atoms independently
selected from unsubstituted or substituted carbon, N, O or S Y and
Z are independently selected to be C or N; and R.sub.2, R.sub.3 and
R4 are independently selected from the group consisting of
hydrogen, hydroxyl, halogen, alkyl, aryl, alkoxyl, cyano, nitro,
alkylthio, arylthio, aralkyl, and heteroaryl
[0016] The new compounds of the invention have many valuable uses
as inhibitors of deleterious telomerase activity, such as, for
example, in the treatment of cancer in mammals, such as humans. The
pharmaceutical compositions of this invention can be employed in
treatment regimens in which cancer cells are killed, in vivo, or
can be used to kill cancer cells ex vivo. Thus, this invention
provides therapeutic compounds and compositions for treating
cancer, and methods for treating cancer and other
telomerase-mediated conditions or diseases in humans and other
mammals (e.g., cows, horses, sheep, steer, pigs and animals of
veterinary interest such as cats and dogs).
DETAILED DESCRIPTION
I. Definitions
[0017] Unless otherwise defined below, the terms used herein have
their normally accepted scientific meanings. Definition of standard
chemistry terms may be found in reference works, including Carey
and Sundberg (1992) "Advanced Organic Chemistry 3.sup.rd Ed." Vols.
A and B, Plenum Press, New York.
[0018] The term "thiazolidinone" or "thiazolidinone derivative" as
used herein refers to compounds of the general formula: 4
[0019] wherein X is O or S. When X is O, the derivatives are
thiazolidinedione derivatives. When X is S, the derivatives are the
thiazolidinonethione derivatives also known as rhodanines.
[0020] The term "alkyl" as used herein refers to a straight,
branched, or cyclic hydrocarbon chain fragment or radical
containing between about one and about twenty carbon atoms, more
preferably between about one and about ten carbon atoms (e.g.,
methyl, ethyl, n-propyl, iso-propyl, cyclopropyl, n-butyl,
iso-butyl, tert-butyl, cyclobutyl, adamantyl, noradamantyl and the
like). Straight, branched, or cyclic hydrocarbon chains having
eight or fewer carbon atoms will also be referred to herein as
"lower alkyl". The hydrocarbon chains may further include one or
more degrees of unsaturation, i.e., one or more double or triple
bonds (e.g., vinyl, propargyl, allyl, 2-buten-1-yl,
2-cyclopenten-1-yl, 1,3-cyclohexadien-1-yl, 3-cyclohexen-1-yl and
the like). Alkyl groups containing double bonds such as just
described will also be referred to herein as "alkenes". Similarly,
alkyl groups having triple bonds will also be referred to herein as
"alkynes". However, as used in context with respect to cyclic alkyl
groups, the combinations of double and/or triple bonds do not
include those bonding arrangements that render the cyclic
hydrocarbon chain aromatic.
[0021] In addition, the term "alkyl" as used herein further
includes one or more substitutions at one or more carbon atoms of
the hydrocarbon fragment or radical. Such substitutions include,
but are not limited to: aryl; heterocycle; halogen (to form, e.g.,
trifluoromethyl, --CF.sub.3); nitro (--NO.sub.2); cyano (--CN);
hydroxyl (also referred to herein as "hydroxy"), alkoxyl (also
referred herein as alkoxy) or aryloxyl (also referred to herein as
"aryloxy")(--OR); thio or mercapto, alkyl- or arylthio (--SR);
amino, alkylamino, arylamino, dialkyl- or diarylamino, or
arylalkylamino (--NRR'); aminocarbonyl, alkylaminocarbonyl,
arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl or
arylalkylaminocarbonyl (--C(O)NRR'); carboxyl, or alkyl- or
aryloxycarbonyl (--C(O)OR); carboxaldehyde, or aryl- or
alkylcarbonyl (--C(O)R); iminyl, aryl- or alkyliminyl
(--C(.dbd.NR)R'); sulfo (--SO.sub.2OR); alkyl- or arylsulfonyl
(--SO.sub.2R); ureido (--HNC(.dbd.O)NRR'); or thioureido
(--HNC(.dbd.S)NRR'); where R and R' independently are hydrogen,
aryl or alkyl as defined herein. Substituents including
heterocyclic groups (i.e., heterocycle, heteroaryl, and
heteroaralkyl) are defined by analogy to the above-described terms.
For example, the term "heterocycleoxy" refers to the group --OR,
where R is heterocycle as defined below.
[0022] The alkyl moiety of "lower alkanoyl", "lower alkoxy", "lower
alkanoyloxy", "lower alkylthio", is the same as "alkyl" defined
above.
[0023] The term "methylene" refers to the group --CH.sub.2--.
[0024] The term "methine" refers to a methylene group for which one
hydrogen atom has been replaced by a substituent as described
above. The term "methine" can also refer to a methylene group for
which one hydrogen atom is replaced by bond to form an sp.sup.2
-hybridized carbon center (i.e., >C.dbd.O).
[0025] The term "halo" or "halogen" as used herein refers to the
substituents fluoro, bromo, chloro, and iodo.
[0026] The term "carbonyl" as used herein refers to the functional
group --C(O)--. However, it will be appreciated that this group may
be replaced with well-known groups that have similar electronic
and/or steric character, such as thiocarbonyl (--C(S)--); sulfinyl
(--S(O)--); sulfonyl (--SO.sub.2--), phosphonyl (--PO.sub.2--), and
methylidene (--C(.dbd.CH.sub.2)--). Other carbonyl equivalents will
be familiar to those having skill in the medicinal and organic
chemical arts.
[0027] The term "aryl" as used herein refers to cyclic aromatic
hydrocarbon chains having twenty or fewer carbon atoms, e.g.,
phenyl, naphthyl, biphenyl and anthracenyl. One or more carbon
atoms of the aryl group may also be substituted with, e.g.: alkyl;
aryl; heterocycle; formyl; halogen; nitro; cyano; hydroxyl, alkoxyl
or aryloxyl; thio or mercapto, alkyl-, or arylthio; amino,
alkylamino, arylamino, dialkyl-, diaryl-, or arylalkylamino;
aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,
dialkylaminocarbonyl, diarylaminocarbonyl or
arylalkylaminocarbonyl; carboxyl, or alkyl- or aryloxycarbonyl;
carboxaldehyde, or aryl- or alkylcarbonyl; iminyl, or aryl- or
alkyliminyl; sulfo; alkyl- or arylsulfonyl; hydroximinyl, or aryl-
or alkoximinyl; ureido; or thioureido. In addition, two or more
alkyl or heteroalkyl substituents of an aryl group may be combined
to form fused aryl-alkyl or aryl-heteroalkyl ring systems (e.g.,
tetrahydronaphthyl). Substituents including heterocyclic groups
(e.g., heterocycleoxy, heteroaryloxy, and heteroaralkylthio) are
defined by analogy to the above-described terms.
[0028] The term "aralkyl" as used herein refers to an aryl group
that is joined to a parent structure by an alkyl group as described
above, e.g., benzyl, .alpha.-methylbenzyl, phenethyl, and the like.
The aralkyl moiety of "aralkylsulfonyl" aralkyloxy is the same as
"aralkyl" defined above.
[0029] The aryl moiety of "aroyl", "arylalkenyl", "arylalkenyl",
"arylsulfonyl", "arylthio", "aryloxy", "arylalkenylsulfonyl",
"arylalkynylsulfonyl" is the same as "aryl" defined above.
[0030] The term "heterocycle" as used herein refers to a cyclic
alkyl group or aryl group as defined above in which one or more
carbon atoms have been replaced by a non-carbon atom, especially
nitrogen, oxygen, or sulfur. Non-aromatic heterocycles will also be
referred to herein as "cyclic heteroalkyl". Aromatic heterocycles
are also referred to herein as "heteroaryl". For example, such
groups include furyl, tetrahydrofuryl, pyrrolyl, pyrrolidinyl,
thienyl, tetrahydrothienyl, oxazolyl, isoxazolyl, triazolyl,
thiazolyl, isothiazolyl, pyrazolyl, pyrazolidinyl, oxadiazolyl,
thiadiazolyl, imidazolyl, imidazolinyl, pyridyl, pyridazinyl,
triazinyl, piperidinyl, morpholinyl, thiomorpholinyl, pyrazinyl,
piperazinyl, pyrimidinyl, naphthyridinyl, benzofuranyl,
benzothienyl, indolyl, indolinyl, indolizinyl, indazolyl,
quinolizinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl,
quinazolinyl, quinoxalinyl, pteridinyl, quinuclidinyl, carbazolyl,
acridiniyl, phenazinyl, phenothiazinyl, phenoxazinyl, purinyl,
benzimidazolyl, benzthiazolyl, and benzoxazolyl.
[0031] The heteroaryl moiety of "heteroarylalkyl",
"heteroarylalkenyl", "heteroarylalkynyl", "heteroarylsulfonyl",
"heteroarylalkylsulfonyl", "heteroarylalkenylsulfonyl",
"heteroarylalkynylsulfonyl", "heteroaryloxy", "heteroarylalkyloxy"
is the same as "heteroaryl" defined above.
[0032] The above heterocyclic groups may further include one or
more substituents at one or more carbon and/or non-carbon atoms of
the heteroaryl group, e.g.: alkyl; aryl; heterocycle; halogen;
nitro; cyano; hydroxyl, alkoxyl or aryloxyl; thio or mercapto,
alkyl- or arylthio; amino, alkyl-, aryl-, dialkyl- diaryl-, or
arylalkylamino; aminocarbonyl, alkylaminocarbonyl,
arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl or
arylalkylaminocarbonyl; carboxyl, or alkyl- or aryloxycarbonyl;
carboxaldehyde, or aryl- or alkylcarbonyl; iminyl, or aryl- or
alkyliminyl; sulfo; alkyl- or arylsulfonyl; hydroximinyl, or aryl-
or alkoximinyl; ureido; or thioureido. In addition, two or more
alkyl substituents may be combined to form fused heterocycle-alkyl
or heterocycle-aryl ring systems. Substituents including
heterocyclic groups (e.g., heterocycleoxy, heteroaryloxy, and
heteroaralkylthio) are defined by analogy to the above-described
terms.
[0033] The term "heterocyclealkyl" refers to a heterocycle group
that is joined to a parent structure by one or more alkyl groups as
described above, e.g., 2-piperidylmethyl, and the like. The term
"heteroaralkyl" as used herein refers to a heteroaryl group that is
joined to a parent structure by one or more alkyl groups as
described above, e.g., 2-thienylmethyl, and the like.
[0034] The compounds of the present invention may be used to
inhibit or reduce telomerase enzyme activity and/or proliferation
of cells having telomerase activity. In these contexts, inhibition
and reduction of the enzyme or cell proliferation refers to a lower
level of the measured activity relative to a control experiment in
which the enzyme or cells are not treated with the test compound.
In particular embodiments, the inhibition or reduction in the
measured activity is at least a 10% reduction or inhibition. One of
skill in the art will appreciate that reduction or inhibition of
the measured activity of at least 20%, 50%, 75%, 90% or 100% may be
preferred for particular applications.
II. Telomerase Inhibitors
[0035] As noted above, the immortalization of cells involves inter
alia the activation of telomerase. More specifically, the
connection between telomerase activity and the ability of many
tumor cell lines, including skin, connective tissue, adipose,
breast, lung, stomach, pancreas, ovary, cervix, uterus, kidney,
bladder, colon, prostate, central nervous system (CNS), retina and
blood tumor cell lines, to remain immortal has been demonstrated by
analysis of telomerase activity (Kim et al.). This analysis,
supplemented by data that indicates that the shortening of telomere
length can provide the signal for replicative senescence in normal
cells (see WO 93/23572), demonstrates that inhibition of telomerase
activity can be an effective anti-cancer therapy. By "inhibition"
is simply meant a reagent, drug or chemical which is able to
decrease the activity of the telomerase enzyme in vitro or in vivo.
Such inhibitors can be readily identified using standard screening
protocols in which a cellular extract or other preparation having
telomerase activity is placed in contact with a potential
inhibitor, and the level of telomerase activity measured in the
presence or absence of the inhibitor, or in the presence of varying
amounts of inhibitor. In this way, not only can useful inhibitors
be identified, but the optimum level of such an inhibitor can be
determined in vitro for further testing in vivo.
[0036] In a related aspect, the invention proves a method for
inhibiting the ability of a cell to proliferate or replicate. In
this method, one or more of the thiazolidinone compounds of the
invention, that are capable of inhibiting telomerase enzyme
activity, are provided during cell replication. As explained above,
telomeres play a critical role in allowing the end of the linear
chromosomal DNA to be replicated completely without the loss of
terminal bases at the 5'-end of each strand. Immortal cells and
rapidly proliferating cells use telomerase to add telomeric DNA
repeats to chromosomal ends. Inhibition of telomerase will result
in the proliferating cells not being able to add telomeres and they
will eventually stop dividing. As will be evident to those of
ordinary skill in the art, this method for inhibiting the ability
of a cell to proliferate is useful for the treatment of a condition
associated with an increased rate of proliferation of a cell, such
as in cancer (telomerase-activity in malignant cells), and
hematopoiesis (telomerase activity in hematopoietic stem cells),
for example.
[0037] Thus, in one aspect, the present invention provides
compounds and compositions for the prevention or treatment of many
types of malignancies. In particular, the compounds of the present
invention can provide a highly general method of treating many, if
not most, malignancies, as demonstrated by the highly varied human
tumor cell lines and tumors having telomerase activity. More
importantly, the thiazolidinone compounds of the present invention
can be effective in providing treatments that discriminate between
malignant and normal cells to a high degree, avoiding many of the
deleterious side-effects present with most current chemotherapeutic
regimes which rely on agents that kill dividing cells
indiscriminately. Representative known thiazolidinedione compounds
include the glitazones, such as, for example, troglitazone (also
known as CS-045 (Sankyo) and CI-991 (Park-Davis)), pioglitazone
(also known as AD-4833 and U-72107E), rosiglitazone (also known as
BRL49653), englitazone (also known as CP-68,722), and
ciglitazone.
[0038] In another aspect, the present invention provides new
compounds, pharmaceutical compositions and methods relating to the
new compounds, or their pharmaceutically acceptable salts, for
inhibiting a telomerase enzyme, comprising contacting the
telomerase enzyme with a compound, or its pharmaceutically
acceptable salt, having the formula (I): 5
[0039] wherein X is O or S; X.sub.1 is O, N or S; L.sub.1 is a
direct bond, --CHR.sub.1--, or .dbd.CR.sub.1--, wherein R.sub.1 is
H or alkyl, such as lower alkyl; A is a direct bond, alkyl, aryl,
aralkyl or heteroaryl; L.sub.2 is a direct bond or a linking group
having from 1 to 3 atoms independently selected from unsubstituted
or substituted carbon, N, O or S W is selected from the group
consisting of O, NR.sub.5, and S, wherein R.sub.5 is selected from
the group consisting of H, alkyl, aryl, and aralkyl; Y and Z are
independently selected to be C or N; and R.sub.2, R.sub.3 and
R.sub.4 are independently selected from the group consisting of
hydrogen, hydroxyl, halogen, alkyl, aryl, alkoxyl, cyano, nitro,
and heteroaryl.
[0040] In the compounds of formula (I) above, L.sub.1 and L.sub.2
may be direct single bonds, or may be linking groups.
Representative linking groups useful in the compounds of the
invention include, for example, --O--, --S--, --NH--, --CH.sub.2--,
--OCH.sub.2--, --OC(O)--, --CO.sub.2--, --NHC(O)--, --C(O)NH--,
--OC(O)CH.sub.2--, --OC(O)NH--, --SO.sub.2--NH--, and --NHC(O)NH--.
In certain embodiments, L.sub.1 is --CHR.sub.1-- or
.dbd.CR.sub.1--, as represented by the formula (II) below: 6
[0041] wherein is a single or double bond and R.sub.1 is hydrogen,
alkyl, or lower alkyl.
[0042] As noted above, A may be phenyl to form, for example, an
aryl moiety. Alternatively, A may be heteroaryl, such as, for
example, pyridine, quinoline, isoquinoline, thiophene, furan,
naphthalene, indene, indole, imidazole, benzimidazole, pyrazole,
and the like, wherein the heteroaryl may be substituted or
unsubstituted. In one embodiment, A is phenyl. In another
embodiment, at least one of R.sub.2 or R.sub.3 is other than
hydrogen. In another embodiment, at least one of R.sub.2 and
R.sub.3 is halo, and preferably both R.sub.2 and R.sub.3 are halo
to form a dihalo-substituted phenyl moiety. In another embodiment,
at least one of R.sub.2, R.sub.3, or R.sub.5 is a thiazolidinone
substituent having the formula (III): 7
[0043] wherein L.sub.1, L.sub.2, and A are as defined above, and n
is 0 or 1. Compounds having the thiazolidinone derivative have the
structure shown in formula (IV) below: 8
[0044] In certain embodiments, the new compounds of the present
invention have the general structure (V) shown below: 9
[0045] and their pharmaceutically acceptable salts, wherein L.sub.1
is a direct bond, --CHR.sub.1--, or .dbd.CR.sub.1--, wherein
R.sub.1 is H or alkyl; A is aryl or heteroaryl; L.sub.2 is a direct
single bond or a linking group having from 1 to 3 atoms
independently selected from unsubstituted or substituted carbon, N,
O or S; W is selected from the group consisting of O NR.sub.5, and
S, wherein R.sub.5 is selected from the group consisting of H,
alkyl, aryl, and aralkyl; Y and Z are independently selected to be
C or N; and R.sub.2 and R.sub.3 and R4 are independently selected
from the group consisting of hydrogen, hydroxyl, halogen, alkyl,
aryl, alkoxyl, cyano, nitro, and heteroaryl. In certain
embodiements, W is NR.sub.5, where R.sub.5 can be H, lower alkyl
such as methyl, ethyl, propyl, butyl, allyl, ethoxycarbonyl methyl,
2-(dimethylamino)ethyl, and the like, aralkyl such as benzyl,
3,4-dichiorobenzyl, 4-formylbenzyl, 4-methoxycarbonyl benzyl,
2-naphtylmethyl, and 5-chlorothiophen-2-ylmethyl, aryl, or
heteroaryl. Some representative compounds of formula (V) wherein
L.sub.1 is .dbd.CH-- and A is phenyl are shown below in Scheme I.
10
[0046] In another embodiment, the new compounds of the present
invention have the general structure (VI) shown below: 11
[0047] and its pharmaceutically acceptable salts, wherein L.sub.1
is a direct bond, --CHR.sub.1--, or .dbd.CR.sub.1--, wherein
R.sub.1 is H or alkyl; A is aryl or heteroaryl; L.sub.2 is a direct
single bond or a linking group having from 1 to 3 atoms
independently selected from unsubstituted or substituted carbon, N,
O or S; W is selected from the group consisting of O, NR.sub.5, and
S, wherein R.sub.5 is selected from the group consisting of H,
alkyl, aryl, and aralkyl; Y and Z are independently selected to be
C or N; and R.sub.2, R.sub.3 and R.sub.4 are independently selected
from the group consisting of hydrogen, hydroxyl, halogen, alkyl,
aryl, alkoxyl, cyano, nitro, and heteroaryl. In certain
embodiments, anyone of R.sub.2, R.sub.3 or R.sub.4 can be a
thiazolidinone substituent represented by formula (III).
[0048] In another embodiment, the new compounds of the invention
have the general structure (VII) shown below: 12
[0049] and its pharmaceutically acceptable salts, wherein X,
L.sub.1, A, L.sub.2, W, Y, Z, R.sub.2, R.sub.3 and R.sub.5 are as
described above.
[0050] Compounds of formula above, wherein L.sub.1 is
.dbd.CR.sub.1--, can be obtained by reacting a thiazolidine
derivative with an aromatic carbonyl compound. The reaction can be
carried out optionally in the presence of a base catalyst and
optionally in a solvent. The base catalyst, usually present in
about 0.1 to about 2 equivalent, may be piperidine, piperidinium
acetate, diethylamine, pyridine, sodium acetate, potassium
carbonate, sodium carbonate, and the like. The solvent may be an
alcohol, such as methanol, ethanol, propanol, or the like, an
ether, such as diethyl ether, tetrahydrofuran, dioxane, or the
like, or a hydrocarbon, such as benzene, toluene, xylene, or the
like, or polar such as N,N-dimethylformamide,
N,N-dimethylacetamide, acetic acid, or the like and mixtures
thereof. The reaction is carried out at a temperature of about room
temperature to about 200.degree. C., preferably about
50-100.degree. C., and completes in about one hour to about 50
hours. Compounds where L.sub.1 is --CHR.sub.1-- can be synthesized
by reducing the double bond of the compound made above. Typically,
reduction is carried out with magnesium in methanol or
hydrogenation is carried out using a noble metal catalyst, such as
palladium, platinum, rhodium, or the like, as is well known in the
art.
III. Synthesis of Telomerase Inhibitors
[0051] The compounds of the present invention can be synthesized
using techniques and materials known to those of skill in the art,
such as described, for example, in March, ADVANCED ORGANIC
CHEMISTRY 4.sup.th Ed., (Wiley 1992); Carey and Sundberg, ADVANCED
ORGANIC CHEMISTRY 3.sup.rd Ed., Vols. A and B (Plenum 1992), and
Green and Wuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS 2.sup.nd Ed.
(Wiley 1991). Starting materials for the compounds of the invention
may be obtained using standard techniques and commercially
available precursor materials, such as those available from Aldrich
Chemical Co. (Milwaukee, Wis.), Sigma Chemical Co. (St. Louis,
Mo.), Lancaster Synthesis (Windham, N.H.), Apin Chemicals, Ltd.
(New Brunswick, N.J.), Ryan Scientific (Columbia, S.C.), Maybridge
(Cornwall, England), Arcos (Pittsburgh, Pa.), and Trans World
Chemicals (Rockville, Md.).
[0052] The procedures described herein for synthesizing the
compounds of the invention may include one or more steps of
protection and deprotection (e.g., the formation and removal of
acetal groups). In addition, the synthetic procedures disclosed
below can include various purifications, such as column
chromatography, flash chromatography, thin-layer chromatography
(TLC), recrystallization, distillation, high-pressure liquid
chromatography (HPLC) and the like. Also, various techniques well
known in the chemical arts for the identification and
quantification of chemical reaction products, such as proton and
carbon-13 nuclear magnetic resonance (.sup.1H and .sup.13C NMR),
infrared and ultraviolet spectroscopy (IR and UV), X-ray
crystallography, elemental analysis (EA), HPLC and mass
spectroscopy (MS) can be used as well. Methods of protection and
deprotection, purification, identification and quantification are
well known in the chemical arts.
[0053] Compounds of the invention can be synthesized using General
Procedures 1, 2, and 3 described in detail in the Examples below.
Detailed protocols from which the individual compounds described
above can be synthesized are also provided in the Examples. The
compounds where L is SO or SO.sub.2 can be synthesized by oxidizing
the corresponsing S compound in an inert solvent. The inert solvent
may be dichloromethane, methanol, tetrahydrofuran, ether, hexane,
toluene, cyclohexane, or the like, and mixtures thereof. The
oxidizing agent may be m-chloroperbenzoic acid, hydrogen peroxide,
or the like. The reaction is carried out at a temperature in the
range of about -78.degree. C. to the boiling point of the solvent,
preferably from about 0.degree. C. to about 30.degree. C. for about
0.5 to about 12 hours.
IV. Anti-Tumor Activity of the Telomerase Inhibitors of the
Invention
[0054] The compounds of the present invention demonstrate
inhibitory activity against telomerase activity in vivo, as has
been and can be demonstrated as described below. The in vitro
activities of the compounds of the invention can also be
demonstrated using the methods described herein. As used herein,
the term "ex vivo " refers to tests performed using living cells in
tissue culture.
[0055] One method used to identify compounds of the invention that
inhibit telomerase activity involves placing cells, tissues, or
preferably a cellular extract or other preparation containing
telomerase in contact with several known concentrations of a test
compound in a buffer compatible with telomerase activity. The level
of telomerase activity for each concentration of test compound is
measured and the IC.sub.50 (the concentration of the test compound
at which the observed activity for a sample preparation was
observed to fall one-half of its original or a control value) for
the compound is determined using standard techniques. Other methods
for determining the inhibitory concentration of a compound of the
invention against telomerase can be employed as will be apparent to
those of skill in the art based on the disclosure herein.
[0056] With the above-described methods, IC.sub.50 values for
several of the compounds of the present invention were determined,
and found to be below 100 .mu.M.
[0057] With respect to the treatment of malignant diseases using
the compounds described herein, compounds of the present invention
are expected to induce crisis in telomerase-positive cell lines.
Treatment of telomerase-positive cell lines, such as HEK-293 and
HeLa cells, with a compound of the invention is also expected to
induce a reduction of telomere length in the treated cells.
[0058] Compounds of the invention are also expected to induce
telomere reduction during cell division in human tumor cell lines,
such as the ovarian tumor cell lines OVCAR-5 and SK-OV-3.
Importantly, however, in normal human cells used as a control, such
as BJ cells of fibroblast origin, the observed reduction in
telomere length is expected to be no different from cells treated
with a control substance, e.g., dimethyl sulfoxide (DMSO). The
compounds of the invention also are expected to demonstrate no
significant cytotoxic effects at concentrations below about 5 .mu.M
in the normal cells.
[0059] In addition, the specificity of the compounds of the present
invention for telomerase can be determined by comparing their
activity (IC.sub.50) with respect to telomerase to other enzymes
having similar nucleic acid binding or modifying activity similar
to telomerase in vitro. Such enzymes include DNA Polymerase I, HeLa
RNA Polymerase II, T3 RNA Polymerase, MMLV Reverse Transcriptase,
Topoisomerase I, Topoisomerase II, Terminal Transferase and
Single-Stranded DNA Binding Protein (SSB). Compounds having lower
IC.sub.50 values for telomerase as compared to the IC.sub.50 values
toward the other enzymes being screened are said to possess
specificity for telomerase.
[0060] In vivo testing can also be performed using a mouse
xenograft model, for example, in which OVCAR-5 tumor cells are
grafted onto nude mice, in which mice treated with a compound of
the invention are expected to have tumor masses that, on average,
may increase for a period following the initial dosing, but will
begin to shrink in mass with continuing treatment. In contrast,
mice treated with a control (e.g., DMSO) are expected to have tumor
masses that continue to increase.
[0061] From the foregoing those skilled in the art will appreciate
that the present invention also provides methods for selecting
treatment regimens involving administration of a compound of the
invention. For such purposes, it may be helpful to perform a
terminal restriction fragment (TRF) analysis in which DNA from
tumor cells is analyzed by digestion with restriction enzymes
specific for sequences other than the telomeric (T.sub.2
AG.sub.3).sub.N sequence. Following digestion of the DNA, gel
electrophoresis is performed to separate the restriction fragments
according to size. The separated fragments are then probed with
nucleic acid probes specific for telomeric sequences to determine
the lengths of the terminal fragments containing the telomere DNA
of the cells in the sample. By measuring the length of telomeric
DNA, one can estimate how long a telomerase inhibitor should be
administered and whether other methods of therapy (e.g., surgery,
chemotherapy and/or radiation) should also be employed. In
addition, during treatment, one can test cells to determine whether
a decrease in telomere length over progressive cell divisions is
occurring to demonstrate treatment efficacy.
V. Telomerase Inhibiting Compositions and Methods for Treating
Diseases
[0062] The present invention also provides pharmaceutical
compositions for inhibiting cell proliferation of telomerase
positive cells, and treating cancer and other conditions in which
inhibition of telomerase is an effective therapy. These
compositions include a therapeutically effective amount of a
telomerase inhibiting compound of the invention in a
pharmaceutically acceptable carrier or salt.
[0063] In one embodiment, the present invention provides methods,
compounds and compositions for inhibiting a telomerase enzyme,
inhibiting proliferation of telomerase postive cells, and for
treating cancer in a mammal. The compositions of the invention
include a therapeutically effective amount of a compound of the
invention (or a pharmaceutically acceptable salt thereof) in a
pharmaceutically acceptable carrier. The compounds and compositions
of the present invention may also be used for the treatment of
other telomerase mediated conditions or diseases, such as, for
example, other hyperproliferative or autoimmune disorders such as
psoriasis, rheumatoid arthritis, immune system disorders requiring
immune system suppression, immune system reactions to poison ivy or
poison oak, and the like.
[0064] In addition, it will be appreciated that therapeutic
benefits for treatment of cancer can be realized by combining a
telomerase inhibitor of the invention with other anti-cancer
agents, including other inhibitors of telomerase such as described
in U.S. Pat. Nos. 5,656,638, 5,760,062, 5,767,278, 5,770,613 and
5,863,936. The choice of such combinations will depend on various
factors including, but not limited to, the type of disease, the age
and general health of the patient, the aggressiveness of disease
progression, the TRF length and telomerase activity of the diseased
cells to be treated and the ability of the patient to tolerate the
agents that comprise the combination. For example, in cases where
tumor progression has reached an advanced state, it may be
advisable to combine a telomerase inhibiting compound of the
invention with other agents and therapeutic regimens that are
effective at reducing tumor size (e.g. radiation, surgery,
chemotherapy and/or hormonal treatments). In addition, in some
cases it may be advisable to combine a telomerase inhibiting agent
of the invention with one or more agents that treat the side
effects of a disease, e.g., an analgesic, or agents effective to
stimulate the patient's own immune response (e.g., colony
stimulating factor).
[0065] In one such method, a pharmaceutical formulation comprises a
telomerase inhibitor of the invention with an anti-angiogenesis
agent, such as fumagillin, fumagillin derivatives, or AGM-1470. The
latter compound is available from Takeda Chemical Industries, Ltd.,
while the former compounds are described in Ingber, et al., Dec. 6,
1990, "Synthetic analogues of fumagillin that inhibit angiogenesis
and suppress tumor growth", Nature 348:555-557. Other combinations
may include, but are not limited to, a telomerase inhibitor of the
invention in addition to one or more antineoplastic agents or
adjuncts (e.g., folinic acid or mesna).
[0066] Antineoplastic agents suitable for combination with the
compounds of the present invention include, but are not limited to,
alkylating agents including alkyl sulfonates such as busulfan,
improsulfan and piposulfan; aziridines, such as a benzodizepa,
carboquone, meturedepa and uredepa; ethylenimines and
methylmelamines such as altretamine, triethylenemelamine,
triethylenephosphoramide, triethylenethiophosphorami- de and
trimethylolmelamine; nitrogen mustards such as chlorambucil,
chlornaphazine, cyclophosphamide, estramustine, iphosphamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichine, phenesterine, prednimustine, trofosfamide, and uracil
mustard; nitroso ureas, such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine and ranimustine. Additional
agents include dacarbazine, mannomustine, mitobronitol, mitolactol
and pipobroman. Still other classes of relevant agents include
antibiotics, hormonal antineoplastics and antimetabolites. Yet
other combinations will be apparent to those of skill in the
art.
[0067] Additional agents suitable for combination with the
compounds of the present invention include protein synthesis
inhibitors such as abrin, aurintricarboxylic acid, chloramphenicol,
colicin E3, cycloheximide, diphtheria toxin, edeine A, emetine,
erythromycin, ethionine, fluoride, 5-fluorotryptophan, fusidic
acid, guanylyl methylene diphosphonate and guanylyl
imidodiphosphate, kanamycin, kasugamycin, kirromycin, and O-methyl
threonine. Additional protein synthesis inhibitors include
modeccin, neomycin, norvaline, pactamycin, paromomycine, puromycin,
ricin, _-sarcin, shiga toxin, showdomycin, sparsomycin,
spectinomycin, streptomycin, tetracycline, thiostrepton and
trimethoprim. Inhibitors of DNA synthesis, including alkylating
agents such as dimethyl sulfate, mitomycin C, nitrogen and sulfur
mustards, MNNG and NMS; intercalating agents such as acridine dyes,
actinomycins, adriamycin, anthracenes, benzopyrene, ethidium
bromide, propidium diiodide-intertwining, and agents such as
distamycin and netropsin, can also be combined with compounds of
the present invention in pharmaceutical compositions. DNA base
analogs such as acyclovir, adenine .beta.-1-D-arabinoside,
amethopterin, aminopterin, 2-aminopurine, aphidicolin,
8-azaguanine, azaserine, 6-azauracil, 2'-azido-2'-deoxynucleosides,
5-bromodeoxycytidine, cytosine .beta.-1-D-arabinoside,
diazooxynorleucine, dideoxynucleosides, 5-fluorodeoxycytidine,
5-fluorodeoxyuridine, 5-fluorouracil, hydroxyurea and
6-mercaptopurine also can be used in combination therapies with the
compounds of the invention. Topoisomerase inhibitors, such as
coumermycin, nalidixic acid, novobiocin and oxolinic acid,
inhibitors of cell division, including colcemide, colchicine,
vinblastine and vincristine; and RNA synthesis inhibitors including
actinomycin D, .alpha.-amanitine and other fungal amatoxins,
cordycepin (3'-deoxyadenosine), dichlororibofuranosyl
benzimidazole, rifampicine, streptovaricin and streptolydigin also
can be combined with the compounds of the invention to provide
pharmaceutical compositions.
[0068] In another embodiment, the present invention includes
compounds and compositions in which a telomerase inhibitor is
either combined with or covalently bound to a cytotoxic agent bound
to a targeting agent, such as a monoclonal antibody (e.g., a murine
or humanized monoclonal antibody). It will be appreciated that the
latter combination may allow the introduction of cytotoxic agents
into cancer cells with greater specificity. Thus, the active form
of the cytotoxic agent (i.e., the free form) will be present only
in cells targeted by the antibody. Of course, the telomerase
inhibitors of the invention may also be combined with monoclonal
antibodies that have therapeutic activity against cancer.
[0069] In addition to the application of the telomerase inhibitors
of the present invention to the treatment of mammalian diseases
characterized by telomerase activity, telomerase inhibitors such as
those disclosed herein, can be applied to agricultural
phytopathogenic organisms that are characterized by telomerase
activity. These organisms include nematodes such as Ceanorhabditis
elegans, in which telomerase activity has been found, and in fungi
which are expected to have telomerase activity based on the
determination that the DNA of the fungus Ustilago maydis exhibits
telomeres having the tandem TTAGGG repeats that are maintained by
telomerase. Also, protozoans have TTAGGG telomeres and cause human
disease. The telomerase-inhibiting compounds of the invention can
be administered to plants and soil infected with phytopathogenic
organisms having telomerase activity alone, or in combination with
other telomerase-inhibiting agents and/or other agents used to
control plant diseases. The determination of the compositions used
to control such phytopathogenic organisms and the appropriate modes
of delivering such compositions will be known to those having skill
in the agricultural arts.
[0070] The determination that nematodes, protozoans and possibly
fungi have telomerase activity also indicates that the telomerase
inhibitors provided by the present invention can be used to treat
nematode infections in humans and animals of veterinary interest
such as dogs and cats. Nematode infection in humans and animals
often is in the form of hookworm or roundworm infection and leads
to a host of deadly secondary illnesses such as meningitis,
myocarditis, and various neurological diseases. Thus, it will be
appreciated that administration of the telomerase-inhibiting
compounds such as those of the invention, alone, or in combination
with other telomerase-inhibiting agents and/or other therapeutic
agents, can be used to control nematode, protozoan and fungal
infections in humans and animals.
[0071] In general, a suitable effective dose of a compound of the
invention will be in the range of 0.001 to 1000 milligram (mg) per
kilogram (kg) of body weight of the recipient per day, preferably
in the range of 0.001 to 100 mg per kg of body weight per day, more
preferably between about 0.1 and 100 mg per kg of body weight per
day and still more preferably in the range of between 0.1 to 10 mg
per kg of body weight per day. The desired dosage is preferably
presented in one, two, three, four, or more subdoses administered
at appropriate intervals throughout the day, or by the action of a
continuous pump. These subdoses can be administered as unit dosage
form, for example, containing 5 to 10,000 mg, preferably 10 to 1000
mg of active ingredient per unit dosage from. Preferably, the
dosage is presented once per day at a dosing at least equal to TID,
or is administered using a continuous pump delivery system.
[0072] The composition used in these therapies can be in a variety
of forms. These include, for example, solid, semi-solid, and liquid
dosage forms, such as tablets, pills, powders, liquid solutions or
suspensions, liposomes, and injectable and infusible solutions. The
preferred form depends on the intended mode of administration and
therapeutic application. The compositions also preferably include
conventional pharmaceutically acceptable carriers and adjuvants, as
is well known to those of skill in the art. See, e.g., REMINGTON'S
PHARMACEUTICAL SCIENCES, Mack Publishing Co.: Easton, Pa., 17th Ed.
(1985). Preferably, administration will be by oral or parenteral
(including subcutaneous, intramuscular, intravenous, and
intradermal) routes. More preferably, the route of administration
will be oral. The therapeutic methods and agents of this invention
can of course be used concomitantly or in combination with other
methods and agents for treating a particular disease or disease
condition.
[0073] While it is possible to administer the active ingredient of
this invention alone, it is preferable to present a therapeutic
agent as part of a pharmaceutical formulation or composition. The
formulations of the present invention comprise at least one
telomerase activity-inhibiting compound of this invention in a
therapeutically or pharmaceutically effective dose together with
one or more pharmaceutically or therapeutically acceptable carriers
and optionally other therapeutic ingredients. Various
considerations for preparing such formulations are described, e.g.,
in Gilman et al. (eds.) GOODMAN AND GILMAN'S: THE PHARMACOLOGICAL
BASES OF THERAPEUTICS, 8th Ed., Pergamon Press (1990); and
REMINGTON'S supra. Methods for administration are discussed
therein, e.g., for oral, intravenous, intraperitoneal,
intramuscular, and other forms of administration. Typically,
methods for administering pharmaceutical compositions will be
either topical, parenteral, or oral administration methods for
prophylactic and/or therapeutic treatment. Oral administration is
preferred. The pharmaceutical compositions can be administered in a
variety of unit dosage forms depending upon the method of
administration. As noted above, unit dosage forms suitable for oral
administration include powders, tablets, pills, and capsules.
[0074] One can use topical administration to deliver a compound of
the invention by percutaneous passage of the drug into the systemic
circulation of the patient. The skin sites include anatomic regions
for transdermally administering the drug, such as the forearm,
abdomen, chest, back, buttock, and mastoidal area. The compound is
administered to the skin by placing on the skin either a topical
formulation comprising the compound or a transdermal drug delivery
device that administers the compound. In either embodiment, the
delivery vehicle is designed, shaped, sized, and adapted for easy
placement and comfortable retention on the skin.
[0075] A variety of transdermal drug delivery devices can be
employed with the compounds of this invention. For example, a
simple adhesive patch comprising a backing material and an acrylate
adhesive can be prepared. The drug and any penetration enhancer can
be formulated into the adhesive casting solution. The adhesive
casting solution can be cast directly onto the backing material or
can be applied to the skin to form an adherent coating. See, e.g.,
U.S. Pat. Nos. 4,310,509; 4,560,555; and 4,542,012.
[0076] In other embodiments, the compound of the invention will be
delivered using a liquid reservoir system drug delivery device.
These systems typically comprise a backing material, a membrane, an
acrylate based adhesive, and a release liner. The membrane is
sealed to the backing to form a reservoir. The drug or compound and
any vehicles, enhancers, stabilizers, gelling agents, and the like
are then incorporated into the reservoir. See, e.g., U.S. Pat. Nos.
4,597,961; 4,485,097; 4,608,249; 4,505,891; 3,843,480; 3,948,254;
3,948,262; 3,053,255; and 3,993,073.
[0077] Matrix patches comprising a backing, a drug/penetration
enhancer matrix, a membrane, and an adhesive can also be employed
to deliver a compound of the invention transdermally. The matrix
material typically will comprise a polyurethane foam. The drug, any
enhancers, vehicles, stabilizers, and the like are combined with
the foam precursors. The foam is allowed to cure to produce a
tacky, elastomeric matrix which can be directly affixed to the
backing material. See, e.g., U.S. Pat. Nos. 4,542,013; 4,460,562;
4,466,953; 4,482,534; and 4,533,540.
[0078] Also included within the invention are preparations for
topical application to the skin comprising a compound of the
invention, typically in concentrations in the range from about
0.001% to 10%, together with a non-toxic, pharmaceutically
acceptable topical carrier. These topical preparations can be
prepared by combining an active ingredient according to this
invention with conventional pharmaceutical diluents and carriers
commonly used in topical dry, liquid, and cream formulations.
Ointment and creams may, for example, be formulated with an aqueous
or oily base with the addition of suitable thickening and/or
gelling agents. Such bases may include water and/or an oil, such as
liquid paraffin or a vegetable oil, such as peanut oil or castor
oil. Thickening agents that may be used according to the nature of
the base include soft paraffin, aluminum stearate, cetostearyl
alcohol, propylene glycol, polyethylene glycols, woolfat,
hydrogenated lanolin, beeswax, and the like.
[0079] Lotions may be formulated with an aqueous or oily base and
will, in general, also include one or more of the following:
stabilizing agents, emulsifying agents, dispersing agents,
suspending agents, thickening agents, coloring agents, perfumes,
and the like. Powders may be formed with the aid of any suitable
powder base, e.g., talc, lactose, starch, and the like. Drops may
be formulated with an aqueous base or non-aqueous base also
comprising one or more dispersing agents, suspending agents,
solubilizing agents, and the like. Topical administration of
compounds of the invention may also be preferred for treating
diseases such as skin cancer and fungal infections of the skin
(pathogenic fungi typically express telomerase activity).
[0080] The topical pharmaceutical compositions according to this
invention may also include one or more preservatives or
bacteriostatic agents, e.g., methyl hydroxybenzoate, propyl
hydroxybenzoate, chlorocreosol, benzalkonium chlorides, and the
like. The topical pharmaceutical compositions also can contain
other active ingredients such as antimicrobial agents, particularly
antibiotics, anesthetics, analgesics, and antipruritic agents.
[0081] The compounds of the present invention can also be delivered
through mucosal membranes. Transmucosal (i.e., sublingual, buccal,
and vaginal) drug delivery provides for an efficient entry of
active substances to systemic circulation and reduces immediate
metabolism by the liver and intestinal wall flora. Transmucosal
drug dosage forms (e.g., tablet, suppository, ointment, pessary,
membrane, and powder) are typically held in contact with the
mucosal membrane and disintegrate and/or dissolve rapidly to allow
immediate systemic absorption. Note that certain such routes may be
used even where the patient is unable to ingest a treatment
composition orally. Note also that where delivery of a telomerase
inhibitor of the invention would be enhanced, one can select a
composition for delivery to a mucosal membrane, e.g., in cases of
colon cancer one can use a suppository to deliver the telomerase
inhibitor.
[0082] For delivery to the buccal or sublingual membranes,
typically an oral formulation, such as a lozenge, tablet, or
capsule, will be used. The method of manufacture of these
formulations is known in the art, including, but not limited to,
the addition of the pharmacological agent to a pre-manufactured
tablet; cold compression of an inert filler, a binder, and either a
pharmacological agent or a substance containing the agent (as
described in U.S. Pat. No. 4,806,356); and encapsulation. Another
oral formulation is one that can be applied with an adhesive, such
as the cellulose derivative hydroxypropyl cellulose, to the oral
mucosa, for example as described in U.S. Pat. No. 4,940,587. This
buccal adhesive formulation, when applied to the buccal mucosa,
allows for controlled release of the pharmacological agent into the
mouth and through the buccal mucosa.
[0083] Parenteral administration is generally characterized by
injection, either subcutaneously, intramuscularly, or
intravenously. Thus, this invention provides compositions for
intravenous administration that comprise a solution of a compound
of the invention dissolved or suspended in an acceptable carrier.
Injectables can be prepared in conventional forms, either as liquid
solutions or suspensions, solid forms suitable for solution or
suspension in liquid prior to injection, or as emulsions. Suitable
excipients are, for example, water, buffered water, saline,
dextrose, glycerol, ethanol, or the like. These compositions will
be sterilized by conventional, well known sterilization techniques,
such as sterile filtration. The resulting solutions can be packaged
for use as is or lyophilized, the lyophilized preparation being
combined with a sterile solution prior to administration. In
addition, if desired, the pharmaceutical compositions to be
administered may also contain minor amounts of non-toxic auxiliary
substances, such as wetting or emulsifying agents, pH buffering
agents and the like, such as for example, sodium acetate, sorbitan
monolaurate, triethanolamine oleate, etc. Such formulations will be
useful in treating ovarian cancers.
[0084] Another method of parenteral administration employs the
implantation of a slow-release or sustained-release system, such
that a constant level of dosage is maintained. See, e.g., U.S. Pat.
No. 3,710,795.
[0085] Liquid pharmaceutically administrable compositions can, for
example, be prepared by dissolving, dispersing, etc., an active
compound as defined above and optional pharmaceutical adjuvants in
an excipient, such as, for example, water, saline, aqueous
dextrose, glycerol, ethanol, olive oil, and other lipophilic
solvents, and the like, to form a solution or suspension. If
desired, the pharmaceutical composition to be administered may also
contain minor amounts of nontoxic auxiliary substances, such as
wetting or emulsifying agents, pH buffering agents, and the like,
for example, sodium acetate, sorbitan monolaurate, triethanolamine
sodium acetate, triethanolamine oleate, etc. Actual methods of
preparing such dosage forms are known and will be apparent to those
skilled in this art; for example, see REMINGTON'S PHARMACEUTICAL
SCIENCES, supra. The composition or formulation to be administered
will contain an effective amount of an active compound of the
invention.
[0086] For solid compositions, conventional nontoxic solid carriers
can be used and include, for example, pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharin,
talcum, cellulose, glucose, sucrose, magnesium carbonate, and the
like. For oral administration, a pharmaceutically acceptable
nontoxic composition is formed by incorporating any of the normally
employed excipients, such as those carriers previously listed, and
generally 0.1-95% of active ingredient, preferably about 20%.
[0087] The compositions containing the compounds of the invention
can be administered for prophylactic and/or therapeutic treatments.
In therapeutic applications, compositions are administered to a
patient already suffering from a disease, as described above, in an
amount sufficient to cure or at least partially arrest the symptoms
of the disease and its complications. An amount adequate to
accomplish this is defined as a "therapeutically effective amount
or dose." Amounts effective for this use will depend on the
severity of the disease and the weight and general state of the
patient.
[0088] In addition to internal (in vivo) administration, the
compounds and compositions of the invention may be applied ex vivo
to achieve therapeutic effects, as for example, in the case of a
patient suffering from leukemia. In such an application, cells to
be treated, e.g., blood or bone marrow cells, are removed from a
patient and treated with a pharmaceutically effective amount of a
compound of the invention. The cells are returned to the patient
following treatment. Such a procedure can allow for exposure of
cells to concentrations of therapeutic agent for longer periods or
at higher concentrations than otherwise available.
[0089] Once improvement of the patient's conditions has occurred,
as, for example, by the occurrence of remission in the case of a
cancer patient, a maintenance dose is administered if necessary.
Subsequently, the dosage or the frequency of administration, or
both, can be reduced, as a function of the systems, to a level at
which the improved condition is retained. When the symptoms have
been alleviated to the desired level, treatment can cease. Patients
can, however, require additional treatment upon any recurrence of
the disease symptoms.
[0090] In prophylactic applications (e.g. chemoprevention),
compositions containing the compounds of the invention are
administered to a patient susceptible to or otherwise at risk of a
particular disease. Such an amount is defined to be a
"prophylactically effective amount or dose." In this use, the
precise amounts again depend on the patient's state of health and
weight.
[0091] As will be apparent to those of skill in the art upon
reading of this disclosure, the present invention provides valuable
reagents relating to human and mammalian telomerase. The above
description of necessity provides a limited and merely illustrative
sampling of specific compounds, and should not be construed as
limiting the scope of the invention. Other features and advantages
of the invention will be apparent from the following examples and
claims.
EXAMPLES
[0092] The following examples describe specific aspects of the
invention to illustrate the invention and also provide a
description of methods that can be used to identify and test
compounds that inhibit the activity of telomerase to aid those of
skill in the art in understanding and practicing the invention. The
examples should not be construed as limiting the invention in any
manner.
General Procedure 1
Condensation of 2,4-thiazolidinedione and aldehyde
[0093] A mixture of appropriately substituted aldehyde (1 eq.),
2,4-thiazolidinedione (1.5 eq.) and piperidine (1.5 eq.) in ethanol
was heated to reflux for 2-24 h. The resulting mixture was
acidified with hydrochloric acid (1 mol/L). The precipitated
products were filtered off and washed with water and/or ether to
afford pure product. Alternatively, the acidified mixture was
extracted with ethyl acetate or chloroform, organic phase washed
with water and brine, dried over anhydrous sodium sulfate and
concentrated under reduced pressure to yield crude product, which
was purified either by column chromatography or recrystallization
from appropriate solvent system.
General Procedure 2
Condensation of aldehyde and diamine To Afford benzimidazole
[0094] A mixture of aldehyde, diamine and 1,4-benzoquinone (1:1:1
molar ratio) in 1,4-dioxane was heated to 100.degree. C. for 2-24
h. The mixture was cooled to room temperature, and the precipitated
products were filtered off and washed with water and/or ether to
afford pure product. Alternatively, the solvent was removed under
reduced pressure and the resulting solid was recrystallized or
triturated with appropriate solvent, such as ethyl acetate, ether,
or 1,4-dioxane to afford pure product.
General Procedure 3
Alkylation of N1-position of 2-(4-formylphenyl)benzimidzole
[0095] To a solution of 2-(4-formylphenyl)benzimidazole (1 eq.) in
dimethylformamide were added sodium hydride (1.5 eq.) and alkyl
halides (2 eq.) at 0_C under nitrogen atmosphere, and the mixture
was stirred at 20-100.degree. C. for 2-24 h. Water was added, and
the precipitated products were collected by filtration and washed
with water and or ether. Alternatively, the mixture was extracted
with ethyl acetate, organic phase washed with water and brine,
dried over anhydrous sodium sulfate and concentrated under reduced
pressure to afford crude product, which was purified either by
column chromatography or recrystallization from appropriate solvent
system.
Example 1
Preparation of
5,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]p-
henyl]benzimidazole
[0096] 13
[0097] To a mixture of terephthalaldehyde mono-(diethyl acetal)
(3.96 mL, 20.0 mmol) and 2,4-thiazolidinedione (2.81 g, 24.0 mmol)
in ethanol (40 mL) was added piperidine (2.0 mL, 20.0 mmol). The
reaction mixture was heated to reflux for 4 h. After cooling on an
ice-bath, hydrochloric acid (0.2 mol/L, 100 mL) was added and the
precipitated products were collected by filtration. The cake was
washed with water and dried. To a solution .of this product (5.50
g) in tetrahydrofuran (180 mL) was added hydrochloric acid (1
mol/L, 18 mL) and the mixture was stirred at room temperature for 2
h. Water was added, and the precipitated products were collected by
filtration to give 5-(4-formylbenzylidene)-2,4-thiazolidined- ione
(3.91 g, 94%).
[0098] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.72 (d,
J=8.1 Hz, 2H), 7.77 (s, 1H), 7.94 (d, J=7.7 Hz, 2H), 9.96 (s,
1H)
[0099] General Procedure 2 was then followed to obtain
5,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]phenyl]benzimid-
azole.
[0100] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.72 (d,
J=8.6 Hz, 2H), 7.74 (br s, 1H), 7.79 (s, 1H), 7.91 (br s, 1H), 8.23
(d, J=8.2 Hz, 2H), 12.7 (br s, 1H), 13.4 (br s, 1H)
[0101] ESI-MS m/z 390, 388 (M-H).sup.-
C.sub.17H.sub.9.sup.35Cl.sub.2N.sub- .3O.sub.2S=389
Example 2
Preparation of
2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]phenyl]benzimi-
dazole
[0102] 14
[0103]
2-[4-[(2,4-Dioxothiazolidin-5-ylidene)methyl]phenyl]benzimidazole
was prepared from 5-(4-formylbenzylidene)-2,4-thiazolidinedione and
1,2-phenylenediamine by following General Procedure 2.
[0104] ESI-MS m/z 320(M-H).sup.-
C.sub.17H.sub.11N.sub.3O.sub.2S=321
Example 3
Preparation of
5,6-dimethyl-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]p-
henyl]benzimidazole
[0105] 15
[0106]
5,6-Dimethyl-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]phenyl]be-
nzimidazole was prepared from
5-(4-formylbenzylidene)-2,4-thiazolidinedion- e and
4,5-dimethyl-1,2-phenylenediamine by following General Procedure
2.
[0107] ESI-MS m/z 348 (M-H).sup.-
C.sub.19H.sub.15N.sub.3O.sub.2S=349
Example 4
Preparation of
2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]phenyl]-5-nitr-
obenzimidazole
[0108] 16
[0109]
2-[4-[(2,4-Dioxothiazolidin-5-ylidene)methyl]phenyl]-5-nitrobenzimi-
dazole was prepared from
5-(4-formylbenzylidene)-2,4-thiazolidinedione and
4-nitro-1,2-phenylenediamine by following General Procedure 2.
[0110] ESI-MS m/z 365 (M-H).sup.-
C.sub.17H.sub.10N.sub.4O.sub.4S=366
Example 5
Preparation of 6-chloro-2-
[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]phen-
yl]-4-(trifluoromethyl)benzimidazole
[0111] 17
[0112]
6-Chloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]phenyl]-4-(tr-
ifluoromethyl)benzimidazole was prepared from
5-(4-formylbenzylidene)-2,4-- thiazolidinedione and
5-chloro-3-trifluoromethyl-1,2-phenylenediamine by following
General Procedure 2.
[0113] ESI-MS m/z 423 (M-H).sup.-
C.sub.18H.sub.9.sup.35ClF.sub.3N.sub.3O.- sub.2S=424
Example 6
Preparation of
5-benzoyl-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]phen-
yl]benzimidazole
[0114] 18
[0115]
5-Benzoyl-2-[4-[(2,4-dioxothiazolidin-5-ylydene)methyl]phenyl]benzi-
midazole was prepared from
5-(4-formylbenzylidene)-2,4-thiazolidinedione and
3,4-diaminobenzophenone by following General Procedure 2.
[0116] ESI-MS m/z 424 (M-H).sup.-
C.sub.24H.sub.15N.sub.3O.sub.3S=425
Example 7
Preparation of
2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]phenyl]-5-(tri-
fluoromethyl) benzimidazole
[0117] 19
[0118]
2-[4-[(2,4-Dioxothiazolidin-5-ylidene)methyl]phenyl]-5-trifluoromet-
hylbenzimidazole was prepared from
5-(4-formylbenzylidene)-2,4-thiazolidin- edione and
4-trifluoromethyl-1,2-phenylenediamine by following General
Procedure 2.
[0119] ESI-MS m/z 388 (M-H).sup.-
C.sub.18H.sub.10F.sub.3N.sub.3O.sub.2S=3- 89
Example 8
Preparation of
5-cyano-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]phenyl-
]benzimidazole
[0120] 20
[0121]
5-Cyano-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]phenyl]benzimi-
dazole was prepared from
5-(4-formylbenzylidene)-2,4-thiazolidinedione and
3,4-diaminobenzonitrile by following General Procedure 2.
[0122] ESI-MS m/z 345 (M-H).sup.-
C.sub.18H.sub.10N.sub.4O.sub.2S=346
Example 9
Preparation of
4,5-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]p-
henyl]benzimidazole
[0123] 21
[0124]
4,5-Dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]phenyl]be-
nzimidazole was prepared from
5-(4-formylbenzylidene)-2,4-thiazolidinedion- e and
3,4-dichloro-1,2-phenylenediamine by following General Procedure
2.
[0125] ESI-MS m/z 390, 388 (M-H).sup.-
C.sub.17H.sub.9Cl.sub.2N.sub.3O.sub- .2S=389
Example 10
Preparation of
4,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]p-
henyl]benzimidazole
[0126] 22
[0127]
4,6-Dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]phenyl]be-
nzimidazole was prepared from
5-(4-formylbenzylidene)-2,4-thiazolidinedion- e and
3,5-dichloro-1,2-phenylenediamine by following General Procedure
2.
[0128] ESI-MS m/z 390, 388 (M-H).sup.-
C.sub.17H.sub.9Cl.sub.2N.sub.3O.sub- .2S389
Example 11
Preparation of
5,6-difluoro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]p-
henyl]benzimidazole
[0129] 23
[0130]
5,6-Difluoro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]phenyl]be-
nzimidazole was prepared from
5-(4-formylbenzylidene)-2,4-thiazolidinedion- e and
4,5-difluoro-1,2-phenylenediamine by following General Procedure
2.
[0131] ESI-MS m/z 356 (M-H).sup.-
C.sub.17H.sub.9F.sub.2N.sub.3O.sub.2S=35- 7
Example 12
Preparation of
2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]phenyl]-4-hydr-
oxybenzimidazole
[0132] 24
[0133]
2-[4-[(2,4-Dioxothiazolidin-5-ylidene)methyl]phenyl]-4-hydroxybenzi-
midazole was prepared from
5-(4-formylbenzylidene)-2,4-thiazolidinedione and 2,3-diaminophenol
by following General Procedure 2.
[0134] ESI-MS m/z 336 (M-H).sup.-
C.sub.17H.sub.11N.sub.3O.sub.3S=337
Example 13
Preparation of
5,6-dichloro-2-[4-(2,4-dioxothiazolidin-5-yl)phenyl]benzimi-
dazole
[0135] 25
[0136] To a mixture of terephthalaldehyde mono-(diethyl acetal)
(2.0 mL, 10 mmol), 4,5-dichloro-1,2-phenylenediamine (1.77 g, 10
mmol) and 1,4-benzoquinone (1.08 g, 10 mmol) in ethanol (50 mL) was
heated to reflux for 6 h. The solvent was removed under reduced
pressure. To the residue were added tetrahydrofuran (5 0 mL) and
hydrochloric acid (1 mol/L, 10 mL), and the mixture was stirred at
room temperature for 2 h. Water was added, and the mixture was
extracted with ethyl acetate. The organic layer was washed with
water and brine, and dried on anhydrous sodium sulfate. The solvent
was removed under reduced pressure, and the residue was triturated
with ether to afford 5,6-dichloro-2-(4-formylpheny- l)benzimidazole
(2.80 g, 97%).
[0137] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.87 (br
s, 2H), 8.04 (d, J=8.6 Hz, 2H), 8.33 (d, J=8.2 Hz, 2H), 10.0 (s,
1H), 13.5 (br s,1H)
[0138] ESI-MS m/z 291, 289 (M-H).sup.-
C.sub.14H.sub.8.sup.35Cl.sub.2N.sub- .2O=290
[0139] To a solution of
5,6-dichloro-2-(4-formylphenyl)benzimidazole (291 mg, 1.00 mmol) in
tetrahydrofuran (40 mL) were added potassium cyanide (260 mg, 4.00
mmol), sodium hydrogen sulfite (500 mg), and water (10 mL). The
mixture was stirred at room temperature for 12 h. Water was added,
and the mixture was extracted with ethyl acetate. The organic layer
was washed with brine and dried on anhydrous sodium sulfate. The
solvent was removed under reduced pressure to afford
2-[4-[cyano(hydroxy)methyl]pheny- l]-5,6-dichlorobenzimidazole (278
mg, 87%).
[0140] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 5.83 (d,
J=6.2 Hz, 1H), 7.17 (d, J=6.2 Hz, 1H), 7.64 (d, J=8.2 Hz, 2H), 7.74
(br s, 1H), 7.91 (br s, 1H), 8.19 (d, J=8.6 Hz, 2H), 13.3 (br s,
1H)
[0141] ESI-MS m/z 318, 316 (M-H).sup.-
C.sub.15H.sub.9.sup.35Cl.sub.2N.sub- .3O=317
[0142] To a suspension of
2-[4-[cyano(hydroxy)methyl]phenyl]-5,6-dichlorob- enzimidazole (275
mg, 0.865 mmol) in tetrahydrofuran (20 mL) was added thionyl
chloride (0.38 mL, 5.2 mmol), and the mixture was heated to reflux
for 3 h. After cooling to room temperature, water was added and the
mixture was extracted with ethyl acetate. The organic layer was
washed with brine and dried on anhydrous sodium sulfate. The
solvent was removed under reduced pressure. The residue was
purified by column chromatography (14:1 chloroform/acetonitrile) to
afford of
2-[4-[chloro(cyano)methyl]phenyl]-5,6-dichlorobenzimidazole (73 mg,
25%).
[0143] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 6.72 (s,
1H), 7.76 (d, J=8.2 Hz, 2H), 7.85 (br s, 2H), 8.25 (d, J=8.2 Hz,
2H)
[0144] ESI-MS m/z 336, 334 (M-H).sup.-
C.sub.15H.sub.8.sup.35Cl.sub.3N.sub- .3=335
[0145] To a solution of
2-[4-[chloro(cyano)methyl]phenyl]-5,6-dichlorobenz- imidazole (22
mg, 0.065 mmol) in ethanol (5 mL) was added thiourea (10 mg, 0.13
mmol), and the mixture heated to reflux for 1 h. Hydrochloric acid
(2 mol/L, 4 mL) was added, and the mixture was heated to reflux for
additional 11 h. After cooling to room temperature, pH was adjusted
to 10, and the mixture was washed with ethyl acetate. The mixture
was acidified, and extracted with ethyl acetate. The organic layer
was washed with brine and dried on anhydrous sodium sulfate. The
solvent was removed under reduced pressure to afford
5,6-dichloro-2-[4-(2,4-dioxothiazolidin-- 5-yl)phenyl]benzimidazole
(7.0 mg, 28%).
[0146] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 5.85 (s,
1H), 7.59 (d, J=8.6 Hz, 2H), 7.73 (s, 1H), 7.91 (s, 1H), 8.14 (d,
J=8.6 Hz, 2H), 12.4 (br s, 1H), 13.3 (br s, 1H)
[0147] ESI-MS m/z 378, 376 (M-H).sup.-,
C.sub.16H.sub.9.sup.35Cl.sub.2N.su- b.3O.sub.2S=377
Example 14
Preparation of
1-(3,4-dichlorobenzyl)-2-[4-[(2,4-dioxothiazolidin-5ylidene-
)methyl]-phenyl]benzimidazole
[0148] 26
[0149] 2-(4-Formylphenyl)benzimidazole was prepared from
terephthalaldehyde mono-(diethyl acetal) and phenylenediamine in a
similar manner as described in Example 13.
[0150] General Procedure 3 was then followed to obtain
1-(3,4-dichlorobenzyl)-2-(4-formylphenyl)benzimidazole from
2-(4-formylphenyl)benzimidazole and 3,4-dichlorobenzyl
chloride.
[0151] General Procedure 1 was then followed to obtain
1-(3,4-dichlorobenzyl)-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]pheny-
l]benzimidazole.
[0152] ESI-MS m/z 480, 478 (M-H).sup.-,
C.sub.24H.sub.15.sup.35Cl.sub.2N.s- ub.3O.sub.2S=479
Example 15
Preparation of
1-(3,4-dichlorobenzyl)-5,6-dimethyl-2-[4-[(2,4-dioxothiazol-
idin-5-ylidene)methyl]phenyl]benzimidazole
[0153] 27
[0154] 5,6-Dimethyl-2-(4-formylphenyl)benzimidazole was prepared
from terephthalaldehyde mono-(diethyl acetal) and
5,6-dimethyl-1,2-phenylenedi- amine in a similar manner as
described in Example 13.
[0155] General Procedure 3 was then followed to obtain
1-(3,4-dichlorobenzyl)-5,6-dimethyl-2-(4-formylphenyl)benzimidazole
from 5,6-dimethyl-2-(4-formylphenyl)benzimidazole and
3,4-dichlorobenzyl chloride.
[0156] General Procedure 1 was then followed to obtain
1-(3,4-dichlorobenzyl)-5,6-dimethyl-2-[4-[(2,4-dioxothiazolidin-5-ylidene-
)methyl]phenyl]benzimidazole.
[0157] ESI-MS m/z 508, 506 (M-H).sup.-,
C.sub.26H.sub.19.sup.35Cl.sub.2N.s- ub.3O.sub.2S=507
Example 16
Preparation of
5,6-dichloro-1-(3,4-dichlorobenzyl)-2-[4-[(2,4-dioxothiazol-
idin-5-ylidene)methyl]phenyl]benzimidazole
[0158] 28
[0159]
5,6-Dichloro-1-(3,4-dichlorobenzyl)-2-(4-formylphenyl)benzimidazole
was prepared from 5,6-dichloro-2-(4-formylphenyl)benzimidazole
obtained in Example 13 and 3,4-diclorobenzyl chloride by following
General Procedure 3.
[0160] General Procedure 1 was then followed to obtain
5,6-dichloro-1-(3,4-dichlorobenzyl)-2-[4-[(2,4-dioxothiazolidin-5-ylidene-
)methyl]phenyl]benzimidazole.
[0161] ESI-MS m/z 548, 546 (M-H).sup.-,
C.sub.24H.sub.13.sup.35Cl.sub.4N.s- ub.3O.sub.2S=547
Example 17
Preparation of
5,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]p-
henyl]-1-methylbenzimidazole
[0162] 29
[0163] 5,6-Dichloro2-(4-formylphenyl)-1-methylbenzimidazole was
prepared from 5,6-dichloro-2-(4-formylphenyl)benzimidazole obtained
in Example 13 and iodomethane by following General Procedure 3.
[0164] General Procedure 1 was then followed to obtain
5,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]phenyl]-1-methy-
lbenzimidazole.
[0165] ESI-MS m/z 404, 402 (M-H).sup.-,
C.sub.18H.sub.11.sup.35Cl.sub.2N.s- ub.3O.sub.2S=403
Example 18
Preparation of
1-butyl-5,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)-
methyl]phenyl]benzimidazole
[0166] 30
[0167] 1-Butyl-5,6-dichloro2-(4-formylphenyl)benzimidazole was
prepared from 5,6-dichloro-2-(4-formylphenyl)benzimidazole obtained
in Example 13 and 1-iodobuthane by following General Procedure
3.
[0168] General Procedure 1 was then followed to obtain
1-butyl-5,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]phenyl]-
benzimidazole.
[0169] ESI-MS m/z 446, 444 (M-H).sup.-,
C.sub.21H.sub.17.sup.35Cl.sub.2N.s- ub.3O.sub.2S=445
Example 19
Preparation of
1-allyl-5,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)-
methyl]-phenyl]benzimidazole
[0170] 31
[0171] 1-Allyl-5,6-dichloro2-(4-formylphenyl)benzimidazole was
prepared from 5,6-dichloro-2-(4-formylphenyl)benzimidazole obtained
in Example 13 and allylbromide by following General Procedure
3.
[0172] General Procedure 1 was then followed to obtain
1-allyl-5,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]phenyl]-
benzimidazole.
[0173] ESI-MS m/z 430, 428 (M-H).sup.-,
C.sub.20H.sub.13.sup.35Cl.sub.2N.s- ub.3O.sub.2S=429
Example 20
Preparation of ethyl
[5,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)m-
ethyl]phenyl]-benzimidazol-1 -yl]acetate
[0174] 32
[0175] Ethyl
[5,6-dichloro2-(4-formylphenyl)benzimidazol-1-yl]acetate was
prepared from 5,6-dichloro-2-(4-formylphenyl)benzimidazole obtained
in Example 13 and ethyl bromoacetate by following General Procedure
3.
[0176] General Procedure 1 was then followed to obtain ethyl
[5,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]phenyl]benzimi-
dazol-1-yl]acetate.
[0177] ESI-MS m/z 476, 474 (M-H).sup.-,
C.sub.21H.sub.15.sup.35Cl.sub.2N.s- ub.3O.sub.4S=475
Example 21
Preparation of
5,6-dichloro-1-(2-dimethyaminoethyl)-2-[4-[(2,4-dioxothiazo-
lidin-5-ylidene)methyl]phenyl]benzimidazole
[0178] 33
[0179]
5,6-Dichloro-1-(2-dimethylaminoethyl)-2-(4-formylphenyl)benzimidazo-
le was prepared from 5,6-dichloro-2-(4-formylphenyl)benzimidazole
obtained in Example 13 and 2-(dimethylamino)ethyl chloride
hydrochloride by following General Procedure 3.
[0180] General Procedure 1 was then followed to obtain
5,6-dichloro-1-(2-dimethyaminoethyl)-2-[4-[(2,4-dioxothiazolidin-5-yliden-
e)methyl]phenyl]benzimidazole.
[0181] ESI-MS m/z 461, 459 (M-H).sup.-,
C.sub.21H.sub.18.sup.35Cl.sub.2N.s- ub.4O.sub.2S=460
Example 22
Preparation of
1-benzyl-5,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene-
)methyl]phenyl]benzimidazole
[0182] 34
[0183] 1-Benzyl-5,6-dichloro2-(4-formylphenyl)benzimidazole was
prepared from 5,6-dichloro-2-(4-formylphenyl)benzimidazole obtained
in Example(138326) and benzyl bromide by following General
Procedure 3.
[0184] General Procedure 1 was then followed to obtain
1-benzyl-5,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]phenyl-
]benzimidazole.
[0185] ESI-MS m/z 480, 478 (M-H).sup.-,
C.sub.24H.sub.15.sup.35Cl.sub.2N.s- ub.3O.sub.2S=479
Example 23
Preparation of methyl
4-[5,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-yliden-
e)methyl]phenyl]benzimidazol-1-ylmethyl]benzoate
[0186] 35
[0187] Methyl
4-[5,6-dichloro2-(4-formylphenyl)benzimidazol-1-ylmethyl]ben- zoate
was prepared from 5,6-dichloro-2-(4-formylphenyl)benzimidazole
obtained in Example 13 and methyl 4-bromomethylbenzoate by
following General Procedure 3.
[0188] General Procedure 1 was then followed to obtain methyl
4-[5,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]phenyl]benzi-
midazol-1 -ylmethyl]benzoate.
[0189] ESI-MS m/z 538, 536 (M-H).sup.-,
C.sub.26H.sub.17.sup.35Cl.sub.2N.s- ub.3O.sub.4S=537
Example 24
Preparation of
5,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]p-
henyl]-1-(2-naphthylmethyl)benzimidazole
[0190] 36
[0191]
5,6-dichloro2-(4-formylphenyl)-1-(2-naphtylmethyl)benzimidazole was
prepared from 5,6-dichloro-2-(4-formylphenyl)benzimidazole obtained
in Example 13 and 2-chloromethylnaphthalene by following General
Procedure 3.
[0192] General Procedure 1 was then followed to obtain
5,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]phenyl]-1-(2-na-
phthylmethyl)benzimidazole.
[0193] ESI-MS m/z 530, 528 (M-H).sup.-,
C.sub.28H.sub.17.sup.35Cl.sub.2N.s- ub.3S=529
Example 25
Preparation of
1-(5-chlorothiophen-2-ylmethyl)-5,6-dichloro-2-[4-[(2,4-dio-
xothiazolidin-5-ylidene)methyl]phenyl]benzimidazole
[0194] 37
[0195]
1-(5-Chlorothiophen-2-ylmethyl)-5,6-dichloro2-(4-formylphenyl)benzi-
midazole was prepared from
5,6-dichloro-2-(4-formylphenyl)benzimidazole obtained in Example 13
and 2-chloro-5-chloromethylthiophene by following General Procedure
3.
[0196] General Procedure 1 was then followed to obtain
1-(5-chlorothiophen-2-ylmethyl)-5,6-4-[(2,4-dioxothiazolidin-5-ylidene)me-
thyl]phenyl]benzimidazole.
[0197] ESI-MS m/z 520, 518 (M-H).sup.-,
C.sub.22H.sub.12.sup.35Cl.sub.3N.s- ub.3O.sub.2S.sub.2=519
Example 26
Preparation of
5,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]p-
henyl]-1-(quinolin-2-ylmethyl)benzimidazole
[0198] 38
[0199]
5,6-dichloro2-(4-formylphenyl)-1-(quinolin-2-ylmethyl)benzimidazole
was prepared from 5,6-dichloro-2-(4-formylphenyl)benzimidazole
obtained in Example 13and 2-chloromethylquinoline by following
General Procedure 3.
[0200] General Procedure 1 was then followed to obtain
5,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]phenyl]-1-(quin-
olin-2-ylmethyl)benzimidazole.
[0201] ESI-MS m/z 531, 529 (M-H).sup.-,
C.sub.27H.sub.16.sup.35Cl.sub.2N.s- ub.4O.sub.2S=530
Example 27
Preparation of
5,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]p-
henyl]-1-(thiazol-4-ylmethyl)benzimidazole
[0202] 39
[0203]
5,6-dichloro2-(4-formylphenyl)-1-(thiazol-4-ylmethyl)benzimidazole
was prepared from 5,6-dichloro-2-(4-formylphenyl)benzimidazole
obtained in Example(138326) and 4-chloromethylthiazole by following
General Procedure 3.
[0204] General Procedure 1 was then followed to obtain
5,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]phenyl]-1-(thia-
zol-4-ylmethyl)benzimidazole.
[0205] ESI-MS m/z 487, 485 (M-H).sup.-,
C.sub.21H.sub.12.sup.35Cl.sub.2N.s- ub.4O.sub.2S.sub.2=486
Example 28
Preparation of
2-[4-[(2,4-dioxothiazolidin-5-yl)methyl]phenyl]-5,6-dichlor-
obenzimidazole
[0206] 40
[0207] A suspension of
2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]phenyl-
]-5,6-dichlorobenzimidazole (100 mg, 0.256 mmol) and magnesium
turnings (125 mg, 5.1 mmol) in methanol (20 mL) was stirred for 40
h at room temperature. Water and hydrochloric acid (1 mol/L) was
added, and the mixture was extracted with ethyl acetate. The
organic layer was washed washed with water and brine, dried on
anhydrous sodium sulfate, and the solvent was removed under reduced
pressure. The residue was purified by silica gel column
chromatography (10:1 chloroform/methanol) and triturated with
ethanol to give 2-[4-[(2,4-dioxothiazolidin-5-yl)methyl]p-
henyl]-5,6-dichlorobenzimidazole (20 mg, 20%).
[0208] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 3.19 (dd,
J=14.1, 9.0 Hz, 1H), 3.42 (dd, J=14.1, 4.3 Hz, 1H), 4.95 (dd,
J=9.0, 4.3 Hz, 1H), 7.40 (d, J=8.2 Hz, 2H), 7.71 (s, 1H), 8.06 (d,
J=8.6 Hz, 2H), 12.1 (br s, 1H), 13.2 (br s, 1H)
[0209] ESI-MS m/z 392, 390 (M-H).sup.-,
C.sub.17H.sub.11.sup.35Cl.sub.2N.s- ub.3O.sub.2S=391
Example 29
Preparation of
5,6-dichloro-2-[3-bromo-5-[(2,4-dioxothiazolidin-5-ylidene)-
methyl]thiophen-2-yl]benzimidazole
[0210] 41
[0211] To a mixture of 4-bromothiophene-2-carboxaldehyde (1.91 g,
10.0 mmol) and 2,4-thiazolidinedione (1.40 g, 12.0 mmol) in ethanol
(30 mL) was added piperidine (1.0 mL, 10.0 mmol). The reaction
mixture was heated at reflux for 3 h, then cooled on an ice-bath,
and the precipitated products were collected by filtration. The
cake was washed with 40% ethanol and dried to afford
5-(4-bromothiophen-2-ylmethylene)-2,4-thiazol- idinedione (2.53 g,
87%).
[0212] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.63 (s,
1H), 7.95 (s, 1H), 8.04 (s, 1H)
[0213] To a solution of diisopropylamine (0.46 mL, 3.3 mmol) in
tetrahydrofuran (5 mL) was added n- butyllithium (1.6 mol/L in
hexanes; 1.9 mL, 3.0 mmol) at 0_C under nitrogen atmosphere, and
the reaction mixture was cooled to -78_C.
5-(4-Bromothiophen-2-ylmethylene)-2,4-thiazo- lidinedione (290 mg,
1.00 mmol) in tetrahydrofuran (8 mL) was added dropwise followed by
the addition of dimethylformamide (0.194 mL, 2.5 mmol), and the
mixture was stirred for 10 min. Water and hydrochloric acid (1
mol/L) were added, and the mixture was extracted with ethyl
acetate. The organic layer was washed with water and brine, dried
on anhydrous sodium sulfate. The solvent was evaporated under
reduced pressure, and the residue was triturated with ethyl
acetate/hexane to afford
5-(3-bromo-2-folmylthiophen-5-ylmethylene)-2,4-thiazolidinedione
(178 mg, 56%).
[0214] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.80 (s,
1H), 7.96 (s, 1H), 9.83 (s, 1H)
[0215]
5,6-Dichloro-2-[3-bromo-5-[(2,4-dioxothiazolidin-5-ylidene)methyl]t-
hiophen-2-yl]benzimidazole was prepared from
5-(3-bromo-2-folmylthiophen-5- -ylmethylene)-2,4-thiazolidinedione
and 4,5-dichloro-1,2-phenylenediamine by following General
Procedure 2.
[0216] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.78 (s,
1H), 7.80 (br s, 1H), 7.95 (br s, 1H), 7.98 (s, 1H), 12.7 (br s,
1H), 12.9 (br s, 1H)
[0217] ESI-MS m/z 478, 476, 474 (M+H).sup.+,
C.sub.15H.sub.6.sup.79Br.sup.-
35Cl.sub.2N.sub.3O.sub.2S.sub.2=473
Example 30
Preparation of
5,6-dichloro-2-[3-bromo-5-[(2,4-dioxothiazolidin-5-ylidene)-
methyl]furan-2-yl]benzimidazole
[0218] 42
[0219] To a solution of 5-bromofuran-2-carboxaldehyde (350 mg, 2.00
mmol) in methanol (15 mL) was added p-toluenesulfonic acid
monohydrate (38 mg, 0.20 mmol), and the mixture was heated at
reflux for 2 h. The reaction mixture was cooled to room
temperature, and aqueous sodium bicarbonate was added. The mixture
was extracted with ether, and the organic layer was washed with
brine, dried on anhydrous sodium sulfate. The solvent was
evaporated under reduced pressure to afford
2-bromo-5-dimethoxymethylfura- n (274 mg, 62%).
[0220] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. (ppm) 3.32 (s,
6H), 5.35 (s, 1H), 6.25 (d, J=3.1 Hz, 1H), 6.36 (dd, J=3.6, 0.6 Hz,
1H) p To a solution of 2-bromo-5-dimethoxymethylfuran (273 mg, 1.24
mmol) in tetrahydrofuran (7 mL) were added n-BuLi (1.6 mol/L in
hexane; 1.6 mL, 2.5 mmol) and dimethylformamide (0.25 mL, 3.0 mmol)
at -78.degree. C. under nitrogen atmosphere, and the mixture was
stirred for 10 min. Water was added and the mixture was extracted
with ether. The organic layer was washed with water and brine,
dried on anhydrous sodium sulfate, and the solvent was removed
under reduced pressure. To the residue were added ethanol (8 mL),
2,4-thiazolidinedione (129 mg, 1.1 mmol), and piperidine (0.092 mL,
0.92 mmol), and the mixture was heated to reflux for 1 h. Water and
hydrochloric acid (1 mol/L; 1 mL) were added, and the mixture was
extracted with ethyl acetate. The organic layer was washed washed
with water and brine, dried on anhydrous sodium sulfate, and the
solvent was removed under reduced pressure. The residue was
purified by silica gel column chromatography (9:1
chloroform/acetonitrile) gave
5-[(5-dimethoxymethyl-2-furanyl)methylene]-2,4-thiazolidinedione
(91 mg, 2 steps 27%).
[0221] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 3.26 (s,
6H), 5.50 (s, 1H), 6.64 (d, J=3.7 Hz, 1H), 7.01 (d, J=3.4 Hz, 1H),
7.54 (s, 1H), 12.4 (br s, 1H)
[0222] ESI-MS m/z 268 (M-H.sup.-, C.sub.11H.sub.11NO.sub.5S=269
[0223] To a solution of
5-[(5-dimethoxymethyl-2-furanyl)methylene]-2,4-thi- azolidinedione
(87 mg, 0.32 mmol) in tetrahydrofuran (5 mL) was added hydrochloric
acid (1 mol/L; 0.4 mL), and the mixture was stirred for 1.5 h at
room temperature. Water was added and the precipitated product was
collected by filtration to afford
5-[(4-formylfuranyl)methylene]-2,4-thaz- olidinedione (40 mg,
56%).
[0224] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.21 (d,
J=4.0 Hz, 1H), 7.63 (d, J=4.0 Hz, 1H), 7.65 (s, 1H), 9.64 (s, 1H),
12.6 (br s, 1H)
[0225] ESI-MS m/z 222 (M-H).sup.-, C.sub.9H.sub.5NO.sub.4S=223
[0226]
5,6-Dichloro-2-[5-[(2,4-dioxothiazolidin-5-ylidene)methyl]furan-2-y-
l]benzimidazole was prepared from
5-[(4-formylfuranyl)methylene]-2,4-thazo- lidinedione and
4,5-dichloro-1,2-phenylenediamine by following General Procedure 2
(48% yield).
[0227] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.24 (d,
J=3.9 Hz, 1H), 7.43 (d, J=3.9 Hz, 1H), 7.63 (s, 1H), 7.76 (br s,
1H), 7.96 (br s, 1H), 12.5 (br s, 1H), 13.2 (br s, 1H)
[0228] ESI-MS m/z 380, 378 (M-H).sup.-,
C.sub.15H.sub.7.sup.35Cl.sub.2N.su- b.3O.sub.3S=379
Example 31
Preparation of
2-[3-Bromo-2-[(2,4-dioxothiazolidin-5-ylidene)methyl]thioph-
en-5-yl]-5,6-dichlorobenzimidazole
[0229] 43
[0230] 2-(4-Bromothiophen-2-yl)-5,6-dichlorobenzimidazole was
prepared from 4-bromo-2-thiophenecarboxaldehyde and
4,5-dichloro-1,2-phenylenediam- ine by following General Procedure
2.
[0231] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.82 (br
s, 2H), 7.83 (d, J=1.6 Hz, 1H), 7.87 (d, J=1.6 Hz, 1H), 13.3 (br s,
1H)
[0232] ESI-MS m/z 349, 347, 345 (M-H).sup.-,
C.sub.11H.sub.5.sup.79Br.sup.- 35Cl.sub.2N.sub.2S=346
[0233] To a solution of diisopropylamine (0.67 mL, 4.8 mmol) in
tetrahydrofuran (6 mL) was added n- butyllithium (1.6 mol/L in
hexanes; 2.7 mL, 4.3 mmol) at 0.degree. C. under nitrogen
atmosphere, and the reaction mixture was cooled to -78.degree. C.
2-(4-Bromothiophen-2-yl)-5,- 6-dichlorobenzimidazole (500 mg, 1.44
mmol) in tetrahydrofuran (14 mL) was added dropwise followed by the
addition of dimethylformamide (0.33 mL, 4.3 mmol), and the mixture
was stirred for 30 min. Water and hydrochloric acid (1 mol/L) were
added, and the mixture was extracted with ethyl acetate. The
organic layer was washed with water and brine, dried on anhydrous
sodium sulfate. The solvent was removed under reduced pressure, and
the residue was triturated with ethyl acetate to afford
2-(3-bromo-2-formylthiophen-5-yl)-5,6-dichlorobenzimidazole (296
mg, 55%).
[0234] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.90 (br
s, 2H), 7.99 (s, 1H), 9.86 (s, 1H), 13.6 (br s, 1H)
[0235] ESI-MS m/z 377, 375, 373 (M-H).sup.-,
C.sub.12H.sub.5.sup.79Br.sup.- 35Cl.sub.2N.sub.2OS=344
[0236] General Procedure 1 was then followed to obtain
2-[3-Bromo-2-[(2,4-dioxothiazolidin-5-ylidene)methyl]thiophen-5-yl]-5,6-d-
ichlorobenzimidazole.
[0237] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.71 (s,
1H), 7.85 (br s, 2H), 7.98 (s, 1H), 12.8 (br s, 1H), 13.5 (br s,
1H)
[0238] ESI-MS m/z 476, 474, 472 (M-H).sup.-,
C.sub.15H.sub.6.sup.79Br.sup.-
35Cl.sub.2N.sub.3O.sub.2S.sub.2=473
Example 32
Preparation of
5,6-dichloro-2-[2-[(2,4-dioxothiazolidin-5-ylidene)methyl]t-
hiophen-5-yl]benzimidazole
[0239] 44
[0240] 2-(2-Bromothiophen-5-yl)-5,6-dichlorobenzimidazole was
obtained from 5-bromo-2-thiophenecarboxaldehyde and
4,5-dichloro-1,2-phenylenediam- ine by following General Procedure
2.
[0241] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.34 (d,
J=3.9 Hz, 1H), 7.65 (d, J=3.9 Hz, 1H), 7.79 (br s, 2H), 13.3 (br s,
1H)
[0242] ESI-MS m/z 349, 347, 345 (M-H).sup.-,
C.sub.11H.sub.5.sup.79Br.sup.- 35Cl.sub.2N.sub.2S=346
[0243] To a solution of
2-(2-bromothiophen-5-yl)-5,6-dichlorobenzimidazole (100 mg, 0.287
mmol) in tetrahydrofuran (6 mL) was added sodium hydryde (60% oil
dispersion; 14 mg, 0.35 mmol) at 0.degree. C. under nitrogen
atmosphere. After cooling to -78.degree. C., n- butyllithium (1.6
mol/L in hexane; 0.36 mL, 0.57 mmol), dimethylformamide (0.085 mL,
1.1 mmol) were added, and the mixture was stirred for 10 min. Water
was added, and the mixture was extracted with ethyl acetate. The
organic layer was washed with water and brine and dried on
anhydrous sodium sulfate. The solvent was evaporated under reduced
pressure, and the residue was purified by silica gel column
chromatography (19:1 to 9:1 chloroform/acetonitrle) to afford
2-(2-formylthiophen-5-yl)-5,6-dichlorob- enzimidazole (36 mg,
42%).
[0244] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.80 (br
s, 1H), 7.95 (br s, 1H), 7.97 (dd, J=3.9, 1.2 Hz, 1H), 8.07 (dd,
J=3.9, 1.2 Hz, 1H), 9.94 (d, J=1.2 Hz, 1H), 13.6 (br s, 1H)
[0245] ESI-MS m/z 297, 295 (M-H).sup.-,
C.sub.12H.sub.6.sup.35Cl.sub.2N.su- b.2OS=296
[0246] General Procedure 1 was then followed to obtain
5,6-dichloro-2-[2-[(2,4-dioxothiazolidin-5-ylidene)methyl]thiophen-5-yl]b-
enzimidazole.
[0247] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.63 (d,
J=3.9 Hz, 1H), 7.75 (br s, 1H), 7.89 (d, J=3.9 Hz, 1H), 7.90 (s,
1H), 8.20 (br s, 1H), 12.3 (br s, 1H), 13.5 (br s, 1H)
[0248] ESI-MS m/z 396, 394 (M-H).sup.-,
C.sub.15H.sub.7.sup.35Cl.sub.2N.su- b.3O.sub.2S.sub.2=395
Example 33
Preparation of
2-[2-[(2,4-dioxothiazolidin-5-ylidene)methyl]-1-methylpyrro-
l-5-yl]-5,6-dichlorobenzimidazole
[0249] 45
[0250] 2-(1-Methylpyrrol-2-yl)-5,6-dichlorobenzimidazole was
prepared from 1-methyl-2-pyrrolecarboxaldehyde and
4,5-dichloro-1,2-phenylenediamine by following General Procedure
2.
[0251] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 4.02 (s,
3H), 6.13 (dd, J=3.9, 2.7 Hz, 1H), 6.88 (dd, J=3.9, 2.0 Hz, 1H),
7.00 (t, J=2.0 Hz, 1H), 7.58 (s, 1H), 7.79 (s, 1H), 12.7 (br s,
1H)
[0252] To a solution of
2-(1-methylpyrrol-2-yl)-5,6-dichlorobenzimidazole (65 mg, 0.24
mmol) in dimethylformamide (2 mL) was added phoshorus oxychloride
(0.067 mL, 0.72 mmol), and the mixture was stirred at room
temperature for 24 h. Water and aqueous sodium hydroxide (1.0
mol/L) were added, and the mixture was extracted with ethyl
acetate. The organic layer was washed with water and brine and
dried on anhydrous sodium sulfate. The solvent was removed under
reduced pressure to afford
2-(2-formyl-1-methylpyrrol-5-yl)-5,6-dichlorobenzimidazole (50 mg,
71%).
[0253] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 4.36 (s,
3H), 7.02 (d, J=4.3 Hz, 1H), 7.14 (d, J =4.3 Hz, 1H), 7.72 (br s,
1H), 7.96 (br s, 1H), 9.65 (s, 1H), 13.2 (br s, 1H)
[0254] ESI-MS m/z 294, 292 (M-H).sup.-,
C.sub.13H.sub.9.sup.35Cl.sub.2N.su- b.3O=293
[0255] General Procedure 1 was then followed to afford
2-[2-[(2,4-dioxothiazolidin-5-ylidene)methyl]-1-methylpyrrol-5-yl]-5,6-di-
chlorobenzimidazole.
[0256] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 4.24 (s,
3H), 6.61 (d, J=4.3 Hz, 1H), 7.12 (d, J=4.3 Hz, 1H), 7.66 (s, 1H),
7.68 (s, 1H), 7.89 (s, 1H), 12.4 (br s, 1H), 13.1 (br s, 1H)
[0257] ESI-MS m/z 393, 391 (M-H).sup.-,
C.sub.16H.sub.10.sup.35Cl.sub.2N.s- ub.4O.sub.2S=392
Example 34
Preparation of
2-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenyl]benzothia-
zole
[0258] 46
[0259] To a suspension of
5-(4-formylbenzylidene)-2,4-thiazolidinedione (50 mg, 0.21 mmol)
and 2-aminothiophenol (0.025 mL, 0.23 mmol) in ethanol (7 mL) was
heated to reflux for 10 h. 1,4-Benzoquinone (24 mg, 0.22 mmol) was
added, and the mixture was heated additional 1 h. The mixture was
cooled to room temperature, ether (3 mL) was added, and the
precipitated product was collected by filtration to afford
2-[4-(2,4-dioxothiazolidin-- 5-ylidenemethyl)phenyl]benzothiazole
(53 mg, 75%).
[0260] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.45 (ddd,
J=8.2, 7.0, 1.2 Hz, 1H), 7.53 (td, J =7.0, 1.2 Hz, 1H), 7.74 (d,
J=8.6 Hz, 2H), 7.81 (s, 1H), 8.05 (d, J=7.8 Hz, 1H), 8.14 (d, J=8.2
Hz, 1H), 8.19 (d, J=8.6 Hz, 2H), 12.7 (br s, 1H)
[0261] ESI-MS m/z 337 (M-H).sup.-,
C.sub.17H.sub.10N.sub.2O.sub.2S.sub.2=3- 38
Example 35
Preparation of
5,6-dichloro-2-[2-[(2,4-dioxothiazolidin-5-ylidene)methyl]p-
yridin-5-yl]benzimidazole
[0262] 47
[0263] To a solution of 2,5-dibromopyridine (2.37 g, 10.0 mmol) in
tetrahydrofuran (15 mL) was added isopropylmagnesium chloride (2
mol/L in tetrahydrofuran; 5.0 mL, 10 mmol) under nitrogen
atmosphere, and the mixture was stirred at room temperature for 1
h. Dimethylformamide (1.6 mL, 20 mmol) was added, and the mixture
was stirred for additional 15 min. Water was added, and the mixture
was extracted with ethyl acetate twice. The organic layer was
washed with brine and dried on anhydrous sodium sulfate. The
solvent was evaporated under reduced pressure, and the residue was
purified by silica gel column chromatography (2:1 hexane/ethyl
acetate) to afford 2-bromo-5-pyridinecarboxaldehyde (1.23 g,
66%).
[0264] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. (ppm) 7.65 (d,
J=8.2 Hz, 1H), 7.99 (dd, J=8.2, 2.3 Hz, 1H), 8.79 (d, J=2.3 Hz,
1H), 10.1 (s, 1H)
[0265] To a solution of 2-bromo-5-pyridinecarboxaldehyde (1.02 g,
5.48 mmol) in methanol (100 mL) was added p-toluenesulfonic acid
hydrate (1.05 g, 5.53 mmol), and the mixture was heated at reflux
for 1.5 h. Diluted aqueous sodium bicarbonate was added, and the
mixture was extracted with ethyl acetate twice. The organic layer
was washed with brine and dried on anhydrous sodium sulfate. The
solvent was evaporated under reduced pressure to afford
2-bromo-5-dimethoxymethylpyridine (1.19 g, 94%) as an oil.
[0266] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. (ppm) 3.28 (s,
6H), 5.39 (s, 1H), 7.45 (d, J=8.2 Hz, 1H), 7.59 (dd, J=8.2, 2.3 Hz,
1H), 8.39 (d, J=2.7 Hz, 1H)
[0267] To a solution of 2-bromo-5-dimethoxymethylpyridine (1.19 g,
5.13 mmol) in tetrahydrofuran (8 mL) was added isopropylmagnesium
chloride (2 mol/L in tetrahydrofuran; 4.2 mL, 8.4 mmol) under
nitrogen atmosphere, and the mixture was stirred at room
temperature for 3 h. Dimethylformamide (0.774 mL, 10.3 mmol) was
added, and the mixture was stirred for additional 10 min. Water was
added, and the mixture was extracted with ethyl acetate twice. The
organic layer was washed with brine and dried on anhydrous sodium
sulfate. The solvent was evaporated under reduced pressure, and the
residue was purified by silica gel column chromatography (5:1 to
3:1 hexane/ethyl acetate) to afford
5-dimethoxymethyl-2-pyridinecarboxaldehyde (413 mg, 44%).
[0268] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. (ppm) 3.32 (s,
6H), 5.50 (s, 1H), 7.9-8.0 (m, 2H), 8.82 (s, 1H), 10.1 (s, 1H)
[0269] To a mixture of 5-dimethoxymethyl-2-pyridinecarboxaldehyde
(192 mg, 1.06 mmol), 2,4-thiazolidinedione (149 mg, 1.27 mmol) in
ethanol (5 mL) was added piperidine (0.11 mL, 1.1 mmol), and the
mixture was heated to reflux for 2 h. Hydrochloric acid (1 mol/L, 2
mL) was added, and the mixture was extracted with ethyl acetate.
The organic layer was washed with brine and dried on anhydrous
sodium sulfate. The solvent was evaporated under reduced pressure.
To the residue were added tetrahydrofuran (10 mL) and hydrochloric
acid (1 mol/L, 1.2 mL), and the mixture was stirred at room
temperature for 14 h. Water was added and the precipitated product
was collected by filtration to afford
5-(5-formylpyridin-2-ylmethylene)-2,4-dioxothiazolidinedione (169
mg, 68%).
[0270] General Procedure 2 was then followed to obtain
5,6-dichloro-2-[2-[(2,4-dioxothiazolidin-5-ylidene)methyl]pyridin-5-yl]be-
nzimidazole.
[0271] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.78 (br
s, 1H), 7.82 (s, 1H), 7.95 (br s, 1H), 7.96 (d, J=8.2 Hz, 1H), 8.52
(dd, J=8.2, 2.0 Hz, 1H), 9.39 (s, 1H), 12.5 (br s, 1H), 13.5 (br s,
1H)
[0272] ESI-MS m/z 391, 389 (M-H).sup.-,
C.sub.16H.sub.8.sup.35Cl.sub.2N.su- b.4O.sub.2S=390
Example 36
Preparation of
5,6-dichloro-2-[5-[(2,4-dioxothiazolidin-5-ylidene)methyl]p-
yridin-2-yl]benzimidazole
[0273] 48
[0274] A mixture of 5-dimethoxymethyl-2-pyridinecarboxaldehyde
obtained in Example 35 (197 mg, 1.09 mmol),
4,5-diamino-1,2-phenylenediamine (192 mg, 1.09 mmol) and
1,4-benzoquinone (117 mg, 1.09 mmol) in methanol (10 mL) was heated
to reflux for 1 h. The solvent was removed under reduced pressure.
To the residue were added tetrahydrofuran (10 mL) and hydrochloric
acid (1 mol/L, 1.2 mL), and the mixture was stirred at room
temperature for 14 h. Water was added, and the mixture was
extracted with ethyl acetate. The organic layer was washed with
brine and dried on anhydrous sodium sulfate. The solvent was
evaporated under reduced pressure, and the residue was triturated
with ether to afford
2-(5-formylpyridin-2-yl)-5,6-dichlorobenzimidazole (225 mg,
71%).
[0275] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.74 (br
s, 1H), 8.00 (br s, 1H), 8.39 (dd, J=8.2, 1.6 Hz, 1H), 8.44 (d,
J=7.8 Hz, 1H), 9.19 (s, 1H), 10.1 (s, 1H), 12.8 (br s, 1H)
[0276] ESI-MS m/z 292, 290 (M-H).sup.-
C.sub.13H.sub.7.sup.35Cl.sub.2N.sub- .3O=291
[0277] General Procedure 1 was then followed to obtain
5,6-dichloro-2-[5-[(2,4-dioxothiazolidin-5-ylidene)methyl]pyridin-2-yl]be-
nzimidazole.
[0278] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.67 (s,
1H), 7.82 (s, 1H), 7.99 (s, 1H), 8.10 (dd, J=8.6, 2.3 Hz, 1H), 8.37
(d, J=8.6 Hz, 1H), 8.96 (d, J=2.0 Hz, 1H), 12.8 (br s, 1H), 13.5
(s, 1H)
[0279] ESI-MS m/z 391, 389 (M-H).sup.-,
C.sub.16H.sub.8.sup.35Cl.sub.2N.su- b.4O.sub.2S=390
Example 37
Preparation of
5,6-dichloro-2-[4-[2,4-dioxothiazolidin-5-ylidene)methyl]-3-
-fluorophenyl]benzimidazole
[0280] 49
[0281] To a solution of 4-bromo-2-fluorobenzaldehyde (3.97 g, 18.8
mmol) in methanol (200 mL) was added p-toluenesulfonic acid
monohydrate (357 mg,1.88 mmol), and the mixture was heated to
reflux for 4 h. Aqueous sodium carbonate (1 mol/L; 2 mL) was added,
and the mixture was concentrated to a volume of ca. 50 mL and
extracted with ether. The organic layer was washed with brine and
dried on anhydrous sodium sulfate. The solvent was removed under
reduced pressure to afford
4-bromo-1-dimethoxymethyl-2-fluorobenzene (4.56 g, 97%).
[0282] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. (ppm) 3.33 (s,
6H), 5.52 (s, 1H), 7.21 (dd, J=9.6, 1.8 Hz, 1H), 7.28 (dd, J=8.4,
1.8 Hz, 1H), 7.41 (t, J=7.8 Hz, 1H).
[0283] To a solution of 4-bromo-1-dimethoxymethyl-2-fluorobenzene
(4.53 g, 18.2 mmol) in tetrahydrofuran (50 mL) were added
n-butyllithium (1.6 mol/L in hexanes; 15 mL) and dimethylformamide
(2.1 mL, 27 mmol) sequentially at -78.degree. C. under nitrogen
atmosphere, and the mixture was stirred for 15 min. Water was
added, and the mixture was extracted twice with ether. The organic
layer was combined, washed with brine and dried on anhydrous sodium
sulfate. The solvent was removed under reduced pressure, and the
residue was purified by column chromatography (6:1 Hexane/ethyl
acetate) to afford 4-dimethoxymethyl-3-fluorobenzaldehyde (2.26 g,
63%).
[0284] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. (ppm) 3.36 (s,
6H), 5.61 (s, 1H), 7.53 (dd, J=9.8, 1.6 Hz, 1H), 7.65 (dd, J=7.8,
1.6 Hz, 1H), 7.73 (t, J=7.8 Hz, 1H), 9.95 (d, J=2.0 Hz, 1H).
[0285] A mixture of 4-dimethoxymethyl-3-fluorobenzaldehyde (321 mg,
1.62 mmol), 4,5-dichloro-1,2-phenylenediamine (283 mg, 1.60 mg) and
1,4-benzoquinone (173 mg, 1.60 mmol) in methanol (10 mL) was heated
to reflux for 2 h. The solvent was removed under reduced pressure.
To the residue were added tetrahydrofuran (15 mL) and hydrochloric
acid (1 mol/L; 1.6 mL), and the mixture was stirred at room
temperature for 16 h. Water was added and the precipitated products
were collected by filtration, followed by trituration with ether to
afford 5,6-dichloro-2-(3-fluoro-4-formylphenyl)benzimidazole (132
mg, 26%).
[0286] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.89 (br
s, 2H), 7.98 (t, J=7.8 Hz, 1H), 8.06 (dd, J=11.2, 1.2 Hz, 1H), 8.11
(dd, J=7.8 1.2 Hz, 1H), 10.2 (s, 1H)
[0287] ESI-MS m/z 309, 307 (M-H).sup.-
C.sub.14H.sub.7.sup.35Cl.sub.2N.sub- .2O=308
[0288] General Procedure 1 was then followed to obtain
5,6-dichloro-2-[4-[2,4-dioxothiazolidin-5-ylidene)methyl]-3-fluorophenyl]-
benzimidazole
[0289] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.68 (t,
J=8.2 Hz, 1H), 7.70 (s, 1H), 7.78 (br s, 1H), 7.94 (br s, 1H), 8.02
(d, J=11.7 Hz, 1H), 8.09 (d, J=8.6 Hz, 1H), 12.8 (br s, 1H), 13.4
(br s, 1H)
[0290] ESI-MS m/z 408, 406 (M-H).sup.-,
C.sub.17H.sub.8.sup.35Cl.sub.2F N.sub.3O.sub.2S=407
Example 38
Preparation of
5,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]--
2-fluorophenyl]benzimidazole
[0291] 50
[0292] To a mixture of 4-dimethoxymethyl-3-fluorobenzaldehyde
obtained in Example 37 (1.12 g, 5.66 mmol) and 2,4-thiazolidindione
(795 mg, 6.79 mmol) in ethanol (20 mL) was added piperidine (0.68
mL, 6.8 mmol), and the mixture was heated to reflux for 13 h.
Hydrochloric acid (1 mol/L) was added, and the mixture was
extracted with ethyl acetate. The organic layer was combined,
washed with brine, dried on anhydrous sodium sulfate, and
concentrated under reduced pressure. To the residue were added
tetrahydrofuran (25 mL) and hydrchloric acid (1 mol/L; 5 mL), and
the mixture was stirred at room temperature for 20 h. Water was
added, and the precipitated products were collected by filtration
to afford 3-fluoro-4-formylbenzylidene-2,4-thiazolidinedione (696
mg, 49%).
[0293] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.53 (d,
J=7.8 Hz, 1H), 7.60 (dd, J=11.7, 1.2 Hz, 1H), 7.79 (s, 1H), 7.92
(t, J=7.8 Hz, 1H), 10.2 (s, 1H), 12.8 (br s, 1H)
[0294] ESI-MS m/z 250(M-H).sup.- C.sub.11H.sub.6FNO3.sub.3=251
[0295] General Procedure 2 was then followed to obtain
5,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]-2-fluorophenyl-
]benzimidazole (58 mg, 71%).
[0296] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.54 (d,
J=8.6 Hz, 1H), 7.66 (d, J=12.5 Hz, 1H), 7.79 (s, 1H), 7.85 (br s,
2H), 8.30 (t, J=8.2 Hz, 1H), 12.7 (br s, 1H), 12.9 (br s, 1H)
[0297] ESI-MS m/z 408, 406 (M-H).sup.-,
C.sub.17H.sub.8.sup.35Cl.sub.2F N.sub.3O.sub.2S=407
Example 39
Preparation of
5,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]n-
aphth-1-yl]benzimidazole
[0298] 51
[0299] To a solution of 1,4-dibromonaphthalene (1.00 g, 3.50 mmol)
in tetrahydrofuran (15 mL) were added n-butyllithium (1.6 mol/L;
3.1 mL) and dimethylformamide (0.54 mL, 7.0 mmol) sequentially at
=78.degree. C. under nitrogen atmosphere, and the mixture was
stirred for 15 min. Water was added, and the mixture was extracted
with ethyl acetate. The organic layer was combined, washed with
brine and dried on anhydrous sodium sulfate. The solvent was
removed under reduced pressure, and the residue was purified by
column chromatography (8:1 hexane/ethyl acetate) to afford
4-bromo-1-naphthalenecarboxaldehyde (458 mg, 56%).
[0300] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. (ppm) 7.6-7.8 (m,
2H), 7.78 (d, J=7.8 Hz, 1H), 7.94 (d, J=7.8 Hz, 1H), 8.34 (dd,
J=8.2, 1.6 Hz, 1H), 9.25 (dd, J=8.2,1.6 Hz, 1H), 10.3 (s, 1H)
[0301] General Procedure 2 was followed to obtain
5,6-dichloro-2-(4-bromo-- 1-naphthyl)benzimidazole.
[0302] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.8-7.9
(m, 2H), 7.87 (d, J=7.8 Hz, 1H), 7.91 (br s, 2H), 8.07 (d, J=7.8
Hz, 1H), 8.25 (d, J=7.0 Hz, 1H), 9.04 (d, J=8.2 Hz, 1H), 13.3 (br
s, 1H)
[0303] ESI-MS m/z 393, 391, 389 (M-H).sup.-
C.sub.17H.sub.9Br.sup.35Cl.sub- .2N.sub.2=390
[0304] To a solution of
5,6-dichloro-2-(4-bromo-1-naphthyl)benzimidazole (160 mg, 0.408
mmol) in tetrahydrofuran (6 mL) were added n-butyllithium (1.6
mol/L; 0.64 mL) and dimethylformamide (0.077 mL, 1.0 mmol)
sequentially at -78.degree. C. under nitrogen atmosphere, and the
mixture was stirred for 15 min. Water was added, and the mixture
was extracted with ethyl acetate. The organic layer was combined,
washed with brine and dried on anhydrous sodium sulfate. The
solvent was removed under reduced pressure, and the residue was
purified by column chromatography (2:1 hexane/ethyl acetate) to
afford 5,6-dichloro-2-(4-formyl-1-naphthyl)benzi- midazole (72 mg,
52%).
[0305] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.7-7.9
(m, 2H), 7.96 (br s, 2H), 8.17 (d, J=7.4 Hz, 1H), 8.31 (d, J=7.4
Hz, 1H), 9.04 (dd, J=8.6, 1.6 Hz, 1H), 9.23 (d, J=8.6 Hz, 1H), 10.5
(s, 1H), 13.4 (br s, 1H)
[0306] ESI-MS m/z 341, 339 (M-H).sup.-
C.sub.18H.sub.10.sup.35Cl.sub.2N.su- b.2O=340
[0307] General Procedure 1 was then followed to obtain
5,6-dichloro-2-[4-[(2,4-dioxothiazolidin-5-ylidene)methyl]naphth-1-yl]ben-
zimidazole.
[0308] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.6-7.7
(m, 2H), 7.83 (d, J=7.8 Hz, 1H), 7.90 (br s, 2H), 7.99 (s, 1H),
8.03 (d, J=7.8 Hz, 1H), 8.21 (d, J=9.4 Hz, 1H), 9.08 (d, J=9.7 Hz,
1H)
[0309] ESI-MS m/z 440, 438 (M-H).sup.-,
C.sub.21H.sub.11.sup.35Cl.sub.2N.s- ub.3O.sub.2S=439
Example 40
Preparation of
5,6-dichloro-2-[6-[(2,4-dioxothiazolidin-5-ylidene)methyl]n-
aphth-2-yl]benzimidazole
[0310] 52
[0311]
5,6-dichloro-2-[6-[(2,4-dioxothiazolidin-5-ylidene)methyl]naphth-2--
yl]benzimidazole was obtained in a similar manner to Example 39 in
4 steps from 2,6-dibromonaphthalene.
[0312] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.72 (d,
J=8.6 Hz, 1H), 7.76 (br s, 1H), 7.89 (s, 1H), 7.93 (br s, 1H), 8.11
(d, J=8.6 Hz, 1H), 8.15 (d, J=8.6 Hz, 1H), 8.18 (s, 1H), 8.2 (d, J
=8.6 Hz, 1H), 8.71 (s, 1H), 12.6 (br s, 1H), 13.4 (br s, 1H)
[0313] ESI-MS m/z 440, 438 (M-H).sup.-,
C.sub.21H.sub.11.sup.35Cl.sub.2N.s- ub.3O.sub.2S=439
Example 41
Preparation of
5,6-dichloro-2-[2,5-dimethyl-6-[(2,4-dioxothiazolidin-5-yli-
dene)methyl]pyridin-3-yl]benzimidazole
[0314] 53
[0315] To a solution of 2,5-dibromo-3,6-dimethylpyridine (989 mg,
3.75 mmol) in tetrahydrofuran (6 mL) was added isopropylmagnesium
chloride (2 mol/L in tetrahydrofuran; 2.0 mL, 4.0 mmol) under
nitrogen atmosphere, and the mixture was stirred at room
temperature for 1.5 h. Dimethylformamide (0.58 mL, 7.5 mmol) was
added, and the mixture was stirred for additional 10 min. Water was
added, and the mixture was extracted with ethyl acetate. The
organic layer was washed with brine and dried on anhydrous sodium
sulfate. The solvent was evaporated under reduced pressure, and the
residue was purified by silica gel column chromatography (7:1
hexane/ethyl acetate) to afford
6-bromo-2,5-dimethyl-3-pyridinecarboxaldehyde (381 mg, 47%).
[0316] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. (ppm) 2.40 (s,
3H), 2.79 (s, 3H), 7.85 (s, 1H), 10.3 (s, 1H)
[0317] To a solution of
6-bromo-2,5-dimethyl-3-pyridinecarboxaldehyde (378 g, 1.77 mmol) in
methanol (25 mL) was added p-toluenesulfonic acid hydrate (342 mg,
1.80 mmol), and the mixture was heated at reflux for 1 h. Diluted
aqueous sodium bicarbonate was added, and the mixture was extracted
with ethyl acetate. The organic layer was washed with brine and
dried on anhydrous sodium sulfate. The solvent was evaporated under
reduced pressure to afford
2-bromo-5-dimethoxymethyl-3,6-dimethylpyridine (423 g, 92%).
[0318] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. (ppm) 2.32 (s,
3H), 2.48 (s, 3H), 3.27 (s, 6H), 5.37 (s, 1H), 7.62 (S, 1H)
[0319] To a solution of
2-bromo-5-dimethoxymethyl-3,6-dimethylpyridine (382 mg, 1.47 mmol)
in tetrahydrofuran (6 mL) were added n-butyllithium (1.6 mol/L; 1,4
mL, 2.2 mmol) and dimethylformamide (0.23 mL, 3.0 mmol)
sequentially at -78.degree. C. under nitrogen atmosphere, and the
mixture was stirred for 40 min. Water was added, and the mixture
was extracted with ethyl acetate. The organic layer was combined,
washed with brine and dried on anhydrous sodium sulfate. The
solvent was removed under reduced pressure, and the residue was
purified by column chromatography (6:1 hexane/ethyl acetate) to
afford 5-dimethoxymetyl-3,6-dimethyl-2-pyridinec- arboxaldehyde
(109 mg, 35%).
[0320] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. (ppm) 2.60 (s,
6H), 3.30 (s, 6H), 5.45 (s, 1H), 7.73 (s, 1H), 10.1 (s, 1H)
[0321] ESI-MS m/z 210 (M+H).sup.+ C.sub.11H.sub.15NO.sub.3=209
[0322] To a mixture of
5-dimethoxymetyl-3,6-dimethyl-2-pyridinecarboxaldeh- yde (53 mg,
0.25 mmol) and 2,4-thiazolidindione (44 mg, 0.38 mmol) in ethanol
(5 mL) was added piperidine (0.038 mL, 0.38 mmol), and the mixture
was heated to reflux for 16 h. Hydrochloric acid (1 mol/L; 0.4 mL)
and water were added, and the mixture was extracted with ethyl
acetate. The organic layer was washed with brine, dried on
anhydrous sodium sulfate, and concentrated under reduced pressure.
To the residue were added tetrahydrofuran (4 mL) and hydrchloric
acid (1 mol/L; 0.5 mL), and the mixture was stirred at room
temperature for 7 h. Water was added, and the precipitated products
were collected by filtration to afford
5-formyl-3,6-dimethylpyridin-2-ylidene-2,4-thiazolidinedione (43
mg, 66%).
[0323] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 2.46 (s,
3H), 2.77 (s, 3H), 7.75 (s, 1H), 8.05 (s, 1H), 10.2 (s, 1H), 12.5
(br s, 1H)
[0324] ESI-MS m/z 261 (M-H).sup.-
C.sub.12H.sub.10N.sub.2O.sub.3S=262
[0325] General Procedure 2 was then followed to obtain
5,6-dichloro-2-[2,5-dimethyl-6-[(2,4-dioxothiazolidin-5-ylidene)methyl]py-
ridin-3-yl]benzimidazole.
[0326] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 2.48 (s,
3H), 2.84 (s, 3H), 7.76 (s, 2H), 7.95 (s, 1H), 8.10 (S, 1H), 12.4
(br s, 1H), 13.1 (br s, 1H)
[0327] ESI-MS m/z 419, 417 (M-H).sup.-,
C.sub.18H.sub.12.sup.35Cl.sub.2N.s- ub.4O.sub.2S=418
Example 42
Preparation of
2-[3,6-dimethyl-5-(2,4-dioxothiazolidin-5-ylidenemethyl)pyr-
idin-2-yl]-5,6-dichlorobenzimidazole
[0328] 54
[0329] A mixture of
5-dimethoxymetyl-3,6-dimethyl-2-pyridinecarboxaldehyde obtained in
Example 41 (57 mg, 0.27 mmol), 4,5-diamino-1,2-phenylenediami- ne
(48 mg, 0.27 mmol) and 1,4-benzoquinone (29 mg, 0.27 mmol) in
methanol (5 mL) was heated to reflux for 6 h. The solvent was
removed under reduced pressure. To the residue were added
tetrahydrofuran (5 mL) and hydrochloric acid (1 mol/L, 0.54 mL),
and the mixture was stirred at room temperature for 18 h. The
mixture was neutralized by aqueous sodium hydroxide. Water was
further added and the precipitated products were collected by
filtration followed by trituration with ether to afford
5,6-dichloro-2-(5-formyl-3,6-dimethylpyridin-2-yl)benzimidazole (43
mg, 50%).
[0330] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 2.81 (s,
3H), 2.84 (s, 3H), 7.74 (s, 1H), 8.00 (s, 1H), 8.14 (s, 1H), 10.2
(s, 1H), 13.1 (br s, 1H)
[0331] ESI-MS m/z 320, 318 (M-H).sup.-
C.sub.15H.sub.11.sup.35Cl.sub.2N.su- b.3O=319
[0332] General Procedure 1 was then followed to obtain
2-[3,6-dimethyl-5-(2,4-dioxothiazolidin-5-ylidenemethyl)pyridin-2-yl]-5,6-
-dichlorobenzimidazole.
[0333] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 2.65 (s,
3H), 2.79 (s, 3H), 7.67 (s, 1H), 7.71 (s, 1H), 7.78 (s, 1H), 7.97
(s, 1H), 12.7 (br s, 1H), 13.0 (br s,1 H)
[0334] ESI-MS m/z 419, 417 (M-H).sup.-,
C.sub.18H.sub.12.sup.35Cl.sub.2N.s- ub.4O.sub.2S=418
Example 43
Preparation of
5,6-dichloro-2-[2-[(2,4-dioxothiazolidin-5-ylidene)methyl]q-
uinolin-6-yl]benzimidazole
[0335] 55
[0336] To a solution of 6-bromoquinaldine (111 mg, 0.500 mmol) in
1,4-dioxane (8 mL) was added selenium dioxide (111 mg, 1.00 mmol),
and the mixture was heated at 100.degree. C. for 1 h. The reaction
mixture was diluted with ethyl acetate, filtered through Celite
pad, and concentrated under reduced pressure. To the residue were
added methanol (15 mL) and p-toluenesulfonic acid monohydrate (99
mg, 0.52 mmol), and the mixture was heated to reflux for 1.5 h.
Diluted aqueous sodium hydroxide was added, and the mixture was
extracted with ethyl acetate. The organic layer was washed with
brine and dried on anhydrous sodium sulfate. The solvent was
evaporated under reduced pressure to afford
6-bromo-2-(dimethoxymethyl)quinoline (127 mg, 90%).
[0337] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. (ppm) 3.43 (s,
6H), 5.44 (s, 1H), 7.65 (d, J=8.6 Hz, 1H), 7.75 (dd, J=9.0, 2.0 Hz,
1H), 7.96 (d, J=2.0 Hz, 1H), 7.99 (d, J=9.0 Hz, 1H), 8.09 (d, J=8.6
Hz, 1H)
[0338] ESI-MS m/z 284, 282 (M+H).sup.+
C.sub.12H.sub.12BrNO.sub.2=281
[0339] To a solution of 6-bromo-2-(dimethoxymethyl)quinoline (1.11
g, 3.94 mmol) in tetrahydrofuran (20 mL) were added n-butyllithium
(1.6 mol/L; 3.7 mL, 5.9 mmol) and dimethylformamide (0.61 mL, 7.9
mmol) sequentially at -78.degree. C. under nitrogen atmosphere, and
the mixture was stirred for 10 min. Water was added, and the
mixture was extracted with ethyl acetate. The organic layer was
combined, washed with brine and dried on anhydrous sodium sulfate.
The solvent was removed under reduced pressure, and the residue was
purified by column chromatography (3:1 hexane/ethyl acetate) to
afford 2-dimethoxymethyl-6-quinolinecarboxaldehyde (344 mg,
38%).
[0340] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. (ppm) 3.46 (s,
6H), 5.47 (s, 1H), 7.75 (d, J=8.6 Hz, 1H), 8.17 (dd, J=9.0, 2.0 Hz,
1H), 8.22 (d, J=9.0 Hz, 1H), 8.32 (s, 1H), 8.33 (s, 1H), 10.2 (s,
1H).
[0341] A mixture of 2-dimethoxymethyl-6-quinolinecarboxaldehyde
(162 mg, 0.701 mmol), 4,5-diamino-1,2-phenylenediamine (124 mg,
0.701 mmol) and 1,4-benzoquinone (75 mg, 0.70 mmol) in ethanol (5
mL) was heated to reflux for 2 h. The solvent was removed under
reduced pressure. To the residue were added tetrahydrofuran (10 mL)
and hydrochloric acid (1 mol/L, 2 mL), and the mixture was heated
to reflux for 2 h. The mixture was neutralized by aqueous sodium
hydroxide. Water was further added and the precipitated products
were collected by filtration followed by trituration with ether to
afford 5,6-dichloro-2-(2-formylquinoline-6-yl)b- enzimidazole (139
mg, 58%).
[0342] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.86 (br
s, 2H), 8.00 (d, J=8.6 Hz, 1H), 8.32 (d, J=8.6 Hz, 1H), 8.57 (d,
J=8.6 Hz, 1H), 8.66 (d, J=8.2 Hz, 1H), 8.83 (s, 1H), 10.1 (s, 1H),
13.6 (br s, 1H).
[0343] General Procedure 1 was then followed to obtain
5,6-dichloro-2-[2-[(2,4-dioxothiazolidin-5-ylidene)methyl]quinolin-6-yl]b-
enzimidazole.
[0344] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.84 (br
s, 2H), 7.94 (s, 1H), 7.96 (d, J=8.6 Hz, 1H), 8.19 (d, J=9.0 Hz,
1H), 8.51 (d, J=9.0, 1H), 8.53 (d, J=9.0 Hz, 1H), 8.73 (s, 1H),
12.5 (br s, 1H)
[0345] ESI-MS m/z 441, 439 (M-H).sup.-,
C.sub.20H.sub.10.sup.35Cl.sub.2N.s- ub.4O.sub.2S=440
Example 44
Preparation of
5,6-dichloro-2-[6-[(2,4-dioxothiazolidin-5-ylidene)methyl]q-
uinolin-2-yl]benzimidazole
[0346] 56
[0347] To a mixture of 2-dimethoxymethyl-6-quinolinecarboxaldehyde
obtained in Example 43 (181 mg, 0.783 mmol), 2,4-thiazolidinedione
(137 mg, 1.17 mmol) in ethanol (5 mL) was added piperidine (0.117
mL, 1.17 mmol), and the mixture was heated to reflux for 26 h.
Hydrochloric acid (1 mol/L, 1.2 mL) and water were added, and the
mixture was extracted with ethyl acetate. The organic layer was
washed with brine and dried on anhydrous sodium sulfate. The
solvent was evaporated under reduced pressure. To the residue were
added tetrahydrofuran (10 mL) and hydrochloric acid (1 mol/L, 2
mL), and the mixture was heated to reflux for 1 h. The mixture was
neutralized by aqueous sodium hydroxide. Water was further added
and the precipitated products were collected by filtration followed
by trituration with ether to afford
2-formylquinolin-6-ylmethylene-2,4-thiazolidindione (80 mg,
36%).
[0348] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.94 (s,
1H), 7.99 (d, J=8.6 Hz, 1H), 8.03 (d, J =8.6 Hz, 1H), 8.27 (d,
J=8.6 Hz, 1H), 8.28 (s, 1H), 8.68 (d, J=8.2 Hz, 1H), 10.1 (s, 1H),
12.7 (br s, 1H)
[0349] ESI-MS m/z 283 (M-H).sup.-,
C.sub.14H.sub.8N.sub.2O.sub.2S=284
[0350] General Procedure 2 was then followed to obtain
5,6-dichloro-2-[6-[(2,4-dioxothiazolidin-5-ylidene)methyl]quinolin-2-yl]b-
enzimidazole.
[0351] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.70 (br
s, 1H), 7.88 (s, 1H), 7.93 (dd, J=9.0, 1.6 Hz, 1H), 7.97 (br s,
1H), 8.13 (d, J=9.0 Hz, 1H), 8.17 (s, 1H), 8.37 (d, J=8.6 Hz, 1H),
8.57 (d, J=8.6 Hz, 1H), 12.7 (br s, 1H), 13.4 (br s, 1H)
[0352] ESI-MS m/z 441, 439 (M-H).sup.-,
C.sub.20H.sub.10.sup.35Cl.sub.2N.s- ub.4O.sub.2S=440
Example 45
Preparation of
5,6-dichloro-2-[5-[(2,4-dioxothiazolidin-5-ylidene)methyl]i-
ndol-2-yl]benzimidazole
[0353] 57
[0354] To a solution of indole-5-carboxaldehyde (2.00 g, 13.8 mmol)
in dimethylformamide (50 mL) were added sodium hydride (60% oil
dispersion; 828 mg, 20.7 mmol) and p-toluenesulfonyl chloride (3.16
g, 16.6 mmol) at 0.degree. C. under nitrogen atmosphere, and the
mixture was stirred for 13 h at room temperature. Water was added,
and the mixture was extracted with ethyl acetate. The organic layer
was washed with brine and dried on anhydrous sodium sulfate. The
solvent was evaporated under reduced pressure, and the residue was
purified by silica gel column chromatography (5:1 hexane/ethyl
acetate) to afford
1-(4-methylphenyl)sulfonylindole-5-carboxaldehyde (2.86 g,
69%).
[0355] 1H NMR (400 MHz, CDCl.sub.3) .delta. (ppm) 2.32 (s, 3H),
6.75 (d, J=3.5 Hz, 1H), 7.22 (d, J=8.2 Hz, 2H), 7.64 (d, J=3.9 Hz,
1H), 7.76 (d, J=8.6 Hz, 2H), 7.82 (dd, J=8.6, 1.6 Hz, 1H), 8.03 (d,
J=1.6 Hz, 1H), 8.08 (d, J=8.6 Hz, 1H), 9.99 (s, 1H)
[0356] To a solution of
1-(4-methylphenyl)sulfonylindole-5-carboxaldehyde (2.30 g, 7.69
mmol) in methanol (120 mL) was added p-toluenesulfonic acid
monohydrate (146 mg, 0769 mmol), and the mixture was heated to
reflux for 2 h. Diluted aqueous sodium hydroxide was added, and the
mixture was extracted with ether. The organic layer was washed with
brine and dried on anhydrous sodium sulfate. The solvent was
evaporated under reduced pressure to afford
5-dimethoxymethyl-1-(4-methylphenyl)sulfonylindole (2.64 g,
99%).
[0357] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 2.24 (s,
3H), 3.33 (s, 6H), 5.36 (s, 1H), 6.80 (d, J=3.5 Hz, 1H), 7.31 (d,
J=7.8 Hz, 3H), 7.56 (s, 1H), 7.76 (d, J=3.5 Hz, 1H), 7.81 (d, J=8.2
Hz, 2H), 7.88 (d, J=8.6 Hz, 1H).
[0358] To a solution of
5-dimethoxymethyl-1-(4-methylphenyl)sulfonylindole (2.37 g, 6.87
mmol) in tetrahydrofuran (35 mL) was added n-butyllithium (1.6
mol/L in hexanes; 6.2 mL, 9.9 mmol) at -78.degree. C. under
nitrogen atmosphere, and the mixture was stirred for 2 h. Then
dimethylformamide (1.1 mL, 14 mmol) was added, and the mixture was
stirred for 20 min. Water was added, and the mixture was extracted
with ethyl acetate. The organic layer was combined, washed with
brine and dried on anhydrous sodium sulfate. The solvent was
removed under reduced pressure, and the residue was purified by
column chromatography (4:1 hexane/ethyl acetate) followed by
trituration with hexane to afford 5-dimethoxymethyl-1-(4-meth-
ylphenyl)sulfonylindole-2-carboxaldehyde (1.89 g, 74%).
[0359] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 2.26 (s,
3H), 3.19 (s, 6H), 5.41 (s, 1H), 7.33 (d, J=8.2 Hz, 2H), 7.54 (dd,
J=8.6, 1.6 Hz, 1H), 7.65 (s, 1H), 7.75 (s, 1H), 7.76 (d, J=8.2 Hz,
2H), 8.10 (d, J=8.6 Hz, 1H), 10.3 (s, 1H).
[0360] A mixture of
5-dimethoxymethyl-1-(4-methylphenyl)sulfonylindole-2-c-
arboxaldehyde (200 mg, 0.536 mmol),
4,5-diamino-1,2-phenylenediamine (94 mg, 0.54 mmol) and
1,4-benzoquinone (57 mg, 0.54 mmol) in ethanol (6 mL) was heated to
reflux for 13 h. The solvent was removed under reduced pressure. To
the residue were added tetrahydrofuran (10 mL) and hydrochloric
acid (1 mol/L, 0.1 mL), and the mixture was stirred at room
temperature for 1 h. Water was further added and the mixture was
extracted with ethyl acetate. The organic layer was washed with
brine and dried on anhydrous sodium sulfate. The solvent was
removed under reduced pressure, and triturated with ether to afford
5,6-dichloro-2-[5-formyl-1--
(4-methylphenyl)sulfonylindol-2-yl]benzimidazole (149 mg, 57%).
[0361] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 2.25 (s,
3H), 7.32 (d, J=8.6 Hz, 2H), 7.4 (s, 1H), 7.74 (d, J=8.6, 2H), 7.94
(d, J=9.0 Hz, 1H), 7.95 (br s, 2H), 8.21 (s, 1H), 8.25 (d, J=9.0
Hz, 1H), 9.99 (s, 1H), 13.5 (br s, 1H)
[0362] ESI-MS m/z 484, 482 (M-H).sup.-,
C.sub.23H.sub.15.sup.35Cl.sub.2N.s- ub.3O.sub.3S=483
[0363] General Procedure 1 was then followed to obtain
5,6-dichloro-2-[5-[(2,4-25
dioxothiazolidin-5-ylidene)methyl]-1-(4-methyl-
phenyl)sulfonylindol-2-yl]benzimidazole.
[0364] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 2.25 (s,
3H), 7.31 (d, J=8.2 Hz, 2H), 7.37 (s, 1H), 7.66 (dd, J=8.6, 1.6 Hz,
1H), 7.70 (d, J=8.6 Hz, 2H), 7.81 (s, 1H), 7.85 (s, 1H), 7.86 (s,
1H), 8.04 (s, 1H), 8.19 (d, J=8.6 Hz, 1H), 12.6 (br s, 1H), 13.5
(br s, 1H)
[0365] ESI-MS m/z 583, 581 (M-H).sup.-,
C.sub.26H.sub.16.sup.35Cl.sub.2N.s- ub.4O.sub.4S.sub.2=582
[0366] To a suspension of
5,6-dichloro-2-[5-[(2,4-dioxothiazolidin-5-ylide-
ne)methyl]-1-(4-methylphenyl)sulfonylindol-2-yl]benzimidazole (77
mg, 0.13 mmol) in methanol (5 mL) was added aqueous sodium
hydroxide (2 mol/L; 1 mL), and the mixture was heated to reflux for
19 h. The reaction mixture was cooled on an ice bath, and pH was
adjusted to 7. The precipitated products were collected by
filtration followed by trituration with tetrahydrofuran/ethyl
acetate to afford 5,6-dichloro-2-[5-[(2,4-dioxothia-
zolidin-5-ylidene)methyl]indol-2-yl]benzimidazole (10 mg, 18%).
[0367] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.32 (s,
1H), 7.40 (d, J=9.0 Hz, 1H), 7.54 (d, J =8.6 Hz, 1H), 7.78 (s, 1H),
7.84 (s, 1H), 7.89 (s, 1H), 7.92 (s, 1H), 12.4 (br s, 2H), 13.4 (br
s, 1H)
[0368] ESI-MS m/z 429, 427 (M-H).sup.-,
C.sub.19H.sub.10.sup.35Cl.sub.2N.s- ub.4O.sub.2S=428
Example 46
Preparation of
5,6-dichloro-2-[2-[(2,4-dioxothiazolidin-5-ylidene)methyl]i-
ndol-5-yl]benzimidazole
[0369] 58
[0370] A mixture of
5-dimethoxymethyl-1-(4-methylphenyl)sulfonylindole-2-c-
arboxaldehyde obtained in Example 45 (200 mg, 0.536 mmol),
2,4-thiazolidinedione (75 mg, 0.643 mmol), and piperidine (0.064
mL, 0.64 mmol) in ethanol (6 mL) was heated to reflux for 6 h.
Hydrochloric acid (1 mol/L; 1 mL) was added to the mixture, and the
precipitated products were collected by filtration followed by
washing with ether to afford 5
-formyl-1-(4-methylphenyl)sulfonylindol-2-ylmethylene-2,4-thiazolidinedio-
ne (155 mg, 68%).
[0371] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 2.25 (s,
3H), 7.32 (s, 1H), 7.32 (d, J=8.2 Hz, 2H), 7.63 (d, J=8.2 Hz, 2H),
7.93 (d, J=9.0 Hz, 1H), 8.24 (s, 1H), 8.27 (s, 1H), 8.28 (d, J=9.0
Hz, 1H), 9.98 (s, 1H), 12.8 (br s, 1H)
[0372] ESI-MS m/z 425 (M-H).sup.-,
C.sub.20H.sub.14N.sub.2O.sub.5S.sub.2=4- 26
[0373] General Procedure 2 was then followed to obtain
5,6-dichloro-2-[2-[(2,4-dioxothiazolidin-5-ylidene)methyl]-1-(4-methylphe-
nyl)sulfonylindol-5-yl]benzimidazole.
[0374] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 2.24 (s,
3H), 7.24 (s, 1H), 7.32 (d, J=8.2 Hz, 2H), 7.64 (d, J=8.2 Hz, 2H),
7.80 (br s, 2H), 8.20 (dd, J=9.0, 1.6 Hz, 1H), 8.26 (d, J=9.0 Hz,
1H), 8.28 (s, 1H), 8.42 (s, 1H), 12.8 (br s, 1H), 13.3 (br s,
1H)
[0375] ESI-MS m/z 583, 581 (M-H).sup.-,
C.sub.26H.sub.16.sup.35Cl.sub.2N.s- ub.4O.sub.4S.sub.2=582
[0376] To a suspension of
5,6-dichloro-2-[2-[(2,4-dioxothiazolidin-5-ylide-
ne)methyl]-1-(4-methylphenyl)sulfonylindol-5-yl]benzimidazole (100
mg, 0.172 mmol) in methanol (7.5 mL) was added aqueous sodium
hydroxide (2 mol/L; 1.5 mL), and the mixture was heated to reflux
for 3.5 h. The reaction mixture was cooled on an ice bath, and pH
was adjusted to 7. The precipitated products were collected by
filtration, washed with water, and dried. To a solution of the
product (50 mg) in dimethylformamide (2 mL) was added
1,1'-carbonyldiimidazole (15 mg, 0.12 mmol), and the mixture was
stirred at room temperature for 30 min. Hydrochloric acid (1 mol/L;
0.5 mL) and water were added to the mixture, and the precipitated
product was filtered off and washed with water and ethanol to
afford
5,6-dichloro-2-[2-[(2,4-dioxothiazolidin-5-ylidene)methyl]indol-5-yl]benz-
imidazole (18 mg, 24%).
[0377] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 7.36 (s,
1H), 7.89 (br s, 1H), 8.10 (s, 1H), 8.23 (dd, J=9.0, 1.6 Hz, 1H),
8.35 (br s, 1H), 8.54 (s, 1H), 8.57 (d, J=8.6 Hz, 1H)
[0378] ESI-MS m/z 429, 427 (M-H).sup.-,
C.sub.19H.sub.10.sup.35Cl.sub.2 N.sub.4O.sub.2S=428
Example 47
Preparation of
5,6-dichloro-2-[5-[(2,4-dioxothiazolidin-5-ylidene)methyl]i-
ndol-2-yl]-1-(methoxymethyl)benzimidazole
[0379] 59
[0380] To a solution of
5,6-dichloro-2-[5-formyl-1-(4-methylphenyl)sulfony-
lindol-2-yl]benzimidazole obtained in Example 45 (824 mg, 1.71
mmol) in dimethylformamide (10 mL) were added diisopropylethylamine
(0.59 mL, 3.4 mmol) and chloromethyl methyl ether (0.16 mL, 2.1
mmol), and the mixture was stirred at room temperature for 15 h.
Water was added, and the mixture was extracted with ethyl acetate.
The organic layer was washed with water and brine, dried on
anhydrous sodium sulfate. The solvent was removed under reduced
pressure. To the residue were added methanol (80 mL) and 2 mol/L
sodium hydroxide (5 mL), and the mixture was heated to reflux for 1
h. Water was added, and the mixture was extracted with ethyl
acetate. The organic layer was washed with water and brine, dried
on anhydrous sodium sulfate. The solvent was removed under reduced
pressure, and the residue was triturated with ether to afford
5,6-dichloro-2-[5-formylindol-2-yl]-1-(methoxymethyl)benzimidazole
(570 mg, 89%).
[0381] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 3.36 (s,
3H), 5.81 (s, 2H), 7.41 (d, J=0.8 Hz, 1H), 7.58 (d, J=8.6 Hz, 1H),
7.71 (dd, J=8.6 Hz, 1H), 7.96 (s, 1H), 8.28 (s, 1H), 8.29 (s, 1H),
9.94 (s, 1H), 12.6 (br s, 1H)
[0382] General Procedure 1 was then followed to obtain
5,6-dichloro-2-[5-[(2,4-dioxothiazolidin-5-ylidene)methyl]indol-2-yl]-1-(-
methoxymethyl)benzimidazole.
[0383] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 3.30 (s,
3H), 5.82 (s, 2H), 7.35 (s, 1H), 7.44 (d, J=8.9 Hz, 1H), 7.58 (d,
J=8.2 Hz, 1H), 7.87 (s, 1H), 7.97 (d, J=1.2 Hz, 1H), 8.16 (s, 1H),
8.29 (s, 1H), 12.4 (br s, 1H), 12.5 (br s, 1H)
[0384] ESI-MS m/z 473, 471 (M-H).sup.-,
C.sub.21H.sub.14.sup.35Cl.sub.2N.s- ub.4O.sub.3S=472
Example 48
[0385] Preparation of
5,6-dichloro-2-[5-[(2,4-dioxothiazolidin-5-ylidene)m-
ethyl]-1-methylindol-2-yl]-1-(methoxymethyl)benzimidazole 60
[0386] To a solution of
5,6-dichloro-2-[5-formylindol-2-yl]-1-(methoxymeth-
yl)benzimidazole obtained in Example 47 (32 mg, 0.086 mmol) in
dimethylformamide (2 mL) were added iodomethane (0.027 mL, 0.43
mmol) and potassium tert-butoxide (19 mg, 0.17 mmol) under nitrogen
atmosphere, and the mixture was stirred at room temperature for 18
h. Water was added, and the mixture was extracted with ethyl
acetate. The organic layer was washed with water and brine, dried
on anhydrous sodium sulfate. The solvent was removed under reduced
pressure to afford 5,6-dichloro-2-
[5-formyl-1-methylindol-2-yl]-1-methoxymethylbenzimidazole (30 mg,
90%).
[0387] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. (ppm) 3.45 (s,
3H), 4.08 (s, 3H), 5.52 (s, 2H), 7.50 (d, J=8.6 Hz, 1H), 7.66 (s,
1H), 7.89 (dd, J=8.6, 1.6 Hz, 1H), 7.92 (s, 1H), 8.21 (d, J=0.8 Hz,
1H), 10.0 (s, 1H)
[0388] General Procedure 1 was then followed to obtain
5,6-dichloro-2-[5-[(2,4-dioxothiazolidin-5
-ylidene)methyl]-1-methylindol-
-2-yl]-1-methoxymethylbenzimidazole.
[0389] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 3.33 (s,
3H), 4.00 (s, 3H), 5.65 (s, 2H), 7.30 (s, 1H), 7.52 (d, J=8.6 Hz,
1H), 7.74 (d, J=9.0 Hz, 1H), 7.90 (s, 1H), 7.98 (s, 1H), 8.07 (s,
1H), 8.26 (s, 1H), 12.5 (br s, 1H)
[0390] ESI-MS m/z 487, 485 (M-H).sup.-,
C.sub.22H.sub.16.sup.35Cl.sub.2N.s- ub.4O.sub.3S=486
Example 49
Preparation of
5,6-dichloro-2-[5-[(2,4-dioxothiazolidin-5-ylidene)methyl]--
1-methylindol-2-yl]benzimidazole
[0391] 61
[0392] To a solution of
5,6-dichloro-2-[5-[(2,4-dioxothiazolidin-5-ylidene-
)methyl]-1-methylindol-2-yl]-1-(methoxymethyl)benzimidazole
obtained in Example 48 (26 mg, 0.053 mmol) in 1,4-dioxane (5 mL)
was added concentrated hydrochloric acid (0.5 mL), and the mixture
was heated to reflux for 1 h. The pH was adjusted to 7 by 2 mol/L
sodium hydroxide and the precipitated products were collected by
filtration to afford 5,6-dichloro-2-[5-[(2,4-dioxothiazolidin-5
-ylidene)methyl]-1-methylindol- -2-yl]benzimidazole (12 mg,
51%).
[0393] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. (ppm) 4.26 (s,
3H), 7.39 (s, 1H), 7.47 (dd, J=8.6, 1.6 Hz, 1H), 7.71 (d, J=8.6 Hz,
1H), 7.74 (br s, 1H), 7.88 (s, 1H), 7.94 (s, 1H), 7.95 (br s, 1H),
12.5 (br s, 1H), 13.4 (br s, 1H)
[0394] ESI-MS m/z 443, 441 (M-H).sup.-,
C.sub.20H.sub.12.sup.35Cl.sub.2N.s- ub.4O.sub.2S=442
Example 50
Preparation of
5,6-dichloro-2-[1-butyl-5-[(2,4-dioxothiazolidin-5-ylidene)-
methyl]indol-2-yl]-1-(methoxymethyl)benzimidazole
[0395] 62
[0396]
5,6-dichloro-2-[1-butyl-5-[(2,4-dioxothiazolidin-5-ylidene)methyl]i-
ndol-2-yl]-1-(methoxymethyl)benzimidazole was prepared from
5,6-dichloro-2-[5-formylindol-2-yl]-1-(methoxymethyl)benzimidazole
obtained in Example 47 and 1 -iodobutane in a similar manner to
Example 48.
[0397] ESI-MS m/z 529, 527 (M-H).sup.-
C.sub.25H.sub.22Cl.sub.2N.sub.4O.su- b.3S=528
Example 51
Preparation of
5,6-dichloro-2-[1-butyl-5-[(2,4-dioxothiazolidin-5-ylidene)-
methyl]indol-2-yl]benzimidazole
[0398] 63
[0399]
5,6-dichloro-2-[1-butyl-5-[(2,4-dioxothiazolidin-5-ylidene)methyl]i-
ndol-2-yl]benzimidazole was prepared in a similar manner to Example
49.
[0400] ESI-MS m/z 485, 483 (M-H).sup.-,
C.sub.23H.sub.18.sup.35Cl.sub.2N.s- ub.4O.sub.2S=484
Example 52
Preparation of
5,6-dichloro-2-[1-[2-(dimethylamino)ethyl]-5-[(2,4-dioxothi-
azolidin-5-ylidene)methyl]indol-2-yl]-1-(methoxymethyl)benzimidazole
[0401] 64
[0402]
5,6-dichloro-2-[1-[2-(dimethylamino)ethyl]-5-[(2,4-dioxothiazolidin-
-5-ylidene)methyl]indol-2-yl]-1-(methoxymethyl)benzimidazole was
prepared from
5,6-dichloro-2-[5-formylindol-2-yl]-1-(methoxymethyl)benzimidazole
obtained in Example 47 and 2-(dimethylamino)ethyl chloride
hydrochloride in a similar manner to Example 48.
[0403] ESI-MS m/z 544, 542 (M-H).sup.-
C.sub.25H.sub.22.sup.35Cl.sub.2N.su- b.5O.sub.3S=543
Example 53
Preparation of
5,6-dichloro-2-[1-[2-(dimethylamino)ethyl]-5-[(2,4-dioxothi-
azolidin-5-ylidene)methyl]indol-2-yl]benzimidazole
[0404] 65
[0405]
5,6-dichloro-2-[1-[2-(dimethylamino)ethyl]-5-[(2,4-dioxothiazolidin-
-5-ylidene)methyl]indol-2-yl]benzimidazole was prepared in a
similar manner to Example 49.
[0406] ESI-MS m/z 500, 498 (M-H).sup.-,
C.sub.23H.sub.19.sup.35Cl.sub.2N.s- ub.5O.sub.2S=499
Example 54
Preparation of
5,6-dichloro-1-[[4-(2,4-dioxothiazolidin-5-ylidene)methyl]p-
henyl]benzimidazole
[0407] 66
[0408] A mixture of 5,6-dichlorobenzimidazole (94 mg, 0.50 mmol),
4-fluorobenzaldehyde (0.064 mL, 0.60 mmol), and potassium carbonate
(83 mg, 0.60 mmol) in dimethylsulfoxide (3 mL) was heated at 100_C
for 3 h. The mixture was cooled to room temperature, and water was
added. The precipitated product was collected by filtration and
washed with water and ethanol to afford
5,6-dicloro-1-(4-formylphenyl)benzimidazole (90 mg, 62%).
[0409] .sup.1H NMR (400 MHz, DMSO-d.sub.6)_(ppm) 7.94 (d, J=8.6 Hz,
2H), 7.97 (s, 1H), 8.08 (s, 1H), 8.11 (d, J=8.6 Hz, 2H), 8.80 (s,
1H), 10.1 (s, 1H)
[0410] General Procedure 1 was then followed to obtain
5,6-dichloro-1-[[4-(2,4-dioxothiazolidin-5-ylidene)methyl]phenyl]benzimid-
azole.
[0411] .sup.1H NMR (400 MHz, DMSO-d.sub.6)_(ppm) 7.80 (d, J=8.6 Hz,
2H), 7.85 (d, J=8.6 Hz, 2H), 7.87 (s, 1H), 7.95 (s, 1H), 8.07 (s,
1H), 8.75 (s, 1H), 12.7 (br s, 1H)
[0412] ESI-MS m/z 390, 388 (M-H).sup.-,
C.sub.17H.sub.9.sup.35Cl.sub.2N.su- b.3O.sub.2S=389
Example 55
Preparation of
5,6-dichloro-1-[[4-(2,4-dioxothiazolidin-5-ylidene)methyl]p-
henyl]-2-methylbenzimidazole
[0413] 67
[0414] 5,6-dichloro-1-[[4-(2,4-dioxothiazolidin-5
-ylidene)methyl]phenyl]-- 2-methylbenzimidazole was obtained from
5,6-dichloro-2-methylbenzimidazole and 4-fluorobenzaldehyde in 2
steps in a similar manner to Example 54.
[0415] ESI-MS m/z 404, 402 (M-H).sup.-,
C.sub.18H.sub.11.sup.35Cl.sub.2N.s- ub.3O.sub.2S=403
Example 56
[0416] Preparation of
5,6-dichloro-1-[[4-(2,4-dioxothiazolidin-5-ylidene)m-
ethyl]phenyl]-2-(methythio)benzimidazole 68
[0417]
5,6-dichloro-1-[[4-(2,4-dioxothiazolidin-5-ylidene)methyl]phenyl]-2-
-(methylthio)benzimidazole was obtained from
5,6-dichloro-2-(methylthio)be- nzimidazole and 4-fluorobenzaldehyde
in 2 steps in a similar manner to Example 54.
[0418] ESI-MS m/z 436, 434 (M-H).sup.-,
C.sub.18H.sub.11.sup.35Cl.sub.2N.s- ub.3O.sub.2S.sub.2=435
Example 57
Preparation of Affinity Purified Extract
[0419] Extracts used for screening telomerase inhibitors were
routinely prepared from 293 cells over-expressing the protein
catalytic subunit of telomerase (hTERT). These cells were found to
have 2-5 fold more telomerase activity than parental 293 cells. 200
ml of packed cells (harvested from about 100 liters of culture)
were resuspended in an equal volume of hypotonic buffer (10 mM
Hepes pH 7.9, 1 mM MgCl.sub.2, 1 mM DTT, 20 mM KCl, 1 mM PMSF) and
lysed using a dounce homogenizer. The glycerol concentration was
adjusted to 10% and NaCl was slowly added to give a final
concentration of 0.3 M. The lysed cells were stirred for 30 min and
then pelleted at 100,000.times.g for 1 hr. Solid ammonium sulfate
was added to the S100 supernatant to reach 42% saturation. The
material was centrifuged; the pellet was resuspended in one fifth
of the original volume and dialyzed against Buffer `A` containing
50 mM NaCl. After dialysis the extract was centrifuged for 30 min
at 25,000.times.g. Prior to affinity chromatography, Triton X-100
(0.5 %), KCl (0.3 M) and tRNA (50 .mu.g/ml) were added. Affinity
oligo (5' biotinTEG-biotinTEG-biotinTE- G-GTA GAC CTG TTA CCA guu
agg guu ag 3'; lower case represents 2' O-methyl ribonucleotides
and upper case represents deoxynucleotides) was added to the
extract (1 nmol per 10 ml of extract). After an incubation of 10
min at 30.degree. C., Neutravidin beads (Pierce; 250 .mu.l of a 50%
suspension) were added and the mixture was rotated overnight at
4.degree. C. The beads were pelleted and washed three times with
Buffer `B` containing 0.3 M KCl, twice with Buffer `B` containing
0.6 M KCl, and twice more with Buffer B containing 0.3 M KCl.
Telomerase was eluted in Buffer `B` containing 0.3 M KCl, 0.15%
Triton X-100 and a 2.5 molar excess of displacement oligo (5'-CTA
ACC CTA ACT GGT AAC AGG TCT AC-3' at 0.5 ml per 125 .mu.l of packed
Neutravidin beads) for 30 min. at room temperature. A second
elution was performed and pooled with the first. Purified extracts
typically had specific activities of 10 fmol nucleotides
incorporated/min/.mu.l extract, or 200 nucleotides/min/mg total
protein.
1 Buffer `A` Buffer `B` 20 mM Hepes pH 7.9 20 mM Hepes pH 7.9 1 mM
MgCl2 1 mM EDTA 1 mM DTT 1 mM DTT 1 mM EGTA 10% glycerol 10%
glycerol 0.5 Triton
Example 58
Telomerase Specific Activity Determination
[0420] Three separate 100 .mu.l telomerase assays are set up with
the following buffer solutions: 50 mM Tris acetate, pH 8.2, 1 mM
DTT, 1 mM EGTA, 1 mM MgCl.sub.2, 100 mM K acetate, 500 .mu.M dATP,
500 .mu.M TTP, 10.mu.M .sup.32P-dGTP (25 Ci/mmol), and a00 nM
d(TTAGGG).sub.3. To the individual reactions 2.5, 5 or 10 .mu.l of
affinity-purified telomerase (see Example 57) is added and the
reactions are incubated at 37.degree. C. At 45 and 90 minutes, 40
.mu.l aliquots are removed from each reaction and added to 160
.mu.l of Stop Buffer (100 MM NaCl, 10 mM Na pyrophosphate, 0.2%
SDS, 2 mM EDTA, 100 .mu.g/ml tRNA). 10 .mu.l trichloroacetic acid
(TCA) (100%) is added and the sample is incubated on ice for 30
minutes. The sample is pelleted in a microcentrifuge (12000.times.g
force) for 15 minutes. The pellet is washed with 1 ml 95% ethanol
and pelleted again in the microcentrifuge (12000.times.g force) for
5 minutes. The pellet is resuspended in 50 .mu.l dH.sub.20 and
transferred to a 12.times.75 glass test tube containing 2.5 ml of
ice cold solution of 5% TCA and 10 mM Na pyrophosphate. The sample
is incubated on ice for 30 minutes. The sample is filtered through
a 2.5 cm wet (dH.sub.2O) GFC membrane (S&S) on a vaccum
filtration manifold. The filter is washed three times under vacuum
with 5 ml ice cold 1% TCA, and once with 5 ml 95% ethanol. The
filter is dried and counted in a scintillation counter using
scintillation fluid. The fmol of nucleotide incorporated is
determined from the specific activity of radioactive tracer. The
activity of extract is calculated based on the dNTP incorporated
and is expressed as fmol dNTP/min/.mu.l extract.
Example 59
Telomerase Activity Assay
[0421] Bio-Tel FlashPlate Assay
[0422] An assay is provided for the detection and/or measurement of
telomerase activity by measuring the addition of TTAGGG telomeric
repeats to a biotinylated telomerase substrate primer; a reaction
catalyzed by telomerase. The biotinylated products are captured in
streptavidin-coated microtiter plates. An oligonucleotide probe
complementary to 3.5 telomere repeats labeled with [.sup.33P] is
used for measuring telomerase products, as described below. Unbound
probe is removed by washing and the amount of probe annealing to
the captured telomerase products is determined by scintillation
counting.
[0423] Method:
[0424] 1. Compounds are stored as concentrated stocks and dissolved
in 100 % dimethylsulfoxide (DMSO).
[0425] 2. For testing, the compounds are diluted to a 15X working
stock in 50% DMSO and 2 .mu.l is dispensed into two wells of a
96-well microtiter dish (assayed in duplicate).
[0426] 3. Telomerase extract is diluted to a specific activity of
0.04-0.09 fmol dNTP incorporated/min./.mu.l in Telomerase Dilution
Buffer and 18 .mu.l added to each sample well to preincubate with
compound for 30 minutes at room temperature.
[0427] 4. The telomerase reaction is initiated by addition of 10
.mu.l Master Mix to the wells containing telomerase extract and
compound. The plates are sealed and incubated at 37.degree. C. for
90 min.
[0428] 5. The reaction is stopped by the addition of 10 .mu.l
HCS.
[0429] 6. 25 .mu.l of the reaction mixture is transferred to a
96-well streptavidin-coated FlashPlate (NEN) and incubated for 2
hours at room temperature with mild agitation.
[0430] 7. The wells are washed three times with 180 .mu.l 2X SSC
without any incubation.
[0431] 8. The counts of probe annealed to biotinylated telomerase
products are detected on a scintillation counter.
[0432] Buffers:
[0433] Telomerase Dilution Buffer
[0434] 50 mM Tris-acetate, pH 8.2
[0435] 1 mM DTT
[0436] 1 mM EGTA
[0437] 1 mM MgCl.sub.2
[0438] 830 nM BSA
[0439] Master Mix (MM)
[0440] 50 mM Tris-acetate, pH 8.2
[0441] 1 mM DTT
[0442] 1 mM EGTA
[0443] 1 mM MgCl.sub.2
[0444] 150 mM K acetate
[0445] 10 .mu.M dATP
[0446] 20 .mu.M dGTP
[0447] 120 .mu.M dTTP
[0448] 100 nM biotinylated primer
(5'-biotin-AATCCGTCGAGCAGAGTT-3')
[0449] 5.4 nM labeled probe [5'-CCCTAACCCTAACCCTAACCC-(.sup.33P)
A.sub.1-50-3']; specific activity approximately 10.sup.9 cpm/.mu.g
or higher
[0450] Hybridization Capture Solution (HCS)
[0451] 12X SSC (1X=150 mM NaCl/30 mM Na.sub.3Citrate)
[0452] 40 mM EDTA
[0453] 40 mM Tris-HCl, pH 7.0
[0454] Using the foregoing assay, the compounds of Examples 1-56
were shown to have telomerase IC.sub.50 values below 100 .mu.M.
Example 60
Anti-tumor Activity
[0455] Ex vivo Studies
[0456] a. Reduction of Telomere Length in Tumor Cells
[0457] Colonies of the tumor cell lines, such as the ovarian tumor
cell lines OVCAR-5 and SK-OV-3, and normal human cells used as a
control (e.g., normal human BJ cells) are prepared using standard
methods and materials. In one test, the colonies are prepared by
seeding 15-centimeter dishes with about 10.sup.6 cells in each
dish. The dishes are incubated to allow the cell colonies to grow
to about 80% confluence, at which time each of the colonies are
divided into two groups. One group is exposed to a subacute dose of
a compound of the invention at a predetermined concentration (e.g.,
between about 5 .mu.M and about 20 .mu.M) for a period of about 4-8
hours after plating following the split; the other group is exposed
to a control (e.g., DMSO).
[0458] Each group is then allowed to continue to divide, and the
groups are split evenly again (near confluence). The same number of
cells are seeded for continued growth. The compound or control is
added every fourth day to the samples at the same concentration
delivered initially. Remaining cells are analyzed for telomere
length. As the untested cell cultures near confluence, the samples
are split again as just described. This sequence of cell doubling
and splitting is continued for about 20 to 25 doublings. Thus, a
determination of telomere length as a function of cell doublings is
obtained.
[0459] Telomere length is determined by digesting the DNA of the
cells using restriction enzymes specific for sequences other than
the repetitive T.sub.2 AG.sub.3 sequence of human telomeres (TRF
analysis). The digested DNA is separated by size using standard
techniques of gel electrophoresis to determine the lengths of the
telomeric repeats, which appear, after probing with a telomere DNA
probe, on the gel as a smear of high-molecular weight DNA
(approximately 2 Kb-15 Kb).
[0460] The results of the telomere length analysis are expected to
indicate that the compounds of the invention have no affect on the
rate of decrease in telomere length for control cells as a function
of progressive cell doublings. With respect to the tumor cell
lines, however, measurable decreases in telomere length are
expected to be determined for tumor cells exposed to the compounds
of the invention. Tumor cells exposed to the control are expected
to maintain steady telomere lengths. Thus, the compounds of the
invention are expected to cause resumption of the normal loss of
telomere length as a function of cell division in tumor cells.
[0461] In another experiment, HEK-293 cells are incubated with a
compound of the invention and a control at concentrations between
about 1 .mu.M and about 20 .mu.M using the protocol just described.
Cells are expected to enter crisis (i.e., the cessation of cell
function) within several weeks following administration of the test
compound of the invention. In addition, TRF analysis of the cells
using standard methodology is expected to show that the test
compounds of the invention are effective in reducing telomere
length. In addition to the HEK-293 cells described above, this
assay can be performed with any telomerase-positive cell line, such
as HeLa cells.
[0462] b. Specificity
[0463] Compounds of the invention are screened for activity
(IC.sub.50) against telomerase and several enzymes having nucleic
acid binding or modifying activities related to telomerase using
standard techniques. The enzymes being screened include Telomerase,
DNA Polymerase I, HeLa RNA Polymerase II, T3 RNA Polymerase, MMLV
Reverse Transcriptase, Topoisomerase I, Topoisomerase II, Terminal
Transferase and Single-Stranded DNA Binding Protein (SSB). The
specificity of a compound of the invention for telomerase is
determined by comparing the IC.sub.50 of the compound with respect
to telomerase with the IC.sub.50 values of the compound for each of
the enzymes being screened. The compound is determined to have high
specificity for telomerase if the IC.sub.50 for telomerase of the
compound is lower than the IC.sub.50 vaules for each of the enzymes
being screened.
[0464] Alternatively, telomerase inhibitory activity of the
compounds was measured in accordance with a known method (U.S. Pat.
No. 5,760,062). That is, a dimethyl sulfoxide (DMSO) solution of
each agent was mixed with partially purified telomerase from a
nuclear extract of HEK293 cells and incubated in the presence of an
oligodeoxynucleotide to be used as the substrate and
deoxynucleotide triphosphate. The obtained reacted product (DNA
having a telomere sequence) was adsorbed on a membrane, and
hybridization was carried out using a labeled oligonucleotide probe
having a sequence complementary to the telomere sequence. The
inhibition ratio was calculated based on the ratio of the signal of
label on the membrane in the presence of the agent to the signal of
label in the absence of the agent (control). Also, concentration of
each agent which inhibits 50% of the enzyme activity based on the
control was used as IC.sub.50. Results of the measurement of
inhibition activity of selected compounds are shown in Table 1.
2TABLE 1 In vitro telomerase inhibition activity Compound of
Example IC.sub.50 (.mu.M) 1 1.86, 1.1, 1.22, 0.85, 1.14 2 1.05,
1.52 3 5.44, 6.2 4 4.3, 3.69 5 4.98, 2.95 6 3.04, 3.35 14 1.61,
1.92 15 3.01, 2.43 20 2.28, 3.26 21 19.2, 15.92 28 14.48, 14.95 29
1.89, 2.26 30 5.19, 2.76 32 1.96, 1.91 33 3.18, 2.47 39 0.44, 0.92,
1.01 40 0.29, 0.55, 0.51 45 0.61 46 5.4
[0465] c. Cytotoxicity
[0466] The XTT assay for cytotoxicity is performed using HeLa
cells. The cell lines used in the assay are exposed to a compound
of the invention for 72 hours at concentrations ranging from about
1 .mu.M to about 1,000 .mu.M. During this period, the optical
density (OD) of the samples is determined for light at 540
nanometers (nm). No significant cytotoxic effects are expected to
be observed at concentrations less than about 5 .mu.M. It will be
appreciated that other tumor cells lines such as the ovarian tumor
cell lines OVCAR-5 and SK-OV-3 can be used to determine
cytotoxicity in addition to control cell lines such as normal human
BJ cells. Other assays for cytotoxicity such as the MTT assay (see
Berridge et al., 1996, Biochemica 4:14-19) and the alamarBlue.TM.
assay (U.S. Pat. No.5,501,959) can be used as well.
[0467] Some compounds may induce G2 arrest at concentrations above
about 5 .mu.M (i.e., at 10 .mu.M-20 .mu.M concentrations or
higher). Preferably, to observe any telomerase inhibiting effects
the compounds should be administered at a concentration below the
level of cytotoxicity. Nevertheless, since the effectiveness of
many cancer chemotherapeutics derives from their cytotoxic effects,
it is within the scope of the present invention that the compounds
of the present invention be administered at any dose for which
chemotherapeutic effects are observed.
[0468] In vivo Studies
[0469] A human tumor xenograft model in which OVCAR-5 tumor cells
are grafted into nude mice can be constructed using standard
techniques and materials. The mice are divided into two groups. One
group is treated intraperitoneally with a compound of the
invention. The other group is treated with a control comprising a
mixture of either DMSO or ethanol and emulphor (oil) and phosphate
buffer solution (PBS). The average tumor mass for mice in each
group is determined periodically following the xenograft using
standard methods and materials.
[0470] In the group treated with a compound of the invention, the
average tumor mass is expected to increase following the initial
treatment for a period of time, after which time the tumor mass is
expected to stabilize and then begin to decline. Tumor masses in
the control group are expected to increase throughout the study.
Thus, the compounds of the invention are expected to lessen
dramatically the rate of tumor growth and ultimately induce
reduction in tumor size and elimination of the tumor.
[0471] In another experiment, each agent is allowed to contact with
human renal carcinoma cell line ACHN for 3 days, and then a cell
extract is prepared by a known method (U.S. Pat. No. 5,629,154) to
measure the enzyme activity. That is, a cell extract is prepared
using a buffer solution containing 0.5% CHAPS. Using the extract,
TRAP (Telomeric Repeat Amplification Protocol) assay is carried out
in vitro (TRAP.sub.EZE.TM. ELISA Telomerase Detection Kit,
manufactured by Intergen). The ratio (%) of the enzyme activity in
the extract from agent-treated cells to the enzyme activity in the
extract from agent-untreated cells is calculated.
[0472] Thus, the present invention provides novel compounds,
compositions and methods for inhibiting telomerase activity and
treating disease states in which telomerase activity has
deleterious effects, especially cancer. The compounds of the
invention provide a highly selective and effective treatment for
malignant cells that require telomerase activity to remain
immortal; yet, without affecting non-malignant cells.
[0473] While the preferred embodiment of the invention has been
illustrated and described, it will be appreciated that various
changes can be made therein without departing from the spirit and
scope of the invention.
* * * * *