U.S. patent application number 14/946368 was filed with the patent office on 2016-05-26 for novel anti-cancer agents.
The applicant listed for this patent is The Walter and Eliza Hall Institute of Medical Research. Invention is credited to Antony Wilks Burgess, Guillaume Lessene, Francesca Walker, Keith Geoffrey Watson, Helen Witchard.
Application Number | 20160145243 14/946368 |
Document ID | / |
Family ID | 45992970 |
Filed Date | 2016-05-26 |
United States Patent
Application |
20160145243 |
Kind Code |
A1 |
Burgess; Antony Wilks ; et
al. |
May 26, 2016 |
Novel Anti-Cancer Agents
Abstract
The present invention provides a compound of Formula (I), or a
pharmaceutical acceptable derivative, salt or prodrug thereof.
Further provided is a method of treatment of cancer in a subject
comprising administering to said subject an effective amount of a
compound of Formula (I), or a pharmaceutical acceptable derivative,
salt or prodrug thereof. Further provided is the use of a compound
of Formula (I), or a pharmaceutical acceptable derivative, salt or
prodrug thereof in the preparation of a medicament for the
treatment of cancer. In addition, the present invention also
provides a pharmaceutical composition comprising a compound of
Formula (I), or a pharmaceutical acceptable derivative, salt or
prodrug thereof.
Inventors: |
Burgess; Antony Wilks;
(Camberwell, AU) ; Walker; Francesca; (West
Brunswick, AU) ; Watson; Keith Geoffrey; (Surrey
Hills, AU) ; Witchard; Helen; (Engadine, AU) ;
Lessene; Guillaume; (Coburg, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Walter and Eliza Hall Institute of Medical Research |
Parkville |
|
AU |
|
|
Family ID: |
45992970 |
Appl. No.: |
14/946368 |
Filed: |
November 19, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14453502 |
Aug 6, 2014 |
9221799 |
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14946368 |
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13878557 |
Jun 11, 2013 |
8835629 |
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PCT/AU2011/001376 |
Oct 27, 2011 |
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14453502 |
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61407265 |
Oct 27, 2010 |
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Current U.S.
Class: |
514/245 ;
544/198 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 31/53 20130101; C07D 403/12 20130101 |
International
Class: |
C07D 403/12 20060101
C07D403/12 |
Claims
1. A compound of Formula I, or a pharmaceutical acceptable
derivative, salt or prodrug thereof, wherein: ##STR00058## E and G
are each independently selected from the group consisting of
C.sub.1-4alkyl, --NH--, --N(C.sub.1-4alkyl)-, --O-- and, in either
orientation, --NH--C.sub.1-4alkyl-,
--N(C.sub.1-4alkyl)-C.sub.1-4alkyl-, and --O--C.sub.1-4alkyl-; and
is a single or double bond; when is a single bond, K is
independently selected from CH and N, and J is independently
selected from NH and CH.sub.2; or when is a double bond, K is C,
and J is independently selected from N and CH; R.sup.1 is 0-2
substituents wherein each substituent is independently selected
from the group consisting of --C.sub.1-4alkyl,
--C.sub.3-6cycloalkyl, --OH, --O--C.sub.1-4alkyl, N(R.sup.4).sub.2,
--C.sub.1-4alkylN(R.sup.4).sub.2,
--O--C.sub.1-4alkyl-N(R.sup.4).sub.2,
--C.sub.3-6cycloalkyl-N(R.sup.4).sub.2, --O-phenyl, --O-benzyl,
--NO.sub.2, halogen, and CF.sub.3; each R.sup.4 is independently
selected from the group consisting of H, OH, --C.sub.1-4alkyl,
--C(O)OC.sub.1-4alkyl, --C.sub.1-4alkyl-OR.sup.7, and
--C(O)R.sup.5, provided that if one R.sup.4 is OH then the other
R.sup.4 cannot be OH; or --N(R.sup.4).sub.2 forms a pyrrolidinyl,
piperidinyl, piperazinyl, or morpholino group optionally
substituted with C.sub.1-4alkyl; R.sup.2 is 0-2 substituents
wherein each substituent is independently selected from the group
consisting of --C.sub.1-4alkyl, --C.sub.3-6cycloalkyl, --OH,
--O--C.sub.1-4alkyl, --N(R.sup.6).sub.2,
--C.sub.1-4alkylN(R.sup.6).sub.2,
--O--C.sub.1-4alkyl-N(R.sup.6).sub.2,
--C.sub.3-6cycloalkyl-N(R.sup.6).sub.2, --O-phenyl, --O-benzyl,
--NO.sub.2, halogen, and CF.sub.3; each R.sup.6 is independently
selected from the group consisting of --H, --OH, --C.sub.1-4alkyl,
--C(O)OC.sub.1-4alkyl, --C.sub.1-4alkyl-OR.sup.7, and
--C(O)R.sup.8, provided that if one R.sup.6 is OH then the other
R.sup.6 cannot be OH; or --N(R.sup.6).sub.2 forms a pyrrolidinyl,
piperidinyl, piperazinyl, or morpholino group optionally
substituted with C.sub.1-4alkyl; wherein each of R.sup.5 and
R.sup.8 are independently selected from the group consisting of
--C.sub.1-4alkyl and phenyl; R.sup.3 is selected from the group
consisting of H, --C.sub.1-4alkyl, aryl, and alkylaryl; and wherein
R.sup.7 is selected from the group consisting of --H and
--C.sub.1-4alkyl.
2. A compound according to claim 1 of Formula II or a
pharmaceutical derivative, salt or prodrug thereof, wherein:
##STR00059## E and G are each independently selected from the group
consisting of C.sub.1-4alkyl, --NH--, --O-- and, in either
orientation --NH--C.sub.1-4alkyl-, and --O--C.sub.1-4alkyl-; and
R.sup.1 is 0-2 substituents wherein each substituent is
independently selected from the group consisting of
--C.sub.1-4alkyl, --C.sub.3-6cycloalkyl, --OH, --O--C.sub.1-4alkyl,
N(R.sup.4).sub.2, --C.sub.1-4alkylN(R.sup.4).sub.2,
--O--C.sub.1-4alkyl-N(R.sup.4).sub.2,
--C.sub.3-6cycloalkyl-N(R.sup.4).sub.2, --O-phenyl, --O-benzyl,
--NO.sub.2, halogen, and CF.sub.3; each R.sup.4 is independently
selected from the group consisting of H, OH, --C.sub.1-4alkyl,
--C(O)OC.sub.1-4alkyl, --C.sub.1-4alkyl-OR.sup.7, and
--C(O)R.sup.5, provided that if one R.sup.4 is OH then the other
R.sup.4 cannot be OH; or N(R.sup.4).sub.2 forms a pyrrolidinyl,
piperidinyl, piperazinyl, or morpholino group optionally
substituted with C.sub.1-4alkyl; R.sup.2 is 0-2 substituents
wherein each substituent is independently selected from the group
consisting of --C.sub.1-4alkyl, --C.sub.3-6cycloalkyl, --OH,
--O--C.sub.1-4alkyl, --N(R.sup.6).sub.2,
--C.sub.1-4alkylN(R.sup.6).sub.2,
--O--C.sub.1-4alkyl-N(R.sup.6).sub.2,
--C.sub.3-6cycloalkyl-N(R.sup.6).sub.2, --O-phenyl, --O-benzyl,
--NO.sub.2, halogen, and CF.sub.3; each R.sup.6 is independently
selected from the group consisting of --H, --OH, --C.sub.1-4alkyl,
--C(O)OC.sub.1-4alkyl, --C.sub.1-4alkyl-OR.sup.7, and
--C(O)R.sup.8, provided that if one R.sup.6 is OH then the other
R.sup.6 cannot be OH; or --N(R.sup.6).sub.2 forms a pyrrolidinyl,
piperidinyl, piperazinyl, or morpholino group optionally
substituted with C.sub.1-4alkyl; wherein each of R.sup.5 and
R.sup.8 are independently selected from the group consisting of
--C.sub.1-4alkyl and phenyl; R.sup.3 is selected from the group
consisting of H, C.sub.1-4alkyl, and aryl; wherein R.sup.7 is
selected from the group consisting of --H and --C.sub.1-4alkyl.
3. A compound according to claim 1 of Formula III or a
pharmaceutical derivative, salt or prodrug thereof, wherein:
##STR00060## E and G are each independently selected from the group
consisting of C.sub.1-4alkyl, --NH--, --O-- and, in either
orientation, --NH--C.sub.1-4alkyl-, and --O--C.sub.1-4alkyl-;
R.sup.1 is 0-2 substituents wherein each substituent is
independently selected from the group consisting of
--C.sub.1-4alkyl, --C.sub.3-6cycloalkyl, --OH, --O--C.sub.1-4alkyl,
--N(R.sup.4).sub.2, --C.sub.1-4alkylNHR.sup.4,
--O--C.sub.1-4alkyl-N(R.sup.4).sub.2,
--C.sub.3-6cycloalkyl-N(R.sup.4).sub.2, --O-phenyl, --O-benzyl,
--NO.sub.2, halogen, and CF.sub.3; wherein each R.sup.4 is
independently selected from the group consisting of --H, --OH,
C.sub.1-4alkyl, --C(O)OC.sub.1-4alkyl, --C.sub.1-4alkyl-OR.sup.7,
and --C(O)R.sup.5, provided that if one R.sup.4 is OH then the
other R.sup.4 cannot be OH; or --N(R.sup.4).sub.2 forms a
pyrrolidinyl, piperidinyl, piperazinyl, or morpholino group
optionally substituted with C.sub.1-4alkyl; R.sup.2 is 0-2
substituents wherein each substituent is independently selected
from the group consisting of --C.sub.1-4alkyl,
--C.sub.3-6cycloalkyl, --OH, --O--C.sub.1-4alkyl,
--N(R.sup.6).sub.2, --C.sub.1-4alkylN(R.sup.6).sub.2,
--O--C.sub.1-4alkyl-N(R.sup.6).sub.2,
--C.sub.3-6cycloalkyl-N(R.sup.6).sub.2, --O-phenyl, --O-benzyl,
--NO.sub.2, halogen, and CF.sub.3; each R.sup.6 is independently
selected from the group consisting of --H, --OH, --C.sub.1-4alkyl,
--C(O)OC.sub.1-4alkyl, --C.sub.1-4alkyl-OR.sup.7, and
--C(O)R.sup.8, provided that if one R.sup.6 is OH then the other
R.sup.6 cannot be OH; or --N(R.sup.6).sub.2 forms a pyrrolidinyl,
piperidinyl, piperazinyl, or morpholino group optionally
substituted with C.sub.1-4alkyl; wherein each of R.sup.5 and
R.sup.8 are independently selected from the group consisting of
--C.sub.1-4alkyl and phenyl; R.sup.3 is selected from the group
consisting of H, C.sub.1-4alkyl, and aryl; and wherein R.sup.7 is
selected from the group consisting of --H and --C.sub.1-4alkyl.
4. A compound according to claim 1 wherein each of E and G are both
independently selected from --NHC.sub.1-4alkyl.
5. A compound according to claim 4 wherein the heteroatoms of E and
G are both bonded to the triazine ring.
6. A compound according to claim 1 wherein R.sup.1 and R.sup.2 are
each independently 1-2 substituents.
7. A compound according to claim 1, wherein each of R.sup.1 and
R.sup.2 is at least one para substituent.
8. A compound according to claim 1, wherein each R.sup.1
substituent is independently selected from the group consisting of
--OH, --O--C.sub.1-4alkyl, and --C.sub.1-4alkyl
9. A compound according to claim 1, wherein each R.sup.2
substituent is independently selected from the group consisting of
--OH, O--C.sub.1-4alkyl, and --C.sub.1-4alkylN(R.sup.6).sub.2
10. A compound according to claim 1, wherein R.sup.3 is selected
from the group consisting of H, methyl, propyl, butyl and
phenyl.
11. A compound according to claim 1, wherein R.sup.3 is
hydrogen.
12. A compound according to claim 1 selected from the group
consisting of: ##STR00061## ##STR00062## ##STR00063## ##STR00064##
##STR00065## ##STR00066## ##STR00067## ##STR00068##
13. A compound according to claim 12 selected from the group
consisting of: ##STR00069##
14. A method of treatment of cancer in a subject comprising
administering to said subject an effective amount of a compound
according to claim 1 or a pharmaceutically acceptable derivative,
salt or prodrug thereof.
15. A method according to claim 14 wherein the cancer is colon
cancer, non-small lung cancer, brain cancer or breast cancer
16. A pharmaceutical composition comprising a compound according to
claim 1 or a pharmaceutically acceptable carrier, diluent or
excipient.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a class of compounds useful
in the treatment of cancer, such as colon cancer, lung cancer,
brain cancer and breast cancer.
BACKGROUND OF THE INVENTION
[0002] Cancer is a major disease that is a leading cause of death
worldwide. A major aspect of the treatment of cancer is
chemotherapy using anti-cancer agents. However, the treatment of
cancer using chemotherapy is rarely straightforward and there is a
general need to develop new and improved anti-cancer agents which
act by different mechanisms and pathways.
SUMMARY OF THE INVENTION
[0003] In a first aspect, the present invention provides a compound
of Formula I, or a pharmaceutical acceptable derivative, salt or
prodrug thereof, wherein:
##STR00001##
E and G are each independently selected from the group consisting
of --C.sub.1-4alkyl-, --NH--, --N(C.sub.1-4alkyl)-, --O-- and, in
either orientation, --NH--C.sub.1-4alkyl-, --N(C.sub.1-4
alkyl)-C.sub.1-4 alkyl-, and --O--C.sub.1-4 alkyl-; and is a single
or double bond; [0004] when is a single bond, K is independently
selected from CH and N, and J is independently selected from NH and
CH.sub.2; or [0005] when is a double bond, K is C, and J is
independently selected from N and CH; R.sup.1 is 0-2 substituents
wherein each substituent is independently selected from the group
consisting of --C.sub.1-4alkyl, --C.sub.3-6cycloalkyl, --OH,
--O--C.sub.1-4alkyl, --N(R.sup.4).sub.2,
--C.sub.1-4alkylN(R.sup.4).sub.2,
--O--C.sub.1-4alkyl-N(R.sup.4).sub.2,
--C.sub.3-6cycloalkyl-N(R.sup.4).sub.2, --O-phenyl, --O-benzyl,
--NO.sub.2, halogen, and CF.sub.3; [0006] each R.sup.4 is
independently selected from the group consisting of --H, --OH,
--C.sub.1-4alkyl, --C(O)OC.sub.1-4alkyl, --C.sub.1-4alkyl-OR.sup.7,
and --C(O)R.sup.5, provided that if one R.sup.4 is --OH then the
other R.sup.4 cannot be --OH; or [0007] --N(R.sup.4).sub.2 forms a
pyrrolidinyl, piperidinyl, piperazinyl, or morpholino group
optionally substituted with --C.sub.1-4alkyl; R.sup.2 is 0-2
substituents wherein each substituent is independently selected
from the group consisting of --C.sub.1-4alkyl,
--C.sub.3-6cycloalkyl, --OH, --O--C.sub.1-4alkyl,
--N(R.sup.6).sub.2, --C.sub.1-4alkylN(R.sup.6).sub.2,
--O--C.sub.1-4alkyl-N(R.sup.6).sub.2,
--C.sub.3-6cycloalkyl-N(R.sup.6).sub.2, --O-phenyl, --O-benzyl,
--NO.sub.2, halogen, and --CF.sub.3; [0008] each R.sup.6 is
independently selected from the group consisting of --H, --OH,
--C.sub.1-4alkyl, --C(O)OC.sub.1-4alkyl, --C.sub.1-4alkyl-OR.sup.7,
and --C(O)R.sup.8, provided that if one R.sup.6 is --OH then the
other R.sup.6 cannot be --OH; or [0009] --N(R.sup.6).sub.2 forms a
pyrrolidinyl, piperidinyl, piperazinyl, or morpholino group
optionally substituted with --C.sub.1-4alkyl; [0010] wherein each
of R.sup.5 and R.sup.8 are independently selected from the group
consisting of --C.sub.1-4alkyl and -phenyl; R.sup.3 is selected
from the group consisting of H, --C.sub.1-4alkyl, aryl, and
alkylaryl; and wherein R.sup.7 is selected from the group
consisting of --H and --C.sub.1-4alkyl.
[0011] In a second aspect, the present invention provides a method
of treatment of cancer in a subject comprising administering to
said subject an effective amount of a compound of the present
invention or a pharmaceutically acceptable derivative, salt or
prodrug thereof.
[0012] In a third aspect, the present invention provides the use of
a compound of the present invention or a pharmaceutically
acceptable derivative, salt or prodrug thereof in the preparation
of a medicament for the treatment of cancer.
[0013] In a fourth aspect, the present invention provides a
pharmaceutical composition comprising a compound of the present
invention or a pharmaceutically acceptable derivative, salt or
prodrug thereof and a pharmaceutically acceptable carrier, diluent
or excipient.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1: Determination of stabilized vs soluble tubulin in
cells treated with G2/M blockers. LIMI215 cells treated with
vehicle, Taxol or compound 3 (198-02) were solubilized in NP-40.
Equal volumes of supernatant and resuspended insoluble material
were analyzed by SDS/PAGE and immunoblotting with antibodies to
.beta.-tubulin. S=soluble and P=polymerized tubulin. Similar
results were obtained on the BaF/3 cell line.
[0015] FIG. 2: Effect of compounds of the present invention on "in
vitro" tubulin polymerization. Tubulin (1 mg/ml in
PIPES/MgCl.sub.2/GTP buffer) was pre-equilibrated in the cuvette of
a Cary 50 spectrophotometer before addition of buffer (A), Taxol
(B), 0.5 mM triazine (C) or 2 mM triazine (D), (arrow). OD[340]
readings were taken at 5 s intervals for 30 min.
[0016] FIG. 3: Representative chromatograms of extracted mouse
plasma. A: Normal mouse plasma with HC. B: Normal mouse plasma
spiked with Compound 2 (156-01) and HC for the standard curve. C:
Plasma obtained 2 hours after injection of Compound 2 (156-01)
shows the recovered Compound 2 (156-01) peak as well as 3 other
putative metabolite peaks.
[0017] FIG. 4: Spectral analysis of putative metabolite peaks. A.
Overlaid chromatograms of standard compound 2 (156-01) in plasma
(dotted line) and the plasma of an injected mouse (solid line). B.
UV spectra of the purified peaks.
[0018] FIG. 5: Anti-mitotic effects of metabolites recovered from
mouse plasma after compound 2 injection. BaF/3 cells were incubated
for 24 hr with increasing amounts of each reconstituted peak. Cell
cycle distribution was assessed by FACS analysis using Propidium
Iodide. The percentage of cells in the G2/M phase of the cell cycle
was determined using ModFi
[0019] FIG. 6: Plots of plasma concentrations of compound 2 in mice
following a single subcutaneous dose of 50 mg/kg. Each point
corresponds to an individual mouse. A: Semi-log plot of calculated
concentrations of Peak 1 values. B: Semi-log plot of values
including all metabolite peaks. C: Mean and se values comparing
Peak 1 only with all peaks.
[0020] FIG. 7: LIM2537 tumor growth in nude mice. Tumor diameters
were measured with external callipers every three days and tumor
volume calculated with the formula: .pi./6.times.(L.times.W.sup.2).
Clear difference between vehicle control treated animals (black
curve) and those treated with compound 2 (red curve) at 15 mg/kg,
three days a week starting on day 3 of the experiment.
[0021] FIG. 8: U87MG(.DELTA.2-7) tumor growth in nude mice. Tumor
diameters were measured with external callipers every three days
and tumor volume calculated with the formula:
.pi./6.times.(L.times.W2). Clear difference between vehicle control
treated animals (black curve) and those treated with compound 2 at
20 mg/kg, three times a week starting on day 5 of the
experiment.
[0022] FIG. 9: Animals treated with compound 2 (labelled "156-01")
(green bars) showed no significant difference in their liver or
spleen weights from vehicle control treated animals (red) whereas
the tumor weight was greatly reduced.
[0023] FIG. 10: H1437 tumor growth in nude mice. Tumor diameters
were measured with external callipers every three days and tumor
volume calculated with the formula: .pi./6.times.(L.times.W2).
Clear difference between vehicle control treated animals (black
curve) and those treated with compound 2 at 20 mg/kg, three times a
week starting on day 5 of experiment.
[0024] FIG. 11: Photograph of the H1437 xenograft tumors removed
from the vehicle control treated animal (top three rows) and
compound 2 treated (20 mg/kg, three times per week) bottom row. A
clear difference in size can be seen.
DETAILED DESCRIPTION OF THE INVENTION
[0025] Naamidine A, an imidazole alkaloid extracted from Leucetta
sponges, has been reported to selectively inhibit the epidermal
growth factor (EGF) signalling pathway and to inhibit tumour growth
in a mouse xenograft model using a human cancer cell line that
greatly over expresses the EGF receptor (Copp et al., J. Med. Chem.
1998, 41:3909).
##STR00002##
[0026] In a search for EGF-receptor-specific small molecules
inhibitors, the present inventors synthesised a number of Naamidine
A analogues and assessed their biological activity.
[0027] Surprisingly, the present inventors identified a class of
molecules related to Naamidine A that show cytostatic and/or
cytotoxic activity and which do not act through the EGF signalling
pathway. This class of triazine compounds show promise as
anti-cancer agents with anticancer activity in the nanomolar
range.
[0028] Accordingly, in a first aspect, the present invention
provides a compound of formula I, or a pharmaceutical acceptable
derivative, salt or prodrug thereof, wherein:
##STR00003##
E and G are each independently selected from the group consisting
of --C.sub.1-4alkyl-, --NH--, --N(C.sub.1-4alkyl)-, --O-- and, in
either orientation, --NH--C.sub.1-4alkyl-,
--N(C.sub.1-4alkyl)-C.sub.1-4alkyl-, and --O--C.sub.1-4alkyl-; and
is a single or double bond; [0029] when is a single bond, K is
independently selected from CH and N, and J is independently
selected from NH and CH.sub.2; or [0030] when is a double bond, K
is C, and J is independently selected from N and CH; R.sup.1 is 0-2
substituents (meaning 0, 1, or 2 substituents or a range comprising
any of two of those integers) wherein each substituent is
independently selected from the group consisting of
--C.sub.1-4alkyl, --C.sub.3-6cycloalkyl, --OH, --O--C.sub.1-4alkyl,
--N(R.sup.4).sub.2, --C.sub.1-4alkylN(R.sup.4).sub.2,
--O--C.sub.1-4alkyl-N(R.sup.4).sub.2,
--C.sub.3-6cycloalkyl-N(R.sup.4).sub.2, --O-phenyl, --O-benzyl,
--NO.sub.2, halogen, and --CF.sub.3; [0031] each R.sup.4 is
independently selected from the group consisting of --H, --OH,
--C.sub.1-4alkyl, --C(O)OC.sub.1-4alkyl, --C.sub.1-4alkyl-OR.sup.7,
and --C(O)R.sup.5, provided that if one R.sup.4 is --OH then the
other R.sup.4 cannot be --OH; or [0032] --N(R.sup.4).sub.2 forms a
pyrrolidinyl, piperidinyl, piperazinyl, or morpholino group
optionally substituted with --C.sub.1-4alkyl; R.sup.2 is 0-2
substituents (meaning 0, 1, or 2 substituents or a range comprising
any of two of those integers) wherein each substituent is
independently selected from the group consisting of
--C.sub.1-4alkyl, --C.sub.3-6cycloalkyl, --OH, --O--C.sub.1-4alkyl,
--N(R.sup.6).sub.2, --C.sub.1-4 alkylN(R.sup.6).sub.2,
--O--C.sub.1-4alkyl-N(R.sup.6).sub.2,
--C.sub.3-6cycloalkyl-N(R.sup.6).sub.2, --O-phenyl, --O-benzyl,
--NO.sub.2, halogen, and --CF.sub.3; [0033] each R.sup.6 is
independently selected from the group consisting of --H, --OH,
--C.sub.1-4alkyl, --C(O)OC.sub.1-4alkyl, --C.sub.1-4alkyl-OR.sup.7,
and --C(O)R.sup.8, provided that if one R.sup.6 is --OH then the
other R.sup.6 cannot be --OH; or [0034] --N(R.sup.6).sub.2 forms a
pyrrolidinyl, piperidinyl, piperazinyl, or morpholino group
optionally substituted with --C.sub.1-4alkyl; [0035] wherein each
of R.sup.5 and R.sup.8 are independently selected from the group
consisting of --C.sub.1-4alkyl and -phenyl; R.sup.3 is selected
from the group consisting of H, --C.sub.1-4alkyl, -aryl, and
-alkylaryl; and wherein R.sup.7 is selected from the group
consisting of --H and --C.sub.1-4alkyl.
[0036] In one embodiment, is a single bond and (i) K is CH and J is
NH, or (ii) K is N and J is CH.sub.2.
[0037] In another embodiment, is a double bond and K is CH and J is
NH.
[0038] Preferably, R.sup.3 is selected from the group consisting of
hydrogen, methyl, propyl, butyl, and phenyl.
[0039] In one embodiment, E is selected from the group consisting
of --NH--C.sub.1-4alkyl-, --N(C.sub.1-4alkyl)-C.sub.1-4alkyl-, and
--O--C.sub.1-4alkyl-; wherein the heteroatom of E is bonded to the
triazine ring.
[0040] In one embodiment, G is selected from the group consisting
of --NH--C.sub.1-4alkyl-, --N(C.sub.1-4alkyl)-C.sub.1-4alkyl-, and
--O--C.sub.1-4alkyl-; wherein the heteroatom of G is bonded to the
triazine ring.
[0041] Preferably, E is --NHC.sub.1-4alkyl, more preferably E is
--NH--CH.sub.2--.
[0042] Preferably, G is --NHC.sub.1-4alkyl, more preferably G is
--NH--CH.sub.2--.
[0043] In one embodiment where R.sup.1 is --N(R.sup.4).sub.2 which
forms a piperazinyl group substituted with --C.sub.1-4alkyl, of the
formula:
##STR00004##
[0044] Preferably, R.sup.1 and R.sup.2 are each independently 1-2
substituents, more preferably R.sup.1 and R.sup.2 are each 1
substituent.
[0045] In one embodiment, each of R.sup.1 and R.sup.2 is at least
one para substituent.
[0046] Preferably, each R.sup.1 substituent is independently
selected from the group consisting of --OH, --O--C.sub.1-4alkyl,
and --C.sub.1-4alkyl, more preferably, each R.sup.1 substituent is
independently selected from the group consisting --OH, --OMe, and
--CH.sub.3.
[0047] Preferably, each R.sup.2 substituent is independently
selected from the group consisting of --OH, O--C.sub.1-4alkyl, and
--C.sub.1-4alkylN(R.sup.6).sub.2.
[0048] More preferably, each R.sup.2 substituent is independently
selected from the group consisting of --OH, --OMe,
--CH.sub.2NH.sub.2, and --CH.sub.2NH--C(O)O(t-Bu).
[0049] In a preferred embodiment, the present invention provides a
compound of Formula II or a pharmaceutical derivative, salt or
prodrug thereof, wherein:
##STR00005##
E and G are each independently selected from the group consisting
of --C.sub.1-4alkyl-, --NH--, --O-- and, in either orientation
--NH--C.sub.1-4alkyl-, and --O--C.sub.1-4alkyl-; and R.sup.1 is 0-2
substituents wherein each substituent is independently selected
from the group consisting of --C.sub.1-4alkyl,
--C.sub.3-6cycloalkyl, --OH, --O--C.sub.1-4alkyl,
--N(R.sup.4).sub.2, --C.sub.1-4alkylN(R.sup.4).sub.2,
--O--C.sub.1-4alkyl-N(R.sup.4).sub.2,
--C.sub.3-6cycloalkyl-N(R.sup.4).sub.2, --O-phenyl, --O-benzyl,
--NO.sub.2, halogen, and --CF.sub.3; [0050] each R.sup.4 is
independently selected from the group consisting of --H, --OH,
--C.sub.1-4alkyl, --C(O)OC.sub.1-4alkyl, --C.sub.1-4alkyl-OR.sup.7,
and --C(O)R.sup.5, provided that if one R.sup.4 is --OH then the
other R.sup.4 cannot be --OH; or [0051] --N(R.sup.4).sub.2 forms a
pyrrolidinyl, piperidinyl, piperazinyl, or morpholino group
optionally substituted with --C.sub.1-4alkyl; R.sup.2 is 0-2
substituents wherein each substituent is independently selected
from the group consisting of --C.sub.1-4alkyl,
--C.sub.3-6cycloalkyl, --OH, --O--C.sub.1-4 alkyl,
--N(R.sup.6).sub.2, --C.sub.1-4alkylN(R.sup.6).sub.2,
--O--C.sub.1-4alkyl-N(R.sup.6).sub.2,
--C.sub.3-6cycloalkyl-N(R.sup.6).sub.2, --O-phenyl, --O-benzyl,
--NO.sub.2, halogen, and --CF.sub.3; [0052] each R.sup.6 is
independently selected from the group consisting of --H, --OH,
--C.sub.1-4alkyl, --C(O)OC.sub.1-4alkyl, --C.sub.1-4alkyl-OR.sup.7,
and --C(O)R.sup.8, provided that if one R.sup.6 is --OH then the
other R.sup.6 cannot be --OH; or [0053] --N(R.sup.6).sub.2 forms a
pyrrolidinyl, piperidinyl, piperazinyl, or morpholino group
optionally substituted with --C.sub.1-4alkyl; [0054] wherein each
of R.sup.5 and R.sup.8 are independently selected from the group
consisting of --C.sub.1-4alkyl and -phenyl; R.sup.3 is selected
from the group consisting of --H, --C.sub.1-4alkyl, and -aryl;
wherein R.sup.7 is selected from the group consisting of --H and
--C.sub.1-4alkyl.
[0055] In one embodiment, E is selected from the group consisting
of --NH--C.sub.1-4alkyl-, --N(C.sub.1-4alkyl)-C.sub.1-4alkyl-, and
--O--C.sub.1-4alkyl-; wherein the heteroatom of E is bonded to the
triazine ring.
[0056] In one embodiment, G is selected from the group consisting
of --NH--C.sub.1-4alkyl-, --N(C.sub.1-4alkyl)-C.sub.1-4alkyl-, and
--O--C.sub.1-4alkyl-; wherein the heteroatom of G is bonded to the
triazine ring.
[0057] Preferably, E is --NHC.sub.1-4alkyl, more preferably, E is
--NH--CH.sub.2--.
[0058] Preferably G is --NHC.sub.1-4alkyl, more preferably, G is
--NH--CH.sub.2--.
[0059] In one embodiment the present invention provides a compound
of formula II wherein R.sup.1 is 1-2 substituents wherein each
substituent is independently selected from the group consisting of
--C.sub.1-4alkyl, --OH, --O--C.sub.1-4alkyl, and
--C.sub.1-4alkylNHR.sup.4; and R.sup.2 is 1-2 substituents wherein
each substituent is independently selected from the group
consisting of --C.sub.1-4alkyl, --OH, --O--C.sub.1-4alkyl, and
--C.sub.1-4alkylNHR.sup.6 [0060] wherein R.sup.4 is selected from
the group consisting of --H, --C.sub.1-4alkyl,
--C(O)OC.sub.1-4alkyl; [0061] wherein R.sup.6 is selected from the
group consisting of --H, --C.sub.1-4alkyl,
--C(O)OC.sub.1-4alkyl;
[0062] Preferably, each R.sup.1 substituent is independently
selected from the group consisting of --OH, --OMe, --CH.sub.3 and
each R.sup.2 substituent is independently selected from the group
consisting of OH, --O--C.sub.1-4alkyl, --C.sub.1-4alkylNHR.sup.6;
[0063] wherein R.sup.6 is selected from the group consisting of
--H, --C.sub.1-4alkyl, and --C(O)OC.sub.1-4alkyl.
[0064] More preferably, R.sup.2 is selected from the group
consisting of --OH, --OMe, --CH.sub.2NH.sub.2, and
--CHNH--C(O)O(t-Bu).
[0065] In another embodiment, the present invention provides a
compound of formula II wherein R.sup.3 is selected from the group
consisting of --H, methyl, propyl, butyl and phenyl.
[0066] In one embodiment, R.sup.3 is a straight alkyl chain.
[0067] In one embodiment, the present invention provides a compound
of Formula III or a pharmaceutical derivative, salt or prodrug
thereof, wherein:
##STR00006##
E and G are each independently selected from the group consisting
of --C.sub.1-4alkyl-, --NH--, --O-- and, in either orientation,
--NH--C.sub.1-4alkyl-, and --O--C.sub.1-4alkyl-; R.sup.1 is 0-2
substituents wherein each substituent is independently selected
from the group consisting of --C.sub.1-4alkyl,
--C.sub.3-6cycloalkyl, --OH, --O--C.sub.1-4alkyl,
--N(R.sup.4).sub.2, --C.sub.1-4alkylNHR.sup.4,
--O--C.sub.1-4alkyl-N(R.sup.4).sub.2,
--C.sub.3-6cycloalkyl-N(R.sup.4).sub.2, --O-phenyl, --O-benzyl,
--NO.sub.2, halogen, and --CF.sub.3; [0068] wherein each R.sup.4 is
independently selected from the group consisting of --H, --OH,
C.sub.1-4alkyl, --C(O)OC.sub.1-4alkyl, --C.sub.1-4alkyl-OR.sup.7,
and --C(O)R.sup.5, provided that if one R.sup.4 is --OH then the
other R.sup.4 cannot be --OH; or [0069] --N(R.sup.4).sub.2 forms a
pyrrolidinyl, piperidinyl, piperazinyl, or morpholino group
optionally substituted with --C.sub.1-4alkyl; R.sup.2 is 0-2
substituents wherein each substituent is independently selected
from the group consisting of --C.sub.1-4alkyl,
--C.sub.3-6cycloalkyl, --OH, --O--C.sub.1-4 alkyl,
--N(R.sup.6).sub.2, --C.sub.1-4alkylN(R.sup.6).sub.2,
--O--C.sub.1-4alkyl-N(R.sup.6).sub.2,
--C.sub.3-6cycloalkyl-N(R.sup.6).sub.2, --O-phenyl, --O-benzyl,
--NO.sub.2, halogen, and --CF.sub.3; [0070] each R.sup.6 is
independently selected from the group consisting of --H, --OH,
--C(O)OC.sub.1-4alkyl, --C.sub.1-4alkyl-OR.sup.7, and
--C(O)R.sup.8, provided that if one R.sup.6 is --OH then the other
R.sup.6 cannot be --OH; or [0071] --N(R.sup.6).sub.2 forms a
pyrrolidinyl, piperidinyl, piperazinyl, or morpholino group
optionally substituted with --C.sub.1-4alkyl; [0072] wherein each
of R.sup.5 and R.sup.8 are independently selected from the group
consisting of --C.sub.1-4alkyl and -phenyl; R.sup.3 is selected
from the group consisting of --H, --C.sub.1-4alkyl, and -aryl; and
wherein R.sup.7 is selected from the group consisting of --H and
--C.sub.1-4alkyl,
[0073] In one embodiment, E is selected from the group consisting
of --NH--C.sub.1-4alkyl-, --N(C.sub.1-4alkyl)-C.sub.1-4alkyl-, and
--O--C.sub.1-4alkyl-; wherein the heteroatom of E is bonded to the
triazine ring.
[0074] In one embodiment, G is selected from the group consisting
of --NH--C.sub.1-4alkyl-, --N(C.sub.1-4alkyl)-C.sub.1-4alkyl-, and
--O--C.sub.1-4alkyl-; wherein the heteroatom of G is bonded to the
triazine ring.
[0075] Preferably E is --NHC.sub.1-4alkyl, more preferably E is
--NH--CH.sub.2--.
[0076] Preferably G is --NHC.sub.1-4alkyl, more preferably G is
--NH--CH.sub.2--.
[0077] In one embodiment the present invention provides a compound
of formula III wherein R.sup.1 is 1-2 substituents wherein each
substituent is independently selected from the group consisting of
--O--C.sub.1-4alkyl, and --C.sub.1-4alkylN(R.sup.4).sub.2; and
R.sup.2 is 1-2 substituents wherein each substituent is
independently selected from the group consisting of
--O--C.sub.1-4alkyl, and --C.sub.1-4alkylN(R.sup.6).sub.2; [0078]
wherein each R.sup.4 is independently selected from the group
consisting of --H, --C.sub.1-4alkyl, --C(O)OC.sub.1-4alkyl; [0079]
wherein each R.sup.6 is independently selected from the group
consisting of --H, --C.sub.1-4alkyl, --C(O)OC.sub.1-4alkyl;
[0080] Preferably, R.sup.1 is --OMe and R.sup.2 is selected from
the group consisting --O--C.sub.1-4alkyl, and
--C.sub.1-4alkylN(R.sup.6).sub.2; [0081] wherein each R.sup.6 is
independently selected from the group consisting of --H,
--C.sub.1-4alkyl, and --C(O)OC.sub.1-4alkyl.
[0082] More preferably, R.sup.2 is selected from the group
consisting of --OMe, --CH.sub.2NH.sub.2, and
--CHNH--C(O)O(t-Bu).
[0083] Preferably, R.sup.3 is hydrogen.
[0084] In one embodiment, the present invention provides a compound
of Formula IV or a pharmaceutical derivative, salt or prodrug
thereof, wherein:
##STR00007##
E and G are each independently selected from the group consisting
of --C.sub.1-4alkyl, --NH--, --O-- and, in either orientation,
--NH--C.sub.1-4alkyl-, and --O--C.sub.1-4alkyl-; R.sup.1 is 0-2
substituents wherein each substituent is independently selected
from the group consisting of --C.sub.1-4 alkyl,
--C.sub.3-6cycloalkyl, --OH, --O--C.sub.1-4alkyl, N(R.sup.4).sub.2,
--C.sub.1-4alkylNHR.sup.4, --O--C.sub.1-4 alkyl-N(R.sup.4).sub.2,
--O--C.sub.1-4alkyl-N(R.sup.4).sub.2,
--C.sub.3-6cycloalkyl-N(R.sup.4).sub.2, --O-phenyl, --O-benzyl,
--NO.sub.2, halogen, and --CF.sub.3; [0085] wherein each R.sup.4 is
independently selected from the group consisting of --H, --OH,
C.sub.1-4alkyl, --C(O)OC.sub.1-4alkyl, --C.sub.1-4alkyl-OR.sup.7,
and --C(O)R.sup.5; or [0086] --N(R.sup.4).sub.2 forms a
pyrrolidinyl, piperidinyl, piperazinyl, or morpholino group
optionally substituted with --C.sub.1-4alkyl; R.sup.2 is 0-2
substituents wherein each substituent is independently selected
from the group consisting of --C.sub.1-4alkyl,
--C.sub.3-6cycloalkyl, --OH, --O--C.sub.1-4alkyl, N(R.sup.6).sub.2,
--C.sub.1-4alkylN(R.sup.6).sub.2,
--O--C.sub.1-4alkyl-N(R.sup.6).sub.2,
--C.sub.3-6cycloalkyl-N(R.sup.6).sub.2, --O-phenyl, --O-benzyl,
--NO.sub.2, halogen, and --CF.sub.3; [0087] each R.sup.6 is
independently selected from the group consisting of --H, --OH,
--C.sub.1-4alkyl, --C(O)OC.sub.1-4alkyl, --C.sub.1-4alkyl-OR.sup.7,
and --C(O)R.sup.8, provided that if one R.sup.6 is --OH then the
other R.sup.6 cannot be --OH; or [0088] --N(R.sup.6).sub.2 forms a
pyrrolidinyl, piperidinyl, piperazinyl, or morpholino group
optionally substituted with --C.sub.1-4alkyl; [0089] wherein each
of R.sup.5 and R.sup.8 are independently selected from the group
consisting of --C.sub.1-4alkyl and -phenyl; R.sup.3 is selected
from the group consisting of --H, --C.sub.1-4alkyl, and -aryl; and
wherein R.sup.7 is selected from the group consisting of --H and
--C.sub.1-4alkyl.
[0090] In one embodiment, E is selected from the group consisting
of --NH--C.sub.1-4alkyl-, --N(C.sub.1-4alkyl)-C.sub.1-4alkyl-, and
--O--C.sub.1-4alkyl-; wherein the heteroatom of E is bonded to the
triazine ring.
[0091] In one embodiment, G is selected from the group consisting
of --NH--C.sub.1-4alkyl-, --N(C.sub.1-4alkyl)-C.sub.1-4alkyl-, and
--O--C.sub.1-4alkyl-; wherein the heteroatom of G is bonded to the
triazine ring.
[0092] Preferably E is --NHC.sub.1-4alkyl, more preferably E is
--NH--CH.sub.2--.
[0093] Preferably G is --NHC.sub.1-4alkyl, more preferably G is
--NH--CH.sub.2--.
[0094] In one embodiment the present invention provides a compound
of formula IV wherein R.sup.1 is 1-2 substituents wherein each
substituent is independently selected from the group consisting of
--O--C.sub.1-4alkyl, and --C.sub.1-4alkylN(R.sup.4).sub.2; and
R.sup.2 is 1-2 substituents wherein each substituent is
independently selected from the group consisting of
--O--C.sub.1-4alkyl, and --C.sub.1-4alkylN(R.sup.6).sub.2; [0095]
wherein each R.sup.4 is independently selected from the group
consisting of --H, --C.sub.1-4alkyl, --C(O)OC.sub.1-4alkyl; [0096]
wherein each R.sup.6 is independently selected from the group
consisting of --H, --C.sub.1-4alkyl, --C(O)OC.sub.1-4alkyl;
[0097] Preferably, R.sup.1 is --OMe and R.sup.2 is selected from
the group consisting --O--C.sub.1-4alkyl, and --C.sub.1-4
alkylN(R.sup.6).sub.2; [0098] wherein each R.sup.6 is independently
selected from the group consisting of --H, --C.sub.1-4alkyl, and
--C(O)OC.sub.1-4alkyl.
[0099] More preferably, R.sup.2 is selected from the group
consisting of --OMe, --CH.sub.2NH.sub.2, and
--CHNH--C(O)O(t-Bu).
[0100] In another embodiment, the present invention provides a
compound of formula IV wherein R.sup.3 is selected from the group
consisting of --H, methyl, propyl, butyl and phenyl. Preferably,
R.sup.3 is hydrogen.
[0101] In one embodiment the present invention provides a compound
selected from the group consisting of:
##STR00008## ##STR00009## ##STR00010## ##STR00011## ##STR00012##
##STR00013## ##STR00014## ##STR00015##
[0102] Preferably, the compound is selected from the group
consisting of:
##STR00016##
[0103] Chang et al (US 2004/0122009) have synthesized triazine
compounds having antiproliferative effects similar to that of
myoseverin. The Chang compounds are structurally distinct from the
compounds of the present invention and are less active against
cancer cell lines by orders of magnitude. In addition, the
compounds disclosed in Chang et at have strikingly different
biological activity to the compounds of the present invention. In
their studies, Chang et al have found that their compounds show a
close correlation between the IC.sub.50 values required for tubulin
polymerisation and for growth inhibition of U937 human leukemia
cells (see Table 1, page 25). By contrast, the present inventors
have found that, for compounds of the present invention, the
concentration required for tubulin polymerisation was more than
1000 times greater than the IC.sub.50 in cell proliferation
assays.
[0104] As used herein, the term "halo" or "halogen" refers to
fluorine (fluoro), chlorine (chloro), bromine (bromo) or iodine
(iodo).
[0105] As used herein, the term "alkyl" either used alone or in
compound terms such as NH(alkyl) or N(alkyl).sub.2, refers to
monovalent straight chain or branched hydrocarbon groups. For
example, suitable alkyl groups include, but are not limited to
methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, and
tert-butyl.
[0106] As understood by a person skilled in the art, the term
"C.sub.1-4alkyl" means a straight or branched chain with 1, 2, 3 or
4 carbon atoms or a range comprising any of two of those
integers.
[0107] The term "aryl" as used herein, refers to a C.sub.6-C.sub.10
aromatic hydrocarbon group, for example phenyl or naphthyl.
[0108] The term "cycloalkyl" as used herein, refers to cyclic
hydrocarbon groups. Suitable cycloalkyl groups include, but are not
limited to cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
[0109] As understood by a person skilled in the art, the term
"C.sub.3-6cycloalkyl" means a cycloalkyl group with 3, 4, 5, or 6
carbon atoms or a range comprising any of two of those
integers.
[0110] The term "alkylaryl" includes, for example, benzyl.
[0111] Amine nitrogens may be protected by suitable nitrogen
protecting groups (see "Protective Groups in Organic Synthesis"
Theodora Greene and Peter Wuts, third edition, Wiley Interscience,
1999).
[0112] The salts of the compound of formula I are preferably
pharmaceutically acceptable, but it will be appreciated that
non-pharmaceutically acceptable salts also fall within the scope of
the present invention, since these are useful as intermediates in
the preparation of pharmaceutically acceptable salts.
[0113] The term "pharmaceutically acceptable derivative" may
include any pharmaceutically acceptable salt, hydrate or prodrug,
or any other compound which upon administration to a subject, is
capable of providing (directly or indirectly) a compound of formula
I or an antibacterially active metabolite or residue thereof.
[0114] Suitable pharmaceutically acceptable salts include, but are
not limited to, salts of pharmaceutically acceptable inorganic
acids such as hydrochloric, sulphuric, phosphoric, nitric,
carbonic, boric, sulfamic, and hydrobromic acids, or salts of
pharmaceutically acceptable organic acids such as acetic,
propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric,
malic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic,
phenylacetic, methanesulphonic, toluenesulphonic, benzenesulphonic,
salicylic, sulphanilic, aspartic, glutamic, edetic, stearic,
palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric
acids.
[0115] Base salts include, but are not limited to, those formed
with pharmaceutically acceptable cations, such as sodium,
potassium, lithium, calcium, magnesium, zinc, ammonium;
alkylammonium (such as salts formed from triethylamine) and
alkoxyammonium (such as those formed with ethanolamine). Base salts
also include salts formed from ethylenediamine, choline; and amino
acids (such as arginine, lysine or histidine). General information
on types of pharmaceutically acceptable salts and their formation
is known to those skilled in the art and is as described in general
texts such as "Handbook of Pharmaceutical Salts" P. H. Stahl, C. G.
Wermuth, 1.sup.st edition, 2002, Wiley-VCH.
[0116] Basic nitrogen-containing groups may be quarternised with
such agents as lower alkyl halide, such as methyl, ethyl, propyl,
and butyl chlorides, bromides and iodides; dialkyl sulfates like
dimethyl and diethyl sulfate; and others.
[0117] Hydroxyl groups may be esterified with groups including
lower alkyl carboxylic acids, such as acetic acid and
2,2-dimethylpropionic acid, sulfonated with groups including alkyl
sulfonic acids, such as methyl sulfonic acid or glycosylated to
form glucuronide derivatives.
[0118] This invention also encompasses pharmaceutical compositions
containing prodrugs of compounds of formula I. This invention also
encompasses methods of treating or preventing cancer in a subject
by administering prodrugs of compounds of the formula I. Compounds
of formula I having free amino, amido, hydroxy or carboxylic groups
can be converted into prodrugs.
[0119] Prodrugs include compounds wherein an amino acid residue, or
a polypeptide chain of two or more (eg, two, three or four) amino
acid residues which are covalently joined to free amino, hydroxy
and carboxylic acid groups of compounds of formula I. The amino
acid residues include the 20 naturally occurring amino acids
commonly designated by three letter symbols and also include,
4-hydroxyproline, hydroxylysine, demosine, isodemosine,
3-methylhistidine, norvlin, beta-alanine, gamma-aminobutyric acid,
citrulline, homocysteine, homoserine, ornithine and methionine
sulfone. Prodrugs also include compounds wherein carbonates,
carbamates, amides and alkyl esters which are covalently bonded to
the above substituents of formula I through the carbonyl carbon
prodrug sidechain. Prodrugs also include phosphate derivatives of
compounds of formula I (such as acids, salts of acids, or esters)
joined through a phosphorus-oxygen bond to a free hydroxyl of
compounds of formula I.
[0120] It will also be recognised that the compounds of formula I
may possess asymmetric centres and are therefore capable of
existing in more than one stereoisomeric form. The invention thus
also relates to compounds in substantially pure isomeric form at
one or more asymmetric centres eg., greater than about 90% ee, such
as about 95% or 97% ee or greater than 99% ee, as well as mixtures,
including racemic mixtures, thereof.
[0121] Such isomers may be prepared by asymmetric synthesis, for
example using chiral intermediates, or by chiral resolution.
[0122] In one embodiment the present invention provides a method of
treatment of cancer in a subject comprising administering to said
subject an effective amount of a compound of the present invention
or a pharmaceutically acceptable derivative, salt or prodrug
thereof.
[0123] In another embodiment the present invention provides the use
of a compound of the present invention in the preparation of a
medicament for the treatment of cancer.
[0124] In another embodiment the present invention provides a
compound for use in the treatment of cancer.
[0125] Preferably, the cancer is colon cancer, non-small lung
cancer, brain cancer or breast cancer.
[0126] The present inventors found that triazines of the present
invention are at least as effective as Taxol in inducing G2/M block
at their optimal concentration. However, whereas Taxol acts by
stabilising tubulin polymerization, further experiments showed that
triazine-induced tubulin polymerization was minimal and occurred at
doses far exceeding their potency as mitotic blockers.
[0127] Therefore, in one embodiment the cancer is resistant to
taxanes, for example paclitaxel, docetaxel and carbazitaxel.
[0128] In one embodiment the cancer is resistant to mitotic
inhibitors, wherein the mitotic inhibitors directly interfere with
the assembly and disassembly of tubulin.
[0129] In one embodiment the present invention provides a
pharmaceutical composition comprising a compound of the present
invention and a pharmaceutically acceptable carrier, diluent or
excipient.
[0130] The compositions of the present invention may contain other
therapeutic agents as described below, and may be formulated, for
example, by employing conventional solid or liquid vehicles or
diluents, as well as pharmaceutical additives of a type appropriate
to the mode of desired administration (for example, excipients,
binders, preservatives, stabilizers, flavours, etc.) according to
techniques such as those well known in the art of pharmaceutical
formulation.
[0131] The compounds of the present invention may be administered
by any suitable means, for example, parenterally, such as by
subcutaneous, intravenous, intramuscular, or intracisternal
injection or infusion techniques (e.g., as sterile injectable
aqueous or non-aqueous solutions or suspensions).
[0132] Pharmaceutical formulations include those for oral, rectal,
nasal, topical (including buccal and sub-lingual), vaginal or
parenteral (including intramuscular, sub-cutaneous and intravenous)
administration or in a form suitable for administration by
inhalation or insufflation. The compounds of the invention,
together with a conventional adjuvant, carrier or diluent, may thus
be placed into the form of pharmaceutical compositions and unit
dosages thereof, and in such form may be employed as solids, such
as tablets or filled capsules, or liquids as solutions,
suspensions, emulsions, elixirs or capsules filled with the same,
all for oral use, in the form of suppositories for rectal
administration; or in the form of sterile injectable solutions for
parenteral (including subcutaneous) use.
[0133] In addition to primates, such as humans, a variety of other
mammals can be treated according to the method of the present
invention. For instance, mammals including, but not limited to,
cows, sheep, goats, horses, dogs, cats, guinea pigs, rats or other
bovine, ovine, equine, canine, feline, rodent or murine species can
be treated. However, the method can also be practiced in other
species, such as avian species (e.g., chickens).
[0134] The subjects treated in the above method are mammals,
including, but not limited to, cows, sheep, goats, horses, dogs,
cats, guinea pigs, rats or other bovine, ovine, equine, canine,
feline, rodent or murine species, and preferably a human being,
male or female.
[0135] The term "effective amount" means the amount of the subject
composition that will elicit the biological or medical response of
a tissue, system, animal or human that is being sought by the
researcher, veterinarian, medical doctor or other clinician.
[0136] As would be understood by those skilled in the art of
treating cancer, the term "treatment" does not necessarily mean
that the cancer is completely cured. The term "treatment"
encompasses any inhibition of replication of cancer cells and/or
reduction in the tumor size in the subject being treated.
[0137] The term "composition" as used herein is intended to
encompass a product comprising the specified ingredients in the
specified amounts, as well as any product which results, directly
or indirectly, from combination of the specified ingredients in the
specified amounts. By "pharmaceutically acceptable" it is meant the
carrier, diluent or excipient must be compatible with the other
ingredients of the formulation and not deleterious to the recipient
thereof.
[0138] The terms "administration of" and or "administering a"
compound should be understood to mean providing a compound of the
invention to the individual in need of treatment.
[0139] The pharmaceutical compositions for the administration of
the compounds of this invention may conveniently be presented in
dosage unit form and may be prepared by any of the methods well
known in the art of pharmacy. All methods include the step of
bringing the active ingredient into association with the carrier
which constitutes one or more accessory ingredients. In general,
the pharmaceutical compositions are prepared by uniformly and
intimately bringing the active ingredient into association with a
liquid carrier or a finely divided solid carrier or both, and then,
if necessary, shaping the product into the desired formulation. In
the pharmaceutical composition the active object compound is
included in an amount sufficient to produce the desired effect upon
the process or condition of diseases. As used herein, the term
"composition" is intended to encompass a product comprising the
specified ingredients in the specified amounts, as well as any
product which results, directly or indirectly, from combination of
the specified ingredients in the specified amounts.
[0140] The pharmaceutical compositions may be in the form of a
sterile injectable aqueous or oleagenous suspension. This
suspension may be formulated according to the known art using those
suitable dispersing or wetting agents and suspending agents which
have been mentioned above. The sterile injectable preparation may
also be a sterile injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example as a
solution in 1,3-butane diol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose any bland fixed oil may be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid find use in the preparation of injectables.
[0141] The pharmaceutical composition and method of the present
invention may further comprise other therapeutically active
compounds which are usually applied in the treatment of the above
mentioned pathological conditions. Selection of the appropriate
agents for use in combination therapy may be made by one of
ordinary skill in the art, according to conventional pharmaceutical
principles. The combination of therapeutic agents may act
synergistically to effect the treatment or prevention of the
various disorders described above. Using this approach, one may be
able to achieve therapeutic efficacy with lower dosages of each
agent, thus reducing the potential for adverse side effects.
[0142] When other therapeutic agents are employed in combination
with the compounds of the present invention they may be used for
example in amounts as noted in the Physician Desk Reference (PDR)
or as otherwise determined by one of ordinary skill in the art.
[0143] In the treatment or prevention of conditions which require
inhibition of replication of cancer cells and/or reduction in the
tumor size, an appropriate dosage level will generally be about
0.01 to 500 mg per kg patient body weight per day which can be
administered in single or multiple doses. Preferably, the dosage
level will be about 0.1 to about 250 mg/kg per day; more preferably
about 0.5 to about 100 mg/kg per day. A suitable dosage level may
be about 0.01 to 250 mg/kg per day, about 0.05 to 100 mg/kg per
day, or about 0.1 to 50 mg/kg per day. Within this range the dosage
may be 0.05 to 0.5, 0.5 to 5 or 5 to 50 mg/kg per day. For oral
administration, the compositions are preferably provided in the
form of tablets containing 1.0 to 1000 milligrams of the active
ingredient, particularly 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0,
75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0,
750.0, 800.0, 900.0, and 1000.0 milligrams of the active ingredient
for the symptomatic adjustment of the dosage to the patient to be
treated. The compounds may be administered on a regimen of 1 to 4
times per day, preferably once or twice per day.
[0144] It will be understood, however, that the specific dose level
and frequency of dosage for any particular patient may be varied
and will depend upon a variety of factors including the activity of
the specific compound employed, the metabolic stability and length
of action of that compound, the age, body weight, general health,
sex, diet, mode and time of administration, rate of excretion, drug
combination, the severity of the particular condition, and the host
undergoing therapy.
[0145] Throughout this specification the word "comprise", or
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated element, integer or step, or
group of elements, integers or steps, but not the exclusion of any
other element, integer or step, or group of elements, integers or
steps.
[0146] Any discussion of documents, acts, materials, devices,
articles or the like which has been included in the present
specification is not to be taken as an admission that any or all of
these matters form part of the prior art base or were common
general knowledge in the field relevant to the present invention as
it existed before the priority date of each claim of this
application.
[0147] In order that the nature of the present invention may be
more clearly understood preferred forms thereof will now be
described by reference to the following non-limiting Examples.
EXAMPLES
Example 1
Synthetic Chemistry
[0148] A rapid, high-yield, synthesis protocol was developed which
allowed modification of the 1,3,5-triazine scaffold to yield a
range of trazine substituted derivatives
Example 1.1
Synthetic method for the preparation of
1-[4,6-Bis-(4-methoxybenzylamino)-{1,3,5}-triazin-2-yl-amino]-imidazolidi-
ne-2,4-dione (compound 1)
Part A Preparation of
1-(4,6-dichloro-[1,3,5]-triazin-2-yl-amino)-imidazolidine-2,4-dione
##STR00017##
[0150] An ice-cold suspension of 1-aminohydantoin hydrochloride
(457 mg, 3.0 mmol), cyanuric chloride (500 mg, 2.73 mmol) and
sodium bicarbonate (505 mg, 6 mmol) in acetonitrile (5 mL) was
stirred under an atmosphere of nitrogen for 2 h and then stirred at
room temperature for 20 h. The reaction mixture was evaporated to
dryness under reduced pressure and the residue was extracted with
ethyl acetate and the ethyl acetate was separated and dried
(MgSO.sub.4). The crude product (770 mg) gave a single spot on thin
layer chromatography [silica; CH.sub.2Cl.sub.2/MeOH (10:1)] and was
used in the next step without further purification. .sup.1H NMR
(CD.sub.3CN): .delta.=4.11 (s, 2H); 8.71 (s, 1H); 8.85 (s, 1H).
Part B Preparation of
1-[4,6-Bis-(4-methoxybenzylamino)-{1,3,5}-triazin-2-yl-amino]-imidazolidi-
ne-2,4-dione (compound 1)
##STR00018##
[0152] To a stirred suspension of
1-(4,6-dichloro-[1,3,5]-triazin-2-yl-amino)-imidazolidine-2,4-dione
(770 mg, 2.93 mmol) and potassium carbonate (2.42 g, 17.6 mmol) in
acetonitrile (20 mL) at room temperature was added
4-methoxybenzylamine (0.96 mL, 7.32 mmol). The reaction mixture was
stirred for 24 h at room temperature under an atmosphere of
nitrogen and then the solvent was removed under reduced pressure.
The residue was extracted with ethyl acetate and the ethyl acetate
was dried (MgSO.sub.4) and evaporated to give the crude product
which was purified by flash chromatography on silica using
CH.sub.2Cl.sub.2/MeOH (50:1.fwdarw.10:1) as eluent. The pure
product was isolated as a colourless solid (587 mg, 43%). .sup.1H
NMR (d.sub.6 DMSO): .delta.=3.67 (s, 6H); 3.96 and 4.05 (2 s, total
2H); 4.20 and 4.32 (2 s, total 4H); 6.80 (bs, 4H); 7.13-7.39 (m,
6H); 8.67, 8.85 and 8.99 (2 s, total 1H); 11.0 (bs, 1H). Mass
spectrum (ES+) 465 (M+H).
Example 1.2
Synthetic method for the preparation of
1-[4-(4-aminomethyl-benzylamino)-6-(4-methoxybenzylamino)-{1,3,5}-triazin-
-2-yl-amino]-imidazolidine-2,4-dione, HCl salt (compound 2)
Part A Preparation of 1-[4-chloro-6-(4-N-Boc-aminomethyl)-benzyl
amino-{1,3,5}-triazin-2-yl-amino]-imidazolidine-2,4-dione
##STR00019##
[0154] To a stirred suspension of
1-(4,6-dichloro-[1,3,5]-triazin-2-yl-amino)-imidazolidine-2,4-dione
(Example 1.1, Part A)(590 mg, 2.24 mmol) and potassium carbonate
(681 mg, 4.94 mmol) in dry dimethylformamide (DMF, 10 mL) was added
4-(N-Boc-aminomethyl)-benzylamine (582 mg, 2.47 mmol). The mixture
was stirred at room temperature for 20 h and then poured into a
mixture of water and ethyl acetate. The resultant emulsion was
filtered and then the organic layer was separated, dried and
evaporated to give the crude product (576 mg, 55%) as a yellow
solid. Mass spectrum (ESI+): 946 (2M+Na), 924 (2M+H).
Part B Preparation of
1-[4-(4-aminomethyl-benzylamino)-6-(4-methoxybenzylamino)-{1,3,5}-triazin-
-2-yl-amino]-imidazolidine-2,4-dione, HCl salt (compound 2)
##STR00020##
[0156] To a stirred suspension of
1-[4-chloro-6-(4-N-Boc-aminomethyl)-benzylamino-{1,3,5}-triazin-2-yl-amin-
o]-imidazolidine-2,4-dione (Example 1.2, Part A) (200 mg, 0.43
mmol) and potassium carbonate (131 mg, 0.95 mmol) in DMF (5 mL) was
added 4-methoxybenzylamine (0.06 mL, 0.48 mmol). The reaction
mixture was stirred at 35.degree. for 18 h and then poured into
water and extracted with ethyl acetate. The resultant emulsion was
filtered and the ethyl acetate layer was separated and evaporated
to give the N-Boc product as an oily residue (90 mg, 37%). Mass
spectrum (ESI+): 1127 (2M+H), 564 (M+H). The N-Boc material (90 mg)
was dissolved in dichloromethane/trifluoroacetic acid (1:1, 3 mL)
and stirred at room temperature under nitrogen for 2 h. The solvent
and excess TFA was removed under reduced pressure and the residue
was dissolved in methanol (5 mL) and treated with a solution of
hydrogen chloride in diethyl ether (0.08 mL of a 2M solution). The
methanol was removed under reduced pressure and the residue was
recrystallised from methanol/acetonitrile to give the crude
hydrochloride salt as an off-white solid (49 mg, 62%). .sup.1H NMR
(d.sub.6 DMSO): .delta.=3.72 (s, 3H); 3.96-4.12 (m, 6H); 4.32-4.52
(m, 4H); 6.84 (brs, 2H); 7.16-7.51 (m, 6H); 8.4 and 8.5 (brd, 6H);
11.3 (s, 1H). Mass spectrum (ES+): 464 (M+H).
Example 1.3
Synthetic Methods for the Preparation of
Triazine-Imidazolidine-Diones
Preparation of
5-[4,6-Bis-(4-methoxy-benzylamino)-[1,3,5]-triazin-2-ylamino]-3-methyl-im-
idazolidine-2,4-dione (compound 3)
Part A Preparation of 1-Methyl-imidazolidine-2,4,5-trione
##STR00021##
[0158] To an ice-cold solution of 1-methylurea (200 mg, 2.70 mmol)
in dry tetrahydrofuran (6 mL) was added oxalyl chloride (0.26 mL,
2.97 mmol) dropwise, under a nitrogen atmosphere. The reaction
mixture was stirred at 0-5.degree. for 2 h and then allowed to warm
to room temperature for 1 h before being diluted with water. The
product was extracted with ethyl acetate, and the extracts were
washed with water, dried and evaporated to give the product as an
off-white solid (326 mg, 94%), mp 146-149.degree. C. (lit.
145-148.degree. C.).
Part B Preparation of
5-Benzylamino-3-methyl-imidazole-2,4-dione
##STR00022##
[0160] To a solution of 1-methyl-imidazolidine-2,4,5-trione
(Example 1.3, Part A) (800 mg, 6.25 mmol), imidazole (467 mg, 6.87
mmol), N,N-dimethylaminopyridine (cat.) and triethylamine (1.82 mL,
13.1 mmol) in chloroform (13 mL) was added chlorotrimethylsilane
(1.67 mL, 13.1 mmol) dropwise at room temperature under a nitrogen
atmosphere, and the reaction mixture was stirred for 2 h.
Benzylamine (0.75 mL, 6.87 mmol) was added and the reaction mixture
was stirred for a further 22.5 h. The reaction mixture was diluted
with chloroform and water and the chloroform extracts were dried
and evaporated. The crude product was purified by column
chromatography (PS with gradient elution to DCM:EtOAc 3:1) to give
the product as a colourless solid (838 mg, 62%), mp 152-154.degree.
C. .delta..sub.H (300 MHz, CD.sub.3CN) 7.95 (v br s, 1H, NH),
7.41-7.27 (m, 5H, PhH), 4.64 (s, 2H, CH.sub.2), 2.95 (s, 3H,
CH.sub.3). Mass spectrum (ESI) m/z 218 (M+H).
Part C Preparation of 5-Amino-3-methyl-imidazolidine-2,4-dione
##STR00023##
[0162] To a solution of 5-benzylamino-3-methyl-imidazole-2,4-dione
(Example 1.3, Part B) (400 mg, 1.84 mmol) in ethanol/ethyl acetate
(50 mL, 1:1) was added 10% Pd/C catalyst (195 mg, 0.18 mmol). The
flask was evacuated and filled with hydrogen three times and then
stirred at room temperature for 23 h. After this period, the
reaction mixture was filtered through celite and the filtrate was
evaporated to give the product as a colourless solid (214 mg, 90%),
mp 122-125.degree. C. .delta..sub.H(300 MHz, CD.sub.3CN) 6.31 (br
s, 1H, NH), 4.68 (d, J 1.5 Hz, 1H, CH), 2.87 (s, 3H, CH.sub.3).
.delta..sub.H (300 MHz, D.sub.2O) 4.95 (s, 1H, CH), 2.96 (s, 3H,
CH.sub.3). .delta..sub.C (75 MHz, D.sub.2O) 175.6, 158.5, 64.0
(CH), 24.3 (CH.sub.3).
Part D Preparation of
5-(4,6-Dichloro-[1,3,5]triazin-2-ylamino)-3-methyl-imidazolidine-2,4-dion-
e
##STR00024##
[0164] An ice-cold suspension of
5-amino-3-methyl-imidazolidine-2,4-dione (Example 1.3, Part C) (266
mg, 2.06 mmol), cyanuric chloride (415 mg, 2.27 mmol) and sodium
bicarbonate (191 mg, 2.27 mmol) in acetonitrile (15 mL) was stirred
over an ice bath under a nitrogen atmosphere for 1 h, and then
allowed to stir at room temperature for 22 h. The solvent was
evaporated, and the crude product was extracted with ethyl acetate.
The organic layer was dried and evaporated, and the crude product
was purified by column chromatography (PS with gradient elution to
DCM:EtOAc 1:1) to afford the product as a cream solid (448 mg,
78%). .delta..sub.H (300 MHz, CD.sub.3CN) 7.50 (br s, 1H, NH), 6.56
(br s, 1H, NH), 5.60 (dd, J 7.8, 1.8 Hz, 1H, CH), 2.97 (s, 3H,
CH.sub.3). Mass spectrum (ESI) m/z 259 [M+H (OH for Cl exchanged
ion)].
Part E Preparation of
5-[4,6-Bis-(4-methoxy-benzylamino)-[1,3,5]-triazin-2-ylamino]-3-methyl-im-
idazolidine-2,4-dione (compound 3)
##STR00025##
[0166] To a stirred suspension of
5-(4,6-dichloro-[1,3,5]triazin-2-ylamino)-3-methyl-imidazolidine-2,4-dion-
e (Example 1.3, Part D) (193 mg, 0.70 mmol) and potassium carbonate
(212 mg, 1.53 mmol) in acetonitrile (15 mL) at 5.degree. was added
4-methoxybenzylamine (0.10 mL, 0.77 mmol) dropwise. The suspension
was stirred at ice temperature for 1 h and then at room temperature
for 24 h. Water was added to the reaction mixture which was then
extracted with ethyl acetate. The crude product was applied to a
silica column and eluted, starting with a mixture of
dichloromethane:ethyl acetate (10:1), then 5:1 and eventually
dichloromethane:methanol (20:1). The early fractions yielded the
intermediate mono-substituted
5-[4-chloro-6-(4-methoxy-benzylamino)-[1,3,5]-triazin-2-ylamino]-3-methyl-
-imidazolidine-2,4-dione (133 mg, 51%) and later fractions gave the
di-benzylamino product (112 mg, 34%) (Found: C, 55.46; H, 4.66; N,
18.39%. C.sub.21H.sub.20N.sub.6O.sub.6 requires C, 55.75; H, 4.46;
N, 18.58). .delta..sub.H (300 MHz, CD.sub.3CN): 7.19 (br d, 4H,
Ar), 6.84 (br d, 4H, Ar), 6.35 (br s, 1H, NH), 5.9-5.8 (br, 3H,
NH), 5.46 (br d, 1H, CH), 4.36 (s, 4H, CH.sub.2), 3.74 (s, 6H,
OMe), 2.9-2.8 (br, 3H, NMe). Mass spectrum (ESI) m/z 479 [M+H].
Example 1.4
Preparation of
5-[4-(4-aminomethyl-benzylamino)-6-(4-methoxybenzylamino)-[1,3,5]-triazin-
-2-ylamino]-3-methyl-imidazolidine-2,4-dione (compound 4)
Part A
5-[4-chloro-6-(N-Boc-{4-aminomethyl}-benzylamino)-[1,3,5]-triazin-2-
-yl amino]-3-methyl-imidazolidine-2,4-dione
##STR00026##
[0168] To a stirred suspension of
5-(4,6-dichloro-[1,3,5]triazin-2-ylamino)-3-methyl-imidazolidine-2,4-dion-
e (Example 1.3, Part D) (300 mg, 1.08 mmol) and potassium carbonate
(329 mg, 2.38 mmol) in dimethylformamide (5 mL) at room temperature
was added N-Boc-(4-aminomethyl)-benzylamine (281 mg, 1.19 mmol).
The reaction mixture was stirred at room temperature for 4 h and
then diluted with water and ethyl acetate. The ethyl acetate layer
was separated, dried and evaporated to give the crude product which
was used directly in the next step.
Part B Preparation of
5-[4-{(N-Boc-4-aminomethyl)-benzylamino}-6-(4-methoxy
benzylamino)-[1,3,5]-triazin-2-ylamino]-3-methyl-imidazolidine-2,4-dione
(compound 4(B))
##STR00027##
[0170] To a stirred suspension of
5-[4-chloro-6-(N-Boc-{4-aminomethyl}-benzylamino)-[1,3,5]-triazin-2-yl
amino]-3-methyl-imidazolidine-2,4-dione (Example 1.4, Part A) (377
mg, 0.79 mmol) and potassium carbonate (240 mg, 1.74 mmol) in
dimethylformamide (5 mL) at 30.degree. was added
4-methoxybenzylamine (0.12 mL, 0.95 mmol). The reaction mixture was
stirred at 30.degree. for 18 h and then diluted with water and
extracted with ethyl acetate. The ethyl acetate layer was washed
with dilute hydrochloric acid and then separated, dried and
concentrated to give the crude product which was purified by
chromatography on silica gel using dichloromethane as initial
eluent and gradually increasing the polarity to
dichloromethane:methanol (20:1). The product was obtained as a
white solid (71 mg, 16%). NMR spectrum .delta..sub.H (300 MHz,
CD.sub.3CN): 7.16 (br s, 6H, Ar), 6.82 (br d, 2H, Ar), 6.5 (br s,
1H, NH), 6.1-5.7 (br m, 4H, NH), 5.47 (br d, 1H, CH), 4.5-4.3 (br,
4H, CH.sub.2), 4.15 (d, 2H, CH.sub.2) 3.73 (s, 3H, OMe), 2.9-23
(br, 3H, NMe), 1.39 (s, 9H, Boc).
Part C Preparation of
5-[4-(4-aminomethyl-benzylamino)-6-(4-methoxybenzylamino)-[1,3,5]-triazin-
-2-ylamino]-3-methyl-imidazolidine-2,4-dione (compound 4)
##STR00028##
[0172] Trifluoroacetic acid (2 mL) was added to a stirred solution
of
5-[4-{(N-Boc-4-aminomethyl)-benzylamino}-6-(4-methoxybenzylamino)-[1,3,5]-
-triazin-2-ylamino]-3-methyl-imidazolidine-2,4-dione (Example 1.4,
Part B) (63 mg, 0.11 mol) in dichloromethane (2 mL) at room
temperature. After 3 h the TFA and solvent were removed by
evaporation under reduced pressure and extra dichloromethane was
added and again removed to help drive off any remaining excess TFA.
The crude product was dissolved in dry methanol and a solution of
hydrogen chloride in ether (2M, 0.05 mL) was added. The reaction
mixture was stirred at room temperature for 15 minutes and then
concentrated and the residue was crystallized by triturating with
methanol/acetonitrile under ice cooling. The resultant pale pink
solid was collected by filtration to give the product as the HCl
salt (48 mg, 86%). NMR spectrum .delta..sub.H (300 MHz,
d.sub.6-DMSO): 8.9-8.2 (br m, 6H, NH), 7.4-7.1 (br m, 6H, Ar), 6.85
(d, 2H, Ar), 5.6 (br, 1H, CH), 4.5-4.2 (br, 4H, CH.sub.2), 3.95 (br
s, 2H, CH.sub.2) 3.70 (s, 3H, OMe), 2.8-2.6 (br, 3H, NMe); Mass
spectrum (ESI) m/z 478 [M+H].
Example 1.5
Preparation of
5-[4-(4-methyl-benzylamino)-6-(4-methoxybenzylamino)-[1,3,5]-triazin-2-yl-
amino]-3-methyl-imidazolidine-2,4-dione (compound 5)
Part A Preparation of
5-[4-chloro-6-(4-methyl-benzylamino)-[1,3,5]-triazin-2-ylamino]-3-methyl--
imidazolidine-2,4-dione
##STR00029##
[0174] To a stirred suspension of
5-(4,6-dichloro-[1,3,5]triazin-2-ylamino)-3-methyl-imidazolidine-2,4-dion-
e (Example 1.3, Part D) (200 mg, 0.72 mmol) and potassium carbonate
(219 mg, 1.59 mmol) in dimethylformamide (5 mL) at room temperature
was added 4-methyl-benzylamine (0.1 mL, 0.79 mmol). The reaction
mixture was stirred at 35.degree. for 24 h and then diluted with
water and ethyl acetate. The ethyl acetate layer was separated,
dried and evaporated to give the crude product which was used
directly in the next step.
Part B Preparation of
5-[4-(4-methyl-benzylamino)-6-(4-methoxybenzylamino)-[1,3,5]-triazin-2-yl-
amino]-3-methyl-imidazolidine-2,4-dione (compound 5)
##STR00030##
[0176] To a stirred suspension of
5-[4-chloro-6-(4-methyl-benzylamino)-[1,3,5]-triazin-2-ylamino]-3-methyl--
imidazolidine-2,4-dione (Example 1.5, Part A) (80 mg, 0.22 mmol)
and potassium carbonate (67 mg, 0.49 mmol) in dimethylformamide (5
mL) at 35.degree. was added 4-methoxybenzylamine (0.03 mL, 0.24
mmol). The reaction mixture was stirred at 35.degree. for 20 h and
then diluted with water and extracted with ethyl acetate. The ethyl
acetate layer was dried and concentrated to give the crude product
as a foam (79 mg, 77%). NMR spectrum .delta..sub.H (300 MHz,
CD.sub.3CN): 7.2-7.0 (br m, 7H, Ar+NH), 6.8 (br d, 2H, Ar), 6.5
(br, 1H, NH), 6.1-5.8 (br m, 2H, NH), 5.45 (br d, 1H, CH), 4.5-4.3
(br, 4H, CH.sub.2), 4.15 (d, 2H, CH.sub.2) 3.74 (s, 3H, OMe),
2.9-2.7 (br, 3H, NMe), 2.3 (s, 3H, Me). Mass spectrum (ESI) m/z 463
[M+H].
Example 1.6
Preparation of
5-[4,6-Bis-(4-methoxy-benzylamino)-[1,3,5]-triazin-2-ylamino]-3-n-butyl-i-
midazolidine-2,4-dione (compound 6)
##STR00031##
[0178] The title compound was prepared using essentially the same
reaction sequence and conditions as described in Example 13, Parts
B to E, but using 1-(n-butyl)-imidazolidine-2,4,5-trione in place
of the 1-methyl-imidazolidine-2,4,5-trione. The product from the
final step was purified by chromatography and characterized by NMR
and Mass spectroscopy. NMR spectrum .delta..sub.H (300 MHz,
CD.sub.3CN): 7.19 (br d, 4H, Ar), 6.84 (br d, 4H, Ar), 6.38 (br s,
1H, NH), 6.0-5.7 (br, 3H, NH), 5.5 (br d, 1H, CH), 4.36 (s, 4H,
CH.sub.2), 3.74 (s, 6H, OMe), 3.38 (t, 2H, NCH.sub.2); 1.52 (m, 2H,
CH.sub.2); 1.28 (m, 2H, CH.sub.2); 0.89 (d, 3H, Me). Mass spectrum
(ESI) m/z 521 [M+H].
Example 1.7
Preparation of
5-[4,6-Bis-(4-methoxy-benzylamino)-[1,3,5]-triazin-2-ylamino]-3-n-propyl--
imidazolidine-2,4-dione (compound 7)
##STR00032##
[0180] The title compound was prepared using essentially the same
reaction sequence and conditions as described in Example 1.3, Parts
B to E, but using 1-(n-propyl)-imidazolidine-2,4,5-trione in place
of the 1-methyl-imidazolidine-2,4,5-trione. The product from the
final step was purified by chromatography and characterized by NMR
and Mass spectroscopy. NMR spectrum .delta..sub.H (300 MHz,
d.sub.6DMSO): 8.31 (s, 1H, NH); 7.5 (br, 1H, NH); 7.1-7.3 (br, 6H,
Ar+NH), 6.83 (br d, 4H, Ar), 5.5 (br d, 1H, CH), 4.32 (s, 4H,
CH.sub.2), 3.78 (s, 6H, OMe), 3.34 (br, 2H, NCH.sub.2); 1.46 (m,
2H, CH.sub.2); 0.79 (br, 3H, Me). Mass spectrum (EST) m/z 507
[M+H].
Example 1.8
Preparation of
5-[4,6-Bis-(4-methoxy-benzylamino)-[1,3,5]-triazin-2-ylamino]-3-phenyl-im-
idazolidine-2,4-dione (compound 8)
##STR00033##
[0182] The title compound was prepared using essentially the same
reaction sequence and conditions as described in Example 1.3, Parts
B to E, but using 1-phenyl-imidazolidine-2,4,5-trione in place of
the 1-methyl-imidazolidine-2,4,5-trione. The product from the final
step was purified by chromatography and characterized by NMR and
Mass spectroscopy. NMR spectrum .delta..sub.H (300 MHz,
CD.sub.3CN): 8.07 (s, 1H, NH); 7.4-7.0 (br, 9H, Ar+NH); 6.9-6.6
(br, 5H, Ar+NH), 6.2-5.5 (br, 3H, NH+CH), 5.5 (br d, 1H, CH), 4.38
(s, 4H, CH.sub.2), 3.73 (s, 6H, OMe). Mass spectrum (ESI) m/z 541
[M+H].
Example 1.9
Preparation of
5-[4,6-Bis-(4-methoxy-phenoxy)-[1,3,5]triazin-2-ylamino]-3-methyl-imidazo-
lidine-2,4-dione (compound 9)
##STR00034##
[0184] A suspension of 5-amino-3-methyl-imidazolidine-2,4-dione
(Example 1.3, Part C) (80 mg, 0.29 mmol), p-methoxyphenol (39 mg,
0.32 mmol) and potassium carbonate (88 mg, 0.64 mmol) in
acetonitrile (5 mL) was stirred at room temperature under a
nitrogen atmosphere for 18 h. The reaction mixture was diluted with
ethyl acetate and water, and the crude product was extracted with
ethyl acetate. The organic layer was dried and evaporated, and the
crude product was purified by column chromatography (silica;
dichloromethane:ethyl acetate, with the solvent ratio increasing
from 10:1 to 1:2) and then recrystallized from acetonitrile to give
the title compound as a colourless solid (17 mg, 13%). (Found: C,
55.5; H, 4.7; N, 18.4%. C.sub.21H.sub.20N.sub.6O.sub.6 requires C,
55.8; H, 4.5; N, 18.6%). .delta..sub.H (300 MHz,
(CD.sub.3).sub.2CO) 7.81 (d, J 8.1 Hz, 1H, NH), 7.40 (s, 1H, NH),
7.08 (d, J 9.0 Hz, 2H, ArH (ortho to OPh attach)), 7.07 (d, J 9.0
Hz, 2H, Aril (ortho to OPh attach)), 6.92 (d, J 9.0 Hz, 4H, ArH
(ortho to OCH.sub.3 attach)), 5.73 (d, J 8.4 Hz, 1H, CH), 3.81 (s,
6H, OCH.sub.3), 2.77 (s, 3H, NCH.sub.3). Mass spectrum (ESI) m/z
453 (72%, MH.sup.+).
Example 1.10
Preparation of
5[4,6-Bis-(4-methoxy-phenylamino)-[1,3,5]triazin-2-ylamino]-3-methyl-imid-
azolidine-2,4-dione (compound 10)
Part A Preparation of
5-[4-Chloro-6-(4-methoxy-phenylamino)-[1,3,5]triazin-2-ylamino]-3-methyl--
imidazolidine-2,4-dione
##STR00035##
[0186] A suspension of 5-amino-3-methyl-imidazolidine-2,4-dione
(Example 1.3, Part C) (228 mg, 0.82 mmol), p-methoxyaniline (111
mg, 0.90 mmol) and sodium bicarbonate (76 mg, 0.90 mmol) in
acetonitrile (7 mL) was stirred at room temperature under a
nitrogen atmosphere for 25 h. The solvent was evaporated, and the
crude product was extracted with ethyl acetate. The organic layer
was dried and evaporated and the crude product was purified by
column chromatography (silica; dichloromethane:ethyl acetate
starting with a solvent ratio of 10:1 and increasing the polarity
to dichloromethane:methanol, 10:1) to afford the title compound as
a cream solid (288 mg, 96%). .delta..sub.H (300 MHz,
(CD.sub.3).sub.2CO) 8.88 (br s, 0.5H, NH), 8.84 (br s, 0.5H, NH),
7.87 and 7.70 (2 br s, 1H, NH), 7.61 (br s, 1H, NH), 7.51 (d, J 9.0
Hz, 2H, ArH (ortho to NH attach)), 6.91 and 6.89 (2 d, J 8.7, 6.6
Hz, 2H ArH (ortho to 0 attach)), 5.82 (br d, J 7.8 Hz, 1H, CH),
3.79 (s, 3H, OCH.sub.3), 2.95 (s, 1.4H, NCH.sub.3), 2.73 (s, 1.6H,
NCH.sub.3). Mass spectrum (ESI) m/z 364 (100%, MH.sup.+).
Part B Preparation of
5-[4,6-Bis-(4-methoxy-phenylamino)-[1,3,5]triazin-2-ylamino]-3-methyl-imi-
dazolidine-2,4-dione (compound 10)
##STR00036##
[0188] A suspension of
5-[4-chloro-6-(4-methoxy-phenylamino)-[1,3,5]triazin-2-ylamino]-3-methyl--
imidazolidine-2,4-di one (Example 1.10, Part A) (50 mg, 0.14 mmol),
p-methoxyaniline (25 mg, 0.21 mmol) and potassium carbonate (48 mg,
0.34 mmol) in anhydrous DMF (5 mL) was stirred under a nitrogen
atmosphere for 18.5 h. The reaction mixture was diluted with water
and the crude product was extracted with ethyl acetate. The organic
layer was dried and evaporated, and the crude product was purified
by column chromatography (silica; dichloromethane:ethyl acetate
starting with a solvent ratio of 10:1 and increasing the polarity
to dichloromethane:methanol, 10:1) to afford the product as a pale
yellow solid (36 mg, 58%). .delta..sub.H (300 MHz,
(CD.sub.3).sub.2CO) 8.14 (br s, 2H, NH), 7.60 (br s, 4H, ArH (ortho
to NH attach)), 7.43 (br s, 1H, NH), 6.91 (d, J 8.7 Hz, 1H, NH),
6.85 (d, J 9.0 Hz, 4H, ArH (ortho to O attach)), 5.91 (d, J 7.8 Hz,
1H, CH), 3.77 (s, 6H, OCH.sub.3), 2.86 (br s, 2.0H, NCH.sub.3),
2.73 (br s, 1.0H, NCH.sub.3). Mass spectrum (ESI) m/z 451 (37%,
MH.sup.+).
Example 1.11
Preparation of
5-[4-(4-Hydroxy-phenylamino)-6-(4-methoxy-phenylamino)-[1,3,5]triazin-2-y-
lamino]-3-methyl-imidazolidine-2,4-dione (compound 11)
##STR00037##
[0190] A suspension of
5-[4-Chloro-6-(4-methoxy-phenylamino)-[1,3,5]triazin-2-ylamino]-3-methyl--
imidazolidine-2,4-dione (Example 1.10, Part A) (76 mg, 0.05 mmol),
p-aminophenol (50 mg, 0.46 mol) and potassium acetate (25 mg, 0.25
mmol) in anhydrous DMF (3 mL) was stirred under a nitrogen
atmosphere over a 35.degree. C. oil bath for 22 h. The reaction
mixture was diluted with water, and the crude product was extracted
with ethyl acetate. The organic layer was dried and evaporated and
the crude product was purified by column chromatography (silica;
dichloromethane:ethyl acetate starting with a solvent ratio of 10:1
and increasing the polarity to dichloromethane:methanol, 10:1) to
afford the product as a fawn solid (24 mg, 33%). (Found: C, 54.1;
H, 4.9; N, 24.3%. C.sub.20H.sub.20N.sub.8O.sub.4.MeOH requires C,
53.8; H, 5.2; N, 23.9%). .delta..sub.H (300 MHz, CD.sub.3CN) 7.49
(br d, J 8.7 Hz, 2H, ArH (ortho to NH attach)), 7.37 (br s, 4H, NH
and ArH (ortho to NH attach)), 6.87 (d, J 9.0 Hz, 2H, ArH (ortho to
0 attach)), 6.75 (d, J 9.0 Hz, 2H, ArH (ortho to O attach)), 6.70
(br s, 1H, OH), 6.48 (s, 1H, NH), 6.10 (br d, J 7.8 Hz, 1H, NH),
5.67 (d, J 7.8 Hz, 1H, CH), 3.78 (s, 3H, OCH.sub.3), 2.88 (br s,
3H, NCH.sub.3). Mass spectrum (ESI) m/z 437 (36%, MH.sup.+).
Example 1.12
Preparation of
5-[4-(4-Benzyloxy-phenylamino)-6-(4-methoxy-phenylamino)-[1,3,5]triazin-2-
-ylamino]-3-methyl-imidazolidine-2,4-dione (compound 12)
##STR00038##
[0192] A suspension of
5-[4-Chloro-6-(4-methoxy-phenylamino)-[1,3,5]triazin-2-ylamino]-3-methyl--
imidazolidine-2,4-dione (Example 1.10, Part A) (93 mg, 0.26 mmol),
p-benzyloxyaniline hydrochloride (132 mg, 0.56 mmol) and cesium
carbonate (184 mg, 0.56 mmol) in anhydrous DMF (5 mL) was stirred
under a nitrogen atmosphere over a 30.degree. C. oil bath for 19 h.
The reaction mixture was diluted with water and the crude product
was extracted with ethyl acetate. The organic layer was dried and
evaporated and the crude product was purified by column
chromatography (silica; dichloromethane:ethyl acetate starting with
a solvent ratio of 10:1 and increasing the polarity to
dichloromethane:methanol, 20:1). Recrystallization from
PS/DCM/EtOAc gave the product as a cream solid (90 mg, 62%).
(Found: C, 61.5; H, 5.1; N, 21.2%. C.sub.27H.sub.26N.sub.8O.sub.4
requires C, 61.6; H, 5.0; N, 21.3%). .delta..sub.H (300 MHz,
CD.sub.3CN) 7.58-7.30 (m, 11H, ArH (ortho to NH attach), PhH and 2
NH), 6.94 (d, J 8.7 Hz, 2H, ArH (ortho to O attach)), 6.87 (d, J
8.7 Hz, 2H, ArH (ortho to O attach)), 6.49 (br s, 1H, NH), 6.14 (m,
1H, NH), 5.67 (d, J 7.2 Hz, 1H, CH), 5.09 (s, 2H, CH.sub.2), 3.78
(s, 3H, OCH.sub.3), 2.88 (br s, 3H, NCH.sub.3). Mass spectrum (ESI)
m/z 527 (100%, MH.sup.+).
Example 1.13
Preparation of
1-((4-((4-methoxybenzyl)amino)-6-((4-(piperidin-1-ylmethyl)benzyl)amino)--
1,3,5-triazin-2-yl)amino)imidazolidine-2,4-di one (compound 13)
Part A Preparation of
1-[4-chloro-6-(4-methoxybenzyl)amino-{1,3,5}-triazin-2-yl-amino]-imidazol-
idine-2,4-dione
##STR00039##
[0194] To a mixture of
1-(4,6-dichloro-[1,3,5]-triazin-2-yl-amino)-imidazolidine-2,4-dione
(Example 1.1, Part A) (3.84 g, 14.6 mmol) and K.sub.2CO.sub.3 (4.4
g, 31.8 mmol) in CH.sub.3CN (50 mL) was added
(4-methoxyphenyl)-methanamine (2.0 g, 14.6 mmol) drop-wise at
0.degree. C. The mixture was stirred at r.t. overnight. TLC showed
the reaction was complete. The mixture was filtered and the cake
was portioned between water and EtOAc. The organic layer was
separated, and the aqueous layer was extracted with EtOAc. The
combined organic layers were washed with brine, dried over
Na.sub.2SO.sub.4 and concentrated in vacuo to give the expected
compound (3.50 g, yield: 66%) as a white solid. It was used for the
next step without further purification. .delta..sub.H (400 MHz,
DMSO-d.sub.6): 11.30-11.23 (m, 1H), 10.06-9.83 (m, 1H), 8.67-8.60
(m, 1H), 7.24-7.13 (m, 2H), 6.90-6.83 (m, 2H), 4.40-4.35 (m, 2H),
4.26-4.05 (m, 2H), 3.73 (s, 3H). Mass spectrum (ESI) m/z 364 (100%,
MH.sup.+).
Part B Preparation of
1-((4-((4-methoxybenzyl)amino)-6-((4-(piperidin-1-ylmethyl)benzyl)amino)--
1,3,5-triazin-2-yl)amino)imidazolidine-2,4-dione (compound 13)
##STR00040##
[0196] A mixture of
1-[4-chloro-6-(4-methoxybenzyl)amino-{1,3,5}-triazin-2-yl-amino]-imidazol-
idine-2,4-dione (Example 1.13, Part A) (150 mg, 0.41 mmol),
4-piperidynylmethylene-benzylamine (101 mg, 0.49 mmol) and
K.sub.2CO.sub.3 (171 mg, 1.24 mmol) in THF (8.0 mL) was heated
under reflux overnight. TLC showed the reaction was complete. The
mixture was cooled to r.t. and poured into water, extracted with
EtOAc repeatedly. The combined organic layers were washed with
brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The
residue solid was purified by flash chromatography (silica gel,
CH.sub.2Cl.sub.2:MeOH=20:1, v/v) to give compound 13 (65 mg, yield:
30%) as an off-white solid. .delta..sub.H (400 MHz, DMSO-d.sub.6):
11.13 (br, 1H), 9.03-8.74 (m, 1H), 7.66-6.77 (m, 10H), 3.91 (s,
1H), 3.72-3.45 (m, 4H), 2.38 (s, 2H), 1.53-1.24 (m, 6H). Mass
spectrum (ESI) m/z 532.3 (100%, MH.sup.+).
Example 1.14
Preparation of 1-((4-((4-methoxybenzyl)amino)-6-((4-(morpholino
methyl)benzyl)amino)-1,3,5-triazin-2-yl)amino)imidazolidine-2,4-dione
(compound 14)
##STR00041##
[0198] Compound 14 was produced using the synthetic procedure set
out in Example 1.13, Parts A and B. .delta..sub.H (400 MHz,
DMSO-d.sub.6): 11.14-11.04 (m, 1H), 7.50-5.50 (m, 10H), 4.41-3.80
(m, 6H), 3.72 (s, 3H), 3.56 (s, 2H), 3.41 (s, 4H), 2.33 (s, 4H).
Mass spectrum (ESI) m/z 534.2 (100%, MH.sup.+).
Example 1.15
Preparation of
1-((4-((4-((diethylamino)methyl)benzyl)amino)-6-((4-methoxybenzyl)amino)--
1,3,5-triazin-2-yl)amino)imidazolidine-2,4-dione (compound 15)
##STR00042##
[0200] Compound 15 was produced using the synthetic procedure set
out in Example 1.13, Parts A and B. .delta..sub.H (400 MHz,
DMSO-d.sub.6): 11.12-11.04 (m, 1H), 9.02-8.92 (m, 1H), 7.62-6.77
(m, 10H), 4.42-4.23 (m, 4H), 4.09, 3.92 (m, 2H), 3.72 (s, 3H), 3.53
(s, 2H), 3.55-3.52 (m, 4H), 0.99 (br s., 6H). Mass spectrum (ESI)
m/z 520.3 (100%, MH.sup.+).
Example 1.16
Preparation of
1-((4-((4-methoxybenzyl)amino)-6-((4-((4-methylpiperazin-1-yl)methyl)benz-
yl)amino)-1,3,5-triazin-2-yl)amino)imidazolidine-2,4-dione
(compound 16)
##STR00043##
[0202] Compound 16 was produced using the synthetic procedure set
out in Example 1.13, Parts A and B. .delta..sub.H (400 MHz,
DMSO-d.sub.6): 11.07 (br s, 1H), 9.04-8.89 (m, 1H), 7.49-6.72 (m,
10H), 4.42-4.21 (m, 4H), 4.09-3.94 (m, 2H), 3.72 (s, 3H), 3.40 (s,
2H), 2.34 (br s, 8H), 1.92 (s, 3H). Mass spectrum (ESI) m/z 547.3
(100%, MH.sup.+).
Example 1.17
Preparation of
1-((4-((4-methoxybenzyl)amino)-6-((4-(piperidin-1-yl)benzyl)amino)-1,3,5--
triazin-2-yl)amino)imidazolidine-2,4-dione (compound 17)
##STR00044##
[0204] Compound 17 was produced using the synthetic procedure set
out in Example 1.13, Parts A and B. .delta..sub.H (400 MHz,
DMSO-d.sub.6): 11.14-11.03 (m, 1H), 8.90-8.70 (m, 1H), 7.56-6.84
(m, 10H), 4.35-4.02 (m, 6H), 3.72 (s, 3H), 3.07 (s, 4H), 1.60-1.51
(m, 6H). Mass spectrum (ESI) m/z 518.3 (100%, MH.sup.+).
Example 1.18
Preparation of 1-((4-((4-methoxybenzyl)amino)-6-((4-morpholino
benzyl)amino)-1,3,5-triazin-2-yl)amino)imidazolidine-2,4-dione
(compound 18)
##STR00045##
[0206] Compound 18 was produced using the synthetic procedure set
out in Example 1.13, Parts A and B. .delta..sub.H (400 MHz,
DMSO-d.sub.6): 10.94 (br s, 1H), 8.89-8.70 (m, 1H), 7.75-6.77 (m,
10H), 4.34-4.06 (m, 6H), 334-3.72 (m, 7H), 3.04 (s, 4H). Mass
spectrum (ESI) m/z 520.2 (100%, MH.sup.+).
Example 1.19
Preparation of
1-((4-((4-methoxybenzyl)amino)-6-((4-(4-methylpiperazin-1-yl)benzyl)amino-
)-1,3,5-triazin-2-yl)amino)imidazolidine-2,4-dione (compound
19)
##STR00046##
[0208] Compound 19 was produced using the synthetic procedure set
out in Example 1.13, Parts A and B. .delta..sub.H (400 MHz,
DMSO-d.sub.6): 8.89-8.70 (m, 1H), 7.40-6.81 (m, 10H), 4.35-4.01 (m,
6H), 3.72 (s, 3H), 3.39 (br s, 4H), 3.10 (s, 4H), 2.27 (s, 3H).
Mass spectrum (ESI) m/z 533.3 (100%, MH.sup.+).
Example 1.20
Preparation of
1-((4-((4-methoxybenzyl)amino)-6-((3-(piperidin-1-yl)benzyl)amino)-1,3,5--
triazin-2-yl)amino)imidazolidine-2,4-dione (compound 20)
##STR00047##
[0210] Compound 20 was produced using the synthetic procedure set
out in Example 1.13, Parts A and B. .delta..sub.H (400 MHz,
DMSO-d.sub.6): 11.12-11.04 (m, 1H), 9.03-8.71 (m, 1H), 7.59-6.60
(m, 10H), 4.37-3.98 (m, 6H), 3.71 (s, 3H), 3.09 (s, 4H), 1.59-1.51
(m, 6H). Mass spectrum (ESI) m/z 518.3 (100%, MH.sup.+).
Example 1.21
Preparation of
1-((4-((4-methoxybenzyl)amino)-6-((3-morpholinobenzyl)amino)-1,3,5-triazi-
n-2-yl)amino)imidazolidine-2,4-dione (compound 21)
##STR00048##
[0212] Compound 21 was produced using the synthetic procedure set
out in Example 1.13, Parts A and B. .delta..sub.H (400 MHz,
DMSO-d.sub.6): 11.13-11.03 (m, 1H), 9.04-8.72 (m, 1H), 7.58-6.66
(m, 10H), 4.37-3.98 (m, 6H), 3.72 (s, 7H), 3.07 (s, 4H). Mass
spectrum (ESI) m/z 520.2 (100%, MH.sup.+).
Example 1.22
Preparation of
1-((4-((4-methoxybenzyl)amino)-6-((3-(4-methylpiperazin-1-yl)benzyl)amino-
)-1,3,5-triazin-2-yl)amino)imidazolidine-2,4-dione (compound
22)
##STR00049##
[0214] Compound 22 was produced using the synthetic procedure set
out in Example 1.13, Parts A and B. .delta..sub.H (400 MHz,
DMSO-d.sub.6): 11.07 (br s, 1H), 9.03-8.71 (m, 1H), 7.56-6.61 (m,
10H), 4.36-3.98 (m, 6H), 3.72 (br s, 7H), 3.12 (s, 4H), 2.27 (s,
3H). Mass spectrum (ESI) m/z 533.3 (100%, MH.sup.+).
Example 1.23
Preparation of
1-((4-((4-(2-(diethylamino)ethoxy)benzyl)amino)-6-((4-methoxybenzyl)amino-
)-1,3,5-triazin-2-yl)amino)imidazolidine-2,4-dione (compound
23)
##STR00050##
[0216] Compound 23 was produced using the synthetic procedure set
out in Example 1.13, Parts A and B. .delta..sub.H (400 MHz,
DMSO-d.sub.6): 11.07 (br s, 1H), 9.03-8.71 (m, 1H), 7.57-6.77 (m,
10H), 4.35-3.98 (m, 8H), 3.72 (s, 3H), 2.81 (s, 2H), 2.60-2.51 (m,
4H), 0.99 (t, J=7.2 Hz, 6H). Mass spectrum (ESI) m/z 550.3 (100%,
MH.sup.+).
Example 1.24
Preparation of
1-((4-((4-methoxybenzyl)amino)-6-((4-(2-morpholinoethoxy)benzyl)amino)-1,-
3,5-triazin-2-yl)amino)imidazolidine-2,4-dione (compound 24)
##STR00051##
[0218] Compound 24 was produced using the synthetic procedure set
out in Example 1.13, Parts A and B. .delta..sub.H (400 MHz,
DMSO-d.sub.6): 11.16-11.06 (m, 1H), 9.03-8.72 (m, 1H), 7.57-6.81
(m, 10H), 4.35-3.99 (m, 8H), 3.72 (s, 3H), 3.58 (s, 4H), 2.67 (s,
2H), 2.52-2.46 (m, 4H). Mass spectrum (ESI) m/z 564.3 (100%,
MH.sup.+).
Example 1.25
Preparation of
1-((4-((4-methoxybenzyl)amino)-6-((4-(2-(pyrrolidin-1-yl)ethoxy)benzyl)am-
ino)-1,3,5-triazin-2-yl)amino)imidazolidine-2,4-dione (compound
25)
##STR00052##
[0220] Compound 25 was produced using the synthetic procedure set
out in Example 1.13, Parts A and B. .delta..sub.H (400 MHz,
DMSO-d.sub.6): 10.78 (br s, 1H), 9.03-8.73 (m, 1H), 7.65-6.77 (m,
10H), 4.41-3.95 (m, 6H), 3.72 (s, 3H), 3.60 (s, 2H), 2.55 (br s,
4H), 1.71 (s, 4H). Mass spectrum (ESI) m/z 518.3 (100%,
MH.sup.+).
Example 1.26
Preparation of
1-((4-((4-methoxybenzyl)amino)-6-((4-(((2-methoxyethyl)amino)methyl)benzy-
l)amino)-1,3,5-triazin-2-yl)amino)imidazolidine-2,4-dione (compound
26)
##STR00053##
[0222] Compound 26 was produced using the synthetic procedure set
out in Example 1.13, Parts A and B. .delta..sub.H (400 MHz,
DMSO-d.sub.6): 9.03-8.73 (m, 1H), 7.52-6.77 (m, 10H), 4.40-4.09 (m,
6H), 3.72 (s, 3H), 3.67 (s, 2H), 3.39 (m, 2H), 3.23 (s, 3H), 2.63
(s, 2H). Mass spectrum (ESI) m/z 522.2 (100%, MH.sup.+).
Example 1.27
Preparation of
1-((4-((4-((dimethylamino)methyl)benzyl)amino)-6-((4-methoxybenzyl)amino)-
-1,3,5-triazin-2-yl)amino)imidazolidine-2,4-dione (Compound 27)
##STR00054##
[0224] Compound 27 was produced using the synthetic procedure set
out in Example 1.13, Parts A and B. .delta..sub.H (400 MHz,
DMSO-d.sub.6): 11.07 (br s, 1H), 9.04-8.75 (m, 1H), 7.66-6.74 (m,
10H), 4.44-3.91 (m, 6H), 3.72 (s, 5H), 2.34 (s, 6H). Mass spectrum
(ESI) m/z 492.2 (100%, MH.sup.+).
Example 1.28
Preparation of
1-((4-((4-methoxybenzyl)amino)-6-((4-(piperazin-1-ylmethyl)benzyl)amino)--
1,3,5-triazin-2-yl)amino)imidazolidine-2,4-dione
2,2,2-trifluoroacetate (compound 28)
##STR00055##
[0226] Compound 28 was obtained by deprotection of the
corresponding Boc-protected 4-(piperazin-1-ylmethyl)benzyl)amino
derivative which was produced using the synthetic procedure set out
in Example 1.13, Parts A and B. .delta..sub.H (400 MHz,
CD.sub.3OD): 7.42-6.83 (m, 8H), 4.64-4.47 (m, 4H), 4.20-3.93 (m,
4H), 3.79 (s, 3H), 3.34-3.32 (m, 4H), 3.12-3.01 (m, 4H). Mass
spectrum (ESI) m/z 533.3 (100%, MH.sup.+).
Example 1.29
Preparation of tert-butyl
4-(((4-((2,4-dioxoimidazolidin-1-yl)amino)-6-((4-methoxybenzyl)amino)-1,3-
,5-triazin-2-yl)amino)methyl)benzylcarbamate (compound 29)
##STR00056##
[0228] Compound 29 was produced using the synthetic procedure set
out in Example 1.4, Parts A and B using
1-(4,6-dichloro-[1,3,5]-triazin-2-yl-amino)-imidazolidine-2,4-dione
(Example 1.1, Part A) as the starting material.
Example 1.30
Preparation of
5-((4,6-bis((4-hydroxybenzyl)amino)-1,3,5-triazin-2-yl)amino)-3-methylimi-
dazolidine-2,4-dione (compound 30)
##STR00057##
[0230] Compound 30 was produced adapting the synthetic procedures
described above.
Example 2
Biological Testing
Example 2.1
Inhibition of Proliferation and Duration of Inhibition in Cancer
Cell Lines
[0231] Triazine compounds of the present invention have been shown
to block the cell cycle at G2/M. Triazines were tested for their
inhibitory activity on the "in vitro" growth of tumor cell lines.
For the initial studies two colon carcinoma cell lines (LIM1215 and
SW480) and two breast cancer cell lines (MDA-MB-231 and MCF-7) were
used. Taxol was used as comparator in these experiments (Table
1).
[0232] All triazines tested were at least as effective as Taxol in
inducing G2/M block at their optimal concentration (Table 1).
TABLE-US-00001 TABLE 1 Antimitotic effects of triazines on
carcinoma cell lines. Inhibitors were added to cells in log-growth
for a period of 24 hrs. Cell cycle phase distribution was monitored
by DNA staining and analysis in ModFit. (a) IC.sub.50.for
progression through the cell cycle and (b) the proportion of cells
in G2/M after exposure to 1 .mu.M Taxol was used as a standard;
triazine potency is expressed as proportion of cells in G2M
(test)/proportion of cells in G2M (Taxol). The maximum number of
cells in G2/M in these experiment were: LIM1215 = 78.5%; SW480 =
72%; MDA-MB-231 = 55%; MCF-7 = 62%. NT = not tested. LIM1215 SW480
MDA-MB-231 MCF-7 IC.sub.50.sup.(a) % of IC.sub.50.sup.(a) % of
IC.sub.50.sup.(a) % of IC.sub.50.sup.(a) % of Inhibitor (.mu.M)
Taxol.sup.(b) (.mu.M) Taxol.sup.(b) (.mu.M) Taxol.sup.(b) (.mu.M)
Taxol.sup.(b) Taxol 0.01 100 0.008 100 0.008 100 0.008 100 Compound
3 3 108 1.45 106 3 93 0.45 102 Compound 1 0.07 105 0.05 120 0.013
94 0.009 108 Compound 2 NT NT 0.25 113 0.009 103 0.009 102
Example 2.2
Effects of Triazines on Cancer Cell Growth "In Vitro"
[0233] The inhibition of proliferation was also measured in a panel
of tumor cell lines by the MTT assay (General Protocol see Example
2.6). The IC.sub.50 determined by this assay which measures cell
proliferation matched closely the values calculated from FACS
analysis of G2/M block, indicating that the G2/M arrest does indeed
prevent cell proliferation. Wash-out experiments after incubation
of the cells with the drug resulted in resumption of proliferation
in breast cancer cell lines, and complete arrest in one colorectal
carcinoma cell line, suggesting that the drug is cytotoxic in at
least some cell types.
Example 2.3.1
Biological Effects of Triazines on Tumor Cell Growth
[0234] Compound 2 was tested on a series of cell lines to determine
its activity and selectivity (Table 2). Cell proliferation was
measured using the MTT assay (for protocol see Example 2.6) after 3
days of incubation with the inhibitor.
TABLE-US-00002 TABLE 2 Inhibition of proliferation of tumor cell
lines by compound 2: Cell line IC.sub.50 (.mu.M) BaF/3 .sup.(a)
0.03 LIM 1215 .sup.(b) 0.09 LIM 2537 .sup.(b) 0.07 LIM 2405
.sup.(b) 0.03 SW 480 .sup.(b) 0.03 MDA-MB-231 .sup.(c) 0.01 MCF-7
.sup.(c) 0.01 .sup.(a) murine B-cell progenitor line; .sup.(b)
human colorectal carcinoma; and .sup.(c) human breast
carcinoma.
[0235] The IC.sub.50 for the different cell lines were very
similar, suggesting that compound 2 targets a common pathway
necessary for completion of G2/M.
Example 2.3.2
Effects of the Triazines on Tubulin Polymerization Both `In Vitro`
and `In Vivo`
[0236] The effects of the triazines on tubulin polymerization both
`in vitro` and `in vivo` were tested. These experiments showed that
triazine-induced tubulin polymerization was minimal and occurred at
doses far exceeding the drug potency as a mitotic blocker.
[0237] To monitor the effects of triazines on tubulin, the present
inventors tested tubulin polymerization in intact cells following
exposure to the drug.
[0238] Cells treated with Taxol or with compound 3 were lysed in
hypotonic buffer containing 0.5% NP-40 and the cellular extracts
were centrifuged at high speed. Polymerized tubulin is
detergent-insoluble: thus after this treatment soluble tubulin is
recovered in the supernatants (S), and polymerized tubulin in the
cellular pellets (P). The relative proportion of the two forms was
monitored by SDS/PAGE and immunoblotting with tubulin-specific
antibodies.
[0239] In this test, Taxol strongly stabilized tubulin in a
dose-dependent manner while the triazines showed negligible effects
even at high concentrations (FIG. 1).
[0240] In an attempt to examine direct interactions of the
triazines with tubulin, `in vitro` tubulin polymerization assays
were used (FIG. 2). In these assays the triazines caused a small
but significant increase in tubulin polymerization. However, the
concentration of triazine required for tubulin polymerization was
more than 1,000 times greater than the IC.sub.50 in cell
proliferation assays.
Example 2.3.4
Dose Escalation in Mice
[0241] Compound 1 was initially tested for tolerability and
toxicity in mice. C57Bl/6 mice (aged 8-12 weeks) were injected
subcutaneously with escalating doses of Compound 1 in a constant
volume of DMSO. Mice were sacrificed 24 hrs post-injection. There
were no adverse effects of doses up to 80 mg/kg within this
time-frame. Thus the compound is well tolerated at doses that far
exceed the IC.sub.50 observed "in vitro".
[0242] Compound 2 showed no adverse effects in outward health or
behaviour from doses up to 64.5 mg/kg for 4 days. At 21.5 and 64.5
mg/kg there were indications of skin thinning at the injection site
in some mice. In BALB/c nude mice Compound 2 caused some skin
lesions at 20 mg/kg. These minor skin lesions healed readily within
2-3 days.
Example 2.4
Pharmacokinetics
[0243] The pharmacokinetic parameters for Compound 2 were
determined following injection into mice.
[0244] When delivered at a dose of 50 mg/kg in aqueous solution,
the drug reaches plateau concentrations in the blood stream of 60
.mu.M and remains at levels of up to 6 .mu.M for 6-10 hours. The
elimination half-life is approximately 1.5 hr. At least two active
metabolites, inducing G2/M cell cycle block `in vitro`, have also
been detected in the plasma, and their structures confirmed by mass
spectrometry. When the active metabolites are included in the
pharmacokinetic calculations, the total concentration of the active
drug averages 30 .mu.M for up to 6 hours before it begins to be
eliminated from the plasma.
Example 2.4.1
Extraction and Quantitation of Triazines from Mouse Blood
[0245] The protocol developed for the extraction of active
triazines from mouse plasma and RP-HPLC quantitation of the
extracted drug, as well as the characterization of the metabolites
and the pharmacokinetic properties of the compounds, is described
below.
Example 2.4.1.1
Extraction of Triazines from Mouse Plasma
[0246] Mouse blood was collected by cardiac puncture after
anaesthesia by CO.sub.2 gas. Mice were subsequently euthanized. The
blood was transferred to an EDTA tube to prevent clotting and the
plasma collected after separation by centrifugation. For each
sample 25 .mu.l plasma was added to 75 .mu.l cold 0.1% TFA (v/v)
with 5 .mu.M hydrocortisone (HC) as an internal standard. These
processed samples were stored at -20.degree. C. until required. On
thawing they were clarified by centrifugation and the extracts were
analyzed on RP-HPLC.
Example 2.4.1.2
Quantitation of Triazines in Mouse Plasma
[0247] Each 100 .mu.l plasma extract was separated on a Zorbax
SB-C18, 2.1.times.150 mm, column using a linear 50 min gradient of
acetonitrile in 0.1% (v/v) TFA at a flow rate of 0.1 ml per minute
and a column temperature of 45.degree. C. Detection was at 250 nm.
Representative chromatograms are shown in FIG. 3.
Example 2.4.1.3
Analysis of Compound 2 Metabolites
[0248] Chromatograms of plasma from mice injected with compound 2,
but not from control mice, contained minor peaks. These peaks
appeared at a later time after injection of compound 2, and are
presumably metabolites. In order to confirm the identity of these
peaks, their absorption spectra were compared with that of the
compound 2 standard.
[0249] As can be seen in FIG. 4, all of the peaks had the
characteristic 250 nm absorbance and appear to be related
compounds. The mouse plasma was fractionated on RP-HPLC and the
peaks indicated above were collected. The pooled peak samples were
concentrated by lyophilization. A portion of each peak sample was
reconstituted in culture media for bioassay (FIG. 5). The remainder
was analyzed by LC-MS to confirm the mass and identify the
structures.
[0250] The IC.sub.50 for recovered compound 2 (Peak 1), Peak 2 and
Peak 3 were 0.1 .mu.M, 0.2 .mu.M and >1 .mu.M, respectively.
Although it was difficult to quantify exactly the concentration of
the recovered peaks because of the very small amounts, the assay
does indicate that all recovered peaks had some biological
activity.
[0251] The results of LC-MS analysis of the recovered compounds are
shown in Table 3.
TABLE-US-00003 TABLE 3 Results of LC-MS analysis of metabolites
recovered from mouse plasma after compound 2 injection. Sample Mass
(Da) Compound 2 464.2 (M + 1) Peak 1 464.1 (M + 1) Peak 2 655.0 (M
+ 1) Peak 3 479.1 (M + 1)
[0252] Analysis of the starting material compound 2 confirmed the
molecular weight of the free base compound to be 464 Da. Peak 1 had
the same mass which confirmed that it is the same compound
recovered from the plasma. Peak 3 had a mass of 479 Da suggesting a
hydroxyl derivative. Peak 2 was a much larger compound with a mass
of 655 Da. The additional mass indicates that it is a glucuronide
derivative of the hydroxyl derivative.
[0253] These results confirm that the recovered compounds are
active metabolites of compound 2 and should be included in the
calculations of active levels of drug in plasma and the elimination
half-life of the compound.
Example 2.4.1.4
Pharmacokinetic Analysis of Compound 2
[0254] The concentration of drug in the plasma was determined by
the internal standard method of calibration by comparing the ratios
of drug to HC peak areas relative to the known standards. Plasma
concentrations of compound 2 in the mice from one pharmacokinetics
experiment are plotted in FIG. 6
[0255] These results suggest that therapeutically significant
concentrations of compound 2 can be achieved in the serum, and
maintained for up to 10 hr.
Example 2.5
Efficacy as Anti-Tumor Drug in Xenograft Experiments
[0256] Xenograft experiments have been used to determine the
efficacy of compound 2 in reducing tumor growth. In initial
experiments, using the established tumor model with the colorectal
carcinoma cell line LIM 2537, compound 2 has significantly reduced
the rate of growth of the tumors.
Example 2.5.1
Tumor Xenograft Studies with LIM 2537 Cells (Colon Carcinoma Cell
Line)
Method:
[0257] The colon carcinoma cell line LIM 2537 was chosen for tumor
xenograft studies in BALB/c nude mice, as its growth was shown to
be inhibited by compound 2 (see "Biological effects of triazines on
tumor cell growth").
[0258] The mice were inoculated with 3.times.10.sup.6 cells per
tumor in 100 .mu.l PBS, one tumor on each flank subcutaneously
(SC). Treatments were started on the third day after inoculation
with either vehicle control (VC) or 100 .mu.l of drug at 3 mg/ml in
aqueous solution giving a dose of 15 mg/kg. Mice were injected SC
in the abdomen, three times per week for the duration of the
experiment. The mice were monitored for body weight, tumor volume
and appearance, and for any adverse reactions to the drug. The
experiment was terminated after 33 days and the control group
euthanized, as the tumor burden in that group had reached the
maximum allowed. The remaining mice were then left without
treatment for a further 9 days to monitor resumption of tumor
growth.
Results:
[0259] Mouse body weights were not affected by the drug treatment.
Differences in tumor size between control and drug groups were
apparent after 12 days, and by the 33.sup.rd day, when the control
group was euthanized, this difference was distinct. Within the
groups, there was one slowly growing tumor in the controls and one
fast-growing tumor in the treated cohort. These anomalous tumors
are responsible for the large standard deviations (FIG. 7).
[0260] From this experiment it can be concluded that the treatment
schedule (3 injections per week of compound 2 at a dose of 15
mg/kg) was successful in slowing the growth of LIM 2537 cells.
There was no long term adverse reaction to the drug at this dose
and formulation.
Example 2.5.2
Tumor Xenograft Studies with U87MG(.DELTA.2-7) (Brain Cancer Cell
Line)
Method:
[0261] Mice: Balb/c nu/nu male; 8 controls and 8 treated.
[0262] Cells inoculated: U87MG(.DELTA.2-7) at 2.13.times.10.sup.6
cells/tumour; two tumours per mouse.
[0263] Dose: Compound 2 at 20 mg/kg=100 .mu.l of 4 mg/ml in water
for injection per 20 g mouse. Control mice were injected with 100
.mu.l water for injection.
[0264] Treatment and monitoring: Treatment commenced at Day 5 after
inoculation. Mice were injected SC in abdomen three times per week
(Mon, Wed, Fri), on alternating sides. Mice were weighed, tumour
diameters measured in two dimensions and the health of the mice was
monitored.
[0265] After mice were sacrificed: the tumours were dissected and
weighed; spleens and livers were also weighed.
Results:
[0266] The tumours grew rapidly and consistently between animals.
The experiment was terminated at 14 days after inoculation because
most of the control mice had reached maximum tumour burden, and
some tumours were beginning to ulcerate. At the end of the
experiment there was a significant difference in volume between
control and treated tumours (FIG. 8).
[0267] Livers and spleens were also weighed post mortem. In FIG. 9
the mean weights of these organs are compared, together with the
mean weights of the tumours.
[0268] Treatment of U87MG(.DELTA.2-7) tumours with compound 2 at 20
mg/kg three times per week has reduced the growth of the tumours
compared with tumours in vehicle control injected mice.
Example 2.5.3
Tumor Xenograft Studies with H1437 (Non-Small Cell Lung Cancer
Line)
Method:
[0269] Mice: Balb/c nu/nu male; 8 controls and 8 treated; 5 weeks
old.
[0270] Cells inoculated: H1437 (non-small cell lung cancer cell
line) at 2.times.10.sup.6 cells/tumour; two tumours per mouse.
[0271] Dose: Compound 2 at 20 mg/kg=100 .mu.l of 4 mg/ml in water
for injection per 20 g mouse. Control mice were injected with 100
.mu.l water for injection.
[0272] Treatment and monitoring: Treatment commenced at Day 5 after
inoculation. Mice were injected SC in abdomen three times per week,
on alternating sides. Mice were weighed; tumour diameters measured
in two dimensions and the health of the mice was monitored.
Injections correspond to data points on the plots, with an
additional treatment on Day 14 when no measurements were made.
Results:
[0273] The tumours in the control group grew rapidly but with some
variation between animals. The measured volume of the tumours
stabilized after 21 days, and the growth rate did not increase as
it had with cell lines in previous experiments. These tumours
generally grew as high round balls rather than spreading along the
flank of the animal. The experiment was terminated at 26 days after
inoculation because most of the control mice had reached maximum
tumour burden. There was no ulceration of the tumours which
remained under the skin and did not appear particularly
vascularised. In contrast, the tumours in the treated group
remained very small and hard throughout the 26 days. At the end of
the experiment there was a significant difference in volume between
control and treated tumours (FIG. 10).
[0274] After the mice were sacrificed the tumours were dissected
and weighed. After dissection the tumours from the two groups were
compared. There is a striking difference in the observed size of
the tumours between the two groups (FIG. 11).
[0275] It will be appreciated by persons skilled in the art that
numerous variations and/or modifications may be made to the
invention as shown in the specific embodiments without departing
from the scope of the invention as broadly described. The present
embodiments are, therefore, to be considered in all respects as
illustrative and not restrictive.
Example 2.6
Protocol for MTT and MTS Assay for all Cells and Inhibitors
Example 2.6.1
General Summary
[0276] Cells plated at 104/well in 100 .mu.l medium+5% FCS
(+Add.sup.1): .sup.1 Additives for culture of LIM cell lines: 10
.mu.M thioglycerol, 0.025 U/ml insulin, 1 .mu.g/ml hydrocortisone.
[0277] Cells incubated overnight: [0278] Titrate inhibitors in a
fresh plate in 150 .mu.l medium [0279] Transfer 100 .mu.l titrated
compounds to matching wells of the cell plate, [0280] Incubate a
further 4 days: [0281] Add 10 .mu.l MTS.sup.2 or MTT.sup.3 for 1.5
to 2 hours and read on Multiscanner (wavelengths 492/690) .sup.2
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo
phenyl)-2H-tetrazolium..sup.3
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Example 2.6.2
Cell Lines Used in MTS Assay
[0281] [0282] LIM2537 human colon carcinoma cell line, grown in
RPMI+Adds.sup.1+5% FCS .sup.1 Additives for culture of LIM cell
lines: 10 .mu.M thioglycerol, 0.025 U/ml insulin, 1 .mu.g/ml
hydrocortisone.
Example 2.6.3
Cell Plating: One Cell Line Per Plate
[0283] Cells are trypsinized and washed .times.2 in medium+5% FCS
and plate out at 10.sup.4 cells per well in 100 .mu.l.
[0284] Incubate plates o/n 37.degree. C.+5%-10% CO2 incubator.
Example 2.6.4
Inhibitors
[0285] Inhibitors are dissolved in DMSO to make 10 mM stocks and
titrated in a 96 well plate in duplicate to use as the stock for
the test plate. Titration range from 40 .mu.M down to 0.01 in 1/2
dilutions and 100 .mu.l of each dilution transferred to the
appropriate wells of the test plate that contains the cells (also
in 100 .mu.l) which results in the final concentration of the
inhibitors being 20 .mu.M down to 0.01 .mu.M across the rows in
duplicate. Three inhibitors are tested per plate (Rows A-F).
[0286] Each plate has the following: [0287] Rows A,B: Inhibitor 1
dilutions, 200 .mu.l per well [0288] Rows C,D: Inhibitor 2
dilutions, 200 .mu.l per well [0289] Rows E,F: Inhibitor 3
dilutions, 200 .mu.l per well [0290] Control row G1-6: Medium+5%
FCS (for maximum growth) [0291] Control row G7-12: Taxol (250 nM
for maximal inhibition) [0292] Control row H: Remove medium from
cells and replace with 200 .mu.l Serum Free medium for serum free
growth rate.
Example 2.6.5
Incubation
[0293] Incubate plates for 4 days in incubator. Process with MTS or
MTT as outlined below.
Example 2.6.6
MTS Assay
[0294] To each well add 10 .mu.l MTS (Sigma) solution. Incubate MTS
for 1.5 to 2 hours and MTT for 4 hours. Stop reaction by adding 10
.mu.l 10% SDS to each well. Read on Multiscan (492/690) plate
reader, plot colour change versus concentration of inhibitor to
establish the IC.sub.50.
Example 2.6.7
MTT Assay
MTT Solution
[0295] MTT (Sigma M-2128)--5 g dissolved in PBS, at 5 mg/ml, filter
sterilized and stored at -20.degree. C. Thaw when required.
MTT Solvent (Acidified Isopropanol)
a. 1M HCl:
[0296] Mix 44.6 ml conc HCl (11.2M) in 500 ml DDW b. Acidified
Isopropanol (Isopropanol with 0.04N HCl): [0297] Mix 20 ml 1M HCl
with 480 ml isopropanol (Propan-2-ol, iso-Propyl Alcohol)
MTT Addition
[0297] [0298] Add 10 .mu.l MTT solution to each well and incubate
for 4 hrs in 37.degree. C. incubator. [0299] Spin plates, 5 min at
1500 rpm and carefully flick out medium, without disturbing the
resultant crystals. Add 200 .mu.l acidified isopropanol (MTT
Solvent) per well. [0300] Place on plate shaker, RT, speed 6.5, for
10 min-30 min. [0301] Read OD of plates on Thermo Multiskan Ex, at
560/690 nm.
Example 2.6.8
Results of MTS Assay on Selected Compounds of the Present Invention
Using LIM2537 Human Colon Carcinoma Cell Line
TABLE-US-00004 [0302] TABLE 4 Results of MTS Assay on selected
compounds of the present invention using LIM2537 human colon
carcinoma cell line Compound Number IC.sub.50 Range (.mu.M) 13
>0.1-1 14 >10 15 1-10 16 .ltoreq.0.1 17 .ltoreq.0.1 18
>0.1-1 19 >0.1-1 20 >0.1-1 21 1-10 22 >10 23 1-10 24
>0.1-1
* * * * *