U.S. patent application number 16/741409 was filed with the patent office on 2020-11-19 for htert modulators and methods of use.
The applicant listed for this patent is Arizona Board of Regents on Behalf of the University of Arizona. Invention is credited to Vijay GOKHALE, Laurence HURLEY, HyunJin KANG, Kui WU.
Application Number | 20200361860 16/741409 |
Document ID | / |
Family ID | 1000005004293 |
Filed Date | 2020-11-19 |
United States Patent
Application |
20200361860 |
Kind Code |
A1 |
HURLEY; Laurence ; et
al. |
November 19, 2020 |
hTERT MODULATORS AND METHODS OF USE
Abstract
The present invention provides hTERT modulators and methods for
producing and using the same. In particular, the present invention
provide a compound of the formula: ##STR00001## where a, n,
R.sup.1, R.sup.2, R.sup.3, R.sup.11 and R.sup.12 are as defined
herein.
Inventors: |
HURLEY; Laurence; (Tucson,
AZ) ; GOKHALE; Vijay; (Tucson, AZ) ; KANG;
HyunJin; (Tucson, AZ) ; WU; Kui; (Tucson,
AZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Arizona Board of Regents on Behalf of the University of
Arizona |
Tucson |
AZ |
US |
|
|
Family ID: |
1000005004293 |
Appl. No.: |
16/741409 |
Filed: |
January 13, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15779132 |
May 25, 2018 |
10556860 |
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PCT/US2016/064287 |
Nov 30, 2016 |
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16741409 |
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62261838 |
Dec 1, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 295/088 20130101;
C07C 275/54 20130101; A61P 35/00 20180101; C07D 239/34 20130101;
A61K 31/505 20130101; C07D 403/04 20130101; A61K 31/17 20130101;
C07D 219/08 20130101; A61K 31/4995 20130101; A61K 31/5377 20130101;
C07D 239/52 20130101; C07D 401/12 20130101; A61K 31/4402 20130101;
C07D 241/18 20130101 |
International
Class: |
C07C 275/54 20060101
C07C275/54; A61K 31/5377 20060101 A61K031/5377; A61K 31/4995
20060101 A61K031/4995; A61K 31/505 20060101 A61K031/505; A61K 31/17
20060101 A61K031/17; A61K 31/4402 20060101 A61K031/4402; C07D
401/12 20060101 C07D401/12; C07D 403/04 20060101 C07D403/04; C07D
239/34 20060101 C07D239/34; C07D 239/52 20060101 C07D239/52; C07D
241/18 20060101 C07D241/18; C07D 295/088 20060101 C07D295/088; A61P
35/00 20060101 A61P035/00; C07D 219/08 20060101 C07D219/08 |
Goverment Interests
STATEMENT REGARDING FEDERALLY FUNDED RESEARCH
[0002] This invention was made with government support under Grant
Nos. R01CA153821 and R01CA177585 awarded by NIH. The government has
certain rights in the invention.
Claims
1. A compound of the formula: ##STR00039## wherein X is O or N such
that when X is O, bond a is absent and when X is N, bond a is
present; n is 0 or 1 such that: when n=0, R.sup.3 is
--NHC(.dbd.NH)NH.sub.2; and when n=1, R.sup.3 is hydrogen, alkyl,
cycloalkyl, optionally substituted heterocycloalkyl, optionally
substituted heteroaryl or optionally substituted aryl, wherein each
substituent is independently selected from the group consisting of
halogen, cyano, nitro, azido, haloalkyl, cycloalkyl, heteroaryl,
aryl, --NR.sup.7R.sup.8, --NR.sup.7C(O)R.sup.8,
--C(O)NR.sup.7R.sup.8, --C(O)R.sup.9, --C(O)OR.sup.9,
--S(O)R.sup.9, --SO.sub.2R.sup.9, --SO.sub.2NR.sup.7R.sup.8,
--OR.sup.9 and --SR.sup.9; R.sup.1 is optionally substituted aryl
or optionally substituted heteroaryl, wherein each substituent is
independently selected from the group consisting of halogen, cyano,
nitro, azido, haloalkyl, cycloalkyl, --NR.sup.4R.sup.5,
--NR.sup.4C(O)R.sup.5, --C(O)NR.sup.4R.sup.5, --C(O)R.sup.6,
--C(O)OR.sup.6, --S(O)R.sup.6, --SO.sub.2R.sup.6,
--SO.sub.2NR.sup.4R.sup.5, --OR.sup.6, and --SR.sup.6; R.sup.2 is
selected from the group consisting of hydrogen, halogen, alkyl,
haloalkyl and cycloalkyl; each of R.sup.4 and R.sup.5 is
independently selected from the group consisting of hydrogen,
alkyl, alkenyl, alkynyl, heteroaryl, aryl and cycloalkyl, or
R.sup.4 and R.sup.5 together with the nitrogen atom to which they
are attached to form an optionally substituted monocyclic or
bicyclic ring with one or more heteroatoms; R.sup.6 is selected
from the group consisting of hydrogen, alkyl, alkenyl, alkynyl,
heteroaryl, aryl, cycloalkyl, wherein the alkyl, alkenyl,
heteroaryl, aryl and cycloalkyl are optionally substituted with one
or more halo, cyano, alkylamino, alkoxy, aryl, heteroaryl or
heterocyclyl groups; each of R.sup.7 and R.sup.8 is independently
selected from the group consisting of hydrogen, alkyl, alkenyl,
alkynyl, heteroaryl, aryl and cycloalkyl, or R.sup.7 and R.sup.8
together with the nitrogen group to which they are attached to form
an optionally substituted monocyclic or bicyclic ring with one or
more heteroatoms; R.sup.9 is independently selected from the group
consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroaryl, aryl
cycloalkyl, wherein the alkyl, alkenyl, heteroaryl, aryl and
cycloalkyl are optionally substituted with one or more halo, cyano,
alkylamino, alkoxy, aryl, heteroaryl or heterocyclyl groups; each
R.sup.11 and R.sup.12 is independently --H, --COR.sup.13 or
--CH.sub.3; and R.sup.13 is alkyl, haloalkyl, alkenyl, alkynyl, or
cycloalkyl.
2. The compound of claim 1, wherein R.sup.1 is selected from the
group consisting of: ##STR00040## ##STR00041## ##STR00042##
3. The compound of claim 1, wherein R.sup.2 is selected from the
group consisting of hydrogen, methyl, ethyl, isopropyl,
trifluoromethyl and a halide.
4. The compound of claim 1, wherein R.sup.3 is selected from the
group consisting of hydrogen, methyl, ethyl, trifluoromethyl,
propyl, cyclopropylmethyl, cyclopentyl, cyclohexyl, benzyl,
2-phenylethyl, 2-methoxyethyl, 3-methoxypropyl, or a moiety
selected from the group consisting of: ##STR00043## wherein m is an
integer from 2 to 4; each of R.sup.14 and R.sup.15 is independently
selected from the group consisting of hydrogen, alkyl, alkenyl,
alkynyl, heteroaryl, aryl and cycloalkyl; or R.sup.14 and R.sup.15
together with the nitrogen atom to which they are attached to form
a substituted or unsubstituted monocyclic or bicyclic
heterocycloalkyl.
5. The compound of claim 1, wherein the compound is selected from a
compound of Table 1 or a pharmaceutically acceptable salt thereof,
or a prodrug thereof.
6. (canceled)
7. A method for treating a patient suffering from a clinical
condition, said method comprising administering to a patient a) a
therapeutically effective amount of compound I: ##STR00044## or a
pharmaceutically acceptable salt thereof, or a prodrug thereof, or
a mixture thereof; or b) a therapeutically effective amount of a
compound of the formula: ##STR00045## wherein X is O or N such that
when X is O, bond a is absent and when X is N, bond a is present; n
is 0 or 1 such that: when n=0, R.sup.3 is --NHC(.dbd.NH)NH.sub.2;
and when n=1, R.sup.3 is hydrogen, alkyl, cycloalkyl, optionally
substituted heterocycloalkyl, optionally substituted heteroaryl or
optionally substituted aryl, wherein each substituent is
independently selected from the group consisting of halogen, cyano,
nitro, azido, haloalkyl, cycloalkyl, heteroaryl, aryl,
--NR.sup.7R.sup.8, --NR.sup.7C(O)R.sup.8, --C(O)NR.sup.7R.sup.8,
--C(O)R.sup.9, --C(O)OR.sup.9, --S(O)R.sup.9, --SO.sub.2R.sup.9,
--SO.sub.2NR.sup.7R.sup.8, --OR and --SR.sup.9; R.sup.1 is
optionally substituted aryl or optionally substituted heteroaryl,
wherein each substituent is independently selected from the group
consisting of halogen, cyano, nitro, azido, haloalkyl, cycloalkyl,
--NR.sup.4R.sup.5, --NR.sup.4C(O)R.sup.5, --C(O)NR.sup.4R.sup.5,
--C(O)R.sup.6, --C(O)OR.sup.6, --S(O)R.sup.6, --SO.sub.2R.sup.6,
--SO.sub.2NR.sup.4R.sup.5, --OR.sup.6, and --SR.sup.6; R.sup.2 is
selected from the group consisting of hydrogen, halogen, alkyl,
haloalkyl and cycloalkyl; each of R.sup.4 and R.sup.5 is
independently selected from the group consisting of hydrogen,
alkyl, alkenyl, alkynyl, heteroaryl, aryl and cycloalkyl, or
R.sup.4 and R.sup.5 together with the nitrogen atom to which they
are attached to form an optionally substituted monocyclic or
bicyclic ring with one or more heteroatoms; R.sup.6 is selected
from the group consisting of hydrogen, alkyl, alkenyl, alkynyl,
heteroaryl, aryl, cycloalkyl, wherein the alkyl, alkenyl,
heteroaryl, aryl and cycloalkyl are optionally substituted with one
or more halo, cyano, alkylamino, alkoxy, aryl, heteroaryl or
heterocyclyl groups; each of R.sup.7 and R.sup.8 is independently
selected from the group consisting of hydrogen, alkyl, alkenyl,
alkynyl, heteroaryl, aryl and cycloalkyl, or R.sup.7 and R.sup.8
together with the nitrogen group to which they are attached to form
an optionally substituted monocyclic or bicyclic ring with one or
more heteroatoms; R.sup.9 is independently selected from the group
consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroaryl, aryl
cycloalkyl, wherein the alkyl, alkenyl, heteroaryl, aryl and
cycloalkyl are optionally substituted with one or more halo, cyano,
alkylamino, alkoxy, aryl, heteroaryl or heterocyclyl groups; each
R.sup.11 and R.sup.12 is independently --H, --COR.sup.13 or
--CH.sub.3; and R.sup.13 is alkyl, haloalkyl, alkenyl, alkynyl, or
cycloalkyl, or a pharmaceutically acceptable salt thereof, or a
prodrug thereof, or a mixture thereof; wherein the clinical
condition is one or more clinical conditions selected from: a
clinical condition associated with hTERT overexpression due to copy
number changes, translocations, missense mutations, and epigenetic
changes or other genetic mechanisms; a clinical condition
associated with a transcription-activating genetic change
associated with the hTERT core promoter region; a cancer selected
from the group consisting of glioblastomas, bladder cancer,
melanoma, thyroid, liver cancer, kidney cancer, stomach cancer,
esophagus cancer, lung cancer and neuroblastoma; a cancer having a
mutation in or associated with a hairpin loop of hTERT in a cancer
cell.
8. The method of claim 7, wherein said clinical condition
associated with said transcription-activating genetic change
associated with the hTERT core promoter region comprises a
tumor.
9. The method of claim 8, wherein said tumor comprises brain tumor,
bladder cancer, melanoma, thyroid, liver cancer, kidney cancer,
stomach, esophagus cancer, lung cancer or neuroblastoma.
10. The method of claim 9, wherein said brain tumor comprises
glioblastomas.
11. (canceled)
12. (canceled)
13. The method of claim 7, wherein said mutation in or associated
with the hairpin loop of hTERT in said cancer cell is in or
associated with a nucleotide sequence of the hairpin loop of hTERT
at a location -146, -139, -138, -125, -124 or a combination
thereof.
14. The method of claim 7, wherein R.sup.1 is selected from the
group consisting of: ##STR00046## ##STR00047## ##STR00048##
15. The method of claim 7, wherein R.sup.2 is selected from the
group consisting of hydrogen, methyl, ethyl, isopropyl,
trifluoromethyl and a halide.
16. The method of claim 7, wherein R.sup.3 is selected from the
group consisting of hydrogen, methyl, ethyl, trifluoromethyl,
propyl, cyclopropylmethyl, cyclopentyl, cyclohexyl, benzyl,
2-phenylethyl, 2-methoxyethyl, 3-methoxypropyl, or a moiety
selected from the group consisting of: ##STR00049## wherein m is an
integer from 2 to 4; each of R.sup.14 and R.sup.15 is independently
selected from the group consisting of hydrogen, alkyl, alkenyl,
alkynyl, heteroaryl, aryl and cycloalkyl; or R.sup.14 and R.sup.15
together with the nitrogen atom to which they are attached to form
a substituted or unsubstituted monocyclic or bicyclic
heterocycloalkyl.
17. The method of claim 7, wherein the compound is selected from a
compound of Table 1 or a pharmaceutically acceptable salt thereof,
or a prodrug thereof.
18. The method of claim 7, wherein the patient is a human
patient.
19. A kit comprising one or more of: a) a composition comprising
compound I: ##STR00050## or a pharmaceutically acceptable salt
thereof, or a prodrug thereof, or a mixture thereof; or b) a
composition comprising a compound of the formula I: ##STR00051##
wherein X is O or N such that when X is O, bond a is absent and
when X is N, bond a is present; n is 0 or 1 such that: when n=0,
R.sup.3 is --NHC(.dbd.NH)NH.sub.2; and when n=1, R.sup.3 is
hydrogen, alkyl, cycloalkyl, optionally substituted
heterocycloalkyl, optionally substituted heteroaryl or optionally
substituted aryl, wherein each substituent is independently
selected from the group consisting of halogen, cyano, nitro, azido,
haloalkyl, cycloalkyl, heteroaryl, aryl, --NR.sup.7R.sup.8,
--NR.sup.7C(O)R.sup.8, --C(O)NR.sup.7R.sup.8, --C(O)R.sup.9,
--C(O)OR.sup.9, --S(O)R.sup.9, --SO.sub.2R.sup.9,
--SO.sub.2NR.sup.7R.sup.8, --OR.sup.9 and --SR.sup.9; R.sup.1 is
optionally substituted aryl or optionally substituted heteroaryl,
wherein each substituent is independently selected from the group
consisting of halogen, cyano, nitro, azido, haloalkyl, cycloalkyl,
--NR.sup.4R.sup.5, --NR.sup.4C(O)R.sup.5, --C(O)NR.sup.4R.sup.5,
--C(O)R.sup.6, --C(O)OR.sup.6, --S(O)R.sup.6, --SO.sub.2R.sup.6,
--SO.sub.2NR.sup.4R.sup.5, --OR.sup.6, and --SR.sup.6; R.sup.2 is
selected from the group consisting of hydrogen, halogen, alkyl,
haloalkyl and cycloalkyl; each of R.sup.4 and R.sup.5 is
independently selected from the group consisting of hydrogen,
alkyl, alkenyl, alkynyl, heteroaryl, aryl and cycloalkyl, or
R.sup.4 and R.sup.5 together with the nitrogen atom to which they
are attached to form an optionally substituted monocyclic or
bicyclic ring with one or more heteroatoms; R.sup.6 is selected
from the group consisting of hydrogen, alkyl, alkenyl, alkynyl,
heteroaryl, aryl, cycloalkyl, wherein the alkyl, alkenyl,
heteroaryl, aryl and cycloalkyl are optionally substituted with one
or more halo, cyano, alkylamino, alkoxy, aryl, heteroaryl or
heterocyclyl groups; each of R.sup.7 and R.sup.8 is independently
selected from the group consisting of hydrogen, alkyl, alkenyl,
alkynyl, heteroaryl, aryl and cycloalkyl, or R.sup.7 and R.sup.8
together with the nitrogen group to which they are attached to form
an optionally substituted monocyclic or bicyclic ring with one or
more heteroatoms; R.sup.9 is independently selected from the group
consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroaryl, aryl
cycloalkyl, wherein the alkyl, alkenyl, heteroaryl, aryl and
cycloalkyl are optionally substituted with one or more halo, cyano,
alkylamino, alkoxy, aryl, heteroaryl or heterocyclyl groups; each
R.sup.11 and R.sup.12 is independently --H, --COR.sup.13 or
--CH.sub.3; and R.sup.13 is alkyl, haloalkyl, alkenyl, alkynyl, or
cycloalkyl, or a pharmaceutically acceptable salt thereof, or a
prodrug thereof, or a mixture thereof; and c) instructions for
administration of compound I or a compound of formula II to a
patient suffering from a clinical condition, wherein the clinical
condition is one or more clinical conditions selected from: a
clinical condition associated with hTERT overexpression due to copy
number changes, translocations, missense mutations, and epigenetic
changes or other genetic mechanisms; a clinical condition
associated with a transcription-activating genetic change
associated with the hTERT core promoter region; a cancer selected
from the group consisting of glioblastomas, bladder cancer,
melanoma, thyroid, liver cancer, kidney cancer, stomach cancer,
esophagus cancer, lung cancer and neuroblastoma; a cancer having a
mutation in or associated with a hairpin loop of hTERT in a cancer
cell.
20. The kit of claim 19, wherein R.sup.1 is selected from the group
consisting of: ##STR00052## ##STR00053## ##STR00054## wherein
R.sup.2 is selected from the group consisting of hydrogen, methyl,
ethyl, isopropyl, trifluoromethyl and a halide.
21. The kit of claim 19, wherein R.sup.3 is selected from the group
consisting of hydrogen, methyl, ethyl, trifluoromethyl, propyl,
cyclopropylmethyl, cyclopentyl, cyclohexyl, benzyl, 2-phenylethyl,
2-methoxyethyl, 3-methoxypropyl, or a moiety selected from the
group consisting of: ##STR00055## wherein m is an integer from 2 to
4; each of R.sup.14 and R.sup.15 is independently selected from the
group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroaryl,
aryl and cycloalkyl; or R.sup.14 and R.sup.15 together with the
nitrogen atom to which they are attached to form a substituted or
unsubstituted monocyclic or bicyclic heterocycloalkyl.
22. The kit of claim 19, wherein the compound is selected from a
compound of Table 1 or a pharmaceutically acceptable salt thereof,
or a prodrug thereof.
23. The kit of claim 19, wherein the patient is a human patient.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/779,132, filed May 25, 2018 which claims
the priority benefit of U.S. Provisional Application No.
62/261,838, filed Dec. 1, 2015, which are incorporated herein by
reference in their entireties.
FIELD OF THE INVENTION
[0003] The present invention relates to hTERT modulators and
methods for producing and using the same.
BACKGROUND OF THE INVENTION
[0004] Activation of telomerase is a hallmark of cancer in the
early stages of tumorigenesis and is associated with telomere
elongation, genetic instability, and subsequent immortalization of
cells. There are several strategies for overcoming activated
telomerase that are potentially useful for therapeutic treatment,
including targeting the telomerase holoenzyme, telomeric
G-quadruplexes with small molecules, human telomerase reverse
transcriptase (hTERT), and human telomerase RNA, and using immune
therapy.
[0005] hTERT is a catalytic subunit of telomerase and a critical
element for telomerase activity. Expression of hTERT is not usually
activated in normal cells, although other components of telomerase
are expressed. In addition, hTERT has various telomere-independent
functions, including enhancement of cellular proliferation, DNA
damage response through change in chromatin structure, and
inhibition of apoptosis by upregulation of BCL2 expression. These
functions are independent of each other.
[0006] Overexpression of hTERT for cell immortalization or
telomerase activation occurs in several ways, including increased
gene copy number and modulation at the transcription level. At the
transcription level, the hTERT promoter does not have TATA or CAAT
boxes but does have several transcription factor binding sites
within 1 Kb of the transcription start site and is controlled by
epigenetic changes, such as chromatin remodeling or methylation of
the CpG islands in the promoter region. With this transcription
machinery, 0.004 RNA molecules per cell in telomerase-negative
cells are elevated to 0.2-6 RNA molecules per cell in
telomerase-positive tumor-derived cells, showing a strong
correlation between telomerase activity and hTERT transcription
level.
[0007] The essential region for activation of transcription is at
the core promoter region, -181 base pairs from the transcription
start site. This region includes the E-box for MYC and other
elements for transcription activation. An additional upstream
region likely contains transcription-repressing elements, because
the longer promoter region shows decreased promoter activity. This
core promoter region becomes nuclease sensitive during cell
proliferation. Because the hTERT core promoter is selectively
activated in cancer cells, it is targeted for gene therapy by
utilizing the promoter for expression of cytotoxic
tumor-suppressing proteins.
[0008] The present inventors have previously shown, by various
biochemical experiments including DMS footprinting experiments,
that end-to-end stacked G-quadruplex structures are formed in the
core promoter element from 12 G-tracts. One of these structures has
a unique 3:26:1 loop configuration; the 26-base internal loop is a
hairpin structure and responsible for the unique cooperative
folding of this G-quadruplex, which is believed to be important in
transcription silencing. Stabilization of this G-quadruplex
structure using small molecules causes repression of hTERT promoter
activity. Significantly the mouse TERT lacks these 12 G-tracts and
has a 16-fold higher transcriptional activation level.
[0009] Several groups have recently demonstrated that many
different kinds of tumors have somatic mutations within the hTERT
promoter region at positions -124, -124/125, -138/139, and -146
from the ATG start site. A G-to-A mutation (G/A) in the antisense
strand is proposed to generate an ETS/TCF element that would
increase binding of the ETS transcription factor for activation of
hTERT transcription. Significantly, these mutations are also
localized in the G-quadruplex with the 3:26:1 loop configuration.
While it has been demonstrated in a number of oncogene promoters
that the G-quadruplex functions as a silencer element, it has also
been shown recently that, in the case of BCL2, the i-motif can act
as a transcription activator, validating both secondary DNA
structures as transcriptional targets for modulation of gene
expression. Therefore, it can be reasonably inferred that DNA
structural changes to either a G-quadruplex or an i-motif as a
result of these mutations would also affect the transcription
activity of the mutated hTERT promoter as well as the binding of
the ETS transcription factor to the duplex form. Accordingly,
modulation of transcription activity of the mutated hTERT promoter
can be used to treat cancer as well as other clinical conditions
associated with a transcription-activating mutationin an hTERT core
promoter region or hTERT overexpression due to genomic
rearrangements such as translocation or amplification.
[0010] Therefore, there is a need for a compound that can modulate
transcription activity of hTERT to treat cancer and other clinical
conditions associated with transcription or overexpression of
hTERT.
BRIEF DISCUSSION OF THE DRAWINGS
[0011] FIG. 1 shows the chemical structures of BRACO-19, GTC365,
GTC260, and Amsacrine.
[0012] FIG. 2 is a graph showing GTC365 dose-dependent FRET change
for WT, G124/125A and G146A.
[0013] FIG. 3 is a graph showing binding affinity (Kd values) with
GTC365 using FAM- and TAMRA-labeled oligomers.
[0014] FIG. 4 is a set of graphs showing GTC365 produces direct
downregulation of the hTERT core promoter (left) while BRACO-19
downregulation of hTERT is mediated via the MYC G-quadruplex
(right), ns=not significant.
[0015] FIG. 5 is a set of graphs showing reversal of mutated
promoter activity of hTERT core promoter by GTC365 (left) and
GTC260 (right).
[0016] FIGS. 6A-6C are a set of graphs showing (A) GTC365 produces
direct repression of hTERT transcription and the downstream
molecule BCL2 but has no effect on MYC mRNA level. (B) GTC260
produces direct repression of hTERT transcription and the
downstream molecule BCL2 but has no effect on MYC mRNA level. (C)
BRACO-19 knocks down MYC and hTERT transcription, but has no
significant effect on BCL2 mRNA levels in MCF7 cells after 72
h.
[0017] FIGS. 7A-7B are a set of graphs showing (A) effects of
compounds (0.25 .mu.M) on hTERT expression in MCF7 cells after 48
hours and (B) effects of compounds (0.125 .mu.M) on hTERT
expression in U87 cells after 48 hours.
[0018] FIG. 8 is a graph showing dose-dependent effect of GTC365 on
hTERT mRNA level in melanoma cells carrying WT, G124A, G124/125A,
G138/139A and G146A.
[0019] FIG. 9 is a set of graphs showing dose- and time-dependent
loss of telomere length following short-term exposure to GTC365 and
BRACO-19. MCF7 cells were treated with different doses of GTC365
for 10 days (left), 0.5 .mu.M of GTC365 for 5 or 10 days (center),
or different doses of BRACO-19 for 5 days (right).
[0020] FIGS. 10A and 10B are a set of graphs showing (A) comparison
of kinetics of initial folding rate of the WT 5-12 G-quadruplex
with GTC365 and BRACO-19 by the temperature-jump method. (B)
Enhanced initial folding rate of the 5-12 G-quadruplexes carrying
different hTERT mutants by GTC365. The time-course CD signal of
preheated oligo with compound or DMSO in a buffer containing 10 mM
Tris-HCl (pH 7.5) and 5 mM KCl was monitored at 262 nm and
25.degree. C.
[0021] FIG. 11 is a graph showing increase of BAX/BCL2 ratios by
GTC365 in mRNA levels. MCF7 cells were treated with GTC365 for 72 h
and then subjected to qPCR and immunoblot analysis. The relative
ratio of BAX/BCL2 was determined compared with a DMSO-treated
sample.
[0022] FIG. 12 is a graph showing activation of caspase-3 by
GTC365.
[0023] FIGS. 13A-13C are a set of graphs showing selective
reduction in viability (A) and hTERT expression (B) in melanoma
cells carrying hTERT core promotor mutations. NHM-002 (normal human
melanocytes) and a G124A mutant cell line were treated with
vehicle, 1.25, 2.5, and 5 .mu.M GTC365 for 72 h. Viability was
reduced in hTERT mutant melanoma cells by 50% (left). GTC365 caused
minimal reduction in NHM-002 viability and was not statistically
significant. hTERT mRNA expression was also reduced in melanoma
cells treated with GTC365 for 72 h. hTERT expression was
undetectable in NHM-002 cells (right). (C) Differential response to
GTC365 in WT and promoter mutant cell lines.
SUMMARY OF THE INVENTION
[0024] Some aspects of the invention are based on the
characterization of the effect of hTERT core promoter region
mutants on the 5-12 G-quadruplex structure and its stability. Other
aspects of the invention are based on identification of compounds
by the present inventors that modulate hTERT activity. Without
being bound by any theory, it is believed that some of the
compounds of the invention bind selectively to the G-quadruplex in
the hTERT core promoter mutant, which results in reversal of the
effect of mutant promoter activation.
[0025] In one specific aspect of the invention, a method is
provided for treating a patient suffering from a clinical condition
associated with a transcription-activating genetic change
associated with the hTERT promoter region. As used herein, the term
"a transcription-activating genetic change associated with the
hTERT promoter region" includes hTERT overexpression due to genomic
rearrangements such as translocation or amplification. Methods of
the invention include administering to a patient suffering from a
clinical condition associated with a transcription-activating
genetic change associated with the hTERT core promoter region a
therapeutically effective amount of compound I, compound II, or a
pharmaceutically acceptable salt thereof, or a prodrug thereof, or
a mixture thereof. Compounds I and II are of the formula:
##STR00002##
respectively, where X is O or N such that when X is O, bond a is
absent and when X is N, bond a is present; n is 0 or 1 such that:
when n=0, R.sup.3 is --NHC(.dbd.NH)NH.sub.2; and when n=1, R.sup.3
is hydrogen, alkyl, cycloalkyl, optionally substituted
heterocycloalkyl, optionally substituted heteroaryl or optionally
substituted aryl, wherein each substituent is independently
selected from the group consisting of halogen, cyano, nitro, azido,
haloalkyl, cycloalkyl, heteroaryl, aryl, --NR.sup.7R.sup.8,
--NR.sup.7C(O)R.sup.8, --C(O)NR.sup.7R.sup.8, --C(O)R.sup.9,
--C(O)OR.sup.9, --S(O)R.sup.9, --SO.sub.2R.sup.9,
--SO.sub.2NR.sup.7R.sup.8, --OR.sup.9 and --SR.sup.9; R.sup.1 is
optionally substituted aryl or optionally substituted heteroaryl,
wherein each substituent is independently selected from the group
consisting of halogen, cyano, nitro, azido, haloalkyl, cycloalkyl,
--NR.sup.4R.sup.5, --NR.sup.4C(O)R.sup.5, --C(O)NR.sup.4R.sup.5,
--C(O)R.sup.6, --C(O)OR.sup.6, --S(O)R.sup.6, --SO.sub.2R.sup.6,
--SO.sub.2NR.sup.4R.sup.5, --OR.sup.6, and --SR.sup.6; R.sup.2 is
selected from the group consisting of hydrogen, halogen, alkyl,
haloalkyl and cycloalkyl; each of R.sup.4 and R.sup.5 is
independently selected from the group consisting of hydrogen,
alkyl, alkenyl, alkynyl, heteroaryl, aryl and cycloalkyl, or
R.sup.4 and R.sup.5 together with the nitrogen atom to which they
are attached to form an optionally substituted monocyclic or
bicyclic ring with one or more heteroatoms; R.sup.6 is selected
from the group consisting of hydrogen, alkyl, alkenyl, alkynyl,
heteroaryl, aryl, cycloalkyl, wherein the alkyl, alkenyl,
heteroaryl, aryl and cycloalkyl are optionally substituted with one
or more halo, cyano, alkylamino, alkoxy, aryl, heteroaryl or
heterocyclyl groups; each of R.sup.7 and R.sup.8 is independently
selected from the group consisting of hydrogen, alkyl, alkenyl,
alkynyl, heteroaryl, aryl and cycloalkyl, or R.sup.7 and R.sup.8
together with the nitrogen group to which they are attached to form
an optionally substituted monocyclic or bicyclic ring with one or
more heteroatoms; R.sup.9 is independently selected from the group
consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroaryl, aryl
cycloalkyl, wherein the alkyl, alkenyl, heteroaryl, aryl and
cycloalkyl are optionally substituted with one or more halo, cyano,
alkylamino, alkoxy, aryl, heteroaryl or heterocyclyl groups; each
R.sup.11 and R.sup.12 is independently --H, --COR.sup.13 or
--CH.sub.3; and R.sup.13 is alkyl, haloalkyl, alkenyl, alkynyl, or
cycloalkyl.
[0026] It should be appreciated that methods of the invention can
include administering a compound of formula I alone, mixture
comprising two or more of compound of formula II, and compound of
formula I with one or more of compounds of formula II. Unless the
context requires otherwise, when referring to a compound of the
invention, the scope of the invention also includes using a
pharmaceutically acceptable salt thereof or a prodrug thereof.
DETAILED DESCRIPTION OF THE INVENTION
Definition
[0027] "Alkyl" refers to a saturated linear monovalent hydrocarbon
moiety of one to twelve, preferably one to six, carbon atoms or a
saturated branched monovalent hydrocarbon moiety of three to
twelve, preferably three to six, carbon atoms. Exemplary alkyl
group include, but are not limited to, methyl, ethyl, n-propyl,
2-propyl, tert-butyl, pentyl, and the like. "Alkylene" refers to a
saturated linear saturated divalent hydrocarbon moiety of one to
twelve, preferably one to six, carbon atoms or a branched saturated
divalent hydrocarbon moiety of three to twelve, preferably three to
six, carbon atoms. Exemplary alkylene groups include, but are not
limited to, methylene, ethylene, propylene, butylene, pentylene,
and the like. "Aryl" refers to a monovalent mono-, bi- or tricyclic
aromatic hydrocarbon moiety of 6 to 15 ring atoms which is
optionally substituted with one or more, preferably one, two, or
three substituents within the ring structure. When two or more
substituents are present in an aryl group, each substituent is
independently selected. "Aralkyl" refers to a moiety of the formula
--R.sup.bR.sup.c where R.sup.b is an alkylene group and R.sup.c is
an aryl group as defined herein. Exemplary aralkyl groups include,
but are not limited to, benzyl, phenylethyl,
3-(3-chlorophenyl)-2-methylpentyl, and the like. "Cycloalkyl"
refers to a non-aromatic, preferably saturated, monovalent mono- or
bicyclic hydrocarbon moiety of three to ten ring carbons. The
cycloalkyl can be optionally substituted with one or more,
preferably one, two, or three, substituents within the ring
structure. When two or more substituents are present in a
cycloalkyl group, each substituent is independently selected. The
terms "cycloalkylalkyl" or "cyclylalkyl" are used interchangeably
herein and refer to a moiety of the formula --R.sup.dR.sup.e where
R.sup.d is an alkylene group and R.sup.e is a cycloalkyl group as
defined herein. Exemplary cycloalkylalkyl groups include, but are
not limited to, cyclopropylmethyl, cyclohexylpropyl,
3-cyclohexyl-2-methylpropyl, and the like. The terms "halo,"
"halogen" and "halide" are used interchangeably herein and refer to
fluoro, chloro, bromo, or iodo. "Haloalkyl" refers to an alkyl
group as defined herein in which one or more hydrogen atom is
replaced by same or different halo atoms. The term "haloalkyl" also
includes perhalogenated alkyl groups in which all alkyl hydrogen
atoms are replaced by halogen atoms. Exemplary haloalkyl groups
include, but are not limited to, --CH.sub.2Cl, --CF.sub.3,
--CH.sub.2CF.sub.3, --CH.sub.2CCl.sub.3, and the like. The terms
"heterocyclyl" and "heterocycloalkyl" are used interchangeably
herein and refer to a non-aromatic monocyclic or bicyclic moiety of
three to eight ring atoms in which one or two ring atoms are
heteroatoms selected from N, O, or S(O). (where n is an integer
from 0 to 2), the remaining ring atoms being C, where one or two C
atoms can optionally be a carbonyl group. The heterocyclyl ring can
be optionally substituted independently with one or more, typically
one, two, or three, substituents. When two or more substituents are
present in a heterocyclyl group, each substituent is independently
selected. Suitable substituents for heterocyclyl group include, but
are not limited to, alkyl, haloalkyl, heteroalkyl, halo, nitro,
cyano, optionally substituted phenyl, optionally substituted
heteroaryl, optionally substituted phenyalkyl, optionally
substituted heteroaralkyl, acyl, -(alkylene).sub.n-COOR (n is 0 or
1 and R is hydrogen, alkyl, optionally substituted phenyl,
optionally substituted phenyalkyl, or optionally substituted
heteroaralkyl), or -(alkylene).sub.n-CONR.sup.aR.sup.b (where n is
0 or 1, and R.sup.a and R.sup.b are, independently of each other,
hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, hydroxyalkyl, aryl,
or R and R' together with the nitrogen atom to which they are
attached form a heterocyclyl ring). Exemplary heterocyclyls
include, but are not limited to, tetrahydropyranyl, piperidino,
piperazino, morpholino and thiomorpholino, thiomorpholino-1-oxide,
thiomorpholino-1,1-dioxide, and the like. The term "heteroaryl"
means a monovalent monocyclic or bicyclic aromatic moiety of 5 to
12 ring atoms containing one, two, or three ring heteroatoms
selected from N, O, or S, the remaining ring atoms being C. The
heteroaryl ring is optionally substituted independently with one or
more substituents, preferably one or two substituents, selected
from alkyl, haloalkyl, heteroalkyl, heterocyclyl, halo, nitro,
cyano, carboxy, acyl, -(alkylene), --COOR (where n is 0 or 1 and R
is hydrogen, alkyl, optionally substituted phenylalkyl, or
optionally substituted heteroaralkyl), or
-(alkylene).sub.n-CONR.sup.aR.sup.b (where n is 0 or 1, and R.sup.a
and R.sup.b are, independently of each other, hydrogen, alkyl,
cycloalkyl, cycloalkylalkyl, hydroxyalkyl, aryl, or R.sup.a and
R.sup.b together with the nitrogen atom to which they are attached
form a heterocyclyl ring). More specifically the term heteroaryl
includes, but is not limited to, pyridyl, furanyl, thiophenyl,
thiazolyl, isothiazolyl, triazolyl, imidazolyl, isoxazolyl,
pyrrolyl, pyrazolyl, pyrazinyl, pyrimidinyl, benzofuranyl,
isobenzofuranyl, benzothiazolyl, benzoisothiazolyl, benzotriazolyl,
indolyl, isoindolyl, benzoxazolyl, quinolyl, isoquinolyl,
benzimidazolyl, benzisoxazolyl, benzothiophenyl, dibenzofuran, and
benzodiazepin-2-one-5-yl, and the like. As used herein, the term
"heteroalkyl" means a branched or unbranched, cyclic or acyclic
saturated alkyl moiety containing carbon, hydrogen and one or more
heteroatoms in place of a carbon atom, or optionally one or more
heteroatom-containing substituents independently selected from
.dbd.O, --OR.sup.a, --C(O)R.sup.a, --NR.sup.bR.sup.c,
--C(O)NR.sup.bR.sup.c and --S(O).sub.nR.sup.d (where n is an
integer from 0 to 2). R.sup.a is hydrogen, alkyl, haloalkyl,
cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl,
aralkyl, heteroaryl, heteroaralkyl, or acyl. R.sup.b is hydrogen,
alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl,
heterocyclylalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, or
acyl. R.sup.c is hydrogen, alkyl, haloalkyl, cycloalkyl,
cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl,
acyl, alkylsulfonyl, carboxamido, or mono- or di-alkylcarbomoyl.
Optionally, R.sup.b and R.sup.c can be combined together with the
nitrogen to which each is attached to form a four-, five-, six- or
seven-membered heterocyclic ring (e.g., a pyrrolidinyl, piperidinyl
or morpholinyl ring). R.sup.d is hydrogen (provided that n is 0),
alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl,
heterocyclylalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, acyl,
amino, monosubstituted amino, disubstituted amino, or hydroxyalkyl.
Representative examples include, for example, 2-methoxyethyl,
benzyloxymethyl, thiophen-2-ylthiomethyl, 2-hydroxyethyl, and
2,3-dihydroxypropyl. "Leaving group" has the meaning conventionally
associated with it in synthetic organic chemistry, i.e., an atom or
a group capable of being displaced by a nucleophile and includes
halo (such as chloro, bromo, and iodo), alkanesulfonyloxy,
arenesulfonyloxy, alkylcarbonyloxy (e.g., acetoxy),
arylcarbonyloxy, mesyloxy, trifluoromethanesulfonyloxy, aryloxy
(e.g., 2,4-dinitrophenoxy), methoxy, N,O-dimethylhydroxylamino,
tosyloxy, and the like. "Pharmaceutically acceptable excipient"
refers to an excipient that is useful in preparing a pharmaceutical
composition that is generally safe, non-toxic and neither
biologically nor otherwise undesirable, and includes excipient that
is acceptable for veterinary use as well as human pharmaceutical
use. "Pharmaceutically acceptable salt" of a compound means a salt
that is pharmaceutically acceptable and that possesses the desired
pharmacological activity of the parent compound. Such salts
include: (1) acid addition salts, formed with inorganic acids such
as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,
phosphoric acid, and the like; or formed with organic acids such as
acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic
acid, glycolic acid, pyruvic acid, lactic acid, malonic acid,
succinic acid, malic acid, maleic acid, fumaric acid, tartaric
acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid,
cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic
acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid,
benzenesulfonic acid, 4-chlorobenzenesulfonic acid,
2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic
acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1 carboxylic acid,
glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid,
tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid,
glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid,
muconic acid, and the like; or (2) salts formed when an acidic
proton present in the parent compound either is replaced by a metal
ion, e.g., an alkali metal ion, an alkaline earth ion, or an
aluminum ion; or coordinates with an organic base such as
ethanolamine, diethanolamine, triethanolamine, tromethamine,
N-methylglucamine, and the like. The terms "pro-drug" and "prodrug"
are used interchangeably herein and refer to any compound which
releases an active parent drug (i.e., a compound of the invention
such as that of Formula I and/or II in vivo when such prodrug is
administered to a mammalian subject. Prodrugs of a compound of the
invention are prepared by modifying one or more functional group(s)
present in the compound of the invention in such a way that the
modification(s) may be cleaved in vivo to release the parent
compound. Prodrugs include compounds of the invention wherein a
hydroxy, amino, or sulfhydryl group in a compound of the invention
is bonded to any group that may be cleaved in vivo to regenerate
the free hydroxyl, amino, or sulfhydryl group, respectively.
Examples of prodrugs include, but are not limited to, esters (e.g.,
acetate, formate, and benzoate derivatives), carbamates (e.g.,
N,N-dimethylaminocarbonyl) of hydroxy functional groups in
compounds of the invention, and the like. "Protecting group" refers
to a moiety, except alkyl groups, that when attached to a reactive
group in a molecule masks, reduces or prevents that reactivity.
Examples of protecting groups can be found in T. W. Greene and P.
G. M. Wuts, Protective Groups in Organic Synthesis, 3.sup.rd
edition, John Wiley & Sons, New York, 1999, and Harrison and
Harrison et al., Compendium of Synthetic Organic Methods, Vols. 1-8
(John Wiley and Sons, 1971-1996), which are incorporated herein by
reference in their entirety. Representative hydroxy protecting
groups include acyl groups, benzyl and trityl ethers,
tetrahydropyranyl ethers, trialkylsilyl ethers and allyl ethers.
Representative amino protecting groups include, formyl, acetyl,
trifluoroacetyl, benzyl, benzyloxycarbonyl (CBZ),
tert-butoxycarbonyl (Boc), trimethyl silyl (TMS),
2-trimethylsilyl-ethanesulfonyl (SES), trityl and substituted
trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl
(FMOC), nitro-veratryloxycarbonyl (NVOC), and the like.
"Corresponding protecting group" means an appropriate protecting
group corresponding to the heteroatom (i.e., N, O, P or S) to which
it is attached. "A therapeutically effective amount" means the
amount of a compound that, when administered to a mammal for
treating a disease, is sufficient to effect such treatment for the
disease. The "therapeutically effective amount" will vary depending
on the compound, the disease and its severity and the age, weight,
etc., of the mammal to be treated. "Treating" or "treatment" of a
disease includes: (1) preventing the disease, i.e., causing the
clinical symptoms of the disease not to develop in a mammal that
may be exposed to or predisposed to the disease but does not yet
experience or display symptoms of the disease; (2) inhibiting the
disease, i.e., arresting or reducing the development of the disease
or its clinical symptoms; or (3) relieving the disease, i.e.,
causing regression of the disease or its clinical symptoms. As used
herein, the term "treating", "contacting" or "reacting" refers to
adding or mixing two or more reagents under appropriate conditions
to produce the indicated and/or the desired product. It should be
appreciated that the reaction which produces the indicated and/or
the desired product may not necessarily result directly from the
combination of two reagents which were initially added, i.e., there
may be one or more intermediates which are produced in the mixture
which ultimately leads to the formation of the indicated and/or the
desired product. As used herein, the terms "those defined above"
and "those defined herein" when referring to a variable
incorporates by reference the broad definition of the variable as
well as preferred, more preferred and most preferred definitions,
if any. As used herein, the term "treating", "contacting" or
"reacting" refers to adding or mixing two or more reagents under
appropriate conditions to produce the indicated and/or the desired
product. It should be appreciated that the reaction which produces
the indicated and/or the desired product may not necessarily result
directly from the combination of two reagents which were initially
added, i.e., there may be one or more intermediates which are
produced in the mixture which ultimately leads to the formation of
the indicated and/or the desired product.
Compounds and Methods of the Invention
[0028] Human telomerase reverse transcriptase (hTERT), highly
activated in most cancer cells, is involved in
telomerase-independent cellular proliferation, apoptosis, DNA
damage response, and telomere maintenance. It has been shown that
many tumors have transcription-activating mutations in the hTERT
core promoter region that form a pair of G-quadruplexes involved in
transcriptional silencing. The present inventors have discovered
the inhibition of the kinetics of the cooperative folding of
activating mutations on the major G-quadruplex structure. This
results in a significant change in the percentages of the different
species of folded intermediates and final functional form of the
silencer element so that the majority of species formed are only
the partially folded form. Significantly, compounds that modulate,
e.g., reduce or inhibit, hTERT activity has been identified through
screening. It is believed that these compounds act as
pharmacological chaperones by restoring the correct folding of the
active G-quadruplex silencer element so that the percentage of the
fully folded form is significantly increased relative to the
mutated promoter sequence. Even when mutations are not present, the
same molecular chaperones increase the kinetics of folding of the
active G-quadruplex silencer element. Thus when the hTERT core
promoter is overexpressed due to other genetic changes, the drugs
are still effective in lowering hTERT levels. Modulator of hTERT,
including a compound named GTC365 herein, directly decreases the
transcription activity of the wild-type ("WT") and the -124,
-124/125, -138/139, and -146 mutants to a similar extent and
suppresses the downstream gene BCL2. Compounds of the invention can
also lower the mRNA level of hTERT in melanoma cells that carry the
mutations. These compounds have been shown to require the
G-quadruplex in the hTERT promoter for activity, and therefore the
compounds are selectively toxic toward cells that overexpress
hTERT. The present inventors have also discovered that in some
instances compounds of the invention, including GTC365, shorten
telomere length after five days of treatment, induce a
senescence-like phenotype, and activate caspase-3 and cell-cycle
arrest, leading to cell death. Compounds belonging to the GTC260
series have similar properties to the GTC365 series and result in
lowering of hTERT and BCL2. An important difference between the
GTC365 and the GTC260 series is that the GTC260 series lacks the
G-quadruplex-interactive moiety but retains the loop-binding
moiety, showing that these pharmacological chaperone properties act
at an early point in the cooperative folding process. In addition,
these compounds have quite distinct properties to compounds such as
BRACO-19, which act through the G-quadruplexes in the telomeric
ends of chromosomes, such as downregulation of BCL2, resulting in
apoptosis, and much more potent inhibition of telomerase, resulting
in much faster telomere degradation.
[0029] One aspect of the invention provides a method for treating a
patient suffering from a clinical condition associated with a
transcription-activating genetic change associated with the hTERT
core promoter region by administering to a patient in need of such
a treatment a therapeutically effective amount of compound I,
compound II, or a pharmaceutically acceptable salt thereof, or a
prodrug thereof, or a mixture thereof. Compounds I and II are of
the formula:
##STR00003##
respectively, where X is O or N such that when X is O, bond a is
absent and when X is N, bond a is present; n is 0 or 1 such that:
when n=0, R.sup.3 is --NHC(.dbd.NH)NH.sub.2; and when n=1, R.sup.3
is hydrogen, alkyl, cycloalkyl, optionally substituted
heterocycloalkyl, optionally substituted heteroaryl or optionally
substituted aryl, wherein each substituent is independently
selected from the group consisting of halogen, cyano, nitro, azido,
haloalkyl, cycloalkyl, heteroaryl, aryl, --NR.sup.7R.sup.8,
--NR.sup.7C(O)R.sup.8, --C(O)NR.sup.7R.sup.8, --C(O)R.sup.9,
--C(O)OR.sup.9, --S(O)R.sup.9, --SO.sub.2R.sup.9,
--SO.sub.2NR.sup.7R.sup.8, --OR.sup.9 and --SR.sup.9; R.sup.1 is
optionally substituted aryl or optionally substituted heteroaryl,
wherein each substituent is independently selected from the group
consisting of halogen, cyano, nitro, azido, haloalkyl, cycloalkyl,
--NR.sup.4R.sup.5, --NR.sup.4C(O)R.sup.5, --C(O)NR.sup.4R.sup.5,
--C(O)R.sup.6, --C(O)OR.sup.6, --S(O)R.sup.6, --SO.sub.2R.sup.6,
--SO.sub.2NR.sup.4R.sup.5, --OR.sup.6, and --SR.sup.6; R.sup.2 is
selected from the group consisting of hydrogen, halogen, alkyl,
haloalkyl and cycloalkyl; each of R.sup.4 and R.sup.5 is
independently selected from the group consisting of hydrogen,
alkyl, alkenyl, alkynyl, heteroaryl, aryl and cycloalkyl, or
R.sup.4 and R.sup.5 together with the nitrogen atom to which they
are attached to form an optionally substituted monocyclic or
bicyclic ring with one or more heteroatoms; R.sup.6 is selected
from the group consisting of hydrogen, alkyl, alkenyl, alkynyl,
heteroaryl, aryl, cycloalkyl, wherein the alkyl, alkenyl,
heteroaryl, aryl and cycloalkyl are optionally substituted with one
or more halo, cyano, alkylamino, alkoxy, aryl, heteroaryl or
heterocyclyl groups; each of R.sup.7 and R.sup.8 is independently
selected from the group consisting of hydrogen, alkyl, alkenyl,
alkynyl, heteroaryl, aryl and cycloalkyl, or R.sup.7 and R together
with the nitrogen group to which they are attached to form an
optionally substituted monocyclic or bicyclic ring with one or more
heteroatoms; R.sup.9 is independently selected from the group
consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroaryl, aryl
cycloalkyl, wherein the alkyl, alkenyl, heteroaryl, aryl and
cycloalkyl are optionally substituted with one or more halo, cyano,
alkylamino, alkoxy, aryl, heteroaryl or heterocyclyl groups; each
R.sup.11 and R.sup.12 is independently --H, --COR.sup.13 or
--CH.sub.3; and R.sup.13 is alkyl, haloalkyl, alkenyl, alkynyl, or
cycloalkyl.
[0030] In some embodiments, the clinical condition associated with
the transcription-activating genetic change associated with the
hTERT core promoter region comprises a tumor. Within these
embodiments, in some instances the tumor comprises brain tumor,
bladder cancer, melanoma, thyroid, liver cancer, kidney cancer,
stomach, esophagus cancer, lung cancer or neuroblastoma. In one
particular instance, the brain tumor comprises glioblastomas.
[0031] Another aspect of the invention provides a method for
treating a patient suffering from glioblastomas, bladder cancer,
melanoma, thyroid, liver cancer, kidney cancer, stomach, esophagus
cancer, lung cancer or neuroblastoma by administering a
therapeutically effective amount of compound I, compound II, or a
pharmaceutically acceptable salt thereof, or a prodrug thereof, or
a mixture thereof.
[0032] Yet another aspect of the invention provides a method for
treating a cancer patient, said method comprising: determining
whether a mutation is present in or associated with a hairpin loop
of hTERT in a cancer cell of a patient; and when a mutation is
present in or associated with the hairpin loop of hTERT in said
cancer cell, treating said cancer patient with a therapeutically
effective amount of compound I, compound II, or a pharmaceutically
acceptable salt thereof, or a prodrug thereof, or a mixture
thereof.
[0033] In some embodiments, the patient is treated using the method
of the invention when the mutation is one of the mutations
discussed herein and in the accompanying Figures. In particular,
when mutation is present at a location -146, -139, -138, -125, -124
or a combination thereof of the nucleotide sequence of hTERT.
[0034] Still another aspect of the invention is directed to a
compound of the formula:
##STR00004##
where X is O or N such that when X is O, bond a is absent and when
X is N, bond a is present; n is 0 or 1 such that: when n=0, R.sup.3
is --NHC(.dbd.NH)NH.sub.2; and when n=1, R.sup.3 is hydrogen,
alkyl, cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted heteroaryl or optionally substituted aryl,
wherein each substituent is independently selected from the group
consisting of halogen, cyano, nitro, azido, haloalkyl, cycloalkyl,
heteroaryl, aryl, --NR.sup.7R.sup.8, --NR.sup.7C(O)R.sup.8,
--C(O)NR.sup.7R.sup.8, --C(O)R.sup.9, --C(O)OR.sup.9,
--S(O)R.sup.9, --SO.sub.2R.sup.9, --SO.sub.2NR.sup.7R.sup.8,
--OR.sup.9 and --SR.sup.9; R.sup.1 is optionally substituted aryl
or optionally substituted heteroaryl, wherein each substituent is
independently selected from the group consisting of halogen, cyano,
nitro, azido, haloalkyl, cycloalkyl, --NR.sup.4R.sup.5,
--NR.sup.4C(O)R.sup.5, --C(O)NR.sup.4R.sup.5, --C(O)R.sup.6,
--C(O)OR.sup.6, --S(O)R.sup.6, --SO.sub.2R.sup.6,
--SO.sub.2NR.sup.4R.sup.5, --OR.sup.6, and --SR.sup.6; R.sup.2 is
selected from the group consisting of hydrogen, halogen, alkyl,
haloalkyl and cycloalkyl; each of R.sup.4 and R.sup.5 is
independently selected from the group consisting of hydrogen,
alkyl, alkenyl, alkynyl, heteroaryl, aryl and cycloalkyl, or
R.sup.4 and R.sup.5 together with the nitrogen atom to which they
are attached to form an optionally substituted monocyclic or
bicyclic ring with one or more heteroatoms; R.sup.6 is selected
from the group consisting of hydrogen, alkyl, alkenyl, alkynyl,
heteroaryl, aryl, cycloalkyl, wherein the alkyl, alkenyl,
heteroaryl, aryl and cycloalkyl are optionally substituted with one
or more halo, cyano, alkylamino, alkoxy, aryl, heteroaryl or
heterocyclyl groups; each of R.sup.7 and R.sup.8 is independently
selected from the group consisting of hydrogen, alkyl, alkenyl,
alkynyl, heteroaryl, aryl and cycloalkyl, or R.sup.7 and R.sup.8
together with the nitrogen group to which they are attached to form
an optionally substituted monocyclic or bicyclic ring with one or
more heteroatoms; R.sup.9 is independently selected from the group
consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroaryl, aryl
cycloalkyl, wherein the alkyl, alkenyl, heteroaryl, aryl and
cycloalkyl are optionally substituted with one or more halo, cyano,
alkylamino, alkoxy, aryl, heteroaryl or heterocyclyl groups; each
R.sup.11 and R.sup.12 is independently --H, --COR.sup.13 or
--CH.sub.3; and R.sup.13 is alkyl, haloalkyl, alkenyl, alkynyl, or
cycloalkyl.
[0035] In some embodiments, R is selected from the group consisting
of:
##STR00005## ##STR00006## ##STR00007##
[0036] In another embodiment, R.sup.2 is selected from the group
consisting of hydrogen, methyl, ethyl, isopropyl, trifluoromethyl
and a halide.
[0037] Still yet in another embodiment, R.sup.3 is selected from
the group consisting of hydrogen, methyl, ethyl, trifluoromethyl,
propyl, cyclopropylmethyl, cyclopentyl, cyclohexyl, benzyl,
2-phenylethyl, 2-methoxyethyl, 3-methoxypropyl, or a moiety
selected from the group consisting of:
##STR00008##
where m is an integer from 2 to 4; each of R.sup.14 and R.sup.15 is
independently selected from the group consisting of hydrogen,
alkyl, alkenyl, alkynyl, heteroaryl, aryl and cycloalkyl; or
R.sup.14 and R.sup.15 together with the nitrogen atom to which they
are attached to form a substituted or unsubstituted monocyclic or
bicyclic heterocycloalkyl.
[0038] Some of the representative compounds of Formula II include,
but are not limited to, compounds shown on table 1.
TABLE-US-00001 TABLE 1 ##STR00009## Compound R.sub.1 R.sub.2
R.sub.3 GTC260 2-NHCOCH(CH.sub.3)NH.sub.2 --CH.sub.3 ##STR00010## 1
2-NH.sub.2 --CH.sub.3 ##STR00011## 2 2-NH.sub.2 --CH.sub.3
--CH.sub.3 3 2-NH.sub.2 --CH.sub.3 ##STR00012## 4 2-NH.sub.2
--CH.sub.3 ##STR00013## 5 2-NH.sub.2 --CH.sub.3 ##STR00014## 6
2-NH.sub.2 --CH.sub.3 ##STR00015## 7 2-NH.sub.2 --H ##STR00016## 8
2-NH.sub.2 --H ##STR00017## 9 2-NH.sub.2 --CH.sub.3 ##STR00018## 10
2-NH.sub.2 --CH.sub.3 ##STR00019## 11 2-NH.sub.2 --CH.sub.3
##STR00020## 12 --H --CH.sub.3 ##STR00021## 13 4-CF.sub.3
--CH.sub.3 ##STR00022## 14 4-F --CH.sub.3 ##STR00023## 15
4-OCH.sub.3 --CH.sub.3 ##STR00024## 16 2-NH.sub.2 --H ##STR00025##
17 2-NH.sub.2 --H ##STR00026## 18 4-OCH.sub.3 --H ##STR00027## 19
4-OCH.sub.3 --Cl ##STR00028## 20 4-OCH.sub.3 --CH.sub.3
##STR00029## 21 ##STR00030## 22 ##STR00031## 23 ##STR00032## 24
##STR00033## 25 4-CF.sub.3 --CH.sub.3 ##STR00034## 26 4-CF.sub.3
--CH.sub.3 ##STR00035## 27 4-CF.sub.3 --F ##STR00036## 28
4-CF.sub.3 --CH.sub.3 ##STR00037## 29 4-CF.sub.3 --CH.sub.3
##STR00038##
[0039] It should be appreciated that combinations of various groups
described herein form other embodiments. As an illustrative
example, compound II can form a wide variety of other compounds of
the invention by combining any one of the disclosed X, R.sup.1,
R.sup.2, R.sup.3 and n independent of each other. In this manner, a
variety of compounds are embodied within the present invention.
[0040] Additional objects, advantages, and novel features of this
invention will become apparent to those skilled in the art upon
examination of the following examples thereof, which are not
intended to be limiting. In the Examples, procedures that are
constructively reduced to practice are described in the present
tense, and procedures that have been carried out in the laboratory
are set forth in the past tense.
EXAMPLES
[0041] Circular Dichroism.
[0042] Oligomers of WT, G124A, G124/125A, G138/139A and G146A were
synthesized and HPSF-purified by MGW Operon, Inc. For CD analysis,
oligomers (5 .mu.M) in a buffer containing 10 mM Tris-HCl (pH 7.5)
and 140 mM KCl were annealed. For the CD analysis of the complex of
oligomers and compounds, oligomers (5 .mu.M) in a buffer containing
10 mM Tris-HCl (pH 7.5) and 5 mM KCl for GTC365 or 1 mM KCl for
BRACO-19 were annealed. Oligomers and GTC365 or BRACO-19 were
incubated overnight at room temperature. For the full-length of
C-rich strand, the oligomer in a buffer of 10 mM Na cacodylate (pH
6.6) was annealed and then incubated with GTC365 overnight. CD
analysis was conducted on a Jasco 810 spectropolarimeter (Jasco,
Easton, Md.) using a quartz cell with 1 mm of path length, 1 nm of
band width, and 1 s of response time for spectra at 20.degree. C.
Melting curves for the determination of T.sub.m were obtained by
recording ellipticity at 262 nm with increasing temperatures from
25.degree. C. to 95.degree. C. at a rate of 1.6.degree. C./min.
T.sub.m of the C-rich strand with GTC365 was obtained from the CD
signal at 286 nm within 10-60.degree. C.
[0043] For the kinetics analysis using the temperature-jump method,
the oligo in a buffer containing 10 mM Tris-HCl (pH 7.5) was heated
for min at 95.degree. C., and then a mixture of KCl and compound
was added and incubated for 1 min. Meanwhile, the CD cuvette was
also heated at 95.degree. C. As soon as the sample in the CD
cuvette at the high temperature was placed in the CD chamber, a
time-dependent CD signal at 262 nm was recorded immediately. The
initial folding rate was determined by one-phase association curve
fitting (eq 1) of the kinetics curve using GraphPad Prism 5:
Y=Y0+(Plateau-Y0)*(1-e.sup.-kx) (1)
where Y is the CD signal at any time point x, Y0 is the Y value
when time (x) is zero, plateau is the Y value at infinite time, and
k is the rate constant.
[0044] Single-Molecule Tweezer.
[0045] Preparation of the DNA construct. The DNA construct that
contains the single-stranded hTERT 5-12 fragment was prepared by
sandwiching the target sequence between two double-stranded DNA
handles. Five deoxythymidines were added at both ends of the hTERT
5-12 fragment to reduce the interference from the double-stranded
DNA handles on the target sequence. Briefly, the 2690-base-pair
double-stranded DNA handle was prepared through restriction enzyme
digestion of the pEGFP vector (Clontech, Mountain View, Calif.). In
the first step, the vector was digested by SacI and EagI
restriction enzymes, followed by purification with agarose gel. The
EagI end of the DNA fragment was labeled with digoxigenin using
terminal deoxynucleotidyl transferase. The other double-stranded
DNA handle (2028 base pairs) was prepared by PCR using the PBR322
plasmid template and a biotinylated primer. The PCR product was
subsequently digested with XbaI restriction enzyme. The middle
section that contains the hTERT 5-12 fragment was hybridized from
three single-stranded DNA targets. Finally, ligation between the
two long double-stranded DNA handles and this double-stranded
DNA/single-stranded DNA middle section was achieved using T4 DNA
ligase.
[0046] To perform the mechanical unfolding experiments, 1 .mu.L of
the 2.10 .mu.M polystyrene beads (0.5% w/v) coated with digoxigenin
antibody was incubated with 0.1 ng (3.5.times.10.sup.-17 mol) of
the DNA prepared above in 5 .mu.L of specific buffers (10 mM Tris
buffer at pH 7.4 or 50 mM MES buffer at pH 5.5) supplemented with
100 mM KCl or LiCl. After 30 min of incubation, the DNA construct
was immobilized on the surface of the beads through affinity
interactions. The incubation mixture was diluted to 800 .mu.L with
the same buffer for injection into a microfluidic chamber. The DNA
construct linked to the 2.10 .mu.M beads was subsequently tethered
to the 1.87 .mu.M polystyrene beads coated by streptavidin through
the biotin-labeled DNA construct.
[0047] The double-stranded hTERT construct was prepared through a
similar strategy. During preparation, two complementary
single-stranded DNA oligomers with respective G-rich and C-rich
hTERT sequences were annealed to form a double-stranded fragment,
which was then ligated with the two double-stranded DNA handles to
produce the final target sequence.
[0048] Single-Molecule Force-Ramp Assay.
[0049] The laser-tweezers instrument has been described
previously..sup.28 Briefly, a home-build dual-trap laser (1064 nm,
4 W, CW mode, BL-106C, Spectra-Physics) was used as the trapping
laser. P- and S-polarized laser light from the same laser source
constituted two traps..sup.28 The mobile laser trap controlled by a
motorized mirror grabbed the 2.10 .mu.M bead ligated with target
DNA and the other trap grabbed the 1.87 .mu.M bead. While moving
the mobile laser trap, the two beads may get close, and a DNA
tether can form between them. After this, the two beads were moved
apart to increase the tension on the DNA tether with a loading rate
of 5.5 pN/s, and the force-extension (F-X) curves were recorded
using the LabVIEW program (National Instruments Corp., Austin,
Tex.).
[0050] The F-X curves were filtered by Savitzky-Golay function with
a 10 ms time constant in the MATLAB program (The Math Works,
Natick, Mass.). The change in extension (.DELTA.x) at a given force
was obtained from the subtraction between the stretching and
relaxing curves at that force. The change-in-contour length
(.DELTA.L) was calculated based on the .DELTA.x through the
worm-like chain model (equation 1).
.DELTA. x .DELTA. L = 1 - 1 2 k B T F P + F S ( 1 )
##EQU00001##
where k.sub.B is the Boltzmann constant, T is temperature, P is
persistence length (50.8 nm), and S is the stretching modulus (1243
pN) for double-stranded DNA handles.
[0051] After calculating .DELTA.L of the structure, the number of
nucleotides (nt) contained in a particular length can be estimated
based on the .DELTA.L (equation 2):
n = .DELTA. L + x L n t ( 2 ) ##EQU00002##
where x is the end-to-end distance for G-quadruplex or i-motif
structures (0.5-1.5 nm) and L.sub.nt is the contour length per
nucleotide. L.sub.nt is located in the range of 0.40-0.45 nm/nt for
single-stranded DNA and 0.30-0.35 nm/bp for double-strand DNA.
[0052] DMS Footprinting.
[0053] FAM-labeled oligomers (WT, G124A, G124/125A, G138/139A, and
G146A) were purchased from MGW Operon Inc. and PAGE purified
(Supplementary Table 1). FAM-labeled oligomers (25 nM) were
dissolved in a buffer (10 mM Tris-HCl, pH 8.1, and 140 mM KCl)
annealed. For the footprinting with GTC365, the methods were
modified from the literature..sup.33 A FAM-labeled oligomer of WT
in a buffer containing 50 mM Na cacodylate (pH 7.6) and 5 mM KCl
was annealed. GTC365 in 20% DMSO was added to oligomers to produce
1, 2, and 4 equiv. and then incubated at 37.degree. C. for 1 h. For
the DMS reaction, oligomers were incubated with 2 .mu.g of salmon
sperm DNA (Sigma, D1626) and 5% DMS in 50% ethanol for 8 min. The
reaction was stopped by 0-mercaptoethanol and then subjected to
ethanol precipitation and cleavage by 10% piperidine with
incubation at 93.degree. C. for 15 min. Cleaved product was washed
twice by water and then separated by 15% denaturing PAGE with 7 M
urea. Fluorescence of separated cleavage product was detected by
Bio-Rad PharosFX.TM. Plus, and the band densitogram was obtained by
ImageJ.
[0054] FRET Assay for Compound Screening and Determination of
K.sub.d Value.
[0055] FRET probes of WT, G124/125A, and G146A of the 5-12
G-quadruplex were synthesized and labeled with FAM (Ex. 490 nm/Em.
520 nm) and TAMRA (Ex. 560 nm/Em. 580 nm) at each end by MGW Operon
Inc. The WT probe (50 nM) was annealed in a buffer containing 10 mM
Tris-HCl (pH 7.5) and 5 mM KCl by heating at 95.degree. C. for 5
min and slowly cooling to room temperature. Compounds (50 .mu.M)
and probe were incubated for 1 h at room temperature. The same
volume of DMSO served as a control. For K.sub.d value
determination, the WT, G124/125A, and G146A probes were annealed,
and then several concentrations of compound were treated for 1 h at
room temperature. Dose-dependent fluorescence intensity at 520 nm
was measured by a microplate reader (BioTek Synergy HT). The data
were corrected with the blank signal of buffer and compound. The
relative fluorescence intensity compared to DMSO was used for
binding curve fitting to determine K.sub.d value using GraphPad
Prism software.
[0056] Cell Cultures.
[0057] MCF7 and melanoma (UACC 383) cells were cultured in media of
RPMI-1640 with 10% FBS and 1% penicillin/streptomycin. For melanoma
cells UACC 2512, UACC 3090, UACC 1729, and UACC 2528, 20% FBS was
included in the RPMI media instead of 10% FBS. Cells were incubated
at 37.degree. C. with 5% CO.sub.2.
[0058] qPCR.
[0059] MCF7 and melanoma cells were treated with GTC365 and
BRACO-19 for 72 h. Total RNA was extracted using an RNeasy
mini-prep kit (Qiagen) and quantitated by measuring absorbance at
260 nm. cDNA was synthesized using a Takara PrimeScript.TM. RT
Reagent Kit with gDNA eraser and then used as a template for qPCR.
The qPCR was performed using Kapa Probe Fast qPCR Master Mix with
ABI Tagman probes of hTERT (Hs00972656_m1), BCL2 (HS00608023_m1,
FAM-labeled) or MYC (Hs00153408_m1), and GAPDH (Hs02758991_g1,
VIC-labeled). The Ct values were measured by running Rotor-Gene Q,
and the relative quantity of hTERT, BCL2, and MYC mRNA was obtained
compared to GAPDH as an internal control.
[0060] Luciferase Assay.
[0061] From gDNA extracted from HeLa cells, the hTERT core promoter
region, including -350 bp to +12 from the transcription start site,
was amplified using a pair of primers, including KpnI and NheI
restriction sites, for cloning of pGL3-hTERT with WT promoter
sequence. Mutant constructs were generated by PCR-based
site-directed mutagenesis. The sequence of each construct was
confirmed by sequencing analysis. MCF7 cells in 24-well plate were
transfected with 200 ng of pGL3 construct and 5 ng of pRL-TK by
Fugene.RTM. HD Transfection Reagent and then incubated for 6 h.
Media were replaced by fresh ones and the cells were treated with
GTC365 or BRACO-19. The same volume of DMSO served as a control.
After 24 h of incubation, cells were lysed by passive lysis buffer
(Promega) and subjected to dual-luciferase assay (Promega) using an
FB12 luminometer. The ratio of firefly to Renilla luciferase
activity was normalized to the DMSO to obtain the relative
luciferase activity.
[0062] Western Blot Analysis.
[0063] GTC365- or DMSO-treated MCF7 cells were lysed by RIPA buffer
and the supernatant of lysate was obtained by centrifugation at
14,000 rpm for 15 min. The concentration of the whole cellular
protein was determined by Bradford assay. The same amount of
proteins (120 .mu.g) was separated on 8% SDS-PAGE and transferred
to PVDF membrane in 20% MeOH/1.times. Tris-glycine. The membrane
was incubated in a blocking buffer containing 5% BSA/5% non-fat
milk with TBS-T (0.1% Tween 20) for 90 min at room temperature
prior to overnight incubation with hTERT antibody against rabbit
(1:200, Santa Cruz #H231, pAb) in 5% BSA/TBS-T buffer at 4.degree.
C. This membrane was incubated with .beta.-actin antibody against
mouse (Cell Signaling #3700, mAb, 1:2,000) for 2 h at room
temperature. The membrane was incubated with secondary antibody,
goat anti-rabbit IgG (H+L) Dylight 680 (1:7,500), and goat
anti-mouse IgG (H+L) Dylight 800 (1:7,500) in 5% non-fat milk/TBS-T
for 90 min at room temperature. LI-COR was used to detect the
immunocomplex band.
[0064] TRAP Assay.
[0065] Compound-treated MCF7 cells were washed with cold D-PBS
twice and then collected using a cell scraper. The cell pellet was
resuspended in lysis buffer including 10 mM Tris-HCl (pH7.5), 1 mM
MgCl2, 1 mM EGTA, 0.5% CHAPS, 10% glycerol, 5 mM
.beta.-mercaptoethanol, 1 .ANG..about.protease inhibitor cocktail
(Sigma P8340), and 0.2 U/.mu.L of RiboLock RNase Inhibitor (Thermo
Fisher, E00381) and kept on ice for 30 min. Supernatant was
obtained by centrifugation at 14,000 rpm for 20 min, and the
concentration of whole protein was determined by Bradford assay.
The whole protein was diluted to the same concentration using the
lysis buffer, and 500 ng of whole protein was incubated with a
mixture of reaction buffer containing 20 mM Tris-HCl (pH 8.3), 1.5
mM MgCl2, 63 mM KCl, 0.05% Tween 20, 1 mM EGTA, 0.5 .mu.M of TS
primer, and 50 .mu.M of dNTPs at 30.degree. C. for 20 min and
95.degree. C. for 2 min for inactivation. As a control reaction,
the same volume of lysis buffer was incubated with the mixture.
After the telomere elongation reaction, the samples were subjected
to purification using a Qiagen nucleotide removal kit (#28304)
because impurity including compounds in cell lysates can inhibit
the PCR reaction. 67 The purified samples were completely dried and
resuspended with 30 .mu.L of nuclease-free water. For the PCR
reaction, 1/10 volume of sample was incubated with a mixture of PCR
mastermix (Thermo Fisher, K0172), 0.125 U/.mu.L Taq DNA polymerase
(Thermo Fisher, EP0402), 62.5 nM TS primer, 62.5 nM ACX primer, and
5 ag of internal standard control (ITAS) in 20 .mu.L. The mixture
was initially incubated at 95.degree. C. for 3 min and then
followed by 32 cycles of 95.degree. C. for 30 s, 61.degree. C. for
30 s, and 72.degree. C. for 30 s. The PCR products were subjected
to 10% Native-PAGE and then stained by SYBR Gold for detection by
Bio-Rad Pharos FX. For preparation of ITAS, myogenin 108 nt (U.S.
Ser. No. 08/423,403) with partial TS and ACX sequence was initially
amplified using TS and ACX primers to generate 156 bp dsDNA and
then purified by gel extraction.
[0066] Telomere Length Assay.
[0067] MCF7 cells were treated with DMSO, GTC365 for 5-10 days, or
BRACO-19 for 25-30 days. Meanwhile, cells were subcultured to
maintain <90% confluent. Cells were collected by cell scraper
and then subjected to gDNA extraction by DNA extraction kit
(Qiagen). Ten ng of gDNA was used for SYBR Green I-based qPCR assay
with 1 M betaine, 700 nM telomere primers, and 200 nM 36B4
single-copy gene. A pair of Tell and Tel2 was used for
amplification of the telomere region and a pair of 36B4F and 36B4R
for amplification of a single-copy gene to normalize data. The PCR
was initiated at 95.degree. C. for 3 min and then 27 cycles at
95.degree. C. for 3 sec and 60.degree. C. for 2 min. The
fluorescence signal at 60.degree. C. was acquired. Triplicate data
was averaged and normalized to 36B4 to obtain .DELTA.Ct. The
relative telomere length was determined compared to the DMSO.
[0068] Senescence .beta.-Galactosidase Assay.
[0069] A colorimetric 0-galactosidase assay was performed. Briefly,
compound-treated MCF7 cells were fixed in 2% formaldehyde and 0.2%
glutaraldehyde for 5 min at room temperature. Fixed cells were
incubated with staining solution containing 40 mM citric acid (pH
6.0), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150
mV NaCl, and 2 mM MgCl2 and 1 mg/ml X-gal (Fermentas R0401) for 7 h
at 37.degree. C. Stained cells were washed with D-PBS and methanol
and subsequently dried in air.
[0070] Establishment of stable cell lines overexpressing hTERT. The
pCDNA-3.times.HA-hTERT plasmid containing hTERT driven by the CMV
promoter was obtained from Addgene (ID: 51637) deposited by Dr.
Steven Artandi. MCF7 cells in a 24-well plate were transfected with
this plasmid (250 ng) using FuGENE HD transfection reagent for 24
h. Cells were treated with several concentrations of G418 (0.1-1
mg/mL) and an antibiotic for colony selection for 4 weeks, and then
one colony was selected for further culture. The expression of
ectopic hTERT with HA-tag was confirmed by immunoblot analysis
using antibody against HA-tag (Santa Cruz, sc-805, 1:250 in TBS-T
buffer with 5% BSA) as described just below (immunoblot analysis)
and qPCR as described above.
[0071] Immunoblot Analysis.
[0072] GTC365- or DMSO-treated MCF7 cells were lysed by RIPA
buffer, and the supernatant of the lysate was obtained by
centrifugation at 14,000 rpm for 15 min. The concentration of the
whole cellular protein was determined by Bradford assay. The same
amount of proteins (120 .mu.g for hTERT and 50 .mu.g for PARP) was
separated on 8% SDS-PAGE and transferred to PVDF membrane in 20%
MeOH/1 .ANG..about.Tris-glycine. The membrane was incubated in a
blocking buffer containing 5% BSA/5% nonfat milk with TBS-T (0.1%
Tween 20) for 90 min at room temperature prior to overnight
incubation with hTERT antibody (1:200, Santa Cruz #H231, pAb) and
PARP antibody (1:1000, Cell Signaling #9542) in 5% BSA/TBS-T buffer
at 4.degree. C. This membrane was incubated with R-actin antibody
against mouse (Cell Signaling #3700, mAb, 1:2,000) for 2 h at room
temperature. The membrane was incubated with secondary antibody,
goat antirabbit IgG (H+L) DyLight 800 (1:7,500), and goat antimouse
IgG (H+L) DyLight 680 (1:7,500), depending on the source of
antibodies, in 5% nonfat milk/TBS-T for 90 min at room temperature.
LI-COR was used to detect the immunocomplex band.
[0073] Immunofluorescence.
[0074] TC365-treated MCF7 cells on coverslip were fixed in 2%
paraformaldehyde in PBS for 10 min at room temperature and then
subjected to permeabilization with 0.2% Triton X-100 in PBS for 10
min at room temperature. Cells were blocked with 4% BSA and 1%
non-fat milk in PBS for 1 h at room temperature and then incubated
with .alpha.-tubulin against mouse (1:200, Cell signaling #3873,
mAb) or ZO-1 against rabbit (1:200, Invitrogen #61-7300, pAb) in
20% blocking solution for 1 h. Afterward, cells were treated with
secondary antibodies (1:1000, goat-anti mouse Dylight 488 conjugate
or goat anti-rabbit IgG Alexa 555 conjugate) in 20% blocking
solution for 1 h. Following three washings with PBS, slides were
mounted in ProLong Gold Antifade solution with DAPI (Life
technologies #P36931). Images were acquired using an Olympus
IX71/DP70 digital microscope camera with blue, green, and red
filters and then processed with Image Studio Lite (LI-COR
Biosciences) and ImageJ.
[0075] Caspase Assay.
[0076] MCF7 cells in a 6-well plate were treated with GTC365 for 48
h. The caspase-3 assay was conducted using an Apoalert Caspase 3
Fluorescent Assay Kit (Clontech) with DEVD-AFC as a substrate of
capase-3 following the manufacturer's instruction. Fluorescence
intensity of released AFC was measured using a BioTek Synergy HT
with excitation at 400 nm and emission at 505 nm. The relative
caspase-3 activity compared to the DMSO was obtained.
[0077] Cell Cycle Analysis by PI Staining.
[0078] MCF7 cells were treated with GTC365 for 48 h. Cells were
trypsinized and washed with cold D-PBS. Cell pellets were subjected
to 70% EtOH fixation at -20.degree. C. overnight. Cells were
incubated with RNase A and PI for 3 h at 37.degree. C. and then
kept in ice before FACS analysis (FACScanto II, BD Biosciences, San
Jose, Calif.).
[0079] Counting of Live Cells Treated with GTC365.
[0080] MCF7 cells were treated with DMSO or 0.5 .mu.M of GTC365.
Cells were trypsinized and then subjected to trypan blue exclusion
staining for counting of live cells using microscopy every three
days. Afterward, 70% of cells were recultured with fresh media and
DMSO or GTC365. Relative cell numbers compared to the DMSO were
obtained.
[0081] Results:
[0082] The somatic mutations of the hTERT core promoter region are
found in G-tracts 5, 7, and 8, which have been demonstrated to be
part of a G-quadruplex with a unique 3:26:1 loop topology. Based
upon DMS footprinting of the full-length oligomer containing all 12
guanine runs, positions -124 and -125 from the ATG site are located
in G-tract 5 (G-quadruplex scaffold), -138 and -139 are in G-tract
7 (stem region of hairpin), and -146 is in G-tract 8 (central loop
of hairpin). To determine the effect of these mutations on the
G-quadruplex structure, circular dichroism (CD), DMS footprinting,
and single-molecule laser tweezer experiments were performed.
[0083] While the CD spectra of the G146A mutant in the full-length
and 5-12 G-rich strands were quite similar to the WT, the G124/125A
and G138/139A mutants both showed decreased CD spectra readings.
The full-length and 5-12 fragments of G124/125A had melting
temperatures (T.sub.m) decreased by 1.9.degree. C. and 4.3.degree.
C. respectively, and G138/139A showed a similar decrease in T.sub.m
of 2.0.degree. C. for the full-length fragment and 3.4.degree. C.
for the 5-12 fragment. On the other hand, T.sub.ms of G124A and
G146A were reduced by 1.5-1.6.degree. C. and 0.9-1.6.degree. C. for
both fragments, which is a smaller decrease compared to G124/125A
and G138/139A; therefore, additional factors beyond the
destabilization of the G-quadruplex might be expected to be
important in the overexpression of hTERT.
[0084] The present inventors have previously demonstrated that
mutations in the hairpin loop result in changes in both the
stability and the folding pattern of the G-quadruplex, based upon
DMS footprinting. Furthermore, single-molecule laser tweezer
experiments showed that the hairpin plays a pivotal role in forming
the fully folded species, since mutation resulted in only 4% of the
fully folded form. On the basis of these results, it was
anticipated that the G124/125A, G138/139A, and, perhaps to a lesser
extent, G124A and G146A mutations would change either the folding
pattern or the cooperative folding of the WT structure.
Importantly, the G124A and G146A mutations, which result in new ETS
transcription factor binding sites, are also likely to contribute
to the transcription activation. Results showed that WT G-tracts 5,
6, 11, and 12 were protected from DMS cleavage. As anticipated, in
comparison to the WT, the footprinting of the G124/G125A mutant
showed the greatest changes in the DMS protection pattern, with a
loss of protection of G-tracts 7, 9, and 10. In addition, G138/139A
and G146A mutants showed more subtle changes in the cleavage of
G-tracts in the stem loop, which may represent conformational
changes in this part of the structure.
[0085] To further understand the influence of the G124/125A
mutation on the 5-12 region, single-molecule experiments were
conducted on a DNA construct that contains these mutations. First,
the effect of the mutation on the DNA secondary structures in the
G-rich strand was studied. Although it is more common to use
single-stranded G-rich hTERT DNA for this goal, double-stranded DNA
in a 10 mM Tris buffer with 100 mM KCl at pH 7.4 were used to allow
the formation of only G-quadruplex structures. Given that the 44-nt
5-12 region contains eight G-rich tracts, multiple G-quadruplex
populations can exist, each requiring a minimum of four G-rich
tracts. Together with partially folded structures, this constitutes
a rather complex array of observable structures in single-molecule
mechanical unfolding experiments. Through mechanical unfolding of
various non-B-DNA structures in a DNA fragment, the present
inventors were able to follow the population dynamics of individual
DNA secondary structures with the statistical method PoDNano
(Population Deconvolution at Nanometer resolution). With this
method, the size of different populations (measured in change in
contour length [.DELTA.L]) and their percentages of formation were
identified. From a comparison of population patterns of different
species, factors such as buffer conditions or mutations can be
evaluated. It was observed that the G124/125A mutations
dramatically changed the population pattern of G-rich structures,
especially for the large species (36 bp and 42 bp), in both
single-stranded and double-stranded 5-12 fragments. These species
are likely fully folded G-quadruplexes. In addition, the overall
formation percentage of G-rich species is reduced compared to the
WT DNA. With the same population pattern analysis it was found that
the G124/125A mutations do not affect the i-motif population as
much as the G-quadruplex, in either the single-stranded or
double-stranded DNA construct.
[0086] The somatic mutation of the hTERT promoter is proposed to
generate an ETS/TCF transcription factor binding site and thus
enhance promoter activity. The present inventors have confirmed the
increased luciferase activity of all the mutants, in comparison to
the WT promoter sequence, using a construct covering -350 to +12
from the transcription start site in MCF7 cells. The extent of
enhanced luciferase activity was dependent on the mutated
sequences. The G124A, G124/125A, G138/139A, and G146A mutants
increased luciferase activity 9.7-, 1.3-, 3.6-, and 3.9-fold,
respectively.
[0087] The hTERT core promoter region includes 12 runs of
consecutive guanines that form two G-quadruplexes in tandem on the
G-rich strand. Since the 5-12 G-quadruplex is primarily responsible
for the thermal stability of the full-length structure, this strand
was labeled with FAM and TAMRA at each end for the FRET assay. When
the G-quadruplex is folded, two fluorophores are in proximity,
which leads to a decrease in FAM fluorescence. Using this oligomer,
the NCI Diversity Set III (.about.1500 compounds) was subjected to
a FRET assay in a buffer containing 10 mM Tris-HCl (pH 7.4) and 5
mM KCl. Forty-five compounds from the NCI Diversity Set decreased
the fluorescence intensity by at least 50%, including compound
GTC365, which showed a very significant reduction of fluorescence
intensity (94%). Compound GTC260 was identified using a similar
strategy. GTC365 is a compound with an acridine scaffold similar to
both the telomeric G-quadruplex-binding compound BRACO-19 and
Amsacrine, whereas GTC260 lacked the acridine scaffold (FIG. 1).
Amsacrine is a topoisomerase II inhibitor used to treat acute
lymphocytic leukemia, whereas BRACO-19 is a
G-quadruplex-interactive compound that produces telomere
shortening, resulting in cellular senescence and cessation of
growth after 15 days. GTC365 also showed a dose-dependent decrease
of fluorescence intensity of the WT, G124/125A, and G146A probes to
a similar extent (FIG. 2). K.sub.d values showed that GTC365
preferentially bound to WT and G124/125A with a similar binding
affinity (.about.400 nM) in contrast to G146A, which showed a
1.5-fold higher K.sub.d value (FIG. 3).
[0088] CD and DMS footprinting analyses were conducted to
characterize the binding of GTC365 and BRACO-19 to the 5-12
G-quadruplex. CD spectra showed that GTC365 and BRACO-19 both
increased the ellipticity at 262 nm with little change at 290 nm.
To verify the effects of compounds on the thermal stability of the
WT and the mutant G-quadruplexes, T.sub.ms were determined by CD.
GTC365 dose dependently increased the .DELTA.T.sub.m of the WT
G-quadruplex by 12.9.degree. C. at 2 equiv. The T.sub.ms of
G124/125A, G138/139A, and G146A mutants were more significantly
increased by 6-7.degree. C. compared to the WT, while that of G124A
was similar to the WT at 2 equiv. On the basis of these results it
was proposed that GTC365 could compensate for thermal instability
derived from mutation or low concentrations of KCl. BRACO-19 also
significantly increased the T.sub.m of the mutants as well as the
WT in a dose-dependent manner. Since acridine derivatives are known
to bind to telomeric G-quadruplex, the effects of GTC365 and
BRACO-19 on telomeric G-quadruplex stability were compared by
CD.
[0089] GTC365 showed an increase in T.sub.m for telomeric
G-quadruplex, with a T.sub.m of 4.4.degree. C. and 7.5.degree. C.
at 1 and 2 equiv., respectively, which is significantly less (42%)
than that of the hTERT G-quadruplex. In contrast, BRACO-19
increased the T.sub.m of telomeric G-quadruplex by 60% compared to
that of the hTERT G-quadruplex at 0.5 and 1 equiv. A summary of the
comparative T.sub.ms of GTC365 and BRACO-19 in hTERT and telomeric
DNA is shown below. This demonstrates that GTC365 and BRACO-19
function in opposite ways by selectively binding to the hTERT core
promoter G-quadruplex and the telomeric G-quadruplex respectively.
There was little change in T.sub.m of the i-motif formed from the
full-length C-rich strand by GTC365 suggesting that GTC365
selectively binds to the G-quadruplex over the i-motif.
TABLE-US-00002 T.sub.ms Summary: Compound: GTC365 BRACO-19
Equivalents 0 1 2 0 0.5 1 hTERT WT G4 0 6.47 12.92 0 2.41 8.73
Telomeric G4 0 4.4 7.5 0 4.24 14.6
[0090] To gain some insight into how GTC365 binds to the 5-12
G-quadruplex, the effect of drug binding on the DMS footprint of
the WT G-quadruplex-forming sequence was examined. Results showed
GTC365 protected the guanine in the 5'-tetrad of G-tract 6 as well
as select bases at the 3'-ends in the G-C and G-G base pairing
between G-tracts 7 and 10 in the stem. These data suggest that the
acridine moiety is positioned on the 5' G-tetrad and the guanidine
side-chain interacts with the four G-C and G-G base pairs formed by
G-tracts 7 and 10 in the stem. In support of this, it is well known
that the guanidinium group of arginine binds to guanines in the
major groove of DNA. Thus the larger increase in .DELTA.T.sub.m of
the hTERT G-quadruplex with GTC365 relative to the telomeric
G-quadruplex is believed to be due to the additional interactions
of the guanidinium with the G-C and G-G base pairs in the hairpin
loop.
[0091] Single-molecule experiments were used to determine the
ability of GTC365 to reverse the effect of the G124/125A mutant on
hTERT G-quadruplex stability. After the G124/125A mutant was
incubated with 2 .mu.M GTC365 in a 10 mM Tris buffer (100 mM KCl,
pH 7.4), in which the formation of a G-quadruplex over an i-motif
is favored, it was observed that the full-length G-rich species
(>36 nt/bp) recovered in both the single-stranded and
double-stranded template. This suggested that GTC365 was acting as
a molecular chaperone in facilitating the cooperative folding of
the functional G-quadruplex silencer element rather than producing
the effects by thermal stabilization of the G-quadruplex.
[0092] Since MYC is a key transcription factor for activation of
the hTERT gene by binding to an E-box in the hTERT promoter region
and is one of the oncogenes having a G-quadruplex structure in the
promoter region, it was important to confirm that GTC365 affects
the repression of hTERT promoter activity directly by binding to
the G-quadruplex structure formed in the hTERT promoter and not by
binding to the G-quadruplex in the MYC promoter. To distinguish
between these possibilities, the luciferase activity of the pGL3-WT
and pGL3-E-box mutant constructs were determined after treatment
with GTC365. As shown in FIG. 4, GTC365 showed a similar
dose-dependent decrease of luciferase activity of the WT and E-box
Mut construct, which suggests that GTC365 acts predominantly by
binding directly to the hTERT G-quadruplex rather than via the MYC
G-quadruplex to downregulate transcription of hTERT. In contrast,
BRACO-19 decreased luciferase activity of the WT but failed to
reduce luciferase activity in the E-box Mut construct. Therefore,
it can be concluded that BRACO-19 downregulates hTERT indirectly
through binding to the MYC G-quadruplex.
[0093] To assess directly whether GTC365 mediates its effects
through the hTERT promoter element, we compared the effect of
GTC365 on hTERT transcription in MCF7 cells versus similar cells
containing a plasmid that overexpresses hTERT but under the control
of the CMV promoter. The results show that although GTC365 has a
significant effect on hTERT transcription in the MCF7 control
cells, where the hTERT promoter is targeted, there was no effect on
hTERT transcription in the MCF7 cell line transfected with the
plasmid that overexpresses hTERT. The results of this experiment
strongly suggest that the molecular target for GTC365 is at the
core promoter level rather than a downstream event.
[0094] The effect of GTC365 on the somatic mutants in the hTERT
promoter region was also examined by transfecting cells with pGL3
constructs that contain the G124A, G124/125A, G138/139A, and G146A
mutations. As shown in FIG. 5A, GTC365 decreased luciferase
activity of the mutants in a dose-dependent manner and therefore
acts broadly to downregulate the hTERT promoter activity of the WT
and mutants. GTC260 had a similar effect on luciferase activity
(FIG. 5B)
[0095] BCL2 is a downstream molecule to hTERT such that BCL2
expression is activated by hTERT to repress apoptosis. To further
define the effect of GTC365 on the transcription of hTERT, the
relative mRNA levels of MYC and BCL2 were determined by
quantitative PCR analysis in MCF7 cells after treatment with GTC365
for 72 h. As shown in FIG. 6A, GTC365 decreased the hTERT mRNA
levels in a dose-dependent manner by up to 67% (at 1 .mu.M), and
BCL2 mRNA level was also dose-dependently decreased by up to 18% at
1 .mu.M, with no significant change in MYC mRNA levels. GTC260 had
a similar effect on hTERT activity (FIG. 6B). A series of GTC260
analogs were synthesized and their effect on hTERT gene expression
in MCF7A and U87 cells determined (FIG. 7). In contrast, as shown
in FIG. 6C, BRACO-19 decreased mRNA levels of both hTERT and MYC to
a similar extent at a somewhat higher concentration range compared
to GTC365. Therefore, GTC365 directly repressed the transcription
of hTERT and then led to a downstream decrease of BCL2 expression.
It was demonstrated using western blot that the protein level of
hTERT protein level was also downregulated.
[0096] To determine the cellular effect of GTC365 on proliferation
as well as suppression of the activation of hTERT by G124A,
G124/125A, G138/139A, and G146A mutants, melanoma cell lines were
used alongside a WT cell line. All mutants increased the mRNA level
of hTERT, compared to WT melanoma cells, in the 6.6-34-fold range.
To evaluate the effect of GTC365 on the hTERT mRNA level in
melanoma cells, cells were treated with several concentrations of
GTC365 for 72 h (FIG. 8). GTC365 showed a similar dose-dependent
decrease (25% at 1 .mu.M) in the mRNA level of hTERT in the WT and
all mutant cells. GTC365 also decreased the mRNA level of BCL2 in
WT and G146A cells as expected. To determine the effect of GTC365
and BRACO-19 on cellular proliferation in melanoma promoter mutant
cell lines, an MTS assay was conducted following 72 h treatment.
GTC365 and BRACO-19 both showed EC.sub.50 values in the 18-40 .mu.M
range.
[0097] hTERT-positive MCF7 breast cancer cell lines were used to
gain mechanistic insight into how the GTC365-induced inhibition of
proliferation occurs. Change in relative telomere length by GTC365
was measured by qPCR with gDNA extracted from GTC365-treated MCF7
cells. After 10 days of treatment GTC365 decreased telomere length
in a dose-dependent manner, and at a dose of 0.5 .mu.M, the
telomere length was reduced by 20%. In contrast, BRACO-19 reduced
telomere length by -12% at the same concentration but required
25-30 days of treatment (FIG. 9).
[0098] To evaluate the comparative effects of GTC365 relative to
BRACO-19 on the cooperative folding process, we carried out two
different experiments. First, we compared the distribution of the
different populations of small and large species using
single-molecule experiments as described previously with GTC365 and
BRACO-19 in the G138/139A mutant. Similar to the G124/125A mutant,
the population of fully folded structures is small in the G138/139A
mutant without addition of ligand. After incubating with 2.mu.
MGTC365, the fully folded structures significantly recovered
demonstrating the population effect of this ligand to form the
fully folded G-quadruplex species. BRACO-19 minimally promoted the
fully folded structure; instead, this molecule increased partially
folded populations. These results provide a rationale for the
discrepancy in the biological activities of GTC365 and BRACO-19.
Second, we used CD kinetic analysis to compare the relative rates
of initial folding for GTC365 and BRACO-19 in the WT and mutant
species with GTC365 (FIG. 10). We then used a temperature-jump
method 84 to compare the initial folding rate of the 5-12 WT
G-quadruplex with GTC365 and BRACO-19 using a time-course CD signal
curve. As shown in FIG. 10A, the initial folding rate of the 5-12
WT G-quadruplex by GTC365 at 2 equiv was increased from 0.051 to
0.093 s.sup.-1, showing an increase of 0.042 s.sup.-1, whereas
BRACO-19 showed a much smaller increase (0.017 s.sup.-1 at 2
equiv). GTC365 also increased the initial folding rates of G124A,
G124/125A, and G146A in a dose-dependent manner by 0.049, 0.042,
and 0.054s.sup.-1 at 2 equiv, whereas the effect on the G138/139A
mutant was insignificant (FIG. 10B). Taken together, these results
strongly suggest that GTC365 and GTC260 both act as pharmacological
chaperones by facilitating the cooperative folding of the
functional silencer element. This role of these compounds in
silencing the transcription activation of hTERT is equally
important for treatment of cancers that overexpress hTERT as a
consequence of mutation in the core promoter element or other
genetic aberrations.
[0099] Since senescence is one of the outcomes resulting from loss
of telomere length, this was measured by the associated
overexpression of lysosomal .beta.-galactosidase, which releases
indigo (blue dye) from X-gal. In addition, the cell morphology will
also be dramatically changed so that cells appear to be flat and
enlarged. The blue-stained MCF7 cells showed a distinct
senescence-like phenotype (SLP), and there was significant
morphological changes in the cells so that they became flat and
enlarged following 5-10 days of treatment with 0.5 .mu.M GTC365.
Abnormal phenotypes such as bridges connecting two adjacent cells
and multinucleated cells were also observed in GTC365-treated
cells. hTERT is known to localize to mitotic spindles and
centromeres and regulate the expression of genes involved in
heterochromatin maintenance. In addition, there is alteration of
expression of some proteins involved in collagen synthesis or
tubulin organization by hTERT transfection. This suggests that
GTC365 induced failure of mitosis and cytokinesis by downregulation
of hTERT.
[0100] Previous observation by the present inventors that GTC365
repressed BCL2 expression suggests that there should be an increase
in the BAX/BCL2 ratio as well as in caspase-3, the final executor
for apoptosis. Indeed at 0.5-1 .mu.M, the BAX/BCL2 ratio was
increased by over 2-fold and the capase-3 activity was increased by
47% (FIGS. 11 and 12).
[0101] Since knockdown of hTERT arrests the cell cycle in the G0/G1
phase, the effects of GTC365 on the cell cycle were evaluated.
GTC365 increased the population of the G0/Gi (from 57% to 71%) and
G2/M phases (from 10% to 19%), whereas the S-phase was decreased
(from 33% to 10%) in a dose-dependent manner with 48 h treatment.
Five days of treatment with GTC365 also showed the same pattern.
Therefore it can be inferred that knockdown of hTERT directly
induced G0/Gi phase arrest, whereas DNA damage by GTC365 induced
G2/M phase arrest. Most of the cells showing SLP underwent cell
death with exposure to a low dose of GTC365 in nine days. It is
known that various anticancer drugs induce SLP in different cancer
cells, leading to mitotic catastrophe as well as apoptotic cell
death. Doxorubicin, one of the representative anticancer drugs,
induced apoptosis and mitotic catastrophe accompanied by multiple
nuclei with SLP. GTC365, like doxorubicin, appears to arrest cell
proliferation and induce cell death, apoptosis, or mitotic
catastrophe through downregulation of hTERT.
[0102] Next, to assess the selectivity of GTC365 for
hTERT-dependent cancer cells relative to normal precursor cells, we
compared the effects of a range of GTC265 concentrations on
viability at 72 h in normal human melanocytes (NHM-002) relative to
those of UACC-903, a G124A mutant (FIG. 13A). Viability was
significantly reduced in a dose-dependent manner in melanoma cells,
dropping below 50% at 5 .mu.M. However, only a minimal viability
change was seen in normal melanocytes and was not statistically
significant. To confirm that the reduction in cell viability was a
result of hTERT downregulation, we performed reverse transcription
and qPCR to measure the mRNA level of hTERT expression in cells
treated with DMSO or 5 .mu.M GTC365. hTERT expression was reduced
by 41% in the melanoma cell line after 72 h but was undetectable in
normal melanocytes, even in the absence of GTC365 (FIG. 13B).
Finally, to broadly assess the selectivity of GTC365 for hTERT
promoter mutant versus WT cells, we conducted a 6-point 72 h MTS
proliferation assay to determine EC.sub.50s in 14 additional
melanoma cell lines with characterized TERT promoter status (19
cell lines in total) (FIG. 13C). GTC365 showed EC.sub.50 values.
Ultimately, this broad screen shows a significant differential
response to GTC365 between WT and mutant melanoma cell lines and
confirms increased GTC365 activity in mutant cells that express
higher levels of hTERT These data support that GTC365 reduces cell
proliferation through downregulation of hTERT expression via
stabilization of the G-quadruplex promoter structure. This
mechanism is selective for hTERT-dependent melanomas such as those
bearing hTERT promoter mutations.
[0103] Discussion:
[0104] Limitless proliferation potential due to telomerase
activation is one of the original hallmarks of cancer, and hTERT is
the key component for maintenance of telomerase activity. While
telomerase has been the major target for drug therapy designed for
destabilization of telomeres by inhibition of telomere elongation
or telomere uncapping, the direct repression of hTERT promoter
activity has distinct advantages associated with impairing various
functions for cell survival as well as telomere maintenance.
Without being bound by any theory, some aspects of the invention
provide a new strategy involving small molecules to directly target
hTERT expression at the promoter level so that short-term effects
are seen that result in apoptosis within 48 h and non-apoptotic
death within 5-10 days.
[0105] Recently it has been shown that specific somatic mutations
in the hTERT core promoter element result in a 2-4-fold increase in
luciferase activity and that these mutations are commonly found in
a number of cancers, most notably melanomas and gliomas, where they
are often associated with poor prognosis. In an attempt to
rationalize the molecular basis for the transcriptional effects of
these somatic mutations of hTERT, it has been proposed that new ETS
transcription factor binding sites are generated in the duplex form
of the promoter. However, while the G124A mutant, which has the
highest transcription activity (9.7-fold), generates an ETS
transcription factor binding site, the G124/125A mutant would be
predicted to diminish the affinity of ETS protein. However,
conflicting results from others strongly suggest that there must be
other factors beyond the duplex sequence information that are
required to understand the effects of promoter mutations on the
transcription activation of hTERT.
[0106] As disclosed herein, the present inventors have shown that
the core hTERT promoter sequence forms a tandem set of
G-quadruplexes that have been demonstrated to inhibit hTERT
transcription when stabilized by compounds that bind to these
structures. Furthermore, all the somatic mutation sites found in
the hTERT promoter are associated with the 5-12 G-quadruplex. These
structural insights provided the present inventors with the
opportunity to evaluate (1) whether the presence of potentially
destabilizing promoter mutations in the major G-quadruplex silencer
element could be used to rationalize the transcription activation
observed in tumors bearing these mutations and (2) whether one can
identify specific compounds that can reverse the effects of these
mutations by stabilizing an otherwise compromised silencer
element.
[0107] The present inventors have discovered that hTERT expression
was enhanced very significantly 6.6-34-fold in the hTERT promoter
mutations melanoma cells. The present inventors have also
identified, through a FRET screening method compounds that can
modulate hTERT, e.g., a guanidine-acridine derivative (GTC365) and
a second compound which lacks the acridine but retains the moiety
which interacts with the hairpin loop (GTC260). These compound
partially reversed the effect of the activating mutations by
binding to the duplex stem of the hairpin. The acridine in the
GTC365 also binds to the tetrad that is in juxtaposition to the
hairpin loop. For comparison the effects of BRACO-19, which has
been previously shown to inhibit telomere elongation, were
evaluated albeit over an extended period of time (15 days). While
GTC365 preferentially stabilized the hTERT G-quadruplex over the
telomeric G-quadruplex, BRACO-19 had the reverse selectivity. The
cellular consequences of the differential binding of the two
acridines to the hTERT promoter versus the telomeric G-quadruplexes
were also quite different: GTC365 directly repressed hTERT
expression and produced induction of apoptosis through lowering of
BCL2, whereas BRACO-19 only exhibited the longer term effects,
mediated through targeting the telomeric G-quadruplexes. Thus while
GTC365, like BRACO-19, produced telomere shortening, this was much
faster than BRACO-19, taking place within 5 days at a significantly
lower concentration. The apoptosis induced within 48 h by GTC365 is
mediated by lowering of BCL2, a known consequence of hTERT
transcription repression. In addition to induction of apoptosis,
GTC365 also induced both G0/G1 and G2/M phase arrest and an SLP,
leading to non-apoptotic cell death accompanied with abnormal cell
division with bridges between cells and multiple nuclei that may
both be related to the short-term telomere-shortening effects or
uncapping of telomeres, as well as telomere-independent activity of
hTERT.
[0108] Drug targeting of the hTERT G-quadruplex represents a
special case where it is possible to gain selectivity based upon
the added complexity of the presence of a large hairpin loop in
juxtaposition to one of the external tetrads. In this case the
present inventors have shown that GTC365 and BRACO-19 produced
different biological consequences based upon their differential
binding to the hTERT and telomeric G-quadruplexes.
[0109] The present inventors have characterized the destabilizing
effect of somatic mutations on the major G-quadruplex, which is the
silencer element in the hTERT core promoter region. The
guanidine-acridine compound GTC365 was then identified as one of
the compounds able to reverse the transcription-activating effect
of these mutations by binding to unique features of this secondary
DNA structure to partially restore the silencing ability of the
mutant G-quadruplexes. In contrast to approaches that target the
telomeric structure, the direct targeting of the hTERT promoter
element produces biological effects, such as apoptosis, telomere
shortening, cell cycle arrest, and failure of cell division leading
to non-apoptotic cell death, that can be observed within 2-5 days,
which makes this an attractive therapeutic strategy for treating
cancer patients with these hTERT mutations or other genetic
aberrations.
[0110] A cartoon illustrating the proposed effect of somatic
mutations on the cooperative folding process, resulting in
activation of hTERT transcription, and how GTC365 is proposed to
act as a pharmacoperone to restore the silencer function is shown
in. For the WT cooperative folding pathway, the loop of the hairpin
is in proximity to a loop in the adjacent G-quadruplex to provide
critical tertiary interactions, leading to the functional silencer
element. For illustrative purposes, G146 is shown making this
interaction, and G124 is base-paired to C134 at the bottom of the
hairpin loop. In the case of any of the mutant promoter elements,
the loss of one of these critical tertiary interactions between the
hairpin and the adjacent G-quadruplex, which is required for
steering the correct folding pathways, then leads to misfolding of
the 5-12 G-quadruplex and a nonfunctional silencer element. The
binding of the pharmacoperone GTC365 to the mutant hairpin loop
restores the folding pathway, leading to a functional silencer
element.
Synthesis of Compounds of the Invention
[0111] Abbreviations used in the present invention: DCM
(Dichloromethane); EtOH (Ethanol); DMSO (Dimethyl sulfoxide); DMF
(N,N-dimethylformamide); Pd(dppf).sub.2C12
[(1,1'-Bis(diphenylphosphino)ferrocene)-palladium(II) dichloride];
TFA (Trifluoroacetic acid); TEA (Triethylamine); TLC (Thin layer
chromatography); and NMR (Nuclear magnetic resonance).
[0112] General Procedure:
[0113] All the chemicals were purchased from commercial vendors.
All the solvents were obtained from Fischer Scientific. Column
chromatography was performed with silica gel 230/400 mesh. All
anhydrous reactions were carried out under positive pressure of
nitrogen. HPLC-MS analyses were performed on a Shimadzu UFLC
instrument with a Phenomenex monolithic Onyx 50.times.2 mm C18
reverse-phase column. HRMS results were obtained on an apex-Qe
instrument. All .sup.1H-NMR and .sup.13C-NMR spectra were recorded
on a Bruker Avance-III 400 MHz NMR instrument, using deuterated
solvents. The spectra are reported in ppm and referenced to
deuterated DMSO (2.49 ppm for .sup.1H, 39.5 ppm for .sup.13C) or
deuterated chloroform (7.26 ppm for .sup.1H, 77 ppm for .sup.13C).
High-resolution mass spectra (HRMS) were acquired on a Bruker 9.4 T
Apex-Qh FTICR mass spectrometer. All the microwave assisted
reactions were performed using Biotage initiator system. All
compounds were analyzed for purity by HPLC using either MS or UV
absorbance detectors.
Synthesis of
2-amino-N-((4-((5-bromopyrimidin-2-yl)oxy)-3-methylphenyl)-carbamoyl)benz-
amide (Compound 1)
[0114] Oxalyl chloride (0.25 g) was added to 2-nitrobenzamide i
(0.1 g) in toluene at 0.degree. C. The solution was allowed to warm
to room temperature, then refluxed with stirring for 24 h. The
solvent was evaporated and product 2-nitrobenzoyl isocyanate ii was
used directly without further purification for the next
reaction.
[0115] A solution of 5-bromo-2-chloropyrimidine (500 mg, 2.6 mmol),
4-amino-2-methylphenol (318 mg, 2.6 mmol), and K.sub.2CO.sub.3 (714
mg, 5.2 mmol) in dry DMSO (20 mL) was stirred at 120.degree. C. for
2.5 h. After cooling to room temperature, the reaction mixture was
poured into water and extracted with ethyl acetate. The organic
layer was washed with water, saturated brine and then dried. The
organic solvent was evaporated to give a residue that was purified
using silica gel column chromatography (ethyl acetate-hexane 1:3)
to give 200 mg product
4-((5-bromopyrimidin-2-yl)oxy)-3-methylaniline v.
[0116] A solution of 2-nitrobenzoyl isocyanate ii (115 mg, 0.6
mmol) in dry DCM (5 mL) was added dropwise to a solution of
4-((5-bromopyrimidin-2-yl)oxy)-3-methylaniline v (168 mg, 0.6 mmol)
in dry 1,4-dioxane (1 mL) with stirring at room temperature. The
reaction mixture was stirred for 18 h and then diluted with water.
The precipitated solid was collected by filtration and washed with
water. The solid was dissolved in ethyl acetate, and the organic
layer was washed with (3.times.30 mL) water, dried and concentrated
to give 280 mg of product,
N-((4-((5-bromopyrimidin-2-yl)oxy)-3-methylphenyl)carbamoyl)-2-n-
itrobenzamide vi. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 11.29 (s,
1H), 10.22 (s, 1H), 8.80 (s, 2H), 8.22 (ddd, J=8.1, 1.2, 0.6 Hz,
1H), 7.94-7.88 (m, 1H), 7.83-7.76 (m, 2H), 7.47 (d, J=19.8 Hz, 2H),
7.13 (d, J=8.6 Hz, 1H), 2.08 (s, 3H).
[0117] Iron powder (160 mg, 3.0 mmol) was added in portions to a
mixture of
N-((4-((5-bromopyrimidin-2-yl)oxy)-3-methylphenyl)carbamoyl)-2-nitrobe-
nzamide vi (283 mg, 0.6 mmol) and ammonium chloride (335 mg, 6
mmol) in 20 mL ethanol at 80.degree. C. The reaction mixture was
refluxed for 30 min and then cooled to room temperature and diluted
with water. The precipitated solid was collected by filtration. The
solid was dissolved in excess ethyl acetate and filtered. The
filtrate was dried and concentrated to give a residue that was
purified by column chromatography (ethyl acetate: hexane 2:3) to
give 45 mg compound
2-amino-N-((4-((5-bromopyrimidin-2-yl)oxy)-3-methylphenyl)carbamoyl)benza-
mide 1. .sup.1H NMR (400 MHz, DMSO) .delta. 10.76 (s, 1H), 10.57
(s, 1H), 8.80 (s, 2H), 7.72 (d, J=8.1 Hz, 1H), 7.50 (d, J=3.6 Hz,
2H), 7.26 (td, J=8.4, 1.5 Hz, 1H), 7.13 (d, J=9.4 Hz, 1H), 6.79 (d,
J=8.4 Hz, 1H), 6.60-6.56 (m, 3H), 2.09 (s, 3H); HPLC-MS: Expected:
442 (MH+); Found: 442.
Synthesis of
2-amino-N-((4-methoxy-3-methylphenyl)carbamoyl)benzamide (Compound
2)
[0118] Compound
N-((4-methoxy-3-methylphenyl)carbamoyl)-2-nitrobenzamide was
prepared using 2-nitrobenzoyl isocyanate and
4-methoxy-3-methylaniline according to the procedure described
above. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 11.23 (s, 1H), 10.07
(s, 1H), 8.20 (d, J=8.0 Hz, 1H), 7.89 (td, J=7.5, 1.2 Hz, 1H),
7.83-7.74 (m, 2H), 7.40-7.28 (m, 2H), 6.91 (d, J=8.7 Hz, 1H), 3.78
(s, 3H), 2.16 (s, 3H).
[0119] 2-Amino-N-((4-methoxy-3-methylphenyl)carbamoyl)benzamide
(Compound 2) was prepared using procedure similar to the synthesis
of compound 1. .sup.1H NMR (400 MHz, DMSO) .delta. 10.60 (s, 1H),
10.48 (s, 1H), 7.71 (d, J=8.1, 1H), 7.39 (dd, J=8.7, 2.6 Hz, 1H),
7.33 (d, J=2.2 Hz, 1H), 7.24 (td, J=4.8, 3.5 Hz, 1H), 6.91 (d,
J=8.8 Hz, 1H), 6.79 (dd, J=8.3, 0.9 Hz, 1H), 6.63-6.45 (m, 3H),
3.78 (s, 3H), 2.17 (s, 3H). HPLC-MS: Expected: 300 (MH+); Found:
300.
Synthesis of
2-amino-N-((3-methyl-4-(pyrimidin-2-yloxy)phenyl)carbamoyl)-benzamide
(Compound 3)
[0120] 3-Methyl-4-(pyrimidin-2-yloxy)aniline was prepared using
2-chloropyrimidine and 4-amino-2-methylphenol according to the
procedure described above.
[0121]
N-((3-Methyl-4-(pyrimidin-2-yloxy)phenyl)carbamoyl)-2-nitrobenzamid-
e was prepared using 3-methyl-4-(pyrimidin-2-yloxy)aniline and
2-nitrobenzoyl isocyanate according to the procedure described
above. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 8.64 (d, J=4.8 Hz,
2H), 8.24-8.20 (m, 1H), 7.93-7.88 (m, 1H), 7.83-7.77 (m, 2H), 7.49
(d, J=2.4 Hz, 2H), 7.26 (t, J=4.8 Hz, 1H), 7.10 (d, J=8.6 Hz, 1H),
2.07 (s, 3H).
[0122]
2-Amino-N-((3-methyl-4-(pyrimidin-2-yloxy)phenyl)carbamoyl)benzamid-
e (Compound 3) was prepared according to the procedure described
for the synthesis of compound 1. .sup.1H NMR (400 MHz, DMSO)
.delta. 10.76 (s, 1H), 10.57 (s, 1H), 8.64 (d, J=4.8 Hz, 2H), 7.72
(dd, J=8.1, 1.4 Hz, 1H), 7.49 (dd, J=6.1, 2.8 Hz, 2H), 7.31-7.22
(m, 2H), 7.10 (d, J=9.4 Hz, 1H), 6.79 (dd, J=8.4, 1.1 Hz, 1H), 6.56
(m, 3H), 2.08 (s, 3H). HPLC-MS: Expected: 364 (MH+); Found:
364.
Synthesis of
2-amino-N-((4-((2-chloropyrimidin-5-yl)oxy)-3-methylphenyl)-carbamoyl)ben-
zamide (Compound 4)
[0123] Compound 4-((5-fluoropyrimidin-2-yl)oxy)-3-methylaniline xi
and 4-((2-chloropyrimidin-5-yl)oxy)-3-methylaniline xii were
prepared using the procedure described for the synthesis of
compound v. Products xi and xii were separated using ethyl
acetate:hexane (1:3) column chromatography. For compound xi:
.sup.1H NMR (400 MHz, Chloroform-d) .delta. 8.39 (s, 2H), 6.88 (d,
J=8.4 Hz, 1H), 6.59-6.57 (m, 1H), 6.56-6.52 (m, 1H), 3.67 (s, 2H),
2.09 (s, 3H). For compound xii: .sup.1H NMR (400 MHz, Chloroform-d)
.delta. 8.20 (s, 2H), 6.77 (d, J=8.5 Hz, 1H), 6.59-6.57 (m, 1H),
6.52 (dd, J=8.5, 2.8 Hz, 1H), 3.73 (s, 2H), 2.08 (s, 3H).
[0124] Compound
N-((4-((2-chloropyrimidin-5-yl)oxy)-3-methylphenyl)carbamoyl)-2-nitrobenz-
amide xiii was prepared according to the procedure described for
the synthesis of compound vi. .sup.1H NMR (400 MHz, DMSO-d6)
.delta. 11.30 (s, 1H), 10.25 (s, 1H), 8.49 (s, 2H), 8.22 (ddd,
J=8.1, 1.2, 0.4 Hz, 1H), 7.90 (dd, J=7.5, 1.2 Hz, 1H), 7.83-7.75
(m, 2H), 7.56 (s, 1H), 7.49 (d, J=9.2 Hz, 1H), 7.12 (d, J=8.8 Hz,
1H), 2.21 (s, 3H).
[0125]
2-Amino-N-((4-((2-chloropyrimidin-5-yl)oxy)-3-methylphenyl)carbamoy-
l)-benzamide (Compound 4) was prepared according to the procedure
described above for the synthesis of compound 1 using compound
xiii. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 10.80 (s, 1H), 10.58
(br, 1H), 8.49 (s, 2H), 7.72 (d, J=8.1 Hz, 1H), 7.57 (d, J=1.5 Hz,
1H), 7.55-7.49 (dd, J=8.1, 1.5 Hz, 1H), 7.25 (dt, J=8.5, 7.0 Hz,
1H), 7.12 (d, J=8.7 Hz, 1H), 6.79 (d, J=8.5 Hz, 1H), 6.57 (br, 2H),
6.55 (dt, J=8.5, 1.5 Hz, 1H), 2.22 (s, 3H). HPLC-MS: Expected: 398
(MH+); Found: 398.
Synthesis of
2-amino-N-((3-methyl-4-(pyrazine-2-yloxy)phenyl)carbamoyl)-benzamide
(Compound 5)
[0126] Compound 3-methyl-4-(pyrazin-2-yloxy)aniline xv was prepared
according to the procedure described above using 2-chloropyrazine
and 4-amino-2-methylphenol. .sup.1H NMR (400 MHz, Chloroform-d)
.delta. 8.39 (d, J=1.4 Hz, 1H), 8.22 (d, J=2.7 Hz, 1H), 8.10 (dd,
J=2.7, 1.4 Hz, 1H), 6.88 (d, J=8.4 Hz, 1H), 6.63 (d, J=2.8 Hz, 1H),
6.58 (d, J=8.4 Hz, 1H), 3.64 (s, 2H), 2.10 (s, 3H).
[0127] Compound
N-((3-methyl-4-(pyrazine-2-yloxy)phenyl)carbamoyl)-2-nitrobenzamide
xvi was prepared using 2-nitrobenzoyl isocyanate and
3-methyl-4-(pyrazin-2-yloxy)aniline according to the procedure
described above. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 11.29 (s,
OH), 10.23 (s, OH), 8.56 (d, J=1.4 Hz, 1H), 8.36 (d, J=2.7 Hz, 1H),
8.24-8.20 (m, 1H), 8.18 (dd, J=2.7, 1.4 Hz, 1H), 7.94-7.88 (m, 1H),
7.80 (td, J=7.7, 1.3 Hz, 2H), 7.51 (d, J=2.6 Hz, 1H), 7.47 (d,
J=8.8 Hz, 1H), 7.12 (d, J=8.6 Hz, 1H), 2.10 (s, 3H).
[0128]
2-Amino-N-((3-methyl-4-(pyrazine-2-yloxy)phenyl)carbamoyl)benzamide
(Compound 5) was prepared according to the procedure described for
the synthesis of compound 1 using compound xvi. .sup.1H NMR (400
MHz, DMSO-d6) .delta. 10.77 (s, 1H), 10.57 (s, 1H), 8.56 (d, J=1.4
Hz, 1H), 8.36 (d, J=2.7 Hz, 1H), 8.18 (dd, J=2.7, 1.4 Hz, 1H), 7.72
(dd, J=8.1, 1.5 Hz, 1H), 7.52-7.49 (m, 2H), 7.26 (dt, J=8.4, 7.0
Hz, 1H), 7.10 (d, J=8.0 Hz 1H), 6.79 (dd, J=8.4, 1.1 Hz, 1H),
6.65-6.51 (m, 3H), 2.10 (s, 3H). Expected: 364 (MH+); Found:
364.
[0129] Compound 6: Compound
N-((4-((5-fluoropyrimidin-2-yl)oxy)-3-methyl-phenyl)carbamoyl)-2-nitroben-
zamide xvii was prepared according to the procedure described for
the synthesis of compound vi. .sup.1H NMR (400 MHz, DMSO-d6)
.delta. 11.30 (s, 1H), 10.25 (s, 1H), 8.70 (s, 2H), 8.22 (ddd,
J=8.1, 1.2, 0.4 Hz, 1H), 7.90 (dd, J=7.5, 1.2 Hz, 1H), 7.83-7.75
(m, 2H), 7.56 (s, 1H), 7.49 (d, J=9.2 Hz, 1H), 7.12 (d, J=8.8 Hz,
1H), 2.21 (s, 3H).
[0130] Compound
2-amino-N-((4-((5-fluoropyrimidin-2-yl)oxy)-3-methylphenyl)-carbamoyl)ben-
zamide 6 was prepared according to the procedure described for the
synthesis of compound 1. .sup.1H NMR (400 MHz, DMSO-d6) .delta.
10.76 (s, 1H), 10.56 (br, 1H), 8.73 (s, 2H), 7.72 (d, J=8.1 Hz,
1H), 7.52-7.46 (m, 2H), 7.26 (dt, J=8.4, 7.0 Hz, 1H), 7.11 (d,
J=9.4 Hz, 1H), 6.80 (d, J=8.4 Hz, 1H), 6.58-6.54 (m, 3H), 2.09 (s,
3H). Expected: 382 (MH+); Found: 382.
[0131] Compound 7: Compound 4-((5-bromopyrimidin-2-yl)oxy)aniline
xix was prepared using 5-bromo-2-chloropyrimidine and 4-aminophenol
according to the procedure described for the synthesis of compound
v. .sup.1H NMR (400 MHz, Chloroform-d) .delta. 8.57 (s, 2H),
7.01-6.95 (dd, J=8.4, 1.2 Hz, 2H), 6.76-6.70 (dd, J=8.4, 1.2 Hz,
2H), 3.70 (s, 2H).
[0132] Compound
N-((4-((5-bromopyrimidin-2-yl)oxy)phenyl)carbamoyl)-2-nitrobenzamide
xx was prepared using 2-nitrobenzoyl isocyanate and
4-((5-bromopyrimidin-2-yl)oxy)aniline according to the procedure
described for the synthesis of compound vi. .sup.1H NMR (400 MHz,
DMSO-d6) .delta. 11.30 (s, 1H), 10.25 (s, 1H), 8.81 (s, 2H),
8.27-8.17 (m, 1H), 7.95-7.88 (m, 1H), 7.84-7.75 (m, 2H), 7.64-7.57
(m, 2H), 7.26-7.18 (m, 2H).
[0133] Compound
2-amino-N-((4-((5-bromopyrimidin-2-yl)oxy)phenyl)carbamoyl)-benzamide
7 was prepared according to the procedure described for the
synthesis of compound 1. .sup.1H NMR (400 MHz, DMSO-d6) .delta.
10.78 (s, 1H), 10.58 (br, 1H), 8.81 (s, 2H), 7.72 (dd, J=8.1, 1.5
Hz, 1H), 7.68-7.60 (m, 2H), 7.29-7.18 (m, 3H), 6.80 (dd, J=8.4, 1.2
Hz, 1H), 6.56 (dt, J=8.1, 1.2 Hz, 1H). Expected: 428 (MH+); Found:
428.
[0134] Compound 8: Compound 4-(pyrimidin-2-yloxy)aniline xxi was
prepared using 2-chloropyrimidine and 4-aminophenol according to
the procedure described for the synthesis of compound v. .sup.1H
NMR (400 MHz, Chloroform-d) .delta. 8.56 (d, J=4.8 Hz, 1H),
7.03-6.99 (m, 2H), 6.76-6.72 (dd, J=8.2 Hz, J=4.8 Hz, 1H).
[0135] Compound
2-nitro-N-((4-((pyrimidin-2-yloxy)phenyl)carbamoyl)benzamide xxii
was prepared using 2-nitrobenzoyl isocyanate and
4-(pyrimidin-2-yloxy)aniline according to the procedure described
for the synthesis of compound vi. .sup.1H NMR (400 MHz, DMSO-d6)
.delta. 11.30 (s, 1H), 10.25 (s, 1H), 8.81 (d, J=4.8 Hz, 2H), 8.222
(d, J=8.2 Hz, 1H), 7.95-7.88 (m, 1H), 7.84-7.75 (m, 2H), 7.64-7.57
(m, 2H), 7.26-7.18 (m, 2H).
[0136] Compound
2-amino-N-((4-(pyrimidin-2-yloxy)phenyl)carbamoyl)benzamide 8 was
prepared from
2-nitro-N-((4-(pyrimidin-2-yloxy)phenyl)carbamoyl)benzamide
according to the procedure described for the synthesis of compound
1. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 10.77 (s, 1H), 10.58 (s,
1H), 8.65 (d, J=4.8 Hz, 2H), 7.72 (dd, J=8.1, 1.5 Hz, 1H),
7.68-7.59 (m, 2H), 7.30-7.23 (m, 2H), 7.23-7.16 (m, 2H), 6.80 (dd,
J=8.4, 1.1 Hz, 1H), 6.65-6.45 (m, 3H). Expected: 350 (MH+); Found:
350.
[0137] Compound 9: Compound
4-((5-chloropyrimidin-2-yl)oxy)-3-methylaniline xxiv was prepared
using 2,5-dichloropyrimidine and 4-amino-2-methylphenol according
to the procedure described for the synthesis of compound v. .sup.1H
NMR (400 MHz, Chloroform-d) .delta. 8.46 (s, 2H), 6.87 (d, J=8.4
Hz, 1H), 6.60-6.49 (m, 2H), 3.66 (s, 1H), 2.08 (s, 3H).
[0138] Compound
N-((4-((5-chloropyrimidin-2yl)oxy)-3-methylphenyl)carbamoyl)-2-nitrobenza-
mide xxv was prepared from 2-nitrobenzoyl isocyanate and
4-((5-chloropyrimidin-2-yl)oxy)-3-methylaniline according to the
procedure described for the synthesis of compound vi. .sup.1H NMR
(400 MHz, DMSO-d6) .delta. 11.29 (s, 1H), 10.22 (s, 1H), 8.80 (s,
2H), 8.22 (ddd, J=8.1, 1.2, 0.6 Hz, 1H), 7.94-7.88 (m, 1H),
7.83-7.76 (m, 2H), 7.47 (d, J=19.8 Hz, 2H), 7.13 (d, J=8.6 Hz, 1H),
2.08 (s, 3H).
[0139] Compound
2-amino-N-((4-((5-chloropyrimidin-2-yl)oxy)-3-methylphenyl)-carbamoyl)ben-
zamide 9 was prepared from
N-((4-((5-chloropyrimidin-2yl)oxy)-3-methylphenyl)carbamoyl)-2-nitrobenza-
mide xxv according to the procedure described for the synthesis of
compound 1. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 10.77 (s, 1H),
10.57 (s, 1H), 8.75 (s, 2H), 7.72 (dd, J=8.1, 1.5 Hz, 1H),
7.53-7.45 (m, 2H), 7.26 (dt, J=8.4, 1.5 Hz, 1H), 7.16-7.08 (m, 1H),
6.79 (dd, J=8.4, 1.2 Hz, 1H), 6.66-6.50 (m, 3H), 2.09 (s, 3H).
Expected: 398 (MH+); Found: 398.
[0140] Compound 11: Compound
4-((5-methoxypyrimidin-2-yl)oxy)-3-methylaniline xxvii was prepared
from 2-chloro-5-methoxypyrimidine and 4-amino-2-methylphenol
according to the procedure described for the synthesis of compound
v. .sup.1H NMR (400 MHz, Chloroform-d) .delta. 8.20 (s, 2H), 6.87
(d, J=8.4 Hz, 1H), 6.60-6.50 (m, 2H), 3.85 (s, 3H), 2.08 (q, J=0.5
Hz, 3H).
[0141] Compound
N-((4-((5-methoxypyrimidin-2-yl)oxy)-3-methylphenyl)-carbamoyl)-2-nitrobe-
nzamide xxviii was prepared from 2-nitrobenzoyl isocyanate and
4-((5-methoxypyrimidin-2-yl)oxy)-3-methylaniline according to the
procedure described for the synthesis of compound vi. .sup.1H NMR
(400 MHz, DMSO-d6) .delta. 11.29 (s, 1H), 10.20 (s, 1H), 8.38 (s,
2H), 8.23-8.18 (m, 1H), 7.94-7.86 (m, 1H), 7.80 (td, J=7.8, 1.2 Hz,
2H), 7.50-7.39 (m, 2H), 7.06 (d, J=8.6 Hz, 1H), 3.86 (s, 3H), 2.07
(s, 3H).
[0142] Compound
2-amino-N-((4-((5-methoxypyrimidin-2-yl)oxy)-3-methyl-phenyl)carbamoyl)be-
nzamide 11 was prepared from
N-((4-((5-methoxypyrimidin-2-yl)oxy)-3-methylphenyl)carbamoyl)-2-nitroben-
zamide according to the procedure described for the synthesis of
compound 1. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 10.75 (s, 1H),
10.56 (s, 1H), 8.39 (s, 2H), 7.72 (dd, J=8.1, 1.5 Hz, 1H),
7.50-7.44 (m, 2H), 7.26 (dt, J=8.5, 1.5 Hz, 1H), 7.09-7.03 (m, 1H),
6.79 (dd, J=8.4, 1.2 Hz, 1H), 6.63-6.50 (m, 3H), 3.86 (s, 3H), 2.08
(s, 3H). Expected: 394 (MH+); Found: 394.
[0143] Compound 12: Compound
N-((4-((5-bromopyrimidin-2-yl)oxy)-3-methyl-phenyl)carbamoyl)benzamide
12 was prepared from benzoyl isocyanate and
4-((5-bromopyrimidin-2-yl)oxy)-3-methylaniline according to the
procedure described for the synthesis of compound vi. .sup.1H NMR
(400 MHz, DMSO-d6) .delta. 11.05 (s, 1H), 10.83 (s, 1H), 8.80 (s,
2H), 8.07-8.02 (dd, J=7.4, 1.2 Hz, 2H), 7.66 (t, J=7.4 Hz, 1H),
7.59-7.48 (m, 4H), 7.14 (d, J=8.3 Hz, 1H), 2.09 (s, 3H). Expected:
427 (MH+); Found: 427.
[0144] Compound 13: Compound 4-(trifluoromethyl)benzoyl isocyanate
xxix was prepared using 4-(trifluoromethyl)benzamide and oxalyl
chloride in situ according to the procedure described for the
synthesis of compound ii.
[0145] Compound
N-((4-((5-bromopyrimidin-2-yl)oxy)-3-methylphenyl)carbamoyl)-4-(trifluoro-
methyl)benzamide 13 was prepared from 4-(trifluoromethyl)benzoyl
isocyanate and 4-((5-bromopyrimidin-2-yl)oxy-3-methylaniline
according to the procedure described for the synthesis of compound
vi. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 11.28 (s, 1H), 10.68 (s,
1H), 8.80 (s, 2H), 8.20 (d, J=9.6 Hz, 2H), 7.92 (d, J=9.6 Hz, 2H),
7.55-7.47 (m, 2H), 7.14 (d, J=8.5 Hz, 1H), 2.10 (s, 3H). Expected:
495, (MH+); Found: 495.
[0146] Compound 14: Compound 4-fluorobenzoyl isocyanate xxx was
prepared from 4-fluorobenzamide and oxalyl chloride in situ
according to the procedure described for the synthesis of compound
ii.
[0147] Compound
N-((4-((5-bromopyrimidin-2-yl)oxy)-3-methylphenyl)carbamoyl)-4-fluorobenz-
amide 14 was prepared from 4-fluorobenzoyl isocyanate and
4-((5-bromopyrimidin-2-yl)oxy)-3-methylaniline according to the
procedure described for the synthesis of compound vi. .sup.1H NMR
(400 MHz, DMSO-d6) .delta. 11.08 (s, 1H), 10.78 (s, 1H), 8.80 (s,
2H), 8.13 (dd, J=8.8, 5.5 Hz, 2H), 7.50 (d, J=8.8 Hz, 2H), 7.39 (t,
J=8.8 Hz, 2H), 7.14 (d, J=8.3 Hz, 1H), 2.09 (s, 3H). Expected: 445,
(MH+); Found: 445.
[0148] Compound 15: Compound 4-methoxybenzoyl isocyanate xxxi was
prepared from 4-methoxybenzamide in situ according to the procedure
described for the synthesis of compound ii.
[0149] Compound
N-((4-((5-bromopyrimidin-2-yl)oxy)-3-methylphenyl)carbamoyl)-4-methoxyben-
zamide 15 was prepared from 4-methoxybenzoyl isocyanate and
4-((5-bromopyrimidin-2-yl)oxy-3-methylaniline according to the
procedure described for the synthesis of compound vi. .sup.1H NMR
(400 MHz, DMSO-d6) .delta. 10.93 (s, 1H), 10.89 (s, 1H), 8.80 (s,
2H), 8.06 (d, J=8.4 Hz, 2H), 7.50 (d, J=8.0 Hz, 2H), 7.14 (d, J=8.0
Hz, 1H), 7.07 (d, J=4.4 Hz, 2H), 3.86 (s, 3H), 2.09 (s, 3H).
Expected: 457, (MH+); Found: 457.
[0150] Compound 16: Compound
N-((4-(2-morpholinoethoxy)phenyl)carbamoyl)-2-nitrobenzamide xxxii
was prepared from 2-nitrobenzoyl isocyanate and
4-(2-morpholinoethoxy)aniline according to the procedure described
for the synthesis of compound vi. .sup.1H NMR (400 MHz, DMSO-d6)
.delta. 11.23 (s, 1H), 3.71-3.56 (m, 4H), 10.12 (s, 1H), 8.21 (dd,
J=8.2, 1.1 Hz, 1H), 7.89 (td, J=7.5, 1.2 Hz, 1H), 7.83-7.70 (m,
2H), 7.46 (d, J=8.5 Hz, 2H), 7.01-6.87 (m, 2H), 4.12 (s, 2H), 2.67
(d, J=82.7 Hz, 8H).
[0151] Compound
2-amino-N-((4-(2-morpholinoethoxy)phenyl)carbamoyl)-benzamide 16
was prepared from
N-((4-(2-morpholinoethoxy)phenyl)carbamoyl)-2-nitrobenzamide
according to the procedure described for the synthesis of compound
1. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 10.61 (s, 1H), 10.50 (s,
1H), 7.71 (dd, J=8.1, 1.5 Hz, 1H), 7.51-7.43 (m, 2H), 7.25 (ddd,
J=8.4, 7.0, 1.5 Hz, 1H), 6.98-6.90 (m, 2H), 6.79 (dd, J=8.3, 1.1
Hz, 1H), 6.60-6.50 (m, 3H), 4.07 (t, J=5.8 Hz, 2H), 3.59 (t, J=4.0
Hz, 4H), 2.69 (t, J=5.8 Hz, 2H), 2.48 (t, J=4.0 Hz, 4H). Expected:
385, (MH+); Found: 385.
[0152] Compound 17: To a solution of
4-((5-bromopyrimidin-2-yl)oxy)aniline xix (200 mg, 751 .mu.mol) and
1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole
xxxiii (234.5 mg, 531.5 mmol) in dioxane: H.sub.2O (5:1, 3 mL),
Na.sub.2CO.sub.3 (239.0 mg, 2.25 mmol) and Pd(dppf).sub.2Cl.sub.2
(61.2 mg, 75.2 .mu.mol) were added sequentially. The mixture was
heated in microwave at 100.degree. C. for 1 h. After completion as
seen by TLC, the mixture was poured into 20 mL water, and extracted
with DCM (3.times.100 mL). The organic layer was combined, washed
with 100 mL water, dried over anhydrous sodium sulfate. Organic
solvent was concentrated and the residue was purified using column
chromatography to give 220 mg product
4-((5-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)oxy)aniline xxxiv as
yellow solid. .sup.1H NMR (400 MHz, Chloroform-d) .delta. 8.62 (s,
2H), 7.67 (s, 1H), 7.62 (s, 1H), 7.02 (d, J=8.7 Hz, 2H), 6.74 (d,
J=8.7 Hz, 2H), 3.98 (s, 3H), 3.75 (s, 2H).
[0153] Compound
N-((4-((5-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)oxy)phenyl)-carbamoyl)-
-2-nitrobenzamide xxxv was prepared from 2-nitrobenzoyl isocyanate
and 4-((5-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)oxy)aniline
according to the procedure described for the synthesis of compound
vi. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 11.30 (s, 1H), 10.26 (s,
1H), 8.86 (s, 2H), 8.27-8.19 (m, 2H), 7.96 (d, J=0.8 Hz, 1H),
7.94-7.86 (m, 1H), 7.80 (ddd, J=8.1, 6.6, 1.6 Hz, 2H), 7.62 (d,
J=8.5 Hz, 2H), 7.23-7.17 (m, 2H), 3.89 (s, 3H).
[0154] Compound
2-amino-N-((4-((5-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)oxy)phenyl)car-
bamoyl)benzamide 17 was prepared from
N-((4-((5-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)oxy)phenyl)carbamoyl)--
2-nitrobenzamide according to the procedure described for the
synthesis of compound 1. .sup.1H NMR (400 MHz, DMSO-d6) .delta.
10.78 (s, 1H), 10.58 (s, 1H), 8.86 (s, 2H), 8.23 (s, 1H), 7.96 (s,
1H), 7.73 (d, J=8.1 Hz, 1H), 7.64 (d, J=9.0 Hz, 2H), 7.26 (ddd,
J=8.5, 7.0, 1.5 Hz, 1H), 7.21 (d, J=9.0 Hz, 2H), 6.80 (d, J=8.4 Hz,
1H), 6.63-6.52 (m, 3H), 3.89 (s, 3H). Expected: 430, (MH+); Found:
430.
[0155] Compound 18: Compound 4-methoxybenzoyl isocyanate xxxi was
prepared from 4-methoxybenzamide in situ according to the procedure
described for the synthesis of compound ii.
[0156] Compound
N-((4-((5-bromopyrimidin-2-yl)oxy)phenyl)carbamoyl)-4-methoxybenzamide
18 was prepared from 4-methoxybenzoyl isocyanate and
4-((5-bromopyrimidin-2-yl)oxy)aniline according to the procedure
described for the synthesis of compound vi. .sup.1H NMR (400 MHz,
DMSO-d6) .delta. 10.95 (s, 1H), 10.90 (s, 1H), 8.81 (s, 2H), 8.07
(d, J=9.0 Hz, 2H), 7.65 (d, J=9.1 Hz, 2H), 7.22 (d, J=8.9 Hz, 2H),
7.08 (d, J=9.0 Hz, 2H), 3.87 (s, 3H). Expected: 443, (MH+) Found:
465.01 (M+Na+).
[0157] Compound 19: Compound
3-chloro-4-((5-chloropyrimidin-2-yl)oxy)aniline xxxvii was prepared
from 2,5-dichloropyrimidine and 4-amino-2-chlorophenol according to
the procedure described for the synthesis of compound v. .sup.1H
NMR (400 MHz, Chloroform-d) .delta. 8.50 (s, 2H), 7.04 (dd, J=8.6,
0.2 Hz, 1H), 6.80 (dd, J=2.7, 0.2 Hz, 1H), 6.63 (dd, J=8.6, 2.7 Hz,
1H), 3.77 (s, 2H).
[0158] Compound
N-((3-chloro-4-((5-chloropyrimidin-2-yl)oxy)phenyl)carbamoyl)-4-methoxybe-
nzamide 19 prepared from
3-chloro-4-((5-chloropyrimidin-2-yl)oxy)aniline and
4-methoxybenzoyl isocyanate according to the procedure described
for the synthesis of compound vi. .sup.1H NMR (400 MHz, DMSO-d6)
.delta. 11.21 (s, 1H), 9.68 (s, 1H), 8.41 (d, J=6.1 Hz, 1H),
7.92-7.86 (m, 2H), 7.48 (d, J=8.7 Hz, 1H), 7.21 (dd, J=8.7, 2.5 Hz,
1H), 4.92 (s, 2H), 3.23 (d, J=0.8 Hz, 3H), 3.10 (s, 3H), 2.90 (s,
3H), 2.76 (d, J=0.7 Hz, 3H). Expected: 433, (MH+); Found: 433.
[0159] Compound 20: Compound
3-methyl-4-((5-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)oxy)aniline
xxxviii was prepared from
4-((5-bromopyrimidin-2-yl)oxy)-3-methylaniline and
1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole
according to the procedure described for the synthesis of compound
xxxiv. .sup.1H NMR (400 MHz, Chloroform-d) .delta. 8.61 (s, 2H),
7.71 (d, J=0.8 Hz, 1H), 7.66-7.59 (m, 1H), 6.92 (d, J=8.4 Hz, 1H),
6.66-6.51 (m, 2H), 3.97 (s, 3H), 3.58-3.15 (m, 2H), 2.12 (s,
3H).
[0160] Compound
4-methoxy-N-((4-((5-(1-methyl)-1H-pyrazol-4-yl)pyrimidin-2-yl)oxy)phenyl)-
carbamoyl)benzamide 20 was prepared from 4-methoxybenzoyl
isocyanate and
3-methyl-4-((5-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)oxy)aniline
according to the procedure described for the synthesis of compound
vi. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 10.94 (s, 1H), 10.88 (s,
1H), 8.84 (s, 2H), 8.22 (s, 1H), 8.07 (d, J=9.0 Hz, 2H), 7.94 (d,
J=0.8 Hz, 1H), 7.51 (d, J=0.8 Hz, 2H), 7.12 (m, 1H), 7.08 (d, J=9.0
Hz, 2H), 3.89 (s, 3H), 3.86 (s, 3H), 2.11 (s, 3H). Expected: 458,
(MH+); Found: 458.
[0161] Compound 21: Compound
4-((5-bromopyrimidin-2-yl)oxy)-N-methylaniline xl was prepared from
5-bromo-2-chloropyrimidine and 4-(methylamino)phenol according to
the procedure described for the synthesis of compound v. .sup.1H
NMR (400 MHz, Chloroform-d) .delta. 8.35 (s, 2H), 7.08 (d, J=8.8
Hz, 2H), 6.74 (d, J=8.8 Hz, 2H), 6.52 (s, 1H), 3.47 (s, 3H).
[0162] Compound
N-((4-((5-bromopyrimidin-2-yl)oxy)phenyl)(methyl)carbamoyl)-4-methoxybenz-
amide 21 was prepared from 4-methoxybenzoyl isocyanate and
4-((5-bromopyrimidin-2-yl)oxy)-N-methylaniline according to the
procedure described for the synthesis of compound vi. .sup.1H NMR
(400 MHz, Chloroform-d) .delta. 8.38 (s, 1H), 8.35 (s, 2H), 7.90
(d, J=8.9 Hz, 2H), 7.35 (d, J=9.0 Hz, 2H), 7.28 (d, J=3.2 Hz, 2H),
7.01 (d, J=8.9 Hz, 2H), 3.91 (s, 3H), 3.52 (s, 3H). Expected: 457,
(MH+); Found: 457.
[0163] Compound 22: Compound picolinoyl isocyanate xli was prepared
in situ from picolinamide according to the procedure described for
the synthesis of compound ii.
[0164] Compound
N-((4-((5-bromopyrimidin-2-yl)oxy)-3-methylphenyl)carbamoyl)-picolinamide
22 prepared from picolinoyl isocyanate and
4-((5-bromopyrimidin-2-yl)oxy-3-methylaniline according to the
procedure described for the synthesis of compound vi. H NMR (400
MHz, Chloroform-d) .delta. 10.59 (s, 1H), 10.11 (s, 1H), 8.69 (dd,
J=4.7, 1.7 Hz, 1H), 8.58 (s, 2H), 8.28 (dd, J=7.8, 1.1 Hz, 1H),
7.98 (td, J=7.7, 1.7 Hz, 1H), 7.62-7.57 (m, 2H), 7.52 (dd, J=8.0,
4.0 Hz, 1H), 7.10 (d, J=8.6 Hz, 1H), 2.22 (s, 3H). Expected: 428,
(MH+); Found: 428.
[0165] Compound 23: Compound
N-((3-methyl-4-nitrophenyl)carbamoyl)benzamide xliii was prepared
from benzoyl isocyanate and 3-methyl-4-nitroaniline according to
the procedure described for the synthesis of compound vi. .sup.1H
NMR (400 MHz, DMSO-d6) .delta. 11.21 (s, 1H), 11.14 (s, 1H),
8.12-8.00 (m, 3H), 7.77 (ddd, J=9.0, 2.4, 0.6 Hz, 1H), 7.72-7.63
(m, 2H), 7.61-7.51 (m, 2H), 2.58 (s, 3H).
[0166] Compound N-((4-amino-3-methylphenyl)carbamoyl)benzamide xliv
was prepared from N-((3-methyl-4-nitrophenyl)carbamoyl)benzamide
according to the procedure described for the synthesis of compound
1. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 10.86 (s, 1H), 10.48 (s,
1H), 8.06-7.97 (m, 2H), 7.68-7.60 (m, 1H), 7.58-7.48 (m, 2H),
7.15-7.06 (m, 2H), 6.60 (d, J=8.2 Hz, 1H), 4.76 (s, 2H), 2.08 (s,
3H).
[0167] To a solution of
N-((4-amino-3-methylphenyl)carbamoyl)benzamide xliv (100 mg, 0.37
mmol) in anhydrous DMF (15 mL) was added
1,3-bis(t-butylcarbonyl)-2-methylthiopseudourea xlv (161 mg, 0.55
mmol), triethylamine (169 mg, 1.67 mmol), and mercury(II) chloride
(151 mg, 0.55 mmol). The suspension was kept stirring at room
temperature for overnight. The reaction mixture was diluted with
DCM, washed with Na.sub.2CO.sub.3 solution. The organic layer was
washed with water and brine, dried over Na.sub.2SO.sub.4, and then
concentrated under vacuum. The residue was treated with 0.5 mL TFA
in 5 mL DCM. The mixture was stirred overnight and basified with
ammonium hydroxide to pH=9. The mixture was extracted with DCM
(3.times.30 mL) and the solvent was evaporated. The product
N-((4-guanidino-3-methylphenyl)carbamoyl)benzamide 23 was purified
using column chromatography to afford 40 mg product 23. .sup.1H NMR
(400 MHz, DMSO-d6) .delta. 11.08 (s, 1H), 10.90 (s, 1H), 9.54 (s,
1H), 8.05 (dd, J=1.9, 0.9 Hz, 1H), 8.03 (t, J=1.0 Hz, 1H), 7.66 (d,
J=7.5 Hz, 1H), 7.60-7.53 (m, 4H), 7.40 (s, 3H), 7.21 (d, J=9.3 Hz,
1H), 2.23 (s, 3H). Expected: 312, (MH+); Found: 312.
[0168] Compound 24: A solution of 1-isocyanato-2-nitrobenzene (100
mg, 0.6 mmol) in dry 1,4-dioxane (5 mL) was added dropwise to a
solution of 4-((5-bromopyrimidin-2-yl)oxy)-3-methylaniline v (168
mg, 0.6 mmol) in dry DCM (3 mL) with stirring at room temperature.
The mixture was stirred for 18 h and then diluted with water. The
precipitated solid was collected by filtration and washed with
water. The solid was dissolved in ethyl acetate, and the organic
layer was washed with water 2-3 times, dried and concentrated to
give 190 mg of
1-(4-((5-bromopyrimidin-2-yl)oxy)-3-methylphenyl)-3-(2-nitrophenyl)urea
xlvi. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.84 (s, 1H),
9.60 (s, 1H), 8.79 (d, J=0.8 Hz, 1H), 8.32 (dt, J=8.6, 1.2 Hz, 1H),
8.11 (dt, J=8.5, 1.2 Hz, 1H), 7.71 (ddd, J=8.6, 7.2, 1.5 Hz, 1H),
7.50-7.39 (m, 1H), 7.35 (ddd, J=8.7, 2.6, 0.7 Hz, 1H), 7.22 (ddt,
J=8.5, 7.0, 1.3 Hz, 1H), 7.08 (d, J=8.7 Hz, 1H), 5.76 (d, J=0.8 Hz,
1H), 2.07 (s, 2H).
[0169] Iron powder (160 mg, 3.0 mmol) was added in portions to a
mixture of
1-(4-((5-bromopyrimidin-2-yl)oxy)-3-methylphenyl)-3-(2-nitrophenyl)ure-
a (221 mg, 0.5 mmol) and NH.sub.4Cl (335 mg, 6 mmol) in EtOH (20
mL) at 80.degree. C. The reaction mixture was refluxed for 30 min
and then cooled to room temperature and diluted with water. The
precipitated solid was collected by filtration. The solid was
dissolved in an excess of ethyl acetate and filtered. Ethyl acetate
was evaporated to give a residue which was then purified using
column chromatography (ethyl acetate:hexanes 2:3) to obtain 85 mg
of
1-(2-aminophenyl)-3-(4-((5-bromopyrimidin-2-yl)oxy)-3-methylphenyl)urea
24. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 8.79 (s, 2H), 8.74 (s,
1H), 7.74 (s, 1H), 7.41-7.38 (m, 1H), 7.36 (dd, J=7.9, 1.5 Hz, 1H),
7.30 (dt, J=8.7, 0.6 Hz, 1H), 7.04 (d, J=8.7 Hz, 1H), 6.85 (ddd,
J=7.8, 7.2, 1.5 Hz, 1H), 4.79 (s, 2H), 2.05 (s, 3H). MS Expected:
414.05, (MH+); Found: 414.05.
[0170] Compound 25: Compound
N-((4-((5-chloropyrimidin-2-yl)oxy)-3-methylphenyl)carbamoyl)-4-(trifluor-
omethyl)benzamide 25 was prepared from 4-(trifluoromethyl)benzoyl
isocyanate and 4-((5-chloropyrimidin-2-yl)oxy)-3-methylaniline
according to the procedure described for the synthesis of compound
vi. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 11.28 (s, 1H), 10.68 (s,
1H), 8.75 (s, 2H), 8.20 (d, J 8.0 Hz, 2H), 7.91 (d, J=8.4 Hz, 2H),
7.56-7.47 (m, 2H), 7.14 (d, J=8.4 Hz, 1H), 2.10 (s, 3H). MS
Expected: 451, Found: 451.07
[0171] Compound 26: Compound
N-((4-((5-methoxypyrimidin-2-yl)oxy)-3-methylphenyl)carbamoyl)-4-(trifluo-
romethyl)benzamide 26 was prepared from 4-(trifluoromethyl)benzoyl
isocyanate and 4-((5-methoxypyrimidin-2-yl)oxy)-3-methylaniline
according to the procedure described for the synthesis of compound
vi. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 11.27 (s, 1H), 10.66 (s,
1H), 8.39 (s, 2H), 8.20 (d, J 8.0 Hz, 2H), 7.93 (d, J=8.4 Hz, 2H),
7.53-7.44 (m, 2H), 7.08 (d, J=8.5 Hz, 1H), 3.86 (s, 3H), 2.09 (s,
3H). MS Expected: 447, Found: 447
[0172] Compound 27: Compound
4-((5-chloropyrimidin-2-yl)oxy)-3-fluoroaniline xlvii was
synthesized from 4-amino-2-fluorophenol and 2,5-dichloropyrimidine
using procedure similar to compound v. .sup.1H NMR (400 MHz,
Chloroform-d) .delta. 8.49 (s, 2H), 7.02 (dd, J=8.6, 0.4 Hz, 1H),
6.55-6.44 (m, 2H), 3.78 (s, 2H). MS Expected: 240, Found: 240
[0173] Compound
N-((4-((5-chloropyrimidin-2-yl)oxy)-3-fluorophenyl)carbamoyl)-4-(trifluor-
omethyl)benzamide 27 was prepared from
4-((5-chloropyrimidin-2-yl)oxy)-3-fluoroaniline and
4-(trifluoromethyl)benzoyl isocyanate according to the procedure
described for the synthesis of compound vi. .sup.1H NMR (400 MHz,
DMSO-d6) .delta. 11.36 (s, 1H), 10.78 (s, 1H), 8.80 (s, 2H), 8.20
(d, J 8.4 Hz, 2H), 7.94 (d, J=8.4 Hz, 2H), 7.79 (dd, J=12.0, 4.0
Hz, 2H), 7.47-7.35 (m, 2H). MS Expected: 455, Found: 455
[0174] Compound 28: Compound
N-((3-methyl-4-(pyrazin-2-yloxy)phenyl)-carbamoyl)-4-(trifluoromethyl)ben-
zamide 28 was prepared from 3-methyl-4-(pyrazin-2-yloxy)aniline and
4-(trifluoromethyl)benzoyl isocyanate according to the procedure
described for the synthesis of compound vi. .sup.1H NMR (400 MHz,
DMSO-d6) .delta. 11.29 (s, 1H), 10.68 (s, 1H), 8.58-8.53 (m, 1H),
8.36 (d, J 2.7 Hz, 1H), 8.24-8.15 (m, 3H), 7.93 (d, J=8.4 Hz, 2H),
7.57-7.47 (m, 2H), 7.13 (d, J 8.6 Hz, 1H), 2.11 (s, 3H). MS
Expected: 417, Found: 417
[0175] Compound 29: Compound
N-((4-((2-chloropyrimidin-5-yl)oxy)-3-methylphenyl)carbamoyl)-4-(trifluor-
omethyl)benzamide 29 was prepared from
4-((2-chloropyrimidin-5-yl)oxy)-3-methylaniline and
4-(trifluoromethyl)benzoyl isocyanate according to the procedure
described for the synthesis of compound vi. .sup.1H NMR (400 MHz,
DMSO-d6) .delta. 11.29 (s, 1H), 10.70 (s, 1H), 8.50 (s, 2H), 8.20
(d, J=8.1 Hz, 2H), 7.91 (d, J=8.1 Hz, 2H), 7.60 (d, J=2. Hz, 1H),
7.13 (d, J 8.6 Hz, 1H), 2.23 (s, 3H). MS Expected: 451, Found:
451
[0176] The foregoing discussion of the invention has been presented
for purposes of illustration and description. The foregoing is not
intended to limit the invention to the form or forms disclosed
herein. Although the description of the invention has included
description of one or more embodiments and certain variations and
modifications, other variations and modifications are within the
scope of the invention, e.g., as may be within the skill and
knowledge of those in the art, after understanding the present
disclosure. It is intended to obtain rights which include
alternative embodiments to the extent permitted, including
alternate, interchangeable and/or equivalent structures, functions,
ranges or steps to those claimed, whether or not such alternate,
interchangeable and/or equivalent structures, functions, ranges or
steps are disclosed herein, and without intending to publicly
dedicate any patentable subject matter. All references cited herein
are incorporated by reference in their entirety.
* * * * *