U.S. patent application number 10/077738 was filed with the patent office on 2002-08-22 for substituted indole compounds and methods of their use.
Invention is credited to Chin, Allison C., Holcomb, Ryan, Nguyen, Mark Q., Tolman, Richard L..
Application Number | 20020115700 10/077738 |
Document ID | / |
Family ID | 22498829 |
Filed Date | 2002-08-22 |
United States Patent
Application |
20020115700 |
Kind Code |
A1 |
Chin, Allison C. ; et
al. |
August 22, 2002 |
Substituted indole compounds and methods of their use
Abstract
5-(3-Isatinylidinyl)thiazolidineones and
3-(thiazolidinon-5-yl)indoles 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 novel methods for assaying or
screening for inhibitors of telomerase activity.
Inventors: |
Chin, Allison C.; (Stanford,
CA) ; Tolman, Richard L.; (Los Altos, CA) ;
Nguyen, Mark Q.; (San Jose, CA) ; Holcomb, Ryan;
(San Carlos, CA) |
Correspondence
Address: |
GERON CORPORATION
230 CONSTITUTION DRIVE
MENLO PARK
CA
94025
|
Family ID: |
22498829 |
Appl. No.: |
10/077738 |
Filed: |
February 13, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10077738 |
Feb 13, 2002 |
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09608861 |
Jun 30, 2000 |
|
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60142173 |
Jul 1, 1999 |
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Current U.S.
Class: |
514/369 ;
548/183 |
Current CPC
Class: |
C07D 277/36 20130101;
A61K 31/506 20130101; C07D 417/10 20130101; C07D 417/06 20130101;
C07D 277/34 20130101; A61K 31/426 20130101; C07D 513/04 20130101;
A61K 31/4439 20130101 |
Class at
Publication: |
514/369 ;
548/183 |
International
Class: |
C07D 417/02 |
Claims
we claim:
1. A compound of formula (I): 65wherein X.sub.1 is chosen from O,
S, CH.sub.2, or NR.sub.5 where R.sub.5 is H, lower alkyl or aryl;
L.sub.1 is a direct single bond, direct double bond, --CH.sub.2--,
or --CH.dbd.; is a single or a double bond; R.sub.1 is selected
from the group consisting of H, OR.sub.5, SR.sub.5,
CR.sub.6R.sub.7R.sub.8, and oxo only when is a single, wherein
R.sub.6, R.sub.7, and R.sub.8 are independently selected from H,
OH, lower alkyl, aryl, or heteroaryl; R.sub.2 and R.sub.3 are
independently selected from the group consisting of H, OH, halogen,
mercapto, nitro, cyano, trifluromethyl, lower alkyl, lower alkoxy,
aryloxy, NR.sub.9R.sub.10, SO.sub.2NR.sub.9R.sub.10,
OCHR.sub.9R.sub.10, COR.sub.9, CO.sub.2R.sub.9, NHCONHR.sub.9,
CONHR.sub.9, NHCOR.sub.9, aryl, and heteroaryl wherein R.sub.9 and
R.sub.10 are independently selected from the group consisting of
hydrogen, lower alkyl, aryl, and heteroaryl, and R.sub.2 and
R.sub.3 further represent replacement in the ring of ring methine
(--CH.dbd.) atoms with aza (--N.dbd.) atoms; 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; and R.sub.4 is H, lower alkyl, alkaryl, aryl, or
heteroaryl; or a pharmaceutically acceptable salt thereof.
2. A compound of claim 1, wherein is a double bond, and L.sub.1 is
a direct single bond, --CH.sub.2--, or --CH.dbd..
3. A compound of claim 1, wherein is a single bond, and R.sub.1 is
H or oxo.
4. A compound of claim 3, wherein L.sub.1 is a direct double
bond.
5. A compound of claim 1, wherein R.sub.2 and R.sub.3 are
halogen.
6. A compound of claim 5, wherein the halogen is Cl.
7. A compound of claim 1, wherein R.sub.3 is hydrogen.
8. A compound of claim 7, wherein R.sub.2 is halogen.
9. A compound of claim 8, wherein the halogen is Cl.
10. A compound of claim 8, wherein R.sub.2 is 4-Cl, 5-Cl, or
6-Cl.
11. A compound of claim 10, wherein R.sub.2 is 4-Cl.
12. A compound of claim 7, wherein R.sub.2 is
SO.sub.2NHR.sub.9.
13. A compound of claim 12, wherein R.sub.9 is aryl.
14. A compound of claim 12, wherein R.sub.9 is alkyl.
15. A compound of claim 7, wherein R.sub.2 is COR.sub.9.
16. A compound of claim 15, wherein R.sub.9 is aryl.
17. A compound of claim 1, wherein L.sub.2 is a direct bond.
18. A compound of claim 1, wherein L.sub.2 is selected from the
group consisting of --O--, --S--, --SO--, --SO.sub.2--, --NH--,
--(CH.sub.2).sub.n--, --(OCH.sub.2).sub.n--,
--(CH.sub.2).sub.nO(CH.sub.2- ).sub.m--, --SCH.sub.2--, --OC(O)--,
--C(O)NH--, --OC(O)CH.sub.2--, --OC(O)NH--, --NHC(O)--, and
--NHC(O)NH--, where n and m are independently selected from an
integer between 0 and 1.
19. A compound of claim 18, wherein L.sub.2 is --CH.sub.2--.
20. A compound of claim 18, wherein L.sub.2 is --SO2--.
21. A compound of formula (II): 66wherein X.sub.1 is O or S;
R.sub.2 is H, OH, halogen, lower alkyl, aryl, or heteroaryl;
L.sub.2 is a direct bond, CH.sub.2, or SO.sub.2; and R.sub.11 is H,
halogen or lower alkoxy; or a pharmaceutically acceptable salt
thereof.
22. A compound of claim 21, wherein X.sub.1 is O, L.sub.2 is
CH.sub.2, and R.sub.2 and R.sub.11 are chloro.
23. A compound of claim 1, further having telomerase inhibiting
activity.
24. A compound of claim 21, further having telomerase inhibiting
activity.
25. A method of inhibiting a telomerase enzyme comprising
contacting the enzyme with a compound of claim 1.
26. A method of inhibiting a telomerase enzyme comprising
contacting the enzyme with a compound of claim 21.
27. A method of inhibiting proliferation of a telomerase positive
cell comprising contacting the cell with a compound of claim 1.
28. The method of claim 27, wherein the cell is a mammalian
cell.
29. The method of claim 28, wherein the cell is a human cell.
30. The method of claim 29, wherein the cell is a cancer cell.
31. A method of treating a tumor comprising contacting the tumor
with a compound of claim 1.
32. A method of treating a tumor comprising contacting the tumor
with a compound of claim 21.
33. A pharmaceutical composition comprising a pharmaceutically
effective amount of a compound of claim 1 and a pharmaceutically
acceptable carrier.
34. A pharmaceutical composition comprising a pharmaceutically
effective amount of a compound of claim 21 and a pharmaceutically
acceptable carrier.
35. The use of a compound of claim 1 to inhibit telomerase
activity.
36. The use of a compound of claim 21 to inhibit telomerase
activity.
37. The use of a compound of claim 1 to inhibit proliferation of a
telomerase positive cell.
38. The use of a compound of claim 21 to inhibit proliferation of a
telomerase positive cell.
39. The use of a compound of claim 1 in the manufacture of a
medicament for inhibition of telomerase activity.
40. The use of a compound of claim 21 in the manufacture of a
medicament for inhibition of telomerase activity.
41. The use of a compound of claim 1 in the manufacture of a
medicament for inhibition of telomerase activity in a cell.
42. The use of a compound of claim 21 in the manufacture of a
medicament for inhibition of telomerase activity in a cell.
43. The use of a compound of claim 1 in the manufacture of a
medicament for the treatment of a telomerase mediated condition or
disease.
44. The use of a compound of claim 21 in the manufacture of a
medicament for the treatment of a telomerase mediated condition or
disease.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The application claims priority from U.S. Application No.
60/142,173, filed Jul. 1, 1999, which is herein incorporated by
reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the synthesis of
substituted indole compounds, to pharmaceutical compositions
containing the compounds and to the use of the compounds and
compositions to inhibit telomerase activity, 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
[0003] 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.
[0004] 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.
[0005] 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). Methods for detecting telomerase activity, as well as for
identifying compounds that regulate or affect telomerase activity,
together with methods for therapy and diagnosis of cellular
senescence and immortalization by controlling telomere length and
telomerase activity, have also been described. See, Feng, et al.,
1995, Science, 269:1236-1241; Kim, et al., 1994, Science,
266:2011-2014; PCT patent publication No. 93/23572, published Nov.
25, 1993; and U.S. Pat. Nos. 5,760,062, 5,767,278, 5,770,613 and
5,863,936.
[0006] U.S. Pat. No. 5,656,638 lists compounds that may have
anti-telomerase activity. U.S. Pat. No. 5,556,874 discloses
substituted 2-thioindoles are inhibitors of the epidermal growth
receptor tyrosine kinase. In addition, 2-oxindole-1-carboxamides
are disclosed to be inhibitors of cyclooxygenase and lipoxygenase
in U.S. Pat. Nos. 4,569,942 and 4,556,672, and as being capable of
inhibiting the biosynthesis of interleukin-1 in U.S. Pat. No.
4,861,794.
[0007] The synthesis of some isatinylidineacetic acid derivatives
is described by Autrey and Tahk (1967) Tetrahedron 23: 901-917, and
the structure elucidation of indolmycin, a antibiotic, is described
by von Wittenau and Els (1963) J. Am. Chem. Soc. 86: 3425-3431.
[0008] 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.
SUMMARY OF THE INVENTION
[0009] In one aspect, the invention relates to the synthesis and
characterization of thiazolidinedione indole and
5-(3-isatinylidenyl)-2,4- -thiazolidinedione compounds and the use
of these compounds as inhibitors of the telomerase enzyme. 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
thiazolidinedione indole and
5-(3-isatinylidenyl)-2,4-thiazolidinedione inhibit the
proliferation of cells that have telomerase activity, such as many
cancer cells. Thus, this aspect of the present invention provides
methods of inhibiting telomerase activity in a cell, such as a
cancer cell. Further, the invention provides methods of inhibiting
telomerase activity in a subject (for example a human or other
mammalian subject) suffering from a telomerase-mediated condition
or disease, comprising administering to the patient a
therapeutically effective amount of a telomerase inhibiting
thiazolidinedione indole or 5-(3-isatinylidenyl)-2,-
4-thiazolidinedione compound, or a pharmaceutically acceptable salt
thereof. Thus, the compounds are useful as inhibitors of telomerase
and as antitumor agents.
[0010] More particularly, the invention comprises compounds, and
their pharmaceutically acceptable salts, of formula (I): 1
[0011] wherein
[0012] X.sub.1 is chosen from O, S, CH.sub.2, or NR.sub.5 where
R.sub.5 is H, lower alkyl or aryl;
[0013] L.sub.1 is a direct single bond, direct double bond,
--CH.sub.2--, or --CH.dbd.;
[0014] is a single or a double bond;
[0015] R.sub.1 is selected from the group consisting of H,
OR.sub.5, SR.sub.5, CR.sub.6R.sub.7R.sub.8, and oxo only when is a
single, wherein R.sub.6, R.sub.7, and R.sub.8 are independently
selected from H, OH, lower alkyl, aryl, or heteroaryl;
[0016] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, OH, halogen, mercapto, nitro, cyano,
trifluoromethyl, lower alkyl, lower alkoxy, aryloxy,
NR.sub.9R.sub.10, SO.sub.2NR.sub.9R.sub.10, OCHR.sub.9R.sub.10,
COR.sub.9, CO.sub.2R.sub.9, NHCONHR.sub.9, CONHR.sub.9,
NHCOR.sub.9, aryl, and heteroaryl wherein R.sub.9 and R.sub.10 are
independently selected from the group consisting of hydrogen, lower
alkyl, aryl, and heteroaryl, and R.sub.2 and R.sub.3 further
represent replacement in the ring of ring methine (--CH.dbd.) atoms
with aza (--N.dbd.) atoms;
[0017] 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; and
[0018] R.sub.4 is H, lower alkyl, alkaryl, aryl, or heteroaryl.
[0019] In another aspect, the invention relates to isatin compounds
comprising formula (II): 2
[0020] wherein
[0021] X.sub.1 is O or S;
[0022] R.sub.2 is H, OH, halogen, lower alkyl, aryl, or
heteroaryl;
[0023] L.sub.2 is a direct bond, CH.sub.2, or SO.sub.2; and
[0024] R.sub.11 is H, halogen or lower alkoxy;
[0025] or a pharmaceutically acceptable salt thereof.
[0026] 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
[0027] 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.
[0028] The term "indole" or "indole derivative" as used herein
refers to compounds of the general formula: 3
[0029] The term "isatin" or "isatin derivative" as used herein
refers to compounds of the general formula: 4
[0030] The term "thiazolidineone" or "thiazolidineone derivative"
as used herein refers to compounds of the general formula: 5
[0031] 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.
[0032] 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
"loweralkyl". 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.
[0033] The term "oxo" means a doubly bonded oxygen.
[0034] 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 ormercapto, 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); carbamido (--HNC(.dbd.O)NRR'); orthiocarbamido
(--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.
[0035] The term "methylene" refers to the group --CH.sub.2--.
[0036] 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).
[0037] The term "halo" or "halogen" as used herein refers to the
substituents fluoro, bromo, chloro, and iodo.
[0038] 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
methine. Other carbonyl equivalents will be familiar to those
having skill in the medicinal and organic chemical arts.
[0039] The term "aryl" as used herein refers to cyclic aromatic
hydrocarbon chains having twenty or fewer carbon atoms, e.g.,
phenyl, naphthyl, biphenyl and anthryl. One or more carbon atoms of
the aryl group may also be substituted with, e.g.: alkyl; aryl;
heterocycle; 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; carbamido; or thiocarbamido.
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.
[0040] 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.
[0041] 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.
[0042] 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; carbamido; or thiocarbamido. 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.
[0043] The term "heterocyclealkyl" refers to a heterocycle group
that is joined to a parent structare 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.
[0044] 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
[0045] 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 may be
determined in vitro for further testing in vivo.
[0046] 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 substituted indole and isatin
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 telomeric repeats 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 (by inhibiting
telomerase enzyme activity in malignant cells), and hematopoiesis
(by inhibiting telomerase activity in hematopoietic stem cells),
for example.
[0047] Thus, in one aspect, the present invention provides
compositions and compounds for the prevention or treatment of many
types of malignancies. In particular, the compounds of the present
invention can provide general method of treating many, if not most,
malignancies, as demonstrated by the highly varied human tumor cell
lines and tumors having telomerase activity. Further, the
substituted indole and isatin compounds of the present invention
are telomerase specific, i.e. they can 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.
[0048] 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. Typically, these methods comprise
contacting the telomerase enzyme with a compound, or its
pharmaceutically acceptable salt, having the formula (I): 6
[0049] wherein
[0050] X.sub.1 is chosen from O, S, CH.sub.2, or NR.sub.5 where
R.sub.5 is H, lower alkyl or aryl;
[0051] L.sub.1 is a direct single bond, direct double bond,
--CH.sub.2--, or --CH.dbd.;
[0052] is a single or a double bond;
[0053] R.sub.1 is selected from the group consisting of H,
OR.sub.5, SR.sub.5, CR.sub.6R.sub.7R.sub.8, and oxo only when is a
single, wherein R.sub.6, R.sub.7, and R.sub.8 are independently
selected from H, OH, lower alkyl, aryl, or heteroaryl;
[0054] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, OH, halogen, mercapto, nitro, cyano,
trifluromethyl, lower alkyl, lower alkoxy, aryloxy,
NR.sub.9R.sub.10, SO.sub.2NR.sub.9R.sub.10, OCHR.sub.9R.sub.10,
COR.sub.9, CO.sub.2R.sub.9, NHCONHR.sub.9, CONHR.sub.9,
NHCOR.sub.9, aryl, and heteroaryl wherein R.sub.9 and R.sub.10 are
independently selected from the group consisting of hydrogen, lower
alkyl, aryl, and heteroaryl, and R.sub.2 and R.sub.3 further
represent replacement in the ring of ring methine (--CH.dbd.) atoms
with aza (--N.dbd.) atoms;
[0055] 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; and
[0056] R.sub.4 is H, lower alkyl, alkaryl, aryl, or heteroaryl.
[0057] In the compounds of formula I, R.sub.2, R.sub.3, and R.sub.4
may be aryl to form, for example, a phenyl moiety. Alternatively,
R.sub.2, R.sub.3, and R may be heteroaryl, such as, for example,
pyridinyl, pyrimidinyl, thiophenyl, and the like. In certain
embodiments, at least one of R.sub.2, R.sub.3, or R.sub.4 is
phenyl. In other embodiments, when is a double bond, R.sub.1 can
not be oxo. In yet other embodiments, at least one of R.sub.2 and
R.sub.3 is other than hydrogen, such as, for example, when at least
one of R.sub.2 and R.sub.3 is halo, most preferably both R.sub.2
and R.sub.3 are halo to form a dihalo-substituted indole
moiety.
[0058] As noted above, L.sub.2 is a linking group that may be a
direct bond, or may be a 1 to 3 atom linking group wherein the
atoms of the linking group independently selected from
unsubstituted or substituted carbon, N, O or S. 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--, and --NHC(O)NH--.
[0059] In certain embodiments, the new compounds of the present
invention have the general structure shown as formula II below:
7
[0060] wherein
[0061] X.sub.1 is O or S;
[0062] R.sub.2 is H, OH, halogen, lower alkyl, aryl, or
heteroaryl;
[0063] L.sub.2 is a direct bond, CH.sub.2, or SO.sub.2; and
[0064] R.sub.11 is H, halogen or lower alkoxy;
[0065] or a pharmaceutically acceptable salt thereof.
[0066] The compounds may be in the form of pharmaceutically
acceptable salts or esters, or may be modified by appending one or
more appropriat , functionalities to enhance selected biological
properties. Such modifications are known in the art and include
those which increase biological penetration into a given biological
system, increase oral bioavailability, increase solubility to allow
administration by injection, and the like.
III. Synthesis of Telomerase Inhibitors
[0067] 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.).
[0068] 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.
[0069] Compounds of the class represented by formulas I and II can
be synthesized using General Procedure 1 and General Procedure 2
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.
[0070] In general, the indole containing compounds of formula (I)
can be prepared by the process described in the following reaction
Scheme 1. 8
[0071] In scheme I, R.sub.2-R4 are designated in Formula (I).
Indole-3-carbaldehyde, having the desired R.sub.2 and R.sub.3
substituents, is reacted with R.sub.4-L.sub.4 having a leaving
group (Lg) in the presence of a base, such as potassium carbonate,
optionally in the presence of a solvent. Appropriate solvents are
those which will at least partially dissolve one or all of the
reactants and will not adversely interact with either the reactants
or the product. Suitable solvents are aromatic hydrocarbons such as
toluene, o-, m- and p-xylene, halogenated hydrocarbons such as
methylene chloride, chloroform and chlorobenzene, ethers such as
diethyl ether, diisopropyl ether, tert-butyl methyl ether, dioxane,
anisole and tetrahydrofuran, nitrites such as acetonitrile and
propionitrile, ketones such as acetone, methyl ethyl ketone,
diethyl ketone and tert-butyl methyl ketone, alcohols such as
methanol, ethanol, n-propanol, isopropanol, n-butanol and
tert-butanol, and also dimethyl sulfoxide (DMSO), dimethylformamide
(DMF) and water, especially preferably DMSO, DMF, acetonitrile, and
toluene. Mixtures of these can also be used. The leaving group on
R4-L.sub.4 is appropriately chosen for the reaction conditions, and
includes halogen or sulfonate groups, preferably chlorine, bromine,
iodine, mesylate, tosylate or triflate. Suitable bases are,
generally, inorganic compounds such as alkali metal hydroxides and
alkaline earth metal hydroxides such as lithium hydroxide, sodium
hydroxide, potassium hydroxide and calcium hydroxide, alkali metal
oxides and alkaline earth metal oxides such as lithium oxide,
sodium oxide, calcium oxide and magnesium oxide, alkali metal
hydrides and alkaline earth metal hydrides such as lithium hydride,
sodium hydride, potassium hydride and calcium hydride, alkali metal
amides such as lithium amide, sodium amide and potassium amide,
alkali metal carbonates and alkaline earth metal carbonates such as
lithium carbonate and calcium carbonate, and also alkali metal
hydrogen carbonates such as sodium hydrogen carbonate, organometal
compounds, in particular alkali metal alkyls such as methyllithium,
butyllithium and phenyllithium, alkylmagnesium halides such as
methylmagnesium chloride, and alkali metal alkoxides and alkaline
earth metal alkoxides such as sodium methoxide, sodium ethoxide,
potassium ethoxide, potassium tert-butoxide and dimethoxymagnesium,
furthermore organic bases, eg. tertiary amines such as
trimethylamine, triethylamine, tri-isopropylamine and
N-methylpiperidine, pyridine, substituted pyridines such as
collidine, lutidine and 4-dimethylaminopyridine, and also bicyclic
amines. Sodium hydride, potassium hydroxide, potassium carbonate
and triethylamine are especially preferred.
[0072] The resulting N-alkylated indole product is dissolved in a
solvent and slowly added to a solution comprising approximately an
1.5 molar equivalent amount of thiazolidinedione. The reaction
mixture is then heated to an elevated temperature, preferably to
near the boiling point of the solvent, until the reaction is
complete. The solvent is preferably a polar aprotic solvent, such
as DMF, dimethylacetamide, N-methylpyrrolidone, DMSO, methanol,
ethanol, propanol, and the like. A preferred solvent is DMF. A wide
variety of basic agents can be used in the condensation of
thiazolidinedione with the indole-aldehyde. However, preferred
basic agents are amines, such as trimethylamine, triethylamine,
tributylamine, N-methylmorpholine, piperidine, N-methylpiperidine,
pyridine and 4-(N,N-dimethylamino)pyridine, with a particularly
preferred basic agent being 4-(N,N-dimethylamino)pyridine. Reaction
times of about 30 minutes to about 72 hours are common. At the end
of the reaction, the volatile components are removed under reduce
pressure. The reaction mixture can be optionally acidified before
workup. The product is precipitated and recovered such as by
filtration. The product can then be washed, dried and further
purified by standard methods such as recrystallization.
[0073] The double bonds in the compounds of Scheme 1 may be reduced
to a single bond by hydrogenation. Typically, hydrogenation is
carried out using a noble metal catalyst, such as palladium,
platinum, rhodium, or the like, as is well known in the art.
[0074] The isatin compounds of formula (I) and (II), having a wide
variety of substitutents, can be prepared by the general method
illustrated in Scheme 2. 9
[0075] Generally, the appropriately substituted isatin compound is
reacted with the R.sub.4 substituent having the appropriate leaving
group to give the N-alkylyated isatin, followed by condensation
with thiazolidinedione, as described above, to give the final
product.
IV. Anti-Tumor Activity of the Telomerase Inhibitors of the
Invention
[0076] 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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] 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.
[0082] 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.
[0083] 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.2AG.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
[0084] 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.
[0085] 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
formulas I to V (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.
[0086] 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).
[0087] In one such method, a pharmaceutical formulation comprises a
telomerase inhibitor of the invention with an anti-angiogenesis
agent, such as fumagillin, fumagillin dirivatives, 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).
[0088] 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.
[0089] 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,
puroinycin, ricin, .alpha.-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.
[0090] 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.
[0091] 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.
[0092] 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.
[0093] 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.
[0094] 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.
[0095] 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.
[0096] 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.
[0097] 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.
[0098] 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, ar
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.
[0099] 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.
[0100] 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.
[0101] 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 fuingi typically express telomerase activity).
[0102] 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.
[0103] 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.
[0104] 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.
[0105] 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.
[0106] 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.
[0107] Liquid pharmaceutically administrable compositions can, for
example, be prepared by dissolving, dispersing, etc., an active
compound as efined 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.
[0108] 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%.
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] All printed patents and publications referred to in this
application are hereby incorporated herein in their entirety by
this reference.
[0114] 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
[0115] 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. Reactions were generally run on a 0.5 mmolar scale.
Example 1
Preparation of
5-benzyloxy-N-(3,4-dichlorophenyl)-3-(2,4-dioxo-thiazolidin-
-5-ylidene-methyl)-indole
[0116] 10
[0117] Step A. [General Procedure 1]: N-alkylation of
isatins/indoles
[0118] 5-Benzyloxyindole-3-carbaldehyde (0.25 g, 1 mmol), potassium
carbonate (150 mg) and 3,4-dichlorobenzyl chloride (0.37 g, 1 mmol)
were dissolved in DMF (10 mL) and stirred at ambient temperature.
Reaction progress was determined by TLC monitoring: silica gel
(1:1) EtOAc/hexanes. When the reaction was be finished (one week),
the mixture was partitioned between water and EtOAc. The organic
layer was washed with water (3.times.) and brine, dried over
Na.sub.2SO.sub.4, and evaporated to a residue. The tan colored oil
was dissolved in EtOAc and hexanes were added to precipitate the
product, which was collected by filtration and air-dried. HPLC (uv
readout at 254 nm): isocratic 35:65 H.sub.2O/CH.sub.3CN showed
greater than 95% purity `System 1`. Identity of the title compound
was confirmed by ms 411 (M+1).
[0119] Step B. [General Procedure 2]: Coupling 2,4
thiazolidinedione (TZD) to indole-aldehydes
[0120] A solution of
5-benzyloxy-N-(3,4-dichlorophenyl)-indole-3-carbaldeh- yde (1 eq.),
2,4-thiazolidinedione (1.5 eq.) and piperidine (1.5 eq.) in EtOH
was heated to 70.degree. C. for 18 hrs. Volatiles were removed
under reduced pressure and the residue was taken up in EtOAc.
Addition of ether precipitated the product, which showed the
appropriate mass ion and was greater than 60% pure by HPLC (uv
readout at 254 nm): isocratic 35:65 H.sub.2O/CH.sub.3CN. Pure
material was obtained by column chromatography on silica gel; (5:3)
EtOAc/hexanes. Purity by HPLC was 85%; m/e 510 (M+1); 44%
yield.
Example 2
Preparation of
5-[N-(2,6-dichlorobenzyl)-isatin-3-ylidene]-2,4-dioxo-thiaz-
olidine-dione
[0121] 11
[0122] Step A: N-(2,6-dichlorobenzyl)-isatin was prepared by the
method of Example 1, Step A from isatin and 2,6-dichlorobenzyl
chloride.
[0123] Step B. [General Procedure 3]: Piperidine-mediated
condensation of 2,4-thiazolidinedione (TZD) with isatin
derivatives.
[0124] Thiazolidinedione (0.5 g, 4.2 mmol) and
N-(2,6-dichlorobenzyl)isati- n were suspended in DCM (10 mL), DIEA
(1.4 mL) was added and the mixture was stirred at ambient
temperature. After 5 min. solution was achieved. Reaction progress
was monitored by NMR analysis of 100 .mu.L aliquots from which
solvent had been evaporated. After 3 days, the reaction was worked
up by washing the organic layer with water (3.times.), drying over
Na.sub.2SO.sub.4, and concentration to a foam. NMR and TLC were
consistent with the formation of the title compound (0.9 g, 2.2
mmol, 70%).
Example 3
Preparation of
5-benzyloxy-3-(2,4-dioxo-thiazolidin-5-ylidenemethyl)-N-(2--
thienylsulfonyl)-indole
[0125] 12
[0126] Step A:
5-Benzyloxy-3-(2,4-dioxo-thiazolidin-5-ylidenemethyl)indole was
prepared by the method of Example 1, Step A from thiazolidinedione
and 5-benzyloxyindole carbaldehyde.
[0127] Step B: [General Procedure 4]: Sulfonylation of
isatins/indoles.
[0128] The product from step A (50 mg) was treated with 1.2
equivalents of NaH in DMF solution with cooling for 1 hr.
2-Thienylsulfonyl chloride (34mg, 1.3 equivaltents) was added
cautiously. After 18 hrs. at ambient temperature and finally at
70.degree. C., the mixture was poured onto ice and the precipitate
was filtered. The title compound was filtered and washed with
ether. HPLC `System 1` (Ex. 1) showed 80% purity; structure
confirmed by mass spec [m.backslash.e 497 (M+1)].
Example 4
Preparation of
5-[5-chloro-N-(p-tolylsulfonyl)-isatin-3-ylidene]-2,4-dioxo-
-thiazolidine-dione
[0129] 13
[0130] Step A. 5-chloro-N-(p-tolylsulfonyl)-isatin was prepared by
the method of Example 3, Step B from 5-chloroisatin and
p-tolylsulfonyl chloride.
[0131] Step B. [General Procedure 5]: Acetic acid/acetic anhydride
condensation of 2,4-thiazolidinedione (TZD) with isatin/indole
derivatives.
[0132] Thiazolidinedione (0.5 g, 4.2 mmol) and
N-(2,6-dichlorobenzyl)isati- n were suspended in DCM (10 mL), DIEA
(1.4 mL) was added and the mixture was stirred at ambient
temperature. After 5 min. solution was achieved. Reaction progress
was monitored by NMR analysis of 100 .mu.L aliquots from which
solvent had been evaporated. After 3 days, the reaction was worked
up by washing organics with water (3.times.), drying over
Na.sub.2SO.sub.4, and concentration to a foam. NMR and TLC were
consistent with the formation of the title compound (0.9 g, 2.2
mmol, 70%).
Example 5
Preparation of
5-[5-(phenylaminosulfonyl)-N-(2,6-dichlorobenzyl)isatin-3-y-
lidene]-thiazolidinedione
[0133] 14
[0134] General Procedure 6: 5-(N-Substituted-sulfonamido)isatin
derivatives via indole chloro-sulfonylation.
[0135] Chlorosulfonic acid (2 mL) was added carefully to of
N-(2,6-dichlorobenzyl)isatin-3-ylidene thiazolidinedion (a foam,
0.9 g, Example 2). The mixture was stirred at RT for 30 min, cooled
in a dry ice/acetone bath and quenched by dropwise addition of
water. The product was extracted with EtOAc, concentrated to a foam
and triturated with hexane/EtOAc. To 10-20 mg of the acid chloride
was added aniline (1 mL), a trace amount of DMAP (1 mg) and
pyridine (1-2 mL). The mixture was stirred at RT for 30 min,
volatiles were removed under reduced pressure and the dark residue
was purified by SG chromatography (gradient elution 0-3% MeOH/DCM).
NMR and ms were consistent with the formation of
5-(phenylaminosulfonyl)-N-(2,6-dichlorobenzyl)isatin-3-ylidene
thiazolidinedione as the product. HPLC (System 1) showed greater
than 92% purity.
Example 6
Preparation of
5-[5-chloro-N-(2-cyanoethyl)isatin-3-ylidene]-thiazolidined-
ione
[0136] 15
[0137] Step A. Acrylonitrile was added dropwise to a mixture of
5-chloroisatin (0.5 g) and Triton B (0.115 mL of a 40% solution)
and stirring was continued for 48 hrs; 470 mg of an orange solid
was filtered. Nmr and ms were consistent with
5-chloro-N-cyanoethyl-isatin.
[0138] Step B. The title compound was prepared by the method of
Example 4, Step B.
Example 7
Preparation of
5-[5-chloro-N-(p-nitrosulfonyl)-isatin-3-ylidene]-2,4-dioxo-
-thiazolidine-dione
[0139] 16
[0140] Prepared by the method of Example 4; NMR was consistent with
structure; ms: m.backslash.e 466 (M+1).
Example 8
Preparation of
5-benzyloxy-N-(3,4-dichlorobenzoyl)-3-(2,4-dioxo-thiazolidi-
n-5-ylidene-methyl)-indole
[0141] 17
[0142]
5-Benzyloxy-3-(2,4-dioxo-thiazolidin-5-ylidene-methyl)-indole
(Example 1, Step A; 10 mg) was dissolved in pyridine (1.5 mL) and
3,4-dichlorobenzoyl chloride (35 mg) and a trace of
dimethylaminopyridine were added. The mixture was stirred at
50-60.degree. for 18 hrs until the reaction was determined to be
complete by TLC. Volatiles were removed under reduced pressure and
the residue was washed with water and ether; 10 mg; consistent nmr
and ms (m.backslash.e 525 (M+1); greater than 95% by HPLC.
Example 9
Preparation of
7-benzyloxy-N-(3,4-dichlorophenyl)-3-(2,4-dioxo-thiazolidin-
-5-ylidene-methyl)-indole
[0143] 18
[0144] Title compound was prepared by the method of Example 1 using
7-benzyloxyindole 3-carbaldehyde (25 mg) affording the desired
product (32 mg, 86% yield, greater than 95% by HPLC); NMR and ms
consistent; ms: m/e 411 (M+1).
Example 10
Preparation of
5-benzyloxy-N-(3,4-dichlorophenyl)-3-(2,4-dioxo-thiazolidin-
-5-ylmethyl)-indole
[0145] 19
[0146] To oven-dried SiO.sub.2(150 mg) in toluene (2 mL) was added
50 mg of the thiazolidinylidene olefin (Example 1) with diethyl
1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate (25 mg). The
mixture was stirred and heated at 70-90.degree. for 3 days. Mass
spectral monitoring indicated the reaction was complete. Silica was
filtered and washed with EtOAc and combined volatiles were removed
under reduced pressure to a residue. The solid dissolved in ether
and solids were filtered and discarded. Ether was evaporated and
residue was washed with hexanes and air-dried to afford the
reduction product; ms: m/e 512 (M+1); HPLC.about.70% pure.
Example 11
Preparation of
2-(4-chlorophenyl)-N-(3,4-dichlorophenyl)-3-(2,4-dioxo-thia-
zolidin-5-ylidene-methyl)-indole
[0147] 20
[0148] Title compound prepared by the method of Example 1 from
2-(4-chlorophenyl)-indole 3-carbaldehyde. Nmr and ms [m/e 515
(M+1)] were consistent; HPLC greater than 95% pure.
Example 12
Preparation of
5-[N-(2-(methoxycarbonyl)thien-3-ylsulfonyl)-isatin-3-ylide-
ne]-2,4-dioxo-thiazolidine-dione
[0149] 21
[0150] Step A: N-(2-(methoxycarbonyl)thien-3-ylsulfonyl)-isatin was
prepared by General Procedure 4.
[0151] Step B: The condensation reaction was performed by General
Procedure 5 to afford the title compound; ms: m/e 484 (M-1). HPLC
greater than 80% purity.
Example 13
Preparation of
5-[N-(3-chlorobenzyl)-isatin-3-ylidene]-2,4-dioxo-thiazolid-
ine-dione
[0152] 22
[0153] Compound was prepared by the method of Example 2; nmr and ms
were consistent with the product; ms m/e 371 (M+1); HPLC showed
greater than 80% purity.
Example 14
Preparation of
5-[5-chloro-N-(2,6-dichlorobenzyl)-isatin-3-ylidene]-2,4-di-
oxo-thiazolidine-dione
[0154] 23
[0155] Compound was prepared by the method of Example 2; nmr and ms
were consistent with the product; ms m/e 440 (M+1); HPLC showed
greater than 80% purity.
Example 15
Preparation of
5-[6-chloro-N-(2,6-dichlorobenzyl)-isatin-3-ylidene]-2,4-di-
oxo-thiazolidine-dione
[0156] 24
[0157] Compound was prepared by the method of Example 2; nmr and ms
were consistent with the product; ms m/e 440 (M+1); HPLC showed
greater than 80% purity.
Example 16
Preparation of
5-[5-methyl-N-(2,6-dichlorobenzyl)-isatin-3-ylidene]-2,4-di-
oxo-thiazolidine-dione
[0158] 25
[0159] Compound was prepared by the method of Example 2; nmr and ms
were consistent with the product; ms m/e 420 (M+1); HPLC showed
greater than 80% purity.
Example 17
Preparation of
5-[5,6-dichloro-N-(3,4-dichlorobenzyl)-isatin-3-ylidene]-2,-
4-dioxo-thiazolidine-dione
[0160] 26
[0161] Compound was prepared by the method of Example 2; nmr and ms
were consistent with the product; ms m/e 475 (M+1); HPLC showed
greater than 80% purity.
Example 18
Preparation of
5-[5-chloro-N-(3-trifluoromethyl)-isatin-3-ylidene]-2,4-dio-
xo-thiazolidine-dione
[0162] 27
[0163] Compound was prepared by the method of Example 2; nmr and ms
were consistent with the product; ms m/e 474 (M+1); HPLC showed
greater than 80% purity.
Example 19
Preparation of
5-[6-benzoyl-N-(3,4-dichloro)-isatin-3-ylidene]-2,4-dioxo-t-
hiazolidine-dione
[0164] 28
[0165] Compound was prepared by the method of Example 2; nmr and ms
were consistent with the product; ms m/e 540 (M+1); HPLC showed
greater than 80% purity.
Example 20
Preparation of
5-[6-benzoyl-N-(3,4-dichloro)-isatin-3-ylidene]-2,4-dioxo-t-
hiazolidine-dione
[0166] 29
[0167] Compound was prepared by the method of Example 2; nmr and ms
were consistent with the product; ms m/e 540 (M+l); HPLC showed
greater than 80% purity.
Example 21
Preparation of
5-[5-(4-chlorophenylaminosulfonyl)-N-(2,6-dichlorobenzyl)is-
atin-3-ylidene]-thiazolidinedione
[0168] 30
[0169] Compound was prepared by General Procedure 6; nmr and ms
were consistent with the product; ms m/e 595 (M+1); HPLC (System 1)
showed greater than 90% purity.
Example 22
Preparation of
5-[5-(4-isopropylphenylaminosulfonyl)-N-(3,4-dichlorobenzyl-
)isatin-3-ylidene]-thiazolidinedione
[0170] 31
[0171] Compound was prepared by General Procedure 6; nmr and ms
were consistent with the product; ms m/e 603 (M+1); HPLC (System 1)
showed greater than 90% purity.
Example 23
Preparation of
5-[5-(phenylaminosulfonyl)-N-(3,4-dichlorobenzyl)isatin-3-y-
lidene]-thiazolidinedione.
[0172] 32
[0173] Compound was prepared by General Procedure 6; nmr and ms
were consistent with the product; ms m/e 561 (M+1); HPLC (System 1)
showed greater than 90% purity.
Example 24
Preparation of
5-[5-(4-trifluoromethoxyphenylaminosulfonyl)-N-(3,4-dichlor-
obenzyl)-isatin-3-ylidene]-thiazolidinedione
[0174] 33
[0175] Compound was prepared by General Procedure 6; nmr and ms
were consistent with the product; ms m/e 645 (M+1); HPLC (System 1)
showed greater than 90% purity.
Example 25
Preparation of
5-[5-(3-trifluoromethylphenylaminosulfonyl)-N-(3,4-dichloro-
benzyl)-isatin-3 -ylidene]-thiazolidinedione
[0176] 34
[0177] Compound was prepared by General Procedure 6; nmr and ms
were consistent with the product; ms m/e 629 (M+1); HPLC (System 1)
showed greater than 90% purity.
Example 26
Preparation of
5-[5-(4-ethylpiperazinylsulfonyl)-N-(3,4-dichlorobenzyl)-is-
atin-3-ylidene]-thiazolidinedione
[0178] 35
[0179] Compound was prepared by General Procedure 6; nmr and ms
were consistent with the product; ms mn/e 582 (M+1); HPLC (System
1) showed greater than 90% purity.
Example 27
Preparation of
5-[5-(4-methylbenzylaminosulfonyl)-N-(3,4-dichlorobenzyl)-i-
satin-3-ylidene]-thiazolidinedione
[0180] 36
[0181] Compound was prepared by General Procedure 6; nmr and ms
were consistent with the product; ms m/e 589 (M+1); HPLC (System 1)
showed greater than 90% purity.
Example 28
Preparation of
5-[5-(3,4-methylenedioxybenzylaminosulfonyl)-N-(3,4-dichlor-
obenzyl)-isatin-3-ylidene]-thiazolidinedione
[0182] 37
[0183] Compound was prepared by General Procedure 6; nmr and ms
were consistent with the product; ms m/e 619 (M+1); HPLC (System 1)
showed greater than 90% purity.
Example 29
Preparation of
5-[5-(4-phenylpiperazinylsulfonyl)-N-(3,4-dichlorobenzyl)-i-
satin-3-ylidene]-thiazolidinedione
[0184] 38
[0185] Compound was prepared by General Procedure 6; nmr and ms
were consistent with the product; ms m/e 630 (M+1); HPLC (System 1)
showed greater than 90% purity.
Example 30
Preparation of
5-[5-(4-morpholinophenylaminosulfonyl)-N-(3,4-dichlorobenzy-
l)-isatin-3 -ylidene]-thiazolidinedione
[0186] 39
[0187] Compound was prepared by General Procedure 6; nmr and ms
were consistent with the product; ms m/e 646 (M+1); HPLC (System 1)
showed greater than 90% purity.
Example 31
Preparation of
5-[5-(naphth-2-ylmethylaminosulfonyl)-N-(3,4-dichlorobenzyl-
)-isatin-3-ylidene]-thiazolidinedione
[0188] 40
[0189] Compound was prepared by General Procedure 6; nmr and ms
were consistent with the product; ms m/e 625 (M+1); HPLC (System 1)
showed greater than 90% purity.
Example 32
Preparation of
5-[2-(3,4-dichlorophenyl)ethylaminosulfonyl)-N-(3,4-dichlor-
obenzyl)-isatin-3-ylidene]-thiazolidinedione
[0190] 41
[0191] Compound was prepared by General Procedure 6; nmr and ms
were consistent with the product; ms m/e 658 (M+1); HPLC (System 1)
showed greater than 90% purity.
Example 33
Preparation of
5-[4-phenoxyphenylaminosulfonyl)-N-(3,4-dichlorobenzyl)-isa-
tin-3-ylidene]-thiazolidinedione
[0192] 42
[0193] Compound was prepared by General Procedure 6; nmr and ms
were consistent with the product; ms m/e 653 (M+1); HPLC (System 1)
showed greater than 90% purity.
Example 34
Preparation of
5-[2-hydroxyphenylaminosulfonyl)-N-(3,4-dichlorobenzyl)-isa-
tin-3-ylidene]-thiazolidinedione
[0194] 43
[0195] Compound was prepared by General Procedure 6; nmr and ms
were consistent with the product; ms m/e 577 (M+1); HPLC (System 1)
showed greater than 90% purity. Meta- and para-hydroxy derivatives
were also prepared and demonstrated similar physical properties and
purity.
Example 35
Preparation of
5-[6-hydroxyhexylaminosulfonyl)-N-(3,4-dichlorobenzyl)-isat-
in-3-ylidene]-thiazolidinedione
[0196] 44
[0197] Compound was prepared by General Procedure 6; nmr and ms
were consistent with the product; ms m/e 585 (M+1); HPLC (System 1)
showed greater than 90% purity.
Example 36
Preparation of
5-[cyclohexylaminosulfonyl)-N-(3,4-dichlorobenzyl)-isatin-3-
-ylidene]-thiazolidinedione
[0198] 45
[0199] Compound was prepared by General Procedure 6; nmr and ms
were consistent with the product; ms m/e 567 (M+1); HPLC (System 1)
showed greater than 90% purity.
Example 37
Preparation of
5-[2-propylphenylaminosulfonyl)-N-(3,4-dichlorobenzyl)-isat-
in-3-ylidene]-thiazolidinedione
[0200] 46
[0201] Compound was prepared by General Procedure 6; nmr and ms
were consistent with the product; ms m/e 603 (M+1); HPLC (System 1)
showed greater than 90% purity.
Example 38
Preparation of
5-[2,6-diisopropylphenylaminosulfonyl)-N-(3,4-dichlorobenzy-
l)-isatin-3-ylidene]-thiazolidinedione
[0202] 47
[0203] Compound was prepared by General Procedure 6; nmr and ms
were consistent with the product; ms m/e 645 (M+1); HPLC (System 1)
showed greater than 90% purity.
Example 39
Preparation of
5-benzyloxy-N-(2-ethoxyethyl)-3-(2,4-dioxo-thiazolidin-5-yl-
idene-methyl)-indole
[0204] 48
[0205] The title compound was prepared by General Procedure 1 with
5-benzyloxyindole 3-carbonitrile and 2-ethoxyethyl chloride; nmr
and ms were consistent (m.backslash.e 423 (M+1); greater than 95%
by HPLC.
Example 40
Preparation of
5-benzyloxy-N-(pivaloyl)-3-(2,4-dioxo-thiazolidin-5-ylidene-
-methyl)-indole
[0206] 49
[0207] The title compound was prepared by the method of Example 8
with 5-benzyloxyindole 3-carbonitrile and pivaloyl chloride; nmr
and ms were consistent (m.backslash.e 421 (M+1); greater than 95%
by HPLC.
Example 41
Preparation of 5-benzyloxy-N-(3-phenoxybenzyl)-3
-(2,4-dioxo-thiazolidin-5- -ylidene-methyl)-indole
[0208] 50
[0209] The title compound was prepared by General Procedure 1 with
5-benzyloxyindole 3-carbonitrile and 3-phenoxybenzyl chlorde; nmr
and ms were consistent (m.backslash.e 535 (M+1); greater than 95%
by HPLC.
Example 42
Preparation of
7-benzyloxy-N-(3-phenoxybenzyl)-3-(2,4-dioxo-thiazolidin-5--
ylidene-methyl)-indole
[0210] 51
[0211] The title compound was prepared by General Procedure 1 with
7-benzyloxyindole 3-carbonitrile and 3-phenoxybenzyl chlorde; nmr
and ms were consistent (m.backslash.e 535 (M+1); greater than 95%
by HPLC.
Example 43
Preparation of
5-methoxy-N-(3,4-dichlorobenzyl)-3-(2,4-dioxo-thiazolidin-5-
-ylidene-methyl)-indole
[0212] 52
[0213] The title compound was prepared by the method of Example 1
from 5-methoxylindole 3-carbonitrile; nmr and ms were consistent
(m.backslash.e 434 (M+1); greater than 95% by HPLC.
Example 44
Preparation of
5-benzyloxy-N-(3-methoxybenzoyl)-3-(2,4-dioxo-thiazolidin-5-
-ylidene-methyl)-indole
[0214] 53
[0215] The title compound was prepared by the method of Example 8
from 5-benzyloxylindole 3-carbonitrile; nmr and ms were consistent
(m.backslash.e 485 (M+1); greater than 95% purity by HPLC.
Example 45
Preparation of
5-benzyloxy-N-(4-ethoxycarbonylbenzyl)-3-(2,4-dioxo-thiazol-
idin-5-ylidene-methyl)-indole
[0216] 54
[0217] The title compound was prepared by the method of Example 1
from 5-benzyloxylindole 3-carbonitrile; nmr and ms were consistent
(m.backslash.e 485 (M+1); greater than 50% purity by HPLC.
Example 46
Preparation of
5-benzyloxy-N-(4-piperidinocarbonylbenzyl)-3-(2,4-dioxo-thi-
azolidin-5-ylidene-methyl)-indole
[0218] 55
[0219] The title compound was prepared by the method of Example 1
from 5-benzyloxylindole 3-carbonitrile; nmr and ms were consistent
(m.backslash.e 552 (M+1); greater than 50% purity by HPLC.
Example 47
Preparation of
5-amino-N-(3,4-dichlorobenzyl)-3-(2,4-dioxo-thiazolidin-5-y-
lidene-methyl)-indole
[0220] 56
[0221] Step A.
5-Nitro-N-(3,4-dichlorobenzyl)-3-(2,4-dioxo-thiazolidin-5-y-
lidenemethyl)-indole was prepared by the method of Example 1 from
5-nitroindole 3-carbonitrile; nmr and ms were consistent with
structure.
[0222] Step B. Reduction of the product of Step 1 was accomplished
by treating 660 mg in aqueous isopropanol (1:3) containing ammonium
chloride (50 mg) with iron filings (400 mg) at 60.degree. for 18
hrs. The yellow solution was filtered and concentrated under
reduced pressure. The aqueous reaction mixture was then partitioned
between EtOAc and water and the organic phase washed additional
water. The organic phase was dried (Na.sub.2SO.sub.4) and
concentrated to a small volume whereupon a solid precipitated. This
material was filtered and air dried; 440 mg (77%); nmr and ms were
consistent [ms: m/e 419 (M+1)]. HPLC (System 1) showed greater than
95% purity.
Example 48
Preparation of
5-(3,4-dichlorobenzoylamino)-N-(3,4-dichlorobenzyl)-3-(2,4--
dioxo-thiazolidin-5-ylidenemethyl)-indole
[0223] 57
[0224] General Procedure 7: Acylation/sulfonylation of
aminoindoles.
[0225] The aminoindole from Example 46 (6 mg) was dissolved in
pyridine (1 mL), 2.5 mg of 3,4-dichlorobenzoyl chloride was added,
and stirring was maintained for 18 hrs. The mixture was stirred at
RT for 30 min, volatiles were removed under reduced pressure and
the dark residue was purified by SG chromatography (gradient
elution 0-3% MeOH/DCM). NMR and ms were consistent with the
formation of 5-(phenylaminosulfonyl)-N-(2,6-d-
ichlorobenzyl)isatin-3-ylidene thiazolidinedione as the product
[ms: m/e 592 (M+1)]. HPLC (System 1) showed greater than 92%
purity.
Example 49
Preparation of
5-[N'-(3,4-dichlorophenyl)ureido]-N-(3,4-dichlorobenzyl)-3--
(2,4-dioxo-thiazolidin-5-ylidenemethyl)-indole
[0226] 58
[0227] The title compound was prepared by General Procedure 7 with
3,4-dichlorophenyl isocyanate and the amino compound from Example
47 as reactants. Nmr and ms data were consistent; ms m/e 607 (M+1).
HPLC (System 1): purity greater than 80%.
Example 50
Preparation of
5-[N'-(2,4-dimethoxyphenyl)ureido]-N-(3,4-dichlorobenzyl)-3-
-(2,4-dioxo-thiazolidin-5-ylidenemethyl)-indole
[0228] 59
[0229] The title compound was prepared by General Procedure 7 with
2,4-dimethoxyphenyl isocyanate and the amino compound from Example
47 as reactants. Nmr and ms data were consistent; ms m/e 598 (M+1).
HPLC (System 1): purity greater than 80%.
Example 51
Preparation of
5-benzoylamino-N-(3,4-dichlorobenzyl)-3-(2,4-dioxo-thiazoli-
din-5-ylidenemethyl)-indole
[0230] 60
[0231] The title compound was prepared by General Procedure 7 with
benzoyl chloride and the amino compound from Example 47 as
reactants. Nmr and ms data were consistent; ms m/e 523 (M+1). HPLC
(System 1): purity greater than 80%.
Example 52
Preparation of
5-succinamido-N-(3,4-dichlorobenzyl)-3-(2,4-dioxo-thiazolid-
in-5-ylidenemethyl)-indole
[0232] 61
[0233] The title compound was prepared by General Procedure 7 with
succinic anhydride and the amino compound from Example 47 as
reactants. Nmr and ms data were consistent; ms m/e 519 (M+1). HPLC
(System 1): purity greater than 80%.
Example 53
Preparation of
5-(3,4-dichlorophthalamido)-N-(3,4-dichlorobenzyl)-3-(2,4-d-
ioxo-thiazolidin-5-ylidenemethyl)-indole
[0234] 62
[0235] The title compound was prepared by General Procedure 7 with
3,4-dichlorophthalic anhydride and the amino compound from Example
47 as reactants. Nmr and ms data were consistent; ms m/e 636 (M+1).
HPLC (System 1): purity greater than 80%.
Example 54
Preparation of
5-benzyloxy-N-[4-(2,4-dioxo-thiazolidin-5-ylidenemethyl)phe-
nyl]-3-(2,4-dioxo-thiazolidin-5-ylidenemethyl)-indole
[0236] 63
[0237] Step A: 5-Benzyloxyindole-3-carbaldehyde (0.25 g, mmol),
potassium carbonate (280 mg, 2 equivs.), copper II oxide (8 mg),
and 4-bromobenzaldehyde (0.37 g, 2 mmol) were dissolved in DMF (10
mL) and heated at 140.degree. for 2-3 days. Reaction progress was
determined by TLC monitoring: silica gel (1:1) EtOAc/hexanes. When
the reaction composition no longer changed, solids were filtered
and discarded, volatiles were removed under reduced pressure, and
the mixture was partitioned between water and EtOAc. The organic
layer was washed with water (3.times.) and brine, dried over
Na.sub.2SO.sub.4, and evaporated to a residue. Column
chromatography on silica (gradient elution 0-5% MeOH/DCM) gave the
dialdehyde [3,4'-(N-phenyl)]. Identity was confirmed by msj; HPLC
greater than 90% purity.
[0238] Step B: Treatment of the dialdehyde with 3 equivalents of
thiazolidinedione by General Procedure 2 gave the title compound.
Nmr and ms [m/e 554 (M+1)] were consistent.
Example 55
Preparation of
5-methoxy-N-[4-(2,4-dioxo-thiazolidin-5-ylidenemethyl)pheny-
l]-3-(2,4-dioxo-thiazolidin-5-ylidenemethyl)-indole
[0239] 64
[0240] The title compound was prepared by the method of Example 54
using 5-methoxyindole 3-carbaldehyde. NMR and ms [m/e 478 (M+1)]
were consistent with the product.
Example 56
Preparation of Affinity Purified Extract
[0241] 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-biotinTEG-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
mmol 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 mmol 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 57
Telomerase Specific Activity Determination
[0242] 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 56) 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.2O 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 vacuum
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 mmol 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 mmol dNTP/min/.mu.l extract.
Example 58
Telomerase Activity Assay
[0243] Bio-Tel FlashPlate Assay
[0244] 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 telome) Ase products is determined by scintillation
counting.
[0245] Method:
[0246] 1. Compounds are stored as concentrated stocks and dissolved
in 100 % dimethylsulfoxide (DMSO).
[0247] 2. For testing, the compounds are diluted to a
15.times.working stock in 50% DMSO and 2 .mu.l is dispensed into
two wells of a 96-well microtiter dish (assayed in duplicate).
[0248] 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.
[0249] 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.
[0250] 5. The reaction is stopped by the addition of 10 .mu.l
HCS.
[0251] 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.
[0252] 7. The wells are washed three times with 180 .mu.l
2.times.SSC without any incubation.
[0253] 8. The counts of probe annealed to biotinylated telomerase
products are detected on a scintillation counter.
[0254] Buffers:
[0255] Telomerase Dilution Buffer
[0256] 50 mM Tris-acetate, pH 8.2
[0257] 1 mM DTT
[0258] 1 mM EGTA
[0259] 1 mM MgCl.sub.2
[0260] 830 nM BSA
[0261] Master Mix (MM)
[0262] 50 mM Tris-acetate, pH 8.2
[0263] 1 mM DTT
[0264] 1 mM EGTA
[0265] 1 mM MgCl.sub.2
[0266] 150 mM K acetate
[0267] 10 .mu.M dATP
[0268] 20 .mu.M dGTP
[0269] 120 .mu.M dTTP
[0270] 100 mM biotinylated primer
(5'-biotin-AATCCGTCGAGCAGAGTT-3')
[0271] 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
[0272] Hybridization Capture Solution (HCS)
[0273] 12.times.SSC (1.times.=150 mM NaCl/30 mM
Na.sub.3Citrate)
[0274] 40 mM EDTA
[0275] 40 mM Tris-HCl, pH 7.0
[0276] Using the foregoing assay, the compounds of Examples 1-55
were shown to have telomerase IC.sub.50 values below 100 .mu.M.
Example 59
Anti-tumor Activity
[0277] Ex vivo Studies
[0278] a. Reduction of Telomere Length in Tumor Cells
[0279] 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).
[0280] 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.
[0281] Telomere length is determined by digesting the DNA of the
cells using restriction enzymes specific for sequences other than
the repetitive T.sub.2AG.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).
[0282] The results of the telomere length analysis are expected to
indicate that the compounds of the invention have no effect 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. 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. Tumor cells exposed to
the control are expected to maintain steady telomere lengths.
[0283] 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.
[0284] b. Specificity
[0285] 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
specificity for telomerase if the IC.sub.50 for telomerase of the
compound is lower than the IC.sub.50 values for each of the other
enzymes being screened.
[0286] 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.
* * * * *